key: cord-008293-5cwb5g3h authors: shaw, shyh-yu; laursen, richard a.; lees, marjorie b. title: analogous amino acid sequences in myelin proteolipid and viral proteins date: 1986-10-27 journal: febs lett doi: 10.1016/0014-5793(86)81502-2 sha: doc_id: 8293 cord_uid: 5cwb5g3h computer analysis of the intrinsic membrane protein, myelin proteolipid, shows strong sequence similarities between the putative extramembrane segments of the proteolipid protein and a number of viral proteins, several of which infect humans. these similarities are even more striking than those reported previously between viral proteins and the encephalitogenic myelin basic protein (mbp). these findings, along with other reports of molecular mimicry by viruses, suggest that immunological cross‐reactions between virus‐induced antibodies or t‐cells and analogous antigenic determinants (epitopes) in myelin proteolipid could be involved in the pathophysiology of multiple sclerosis or post‐infectious demyelinating syndromes. current evidence suggests that the etiology of multiple sclerosis and other demyelinating diseases involves a combination of viral and autoimmune factors [1] . a particularly well studied model for multiple sclerosis is eae which can be induced by immunization of laboratory animals with cns tissue or with mbp, one of the major myelin proteins [2] . recently it has been reported that mbp shows homology with certain viral proteins [3] . furthermore, immunization with peptides having regions common to mbp and hepatitis b virus dna polymerase showed histological eae in rabbits [4] . however, it has long been recognized that components other than mbp contribute to the encephalitogenic response to cns tissue [5] . * to whom correspondence should be addressed abbreviations: mbp, myelin basic protein; eae, experimental allergic encephalomyelitis; cns, central nervous system; mlv, murine leukemia virus; aids, acquired immune deficiency syndrome; htlv-iii/lav, human t-lymphotropic retrovirus the most abundant protein of cns myelin is myelin proteolipid protein, which is embedded in the myelin membrane. its amino acid sequence has been reported recently [6, 7] , and two different models have been proposed for its conformation in the myelin membrane [8, 9] . immunization of rabbits with the proteolipid apoprotein leads to the development of a chronic, progressive or relapsing form of eae [10] . however, the epitopes in myelin proteolipid which canse the encephalitogenic response are not known. in the present study we compared decapeptide sequences from proposed [8, 9] extramembrane hydrophilic regions of myelin proteolipid with various viral proteins by computer analysis, with the aim of searching for similarities, such as found between mbp and viral proteins, that might shed light on the origin of demyelinating diseases. decapeptides were chosen for comparison with the extramembrane segments of the proteolipid protein, the rationale being that decapeptides are more than sufficient to induce an immune response [11] and that epitopes of membrane proteins are generally located at extramembrane hydrophilic regions. comparisons and statistical scorings were made using the database (release 6.8 of 28 august 1985, containing 3309 sequences) and programs of the protein identification resource of the national biomedical research foundation. the search program compared every decapeptide in bovine brain myelin proteolipid with every decapeptide in the database. the calculation of similarity scores was accomplished using the mutation data matrix [12] program. for each proteolipid decapeptide, 709253 comparisons were made and the highest scoring segments and their standard deviations were printed out. we selected those viral decapeptides, similar to extramembrane hydrophilic segments of myelin proteolipid, with scores higher than 30, and which also exceed the average by 5 standard deviations; i.e., the probability that the match is due to random chance is <3 x 10 -7 . a vax 11/750 computer was used for searching and calculations. sequence alignments with scores of 30 or greater were observed between proteolipid and 249 viral decapeptides, excluding identities seen with proteins from similar strains of virus. these include decapeptides from a variety of types of viruses, such as adenoviruses and epstein-barr, influenza and measles viruses. only those with a score of 38 or greater, or which show at least 60% identity are shown in table 1. the degree of similarity between proteolipid and viral proteins is significantly greater than that observed [3] between mbp and viral proteins. furthermore, although only 10% of the proteins in the protein sequence database are viral proteins, they account for over 30% of the similarities. it is interesting and perhaps significant that many of the strong similarities lie between residues 135 and 150 in the proteolipid. this region is predicted (shaw, s.-y., unpublished) to have a strong tendency to form an amphipathic helix (hydrophobic on one side and polar on the other), and thus might be regarded as a prime candidate for an antigenic site. likewise, many similarities are found with the n-terminus of the proteolipid, a region which has also been predicted to be an amphipathic helix [8l. the two proposed models [8, 9] for the organization of the proteolipid in the myelin membrane are table 1 comparison of myelin proteolipid decapeptide sequences with those of certain viral proteins all of the peptides shown have alignment scores of 38 or greater and/or have at least 6 identities. amino acids which are identical with the corresponding residues in the proteolipid are underlined similar in that they depict the protein as threading through the lipid bilayer several times with the polar segraents external to the bilayer; they differ, however, in the sidedness of some of the extramembrane segments. while both models put residues 1-10 and 220-235 on the extracellular face of the membrane, the model of laursen et al. [8] puts residues 35-60 on the extracellular face and 90-150 on the cytoplasmic face, whereas the model of stoffel et al. [9] predicts the opposite orientation for the latter two segments. regardless of which (if either) model is correct, similarities between viral proteins and proposed extracellular and cytoplasmic segments of the proteolipid are seen. it has been proposed [3, 4] that eae-like diseases can arise when virus-evoked antibodies and/or sensitized t-cells cross-react with homologous amino acid sequences (epitopes) in mbp. however, since mbp is located entirely on the cytoplasmic face of the membrane and is therefore within the oligodendroglial cell, it would be expected to be relatively inaccessible to immune surveillance. on the other hand, the proteolipid has potential antigenic sites not only on the cytoplasmic face but also on the outer surface of the membrane. recognition of these sites by antivira[ antibodies or sensitized cells could induce a cascade of immunological events leading to cell destruction. in the process, mbp would be released, which might result in further inflammatory consequences. the similarities between mbp and virus proteins have been discussed [3, 4] previously in terms of 'homology', which implies a common ancestor for the proteins. when comparing myelin proteins with viral proteins, it is difficult to make a compelling case for homology, since the similarities fall off after a few residues. while it is not uncommon to encounter five or six identical residues in a stretch of ten (see table 1), extended regions of similar sequence are encountered only rarely. for example, although a 55% identity is seen between the first 20 residues of the proteolipid and the late 100 kda protein of adenovirus 2 or 5, i.e., gia.e('ca r('i v(;ap.i asi.va illll ii ! ii~ adenovirus (561-580) gi.i.i;('hcr('ni_(.'tpi, trsi.vc, more likely that the virus and myelin proteins are 'analogous', i.e., that similar portions arose by convergent evolution to a common sequence or secondary structure. this idea is consistent with the well-known propensity of viruses to undergo rapid mutation, particularly of their surface antigenic sites [13] . the tryptophan containing region of mbp has long been recognized as being highly encephalitogenic [2, 5] . in particular, the nonapeptide, phe-ser-trp-gly-ala-glu-gly-gin-lys, has been shown to be highly effective in inducing eae in guinea pigs, and studies with peptide analogs indicate that the essential features of an encephalitogenic determinant are x-x-trp-x-x-x-x-gln/asn-lys/arg. the proteolipid contains two regions that have a trp and a lys residue separated by 5 residues, but otherwise they are not remarkably similar to the encephalitogenic mbp peptide. it is interesting, however, that one of the tryptophans (trp-144) occurs in a region that shows several similarities with viral proteins (table 1). in the course of our analysis, we also found a strong similarity, not previously reported [3] , between the encephalitogenic mbp protein and the gag-pol polyprotein of murine leukemia virus it would not be surprising if antibodies against the mlv peptide were found to cross-react with mbp. there are now at least two reports of mimicry of normal human proteins by viruses: mbp by hepatitis b virus polymerase [4] and c~-thymosin by the aids virus htlv-iii/lav [14] . in addition pruijn et al. [15] have reported that sera from patients suffering from autoimmune diseases can inhibit adenovirus dna replication, suggesting a similar phenomenon. the similarities between viral proteins and myelin proteolipid may represent yet another case. in this instance, it seems likely that portions of the protein which are potential antigenic determinants are located on the outer surface of the cell membrane, making it more understandable how the cell membrane could come under immunological attack. the similarity does not extend further. it seems viral infections of the nervous system experimental allergic encephalomyelitis: a useful model for multiple sclerosis hoppc seyler's proc. natl. acad. sci. usa proc. natl. acad. sci. usa this work was supported by grants from the national institutes of health (ns-13649 and ns 16945) and national science foundation (dmb 85-03940). key: cord-024989-0o6agnrc authors: li, qihao; peng, wen; ou, yu title: prediction and analysis of key protein structures of 2019-ncov date: 2020-05-12 journal: nan doi: 10.2217/fvl-2020-0020 sha: doc_id: 24989 cord_uid: 0o6agnrc aim: the purpose of this study was to predict and analyze the structure and function of 2019-novel coronavirus (ncov) key proteins. materials & methods: we obtained the structure and sequence of proteins from related databases and studied them through multiple sequence alignment, homology modeling, sequence analysis, virtual screening, reverse mutation, protein structure overlap and surface property analysis. results & conclusion: we found no significant changes in envelope protein, membrane protein, nucleocapsid protein and key proteases in open reading frame 1ab, and predicted results of proteins and performed molecular dynamics simulations. based on the surface properties of spike protein and docking results with angiotensin-converting enzyme 2, we believe that the binding ability of spike protein to angiotensin-converting enzyme 2 may be similar to sars. these studies will help us in fighting 2019-ncov. we searched the complete genomes of wuhan seafood market pneumonia virus (2019-ncov) and other bat cov in ncbi (ncbi id is as follows: 2019-ncov: nc 045512.2, bat sars cov ratg13: mn996532.1, bat sars cov rs672 / 2006: fj588686.1 bat sars cov zc45: mg772933.1, bat sars cov bj01 ay278488.2, bat sars cov exon1 fj882956.1 and bat sars cov gz02 ay390556.1), and downloaded the amino acid sequence of each key protein. multisequence alignments are analyzed using clustal omega in the european bioinformatics institute. clustal omega is a new multiple sequence alignment program that is used to seed guide trees and hidden markov model profile-profile techniques to generate alignments between three or more sequences. predicting key protein structures using homology modeling after obtaining the amino acid sequence of each key protein in ncbi, the spatial structure of each protein was predicted using the swiss-model homology modeling method. we entered the amino acid sequence and selected the protein structure of the amino acid sequence with the highest homology to the input sequence as a template, which has been experimentally determined for protein structure. the designated template protein data bank (pdb) ids for each key protein are 3cl hydrolase (id: 2z9j), e protein (id: 5x29), plpro (id: 5tl6) and s protein (id: 6acd and 6acc). when the sequence homology of each template reached more than 75%, the predicted structure obtained was highly reliable. through discovery studio 2016 software, we conducted a molecular dynamics simulation of the predicted key protein structure of the virus. the protein molecules were put into physiological saline solvent environment. in the process of minimization and molecular dynamics simulation, the particle-mesh ewald is used to deal with long-range electrostatic interactions. all chemical bonds related to hydrogen atoms are fixed using the shake algorithm. the standard dynamics cascade process includes five stages: minimization, minimization 2, heating, equilibration and production. minimization and minimization 2 is minimized by the 2000-step steepest descent method and the 2000-step conjugate gradient method. the heating step is slowly heating up to 300 k. next, the system performs the simulation of the balancing step under the ensemble of constant pressure normal pressure and temperature (npt). simulation time is set to 200 picosecond (ps) and so on, and 100 conformations are obtained in the production item. for the files generated by the molecular dynamics simulation, the trajectory analysis was carried out. in order to evaluate the structure of the simulation system, we performed the calculation of root mean square deviation. a full-sequence tripeptide library containing 8000 peptides was constructed, and the plpro-predicted structure obtained by homology modeling was used to virtually screen the peptide library. the specific operation is using protonate 3d to protonate the structure of the protein. energy minimize minimizes the added hydrogen bond energy, specifies the catalytic site in the active pocket as a virtual screening target and finally obtains the best-scoring tripeptide. overlapping alignment of protein spatial structure by overlapping the predicted 2019-ncov s protein structure with the template bat sars cov s protein (pdb id: 6acc) structure, we found that there was a spatial structural difference in the s protein between 2019-ncov and previous bat sars covs. we used swiss pdb viewer (spdbv) to open the predicted s protein structures of 2019-ncov (yellow) and bat sars cov (blue) at the same time, and the s protein of bat sars cov was used as the template. according to the sequence alignment results, the identical amino acid sequence from ala 930 to gln 1040 was designated as the overlap position. fit molecules were used for intelligent overlap and finally the overlap result was analyzed. back-mutate study of receptor binding domain of s protein according to the sequence alignment of s protein and docking results of s protein with ace2, there are two obvious changes in receptor binding domain (rbd) of s protein: first, three of the six amino acids that interact with ace2 are mutated; second, a large number of prolines in the proline concentrated region of rbd are replaced. 10 therefore, we reversed the mutated amino acids or replaced them with other amino acids, then docked with ace2, and used pdbepisa of european bioinformatics institute to analyze the docking results. we compared homology of 2019-ncov five key protein structures with other bat sars covs in this section [13] . five key proteins include open reading frame 1ab (orf1ab) in the nonstructural region and four structural proteins: s protein, e protein, m protein and n protein. the results show that the four main proteins of 2019-ncov have the highest homology with that of bat sars cov ratg13 and bat sars cov zc45. e protein of 2019-ncov has 100% homology with that of both bat sars covs ( figure 1a) , and the identity of orf1ab, m and n proteins between 2019-ncov and bat sars covs has also been reached at 94.27% or more ( figure 1b-d) . the comparison results of the s protein showed that it still has the highest homology with bat sars cov ratg13 and bat sars cov zc45 reached 97.71 and 81.85%, respectively ( figure 1e ). overall, 2019-ncov is basically developed from bat sars cov, with the highest degree of homology to bat sars cov ratg13, which is consistent with the results of existing studies [14] , and the degree of agreement with other sars covs is also greater than 75%, suggesting that we can use the homology modeling method to predict 2019-ncov protein structure, which can be used for virtual screening, molecular docking and drug design [15, 16] for accelerating the development of anti-2019-ncov drugs. key protein structures of 2019-ncov are predicted with homologous modeling we selected the existing crystal structure of pdb, which is more than 75% consistent with 2019-ncov's plpro, 3cl hydrolase, structural protein s and e, and used swiss-model's homology modeling method to predict the structure of each protein of 2019-ncov [17] , as shown in figure 2a -d. among them, 3cl protease is the main protein processing enzyme of cov, which is essential for virus replication and proliferation ( figure 2a ) [18] . e protein is also a membrane integrin, consisting of a highly hydrophobic n-terminus (the transmembrane region of e protein) and a c-terminus that extends into the body of the virus ( figure 2b ) [19] . the s protein is the main protein that interacts with host cells on the viral coat ( figure 2c ).the protein produced by plpro digestion is necessary for the virus, because it can activate the synthesis of viral mrna ( figure 2d ) [20] . we separately predicted the structure of these key proteins. ramachandran plot and profile-3d were used to evaluate the quality of the predicted structure ( supplementary figure 1) , and by using two different s protein templates, we predicted two different states conformation of the s protein during its interaction with ace2. this is consistent with the research by wrapp et al. [21] . at the same time, we conducted a molecular dynamics simulation, and the root mean square deviation trajectory of each conformation was basically stable, as shown in figure 2a -d. the structures of these proteins are common targets for drug development. taking the plpro structure obtained by homology modeling as a target, and using a peptide with low toxicity and favorable for clinical acceleration as an example [22] , the full-sequence library of tripeptides was subjected to screening of antivitual drugs. finally, the tripeptide with amino acid sequence val-val-asn (tp8) with strong binding ability to ace2 was obtained. the results show that tp8 can contact and form hydrogen bonds with the catalytic sites his 272 and asp 286 of the plpro ( figure 2e ). differential key protein structure analysis of 2019-ncov although some amino acids were inserted in two positions of nsp3 in orf1ab [23] , the insertion sites were in the nsp3b and nsp3c regions, which are mainly related to the binding reaction of nucleic acids. because the insertion sites are not in the nsp3d region that contain plpro ( figure 3a) , the inserted sequence has little effect on the structure and function of plpro. however, the two transmembrane domains contained in nsp3 are localized on nsp3b and nsp3c [24] . it may affect the localization of the nsp3 protein on the endoplasmic reticulum membrane [25] . the results of s protein sequence comparison showed the largest differences among all proteins. in the rbd site [26] , three of the six key amino acid residues that interact with ace2 have been changed. pro 470 , tyr 484 and thr 487 are converted to glu, gln and asn, respectively ( figure 3b ). it is worth mentioning that the 470th amino acid was changed from nonpolar to polar amino acid. by analyzing the surface properties of the rbd structure of 2019-ncov and bat sars cov (pdb id: 6acc), we found that the rbd region polarity of the 2019-ncov was more dense than the bat sars cov after mutation ( figure 3c ). at the same time, four insert boxes (ibs; 1-4) were inserted into the n-terminus and s2 region of s protein in 2019-ncov ( figure 3b ). we selected the 2019-ncov s protein with a low degree of homology comparison and compared it with the s protein of bat sars cov [27] . it was found that the insertion of ib3 increased the lateral expansion area of the s1 portion of the 2019-ncov s protein, and a loop structure is extended at the overlap with the bat sars cov. the insertion of ib4 also adds a loop structure to the envelope region of s2 ( figure 3d ), and the loop structure of proteins is often closely related to the structure and function of proteins [28, 29] . back-mutating mutant amino acids to study the functional change of rbd of s protein in order to study the effect of interactional amino acid changes in 2019-ncov-ace2 binding region rbd, we mutated the changed three amino acid residues (glu 470 , gln 484 and asn 487 ) within the rbd structure back to the original amino acids. then new, predicted structure is used to analyze the interaction between rbd and ace2. we found that based on the original hydrogen bond, arg 170 of ace2 and thr 486 of rbd added a new hydrogen bond. gln 81 of ace2 forms hydrogen bonds with tyr 484 while forming hydrogen bonds with tyr 435 of rbd ( figure 4a & b). it is suggested that the mutation of three amino acid residues in rbd may weaken the 2019-ncov interaction with ace2. at the same time, we found that compared with ordinary bat sars cov, the four in the five prolines (pro 458 , pro 461 , pro 465 , pro 468 and pro 470 ) of 2019-ncov rbd were replaced with other amino acids ( figure 3b ), so we changed the four amino acid residues to the original proline. the interaction between rbd and ace2 was also analyzed to study the impact of this change on 2019-ncov, but the results showed that the replacement of prolines has little effect on the interaction between 2019-ncov and ace2 ( figure 4a & c) . through homology alignment, we elaborated the sequence differences of each key protein between 2019-ncov and other bat sars covs, and analyzed whether the new sequence changes in 2019-ncov affected the function of each key protein. it was found that the sequence and protein structure of the structural proteins e, m and n of 2019-ncov are basically consistent with that of bat sars cov. considering that the structure determines the function, we believe that these three proteins should not be mutated. although the orf1ab region has two large changes in the sequence, these changed positions are in nsp3b and nsp3c, not in the plpro and 3cl hydrolase regions. therefore, it has little effect on the two proteins that play a key role in the virus replication process. at the same time, we give examples of its application in the screening of peptide drugs after predicting the structure of plpro. among all proteins, the s protein has the largest variation, and most of the changes are located in the s1 region that interacts with ace2. we also predicted two possible conformational changes of the s protein. they are similar to the changes of bat sars cov in the process of binding with ace2, which suggests that the interaction mechanism between 2019-ncov and ace2 may be the same as bat sars cov. based on the amino acid sequence and protein structure alignment, we found that the periphery of the s1 region is more extended than the general bat sars covs. as the most direct structure with the outside world, this may eventually affect its binding to the receptor or its adsorption to objects. the three amino acids that interact with ace2 are altered in the rbd of 2019-ncov. by analyzing the surface properties of the protein, it was found that this change made the region more polar. in order to further study the effect of changed amino acids on the rbd, we back mutated these three amino acids and found that the mutated rbd structure has a stronger effect on ace2. because the rbd is more polar, and the number of hydrogen bonds it interacts with ace2 is reduced, the strength of 2019-ncov binding to ace2 may be similar to common bat sars cov. the results presented in this manuscript demonstrate that the e, m and n protein of 2019-ncov are not significantly different compared with the original bat sars covs. the new fragment inserted in orf1ab has no effect on plpro and 3cl hydrolase. our predicted protein structure is highly reliable and can be used for drug development. we took the low-toxicity peptide library as an example, and successfully screened a tripeptide for potential drugs. for s proteins with large sequence differences, changes of key amino acid residues in rbd reduce the number of hydrogen bonds binding to ace2. however, due to the extension of the loop structure in s1 and future science group 10.2217/fvl-2020-0020 the increase of polarity, this may make the binding ability of 2019-ncov and ace2 similar to that of bat sars cov. from the appearance of the first 2019-ncov infected patient to now, due to the convenience of modern transportation, its global impact far exceeded the sars that occurred in guangdong, china in 2003. cov is an ssrna structure, based on its instability, multiple virus mutations have been found in the past few years. however, because the experimental structure analysis of viral proteins requires time, it is difficult to meet the urgent need for drug development in emergencies. although the virus mutates, it has a high degree of homology with the original bat sars covs. the structure obtained by homology modeling has the potential to replace the crystal structure, and this will help to speed up drug development and facilitate structural and functional analysis of mutant viral proteins. it will play an important role in the future fight against different mutant viruses. • we elaborated the sequence and structure differences in each key protein of 2019-ncov and other bat sars coronaviruses (covs). we found no significant changes in envelope proteins, membrane proteins, nucleocapsid proteins and key proteases in open reading frame 1ab. • we used the method of homology modeling to predict the structure of each key protein, then used molecular dynamics simulation to further process the predicted protein structure. on the basis of predicting a key protein structure, we also predicted two different state changes of s protein structure when it interacts with ace2, and gave an example of the application of papain-like protease structure in peptide drug screening. • we analyzed whether the new sequence changed in 2019-ncov affected the function of each key protein. s protein has the largest variation among all proteins, and we used a virtual back-mutation method to study whether the new amino acid mutation of receptor binding domain of s protein in 2019-ncov has an effect on the interaction between s protein and ace2. through a series of analyses, combined with the docking and simulation of s protein, we believe that the binding ability and mechanism of action between 2019-ncov and ace2 may be similar to that of bat sars covs. • this study combines bioinformatics tools and previous relevant experimental studies on the basis of viral sequences. it can overcome the problem of limited time and lack of experiments in the early stage of the disease, and has a good theoretical and practical basis. financial & competing interests disclosure bat-origin coronaviruses expand their host range to pigs update: severe respiratory illness associated with a novel coronavirus-worldwide potential of large 'first generation' human-to-human transmission of 2019-ncov novel wuhan (2019-ncov) coronavirus updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan the fight against the 2019-ncov outbreak: an arduous march has just begun note from the editors: novel coronavirus (2019-ncov) the genome sequence of the sars-associated coronavirus epidemiology, genetic recombination and pathogenesis of coronaviruses epub ahead of print) future science group cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding pre-fusion structure of a human coronavirus spike protein structure, function and evolution of coronavirus spike proteins clustal omega for making accurate alignments of many protein sequences discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin protein structure homology modeling using swiss-model workspace the swiss-model repository: new features and functionalities swiss-model: an automated protein homology-modeling server potential broad spectrum inhibitors of the coronavirus 3clpro: a virtual screening and structure-based drug design study coronavirus envelope protein: current knowledge the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds cryo-em structure of the 2019-ncov spike in the prefusion conformation progress in regulatory peptide research nsp3 of coronaviruses: structures and functions of a large multi-domain protein nuclear magnetic resonance structure shows that the severe acute respiratory syndrome coronavirus-unique domain contains a macrodomain fold sars coronavirus replicase proteins in pathogenesis structure of sars coronavirus spike receptor-binding domain complexed with receptor swiss-pdb viewer (deep view) modeling of loops in protein structures ab initio construction of all-atom loop conformations the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.no writing assistance was utilized in the production of this manuscript. key: cord-007092-ukqvhzws authors: themsakul, sirintra; suebwongsa, namfon; mayo, baltasar; panya, marutpong; lulitanond, viraphong title: secretion of m2e:hbc fusion protein by lactobacillus casei using cwh signal peptide date: 2016-09-08 journal: fems microbiol lett doi: 10.1093/femsle/fnw209 sha: doc_id: 7092 cord_uid: ukqvhzws the ability to serve as a delivery vehicle for various interesting biomolecules makes lactic acid bacteria (lab) very useful in several applications. in the medical field, recombinant lab expressing pathogenic antigens at different cellular locations have been used to elicit both mucosal and systemic immune responses. expression–secretion vectors (esvs) with a signal peptide (sp) are pivotal for protein expression and secretion. in this study, the genome sequence of lactobacillus casei atcc334 was explored for new sps using bioinformatics tools. three new sps of the proteins cwh, sura and sp6565 were identified and used to construct an esv based on our escherichia coli–l. casei shuttle vector, prceid-lc13.9. functional testing of these constructs with the green fluorescence protein (gfp) gene showed that they could secrete the gfp. the construct with cwhsp showed the highest gfp secretion. consequently, cwhsp was selected to develop an esv construct carrying a synthetic gene encoding the extracellular domain of the matrix 2 protein fused with the hepatitis b core antigen (m2e:hbc). this esv was shown to efficiently express and secrete the m2e:hbc fusion protein. the identified sps and the developed esvs can be exploited for expression and secretion of homologous and heterologous proteins in l. casei. lactic acid bacteria (lab) are a heterogeneous group of bacteria that are used in various biotechnological applications. with respect to human and animal health, many lab are being used as mucosal delivery vehicles for therapeutic and prophylactic molecules because of their gras (generally regarded as safe) status (eijsink et al. 2002) , their probiotic properties and the ease with which they can be engineered to express heterologous proteins (ouwehand et al. 2002) to elicit an effective immune response (lee et al. 2006 ). up to now, a definite conclusion on the best cellular location for optimal immunization, i.e. intracellular, cell wall anchored or secreted, has yet to be reported. this seems to depend on parameters such as the bacterial type, the amount and localization of the antigen, the route of administration, etc. (wells and mercenier 2008) . many expression and secretion systems for lab to deliver protective antigens have already been developed (kruger et al. 2002; pusch et al. 2005) . such systems require the use of expression-secretion vectors (esvs) with a signal peptide (sp) to translocate the protein after synthesis across the bacterial cell membrane. several sps have been identified and incorporated into esvs (hols et al. 1997; hazebrouck, pothelune and azevedo 2007) . the secretion efficiency of different sps seems to be host specific. for this reason, homologous sps are thought to drive protein secretion more efficiently than heterologous sps (mathiesen et al. 2008) . the availability of the complete genome sequences of many lactobacillus casei strains (makarova et al. 2006) makes it easy to search for new and efficient sps that can be used in the construction of new esvs for this species and maybe other lactobacilli. in this study, putative sps were selected from the genome of l. casei atcc334 (accession no. cc000423.1) using bioinformatics analysis. the selected sps were tested for the secretion of a gene product encoding a green fluorescent protein (gfp) as a reporter protein and using the esv based on escherichia coli-l. casei shuttle vectors previously developed in our group (panya et al. 2012; suebwongsa et al. 2013) . the sp with the highest efficiency was then used to drive the secretion of a synthetic gene coding for an m2e:hbc fusion protein. m2e is a highly conserved protein of the influenza a virus that has been incorporated as an antigen into many influenza vaccine formulations. the immunogenicity of m2e has been reported to be enhanced when fused with the hepatitis b core antigen (hbc) (de filette et al. 2005) . the final construct, plc-cwh:m2e:hbc, successfully expressed and secreted the fused antigenic protein. the bacterial strains, plasmid vectors and oligonucleotide primers used in this study are listed in table 1 . lactobacillus strains were grown statically in de man rogosa and sharpe (mrs) medium (labm, heywood, lancashire, uk) at 37 • c. escherichia coli dh5α was cultured in luria-bertani (lb) broth (labm) at 37 • c with shaking. agarified media were prepared by adding 15 g l −1 bacteriological agar to the corresponding broth. when required, appropriate antibiotics (sigma-aldrich, st louis, mo, usa) were added to the media as follows: erythromycin for l. casei at 2.5 μg ml −1 , and ampicillin for e.coli at 100 μg ml −1 . all dna manipulation procedures were performed as described by sambrook and russell (2001) . plasmids from e. coli were isolated and purified using the hiyield plasmid mini kit (rbc bioscience, new taipei city, taiwan). plasmids from l. casei were extracted as described by o'sullivan and klaenhammer (1993) . genomic dna from l. casei was extracted using the genelute bacterial genomic dna kit (sigma-aldrich). pcr amplification was performed using taq dna polymerase (invitrogen, carlsbad, ca, usa). amplicons were purified from agarose gels using the hiyield gel pcr dna fragments extraction kit (rbc bioscience). the nucleotide sequences of cell surface-associated proteins of l. casei atcc334 were retrieved from the ncbi microbial genomes database (http://www.ncbi.nlm.nih.gov/genomes/ lproks.cgi). the signalp4.1 program (http://www.cbs.dtu.dk/ services/signalp/) was used to predict the putative signal peptides. the likely location of the proteins was predicted with the programs psortb v 3.0 (http://www.psort.org/psortb/), subloc v 1.0 (http://www.bioinfo.tsinghua.edu.cn/subloc/pro predict.htm) and gpos-mploc (http://www.csbio.sjtu.edu.cn/ bioinf/gpos-multi/). dna sequences encoding sps of cell surface-associated proteins with a clear sp cleavage site and an extracellular location were selected for the construction of esvs. to determine whether the three selected sps (i.e. cwhsp, surasp and sp6565) could drive heterologous protein secretion in l. casei, an esv based on each of these was constructed. sp strength was analyzed using the gfpuv-encoding gene as a reporter. sequences encoding the three sps were amplified from genomic dna of l. casei atcc334 under the following conditions: one cycle of 95 • c for 3 min; 30 cycles of 95 • c for 30 s, 60 • c for 30 s, 72 • c for 14 s; and 72 • c for 3 min. the lengths of the amplicons, cwhsp, surasp and sp6565, were 129 bp, 105 bp and 126 bp, respectively. the gfpuv gene fragment, recovered as a kpni/spei-digested fragment from pgfpuv, was cloned into pcwh, psura and psp6565 to generate pcwhsp:gfpuv, psursp:gfpuv and psp6565:gfpuv, respectively. the cwh:gfpuv fusion gene was obtained by double digestion of the pcwh:gfpuv with hindiii/spei and cloned into hindiii/spei-digested pldh-pro1, a vector containing the constitutive promoter (p ldh ) and ribosome binding site (rbs ldh ) of the lactate dehydrogenase gene (genbank accession no. d12591.1), resulting in pldh:cwh:gfpuv. using the same procedure, pldh:sur:gfpuv and pldh:sp6565:gfpuv were also obtained. the ldh:cwh:gfpuv dna fragment obtained as a aatii/spei-digested pldh:cwh:gfpuv was cloned into aatii/speidigested prceid-lc13.9 resulting in plc-cwh:gfpuv. the same strategy was followed to obtain the recombinant esvs containing gfpuv with sursp and sp6556, designated as plc-sur:gfpuv and plc-sp6565:gfpuv. figure 1 shows the construction diagram of plc-cwh:gfpuv and a similar procedure was used for the generation of the pldh:sur:gfpuv and pldh:sp6565:gfpuv. all three recombinant plasmids were verified by dna sequencing (data not shown) and were independently electrotransformed into l. casei rceid02. the preparation of competent cells and the electroporation protocol were as described elsewhere (chassy and flickinger 1987) . to determine the secretion efficiency, the fluorescence intensity of culture supernatants derived from recombinant l. casei rceid02 harboring plc-cwh:gfp, plc-sur:gfp and plc-sp6565:gfp was determined in triplicate. cultures of the above three recombinant constructs were incubated at 37 • c for 20 h with shaking at 200 rpm until an optical density of 3.0 at 600 nm (od600) was reached. supernatants of the cultures were harvested and the fluorescence intensity was measured with a fluorometer (cary eclipse, victoria, australia), as previously described (wu and chung 2006) . as a negative control, the culture supernatant of l. casei rceid02 harboring the expression vector without signal peptide (plc-gfpuv) was used. transformants containing plc-cwh:m2e:hbc were cultured at 37 • c for 20 h with shaking at 200 rpm until an od600 of 3.0 was reached. the supernatant and cell pellet were collected. the supernatant was concentrated 10-fold using centricons with a molecular weight cut-off of 10 kda (pall life sciences, ny, usa). the total cell extract and concentrated supernatant were electrophoresed in 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page) followed by western blotting. the m2e:hbc fusion protein on the membrane was detected with mouse anti-m2 monoclonal antibody (abcam, cambridge, uk) at a dilution of 1:1000. horseradish peroxidase-conjugated goat anti-mouse igg (abcam) was used as a secondary antibody at a dilution of 1:10 000. the signal was developed with a chemiluminescent substrate reagent (thermo fisher scientific, rockford, il, usa) and recorded with a digital camera (image quant tm las 4000, uppsala, sweden). total cell extracts from the transformants with plc-cwh:m2e:hbc and those with plc-m2e:hbc were used as positive controls for m2e:hbc expression. supernatants from the transformants with the plc-m2e:hbc construct and those with prceid-lc13.9 were used as negative controls. seventy-seven deduced amino acid sequences of l. casei atcc334 genes encoding cell surface-associated proteins were retrieved from the ncbi database and analyzed with the sig-nalp 4.1 program to predict likely sp sequences (data not shown). the program generates a parameter called the d score, which is used to discriminate signal peptide from non-signal peptide sequences. it also indicates the presumptive location of the signal peptide cleavage site. six proteins with a d score above the cutoff value of 0.450 contained good predictions for sp sequences ( table 2 ). the d score of these proteins and the likely cleavage sites are summarized in table 2 . cellular location of each of the six proteins were predicted using three different programs. five, three and one of six proteins were predicted to be extracellular by gpos-mploc, psortb and subloc programs, respectively. based on this bioinformatics analysis, sps of the proteins encoded by the orfs yp˙805583.1, yp 805328.1 and yp 806565.1 were selected. these were designated as cwhsp, sursp and sp6565, respectively. to determine the secretion efficiency of the selected sequences, esvs based on each sp with the gfpuv gene as a reporter were constructed and designated plc-cwh:gfpuv, plc-sur:gfpuv and plc-sp665:gfp, respectively. these esvs were independently transformed into l. casei rceid02, resulting in the recombinant strains rceid02:cwhsp, rceid02:sursp and rceid02:sp6556. the fluorescence intensity of culture supernatants of these strains, and that of l. casei rceid02 plc-gfpuv (used as a negative control), was determined. the highest fluorescence intensity value was shown by the cwhsp (164.80), followed by that of surasp (118.80) and sp6565 (113.49) (fig. 3) . the cwhsp was selected for constructing the esv containing the m2e:hbc fusion gene, i.e. plc-cwh:m2e:hbc. western blot analysis of the supernatant from l. casei cells containing plc-cwh:m2e:hbc showed a prominent band with an apparent molecular weight of around 27.6 kda (fig. 4, lane 6) , which presumably corresponded to the expected fusion protein. the supernatant from an intermediate construct lacking cwhsp showed no band (fig. 4, lane 4) . the m2e:hbc fusion protein was detected in total cell extracts of both l. casei carrying plc-m2e:hbc (fig. 4 lane 3) and plc-cwh:m2e:hbc (fig. 4 lane 5) . no protein band was detected in either total cell extracts (fig. 4 , lane 1) or supernatant (fig. 4, lane 2) of the negative control strain. engineered lab strains constitute bacterial systems that may serve as alternative mucosal delivery vehicles for a variety of biomolecules (wells and mercenier 2008) . recombinant lab can be used as vaccine vehicles to elicit both secretory iga and systemic antibody responses against the delivered antigen (neutra and kozlowski 2006) . in this study we constructed recombinant l. casei cells expressing m2e:hbc in a secreted form with the use of novel sps derived from the l. casei genome. several sps derived from cell surface-associated proteins and secreted proteins of lab have been identified and used to construct distinct esvs (wu and chung 2006; mathiesen et al. 2008) . of these, the sp of usp45sp from lactococcus lactis (daniel et al. 2011) is the most widely used heterologous sp for protein secretion in lab species (dieye et al. 2001; mathiesen et al. 2008) . previous studies in gram-positive bacteria have shown that the secretion efficiency depends not only on the sp but also on the protein to be secreted and on bacterial host (dieye et al. 2001; mathiesen et al. 2008) . nonetheless, studies on the sp functionality for heterologous protein secretion in lactobacillus plantarum wcfs1 found that the homologous sps have similar or higher secretion efficiencies than those of heterologous origin (mathiesen et al. 2008) . aimed to express and secrete m2e:hbc in l. casei, we screened for homologous sps using a bioinformatics approach. in our hands, the best sp predictor is signalp4.1 (petersen et al. 2011) . this program has been successfully used to select efficient sps from the protein pool of l. plantarum wcsf1 (mathiesen et al. 2008) . the subloc, psortb and gpos-mploc programs were also used to predict the localization of the proteins produced. using these programs, three putative sps (cwhsp, surasp and sp6565) were selected from cell surface-associated proteins of l. casei atcc334 and used for construction of esvs with gfp as reporter protein. the reason that the subloc program predicts cwhsp and sp6565 as periplasmic might be due to the fact that this program uses a dataset based on that of reinhardt and hubbard (1998) . this dataset contains a small number of sequences of both extracellular and periplasmic groups for training the neural network, which may result in a less accurate prediction. based on these esv constructs, it was found that all three sps can function for gfp secretion in l. casei rceid02, and that the construct using cwhsp provides the highest gfp secretion. previous studies found that an efficient signal peptide in gram-positive bacteria generally contains the consensus sequence vla-x-ala↓ala or ala-x-ala↓ala as the sp cleavage site (nielsen et al. 1997; mathiesen et al. 2008) . in this study, all three selected sps have the consensus motif val-x-ala at position −3 to −1 relative to the putative cleavage site. however, none of the selected sps contained ala at position +1. in addition, the most efficient sp, cwhsp, harbors ser at position +1, which indicates ala at this position is not required. cwhsp was further used to construct an esv containing m2e:hbc. recombinant l. casei carrying this construct expressed and secreted m2e:hbc successfully, demonstrating the usefulness of bioinformatics to predict and select novel sps for l. casei. however, this approach does not guarantee that the sps selected will function, since protein secretion depends on the sp itself, the genetic background of the host strain and the target protein. in addition, secretion efficiency may be affected by the extent to which protein production levels and rates are adapted to the capacity of the translocation machinery, the influence of sp sequence on mrna stability and its translation efficiency (mathiesen et al. 2008) . for these reasons, to maximize the production of a secreted protein in lactobacillus, and before optimization and balancing of other factors, it might be worthwhile selecting an optimal sp for each individual protein. besides conferring only short-term immunity, a major drawback of the current influenza virus a vaccines is that they confer only short-term subtype-specific protection. thus, keeping vaccines up-to-date with influenza a antigens requires constant monitoring of the subtypes of the circulating viruses (johansson and brett 2007) . this inconvenience has led to much discussion about the development of a universal influenza a vaccine, able to provide protection against all virus subtypes (du, zhou and jiang 2010) . such a vaccine would require identification of a viral component conserved through all subtypes of influenza a virus for eliciting a broad-spectrum immunity (stanekova and vareckova 2010) . the extracellular domain of the m2 protein (m2e) of the influenza virus is one such conserved component (ebrahimi and tebianian 2011) . however, this is a short peptide of only 24 amino-acid residues. in this study, the m2e-encoding gene was fused with the gene coding for the hepatitis b core antigen (hbc) protein in order to increase its immunogenicity. in a previous study, strong immunogenicity and full protection were obtained in mice after either intraperitoneal or intranasal administration of the m2e:hbc fusion protein (de filette et al. 2005) . considering their gras status, the use of l. casei as an antigen delivery vehicle can confer great advantage for the expression of the m2e:hbc fusion protein. as a conclusion, in this study, we used a bioinformatics approach to select potential sps from the l. casei atcc334 genome. the three selected sps, which include those of cwh, sura and 6565 proteins, proved to direct secretion of the gfp in l. casei. cwhsp, which showed the highest secretion efficiency, was then used to develop an esv construct expressing and secreting the fused m2e:hbc protein. this sp and the esv developed may further serve for the expression and secretion of a variety of homologous and heterologous proteins in l. casei and surely in other lab species. transformation of lactobacillus casei by electroporation recombinant lactic acid bacteria as mucosal biotherapeutic agents universal influenza a vaccine: optimization of m2-based constructs design of a protein-targeting system for lactic acid bacteria research and development of universal influenza vaccines influenza a viruses: why focusing on m2e-based universal vaccines production of class ii bacteriocins by lactic acid bacteria; an example of biological warfare and communication efficient production and secretion of bovine β-lactoglobulin by lactobacillus casei. microb efficient secretion of the model antigen m6-gp41e in lactobacillus plantarum ncimb 8826 changing perspective on immunization against influenza in situ delivery of passive immunity by lactobacilli producing single-chain antibodies mucosal immunization with surfacedisplayed severe acute respiratory syndrome coronavirus spike protein on lactobacillus casei induces neutralizing antibodies in mice comparative genomics of the lactic acid bacteria heterologous protein secretion by lactobacillus plantarum using homologous signal peptides mucosal vaccines: the promise and the challenge identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites high-and low-copy-number lactococcus shuttle cloning vectors with features for clone screening probiotics: an overview of beneficial effects sequencing and analysis of three plasmids from lactobacillus casei tistr1341 and development of plasmid-derived escherichia coli-l. casei shuttle vectors signalp 4.0:discriminating signal peptides from transmembrane regions bioengineering lactic acid bacteria to secrete the hiv-1 virucide cyanovirin using neural networks for prediction of the subcellular location of proteins molecular cloning: a laboratory manual conserved epitopes of influenza a virus inducing protective immunity and their prospects for universal vaccine development cloning and expression of a codon-optimized gene encoding the influenza a virus nucleocapsid protein in lactobacillus casei coli host strains significantly affect the quality of small scale plasmid dna preparations used for sequencing mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria green fluorescent protein is a reliable reporter for screening signal peptides functional in lactobacillus reuteri we would like to acknowledge prof. david blair for editing the manuscript via the kku publication clinic. this study was supported by the higher education research promotion and national research university project of thailand, office of the higher education commission, thailand and by the invitation research grant (i57301) from the faculty of medicine, khon kaen university. none declared. key: cord-004673-c8qcjve9 authors: faaberg, k. s.; plagemann, p. g. w. title: membrane association of the c-terminal half of the open reading frame 1a protein of lactate dehydrogenase-elevating virus date: 1996 journal: arch virol doi: 10.1007/bf01718835 sha: doc_id: 4673 cord_uid: c8qcjve9 orf 1a of lactate dehydrogenase-elevating virus, strain p (ldv-p), encodes a protein of 2206 amino acids. eisenberg hydrophobic moment analysis of the protein predicted the presence of eleven transmembrane segments in the c-terminal half of the molecule (amino acids 980–1852) that flank the serine protease domain. cdnas encoding orf 1a protein segments encompassing transmembrane segments 5 to 11 and its amphipathic c-terminal end as well as the n-terminal 80 amino acids of the downstream orf 1b protein were transcribed and the transcripts in vitro translated in the absence and presence of microsomal membranes. the synthesis of the protein products with putative transmembrane segments was enhanced by the presence of the microsomal membranes and the proteins became membrane associated. when synthesized in the absence of membranes they were recovered in the supernatant upon ultracentrifugation of the translation reaction mixtures, whereas they were recovered in the membrane pellet when synthesized in the presence of membranes. furthermore, the latter proteins were not released from the membranes by disruption of the membrane vesicles in carbonate buffer, ph 11.5, and large portions of the proteins were resistant to digestion by trypsin, chymotrypsin and proteinase k. no n-glycosylation was observed and only little, if any, processing of the protein by the putative serine protease. the results indicate that the c-terminal half of the orf 1a protein represents a non-glycosylated integral membrane protein. potential modes of synthesis and function of the protein are discussed. in addition, the results showed that the synthesis of the orf 1a protein was generally terminated at its termination codon, but that read-through into the orf 1b gene occurred with low frequency. summary. orf la of lactate dehydrogenase-elevating virus, strain p (ldv-p), encodes a protein of 2206 amino acids. eisenberg hydrophobic moment analysis of the protein predicted the presence of eleven transmembrane segments in the c-terminal half of the molecule (amino acids 980-1852) that flank the serine protease domain, cdnas encoding orf la protein segments encompassing transmembrane segments 5 to 11 and its amphipathic c-terminal end as well as the n-terminal 80 amino acids of the downstream orf lb protein were transcribed and the transcripts in vitro translated in the absence and presence of microsomal membranes. the synthesis of the protein products with putative transmembrane segments was enhanced by the presence of the microsomal membranes and the proteins became membrane associated. when synthesized in the absence of membranes they were recovered in the supernatant upon ultracentrifugation of the translation reaction mixtures, whereas they were recovered in the membrane pellet when synthesized in the presence of membranes. furthermore, the latter proteins were not released from the membranes by disruption of the membrane vesicles in carbonate buffer, ph 11.5, and large portions of the proteins were resistant to digestion by trypsin, chymotrypsin and proteinase k. no n-glycosylation was observed and only little, if any, processing of the protein by the putative serine protease. the results indicate that the c-terminal half of the orf la protein represents a non-glycosylated integral membrane protein. potential modes of synthesis and function of the protein are discussed. in addition, the results showed that the synthesis of the orf la protein was generally terminated at its termination codon, but that read-through into the orf lb gene occurred with low frequency. lactate dehydrogenase-elevating virus (ldv) belongs to a new group of positive-strand rna viruses, presently classified as genus arterivirus [3] , which also includes equine arteritis virus (eav), simian hemorrhagic fever virus, and porcine reproductive and respiratory syndrome virus (prrsv) [22, 23] . expression of the viral genomes of 12-15 kb in infected cells is via the formation of 3'coterminal nested sets of 6 or 7 subgenomic mrnas. the major structural proteins of these viruses are translated from subgenomic mrnas 5, 6, and 7. the 5' three-quarters of the viral genome encodes two large proteins, la (1727-2396 amino acids) and lb (1410-1448 amino acids) which are translated from genomic rna. the orf lb protein is expressed via a frameshift mechanism involving a slippery sequence and a pseudoknot [8] . the orf lb proteins of these viruses possess several common functional motifs, i.e. replicase, helicase, and zinc finger motifs, and probably represent the rna replicases of these viruses. the orf la protein of eav possesses an n-terminal papain-like cysteine proteinase (pcp) that autocatalytically cleaves off the n-terminal 29-kda end of the protein; (nonstructural protein-l; nsp-1) downstream of the catalytic pcp residues [26] . the orf la proteins of ldv and prrsv possess two pcps. both are functionally active in cleaving off n-terminal products of about 21 (nsp-la) and about 26kda (nsp-l[3), respectively [7] (see fig. 1 ). in the case of eav, a 61 kda product (nsp-2) is then removed by another cysteine proteinase cp [7] . in addition, the orf la proteins of these viruses possess a serine proteinase (sp) motif in the c-terminal half of the molecule (see fig. 1 ), and the sp has been suggested to be responsible for the further cleavage of the protein into functional units [7, 26] . the function of the orf la proteins is unknown. however, hydrophobic moment analyses of the orf la proteins of ldv, eav, and prrsv have identified similar 11 or 12 potential transmembrane segments in their c-terminal segments of about 1200 amino acids [20] which flank the sp motif, 4 to 7 segments on either side (see fig. 1 ). the predicted structure is unique among virus-encoded proteins and implies some specific function of the orf la proteins in arterivirus replication. the only other proteins with 11 or more transmembrane segments are membrane-associated transport proteins [2, 6, 15, 19] . in the present study we provide strong evidence that the segment of ldv orf la protein with transmembrane segments 5-11 (see fig. 1 ) becomes intimately associated with endoplasmic reticulum (er) membranes during synthesis and that none of its potential n-glycosylation sites becomes glycosylated. our approach has been to in vitro translate mrnas representing portions ofldv orf la in the absence and presence of microsomal membranes and then to examine the membrane association of the protein products and their susceptibility to degradation by proteinases. at the same time we have examined the efficiency of termination of the orf la protein and of frameshift/readthrough at the slippery sequence at the end of ldv-p orf la (6763gcu uua aac uguuga... ; slippery sequence and orf la termination codon are underlined; [20] ). plasmids pbsk-a66, pbks ÷ 181-3 (g3), and pbks ÷4-6 carry overlapping segments of the 3' end of ldv-p orf la (see fig. 1 ; nt 4202-4934, 4805-5945 and 5833-7043, respectively; genbank accession number u15146) and have been generated in previous studies [20] . cdna 4-6 also encompasses the y-terminal end of orf lb including the putative pseudoknot that is postulated to play a role in frame-shifting between orf la and lb. cdna 4-6 was excised from its bluescript vector with restriction endonucleases bamhi and aatii, incubated with klenow dna polymerase in the presence of deoxynucleotide triphosphates to obtain blunt ends and ligated into the msci site of the pcite-1 vector (novagen, madison, wi) which supplied an aug initiation codon 12 nucleotides upstream of the orf-la segment in pc4.6. the dnas of pbsk-a69, pbsk÷181-3 and pc4.6 were linearized and transcribed with t7 rna polymerases or with t3 rna polymerase in the case of pbsk-a69 as described previously [11] . the integrity of the rna was verified by agarose gel electrophoresis (see later, fig. 3a ). the transcribed rnas were in vitro translated in a rabbit reticulocyte lysate system in the absence and presence of canine pancreatic microsomal membranes as also described previously [10, 11] . for investigating their association with microsomal membranes, the products of translation were incubated and centrifuged under different buffer conditions 1-5, 13, 21] . the translation reaction mixtures were diluted about t00-fold with tris-buffered sucrose (250ram sucrose, 25 mm tris-hct, ph 7.5) or 100mm sodium carbonate, ph 11.5. the mixtures were incubated on ice for 1 h and then centrifuged for 1 h at about 100 000 x g at 4 °c. when incubated in isotonic sucrose buffer, the microsomal membranes remain closed vesicles and all proteins associated with the membrane and those located within the vesicles will be located in the membrane pellet (p), whereas proteins synthesized on free ribosomes will be recovered in the supernatant (s). in contrast, when translation products are incubated in 100mm na÷-carbonate at ph 11.5, the membrane vesicles are disrupted and only integral membrane proteins remain associated with the pelleted membranes, whereas secreted glycoproteins and peripheral proteins are recovered in the supernatant [13] . after centrifugation, the proteins in the supernatant (s) were precipitated with trichloroacetic acid, washed with acetone, and air dried. the pelleted material (p) was washed once in sterile water. the s and p proteins were suspended in reducing sample buffer and analyzed by tricine sodium dodecylsulfate polyacrylamide gel electrophoresis (sds-page) [25] using 16.5%t: 3%c or 12.5%t: 3%c gels (t refers to total percentage of acrylamide and bisacrylamide monomers and c refers to the percentage of crosslinker relative to t [ 17] ) as described previously [11] . only the orf la segment present in each transcript could yield a protein of significant size since the other two reading h-ames in each transcript did not encode any protein longer than 34 amino acids. the main product of the a69 transcript whether translated in the absence or presence of microsomat membranes was a protein of about 2022 kda ( fig. 2a, lanes 1 and 4) . however, the presence of membranes enhanced translation of the a69 transcript and the product was almost exclusively recovered in the membrane pellet and thus associated with membranes, whereas the protein synthesized in the absence of membranes was mainly recovered in the s fraction. incubation in carbonate buffer, ph 11.5, did not cause a significant release of the protein from the membranes (fig. 2b, lanes 2 and 5) . thus the protein product became integrated into the membranes during membrane-associated synthesis. translation must have been initiated at one of the three aug codons of orf la located close together at the 5' end of the transcript, but which one is not clear. none of the three augs are in favorable context for translation [18] with c rather than a purine at the -3 position, relative to the a(+ 1) of the aug codon but the same is the case for the initiation codons of most of orfs 2-7 which function efficiently and specifically in translation initiation [11] . the termination codon was provided by the vector so that the six terminal amino acids in the protein product were derived from vector sequences. initiation at one of the three aug codons would yield proteins with 246, 234 or 205 amino acids, respectively. another minor product of about 45-kda was consistently produced from the a69 transcript with properties similar to those of the about 22-kda protein. its nature is unknown. the results for the translation of the 183-1 transcript, which also encodes a protein sequence with potential transmembrane segments, were similar to those described for the a69 transcript. the main product of about 36 kda was recovered in the s fraction when synthesized in the absence of membranes, whereas it was recovered in the p fraction when synthesized in the presence of membranes ( fig. 2a, lanes 5 and 8) . incubation in carbonate buffer, ph 11.5, did not cause a significant dissociation of the protein from the membranes (fig. 2b, lanes 8 and 9) . translation was markedly enhanced by the presence of membranes. since the molecular weight of the product was the same whether the protein was syl~thesized in the absence or in the presence of membranes, none of three potential n-glycosylation sites in the predicted protein (see fig. 1 ) became fig. 2 . analysis of the membrane association of the in vitro translated proteins a69, 183-1, and 4-6. the transcripts of the appropriate plasmids were in vitro translated in the absence and presence of pancreatic membranes (-or +, respectively). the reaction mixtures were further incubated in trissucrose, ph 7.5 (a), or carbonate buffer, ph 11.5 (b) and centrifuged. the proteins in the supernatant (s) and the pellet (p) were analyzed by tricine sds-page using 16.5%t: 3% gels glycosylated. in contrast, we have shown previously [11, 12] , that under the same experimental conditions the orf 2-5 glycoproteins of ldv-p become nglycosylated in vitro when synthesized in the presence of membranes. there were also membrane-associated proteins of about 28, 24, and 16 kda synthesized from the 183-1 transcript (fig. 2a, lane 8) . since the putative transmembrane segments fall in the middle of the expected product (see fig. 1 ), the smaller products could have resulted from premature termination or initiation at more downstream aug codons. the former was probably the case, since there is only one additional in-frame aug codon that could yield a product with putative membrane segments and approximately the size (22 kda) of one of the smaller products and it is in very unfavorable context for initiation. processing by the serine proteinase could also not play a role in generating the smaller products since the potential 183-1 product does not contain the complete sp motif. the primary findings concerning the a69 and 183-1 proteins were that they are, as predicted by their hydrophobicity profiles, integral membrane proteins and that they are not n-glycosylated. as also predicted by the hydrophobic moment analyses (fig. i) , the pc4.6 product did not become membrane-associated. a transcript of pc4.6 could potentially yield two protein products if a frame-shift occurred at the slippery sequence at nt 6765-6771 (see earlier and fig. 1 ). one protein would be composed of 321 amino acids (35.2 kda), 4 nterminal amino acids encoded by the vector plus the 317 amino acid long c-terminal end of the orf la protein. the second protein would possess an additional 80 amino acids (about 10 kda), 76 representing the n-terminal end of the orf lb protein plus c-terminal 4 amino acids encoded by the vector. both products were generated. since the 44 kda product possessed twice as many met residues as the 35 kda protein, namely six, the level of radioactivity in the two proteins indicates that the read-through product was synthesized in considerably lower amounts than the terminated orf la protein ( fig. 2a, lane 9 ). since significant amounts of a 10 kda protein were not produced, little, if any, cleavage of the 10 kda orf lb protein segment from the 44 kda read-through product occurred. the presence of membranes had no significant effect on the translation of the pc4.6 transcript and the products were recovered in the soluble supernatant fraction whether synthesized in the presence or absence of membranes ( fig. 2a, lanes 9-12) . the predicted catalytic triad of the sp of the ldv-p orf la protein consists of his-1551, asp-1576, ser-1646 [14, 20] . his-1551 and asp-1576 are present in the c-terminal end of the a69 protein. ser-1646 is encoded in cdna 183-1. in order to examine the functionality of the sp and to further examine the membrane association of the orf la protein, we have joined a69 and 183-1 at a styi site in their overlapping segment (see fig. 1 ) and in vitro transcribed/translated the construct. a69 was excised from pbsk-a69 using restriction endonucleases styi and ncoi releasing a segment of 711 nt (nt 4215-4926). pbks ÷ 183-1 was cut with styi and the a69 segment ligated to it resulting in a clone which represented ldv nts 4779-4926 followed by ldv nts 4215-5945. this clone was then digested with bamhi and ncol (removing ldv nt 4779-4926 from the 5' end), blunt ended and ligated. the resulting construct pbks ÷ a69/183-1 possessed the 5 r terminal aug codons of a69 and potentially encoded a protein of 592 amino acids (63.7 kda), orf la amino acids 1354-1930 plus 15 c-terminal amino acids encoded by the vector. it encompassed the sp-motif and putative transmembrane segments 5---11 (see fig. 1 ). transcription of the construct yielded a transcript of the appropriate size (fig. 3a) . translation of the transcript yielded in some experiments a protein of the expected size of about 64 kda (see fig. 4 ), but in many others, the main product had a size of about 44 kda whether or not membranes were present (fig. 3b, lanes 1 and 4) . the reason for this difference is not known. the 44-kda protein could have resulted from processing of the 64-kda protein, but, if this was the case, processing must have occurred very rapidly since a time course experiment showed that maximum amounts of product were produced by 30 min fig.3 . agarose gel electrophoretic analysis of the transcripts of pbks+a69/183-1, pbsk a69, pbks÷183-1 and pc4.6 (a) and in vitro synthesis of protein a69/183-1 and analysis of its membrane association (b). the a69/183-1 proteins synthesized in the absence and presence of microsomal membranes were analyzed as described in the legend to fig. 2 of incubation. the protein profile changed little, if at all, during the 15-180 min period of translation (data not shown) and there was no indication of the formation of an 18-20 kda protein that would have been expected to be formed in such a processing step (figs. 3b and 4) . only a minor product of 27 to 29 kda was consistently produced (figs. 3b and 4) . furthermore, the formation of the protein products was the same when all proteinase inhibitors (1 gg pepstatin a, 2 gg leupeptin and 2 gg aprotonin per ml, and 50 iam phenylmethylsulfonyl fluoride) were omitted from the translation reaction mixture (data not shown). thus very little, if any, processing by the sp was observed. regardless, when synthesized in the absence of membranes the protein products were recovered in the supernatant fraction, whereas when synthesized in the presence of pancreatic membranes they were recovered in the membrane fraction whether the reaction products were incubated in the buffered sucrose solution or in carbonate buffer, ph 11.5, prior to centrifugation (fig. 3b, lanes 1 and 4 and 5 and 8, respectively) . the results clearly indicate that the primary protein products become membrane associated when synthesized in the presence of membranes. furthermore, the lack of increase in size of the primary products when synthesized in the presence of membranes indicates that the proteins were not significantly glycosylated at any of three potential glycosylation sites. in order to further investigate the membrane association of the a69/183-1 protein we determined its proteinase resistance after synthesis in the presence of microsomal membranes. large portions of the protein seemed to be protected by membrane association from digestion by typsin, chymotrypsin, or proteinase k. the main digestion product was about 18 kda but there were lower amounts of products with apparent molecular weights of about 29kda and 13 14kda k.s. faaberg and p. g. w. ptagemann fig. 4 . sensitivity of the membraneassociated a69/183-t proteins to proteinase digestion. the products synthesized in vitro in the presence of membranes were incubated in trissucrose, ph 7.5 (a), or carbonate, ph 11.5 (b), and then digested for 1 h on ice with trypsin, chymotrypsin or proteinase k (each at a concentration of 0.1~tg/ml), or without them (control) before analysis by tricine sds-page using a 16.5%t: 3%c gel (fig. 4, lanes 2-4) . the same products were generated by proteinase digestion after incubation of the primary product in carbonate buffer, ph 11.5 (fig. 4b) . the lower recovery of some products after the carbonate treatment does not indicate additional protein digestion since a variable loss was also frequently observed with non-proteinase-treated proteins after carbonate, ph 11.5, treatment, apparently resulting from sticking of the proteins to test tube walls [1 ii. the smallest proteinase digestion products varied in size depending on the proteinase used (fig. 4) ; it was the smallest after proteinase k digestion. the proteinase digestion products, except for the 13-14-kda proteins, were larger than the segments making up the transmembrane segments 5-7 (about llkda) or 8--11 (about 15kda). this finding indicates that portions of the a69/183-1 protein in addition to the transmembrane segments became proteinase resistant as a result of membrane association of the proteins. similar findings have been reported for coronavirus m proteins; protein segments upstream and downstream of their transmembrane segments seem to be resistant to chymotrypsin and protein k attack [4, 24] . the same applies to the m/vp-2 and vp-3p proteins of ldv [11] . in contrast, the ldv proteins when synthesized in vitro in the absence of membranes were completely digested by the three proteinases under the same experimental conditions [11] . the authenticity of the in vitro synthesized proteins as ldv proteins was confirmed by immunoprecipitation of the products by anti-ldv antibodies. both the about 44kda and 27-29 kda products of the a69/183-1 rna were precipitated by incubation with plasma from 5-month ldv-infected mice (imp) but not by normal mouse plasma (nmp), or monoclonal antibodies to ldv n/vp-1 or vp-3 (fig. 5, lanes 1-5) . the same applied to the about 44kda and fig. 5a , b. immunoprecipitation of in vitro synthesized products of the a69/183-1 transcript mouse plasma (imp), anti-vp-1 mab c350201.7 or anti-vp-3pmab 159-12 as described previously [ 11 ] 35 kda products of the 4.6 rna (fig. 5, lanes 6-10) . the results are also of interest in that they demonstrate that mice mount antibody responses to the c-terminal half of the orf la protein. in conclusion, the present study shows that at least a large portion of the c-terminal half of the ldv orf la protein represents an integral membrane protein(s). the in vitro synthesis of the portion of the orf la protein with the putative transmembrane segments is enhanced by the presence of microsomal membranes and the protein(s) synthesized in the presence of the membranes becomes inserted into the membrane in a form that is not released by disruption of the membrane vesicles in a carbonate buffer, ph 11.5, and that is largely protected from proteinase digestion. much larger portions of the protein seem to become proteinase resistant than strictly the portions making up the putative transmembrane segments, suggesting intimate association with the membrane. the membrane association of a large portion of the orf la protein suggests that the orf la protein or at least its c-terminal half is synthesized in vivo on rough er. one scenario suggests that the synthesis of the orf la protein starts on free ribosomes, that the n-terminal end is processed autocatalyticalty by the two paps releasing products of 22 and 26 kda, (fig. 1 ; nsp-l~ and 113, respectively) [7-1. this might be followed by the autocatalytic release of another protein (nsp-2) by the action of cysteine protease [7] . one of the putative transmembrane segments then could function as a processed or uncleaved signal peptide so that the synthesis of the remainder of the orf la protein occurs on membrane vesicles, however, in a manner that prevents n-glycosylation. in contrast, all the n-glycosylation sites in the ectodomains of the orf 2 and orf 5 glycoproteins become glycosylated during membrane-associated in vitro synthesis and the oligosaccharide chains seem to become processed during traverse through golgi membranes in vivo [11, 12] . nothing is known about the functions of the orf la protein. however, the integration of at least a large portion of the protein into er membranes suggests one possibility, namely that this process results in the formation of unique double-membrane vesicles that are invariably associated with the replication of all arteriviruses regardless of the nature of the host cell [23, 27, 28] . electron micrographs suggest that these double membrane vesicles originate from rough er by protrusion and detachment [28] . their formation is a very early event in ldv replication in macrophages and free nucleocapsids first appear among these vesicles before budding into single-membrane cisternae located generally next to golgi membranes [23, 27] . these findings combined with the uniqueness of these structures suggests that these double membrane vesicles may play an important role in virus replication. the possibility that comes to mind is a role in the synthesis of viral genomic and subgenomic lnrnas since viral rna synthesis in general seems to be membrane associated. the function of membranes and their associated proteins in viral nucleic acid synthesis is not known, but they might supply some organizational component to the replication complex and/or facilitate rna folding as suggested for rna chaperones [16] . in the case of the arteriviruses, the orf lb replicase protein is not an integrai membrane protein and probably supplies the catalytic rna replicase functions since it possesses helicase, replicase, and zinc binding motifs [22] . our results as well as those for eav [8] suggest that the orf lb protein is synthesized in much smaller amounts than the orf la protein which is typical for ribosomal frame shifting [1] . our results are consistent with the hypothesis that the orf la and lb proteins provide structural and catalytic functions, respectively. ribosomal frame shifting on viral rnas structure and function ofcytochrome c oxidase revision of the taxonomy of the coronavirus, torovirus and arterivirus genera coronavirus ibv glycopolypeptides: locational studies using proteases and saponin, a membrane permeabilizer new variation on the translocation of proteins during early biogenesis of apolipoprotein b structure and function of mammalian facilitated sugar transporters processing and evolution of the n-terminal region of the arterivirus replicase orf 1 a protein: identification of two cysteine proteases equine arteritis virus is not a togavirus but belongs to a coronavirus-like superfamily analysis of membrane and surface protein sequencers with the hydrophobic moment plot disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity the envelope proteins of lactate dehydrogenaseelevating virus and their membrane topography open reading frame 3 of lactate dehydrogenaseelevating virus encodes a soluble, non-structural highly glycosylated and antigenic protein isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum complete genomic sequence and phylogenetic analysis of the lactate dehydrogenaseelevating virus (ldv) the multidrug transporter, a double-edged sword rna chaperones and the rna folding problems molecular-sieve" chromatography of proteins on columns of cross-linked polyacrylamide the scanning model for translation: an update the high amnity na+/glucose cotransporter sequence ofgenome of lactate dehydrogenase-elevating virus: heterogeneity between strains p and c a former amino terminal signal sequence engineered to an internal location directs translocation of both flanking protein domains lactate dehydrogenase-elevating virus and related viruses lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand rna viruses assembly in vitro of a spanning membrane protein of the endoplasmic reticulum: the e1 glycoprotein of coronavirus mouse hepatitis virus a59 orf la protein ldv tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100kda proteolytic processing of the replicase orf la protein of equine arteritis virus replication of lactate dehydrogenase-elevating virus in macrophages. 1. evidence for cytocidal replication electron microscopic studies on the morphogenesis of prrsv in infected cells -comparative studies we thank brent langley for competent secretarial help. during part of the work ksf was supported by usphs training grant ca 09138. received december 8, 1995 key: cord-002711-b7mlt19n authors: jacomin, anne-claire; samavedam, siva; charles, hannah; nezis, ioannis p. title: ilir@viral: a web resource for lir motif-containing proteins in viruses date: 2017-08-14 journal: autophagy doi: 10.1080/15548627.2017.1356978 sha: doc_id: 2711 cord_uid: b7mlt19n macroautophagy/autophagy has been shown to mediate the selective lysosomal degradation of pathogenic bacteria and viruses (xenophagy), and to contribute to the activation of innate and adaptative immune responses. autophagy can serve as an antiviral defense mechanism but also as a proviral process during infection. atg8-family proteins play a central role in the autophagy process due to their ability to interact with components of the autophagy machinery as well as selective autophagy receptors and adaptor proteins. such interactions are usually mediated through lc3-interacting region (lir) motifs. so far, only one viral protein has been experimentally shown to have a functional lir motif, leaving open a vast field for investigation. here, we have developed the ilir@viral database (http://ilir.uk/virus/) as a freely accessible web resource listing all the putative canonical lir motifs identified in viral proteins. additionally, we used a curated text-mining analysis of the literature to identify novel putative lir motif-containing proteins (lircps) in viruses. we anticipate that ilir@viral will assist with elucidating the full complement of lircps in viruses. autophagy is a multistep process that consists of the isolation of cytoplasmic components into double-membrane vesicles, called autophagosomes, that shuttle to lysosomes, which serve as end-point degradative organelles. it is a catabolic mechanism that enables the removal of damaged or excess cellular organelles and proteins, thereby contributing to the maintenance of cell homeostasis and survival. 1 the autophagic machinery is highly conserved from unicellular eukaryotes to metazoans. among the proteins that take part in this process, the atg8-family proteins play a central role. 2 indeed, these proteins are involved in the elongation and maturation of the autophagosome and its fusion with lysosomes. 1, 3 phosphatidylethanolamine-conjugated atg8-family proteins reside on autophagosomal membranes where they can contribute to the recruitment of other core autophagy machinery proteins essential for the effective course of the autophagy process. 1, [4] [5] [6] [7] although originally considered to be a nonselective bulk degradation mechanism, a gathering body of evidence over the past decade suggests that autophagy is much more selective than initially appreciated. selective targeting of cellular components to autophagosomes for degradation relies on the existence of selective autophagy receptors able to recognize and tether cargos toward nascent autophagosomes. 8, 9 examples of selective autophagy include aggrephagy, mitophagy, lipophagy, and xenophagy. [10] [11] [12] [13] the interaction between selective autophagy receptors and atg8-family proteins is essential for the proper steering of the cargo for degradation. these receptors typically contain an lc3-interacting region (lir, also known as lrs, aim or gim; the latter correspond to lc3 recognition sequence, atg8-interacting motif and gabarap interaction motif respectively) critical for the binding to atg8-family proteins. 7, [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] the lir motif consists of a short amino acid sequence with a core motif originally described as w/f/yxxi/l/ v (where 'x' represents any amino acids, and referred to as wxxl hereafter). 7, 18 this sequence has lately been relaxed and extended to 6 amino acids to integrate most of the experimentally verified lirs. [ilv] , where the residues in positions 3 and 6 correspond to the most crucial ones for the interaction with atg8-family proteins. [26] [27] [28] besides its role in cellular homeostasis, autophagy is also involved in the innate immune response against pathogens. 13, 29, 30 recent years have seen an outburst of studies on autophagy and viral infections. autophagy may exert a variety of antiviral functions, including the degradation of viral components (known as virophagy), the activation of innate immunity by the delivery of viral nucleic acids to the toll-like receptors, and adaptive immunity through the major histocompatibility complex ii (mch-ii/hla class ii), or the control of the production of reactive oxygen species (ros). [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] however, to be successful, viruses have evolved mechanisms to evade host defense. several viruses have thus developed strategies to use the autophagy machinery or even thrive in the autophagosomes and promote their replication, spread, and survival. [44] [45] [46] [47] other viruses susceptible to autophagy have evolved mechanisms to counteract autophagy activation by expressing proteins that interfere with the cellular machinery, essentially inhibiting the autophagosome-lysosome fusion or interfering with early stages of autophagy activation. [48] [49] [50] [51] [52] [53] [54] [55] [56] a few proteins from viruses infecting mammals and plants that interfere with the host autophagy process have been shown to associate with atg8-family proteins. 52, [57] [58] [59] [60] [61] [62] yet, only one lirdependent interaction has been reported. 62 we have developed a database, ilir@viral (http://ilir.uk/ virus/), that organizes information on the presence of lir motifs in viral proteins. additionally, a curated text-mining analysis of the literature permitted us to predict functional lir motifs in viral proteins that have already been shown to associate with atg8-family members. the ilir@viral database is a web resource freely available to the academic community at http://ilir.uk/virus/. various functionalities are accessible under specific menus. the 'classification' menu gives access to the complete list of putative lir motifs in viral proteins. two virus taxonomic systems have been used: the nomenclature used by the international committee on taxonomy of viruses (ictv) to name the species, genus and families of each of the viruses cited in the database, and the baltimore classification system that groups viruses depending on their genome and kind of replication (dsdna, dsrna, ssdna, ssrna and reverse-transcribing viruses) (see methods). 63, 64 for each specific family or group of viruses, the data are presented in a table containing (i) the clickable uniprotkb accession number of the protein, (ii) the information related to the lir-motif (position, sequence, pssm score, if the pattern is recognized as an xlir or wxxl motif and the presence of the motif in an intrinsically disordered region (anchor)), (iii) the name of the protein and (iv) the name of the species. for some genera, no putative lir-containing protein (lircp) could be identified; for accuracy in the classification, these are listed but appear in red (fig. 1) . the 'search' menu allows the user to look in the database for a specific protein or virus order, family, subfamily, genus, species or common name. uniprot identifiers can also be used in the search function. the blast (basic local alignment search tool) menu offers the user to search the database using a protein sequence as query against the reviewed set of viral proteins from uniprot database. we have used position-specific iterative (psi) blast to search against the database. 65 this search can be run against any of the viral classification systems described above. by default, the blast search runs against all the data available in the database with a default e-value 0.01; nevertheless, the user has the possibility to run the blast search against a specific category (baltimore classification) or order taxonomic rank (ictv classification), and define a different e-value. the results page displays the subject sequences from the database that match the query sequence. the lir patterns are highlighted with red asterisks. the menus 'bibliography' and 'help' provide users with additional information. finally, the 'links' menu gives access to other ilir web resources, inducing the ilir search tool and the ilir database for eukaryote model organisms. 27, 28 analysis of the content of the database we have used the ilir web resource (https://ilir.warwick.ac.uk) to identify lircps in viruses (see methods for details). 27, 28 out of 16,609 reviewed viral sequences available from uniprot across 2569 individual viral species we found that 15,589 of them contain either xlir or wxxl motifs. 6376 proteins figure 1 . screenshot of the ilir@viral database 'classification' menu. example for the ictv classification system. the genera for which no lircps were found appear in red. contain xlir motifs whereas 15,460 contain wxxl motifs. 6247 proteins contain both xlir and wxxl motifs whereas 129 proteins contain only xlir motifs (without containing wxxl patterns) and 9213 proteins contain only wxxl motifs (without xlir patterns) (table s1 ). we found a correlation between the total number of putative lir motifs identified in a family and number of sequences ( fig. 2a) . on average, we found 8.3 lir motifs per sequence. the fact that viral sequences often refer to polyproteins instead of individual proteins is possibly an explanation of the high proportion of patterns identified. the ilir web resource can make the distinction between xlir and wxxl patterns. 27, 28 we noticed that a vast majority of the motifs identified in viral proteins correspond to the wxxl pattern ( fig. 2b and table s1 ). the identification of putative lir motifs has been done for all the reviewed sequences for viral proteins. among the sequences sorted as putative lir-containing proteins, 1517 sequences belong to 188 species of bacteriophages (table s2 ). however, it is likely that these sequences correspond to false-positive hits as bacteria don't have an autophagy process. using a hypergeometric test (see supplementary information), we compared the enrichment fold of proteins containing lir motifs (lircps) in bacteriophages with viruses infecting eukaryotes. we noticed that the enrichment fold for both xlir and wxxl patterns in lircps was higher in viruses infecting eukaryotes than in bacteriophages (table s2) which is in line with the fact that autophagy has been reported only in eukaryotes. we have previously identified all the verified and putative lir motifs in eukaryotic model organisms. 28 we also compared the enrichment of putative lir motifs in viruses infecting some of the model organisms (human, mouse, rat, and chicken) (table s3) . two hypergeometric tests have been conducted to compare the enrichment fold of lircps in viruses against the host: one for proteins containing all combination of lir patterns (i.e., either xlir or wxxl, or both kind) and another considering only the proteins containing at least one xlir motif (i.e., xlir patterns alone or along with one or more wxxl patterns). we observed that when all the possible combinations of lir patterns are taken into account, there is an enrichment of putative lir motifs in viruses compared with the host organism for all 4 model organisms tested. however, there is an enrichment of xlir patterns in the host compared with the infecting viruses. the lir motifs can be divided into 3 subtypes depending on the residue at the first position: w-, f-and y-types. 8 it has been shown that the f-type lir motif of mammalian ulk1 and atg13 has a preference for gabarap proteins, thus suggesting that the subtype of the lir motifs could be related to a specificity toward atg8family proteins. 7 additionally f-type and y-type lir motifs are mostly contained in selective autophagy adaptor proteins. 8 we thus analyzed the distribution of w-, f-and y-type of wxxl and xlir patterns at the viral order and family levels (table s4 and fig. 2c, d) . we observed that 45% and 38% of the putative lir motifs matching the wxxl pattern are of f-type and y-type respectively. w-type motifs are the least represented, with about 17% of the patterns (fig. 2c) . similar distribution could be observed for the putative xlir motifs with a higher variability across families, probably due to the low representation of xlir motifs compared with wxxl patterns (fig. 2d) . to assess the trustworthiness of our in silico screening for lircps in viruses, we compared our data with the already published viral proteins listed on the web resource viral-zone as modulators of autophagy. 66 viralzone classifies 180 entries related to the activation of the host autophagy, and 163 entries linked to the inhibition of host autophagy. we found all these entries in our database. among viruses that inhibit autophagy, only 2 proteins have been shown to interact directly with mammalian atg8also table s4 ). (d) distribution of the w-, f-and y-type of xlir patterns across the different families (see also table s4 ). family proteins: viral infectivity factor (vif) from hiv-1 binds to all atg8-family proteins, and matrix protein 2 (m2) from influenza was shown to bind to lc3. yet, influenza m2 protein is the only one that contains a lir motif that has been experimentally validated. 52, 62 other viral proteins listed in viralzone as being related to inhibition of the autophagy process are the neurovirulence factor icp34.5 and rna-binding protein us11 from human herpesviruses, the protein nef from hiv-1, and the protein trs1 from human cytomegalovirus. 49, 61, 62, 67 negative regulation of autophagy by icp34.5 and trs1 proteins depends on their ability to interact with becn1/beclin 1; 68-70 while us11 function has been linked to the protein kinase eif2ak2/pkr. 71 putative, or functional, lir motifs could be identified for all these proteins using the ilir web resource, except for the rna-binding protein us11. the protein m2 from influenza a virus is necessary and sufficient for the inhibition of the autophagic degradation of the virus by blocking the fusion between the autophagosomes and lysosomes. 49 these results were further confirmed and extended by beale and colleagues who show that the cytoplasmic tail of m2 interacts in a lir-dependent manner with lc3 and promotes the relocalization of lc3 at the plasma membrane. 62 we have identified a wxxl motif at positions 89 to 94 that has the highest pssm score (8 to 9), and corresponds to the one experimentally verified (fvsi). 27, 72 it is an f-type lir motif and the fact that m2 protein has been shown to block the autophagosome-lysosome fusion suggests that it may act as a an adaptor protein. we were also able to identify one xlir motif in the sequence of the accessory viral protein nef of the virus hiv-1 group m subtype b (strain 89.6). nef colocalizes with lc3 and becn1, and contributes to the inhibition of autophagic maturation, thus protecting the virus from elimination by autophagy. 61, 67 the proteins trs1 from human cytomegalovirus and neurovirulence factor icp34.5 from human herpesvirus (hsv) both interact with becn1 via a specific becn1-binding domain. this interaction is required for the inhibition of autophagosome maturation and fusion with the lysosomes. [68] [69] [70] 73 while ilir could detect several wxxl motifs in trs1 sequence, a single one in an intrinsically disordered region was identified for icp34.5 whose sequence (64-rqwlhv-69) is quite well conserved among 4 strains of hsv-1 and one strain of hsv-2. however, to date, there is no evidence of association between icp34.5 and lc3 proteins. we observed that 7% of the reviewed sequences that contains at least one putative lir motif correspond to the genome polyprotein from various viruses. because polyproteins are processed co-and post-translationally by both host and viral proteases, we ran a systematic pubmed search with the terms 'name of the virus c autophagy' followed by 'name of the virus c lc3' to restrain the result outcome as necessary, finally we looked for papers (excluding reviews and commentaries) that specifically mention proteins derived from the processing of the viral genome polyprotein. our literature searching strategy pinpointed several nonstructural viral proteins; one of those was the protein ns1 from dengue viruses. studies have shown that ns1 protein from dengue virus type 2 (denv-2) colocalizes with lc3 and that denv-2 particles and autophagosomes travel together during viral infection. 58, 74 in contrast to denv-2, ns1 protein from denv-3 displays a low level of colocalization with lc3. 75 sequence alignment of ns1 proteins from denv-2 and denv-3 showed that they are highly conserved. however, checking for the presence of lir motifs revealed a discrepancy between them. we observed that denv-2 ns1 has an xlir motif (asfiev) that is not recognized in any denv-3 ns1 sequences due to the substitution f to l, as well as an additional wxxl motif with a pssm score 12 (rawnsl) that is absent in denv-3 (sl to vw) (fig. s1 ). it is possible that the absence of either the xlir or wxxl motifs (or both) in denv-3 is responsible for its lower affinity to lc3. other nonstructural proteins from different viruses interact with lc3 proteins. for instance, the nonstructural protein ns5a from hepatitis c virus that colocalizes and can be coimmunoprecipitated with lc3 proteins when ectopically expressed in various hepatoma cell lines. 59, 76, 77 also, the viral peptide 2bc and the protein 3a encoded by the genome polyprotein from poliovirus type 1 interact with lc3-ii. 60, 78 all these proteins contain wxxl motifs. finally, we were able to identify proteins from zika virus that have been just recently related to autophagy. independent studies have shown that zika virus activates autophagy and that the formation of autophagosomes is crucial to the replication of the virus. [79] [80] [81] it appears that the nonstructural proteins ns4a and ns4b are responsible for the induction of autophagy in infected cells by inhibiting the akt-mtor signaling, and both of them contain 3 wxxl motifs. 80, 82 very little is known about the relation between zika virus infection and autophagy modulation. we have found several proteins encoded by zika genome polyprotein that contain lir motifs, that could be good candidates for experimental validation. autophagy is an evolutionarily conserved and highly regulated, intracellular catabolic mechanism that is essential for maintaining homeostasis and coping with nutrient starvation. it is increasingly appreciated that autophagy can be highly selective, and that xenophagy, the selective autophagy of pathogens, is an important aspect of the immune response, which protects against infection. a vast array of viruses are associated with autophagy, and we have found several viral proteins containing putative lir motifs that are thought to interact with the autophagic machinery via atg8-family proteins. a continued research effort to better understand how these viral proteins interact with the autophagic machinery may provide therapeutic strategies and ultimately lead to the discovery of novel pharmacological agents to fight viral infections. protein sequences of all reviewed viral proteins were downloaded from uniprot database vailable at: http://www.uni prot.org/ [accessed 20 september 2016]. xlir and wxxl patterns for these proteins were identified using the approach suggested previously. 27, 28 for a given protein, information related to the start and end of lir pattern, actual lir sequence, pssm score (position specific scoring matrix), similar lircps and presence or absence xlir and wxxl in intrinsically disordered region were obtained. the taxonomic lineage obtained from uniprot for all the reviewed viral proteins correspond to the baltimore classification system. to do the conversion between the baltimore and ictv classification systems, we matched the organism name for each reviewed sequence from uniprot with the organism names obtained from the ictv master species list 2015 v1, available at: https://talk.ictvonline.org/files/master-species-lists/ m/msl/5945 [accessed 20 september 2016]. the differences between the classification systems were identified through a battery of sql queries. the details of these are attached in tables s5, s6 and s7. levenshtein distance (also known as edit distance) was used to compare the species name that belongs to a particular genus and family between both the classification systems. levenshtein distance is defined as the minimal number of characters required to replace, insert or delete to transform one string into another. the levenshtein distance is symmetric and it holds: here, 'x' and 'y' are 2 strings and d(x,y) is the distance between 'x' and 'y' put as minimal cost of operations to transform 'x' to 'y'. the complexity of the algorithm is o(m ã� n), where n and m are the lengths of 2 strings. 83 we used perl extension for approximate matching (search.cpan. org, (2016). string-approx-3.27. retrieved from: http://search.cpan.org/ cpan/authors/id/j/jh/jhi/string-approx-3.27.tar.gz) the closer the value of levenshstein distance to zero, the closer are the species names. using this approach, we could reliably justify the ictv classification of 14055 out 15589 proteins loaded into the database. to share the information beyond our mysql (v5.6.33) dbms (database management system), we built a website using html, css, javascript and php (v5. 6 no potential conflicts of interest were disclosed. the machinery of macroautophagy network organization of the human autophagy system atg8 family lc3/gabarap proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during pink1/parkin mitophagy and starvation role of the mammalian atg8/lc3 family in autophagy: differential and compensatory roles in the spatiotemporal regulation of autophagy lc3 and gate-16/gabarap subfamilies are both essential yet act differently in autophagosome biogenesis plekhm1 regulates autophagosome-lysosome fusion through hops complex and lc3/gabarap proteins atg8 family proteins act as scaffolds for assembly of the ulk complex: sequence requirements for lc3-interacting region (lir) motifs the lir motif -crucial for selective autophagy the lc3 interactome at a glance aggrephagy: selective disposal of protein aggregates by macroautophagy mechanisms of mitophagy lipophagy: selective catabolism designed for lipids the role of 'eat-me' signals and autophagy cargo receptors in innate immunity p62/sqstm1 binds directly to atg8/lc3 to facilitate degradation of ubiquitinated protein aggregates by autophagy structural basis for sorting mechanism of p62 in selective autophagy the autophagy-related protein kinase atg1 interacts with the ubiquitin-like protein atg8 via the atg8 family interacting motif to facilitate autophagosome formation structural basis of target recognition by atg8/lc3 during selective autophagy atg8-family interacting motif crucial for selective autophagy the structure of atg4b-lc3 complex reveals the mechanism of lc3 processing and delipidation during autophagy a role for nbr1 in autophagosomal degradation of ubiquitinated substrates nix is a selective autophagy receptor for mitochondrial clearance the tbk1 adaptor and autophagy receptor ndp52 restricts the proliferation of ubiquitin-coated bacteria nix directly binds to gabarap: a possible crosstalk between apoptosis and autophagy phosphorylation of the autophagy receptor optineurin restricts salmonella growth structural and functional analysis of the gabarap interaction motif hfaim: a reliable bioinformatics approach for in silico genome-wide identification of autophagy-associated atg8-interacting motifs in various organisms ilir: a web resource for prediction of atg8-family interacting proteins ilir database: a web resource for lir motif-containing proteins in eukaryotes autophagy in antiviral innate immunity autophagy in infection, inflammation and immunity autophagy protects against sindbis virus infection of the central nervous system image-based genome-wide sirna screen identifies selective autophagy factors pkr-dependent autophagic degradation of herpes simplex virus type 1 autophagydependent viral recognition by plasmacytoid dendritic cells replication-independent activation of human plasmacytoid dendritic cells by the paramyxovirus sv5 requires tlr7 and autophagy pathways production of interferon alpha by human immunodeficiency virus type 1 in human plasmacytoid dendritic cells is dependent on induction of autophagy antigen-loading compartments for major histocompatibility complex class ii molecules continuously receive input from autophagosomes autophagy enhances the presentation of endogenous viral antigens on mhc class i molecules during hsv-1 infection absence of autophagy results in reactive oxygen species-dependent amplification of rlr signaling autophagy facilitates ifngamma-induced jak2-stat1 activation and cellular inflammation hepatitis c virus upregulates beclin1 for induction of autophagy and activates mtor signaling peroxisomal protein pex13 functions in selective autophagy fam134b, the selective autophagy receptor for endoplasmic reticulum turnover, inhibits replication of ebola virus strains makona and mayinga sustained autophagy contributes to measles virus infectivity autophagy and the effects of its inhibition on varicella-zoster virus glycoprotein biosynthesis and infectivity host cell autophagy modulates early stages of adenovirus infections in airway epithelial cells the autophagy elongation complex (atg5-12/16l1) positively regulates hcv replication and is required for wild-type membranous web formation herpes simplex virus gamma34.5 interferes with autophagosome maturation and antigen presentation in dendritic cells matrix protein 2 of influenza a virus blocks autophagosome fusion with lysosomes coronavirus membrane-associated papain-like proteases induce autophagy through interacting with beclin1 to negatively regulate antiviral innate immunity human immunodeficiency virus type-1 infection inhibits autophagy hiv-1 viral infectivity factor interacts with microtubule-associated protein light chain 3 and inhibits autophagy irgm is a common target of rna viruses that subvert the autophagy network multi-layered control of galectin-8 mediated autophagy during adenovirus cell entry through a conserved ppxy motif in the viral capsid foot-and-mouth disease virus infection suppresses autophagy and nf-small ka, cyrillicb antiviral responses via degradation of atg5-atg12 by 3cpro berlioz-torrent c. lc3c contributes to vpu-mediated antagonism of bst2/tetherin restriction on hiv-1 release through a non-canonical autophagy pathway autophagy functions as an antiviral mechanism against geminiviruses in plants co-localization of constituents of the dengue virus translation and replication machinery with amphisomes replication of hepatitis c virus rna on autophagosomal membranes modification of cellular autophagy protein lc3 by poliovirus autophagy pathway intersects with hiv-1 biosynthesis and regulates viral yields in macrophages a lc3-interacting motif in the influenza a virus m2 protein is required to subvert autophagy and maintain virion stability taxonomy of viruses., king amq. virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses expression of animal virus genomes gapped blast and psi-blast: a new generation of protein database search programs viralzone: a knowledge resource to understand virus diversity human immunodeficiency virus type 1 nef inhibits autophagy through transcription factor eb sequestration hsv-1 icp34.5 confers neurovirulence by targeting the beclin 1 autophagy protein the human cytomegalovirus protein trs1 inhibits autophagy via its interaction with beclin 1 analysis of the role of autophagy inhibition by two complementary human cytomegalovirus becn1/beclin 1-binding proteins the herpes simplex virus 1 us11 protein inhibits autophagy through its interaction with the protein kinase pkr analysis of the native conformation of the lir/ aim motif in the atg8/lc3/gabarap-binding proteins herpes simplex virus type i induces an incomplete autophagic response in human neuroblastoma cells single-virus tracking approach to reveal the interaction of dengue virus with autophagy during the early stage of infection a role for autophagolysosomes in dengue virus 3 production in hepg2 cells interferoninducible protein scotin interferes with hcv replication through the autolysosomal degradation of ns5a a functional role for ns5atp9 in the induction of hcv ns5a-mediated autophagy subversion of cellular autophagosomal machinery by rna viruses biology of zika virus infection in human skin cells zika virus ns4a and ns4b proteins deregulate akt-mtor signaling in human fetal neural stem cells to inhibit neurogenesis and induce autophagy zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells anchor: web server for predicting protein binding regions in disordered proteins a guided tour to approximate string matching we would like to thank professor andrew easton (university of warwick) and professor keith leppard (university of warwick) for helpful discussions. this work is supported by bbsrc grants bb/l006324/1 and bb/p007856/1 awarded to i.p.n. key: cord-001866-s5otdtwq authors: mandal, nakul; lewis, geoffrey p.; fisher, steven k.; heegaard, steffen; prause, jan u.; la cour, morten; vorum, henrik; honoré, bent title: proteomic analysis of the vitreous following experimental retinal detachment in rabbits date: 2015-11-18 journal: j ophthalmol doi: 10.1155/2015/583040 sha: doc_id: 1866 cord_uid: s5otdtwq purpose. the pathogenesis of rhegmatogenous retinal detachment (rrd) remains incompletely understood, with no clinically effective treatment for potentially severe complications such as photoreceptor cell death and proliferative vitreoretinopathy. here we investigate the protein profile of the vitreous following experimental retinal detachment using a comparative proteomic based approach. materials and methods. retinal detachment was created in the right eyes of six new zealand red pigmented rabbits. sham surgery was undertaken in five other rabbits that were used as controls. after seven days the eyes were enucleated and the vitreous was removed. the vitreous samples were evaluated with two-dimensional polyacrylamide gel electrophoresis and the differentially expressed proteins were identified with tandem mass spectrometry. results. ten protein spots were found to be at least twofold differentially expressed when comparing the vitreous samples of the sham and retinal detachment surgery groups. protein spots that were upregulated in the vitreous following retinal detachment were identified as albumin fragments, and those downregulated were found to be peroxiredoxin 2, collagen-iα1 fragment, and α-1-antiproteinase f. conclusions. proteomic investigation of the rabbit vitreous has identified a set of proteins that help further our understanding of the pathogenesis of rhegmatogenous retinal detachment and its complications. rhegmatogenous retinal detachment (rrd) is characterized by the accumulation of subretinal fluid between the neurosensory retina and retinal pigment epithelium following the formation of a retinal break [1] . the pathogenesis of rrd is complex and incompletely understood, involving age-related and/or inherited structural and molecular changes of the vitreous extracellular matrix and vitreoretinal interface, and the process of posterior vitreous detachment [2] . the annual incidence of the condition has been estimated at 12.05 per 100,000, and although primary surgical reattachment is successful in the great majority of cases, photoreceptor cell death, growth factors as a result of rrd significantly contributes to the pathogenesis of pvr, though the basic cause as well as a clinically effective therapeutic approach for this condition remains elusive [5, 6] . proteomics studies proteins on a large scale in pursuit of a global and integrated view of disease processes at the protein level, which may potentially lead to the identification of novel biomarkers and therapeutic targets useful in clinical practice [7] [8] [9] [10] [11] . rrd would likely be associated with alterations in the proteomic profiles of both the retina and vitreous. indeed, we initially undertook the first such retinal study from which a number of potentially important proteins were identified [12] . the present study extends the proteomic investigation to the vitreous of this rabbit model of retinal detachment, building upon previous such analyses of human vitreous [13] [14] [15] [16] , in order to add further knowledge of the underlying pathophysiology [10, 17] . inferior retinal detachment was created in the right eyes of six new zealand red pigmented rabbits. the eyes were normal with no evidence of disease on examination. combined injections of xylazine (6.7 mg/kg) and ketamine (33.3 mg/kg) were administered intramuscularly to induce anesthesia and analgesia. the pupils were dilated with topical drops of atropine and tropicamide (1% solutions). a pipette tip, with an external diameter of approximately 100 m, was inserted into the eye through a pars plana incision. sodium hyaluronate (healon, 0.25% in a balanced salt solution; pharmacia, piscataway, nj) was infused via a glass pipette between the neurosensory retina and retinal pigment epithelium. healon was necessary to prevent spontaneous retinal reattachment, and 0.25% is the most dilute solution that maintains the detachment for extended periods. approximately 50% of the retina beneath the medullary rays, which included the central retina, was detached ( figure 1 ). sham surgery was performed in the right eyes of five other rabbits that were used as controls, which involved surgical entry of the vitreous cavity without disruption of the retina. scleral incisions were closed with 8-0 nylon suture. seven days postoperatively the animals were euthanized by the administration of sodium pentobarbital (120 mg/kg; butler schein, dublin, oh) and the eyes enucleated. after removal of the cornea and lens, the associated vitreous of the sham and detached retinas was extracted and immediately snap-frozen in liquid nitrogen within separate vials. there was no gross evidence of blood or other contamination of the vitreous samples at the time of tissue harvesting. the vitreous samples were stored at −80 ∘ c until further use. all of the animal experiments undertaken in this study were in accordance with the standards of the national institutes of health animal care and use committee protocols, the arvo statement for the use of animals in ophthalmic and vision research, and the guidelines of the animal resource center, university of california, santa barbara. the rabbit vitreous samples were homogenized and dissolved in a lysis buffer containing 9 m urea, 2% (v/v) triton x-100, 2% (v/v) immobilized ph gradient (ipg) buffer (ph 3-10 nonlinear), and 2% (w/v) dithiothreitol (dtt). the total protein content in each vitreous sample was determined with non-interfering protein assay (calbiochem, san diego, ca). the protein samples were stored at −80 ∘ c until further use. the extracted proteins were first fractionated by isoelectric focusing (ief) using ph 3-10 nonlinear 18 cm ipg strips (ge healthcare, chalfont st. giles, buckinghamshire, uk). the ipg strips were rehydrated for 20 h at room temperature in 200 l lysis buffer each containing 20 g protein from individual vitreous samples and 150 l rehydration buffer (8 m urea, 2% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (chaps), 0.3% (w/v) dtt, and 2% (v/v) ipg buffer), using the immobiline drystrip reswelling tray (ge healthcare). the ief was undertaken on a multiphor ii electrophoresis system (ge healthcare) at 500 v for 5 h and 3500 v in two steps for 5 h and 9.5 h in a gradient mode at 17 ∘ c with the use of a multitemp iii thermostatic circulator (ge healthcare). before the second-dimension sodium dodecyl sulfate (sds) polyacrylamide gel electrophoresis (page), the ipg strips were equilibrated firstly for 10 min with gentle agitation in 20 ml of equilibration solution (0.6% (w/v) tris-hcl, ph 6.8, 6 m urea, 30% (v/v) glycerol, 1% (w/v) sds, and 0.05% (w/v) dtt) and secondly using 4.5% (w/v) iodoacetamide and bromophenol blue. the ipg strips were then transferred to 12% polyacrylamide gels for electrophoresis, which was performed at a maximum voltage of 50 v for approximately 20 h to separate the proteins vertically on the basis of molecular mass. staining. the two-dimensional (2d) gels were silver stained using a protocol optimized for protein identification with mass spectrometry [18] . in brief, the gels were fixed overnight in 50% (v/v) ethanol, 12% (v/v) acetic acid, and 0.0185% (v/v) formaldehyde. the gels were washed 3 times for 20 min in 35% (v/v) ethanol and pretreated for 1 min in 0.02% (w/v) na 2 s 2 o 3 ⋅5h 2 o. they were then rinsed in water and stained for 20 min in 0.2% (w/v) agno 3 and 0.028% (v/v) formaldehyde. following further rinsing with water, development was undertaken for approximately 3 min in 6% (w/v) na 2 co 3 , 0.0185% (v/v) formaldehyde, and 0.0004% (w/v) na 2 s 2 o 3 ⋅5h 2 o. the development was arrested in a fixative solution of 40% (v/v) ethanol and 12% (v/v) acetic acid. the 2d gels were then dried between cellophane sheets and sealed in plastic envelopes. silver stained 2d gels were scanned on a gs-710 calibrated imaging densitometer (bio-rad, hercules, ca) using the quantity one program (bio-rad), and the pdquest software (bio-rad) was used to define, quantify, and match the protein spots on each of the 2d gels. all well-defined protein spots that were at least twofold (mann-whitney test, < 0.05) differentially expressed between the sham and retinal detachment vitreous groups were selected for identification with nanoliquid chromatographyelectrospray ionization tandem mass spectrometry (lc-ms/ms). the 2d gels were removed from their plastic envelopes and rehydrated in water. the selected protein spots were carefully excised from the gels with a scalpel and subjected to in-gel digestion with trypsin gold (mass spectrometry grade; promega, madison, wi). the peptide samples that were obtained were analyzed by lc-ms/ms as previously described [12] . in brief, peptides generated by trypsin digestion were separated on an inert nano-lc system (lc packings, san francisco, ca) connected to a q-tof premier mass spectrometer (waters, milford, ma). the masslynx 4 sp4 (waters) was used to obtain spectra and the raw data was processed using proteinlynx global server 2.1 (waters). the processed data were used to search the total part of the swiss-prot database using the online version of the mascot ms/ms ions search facility (matrix science, ltd.). the search was undertaken with doubly and triply charged ions with up to two missed cleavages, a peptide tolerance of 50 ppm, one variable modification, carbamidomethyl-c, and a ms/ms tolerance of 0.05 da. contaminating peptides such as trypsin, keratin, bovine serum albumin, and all peptides originating from previous samples were disregarded. at least one "bold red" (matrix science ltd., http://www .matrixscience.com/) peptide match was required in the search for protein hits. individual peptide ions scores above approximately 36 indicated identity or extensive homology giving a less than 5% probability that the observed match was a random event. all peptides for the protein hits are reported (table 1) . blotting. in each case three micrograms of vitreous sample protein was separated on novex 10-20% gradient tris-glycine polyacrylamide gels (invitrogen corporation, carlsbad, ca) and subsequently transferred to nitrocellulose hybond-c extra membranes (ge healthcare). the membranes were blocked overnight with 5% skimmed milk in 80 mm na 2 hpo 4 , 20 mm nah 2 po 4 , 100 mm nacl, and 0.05% tween 20 buffer, ph 7.5. membranes were incubated with anti-albumin (genway biotech, ca, usa; 1 : 5000) and anti-peroxiredoxin 2 (abcam, cambridge, uk; 1 : 200). no suitable antibodies were commercially available for the rabbit f isoform of -1-antiproteinase or the rabbit collagen-i 1 fragment that was identified with lc-ms/ms. following washing, the membranes were further incubated with appropriate horseradish peroxidase-conjugated secondary antibodies: p0163 sheep and p0260 mouse (both 1 : 1000; dako, glostrup, denmark). proteins were visualized with the enhanced chemiluminescence system (ge healthcare) and imaging system (fujifilm las-3000, tokyo, japan). up to approximately 340 protein spots were clearly resolved on each of the 11 2d gels. ten protein spots were found to be significantly and at least twofold differentially expressed between the sham and detachment vitreous groups (figure 2 ). three protein spots were upregulated and seven spots were downregulated. from the three upregulated protein spots, two were identified as fragments of albumin (spots 5104 and 6101), whilst spot 6205 could not be identified. four of the seven downregulated protein spots were identified as fragment of collagen-i 1 (spot 0503), -1-antiproteinase f (spots 0703 and 1707), and peroxiredoxin 2 (spot 0705). protein spots 0102, 0815, and 1302 could not be identified ( figure 2 ; table 1 ). western blotting developed with anti-albumin showed a heavy band at approximately 60 kda, which is likely to represent the full length protein, whilst multiple bands below this suggest the presence of several fragments, some of which may correspond with those identified with the 2d-page analysis (figure 3, left) . peroxiredoxin 2 has a deduced molecular mass of approximately 22 kda. however, spot 0705 containing peroxiredoxin 2 migrates with a molecular mass around 60 kda with 2d-page (figure 2) , and this size was verified by western blot analysis (figure 3 , right). though a single and specific band was achieved with anti-peroxiredoxin 2, western blotting could not be reliably used for quantification due to a weak signal near the detection limit and variable background reaction. analysis with 2d-page revealed fragments of albumin to be upregulated in the vitreous following retinal detachment. albumin is the most abundant protein in plasma, aqueous, and vitreous humor, where in the latter it constitutes around 60-70% of total protein [19] [20] [21] . serum proteins such as albumin are present in the aqueous and vitreous humor at a relatively lower level compared to the vascular circulation from where they may have in part originated [21, 22] . western blot analysis showed an intense band at approximately 60 kda corresponding with the full length albumin protein, with multiple lower molecular mass bands that are likely its fragments. increase of albumin and its fragments may signify increased proteolysis and the passage of albumin into the vitreous. indeed, the breakdown of the blood-retinal barrier that occurs with retinal detachment has also been implicated in the increase of other such proteins in the vitreous [12, [23] [24] [25] [26] [27] [28] . it is also possible that albumin in the vitreous may arise from de novo synthesis in the retina, similar to the reported increased gene and protein expression of albumin in the corneal epithelium during wound healing [29, 30] . extraocular albumin is known to have diverse and important functions, which include maintenance of colloid osmotic pressure, transport of biomolecules, and inactivation of toxins through intermolecular binding [31, 32] . albumin can also act as an antioxidant by scavenging reactive oxygen species and sequestration of metal ions and has anti-inflammatory and apoptotic regulatory abilities [32] [33] [34] [35] . vitreal albumin has been proposed to transport long chain fatty acids into the lens for biosynthesis of lenticular lipids [21, 31, 36] . indeed, albumin is likely to have many such important roles in the eye, which requires further investigation. in the present study we observed peroxiredoxin 2 to have a molecular mass above 60 kda using both 2d-page and western blot analyses. however, the predicted molecular mass of the peroxiredoxin family of proteins is approximately 22 kda-31 kda. this variation may represent the well-studied property of these proteins to undergo oligomerization, which can be promoted by a number of factors including overoxidation of cysteine residues of peroxiredoxin [37] . although the present experiments were conducted in standard reducing conditions that aim to break cysteine bonds, we obtained a band well above 20 kda. this is in keeping with another study, which also showed some peroxiredoxin 2 western blot bands appearing at molecular mass much higher than 20 kda that was suggested to result from oligomerization or posttranslational modification [38] . our finding could also represent a novel alternative splicing variant of peroxiredoxin 2, as reported for peroxiredoxin 5 [39] . the peroxiredoxins are a group of ubiquitous antioxidant proteins that currently comprise six members in mammals [40] . these proteins are primarily found at high levels intracellularly, mainly within the cytosol, but are also present in the mitochondria, peroxisomes, and nuclei, and they may be exported [37] . furthermore, presence of peroxiredoxin 2 has been shown in plasma, not only as a result of hemolysis but also possibly by secretion from the t lymphocytes [41, 42] . these multifunction enzymes act as antioxidants by using redox active cysteines for the reduction and degradation of hydrogen peroxide, peroxynitrite, and organic hydroperoxides [37, 43] . oxidative stress is thought to result from an imbalance between reactive oxygen species production and antioxidant ability and is recognized to be an important factor in the pathogenesis of a number of age-related and neurodegenerative diseases, which include age-related cataract, agerelated macular degeneration, glaucoma, diabetic retinopathy, retinal detachment, and pvr [44] [45] [46] [47] . indeed, the present study showed a decrease in the vitreal levels of peroxiredoxin 2 following retinal detachment. this may be in keeping with reported reductions in the levels of other members of the antioxidant defense system such as glutathione and ascorbic acid both in vitreal and in blood samples of patients suffering from pvr [44, 45] . furthermore, apart from their role as antioxidants, the peroxiredoxins can affect a diverse range of biological processes that include cellular proliferation, differentiation, and apoptosis by influencing signal transduction pathways that employ hydrogen peroxide as a secondary messenger [43, 48] . recent studies on tears from patients with glaucoma have also identified peroxiredoxin 1 as having a possible involvement in inflammation [49, 50] . indeed, peroxiredoxin 2 and other members of this family of proteins are liable to have a significant role in the pathophysiology of retinal detachment. a fragment of collagen-i 1 was identified in the vitreous of the rabbit; however, type i collagen has not previously been identified as a natural component of the mammalian vitreous and is rather known to be a constituent of early pvr membranes [51] [52] [53] and retinal blood vessels [54, 55] . a mixture of type ii, ix, and v/xi hybrid collagen fibrils, which are separated out mainly by water and ions attracted to hyaluronan, characterizes the vitreous body [56] . collagen, possibly with the aid of adhesive-like intermediate molecules, may provide the basis of vitreoretinal adhesion by connecting the vitreous with the retinal inner limiting membrane (ilm). this attachment is extremely strong in the vitreous base since the fibrils pass through the ilm to merge into underlying collagen networks and crypts [2, 56, 57] . collagen is also a significant component of both epiretinal and subretinal pvr membranes [53, 58] , and type i collagen is recognized to be a principal constituent during their early development [51] [52] [53] . the presence of collagen in the subretinal space, a place normally devoid of this protein, suggests that certain cells, particularly the rpe and müller cells associated with membranes, are able to synthesize collagen under certain pathological conditions such as retinal detachment and pvr [58] [59] [60] [61] . however, the present analysis suggests collagen-i 1 fragment to be found in sham vitreous, which furthermore showed a decreased concentration following retinal detachment that may indicate perturbed proteolytic activity. matrix metalloproteinases (mmp) and other proteolytic enzymes that are able to degrade and remodel vitreal collagen have been found to be increased with rrd and pvr [62] [63] [64] [65] , which could be in keeping with the decrease in -1-antiproteinase shown in the present study. further studies will be necessary to confirm the source and nature of collagen-i 1 in the vitreous and the possible mechanisms of collagen fragmentation that may be an important feature of vitreous liquefaction and rrd [2, 62, 66] . alpha-1-antiproteinase. 2d-page showed -1-antiproteinase (also called -1-antitrypsin or -1-proteinase inhibitor) at two closely positioned spots, which were largely in keeping with their predicted molecular mass but differing by their charge. currently, four isoforms of -1-antiproteinase have been identified in the rabbit, termed f, s1, s2, and e, which is a similar picture to the multiple variants identified in humans [67, 68] . alpha-1-antiproteinase is an acute phase protein and archetypal member of the superfamily of serine protease inhibitors (serpin), which are involved in a wide range of biological processes that includes inflammation, angiogenesis, blood coagulation, ecm remodeling, and tumor suppression [69] . this protein has the ability to inhibit a large number of serine proteases though its principle target is neutrophil elastase [68] . indeed, -1-antiproteinase originally received much attention because its deficiency increases the risk of a variety of clinical conditions, such as chronic obstructive pulmonary disease, which can result from unrestrained elastase activity. we found the f isoform of rabbit -1-antiproteinase to be downregulated in the vitreous following retinal detachment. the f isoform of -1-antiproteinase is the only one of the rabbit isoforms so far identified that has been shown to have the oxidizable methionine residue site that is present in human -1-antiproteinase [67] . the oxidation of methionine to methionine sulfoxide, which can occur during episodes of inflammation as a result of oxygen-free radicals secreted by leucocytes, has an inhibiting effect upon -1-antiproteinase function. this process is thought to enhance the ability of proteinases such as elastase to locally degrade tissue debris that occurs at sites of inflammation [67, 68] . alpha-1-antiproteinase is primarily produced in the liver and circulated to the rest of the body tissues via the blood; however, extrahepatic sites of its synthesis have been identified, which include blood monocytes, alveolar macrophages, bronchial and gastrointestinal epithelial cells, and the cornea [70] [71] [72] [73] . the protein has also been localized to the tear film, aqueous humor, and vitreous, where in the latter a phosphorylated form of -1-antiproteinase has been suggested as a potential biomarker of idiopathic macular hole and rhegmatogenous retinal detachment [73] [74] [75] . it has been postulated that one of the main functions of corneal -1-antiproteinase is to protect against the damaging effects of neutrophil elastase produced during corneal inflammation [73] , and it may be expected that a similar role in addition to others is applicable to vitreal -1-antiproteinase, though this requires further investigation. this proteomic investigation of the rabbit vitreous has identified a set of proteins that assist our understanding of the pathogenesis of rhegmatogenous retinal detachment and its journal of ophthalmology 7 complications. further studies will be necessary to clarify the role of these proteins. certain proteins, such as those of low abundance and at the extremes of molecular mass, together with membrane proteins, can be difficult to resolve and detect using the 2d-page technique. therefore, complementary proteomic methods such as gel-free mass spectrometry should be considered in future work in order to help address these limitations. the authors alone are responsible for the content and writing of the paper. recent trends in the management of 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levels with the grade of proliferative vitreoretinopathy in the subretinal fluid and vitreous during rhegmatogenous retinal detachment age-related liquefaction of the human vitreous body: lm and tem evaluation of the role of proteoglycans and collagen various forms of rabbit plasma -1-antiproteinase mammalian 1 -antitrypsins: comparative biochemistry and genetics of the major plasma serpin the serpins are an expanding superfamily of structurally similar but functionally diverse proteins. evolution, mechanism of inhibition, novel functions, and a revised nomenclature expression of the alpha 1-proteinase inhibitor gene in human monocytes and macrophages biosynthesis of 1-proteinase inhibitor by human lung-derived epithelial cells regulation of 1-proteinase inhibitor release by proinflammatory cytokines in human intestinal epithelial cells corneal synthesis of 1-proteinase inhibitor ( 1-antitrypsin) alpha-1-antitrypsin in aqueous humour from patients with corneal allograft rejection identification of phosphotyrosyl proteins in vitreous humours of patients with vitreoretinal diseases by sodium dodecyl sulphate-polyacrylamide gel electrophoresis/western blotting/ matrix-assisted laser desorption time-of-flight mass spectrometry the authors appreciate the excellent technical support pro the authors report no conflict of interests. key: cord-013348-lsksys56 authors: goto, keiko; yamaoka, yutaro; khatun, hajera; miyakawa, kei; nishi, mayuko; nagata, noriko; yanaoka, toshikazu; kimura, hirokazu; ryo, akihide title: development of monoclonal antibodies and antigen-capture elisa for human parechovirus type 3 date: 2020-09-19 journal: microorganisms doi: 10.3390/microorganisms8091437 sha: doc_id: 13348 cord_uid: lsksys56 human parechovirus type 3 (hpev3) is an etiologic agent of respiratory diseases, meningitis, and sepsis-like illness in both infants and adults. monoclonal antibodies (mabs) can be a promising diagnostic tool for antigenic diseases such as virus infection, as they offer a high specificity toward a specific viral antigen. however, to date, there is no specific mab available for the diagnosis of hpev3 infection. in this study, we developed and characterized mabs specific for hpev3 capsid protein vp0. we used cell-free, wheat germ-synthesized viral vp0 protein for immunizing balb/c mice to generate hybridomas. from the resultant hybridoma clones, we selected nine clones producing mabs reactive to the hpev3-vp0 antigen, based on enzyme-linked immunosorbent assay (elisa). epitope mapping showed that these mabs recognized three distinct domains in hpev3 vp0. six mabs recognized hpev3 specifically and the other three mabs showed cross-reactivity with other hpevs. using the hpev3-specific mabs, we then developed an elisa for viral antigen detection that could be reliably used for laboratory diagnosis of hpev3. this elisa system exhibited no cross-reactivity with other related viruses. our newly developed mabs would, thus, provide a useful set of tools for future research and ensure hpev3-specific diagnosis. human parechoviruses (hpevs) belong to the parechovirus genus of the picornaviridae family [1, 2] . the two serotypes (hpev1 and hpev2) of parechoviruses were initially isolated in 1956 from children with diarrhea, and were assigned to the enterovirus genus. however, it became evident that they were genetically distinct from the enteroviruses, and reclassified as the parechovirus genus in 1999. at present, 19 hpevs are reported and categorized, based on the nucleic acid sequences of the vp1 gene, not on the classification as enteroviruses serotype. hpev3 was first identified in japan. hpev3 was isolated from a stool sample provided by a 1-year-old infant who was experiencing fever, gastritis like symptoms, and transient lower extremity paralysis [3] . hpev types 1 to 8 are the common identified strains; among them, hpev type 1, 3, and 6 account for the majority of infectious strains worldwide [4] . infection with hpevs is associated with a broad spectrum of clinical manifestations, ranging from respiratory symptoms and mild gastrointestinal illness to sepsis-like diseases, meningitis, and encephalitis in children [5] . while most hpevs cause mild symptoms in children between 1 and 5 years of age, human parechovirus 3 (hpev3) is clinically the most important genotype, owing to its association to severe diseases in younger infants under 3 months of age [6] [7] [8] . hpev3 infection in infants can trigger a sepsis-like dysregulated host response involving the central nervous system [9] [10] [11] [12] . in cases of acute meningitis or encephalitis, patients might develop abnormal white matter lesions and neurological sequelae, and even death might occur [13] [14] [15] [16] [17] . apnea can occur in children regardless of encephalitis [17] . hpev3 is known to cause myalgia and myositis in adult patients and a similar pattern is also sporadically seen among pediatric patients. [18, 19] . an epidemic of hpev3 occurs every 3 to 4 years in japan. as respiratory disease or meningitis cases due to hpev3 are not subject to notifiable disease surveillance in japan, the actual number of the patients is not known [1, 6, [20] [21] [22] [23] . the appropriate diagnosis tool for hpev3 detection might be able to rule out infectious etiology and avoid unnecessary antibiotics use, which is a given because hpev3 leads to septic shock-like symptoms. for these reasons, the establishment of a method to detect hpev3 plays a vital role in healthcare fields. importantly, hpev3 s epidemic cycle occurs in summer time and is concurrent with enteroviral infection. thus, it is essential to develop a detection method that does not cross-react with enterovirus. hpev3 contains a small, non-enveloped, single-stranded positive-sense rna genome of approximately 7.3-kb nucleotides [24, 25] . the hpev3 virion is composed of 60 copies of three structural proteins (vp0, vp1, and vp3) that fit together to form a 28-nm-diameter icosahedral shell around the viral genome [26] . the genome encodes a single polyprotein that, during infection, is subsequently cleaved into all essential capsid components and non-structural proteins [24] . vp0 is an important protein for stabilizing the surface of the viral capsid, and the assembly of hpev is controlled by multiple interactions of the genome with the capsid, through conserved amino acids in vp1 and vp3 [25] . although rt-pcr-based diagnostic tests targeting 5 -utr of the hpev3 genome were developed for hpev3 detection in clinical samples, there is currently no diagnostic method for detecting the viral antigens. recently, chen et al. generated polyclonal antibodies for hpev3 vp0, and proposed an immunofluorescence-based diagnostic assay [27] . however, this method requires virus isolation by cell culture and takes several weeks for the identification of viral genotype/serotype. abed et al. developed a serological enzyme-linked immunosorbent assay (elisa), using a synthetic peptide from the vp0 protein of hpevs [28] . although it can provide a definitive diagnosis, serological test requires paired serum samples from acute and recovery phases, which makes it difficult to diagnose immediately as a point of care testing (poct). to develop a rapid and effective diagnostic strategy, there is an urgent need to produce highly specific monoclonal antibodies (mabs) toward hpev3 antigens. in this study, we sought to generate mabs specific to the capsid protein vp0 of hpev3. we prepared the viral vp0 antigen using the wheat germ cell-free system, which has the advantage of producing properly folded functional proteins [29, 30] , to immunize mice. as a result, we obtained nine mab clones for characterization, and thereafter, generated an elisa system that is specifically able to detect the hpev3 vp0 antigens. complementary dna encoding hpev3-vp0 (genbank no. ab084913) was used to generate the expression vector for antigen production with the wheat germ cell-free system. the hpev3-vp0 open reading frame was amplified by pcr, using the corresponding primer pairs. the amplified fragment was cloned into vector peu-e01-his-tev-mcs-n2 (cellfree sciences, yokohama, japan), using restriction enzymes xhoi and spei. in vitro wheat germ cell-free protein synthesis was carried out as previously described [29, 30] . for cell-free protein synthesis, wepro7240h wheat extract (cellfree sciences, yokohama, japan) was used in the bilayer translation reaction, as previously described. synthesized proteins were confirmed by immunoblotting. the his-hpev3-vp0 (full length, 1-298) protein was synthesized using a proteomist xe robotic protein synthesizer (cellfree sciences, yokohama, japan) for mouse immunization. the cell-free translation reaction mixture was separated into soluble and insoluble fractions by centrifugation at 18,000× g for 15 min. the soluble fraction was mixed with ni-sepharose high performance beads (ge healthcare, waukesha, wi, usa) in the presence of 20 mm imidazole. the beads were washed thrice with a washing buffer [20 mm tris-hcl (ph 7.5), 500 mm nacl] containing 40 mm imidazole. his-hpev3-vp0 was then eluted in another washing buffer containing 500 mm imidazole. amicon ultra centrifugal filters (millipore, bedford, ma, usa) were used to concentrate the purified his-hpev3-vp0. protein concentration was determined using the bradford method, with bovine serum albumin (bsa) as a protein standard. immunization of balb/c mice and generation of anti-hpev3-vp0 mab-producing hybridomas were carried out as previously described [29, 30] . briefly, his-tagged full-length hpev3-vp0 protein was injected into the footpad of the balb/c mice, using keyhole limpet hemocyanin as an adjuvant. four weeks later, the spleen cells were isolated and fused to the myeloma cell line, sp2/o, using polyethylene glycol 1500 (peg 1500). monoclonal antibodies in the hybridoma culture supernatant were tested using elisa with his-tagged recombinant hpev3-vp0 protein. isotype determination was performed using isostrip mouse monoclonal antibody isotyping kit, following the manufacturer's instructions (roche diagnostics, basel, switzerland). vero cells were grown in dmem containing 10% fbs. hpev3 was provided by dr. masaki takahashi (iwate prefectural institute of public health). hpev3 was propagated in vero cells and quantified by qrt-pcr. the sequence information was as follows: 5 -gtaacaswwgcctctgggs ccaaaag-3 (forward primer), 5 -ggccccwgrtcagatccayagt-3 (reverse primer), and 5 -vic-cctrygggtacctycwgggcatccttc-bhq-3 (probe). wheat germ-synthesized recombinant his-tagged hpev3-vp0 proteins were separated by 10% sds-page in running buffer (250 mm glycine, 25 mm tris, 0.1% sds). the separated proteins were transferred to a pvdf membrane (millipore). the membranes were washed with blotting buffer tbst (tbs containing 0.05%-tween 20), and then blocked for 1 h at room temperature in 5% non-fat powdered milk in tbst. thereafter, the membranes were incubated overnight with generated hybridoma supernatant (1:50 dilution in tbst) at 4 • c. next, after washing thrice in tbst, the membranes were incubated for 1 h at room temperature with anti-mouse igg-hrp secondary antibody (1:10000 dilution in tbst). finally, after washing thrice in tbst, the target protein was detected with the immobilon western chemiluminescence detection system (ge healthcare) using fluor chem fc2 (alpha innotech corp. tokyo, japan). for epitope mapping, we prepared deletion mutants of hpev3-vp0 using pcr mutagenesis with template vector peu-his-hpev3-vp0, followed by wheat germ cell-free protein synthesis. the proteins were analyzed by immunoblotting using our generated mabs. to examine the amino acid variability among the vp0 proteins of other hpev genotypes, vp0 sequences for hpev1-8, 14, and 17-19 were accessed from the genbank and aligned with the hpev3-vp0 sequence using the mega software. k d values were determined by bio-layer interferometry (bli) using octet red96 (fortebio, usa). anti-mouse igg capture biosensor tips (amc, fortebio) were loaded with 20 µg/ml of mab #8 and #39 for 5 min, in pbs containing 0.1% bsa and 0.01% tween20. the association of recombinant vp0 at concentrations of 50, 25, 12, and 5 nm for #8 mab, and 14, 7, 3.5, and 1.75 nm for #39 mab, was measured for 5 min, followed by a 10-min-long dissociation phase. all measurements were corrected for baseline drift by subtracting a reference well. the operating temperature was maintained at 30 • c. data were analyzed using a 1:1 binding model with global fitting algorithms in the fortebio data analysis software. each mab was diluted in 50 mm of carbonate buffer (ph 9.6) to a concentration of 10 µg/ml, and then added to an elisa plate (agc techno glass, shizuoka, japan). to immobilize the antibodies, the plate was incubated overnight at 4 • c. wells were blocked with pbs containing 2% (w/v) skim milk for 1 h at room temperature (rt). after three washes with pbs containing 0.05% (v/v) tween-20 (pbs-t), 100 µl of antigen protein (8 ng/ml) diluted with pbs-t or blank (pbs-t alone) was added, and the mixture was incubated for 60 min at rt. after three washes with pbs-t, 100 µl of each mab, conjugated with horseradish peroxidase (hrp), was added into each well and incubated for 60 min at rt. antibody labeling was performed using the peroxidase labeling kit-nh2 (dojindo laboratories, kumamoto, japan). after three washes with pbs-t, 100 µl of abts substrate solution (kirkegaard and perry laboratories, washington, dc, usa) was added and the mixture was incubated for 30 min at rt. absorbance at 405/490 nm was measured on glomax discover system (promega), and the signal-to-noise ratio (s/n) was calculated. for generation of mabs, we produced n-terminal his-tagged full-length vp0 protein of hpev3 as an antigen. as a result, hpev3-vp0 protein was produced with high aqueous solubility (figure 1a) . the protein was subsequently purified using ni-sepharose beads, followed by elution with imidazole. balb/c mice were then immunized with the purified protein. after 4 weeks of immunization, the mice splenocytes were isolated, fused with myeloma cells, and hybridomas were produced. as a result, 48 stable hybridomas were generated and designated as #1 to #48. among these 48 clones, nine were selected (#3, #6, #8, #12, #27, #30, #34, #39, and #41), based on the reactivity in elisa to the target antigens vp0 proteins derived from hpev1 and hpev3 (figure 1b) . isotype analysis revealed that #3 mab belongs to igg1, kappa isotype, #6 and #12 mabs belong to igg2a, kappa isotype, while the others belongs to igg2b, kappa isotype ( figure 1c) . to demonstrate the antibody-binding sites within the antigen, we next performed epitope mapping. for epitope mapping, we produced five deletion mutants of vp0 and performed immunoblotting analysis. we found that our newly developed mabs recognized three distinct domains in hpev3 vp0: #30 and #34 mabs bind to 68-121 amino acid (aa), #3, #8, and #27 mabs bind to 127-172 aa, and remaining four mabs (#6, #12, #39, and #41) bind to 225-289 aa within c-terminal region of the vp0 protein of hpev3 (figure 2a) . we further created deletion mutants for a more precise epitope determination for these mabs, and found that #30 and #34 mabs bind to 82-95 aa, #3, #8, and #27 mabs bind to 133-159 aa, and #6, #12, #39, and #41 mabs bind to 275-289 aa (figure 2b) . we next examined whether the antigenic epitopes were located on the surface of hpev3-vp0. the ucsf chimera software revealed that, except for #30 and #34, the binding regions of all mabs were located on the molecular surface of vp0 protein (figure 2c) . the binding region of mabs #30 and #34 was relatively conserved among the analyzed hpevs (figure 2d ). to demonstrate the antibody-binding sites within the antigen, we next performed epitope mapping. for epitope mapping, we produced five deletion mutants of vp0 and performed immunoblotting analysis. we found that our newly developed mabs recognized three distinct domains in hpev3 vp0: #30 and #34 mabs bind to 68-121 amino acid (aa), #3, #8, and #27 mabs bind to 127-172 aa, and remaining four mabs (#6, #12, #39, and #41) bind to 225-289 aa within c-terminal region of the vp0 protein of hpev3 (figure 2a) . we further created deletion mutants for a more precise epitope determination for these mabs, and found that #30 and #34 mabs bind to 82-95 aa, #3, #8, and #27 mabs bind to 133-159 aa, and #6, #12, #39, and #41 mabs bind to 275-289 aa (figure 2b ). we next examined whether the antigenic epitopes were located on the surface of hpev3-vp0. the ucsf chimera software revealed that, except for #30 and #34, the binding regions of all mabs were located on the molecular surface of vp0 protein (figure 2c) . the binding region of mabs #30 and #34 was relatively conserved among the analyzed hpevs (figure 2d ). we next investigated the specificity of our newly developed mabs. for this, we created vp0 proteins encoded by the six different hpev genotypes and vp4-vp2 proteins derived from enteroviruses 71 and d68. immunoblotting analysis showed that six mabs (#3, #6, #8, #12, #27, and #39) react specifically with hpev3 vp0, while three mabs (#30, #34, and #41) showed some crossreactivity to other hpevs (figure 3) . no mabs showed cross-reactivity to enterovirus vp4-vp2 proteins. we next investigated the specificity of our newly developed mabs. for this, we created vp0 proteins encoded by the six different hpev genotypes and vp4-vp2 proteins derived from enteroviruses 71 and d68. immunoblotting analysis showed that six mabs (#3, #6, #8, #12, #27, and #39) react specifically with hpev3 vp0, while three mabs (#30, #34, and #41) showed some cross-reactivity to other hpevs (figure 3) . no mabs showed cross-reactivity to enterovirus vp4-vp2 proteins. sandwich elisa systems with highly specific matched antibody pairs are commonly used to detect and quantify viral antigens in immunoassay. hence, we next determined the optimal pair of mabs for antigen-capture using elisa, by evaluating all possible combinations of immobilized and labeled mabs. among the 36 possible pair combinations, only the #8 and #39 mab pair represented a combination of antibodies specific for hpev3-vp0 (figure 4a ). therefore, we selected this combination for further analysis. sandwich elisa systems with highly specific matched antibody pairs are commonly used to detect and quantify viral antigens in immunoassay. hence, we next determined the optimal pair of mabs for antigen-capture using elisa, by evaluating all possible combinations of immobilized and labeled mabs. among the 36 possible pair combinations, only the #8 and #39 mab pair represented a combination of antibodies specific for hpev3-vp0 (figure 4a ). therefore, we selected this combination for further analysis. to characterize the equilibrium dissociation constant (k d ) of the selected antibodies and the target vp0 antigen, we used the bli octet assay system. k d for antibody-antigen binding in mabs #8 and #39 was calculated as < 1 × 10 −12 and 3.72 × 10 −10 , respectively, suggesting that both mabs showed high binding affinity to the hpev3-vp0 (figure 4b) . we next performed antigen-capture elisa with recombinant vp0 protein and virions released into the cell-culture supernatant of the hpev3-infected cells. using the optimal antibody pair (#8 and #39 mabs) identified above, we determined the detection threshold for antigen recognition by antigen-capture elisa. our results revealed that our system was highly sensitive to the recombinant antigen, capable of detecting the protein at a concentration of 3 ng/ml (figure 4c, left) . in parallel, we investigated the detection limit of elisa for the hpev3 virion. this elisa system could detect heat-treated hpev3, but not non-heated virions, and its detection limit of the system was 1 × 10 9 copies/ml (figure 4c, right) . we also found that this elisa system exhibited no cross-reactivity with enteroviruses ( figure 4d ) to characterize the equilibrium dissociation constant (kd) of the selected antibodies and the target vp0 antigen, we used the bli octet assay system. kd for antibody-antigen binding in mabs #8 and #39 was calculated as < 1 × 10 −12 and 3.72 × 10 −10 , respectively, suggesting that both mabs showed high binding affinity to the hpev3-vp0 (figure 4b) . we next performed antigen-capture elisa with recombinant vp0 protein and virions released into the cell-culture supernatant of the hpev3-infected cells. using the optimal antibody pair (#8 and #39 mabs) identified above, we determined the detection threshold for antigen recognition by antigen-capture elisa. our results revealed that our system was highly sensitive to the recombinant antigen, capable of detecting the protein at a concentration of 3 ng/ml (figure 4c, left) . in parallel, we investigated the detection limit of elisa for the hpev3 virion. this elisa system could detect heattreated hpev3, but not non-heated virions, and its detection limit of the system was 1 × 10 9 copies/ml hpev3 is increasingly being highlighted as a potentially severe viral infection in neonates and young infants. therefore, there is an urgent need to develop assays for early diagnosis of hpev3 infection for reducing inappropriate antimicrobial use, unnecessary investigations, and prolonged hospitalization. it is also likely to lead to follow-up for potential complications in infants who are severely affected [1] . in this context, a real-time pcr-based molecular test to detect virus from patients was recently developed [31] . however, pcr tests are extremely sensitive and need extensive controls, whereas antigen detection by mabs has the advantage of the relative ease of sample handling and the use of less stringent procedures. specific mabs could then be used to develop a rapid test such as elisa. in this study, we sought to generate specific mabs and develop an elisa test for the detection of hpev3 vp0 antigen. hpev3 genome encodes for three structural proteins, namely vp0, vp3, and vp1, which assemble to create the virus particle [10] . among these, vp0 was identified as an antigenic determinant and it might be relatively more useful for diagnostic purposes, due to a higher level of sequence conservation [32] . furthermore, it possesses high immunogenicity [7] . therefore, the selection of vp0 as an antigen is both practical and reasonable. in addition, the mab quality was determined mostly by preparation of high-quality antigen. here, we used a wheat germ, cell-free protein production system for synthesizing recombinant vp0 proteins, as this system produces properly folded, soluble, and biologically active native proteins similar to those expressed in mammalian cells [29, 30, 33] . in our current study, we newly produced nine different mabs that recognized hpev3-vp0 as an antigen. we then performed epitope mapping of our generated mabs, as identification of the epitope is a key step in the characterization of monoclonal antibodies [34] . based on the epitope analysis, the mabs were able to recognize three different areas of hpev3-vp0 and specify 12-14 aa length epitopes within the hpev3-vp0. interestingly, two mab clones (#30 and #34) exhibiting cross-reactivity to vp0 proteins of other hpevs, bound a distinct epitope (82-95 aa), which partially overlapped with the recognition site for the polyclonal antibody (79-99 aa) created by chen et al. [27] . nevertheless, we obtained mabs specific to hpev3, which recognized sites 133-159 aa and the 275-280 aa site of vp0, which was not reported earlier. moreover, we also developed an elisa for detecting hpev3 antigen using two of our newly generated hpev3-specific mabs (#8 and #39), as mab-based elisa is highly specific and sensitive towards viral antigen detection [35] . our octet assay suggested that both mabs show a high binding affinity to the full-length hpev3-vp0 recombinant protein. vp0 protein can be folded into the correct native structure and is likely to form the capsid-like structure via oligomerization. in this situation, #8 mab could bind various sites on polymerized vp0 proteins, as a result of an allosteric effect or "avidity", owing to which the k d value of #8 mab was calculated to be much lower than that of #39 mab. our newly developed elisa system requires heat treatment of hpev3 to detect vp0 antigen. based on previous studies [7] , the binding area of mab #39 was estimated to localize to the interface between vp0 and vp3. on the other hand, three-dimensional modeling of viral particles revealed that the antigen-recognizing sites of mabs #8 and #39 in vp0 protein are proximally located. moreover, a previous study showed that glu285 (in the epitope region of #39) and ser28 (in the vp0 protein of hpev3) bind together by a hydrogen bond [24] . thus, a possible explanation for our observation is that heat treatment helps antigen retrieval for the interface between vp0 and vp3, resulting in efficient access of mabs to the epitopes. according to a previous study [36] , an increase in antibody-binding capacity was exhibited when glycosylation of capsid protein was removed. based on this finding, we carried out pretreatment by resecting the connection between o-linked and n-linked glycosylation of hpev3. however, there was no obvious effect on antibody recognition in our sandwich elisa assay (data not shown), indicating that glycosylation might not affect the antigenicity for mab recognition. the detection limits of our newly developed sandwich elisa were 3 ng/ml for recombinant vp0 protein and 1 × 10 9 copies/ml for viral particles, respectively. assuming that one viral particle contains a single copy of a viral genome, there are 60 copies of vp0 protein per viral particle. for a viral particle detection sensitivity of 1 × 10 9 copies/ml, the vp0 protein detection sensitivity is presumed to be 3 ng/ml. therefore, we conclude that the detection sensitivity for the recombinant vp0 protein and the virus particles was almost equivalent, suggesting the feasibility of our method for use in actual clinical settings. other than the elisa method, several antigen-detecting tests using an antigen-antibody interaction are now available. for instance, the multi-array technology using electrochemiluminescence immunoassay (eclia) and chemiluminescent enzyme immunoassay (cleia) can provide several hundred times more sensitivities than the conventional elisa method [37] . another example is an influenza-testing kit using a highly sensitive immunochromatographic detection method based on silver amplification [38] . the limitation of sensitivity might be overcome by utilizing these sophisticated technologies combined with our monoclonal antibodies for hpev3 vp0 antigen. furthermore, we can potentially improve the detection sensitivity by altering the second antibody to recognize the poly-hrp complex [39] or by adding other antibodies, which can target vp1 or vp3 proteins. in summary, we utilized the wheat germ cell-free protein production system to synthesize the hpev3 vp0 protein and produced mabs that could specifically detect hpev3 but not other hpevs. we further explored the feasibility of these mabs in terms of their utility in various immunological applications. to the best of our knowledge, the hpev3 vp0 antibody as well as the elisa-based viral detection system reported here is the first of its kind ever reported. with the implementation of more sophisticated applications, our newly developed mabs could be useful for further development of diagnostic methods for hpev3 infection. human parechovirus: an increasingly recognized cause of sepsis-like illness in young infants detection and characterization of a novel human parechovirus genotype in thailand isolation and identification of a novel human parechovirus identification and molecular characterization of the first complete genome sequence of human parechovirus type 15 enterovirus and parechovirus infection in children: a brief overview sepsis-like disease in infants due to human parechovirus type 3 during an outbreak in australia strain-dependent neutralization reveals antigenic variation of human parechovirus 3 human parechovirus 3 causing sepsis-like illness in children from midwestern united states human parechovirus 3 and neonatal infections human parechovirus-3 infection: emerging pathogen in neonatal sepsis prevalence and characteristics 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improve detection and management of human parechovirus infection in young infants wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody monoclonal antibody-based capture elisa in the diagnosis of previous dengue infection identification of two n-linked glycosylation sites within the core of the simian immunodeficiency virus glycoprotein whose removal enhances sensitivity to soluble cd4 measurement of sirolimus concentrations in human blood using an automated electrochemiluminescence immunoassay (eclia): a multicenter evaluation development of a highly sensitive immunochromatographic detection kit for h5 influenza virus hemagglutinin using silver amplification improving the sensitivity of traditional western blotting via streptavidin containing poly-horseradish peroxidase (polyhrp) we thank naohito nozaki and satoko matsunaga for antibody production and technical assistance, and masaki takahashi (iwate prefectural institute of public health) for providing regents.conflicts of interest: y.y. is a current employee of kanto chemical co., inc. the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-023647-dlqs8ay9 authors: nan title: sequences and topology date: 2003-03-21 journal: curr opin struct biol doi: 10.1016/0959-440x(91)90051-t sha: doc_id: 23647 cord_uid: dlqs8ay9 nan . garrell j, modolell j: the drmm~hila locus, am antagonist of proneural geors that, lilac these genes, ~.ncodes a helix-loop-helix protein. ce/11990, 61:39-48 in crystals of ~or-nib-glu(oz~)-leu-mb-ala-leu-an~alm-lys(z)-alb.ome. pro~ natl acx*d sci usa 1990, 87:7921-7925 cloning and expre~inn of two distinct high-afl~nlty p~eptot~ ~geat¢ting with acidic and b~ic ~last growth imctogs. embo j 1990 . emboj 1990 emboj , 9:1957 emboj -1962 . he~gst l~ lf~w~m~ t, g~lwr~ i~. the ryb l gene in the l~lmion ye~tt sd~m~acctm~ pombe lfalcodin 8 a gtp-bindin s protein belated to rho and ypt: structot~, expfemflon and identificetion of its human homulogue. embo j 19~0, 9:1949 embo j 19~0, 9: -1956 serotouln receptor that activates adenyiate cyclase domain • of lutropin/choriogonadotropin receptor expressed in t~ cells binds choringonadotropin with ltlgh /tmnlty cllgol~o0onlal orsanization of adrenergic receptor genes molecoiat chagactegization of a rat ~2n-adrenerl#c receptor identificatlon of rpo30, a vaccinia virus rna polymerase gene with structut~ similarity to a eucaryotic transcription elon~ation factor nucleotide sequence analysis of the l g~ne of vesicular stomafltia virus (new jersey serotype) --identification of conserved domai~l~ in l proteins of nonsegmented negative-strand rna viruses a novel u,man immunodeflclency virus type-1 protein, try, shares ~'quences with tat, ent~ and rev proteins phosphoprotein and nucleo~psid protein evolution of vesicular stomatitis virus new jersey identification of a conserved region common to cadherius and hl~u~llzat s~ a hema~u!tinin~ sequence and evolutionary relationships of african swine fever vh*es thymidine kinase all unusual stnlctul~ of a putative t cell oncugene whlch allows production of similar proteh~ from distinct messengeg rnas ~ ldentilfication of a 3rd protein factor which binds to the rolls sarcoma virus ltr enhancer --po~lble homology with the serum response factor genetic variation and multigene fannllles in aj~rics~t swine fever virus sequence of the genome rna of rubella vh'us --evidence for genetic rearrangement du~n~ tosavirus evolution derse i~ equine infectious anemia virus tat--insights into the structure, function, and evolution of lentivtrus tran.~activator proteins a colrpoeison of the genome organization of capripoxvirns with that of the orthopo~ ~golutionary orlffln of human and s imian lmtmo~odeflciency vir~jes a new supe~mlly of putative ntp-bindin 8 domaan,t encoded by genomes of small dna and rna viruses envelope gene sequence of htlv-1 isolate mr-2 and its comlmrlann with other htlv-i isolates evolutionary relationship between luteovtruses and other rna plant viruses based on sequence motifs in their putative rna pulymet-am~ and nucleic acid hellcases isolation and sequence analysis of caenothabd/~s br/~w~e repetitive elements related to the ~ dqana transposon tcl jmolevo11990 selective clo~ sequence analysis of the hum l1 sequence* which t~ in the rehttively recent l~tt jowcs m& sequences related to the matte tra~acmable element acin the genum zea. j m0/ evo/1990 evulutiotmtv pattern of the hemas~utinin gene of inflmmza-b viru~s ~ulated in japan ~ cocir~lating linesses in the same epidemic semon the dna binding subuult of nf-kaplm-b is identical to factor kbfl and homologous to the rel oncogene product sequencins analyses and com~ of pmrainfluenza virus type-4a and type-4b np protein genes. virok>gie complete sequence of the gcnomic gna of o'nyon8-nyong virus and its use in the constroctton of alphavir~ phylogenetic trecs molecular clouln s of the rinderpest virus matrix gene --comparative sequence analysis with other paramyxorirm~. vi~logy cautd~an p~ ancestry of a human endogenous retrovirus ~ determination of an epitope of the diffuse systemic sclerosis marker antisen dna topoisomerase-l: sequence $1mllagity with retroviral ~ protein suggests a possible cause for autoimmunity in systemic sclerosis. pro6 natlacad s6i u&11989, 86:8492~96. mcgeoch dj: pgotein sequence cota~lxs show that the 'psuedoprotesses' encoded by poxviruses and certain retrovirus~ belong to the deoxyoridine triphtmphate family ~sk1 life: ~es of comme//na yellow mottle vlrus's complete dna sequence, genomlc discontinuities and transcript su88est that it is a pararetrovlnm i~l~titis c vllrll~ sborl~ amino acid sequence similarity with pe~tivirutu~ and flavivirus~ as well as members of two plant vlgus superggoupo mo~mann ti~ homology of cy~kine synthesis inhibitory factor (el-10) to the epstein-barr virus gene bcrfi nucleotlde sequence analysis of sa-omvv, a vlsna-related ovine lentivirus --phylo~-netic history of lentivirmms single copy seqoences in g~qgo dna retmmable a repetitive hnman aetrotrmmposon-llke family. y mo/e~/1990, 31:92 100 re¢otnblnation resulting in unusual features in the polyomavlrus genome isolated from a murine tumor cell line sequence anal~is of rice dwarf elxytoreovirus genome sewments s4, s5, and s6 -comparison with the equivalent wound tumor virus segments ho~tu~ ~ s71 is a ehylngcueticellly distinct human endogenous reteovtgal 1rlement with structural mad sequence homology to simian sarcoma virus (ssv). vi~ologie identification of a novel 65-kl)a cell surface receptor common to pm~cee~flc polypeptide molybdenum hydroxylas~ ~ the amino acid seqoence of chicken hepatic solfite oxidase frequency of a]mloglnai h |lm~tn haemoglobins ~ by c ~ t trmmitions in cpg dlnucleotid~ evidence for conservation of ferritin sequences amon 8 plants and animctbt and for a transit peptlde in soybean a 32-kda llpo~ortin from human mononuclear cells appears to be identical with the placental inhibitor of blood coagulation distinct fercedoxins from rhodobacter-capsulstus -complete amino acid sequences and molecular evolution n~ptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membgane cloning and characterization of a novel member of the cytochrome-p450 subfamily iva in rat prostate a directiy repeated sequence in the ~-globin promoter resulates transcription in murine efythroleukemla cells isolation and chamcterizatinn of the alkane-inducibie nadph-cytochrome-p-450 olf, idoreductsse gene from candida-tropicalls -identification of invarlant residues wlthin slmilmr amino acid sequences of direr'sent flavoproteins protein klnase-c inhibitor proteins -purification from sheep brain and sequence similarity to lipocortins and 14-3-3 mci~ aveml~ b& sequence homology between purple acid phosphatases and phusphoprotein pho*phatsses --are phesphoprotcin phosphatatms metalloproteins collt~|nln~ oil~-bridged dinuclcar metal centers negative regulation of the human ~-globin ca~ne by transcriptional interference: role of an mu repetitive ~lement amino acid sequence of chicken catisequestrin deduced from c dna -comp~rison of caisequestrin and aspartactin caisequestrin, an intesccilular calciumbinding protein of skeletal muscle sarcoplssmic reticulm, is homolokous to ~, a putstive latminin-binding protein of the exteac¢llular matr~ bovsm~ ]prote~ c inhihl.gog with structugll and fun~ hotdoio~ou~ ]~-.gtl~ to hum~zn plum~ protein c inhibitor sequence of silkworm hemolymph antitrypsin deduced from its cdna nucleoude sequence --~on of its homology with ~.rplus. l b~cbem (tokyo) human mm~t cell tryptm~e multiple cdnas and genes reveal • multigene serlne protemje lmmlly howam> jc: msc ore. n k#on encoding protehm iteleted to the multidtog ite~letance family of tra~membt'mne tratmpofters m~, a tks~me-speclfi¢ b•tmment membrane protein, is a ia.minin.like protein commrvation of a cytoplasmic ~xy-termitml domain of couexin 43, a gap junctional protein, in mammal heart and brain the a~lba//a~ plasma membrane h+-a~ multigene ~ -genomle sequence and expression of • 3rd lsoform, f b/0/owra op#n of calliphora peripheral l~otoreceptors r1-6 --homology with d~ rhl and po~tmnsi~domd processing evolution of rhodopsin supergene family --independent divergence of visual pibments in vertebrates and insects and po~ibly in mollusks ct~tpl¢ the g~ne~ amino acid ~'~m~me gene of sac~baromy~wcet~-v/~ae --nucleotide seque~tce, protein similarity with the other i~kers yeast amino acid petmme~mes, and nitrogen cataboht~ repreulon the 70-kda peroxlsomal membrane protein is a member of the mdr (p-glycoprotein)-related atp-bindin~g protein superfamlly a new clam of lym~o-real/vacuolar protein sorelng signais. l b~/chem complete amino acid sequence and homologies of human erythrocyte membrane protein band 4.2. proc natl acad scd us a the primary structure of a halorhodopsin from n pbaraom~--structural, functional and evolutionary impnoations for bacterial rhod~ and haloghodopslns soluble lactose. blndln~ vertelmue lectlns: a ~ family the a regulatory subunit of the mltochondrlal fi-atpa~ complex is a heat shock protein. identification of two highly conserved amino acid sequences amon~ the ~x-subunits and molecular ~ sequence of h ilmlfl ~ l~ieat~ • novel gene family of integral membrane proteoglycans a protein with homology to the c-termimml relationaxip~ between/m~-nylate cycla~ and n•+,k+-ati~se lit pat pancgtmti¢ islets human na+ ,k+-ati~¢ genes ~ beta~ubunit gene family conmina at lcest one gene and one ~ evolution of the mltc cles~l genes of a new world lh'imate from ancestral homologues of human non-clessical genes the cdna sequence of mouse pllp-1 arid homololgy to hntman cd44 c~ll s~e antitpm and promot#ycen core unk proteins ~tjott of cdna encodin~ a hnman sperm membr~e protein related to a4 amyloid protebm ptwlflcstlo~ c~mu'actet~muon, and con with memb~ne carbonic anhydrase from human kidney hypermumbility of cpg dinucleoudes in the propcptide-enced/ng sequence of the human alb~tmi~ gene dystt'ophhl in electric or~n of to, pedo-~ homologous to that in h,ml~ muscle botste~ i~ homolosy of a yeast acun-binding protein to signal trmmductlon proteins slid myosin-1 the complete sequence of drosophila alpha-spectrin --conservation of structurml domahm between alplm-~ and alpl~t4cttnin •~ettaatflon of a lqbrilisr collqwn gene ~ spruces reveais the etdy evolutionary appearance of two collqwn gene fmmilk~ the predicted amino acid sequence of ct-lnternexin is that of a novel neuronal lntegmedla~ ~ent protein otsen bl~ type xil collm~n. a larbe multidomnln molecule with partial homology to type ix cousllem / b/d aera 1989 amyioid protein in i~mni~l amyloidmfls (plmnlah type) ks homolollotm to gd$oilmb an ac, tht-i~h,.da~g protein. b/~bera b/q0b~ res commun key ji~ ~ of a proline-rich cell wall protein gene ~ of soybesn. a ~ ana/ysis. j b/o/~em chicken liver evolutionary rehttinnships and impflcations for the resulation of phoophohpsse-a2 from snake venom to human secreted forms identification of a locality in snake venom a-ncurotoxins with a slsnlficant comlm*itinmd similarity to marine smdl ct-conotoxins: implications for evolution and structure activity al~ph[biml~ albmtm|nm ~s members of the albumin, alpha-l~toprotein. vitamin-d-binding protein mul~ flmily ~ni~on of the hnm~n llpoprotein lllmse gene and evolution of the llpase gene family e~'t~ion of cloned human reticulocyte 15-1ipoxygenase and immunological evidence that 15-hpoxygetmses of different cell types are related identification of a protein alt~ inttaspecific evolution of a gene family coding for urinary proteins conservation between yeast and man of a protein a~ociated with i35 small nuclear rlbonucleoprotein stl~ctute and partial amino acid sequence of calf thymus dna topobmmaertt~-ii -coml~on with other type-h emmyme~ ol~nudeotide correlations between infector and hem genomes hint at evolutiotmry relationships. nu6/e~ scot/~ik p& carotenoid desmurases fi, om ~ ~and nmoowo~craua are stru~ and l~n~'tinnally comerved and eonmin domains homolosons to flavoprotein dimdflde oxldoreductm~ deininger pi2 stt'uc~uee and vsrisbihty of recently inserted alu family members a novel neutrolphfl chemmtttactant generated duan8 an ln~ammmtory reaction in the l~mt peritoneal cal~lt~ tt~ t~t~o -l~tl'~t~tloil~ ~ amino acid seque~tce and structural relmtmmhip to interkukin-& b~ffx~m j the multlfimctinna 6-methylmllcyllc acid syn~ ge~e of ~~ ~ its ge~e structmm ieimive to tl~t of other po~lyketide symhase~. f.urj b/odaem 1990 mammalkm ublquitin carrier prmmtmh but not i~:i~k, ame ltdated to the 20-kda yeast 182, rad6. bk~chem b/qohys res commun chambers gk: sequence. structure and evolution of the c.ene codin b for ~t-gi~erol-3-phe~plmte ~rdrotfm~ in om,qt~ the cotaplete sequence of bogu/ktmm nenrotoxin type-#, and com~ with other clostrldhtl neugoto~hm if: a pamlly of cxam~fltutive c/bbp-llkc dna blndln~ proteins attenuate the il-l~t induced, ni~b mediated trans-activation of the ansiotemflnogen gene acute-phase response element different fort~ of ultmhithomx proteim generated by alternative spttcim~ are functionally equivalent evolution of collagen-iv genes from a 54-batm pair faton --a role for lntrmm ht gem~ evolution evolution of the insulin superfamlly tcetins are structoraily related sertoli cell proteim who~ ~on is tightly coupled to the iprtsence of germ cells ivarie r~ a bovine homolo s to the human myolletti c determination factor myf~ sequence conservation and 3' proce~ing of transcripts proteiu sertne threonine phoephatmes -an expanding family coppes zl divergence of duplicate genes in three sciaenid species (perciformes) from the south co~t of uruguay coasfaneda m: rrs~j~o~a (mu-~--a~) repetitive dna seqmmce l~vointion in 3 ~hically mstinct isolates. cor~0 bnz~n physiol repetitive seq~ce involvement in the duplication and divergence of mouse lysozyme genes the structure of a subtermlnal nut/e/6 a6/ds res 1990 schoofs i~ h~ between amino acid sequenc~ of ~ v~'lt~tm'stte peptide hormones and peptides ~mlated fi-on~ invertebrate sources. corn# bm&.n mg~ol bun'nng s, ~us r& lqatelet gtycoprotetn nb-ma protein antssonim from snake venoms ---evidence for s fumlly of p~telet-~sgqpttlon lnhll~tol~ hikher plant orilgins and the whylogeny of gt~en allpte simihtrity between the t~ ~ sindln s proteins abf1 how big is the univet~ of e~otm worklwide diffegences in the ~ncideace of type ! diabetes are ammciated with amino acid variation at pos/tion 57 of the hi~-dq ~ chain yeast general trtnscelptimt l~ctor gf! --sequence requirements for binding to dna mad evointhmky commrvttion. nudeg m/ds res concerted ]rv~ution of primate mplm smelllte dna. e'~kmce foe tm an~mt~ sequence sbm'ed by goal~ md human x ~e alpha ~ttdllte the nuchl~m~ sequence of etve ribommaal protein genea from the o/anene. of ~~ impacattom concem~ the mtytosene~ relationship bet~-en cyanelles and chloropluts wmslanoer l~ a new member of a secretory protein gene family in the dipteran c~t~onomot~ tentaus ~ a variant repeat stracture the ~r sequence ~ --die.inn on the x-chromosome and y-chromosome of a large set of closely related sequence~, most of wmda are i~eudogene~ ba~ttmo~e l~ cloning of the pso dna binding subutdt of nf-kapi~-b -homolo~" to gel and dortml l-~te two-monooxr~muse from m~ --clon~ nucleotide sequence, and primary structu~ homology within an enzyme family genetic hot~o~n~ty ~ acute and chronic acute forms of spinal muscular atrophy genetic variants of bovine ~-lactogiobulin --a novel wild.type ~-lacto#obulin w and ~ts primary sequence. b/or (~rn h0tt0e sey/er l~ltogh~ dna evolution in the olmcm species subgroup of drooophll~ f mot evot lovell-badge l~ a gene mapldng to the sex-determining gegion of the mouse y chromommae ~ a member of a novel ~ of zmbryonk~ly genes ~titmte 1,2-dioxy~mm~ from p~.udomotm~ pustfi~mtion, characterization, ~md compm'tson of the f.mtymes from psemffmmm~m ta~o~k-ron/and aaammms~ spec~clties of the peptidyl prolyl cis-tratm isomeric activities of cydophmn and fk-506 bindh~ protein --evidence for the existence of a family of distinct enzymes. b~x/aem/ary mltochondrl~ dna evolution in primates -tt-atmltion gate has been extremely low in the lemug homeobox containing genes in the nematode ~enorbabd/f~ elk.gamin nucleic ac shdic add fateesses of ~ • voluttomu.y origins have serine active sites f~entlal arginlne residues dewact-rrer l~ the 188 ltilm0omal rna ~-quence of the s~t anemone anemom~s ssdcmta and its evolutionary intuition amomqg other eukaryotes inferred b'om s~l,.m.~ comlmrttmas of a heat shock g~ae in two nematorl~ the l~'/o multtgene family of ok~hag of cdna ~ for the ~ omin of human complement component ca~bi~una protein, seqaenoe homolo~ with thc a c~t~:~a~h proc natl acad s¢t usa1990 highly conserved core domain and unique n terminus with presumptive regulatory moti~ in a hmman tata factor (l'lql~) [letter] identification cimractertzaflon of a novel member of the nerve growth fmctor/besln.dertved neurotrophic factor family ~ bind8 to s~dlfmme [eal(~-so4)l~l-lcer ] and has a sequence homology with other pt'otelns that bind sulfated glycoconjut~tes anllllo acid seqmmce of clnnamomin, a new member of the elicitin family, and its comparison to cryptogein and capsicetn soluble and mtmo[~tle~ioc~ta~l h~ low-ml~n|ty adenomne binding protein (adenotin) --properties and homology with mtmmall~la and avian stress protelus. b~-/~om/stry edolatlon of complementary dna$ f~lcoding a cerebellum-enriched nuclear factor-i family that activates tt'anscription from the mouse m~.lin basic protein promoter ye~mt mltochondrlal dna polymet'ase is related to the family a dna polymerases nudeotide and deduced amino add sequence of a human cdna (nqo2) corresponding to a second membeg of the nad(p)h --quinone oxldoreductase gene family --extensive polymorphism at the nqo 2 gene locus on chgomo~ome-6. b/oc.heraistry ult~ sltnlltt'leles a~llolltll enzyme pterin binding sites as demonstrated by a monoeinnal amiidiotypic antibody blundell tl molecular anatomy: phylogenetic relationship* derived from three~limenslonal structure~ of proteins subfamily structure and evolution of the hnmtn 1.1 family of repetitive scquence~. f mot evo 3 selmt~te mltochondrlal dna sequences are contiguous in htlmsa~ genol~ic dna l~t~lit~ within mmmm~lla~ sogl~tol deh~ --the prlmm'y structure of the human liver enzyme heterogeneous modifications of the l14/alo ltrote~a of ibtegleuldn-~t cells are concentrated in a/,ti~hly r~qg~.titlv ~ amino-t~ vaults.ell rebofmcleoprotein structures are msl~ conserved among higher and lower e~tes rnas le~d support to the monophyletic nature of the ~erla lmmunoloslcal ~lmllmtties ~etween cytosolic and partictdate tissue trans#utamilsc. febs lat mans~ti x#tope m~w~zed by a protective m~aodonm antibody is identical to the sta~e-specific embryonic antlgen-l. proc naa acad sa o~ 1990 the murg3 gene of t-brucei contains multiple dom.l.m of extensive editinil and is hofaoin~m~ to a subultit of nadh dehy~ neparm-bindl~ nenrotrophtc x~tor (hbnf) and mk, member's of z new i~mily of homolosous~ developmentally l~ted proteitm pugmattion and strucrmml ~on of pttcentel nad + .mtked 15-hydroxyproma#andm dehydtoffmase ~ the primary structure reveals the enzyme to belon 8 to the short-alcohol l)ehydrogena~ l~mlly. b/ochemistry structores and homologies of carbohydrate ~pho~ system ep~l~[ln, a ~o~a-gmjoclated mudn, is generated by a polymorphlc gene encodin8 splice variants with alternative amino termini a new member of the leucine zipper class of proteins that binds to the hia drct promoter. sc/ence attalysi~ of cdna for human ~ ajudgyrin i~dicltes a repeated structure with homology to tissue-differentiation a~td cell-cycle control protein the b subunlt of a rat hetefomeric ocaat-binding transcription factor shoes a striking sequence identity with the yeast hap2 transcription factor homology to mouse s-if and sequence similarity to yeast pt~2 stgucttu'e and evolution of the 02 small nuclear rna multigene family in primates: gene amplification under nat-¢wal selectinn? ident~catinn of an additional member of the proteln.tyrushle-phosp~ family --l*vidence f~ alternative spliclog in the tyrmine phosphzmme domain a 8~le am~o acid difference dis~ishes the human and the rat sequences of statlmaln, a ubiquitous intracehular pho~phoproteln ~ with cell item comp~ison of the seve~le~ gene* of drosop~ffa t~'ff~ end ma4~ muty, an adenine ~ active on g-a mislmirs, has homology to ~t evolution of largesubunit iutna structuge --the ~cation of imvetbe~t d3 dommin amon8 mmjor phyiolpmetic groups discrepancy in diveqlenoe of the mltodtondrlal and nuclear genomes of m sensor/and y~ j mot evot 1~90 adenylate deamll~t~. a mt~flige~e fam~ in p..m~,n, and rats isolmion and structure of ceerol#m, itna,~le hat~ peptmes, from the smm~m, ~ mo~ comp a~a rmm~ i~ vmotocin ge~ of the teleom f.,xott intro~ botany. ~ hot~ ot'l~mization. b~hemioy the adb gene areal share features of sequence structure and nudeast~protected sites. m0/cell bto/1990 the amino-acid sequence of multip/e lectins of the #.corn barnacle m~us-lgo~ and its homology with .animal ]~'tllls. bioclx'm btqobys acta amino add ~.-quence of mtmkey erythrocyte glycophorba mk. its amino acid ~'qu~'~icc ]f][~ a stri~tl~ homology with that of human glycophorin a flsp~r p& drtmophila proliferating cell nuclear antigen. structural and functional homology with its mammalian coonterpart phylogeny of n|trogen*me s~queac~ in ][~mnkla and other nlteogen-fixing ml~m$ vertebrate prot~mlne c~ne evolution.1. sequence alignments and gene structure florin l~ a major styl~ matrix polypeptid~ (sp41) is a member of the f~thogenesia-reiated proteins superciass complete amino acid sequence of rat kidney ornithine aminoteat~fet-~e --identity with ijver omithine aminotransferme. l bnxl;em (tokyo) rlbonuclease p --function and variation. j b/o/~bem the primary strum of glycoprotein-m from bovine adrenal medullary granules --sequence similarity with bnmmn serum protein-40,40 and rat sertoli cell giycoprotein-2 compm'ative ~quence/umlysis of m~mmantan f'a~or ix protaotegs the amino acid sequence of the b nman l~ia polymet'a~-h 33-kda subunit hrpb 33 is highly cotmerved among eukaryotes phylogenetic conservation of atylsulfatases --cdna cloturing and expre~ion of hnman aryisul~t~e-b. j b/o/cbem c.oll/l~'vlltion and diversity in fatnllies of coated vemcle adaptlns cllaracterizaflon of petel porcine bone sialoproteins, soca'~ted phosphopgotein ! (sppi, osteopontin), bone siaioprotein, and a 23.kda glycoprotetn ~ demonstration that the 23-kda glycoprotein is derived from the carboxyl terminus of sppi characterization of matteuccin, the 2.2s storag~ prote~ of the ostcich fern -evolutionary iteiatinnshlp to angiosperm seed storage ~ a new mmber of the glutamine-rlch protein gene family is characterized by the absence of internal lgepe~ts and the androgen control of its expression in the subm*ndlbuiar gland of pad 2 novel insect n~ with homology to peptides of the vea'te~ tachykinin family identircation of a novel platelet-derived neutrophli-chcmaotgctic po~ with structural homology to piatelet-factor-4 a novel repeated dna sequoncc located in the intergenic regions of ba~tceial chromosomes. nuc2eic.,k:ids res the proianlin storage protellx¢ of cere~ seeds ~ structure and evolution functional analysis of the 3'-terminal part of the balbiani ring gene by hlterspecies sequence comparison dr= mammaban ~yl phosphate symhetase (cp*) --cdna sequence and evolution of the cl m domain of the syrian hamster multifunctional protein cad mammalian dihydroorotase --nudeotide sequence, peptide sequences, and evolution of the imhydroorotsse domain of the multifunctinnal protein cad a receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins the control of flower morphogenesis in a~..ffd~um majusthe protein shows homoinff~ to transcription factors an element of symmetry in ytmst tata-box binding protein transcription factor-lid --consequence of an ancestra/ duplication? c-type natciuretic peptide (cnp): a new member of nateinretic peptide family identified in porcine brain evolution of antioxidant m~: ediol-dependent petoxidm~.s and thiol~ ~umong ptocaryotes towards the evolution of ribozymes alkyl hydroperoxide reductase from sa/mone/ta ~ur/um --sequence and homology to thinredoxin reductase and other fiavoprotein disuliide oxidoreducmses fc: nonuniform evolution of duplicated, developmentally controlled c~azrion genes in a sillumoth the fission yeast cutl + gene regulates spindle pole body duplication and has homolosy to the buddin structural homology b~ween the hnmmn fur gene product mad the sub---like protea~ encoded by ye~t/~x2. nuc~ a¢/ds res 1990 nudeotide sequences and novel steuctut~ features of hnm=. and cimm~ lighter ~# primary stt~t~ and expression of a nuclear-coded subunit of complex-n n~ to protetm specified by the chtoropiast genome. b/0chera bnfhys r~ commun a novel gene member of the human giycophorin-a and glycophorin-b genc fatuily -molecular cloning and expression the x-chromosome of monotremes shares a highly conserved region with the eutherlan and marsupial x-o~romosomes despite the absence of x-chromosome ittactt~tion c~lract~tion and or~= nl~tion of dna sequences adjacent to the evidence for a new fmily of evolutionarily conserved homeobox genes elellatltlll and albolabrin purified peptides from viper venoms --homologies with the rgds domain of flbrinogen and yon willebrand pactor measurement of $~tiv~-site homology between potato and l~bbit muscle alpha-glum phosphoryiases through use of a iane~r free energy relationship white 1~ weiss 1~ the neuroflbromatosis typed gene encodes a protein related to gap the dna damage-inducible gcne-dinl of saocbarom3q~ewcet~#.s/ae encodes a regulatory subunit of elbonucleotide reductase and is identical to gnr3 fhlgegprinting of ne~lr-homogeneous dna hgase-i and ligase-h from eh,m~n cells --similarity of their amp-binding domains control of m11na st~mlity in • chnoc~qg.~um, by 3'inverted ltepeats: effects of stem and loop mutations on degradation ofxtmba mlna/n vt~ nuc/e~ ac alternative messenger rna structures of the ciil-gene of bacteriophage ~. determine the rate of its tt'ansbttion initiation alternative mrna structures of the cm genc of bacta~ophage ~ detc:'mine the rate of its translation initiation. j mo/b~0/1989 a model fog iina editing in klnetopiastid mltochondrla --guide rna molecules transcribed from max/circle dna provide the edited information elements and coding sequences. j mol bio11989, 210:417-427 . chang c-y, ~ d-a, mohandas til chung b-c: stt~ctut~e, ~-quence, chromo~maal location, and evolution of the human fercedoxin gene family. dna cell b/o/1990, 9:205-212 key: cord-002973-bkr4ndl2 authors: seifi, morteza; walter, michael a. title: accurate prediction of functional, structural, and stability changes in pitx2 mutations using in silico bioinformatics algorithms date: 2018-04-17 journal: plos one doi: 10.1371/journal.pone.0195971 sha: doc_id: 2973 cord_uid: bkr4ndl2 mutations in pitx2 have been implicated in several genetic disorders, particularly axenfeld-rieger syndrome. in order to determine the most reliable bioinformatics tools to assess the likely pathogenicity of pitx2 variants, the results of bioinformatics predictions were compared to the impact of variants on pitx2 structure and function. the mutpred, provean, and pmut bioinformatic tools were found to have the highest performance in predicting the pathogenicity effects of all 18 characterized missense variants in pitx2, all with sensitivity and specificity >93%. applying these three programs to assess the likely pathogenicity of 13 previously uncharacterized pitx2 missense variants predicted 12/13 variants as deleterious, except a30v which was predicted as benign variant for all programs. molecular modeling of the pitx2 homoedomain predicts that of the 31 known pitx2 variants, l54q, f58l, v83f, v83l, w86c, w86s, and r91p alter pitx2’s structure. in contrast, the remaining 24 variants are not predicted to change pitx2’s structure. the results of molecular modeling, performed on all the pitx2 missense mutations located in the homeodomain, were compared with the findings of eight protein stability programs. cupsat was found to be the most reliable in predicting the effect of missense mutations on pitx2 stability. our results showed that for pitx2, and likely other members of this homeodomain transcription factor family, mutpred, provean, pmut, molecular modeling, and cupsat can reliably be used to predict pitx2 missense variants pathogenicity. paired-like homeodomain transcription factor 2 (pitx2, refseq nm 000325.5, mim# 601542) is located at 4q25 and is expressed in the developing eye, brain, pituitary, lungs, heart, and gut [1] . mutations in human pitx2 or the forkhead box transcription factor c1 (foxc1; 6p25, refseq nm 001453.2, mim# 601090) underlie the autosomal dominant disorder called axenfeld-rieger syndrome (ars; mim# 602482) [2] [3] [4] [5] . ars is a full penetrant, but clinically and genetically heterogeneous disorder characterized by developmental anomalies involving both ocular and non-ocular structures [6] . to date, 87 identified including deletions, insertions, splice-site mutations, and coding region frameshift, nonsense and missense mutations [7] [8] [9] [10] [11] [12] [13] . identifying new disease-associated variants is becoming increasingly important for genetic testing and it is leading to a significant change in the scale and sensitivity of molecular genetic analysis [14] . one of the most frequent approaches for detecting novel variants in target genes is using direct gene sequencing. however, due to increasing number of newly identified missense variants, it is often difficult to interpret the pathogenicity of these variants as not all the mutations alter protein function, and the ones that do may also have different functional impacts in disease [15, 16] . thus, prior to detailed analyses, novel variants cannot be easily classified as either deleterious or neutral, because of their unknown functional and phenotypic consequences. therefore, further research should be conducted to validate the genetic diagnosis when a novel missense variant is discovered. preferably, in vitro characterization of novel variants should be undertaken; however, due to facility limitation, it is often not practicable to experimentally verify the impact of large number of mutations on protein function [17] . another robust approach to substantiate the pathogenicity is using animal models by generating the homologous mutation that recapitulates the human phenotype; but, similar to in vitro studies, these are time-consuming, labor-intensive, difficult and expensive, making this approach unfeasible to experimentally determine the pathogenicity effects of all novel identified variants [18] . to circumvent the above mentioned limitations and to provide fast and efficient methods for predicting the functional effect of nonsynonymous variants on protein stability, structure, and function, several computational tools have been developed [19] [20] [21] . protein stability and structure are key factors affecting function, activity, and regulation of proteins. conformational changes are necessary for many proteins' function and disease-causing variants can impair protein folding and stability. missense variants are also capable of impairing protein structure, likely by affecting protein folding, protein-protein interaction, solubility or stability of protein molecules. the structural effect of mutational changes can be examined in silico on the basis of three-dimensional structure, multiple alignments of homologous sequences, and molecular dynamics [22] [23] [24] . therefore, analysing sequence data in silico first and detecting a small number of predicted deleterious mutations for further experimental characterization is a key factor in today's genetic and genomic studies. in general, bioinformatics prediction methods obtain information on amino acid conservation through alignment with homologous and distantly related sequences. the most common criteria considered in many bioinformatics programs for predicting the functional effect of an amino acid substitution are amino acid sequence conservation across multiple species, physicochemical properties of the amino acids involved, database annotations, and potential protein structural changes [23, 25, 26] . as mentioned above, resources for in vitro and in vivo functional analysis of novel variants are constrained in most clinical laboratories. therefore, identifying and reporting novel variants that are likely to be pathogenic often requires accurate prediction using computational tools. in a previous study, we examined the effect of foxc1 variants on protein structure and function by combining laboratory experiments and in silico techniques. our results showed that integration of different algorithms with in vitro functional characterization serves as a reliable means of prioritizing, and then functional analyzing, candidate foxc1 variants [27] . unlike most previous studies that focused on using only polyphen and sift to predict the pathogenicity of missense mutations, here, we investigated the predictive value of sift, poly-phen and nine other prediction tools by comparing their predictions to in vitro functional data for pitx2 variants. the bioinformatics programs found to be most reliable were then used to predict the likely consequences of 13 functionally-uncharacterized pitx2 variants. we also performed molecular modeling on all the pitx2 missense mutations located in the homeodomain and compared the results with the findings of protein stability algorithms to identify the most reliable tools in predicting the effect of missense mutations on pitx2 stability. to the best of our knowledge, this is the first study that incorporates the results of functional studies in conjunction with bioinformatics approaches for predicting the pathogenicity of mutations in pitx2 gene. lists of pitx2 missense variants were assembled from the previous literature and a search using the clinvar [28] , human gene mutation database (hgmd) [29], the genome aggregation database (gnomad), and the single nucleotide polymorphism database (dbsnp). this study found 47 pitx2 missense variants; 31 of which were described in the literature as being associated with ars or coronary artery disease (cad), while the remaining 16 variants, were considered as benign variants (fig 1) . eighteen of the 31 variants were classified as pathogenic based on functional studies utilizing site-directed mutagenesis, expression studies, and other functional analysis ( table 1) . thirteen of 31 variants were described as associated with ars and cad in the absence of functional analyses on pitx2 structure or function. sixteen snps, with population allele frequencies > 0.0005 were identified from the gnomad and the clin-var. based upon the allele frequency (approximately 10-fold greater than the disease frequency of ars) these have been considered benign polymorphisms. nucleotide numbering of the mutations herein indicates cdna numbering with +1 as the a of the atg translation initiation codon in the ncbi reference sequence nm_000325.5, while the amino positions are based on the corresponding ncbi reference sequence np_000316.2. this study is a retrospective case report that does not require ethics committee approval at our institution. all patients' mutations and phenotypes were obtained from previously published studies. these programs were used to analyse 18 functionally characterised pitx2 missense variants plus 13 additional, functionally uncharacterized pitx2 missense variants. sift program provides functional predictions for coding variants, based on the degree of conservation of amino acid residues in sequence alignments derived from closely related sequences, collected by psi-blast algorithm [60]. the polyphen-2 (polymorphism phenotyping-2) server predicts possible effect of an amino acid change on the structure and function of a protein using several sources of information such as straightforward physical and comparative considerations [61] . panther-psep is a new application that analyses the length of time a given amino acid has been conserved in the lineage leading to the protein of interest. there is a direct association between the conservation time and the likelihood of functional impact [62] . mutpred is a free web-based application that utilizes a random forest algorithm with data based upon the probabilities of loss or gain of properties relating to many protein structures and dynamics, predicted functional properties, and amino acid sequence and evolutionary information [52] . mutationtaster is a tool that combines information derived from various biomedical databases and uses established analysis programs. unlike sift or poly-phen-2 which work on dna level, mutationtaster processes substitutions of single amino table 2 for more information on the prediction tools used in this study. the nmr structure of the homeodomain of pitx2 complexed with a taatcc dna binding site (pdb: 2lkx) were analyzed by the swiss-model server (http://www.expasy.org/spdbv/ ; provided in the public domain by the swiss institute of bioinformatics, geneva, switzerland). model structures of wild-type and mutants were created in swiss-pdb viewer and investigated using the anolea server (http://melolab.org/anolea). for structure predictions of pitx2, sequence in fasta format was obtained from ncbi database (np_001191327.1). eight different protein stability programs (duet, sdm, mcsm i-mutant3.0, mupro, iptree-stab, cupsat, and istable) were used to predict the effects of missense mutations on the stability of pitx2 protein. duet is a web server that uses integrated computational approach to predict effect of missense mutations on protein stability [66] . duet calculation is based on complementary data regarding the mutation including secondary structure [67] and a pharmacophore vector [68] . sdm, a computational method, has been demonstrated as the most appropriate method to use along with many other programs. sdm assesses the amino acid substitution occurring at specific structural environment that are tolerated within the family of homologous proteins of defined three dimensional structures and change them into substitution probability tables [69] . mcsm relies on graph-based signature concept and predicts not only the effect of single-point mutations on protein stability, but also protein-protein and protein-nucleic acid binding [70] . i-mutant3.0 is a neural-network-based web server that predicts automatically protein stability changes upon single point protein mutations based on either protein sequence or protein structure. i-mutant3.0 can predict the severity effect of a mutation on the stability of the folded protein [71] . mupro is a set of machine learning programs that accurately calculates protein stability alterations based on primary sequence information particularly where the tertiary structure is unrevealed, overcoming a major restriction of previous methods which are based on the tertiary structure [72] . iptree-stab is a web service and mainly provides two function modules of services including discriminating the stability of a protein upon single amino acid substitutions and predicting their numerical stability values [73] . cupsat uses protein environment specific mean force potentials (through solvent accessibility and secondary structure specificity) to analyse and predict protein stability changes upon point mutations [74] . istable, a combined predictor, was designed by using sequence information and prediction data from various element predictors. istable is available with two different input types: structural and sequential [75] . please see table 3 for more information on the stability predictors used in this study. previous analyses of missense variations in different human diseases predicted that the stability margin without any immediate effect on protein fitness is 1-3 kcal mol -1 [77] [78] [79] . mutations that reduce the protein stability by >2 kcal mol -1 contribute to severe disease phenotypes [80, 81] . therefore, in this study, all variations were classified as predicted to be neutral (-1.5 < δδg < 1.5), stabilizing (δδg > 1.5) or destabilizing (δδg < -1.5). the protein sequence and/or protein structure with mutational position and amino acid residue of 18 previously functionally characterized pathogenic pitx2 missense variants, plus 16 snps with a population frequency of higher than 0.05% (thus considered benign polymorphisms), were used to test the predictive value of eleven common bioinformatics prediction programs; sift, polyphen-2, panther-psep, mutpred, mutationtaster, provean, pmut, fathmm, nssnpanalyzer, align gv-gd, and revel (table 4 and table 5 ). to evaluate the performances of the programs, seven measures (sensitivity, specificity, accuracy, precision, positive predictive value (ppv), negative predictive value (npv), and matthews correlation coefficient (mcc)) were calculated by comparing the results of all programs with previously generated functional data. for pitx2, mutpred, provean, and pmut were the most reliable of the bioinformatics tools in predicting the pathogenicity effects of all 18 functionally characterized missense variants in pitx2, with sensitivity and specificity of > 93% (fig 2) . then, revel tool showed high sensitivity and specificity, 94.44% and 87.50%, respectively. analysis of the sensitivity and specificity sift showed that this program had good sensitivity (72.22%) but low specificity (43.75%). although polyphen-2, mutationtaster, panther-psep, fathmm, and align gv-gd exhibited over 83% sensitivity, they were unable to identify the benign polymorphisms, showing specificity of 37.50%, 6.25%, 43.75%, 6.25%, and 6.25%, respectively. the predictive value of nssnpanalayzer was similar to that of sift program, with sensitivity and specificity of 66.67% and 43.75%, respectively. the most reliable programs found in this study's analyses (mutpred, provean, and pmut) were then used to predict the likely pathogenicity of 13 pitx2 missense variants for which functional testing has not been performed (table 6) . interestingly, the a30v variant unanimously was predicted as benign by all three programs. the remaining 12 pitx2 variants were predicted to be disease-associated mutations by all programs. molecular models for the homeodomain of wild-type and variant-containing pitx2 proteins were designed using threading algorithms to assess impairment of pitx2 structure by missense variants. three functionally characterised variants, n100d, l105v, and n108t, were excluded from these molecular modeling analyses since they are not located in the homeodomain, which is the only portion of pitx2 with a known structure. wild-type amino acids were changed to variant residues to determine putative structural effects of the remaining 15 functionally analysed pitx2 variants through anolea mean force potential calculations. the molecular modeling identified three mutations as high-risk (l54q, v83l, and r91p) to change the structure of pitx2, particularly in the h1, h2, and h3 subdomains (fig 3) . the r91p variant was predicted to grossly disrupt the non-local amino acid side chain contacts. similar, although less profound, effects were predicted when l54 and v83 were altered to glutamine and leucine, respectively. in contrast, the remaining twelve amino acid variants showed no predicted substantially altered pairwise interactions, indicating that these missense variants are predicted to have minor or no effects on pitx2's structure (s1 fig). molecular modeling was also performed on the nine functionally uncharacterised pitx2 missense mutations located in the homeodomain. four mutations (f58l, v83f, w86c, w86s) were predicted to change the structure of pitx2 (fig 4) , while, the remaining five variants (r62h, p64l, p64r, r69c, and r90p) were predicted to have minor or no impact on pitx2's structure (s2 fig). to assess the performance of eight different stability predictor programs (duet, sdm, mcsm, i-mutant3.0, mupro, iptree-stab, cupsat, and istable) in predicting the effect of missense mutations on pitx2 protein stability, the change in protein stability (δδg) were computed for all 24 pitx2 homeodomain variants (15 functionally characterised and 9 functionally uncharacterised mutations) (table 7) . of these eight programs, cupsat was the most consistent with the results of our molecular modeling, by identifying 5 of 7 destabilizing mutations that were also predicted to be destabilizing by molecular modeling (v83l, v83f, w86s, w86c, and r91p). computational analysis of pitx2 mutations sdm also showed high consistency with the results of our molecular modeling, by detecting 4 of 7 destabilizing mutations that were also predicted to be destabilizing by molecular modeling (l54q, r91p, w86s, and w86c). duet, mcsm, and i-mutant3.0 identified 3 and iptree-stab detected 2 of 7 destabilizing mutations detected by molecular modeling. mupro and istable were unable to identify any of the 7 destabilizing mutations predicted by molecular modeling. although in silico programs are not a substitute for wet-lab experiments, they can provide a supportive role in the experimental validation of disease-associated alleles and can help further diagnostic strategies by prioritizing the most likely pathogenic novel variants. while many tools are available for assessing the functional significance of variants, determining the reliability of prediction results is challenging. in this context, the current study investigated the combination of experimental findings, molecular modeling, in silico mutation prediction programs, and stability prediction software to assess the pathogenicity of pitx2 missense variants. in silico methods that correctly identify deleterious variants do not always inevitably work well for benign predictions. the methods determined by this study to be preferred for analyses of pitx2 variants were those best able to distinguish both pathogenic and benign variants, thus yielding the highest accuracy. our results showed that mutpred, provean, and pmut tools were the most accurate in predicting pathogenicity of pitx2 missense variants (fig 2) . the sensitivity and specificity of these three tools in recognizing pitx2 disease-causing variants were over 93%, indicating the strong performance of these programs in identifying as pathogenic only pitx2 variants with significant functional defects. after these three tools, revel showed highest sensitivity and specificity, 94.44% and 87.50%, respectively. sift showed good sensitivity (72.22%) but low specificity (43.75%). polyphen-2, mutationtaster and panther-psep, fathmm, and align gv-gd demonstrated > 83% sensitivity, but, they were unable to identify the benign polymorphisms, showing the specificity of 37.50%, 6.25%, 43.75%, 6.25%, and 6.25%, respectively. the predictive value of nssnpanalayzer was similar to that of sift program, with sensitivity and specificity of 66.67% and 43.75%, respectively. our results showed, therefore, that computational analysis of pitx2 mutations mutpred, provean, and pmut can be utilized with high confidence to test whether or not a pitx2 missense variant is likely to be deleterious. interestingly, mutpred was the only in silico program that ranked in the top three programs in identifying both pathogenic and benign pitx2 and foxc1 variants [27] . a likely explanation for mutpred's high ranking is that it evaluates the most factors in making assessments. however, since the number of variants available for testing in this study were small, a larger dataset would confirm that our results are reproducible and generally applicable. the three programs that were found to be the most reliable (mutpred, provean, and pmut) were then used to assess the likely pathogenicity of thirteen pitx2 missense variants for which functional analyses have not been performed, but which have been associated with ars or cad (table 6 ). our results showed that mutpred, provean, and pmut predicted as pathogenetic 12/13 of the variants. the a30v variant was scored as non-pathogenetic/benign by all three programs. while it is possible that a30v is an example of a false negative for all three programs, it is likely that this variant is instead benign. functional testing of the a30v variant is needed to determine which of these possibilities is accurate. various intramolecular interactions are involve in stabilizing and folded state of protein, including hydrophobic, electrostatic, and hydrogen-bonding [95] [96] [97] [98] . the stability state of a protein is key factor in its proper functionality. in fact, up to 80% of mendelian disease-causing mutations in protein coding regions are predicted to be caused by altering protein stability [99] . in recent years, due to the availability of high-throughput array-based genotyping methods [100] and next generation sequencing platforms [101, 102] , a large number of snps has been reported. however, the association of missense variants with protein stability has often been difficult to predict. fortunately, recent advances in computational prediction of protein stability offers potential insight into this question. we used two parallel prediction methods to investigate the possible effects on pitx2 protein structure and stability of missense variants. knowledge of a protein's 3d structure can be used to predict the functionality of protein and the possible impact of variants on protein conformation and structure. we thus first used molecular modelling analyses to assess and compared the total energy difference between native and mutated modeled structure of pitx2 proteins. the results predicted that while most pitx2 variants did not dramatically affect the protein tertiary structure, seven variants (l54q, f58l, v83f, v83l, w86c, w86s, and r91p) altered the total energy level in comparison with the native structure, suggesting that these amino acid substitutions changed the structure of the pitx2 protein. molecular modeling of the pitx2 homeodomain predicted that these variants impair the required energy to maintain the proper folding of helix 1-3 and cause global destabilization of the structure of pitx2. these seven amino acids are either invariant (e.g., w86) or highly conserved in the approximately 300 homeobox proteins analyzed, consistent with a pivotal role of these residues in the homeodomain [103] [104] [105] . these seven amino acids are tightly packed hydrophobic amino acids responsible for holding helices of the pitx2 homeodomain together, supporting our molecular modeling predicting that mutations of these amino acids disrupt pitx2 structure. for f58l, v83f, and v83l, the native wild-type residues and the introduced mutant residues differ in size, probably causing loss of hydrophobic interactions in the core of the protein, particularly involving helix 1-3. for l54q, w86c, w86s, and r91p, the wild-type residues and the mutant residues are different in both size and charge, likely disturb the local structure of protein thereby altering protein structure and function. residues v83, w86, and r91 are located within the third helix which is specifically responsible for binding with the major groove of the dna [106] . thus, the prediction that these mutations impair the capacity of this helix to interact with dna is consistent with this knowledge and with previous functional characterizations that showed reduced dna-binding capacities of the v83l and r91p mutant pitx2 proteins [5, 107] . consistent with bioinformatics predictions of deleterious affects of mutation of w86, mutations of the neighboring amino acids (r84w and k88e) have been shown to decrease the ability of the mutant proteins to interact with dna [39, 108] . residues l54 and f58 are located in helix 1 of the homeodomain, responsible for contacting with the minor groove of the dna. molecular modeling of l54q is consistent with the computational analysis of pitx2 mutations suggestion that mutations in these highly-conserved residues in helix 1 of the homeodomain might disturb the dna-protein binding affinity. our prediction is supported by the fact that changing the leucine to a glutamine (l54q) disrupts dna-protein complex, indicating the necessity of leucine at position 54 for pitx2 binding ability [109] . thus, consistent with our recent studies on foxc1 protein [110] , the results of molecular modeling of pitx2 are strongly consistent with the functional characterization of pitx2 missense variants. the results from our molecular modeling analysis were also compared to the predictions of eight stability predictor methods (duet, sdm, mcsm, i-mutant3.0, mupro, iptree-stab, cupsat, and istable). based on our analyses, it appears that cupsat performs the best of the seven methods evaluated here in predicting the effect of missense mutations on pitx2 protein stability, with sdm, duet, mcsm, and i-mutant3.0, performing weaker, consistent with the results of previous studies [111, 112] . our results indicate that further studies are required to improve δδg predictions, especially for buried amino acids. in this study, for the first time, we evaluated the impact of missense variants on pitx2 stability, structure and function by integrating stability prediction algorithms, bioinformatics mutation prediction tools, and molecular modeling. our results showed that mutpred, provean, pmut, molecular modeling, and cupsat are reliable methods to assess pitx family missense variants in the absence of laboratory experiments. however, for our analyses, it must be noted that we used sixteen snps as non-pathogenetic control variants to investigate the performance of prediction programs. although we considered snps with a population frequency of >0.05% as benign, we cannot formally exclude that these snps might have un-documented pathogenic effects on pitx2. in addition, while the prediction methods used in this study are not gene-specific, generalization of the performance of these programs to other human genes may be inappropriate without additional study. when assessing the pathogenicity of missense variants, it is necessary to be cautious on depending merely on in silico programs without wetlab experiments. according to standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the american college of medical genetics and genomics (acmg) and the association for molecular pathology, in silico predictions only serve as one supporting factor, whereas functional tests are frequently needed to assess the pathogenicity of missense variants. in particular, as per clinical guidelines for the interpretation of single substitution variants, the output of computational tools should be interpreted in the light of functional studies results, population frequency data and segregation in affected families. evidence that rieger syndrome maps to 4q25 or 4q27 expanding the spectrum of foxc1 and pitx2 mutations and copy number changes in patients with anterior segment 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syndrome the molecular basis of rieger syndrome. analysis of pitx2 homeodomain protein activities comparison of bioinformatics prediction, molecular modeling, and functional analyses of foxc1 mutations in patients with axenfeld-rieger syndrome screening of mutations affecting protein stability and dynamics of fgfr1-a simulation analysis performance of protein stability predictors the authors would like to thank the members of the walter laboratory for critical reading of the manuscript and for helpful comments. key: cord-004181-exbs3tz7 authors: pumchan, ansaya; krobthong, sucheewin; roytrakul, sittiruk; sawatdichaikul, orathai; kondo, hidehiro; hirono, ikuo; areechon, nontawith; unajak, sasimanas title: novel chimeric multiepitope vaccine for streptococcosis disease in nile tilapia (oreochromis niloticus linn.) date: 2020-01-17 journal: sci rep doi: 10.1038/s41598-019-57283-0 sha: doc_id: 4181 cord_uid: exbs3tz7 streptococcus agalactiae is a causative agent of streptococcosis disease in various fish species, including nile tilapia (oreochromis niloticus linn.). vaccination is an effective disease prevention and control method, but limitations remain for protecting against catastrophic mortality of fish infected with different strains of streptococci. immunoproteomics analysis of s. agalactiae was used to identify antigenic proteins and construct a chimeric multiepitope vaccine. epitopes from five antigenic proteins were shuffled in five helices of a flavodoxin backbone, and in silico analysis predicted a suitable rna and protein structure for protein expression. 45f2 and 42e2 were identified as the best candidates for a chimeric multiepitope vaccine. recombinant plasmids were constructed to produce a recombinant protein vaccine and dna vaccine system. overexpressed proteins were determined to be 30 kda and 25 kda in the e. coli and tk1 systems, respectively. the efficacy of the chimeric multiepitope construct as a recombinant protein vaccine and dna vaccine was evaluated in nile tilapia, followed by s. agalactiae challenge at 1 × 10(7) cfu/ml. relative percentage survival (rps) and cumulative mortality were recorded at approximately 57–76% and 17–30%, respectively. these chimeric multiepitope vaccines should be applied in streptococcosis disease control and developed into a multivalent vaccine to control multiple diseases. immunogenic protein characterization. proteins bound to a s. agalactiae antibody were eluted from protein a agarose and divided into two fractions. the first fraction was subjected to 4-20% gradient sds-page to observe the protein features and compare the protein profile from serotypes ia and iii. the second fraction was subjected to lc-ms/ms mass spectrometry to identify the immunogenic proteins. the protein profile from the immunoprecipitation on 1d-sds-page demonstrated that the major protein (approximately 55 kda) corresponded to rabbit immunoglobulin. however, several bacterial proteins could not be bound to rabbit immunoglobulin and were removed through the flow-through fraction (ft), whereas the protein that specifically bound to the anti-s. agalactiae antibody could be detected in the eluted fraction (fig. 1) . comparative immunoproteomics analysis of s. agalactiae serotypes ia and iii was determined by lc-ms/ ms and assessed by a venn diagram ( supplementary fig. 1 ). one hundred proteins were matched and identified between serotype ia and serotype iii via in-house protein databases, resulting in 79 shared proteins between serotype ia and serotype iii. the protein expression levels of the 79 common proteins were determined by hierarchical clustering (hcl). two groups of immunogenic proteins were demonstrated based on their abundance, and 37 proteins were overexpressed in serotype iii, whereas there was a lower abundance of 39 immunogenic proteins in serotype iii than in serotype ia (fig. 2) . regarding specific antigen-antibody interactions, 10 and 11 proteins were uniquely identified in serotypes ia and iii, respectively (supplementary figs. 1, 2) . linear β-cell epitope prediction and chimeric vaccine design. the epitopes of immunogenic proteins were predicted by the bcpreds server based on b cell epitopes to be used in chimeric multiepitope vaccine construction. in this study, not only immunogenic proteins from the immunoproteomics analysis were used but also other subunit vaccine candidates were subjected to epitope prediction and combined to produce a chimeric multiepitope vaccine. the amino acid sequences of the c-β protein (bac), surface protein rib (rib), lpxtg cell wall anchor domain-containing protein (spb1), surface immunogenic protein (sip), and cell surface protein heat map with hierarchical clustering (hcl) of normalized protein abundance reveals the 79 differentially expressed immunogenic proteins. the expression value showed in the relative intensities ranges from the highest protein abundance (red) to the lowest protein abundance (green) expression value. alternatively throughout the backbone likely provides potential bioactivity 22 . considering α/β fold structure, flavodoxin from escherichia coli [pdb accession code: 3chy] 23 was utilized as a linker to combine the epitope fragments from five antigenic proteins. predicted epitopes were randomly displayed on the α-helix structure of flavodoxin, generating 1,440 designed models due to the variance of 6 epitopes of bac and of 2 epitopes of sip protein. after joining, protein conformation was examined by molecular modeling with 7,200 constructs. i-tasser and stereochemical qualitative allowance manifested from 45f2 and 42e2 showed appropriate potential tertiary structure with optimal c-scores between −5 and 2. 45f2 and 42e2 also demonstrated the highest score of the amino acid allowance region in the ramachandran plot. the 45f2 multiepitope model represented 90.2%, 8.5%, and 0.7% of residues located in the most favored, allowed, and disallowed regions, respectively. meanwhile, the ramachandran plot regions for the 42e2 designed model comprised 83.0%, 11.6%, and 0.7%, respectively, of the residues ( supplementary fig. 3 ). the epitope arrangements in 45f2 and 42e2 were represented in a 3d structure of chimeric proteins, showing that all chosen epitopes were exposed to the protein surface. the five epitopes were displayed as α-helical layers surrounding 3chy linkers, which appeared as five-stranded parallel β-sheets at the structure's center, with the order 21345 (fig. 3 ). codon optimization of chimeric multiepitope vaccines and plasmid construction. the ectopic expression of bacterial protein in the fish cells may not be achieved due to different codon utilization in the bacterial system. subsequently, codon optimization of the chimeric multiepitope vaccine was analyzed by geneart ™ 's gene optimization according to iso 9001 standards (registration no. 1210024212) to apply the codon bias of oreochromis niloticus. the region of an ideal gc content range-between 30% to 70%-was well optimized. moreover, negative cis-acting sites included internal tata-boxes, chi-sites and ribosomal sites; at-rich or gc-rich sequence stretches; rna instability motifs; repeat sequences; rna secondary structures; and splice donor and acceptor sites in higher eukaryotes, which were successfully removed from these chimeric multiepitope dna vaccine sequences. the best two predicted chimeric multiepitope vaccines were designated 45f2 and 42e2. codon adaptation index (cai) presented 45f2 and 42e2 scores that matched in codon utilization with that of nile tilapia of 0.92 and 0.93, respectively. the codon quality distribution index of 45f2 and 42e2 demonstrated that the codons within the dna sequence were distributed frequently in 90-100 positions at 77% and 78% ( supplementary fig. 4a-d) . the average gc content of both chimeric multiepitope vaccines was 56% ( supplementary fig. 4e ,f). single-stranded rna-folding prediction revealed the minimum free energy (mfe) secondary structure of 45f2 and 42e2 ( supplementary fig. 5 the protparam server demonstrated a theoretical pi of 4.1 and a molecular mass of 20 kda for 45f2 and 42e2. the total number of negatively (asp and glu) and positively (arg and lys) charged amino acid residues of 45f2 was 33 and 13 residues, while for 42e2, there were 31 and 12 residues, respectively. the estimated half-life of both chimeric multiepitope constructs was approximately 30 h in mammalian reticulocytes (in vitro), more than 20 h in yeast (in vivo), and over 10 h in e. coli (in vitro). 45f2 showed aliphatic index and grand average of hydropathicity values of 65.32 and −0.401, respectively, whereas 42e2 showed values of 67.93 and −0.296, respectively. the 45f2 and 42e2 proteins were indicated to be stable proteins, as represented by instability indexes of 31.31 and 25.53, respectively. antigenicity of the 45f2 and 42e2 chimeric multiepitope vaccines was predicted as 0.7538% and 0.7424% at a 0.4% threshold for the bacterial model, consistent with antigenpro server prediction by representing 0.936 and 0.923, respectively. these results indicate that both vaccine candidates have high potential antigenic www.nature.com/scientificreports www.nature.com/scientificreports/ properties. conformational b cell epitopes from the 3d protein structure computed by the discotope server demonstrated 11 b cell epitope residues in both 45f2 and 42e2 at a −3.7 threshold ( table 2) 24 . interestingly, the number of epitopes was reduced when computed at the −2.5 and −1.0 thresholds, with 45f2 showing 6 and 3 b cell epitope residue regions, respectively, while 42e2 contained only 2 and 1 b cell epitope residue regions, respectively (table 2 ). recombinant plasmids harboring 42e2 and 45f2 were constructed, namely, pet28a (+)_42e2 or _45f2 and pcdna3.1 (+)_42e2 or _45f2, which were used to determine the recombinant chimeric multiepitope vaccine expression (fig. 4) . chimeric multiepitope protein expression was tested in a bacterial expression system and a fish cell (tk-1) culture expression system. these results demonstrated that both chimeric multiepitope proteins could be expressed in both systems, with the expression detectable within 3 h in e. coli (30 kda) and within 7 days post-transfection in tk-1 cells (25 kda) (fig. 5 ). larger-sized chimeric multiepitope proteins in the e. coli expression system resulted from an additional tag at the n-terminus, which was contained in the pet28 expression vector. vaccine efficacy. after vaccination, fish were challenged with s. agalactiae, and infected fish showed clinical signs of streptococcosis disease, such as swirling swimming, opaque eye, exophthalmia and abscess. these moribund fish were collected, and bacteria were re-isolated, showing that they were infected with s. agalactiae serotype iii ( supplementary fig. s7) . www.nature.com/scientificreports www.nature.com/scientificreports/ dna vaccine efficacy testing showed that fish immunized with either 45f2 or 42e2 had cumulative mortality rates of 16.67 ± 5.77% and 16.67 ± 15.27%, respectively, which were not significantly different from those of the fkc-vaccinated fish (p > 0.05). however, in the control group [empty vector; pcdna3.1(+)], 70.00 ± 10.00% mortality was observed at 21 days post-challenge (fig. 6a ). the recombinant chimeric multiepitope protein vaccination showed that 45f2 and 42e2 produced cumulative mortality rates of 30.00 ± 10.00% and 26.67 ± 5.77%, respectively, which were significantly lower than those of the negative control group, at 70% (p < 0.05) (fig. 6a) . the 45f2 and 42e2 dna vaccines demonstrated similar patterns of rps, with 76.19 ± 8.24% and 76.19 ± 21.82%, respectively, which were not significantly different from those of the fkc-immunized fish (76.19 ± 21.82%). however, they were significantly higher than those of the recombinant protein vaccines, which showed 61.90 ± 8.24% and 57.14 ± 14.28% for 42e2 and 45f2, p < 0.05, respectively (fig. 6b ). immune response. to determine the immune response, dot blot analysis of serum prepared from 42e2-or 45f2-vaccinated fish was used. it was demonstrated that the dna vaccine could gradually activate the production of fish antibodies from the 1 st to the 4 th week. the pattern of antibody response differed from that for the recombinant protein vaccine, with the highest activation of antibody production being significantly produced in the 2 nd -3 rd week and suddenly dropping in the 4 th week. the highest induction was observed in fkc-immunized fish (fig. 7) . dot blot analysis of vaccinate fish sera against whole cell lysate of s. agalactiae serotype ia and iii demonstrated that fish vaccinated with recombinant protein vaccine 42e2 and 45f2 showed cross-reactivity to whole cell lysate of s. agalactiae serotype ia and iii ( supplementary fig. s8 ). for the reverse vaccinology approach, computational analysis using a variety of bioinformatics tools is robust and beneficial when identifying appropriate vaccine candidates 18 . bacterial genomics and proteomics analysis indeed help researchers analyze proteins, short domains, and pathogenic epitopes that provide high immunogenicity and high antigenicity for multimeric vaccine development 25 . therefore, immunoproteomics should be applied as a preliminary process to screen antigenic proteins and minimize potential candidates for vaccine development 21 . several immunogenic proteins in this study were described previously, such as c5a peptidase and laminin-binding surface protein (lmb), which are cell surface proteins that have an important function in www.nature.com/scientificreports www.nature.com/scientificreports/ chemoattractant activities and are proteins promoting invasion of group b streptococcus (gbs) 26, 27 . however, the current immunoproteomics analysis from this study identified new immunoreactive proteins, such as bacteriocin transport accessory protein, dihydrofolate reductase, ssu ribosomal protein s8p, transposase tnpa, 1,4-alpha-glucan, cell wall surface anchor family protein, and the gtp-binding protein era. as expected, most of these are cell surface proteins, which are suggested to be associated with bacterial virulence 27, 28 . subsequently, the identified immunoreactive proteins may be used in further vaccine development. multiepitope vaccines are an interesting issue since constructed vaccines designed by in silico analysis may elicit cellular immunity and provide effective responses 25, 29 . it is known that immunodominant b cells could strongly induce both cellular and humoral immunity; thus, evaluation of b cell epitopes was performed to identify potent epitopes before integrating them to produce a multiepitope vaccine. moreover, this vaccine type is more efficient than whole antigens for controlling staphylococcus spp. infections 20, 30 . from the present study, table 2 . predicted conformational b-cell epitopes from 3d structure of designed chimeric multiepitope vaccines using discotope 2.0 server. www.nature.com/scientificreports www.nature.com/scientificreports/ linear b cell epitope prediction was assessed and identified 11 potent epitopes from 5 common immunogenic and virulence proteins that were present in serotypes ia and iii. the previous studies supported that one of the chosen proteins, sip, represented a highly conserved protein among gbs isolates and showed cross-protective immunity against gbs infections 11, 13, 14, 31 . prediction of candidate antigenic proteins can be used to select the bacterial strains that carry antigenic genes, as well as to determine high expression levels in the target host and the accessibility of particular antigens in host organisms 16 . therefore, these selected immunogenic proteins might be suitable for consideration in a rational vaccine design. rational chimeric multiepitope vaccine design was achieved by randomly combining epitopes from 5 immunogenic proteins and conjugating with core structures of flavodoxin (β-1-5-3chy) to produce a secondary structure with α/β folding. in addition to the α/β-type folding of flavodoxin, it was also useful to construct our chimeric multiepitope vaccine by forcing the 5 chosen epitope segments to fit within 5 α-helix loops and protrude out of the 3d-folded structure since that configuration benefits protein solubility by exposure to water molecules 23 . additionally, this linker may promote the solubility of the constructed vaccine and help enhance the recognition of the vaccine by the host's immune system, which contributes to vaccine efficacy. 45f2 and 42e2 presented the most favored region of protein folding, with the stereochemical quality representing the disallowed region at only 0.7%, which is acceptable since the minimum quality should be less than 2% 32 . it is suggested that in silico analysis could design a chimeric multiepitope vaccine that could probably manifest effective properties 33,34 . to achieve a high level of protein expression in nile tilapia, codon optimization was conducted to improve the transcription and translation capability by removing all possible cis-acting sequence motifs, which may have a negative impact on protein expression. both proteins had a cai > 0.8 and a codon with frequent distribution (cfd) > 30%, which are acceptable for high expression in the target organism 18, 21 . the gc content of 45f2 and 42e2 was optimized between 30-70% and had a suitable thermodynamic ensemble free energy, which allowed rna folding and thermodynamic stability 35, 36 . the overall points suggested that the modeled 45f2 chimeric multiepitope vaccine was clearly the best candidate vaccine. numerous effective single-serotype gbs vaccines have been reported, including vaccines for controlling streptococcosis in tilapia 10, 11, 13, 14, 17, 37, 38 . however, it is known that single-serotype whole-cell inactivated vaccines have limitations during cross-prevention against different serotypes. for instance, a s. iniae vaccine (serotype i) could not protect atlantic salmon from infection by s. iniae (serotype ii) 39 . meanwhile, mixed-serotype vaccines (serotypes iv and vii) could promote antiserum levels and enhance the survival rate of newborn pups against streptococcal infection 40 . although formalin-killed vaccines generally provide highly protective effects compared with those of subunit vaccines and dna vaccines, the subunit and dna vaccines may replace the original formalin-killed vaccines or inactivated vaccines due to their promising efficacy, which are similar to those of inactivated vaccines, and longer shelf life 18, 33 . evidence suggests that dna and subunit vaccines can efficiently trigger the immune system and promote protective efficacy, with an rps value greater than 50% 11, 13, 14, 17 . nevertheless, these vaccines have limitations, such as their mass production costs, and they may require various optimizations to obtain the highest stable storage conditions 18, 41 . regarding this idea, a chimeric multiepitope vaccine composed of different epitopes from different proteins common in both serotypes ia and iii was generated to achieve broad protection against different serotypes and increase their stability. interestingly, the designed chimeric multiepitope dna vaccine and protein vaccine exhibited effective prevention in nile tilapia against s. agalactiae, with efficacy similar to that of the whole-cell inactivated vaccine. this evidence supports the strategy of rational vaccine design through b cell recognition using in silico analysis. importantly, immunoproteomics analysis could assist the preliminary determination of suitable immunogenic proteins for vaccine development due to the distinct antigenic determinants that can mediate dissimilar immune responses. the criterion in immunogenic protein selection for vaccine development has focused on the ability of a particular protein to induce an immune response. among 79 identified proteins shared in both serotypes, in addition to providing the highest bcpred scores (table 1) , the 5 proteins chosen were also reported as virulence proteins and used as vaccine candidates for streptococcosis disease prevention 13 . for example, c-β protein (bac) can lead to antibody production through fc region binding of human iga 42 . sip protein has been shown to mediate protection against streptococcal infection 11, 13, 14 . additionally, the chosen immunogenic protein should be conserved among streptococcus spp., so it would be suitable for application in cross-reactive prevention among s. agalactiae serotypes 11, 43 . moreover, it should be mentioned that peptide vaccines or epitopes with only 30 amino acid residues may trigger immune responses through binding directly to mhc-i or mhc-ii molecules. these molecules localize to nonprofessional antigen-presenting cells. vaccines containing proteins with longer amino acid sequences can enhance the presentation of epitopes to dendritic cells due to t cell induction 25, 44, 45 . herein, the comparative efficacy of both the 45f2 and 42e2 dna and recombinant protein vaccines indicated that the dna vaccine provided a higher efficacy than the recombinant protein vaccine. this result suggests that the dna vaccine can prolong the activation of the immune response by triggering both humoral and cellular immune responses 46, 47 . moreover, the clearance rate of the recombinant protein vaccine in the host system may be faster than that of the dna vaccine. this difference implies that the dna vaccine can enter the host cell to produce chimeric multiepitope protein, with that protein existing in the host system for longer than the recombinant protein vaccine, thus enhancing its bioavailability. taken together, these data indicate that the antigen combination has shown promise for streptococcosis disease control in nile tilapia. this research demonstrated a novel platform for rational vaccine design based on chimeric vaccine development that used flavodoxin with a tim-barrel structure as a template. our chimeric protein backbone is suitable for presenting epitopes to be recognized by the host immune system. with 5 epitopes, it could activate antibody production and demonstrated promising protection against bacterial disease similar to that of a whole-cell inactivated vaccine. this platform will promote the production of multivalent vaccines to control multiple diseases and for other applications in the future. experimental fish, bacterial strain and antibody. all male s. agalactiae-free nile tilapia (oreochromis niloticus linn.) were obtained from a commercial gap farm in thailand. the experiments were conducted in accordance with guidelines approved by the national research council of thailand. the experimental fish were anesthetized with clove oil to minimize stress during vaccination and challenge testing. s. agalactiae serotypes ia and iii were cultured as described previously 7 . s. agalactiae serotype iii was used for polyclonal antibody (pab) production, which was kindly provided by prof. ikuo hirono, tumsat, japan. antibody against igm of nile tilapia was kindly provided by assist. prof. eakapol wangkahart. mahasarakham university, thailand. immunoproteomics analysis. s. agalactiae was grown in bhi broth at 30 °c with agitation until reaching exponential phase. bacterial cells were collected by centrifugation, lysed in 100 µl of lysis buffer [tris-buffered saline (tbs) with 1% tween-20 and 0.01% lysozyme] and incubated at 50 °c for 20 min following sonication on ice. protein a agarose beads (cell signaling, usa) were added to the bacterial protein lysate, and nonspecific proteins were removed by 10 min of centrifugation at 10,000 × g at 4 °c. clarified supernatant was supplemented with 5% glycerol and then with a pab specific to s. agalactiae serotype iii (1:500 dilution). then, 30 µl of protein a agarose beads were added to separate bound immunogenic proteins, and the bound proteins were separated by acetone precipitation [1:5 (v/v)]. precipitated proteins were solubilized in 20 mm tris-hcl with 0.5% sds, and a lowry assay was used to measure the protein concentration. the protein profile was assessed by fractionating 25 µg of protein on a nupage 4-12% bis-tris protein gel (thermofisher, usa). 3 µg of immunogenic protein was mixed with a lysis buffer (0.1% rapidgest sf in 20 mm ammonium bicarbonate) and 5 mm dtt in 10 mm ammonium bicarbonate at 60 °c for 3 h. this step was followed by incubation with 15 mm iodoacetamide (iaa) in 10 mm ammonium bicarbonate at room temperature for 45 min in the dark. the protein solution was cleaned up by a zeba spin desalting column before digestion with 50 ng of sequencing-grade trypsin (promega, germany) at 37 °c for 6 h. tryptic peptides were dried at 44 °c under a vacuum and then protonated with 0.1% formic acid in lc water before injection into an lc-ms/ms. the tryptic peptides' immunoproteomics profiles were analyzed using an ultimate ™ 3000 nano/capillary lc system (dionex, uk) and hybrid quadrupole q-tof impact ii ™ (bruker daltonics gmbh, germany) equipped with a nano-captivespray ion source. first, 500 nl of extracted peptide was subjected to a trapping column (thermo scientific, pepmap100, c18, 300 μm i.d. × 5 mm) through a full loop injection before being resolved in an analytical column (pepswift c18 nano column, 100 μm × 15 cm, i.d.) at 60 °c. the linear gradient method was used to elute peptides with mobile phase a (0.1% formic acid in water) and mobile phase b (0.1% formic acid in 80% acetonitrile) at a 0.35 µl/min constant flow rate into the mass spectrometer. electrospray ionization was conducted at 1.6 kv using captivespray. mass spectra (ms) and ms/ms spectra were fully acquired in positive ion mode (compass 1.9 for otofseries software, bruker daltonics). mass accuracy was assessed using positive detection mode after internal calibration with sodium trifluoroacetate (na-tfa) within 1.6 ppm. raw lc-ms/ ms spectra were collected using compassxport version 3.0.9.2 (bruker daltonics gmbh, germany) to convert all spectra into the mzxml data format. the mzxml files of the lc-ms/ms datasets for label-free quantification of peptides were evaluated based on the ms profile by maxquant software. chimeric multiepitope vaccine design. the linear b cell epitope was predicted by bcpred 48 . the scop and cath databases were used to design an appropriate chimeric multiepitope vaccine structure 49 (an: wp_000913277). a 3d structure was rendered by i-tasser (iterative threading assembly refinement) using the qualifying c-score value as a confidence score 32 . to refine the tertiary structure, the derived i-tasser results in the pdb files were prepared using the galaxyrefine server, which performed a repeated structure perturbation, and the best structural relaxation candidates were chosen 19 . moreover, to obtain the best chimeric multiepitope vaccine candidates, the residues were determined according to residue stereochemical quality for all the refined chimeric multiepitope models and validated by the procheck program v.3.5.4 to generate ramachandran plots 50 . codon optimization. amino acid sequences were reverse-translated to nucleotide sequences using nile tilapia codon usage (oreochromis niloticus [gbvrt]: 113). the codon adaptation index (cai) of the designed vaccine candidates' nucleotides was analyzed by an optimizer program (http://genomes.urv.es/optimizer/) and combined with geneart tm 's gene optimization process (thermo fisher scientific, usa). the secondary structure of the single-stranded rna folding and free energy of the thermodynamic ensemble were calculated by the rnafold web server 51 . the optimized dna sequence was synthesized by geneart ® gene synthesis (thermo www.nature.com/scientificreports www.nature.com/scientificreports/ to verify the ectopic expression of the chimeric multiepitope dna vaccine, pcdna3.1(+)_42e2 or _45f2 was transfected into tk1 (tilapia kidney 1) tilapia cells using effectene transfection reagent (qiagen, germany). the transfected fish cell cultures were maintained with leibovitz's l-15 media containing 10% fbs and penicillin-streptomycin at 25 °c, and dna vaccine expression was determined after 1 week. recombinant chimeric multiepitope protein was purified by ni-nta agarose beads (qiagen) with a gradient concentration buffer of imidazole ranging from 5 mm to 500 mm. subsequently, the gel filtration chromatography method was performed by fast protein liquid chromatography (fplc) incorporated with a hiprep 16/60&26/60 sephacryl s-300 high-resolution column (ge healthcare, usa) using a 1 × pbs buffer with a 1 ml/min flow rate. recombinant protein detection was confirmed by sds-page analysis and western blot analysis using an anti-his tag antibody (recombinant protein vaccine) or an anti-flag (rabbit igg) (dna vaccine) and anti-rabbit antibody conjugated to ap (alkaline phosphatase). vaccine efficacy analysis. to evaluate vaccine performance, nile tilapia (o. niloticus) were immunized with chimeric multiepitope vaccines (recombinant protein and dna vaccines), followed by bacterial challenge. a total of 6 experimental groups, namely, 1) the 45f2 recombinant protein vaccine, 2) 42e2 recombinant protein vaccine, 3) 45f2 dna vaccine, 4) 42e2 dna vaccine, 5) formalin-killed (fkc) s. agalactiae vaccine 58 , and 6) pcdna3.1(+) [empty vector], were conducted in triplicate. before vaccination, 25 streptococcosis-free nile tilapia (60 ± 5 g) were transferred into 18 glass aquarium tanks containing 30 l of water for one week. after a week of acclimatization, fish were vaccinated according to above mentioned groups. all fish were maintained under running and aerated water at 30 ± 3 °c and fed with commercial pellet feed twice a day. for the chimeric multiepitope protein vaccination, purified 45f2 and 42e2 proteins were mixed with montanide isa 763 (seppic, france) in a 7:3 ratio prior to intraperitoneal injection with 200 µg of protein per fish. for the chimeric multiepitope dna vaccine, plasmid dna of 45f2 and 42e2 were purified by ultracentrifugation using a cscl gradient 59 and dissolved in te buffer (ph 8.0) to obtain a concentration of 0.1 µg/µl. the dna vaccine was applied to the fish with 10 µg of dna through intramuscular injection. fkc and pcdna3.1(+) were used as positive and negative controls, respectively. the schedule of vaccine efficacy analysis and immune response analysis was demonstrated in supplementary fig. 9 . for the immune response analysis, blood was drawn from the caudal vein to separate serum for the immunoblotting assay, and those fish were transferred to another separate tank. the analysis was performed every week, using 3 fish in each treatment from the 1 st week to the 4 th week. after one month of vaccination, 10 vaccinated fish in each treatment group were taken from among the remaining fish for serum collection and anesthetized with eugenol before challenge with s. agalactiae (serotype iii) at 1 × 10 7 cfu/ml through ip administration. mortality and clinical signs of infected tilapia were recorded daily for 3 weeks. the brain, head kidney, and liver were collected from moribund fish for bacterial isolation and identification 7 . cumulative mortality and relative percentage survival (rps) were calculated 60 . a one way analysis of variance (anova) was used for statistical analysis and p < 0.05 was considered significant. to detect the antibody response after immunization, antibody production was evaluated through dot blot analysis using the minifold ® i dot blot system (ge healthcare, germany). briefly, 20 µl of purified 42e2, 45f2 proteins, or a whole-cell lysate of s. agalactiae (10 µg/ml) were spotted on a nitrocellulose membrane and blocked with blocking solution (0.1% bsa in tbst) before adding 10 µl of serum of the different treatment groups as above mentioned. then, the membrane was probed with a primary antibody (anti-igm at 1:5,000) for 1.5 h, followed by washing 3 times with tbst buffer and 45 min of incubation with an anti-mouse igg hrp-linked ab (1:10,000). subsequently, the signal was detected with a chemidoc ™ imaging system (bio-rad) after adding a substrate reagent (perkinelmer, usa). the integrated density of the dot blot was analyzed by imagej (version 1.x) 61 . five different piscidins from nile tilapia, oreochromis niloticus: analysis of their expression and biological functions prevention and control of viral disease in aquaculture development of a quantitative pcr assay for monitoring streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia parasites and diseases streptococcus agalactiae serotype distribution and antimicrobial susceptibility in pregnant women in gabon microevolution of streptococcus agalactiae st-261 from australia indicates dissemination via imported tilapia and ongoing adaption to marine hosts or environment molecular 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subunit vaccines high-throughput prediction of protein antigenicity using protein microarray data first report of streptococcus agalactiae isolated from oreochromis niloticus in piura, peru: molecular identification and histopathological lesions dna extraction from 0.22 µm sterivex filters and cesium chloride density gradient centrifugation potency and efficacy test of a vaccine in addition with adjuvant against koi herpesvirus in koi (cypronus carpio) nih image to imagej: 25 years of image analysis the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/10.1038/s41598-019-57283-0.correspondence and requests for materials should be addressed to s.u. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-000182-ni6iyzdn authors: he, zhisong; zhang, jian; shi, xiao-he; hu, le-le; kong, xiangyin; cai, yu-dong; chou, kuo-chen title: predicting drug-target interaction networks based on functional groups and biological features date: 2010-03-11 journal: plos one doi: 10.1371/journal.pone.0009603 sha: doc_id: 182 cord_uid: ni6iyzdn background: study of drug-target interaction networks is an important topic for drug development. it is both time-consuming and costly to determine compound-protein interactions or potential drug-target interactions by experiments alone. as a complement, the in silico prediction methods can provide us with very useful information in a timely manner. methods/principal findings: to realize this, drug compounds are encoded with functional groups and proteins encoded by biological features including biochemical and physicochemical properties. the optimal feature selection procedures are adopted by means of the mrmr (maximum relevance minimum redundancy) method. instead of classifying the proteins as a whole family, target proteins are divided into four groups: enzymes, ion channels, g-proteincoupled receptors and nuclear receptors. thus, four independent predictors are established using the nearest neighbor algorithm as their operation engine, with each to predict the interactions between drugs and one of the four protein groups. as a result, the overall success rates by the jackknife cross-validation tests achieved with the four predictors are 85.48%, 80.78%, 78.49%, and 85.66%, respectively. conclusion/significance: our results indicate that the network prediction system thus established is quite promising and encouraging. identification of drug-target interaction networks is an essential step in the drug discovery pipeline [1] . the emergence of molecular medicine and the completion of the human genome project provide more opportunity to discover unknown target proteins of drugs. many efforts have been made to discover new drugs in the past few years. however, the number of new drug approvals remains quite low (around only 30 per year). this is partially because many compounds or drug candidates have to be withdrawn owing to unacceptable toxicity. such failures have wasted a lot of money. it would be beneficial to develop computational methods for predicting the sensitivity and toxicity before a drug candidate was synthesized [2, 3, 4] . however, a number of problems need to be overcome in order to find out the exact effects of a drug. firstly, drugs could have numerous effects including positive and negative effects, and it is hard to find out and elucidate the possible effects; secondly, different people would have completely different responses to a drug even though the same gene products are only slightly different [5, 6, 7, 8] ; thirdly, it is very hard to trace the drug effects since the biological interaction pathways are extremely complicated in human beings. therefore, it would be very helpful for drug development if the interactions between drugs and target proteins could be predicted more accurately and the underlying mechanisms could be better understood. several computational approaches have been developed for analyzing and predicting drug-protein interactions. the most commonly used are docking simulations [9, 10, 11, 12] , literature text mining [13] , and combining chemical structure, genomic sequence, and 3d structure information [14] , among others (see, e.g., [15, 16, 17] ). machine learning and data mining methods have been widely used in the computational biology and bioinformatics area. many researchers have made lots of efforts to develop useful algorithms and softwares to investigate various drug-related biological problems, such as hiv protease cleavage site prediction [18, 19] , identification of gpcr (g protein-coupled receptors) type [20, 21] , protein signal peptide prediction [22] , protein subcellular location prediction [23, 24, 25] , analysis of specificity of galnac-transferase protein [26] , identification of protease type [27, 28] , membrane protein type prediction [29, 30, 31, 32] , and a series of relevant webserver predictors as summarized in a recent review [33] . here we propose a predictor for drug-target interactions based on the nearest neighbor algorithm [34] . since biochemical and physicochemical features [35] are important for characterizing proteins, in this study they are used to represent proteins as done by many previous investigators (see, e.g., [36, 37, 38] . to improve the predictor's performance, minimum redundancy maximum relevance (mrmr) algorithm [39] is used to rank the features. meanwhile, the incremental feature selection and forward feature selection are applied for feature selection. the protein targets for drugs are divided into enzymes, ion channels [40, 41, 42, 43] , gpcrs [44, 45] , and nuclear receptors [14] in this study. finally, four predictors for predicting the interactions of drugs with each of the four protein families are developed in hopes that they can help provide useful information for drug design. in addition to the dataset used by yamanishi et al. [14] , information about drug compounds and genes can be obtained from kegg [46, 47] by the ftp operations: ftp://ftp.genome.jp/ pub/kegg/ligand/drug/drug for the drugs, and ftp://ftp.genome. jp/pub/kegg/genes/fasta/gene.pep for the genes. after excluding the drug-target pairs that lack experimental information, we finally obtained a total of 4,797 drug-target pairs, of which 2,719 for enzymes, 1,372 for ion channels, 630 for gpcrs, and 82 for nuclear receptors. all these datasets were used as the positive datasets in the current study. the corresponding negative datasets were derived from the above positive datasets via the following steps: (1) separate the pairs in the above positive dataset into single drugs and proteins; (2) re-couple these singles into pairs in a way that none of them occurs in the corresponding positive dataset; (3) randomly picked the negative pairs thus formed until they reached the number two times as many as the positive pairs. the drug-target benchmark datasets thus obtained for enzymes, ion-channels, gpcrs, and nuclear receptors are given in online supporting information s1, s2, s3, and s4, respectively. representing drugs with chemical functional groups composition. the number of drugs is extremely large. however, most of them are small organic molecules and are composed of some fixed small structures, called functional groups. since functional groups usually represent the characteristics of a compound as well as its reaction mechanism with other molecules, features derived from its functional groups could be very effective in characterizing a drug. moreover, the number of common functional groups is quite small, and hence it is possible to use the functional group composition to uniquely represent a drug [48] . a number of functional groups are available in nature, and we selected the following 28 common groups for the current study: (1) alcohol, (2) aldehyde, (3) amide, (4) amine, (5) hydroxamic acid, (6) phosphorus, (7) carboxylate, (8) methyl, (9) ester, (10) ether, (11) imine, (12) ketone, (13) nitro, (14) halogen, (15) thiol, (16) sulfonic acid, (17) sulfone, (18) sulfonamide, (19) sulfoxide, (20) sulfide, (21) a_5c_ring, (22) ar_6c_ring, (23) non_ar_5c_ring, (24) non_ar_6c_ring, (25) hetero ar_6_ring, (26) hetero non_ar_5_ring, (27) hetero non_ar_6_ring, and (28) hetero ar_5_ring. thus, following the same treatment as in [23] , a drug compound can now be formulated as a 28-d (dimensional) vector given below: where g i (i~1, 2, á á á , 28) is the occurrence frequency of the i-th functional group in the drug d, and t the matrix transpose operator. representing target proteins with pseudo amino acid composition by incorporating biochemical and physicochemical features. now the problem is how to effectively represent a target protein. two kinds of representations are generally used in this regard: the sequential representation and the non-sequential representation. the most typical sequential representation for a protein sample is its entire amino acid sequence, which can contain the most complete information of a protein. to deal with this model, the sequence-similarity-searchbased tools, such as blast [49] , are usually used to find the desired results. unfortunately, this kind of approach failed to work when the query protein did not have significant homology to the proteins in the training dataset. thus, various non-sequential representations or discrete models were proposed. the simplest discrete model was based on the amino acid composition (aac) (see, e.g., [50] ). however, if using the aac model to represent a protein, all its sequence-order information will be lost. to avoid completely losing the sequence-order information, the pseudo amino acid composition (pse-aac) was proposed [36] to represent the sample of a protein. the pseaac can be used to represent a protein sequence with a discrete model yet without completely losing its sequence-order information. for further information about pseaac, see the web-page by clicking the link http://en. wikipedia.org/wiki/pseudo_amino_acid_composition. ever since the concept of pseaac was introduced, it has been widely used to study various problems in proteins and protein-related systems (see, e.g., [37, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66] ). meanwhile, many different forms of discrete models were also proposed (see, e.g., [20, 30, 32, 51, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82] ). however, regardless of how much different these models are, they just belong to different forms of pseaac, as elucidated in a recent comprehensive review [83] . here, we are to propose a different pseaac to represent drug-targeted proteins in terms of their biochemical and physicochemical features [84] . six different types of features were considered: (1) hydrophobicity, (2) polarizability, (3) polarity, (4) secondary structure, (5) normalized van der waals volume, and (6) solvent accessibility. each amino acid residue in a protein sequence can be represented by a set of different states according to its features. for instance, its hydrophobicity feature can be marked by one of the following three states: ''polar'', ''neutral'', or ''hydrophobic'' [85] ; its solvent accessibility feature by one of the two: ''buried'' or ''exposed to solvent'', as predicted by predacc [35] ; its secondary structure feature by one of the three: ''helix'', ''sheet'', or ''coil'', as predicted by the method in [86] ; and so forth. thus, a protein sequence can be translated to a series of codes according to the biochemical and physicochemical properties of its constituent amino acid residues. for example, if using ''p'', ''n'' and ''h'' to represent the three states of hydrophobicity: ''polar'', ''neutral'', and ''hydrophobic'', the protein sequence ''dmaeimsdkp-qagml'' can be translated to ''phnphhnppnpnnhh'' according to the codes of the hydrophobic property feature. the encoded sequences thus obtained would have different length for proteins of different sizes, which will make the prediction engine difficult to handle. to make the feature-encoded sequence to be a vector with a fixed number of dimensions, three properties of a sequence was used: composition (c), transition (t), and distribution (d). c represents the global composition of each letter in the sequence; t, the frequency of a code letter changing from one to another; d, the distribution pattern of the code letters along the sequence, measuring the percentage of the sequence length within which the first, 25%, 50%, 75%, and 100% of the amino acids of each code letter is located. take the above hydrophobic property sequence as an example: its c feature is 5/15 = 33.3% for all of p, h, and n, while the t feature is 2/10 = 20%, 3/10 = 30% and 5/10 = 50% for the changes between h and p, n and h, n and p, respectively. the measurement of feature d is a little more complicated. for the letter h, the first, 25%, 50%, 75% and 100% of hs in the sequence is located at the position of 2, 5, 6, 14, and 15. thus its d feature is ( , with a total of 21 components. likewise, for the sequences encoded by the other four biochemical properties, each is also corresponding to 21 components. but for the sequence encoded by the solvent accessibility with only two states (''buried'' or ''exposed to solvent''), the encoded sequence is corresponding to only 14 components. finally, by adding the 20 components of aac [87] into the vector concerned, the total number of components thus obtained for a given protein is 5|21z20z14~139; i.e., the protein can be formulated as a 139-d vector given by where p i (i~1, 2, á á á , 139) is the i-th component of the protein p. of the 139 components, 119 are derived according to the codes of the above six biochemical and physicochemical features, and 20 are the aac components of p. with all samples represented by a feature vector, now it is possible for us to construct our predictor using the machine learning approach. the nn (nearest neighbor) algorithm is quite popular in pattern recognition community owing to its good performance and simple-to-use feature. according to the nn rule [88] , the query sample should be assigned to the subset represented by its nearest neighbor. in this study, if the drug-target pair with the shortest distance is a positive sample, meaning that they can interact with each other, the sample for test is seen as a positive drug-target pair. otherwise, the test sample is seen as a negative one. there are many different definitions to measure the ''nearness'' for the nn algorithm, such as euclidean distance, hamming distance [89] , and mahalanobis distance [50, 90, 91] . in the current study, the following equation was adopted to measure the nearness between samples v x and v y where v x : v y is the dot product of the two vectors, and v x k k and v y their modulus, respectively. when v x :v y we have d(v x ,v y )~0, indicating the ''distance'' between these two sample vectors is zero and hence they have perfect or 100% similarity. after constructing the drug-target interaction predictor, we have to evaluate its performance. in statistical prediction, the following three cross-validation methods are often used to examine a predictor for its effectiveness in practical application: independent dataset test, subsampling (k-fold cross-validation) test, and jackknife test [92] . however, as elucidated by [24] and demonstrated by eq.50 in [93] , among the three cross-validation methods, the jackknife test is deemed the most objective that can always yield a unique result for a given benchmark dataset, and hence has been increasingly used and widely recognized by investigators to examine the accuracy of various predictors (see, e.g. [51, 53, 54, 55, 56, 57, 59, 62, 63, 64, 94, 95, 96] ).'' accordingly, in this study the jackknife cross-validation was adopted to calculate the success prediction rates as well. although we've constructed the drug-target predictor based on the original feature set described above, it is possible to improve its performance with a better feature set. apparently, not every feature in the feature set is equally relevant to the drug-target interaction. what's more, features may not be independent with each other. the ''bad'' will have negative impact on the accuracy and efficiency of the predictor, so it is possible to do the feature selection process to construct a more compact and effective feature set. the first step is using maximum relevance minimum redundancy (mrmr) [36] to do feature evaluation. maximum relevance minimum redundancy (mrmr) [39] was firstly developed for analysis of microarray data. it ranks each feature according to its relevance to the target and redundancy to other features. the better a feature is deemed to be, the higher the rank it will be assigned to. mutual information (mi), denoted by i to indicate the dependence of two features used to quantify the relevance and redundancy. mi is defined as following: based on mi, we can quantify relevance (d) and redundancy (r) as: where f candidate is the feature to be calculated, and c is the target variable. by combining the above two equations to maximize relevance and minimize redundancy, the following mrmr function is constructed: where v s and v t are the already-selected feature set and to-beselected feature set, respectively, and m and n are the sizes of these two feature sets, respectively. the earlier a feature is selected, the better it would be though of. finally, we can get an ordered feature list with a rank for every feature to indicate its importance in the feature set. in our study, the mrmr program is obtained from: http://research. janelia.org/peng/proj/mrmr/index.htm. to calculate mi, the joint probabilistic density and the marginal probabilistic densities of the two vectors were used. a parameter t is introduced here to deal with these variables. suppose mean to be the average value of one feature in all samples, and std to be the standard deviation, the feature of each sample would be classified into one of the three groups according to the boundaries: mean+(t : std). in our study, t was assigned to be 1. as mentioned above, the importance of each feature is rated according to its rank in the mrmr analysis. the next step is to determine which features should be selected as the optimal feature set for our drug-target predictor. here the ifs (incremental feature selection) procedure is used to solve the problem. each feature in the mrmr feature list was added one by one, and n different feature sets are obtained if the total feature number is n, while the i-th feature set is: based on each of the n feature sets, an nn algorithm predictor was constructed and tested with the jackknife cross-validation test. with all the n overall accurate rates calculated, we could draw an ifs curve with the index i to be the x-axis and the corresponding overall accurate rate to be the y-axis. thus, s opt~f f 1 , f 2 , :::, f n g is regarded as the optimal feature set if the curve reach its peak where the value of its x-axis is nƒn. because four independent predictors are needed for the four different classes of drug-target pairs, the ifs analysis procedure will be processed four times with each for a specific predictor. to refine feature selection, the ffs (forward feature selection) procedure based on the result of ifs was used. ffs is a feature selection method based on ifs results which tries every feature in the candidate feature set and adds the feature that achieves the highest prediction accuracy into the already-selected feature set in each goes. suppose the ifs curve reaches its peak with apex as its x-axis, the initial ffs-selected feature set was constructed as: more features in ffs-to-be-selected feature set would be added into the ffs-selected feature set one by one. the ffs-to-beselected feature set with m features covers the features with mrmr ranks between k+1 and k+1+m, where m is a user-defined positive integer smaller than n{k with n to be the size of the original feature set. in each round of ffs, each feature in ffs-tobe-selected feature set would be taken out and added to the ffsselected feature set. each predictor based on each new ffsselected feature set would be tested, and the feature set obtained the highest overall accurate rate would be used as the new ffsselected feature set. this process would be run for m times, until the ffs-to-be-selected feature set becomes a null set. an ffs curve similar to the ifs curve could be drawn with x-axis as the index and y-axis as the overall accurate rate. in this study, ffs was run for each of the four benchmark datasets based on the corresponding ifs result. m for all these processes was set to 50, while k for each ffs was set to be the index of the point with the first maximum value (i.e. the maximum point with the smallest index) in the corresponding ifs curve. to improve performance of the predictor of drug-target interaction, feature selection process was carried out. the first step of feature selection is feature evaluation. in this study, mrmr was used to evaluate every feature in original feature set. listed in online supporting information s5 are two kinds of outputs: the first one is the maxrel list which shows ranks of features for their relevance to the target; the second is mrmr list showing the mrmr ranks according to the feature order satisfying eq. 3. in this study, only the mrmr list was used as the results of feature evaluation. since there are four groups of samples, mrmr was run four times with each for one of them. with the four mrmr lists, ifs was processed for each of the four sample groups, generating four ifs curves. based on these results, we set k in ffs to be 16, 15, 14 and 19 for the data of enzymes, ion channels, gpcrs and nuclear receptors, respectively. each of these figures is the index of the point of the first maximum value in the corresponding ifs curve. shown in fig. 1 are the four ifs curves with their corresponding ffs curves. the peaks of the four ffs curves finally reach the overall success rates of 85.48% with 32 features, 80.78% with 37 features, 78.49% with 30 features, and 85.66% with 32 features for enzyme group, ion channel group, gpcr group and nuclear receptor groups, respectively. features selected by mrmr+ffs for the four different groups are quite different from each other, showing the intrinsic differences between them. although there are more features for target than those for drug in the original feature set, more drug features were selected, showing the important role of drugs. many of the selected target features are for protein secondary structure, especially for enzyme group (half of selected target features are for this). all types of features are selected in at least one group, showing that all biochemical and physicochemical features have their irreplaceable positions in drug-target interaction process. for the details of the optimal feature-set outputs by ffs for the four benchmark datasets, see the online supporting information s6. for the specificity and promiscuity, we divided the drug-protein interactions into four groups according to the targets of drugs: enzymes, ion channels, gpcrs, and nuclear receptors. we used all the known drugs and target proteins in the gold standard data as training data to predict the potential interactions between all human proteins annotated as members of the four classes in kegg genes and all compounds in kegg ligands. enzyme recognition is the primary event involved in the interaction of proteins with other proteins and with small molecules such as metabolites and therapeutics. predicting drugenzyme interactions has direct application for completing genome annotations, finding enzymes for synthetic chemistry, and predicting drug specificity, promiscuity and pharmacology. it is suggested that the secondary structure information plays the major role in determining the drug-enzyme interactions activity. for example, cytochrome p450 (cyp) induction-mediated interaction is one of the major concerns in clinical practice and for the pharmaceutical industry [97] . induction of cyp1a enzymes with a specific structure-stable state may activate some xenobiotics to their reactive metabolites, leading to toxicity [98, 99] . amino acid composition and hydrophobicity also contribute considerably to these interactions. an insertion/deletion (i/d) polymorphism of the angiotensin i-converting enzyme (ace) have an influence on the antihypertensive response, particularly when using ace inhibitors (acei) [100] , mirroring that the amino acid composition did contribute to the interactions. hydrophobicity plays a role in determining the coefficients of drug-enzyme interaction energy with the application to drug screening as well as in silico target protein screening [101, 102] . the g-protein coupled receptor (gpcr) superfamily, which is comprised of estimated 600-1,000 members, is another largest known class of molecular targets with varieties of physiological activities and proven therapeutic value [103] . they are integral membrane proteins sharing a common global topology that consists of seven transmembrane alpha helices, intracellular cterminal, an extracellular n-terminal, three intracellular loops and three extracellular loops [33, 44] . it is suggested that secondary structure and polarity would play a major role in determining the drug-gpcrs interactions activity. small secondary structures such as helices and loops are identified as entities potentially involved in stabilizing interactions with ligands [33] . these motifs were situated mainly in the apical region of transmembrane segments and included a few extracellular residues [104] . crystal structures of engineered human beta 2-adrenergic receptors (ars) in complex with an inverse agonist ligand, carazolol, provide threedimensional snapshots of an important g protein-coupled receptor (gpcr) with a beta-sheet structure and forms part of the chromophore-binding site [105] . glida provides interaction data between gpcrs and their ligands, along with chemical information on the ligands, as well as biological information regarding gpcrs [106] . some of the features reflect physical interactions that are responsible for the structural stability of the transmembrane, the formation of extensive networks of interhelical h-bonds and sulfur-aromatic clusters that are spatially organized as ''polarity'', the close packing of side-chains throughout the transmembrane domain. when more experimental 3d structures become available for gpcrs in the future, this will help building reliable models for a wider range of gpcrs that would be suitable for docking studies. joint use of ligand-based chemogenomic and docking would certainly improve the prediction. ion channels are a large superfamily of membrane proteins that pass ions across membranes and are critical to diverse physiological functions in both excitable and nonexcitable cells and underlie many diseases. as a result, they are an important target class which is proven to be highly ''druggable''. according to our analysis, secondary structure and polarity play the major role in determining the drug-ion channels interactions activity. secondary structure controls the membrane potential and interrogates ion channels in different conformational states. the drug-ion channels interaction needs gated state where they can switch conformation between a closed and an open state [42, 43] . simulations on model nanopores reveal that a narrow hydrophobic region can form a functionally closed gate in the channel and can be opened by either a small increase in pore radius or an increase in polarity [107, 108] . nowadays, intense research is being conducted to develop new drugs acting selectively on ion channel subtypes and aimed at the understanding of the intimate drug-channel interaction [109] . nuclear receptors (nr) are ligand-activated transcription factors that regulate the activation of a variety of important target genes, which are the most important drug targets in terms of potential therapeutic application. according to our results, secondary structure and polarizability play the major role in determining the drug-nrs interactions. the conservative motif of the nr is typically described as three stacked alpha-helical sheets. the helices that make up the ''front'' and ''back'' sheets are aligned parallel to one another. the helices in the middle sheet run across the two outer sheets and only occupy the space in the upper portion of the domain. the space in the lower part of the domain is relatively void of protein, and for most nrs, this creates an internal cavity for small-molecule ligands [110] . hydrogen bonds with polarizability activity play a crucial role in protein-drug interactions (see, e.g., [11] ). our approaches and the results thus obtained could be used to demonstrate how nuclear hormone receptors form a network of direct interactions. and this network increases in complexity to describe the interactions with target genes as well as small molecules known to bind a receptor, enzyme, or transporter. a comprehensive drug-target interaction network system has been established that contains four classifiers for predicting the drugable interaction of compounds with enzymes, ion-channels, gpcrs, and nuclear receptors, respectively. it is anticipated that the network predictor system may become a very useful tool for drug development. particularly it may help us find new or potential drug-target interactions. online supporting information s1 the benchmark dataset for the drug-target enzyme interaction system. it contains 8,157 genedrug pair samples, of which 2,719 are positive and 5,438 negative. the 1st column of the table indicates the nature of samples with 1 for positive and 2 for negative; the 2nd column shows the code of target gene; and the 3rd column shows the code of drug. all the detailed information for the genes and drugs listed here can be found in a guide to drug discovery: target selection in drug discovery predicting human safety: screening and computational approaches assessment of chemical libraries for their druggability review: progress in computational approach to drug development against sars 3d structure modeling of cytochrome p450 2c19 and its implication for personalized drug design molecular modeling of two cyp2c19 snps and its implications for personalized drug design review: pharmacogenomics and personalized use of drugs review: structure of cytochrome p450s and personalized drug structure-based maximal affinity model predicts small-molecule druggability a fast flexible docking method using an incremental construction algorithm review: structural bioinformatics and its impact to biomedical science binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against sars a probabilistic model for mining implicit 'chemical compound-gene' relations from literature prediction of drug-target interaction networks from the integration of chemical and genomic spaces statistical prediction of protein chemical interactions based on chemical structure and mass spectrometry data integrating statistical predictions and experimental verifications for enhancing protein-chemical interaction predictions in virtual screening alignment-free prediction of a drug-target complex network based on parameters of drug connectivity and protein sequence of receptors a vectorized sequence-coupling model for predicting hiv protease cleavage sites in proteins review: prediction of hiv protease cleavage sites in proteins gpcr-ca: a cellular automaton image approach for predicting g-protein-coupled receptor functional classes gpcr-gia: a web-server for identifying gprotein coupled receptors and their families with grey incidence analysis signal-cf: a subsite-coupled and window-fusing approach for predicting signal peptides using functional domain composition and support vector machines for prediction of protein subcellular location cell-ploc: a package of web-servers for predicting subcellular localization of proteins in various organisms euk-mploc: a fusion classifier for large-scale eukaryotic protein subcellular location prediction by incorporating multiple sites a sequence-coupled vector-projection model for predicting the specificity of galnac-transferase protident: a web server for identifying proteases and their types by fusing functional domain and sequential evolution information prediction of protease types in a hybridization space prediction of membrane protein types and subcellular locations low-frequency fourier spectrum for predicting membrane protein types memtype-2l: a web server for predicting membrane proteins and their types 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genomics drug discovery-database and tools update investigation into adamantane-based m2 inhibitors with fb-qsar an in-depth analysis of the biological functional studies based on the nmr m2 channel structure of influenza a virus ion channel pharmacology the nuclear receptor superfamily and drug discovery key: cord-002739-7t1o19kn authors: yu, xiaobo; song, lusheng; petritis, brianne; bian, xiaofang; wang, haoyu; viloria, jennifer; park, jin; bui, hoang; li, han; wang, jie; liu, lei; yang, liuhui; duan, hu; mcmurray, david n.; achkar, jacqueline m.; magee, mitch; qiu, ji; labaer, joshua title: multiplexed nucleic acid programmable protein arrays date: 2017-09-20 journal: theranostics doi: 10.7150/thno.20151 sha: doc_id: 2739 cord_uid: 7t1o19kn rationale: cell-free protein microarrays display naturally-folded proteins based on just-in-time in situ synthesis, and have made important contributions to basic and translational research. however, the risk of spot-to-spot cross-talk from protein diffusion during expression has limited the feature density of these arrays. methods: in this work, we developed the multiplexed nucleic acid programmable protein array (m-nappa), which significantly increases the number of displayed proteins by multiplexing as many as five different gene plasmids within a printed spot. results: even when proteins of different sizes were displayed within the same feature, they were readily detected using protein-specific antibodies. protein-protein interactions and serological antibody assays using human viral proteome microarrays demonstrated that comparable hits were detected by m-nappa and non-multiplexed nappa arrays. an ultra-high density proteome microarray displaying > 16k proteins on a single microscope slide was produced by combining m-nappa with a photolithography-based silicon nano-well platform. finally, four new tuberculosis-related antigens in guinea pigs vaccinated with bacillus calmette-guerin (bcg) were identified with m-nappa and validated with elisa. conclusion: all data demonstrate that multiplexing features on a protein microarray offer a cost-effective fabrication approach and have the potential to facilitate high throughput translational research. protein microarrays display individual proteins at high density on a chemically-modified slide that can be tested simultaneously with high sensitivity, high specificity, and low reagent consumption. they have been widely applied in basic and translational research, such as protein interaction studies, immune profiling, vaccine development, biomarker discovery and clinical diagnostics, etc. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . for example, zhang et al. used a human protein microarray to better understand how arsenic, which is used in chemotherapy, disrupts cancer signaling pathways and, further, to identify potential targets of novel therapeutic treatments. of the 16,368 proteins that were screened, 360 arsenic binding proteins were identified, which may be novel targets for cancer treatment [7] . anderson et al. used protein ivyspring international publisher microarrays to discover a 28-autoantibody biomarker signature of early stage breast cancer with a sensitivity and specificity of 80.8% and 61.6%, respectively [13] . by combining those autoantibodies with several protein biomarkers, provista diagnostics developed the first protein-based blood test for early breast cancer detection called videssa® breast [14] . ayoglu et al. screened sera from multiple sclerosis (ms) patients using protein microarrays containing 11,520 purified protein fragments and then validated those results using bead-based arrays [15] . the arrays indicated that anoctamin 2 autoantibodies and the ms-associated hla complex drb1*15 allele were strongly associated. additional experiments showed that anoctamin 2 aggregates near and inside lesions within human ms brain tissue [15] . protein microarrays can be classified into two different types, purified or cell-free, based on whether the proteins are produced in vivo or in vitro, respectively [16] . purifying proteins is labor-intensive, requires method optimization and multiple manipulations, exhibits highly variable yields of different proteins, and may not result in naturally-folded or functional mammalian products due to expression in non-mammalian systems (e.g., e. coli, yeast). cell-free protein microarrays overcome these challenges by depositing rna or dna on the slide surface and rapidly expressing them just before an experiment (~2 h) through the use of various cell-free expression systems (e.g., lysate from wheat germ, insect cells, rabbit reticulocyte and human cells). compared to purified protein microarrays, cell-free protein microarrays are more likely to produce naturally-folded mammalian proteins due to the decreased sample manipulation and use of enhanced cell extracts with native chaperone proteins. moreover, the use of nucleic acids vastly simplifies the production of custom arrays since any protein can be produced as long as the gene-of-interest is synthesized; for example, arrays can be produced that represent a specific proteome or signaling pathway [17] [18] [19] [20] . a primary disadvantage of planar-based cell-free protein microarrays is the diffusion of mrna or expressed proteins during in vitro transcription and translation (ivtt), which can then be captured by neighboring features (i.e., cross-talk). thus, the closer the features are to each other, the higher the cross-talk [21] [22] [23] [24] . planar-based cell-free protein microarrays include the protein in situ array (pisa) [22] , dna array to protein array (dapa) [25] , nucleic acid programmable protein array (nappa) [18, [26] [27] [28] , and in situ puromycin-capture array [29] . dapa, nappa and puromycin-capture arrays employ a probe (e.g., ni-nta or anti-tag antibody) on a microarray surface that captures the expressed recombinant proteins in situ during ivtt. of the cell-free approaches, nappa has achieved the highest densities with ~ 2,300 plasmids per slide where the distance between neighboring spots is 625 µm) and the cross-talk is less than 2%. however, cross-talk is increased when the feature spacing is reduced to 375 µm [21] . with ~ 2,300 plasmids per slide, five nappa slides are needed to screen a proteome-scale array with over 10,000 genes [18, 30] . therefore, an increase in spot density would reduce the amount of labor, time, reagents, and cost needed for large-scale proteome analyses like target discovery and validation experiments. to address this issue, angenendt et al. printed cdna and expressed the proteins in nanowells using piezoelectric dispensers [31] . takulapalli et al. demonstrated the fabrication of high-density cell-free protein arrays by combining photolithographicallyetched silicon nanowells (n=8,000/slide), nappa, and a piezo-inkjet printer [21] . here we utilized a different strategy to produce high density arrays that does not require any specialized equipment or substrates. we developed the multiplexed nucleic acid programmable protein array (m-nappa) method by combining as many as five different dna plasmids within one spot, which increases the number of displayed proteins per microarray by five-fold. we first demonstrate that multiplexed proteins are displayed on m-nappa using protein-specific antibodies. second, we compare the ability of m-nappa with non-multiplexed nappa to detect different protein-protein interactions and the serological antibody reactivity against 646 viral proteins. next, we show the feasibility of m-nappa in performing high throughput screening for immune-dominant tuberculosis (tb) antigens through the use of an ultra-high density m-nappa tb proteome array containing four subarrays with 4,045 tb open reading frames (orfs) on one slide. using m-nappa tb protein microarrays, four new immune-dominant antigens in the sera of bcg-vaccinated guinea pigs were identified, which were then validated using elisa. finally, we propose a high throughput target discovery and verification pipeline based on the m-nappa approach. all sera samples were collected with written informed consent with the approval of institutional review boards (irb) at university of florida (gainsville, fl), arizona state university (tempe, az) and albert einstein college of medicine (bronx, ny). detailed sample information was provided in our previous work [17, 20] . the sera from guinea pig tb models vaccinated with bcg were kindly provided by dr. david n. mcmurray from texas a&m college of medicine [32] . all experiments using clinical sera samples were executed according to the declaration of helsinki. a mathematical model was built based on a two-step analysis process. the first round of screening would use multiplexed plasmids with the primary objective of identifying potential protein "hits." the second round would be non-multiplexed, in which each multiplexed "hit" from the first round would be printed separately, with the primary goals of validating and identifying specific individual hits. the total number of printed spots (n) needed for the combined two-round screening of 10k proteins was determined by the number of plasmids printed per spot and the anticipated hit rates (i.e., percentage of displayed proteins that will be identified as significant in the study). the probability p of an individual protein being a true hit can be estimated from previous studies of a similar nature (e.g., antibody biomarkers). the following equation assumes that p follows a bernoulli distribution and that its corresponding plasmid is randomly multiplexed where the number of different plasmids per spot is k: the number of spots needed in the first round is , the probability that a multiplexed spot would be a hit (i.e., containing at least one immune-dominant the optimal level of multiplexing of k different plasmids per spot results in the smallest n. all human and viral orf plasmids were obtained from dnasu (https://dnasu.org/), and transferred into a t7-based mammalian expression vector, pant7-cgst, as previously described [18, 30, 33] . purified dna plasmids were prepared by our automated dna factory robot as previously described [18, 30, 33] , and were normalized to 1,200 ng/μl, such that multiplexed plasmids contributed equally to the final concentration. in other words, a plasmid in a five-multiplexed spot would represent 240 ng/μl. five (5) different plasmids containing a different gene-of-interest were mixed with a master printing mixture containing bsa (sigma), bs 3 cross-linker (thermo fisher scientific, il) and polyclonal α-gst antibody (thermo fisher scientific, il) [26] , and subsequently incubated at 4 o c for 2 h. m-nappa and nappa were printed by the nappa protein array core (http://nappaproteinarray.org/) according to published protocols [18, 30, 33] . the quality of printed plasmid dna on m-nappa and nappa was determined using picogreen dna staining [26] . each m-nappa microarray was blocked with superblock solution (pierce, rockford, il) for 1 h at 23 °c, briefly washed with water, centrifuged at 1000 rpm for 3 min to dry, and covered with a hybridization chamber (grace biolabs, or). the array was then incubated with 160 μl of human in vitro transcription & translation (ivtt) solution containing human hela cell lysate, accessory proteins, reaction mixture, and nuclease-free water (thermo fisher scientific, il) for 1.5 h at 30 °c and 0.5 h at 15 °c to express the gst-tagged proteins-of-interest. the gst-tagged proteins were displayed on the slide surface via the polyclonal α-gst antibody that was included in the printing mixture. then, the resulting protein microarray was incubated with 5% (w/v) milk in 1xpbs with 0.2% (v/v) tween-20 (pbst) for 1 h at 23 °c, followed by three brief washes with pbst. the protein specific antibodies were diluted with 5% milk-pbst at 1:50 or 1:100, respectively, and incubated with the protein microarray for 16 h at 4 °c followed by a 1 h incubation at 23 °c with an alexa fluor 555 labeled secondary antibody (jackson immunoresearch laboratories, pa). after washing three times with pbst, the m-nappa slides were briefly rinsed with water and dried by centrifugation (2,000 rpm, 2 min). the arrays were scanned by a tecan scanner (männedorf, switzerland). after proteins were expressed on m-nappa, the resulting protein arrays were blocked with blocking buffer (1×pbs, 1%tween 20 and 1% bsa, ph 7.4) for 1 h at 4 o c. in parallel, the query proteins (e.g., rb1, jun, fos, lida) fused to a halotag were produced by incubating 90 ng of dna in 180 µl human cell-free expression system (thermo fisher scientific, il) for 2 h at 30 o c. to screen protein-protein interactions, the protein array was incubated with unpurified rb1-halo protein in human hela lysate for 16 h at 4 o c, and then washed with cold washing buffer (pbs, 5 mm mgcl2, 0.5% tween20, 1% bsa and 0.5% dtt, ph 7.4 at 4 o c) three times to remove unbound molecules. the arrays were consecutively incubated with a chicken anti-halo tag antibody (genetel, wi) and alexa fluor 555 goat anti-chicken secondary antibody (jackson immunoresearch laboratories, pa) for 2 h at 4 o c. arrays were washed and dried with brief centrifugation at 1,000 rpm for 1 min, and scanned as described above. the protein binding signal was quantified using array-pro analyzer (media cybernetics) software as previously reported [20, 33] . for a given experiment, a tab-separated file with the interaction information was generated and loaded into the cytoscape software [34] with an attribute file that contained signal intensities of features on m-nappa and nappa. in figures 4 and 5 , proteins within a multiplexed m-nappa feature and its five corresponding non-multiplexed proteins on nappa were displayed as connecting large and small nodes, respectively, with color gradients depicting signal intensities. after proteins were expressed on m-nappa, the arrays were blocked with 5% milk-pbst for 1 h and then incubated with sera at 1:300 dilution in 5% milk-pbst for 16 h at 4 °c. after washing three times with pbst, the resulting arrays were incubated with alex fluor 555 labeled anti-human igg antibody (jackson immunoresearch laboratories, pa) 1 h at 23 °c. the slides were washed with pbst, briefly rinsed with water, and dried by centrifugation (2,000 rpm, 2 min). the fluorescent scanning was performed using a tecan scanner (männedorf, switzerland). the antibody binding event was quantified by fluorescence signal intensity using array-pro analyzer (media cybernetics) software as previously reported [20, 33] . protein microarrays have been used in functional protein and antibody biomarker studies to screen for target(s)-of-interest, which are generally rare in the tested protein population ( figure 1b) . for example, the median hit rates (± standard deviation, sd) of studies employing protein microarrays in the past five years for screening protein function and autoantibody biomarkers (table s1) were 0.49% ± 1.23% and 1.02% ± 4.46%, respectively ( figure 1b) . since false positives are not uncommon during initial screens, all initial candidates require an independent verification step performed using different samples [5, 7, 8, 19, [35] [36] [37] . considering that a two-step approach for target discovery and verification often uses hundreds to thousands of samples, the cost of such studies using full-scale arrays can be inhibitory. to decrease the cost of high throughput screening experiments, we hypothesized that the plasmid cdna encoding for different proteins could be multiplexed (by combining m different plasmids) within each feature to create a high-density array, m-nappa ( figure 1a) . this multiplexed array could be implemented during the initial functional screen, testing entire proteomes (p proteins) using only a fraction of the features (p/m). multiplexed hits identified during the screening step could then be de-convoluted in the subsequent verification step using the standard, non-multiplexed nappa array where each feature displays only one protein (i.e., m=1). the objectives of the second step would be to identify which proteins were responsible for the positive multiplexed signal and to verify whether the hits were real. this approach exploits the high flexibility of cell-free microarrays, in which arrays can be customized by simply re-arraying individual plasmids encoding for the multiplexed features-of-interest. the schematic illustration of how m-nappa arrays are processed is shown in figure 1a . using a standard pin-based arrayer, each spot on m-nappa contains plasmids encoding for different proteins-of-interest with the same fusion tag. the genes are then transcribed and translated into recombinant proteins in two hours using a cell-free expression system, and captured to the slide surface in situ via a fusion tag antibody. the optimal number of proteins to multiplex depends upon several factors, including hit frequency, cost, array space, and number of proteins. as the frequency of hits in the screen increases, more proteins will need to be tested as individual features during the verification step. taken to the extreme, if one protein per multiplexed feature were a hit (hit rate = 1/m), all multiplexed features would require deconvolution, making the multiplexing approach impractical. however, such a high hit rate is not reflected by data collected by numerous studies; for example, the hit rate was < 5% in most of our previous nappa-based screening studies with 10k human genes ( figure 1b) . we generated a mathematical model (materials and methods) to find the optimal m that would take into consideration array space and the cost of screening and verifying hits using our 10k protein human collection at different hit rates. in figure 1c , the x-axis represents the number of genes per spot (m) while the y-axis represents the number of spots or proteins that are needed for the two-step screening process. notably, when the hit rate is < 5% for 10k proteins, a relatively small number of spots would be needed for the entire study (screening + verification) with 5 proteins multiplexed per feature in the initial screen, thus representing a good compromise between the number of initial features screened and the subsequent number of features that would be needed for deconvolution and verification. to assess the difference in transcriptional/translational efficiency as well as display competition between large and small proteins within one feature, we multiplexed proteins of varying molecular weights (mw; 20 -124 kda) covering 80% of the size range in our human protein collection ( figure s2 ) on m-nappa. as indicated in figure 1 , we prepared nappa and m-nappa slides in parallel where nappa had only one plasmid per spot and m-nappa multiplexed five plasmids per spot. after ivtt, the protein arrays were probed with eight antibodies that bound targets ranging in size from 20 to 106 kda (methods and figure s1 ). these antibodies were specific to ia-2 (106 kda), gad2 (65 kda), clusterin (52 kda), p53 (44 kda), fos (40 kda), pp2a (36 kda), sfn (28 kda) and bcl2l2 (20 kda). we compared the protein display between the two array types using groups of five proteins with either similar (figure 2a and figure 2b ) or varied molecular weights (figure 2a and figure 2c) , and then calculated the signal ratio of m-nappa to nappa. in both cases, all of the antibodies readily detected their corresponding antigens. for the spots with similarly-sized proteins (36 kda to 85 kda), the signal ratio of m-nappa to nappa was 0.78±0.44. for the spots containing five proteins covering a wide range of molecular weight, from 29 kda to 106 kda (figure 2c) , the binding signal ratio of m-nappa to nappa was 1.03±0.75 ( figure s3) . thus, multiplexing proteins of similar size did not confer any advantage over random multiplexing. to further demonstrate that there were no biases in the expression of different proteins produced from mixed plasmids, five-plasmid mixtures containing various combinations of seven different genes (abl1, ia-2, gad2, jun, rhou, bcl2l2 and mt3033) were co-expressed in ivtt solution and analyzed via western blot. despite a wide range of protein sizes, all proteins were expressed at similar amounts in their relevant combinations ( figure s4) . these data indicate that, although each plasmid in m-nappa is present at one-fifth the amount present in standard nappa, there was no significant difference of protein display levels between the arrays (p-value = 0.36, paired sample t-test). in addition, background signals that resulted from non-specific antibody binding were comparable between the platforms, demonstrating that multiplexing does not result in an accumulation of background signal that could contribute to the identification of false positives ( figure s5) . therefore, we randomly mixed different gene plasmids in the following m-nappa studies. to demonstrate that the signal intensity for m-nappa was reproducible, we also tested the spot-to-spot, zone-to-zone and slide-to-slide variations by printing 80 gene plasmids on different locations across the m-nappa and nappa slides. protein display was then examined with anti-gst antibody staining (materials and methods). the coefficient of variations (cvs) for spot-to-spot, zone-to-zone and slide-to-slide were 3.64±3.27%, 7.57±3.41% and 7.27±4.00% for m-nappa, respectively, and 7.63±10.58%, 12.13±7.56%% and 13.25±9.42%% for nappa, respectively (table s4) . we purified 646 viral orf plasmids from ~23 viruses, normalized their concentrations to 1,200 ng/µl, and printed viral nappa and m-nappa arrays in duplicate [20] . analyses of the deposited dna and displayed protein levels indicate that most viral dna plasmids were successfully printed, expressed, and captured onto the microarrays in a reproducible manner (figure 3a) . for example, plasmid dna deposition across technical replicates of nappa and m-nappa had correlations (r) of 0.95 and 0.96, respectively. the protein display correlation (r) across technical replicates of nappa and m-nappa were 0.90 and 0.93, respectively ( figure 3b, figure s6 ). "non-spots" containing printing buffer alone without plasmid dna was used as a negative control. 94% and 93% of the spots on nappa and m-nappa viral arrays, respectively, produced signal that was at least two sds above the average signal intensity of these "non-spots" (figure s7 ). together with figure 2 , the results indicate that the majority of viral proteins can be displayed on m-nappa arrays. in addition, we compared the s/b (signal to background) ratios between direct fluorescence and tyramide signal amplification (tsa) using a fluorophore-linked or hrp-conjugated anti-p53 antibody, respectively. using the signal from "non-spots" as background, we found that the s/b ratio of fluorescence detection using an antibody with a directly-conjugated fluorophore, the dylight649 rabbit anti-mouse igg, was higher (s/b ratio = 431±38) than the tsa method with the hrp-labeled goat anti-mouse igg (s/b ratio = 323±18). thus, directly-conjugated fluorescent secondary antibodies were used for the following assays ( figure s8 ). to determine whether nappa and m-nappa detect similar protein-protein interactions, both arrays were programmed to display proteins that are known to interact with the tumor suppressor protein rb1. the arrays were then probed with a rb1 query protein fused to halotag, and interactions were detected using an anti-halotag antibody. the rb1-halotag query protein bound to several targets (red arrow) on nappa and m-nappa arrays; the query also bound to diffused targets outside of each spot, which appear as a "ring" around each feature (figure 4a) . in figure 4b , we used a flower pattern diagram to depict the multiplexed proteins on m-nappa (large central circle) and the deconvoluted individual proteins on nappa (five small connecting circles) (materials and methods). the blue gradient within the spot indicates target binding to the rb1 query protein, whereas reactivity to the "ring" [20, 33] is indicated by a red circle around the spot. using custom defined criteria, where the target-to-"non-spot" signal ratio is > 2 and the ring score is > 3, we found that 5 and 6 hits were identified on nappa and m-nappa, respectively, out of the 30 possible candidate target proteins ( table s2, table s3 ). five of the 6 hits on m-nappa were e1a, hpv11-e7, hpv16-e7, hpv18-e7 and hpv33-e7, which agrees with previous studies [38] [39] [40] . the sixth hit on m-nappa was not detected with nappa, thus suggesting that the hit may be a false positive. to further examine the utility of m-nappa to test protein-protein interactions, additional interactions were analyzed with 35 displayed proteins on nappa and m-nappa using halotagged-jun, -fos, and -lida queries. jun, fos and lida bound to their expected interaction partners (i.e., fos, jun and three rab family proteins, respectively) on both m-nappa and nappa arrays (figure s9) . regarding the protein interactions that were identified, the spot-to-spot and zone-to-to zone cvs were 5.65±2.69% and 5.75±3.86% for nappa, respectively, and 2.55±2.56% and 3.11±3.46% for m-nappa, respectively (table s5) . these results indicate that m-nappa can be used for preliminary high throughput (ht) screening of novel protein-protein interactions. the screen can then be followed by a verification step using deconvoluted spots via nappa to identify the specific proteins that are involved. to test whether m-nappa can be used to detect proteomic serological response, we screened ten serum samples from patients with type 1 diabetes that had been previously characterized using nappa arrays [20] . a dozen hits were observed with m-nappa and nappa (figure 5) . forty-nine of the 53 antigens (92.5%) identified by nappa were also detected by m-nappa. four antigens, however, were detected with only one platform (i.e., two with nappa, two with m-nappa). these uncommon discrepancies may be due to variations in surface chemistry, plasmid concentration, printing or array processing. since the multiplex concept to increase feature density was successful in detecting protein-protein interactions and serological antibody responses on planar microarrays, we wanted to determine whether m-nappa could also be applied to a nano-well microarray platform. we previously increased feature number by printing plasmids into photolithography-etched silicon nano-wells to create a high-density nappa (hd-nappa) platform [41] . hd-nappa can have as many as 10k features per slide, and has successfully detected antiviral antibodies in autoimmune diseases with 761 different proteins displayed on the array in quadruplicate. these tiny wells hold only 1200 pl and use only 0.12 ng of plasmid dna. we applied the multiplex concept to hd-nappa using a mixture of plasmids encoding for ia-2, gad2 and p53 proteins. we then detected their expression and display using specific antibodies; all of these proteins were readily detectable when printed as a three-plexed mixture ( figure s10) . we then multiplexed 4,045 tuberculosis (tb) orfs [32] onto hd-nappa microarrays as four separate subarrays using three gene plasmids per well (m=3), resulting in an m-hd-nappa microarray displaying > 16k proteins on a single slide. this lower multiplicity was based on the mathematical model ( figure 1c ) that took into account that the high number of conserved proteins in endemic, non-pathogenic mycobacterial species results in a higher hit rate (~10% [37] ). over 95% of the spots generated a signal that was at least 10 sds above the background, which indicates that the vast majority of proteins were well-expressed and displayed (figure 6a) , with a correlation of r = 0.90 across technical replicates (figure 6a-c) . antibody reactivity from tb patient sera was observed with m-hd-nappa (figure 6d) . the technical reproducibility of these immune-dominant antigens across different m-nappa arrays using the same sera was very high, with a correlation of r = 0.98 (figure 6d-f) . all immune-dominant antigens identified with m-hd-nappa screening were then deconvoluted in the verification step using single protein nappa ( figure 6g ) and validated with rapid-elisa as previously described [19] (figure 6h ). we screened the sera from guinea pigs immunized with bacillus calmette-guerin (bcg), a tb vaccine, using m-hd-nappa tb proteome microarrays. the aim of this experiment was to identify potential protective antibodies induced with bcg. the representative fluorescence images are shown in figure 7a . compared to the control mock sera pool using pbs buffer (n=5), four features on m-nappa arrays showed increased signals with the bcg samples (n=4) (figure 7b) . to deconvolute and validate those targets, we repeated the serological assay for those candidate proteins, along with two non-responsive control proteins (rv2077a and rv2682c), using rapid-elisa and the individual sera from the guinea pigs. the antibody levels of four antigens (rv3405c, rv1078, rv2853 and rv0928) in bcg-vaccinated guinea pigs were significantly higher than that of the pbs control with a p-value <0.01 ( figure 7c, figure s11 ). according to the tuberculist database (http://tuberculist.epfl.ch/), these proteins are involved in regulation, cell wall and cell processes, and are considered to be in the proline-glutamic acid / proline-protein-glutamic acid (pe/ppe) protein families ( figure 7d ). a primary advantage of cell-free protein microarrays is that the arrays have a long shelf life. we compared the protein expression of m-nappa tb arrays immediately after printing and then again after 6 months of storage at room temperature in a nitrogen atmosphere. a gst-tagged protein, detected with an anti-gst antibody, was considered to be displayed if it had a signal that was two sds above the signal of the "non-spots." over 99% of the proteins were displayed on new m-nappa arrays; this number, as well as the anti-gst signal intensity, did not change even after 6 months of storage ( figure s12 ). nappa has been widely applied in protein-protein interactions, post-translational modifications (ptms), antibody epitope mapping and discovery of (auto) antibody biomarkers for a variety of human diseases, including markers that are currently being used in the clinic for the detection of breast cancer [13, 14, 18, 20, 32, 36, [42] [43] [44] . due to mrna and protein diffusion during ivtt, the number of features per planar microscope slide has been limited to ~2,300 to minimize cross-talk to neighboring spots. the feature density limit has thus required that multiple slides be used to study large proteomes. here, we developed a new strategy, m-nappa, that significantly increases the number proteins that can be tested per slide multiple-fold. by combining five different plasmids within one feature, >10k proteins can be printed on one microscope slide for ht, low cost analyses when compared to studies using one-plasmid-per-feature arrays. the multiplexed hits that are identified with m-nappa can then be deconvoluted during the subsequent verification step (figure 8) . first, we constructed a mathematical model to determine the optimal level of multiplexing, which considers the number of proteins, cost, array size, and hit rate to predict the number of arrays that would be needed for a two-step screening and verification study. a survey of ht unbiased target screening studies that used protein microarrays, both in the literature and our own results using nappa, revealed that hits are rare (typically <5%) (figure 1b) . for 10k proteins and a hit rate of 5%, the mathematical model indicated that multiplexing 5 proteins per spot (figure 1c) would provide a good balance of maximizing the number of features, minimizing the number of arrays, and yielding the minimum overall workload when compared to using non-multiplexed arrays for both the screening and verification steps. second, we demonstrated that there was nothing inherent to printing plasmids as a five-plasmid mixture that prevented their expressed proteins from routine detection regardless of protein size. however, display levels of large proteins (> 65 kd) were decreased by 60±0.1% for gad2 and 63±11% for ia-2 ( figure 2c ). this may be because the plasmids were mixed equally together based on their masses, a requirement imposed by the printing chemistry; this would result in a lower molarity of large plasmids (i.e., large proteins) relative to small plasmids in the printing mixture. another possible reason is that larger proteins are produced more slowly than smaller proteins due to their longer mrna sequences. third, we showed that m-nappa can be used in protein-protein interaction and serological screening studies. the results from m-nappa agreed strongly with those observed with non-multiplexed nappa (figure 3-5, table s2 and s3, figure s9 ). these data indicate that m-nappa presents a labor-and cost-effective strategy to initially screen for hits. fourth, we further increased the feature density by applying this method to our previously-published nano-well platform [21] . with m-hd-nappa, the entire tb proteome containing 4,045 genes was successfully printed on a nano-well array in quadruplicate [20] (figure 6 ). this generates the highest density nano-well protein microarray to date and increases the previously demonstrated content by more than five-fold [20] . our data indicate that the multiplexing strategy has great potential value for use with different microarray platforms (figure 6 and figure 7 ) [45] . finally, we evaluated the reproducibility of m-nappa arrays for protein array preparation and protein-protein interactions. we found m-nappa can be reproducibly fabricated with spot-to-spot, zone-to-zone and slide-to-slide cvs that are similar to those obtained with nappa ( table s4) . the spot-to-spot and zone-to-to zone cvs for protein-protein interactions were also similar between the two array platforms (table s5) . while the correlations within and between different m-nappa slides were good (i.e., r = 0.93 for both) (figure 3 and figure s6 ), with some size adaptation, the reproducibility could eventually be further improved with the use of automation equipment like the hs 4800 pro hybridization station (tecan trading ag; männedorf, switzerland). in some ways, m-nappa resembles "natural protein" microarrays that print unpurified or partially fractionated proteins from lysates of human cells, tissues or body fluids, but in a much more controlled manner. each feature of a natural protein microarray typically represents a mixture of unknown proteins. thus, responsive hits on natural protein arrays require a challenging and time-consuming process to determine the identity of the protein responsible for the response. this may require further purification, identification by mass spectrometry and additional response testing of recombinant proteins [46, 47] . in the case of m-nappa, the identities of the proteins in each mix are known in advance and the plasmids encoding for each protein are available for secondary testing. m-nappa would be useful in unbiased ht screening studies, such as protein-protein interactions, protein-dna interactions, discovery of drug binding target as well as (auto)antibody biomarkers for a variety of human diseases. however, it should be noted that there are situations in which using a non-multiplexed array format would be more appropriate. for example, nappa should be used when investigating protein functions or when the number of proteins to be screened is low. additional attributes of m-nappa should be considered as well. large, multiplexed proteins (> 65 kda) on m-nappa are displayed at a lower level (37 -40%) than their non-multiplexed counterparts ( figure 2c ). this issue could be resolved by increasing plasmid dna concentration before printing or reducing multiplicity per spot. alternatively, since large proteins represent a small fraction of the proteome, a hybrid array containing multiplexed spots with plasmids encoding for proteins with low to moderate mws and non-multiplexed spots for large proteins (> 65 kda) could be employed. in addition, ptms that occur during cell-free protein expression may affect the protein display or activity on m-nappa arrays [42] . we have observed that the human expression system contains the ability to phosphorylate some proteins (data not shown); other types of ptms (e.g., glycosylation, acetylation) by the expression system are not well known or reported. in our studies, ptms did not appear to affect protein expression, protein-protein interactions, or the identification of serological antigens on m-nappa when compared to nappa (figure 2, figures 4-7 and figure s7 ). we developed a method that multiplexes five different proteins within the same feature, called m-nappa, which significantly increases array density while decreasing experimental time and cost. although we used this approach with nappa and hd-nappa, the same concept could be applied toward other microarray technologies or platforms. our results show that m-nappa identified hits in protein interaction and serum screening studies, thus highlighting its potential to be employed in high throughput proteomics studies. supplementary table s1-s5 and supplementary figures s1-s12. http://www.thno.org/v07p4057s1.pdf protein analysis on a proteomic scale protein microarray technology severe acute respiratory syndrome diagnostics using a coronavirus protein microarray a burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens protein acetylation microarray reveals that nua4 controls key metabolic target regulating gluconeogenesis protein microarrays for personalized medicine systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic identification of serum biomarkers for gastric cancer diagnosis using a human proteome microarray aagatlas 1.0: a human autoantigen database advancing translational research with next-generation protein microarrays nappa as a real new method for protein microarray generation emerging protein array technologies for proteomics protein microarray signature of autoantibody biomarkers for the early detection of breast cancer integration of serum protein biomarker and tumor associated autoantibody expression data increases the ability of a blood-based proteomic assay to identify breast cancer anoctamin 2 identified as an autoimmune target in multiple sclerosis advancing translational research with next generation protein microarrays identification of antibody targets for tuberculosis serology using high-density nucleic acid programmable protein arrays high-throughput identification of proteins with ampylation using self-assembled human protein (nappa) microarrays plasma autoantibodies associated with basal-like breast cancers immunoproteomic profiling of anti-viral antibodies in new-onset type 1 diabetes using protein arrays high density diffusion-free nanowell arrays single step generation of protein arrays from dna by cell-free expression and in situ immobilisation (pisa method) optimised 'on demand' protein arraying from dna by cell free expression with the 'dna to protein array' (dapa) technology cell free expression put on the spot: advances in repeatable protein arraying from dna (dapa) printing protein arrays from dna arrays next-generation high-density self-assembling functional protein arrays self-assembling protein microarrays mapping transcription factor interactome networks using halotag protein arrays protein chip fabrication by capture of nascent polypeptides copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based detection of global pathogen-host ampylation on self-assembled human protein microarrays cell-free protein expression and functional assay in nanowell chip format mycobacterial membrane vesicles administered systemically in mice induce a protective immune response to surface compartments of mycobacterium tuberculosis host-pathogen interaction profiling using self-assembling human protein arrays cytoscape: a software environment for integrated models of biomolecular interaction networks mapping transcription factor interactome networks using halotag protein arrays autoantibody signature for the serologic detection of ovarian cancer dynamic antibody responses to the mycobacterium tuberculosis proteome systematic identification of interactions between host cell proteins and e7 oncoproteins from diverse human papillomaviruses functional studies of e7 proteins from different hpv types adenovirus e1a, simian virus 40 tumor antigen, and human papillomavirus e7 protein share the capacity to disrupt the interaction between transcription factor e2f and the retinoblastoma gene product antiviral antibody profiling by high-density protein arrays a contra capture protein array platform for studying post-translationally modified auto-antigenomes exploration of panviral proteome: high-throughput cloning and functional implications in virus-host interactions serological autoantibody profiling of type 1 diabetes by protein arrays mycobacterium tuberculosis proteome microarray for global studies of protein function and immunogenicity the identification of phosphoglycerate kinase-1 and histone h4 autoantibodies in pancreatic cancer patient serum using a natural protein microarray development of natural protein microarrays for diagnosing cancer based on an antibody response to tumor antigens we thank for dr. mark atkinson (university of florida) for providing sera samples. this work was supported by the national natural science the authors have declared that no competing interest exists. key: cord-012682-7goljir4 authors: yuan, meng; song, zi-han; ying, mei-dan; zhu, hong; he, qiao-jun; yang, bo; cao, ji title: n-myristoylation: from cell biology to translational medicine date: 2020-03-18 journal: acta pharmacol sin doi: 10.1038/s41401-020-0388-4 sha: doc_id: 12682 cord_uid: 7goljir4 various lipids and lipid metabolites are bound to and modify the proteins in eukaryotic cells, which are known as ‘protein lipidation’. there are four major types of the protein lipidation, i.e. myristoylation, palmitoylation, prenylation, and glycosylphosphatidylinositol anchor. n-myristoylation refers to the attachment of 14-carbon fatty acid myristates to the n-terminal glycine of proteins by n-myristoyltransferases (nmt) and affects their physiology such as plasma targeting, subcellular tracking and localization, thereby influencing the function of proteins. with more novel pathogenic n-myristoylated proteins are identified, the n-myristoylation will attract great attentions in various human diseases including infectious diseases, parasitic diseases, and cancers. in this review, we summarize the current understanding of n-myristoylation in physiological processes and discuss the hitherto implication of crosstalk between n-myristoylation and other protein modification. furthermore, we mention several well-studied nmt inhibitors mainly in infectious diseases and cancers and generalize the relation of nmt and cancer progression by browsing the clinic database. this review also aims to highlight the further investigation into the dynamic crosstalk of n-myristoylation in physiological processes as well as the potential application of protein n-myristoylation in translational medicine. lipids are one of the principal components that structure the cell membrane and provide the barrier required for cells to survive. in addition, various lipids and lipid metabolites are also generated for modifying proteins in eukaryotic cells in a process known as 'protein lipidation'. generally, there are four major types of protein lipidation: myristoylation, palmitoylation, prenylation, and glycosylphosphatidylinositol (gpi) anchoring. these lipid modifications have been defined by different functional properties that are classified according to the characteristics of lipid attachment, the covalent bond, the specific sequence on the protein and the enzymes involved [1, 2] . these lipidation distinctions impact the charge, hydrophobicity, and other aspects of targeted-protein chemistry, resulting in marked differences in the physiology of the targeted protein, such as its conformation, trafficking, localization, and binding affinity for cofactors. therefore, due to the deregulated lipid metabolism that occurs, protein lipidation may contribute to various diseases. this review primarily summarizes the current knowledge of nmyristoylation with updated study results and discusses the strategy of using n-myristoylation in the treatment of diseases. n-myristoylation consists of the addition of the 14-carbon fatty acid, myristate, to the n-terminal glycine residue of a protein via a covalent amide bond. in rare cases, including those of ras gtpases and tnf [3, 4] , myristic acid is attached to a lysine residue [5] through an amide bound, a process named lysine myristoylation. previously, it was observed that the myristate attaches to the nascent peptide in the first 10 min of translation during protein biosynthesis. therefore, n-myristoylation is considered a cotranslational modification with the most accurate step occurring after the removal of the methionine initiator by methionine aminopeptidase (metap) (fig. 1a, b) . furthermore, it is recognized that nmyristoylation can also occur posttranslationally on an internal glycine exposed by caspase cleavage during apoptosis (fig. 1c) . for many protein signaling systems, the n-myristoyl moiety represents an essential feature and contributes to numerous effects, including changing protein stability, influencing protein-protein interactions, enhancing subcellular targeting to organelles or the plasma membrane, and so on [6] [7] [8] [9] [10] . two nmt isozymes during the multiple enzymatic steps of n-myristoylation, the process of myristate transfer is completed by nmt, which is classified as a member of the gcn5-related n-acetyltransferase (gnat) superfamily. some well-studied structural and biochemical investigations of yeast and human nmts revealed an ordered bi bi kinase mechanism (fig. 1d ) that involves a structural transformation of nmt1 during nmt catalysis. in fact, nmt is ubiquitous in eukaryotes while no protozoans possessed it. typically, lower eukaryotes (e.g., s. cerevisiae and drosophila) have only one isoform of nmt. however, for some mammalian species, including humans and mice, two isozymes have been identified, referred to as nmt1 and nmt2. these isozymes are encoded by distinct genes but share approximately 77% peptide sequence identity with unique substrate specificities in the n-terminus, suggesting a distinct physiologic role of each isozyme in mammals. additionally, each isozyme has a conserved sequence in the catalytic domain of divergent species, implicating an essential role for each gene family throughout evolution. in humans, there are four isoforms of nmt1 and two isoforms of nmt2, which are translated from splice variants of mrna with different reading frames. the main differences among the nmt isoforms are found in the n-termini. although the n-terminal structure of these nmts does not contribute to the construct of the kinase pocket, some investigations have characterized an nterminal truncation that increases kinase activity without affecting enzyme stability [11] . in addition, the isoforms of these nmts may have specific effects on their intracellular localization and substrate selectivity [12, 13] , suggesting that nmts are involved in diverse physiological processes. physiological roles of nmts unequivocally, nmts play essential roles in the survival and cell proliferation of diverse species [14, 15] . some evidence suggests a principal role of nmt1 in the embryonic development of mice. in normal mice, the expression level of nmt1 is similar to that of nmt2 in a wide variety of tissues, but it is higher than the nmt2 levels in embryos. knocking out nmt1 severely impaired the differentiation ability of embryonic stem cells. homozygous nmt1 −/− -knockout mice were not born alive, and induction of nmt2 activity alone was unable to elicit the survival of heterozygous nmt1 +/− mice. in addition, it was reported that the subcellular localization and catalytic activity of both nmt1 and nmt2 were altered during apoptosis. nmt1 was transported to the cytosol from ribosomes and membranes following caspase-8-and caspase-3-mediated nmt1 cleavage, and 40% of the nmt1 activity was eliminated 8 h after the induction of apoptosis. however, the relocalization of the cytosolic fraction to the membrane and reduced activity of nmt2 were also found under the same conditions. in addition, the depletion of nmt2 caused a 2.3-fold increase in the apoptosis rate compared to the apoptosis rate upon depletion of nmt1 [16] . this evidence led to the speculation that nmt1 may be responsible for ribosome-based cotranslational n-myristoylation, while nmt2 may be the major contributor to apoptosis-related posttranslational n-myristoylation. in summary, the widespread and conservative presence of nmt1 in different species emphasizes its importance in basic physiological processes, such as embryonic development, while nmt2 appears only in higher-level organisms, suggesting that it is necessary for sophisticated physiological processes. in the future, in-depth biochemical and pharmacological research is expected to improve the understanding of the unique roles of nmt1 and nmt2 as they apply to the clinic. protein demyristoylation some reports have revealed the demyristoylation ability of some proteins. human sirtuin 2 (sirt2), which is a member of the lysine deacetylase sirtuin protein family, was found to exhibit more efficient demyristoylase activity than deacetylase activity [17] . the crystal structure implied that, in complex with a thiomyristoyl peptide, sirt2 has a dominant hydrophobic pocket that can adopt a myristoyl group. the hydrophobic acyl pocket of sirt2 resembles that of sirt6, which has been previously demonstrated to possess efficient fatty acid deacylase activity [18] . in addition, the two pockets are different in certain aspects, suggesting differential adoption of fatty acid chains. moreover, the other homologs, sirt1 and sirt3, each have a hydrophobic acyl pocket very similar to that of sirt2, hinting at analogous demyristoylation activities among them. the shigella virulence factor ipaj was identified as an irreversible demyristoylase [19] that cleaves the peptide bond between nmyristoylated gly-2 and asn-3 in some n-myristoylated proteins such as human adp-ribosylation factor 1p (arf1) and c-src. this irreversible demyristoylation mechanism provides a new approach to exploring the functional effects of n-myristoylated proteins in human health and diseases. in most cases, n-myristoylation on a protein is irreversible, indicating that the myristoyl motif may orient the protein toward a specific destiny, as if it is pressing a button that will irrevocably affect the dynamics of the protein and its subsequent pathway. therefore, it is reasonable to study the interactions among the factors of n-myristoylation and those of biological signaling pathways to understand the significant role of n-myristoylation. although n-myristoylation is irreversible, it cannot shield the myristoylated protein from cross talk. in contrast, cross talk is regarded as a means of interfering with n-myristoylation functions. it has been proposed that one protein modification might initiate the signaling that leads to the addition or removal of a second protein modification or the binding of another protein, suggesting that cross talk between protein modification components may serve as an important bypass of regulating protein functions. for example, both methylation and phosphorylation are able to trigger acetylation of histones [20] . here, while introducing the physiological functions of nmyristoylation, we also delineate the cross talk of nmyristoylation components with signaling constituents in light of well-established studies to explore the robust role of nmyristoylation in cell biology. dynamic structural changes in membrane anchoring and intracellular trafficking one of the major functions of n-myristoylation is to facilitate protein binding in membranes. in fact, peitzsch and mclaughlin established a tenet stating that the myristoyl motif is insufficient for the stable anchorage of a protein to a lipid bilayer [21] . therefore, a second signal, comprising a group of hydrophobic residues, positively charged amino acids or another lipid moiety, is required for stable membrane attachment. in one scenario called the 'ligand-dependent switch' (fig. 2a) , the conformation of a protein is changed upon ligand binding, exposing the myristoyl motif that attaches to a component in the lipid bilayer. for example, the gtp-myristoyl switch facilitates the membrane interaction of arf [22, 23] . the exposed myristoyl motif and the basic hydrophobic residues in the n-terminus facilitate the interaction of arf1-gtp with the membrane. the second scenario refers to a cluster of positively charged amino acids that are associated with a cofactor, such as calcium (fig. 2b) , or are phosphorylated (fig. 2c) ; the former cluster accumulates a positive charge to strengthen membrane binding, while the latter attenuates the positive charge to weaken membrane binding and cause membrane dissociation. the binding of two calcium ions to ef-hand motifs in the recoverin protein facilitates the exposure of a myristoyl group from a hydrophobic cavity to solvent (fig. 2b ) [24] . another example is the myristoylated alanine-rich c kinase substrate (marcks) protein. the phosphorylation of serine residues contributes to its membrane dissociation, since the phosphate moiety reduces the positive charge ( fig. 2c ) [25] . ece c. gaffarogullari et al. [26] proposed a novel myristoyl/phosphorylation switch in a pka-c model of membrane attachment. pka-c maintains conformational equilibrium between a myr-in state and a myr-out state, which refer to the myristoyl group tucked into the hydrophobic pocket of the pka-c or extruded from the hydrophobic pocket, respectively. in the myr-out sate, the exposed myristoyl group inserts into the lipid bilayer and facilitates pka-c binding to the membrane. therefore, it was proposed that a large population of pka-c maintains the myr-in state distal to the membrane and that pka-c shifts to the myr-out state in the proximity of the plasma membrane or when phosphorylated at ser10 independent of the regulatory subunit. the palmitoyl moiety, which consists of a 16-carbon fatty chain, also induces signal transduction (fig. 2d) . it was observed that h-ras requires both a myristoyl group and palmitoyl group to target the membrane and stimulate kinase activity, whereas only n-myristoylated h-ras acts a substrate for palmitoyltransferases and therefore is palmitoylated [27, 28] . the n-myristoylationnegative mutants of cdpk2 [29] and fibroblast growth factor receptor substrate 2α (frs2α) [30] do not incorporate palmitate and therefore do not attach to the plasma membrane. moreover, proteins with myristoyl moieties show a tendency for inclusion in the membrane fraction. for example, it was reported that both n-myristoylation and palmitoylation appear to have opposing roles and different membrane lipid microdomain preferences for the g protein-membrane interactions i (gαi1) monomer, which are likely due to the conformational differences in the presence of different fatty acids [31] . the gαi1 protein that is n-myristoylated but not palmitoylated preferentially anchors to lamellar-prone membrane domains with a net negative charge. in contrast, the gαi1 protein that is both n-myristoylated and palmitoylated preferentially localizes to raft-like lamellar membranes without negative charges. n-myristoylated akt1 (myrakt1) exhibits a distinct substrate preference that is not exhibited by the cytosolic or the membrane fractions that do not include lipid rafts, indicating that akt1 for which oncogenicity is conferred by nmyristoylation is enriched in lipid rafts [7] . in addition, the evidence that simvastatin or cholesterol depletion preferentially ablates the phosphorylation of myrakt1 in lipid rafts suggests that myrakt1 is modified in a cholesterol-sensitive manner (fig. 2e ). it is well known that the most organelles in cells, such as the endoplasmic reticulum (er), mitochondrion and endosome, are bound to the plasma membrane and that these membrane interactions produce different effects. in addition, some proteins must undergo n-myristoylation for subcellular trafficking and localization. some mitochondria-related proteins, such as tomm40, samm50 [32] and, clpabp [33] , were demonstrated to require the function of myristoylated proteins to bind to the mitochondrial outer membrane. in one mechanism, the hydrophobic myristoyl group motif increases dependence of the protein to reduce cytoplasmic shuttling. for example, in mice, binding domain-containing protein 1 (stbd1) was observed to be a transmembrane resident protein, and nonmyristoylated stbd1 was shuttled with ease between the er and mitochondria [34] . ring finger protein 11 (rnf11) colocalizes with both early and recycling endosomes. while rnf11 requires n-myristoylation and spalmitoylation for membrane binding, n-myristoylation plays a greater role [35] . the removal of myristoylate results in protein diffusion. in another mechanism, the hydrophobicity of the myristoyl moiety increases protein flexibility, facilitating its shuttling through hydrophobic areas. in a yeast model [36] , the proteasome subunit rpt2 is cotranslationally n-myristoylated. loss of rpt2 n-myristoylation causes abnormal dynamic nucleuscytoplasm translocation of proteasomes and the aberrant accumulation of ubiquitinated protein levels (fig. 2f) . requirements for protein assembly and protein-protein interaction many proteins require assembly for maturity and function, and some evidence indicates that n-myristoylation drives the aggregation of proteins in some cases [37] . spassov et al. reported that src dimerization is mediated by the myristoylated n-terminal region of one partner interacting with the hydrophobic kinase domain of its counterpart [38, 39] . both y419 autophosphorylation and dimerization are codependent and activate src kinases (fig. 3a) . the functions of src kinases are disrupted by interfering dimerization, emphasizing the importance of n-myristoylation to src activity. in addition, the myristoyl-group-driven aggregation in lipid bilayers is also observed for h-ras [37] . some protein-protein interactions require a myristoyl group. the brain-specific protein cap-23/nap22 binds calmodulin (cam) with high affinity even though it lacks any canonical cam-binding domain. a crystal structure of ca 2+ -cam-cap-23/nap22-cam complex showed that the myristoyl group of cap-23/nap22 passes through a hydrophobic tunnel created by two hydrophobic components exclusively in the pockets of the terminus of cam, implying the direct involvement of the myristoyl group in this interaction. further, the interaction of calmodulin induces the phosphorylation of cap-23/nap-22 [40] . in hiv infections, both nef and gag are myristoylated by the host nmt cell to execute proper functions. in the assembly of hiv, nef and gag proteins are essential for infection. nef has many virulence factors that attenuate the immune system recognition of infected cells and enhance infectivity and viral replication [12] , and gag is a precursor protein for structural components of the viral capsid. because this virus lacks nmt proteins, both nef and gag are myristoylated by the host nmt cell, and the gag-gag interaction triggers an entropic switch toward a myristoyl-exposed conformation, providing the impetus for protein assembly (fig. 3b) . in contrast, in beta-retroviruses, such as mouse mammary tumor virus (mmtv), the oligomerization of the matrix (ma) domain, which contains the n-terminal residue in gag, adopts a myristoylsequestering conformation [41, 42] . moreover, the results from screens of the interaction of the host factors with the hiv-1 ma domain showed that heme oxygenase 2 (ho-2) specifically binds the myristoyl moiety of gag via a hydrophobic channel adjacent to its heme-binding pocket, which inhibits virus production. in addition, ho-2 binds n-myristoylated tram, an adaptor protein for toll-like receptor 4 (tlr4) [43] , which inhibits the tramdependent lipopolysaccharide(lps)-tlr4 pathway. these findings suggest ho-2 is a novel cellular n-myristoylated protein that negatively regulates both virus replication and host inflammatory responses [44] . recent studies have revealed that a glycine positioned in the nterminus can act as a potent degron, indicating that nmyristoylation may contribute to the removal of proteolytic cleavage products. richard t. timms et al. [45] identified two cul2 cullin-ring e3 ubiquitin ligase complexes called cul2 zyg11b and cul2 zer1 , both of which target n-myristoylated proteins for proteasomal degradation by recognizing n-terminal glycine degrons, which presumably play important roles in the quality control of protein n-myristoylation. nonmyristoylated c-src has enhanced stability compared to that of soluble n-myristoylated c-src [46] . borja belda-palazon et al. [47] found that rglg1, an e3 ligase in arabidopsis, is n-myristoylated by nmt1. n-myristoylation facilitates the attachment of rglg1 to the plasma membrane. the phytohormone abscisic acid (aba) induces the degradation of pp2ca, which is predominantly localized in the nucleus, through rglg1/5 e3 ligases. this degradation mechanism was found by aba downregulation of nmt1, which hindered the nmyristoylation of rglg1 and promoted its translocation to the nucleus, where it interacted with pp2ca, increasing pp2ca degradation. in addition, in aspergillus nidulans [48] , the swof gene was found to encode an nmt. the enhanced activity of the 26s proteasome and the accumulation of ubiquitinated substrates in the n-myristoylate-deficient swof mutant, compared with the activity and accumulation in wild-type strain, resulted in impaired cell morphogenesis. alterations to signaling pathways n-myristoylation influences downstream kinase activity directly or indirectly, usually by the mechanisms described above, through ras and src. c-abl is a member of the src family of tyrosine kinases. a 'myristoyl/phosphotyrosine' switch has been identified in the regulation of the kinase activity of c-abl [49] . nmyristoylation locks the protein into an autoinhibitory conformation when the sh2 domain docks to the kinase domain. in contrast, myristoylation leads to an unpredicted function: c-src is induced into a conformation optimal for kinase activity. a series of studies have unequivocally demonstrated that n-myristoylation of the β-subunit is a prerequisite for the initiation of ampk signaling in response to amp [26, 50, 51] . therefore, in the case of nmt deficiency or upon cell treatment with an nmt inhibitor, suppressed n-myristoylation diminished the extent of α-thr172 phosphorylation of amp and abolished its activation, thereby causing multiple morbid physiological outcomes. moreover, the myristoyl switch regulated by amp may affect the selectivity of the substrates and serve as a gatekeeper for transducing signals of metabolic stress [52] . recently, it was suggested that pka-c n-myristoylation may account for the enriched kinase activity at the membrane and the preferential phosphorylation of membrane substrates. these findings indicate an important role for n-myristoylation in synaptic function normality and plasticity during normal pka regulation [53, 54] . in another pattern of results from recent studies, several proteins were found to phosphorylate nmt, resulting in the activation of nmt. rajala et al. [55] verified that lyn and fyn are nmyristoylated by nmt before they phosphorylate nmt. in addition, it has been hypothesized that the interaction between nmt1 and the lyn tyrosine kinase proceeds in a phosphorylationdependent manner [56] . manipulation of substrates during apoptosis apoptosis, or programmed cell death, is the physiologically and tightly controlled process for eliminating unnecessary or redundant cells in eukaryotic organisms. in addition, dysfunctional apoptosis is one of the characteristics of malignant cells. apoptosis is orchestrated by a signaling cascade of proteases called caspases (cysteine-containing aspases), which leads to the exposure of novel n-termini susceptible to various posttranslational modifications. since the initial discovery of myristoylated bid, it has been revealed that multiple proteins are modified posttranslationally by n-myristoylation upon caspase cleavage. using a myristoylated protein-labeling method in jurkat t cells and mcf-7 cells, at least 15 and 7 posttranslationally nmyristoylated proteins were discovered, respectively, during apoptosis [57] . further characterization of these proteins revealed that caspase truncated (ct)-gelsolin and ct-pkc have antiapoptotic roles while ct-bid and ct-pak2 have proapoptotic roles upon posttranslational n-myristoylation, suggesting that posttranslational n-myristoylation plays an important role in maintaining a balance between cell death and survival. moreover, it has been found that nmt1 and nmt2 are substrates of different caspases. furthermore, n-myristoylation may be involved in sphingolipid biosynthesis pathway, which includes ceramide synthesis and is involved in the induction of apoptosis. in the last step of ceramide biosynthesis, a trans △4-double bond in the carbon chain of dihydroceramide is specifically catalyzed by dihydroceramide △4desaturase 1 (des1), which is n-myristoylated by nmt1 [58] . myristic acid upregulates novel des1 activity, which differs from that of saturated fatty acids, possibly because of the accumulation of n-myristoylated des1. n-myristoylation causes a proportion of des1 to be translocated from the er to the mitochondrial outer membrane, leading to an increase in ceramide levels. since the production of ceramide can induce apoptosis by inducing mitochondrial dysfunction [59] , the myristic acid-associated increase in des1 activity can enhance apoptosis induction in cells, which shows the importance of n-myristoylation in the process of apoptosis. n-myristoylated lancl2, which belongs to the eukaryotic lanc-like protein family, is known as the testisspecific adriamycin sensitivity protein (tasp). one biochemical study identified a phosphatidylinositol phosphate (pip)-binding site in the n-terminus of lancl2. in addition, both nmyristoylation and pip binding are crucial for lanc2 binding to the membrane. it is possible that the overexpression of lancl2 increases cell sensitivity to adriamycin, which is dependent on both n-myristoylation and membrane association [60] . in summary, we can conclude that a myristoyl group as a hydrophobic motif always stably binds to substrates to change the conformation and increase the hydrophobicity of the protein. further, it affects protein localization and the ease with which a protein binds to substrates. however, insufficient hydrophobicity of the 14-carbon myristoyl group leads to it being influenced by its surroundings. the hydrophilic group can ease protein binding, and the hydrophobic group can stabilize the state of the n-myristoylated protein. it seems that n-myristoylation triggers consequential functions of protein necessarily but insufficiently. accumulating evidence has demonstrated that n-myristoylation is an evolutionarily conserved lipidation that is essential for cell viability in different organisms. currently, interventions of protein n-myristoylation are principally achieved through nmt inhibitors. therefore, nmt inhibitors are very suitable for use against diseases caused by proliferating cells or pathogens, such as infectious diseases caused by various pathogens and malignancies. in fact, broadly classified inhibitors for each process in nmt-induced disease are designated as antifungal and antiviral agents. typically, these inhibitors can be divided into three categories [56] : (1) myristoyl-coa and myristate derivatives. although the myristoyl-coa binding sites in human nmt and fungal nmt are highly conserved, their peptide-binding sites are divergent, which provides an explanation for the selectivity of inhibitors that do not induce adverse toxicity in humans. (2) histidine analogs. in most conserved regions in the catalytic domain eeveh (289-293), histidine is critical for the myristoyl-coa transfer [61] . (3) myristoyl peptide derivatives. as previously mentioned, lipid metabolism disorders can affect protein lipidation [1] . various saturated and unsaturated fatty acids have been evaluated as potent inhibitors of human nmt1. indeed, the development of nmt inhibitors is not limited to proteins involved in infectious diseases because they are great prospects as immunodeficiency and cancer treatments. as mentioned previously, nmt1 has been characterized as the principal enzyme for the early development of embryogenesis. further investigation was focused on the role of nmt1 in myelopoiesis through which blood cell types are matured. bone marrow-derived macrophages (bmdms) from nmt1 +/− -deficient mice have defective morphology compared with that of mature bmdms in wild-type mice. during bmdm maturation, the nmt activity increased during the initial period and then decreased for the remaining time due to differential nmt expression. although the nmt activity in the bmdms of nmt1 +/− -deficient mice followed a trend similar to that of the bmdms in the wild-type mice, the nmt expression levels were reduced to approximately one-half in the mutant mice. this report addressed an essential role of nmt1 during monocytic/macrophage differentiation. of course, studies are continuously uncovering valuable evidence not only for nmt1 but also for nmt2 in both normal and malignant hematopoiesis. a bioinformatic database of gene expression in different cancer cell lines revealed decreased nmt2 expression and preserved nmt1 expression in some hematological cancer cell lines, such as burkitt lymphoma, diffuse large b-cell lymphoma and acute myeloid leukemia (aml) cell lines. a study performed by ryan stubbins et al. suggested a correlation between nmt2 and aml. nmt1 and nmt2 protein levels in the marrow aspirates or peripheral blood of aml patients were assayed by fluorescence-activated cell counting with intracytoplasmic staining, expressed as the mean fluorescent intensity (mfi). the evidence showed that the nmt2 mfi was higher in the lymphocytes and lower in the monocytes, suggesting that the regulation of nmt2 protein levels may influence early lymphoid/myeloid lineage commitment. further, the overall trend revealed by a survival analysis showed higher nmt2 mfi values, portending a worse prognosis for aml patients and suggesting a role for nmt2 as a novel prognostic biomarker for intermediate-risk aml [62] . in terms of adaptive immunity, it has been reported that nmyristoylation is indispensable for t cell development [63, 64] . a comparative analysis of thymuses showed that mice with deficient nmt1 or nmt2 levels had reduced medullary volume, which was 30% and 25% lower, respectively, than it was in wild-type mice, and mice with double nmt1 and nmt2 deficiency had a much greater medullary volume reduction (83%). these findings suggest nonredundant roles for both nmts in maintaining the development of thymocytes, since the thymus has high nmt activity levels [65] . in agreement with these results, it was demonstrated that n-myristoylation and its potential applications m yuan et al. t-cell receptor (tcr) signaling is disrupted in mice with t-cell lineages characterized by specific nmt1 and nmt2 activity deficiencies and results in retarded thymocyte development. these outcomes may be attributable to the mislocalization of nmyristoylation-deficient src family tyrosine kinases, such as lck or fyn [66] . for example, n-myristoylation contributes to the cytomembrane targeting of fyn and facilitates its binding with the z chain of tcr. non-n-myristoylated lck is in the cytoplasm and unable to facilitate tcr signaling. however, the regulation of n-myristoylation or nmt activity in these processes during adaptive immunity has not been well established to date. to gain in-depth understanding of the regulation of immune responses, further investigation into the specific roles of nmyristoylation, which may involve nmt as a potential target of immune modulation, is warranted. parasitic and other infectious diseases it is known that some n-myristoylated proteins in small rna viruses and retroviruses are essential for virus assembly during viral replication or production of infectious viral particles, suggesting that n-myristoylation may be related to the survival and propagation of pathogens. in addition, some pathogens need to utilize host cellular machinery to replicate within host cells due to deficiency in viral nmts. in immunosuppressed patients, cryptococcus neoformans can easily cause chronic meningitis; however, it is unable to survive at 37°c if it harbors mutant nmt with reduced activity. some disease-causing parasitic protozoa such as falciparum (malaria), leishmania donovani (leishmaniasis), and trypanosoma brucei (african sleeping sickness) retain the nmt necessary for their survival. given that the peptide-binding pocket of nmt is not strictly conserved across species, the search for species-specific nmt inhibitors focused on the binding pocket is worthwhile [67] . notwithstanding, current anti-infectious agents have some drawbacks, such as drug resistance, poor oral bioavailability, and renal toxicity. a group of studies have suggested nmt as a novel target for use in anti-infective drugs. theoretically, according to the respective skeletal structures, nmt inhibitors are classified into four categories (table 1) . among these inhibitors, benzofuran and, benzothiazole have high species selectivity [68] . anti-infective drugs targeting nmt have certain advantages. first, myristoyl-coa is found at very low accumulation levels, at approximately 5 nm, in mammalian cells. in addition, the strict hydrophobic structure of the fatty carbon chains reduces the likelihood that incompatible fatty acid groups will be recognized by the nmt, even in cases where the palmitic acid concentration is higher than the myristic acid in vitro [69] . these physiological phenomena suggest that a small amount of inhibitor can have a good inhibitory effect. second, nmt inhibitors can be designed for high selectivity because of the significant differences in substrate specificity in the human and parasitic organisms. moreover, many fungal and parasites must use the nmt of the host to synthesize essential proteins for their own reproduction. potential targets of cancer treatments given that altered nmt expression is observed in many types of cancer tissues and because many n-myristoylated proteins are involved in signaling processes that regulate cell proliferation, growth and death, it has been proposed that n-myristoylation or nmts can be considered as therapeutic targets for cancer. the premise for their use is based on a thorough understanding of the abnormal regulation of n-myristoylation in carcinogenesis. for instance, given that n-myristoylation can facilitate src-mediated prostate tumorigenesis, the myristoyl-coa analog b13 and its derivative lcl204 [68, 70] have been identified as inhibitors of nmt1 enzymatic activity and blockers of src n-myristoylation; their actions are based on competing for myristoyl-coa binding site, which provides a promising approach to inhibit src family kinase-mediated oncogenic activity, and offers preclinical support for the use of protein n-myristoylation inhibitors in treating cancer [71] . an organopalladium compound, tris(dibenzylideneacetone) dipalladium (tris dba) was identified as a novel human nmt1 inhibitor that not only blocks the kinase activity of nmt1 but also reduces its expression. it showed potent antiproliferative activity against melanoma cells by inhibiting several proliferation-related signaling pathway proteins including mapk, akt, and stat-3 [72] . the most successful examples of myristoylation-related anticancer inhibitors are allosteric abl tkis such as abl001 [73] [74] [75] , gnf2 and gnf5 [76] (table 1) . these allosteric inhibitors selectively target the myristoyl-binding pocket in the c-lobe of the abl kinase domain and not the atp-binding pocket. moreover, these inhibitors increase the sensitivity of a bcr-abl (t315i) mutant to atp-competitive tkis. a series of phase i clinical trials of abl001 and tkis (nilotinib, imatinib, and dasatinib) therapy have been ongoing for cml and ph+all patients (https://clinicaltrials.gov/ ct2/show/nct02081378). some studies have shown the potential targets of nmt in cancer cells. the cancer genome atlas (tcga) reports a group of genomic alterations induced by nmt1 and nmt2 in several cancers. although the occurrence of somatic mutations is more common than the occurrence of genomic amplification in nmt1 and nmt2, the roles of these mutations in regulating pathological mechanisms remain to be determined (https://www.cbioportal. org/). moreover, patients with diseases such as liver cancer, cervical cancer, and lung cancer and high expression levels of nmt1 or nmt2 are more likely to have a poor prognosis, as indicated consistently in some reports. in addition, it is noteworthy that a cohort with renal cell carcinoma with either high expression of nmt1 or low expression of nmt2 was found to have worse overall survival, suggesting the unique roles of these proteins in kidney cancer. however, no evidence showed that their catalytic activity is linked to tumor progression in kidney cancer (http:// kmplot.com/analysis/). in addition, several nmt activators that enhance nmt enzyme activity have been identified. l-histidine and d-histidine can activate human nmt activity in a concentration-dependent manner. this finding suggests that two analogs may be involved in myristoyl-coa transfer by interacting with his-293 of nmt. naf45 was identified as an nmt activator factor in bovine brain [77] . the reconstitution of naf45 and nmt likely resulted in an open conformation where the active site is expanded. a novel stratagem of nmt activation is enhanced nmt specificity for specific myristoyl-coa substrates. on the basis that nmt can transfer abundant palmitoyl-coa but can use only the rare myristoyl-coa for acylation of a substrate protein, eric soupene et al. [78] determined that the acyl-coa-binding protein domain 6 (acbd6) stimulates the activity of nmt2. acbd6 can block the access of palmitoyl-coa to the nmt2-binding site and enhance its catalytic activity, which requires interaction between nmt2 and acbd6. a mutant acbd6 unable to interact with nmt2 or with deficient ligand binding at its own n-terminus does not stimulate nmt2 activity. in this review article, we outlined advanced studies of nmyristoylation, focusing on the role of protein n-myristoylation in physiology and pathology. the important role of nmyristoylation underlies the early stages of protein maturation. after the protein is folded in the golgi apparatus or the er, cotranslationally n-myristoylation is likely to influence the transport and localization of the protein, which can affect the biofunction of protein. the insufficient nmt kinase activity in pathogens and the variations in different species emphasize the selective lethality of nmt inhibitors for infectious diseases for which either no valid drug is qualified or for which available drugs induce drug resistance. the breakthroughs of nmt inhibitors will likely be as tumor treatments. abl001 is being pioneered continuously for cml and ph+all patients in phase i trials, which suggests a strategy focused on the n-myristoylation of oncoproteins. furthermore, posttranslational n-myristoylation in the apoptotic process suggests the participation of nmts, specifically nmt2, in cell death. the function of the n-myristoylated protein in the apoptotic process, whether pathogenically or physiologically normal, can further indicate the orientation of the treatment strategy for targeting nmt. the knowledge of other protein modifications, such as ubiquitination or palmitoylation, which involves a multimember kinase family, can inform many specific enzyme-substrate studies and the design of selective inhibitors. in contrast, scientists need more precise approaches for analyses of nmts and nmyristoylation. nevertheless, traditional methods based on radioactively labeled probes to detect n-myristoylation are insensitive and time consuming, and the technical difficulties in obtaining three-dimensional structures of myristate-attached proteins need to be overcome. further, bioinformatics on n-myristoylation and nmts is still in its infancy; however, since databases are integrated, these data provide many clues linked to other biological fields. regardless of the technical difficulties, it is worth exploiting novel nmt inhibitors as single agents and exploring the potential drug synergies that might improve multiple clinical applications and enhance therapeutic efficacy, reverse drug resistance, or extend the therapeutic index for drugs already used in the clinic. it is likely that two approaches [79] can be used to reveal the prospects of nmt inhibitors for oncology therapy in the future: (1) identify currently unknown sensitivities of certain cancer types by widely screening cancer cell line panels and (2) discover the essential sensitivity or resistance mechanisms in resistant cell lines and wild-type cell lines by proteomic analyses of nmt substrate profiles and proteomic changes. as our knowledge of the biochemistry and cell biology of n-myristoylation continues to grow, more substrate proteins will be identified, and scientists will continue to deduce the effects of this lipid attachment on protein structure and function. protein lipidation in cell signaling and diseases: function, regulation, and therapeutic opportunities protein lipidation sirt2 and lysine fatty acylation regulate the transforming activity of k-ras4a hdac11 regulates type i interferon signaling through defatty-acylation of shmt2 protein lysine acylation and cysteine succination by intermediates of energy metabolism protein myristoylation in health and disease cholesterol sensitivity of endogenous and myristoylated akt myristoylated naked2 antagonizes wnt-beta-catenin activity by degrading dishevelled-1 at the plasma membrane myristoylation of the dual-specificity phosphatase c-jun n-terminal kinase (jnk) stimulatory phosphatase 1 is necessary for its activation of jnk signaling and apoptosis inhibition of enterovirus vp4 myristoylation is a potential antiviral strategy for hand, foot and mouth disease n-terminal region of the catalytic domain of human nmyristoyltransferase 1 acts as an inhibitory module hiv-1 production is specifically associated with human nmt1 long form in human human n-myristoyltransferase amino-terminal domain involved in targeting the enzyme to the ribosomal subcellular fraction fatty acids regulate germline sex determination through acs-4-dependent myristoylation comparison of myristoyl-coa: protein n-myristoyltransferases from three pathogenic fungi: cryptococcus neoformans, histoplasma capsulatum, and candida albicans two n-myristoyltransferase isozymes play unique roles in protein myristoylation, proliferation, and apoptosis efficient demyristoylase activity of sirt2 revealed by kinetic and structural studies sirt6 regulates ras-related protein r-ras2 by lysine defatty-acylation nmyristoyltransferase 1 is essential in early mouse development protein kinase c coordinates histone h3 phosphorylation and acetylation molecular determinants of the myristoyl-electrostatic switch of marcks structural basis for activation of arf gtpase: mechanisms of guanine nucleotide exchange and gtp-myristoyl switching structure and membrane interaction of myristoylated arf1 sequestration of the membranetargeting myristoyl group of recoverin in the calcium-free state myristoylation-dependent n-terminal cleavage of the myristoylated alanine-rich c kinase substrate (marcks) by cellular extracts a myristoyl/phosphoserine switch controls camp-dependent protein kinase association to membranes c-terminal 15 kda fragment of cytoskeletal actin is posttranslationally n-myristoylated upon caspase-mediated cleavage and targeted to mitochondria n-terminally myristoylated ras proteins require palmitoylation or a polybasic domain for plasmamembrane localization membrane localization of a rice calcium-dependent protein kinase (cdpk) is mediated by myristoylation and palmitoylation myristoylationdependent palmitoylation of the receptor tyrosine kinase adaptor frs2alpha g proteinmembrane interactions i: galphai1 myristoyl and palmitoyl modifications in protein-lipid interactions and its implications in membrane microdomain localization identification and characterization of protein n-myristoylation occurring on four human mitochondrial proteins, samm50, tomm40, mic19, and mic25 role of n-myristoylation in stability and subcellular localization of the clpabp protein mouse stbd1 is n-myristoylated and affects er-mitochondria association and mitochondrial morphology multiple modification and protein interaction signals drive the ring finger protein 11 (rnf11) e3 ligase to the endosomal compartment n-myristoylation of the rpt2 subunit of the yeast 26s proteasome is implicated in the subcellular compartment-specific protein quality control system aggregation of lipid-anchored full-length h-ras in lipid bilayers: simulations with the martini force field a dimerization function in the intrinsically disordered n-terminal region of src a myristoyl-binding site in the sh3 domain modulates c-src membrane anchoring crystal structure of a myristoylated cap-23/nap-22 n-terminal domain complexed with ca2+/calmodulin a myristoyl switch regulates membrane binding of hiv-1 gag myristoylation drives dimerization of matrix protein from mouse mammary tumor virus the myristoylation of trif-related adaptor molecule is essential for toll-like receptor 4 signal transduction heme oxygenase 2 binds myristate to regulate retrovirus assembly and tlr4 signaling a glycine-specific ndegron pathway mediates the quality control of protein n-myristoylation myristoylation and membrane binding regulate c-src stability and kinase activity aba inhibits myristoylation and induces shuttling of the rglg1 e3 ligase to promote nuclear degradation of pp2ca a novel interaction between n-myristoylation and the 26s proteasome during cell morphogenesis a myristoyl/phosphotyrosine switch regulates c-abl beta-subunit myristoylation is the gatekeeper for initiating metabolic stress sensing by ampactivated protein kinase (ampk) n-myristoyltransferase deficiency impairs activation of kinase ampk and promotes synovial tissue inflammation exploring protein myristoylation in toxoplasma gondii liberated pka catalytic subunits associate with the membrane via myristoylation to preferentially phosphorylate membrane substrates myristoylated alanine-rich c-kinase substrate effector domain phosphorylation regulates the growth and radiation sensitization of glioblastoma phosphorylation of human n-myristoyltransferase by n-myristoylated src family tyrosine kinase members potential role of n-myristoyltransferase in cancer rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog myristic acid increases the activity of dihydroceramide delta 4-desaturase 1 through its nterminal myristoylation monounsaturated fatty acid modification of wnt protein: its role in wnt secretion myristoylation of human lanc-like protein 2 (lancl2) is essential for the interaction with the plasma membrane and the increase in cellular sensitivity to adriamycin effects of l-histidine and its structural analogues on human n-myristoyltransferase activity and importance of eeveh amino acid sequence for enzyme activity expression and activity of n-myristoyltransferase in lung inflammation of cattle and its role in neutrophil apoptosis deficiency of n-myristoylation reveals calcineurin activity as regulator of ifn-gammaproducing gamma delta t cells myristoylation: an important protein modification in the immune response n-myristoylation of ampk controls t cell inflammatory function the residue at position 5 of the n-terminal region of src and fyn modulates their myristoylation, palmitoylation, and membrane interactions vhy, a novel myristoylated testis-restricted dual specificity protein phosphatase related to vhx novel analogs of d-e-mapp and b13. part 2: signature effects on bioactive sphingolipids role of long-chain fatty acyl-coa esters in the regulation of metabolism and in cell signalling novel analogs of de-mapp and b13. part 1: synthesis and evaluation as potential anticancer agents blocking myristoylation of src inhibits its kinase activity and suppresses prostate cancer progression tris (dibenzylideneacetone) dipalladium, a n-myristoyltransferase-1 inhibitor, is effective against melanoma growth in vitro and in vivo discovery of asciminib (abl001), an allosteric inhibitor of the tyrosine kinase activity of bcr-abl1 allosteric inhibitors of bcr-abl: towards novel myristate-pocket binders the allosteric inhibitor abl001 enables dual targeting of bcr-abl1 inhibitors of the abl kinase directed at either the atp-or myristate-binding site differential activation of bovine brain n-myristoyltransferase(s) by a cytosolic activator acbd6 protein controls acyl chain availability and specificity of the n-myristoylation modification of proteins n-myristoyltransferase inhibition induces er-stress, cell cycle arrest, and apoptosis in cancer cells this work was supported by grants from the national natural science foundation of china (81872885 to j.c.), zhejiang provincial natural science foundation (y18h310005 to j.c.), and the talent project of zhejiang association for science and technology (no. 2018ycgc002 to j.c.). my and jc conceived and designed the review article. my, zhs, and jc collected the related research articles contributed to the paper. my, mdy, hz, by, qjh, and jc made amendments to the paper. competing interests: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential competing interests. key: cord-033010-o5kiadfm authors: durojaye, olanrewaju ayodeji; mushiana, talifhani; uzoeto, henrietta onyinye; cosmas, samuel; udowo, victor malachy; osotuyi, abayomi gaius; ibiang, glory omini; gonlepa, miapeh kous title: potential therapeutic target identification in the novel 2019 coronavirus: insight from homology modeling and blind docking study date: 2020-10-02 journal: egypt j med hum genet doi: 10.1186/s43042-020-00081-5 sha: doc_id: 33010 cord_uid: o5kiadfm background: the 2019-ncov which is regarded as a novel coronavirus is a positive-sense single-stranded rna virus. it is infectious to humans and is the cause of the ongoing coronavirus outbreak which has elicited an emergency in public health and a call for immediate international concern has been linked to it. the coronavirus main proteinase which is also known as the 3c-like protease (3clpro) is a very important protein in all coronaviruses for the role it plays in the replication of the virus and the proteolytic processing of the viral polyproteins. the resultant cytotoxic effect which is a product of consistent viral replication and proteolytic processing of polyproteins can be greatly reduced through the inhibition of the viral main proteinase activities. this makes the 3c-like protease of the coronavirus a potential and promising target for therapeutic agents against the viral infection. results: this study describes the detailed computational process by which the 2019-ncov main proteinase coding sequence was mapped out from the viral full genome, translated and the resultant amino acid sequence used in modeling the protein 3d structure. comparative physiochemical studies were carried out on the resultant target protein and its template while selected hiv protease inhibitors were docked against the protein binding sites which contained no co-crystallized ligand. conclusion: in line with results from this study which has shown great consistency with other scientific findings on coronaviruses, we recommend the administration of the selected hiv protease inhibitors as first-line therapeutic agents for the treatment of the current coronavirus epidemic. the first outburst of pneumonia cases with unknown origin was identified in the early days of december 2019, in the city of wuhan, hubei province, china [1] . revelation about a novel beta coronavirus currently regarded as the 2019 novel coronavirus [2] came up after a high-throughput sequencing of the viral genome which exhibits a close resemblance with the severe acute respiratory syndrome (sars-cov) [3] . the 2019-ncov is the seventh member of enveloped rna coronavirus family (subgenus sarbecovirus, orthocoronavirinae) [3] , and there are accumulating facts from family settings and hospitals confirming that the virus is most likely transmitted from person-to-person [4] . the 2019-ncov has also recently been declared by the world health organization as a public health emergency of international concern [5] and as of the 5th of february 2020, over 24,000 cases has been confirmed and documented from laboratories around the world [6] while more than 28, 000 of such cases were documented in china through laboratory confirmation as of the 6th of february 2020 [7] . despite the fast rate of global spread of the virus, the characteristics clinically peculiar to the 2019-ncov acute respiratory disease (ard) remain unclear to a very large extent [8] . over 8000 infections and 900 deaths were recorded worldwide in the summer of 2003 before a successful containment of the severe acute respiratory syndrome wave was achieved as the disease itself was also a major public health concern worldwide [9, 10] . the infection that led to a huge number of death cases was linked to a new coronavirus also known as the sars coronavirus (sars-cov). coronaviruses are positive-stranded rna viruses and they possess the largest known viral rna genomes. the first major step in containing the sars-cov-lined infection was to successfully sequence the viral genome, the organization of which was found to exhibit similarity with the genome of other coronaviruses [11] . the main proteinase crystal structure from both the transmissible gastroenteritis virus and the human coronavirus (hcov 229e) has been determined with the discovery that the enzyme crystal structure exists as a dimer and the orientation of individual protomers making up the dimer has been observed to be perpendicular to each other. each of the protomers is made up of three catalytic domains [12] . the first and second domains of the protomers have a two-βbarrel fold that can be likened to one of the folds in the chymotrypsin-like serine proteinases. domain iii have five α-helices which are linked to the second domain by a long loop. individual protomers have their own specific region for the binding of substrates, and this region is positioned in the left cleft between the first and second domain. dimerization of the protein is thought to be a function of the third domain [13] . the main proteinase of the sars cov is known to be a cysteine proteinase which has in its active site, a cysteine-histidine catalytic dyad. conservation of the sars cov main proteinase across the genome sequence of all sars coronaviruses is very high, likewise the homology of the protein to the main proteinase of other coronaviruses. on the basis that high similarity exists between the different coronavirus main proteinase crystal structures and the conservation of almost all the amino acid residue side chains involved in the dimeric state formation, it was proposed that the only biologically functional form coronavirus main proteinase might be is its existence as a dimer [14] . more recently, chen et al. in his study which involved the application of molecular dynamic simulations and enzyme activity measurements from a hybrid enzyme showed that the only active form of the proteinase is in its dimeric state [15] . recent studies based on the sequence homology of the coronavirus main proteinase structural model with tgev as well as the solved crystal structure has involved the docking of substrate analogs for the virtual screening of natural products and a collection of synthetic compounds, alongside approved antiviral therapeutic agents in the evaluation of the coronavirus main proteinase inhibition [16] . some compounds from this study were identified for the inhibitory role played against the viral proteinase. these compounds include the l-700,417, which is an hiv-1 protease inhibitor, calanolide a, and nevirapine, both of which are reverse transcriptase inhibitors, an inhibitor of the α-glucosidase named glycovir, sabadinine, which is a natural product and ribavirin, a general antiviral agent [17] . ribavirin was shown to exhibit an antiviral activity in vitro, at cytotoxic concentrations against the sars coronavirus. at the start of the first outbreak of the sars epidemic, ribavirin was administered as a first-line of defense. the administration was as a monotherapy and in combination with corticosteroids or the hiv protease inhibitor, kaletra [18] . according to reports from a very recent research conducted by cao et al., where a total of a 199 laboratoryconfirmed sars-cov-infected patients were made to undergo a controlled, randomized, open-labeled trial in which 100 patients were assigned to the standard care group and 9 patients assigned to the lopinavirritonavir group. 48.4% of the patients in the lopinavir-ritonavir group (46 patients) and 49.5% of the patients in the standard care group (49 patients) exhibited serious adverse events between randomization and the 28th day. the exhibited adverse events include acute respiratory distress syndrome (ards), acute kidney injury, severe anemia, acute gastritis, hemorrhage of lower digestive tract, pneumothorax, unconsciousness, sepsis, acute heart failure etc. patients in the lopinavir-ritonavir group in addition, specifically exhibited gastrointestinal adverse events which include diarrhea, vomiting, and nausea [19] . our current study took advantage of the availability of the sars cov main proteinase amino acid sequence to map out the nucleotide coding region for the same protein in the 2019-ncov. two selected hiv protease inhibitors (lopinavir and ritonavir) were then targeted at the catalytic site of the protein 3d structure which was modeled using already available templates. the predicted activity of the drug candidates was validated by targeting them against a recently crystalized 3d structure of the enzyme, which has been made available for download in the protein data bank. lopinavir is an antiretroviral protease inhibitor used in combination with ritonavir in the therapy and prevention of human immunodeficiency virus (hiv) infection and the acquired immunodeficiency syndrome (aids). it plays a role as an antiviral drug and a hiv protease inhibitor. it is a member of amphetamines and a dicarboxylic acid diamide (fig. 1 ). the complete genome of the isolated wuhan seafood market pneumonia virus (2019-ncov) was downloaded from the genbank database with an assigned accession number of mn908947.3. the nucleotide sequence of the full genome was copied out in fasta format. the gen-bank sequence database is an annotated collection of all nucleotide sequences which are publicly available with their translated protein segments and also open access. this database is designed and managed by the national center for biotechnology information (ncbi) in accordance with the international nucleotide sequence database collaboration (insdc) [20] . nucleotides between the 10055 and 10972 sequence of the 2019-ncov genome was selected as the sequence of interest. translation of the nucleotide sequence of interest in the 2019-ncov and the back-translation of the sars cov main proteinase amino acid sequence was achieved with the use of emboss transeq and backtranseq tools, respectively [21] . transeq reads one or more nucleotide sequences and writes the resulting translated sequence of protein to file while backtranseq makes use of a codon usage table which gives the usage frequency of individual codon for every amino acid [22] . for every amino acid sequence input, the corresponding most frequently occurring codon is used in the nucleotide sequence that forms the output. the corresponding amino acid sequence generated as a product of the transeq translation of the nucleotide sequence of interest had no stop codons and as such was used directly for protein homology modeling without the need for any deletion. two sets of sequence alignments were carried out in this study. the first was the alignment between the translated nucleotide sequence copy of the 2019-ncov genome which was used for the reference protein homology modeling and the amino acid sequence of the sars cov main proteinase while the second alignment was between the back-translated sars cov main proteinase nucleotide sequence and the 2019-ncov full genome. the latter was used in mapping out the protein coding sequence in the 2019-ncov full genome. these alignments were carried out using the clustal omega software package. clustal omega can read inputs of nucleotide and amino acid sequences in formats such as a2m/fasta, clustal, msf, phylip, selex, stockholm, and vienna [23] . template search with blast and hhblits was performed against the swiss-model template library. the target sequence was searched with blast against the primary amino acid sequence contained in the smtl. a total of 120 templates were found. an initial hhblits profile was built using the procedure outlined in remmert et al. [24] followed by 1 iteration of hhblits against nr20. the obtained profile was then searched against all profiles of the smtl. a total of 192 templates were found. models were built based on the targettemplate alignment using promod3. coordinates which are conserved between the target and the template are copied from the template to the model. insertions and deletions are remodeled using a fragment library. side chains were then rebuilt and finally, the geometry of the resulting model, regularized by using a force field [25] . for the estimation of the protein structure model quality, we used the qmean (qualitative model energy analysis), a composite scoring function that describes the main aspects of protein structural geometrics, which can also derive on the basis of a single model, both global (i.e., for the entire structure) and local (i.e., per residue) absolute quality estimates [26] . an appreciable number of alternative models have been produced which formed the basis on which scores produced by the final model was selected. the qmean score was thus used in the selection of the most reliable model against which the consensus structural scores were calculated. molprobity (version 4.4) was used as the structurevalidation tool that produced the broad-spectrum evaluation of the quality of the target protein at both the global and local levels. it greatly relies on the provided sensitivity and power by optimizing the placement of hydrogen and all-atom contact analysis with complementary versions of updated covalent-geometry and torsion-angle criteria [27] . the torsion angles between individual residues of the target protein were calculated using the ramachandran plot. this is a plot of the torsional angles [phi (φ) and psi (ψ)] of the amino acid residues making up a peptide. in the order of sequence, the torsion angle of n(i-1), c(i), ca(i), n(i) is φ while the torsion angle of c(i), ca(i), n(i), c(i+1) is ψ. the values of φ were plotted on the x-axis while the values of ψ were plotted on the y-axis [28] . plotting the torsional angles in this way graphically shows the possible combination of angles that are allowed. the quaternary structure annotation of the template is employed to model the target sequence in its oligomeric state. the methodology as proposed by bertoni et al. [29] was supported on a supervised machine learning algorithm rule, support vector machines (svm), which mixes conservation of interface, clustering of structures with other features of the template to produce a quaternary structure quality estimate (qsqe). the qsqe score is a number that ranges between 0 and 1, and it is a reflection of the accuracy expected of the inter-chain contacts for a model engineered based on a given template and its alignment. the higher score is an indication of a more reliable result. this enhances the gmqe score that calculates the accuracy of the 3d structure of the resulting model. the 3d structural homology modeling of the 2019-ncov genome translated segment was followed by a structural comparison with the sars cov main proteinase 3d structure (pdb: 1uj1). this was achieved using the ucsf chimera which is a highly extensible tool for interactive analysis and visualization of molecular structures and other like data, including docking results, supramolecular assemblies, density maps, sequence alignments, trajectories, and conformational ensembles [30] . high-quality animation videos were also generated. the amino acid constituents of the target protein secondary structures were colored and visualized in 3d using the pymol molecular visualzer which uses opengl extension wrangler library (glew) and freeglut. the py aspect of the pymol is a reference to the programming language that backs up the software algorithm which was written in python [31] . the percentage composition of each component making up the secondary structure was calculated using the chou and fasman secondary structure prediction (cfssp) server. this is a secondary structure predictor that predicts regions of secondary structure from an amino acid input sequence such as the regions making up the alpha helix, beta sheet, and turns. the secondary structure prediction output is displayed in a linear sequential graphical view according to the occurrence probability of the secondary structure component. the cfssp implemented methodology is the chou-fasman algorithm, which is based on the relative frequency analyses of alpha helices, beta sheets, and loops of each amino acid residue on the basis of known structures of proteins solved with x-ray crystallography [32]. the expasy server calculates protein physiochemical parameters as a part of its sub-function, basically for the identification of proteins [33] . we engaged the function of the protparam tool in calculating various physiochemical parameters in the model and template protein for comparison purposes. the calculated parameters include the molecular weight, theoretical isoelectric point, amino acid composition, extinction coefficient, instability index, etc. the inference on evolutionary relationship was made utilizing the maximum likelihood methodology which is the basis of the jtt matrix-based model [34] . the corresponding consensus tree on bootstrap was inferred from a thousand replicates, and this was used to represent the historical evolution of the analyzed taxa. the tree branches forming partitions that were reproduced in bootstrap replicates of less than 50% were automatically collapsed. next to every branch in the tree is the displayed percentage of tree replicates of clustered associated taxa in the bootstrap test of a thousand replicates. initial trees were derived automatically for the search through the application of the neighbor-join and bionj algorithms to a matrix of pairwise distances calculated using a jtt model and followed by the selection of the most superior log likelihood value topology. the phylogenetic analysis was carried out on 12 amino acid sequences with close identity. the complete dataset contained a total of 306 positions. the whole analysis was conducted using the molecular evolutionary and genetics analysis (mega) software (version 7) [35] . ligand preparation and molecular docking protocol 2d structures of the experimental ligands were viewed from the pubchem repository and sketched using the chemaxon software [36] . the sketched structures were downloaded and saved as mrv files which were converted into smiles strings with the openbabel. the compounds prepared as ligands were docked against each of the prepared protein receptors using autodock vina [37] . blind docking analysis was performed at extra precision mode with minimized ligand structures. after a successful docking, a file consisting of all the poses generated by the autodock vina along with their binding affinities and rmsd scores was generated. in the vina output log file, the first pose was considered as the best because it has stronger binding affinity than the other poses and without any rmsd value. the polar interactions and binding orientation at the active site of the proteins were viewed on pymol and the docking scores for each ligand screened against each receptor protein were recorded. the same docking protocol was performed against the sars-cov main proteinase 3d structure that was downloaded from the protein data bank with a pdb identity of 6m2n. obtained outputs were visualized, compared, and documented for validation purpose. the full genome of the 2019-ncov (https://www.ncbi. nlm.nih.gov/nuccore/mn908947.3?report=fasta) consists of 29903 nucleotides, but for the purpose of this study, nucleotides between 10055 and 10972 were considered to locate the protein of interest. the direct translation of this segment of nucleotides produced a sequence of 306 amino acids (fig. 2 ). this amino acid count was reached after the direct translation of the nucleotide sequence of interest as there were no single existing stop codons hence, deletion of any form was needless. as depicted in fig. 3 , few structural differences were noticed. the amino acid sequences making up these nonconserved regions were clearly revealed in fig. 4 . notwithstanding, a 96% identity was observed between both sequences showing the conserved domains were predominant. figure 4 represents the percentage amino acid sequence identity between the target and the template protein, where the positions with a single asterisk (*) depicts regions of full residue conservation while the segments with the colon (:) indicates regions of conservation between amino acid residues with similar properties. positions with the period (.) show regions of conservation between amino acids with less similar properties. the amino acid sequence of the sars coronavirus main proteinase was back-translated to generate the corresponding nucleotide sequence which was then aligned with the 2019-ncov full genome. this was carried out for the purpose of mapping out the region of the 2019-ncov full genome where the proteinase coding sequence is located. as depicted in fig. 5 , the target protein coding sequence is located between 10055 and 10972 nucleotides of the viral genome the outcome of a qmean analysis is anchored on the composite scoring function which calculates several features regarding the structure of the target protein. the expression of the estimated absolute quality of the model is in terms of how well the score of the model is in agreement with the values expected of a set of resultant structures from high-resolution experiments. the global score values can either be from qmean4 or qmean6. qmean4 is a combination of four statistical potential terms represented in a linear form while qmean6 in addition to the functionality of qmean4 uses two agreement terms in the consistency evaluation of structural features with sequence-based predictions. both qmean4 and qmean6 originally are in the range of 0 to 1 with 1 being the good score and are by default transformed into z-scores (table 1) for them to be related to what would have been expected from x-ray structures of high resolution. the local scores are also a combination of four linear potential statistical terms with the agreement terms of evaluation being on a per residue basis. the local scores are also estimated in the range of 0 to 1, where one is the good score (fig. 6) . when compared to the set of non-redundant protein structures, the qmean z-score of the target protein as shown in fig. 7 was 0. the models located in the dark zone are shown in the graph to have scores less than 1 while the scores of other regions outside the dark zone can either be 1 < the z-score < 2 or z-score > 2. good models are often located in the dark zone. whenever such values are found, they result in some strains in the polypeptide chain and in cases of such, the stability of the structure will depend greatly on additional interactions but this conformation may be conserved in a protein family for its structural significance. another αand β-regions clustering principle exemption can be viewed on the right side plot of fig. 8 where the distribution of torsion angles for glycine are the only displayed angles on the ramachandran plot. glycine has no side chain, and this gives room for flexibility in the polypeptide chain hence making accessible the forbidden rotation angles. glycine for this reason is more concentrated in the regions making up the loop where sharp bends can occur in the polypeptide. for this reason, glycine is highly conserved in protein families as the presence of turns at specific positions is a characteristic feature of particular structural folds. the comparative physiochemical parameter computation of the template and target proteins by protparam were deduced from the amino acid sequences of the individual proteins. no additional information was required about the proteins under consideration and each of the complete sequences was analyzed. the amino acid sequence of the target protein has not been deposited in the swiss-prot database. for this reason, inputting the standard single-letter amino acid sequence for both proteins into the text field was employed in computing the physiochemical properties as shown in tables 3, 4 the two hiv protease inhibitors (lopinavir and ritonavir) when targeted at the modeled 2019-ncov catalytic site gave significant inhibition attributes; hence, the in silico study was planned through molecular docking analysis with autodock vina. the binding orientation of the drugs to the protein active site as viewed in the pymol molecular visualizer (fig. 11) showed an induced fit model binding conformation. the same compounds were targeted against the active site of the downloaded pdb 3d structure of the sars-cov main proteinase (pdb 6m2n) for comparison purposes fig. 12 . the active site residues as visualized in pymol are shown in fig. 13 . the binding of lopinavir to the target protein which produced the best binding score was used as the predictive model. residues at the distance of < 5 angstroms to the bound ligand were assumed to form fig. 13 the combined view of the 3d structural comparison between the modeled target protein and the downloaded pdb structure of the viral protein (left column) and their primary sequence alignment (right column). the target protein is colored in grey while its protein data bank equivalence is colored in red. the high structural similarity between the two proteins was validated through their sequence alignment which produced 99.34% sequence identity score. homology modeling which is a computational method for modeling the 3d structure of proteins and also fig. 8 depicted here are two ramachandran plots. the plot on the left hand side shows the general torsion angles for all the residues in the target protein while the plot on the right hand side is specific for the glycine residues of the protein fig. 9 the target protein secondary structures with bound lopinavir. at the top is the secondary structure visualization on pymol with regions making up the alpha helix, beta sheets, and loops shown in light blue, purple, and brown, respectively. below is the prediction by cfssp where the red, green, yellow, and blue lines depict regions of the helices, sheets, turns, and coils (loops), respectively. the predicted secondary structure composition shows a high degree of alpha helix and beta sheets, respectively, occupying 45 and 47% of the total residues with the percentage loop occupancy at 8% regarded as comparative modeling, constructs atomic models based on known structures or structures that have been determined experimentally and likewise share more than 40% sequence homology. the backing principle for this is that there is likely to be an existing three-dimensional structure similarity between two proteins with high similarity in their amino acid sequence. with one of the proteins having an already determined 3d structure, the structure of the unknown one can be copied with a high degree of confidence. there is a higher degree of accuracy for alpha carbon positions in homology modeling than the side chains and regions containing the loop which is mostly inaccurate. as regards the template selection, homologous proteins with determined structures are searched through the protein data bank (pdb) and templates must have alongside a minimum of 40% identity with the target sequence, the highest possible resolution with appropriate cofactors for selection consideration [29] . in this study, the target protein was modeled using the sars coronavirus main proteinase as template. this selection was based on the high resolution and its identity with the target protein which is as high as 96%. qualitative model energy analysis (qmean) is a composite scoring function that describes protein structures on the basis of major geometrical aspects. the scoring function of the qmean calculates the global quality of models on six structural descriptors linear combination basis, where four of the six descriptors are statistical potentials of mean force. the analysis of local geometry is carried out by potential of torsion angle over three consecutive amino acids. in predicting the structure of a protein structure, final models are often selected after the production of a considerable number of alternative models; hence, the prediction of the protein structure is anchored on a scoring function which identifies within a collection of alternative models the best structural model. two distance-dependent interaction potentials are used to assess long-range interactions based on c_β atoms and all atoms, respectively. the burial status of amino acid residues describes the solvation potential of the model while the two terms that reflect the correlation between solvent accessibility, calculated and predicted secondary structure are not excluded [38] . the resultant target protein can be considered a good model as the z-scores of interaction energy of c_β, pairwise energy of all atoms, solvation energy, and torsion angle energy are − 0.35, − 0.65, − 0.77, and 0.36, respectively, as shown in table 1 . the quality of a good model protein can be compared to high-resolution reference structures that are products of x-ray crystallography analysis through z-score where 0 is the average z-score value for a good model [26] . according to benkert et al., qmean z-score provides an estimate value of the degree of nativeness of the structural features that can be observed in a model, and this is an indication that the model is of a good quality in comparison to other experimental structures [26] . our study shows the z-score of the target is "0" as indicated in fig. 6 and such a score is an indication of a relatively good model as it possesses the average z-score for a perfect model. properties of the model that is predicted determines the molprobity scores. work initially done on all-atom contact analysis has shown that proteins possess exquisitely well-packed structures with favorable van der waals interactions which overlap between atoms that do not form hydrogen bonds [39] . unfavorable steric clashes are correlated strongly with the quality of data that are often poor where a near zero occurrence of such steric clashes occurs in the ordered regions of crystal structures with high resolution. therefore, low values of clash scores are indications of a very good model which likewise has been proven by the clash score value exhibited by the target protein that was modeled for the purpose of this study (table 2 ). in addition to the clash score, the protein conformation details are remarkably relaxed, such as staggered χ angles and even staggered methyl groups [40] . applied forces to a given local motif in environments predominantly made up of folded protein interior can produce a locally strained conformation but significant strain are kept near functionally needed minimum by evolution and this is on the presumption that the stability of proteins is too marginal for high tolerance. in traditional validation measures updates, there has been a compilation of rigorously quality-filtered crystal structures through homology, resolution, and the overall score validation at file level, by b-factor and sometimes at residue level, by all-atom steric clashes. the resulting multi-dimensional distributions generated after an adequate smoothing are used in scoring the "protein-like" nature of each local conformation in relation to known structures either for backbone ramachandran values or the side chain rotamers [41] . rotamer outliers are equivalent to < 1% at high resolution while general-case ramachandran outliers to a high-resolution equivalence of < 0.05%, and ramachandran favored to 98%. in this regard, the definition of the molprobity score (mpscore) was given as mpscore = 0.426 *ln(1+clashscore) + 0.33 *ln(1+max(0, rota_out|-1)) + 0.25 *ln(1+ max(0, rama_iffy|-2)) + 0.5 where the number of unfavorable all-atom steric overlaps ≥ 0.4 å per 1000 atoms defines the clashscore [38] . the rota_out is the side chain conformation percentage termed as the rotamer outliers, from side chains that can undergo possible evaluation while rama_iffy is the backbone ramachandran percentage conformations that allows beyond the favored region, from residues that can undergo possible evaluation. the derivatives of the coefficients are from a log-linear fit to crystallographic resolution on a set of pdb structures that has undergone filtration, so that the mpscore of a model is the resolution at which each of its scores will be the values expected thus, the lower mpscores are the indications of a better model. with a clash score of 2.06 and a 95.66% value for the ramachandran favored region as compared to the ramachandran outliers and rotamer outliers with individual values of 0.83% and 5.21% respectively, we arrived at a molprobity score of 1.82 which is low enough to indicate the quality of a good model in our experimental protein. the characteristic repetitive conformation attribute of amino acid residues is the basis for the repetitive nature of the secondary structures hence the repetitive scores of φ and ψ. the range of φ and ψ scores can be used in distinguishing the different secondary structural elements as the different φ and ψ values of each secondary structure elements map their respective regions on the ramachandran plot. peptides of the ramachandran plot have the average values of their α-helices clustered about the range of φ = − 57°and ψ = − 47°while the average values of 130°and ψ = + 140°describes the ramachandran plot clustering for twisted beta sheets [42] . the core region (green in fig. 8 ) on the plot has the most favorable combinations for the φ and ψ values and has the highest number of dots. the figure also shows in the upper right quadrant, a small third core region. this is known as the allowed region and can be found in the areas of the core regions or might not be associated with the core region. it has lesser data points compared to the core regions. the other areas on the plot are regarded as disallowed. since glycine has only one hydrogen atom as side chain, steric hindrance is not as likely to occur as φ and ψ are rotated through a series of values. the glycine residues having φ and ψ values of + 55°and − 116°, respectively [43] do not exhibit steric hindrance and for that reason positioned in the disallowed region of the ramachandran plot as shown in the right hand side plot in fig. 8 . the extinction coefficient is an indication of the intensity of absorbed light by a protein at specific wavelength. the importance of estimating this coefficient is to monitor a protein undergoing purification in a spectrophotometer. woody in his experiment [44] has shown the possibility of estimating a protein's molar extension coefficient from knowledge of its amino acid composition which has been presented in table 3 . the extinction coefficient of the proteins (both the template and the target proteins) was calculated using the equation: e(prot) = numb(tyr) × ext(tyr) + numb(trp) × ext(trp) + numb(cystine) × ext(cystine)the absorbance (optical density) was calculated using the following formula: for this equation to be valid, the following conditions must be met: ph 6.5, 6.0 m guanidium hydrochloride, 0.02 m phosphate buffer. the n-terminal residue identity of a protein is an important factor in the determination of its stability in vivo and also plays a major role in the proteolytic degradation process mediated by ubiquitin [45] . βgalactosidase proteins with different n-terminal amino acids were designed through site-directed mutagenesis, and the designed β-galactosidase proteins have different half-lives in vivo which is striking, ranging from over a hundred hours to less than 2 min, but this is dependent on the nature of the amino terminus residue on the experimental model (yeast in vivo; mammalian reticulocytes in vitro, e. coli in vivo). the order of individual amino acid residues is thus in respect to the conferred half-lives when located at the protein's amino terminus [46] . this is referred to as the "n-end rule" which was what the estimated half-life of both the template and target proteins were based on. the instability index provides an estimate of the protein's stability in a test tube. statistical analysis of 32 stable and 12 unstable proteins has shown [47] that there are specific dipeptides with significantly varied occurrence in the unstable proteins as compared with those in the stable proteins. the authors of this method have assigned a weight value of instability to each of the 400 different dipeptides (diwv). the computation of a protein's instability index is thus possible using these weight values, which is defined as: table 3 amino acid composition table for both template and target protein amino acid residues in one letter codes template 17 12 19 17 12 14 9 26 8 12 30 11 10 16 13 16 26 3 11 24 target 17 11 21 17 12 14 9 26 7 11 29 11 10 17 13 16 24 3 11 27 durojaye et al. where l is the sequence length and diwv(x[i]x[i + 1]) is the instability weight value for the dipeptide starting from position i. a protein that exhibits an instability index value less than 40 can be predicted as a stable protein while an instability index value that exceeds the 40 threshold is an indication that the protein may be unstable. the comparative instability index values for the template and target proteins were 29.67 and 27.65 (table 4) , respectively, showing both are stable proteins. the relative volume occupied by aliphatic side chains (valine, alanine, leucine, and isoleucine) of a protein is known as its aliphatic index. it may be an indicator of a positive factor for an increment in globular proteins thermostability. the aliphatic index of the experimental proteins was calculated according to the following formula [48] : where x(ala), x(val), x(ile), and x(leu) are the mole percent (100 × mole fraction) of alanine, valine, isoleucine, and leucine. the coefficients "a" and "b" are the relative volume of the valine side chain (a = 2.9) and of leu/ile side chains (b = 3.9) to the alanine side chain. the calculated aliphatic index for the experimental protein shows that the thermostability of the target protein is slightly higher than the template. the most common secondary structures are the alpha helices and beta sheets although the beta turns and omega loops also occur. elements of the secondary structures spontaneously form as an intermediate before their folding into the corresponding three-dimensional tertiary structures [49] . the stability and how robust the α-helices are to mutations in natural proteins have been shown in previous studies. they have also been shown to be more designable than the beta sheets; thus, designing a functional all-α helix protein is likely to be easier than designing proteins with both α helix and strands, and this has recently been confirmed experimentally [50] . the template and target proteins both have a total of 306 amino acid residues (table 4 ) with the composition of individual residues shown in table 3 . as shown in fig. 9 , the target protein which shares a structural homology with the template (fig. 3 and the animation video) is predominantly occupied by residues forming alpha helix and beta sheets, with very low percentage of the residues forming loops. the stability of these two proteins is revealed in their physiochemical characteristics which can therefore be linked to the high percentage of residues forming alpha helix. the ultimate goal of genome analysis is to understand the biology of organisms in both evolutionary and functional terms, and this involves the combination of different data from various sources [51] . for the purpose of this study, we compared our protein of interest to similar proteins in the ncbi database to predict the evolutionary relationships between homologous proteins represented in the genomes of each divergent species. this makes the amino acid sequence alignment the most suitable form of alignment for the phylogenetic tree construction. organisms with common ancestors were positioned in the same monophyletic group in the tree, and the same node where the protein of interest (the 2019-ncov main proteinase) is positioned also houses the non-structural polyprotein of the 1ab bat sars-like coronavirus. this shows that the two viral proteins share a common source with shorter divergence period. bootstrapping allows evolutionary predictions on the level of confidence. one hundred is a very high level of confidence in the positioning of the node in the topology. the lower scores are more likely to happen by chance than it is in the real tree topology [52] . the bootstrap value of the above-mentioned viral proteins which is exactly 100 is a very high level of statistical support for their positioning at the nodes in the branched part of the tree. the length of the branches is a representation of genetic distance. it is also the measure of the time since the viral proteins diverged, which means, the greater the branch length, the likelihood that it took a longer period of time since divergence from the most closely related protein [53] . the tw9 and tjf strains of the sars coronavirus orf1a polyprotein and replicase, respectively, are the most distantly related, based on their branch length and as such can be regarded as the out-group in the tree. structure-based drug discovery is the easiest molecular docking methodology as it screens variety of listed ligands (compounds) in chemical library by "docking" them against proteins of known structures which in this study is the modeled 3d structure of the 2019-ncov main proteinase and showing the binding affinity details alongside the binding conformation of ligands in the enzyme active site [54] . ligand docking can be specific, that is, focusing only on the predicted binding sites of the protein of interest or can be blind docking where the entire area of the protein is covered. most docking tool applications focus on the predicted primary binding site of ligands; however, in some cases, the information about the target protein binding site is missing. blind docking is known to be an unbiased molecular docking approach as it scans the protein structure in order to locate the ideal binding site of ligands [55] . the autodock-based blind docking approach was introduced in this study to search the entire surface of the target and template protein for binding sites while optimizing the conformation of peptides simultaneously. for this reason, it was necessary to set up our docking parameters to search the entire surface of the modeled main proteinase of the 2019-ncov. this was achieved using the autogrid to create a very large grid map (center 77 å × − 10 å × 15 å and size 30 å × 60 å × 35 å) with the maximum number of points in each dimension in order to cover the whole protein. we observed a partial overlap in the docking pose of lopinavir to the active site of both template and target protein as compared to the conspicuous difference observed in the binding orientation of ritonavir to the protein active sites. these differential poses can be viewed distinctively in the attached animation video. a keen view of the binding orientation of the two drug candidates to the 2019-ncov virus main proteinase active site (fig. 11 ) is also consistent with the proposed induced fit binding model. in a comparative docking study, the same drug candidates (lopinavir and ritonavir) were docked against the active site of the pdb downloaded version of the viral main proteinase. the docking grid for this purpose was set with precision as the solved pdb structure of the virus included a cocrystalized ligand at the enzyme active site (center -32 å × − 65 å × 42 å and size 25 å × 30 å × 25 å) and experimental ligands bind to this site with precision and variation in poses (fig. 12) . the binding energy results table 5 here, the docking results of lopinavir and ritonavir against the template and target protein are shown. the binding of ritonavir to the template protein produced the highest number of inter model hydrogen bonds while the binding of lopinavir to the target protein formed polar interaction with three residues at the active site as compared to the two formed by the other interactions table 6 the amino acid residues involved in polar interaction, the number of inter-model hydrogen bonds and the docking score of lopinavir and ritonavir upon binding to the 3d pdb download of the sars-cov main proteinase (pdb 6m2n) showed a difference of − 0.3 kcal/mol upon the binding of lopinavir to the template and the pdb 3d structure of the enzyme (pdb 6m2n), and a difference of − 0.5 kcal/ mol between the pdb 3d structure of the enzyme and the target protein (table 5 and 6). the same comparative study was repeated for the binding of ritonavir and a difference of − 0.1 and − 1.0 kcal/mol was observed upon the binding of drug to the template and target proteins, respectively, in comparison with the binding to the downloaded 3d structure of the enzyme from the pdb. the observed consistency in the binding energy of the drug candidates can also serve as a reference to the validity and quality of the modeled protein, which has exhibited a high sequence and structural similarity with the downloaded 3d structure from the protein data bank (fig. 13 ). in an effort to make available potent therapeutic agents against the fast rising 2019 novel coronavirus epidemic, we identified from the viral genome the coding region and modeled the main proteinase of the virus coupled with the evaluation of the efficacy of existing hiv protease inhibitors by targeting the protein active site using a blind docking approach. our study has shown that lopinavir displays a broader spectrum inhibition against both the sars coronavirus and 2019-ncov main proteinase as compared to the inhibition profile of ritonavir. the modeled 3d structure of the enzyme has also provided interesting insights regarding the binding orientation of the experimental drugs and possible interactions at the protein active site. the conclusion from the study of cao et al. as previously discussed however has shown that the administration of the lopinavir-ritonavir therapy might elicit additional health concerns as a result of the extreme adverse events exhibited by the experimental subjects for the purpose of their study. it was also observed that the drugs showed no increased benefit when compared with the standard supportive care. in view of this findings, we therefore suggest a drug modification approach aimed at avoiding the health concerns posed by the lopinavir-ritonavir combined therapy while retaining their proteinase inhibitory activity. supplementary information accompanies this paper at https://doi.org/10. 1186/s43042-020-00081-5. additional file 1. supplementary information to this article can be found online at https://www.rcsb.org/structure/6m2n clinical features of patients with 2019 novel coronavirus in wuhan genomic characterization and epidemiology of 2019 novel coronavirus: implications of virus origins and receptor binding a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster importation and human-tohuman transmission of a novel coronavirus in vietnam national health commission of the people's republic of china transmission of 2019-ncov infection from an asymptomatic contact in germany alert, verification and public health management of sars in the post-outbreak period coronavirus in severe acute respiratory syndrome (sars) a novel coronavirus and sars crystal structures of the main peptidase from the sars coronavirus inhibited by a substrate-like aza-peptide epoxide dissection study on the sars 3c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly-specific protease inhibitors 3c-like proteinase from sars coronavirus catalyzes substrate hydrolysis by a general base mechanism only one protomer is active in the dimer of sars 3c-like proteinase biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 emboss: the european molecular biology open software suite srs, an indexing and retrieval tool for flat file data libraries issues in bioinformatics benchmarking: the case study of multiple sequence alignment hhblits: lightning-fast iterative protein sequence searching by hmm-hmm alignment the swiss-prot protein knowledgebase and its supplement trembl in 2003 toward the estimation of the absolute quality of individual protein structure models molprobity: more and better reference data for improved all-atom structure validation chapter 2: protein composition and structure modeling protein quaternary structure of homo-and hetero-oligomers beyond binary interactions by homology ucsf chimera-a visualization system for exploratory research and analysis fasman gd (1974) prediction of protein conformation protein identification and analysis tools on the expasy server the rapid generation of mutation data matrices from protein sequences mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets chemoinformatics: theory, practice, & products critical assessment of the automated autodock as a new docking tool for virtual screening critical assessment of methods of protein structure prediction (casp) round 6 visualizing and quantifying molecular goodnessof-fit: small-probe contact dots with explicit hydrogen atoms a test of enhancing model accuracy in high-throughput crystallography the penultimate rotamer library protein geometry database: a flexible engine to explore backbone conformations and their relationships to covalent geometry circular dichroism spectrum of peptides in the poly(pro)ii conformation calculation of protein extinction coefficients from amino acid sequence data universality and structure of the n-end rule the n-end rule in bacteria correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence thermostability and aliphatic index of globular proteins alpha helices are more robust to mutations than beta strands global analysis of protein folding using massively parallel design, synthesis, and testing time of the deepest root for polymorphism in human mitochondrial dna intraspecific nucleotide sequence differences in the major noncoding region of human mitochondrial dna limitation of the evolutionary parsimony method of phylogenetic analysis efficient docking of peptides to proteins without prior knowledge of the binding site molecular recognition and docking algorithms we appreciate the leadership of the laboratory of cellular dynamics (lcd), university of science and technology of china, for the all-around support and academic advisory role. we also acknowledge the strong support from the ustc office of international cooperation all through the challenging period of the coronavirus epidemic. the authors received no funding for this project from any organization. ethics approval and consent to participate not applicable the authors declare that they have no competing interests. key: cord-016442-3su3x6ed authors: aiking, harry; de boer, joop; vereijken, johan m. title: transition feasibility and implications for stakeholders date: 2006 journal: sustainable protein production and consumption: pigs or peas? doi: 10.1007/1-4020-4842-4_7 sha: doc_id: 16442 cord_uid: 3su3x6ed nan role of citizens who support more stringent environmental standards might be greater than their economic role as consumers when they make trade-offs between the prices of pork and npfs. third, the ecological approach argued that for developing a coherent set of indicators no framework turned out to be available, which (1) links pressures to environmental consequences, and (2) focuses on how each of the indicators can contribute to the problem solving logic required to answer questions of environmental sustainability. rather, the existing frameworks take a one-dimensional perspective. therefore, causal networks were recommended, describing how individual indicators are interrelated, thus providing a more complete, holistic picture of what is happening in the environment. in short, process knowledge provides insight into cause-andeffect networks and is put forward to guide the selection of appropriate indicator sets. together, irrespective of the approach, a clear consensus was provided (1) that npfs are environmentally more sustainable than pork and (2) that the most important impacts are due to tampering with (a) land use and the cycling of (b) water, (c) carbon and (d) nitrogen, leading to biodiversity loss, climate change, eutrophication and acidification (chapter 2). given the convergent results concerning the relative advantages of npfs, the question emerges whether this information will be enough for policymakers who want to choose between alternative protein chains. as we shall see in greater detail later, policymakers should try to avoid suboptimal solutions and that makes it important for them to start with a comparative analysis of a large number of options. presently, the environmental assessment tool is limited to the agricultural production phase. after extension to the rest of the production and consumption chain the method should be generalised beyond comparing just protein food options. in view of the increasing relevance of linkages between transitions, it should be extended sufficiently to become a tool to assess the environmental sustainability of transitions in general. because a large part of the environmental impact of the pork chain takes place outside western europe, it is important to also look at the global dimension. reducing this environmental impact in developing countries of asia and south america by shifting to a more plant protein centred consumption pattern in western europe may have important economic repercussions in those developing countries. in turn, this may or may not cause negative environmental impacts there, depending on what alternative livelihood strategies will be developed to compensate reduced feed crop exports to western europe. so north-south issues clearly require further study. six projects aimed at assessing aspects of the technological feasibility of pea-derived npfs were performed in profetas. two of them were directed at aspects concerning primary production of peas and two at aspects concerning processing of peas to npfs. in addition, in one project a tool is being developed to optimise the npf chain. last but not least, in one project the options for the non-protein fractions were studied. in the breeding project it was shown that between different varieties there is a large variation in protein composition. this variability can be exploited in classical breeding programmes to obtain pea varieties that are optimised towards npf production. furthermore, a tool has been developed for genetic modification of pea and other legumes. its application will enable selective removal of certain storage proteins (which affect texture formation) and of certain enzymes (which affect flavour compounds) in order to improve the quality of the raw material. as an alternative, genetic modification may be used in r&d for screening the effects of alterations in protein and flavour composition. such an approach will speed up classical breeding while field crops are kept gm free. in the cultivation project, a crop growth model has been developed that predicts yield of peas and pea protein as a function of genotype and environmental variables (such as solar radiation, temperature and rainfall). the model is generic; it can be extended to any arable crop. the model can be used to optimise production (e.g. with respect to resource use efficiency such as water) or to define the characteristics a variety should have for a given environment. an important insight resulting from this project is that using varieties with a high protein content cannot increase the protein yield per hectare. to achieve the latter, other traits (e.g. plant architecture to sustain leaf area duration during the grain filling period) should also be changed. furthermore, attention was drawn to agronomic aspects, such as resistance to lodging and soil-borne diseases, which indicate that peas may not be the best choice to function as starting material for npf production. the texture formation studies yielded new insights into a process that is of prime importance in texture formation, i.e. heat-induced gelling behaviour of pea proteins. this behaviour was shown to be affected by the protein 7.3.1 composition and by the rate of cooling during processing. this implies that a range of textures can be produced (a) by varying the protein composition by selecting appropriate pea varieties and (b) by varying the processing parameters. this provides basic tools to food technologists to direct the texture of pea-based npfs towards consumer demands. comparative studies using soy and pea proteins led to the insight that despite similarities between proteins on the molecular level, their gelling behaviour may be different. on the one hand, this could mean that a large range of textures can be made using a limited set of proteins. on the other hand, this observation likely sets limits to the interchangeability of proteins. protein-flavour interactions studies using model volatile flavour compounds provided information on the amounts of flavour compounds associated with the pea proteins under various conditions. they also showed that the interactions with flavour compounds differ per type of pea protein. so again, varying protein composition or process parameters offers possibilities to tune the flavour of npfs. furthermore, it was shown that saponins, not only present in peas but also in numerous other legumes (including soy) are responsible for a bitter taste. this bitterness may be reduced by heating, thus providing a technological tool to adjust taste. it was also found that in pea protein preparations "off-flavours" seem to be present that are derived from fat oxidation. similar "off-flavours" have been found in other plant protein preparations too; very well known examples are soy protein preparations. the project on chain design will not be completed before 2006. tools developed in this project are expected to be useful in the design of food chains to optimise these chains either towards a single goal (such as product quality, costs, or environmental load), or even towards multiple goals. preliminary results are that the cost price of npfs could be less than or at least comparable to that of pork. comparison of the environmental load by using exergy analysis showed that an npf chain is more efficient than a pork chain only if the non-protein fractions of pea seeds (mainly starch) are put to use. options for the non-protein fractions were investigated in a project added later to study the feasibility of plant-derived npfs from a different perspective. a tool was developed to assess different protein crops on the potential uses of the non-protein fractions. only bulk fractions were distinguished and evaluated on their suitability to be used as food, feed, stock and biofuel. the conclusion was drawn that -at present -oil crops may be at an advantage, because the oil fractions have better application perspectives than other non-protein fractions. interestingly, a transition from intensive meat production to npf production from any crop would lead to a shortage of soy oil for food applications, rather than to a surplus of this non-protein fraction. if it is assumed that all intensive livestock farming disappears and only grass-fed and other extensive meat production would remain, then this shift -irrespective of the crop (pea or soy) -would release an enormous area (over 300 million ha) of highly productive land for other uses. for example, if used to grow biomass, this would be sufficient to cover approximately 25% of the current world energy demand. the research on technological feasibility has focussed primarily on basic problems and on issues that needed to be solved before products could be designed. with respect to processing some important insights and tools are still missing. for instance, neither have texturisation processes been studied, nor have products (or product concepts) actually been made. such research is indispensable to allow a consumer-oriented development of npfs. at present it is still unknown whether more traditional texturing processes such as extrusion can deliver the range of textures desired by consumers. it may well be that novel techniques have to be deployed and further developed, such as techniques based on phase separation. furthermore, in actual npfs other components (e.g. hydrocolloids, fats) will be present. no information is available about the effects of such components on the texture and flavour of the npfs. with respect to flavour, stakeholders from industry have indicated that starting materials for npf production should preferably be free from "off-flavours". the prevention of formation of such compounds or ways to remove them should be investigated. a lot of knowledge on this subject is already available from research on soy proteins. missing insights with respect to the use of the newly developed protocol to genetically modify peas include, among others, its effectiveness and efficiency as well as its effects on cultivation. these effects not only include those relevant for npf processing (e.g. effects of protein composition on texture and flavour) but also those relevant for primary production. questions have to be answered regarding the effects of the modification on for example (a) germination power of the seeds, (b) viability of the modified peas in the field and (c) an eventual yield penalty for total protein content because of the modification. furthermore, the applicability of this new protocol to modify crops other than peas needs to be further explored. to exploit the interesting possibilities offered by the crop growth model, it has to be validated and to be extended to crops other than peas. then it can be a powerful tool in identifying differences among various crops in resource use efficiency for e.g. biomass and protein production, hence in contributing to a sustainable primary production of raw materials for npfs. in summary, quite a lot of research is still required to actually develop and produce npfs that meet consumer preferences. in addition to the technological issues discussed above, the project on chain design will most likely result not just in answering questions but also in raising others. furthermore, options for the non-protein fractions should be detailed by means of scenario studies, with particular attention for the projected shortage of soy oil for food applications. finally, the issue of the crops to be used for npf production is still open. as argued in section 6.2, even in europe peas may not be the preferred crop for npf production. more research concerning crop choice is required. in addition to sustainability -which is an evident societal preferencesocietal desirability was studied in profetas with respect to (1) the behaviour and preferences of consumers and (2) the food-related political and economic developments of the next decades. the underlying notion of this work was that the desirability of a diet shift is in proportion to its fitting in with the behavioural patterns of current and future generations. in addition, it was argued that a lack of fit would create less serious problems if mitigating measures can be taken. based on a long-term view on behaviour, the potential for a diet shift in relation to socio-cultural changes was examined. a newly developed analytical framework sorts insights on influences on behaviour into a logical order. its cascade-like structure embodies the view that a long-term development will create opportunities for food choices that match its general direction, whereas it will put constraints on others. the analysis of long-term changes indicated that there is a favourable socio-cultural context for decisions that make consumers and producers less dependent on meat proteins. one of the most salient results of this work, however, is the contrast between, on the one hand, a series of impressive changes in dietary choices over the last few centuries and particularly over the last few decades and, on the other hand, the observation that an individual will not easily change his or her food preferences from one day to the next. this contrast underlines the value of the analytical framework in combination with smallscale consumer research. by using currently available meat substitutes as a model to analyse consumer behaviour and food choices, it was possible to develop several 7.4 social desirability tools that may guide npf product development. it appears that the currently available meat substitutes will not become popular without additional measures. analysis of consumers and consumption behaviour with respect to meat and meat substitutes provided insights such as (a) non-vegetarian consumers of meat substitutes are not impressed by environmental arguments, (b) only a small group of consumers is open to completely new products due to so-called "neophobia", (c) consumers would like more information on usage and preparation of meat substitutes and on ingredients used. consumer sensory preferences were analysed and their translation into product characteristics was attempted, yielding insights such as (a) attention should be paid to satiating properties of npfs, which are related to protein content, (b) most people want soft, smooth or crispy meat substitutes to have a seasoned, meat-like flavour and a brown colour, (c) meat substitutes should have the same place in the dish as meat. other than originally anticipated, the latter results clearly show that people want npfs to have meat-like characteristics. they confirm the notion that people will habitually look for what is familiar when they are trying to make sense of something, such as an invitation to try a product. the retrospective character of sense making can explain why non-vegetarian consumers keep relying on distinctions drawn in the past and why they evaluate meat substitutes by using meat-based criteria. the selection environment in which npfs have to survive is partly dependent on their positioning in the market, for example as cheap (bulk) proteins or as quality products (specialties). the prospects of the new products may also depend on linkages with other issues, such as increasing meat prices or health promotion. these prospects are not only dependent on consumer responses in a potential usage situation, but also on processes at the level of markets and public institutions. given the fact that technology development takes place in a changing and malleable world, the stakeholders of a new technology may opt to monitor and influence its selection environment. profetas applied some advanced tools such as policy analysis and econometric modelling, as well as a skilled examination of the rules of international institutions with regard to novel foods. in a political analysis it was investigated how politics and public policy affect the possibilities of a diet shift. as expected, it appeared that governmental policy does not have many direct influences on food choices (i.e. the proportion of meat vs. plant proteins in the nation's diet), but mostly indirect influences. these influences demonstrate that production and consumption are, at least partially, facilitated by a political "infrastructure", implicitly favouring certain products or production processes over others. to uncover indirect influences, it was analysed how current food practices have developed in the netherlands since 1850 (i.e. when the physician g.j. mulder brought up the issue of proteins and the societal effects of the lack of proteins in most people's diets). given the current sustainability-related issues, the political infrastructure still seems to favour the option of more protein production and consumption, although that option is not actively promoted by governmental action. from a sustainability perspective, however, it may be desirable to have a political infrastructure that favours a more divergent food system with different approaches to food. this would mean, for example, that the option of producing more plant protein does not necessarily get more emphasis than the option of simply "eating less meat". two complementary econometric analyses studied agricultural production and consumption and the patterns exhibited (by adaptations to the existing gemat model), and institutional arrangements affecting agricultural production and trade (adapting the gtap model), respectively. it was shown that a protein transition results in a decrease of environmental pressure on land under all scenarios (gtap) and that the potential is even larger (gemat). though crucially important to the feasibility of a transition, interestingly, the two models employed disagree on future meat price development. from gtap a price decrease may be inferred, but from gemat a price increase. the underlying cause of this disagreement is the implicit assumption, deeply embedded in the models, to what extent agricultural efficiency will continue to increase in the future. these results show the added value of employing two complementary models. a study of institutional aspects provided the insight that even though international institutions (such as the eu and the wto) may provide both incentives and barriers for the introduction, marketing and promotion of npfs, on balance the barriers exceed the incentives. since these barriers (primarily intended to resist protectionist practices in international trade) cannot be circumvented, a successful introduction and marketing of npfs should be taking them into proper account. in short, it is easier to start from already authorised foods and ingredients than to start from foods and ingredients that still need to be authorised, especially in europe if they are gm crop derived. for the promotion of npfs not too much should be expected from traditional government instruments such as taxes (on meat) and subsidies (on npfs). subsidies have already lost their appeal in most eu countries for purely domestic reasons, plus they are heavily restricted by eu regulations concerning state aid and the single market. if npfs are to become a success, it should primarily be through private, commercial means and action. international institutions can protect and support commercial interests, however, through the international protection of intellectual property rights. each of the tools has provided relevant information about opportunities and barriers for npfs, but, under the present conditions of uncertainty, none could lead to conclusive answers. for example, one of the drawbacks of consumer research is that the currently available meat substitutes are, in fact, sold in a niche market and that they are almost twice as expensive as the cheapest meats. in order to improve the relevance of consumer research (sensory research, in particular), actual pea-derived npf products -in the form of cheap protein products or as quality products -are indispensable. since such products are not available yet, they should be crafted for this purpose. these products could also be used in assessing the relation between sensory properties of npfs and their physical characteristics, a very difficult field of research, but of great importance for consumer-oriented product development. evidently, it is important for product developers to realize that "the average consumer" does not exist. subsequently, it is important to realize that different npfs will have to be developed to fulfil the needs of different consumer groups. all of the tools may seriously be influenced by the use of pre-conceptions in thinking about the future. the role of pre-conceptions is particularly great when questions have to be answered about the social desirability of policy options. it was shown that pre-conceptions lead to hidden assumptions that blur the arguments for or against an option. for instance, one of the preconceptions defined a diet shift primarily as an opportunity to develop products with a larger profit margin than meat. in contrast, it may be more sensible to develop plant protein ingredients that can serve as a low price alternative to meat protein ingredients. another example is the implicit assumption mentioned above that meat prices will continue to decrease in the future, in consequence of continuing agricultural efficiency. probably, policymakers may show wisdom by expecting that the meat prices will not decrease but increase as a result of changes in the world market and agroecological constraints on production. in fact, it seems inevitable that the present growth of spending power in china will put the world market prices of meat and feed under pressure. in addition, it should be emphasized that not too much should be expected of traditional government instruments such as taxes and subsidies. although the latter are effective tools, their application is fraught with political difficulties. although the various tools to analyse the social desirability of a diet shift have not identified strong arguments against it, a critical reflection on the results may lead to the conclusion that the corresponding profetas hypothesis deserves some more stringent tests than the arguments that have been described. in addition to the limitations mentioned above, it should be 7.4.2 noted that the present analyses might have overstated the importance of agriculture to the political and economic processes in modern society. other linkages, in particular to non-food issues, may have been overlooked or underestimated. it is clear that health issues -which have not been directly studied in profetas -require further attention. for example, it has emerged recently that intensive production of poultry and pigs in close proximity with people may play an important role in the adaptation of originally poultry-specific viruses via pigs to human beings as suitable hosts (pilcher, 2004; chen et al., 2004) . under such conditions -primarily extant in south east asia -new viruses are frequently spawned. it seems more and more likely that recent incidents such as with sars and avian influenza are correlated with the intensive meat production there, and that the frequency of such incidents will continue to rise in parallel in the future. apart from animal welfare issues, this health aspect, in itself, seems a good reason to reduce intensive meat production in general, and that of poultry and pigs in particular, at least under conditions such as in south east asia, which is globally an important producing and exporting area. from profetas the conclusion emerges that there are several sound reasons that support the triple hypothesis. that is, a shift in the western diet from meat proteins to plant-derived protein products appears to be (1) environmentally more sustainable than present trends, (2) technologically feasible, and (3) socially desirable. interestingly, the citizen generally seems to consider sustainability to be socially desirable, but the consumer does not like the taste of present meat substitutes. nevertheless, the main evidence is in support of a transition to make food production and consumption more sustainable. given the aims of profetas, the programme has not included specialized transitions research. nevertheless, many insights have been generated on the protein transition and on its linkages with the energy and freshwater transitions. these insights are described below. first, it has become clear that the protein transition is a necessity at the global level. without it, food production and sustainability are on a collision course. from time to time, there will be signals, alarming reports or dramatic events, which may contribute to the opening of a policy window. if the window opens, however, the market system will not simply guarantee that 7.5 transition feasibility 7.5.1 the most sustainable alternative technology will win. market failures, but also government failures, may result in the selection of a suboptimal solution. second, study of the technological feasibility has revealed a number of options, but also some gaps in the required knowledge, such as the preferred crop for npf production. although many criteria for such a crop have been established (such as low fertiliser requirement, high protein yield, and preferably already part of the established food system), a more conclusive choice cannot be made yet, for criteria in other areas (such as concerning the non-protein fraction) have not yet fully materialised. presently, the top choice in europe may be rapeseed or pea yet, but in asia it will probably be soy. third is the insight that the protein, energy and freshwater transitions are inextricably intertwined and that all parts of the crop or seed -protein and non-protein -should be considered one combined chain. although the former, in particular, is a novel insight, both the former and the latter views fit in nicely with the present trend towards a biobased economy, aiming to derive food and feed, chemicals and other non-food materials, and energy from plant crops in the most efficient way. fourth, there is support from various actors, although this depends on linkages with other issues. feedback from dutch governmental actors and ngos revealed enthusiasm for a protein transition, however, they are not inclined to initiate one themselves. in contrast, they do feel committed towards an energy transition. for most western consumers, sustainability or the environment is not an incentive for food choice, however, health is. due to a number of meat crises and other food scares, health and animal welfare are valued as increasingly important issues by western consumers. in fact, sales of meat substitutes are increasing every year and they are being bought and eaten by non-vegetarian consumers. in conclusion, several trends in different areas (protein, biofuel, water saving), on different geographic levels (local to global; western to developing countries) and concerning different actors (consumer, government, industry, ngos) have been identified by profetas, whichtaken together -may lead towards a protein transition. a major step is raising the awareness that all these trends are linked up and cannot be seen in isolation. so a major achievement of profetas is the insight to propose bringing together all these different actors, all with their own agendas and multiform aims. this is, we believe, the true definition of a societal transition. for pragmatic reasons, in profetas many boundary conditions were assumed, such as the focus on the western consumer and the focus on peas. at this stage, it seems timely to start focusing the attention on how -and where -a protein transition may be achieved in a dynamic, multiform society. alternative options such as deriving proteins from algae should be investigated, as well as the feasibility of reducing the present protein overconsumption in western countries (the "eating less protein" option). more direct research on transitions seems in order. the latter requires better insight into the role of explicit as well as implicit pre-conceptions in thinking about the future. in this respect, the use of multi-method strategies appears to be indispensable. multi-method strategies may not only validate each other or specify complementary aspects of a complex phenomenon, but they may also show interesting divergences. profetas has shown several divergences that are extremely important for further research into transitions. some examples are: the divergence between pre-conceptions that see a diet shift primarily as an opportunity to develop products with a larger profit margin than meat and pre-conceptions about developing plant based products that can serve as a low price alternative to meat protein ingredients. the contrast in the studies of consumer behaviour between, on the one hand, a series of impressive changes in dietary choices over the last few centuries and decades and, on the other hand, the observation that an individual will not easily change his or her food preferences from one day to the next. the assumption that meat prices will continue to decrease in the future, in consequence of continuing agricultural efficiency, versus the assumption that meat prices will increase as a result of changes in the world market and agro-ecological constraints on production. in spite of historical trends, it seems inevitable that the strong increase in meat consumption in countries such as china will put the world market prices of meat and feed under pressure. further research may be necessary into historical transitions, as well as into future general trends in global society. with respect to the latter, developing a number of contrasting visions of a potential global future is often considered helpful. in order to arrive at a robust transition strategy, these visions can be confronted with desirable options for protein food development. such an approach should not be too general, for example, by focussing on aggregated parameters such as the growth rate of the world economy. more specific factors, such as global public health, should be an explicit part of the picture. so does taking geographic and cultural 7.5.2 inhomogeneities into account, which clearly requires international cooperation. for the purpose of dutch and european policy making, it can be argued that protein sustainability is a medium to long-term issue on a global scale. in the not too distant future, intensive meat production worldwide should be discontinued, or at least strongly reduced. in practice, the notion of an approaching collision between current food production and sustainability means that imminent disturbances of the protein production chain will let themselves be known to policymakers through all kinds of signals. obviously, some of the weak signals could be a gradual rise of meat and feed prices. stronger signals may include human and animal health incidents, such as with sars and avian influenza, which are probably correlated with intensive meat production. against the background of irregular signals that vary in strength, governmental policymakers may have to manage at least three emerging goals: the first goal is to detect and interpret the signals correctly and to attribute them without delay to the relevant disturbances of the protein production chain. the bse case has shown that a delay might have disastrous consequences on the controllability of the whole policy making process. the second goal is to minimize any negative effects that the disturbances may have on society. for instance, it may be wise to be prepared for a large-scale vaccination campaign. the third goal is to prevent the opportunity to take action getting lost as a result of counteracting processes that favour suboptimal solutions. that is, solutions that are suboptimal from the perspective of long-term sustainability objectives, not aimed at the root of the problem, such as temporary bans on certain types of meat. the prevention of suboptimal solutions may require that governmental policymakers take action to correct market failures, for example, where private actors do not take full responsibility for the societal consequences of their activities. however, suboptimal solutions may also result from government failures, such as subsidies given to the wrong group or at the wrong moment. moreover, the various linkages between the protein transition and other transitions on different levels of scale will seriously complicate any attempt to apply straightforward forms of strategic planning. the least a robust policy should entail is an open mind to problem solving. for governmental policymakers it would be wise to avoid fixation on one particular technological option. in contrast, this might involve a decision to actively stimulate a range of divergent potential solutions to be developed, for example, by supporting research in the pre-competitive stage of technology development. this may especially be necessary where private parties have difficulties in making sense of the linkages between transitions, i.e. protein, energy and water. this approach would entail: slighting the mental barriers between thinking about food, energy and water policy, or about natural resources in general. trying to identify links with specific other policies, such as with the fight against obesity, the aims for renewable energy (such as the eu biomass action plan) and the aims to promote organic farming. considering alternatives such as npfs as sustainable successors to animal products (such as pork) with regard to export of products and know-how and considering incentives to r&d in that area. in combination with an open-minded approach, it is extremely important that governmental policymakers pay attention to the selection environment in which newly developed npfs have to survive. governmental policymakers can do a lot to prevent suboptimal solutions by monitoring and influencing the selection environment npfs are facing. some points in need of attention are the following: initiating removal of national, eu and international (wto) institutional barriers to the introduction of npfs on the market. increasing the transparency of the food chain in order to enable citizens and consumers to make sound choices. continuing and internationally promoting "green" thinking by acknowledging the role of plants for improving sustainability. in fact, the presently emerging trend towards a "biobased economy" is a first step toward the latter. in addition to deriving materials and energy from plant crops in combination, by the same token, food can be added to the list because crops are the ultimate renewables, degradable and all, and particularly sustainable if little fertiliser is required (such as with nitrogenfixing protein crops). european (eu) policymakers are in much the same boat as their dutch counterparts, where they have to deal with global environmental changes. the goals for biofuels and organic farming are easily linked to the protein transition. in addition, recovering the presently lacking self-sufficiency in protein rich feed crops may be an incentive for eu policy towards striving for sustainability by means of a protein transition. this may be even more so in view of the expected increase in demands (and consequently, competition) for protein rich feed crops by industrialising countries such as china. an additional incentive may be provided by the potential savings on freshwater use by agriculture, given the rising need expressed by the world water forum. for industrial policymakers an important question will be whether or not they want to be among the first movers in the market of new protein products. a company's decision on this topic will be governed by strategic and political circumstances at the time the options are contemplated, such as the ripeness of an issue. these circumstances depend on its own capabilities, its position in the industry, the economic situation of this industry and the industry's public image. the ripeness of an issue will in particular be influenced by the technological state-of-the-art (including innovative power), by the durability of the issue (a trend or a hype), by public opinions (including campaigns that emphasize the "ills" of an industry), and by linkages that improve the market success of a particular product segment. in view of the many potential linkages between a protein transition and other transitions, it should be emphasized that developing alternatives to an established technology may require many innovations and that these will not occur automatically as the outcome of a linear process. the currently predominating policy of multinational companies seems to be rather riskaversive by doing r&d themselves only on a moderate scale and, when deemed necessary, strategically buying emerging small companies with innovative products. the progress of small companies may be highly dependent on the relational context in which an innovation is located, such as the other innovations on which it builds, the status of the actors that own competing innovations, and the underlying community of experts involved in its elaboration. in addition, many chance factors may affect the content and the timing of innovation decisions. accordingly, the prospects of npfs may depend on many initiatives. as a start, they may entail: implementing "green" thinking in policies towards stepwise innovation and stepwise improving sustainability. slighting the mental barriers between thinking about raw materials, energy and water. integral thinking is a cornerstone of the emerging "biobased economy". considering plant-derived alternatives as sustainable successors to animal products with regard to export of products and of know-how, and increasing r&d in that area. this way of thinking coincides very well with parts of the mission statements of many companies: to create innovative products that promote a sustainable future. next to contributing to a sustainable future, npfs could also meet another important issue for industry: making profit. preliminary profetas calculations indicate that cost price of npfs could be lower or at least comparable to that of pork. furthermore, meat prices are expected to increase. this increase will be higher than the increase in the cost of plant proteins because of the low conversion factor of plant protein to meat protein. hence the gap between the costs of npfs and meat will become bigger, thereby increasing the profit margin. furthermore, npfs are directed towards a growing market. among others, the increase will evidently depend on the extent to which the products meet consumer demands and preferences, not only with respect to sensory properties but also to other issues such as health and animal welfare. this will be discussed in section 7.8. those industrial policymakers who want to be among the first movers on the npf market may start thinking about questions such as: which markets are the most interesting options, specialty or bulk, which products and how to position the products on the market? what are the preferences of the different consumer groups and which ones can be translated to product characteristics? what techniques are required to produce the desired products? which proteins or, even more basic, which crops seem feasible where? what chains should be developed and optimised, and with whom? this sounds partly like revisiting technological projects of profetas and indeed it is, but now on the level of actual and competitive products, rather than on the level profetas was directed at, i.e. the development of a toolkit on a basic, pre-competitive level. however, to answer the questions asked above in a science-based way and to provide a sound base for future npf development, further pre-competitive research on technological issues is still required. a number of subjects for such research have been discussed for example in section 7.3. in addition to technological issues, questions should be addressed such as: cooperation with what other parties is useful (e.g. for optimal use of the crop)? how may a protein transition spread across the world (e.g. which countries are best suited for introducing and testing npfs)? what may be the side effects of a protein transition (e.g. with respect to the north-south divide)? what trends in other areas (transport, technologies, lifestyles) may be relevant? for smaller companies it may pay to closely watch the emerging trend towards a "biobased economy" and start thinking about strategic alliances. as long as innovation is not "directed" by larger companies (cf. transition "management" as proposed by the dutch government), niches for innovative products will open, be it plant-derived protein foods or products made from the non-protein fraction of crops, products for saving water and/or energy, or others. the consequences of a protein transition will affect many stakeholders, such as consumers, retailers, farmers and ngos. without addressing each of these groups individually, the present section intends to sketch the most pertinent implications. what consumers should ideally do in the context of a protein transition may boil down to (a) eating one third less protein (the average dutch over-consumption), (b) replacing one third by plant-derived proteins, and (c) replacing the remaining third by extensively produced meat (such as most beef and lamb). although in the netherlands intensively produced pork converts much food industry waste in a sustainable way, globally this is not a representative example. theoretically, the proposed threefold option has both environmental and health benefits. in practice, few consumers will be convinced immediately, but they may do so in the long run. at present, the environmental benefits may not appeal to many consumers. the well-known activist storyline of "global nature" under threat and in need of protection from a global community has become too simplistic. modern westerners do not tend to think in terms of one big environment that is the same for everyone. they want credible solutions that give them the feeling that they are "doing the right thing." in contrast, the fact that many people are no longer aware of the animal origin of meat indicates that there is an increasing indifference toward the origins of proteins. at first sight, this seems to open possibilities for npfs. some producers might be tempted to change the protein chain in a way that does not have to be noticed by the people who are eating it, which may create a substantial shift from meat to plant protein foods without much consumer involvement. on second thought, however, a low-involvement approach may not be the optimal strategy to pursue more sustainable food choices. if people are no longer aware of meat's animal origin, they will also be less inclined to 211 transition feasibility and implications for stakeholders 7.8 pay attention to animal welfare. this may have negative consequences for attempts to stimulate sustainable agriculture by promoting high quality meat from well-treated animals or by simply eating less meat. an additional reason to not opt for a low-involvement approach refers to the societal value conflicts that are expected in many technology-related areas. in europe, the recent example of genetically modified food has shown that a lowinvolvement approach may backfire if people get the impression that they are part of a "hidden" transition. therefore, it is essential that all the people concerned are mindful of any transitions of food production methods. one of the ways to involve people is a discussion on personal health aspects, which have many links to the protein transition. in contrast to plantderived diets, meaty diets generally contain more saturated fats -associated with heart and coronary disease -and are sometimes associated with overconsumption of calories -leading to obesity. npfs may not only exert a beneficial effect on health indirectly, via these relationships, but maybe also directly. that plant proteins may provide such an effect is indicated, for example, by the claim approved by the american food and drug administration: "diets low in saturated fat and cholesterol that include 25 grams of soy protein a day may reduce risk of heart disease" (fda claim 21 cfr 101.82, october 1999). proteins from crops other than soy seem to exert the same protective effect, according to the fda, though this protective effect does not go unchallenged. last but not least, npfs are complex foods for which the amount of calories can be set via the choice of ingredients, which may be an important tool in view of the increase in obesity. admittedly, certain plant-derived foods may generate more allergies than meat, but this affects a minority of people compared to the stifling incidence of heart and coronary disease, let alone the downright epidemic incidence of obesity (800 million people afflicted worldwide). public health aspects, such as food safety, add to personal health aspects perceived by consumers. due to recent meat crises, both with chemical contaminants (such as dioxins, antibiotics, growth hormones) and pathogenic microbes (such as foot-and-mouth disease, avian influenza) european consumers are rather keen on food safety. other aspects -such as the proposed relationship between intensive pork and poultry production in south east asia and the increasing outbreaks of avian influenza -may not be topics consumers think about when they are shopping for food, but reminders of these issues may gradually induce "green" thinking by acknowledging the role of plants for improving their quality of life. the preceding chapters and sections have once again made it crystalclear how large the environmental burden of meat production is. conservatively estimated, it requires a 3-10 fold larger agricultural area and energy input and produces 3-10 fold more eutrophication than the production of plant protein. not only does meat production bring about over 60 fold more acidification, but it also appropriates 30-40 fold more of the dwindling freshwater resources. in addition, it produces a lot more pollution by pesticides, heavy metals and antibiotics. as an entirely novel finding, profetas has clearly demonstrated that the protein transition is coupled inseparably to two other societal transitions, namely those towards sustainable energy production and towards sustainable water use (section 6.3). the freshwater link is evident from the resource difference indicated above. the energy production link primarily regards the release of agricultural area, which is freed for biomass production. furthermore, the considerable proportion (60-80%) of non-protein biomass released as a byproduct during npf production may be utilised very efficiently for energy production. an evident case of win-win-win, with a triple environmental gain due to combined savings on protein, energy and water (section 6.3). concerning technological feasibility many questions have been answered, both in primary production, processing and chain development. generic as well as dedicated tools have been developed or are under development. among the generic tools is the crop growth model (which still has to be validated) and the model for chain design (which is still under development). applied knowledge concerning flavour-binding properties of pea proteins should be rated among the specialised tools. as might be expected, in addition to answers, many new questions have been raised as well (see chapter 3). these concern, among others, the way in which process and ingredient selection may fulfil consumer wishes for npfs within certain target groups (sections 3.2, 3.3 and 6.4). this puts forward three major challenges: how to obtain a wide range of textures by using plant proteins, how these textures are affected by ingredients other than proteins and how to obtain the desired flavour? next to questions regarding processing, also questions with respect to primary production remain to be answered. these questions concern among others the extent to which protein composition may be manipulated without affecting the viability of the plant in the field, and which crops are the most suitable to yield raw materials with the desired specifications (section 6.2). another major issue is the extent to which new 213 transition feasibility and implications for stakeholders 7.9 conclusions developments in molecular biology and breeding may affect the suitability of crops for npf production. from the sustainability perspective, the societal desirability has been established beyond doubt, particularly concerning resource and pollution aspects (land, water and energy uses, and their implications for biodiversity loss and climate change). furthermore, increased availability of plant protein based foods will undoubtedly make an important contribution to food protein security, which is presently under increasing pressure from world-wide increasing meat consumption. doubtlessly, such a transition will have a significant impact on north-south relationships and the poverty issue in the world (section 6.5). concerning societal desirability it has also become apparent that different actors can hold different interests and opinions here (section 6.4). it has been clearly established that the consumer is the player who holds the key to a short-term protein transition and that western consumers currently rate health above sustainability. for consumer-oriented product development, much more interaction is necessary between product developers and various user groups, such as consumers leaning towards health, convenience or culinary traditions. although profetas was originally designed from the western perspective, it has become clear that in developing countries the incentives to achieve a protein transition are not just different (section 6.6), but generally much stronger (section 6.5). in china, for example, meat production generates pressure by its inability to meet the national demand on top of the pressure generated by severe local pollution. crops tailored to climatic (section 6.2) and cultural (section 6.6) characteristics are available. it can be concluded that, although the developed countries are primarily responsible for the unbalanced meat consumption referred to earlier, it is primarily the developing countries that are confronted with the effects (see section 2.5). the latter, therefore, may experience stronger and more direct incentives to strive for a protein transition. however, the required technological expertise may be available in developed countries mainly. at any rate, if mineral oil and meat prices continue to rise, particularly in developing countries there will be opportunities for the onset of a combined protein plus biomass transition. since the majority of the developing countries are particularly short of freshwater resources, the additional implicit water conservation will be considered an important bonus. in profetas, neither transitions, nor their feasibility have been studied directly. rather, insights and tools have been developed to facilitate a potential transition from meat protein to plant-derived protein products in the near future. these have been summarised in the present chapter. for the reasons outlined above we feel that a protein transition -reducing intensive livestock farming and cultivation of feed crops -is not just beneficial to the environment, but also more sustainable, most certainly socially desirable, and in the long run inevitable. it is yet unclear whether npfs will be able to replace meat, to what extent, on what scale internationally, and how rapidly. when the window of opportunity opens, however, it will be crucial to have the technology available for more sustainable alternatives. facilitating a transition does not seem easy. however, it is interesting to note that -while exclusively working on an approach to make protein production and consumption more sustainable -the profetas research community has yielded an integrated solution for various global problems far beyond its original scope. in addition to promising much more sustainable protein production -directly contributing to biodiversity and resource conservation and probably indirectly to animal welfare and human health -profetas simultaneously indicated realistic options to produce a significant amount of biomass for sustainable energy production, and to save an immense volume of freshwater. this suggests that integral thinking combined with an even further extension of the disciplines involved (including health aspects, in particular) may be a promising avenue to meet the foreseeable challenges of the next few decades. the evolution of h5n1 influenza viruses in ducks in southern china increasing virulence of bird flu threatens mammals key: cord-252304-lwiulri7 authors: fragnoud, romain; flamand, marie; reynier, frederic; buchy, philippe; duong, vasna; pachot, alexandre; paranhos-baccala, glaucia; bedin, frederic title: differential proteomic analysis of virus-enriched fractions obtained from plasma pools of patients with dengue fever or severe dengue date: 2015-11-14 journal: bmc infect dis doi: 10.1186/s12879-015-1271-7 sha: doc_id: 252304 cord_uid: lwiulri7 background: dengue is the most widespread mosquito-borne viral disease of public health concern. in some patients, endothelial cell and platelet dysfunction lead to life-threatening hemorrhagic dengue fever or dengue shock syndrome. prognostication of disease severity is urgently required to improve patient management. the pathogenesis of severe dengue has not been fully elucidated, and the role of host proteins associated with viral particles has received little exploration. methods: the proteomes of virion-enriched fractions purified from plasma pools of patients with dengue fever or severe dengue were compared. virions were purified by ultracentrifugation combined with a water-insoluble polyelectrolyte-based technique. following in-gel hydrolysis, peptides were analyzed by nano-liquid chromatography coupled to ion trap mass spectrometry and identified using data libraries. results: both dengue fever and severe dengue viral-enriched fractions contained identifiable viral envelope proteins and host cellular proteins. canonical pathway analysis revealed the identified host proteins are mainly involved in the coagulation cascade, complement pathway or acute phase response signaling pathway. some host proteins were overor under-represented in plasma from patients with severe dengue compared to patients with dengue fever. elisas were used to validate differential expression of a selection of identified host proteins in individual plasma samples of patients with dengue fever compared to patients with severe dengue. among 22 host proteins tested, two could differentiate between dengue fever and severe dengue in two independent cohorts (olfactomedin-4: area under the curve (auc), 0.958; and platelet factor-4: auc, 0.836). conclusion: a novel technique of virion-enrichment from plasma has allowed to identify two host proteins that have prognostic value for classifying patients with acute dengue who are more likely to develop a severe dengue. the impact of these host proteins on pathogenicity and disease outcome are discussed. electronic supplementary material: the online version of this article (doi:10.1186/s12879-015-1271-7) contains supplementary material, which is available to authorized users. infection by one of the four serotypes of the dengue virus (dv), a member of the flaviviridae family, can cause a wide spectrum of clinical manifestations. although the majority of symptomatic patients develop a febrile illness known as dengue fever (df) with nonspecific symptoms such as headache, fever or myalgia, around 10 % of patients develop a more severe form of disease, severe dengue (sd), that may include plasma leakage, severe hemorrhage and organ failure [1] . the dv genome is a positive single-stranded rna molecule encoding a polyprotein that is processed into three structural proteins (the capsid protein c, the membrane protein m, and the envelope proteins e) and seven non-structural (ns) proteins involved in replication and pathogenicity [2] . dv enters target cells via receptor-mediated endocytosis and traffics via the endosome, where the acidic environment triggers fusion of viral and host cell membranes. once within the target cell, the virus manipulates the host cell membrane to create an optimal environment for the assembly of its replication complex and subsequent rna amplification [3] . virion assembly occurs on the surface of the endoplasmic reticulum (er), followed by budding of an immature particle into the er lumen. the immature virion is then transported to the trans-golgi network, matured via proteolytic cleavage, and finally released by exocytosis into the extracellular medium. no specific antiviral treatment against dv currently exists. the available therapies are symptomatic and are administered to control the clinical manifestations. the ability to diagnose dv infection at an early stage and successful prognosis of the resulting complications of dengue are urgently required to improve the management of patients. it is possible that the identification of proteins specifically present in plasma before the onset of severe symptoms may ultimately lead to the discovery of new prognostic biomarkers. despite an incomplete understanding of the mechanisms of pathogenicity, several hypotheses have been formulated to explain the disease process in patients infected with dv. however, the lack of animal models capable of reproducing the features of human disease has hampered the identification of reliable parameters and indicators to explain or predict the development of sd. a number of host immune components, especially antibodies, are associated with the pathogenicity of viral infections. such mechanisms include antibody-dependent enhancement of a secondary infection or cross-reactivity with proteins such as endothelial cell or coagulation proteins [4] [5] [6] [7] [8] . other immune components, including memory t-cells, innate immunity effectors and complement factors have been shown to modulate the outcomes [9, 10] . the dv non-structural protein 1 (ns1) may also play a major pathogenic role, as it interacts with host complement proteins [11, 12] . numerous studies have investigated associations between altered levels of circulating cytokines/chemokines and dengue severity [13, 14] . alternatively, as there is a strong biological rationale for investigating markers implicated in vascular pathologies, many markers such as the soluble intercellular adhesion molecule-1 (sicam-1), the soluble vascular cell adhesion molecule-1 (vcam-1), the e-selectin or the thrombomodulin have been identified and seemed to correlate with disease severity [15] . unfortunately, no clear consensus has emerged from these studies. predicting outcome in dengue remains challenging, and the search for robust markers remains crucial. previous studies have demonstrated patients with sd have higher viremia than patients with df [9, 16, 17] . additionally, reports from cuba and australia have suggested that circulating dv may become more virulent through passage in successive hosts during an epidemic [18] [19] [20] [21] . as the virus cycle and the virus pathogenicity are strongly linked with the host metabolism, it is assumed that host proteins interacting with virions are a reflection of pathological status of the patient. therefore, in the present study, a dv-enrichment procedure combining sucrose gradient ultracentrifugation and a polymer-based technique was developed for differential proteomic analysis of plasma pools from patients with df and sd by liquid chromatography coupled with tandem mass spectrometry (lc-ms/ms). the objective was to identify the viral proteins and the host proteins incorporated into virions, and also the host proteins that interact/are associated with virions. the identified markers were validated by quantitative elisas using samples from dengue patients from two different geographical regions. the possible role of the co-identified host proteins in disease pathogenicity and the potential of these proteins as fingerprints of disease severity in patients infected with dv are discussed. plasma samples were provided by the universidad industrial de santander, bucaramanga, colombia and the institut pasteur, phnom-penh, cambodia. samples were collected from dengue patients as part of retrospective (colombia) or prospective study (cambodia). both studies were reviewed and approved by the local medical ethics committees (universidad industrial de santander, colombia; national ethic committee, cambodia) and performed in compliance with the ethical standards set out by the declaration of helsinki. all patient plasma samples were anonymized after a physical examination and obtaining informed consent. dr villar-centeno (universidad industrial de santander, bucaramanga, colombia) and dr philippe buchy (institut pasteur, phnom-penh, cambodia) granted the authors permission to use the samples. all samples were collected between the onset of symptoms and defervescence. the cases were classified as primary or secondary infections by the physician based on hemagglutination assays performed on different dv serotypes and on japanese encephalitis virus. the serotype and the copy number were determined by real-time quantitative rt-pcr (qrt-pcr). dv-negative plasma specimens from healthy donors were obtained from the french national blood bank (etablissement français du sang, lyon, france). dengue samples were tested for the presence of the viral ns1 protein using the platelia™ elisa (biorad, marnes-la-coquette, france) following the manufacturer's instructions. rna was extracted from the plasma using the qiamp viral rna kit (qiagen, hilden, germany). viremia was measured using a qrtpcr kit (primerdesign southampton, uk) according to the manufacturer's instructions. qrt-pcr was performed in a final volume of 20 μl, containing 5 μl of extracted rna, 10 μl of 2x precision onesteptm qrt-pcr mastermix, and 1 μl of dengue primer/probe mix. assays were carried out using a lightcycler® 1.2 (roche applied science, bâle, switzerland) using the onestep amplification protocol recommended by the manufacturer. hepg2 cells (atcc hb-8065) were cultivated at 37°c in 5 % co 2 in dmem supplemented with 10 % decomplemented fetal calf serum, 5 × 10 4 iu penicillin, 50 mg streptomycin and 10 mm l-glutamine (invitrogen, paisley, uk). the cells were infected as previously described [22] with a serotype 3 dv (dv3, strain d78-878 thailand) graciously provided by dr v. barban (sanofi-pasteur, lyon, france). sub-confluent hepg2 cell cultures (approx. 10 7 cells/75 cm 3 flask) were incubated with virus diluted in serum-free culture medium at various multiplicities of infection (mois) for 90 min, the supernatant was removed, the cells were washed once with pbs (invitrogen) and 10 ml of fresh complete medium was added to the cells. after 5 days of culture, the supernatant was harvested and clarified by centrifugation at 10,000 g for 5 min at 4°c. denaturing polyacrylamide gel electrophoresis, western blotting and silver-staining following denaturation in sds sample-buffer (novex invitrogen, paisley, uk) at 37°c and denaturing polyacrylamide gel electrophoresis (page) on 4-12 % polyacrylamide gels in sds-mops buffer (nupage invitrogen), samples were electro-transferred to pvdf membranes (millipore, billerica, mt, usa) in 10 % caps-10 % methanol buffer, blocked in tbs-0.1 % tween 20-5 % skimmed dried milk (régilait, macon, france), incubated with anti-e monoclonal antibody (diluted to 1 μg/ml; biomerieux, marcy-l'etoile, france) followed by horseradish peroxidase-labeled conjugate (diluted at 0.1 μg/ml; p.a.r.i.s., compiegne, france) for 1 h each at room temperature (rt). after washing with tbs-0.1 % tween 20, the proteins were revealed using the supersignal west dura kit (thermo scientific, rockford, il, usa) and imaged using the versadoc™ imaging system (biorad, hercules, ca, usa). alternatively, the gels were silver-stained after electrophoresis using the silverxpress kit (life technologies, paisley, uk). densitometry analysis was carried out using quantity-one software (biorad). five milliliters of pre-clarified plasma pools (5000 g/ 10 min/4°c; cf. table 1) or 5 ml of pre-clarified cell culture supernatant were centrifuged for 2 h at 200,000 g using a beckman sw41 rotor in an optima l90 ultracentrifuge (beckman, fullerton, ca, usa). after centrifugation, the pellet was dissolved in a small volume of cold pbs (euromedex, souffelweyersheim, france), loaded on a discontinuous sucrose gradient constituted of 5 ml of 60 % sucrose in pbs (w/w) and 5 ml of 20 % sucrose in pbs (w/w), and centrifuged for 2 h at 200,000 g. the fraction containing virions, located at the interface of the two sucrose solutions, was collected, diluted ten-fold in cold pbs, centrifuged for 2 h at 200,000 g and the pellet was resuspended in 200 μl of cold pbs. all centrifugation steps were performed at 4°c. ultracentrifugation was complemented by additional purification steps using viraffinty™ (biotechsupportgroup, monmouth, usa), a water insoluble elastomeric polyelectrolyte developed for the capture and recovery of viruses and 100 μl of viraffinity™ were added to the resuspended pellet obtained after ultracentrifugation. the mixture was incubated for 5 min at rt and centrifuged at 1000 g for 10 min. the supernatant was discarded and the pellet was rinsed three times with mn buffer. finally, the viral fraction was separated from the polymer by heating for 5 min at 70°c in sds-buffer (novex invitrogen). after a final centrifugation step at 1000 g, the supernatant was harvested and stored at −80°c for immunoblotting and lc-ms/ms analysis. the following experiments were conducted by the edyp laboratory (edyp-service, cea grenoble, france). the virion-enriched preparation was loaded on a 10 % polyacrylamide gel and electrophoresed until all proteins entered the gel. the band containing the proteins was manually excised, washed three times in buffer containing 50 % acetonitrile and dried using 100 % acetonitrile. proteins with a mascot score higher than 40 (p < 0.05) were selected for further analysis [24] . the mascot score is a measure of the reliability of identification: the higher the score, the better the identification. in order to eliminate false matches and incorrect protein identification, consecutive searches against the concatenated swiss-prot and trembl_decoy databases (versions 57.6 and 40.6 decoy database, respectively; homo sapiens taxonomy, 164,620 entries) were performed for each sample using mascot 2.3 software (matrix science). irma software [25] was used to filter the results to achieve a false positive rate lower than 1 %. in-gel digestion and lc-ms/ms analysis was performed twice for each sample. ingenuity pathways analysis (ipa) software (ingenuity systems, redwood city, ca, usa) was used to investigate the interactions among all of the host proteins identified. interactive pathways were generated to observe the potential direct and indirect relationships among the proteins that were differentially expressed in the df and sd samples. commercial elisas (uscn, wuhan, china) targeting potential severity markers were used to measure in duplicate the levels of the proteins in individual df and sd plasma specimen, using the protocols recommended by the manufacturer. for each test, a standard curve was established by serial dilution of the calibrator provided in the kit to determine the protein concentration. the optical density values were determined at 450 nm using a microplate reader (eon; biotek, vinooski, vt, usa). statistical analysis (mann-whitney u test, chi-square test) and receiver operating curve analysis were performed using graphpad prism v.4.03 software (graphpad software, san diego, ca, usa). the mann-whitney and the chi-square tests were used to examine differences in demographic and clinical characteristics between patients and to assess potential confounding variables. comparisons of continuous variables were performed using mann-whitney u test that can be applied on unknown distributions for two small sets of observation (n < 30). comparisons of proportion were performed using chisquare test. p < 0.05 was considered significant [26] . plasma obtained from patients with df or sd were pooled to create two samples ( table 1) ; all of the plasma samples used for this step came from colombian patients who had secondary dv serotype 2 or 3 infections, and were collected between the onset of symptoms and the development of severe symptoms [27] . classification of the infections as secondary infections and the degree of severity were based on review of the patients' medical records. in these medical records, the estimation of the severity was based on the who criteria of 1997 [1]. df and dhf grade i (dengue haemorrhagic fever, minor haemorrhages) were considered as classicdengues (not severe). dhf grade iii/iv (dss, dengue shock syndrome) were considered as severe dengues. each medical records compiled details on platelet and blood cell counts, transaminase levels, the presence/absence of warning signs (persistent vomiting, abdominal pain…), haemorrhagic signs (petechiae, ecchymosis, epistaxis…) or shock signs (cold extremities, cyanosis…) that help to the patients classification by the physician. the proportion of samples from male patients was higher for the df pool (75 %) than the sd pool (43 %). the age of the patients (mean, 30 years) and the number of days the samples were collected after the onset of symptoms (mean, approximately 3 days) were similar between the two groups. no comorbidity was reported for any patient in either group. each of the individual plasma samples was confirmed as ns1-positive. the viremia of each pool was estimated using a commercial qrt-pcr kit. all patients were positive for dv, indicating all samples were collected during the acute phase of disease. the average number of rna copies per ml was 4.05 × 10 6 and 4.13 × 10 7 for the samples in the df and the sd pools, respectively. a technique based on ultracentifugation (step 1) followed by concentration using a commercial water-insoluble elastomeric polyelectrolyte specially engineered for the capture and recovery of viruses (viraffinity™; step 2), was created to obtain a fraction of plasma enriched with dv particles. initially, this technique was developed using the cell culture supernatant of hepg2 cells harvested five days after infection with dv at a moi of 1 or 10. the samples obtained after each step of the purification process (step 1 and step 1 + 2) were analyzed by western blotting using an anti-e monoclonal antibody (fig. 1a) . a positive signal corresponding to monomeric (60 kda) and dimeric e protein (120 kda) was observed in the dv-infected samples, and became more intense after the viraffinity™ step (fig. 1a, step 1 + 2). the purity of the step 1 + 2 sample was assessed by electrophoresis using denaturing page and silver-staining (fig. 1b) . bovine serum albumin (bsa), an intense protein band of approximately 66 kda was observed in the sample before purification ("not purified" lanes); however, this band was almost absent from the final purified sample ("step 1 + 2" lane). only a small number of bands were detected in the purified sample. densitometry indicated that the protein complexity of the purified samples was reduced by roughly 350-fold compared to the unpurified samples. fig. 1 characterization of the viral-enriched fraction of dv-infected hepg2 supernatant or plasma obtained from patients with dengue. a after infection with dv at a moi of 1 or 10, hepg2 cell culture supernatants were purified by ultracentrifugation (step 1) followed by the use of viraffinity™ (step 1 + 2). b silver-staining assessment of the protein complexity of dv-infected hepg2 cell culture supernatant before and after purification; not diluted (lane "not purified"), 1/10 diluted (lane "not purified 1/10") dv-infected cell supernatant before purification or the same sample as shown in lane 1 after purification (lane "step 1 + 2") were separated by denaturing polyacrylamide gel electrophoresis and silver-stained. c electron microscopy of dv-infected hepg2 culture supernatant after ultracentrifugation. grids were negatively stained using uranyl acetate. bars: 50 nm, 200 nm. d representative western blotting analysis of plasma pools obtained from patients with dengue after purification by ultracentrifugation and viraffinity™ using an anti-e monoclonal antibody. mw: molecular mass the presence of intact viral particles was also confirmed by electron microscopy, which was performed by the ezus laboratory (université claude bernard, villeurbanne, france). after negative staining, virions could be visualized in the purified samples obtained from the supernatant of cells infected at a moi of 10 (fig. 1c) . the viral membrane, including the e spikes, was visible. the spikes observed on the virion surface are probably due to the slightly basic ph (ph 7.5) of the pbs-buffered solutions used during centrifugation and electron microscopy. virions tended to aggregate in clusters of 10-20 viral particles as seen in images taken at a lower magnification. little background signal was observed around the virion clusters. following the two-step purification process, the moi 10 sample was also analyzed by nano-lc ms/ms. all structural viral proteins were identified ( table 2 ). the e protein peptides were the most largely represented (21 peptides; 15 single peptides). the sequence coverage for the e protein represented 44 % of the entire protein. the protein m, which is part of the external viral layer, along with the e protein, was identified three times (3 single peptides) with coverage of more than 58 % of the protein sequence. the pr peptide and capsid were also identified (one peptide each). in conclusion, combining ultracentrifugation with viraffinity polymer yielded a fraction with a considerably reduced protein complexity that contained a large quantity of enveloped virions. this technique was then applied to the pooled plasma samples obtained from the colombian patients (table 1) . after purification, the preparations were analyzed by western blotting using a monoclonal antibody directed against the e viral protein (fig. 1d , sd pool as an example). strong signals corresponding to monomeric (60 kda) and dimeric e protein (120 kda) were observed. the e protein was not detected in purified plasma obtained from healthy individuals, which was tested as a mock-control. to identify the proteins present in the purified samples and to compare their composition, nano-lc-ms/ms was conducted on the purified df and sd plasma pools. this experiment was also performed on the purified mock-control sample to determine the host background. the same quantity of total protein was analyzed for each sample. these experiments were performed independently twice. both viral and host peptides were identified in the purified pooled plasma samples from the patients with df and sd. one peptide corresponding to the viral envelope protein was detected twice in the sd sample; this peptide (gwgngcgllfgk) was also identified in the purified dv-infected hepg2 supernatant (table 2) . after removal of the mock-control background, the remaining peptides identified in the df and sd samples were analyzed. in order to consolidate the results obtained by nano-lc ms/ms, only proteins for which the variance of the average number of peptides was lower than 25 % were selected [28] ; 188 host proteins met this criterion. the sd/df peptide ratio was calculated for these 188 proteins (see additional file 1). the highest sd/df ratio was obtained for c1s esterase (sd/df peptide ratio = 4.14) and the vitamin k-dependent protein s (ratio = 4). the lowest ratio was obtained for beta-1 spectrin (ratio = 0.13). six proteins were only identified in the sd sample; consequently, the sd/df ratio could not be calculated for these proteins. ingenuity pathway analysis (ipa) was conducted to further elucidate the specific pathways associated with the identified host proteins. the specific location and function of each pathway was attributed by ipa for 174 of the 188 proteins; the most prominently represented pathways are illustrated in fig. 2 . the ipa diagram shows that the highest p-values were attributed to acute phase response signaling [−log(p-value), lp = 31], the lxr/rxr activation pathway (lp = 18.4), the complement system (lp = 16.6), the coagulation system (lp = 16.2) and the extrinsic prothrombin activation pathway (lp = 12.6). the p-values indicate the likelihood that the focus genes in a network are found in these pathways by random chance alone. although represented to a lesser degree, three pathways related to host proteins involved in the dv cell cycle, clathrin-mediated endocytosis signaling (lp = 10.3), caveolar-mediated endocytosis signaling (lp = 9.2) and the virus entry via endocytic pathway (lp = 6.05), were also identified. other identified pathways, including integrin (lp = 6.96) and paxillin signaling (lp = 6.75), correspond to proteins involved in cell-matrix interactions and cell-to-cell communication. figure 2 also illustrates the ratio of identified proteins for each canonical pathway. higher proportions of proteins mapped to the complement system (ratio = 0.34), coagulation system (ratio = 0.32) and extrinsic prothrombin activation pathway (ratio = 0.4) than the other pathways (ratios between 0.16 and 0.05). ipa software was also used to investigate possible interactions among all of the identified proteins and to assess the representation of the identified proteins in acute phase response signaling, the complement system and the coagulation system with prothrombin activation (see additional file 2). twenty of the 50 (40 %) secreted proteins identified by ipa as part of the acute phase response signaling pathway, which is activated in macrophages and endothelial cells upon infection and inflammation, were over-represented in the pooled sd plasma sample compared to the pooled df plasma sample. thirteen complement component proteins were also over-represented in sd plasma, with high numbers of peptides identified for c1r and c1s in particular. complement factor c8 and complement factor b (cfb), which are involved in the complement alternate pathway, were only identified in the pooled sd plasma sample. the c9 protein was enriched in the pooled df plasma sample. fig. 2 ingenuity pathway analysis for host proteins identified in the viral-enriched plasma fraction of patients with dengue. pathway classification according to canonical pathways was performed using ipa software. the x-axis represents the pathways identified. the y-axis (left) shows the − log of the p-value calculated using fisher's exact test. the ratio (y-axis, right) represented by the line is calculated as follows: number of proteins in a given pathway that meet the cutoff criteria divided by total number of proteins that make up that pathway the extrinsic and intrinsic prothrombin activation pathways are part of the coagulation system. the majority of the proteins involved in the fibrinogen/fibrin cascade (i.e. 10 proteins out of 11) were over-represented in the sd sample compared to the df sample. overall, the network analysis also provided evidence of strong links between these three pathways. the coagulation factor 2 (f2) and the c3 and c5 proteins are located at the interface of the coagulation system and the complement system (see additional file 3). to validate the mass spectrometry data, the levels of selected host proteins were assessed by quantitative elisa both in the virus-enriched fraction and in individual plasma samples from df or sd patients. for the elisa, proteins with a sd/df ratio higher than 1.3 or lower than 0.78 were selected (see additional file 1); other criteria, such as the number of peptides identified in the sd sample and the availability of a commercial elisa kit were also considered. some proteins, such as ribosomal protein p2 (accession number p05387; ratio = 0.6) and histone h4 (accession number p62805; ratio = 0.4) were not deemed relevant enough for the study and were consequently not tested. the proteins readily found at high concentrations in plasma and known to be frequent contaminants in proteomic experiments, such as the immunoglobulin heavy chain (accession number p01779; ratio = 2.4), were not tested. finally, a non-exhaustive list of 22 proteins to assay was established. these 22 proteins were ceruloplasmin (cp), vitamin k-dependent protein s, complement factor properdin, antithrombin iii, secretory component p85, complement factor c1r (c1r), complement factor c1s, angiotensin, factor 2, anti-factor viii, serum amyloide p-component, olfactomedin-4 (olfm4), thrombospondin, gelsolin, platelet factor 4 (pf4), complement factor c1q, complement factor b, moesin and complement factor c8. multimerin-1, apolipoprotein b-100 and von willebrand factor were also tested. these 22 proteins were first quantified in the virusenriched fraction of the individual plasma samples obtained from df or sd patients of the colombian cohort. the elisa signal levels for these samples were close to background levels (purified mock-control sample); the results were not interpretable. to investigate the potential interaction of these proteins with the virion, fresh plasma was incubated for various times with purified viral particles. the change of protein concentration before and after the incubation was assessed by elisa. this experiment was performed for 4 proteins out of 22 (olfm4, cp, c1r, pf4) and showed that no significant signal change was observed for these proteins, whatever the condition tested (data not shown). the virus preparation is probably not concentrated enough to induce a significant variation in host protein concentration. furthermore, the plasmatic concentration of the four proteins is too high to be significantly affected by the interactions with the virus coated in the microplates. the elisas were then performed on the whole plasma samples obtained from colombian patients ( table 3 , colombian patients). all patients had secondary infections associated with various dv serotypes (dv1, dv2 and dv3); all samples were collected between onset and defervescence (between days 2 and 5). the male/female comorbidity 0 (0 %) 0 (0 %) -0 (0 %) 0 (0 %) -iu international units, ns not significant, na not applicable chi-square or mann-whitney tests were used to analyze the differences between groups sex ratio and age were similar between the groups of patients with df or sd. the samples were confirmed positive for both dv by qrt-pcr and the viral ns1 protein using a commercial elisa (ns1 platelia™). no comorbidity was recorded in any patient. among the 22 proteins tested, only cp, c1r, olfm4 and pf4, had different plasma concentrations between df and sd patients. the average concentration of c1r was higher for patients with sd than patients with df (p = 0.049). moreover, the variance between the two groups was significantly different (p = 0.019). the protein concentration of olfm4 was considered higher for sd patients (p = 0.051). for cp, the difference between the two groups was significant (p = 0.045). the largest difference was observed for pf4 with a higher concentration for patients with df (p < 0.001; fig. 3 ). to further confirm the relevance of c1r, cp, olfm4 and pf4 as potential markers to differenciate df and sd, plasma samples from patients with acute dengue from cambodia, another dengue-endemic area, were tested. as for colombian patients, the cambodian patients were classified by the local physician using the who classification of 1997. for the present study, patients classified df or dhf grade i were considered as classic dengue. patients classified dhf grade iii and iv were considered as severe dengue. clinical data were collected and included details on the platelet count, transaminases level, blood cells count, biochemistry (cholesterol, triglycerides, bilirubin…), ultrasound imagery, hemorrhagic signs (petechiae, ecchymosis, epistaxis…), the presence/or absence of warning signs (persistent vomiting, abdominal pain…), plasma leakage and shock signs (cold extremities, cyanosis…). the main characteristics of the cambodian patients are detailed in table 3 (cambodian patients). the mean age of patients from cambodia (about 8 years-old) was lower than that of the colombian patients. the male/female ratios of the df and sd groups were similar. all cambodian patients were infected with serotype 1 viruses. the plasma samples were collected approximately 3 days after the onset of symptoms (=acute samples) and also just before the discharge from the hospital (=discharge samples). in contrast to the samples obtained from the colombian patients, elisas showed that the levels of c1r and cp were not significantly different between df and sd patients in the cambodian cohort (see additional file 4); however, concentrations of olfm4 (p < 0.0001) and pf4 (p <0.0001) during the acute phase were significantly different between df and sd plasma (fig. 4) . the olmf4 concentration in df patients was lower than in sd patients. in contrast, pf4 concentrations were higher in df patients than in sd patients and sd patients showed no significant difference with healthy controls (n = 8). for these two markers, at the time of discharge, concentrations tended to decline back to the levels observed in controls. receiver operating characteristic (roc) curves compare sensitivity versus specificity across a range of values. roc curves are used to assess the discriminatory ability of each markers. area under the roc curve (auc) is another measure of test performance. the auc quantifies the fig. 3 assessment of the protein concentrations of c1r, cp, olfm4 and pf4 in the individual plasma samples of colombian patients using specific quantitative elisas. each value corresponds to the mean of two independent experiments, each performed in duplicate. *: 0.01 < p < 0.05; **: 0.005 < p < 0.01; ***: p < 0.005 overall ability of the test to discriminate between individuals with df and those with sd. a perfect test (zero false positives and zero false negatives) has an auc of 1.00. roc curve analysis was used to determine aucs and specificity/sensitivity values for olfm4 and pf4 as prognostic biomarkers of disease severity in cambodian acute patients with dengue. the roc curves indicated good discrimination between df and sd as the auc were 0.958 for olfm4 and 0.836 for pf4. the highest specificity/sensitivity values obtained using this model are summarized in table 4 . for a sensitivity of 96 %, the specificity exceeded 62 % (pf4) or 86 %(olmf4). when the sensitivity reached 100 %, the specificity dropped to 56.25 % (pf4) or 82.6 % (olfm4). dengue viral infections are prevalent in tropical and subtropical areas, and are associated with substantial morbidity and mortality. the pathogenesis of dengue remains unclear. various proteomic approaches have been used to characterize host-protein changes during dv infection and identify prognostic biomarkers of disease severity; several studies have been conducted on cells infected in vitro [29] [30] [31] . studies on plasma specimens have identified a number of candidate biomarkers [32] [33] [34] . as the dynamic range for plasma proteins is large (over 10 orders of magnitude) and the most abundant proteins (0.1 % of the total number) constitute up to 95 % of the plasmatic protein mass, identification of biomarkers present at low concentrations is a major challenge in proteomic studies dealing with plasma. however, proteomic techniques are constantly being improved to assess low abundance plasma proteins [35] . viruses facilitate their replication and propagation by subverting host cellular pathways and processes, and constantly adapt to and modulate their host's environment. enveloped viruses are able to incorporate numerous host proteins, both into virus particles as well as the host-derived viral envelope. as the genomes of rna viruses only encode a small number of proteins, they must rely on host proteins during an infection. interactions between the virus and host may have unforeseen consequences, depending on viremia level, the host's genetic background and other relevant factors [36] . host proteins can be incorporated into virions either randomly, by being present at the site of budding, or specifically, as a result of interacting with viral proteins. the functional significance of the host proteins that associate with viral particles remains to be thoroughly investigated; however, it is very likely that such interactions contribute to pathogenicity if they disturb the metabolism of the host cell [37] . for instance, the incorporation of cellular proteins such as integrins or hla class ii proteins can affect the ability of hiv-1 to infect host cells and contributes to immunopathogenesis [38, 39] . viroproteomic analyses have already been conducted on dna viruses [40] and rna viruses, such as retroviruses [39, 41] , paramyxoviruses [42] , coronaviruses [43] and flaviviruses [44] ; these studies have proven that many cellular proteins are incorporated into the virion or associate with the viral membrane. usually, prior to ms analysis, protease hydrolysis is combined with ultracentrifugation to remove host proteins that may co-purify with virions. in the present study, as we aimed to characterize the host proteins that interact with viral membrane proteins and particles, enzymatic hydrolysis of the proteins outside of the virion could not be performed. therefore, a new technique combining ultracentrifugation with water-insoluble polyelectrolyte-based enrichment was developed to enable proteomic characterization of a fraction of human plasma enriched with virus particles and depleted of the host proteins predominantly expressed in plasma. previous studies measuring viremia have demonstrated that patients with sd have higher viremia than patients with df [9, 16, 17] . higher viremia may lead to biosynthesis of virions with an altered host protein composition, which may reflect an increased level of cellular stress, or subtle changes in the assembly pathway. these host proteins may be packaged into the virus particle along with the viral components or incorporated into the viral membrane. alternatively, they can be simply co-purified along with virions. such proteins may potentially be fingerprints of the virus assembly pathway and may also play a role in viral pathogenicity. nano lc-ms/ms was used in this study to analyze the protein composition of the virion-enriched samples purified from the plasma pools. some proteins had a higher average number of peptides in the sd sample than the df and control samples, suggesting the presence of greater amounts of these proteins associated to viruses purified from the plasma of patients with sd; conversely, other proteins were mainly identified in the samples purified from the df plasma pool. interestingly, the identified host proteins belong to three main pathways: the complement system, the coagulation system and the acute response signaling pathway. the complement system plays an important role in both innate and adaptive immune responses, is first line of defense against infectious agents, and modulates b-and t-cell responses. in flavivirus infections, excessively-activated complement proteins have been reported to induce a deleterious, exacerbated inflammatory response [45] [46] [47] . recently, it has been shown that the level of complement proteins positively correlates with the severity of dengue in indonesian patients [48] . coagulation is the process by which the blood changes from a liquid to a gel. coagulation is required to stop blood loss and enable the subsequent repair of damaged vessels. hemorrhage is one of the major symptoms of sd, and is probably caused by vasculopathy and a deficiency in coagulation and fibrinolysis. the normal vascular endothelium produces inhibitors of coagulation and fibrinolysis. hemostasis can be impaired if the endothelium is stimulated by excessive levels of cytokines or by a pathogen, which may in turn result in thrombosis and bleeding [49, 50] . the acute phase response corresponds to the inflammatory response observed in response to an infection, tissue injury or an immunological disorder [51] , and is mainly characterized by increased levels of inflammatory factors and a change in the protein composition of plasma. interestingly, these changes can inhibit complement activation [52] . interconnections between the coagulation process and complement cascade have been reported by many authors [53, 54] . deregulation of one or both systems can result in the clinical manifestations of diseases with inflammatory complications. in a previous study, differentiallyexpressed genes associated with the immune response were identified by microarray analysis of peripheral blood mononuclear cells isolated from colombian children with dengue fever or dengue hemorrhagic fever. these results indicated that the complement and numerous cytokines are deregulated in patients with dengue-hemorrhagic fever. such changes may enhance disease severity by disturbing coagulation and inducing endothelial cell damage [55] . in another recent study, sera from indian patients with df were compared with that of healthy controls using 2d-dige associated with maldi tof/tof ms. the authors reported that dv infection led to altered expression of multiple serum proteins involved in complement cascades, blood coagulation and acute phase response signaling, providing further clues regarding the pathogenesis and host immune response to dv infection [56] . the complement system, coagulation system and acute response signaling pathway are strongly interconnected and share proteins whose expression is modulated during an infection. they are all related to the inflammatory process and the innate immune response, and act upstream of activation of the adaptive immune response. expression deregulation of these proteins could induce deleterious effects in endothelial cells [57] . in our hands, none of the seven complement proteins tested by elisa showed a difference in concentration levels between sd and df acute samples, minimizing the potential role of these proteins in dengue pathogenesis, at least in the early phase of the disease. several authors have reported proteomic analysis of samples from patients with dengue using different approaches [29, 30, 32] . to our knowledge, this study is the first assessment of the proteins that are differentially present in a virus-enriched fraction purified from plasma specimens obtained from patients during the acute stage of dv infection. we were unable to confirm that proteins identified by lc-ms/ms were directly associated with the viral particles. neither elisas conducted on virions purified from plasma, nor electron microscopy using specific goldlabeled antibodies succeed to yield reliable results. consequently, we cannot discard the possibility that the host proteins identified in this study may be contaminants that co-purify reproducibly with viral particles. elisas carried out on individual plasma specimens from the colombian cohort indicated trends towards higher levels of cp, c1r and olfm4 in patients with sd compared to patients with df. cp, an acute phase protein, is a ferroxidase involved in iron metabolism. elevated levels of cp are generally observed during inflammation. c1r is a complement protein that interacts with c1s at the beginning of the complement cascade. olfm4 is an anti-apoptotic factor that promotes tumor growth and facilitates cell adhesion, probably via interacting with cell surface lectins and cadherin [58] . the associations between these proteins and disease severity in dengue remain to be explored. interestingly, a recent paper mentioned the interest of olfm4 as a marker of disease severity in respiratory syncytial virus infection in children [59] . elisas also showed that the concentration of pf4 was higher in the plasma samples of colombian patients with df than patients with sd. pf4 is a small cytokine released by platelets that promotes blood coagulation by moderating the effects of heparin-like molecules. pf4 also plays a role in wound repair and inflammation. alterations in platelet function have been associated with plasma leakage, which is one of the major features of severe disease in patients with dengue [60] . recently, it has been demonstrated that dv replicates and produces infectious virus in platelets [61] . in 1989, srichaikul t. et al. demonstrated that the level of pf4 increases during acute phase in both shock or non-shock dhf children. it is difficult to compare these results with the results presented here because there is no strict correspondence between the patient classification used by srichaikul t. et al. and the classification of 1997 used in the present work [62] . interestingly, among the six proteins involved in coagulation and having a sd/df peptide ratio higher than 1.3 or lower than 0.78 (see additional file 1), pf4 is the only one that was confirmed by elisa. pf4 is significantly less concentrated in sd acute patient plasma. the trends observed for olfm4 and pf4 by elisas for the colombian patients were also confirmed in the cambodian patients. by testing different time points during the course of the disease, we showed that these markers evolved through time and tended, during the process of healing, to get close to the protein concentration found in healthy patients. pf4 was overabundant in df acute patients, but remained surprisingly low in sd patients, with levels remaining similar to control individuals. it has previously been proposed that pf4 interacts with the vasculature and is involved in thrombus formation at sites of vascular injury [63] . murine studies suggest that pf4 may have an overall salutary effect in sepsis [64] . therefore, a basal level of pf4 expression during the acute phase of dv infection could be an interesting marker of a future severe dengue. the severity of dengue is modulated by multiple risk factors such as the age, genetic background and nutritional status of the host, as well as the genotype and serotype of the virus. these factors could explain the differences in the concentrations of specific markers observed between the plasma samples from the colombian and cambodian patients. for example, sd in south-east asia is mainly observed in children, whereas adults are predominantly affected in south america [65] . as a consequence, results should be extrapolated with caution to different geographic areas or different demographic groups. in conclusion, we describe the development of a novel technique of dv-enrichment from complex biological fluids based on centrifugation combined with a waterinsoluble polyelectrolyte-based technique, for subsequent proteomic analyses. we found no evidence that the identified host proteins are specifically associated with virions. however, this purification technique enables the analysis of a plasma fraction enriched in virions and from which the most abundant plasma proteins were removed. the host proteins characterized in this study may potentially reflect how dv infection disturbs the function of the cellular proteome. in this regard, further studies are required to assess the prognostic value of host proteins associated with inflammation, complement cascade and coagulation for disease severity by analysis of additional biological samples from patients infected with dv. analysis of a selection of the identified proteins using elisas identified two host proteins, olfm4 and pf4, which had significant prognostic value for classifying patients with dengue who were likely to develop sd. further prospective studies are warranted to confirm and validate the prognostic value of olfm4 and pf4 as potential biomarkers of disease severity in larger cohorts of patients from a variety of dengue-endemic areas around the globe. complement factor 1r; ci: confidence interval; cp: ceruloplasmin; df: dengue fever; dige: difference gel electrophoresis; dv: dengue virus; elisa: enzyme linked immunosorbent assay; er: endoplasmic reticulum; iu: international unit; lc-ms/ms: liquid chromatography coupled to mass spectrometry chain; mes: 2-(n-morpholino)ethanesulfonic acid; moi: multiplicity of infection; na: not 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associated with plasma leakage dengue virus binding and replication by platelets platelet function during the acute phase of dengue hemorrhagic fever interactions of platelet factor 4 with the vessel wall role of the platelet chemokine platelet factor 4 (pf4) in hemostasis and thrombosis dengue viruses -an overview we would like to thank dr. y. coute data supporting the findings and materials are available upon request to frederic bedin, biomerieux sa, chemin de l'orme, 69280 marcy l'etoile (france). additional file 1: virus-enriched fraction proteome change in purified plasma pools obtained from acute dengue patients. authors' contributions rf, mf, fr and fb carried out all the experiments and performed the statistical analysis. rf and fb drafted the manuscript. ap, fr and gb participated in the design of the study. rf, fb, gb participated to draft the manuscript. dv and pb collected cambodian samples and managed the patient data and ethics. all authors read and approved the final 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the activity of antioxidant enzymes in fenneropenaeus indicus phosphorylation and dephosphorylation events that regulate viral mrna translation identification of two amino acid residues on ebola virus glycoprotein 1 critical for cell entry sindbis virus infection of two model insect cell systems-a comparative study the amino-terminal residue of glycoprotein b is critical for neutralization of bovine herpesvirus an endornavirus from a hypovirulent strain of the violet root rot fungus importance of the extracellular and cytoplasmic/transmembrane domains of the haemagglutinin protein of rinderpest virus for recovery of viable virus from cdna copies envelope gene capture and insect retrovirus evolution: the relationship between errantivirus and baculovirus envelope proteins multiclonal pattern of jaagsiekte sheep retrovirus integration sites in ovine pulmonary adenocarcinoma venezuelan equine encephalitis virus complex-specific monoclonal antibody provides broad protection, in murine models, against airborne challenge with viruses from serogroups i, ii and iii patterns of sequence evolution at epitopes for host antibodies and cytotoxic t-lymphocytes in human immunodeficiency virus type phylogenetic analysis of the gag region encoding the matrix protein of small ruminant lentiviruses: comparative analysis and molecular epidemiological applications gene expression array analyses predict increased proto-oncogene expression in mmtv induced mammary tumors evolutionary genomics of archaeal viruses: unique viral genomes in the third domain of life hiv-l and the microrna-guided silencing pathway: an intricate and multifaceted encounter a modified viral satellite dna-based gene silencing vector is effective in association with heterologous begomoviruses tatabinding protein and tbp-associated factors during herpes simplex virus type 1 infection: localization at viral dna replication sites immunohistochemical examination of the role of fas ligand and lymphocytes in the pathogenesis of human liver yellow fever complete sequence and organization of the human adenovirus serotype 46 genome molecular characterization and phylogenetic study of maedi visna and caprine arthritis encephalitis viral sequences in sheep lymphocytopathogenic activity in vitro correlates with high virulence in vivo for bvdv type 2 strains: criteria for a third biotype of bvdv inactivation of white spot syndrome virus (wssv) by normal rabbit serum: implications for the role of the envelope protein vp28 in wssv infection of shrimp low prevalence of primary antiretroviral resistance mutations and predominance of hiv-1 clade c at polymerase gene in newly diagnosed individuals from south brazil characterization of a highly virulent feline calicivirus and attenuation of this virus interaction of the hepatitis b virus protein hbx with the human transcription regulatory protein p120e4f in vitro translation reinitiation and leaky scanning in plant viruses phylogenetic analysis of recent isolates of classical swine fever virus from colombia inhibition of severe acute respiratory syndrome-associated coronavirus (sars-cov) infectivity by peptides analogous to the viral spike protein rabies virus-induced apoptosis involves caspase-dependent and caspase-independent pathways translational control during virus infection epstein-barr virus immunossuppression of innate immunity mediated by phagocytes binding of shrimp cellular proteins to taura syndrome viral capsid proteins vp1 collaborative study to evaluate a new elisa test to monitor the effectiveness of rabies vaccination in domestic carnivores efficient inhibition of hepatitis b virus replication by small interfering rnas targeted to the viral x gene in mice preparation and characterization of a novel monoclonal antibody specific to severe acute respiratory syndrome-coronavirus nucleocapsid protein the kinetics of proinflammatory cytokines in murine peritoneal macrophages infected with envelope protein-glycosylated or non-glycosylated west nile virus members of adenovirus species b utilize cd80 and cd86 as cellular attachment receptors exogenous nitric oxide inhibits crimean congo hemorrhagic fever virus structural and antigenic analysis of the yellow head virus nucleocapsid protein p20 efficient expression of the 15-kda form of infectious pancreatic necrosis virus vp5 by suppression of a uga codon phylogenetic relationships of brazilian bovine respiratory syncytial virus isolates and molecular homology modeling of attachment glycoprotein genetic manipulation of two fowlpox virus late transcriptional regulatory elements influences their ability to direct expression of foreign genes a new rna virus found in black tiger shrimp penaeus monodon from thailand conformational maturation of the nucleoprotein synthesized in influenza c virus-infected cells hiv-1-mediated syncytium formation promotes cell-to-cell transfer of tax protein and htlv-i gene expression lack of a mechanism for faithful partition and maintenance of the kshv genome studies on the activity of a bidirectional promoter of mungbean yellow mosaic india virus by agroinfiltration expression, purification, and in vitro activity of an arterivirus main proteinase genetic characterization of the capra hircus papillomavirus: a novel close-to-root artiodactyl papillomavirus transient expression of human papillomavirus type 16 l1 protein in nicotiana benthamiana using an infectious tobamovirus vector a deletion and point mutation study of the human papillomavirus type 16 major capsid gene hearsnpv orf83 encodes a late, nonstructural protein with an active chitin-binding domain molecular epidemiology of bluetongue virus in northern colorado amino acid sequence of the amur tiger prion protein involvement of endoplasmic reticulum in hepatitis b virus replication systemic antiviral silencing in plants sequencing and comparative analysis of a pig bovine viral diarrhea virus genome esirnas inhibit hepatitis b virus replication in mice model more efficiently than synthesized sirnas chronological and geographical variations in the small rna segment of the teratogenic akabane virus hcv ns2 protein inhibits cell proliferation and induces cell cycle arrest in the s-phase in mammalian cells through down-regulation of cyclin a expression pepper mild mottle virus pathogenicity determinants and cross protection effect of attenuated mutants in pepper hepatitis e virus genotyping based on full-length genome and partial genomic regions location and phylogenetic analysis of the region immediately upstream of the granulin gene of the clostera anachoreta granulovirus molecular characterization of rabies virus isolates in china during proteolytic cleavage and shedding of the bovine prion protein in two cell culture systems antigenic structure analysis of glycosylated protein 3 of porcine reproductive and respiratory syndrome virus generation of virus-like particles consisting of the major capsid protein vp1 of goose hemorrhagic polyomavirus and their application in serological tests amino acid changes in the recombinant dengue 3 envelope domain iii determine its antigenicity and immunogenicity in mice key: cord-004435-l66ost6q authors: oli, angus nnamdi; obialor, wilson okechukwu; ifeanyichukwu, martins ositadimma; odimegwu, damian chukwu; okoyeh, jude nnaemeka; emechebe, george ogonna; adejumo, samson adedeji; ibeanu, gordon c title: immunoinformatics and vaccine development: an overview date: 2020-02-26 journal: immunotargets ther doi: 10.2147/itt.s241064 sha: doc_id: 4435 cord_uid: l66ost6q the use of vaccines have resulted in a remarkable improvement in global health. it has saved several lives, reduced treatment costs and raised the quality of animal and human lives. current traditional vaccines came empirically with either vague or completely no knowledge of how they modulate our immune system. even at the face of potential vaccine design advance, immune-related concerns (as seen with specific vulnerable populations, cases of emerging/re-emerging infectious disease, pathogens with complex lifecycle and antigenic variability, need for personalized vaccinations, and concerns for vaccines' immunological safety -specifically vaccine likelihood to trigger non-antigen-specific responses that may cause autoimmunity and vaccine allergy) are being raised. and these concerns have driven immunologists toward research for a better approach to vaccine design that will consider these challenges. currently, immunoinformatics has paved the way for a better understanding of some infectious disease pathogenesis, diagnosis, immune system response and computational vaccinology. the importance of this immunoinformatics in the study of infectious diseases is diverse in terms of computational approaches used, but is united by common qualities related to host–pathogen relationship. bioinformatics methods are also used to assign functions to uncharacterized genes which can be targeted as a candidate in vaccine design and can be a better approach toward the inclusion of women that are pregnant into vaccine trials and programs. the essence of this review is to give insight into the need to focus on novel computational, experimental and computation-driven experimental approaches for studying of host–pathogen interactions and thus making a case for its use in vaccine development. vaccination has been undeniably very helpful in promoting a healthy global population. it has severally saved lives, reduced healthcare costs and raised man's quality of life. 1 it greatly reduces disease burden, disability and death. however, newly emerging and reemerging infectious diseases (erid), infectious agents with complex lifecycle and antigenic variability and the need for personalized vaccination present additional challenges in vaccine development. 2, 3 for many pathogens (especially the emerging and those with antigenic variability), their genomes are known but their immune correlates of protection remain unclear. 1, 4 some of these reasons are why vaccine development for erid and multi-lifecycle pathogenic diseases is a tall order. serendipitous discoveries in immunology coupled with knowledge of bioinformatics tools for epitope predictions have resulted in the emergence of new pattern of vaccine design. 5, 6 the art and science of efficient and comprehensive information extraction and analysis of data deposited in relevant databases is now increasingly essential in researches related to immunology. 7 even with this capacity (efficient information extraction), some challenges in the application of bioinformatics in immunology include structure and/or function analysis and immune process analyses as concern the immune interaction specificity. fortunately, although researches in immunology are experimentally costly and very intensive, colossal amounts of data are usually generated. such data can only be analyzed with high precision and speed using bioinformatics tools. for instance, genome sequencing as well as in vitro t-cell confirmation is done in few months as opposed to years using the conventional vaccine design. 8 also, computational immunological methods drastically reduce both time and labor needs in epitopes screening. 5, 9 with computational immunology techniques, it is possible to discover vaccine candidate epitopes simply by scanning the protein sequences in a pathogen of interest. 5 many of these proteins are yet to be isolated or at least cloned. being pathogens specific and unique, they present ready candidates in vaccine construct. this review describes the need to use immunoinformatics-based techniques to unveil vital determinants of immunity made available in the genome sequence database and design vaccines. also, this review gives insight into the need to focus on novel computational, experimental and computation-driven experimental approaches for studying of host-pathogen interactions and thus making a case for its use in vaccine development. this review will further show the need for new approaches for effective drugs or vaccine design so as to combat the antigenic variability of some pathogens. the process of generating vaccine-induced immunity is somewhat challenging in immunology. current conventional vaccines came empirically when there were vague or no knowledge of vaccine immune system activation. a lot of research [10] [11] [12] has been geared toward the understanding of this challenge, but the complexity of it requires a different dimension of approach. 13 an approach that must accommodate many factors affecting vaccine development like pathogen antigenic variability, the emergence of infectious disease, human genetic variation is the goal of immunoinformatics [ figure 1 ]. activation of the immune system involves, among many processes, induction of the immune memory. the strength of this induction determines the efficacy of a vaccine. hence, vaccine efficacy in the long run is influenced by the determinants of immunological memory stimulation, persisting antibodies and kind and type of immune memory cells induced. 14 the primary vaccine-mediated immunological effectors (table 1 ) are mainly the antibodies (from b lymphocytes/ cell) 15, 16 and sometimes cd8+ and cd4+ t cells. these antibodies bind specifically to a particular kind of toxin or pathogen. vaccines and most antigens evoke humoral as well as cell-mediated immune responses. 17 vaccines that mediate immune responses through these systems (b and t cell responses) are said to be more effective. although b cells are regarded as the primary vaccine immune effectors, t cells induce immune memory cells and high-affinity antibodies. studies in reverse vaccinology and immunomics had also proved t cells as prime immune effectors following the discovery of novel vaccine targets with epimatrix. [18] [19] [20] this change of immune target has led to successful advances in vaccine design. even at the face of potential vaccine design advances, immune-related questions are now focused on specific vulnerable populations such as the young, elderly and immunocompromised. 21, 22 these concerns have propelled a better understanding of the efficacy of current vaccines on this vulnerable population and have also paved way for the application of new approaches that can put into consideration the differences of population and better targets that can generate optimum immune induction [23] [24] [25] with the exception of type ii t-cell-independent (ti-2) antigens (i.e., polysaccharide antigens). antigens that could provoke the b lymphocytes as well as the t lymphocyte responses stimulate the germinal centers causing antigen-specific highly efficient b-cell multiplication and eventual differentiation into antibodyforming plasma cells and memory b cells. all existing protein and dna antigens induce immunological memory b cells unlike type ii t-cell-independent (ti-2) antigens (i.e., polysaccharide antigens). these polysaccharide antigens do not generate memory b cells but can induce longlasting humoral immunity even when recall responses are lacking. 26 vaccine efficacy may be short term 27 if only the b cells are activated. the traditional approach for developing vaccines for infectious disease threats has shifted to include other vaccine design techniques like cloning and expressing major surface antigens 28 although this frequently resulted in the formulation of vaccines with poor immunogenicity, requiring strong adjuvantation. 29 this approach is particularly likely to be less specific for pathogens with complex lifecycles (e.g., parasites) or very high mutability (e.g., rna viruses). these pathogens do not depend on one route for their virulence of pathogenesis in human and thus to alter this process, increasing the specificity of the vaccine should be the aim and not just the effectiveness as seen in the current conventional vaccines. 28, 29 vaccines for several neglected tropical diseases are in various stages of development, 30 thanks to mega drug companies that have continued to demonstrate a willingness to invest money in the research and development as regards to diseases plaguing the developing nations. 16, [30] [31] [32] it is very pertinent to invest in researches that have an interest in vaccine specificity on the pathogen antigens than totally 33 computational vaccinology may now be applied to screen these genomes for possible vaccine target. with these tools, many proteins of virulence interest can be sequenced and the most essential gene of interest modeled for a potential vaccine candidate specific for that pathogen. immunoinformatics is the way forward in the identification of vaccine candidates for these tropical erid, for pathogens with varying antigens and for individualized therapy. immunology studies produce data in colossal quantities. also, with proteomics and genomics projects, extensive screening of pathogens and/or pathogen-host interaction, it has become increasingly necessary to store, manage and analyze these data, hence the birth of immunoinformatics. immunoinformatics deals with computational techniques and resources used to study the immune functions. statistical, computational, mathematical and biological knowledge and tools are applied in immunoinformatics in order to accurately and specifically store, and analyze data concerning the immune system and its functions. to handle evidence diversity, immunoinformatics uses tools that cut across several aspects of bioinformatics such as creation and management of databases, 34, 35 use and definition of both structural and functional signatures and the formation and application of predictive tools. [35] [36] [37] these strategies can synergize toward a better understanding of the immune system of both man and animals and fight against some less predictable pathogenesis. the complex nature of vertebrates' immune system, the variable nature of pathogens and environmental antigens coupled with the multi-regulatory pathways show that colossal quantities of data will be needed to unveil how the human immune systems work. conventionally, much cannot be achieved based on the complexity of the immune system and the virulent antigen but with the application of computational vaccinology, researches on vaccine design have been made easier, accurate and specific. applying immunoinformatics in disease study (table 2 ) requires the knowledge of disease pathogenesis, the immune system dynamics, and computational vaccinology, painstaking searches of the database, sequence comparison, structural modeling as well as motif analysis. 35, 38 these methods can go a long way in analyzing the pathogenesis of a disease and identification of vaccine candidates. in order to help understand complex pathogenrelated processes, computational models were developed for viral 46, 47 bacterial, 48 parasitic 49 and fungal pathogens. 50 the bioinformatics tools (table 3 ) are used to identify possible epitopes for vaccine formulation. each tool can screen protein sequences and identify aggregates of mhc binding and supertype motifs for possible use in epitopebased vaccine development and for use among human populations with genetic variability. there are several databases ( table 4 ) that can provide a wide range of information for all forms of immunological studies. generated data from the studies are further organized and stored in the databases (table 4) to provide a means for the development and advance in immunotargets and therapy 2020:9 immunological research. a tour on these databases will actually stimulate some interest in the vaccinology of emerging and re-surging disorders attributable to pathogen including cancer. emerging infections (eis) include infections that are entirely new in a population or that may have existed before in the population but are now gaining rapid and continued spread and/or wide geographical range. re-emerging or re-surging infections represent the infections that were previously of historical relevance but are now quickly becoming relevant because of either increasing incidence or increasing geographical and/or human host range while emerging infections represent the infections that were not originally observed in man. 66 several factors such as human behavioral changes, environmental changes, and host/intermediate factors, animal-human switching and microbial genetic changes all affect infectious disease emergence and spread. 67 these factors interact to promote the evolution of pathogens into new ecosystems, infect, spread and thrive in their new hosts. 68 the overall consequences of these are continued epimatrix this is an in-silico product of epivax developed for predicting and identifying the immunogenicity of therapeutic proteins and epitopes. it is also used to re-design proteins and in designing t-cell vaccine 51 has been applied in comparing strings from different strains of same pathogens and for pathogens identification. configuration of conservatrix allows for amino acid replacement at unusual positions. highly conserved t-cell epitopes in variable genomes such as some viruses are amenable to the algorithm 51,52 clustimer potential t-cell epitopes usually aggregate in specific immunogenic consensus sequence (ics) regions as clusters of 9-25 amino acids with 4-40 binding motifs instead of randomly distribute throughout protein sequences. in combination with epimatrix, the clustimer algorithm may be used to identify those peptides with epimatrix immunogenicity cluster scores ≥ +10. such peptides are usually immunogenic and tend to make a promising vaccine candidate. blastimer using blastimer program, one may also choose to automatically blast "putative epitopes against the human sequence database at genbank". blasting screens off those epitopes with possible autoimmunity and cross reactivity questions and locates the epitopes that can safely be used in developing human or animal vaccine. blastimer can also blasts sequences against pdb, swissprot, pir, prf and non-redundant genbank cds translations. vaccine cad this algorithm evaluates junctional epitopes for possible immunogenicity and inserts "spacers and breakers into the design of any string-of-beads construct". infectious disease emergence and re-emergence, epidemics and public health challenges. emerging infections and multi-antibiotic-resistant strains of pathogenic bacteria usually surge from one geographic location from where it spreads to other places due to immigration of people. 67, 69 most emerging infections originate from a specific population and can spread to a new population or become selectively advantaged that it can lead to the emergence of new strains of the pathogen. 67, 70, 71 also, there could be microbial traffic, in which case, an infectious agent transfers from animals to humans or spreads from isolated groups to new populations. 67, 71, 72 several factors, including ecology, are known to be associated with infectious disease outbreaks. these factors bring man into close contact with a natural disease reservoir/host. 70 with an increasing world population and poor infection control, the emergence of infection and increased microbial populations are sure. the human growth population will only increase the spread of the infection across populations. the information provided in table 5 is the list of remerging infections and current emerging diseases put forward during the who 2018 annual review. 73 the review noted that these infections, if not well controlled, can cause disease outbreaks, bioterrorism and similar occurrences requiring urgent public health attention and that with the dearth of efficacious medicines or vaccines, there is a compelling demand for continuous as well as accelerated research and development in those areas. advances in genomics, proteomics, immunomics, vaccinomics and nanotechnology are being continually exploited in diagnostic, therapeutic and in rational drug and vaccine development. these advances have also served in the control of the afore-mentioned emergences. 74, 75 the knowledge of the emerging pathogen's genome, protein make-up, pathogen-immune system interactions and researching the possible therapeutics will go a long way in directing the optimum path to containing the infection spread and controlling potential re-emergence or emergence in a different population. approaches in direct and computer-based structural determinations, 76 protein-protein interactions predicting, and bioinformatics tools now exist and are used in modern-day development of drug and biologics. 77 vaccine development has been sped up through the advance in the knowledge of the immune system of man. researches in the traditional targets of vaccines (adaptive immune response) and the less specific and fast-acting innate immune responses have been clear evidences for this advance. [78] [79] [80] as our understanding of the intercourse between innate and adaptive immunity increases, reasons and opportunities for more effective vaccine adjuvants will open up. this can be a step forward in solving a critical world's health challenge per population. following the conventional approach of vaccine design, much cannot be achieved but when the knowledge of immunoinformatics is applied, population safety and disease control can be achieved through pathogen's genome sequencing leading to optimum new vaccine design or development of a novel vaccine for the infection. antigenic variability is an important mechanism pathogens use to evade their host immunity. the surface proteins of pathogens are normally variable. this assists them to escape recognition by the immune system. a successful infectious agent presents to the host immune system information that differs from that of its virulence. pathogenic organisms have organized systems of escaping destruction by the immune system of their hosts. for instance, toxoplasma invades and appropriates the host cells thereby circumventing phagocytosis and then spread within their host to establish infection. 81 vertebrates on their own are endowed with immune system robust enough to efficiently and effectively surmount the non-self-attacks. yet the more the host's immune system elaborates, the better the organisms in their evasion of immune effector cells. antigenic variation refers to a pathogen's ability to modify its surface proteins such that it can circumvent the host's immunological attacks. it involves several mechanisms including the varying of surface protein's phase, shifting and drifting of surface protein antigens and/or any other form of alteration of antigenic protein. 82 antigenic variation plays significant roles in the pathogenicity of microorganisms by evasion of the host immune responses and establishment of re-infection. when a pathogen alters its surface antigens, it can evade the host's adaptive immunity and so reestablishes infection. the immune system may battle to generate new immunoglobulins against the new antigen. certain bacteria like neisseria gonorrhea, neisseria meningitides, mycoplasma and species of the genus streptococcus show antigenic diversity. 83 in eukaryotic pathogens, antigenic variation is shown by trypanosoma brucei and plasmodium falciparum. 81, 84 another vital cause of antigenic variation in bacteria is horizontal gene transfer (more important than point mutation) through plasmid acquisition and transduction via bacteriophages. virulence genes are normally acquired by non-virulent organisms via these routes. once this occurs, the new bacteria may quickly get established and cause fresh epidemic outbreak. species of the genus neisseria are champions in the rapid change of surface antigens amongst bacterial pathogens. pathogenic forms exhibit an amount of phenotypic variability not found in the commensal species. the pathogenic forms are implicated in std and meningitis. they employ amazing varieties of antigenic variability mechanisms. • they can recombine their pilin genes in a similar manner that eukaryotes recombine their own genes, such that they can express variable surface protein. 85 • some cell-surface proteins and enzymes synthesizing bacterial cell-surface carbohydrates are expressed in a variety of ways. this is as a result of replication slipping or slippage errors and repairs of simple tandem nucleotide repeats involving either the di-, or tri-or tetra-nucleotides. 86 • neisseria is able to take up and incorporate environmental dna into its genomes. 83 these are why an effective vaccine against neisseria infections is not yet developed. neisseria may be considered as an extreme example. however, many other bacterial pathogens like streptococcus and mycoplasma in promoting their antigenic variation tend to utilize one or more of these techniques. additionally, there are reports that dna-related defects have a much greater association with bacterial pathogen from symptomatic patients than samples of the same bacterial species isolated from environmental sources. [87] [88] [89] pneumococcus streptococcus pneumoniae, gram-positive cocci bacteria that cause otitis media, bacteremia and pneumonia, are a public health concern, causing morbidity and mortality in adults and children. 90 two forms of vaccines (polysaccharide and conjugate vaccines) are currently marketed for the prevention of pneumococcal infections. while the polysaccharide vaccines are for vaccination in the adult population, conjugate vaccines have an added immunogenic non-pneumococcal protein conjugated to the pneumococcal polysaccharides for enhanced immunogenicity in children. it is not yet known that these vaccines can evoke complete immunity against the infection. a polysaccharide capsule is a major virulence factor in the bacteria. several of these capsule types have been identified, and these form the basis of pathogen's antigenic serotyping. 91, 92 current pneumococcal vaccines are combinations of various capsular (polysaccharide) antigens from the serotypes most common in a particular population. currently, over 100 different serotypes are known but are not all covered in the available vaccines. 92 the discovery of a common antigen(s) will produce an effective vaccine. knowledge of the genome of the organism and the different strains has led to a possible advance in driving the pneumococcal potential vaccine search through a different approach. and this consideration will help solve a lot of concerns about the current vaccines. with this knowledge, many methods are been tried to determine whether they can be a source of effective vaccine design that can accommodate all the serotypes of the organism. search for antigen that is common to all the serotypes can be achieved with the knowledge of genomics and immunoinformatics. the introduction of genomic and computational technologies has given new directions in the study of bacterial pathogenesis and vaccine design. 93, 94 plasmodium plasmodium falciparum undergoes two life cycles: one in humans and the other in mosquitoes. the human host's erythrocytes and hepatocytes usually display modified parasite proteins called plasmodium falciparum erythrocyte membrane protein 1 (pfemp1) and plasmodium falciparum hepatocyte membrane protein 1 (pfhmp1), respectively. these proteins function to assist the parasite to evade destruction by the host immune systems. 95, 96 the pfemp1 proteins were identified as the prime ligands responsible for cytoadherence and resetting. 97 they cause the infected rbcs of host tissues to sequester thus helping the parasite to circumvent clearance by the host's spleen. 98 the membrane proteins also shield infected host cells from destruction by the spleen by adhering to the endothelium. luckily, if the pfemp1 protein is expressed for a long while; it comes under attack by the naturally acquired immunity. 98, 99 in defence to this, the parasite has expanded the var genes coding for pfemp1 such that the genes can exist as a polymorphic family of as much as over 50 members in every genomic haploid. antigenic switches work well here in that members of the polymorphic family (also called antigenic-variant-protein family) can be interchanged and cannot express their proteins at the same time. in this way, only one particular protein at the surface of the infected rbc is expressed at any given time. 97, 100 when studying antigenic-variant-protein families, it is pertinent to understand if grouping them into single-family results in any meaningful antigenic activity. studies have tried to understand the "languages" of the antigenic variant of pfemp1 proteins. 97, 101, 102 they sought to know the pfemp1 proteins binding properties or search to understand the correlation between motifs and infection severity. the vardb database is a repository for protein sequences involved in antigenic variation and their associated functionalities. 103 antigenic variant data obtained from several pathogens may be regrouped into a unified database. this will enable researches from several multicopy gene families to be accessed and compared swiftly in a single moment. updated vardb database contains close to 10,000 dna sequences, several protein translations, tens of infectious diseases and pathogens with their gene families. with a novel sequencing-based approach, pacbio, the different pfemp1 proteins can be sequenced and the related sequences used as potential vaccine targets. 104, 105 trypanosoma for many pathogens, antigenic variability occurs during the infection pathogenesis and is to enable them to escape destruction by the host antibodies. for instance, some eukaryotic parasites take to genetic assortment and rearrangement thereby changing their surface antigens. a ready example is seen in trypanosoma brucei, the causative organism for sleeping sickness. trypanosoma brucei replicates in the bloodstream (outside the cells) of their host, but at maturity, it crosses the blood-brain barrier to cause several fatal complications. during replication in the bloodstream, the parasites are subjected to humoral as well as cellular immune responses. it evades the host defenses by encasing itself in homogeneous coat of glycoprotein called the variant surface glycoprotein (vsg). 106, 107 though at initial invasion, the protein coat tends to protect the microbe from the immune system but on constant exposure, the coat will be identified as a foreign matter, and at this point the immune effectors can launch an attack against it. in a particular trypanosoma brucei, there are diversities of the vsg protein being coded by more than a thousand genes in the parasite's genome. unfortunately for the host, the expression of these genes is mutually exclusive. expressed vsg gene is normally due to genetic reassortments causing new alleles to be copied into the sites of expression. some trypanosomes with these abnormal vsg genes evade humoral immunity and multiply thereby causing re-infection and chronic recurring infections. 107 influenza is a viral infectious disease due to infection by any of the three types of rna viruses, namely influenza types a, b and c. current vaccines contain double type a and single type b strains and induce strong antibody responses to neuraminidase and the surface glycoprotein hemagglutinin. these vaccines, however, cannot effectively protect against newly emerging viruses with antigenic shift and drift. 108, 109 antigenic drift results in changes in the antigenic site (a minor change) while antigenic shift results in a new virus subtype. hemagglutinin and neuraminidase are the two enzymes dictating the antigenic properties of the viruses. while inside its host, defined host proteases break the peptide bonds in the hemagglutinin molecule to form hemagglutinin 1 and 2 subunits. virulence tendencies are decreased when the amino acids at the cleavage sites are lipophobic, the virus exhibits high virulence tendencies. 110 the surface glycoprotein can be regarded as antigen and hence can serve as a target for the immune system which if sequenced, using the new immunoinformatics approach and a common site for the varying proteins identified, a potent vaccine can be developed which can accommodate the antigenic drift/shifts of the virus. influenza viruses are able to thrive for a long while in a given human population. 111, 112 the virus has a high mutation rate such that a once effective vaccine can easily lose efficacy. antigenic variability is only one of the evidences of phenotypic variation in the biology of the influenza virus. the use of immunoinformatics in vaccine development has been accelerated toward the design of a multiepitope vaccine construct which has and will fully address the challenges faced with pathogens with mutagenic antigens. previous vaccines developed by conventional approaches consist of several proteins or a whole pathogen. this constitutes unwarranted antigenic load and increases the chances of inducing allergy. the use of peptide-based vaccines surmounts these challenges. the vaccines are made from short peptide fragments capable of eliciting highly specific immune responses, precision targeting and multiepitope constructs, in the case of varying antigenic peptides, which has been made feasible with the advancements in the field of computational biology. [113] [114] [115] [116] vaccines for pathogens with immune escape potentials can basically be constructed by using most, if not all, of their immunogenic peptides 116, 117 because such vaccines prove to be better than single-epitope and classical vaccines. multiepitope vaccines enjoy the following advantages over single-epitope and classical vaccines: a) they are an assemblage of several epitopes obtained from distinct protein targets/antigens of an intended infection; b) the multiple t-cell receptors (tcrs) in the vaccine recipient can easily recognize vaccines with multiple hla epitopes; c); they can be easily adjuvanted to improve their immunogenicity; d) they can activate antibody-mediated and cell-mediated immunological responses because of their overlapping helper t lymphocytes (htl), cd8+ t-cell and b-cell epitopes; and e) unwanted protein antigens are excluded in such construct thereby reducing the chances of untoward effects and/ or immune responses likely to cause disease(s). [118] [119] [120] [121] [122] [123] thus producing a vaccine with these qualities can provide chances of combating most infections such as streptococcus pneumoniae and hiv infections. immunoinformatics can be employed in the docking of single and multiepitope vaccines and subsequently to predict their properties (physicochemical, allergenic and antigenic). this approach has seen the use of diverse tools and database in the analysis of ligands with their targets and has greatly helped to predict the binding score of antigenic peptides with the immune proteins like hla. peptides and hla allele modeling can be done by the 3d structural designing of the epitopes using pepfold3 (an online server), 124 retrieving from protein data bank (pdb) the x-ray crystallographic structure of human population most occurring hla alleles (hla-drb1 01:01, 15:01 and hla-a 02:01) followed by filtration of previously bound ligands. the following is a step-wise detail on how to construct a single or multiepitope-based vaccine and its property prediction; • molecular docking analysis: to determine the interaction pattern of the screened out epitopes with the hla alleles by employing cluspro v.2 (a proteinprotein docking web server). this server performs this task by energy minimization, calculation of both the binding energy scores of the docked complex and electrostatic/shape complementarity. • target-protein comparative modeling and associated structure validation: the sequence of the amino acid in the target protein (e.g., tlr-9) can be retrieved from uniprot and the tertiary structure with raptor-x and i tasser (online comparative modeling tools). the server constructs and creates a 3d model immunotargets and therapy 2020:9 (mathematical representation) of the target protein using hierarchical algorithms. 118 personalized vaccine refers to vaccines "targeted" toward an optimized outcome. immunogenicity is maximized while either the risk of vaccine failure or reactogenicity and side effect is minimized. personalized are developed in the following cases; the individual level vaccines are developed to take care of haplotype and polymorphism knowing that they can retard the formation of a protective immune response or become pointers to the risk of an adverse vaccine reaction. this is needed when it is clear that females produce a higher antibody titre against a particular vaccine antigen than do their male counterparts. where it is clear that a particular human race or ethnic group has a higher or lower immune response to a particular vaccine antigen. personalized vaccines arise due to known complex interactions between host environmental, genetic and some other factors that may be influencing the vaccine immune responses. the associations between the immune response gene polymorphisms and variations in immune responses to a particular gene must be pine-pointed when it is clear that a particular drug either suppresses or augments the transcription of an immune response gene. 127, 128 this could help in designing vaccines or vaccine adjuvants that can circumvent restrictions due to immunological differences arising from varying genetic compositions. 129, 130 personalized vaccines stem from our understanding of how, within the human leukocyte antigen (hla) systemalso referred to as the major histocompatibility complex (mhc), the t cells are able to recognize peptides of pathogenic origin. 131, 132 hla molecules enjoy the double advantages of having stable polymorphisms and being fully characterized. 133 these advantages make good candidates for personalized vaccine design. hla polymorphism, although stable, is complex. for instance, more than 12,000 alleles of hla class i molecules and greater than 4000 class ii molecules have been identified among human populations. 133, 134 hla class i and ii molecules have heterodimeric character comprising of α and β chains, three highly variable extracellular domains (α1, α2, and α3) and then transmembrane and intracytoplasmic domains that are less variable. 133, 135 hla genes contain eight exons. exon 1 is responsible for producing the leader peptide; exons 2,3,4 produce α1, α2, and α3 extracellular domains, respectively, for mhc class 1 or α1, β1and α3, respectively, for mhc class ii; exon 5 encodes transmembrane anchor; exons 6 and 7 encodes the cytoplasmic tail while exon 8 encodes the 3ʹ-untranslated region. 135 most of the several forms associated with the class i and ii genes are seen in α-1 and α-2 (as known as class i) and in α-1 and β-1 (as known as class ii) domains. 133 mhc i and ii bind and present the peptide to t cells. t cell responses to viral pathogens differ from one patient to the other, basically because of the expression of differing hla (mhc) alleles which determine the several viral amino acid sequence brought to the t cells to read. 136, 137 it is most likely that during an infection, diverse epitopes are usually presented to the t cells to read owing to the several forms of hla alleles and also because each human person expresses six hla class i and six hla class ii alleles. 138 now, antibody-binding sites in a given hla (mhc) molecule are mostly prediction-servers predetermined on the basis of particular binding motifs and the anchor residues. 139, 140 these residues refer to known amino acids located at defined locations in the peptide chain and which are peculiar to each mhc molecule. 141, 142 prediction-server database of peptide motifs and/or of mhc ligands may be obtained from web-based and/or from prediction-servers dedicated to netmhc family. 143, 144 in another example, sequence analysis of lassa fever virus (the lasv) and other viruses' immunoproteomic was used to identify the best immunogenic protein predicting t-cell as well as b-cell epitopes and also target sequence and binding sites. 145, 146 the ssnlykgvy peptide sequence at aa41-49 of glycoprotein 1 (produced by the l segment) was the best candidate epitope for the induction of humoral as well as the cell-mediated immunity for lassa fever vaccine construct. 17 hla-i and 16 hla-ii molecules have been proven in sizable african populations and their combination with the ssnlykgvy peptide sequence may prove useful in such lassa fever virus endemic areas. 145 this approach will strongly improve individualized vaccination and help combat emerging infections. the hla region is suspected to contribute, to a large extent, to genetic propensity to infections and differences in vaccine expected immune responses. 132, 147 studies show that females exhibit stronger immune responses to immunization compared to males. 148, 149 there are differential antibody responses to rubella and measles viral protein between males and females and that both hormonal and genetic difference may be influencing the immune responses. 148, 150, 151 practical issues may stand in the way of achieving this new development (personalized vaccinology). having to use different vaccines for different persons based because of personal genetic composition requires more time and labor during the vaccination process. also, screening for individual factors for targeted vaccination can significantly increase vaccination cost. but with all these challenges, personalized vaccination is the new age approach in achieving an optimum immunization that takes into consideration the individual immune differences in a particular population and it is a new dawn for vaccine development. personalized vaccine development is strongly improved by vaccinomics. the field of vaccinomics looks at how immune response gene polymorphisms affect the cellmediated, humoral and innate immune responses to vaccine antigens at population and specific individual levels. "vaccinomics" encompasses both immunogenomics and immunogenetics as it concerns immune responses to vaccine antigen. 152 the fields of personalized vaccinology and vaccinomics were the products of phase i of the international hapmap and that of the human genome project. also, modern molecular assay techniques permitting highthroughput detection of variations at gene level, in particular linkage disequilibrium maps and single nucleotide polymorphism (snp), played significant roles in the development of personalized vaccinology and vaccinomics. it has also been shown that polymorphisms at vital immune response genes can bring about differing immune responses to biopharmaceutical products including vaccines. [152] [153] [154] newer, accurate, cheap and reproducible sequencing technologies; validated databases containing genotypephenotype data; statistical and bioinformatics tools are needed in order to analyze and interpret data that will help and improve vaccine adverse and immune response quantifiability and predictability. 155 the information will enhance clinical practice and accelerate rational and directed vaccine development. safe vaccines are a critical requirement for any immunization program. 156 conventional vaccination has been an approach targeted at all groups and individuals but has failed toward the enrolment of pregnant women into vaccination programs because of presumed fetal and maternal harms. 157, 158 evidence on the safety of vaccination in pregnancy is very small because pregnant women were usually excluded from participating in vaccine trials. 159 pregnancy can alter the maternal as well as fetal immunological responses. 160 it is pertinent to explore research opportunities presented in advanced vaccine designs such as immunoinformatics (multiepitope vaccine docking) by studying human immune system functions and responses specific to pregnant women and their unborn children. 157 according to a report 161 from the dominican republic of congo, during the 2016-2017 zika virus outbreaks, over a thousand pregnant women were suspected of being infected with the virus and a sizable number were at their first trimester. the report further stated that fetal loss was approximately one-tenth of the pregnancies and that there were up to 3 cases of fetus with head circumferences smaller than normal. the widespread morbidity during the epidemic showed that zika virus infection adversely affects pregnancy outcome. 160, 161 currently, there is no proof that pregnancy predisposes to ebola virus infections in comparison with the non-pregnant population, but there is some evidence suggesting pregnancy to worsen the disease prognosis including fetal loss. also, evidence showed that the virus can pass the placental barriers to establish infection in the unborn child. 162 designing, implementing and enrolling pregnant women as well as perspective pregnant women into vaccine trials and programs is imperative in order to protect this group and ensure good vaccine uptake by them during infection outbreaks and epidemics. 157, 163 these recommendations will give an informed decision to be investigated using the immunoinformatic tools to determine the immunogenic responses worthy of safe vaccine development for the pregnant women and perspective pregnant women group. maternal immunization offers palpable benefits in several ways. some vaccines are primarily administered to shield these pregnant women from morbid conditions and/ or death including fetal death. 164, 165 pregnant women stand the risk of being exposed to virulent pathogens and may be at a higher risk of morbidity and/or mortality when compared to the general population. 166 there has been an explosion of new immunological data (table 4 ) due to an increase in research to understand the immune system pathway in infectious disease pathogenesis and the application of the knowledge of bioinformatics has led to a better exposition of the immune system importance through immunoinformatics. the knowledge of immune system and the cost-effective, specific and effective approach like immunoinformatics, the concerns for emerging and re-surging diseases caused by pathogenic organisms, antigenic variability/complex lifecycle of pathogens and the need of personalized vaccination can be combated on a molecular level. the future of immunological research is sharpened by the ability to make discoveries in biologics (e.g., vaccines) more effectively and efficiently. this will mean reduction and better targeting of wet laboratory experiments and will only be possible if wet laboratory experimentation is combined with bioinformatics techniques. • immunoinformatics depends on experimental science (wet laboratory) to produce raw data for analysis. the predictions are not formal proofs of any concepts. they do not replace the traditional experimental research methods of actually testing hypotheses. • the quality of immunoinformatics predictions depends on the quality of data and the sophistication of the algorithms being used. sequence data from high-throughput analysis often contain errors. if the sequences are wrong, or annotations incorrect, the results from the downstream analysis are misleading as well. 167 immunotargets and therapy is an international, peer-reviewed open access journal focusing on the immunological basis of diseases, potential targets for immune based therapy and treatment protocols employed to improve patient management. basic immunology and physiology of the immune system in health, and disease will be also covered. in addition, the journal will focus on the impact of management programs and new therapeutic agents and protocols on patient perspectives such as quality of life, adherence and satisfaction. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. immunotargets and therapy 2020:9 time for t? immunoinformatics addresses vaccine design for neglected tropical and emerging infectious diseases vaccinology in the third millennium: scientific and social challenges antigenic variability: obstacles on the road to vaccines against traditionally difficult targets complex immune correlates of protection in hiv-1 vaccine efficacy trials immunoinformatics approach to design a novel epitope-based oral vaccine against helicobacter pylori immunoinformatics approach for multiepitopes vaccine prediction against glycoprotein b of avian infectious laryngotracheitis virus transcriptome and proteome: the rise of omics data and their integration in biomedical sciences neoantigen vaccine: an emerging tumor immunotherapy designing of cd8 + and cd8 + -overlapped cd4 + epitope vaccine by targeting late and early proteins of human papillomavirus immunization with a recombinant antigen composed of conserved blocks from tsa56 provides broad genotype protection against scrub typhus vaccine mediated protection against zika virus-induced congenital disease vaccination with sporozoites: models and correlates of protection novel approaches for the design, delivery and administration of vaccine technologies vaccination to gain humoral immune memory humoral and cell-mediated immune responses after a booster dose of hbv vaccine in hiv-infected children, adolescents and young adults an evaluation of the cold chain technology in south-east, nigeria using immunogenicity study on the measles vaccines a review of the immunological mechanisms following mucosal vaccination of finfish reverse vaccinology 2.0: human immunology instructs vaccine antigen design large screen approaches to identify novel malaria vaccine candidates reverse vaccinology and subtractive genomics reveal new therapeutic targets against mycoplasma pneumoniae: a causative agent of pneumonia evolution of the immune system in humans from infancy to old age the twilight of immunity: emerging concepts in aging of the immune system pneumococcal vaccination strategies. an update and perspective progress and challenges in tb vaccine development vaccines for the elderly: current use and future challenges remembrance of things past: long-term b cell memory after infection and vaccination global practices of meningococcal vaccine use and impact on invasive disease new vaccine technologies to combat outbreak situations what are the most powerful immunogen design vaccine strategies? reverse vaccinology 2.0 shows great promise the new malaria vaccine program for african children is promising but still quite limited. quartz africa merck's ebola vaccine helps combat deadly outbreak in the congo as the virus spreads. biotech and pharma innovation for the 'bottom 100 million': 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protein vibrations, classification and cross-paradigm de novo image generation using deep neural networks date: 2020-04-16 journal: nan doi: nan sha: doc_id: 136540 cord_uid: 2h2braww in recent work we reported the vibrational spectrum of more than 100,000 known protein structures, and a self-consistent sonification method to render the spectrum in the audible range of frequencies (extreme mechanics letters, 2019). here we present a method to transform these molecular vibrations into materialized vibrations of thin water films using acoustic actuators, leading to complex patterns of surface waves, and using the resulting macroscopic images in further processing using deep convolutional neural networks. specifically, the patterns of water surface waves for each protein structure is used to build training sets for neural networks, aimed to classify and further process the patterns. once trained, the neural network model is capable of discerning different proteins solely by analyzing the macroscopic surface wave patterns in the water film. not only can the method distinguish different types of proteins (e.g. alpha-helix vs hybrids of alpha-helices and beta-sheets), but it is also capable of determining different folding states of the same protein, or the binding events of proteins to ligands. using the deepdream algorithm, instances of key features of the deep neural network can be made visible in a range of images, allowing us to explore the inner workings of protein surface wave patter neural networks, as well as the creation of new images by finding and highlighting features of protein molecular spectra in a range of photographic input. the integration of the water-focused realization of cymatics, combined with neural networks and especially generative methods, offer a new direction to realize materiomusical"inceptionism"as a possible direction in nano-inspired art. the method could have applications for detecting different protein structures, the effect of mutations, or uses in medical imaging and diagnostics, with broad impact in nano-to-macro transitions. proteins are the basic building blocks of life. they form materials as diverse silk, cells, and hair, but also offering other functions from enzymes to drugs, and pathogens like viruses [1] [2] [3] [4] [5] [6] . while we cannot see small nanoscopic objects like proteins or other molecules, a common feature is their continuous motion, or vibration, that can be understood as an overlay of fundamental normal modes each consisting of harmonic waves. in recent work [1] we reported the vibrational spectrum of more than 100,000 known protein structures, and a self-consistent sonification method to render their complex vibrational spectrum in the audible range of frequencies. the sonification work has also been used to train generative neural networks to facilitate the design of de novo proteins using machine learning [2, 7] . this article focuses on a different perspective and reports a distinct, complementary and translational approach, in which we transform these molecular vibrations into vibrations of thin water films using acoustic actuators, leading to visual images of complex materialized patterns of surface waves. this approach follows the pioneering concept developed by chladni [8] and is similar to the cymatics method [9] [10] [11] [12] [13] [14] , offers a distinct means to assess the molecular details of protein vibrations in the form of macroscopic vibrations visible to our eyes. in addition to potential applications in outreach, the physical manifestation of molecular vibrations at the macroscale provides a novel avenue to render sound visible, providing an alternative method to conventional musical notation [11] and novel interactive approaches to interact with sound using senses other than our ears [15] [16] [17] [18] . moreover, the use of artificial intelligence provides an exciting avenue to classify, process and understand, or augment images generated by sound. we will explore some of these augmentation concepts in this paper, by highlighting key features of images as detected by distinct layers in deep neural networks. these connections between sound, materialization and images can offer many avenues for future research, enabled by significant progress made in recent years in computer vision [2, [19] [20] [21] [22] [23] [24] [25] . figure 1a shows the overall flow of the research reported here. the approach includes the calculation of molecular vibrational spectra transposed to audible frequencies and made audible (see earlier work for details, [1, 6] ), which are then used to excite a thin film of water. images of surface wave patterns are collected, building a training set for a machine learning model. the predictive power of the classifier model is validated, showing that the model can correctly determine the protein structure solely based on images of the surface wave patterns. figure 1b depicts an overview of eight distinct protein structures (chosen to reflect different complexity and size in its molecular structure) that are investigated in this paper. sonification is a method to translate data structures into audible signals, which has been explored widely as a means to better understand scientific data in a range of areas of application including spider webs, proteins, and other systems [26] [27] [28] [29] [30] [31] [32] [33] [34] . here, we utilize the protein synthesizer published in earlier work [1] and generate various audio signals that are fed into an actuator attached to a petri dish with a thin layer of water. all protein codes are expressed as protein data bank (pdb) [35] identifier, and can be downloaded and further explored at www.rcsb.org. the experimental setup is shown in figure 2 . the system consists of an actuator attached to a petri dish. the actuator (dayton audio daex25 sound exciter, driven by an analog amplifier) is driven by an amplifier, who receives the analog audio signal directly from a digital-to-analog (d/a) audio interface in a laptop computer. a 2,100 lumens led light source is used for illumination during image capturing (winplus, led folding worklight). the actuator is fixed permanently to a solid and immobile substrate with high mass. the petri dish has been spray-painted white for a clear optical signal (unless indicated otherwise; as we generated some images with a reflective aluminum foil background for better contrast). water is inserted into the petri dish with a height of 1 mm. the higher the water level, the more actuation energy is needed, so we pick a level that provides with significant surface wave generation at the available actuation energy. a camera is mounted at a fixed location for consistent images. the digital audio workstation (daw) ableton live [36] and max/msp [37] patches, as described in earlier work [1] , are utilized here to generate the audio signals; these provide implementations of the protein synthesizer. a few comparative simple, pure sine wave forms are generated as well. images are taken in the form of a continuous video, recorded at 60 fps, and with 1920x1080 resolution, recorded with a samsung galaxy s10+ 5g camera (the camera combines a primary 12 mp camera and variable-aperture lens with a 16 mp ultra-wide-angle component). the video is stored, and subsequently individual frames are extracted, using a python code. the images are cropped consistently so as to focus on the surface wave structures inside the petri dish, without showing a boundary. each image has a size of 585x788 after such processing, and before being fed into the neural network. no additional image processing is conducted. figure 3b shows an example of the surface wave structures that emerges in the experiment, as used in the deep neural network training. the majority of images are used for training (80%), and 20% for validation. to test the prediction of the model, new data from additional imaging of the experiment is used. two neural networks are considered in this work. first, an existing resnet neural network model as reported in [38] that has been pre-trained against a large dataset (https://tfhub.dev/tensorflow/resnet_50/feature_vector/1). this tensorflow hub model uses the implementation of resnet with 50 layers, and contains a pre-trained instance of the network, arranged to get feature vectors from images. we use transfer learning to retrain all parameters in the neural network, by adding a dense layer after the feature layer to add a new set of classifications to reflect the distinct audio sources of molecular spectra. the resnet model features 23,587,789 parameters [38] . second, a custom designed neural network with much fewer parameters (consisting of alternating convolutional and pooling layers), as a comparative approach. figure 4 depicts a summary of this neural network design. the model consists of multiple sequences of convolutional and pooling layers, following a standard image classifier design. the custom designed neural network features a total of 945,165 parameters, and 20 layers. we use deepdream [39] to generate novel images by activating select layers in the deep neural network, based on the model trained against the water surface images. this method is an approach to visualize the patterns learned by a neural network, and results in novel images. it can be understood similar to the process of an interpretation of structures in clouds, for instance, in which we try to interpret shapes and forms from the abstract patterns. in the case of the neural network, patterns it sees in a given image are interpreted and then accentuated. the method works by processing an image through a neural network, then calculating the gradient of the image with respect to the activations of a set of layers. the image is then adapted to enhance the patterns seen by the neural network. in processing the images, we explore different layers for the activation algorithm, each resulting in distinct features being highlighted in the image (e.g. internal convolutional layer vs. internal pooling layers). the purpose of this computation is to explore the inner structures and features learned by the neural network, and make them visible. we collect a number of images, around 1,000 for each case, and feed the labeled images into the neural network training process. a total number of 13 labels are used, reflecting 13 distinct frequency spectra corresponding to the individual protein structures considered here. the audio signals of a consistent, stationary spectrum corresponding to the molecular vibrational signature of each of these structures are fed into the actuator. the cases included in the training set are: -flat: no actuation (i.e., no audio signal) -pure sine waves: 3 distinct frequencies (sine33 = 50 hz, sine43 = 98 hz, sine65 = 349 hz) -various protein structures, including protein data bank ids: 6vsb, 107m, 5xdj, 6m17, 6m18 (and others). figure 1b depicts an overview of all proteins considered in this study, where the first row represents relatively simple structures (added complexity from left (short alpha-helix) to right (more complex alpha-helical fold). the lower row features more complex protein structures, including two spike proteins from the pathogen of covid-19 (two distinct molecular states -6vxx and 6vsb of covid-19), as well as the covid-19 spike protein bound (6m17) and unbound (6m18) to the human ace2 receptor. figure 5 depicts the results of the training process for both neural network formulations. both cases show reasonable convergence. the pre-trained resnet case ( figure 5a ) converges faster and in a more stable manner, however. this makes sense given that the model starts from pre-trained parameters. figure 6 shows results of a classification test suite conducted with the two trained models, depicting averaged values for a series of unknown, new images, fed to the neural network. the radar plot shows the score for each of the 13 labels (each is denoted out of 1.00). the data shows that both models predict the case very well, and without error. it can also be seen that the score for the correct prediction is quite pronounced, showing that the classifier works very well and can distinctly classify the correct protein structure at the origin of the acoustic actuation. this can be verified by the fact that the highest score in the plot is close to 1.00, and that there is a distinct peak for each of the cases. going into more detail, we analyze a few specific cases. for instance, the orange color represents the scores for protein 1akg. the highest score, in both models, is for the label 1akg, which indicates correct classification. for 3tnu, the highest score is 3tnu, and so on. notably, both models are capable of predicting the correct labels, solely from images of surface waves, for all cases studied. it is remarkable that the method works for a variety of protein structures with distinct frequency spectra from complex to simple including the pure sine wave cases at different frequencies. it also renders correct predictions for proteins with distinct geometric dimensions. for instance, 1akg is a very small protein that features only a simple fold and a short alpha-helix segment. 3tnu is a small alpha-helix fragment without any folding. in contrast, 6vsb is a very large protein. other interesting cases are 6m17 and 6m18. the protein 6m17 shows only minute distinctions from 6m18 in terms of its vibrational spectrum, as is shown in figure 7 . yet, the model can distinguish the cases very well. figure 10 shows a similar experiment, this time applied to the same image, but considering the result for using individual layers to generate features. the collection of examples of images visualize features seen by the internal layers of the deep neural network, by selecting different layers. figure 11 shows an analysis of images with deepdream,using the inception deep neural network [19] . unlike in the cases discussed above, here the neural network model used is taken "as trained" against a large number of conventional images (and has not been trained against the dataset reported here). it is clear that the conventional image classification model features very distinct patterns, reflecting the characteristic features learned in a conventional image classifier. it offers interesting insights into what image features are seen by a model like inception in the abstract representation of surface wave patterns induced by proteins. the new experimental-computational method reported here provides a new way to analyze and visualize molecular vibrations expressed in sound. while the method could in principle be used to predict visuals of conventional audio signals (e.g. specific musical instruments, songs, varied classes of music, etc.) the focus in this paper was on determining the protein structure solely from the pattern of surface waves. while cymatics, or the general attempt to make sound into images is a concept explored since ancient times (since the original works in the late 1700s [8, 9] ), here we report for the first time a method to visualize molecular vibrations in protein structures in that way, and offer a path to integrate it with artificial intelligence methods. the integration of the water-focused rendering of cymatics, combined with neural networks and especially generative methods, offer a new direction to realize materiomusical "inceptionism" [39] as a possible future direction in nano-inspired art. our method shows that even minute changes in the molecular vibrational spectra (e.g. 6m17 vs 6m18, as shown in figure 6 ) can be detected through this method, as well as different states of proteins (e.g. different molecular states of the covid-19 virus spike protein, 6vxx vs 6vsb).since we now have the vibrational spectra of all known protein structures available [1] , the approach reported here could potentially be used to build a very large dataset to render visual macroscopic representations of all known proteins. while this is beyond the scope of the present study, which focused on the establishment of the basic methodology, such nano-to-macro translations could find applications in a variety of fields. further work could also be done by providing detailed fluid-structure-interaction models of how the specific patterns emerge. such research may provide a theoretical basis to complement the experimental research done here. in terms of limitations, the method reported here can only classify protein structures that the neural network has been trained against. however, future work could seek to generalize the method. other limitations are the dependence of the images on the experimental setup. it is likely that the patterns of surface waves depend on the particular geometries of the experimental setup, such as the water level, the size of the contained, the power output of the actuators and so on. more broadly, looking ahead, the method can find applications as a means to accentuate images, find patterns in other biomaterials, or to use the approach in detecting patterns across manifestations of domains. figure 1 : a, overall flow of this research, ranging from the molecular vibrational spectra transposed to audible frequencies, and used to excite a thin film of water using an actuator. images of surface wave patterns are collected, building a training set for a machine learning model. the predictive power of the classifier model is validated, showing that the model can correctly determine the protein structure solely based on images of the surface wave patterns. b, overview of the various proteins studied in this paper. the first row represents relatively simple structures (added complexity from left (short alpha-helix) to right (more complex alpha-helical fold). the lower row features more complex protein structures, including two spike proteins from coronaviruses (6vxx and 6vsb of covid-19), as well as the covid-19 spike protein bound (6m17) and unbound (6m18) to the human ace2 receptor. experimental setup used in this study. a think water film in a petri dish is excited by an actuator, resulting in surface waves, as can be seen in the right column labelled "top". the resulting structures are captured using a high-resolution camera, and collected as a training set for machine learning. : results of the classifier as applied to a new data set that wasn't included in training or validation. the data is plotted in a radar plot (1.0 = the neural network is capable of correctly labeling the image, 0.0 reflects worst performance). we plot averaged scores, providing average values over classifications for multiple images. the data shows that both neural networks are capable of identifying different protein structures, as well as the test cases of sine waves (at different frequencies), as well as silence. for instance, the orange color represents the scores for 1akg. the highest score, in both models, is for the label 1akg, which indicates correct classification. for 3tnu, the highest score is 3tnu, and so on. both models are capable of predicting the correct labels, solely from images of surface waves, for all cases studied. in spite of the small differences in the structure and the frequency spectrum as depicted in the figure, the machine learning model is capable of easily distinguishing the patterns generated from these audio signals. a, normal mode frequencies of 6m17 and 6m18 over the mode number. b, spectrogram of the resulting audio signal, using the method reported in [1] . an application of deepdream [39] to generate novel images by activating select layers in the deep neural network, based on the model trained against the water surface images. this approach offers a new way to design generative art from molecular vibrations. the top image is the original, the bottom the processed image. this example shows how the method can be a powerful method to better understand features seen and learned in the neural network, which can be used to accentuate key elements in images. : analysis of images with deepdream, using the inception deep neural network [19] (note, this model is taken "as is", and has not been trained against the dataset reported here). it is clear that the conventional image classification model features very distinct patterns, reflecting the characteristic features learned in a conventional image classifier. the left column shows the processing resulting from a mountain landscape (same as in figure 9 , right column) for comparison. the right column shows the water surface wave pattern as source image. analysis of the vibrational and sound spectrum of over 100,000 protein structures and application in sonification a self-consistent sonification method to translate amino acid sequences into musical compositions and application in protein design using ai reoccurring patterns in hierarchical protein materials and music: the power of analogies materials by design -a perspective from atoms to structures mater. impuls. natur, acatec nanomechanical sonification of the 2019-ncov coronavirus spike protein through a materiomusical approach sonification based de novo protein design using artificial intelligence, structure prediction and analysis using molecular modeling entdeckungen ã¼ber die theorie des klanges cymatics for the cloaking of flexural vibrations in a structured plate experimental study of cymatics, wcse 2012 -int cymasense: a real-time 3d cymatics-based sound visualisation tool discovering an uncanny world: cymatics software and the journey to the creation of knowledge within the field of contemporary cymatics nanoscale self-organization using standing surface acoustic waves cymatics" of selenium and tellurium films deposited in vacuum on vibrating substrates interacting with 3d reactive widgets for musical performance imaging soundscapes : identifying cognitive associations between auditory and visual dimensions synesthesia and cross-modality in contemporary audiovisuals deep residual learning for image recognition accelerated search and design of stretchable graphene kirigami using machine learning human-level control through deep reinforcement learning bioinspired hierarchical composite design using machine learning: simulation, additive manufacturing, and experiment convolutional lstm network: a machine learning approach for precipitation nowcasting gradient-based learning applied to document recognition world cloud: a prototype data choralification of text documents sonification report: status of the field and research agenda sublime frequencies: the construction of sublime listening experiences in the sonification of scientific data research set to music: the climate symphony and other sonifications of ice core, radar, dna, seismic and solar wind data a systematic review of mapping strategies for the sonification of physical quantities tuning the instrument: sonic properties in the spider's web decoding the locational information in the orb web vibrations of araneus diadematus and zygiella x-notata tangled web imaging and analysis of a three-dimensional spider web architecture a series of pdb related databases for everyday needs deep residual learning for image recognition inceptionism: going deeper into neural networks acknowledgements: this work was supported by mit cast via a grant from the mellon foundation, with additional support from onr (grant # n00014-16-1-2333) and nih u01 eb014976. key: cord-018437-yjvwa1ot authors: mitchell, michael title: taxonomy date: 2013-08-26 journal: viruses and the lung doi: 10.1007/978-3-642-40605-8_3 sha: doc_id: 18437 cord_uid: yjvwa1ot this chapter addresses the classification and taxonomy of viruses with special attention to viruses that show pneumotropic properties. information provided in this chapter supplements that provided in other chapters in parts ii–v of this volume that discuss individual viral pathogens. taxonomy may be defi ned as a logical discipline for the identifi cation and classifi cation of biological entities based on objective, measurable characteristics of relevant entities. useful taxonomic systems should be broadly applicable across diverse types of biological groups. they should also be fl exible, so that new data from technological advances may be integrated into the classifi cation scheme. primary goals of systemic taxonomy, regardless of biological discipline, include the following: • establishing groups (taxa) that refl ect varying degrees of evolutionary relatedness among the different biological entities studied • establishing criteria for assignment of known or unknown clinical isolates to a given group • establishing a clear and unequivocal nomenclature the origins of biological taxonomy are fi rmly rooted in botany and zoology. early taxonomic systems relied on gross characteristics, like biological niche, internal and external morphology, reproductive strategies and compatibilities, and fossil records. the seminal works of the swedish botanist carl linnaeus used a hierarchical scheme to represent biological relatedness and established the simplifi ed binomial system of nomenclature that serves as the basis for modern classifi cation systems. the modern scientifi c classifi cation in biology is designed to describe all biological entities within a hierarchy consisting of the following taxa: a basic assumption for the establishment of such a hierarchy assumes that all biological entities have evolved from a single common cellular life-form. different biological entities have evolved as a result of accumulated changes in dna that have provided survival advantages in different ecological niches. species may be classifi ed on the basis of phylogenetic and evolutionary relatedness: members of a given species are the most closely related, different species within a single genus are more closely related to each other than to a species within a different genus, and so on. newer technologies like microscopy, improved biochemical and physiological analysis, and advanced protein and molecular analytical methods have resulted in an enormous expansion of characteristics that may be studied for the classifi cation of biological entities and validation of taxonomic systems (woese et al. 1990 ). there are a number of excellent texts that discuss the clinical and laboratory aspects of virus biology (knipe and howley 2007 ; richman et al. 2009 ; versalovic 2012 ) . though viruses are certainly "biological entities," they are fundamentally different from the cellular life-forms classifi ed by previous taxonomic schemes. viruses have no autonomous metabolic or replicative ability; they are completely dependent on cellular life-forms. however, within their biological milieu, viruses do replicate and evolve, and they are composed of the same types of organic macromolecules as are cellular lifeforms. because of their intimate relationship with cellular life-forms, it seems legitimate to integrate the schemes for classifi cation of viruses with the schemes used for biological classifi cation of cellular life-forms (lefkowitz 2012 ) . initially, various features, like host range, crossimmunity, clinical disease, and pathologic features, were used to classify viruses. technological advances have led to more detailed and integrated classifi cation, taxonomy, and phylogenetic characterization (evolutionary relatedness) of viruses. sophisticated nucleic acid sequence analysis has emerged as a powerful tool for virus classifi cation and phylogenetic determination, in spite of some limitations (holmes 2008 ; mccormack and clewley 2002 ; zanotto et al. 1996 ) . a robust system for classifi cation of viruses developed by david baltimore has gained wide acceptance (baltimore 1971 ) . classifi cation is based on the genomic nucleic acid used by the virus (dna or rna), strandedness (single or double stranded), and method of replication. the system has been used to defi ne seven classes of viruses: class i: double-stranded dna (dsdna) class ii : single-stranded dna (ssdna) the primary classifi cation of viruses is into species. a virus species is defi ned as a polythetic class of viruses that constitute a replicating lineage and occupy a specifi c ecological niche (international committee on taxonomy of viruses 2002 ). in polythetic classifi cations, group members share a number of characteristics, but no single characteristic is necessary or suffi cient to defi ne members of the group. higher-level taxa are monothetic, i.e., there are characteristics that are necessary and suffi cient to defi ne members of the class. it is important to note that not all viruses can be assigned through all taxonomic levels. virus species may be assigned to a genus or remain unassigned. similarly, a genus may be assigned to a family or subfamily, or remain unassigned, and so on up the taxonomic hierarchy. each genus has a type species . the type species is the virus that necessitated the creation of the genus; it is always linked to the genus. in the most recent publication (2012), the ictv recognized 7 orders, 96 families, 22 subfamilies, 420 genera, and 2,618 species. important characteristics used by the ictv to defi ne and classify viruses within these taxa include the following: • susceptible host range : most viruses have a restricted range of hosts which they are able to infect. • virus structure : the viral genome is surrounded by a protective shell of proteins called a capsid. the capsid may also enclose proteins, like reverse transcriptase or proteins required for organization of the nucleocapsid. a nucleocapsid refers to a viral nucleus surrounded by an intact capsid. the nucleocapsids of certain viruses are also surrounded by an envelope of host-derived membranes. the complete virus particle is referred to as a virion. icosahedral capsids are very common; these quasi-spherical shells are composed of 20 identical equilateral triangles with 30 edges and 12 vertices. icosahedral capsids are very effi cient geometrically (internal volume versus protein content) and genetically (many small sides require fewer and smaller genes to code for capsid proteins). the nucleocapsid proteins of some viruses, like the infl uenza viruses, form helical tubes with the nucleic acid incorporated directly into the helical structure. the nucleocapsids of some viruses are surrounded by envelopes composed of lipid bilayers and host-or viral-encoded proteins. envelopes are typically acquired by budding of the nucleocapsid through a virally modifi ed portion of a specifi c host-cell membrane (plasma, endoplasmic reticulum, golgi, nucleus) . the shape of the virus nucleocapsid or intact virion is usually determined by electron microscopy. the shape and dimensions of the nucleocapsid and intact virion, and the presence or absence of an envelope, are useful characteristics for classifying viruses. • genome : the viral genome is either dna or rna; the nucleic acids may be single or double stranded. the genome size may be expressed in terms of kilobases (kb) for singlestranded genomes or kilobase pairs (kbp) for double-stranded genomes. the sequence of genes of positive-sense ssrna may be directly translated by the host into viral proteins. the sequence of negative-sense ssrna is complementary to the coding sequence for translation, so mrna must be synthesized by rna polymerase, typically carried within the virion, before translation into viral proteins. the sequence of positive-sense ssdna is the same as that of the mrna coding for viral proteins; negative-sense ssdna is complementary to mrna and may be transcribed into mrna for viral protein synthesis. ambisense single-stranded nucleic acids use both positive-sense and negative-sense sequences. the viral nucleic acid may be linear or circular; the nucleic acid may be in the form of a single molecule or broken into two or more segments. in addition to the type of nucleic acid, the size of the viral genome, measured in number of bases or base pairs, is an important characteristic used for classifi cation. • nucleic acid sequence analysis : the analysis of specifi c viral nucleic acid sequences is increasingly used as a powerful tool for taxonomic assignment and assessment of evolutionary relatedness. the utility is greatest for related groups of viruses (lauber and gorbalenya 2012a , b ) , but has been challenging for more divergent groups of viruses. sequence analysis alone has not provided a reliable single criterion on which all viruses may be classifi ed. construction of a universal phylogenetic tree for viruses, as has been proposed for cellular life-forms, may not be possible for viruses. it is not clear that all viruses emerged from a single progenitor virus; there is evidence for multiple, independent origins of existing viruses. phylogenetic analysis using nucleic acid sequences is further complicated by recombination, reassortment, incorporation of host nucleic acid sequences, and other factors (domingo 2007 ; holmes 2011 ) . currently, expert consensus, considering laboratory, phenotypic, clinical, and other characteristics, remains the most accurate and robust method for the classifi cation and taxonomic assignment of viruses. note that the formal names assigned at all taxonomic levels are italicized, while the common names, which are often used clinically, are not italicized. the viruses that have been associated with human infections are shown in table 3 .1 . among the families of viruses able to infect humans and other vertebrate hosts, there are many species that target and cause disease in the lung. these viruses commonly use airborne transmission as an effective mode of transmission between an infected host and a new susceptible host. characteristics of viruses that directly or indirectly cause pulmonary disease are discussed in this section. adenoviridae: adenoviruses are pathogenic for humans and other vertebrate species. a structural protein at each of the 12 of the icosahedral nucleocapsid vertices anchors a rodlike projection with a terminal knob, which interacts with specifi c host surface receptor molecules and which confers the hemagglutination pattern and tissue tropism for the different groups of adenoviruses. the genome encodes ~40 genes (davison et al. 2003a ) , including common genes and species-specifi c genes. genes are grouped into early, delayed early, and late transcribed genes. the genome contains inverted repeat sequences at both ends. sequences of both dna strands are transcribed to mrna; mrna splicing is used for expression of many adenovirus genes. the family adenoviridae has not been assigned to an order. within this family, there are fi ve genera. the seven species that cause human infection are human adenovirus a, b, c, d, e, f, and g , all within the mastadenovirus genus; there are 57 accepted serotypes (buckwalter et al. 2012 ) . endemic respiratory infections are most commonly caused by serotypes of human adenovirus c (the type species of the genus); most epidemic respiratory infections are caused by serotypes within species adenovirus b and adenovirus e . arenaviridae : arenaviruses may cause several hemorrhagic fever syndromes. specifi c rodents are the reservoir for each arenavirus; human disease is incidental and is usually transmitted by infectious aerosols. viruses of this family are enveloped; evenly spaced glycoprotein complexes (a tetramer of viral gp2 with viral gp1 ionically bound as a globular head) are attached to the envelope giving complete virions a studded spherical morphology. complete virions are ~100 nm in diameter, but show signifi cant pleomorphism (range, 60-300 nm). the genome is divided into two segments which are complexed with nucleoproteins (peters 2009 ) . complementary sequences at the 3′ and 5′ ends of each segment result in the formation of two circular nucleocapsids. arenaviruses use both negative-sense and ambisense coding strategies. host ribosomes are often incorporated within the envelope of complete virions. this family of viruses is not assigned to an order. there is one genus, arenavirus , with 25 species that fall into two complexes on the basis of serologic and genetic relatedness. the old world, or african, species include lassa virus (lassa fever) and lujo virus. the new world species include guanarito virus (venezuelan hf), junín virus (argentine hf), and machupo virus (bolivian hf). the type species of the genus arenavirus is lymphocytic choriomeningitis virus . bunyaviridae : bunyaviruses may cause several hemorrhagic fever syndromes. viruses coronaviridae : transmembrane proteins produce blunt projections from the surface of coronaviruses, resulting in a "crown-like" appearance on electron microscopic studies (100-160 nm in diameter). translation of the coronavirus genome is unique and includes production of polyproteins, discontinuous synthesis, overlapping reading frames, ribosomal frame shifting, and post-translational proteolytic processing (marra et al. 2003 ; rota et al. 2003 ; theil et al. 2003 ) . the major structural proteins, spike glycoprotein (s), membrane glycoprotein (m), nucleocapsid phosphoprotein (n), hemagglutinin-esterase glycoprotein (he), and envelope protein (e), are present in all coronaviruses. nonstructural proteins are encoded in 5-10 unique or overlapping reading frames (lai et al. 2007 ). the human coronaviruses are assigned to the order nidovirales , family coronaviridae , and subfamily coronavirinae . there are four genera and three serological groups. relevant viruses include human coronavirus 229e and human coronavirus nl63 of the genus alphacoronavirus (antigenic group i), human coronavirus hku1, betacoronavirus 1 and severe acute respiratory syndrome-related coronavirus of the genus betacoronavirus (antigenic group ii). filoviridae : filoviruses may cause several hemorrhagic fever syndromes. the fi loviruses have a unique threadlike morphology. the helical nucleocapsids are surrounded by an envelope studded by spikes formed by a single type of glycoprotein (gp). the genome consists of a single segment of negative-sense ssrna that encodes for seven proteins (kuhn et al. 2010 ) . the presence of gene overlap for several genes is an unusual feature of fi loviruses. in ebolaviruses, the surface glycoprotein is encoded by two adjacent reading frames. a truncated version (sgp), which lacks the hydrophobic anchor, results from translation of the upstream reading frame only. this protein is secreted from cells and may serve as a decoy for the host's immunological response. the full-length gp is formed only when the rna polymerase misreads a poly-u editing site between the reading frames. the fulllength gp is inserted, as homotrimers, into the host membranes that will form the virion envelope. a helical nucleocapsid is formed by association of the ssrna with nucleoproteins. the nucleocapsid is ~50 nm in diameter, with a central axial space ~20 nm in diameter. the nucleocapsid is attached to the envelope by matrix protein. the complete virions are ~80 nm in diameter, but the virion length may vary from 800 to 10,000 nm. the family filoviridae is assigned to the order mononegavirales . there are two genera within the family ebolavirus and marburgvirus . there are fi ve ebolavirus species, including sudan ebolavirus and zaire ebolavirus (the type species). the genus marburgvirus consists of one species, marburg marburgvirus . humans and nonhuman primates are susceptible to ebolavirus and marburgvirus infection; the host reservoirs for these viruses are unknown. humans may be infected sporadically by presumed contact with the host species or by direct contact with virus containing body fl uids taken from acutely infected humans or nonhuman primates. nosocomial and laboratoryacquired infections are well described. flaviviridae : flaviviruses may cause several hemorrhagic fever syndromes. hepatitis c virus is also a fl avivirus species. flaviviruses are surrounded by an envelope studded with dimers of viral e glycoprotein and m protein which give the mature virion a herringbone appearance with icosahedral symmetry. the genome consists of a single segment of positive-sense ssrna (chambers et al. 1990 ; osatomi and sumiyoshi 1990 ) . cyclization of the genome, through hybridization of rna sequences of the 5′ and 3′ ends of the genome, may be required for mrna synthesis (alvarez et al. 2005 ). there is a long open reading frame that codes for three structural proteins at the 5′ end; downstream of this region are genes for seven nonstructural proteins (thurner et al. 2004 ). the positive-sense genome is directly translated into a large polyprotein, which undergoes intra-and post-translational cleavage. strain evolution and clinical diversity have been driven by a high rate of mutation at replication and through molecular recombination. the nucleocapsid is formed by interaction of genomic rna with capsid proteins. the complete virion has a spherical morphology approximately 50 nm in diameter. this family of viruses is not assigned to an order. there are four genera within the family flaviviridae . within the genus flavivirus , there are 53 species, including dengue virus (simmons et al. 2012 hepatitis c virus (hcv) is the type species of the genus hepacivirus in the family flaviviridae . the physical properties of hcv have not been as well defi ned as other fl aviviruses because there is no effi cient method for in vitro replication of hcv. virion morphology is consistent with other fl aviviruses; complete, enveloped virions have a diameter of 55-65 nm. the single segment positive-sense ssrna is ~9.6 kb in length (hijikata et al. 1991 ) . a single open reading frame is fl anked by highly conserved regions at the 5′ and 3′ ends. cap-independent protein synthesis, typical of flavivirus species, is initiated at an internal ribosomal entry site (ires) within the 5′ untranslated region. this results in synthesis of a polyprotein that undergoes cleavage and further processing during and after translation. a unique and highly conserved sequence upstream of the ires interacts with liver-specifi c microrna and is required for effi cient replication. circulating hcv is associated with host ldl/vldl, which may play a role in delivery of virions to hepatocytes. the error-prone rna polymerase and high replication rate of hcv has resulted in a great genetic diversity and heterogeneity of clinical isolates. hcv isolates can be grouped by genotypic analysis into six groups and many subgroups. there are differences with respect to responses to antiviral therapy among the genotypes, but intrinsic virulence is similar. the vast majority of strains in the united states are genotypes 1a, 1b, and 2, whereas central african strains are almost exclusively genotype 4. hemorrhagic fever (hf) syndromes : viral hemorrhagic fever syndromes may be caused by many species of viruses from four different families: arenaviridae, bunyaviridae, flaviviridae and filoviridae ; all are single-stranded rna viruses. see the discussions above for specifi c information related to these virus families. typical symptoms of viral hemorrhagic fever infection include fever, malaise, hypotension, and coagulation defects. with the exception of dengue, the other hf viral agents are maintained in nonhuman vertebrate hosts; humans are coincidental, dead-end hosts. in dengue, human infection is maintained through a mosquito vector. the epidemiologic distribution of disease refl ects the geographic range of the reservoir host. hf viruses primarily infect dendritic cells, macrocytes, and monocytes, which are present in virtually all tissues and organ systems; parenchymal cells may also be susceptible to infection, depending on the virus. infected cells release mediators that result in marked increased vascular permeability, compromising the function of critical organ systems. suppression of cellular type 1 interferon response is a signifi cant contributor to pathogenesis (habjan et al. 2008 ) . hepadnaviridae: in the family hepadnaviridae , there are two genera, avihepadnavirus (two species) and orthohepadnavirus (four species); hepatitis b virus (hbv), the type species of orthohepadnavirus , is only human pathogen in family. the family hepadnaviridae is not assigned to an order. eight distinct hbv genotypes (a-h) and subtypes can be recognized on the basis of antigenic or sequence variation. the genotypes show geographic and ethnic variability; the hbv genotype infl uences the severity and outcome of disease (garfein et al. 2004 ; lin and kao 2008 ) . the complete, enveloped hbv virion (dane particle) is 42-47 nm in diameter. the icosahedral nucleocapsid (~28 nm in diameter) of the virion contains a single molecule of partially double-stranded dna with a dna-dependent polymerase covalently linked to the 5′ end of the complete dna strand, hepatitis b e antigen (hbeag) and hepatitis b core antigen (hbcag). the nucleocapsid is surrounded by an envelope derived from host-cell membrane and viral envelope proteins, including hepatitis b surface antigen. the genome of hbv is a circular, partially double-stranded dna molecule which is replicated by a unique process of reverse transcription of an rna intermediate. the minus dna strand runs the entire length of the hbv genome; the plus strand covers only about two-thirds of the genome. the genome is replicated by synthesis of a fulllength ssrna transcript (pre-genomic rna), followed by dsdna synthesis by reverse transcription of the ssrna by viral-encoded reverse transcriptase/dna polymerase. all viral proteins are also transcribed from the minus dna strand. there are four overlapping open reading frames, all read in the same direction (liang 2009 ) . herpesviridae : the herpesvirus species associated with human infections (hsv-1, hsv-2, cmv, ebv, vzv, hhv-6, hhv-7, and hhv-8) belong to the family herpesviridae within the order herpesvirales . there are four subfamilies of the herpesviridae : alphaherpesvirinae (5 genera), betaherpesvirinae (4 genera), gammaherpesvirinae (4 genera), and a single genus in an unassigned subfamily. specifi c human herpesviruses are discussed in the sections below. the herpesviruses are double-stranded dna viruses. the icosahedral capsid (~100 nm diameter) is surrounded by an envelope studded by a variety of short glycoproteins. the nucleocapsid is a dense toroid complex with an outer diameter ~70 nm and inner diameter ~18 nm. an irregular "tegument" fi lls the space between the envelope and capsid. depending of the thickness of the tegument layer, complete virions range in size from ~125 to >250 nm. the size and organization of the dsdna genome varies among the species causing human disease (mcgeoch et al. 2006 ) . the genomes of human herpesviruses include unique sequences and repeated sequences. though the genomes are linear in virions, they circularize in the nucleus of infected cells, which is mediated through repeat sequences at both ends of the dsdna genome. for hhv6 and hhv7 (class a genome), a large unique sequence region is fl anked by a region that is repeated at both ends of the linear strand of dsdna. the genome of ebv and the kaposi's sarcoma-associated herpesvirus (class c genome) have smaller left and right terminal repeat sequences, while repeat sequences r1 to r4 divide the unique sequence nucleic acid into four discrete regions. for vzv (class d genome), a large terminal sequence is inverted and inserted into the genome, resulting in a large unique sequence region (ul) and a small unique sequence region (us). hsv-1, hsv-2, and cmv (class e genomes) are the most complex. there are repeat sequence regions at both ends of the linear dsdna molecule. the unique sequence dsdna is divided into ul and us regions by a sequence composed of juxtaposed copies of the terminal repeat sequences inserted in an inverted orientation. typical of dsdna viruses, a large number of proteins are produced by various herpesviruses. the organization of the coding regions is complex, with 3′ and 5′ reading frames, gene overlap, spliced genes, and intron regions. forty genes are conserved among the α-, β-, and γ-herpesviruses. these core genes are divided among seven gene blocks (albà et al. 2001 ) ; within each block the order and polarity of genes are conserved, including genes for gene regulation, nucleotide metabolism, dna replication, virion maturation, envelope glycoprotein synthesis, and capsid, fusion and tegument protein synthesis. diseases caused by human herpesviruses range from systemic to localized infection of virtually all organ systems, although the hostcell range and typical disease characteristics vary by species. a characteristic of herpesvirus infections is latency, which is commonly associated with reactivation and symptomatic infections (e.g., shingles). while active infection with herpesviruses results in the destruction of the infected host cell, latently infected cells remain viable. in latently infected cells, the viral genome forms circularized molecules within the host nucleus with limited expression of viral genes. (gompels et al. 1995 ) of these viruses has the simplest organization and lowest %g + c content compared to the other herpesviruses. reading frames are present on each strand of the dsdna. core herpesvirus proteins are clustered near the center of the strands, while species-specifi c genes are located toward the ends of the strands (braun et al. 1997 ). hhv-6 and hhv-7 are assigned to the genus roseolovirus in the subfamily betaherpesvirinae , family herpesviridae , and order herpesvirales . there are two distinct hhv-6 species: human herpesvirus 6a (the roseolovirus type species) and human herpesvirus 6b . hhv-6b is the agent of exanthem subitum. there is a single hhv-7 species, human herpesvirus 7 , which is also a cause of exanthem subitum. t-lymphocytes are the primary target cell of hhv-6 and hhv-7 viruses. • kaposi's sarcoma-associated herpesvirus : the complete virions of kaposi's sarcoma-associated herpesvirus (kshv) have a diameter ~100 nm. in addition to virusspecifi c proteins, the tegument also carries viral mrnas, probably the result of passive incorporation during the cytoplasmic envelopment process (bechtel et al. 2005 ) . the envelopes of complete virions bear kshv-specifi c glycoproteins. the genome (~170 kbp) has class c organization (russo et al. 1996 ) typical of gammaherpesvirinae . the conserved herpesvirus genes are clustered in four blocks; kshv-specifi c genes are typically distributed in the regions outside and between these blocks (renne et al. 1996 ) . the kshv species designation is human herpesvirus 8 , which is assigned to the genus rhadinovirus within the subfamily gammaherpesvirinae . the virus has tropism for b-lymphocytes and is implicated in all forms of kaposi's sarcoma. four clades, a-d, with distinctive geographical distributions, have been identifi ed by genotypic analysis; the a and c clades cluster together and are most typical for isolates from europe and the united states. • varicella-zoster virus (vzv) : the dense core of vzv is enclosed in an icosahedral capsid (80-120 nm diameter), which is surrounded by an amorphous tegument. the envelope may be derived from multiple types of hostcell membranes during transit from the nucleus through the cytoplasm; specifi c viralencoded glycoproteins are embedded in the envelope of the complete virions, which may be spherical or pleomorphic (180-200 nm in diameter). vzv has a class d dsdna genome (~125 kbp) (clarke et al. 1995 ; davison 1984 ) , resulting in production of two isomeric genomic forms by infected cells through inversion of the us region (ecker and hyman 1982 ) . the genome encodes more than 70 proteins. the organization includes grouping of several genes into single transcription units, genes with overlapping reading frames, and spliced segments (davison and scott 1986 ) . the species designation for vzv is human herpesvirus 3 . it is the type species of the genus varicellovirus within the subfamily alphaherpesvirinae . there is only a single serotype of vzv. for epidemiologic purposes, vzv isolates may be genotyped on the basis of minor differences in dna sequence; different genotypes may be classifi ed as european, japanese, or mosaic (loparev et al. 2004 ). the host range of vzv is restricted to cells of humans or other primates; in humans, vzv has tropism for human t-lymphocytes and establishes latent infection in the cells of the dorsal root ganglia. orthomyxoviridae : infl uenza viruses belong to the family orthomyxoviridae . they are polymorphic; viruses may be spherical (~100 nm diameter) or fi lamentous. complete virions are surrounded by an envelope derived from the host cytoplasmic membrane. viral hemagglutinin and neuraminidase proteins are embedded in the envelope resulting in characteristic 10-14 nm spikes projecting from the surface of virions. in addition to the ha and na protein, m2 protein is embedded into the envelope of infl uenza a viruses; nb and bm2 proteins are embedded into the envelopes of infl uenza b viruses. the matrix protein (m1) is located just below the envelope. the nucleocapsid is composed of viral rna and nonstructural proteins, including ribonucleoproteins and polymerases. the genome of infl uenza viruses is composed of negative-sense ssrna. all viral rna synthesis occurs in the nucleus of the host cell. the a and b infl uenza virus genomes are composed of eight segments, while the infl uenza c virus genome consists of seven segments (hayden and palese 2009 ) . the segments range in size from ~900 to 2,300 nucleotides in length. each segment codes for one or more viral proteins (mccauley et al. 1983 ). the 3′ and 5′ ends of each segment contain noncoding, regulatory regions (fujii et al. 2005 ) . the three largest segments code for various components of rna polymerase; the pb1 segment of infl uenza a virus has a second open reading frame that encodes the pro-apoptotic protein pb1-f2. in infl uenza types a and b, the fourth and sixth segments encode for the surface hemagglutinin (ha) and neuraminidase (na) glycoproteins, respectively. the infl uenza a surface protein m2 is encoded by the seventh segment; infl uenza b surface protein nb is encoded by the sixth segment, while the bm2 is encoded by the seventh segment. the fi fth segment of both a and b infl uenza viruses encodes for the rna-binding nucleoprotein (np). the matrix protein m1 is encoded by the seventh segment of both viruses. the eighth and smallest rna segment of infl uenza a and b viruses encodes for ns1, a multifunctional protein with interferon antagonistic properties and nep/ ns2 protein which is involved in transport of vrnps across the nuclear membrane of the host cell. the names of clinical isolates of human infl uenza isolates include the species of origin, isolation location, number of the isolate, and year of isolation; infl uenza a virus isolates also include the hemagglutinin (h1 to h16) and neuraminidase (n1 to n9) subtypes (atmar and lindstrom 2012 ) . for example, a/california/7/2009 (h1n1), a/victoria/361/2011 (h3n2), and b/ wisconsin/1/2010 viruses were recommended for the 2012-2013 seasonal infl uenza vaccine. large outbreaks have only occurred with h1, h2, and h3 and neuraminidases n1 and n2 viral subtypes. antigenic drift and antigenic shift contribute to reinfection with infl uenza viruses (taubenberger and kash 2010 ) . antigenic drift is caused by a gradual accumulation of point mutations in hemagglutinin and neuraminidase genes, which result in minor antigenic changes in these proteins. antigenic shift is caused by a virus created by reassortment of infl uenza virus rna segments during coinfection of a host, usually with a human infl uenza virus and an avian or swine infl uenza virus or through introduction of a nonhuman infl uenza virus strain into human populations after mutation during a host-species infection creates a new isolate permissive for interspecies transmission. papillomaviridae: the papillomaviruses (pvs) represent a large (and growing) family of viruses that currently includes 30 different genera and 69 species; the taxonomy has undergone signifi cant reorganization in recent years (bravo et al. 2010 ) . the oncogenic potential of human papillomaviruses is well established. pvs are non-enveloped; virions are icosahedral with diameters of 50-55 nm. the capsid contains two structural proteins, l1, the most abundant viral protein, and l2. the pv genome consists of a single molecule of circularized dsdna (zheng and baker 2006 ) . the open reading frames for all viral genes are located on only one of the dna strands, and transcription proceeds in a single direction. there are eight early (e) open reading frames that encode for regulatory proteins that control viral metabolism and dna synthesis. the e6 proteins of high-risk hpv types have anti-apoptotic effects and interfere with p53 regulatory function in infected host cells (howley et al. 1990 ) . two late (l) reading frames encode for synthesis of the structural proteins l1 and l2. epithelial cells of a wide variety of vertebrate hosts are susceptible to papillomavirus infection, but the different host species are only susceptible to species-specifi c viruses. papillomaviruses have been classifi ed according to susceptible host species and the type of disease produced, but comparison of sequence differences of the l1 reading frame has provided a more detailed description of papillomavirus phylogeny (de villiers et al. 2004 ) . the family papillomaviridae is not assigned to an order. human pathogens are clustered within fi ve papillomavirus genera. paramyxoviridae : the paramyxoviruses are enveloped (host cytoplasmic membrane) with an unsegmented negative-sense ssrna genome (15-19 kb) . the viral rna serves as template for synthesis of mrna and for synthesis of antigenomic (positive-sense) rna for synthesis of new viral negative-sense rna for new virions. there are six to ten genes; genes for the six major proteins are linked in the following 3′ to 5′ order: nucleocapsid (n) → phosphoprotein (p) → matrix (m) → fusion (f) → hemagglutinin/neuraminidase (hn) → large polymerase (l). there is an untranslated leader sequence at the 3′ end and untranslated trailer sequence at the 5′ end. the genes are separated by untranslated sequences and do not overlap, with the exception of the m and l genes of human metapneumovirus . translation is initiated at the 3′ end and proceeds directly through to the 5′ end. because the rna polymerase is unstable and may detach at the untranslated regions between genes, there is a gradient in the concentration of gene products from 3′ to 5′. in different species, other proteins are produced by additional small genes, mrna editing, or overlapping reading frames within the p gene. the v and c proteins regulate viral rna transcription and also interfere with host interferon signaling and other aspects of the immune response to paramyxovirus infection (andrejeva et al. 2004 ; durbin et al. 1999 ; swedan et al. 2009 ). formation of the nucleocapsid core is constrained by a required association of one n protein to every six genomic nucleotides (kolakofsky et al. 2005 ; skiadopoulos et al. 2003 ) . the resulting helical structure has a diameter of 18 nm with a 4 nm central core. p proteins (a polymerase cofactor) are attached to this rigid rod and serve as attachment of l proteins, which interact to provide enzymatic activity for rna synthesis. this core structure, rather than free genomic rna, serves as the template for mrna and antigenomic rna synthesis. the paramyxovirus m proteins surround and organize the nucleocapsid and interact with the cytoplasmic tails of transmembrane envelope proteins. the envelope formed from modifi ed host-cell plasma membranes is studded by viral protein complexes, including hn proteins, which mediate virion attachment to target cells, and f protein, which mediates ph-independent fusion of the viral envelope and cell cytoplasmic membrane. the paramyxoviridae are one of the four families within the order mononegavirales and include signifi cant and frequent pathogens of humans and animals. there are two subfamilies: the paramyxovirinae and the pneumovirinae . there are seven genera and thirty-one species in the subfamily paramyxovirinae and two genera and fi ve species in the pneumovirinae subfamily. • henipahviruses: in the subfamily paramyxovirinae , there are two species within the genus henipahvirus : hendra virus (hev) and nipah virus (niv); hev is the type species. henipahvirus virions are pleomorphic (spherical to helical forms). electron microscopy of hendra virus shows a "double-fringe" appearance due to short and long surface projections (hyatt et al. 2001 ) . complete virions range in size from 40 to 1,900 nm in longest dimension. the genome (~18 kb) includes genes typical of paramyxoviridae . long untranslated sequences are attached to the 3′ end of fi ve of the six genes, resulting in the larger genome size of henipahviruses compared to other paramyxoviruses (eaton et al. 2007 ; wang et al. 2000 ) . the p gene also codes for v and w proteins by mrna editing and c protein by a shifted reading frame. henipahviruses are assigned to the family paramyxoviridae and subfamily paramyxovirinae . henipahvirus infections are zoonotic; fruit bats are the presumed reservoir for hendra virus infections, while fruit bats (johara et al. 2001 ) includes two open reading frames; the function of m2-1 protein is undefi ned, while m2-2 protein is a regulator of viral transcription. there is no gene for hemagglutinin-neuraminidase; the product of the g gene serves as the major attachment protein. metapneumoviruses are members of the subfamily pneumovirinae . human metapneumovirus is a species in the genus metapneumovirus . there is a single hmpv serotype, with two antigenic subtypes a and b. • human respiratory syncytial viruses: the envelope of human respiratory syncytial virus (hrsv) is studded with three viral glycoproteins: g protein (the major attachment protein), f protein, and sh protein (a small hydrophobic protein). complete virions are pleomorphic (spherical to fi lamentous forms). the nucleocapsid diameter, 12-15 nm, is smaller than typical for other paramyxoviruses (hall 2001 ) . the hrsv genome is ~15 kb and includes ten genes (collins and wertz 1983 ) . the fi rst eight reading frames are nonoverlapping; the last two genes, m2 and l, overlap by 62 nucleotides. in addition to n, p, and l proteins, m2-1 protein, a transcription factor, is associated with the nucleocapsid. the overall organization of the hrsv genome is similar to other paramyxoviruses. in addition to the typical genes, the hrsv genome includes genes ns1 and ns2 (nonstructural proteins that interfere with interferon induction and signaling), sh, and m2-1 and m2-2 (nonstructural proteins involved in regulation of transcription). there is no nh gene; attachment is mediated by the g gene product. human respiratory syncytial virus is the type species of the genus pneumovirus in the subfamily pneumovirinae . there is a single hrsv serotype, with two antigenic subtypes a and b. (henrickson 2003 ) . the genome of human parainfl uenza viruses is ~15 kb in length with an organization and six reading frames (n, p, m, f, hn, l) typical of the paramyxoviridae (karron and collins 2007 ) . there are no overlapping reading frames. accessory proteins, c (hpiv1 and 3), v (hpiv 2 and 4), and d (hpiv3), are produced by mrna editing of the p gene. n proteins are tightly bound to viral and antigenomic rna; p and l proteins are also bound to the nucleocapsid, forming functional complexes for rna polymerization and processing. human parainfl uenza viruses are assigned to two genera in the subfamily paramyxovirinae . (berns 1990 ). virions are stable in the environment and thought to transmit infection by attachment to specifi c receptors of actively dividing cells. the parvovirus genome is composed of unsegmented ssdna (cotmore and tattersall 1984 ; shade et al. 1986 ; zhi et al. 2004 ) . complete virions of different species may contain negativesense or both negative-and positive-sense dna in various proportions. there are two major reading frames: one encoding capsid proteins and the other coding for nonstructural proteins. noncoding sequences at the 3′ and 5′ ends include complementary sequences which result in the formation of hairpin structures that serve to regulate nucleic acid synthesis (deiss et al. 1990 ) . various host-cell molecules mediate attachment and infection by parvoviruses. erythrocyte p antigen is the major receptor for human parvovirus b19 . viruses are taken up by endocytosis, followed by transport into the host-cell nucleus. viral dna replication depends on host-cell polymerases during the s phase of host-cell replication. human infections are caused by parvovirus b19 and bocavirus (schildgen et al. 2008 ; vicente et al. 2007 ) . the family parvoviridae is not assigned to an order; there are two subfamilies, the densovirinae and the parvoviridae . there are fi ve genera in the subfamily parvoviridae : amdovirus (1 species), bocavirus (2 species including the type species bovine parvovirus ), dependovirus (12 species, including adenoassociated viruses), erythrovirus (4 species including the type species human parvovirus b19 ), and parvovirus (12 species). picornaviridae : infections of the respiratory tract and other organ systems by enteroviruses and parechovirus are well described. enteroviruses were initially classifi ed on the basis of clinical disease and epidemiology, suckling mouse inoculation, replication in cell culture, electron microscopic studies, physical properties, and the vast range of specifi c antigenic differences. the major subgroups were poliovirus, coxsackievirus (a and b), and echovirus. a characteristic of these viruses is their relative stability in acidic media and nonionic detergents. translation of the positive-sense ssrna genome is regulated by a 5′ non-translated region (lindberg and polacek 2000 ) that is covalently linked to protein vpg (virion protein, genome linked); the short 3′ noncoding region is polyadenylated. translation results in synthesis of a single polyprotein, which is cleaved into functional proteins by post-translational processing (nicklin et al. 1987 ; pallansch and roos 2007 ) . there are three functional regions delimited by ribosomal entry sites. the p1 region codes for capsid proteins, while regions p2 and p3 code for nonstructural proteins. capsid proteins vp1, vp2, and vp3 are exposed externally and account for the serological diversity of the viruses. with the advent of molecular phylogenetic analysis, the enteroviruses have been reclassifi ed by the ictv. enteroviruses are in the order picornavirales , family picornaviridae, and genus enterovirus . the enteroviruses have been assigned to 12 species, including human enterovirus a (17 serotypes including coxsackieviruses and enteroviruses), human enterovirus b (56 serotypes, including coxsackieviruses, echoviruses, and enteroviruses), human enterovirus c (the type species; 16 serotypes including coxsackieviruses, all human polioviruses, and enteroviruses), and human enterovirus d (3 enterovirus serotypes). in addition to the enteroviruses, the genus enterovirus also includes 3 rhinoviruses species, human rhinovirus a , b, and c , and more than 100 serotypes. also within the family picornaviridae is the genus parechovirus . human parechovirus is the type species for the genus. there are 14 parechovirus serotypes. polyomaviridae : polyomaviruses may infect a variety of primate and non-primate vertebrate host species; the oncogenic potential of polyomaviruses is well established (white and khalili 2004 ) . sialic acid and/or gangliosides on the hostcell membranes serve as receptors for attachment of human polyomavirus. though these molecules are widespread on human cells, there is a restricted tropism. respiratory epithelial cells and cells of lymphoid origin are the likely targets for initial infection, followed by hematogenous spread to target organs. the virions are non-enveloped; the icosahedral capsids (40-45 nm diameter) are composed of three proteins (vp1, vp2, and vp3), which enclose the circular dsdna genome (~5 kbp). the genome is divided into three regions. the early region encodes for proteins involved in viral processes that occur prior to dna replication, including t (tumor) antigens (benjamin 2001 ) . the late region encodes for proteins involved in processes that primarily occur after dna replication. the early and late regions do not overlap and are transcribed from opposite strands of the viral dna and in opposite directions. a number of viral proteins are encoded as a result of alternative splicing and other posttranslational modifi cations of mrna. polyomaviruses are members of the family polyomaviridae , which is not assigned to an order. there is 1 genus, polyomavirus , and 13 species, including the human pathogens bk polyomavirus and jc polyomavirus and simian virus 40 (type species). retroviridae : the retroviruses are a unique group of viruses, including human immunodefi ciency virus types 1 and 2 and human t-cell leukemia virus type 1; they may infect a wide range of vertebral host species. the human immunodefi ciency viruses and human t-cell leukemia virus 1 are able to cause disease in humans. these rna viruses use a unique replication cycle that uses a "reverse fl ow" of genetic information from rna to dna: viral rna is reverse transcribed and converted into a dsdna copy of the viral genome which is integrated into the host-cell genome. integration of the proviral dna allows the viruses to establish persistent, presumably lifelong, infection. another consequence of insertion of the viral dna is functional mutation of the host genome at the site of insertion which may alter the host gene or regulation of a gene's expression; the oncogenic potential of retroviral infection is well described in humans and other vertebrate host species. the electron microscopic morphology of retroviruses shows a dense nucleocapsid core (cylindrical or cone shaped) (chrystie and almeida 1988 ; gelderblom et al. 1989 ) . viruses are functionally diploid: the core includes two copies of the positive-sense ssrna genome, which are closely complexed with viral nucleoproteins. the sequences of the two ssrna molecules may differ because of errors in transcription of new genomic ssrna molecules during replication. the core also includes several functional viral proteins, including reverse transcriptase, integrase, and protease. the core is surrounded by capsid proteins; the nucleocapsid is surrounded by viral matrix protein. complete virions are surrounded by an envelope derived from virus-modifi ed host-cell cytoplasmic membranes; the envelope is studded by viral glycoproteins. the transmembrane protein extends from the matrix layer through the lipid bilayer to the external surface. the receptorbinding complex is anchored to the external portion of the transmembrane protein. mature virions are spherical (~100 nm diameter). the ssrna genomes of retroviruses are similar to the host-cell mrna. a repeat sequence is present at both ends of the ssrna; the 5′ end is capped and the 3′ end polyadenylated. the order of sequences from the 5′ end to the 3′ end is cap → repeat sequence → unique sequence (u5) → the initiation site for initiation of minus-strand dna synthesis → gag gene → pol gene → env gene → the initiation site for plus-strand dna synthesis → a unique sequence (u3) → repeat sequence → poly(a) sequence. after entry into the cytoplasm of a susceptible cell, double-stranded dna is synthesized by reverse transcription of both copies of the retroviral ssrna. the viral-encoded dna is transported into the nucleus, after which it is integrated into the host's genomic dna. the process of forming new virions is initiated by transcription of the proviral dna. the processed viral rna is exported into the cytoplasm and genes for precursor viral proteins are translated. virions are assembled at the cytoplasmic membrane and then released by budding; fi nal virion maturation occurs by extracellular processing of viral proteins. a characteristic of retroviruses is the high mutation rate and marked genomic heterogeneity of isolates. the major factors that contribute to this phenomenon include (1) error-prone reverse transcription, without proofreading correction, of the infecting virus genome; (2) recombination between the two genomic ssrna strands during reverse transcription; and (3) the very high-level production of progeny viruses from infected cells. retroviruses are not assigned to a taxonomic order. the family retroviridae has two subfamilies. the orthoretrovirinae includes six genera, including deltaretrovirus and lentivirus . htlv-1 is assigned the species name primate t-lymphotropic virus 1 in the deltaretrovirus genus. human immunodefi ciency virus 1 and hiv-2 are named human immunodefi ciency virus 1 (type species) and human immunodefi ciency virus 2 , respectively, in the genus lentivirus (clavel et al. 1986b ). • human immunodefi ciency viruses : the human immunodefi ciency viruses have a conical core surrounded by an envelope derived from viralmodifi ed host-cell cytoplasmic membrane. binding and entry of hiv into susceptible cells requires several specifi c receptors: cd4 (present on host helper t cells, cd4+ macrophages, and some dendritic cells) plus chemokine receptors, including ccr5 and cxcr4 (klatzman et al. 1984 ; simmons et al. 1998 ). the biological properties of hiv-1 isolates depend on the chemokine coreceptor(s) used by the virus (berger et al. 1998 ). isolates that exclusively use cxcr4 are t-cell tropic with rapid replication and syncytium formation. isolates that use ccr5 exclusively are tropic to macrophages, replicate more slowly, and do not induce syncytium formation. isolates that can use either cxcr4 or ccr5 have intermediate phenotypes. the gag , pro , pol, and env genes are translated from full-length mrna transcripts of the proviral genome: gag and env in one reading frame and pro and pol from a second reading frame. in addition, several genes are transcribed from overlapping or unique reading frames, including several spliced gene products. human immunodefi ciency type 1 and 2 viruses evolved from simian viruses (gao et al. 1999 ; peeters et al. 1989 ; daniel et al. 1985 ; marx et al. 1991 ) . these viruses may be distinguished by a number of characteristics, including clinical disease, specifi c antigens, and gene sequences (clavel et al. 1986a ) . hiv-1 isolates may be further characterized into genetic groups and subtypes or clades (wainberg 2004 ) . most hiv-1 isolates are in the m (main) group, which has a number of well-defi ned subgroups and recombinant forms with heterogeneous global distribution; clade b viruses are the predominant isolates in north america and europe (hemelaar et al. 2006 ; osmanov et al. 2002 ) . group o (outlier) strains have mainly been isolated or acquired in western africa. group n (non-m, non-o) and recombinant forms are also most commonly isolated from western africa. • human t-cell leukemia virus type 1 : mature htlv virions have a spherical core, symmetrically placed within the envelope. the host-cell receptor is glut-1, a surface glucose transport molecule (manel et al. 2003 ) . the gag , pro , pol, and env genes are translated from fulllength mrna transcripts of the proviral genome: gag in one reading frame, pro and env from a second, and pol from a third reading frame. in addition, several spliced genes are transcribed from overlapping reading frames. recent and continuing progress to develop and use standardized and widely accepted methods for biological and taxonomic classifi cation of viral pathogens has resulted in improvement in the medical response to viral illnesses. at a very basic level, these systems allow clinicians and scientists to communicate effectively and ensure the comparability of data generated by clinical or basic scientifi c studies. further, accurate and standardized data is critical for understanding issues related to transmission, prevention, and treatment of viral illnesses. establishing phylogenetic similarity to known viral pathogens may allow clinicians to anticipate the clinical behavior of new and emerging viral pathogens, as may be seen when virus mutation results in acquisition of new pathogenic mechanisms, like changes to antigens associated with evasion of the immune response of the host species or changes that allow a viral pathogen to jump from one species into new, susceptible species. as analytical tools improve, even more informative data relevant to clinical and pathologic characteristics of viral pathogens is anticipated. genomewide function conservation and phylogeny in the herpesviridae longrange rna-rna interactions circularize the dengue virus genome the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the inf-beta promoter infl uenza viruses, chap 81. in: versalovik j (ed) manual of clinical microbiology dna sequence and expression of the b95-8 epstein-barr virus genome expression of animal virus genomes rnas in the virion of kaposi's sarcoma-associated herpesvirus polyoma virus: old fi ndings and new challenges a new classifi cation for hiv-1 human herpesvirus 6 the clinical importance of understanding the evolution of papillomaviruses real-time qualitative pcr for 57 human adenovirus types from multiple specimen sources flavivirus genome organization, expression, and replication the morphology of human immunodefi ciency virus (hiv) by negative staining confi guration and terminal sequences of the simian varicella virus genome isolation of a new human retrovirus from west african patients with aids molecular cloning and polymorphism of the human immune defi ciency virus type 2 cdna cloning and transcriptional mapping of nine polyadenylylated rnas encoded by the genome of human respiratory syncytial virus characterization and molecular cloning of a human parvovirus genome isolation of t-cell tropic htlv-iii-like retrovirus from macaques structure of the genome termini of varicella-zoster virus the complete dna sequence of varicella-zoster virus genetic content and evolution of adenoviruses the human cytomegalovirus genome revisited: comparison with the chimpanzee cytomegalovirus genome classifi cation of papillomaviruses cloning of the human parvovirus b19 genome and structural analysis of its palindromic termini the genome sequence of herpes simplex virus type 2 et al (1986) transcriptional map of the measles virus genome functional profi ling of a human cytomegalovirus genome mutations in the c, d, and v open reading frames of human parainfl uenza virus type 3 attenuate replication in rodents and primates henipaviruses, chap 45 virus taxonomy: one step forward, two steps back varicella-zoster virus dna exists as two isomers importance of both the coding and the segment-specifi c noncoding regions of the infl uenza type a virus ns segment for its effi cient incorporation into virions origin of hiv-1 in the chimpanzee pan troglodytes troglodytes factors associated with fulminant liver failure during an outbreak among injection drug users with acute hepatitis b morphogenesis and morphology of hiv. structure-function relations the dna sequence of human herpesvirus-6: structure, coding content, and genome evolution processing of genome 5′ termini as a strategy of negative-strand rna viruses to avoid rig-idependent interferon induction respiratory syncytial virus and parainfl uenza virus infl uenza virus, chap 42 global and regional distribution of hiv-1 genetic subtypes and recombinants in parainfl uenza viruses gene mapping of the putative structural region of the hepatitis c virus genome by in vitro processing analysis evolutionary history and phylogeography of human viruses what does virus evolution tell us about virus origins? association of human papillomavirus types 16 and 18 e6 proteins with p53 ultrastructure of hendra virus and nipah virus within cultured cells and host animals the international code of virus classifi cation and nomenclature nipah virus infection in bats (order chiroptera) in peninsular malaysia t-lymphocyte t4 molecule behaves as the receptor for human retrovirus lav paramyxovirus mrna editing, the "rule of six" and error catastrophe: a hypothesis proposal for a revised taxonomy of the family filoviridae: classifi cation, names of taxa and viruses, and virus abbreviations partitioning the genetic diversity of a virus family: approach and evaluation through a case study of picornaviruses toward genetics-based virus taxonomy: comparative analysis of a geneticsbased classifi cation and the taxonomy of picornaviruses taxonomy and classifi cation of viruses, chap 75. in: versalovic j (ed) manual of clinical microbiology hepatitis b: the virus and disease hepatitis b viral factors and clinical outcomes of chronic hepatitis b molecular analysis of the prototype coxsackievirus b5 genome global identifi cation of three major genotypes of varicella-zoster virus: longitudinal clustering and strategies for genotyping the ubiquitous glucose transporter glut-1 is a receptor for htlv the genome sequence of the sars-associated coronavirus isolation of a simian immunodefi ciency virus related to human immunodefi ciency virus type 2 from a west african pet sooty mangabey structure and function of the infl uenza virus genome the application of molecular phylogenetics to the analysis of viral genome diversity and evolution topics in herpesvirus genomics and evolution bunyaviridae: bunyaviruses, phleboviruses, nairoviruses, and hantaviruses, chap 43 site-specifi c inversion sequences of the herpes simplex virus genome: domain and structural features poliovirus polypeptide precursors: expression in vitro and processing by exogenous 3c and 2a proteinases complete nucleotide sequence of dengue type 3 virus genome rna estimated global distribution and regional spread of hiv-1 genetic subtypes in the year 2000 enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses, chapter 25 isolation and partial characterization of an hiv-related virus occurring naturally in chimpanzees in gabon arenaviruses, chap 44 the size and conformation of kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) dna in infected cells and virions characterization of a novel coronavirus associated with severe acute respiratory syndrome nucleotide sequence of the kaposi sarcoma-associated herpesvirus (hhv8) human bocavirus: passenger or pathogen in acute respiratory tract infections? nucleotide sequence and genome organization of human parvovirus b19 isolated from the serum of a child during aplastic crisis cxcr4 as a functional coreceptor for human immunodeficiency virus type 1 infection of primary macrophages the genome length of human parainfl uenza virus type 2 follows the rule of six, and recombinant viruses recovered from non-poly-hexameric-length antigenomic cdnas contain a biased distribution of correcting mutations respiratory syncytial virus nonstructural proteins decrease levels of multiple members of the cellular interferon pathways infl uenza virus evolution, host adaptation, and pandemic formation mechanisms and enzymes involved in sars coronavirus genome expression conserved rna secondary structures in flaviviridae genomes analysis of the genomic sequence of a human metapneumovirus manual of clinical microbiology human bocavirus, a respiratory and enteric virus hiv-1 subtype distribution and the problem of drug resistance the exceptionally large genome of hendra virus: support for the creation of a new genus within the family parmyxoviridae polyomaviruses and human cancer: molecular mechanisms underlying patterns of tumerogenesis towards a natural system of organisms: proposal for the domains archaea, bacteria and eucarya a reevaluation of the higher taxonomy of viruses based on rna polymerases papillomavirus genome structure, expression, and post-transcriptional regulation construction and sequencing of an infectious clone of the human parvovirus b19 key: cord-014864-0d682m0n authors: nan title: biomedical vignette date: 2008-10-26 journal: j biomed sci doi: 10.1007/s11373-008-9279-2 sha: doc_id: 14864 cord_uid: 0d682m0n nan connective tissue growth factor (ctgf) was initially discovered in 1991 as a secreted protein in the conditioned media of cultured human umbilical vascular endothelial cells [1] . ctgf is a member of the cnn family of secreted, matrix-associated proteins encoded by immediate early genes that play various roles in angiogenesis and tumor growth [2] . although papers and reviews on ctgf have been published, this review [3] focuses on the functional role of ctgf in cancer progression. the influence of ctgf expression on the behavior and progression of various cancer cells, as well as its regulation on various types of protein signals and their mechanisms are highlighted. although ctgf expression seems to be associated with progression of many kinds of cancers, its expression may have tumor suppressive effects in a few cases such as lung adenocarcinoma cells, colorectal cancer cells and oral squamous carcinoma cells. cgcgh: a tool for molecular karyotyping using dna microarray-based comparative genomic hybridization (array-cgh) to analyze the rare events of single-copy dna aberrations, a matlab-based, array cgh analyzing program, chang gung comparative genomic hybridization (cgcgh) was employed to survey chromosomal amplifications and deletions in fetal aneuploidies or cancer tissues [4] . the analyzed chromosomal data are displayed in a graphic interface, and cgcgh allows users to launch a corresponding g-banding ideogram. in 15 karyotyped samples, the cgcgh program outperformed other programs and cgcgh supported the data generated from cdna microarrays, spotted oligonucleotide microarray and affymetrix human mapping arrays. a computational screen for c/d box snornas in the human genomic region associated with prader-willi and angelman syndromes to identify snornas (small nuclear rnas) in the deletion of human chromosomal region 15q11-q13 related to prader-willi syndrome (pws) and angelman syndrome (as), computational scanning and screening and a novel hybridization energy test were used to identify all the snornas. three previously unknown methylation snor-nas targeting ribosomal 18s and 28s rnas and two snornas targeting serotonin receptor 2cmrna were identified [5] . the application of the present method to the pws/as region of human genome identified 11 snornas, three of which pass the hybridization test. these snornas require experimental confirmation. present hybridization test can be incorporated and automated with motif scanning for genome-wide studies. interactions between m protein and other structural proteins of severe acute respiratory syndrome coronavirus the study by hsieh et al. [6] in the current issue focuses on the protein-protein interactions that regulate sars coronavirus assembly. this was performed by co-localization studies in cells that express structural viral proteins either individually or combined. changes in localization induced by the presence of other virus components indicate direct protein-protein interactions. these experiments indicate that the sars m protein plays a pivotal role in virus assembly, similar to findings with other coronaviruses (vennema et al., reference 14 in the paper). a model is presented to explain how the m protein interacts with multiple other structural proteins (s, e and nc) to facilitate virion assembly. severe acute respiratory syndrome coronavirus nucleocapsid protein confers ability to efficiently produce virus-like particles when substituted for the human immunodeficiency virus nucleocapsid domain the assembly of virus particles is a complex, but illunderstood process. some viruses like the human immunodeficiency virus type 1 (hiv-1) require only a single protein for the assembly of virus-like particles (vlps) (gheysen, reference 14 in the paper). the situation is more complex for the sars coronavirus, which requires at least two structural proteins for vlp formation. the current study by wang et al. [7] asked whether the sars nucleocapsid (n) protein and fragments thereof can functionally replace the nucleocapsid domain of the self-assembling hiv-1 gag protein. using this novel chimeric vlp approach, the authors were able to map more precisely the sars n domain that facilitates self-association and vlp formation. host factors, such as iron concentration at the site of infection, may influence the virulence of pseudomonas aeruginosa and hence the outcome of infection. here, mittal et al. [8] reported that the depletion of iron in the growth medium led to increased adherence of p. aeruginosa to uroepithelial cells and decreased phagocytosis of the bacteria. furthermore, p. aeruginosa growth in irondepleted medium showed increased renal bacterial load and tissue pathology in the challenged mice. the current study may provide insight into the pathogenesis of p. aeruginosa and facilitate the development of a preventive approach against p. aeruginosa-induced urinary tract infections. in vertebrate retinas, glycinergic synapses regulate glutamate transmissions in both synaptic layers, the inner plexiform layer (ipl) and outer plexiform layer (opl). the function of glycinergic synapses in the inner retina has been known to inhibit glutamate transmissions, shaping light-evoked response in ganglion cells [9, 10] . however, little is known about the function of glycinergic synapses in the outer retina. in this issue, shen et al. [11] reported that glycine depolarized rods and activated voltage-gated ca 2+ channels in the neurons, resulting in facilitation of glutamate release in photoreceptors and increase of the spontaneous excitatory postsynaptic currents in off-bipolar cells. furthermore, these authors reported that inhibition of a cluptake transporter nkcc1 effectively eliminated glycine-evoked depolarization in rods suggesting that nkcc1 maintains a high cllevel in rods, which is responsible for glycine-induced depolarization. this finding is quite significant since it reveals a new function of glycine in retinal synaptic transmission. dose-finding study with nicotine as an anti-antiseizure agent in ptz-induced seizure model in mice nicotine exerts agonistic effect on neuronal nicotinic acetylcholine receptors and is reported to enhance release of excitatory neurotransmitter glutamate into the synapses [12] . pentylenetetrazole (ptz), on the other hand, produces seizures in small rodents [13] and is believed to act by antagonizing the inhibitory gabaergic [14] neurotransmission in central nervous system. in this issue, medhi et al. [15] reported that subthreshold dose of nicotine pretreatment significantly decreased the cd50 value for ptz. sodium valproate but not topiramate, significantly inhibited ptz-induced seizure. nonconvulsive dose of nicotine significantly antagonized the protective efficacy of sodium valproate against ptz-induced seizures. these findings are quite significant since they bear clinical relevance particularly amongst epileptic smokers who may show failure of efficacy of antiseizure agents and present with breakthrough seizure attacks due to nicotine. benzyl alcohol inhibits n-methyl-d-aspartate receptor-mediated neurotoxicity and calcium accumulation in cultured rat cortical neurons benzyl alcohol has been used as a preservative in some small multiple-dose vials of bacteriostatic sodium chloride or water for injection. however, there is concern that excipients such as benzyl alcohol may cause adverse reactions to neurons in premature infants [16, 17] . in this issue, takadera and ohyashiki [18] reported that benzyl alcohol inhibited nmda-induced cytotoxicity. furthermore, these authors showed that the protective effect of benzyl alcohol on nmda-induced toxicity is due to its effect in reducing nmda receptor-mediated calcium accumulation, indicating that benzyl alcohol inhibits nmda receptor activity. this finding is significant since it shows potential beneficial effect of benzyl alcohol on mature neurons against glutamate-induced neurotoxicity although it may have adverse effect on immature neurons. bone morphogenetic proteins (bmps) are the most potent class of all osteoinductive proteins, and bmp-2 has already been clinically applied to accelerate bone regeneration in both fracture and spinal fusion [19] . bone narrow-derived mesenchymal stem cells (bmmscs) have been shown to be able to differentiate in vitro toward osteogenic lineages, when treated with established lineage-specific factor [20] . kim et al. [21] studied if a combination of the undifferentiated bmmscs and bmp-2 delivered via heparin-conjugated plga nanoparticles (hcpn) would extensively regenerate bone in vivo. in the in vivo testing, the undifferentiated bmmscs with bmp-2 with bmp-2loaded hcpn induced far more extensive formation, indicating the feasibility of entensive in vivo bone regeneration by transplantation of undifferentiated bmmscs and bmp-2 delivery via hcpn. previous studies indicated that t-box genes play an essential role in cell specification and morphogenesis [22, 23] . in vertebrates, tbx5 plays a critical role in cardiac and upper limb development. expression of tbx5 at the appropriate dose, time and position is important for normal cardiac development. zebrafish appears to be a well-established model used in studying the development of vertebrates. the zebrafish is an ideal model and has been receiving attention as a human disease model, because the species fertilizes embryos externally, the fetus develops rapidly, and it manages to survive without cardiac function for days. thus, zebrafish was used as the model organism to facilitate our investigation on the causal relationship between tbx5 and cardiac myogenesis during cardiac looping. results demonstrated that in zebrafish, injection of tbx5 morpholino antisense rna caused changes of heart conformation, defect of heart looping, pericardium effusion, dropsy of ventral position and decreased heart rate. in conclusion, this study showed deficiency of tbx5 might perturb cardiac looping progress, as well as the formation of atrium and ventricle, possibly through down-regulating cardiacmyogenesis genes such as amhc, vmhc and cmlc2 [24] . the discovery of the naturally occurring cardiac nonfunction (c) animal strain in ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. it was shown that this recessive mutation, in homozygous animals, causes incomplete differentiation of the embryonic heart at heartbeat initiation stages [25] due to a lack of organized myofibril formation in homozygous mutant hearts [26, 27] . in this issue, jia et al. [28] reported cloning of a peptide cdna (n1) from an anterior-endoderm-conditioned-medium rna library. furthermore, the authors have shown a dramatic decrease of expression of n1 mrna in mutant (c/c) embryos, indicating that the n1 gene is involved in heart development. these findings are quite significant since revealing the underlying molecular mechanisms of heart development will be an important step in finding cures for heart diseases. immunohistochemical assessment of cyclic guanosine monophosphate (cgmp) and soluble guanylate cyclase (sgc) within the rostral ventrolateral medulla the rostral ventrolateral medulla (rvlm) in the caudal medulla oblongata is a major site for generation of neurogenic vasomotor tone. nitric oxide (no) in the rvlm plays an important role in central cardiovascular regulation by regulating the sympathetic vasomotor outflow [29, 30] , and cgmp-dependent no signaling in the rvlm is impaired in hypertension [31] . to date, no studies have described the immunohistochemical localization of neurons capable of expressing cgmp within the rvlm. in this communication, powers-martin et al. [32] sought to identify the cellular targets for no in the rvlm by visualizing anatomical relationship of cgmp with the tyrosine hydroxylase (th) or phenylethanolamine n-methyltransferase (pnmt) cell group. double label immunohistochemistry for cgmp-immunoreactivity (ir) and th or pnmt neurons failed to reveal cgmp-ir neurons in the rvlm of either normotensive wistar-kyoto rats or the spontaneously hypertensive rats. in addition, soluble guanylate cyclase (sgc)-ir was found throughout neurons of the rvlm, but did not co-localize with pnmt-or th-ir neurons. these results indicate that within the rvlm, cgmp is not detectable using immunohistochemistry in the basal state and this raises the hypothesis that functional network inputs, such as the sympathetic baroreflex pathway are required to drive a sgc/cgmp cascade in the rvlm. in vivo gm-csf promoter-based assay for drug screening in drug discovery research, in vitro cell-based screening systems are well established as methods for evaluation of candidate lead compounds. for example, in vitro assays of nf-jb [33] and cox-2 [34] , two examples of drug targets, are employed to develop therapeutic strategies to counter inflammation. since the regulation of immuno-modifiers is highly dependent on three-dimensional microenvironments, an in vivo assay can more accurately evaluate the effects of drugs on the expression of key cytokines. su et al. [35] devised an in vivo, transgenic, human cytokine (e.g. gm-csf) gene promoter assay using defined epidermal skin cells as test tissue. test compounds were topically applied to mouse skin before or after gene gun transfection, using a cytokine gene promoter-driven luciferase reporter. croton oil, an inflammation inducer, induced six-folds transgenic gm-csf promoter activity in skin epidermis, and this effect was drastically inhibited by the phytocompound shikonin. these results demonstrate that this in vivo transgenic promoter assay system is cytokine gene-specific, and highly responsive to pro-inflammatory stimuli. arecoline and the 30-100 kda fraction of areca nut extract differentially regulate mtor and respectively induce apoptosis and autophagy-a pilot study in taiwan, chewing of betel quit causes oral cancer, which is the top sixth killer of all types of cancer. betel quit consists of areca nut (an), lime and influorescene of piper betel. it was demonstrated previously that extracts of an (ane), but not those of lime, inflorescence of piper betel, induced rounding of cell morphology and nuclear shrinkage in several carcinoma cell lines. in the present study [36] , the mw of active principle was found to be between 30-100 kda, which induced nuclear shrinkage, clearance of the cytoplasm, cleavage of lc3-1q and appearance of autophagic vacuoles and acid vesicles. on the other hand, arecoline (are) triggered caspase-3 activation, peri-nuclear condensation and micronucleation. furthermore, ane 30-100 k but not are, inhibited the phosphorylation of rapamycin (m-tor)-ser2448. it indicates that different constituents of an induces apoptosis or autophage of an oral cancer cell line. to the present, utilization of high-resolution gas chromatography with high-resolution mass spectrometry (hrgc/ ms) is recognized as the most efficient way for determining dioxin compounds [37] . however, the procedures of this detection system are not easy to carry out. the fluorescence resonance energy transfer (fret) technique can precisely evaluate interaction between two molecules in cells, and the use of cyan-fluorescent protein (cfp) as an energy donor and yellow-fluorescent protein (yfp) as an energy acceptor has been reported as the most suitable combination of fret signal detection [38] . lin et al. [39] therefore established a fret-based dioxin-detection assay. aryl hydrocarbon receptor (ahr) and ahr nuclear translocate (arnt) fused-cyan fluorescent protein 9cfp) and-yellow fluorescent protein (yfp) constructs were transiently cotransfected into rat hepatoma cell line h4iiec3 cells. no fret signals were detected in ahr-cfp-and arnt-yfptransfected cells. however, dioxin treatment upregulated fret signals in these transfected cells in a dose-dependent manner, indicating the potential of fret technique in the detection of dioxin-like compounds. connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the src-induced immediate early gene product cef-10 nov (nephroblastoma overexpressed) and the ccn family of genes: structural and functional issues connective tissue growth factor (ctgf) and cancer progression cgcgh: a tool for molecular karyotyping using dna microarray-based comparative genomic hybridization (array-cgh) a computational screen for c/d box snornas in the human genomic region associated with prader-willi and angelman syndromes interactions between m protein and other structural proteins of severe, acute respiratory syndrome-associated coronavirus severe acute respiratory syndrome coronavirus nucleocapsid protein confers ability to efficiently produce virus-like particles when substituted for the human immunodeficiency virus nucleocapsid domain iron dictates the virulence of pseudomonas aeruginosa in urinary tract infections synaptic mechanisms that shape visual signaling at the inner retina glycinergic synaptic inputs to bipolar cells in the salamander retina glycine input induces the synaptic facilitation in salamander rod photoreceptors nicotine enhancement of fast excitatory synaptic transmission in cns by presynaptic receptors laboratory evaluation of antiepileptic drugs. review of laboratory methods the gaba postsynaptic membrane receptorionophore complex dose-finding study with nicotine as a proconvulsant agent in ptz-induced seizure model in mice fatal benzyl alcohol poisoning in a neonatal intensive care unit the gasping syndrome and benzyl alcohol poisoning benzyl alcohol inhibits n-methyl-d-aspartate receptor-mediated neurotoxicity and calcium accumulation in cultured rat cortical neurons bmp-2 evaluation in surgery for tibial trauma-allgraft (bestt-all) study group (2006) recombinant human bmp-2 and allograft compared with autogenous bone graft for reconstruction of diaphyseal tibial fractures with cortical defects. a randomized, controlled trial epidermal growth factor as a candidate for ex vivo expansion of bone marrow-derived mesenchymal stem cells enhancement of ectopic bone formation by bone morphogenetic protein-2 delivery using heparin-conjugated plga nanoparticles with transplantation of bone marrow-derived mesenchymal stem cells t-box genes: what they do and how they do it t-targets: clues to understanding the functions of t-box proteins cascade effect of cardiac myogenesis gene expression during cardiac looping in tbx5 knockdown zebrafish embryos genetic and experimental studies on a mutant gene (c) determining absence of heart action in embryos of the mexican axolotl (ambystoma mexicanum) morphology of developing heart in cardiac lethal mutant mexican axolotls imunofluorescent confocal analysis of tropomyosin in developing hearts of normal and cardiac mutant axolotls a novel protein involved in heart development in ambystoma mexicanum is localized in endoplasmic reticulum differential cardiovascular responses to blockade of nnos or inos in rostral ventrolateral medulla of the rat the nnos/cgmp signal transducing system is involved in the cardiovascular responses induced by activation of nmda receptors in the rostral ventrolateral medulla of cats pressor and sympathoexcitatory effects of nitric oxide in the rostral ventrolateral medulla immunohistochemical assessment of cyclic guanosine monophosphate (cgmp) and soluble guanylate cyclase (sgc) within the rostral ventrolateral medulla nf-jb: a key role in inflammatory diseases development and use of a gene promoter-based screen to identify novel inhibitors of cyclooxygenase-2 transcription immunomodulatory effects of phytocompounds characterized by in vivo transgenic human gm-csf promoter activity in skin tissues arecoline and the 30-100 kda fraction of areca nut extract differentially regulate mtor and respectively induce apoptosis and autophagy: a pilot study a hybrid hrgc/ms/ms method for the characterization of tetrachlorinated-p-dioxins in environmental samples reducing the environmental sensitivity of yellow fluorescent protein. mechanism and applications establishment of a fluorescence resonance energy transfer-based bioassay for detecting dioxin-like compounds key: cord-010681-tmpxs9og authors: dondapati, srujan kumar; stech, marlitt; zemella, anne; kubick, stefan title: cell-free protein synthesis: a promising option for future drug development date: 2020-03-20 journal: biodrugs doi: 10.1007/s40259-020-00417-y sha: doc_id: 10681 cord_uid: tmpxs9og proteins are the main source of drug targets and some of them possess therapeutic potential themselves. among them, membrane proteins constitute approximately 50% of the major drug targets. in the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis. cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. in a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. in the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. in this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. further, this article discusses some recent progress in cell-free systems derived from chinese hamster ovary and sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. we particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. we also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development. proteins whose functionality is not well characterized form a large percentage of entries in many of the currently available biological databases, including the protein data bank (pdb), and there is a constantly growing demand for reliable and fast synthesis and characterization methods. when it comes to drug discovery, proteins are key components as they can have therapeutic potential themselves (e.g., antibodies, coagulation factors, hormones, growth factors, enzymes, and antimicrobial peptides), but also because they could serve as drug targets for diverse diseases (as ion channels, receptors, enzymes, and transporters, for example) [1] [2] [3] [4] [5] [6] [7] . a large proportion of approved pharmaceutical drugs target human proteins. beyond that, protein-based therapeutics, such as antibody-drug conjugates, represent a significant percentage of total drug molecules currently approved. they are poised to grow further with increased gene expression technology, improved protein engineering, and refined bioinformatics tools. some proteins are very difficult to express in traditional cell-based systems and this can hamper our ability to define the mechanism of action and structure-function relationship of the individual protein, knowledge which aids the development of drugs targeting these proteins [1] [2] [3] [4] . generally, to exploit and fine-tune the structural and functional characteristics of a protein, it needs to be expressed and purified with high quality by using recombinant expression technology. traditionally, escherichia coli-based systems were widely used for the production of recombinant proteins due to simplicity in preparation and operation, and cost effectiveness. as a result, broad research and standardization from several years was performed using e. coli-based expression systems, resulting in their often-cited utilization as a state-of-the-art protein expression system [8] . for complex therapeutic proteins, membrane proteins (mps) originating from humans, and virus-like proteins (vlps), mammalian expression systems fulfill all the requirements like post-translational modifications (ptms), cofactors, and chaperones for correct folding and efficient production. however, batch-tobatch variation in cell culture may be a source of process variation. additionally, overexpression of mps might be toxic for the cultivated cells, resulting in cell death or truncated and misfolded proteins [9] . ideally, synthesized proteins are functionally folded and exhibit appropriate ptms. due to the lack of extensive research and low yields in recombinant protein expression, many mps are not yet crystallized, thus limiting the computer-aided drug discovery efforts. due to the growing demand for the production of protein biologics and drug discovery targeting proteins, alternative strategies for protein synthesis should be developed. new expression technologies where proteins can be expressed in a simple way and which allow high throughput screening of different reaction conditions, different genes, and different supplements in a cost-effective manner are extremely important for future drug development. in this review, we give an overview of recent advances in cell-free (cf) synthesis platforms and their diverse applications. additionally, we focus on human and therapeutic proteins produced by different types of cf systems and how these cf protein synthesis (cfps) methods can further play a prominent role in future drug development. cfps systems use crude cell extracts prepared from cells of choice by lysis followed by many steps of washing to remove the cell debris and genomic dna [10, 11] . these cell extracts can be stored at − 80 °c for years and can be used by thawing just before the reaction. such extracts contain all the principal components necessary for transcription and translation, such as aminoacyl-trna synthetase (aas), ribosomes, and factors necessary for elongation, initiation, and transcription. protein synthesis can be realized by combining cell extracts with necessary substrates like amino acids, energy substrates, dna, cofactors, salts, and nucleotides. depending on the biochemical properties of the protein and its end application, the appropriate cf system can be selected. cfps is a fast protein production system since it does not require transfection or cell culture and lacks cell viability constraints. due to its openness, cfps platforms offer additional advantages when compared with cell-based expression methods. a comparative analysis of cf and cellbased approaches is shown in table 1 . for complex proteins, eukaryotic cf systems are ideal as they contain the endogenous microsomes derived from the endoplasmic reticulum (er), enabling co-translational translocation of proteins and er-based ptms [10, 11, 18, 19] . there has been a constant improvement in the quality of lysate preparation, system optimization, linear templatebased protein synthesis, and reduction of process costs, which has led to the preparation of cost-effective systems suitable for commercial purposes. a general scheme of cf protein production is depicted in fig. 1 . cfps platforms are based on either prokaryotic or eukaryotic origin. among the prokaryotic cf systems, extracts based on e. coli are regularly used and are available commercially for cfps of a diverse range of proteins. very recently, cf systems based on bacillus subtilis [32] , pseudomonas putida [33] , streptomyces [34] , and vibrio [12] have been optimized well at the laboratory level due to the ease of preparation of cf lysates. a wide range of detailed protocols is currently available for the preparation of e. coli-based lysates. among the eukaryotic cf systems, extracts based on rabbit reticulocyte lysate (rrl), wheat germ, insect spodoptera frugiperda 21 (sf21), chinese hamster ovary (cho), and cultured human cells are regularly used. an increasing number of eukaryotic cf systems have so far reached technical maturity and become commercially available. requires dna template to be cloned in a plasmid [15] toxic proteins ideal choice for the synthesis of most of the toxic proteins due to high toxic tolerance of cf systems toxic proteins may be difficult to synthesize [16] membrane proteins suitable for a wide range of mps of different sizes mp overexpression can lead to cell toxicity and death [17] [18] [19] membrane protein solubilization to solubilize mps, supplements can be added directly to the reaction mixture in the form of nanodiscs, detergents or liposomes (prokaryotic systems and wheat germ systems) or by using endogenous microsomes or proteoliposomes (eukaryotic systems) not possible to add supplements externally during translation mps have to be purified and reconstituted into liposomes or detergents for functional analysis [9, 20] reliability most of the reports related to cfps are currently limited to the research and laboratory level, but progress towards the drug discovery pipeline has been made recently most reliable and state of the art for protein production and drug discovery purposes, and approved by drug authorities [21] functional characterization compared with cell-based systems, standardized biophysical, biochemical assays are limited, but progress has been made recently a wide range of standardized biophysical and biochemical techniques are available for proteins synthesized by cell-based systems [22] protein yields and downstream applications yields range from µg/ml (complex proteins) to several mg/ml (cytosolic proteins and few mps) with more complex proteins downstream applications are simple and protein can be purified and reconstituted immediately after synthesis yields can be very high, in the range of mg/ml downstream applications are possible but need to additionally lyse the cells for mps [11, 21, 22] post-translational modifications (ptms) ptms possible (mostly in eukaryotic cf systems with translationally active microsomes) limited ptms in prokaryotic and eukaryotic lysates lacking endogenous microsomes. o-glycosylation not possible all ptms are possible including o-glycosylation [13] incorporation of non-standard amino acids ideal choice for the incorporation of single and multiple noncanonical amino acids difficult to incorporate non-canonical amino acids due to cell membrane barrier and cytotoxic effects [23, 24] scale of reaction volume ranging from few µl (chip-based and batch-based in an eppendorf tube) to 100 l reaction (in a fermenter) typical reactions require a minimum of 5 ml. there are exceptional cases where it is performed in 60 µl spots [25] [26] [27] [28] flexibility completely open system and easy to manipulate the reaction conditions with lack of cell membrane constraints completely closed system and difficult to manipulate [22] automation cf systems can be automated with high throughput screening of multiple templates, starting in an elisa plate format generally difficult to automate due to the requirement of larger volumes and aseptic techniques [29] point of care production of biologics lyophilized cf lysates are suitable for the production of therapeutic proteins next to the emergency settings very difficult due to its time-consuming process and requirements of large infrastructure including manufacturing facilities, transport, and cold storage facilities [30, 31] recently, several eukaryotic cf extracts based on tobacco [35] , leishmania [36] , neurospora [37] , yeast cells [38] , and human blood cells [39] were characterized and optimized for a limited number of proteins at the laboratory level. there is a growing trend in the development of novel cf platforms for taking advantage of the genetic tools available in the literature and the abundant literature available on the in vivo expression of proteins. prokaryotic cf systems based on e. coli are most commonly used for protein production towards drug development due to their simplicity and a vast literature available on the utilization of these cells. protein synthesis starts with crude cell extracts prepared from e. coli cells that contain the translation machinery along with all the essential components required for translation. a modified and reconstituted cf synthesis system known as the pure system (protein synthesis using recombinant elements), where all the components of the translation machinery are purified and added individually along with the dna template to produce the protein, has been reported [40] . this is a highly controlled system compared with crude extract methods. a major advantage of the pure system is that protein factors participating in the initiation, elongation, and termination of the protein synthesis process are identified and can be adapted individually to the cf system's requirements. although the naturally occurring ptm machinery is not available in the e. coli lysates, recently proteins with n-glycosylation were synthesized by using e. coli extracts enriched with glycosylation components, including oligosaccharyltransferases (osts) and lipid-linked oligosaccharides (llos) [41] . using release factor (rf1) deficient e. coli lysates, proteins were phosphorylated by incorporation of non-canonical amino acids, which will be addressed in a later part of this review [42] . due to a constantly growing demand for more complex proteins of pharmaceutical value, cf systems based on eukaryotic lysates have been developed to produce highquality proteins. cf systems based on wheat germ lysates (wgl) are among the most popular eukaryotic platforms due to their capacity to produce eukaryotic proteins with high yields [43] . cfps based on wgl have been used frequently for the discovery of novel vaccine candidates as well as for producing several proteins of high quality for structural analysis. despite the high yields and quality of the lysate, this system does not offer all the ptms like glycosylation and does not support the solubilization of complex mps [13] . in the case of wheat germ and rrl, there are no translationally active endogenous microsomes present in the system. in the case of rrl, exogenous microsomes are typically supplied from the canine pancreas for protein translation [13, 44] . it is quite laborious and difficult to enrich rrls with heterologous microsomes. cf systems derived from cultured insect (sf21) cells represent the most popular eukaryotic-based approach for synthesizing a wide variety of proteins. sf21 lysates contain translationally active endogenous er membranes, thereby supporting the signal peptide-mediated translocation of proteins across the membrane, and further provides functions such as signal peptide cleavage, post-translational modifications like n-glycosylation, and lipid modification [13, 14, 45] . cho cell-based expression is well established and is approved for the large-scale synthesis of several biologics by the fda because it undergoes human-compatible ptms. nearly 70% of the approved mammalian therapeutic proteins are currently expressed in cho cells. however, these cells have limitations when it comes to difficult-to-express proteins like overexpression of complex mps, toxic proteins, and multi-subunit proteins as discussed above. cf systems based on cho lysates are evolving as an alternative strategy for the expression of difficult-to-express proteins [13, [17] [18] [19] 46] . apart from many general advantages of cf systems, cho-based cf systems retain most of the features of cho cells while being more flexible due to the lack of cell membrane boundaries. cho-based lysates harbor endogenous microsomal vesicles enabling translocation of transmembrane proteins and secretory proteins. furthermore, ptms of de novo synthesized mps, such as glycosylation, are possible using cho lysate. thus, using cho cell lysate for cfps has a potential value and enables new opportunities, in particular, the high-yield production of pharmaceutically relevant mps [13, [17] [18] [19] . there is a significant increase in the number of publications based on cho lysates for cfps. table 2 compares different cf systems and their advantages and limitations with some selected examples. fig. 1 general scheme depicting the overall process of cell-free protein production. aatrna aminoacyl-trna, aas aminoacyl-trna synthetase, atp adenosine triphosphate, ef elongation factor, gsh glutathione, gssg glutathione-disulfide, gtp guanosine-5'-triphosphate, if initiation factor, ires internal ribosome entry site, mp membrane protein, ncaa non-canonical amino acid, pdi protein disulfide isomerase, peg polyethylene glycol, ptm post-translational modification, r ribosomes, t-rna transfer rna, tf transcription factor, utr untranslated region, vlp virus like particle chinese hamster ovary (cho) mimic the cho cell-based production ptms (n-glycosylation, disulfide bridging, and lipidation) suitable for a wide range of eukaryotic and complex proteins presence of translational active endogenous microsomes [45] high yields in cecf mode endotoxin free lysates used for point-of-care testing [30] low yields especially in the batch mode [58] cost ineffective and difficult to establish unlike e. colibased system streptokinase (cecf): 500 µg/ml [59] human tlr9 receptor [18] (3-h batch): 21 µg/ml (48-h cecf): 900 µg/ml hegfr [46] (batch): 40 µg/ml (cecf): 800 µg/ml cfps can be performed in different formats. the batchbased format is the most commonly used method both in the prokaryotic and eukaryotic systems. this method is relatively fast and cheap, and synthesis can be performed within 1.5-3 hours depending on the system. e. coli-based systems can provide protein yields ranging from 100 µg/ ml to 2-3 mg/ml. although the yields from batch-based eukaryotic systems are comparatively low, mps are automatically incorporated into microsomal membranes and the functionality can be addressed immediately after the synthesis [62] . for researchers who would like to further scale up the protein yields via batch-based eukaryotic systems, a repetitive batch-based synthesis format has been proposed where the microsomes incorporating the mp of interest generated in an initial synthesis reaction can be added to a fresh cf synthesis reaction that has been depleted of its microsomes [19, 45] . another popular cf synthesis format that has been used for a rapid increase in the protein yields is the so-called continuous exchange cell-free synthesis platform (cecf). in this format, a semi-permeable dialysis membrane separates the reaction chamber and a feed chamber and thereby a feed chamber provides the fresh reaction components and enriches the reaction chamber. in exchange, the inhibitory components accumulated during the reaction are removed [14, 17, 18, 46] . typically, the cecf format prolongs the reaction time and increases the protein yields. until now, the cecf format has been used to increase the protein yield by multiple fold, and is widely used as cf platforms (table 2 ). this section highlights some of the key parameters that might influence the protein production using cf lysates. designing synthetic dna and sequence manipulation for cf synthesis by adding regulatory elements plays a significant role in high-yield protein production. in eukaryotic cf systems, initiation factors (ifs) in particular limit the initiation of protein synthesis, thereby leading to low protein yields. one alternative is to use internal ribosome entry site (ires) elements found in the 5′-untranslated region (5′utr) of the different viral genomes upstream of the start codon for cap-independent translation initiation [14, 18, 62] . ires elements from three different viral sources were compared for their translational efficiency in sf21, cho, and human leukemia k562 cf lysates. the ires from the cricket paralysis virus (crpv) typically increased protein yields by a factor of 3-5 [62] . inserting the crpv-ires into the corresponding vector upstream of the epidermal growth factor receptor (egfr) gene, and using the cecf reaction format, egfr yields were significantly increased to more than 100-fold compared with batch reaction format without crpv-ires [14] . additionally, replacement of the initiator codon (atg) to a gcu-codon in combination with the crpv-ires resulted in a further improvement of protein expression levels in cho and k562 cf systems [62] . the vector backbone also plays an important role in cfps. a detailed study comparing commercially available vectors harboring the luciferase gene in combination with crpv-ires showed that there is a significant 5-fold increase in protein yield with a change in the vector backbone [19] . species-independent translational sequences (sits) are another group of synthetic 5′utrs capable of initiating cap-independent translation in multiple prokaryotic and eukaryotic cf systems [66] . typically, polymerase chain reaction (pcr) products are generated with sits downstream of the t7 promoter and upstream of the start codon atg [66] . the 3′ hairpin region of the sits increases the residence time of the preinitiation complex in the vicinity of the start codon [66] . using l. tarentolae cfps in the presence of genes encoding 58 rab encoding variable fragments in combination with a universal sits, nearly a full complement of human rab gtpases were produced with a yield of around 30 µg/ml [36] . similarly, egfp with a yield of around 300 µg/ml [66] , and an active multisubunit enzyme heterodimeric farnesyl transferase (ftase) [36] were synthesized using the l. tarentolae cfps [36] . codon optimization is another important parameter that plays a crucial role in increasing the expression yields of proteins. codon optimization has been shown to influence the translation efficiency of several proteins [71] . by taking advantage of the cf lysates derived from n. crassa and s. cerevisiae, transcription and translation reactions were uncoupled for ribosome profiling, which provided strong biochemical evidence that codon optimization enhances the rate of translational elongation, thereby affecting the ribosome traffic on the mrna [72] . on the one hand, codon optimization usually improves protein yields, but on the other hand, it was shown that faster translation rates might negatively affect the protein folding and function of the individual protein [72, 73] . this problem often cannot be solved even by altering the trna population in the case of cfps. the addition of anti-spliced leader oligonucleotide to l. tarentolae cell extracts suppressed the translation of endogenous l. tarentolae mrnas, thus increasing the translation efficiency of exogenously supplied mrna [65] . using the er-specific signal sequence of honeybee melittin (melittin signal peptide) instead of the native signal peptide increased the translocation of synthesized proteins such as wnt proteins, single-chain antibody variable fragments, and the htlr9-ectodomain into microsomes in the case of sf21 and cho-based cf systems [18, 59, 74, 75] . iterative optimization processes are required to develop highyield cfps. factors that influence both protein quality and quantity include reaction temperature, reaction time, plasmid concentration, salt concentration, t7 polymerase, and other supplements. the influence of these factors on the synthesis rates is also protein specific. very recently, cfps of human toll-like receptor protein (htlr9) in cho-based lysates has been reported by using a cecf method with high yields of around 0.9 mg/ml. by increasing the temperature from 27 to 30 °c, the protein yields were increased by almost 50%. stable monitoring and maintenance of ph throughout the entire cf reaction along with sufficient adenosine triphosphate (atp) supply are essential for efficient and maximum yield protein production. by using amino acid decarboxylase, the ph is controlled throughout the cf reaction [76] . supplementation of chaperones influences the functional folding of many proteins. supplementation of chaperones such as groes/el and dnak/dnaj/grpe in prokaryotic cf systems was used to increase the yield and solubility of colicin m from 16 to 100%, resulting in enhanced cellkilling activity [77] . li et al. demonstrated that by using cfps based on wheat germ extracts, expression of j-domain containing chaperone proteins (dnajb12 and dnajb14) along with potassium channels plays a critical role in the folding, stabilization, and tetramerization of k+ channels [78] . ion concentrations (potassium and magnesium) in the cf reaction have a significant effect on protein production. in the case of cho-based cecf reactions, an increase in the magnesium ion (mg 2+ ) concentration from 3.9 to 22.5 mm led to a 3.9-fold increase in egfr yield [46] . for efficient regeneration of atp, several methods have been developed in cf systems. in prokaryotic systems, compounds like phosphoenolpyruvate (pep), glucose + glutamate decarboxylase, glucose-6-phosphate, fructose-1,6-biphosphate, acetyl phosphate, maltodextrin, and creatine phosphate are widely used as energy sources [79] . in eukaryotic cf systems, a combination of creatine phosphate and creatine kinase is typically used for energy regeneration. apart from these, phosphoglycerate (b. subtilis), and polyphosphate are used in cf systems [80, 81] . cf systems have evolved over the last decade from their use as a prototype method in research laboratories to commercial and large-scale applications. in this section, the utility of cf systems in mp synthesis, antibody production, vaccine development, protein labeling, and antimicrobial peptide synthesis are addressed. mps are structurally and functionally diverse, and constitute 30% of the proteins encoded in the human genome. drugs targeting mps such as ion channels, transporters, and g-protein coupled receptors (gpcrs) represent 12 out of the top 20 global revenues in the pharmaceutical industry [3] . due to the presence of transmembrane domains, ranging from 1 to 24, these proteins are highly hydrophobic and are very challenging to express by traditional cell-based systems. expression of human proteins in heterologous cellular hosts is very much limited due to the difference in their lipid composition, which can prevent the mps from attaining maximum functionality [9] . synthesis of mps by cell-based methods often leads to cytotoxicity, aggregation, and improper folding [9] . to analyze mp functionality, the protein needs to be folded properly and in the appropriate hydrophobic environment. cf systems derived from prokaryotic, as well as eukaryotic lysates lacking endogenous microsomes, require specific supplements for the solubilization of mps in the form of detergents, nanodiscs, or liposomes. non-ionic and zwitterionic detergents are commonly used as supplements in the majority of cfps reactions for the solubilization of mps during their production. detergent-solubilized mps can be either used directly for functional analysis or may be reconstituted into liposomes by mixing with artificial lipids followed by detergent removal [9, 82, 83] . alternatively, nanodiscs (nds) and liposome-based reconstitution are detergent-free strategies where nds and liposomes, prepared and characterized externally, could be supplemented directly into the reaction mixture for the reconstitution [9, 84] . a detailed review of the cf synthesis of mps and the usage of solubilization supplements for isolation and functional analysis is presented in the literature [9] . some of the advantages of nds are easy purification and flexibility in using different lipids and membrane scaffold proteins for creating different sizes, and their availability as monodisperse and homogenous nds. nonetheless, nds have their limitations, particularly when working with a protein whose functionality depends on its orientation and also working with transporter proteins. liposome-based reconstitution covers the limitations of the nds, but the separation of liposomes after the cf reaction is quite challenging and often suffers from disruption due to osmotic instability. further, such passive reconstitution strategies do not offer the advantages of post-translational modifications within native membranes and are limited for mps whose function does not depend on active translocon-based translation. cf systems derived from eukaryotic lysates equipped with endogenous microsomes (e.g., sf21, cho, cultured human cells, and tobacco-by2) satisfy all the necessary requirements for proper folding of mps. the microsomes offer a native environment and intact translocon machinery for a proper embedment and folding of mps [13, 18, 19, 45, 46, 51, 59] . there are continuing efforts in analyzing the functionality of microsomal reconstituted mps, indicated by a good number of publications reporting on this reconstitution strategy, which should help the pharmaceutical industry to develop more dynamic drug screening assays involving mps [9, 46, 83] . here we present recent works on ion channels, transporters, and gpcrs, which constitute more than 40% of the major drug targets in the pharma industry [85] . ion channels constitute approximately 19% of all currently existing human drug targets and play a crucial role in diverse physiological processes involving cell excitability, neuronal transmission, metabolism, sensory transduction, cognition, and electrolyte homeostasis. transporters mediate the translocation of a variety of substrates across biological membranes [86] . the solute carrier (slc) family is the largest class of transporters and is implicated in metabolic conditions and diseases, and in the transport of drugs. these proteins typically have 9-12 transmembrane domains and are difficult to express by traditional methods [2, 9, 50, 87] . slc transporters are an emerging drug target class and the molecular target of several approved inhibitor drugs [2] . despite this, these classes of proteins remain largely unexplored in recent years due to the high costs involved and lack of proper expression methods [9, 53] . table 3 highlights some of the selected publications using cf methods for synthesis, reconstitution, and functional analysis of ion channels and transporters. the most widely used method of reconstitution for functional analysis is detergent-based reconstitution into liposomes or passive integration of mps into liposomes and nds. the majority of the functional assays were performed with plbe in the case of ion channels and substrate uptake assays in the case of transporters. gpcrs transduce extracellular stimuli to the inside of cells, after activation by a variety of different molecules such as neurotransmitters, hormones, odorants, and peptides, thereby triggering several signal transduction cascades. the involvement of gpcrs in almost all processes in living cells has resulted in significant pharmaceutical interest in this protein class, and the development of robust and high-throughput-suitable assays for the discovery of novel ligands and drugs targeting these proteins. in principle, the screening of ligands can be performed in whole-cell assays by measuring a downstream signaling event, or in cf assays, which are decoupled from the living organism. usually, these decoupled methods are preferable for high-throughput screenings, as they are easy to handle and therefore amenable for automation and downsizing. these parameters can be well combined with cfps. an automated cf synthesis procedure for the production of different mps is already reported [97] . this procedure might be further expanded for the parallel analysis and identification of molecules that target different gpcrs. to date, only a few studies have analyzed in detail the activity of receptors produced by cf systems. the main reason for this is that there are limited well established activity assays. this section addresses possible activity assays that might be transferred to cf systems in the future. radioligand binding assays, the gold standard for identifying binding molecules, are already adapted for gpcrs that have been synthesized in eukaryotic and prokaryotic cf systems, and demonstrate similar binding affinities in comparison with in vivo produced gpcrs [20, [98] [99] [100] . alternatively, fluorescently labeled ligands can be analyzed by an optical read-out system using eukaryotic cf systems harboring endogenous membrane structures [101] . nevertheless, for these systems, radiolabeled or fluorescently labeled ligands are required, thereby limiting the analysis mainly to gpcrs with already known ligands. in addition, simple ligand binding assays usually do not differentiate between an agonistic and an antagonistic effect of the bound substance. in this context, measuring downstream signaling to distinguish between an activating and inhibitory ligand is preferable. one possible method of choice is the receptor-mediated coupling of g proteins [102] . this early event immediately follows after gpcr activation and is detected by the binding of [ 35 s]gtpγs to gα subunits. this method is not yet established in cf systems but might be transferable assuming the presence of gα proteins in the eukaryotic lysate. alternatively, the gα proteins can be additionally co-synthesized to the target gpcr or directly applied to the reaction based on the open nature of cf systems. after gpcr activation, gtp binding and hydrolysis should be detectable. in addition to ligand binding and g protein coupling, intra-and intermolecular interactions can be visualized by förster and bioluminescence resonance energy transfer techniques. different sensor models are known in living cells [103] . the monitoring of intermolecular interactions can be performed as well in cf systems using the already established in vivo models. one model includes the tagging of the c-terminus of a gpcr of interest to a fluorophore (gfp/yfp) and fusing a binding partner such as β-arrestin to luciferase or a second fluorophore [104] . upon activation of the gpcr, β-arrestin binds to the receptor and both tags are in close proximity, resulting in a measurable energy transfer. this model requires active g protein-coupled receptor kinases for the phosphorylation of the c terminus of the synthesized gpcr to get recognized by β-arrestins. this requirement has to be analyzed in detail in the individual cf systems. the second known in vivo model visualized intramolecular changes after agonist and antagonist binding by introducing fluorophores into the third extracellular loop and the c terminus of different gpcrs. upon activation, the distance between both fluorophores changes and an alteration in the energy transfer can be measured [105, 106] . initial experiments to transfer these energy transfer-based sensors were recently performed in cf systems [107] . both models can be applied to high-throughput analyses. in summary, the successful cf synthesis of a variety of gpcrs has been demonstrated in recent years and a transfer of these gpcr production systems to a drug discovery format in a high-throughput manner has recently started. in the near future, we might see novel technologies for ligand screenings, thereby utilizing the advantage of the automatization and downsizing capacity of cf systems. the gold standard for synthesis, development, and production of antibody-based drugs (based on full-length antibodies) is mammalian cell culture-based expression systems. although cultivation of mammalian cells is well established and widely used, the development of monoclonal antibodies (mabs) and antibody-drug conjugates (adcs) remains time-consuming and challenging. thus, methods for highthroughput screenings, especially in the early-stage evaluation of antibody candidates, are valuable. in view of this, the use of the cf technology constitutes a promising strategy to shorten the time from antibody discovery to production. using cf technology, antibodies can be produced in a flexible scale within a couple of hours. besides the synthesis of individual antibodies, cf technology can be used to display libraries of antibodies. in contrast to phage and yeast display, in vitro cf systems such as ribosome and mrna display are open, and thus result in higher library sizes. in theory, the size of the library is only limited by the quantity of supplemented mrna/dna, the volume of the cf reaction, and the number of ribosomes within the system, resulting in library sizes of ~ 10 12−15 /ml cf reaction [108] . in comparison, phage and yeast display exhibit library diversities of ~ 10 6 -10 10 . selection technologies such as ribosome display [109] , mrna display [110] , and cis display [111] have been developed based on reticulocyte lysate [110] and e. coli cf systems [109] . these systems focused on smaller antibody fragments because their functionality does not rely on the assembly of multiple polypeptide chains. nonetheless, recently two groups have succeeded in developing completely cf display technologies that allow the selection of fab fragments [112] . the challenge to assemble the heavy and light polypeptide chain (hc/lc) of the fab fragment was approached in different ways. while sumida et al. succeeded by combining mrna display based on two mrna sub-libraries, one encoding hc, the other one encoding lc, with in vitro compartmentalization pcr to link and then amplify hc and lc gene pairs [112] , stafford et al. developed a ribosome display method where they displayed only one of the two fab chains, while the other one was not presented in display format [113] . successful synthesis of different antibody formats, including single-chain variable fragments (scfvs), fab fragments, as well as complete iggs, has already been shown in e. coli [114, 115] , sf21 [10, 116] , reticulocyte [110] , wheat germ [117] , and cho cf systems [46, 75, 118] . furthermore, the upscaling of cf reactions to the liter-scale [25, 115] as well as downscaling [119] and high-throughput applications [120] have been demonstrated. in addition, advances in bioorthogonal reaction chemistries have paved the way to expand the possibilities for adc development. the site-specific introduction of non-canonical amino acids into a genetically engineered sequence can be used to create site-specifically labeled adcs [121] . currently, seven adcs are approved for therapy. to date, all of these adcs have been generated by coupling of mabs to the cytotoxic linker-payload via surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, which typically results in a controlled but heterogeneous adc population with varying numbers and positions of drug molecules attached to the mab [122] . homogeneous adc populations can be achieved by introducing the payload at one or more defined positions. by developing a bioorthogonal trna/synthetase pair, zimmerman et al. have shown that the optimized non-canonical amino acid para-azidomethyll-phenylalanine (pamf) can be site-specifically incorporated into the tumor-specific, her2-binding igg trastuzumab [123] . subsequently, the cytotoxic linker payload dbco-peg-monomethyl auristatin (dbco-peg-mmaf) was conjugated to pamf via strain-promoted azide-alkyne cycloaddition (spaac) copper-free click chemistry. in the context of dual-functioning molecules, bispecific antibodies have also emerged as promising anti-cancer agents. one of the advantages of these proteins is their capability to target two different epitopes simultaneously, thereby increasing target engagement, where mono-specific antibodies might fail [124] . due to their open design, cfps reactions can easily be manipulated, for example by varying the template ratios and concentrations of hc and lc. for example, xu et al. showed the successful assembly of bispecific 'knobs-into-holes' antibodies in multiple scaffolds by using an e. coli-based cf expression platform [125] . taken together, antibody evolution, selection, and engineering can dramatically benefit from the technological advances in the field of cfps. (1) novel display technologies based on cf methods enable the in vitro evolution of multimeric proteins and allow for more sophisticated protein engineering. (2) due to the very short time frame from synthesis to functional testing, cf systems can accelerate antibody construct evaluation by a repetitive (one after one) and/or parallel screening. (3) the introduction of non-canonical amino acids expands the chemical repertoire and thus the possibilities to modify and improve antibody-based therapeutics. advanced labeling technologies allow for a very fast qualitative analysis of drug-to-antibody ratio (dar), linker, linker/position, drug, drug/position (research application), and allow full control of the adc design (commercial application). cf systems are becoming a potential option for synthesizing vaccine antigens. most of the vaccine antigens produced by cf systems to date have used e. coli and wgl. recent progress on eukaryotic cf systems may offer additional advantages. in this context, eukaryotic cf systems are endotoxin free and lack the complex plasma membrane that makes the protein purification simple. some of the antigens synthesized by using cf systems are highlighted in table 4 . they are able to induce a strong immune response in experimental animals and could serve as a proof of concept for future vaccine development. using recent advances in cfps technology, a freeze-dried, cell-free (fd-cf) expression system was created based on e. coli cf lysates [31] . using this fd-cf technique, diphtheria toxoid antigen variants (dt5 and dt6) were produced following rehydration with water and functional characterization of the synthesized proteins was verified following administration in mice and measuring the immune response [31] . the fd-cf method could enable the production of on-demand, point-of-care biologics requiring just the simple addition of water for activation and synthesis. recently, cf-based expression has proven successful in producing difficult-to-express proteins like major outer membrane protein (mmomp) of chlamydia spp., a major vaccine antigen. using e. coli-based cfps, mmomp was synthesized in a native trimeric form in the presence of nanolipoproteins (nlps) with a yield of around 1.5 mg/ml. when injected into mice in the presence of an adjuvant, the protein elicited an enhanced humoral immune response [126] . this method of synthesizing and simultaneous incorporation of antigens into nlps using a cf approach is a promising method for future vaccine development. conjugate vaccines are one of the safest and most effective biologics [127] . bioconjugate vaccines are produced using protein glycan coupling technology (pgct). however, pgct has its own limitations such as time-consuming in vivo processes. additionally, fda-approved carrier proteins, such as toxins derived from clostridium tetani and corynebacterium diphtheria, have not yet been demonstrated to be compatible with an e. coli-based production process. relevant ptms are often difficult to synthesize in e. coli cf systems. meanwhile, there have been further advances in using bacterial glycoengineering combined with cfps for producing bioconjugate vaccines. this cf glycoprotein synthesis (cfgps) used glycooptimized e. coli extracts integrating both n-linked glycosylation and protein synthesis. using cfgps, two bioconjugate vaccines were synthesized against f. tularensis and e. coli o78 [146, 147] . besides posttranslational modification, the assembly of macromolecular structures in cf systems is highly ambitious. virus-like particles (vlps), for example, are nanoscale structures that are formed from the self-assembly of viral proteins without the viral genome responsible for the infection. usually, vlps mimic the capsid structure of the real virus. vlp antigens are vaccine candidates for several diseases [148] . one of the vaccine candidates, which is currently in clinical trials, contains vlp antigens addressing noroviruses responsible for gastroenteritis in humans [149] . cf synthesized vlps were structurally confirmed by electron microscopy [150] . using e. coli cf systems, antimicrobial colicins (colicin m, la, e1, and e2) have been synthesized with high yields (around 300 µg/ml) and solubility. the synthesized colicins are able to effectively kill the target cells without any purification [151] . antimicrobial peptides (amps) are another class of defense molecules that have a wide spectrum of targets; for example, bacteria, viruses, fungi, parasites, and cancer. amps are evolving as alternatives to antibiotics [152] . using lyophilized e. coli cf lysates, ten different amps have been synthesized successfully and the functionality of plasmodium falciparum wheat germ interaction of pfmsa180 with cd47 was confirmed by erythrocyte binding assay [133] . antigen-specific igg responses to lsa3-c were profiled by an alphascreen assay [134] . western blotting and elisa confirmed the interaction of purified recombinant msp11 with human sera [135] . immunization of purified pfron12 with freund's complete adjuvant into japanese white rabbits generated pfron12 antisera [136] . rabbits immunized with expressed pfripr produced specific antibodies [137] . anti-exp1 antibodies were generated by immunization of recombinant exp1 [138] . wheat germ proteoliposomal engineered cldn-5 antigens induced anti-cldn5-5-ecr antibodies in mice [145] αcd19-id small diabody (db) molecule containing both a b-cell-targeting moiety (anti-cd19) and a lymphoma id, 38c13-scfv idiotype-specific single-chain variable fragment of the immunoglobulin from the 38c13 mouse b-cell lymphoma, cldn-5 claudin-5, when non-canonical amino acids (ncaas) are incorporated into proteins, novel functional, structural, and imaging properties can be generated. this synthetic biology application is fast emerging and has wide applications such as incorporating precise ptms and adding novel functions to proteins [23, 24, 42, 45] . by taking advantage of the openness of the cfps, one can add the machinery responsible for the co-translational incorporation of ncaas directly to the standard reaction components. one possible method to incorporate ncaa is to use precharged trnas harboring the ncaa. one of the most commonly used methods is the amber suppression technology using an orthogonal pair of aminoacyl-trna synthetase/trna (o-trna/aars pairs from distinct organisms), which functions independent of endogenous aarss and trnas in the host and is used to direct the incorporation of ncaas to specific positions such as the amber stop codon (uag). after incorporation of an ncaa with a reactive group, bioorthogonal click reactions can be performed to conjugate a molecule of interest. the most general biorthogonal click reactions for conjugating molecular probes or polymers are the copper-catalyzed azide-alkyne cycloaddition (cuaac), staudinger-ligation, photo click cycloaddition, strain-promoted azide-alkyne cycloaddition (spaac) and inverse electron-demand diels-alder cycloadditions (iedda + spiedac). using e. colibased cf systems, cui et al. showed the incorporation of two fluorescent labels, bodipy fluorophore and tamra-dibo, by using a precharged trna + orthogonal system for fret measurements [153] . using sf21-based cf systems, quast et al. demonstrated the incorporation of p-azido-l-phenylalanine at defined amber positions in parallel in the two subunits of the human egfr protein dimer. later, the azido group of the incorporated azfs was coupled by photoaffinity cross-linking using a bis-combo linker to create a stable synthetic dimer of egfr [14] . the dimerized protein shows autophosphorylation in the presence of tyrosine kinase. in general, release factor 1 (rf1) competes with orthogonal ncaa-trna for the amber codon, which results in truncated products along with successfully suppressed products. so, cf lysates derived from genetically modified e. coli lacking release factor 1 (rf1) can be used to enhance the incorporation efficiency of ncaas. using the orthogonal system and e. coli-based cfps, human mek1 kinase with ptms was synthesized up to milligram quantities by site-specific, co-translational incorporation of phosphoserine at specific positions [154] . various polyethylene glycol (peg) moieties have been widely used to decorate therapeutic proteins. the peg moiety usually offers high stability and extends the half-life of proteins while in circulation inside the body. the food and drug administration (fda) has recognized peg moieties as safe due to their structural flexibility, hydrophilicity, and minimal toxicity, and several pegylated drugs have been approved by the fda. using sf21-based cf systems, a site-specific pegylated human epo was produced and characterized by autoradiography [45] . apart from the amber suppression strategy, there are other strategies like frameshift suppression, sense codon reassignment, and unnatural base pairing. a detailed review of prominent methods for the incorporation of ncaas into proteins using cfps has been recently published [23] . a wide range of commercial cf systems is available in the market based on lysates derived from diverse sources. as well, a few companies provide services for cf synthesis of proteins. some of the products derived from the cf systems based on e. coli lysates are already in clinical trials, such as adcs targeting cd74 and folate receptor alpha highly expressed in myeloma and cancer cells (sutro biopharma, inc, usa). table 5 lists commercial systems currently available for the cf production of proteins. evolving cf systems from a laboratory level to a robust production platform is necessary to fulfill their potential. prior to full realization of cf systems as emerging tools for drug discovery and evaluation, several factors need to be addressed, like synthesis of the high-quality functional protein with proper folding and ptm, cost of production, scalability, and safety issues. a more detailed understanding of the components in the cf lysates will substantially improve the quality and stability of the extract preparation. the quantity of the protein depends on the translation efficiency of the cf system. the most important factors that influence the protein yields are quality of the celllysate, reaction conditions, and template optimization as addressed in section 3.2. to increase the translation efficiency, further efforts are required to increase the quality of lysate production. this can be achieved by using genetic engineering tools to remove the factors responsible for nucleic acid degradation, ribosome inactivation, and protein degradation. brodiazhenko et al. showed that genomic disruption of genes encoding ribosome-inactivating factors (hpf in b. subtilis and stm1 in s. cerevisiae) has improved the activities of bacterial and yeast translation systems [54] . in this context, advanced engineering tools like crispr cas could help to improve the translation efficiency of the cf systems [155] . activation and enrichment of translation-relevant factors could also increase translation efficiency [63] . when it comes to eukaryotic cfps platforms, translocation through microsomes currently remains a black box. optimizing the efficiency of coupling translation and translocation needs to be addressed. the most important issue with cf systems, especially when working with cecf systems, is to maintain the balance between the amount of protein synthesized and the stability and quality of the protein. although cecf has been capable of producing 0.6-1 mg protein per ml, especially with the mammalian expression systems, only a small fraction of the produced protein was subject to detailed functional analysis [15, 155] . this is one of the reasons why the functional assays are limited to binding assays (gpcr, tlr, antibody), plbe (ion channels), and colorimetric assays (enzymes). by optimizing the redox conditions, the problem of ab translocation into the lumen of microsomes is addressed already [75, 155] . however, when it comes to the synthesis of complex transmembrane proteins in mammalian systems, the insertion efficiency might be already saturated at the low synthesis rates due to restrictions on the level of the translocon's functionality. a more detailed analysis of lipid composition and proteins constituting the microsomes present in the insect, cho, and human-derived lysates will help to improve the quality of synthesized membrane proteins. one could use alternative supplements like nanodiscs or liposomes reconstituted from microsomal membranes to support mp integration [156] . intense efforts on designing novel and improved mammalian cf systems should be maintained as the majority of the drug targets are related to complex eukaryotic proteins. optimizing cf reactions in order to decrease protein aggregation during the purification processes and increasing the quality of the protein purification, especially when using the cecf method, is strongly required. another point to address in the field of cfps is to decrease the costs of production, especially in the preparation of cf lysates and the individual reaction components. substantial costs arise from the usage of phosphorylated energy systems, cofactors, nucleotides, amino acids, and dna. alternative energy regeneration systems are available in the place of phosphorylated substrates (e.g., glucose, maltodextrin, etc.) for sustainable atp regeneration throughout the synthesis reaction [157] [158] [159] . use of nucleoside monophosphates instead of nucleoside triphosphates as the nucleotide source in the cf systems could be another cost-effective parameter [159] . avoiding the use of exogenous trnas and cyclic amps and reducing the concentration of amino acids and nucleotides are some of the cost-effective parameters one could optimize during protein synthesis. additionally, new high-cell-density cultivation strategies and improvement in the quality of cell lines by genetic engineering could help to produce cost-effective high-quality cf systems. costs can also be decreased by engineering and optimization of eukaryotic lysates to extend the lifetime of these systems, thereby increasing the yield of the produced protein. there has been considerable progress in the point-ofcare production devices for on-demand biologic synthesis of small quantities of therapeutic proteins using cho lysates and e. coli lysates through on-site good manufacturing practice (gmp) [30] . this type of miniaturized device could be useful for quick testing of proteins and thus help in treating common and rare diseases, and cfps could help solve the challenges associated with in vivo expression. due to the open nature of the cf systems, proteins can be modified with chemically synthesized glycans by bioconjugate chemistries. this will help to increase the quality and therapeutic efficiency of the synthesized proteins. there is an exponential increase in the number of publications from the last 5 years using cf lysates for producing a wide range of proteins [160] . due to the increased awareness of the biosynthetic potential of the cf systems, protocols becoming simpler, improvement in the lysate quality, and its applicability in the preparation of a diverse range of proteins, there will be unexpected outcomes in the field of protein production towards future drug development. funding this research is supported by the european regional development fund (efre) and the german ministry of education and research (bmbf, no. 031b0078a). open access this article is licensed under a creative commons attribution-noncommercial 4.0 international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to 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cell-free protein synthesis using glucose and nucleoside monophosphates cell-free biosensors for biomedical applications key: cord-000012-p56v8wi1 authors: bigot, yves; samain, sylvie; augé-gouillou, corinne; federici, brian a title: molecular evidence for the evolution of ichnoviruses from ascoviruses by symbiogenesis date: 2008-09-18 journal: bmc evol biol doi: 10.1186/1471-2148-8-253 sha: doc_id: 12 cord_uid: p56v8wi1 background: female endoparasitic ichneumonid wasps inject virus-like particles into their caterpillar hosts to suppress immunity. these particles are classified as ichnovirus virions and resemble ascovirus virions, which are also transmitted by parasitic wasps and attack caterpillars. ascoviruses replicate dna and produce virions. polydnavirus dna consists of wasp dna replicated by the wasp from its genome, which also directs particle synthesis. structural similarities between ascovirus and ichnovirus particles and the biology of their transmission suggest that ichnoviruses evolved from ascoviruses, although molecular evidence for this hypothesis is lacking. results: here we show that a family of unique pox-d5 ntpase proteins in the glypta fumiferanae ichnovirus are related to three diadromus pulchellus ascovirus proteins encoded by orfs 90, 91 and 93. a new alignment technique also shows that two proteins from a related ichnovirus are orthologs of other ascovirus virion proteins. conclusion: our results provide molecular evidence supporting the origin of ichnoviruses from ascoviruses by lateral transfer of ascoviral genes into ichneumonid wasp genomes, perhaps the first example of symbiogenesis between large dna viruses and eukaryotic organisms. we also discuss the limits of this evidence through complementary studies, which revealed that passive lateral transfer of viral genes among polydnaviral, bacterial, and wasp genomes may have occurred repeatedly through an intimate coupling of both recombination and replication of viral genomes during evolution. the impact of passive lateral transfers on evolutionary relationships between polydnaviruses and viruses with large double-stranded genomes is considered in the context of the theory of symbiogenesis. approximately two-thirds of these wasps are endoparasites, meaning that the larval stages develop within the body cavity of their hosts, typically other insects. among the most successful of these endoparasitic wasps are those that use lepidopteran larvae as hosts. owing to the economic importance of these insects and the utility of their wasp parasites as biological control agents, the ability of these parasites to develop within lepidopteran hosts without triggering an intense immune response has been the subject of numerous studies over the past forty years. early studies of the mediterranean flour moth, ephestia kuhniella, parasitized by the ichnemonid, venturia canescens, showed that eggs of this species are coated with particles that resemble virions [2] [3] [4] and contain surface proteins that mimic host proteins, thus keeping the eggs and larvae from being recognized as foreign material by their host. these particles lack dna, and thus are not considered virions [5] . with respect to both species number and mechanisms that lead to successful parasitism, endoparasitic wasps are known to inject secretions at oviposition, but only a few lineages use viruses or virus-like particles (vlps) to evade or to suppress host defences. in the family ichneumonidae, for example, four types of host defence suppression mediated by the injection of fluids or suspensions are known that lead to successful parasitism. 1) fluid injected with eggs bypasses host defences without the aid of viruses or vlps [6] . 2) wasps inject a virus that replicates in both the wasp and lepidopteran host. one example is the wasp diadromus pulchellus, which injects an ascovirus, dpav4 [7] into host pupae to circumvent host defence response. 3) the wasp injects vlps capable of molecular mimicry and/or direct defence suppression. 4) the wasp injects polydnavirus particles that contain genes coding for proteins that interfere with host defence responses. the last mechanism is by far the best-studied type of direct immune suppression by ichneumonid wasps, and occurs in many species belonging to genera campoletis, hyposoter and tranosema (ichneumonidae, campopleginae), and glypta (ichneumonidae, banchinae) [8] . in these cases, female wasps inject eggs along with ichnovirus particles into their hosts. similarly, in certain lineages of endoparasitic braconid wasps, other types of immunosuppressive particles containing dna occur in the fluid injected along with eggs [ [9] ; for a review, [10] ]. once in the host, ichneumonid and brachonid particles enter host nuclei and their dna is transcribed, producing proteins that selectively suppress various steps in the host defence response. as a result of this unusual biology, these particles were described as symbiotic viruses belonging to new viral family, polydnaviridae [10] [11] [12] since the 1970's, it was assumed that the dna in the polydnavirus particles, as with all other viruses, encoded typical enzymes and proteins for viral replication and virion assembly and structure. however, several recent genomic studies have shown that only a small number of the genes vectored into lepidopteran hosts, less than 2%, have homologs in other viruses. most viral dna is noncoding, except that which codes for wasp proteins involved in suppression of immune pathways, such as phenoloxidase activation and the toll pathways [8, 13, 14] . even before these genomic studies, it was suggested that these particles were more similar to organelles than viruses [15] . the similarities between particle structure and virions of known types of complex dna insect viruses are striking, and suggest these immunosuppressive particles originated by symbiogenesis between viruses and endoparasitic wasps, the same evolutionary process by which mitochondria and plastids originated from symbiotic bacteria [16] . for example, most braconid wasps produce enveloped bacilliform particles classified as bracoviruses, and these resemble baculovirus and nudivirus virions [10, 15] . similarly, ichneumonid wasps produce enveloped spindle-shaped particles classified as ichnoviruses that resemble virions of ascoviruses, viruses lethal to lepidopterans, which, interestingly, are vectored by endoparasitic wasps [15] . it must also be noted that ichnoviruses resemble other true virus particles that are structurally very similar to virions of ascoviruses, but which remain unclassified because the lack of information about their genomes [17] [18] [19] [20] [21] . however, ascoviruses and ichnoviruses display very different genome properties; similar genomic differences occur between bracoviruses and baculoviruses or nudiviruses, suggesting that convergent evolution led to the origin the different polydnavirus types from at least two different types of viruses. in ascoviruses, the genome consists of a single circular dna molecule ranging from 119-to 180-kpb in size [7] . phylogenetic analyses of several viral genes have revealed that ascoviruses are closely related to iridoviruses [22] , and likely evolved from them. in contrast, the genome of ichnoviruses is composed of multiple circular dna molecules (25 to 105) representing a total size of 250 to 300kbp, all of which are replicated from the wasp chromosomes. the ichnovirus proviral genome is specifically excised and amplified in several segments in the female calyx cells, the only wasp tissue in which ichnovirus virogenesis occurs. after assembly, these particles are secreted into the female genital tract. once injected into the host, the ichnovirus genome does not replicate, and does not lead to the production of a new virus generation. the third characteristic of ichnoviruses is that most of the genes borne by the particles are not related to viral genes. among the 7 annotated ichnovirus gene families, there are four (rep, prrp, n, and trv) for which no homology with known eukaryotic (or prokaryotic) proteins has been detected and for which no function has been proposed. among the remaining three (cys, ank and inx), cys-motif proteins have no clear homologs among eukaryotic (or prokaryotic) proteins, although the "cysteine knot" that they form is a folding domain found in many proteins, but not one that is necessarily related to eukaryotic host immune systems [10, 14] . however, some protein domains and their putative functions suggest that they might be related to regulatory components of eukaryotic host defence systems that are not sufficiently elucidated. although the resemblance of the polydnavirus virions to those of conventional insect viruses suggests that the former evolved from the latter, to date no molecular evidence supports this hypothesis. in the case of ascoviruses and ichnoviruses, well-conserved genes found among the three ascoviruses sequenced so far (sfav1a [23] , tnav2c [24] , and hvav3e [25] ) are not found in ichnovirus genomes. as noted above, the principal reason for this is that the genomes of the latter viruses appear to contain mainly wasp genes, not viral genes. this highlights the need for new and alternative types of sequence data obtained from pertinent biological systems. in this regard, dpav4 has features that could provide important insights. indeed, it is the only ascovirus known to replicate in both its wasp and caterpillar hosts. it is transmitted vertically from wasp to caterpillars to suppress the defence response of the latter host, thereby enabling parasite development [26, 27] . moreover, in males and females of d. pulchellus, the dpav4 genome resides in the nuclei of all hosts cells, providing a possible example of what may have been an intermediate stage in the symbiogenesis that led to the evolutionary origin of ichnoviruses. we recently sequenced the dpav4 genome, and a combination of our analysis of this genome and recent data from new types of ichnoviruses, as well as new software programs that elucidate protein relationships based on structural analysis, have enabled us to detect phylogenetic relationships between proteins encoded by open reading frames of dpav4 and the glypta fumiferanae (gfiv) and campolitis sonorensis (csiv) ichnoviruses. in support of the symbiogenesis hypothesis for the origin of ichnoviruses, data and analyses suggest two independent symbiogenic events, in agreement with what was previously proposed [28] . the first led to the ichnoviruses in banchinae lineage. this hypothesis is based on the occurrence of a gene cluster present in gfiv and dpav4. the second symbiogenic event led to ichnoviruses in the campopleginae wasp lineage. this hypothesis is based on relationships of the major capsid proteins among csiv, ascoviruses and iridoviruses. extending our investigations to proteins encoded by open reading frames of certain ascoviruses and bracoviruses, hosts and bacteria, in the light of recent analyses about the involvement of the replication machinery of virus groups related to ascoviruses in lateral gene transfer [29] , we discuss the robustness and the limits of the molecular evidence supporting an ascovirus origin for ichnovirus lineages. the dpav4 genome sequenced by genoscope (france) is 119,334-bp in length. its organization, gene content and evolutionary characteristics will be detailed in a separate publication (manuscript in preparation; additional file 1). however, blast results obtained with several orfs in the dpav4 genome provide evidence that certain ichnovirus orfs have their closest relatives in an ascovirus genome. specifically, we identified a 13-kbp region that contains a cluster of three genes ( fig. 1 , orf90, 91 and 93; additional files 1 and 2) that have close homologs in a gfiv gene family composed of seven members [28] . all contain a domain similar to a conserved domain found in the pox-d5 family of ntpases. to date, this pox-d5 domain has been identified as a ntp binding domain of about 250 amino acid residues found only in viral proteins encoded by poxvirus, iridovirus, ascovirus and mimivirus genomes. these genes seem to be specific to gfiv, as they are absent in the three sequenced genomes of other ichnoviruses, namely csiv, tranosema rostrales ichnovirus (triv), and hyposoter fugitivus ichnovirus (hfiv). more specifically, in dpav4, orf90 encodes a protein of 925 amino acid residues that is 40% similar from position 140 to 925 to a protein of 972 amino acid residues encoded by the orf1 contained in the segment c20 in the gfiv genome (fig. 2) . these two proteins can therefore be considered putative orthologs. the 480 c-terminal residues of this dpav4 protein are also 42% similar to the cterminal domain of the protein homologs encoded by the orf1 of the d1 and d4 gfiv segments, 36% similar to the n-terminal and the c-terminal domains of the protein encoded by the orfs 184r and 128l of the iridovirus civ and lcdv, and 30% similar with those encoded by orfs 119, 99 and 78 in the ascovirus genomes of hvav3e, sfav1a and tnav2c, respectively. overall, this indicates that this dpav4 protein is more closely related to that of gfiv than to those found in other ascovirus and iridovirus genomes currently available in databases. orf091 encodes a protein of 161 amino acid residues similar only with the c-terminal domain of three proteins encoded by the orfs 1, 1 and 3, contained, respectively, in gfiv segments d1, d4 and d3. in contrast, orf93 is closer to iridovirus and ascovirus genes than to gfiv genes. this protein of 849 amino acid residues is 43% similar over all its length to civ orf184r orthologs in all iridoviral and ascoviral genomes and is only 36% similar over 350 amino acid residues to the c-terminal domain of the gfiv protein homologs encoded by the orf1, 2, 1, 1, 1 and 1 in, respectively, the c20, c21, d1, d2, d3 and d4 segments of this virus. analysis of the genes surrounding the dpav4 orf-90-91-93 cluster confirms that this virus has an ascovirus origin since this region contains orfs that are close homologs of genes in iridovirus and ascovirus genomes. upstream from the orf-90-91-93 cluster, an orf encoding the dna-dependent rna polymerase 1 subunit c is present, which is an ortholog of the iridoviral civ orf176r and the ascoviral sfav1a orf008. downstream from this cluster, there are two genes, absent in known ascoviral genomes, but similar to the iridoviral civ orf115l and civ orf132l. these two genes encode, respectively, a chromosomal replication initiation protein and zinc finger protein. in between them, a gene encoding a small protein is present that is similar to that encoded by the orf069l of the iridovirus civ, and which corresponds to the ali-like protein also found in entomopoxviruses [30] . since the three dpav4 genes have relatives in all ascovirus and iridovirus genomes sequenced so far, their presence in the dpav4 genome cannot result from a lateral transfer that occurred from an ichnovirus genome related gfiv to dpav4. thus, as these dpav4 genes are the closest relatives of the pox-d5 gene family present in gfiv identified so far, they could be considered a landmark of the symbiogenic ascovirus origin of the ichnovirus lineage to which this polydnavirus belongs. an alternative explanation is that the presence of dpav4-like genes in the genome of gfiv resulted from a lateral transfer from viral genomes closely related to those of gfiv and dpav4. indeed, this might have happened when a glypta wasp was infected by an ancestral virus related to dpav4. nevertheless, the symbiogenic origin of gfiv from ascoviruses is also supported by morphological features of its virions [28] , which, aside from similarities in shape, also show reticulations on their surface in negatively stained preparations, a characteristic of the virions of all ascovirus species examined to date [7] . because ascovirus virions and ichnovirus particles display structural similarities, we developed an approach to search for homologs of virion structural proteins in ichnoviruses. these approaches were initiated in 2000 and recently finalized, but some of the conclusions have been published [14] . to date, only two virion proteins from the campoletis sonorensis ichnovirus (csiv) have been characterized [31, 32] . the first is the p44 (acc n° aad01199), a structural protein that appears to be located as a layer between the out envelope and nucleocapsid, and the second, p12, a capsid protein (acc n° af004367). presently, there are more than one hundred ascoviral or iridoviral mcp sequences in databases. blast searches using these sequences failed to detect any similarities between csiv virion proteins and ascoviral or iridoviral mcps, or any other proteins [33] . to evaluate the possibility that homology between ichnovirus and ascovirus virion proteins may simply not be detectable by conventional blastp searches, we used a different method, wapam (weighted automata pattern matching; [34] ). the models were designed on the basis of a previous study [22] demonstrating that mcp encoded by ascovirus, iridovirus, phycodnavirus and asfarvirus genomes are related, and all contain 7 conserved domains separated by hinges of very variable size. we investigated these conserved domains further using hydrophobic cluster analysis (hca, [35] ). this map of the 13-kbp region of the dpav4 genome (embl acc. n° cu469068 and cu467486) that contains the gene cluster with direct homologs in the genome of the glypta fumiferanae ichnovirus amino acid sequence comparison resulting from a blast search done with the dpav4 orf90 as a query, and the best hit corresponding to the protein encoded by the orf1 of the ichnovirus segment gfv-c20 (subject; genbank acc. n° yp_001029423) figure 2 amino acid sequence comparison resulting from a blast search done with the dpav4 orf90 as a query, and the best hit corresponding to the protein encoded by the orf1 of the ichnovirus segment gfv-c20 (subject; genbank acc. n° yp_001029423). analysis revealed that most conservation occurred at the level of hydrophobic residues, as expected for structural proteins (additional file 3a and 3b). the size variability of the hinges between conserved domains and the conservation of hydrophobic residues might explain why blast searches using iridoviral and ascoviral mcp sequences have limited ability to detect mcp orthologs in phycodnavirus and asfarvirus genomes. we designed two syntactic models (see materials and methods), which together were able to specifically align all mcp sequences of the four virus families. importantly, wapam aligned the csiv ichnovirus p44 structural protein with both models. complementary structural and hca confirmed the presence of the seven conserved domains in this csiv structural protein ( fig. 3a and additional file 3c). in addition to the above analysis, ten syntactic models were developed using proteins conserved in the three sequenced ascovirus species (sfav1a, tnav2c, and hvav3a) and twelve iridoviruses [36] . none of these 1 and 4, typed in black) , dpav4 (lanes 2 and 5, typed in blue) and sfav1a (lanes 3 and 6, typed in purple) . conserved positions among the amino acid sequence of csiv and those of dpav4 and sfav1a are highlighted in grey. secondary structures in the three sfav1a orf061 orthologs were calculated with the network protein sequence analysis at http://npsa-pbil.ibcp.fr/ and the statistical relevance of the secondary structures were evaluated with psipred at http://bioinf.cs.ucl.ac.uk/psipred/. c, e and h in lanes 4 to 6 respectively indicated for each amino acid that it is involved in a coiled, b sheet or a helix structure. using default parameters of psipred, upper case letters indicate that the predicted secondary structure is statically significant in psipred results. significant secondary structures are highlighted in yellow. in (a), the comparisons were limited to three of the seven conserved domains (additional file 3a, b and 3c), the 2, 5 and 7. indeed, classical in silico methods appeared to be inappropriate to predict statistically significant secondary structures in conserved structural protein rich in b strand such as iridovirus and ascovirus mcp. in contrast, a complete and coherent domain comparison was obtained by hca profiles (fig. s3b, c) . , developed from small proteins encoded by the dpav4 orf041, sfav1a orf061, hvav3a orf74, and tnav2c orf118 in the ascovirus genomes, and iridovirus civ orf347l and mimivirus miv orf096r genomes, respectively. importantly, these proteins have orthologs in vertebrate iridoviruses, phycodnaviruses, and asfarvirus. in sfav1a, the peptide encoded by orf061 is one of the virion components. in ascoviruses, iridoviruses, phycodnaviruses, and the asfarvirus, they have been annotated as thioredoxines, proteins that play a role in initiating viral infection [37] [38] [39] . database mining with our model revealed four hits with csiv sequences (acc n°. m80623, s47226, af236017, af362508) each a homolog orf of sfav1a orf061. in fact, these sequences correspond to several variants of a single region contained in the b segment of the csiv genome. to date, these have not been annotated in the final csiv genome, probably because they overlap a recombination site. hca analyses confirmed that the hydrophobic cores were conserved ( fig. 3b and additional file 3d and 3e). the chromosomal locations of genes encoding these two csiv proteins, i.e., p44 and p12, were also consistent with the symbiogenesis hypothesis. in fact, the orf encoding p44 is not found in proviral dna. it is notable that no orfs encoding orthologs of p44 or other structural proteins such as mcps are found in any of the other three ichnovirus genomes sequenced -triv, gfiv, hfiv [8, 14] . therefore, this indicates that the orthologs of ichnovirus mcps and other virion structural proteins are also probably located in the genomes of these wasps, i.e., not in proviral dna. in contrast to this, we found that the gene encoding the csiv ortholog of sfav1a orf061 is located within the proviral dna. whether ortholog proteins are similarly involved in the triv, gfiv and hfiv biology, their genes are not found in proviral dna, since no matches were detected in their viral genomes. the phylogenetic analysis performed previously on p44 and the sfav1a orf061 orthologs [15] indicated that they have an ancestor close to that of the ascoviruses and iridoviruses. as in the case of genes encoding pox-d5 family of ntpases in all ascoviruses, iridoviruses, and gfiv, genes encoding virion proteins cannot result from a horizontal transfer from a campoplegine or banchine ichnovirus genome to all ascovirus, iridovirus, phycodnaviruses and asfarvirus genomes. as the ascovirus genes encoding the two virion proteins investigated here are the closest relatives of virion proteins in csiv, they can be considered a landmark reflecting the symbiogenic origin of the two ichnovirus lineages from ascoviruses closely related to dpav4. in fact, the difficulty encountered in elucidating their sequence relationships can be explained by a combination of the marked transition from ascovirus to ichnovirus, and the significant selection constraints that resulted as the latter virus type evolved from the former. analysis of available ascovirus, iridovirus and ichnovirus genomes provides some of the first molecular support for the hypothesis that ichnoviruses evolved from ascoviruses by symbiogenesis. however, examining genes shared only by ascovirus, iridovirus and ichnovirus genomes likely limits the sources of genes that contributed to the evolution and complexity of these viruses, especially of the role of lateral gene transfer. relevant to this is the recent finding that an important part of the mimivirus and phycodnavirus genomes had a bacterial origin [28] . obviously, this did not lead to the conclusion that these viruses had a bacterial origin. the cytoplasmic environment in which these viruses replicate is rich in bacterial dna because their amobae and unicellular algae hosts feed on bacteria that they digest in their cytoplasm. thus, it has been proposed [28] that lateral transfers of bacterial dna within these viral genomes were driven by intimate coupling of recombination and viral genome replication. indeed, replication of these viruses is similar to that of bacteriophage t4. this mode of replication has been called recombination-primed replication. it permits integration of dna molecules with sequence homology as short as 12-bp [28, 40] . the replication machinery used by ascoviruses, iridoviruses, mimiviruses, phycodnaviruses, and other nucleocytoplasmic large dna viruses (ncldv) [41, 42] is common to all of them, despite differences in the specifics of replication in each virus family. it can therefore be expected that recombination-primed replication occurred repeatedly during evolution of both these viruses and the genome of their eukaryotic hosts. in an eukaryotic cellular environment in which bacteria, chromosomes, ncldv viruses and non-ncldvs (such as baculoviruses) intimately cohabit temporarily or permanently, recombination-primed replication is able to allow reciprocal passive lateral transfers between viral genomes, host chromosomes, and bacterial dna. under these conditions, lateral transfers are considered passive since they just result from the intimate environment and not from an active mechanism dedicated to genetic exchanges. in ascoviruses and iridoviruses, the occurrence of such lateral transfers is supported by blastp searches that detected the presence of orfs whose closest relatives have their origin within eukaryotic genomes (e.g., for dpav4, in additional data 1, orfs 029, 049, 077, 080, 083, 118), bacterial genomes (e.g., for dpav4, in additional data 1, orfs 056, 057, 059, 112, 115 and119) or viruses belonging to other ncldv and non-ncldv families (e.g., for dpav4, in additional data 1, orfs 007, 037, 062, 068). the transmission of ascoviruses is unusual in that they are poorly infectious per os and appear to be transmitted among lepidopteran hosts by parasite wasp vectors at oviposition [7, 43] . the genome of the ascoviruses can be replicated in presence of polydnavirus dna either within the reproductive tissues of female wasps or within the body of the parasitized hosts infected by both polydnavirus and ascovirus. consequently, integrated sequences of ascovirus origin can be expected within wasp and polydnavirus genomes. reciprocally, sequences of polydnavirus origin may have been integrated in ascovirus genomes, whatever the wasp origin, ichneumonid or braconid. one gene family related to a bacterial family of n-acetyl-l-glutamate 5-phosphotransferase (acc. n° of the closest bacterial relatives yp_001354925, cam32558, zp_00944224, zp_02006449), identified only within the sfav1a, hvav3e and tnav2c genomes, supports this conclusion. it has been found in the genome of a bracovirus, cotesia congregata bracovirus (ccbv [13] ; fig. 4 ). since this gene is absent in the genome of microplitis demolitor bv, a related bracovirus [8] , it is difficult to infer the direction of the lateral transfer between the common ancestors of the three ascoviruses and of the wasp c. congregata. however, they unambiguously indicate that there was at least one lateral transfer for this gene between the common ancestor of ascoviruses and the parasitic wasp. since iridoviruses, like ascoviruses and other virus species [44, 45] , are, in some cases, vectored by parasitic wasps, databases were mined using all the available ichnovirus virus proteins as queries. we found no significant relationships between csiv, hfiv and triv genomes and genomes of their putative closest relatives ncldv and non-ncldv relatives. this indicates that passive lateral gene transfers from virus to eukaryotes that are successfully spread and maintained in ichnovirus genomes remain rare events. one case of such lateral transfer was described in the ccbv genome. in this genome, aside from the presence of cardinal endogenous eukaryotic retrotranposon and polintons that transposed in the chromosomal dna of the proviral form of ccbv [46] [47] [48] , two genes encoding acmnpv p94-related proteins, which have their closest relatives among granuloviruses (xcgv), were found. this suggests that ccbv contained at least two cases of lateral transfers between non-ncldv and a bracovirus. our results provide another source of evidence that passive lateral gene transfers have occurred regularly during evolution from bacteria to viruses and eukaryotes, and between viruses and eukaryotes [49] [50] [51] [52] . even if the pox-d5 ntpase genes in the gfiv genome, and the mcp and sfav061-like genes in the csiv genome, indicate that they have an ascovirus origin, they provide only limited evidence supporting an ascovirus origin of ichnoviruses. indeed, their sequence conservation and biological characteristics suggest that there were repeated lateral transfers during evolution between ascoviruses and wasp genomes, including the proviral ichnovirus loci. this raises an important issue about the role of lateral transfers during co-evolution of the ncldvs and non-ncldvs, ichnovirus, wasp and parasitized host. indeed, genetic materials of various origins have been exchanged and maintained during co-evolution. this therefore suggests that ichnoviruses might be chimeric entities partly resulting from sevsymbiogenesis was first proposed as an evolutionary mechanism when it became widely recognized that mitochondria and plastids originated from free-living prokaryotes [7] . the genomes of the endosymbiotic cyanobacteria and proteobacteria, respectively, at the origin of chloroplasts and mirochondria have evolved by reduction of several orders of magnitude to the approximate size of plasmids. concurrently, nuclear genomes have been the recipients of plastid genomes. this relocation of the genes encoding most proteins of the endosymbiotic bacteria to the host nucleus is the ultimate step of this evolutionary process, so-called endosymbiogenesis [7, 53] . recent studies of plants have revealed a constant deluge of dna from organelles to the nucleus since the origin of organelles [54] . this allows the host cell to have the genetic control on its organelles, in a relationship that is closer to enslavement or domestication than to a symbiosis or a mutualism in which the organelles would recover benefits from their contribution to the eukaryotic cell well-being. to date, this deluge of dna is considered to correspond to passive lateral transfers that result from the interactions between the life cycle of the organelle and nuclear replication. numerous cases of symbiogenesis between endocellular bacteria and a wide variety of eukaryotic hosts have been characterized. however, recent work has demonstrated that this evolutionary process was not restricted to bacteria. it also occurred between endocellular eukaryotes such as unicellular algae and fungal endophyte in plants [55, 56] . endosymbiogenesis was also proposed as the evolutionary mechanism that allowed some invertebrate viruses with a large double-stranded dna genome related to the nudiviruses and the ascoviruses [22] , to have led, respectively, to the origin of bracoviruses and ichnoviruses, which are currently recognized as forming two genera within the family polydnaviridae. although presently there is no definitive evidence ruling out the hypothesis that the resemblance between ichnovirus and ascovirus virions is only an evolutionary convergence, the genomic differences between ascovirus and ichnoviruses are in good agreement with the symbiogenetic hypothesis. indeed, they match an evolutionary scenario of endosymbiogenesis during which, from a single integration event of symbiotic virus genome, viral genes were lost and/or translocated from the provirus to other chromosomal regions (fig. 5 ). in parallel, host genes of interest for the wasp parasitoid were integrated and diversified by selection and gene duplication in the proviral dna. in this scenario, the more ancient symbiogenesis, the rarer the traces of genes from viral origin in the ichnovirus genome would be. this constitutes a constraint that dramatically limits the possibility to investigate the evolutionary links between ascovirus and ichnovirus. results of our analyses demonstrate that the situation is also complicated by the fact that lateral gene transfers unrelated to the origin of ichnoviruses cause important misleading background noise. moreover, the scenario in figure 5 is close to a previously proposed version [57] , but is not consistent with results presented here, nor with recently accumulated knowledge on dna transfer from organelles into the nucleus. since endocellular environments favour lateral transfers between virus and wasp nucleus, it can be proposed that genes of virus origin that are involved in the ichnovirus biology were passively integrated in one or several loci, step by step over time, alone or through transfers of gene clusters, or even the entire viral genome. since parasitoid wasps are able to vector different viruses [44, 45] , this second scenario opens the exciting possibility that virus genes involved in the ichnovirus biology might correspond to a gene patchwork resulting from transfers from viruses belonging to different ncldv and non-nclvd families. because of the background noise due to lateral gene transfers found in these systems, elucidating the origins of ichnoviruses will be very time-consuming, requiring new accurate experimental approaches to generate more robust evidence. sequencing wasp genomes to identify proteins of viral origin that are components of virions and involved in the assembly of these may well contribute to our understanding of how ichnoviruses and bracoviruses evolved from other insect dna viruses. searches for similarities were mainly developed using facilities of blast programs at two websites http:// www.ncbi.nlm.nih.gov/blast/blast.cgi and http:genoweb.univ-rennes1.fr/serveur-gpo/out ils.php3?id_rubrique=47. for dpav4 genes having their origin within eukaryotic, bacterial or virus genomes belonging to ncldv and non-ncldv families, the closest gene was located using the distance trees supplied with each blast search at the ncbi website. construction of syntactic models: conserved amino acid blocks and positions described previously [15, 22] and with new data sets were verified or determined using meme at http://meme.sdsc.edu/meme/meme.html. in the first step, we used motifs resulting from meme to make mast minings in databases at http:// meme.sdsc.edu/meme/mast.html. since meme motifs depend significantly on the data set use to calculate them, this approach did not enable an exhaustive detection of homologs among ascoviruses, iridoviruses, phycodnaviruses, mimiviruses and asfarviruses, and the detection sensitivity was ultimately very similar to that obtained with blast. to reach our detection objectives, we therefore constructed syntactic models that only included the most conserved positions and their variable spacing using wapam at the website. http://genoweb.univ-rennes1.fr/ serveur-gpo/ outils_acces.php3?id_syndic=185&lang=en. defining these models was obtained empirically until they allowed an exhaustive detection in refseq-protein and genbank databases of the homologs among ascoviruses, iridoviruses, phycodnaviruses, mimiviruses and asfarviruses. the procedures were done until we were only able to detect exact match with the syntactic model. whatever obtained with wapam, they required a confirmation with other approaches. here, we used psipred result comparison for regions with scores over 7 and hca analyses for regions having scores lower than 7 with psipred. this simplified the statistical treatment of the result obtained with wapam, since all exact matches have significance or a score of 100%. syntactic hypothetical mechanism for the integration and evolution of ascovirus genomes in endoparasitic wasps figure 5 hypothetical mechanism for the integration and evolution of ascovirus genomes in endoparasitic wasps. schematic representation of the three-step process of symbiogenesis, and dna rearrangements that putatively occurred in the germ line of the wasp ancestors in the banchinae and campopleginae lineages, from the integration of an ascoviral genome to the proviral ichnoviral genome. sequences that originate from the ascovirus are in blue, those of the wasp host and its chromosomes are in pink. genes of ascoviral origin are surrounded by a thin black or white line, depending on their final chromosomal location. two solutions can account for the final chromosomal organisation of the proviral ichnovirus genome, monolocus or multilocus, since this question is not fully understood in either wasp lineage. more complex alternatives to this three-step process might also be proposed and would involve, for example, the complete de novo creation of a mono or multi locus proviral genome from the recruitment by recombination or transposition of ascoviral and host genes located elsewhere in the wasp chromosomes. this model for the chromosomal organization of proviral dna in polydnaviruses is consistent with data recently published [58] . immune surface of eggs of a parasitic insect the resistance of insect parasitoids to the defense reactions of their hosts an insect glycoprotein: a study of the particles responsible for the resistance of a parasitoid's egg to the defence reactions of its insect host role of virus-like particles in parasitoid-host interaction of insects venom from the endoparasitic wasp pimpla hypochondriaca adversely affects the morphology, viability, and immune function of hemocytes from larvae of the tomato moth, lacanobia oleracea characteristics of pathogenic and mutualistic relationships of ascoviruses in field populations of parasitoid wasps polydnavirus genomes reflect their dual roles as mutualists and pathogens particles containing dna associated with the oocyte of an insect parasitoid family polydnaviridae. in virus taxonomy. eighth report of the international commitee on taxonomy of viruses edited by: fauquet cm virus in aparasitoid wasp: suppression of the cellular immune response in the parasitoid's host polydnaviridae -a proposed family of insect viruses with segmented, doublestranded, circular dna genomes genome sequence of a polydnavirus: insights into symbiotic virus evolution shared and species-specific features among ichnovirus genomes origin and evolution of polydnaviruses by symbiogenesis of insect dna viruses in endoparasitic wasps symbiosis in cell evolution hyenoptera: formicidae) from brazil the ultrastructure of microorganisms in the tissues of casenaria infesta (hymenoptera: ichneumonidae) apparent replication of an unusual viruslike particle in both parasitoid wasp and its host an unusual virus from the parasitic wasp cotesia melanoscela. virology viruslike particles in the ovaries of microctonus aethiopoides loan (hymenoptera: braconidae), a parasitoid of adult weevils (coleoptera: curculionidae) evidence for the evolution of ascoviruses from iridoviruses genomic sequence of spodoptera frugiperda ascovirus 1a, an enveloped, double-stranded dna insect virus that manipulates apoptosis for viral reproduction sequence and organization of the trichoplusia ni ascovirus 2c (ascoviridae) genome. virology sequenceand organization of the heliothis virescens ascovirus genome biological and molecular features of the relationships between diadromus pulchellus ascovirus, a parasitoid hymenopteran wasp (diadromus pulchellus) and its lepidopteran host, acrolepiopsis assectella dpav-4, on thehemocytic encapsulation response and capsule melanization of the leek-moth pupa, acrolepiopsis assectella genomic and morphological features of a banchine polydnavirus: comparison with bracoviruses and ichnoviruses i am what i eat and i eat what i am: acquisition of bacterial genes by giant viruses the genome of melanoplus sanguinipes entomopoxvirus cloning and expression of a gene encoding a campoletis sonorensis polydnavirus structural protein a gene encoding a polydnavirus structural polypeptide is not encapsidated what does structure tell us about virus evolution? cluster of re-configurable nodes for scanning large genomic banks deciphering protein sequence information through hydrophobic cluster analysis (hca): current status and perspectives comparative genomic analysis of the family iridoviridae: reannotating and defining the core set of iridovirus genes the thioredoxin system in retroviral infection and apoptosis mimivirus giant particles incorporate a large fraction of anonymous and unique gene products cell entry by enveloped viruses: redox considerations for hiv and sars-coronavirus genetic recombination of the dna plant virus pbcv-1 in a chlorella alga common origin of four diverse families of large eukaryotic dna viruses evolutionary genomics of nucleo-cytoplasmic large dna viruses effects of the nonoccluded virus of spodoptera frugiperda (lepidoptera: noctuidae) on the development of a parasitoid parasitoid-mediated transmission of an iridescent virus non-poly-dna viruses, their parasitic wasp, and hosts the few virus-like genes of cotesia congragata self-synthesizing dna transposons in eukaryotes marvericks, a novel class of giant transposable elements widespread in eukaryotes and related to dna viruses evolution of viruses by acquisition of cellular rna or dna nucleotide sequences and genes: an introduction microbialgenes in the human genome: lateral transfer or gene loss? science are there bugs in our genome? science express genome-wide survey for genes horizontally transferred from cellular organisms to baculoviruses morphogenesis by symbiogenesis endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes a cryptic intracellular green alga in ginkgo biloba: ribosomal dna markers reveal worldwide distribution forest succession suppressed by an introduced plant-fungal symbiosis unfolding the evolutionary story of polydnaviruses structure and evolution of a proviral locus of glyptapanteles indiensis bracovirus this research was funded by grants from the c.n.r.s. (pics n°204343), the genoscope, the a.n.r. project in bioinformatics modulome, the ministère de l'education nationale, de yb is the leader of all aspects of the research on the biology, genomics, and evolution of dpav4. ss coordinated the sequencing, assembly, and sequence quality control of the dpav4 genome. cag participated in the bioinformatics analysis of the dpav4 genome development of the manuscript. baf contributed original concepts regarding the evolutionary origins and role of polydnaviruses in endoparasitoid biology, provided virological expertise to optimize data interpretation, and participated in writing the manuscript. predicted orfs in dpav4 genome. key: cord-021772-5v4gor2v authors: levine, gwendolyn j.; cook, jennifer r. title: cerebrospinal fluid and central nervous system cytology date: 2019-05-31 journal: cowell and tyler's diagnostic cytology and hematology of the dog and cat doi: 10.1016/b978-0-323-53314-0.00014-6 sha: doc_id: 21772 cord_uid: 5v4gor2v nan analysis of csf is an important adjunctive diagnostic tool in the workup of patients with cns disease and must be interpreted within the context of the patient's history, clinical signs, clinicopathological data, imaging studies, and other ancillary diagnostics. rarely is csf solely used to provide an etiological diagnosis (exceptions include cytological visualization of infectious agents or overtly neoplastic cells), but analysis may significantly narrow the field of pathophysiological differentials, guiding further diagnostic and therapeutic options. csf analysis is most sensitive in detecting inflammatory disease. 3 positive findings in csf tend to be more diagnostically helpful compared with negative findings but are often nonspecific because many different diseases may cause a common csf pathology (e.g., neutrophilic pleocytosis). 4 occasionally, the magnitude of change within the csf may be as instructive as the character of the change (e.g., a marked increase in protein concentration raising diagnostic concern for feline infectious peritonitis, marked neutrophilic pleocytosis raising diagnostic concern for steroid-responsive meningitis-arteritis in a young dog in pain). 4 more frequently, however, specific disease etiologies will present with csf changes of variable character and magnitude. csf that falls within laboratory reference intervals should never be used to rule out a differential diagnosis because negative findings may represent early or mild disease, disease suppressed or masked by therapeutic intervention, or a disease process that does not present within the particular area of the extracellular space being sampled. csf analysis may or may not correlate with imaging studies; a retrospective study of 92 cats receiving magnetic resonance imaging (mri) for spinal signs showed that abnormal csf was not a predictor for abnormal mri. 5 in another study, approximately 25% of dogs with intracranial signs and inflammatory csf had normal brain mri results. 6 the conventionally accepted theory of csf secretion and transport is based on the concept of active transport of ions within the ventricular ependymal cells and choroid plexi, subsequent passive flow of fluid, and circulation and drainage of csf into dural venous sinuses. these ideas have recently come under scrutiny as potentially simplistic and inconsistent with the past 100 years of experimental evidence. 7 analysis of past experiments, coupled with new data, supports a "global production" hypothesis-that instead of exclusive formation within the ventricles, csf is continually created and reabsorbed diffusely by cerebral capillaries that have slight variances in hydrostatic and osmotic pressure. canine studies have documented csf production within the ventricular system and the sas. 2 a study of 158 dogs with focal, noninflammatory disease showed that in cases of spinal lesions, csf was more likely to be abnormal if collected from the lumbar cistern, that is, caudal to the lesion. 12 this observation may be explained by presupposing cranial to caudal flow of csf, but the traditionally held theory of csf flow has recently been contested. 7 in canines, csf collected from the cerebromedullary cistern generally has lower microprotein concentrations compared with samples collected from the lumbar cistern. 13 blood contamination may be more pronounced in lumbar collection, as the desired subarachnoid space is more difficult to enter and yields a smaller volume of fluid that tends to flow more slowly. 9, 10 moreover, hemodilution may contribute to increased measured protein concentration. 13 rare instances of csf contamination with hematopoietic precursors have only been reported from lumbar sites. 14 a low, but potentially catastrophic, risk for puncturing the cervical spinal cord or caudal brainstem exists during cerebromedullary collection. because the spinal cord length is variable, spinal cord puncture is a possibility during lumbar collection, but it is associated with less severe adverse effects compared with injury following cisternal puncture. in a case series of four accidental cisternal parenchymal punctures (documented by using mri), three of the four patients suffered neurological decompensation and subsequently had to be euthanized. 15 the following equipment should be assembled: anesthesia and monitoring equipment, clippers, aseptic preparation materials for the skin, sterile gloves, and a spinal needle with stylet. for cerebromedullary cistern collection in dogs weighing less than 25 kg and for cats, a 22-gauge, 1.5-inch spinal needle is usually adequate, and a 22-gauge, 2.5-inch spinal needle is recommended for dogs weighing greater than 25 kg. for lumbar puncture, a 22-gauge spinal needle up to 6 inches long may be required for obese or extremely large patients. if available, fluoroscopic equipment may aid in the acquisition of cisternal or lumbar csf. at the authors' institution, fluoroscopy is often used before cisternal csf acquisition in toy-breed dogs to exclude the possibility of subclinical atlantoaxial subluxation. the anesthetized patient is placed in lateral recumbency (it is generally easier for a right-handed clinician to have the patient in right-lateral recumbency, and vice versa), with the neck and back flush to the edge of a sturdy table. for collection from the cerebromedullary cistern, the neck is flexed such that the dorsum of the muzzle is 90 degrees to the long axis of the body (if needed, stabilizing the endotracheal tube to prevent kinking and deflating the cuff to prevent tracheal trauma), and the snout is propped up slightly, if necessary, to keep it parallel with the table and not angulated from the sagittal plane. 10 a wide area (3-5 cm) around the atlanto-occipital joint (beyond atlas wings and axis spinous process and to the external occipital protuberance) is shaved and aseptically prepared, and landmarks are palpated with a gloved, nondominant hand. 9 the needle is inserted at the intersection of two imaginary perpendicular lines that run (1) along the dorsal midline (dividing the patient sagittally) from the occipital protuberance to the cranial spinous process of the axis (c2) and (2) across the craniolateral aspects of the wings of the atlas (c1) (dividing the patient craniocaudally). for lumbar collection, the pelvic limbs are brought forward into full flexion, and the needle is inserted cranial and parallel to the dorsal spinous process of l6 for dogs and l7 for cats, advancing the needle until the ventral aspect of the vertebral canal is encountered; the needle is then retracted slightly and csf is collected from the ventral sas. 10, 16 the pelvic limbs may be kicked or may twitch slightly during collection because of irritation of the cauda equina or spinal cord parenchyma. for either location, once landmarks are palpated, the needle is held stably with the dominant hand and very slowly advanced, stylet in place. the heel of the dominant hand may be supported against the table. for cisternal collection, it is important to advance the needle toward the point of the nose without angulation. the stylet is removed with the nondominant hand every 2 to 3 mm to check for fluid within the needle hub, waiting a few seconds. it is common to feel a decrease in resistance to forward needle movement once the thecal space is entered. if bone is hit or frank hemorrhage is observed from the needle, it should be withdrawn slowly and collection reattempted. 9 if clear or slightly blood-tinged fluid is observed, advancement of the needle is stopped, and open tubes are placed directly under the needle hub to collect freely falling drops. csf is collected passively and should not be aspirated. there are no significant objective data regarding the maximal amount of csf that may be collected in dogs. several authors claim that it is safe to collect 0.2 milliliters (ml) of csf per kilogram of body weight (1 ml/5 kg); in other species much higher volumes of csf per body weight are acquired standardly. 17 in general, 0.5 to 1 ml of csf is adequate for routine diagnostic tests, including cell counts, protein concentration, and cytological analysis. larger volumes are necessary for additional diagnostics (cultures, titers, polymerase chain reaction [pcr], flow cytometry, protein electrophoresis, etc.). two sets of tubes should be readied and ideally handled by an assistant. an ethylenediaminetetraacetic acid (edta)-treated (purple-top) tube is used for cell counts, flow cytometry, and pcr testing for organisms, and plain (red-top) tubes are used for protein concentration, culture, or immunologic assays. 10 some sources indicate that plain tubes are recommended, as edta could increase protein concentration. if csf analysis will occur rapidly (within 1 hour), collection into a plain tube is adequate, whereas preservation of cells may be improved with collection into edta if analysis will be delayed. if low volume is present, priority is given to the edta tube. if csf appears red, then iatrogenic hemorrhage (puncture of a dural vessel) or actual cns hemorrhage has occurred. in this instance, the first few drops are allowed to collect into the first set of tubes, and the second set of tubes are reserved for the latter portion of the sample, as iatrogenic hemorrhage tends to clear over time. if the hemorrhage does clear, a decision may be made about discarding the first set of tubes or keeping them for ancillary testing not affected by the hemorrhage. after collection, the needle is withdrawn without the stylet, and the csf within the needle is allowed to drip into one of the tubes or is placed in an additional plain tube and saved for culture. as with other clinicopathological and cytological samples, evaluation of a fresh specimen is preferred to minimize cellular degradation, to which csf is particularly vulnerable because of its relatively low protein concentration. sample degradation will affect cell differential count to a greater extent than the total nucleated cell count or the protein concentration. 18 a study of 30 canine csf samples with pleocytosis concluded that delay of analysis up to 8 hours was unlikely to alter interpretation, especially in samples with protein concentrations above 50 milligrams per deciliter (mg/dl). 18 preservative should be added to low protein samples unless analysis is to be completed within 60 minutes (see next section), and a dilutional effect must then be factored into cell counts. 18 samples to be shipped to a reference laboratory overnight should be kept at refrigeration temperature and shipped with ice packs for analysis within 48 hours. 9, 16 the reference laboratory should be prenotified to ensure prompt analysis. if analysis is likely to be delayed by more than 1 hour and the csf sample has a protein concentration less than 50 mg/dl, one of the following may be added as a protein source to maintain cellular integrity: (1) hetastarch (add 1:1 volume), (2) fetal calf serum (3.7 g/dl protein; add 20% by volume), or (3) autologous plasma or serum (fresh or frozen; 11% by volume ≡ one drop from 25-gauge needle (approximately 0.03 ml) mixed into 0.25 ml csf). 10, 19, 20 the sample should be labeled with the protein source and amount added to the sample. one study demonstrated better preservation of mononuclear cells in canine samples when fetal calf serum was used instead of hetastarch. 18 all samples should be refrigerated at 4°c to minimize cellular degradation. a hemocytometer may be employed in practice to count nucleated cells and erythrocytes. both sides of the cover-slipped hemocytometer are loaded with unstained csf, which is then placed in a humidified container for 10 to 15 minutes to allow cells to settle on the glass. because the fluid is unstained, the microscope condenser is lowered to improve contrast. erythrocytes and nucleated cells are differentiated by size, refraction, granularity, and smoothness of plasma membrane. 21 some laboratories stain csf samples with new methylene blue (nmb), as leukocytes will take up stain, whereas erythrocytes remain unstained, making differentiation of leukocytes (specifically small lymphocytes) and erythrocytes easier (fig. 14.1) . 22 a small volume of csf is drawn into a capillary tube coated with nmb or a tube that has a small volume of nmb followed by an air pocket. 22 the tube containing nmb and csf is gently rocked back and forth, allowing the cells to take up some stain without diluting the csf with a volume of nmb. 22 the hemocytometer is then loaded, and each population is counted and totals are calculated, as follows: neubauer chamber: (1) both areas of large nine squares are counted, and the average of the number of leukocytes and erythrocytes is found; (2) the average is multiplied by 9 to get the cells per microliter (cells/μl). 10 the advia 120 (siemens medical solution, fernwald, germany) hematology instrument has been validated for analyzing canine csf samples and shows excellent correlation with manual methods used in dogs with increased total cell counts (pleocytosis), but the instrument may overestimate the cell count in samples without pleocytoses and has not been validated for the identification of eosinophils. 23 the automated differential count is also more accurate at higher cell numbers and thus should be compared with a traditional manual differential. the advia 2120 hematology analyzer displayed satisfactory agreement with the standard hemocytometer method. 24 validation experiments using 67 canine samples showed a sensitivity of 100% and specificity of 89% for accurately identifying samples with pleocytosis when manual counting was considered the gold standard (>5 cells/μl). 24 the instrument tended to be less accurate at lower (within reference interval) nucleated cell counts. 24 erythrocytes may be a source of interference, as a red blood cell (rbc) count of 250 cells/μl was shown to elevate the nucleated cell count. 24 with regard to differential cell count, the instrument performed better in the presence of pleocytosis, whereas monocytes were overcounted at lower nucleated cell counts. 24 automated cell counts thus should not replace a manual differential but may be used as another level of quality control. automated instruments cannot recognize altered cell types, such as atypical neoplastic cells. measurement of csf specific gravity is not considered to be helpful because of low sensitivity for detecting abnormalities. 12 csf microprotein may be semiquantitatively measured by using urine dipsticks that detect albumin. this assay has a lower detection limit of 100 mg/ dl; therefore, it has low sensitivity for mild to moderate csf protein concentration elevations (30 mg/dl to 100 mg/dl). false-positive or false-negative reactions may occur if the dipstick reads at trace or 1+, but this method is useful if other techniques are not available. 11 reference laboratories apply a similar but more sensitive methodology to measurement of csf microprotein as that of serum protein, using the trichloroacetic acid method, the ponceau s red dye-binding method, or the coomassie brilliant blue method. 22 csf globulin production is typically screened for with the pandy reaction. in this test, a few drops of csf are added to 1 ml of 10% carbolic acid solution, and the resulting turbidity is graded 0 to 4+. any pandy score above zero is considered elevated. globulin concentration below 50 mg/dl will be undetectable with either test. 10, 21 protein electrophoresis and immunoelectrophoresis may be performed on csf and serum for maximum fractionation. 25 the utility of protein electrophoresis or immunoelectrophoresis of csf lies in discriminating altered blood-brain barrier (bbb) permeability from increased localized production of immunoglobulin, which may be suggestive of (but not specific for) a disease entity for which an electrophoretic pattern has been established. cytological analysis is a critical component of csf evaluation because the differential count (percentages) of cells may be abnormal, even if the total nucleated count is within reference interval. cytology also enables examination for neoplastic cells, infectious agents, and evidence of prior hemorrhage. it may also serve as a quality control point, allowing for correlation between observed cellularity and the total count generated by a hemocytometer or an automated analyzer. because of its low cellularity, csf must be concentrated before cytological smear preparation. use of an in-house sedimentation chamber (sörnäs procedure) may be very useful and preserves cell-free fluid for ancillary testing. 10 this technique will recover approximately 60% of total cells, which is sufficient for analysis. 16 a syringe barrel (with the tip and needle aseptically removed with a scalpel blade) is turned upside down and the smooth, top side is placed in warm petroleum jelly and then onto a clean slide. once a seal has formed, fresh csf (at least 0.5 ml) is placed in the syringe and allowed to sit for 30 minutes. 16, 21 then, the supernatant is aspirated carefully with a pipette so as not to disturb the bottom layer contacting the slide. the syringe barrel is removed, and any excess csf is carefully absorbed with a small piece of filter paper or paper towel. the slide is completely and rapidly air-dried without heat (inadequate drying results in cellular distortion), excess petroleum jelly removed with a scalpel blade, and the slide is stained with routine romanowsky stains (e.g., diff-quik). if csf is sent to a reference laboratory, a cytological slide will likely be prepared using cytocentrifugation (500-1000 revolutions per minute [rpm] for 5-10 minutes, either onto a slide coated with albumin or with the addition of 0.05 ml of 30% albumin for improved cell capture) for maximal concentration of nucleated cells onto one slide. 16 cytocentrifuged cytology may show excellent cellular detail, but the preparation may enlarge cells slightly and create an artifactual foamy or vacuolated appearance. 16 slides are air-dried and stained with conventional romanowsky stains. multiple cytospin preparations may be made to yield 200 intact nucleated cells for classification. as it is rare for etiologic agents to localize only within the cns, all cases of suspected infection may be aided diagnostically by fine-needle aspiration (fna) cytology, biopsy with histopathology, culture of nonneural lesions, or all of these. 21 bacterial culture and sensitivity testing of csf is recommended for most cases of neutrophilic pleocytosis, given the appropriate clinical index of suspicion for a septic lesion. even when organisms are visualized on csf cytology, speciation and susceptibility testing may help guide prognostic and treatment decisions. alternatively, bacterial or fungal culture may be negative regardless of cytological observation of organisms. 10, 20 it must be remembered that bacterial cns infection is highly uncommon in dogs and cats compared with other domestic animal species. 26 advanced techniques for neurological disease diagnosis are expanding rapidly. enzyme-linked immunosorbent assay (elisa)-based assays for antibody detection and pcr-based assays for nucleic acid detection of several medically important microbes have been developed for use on csf and may be instructive in the diagnosis of viral, rickettsial, protozoal, or fungal diseases. 20 a large canine study that included a subset of 16 dogs with neoplastic or inflammatory disease showed that csf titer provided diagnosis in 25% of cases. 3 antibody assays should be interpreted cautiously because the presence of antibody may indicate prior exposure or vaccination rather than active infection. moreover, compromise to the bbb in states of inflammation may translate to the presence of antibodies within the csf without local production. occasionally cross-reactive antibodies may be present that do not represent presence of the disease agent under assessment. similarly, specimens for pcr should be submitted to a laboratory with strict quality control to minimize false-negative and false-positive results. poor collection technique may result in false-positive results, especially for bacterial species that are ubiquitous in the environment. 27 as with other aspects of csf analysis, a negative pcr result does not definitively rule out the presence of a pathogen because of the sampling limitation of a small portion of the extracellular space. 20 csf contains glucose, electrolytes, neurotransmitters, and enzymes, but these substances are not measured routinely, although this measurement represents a rapidly expanding area of research in the effort to give clinicians better tools for diagnosing patients and determining prognoses. csf enzymes originate from the bloodstream, the cns, or cells within csf. 10 one study of 34 cats with noninflammatory cns disease showed that measurement of csf activities of lactate dehydrogenase (ldh), aspartate aminotransferase (ast), and creatine kinase (ck) were not diagnostically sensitive but may be useful in detection of acute injury. 28 multiple studies have correlated elevations in csf ck activity with poor prognosis in dogs with neurological disease or spinal cord injury. 29, 30 immunoassays for vascular endothelial growth factor (vegf) and s-100 calcium-binding protein have shown elevations of both molecules in the csf of experimentally induced hypothyroid dogs, suggesting endothelial and glial contribution to increased bbb permeability in this population. 31 myelin basic protein (mbp) has been found to be elevated in lumbar csf in dogs with degenerative myelopathy, supporting the conclusion that it is a demyelinating lesion. 32 mbp concentration is elevated in the csf of dogs affected by intervertebral disk herniation (ivdh) and has been found to be an independent predictor of poor prognosis. 33 beta-2-microglobulin, a major histocompatibility complex i (mhc-i)-associated molecule, has been assayed by using elisa and found to be elevated in the csf of dogs with ivdh and inflammatory disease and also positively correlated with normal total nucleated cell count (tncc). 34 the amino acids tryptophan and glutamine have been found to be elevated in the csf of dogs with portosystemic shunts because of abnormal ammonia metabolism. 35 one study found increased oxytocin in the csf of dogs with spinal cord compression, where it is believed to have an analgesic effect. 36 gamma-aminobutyric acid (gaba) and glutamate neurotransmitter concentrations have been measured in dogs with epilepsy. 37 normal csf is clear and colorless, with few cellular elements and a protein concentration approximately 200 to 300 times less than that of plasma or serum. red or yellowish coloration indicates prior lesional hemorrhage or iatrogenic hemorrhage during collection. in the latter case, a pellet of rbcs will be present after centrifugation. true xanthochromia (yellowish color of hemoglobin breakdown products) that does not clear on centrifugation, cytological evidence of erythrophagia, or both indicate prior hemorrhage into the subarachnoid space. 20 increased bilirubin leakage into the sas or high concentrations of csf protein (>100-150 mg/dl) may cause xanthrochromia. 21 increased turbidity of the sample may be caused by increased number of cells present (>400 rbcs/μl or >200 nucleated cells/μl) but is usually not affected by mild changes. 10, 11 cell counts tncc is fewer than 5 cells/μl in the dog and fewer than 8 cells/μl in the cat, and elevation above this range is termed pleocytosis. 10 grading of pleocytosis is somewhat subjective: in one reference, "mild" was defined as 6 to 50 cells/μl; "moderate" as 51 to 1000 cells/μl; and "marked" as more than 1000 cells/μl. 4 depending on laboratory-specific reference intervals, normal protein concentration is usually less than 25 to 30 mg/dl for cisternal csf and less than 45 mg/dl for lumbar csf. 10, 20 approximately 80% to 95% of csf protein is albumin, and 5% to 12% of csf total protein comprises gammaglobulins. 2 eighty percent of csf protein is transferred from plasma, with the remainder produced within the cns. the latter population includes molecules also produced by other organs and proteins unique to the csf that may potentially be used as markers of cns tissue damage. experimental evidence and earlier literature support a gradient of increasing protein concentration from cranial to caudal within the subarachnoid space, which has been attributed to slower flow and greater blood-csf permeability caudally. 12 normal csf is acellular or contains small numbers of small lymphocytes (figs. 14.2 and 14.3) and large mononuclear cells (macrophages, ependymal lining cells, meningothelial lining cells, choroid plexus cells) (figs. 14.4 and 14.5). large mononuclear cells may be vacuolated and contain phagocytized material ( fig. 14.6) . a low frequency of nondegenerate neutrophils (<25%), which are usually indicative of blood contamination during collection, may be present. 38 a study of 359 samples of canine csf found a 7.5% incidence of meningeal, choroid plexus, ependymal, endothelial cells, or all of these. 39 no correlation existed between the presence of these cells and the presence of pleocytosis, elevated protein concentration, or the primary disease etiology. 39 thus it is postulated that the presence of these cells is an artifact of collection and should not be overinterpreted. the authors recommended the term "surface epithelial cells" for the combined grouping (which cannot be distinguished cytologically), although not all of these cells (meningeal, endothelial) are of epithelial origin. 39 occasionally, anucleate superficial squamous epithelial cells may be seen; these may be caused by contamination from the skin (fig. 14.7 ). occasionally, small amounts of granular, foamy extracellular material are present and are consistent with myelin or myelin-like material, which will stain positively with luxol fast blue stain. this material may consist of myelin fragments, which are generated from demyelination, or may consist of myelin figures (a nonspecific term for layered phospholipids exfoliated from damaged cells). 40 the two cannot be distinguished with light microscopy. the significance of this material remains unclear because it may be observed in samples from patients with no discernible cause. a study of 98 canine cerebromedullary and lumbar csf samples showed 20% incidence of myelin-like material, with a higher percentage in samples from the lumbar cistern or from small dogs (<10 kg). 41 the presence of the material was not correlated with case outcome. 41 similarly, in a study of 61 cavalier king charles spaniels with chiari-like malformations, myelinlike material was observed in 57% of lumbar csf collections and 12% of cerebromedullary collections. 42 thus myelin-like material may be a procedural artifact or may be consistent with a demyelinating (e.g., canine distemper virus, degenerative myelopathy) or potentially necrotizing disorder (e.g., ivdh, other spinal trauma, or a necrotic neoplasm). 40, 41 normal csf should not contain erythrocytes, but hemodilution is a common occurrence. varying reports on the effect of blood contamination on tncc, leukocyte differential, and protein concentration have been published. [43] [44] [45] [46] deciding whether increased tncc or protein concentration is the result of hemodilution alone or a significant change concurrent with hemodilution necessarily remains, to an extent, a subjective assessment and must be critically evaluated in light of the magnitude of csf findings along with the other pertinent facts of the case. correction formulas for csf parameters in the face of hemodilution (e.g., adding 1 nucleated cell/μl per 100 or 500 rbcs/μl) are unreliable. 45, 46 in a recent study of 106 canine csf samples without pleocytosis (tncc <5/μl) but containing at least 500 rbcs/μl, the mean percentage of neutrophils (45.2% versus 5.7%), percentage of samples with eosinophils present (36.8% versus 6.8%), and mean protein concentration (40 mg/dl versus 26 mg/dl) were found to be significantly increased in the samples with blood contamination when compared with controls. 47 significant rbc contamination warrants repeat sampling, if possible. marked hemorrhage or evidence of prior hemorrhage (erythrophagocytosis, xanthochromia, hemosiderin-laden macrophages) may be useful in the diagnosis of cns trauma, which may be accompanied by neutrophilic to mixed cell pleocytosis and mild increase in protein concentration. 4 elevated protein concentration in csf (>30 mg/dl) may occur with or without pleocytosis, and in the absence of pleocytosis is termed albuminocytological dissociation (acd). high protein concentration may be the result of leakage of plasma or cellular proteins across the bbb, localized production of immunoglobulin, localized tissue damage or necrosis, decreased clearance of protein into the venous sinuses, obstruction of csf circulation, or all of the above. as such, it is a nonspecific change that indicates cns damage or hyperproteinemic disease and is consistent with disease of any etiology (e.g., trauma, metabolic, infectious, inflammatory, degenerative, or neoplastic). caution should be exercised when diagnosing acd if the sample is hemodiluted (>500 rbcs/μl). 47 as is true for pleocytosis, inflammation of the meninges and superficial regions of parenchyma will result in greater csf protein elevations than for lesions that are more remote from the sas. occasionally, an abnormal leukocyte differential (shifted from mononuclear predominance to neutrophil predominance) without pleocytosis occurs. this may only be detected if cytological analysis (after sedimentation or cytocentrifugation of csf) is performed. increased percentages of neutrophils may occur in early or mild inflammatory disease, noninflammatory cns disease, disease that is remote from the sas or sampling site, or in cases of hemodilution. an increased proportion of neutrophils is present when neutrophils comprise greater than 25% of all nucleated cells, and increased percentage of eosinophils occurs when eosinophils comprise greater than 1% of the differential. 10 when present (with or without increased tncc), neutrophils should be evaluated for toxic change, degenerative change, and intracellular organisms or other inclusions ( fig. 14.8 ). increased percentage of neutrophils without pleocytosis has been associated with healthy dogs, blood contamination, degenerative disk disease, neoplasia, cerebrovascular accident, fracture, cns aspergillosis, and fibrocartilaginous embolism (fce). 10, 42, 48 a study of 61 cavalier king charles spaniels with chiari-like malformation documented that those with syringomyelia were more likely to have an increased percentage of neutrophils, but it was not reported whether this subpopulation also had a concurrent pleocytosis. 42 in another study, cats with cns neoplasia had increased percentage of neutrophils or lymphocytes without a pleocytosis. 28 although not a classic pattern, infectious or inflammatory disease should not be ruled out if increased neutrophils are visualized without pleocytosis. increased percentage of eosinophils has been reported in parasitic and protozoal diseases, such as neospora caninum infection. 38 one cat with eosinophilic meningoencephalitis (eme) of unknown etiology had an increased percentage of eosinophils and lymphocytes without pleocytosis. 49 the specific diseases mentioned in the next section on various categories of pleocytosis are a survey of the current literature and meant to be a helpful starting point in the generation of particular differential diagnoses. thus disease entities are listed in the section under which they are most commonly present, but it is important to note that for all disease entities, variability in the nature and the magnitude of pleocytosis may emerge in a particular patient at a particular point in time. wherever possible, other categories of pleocytosis that have been reported for a disease have been mentioned. generally, pleocytoses are defined by the cell type that comprises 70% or more of the nucleated cell population. if all cell types are 50% or less, the pleocytosis is classified as a mixed cell pleocytosis. and if, for example, lymphocytes are greater than 50% but less than 70%, some pathologists will classify the pleocytosis as mixed cell, lymphocyte predominant. a pleocytosis will be classified as eosinophilic if eosinophils compose at least 10% to 20% of the nucleated cell population. 11 bacterial meningoencephalomyelitis. bacterial infections of the cns are unusual and represent a small portion of neutrophilic pleocytoses. typically, this pleocytosis is severe (could be over 1000 cells/μl), neutrophilic, and accompanied by significantly elevated protein concentration, but the cell population may change to mononuclear during the course of treatment. 10, 20, 50 rare instances of brain abscessation secondary to sepsis (which may be a sequela of iatrogenic immunosuppression) may result in marked neutrophilic pleocytosis, markedly elevated protein concentration, visualization of bacterial organisms (see fig. 14.8) , and abnormal mri findings. 51 staphylococcus intermedius was cultured from the csf of a dog presenting with a retrobulbar abscess and neurological signs. 52 the csf showed a moderate neutrophilic pleocytosis (75 cells/μl) and borderline elevation in protein concentration (30 mg/dl). 52 local extension of severe otitis interna resulting in meningoencephalitis and ventriculitis in a dog has been reported. 53 this patient exhibited a severe neutrophilic pleocytosis (3672 cells/μl) and protein elevation (>400 mg/dl). 53 pasteurella multocida meningoencephalomyelitis in a kitten was characterized by marked neutrophilic pleocytosis (981 cells/μl) with mild protein elevation (31 mg/dl) and rare extracellular and intracellular bacterial rods. 54 bacterial culture and susceptibility testing are recommended but may yield false-negative results if organisms are not circulating in the extracellular space or if prior antibiotic therapy had been given. serology and csf-pcr (using organism-specific or universal bacterial [ub] pcr) are recommended. 27, 54 cryptococcosis in dogs. cryptococcus spp. are a large genus of systemic dimorphic fungi with a predilection for cns tissue, which is infected hematogenously or via direct penetration of the cribriform plate. only two species at this time are medically important: (1) cryptococcus neoformans (var. neoformans and var. grubii) and (2) cryptococcus gattii. in a recent study of 31 dogs with cryptococcosis, 68% had cns infection, with neurological signs being the most common reason for presentation. 55 dogs and cats with cryptococcosis typically have pleocytoses and elevated protein concentrations, but pleocytoses may be variably neutrophilic, eosinophilic, mononuclear, or mixed. in a recent study of 15 dogs with cns cryptococcosis, organisms were found in 11 of 15 csf samples (figs. 14.9 and 14.10). 56 all affected dogs had pleocytoses that were mixed to mononuclear, whereas cats tended to have neutrophilic pleocytoses. 56 of the samples, 11 of 12 also had increased protein concentrations (mean 494 mg/dl), which were significantly higher than in cats in the same study (mean 45 mg/ dl). 56 capsular antigen latex agglutination testing on serum or csf is highly sensitive and specific and is recommended if cryptococcosis is suspected but organisms are not visualized cytologically. 57 this test may yield negative results if disease is present but localized (i.e., within the respiratory tract), so appropriate clinical signs should guide testing. culture of csf may also be helpful and may distinguish c. neoformans from c. gattii with the use of selective media. the finding of inflammatory foci on mri may be supportive of the presence of fungal disease; cryptococcosis may result in mass lesions, meningitis, or pseudocyst formation. cryptococcosis is the most common systemic fungal disease of cats and is believed to infect the cns less frequently than in the dog. a recent study found that 42% of 62 cats with cryptococcosis had cns infection, but respiratory signs were still a more common reason for presentation. 55 mild to marked neutrophilic or mononuclear pleocytosis may occur, with variable and occasionally normal protein concentrations. 4 a study of cats with cns cryptococcosis showed organisms in 9 of 11 of the csf samples, and a majority of cases (9 of 10) had neutrophilic pleocytosis and increased protein concentration (8 of 10). 56 eosinophilic pleocytosis may also occur. capsular antigen latex agglutination testing on serum or csf is recommended for confirmation of cryptococcus spp. infection, with rare false-negative reactions if disease is highly localized. fungus that has been visualized in canine csf and may be extracellular or within leukocytes. 58 ehrlichiosis. neutrophilic pleocytosis has been reported in cases of granulocytic ehrlichia spp. in dogs (fig. 14.11 ). 61 neurological signs are uncommon in this disease, and affected dogs may display features ranging from ataxia to seizures. (fip) has been traditionally linked to marked csf changes, but the current literature paints a somewhat more varied picture. one study of natural fip infection showed neutrophilic pleocytosis (as defined by >50% neutrophils) in the majority (7 of 11) of cases, with fewer cases of mononuclear (3 of 11; as defined by >80% mononuclear cells) and mixed cell (1 of 11) pleocytosis, all of variable severity. 4 most cases (7 of 9) also had differing degrees of elevated protein concentrations. 4 diagnosis was confirmed by histopathology or suggested by elevated feline coronavirus antibody titers and reduced albumin-to-globulin ratios in both serum and body cavity effusions. 4 a slightly older study of 16 csf samples (natural and experimental infections) showed pleocytosis in 2 of 16 cases (neutrophilic and lymphocytic) and elevated protein concentration in 4 of 16 cases. 62 in a larger study of 67 cats with fip or non-fip disease, incidence of pleocytosis was highest in the neurological fip group, but 20% of these patients did not have a pleocytosis. 63 additionally, protein concentrations were variably elevated and not statistically different in fip compared with non-fip neurological disease. 63 another study of 12 cats with cns fip showed 8 of 12 with unspecified pleocytosis and 3 of 12 with elevated protein concentration. 64 in cats with cns disease, sensitivity of feline coronavirus (fecov) immunoglobulin g (igg) in csf for the diagnosis of fip was 60%, and specificity was 93%, with a positive predictive value of 75% and a negative predictive value of 87% (fip prevalence in this population was 25.6%). 63 definitive diagnosis of this disease remains challenging, with virus identification (pcr or immunohistochemistry) accompanied by pyogranulomatous inflammation in tissues being the gold standard. hypergammaglobulinemia, elevated serum α 1 -acid glycoprotein (agp), mri abnormalities (typically involving the ventricular lining and meninges), and positive feline coronavirus igg titer or pcr from serum, tissue, or csf are supportive but not specifically diagnostic, and negative findings do not rule out disease. 20, 63, 65 toxoplasmosis in cats. cats are the definitive hosts for toxoplasma gondii and may be subclinically infected; thus, diagnostics should only be performed on patients with appropriate clinical signs. cats typically present with mild neutrophilic or mononuclear pleocytosis and normal to mildly elevated protein concentration, but marked protein elevation may occur. 4 mild lymphocytic pleocytosis is also reported. 65 diagnosis may be confirmed by direct visualization of organisms in csf, aspirates of other inflammatory foci, histopathology of affected tissues, or fecal examination. serology must be interpreted cautiously because igg may remain elevated for up to 6 years after exposure. therefore, paired serum igm-igg titers, indicating acute exposure, or documentation of rising serum igg titers are more useful, but the latter is difficult to document in the advanced state of disease. 65, 66 spinal epidural empyema in dogs. epidural empyema is an uncommon disease in dogs, resulting from pyogenic infection in the epidural space. one study showed 4 of 5 dogs with neutrophilic pleocytosis of variable magnitude (11-342 cells/μl). 67 no organisms were visualized on any of the samples. 67 except for one case with a lumbar csf protein concentration of 726 mg/dl, protein elevations were modest. 67 three csf samples were cultured with no growth, and two dogs for which follow-up csf was obtained showed resolution of pleocytosis. 67 these results are not surprising, as the dura likely provides a barrier to prevent infection extending from the epidural space to the subarachnoid space. reported in a young cat with a marked neutrophilic pleocytosis with intracellular and extracellular merozoites observed on csf cytology. 68 diagnosis was confirmed with decreasing paired serologic titers, and speciation to the level of sarcocystis dasypi or sarcocystis neurona was conducted with pcr from blood. 68 a case of systemic acanthamoeba spp. infection in a young boxer, diagnosed post mortem, had antemortem csf with marked neutrophilic pleocytosis (4956 cells/μl), marked increase in protein concentration (259 mg/ dl), and subnormal csf iga concentration (33 mg/dl; reference interval 35-270 mg/dl). 69 postmortem pcr for the organism was positive on extraneural tissue but not on csf or spinal cord. 69 the patient had been deliberately immunosuppressed on the basis of a preponderance of evidence of steroid-responsive meningitis arteritis at initial presentation and thus may have been infected either before or opportunistically after treatment. 69 another case report of canine cerebellar balamuthia mandrillaris infection (diagnosed post mortem with immunohistochemistry) displayed a marked neutrophilic pleocytosis (234 cells/μl), but other cases with lymphocytic pleocytosis have been reported. 70 because of tissue encystment, it is suggested that extraneural tissue be used for immunohistochemistry or pcr for antemortem confirmation of amoebic infection; pcr of csf may be diagnostic but is not widely available. 69, 70 two dogs with aberrant spinal migration of spirocirca lupi nematodes had moderate to marked neutrophilic to mixed or eosinophilic pleocytoses (800 cells/μl with 91% neutrophils; 180 cells/μl with 60% neutrophils, 30% eosinophils). 71 steroid-responsive meningitis arteritis. steroid-responsive meningitis arteritis (srma) is presumptively an immune-mediated disease of mainly young, medium-and large-breed dogs: beagles, boxers, bernese mountain dogs, weimaraners, and nova scotia duck tolling retrievers are overrepresented. 20 csf analysis is important in diagnosis and typically features a moderate to marked neutrophilic pleocytosis (a left shift may be present) and markedly elevated protein concentration. chronically, pleocytosis may change to a more mononuclear or mixed population (fig. 14.12 ) and may become mild or even fall into reference intervals. 72 a study of 20 affected dogs showed neutrophilic pleocytosis in 12 of 20 cases and mononuclear pleocytosis in 8 of 20 cases. 72 concurrent elevations of serum and csf iga titers (elevated igg and igm fractions may be present), serum concentration of cross-reactive protein (crp), or serum α 2 -macroglobulin is diagnostically supportive but not specific. 20, 50 increases in iga have been linked to a t-helper 2 (th2)-dominated immune response driven by elevated interleukin-4 (il-4) and decreased il-2 and interferon-gamma (ifn-γ). 73 serum amyloid a (saa), serum agp, and serum haptoglobin may also be elevated. 74 another study of 36 dogs with srma reported statistically significant elevations of csf and serum crp, but not serum α 2 -macroglobulin, in dogs with srma compared with other neurological diseases. 75 in a study of 20 dogs, serum crp was positively correlated with csf tncc. 72 additionally, serum haptoglobin and serum and csf iga remained increased throughout successful treatment, indicating that these parameters are more useful for diagnosis than for monitoring therapy. 72 serum and csf concentrations of crp and saa have been documented to fall significantly during treatment, and repeat measurement of serum crp or saa may be used to guide therapy and predict relapse, which is less invasive and more sensitive than repeat csf sampling. 72, 74, 75 rare cases have been documented in cats with marked mononuclear or mixed pleocytosis and mild to moderate protein concentration elevations. 4 intervertebral disk herniation. csf from patients with ivdh may be extremely variable; data indicate that csf findings correlate with location of sampling, disk herniation location, chronicity of the lesion, and severity of spinal cord injury. bearing this in mind, it is no surprise that some reports in the literature state that neutrophilic, lymphocytic, mixed, and mononuclear pleocytoses are most common in dogs with ivdh. 12, 30, 76 a study of 423 cases of ivdh showed 51% with pleocytosis, of which 31% were neutrophilic, 41% were lymphocytic, 20% were mixed, and 7.4% were mononuclear. 76 of all cases, 71% had elevated protein concentrations. 76 interestingly, a larger number of cases of lymphocytic pleocytosis were observed in the samples analyzed more than 7 days after onset of clinical signs. 76 the magnitude of pleocytosis, in general, was also shown to decrease with increasing time between clinical onset and sampling, and this observation has been corroborated by other studies. 12, 76 prior treatment with corticosteroids was observed to reduce the number of observed lymphocytes in csf. 76 the authors also found a higher incidence of pleocytosis in thoracolumbar disease (61%) compared with cervical disease (23%), but this may have been caused by exclusive sampling of lumbar csf closer to the lesion. 76 ivdh is rare in cats and has been reported to feature mild mixed cell pleocytosis and elevated protein concentration. 4 patients typically present with nonpainful, progressive, asymmetrical neurological signs. as only histopathology is confirmat ory, it is a multimodal diagnosis of exclusion. a study of 32 dogs with presumptive fce, based on history, clinical signs, imaging, and outcome, showed 53% with normal csf, 25% with acd, and 19% with mild to moderate pleocytosis (7-84 cells/μl; median 12/μl). 77 pleocytoses were neutrophilic or mixed. 77 one study of 36 confirmed cases in dogs showed that 64% had normal csf and the remainder displayed mild changes. 78 another study looking at five dogs suggested that pleocytosis may be marked, up to 529 cells/μl. 3 fce is much less common in cats. in general, the disease process and clinical signs are similar to those in dogs, with the exception that the disease presents in cats in middle or older age, usually with cervical spinal cord signs. a case series of five cats showed csf ranging from normal to marked neutrophilic pleocytosis with moderately elevated protein concentration and variable correlation to clinical outcome. 79 the case with the most severe csf changes had extensive myelomalacia at necropsy. 79 it was suggested in this study that csf is more likely to be abnormal if collected closer to the lesion and that mri is helpful for localization and in supporting the diagnosis. 77, 79 thiamine deficiency in cats. thiamine deficiency is a rare nutritional disorder of patients fed noncommercial, misformulated commercial, or irradiated diets. two case reports showed increased percentage of neutrophils or mild neutrophilic pleocytosis, presumptively from cerebrocortical necrosis. 4 diagnosis is based on history, response to treatment, mri features compatible with the disease (cortical and brainstem hyperintensities), or histopathology. 80 spaniels with chiari-like malformation showed that 40% of dogs with concurrent syringomyelia and cisternal csf sampling had mild (up to 15 cells/μl) pleocytoses and increased percentages of neutrophils compared with the subpopulation without syringomyelia, but it was not specifically documented whether pleocytoses were, in fact, neutrophilic or mixed with an increased percentage of neutrophils. 42 a positive correlation was also seen to exist between tncc and syrinx size. 42 neoplasia. it is important to perform csf in neurology patients with suspected neoplasia, as definitive diagnosis may be achieved if neoplastic cells are directly observed via cytology. inflammatory pleocytoses or elevated protein concentrations are common in patients with cancer, tend to be mild to moderate in magnitude, and may represent paraneoplastic inflammation, compromise of the bbb, lesional necrosis, or all of these. 28 normal csf is also a common finding in cases of neoplasia. moreover, in the absence of overtly neoplastic cells, no defined patterns connect specific tumors with specific types of inflammatory pleocytoses. neutrophilic pleocytosis of unspecified magnitude was found in the csf of 2 of 11 cats with spinal lymphoma and in 3 of 7 cats with nonlymphoma spinal neoplasia (astrocytoma or osteosarcoma). 81 additionally, the remaining four cats with nonlymphoma spinal tumors (meningioma, peripheral nerve sheath tumor, plasma cell tumor) had either normal csf or acd of unspecified magnitude. 81 metastatic tumors to the cns should also be considered in a patient with neurological signs. eosinophilic meningoencephalitis of dogs. eme is an idiopathic diagnosis of exclusion that is typically steroid responsive and is postulated to be triggered by an underlying hypersensitivity, allergy, or self-limiting infection. the disease may be overrepresented in rottweilers and golden retrievers. 82 a study of 23 dogs with eosinophilic pleocytosis (defined by >20% eosinophils) showed 16 cases of idiopathic eme, 4 cases of infectious disease (c. neoformans, n. caninum, baylisascaris procyonis), and 3 cases of ivdh. 83 the magnitude of pleocytosis or the percentage of eosinophils could not be used to distinguish infectious versus eme cases, although ivdh cases tended to have milder pleocytoses (<84 cells/μl). 83 in about half the eme cases, mri showed abnormal findings. 83 peripheral eosinophilia may or may not be present. highly suggestive of protozoal (toxoplasmosis, neosporosis), fungal (cryptococcosis), parasitic (including cuterebra spp., dirofilariasis), and algal (protothecosis) infections and also rarely in cases of canine distemper and rabies viruses. 10, 84 eosinophils have also been found in cases of granulomatous meningoencephalomyelitis (gme). 85 eosinophilic pleocytosis has been documented in bacterial encephalitis as well. 21 gondii infection tend to have neurological or neuromuscular signs. case reports are sporadic; documentation of mild acd (58 mg/dl) and also a report of mild lymphocytic or eosinophilic pleocytosis (35 cells/μl) with an elevated protein concentration of 77 mg/dl exist in the literature. 86 it is important to rule out other potential causes of the neurological signs because immunocompetent dogs tend to clear subclinical infections, and therefore paired serum igm-igg titers or sequential serum igg titers are preferable to a single serum igg titer. to the author's knowledge, no data on the life span of canine igg antibodies exist. reports in the literature are conflicting with regard to the cross-reactivity of t. gondii antibodies to other agents, such as n. caninum. 20,86 pcr testing for toxoplasma in serum, tissue, or csf is diagnostic. 86, 87 rabies. pleocytoses may be lymphocytic and of varying severity. ancillary antemortem diagnostics include viral pcr on saliva or csf and the saliva antigen latex agglutination test. in a study of 15 dogs under quarantine for suspected natural infection (subsequently confirmed positive), 13 of 15 were saliva-pcr positive, and 4 of 15 (27%) were csf-pcr positive. 88 all animals with positive results on csf were also positive on saliva, and interestingly 100% correlation was seen between positive csf-pcr and the dull clinical presentation (all aggressive clinical presentations were csf-pcr negative). 88 negative testing should never exclude diagnosis because viral load is highest within salivary glands and brain parenchyma. 88 frequently made by exclusion when coupled with appropriate clinical signs. csf and mri findings are variable and may be normal in the acute stage of disease before inflammation has peaked. 89 in a study of 32 dogs with noninflammatory distemper, half (15 of 32) had normal csf. 85 a study of eight dogs with natural infection (confirmed by cns tissue-pcr and histopathology) showed lymphocytic pleocytosis in all samples and normal protein concentrations. 90 another case (confirmed by tissue-pcr and csf-pcr) in a 7-month-old dog displayed marked (554 cells/μl) lymphocytic pleocytosis and a normal protein concentration. 89 because this is a demyelinating disease, myelin-like material, which is amorphous, granular, pink, foamy, and stains positively with luxol fast blue, may be present. 40 pcr testing of csf, serum, urine, epithelial or tonsillar tissue is available, and immunohistochemistry on biopsy specimens of nasal mucosa, haired skin, or footpad is 88% to 96% sensitive for detection of viral antigen. 91 is present, it is likely to be lymphocytic. pleocytoses are typically mild to moderate, but severe lymphocytic pleocytoses have been reported. 89 main differential diagnoses include other viral diseases, gme, or chronic bacterial infection. extranigral signs related to the gastrointestinal or the respiratory system, if present, may be helpful in distinguishing this disease from gme. 89 in a study comparing four dogs with chronic cdv, six dogs with acute cdv, and controls, dogs with chronic cdv had markedly elevated csf igg concentration. 92 the igg region was polyclonal, including a population of neutralizing antibodies for cdv. 92 fungus acquired through inhalation, and most cases in the united states are observed in the southwestern region of the country. signs tend to involve respiratory or skeletal systems, and cns involvement is rare. one dog had a mild to moderate lymphocytic pleocytosis. 57 complement fixation (detecting igg) or tube precipitation (detecting igm), or agar-gel immunodiffusion serological testing is recommended for confirmation. necrotizing meningoencephalitis. meningoencephalitis has been subcategorized as necrotizing meningoencephalitis (nme) and necrotizing leukoencephalitis (nle) on the basis of histopathological appearance. both nle and nme are believed to have an immune-mediated basis, and recent data support that in pugs with nme, canine leukocyte antigen gene aberrations exist. 93 meningoencephalitis is rapidly progressive and affects a variety of generally young to middle-aged toy-breed dogs, including the pug, shih tzu, papillon, maltese, chihuahua, yorkshire terrier, french bulldog, pekingese, west highland white terrier, boston terrier, japanese spitz, and miniature pinscher breeds. 20, 94 a study of csf from 14 pugs with nme showed 12 of 14 with pleocytoses of varying severity (mean 120 cells/μl). 95 of these dogs, 66% had a lymphocytic pleocytosis, 17% had a mononuclear pleocytosis, and 17% had a mixed cell pleocytosis (fig. 14. 13) 95 ; 11 of 14 dogs had elevated protein concentrations (mean 88.4 mg/dl). 95 another study of three dogs showed one with acd and two with moderate to marked (40-220 cells/μl) neutrophilic to lymphocytic pleocytosis. 94 mri findings may help support a diagnosis, but only histopathological examination of lesions provides definitive proof. other. four cats with ischemic encephalopathy had mild (<10 cells/μl) lymphocytic pleocytosis. 28 another study of feline ischemic encephalopathy reported one cat with normal csf and another with mononuclear to mixed pleocytosis (26 cells/μl). 96 a report of two cats with cerebrovascular disease (infarction or stroke) showed one with mononuclear pleocytosis and the other with acd. 97 cerebrovascular disease was correlated in several other cases (without csf data) to hepatic lipidosis or fip. 97 other. a case report of a pug with a mild mononuclear pleocytosis (8 cells/μl), mild elevation in protein concentration (89 mg/dl), evidence of hemorrhage, and direct visualization of angiostrongylus vasorum helminth larvae is found in the literature. 102 eosinophilia was not observed within the csf or peripheral blood. 102 the organism is endemic in europe and canada among foxes and canids, mainly causing respiratory signs or coagulopathy; neurological signs are typically caused by hemorrhage. 102 two dogs with paraparesis and pyogranulomatous lumbar masses (one intradural, one extradural) had lumbar csf with mild mixed cell pleocytosis (lymphocytes and nondegnerate neutrophils) or lymphocytic pleocytosis. 103 these patients were serologically pcr positive for bartonella vinsonii subsp. berkhoffi and presented with a nodular dermatosis. 103 a dog with neurological signs and hepatozoon canis infection showed marked lymphocytic pleocytosis (243 cell/μl) with mildly elevated protein concentration (37 mg/dl). 104 organisms were not visualized in csf but were found on cytology of peripheral blood, lymph node, bone marrow, and bony lesions. serology and positive pcr from a bone marrow sample were diagnostic. 104 intrathecal contrast administration. contrast media or pharmacological agents, such as epidural anesthetics, may introduce preanalytical error into csf samples, artificially raising tncc and protein concentrations. 105 in a study of 17 healthy dogs given either iopamidol or metrizamide for emg, same-day csf sampling showed that 8 of 17 developed a mild to moderate mononuclear to mixed mononuclear or neutrophilic pleocytosis (6 of 8 were from iopamidol). 106 in the same study, 3 of 17 (all metrizamide) developed mild protein elevation, but mean protein concentration for both groups stayed within the reference interval. 106 in dogs given metrizamide, 7 of 8 had an increased pandy score after emg, which was considered a false-positive result because of the contrast agent. 106 these data, plus histopathology from the same population, showed that the contrast agents caused low-grade leptomeningeal inflammation with no statistical difference between the two agents studied. 106 another similar study over 30 days showed that post-emg csf changes reversed after approximately 5 days. 107 granulomatous meningoencephalitis. gme is a progressive immune-mediated disease that is overrepresented in females, toybreed dogs, and terriers. 20 it is a diagnosis of exclusion and has clinical presentations and mri findings that may be similar to various infectious and neoplastic diseases. csf may be unaffected or may display a mononuclear to mixed pleocytosis and protein concentration elevations, both of varying severity (figs. 14.14 and 14.15). in a study of 188 csf samples from dogs with inflammatory neurological diseases, marked pleocytosis (>1000 cells/μl) was found in cases of srma, bacterial encephalitis, or gme. 85 pleocytosis may also be lymphocytic or neutrophilic. 10 csf protein electrophoresis may be helpful, as several cases have been shown with increased β-globulin and gammaglobulin fractions. 108 most of the diseases described previously in this chapter may manifest as mixed cell pleocytoses, depending on the time interval between disease onset and csf sampling, disease severity, and previous treatment administered. a mixed cell pleocytosis would be expected to occur during transition between different phases of the inflammatory response, where certain cells may predominate at specific times after injury. is typically rare and may involve chorioretinitis or focal cerebral granuloma in the cat. 109 a study of two dogs with systemic blastomycosis and neurologic signs showed mild mixed cell pleocytosis (8 cells/μl, mononuclear predominant; and 15 cells/μl, lymphocytic predominant). 110 using csf cytology or culture to diagnose the organism may be unrewarding. agar-gel immunodiffusion serologic testing has high sensitivity and specificity for canine antibodies and is recommended if appropriate clinical signs (respiratory signs or lymphadenopathy) are present. 57 agar-gel immunodiffusion testing is less sensitive (25%-33%) in the cat, as indicated by a limited number of reports. 57 urine antigen enzyme immunoassay (eia) has good sensitivity for dogs and has been used successfully on at least one cat. 109 eia may also be performed on csf. cytology of nasal, pulmonary, or dermal lesions is more likely to yield direct visualization of organisms. lymphoma. lymphocytic pleocytosis of inflammatory origin may be difficult to distinguish from lymphoma exfoliating into the csf (fig. 14.16 ). the size of lymphocytes and morphological atypia may be helpful, although these may be challenging to differentiate from artifactual morphological changes secondary to cytospin preparation. cats with neoplasia may have lymphocytic pleocytoses (suggestive of lymphoma), mild to moderate mononuclear to mixed cell pleocytoses (suggestive of nonlymphoma tumors), or normal csf. one study examined six cases of feline cns or multifocal lymphoma, which displayed pleocytoses of variable magnitude, absent to mildly elevated protein concentrations, and neoplastic cells visualized in 5 of 6 of the csf samples. 4 in this study, eight cats with cns signs that were ultimately diagnosed with nonlymphoma tumors (e.g., meningioma, carcinoma, nerve sheath tumor) had mild csf protein elevations and either normal tncc (1 of 8) or mild to moderate mononuclear or mixed cell pleocytosis (7 of 8). 4 another study of 11 cats with spinal lymphoma showed neoplastic cells visualized in one case and hemodilution, acd, or neutrophilic pleocytosis in the remainder of cases. 81 a case report of feline multiple myeloma involving lumbar vertebrae and associated soft tissues exhibited cisternal csf with an elevated protein concentration of 290 mg/dl and mild pleocytosis (8 cells/μl) consisting of a majority of neoplastic plasma cells. 111 diagnosis was further confirmed by abnormal urine protein electrophoresis and bone marrow aspiration. 111 histiocytic malignancies. malignant histiocytosis or histiocytic sarcoma tumor cells in canine csf have been documented in two recent case reports; csf cytology displayed marked mononuclear pleocytoses (>500 cells/μl) and mild to moderately elevated protein concentrations (<135 mg/dl). 112, 113 tumor cells phenotypically resembled macrophages, displayed multiple criteria of malignancy, and reacted positively to cd1c on immunocytochemistry, compatible with interstitial dendritic cell origin. 112, 113 necropsy was confirmatory and found no evidence of neoplasia outside of the cns. 112, 113 a case report of a gliomatosis cerebri (gc) neoplasm in a middle-aged poodle showed csf with a mild lymphocytic pleocytosis (20 cells/μl) and protein concentration elevation. 114 on histopathology, lymphocytelike perivascular cuffing and meningitis were noted. other case studies of canine gc have reported normal csf or mild acd. 115 in a study of 56 dogs with intracranial meningioma, in which csf analysis was performed, 29% had normal csf, 45% had acd, and 27% had pleocytosis (2 of 3 of these neutrophilic pleocytosis; 1 of 3 unspecified), with the overall incidence of neutrophilic pleocytosis at 18%. 116 in this study, a positive correlation existed between elevated tncc and anatomical localization of the lesion to the caudal (versus middle or rostral) portion of the cranial fossa, and no association between pleocytosis and necrosis within the lesion was found. 116 these findings contradict prior reports of a high percentage of abnormal csf findings in meningioma, and the authors reported that concurrent glucocorticoid therapy in some of the patients may have negatively biased the data. 11, 116 a study of 26 dogs with spinal meningioma showed no cases with exfoliating tumor cells, 62% with mild pleocytosis up to 47 cells/μl (mean 11 cells/μl), and normal or variably elevated protein concentrations up to 836 mg/dl (mean 212 mg/dl). 117 both cisternal and lumbar csf samples were evaluated in this study and not found to be significantly different. 117 interestingly, tumors of the lumbar region displayed higher mean tncc and protein concentrations compared with tumors of the cervical area (24 versus 4 cells/μl and 158 versus 98 mg/dl, respectively), which the authors postulated may be reflective of a higher number of lumbar csf samples with proximity to the lesion. 117 other neoplasms. a case report of canine csf with 240 cells/μl was characterized by atypical neoplastic round cells that were confirmed on immunocytochemistry and immunohistochemistry to be from a metastatic mammary carcinoma. 118 inflammatory cells were of low numbers and were of a mixed population. 118 a study of csf from 25 dogs with choroid plexus tumors showed direct observation of tumor cells in 47% of the cases of carcinoma. 119 mild to moderate mixed-cell pleocytosis was present in all cases of papilloma and in half of the carcinomas; when pleocytosis was present, no difference in magnitude existed between benign and malignant tumors. 119 all cases had elevated protein concentrations, with median concentration for carcinoma being significantly higher (108 mg/dl) than median concentration for papilloma (34 mg/dl). 119 a cutoff protein concentration of 80 mg/dl yielded a sensitivity of 67% and a specificity of 100% for detection of choroid plexus carcinomas. 119 another case report of canine choroid plexus carcinoma had a mononuclear pleocytosis of 165 cells/μl, mildly elevated protein concentration of 30 mg/dl, and numerous tumor cells visualized. 120 a rise in the availability of stereotactic brain biopsy has facilitated increased cytological assessments of cns lesions. this technique offers several advantages, although significant equipment investment and time to perfect techniques is required. stereotactic biopsy often offers application accuracy for targeting lesions that approximate 3 mm or less in all directions. in one study, diagnostic accuracy of stereotactic biopsy specimens submitted for histopathology (i.e., agreement with specimens obtained via open approaches) exceeded 90%. 121 in experienced hands, stereotactic biopsy is believed to be a relatively low-morbidity procedure. a b 20um 20um cytological interpretation of brain biopsy specimens acquired via stereotaxy or open approaches may be challenging and does require a tumor that exfoliates well, a surgeon willing to provide multiple samples, and a cytologist with expertise in this area. 122 a study of 42 canine and feline cases of biopsy-or necropsy-confirmed cns lesions showed squash-prep smear cytology to have 76% sensitivity in accurately determining diagnosis, with an additional 14% of cases having partial correlation between cytology and histopathology. for the remaining 10% of cases, cytological interpretation did not correlate with final diagnosis. 123 cytological interpretation of cns lesions may be very difficult, and biopsy with histopathological examination is recommended to confirm all diagnoses. it is important for cytological samples to be prepared in the same manner each time to avoid introducing additional cytological variations that the pathologist has to read through. some authors recommend wet-fixation of tissues followed by staining with hematoxylin and eosin (h&e). 122 at the authors' institution, cns cytological samples are air-dried and stained with diff-quik or a modified wright stain. the reader is referred elsewhere for a complete discussion of normal cns cytology. 124 clinical imaging findings, and signalment, should be considered carefully and may help the pathologist to formulate a list of potential differential diagnoses. it must be kept in mind that primary tumors may metastasize to the cns, and these should be included in the differential diagnoses, where appropriate. meningiomas are composed of neoplastic cells arising from the meningothelial cells of the leptomeninges of the cns. 125 these tumors are the most common primary cns tumors of dogs and cats. 126 histologically, these neoplasms are classified into at least nine subtypes based on appearance, and some tumors may be characterized by more than one pattern (fig. 14.17 ). 125 cytologically, smears are often characterized by spindle-shaped cells draped around vessels and arranged in large whorling structures (fig. 14.18 ). some cells may contain nuclei that display intranuclear cytoplasmic pseudoinclusions, but this is not a feature reliably seen on a majority of tumors (fig. 14.19 ). 127 as a whole, this group represents the second most common primary cns neoplasm seen in dogs and cats. 126 glial tumors are more a b common than meningiomas in brachycephalic breeds. 126 glial tumors arise from the supporting cells of the cns. astrocytomas are found most frequently in the cerebral hemispheres, although they have been reported to occur in various locations throughout the cns. 125 astrocytomas arise from transformed astrocytes and are characterized cytologically by high cellularity, a high degree of nuclear pleomorphism, and fibrillar cytoplasmic processes. 125 tumor cells will stain positively for glial fibrillary acid protein (gfap). 125 oligodendrogliomas are derived from transformed oligodendrocytes and are found within the gray or white matter of the cns, with the highest incidence in the cerebral hemispheres. 125 cytological preparations are characterized by large numbers of blood vessels surrounded by neoplastic cells (fig. 14.20) . 125 neoplastic oligodendrocytes have small amounts of eosinophilic cytoplasm surrounding uniformly round nuclei. 125 ependymomas are derived from the ependymal lining cells found on the surface of the ventricular 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prednisone and cyclosporine therapy necrotizing cerebellitis and cerebellar atrophy caused by neospora caninum infection: magnetic resonance imaging and clinicopathologic findings in seven dogs angiostrongylus vasorum causing meningitis and detection of parasite larvae in the cerebrospinal fluid of a pug dog bartonella-associated meningoradiculoneuritis and dermatitis or panniculitis in 3 dogs hepatozoonosis in a dog with skeletal involvement and meningoencephalomyelitis cerebrospinal fluid response following metrizamide myelography in normal dogs: effects of routine myelography and postmyelographic removal of contrast medium cerebrospinal fluid changes after iopamidol and metrizamide myelography in clinically normal dogs transient leakage across the blood-cerebrospinal fluid barrier after intrathecal metrizamide administration to dogs cerebrospinal fluid protein electrophoresis: a clinical evaluation of a previously reported diagnostic technique cerebral blastomyces dermatitidis infection in a cat clinical and magnetic resonance imaging features of central nervous system blastomycosis in 4 dogs multiple myeloma with central nervous system involvement in a cat antemortem diagnosis of localized central nervous system histiocytic sarcoma in 2 dogs cerebrospinal fluid from a 10-yearold dog with a single seizure episode oligodendroglial gliomatosis cerebri in a poodle gliomatosis cerebri in six dogs characteristics of cisternal cerebrospinal fluid associated with intracranial meningiomas in dogs: 56 cases (1985-2004) canine intraspinal meningiomas: imaging features, histopathologic classification, and long-term outcome in 34 dogs neoplastic pleocytosis in a dog with metastatic mammary carcinoma and meningeal carcinomatosis choroid plexus tumors in 56 dogs (1985-2007) choroid plexus carcinoma cells in the cerebrospinal fluid of a staffordshire bull terrier ct-guided brain biopsy using a modified pelorus mark iii stereotactic system: experience with 50 dogs primary canine and feline nervous system tumors: intraoperative diagnosis using the smear technique squash-prep cytology in the diagnosis of canine and feline nervous system lesions: a study of 42 cases canine and feline cytology-e-book: a color atlas and interpretation guide tumors in domestic animals what is your diagnosis? intracranial mass in a dog a true "triphasic" pattern: thoracolumbar spinal tumor in a young dog key: cord-017866-h5ttoo0z authors: bowman, grant r.; cowan, andrew t.; turkewitz, aaron p. title: biogenesis of dense-core secretory granules date: 2010-05-27 journal: trafficking inside cells doi: 10.1007/978-0-387-93877-6_10 sha: doc_id: 17866 cord_uid: h5ttoo0z dense core granules (dcgs) are vesicular organelles derived from outbound traffic through the eukaryotic secretory pathway. as dcgs are formed, the secretory pathway can also give rise to other types of vesicles, such as those bound for endosomes, lysosomes, and the cell surface. dcgs differ from these other vesicular carriers in both content and function, storing highly concentrated cores’ of condensed cargo in vesicles that are stably maintained within the cell until a specific extracellular stimulus causes their fusion with the plasma membrane. these unique features are imparted by the activities of membrane and lumenal proteins that are specifically delivered to the vesicles during synthesis. this chapter will describe the dcg biogenesis pathway, beginning with the sorting of dcg proteins from proteins that are destined for other types of vesicle carriers. in the trans-golgi network (tgn), sorting occurs as dcg proteins aggregate, causing physical separation from non-dcg proteins. recent work addresses the nature of interactions that produce these aggregates, as well as potentially important interactions with membranes and membrane proteins. dcg proteins are released from the tgn in vesicles called immature secretory granules (isgs). the mechanism of isg formation is largely unclear but is not believed to rely on the assembly of vesicle coats like those observed in other secretory pathways. the required cytosolic factors are now beginning to be identified using in vitro systems with purified cellular components. isg transformation into a mature fusion-competent, stimulus-dependent dcg occurs as endoproteolytic processing of many dcg proteins causes continued condensation of the lumenal contents. at the same time, proteins that fail to be incorporated into the condensing core are removed by a coat-mediated budding mechanism, which also serves to remove excess membrane and membrane proteins from the maturing vesicle. this chapter will summarize the work leading to our current view of granule synthesis, and will discuss questions that need to be addressed in order to gain a more complete understanding of the pathway. in eukaryotes, newly-synthesized proteins destined for secretion are first transferred from the cytoplasm to the lumen ofthe endoplasmic reticulum, and then progress through the golgi apparatus to the trans-golgi network (tgn). at the tgn, the choice of secretory pathways broadens. one route, which appears to be present in all cells, is constitutive in the sense that secretion does not depend on extracellular signals. such secretion involves the budding of vesicles or tubular elements from the tgn and their subsequent transport to and fusion with the plasma membrane, and is essential for cell growth since, among other functions, it provides new material to expand the cell surface. 1 in addition to a constitutive route , many cells maintain a secretory mode that is adapted for the tight coupling of protein release to extracellular stimuli. for such regulated exocytosis, the vesicles that carry newly-synthesized protein from the tgn accumulate in the cytoplasm until specific extracellular events trigger their fusion with the plasma membrane, resulting in the release of vesicle contents.f the vesicles involved are called dense-core granules (dcgs), the name reflecting the fact that the contents are so highly condensed that they form a large electron-dense plug in the vesicle lumen. a large amount ofprotein, as well as other molecular cargo, is thus efficiently stored in vesicular reservoirs and later released on demand. this pathway therefore permits larger and more rapid secretory responses than can be generated via constitutive secretion. classical dcgs in endocrine, exocrine and neuroendocrine cells are responsible for storage of a wide array of signaling molecules (e.g., peptide hormones) and secreted enzymes, and related vesiclesare found in metazoan cells of other lineages as well as in numerous unicellular organisms. the secreted proteins and macromolecules playa vast range of functions, from tissue coordination in metazoans to cyst formation in protists . regulated secretion also depends upon mechanisms for controlling the timely release of dcg contents, and this is accomplished by regulating the fusion of the vesicle membrane with the plasma membrane. much of the progress in understanding the mechanisms that mediate this step has been preceded or aided by studies of synaptic vesicles (reviewed in ref 3) , which undergo regulated fusion with the plasma membrane, but differ from dcgs in their biogenesis and acquisition of contents. comparable work in dcg secretion has shown that many of the molecular components involved in regulating exoeytosis and achieving membrane fusion are shared by these two vesicle types. 2 in addition to proteins that appear to be specific for regulated fusion with the plasma membrane, the mechanisms include factors, such as snare s and rab proteins, which are members of families of proteins that are of central importance to vesicular trafficking at multiple stages in the eukaryotic secretory pathway. thus, regulated exocytosis appears to be accomplished by the coupling of a regulatory mechanism to a universal core of membrane trafficking machinery. although many of the protein components have been identified, and a more complete understanding of the process remains an important goal for ongoing research. the mechanistic studies of regulated membrane fusion are too extensive to be included in this chapter, but have been covered in many reviews,4-8 and above. figure1.at least3 pathways diverge at the tgn in neuronal, endocrineand exocrinecells. a) a subsetof proteinsare destinedfor densecoresecretorygranules for release via regulatedexocytosis, these are found asaggregates in distendedareasofthe cisterna. b)proteinsdestinedforconstitutivesecretionaretransported via vesicles or tubules. c) proteinsdestinedfor lysosomes are concentrated,via the mannose-6-phosphate receptor, into clathrin coated pits and vesicles. darkly-shaded squaresand circlesrepresentproteins that tend to coaggregate under tgn conditions, and that are subsequentlystored in dcgs. lightly-shaded formsrepresentproteinsthat primarilyexitthetgn viaother pathways.arrowheads representproteinsthat areligandsforthe mannose-6-phosphatereceptor. crescents representproteinsthat arefound in a relatively evendistribution throughout the lumen. the extent to whichsomeconstitutively secretedproteinsmaybe concentrated within specific regionsof individualcisternaeis not incorporated into this model. this chapter will instead focus on a part of the pathway that precedes regulated exocytosis, namely the synthesis steps that lead to the formation of dcgs, beginning at the tgn. the tgn is a complex compartment that gives rise not only to the regulated and constitutively released classes of secretory vesicles but also to vesicles that carry hydrolytic enzymes to lysesomes. 9 -11 therefore, we seek an understanding ofthe signals that guide outbound proteins in a 3-way tgn sorting problem (fig. 1) . dcg protein sorting also continues in a post-tgn compartment, where additional factors come into play. this pathway has been the subject of numerous valuable reviews,12-19 with a particularly thorough treatment by arvan and cascle. 20 the anatomy ofdcg formation a number of important insights into the pathway of dcg formation have come from electron microscopy, providing a context for molecular and genetic studies. first , the fact that dcgs appear dense implies the existence of mechanisms to drive a degree of macromolecular aggregation that is unusual within the secretory pathway. many lines of research have led to the conclusion that protein sorting and concentration are intimately linked in this pathway, both relating to the self-aggregating tendency of dcg proteins that will be discussed below. rambourg and colleagues have investigated the localization of protein aggre:fiates, using serial thin sections to reconstruct the golgi apparatus during granule formation. -27 in cells producing mucous-containing dcgs, the cis and medial golgi appear as flat cisternae, and secretory proteins are evenly distributed in their lumina. in contrast, cisterna in trans regions are marked by multiple perforations and are dilated in regions that accumulate aggregates of secretory material. those dilations grow progressively larger in the more distal regions, while the nondilated portions take on a tubular appearance. at the trans-most cisterna, the dilated regions with their concentrated secretory cargo appear to exist as independent bodies, separate from a residual network oftubular membranes. several points were established or reinforced by these images. the first is that the visible concentration of dcg proteins begins within golgi cisternae . a second point is that the tgn, the vesicle donor, appears to be undergoing large-scale changes itself. the images also indicate that the vesicles do not bud conventionally in the manner that is well-established for coat (e.g., clarhrinl-mediared steps, since no coats are seen. electron microscopy also suggested that the aggregates undergo progressive changes, and are therefore likelyto be dynamic in nature. in pancreatic cellsthat are synthesizing insulin-storage granules, the proteinaceous cores seen in golgi dilations appeared less dense than the cores of insulin granules in the cell cyroplasm. 28 since the latter are derived from the former, this implied that proteins reorganize during an organellar maturation process. an important conclusion is that dcg formation should be considered as a multi-step process that plays out in sequential compartments. an early phase occurs at the trans face ofthe golgi and results in the production of vesicles bearing concentrated secretory proteins. these are called immature secretory granules (isgs).29subsequently those vesiclesare remodeled, as reflected morphologically by cargo condensation and biochemically by changes in protein composition, to become mature dcgs. 30. 31 for simplicity, we will refer to the first process as budding, and the second as maturation. central issues to be considered in this chapter are the mechanisms responsible for protein sorting during those successivesteps. again for simplicity, we will largely confine our discussion to the dcgs found in neuronal and endocrine cells. many of the same mechanisms are likely to apply to other classes of dcgs, among which are those in hematopoetic cells; for example, see references 32 and 33. genetics frequently offers a natural complement to morphological studies for developing an overview ofa pathway. unfortunately, a weakness in current approaches to analyzing dcg biogenesis is the absence of developed genetic models, although several systems show promise. no human diseases are known to stem from an inability to synthesize neuroendocrine dcgs. presumably, strong defects in dcg formation would result in embryonic lethality in a complex multicellular organism, since such a defecr would preclude regulated secretion of many peptides involved in tissue coordination. however, this has not prevented the generation of regulated exocytosis mutants in more simple systems, such as drosophila. in flies, the null phenotype of a gene called dcaps (calcium-activated protein for secretion), is embyronic lethal, but analysis of the larva has shown that the gene product is necessary for dcg exocytosis,34 as predicted from earlier work in mammalian chromaffin cells. 35. 36 although mutations affecting earlier stages in the pathway (i.e., dcg synthesis) have not been characterized in this organism, the characterization of caps mutants in this system provides hope that the earlier steps will be accessible by further mutational analysis. c elegans offers another potentially useful system for the genetic analysis of dcg synthesis, and several mutations affecting regulated exocytosis have been identified in this organism (reviewed in ref. 37) . currently, the only examples of dcg synthesis mutants are found in single-cell systems: the unicellular ciliates tetrahymena thermophila and paramecium tetraurelia, in which the mutations were chemically induced, and spontaneously-arising clones of the rat pheochromocytoma line pci2. [38] [39] [40] [41] [42] [43] [44] [45] [46] the viability ofthese mutants substantiates the idea that regulated exocytosis, unlike constitutive secretion, is not involved in basal cell growth . that is, dcgs are essential for organismal survival in metazoans, but not for individual cell viability. in the pcl2 lines, some mutations appear to disrupt the transcription ofnumerous granule protein genes. 45, 46 in the ciliate mutants, which appear to be due to single recessivealleles, the cargo genes are still expressed though no granules are synthesized. in one tetrahymena line, normal granule cargo appears to be shunted to the constitutive secretory pathway.47this phenotype indicates that dcg cargo proteins are not sufficient to direct granule formation, a result which was particularly interesting in the context of experiments in which mammalian dcg cargo proteins were expressed in tissue culture cells that do not normally make dcgs. 48 -51 such cells make vesicles with dense cores, presumably because cargo proteins expressed in nonspecialized cells can induce the formation oftheir own carriers from the tgn. these results implied that the capacity to make dcgs was inherent in the basic organization of the golgi/tgn since it could also occur in such nonspecialized cells. since this capacity appears to have been lost in the tetrahymena mutant, the defect in that line may point to an aspect of goigi/tgn function that is critical for regulated but not constitutive secretion. the full relevance of the ciliate or pc12 cell mutants to dcg biosynthesis will only be known when the mutations themselves havebeen identified. such geneticapproaches provide an unbiasedmethod for the identification of novel genes, and mayprovecritical in broadening our understanding of the granule synthesis pathway. although many of the dcg cargoproteins themselves have been cloned and characterized, much less is known about the mechanismsthat control protein sorting and condensation. geneticsystems may help to identifythe regulatory factors that are involved in theseprocesses. proteinsortingtakes place in thetgn and duringmaturation. in eachcase, a single compartment gives rise to multiple pathways, and the challenge is in understanding how dcg proteins, both in the lumen and the membrane, are cosorted from a larger cohort that includes proteins destined for other pathways. the relevant contributions oftgn vs, isg sortingarelikely to be cell-type specific and aregenerally difficult to quantify experimentally. however, the mechanisms for controlling sorting at both stages may be fundamentally similar. in particular, the considerationsthat arise from protein aggregation are relevant for both compartments. a long-standing issue is whether the primary mode of dcg protein sorting is active or passive. the model of active sorting was initially inspired by the paradigmof sorting to lysosornes, in which sorting derives from recognition of a set of soluble lumenal proteins by a transmembranereceptor. extendingthis to dcg biogenesis, the modelpositedthat a subsetof proteinshavepositive sorting signals for inclusionin isgs. 52 • 53 in this scheme, proteinsin the tgn lumen that lack targetingsignals are presumedto follow an alternative, default pathway of constitutive secretion. this model has been called "sorting for entry" (fig. la) . an alternative model posits that newly synthesized proteins can be targeted to isgs by default , even in the absence of specific targeting signals, if the flux of bulk membrane traffic toward isgs is greater than that to constitutive or lysosomal carriers. this may indeed be the case for cells that are highly committed to regulated exoeytosis. 54 ,55 in this case, the major sorting events occur in the isg, which becomes a functional extension of the tgn. proteins that are retained as isgs undergo maturation end up as the contents of mature granules. nongranule proteins can be selectivelywithdrawn from isgs during this period, and this model is termed "sorting by retention" (fig. 2b) . in evaluating either model, the sorting of dcg proteins cannot be considered in precisely the same terms that apply in other pathways, because the tendency of such proteins to self-aggregate facilitates a unique mode of targeting. among other things, it allows a large group of proteins to be sorted together in a single step. one implication is that sorting receptors, if present, could presumably function at concentrations that are dramatically sub-stoichiometric to their dcg protein ligands. furthermore, such receptors would only have to recognize some subset ofdcg proteins , since the remainder could be sorted indirectly via aggregation. in fact, no receptor has ever been unambiguously identified in this pathway. this does not by itself eliminate a "sorting for entry" model, because a second unusual feature of many dcg proteins is a tendency to bind to membranes. this has implications for sorting that will be discussed in a later section. many isolated dcg proteins will self-associate under in vitro conditions believed to approximate the tgn; namely, a slightly acidic flh and high calcium concentration relative to earlier compartments in the secretory pathway. 56 57,58 this can serve as a mechanism for sorting because it is selective: proteins that are constitutively secreted tend to remain soluble under conditions that promote dcg protein aggregation. this first sorting step can therefore be imagined as the evolutionary version of ammonium sulfate precipitation, with the collective behavior based on the proteins' individual biophysical properties, for example their surface charge. while the ability of individual proteins to aggregate is variable,59 mixtures of proteins may show cooperativiry in vitro, thereby increasing the efficiency of the step (fig. 3a) . 60 efficient protein aggregation might be expected to show concentration-dependence, and indeed isolated dcg proteins only self-associate above a threshold concentration.57 this in turn suggests that minor constituents of dcgs may depend for their efficient sorting on coassociation with more abundant species, whose concentrations must be sufficiently high to drive their independent self-aggregation. the sorting efficiency of individual proteins can be experimentally measured as the fraction that is stored in dcgs as opposed to being mistargeted to the constitutive pathway. asexpected from coassociation models, the sorting efficiency of a protein may vary widely between different cell lines. one would also predict that the sorting efficiency ofa protein could be boosted by increasing the expression levelof other proteins with which it coaggregates, particularly those which are most abundant. chiefamong the abundant metazoan dcg proteins are the chromogranin/secretogranins, a group of proteins with shared physical characteristics despite their very limited sequence similariry.61,62 indeed, the overexpression ofchromogranin b (cgb) in the att-20 neuroendocrine cell line increased the sorting efficiency of a second dcg protein, pro-opiomelanocortin (pomc). 63 nonetheless , it is inherently difficult to test the proposition that self-or coaggregation is a primary sorting determinant using conventional structurefunction analysis, since aggregation is thought to be directed by gross biophysical properties of dcg proteins , and there are no clear "aggregation signals" at the amino acid sequence level. however, recent studies have shown that sorting efficiency can be increased by providing an artificial aggregation signal. heterologous expression of a 6his-tagged secretory protein enhanced the aggregation and dcg storage, in a calcium-dependent fashion, of cga.64,65the authors speculate that the tag functions as an "aggregation chaperone" by providing a local site for the binding of divalent cations, thereby nucleating the aggregation process. curiously stably incorporated into the aggregates, suggesting that dcg proteins in their aggregated form interact more strongly with other dcg proteins than with the his tagged peptide. whether endogenous proteins have similar nucleation-promoting properties remains to be determined. identifying the role of any single protein or protein domain in dcg sorting is complicated by the high degree ofcooperativity that is hypothesized to exist within dcg protein aggregates. colo mer et al took advantage of the observation that two exocrine dcg proteins, amylase and gp2, do not coaggregate with neuroendocrine dcg proteins in solution,66 to study the sorting of dcg proteins the absence of coaggregation. when expressed in the neuroendocrine cells, the exocrine proteins were not stored in dcgs but instead secreted constitutively. 67 in similar experiments, an endothelial dcg protein, von willebrand factor, was expressed in neuroendocrine att20 cells. 49 this resulted, however, not in the constitutive secretion ofvon willebrand factor but instead in the formation of two morphologically-distinct classes of granules. one contained endogenous chromogranins, while the other contained von willebrand factor. a possibility is that two sets ofproteins aggregate independently in the tgn, which could be determined by a number of factors. for example, the two sets could precipitate at relatively distinct ph and/or calcium concentrations and thus be spatially or temporally separated. specific aggregate formation can also arise from conventional protein-protein interactions. in pituitary and pancreatic islet cells, for example, efficient sorting ofcga to dcgs depends on its association with secretogranin iii, and an essential targeting sequence in cga has been determined by gene truncation. 68 cga sorting in pc12 cells also depends on a specific sequence in the protein, which overlaps with, but is not identical to, that region which is required in pituitary cells. 69 this difference suggests that cga may be interacting with a different partner in pc12 cells, and indeed these cells do not express secretogranin iii. one possibility is that different surfaces of a cga domain can interact specifically with a range of partners, like a good host at a cocktail party. in summary, the data indicate that the aggregation of a particular protein depends on a number offactors, including its attraction to other potential binding partners within the aggregate, and the physiologic qualities of the lumenal environment, such as ph and calcium concentration, which affect the strengths of those interactions. the expression of proteins that are differentially sensitive to lumenal conditions or that form exclusive sets of protein-protein interactions can potentially result in the formation of multiple distinct aggregates in the same tgn compartment, each comprised of different proteins . these mechanisms could underlie the natural ability ofsome cell types to produce more than one classofdcgs, as is observed in aplysia bag cell neurons, bovine pituitary cells, as well as some protozoa. 7o-n though the model of sorring-by-aggregation is well established, the actual nature of the molecular interactions within such aggregates is difficult to define. the process of aggregation must be reversible so that the contents can be released into solution following exocyrosis, and moreover, it must be dynamic enough to permit the reorganization oftheir substituents during maturation.v' the latter isparticularly clear in pancreatic~-cells, in which the insulin-containing dcgs exhibit a crystalline ultrastructure, observed by electron microscopy, that is not found in isgs. in comparison to the production of insulin crystals, which involves the assembly of a single protein, the formation of dcg ultrastructure in protozoa may be significantly more complex. in these cells, the lumen ofmature dcgs is filled by a crystalline core that consists of multiple varieties ofproteins,?4,75 indeed , the localization of different proteins within the cores of paramecium dcgs has revealed that the crystals contain at least two distinct layers, each with a different set of protein componenrs. f" images ofisgs reveal that the components of the two layers are interspersed in this compartment, indicating that the layersare formed during a subsequent reorganization phase. thus, there is a significant amount of reorganization that must occur during crystal assembly. overall, the term "aggregation" may be misleading insofar as it suggests a phenomenon based on "stickiness", as for example for misfolded proteins in the endoplasmic reticulum. 77 instead, the interactions that occur between individual proteins in an aggregate may be transient and weak, stimulating formation of aggregates in the tgn due to stabilizing effects provided by multivalent interactions while also allowing for reorganization of the proteins during crystallization, as in figure 3 . some of the nonspecific, low-affinity interactions that occur in aggregates are likely to be mediated by the effects of calcium and ph in charge neutralization, leading to intermolecular interactions of acidic proteins by coordinate association with calcium ions (fig. 3a) . it is noteworthy that the chromogranins/secretogranins contain a preponderance of acidic amino acids, which endow these proteins with the capacity to bind large numbers of calcium ions with low affinity.6! acidic calcium-binding proteins also form the core of some lrotist dcgs, though they show little overall sequence homology with mammalian proreins.f . 79 an attractive explanation for the similarities is that they reflect a common aggregation-based dcg synthesis mechanism between protozoa and multicellular organisms, and that the amino acid sequences have evolved under similar constraints. following dcg synthesis, the regulated secretion of dcg cargo proteins is dependent on mechanisms that bring the vesicles to the cell surface and control their fusion with the plasma membrane. these activities are dependent on the activity of dcg membrane proteins, for example those that interact with cytoskeleton-based motors for intracellular transpon 80 and those that mediate regulated exocytosis.f it follows that the aggregation ofcore proteins during dcg synthesis cannot by itself be sufficient to form functional dcgs, and that there must be specific, though not necessarily direct, interactions between the lumenal proteins and the membrane constituents in order to ensure efficient sorting of these proteins to the same vesicles. these interactions have been difficult to detect, although some possible examples are discussed in a later section. what is clear, however, is that many lumenal proteins can themselves associate with membranes in unconventional ways. however, the nature and the functional significance of those associations are largely unsettled. five to ten percent of cgb adheres tightly, in a calcium and ph sensitive manner, to mernbranes. 8! whether this fraction is in dynamic equilibrium with the remaining~90% is not known, but there is no known chemical difference between the two cohorts. the membrane binding ofcgb is associated with an n-terminal domain defined by a disulfide-anchored loop, which is sufficient to confer membrane association when linked to an otherwise soluble protein. 82 importantly, the chimeric protein was sorted to dcgs in spite of the fact that it did not appear to aggregate, suggesting that the n-terminal domain constitutes an independent targeting signal. that same domain may promote homodimerization at neutral~h, implying that it may mediate different interactions in sequential secretory compartments. 3 cgb, as discussedearlier,also showsa strong tendency to aggregatein a controlled fashion. the coexistence in a single protein of domains that facilitate both protein-membrane binding and homo-or heterotypic protein-protein aggregation, offers the potential to generate cooperative networks with physiologically-useful properties (fig. 3b) . first, the total concentration of dcg proteins needed to reach the aggregation threshold in the tgn may be reduced for any proteins that interact with the membrane, since the local concentration may be increased depending on local membrane geometry. secondly, the avidity of a cgb aggregate for the membrane will be greater than that ofa monomer, since multiple n-terminal domains are availablefor independent membrane binding. validation of this came from an extension of the experiments with cgb chimeras outlined above. while a single n-terminal cgb domain was able to direct sorting to dcgs, efficient sorting only occurred when two such domains were present.82 this suggests that the membrane affinity of a single domain may be only marginally sufficient, but is more than adequate if two or more such domains are linked, as would be the case in a cgb aggregate. in a nonconventional sense, cgb could be considered as a dcg sorting receptor: a membrane-associated protein that is itself targeted to dcgs, and that can potentially cotranspon any proteins with which it associates. a similar argument has been made for the enzyme carboxypeptidase e (cpe), which is targeted to dcgs by a c-terminal amphipathic alpha helical domain. 84 • 85 in addition to acting as an enzyme to modify dcg cargo, cpe can also bind a subset ofdcg proteins , for example the hormone precursor pro-opiomelanocortin (pomc). 86 the cpe recognition site involved is different from the enzymatic cleft,87 and binding may be important for efficient sorting ofpomc, a conclusion based on experiments with cpe knockout mice and from cpe -deficient cell lines.86.88 cpe has been called a receptor for pomc and perhaps for other cargo proteins, though use of the term "receptor" has remained contentious since cpe can also aggregate with pome, chromogranins, and other cargo proteins in a conventional ca 2 + and ph-dependent fashion. 89,9o membrane association of cgb and cpe may be a property that has arisen convergently in these proteins, albeit by different mechanisms, reflecting the importance of this activity in dcg cargo sorting. an n-terminal disulfide bonded loop such as that found in cgb is found in several dcg proteins, including pomc and chromogranin a (cga), though the homology does not extend beyond the structural level,84 and evidence to date suggests that its role in sorting may be protein-specific. as in cgb , n -terminal disulfide loop domain in pomc is both necessary and sufficient for sorting, but sur~risingly,it appears to interact with the membrane indirectly, through interaction with cpe. 4 the disulfide loop in chromogranin a was not necessary for the sorting of this protein in pcl2 cells,69 and instead an interior domain is essential for sorting in these cells,via interaction with membrane-associated secretogranin 111. 68 • 91 these studies find no evidence for a conserved dcg targeting signal, but they do indicate that specific protein-protein interactions can be important for efficient sorting oflumenal cargo. precisely how cgb, cpe, secretogranin iii, and other ostensibly soluble lumenal proteins associate with membranes is not resolved. there is some evidence that they associate preferentially with cholesterol-rich membranes, so-called lipid rafts. 91 . 92 consistent with this, depletion of cholesterol from tissue culture cells decreased the sorting efficiency of both cpe and cgb, though it is difficult to distinguish direct from indirect effects in such experiments. 19. 93 in addition, because both constitutive and regulated secretion were inhibited by cholesterol withdrawal, the results do not demonstrate a specific role for cholesterol in dcg formation. the experimental limitations notwithstanding, these data suggest that the association of cpe and cgb with specific membrane sub-domains could be an important aspect ofsorting. iflipid rafts are indeed involved in this pathway, it could add another level of complexity to the cooperative mechanisms that may perrain (fig. 4) . interestingly, cgb is also differentially sorted between the apical and basolateral pathways in polarized epithelial cells, which do not make figure4. selective association ofocg proteinswithlipidraftsin thetgn. implications ofsuchassociation includethe following possibilities: 1. independentassociation ofproteinswith a singleraftwouldpromote protein-protein aggregation . 2. protein aggtegates could stabilize rafts with which they associate. large aggregates couldleadto formationofextensive rafis, in principle, thisprocess couldbesufficient to generate ocgs with a highly biased lipid composition, which is indeed observed. l9l the thickened, patterned regions of the cisternal membrane representputativelipid subdomains. dcgs, and this also requires signals within the n-terminal domain. 94 this may suggest a similarity in sorting mechanisms used in epithelial and regulated secretory cells. a final complication in dissecting dcg sorting signals is that the requirement for the disulfide loop in cgb depends on cell type. disulfide bond reduction led to the constitutive secretion of newly synthesized cgb in pcl2 cells. 95 as expected, this treatment did not affect the sorting of secretogranin ii, a protein that undergoes aggregation but does not contain cysteine residues. however, in gh4ci cells, the same treatment did not perturb the sorting of cgb. 96 similarly, cga soning also appears to exhibit cell~e specificity: a c-terminal truncation was correctly sorted in pcl2 but not in gh4ci cells, and an n-terminal region, which does not contain a disulfide loop, was important for sorting in pcl2 cells. 69 thus, the sorting requirements for cga, cgb, and granule proteins more generally, may depend on the cell type, specificallybecause the efficiency of any protein's sorting will depend on the available interacting parmers . in some cases, a protein's interacting parmer could be a membrane raft, whereas in other cases, the same protein may be delivered to dcgs by virtue of its ability to aggregate with other lumenal cargo proteins. our current understanding of signals involved in dcg membrane protein targeting is relatively primitive. in principle, membrane proteins could be targeted by signals in their lumenal, transmembrane and/or cytoplasmic domains; however the characterization ofsuch signals has not been straightforward. a significant obstacle has been the fact that relatively few membrane proteins have been identified that are exclusivelylocalized to granules. 20 phogrin (phosphatase homolog in granules of insulinoma) localizes to dcgs in a range of neuronal and endocrine tissues. 98 it is a transmembrane protein with an n-terminallumenal domain and c-terminal cytoplasmic domain, and is synthesized with a large n-terminal proregion that is later cleaved within isgs. either the pro-domain or the lumenal domain of the processed protein can be independently stored in dcgs, indicating that each contain signals sufficient for rargeting. 99 one possibility is that these, and by implication the full length phogrin as well, can be sorted by associating with the condensing core of granule cargo in the tgn. this may also be true for two dcg membrane proteins of the anterior pituitary and adrenal medulla, peptidylglycine a-amidating monooxygenase (pam) and dopaminẽ -hydroxylase.60in these cases, there is physiological evidence that the lumenal domains can sort independently of the transmembrane or cytosolic domains, since both the soluble forms and the transmembrane forms occur naturally in dcgs. loo nonetheless, efficient storage of the transmembrane form of pam also requires signals within the cytoplasmic tail101 the idea that sorting of transmembrane proteins in dcg involves cytosolic signals is also supported by analyses ofvamp2, a widely distributed dcg v-snare,4 and p-selectin, a protein of platelets and endothelial cells. 102 the sorting ofvamp2 to insulin-containing dcgs is impaired by a point mutation in the cytosolic portion of the protein, and the expression of this incorrectly sorted mutant protein is unable to support regulated exocytosis in the absence of wildrype vamp2. 103 analysis ofp-selectin targeting is complicated by the fact that it can be found in more than one intracellular compartment, suggesting that it contains hierarchical targeting signals. 104 in addition, the dcgs of platelets and endothelial cells share some properties with lysosomes, and mechanisms involved in their biogenesis may differ from those in neuronal and endocrine cells.102.105·107 nonetheless, p-selectin expressed heterologously in the neuroendocrine cell line att-20 was targeted to dcgs, and this depended on a tyrosine-containing motif in the cytoplasmic domain.l08. 109 the same motif is important in the endogenous endothelial cell context, indicating that the rargeting mechanisms may be similar. the tyrosine-based motif suggests that this protein can interact with a coat-associated adaptor, and indeed a functional role for ap-3 in the sorting ofp-selectin to dcgs has been suggested,llo but no ident ified coats are involved in the formation ofisgs in the tgn. one possibility is that conventional adaptor/coat-mediated sorting of p-selectin occurs at a step distinct from the known budding and maturation steps in dcg bio~enesis; a second is that adaptors may have noncanonical roles unrelated to coat recruitment.i i the studies ofp-selectin have revealed clear evidence that a cytosolic signal can be important for the sorting of transmembrane proteins to dcgs. further analysis of the targeting mechanism will likely be an important topic in future research, as it represents an activity that is topologically distinct from the relatively well characterized aggregation-based sorting events in the lumen. in an intriguing set of experiments, cutler and colleagues found evidence that the function ofthis cytosolic sorting determinant can be coupled to the expression ofa lumenal dcg protein. when the lumenal dcg protein von willebrand factor (vwf) was coexpressed with p-selectin in neuroendocrine att-20 cells, the vwf was stored in vesicles that were distinct from dcgs containing endogenously-expressed cgb, 1l2,1l3 a finding that is consistent with previous results.i14the novel and intriguing finding was that p-selectin was preferentially targeted to the vwf-containing vesicles, indicating that vwf and pvselectin, which are normally expressed in platelet and endothelial cells, could be cosorred in a cell type in which they are heterologously expressed (fig. 5) . there was no indication, however, of a direct interaction between the two proteins, and the sorting ofp-selectin in this context was instead dependent on the same tyrosine-containing cytoplasmic motif that had previously been shown to be necessary for targeting to dcgs. the targeting of one class of membrane proteins, those linked via a gpi-anchor, cannot depend on cytosolic signals, since anchors of this type do not penetrate the cytoplasmic membrane leaflet.i15 for gp-2, the major membrane protein of zymogen granules in pancreatic acinar cells, sorting may occur via a coaggregation mechanism. its lumenal domain has been found to associate with a lectin (zg 16p), sulphated matrix proteoglycans, and syncollin, the last a lumenal protein that may itself interact with the membraney6 these proteins have been postulated to form a membrane-associated matrix that could serve as a sorting intermediary between the membrane and the zymogen core contents. 1l7 this is a variation on the model described for cgb and cpe as sorting receptors , and suggests by analogy that gp-2 or syncollin might serve as the membrane anchor for the zymogen core. however, dcg assembly is normal in the absence of either protein, indicating that neither is playing a unique role in that regardys,1l9 in summary, the relatively limited evidence to date suggests that a mechanism similar to that involved in cargo protein condensation is involved in sorting of some, but not all membrane proteins with lumenal dcg contents. in principle, indirect interactions between membrane and core proteins may be equally important in the cosorting of membrane and lumenal cargo. iflumenal proteins like cpe preferentially insert into membrane sub-domains based on their lipid composition, then any membrane proteins that independently partition into the same sub-domains would be cosorted. in support of this hypothesis, recent studies have suggested that prohormone convertases 1 and 2, which are responsible for proteolytic cleavageof lumenal proteins during granule maturation, are sorted to isgs by virtue of c-terminal membrane raft-associated tails, which are by themselves necessary and sufficient for targeting to dcgs,uo 120 additionally, cytoplasmic signals on some transmembrane proteins appear to play important roles in sorting, but the mechanisms are unknown. the canonical mechanism of vesicle budding, as for example that involved in the emergence oflysosome-bound carriers from the tgn, involves transmembrane receptors, adaptors , and coat proteins . since there is no evidence that transmembrane proteins or coat proteins are relevant in isg budding, other mechanisms are likely to apply. there has been some progress in reconstituting this process using cell-free systems, though the field has generally suffered from a lack of in vivo models, for example a well developed genetic system with mutations that affect this step in the pathway. the general approach has been to start with labeled dcg protein in the tgn of permeabilized cells or in golgi-enr iched fractions, then measure the transfer of the label from the relatively large and pelletable golgi membranes to nonpelletable vesicles,using medium speed centrifugation to separate the two pools. the appearance oflabel in smaller vesiclesis taken as an indication of cargo transfer to isgs via vesicle budding. since little is known about dcg biogenesis, it is important to note that "budding" as defined by this assay may include a large number of steps, including the establishment of golgi/tgn microdomains, and the release of previously budded, weakly associated vesicles. thus, the results of these experiments could depend upon on the nature of the starting material. in addition, it has not yet been rigorously demonstrated in any system that the released vesicles are bona fide isgs, for example by testing whether they are competent to fuse with their appropriate target membrane. the reconst ituted buddin~reactions utilize atp, as expected , and most but not all require a cytosol extract . 31 ,121-1 4 the small gtpase arf is required, although the targets for this regulatory protein are not yet clear. one potentially relevant arf target is phosfholipase d (pld) , the binding of which to the membrane can enhance isg buddingp pld converts phospharidyl choline to phosphatidic acid, perhaps thereby effecting a change in membrane curvature.126 this idea is appealing because, in the absence of coat proteins, the membrane curvature required for isg budding must be induced by other mechanisms.127in addition, the indirect products of pld activity may recruit additional effectors to the budding site, including the unconventional gtpase dynamin_2. 128 dynamin mediates membrane scission events, such as pinching off vesicle buds . however, pld does not stimulate budding in all reconstituted systems; the differences may reflect the variery of ways in which donor fractions are prepared. there is evidence that kinases and phosphatases, heterotrimeric g-proteins, and a phosphatidyl inositol transfer protein (pitp) are involved in isg budding, but the enzymatic substrates have not been established. [129] [130] [131] [132] [133] an important unanswered question is whether any of these activities, the majoriry of which are as yet unidentified at the molecular level, is specifically required for the formation ofdcgs and not other membrane carriers. pld, for example, has been implicated in tgn tubularion, but the downstream effectors, as for dcg budding, the reaction depends upon ap-l, which can be recruited by the cytoplasmic tails of furin , the mannose-o-phospharereceptor,and other membrane proteins. the mannose-6-phosphatereceptorcan in turn bind any soluble lysosomal enzymes in the isg lumen, so thesewillalsobewithdrawn in the budding vesicles. other soluble proteins may also be included based on random partitioning, but the aggregated deg proteins will be excluded.at the end of this process, the mature secretory granule is no longer a budding donor compartment, perhaps becauseit no longer contains membrane proteins that can recruit ap-l (representedby stars). are unknown. 134 the noncanonical gtpase dynamin has been implicated in dcg budding as well as in constitutive secretion. 128.135 one possibility is that these activities are only indirectly involved in dcg synthesis. according to the "sort ing by exclusion" model ( fig. 6a ), isgs are created by a passive process, as aggregation prevents dcg cargo from entering into outbound vesicles and tubules that bear lysosomal or constitutively secreted proteins. instead of being actively budded from the tgn, aggregated proteins would be enriched in a separate subset of relatively large membrane carriers, while non-dcg proteins are removed from the compartment by active coat-dependent processes . thus, the cyeosolic components identified as isg budding factors by in vitro reconstitution assays may really be parts of the mechanisms for other secretory pathways. according to this model, the so-called sorting receptors need only act as membrane tethers in associating the lumenal aggregates with membrane rafts. as there is no need to transport this material to a new compartment, the receptors do not recruit cytosolic coat proteins for vesicle budding, as the traditional membrane receptor proteins do in sorting proteins to other pathways. an alternative to the "sorting by exclusion" model proposes that isg budding is indeed an active process, and that the same mechanism is also involved in driving the budding and tubulation of constitutive secretory carriers from the tgn. although the two pathways give rise to vesicles of vastly different sizes, it is possible that the difference is caused by the cargo proteins (large aggregates versus soluble material) and is not a reflection of different cytosolic budding machinery. the formation ofconstitutive secretory carriers, like isg budding, differs from clathrin-dependent transport at the tgn in that is not associated with the appearance of vesicle coats. there are similarities between the budding of constitutive and regulated vesicles at the molecular level as well: in addition to rab proteins, constitutive traffic has been shown to rely on the activity of dynamin-2,128 protein kinase d,136and heterotrimeric g-proteins ,130 factors which may also be associated with isg budding (above). cholesterol depletion has been shown to inhibit both pathways,93 however it is difficult to know whether the treatment has a direct effect on both pathways, or whether the inhibition of one pathway could inhibit the second via some indirect mechanism. thorough testing ofthis model requires experiments that avoid this problem. the only concrete indication that there are dcg-specific budding factors is that, at least in one reconstituted system, the cytosol re~uirement cannot be substituted for by an extract from hela cells, which do not make dcgs. 1 7 one possibility that is compatible with both sorting models is that specific cytosolic proteins are involved in establishing or facilitating golgi subdomains in which dcg proteins condense. the structural and functional analysis of the tgn is at a very early stage, but the existence of sub-domains is consistent with the observed nonuniform protein distribution within a single cisterna, as well as with live imaging of heterogenous budding structures. 94 ,138 however, the cisternal dilations involved in isg budding do not necessarily reflect the active maintenance of sub-domains. a simpler view is that the cisterna are passively stretched around the forming granule protein cores, like the bulges in a pancake around blueberries. future work with in vitro systems may provide molecular identification ofactivities that are required for isg budding, but the question ofwhether cells have machinery that is specifically used for this purpose will need to be addressed by other types of analyses. if the budding mechanism is specific to isgs, and not indirectly required for isg production, as in the "sorting by exclusion" model, the prediction is that knocking out individual components would inhibit isg formation without also inhibiting the exit of lysosomal or constitutive proteins from the tgn. depending on cell type, the importance of isgs as a locus of protein sorting may be as important as that of the tgn. sorting at this level involves the budding of vesicles from isg membranes, resulting in the remodeling of membrane and lumenal contents by selective withdrawal (fig. 6b ). this targeted removal occurs via clathrin-coat recruitment to isgs occurs via the ap-1 adaptor, wh ich is recruited by membrane proteins in an arf-dependent, bfa-inhibitable step.139 proteins known to be withdrawn from isgs include the cargo protease furin and the mannose-6-phosphate receptor, both of which can interact directly with ap_l.140.143 the mannose-s-phosphate receptor can bind any lysosomal enzymes that may have been incorrectly sorted upon exit from the tgn, and this step therefore leads to selective withdrawal of some lumenal proteins by classical receptor-based sorting. 144 mature dcgs do not support ccv formation, the simplest explanation for which is that isgs become progressivelydepleted ofproteins that act in the recruitment ofap-1. consistent with this, myristoylated arf 1 binds to isgs but not mature granules in vitro. 139 recent evidence suggests that the full cohort of arfs and adaptors present on isgs includes arf1, 5 and 6, and ap-1 and _3. 145 these may all be present on a uniform population of vesicles, or may reflect heterogeneity within isgs. 143 coated vesicles budding from the isg will also withdraw any soluble proteins that randomly partition by diffusion into the vesicle lumen during budding. however, large aggregates of proteins that are condensing in the isg are too large to fit into the buds , and are therefore selectively retained. 146 the efficiency of this separation is increased by the tendency of trafficking imide cells: pathways, mechanisms and regulation nonaggregating proteins to be concentrated at the periphery of the vesicle lumen , as they are excluded from the dense core forming in the center ofthe isg. as a result, the soluble proteins accumulate in a place where they can readily enter the vesicles that are budding from the membrane. these may include proteins that randomly partition at the tgn into budding isgs, but will also include some soluble products of dcg proprotein processing. the best characterized of these is derived from proinsulin, which is processed into and a, b, and c peptides. 147 the first two are disulfide linked, and crystallize to form the granule core. the c peptide is soluble and is largely excluded from the core, and is selectivelywithdrawn. 148.149 a collateral consequence of isg maturation is the generation of a set of coated vesicles bearing newly synthesized proteins, some ofwhich have undergone processing by isg-specific enzymes. at least in some cell types, these can deliver their cargo to the plasma membrane, probably via an endosomal intermediate. 150 this has been called "constitutive-like" secretion: constitutive-like in that it is independent ofextracellular stimulation, but with kinetics that are slower than those of true constitutive secretion. in pancreatic~cells, the c peptide that is withdrawn from isgs is secreted via this route . the model that describes the progressive enrichment of granule cargo during isg maturation has been given the name "sorting by retention" and essentially posits that sorting in isgs can be based on a protein's ability to aggregate, rather than depending on specific targeting signals. the concepts are like those of the "sorting by exclusion" model that may apply at the tgn, and the similarity in models may be a reflection of similar molecular mechanisms in vivo. thus, isgs may simply be a functional extension of the tgn, which becomes progressivelyenriched in dcg contents as nonaggregating proteins are actively removed during maturation. thus, there may not be any mechanistic differences between coat mediated sorting at the tgn versus the isgs, though the material that is included in the budding vesicles could change as the compartment matures. alternatively, modification of the coat mediated sorting machinery may be required in order to facilitate sorting from a compartment that is progressively changing. for example, such modifications may be necessary for the trafficking of proteins that are allowed to enter isgs but are not stored in mature dcgs, such as proteases (see "structural maturation of isgs" section) , or for adapting to differences in membrane composition between the tgn and isgs. indirect evidence in support of this possibility comes from the study of the membrane lipid component phospharidyl inosirol-i-phosphare (pi-4-p) and its derivatives. in the tgn, these molecules play important modulating roles, including the recruitment of ap-l/clathrin coat proteins for vesicle budding. 151 the levels on pi-4-p in the tgn are affected by the activity of pi-4 kinase, which is stimulated by myristoylated arf1-gti~a part of the coat formation machinery.152,153 interestin~r: isgs have been found to contain a pi-4-k activity that is not stimulated by arfi-gtp' 1 the tgn has two different pi-4 kinases (ii and iii) , and it is possible that isgs only recruit one of these. 152.153 coat recruitment at the tgn vs. isgs may also be differentially regulated by modification of the vesicle cargo, since the binding of ap-l to the cytoplasmic tails of both furln and the mannose-6~hosphate receptor is stimulated following their phosphorylation by casein kinase ii.141 ,1 in this regard, a very interesting observation is that newly-budded isgs are rapidly transported to the cell periphery, at least in some cell types, and therefore primarily inhabit a different cellular microenvironment from the tgn. 155 this may be relevant for differential regulation of similar activities at the tgn vs. isgs, for example if receptors in isgs are selectively modified. although the data is not yet conclusive, the emerging view of sorting from isgs is that it is directed by the core elements of a "flexible" ap-l/clathrin dependent sorting mechanism that is differentially controlled at the isgs versus the tgn. the model holds that the sorting events ofisg maturation are not mediated by a unique vesicle trafficking mechanism, but are instead accomplished by pathway-specific modifications ofmachinery that is common to all cell rypes. a similar phenomenon may occur at an earlier stage ofthe pathway, where the coat-independent machinery that drives the formation ofconstitutive carriers from the tgn may be adapted for the budding of isgs, as discussed in the "mechanisms of immature secretory granule (isg) budding" section. this apparent mechanistic conservation may explain the abiliry offibroblast cells, which do not normally make dcgs, to make dense-cored vesicles when expressing heterologous chromogranin genes or vonwillebrand factor.48.50.51 however, these observations do not preclude the possibility that specialized dcg-producing cells express proteins that specifically modify parts of the conserved cellular trafficking machinery to enhance dcg synthesis. the cores of newly-budded isgs apftear less electron-opaque than those in mature dcgs, and are also lower in buoyant density, 0 indicating that granule cargo becomes increasingly condensed during granule maturation. this is one reflection of the larger remodeling of protein and lipid constituents during the maturation process, which includes the selective withdrawal of components that are present in im matu re, but not mature, granules . this overall process serves important structural functions. the tighter packing offers increasingly efficient storage, and not simply because more material can be contained in a fixed vesicle volume. protein condensation overcomes an energetic barrier that is posed by a vesicle filled with concentrated soluble macromolecules, which is hyperosmolar when compared to cytosol. maintaining such a vesiclewould require constant pumping ofosmolytes to counter vesicle swelling, an expensive cellular proposition. within dcgs, aggregated proteins are no longer solvated, and are therefore osmotically inert. the progressive condensation durin~maturation parallels, and is likely to be controlled by, changes in the lumenal environment. 1 in neuroendocrine cells, the tgn is acidified to ph 6.4 by vacuolar atpases.157these are also present in the isg membrane, with the result that the isg continues to acidify 158-160 at the same time there is an increase in calcium that , along with other cations,161 is important for charge neutralization of the largely acidic core proteins. this calcium may be cotransported from the endoplasmic reticulum with calcium-binding dcg cargo proteins, or imported via isg membrane ion exchangers. 162 the ionic changes can trigger changes in dcg protein conformations or interactions . for example, cgb forms horno-oligomers under the conditions found in isgs. 56 .163the functional significance is as yet unknown, but these are presumably based on contacts different from those involved in aggregative sorting. one well-established consequence ofisg acidification, in combination with increased ca 2 +, is the activation of proteases that are specifically localized to dcgs. the contents of neuronal and endocrine dcgs are largely synthesized as proteins that are proteolytically processed to generate bioactive peptides, the species that are eventually released during exocytosis. l64 proteolytic processing involves a variety of enzymes including amino-and carboxypeptidases, and a family ofaspartyl proteases called prohormone convertases. [165] [166] [167] [168] members of this family are differentially active over a range of proton and calcium concentrations, and may thus act sequentially on their substrates during isg maturation, in a cell type-dependent fashion [davidson, 1988 #572 ;laslop, 1998 #2270;goodge, 2000 #1981;.169 though isgs are considered to be the major compartment of proprotein processing, in some cell types processing may begin in the tgn, and moore and colleagues have begun to resolve the requirements for isg budding from those required for the onset of processing. 137.170.171 in their cell-free system, the onset of processing precedes budding. both require hydrolyzable gtp, but at two distinct concentrations . this difference suggested a model in which the former requires arf, while the latter depends upon a heterotrimeric g-protein. in addition to generating mature peptides, proprotein processing may drive the physical reorganization of the core, in cases where mature peptides can pack more tightly than the precursors . the best example of this is found in~-cell granules, in which mature insulin but not proinsulin can assemble into hexagonal crysrals, simply because processing relieves a packing constraint 147 . 172 ,173 (fig. 7) . the control of assembly via proteolytic processing is strongly reminiscent of mechanisms involved in viral capsid formation. 174 the process ofdcg maturation, which includes the generation of active peptides by proteolytic processing and the condensation of cargo into a densely packed, osmotically inert form, serves to increase the efficiency of the regulated secretory pathway in several ways. first, the condensation ofmaterial allows great quantities of protein to be stored in the vesicles,with the consequence that a small number ofexocytic events can generate a relativelylarge secretory response. second, proteolytic processing in isgs allows the cell to combine multiple dcg peptides into a single proprotein, thereby linking the sorting of these proteins at earlier stages of the pathway. in neuroendocrine isgs, for example, the chromogranin proteins are cleaved into multiple biologically active peptides with different postexocytic functions. 62 ,175 furthermore, limiting the site of proteolytic processing to isgs may provide a failsafe mechanism, ensuring that the active forms of the proteins are only found in a compartment that is under direct control of the regulated secretory pathway, therefore leaving any incorrectly sorted proteins as uncleaved precursors. the remodeling of the membrane attending the budding of clathrin coated vesicles does not simply serve to remove proteins that may have been incorrectly targeted at the tgn. rather, it also underlies differences in the activity ofisgs and mature granules. this was suggested by the observation that isgs and mature granules differ dramatically with regard to exocytosis: whereas mature dcgs undergo efficient exocytic fusion with the plasma membrane in a stimulus-dependent fashion , isgs exhibit an increased tendency to fuse with the plasma membrane in the absence of stimulation. in att20 cells, unregulated release ofdcgs from isgs proceeds for 2-3 hours after isg budding from the tgn. 176 these isgs contain two snares, vamp4 and synaptota~min iv (syt iv) , which are withdrawn during maturation in a brefeldin a-inhibitable step.i 9,171,177 during the same period , the maturing granules become responsive to exocytic stimuli, a process also blocked by bfa. that the two phenomena may be linked is suggested by the observation that overexpression ofsyt iv itself decreased the responsiveness of maturing granules to secretory stimuli. 17l syt iv is thought to act as a negative regulator of calcium-induced exocytosis,178 and the withdrawal of this inhibitory factor from isgs may foster maturation. a recent study showed that the removal ofvamp4 from isgs depends upon interactions with ap-l and the coat protein pacs-l,179 thereby providing genetic confirmation and molecular detail to this model. however, a complication ofthis model is that syt iv is thought to inhibit membrane fusion by forming inactive heterodimers with synaptotagmin i, and the mechanism by which the heterodimers are separated and syt n is selectively removed from the isgs is unknown. another functional characteristic that may, in some cell types, distinguish isgs from dcgs, is that isgs can undergo homotypic fusion, a reaction that has been more extensively characterized in vitro than in vivo. 177 ,180,181 the specific function ofthis reaction is not clear. in some systems homotypic-like fusion might allow for the synthesis ofspecialized dcg cores in which the contents are not randomly distributed. in pseudomicrotborax dubius, two kinds of isgs, containing morpholo §ically-distinguishable cargo, fuse during the process ofassembling a complex core structure. 18 more generally, consolidation could potentially define the size of the granules, which in many systems appear to be controlled. 183 disruption of the gene encoding rab3d, an exocrine granule-associated small gtpase, resulted in a doubling ofmature granule volume, and one possibility is that rab3d acts as a negative regulator of homotypic fusion. 184 at some level, membrane remodeling must account for the difference in the fusogenic behavior of isgs vs. mature granules, and attention has focused on the snares, due to their importance in regulating membrane fusion. isgs from pcl2 cells contain syntaxin 6, which must be present on both donor and acceptor membranes for efficient homotypic fusion in vitro.in syntaxin6 is also present in clathrin-coated vesicles which bud from the isg rnernbrane ,142 consistent with the idea that it is selectively removed during maturation via several likely ap-i binding sites in its cytoplasmic domain. 140 as isg maturation appears to involve the removal of specific factors via the budding of clathrin coated vesicles, it is possible that more thorough analyses of the target proteins and their interacting partners will help to uncover isg-specific machinery that regulates clathrin-dependenr sorting in this compartment. more broadly, the identification of the molecules that define the functional maturity ofdcgs by their presence or absence in the vesicle will provide insights into the nature of organelle identity, a topic that is central to an understanding the general principles of vesicular traffic. finally, recent evidence hints at aspects of granule maturation that have not previously been recognized. functional maturation of secretory granules may extend beyond the period of morphological change, based on the observation that the distribution and fusogenic activity of granules may change with vesicle age.185 the majority of the work on dcg synthesis has focussed on the sorting of the lumenal content proteins in the tgn and isgs. these studies have, for the most part, supported the nonspecific aggregation-based model for sorting that was proposed by chanat and huttner in 1991.57 not surprisingly, studies of many granule cargo proteins in multiple systems have revealedsome casesthat are possible exceptions to this general rule, where specificprotein-protein interactions are required for the sorting of a particular protein, as discussed in the "protein sorting into isgs" section of this chapter. overall, the precise requirements for the sorting of any particular protein is likely to be both context (which other granule cargo proteins are being expressed, and in what quantities) and cell type dependent (protein aggregation is sensitive to physiological properties of the lumen, such as calcium concentration and ph, which may vary between cell types), though it is likely that the general principles of aggregation-based sorting apply in all cells that produce dcgs. further analysis of the specific sorting requirements for individual proteins may lead to a greater knowledge of the details ofaggregation-based sorting, but the next leap forward in our understanding of they system will more likely come from experimental approaches that expand beyond the level of individual proteins and consider the dcg synthesis pathway more broadly. for example, cargo protein aggregation is known to be sensitive to lumenal calcium concentration and ph levels, but the mechanisms that control these physiologic parameters have not been elucidated. secondly, how are granule cargo proteins sorted to the same destination as other proteins that are essential for dcg function, such as membrane fusion machinery? the answers to these questions may be learned from studies in genetic systems, such as c. elegans, drosophila, and ciliated protozoans, which offer promising avenues for further experimentation. these orf.anisms have recently been used to identify elements of the regulated exocytosis machinery.' ,186,187 and similar studies could uncover genes that are involved in vesicle synthesis. another major gap in our understanding of the granule synthesis pathway is the extent of its functional relationship with other branches of the secretory pathway. two decades ago, dgc formation was considered to be one of a small number of distinct, post-tgn secretory pathways. this carried the assumption that vesicles bound for constitutive or regulated exocytosis, or toward lysosomes, would rely on distinct mechanisms for their biogenesis. that view now seems, paradoxically, to have been both too simple and too complex. it was too simple because post tgn traffic cannot be neatly divided into three branches: for example, what was called the constitutive pathway may in fact consist of multiple branches.188.189 this was initially established for apical vs. basolateral targeting in polarized epithelia, but there is evidence in other cell types as well. furthermore, the mechanisms for dcg formation are not easily separated from those that are directly involved in other pathways, implying that the secretory pathway cannot be divided into distinct, independently functioning branches. for example, ap-1 dependent sorting of proteins to the lysosomal pathway is associated with isg maturation, and may also be part of the driving force for the "sorting by exclusion" of dcg contents in the tgn (see "protein sorting in isgs" section). at the same time, the fact that the dcg synthesis pathway and lysosomal pathway use some of the same machinery argues that the historical view of distinct mechanisms was too complex. similarly, the historical view that constitutive and regulated secretory carriers are fundamentally different may also be incorrect. the idea that constitutive traffic is based on small vesiclesis being modified by the recognition that tgn tubularion may be as, if not more, important in this pathway, at least in some cell types (referencesin ref 190) . thus coat-mediated vesicle formation may be the exception rather than the rule for anterograde traffic to the plasma membrane, and the formation of constitutive and regulated secretory carriers may share common mechanisms . in the extreme, the mechanisms may be mostly conserved, and the end products depend upon the behavior of the vesicle cargo. addressing these issues directly will require identification of factors required for isg budding and tgn rubulation. while progress has recently been made toward the latter, details regarding the former are extremely limited. success in this may depend on further exploitation of cell-free systems, strengthened by development of new genetic models. secretion and cell-surface growth are blocked in a temperaturesensitive mutant of saccharomyces cerevisiae secretory granule exocytosis the synaptic vesicle cycle snares and snare regulators in membrane fusion and exocytosis calcium sensors in regulated exocytosis principles of exoeytosis and membrane fusion regulated exocytosis and snare function (review) the trans-most cisternae of the golgi complex: a compartment for sorting of secretory and plasma membrane 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the similar role of chromogranin a molecular sorting in the secretory pathway protein secretion: puzzling receptors proteins synthesized and secreted during rat pancreatic development phasic release of newly synthesized secretory proteins in the unstimulated rat exocrine pancreas ph-and ca2+-dependent aggregation properry of secretory vesicle matrix proteins and the potential role of chromogranins a and b in secretory vesicle biogenesis milieu-induced, selective aggregation of regulated secretory proteins in the trans-golgi network signal-mediated sorting to the regulated pathway of protein secretion 2+)-induced conformational change and aggregation of chromogranin b. comparison with chromogranin a and implication in secretory vesicle biogenesis secretory granule content proteins and the luminal domains of granule membrane prot eins aggregate in vitro at mildly acidic ph the granin (chromagranin/secretogranin) family the chromogranins: th eir roles in secretion from neuroendocrine cells and as markers for neuroendocrine neoplasia chro mogranin b (secretogranin i) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor gorr suo aggregation chaperones enhance aggregation and storage of secretory proteins in endocrine cells in vitro aggregation of the regulated secretory protein chromogranin a reconstitution in vitro of the ph-dependent aggregation of pancreatic zymogens en route to the secretory granule: implication of gp-2 exocrine granule specific packaging signals are present in the polypeptide moiery of the pancreatic granule membrane protein gp2 and in amylase: implications for protein targeting to secretory granules identification of a chromogranin a domain that mediates binding to secretogranin iii and targeting to secretory granules in pituitary cells and pancreatic beta-cells identification of a novel sorting determinant for the regulated pathway in the secretory protein chromogranin a sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary multiple neuropept ides derived from a common precursor are differentially packaged and transported structure et ultrastructure de lacrymaria olor (o.f.m. 1786) cocrystallization of proinsulin and insulin vayssie let ai. a large multigenic family codes for the polypeptides of the crystalline trichocyst matrix in paramecium protein secretion in tetrahymena thermophila. characterization of the major proteinaceous secretory proteins growth and form of secretory granules involves stepwise assembly but not differential sorting of a family of secretory proteins in paramecium quality control in the endoplasmic reticulum proteolytic processing and ca 2+-binding activity of dense-core vesicle polypeptides in tetrahymena identification and characterization of a novel secretory granule calcium-binding protein from the early branching eukaryote giardia lamblia myosin va facilitates the distribution of secretory granules in the f-actin rich cortex of pc12 cells chromogranin b (secretogranin i) a secretory protein of the regulated pathway, is also present in a tightly membrane-associated form in pc12 cells the disulfide-bonded loop of chromogranin b mediates membrane binding and directs sorting from the trans-golgi network to secretory granules the disulfide-bonded loop of chrornogranins, which is essential for sorting to secretory granules, mediates hornodimerizarion identification of the sorting signal motif within pro-opiomelanocortin for the regulated secretory pathway carboxypeptidase e, a prohormone sorting receptor, is anchored to secretory granules via a c-terminal transmembrane insertion carboxypeptidase e is a re~lated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in cpe at mice identification of a novel prohormone sorting signal-binding site on carboxypeptidase e, a regulated secretory pathway-sorting receptor depletion of carboxypeptidase e, a regulated secretory pathway sorting receptor, causes misrouting and constitut ive secretion of proinsulin and proenkephalin, but not chromogranin a proinsulin targeting to the regulated pathway is not impaired in carboxypeptidase e-deficient cpefat/cpefat mice carboxypeptidase e, a peripheral membrane protein implicated in the targeting of hormones to secretory granules, coaggregates with granule content proteins at acidic ph secretogranin iii binds to cholesterol in the secretory granule membrane as an adapter for chromogranin a sorting of carboxypeptidase e to the regulated secretory pathway requires interaction of its transmembrane domain with lipid rafts cholesterol is required for the formation of regulated and constitutive secretory vesicles from the trans-golgi network selective delivery of secretory cargo in golgi-derived carriers of nonepithelial cells reduction of the disulfide bond of chromogranin b (secretogranin i) in the trans-golgi network causes its missorting to the constitutive secretory pathways disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins n-and c-terminal domains direct cell type-specific sorting of chromogranin a to secretory granules molecular cloning of phogrin, a protein-ryrosine phosphatase homologue localized to insulin secretory granule membranes the lumenal domain of the integral membrane protein phogrin mediates targeting to secretory granules differential trafficking of soluble and integral membrane secretory granule-associated proteins identification of routing determinants in the cytosolic domain of a secretory granule-associated integral membrane protein biogenesis of weibel-palade bodies mutational analysis of vamp domains implicated in ca2+-induced insulin exocytosis targeting of p-selectin to two regulated secretory organelles in pc12 cells p-selectin targeting to secretory iysosomes of rbl-2h3 cells regulated secretion of conventionallysosomes angelica ec the molecular machinery for the biogenesis of lysosome-related organelles: lessons from hermansky-pudlak syndrome p-selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in att-20 cells a complex web of signal-dependent trafficking underlies the triorganellar distribution of p-selectin in neuroendocrine pc12 cells ap-3 adaptor functions in targeting p-selectin to secretory granules in endothelial cells coat proteins: shaping membrane transport selective and signal-dependent recruitment of membrane proteins to secretory granules formed by heterologously expressed von willebrand factor weibel-palade bodies recruit rab27 by a content-driven , maturation-dependent mechanism that is independent of cell type assembly of multimeric von willebrand factor directs sorting of p-selectin how glycosylphosphatidylinositol-anchored membrane proteins are made interaction of syncollin with gp-2, the major membrane protein of pancreatic zymogen granules, and association with lipid microdomains a submembranous matrix of proteoglycans on zymogen granule membranes is involved in granule formation in rat pancreatic acinar cells the major zymogen granule membrane protein gp-2 in the rat pancreas is not involved in granule formation loss of the zymogen granule protein syncollin affects pancreatic protein synthesis and transport but not secretion recycling of raft-associated prohormone sorting receptor carboxypeptidase e requires interaction with arf6 requirement for gtp hydrolysis in the formation of secretory vesicles the use of permeabilized cells to investigate secretory granule biogenesis exocytic transport vesicles generated in vitro from the trans-golgi network carry secretory and plasma membrane proteins reconstitution of constitutive secretion using semi-intact cells: regulation by gtp but not calcium phospholipase d stimulates release of nascent secretory vesicles from the trans-golgi network secretory vesicle budding from the trans-golgi network is mediated by phosphatidic acid levels implications of lipid microdomains for membrane curvature, budding and fission role of dynamin in the formation of transport vesicles from the trans-golgi network a role for phosphatidylinositol transfer protein in secretory vesicle formation multiple trimeric g-proteins on the trans-golgi network exert stimulatory and inhibitory effects on secretory vesicle formation t rimeric g-proteins of the trans-golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules formation of nascent secretory vesicles from the trans-golgi network of endocrine cells is inhibited by tytosine kinase and phosphatase inhibitors cooperativity of phosphatidylinositol transfer protein and phospholipase d in secretory vesicle formation from the tgn-phosphoinositides as a common denominator? role of diacylglycerol in pkd recruitment to the tgn and protein transport to the plasma membrane functional diversity in the dynamin family protein kinase d regulates the fission of cell surface destined transport carriers from the trans-golgi network biogenesis of processing-competent secretory organelles in vitro two independent targeting signals in the cytoplasmic domain determine trans-golgi network localization and endosomal trafficking of the proprorein convertase furin direct and gtp-dependent interaction of adp-ribosylation factor 1 with clathrin adaptor protein ap-l on immature secretory granules the ap-l adaptor complex binds to immature secretory granules from pc12 cells, arid is regulated by adp-ribosylation factor interaction of furin in immature secretory granules from neuroendocrine cells with the ap-1 adaptor complex is modulated by casein kinase ii phosphorylation mannose 6-phosphate receptors are sorted from immature secretory granules via adaptor protein ap-1, clathrin, and syntaxin 6-positive vesicles differential distribution of mannose-e-phosphare receptors and furin in immature secretory granules differential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic beta cells site-specific cross-linking reveals a differential direct interaction of class 1, 2, and 3 adp-ribosylation factors with adaptor protein complexes 1 and 3 proinsulin endoproteolysis confers enhanced targeting of processed insulin to the regulated secretory pathway the role of assembly in insulin's biosynthesis protein discharge from immature secretory granules displays both regulated and constitutive characteristics protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment protein traffic from the secretory pathway to the endosomal system in pancreatic beta-cells phospharidyiinosirol 4 phosphate regulates targeting of clathrin adaptor ap-l complexes to the golgi arf mediates recruitment of ptdlns-4-0h kinase-beta and stimulates synthesis of ptdlns(4,5)p2 on the golgi complex type i phosphatidylinositol 4-phosphate 5-kinase directly interacts with adp-ribosylation factor 1 and is responsible for phosphatidylinositol 4,5-bisphosphate synthesis in the golgi compartment regulation and recruitment of phosphatidylinositol 4-kinase on immature secretory granules is independent of adp-ribosylation factor 1 dynamics of immature secretory granules: role of eytoskeletal elements during transport, cortical restriction, and f-actin-dependent tethering mechanisms of ph regulation in the regulated secretory pathway endoproteolytic cleavage is mediated by a vacuolar atpase that generates an acidic ph in the trans-golgi network ph-independent and -dependent cleavage of proinsulin in the same secretory vesicle biosynthesis and secretion of pituitary hormones: dynamics and regulation inhibition of the vacuolar h+-atpase perturbs the transport , sorting, processing and release of regulated secretory proteins low-molecular-weight constituents of isolated insulin-secretory granules . bivalent cations , adenine nucleotides and inorganic phosphate endoplasmic reticulum ca2+ is important for the proteolytic processing and intracellular transport of proinsulin in the pancreatic beta-cell effects of ph and ca2+ on heterodimer and heteroretramer formation by chromogranin a and chromogranin b prohormone and proneuropeptide processing calcium-and ph-dependent aggregation and membrane association of the precursor of the prohormone convertase pc2 ionic milieu controls the compartment-specific activation of pro-opiomelanocortin processing in att-20 cells molecular and cellular regulation of prohormone processing the proprotein convertases furin and prohormone convertase 1/3 are major convertases in the processing of mouse pro-growth hormone-releasing hormone an antibody specific for an endoproteolytic cleavage site provides evidence that pro-opiomelanocortin is packaged into secretory granules in att20 cells before its cleavage lau 0 et ai. biogenesis of regulated exocytotic carriers in neuroendocrine cells comparison of secondary structures of insulin and proinsulin by ftir nmr and photo-cidnp studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition the role of proteolytic processing in the morphogenesis of virus particles peptides derived from the granins (chromograninslsecretogranins) distinct molecular events during secretory granule biogenesis revealed by sensitivities to brefeldin a homotypic fusion of immature secretory granules during maturation requires syntaxin 6 synaptic function modulated by changes in the ratio of synaptoragmin i and iv ap-l recruitment to vamp4 is modulated by phosphorylationdependent binding of pacs-1 cytoplasmic granule formation in mouse pancreatic acinar cells. evidence for formation of immature granules (condensing vacuoles) by aggregation and fusion of progranules of unit size, and for reductions in membrane surface area and immature granule volume during granule maturation homotypic fusion of immature secretory granules during maturation in a cell-free assay immunological characterization of trichocyst proteins in the ciliate pseudomicrothorax dubius lysosome function in the regulation of the secretory process in cells of the anterior pituitary gland rab3d is not required for exocrine exocytosis but for maintenance of normally sized secretory granules functional and spatial segregation of secretory vesicle pools according to vesicle age genetic approach to regulated exocyrosis using functional complementation in paramecium: identification of the nd7 gene required for membrane fusion novel secretory vesicle proteins essential for membrane fusion display extracellular-marrix domains post-golgi biosynthetic trafficking multicolour imaging of post-golgi sorting and trafficking in live cells gaip participates in budding of membrane carriers at the rrans-golgi network macro-and micro-domains in the endocrine pancreas key: cord-016652-x8t3lf1x authors: matthews, david; emmott, edward; hiscox, julian title: viruses and the nucleolus date: 2011-05-23 journal: the nucleolus doi: 10.1007/978-1-4614-0514-6_14 sha: doc_id: 16652 cord_uid: x8t3lf1x the nucleolus is a dynamic sub-nuclear structure integral to the function of a eukaryotic cell. some of its major roles involve ribosome subunit biogenesis, rna processing, cell cycle control and responding to cellular stress, such as infection. our understanding of the relationship between viruses and the nucleolus has moved from a phenomenological approach describing protein localisation to functional studies involving genetic analysis and proteomic approaches. these advances have provided fundamental insights as to how and why the nucleolus is targeted by many different viruses both to usurp normal functioning and to recruit nucleolar proteins to facilitate virus replication. this knowledge has been exploited for therapeutic strategies involving targeted inhibition of virus replication and live-attenuated recombinant vaccines. ; lee et al. (2003) ; lutz and kedinger (1996) ; tollefson et al. (2007) ; matthews and russell (1998) asfaviridae lower et al. (1995) many of these proteins have been shown to interact with nucleolar proteins the reason why rna viruses, and positive-strand rna viruses in particular, interact with the nucleolus when the site of genome replication is in the cytoplasm is less intuitive. in this latter case, viral proteins that are normally required in the cytoplasm must transit through the nuclear pore complex both to and from the nucleus. this process is crucial for virus biology because if the viral proteins that are required for cytoplasmic functions such as rna synthesis and encapsidation are sequestered in the nucleolus or nucleus, then progeny virus production will be affected as has been revealed by inhibitor and genetic studies (lee et al. 2006; tijms et al. 2002) . viruses may interact with the nucleolus to usurp host cell functions and recruit nucleolar proteins to facilitate virus replication. investigating the interactions between viruses and the nucleolus may facilitate the design of novel anti-viral therapies both in terms of recombinant vaccines (pei et al. 2008 ) and molecular intervention (rossi et al. 2007) , and also contribute to a more detailed understanding of the cell biology of the nucleolus. for many years our understanding of the interaction of viruses and the nucleolus was phenomenological and focused on identifying viral proteins that localised to this structure, their mechanisms of trafficking and potential interaction with nucleolar proteins (e.g. see table 14 .1). however, recent research capitalising on advances in proteomics, viral genetics and cellular imaging techniques are beginning to increase our understanding of the mechanisms viruses use to subvert host cell nucleoli and facilitate virus biology . new data are now emerging that support the view that many viruses interact with the nucleus and nucleolus, particularly to facilitate virus replication. one of the best-studied viruses in terms of viral interactions with the nucleolus is hiv-1 and is described in detail in chap. 17. although hiv has clearly defined cytoplasmic and nuclear replication strategies, the virus has a positive-sense rna genome in the sense that the viral capsid contains two copies of positive-sense rna, but these are reverse transcribed in the cytoplasm and then trafficked to the nucleus, where ultimately the new genome is transcribed and trafficked back to the cytoplasm. part of the reasoning for the interaction of hiv-1 with the nucleolus is the trafficking of intronless mrna from the nucleus into the cytoplasm (michienzi et al. 2000) . this is a property shared with herpes viruses and indicated that different viruses have evolved similar strategies involving subversion of nucleolar function for the benefit of virus biology (boyne and whitehouse 2006) . in the case of hiv-1, this knowledge has also led to the design and implementation of effective genetic therapies against the virus (unwalla et al. 2008 ). a large number of viruses with dna genomes have been shown to interact with nucleolus, and this perhaps is not surprising as most dna viruses replicate in the nucleus. a genome-wide screen of three distinct herpesviruses, herpes simplex virus 1 (hsv-1), cytomegalovirus (cmv) and epstein-barr virus (ebv), has shown that at least 12 herpesvirus-encoded proteins specifically localise to the nucleolus (salsman et al. 2008) , which are implicated in many aspects of the herpesvirus life cycle. therefore, a number of proteomic studies are currently being undertaken to study changes, in a global context, within the nucleolar proteome during virus infections, and are discussed later (lam et al. 2010) . several different herpes virus proteins have been shown to cause the redistribution of nucleolar proteins and hence disruption of the nucleolus. these include herpes simplex virus 1, the major tegument structural protein vp22 (lopez et al. 2008) , and the us11 (xing et al. 2010) and ul24 proteins (bertrand and pearson 2008; lymberopoulos and pearson 2007) . such disruption in many cases may have a direct effect on nucleolar function. a significant area of virus biology that has been investigated is the role of viral proteins that traffic through the nucleolus. for example, a number of hiv proteins that traffic through the nucleolus have been implicated in virus mrna processing (dundr et al. 1995) . similar observations have also been made in herpesviruses whitehouse 2006, 2009; leenadevi and dalziel 2009) . initial studies utilising the prototype g-2 herpesvirus, herpes virus saimiri (hvs), demonstrated that the hvs nucleolar trafficking orf57 protein induces nucleolar redistribution of the host cell human trex proteins, which are involved in mrna nuclear export (boyne and whitehouse 2006) . intriguingly, ablating orf57 nucleolar trafficking led to a failure of orf57-mediated viral mrna nuclear export (boyne and whitehouse 2006) . the precise role of this nucleolar sequestration is yet to be determined, but possible effects on viral mrna/protein processing and viral ribonucleoprotein particle assembly are currently being investigated. this property may also be conserved in other orf57 homologues as recent analysis has shown that the orf57 protein from kaposi's sarcoma associated herpesvirus (kshv) also dynamically traffics through the nucleolus (boyne et al. 2008b) . moreover, on the rapid disorganisation of the nucleolus a reduction is observed in virus mrna nuclear export (boyne and whitehouse 2009 ). the formation of an orf57-mediated export competent ribonucleoprotein particle within the nucleolus may also have implications for the translation of viral mrnas. for example, it has recently been demonstrated that the cellular nucleo-cytoplasmic shuttle protein, pym, which is involved in translation enhancement, is redistributed to the nucleolus in the presence of the kshv orf57 protein (boyne et al. 2010 ). this interaction effectively enhances the translation of the predominantly intronless transcripts made by kshv, and draws parallels with potential translation enhancement of positive strand rna virus genomes through their interaction with the nucleolus (discussed later). a second area of virus replication where nucleolar proteins are sequestered involves the replication of the virus dna genome. for example, we (matthews) and others have observed that nucleolar antigens upstream binding factor (ubf) and nucleophosmin (b23.1) are both sequestered into adenovirus dna replication centres where they promote viral dna replication (hindley et al. 2007; lawrence et al. 2006; okuwaki et al. 2001) . similarly, in hsv-1 infected cells, a number of nucleolar proteins including nucleolin and ubf are recruited into viral dna replication centres . these are specific sites where replication and encapsidation of the hsv-1 genome occurs. evidence suggests that sequestration of ubf is essential for viral dna replication as overexpression of tagged version of ubf acts in a dominant-negative manner inhibiting virus dna replication (stow et al. 2009 ). moreover, depletion of nucleolin results in reduced virus gene expression and infectious virion production (calle et al. 2008; sagou et al. 2010) . in addition to enhancing virus replication, nucleolar proteins are redistributed to alter cellular pathways during infection. for example, the nucleolar targeted hsv-1 us11 protein has been shown to interact with homeodomain-interacting protein kinase 2 (hipk2), which plays a role in p53-mediated cellular apoptosis and hypoxic response (calzado et al. 2009 ) and also participates in the regulation of the cell cycle (calzado et al. 2007 ). this interaction alters the sub-cellular localisation of hipk2 and protects against hipk2-mediated cell cycle arrest (giraud et al. 2004) . in contrast, the cellular protein, protein interacting with the carboxyl terminus-1 (pict-1), can sequester the virally encoded apoptosis suppressor protein, ks-bcl-2 protein, from the mitochondria into the nucleolus to down-regulate its anti-apoptotic activity (kalt et al. 2010 ). this is a potential interesting interplay between two sub-cellular structures involved in the viral stress response (olson 2009 ), and maybe more common and widespread. for example, bacterial infection has been shown to disrupt the nucleolus through regulating mitochondrial dysfunction (dean et al. 2010). although many rna virus proteins have been shown to localise to the nucleolus, most attention has focused on viral capsid proteins. these are proteins that associate with the viral genome for encapsidation and assembly of new virus particles. these proteins may also modulate replication (and transcription, where appropriate) of the viral genome. increasingly, capsid proteins have also been shown to have a number of roles in modulating host cell signalling pathways and functions. these capsid proteins are referred to as capsid, nucleoproteins or nucleocapsid proteins, depending on the virus. in many cases, they are phosphorylated (chen et al. 2005) , which can modulate activity (spencer et al. 2008) . many examples of these proteins have been shown to localise to the nucleolus both when over-expressed and also in infected cells. these include proteins from positive-strand animal and plant rna viruses, including the coronavirus nucleocapsid protein (chen et al. 2002; hiscox et al. 2001; wurm et al. 2001) , the arterivirus nucleocapsid protein (rowland et al. 1999) , the alphavirus capsid protein (jakob 1994 ) and non-structural protein nsp2 (rikkonen et al. 1992 (rikkonen et al. , 1994 and the umbravirus orf3 protein (ryabov et al. 2004 ). capsid proteins from negative-strand rna viruses also localise to the nucleolus. these have strain dependent localisation of a number of different influenza virus proteins (emmott et al. 2010c; han et al. 2010; melen et al. 2007; volmer et al. 2010) . for many years this has followed a phenomenological pattern and viral capsid and rna-binding proteins might simply localise to the nucleolus because they diffuse through the nuclear pore complex and associate with compartments in the nucleus that have high rna contents -the nucleolus in particular because it is transcriptionally active. in this case, sub-cellular localisation to the nucleolus would have no physiological consequence for the virus or the cell. however, rna virus replication is error prone and selection pressure might apply to such a fortuitous localisation (given the ~4,500+ nucleolar proteins and their diverse roles (ahmad et al. 2009 )), with the concomitant effect that the virus could select for changes that ultimately disrupt nucleolar function and/or recruit nucleolar proteins to aid virus replication. there is a potential correlation between the nucleolar localisation of a viral protein and the loss of an essential nucleolar function. the molecular mechanisms responsible for this effect are unknown, but the displacement and re-localisation of nucleolar proteins by viral proteins could increase or decrease the nucleolar, nuclear and/or cytoplasmic pool of these proteins. certainly, the accumulation of viral proteins in the nucleolus could potentially cause volume exclusion or crowding effects, which have been proposed to play a fundamental role in the formation of nuclear compartments including the nucleolus, and can be addressed by proteomic strategies. therefore, disruption of nucleolar architecture and function might be common in virus-infected cells if viral proteins target the nucleolus or a stage of the virus lifecycle disrupts nucleolar proteins. for example, poliovirus infection results in the selective redistribution of nucleolin from the nucleolus to the cytoplasm (waggoner and sarnow 1998) and inactivation of ubf, which shuts off rna polymerase i transcription in the host cell. the infection of cells with ibv has been shown to disrupt nucleolar architecture (dove et al. 2006b ) and cause arrest of the cell cycle in the g2/m phase and failure of cytokinesis (dove et al. 2006a) . the ibv and arterivirus nucleocapsid proteins associate with nucleolin and fibrillarin, respectively. similarly, the hiv-1 rev protein has been shown to localise to the dfc and gc and over-expression of rev protein alters the nucleolar architecture and is associated with the accumulation of nucleophosmin (dundr et al. 1995) . many different virus proteins localise to the nucleolus (table 14 .1). however, predicting viral (and cellular) nucleolar targeting signals has historically been problematic and only recently has bioinformatic software been developed to fascilitate this (scott et al. 2011) . nucleolar trafficking might be mediated by virtue of the fact that viral proteins that are trafficked to the nucleolus contain motifs that resemble host nucleolar targeting signals, that is, a form of molecular mimicry is used . the discovery of specific nucleolar trafficking signals in viral proteins has indicated a functional mechanism behind this observed localisation (lee et al. 2003; reed et al. 2006; . analysis of the different nucleolar trafficking signals identified in viral proteins using dynamic live-cell imaging has certainly demonstrated that different proteins can confer differential trafficking rates and localisation patterns (emmott et al. 2008) . this is very similar to cellular nucleolar proteins (lechertier et al. 2007 ). in some virus proteins, both nlss and nucleolar targeting signals act in concert to direct a protein to the nucleolus. the arterivirus porcine reproductive and respiratory syndrome virus (prrsv) nucleocapsid protein localises to the nucleolus and has been shown to contain two potential nlss, a pat4 and a downstream pat7 motif (rowland et al. 1999 . analysis revealed that a 31 amino acid sequence incorporating the pat7 motif could direct the nucleocapsid protein to both the nucleus and nucleolus. the protein also contains a predicted nes, presumably to allow the protein to traffic back into the cytoplasm to contribute to viral function in this compartment. this is common with other similar related proteins. for example, in the avian coronavirus nucleocapsid protein an eight amino acid sequence is necessary and sufficient to target the protein to the nucleolus (reed et al. 2006 ) and contains an nes (reed et al. 2007 ). intriguingly, genetic analysis (lee et al. 2006) , dynamic livecell imaging (you et al. 2008 ) and use of trafficking inhibitors (tijms et al. 2002) paint a picture of the requirement of these positive sense rna virus capsid proteins localising to the nucleolus as soon as they are translated, prior to their involvement in virus replication or assembly. this may be related to subversion of host cell function, protein modification (e.g. phosphorylation) or recruitment of nucleolar proteins. viral proteins might also traffic to the nucleolus through association with cellular nucleolar proteins . for example, the hepatitis delta antigen has been shown to contain a nucleolar targeting signal that also corresponded to a site that promoted binding to nucleolin (lee et al. 1998) . mutating this region prevented nucleolin binding to the delta antigen and nucleolar trafficking. by implication, this relates nucleolin binding to nucleolar trafficking (lee et al. 1998) . certainly, interaction with nucleophosmin and hepatitis delta antigens can modulate viral replication (huang et al. 2001 ) and more recently combined proteomic-rnai screens have revealed many other nucleolar proteins that can be associated with this viral protein (cao et al. 2009 ). trafficking and accumulation of viral proteins to and from the nucleolus, similar to cellular proteins, may also be cell cycle related. for example, the coronavirus nucleocapsid protein localises preferentially to the nucleolus in the g2 phase of the cell cycle (cawood et al. 2007) , as does the human cytomegalovirus protein ul83 in the g1 phase (arcangeletti et al. 2011) . again these trafficking profiles may be related to the interaction with cellular nucleolar proteins (emmott and hiscox 2009). semliki forest virus non-structural protein nsp2 can localise to the nucleolus (peranen et al. 1990; rikkonen et al. 1992 rikkonen et al. , 1994 and disruption of this localisation through a single amino acid change results in a reduction in neurovirulence (fazakerley et al. 2002) . such in vitro data has also been backed up by persuasive in vivo data. mutation of the arterivirus nucleocapsid protein pat7 nls motif in the context of a full-length clone revealed that this sequence could have a key role in virus pathogenesis in vivo, as animals infected with mutant viruses had shorter viraemia than wild-type viruses (lee et al. 2006; pei et al. 2008) . interestingly, reversions occurred in the mutated nucleocapsid gene sequence and although the amino acid sequence of the pat7 motif was altered, its function was not; this new signal was defined as a pat8 motif (lee et al. 2006) . the clear implications of this groundbreaking work is that disruption of nucleolar trafficking of a viral protein proves functional relevance and illustrates the potential of exploiting this knowledge for the generation of growth attenuated recombinant vaccines (pei et al. 2008; reed et al. 2006 reed et al. , 2007 . similarly, point mutations in the japanese encephalitis virus (jev) core protein that abolished nuclear and nucleolar localisation resulted in recombinant viruses with impaired replication in mammalian cells, compared to wild type virus (mori et al. 2005; tsuda et al. 2006) . curiously, replication of recombinant viruses was not impaired in insect cells, illustrating this could potentially be related to differences in nucleolar architecture and proteomes between these cell types (thiry and lafontaine 2005) . the jev core protein has been shown to interact with nucleophosmin and is translocated from the nucleolus to the cytoplasm. flaviviruses in general (jev, dengue virus and west nile virus) appear to have a part-nuclear stage to the synthesis of viral rna and several components of the viral replicase together with newly synthesised rna have been found in the nucleus of infected cells (uchil et al. 2006) . one intriguing question that has yet to be elucidated is how such viral rna traffics from the nucleus to the cytoplasm. most cellular mrnas are spliced and it is part of the splicing process that signals nuclear export. certain dna viruses, such as herpesvirus saimiri, produce intron-less mrna and these viruses have evolved specific viral proteins (such as herpesvirus saimiri orf57 (boyne et al. 2008a) ), which interact with the cellular mrna export machinery (e.g. the mrna processing and export factor aly) to traffic viral mrna from the nucleus to the cytoplasm (boyne et al. 2008b (boyne et al. , 2010 boyne and whitehouse 2006) and a similar process might be required by rna viruses. for example, tomato bushy stunt virus (tbsv) redistributes aly from the nucleus to the cytoplasm, and this might be a way the virus mediates host cell protein synthesis (uhrig et al. 2004 ). in plants rna silencing, a host defence mechanism targets virus rnas for degradation in a sequence-specific manner and viruses use several mechanisms to counteract this system (canto et al. 2006) . tbsv encodes a protein, p19, which interferes with this pathway. however, aly might transport p19 from the cytoplasm to the nucleus or nucleolus and disrupt its silencing suppression activity. nucleolin has also been shown to be involved in the trafficking of herpes simplex virus type 1 nucleocapsids from the nucleus to the cytoplasm (sagou et al. 2010) , drawing parallels with the involvement of nucleolar proteins in the movement of plant viruses (kim et al. 2007a, b) . different plant virus proteins involved in long-distance phloem-associated movement of virus particles or with roles in binding to the rna virus genomes localise to the nucleolus and other sub-nuclear structures (kim et al. 2007b; ryabov et al. 2004 ). this may be mediated by association with nuclear proteins, as is the case with fibrillarin and the orf3 protein of plant umbraviruses (kim et al. 2007a) . hijacking the nucleolus is not exclusive to plant viruses and may also occur with mammalian viruses. similar to the plant rhabdovirus maize fine streak virus (mfsv), whose nucleocapsid and phosphoproteins localise to the nucleolus (tsai et al. 2005) , the animal negative-stranded rna virus borna disease virus has been reported to use the nucleolus as a site for genome replication, and its rna-binding protein has the appropriate trafficking signals for import to and export from the cytoplasm to the nucleus (pyper et al. 1998 ). the hepatitis delta virus genome also has differential synthesis in the nucleus with rna being transcribed in the nucleolus (huang et al. 2001) ; this is similar to the potato spindle tuber viroid where rnas of opposite polarity are sequestered in different nuclear compartments, with the positive-sense rna being transported to the nucleolus. again localisation to different sub-nuclear strcutures may have different roles in the virus life cycle (li et al. 2006 ). an intriguing recent discovery has been made showing that adenoassociated virus (aav) encodes an additional protein called assembly-activating protein (aap) that localises to the nucleolus and promotes assembly of the viral capsid (sonntag et al. 2010) . as a result of their limited genomes and coding capacities, recruitment of cellular proteins with defined functions in rna metabolism would be a logical step to facilitate rna virus infection. as nucleolar proteins have many crucial functions in cellular rna biosynthesis, processing and translation, it comes as no surprise that nucleolar proteins are incorporated into the replication and/or translation complexes formed by rna viruses. given that some nucleolar proteins have many different functions, the same nucleolar protein might be used by a virus for different aspects of the replication pathway. studies suggest that the human rhinovirus 3 c protease (3cpro) pre-cursors, 3cd' and/or 3cd, localise in the nucleoli of infected cells early in infection and inhibit cellular rna transcription via proteolytic mechanisms (amineva et al. 2004 ). this general property is not restricted to human rhinovirus and in terms of the inhibition of cellular translation has also been described for encephalomyocarditis virus (aminev et al. 2003a, b) , again suggesting roles in translational regulation. given the many roles of the nucleolus in the life cycle of the cell, including as stress sensor (boulon et al. 2010; mayer and grummt 2005) , it would seem reasonable that comprehensive unbiased analysis of the nucleolar proteome would yield interesting data, particularly, with providing clues as to what cellular nucleolar functions may be altered by virus infection and what mechanisms the nucleolus may use to respond to this. how the nucleolar proteome changes in response to virus-infection has been investigated using stable isotope labelling with amino acids in cell culture (silac) coupled to lc-ms/ms and bioinformatics (fig. 14.1) . these studies, led by our laboratories, have analysed purified nucleoli and the nucleus, and have directly stemmed from the pioneering work of the lamond laboratory in analysing purified nucleoli using quantitative proteomics (andersen et al. 2005 (munday et al. 2010) . overall, our data indicates that only a small proportion of nucleolar proteins change in abundance in virus-infected cells, fig. 14. 1 diagram of a "classic" silac experiment. this technology allows high-throughput quantitative proteomics and has been readily applied to the nucleolus, especially when coupled with dynamic live-cell imaging (andersen et al. 2005) . the ability to simultaneously compare up to three different conditions through selection of the appropriate isotope label has enabled the recent studies of how the nucleolar proteome changes in virus-infected cells (emmott et al. 2010a; emmott et al. 2010b; emmott et al. 2010c; hiscox et al. 2010; lam et al. 2010) and these tend to be virus-specific. for example, in adenovirus infected cells just 7% of proteins identified show a twofold or greater change compared to almost a third of nucleolar antigens showing a greater than twofold change when cells are treated with actd which inhibits rrna synthesis (lam et al. 2010) . what is notable is that direct comparison between the adenovirus data set and the actd dataset shows no clear correlation lam et al. 2010) , further supporting the case that adenovirus induces effects on the nucleolus distinct from that of a generalised, non-specific shut down of nucleolar function. this fits well with a previous observation that adenovirus infection does not affect rrna synthesis even 36 h post-infection (lawrence et al. 2006 ). these results were initially surprising given the number of different viral proteins that can localise to this structure and how they interact with nucleolar proteins. this suggests that the nucleolar proteome and architecture is resilient during early stages of infection but may become disrupted as more and more damage accumulates inside cells because of virus activity, as clearly evidenced in live-cell imaging experiments (bertrand and pearson 2008; dove et al. 2006b; ). coupling quantitative proteomic analysis of the nucleolus and deep sequencing throughout infection in time-course experiments of lytic, latent, acute and persistent viruses would reveal valuable insights into the response of the nucleolus to virus infection. likewise, being able to move from studying cell culture-adapted laboratory strains into clinical isolates replicating in primary cells would yield more biologically relevant information, particularly with regard to the severity of disease and nucleolar changes. these technologies could also be applied to large-scale analysis of viral proteins that traffic to the nucleolus and the cellular nucleolar proteins that they associate with (e.g. using silac and egfp-traps (trinkle-mulcahy et al. 2008)), thus generating and integrating interactome networks with the nucleolar proteome during infection. nopdb: nucleolar proteome database-2008 update encephalomyocarditis viral protein 2a localizes to nucleoli and inhibits cap-dependent mrna translation encephalomyocarditis virus (emcv) proteins 2a and 3bcd localize to nuclei and inhibit cellular mrna transcription but not rrna transcription rhinovirus 3 c protease precursors 3cd and 3cd' localize to the nuclei of infected cells nucleolar proteome dynamics human cytomegalovirus proteins pp65 and iep72 are targeted to distinct compartments in nuclei and nuclear matrices of infected human embryo fibroblasts cell-cycle-dependent localization of human cytomegalovirus ul83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro np9 protein of human endogenous retrovirus k interacts with ligand of numb protein x functional role of px open reading frame ii of human t-lymphotropic virus type 1 in maintenance of viral loads in vivo a temporal study of the expression of the capsid, cytoplasmic inclusion and nuclear inclusion proteins of tobacco etch potyvirus in infected plants visualization of the interaction between the precursors of vpg, the viral protein linked to the genome of turnip mosaic virus, and the translation eukaryotic initiation factor iso 4e in planta the conserved n-terminal domain of herpes simplex virus 1 ul24 protein is sufficient to induce the spatial redistribution of nucleolin m148r and m149r are two virulence factors for myxoma virus pathogenesis in the european rabbit the nucleolus under stress nucleolar trafficking is essential for nuclear export of intronless herpesvirus mrna nucleolar disruption impairs kaposi's sarcoma-associated herpesvirus orf57-mediated nuclear export of intronless viral mrnas herpesvirus saimiri orf57: a post-transcriptional regulatory protein recruitment of the complete htrex complex is required for kaposi's sarcoma-associated herpesvirus intronless mrna nuclear export and virus replication kaposi's sarcoma-associated herpesvirus orf57 protein interacts with pym to enhance translation of viral intronless mrnas nucleolin is required for an efficient herpes simplex virus type 1 infection hipk2: a versatile switchboard regulating the transcription machinery and cell death from top to bottom: the two faces of hipk2 for regulation of the hypoxic response nuclear localization of nucleocapsid-like particles and hcv core protein in hepatocytes of a chronically hcv-infected patient a single amino acid change in the nuclear localization sequence of the nsp2 protein affects the neurovirulence of semliki forest virus analysis of the subcellular localization of the proteins rep, rep' and cap of porcine circovirus type 1 bovine immunodeficiency virus tat gene: cloning of two distinct cdnas and identification, characterization, and immunolocalization of the tat gene products human t-cell leukemia virus type i p30 nuclear/nucleolar retention is mediated through interactions with rna and a constituent of the 60 s ribosomal subunit us11 of herpes simplex virus type 1 interacts with hipk2 and antagonizes hipk2-induced cell growth arrest nuclear and nucleolar localization of an african swine fever virus protein, i14l, that is similar to the herpes simplex virus-encoded virulence factor icp34.5 the nucleoprotein and the viral rna of infectious salmon anemia virus (isav) are localized in the nucleolus of infected cells the bovine immunodeficiency virus rev protein: identification of a novel lentiviral bipartite nuclear localization signal harboring an atypical spacer sequence cucumber mosaic virus 2b protein subcellular targets and interactions: their significance to rna silencing suppressor activity involvement of the nucleolus in replication of human viruses identification of nucleolus localization signal of betanodavirus ggnnv protein alpha characterization of the nuclear and nucleolar localization signals of bovine herpesvirus-1 infected cell protein 27 new regulatory mechanisms for the intracellular localization and trafficking of influenza a virus ns1 protein revealed by comparative analysis of a/pr/8/34 and a/sydney/5/97 imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy distinctions between bovine herpesvirus 1 and herpes simplex virus type 1 vp22 tegument protein subcellular associations nucleolar localization of potato leafroll virus capsid proteins relationship between adenovirus dna replication proteins and nucleolar proteins b23.1 and b23.2 direct interaction between nucleolin and hepatitis c virus ns5b brief review: the nucleolus -a gateway to viral infection? the interaction of animal cytoplasmic rna viruses with the nucleus to facilitate replication rna viruses: hijacking the dynamic nucleolus the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus nucleolar proteomics and viral infection the hbz-sp1 isoform of human t-cell leukemia virus type i represses junb activity by sequestration into nuclear bodies nucleolar localization of mouse mammary tumor virus proteins in t-cell lymphomas identification and characterization of the ul24 gene product of herpes simplex virus type 2 the nucleolar phosphoprotein b23 interacts with hepatitis delta antigens and modulates the hepatitis delta virus rna replication expression and processing of a small nucleolar rna from the epstein-barr virus genome a novel, mouse mammary tumor virus encoded protein with rev-like properties nucleolar accumulation of semliki forest virus nucleocapsid c protein: influence of metabolic status, cytoskeleton and receptors gltscr2/pict-1, a putative tumor suppressor gene product, induces the nucleolar targeting of the kaposi's sarcoma-associated herpesvirus ks-bcl-2 protein interaction of a plant virus-encoded protein with the major nucleolar protein fibrillarin is required for systemic virus infection cajal bodies and the nucleolus are required for a plant virus systemic infection electron microscopy of ribonucleic acid in nuclear particulate aggregates of hepatitis d using nuclease-gold complexes functional similarity of hiv-i rev and htlv-i rex proteins: identification of a new nucleolar-targeting signal in rev protein nucleo-cytoplasmic redistribution of the htlv-i rex protein: alterations by coexpression of the htlv-i p21x protein proteomics analysis of the nucleolus in adenovirus-infected cells immunocytology shows the presence of tobacco etch virus p3 protein in nuclear inclusions nucleolar protein upstream binding factor is sequestered into adenovirus dna replication centres during infection without affecting rna polymerase i location or ablating rrna synthesis a b23-interacting sequence as a tool to visualize protein interactions in a cellular context the nucleolin binding activity of hepatitis delta antigen is associated with nucleolus targeting adenovirus core protein vii contains distinct sequences that mediate targeting to the nucleus and nucleolus, and colocalization with human chromosomes precursor of human adenovirus core polypeptide mu targets the nucleolus and modulates the expression of e2 proteins mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication the alcelaphine herpesvirus-1 orf 57 encodes a nuclear shuttling protein functional interaction and colocalization of the herpes simplex virus 1 major regulatory protein icp4 with eap, a nucleolar-ribosomal protein rna-templated replication of hepatitis delta virus: genomic and antigenomic rnas associate with different nuclear bodies capsid protein of cucumber mosaic virus accumulates in the nuclei and at the periphery of the nucleoli in infected cells nucleolar and nuclear localization properties of a herpesvirus bzip oncoprotein, meq the major tegument structural protein vp22 targets areas of dispersed nucleolin and marginalized chromatin during productive herpes simplex virus 1 infection identification of a rev-related protein by analysis of spliced transcripts of the human endogenous retroviruses htdv/ herv-k properties of the adenovirus iva2 gene product, an effector of late-phase-dependent activation of the major late promoter involvement of ul24 in herpes-simplex-virus-1-induced dispersal of nucleolin relocalization of upstream binding factor to viral replication compartments is ul24 independent and follows the onset of herpes simplex virus 1 dna synthesis involvement of the ul24 protein in herpes simplex virus 1-induced dispersal of b23 and in nuclear egress the products of gene us11 of herpes simplex virus type 1 are dna-binding and localize to the nucleoli of infected cells stable expression of hepatitis delta virus antigen in a eukaryotic cell line adenovirus core protein v is delivered by the invading virus to the nucleus of the infected cell and later in infection is associated with nucleoli cellular stress and nucleolar function identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein icp27 nuclear and nucleolar targeting of influenza a virus ns1 protein: striking differences between different virus subtypes karyophilic properties of semliki forest virus nucleocapsid protein ribozyme-mediated inhibition of hiv 1 suggests nucleolar trafficking of hiv-1 rna localization and importance of the adenovirus e4orf4 protein during lytic infection the lactate dehydrogenase-elevating virus capsid protein is a nuclear-cytoplasmic protein the protein icp0 of herpes simplex virus type 1 is targeted to nucleoli of infected cells nuclear localization of japanese encephalitis virus core protein enhances viral replication quantitative proteomic analysis of a549 cells infected with human respiratory syncytial virus functional domain structure of human t-cell leukemia virus type 2 rex nucleolar localization of human hepatitis b virus capsid protein nucleolar targeting signal of human t-cell leukemia virus type i rex-encoded protein is essential for cytoplasmic accumulation of unspliced viral mrna identification of nucleophosmin/b23, an acidic nucleolar protein, as a stimulatory factor for in vitro replication of adenovirus dna complexed with viral basic core proteins induction of apoptosis by viruses: what role does the nucleolus play? inhibition of human immunodeficiency virus type 1 and type 2 tat function by transdominant tat protein localized to both the nucleus and cytoplasm nuclear entry and nucleolar localization of the newcastle disease virus (ndv) matrix protein occur early in infection and do not require other ndv proteins functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association nuclear localization of semliki forest virus-specific nonstructural protein nsp2 the nucleolus is the site of borna disease virus rna transcription and replication the complex subcellular distribution of satellite panicum mosaic virus capsid protein reflects its multifunctional role during infection control of nuclear and nucleolar localization of nuclear inclusion protein a of picorna-like potato virus a in nicotiana species proapoptotic effect of hepatitis c virus core protein in transiently transfected cells is enhanced by nuclear localization and is dependent on pkr activation delineation and modelling of a nucleolar retention signal in the coronavirus nucleocapsid protein characterization of the nuclear export signal in the coronavirus infectious bronchitis virus nucleocapsid protein nuclear and nucleolar targeting signals of semliki forest virus nonstructural protein nsp2 nuclear targeting of semliki forest virus nsp2 intracellular transport of the murine leukemia virus during acute infection of nih 3t3 cells: nuclear import of nucleocapsid protein and integrase functional analysis of proteins involved in movement of the monopartite begomovirus, tomato yellow leaf curl virus genetic therapies against hiv nucleolar-cytoplasmic shuttling of prrsv nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences the localisation of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus structural and functional characterization of human immunodeficiency virus tat protein human endogenous retrovirus herv-k(hml-2) encodes a stable signal peptide with biological properties distinct from rec intracellular location of two groundnut rosette umbravirus proteins delivered by pvx and tmv vectors identification of a nuclear localization signal and nuclear export signal of the umbraviral long-distance rna movement protein nucleolin is required for efficient nuclear egress of herpes simplex virus type 1 nucleocapsids genome-wide screen of three herpesviruses for protein subcellular localization and alteration of pml nuclear bodies identification of the caprine arthritis encephalitis virus rev protein and its cis-acting rev-responsive element the rev protein of visna virus is localized to the nucleus of infected cells pnac: a protein nucleolar association classifier characterization of signals that dictate nuclear/nucleolar and cytoplasmic shuttling of the capsid protein of tomato leaf curl java virus associated with dna beta satellite sequence requirements for nucleolar localization of human t cell leukemia virus type i px protein, which regulates viral rna processing a viral assembly factor promotes aav2 capsid formation in the nucleolus role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein upstream-binding factor is sequestered into herpes simplex virus type 1 replication compartments nucleolin associates with the human cytomegalovirus dna polymerase accessory subunit ul44 and is necessary for efficient viral replication reversible nucleolar translocation of epstein-barr virus-encoded ebna-5 and hsp70 proteins after exposure to heat shock or cell density congestion birth of a nucleolus: the evolution of nucleolar compartments nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus identification of a new human adenovirus protein encoded by a novel late l-strand transcription unit identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes complete genome sequence and in planta subcellular localization of maize fine streak virus proteins nucleolar protein b23 interacts with japanese encephalitis virus core protein and participates in viral replication nuclear localization of flavivirus rna synthesis in infected cells relocalization of nuclear aly proteins to the cytoplasm by the tomato bushy stunt virus p19 pathogenicity protein use of a u16 snorna-containing ribozyme library to identify ribozyme targets in hiv-1 avian reovirus sigmaa localizes to the nucleolus and enters the nucleus by a nonclassical energy-and carrier-independent pathway nucleolar localization of influenza a ns1: striking differences between mammalian and avian cells viral ribonucleoprotein complex formation and nucleolarcytoplasmic relocalization of nucleolin in poliovirus-infected cells interactions of minute virus of mice and adenovirus with host nucleoli intracellular localization and determination of a nuclear localization signal of the core protein of dengue virus proteins c and ns4b of the flavivirus kunjin translocate independently into the nucleus subcellular compartmentalization of adeno-associated virus type 2 assembly the n-terminal domain of pmtv tgb1 movement protein is required for nucleolar localization, microtubule association, and long-distance movement localisation to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division molecular anatomy of subcellular localization of hsv-1 tegument protein us11 in living cells nucleolar localization of the ul3 protein of herpes simplex virus type 2 colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rna-associated protein fibrillarin subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein a model for the dynamic nuclear/ nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (prrsv) nucleocapsid protein based on live cell imaging g0/g1 arrest and apoptosis induced by sars-cov 3b protein in transfected cells intracellular localization of the ul31 protein of herpes simplex virus type 2 acknowledgements dam and jah would like to acknowledge their co-workers and collaborators over the years for developing viral interactions with the nucleolus. dam's research on the nucleolus is supported by the wellcome trust and jah's by the bbsrc and a leverhulme trust research fellowship. ee is supported by a bbsrc astbury dtg studentship. key: cord-023740-g84fa45m authors: oldstone, michael b.a.; schwimmbeck, peter; dyrberg, thomas; fujinami, robert title: mimicry by virus of host molecules: implications for autoimmune disease date: 2014-06-27 journal: progress in immunology doi: 10.1016/b978-0-12-174685-8.50079-2 sha: doc_id: 23740 cord_uid: g84fa45m molecular mimicry defines the shared identity of molecules from disparate genes or proteins. thus, although their origins are as separate as a virus and the self-determinant of a human or lower animal, two molecules' linear amino acid sequences or their conformational fits may be shared. such molecular homologies between proteins occur frequently and likely play roles in the processing of viral proteins inside cells. the homologies shared between viruses and host cytoskeletal proteins likely indicate that shared determinants on cell linker proteins guided viral proteins along highways and stop points inside cells. most importantly, these unexpected cross-reactivities have broad and major implications for understanding autoimmune disease. molecular mimicry is detected either by using humoral or cellular immune components, that cross-react with two presumably unrelated protein structures, or by computer searches to match descriptions of proteins in storage banks. the use of both these approaches to define molecular mimicry and establish its potential role in autoimmune disease is the topic of this chapter. by molecular mimicry we mean the shared identity of molecules from disparate genes or proteins . thus, although their origins are as separate as a virus and the self-determinant of a human or lower animal, two molecules' linear amino acid sequences or their conformational fits may be shared. for a variety of reasons including false signals from enriched guaninecytosine sequences or as introns designed to be spliced away may provide false hybridization signals, we have focused on molecular mimicry at the protein level. such molecular homologies between proteins occur frequently and have broad and major implications for understanding autoimmune disease. further, such homologies likely play roles in the processing of viral proteins inside cells. this realization came from repeated observations of homologies shared between viruses and host cytoskeletal proteins. this phenomenon led dales et al. (1983) to hypothesize that shared determinants on cell linker proteins guided viral pro-teins along highways and stop points inside cells. additionally, these unexpected cross-reactivities attendant to mimicry warrant cautious use of reagents in diagnostic virology, microbiology laboratories, and synthetic vaccines, even though these materials originated from hybridomas or from animals immunized with predetermined (peptide) amino acid sequences. molecular mimicry is detected either by using humoral or cellular immune components that cross-react with two presumably unrelated protein structures, or by computer searches to match descriptions of proteins in storage banks. the use of both these approaches to define molecular mimicry and establish its potential role in autoimmune disease is presented in this chapter. several independent reports appeared in the early 1980s noting molecular mimicry between the viral antigen sv40-t and host cell proteins (lane and hoeffler, 1980) , measles virus phosphoprotein and the cytoskeleton component keratin , and a herpes simplex glycoprotein of 140k and a separate epitope on keratin from that recognized by the measles virus phosphoprotein . to better determine the frequency of molecular mimicry, srinivasappa and his colleagues at the nih acquired from many laboratories over 600 monoclonal antibodies raised against viral polypeptides (srinivasappa et al., 1986) . these investigators then mapped the incidence of monoclonals cross-reactivity with host proteins expressed on a large panel of normal tissues (table i ). in the analysis were antibodies against 11 different viruses including dna and rna viruses known to cause human infection from the herpes virus group, vaccinia virus, myxoviruses, paramyxoviruses, arenaviruses, flaviviruses, alphaviruses, rhabdoviruses, and coronaviruses. the results indicated that over 3.5% of such monoclonals cross-reacted with host cell determinants expressed on uninfected tissues. these determinants occurred at a single site or in widely diverse places located in a wide panel of cells found in the nervous system, endocrine system, immune system, gut, heart, and muscle (fig. 1 ). these and our other observations indicated that certain monoclonal antibodies stained restricted subsets of neurons in selected areas of the nervous system or unique subsets of lymphocytes within a defined functional lymphocyte class were stained. the above data indicate common cross-reactivity at the monoclonal level between viral protein and host self-proteins. in this situation, antibodies to hor"listed are the analyses of over 600 monoclonal antibodies done by srinivasappa et al. (1986) . monoclonal antibodies against 11 different viruses including dna and rna viruses known to cause human infection from the herpes virus group, vaccinia virus, myxoviruses, paramyxoviruses, arenaviruses, flaviviruses, alphaviruses, rhabdovirus, and coronaviruses cross-react with host cell determinants expressed on uninfected tissues. twenty-two of such monoclonal antibodies, or 3.5% provide evidence of molecular mimicry. mones, lymphocyte subsets, or cells of the nervous system, etc., can develop as a consequence of virus infection, with all the inherent potential for participating in disease. the cross-reactivity between viruses and particular tissues offers some interesting insights into the association of virus infections with specific diseases. for example, coxsackie b virus has been found in individuals with myocarditis, an inflammatory disease of the heart muscle. one of the monoclonal antibodies described by srinivasappa et al. (1986) was directed against the neutralizing domain of coxsackie virus but also interacted with heart muscle . equally intriguing was the recent finding by fujinami and powell (1986) of a link between theiler's virus and the demyelinating disease it causes. in this instance, a monoclonal antibody directed against the major neutralizing domain of theiler's virus also reacted with galactocerebroside, the main component on the surface of oligodendrocytes. because oligodendrocytes are cells that make the myelin lamella that wraps around axons, their destruction leads to demyelination. interestingly, inoculation of this monoclonal antibody into the sciatic nerve results in several fingerprints of demyelination. these and table i ). note that these monoclonal antiviral antibodies cross-react with one or more groups of uninfected cells representative of the nervous system, endocrine system, immune system, gut, heart, and muscle. see srinivasappa etal. (1986) for experimental data. other examples (reviewed in oldstone and notkins, 1986 ) suggest a mechanism whereby immune reactants directed against a viral or microbial component may cross-react with a host component and generate autoimmune disease. a second immunopathologic sequella associated with molecular mimicry is the formation and trapping of immune complexes (dales et ai, 1983; oldstone and notkins, 1986) . in this instance, antibodies induced against proteins of the infecting virus, but cross-reactive with host proteins such as cytoskeletal or other self-proteins released into fluids during normal cell turnover or enhanced turnover and lysis occurring during virus infection, form complexes with antigen in the circulation. these complexes may become trapped in vessels with fenestrated endothelial linings such as the renal glomeruli, small arteries and capillaries, and the choroid plexus. here, they accumulate to set in motion the events of immune complex disease. such events may well account for the high incidence of antiself (myosin, actin, smooth muscle, nucleic acids, etc.) antibodies producing during virus infection and the formation of immune complexes during such infections (reviewed in oldstone and notkins, 1986 ). the analysis discussed above indicates that 3-4% of monoclonal antibodies generated against specific virus determinants also bind to host "self-determinants. other experiments have established that a minimum of five to six peptides is required for the induction of monoclonal antibodies (wilson et al., 1984) . since, on the basis of antibody cross-reactivity, many viruses share antigenic sites with normal host cell components, the next step was to look for crossreactive capability in eliciting autoimmunity and related disease. this was approached by using a computer-assisted search of the dayhoff data bank. after examining the 2511 amino acid sequences including 470,158 residues deposited in the protein data bank to look for overlapping peptides, homologies were noted among 2469 hexomers, 186 septomers, and 17 octomers; however these may not have been in tandem. since the probability of the requisite 20 amino acids occurring in six identical sequences in a row between two proteins, if amino acids occur at a random frequency, is 20 6 or one to 64 million, it is unlikely that such a homology would occur by chance. to provide evidence that molecular mimicry could cause autoimmunity we chose to study the encephalitogenic site of myelin basic protein and the disease allergic encephalomyelitis (eae). the entire amino acid sequence of myelin basic protein is known, and its encephalitogenic sites have been mapped in several animal species (hashim, 1978) . computer-assisted analysis located several viral proteins in the dayhoff files that have significant homology with the encephalitogenic site of myelin basic protein (hashim, 1978) ; these are the nucleoprotein and hemagglutinin of influenza virus, coat protein of polyoma virus, core protein of adenovirus, polyprotein of poliomyelitis virus, ec-lf2 protein of epstein-barr virus, rabies virus glycoprotein, measles virus nucleoprotein, and hepatitis b virus polymerase. however, of the banked sequences, the myelin basic protein encephalitogenic site in the rabbit fit best with hepatitis b virus polymerase (hbvp): 66 75 thr-thr-his-tyr-gly-ser-leu-pro-gln-lys encephalitogenic site, rabbit myelin basic protein 589 598 ile-gly-cys-tyr-gly-ser-leu-pro-gln-glu hbvp as seen in fig. 2 , immune responses, both humoral and cellular, were generated in rabbits inoculated with the dexomer viral peptide reacted with myelin basic protein. further, inoculation of the hbvp peptide into rabbits caused perivascular infiltration localized to the central nervous system reminiscent of the disease induced by inoculation of either whole myelin basic protein or the encephalitogenic site of myelin basic protein (fig. 2) . this outcome provided the first evidence for the potential of molecular mimicry to cause both autoimmune responses and autoimmune disease (fujinami and oldstone, 1985) . conceptually, molecular mimicry can produce autoimmunity when virus and host determinants are sufficiently similar to induce a crossreactive response, yet different enough to break immunologie tolerance. such principles for molecular mimicry undoubtedly follow those mapped for the induction and breaking of tolerance at both the b and t lymphocyte levels by heterologous serum protein (weigle, 1980) . with current technology allowing cloning and rapid sequencing of genes, and utilizing nucleic acid sequences of the open reading frame to determine the protein sequences encoded by a gene, more data on viral polypeptides will soon be available. such information with respect to homologies between microbial agents and the acetylcholine receptor, insulin receptor, and/or encephalitogenic receptor will likely show similarities and should improve understanding of the postinfectious encephalopathies and demyelination following virus infections, potential causes of myasthenia gravis and perhaps endocrine disorders such as diabetes. important will be the recognition that unless homology and the subse-quent immunologie cross-reactivity involve a host protein that precipitates disease, e.g., the restricted encephalitogenic site of myelin rather than multiple other sites of myelin basic protein, disease will be unlikely to follow despite autoimmune response. we have uncovered several other interesting examples of molecular mimicry, and these are currently under study in our laboratory. included are mimicry between viruses and the α-chain of the human acetylcholine receptor (fig. 3) and between a number of microbial agents and the human major histocompatibility (mhc) marker hla b.27 (fig. 4) . in the first instance, 10% of sera samples tested from myasthénie patients bound to the selected amino acid sequences for the α-chain of the acetylcholine receptor depicted in fig. 3 . affinity purification of such antibodies is underway, and their ability to cause depolarization of the receptor is being evaluated. similarly, sera from hla b.27 patients with and without ankylosing spondylitis are being studied for their cross-reactivity with amino acid sequences from several microbial agents, depicted in fig. 4 . other interesting molecular mimicries under study are those between myelin and aids virus and between sequences representing other mhc and viruses. the most likely mechanism by which molecular mimicry would cause disease is by eliciting an immune response against a determinant shared between the host and the virus to bring forth a tissue-specific immune response, presumably capable of destroying cells and eventually the tissue. the probable mechanism is the generation of cytotoxic cross-reactive effect of lymphocytes and/or antibodies that recognize "self-determinants" localized on target cells. interestingly, the induction of cross-reactivity would not require a replicating agent, and the immunologically mediated injury could occur after removal of the immunogen-a hit and run event. by such a mechanism, the microbial infection that initiates the autoimmune phenomenon need not be present at the time overt disease develops. the likely picture would be that the virus responsible for inducing a cross-reactive immune response is cleared initially, but the components ofthat immunity continue to assault host elements. this cycle continues as the autoimmune response itself leads to tissue injury that, in turn, releases more self-antigen, thereby incuding more antibodies, and so on. such a sequence of events would render the isolation or identification of an initiating infectious agent difficult or impossible. indeed, such events likely occur with the encephalopathies that follow measles, mumps, vaccinia, or herpes zoster virus infections of humans. in such postinfectious diseases, the infected host develops encephalitis, frequently associated with such other symptoms of autoimmune disease as rash when the viral amino acid sequences were inoculated into rabbits and then tested for their binding to the a-chain of the acetylcholine receptor note that herpes simplex virus (hsv) glycoprotein d residues 286 to 293 showed a higher degree of cross-reactivity then those of polyoma virus, which showed a greater degree of sequence homology. specificity of binding was checked by quantitative blocking experiments. analysis of sera from over 40 patients with myasthenia gravis indicated that their antibodies bound to the acetylcholine receptor sequence shown. see dyrberg and oldstone (1986) for details. oldstone.) and pain in the joints and skeletal muscles. recovery of the viral agent at this time is exceedingly rare, although the agent has been recovered easily several days earlier. this link between molecular mimicry and host proteins is further supported by studies showing that, after several types of acute virus infections, mononuclear cells from peripheral blood or cerebral spinal fluid proliferate in response to host antigens, one of which is myelin basic protein. further, several clonal populations of lymphocytes have been harvested from the central nervous system fluid of patients with encephalitis, and these lymphocytes proliferate when incubated with the infecting virus, its antigens, or nervous system antigens. these and other issues are likely to be studied increasingly to provide insights as to both potential etiologic agents and pathogenic mechanisms responsible for a variety of autoimmune disorders of man. proc. natl. acad. sei concepts in viral pathogenesis this is publication number 4393-imm from the department of immunology, scripps clinic and research foundation, la jolla, ca 92037. this work was supported in part by usphs grants ai-07007 and ag-04342 and nms jf2009 from the national multiple sclerosis society. p.s. is the recipient of a fellowship from the deutsche forschungsgemeinschaft (dfg). r.s.f. is a harry weaver scholar of the national multiple sclerosis society. key: cord-003070-6oca1mrm authors: shen, wen-jun; cui, wenjuan; chen, danze; zhang, jieming; xu, jianzhen title: rpirls: quantitative predictions of rna interacting with any protein of known sequence date: 2018-02-28 journal: molecules doi: 10.3390/molecules23030540 sha: doc_id: 3070 cord_uid: 6oca1mrm rna-protein interactions (rpis) have critical roles in numerous fundamental biological processes, such as post-transcriptional gene regulation, viral assembly, cellular defence and protein synthesis. as the number of available rna-protein binding experimental data has increased rapidly due to high-throughput sequencing methods, it is now possible to measure and understand rna-protein interactions by computational methods. in this study, we integrate a sequence-based derived kernel with regularized least squares to perform prediction. the derived kernel exploits the contextual information around an amino acid or a nucleic acid as well as the repetitive conserved motif information. we propose a novel machine learning method, called rpirls to predict the interaction between any rna and protein of known sequences. for the rpirls classifier, each protein sequence comprises up to 20 diverse amino acids but for the rpirls-7g classifier, each protein sequence is represented by using 7-letter reduced alphabets based on their physiochemical properties. we evaluated both methods on a number of benchmark data sets and compared their performances with two newly developed and state-of-the-art methods, rpi-pred and ipminer. on the non-redundant benchmark test sets extracted from the pridb, the rpirls method outperformed rpi-pred and ipminer in terms of accuracy, specificity and sensitivity. further, rpirls achieved an accuracy of 92% on the prediction of lncrna-protein interactions. the proposed method can also be extended to construct rna-protein interaction networks. the rpirls web server is freely available at http://bmc.med.stu.edu.cn/rpirls. the interactions of proteins with other proteins, peptides, dnas and rnas govern most the essential molecular function. rna-protein interactions (rpis) have a critical influence on post-transcriptional gene regulation [1] [2] [3] , viral assembly [4] [5] [6] , cellular defence [7] , protein synthesis [8, 9] and various other fundamental biological processes [10, 11] . a significant portion of transcripts is long non-coding rnas (lncrnas) which are not translated into proteins and are longer than 200 nucleotides [12] . lncrnas normally function with their interacting proteins [13] . for instance, the lncrna hotair regulated the hoxd locus in trans by interacting with pcg proteins [14] ; several lncrnas were shown to be able to interact with auf1, a protein linked to aging and cancer [15] ; lncrnas binding to jarid2 protein were essential for the recruitment of prc2 to the chromatin [16] ; lncrna gas5 inhibited hepatitis c virus replication by decoying hcv ns3 protein [17] . hence, the study of rpis is essential for understanding their functions. compared to those of protein-protein interactions and dna-protein interactions, current knowledge regarding rna-protein interactions, especially lncrna-protein interactions is still limited. in this study, we propose a novel machine learning method, which we call rna-protein interaction prediction based on regularized least squares (rpirls), to quantitatively predict the potential rna-protein interactions. the experimental determination of rpis remains expensive and time-consuming [18] [19] [20] , but fortunately, the accumulated rpi experimental data facilitate the development of computational models for rpi prediction [21] [22] [23] . in 2011, pancaldi and b .. ahler [24] introduced a computational approach for rbp (rna binding protein)-mrna interaction prediction. they employed support vector machines (svms) and random forests (rfs) based on more than 100 physical and functional features of rpis, including gene ontology, chromosomal position, gene and protein physical properties, protein localization, experimental translation, mrna properties, predicted protein structure, utr properties and genetic interactions. bellucci et al. [25] proposed a method called catrapid for the prediction of protein lncrna interaction. they evaluated the interaction propensities of protein-rna based on their physicochemical properties, including secondary structure, hydrogen bonding and van der waals. muppirala et al. [26] developed a method called rpiseq, which predicted rpis solely based on primary sequences. the rpiseq method still employed svms and rfs but exploited different features. they represented each sequence of proteins and rnas as the normalized frequencies of the corresponding 3-mer and 4-mer, respectively. in 2013, based on the same feature vectors presented in muppirala et al., wang et al. [27] first reduced the dimensionality of feature vectors, and then performed the rpis prediction by using naive bayes classifier which assumed the independence of attributes and by using extended naive bayes classifier which considered the correlation between attributes. lu et al. [28] integrated the information on the secondary structure, hydrogen bonding propensities and van der waals of lncrnas and proteins with fisher's linear discriminant model. in 2015, suresh et al. [29] developed a method called rpi-pred to predict rpis by considering the high-order 3d protein and rna structure information. in 2016, pan et al. [30] proposed a new method named ipminer that integrated deep learning with stacked ensembling to improve the prediction performance of ncrna-protein interactions. in this paper, we classified rna-protein pairs as interacting or non-interacting by integrating derived kernel with regularized least squares (rls) [31] . the motivation is to relate the sequence information of proteins and rnas to their biological functions, i.e., interactions. our method attempted to extract discriminant subsequence features from amino acid sequences and nucleotide sequences. the derived kernel measures the similarity between two biological sequences by capturing nucleic acid or amino acid compositions and repetitive sequence patterns. we used regularized least squares in learning as the computations performed by rls algorithms can be expressed using just inner products, hence allowing efficient implementation of kernel-based learning, in addition the rls algorithms often perform comparable to the best batch classifiers [32] . since the dimensionality of feature space increases exponentially with the template size, for computational sake, we set upper limit for template size. on the other hand, we categorized 20 amino acids into several groups based on their physiochemical properties [33] [34] [35] , the reduced alphabet representation of the protein sequence allows larger template size and also decreases the dimensionality of feature space. we considered the derived kernel with two-layer architecture, hence there were two template sets, denoted as t p and t r needed to be constructed for protein and rna, respectively. here we considered all possible substrings of the same length making up a template set. the template set t p for amino acid sequences was composed of substrings with k continuous amino acids, while the template set t r for nucleic acid sequences was composed of substrings with l continuous nucleic acids. in order to extract discriminant subsequence features from amino acid sequences and nucleotide sequences, we explored the effect on rpi prediction over a range of choices for the template sizes of protein and rna. the training set rpi2662 was used to determine these parameters. in our case, we used different template sizes of protein and rna chosen from set {1, 2, . . . , 4} ∪ {1, 2, . . . , 8} for rpirls and {1, 2, . . . , 6} ∪ {1, 2, . . . , 8} for rpirls-7g. with different combination of protein template size and rna template size, the combined kernelk dk 2 was integrated with rls to predict rna-protein interactions. the ten-fold stratified cross-validation has been verified to be the best algorithm for model selection on a large scale experiments [36] , therefore on the data set rpi2662, we tuned the parameter λ by ten-fold stratified cross-validation with the optional parameter set {λ = e n , n = −15, · · · , 15}. the data set rpi2662 was divided into ten mutually exclusive folds and the mean response of each fold was approximately equal. in each test we merged 9 parts of the samples as the training set and left the other part as the test set. the parameter λ was chosen by leave-one-out cross-validation on the training set. for rpirls, in all the ten sets, λ = e −2 uniformly achieved the best performance in the training data. tables 1 and 2 showed the performance of the proposed method in terms of auc and accuracy with different combination of parameters k and l, respectively. the experiment results showed that when the protein template size k = 2 and the rna template size l = 5, the model performs best with auc score of 0.926 and accuracy of 0.830. the other measurements of specificity (sp) and sensitivity (se) were 0.771 and 0.890, respectively. while for rpirls-7g, λ = e −3 uniformly performed best in all the ten sets. table 3 showed that the method achieved the best prediction accuracy of 0.823 when the protein template size k = 3 and the rna template size l = 4. the other measurements (auc, sp and se) were observed as 0.902, 0.761 and 0.884, respectively. the computational results showed that the rpirls classifier outperformed the rpirls-7g classifier in terms of various performance measurements, indicating that the diversity of amino acids at a sequence is important for the prediction of rpis. the performance of predicting rpis was evaluated by using 10-fold stratified cross-validation on the rpi2662 data set. different combinations of parameters k and l were evaluated. remark on the symbols of template sizes: k stands for template size of amino acid sequences; l stands for template size of nucleic acid sequences. the best auc in the table is marked in bold. remark on the symbols of template sizes: k stands for template size of amino acid sequences; l stands for template size of nucleic acid sequences. the best accuracy in the table is marked in bold. in order to evaluate the reliability and robustness of rpirls and rpirls-7g, we compared them with other two state-of-the-art methods rpi-pred and ipminer. the rpi2241 and rpi369 data sets after removing overlapping rpis with the training data were evaluated. both the rpirls and rpirls-7g classifiers were trained on the rpi2662 data set, and tested on the rpi2241 and rpi369 data sets, respectively. as shown in tables 4 and 5 , rpirls outperformed the rpirls-7g, rpi-pred and ipminer methods on both data sets. for the rpi369 data set as shown in table 4 , the performance of the rpirls method was 0.85, 0.92, 0.84 and 0.86 for predictive accuracy, auc, specificity and sensitivity, respectively. while the predictive accuracy of the rpi-pred and ipminer methods were just 0.49 and 0.5, respectively which were much lower than rpirls's. the remaining measurements (specificity and sensitivity) were observed as 0.34 and 0.63, respectively for rpirls, and 0.52 and 0.48, respectively for ipminer. the rpirls method outperformed rpi-pred and ipminer in terms of accuracy, specificity and sensitivity on the rpi369 data set. table 3 . predictive performance of, rpirls-7g in terms of the accuracy on the, rpi2662 training data set over varying template sizes. the performance of predicting rpis was evaluated by using 10-fold stratified cross-validation on the rpi2662 data set. different combinations of parameters k and l were evaluated. remark on the symbols of template sizes: k stands for template size of amino acid sequences; l stands for template size of nucleic acid sequences. the best accuracy in the table is marked in bold. similar results were observed on the rpi2241 data set in table 5 . the specificity of the rpi-pred and ipminer methods was just 0.38 and 0.20, respectively, indicating there was a positive bias in their predictions of performance. a low specificity increases the labor, cost, and time needed to perform the required experimental tests, but our rpirls method achieved both reasonable specificity and sensitivity. furthermore, we evaluated the rpirls classifier on large-scale rna-protein pairs in the currently available rpintdb data base. the rpirls method correctly predicted 35980 out of 43010 rpis, reaching the predictive accuracy of 84%. to explore the effectiveness of the proposed method on predicting ncrna-protein interactions, a large-scale ncrna-protein interaction data set (we called nrpi13153) was retrieved from the npinter data base [37] . we trained rpirls and rpirls-7g on the rpi2662 data set, and tested it on the nrpi13153. table 6 showed the prediction results compared with the rpi-pred classifier on the nrpi13153 data set. the ipminer method showed a significantly positive bias on predicting ncrna-protein interactions, thus here we didn't include it into the comparison. the predictive accuracy for different organisms were separately computed. for the six organisms, our method rpirls performed best for the homo sapiens and saccharomyces cerevisiae, rpi-pred performed best for drosophila melanogaster, escherichia coli and mus musculus, and both methods obtained the same predictive accuracy for the caenorhabditis elegans. rpirls outperformed rpirls-7g over all six organisms. for 13153 ncrna-protein pairs, the rpirls method achieved an accuracy of 91% compared to 76% for rpirls-7g and 88% for the rpi-pred method. we further tested rpirls and rpirls-7g on the lnrpi12114 data set which was a subset of the nrpi13153 data set and consisted of only lncrna-protein interactions (lncrpis). our model achieved an overall accuracy of 92% compared to 77% for rpirls-7g and 89% for the rpi-pred classifier as shown in table 7 . the predictive performance of rpirls was improved in 5 out 6 organisms compared with its performance on the nrpi13153 data set. the results indicated the effectiveness of our method to predict lncrna-protein interactions only by using primary sequences of proteins and rnas. predicting lncrna-protein interaction networks is useful to explore the molecular mechanisms that are regulated by lncrnas [38, 39] . in this experiment, we evaluated the performance of rpirls on building lncrpi networks and further compared its performance with rpi-pred. on the basis of the data in npinter, we analyzed the results of four organisms, i.e., caenorhabditis elegans, drosophila melanogaster, escherichia coli and saccharomyces cerevisiae, consisting of 4, 61, 78 and 437 lncrpis, respectively. for caenorhabditis elegans, the rpirls method correctly identified all 4 lncrpis (blue edges) while the rpi-pred method correctly identified 3 out of 4. as shown in figure 1 , rpi-pred made incorrect prediction for the pair of n342950-g5ebf5 (red edges). in figure 2 , rpi-pred correctly predicted all 61 lncrpis, whereas rpirls missed 7 out of 61 lncrpis for drosophila melanogaster. these 6 out of 7 incorrect predictions which were observed between two proteins p49963 and q9vss2 (yellow rectangle) and three signal recognition particle (srp) rnas n5330, n5333 and n389 (green ellipse), formed the srp rna-protein interactions. for escherichia coli, the rpirls classifier made much more errors than the rpi-pred method, with predictive accuracies of 45% vs. 86%. the performance of rpirls for escherichia coli was much poorer than that for the other five organisms. in order to analyze why rpirls had relative poor performance on escherichia coli, we estimated the amino acid composition of escherichia coli compared with that of the other five organisms. as shown in figure 3 , we found that escherichia coli had relative higher observing frequencies of alanine and valine as well as much lower content of serine compared with that of the other five organisms. the amino acid composition bias in escherichia coli probably leaded to poor results. as shown in figure 4 , among 43 incorrect predicted pairs, 39 rpis corresponded to 7 protein hubs, e.g., p0a6h1, p0afz3, p0ag67, p0ce47, p0ce48, p21499 and p77398, each of which appearing as a yellow rectangle node was shown to interact with six transfer-messenger rnas (tmrnas), e.g., n3828, n1877, n5000, n435, n329 and n4292 (green ellipse). for saccharomyces cerevisiae, as showed in figure 5 , among 27 incorrect predicted pairs, 10 rpis were involved in 3 protein hubs (p57743, q03338 and q06819), in which each protein interacted with 4 small nuclear rnas (snrnas: n4610, n6134, n4606 and n6136), and other 13 rpis corresponded to 7 protein hubs (p15646, p47083, p53941, q04217, q04500, q08492 and q12136), each of which interacted with three small nucleolar rnas (snornas: n5819, n4618 and n6159). the rpirls classifier correctly identified 410 out of 437 rpis, achieving a high accuracy of 94%, compared of 87% (correctly predicted 379 out of 437 pairs) for rpi-pred. in this work, we illustrated the effectiveness and reliability of rpirls in predicting rpis for eukaryotic organisms in networks which comprised a variety protein hubs and rna hubs. mammalian cells contain more than 1000 different proteins interacting with rna [3] . normally, any individual rna can interact with multiple proteins [11, 40] . conversely, most proteins are capable of interacting with multiple rnas [41] . given the number of rnas and rna-binding proteins, the number of possible rpis is enormous. high-throughput sequencing methods have accumulated huge amount of rna-protein binding experimental data and opened new possibilities to measure and understand rna-protein interactions by computational methods. most of the previous computational works on rpis focus on the prediction of rna-binding proteins or rna-binding residues in a protein sequence [34, [42] [43] [44] . to our knowledge, very limited works have been developed to predict the specific associations between rnas and proteins, which play a critical role in post-transcriptional gene regulatory networks. complex networks of rpis mediate post-transcriptional gene regulation and therefore prediction of rpis helps us to gain insight into regulatory networks [45, 46] . the work presented here provided a computational method, called rpirls, to classify rna-protein pairs as interacting or non-interacting by integrating a sequence-based derived kernel with regularized least squares. the derived kernel exploited the contextual information around an amino acid or a nucleic acid as well as the repetitive conserved motif information. our results demonstrated that only the sequence structures of rnas and proteins provide sufficient information to accurately predict rna-protein interactions, especially long non-coding rna-protein interactions. specifically, the rpirls classifier considered each protein sequence comprising up to 20 diverse amino acids, while the rpirls-7g classifier encoded each protein sequence by using the 7-letter reduced alphabets according to amino acid physiochemical properties. the computational results showed that the rpirls classifier was superior to the rpirls-7g classifier in reliability and effectiveness, indicating that the diversity of amino acids at a sequence has critical impact on the function of rna-binding proteins. on two non-redundant benchmark data sets extracted from the pridb, the rpirls method outperformed rpi-pred and ipminer in terms of accuracy, specificity and sensitivity. compared with rpi-pred and ipminer, the rpirls method obtained a reasonable sensitivity at a lower false positive rate. further, rpirls achieved an accuracy of 92% compared to 89% for rpi-pred on the prediction of lncrna-protein interactions. the rpirls method can be extended to construct rna-protein interaction networks and therefore helps us to gain insights into post-transcriptional gene regulation. the reason for the good performance of the proposed method may be due to several factors. firstly, the use of similarity scores is a significant conceptual change in protein/rna evaluation, quantifying the overall similarity between proteins, rnas and their interactions. combining kernels by tensor product for the set of rna-protein pairs allowing to share information across the rna-binding proteins considerably improved the prediction, especially in the case of rnas with few known binding proteins. secondly, we have found that contiguous k-mer frequencies alone captured rich statistical information on the repetitive conserved motif of rna-protein pairs and the diversity of amino acids at a sequence has also contributed to an observed improvement contrast to rpi-pred which just applied 1-letter frequency for both protein and rna. finally, a kernel works as a measure of similarity and supports the application of powerful machine learning algorithms such as regularized least squares which we used in this paper. the rls mehod enables us to efficiently search for an optimized parameter λ at essentially no additional cost [31] . further, our model was trained on a large data set which contained 2662 rna-protein pairs, and yielded more robust results. in contrast, ipminer had much more model parameters to fit as combining deep learning with stacked ensembling, however, was trained on a small data set of just 488 rna-protein pairs, and thus showed a significantly positive bias on predicting ncrna-protein interactions. the main disadvantage of the proposed method is that the method is purely data-driven, in the sense that it relies solely on information derived from amino acid sequences and nucleic acid sequences, and thus does not consider higher structural information of protein and rna. while this may be seen as an advantage, since it can predict any rna-protein pair of known sequences. the increase of the number of protein-rna complexes in protein data bank [47] has opened possibilities for researchers to develop secondary data bases and to gain valuable insight into the structure and function of these complexes. the protein-rna interface data base (pridb) v2.0 [48] identifies interfacial residues in rna-protein complexes and also calculates atomic distances between interfacial residues. the rb344 and rb1179 are two precalculated data sets in the pridb, which respectively consist of 344 and 1179 rna-binding protein chains. after combining the rb344 and rb1179 data sets, we obtained a total of 1750 experimentally validated non-redundant rna-protein pairs, which had at least two atoms respectively coming from rna and protein with distance no more than 4 å. next, we removed redundant rna-protein pairs, which are the same protein chains interacting with the same rna chains. further, we removed those rna-protein pairs with amino acid sequence length < 25 or nucleic acid sequence length < 15. finally, we obtained a positive sample set which consisted of 1331 experimentally validated rna-protein pairs. so far there are no definite negative samples of rna-protein interactions that are available. to construct a balanced negative sample set ("rna-protein non-interacting pairs"), we made it by randomly permute the proteins in the positive sample set but kept the rna fixed. we repeated the permutation process until no negative pairs existed in the positive sample set. as a result, the training set, called rpi2662, was composed of 1331 rna-protein interacting pairs and 1331 rna-protein non-interacting pairs. several data sets were employed to evaluate the performance of the proposed methods. our rpirls method was first evaluated using two popular non-redundant data sets of rpis studied in [27] . the rpi2241 data set consisted of 2241 experimentally validated rna-protein pairs extracted from the pridb data base. while the rpi369 data set eliminated all rpi pairs with ribosomal proteins or ribosomal rnas from the rpi2241 data set. to avoid overlapping between training and testing data sets, those rpis overlapping the training data were removed, leaving the rpi2241 data set of 1832 rpis and the rpi369 data set of 204 rpis. their corresponding negative pairs were generated by following the same steps as developing the training negative pairs. next, we tested the performance of the rpirls method on a large scale data set extracted from the rna-protein interaction data base (rpintdb) (http://pridb.gdcb.iastate.edu/rpiseq/download.php). this data set consisted of 43,010 experimentally validated rpis from several sources, including the rpidb, npinter data base and high-throughput experiments published in literature. the fourth data set were extracted from the npinter data base which we called nrpi13153. the nrpi13153 data set consisted of 13,153 experimentally validated ncrna-protein pairs from six model organisms, i.e., caenorhabditis elegans, drosophila melanogaster, escherichia coli, homo sapiens, mus musculus and saccharomyces cerevisiae. we constructed the fifth data set called lnrpi12114 by extracting only lncrna-protein pairs from the npinter data base. this data set contains 12,114 experimentally validated lncrna-protein pairs. in this paper, we proposed two classifiers for predicting rpis based on different representations of protein sequences. for the rpirls classifier, each amino acid sequence comprised up to 20 different amino acids. while for the rpirls-7g classifier, we adopted the same amino acid classification approach as [26, 29] . the derived kernel was proposed by smale et al. [49] on images inspired by neuroscience of visual cortex. in what follows, we briefly described the construction of derived kernel on sequences. suppose a is a finite set called the alphabet. in the work here a is the set of 20 amino acids (for rpirls), 7 alphabets (for, rpirls-7g) or 4 nucleic acids. let a 1 = a and define a k+1 = a k × a recursively for any k ∈ n. we say s is a string if s ∈ ∪ ∞ k=1 a k , and s = (s 1 , . . . , s k ) is a k-mer (e.g., a sequence of length k) if s ∈ a k for some k ∈ n with s i ∈ a . the process of computing the derived kernel mainly includes three steps as below: step 1. set an initial kernelk 1 at the first layer. here the initial kernel is defined as: where x, y ∈ a k . x = {x 1 , . . . , x k } and y = {y 1 , . . . , y k } are substrings of the same length k. x = y if and only if x i = y i for i = 1, . . . , k. step 2. let f = ( f 1 , . . . , f n ), denote by | f | the length of f , so here | f | = n. then define the second layer neural response of f at t : where t 1 is the template set at the first layer, here we consider all possible substrings of length k making up the template set t 1 , so here t 1 = a k . h 1 is the transformation set at the first layer. step 3. compute the second layer derived kernel by normalizing the inner product of two neural responses: where ·, · l 2 (t 1 ) denotes the l 2 inner product with respect to the uniform measure 1 |t 1 | ∑ t∈t 1 δ t , where |t 1 | is the cardinality of the template set t 1 and δ t is the dirac measure; with correlation normalization: . this process can continue if appropriate higher level templates are defined. at each layer (local) derived kernels are built by recursively pooling over previously defined local kernels. here, for the 2-layer derived kernel, pooling is accomplished by taking an average over a set of transformations which calculating the frequency of a template that occurs in a sequence. in this paper we deal with inner product kernels k which satisfies the mercer condition, are known to be an instance of reproducing kernels. next with correlation normalization,k is also a reproducing kernel andk(x, x) = 1 for all x ∈ x. the kernel function is symmetric (i.e., k( f , g) = k(g, f )), and non-negative (i.e., k( f , f ) ≥ 0), therefore it can be interpreted as a measure of similarity. we first apply the kernel to the set r which contains nucleic acid sequences, and denote it byk r 2 , and then apply the kernel to the set of amino acid sequences p, denote it byk p 2 , and lastly combine two kernels in a natural way by tensor product for the set of rna-protein pairs . the reproducing kernel for two rna-protein pairs (r, p), (r , p ) ∈ r × p is defined by: k dk 2 ((r, p), (r , p )) =k r 2 (r, r )k p 2 (p, p ). since bothk r 2 (r, r ) andk p 2 (p, p ) are positive definite kernels,k dk 2 ((r, p), (r , p )) is obviously a positive definite kernel too [50] . after combining the kernel with other kernel-based supervised learning algorithm, we can predict rpis to any rna-protein pairs with known primary sequences. the rls algorithm is one of the most widely used models for regression. let k be a kernel on a finite set x. write h k to denote the inner product space of functions on x defined by k. supposez = {(x i , y i )} m i=1 is a sample set (called the training set) with x i ∈ x and y i ∈ r for each i. the rls can be written as follows: we integrated rls with the combined kernel k =k dk 2 , hence the main construction is to computē herein, we aim to develop a novel method to distinguish rna-protein interaction pairs from non-rna-protein interaction pairs. therefore, for the binary classification case with y i ∈ {−1, 1} for each i, iff ≤ 0, the predicted class is −1 ( denotes non-interaction), otherwise it is 1 (denotes interaction). one important step of rls is to find a "good" value of the regularization parameter λ > 0 in equation (7). they were selected from an optional set λ by leave-one-out cross-validation [51] on the training data. we never used testing data for parameter selection which is under the risk of over-fitting. the sensitivity (se) and specificity (sp) are used to measure the ability of identifying positive and negative instances, respectively. they are defined by the accuracy which is used to measure the prediction quality, is defined by accuracy = tp + tn tp + tn + fp + fn . the auc (area under the receiver operating characteristic curve) is further employed to measure the predictive performance, which is 1 for perfect prediction and 0.5 for random prediction. rna regulons: coordination of post-transcriptional events rpicool: a tool for in silico rna-protein interaction detection using random forest a census of human rna-binding proteins sequence-specific interaction of r17 coat protein with its ribonucleic acid binding site rna-rna and rna-rotein interactions in coronavirus replication and transcription diverse roles of host rna binding proteins in rna virus replication rna-protein 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stacked autoencoder for accurate computational prediction applications of regularized least squares to pattern classification simulations of the dynamics at an rna-protein interface prediction of rna-binding proteins from primary sequence by a support vector machine approach prediction of rna binding sites in proteins from amino acid sequence a study of cross-validation and bootstrap for accuracy estimation and model selection the noncoding rnas and protein related biomacromolecules interaction database molecular mechanisms of long noncoding rnas function of lncrnas and approaches to lncrna-protein interactions principles and properties of eukaryotic mrnps transcriptome-wide analysis of protein-rna interactions using high-throughput sequencing a neural network method for identification of rna-interacting residues in protein a server for the computational prediction of rna-binding residues in protein sequences prediction of protein-rna binding sites by a random forest method with combined features dissecting the expression dynamics of rna-binding proteins in posttranscriptional regulatory networks deciphering the role of rna-binding proteins in the post-transcriptional control of gene expression the protein data bank pridb: a protein-rna interface database introduction to the peptide binding problem of computational immunology: new results. found generalized cross-validation as a method for choosing a good ridge parameter this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license author contributions: w.s. and j.x. conceived and designed the experiments; w.s., w.c. and j.z. performed the experiments; w.s. and w.c. analyzed the data; w.s. and d.c. contributed web tools; w.s. and j.x. wrote the paper. the authors declare no conflict of interest. key: cord-000003-ejv2xln0 authors: crouch, erika c title: surfactant protein-d and pulmonary host defense date: 2000-08-25 journal: respir res doi: 10.1186/rr19 sha: doc_id: 3 cord_uid: ejv2xln0 surfactant protein-d (sp-d) participates in the innate response to inhaled microorganisms and organic antigens, and contributes to immune and inflammatory regulation within the lung. sp-d is synthesized and secreted by alveolar and bronchiolar epithelial cells, but is also expressed by epithelial cells lining various exocrine ducts and the mucosa of the gastrointestinal and genitourinary tracts. sp-d, a collagenous calcium-dependent lectin (or collectin), binds to surface glycoconjugates expressed by a wide variety of microorganisms, and to oligosaccharides associated with the surface of various complex organic antigens. sp-d also specifically interacts with glycoconjugates and other molecules expressed on the surface of macrophages, neutrophils, and lymphocytes. in addition, sp-d binds to specific surfactant-associated lipids and can influence the organization of lipid mixtures containing phosphatidylinositol in vitro. consistent with these diverse in vitro activities is the observation that sp-d-deficient transgenic mice show abnormal accumulations of surfactant lipids, and respond abnormally to challenge with respiratory viruses and bacterial lipopolysaccharides. the phenotype of macrophages isolated from the lungs of sp-d-deficient mice is altered, and there is circumstantial evidence that abnormal oxidant metabolism and/or increased metalloproteinase expression contributes to the development of emphysema. the expression of sp-d is increased in response to many forms of lung injury, and deficient accumulation of appropriately oligomerized sp-d might contribute to the pathogenesis of a variety of human lung diseases. surfactant protein-d (sp-d) is a member of the collagenous subfamily of calcium-dependent lectins (collectins) that includes pulmonary surfactant protein a (sp-a) and the serum mannose-binding lectin [1] [2] [3] . collectins inter-act with a wide variety of microorganisms, lipids, and organic particulate antigens, and can modulate the function of immune effector cells and their responses to these ligands. this article reviews what is currently known about the sites of production, structure, function, and regulated expression of sp-d. emphasis will be placed on functional attributes, known ligand interactions, and structure-function relationships believed to be important for host defense. for additional information on sp-a and other members of the collectin family, the reader is referred to other recent reviews [4] [5] [6] . sp-d is synthesized and secreted into the airspaces of the lung by the respiratory epithelium [1] . at the alveolar level, sp-d is constitutively synthesized and secreted by alveolar type ii cells. more proximally in the lung, sp-d is secreted by a subset of bronchiolar epithelial cells, the non-ciliated clara cells. because sp-d is stored within the secretory granules of clara cells [7, 8] , it seems likely that sp-d is subject to regulated secretion via granule exocy-tosis at this level of the respiratory tract. in some species, sp-d is also synthesized by epithelial cells and/or submucosal glands associated with the bronchi and trachea [9] . although many alveolar macrophages show strong cytoplasmic and/or membrane labeling with antibody against sp-d, they do not contain detectable sp-d message. the lung seems to be the major site of sp-d production. however, there is increasing evidence for extrapulmonary sites of expression as assessed with monoclonal or affinity-purified antibodies, reverse-transcriptase-mediated pcr (rt-pcr), and/or hybridization assays of tissues from humans and other large mammals [10 • ,11-14] (summarized in table 1 ). it is difficult to entirely exclude crossreactions or amplification of related sequences; however, localization to many of these sites in human tissues was confirmed by using monoclonal antibodies in combination with rt-pcr with sequencing of the amplified products [10 • ]. non-pulmonary expression seems to be largely restricted to cells lining epithelial surfaces or ducts and certain glandular epithelial cells that are in direct or indirect continuity with the environment. notable exceptions to this generalization might include heart, brain, pancreatic islets, and testicular leydig cells. sp-d has also been identified in amnionic epithelial cells by immunohistochemistry [15] ; however, it is unclear whether this is synthesized locally or derived from the lung by way of the amniotic fluid. interestingly, in many of these sites sp-d microscopically co-localizes with gp-340, an sp-d binding protein and putative sp-d receptor [10 • ]. sites of extrapulmonary expression have also been described in small mammals. in the rat, sp-d message was identified in rna extracted from skin and blood vessel [16] , and both protein and message were identified in gastric mucosa [17] and mesentery [13] . using rt-pcr, sp-d message has also been identified in mouse stomach, heart, and kidney [14] . sp-d (43 kda, reduced) consists of at least four discrete structural domains: a short, n-terminal domain; a relatively long collagenous domain, a short amphipathic connecting peptide or coiled-coil neck domain, and a c-terminal, ctype lectin carbohydrate recognition domain (crd). each molecule consists of trimeric subunits (3 × 43 kda), which associate at their n-termini (fig. 1) . although most preparations of sp-d contain a predominance of dodecamers (that is, four trimeric subunits), the proportions of various oligomers vary between species. for example, rat lavage and recombinant rat sp-d are almost exclusively assembled as dodecamers (four trimers), whereas recombinant human sp-d is secreted as trimers, dodecamers and higher-order multimers [18] . sp-d isolated from the lavage of some patients with alveolar proteinosis consists predominantly of higher-order multimers, which can contain up to 32 (or more) trimeric subunits (fig. 1 ). recent crystallographic and mutagenesis studies suggest that the structural determinants of saccharide binding are similar to those originally described for mannose-binding lectin [19,20,21 • ,22 • ]. at least two bound calcium ions and two intrachain disulfide crosslinks stabilize the required tertiary structure, and glu321 and asn323 within the crd participate in glucose/mannose type recognition. interactions with at least one glycolipid ligand, phosphatidylinositol (pi), require the participation of the c-terminal end of the protein [23, 24] . a trimeric cluster of crds is necessary for high-affinity binding to carbohydrate ligands [21 • ,25]. the crystal structure of human sp-d suggests that the spatial distribution of crds within a trimeric subunit permits simultaneous and cooperative interactions with two or three glycoconjugates displayed on the surface of a particulate ligand [21 • ]. furthermore, solid-phase binding studies have shown that monomeric crds have an approximately 10-fold lower binding affinity for multivalent ligands than trimeric crds. crystallographic studies of human sp-d further suggest that the spatial organization of crds within a trimer is stabilized by interactions of the c-terminal sequence with the trimeric neck domain [21 • ,26]. interestingly, the three crds show a deviation from threefold asymmetry, suggesting some flexibility of the crds in relation to the neck. thus, the dependence of the binding of pi on the c-terminal sequence could reflect conformational effects, rather than the direct participation of this sequence in ligand interactions. the collagen domain length of sp-d is highly conserved and lacks interruptions in the repeating gly-x-y sequence (in which x and y are different amino acids). as for other collagenous proteins, this domain is enriched in imino acids and contains hydroxyproline. unlike sp-a, sp-d also contains hydroxylysine. although the collagen domain of rat, human, bovine, and mouse sp-d lacks cysteine residues, cdna sequencing has identified a codon for cysteine within the collagen domain of pig sp-d [27 • ]; this suggests the possibility of alternative patterns of chain association and oligomeric assembly for pig sp-d. the first translated exon of sp-d contains a highly conserved and unusually hydrophilic gly-x-y sequence that shows little homology with the remainder of the collagen sequence. the functional significance of this region is unknown. however, it has been suggested that this region contributes to oligomer assembly or mediates interactions with cellular receptors. the collagen domain determines the maximal spatial separation of trimeric, c-terminal lectin domains within sp-d molecules, but might also contribute to normal oligomeric assembly and secretion. for example, deletion of the entire collagen domain of rat sp-d results in the secretion of trimers rather than dodecamers [28] . in addition, 2,2-dipyridyl, an inhibitor of prolyl hydroxylation that interferes with the formation of a stable collagen helix, causes the intracellular accumulation of 43 kda monomers and dimers [29] . in any case, the complete conservation of the number of gly-x-y triplets suggests that the spatial separation of trimeric crds is critical for normal sp-d function. the n-terminal peptide of the mature protein contains two conserved cysteine residues at positions 15 and 20. these residues participate in interchain disulfide crosslinks that stabilize the trimer, as well as the n-terminal association of four or more trimeric subunits. stable oligomerization of trimeric subunits permits cooperative or bridging interactions between spatially separated binding sites on the same surface or on different particles. the process of forming interchain disulfide bonds is complex, and appropriate crosslinking of the n-terminal domains might be rate limiting for secretion [30] . subcellular fractionation studies suggest that interchain bonds form initially between the three chains of a trimeric subunit. subsequent rearrangements within the rough endoplasmic reticulum might allow the covalent crosslinking of a single chain from one subunit and two crosslinked chains of another, with the associated elimination of free thiol groups. mutant proteins that contain unpaired n-terminal cysteine residues are not secreted. however, it is unclear whether this results from abnormalities in disulfide bonding itself, or the failure to stabilize the required n-terminal conformation. the collagen domain contains hydroxylysyl-derived glycosides and a single n-linked oligosaccharide. in most species (human, rat, mouse, and cow) the site of n-linked glycosylation is located near the n-terminal end of the collagenous domain. recently, it was shown that pig sp-d has an additional potential site of n-linked glycosylation within the crd [27 • ]. although rat and human lung lavage sp-d seem to be sialylated, as suggested by charge heterogeneity and cleavage with highly purified neuraminidase, preparations of human amniotic fluid and bovine lavage sp-d recovered from amniotic fluid showed predominantly complex type biantennary structures and no sialic acid [31] . a variant form of sp-d (50 kda) has been identified in lavage from a subset of human lavage samples; this protein shows o-linked glycosylation of threonyl residues within the n-terminal peptide domain [32 • ]. at present, the functional significance of these sugars is not known. the presence of o-linked glycosylation within the n-terminal domain might be predicted to interfere with normal dodecamer assembly. in this regard, the o-glycosylated 50 kda form of human sp-d is recovered as trimeric subunits or smaller species. as for many glycoproteins, the functional role of the attached carbohydrate is unknown. mutational analysis has shown that the n-linked sugar on rat sp-d is not required for secretion, for dodecamer formation, or for interactions with a variety of microorganisms [29,33]. consistent with its designation as a 'mannose-type' c-type lectin, sp-d preferentially binds to simple and complex saccharides containing mannose, glucose, or inositol [34, 35] . sp-d also interacts with specific constituents of pulmonary surfactant including pi [36-38] and glucosylceramide [39] . binding to glucosylceramide involves interactions of the carbohydrate-binding sequences of the crd with the glucosyl moiety. however, the interaction of sp-d with pi involves interactions with the lipid, as well as crd-dependent interactions with the inositol moiety [24, 40] . microorganisms are surfaced with a diverse and complex array of polysaccharides and glycoconjugates, and most classes of microorganism contain one or more sugars recognized by sp-d. however, the outcome of this interaction depends on the specific organism and can be modified by the conditions of microbial growth. the potential consequences of this interaction include the following: varying degrees of lectin-dependent aggregation (namely, microbial agglutination), enhanced binding of microorganisms or microbial aggregates to their 'receptors' on host cells, phagocyte activation, and opsonic enhancement of phagocytosis and killing, potentially involving one or more cellular receptors for sp-d. binding to organisms in suspension is often -but not always -accompanied by some degree of aggregation. sp-d binds to purified lipopolysaccharide (lps) isolated from a variety of gram-negative organisms [35, 41] . in addition, lps is the major cell wall component that is labeled on lectin blotting of outer membranes isolated from escherichia coli [41] . the latter interactions involve the recognition of the core oligosaccharide domain, which contains glucose and heptose [41] . sp-d interacts preferentially with purified lps molecules characterized by short or absent o-antigens and preferentially agglutinates bacterial strains expressing a predominance of rough (o-antigen-deficient) lps [41, 44] . although the core oligosaccharide domain of lps constitutes the major ligand for sp-d on at least some gram-negative bacteria, the mechanism of interaction with this group of microorganisms is probably heterogeneous. sp-d binds to some smooth, unencapsulated strains of gram-negative bacteria by immunofluorescence. the mechanism is uncertain; the quantity or quality of binding differs from that observed for rough strains and does not necessarily result in agglutination. lps molecules on the surfaces of bacteria show heterogeneity in the extent of maturation, so it is possible that this interaction is mediated by a subpopulation of lps with deficient o-antigens and that the density of binding sites is too low for high-affinity binding. the recognition of the surface glycoconjugates on gramnegative bacteria by sp-d depends not only on the expression of lectin-specific residues by a given strain or species, but also on the accessibility of these residues [1, 45] . for example, sp-d binds inefficiently to the core region of lps of encapsulated klebsiella, but efficiently agglutinates the corresponding unencapsulated phase variants. interactions of sp-d with the core oligosaccharides of gram-negative organisms are also influenced by the number of repeating saccharide units associated with the terminal o-antigen of the lps [41,44]. other potential ligands include the o-antigen domain of lps, certain capsular polysaccharides, and membraneassociated glycoproteins. in this regard, sp-d can bind to di-mannose containing o-antigens expressed by a subset of klebsiella serotypes (i ofek, h sahly and ec crouch, unpublished data). although other c-type lectins, specifically sp-a and the mannose receptor, can interact with specific capsular polysaccharides [46], a specific interaction of sp-d with capsular glycoconjugates or exopolysaccharides has not been described. the mechanism of interaction with gram-positive organisms has not been elucidated. lipoteichoic acids, which are the major glycolipids associated with the gram-positive cell wall, do not detectably compete with lps for binding to sp-d (i ofek, a mesika, m kalina, y keisari, d chang, d mcgregor and ec crouch, manuscript submitted). in preliminary studies we observed that binding was competed only partly with maltose and/or edta, raising the possibility that binding might be more complex than for some gram-negative organisms. . however, similar effects were observed when the neutrophils were preincubated with sp-d, and there was only a slight enhancement of uptake when bacteria were incubated with human sp-d and washed before their addition to neutrophils. notably, the extent of binding and internalization was dependent on the extent of multimerization, with human sp-d multimers demonstrating the highest potency. differences in cell type, the extent of sp-d multimerization, or differences in size or organization of bacterial aggregates could account for some of the apparent inconsistencies. although lps mediates the binding of sp-d to at least some gram-negative bacteria, sp-d can also bind to spein the latter study the authors suggested that fungal aggregation inhibits phagocytosis. interestingly, sp-d binding directly inhibited fungal growth and decreased the outgrowth of pseudohyphae, the invasive form of the fungus, in the absence of phagocytic cells [57] . it is possible that these effects are also secondary to agglutination, possibly as result of nutrient deprivation. purified rat and human sp-d inhibit the infectivity and hemagglutination activity of influenza sp-d can interact with host cells, both directly and indirectly. as indicated above, sp-d can enhance the phagocytosis and killing of certain microorganisms and enhance the oxidant response to microbial binding. however, at present there is only one study that suggests that the enhancement of phagocytosis by sp-d might involve the participation of an opsonic receptor. furthermore, the enhanced uptake of iav seems to be mediated by viral aggregation, with enhanced interactions of the virus with its natural receptors on the host cell. in any case, sp-d can interact directly with host cells, and in some cases can influence their behavior. sp-d is chemotactic and haptotactic for neutrophils and certain mononuclear phagocytes [59 • ,67-69] and can elicit directional actin polymerization in alveolar macrophages [69] . in this regard, sp-d is considerably more potent than sp-a. early studies with natural proteins isolated from silicotic animals reported directed effects on the oxidant metabolism of isolated alveolar macrophages [70] . however, such effects can probably be attributed to endotoxin contamination and/or aggregation. purified dodecamers do not significantly increase the production of nitric oxide [71] or of proinflammatory cytokines such as tumor necrosis factor-α (y kesari, h wang, a mesika, e crouch and i ofek, unpublished data). interestingly, purified sp-d has been reported to increase the production of several metalloproteinases in the absence of a significant effect on proinflammatory cytokine production [72] . despite the ability of sp-d to modulate a variety of cellular functions, little is currently known about potential cellular receptors for this protein. compartments [73] , but it is unclear whether the uptake is receptor dependent and whether sp-d is being internalized in association with specific ligands. there are at least two classes of binding to host cells: crd-dependent and crd-independent. some studies have demonstrated crd-dependent binding to phagocytes that can be inhibited with edta or competing saccharides, both in vitro and in vivo. as indicated above, the ability of sp-d to elicit the chemotaxis of neutrophilic and monocytic cells depends on the lectin activity of sp-d [68] . in addition, kuan and coworkers reported that extracting formaldehyde-fixed alveolar macrophages with detergents largely eliminates the binding of purified sp-d, suggesting a membrane-associated ligand or glycolipid receptor [73] . dong and wright have extended these findings and suggest that pi can contribute to sp-d binding by alveolar macrophages [74] . it is of interest that sp-d can bind to recombinant scd14 through interactions with n-linked oligosaccharides [51 • ]. given that the membrane-associated form of cd14 is widely expressed on host cells, it is possible that cd14 can serve as a binding site on macrophages and other cell types. the phagocytic uptake of certain bacteria by neutrophils is also inhibited by calcium chelation or competing sugars [42]; however, this could result from the inhibition of microbial agglutination rather than lectin-dependent interactions with the phagocyte. wang et al suggested that sp-d can bind to lymphocytic cells by a lectin-dependent mechanism [75 •• ] . in this regard, it is interesting to note that glucosylceramide, a ligand for sp-d in vitro, is one of the most abundant neutral glycolipids expressed by lymphoid cells. reid and co-workers were the first to present evidence for lectin-independent binding [76] . these and other studies suggested that binding does not involve known c1q or collectin receptors. the only putative receptor protein, gp-340, is a widely expressed member of the scavenger receptor superfamily [77,78 • ]. it binds to the crd of sp-d in a calcium-dependent manner that does not require the lectin activity of sp-d. although the protein has been immunolocalized to alveolar macrophage membranes and distributes together with sp-d in many different human tissues [10 • ,77], it has not yet been shown to mediate the binding of sp-d to these cells or to participate in signal transduction events. the cdnas isolated from lung have not shown a membrane-spanning region [77] , and the protein is abundant as a soluble component in bal. given that gp-340 is a highly multimerized protein that contains numerous potential ligand binding domains (fig. 1b) , it is possible that the protein cooperates with sp-d in the neu-tralization or clearance of certain ligands rather than specifically mediating the interactions of sp-d with host cells. wright and co-workers have demonstrated the binding of sp-d to isolated type ii pneumocytes. the mechanism seems distinct from the binding to macrophages [79 • ]. the binding was dependent on concentration, time, and temperature and required calcium; it was not sensitive to protease treatment or to pi-phospholipase c. although the internalized sp-d was degraded or recycled to lamellar bodies, sp-d binding did not alter the uptake of surfactant lipids. sp-d has demonstrated comparatively few direct effects on the metabolism of host cells, at least in situations where self-aggregation and endotoxin contamination have been excluded. one possible explanation is that modulation of cellular function requires the prior interaction of sp-d with a ligand. this would have numerous potential physiological advantages, because the presence of 'active' protein might be restricted to sites of microbial or antigenic deposition. the binding of complex, multivalent, particulate antigens to two or more crds could markedly alter the conformation of sp-d molecules, with respect to the spatial orientation of the arms in relation to the n-terminal crosslinking domain and/or with respect to the spatial orientation of the crds within a given trimeric subunit. thus, the 'charging' of sp-d with a particulate ligand could lead to local or distant conformational changes that expose 'cryptic' binding sites for cellular receptors. there is some preliminary evidence consistent with the notion that the interaction of sp-d with a ligand alters its capacity to activate host cells. table 3 and discussed below. sp-d can be isolated in different multimeric forms from proteinosis lavage [32 • ] and are produced by chinese hamster ovary k1 cells transfected with human sp-d cdna [18] . as described previously, the effects of sp-d on the neutrophil response to influenza virus are highly dependent on the ability of sp-d to agglutinate the viral particles, and the agglutination activity is directly correlated with the extent of multimerization. trimers can bind to the virus but have little capacity to modulate neutrophil interactions. by contrast, highly multimerized proteins show greater activity than dodecamers [81] . given these observations, factors that favor enhanced oligomerization or lead to the accumulation of trimeric subunits promote might influence sp-d function. for example, the liberation of active trimers by a hypothetical microbial protease could lead to the accumulation of molecules that might inhibit the aggregation-dependent activities of sp-d. in contrast, recombinant trimeric crds can stimulate chemotaxis [67] and decrease viral infectivity [65 • ]. although higher-order oligomers of sp-d can self-aggregate and precipitate in the presence of calcium in vitro, the functional consequences are not known. the lectin activity of sp-a is decreased after the nitric oxide-dependent nitration of tyrosine residues [82] , and nitration decreases the ability of sp-a to enhance the adherence of pneumocystis to alveolar macrophages [83] . however, similar findings have not yet been reported for sp-d. conditions of mildly acidic ph, as might be found in endocytic compartments, are predicted to disrupt the lectin-dependent activities of sp-d [34]. proteolytic degradation remains an important possibility. however, sp-d is highly resistant to degradation by a wide variety of neutral proteases in vitro, and degradation products have not yet been shown to accumulate under pathological conditions in vivo. glucose concentrations at levels encountered in diabetes can interfere with sp-d's ability to interact with specific strains of iav or other microorganisms in vitro [84 • ]. many microorganisms release cell wall polysaccharides or glycoconjugates, which might interfere with the binding of collectins to the same or other organisms. in this regard, sp-d recovered from rats after the instillation of lps into the airway shows decreased lectin activity, which is attributed to occupancy of the crd with lps [49 • ]. it seems reasonable to speculate that some organisms might compete with other organisms for binding to sp-d. such a situation could conceivably predispose to secondary infections. lastly, the potential inhibitory effects of competing saccharide ligands presents important methodological considerations for experiments using carbohydrate-containing cell culture medium or buffers. non c-type lectins (such as ficolins) it is difficult to predict the functions of sp-d within the airspace. other lectins with overlapping specificity are also present. although the levels of mannose-binding lectin are probably low in the absence of increased vascular permeability, sp-a and the macrophage mannose receptor could conceivably interact with the same ligands in the distal airways and alveoli. such interactions could lead to antagonistic or cooperative effects. furthermore, we have little knowledge regarding the microanatomic distribution of these molecules in specific circumstances in vivo. although most sp-a is probably associated with the insoluble phase of the alveolar lining material, and the macrophage mannose receptor is membrane-associated, the distribution might be altered in the setting of lung injury. models of sp-d deficiency show no detectable anatomical or physiological abnormalities at birth. however, the animals gradually develop a patchy, subpleural alveolar lipidosis with associated type ii cell hypertrophy, the accumulation of enlarged and foamy macrophages, and an apparent expansion of peribronchial lymphoid tissue [85 • ,86 • ]. interestingly, the mice eventually develop distal-acinar emphysema and areas of subpleural fibrosis, which could reflect a continuing inflammatory reaction associated with abnormal oxidant metabolism and metalloproteinase activity [87 • ]. by contrast, sp-a-deficient mice (-/-) show essentially normal respiratory function and surfactant lipid metabolism [88, 89] but numerous apparent host defense abnormalities [90] . the capacity of sp-d to bind to specific strains of influenza a in vitro is highly correlated with the capacity of the virus to proliferate in mice in vivo [62] . specifically, strains with more oligosaccharide attachments on the ha are preferentially neutralized by sp-d in vitro and show decreased proliferation in mice. because the administration of mannan together with the virus increased the replication of iav in the lung, the involvement of a mannose-type, c-type lectin was implicated. sp-d-sensitive iav strains also replicate to higher titers in the lungs of diabetic mice than in nondiabetic controls [84 • ]. replication of the virus is positively correlated with blood glucose level, and decreases in response to insulin treatment. significantly, blood glucose levels comparable to those measured in the diabetic mice were sufficient to inhibit the interaction of sp-d with these viral strains in vitro. pr-8, a strain that does not interact with sp-d but does interact with sp-a, replicated to the same extent in diabetic and control mice. sp-d levels increase in association with certain infections. for example, sp-d levels, but not the levels of serum mannose-binding lectin, increase markedly after iav infection [62] . impressive increases in sp-d have also been observed in murine models of pneumocystis carinii [91] and p. aeruginosa infection [92] . sp-d-deficient mice have not yet been extensively characterized with respect to host defense function. however, they show decreased viral clearance and enhanced inflammation after challenge with respiratory syncytial virus [93] and iav (am levine, personal communication). in addition, they show increased inflammation, increased oxidant production, and decreased macrophage phagocytosis in response to intratracheally instilled group b streptococcus and haemophilus influenzae (am levine, personal communication). although the overexpression of wild-type sp-d in type ii pneumocytes with the sp-d-deficient mice can prevent the lipidosis and inflammatory changes [94] , the ability of overexpressed wild-type sp-d or exogenous sp-d to ameliorate these abnormalities has not yet been described. the coexisting pulmonary abnormalities also complicate the interpretation of challenge models. for example, macrophage activation might enhance killing and offset any decrease that results more directly from sp-d deficiency. sp-d deficiency modifies the host response to instilled lps with decreased lung injury and inflammatory cell recruitment [50]. molecules that can bind to potential antigens and deliver them to macrophages and other antigen-presenting cells might contribute to the development of acquired immunity. in this regard, a few published observations suggest possible roles in the development of humoral and/or cellular immunity in response to microorganisms or complex organic antigens. for example, sp-d can decrease interleukin-2dependent t-lymphocyte proliferation [95 • ]. interestingly, single-arm mutants were at least as potent as intact dodecamers in mediating this effect. sp-d also binds to oligosaccharides associated with dust mite allergen [96 • ], and can inhibit the binding of specific ige to these allergens, possibly through direct, crd-dependent binding to lymphocytes [96 • ]. thus, alterations in the level of sp-d (or the state of oligomerization) might influence the development of immunological responses and contribute to the pathogenesis of asthma and other hypersensitivity disorders. there are other potential interplays between humoral immunity and collectins with regard to antimicrobial host defense. for example, increased glycosylation of iav coat proteins, an adaptation that is believed to help the virus to evade antibody-mediated neutralization, is associated with increased reactivity with sp-d and other collectins [62]. thus, the relative potential importance of antibody and collectin-mediated host defenses might be influenced by subtle variations in the structure of the microbial surface. there is little recent information on the developmental regulation of sp-d expression. in general, sp-d increases rapidly late in gestation [97] [98] [99] [100] . the production of sp-d increases during the culture of fetal lung explants, and expression can be increased with glucocorticoids [98, 100, 101] . the exposure of fetal rats to glucocorticoids in vivo leads to precocious expression with increased numbers of sp-d-expressing cells and increased cellular levels of sp-d message [98, 101, 102] . although sp-d is produced constitutively within the lung, protein accumulation and gene expression are inducible and increases in sp-d expression have been observed in a number of disease states or models (tables 4 and 5 ). in general, the synthesis and secretion of sp-d increase in association with lung injury and activation of the respiratory epithelium [1] . for example, levels of sp-d mrna and sp-d accumulation are increased within 24-72 h after intratracheal instillation of lps [103 • ], and sp-d expression by alveolar and bronchiolar epithelial cells increases after exposure of rats to 95% o 2 for 12 h [104] . keratinocyte growth factor (kgf) increases sp-d expression and protein production in association with pneumocyte hyperplasia and after injury caused by bleomycin [105] . in addition, the levels of sp-d can increase markedly in response to the overexpression of certain cytokines, such as interleukin-4, or in response to microbial challenge [91, 92] . studies of the upstream regulatory region of the sp-d gene have demonstrated increased promoter activity in the presence of glucocorticoids, which is consistent with the findings in vivo and in lung organ culture [106] . however, no functional glucocorticoid response elements have been identified, and the effects of dexamethasone seem to be secondary and involve the effects of other transregulatory molecules. the activity of the human sp-d promoter is dependent on a conserved activator protein-1 (ap-1) element (-109) that binds to members of the fos and jun families of transcriptional factors [107] . in addition, the promoter contains multiple functional binding sites for ccaat-enhancer-binding protein (c/ebp) transcription factors. mutagenesis experiments suggest that these are required for basal and stimulated promoter activity, and promoter activity is markedly increased in h441 cells after co-transfection with c/ebpβ cdna (yc he and e crouch, unpublished data). the importance of the conserved ap-1 element and the presence of multiple binding sites for c/ebp transcription factors is consistent with the observed modulation of sp-d expression in the setting of tissue injury. sp-d promoter activity is not dependent on the binding of thyroid transcription factor 1 (ttf-1) [107] . however, promoter activity is dependent on two interacting forkhead binding sites, upstream and downstream of the ap-1 element; these sites bind to hepatic nuclear factor-3α and apparently other forkhead box proteins in h441 lung adenocarcinoma nuclear extracts [107] . initial comparison of genomic and cdna sequence suggested the existence of genetic polymorphisms in the sp-d coding sequence, including one in the n-terminal propeptide domain (thr11 compared with met11 in the mature protein) and three additional differences within the collagen domain at positions 102, 160, and 186 [108] . the latter substitutions are conservative to the extent that they are not expected to disrupt the collagen helix. floros table 5 increased sp-d accumulation or expression in animal models silicosis rat [118] hyperoxia rat [104] endotoxin (lps) rat [103] challenge with p. aeruginosa mouse [92] challenge with iav mouse [62] challenge with pneumocystis carinii scid mouse [91] rat [119] overexpression of interleukin-4 mouse [120] scid, severe combined immunodeficiency. and co-workers have recently confirmed the existence of polymorphisms at positions 11 and 160 of the mature protein [109] . the potential biological significance, if any, is not known. interestingly, the 50 kda variant of sp-d showed o-linked glycosylation of thr11 [32 • ], suggesting that this polymorphism might be associated with altered glycosylation. interestingly, the 50 kda variant was recovered as trimeric subunits, raising the possibility that differences in the glycosylation of residue 11, which is immediately n-terminal to cys15, could influence multimerization and the capacity of sp-d to participate in bridging interactions. there is increasing evidence that sp-d interacts specifically with a wide variety of respiratory pathogens, modulates the leukocyte response to these organisms, and participates in aspects of pulmonary immune and inflammatory regulation (table 6) . sp-d can influence the activity of phagocytes through crd-dependent and crd-independent interactions. at least some of the effects of sp-d result from aggregation with enhanced binding of the agglutinated ligand to their natural 'receptors'. although the lung is the major site of sp-d expression, it is likely that the protein has more generalized roles in host defense and the acute response to infection and tissue injury. 16 collectins and pulmonary host defense immunomodulatory functions of surfactant lung surfactant proteins involved in innate immunity interactions of surfactant protein a with pathogens the role of collectins in host defense structural aspects of collectins and receptors for collectins immunocytochemical localization of surfactant protein d (sp-d) in type ii cells, clara cells, and alveolar macrophages of rat lung surfactant protein d: subcellular localization in nonciliated bronchiolar epithelial cells localization and developmental expression of surfactant proteins d and a in the respiratory tract of the mouse localiza-• tion of lung surfactant protein d (sp-d) on mucosal surfaces in human tissues this recent study was the first to systematically investigate the extrapulmonary expression of sp-d in human tissues immunolocalization of sp-d in human secretory tissues surfactant protein a and d expression in the porcine eustachian tube expression of hydrophilic surfactant proteins by mesentery cells in rat and man mouse surfactant protein-d. cdna cloning, characterization, and gene localization to chromosome 14 surfactant proteins a (sp-a) and d (sp-d): levels in human amniotic fluid and localization in the fetal membranes antiviral activity of bovine collectins against rotaviruses recombinant sp-d carbohydrate recognition domain is a chemoattractant for human neutrophils interactions of pulmonary surfactant protein d (sp-d) with human blood leukocytes surfactant proteins a and d specifically stimulate directed actin-based responses in alveolar macrophages rat surfactant protein d enhances the production of oxygen radicals by rat alveolar macrophages effects of endotoxin on surfactant protein a and d stimulation of no production by alveolar macrophages induction of matrix metalloproteinase biosynthesis in human alveolar macrophages exposed to surfactant protein d (sp-d): possible roles in pulmonary host defense binding of surfactant protein d (sp-d) to membrane glycolipids on alveolar macrophages inhibitory effect of •• pulmonary surfactant proteins a and d on allergen-induced lymphocyte proliferation and histamine release in children with asthma surfactant protein d binding to alveolar macrophages cloning of gp-340, a putative opsonin receptor for lung surfactant protein d isolation and characterization of a new member of the scavenger receptor superfamily, glycoprotein-340 (gp-340), as a lung surfactant protein-d binding molecule gp340 was the first protein shown to bind to sp-d in a crd-independent manner. although it can be found on the surface of alveolar macrophages, it remains uncertain whether it can function as a cellular receptor binding and uptake of surfactant • protein d by freshly isolated rat alveolar type ii cells this study describes the direct binding of sp-d to type ii pneumocytes. the mechanism appears distinct from that observed for alveolar macrophages this study describes specific ultrastructural alterations in the organization of phospholipid mixtures containing phosphatidylinositol. similar tubular structures are found in type ii cell cultures collectins and pulmonary innate immunity nitration of surfactant protein a results in decreased ability to aggregate lipids nitrated sp-a does not enhance adherence of pneumocystis carinii to alveolar macrophages increased suscepti-• bility of diabetic mice to influenza virus infection: compromise of collectin-mediated host defence of the lung by glucose? is uncontrolled diabetes mellitus associated with defective collectin function? surfactant protein-d regulates surfactant phospholipid home ostasis in vivo this paper and the following paper by botas and coworkers [86 • ] describe the phenotype of sp-d deficient transgenic mice altered surfactant homeostasis and alveolar type ii cell morphology in mice lacking surfactant protein d increased metalloproteinase activity, oxidant production, and emphysema in surfactant protein d gene-inactivated mice altered surfactant function and structure in sp-a gene targeted mice surfactant metabolism in surfactant protein a-deficient mice surfactant protein a (sp-a) gene targeted mice p. carinii induces selective alterations in component expression and biophysical activity of lung surfactant interleukin-4 enhances pulmonary clearance of pseudomonas aeruginosa surfactant protein-d modulates lung inflammation with respiratory syncytial virus infection in vivo pulmonary-specific expression of sp-d corrects pulmonary lipid accumulation in sp-d gene-targeted mice • recombinant rat surfactant-associated protein d inhibits human t lymphocyte proliferation and il-2 production this paper was the first to describe direct, inhibitory effects of sp-d on the stimulated proliferation of blood lymphocytes interaction of • human lung surfactant proteins a and d with mite (dermatophagoides pteronyssinus) allergens this study was the first to suggest that interactions of sp-d with glycoconjugates on particulate allergens might influence the immune response developmental expression of pulmonary surfactant protein d (sp-d) modulation of surfactant protein d expression by glucocorticoids in fetal rat lung ontogeny of surfactant apoprotein d, sp-d, in the rat lung regulation of surfactant protein d in human fetal lung regulation of surfactant protein d expression by glucocorticoids in vitro and in vivo pre-and postnatal stimulation of pulmonary surfactant protein d by in vivo dexamethasone treatment of rats surfactant proteins a • and d increase in response to intratracheal lipopolysaccharide this important paper suggests that sp-a and sp-d participate in the acute response to lung injury brief exposure to 95% oxygen alters surfactant protein d and mrna in adult rat alveolar and bronchiolar epithelium kgf increases sp-a and sp-d mrna levels and secretion in cultured rat alveolar type ii cells characterization of the human surfactant protein d promoter: transcriptional regulation of sp-d gene expression by glucocorticoids proximal promotor of the surfactant protein d (sp-d) gene: regulatory role of ap-1, forkhead box, and gt-box binding proteins genomic organization of human surfactant protein d (sp-d). sp-d is encoded on chromosome 10q22.2-23.1 novel, non-radioactive, simple and multiplex pcr-crflp methods for genotyping human sp-a and sp-d marker alleles recognition of klebsiella pneumoniae by pulmonary c-type lectins deficient hydrophilic lung surfactant proteins a and d with normal surfactant phospholipid molecular species in cystic fibrosis serial changes in surfactant-associated proteins in lung and serum before and after onset of ards decreased contents of surfactant proteins a and d in bal fluids of healthy smokers surfactant proteins a and d in premature baboons with chronic lung injury (bronchopulmonary dysplasia). evidence for an inhibition of secretion deficiencies in lung surfactant proteins a and d are associated with lung infection in very premature neonatal baboons pulmonary surfactant protein d in sera and bronchoalveolar lavage fluids surfactant protein-a concentration in bronchoalveolar lavage fluids of patients with pulmonary alveolar proteinosis surfactant protein d. increased accumulation in silica-induced pulmonary lipoproteinosis surfactant protein-d mediates aggregation of pneumocystis carinii il-4 increases surfactant and regulates metabolism in vivo this paper was the first to definitively demonstrate a classical opsonic activity of sp-d. the studies further suggest the existence of an opsonic receptor on alveolar macrophages. key: cord-022235-6ircruag authors: pugsley, anthony p. title: later stages in the eukaryotic secretory pathway date: 2012-12-02 journal: protein targeting doi: 10.1016/b978-0-12-566770-8.50009-4 sha: doc_id: 22235 cord_uid: 6ircruag nan the secretory pathway of eukaryotic cells comprises a succession of compartments, the secretory organelles, through which proteins pass en route to their final destinations. although each secretory organelle has its own special characteristics, a number of basic features are common to all of them. some proteins pass more or less unimpeded through the entire length of the secretory pathway, whereas others are retained in secretory organelles. this raises two of the fundamental questions which will be addressed in this chapter, namely, what determines whether a protein will be taken out of circulation at a particular step in the pathway, and how tight is the separation between secretory organelles? ultrastructure and cell fractionation studies indicate that there is little or no physical link between the rer and the golgi. furthermore, specific proteins are known to reside in individual compartments of the golgi apparatus. as we shall see, the physical separation of golgi enzymes is also indicated by the succession of posttranslational modifications to which secretory proteins are subjected, by in situ immunocytochemistry, and by the separation of golgi-derived vesicles containing different en zymes. how then do secretory proteins move between these compart ments, and is the secretory pathway a continuous gradient of secretory organelles or are they functionally and structurally independent? a feature common to all stages of the secretory route beyond the rer is that proteins move between secretory organelles in specific classes of transport vesicles. consequently, soluble secretory proteins never come into direct contact with the outer face of the organelle to which they are being targeted and therefore can play no direct part in sorting. integral membrane proteins usually have segments exposed on the cytoplasmic face of the transport vesicle which could be recognized by receptors on the target organelle. microinjected antibodies recognizing the c-terminal, cytoplasmic tails of plasma membrane proteins can prevent their trans port to the cell surface (25, 590) , but this is probably due to antibodyinduced changes in protein conformation which make the protein incom petent for transport along the secretory pathway, rather than to inhibition of receptor-secretory protein interactions. in this chapter, we will consider three different ways in which sorting of secretory proteins might occur: (i) all secretory proteins have signals which target them successively through the secretory organelles and then on to their final target; some proteins remain in different organelles because they lack the signal necessary for targeting to the next organelle in the pathway. (ii) secretory proteins only have sorting signals for the last stage in the secretory pathway; some proteins have retention signals which pre vent them joining the bulk flow through the secretory pathway, thereby causing them to be retained in specific organelles. (iii) as (ii), except that secretory organelle proteins pass through the secretory pathway in the bulk flow and are recycled via signaldependent counterflow. if either of the last two models are correct, then molecules devoid of retention or sorting signals should travel through the secretory pathway as part of the bulk flow. the rate at which molecules are transported out of the cell will thus be determined by the rate at which they diffuse to sites within the rer and golgi where transport vesicles are formed and depart en route to the next compartment in the chain. this flow rate has recently been measured with the n-glycosylation acceptor tripeptide asn-tyr-thr (section v.b.i). if this tripeptide contains a radioiodinated tyr residue, its progress through the secretory pathway, which it seems to enter by diffusing across the rerm, can be followed by chromatography and autoradiography. wieland et al. (1190) found that the tripeptide was gly cosylated in the er (this prevented it from leaking back into the cytosol), then trimmed by mannosidases located in the golgi (section v.d), and finally secreted into the medium. tripeptide was detected in the medium after 5-10 min, depending on the cell type, which is considerably faster than the time required for the transport of most secretory proteins through the secretory pathway. thus, bulk flow is very efficient, implying that diffusion through the lumen of the er and golgi can occur relatively easily and that there is massive, vectorial movement of vesicles between the organelles and the cell surface. the way in which this result influences our understanding of protein sorting in the secretory pathway is discussed in following sections. soluble and integral membrane secretory proteins fold once they have crossed the rerm; they are also often covalently modified. the follow ing sections deal with the types of modifications which occur in the er and their role, if any, in protein sorting. palmitoylation and phosphatidyl inositol modification of secretory proteins are discussed in section iii.f.4.c; only their role in protein transit through the secretory route will be discussed here. most secretory proteins are nitrogen (n)-glycosylated in the rer. be cause we are primarily concerned with the effects of glycosylation on protein targeting, only general details of the glycosylation reactions will be given here. a. the sequence of reactions shown in fig. v .l, which is common to both yeasts and higher eukaryotes, results in the addition of a (glucose) 3 -(mannose) 9 -( j /v-acetylglucosamine) 2 complex onto asn residues in the sequence asn-x-ser or thr (the acceptor peptide, in which x can be any i i i i amino acid except possibly proline or aspartate). isolated tripeptides can act as acceptors only when the two extremities are blocked. longer ac ceptor peptides are more rapidly glycosylated (577). as shown in fig. v .l, the entire complex is assembled before it is attached to the asn residue in the lumen of the rer. at least some sugars cross the rerm as intermediates complexed with the long-chain lipid dolichol, whereas others may cross the rerm as nucleotide 5 '-diphosphate-sugar complexes. however, it is by no means certain that the complex is assembled entirely within the lumen of the rer. for example, there is no evidence that gdp-mannose can be trans ported across the rerm, leading to the proposal that the five mannose residues are added via gdp-mannose donors on the cytoplasmic face of the rerm, whereas later modifications, and possibly earlier modifica tions, occur in the lumen. this presupposes that the dolichol-pp-(nagn) 2 complex can "flip" from the lumen to the cytoplasmic face and then back again once the five mannose residues have been added [see (452) for further discussion of the topology of glycosylation reactions in the rer]. the preferred donor lipid-carbohydrate complex is the com plete complex shown in fig. v.l (1129) . however, truncated versions lacking glucose and even mannose residues can act as donors in vitro, and under-glycosylated complexes can act as donors in vivo in yeasts (598) and protozoa (577) . variations in the mannose content of transferred oligosaccharides have also been reported (577). the transfer of the first n-acetylglucosamine (nagn) residue from the udp complex to the dolichol, is inhibited by the antibiotic tunicamycin, which therefore blocks all n-glycosylation. this provides a valuable tech nique for studying the role of n-glycosylation in protein traffic. the yeast (s. cerevisiae) gene (alg7) coding for the rerm-associated, tunicamycin-sensitive enzyme (udp-nagn : dolichol-p-transferase) was cloned by virtue of its ability to rescue tunicamycin-treated cells when present on multiple copy number plasmids (931). null mutations in alg7 are lethal. mutants affected in other stages in the yeast glycosylation pathway have been isolated by mannose suicide selection (section ii.f.l.b). only muta tions blocking the earliest stages in the pathway are lethal; incomplete dolichol-linked oligosaccharides containing a minimum of four mannose residues allow normal growth, presumably because they can be attached to secretory proteins if the full-length lipid-oligosaccharide complex is absent (see above) (598). the complete oligosaccharide chain is transferred onto the asn accep tor site in the lumen of the rer. n-glycosylation is generally thought to occur as the polypeptide is being threaded through the rerm, while it is still in its unfolded state. however, some acceptor sites are not glyco-sylated. this may be because they rapidly become inaccessible within the structure of the folded polypeptide, although other explanations are possi ble (577, 968) . an unexplained anomaly is that some er membrane glyco proteins have glycosylated residues exposed on the cytoplasmic face of the er (1). one explanation could be that additional nagn transferases are present on the cytoplasmic face of the smooth er, where these glyco proteins are predominantly located. the initially homogeneous oligosaccharide is processed immediately fol lowing its attachment to the polypeptide chain, initially by the removal of glucose residues by one or more glucosidases. glucose trimming of glyco sylated vesicular stomatitis virus (vsv) g protein has been reported to occur cotranslationally (27). further processing steps are different in yeasts and in animal cells. in s. cerevisiae cells, a single al-2-linked mannose residue is replaced by αϊ-3 mannose. a mutation in the gls1 gene coding for α 1-2 glucosidase does not affect removal of the mannose residue or subsequent chain elongation, whereas removal of the ninth al-2-linked mannose residue may be essential for outer chain elongation, which occurs in the golgi (section v.d.i) (598). further processing of animal cell glycoproteins also occurs in the golgi, although further man nose trimming of resident er proteins and reglycosylation may both oc cur in the er (577,847). c. ser and thr residues on secretory proteins of yeasts and possibly other fungi can be o-glycosylated in the er (429). the process is less well characterized than n-glycosylation but seems to involve the direct trans fer of mannose residues from dolichol-1-mannose onto acceptor amino acids. studies with acceptor oligopeptides suggest that no particular se quence is required around the modification site (54,621). further process ing of the mannose residue occurs in the golgi apparatus (429) (section v.d.2). yeast secretory proteins may be both n-and o-glycosylated. most studies indicate that o-glycosylation occurs exclusively in the golgi apparatus (section v.d.2). secretory proteins apparently fold spontaneously as they are extruded .across the rer membrane (194, 1222) . however, disulfide bridge forma tion is probably catalyzed by protein disulfide isomerase (pdi), which is loosely associated with the rerm (325,326). the formation of soluble protein complexes occurs shortly after synthesis (604), but trimerization of vsv g protein and influenza virus hemagglutinin (ha), both of which are integral membrane proteins, occurs only 7-10 min after synthesis (193, 194, 364, 589) and involves the selection of polypeptides from a ran dom pool of prefolded monomers rather than from a restricted pool of monomers synthesized on a single polysome (108). kreis and lodish (589) suggest that this delay may be due to the segregation of pdi in a late "compartment" of the er. according to the papers discussed above, oligomerization of g and ha occurs just before they leave the er, 5-10 min after synthesis. this view is challenged, however, by yewdell et al. (1222) , who found that mono clonal antibodies specific for oligomerized ha reacted only with proteins in the golgi apparatus, and not with ha in the er (see next section). a possible explanation for this ambiguity is that ha monomers fold and trimerize in the er and are then transported to the golgi apparatus, where further modifications alter the conformation of the ha trimers to produce the antigenic sites recognized by the antibody used by yewdell et al. different proteins transit through the secretory pathway together, but the rates at which they are secreted may vary considerably (335, 343, 615, 1221) . the site at which the secretion lag is most prominent seems to be transit from the rer to the golgi (646). it has been suggested that this delay might reflect the need for secretory proteins to interact with specific receptors in the rer for transport to the golgi and that carbohydrates might form part of the recognition signal (320,647,648). alternatively, proteins may be retained in the er until they have folded correctly; different folding kinetics may result in different retention times in the er (589,1192). indeed, exit from the er of one of the proteins studied by fitting and kabat (320) coincided with a partial proteolysis event, and polypeptides were "selected" for exit from a random pool of "new" and "old" proteins. furthermore, studies discussed in the preced ing section showed that oligomerization of virus-encoded membrane pro teins occurs just before they are transported to the golgi. if secretory proteins are indeed retained in the er until they are folded and oligomerized in the correct way, then exit-incompetent proteins should either remain indefinitely in the er or be secreted at very much reduced rates. this could explain why genetically altered or hybrid pro teins are sometimes translocated into the rer without difficulty and yet do not transit further through the secretory pathway (104, 281, 364) , and why incomplete glycosylation or glucose trimming and failure to process signal peptides sometimes affect secretion kinetics (96, 577, 649, 1045) . even minor sequence changes have been reported to affect protein con formation and exit from the er (1192). what happens to incorrectly folded proteins in the er? results from numerous studies indicate that they stay in the er (604,681) or are de graded either in the er (642a) or in the lysosome (723). these proteins do not seem to precipitate in large aggregates. instead, a specific, major er protein seems to bind to some incorrectly folded proteins, thereby pre venting their exit from the er. haas and wabl (413) first detected this protein (bip) complexed with immunoglobulin heavy chains synthesized in the absence of light chains (heavy and light chains are only transported to the golgi as complexes) (97), and it was subsequently found to be complexed with incorrectly oligomerized or monomeric ha (194, 364) , nonglycosylated invertase (in vitro in dog pancreatic microsomes) and incorrectly folded prolactin (557), and recombinant human factor vii in chinese hamster ovary cells (561). it was not detected in association with aggregated vsv g protein (250) or with incorrectly folded (mutant) class i histocompatibility antigen (1192). the dissociation of secretory protein-bip complexes requires atp (758), which might explain part of the atp requirement for protein movement along the secretory pathway, but be cause atp is usually present in cell ly sates, some bip complexes (such as g-bip) may dissociate during extraction. bip has also been shown to associate with nascent polypeptides as they enter the lumen of the er (557). two interesting features of bip are that its synthesis is stimulated by glucose deprivation and that it is structurally related to a heat shock protein (758,857). glucose starvation is likely to reduce glycosylation, thereby increasing the proportion of incorrectly folded secretory proteins in the er. studies have confirmed the idea that the extent of glycosylation can affect the association between secretory proteins and bip, as well as their rate of secretion (251). the accumulation of misfolded (mutant) pro teins in the er also increases bip synthesis (583), possibly because the cell senses that its store of bip has been sequestered into protein com plexes. thus, synthesis of bip may be increased according to require ments (857), but studies on the "proofreading" or quality control role of bip are still at an early stage. a different, cytoplasmic quality control protein may be responsible for proofreading cytoplasmic domains in transmembrane proteins to ensure that they too are correctly folded and oligomerized (406a). protein conformation, rather than any specific structural feature such as glycosylated residues, is thus likely to determine whether a protein is competent to leave the er. this feature is illustrated by studies on the effects of glycosylation on vsv g protein. machamer et al. (672) used site-directed mutagenesis to replace the glycosylated asn residues. nonglycosylated g protein was transported from the er to the golgi but did not reach the cell surface. when only one of the asn residues was de leted, the protein reached the cell surface; thus g protein with one glyco sylated asn residue can be transported through the entire secretory path way. g protein export became temperature-sensitive when new glycosylation sites were added (671). kotwal et al. (581) noted that some natural g protein variants have only one oligosaccharide whereas others have none at all and suggested that compensatory changes in primary structure may allow nonglycosylated protein to fold into a secretion-com petent conformation. thus, glycosylation is probably needed for correct protein folding but is not directly implicated in the formation of secretion competence signals. at least one secreted protein, ovalbumin, is not gly cosylated. the er contains a large number of proteins, some of which it shares with the contiguous nuclear membrane (117). they include proteins involved in secretory protein translocation through the rerm, signal peptidase, ribophorins, bip, enzymes involved in lipid synthesis and in protein glycosylation, and cytochrome p 4 5 0 . the characterized proteins are similar to other secretory proteins: they often have signal peptides (198, 630, 1089) , are glycosylated, and may be located in the lumen or in the rerm. rothman (965) has argued that it may be physically impossible for the er to prevent the escape of endogenous proteins, and particularly mem brane proteins, to the cis golgi. he cited two observations in support of the idea that these proteins could leave the rer as part of the bulk flow through the secretory pathway, and then recycle from the golgi to the er: (i) some er proteins are detected in significant amounts in vesicles derived from the cis golgi when cells are fractionated (102). (ii) bulk lipid flow out of the er necessitates efficient recycling, possi bly from the cis golgi, which could provide "carriers" for the recy cling of er proteins. most er proteins are, however, almost completely excluded from the golgi (663,697). furthermore, er proteins are not terminally glycosyla ted (117,1089), which means that they do not reach the medial or trans golgi (section v.e). mannose residues on some er proteins are not trimmed beyond the stage catalyzed by er mannosidase (117,954,1089), but a lysosomal protein carrying an er retention signal (see below) is phosphorylated by nagn phosphotransferase, indicating that it reaches the early cis golgi (858). significantly, the phosphate groups are not modi fied by nagn phosphodiesterase, which may be located in a different, er-distal golgi compartment (table v.l). warren (1166) has proposed that er proteins are salvaged from an intermediate compartment, the socalled transitional element (section v.c), located between the er and the cis golgi. recycling of "escaped" endogenous er proteins, rather than receptormediated retention in the er, is the currently favored model for the specific accumulation of proteins in the er (151). it is not clear whether soluble and er membrane proteins are both subject to the same retention mechanism, but studies on the rates of diffusion of membrane proteins in the er indicate that rerm proteins are more restricted than bip and that the mobility of bip devoid of its er retention-salvage signal (see below) cannot be distinguished from that of normal bip (151). several studies have sought to determine the nature of the er reten tion-salvage signal(s). the rotaviral type ii er membrane protein vp7 was found to be secreted when the two potential n-terminal transmem brane segments were deleted (884), suggesting that retention-salvage de pended on a membrane anchor domain. however, subsequent studies showed that the entire n-terminus was absent from mature vp7 (1084) and thus that some other feature must account for vp7 retention in the er (883c). although deletion of the c-terminal transmembrane and cyto plasmic tails of the type i er membrane protein ε19 of adenovirus also causes the protein to be secreted, deletion of the last eight residues (fidekkmp) of the c-terminal, cytoplasmic tail alone causes the protein to appear on the cell surface, suggesting that the c-terminus contains the er retention-salvage signal. furthermore, cell-surface interleukin 2 re ceptor protein β chain was converted into a resident er protein following fusion of fidekkmp to its cytoplasmic c-terminus (832). the cytoplasmically exposed signal could be recognized by "salvage receptors" in the transitional element. these studies on the ε19 protein are reminiscent of earlier, seminal work on the soluble er protein bip which resulted from the observation that three soluble proteins in the lumen of the er, including bip, had the same four residues, lys-asp-glu-leu (kdel), at their c-termini. munro and pelham (759) found that if this sequence was deleted or extended, bip was secreted into the medium. this suggests that kdel is the er reten-tion signal and that it must be at the extreme c-terminus, which is pre sumably the last segment of the polypeptide to fold and may therefore be exposed on the surface of the protein. in a complementary study, dna coding for kdel was fused to the end of a cdna clone coding for secreted lysozyme. the resulting hybrid protein was retained in a perinu clear region probably corresponding to the er (759). further studies are required to determine whether kdel is the "uni versal" er retention signal. preliminary studies suggest that some mam malian er proteins have the sequence rdel at the c-terminus, whereas yeast er proteins have the sequence hdel (858a). hdel or kdel is presumably recognized either by an endogenous er membrane protein, which could anchor soluble proteins in the er, or, more likely, by the recycling receptor located in the transition element or cis golgi. indeed, a putative hdel receptor gene (erd1) has recently been identified in yeast cells (858,858a). it is not clear what triggers the release of bip from its receptor once it is recycled to the er. β-glucanase is a typical lysosomal protein; small but significant amounts of it, however, are retained in the er through a specific interac tion with an endogeneous er protein, the esterase egasyn. studies by medda et al. (704) indicate that /3-glucanase-egasyn interaction is blocked by inhibitors of esterase activity, leading to β-glucanase secretion (rather than sorting to the lysosome). whether this is a physiologically significant mechanism for retaining proteins in the er remains to be de termined. only about 10% of egasyn is complexed with /3-glucanase (666), so perhaps it also binds to other resident er proteins. it would be inter esting to see whether egasyn itself has a c-terminal kdel-like sequence. as discussed above, secretory proteins do not move to the cis golgi until they attain an exit-competent state, and some proteins are specifically retained in the er, probably by recycling of escaped proteins. both nor mal secretory proteins (1223) and exit-incompetent proteins have been reported to accumulate in a specific region of the smooth er, the vacuolar transitional element (79,996), from which vesicles either migrate to and fuse with the cis golgi (41) or coalesce to form new golgi cisternae (797). specific membrane proteins [e.g., the product of the sec 12 gene in yeasts (767a)] may be required to form this specialized domain of the er and hence directly or indirectly assist vesicle formation and protein transport to the golgi. secretory pathway "shuttle" vesicles are difficult to isolate because (533) also showed that atp depletion caused secretory pro teins to accumulate in transitional elements, but this study did not distin guish between energy requirements for protein folding and for vesicular transport from the transitional element to the golgi. a different in vitro assay was developed by haselbeck and schekman (428) to study protein movement from the er to the golgi in yeast cell extracts. their assay uses donor er vesicles derived from a strain carry ing the secl8ts mutation, which blocks protein movement from the er, and the mnnl mutation, which prevents terminal (golgi) mannosylation of secretory proteins. invertase accumulated in the er grown at the restric tive temperature was mannosylated after transfer to recipient golgi from wild-type cells. the transfer efficiency was low, however, possibly be cause invertase accumulated at the restrictive temperature did not be come exit-competent at the permissive temperature in vitro, or because recipient golgi were saturated. as in the mammalian system, protein transfer was atp-dependent and required soluble cofactors including sec 18 protein (269b) as well as proteins on the surface of the recipient golgi. a similar but more efficient reconstitution system using gently lysed yeast-cells has recently been developed (35a). results obtained with this system indicate the probable requirement for gtp in er to golgi traffic. secretory proteins are subjected to further chemical modification as they transit through the golgi. these processes are discussed in the following sections (except palmitoylation, which was covered in section iii.f.4.c); their significance with regards to compartmentalization of the golgi appa ratus and their role in protein targeting will be discussed in later sections of this chapter. in section v.b.i, we saw that the basic oligosaccharide core on asn residues is trimmed by glucose-and mannosidases before secretory pro teins leave the er. following their arrival in the golgi, mannose residues on secretory proteins may be processed in one of two ways, depending on whether they are targeted to the lysosome. soluble lysosomal proteins carry phosphorylated mannose residues which function as lysosomal sorting signals (section v.g.5). they un dergo specific mannose phosphorylation catalyzed by one or two nacetylglucosamylphosphotransferases and 7v-acetylglucosamine-1 -phosphodiester-a-n-acetylglucosaminidase (phosphodiesterase) in different cis golgi compartments (614a). sequential action of these enzymes results in the transfer of tv-acetyl glucosamine-1-phosphate from udp-nagn to any one of five mannose residues in the oligosaccharide core, followed shortly afterwards by the removal of the jv-acetylglucosamine to expose the phosphomannosyl group (577). goldberg and kornfeld (379) found three partially phosphorylated peptides in β-glucuronidase, indicating that lysosomal enzymes may not be uniformly phosphorylated. lysosomal enzymes presumably contain signals which are recognized by the phosphorylating enzymes (919). deglycosylated (endoglycosidase η-treated) lysosomal cathepsin d is not a substrate for the phosphoryl ation reaction in vitro, but it inhibits phosphorylation of intact lysosomal enzymes. proteolytic fragments of glycosylated cathepsin d are also not phosphorylated, and do not inhibit phosphorylation when they are dephosphorylated, indicating that the "phosphorylation signal" is probably not a linear sequence of amino acids (612). frog oocytes are also able to recognize the phosphorylation signal of human cathepsin d (301). renin is closely related to cathepsin d and yet is secreted by mammalian cells, presumably because it lacks the phosphorylation signal. however, renin produced in oocytes is phosphorylated, remains intracellular, and is de graded (presumably in the lysosome) (302). this suggests that renin has a phosphorylation signal which is recognized by amphibian but not by mam malian phosphorylating enzymes. outer chains on lysosomal and nonlysosomal secretory proteins of com plex eukaryotes may be further trimmed by golgi mannosidase i to leave five mannose residues on the core oligosaccharide. further modifications involve the addition of an n-acetylglucosamine residue by n-acetylglucosaminyltransferase i, further removal of two mannose residues by golgi mannosidase ii, fucosylation of the innermost tv-acetylglucosamine by fucosyltransferase, and the addition of galactose, n-acetylglucosamine, and sialic acid residues by appropriate transferases (fig. v. 2). oligosaccharides of both lysosomal and nonlysosomal proteins may be further modified by sulfation of mannose and n-acetylglucosamine resi dues and o-acetylation of sialic acid residues (577). outer chain modification in yeasts is markedly different. golgi mannosyltransferases extend the basic (man) 8 core oligosaccharide to produce large mannan structures typical of many yeast mannoproteins, which may carry as many as 150 mannose residues (598). the single o-linked mannose residue on yeast glycoproteins (section v.b.l.c) may be further modified by the addition of up to four more mannoses transferred from gdp-mannose (598,1107 blocked in sec mutants, which prevent secretory proteins from reaching the golgi (429). in complex eukaryotes, hydroxyl groups of ser or thr residues are oglycosylated by enzymes thought to be located exclusively in the golgi (418,786). acceptor octapeptides can be o-glycosylated. 7v-acetylgalactosamine is the primary sugar in o-linked oligosaccharides; further resi dues of galactose, sialic acid, fucose, ^-acetylgalactosamine, and nacetylglucosamine can then be added. lipid intermediates are not in volved in o-glycosylation, and the reaction is not inhibited by tunicamy cin (418), but it is not known whether other sugar transferases are in volved in both n-and o-glycosylation. glycosylated and unglycosylated secretory proteins are major substrates for tyrosine sulfation (501). tyrosynylprotein sulfotransferase is enriched in golgi-derived membrane fractions and has its active site oriented to ward the golgi lumen (619). the sulfate donor is 3 '-phosphoadenosine 5 phosphosulfate. almost all tyrosinated proteins have an acidic residue, a glycine or proline residue, and no cysteines, basic residues, extended secondary structure, or n-glycosylation sites close to the modified tyro sine, but there does not appear to be a strict consensus sequence around the modification site (474,619). inhibition of tyrosine sulfation is reported to retard the exit of a secretary protein from the tgn (331b). many secretory proteins undergo secondary proteolytic processing fol lowing removal of the signal peptide. the best characterized examples of this class of processed secretory proteins are α factor and killer toxin of yeasts. the α-factor peptide is present four times in the pro-a-factor protein which reaches the golgi apparatus. this probably represents a way of reducing "shipping costs" since α factor itself may be too small to be efficiently transported to the rerm and it would be inefficient to pad the polypeptide out with redundant sequences (602). julius et al. (545) found that pro-a-factor processing was blocked by sec mutations, which prevented protein movement through the golgi, and that mature α factor was normally present in secretory vesicles en route to the cell surface. thus, processing occurs in the golgi. some viral coat proteins such as influenza virus ha are also proteolytically processed in the trans-golgi network (tgn) or trans golgi. processing of mammalian prohormones, which is similar to that of pro-α factor, occurs in secretory granules budding from the trans golgi [see section v.g.4.c and (1003)]). the four 13-residue-long α-factor peptides in pro-α factor were found to be separated by 6-8 residues (lys-arg-glu-ala-asp-ala-glu-asp) (602). this suggests that at least one processing protease has trypsin-or chymotrypsin-like activity. killer toxins are processed at similar sites (245,1074). in fact, the enzyme which performs this initial processing step, the product of the kex2 gene, is a ca 2 + -thiol protease which cleaves between basic residues. the enzyme is inhibited by anti-αΐ-trypsin and can correctly process mammalian proalbumin (53). strains mutated at kex2 secrete unprocessed pro-α factor (546). the product of the ste13 gene, a membrane-associated aminopeptidase (544), processes the nterminal tetrapeptide, and further processing of the c-terminus is per formed by kex1 -encoded carboxypeptidase (245). other secretory pro teins produced by different species of yeasts may also be processed by one or more of these enzymes (695b). results from a number of experimental approaches (summarized below) show quite conclusively that individual golgi cisternae are separate, bio chemically and functionally distinct entities organized according to a very strict pattern and that secretory proteins progress in a synchronized wave from one end of the stack of cisternae to the other en route to their final destinations. according to these data, golgi stacks must contain at least three distinct cisternae (generally referred to as cis, medial, and trans according to their orientation with respect to the er). heterogeneity within these "domains" indicates that the actual number of cisternae is probably higher than three (see table v .l, fig. v .3) (299). cells normally have one or a very limited number of golgi stacks. the stacks break up into clusters of many vesicles during mitosis, apparently to ensure equal partitioning of golgi components to daughter cells, although the number of golgi clusters produced is far in excess of that required for this pur pose. golgi breakdown presumably involves membrane fission, so each cluster of vesicles contains cis, medial, and trans components (662). pro tein secretion is usually shut down during mitosis or meiosis, but the yeast s. cerevisiae continues to process and secrete invertase during mitosis, implying that the golgi fragments remain active (685). in general, results from different analyses give a coherent picture of the cis to trans organization of golgi cisternae (see table v . 1), on the basis of which a simple map can be drawn (fig. v.3) . the segregation of modify ing enzymes presumably allows prosthetic groups to be added or removed according to a strict sequence and prevents competition between process ing enzymes which could act on the same substrate (266). table v .l is five, but the actual number of golgi cisternae may be higher than that shown. nagn, iv-acetylglucosamine; tgn, trans-golgi network. early studies showed that one or two cis (er-proximal) golgi cisternae were preferentially stained during prolonged exposure to osmium [pre sumably due to strong reducing conditions in these cisternae (333)]. sub sequent histological and cytochemical tests showed that many enzymes involved in protein glycosylation and other reactions, as well as some proteins with unknown function, were not evenly distributed through the golgi stock but were compartmentalized in one or two cisternae (table v .l). the techniques employed include immunocytological detection of proteins using specific antibodies, enzymatic cytochemical reagents for specific enzymes, and the detection of lectin-specific sugar residues on terminally modified glycoproteins in transit through the golgi (table v .l). as noted by farquhar (299), some studies with different cell lines give conflicting results, suggesting that the organization of the golgi stack may vary depending on cell type and function. intermediates in the secretion pathway can be detected by pulse-chase experiments in which oligosaccharides or other prosthetic groups are la beled by the metabolic incorporation of radioactive precursors. the se quence in which processing occurs can thus be determined and correlated with the location of processing enzymes in the golgi cisternae. golgi enzymes involved in n-and o-glycosylation, sulfation, and palmi toylation of secretory proteins can be separated by density gradient cen trifugation of golgi-derived vesicles, which apparently differ in density due to differences in cardiolipin content (817). enzymes identified in cis golgi by kinetic and histological or cytochemical tests are generally found in heavier golgi membrane fractions, and density is now widely used to define the approximate location of golgi proteins in the stack (table v .l). the ionophore monesin slows or arrests intra-golgi transport and inhibits late (trans) golgi functions, and can thus be used to distinguish between early and late golgi processing events. monesin also causes secretory proteins to accumulate in medial or late golgi compartments, which be come distended and vacuolated, aiding their identification by cytochemi cal methods (400,905,1108). however, as discussed by dunphy and rothman (266), studies on the effects of monesin in different cell types often give conflicting results and do not necessarily indicate the precise site of secretory protein accumulation or processing. oligosaccharides on secretory proteins are only cleaved by endoglycosi dase η before they are processed by nagn transferase i and mannosi dase ii. these enzymes are probably located in medial golgi cisternae. thus, endoglycosidase η-sensitivity provides a simple test for determin ing whether secretory proteins have reached these cisternae (267). rothman et al. (969, 970) demonstrated that pulse-labeled vsv g protein present in the golgi of a cell lacking a particular modification enzyme can be modified upon fusion with a cell producing the modifying enzyme. the golgi cisternae from donor and recipient cells did not fuse, and the g protein, rather than the modifying enzyme, was transferred from one cisterna to the other. these observations have implications for the mecha nisms of protein movement between cisternae (section v.f) but, like in vivo kinetic experiments, may also indicate the sequence in which pros thetic groups are added or removed ( having established that secretory proteins are progressively processed as they migrate through the golgi stacks, we now turn our attention to how the proteins themselves migrate between cisternae, and how golgi pro teins can be specifically retained within specific cisternae. of all of the models explaining intra-golgi movement of secretory pro teins (300), only that invoking shuttle vesicles fits the experimental data showing that golgi cisternae are distinct entities. numerous proteincoated vesicles are produced by golgi stacks in vitro under conditions which favor the intra-golgi movement of secretory proteins (see below) (40,820). these uniformly sized vesicles were shown to contain at least one secretory protein, vsv g protein, indicating that they could be bona fide intra-golgi shuttle vesicles (820). similar vesicles were also found around the golgi apparatus in situ (996, 1088) . furthermore, different se cretory proteins destined to different locations and exported or secreted at different rates were present in the same vesicles (1088), which agrees with the idea that secretory proteins are not segregated from each other during intermediate stages in the secretory pathway. the protein coat on these vesicles is distinct from the clathrin-containing coats present on endocytic and lysosomal vesicles and on budding secretory granules (see section v.g.6). the only regions of the golgi apparatus which have pro tein coats are those from which the golgi shuttle vesicles bud. a number of different conditions prevent intra-golgi movement of secre tory proteins in vivo and in vitro. the ionophore monesin (section v.e.4) probably prevents movement though the trans cisternae by disrupting a proton gradient maintained by an atp-dependent protein pump present in golgi membranes (48), thus causing the ph of the normally acidic trans golgi cisternae to rise. saraste and hedman (994) demonstrated that mi gration between different golgi cisternae was blocked at different critical temperatures and suggested that this might be caused either by atp depletion or by changes in membrane fluidity. atp may be required to maintain the acidic ph of trans cisternae, and vesicle fission and fusion, which must occur at the cisternal membranes, can probably only occur when membrane lipids are in a "fluid" state above the phase transition temperature. atp and soluble cytosolic factors including a 74-kda, n-ethylmaleimide-sensitive protein (93a) are required in both early and late stages of intra-golgi movement in vitro (39,40,685a,966) and in permeabilized cells (62). intriguingly, soluble yeast cell extracts can replace endogenous cy toplasmic components in a mammalian cell-derived assay system for in tra-golgi movement of proteins (268), raising the possibility of using ex tracts from yeast sec mutants blocked at different stages in the secretory pathway to define the role of the corresponding wild-type gene products in intra-golgi transport. indirect evidence based on the effects of gtp analogs suggests that gtp is also needed for intra-golgi movement of secretory proteins (710), and studies with antibodies against a yeast gtp binding protein indicate that a similar protein is apparently located in the golgi apparatus in multicellular eukaryotes (1030). the significance of this observation is not understood, but gtp and gtp binding proteins (g proteins) may be involved in maintaining vectorial movement of shuttle vesicles or in vesicle recycling (see below). surface components on golgi membranes are also required for intra-golgi transport (3,39,40). a recep tor may recognize the protein coat on the golgi transport vesicles. balch et al. (40) considered that the migration of secretory proteins from one cisterna to another depends on three separate events: (i) secretory proteins are primed to make them competent or available for transport. priming probably involves the migration of secretory proteins to regions of the cisternae where budding occurs, princi-pally at the outer rims. this step might depend on interactions be tween the secretory protein and receptors migrating to the budding areas or may rely on free diffusion within the cisternal membrane or lumen. segregation may depend on signals generated by processing enzymes in individual golgi cisternae, but most experiments in which processing inhibitors have been used do not support this idea and show instead that processing is nonessential for secretory pro tein targeting. some mutationally altered secretory proteins, how ever, transit normally through the early stages of the secretory path way and yet are not transported through the golgi (344,1040). perhaps secretory proteins must fold into a particular conformation to be competent for movement between golgi cisternae. the accu mulation of such abnormal proteins in the golgi may cause it to become distended, but this does not drastically affect intra-golgi movement and secretion of other secretory proteins (344). (ii) vesicles move from one cisterna to another. vesicle migration be tween golgi cisternae may be either vectorial or random. vectorial movement is more compatible with traditional views on the strict sequence of events in secretory protein processing and is supported by the observation that the likelihood of forward transfer is at least five times greater than that of lateral movement in golgi of fused cells (969, 970) . this implies that there are receptors on the surfaces of golgi cisternae which recognize vesicles budding from the pre ceding cisterna in the chain. nonetheless, the topology in the golgi complex is well maintained, possibly because the cytoskeleton pre vents cisternae from coming into direct contact. another puzzling feature of vesicular intra-golgi transport is that small transport vesi cles would be expected to diffuse away from the golgi complex. the cytoskeleton may restrict the movement of golgi vesicles and may even play a more positive role in directing vesicles between cister nae. however, although movement along microtubules has been well documented for large organelles (1136) and may be important in the sorting of some proteins leaving the golgi complex (section v.g.3), there is no evidence that it could play more than a minor role in the movement of vesicles over the very short distances which separate golgi cisternae (139). furthermore, cell fusion studies by rothman et al. (969) show that inter-golgi movement of vsv g protein can occur, indicating either fusion of the two golgi com plexes or, more likely, that movement between golgi cisternae is dissociative; i.e., the vesicles do indeed diffuse into the cytoplasm. (iii) vesicle fusion with the membrane of the acceptor cisterna and re lease of vesicle contents into the lumen of the cisterna occurs. this model for intra-golgi transport fits many experimental observa tions, but further studies are required to define clearly the steps involved. for example, atp may be required for vesicle fission and fusion, as well as for reducing the ph in trans golgi compartments, but this has not been proven, and the nature and role of some of the cytosolic components required in in vitro assays have yet to be determined. further work is needed to define how proteins find their way to budding regions of the golgi membrane and what triggers vesicle formation and vesicle fusion with acceptor membranes. how are proteins specifically retained in individual golgi cisternae? we saw earlier that receptor-dependent recycling of er proteins from the cis golgi or an intermediate compartment could explain how these proteins remain almost entirely in the er (section v.b.4). golgi residents proba bly also have signals which are recognized by some kind of receptor. indeed, coronavirus el membrane glycoprotein appears to have a golgi retention signal in one of its transmembrane domains (670). however, recycling of escaped proteins is a far less attractive model for explaining how golgi proteins are retained than it is for the case of er proteins. one possibility, proposed by pfeffer and rothman (872) , is that the membranes of golgi cisternae have two domains: a fluid domain, close to the budding rims, and an immobile phase, in which endogenous membrane proteins are anchored. a protein would require a signal to associate with a recep tor in the immobile phase or to become anchored to it, whereas all other proteins would enter the mobile membrane phase or the bulk phase of the cisternal lumen. pfeffer and rothman point out that this model reduces the number of proteins which need to have signals for routing through or retention in the golgi, because fewer proteins are retained in the golgi than transit through it. cytoskeletal structures, including possibly the cytoplasmic matrix, which "glues" the cisternae together, were proposed to limit movement in the immobile phase. an alternative idea, also con sidered by pfeffer and rothman, is that endogenous golgi proteins inter act to form patches which, by virtue of their size, are too small to fit into transport vesicles. secretory proteins transit through the golgi apparatus and arrive in the trans cisternae together. the trans golgi compartment is therefore the point at which the different branches of the secretory pathway diverge. the following sections deal with the site at which sorting occurs, the ways in which proteins are sorted, and what happens when vesicles carrying secretory proteins arrive at their destinations. as its name suggests, the trans-golgi network (tgn, also called golgi endoplasmic reticular lysosomes or gerl) is the most distal compart ment of the golgi apparatus (relative to the rer). it differs from the golgi cisternae in that it has a distended, reticular appearance rather than that of a flattened dish. early morphological studies suggested that the tgn was a reticular adjunct of the golgi specifically involved in lysosome biosynthesis [lysosomal enzymes were originally thought to bypass the golgi (398)]. the tgn is now known to be distinct from endosomes or lysosomes. endocytosed horseradish peroxidase does not accumulate in the tgn (401), although some endocytosed proteins may be recycled to the cell surface via the trans golgi and especially via the tgn (319) (see chapter viii). the tgn probably contains tyrosinyl sulfotransferase (33), acid phosphatase, sialotransferase, and galactosyltransferase (365,957) (table v .l, fig. v.3) , although tgn-derived vesicles are diffi cult to distinguish from those derived from the trans cisternae. the tgn also marks the site at which assembled clathrin, one of the proteins which coat some secretory and endocytic vesicles, appears along the secretory pathway (see section v.g.6). the tgn is the most acidic golgi compartment, although the ph is almost certainly not as low as in secretory granules (823) or in lysosomal sorting vesicles, in which low ph causes the dissociation of lysosomal proteins from the mannose-6-phosphate receptor (577) (see section v.g.5). furthermore, influenza virus hemagglutinin, which is activated at low ph, reaches the cell surface as a nonactivated form, indicating that it does not spend an appreciable period (more than 2 min) in a compartment with a ph of less than 6 (107). different secretory proteins accumulate together in the tgn (1088,1123), which can become further distended when the load of secre tory or lysosomal proteins increases (398). transport of secretory pro teins from the tgn is blocked at 20°c, and numerous protein-coated and naked vesicles accumulate as buds on the surface of the tgn (401). different types of vesicles seem to be involved in sorting secretory pro teins into different branches of the secretory pathway. thus, different classes of soluble secretory proteins may be segregated into different domains of the tgn according to their interaction with specific receptors (fig. v.4 ). although this model does not explain how receptor proteins note that constitutive default sorting is not receptor-mediated and that sorting into the regulated secretory branch of the pathway may result from protein aggregation and granule formation rather than receptor interactions. tgn, trans-golgi network; l, lysosome; pl, prelysosome. segregate into these domains, it does serve to illustrate the possible mech anisms involved in receptor-dependent segregation and packaging secre tory proteins discussed in the following sections. default sorting is the final stage in the secretion of proteins which lack specific sorting (lysosomal, vacuolar, or polarity) signals and which are not accumulated within specific secretory storage granules of the regu lated branch of the secretory pathway, i.e., those which are constitutively secreted (fig. v.4) . (note, however, that some constitutively secreted proteins carry sorting signals.) immunocytochemical studies show that proteins secreted by the default pathway accumulate in secretory vesicles which do not have protein coats (401,821). plasma membrane proteins are transported to the cell surface in the same vesicles as secreted proteins (114, 462, 1088) . (1204) (see section ii. f. 3) required atp and was sensitive to trypsin, implying that at least one cytoplasmic or vesicle-cytoplasmic membrane surface protein is involved. atp might be needed for fusion between the vesicle and cytoplasmic mem brane. saccharomyces cerevisiae strains carrying a temperature-sensitive mu tation in the sec4 gene accumulate large numbers of secretory vesicles. these were purified by walworth and novick (1165), who found them to contain three dominant proteins (110 kda, 40-45 kda, and 18 kda) to gether with invertase, the secretory marker protein. these three proteins were made during the period in which the vesicles accumulated, i.e., after secretion had been shut down at the nonpermissive temperature. the 110-kda protein was the most abundant protein in the lumen of the secretory vesicles, whereas the other two proteins were membrane-associated and had cytoplasmic domains which could interact with the cytoskeleton, the cytoplasmic membrane, or cytoplasmic components (1165). it should be noted, however, that exocytosis in s. cerevisiae might be considered as polarized rather than default sorting because secretory vesicles are di rected towards the growing bud rather than being randomly distributed over the entire cell surface (section v.g.2 and fig. ii.5) . the sec4 gene was cloned and sequenced by salminen and novick (987), who found it to be homologous to gtp-binding ras regulatory proteins of higher eukaryotes. whether this is significant for the role of sec4 in secretion is unclear, especially since it is not known whether gtp is required for exocytosis in yeasts. recent studies show that sec4 protein binds gtp (391). overexpression of sec4 suppresses the effects of mutations in three other sec genes, but strains carrying sec4ts muta tions rapidly become secretion-defective at the nonpermissive tempera ture, suggesting a direct role in secretion. the cytoplasmic and secretory vesicle membrane-associated sec4 product does not itself appear to be a secretory protein because its predicted primary sequence does not in clude a potential secretory routing signal. one possibility raised by salminen and novick is that the c-terminal cysteine residue of sec4 is acylated, as are the gtp-binding ras proteins and that the acyl groups anchor the polypeptide in the membrane, but this could not be confirmed directly. another gtp binding protein, ypt1 (which is 50% homologous to sec4) was detected close to the bud as well as in ill-defined structures (possibly the er and golgi) of s. cerevisiae cells (1030). mutations in the ypt1 gene affect several stages in the secretory pathway, but ypt1 pro tein probably plays an indirect role in protein transport as a result of its role in ca 2 + regulation (1014). the plasma membrane of epithelial cells is divided into two distinct do mains. the apical surface, which may have microvilli, is oriented toward the outside (e.g., lumen of the intestine), whereas the basolateral surface is on the inside, facing the basolateral surface of other cells or resting on an extracellular matrix of basal lamina (fig. v.5) . the two membrane domains have distinctly different lipid contents in their outer leaflets [the apical membrane has a higher glycolipid and cholesterol content, and a lower phosphatidylcholine content than the basolateral membrane (1050)], although the lipid contents of their inner leaflets may be identical (inset to fig. v.5 ). this implies that only the outer leaflets of the two membranes are separated by tight junctions, morphologically distinct structures rich in nonbilayer lipids and containing unique proteins which form the junction between apical and basolateral surfaces and probably prevent the movement of outer leaflet lipids and proteins between them, as well as acting as ion gates between adjacent cells (fig. v .5) (177, 260, 409, (706) (707) (708) 1050) . there may also be differences in basal and lateral membrane composition. cell-cell interactions are required for effi cient formation of the basolateral surface of polarized cells, but not for sorting to the apical zone (1143). adjacent cells may be held together by desmosomes. the major breakthrough in studies on protein sorting in polarized cells (878). in general, the steady-state distribution of polarized membrane proteins indicates that sorting of basolateral pro tein is highly efficient (>97% fidelity) (873), whereas apical proteins may be found in significant amounts in the basolateral membrane. however, the surface area of the apical membrane is usually much smaller than that of the basolateral membrane, which means that the fidelity of apical tar geting is actually quite high (about 88%) (873). basolateral and apical sorting seem to represent two distinct pathways, but most polarized cells also secrete some proteins from both surfaces, implying that neither basolateral nor apical sorting are default pathways (390,574). furthermore, lysosomal proteins are secreted by the default pathway from both apical and basolateral surfaces when lysosomal sort ing is blocked (140). this may not be the case in caco-2 cells in which high-level basolateral secretion of normally nonpolarized lipoproteins im plies that the basolateral sorting predominates over all other sorting path ways, including the default pathway (136,930a). do polarized cells sort and direct proteins directly to their target mem branes, or are they all first randomly targeted to both domains, or specifi cally to one or other domain, and then transcytosed? although some studies designed to answer this question have been conducted with en dogenous proteins, the most detailed results come from studies on the basolaterally targeted vsv g protein and the apically targeted influenza virus hemagglutinin (ha) in mdck cells. furthermore, it is through derivatives of these proteins that most recent studies have attempted to identify apical and basolateral targeting signals (see next section). the results of pulse-chase experiments combined with a trypsin sensi tivity assay and tests with anti-ha antibodies applied to the basal surface of mdck cells led matlin and simons (695) to conclude that ha was targeted directly to the apical surface. conversely, pfeffer et al. (873) showed that pulse-labeled g protein went directly to the basolateral mem brane, where it could be detected with specific monoclonal antibodies. another approach to studying the sorting of ha and g proteins is to ''freeze" them in the golgi apparatus by cooling the cells to 20°c (which specifically blocks protein exit from the tgn) or to use cells infected with viruses carrying temperature-sensitive mutations in the ha or g genes at the restrictive temperature. the bulk movement of these proteins can thus be followed by immunocytochemistry when the cells are restored to the permissive temperature. rindler et al. (929) found that gts protein was transported directly from the golgi to the adjacent lateral cell sur face, whereas wild-type ha accumulated at 20°c was sorted directly to the region of the apical surface closest to the tgn. similarly, pfeiffer et al. (873) found that g protein accumulated in the tgn went to the baso lateral membrane 67 times faster than to the apical surface when the cells were warmed to 37°c. these studies are compatible with the idea that apical and basolateral proteins go directly to their respective target mem branes. different results were reported by bartles et al. (50) , who found that endogenous apical proteins in hepatocytes appeared first in the baso lateral membrane and were subsequently redistributed to the apical sur face by transcytosis (section viii.a.5.b). whether this result indicates the existence of totally different mechanisms for sorting apical proteins in kidney and liver cells could be tested by expressing hepatocyte genes for apical proteins in mdck cells. sorting of polarized secreted and membrane proteins is assumed to de pend on sorting signals (probably signal patches) [see section i.d.2 and (88)] present in the sorted proteins and on their interaction with receptors. sorting presumably occurs in the tgn, where at least one set of cognate receptors are probably located. to date, the primary approach used to locate sorting signals has been to introduce major sequence alterations including the deletion of cytoplasmic or transmembrane domains from the ha, g, or other polarized viral glycoproteins, or to create hybrids be tween them. even these relatively unsophisticated studies give conflicting results. for example, mcqueen et al. (702) and roth et al. (959) found that either deleting the transmembranous and cytoplasmic tail of ha (to make a soluble, secreted protein) or replacing them with the correspond ing regions of g did not affect apical targeting and concluded that the apical sorting signal was located in the n-terminal, extracytoplasmic do main of ha. gonzalez et al. (387) reported, however, that truncated ha was secreted by the default pathway and concluded that the sorting signal was in the c-terminal transmembranous or 10-amino-acid cytoplasmic domains. there is also disagreement concerning the location of the g protein basolateral sorting signal, which, according to the deletion and ha gene fusion studies of paddington et al. (835) and gonzalez et al. (387) is located in the 29-amino-acid, c-terminal cytoplasmic domain. stephens and compans (1080) mcqueen et al. (703) have suggested that the origin of these conflicting results may be the fact that recombinant genes are not stably expressed in mdck cells. their studies (702,703) were carried out shortly after infec tion by recombinant viruses and are, they claim, more likely to reflect the true sorting pathway of the recombinant gene product. other groups may in fact be studying the sorting of hybrid or truncated proteins which have been further modified by genetic selection for more stable cell lines. an other argument in favor of the idea that sorting signals are in the extracy toplasmic domain is that this part of the signal is initially localized in the lumen of the tgn, where sorting is presumably initiated, and could there fore bind to hypothetical sorting receptors which segregate sorted pro teins from those destined for default export or secretion. these receptors may themselves have cytoplasmic domains recognized by receptors on the basolateral or apical surfaces. furthermore, such a system could han dle both secreted and exported (membrane) proteins. however, there is some evidence, based on the effect of nh 4 c1 (or ph) on basolateral sorting of laminin and heparin sulfate proteoglycan, that soluble and membrane proteins may be sorted by different mechanisms (142). clearly, more work remains to be done before we can identify polarity sorting signals with any precision. one of the difficulties may be that these signals are probably not linear sequences of amino acids and are therefore less amenable to gene fusion studies, which have been so useful in identi fying routing signals. even very subtle conformational changes may alter sorting signals and render them nonfunctional; this could explain why prevention of glycosylation with tunicamycin may cause normally polar ity-sorted proteins to enter the default pathway (1135). another aspect of the work on polarity sorting signals which needs to be pursued is the identity of receptors and the stage at which sorted proteins dissociate from them. sorting receptors are presumably re cycled. little is known about how the vesicles are targeted to different regions of the plasma membrane. one possibility is that sorting of lipids ( fig. v.5) is involved (709,1050a) . alternatively, sorting vesicles could interact with components of the cytoskeleton, along which they are driven by atp-dependent motors attached to microfilaments; further receptor-ligand interactions might complete the sorting process when vesi cles arrive at the plasma membrane (1136). although there is no evidence that microfilaments are required for default sorting of secretory proteins (1136), rindler et al. (930) have reported that colchicine and other microtubule-disorganizing agents abolished specific apical sorting of ha and caused influenza virus to bud from both cell surfaces in polarized cells. salas et al. (986) obtained the opposite result, however, and neither group observed any effect on basolateral targeting of vsv g protein. rogalski (944), however, found that agents which caused microtubule disassembly caused random sorting of g protein. thus, the role of the cytoskeleton in the targeting of polarity-sorted secretory proteins in complex eukaryotes' proteins remains unclear. a temperature-sensitive mutation in the s. cerevisiae actin gene results in abnormal exocytosis and bud formation at the nonpermissive temperature, suggesting that actin filaments may direct secretory vesicles to the growing bud in yeast cells (793). there is evidence to show that the cytoskeleton may be important in maintaining polarized distribution in complex eukaryotic cells (410). a polarized atpase has recently been shown to bind directly to ankyrin, a protein which is known to link an integral erythrocyte membrane protein to spectrin and actin of the erythrocyte cytoskeleton (770). thus, ankyrin might link the atpase to microfilaments and thereby maintain its polar ized distribution. two features distinguish regulated exocytosis from other branches of the secretory pathway: (i) protein release only occurs when the cells are stimulated by secretagogues (e.g., cyclic amp or ca 2 + ). (ii) proteins accumulate within the cell before their release. proteins accumulate in a special class of secretory vesicles called secre tory granules, wherein protein concentrations reach such high levels that they become electron-dense and can be clearly identified by electron mi croscopy. the secretory pathway is restricted to certain cell types (e.g., endocrine and exocrine cells, mast cells), and only certain types of pro teins (examples include hormones, albumin, and some degradative en zymes including proteases and lipases) enter into the pathway. another feature of the regulated secretory pathway is that proteins may be pro-teolytically processed before release or as they are released from the cell, although this feature is also found in some constitutively secreted pro teins. regulated and constitutive branches of the secretory pathway can coexist in the same cell (395, 567, 1109) , implying that proteins destined for storage in secretory granules must be sorted from other secretory proteins. elec tron microscopic studies show that both classes of secreted proteins tran sit through the golgi cisternae together and are segregated in the tgn, where proteins destined for the regulated branch of the pathway condense into specific areas coated with the protein complex clathrin (see figs. v.4 and 6; see also section v.g.6) (824,1122). the clathrin coat is subse quently removed as the condensing granules bud from the tgn and ma ture (818) (see fig. v.6 ). these areas probably represent the sites at which secretory granules mature and are released from the tgn. it should be noted, however, that some proteins which are normally se creted by the regulated pathway may 4 'escape" and be secreted 4 'consti tutively" (31). it remains to be determined whether this is due to incorrect sorting or to low-level, secretagogue-independent secretion from storage granules, as suggested by studies by von zastrow and castle (1231). bur gess and kelly (136) propose that this "spillover" secretion may be due to inefficient sorting in the specific cell lines tested, since rhodes and halban (921) observed much more efficient sorting into the regulated pathway. efficient segregation of proteins into regulated and constitutive branches of the secretory pathway implies that the former have sorting signals (see section v.g.4.b for an alternative explanation). the exis tence of these signals was suggested by moore and kelly (731), who transfected a pituitary tumor cell line with a hybrid gene comprising the 5 ' end of the vsv g protein and the 3 ' end of the gene for human growth hormone. the hybrid protein was diverted into the regulated secretory pathway, indicating that the constitutive pathway had been bypassed due to sorting signals present in the growth hormone part of the hybrid. there are no obvious sequence similarities between proteins secreted by the regulated pathway, implying that the sorting signal is probably a patch signal rather than any identifiable linear stretch of amino acids. several proteins secreted via the regulated pathway are proteolytically processed prior to their release from the cell (see below). this raises the possibility that the sorting signal could reside in that part of the secretory polypep tide which is eventually cleaved off. this possibility was tested by bur gess et al. (135) , who found that deleting dna coding for the propeptide part of trypsinogen had only a minor effect of the targeting of the enzyme into secretory granules. thus, they concluded, there must be at least one sorting signal in the mature part of the trypsinogen. one surprising feature of regulated pathway sorting is that the putative sorting signal seems to be universal. moore et al. (732) , for example, found that human proinsulin was packaged into secretory granules in mouse adrenocorticotropic hormone (acth)-secreting cells, that it was correctly processed (see below), and that its release from these cells was stimulated by the same secretagogues that stimulated acth release. similar results were obtained in studies on the expression of human kid ney renin dna in the same cell line (337). fibroblast l cells, however, which do not have the regulated pathway, secreted (unprocessed) proin sulin via the constitutive pathway (732). receptors localized in specific domains of the tgn membrane may segregate proteins into the regulated branch of the secretory pathway, but this has not been proven, and other factors may be important. for exam ple, treatment with the weak base chloroquinone causes acth to be secreted by the constitutive pathway, indicating that low ph is required for sorting into the regulated pathway (733). low ph in condensing secre tory granules may dissociate proteins from their receptors, which can then be reused (but see section v.g.4.b). another feature of secretory granules which appears to have been largely overlooked is that proteases involved in post-tgn processing of secretory proteins must also carry sorting signals. it remains to be seen whether the membrane content of secretory granules differs significantly from that of vesicles of the consti tutive branch of the secretory pathway. in certain exocrine cells, the concentration gradient of a secretory protein between the rer and secretory granules can be as high as 200 (988). the dense core of aggregated protein is sometimes seen to be separated from the membrane of the secretory granule (136) and may remain intact when the membrane is removed (1230) or upon exocytosis (19, 1122) . although receptor-mediated sorting into specific regions of the tgn may assist the condensation process, proteins secreted by the regulated pathway may aggregate spontaneously and be packaged into secretory granules when they reach a critical size. thus, the packaging of g protein-growth hor mone hybrids into secretory granules discussed above (731) may be due to the presence of "aggregation" sequences in the growth hormone segment of the polypeptide, rather than to the presence of a specific sorting signal. the low ph of the condensing granule (19,824) may be important for protein aggregation, since aggregates dissociate at high ph (534). pfeffer and rothman (872) suggest that this could explain the failure of chloroquinone-treated cells to package secretory proteins into secretory gran ules (see above). secretory granules of exocrine (hormone-secreting) cells remain acidic during maturation, but those of endocrine (enzyme-secret ing) cells return to neutral ph as they mature (534). atp is also needed for secretory granule formation. there are conflicting views as to whether different secretory proteins cosegregate and coaggregate into the same secretory granule. detailed studies by fumagalli and zanini (341) revealed that bovine growth hor mone and prolactin could be present either in different aggregates in the same secretory granule or in mixed aggregates in the same granule or in pure aggregates in different granules. the ratios of the three type of granules varied from animal to animal. similar results were reported by mroz and lechene (744) , who showed that the enzyme content of individ ual secretory granules derived from single cells from the same gland can vary enormously. the simplest interpretation of these data seems to be that segregation is a random process and that the formation of mixed or pure aggregates depends on the local concentration of the respective pro teins and on their preference for forming homo-rather than heteroaggregates. packaging of proteins into different secretory granules, however, might permit their release to be stimulated by different secretagogues, but there is only limited evidence for such a phenomenon at the level of individual cells (11,136,317a) . many of the proteins secreted by the regulated branch of the secretory pathway are proteolytically processed and activated, usually in secretory granules. processing often involves the proteolytic removal of an n-terminal propeptide and can be mimicked by exogenous proteases such as trypsin (336). alternatively, short spacer peptides may be removed from polyprotein precursors (187). in the latter cases, cells in different tissues can process the precursors to give different "mature" forms. this is the case for prosomatostatin, which is processed to a 28-amino-acid form by cells in the gut and to a 14-amino-acid form by brain and pancreatic cells (790). islet tissue from angler fish pancreas contains at least two proteases which process prosomatostatin to give products of different lengths. one of these proteases can also process proinsulin (674) . other examples of processing of heterologous secretory proteins (188, 444, 732) indicate the existence of only a limited number of processing proteases, which may also be found in some cell types which do not have a regulated secretory pathway (444, 1168) . when secretory proteins which would normally use the regulated pathway are produced in cells which do not have the regu-lated pathway, however, they are constitutively secreted as unprocessed (pro-) forms (889). the site of proinsulin processing was determined cytochemically by orci et al. (816, 825) , using monoclonal antibodies specific for mature insulin. fully processed insulin was first detected in clathrin-coated vesi cles budding from the tgn, and subsequently in naked granules. process ing was coincident with condensation and acidification and was inhibited at higher ph, indicating that the two proinsulin-processing proteases have low ph optima (821). therefore, this and other proteolytic processing steps probably occur in a late "compartment" of the tgn. although propeptides may not play a role in protein sorting, they may prevent enzymes such as proteases from folding into active conforma tions prior to their release, thereby protecting secretory granules from endoproteolytic attack. proinsulin and other prohormones may be loosely membrane-associated (789,819). in these cases, bridge sequences may contribute to the patch signal which shunts proteins into budding secre tory granules. relatively little appears to be known about the events which accompany protein release from secretory granules. the general view seems to be that secretagogues directly, or more likely indirectly, stimulate fusion between the granule membrane and the cytoplasmic membrane, resulting in the release of granule contents to the outside of the cell. secretagogues bind to specific cell-surface receptors and promote ca 2 + influx, which seems to be intimately related to the fusion event (756). gtp binding proteins also seem to be involved in generating the signal which stimu lates secretion (138), and microtubules may play a minor role in directing storage granules to the cell surface (112). inhibitors of metalloprotease and dipeptide protease substrates inhibit exocytosis, suggesting that pro teolytic cleavage of a membrane protein may be essential for exocytosis. mundy and strittmatter (756), who found that metalloprotease activity was highest in the plasma membrane, propose that proteolysis may un mask the active site on a fusogenic membrane protein. breckenbridge and aimers (121) have recently studied exocytosis-associated changes in membrane capacitance in a mouse mast cell mutant with enlarged secretory granules. small fluctuations in capacitance preceded larger increases, which themselves preceded granule swelling and the release of a fluorescent tracer dye from the lumen of the granule. the large increase in capacitance probably results from productive fusion be tween the plasma and granule membranes (312), whereas capacitance "flutters" may represent nonproductive membrane association. the most plausible interpretation of these data is that release of secretory granule contents is preceded by the formation of a narrow channel, the fusion pore, (121) between the two membranes, leading eventually to the open ing of the granule membrane to the outside of the cell and the subsequent swelling and dissociation of the granule contents and their release as soluble proteins. lysosomes (in animal cells) and vacuoles (in plant and fungal cells) con tain most of the cells' degradative enzymes, which function not only in general "housekeeping" but also in the degradation of endocytosed mate rial (chapter viii). the following sections review the evidence for lysoso mal and vacuolar sorting signals, special features of the sorting pathways, and differences between the lysosomal and vacuolar routes. specific sorting of soluble lysosomal enzymes is determined by mannose-6-phosphate (m6p) residues on n-linked core oligosaccharides (section v.d.i.a). two receptors have been identified. the major m6p receptor (275 kda, formerly called the 215-kda receptor) was detected predomi nantly in the cis golgi compartment (131), leading to the proposal that the lysosomal pathway diverged from the main secretory pathway at the cis end of the golgi stack rather than in the tgn. this idea is incompatible with the observation that some lysosomal proteins are terminally pro cessed by enzymes located in medial and trans golgi compartments. other cytological studies indicate that m6p receptors are located in the tgn, as well as throughout the golgi stack (208), in coated vesicles and the plasma membrane (365,366) (see below for explanation), and in a golgi-proximal vesicular structure (402), but not in lysosomes. thus, ly sosomal enzymes probably do transit through the tgn, possibly already complexed with their receptor, although farquhar (299) argues strongly in favor of multiple lysosomal sorting pathways and in particular for sorting from the cis golgi in certain cell lines. thus, the site of accumulation of m6p receptors along the secretory pathway may have little relevance for lysosomal enzyme sorting. two m6p receptors (275 kda and 46 kda) have been identified and characterized. the receptor activity of the 275-kda protein, but not that of the 46-kda protein is cation-independent, and the 46-kda receptor recognizes only phosphomonoesters whereas the 275-kda protein also binds methylphosphomannosyl residues (461). the 275-kda protein ap pears to be present in most mammalian cell lines tested so far (982), but the distribution of the 46-kda receptor has not been determined. some mutant cell lines lack the 275-kda m6p receptor yet still target lysosomal enzymes normally, which suggests that both receptors are involved in lysosomal sorting (461). studies with such cell lines revealed another difference between the two receptors, however. although part of the cellular pool of both receptors is located on the cell surface, only cells with the 275-kda protein can endocytose secreted lysosomal proteins (1077). this "defect" in the 46-kda protein appears to be due to a failure to bind the ligands, since antibodies against the 46-kda protein are endocytosed normally (see chapter viii for more details on endocytosis). thus, the 46-kda protein seems to be specifically involved in the sorting of endogenous lysosomal enzymes. the genes for both m6p receptors have been cloned and sequenced. although the predicted sequence of the two gene products are generally different, the two proteins have a region of moderate sequence similarity in their lumenal domains (645,826) which dahms et al. (208) propose could be the m6p binding domain. the sorting of the lysosomal enzymes cathepsin c and cathepsin d was studied directly by lemansky et al. (626) , who found that lysosomal enzyme precursors occurred only in coated vesicles. proteolytically ma tured forms were found in lysosomes. schulze-lohoff et al. (1022) also observed the transient accumulation of one of these enzymes in coated vesicles, which, they proposed, are specifically involved in the sorting of lysosomal proteins from the secretory pathway. lemansky et al. (626) devised procedures which allowed vesicles derived from the secretory pathway to be separated from those derived from the endocytotic path way. they found that both classes of vesicles contained precursor forms of cathepsin. these studies have two profound implications: (i) the fact that vesicles involved in direct sorting of cathepsins to the lysosome contain clathrin implies that they had passed through the tgn, the first site along the secretory pathway at which clathrin is detected (section v.g.i). (ii) the fact that some lysosomal enzymes are "fished" out of the sur rounding medium and retargeted to the lysosome implies that some lysosomal enzymes are incorrectly sorted, probably into the consti tutive secretory pathway (see also section v.d. 1 .a). higher levels of incorrect sorting occurs in nh 4 -cl-treated cells, probably because low ph is required to dissociate m6p from its receptor in the prelysosome ( another interesting observation concerning the m6p receptor is that it specifically binds to one of the components (the 100-kda accessory pro tein) of the clathrin cage which coats the sorting vesicles (854) (see be low). this may have particular relevance for the sorting of lysosomal enzymes because different classes of clathrin-coated vesicles appear to have different types of accessory proteins (855). coated vesicles almost certainly do not transport proteins directly into lysosomes. instead, the vesicles are targeted to endosome-like reticular organelles (prelysosomes or secondary endosomes), where the receptor probably dissociates and recycles back to the golgi cisternae. this organ elle is also the site to which endocytosed lysosomal proteins are targeted (134,402) ( fig. v.6 and section viii. a.4.b). a different class of vesicles may complete the transport of lysosomal enzymes once they have dissoci ated from their receptor, but von figura and hasilik (313) consider it more likely that there is a gradual transition from tubular prelysosomes to lyso somes proper. although m6p is undoubtedly the major, and in some cells the only, sorting signal on lysosomal enzymes, some lysosomal proteins do not have m6p residues. owada and neufeld (829) found that a human liver cell line devoid of iv-acetylglucosamine-l-phosphotransferase [and there fore unable to phosphorylate mannose residues (see section v.d.i.a)], still targeted some lysosomal enzymes correctly with, at most, only slightly reduced efficiency. these cells may have a completely m6p-independent system for sorting lysosomal enzymes. all lysosomal membrane proteins are also devoid of m6p residues. therefore, some lysosomal enzymes may have membrane-associated intermediates which are sorted to the lysosome together with authentic lysosomal membrane proteins. barriocanal et al. (49) used immunocytochemistry to follow the fate of three lysosomal membrane proteins which they detected in lysosomes, the golgi apparatus, and coated and uncoated vesicles in the region of the tgn. they found that oligosaccharide modifications were not required for lysosomal targeting (although they may be required to protect against proteolysis). thus the sorting pathway for these proteins remains to be determined; they could be sorted completely independently of lysosomal enzymes or could be colocalized to the same coated vesicles by an m6pindependent receptor and then segregated from recycling vesicle mem brane components in the prelysosome. green et al. (397) have recently found that newly synthesized lysosomal membrane proteins appear in lysosomes with the same kinetics as newly synthesized plasma membrane proteins appear at the cell surface, making it unlikely that the former pass via the plasma membrane en route to the lysosome. although vacuoles are the functional equivalents of lysosomes in animal cells, the sorting of vacuolar enzymes is completely independent of m6p receptors. this was demonstrated most simply by the fact that tunicamy cin treatment did not affect vacuolar protein targeting (572,1025). how ever, at least one vacuolar protein does have phosphorylated mannose residues. most of the work on the sorting of vacuolar proteins has concentrated on the identification of sorting signals in yeast vacuolar proteins. early studies showed that vacuolar proteases were proteolytically processed in two distinct stages, the first of which corresponded to the removal of a signal peptide (711). the second processing step is catalyzed by a vacuo lar protease, proteinase a, which removes an additional n-terminal poly peptide segment, the propeptide, from other vacuolar enzymes. protein ase a is also autoactivated by the same mechanism (15,1206). the second processing step is blocked by certain sec mutations, which cause secre tory proteins to accumulate in the rer or golgi apparatus, whereas mutations which affect the final stage of the secretory pathway from the golgi to the cell surface do not affect vacuolar protein sorting or process ing (1082). this implies that the golgi is the site of sorting of vacuolar and secreted or plasma membrane proteins in yeast cells. gene fusion studies conclusively demonstrated that propeptides are vacuolar sorting signals. bankaitis et al. (44) and johnson et al. (541) found that at most 50 n-terminal residues of preprocarboxypeptidase y (cpy), including the 20-residue propeptide, could target the normally secreted enzyme invertase into the vacuole. similar results were obtained with proteinase a-invertase hybrids (572). vails et al. (1137) subse quently found that mutations affecting the sequence of the procpy pro peptide caused the enzyme to be secreted in an inactive form. there appears to be no sequence similarity between the propeptides of different yeast vacuolar enzymes, even though genetic studies described below suggest that they are sorted into the vacuole via a common pathway. part of the propeptide may be required to maintain vacuolar enzymes in an inactive form until they reach the vacuole and may also maintain the precursors in a competent conformation for transport through the secre tory pathway. the overproduction of vacuolar proteinase a (972) and of cpy-inver-tase hybrids (44) causes them to be secreted into the medium, suggesting that some component of the vacuolar sorting pathway (e.g., a receptor) had been saturated. these observations led to the development of tech niques for selecting mutants which secreted cpy or cpy-invertase with out overproduction (section ii.f.l.b). mutations in over 50 different genes (called vpl or vpt) have been identified (44,939a,971) . the extent of the sorting defect varied in different mutants: some of them, for exam ple, did not affect the targeting of proteinase a, and none of them affected the sorting of the vacuolar membrane protein α-mannosidase, which is presumably sorted to the vacuole by an alternate pathway (971). it is unlikely that any of the mutations affected protein retention in the vacuole because vacuolar enzymes were not terminally processed, and the kinet ics of cpy secretion were comparable to those of a normally secreted protein. the characterization of the vpt or vpl gene products and their localization in the cell could provide revealing insights into the mecha nisms of vacuolar protein targeting, but at present we can only speculate on their roles. obvious candidates are the propeptide receptor, vacuolar or golgi atpases which might produce a low ph environment necessary for sorting or receptor dissociation, or proteins involved in vesicle fission and fusion. much less work has been done on plant vacuolar proteins. tague and chrispeels (1100) found that the plant vacuolar storage protein phytohemagglutinin was targeted mainly to the vacuole when its structural gene was expressed in yeast cells. this protein does not have a cleavable propeptide (it does have a signal peptide, which was at least partially processed in yeast cells), which means that the vacuolar plant sorting signal which is recognized by the yeast vacuolar sorting pathway is lo cated in the mature part of the phytohemagglutinin polypeptide. mrna coding for a second plant storage protein, globulin p, has been microinjected into frog oocytes, which secreted the protein into the medium (51). this confirms that plant vacuolar proteins are bona fide secretory proteins and that plant cells have a special branch of the secretory pathway which shunts storage proteins into vacuoles. as we have seen, protein-coated vesicles are involved in transporting secretory proteins at various stages of the secretory pathway. some vesi cles (e.g., lysosomal sorting vesicles and immature secretory granules), are coated with a protein complex called clathrin, which also coats endocytotic vesicles (chapter viii). other secretory vesicles (e.g., those me diating protein transport through the golgi) have a different type of pro tein coat (797). clathrin, which is composed of equimolar amounts of heavy and light chains, forms a three-layered cage which envelops vesicles in a shell with fibrous interconnections, which give it mechanical strength and stability. the vesicle membrane is thought to have receptors which anchor the clathrin cage to the surface via a number of ancillary assembly proteins which probably act as bridges (1148, 1149) . different assembly proteins are found in different classes of clathrin-coated, tgn-derived, and endocytic vesicles, suggesting that they might contribute to their respective specificity for particular membrane targets (7). the clathrin coat probably prevents intimate contact between fusing membranes and must thus be removed to allow fusion to occur. this may explain, for example, the disappearance of the clathrin coat from matur ing secretory granules (section v.g.4.b) and the presence of lysosomal protein precursors in naked as well as coated vesicles. vesicles coated with other proteins are also presumably uncoated to allow fusion to oc cur. the uncoating of clathrin-coated vesicles is mediated by the atpdependent cytosolic "uncoating" protein, which remains attached to the released clathrin (1010). soluble clathrin retains its typical triskelion con formation. uncoating protein is a member of a highly conserved group of stress proteins and may be related to hsp70 heat shock proteins involved in other stages in secretory protein transport and in mitochondrial protein import (sections iii.c.3 and vlb.5). the significance of this observation remains to be determined (855,967), but one possibility is that the atpase (hsp70) is required to activate an uncoating enzyme which is already present in the clathrin complex. the action of uncoating protein must be triggered in some way to pre vent it from destroying protein coats on immature vesicles or on coated buds, but the nature of the signal remains to be determined. the require ment for atp for uncoating activity could in part explain the observed requirement for the same nucleotide during the transport of proteins be tween different stages of the secretory pathway if a similar activity is required to remove other, nonclathrin coats. attempts to determine the role of clathrin in protein targeting in yeast cells have given ambiguous results. yeasts are known to have clathrin, and coated vesicles have been observed, but it is not known whether clathrin coats vesicles involved in secretory protein targeting (1165). the gene for the clathrin heavy chain was independently cloned by two groups who used it to inactivate the chromosomal gene to study the effect of the absence of clathrin on cell growth and protein secretion. payne and schekman (852) and payne et al. (853,853a) reported that their mutants grew somewhat more slowly than wild-type cells, secreted invertase at a slightly reduced rate, and were partially defective in prepro-a-factor pro-cessing. the mutants accumulated unusual vacuoles, vesicles, and golgiderived structures. these results suggested that the absence of clathrin did not completely impair plasma membrane growth and protein secretion but that there was nevertheless a reduced rate of transit of secretory proteins through the later stages of the secretory pathway. different results were obtained using exactly the same approach by lemmon and jones (627). they found that cells lacking the clathrin heavy chain were not viable unless they also carried a suppressor mutation. even with this mutation, the cells grew slowly, were larger and rounder, had an unusual granular appearance, tended to aggregate in liquid culture, and were poly ploid. these results suggest that the absence of clathrin is highly detri mental to yeast cells, making it difficult to determine whether clathrin plays a specific role in protein secretion in this organism. the secretory pathway is almost certainly the route by which the vast majority of secreted and plasma membrane proteins are exported by eukaryotic cells. however, there is increasing evidence that some plasma membrane and secretory organelle proteins may reach their final destina tions directly rather than via the secretory pathway. examples of this class of proteins include ras-like gtp binding proteins such as sec4 (987) and ras2 (232) of the yeast s. cerevisiae and similar proteins from complex eukaryotes (1194), mating pheromones in yeast and some fungi (726, 886, 983) , capsid proteins of picornaviruses (851), and src proteins of rous sarcoma and other transducing viruses (1029). most if not all of these proteins are fatty acylated. two different amino acids seem to be modified: n-terminal glycines, which are substrates for myristoyl coa protein 7v-myristoyltransferase (1124), and c-terminal cysteines in raslike proteins (232). these cys residues are reported to be palmitoylated, but a farnecyl residue has been found in basidiomycete pheromones (983). the absence of the fatty-acylated amino acid disrupts membrane associa tion of ras and src proteins (232, 555, 1029, 1194) , indicating that fatty acids probably anchor these proteins in their respective membranes. it remains to be determined how fatty-acylated proteins actually cross the plasma membrane (as in the case of the fungal pheromones) or what determines their specificity for certain membranes. a further example of a secreted protein which does not have a secretory routing signal is interleukin 1, but very little appears to be known about how this protein crosses the plasma membrane. there is general agreement concerning the events which lead to the sort ing of secretory proteins into different terminal branches of the secretory pathway, as illustrated in fig. v. 6, but we are clearly a long way from understanding exactly what directs proteins to their specific targets. patch signals are undoubtedly necessary for sorting soluble proteins (other than lysosomal enzymes) into the various branches of the secretory pathway, but these will be difficult to identify by gene fusion techniques. at present, we have no clear idea of the extent to which the sorting vesicles have different membrane contents, but it seems probable that specific groups of membrane proteins (lysosomal, secretory granule, apical, and basolateral) accumulate at different sites in the membrane of the tgn from which sorting vesicles bud. this specialization is presumably also determined by protein-protein interactions, but the possibility that other interactions (e.g., protein-lipid) might be involved should not be over looked. another interesting observation is that atp seems to be required at almost every stage in the secretory pathway. atp has been proposed to act in a variety of ways, including acidification of secretory organelles and vesicles, phosphorylation of receptors or ligands, protein folding and "proofreading," activation of cytoskeletal motors, and vesicle uncoating activity. there is increasing evidence that gtp and gtp binding proteins (g proteins) are also involved at several stages in the secretory pathway. gtp binding proteins are also known to be involved in the generation of other intracellular signals, such as the activation or inactivation of adeny late cyclase, the activation of cyclic gmp phosphodiesterase, and the control of phospholipase c action (769). by analogy, gtp binding pro teins may act as signals or to activate receptors or ligands to ensure vectorial transport through the secretory pathway (109). constitutive and regulated secretion of proteins progress in unravelling pathways, of golgi traffic assembly of asparagine-linked oligosaccharides the sorting of proteins to the plasma membrane in epithelial cells biosynthetic protein transport and sorting by the endoplasmic reticulum and golgi protein localization and membrane traffic in yeast lysosomal enzymes and their receptors key: cord-018969-0zrnfaad authors: giese, matthias title: types of recombinant vaccines date: 2015-09-24 journal: introduction to molecular vaccinology doi: 10.1007/978-3-319-25832-4_9 sha: doc_id: 18969 cord_uid: 0zrnfaad the original scientific strategy behind vaccinology has historically been to “isolate, inactivate, and inject,” first invoked by louis pasteur. new vaccines and vaccination strategies are being developed including the use of attenuated live mycobacteria, recombinant microorganisms, and subunits, prime-boost strategies based on the successive administration of a certain mycobacterial antigen under two different vaccine vectors, and dna vaccines [ 1 ] . the various types of vaccines differ in eliciting an immune response. live attenuated vaccines (lavs) mimic a natural infection without being virulent and trigger the activation of the innate immune system through pamps. following injections, lavs rapidly disseminate throughout the vascular network to the draining lymph nodes. therefore, the route of application of lavs does not specifi cally infl uence the immune response. lavs also don't need an adjuvant; they possess a natural intrinsic adjuvancy. safety concerns exist because of the replication competence and the possibility of recombination with a wild type. non-live vaccines, inactivated and most recombinant vaccines, whether containing proteins or carbohydrates (−conjugates), are less effective. in the absence of replication, vaccine-induced immune reactions remain more limited, and therefore the route of vaccination infl uences the effi cacy and the duration of the immune reaction. nonlive vaccines induce a lower antibody response and generally no cytotoxic t lymphocyte activation. compared to lav, all non-live vaccines are regarded as biologically safe ( fig. 9 .2 ). dna vaccines entail the direct, in situ inoculation of dnabased eukaryotic expression vectors that encode the sequence of a pathogenic protein antigen. the constructed plasmids are then subsequently grown in bacteria like e. coli and highly purifi ed via chromatographic methods. lps contamination of plasmids has to be prevented because of the immunotoxic properties of natural lps. after purifi cation the circular double-stranded dna plasmids are ready for vaccination. the de novo production of the encoded antigens in the host results in the elicitation of both the antibody and the cellular response by activating cytotoxic t lymphocytes (ctls). vaccine proteins made by the host are natural proteins and contain important posttranslational modifi cations such as the correct glycosylation. but like subunit vaccines, dna vaccines must be adjuvanted. naked dna does not work. the unique advantage of dna vaccines is their ability to mimic the effects of live attenuated vaccines without the risk associated with the administration of infectious albeit attenuated material. dna vaccines are able to stimulate a complete, humoral and cellular immune response. peptide fragments are processed via the endogenous pathway, resulting in the presentation of antigen on the cell surface by mhc class i molecules. plasmid dna is very stable also beyond a cold chain. therefore, the storage, transportation, and distribution of dna vaccines are more practical and also cheaper [ 2 ] . mostly all plasmid dna constructs ( fig. 9 .3 ) used for vaccination share fi ve main characteristics: • strong promoter/enhancer sequence for driving the incorporated foreign gene • convenient cloning site for insertion of foreign genes • origin of replication for initiation of plasmid replication • polyadenylation/termination sequence for production of mature mrna • resistance/antibiotic marker for selection • immunomodulators, e.g., cpgs, interleukins, ubiquitin, etc. • (on the same plasmid or on extra plasmids) uptake of plasmid dna. some biological barriers have to be overcome by dna vaccines on the way to the cell nucleus where the plasmid dna is translated into cellular mrna. after delivery of plasmid dna to the target tissue, e.g., skeletal muscle or skin, lots of tissue nucleases attack 9.3 dna vaccines and degrade a large amount of the applicated dna. also the extracellular matrix with collagen and hyaluronic acid infl uences the passage from the application site to the cell membrane. only a small portion (1 % estimated) of the still intact plasmid dna will cross the cell membrane by phagocytosis or pinocytosis. inside the cell the route toward the nucleus is also spiked with exo-and endonucleases so that probably only 0.1 % (estimated) is successfully and actively transported through the nucleus pore membrane (npc). small particles (<~40 kda) are able to pass through the nuclear pore complex (npc) by passive diffusion; larger particles need the support of carrier proteins for effi cient passage through the complex. because of this enormous loss of plasmid dna (up to 99.9 %), various tools were developed to protect the plasmid dna and thus increase the effi cacy such as encapsulation into liposomes or binding of dna to dendrimers. figure 9 .4 illustrates the passage of plasmid dna from the extracellular matrix (ecm) to the nucleus. whereas in human medicine clinical trials with dna vaccines are still ongoing without any registered product on the market, the fi rst approved dna vaccines for the veterinarian medicine are available since 2005 and are discussed now. the fi rst veterinarian dna vaccines were developed for horses (davis b.s., 2001 for wnv [ 3 ] ; giese m., 2002 for eav [ 4 ] ). today the number of current clinical trials worldwide with veterinary dna vaccines is unmanageable and probably all species are hit. canine malignant melanoma (cmm) typically begins in the mouth or around the toes and can spread within the body to the heart, lungs, intestines, and other organs. canine malignant melanoma is known for being one of the most aggressive cancers in dogs and deadly. cmm is most commonly seen in golden retrievers, scottish terriers, dachshunds, labradors, and poodles ( fig. 9 .5 ). metastases of the tumors will be found very often in distant parts of the body. the overall biology of cmm is similar to the biology of human melanoma. however the melanomas in dogs have diverse biologic behaviors due to the race and a variety of factors. standardized treatments such as surgery, radiation, and chemotherapy are the common tools to fi ght canine malignant melanoma. these traditional tools have afforded minimal to modest stage-dependent clinical benefi ts. xenogeneic dna vaccine. the plasmid dna contains a cdna for the human tyrosinase, hutyr, a tumor antigen (ta). this is a non-mutated differentiation antigen and specifi c to melanoma. tyrosinase is a glycoprotein and essential in the process of melanin synthesis ( fig. 9 .6 ). like other tas tyrosinase is overexpressed in tumor cells and therefore an ideal target in cancer therapy. normally there is no strong immune reaction against the body's own protein. but immunization of dogs with xenogeneic hutyr cdna can break the immune tolerance against this self tumor differentiation antigen and induce antibody and cytotoxic t cell response against melanoma cells [ 5 ] . tyrosinase is highly conserved from dog to mouse to man. radiotherapy in cases with positive surgical margins or positive regional lymph nodes. one dose contains 102 μg dna given in a volume of 0.4 ml by the transdermal route via a needle-free vaccination device. booster immunizations were given at 6-month intervals. in march 2007 the drug manufacturer received a conditional license for oncept from the usda and a full license in 2010. the results of the xenogeneic immunization of dogs with hutyr cdna as an adjunct therapy for cmm demonstrate a signifi cant increase of survival time compared to the control group. none of the dogs developed systemic adverse reactions; no toxicity was seen. the overall safety of this dna vaccine is confi rmed. this vaccine development represents a tremendous milestone in dna science and technology. virus. west nile virus (wnv) is a mosquito-borne member of the family flaviviridae , genus flavivirus , and was fi rst identifi ed in 1937 in uganda, africa. it is a positive-sense, single-strand rna virus, (+)ssrna, of about 11 kb that encodes a single polyprotein with seven nonstructural proteins and three structural proteins. the rna strand is held within a nucleocapsid. wnv replicates in the cytoplasm of infected cells. wnv is a zoonotic virus. the primary reservoir is birds with a signifi cant impact to spread the infection across countries and continents. more than 170 different species are described as carrier of this virus. wnv is spread from bird to bird by mosquitoes when they bite, or take a blood meal, from birds that are infected with the virus. birds from some species get ill and die; others have no clinical signs and survive. mosquitoes are also capable of spreading the virus to horses, dogs, cats, mice, alligators, and lots of other mammals but also to humans. one-third of all horses bitten by carrier mosquitoes develop the disease and die or are so affected that euthanasia is required. the incubation period ranges from 3 to 14 days. horses that do become ill vary in symptoms: muscle trembling, skin twitching, ataxia, sleepiness, dullness, and listlessness. wnv may cross the placenta from mother to gestating foal. horses cannot spread the disease to humans. wnv produces different outcomes in humans like in horses: fever, headache, chills, diaphoresis, weakness, swollen lymph nodes, drowsiness, and pain in the joints comparable to symptoms of infl uenza. more severe neuroinvasive infection includes meningitis and encephalitis. wnv-dna vaccine. the surface envelope protein e is the main target for the antibody response. there are more than 180 copies of the e protein in a mature wnv virion. the e function is the interaction between the cell surface and the fusion between virus and cellular membrane. the premembrane protein prm is cleaved during viral maturation into a smaller membrane m peptide. the expression of prm and e protein in cells results in the formation of virus-like particles, vlp. these vlp share many of the antigenic and structural properties of fully mature viruses and are of special interest for a vaccine development ( fig. 9.7 ) . the fi nal expression plasmid for immunization of horses contains the human cytomegalovirus early gene promoter, signal sequences from japanese virus, and a fusion gene of part of the fi shing industry is aquaculture, also known as aqua farming, but it can be contrasted with commercial fi shing, which is the harvesting of wild fi sh. aquaculture involves cultivating freshwater and saltwater fi sh and other populations (shrimp, oyster) under controlled conditions. salmon is one of the main food-producing fi sh in the world. a dna vaccine for fi sh must be not only safe for the animal but especially safe for the fi sh consumer. salmon is the major economic contributor to the world production of farmed fi sh, representing over u$1 billion annually in the united states. salmon farming is also very big in norway, scotland, canada, and chile and is the source for most salmon consumed in the united states and europe. like all other animals also fi sh is threatened by viruses, bacteria, and parasites. one major problem for salmons is the infectious hematopoietic necrosis (ihn) virus [ 6 ] . virus. infectious hematopoietic necrosis (ihn) virus is a common viral pathogen of both wild and farmed salmonids, in particular pacifi c salmonids, rainbow trout, and atlantic salmon. ihn virus is enzootic to the pacifi c northwest; however it has varying effects on different pacifi c salmonids. it is a negative-sense single-stranded, (−)ssrna virus that is a member of the rhabdoviridae family, genus novirhabdovirus . the rna genome is 11,133 nucleotides long and contains a leader (l) and trailer (t) sequences at its 3′-end and 5′-end, respectively. the coding regions are n, p, m, g, nv, and l genes. g encodes the surface glycoprotein, so-called spikes, main target for the immune response. transmission. ihnv is transmitted following shedding of the virus in the feces, urine, sexual fl uids, and external mucus and by direct contact or close contact with surrounding contaminated water. the virus gains entry into fi sh at the base of the fi ns. salmons are carnivorous and are currently fed a meal produced from catching other wild fi sh and other marine organisms -a permanent origin of possible infections with ihnv. clinical signs of infection with ihnv include anemia, skin darkening, bulging of the eyes, fading of the gills, and abdominal distension. infected fi sh commonly hemorrhage in several areas, like the mouth, the pectoral fi ns, muscles near the anus, and the yolk sac of fry. diseased fi sh weaken, eventually fl oating on the surface of the water. necrosis is common in the kidney and spleen and sometimes in the liver. mortality rates in older fi sh (2-3 kg) tend to range from 10 to 20 %; in smolts the mortality rate often exceeds 85 %. the average cumulative mortality following an outbreak is estimated at 47 %. ihnv-dna vaccine. the antigen is the viral surface glycoprotein (g) capable of eliciting neutralizing antibody and the production of a protective immune response. the g gene was cloned into a eukaryotic expression vector by insertion of an intermediate-early promoter and a polyadenylation signal. but the speciality of this vaccine is to be prepared as a two-component vaccine in a single vaccine, one plasmid or more. the second component is a portion of the nucleic acid sequence encoding a second peptide, derived from a fi sh pathogen other than the said rhabdovirus resulting in a fusion. this second pathogen can be any fi sh pathogen, e.g., isav, ipnv, iridovirus, nnv, spdv, svcv, vhsv, koi herpes virus, and more. the rationale behind this is that the presence of the ihnv g protein boosts the immune response to the second protein, resulting in a protective effect against infection by this fi sh pathogen. the vaccine is given intramuscularly with a dosage of only 10 μg in 50 μl on the left dorsal fl ank, in the area just below the dorsal fi n [ 7 , 8 ] . this fi rst dna fi sh vaccine was licensed in 2005 in canada by the veterinary biologics section (vbs), animal health and production division, canadian food inspection agency (cfia) and is also used now in studies in norway. there are many environmental stressors and diseases which infl uence and seriously threat the life of european honey bees, apis mellifera. the european honey bee is professionally managed worldwide for honey production and pollination. the bee was imported to the united states 400 years ago with the fi rst european settlers and called "white man's fl y" by the native americans, the indians. first reported in the united states, a mysterious socalled colony collapse disorder (ccd) decimated the bee colonies there between 50 and 90 %, fi rst observed during the winters of 1995-1996 and then 2000-2001 and without interruptions up to now. a similar situation is also given in europe. about 20,000-60,000 bees live in a colony. the fi rst description originated from the 1950s. in the early nineteenth century, the colony losses were known in england as "isle of wight disease," and the americans called this phenomenon "disappearing disease" in the 1960s, whereas these colony losses in france in the late 1990s were called "mysterious bee losses." where have all the bees gone? economic value. the huge loss of honey bees as pollinators has a dramatic impact on agricultural pollination. about 130 crops, nuts, fruits, and vegetables are pollinated by a. mellifera , with an overall value of more than $ 15 billion in the united states and more than € 14 billion for the eu in 2005. a bee colony produces some 1 kg/2205 lb honey per day. in return, these bees have to pollinate 10-15 million fl owers. one should keep in mind that besides european honey bees, wild insects, among them 30,000 species of wild bees, have also a very great impact on pollination and seem to be more effi cient in pollination as managed honey bees [ 9 ] . the industrial farming threatens also the natural biotope of wild insect pollinators. ecologic value. the total global economic value of honey bee pollination was calculated in 2005 to more than € 150 billion or $ 202 billion. the food and agriculture organization (fao) of the united nations estimates that there are 65 million managed honey bee colonies worldwide. beside this professional agriculture, honey bees are irreplaceable for the biodiversity. this organism appeared during evolution with the fi rst fl ower plants and exists since 100 million years as described in chap. 2 , fig. 2 .10 . after swine and cattle, bees are in europe and north america the third important farm animal and since 2007 formally listed as farm animal in switzerland. therefore, ccd is not only an economical but also an essential global ecological problem which urgently must be solved in the future. "the bee is more than honey." what is causing ccd? the colony collapse disorder of the last years seems to differ from past outbreaks: the worker bees disappear instead of dying in place, leaving behind the queen and young bees. high levels of bacteria, viruses, and fungi are measured in the gut of the remaining bees. collapses can occur within 2 days. a complex problem. different theories are discussed about what is causing ccd. pesticide contamination, hotly debated to interfere with the nerve system affecting foraging behavior of bees, lead them to abandon their hives. fungal diseases such as nosema spp. is known for big bee losses in spain. monocultures or gene-manipulated crops. electro smoke (radio waves) caused by cell phones destroys the bee's compass. the rigors of travelling in trucks from crop to crop in the usa. down from february professional us beekeepers travel with their colonies through the country until december. thereby the bees must relocate up to 15 times. in europe the bee colonies begin the winter sleep around september. also the climate change, the temperature sensitivity is discussed to have an impact on crop pollination. ccd is likely caused by a combination of factors [ 10 , 11 ] . varroa destructor . but in all ccd cases, an overload of bloodsucking varroa mites is detectable and varroa is currently considered the major threat for apiculture. the infection and disease is called varroosis. varroa destructor is an ectoparasite, has a reddish-brown fl at shape, and is 1-1.8 mm long and 1.5-2 mm wide, with eight legs. v. destructor infest worker bees and drones and its brood. the mite develops inside the brood cells. varroa is a real colossus compared to the size of bees as can be seen in fig. 9 .1 . varroa mites belong to the scientifi c class of arachnida, subclass acari. there are 50,000 species described alone from mites. some mites prefer carbohydrates as food such as meal or crops. the house dust mites feed fl akes of shed human skin. varroa mites prefer fresh "blood" and the hemolymph of bees and can feed 0.1 mg/0.0000002205 lb within 2 h. varroa is transported into the hives via piggyback by worker bees. the female mite enters broad cells, preferentially drone cells. once the cell is capped, varroa lays eggs on the larvae. the development from egg to insect takes 7 days. bee larvae and mites hatch in about the same time and the newborn varroa mites spread to other bees [ 12 , 13 ] . the lifetime of summer mites are 3-6 weeks, whereas fall mites can live for several months. varroa can only reproduce in honey bees and thus are considered harmless to other insects. varroa is more than a disease. it is a global pest having devastating effects on bees ( fig. 9 .8 ). varroa as vector. varroa may be not considered as isolated agent for the disease. the mortality of adult bees and its brood must be considered in the context with secondary viral infections. at least 18 various viruses are able to infect honey bees, mostly ssrna viruses. eight viruses are known to be associated with varroa mites: acute bee paralysis virus (abpv), black queen cell virus (bqcv), chronic bee paralysis virus (cbpv), deformed wing virus (dwv), kashmir bee virus (kbv), sacbrood bee virus (sbv), cloudy wing virus (cwv), and slow bee paralysis virus (sbpv) [ 14 -17 ] . varroa control. a number of natural and synthetic chemicals are commercially available for the control of varroa infestations. the fi rst compounds were bromopropylate, fl uvalinate, or other pyrethroid insecticides. and to make a long story short, varroa mites became resistant not only against one product of a given chemical class; the resistance was against the entire class with several related synthetic products. also the use of natural products, such as formic acid, mineral oil, or thymol, is only partially and temporally effective and show adverse effects [ 18 ] . there is no successful chemical treatment. mites will quickly develop resistance to all chemicals. the immune system of insects. the basic difference between insect and vertebrate immunity is the missing highly specifi c antigen response of the acquired immune system in insects. nevertheless, in the 400 million years of evolution, insects developed a powerful defense strategy against bacteria, fungi, viruses, and parasites. only protected by this "primitive" immune system insects were so successful that they colonized all terrestrial ecosystems. the insect innate immunity shows many similarities to the vertebrate and to the human innate immunity, is multifaceted, and involves both humoral and cellular components [ 19 ] . most insights on insect immunity are provided by drosophila melanogaster research. the key mechanism is also observed in honey bees. the humoral and systemic response to bacterial and fungal infections is controlled by antimicrobial peptides (amps). there are circulating receptors sensing a danger signal and activating the toll pathway, whereas membranebound receptors activate the imd pathway. both pathways lead to the translocation of nf-kb-like transcription factors and the production of amps. nf-kb response elements can be detected in the promoter region of the diptericin gene. the cellular immune response is mediated by specialized blood cells, the hemocytes, plasmatocytes, crystal cells, and lamellocytes [ 20 ] . plasmatocytes represent 95 % of the majority of hemocytes. they express phagocytic receptors and patrol through the body, clear microorganism and cell a b c debris, and signal infections to the fat bodies. the bee genome was completely sequenced in 2006 [ 21 ] . the bee dna vaccine. an expression plasmid was constructed with a cmv promoter. surprisingly, no bee or other insect specifi c promoter was essential to drive the expression of the protein. the enhanced green fl uorescent protein (egfp) was chosen as reporter gene and inserted into the multiple cloning site, together with an sv40 enhancer element. the plasmid construct was produced in e. coli and highly purifi ed by standard techniques. european honey bees ( apis mellifera ) were obtained from local beekeepers and cultivated under lab conditions. varroa mites were collected from infested bees. the oral vaccination of the egfp plasmid was operated by feeding the bees with a mixed solution of sugar and plasmid dna (vaccine sugar). standard sugar solutions made by the beekeeper are the normal food for winter bees. results. over 10 days after onset of feeding, we measured the expression of egfp by immunofl uorescence and western blot analysis with egfp antibodies. between day 3 and 10, a clear egfp signal was detected in the thorax and especially in the malpighian tubules. control bees fed with dna lacking the reporter did not show any signal. in parallel, control experiments with transformed e. coli were done to study the possibility of egfp expression in gut bacteria instead of bee cells. no egfp signal was detected in transformed bacteria. most surprisingly, we found the egfp signal after 5 days in varroa mites sucking hemolymph of bees which were fed by the vaccine sugar solution and no signals in control mites of infested control bees. feeding of plasmid dna results in expression of a reporter gene in different bee tissues over a period of several days, and fi nally varroa absorbs this protein via bloodsucking. the bee blood is not carried by arteries and veins but fl ows loosely around the body. no egfp signals were detected either in the honey stomach or in the feces. figure 9 .9 illustrates the egfp passage through the bee body and toward the varroa mite. we started with the simple idea that the biochemistry in eukaryotic cells remains the same, irrespective of the organism. a difference is given in the confi guration of the immune system. that means, an insect can successfully fi ght against parasites and infections but with different weapons. no t cells, no b cells, and consequently no antibodies and no memory. we are able to stimulate targeted immune genes of bees and measure an insect typical immune response. a standard plasmid dna vaccine, fi rst developed for horses, bridges the evolution from fi sh to insects to mammals. no other vaccine type is able to do this job. how fascinating biology is! a protein subunit is based on a single protein molecule and able to stimulate a humoral immune response, but usually not a cellular response. after phagocytosis proteins are degraded by acid-dependent proteases in endosomes (endosomal or exogenous pathway), resulting in an mhc ii presentation of the antigenic peptides. a peptide is one form of a subunit. carbohydrates are also used as subunits with a poor and age-dependent immunogenicity. carbohydrate antigens induce a t cell-independent b cell response as discussed in chap. 6 . therefore carbohydrates are mainly linked to a protein (conjugation) to enhance toe immune reaction as discussed here with the hib conjugate vaccine. conjugate vaccines. the polyribosylribitol phosphate (prp) capsule of haemophilus infl uenzae type b (hib) is a major virulence factor for the organism. prp is a t cellindependent antigen characterized by, e.g., induction of a poor antibody response in less than 18-month-old infants and children and the inability to induce a booster response. polysaccharide vaccines based on prp alone were developed in the 1970s. by covalent linkage of prp with t cell dependent protein antigens, a conjugated vaccine was created to overcome the t cell independent characteristics of prp. at present three different licensed protein carriers are linked to prp: • hboc: diphtheria crm protein 197, mutant corynebacterium -linkage: no spacer • hbomp: outer membrane protein, omp, neisseria meningitidis -linkage: spacer • prp-d: diphtheria toxoid, d -linkage: spacer these hib conjugate vaccines differ by protein carrier, polysaccharide size, and method of chemical conjugation, including use of a spacer between the prp and protein carrier. a standard chemical conjugation between a polysaccharide and a protein is illustrated in fig. 9 .10 . subunit vaccines, while offering greater safety, are intrinsically poorly immunogenic and strong adjuvants are essential to boost the activation of immune responses. serotype variability is dictated by modifi cations of the o-antigen portion of lps. o antigens vary in the number of oligosaccharide unit repeats, the types and distribution of carbohydrates, and the intra-and intermolecular linkages [ 22 ] . in s. fl exneri , these genes are encoded in the bacterial chromosome. in contrast, s. sonnei , which shows no serotype variability, expresses plasmid-encoded o-antigen modifi cation enzymes. the o antigen is one of the major immunogenic components of shigella and is a virulence factor, in part, due to masking the exposure of type three secretion apparatus [ 23 ] . the inclusion of conserved proteins in vaccine compounds potentially solves the issue of serotype specifi city, thus allowing the generation of a highly desirable pan-shigella vaccine. in addition, recombinant proteins usually have increased safety profi les. another important impact in shigella epidemiology that prompts vaccine development is the increasing frequency of antibiotic-resistant strains. antibiotic resistance is continually rising for this pathogen [ 24 ] . shigella spp. as causative agent of shigellosis. first defi ned as a causative agent of bacillary dysentery by shiga in japan, shigella is a gram-negative bacillus that is noncapsulated and nonmotile. diagnosis is generally based on symptoms [ 25 ] since bloody, mucoid stools are indicative of shigella infections. however, because several diarrheal infections caused by other microorganisms share these symptoms (enteroinvasive e. coli and campylobacter , among others), the sole analysis of symptoms is insuffi cient for an accurate diagnosis. therefore, clinical diagnosis must be complemented with microbiological isolation from culture. shigella invasion and pathogenesis. shigella is transmitted through the fecal-oral route by consumption of contaminated food and water. following ingestion, the acid-tolerant shigella passes through the stomach and small intestine into the large intestine [ 26 ] (fig. 9.11 ). here, they are taken up by m cells, transcytosed to the basolateral face of the colonic epithelium, and presented to resident macrophages wherein ipab of the type three secretion system (t3ss) induces apoptosis by caspase 1 activation, thereby escaping killing by the macrophage [ 27 ] . shigella then invades epithelial cells using its t3ss to create a translocation pore in the host cell membrane to initiate an orchestrated fl ow of effectors into the host cell cytoplasm to induce actin rearrangements that ultimately result in uptake of bacteria. once inside, shigella quickly escapes its vacuole, replicates, and moves about the cytoplasm via actin-based motility. in a t3ssdependent manner, the shigella then forms a protrusion into a neighboring uninfected cell with the resulting vacuole being quickly lysed to complete the process of intercellular spread. the genes associated with the t3ss are encoded on a 220-kb plasmid which is highly conserved among the shigella species. at the heart of the t3ss is the type three secretion apparatus (t3sa) which is composed of a basal body similar to that of fl agellar systems and an extracellular needle [ 28 ] . invasion plasmid antigen d (ipad) is a 37 kda protein that forms a pentameric ring at the tip of the needle. it controls secretion of effector proteins and is the environmental sensor for mobilization of ipab to the t3sa tip complex. ipab is a 64 kda translocator that forms a ring atop the ipad ring and is responsible for host cell contact. this contact is required for mobilization of ipac to the needle tip and formation of a complete unidirectional conduit from the bacterial cytoplasm to the host cell cytoplasm. the initiation of infl ammation and invasion processes occurs exclusively at the basolateral side of host cells, highlighting the importance of the previous steps of macrophage subversion in shigella colonization of the gut. animal models. shigellosis is strictly a human disease. while the basis of this restriction is unknown, it complicates the ability to investigate the pathogenesis of shigella . however, several animal models have been developed to study the pathogenesis of shigella , the resulting immune response against shigella antigens, and the protection efficacy of candidate vaccines against shigellosis: [ 31 ] . nonhuman primate (nhp) models : nhp models have been used to defi ne the ability of vaccines to elicit immune responses and protection (rhesus and cynomolgus monkeys) [ 32 ] . the main advantage of this model is that shigella is able to colonize the large intestine and generate symptoms that these bacteria generate in human infection. o antigen/proteosome : o antigen represents the variable portion of shigella lps ( fig. 9 .12 ). administration of lps or o antigen alone in animal models is not enough to elicit immune responses, making them ineffective immunogens. to solve this limitation, these molecules have been used in conjunction with different proteins as carriers. several variants of lps/oantigen mixtures have been developed and characterized. one of these protein combination approaches uses s. fl exneri and s. sonnei lps complexed with neisseria meningitidis outer membrane protein proteosomes [ 33 , 34 ] . lps is extracted from s. fl exneri or s. sonnei by hot phenol extraction and mixed with detergent-extracted outer membrane proteins from n. meningitidis . the complex was then separated from free lps present in the mixture by gel fi ltration chromatography. the concept behind this vaccine is that the proteins present in the n. meningitidis proteosome are able to act as carriers for t cell stimulation, thus allowing the recognition of lps. shigella outer membrane vesicles. outer membrane vesicles (omvs) are particles composed of lps, proteins, and nucleic acids. in a proposed vaccine formulation, these particles were purifi ed from liquid cultures of s. boydii by centrifugation with subsequent fi ltering ( fig. 9.13 ). the precise identity and amount of the proteins included in this preparation is not currently known, although the presence of proteins having the same mass as ipab, ipac, and ipad suggests its composition includes these proteins. when these omvs are administered orally to mice, antibodies are generated against omv lysates. this vaccine has the advantage of heterologous protection (as shown by challenge against strains from each shigella serogroup) and the absence of adjuvant dependency. in addi-tion, immunity can be passively transferred to offspring, suggesting that the protective mechanism involves antibodies and raising the possibility that this vaccine can be used in infants, which is the main target population for a shigella vaccine. the use of live, fully virulent shigella during its formulation process, the presence of lps, and lot-to-lot consistency are possible downsides of this preparation. invaplex. another vaccine candidate that uses t3ss proteins and lps as part of the formulation is the invaplex [ 35 ] . these complexes are obtained by aqueous extraction followed by ion exchange chromatography (fig. 9.13 ). the precise composition of these extracts has not been completely characterized but includes lps, ipab, and ipac [ 36 ] . these complexes are able a b d c fig. 9 .12 depiction of lps/o-antigen-based vaccines. lps ( a ) extracted from shigella fl exneri or sonnei is admixed with protein preparations from n. meningitides and used as a carrier. when this complex is administered orally and intranasally to mice and guinea pigs [ 33 ] , serum igg and mucosal iga in intestines and lungs are vaccine com-pound ( b ). o antigen purifi ed from lps is delivered in combination with exoprotein a from p. aeruginosa ( c ). finally, o antigen from different shigella serogroups is combined with ribosomes from shigella and is depicted in ( d ) to elicit igg and iga responses against ipa proteins as well as lps in both mice and guinea pigs. in addition, they are protective against the shigella species/serotype used for extract generation [ 37 ] in the mouse and guinea pig challenge models. two phase one studies have been performed using the invaplex vaccine on adult volunteers [ 38 ] and showed no major side effects to delivery of intranasal doses of up to 690 μg. the highest dose employed in these studies generated an asc response to lps in 58 % of the volunteers. an advantage of this approach is that, other than the invaplex itself, no additional adjuvants need to be administered. a drawback of this vaccine consists in a challenging production process that includes cultures of virulent shigella as well as the presence of bacterial lps products in the intermediate steps and fi nal formulation. another possible caveat is the uniformity of protein composition in these complexes through manufacturing lots. finally, this vaccine was not designed to protect against multiple serotypes. a solution for this possible drawback, however, is the generation of formulations that include invaplex complexes generated from more than one particular serotype, which increases an already diffi cult manufacturing process. this would allow the generation of vaccine formulations specifi c for the serotypes prevalent in a particular region. a vaccine candidate that targets conserved shigella virulence proteins includes some of the t3ss ipa proteins (fig. 9.14 ) . recombinant ipab and ipad can be expressed in e. coli at high levels. ipad is then easily purifi ed from the e. coli cytosol while ipab must be purifi ed as a complex with its cognate chaperone ipgc. the chaperone is needed to maintain the hydrophobic ipab in a soluble state and to provide stability for ipab from proteolytic degradation. ipab can then be further purifi ed after separation from ipgc in low concentrations of detergent. analyses have indicated that ipab is greater than 90 % pure following this scheme. in its fi nal formulation, this ipa-based vaccine also contains a double mutant of heat-labile enterotoxin from e. coli (dmlt) [ 39 ] as an adjuvant. the mechanism of protection for this vaccine has not yet been worked out. nevertheless, it was tested in the mouse lethal pulmonary model [ 40 ] where it exhibited over 90 % homologous protection (against s. fl exneri ) and greater than 60 % heterologous protection (using s. sonnei during the challenge experiments). igg and mucosal iga were generated after intranasal administration along with antigen-specifi c ifn-γ-secreting cells. ompa. a 34-kda outer membrane protein (omp) was purifi ed from s. fl exneri 2a using ion exchange chromatography. incubation of macrophages with this 34-kda protein induced the production of nitric oxide and increased production of il-12 and tnf-α. this protein was delivered parenterally fi ve times in rabbits, giving protection against challenge by s. fl exneri in the rabbit cecal ligation model [ 41 ] . subsequent work using a recombinant protein purifi ed by affi nity chromatography identifi ed this 34-kda omp as ompa, part of a family of immunomodulating proteins present in numerous gram-negative bacteria. this protein showed high protective effi cacy in the mouse lethal pulmonary model [ 42 ] where it elicited serum igg and mucosal iga. ticks are widely distributed throughout the world, affecting 80 % of the world's cattle population [ 43 ] . the economic importance of ticks and tick-borne diseases (tbds) has been estimated by a number of studies; however they most likely represent an underestimation of the real impact of these arthropod vectors and their transmitted diseases. tick feeding has devastating effects including disease transmission, paralysis, toxicosis, and secondary infections of the tick-feeding site [ 44 ] . the effect of ticks and tickborne diseases is particularly pronounced in the livestock sector where it is repeatedly rated highly for its impact on the livelihood of farmers, particularly in countries of the south which are heavily dependent on agricultural production. there are six genera of ixodid ticks of importance, namely, amblyomma , dermacentor , haemaphysalis , hyalomma , rhipicephalus , and ixodes . historically, tick and tick-borne disease control has focused on the control of ticks at tolerable levels through acaricide use and treatment of disease with appropriate drugs. in some cases acaricide-based tick control is often the only method of reducing tick populations without sacrifi cing productivity [ 45 ] . acaricides are commercially available in a number of formulations that are applied either directly onto livestock or in dipping vats where multiple animals can be passed through at regular time intervals. acaricide application relies heavily on correct formulation and administration to be effective. a large number of chemical compounds have been found to be effective against ticks including arsenic (introduced ~1983), ddt (~1946), cyclodienes and toxaphene (~1947), organophosphates-carbamate group (~1955), formamides (~1975), and macrocyclic lactones (~1981). the potency and usefulness of many of the abovementioned compounds is gradually eroding with resistance developing in many tick species of rhipicephalus , amblyomma , and hyalomma . multiple acaricide-resistant tick stocks have been identifi ed, limiting or entirely excluding the use of many acaricides [ 46 ] . in addition to resistance, chemical control through the guiding principle for anti-tick vaccination stems from early studies conducted on acquired host resistance to tick infestations. repeated exposure of hosts to ticks or tick organ homogenates induced resistance to tick re-infestation. while the degree of resistance may vary between different tick and host species, evidence strongly suggests that natural resistance against tick infestation develops based on adaptive immune response mechanisms [ 47 ] . ticks feeding from hosts vaccinated with tick components take up effector molecules during feeding that mediate deleterious effects on the ticks. this effect manifests as reduction of feeding time, tick mortality (during or after feeding), reduced engorgement weights, and reduced reproductive capacity of adult females. eggs laid from ticks fed on vaccinated hosts may also show reduced hatching rates. the overall result culminates in reduction of tick populations and tick-borne diseases. many of the anti-tick vaccine targets have been identifi ed using conventional immune-screening techniques. immunization of vertebrate hosts with tick homogenates or purifi ed tick extracts generates immune sera. these sera are used to screen for tick antigens detected by the host. the identifi cation of tick proteins essential for tick survival is a useful method for more targeted antigen discovery, which is made increasingly possible as information is gathered on tick biology. with the availability of genome sequences for a number of tick species, the number of candidates for discovery is expanding through reverse vaccinology. the use of other techniques such as rna interference (rnai) has been useful in confi rming the importance of antitick vaccine candidates and is likely to play a role in future anti-tick vaccine antigen discovery [ 48 ] . exposed or concealed antigens. anti-tick vaccine candidates have been classifi ed into two categories: exposed or concealed antigens. exposed antigens are secreted in tick saliva during attachment and feeding on a host while concealed antigens are normally hidden from the host immune response. molecular mimicry by ticks of host components has been observed, and vaccination may induce host sensitivity and autoimmune reactions when exposed antigens are used [ 49 ] . one advantage of using exposed antigens is that natural boosting occurs through tick feeding. mechanistically, vaccination with exposed antigens is thought to induce a focal hostile environment unsupportive for tick attachment and feeding. concealed antigens do not come into contact with the host immune response during natural tick feeding. although often contained within the thoracic cavity of the tick, some salivary gland proteins can be characterized as concealed if they are not secreted into the tick-feeding site. one diffi culty in the development of concealed anti-tick vaccines is that the antigen must be accessible to the induced humoral vaccine response. this often limits the number of candidates to those coming into prolonged and direct contact with the blood meal or where the humoral response can be transported over the gut barrier into the hemolymph [ 50 -52 ] . the second limitation of concealed antigens relates to natural boosting of the immune response. as the antigens do not come into contact with the immune response within the host, suffi ciently high antibody levels must be induced through repeated vaccination. as the blood meal acts as the carrier for the effector immune responses, the anti-tick effect can take place over a longer period of time compared to exposed antigens. this effect may even extend beyond the mere feeding period into the inactive stages where digestion and molting/egg laying takes place. bm86 anti-tick vaccine. the bm86-based anti-tick vaccine remains the only anti-tick vaccine commercially produced and has become the benchmark for future anti-tick vaccine development and evaluation. the gut-associated bm86 glycoprotein was fi rst identifi ed in r. microplus although homologues in other tick species have since been identifi ed [ 53 -55 ] . the biological function of bm86 remains unknown although it is thought to play a role in the digestion of the blood meal [ 56 ] . in r. microplus , expression of bm86 is increased during embryogenesis, reaching the highest level in unfed larvae. expression decreases during feeding and molting with lowest levels of expression detected during the resting stages of the tick. bm86 has a translated coding sequence of 650 amino acids and a size of 71.7 kda. the protein contains four potential n-linked glycosylation sites and a leader peptide suggesting transport to the cell surface. localization studies have shown the molecule is located predominantly on the microvilli of gut epithelial cells. a single c-terminal transmembrane sequence is present in the unprocessed protein which is replaced by a glycosylphosphatidylinositol anchor in the mature protein. the protein also contains multiple predicted egf repeats rich in cysteine residues. vaccination has been performed mostly with the whole molecule, and protective epitopes for bm86 have not been well determined. the site of a protective b cell epitope was defi ned and additional epitopes are likely to exist. overlapping cross-reactive immune-reactive epitopes have been found between bm86 and the r. decoloratus homologue, bd86. vaccine effi cacy is directly related to anti-bm86 antibody titer and the ability to control tick populations is directly related to achieving a strong antibody response. substantial animal-to-animal variation has been observed in the ability to generate anti-bm86 antibody titers which is likely related to the mhc class ii haplotypes expressed. antibodies to bm86 and cattle complement system are taken up during the blood meal. antibody binding results in lysis of the gut epithelial cells culminating in impaired blood meal digestion. strong antibody responses may induce tick mortality due to blood leakage from the gut into the hemolymph and ticks may turn reddish instead of gray. the development of the antibody response in cattle [ 57 ] after immunization with rbm86 is demonstrated in fig. 9.19 . recombinant expression of bm86 has been attempted in several expression systems including escherichia coli , aspergillus nidulans , aspergillus niger , and pichia pastoris . vaccine trials showed that bm86 vaccination targeted mainly the adult stage of r. microplus , particularly the number of adult females fully engorging and post-engorgement mortality. reproductive capacity of adult r. microplus females was affected in terms of egg-laying capacity and hatching of eggs [ 58 ] . under fi eld situations, vaccination of cattle reduced tick numbers by 56 % within a single generation and reduced the reproductive capacity by 72 %. reversal of negative effects of tick feeding on live weight of vaccinated animals by an average increase in live weight of 18.6 kg over a 6-month period was observed. extensive fi eld trials in cuba, brazil, argentina, and mexico showed between 55 and 100 % control of r. microplus ticks within a 36-week period [ 59 ] . importantly, complete control of acaricide-resistant ticks could be accomplished by integrating bm86 vaccination with acaricide use [ 52 ] , showing that integrated control systems are effective in controlling tick populations. vaccination also decreased the amount of acaricides required to control tick populations and prolonged the time interval between cattle dipping. bm86 vaccination has been extensively evaluated for its ability to control other tick species. almost complete cross-protection against rhipicephalus annulatus has been reported [ 60 ] . signifi cant protection against hyalomma anatolicum , h. dromedarii , and r. decoloratus has been observed; however no cross-protection was seen against r. appendiculatus or amblyomma variegatum . genetically attenuated microorganisms, viruses and bacteria, can be engineered to deliver recombinant heterologous antigens to stimulate the host immune system. some experimental vector systems are summarized in work with vectors classifi ed as bsl-1 does not require biosafety program approval. work with vectors classifi ed as bsl-2 or higher requires approval by the local biosafety committee. safety concerns. as demonstrated for adenovirus 5 (ad5), following i.m. injection, the vector persisted mainly near the injection site and in draining lymph nodes for up to 6 months. low levels of integration into chromosomal dna were observed, with a calculated mutation rate of 2 × 10 −7 mutations per cell. the spontaneous mutation rate of a cell is 2 × 10 −6 and therefore tenfold higher. ad5 is classifi ed as biosafety level 2 (bsl-2). live vectors are able to stimulate the mucosal as well as a systemic humoral and cellular immunity. a severe drawback of the vector technology is that, once used, the vector cannot be effectively used in the patient again because it will be recognized by antibodies. repeated booster immunization will fail. also preexisting immunity in the patient against the vector could render the vaccination ineffective. a heterologous prime-boost and vector priming as described in chap. 2 could circumvent this barrier. disease 4 lactic acid bacteria have generally recognized as safe (gras) status and have been developed in the past decade as potent adjuvants for mucosal delivery of vaccine. a platform technology based on lactobacillus plantarum ( fig. 9.21 ) was developed to deliver antigens against plague disease caused by yersinia pestis , an aerobic, nonmotile, gram-negative bacillus belonging to the family enterobacteriaceae , which is transmitted to humans via fl eabite or via aerosol droplet, causing bubonic or pneumonic plague, respectively [ 61 ] . most human plague cases present as one of three primary forms -bubonic, septicemic, or pneumonic. secondary plague septicemia, pneumonia, and meningitis are the most common complications. the pathogenicity of y. pestis results from its impressive ability to overcome the defenses of the mammalian host and to overwhelm it with massive growth. plague is enzootic in rodents in africa, asia, south america, and north america. y. pestis is transmitted from host to host by fl eas via blood feeding, through consumption or handling of infectious host tissues, or through inhalation of infectious materials. y. pestis infects an astonishingly broad range of mammals and uses rats, squirrels, mice, prairie dogs, marmots, or gerbils as reservoirs and several arthropod vectors for transmission [ 62 ] (fig. 9.20 ) . humans acquire this zoonotic infection via an atypical bite from animal fl eas, sometimes prompted by an animal's death from plague, after which the fl ea seeks a new source of blood. most infected fl eas come from the domestic black rat rattus rattus or the brown sewer rat rattus norvegicus. y. pestis cells spread from the site of the infected fl eabite to the regional lymph nodes, grow to high numbers causing the formation of a bubo, and spill into the bloodstream where bacteria are removed in the liver and spleen. growth continues in these organs, spreads to others, and causes septicemia. fleas feeding on septicemic animals complete the infection cycle. in humans, bubonic plague can develop into an infection of the lung (secondary pneumonic plague) that can lead to aerosol transmission (primary pneumonic plague) [ 63 ] . multiple antibiotic-resistant strains of y. pestis occur naturally and they can be easily bioengineered. thus, plague is a category a bioterrorism agent in need for novel strategies for its prevention. bubonic plague is the classic form of the disease. patients usually develop symptoms of fever, headache, chills, and swollen, extremely tender lymph nodes (buboes) within 2-6 days of contact with the organism either by fl eabite or by exposure of open wounds to infected materials. primary septicemic plague is generally defi ned as occurring in a patient with positive blood cultures but no palpable lymphadenopathy. patients are febrile, and most have chills, headache, malaise, and gastrointestinal disturbances. primary pneumonic plague is a rare but deadly form of the disease that is spread via respiratory droplets through close contact (2-5 ft) with an infected individual. it progresses rapidly from a febrile fl u-like illness to an overwhelming pneumonia with coughing and the production of bloody sputum. the incubation period for primary pneumonic plague is between 1 and 3 days. in general, patients who develop secondary plague pneumonia have a high fatality rate. fig. 9 .20 schematic of the plague cycle with small mammals as hosts and fl eas as vectors. arrows represent connections affected by climate with a color coding depending on the most infl uential climate variable on this link (i.e., precipitation, temperatures, and other variables indirectly depending on them such as soil characteristics and soil moisture). grey rectangles somewhat arbitrarily delimit epizootic, enzootic, and zoonotic cycles. note that despite their location at the far end of the cycle, humans often provide the only available information on plague dynamics the laboratory diagnosis of plague is based on bacteriological and/or serological evidence [ 64 ] . samples for analysis can include blood, bubo aspirates, sputum, cerebrospinal fl uid in patients with plague meningitis, and scrapings from skin lesions, if present. staining techniques such as the gram, giemsa, wright, or wayson stain can provide supportive but not presumptive or confi rmatory evidence of a plague infection. lactic acid bacteria (lab) are a group of gram-positive, nonpathogenic, non-sporulating bacteria that include species of lactobacillus (fig. 9.21 ) , lactococcus , leuconostoc , pediococcus , and streptococcus . they have limited biosynthetic abilities and require preformed amino acids, b vitamins, purines, pyrimidines, and a sugar as a carbon and energy source. these nutritional requirements restrict their habitats to those in which the required compounds are abundant. thus, these highly specialized bacteria occupy a range of niches including milk, plant surfaces, the oral cavity, the gastrointestinal tract, and the vagina of vertebrates [ 65 ] . lab have been consumed for centuries by humans in fermented foods and have an extraordinary safety profi le. these intrinsic advantages turn lab into excellent delivery vectors of novel preventive and therapeutic molecules for humans. a number of studies of oral vaccines generated from genetically engineered pathogenic or commensal bacteria have been reported [ 66 , 67 ] . live attenuated pathogenic bacteria, such as derivatives of mycobacterium , salmonella , and bordetella spp., are the most popular live delivery vectors used currently. they are particularly well adapted to interact with mucosal surfaces as they have specialized machinery to initiate the infection process. the major disadvantages of live vaccines include inadequate attenuation and the potential to revert to virulence. lactic acid bacteria-based vaccines act as live attenuated vaccines but without the safety concern. lab have a generally recognized as safe (gras) status and thus are not likely to cause harm. the production of a desired antigen by lab can occur in three different cellular locations: • intracellular , which allows the protein to escape harsh external environmental conditions (such as gastric juices in the stomach) but requires cellular lysis for protein release and delivery • extracellular , which allows the release of the protein into the external medium, resulting in direct interaction with the environment (food product or the digestive tract) • cell wall anchored , which combines the advantages of the other two locations (i.e., interaction between the cell wall-anchored protein and the environment, in addition to protection from proteolytic degradation) in this context, several studies have compared the production of different antigens in lab, using all three locations, and evaluated the subsequent immunological impact. these studies demonstrated that the highest immune response was obtained with cell wall-anchored antigens exposed on the surface of lab. therefore, most of the recent lab vaccination studies have selected surface exposure of the antigen of interest, rather than intra-or extracellular production [ 68 ] . dendritic cells (dcs) play a central role in bridging the innate immune system with the adaptive immune system. dcs are found throughout the body and are especially common at mucosal surfaces. with only a single layer of epithelial cells separating the external from the internal world amid the constant need for particle exchange, intestinal dendritic cells (dcs) play a key role in maintaining intestinal homeostasis as well as governing protective immune responses against invading pathogens. to avoid activation of selfreactive t cells and to limit unnecessary responses, such as those against commensal fl ora, dcs can imprint tolerance onto t cells (fig. 9.22 ). immature-type dcs are enriched underneath the epithelium of mucosal inductive sites and are poised to capture antigens. they extend protrusions between epithelial cells, enabling direct sampling of luminal antigens [ 69 ] . through upregulation of mhc and co-stimulatory molecules, matured dcs convert into highly effi cient antigen-presenting cells. successful antigen presentation to cd4+ t cells requires recognition of cognate peptide in the context of mhc class ii molecules, whereas epitopes presented on mhc class i molecules stimulate ag-specifi c cd8+ t cells. when antigen uptake occurs, these dcs change their phenotype by expressing higher levels of co-stimulatory molecules and move to t cell areas of inductive sites for antigen presentation. thus, dcs and their derived cytokines play key roles in the induction of antigen-specifi c effector th cell responses. in this regard, targeting mucosal dcs is an effective strategy to induce mucosal and systemic immune responses. lab persistence. the ability of some lab to persist in the gastrointestinal tract may be critically important in the effectiveness of lab-based vaccines. a comparison of a persisting lab strain, l. plantarum , with a nonpersisting lab strain, l. lactis , identifi ed l. plantarum to be more effective at eliciting antigen-specifi c immunity, suggesting that persistence promoted immunogenicity [ 70 ] . furthermore, it has been shown that particular lactobacillus species induced critical infl ammatory cytokines and induced activation and maturation of dendritic cells [ 71 ] . it has also been shown that immature dcs effi ciently capture lactobacillus species, and these bacteria activated human dcs, resulting in the production of pro-infl ammatory cytokines like il-12, increased proliferation of cd4+ and cd8+ cells, and skewed t cell response toward a th1 pathway believed to be involved in effective clearance of microbial pathogens [ 72 , 73 ] ( fig. 9.23 ). evidence suggests that the peptidoglycan layer of some lab promote natural immuno-adjuvanticity [ 74 ] , and antigen localization on the cell wall makes it more accessible to the immune system as compared to intracellular or secreted proteins. leader peptides mark proteins for translocation across the cytoplasmic membrane, and lipid modifi cation is of major importance both for anchoring exported proteins to the membrane and for protein function [ 75 ] . it has been shown that lipidation at the fi rst amino acid of the mature borrelia burgdorferi ospa protein is essential to induce an immune response via tlr2 [ 76 ] . the leader peptide of ospa targets the protein to the cell envelope of lactobacillus and that the cys [ 17 ] is recognized by the l. plantarum cell wall-sorting machinery that lipidates and anchors the protein to the cell envelope. the end result is a delivery system that exerts a potent adjuvant effect [ 77 ] . virus-like particles (vlps) that mimic the antigenic architecture of authentic virions, however, can be produced in insect, mammalian, and plant cells by the expression of the capsid protein. the particulate nature and high-density presentation of viral structure proteins on their surface render vlps as a premier vaccine platform with superior safety, immunogenicity, and manufacturability. therefore, this chapter focuses on the development of effective nov vaccines based on vlps of capsid proteins. the expression and structure of nov vlps, especially vlps of norwalk virus, the prototype nov, are extensively discussed. the ability of nov vlps in stimulating a potent systemic and mucosal anti-nov immunity through oral and intranasal delivery in mice is presented. gastroenteritis (ge) is a worldwide health problem that affects people of all ages. as its name implies, ge is characterized by infl ammation of the gastrointestinal tract and often associated with symptoms of diarrhea, nausea, vomiting, and abdominal cramping and pain. ge and its associated diarrheal diseases remain as one of the top causes of death in the world especially in developing countries and in young children with an estimated death toll of four to six million per year [ 78 ] . ge can be caused by a variety of pathogens including viruses, bacteria, and parasites and by ingestion of noninfectious toxins or medications, with viruses as the most common offending agents. norovirus (nov) and rotavirus are the most common viruses that cause viral ge, while adenovirus, astrovirus, coronavirus, and parechovirus are also known to cause ge in humans. novs are a group of genetically diverse rna viruses that belong to the genus of norovirus in the caliciviridae family [ 79 ] . they were fi rst discovered and characterized in their prototype virus, the norwalk virus (nv), in 1972 [ 80 ] . studies of nv revealed that novs are non-enveloped viruses with a rna genome surrounded by a round capsid protein shell approximately 38 nm in diameter. novs are divided into 5 genogroups and 29 clusters with 8 clusters in genogroup i (gi), 17 in gii, 2 in giii, and 1 each in giv and gv. within the fi ve genotypes, gi and giv strains are found to infect humans exclusively and gii are found in both humans and pigs, while giii and gv strains are animal viruses that infect cattle and murine species, respectively [ 81 ] . currently, strains in cluster 4 of gii (gii 4) are the most prevalent novs in human population [ 82 ] . the genome of novs, which was fi rst characterized in nv, contains a single-stranded positive-sense rna of 7.5-7.7 kb with three open reading frames (orfs) and a poly a tail at its 3′ end [ 83 ] (fig. 9.24 ). orf1 encodes a polyprotein that is processed by viral protease 3clpro into the rnadependent rna polymerase and approximately fi ve other nonstructural proteins including p48, the nucleoside triphosphatase, p22, vpg, and 3clpro. the two structural proteins, the major (vp1) and minor (vp2) capsid proteins, are encoded by orf2 and orf3, respectively [ 84 ] . structural analysis of nov has revealed that each viral capsid is composed of 90 dimers of vp1 in a t = 3 icosahedral symmetry. vp1 folds into two domains: a shell (s) domain that is responsible for initiating capsid assembly and icosahedral contacts and a protruding domain (p), containing two subdomains of p1 and p2, that enhance the stability of the capsid by providing intermolecular contacts between vp1 dimers. studies of nv also indicate that the vp2 protein enhances the expression level of vp1 and stabilizes the vp1 in the viral capsid. novs are highly contagious and spread rapidly, and their outbreaks commonly occur in various social places and settings where people share common food and water sources or are in close physical proximity, such as cruise ships, schools, military units, nursing homes, daycare centers, hospitals, restaurants, and catered events . the life cycle of nov has not been fully understood due to the lack of an in vitro cell culture system and a small animal model of infection. the failure of nov replication in mammalian cell cultures is not due to the lack of host factors to support intracellular expression of nov rna. instead, the problem may lie in the steps of viral binding to cellular receptors, virus entry into cells. nov [ 85 ] . while serum antibodies to nov can be readily detected, this method has little clinical relevance due to the cross-reactivity of antibodies. since there is no culture system available for nov, the detection of virus in stool samples has become the preferred method of diagnosis. traditionally, nov infection was diagnosed by detecting the virus by immune transmission electron microscopy (tem). tem offers the advantage of direct visualization of any potentially responsible virus particles in stool samples. however, it does have the disadvantage of requiring sophisticated and expensive equipment and highly specialized technicians for its operation. several enzyme-linked immunosorbent assays (elisas) that detect nov antigens were later developed for nov diagnosis. studies have shown that nov antigen-detecting elisas have high specifi city (94-96 %) but poor sensitivity (40-60 %), most likely due to the antigenic diversity of nov strains [ 86 ] . similar to elisa-based assays, rt-pcr is rapid and robust, because it can process large numbers of samples simultaneously and results can be obtained within a working day. however, it requires rna extraction from fecal samples and needs expensive equipment and skilled workers to operate. therefore, rt-pcr is more labor intensive and less economical than elisas. overall, tem, elisa, and rt-pcr-based methods all have their advantages and challenges. the three methods detect different components of the virus and therefore are complementary to each other. immunology of nov infection. the immunological knowledge of nov is mostly obtained from human challenge studies and natural outbreaks due to the lack of small animal models. observations of repeat infections in adults suggest the scarcity of long-term immunity against these viruses. however, other studies showed that close to 50 % of the genetically susceptible subjects were not infected by nov challenge, which support the possibility of long-term immunity [ 87 ] . nov. the lack of a tissue culture system also impedes the development of vaccines against nov. fortunately, the discovery of the spontaneous assembly of expressed vp1 into virus-like particles (vlps) that are morphologically and antigenically similar to the native viruses has facilitated vaccine development. vlps combine the best traits of wholevirus and subunit antigens for vaccine development. vlps are noninfectious, therefore, safer than inactivated or attenuated virus due to the lack of viral nucleic acid genome. importantly, vlps can induce potent cellular and humoral immune responses without adjuvants and are more effective vaccines than other subunit antigens because their architectures mimic infectious viruses. vlps can be produced by recombinant technology in heterologous expression systems without requiring the ability to support viral replication. this is particularly important for nov because no such culture system has been developed to support the growth of these viruses. studies have demonstrated that viruses and corresponding vlps have a particle size ideal for dc and macrophage uptake to initiate antigen processing [ 88 ] . thus, the particulate nature of vlps favors their targeting to relevant apcs for optimal induction of t cell-mediated immune responses. vlps can also be presented effi ciently to b cells and induce strong antibody responses. like live viruses, the quasicrystalline surface of vlps, with its arrays of repetitive epitopes, presents a prime target that vertebrate b cells have evolved to specifi cally recognize. this recognition triggers the cross-linking of surface membrane-associated immunoglobulins (ig) on b cells and leads to their proliferation and migration, t helper-cell activation, antibody production and secretion, and the generation of memory b cells. thus, vlps can directly activate b cells at much lower concentrations than other subunit antigens and induce high titer and durable b cell responses in the absence of adjuvants. these inherent advantages of vlps have made them one of the most successful recombinant vaccine platforms. for example, fi ve vlp-based vaccines for hepatitis b virus characterization of nov vlps. vlps of novs were fi rst produced in insect cells using baculovirus vectors [ 89 ] and then in plants using tobamovirus and geminivirus vectors and in mammalian cells using the venezuelan equine encephalitis (vee) replicon system [ 90 ] . these studies demonstrated that expression of the major capsid protein vp1 alone can drive the self-assembly of vlps that morphologically and antigenically resemble native virus particles (fig. 9.25 ) . vlps generated by all three expression systems are similar to each other. the structure of nov vlps is exemplifi ed by the vlp of nv capsid protein (nvcp). studies of insect cellbaculovirus-derived nvcp vlps by cryo-electron microscopy and x-ray crystallography reveal that the nv capsid is a 38 nm icosahedral arrangement of 180 copies of the 58 kda capsid protein vp1 organized into 90 dimers in a t = 3 symmetry. while all dimers are formed from two identical nvcp monomers, two different dimer confi gurations are required to correctly form the complete assembled capsid [ 91 ] . as in native nv particles, the nvcp also folds into two distinctive domains in vlps, with s domain forming the inner core of the shell and p domain protruding out from the capsid [ 92 ] . similarly, the p2 subdomain is also the most surface-exposed region in nv vlps and may contain hbga and neutralizing antibody-binding sites and determinants of strains specifi city [ 93 , 94 ] . the similarity between vlps of nv and other novs including gii.4 viruses has been demonstrated [ 95 ] . insect cell-baculovirus vector-produced nvcp vlps. it was shown that four oral doses of as little as 5 μg nvcp vlps without any adjuvant triggered serum nv-specifi c anti-igg response in the majority (8/11) of vlpfed icd1 outbred mice [ 90 ] . systemic igg response was observed after two oral dosages and the highest titer was induced by four doses of 200 μg vlps. moreover, mice in the 200 μg dosage group developed nv-specifi c intestinal iga in a level up to 0.1 % of total iga. inclusion of the mucosal adjuvant cholera toxin (ct) did not signifi cantly change the number of positive responders of serum igg or intestinal iga but signifi cantly enhanced the amplitude of serum igg response, especially for higher doses of vlps. thus, nvcp vlp is clearly a potent oral immunogen and can induce both systemic and gut mucosal antibody responses. nanovaccines: gas infections 6 group a streptococcus ( streptococcus pyogenes) (gas) is an important human mucosal pathogen that is responsible for a wide spectrum of diseases with varying clinical manifestations and severity [ 96 , 97 ] : pharyngitis (strep throat) is a common minor complication of gas infection but when left untreated can lead to life-threatening diseases including the autoimmune sequelae rheumatic fever (rf) and rheumatic heart disease (rhd). rhd results in permanent damage to the heart tissues and valves. gas infections cause >500,000 deaths each year mostly in developing countries and indigenous populations within developed nations where poor socioeconomic conditions and overcrowding contribute to the high rates of gas diseases. in developing countries, rf is the leading cause of heart disease among children [ 6 ] . there is currently no available vaccine to prevent infection with gas and consequently prevent gas diseases. a successful mucosal gas vaccine would need to stimulate the appropriate humoral and cellular immunity for protection against gas infection ( fig. 9.26 ). this is especially diffi cult due to a lack of human-compatible mucosal vaccine adjuvants that are essential to boost immune responses. researchers have therefore focused mainly on parenteral gas vaccine delivery approaches, for which suitable adjuvants are available, designed to provide protection against systemic infection via the induction of opsonic igg antibodies. antigen. an effective gas vaccine needs to have broad antigenic reach because of the many different gas strains (>150 different m types) circulating in a population fig. 9 .26 gas vaccination approaches. gas that breaches the physical barrier of the mucosal epithelium of the nasal-associated lymphoid tissue, functionally analogous to human tonsils, is purported to be transported to the underlying lymphoid tissue via association with membranous (m) cells. cells of the innate immune system sense gas and produce cytokines and chemokines to contain the infection to the mucosa. gas antigens are delivered to antigen-presenting cells such as dcs and b cells. iga-committed b cells are activated and initiate antigen-specifi c iga responses. dcs play a fundamental role in the development of immunity to gas and present antigen to t cells to induce a th17 response that is integral along with iga for mucosal defense against pharyngeal gas colonization. mucosal vaccination is designed to mimic these responses and effectively clear gas from the mucosal surface upon infection to prevent gas colonization and carriage. gas that escapes the host's defense mechanisms can disseminate into the lymphatics and blood, leading to systemic infection. mucosal vaccination is also able to induce a systemic immune response characterized by the induction of opsonic igg antibodies, which destroy the pathogen by opsonophagocytosis. parenteral gas vaccination induces serum igg but is not able to induce mucosal immunity and should not induce immune responses that are potentially cross-reactive with self tissue proteins. the gas m protein is the major protective antigen and an ideal target for vaccine development; however it contains heart tissue cross-reactive epitopes particularly in the conserved region [ 98 ] . evidence suggests that cross-reactive t cells especially play a pivotal role in the pathogenesis of rhd. the m protein is an α-helical coiled-coil surface protein consisting of a hypervariable amino-terminal region and a highly conserved (>98 % sequence identity) carboxyterminal c-repeat region [ 99 ] (fig. 9.27 ) . functionally, the m protein is important in preventing bacterial clearance by complement-mediated phagocytosis, which limits host defense mechanisms. previous studies indicate that protective immunity to gas can be evoked by opsonic antibodies to serotypic epitopes at the amino-terminal region that are m-type specifi c [ 100 ] . nanovaccine. combined vaccine/adjuvant delivery systems offer the potential of mucosal vaccine delivery. for example, the lipid-core-peptide (lcp) system is a novel, synthetic, selfadjuvanted vaccine delivery system that incorporates the adjuvant (prr agonist), carrier, and antigenic peptides of a vaccine into a single molecular entity ( fig. 9 .28 ). this system has been previously shown to effi ciently deliver gas vaccines and induce immunity [ 101 ] . evidence suggests the adjuvant activity of lcp involves the induction of dc activation. preclinical developments. three approaches are currently being investigated in the development of a subunit gas vaccine based on the m protein: 1. multivalent gas vaccine . a multivalent approach employs a combination of amino-terminal protein fragments representing different m types and is designed to target prevalent gas strains in a population. using this approach, a recombinant multivalent gas vaccine containing m protein peptides from 26 different gas serotypes prevalent in north america was demonstrated to evoke opsonic antibodies in animals [ 102 ] . from epidemiological data, the 26-valent vaccine would cover the majority of pharyngitis and invasive gas diseases, including rf, invasive fasciitis, and toxic shock syndrome. recently, a new 30-valent gas vaccine was shown to be immunogenic in rabbits and evoked opsonic antibodies against "non-vaccine" serotypes [ 103 ] potentially creating a vaccine with much broader coverage. this type of vaccine is population specifi c and therefore may not be effective universally. it may also need to be re-designed periodically to refl ect changes in the epidemiology of gas infections. (ch 2 ) 7 (ch 2 ) 7 ch 3 ch 3 ch 3 fig. 9.28 the lipid-core-peptide (lcp) system. the lcp system contains an adjuvant component (lipid-core made of lipoamino acids) and a polylysine carrier onto which peptide epitopes are attached. the example shows a gas vaccine candidate containing j8 and an amino-terminal serotypic epitope called 8830. the adjuvant has three 2-amino-dodecanoic ( n = 9) lipoamino acids separated by glycine amino acid spacers 2. j8 vaccine . a gas vaccine that employs peptide epitopes from the conserved c-repeat region of the m protein is the second approach and has the potential in theory for greater coverage of m types. immunization of mice with a c-region peptide gas vaccine candidate called j8 conjugated to the carrier protein diphtheria toxoid (dt) and co-delivery with an appropriate adjuvant led to protection against systemic and mucosal gas infection [ 104 ] (fig. 9.29 ). j8 also elicited protective immunity against gas when linked to lipopeptides [ 105 , 106 ] . other studies have shown that intranasal immunization of mice with c-region peptides conjugated to the experimental mucosal adjuvant cholera toxin b subunit (ctb) evoked protective immunity against gas at the mucosal level [ 107 ] . ctb could possibly enter olfactory regions of the central nervous system and cause neuronal damage following intranasal delivery [ 108 ] and therefore is not suitable for human use. vector delivery approaches have included expressing the c-repeat region on vaccinia virus [ 109 ] , the commensal bacterium lactococcus lactis [ 110 ] , or streptococcus gordonii [ 111 ] . 3 . j14 . the third combination vaccine approach uses both serotypic and conserved m protein peptide epitopes. initially, a heteropolymer gas vaccine construct was synthesized by free radical-induced polymerization of acryloyl peptides to combine seven serotypic epitopes and a highly conserved c-region peptide epitope called j14 [ 112 ] . the m types that were targeted in the heteropolymer represented gas infections prevalent in the northern territory of australia -a region highly endemic for gas. immunization of mice with the heteropolymer demonstrated excellent immunogenicity and protection fig. 9 . 29 preclinical evaluation of gas vaccine candidates. intranasal immunization of mice with the j8-dt vaccine candidate led to significantly greater survival after intranasal challenge with gas versus control groups but the mucosal adjuvant ctb was essential for protection ( a ). a multiepitope lcp-based gas vaccine candidate elicited protective immunity against mucosal gas infection even in the absence of ctb ( b ) against homologous and heterologous gas strains, indicating its potential to provide broad coverage. however, batch-to-batch variation led to altered immune responses, which limited its applicability for human use. the vaccine also required the addition of an adjuvant to be effective, further limiting its use as a mucosal vaccine due to a lack of safe and effective mucosal adjuvants. later, multiepitope gas vaccine candidates were synthesized based on the lcp system that induced highly opsonic antibodies following parenteral delivery to mice [ 113 ] , as well as protection against mucosal gas infection following intranasal immunization [ 114 ] (fig. 9.29 ). safety and effi cacy. the main concern when using large regions of the m protein in a gas vaccine is the potential for inducing an autoimmune response due to immunological cross-reactivity with host proteins. it is therefore important to identify protective antigenic determinants and to separate the biological relevant epitopes from those that are host tissue cross-reactive and potentially harmful. epitope mapping studies were used to identify the conserved gas vaccine candidate j8, which contains a conformational protective b cell epitope and was designed to lack a human heart cross-reactive t cell epitope [ 115 , 116 ] . tb vaccines -state of the art and progresses dna-antiviral 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highly divergent gut-expressed bm86 antigen gene homologues in the tick rhipicephalus appendiculatus (acari: ixodida) cloning and expression of a protective antigen from the cattle tick boophilus microplus bovine immunoprotection against rhipicephalus (boophilus) microplus with recombinant bm86-campo grande antigen tick vaccines and the transmission of tick-borne pathogens vaccination against ticks (boophilus spp.): the experience with the bm86-based vaccine gavac immunity against boophilus annulatus induced by the bm86 (tick-gard) vaccine developing live vaccines against plague yersinia pestis--etiologic agent of plague yersinia: strategies that thwart immune defenses plague . in: crc handbook series in zoonoses . section a. bacterial, rickettsial, chlamydial and mycotic diseases mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria the induction of hiv gag-specifi c cd8+ t cells in the spleen and gutassociated lymphoid tissue by parenteral or mucosal 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streptococcal infections group: a streptococcal infections and acute rheumatic fever multiple cross reactive epitopes of streptococcal m proteins streptococcal m protein: molecular design and biological behavior localization of protective epitopes of the amino terminus of type 5 streptococcal m protein towards the development of a broadly protective group a streptococcal vaccine based on the lipid-core peptide system immunogenicity of a 26-valent group a streptococcal vaccine new 30-valent m protein-based vaccine evokes cross-opsonic antibodies against non-vaccine serotypes of group a streptococci protection against group a streptococcus by immunization with j8-diptheria toxoid: contribution of j8-and diphtheria toxoid-specifi c antibodies to protection intranasal vaccination with a lipopeptide containing a minimal, conformationally constrained conserved peptide, a universal t-cell epitope and a self-adjuvanting lipid protects mice from streptococcus pyogenes and reduces throat carriage immunisation of mice with a lipid core peptide construct containing a conserved region determinant of group a streptococcal m protein elicits heterologous opsonic antibodies in the absence of adjuvant epitopes of group a streptococcal m protein that evoke cross-protective local immune responses cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues protection against streptococcal pharyngeal colonization with a vaccinia: m protein recombinant mucosal vaccine made from live, recombinant lactococcus lactis protects mice against pharyngeal infection with streptococcus pyogenes clinical and microbiological responses of volunteers to combined intranasal and oral inoculation with a streptococcus gordonii carrier strain intended for future use as a group a streptococcus vaccine new multi-determinant strategy for a group a streptococcal vaccine designed for the australian aboriginal population immunization with a tetraepitopic lipid core peptide vaccine construct induces broadly protective immune responses against group a streptococcus intranasal administration is an effective mucosal vaccine delivery route for self-adjuvanting lipid core peptides targeting the group a streptococcal m protein mapping a conserved conformational epitope from the m protein of group a streptococci mapping the minimal murine t cell and b cell epitopes within a peptide vaccine candidate from the conserved region of the m protein of group a streptococcus key: cord-000708-iuo2cw23 authors: lippé, roger title: deciphering novel host–herpesvirus interactions by virion proteomics date: 2012-05-28 journal: front microbiol doi: 10.3389/fmicb.2012.00181 sha: doc_id: 708 cord_uid: iuo2cw23 over the years, a vast array of information concerning the interactions of viruses with their hosts has been collected. however, recent advances in proteomics and other system biology techniques suggest these interactions are far more complex than anticipated. one particularly interesting and novel aspect is the analysis of cellular proteins incorporated into mature virions. though sometimes considered purification contaminants in the past, their repeated detection by different laboratories suggests that a number of these proteins are bona fide viral components, some of which likely contribute to the viral life cycles. the present mini review focuses on cellular proteins detected in herpesviruses. it highlights the common cellular functions of these proteins, their potential implications for host–pathogen interactions, discusses technical limitations, the need for complementing methods and probes potential future research avenues. over the last decades, many host-pathogen interactions have been characterized using genetics, biochemical, and microscopy approaches. these discoveries relied on mutants, pharmacological reagents, immunoprecipitations, immunofluorescence, electron microscopy, cell fractionation, and western blotting to name a few of the methods employed. these approaches provided much precious information but, given the typical focus of these approaches on individual molecules, likely only revealed a small portion of the proteins involved. other methods such as high throughput two-hybrid and genetic screens, nucleic acid arrays, rna interference, and proteomics are now proving essential tools to tackle the complexity of these interactions. the main advantages of mass spectrometry, for instance, are that it is a fast, sensitive and potentially a quantitative approach to identify putative novel players, particularly when coupled to efficient purification schemes. already, proteomics revealed how viruses modulate the expression of host proteins (rassmann et al., 2006; sun et al., 2008; tong et al., 2008; antrobus et al., 2009; pastorino et al., 2009; thanthrige-don et al., 2009; zandi et al., 2009; zhang et al., 2009 zhang et al., , 2010 coombs et al., 2010; emmott et al., 2010; lu et al., 2010 lu et al., , 2012 munday et al., 2010; bartel et al., 2011; lietzen et al., 2011; ramirez-boo et al., 2011; chou et al., 2012) . a relatively new and interesting field is the characterization of host-pathogen interactions within mature purified virions. as reviewed on several occasions, several studies reported the presence of individual cellular proteins in viral particles (bernhard et al., 2005; maxwell and frappier, 2007; viswanathan and fruh, 2007; friedel and haas, 2011; zheng et al., 2011) . this includes vaccinia virus (krauss et al., 2002) , influenza virus (shaw et al., 2008) , hiv (gurer et al., 2002; cantin et al., 2005; ott, 2008) , vesicular stomatitis virus (moerdyk-schauwecker et al., 2009) , and several herpesviruses (see below). though these cellular components have often been considered purification contaminants, the presence of similar proteins in both related and unrelated viruses suggests that some of them may be biologically relevant. the identification of virion-associated host proteins could thus lead to the discovery of novel therapeutic tools against viruses. the present review focuses on their identification and putative roles with respect to the proteomics of herpesviruses. thus far, the protein composition of eight different herpesvirions has been studied by mass spectrometry. these studies include the alphaherpesvirinae herpes simplex virus type 1 (hsv-1) and pseudorabies virus (prv; loret et al., 2008; kramer et al., 2011) , the betaherpesvirinae human and murine cytomegaloviruses (hcmv and mcmv, respectively; kattenhorn et al., 2004; varnum et al., 2004) and the gammaherpesvirinae kaposi sarcoma herpesvirus (kshv), gamma herpesvirus 68 (γhv68), epstein-barr virus (ebv), and alcelaphine (bortz et al., 2003; johannsen et al., 2004; bechtel et al., 2005; zhu et al., 2005; dry et al., 2008) . interestingly, host proteins were detected in all herpesvirions analyzed so far, as summarized in table 1 . for instance, our laboratory previously reported the protein composition of mature extracellular hsv-1 viral particles and identified as many as 49 cellular proteins (loret et al., 2008) . similarly, studies focusing on prv and ebv reported up to 48 and 43 cellular proteins, respectively (johannsen et al., 2004; kramer et al., 2011) . meanwhile, varnum et al. (2004) found as many as 70 different host proteins in extracellular hcmv virions. while fewer cellular proteins were reported for other viral particles, it is clear that herpesviruses can potentially incorporate many proteins from its host. moreover, of the 173 different proteins detected in herpesvirions, nine protein groups are present in at least four distinct herpesvirions. this includes 14-3-3, actin, annexins, cofilin, translation factors, gapdh, heat shock proteins, pyruvate kinase m2, and various rab gtpases. these results indicate that, first of all, it is common for herpesviruses to incorporate cellular proteins into their viral particles and, secondly, that www.frontiersin.org ? different viruses share similar host proteins. most excitingly, it also suggests that these host proteins may play common roles throughout the herpesviral family. this defines an interesting and novel set of host-pathogen interactions taking place within the virus itself, rather than the cell. it is tempting to speculate that some viruses might have a higher capacity to steal cellular proteins because of their size and symmetry. herpesviruses are indeed large viruses containing a layer called the tegument between their capsids and envelopes that could accommodate non-viral proteins. though some host proteins may randomly be incorporated into virions, others may rather be selected to insure the optimal replication of the viruses that carry them. bioinformatics databases such as the kegg, gene ontology, or david are useful tools to get an overview of the functional interplay of proteins (ashburner et al., 2000; huang da et al., 2009; kanehisa et al., 2010) . as pointed out by friedel and haas (2011) , complex statistical tools are available to quantitatively evaluate the implication of proteins in various processes but these are beyond the scope of the present review. here an analysis of the proteins identified in herpesvirions was instead performed with the ingenuity pathways analysis database (ingenuity ® systems), which contains all the known physical and functional links among cellular proteins and defines their most significant functions. that analysis indicates that many of the cellular proteins found in herpesvirions normally modulate trafficking, cell proliferation, cell death, cell migration, cell metabolism, or the cytoskeleton (figure 1 , upper pie chart). though subtle differences between family members are noticeable when looking at individual viruses, similar functions are found (figure 1, other charts) . immune-related molecules are also important constituents for several viruses, including hsv-1, kshv, γhv68, alcelaphine, and mcmv. altogether, this provides an overall picture whereby herpesviruses, not surprisingly, modulate all of the important aspects of the cell but where each virus might deploy its energies slightly differently. the main surprise is that so many cellular proteins are detected within assembled viral particles, which raises an important question as to their biological significance and mode of action. the overall picture that several important cellular functions might be modulated by the host proteins incorporated into viral particles is intriguing. this clever strategy is consistent with the parasitic nature of all viruses, including herpesviruses, which would presumably gain some replication advantage from stealing cellular modulators rather than coding for them in their own genomes. the most critical question is the benefit for the viruses to incorporate these cellular proteins in their assembled particles, particularly since these proteins also exist in the cells. while this is open to discussion, one possibility is that some of the incorporated cellular proteins may be remnants of the final capsid envelopment process. alternatively, this may allow the prompt action of some of these proteins immediately upon viral entry. this could jumpstart the expression and/or duplication of the viral genome, as it is the case for the herpesviral vhs, vp16, icp0, and icp4 proteins that are present in virions (lam et al., 1996; everett, 2000; halford and schaffer, 2001; ellison et al., 2005; hancock et al., 2006; loret et al., 2008; sarma et al., 2008; loret and lippe, 2012) . other early potential sites of action are the process of viral entry itself, intracellular capsid transport, import of the viral genome through the nuclear pore or immune modulation, all common steps among herpesviruses. whatever the case might be, the question remains as to why the cellular pool of these proteins would not suffice. several options may be considered. first, it may be that the virions incorporate specific isoforms, splice variants or post-translationally modified proteins that could have properties or functions distinct than their cellular counterparts. second, the incorporation of a host protein from one cell type might permit the infection of a different cell type that does not express such protein. for example, alpha herpesviruses initially infect mucosal cells and could acquire host proteins that are beneficial to infect dormant neuronal cells. finally, the host proteins might be in complex with viral proteins and it is those complexes that are active to promote the infection. these possibilities are of course speculative at this point and need to be explored. one aspect where the incorporation of host proteins in mature virions might be beneficial is molecules involved in intracellular trafficking. work by numerous laboratories demonstrated that the transport machinery used to move cellular proteins is also employed by viruses (simons and warren, 1984; lodish et al., 2000; sollner, 2004; greber and way, 2006; mercer et al., 2010) . this is essential for their proteins and particles to reach their final destination, for example, the site of viral replication, assembly, and/or envelopment. along with snares proteins, rab and arf gtpases are master regulators of molecular trafficking throughout the cell (sollner and rothman, 1996; zerial and mcbride, 2001; mizuno-yamasaki et al., 2012) . so far,vamp3, a snare, was identified in prv virions (kramer et al., 2011) but it may only be a matter of time until other snares are discovered in other members of the herpes family. this is relevant as another snare was reported to facilitate the envelopment of mcmv capsids (cepeda and fraile-ramos, 2011) . in contrast, a great number of rab proteins have been identified in herpesvirions, particularly hsv-1 and prv (table 1) . one stimulating option is that these proteins regulate the displacement of viral capsids in the cell, which could justify their incorporation in the viral particles. as rab and arf proteins collectively modulate several intracellular transport steps within the cell, it is anticipated they may be involved in various stages of the infection. for instance, rab1, which is present in hsv-1 extracellular virions (loret et al., 2008) , and rab43 were recently demonstrated to modulate the final envelopment of the virus (zenner et al., 2011) . similarly, rab6, found in hsv-1 and prv (loret et al., 2008; kramer et al., 2011) , is also necessary for the efficient assembly of the related hcmv (indran and britt, 2011) . it will now be of interest to determine if the virion-associated pool of these gtpases actively participates in the viral life cycle. interestingly, several rab proteins have been implicated in autophagosome formation and maturation (chua et al., 2011) . while it is difficult to consider how virion-incorporated rab proteins play a role at that stage, they might rather be incorporated into the virions as mcmv figure 1 | the proteins from table 1 were analyzed with the ingenuity database to define their putative functions in the context of an infection. to this end, the protein accession numbers (or gi numbers) were queried from the ingenuity database. for the purpose of this figure, all known functions associated with these proteins were exported to microsoft excel and regrouped. in the top pie chart, the cellular proteins found in all the herpesvirions were analyzed collectively, while the other pie charts depict the host proteins incorporated into each virus. since each protein can be associated with multiples functions in the database, the results of those analyses are expressed as relative values instead of raw numbers, which consequently exceeds the original number of proteins analyzed. the percentages therefore represent the number of proteins falling into a given category with the total of each pie chart being 100%. a graphical legend of the categories is provided at the bottom right corner of the figure. a consequence of their involvement in autophagosome formation and concomitant viral envelopment. given the vast impact of rab proteins on the cell, it will be a major challenge to decipher all their roles in the life cycle of herpesviruses, particularly for the pool present in mature virions. molecular trafficking is not only dependent on snares, rab, and arf proteins, it is also intimately linked to the cytoskeleton. it is thus not surprising that herpesviruses devote some of their resources toward regulating this central cellular machinery. for instance, herpesviruses significantly reorganize both cellular and nuclear actin as well as microtubules (norrild et al., 1986; avitabile et al., 1995; sharma-walia et al., 2004; simpson-holley et al., 2005; de regge et al., 2006; saksena et al., 2006) . they also travel along microtubules during both entry and egress and interact with several cellular molecular motors (sodeik et al., 1997; smith et al., 2001; dohner et al., 2002; marozin et al., 2004; lee et al., 2006; wolfstein et al., 2006; radtke et al., 2010) as well as cortical and nuclear actin filaments (forest et al., 2005; feierbach et al., 2006; roberts and baines, 2011) . furthermore, some members incorporate in their viral particles tubulin or actin-related components ( table 1 ; wong and chen, 1998; grunewald et al., 2003) . actin has been reported to compensate the loss of various viral tegument proteins in prv (del rio et al., 2005; michael et al., 2006) and may thus act as an abundant filling agent, so its significance in herpesviral particles remains enigmatic. similarly, the relevance of intermediate filament components vimentin and keratins in some herpes virions ( table 1) is difficult to assess given these filaments are not as well characterized as other cytoskeletal elements. it may nevertheless be important for herpesviruses, particularly since they are not all associated with the common skin or hair contaminants often detected in mass spectrometry (hertel, 2011) . viruses tend to monopolize for their own purpose their host expression apparatus, including protein translation (bushell and sarnow, 2002) . for example, the prototypic hsv-1 icp27 viral protein regulates all aspect of mrnas including transcription, splicing, nuclear export, and translation for the benefit of the virus (rice and knipe, 1988; sekulovich et al., 1988; sandri-goldin and mendoza, 1992; smith et al., 1992; hardwicke and sandri-goldin, 1994; hardy and sandri-goldin, 1994; brown et al., 1995; soliman et al., 1997; chen et al., 2002; lindberg and kreivi, 2002; ellison et al., 2005; larralde et al., 2006; fontaine-rodriguez and knipe, 2008) . as these cellular functions are highly regulated, the inclusion of ddx3x, a multifunctional rna helicase that also regulates transcription, nuclear export, and translation that is used by several viruses (schroder, 2010 (schroder, , 2011 ) may be relevant. its incorporation into mature virions could thus accelerate viral gene expression in the early stages of the infection. similarly, the presence of translation initiation or elongation factors in virions (table 1 ) may also jumpstart gene expression in favor of the viruses. interestingly, hsv-1 does not require cells to be in the s-phase and even arrests the cell cycle at the g1/s transition step (shadan et al., 1994; song et al., 2000) , which partly explains why it can grow in non-dividing neurons. while the precise mechanism of this arrest is unclear, it is known that the viral icp0 protein and the vp16 cellular partner hcf modulate the cell cycle (hobbs and deluca, 1999; lomonte and everett, 1999; piluso et al., 2002) . moreover, icp0 interacts with the host cyclin d3 (kawaguchi et al., 1997) . however, it was recently reported that stress, rather than the cell cycle per se, may be a critical feature (bringhurst and schaffer, 2006) . clearly, the interaction of herpesviruses with the cell proliferation apparatus is complex and likely involves several host and viral proteins. identifying novel players that might be incorporated into mature virions may thus be very useful to clarify this process. an interesting scenario is the possible regulation of apoptosis by host proteins loaded onto viral particles. apoptosis is regulated both negatively and positively by several viruses (teodoro and branton, 1997; goodkin et al., 2004) , presumably to insure their survival at the early stages of the infection but their efficient release later on. conceptually, the presence of anti-apoptotic proteins in herpes particles might thus provide a mean to quickly evade death upon entry, while the presence of pro-apoptotic proteins on newly assembled/enveloped viral particles may trigger or stimulate their extracellular release. only further work will resolve this open question. several factors generally contribute to variation among proteomic studies. hence, the preparation of the samples (e.g., in-gel trypsin digestion versus liquid digestion and chromatography) may lead to the detection of different populations of tryptic peptides. moreover, the sensitivity of the mass spectrometers and the abundance of the proteins in the samples also impact peptide detection. the relative abundance of a peptide is itself influenced by the complexity of the samples, where some proteins may evade identification. finally, each protein differs in its properties (ionization, resolution in sds-page gels), which will be reflected in their detection. this includes snares, which are transmembrane proteins resistant to sds extraction (yang et al., 1999; kubista et al., 2004) . it is thus likely that some of the proteins in table 1 are present in more viral particles than reported and that additional proteins are indeed incorporated in herpesvirions. more specific aspects regarding herpesviruses includes the purification schemes employed to enrich the viral particles, which will directly influence the purity of the samples and hence the potential detection of contaminants. one important caveat is that some host proteins may simply stick to the large viral particles. another one is common contaminants such as some hair/skin associated keratins or as mentioned above actin, which may simply fill the virions. however, even potential contaminants cannot simply be discarded since actin and even some keratins may indeed participate in viral life cycles. moreover, the relative abundance of all the cellular proteins within the cell is unknown, so it is not possible to rule out potential contaminants on the sole basis of abundance. it is thus critical to orthogonally validate all proteomics hits. various tools are available to define the biological relevance of host proteins identified in viral particles, including western blotting, immuno-electron microscopy or functional screens. one powerful method is rna interference. however, given the dual presence of the host proteins within the viral particles and the cell itself, this becomes a challenging task. rna interference also has its own caveats (false positives and negatives). another common step is the expression of dominant positive or negative mutants. in all cases, one major difficulty is that the host proteins may be essential for the cells and their depletion may lead to cytotoxicity, thus proper controls are needed. in addition, the host proteins might be essential for the virus within the cells but only accessory within the virions. consequently, depletion of a protein may have limited impact on the virus since complemented by the other pool of that protein in the virus or the cell. small reduction or stimulation in viral yields may thus result. it such cases, it may be necessary to produce the virus on cells that lack these proteins to see if this makes a difference. one should also consider animal models since tissue culture based screens may miss important players, for instance modulators of the immune system or virulence factors. clearly, multiple experimental strategies are needed to ultimately insure the biological significance of the host proteins found in viral particles. the identification and functions of host proteins in viral particles is an important step toward the elucidation of novel host-pathogen interactions. in the case of herpesvirions, this is well under way with eight different family members analyzed so far. one main aspect is to sort biologically relevant cellular proteins from sticky contaminants. the orthogonal validation of the host proteins found in herpesvirions using biologically relevant assays is thus critical. as pointed out above, it will necessary to analyze all these proteins in the background of two pools, one cellular and one virion-associated, which are likely to complement one another. an interesting possibility is that some isoforms or specific posttranslationally modified host proteins may be loaded into the capsids. thus a detailed analysis of the host proteins present in viral particles will be important and a potential way to distinguish them from their cell-associated counterparts. another issue is the expected variation among cell types. in that respect, it would be most interesting to examine the cellular protein content of hsv-1 produced in neurons in opposition to the virions produced on other cell types. finally, the mechanisms by which all these host proteins are recruited to the viral particles will also need to be explored. thus the proteomics of viral particles is only the beginning of the adventure, which should prove most exciting yet challenging. i am indebted to the canadian institutes of health research (grant # mop82921) for funding our proteomics research. i also wish to thank kerstin radtke for excellent suggestions and daniel henaff for critical reading of the manuscript. proteomic analysis of cells in the early stages of herpes simplex virus type-1 infection reveals widespread changes in the host cell proteome gene ontology: tool for the unification of biology redistribution of microtubules and golgi apparatus in herpes simplex virus-infected cells and their role in viral exocytosis proteome analysis of vaccinia virus ihd-w-infected hek 293 cells with 2-dimensional gel electrophoresis and maldi-psd-tof ms of on solid phase support n-terminally sulfonated peptides host and viral proteins in the virion of kaposi's sarcomaassociated herpesvirus new insights into viral structure and virus-cell interactions through proteomics identification of proteins associated with murine gammaherpesvirus 68 virions cellular stress rather than stage of the cell cycle enhances the replication and plating efficiencies of herpes simplex virus type 1 icp0-viruses herpes simplex virus trans-regulatory protein icp27 stabilizes and binds to 3 ends of labile mrna hijacking the translation apparatus by rna viruses plunder and stowaways: incorporation of cellular proteins by enveloped viruses a role for the snare protein syntaxin 3 in human cytomegalovirus morphogenesis icp27 interacts with the rna export factor aly/ref to direct herpes simplex virus type 1 intronless mrnas to the tap export pathway an overview of the vaccinia virus infectome: a survey of the proteins of the poxvirus-infected cell involvement of members of the rab family and related small gtpases in autophagosome formation and maturation quantitative proteomic analyses of influenza virus-infected cultured human lung cells alpha-herpesvirus glycoprotein d interaction with sensory neurons triggers formation of varicosities that serve as virus exit sites actin is a component of the compensation mechanism in pseudorabies virus virions lacking the major tegument protein vp22 function of dynein and dynactin in herpes simplex virus capsid transport proteomic analysis of pathogenic and attenuated alcelaphine herpesvirus 1 control of vp16 translation by the herpes simplex virus type 1 immediateearly protein icp27 quantitative proteomics using stable isotope labeling with amino acids in cell culture reveals changes in the cytoplasmic, nuclear, and nucleolar proteomes in vero cells infected with the coronavirus infectious bronchitis virus icp0, a regulator of herpes simplex virus during lytic and latent infection alpha-herpesvirus infection induces the formation of nuclear actin filaments herpes simplex virus icp27 increases translation of a subset of viral late mrnas active intranuclear movement of herpesvirus capsids virus-host interactomes and global models of virus-infected cells herpes simplex virus infection and apoptosis a superhighway to virus infection three-dimensional structure of herpes simplex virus from cryoelectron tomography specific incorporation of heat shock protein 70 family members into primate lentiviral virions icp0 is required for efficient reactivation of herpes simplex virus type 1 from neuronal latency herpes simplex virus regulatory proteins vp16 and icp0 counteract an innate intranuclear barrier to viral gene expression the herpes simplex virus regulatory protein icp27 contributes to the decrease in cellular mrna levels during infection herpes simplex virus inhibits host cell splicing, and regulatory protein icp27 is required for this effect herpesviruses and intermediate filaments: close encounters with the third type perturbation of cell cycle progression and cellular gene expression as a function of herpes simplex virus icp0 systematic and integrative analysis of large gene lists using david bioinformatics resources a role for the small gtpase rab6 in assembly of human cytomegalovirus proteins of purified epstein-barr virus kegg for representation and analysis of molecular networks involving diseases and drugs identification of proteins associated with murine cytomegalovirus virions herpes simplex virus 1 alpha regulatory protein icp0 interacts with and stabilizes the cell cycle regulator cyclin d3 proteomic characterization of pseudorabies virus extracellular virions an investigation of incorporation of cellular antigens into vaccinia virus particles evidence for structural and functional diversity among sds-resistant snare complexes in neuroendocrine cells herpes simplex virus vp16 rescues viral mrna from destruction by the virion host shutoff function direct stimulation of translation by the multifunctional herpesvirus icp27 protein reconstitution of herpes simplex virus microtubule-dependent trafficking in vitro quantitative subcellular proteome and secretome profiling of influenza a virus-infected human primary macrophages splicing inhibition at the level of spliceosome assembly in the presence of herpes simplex virus protein icp27 viruses: structure, function, and uses herpes simplex virus type 1 immediate-early protein vmw110 inhibits progression of cells through mitosis and from g(1) into s phase of the cell cycle comprehensive characterization of extracellular herpes simplex virus type 1 virions biochemical analysis of icp0, icp4, ul7 and ul23 incorporated into extracellular herpes simplex type 1 virions two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (itraq) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine reproductive and respiratory syndrome virus proteomic analysis of the host response in the bursa of fabricius of chickens infected with marek's disease virus herpes simplex virus type 1 infection of polarized epithelial cells requires microtubules and access to receptors present at cell-cell contact sites virus entry by endocytosis composition of pseudorabies virus particles lacking tegument protein us3, ul47, or ul49 or envelope glycoprotein e gtpase networks in membrane traffic analysis of virion associated host proteins in vesicular stomatitis virus using a proteomics approach quantitative proteomic analysis of a549 cells infected with human respiratory syncytial virus subgroup b using silac coupled to lc-ms/ms organization of cytoskeleton elements during herpes simplex virus type 1 infection of human fibroblasts: an immunofluorescence study cellular proteins detected in hiv-1 isolation and preliminary characterization of herpes simplex virus 1 primary enveloped virions from the perinuclear space host cell factor-1 interacts with and antagonizes transactivation by the cell cycle regulatory factor miz-1 plus-and minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures quantitative proteomics by 2-de, 16o/18o labelling and linear ion trap mass spectrometry analysis of lymph nodes from piglets inoculated by porcine circovirus type 2 proteome alterations in human host cells infected with coxsackievirus b3 gene-specific transactivation by herpes simplex virus type 1 alpha protein icp27 actin in herpesvirus infection herpes simplex virus type 1 accumulation, envelopment, and exit in growth cones and varicosities in mid-distal regions of axons a herpesvirus regulatory protein appears to act posttranscriptionally by affecting mrna processing small interfering rnas that deplete the cellular translation factor eif4h impede mrna degradation by the virion host shutoff protein of herpes simplex virus human deadbox protein 3 has multiple functions in gene regulation and cell cycle control and is a prime target for viral manipulation viruses and the human dead-box helicase ddx3: inhibition or exploitation? the herpes simplex virus type 1 alpha protein icp27 can act as a trans-repressor or a trans-activator in combination with icp4 and icp0 n-butyrate, a cell cycle blocker, inhibits the replication of polyomaviruses and papillomaviruses but not that of adenoviruses and herpesviruses kaposi's sarcomaassociated herpesvirus/human herpesvirus 8 envelope glycoprotein gb induces the integrin-dependent focal adhesion kinase-srcphosphatidylinositol 3-kinase-rho gtpase signal pathways and cytoskeletal rearrangements cellular proteins in influenza virus particles semliki forest virus: a probe for membrane traffic in the animal cell identification and functional evaluation of cellular and viral factors involved in the alteration of nuclear architecture during herpes simplex virus 1 infection herpesviruses use bidirectional fast-axonal transport to spread in sensory neurons evidence that the herpes simplex virus immediate early protein icp27 acts post-transcriptionally during infection to regulate gene expression microtubulemediated transport of incoming herpes simplex virus 1 capsids to the nucleus shuttling of the herpes simplex virus type 1 regulatory protein icp27 between the nucleus and cytoplasm mediates the expression of late proteins intracellular and viral membrane fusion: a uniting mechanism molecular machinery mediating vesicle budding, docking and fusion herpes simplex virus infection blocks events in the g1 phase of the cell cycle proteomic alteration of pk-15 cells after infection by classical swine fever virus regulation of apoptosis by viral gene products analyses of the spleen proteome of chickens infected with marek's disease virus proteomic analysis of cellular protein alterations using a hepatitis b virus-producing cellular model identification of proteins in human cytomegalovirus (hcmv) particles: the hcmv proteome viral proteomics: global evaluation of viruses and their interaction with the host the inner tegument promotes herpes simplex virus capsid motility along microtubules in vitro evidence for the internal location of actin in the pseudorabies virion snare interactions are not selective. implications for membrane fusion specificity proteomics analysis of bhk-21 cells infected with a fixed strain of rabies virus analysis of rab gtpase-activating proteins indicates that rab1a/b and rab43 are important for herpes simplex virus 1 secondary envelopment rab proteins as membrane organizers proteomic analysis of pbmcs: characterization of potential hiv-associated proteins differential proteome analysis of host cells infected with porcine circovirus type 2 mass spectrometry based proteomic studies on viruses and hosts -a review virion proteins of kaposi's sarcoma-associated herpesvirus the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-014901-d9szap94 authors: permyakova, n. v.; uvarova, e. a.; deineko, e. v. title: state of research in the field of the creation of plant vaccines for veterinary use date: 2015-01-04 journal: russ j plant physiol doi: 10.1134/s1021443715010100 sha: doc_id: 14901 cord_uid: d9szap94 transgenic plants as an alternative of costly systems of recombinant immunogenic protein expression are the source for the production of cheap and highly efficient biotherapeuticals of new generation, including plant vaccines. in the present review, possibilities of plant system application for the production of recombinant proteins for veterinary use are considered, the history of the “edible vaccine” concept is briefly summarized, advantages and disadvantages of various plant systems for the expression of recombinant immunogenic proteins are discussed. the list of recombinant plant vaccines for veterinary use, which are at different stages of clinical trials, is presented. for many thousands of years plants have served humanity as a source of medicinal substances. how ever, only at the turn of the century xxi with the appearance of dna technologies, it became possible to modify plant genomes and to create new types of plants (transgenic plants), which are capable of syn thesizing and accumulating in their tissues recombi nant proteins from various heterologous systems. to date transgenic plants in which nuclear and chloro plast genomes have been transformed with genes encoding heterologous proteins that are important in the treatment of various diseases -antigens of infec tious agents, antibodies, immunomodulators, etc. have been created [1, 2] . of principal importance of this work development was the creation of the "edible vaccine" concept, the essence of which is the use of genetically modified plants containing protein anti gens of infectious agents for oral delivery of relevant antigens to the mucosa of the gastrointestinal tract of warm blooded animals. vaccination based on the programming of the spe cific mechanisms of warm blooded animal protection against pathogens is the most efficient method for the struggle against infectious diseases, which often result in a mass mortality. in agriculture, there is no alterna tive to livestock vaccination, because there are no anti viral drugs that are suitable for a wide use in animal husbandry. the importance of animal vaccination indirectly affects human health, because the use of vaccines significantly reduces the amount of pharma ceuticals in the food chain. as a rule, animal immune mechanisms are acti vated by the direct introduction of infectious agents or their components. at present, most of used vaccines are preparations on the basis of inactivated agents. although these vaccines manifest the high immunoge nicity, they are not without serious shortcomings. among such disadvantages are the increased sensitiv ity of the organism to them, the large load on the immune system, the reactogenicity of vaccines (side effects), their toxicity. etc. the application of molecular biology and genetic engineering methods opened wide prospects for the manufacturing of vaccines of new generation, which immunogenic components can be biological mole cules or their fragments. dna fragments or proteins of the infectious agent cell envelopes can serve as immu nogenic components. when the gene encoding the envelope protein of the infectious agent is transferred into the genome of another organism, for example, plant, then the cells of such plant will synthesize the protein antigen capable of formation of resistance to this agent. thus, the introduction into the organism not the whole pathogen but only its part, which is not capable of inducing infection development, will pro vide for the effect of vaccination. one fourth of the total pharmaceutical market of drugs for veterinary use, and it is constantly expanding. [3] . preparation of medicinal substances for the pro duction of veterinary products is based on various approaches, including biotechnology using genetically modified (transgenic) organisms for these purposes; such expression systems as bacteria, yeast, cells of insects and mammals are used. the application of genetically modified plants with genes encoding phar maceutic proteins inserted in the genome opens new prospects for obtaining recombinant proteins, includ ing plant vaccines [4] . this review is devoted to the analysis of possibilities of producing recombinant immunogenic proteins for veterinary use on the basis of plant expression systems, and the history of the concept of "edible vaccines" for animal immunization. of plant vaccines the idea of the usage of plant cells for the synthesis and accumulation of recombinant protein antigens was for the first time successfully realized in 1992 by c. arntzen and his colleagues [5] . just this team of researchers not only demonstrated a possibility of the accumulation of the surface hbsag antigen of hepati tis b virus but also its capability for self assembling in the virus like particles in transgenic tobacco plants. the virus like particles isolated from plant tissues were identical to the particles of hbsag antigen of indus trial recombinant vaccine obtained in the yeast expres sion system and also to virus like particles from the blood plasma of patients infected with hepatitis b virus. thus, it became obvious that genetically modi fied plants producing and accumulating protein anti gens of various infectious agents can be used for oral delivery of corresponding agents to the mucosa of the gastrointestinal tract of warm blooded animals, i.e., as "edible vaccines." the next important step in the development of the "edible vaccine" concept on the basis of genetically modified plants was the creation of transgenic plants producing the heat labile enterotoxin of escherichia coli [6, 7] and b subunit of the cholera toxin [8] . heat labile toxin of e. coli consists of two parts: lt a (enzyme) and lt b (pentamer of receptor binding polypeptides). lt b binds to the receptors on the sur face of membranes of epithelial cells of the mammal small intestine and transports lt a in the intestinal cells, where it induces changes in the cell metabolism and cell dehydration. when the two parts of the heat labile enterotoxin are separated, the appearance of the lt b protein complex on the surface of epitheliocytes will stimulate a strong immune response of intestine mucosa without the appearance of any disease signs. just this feature was the basis for the research of c. arntzen [6] team on the creation of plant vaccine providing for the resistance to enterotoxigenic e. coli toxins. the authors established that lt b synthesized in transgenic tobacco and potato plants and also lt b isolated from e. coli delivered orally to mice induced similar immune responses. later, lt b sequence was optimized for expression in plant cells and transferred into the potato genome [7] . in potato tubers, the protein assembled correctly in oligomers and was accumulated in amounts suffi cient for the induction of the immune response at oral delivery to the organism. on the basis of clinical trials of the "candidate" plant lt b vaccine, it was estab lished that the consumption of raw potato tubers con taining 0.3-10 mg lt b by volunteers resulted in the formation of serum and mucosal immune responses with high titers of antibodies [9] . the initial concept of "edible vaccine" was heavily criticized by researchers who believed that in the aggressive medium of the gastrointestinal tract the recombinant protein should be destroyed. however, later it was experimentally established that the recom binant b subunit of the cholera toxin fused with green fluorescent protein (gfp) protected by the plant cellu lose cell wall at oral delivery is capable of passing through the gastrointestinal tract and reaching the antigen containing cells of the mouse intestine [10] . the results obtained confirmed the possibility of using plants synthesizing protein antigens of various infec tious agents for oral delivery of antigens to the mucosa of the gastrointestinal tract and experimentally con firmed a consistency of the "edible vaccine" concept. it became evident that genetically modified plants can be used for the creation of plant vaccines as a raw mate rial, and separate plant parts (fruits, roots, berries, leaves, etc.) can be used directly in food without pre liminary heat treatment. response formation in the process of evolution, mammals developed secondary lymphoid mucosal tissue capable of antigen absorption, processing them, and using for the induc tion of the mucosal response. it was established that in this case both cellular and humoral immunity were formed. it is of importance that adaptive mucosal immunity can distinguish between usual food and symbiotic antigens and infectious agents [11] . the scheme of the mechanism of mucosal immune response formation is presented in fig. 1 . in the digestive tract, which is one of the pathways for the penetration of a variety of pathogens into organism, the main associated lymphoid tissue is the peyer's patches. peyer's patches, inductive sites of the intestine, which contain the dome, underlying the fol licle (b zones with germinal center) and interfollicu lar region containing t cells. the surface of the dome is covered by a specialized follicle associated epithe lium containing the folded cells (m cells), which are able to absorb and transport antigens from the intesti nal lumen. after successful capture, the antigen is par tially cleaved and enters into dendritic cells (see fig. 1 , step 1). dendritic cells are especially important in the initiation of adaptive immune responses, since they migrate to the lymph nodes (see fig. 1 , step 2), and the mediators act in the development of various subpopu lations of t helper cells from naive t lymphocytes and also can interact with b lymphocytes (see fig. 1 , step 5). the activated b and t cells leave the peyer's patches and penetrate into the circulatory system. mature t helper cells then return to the mucosa sur pathogens (antigen) epithelium t helper cells face to function as effectors (see fig. 1 , step 3). t helper 17 expressing interleukin 17 (il 17) increases the expression of the receptors of polymeric immunoglobulin (pig) and secretion of antigen spe cific iga (fig. 1, step 4) . subsequent generation of mature plasma cells producing iga leads to the induction of antigen specific protection of local and distal mucosal surfaces. since the mucosal immune response has a general ized nature, oral (i.e., mucosal) vaccination is not only the immune response of the mucous membranes, but also the overall immune response of the organism [11] [12] [13] [14] . it is proved that vaccination via the surface of mucosa membrane can specifically activate the immune response to infection without the develop ment of such processes related to the disease as inflammation or toxicity [14, 15] . of plant vaccines in comparison with traditional expression systems, plant systems are attractive to researchers in many ways, primarily due to the absence of the risk of plant cell infection with animal pathogens, viruses, prions, etc. plants are capable of the synthesis of most recom binant antigens with the same posttranslational modi fications as in animal cells [16] . plant vaccines can play especially important role in the protection of ani mals against diarrheal diseases and diseases, which infectious agents penetrate into the organism through the mucosal tissues. modern techniques of genetic engineering allow to selectively direct the recombinant proteins expressed in plant cells to various plant organs (seeds, tubers, fruits, etc.) [17] . this possibility greatly simplifies the large scale production of plant vaccines and reduces their cost. according to the experts, the final price of the product (recombinant protein) produced in a plant expression system will be much less than the price of a similar protein produced, for example, in mammalian cell culture [18] . since recombinant proteins can be accumulated in the storage organs or seeds, they are able to be main tained without any changes and the loss of biological activity for a long time (months and years). it was established that the recombinant protein of cholera toxin b subunit remained stable in transgenic rice grains for at least 18 months, when grains were stored under room temperature conditions [19] . recombi nant protein antigens remained stable in rice grains during three years and provided for the formation of the protective immunity in mice against cholera agent or against enterotoxigenic e. coli [20] ; in soybean seeds and soy milk, the preservation of antigen stabil ity was observed for four years [21] .thus, grains of transgenic plants can be transported to the site of final destination without additional freezing and treatment, and this ensures the retention of activity of the recom binant protein activity, its stability, and the constancy of dosage. a somewhat different picture is observed when lyo philization is used as a method of the conservation of protein antigens synthesized by plant cells. it was established that the recombinant protein of the norovirus envelope retained its immunogenicity in tis sues of both lyophilized and air dried tomato fruits [22] , but the tested samples differed in immunogenic ity. the immunogenicity of this recombinant protein in air dried tomato fruits was somewhat higher in comparison with lyophilized fruits. similar results were obtained in experiments on the lyophilization of potato tubers synthesizing the protein of norovirus envelope, e.g., the immunogenicity of lyophilized tubers was lower than that of fresh tubers [22] . how ever, additional studies are required for the final solu tion of this question. despite these advantages, plant vaccines are not without disadvantages. one of them is differences in protein posttranslational modifications in plants and animals, e.g., in glycosylation of the recombinant pro tein [23] . it is known that more than a half of proteins synthesized by eukaryotic cells are glycosylated and more than a third of currently applied biopharmaceu tics are glycoproteins [24] . although the activity of most proteins does not depend on glycosylation, in some cases it may be critical. specific features of pro tein glycoforms can affect their folding, stability, trans portation, and changes in their functional activity and immunogenicity. examples of biopharmaceuticals, which functional activity depends on the specific gly coform, are erythropoietin, antibodies, blood anti gens, some interferons and hormones [24] . scheme of the n glycan complex formation in plants and animals (humans, for example) is shown in fig. 2 . the most significant difference in glycosylation is that the plant β 1,2 xylose is attached to the core mannose residue and α 1,3 fucose -to n acetylglu cosamine residue of the core glycan. in human cells xylose is not used at all in glycosylation and a proximal fucose residue is attached to glycans through the α 1,6 bond. it was established that sugar residues attached at posttranslational modifications of recom binant proteins in plant cells themselves were capable of exhibiting the immunogenicity. approximately in one quarter of patients with allergy symptoms, ige antibodies specific for complex glycans, which include xylose or fucose, were revealed [25] . differ ences in glycosylation during posttranslational modi fications of recombinant proteins in plant and mam mal cells can be eliminated by genetic engineering techniques, which was successfully demonstrated on the moss physcomitrella patens, in which the genes encoding the enzymes β 1,2 xylosyltransferase and α 1,3 fucosyltransferase responsible, respectively, for xylosylation and fucosylation of proteins were switched off by the "knock down" method [25] . it is known that when the foreign gene is inserted in the genome of the transgenic plant, the level of its expression depends on the site of its insertion, which determines the level of protein (antigen in particular) accumulation in plant tissues. in this connection, one of disadvantages of plant vaccines is the difficulty in the standardization of their dosage. it is at this stage the development of the "edible vaccine" concept has undergone substantial revision, since the possibility of oral delivery of the recombinant antigen with the raw plant material was untenable because of the variability of the recombinant protein content. according to the researchers involved in the development of "candi date" plant vaccines, the antigen dosage problem in plant tissues can be successfully solved by the intro duction of additional treatment of the plant material: its refinement (to equalize the concentration of the antigen), drying or lyophilization. it is also necessary to introduce an additional stage associated with the development of rapid methods for determining and monitoring the dosage of the recombinant antigen. after appropriate preparation, plant vaccines can be encapsulated, tableted, and used in practice under corresponding medical supervision [14, 26] . thus, performed studies allow a suggestion that the plant vaccine based on the genetically modified plants is capable of inducing protective immunity and open new opportunities for the creation of low cost and easy handling vaccines against infectious diseases of animals. it is of importance that developed to date, highly effective methods of cultivation of agro eco nomically important plant species, as well as the seed production system for a particular culture make the plants attractive to be used as "biofactories" for man ufacturing cheap recombinant proteins for medical purposes. roplasts). among plant expression systems with stable integration of the transgene in the nuclear genome, a separate group includes the cultivated duckweed, microalgae, and cell culture systems in vitro: cell sus pensions, cultures of "hairy roots", moss protonemas, which cultivation conditions are a completely closed environment (bioreactors). promising is the transient expression system, in which the target gene is intro duced into plant cells and is expressed for a short period of time (several days), but is not integrated into the genome. each of these expression systems has its advantages and disadvantages; the main details of these systems are considered below. the most widely used system of heterologous gene expression, in particular for plant vaccine production, are transgenic plants with the stable integration of the transgene into the nuclear genome. the creation of these plants involves the transfer of foreign genes into the genome of plant cells using agrobacterium tumefa ciens or bioballistics and subsequent regeneration of transformed plants from these cells. being inserted into the nuclear genome, transgene becomes its resi dent part, is stably expressed, and is maintained in subsequent generations. using this expression system for producing recom binant proteins, including plant vaccines, is still ham pered by relatively low levels of transgene expression, which is, as a rule, less than 1% of total soluble protein (tsp) and also by its variability in different plant organs and tissues within a single plant and in different plants. most often, the variability in expression of the transgene is due to the random nature of its insertion into the nuclear genome (effect of position) and may be associ ated with partial or complete transgene inactivation [27, 28] . experts believe that the use of plant expression sys tems for obtaining plant vaccines is economically bene ficial at the level of expression of a target gene, which allows the accumulation of recombinant protein in an amount not less than 1% of tsp [29] . a lot of ways to increase the level of foreign gene expression in transgenic plants is developed; they are very fully discussed in the reviews [17, 30, 31] . among them are the optimization of the codon composition of the target sequence, the usage of strong promoters, the addition of introns or regions of binding with the nuclear matrix (sar), and others. the search for tis sue and organ specific promoters, i.e., promoters providing for target gene expression in definite plant tissues or organs, is of a special interest. for example, the usage of promoters directing transcription of the target gene predominantly in the seeds can increase the yield of the target protein by an order or several fold: up to 13-14% of tsp in rice grains and up to 25% of tsp in tobacco seeds [17] . it is of importance that as compared with leaves, seeds contain less pro teases and much less water; therefore, recombinant protein is saved better. despite the fact that transgenic plants with stable transgene expression in the nuclear genome are used most widely, it is just this expression system that induces a cautious attitude of human society. one of the fears is associated with the possibility of transgene transfer from the cross pollinated plants into the genomes of wild relatives at growing biotechnological crops in open field. to solve this problem, researchers developed various agricultural technologies as well as fixing male sterility in transgenic plants to prevent unwanted cross pollination of cultivated and wild spe cies. examples of production of various immunogenic proteins using for this purpose transgenic plants with stable transgene integration into the nuclear genome are presented in table 1 . chloroplasts are most attractive among plant expression systems. genetically modified plants with the stable transgene integration into the chloroplast genome were called transplastome plants. the specific organization of chloroplasts allows achieving a high dose of foreign gene in transplastome plants, which provides for the efficient production of the target pro tein. the record of the recombinant protein yield was achieved by transplastomic tobacco plants with the bacteriophage lysin gene plygbs, encoding a hydro lase of the bacterial cell wall, the level of which accu mulation in the leaves amounted to 70% of tsp [32] . although in some cases, negative physiological changes were observed in plants with such high level of foreign gene expression, usually there were no devia tions in the development of such plants. problems of transplastome plant adaptation to the high level of for eign gene expression are discussed in the review of bally et al. [33] . transgene delivery to chloroplasts is performed using bioballistics (gene gun); its integration into the chloroplast genome occurs via homological recombi nation [34] . the advantage of chloroplast expression system is in the absence of the effect of position observed in the case of random pattern of transgene distribution in the nuclear genome. in plastids, there is no transgene splicing (inactivation); therefore, its expression is stably preserved in subsequent genera tions. due to the prokaryotic organization of chloro plast genome, there is a possibility of co expression of several genes within a single operon [35] . an impor tant specificity of plastids is that they are inherited through the maternal line and usually are absent from pollen. therefore, as distinct from usual transgenic plants, transplastome plants are safe for environment because the uncontrolled spread of the transgene into other plants is prevented [36, 37] . it should be noted that protein posttranslational modifications, e.g., assembling multimer proteins, the tmv-tobacco mosaic virus; camv-cauliflower mosaic virus; tsp-total soluble protein; (+) immune response is revealed at oral immunization; (++) immune response is revealed at oral immunization, animals did not die after virus infection. species of immunized animals are indicated, the way of antigen delivery is indicated in the cases when it was not oral; the amount of survived infected animals is indicated in the cases when the survival was less than 100%. no. 1 2015 formation of disulfide bridges, lipid modifications, etc., occur successfully in chloroplasts. it is estab lished that recombinant proteins synthesized in chlo roplasts do not differ from native ones in their func tional activities [38, 39] . chloroplast attractiveness as the system of recombinant protein expression is in the fact that they are closed structures and it preserves the metabolites, which when released into the cytosol are toxic to plant cells, such as b subunit of cholera toxin [38] and trihalose in tobacco cells [40] . on the basis of the above works, it becomes appar ent that transplastome plants can be regarded as the most promising system for efficient production of pro tein antigens. however, the main disadvantage of such expression system at plant vaccine manufacturing is that chloroplasts cannot glycosylate proteins. at present, the creation of transplastome plants is also associated with some technological problems related to the absence of the efficient system of regeneration for most plant species and also with the absence of the efficient system of transgen delivery to the chloroplast genome providing for the high percent of transformed plant yield. the suspension cell cultures on the basis of geneti cally modified plants attract the attention of research ers as promising potential systems for biopharmaceu tics production. such cultures can be obtained from loose callus tissues induced from genetically modified explants or on the basis of co culturing of the cell sus pension and a. tumefasciens. after the assessment of growth characteristics and cell line screening to obtain promising lines capable of the accumulation of great amounts of recombinant proteins, such lines can be cultivated in bioreactors for target protein obtaining. an attractive feature of the cell cultures as expres sion systems for recombinant protein production, as compared to the use of whole plants for this purpose, is that cell culture can be unified in growth character istics, cell dimensions and types. moreover, cells are grown under strictly controlled conditions, when the product accumulation does not depend on the sea sonal weather changes and allows the permanent product obtaining in bioreactors. the additional insertion of signal peptide nucleotide sequences into the construct permits a protein secretion into the intercellular space, which allows target protein isola tion directly from the culture liquid. the addition of recombinant protein stabilizers to the suspension cul ture increases the yield of the target protein [41] . by their capabilities, plant cell cultures are compa rable in production of therapeutic recombinant pro teins with the conventionally used mammalian cell cultures, such as chinese hamster ovary cells. how ever, as distinct from the mammal suspension cultures, they are not infected by any animal pathogens. to data, there are many examples of plant cell cultures with the yield of recombinant protein in the amount more than 10 mg/l, which is a threshold value for starting the commercial manufacture of the product [42] . an example of a commercially successful produc tion of veterinary vaccine products is developed by dow agro sciences company (united states) system concert™, patented as an effective and safe system for the production of vaccine proteins in cultured plant cells, cultured in a bioreactor. the first plant vaccine against newcastle disease virus of birds obtained in the tobacco cell culture was approved for use by the min istry of agriculture of the united states in 2006. the disadvantages of this expression system are still insufficiently high yield of the target recombinant pro tein and the instability of foreign genes in cultured plant cells due to the epigenetic silencing of the trans gene transcription [43] . advantages, disadvantages, and specific features of recombinant protein produc tion in the plant cell cultures are discussed in reviews [42, 44] . there are many examples of successful use of aqueous plants, such as duckweed, unicellular algae, and mosses, which are cultivated similarly as plant cell suspensions in bioreactors, for recombinant pro tein expression. duckweed attracts the attention of researchers as a potential highly efficient system for recombinant pro tein expression due to its capability of a rapid biomass accumulation: it can be doubled for 24-48 h. geneti cally modified duckweed as a potential producer of biopharmaceutic proteins can be used by animals as a raw or dried food. duckweed is a monocotyledonous angiosperm; for foreign gene transfer into its genome, the methods of agrobacterial transformation and bioballistics are used. examples are known when genetically modified duckweed accumulated recombi nant protein in the amounts up to 25% of tsp, as assessed after the accumulation of gfp [45] . sequenc ing the duckweed chloroplast genome is close to com pleting, which opens up some prospects for a signifi cant increase in the yield of recombinant proteins. the systems of biopharmaceuticals production using genetically modified microalgae are actively developed. algae combine advantages of both bacteria (rapid growth and simplicity of cultivation) and higher plants (a capability of posttranslational modifications and photosynthesis). chlamydomonas reinhardtii is most promising among algae: it has a short time of bio mass doubling (about 10 h), it is easily subjected to nuclear and chloroplast transformation, it can be grown under photoautotrophic conditions or with the addition of acetate as the source of carbon. nuclear transformants usually give rather low yield of protein product; therefore, recombinant protein production by this alga is based on the transformation of chloro no. 1 2015 plast, which occupies about 40% of the cell volume [46] . c. reinhardtii nuclear and chloroplast genomes are sequenced, and this simplifies substantially any genetic engineering manipulations. known examples of protein antigen production in chloroplasts of c. reinhardtii are b subunit of cholera toxin fused with the coat protein of foot and mouth disease virus [47] or with d2 fibronectin binding domain of staphylococ cus aureus [48] , as well as protein 28 virus cryptokary osis (shrimp disease) [49] and e2 protein of swine fever [50] . the green moss physcomitrella patens is the only representative of bryophytes, the genome of which is currently completely sequenced and approaches to its transformation are developed. the peculiarity of this moss is that at the stage of the haploid juvenile game tophyte (protonema) this moss is morphologically similar to filamentous algae and easily enough culti vated in a bioreactor [25, 42] . under certain culture conditions the moss can be in the stage of protonema indefinitely long. the attractiveness of this moss spe cies as the system for recombinant protein expression is that, as distinct from plants, fragments of foreign dna can be integrated in its genome through homo logical recombination, which reduces substantially a possibility of transferred gene inactivation. the p. pat ens cells are capable of postranslational modification of proteins of eukaryotic origin. since at the step of gametophyte the moss has the haploid number of chromosomes, it becomes possible to modify the func tioning of individual genes, in particular the moss lines conducting glycosylation of recombinant proteins as in mammalian cell type were obtained [25] . the firm grenovation (germany) is developing the technology of biopharmaceutical protein production on the basis of p. patens in bioreactors. expression system for pro ducing biopharmaceuticals based on the green moss is not the part of the food chain and is characterized by a high degree of biosafety. as distinct from above described systems based on the stable expression of foreign genes integrated into nuclear or chloroplast genomes, during transient expression target proteins are synthesized in the plant cell during relatively short time (several days) without insertion into the plant genome. at present, the fol lowing approaches are used for transient gene expres sion in plants: gene delivery with the help of agrobac terium, the use of plant virus vectors, and magnifec tion [51] [52] [53] . tobacco mosaic virus (tmv), potato x virus, alfalfa mosaic virus, and cowpea mosaic virus are used as virus vectors [52] . the availability of infec tious cdna clones, the small size of the viral genomes, the short time required for the expression of a target gene, and a high level of expression provides for a high attraction of this system. the rapid development of this expression system led to substantial modifications of the first gene inser tion vectors or full virus vectors, which is a recombi nant virus that behaves as wild type virus but is capable of expressing additional genes. the next step was the creation of "disarmed vectors" (deconstructed vec tors) lacking a number of original virus genes, and gene replacement vectors, in which a portion of the viral genes is replaced by alien genes [51] . viral vectors have several substantial disadvantages: a tendency to the loss of foreign insertion in the process of virus spreading over the plant and a potential risk for envi ronment related to the presence of infectious recombi nant viral particles. launch vectors represent an alternative to recom binant plant viruses; cdna of these viruses is deliv ered to plants within t dna region of agrobacterial ti plasmid. firstly, primary transcription of t dna occurs in the nucleus; then viral rna is released into the cytoplasm, where its further amplification, trans lation, and protein synthesis occur [52] . by 2005, the system of agroinfiltration based on the use of plant viruses and agrobacterial binary plasmids was upgraded and named as magnifection [54] . at magnifection, multiple agrobacterial lines carrying different parts of the tmv genome are used simulta neously. after agrobacterial transfer into the plant cell nucleus, separate parts of viral genome are assembled in plants in the completely functional viral replicon [51] . the substantial modification of the viral genome, including numerous point mutations for the removal of potential sites of splicing, intron insertion, and the removal of the gene encoding envelope proteins, pro vided for the highly efficient system capable of recom binant protein synthesis (up to 5 g/kg of fresh tissue), which is more than 50% of tsp [51] . among disadvantages of transient system is a necessity for recombinant protein isolation and purifi cation immediately after its accumulation in the plant, because, as distinct from seeds and fruits, plant leaves and stems cannot be stored for a long time. the systems of transient expression of recombinant proteins are rather promising in the cases when a rapid production of a small amount of proteins is required. experiments with transient expression in plants are held indoors, which reduces the risks associated with biosafety to almost zero. examples of manufacturing immunogenic proteins for veterinary using transient expression systems are presented in table 1 . the sys tems of transient expression for the production of recombinant proteins are described in more details in reviews [1, 41, 51, 52, 54] . "candidate" plant vaccines for veterinary use table 1 presents examples of using various expres sion systems for the production of "candidate" plant vaccines for veterinary use. main specific immunogenic no. 1 2015 proteins synthesized at respective diseases (structural proteins, hemagglutinins, glycoproteins) are usually used as antigens. the most commonly used method of transgene construct delivering into plant cells is still the agrobacterial transformation. in some cases, the level of target protein expression was rather high [55] [56] [57] [58] , espe cially in the systems of transient expression [59] [60] [61] , and suitable for product commercialization. in all experiments using the "candidate" plant vac cines, the formation of a specific immune response was demonstrated in vivo, and in most experiments immunogenic proteins were delivered to animals just orally. "candidate" plant vaccines were usually tested on mice, but in approximately a quarter works the ani mals subjected to the disease were tested. protein s of the transmissible swine gastroenteritis virus synthe sized in the cells of transgenic maize [55, 62, 63] or tobacco [64] and delivered into the body of pigs as a food supplement, provided for 100% survival of ani mals after infection [62] . rabbit protein vp60 virus synthesized in potato [65] and other plants (tobacco, pea, rape) [66] and delivered orally enhanced protective immunity: after infecting rabbits with this virus all ani mals survived [66] . the effect of plant recombinant antigen was comparable with commercially used vac cines. the use of plant vaccines for the vaccination of wild animals using edible baits (e.g., vaccine against rabies) will lead to an increase in the proportion of wild animal populations having immunity to the rabies virus. a potential possibility to reduce the cost of produc tion of biopharmaceutics using genetically modified plants served at the end of the xx century as an impe tus for more than twenty biotechnological companies to initiate commercial programs. as seen from the table 1 , many biological products of plant origin are developed, expressed in different types of plants and plant cell cultures. for a variety of reasons, including the still skeptical attitude of the human community to the biosafety of genetically modified plants, many of these works remained in the framework of laboratory tests. at the moment three companies function on the biotechnology market of veterinary preparations, two from the united states and one from canada (table 2 ). in the united states the dow agro sciences company presented a recombinant plant viral hn protein of the newcastle disease virus (approved by usda) and a mixture of antiviral vaccines at the first stage of clinical trials. the second american company at thomas jef ferson university has developed a plant anti rabies vaccine (completion of phase 1). the canadian guardian biosciences company presented plant vac cine against chicken coccidiosis at the second phase of clinical trials. production and wide distribution of biopharmaceu ticals is hampered by a number of circumstances. the first of them is related to the problem of biosafetythe cultivation of genetically modified plants in the field can lead to the accidental introduction of foreign genes into crops grown for human consumption. therefore, most companies producing biopharma ceutics focused on plant species, which are absent from the food chain of humans and animals and also on growing of genetically modified plants preventing their cross pollination with other crops. the second difficulty is related to the necessity of plant material treatment for the removal of various undesired com pounds, such as lignin, proteases, phenolic com pounds, and pigments, especially in the case of plant species, which are not consumed. all these facts result in the requirement of additional studies. the third cir cumstance is due to the fact that until now all aspects of maintaining and growing of plants producing biop harmaceuticals are not settled at the legislative level. ambiguity and vagueness of the existing legislation in this area lead to the fact that large biopharmaceutical and biotechnology companies do not tend to invest in the development of technological lines and research programs in this area, which significantly inhibits the development of the industry. a significant problem for the development of vac cines for veterinary use, especially those used in agri culture, is the need to minimize the price of the final product. the vaccine should be inexpensive for entre preneurs engaged in commercial animal breeding and fully subsidized, if you intend to use the vaccine for mass immunization and the prevention of the disease spread in underdeveloped regions. as a result, the potential income of manufacturers of vaccines for ani mals is much less than that for vaccines intended for humans. for example, in 2007, the volume of the mar ket for the vaccine against human papilloma virus was estimated at more than 1 billion dollars, but the mar ket for the most popular animal vaccines (against foot and mouth disease of cattle and against mycoplasma hyopneumoniae in pigs) together amounted to only 10-20% of this amount [3] . animal vaccines are cheaper and the volume of market for them is less; therefore, the investments in their development are substantially less as compared with investments in the production of vaccines for humans, whereas the com plexity and diversity of both hosts and pathogens in the case of vaccines for animals is much higher. expression systems based on the use of plant cells still have a limited application or are used primarily in some laboratories. nevertheless, biopharming (the biotechnological production of various substances for medicine) in plants has attracted the attention of researchers and manufacturers in developed countries. first biopharmaceuticals of plant origin, such as anti bodies (anti hbsag required to purify the hepatitis b vaccine), therapeutic and dietary proteins ("intrinsic factor" required at vitamin b12 deficiency, gastric lipase), have entered the market, that is an excellent illustration of this progress [67] . despite the fact that today the number of biophar maceutical proteins expressed in plant cells is enor mous, many questions still remain unresolved. the methods for target recombinant protein quantification and purification are still not developed for most of products. the problems of transgene silencing and increased expression of target protein genes are still at the stage of research. the important task that has yet to be solved is to achieve a stable level of expression in different batches of plant raw material. not much work appeared for judging about maintaining the sta bility of recombinant proteins after the harvest, pro cessing, and storage. all of these problems require additional expenses for further research. one of the main obstacles for the leading research groups working on the development and production of plant vaccines, given the financial constraints, is the fulfillment of the relevant official regulations govern ing the use of oral medications. to date, the purified vaccines and therapeutic proteins of plant origin must meet the same standards relating to the production, biosafety, purification, storage, dosage, etc. as any other recombinant proteins for medical purposes. nevertheless, despite these difficulties, there were first biopharmaceuticals of plant origin that passed all the necessary tests and were approved for use by the relevant authorities. some new products having spe cific advantages over similar products obtained in mammalian cell cultures were developed. such com panies as sembiosys genetics inc. (calgary, can ada), medicago inc. (quebec, canada), protalix bio therapeutics (karmiel, israel), and orf genetics (iceland) proved the possibility of quick establishing of the production of purified plant proteins, which are quite competitive in today's market. progress has been made in the formation of the legal framework related to the cultivation of transgenic plants, testing and use of plant biopharmaceuticals. several production pro cesses based on transgenic plants have already received a brand gmp (good manufacturing practice), the interest of manufacturers to this field of biotechnology began to increase again. this work was performed within the framework of the project vi.62.1.5 (no. 01201280334) development and improvement of genetic constructs to optimize the expression of target genes and the production of recombi nant proteins for medical purposes in transgenic plants and animals. plant produced vaccines: promise and reality evolution of plant made pharmaceu ticals current status of veterinary vac cines clinical trials fuel the promise of plant derived vaccines expression of hepatitis b surface antigen in transgenic plants oral immunization with a recombinant bacterial anti gen produced in transgenic plants edible vaccine protects mice against escher ichia coli heat labile enterotoxin (lt): potatoes express ing a synthetic lt b gene effi cacy of food plant based oral cholera toxin b subunit vaccine immunogenicity in humans of a recombinant bacte rial antigen delivered in a transgenic potato receptor mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chlo roplasts into the mouse circulatory system delivery of plant made vaccines and therapeutics defending the mucosa: adjuvant and carrier formula tions for mucosal immunity induction of secretory immunity and memory at mucosal surfaces, vaccine oral delivery of human biopharmaceuti cals, autoantigens and vaccine antigens bioencapsu lated in plant cells the mucosal immune response to plant derived vaccines posttranslational modifica tion of therapeutic proteins in plants seed based expression systems for plant molecular farming the economic potential of plant made pharmaceuticals in the manu facture of biologic pharmaceuticals rice based mucosal vac cine as a global strategy for cold chain and needle free vaccination secretory iga mediated protection against v. cholerae and heat labile enterotoxin producing enterotoxigenic escherichia coli by rice based vaccine stability of a soybean seed derived vaccine antigen following long term storage, processing and transport in the absence of a cold chain tomato is a highly effective vehicle for expression and oral immunization with norwalk virus capsid protein production of plant made pharma ceuticals: from plant host to functional protein post translational modifica tions in the context of therapeutic proteins current achievements in the production of complex biopharmaceuticals with moss bioreactors low dose oral immunization with lyophilized tissue of herbicide resistant lettuce expressing hepatitis b surface antigen for prototype plant derived vaccine tablet formulation rna mediated chromatin based silencing in plants transcriptional gene silencing in plants plant based production of biopharma ceuticals production of heterologous proteins in plants: strategies for optimal expression expression of heterologous genes in plant systems: new possibilities exhaustion of the chloroplast protein synthesis capac ity by massive expression of a highly stable protein anti biotic metabolic adaptation in transplastomic plants massively accumu lating recombinant proteins transplastomic plants overexpression of the bt cry2aa2 operon a ¸n´l in chloroplasts leads to formation of insecticidal crys tals chloroplast vector systems for biotechnology applications determining the transgene containment level provided by chloroplast transformation expression of the native cholera toxin b subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts chloroplast expression of his tagged gus fusions: a general strategy to over produce and purify foreign proteins using transplas tomic plants as bioreactors accumulation of trehalose within transgenic chloro plasts confers drought tolerance plants as bioreactors for the production of vaccine anti gens towards high yield production of pharmaceutical proteins with plant cell suspension cultures position effects and epigenetic silencing of plant transgenes bioreactor systems for in vitro production of foreign proteins using plant cell cultures high expression of transgene protein in spirodela micro algae come of age as a platform for recombinant protein production foot and mouth disease virus vp1 pro tein fused with cholera toxin b subunit expressed in chlamydomonas reinhardtii chloroplast heat stable oral alga based vaccine pro tects mice from staphylococcus aureus infection factors effecting expression of vaccines in microalgae recombination and expression of classical swine fever virus (csfv) structural protein e2 gene in chlamydomonas rein hardtii chroloplasts viral vec tors for the expression of proteins in plants agrobacterium mediated transient expression as an approach to production of recombi nant proteins in plants a novel two component tobacco mosaic virus based vector system for high level expression of multiple ther apeutic proteins including a human monoclonal anti body in plants magnifec tion -a new platform for expressing recombinant vac cines in plants plant based vaccines: unique advan tages expression of the newcastle disease virus fusion protein in transgenic maize and immunological studies multimerization of peptide antigens for pro duction of stable immunogens in transgenic plants generation and immunogenicity of japanese encepha litis virus envelope protein expressed in transgenic rice induction of protective immunity in swine by recombinant bamboo mosaic virus express ing foot and mouth disease virus epitopes in planta production of two peptides of the classical swine fever virus (csfv) e2 glycoprotein fused to the coat protein of potato virus x expression in plants and immunogenicity of plant virus based exper imental rabies vaccine delivery of subunit vaccines in maize seed a corn based deliv ery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immu nity in swine immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants oral immunization using tuber extracts from transgenic potato plants expressing rabbit hemorrhagic disease virus capsid protein pea derived vaccines demonstrate high immunoge nicity and protection in rabbits against rabbit haemor rhagic disease virus plant made pharmaceuti cals: leading products and production platforms pro duction of immunogenic vp6 protein of bovine group a rotavirus in transgenic potato plants rotavirus vp6 expressed by pvx vectors in nicotiana benthamiana coats pvx rods and also assembles into viruslike parti cles expression of rotavirus capsid protein vp6 in transgenic potato and its oral immuno genicity in mice protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants bovine herpes virus gd protein pro duced in plants using a recombinant tobacco mosaic virus (tmv) vector possesses authentic antigenicity induction of a protective antibody response to foot and mouth disease virus in mice fol lowing oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein vp1 induction of a protective antibody response to fmdv in mice following oral immunization with transgenic stylosanthes spp. as a feedstuff additive expression of hemagglutinin protein of rinderpest virus in transgenic tobacco and immunogenicity of plant derived protein in a mouse model systemic and oral immunogenicity of hemagglutinin protein of rinderpest virus expressed by transgenic peanut plants in a mouse model expression of hemagglutinin protein of rinderpest virus in trans genic pigeon pea oral immunogenicity of the plant derived spike protein from swine transmissible gastroenteritis coronavirus cloning and sequence analysis of the korean strain of spike gene of porcine epidemic diarrhea virus and expression of its neutraliz ing epitope in plants successful oral prime immunization with vp60 from rabbit haemorrhagic disease virus pro duced in transgenic plants using different fusion strate gies mucosal and sys temic immunization elicited by newcastle disease virus (ndv) transgenic plants as antigens expres sion of the fusion glycoprotein of newcastle disease virus in transgenic rice and its immunogenicity in mice expression of immunogenic s1 glycoprotein of infectious bronchitis virus in trans genic potatoes transient expression of the ectodomain of matrix protein 2 (m2e) of avian russian immunization with plant expressed hemagglutinin protects chickens from lethal highly pathogenic avian influenza virus h5n1 challenge infection immunogenicity study of plant made oral subunit vaccine against porcine reproductive and res piratory syndrome virus (prrsv) expression of the rabies virus glycoprotein in transgenic tomatoes immunization against rabies with plant derived antigen development of an edible rabies vaccine in maize using the vnukovo strain induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein expression of the rabies virus nucleoprotein in plants at high levels and evaluation of immune responses in mice expression of rabies virus g pro tein in carrots (daucus carota) key: cord-023770-ymxapsv6 authors: nan title: closteroviridae date: 2011-11-23 journal: virus taxonomy doi: 10.1016/b978-0-12-384684-6.00085-9 sha: doc_id: 23770 cord_uid: ymxapsv6 this chapter focuses on closteroviridae family whose member genuses are closterovirus, ampelovirus, and crinivirus. the virions are helically constructed filaments with a pitch of the primary helix in the range of 3.4–3.8 nm, containing about 10 protein subunits per turn of the helix and showing a central hole of 3–4 nm. the very flexuous and open structure of the particles is the most conspicuous trait of members of the family. the virions have a diameter of about 12 nm and their length ranges from 650 nm in case of species with fragmented genome, to over 2000 nm in case of species with monopartite genome. the fragility of virions and a tendency to end-to-end aggregation contribute to the fact that a range of lengths is often given for single viruses. the virions of several species are degraded by cscl and are unstable in high salt concentration, resist moderately high temperatures and organic solvents, but are sensitive to rnase and chelation. regardless of the genome type, monopartite or fragmented, virions contain a single molecule of linear, positive sense, single stranded rna, constituting 5–6% of the particle weight. the structural proteins of most members of the family consist of a major cp and of a diverged copy of it denoted minor cp (cpm), with a size ranging from 22 to 46 kda (cp) and 23 to 80 kda (cpm). the members of the family have one of the largest genomes among plant viruses because of sequence duplication and acquisition of nonviral coding sequences such as protease, and hsp70 protein via rna recombination. recombination. recombination may also explain differences in genome organization between genera and members of the same genus. genome organization, i.e. the number and relative position of the orfs differs between the genera and/or individual viral species. however, the complex orf1a-orf1b invariably encodes the replication-related proteins, with methyl-transferase (mtr), helicase (hel), and rna-dependent rna polymerase (rdrp) conserved domains. downstream orfs, which encode in 5→3 direction a 6k small hydrophobic protein, the hsp70h, the ~60 kda protein, the cp and cpm, form a five-gene module which is conserved, with few modifications, among most members of the family analysed so far. the hsp70h and the ~60 kda proteins are integral virion components present in all the sequenced members of the family. the functions postulated for hsp70h are: mediation of cell-to-cell movement through plasmodesmata, involvement in the assembly of multisubunit complexes for genome replication and/or subgenomic rnas synthesis, and assembly of virus particles. the ~60 kda protein is required for incorporation of both hsp70h and cpm to virion heads. the duplication of the capsid protein gene seems to be the only example of such condition among viruses with elongated particles. in general, capsid proteins and their homologs (cpm) show a significant degree of sequence conservation and the duplicate copies probably retain the general spatial folding and some crucial properties of the cps. notable exception are a group of ampeloviruses with the smallest genomes in the family [e.g. grapevine leafrollassociated virus 4 (glrav-4), glrav-5, glrav-6, glrav-9, pineapple mealybug wilt-associated virus 1(pmwav-1) and pmwav-3] which do not appear to possess cpm. the genome expression strategy is based on: (i) proteolytic processing of the polyprotein encoded by orf1a; (ii) 1 pos. ssrna ribosomal frameshift for the expression of the rdrp domain encoded by orf1b, a mechanism not found in other ()rna plant viruses; (iii) expression of the downstream orfs via the formation of a nested set of 3 co-terminal sub-genomic rnas (sgrnas). the dsrna patterns are very complex and variable among species, reflecting the different numbers and sizes of the orfs present in individual genomes and, in some cases, the existence of defective rnas. replication occurs in the cytoplasm, possibly in association with endoplasmic reticulum-derived membranous vesicles and vesiculated mitochondria. from an evolutionary point of view, closteroviruses represent a monophyletic virus lineage that might have evolved from a smaller filamentous virus when higher plants have differentiated. this progenitor virus, thought to be composed of three genes encoding replication-associated proteins, a protein (p6) with affinity for cell membranes, and a single coat protein, acquired the hsp70h and a ~60 kda protein derived from a fusion of two domains, n-terminal domain of unknown evolutionary provenance, and a duplicated capsid protein domain. under the pressure of further modular evolutionary events, i.e. duplication of the coat protein gene, acquisition of diverse suppressors of rna silencing and of additional genes acquired via horizontal gene transfer (e.g. papain-like cysteine proteinase and alkb domains), this family ancestor gave rise to the progenitors of the three extant genera of the family. one of these genera (crinivirus), differentiated further by splitting its genome in two or three genome components. virion proteins are moderately antigenic. most virus species within genera are serologically unrelated or distantly related to one another. no intergeneric serological relationship has been detected. the natural and experimental host ranges of individual virus species are usually restricted, except for a few members of the genus crinivirus. disease symptoms are of the yellowing type (i.e. stunting, rolling, yellowing or reddening of the leaves, small and late ripening fruits), or pitting and/ or grooving of the woody cylinder of woody hosts. infection is systemic, but usually limited to transmission is semi-persistent regardless of the type of vector. geographical distribution varies from restricted to widespread, depending on the virus species, most of which occur in temperate or subtropical regions. virions are usually found in the phloem (sieve tubes, companion cells, phloem parenchyma), occasionally in the mesophyll and epidermis. ultrastructural modifications arise by membrane proliferation, degeneration and vesiculation of mitochondria, and formation of inclusion bodies. these are made up of aggregates of virions or membranous vesicles, or a combination of the two. virions accumulate in conspicuous cross-banded fibrous masses or, more typically, in more or less loose bundles intermingled with single or clustered membranous vesicles. inclusions of this type are one of the hallmarks of the family. the vesicles contain a fibrillar network and derive either from the endoplasmic reticulum, or from peripheral vesiculation of mitochondria. traits that largely characterize the family and that are the basis of the current classification are: l natural transmission by aphids, mealybugs or whiteflies in a semi-persistent manner; experimental transmission by mechanical inoculation very difficult or not possible. see table 1 . the genus comprises species with particle length above 1200 nm, and monopartite rna genome, 14.5-19.3 kb in size, in which cpm is located upstream of the cp gene. natural transmission by aphids. particle morphology largely conforms to that of other members of the family. virions are of one size, ranging from 1350 to 2000 nm in length. ctv has also smaller than full-length particles that may encapsidate subgenomic or multiple species of defective rnas (d-rna) containing all of the cis-acting sequences required for replication. sgrnas may be involved in the construction of recombinant d-rnas. according to the species, infectivity is inactivated at temperatures between 40 and 55 °c, is retained for 1 to 4 days at room temperature, up to 1 year in frozen sap, 2 years in dried leaf material, 5 years in lyophilized preparations stored at 20 °c, and is destroyed at ph lower than 6. a 260 /a 280 ratio is around 1.20 but some members [byv, carnation necrotic fleck virus (cnfv), burdock yellows virus (buyv)] lack tryptophan, which results in a higher ratio (1.4-1.8) for the virions. s 20,w ranges from 130 (byv) to 140 (ctv), buoyant density is 1.33 g cm 3 in cscl (byv and ctv) and 1.257 g cm 3 in cs 2 so 4 (ctv). virions contain a single molecule of linear, positive sense, single stranded rna from 14.5 to 19.3 kb in size. multiple double stranded rna (dsrna) species occur in infected tissues, the largest of which is usually the replicative form of the entire genome. sgrnas generate a range of smaller dsr-nas. with ctv, the presence of d-rna makes the dsrna pattern of virus isolates more complex than that of other members of the genus. sequenced members of the genus closterovirus show three types of genome organization exemplified by byv (figure 2 ), ctv ( figure 3 ) and bysv: (i) byv contains eight orfs flanked by 5 and 3 utrs of 107 and 181 nt, respectively; (ii) ctv has 12 orfs and utrs of 107 nt at the 5 end and 275 nt at the 3 end. it differs from the byv genome in having two papain-like protease domains in orf1a, an extra 5 proximal orf (orf2) encoding a 33 kda product with no similarity to any other protein in databases, and two extra 3 proximal orfs (orf9 and orf11); (iii) bysv has 10 orfs and a 3 utr 241 nt in size, a length intermediate between that of the bysv and ctv utrs. a further difference with the byv genome rests in the presence of an extra orf (orf2) encoding a 30 kda polypeptide with no similarity to any other protein in databases. this orf is located downstream of orf1b, i.e. in the same position as the unrelated ctv orf2. thus, the organization of bysv genomes is intermediate between that of byv and ctv, suggesting that these three viruses might represent three distinct stages in closterovirus evolution. non-structural proteins common to all members of the genus are: (i) a large polypeptide (over 300 kda) containing the conserved domains of papain-like protease (p-pro), methyltransferase (mtr), and helicase (hel); (ii) a ~50 kda protein with all sequence motifs of viral rdrp of the "alpha-like" supergroup of positivestrand rna viruses; (iii) a 6 kda hydrophobic protein with membrane-binding properties; (iv) the homolog of the cellular hsp70 heat-shock proteins (hsp70h); (v) a 55-64 kda product, referred to as the ~60 kda protein. some of the structural and non structural proteins function as suppressors of the rna silencing plant defence machinery. for instance, cp, p20 and p23 proteins of ctv have suppressor activity, much the same as the homologs of p21 of bysv, byv, and glrav-2. ctv p23 is a unique protein in the family and has a nucleolar localization. silencing suppressors contribute to the accumulation of virus particles and are important determinants of pathogenesis. no serological relationships reported among different virus species of the genus. monoclonal antibodies have been produced to byv, ctv and glrav-2 and polyclonal antisera have been raised to byv, ctv and cylv from fusion proteins obtained in bacterial expression systems. polyclonal antisera have been raised to normal capsid proteins of byv, bysv, glrav-2 and buyv. most of the members of the genus infect herbaceous hosts (weeds, vegetable and flower crops) or shrubs ( the criteria demarcating species in the genus are: the genus comprises species with particles 1400-2000 nm long, monopartite genome 13.0-18.5 kb in size, transmitted by pseudococcid mealybugs and soft scale insects. particle morphology largely conforms to that of other members of the family. information is very limited, except for the size of cp and cpm, as deduced from sequence data. virions contain a single molecule of linear, positive sense, single stranded rna from 13.0 to 18.5 kb in size. multiple double stranded rna (dsrna) species occur in infected tissues, the largest of which is usually the replicative form of the entire genome. smaller dsrna are thought to be replicative forms of subgenomic rnas. the genus ampelovirus shows a wide variation in genome size and organization. at one extreme there are grapevine leafroll-associated virus 1 (glrav-1) and glrav-3, which has the largest genome of all (18,498 nt). glrav-3 has 12 orfs, coding for the replication related proteins (orfs 1a and 1b), two small hydrophobic proteins (6 kda), the hsp70h, the ~60-kda protein, cp, cpm and five additional proteins 21, 20, 20, 4 and 7 kda in size, respectively (figure 4) . the 5' utr and 3' utr are 737 and 277 nt in size, respectively. glrav-1 differs from all other members of the genus in encoding two copies of the cpm. at the other extreme there is a group of viruses infecting grapevine [e.g, grapevine leafroll-associated viruses 5 and 9 (glrav-5 and -9)] and pineapple [e.g. pineapple mealybug wilt-associated viruses 1 and 3 (pmwav-1 and -3)]. all these viruses have a genome made up of seven orfs and lack the cpm. pmwav-1, a representative of this group, has a genome 13,071 nt in size, beginning with a 535 nt utr at the 5 end, followed by the orfs expressing, respectively, the replication related proteins, a 6 kda hydrophobic protein, the hsp70h, the ~60 kda protein, the cp and a 24 kda protein. a utr 132 nt in size terminates the genome. replication occurs in the cytoplasm, likely in association with membranous vesicles, derived either from the endoplasmic reticulum or from peripheral vesiculation and disruption of mitochondria (glrav-1, glrav-3). structural and non-structural proteins are similar in type and function to those reported for the genus closterovirus. polyclonal antisera and monoclonal antibodies have been raised to most of the members of the genus. a recombinant single-chain variable fragment antibody was synthesized to glrav-3. glrav-1 and glrav-3 are distantly serologically related based on cross-reactivity to a monoclonal antibody to glrav-1. glrav-4, -5, -6 and -9 show serological interrelations when tested with polyclonal antisera or monoclonal antibodies. grapevine leafroll-associated virus 7 (glrav-7), an unassigned member of the family, is also distantly related to the four above species. the three pineapple mealybug wilt-associated viruses are serologically unrelated to one another. the majority of extant ampelovirus species are recorded from woody hosts (grapevine, plum, fig) and pineapple. according to the host, they induce rolling yellowing and reddening of the leaves (grapevine), stem pitting (plum), wilting or symptomless infections (pineapple). natural vectors are mealybugs which transmit with a semipersistent modality. the range of vectors varies for individual viruses from rather wide to restricted. for instance, glrav-1 is transmitted by species of several genera of pseudococcid mealybugs (heliococcus, phenacoccus, pseudococcus) and soft scale insects (pulvinaria, neopulvinaria and parthenolecanium); glrav-3 by pseudococcid mealybugs (planococcus, pseudococcus, heliococcus, phenacoccus) and soft scale insects (pulvinaria, neopulvinaria, parthenolecnium, coccus, saissetia, parasaissetia and ceroplastes), whereas glrav-5 is transmitted by pseudoccocus, planococcus and ceroplastes spp. vectors of pineapple ampeloviruses are two species of the genus dysmicoccus, and lchv-2 is transmitted by phenacoccus aceris. none of the viruses is transmitted through seed or mechanically. all persist in plant parts used for propagation and are disseminated with them over long distances. geographical distribution is very wide. the criteria demarcating species in the genus are: l particle size. l size of cp, as determined by deduced amino acid sequence data. pro mtr hel figure 4 : genome organization of grapevine leafroll-associated virus 3, showing the relative position of the orfs and their expression products. pro, papain-like protease; mtr, methytransferase; hel, helicase; rdrp, rna polymerase; hsp70h; ~60 kda protein; cpm, minor capsid protein; cp, capsid protein. l serological specificity using discriminatory monoclonal or polyclonal antibodies. l genome structure and organization (number and relative location and size of the orfs). l amino acid sequence of relevant gene products (polymerase, cp, hsp70h) differing by more than 25%. l vector species and specificity. l magnitude and specificity of natural and experimental host range. l cytopathological features (i.e., aspect of inclusion bodies and origin of cytoplasmic vesicles). genus crinivirus the genus comprises species transmitted by whiteflies. virions usually have two modal lengths (650-850 and 700-900 nm) and a bipartite genome, but potato yellow vein virus (pyvv) has a tripartite genome. particle morphology largely conforms to that of other members of the family. information is very limited, except for the size of cp and cpm, as deduced from sequence data. virions contain a single molecule of linear, positive sense, single stranded rna with size ranging from 7801 to 9127 nt (rna-1) and from 7903 to 8530 nt (rna-2) in species with bipartite genome. the rna size of pyvv, the only species with a tripartite genome, is 8035 nt (rna-1), 5339 nt (rna-2) and 3892 nt (rna-3). the genome of most criniviruses (e.g. liyv) is divided between two linear, positive sense, single stranded rnas totalling 15.6-17.9 kb in size ( figure 5 ), but pyvv possesses a tripartite genome. all molecules are needed for infectivity and are separately encapsidated. rna-1 of liyv contains three orfs, i.e. the orf1a-orf1b complex plus a 3-most orf coding for a 32 kda protein with no similarity to any protein in databases. this orf is similar in size and location to orf2 of ctv and bysv but the respective expression products are not related. rna-1 has 5 and 3 utrs of 97 and 219 nucleotides, respectively. as with other members of the family, the orf1a-orf1b complex codes for the replication-related proteins including the rna-dependent rna polymerase (rdrp). rna-2 has seven orfs flanked by a 5 utr of 326 nt and a 3 utr of 187 nt. rna2 contains the five-gene module which, however, differs from that of members of the genus closterovirus by the insertion of an extra gene (orf4) upstream of the cp gene. as to pyvv: (i) rna-1 (8035 nt in size) is composed of three orfs, i.e. the orf1a-orf1b complex and a 7 kda hydrophobic protein containing a potential transmembrane helix; (ii) rna-2 (5,339 nt in size) comprising five predicted orfs that encode, in the order, the hsp70h; a 7 kda protein similar to a comparable protein of cucurbit yellow stunting disorder virus (cysdv); the ~60 kda protein; a 9.8 kda product with no significant similarity to any other sequence in database; the 28.2 kda putative cp; (iii) rna-3 (3892 nt) has three potential orfs coding for a protein 4 kda in size with no counterpart with other proteins in the family and no significant sequence homology in database; the 77.5 kda cpm, and a 26.4 kda protein present in other members of the genus. in all criniviruses, the order of the cp and cpm orfs is reversed compared to that of species in the genus closterovirus. sweet potato chlorotic stunt virus (spcsv) and tomato chlorosis virus (tocv) have a particularly large cpm (75-80 kda), compared to liyv (53 kda). replication occurs in the cytoplasm, likely in association with membranous vesicles, derived from the endoplasmic reticulum or from vesiculated mitochondria (cysdv). structural and non-structural proteins are similar in type and function to those reported for the genus closterovirus. both genomic rnas of tocv encode rna silencing suppressors, e.g. the p22 protein in rna-1 and cp and cpm in rna-2. the p25 protein of cysdv, the viral rnase iii and the p22 gene present in a few isolates of spcsv also have suppressor activity. monoclonal antibodies have been produced to proteins of spcsv. antisera have been raised from structural and non structural proteins produced as fusion proteins in bacterial expression systems (spcsv and liyv) phylogenetic relationships within the family are depicted in figure 6 . virions of some of the genera of the families alphaflexiviridae (allexivirus) and betaflexiviridae (capillovirus, trichovirus, vitivirus, citrivirus and foveavirus) have the same particle morphology as those of the family closteroviridae. however, the sequence of the cp of members of this family has little similarity with that of cps of viruses in the above genera, and major differences exist in genome size and organization, and in strategy of expression. replication-associated proteins (rdrp, mtr and hel) contain signature sequences homologous to those of other taxa of the "alphalike" supergroup of ssrna viruses, the closest affinity being with the families bromoviridae and virgaviridae. the replication strategy, based on polyprotein processing, translational frameshifting and multiple sgrna generation, closely resembles that of viruses in the families coronaviridae and picorna-like" supergroup of polymerases. hence, the transcriptional strategy of members of the family closteroviridae follows the mechanism of other "alpha-like" viruses, and is dissimilar from the discontinuous, leader-primed transcription of coronaviruses and arteriviruses. derivation of names ampelo: from greek ampelos, "grapevine principles of molecular organization, expression and evolution of closteroviruses: over the barriers closterovirus group". cmi/aab description of plant viruses molecular biology and evolution of closteroviruses: sophisticated build up of large rna genomes comparative and functional genomics of closteroviruses genetic diversity and evolution of closteroviruses glrav-3 glrav-5 glrav-car pos. ssrna molecular biology of the citrus tristeza virus closteroviruses the family closteroviridae revised citrus tristeza virus: a pathogen that changed the course of the citrus industry ecology and epidemiology of whitefly-transmitted closteroviruses criniviruses infect primarily herbaceous hosts, in which they induce extensive chlorosis to yellow discoloration of the leaves, often accompanied by stunting. they are transmitted semi-persistently by whiteflies of the genera trialeurodes and bemisia. persistence and specificity of transmission by their respective vectors have been used as characters for species differentiation. thus, the viruses of group 1 [pyvv, blackberry yellow vein-associated virus (byvav), beet pseudoyellows virus (bpyv) and strawberry pallidosis-associated virus (spav)] are transmitted by t. vaporariorum, viruses of group 2 [tocv, spcsv, cysdv and bean yellow disorder virus (bydv)] by b. tabaci, whereas one of the viruses of group 3 is transmitted by b. tabaci (liyv) and the other by t. vaporariorum (ticv). these groups were identified by comparative phylogenetic analyses of rdrp amino acid sequences. none of the viruses is transmitted through seed or mechanically. geographical distribution varies from restricted (e.g. byvav) to very wide. some emerging viruses (e.g. cysdv, ticv and tocv) are being increasingly recorded from a number of european, american and asiatic countries. the membranous vesicles with a fibrillar content derive from the endoplasmic reticulum and/or from vesiculated mitochondria (cysdv). the criteria demarcating species in the genus are: martelli, g.p., agranovsky, a.a., bar-joseph, m., boscia, d., candresse, t., coutts, r.h.a., dolja, v.v., hu, j.s., jelkmann, w., karasev, a.v., martin, r.r., minafra, a., namba, s. and vetten, h.j. key: cord-017326-1caeui30 authors: seay, montrell; dinesh-kumar, savithramma; levine, beth title: digesting oneself and digesting microbes: autophagy as a host response to viral infection date: 2005 journal: modulation of host gene expression and innate immunity by viruses doi: 10.1007/1-4020-3242-0_11 sha: doc_id: 17326 cord_uid: 1caeui30 although research in this area is still in a stage of infancy, it seems likely that the lysosomal degradation pathway of autophagy plays an evolutionarily conserved role in antiviral immunity. the interferon-inducible, antiviral pkr signaling pathway positively regulates autophagy, and both mammalian and plant autophagy genes restrict viral replication and protect against virus-induced cell death. given this role of autophagy in innate immunity, it is not surprising that viruses have evolved numerous strategies to inhibit host autophagy. different viral gene products can either modulate autophagy regulatory signals or directly interact with components of the autophagy execution machinery. moreover, certain rna viruses have managed to “co-apt” the autophagy pathway, selectively utilizing certain components of the dynamic membrane rearrangement system to promote their own replication inside the host cytoplasm. in addition to this newly emerging role of autophagy in innate immunity, autophagy plays an important role in many other fundamental biological processes, including tissue homeostasis, differentiation and development, cell growth control, and the prevention of aging. accordingly, the inhibition of host autophagy by viral gene products has important implications not only for understanding mechanisms of immune evasion, but also for understanding novel mechanisms of viral pathogenesis. it will be interesting to dissect the role of viral inhibition of autophagy in acute, persistent, and latent viral replication, as well as in the pathogenesis of cancer and other medical diseases. the cellular pathway of autophagy is as ancient as the origins of eukaryotic life. derived from the greek and meaning to eat ("phagy") oneself ("auto"), the term autophagy refers to a lysosomal pathway of selfdigestion, involving dynamic membrane rearrangement to sequester cargo for delivery to the lysosome, where the sequestered material is degraded and recycled. for decades, it has been known that autophagy is the primary intracellular catabolic mechanism for the degradation and recycling of longlived cellular proteins and organelles. for decades, it has also been known that the recycling function of autophagy is an important adaptive response to nutrient deprivation and other forms of environmental stress. however, only recently have we discovered that autophagy may also be an important mechanism for the degradation of intracellular pathogens and that autophagy may also be important in cellular protection against the stress of microbial infection. not surprisingly, we have also recently learned that some successful intracellular pathogens have devised strategies either to block host autophagy or to subvert the host autophagic process to foster their own replication. in this chapter, we will review recent progress in understanding the interrelationships between viruses, autophagy, and innate immunity (figures 1 & 2) . before discussing the interrelationships between viruses, autophagy, and innate immunity, we will provide a brief overview of the molecular and cell biology of autophagy. while this subject has been covered extensively in a recent book and numerous recent review articles 2-5 we will highlight the aspects of this subject that may have particular relevance for viral infections. r r the process of autophagy was first described more than forty years ago, however, for many decades our understanding of autophagy was based largely on morphological observations from electron microscopy (reviewed in 6 ). the field has expanded considerably within the last 15 years after the cloning and molecular characterization of the yeast autophaggy (atg)related genes (reviewed in 7 ) . the analysis of sequenced genomes of higher eukaryotes has identified atg homologues in mammals, c. elegans, drosophila, dictyostelium, and plants, and many of these genes have been shown to be essential for autophagy function in higher eukaryotes (reviewed in 2 ) ( table 1 ). in addition to the identification of the autophagy genes, significant progress has been made in the past decade in understanding some of the signaling events that regulate autophagy (reviewed in 8, 9 ) . interestingly, some of the signaling molecules that regulate autophagy, as well as some of the autophagy genes, play a role in the host antiviral innate immune response (table 1, figure 2 ). the initial step of autophagy is the formation and elongation of the isolation membrane. the isolation membrane invaginates and sequesters cytoplasmic constituents including mitochondria, endoplasmic reticulum (er) and ribosomes, and the edges of the membrane fuse with each other to form a double-membrane structure called an autophagosome. the outer membrane of the autophagosome fuses to the lysosome/vacuole with subsequent delivery of the inner vesicle or autophagic body into the lumen of the degradative compartment. the source of the autophagosomal membrane is still unclear, but presently, it is thought that the preautophagosomal structure (pas) acts as the site of vesicle formation during autophagy [10] [11] [12] . the pas is thought to form de novo, but the source of the vesicle membrane is not known. it seems likely that the "typical" autophagosomes observed during viral infection that contain a mix of virions and self-cytoplasmic constituents, originate from the pas. however, it is not yet known whether the pas also serves as the site of vesicle formation for the formation of "atypical" autophagic-like double-membrane vesicles that function as replication sites for certain rna viruses (e.g. poliovirus, mouse hepatitis virus, equine arterivirus) (reviewed in 13 ). more likely, these double-membrane vesicles arise directly from the endoplasmic reticulum (er) 13 . autophagosomes are lipid-rich, protein-poor vesicles that vary in size and membrane thickness depending on the organism and cell type. the composition and abundance of proteins sequestered within autophagosomes reflects the relative composition and abundance of proteins in the surrounding cytoplasm 14 . this observation has led to the concept that autophagosomes indiscriminately sequester cytoplasmic content. however, in yeast, there are well-established pathways of specific autophagy, including the biosynthetic cytoplasm-to-vacuole targeting pathway and pexophagy (reviewed in 15 ), and in mammalian cells, mitochondria-specific autophagy has been reported 16 . although molecular determinants of cargo recognition have been identified in yeast pathways of specific autophagy, virtually nothing is known about the specificity of cargo recognition in higher eukaryotes. in circumstances where there is degradation of viruses observed inside "typical" autophagosomes that also contain cellular constituents, the sequestration step may lack specificity. however, in circumstances where viruses utilize components of the autophagic machinery for the formation of "autophagic-like" double-membrane structures that exclusively contain viral constituents, the sequestration step is likely to have exquisite specificity. the identification of the viral and cellular determinants of this specificity will be an important advance in understanding the cell biology of these types of rna virus infections and may eventually lead to the identification of novel antiviral therapeutic targets. autophagy is tightly regulated by nutritional, hormonal, and other environmental cues. it occurs as a cellular response to extracellular stimuli (e.g. nutrient starvation, hypoxia, overcrowding, high temperature, hormonal or chemotherapeutic treatment), and intracellular stimuli (e.g. accumulation of damaged, superfluous or unwanted organelles, accumulation of misfolded proteins, invasion of microorganisms). although it is not yet known whether different stimuli act through parallel, convergent, or divergent pathways to trigger autophagy, significant progress has been made within the past decade in identifying different signaling molecules that function in the positive (e.g. eif2 kinases, class iii pi-3 kinases, pten, death-associated protein kinases) or negative (e.g. tor, insulin-like growth factor signals, class i pi-3 kinase, rho/ras family of gtpases) regulation of autophagy (reviewed in 8, 9 ) . the identification of a role for these signaling molecules in autophagy regulation has implications for understanding antiviral immunity, and more speculatively, generates hypotheses about novel principles of virus-host interactions. the recently defined evolutionarily conserved role of the eif2 kinase signaling pathway in autophagy induction suggests that autophagy regulation may contribute to the antiviral function of the interferon-inducible eif2 kinase, pkr. pkr and other eif2 kinases induce a general translational arrest by phosphorylating the serine 51 residue of eif2 (reviewed in 17 ) . genetic studies in yeast and mammalian cells have also shown that the eif2 kinase signaling pathway is required for starvation and herpes simplex virus-induced autophagy 18 . while further analyses are required to dissect the relative contributions of autophagy induction vs. translational arrest in mediating the antiviral effects of pkr, these findings link a new cellular function (i.e. autophagy) with interferon signaling. although the eif2 kinase signaling pathway is the only as-of-yet defined autophagy regulatory signaling pathway that has known antiviral functions, it is interesting to note that most autophagy regulatory signals play a role in other important cellular processes, including cell growth control, cell death, and aging. some of these effects may be the consequence of divergent downstream targets of these regulatory signals and some of these effects may be directly mediated through autophagy. as will be discussed below, given the evidence for a role of autophagy in innate immunity, it is likely that viruses have evolved different strategies to antagonize host f f autophagy, which, at least in the case of herpes simplex virus (see sections 4.1 and 5) include the targeting of upstream autophagy regulatory signals f 18 . therefore, it is tempting to speculate that some of the effects of viruses on cell growth control and cell death may be either direct or indirect consequences of the evolutionary pressure that viruses face to modulate host autophagy. as one example, the insulin-like/class i pi-3k/akt signaling pathway inhibits autophagy 19, 20 , promotes oncogenesis (reviewed in 21 ) , and decreases lifespan (most likely through autophagy-inhibitory effects) 20 . certain retroviruses have recruited the catalytic subunit of pi3-k and its downstream target akt and these viral gene products function as oncoproteins (reviewed in 22 ) . the emerging link between these signaling molecules and autophagy inhibition raises the interesting hypothesis that the initial acquisition of these molecules by viruses was perhaps related to the selective advantage of autophagy inhibition in viral growth. in view of recent evidence supporting a role of autophagy in tumor suppression [23] [24] [25] [26] , the presence of these genes in retroviral genomes could contribute to oncogenesis at least, in part, through inhibition of autophagy signaling, as well as through modulation of other downstream pathways. the atg genes encode proteins important for responding to upstream signaling pathways as well as proteins needed for the generation, maturation, and recycling of autophagosomes (reviewed in 4, 5, 15, 27 ). the atg proteins can be grouped into four functional groups, including a protein kinase cascade important for responding to upstream signals, a lipid kinase signaling complex important for vesicle nucleation, ubiquitin-like conjugation pathways important for vesicle expansion, and a recycling pathway important for the disassembly of atg protein complexes from matured autophagosomes. the role of some of the atg proteins, including ones that act in the lipid kinase signaling complex and in the ubiquitin-like conjugation pathways, has been studied in plant and mammalian viral infections (see table 1 ). autophagy is a dynamic process that is tightly regulated by protein kinases and phosphatases. one of the first atg genes identified in yeast, atg1, encodes a serine/threonine kinase 28 . the atg1 kinase maintains a weak interaction with a hyperphosphorylated atg protein, atg13, in nutrientrich conditions. upon starvation conditions or stress, atg13 is dephosphorylated resulting in a tighter association with atg1 29 . atg13 binding is essential for autophagy since atg13 mutants unable to bind to atg1 are completely defective in autophagy 29 . atg17, which is also thought to play a role in atg1 activation, also interacts with atg1 29 . downstream targets of atg1 have not been identified, although atg1 interacts with other proteins independently of its kinase activity 30 . the upstream kinase, tor (target of rapamycin), indirectly or directly results in atg13 hyperphosphorylation, which is one presumptive mechanism by which tor kinase inhibits autophagy. of note, the atg1 component of the yeast autophagy induction complex plays a conserved role in autophagy in higher eukaryotes. however, as-of-yet, the role of atg1 in antiviral immunity has not been evaluated. lipid kinase signaling vps34, which encodes a phosphatidylinositol-3 kinase (pi3-k), d d phosphorylates the 3' hydroxyl group inosotiol ring of phosphoinositides u u 31 . although there is only one pi3-k in yeast, there are three classes of pi3-k in higher eukaryotes; class iii pi3-k has been shown to be analogous to yeast vps34. the importance of class iii pi3-k signaling in autophagy has been demonstrated pharmacologically and genetically. the nucleotide derivative, 3-methyladenine, inhibits class iii pi3-k activity and is widely used to inhibit autophagosome formation in mammalian cells 32, 33 . a null mutation in vps34 causes defects in autophagosome formation in yeast 34, 35 and microinjection of an inhibitory antivps34 antibody blocks autophagy in cultured mammalian cells 36 . vps34 functions through the association with other atg proteins in a large complex that includes vps15, atg6/vps30 and atg14 35 . this complex is thought to be important in vesicle nucleation by mediating the localization of other atg proteins at the pas 10, 35 . vps34 and atg6/vps30 are conserved in higher eukaryotes. importantly, the mammalian (beclin 1) and plant (beclin 1 ( ( ) homologues of yeast atg6/vps30 have been the most extensively studied atg genes in viral infections. as will be discussed in more detail below, mammalian beclin 1 restricts viral replication, protects against virus-induced cell death, and is a target of inhibition by different virally-encoded gene products [37] [38] [39] . furthermore, both plant beclin 1 and its binding partner, class iii pi3-k/vps34 prevent the spread of programmed cell death during the plant antiviral hypersensitive response 40 . thus, the lipid kinase complex plays an evolutionarily conserved role in antiviral innate immunity. autophagic vesicle expansion and completion involves conjugation machinery analogous to the ubiquitin conjugation needed for proteasomemediated protein degradation. autophagy utilizes an e1-like enzyme (atg7), two e2-like enzymes (atg10 and atg3) that facilitate the conjugation, and activation and localization of different ubiquitin-like modifiers (atg5 and t atg8). the conjugation modification of atg proteins is necessary for the formation of an autophagosome of appropriate size and shape 41 . however, the precise molecular functions of the conjugation reactions are not known and remain a critical unanswered question in autophagy research. the first conjugation system involves the lipidation of atg8, a ubiquitinlike protein whose close mammalian homologues have three-dimensional structures very similar to ubiqutin [42] [43] [44] [45] . both atg8 and the mammalian homomlogue lc3 are cleaved post-translationally by the cysteine endopeptidase atg4 46, 47 . the cleavage of atg8/lc3 is essential for conjugation and further maturation of the autophagosomes 48 . in yeast and mammalian systems, the cleaved atg8/lc3 is immediately activated by atg7, an e1-like enzyme; transferred to atg3, an e2-like enzyme; and finally conjugated to the lipid molecule phosphatidylethanolamine (pe) 42, 47, 49, 50 . the second ubiquitin-like reaction is the conjugation of atg12 to atg5. atg12 is an ubiquitin-like protein that is activated by atg7 (e1-like enzyme), transferred to atg10 (e2-like enzyme), and subsequently conjugated to atg5 through an isopeptide bond 41, 49 . the conjugation of atg5 to atg12 is necessary for autophagosome formation but not necessary for localization to the pas 10 . almost all of the components of the autophagy machinery that participate in the protein conjugation systems have orthologs in at least some higher eukaryotes. however, only mammalian atg5 has been studied in the context of its role in viral infections. the contrasting phenotypes of atg5 null cells infected with two different rna viruses illustrate two distinct mechanisms by which viruses interact with the autophagic machinery. the murine coronavirus, mouse hepatitis virus, which replicates in association with double-membrane vesicles, has severely impaired growth in atg5 null embryonic stem (es) cells 51 , suggesting that atg5 is required for the formation of coronavirus replication complexes. in contrast, the prototype alphavirus, sindbis virus, replicates to higher titers in atg5 null murine embryonic fibroblasts (mefs) than in wildtype controls 38 , suggesting that the autophagic machinery functions to restrict sindbis virus replication. in yeast, atg proteins that act at the stage of vesicle formation are not associated with the completed autophagosome, with the exception of atg8. this suggests that atg proteins are retrieved at some point prior to, or upon, vesicle completion, and then reutilized in the generation of new autophagosomes. the process of recycling requires the action of atg2 and atg18, which allow the recycling of atg9, the only transmembrane protein that is part of the autophagic machinery 52, 53 . atg9 and atg18 have orthologues in higher eukaryotes, but their function in antiviral responses has not been studied. the unique association of atg8/lc3 with the mature autophagosome has led to an important technical advance in autophagy research. atg8/lc3 is presently the most widely used and reliable marker for labeling autophagosomes 10, 11, 20, 50, 54 and with the recent availability of transgenic mice that express gfp-tagged lc3 54 , it is now possible to study autophagy induction in vivo during viral infections. in addition to its emerging role in innate immunity, autophagy plays a role in diverse other biological processes, including survival during starvation, differentiation and development, tissue homoeostasis, aging, cell growth control, and certain forms of programmed cell death. these biological functions of autophagy have been reviewed in detail elsewhere 2, 55, 56 . in this section, we will however, briefly discuss selected biological functions of autophagy that have relevance either to understanding the mechanisms by which autophagy protects cells against virus infections ( figure 1 ) or to understanding the potential consequences for the host of viral evasion of autophagy ( figure 3 ). perhaps the primordial function of autophagy is its ability to recycle nutrients and help sustain life during periods of starvation. several decades ago, starvation was noted to be a potent inducer of autophagy in rodent liver (reviewed in 57 ), leading to the hypothesis that autophagy is an adaptive response to starvation. following the identification of the conserved autophagy genes, genetic studies in different species have confirmed that autophagy genes are required for the maintenance of eukaryotic life in the face of limited environmental nutrient supply. this principle was first demonstrated in yeast, i.e. all atg gene mutant yeasts grow normally in nutrient rich conditions, but unlike wild-type yeasts, die rapidly during carbon or nitrogen starvation 28 . similarly, dictyostelium discoideum that lack atg genes also grow normally in the presence of their food, nonpathogenic bacteria, but die rapidly when subjected to starvation 58, 59 . during nitrogen or starvation, atg7 and atg9 mutant plants display two phenotypes that are thought to result from a defective ability to mobilize nutrients through autophagic delivery, including enhanced chlorosis (yellowing of leaves due to a loss of chlorophyll) and accelerated leaf senescence 60, 61 . in addition, mammalian cells deleted of the autophagy genes, atg5 or beclin 1, also undergo accelerated cell death in response to starvation as compared to their wild-type counterparts 62 . the pro-survival function of autophagy during starvation is thought to be related directly related to its ability to recycle nutrients to generate a sufficient pool of amino acids required for the synthesis of essential proteins. while the eif2 kinase signaling pathway shuts off general translation during starvation, at least in yeast, gcn2 signaling simultaneously stimulates the transcription of essential starvation response genes, including autophagy genes 63, 64 . thus, this signaling pathway provides a coordinated method to effectively generate new amino acids by autophagy and redirect the host cell synthetic machinery to use its limited amino acid supply specifically for the synthesis of essential starvation response proteins. although a downstream transcription factor like yeast gcn4 (which is downstream of yeast eif2 and transcriptionally transactivates autophagy genes), has not yet been identified for mammalian pkr signaling initiated during virus infection, it seems likely that there are functionally homologous molecules that direct virus-infected cells to mount an adaptive and selective transcriptional and translational response during virus infection. even in the absence of this postulated arm of pkr signaling, the mere recycling of nutrients in virus-infected cells would be predicted to have a beneficial function for the host. although few studies have compared cellular amino acid pools during nutrient starvation and virus infection, acute viral replication involves the parasitism of not only the host cell's translational machinery, but also the host cell's translational building blocks. therefore, it seems likely that acute viral replication induces what can be thought of as a state of "pseuodostarvation". according to this model, the prediction is that the nutrient recycling function of autophagy plays a similar protective function during viral infection as it plays during nutrient deprivation. differentiation and development both require cells to undergo significant phenotypic changes and must entail a mechanism for the breakdown and recycling of obsolete cellular components. genetic studies have revealed an essential role for components of the autophagic machinery in differentiation and developmental processes in several different organisms, including sporulation in yeast, multicellular development in dictyostelium, dauer development in c. elegans, and embryonic development in mice (reviewed in 2 ). in addition, the mammalian autophagy gene, beclin 1, appears to play a role in epithelial cell differentiation, since the mammary glands in beclin 1 heterozygous-deficient mice display striking morphological abnormalities 23 . since viral gene products can inhibit the autophagy function of beclin 1 (see below) and potentially other autophagy proteins 65 , it is possible that autophagy blockade represents a mechanism by which viruses can affect cellular differentiation. for example, the bcl-2-like bhrf1 protein encoded by ebv binds to beclin 1 39 blocks its autophagy function 39 , and also perturbs epithelial cell differentiation 66 . further studies are needed to determine the role of beclin 1 binding in the perturbation of epithelial cell differentiation by bhrf1, as well as to investigate the effects of other viral inhibitors of autophagy on cellular differentiation and multicellular development. many different viruses, including retroviruses, gammaherpesviruses, papillomaviruses, and hepatitis viruses are oncogenic. studies of the mechanisms of viral oncogenesis have largely focused on the ability of viruses to alter mitogenic signaling, cell cycle regulation, and/or apoptosis. however, new evidence is emerging that autophagy plays a role in tumor suppression and that autophagy is antagonized by gene products encoded by certain oncogenic viruses. accordingly, it will be important to evaluate the role of viral inhibition of autophagy in viral oncogenesis. normal cell growth requires a well-coordinated balance between the cell's biosynthetic machinery (e.g. protein synthesis and organelle biogenesis) and its degradative processes (e.g. protein degradation and organelle turnover). in the 1970's, it was first proposed that protein catabolism through autophagy is a major determinant of cell growth 67, 68 . according to this model, both cell mass and the rate of cell growth is a balance between the amount of protein synthesized and the amount of autophagic protein degradation. although this model has received little attention in recent years, interest in the role of autophagy in cell growth control has reemerged in light of new biochemical and genetic links between autophagy and the negative regulation of tumorigenesis. as stated above in section 2.2, several different oncogenic signaling molecules, including members of the insulin signaling pathway (e.g. class i pi-3k, akt) and members of the rho and ras family of gtpases negatively regulate autophagy in mammalian cells and the pten tumor suppressor positively regulates autophagy (reviewed in 69 ). furthermore, the autophagy inhibitor, tor, is an important positive regulator of cell growth in diverse organisms, and the tor inhibitor, rapamycin, has promising anti-tumor effects in human clinical trials (reviewed in 70 ). oncogenic viruses have developed multiple different strategies to activate autophagy-inhibitory signaling pathways. these strategies include encoding viral oncoproteins that represent activated forms of the corresponding cellular proto-oncogene (reviewed in 22 ) or upregulating rho/ras or class i pi-3k/akt/tor signaling through alternative mechanisms [71] [72] [73] [74] [75] [76] [77] . components of the autophagic machinery may also play a direct role in tumor suppression. the beclin 1 gene is monallelically deleted in a high percentage of cases of human breast, ovarian, and prostate cancer (reviewed in 78 ) and has tumor suppressor function in cultured mammary carcinoma cells 79, 80 . heterozygous disruption of beclin 1 in mice increases the frequency of spontaneous tumorigenesis (including papillary lung carcinomas, b cell lymphomas, and hepatocellular carcinomas) and accelerates the development of hepatitis b virus-induced pre-malignant lesions 23, 24 . in addition, atg5 null es cells are more tumorigenic in mice than their wild-type counterparts and result in teratomas that are less welldifferentiated 81 . together, these findings lead to the concept that autophagy genes may represent a novel class of tumor suppressor genes and that genetic disruption of autophagy may represent a novel mechanism of tumorigenesis. as will be discussed in more detail below, two different classes of viral gene products have been identified thus far that bind to beclin 1 and inhibit its autophagy function, including the alphaherpesvirus-encoded neurovirulence protein, hsv-1 icp34.5, and the gammaherpesvirus-encoded bcl-2-like proteins, kshv vbcl-2 and ebv bhrf1 38, 39 . the gammaherpesviruses are oncogenic viruses that are etiologically linked to a variety of different malignancies, including lymphoma, nasopharyngeal carcinoma, and kaposi's sarcoma. at present, the precise role of gammaherpesvirus bcl-2like proteins in viral oncogenesis is uncertain. nonetheless, given the welldefined role of cellular bcl-2 in oncogenesis and the emerging evidence that beclin 1 is a tumor suppressor protein, it will be important to evaluate whether viral bcl-2 antagonism of beclin 1 function plays a role in gammaherpesvirus oncogenesis. of note, preliminary data indicates that kshv may also encode other gene products that interact with other components of the autophagic machinery 65 . thus, oncogenic gammaherpesviruses may have multiple mechanisms to disarm host autophagy. it will be of interest to determine whether other oncogenic dna viruses, especially human papillomavirus, also directly inhibit the host autophagic machinery. in many tissues in the adult organism (especially post-mitotic cells), protein and organelle turnover by autophagy plays an essential cellular homeostatic or housekeeping function, removing damaged or unwanted organelles and proteins. for many decades, it has been presumed that this homeostatic function of autophagy represents an anti-aging mechanism, perhaps by reducing reactive oxidative species and other toxic intracellular substances that contribute to genotoxic stress (reviewed in 82 ). the conserved effects of protein caloric restriction (a dietary inducer of autophagy) on lifespan extension has provided further fuel for this concept (reviewed in 83 ). recent genetic studies, especially those performed in c. elegans, provide more direct evidence for a role of both autophagy regulatory signals and components of the autophagic machinery in anti-aging pathways. loss-offunction mutations in autophagy-inhibitory insulin-like signaling pathway extend lifespan (reviewed in 84 ) , and inactivation of the c. elegans ortholog of yeast autophagy gene, atg6/vps30, blocks this lifespan extension 20 . while the precise mechanisms by which autophagy extends lifespan are unknown, one theory is that autophagy selectively removes damaged mitochondria, resulting in decreased levels of intracellular reactive oxygen species and cellular protection against oxidative damage. viral infections, as well as the inflammatory response to viral infections, can damage mitochondria and/or increase the intracellular generation of reactive oxygen species, and these effects may contribute to viral pathogenesis. for example, the mitochondrial damage that occurs in hiv infection (even in the absence of antiretroviral treatment) is thought to be a major contributory factor to the metabolic abnormalities and cardiomyopathy that occur in patients with aids (reviewed in 85 ). as another example, in a transgenic mouse model of hepatitic c virus (hcv) infection, oxidative stress in the absence of inflammation has been implicated in hcv-associated hepatocarcinogenesis 86 . similarly, studies in transgenic mice and cultured cells indicate that pre-s1/s2 mutant hepatitis b virus surface antigens, which accumulate in late stages of hbv infection, cause oxidative stress and dna damage 87 . therefore, it is possible that the mechanisms by which autophagy functions as an anti-aging pathway may be relevant to potential roles that autophagy may play in protecting cells against adverse sequelae of oxidative stress during virus infection. diseases associated with an accumulation of misfolded and aggregated proteins, including neurodegenerative disorders and 1 -anti-trypsin liver disease, are associated with an increase in the accumulation of autophagic vacuoles (reviewed in 56 ). in these diseases, it has both been argued that autophagy plays a protective role (i.e. by removing protein aggregates and damaged mitochondria) and a pathologic role (i.e. by promoting liver dysfunction in 1 -anti-trypsin deficiency through excessive mitochondrial autophagy 88 or by promoting autophagic cell death). although both of these roles may be operative in different diseases or even in different facets of a single disease, recent studies provide compelling evidence that autophagy plays a protective role against the toxic effects associated with protein aggregation. for example, mutant -synuclein (associated with early onset parkinson's disease), and aggregate-prone proteins with polyglutamine expansions (associated with huntington's disease) are targeted for autophagic degradation 88, 89 . rapamycin, which stimulates autophagy, not only enhances the clearance of aggregate-prone proteins but also reduces the appearance of the aggregates and the cell death associated with expression of mutant huntington's proteins 89 . furthermore, induction of autophagy with rapamycin protects against neurodegeneration in both a fly and mouse model of huntington's disease 90 . recent advances have also been made in understanding the mechanisms by which autophagy is induced in response to misfolded protein aggregates. in cell models, transgenic mice, and samples from human brains of patients with huntington's disease, mtor is sequestered into polyglutamine aggregates. this sequestration impairs its kinase activity, leading to induction of autophagy 90 . although it has not yet been evaluated, it is likely that the accumulation of misfolded protein aggregates also induces autophagy through activation of the er stress response, which is mediated by the eif2 kinase, pkr-like er resident kinase (perk) 91, 92 , since other stress stimuli (e.g. starvation and virus infection) that activate other eif2 kinases (e.g. gcn2 and pkr) induce autophagy through this same signaling pathway 18 . these observations are potentially relevant to understanding the role of autophagy in protection against virus-induced diseases in which protein misfolding and er stress are thought to play pathogenetic roles. similar to genetic neurodegenerative disorders, there is increasing evidence that murine retrovirus-associated spongiform-like neuronal degeneration is also associated with protein misfolding and er stress. for example, viral envelope proteins from avirulent strains are processed normally and fail to induce er stress, whereas envelope proteins from neurovirulent strains are misfolded and activate er stress response pathways [93] [94] [95] . in addition, it has been proposed that the mechanism by which pre-s mutant hbv surface antigens promote oxidative stress and dna damage is through the accumulation of misfolded mutant proteins and activation of er stress 87, 96 . thus, based on recent studies with non-viral associated neurodegenerative disorders, the prediction is that autophagy induction might be beneficial in attenuating diseases associated with misfolded viral proteins, such as retrovirus-associated spongiform encephalopathy and hepatitis b virusinduced liver damage. as a corollary, the possibility that these viruses might possess mechanisms to evade host autophagy could be an exacerbating factor in the pathogenesis of these infections. an interesting question is whether viral protein aggregates trigger autophagy by mechanisms that are similar to those involved in autophagy induction initiated by cellular protein aggregates. different viral glycoproteins are known to activate the er stress-related eif2 kinase, perk 97,98 , although a role for perk in autophagy induction has not yet been formally demonstrated. it is completely unknown, however, whether viral protein aggregates, like polyglutamine aggregates in huntington's disease, sequester and thereby inactivate the autophagy-inhibitory kinase, mtor. if so, this would represent a highly novel mechanism by which viruses trigger intracellular innate immune responses. autophagy is emerging as a newly described mechanism of antiviral innate immunity that is targeted by viral virulence gene products. although there are not yet many published articles in this area, there are several observations that support this concept. first, during herpes simplex virus infection, the interferon-inducible antiviral pkr signaling pathway regulates the autophagic degradation of cellular and viral components 18, 99 . second, mammalian autophagy execution genes, including beclin 1 and atg5, regulate sindbis virus replication and sindbis virus-induced cell death 37, 38 . third, plant autophagy execution genes, including beclin 1, class iii pi3-k/vps34, atg3, and atg7, restrict tobacco mosaic virus replication and limit the spread of cell death during the innate immune response 40 . in this section, each of these observations will be described in more detail. the interferon-inducible dsrna-dependent protein kinase r (pkr) plays an important role in innate immunity against viral infections. pkr activation leads to phosphorylation of the subunit of eukaryotic initiation factors 2 (eif2 ) and a subsequent shutdown of host and viral protein synthesis and viral replication (reviewed in 100 ). to avoid this translational shutdown, many viruses have evolved different strategies to antagonize pkr function. these include interference with the dsrna-mediated activation of pkr or pkr dimerization; blockade of the kinase catalytic site or pkr-f substrate interactions; alterations in the levels of pkr; direct regulation of eif2 phosphorylation; and effects on components downstream of eif2 (reviewed in 100,101 ). the importance of viral antagonism of pkr function in viral pathogenesis has been most clearly demonstrated using a herpes simplex virus type 1 (hsv-1) model system 102, 103 . the hsv-1 neurovirulence protein, icp34.5, binds to protein phosphatase 1 and causes it to dephosphorylate eif2 , thereby negating the activity of pkr 104, 105 . a neuroattenuated hsv-1 mutant lacking icp34.5 exhibits wild-type replication and virulence in mice genetically lacking pkr 103 , proving that the icp34.5 gene product mediates neurovirulence by antagonizing pkrdependent functions. in addition to regulating host translation during viral infection, the pkr signaling pathway also regulates the autophagic degradation of host proteins 18 . as mentioned above, molecules in the yeast eif2 kinase signaling pathway (e.g. the eif2 kinase, gcn2, the eif2 ser51 residue, and the transcriptional transactivator, gcn4) are required for nitrogen starvationinduced autophagy. interestingly, the autophagy defect of gcn2 null yeast can be rescued by mammalian pkr transformation 18 . direct evidence that viruses can induce pkr-dependent autophagy has been provided by studies done with herpes simplex virus type 1 (hsv-1) infection in genetically engineered mefs 18 . a herpes simplex virus (hsv-1) mutant virus lacking the icp34.5 inhibitor of pkr signaling (termed hsv-1 34.5), but not wild-type hsv-1, is able to induce the autophagic breakdown of long-lived cellular proteins in wild-type mefs. however, hsv-1 34.5 infection is not able to induce the autophagic breakdown of long-lived cellular proteins in mefs lacking pkr or with a nonphosphorylatable mutation in ser-51 of eif2 . these findings indicate the pkr-dependent signaling events regulate the autophagic breakdown of host proteins during viral infection and that this function of pkr is antagonized by the hsv-1 icp34.5 neurovirulence gene product. as discussed in section 3.1, the breakdown of cellular proteins may help protect host cells against the effects of "pseudostarvation" induced by viral infection. more recent studies indicate that pkr-dependent signaling also regulates the breakdown of viral components during hsv-1 infection 99 . ultrastructural analyses of wild-type and pkr-deficient mefs and sympathetic neurons infected with wild-type and hsv-1 34.5 demonstrate that hsv-1 is degraded in autophagosomes by a pkr-dependent process. in wild-type cells infected with wild-type hsv-1, the majority of intracytoplasmic virions are a either randomly dispersed in the cytoplasm or are contained within "viral vacuoles," a structure thought to represent an important intermediate in the egress of hsv-1 from the nucleus out of the cell 106 . in contrast, in wild-type cells infected with hsv-1 34.5, most cytoplasmic virions are localized within autophagosomes that contain a mix of different cytoplasmic constituents ( figure 4a ). pkr-deficient mefs or pkr-deficient neurons infected with hsv-1 34.5 have very few autophagosomes, and appear similar to wild-type hsv-1-infected wild-type cells, with randomly dispersed intracytoplasmic virions and numerous viral vacuoles. together, these observations demonstrate that hsv-1 is degraded by autophagy, that hsv-1 icp34.5 antagonizes the cellular autophagic degradation of hsv-1, and that this process requires pkr. recent biochemical analyses confirm that pkr signaling and hsv-1 icp34.5 regulate viral protein degradation 99 . hsv-1 protein degradation is significantly accelerated in wild-type mefs infected with hsv-1 t t 34.5 as compared to wild-type mefs infected with wild-type hsv-1, indicating that icp34.5 delays viral protein degradation. however, in autophagy-deficient pkr -/-mefs or eif2 s51a mutant mefs, the rate of hsv-1 protein degradation is similar in hsv-1-and hsv-1 34.5-infected cells, indicating that hsv-1 protein degradation is positively regulated by the pkr signaling pathway. thus, the eif2 kinase-dependent autophagy signaling pathway not only regulates the degradation of long-lived cellular proteins but also regulates the degradation of viral proteins. accordingly, it seems logical to speculate that pkr-dependent autophagic degradation of viruses inhibits viral replication and is an antiviral defense mechanism. however, the relative contributions of the effects of pkr on viral protein synthesis and the effects of pkr on viral protein degradation in the regulation of the hsv-1 replication have not yet been assessed. for this purpose, it will be necessary to selectively inhibit the autophagic protein degradation machinery and/or have hsv-1 mutant viruses that selectively block specific downstream functions regulated by pkr. it will also be important to determine whether pkr-dependent autophagy degrades and inhibits the replication of viruses other than hsv-1. preliminary observations indicate that pkr-dependent autophagy does lead to the degradation of another neurotropic virus, the enveloped, positive-strand rna virus in the alphavirus genus, sindbis virus 107 ( figure 4b ). the role of pkr in antiviral innate immunity is well established, but pkr regulates many different cellular processes, and it is not yet known exactly what role autophagy induction plays in the antiviral effects of pkr. however, the concept that autophagy is important in innate immunity is more directly supported by studies involving components of the mammalian and plant autophagic machinery. the first identified mammalian autophagy gene product, beclin 1, was isolated in a yeast two-hybrid screen, in the context of studies of the mechanism by which the antiapoptotic protein, bcl-2, protects mice against lethal sindbis virus encephalitis 37 . similar to the neuroprotective effects of bcl-2 108 , enforced neuronal expression of wild-type beclin 1 in a recombinant chimeric sindbis virus vector reduces sindbis virus replication, reduces sindbis virus-induced apoptosis, and protects mice against lethal sindbis virus encephalitis 37 . mutations in the bcl-2-binding domain of beclin 1 and mutations in other regions of beclin 1 that block its autophagy function also block its protective effects during sindbis virus infection 37, 62 . thus, it appears that both the interaction with bcl-2 and the autophagy function may be required for the antiviral effects of beclin 1. however, further studies are required to define the precise mechanism of how beclin 1 inhibits viral replication and virus-induced apoptosis and to identify the precise role of bcl-2-beclin 1 interactions in these processes. preliminary studies with beclin 1 null es cells and atg5 null mefs indicate a role for these two endogenous autophagy genes in innate immunity against sindbis virus infection. sindbis virus replicates to higher titers and results in accelerated death in beclin 1 null es cells as compared to wild-type control es cells and in atg5 null mefs as compared to wild-type control mefs 38 . in the case of sindbis virus infection, it is not known whether the acceleration of virus-induced death in beclin 1 null or atg5 null cells is a result of increased viral replication or of independent effects of atg gene deficiency on cell death. however, studies comparing hsv-1 infection in wild-type and beclin 1 -/-es cells suggest that beclin 1 can protect against virus-induced cell death in the absence of inhibitory effects on viral replication 38 . together, the studies of sindbis virus infection in neurons overexpressing beclin 1 or in cultured cells lacking beclin 1 or atg5 demonstrate a role for mammalian autophagy genes in both restricting viral replication and in protection against virus-induced cell death. it will be important to examine whether other autophagy genes have a similar antiviral function and to examine whether autophagy genes also protect against other types of virus infections. the mechanisms by which autophagy genes exert protective effects in sindbis virus infection are not yet known. presumably, the autophagic breakdown of viral components leads to decrease viral yields. however, it is also possible that autophagy leads to the breakdown of cellular components required for viral replication. as noted above, the protective effects against cell death may be secondary to inhibitory effects on viral replication. alternatively, the protective effects may relate to the nutrient recycling functions or "damage control" functions of autophagy, or in the case of beclin 1, to interactions between autophagy proteins and antiapoptotic pathways. it is also possible that autophagy may protect against cell death by degrading specific viral proteins (e.g. the sindbis virus e1 and e2 envelope glycoprotein's 109 ) that are involved in triggering the apoptotic pathway. the protective effects of beclin 1 and atg5 on virus-induced cell death are consistent with the "pro-survival" effects of autophagy during nutrient starvation and other forms of environmental stress. it is not yet clear how to reconcile these pro-survival effects with the view that autophagy represents an alternative form of non-apoptotic programmed cell death (reviewed in 2, 25, 110 ). while the primary basis for this view has been morphologic correlations between the presence of autophagic vacuoles and dying cells (reviewed in 2,111 ), recent genetic experiments establish a more direct role for autophagy genes in certain types of programmed cell death. mammalian atg7 and beclin 1 rnai blocks cell death in fibroblast and macrophage cell lines treated with the caspase inhibitor, zvad 112 , and atg 6 , 7 , and 12 rnai blocks salivary gland destruction during drosophila development 113 . thus, the relationship between autophagy, cell survival, and cell death is quite complex and likely varies according to the cell type and the specific physiological or pathophysiological setting. it remains to be determined whether autophagy genes primarily play a protective role in preventing cell death during virus infection, or whether they also participate directly in cell death that is induced by certain viruses. the plant homologue of beclin 1 also functions in antiviral host defense in plants. similar to mammalian beclin 1, plant beclin 1 restricts viral replication; tobacco mosaic virus replication is increased in beclin 1silenced tobacco plants as compared to vector-treated control plants 40 . however, in contrast to mammalian beclin 1, which prevents the death of sindbis virus-and hsv-1-infected cells, plant beclin 1 plays an interesting role in preventing the death of uninfected cells 40 . in plants, the innate immune response during virus infection is characterized by a hypersensitive response which is a programmed cell death response that occurs around the infected areas (reviewed in [114] [115] [116] ). this hypersensitive response limits virus spread and confers pathogen resistance. it is triggered by a pathogen-encoded avirulence protein, which is recognized by a specific plant cognate resistance protein, termed an r protein. in plants lacking r proteins, there is uncontrolled virus spread and pathogen sensitivity. a tobacco mosaic virus infection model has recently been used to study the role of autophagy genes in plant innate immunity. during tobacco mosaic virus infection, the hypersensitive response is triggered by tobacco mosaic virus protein, tmv p50, which is the helicase domain of the viral replicase 117 . tmv p50 is recognized by an r protein (called the n protein) of n. benthamiana, which is composed of a toll/interleukin 1 receptor domain, a nucleotide binding domain, and a leucine-rich repeat domain 118 . therefore, in tobacco plants containing the n protein (n ( ( +/+ n n ), there is local cell death in cells that are either infected with tmv or that express the tmv p50 protein but no systemic illness is observed. beclin 1 silencing in n +/+ n n tobacco plants reveals a striking role for this autophagy gene in limiting the spread of cell death during the hypersensitive response 40 . during tmv infection of n n plants, cell death begins as discreet and defined foci but continues to spread beyond the site of tmv infection until there is death of the entire inoculated leaf and other uninoculated leaves. a similar spreading cell death phenotype is seen with local expression of the tmv p50 protein, suggesting that the cell death occurs in response to a specific signal triggered by the pathogen encoded avirulence protein, and is not due to increased tmv replication or altered virus movement. in addition, in plants that lack the n gene, beclin 1 silencing does not lead to cell death after tmv infection. moreover, beclin 1 silencing also results in spreading cell death during the hypersensitive response triggered by bacterially-encoded pathogen avirulence proteins. together, these observations demonstrate that the spreading cell death phenotype in beclin 1-silenced plants is mediated by r gene-mediated innate immune responses and that beclin 1 is an important negative regulator of cell death during the plant innate immune response. a similar role for other autophagy genes in limiting the spread of programmed cell death during tmv infection has also been observed. as discussed in section 2.3.3, pi3-k/vps34 is a protein that physically interacts with atg6/beclin 1 in yeast and mammals and is essential for proper autophagosome formation. interestingly, silencing of the plant class iii pi-3k/vps34 in n +/+ n n plants results in a spreading cell death phenotype during tmv infection that is similar to that observed with beclin 1 silencing. as discussed in section 2.3.3, yeast and mammalian atg3 and atg7 are essential for conjugation reactions needed for autophagosome formation, and d silencing of the plant homologues of these genes also results in a spreading cell death phenotype after tmv infection. thus, multiple different f f autophagy genes, including those that act in the vesicle nucleation stage (e.g. t beclin 1, class iii pi3-k/vps34) and those that act in the vesicle expansion stage (e.g. atg3 and atg7) are necessary to prevent the spread of cell death during the plant innate immune response. while plant autophagy genes protect uninfected cells against death whereas mammalian autophagy genes protect infected cells against death during virus infection, the plant data nonetheless further support a "prosurvival", rather than "pro-death" function of autophagy genes during viral f f infections. at present, it is not yet clear how autophagy genes protect uninfected cells against death during the plant hypersensitive response. one possibility is that the absence of autophagy genes in uninfected cells somehow modifies the r gene-mediated signal transduction pathway in a way that instructs uninfected cells to die. an alternative, perhaps more likely, possibility is that the absence of autophagy genes in uninfected cells f renders them more susceptible to pro-death signals emitted from infected cells. regardless of the mechanism, this newly defined role for autophagy genes in preventing the spread of cell death during plant innate immunity has significant implications for understanding the role of autophagy in systemic protection against viral infections. an important question is whether autophagy genes play a similar role during animal virus infections. the evolutionarily conserved function of both mammalian and plant autophagy genes in restricting viral replication and/or protection against cell death suggests an essential role for autophagy in innate immunity. this concept is further supported by recent observations indicating that the herpes simplex virus neurovirulence protein, icp34.5, possesses multiple mechanisms to disarm host autophagy. it can both antagonize the pkr signaling pathway required for autophagy induction and inhibit the function of one component of the autophagic machinery, beclin 1. as discussed in section 4.1, icp34.5 blocks pkr-dependent, eif2 ser-51-dependent autophagic degradation of cellular and viral components in f hsv-1-infected mefs and neurons 99 . one predicted mechanism by which icp34.5 blocks pkr-dependent autophagy is through its known ability to promote the dephosphorylation of eif2 via interactions with pp1 105 . however, new evidence also suggests a second potential mechanism. roizman et al. isolated the mammalian autophagy protein, beclin 1, in a yeast two-hybrid screen using icp34.5 as a bait 119 . subsequent studies have shown that icp34.5 directly interacts with beclin 1 in mammalian cells and inhibits the ability of beclin 1 to rescue autophagy in autophagy-defective atg6 null yeast and in autophagy-defective human mcf7 breast carcinoma cells 38 . since icp34.5 binds to beclin 1 via a domain that is distinct from its pp1 -binding domain, it should be possible to construct hsv-1 viruses containing mutations in icp34.5 that help differentiate between the role of pp1 -binding (and eif2 phosphorylation) and the role of beclin 1-binding in hsv-1 icp34.5-mediated neurovirulence. besides hsv-1 icp34.5, there are numerous other viral proteins or rnas that suppress pkr signaling through a variety of different mechanism (reviewed in 100,101 ). for example, vaccinia virus e3, influenza virus ns1, hsv-1 us11, reovirus 3, and rotavirus nsp3 encode double-stranded (ds) rna-binding proteins that prevent pkr activation. adenovirus vai rnas and hiv tar rnas bind to dsrna substrates and inhibit pkr. hepatitis c virus ns5a protein inhibits the dimerization of pkr, and influenza virus recruits a cellular protein, p58 ipk , that directly interacts with pkr and inhibits its dimerization. the vaccinia virus k3l, hepatitis c virus e2, and hiv tat proteins act as pseudosubstrates of pkr. as-of-yet, the role of these other viral rnas and proteins in autophagy inhibition has not been investigated. however, given the evolutionarily conserved requirement for an intact eif2 kinase signaling pathway in autophagy induction, the prediction is that these other viral inhibitors of pkr, like hsv1 icp34.5, also function as antagonists of host autophagy. further studies are needed to test this prediction and to study the role of this predicted antagonism of host autophagy in the pathogenesis of diseases caused by these other important viral pathogens that encode putative autophagy inhibitors. not only may other viruses antagonize the autophagy function of pkr, but other viral gene products may also antagonize the autophagy function of specific mammalian atg genes. beclin 1 was originally isolated in a yeast two-hybrid screen with the cellular anti-anti-apoptotic protein, bcl-2 37 . subsequently, beclin 1 has also been shown to interact with viral bcl-2-like proteins encoded by different gammaherpesviruses, including ebv-encoded bhrf1, kshv-encoded v-bcl-2, and murine hv68-encoded m11 39 . like icp34.5, these viral proteins can also inhibit the autophagy function of beclin 1 in yeast and mammalian assays. in addition, preliminary evidence indicates that other kshv-encoded proteins may interact with other specific atg proteins 65 . an as-of-yet explored area is whether viruses also inhibit autophagy by activating autophagy inhibitory signaling pathways. as noted in section 2.2 and 3.2, the class i pi-3k/akt signaling pathway negatively regulates autophagy in both mammalian cells and c. elegans 19, 20, 120 and several different viruses activate this pathway. certain oncogenic retroviruses encode the catalytic subunit of pi3-k and akt (reviewed in 22 ). in addition, the ebv latent membrane proteins, lmp1 and 2a, the hepatitis b virus protein, hbx, the kaposi's sarcoma virus protein, k1, and the hepatitis c virus protein, ns5a all activate the pi3-k/akt signaling pathway [72] [73] [74] [75] [76] [77] . presumably, such activation plays a role in autophagy inhibition, although this has not yet been formally tested. while further studies are required to more precisely define the interactions between viral gene products and autophagy regulatory signals and autophagy proteins, there is, however, accumulating evidence that viruses do target multiple different steps of the host autophagy pathway. this observation strongly suggests an evolutionary advantage for viruses to inhibit host autophagy, and by extrapolation, a beneficial role for host autophagy in defense against viral infections. some viruses appear to have even further outsmarted host autophagy. rather than merely devising strategies to block host autophagy, certain positive-strand rna viruses have figured out ways to "co-opt" elements of the autophagy pathway to promote their own replication. this subject has been recently reviewed in detail elsewhere 13 and will therefore only be briefly summarized in this section. as early as 1965, electron microscopic studies of poliovirus-infected cells demonstrated the presence of large numbers of membranous vesicles that were postulated to develop by an autophagic-like mechanism 121 . more recently, work by kirkegaard et al, has extended these findings to further show that poliovirus replication complexes are associated with doublemembrane vesicles that resemble autophagosomes, in that they (1) have similar double membrane-bound morphology; (2) have low buoyant density; and (3) label with the autophagosome marker, gfp-lc3, and the lysosome marker, lamp1 13, 122 . unlike classical autophagosomes, these autophagiclike vesicles do not appear to have a destructive role or mature into degradative compartments. in support of this, treatment with autophagy r r inducers, rapamycin or tamoxifen, both increase, rather than decrease poliovirus growth 13 . furthermore, these double-membrane vesicles are also different from classical autophagosomes in that they contain sec13 and sec31, components of the anterograde transport system that bud from the er 123 . therefore, it is possible that poliovirus-induced vesicles arise from an alternate source rather than the pas, but still share some of the same characteristics of classical autophagosomes (e.g. gfp-lc3 and labeling, augmentation with rapamycin treatment). similar to the replication vacuoles that are associated with certain intracellular bacterial pathogens (e.g. legionella pneumophila) 124 , the poliovirus-induced double-membrane vesicles likely originate from the er 123 . furthermore, these poliovirusinduced vesicles seem to have an alternate function than autophagosomes (i.e. they are pro-replicative, rather than degradative compartments). these r observations suggest that poliovirus may promote its own replication by inducing dynamic membrane rearrangements that share in common certain features of the autophagy pathway (e.g. formation of sequestering doublemembrane vesicles, presence of overlapping markers) but avoid, other unwanted features of the autophagy pathway (e.g. maturation into degradation compartments). of note, specific poliovirus proteins, including 2bc and 3a, have been identified that are sufficient for the induction of t these "autophagic-like" double-membrane bound vesicles 122 . however, the mechanisms by which these proteins induce the formation of such vesicles are not yet known. a recent study with the coronavirus, murine hepatitis virus, has provided more direct evidence that components of the autophagic machinery can be utilized for rna virus replication 51 . mhv replication complexes localize to double-membrane vesicles (that are also thought to arise from the er) ( figure 5a ) and they co-localize with certain autophagy proteins, including lc3 and atg12. in mhv-infected atg5 -/-es cells, double-membrane vesicles are not detected ( figure 5b) , and viral replication is dramatically reduced. these observations provide the first genetic demonstration that proteins necessary for autophagic vacuole formation are also required for maximal levels of viral replication. thus, mhv, and potentially other viruses that replicate in association with double-membrane vesicles (e.g. poliovirus, equine arterivirus), utilize components of cellular autophagy to foster their f own growth. presumably, the atg protein conjugation system (involving atg5) that plays a role in autophagic vesicle expansion and completion also plays a role in the formation of double-membrane vesicles involved in viral replication. it is not yet known whether the entire autophagic machinery or only selective components of the autophagic machinery are used for the formation of double-membrane vesicles that are associated with viral replication complexes. these observations with poliovirus and mhv represent two examples of how viruses can "subvert" elements of the host autophagy pathway to promote their own intracellular growth. in these infections, rna replication complexes are observed in association with "autophagic-like" doublemembrane vesicles but not in association with degradative autophagosomes. it is not clear whether this represents fundamental differences in the host pathways leading to the formation of "autophagic-like" double-membrane vesicles and classical degradative autophagosomes, the diversion of the autophagic machinery towards the formation of "autophagic-like" doublemembrane vesicles from the formation of classical degradative autophagosomes, or specific viral mechanisms to antagonize the maturation of "autophagic-like" double-membrane vesicles into mature degradative autophagosomes. however, interestingly, mhv infection does lead to the induction of atg-5-dependent long-lived cellular protein degradation, ruling out the hypothesis that the autophagic machinery is entirely diverted to form membranes required for viral replication complexes. perhaps mhv possesses as-of-yet defined mechanisms to shield its replication complexes from autophagic degradation. although research in this area is still in a stage of infancy, it seems likely that the lysosomal degradation pathway of autophagy plays an evolutionarily conserved role in antiviral immunity. the interferon-inducible, antiviral pkr signaling pathway positively regulates autophagy, and both mammalian and plant autophagy genes restrict viral replication and protect against virus-induced cell death. given this role of autophagy in innate immunity, it is not surprising that viruses have evolved numerous strategies to inhibit host autophagy. different viral gene products can either modulate autophagy regulatory signals or directly interact with components of the autophagy execution machinery. moreover, certain rna viruses have managed to "co-apt" the autophagy pathway, selectively utilizing certain components of the dynamic membrane rearrangement system to promote their own replication inside the host cytoplasm. in addition to this newly emerging role of autophagy in innate immunity, autophagy plays an important role in many other fundamental biological processes, including tissue homeostasis, differentiation and development, cell growth control, and the prevention of aging. accordingly, the inhibition of host autophagy by viral gene products has important implications not only for understanding mechanisms of immune evasion, but also for understanding novel mechanisms of viral pathogenesis. it will be interesting to dissect the role of viral inhibition of autophagy in acute, persistent, and latent viral replication, as well as in the pathogenesis of cancer and other medical diseases. development by self-digestion: molecular mechanisms and biological functions of autophagy development by self-digestion: molecular mechanisms and biological functions of autophagy autophagy: a regulated bulk degradation process inside cells. d the molecular mechanism of autophagy functions of lysosomes a unified nomenclature for yeast autophagy-related genes diversity of signaling controls of macroautophagy in mammalian cells the pre-autophagosomal structure organized by concerted functions of 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ras-map kinase pathway and induce cell proliferation epstein-barr virus lmp2a transforms epithelial cells, inhibits cell differentiation, and activates akt latent membrane protein 2a inhibits transforming growth factor-beta 1-induced apoptosis through the phosphatidylinositol 3-kinase/akt pathway differential signaling pathways are activated in the epstein-barr virus-associated malignancies nasopharyngela carcinoma and hodgin lymphoma hepatitis b viral hbx induces matrix metalloproteinase-9 gene expression trhough activation of erk and pi-3k/akt pathways: involvement of invasive potential the k1 protein of kaposi's sarcoma-associated herpesvirus activates the akt signaling pathway the hepatitis c virus ns5a protein activates a phosphoinosite 3-kinase-dependent survival signaling cascade cloning and genomic organization of beclin 1, a candidate tumor suppressor gene on chromosome 17q21 induction of autophagy and inhibition of tumorigenesis by beclin 1 beclin 1 contains a leucine-rich nuclear export signal that is reguired for its autophagy and tumor suppressor function unpublished data autophagy and aging--when "all you can eat" is yourself the anti-ageing effects of caloric restriction may involve stimulation of macroautophagy and lysosomal degradation, and can be intensifid pharmacologically genetic pathways that regulate ageing in model organisms mitochondria and hiv infection: the first decade oxidative stress in the absence of inflammation in a mouse model for hepatitis c virus-associated carcinogenesis pre-s mutant surface antigens in chronic hepatitis b virus infection induce oxidative stress and dna damage mitochondrial autophagy and injury in the liver in 1 -antitrypsin deficiency aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy inhibition of mtor induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models off huntington disease identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, pek, involved in translational control protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase endoplasmic reticulum stress is a determinant of retrovirus-induced spongiform neurodegeneration activation of endoplasmic reticulum stress signaling pathway is associated with neuronal degeneratio in momulv-ts1-induced spongiform encephalopathy endoplasmic reticulum (er) stress induced by a neurovirulent mouse retrovirus is associated with prolonged bip binding and retention of a viral protein in the er different types of ground glass hepatocytes in chronic hepatitis b virus infection contain specific pre-s mutants that may induce endoplasmic reticulum stress replication of a cytopathic strain of bovine viral diarrhea virus activates perk and induces endoplasmic reticulum stress-mediated apoptosis of mdbk cells possible involvment of both endoplasmic reticulum-and mitochondriadependent pathways in 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synthesis by double-stranded rna-activated protein kinase electron microscopic observations on the development of herpes simplex virus unpublished data bcl-2 protects mice against fatal alphavirus encephalitis the transmembrane domains of sindbis virus envelope glycoproteins induce cell death the autophagosomal-lysosomal compartment in programmed cell death regulation of an atg7-beclin 1 program of autophagic cell death by caspase 8 american society for cell biology 44th annual meeting plant pathogens and integrated defence responses to infection plant innate immunity -direct and indirect recognition of general and specific pathogen-associated molecules controlled cell death, plant survival and development the helicase domain of the tmv replicase proteins induces the nmediated defense response in tobacco the product of the tobacco mosaic virus resistance gene n: similarity to toll and the interleukin-1 receptor the tumor suppressor pten positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase b pathway electron microscopic study of the formation of poliovirus remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles cellular coopii proteins are involved in production of the vesicles that form the poliovirus replication complex apg1p, a novel protein kinase required for the autophagic process in saccharomyces cerevisiae apg14p and apg6/vps30p form a protein complex essential for autophagy in the yeast, saccharomyces cerevisiae the drosophila homolog of aut1 is essential for autophagy and development human apg3p/aut1p homologue is an authentic e2 enzyme for multiple substrates, gate-16, gabarap, and map-lc3, and facilitates the conjugation of hapg12p to hapg5p the loss of drosophila apg4/aut2 function modifies the phenotypes of cut and notch signaling pathway pathway mutants a single protease, apg4b, is specific for the autophagy-related ubiquitin-like proteins gate-16, map1-lc3, gabarap, and apg8l dissection of autophagosome formation using apg5deficient mouse embryonic stem cells a new protein conjugation system in human. the counterpart of the yeast apg12p conjugation system essential for autophagy structural and functional analyses of apg5, a gene involved in autophagy in yeast the human homolog of saccharomyces cerevisiae apg7p is a protein-activating enzyme for multiple substrates including human apg12p, gate-16, gabarap, and map-lc3 glucose-induced autophagy of peroxisomes in pichia pastoris requires a unique e1-like protein mouse apg10 as an apg12conjugating enzyme: analysis by the conugation-mediated yeast two-hybrid method apg10p, a novel protein-conjugating enzyme essential for autophagy in yeast murine apg12p has a substrate preference for murine apg7p over three apg8p homologs apg16p is required for the function of the apg12p-apg5p conjugate in the yeast autophagy pathway cvt18/gas12 is required for cytoplasm-to-vacuole transport, pexophagy, and autophagy in saccharomyces cerevisiae and pichia pastoris the work done in the authors' laboratories was supported by nih ro1 grants ai51367 and ai44157 to b.l, and an nsf plant genome grant dbi-0116076 and nih ro1 grant gm62625 to s.p.d-k. key: cord-003297-fewy8y4a authors: wang, ming-yang; liang, jing-wei; mohamed olounfeh, kamara; sun, qi; zhao, nan; meng, fan-hao title: a comprehensive in silico method to study the qstr of the aconitine alkaloids for designing novel drugs date: 2018-09-18 journal: molecules doi: 10.3390/molecules23092385 sha: doc_id: 3297 cord_uid: fewy8y4a a combined in silico method was developed to predict potential protein targets that are involved in cardiotoxicity induced by aconitine alkaloids and to study the quantitative structure–toxicity relationship (qstr) of these compounds. for the prediction research, a protein-protein interaction (ppi) network was built from the extraction of useful information about protein interactions connected with aconitine cardiotoxicity, based on nearly a decade of literature and the string database. the software cytoscape and the pharmmapper server were utilized to screen for essential proteins in the constructed network. the calcium-calmodulin-dependent protein kinase ii alpha (camk2a) and gamma (camk2g) were identified as potential targets. to obtain a deeper insight on the relationship between the toxicity and the structure of aconitine alkaloids, the present study utilized qsar models built in sybyl software that possess internal robustness and external high predictions. the molecular dynamics simulation carried out here have demonstrated that aconitine alkaloids possess binding stability for the receptor camk2g. in conclusion, this comprehensive method will serve as a tool for following a structural modification of the aconitine alkaloids and lead to a better insight into the cardiotoxicity induced by the compounds that have similar structures to its derivatives. the rhizomes and roots of aconitine species, a genus of the family ranunculaceae, are commonly used in treatment for various illnesses such as collapse, syncope, rheumatic fever, joints pain, gastroenteritis, diarrhea, edema, bronchial asthma, and tumors. they are also involved in the management of endocrinal disorders such as irregular menstruation [1, 2] . however, the usefulness of this aconitine species component intermingles with toxicity after it is administered to a diseased patient. so far, few articles have recorded the misuse of aconitine medicinals with strong emphasis and thus have referenced that the misuse of this medicinal can result in severe cardio-and neurotoxicity [3] [4] [5] [6] [7] . in our past research, it was evidenced that the aconitine component is the main active ingredient in this species' root and rhizome, and is responsible for both therapeutic and toxic effects [8] . this medicinal has been tested for cancerological and dermatological activities. its application to disease conditions proved to exhibit an activity that slowed down cancer tumor growth and to cure serious cases of dermatosis. it was also found to have an effect on postoperative analgesia [9] [10] [11] [12] . however, a previous safety study has revealed that aconitine toxicity is responsible for its restriction in clinical settings. further studies are needed to explain the cause of aconitine toxicity as well as to show whether the toxicity supersedes its usefulness. a combined network analysis and in silico study was once performed to obtain insight on the relationship between aconitine alkaloid toxicity and the aconitine structure, and it was found that the cardiotoxicity of aconitine is the primary cause of patient death. the aconitine poison is similar to the poison created by some pivotal proteins such as the ryanodine receptor (ryr1 and ryr2), the gap junction α-1 protein (gja1), and the sodium-calcium exchanger (slc8a1) [9] [10] [11] [12] . however, among all existing studies about the aconitine medicinal, no one has reported detail of its specific binding target protein linked to toxicity. protein-protein interactions (ppis) participate in many metabolic processes occurring in living organisms such as the cellular communication, immunological response, and gene expression control [13, 14] . a systematic description of these interactions aids in the elucidation of interrelationships among targets. the targeting of ppis with small-molecule compounds is becoming an essential step in a mechanism study [14] . the present study was designed and undertaken to identify the critical protein that can affect the cardiotoxicity of aconitine alkaloids. a ppi network built by the string database is a physiological contact for the high specificity that has been established for several protein molecules and has stemmed from computational prediction, knowledge transfer between organisms, and interactions aggregated from other databases [15] . the analysis of the ppi network is based on nodes and edges and is always performed via cluster analysis and centrality measurements [16, 17] . in cluster analysis, highly interconnected nodes and protein target nodes are divided and used to form sub-graphs. the reliability of the ppi network is identified by the content of each sub-graph [18] . the variability in centrality measurements is connected to the quantitative relationship between the protein targets and its weightiness in the network [18] . hence, ppi networks with protein targets related to aconitine alkaloid cardiotoxicity must enable us to find the most relevant protein for aconitine toxicity and to understand the mechanism at the network level. in our research, the evaluation and visualization analysis of essential proteins related to cardiotoxicity in ppis were performed by the clusterone and cytonca plugins in cytoscape 3.5, designed to find the potential protein targets via combination with conventional integrated pharmacophore matching technology built in the pharmmapper platform. structural modification of a familiar natural product, active compound, or clinical drug is an efficient method for designing a novel drug. the main purpose of the structural modification is to reduce the toxicity of the target compound while enhancing the utility of the drug [19] . the identification of the structure-function relationship is an essential step in the drug discovery and design, the determination of the 3d protein structures was the key step in identifying the internal interactions in the ligand-receptor complexes. x-ray crystallography and nmr were the only accepted techniques of determining the 3d protein structure. although the 3d structure obtained by these two powerful techniques are accurate and reliable, they are time-consuming and costly [20] [21] [22] [23] [24] . with the rapid development of structural bioinformatics and computer-aided drug design (cadd) techniques in the last decade, computational structures are becoming increasingly reliable. the application of structural bioinformatics and cadd techniques can improve the efficiency of this process [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] . the ligand-based quantitative structure-toxicity relationship (qstr) and receptor-based docking technology are regarded as effective and useful tools in analysis of structure-function relationships [35] [36] [37] [38] . the contour maps around aconitine alkaloids generated by comparative molecular field analysis (comfa) and comparative molecular similarity index analysis (comsia) were combined with the interactions between ligand substituents and amino acids obtained from docking results to gain insight on the relationship between the structure of aconitine alkaloids and their toxicity. scores from functions were used to evaluate the docking result. the value-of-fit score in moe software reflects the binding stability and affinity of the ligand-receptor complexes. when screening for the most potential target for cardiotoxicity, the experimental data was combined with the value-of-fit score by the ndcg (normalized discounted cumulative gain). the possibility of a protein being a target of cardiotoxicity corresponds with the consistency of this experimental data. since the pioneering paper entitled "the biological functions of low-frequency phonons" [39] was published in 1977, many investigations of biomacromolecules from a dynamic point of view have occurred. these studies have suggested that low-frequency (or terahertz frequency) collective motions do exist in proteins and dna [40] [41] [42] [43] [44] . furthermore, many important biological functions in proteins and dna and their dynamic mechanisms, such as cooperative effects [45] , the intercalation of drugs into dna [42] , and the assembly of microtubules [46] , have been revealed by studying the low-frequency internal motions, as summarized in a comprehensive review [40] . some scientists have even applied this kind of low-frequency internal motion to medical treatments [47, 48] . investigation of the internal motion in biomacromolecules and its biological functions is deemed as a "genuinely new frontier in biological physics," as announced in the mission of some biotech companies (see, e.g., vermont photonics). in order to consider the static structural information of the ligand-receptor complex, dynamical information should be also considered in the process of drug discovery [49, 50] . finally, molecular dynamics was carried out to verify the binding affinity and stability of aconitine alkaloids and the most potential target. this present study may be instrumental in our future studies for the synergism and attenuation of aconitine alkaloids and for the exploitation of its clinical application potential. a flowchart of procedures in our study is shown in figure 1 . of-fit score by the ndcg (normalized discounted cumulative gain). the possibility of a protein being a target of cardiotoxicity corresponds with the consistency of this experimental data. since the pioneering paper entitled "the biological functions of low-frequency phonons" [39] was published in 1977, many investigations of biomacromolecules from a dynamic point of view have occurred. these studies have suggested that low-frequency (or terahertz frequency) collective motions do exist in proteins and dna [40] [41] [42] [43] [44] . furthermore, many important biological functions in proteins and dna and their dynamic mechanisms, such as cooperative effects [45] , the intercalation of drugs into dna [42] , and the assembly of microtubules [46] , have been revealed by studying the low-frequency internal motions, as summarized in a comprehensive review [40] . some scientists have even applied this kind of low-frequency internal motion to medical treatments [47, 48] . investigation of the internal motion in biomacromolecules and its biological functions is deemed as a "genuinely new frontier in biological physics," as announced in the mission of some biotech companies (see, e.g., vermont photonics). in order to consider the static structural information of the ligand-receptor complex, dynamical information should be also considered in the process of drug discovery [49, 50] . finally, molecular dynamics was carried out to verify the binding affinity and stability of aconitine alkaloids and the most potential target. this present study may be instrumental in our future studies for the synergism and attenuation of aconitine alkaloids and for the exploitation of its clinical application potential. a flowchart of procedures in our study is shown in figure 1 . the whole framework of the comprehensive in silico method for screening potential targets and studying the quantitative structure-toxicity relationship (qstr). the 33 compounds were aligned over, under the superimposition of the common moiety and template compound 6. the statistical parameters for database alignment-q 2 , r 2 , f, and see-were the whole framework of the comprehensive in silico method for screening potential targets and studying the quantitative structure-toxicity relationship (qstr). the 33 compounds were aligned over, under the superimposition of the common moiety and template compound 6. the statistical parameters for database alignment-q 2 , r 2 , f, and see-were table 1 . the comfa model with the optimal number of 6 components presented a q 2 of 0.624, an r 2 of 0.966, an f of 124.127, and an see of 0.043, and contributions of the steric and electrostatic fields were 0.621 and 0.379, respectively. the comsia model with the optimal number of 4 components presented a q 2 of 0.719, an r 2 of 0.901, an f of 157.458, and an see of 0.116, and the contributions of steric, electrostatic, hydrophobic, hydrogen bond acceptor, and hydrogen bond donor fields were 0.120, 0.204, 0.327, 0.216, and 0.133, respectively. the statistical results proved that the aconitine alkaloids qstr model of comfa and comsia under the database alignment have adequate predictability. experimental and predicted pld 50 values of both the training set and test set are shown in figure 2 , and the comfa ( figure 2a ) and comsia ( figure 2b ) model gave the correlation coefficient (r 2 ) value of 0.9698 and 0.977, respectively, which demonstrated the internal robustness and external high prediction of the qstr models. experimental and predicted pld50 values of both the training set and test set are shown in figure 2 residuals vs. leverage williams plots of the aconitine qstr models are shown in figure 3a ,b. all values of standardized residuals fall between 3σ and −3σ, and the values of leverage are less than h*, so the two models demonstrate potent extensibility and predictability. residuals vs. leverage williams plots of the aconitine qstr models are shown in figure 3a ,b. all values of standardized residuals fall between 3σ and −3σ, and the values of leverage are less than h*, so the two models demonstrate potent extensibility and predictability. under mesh (medical subject headings), a total of 491 articles (261 articles were received from web of science, and others were received from pubmed) were retrieved. after selecting cardiotoxicity-related and excluding repetitive articles, 274 articles were used to extract the correlative proteins and pathways for building a ppi network in the string server. the correlative proteins or pathways are shown in table 2 . all proteins were taken as input protein in the string database to find its direct and functional partners [51] , and proteins and its partners were then imported into the cytoscape 3.5 to generate the ppi network with 148 nodes and 872 edges ( figure 4 ). potassium voltage-gated channel h2 7 scn3a sodium voltage-gated channel type 3, 3 scn2a sodium voltage-gated channel type 2 3 scn8a sodium voltage-gated channel type 8 2 scn1a sodium voltage-gated channel type 1 2 scn4a sodium voltage-gated channel type 4 1 kcnj3 potassium inwardly-rectifying channel j3 1 during the case of screening of the essential proteins in ppi network, three centrality measurements (subgraph centrality, betweenness centrality, and closeness centrality) in cytonca were utilized to evaluate the weight of nodes. after removing the central node "ac," the centrality measurements of 147 nodes were calculated by cytonca and documented in table s1 . the top 10% of three centrality measurement values of all node are painted with a different color in figure 4a . to screen the node with the high values of each three centrality measures, nodes with three colors were overlapped and merged into sub-networks in figure 4b . under mesh (medical subject headings), a total of 491 articles (261 articles were received from web of science, and others were received from pubmed) were retrieved. after selecting cardiotoxicity-related and excluding repetitive articles, 274 articles were used to extract the correlative proteins and pathways for building a ppi network in the string server. the correlative proteins or pathways are shown in table 2 . all proteins were taken as input protein in the string database to find its direct and functional partners [51] , and proteins and its partners were then imported into the cytoscape 3.5 to generate the ppi network with 148 nodes and 872 edges ( figure 4 ). table 2 . proteins related to aconitine alkaloids induced cardiotoxicity extracted from 274 articles. classification frequency ryanodine receptor 2 19 ryr1 ryanodine receptor 1 15 gja1 gap junction α-1 protein (connexin43) 13 slc8a1 sodium/calcium exchanger 1 11 atp2a1 calcium transporting atpase fast twitch 1 9 kcnh2 potassium voltage-gated channel h2 7 scn3a sodium voltage-gated channel type 3, 3 scn2a sodium voltage-gated channel type 2 3 scn8a sodium voltage-gated channel type 8 2 scn1a sodium voltage-gated channel type 1 2 scn4a sodium voltage-gated channel type 4 1 kcnj3 potassium inwardly-rectifying channel j3 1 during the case of screening of the essential proteins in ppi network, three centrality measurements (subgraph centrality, betweenness centrality, and closeness centrality) in cytonca were utilized to evaluate the weight of nodes. after removing the central node "ac," the centrality measurements of 147 nodes were calculated by cytonca and documented in table s1 . the top 10% of three centrality measurement values of all node are painted with a different color in figure 4a . to screen the node with the high values of each three centrality measures, nodes with three colors were overlapped and merged into sub-networks in figure 4b . in the sub-networks, the voltage-gated calcium and sodium channel accounted for a large proportion, which is consistent with our research in clustering the network (clusters 1, 2, and 9). all proteins in the sub-networks will be utilized to predict the results of the pharmmapper server to receive the potential target of cardiotoxicity induced by aconitine alkaloids (in figure 5a ,b). in the meantime, 2v7o (camk2g) and 2vz6 (camk2a) were identified as the potential targets with higher fit scores. in the sub-networks, the voltage-gated calcium and sodium channel accounted for a large proportion, which is consistent with our research in clustering the network (clusters 1, 2, and 9). all proteins in the sub-networks will be utilized to predict the results of the pharmmapper server to receive the potential target of cardiotoxicity induced by aconitine alkaloids (in figure 5a ,b). in the meantime, 2v7o (camk2g) and 2vz6 (camk2a) were identified as the potential targets with higher fit scores. all compounds were docked into three potential targets. the values of ndcg are shown in table 3 . the dock study of three proteins with an ndcg of 0.8503 and 0.9122, respectively (the detailed in the sub-networks, the voltage-gated calcium and sodium channel accounted for a large proportion, which is consistent with our research in clustering the network (clusters 1, 2, and 9). all proteins in the sub-networks will be utilized to predict the results of the pharmmapper server to receive the potential target of cardiotoxicity induced by aconitine alkaloids (in figure 5a ,b). in the meantime, 2v7o (camk2g) and 2vz6 (camk2a) were identified as the potential targets with higher fit scores. all compounds were docked into three potential targets. the values of ndcg are shown in table 3 . the dock study of three proteins with an ndcg of 0.8503 and 0.9122, respectively (the detailed all compounds were docked into three potential targets. the values of ndcg are shown in table 3 . the dock study of three proteins with an ndcg of 0.8503 and 0.9122, respectively (the detailed docking result is shown in table s2 ) proves that the result of the dock study of 2v7o is consistent with the experimental pld 50 , so the protein 2v7o was utilized for the ligand interaction analysis. table 3 . ranking results by experimental and predicted pld 50 and fit score. experimental pld 50 fit score (2v7o) fit score (2vz6) 6 1 3 3 20 2 1 12 12 3 4 9 1 4 2 4 11 5 7 2 14 6 8 13 16 7 5 6 7 8 17 15 8 9 10 11 27 10 23 17 13 11 12 19 15 12 11 5 32 13 18 18 5 14 22 8 33 15 13 29 21 16 15 1 25 17 9 20 22 18 25 25 17 19 20 16 28 20 24 30 9 21 16 32 29 22 32 14 2 23 30 24 30 24 31 26 18 25 21 27 10 26 26 21 23 27 29 31 31 28 33 7 26 29 14 23 4 30 28 33 3 31 6 10 19 32 27 28 24 33 19 22 ndcg 1 0.9122 0.8503 the 3d-qstr contour maps were utilized to visualize the information on the comfa and comsia model properties in three-dimensional space. these maps used characteristics of compounds that are crucial for activity and display the regions around molecules where the variance of activities is expected based on physicochemical property changes in molecules [52] . the analysis of favorable and unfavorable regions of steric, electrostatic, hydrophobic, hbd, and hba atom fields contribute to the realization of the relationship between the aconitine alkaloid's toxic activity and its structure. steric and electrostatic contour maps of the comfa qstr model are shown in figure 4a ,b, respectively. hydrophobic, hbd, and hba contour maps of the comsia qstr model are shown in figure 4c -e. compound 6 has the most toxic activity, so it was chosen as the reference structure for the generation of the comfa and comsia contour maps. in the case of the comfa study, the steric contour map around compound 6 is shown in figure 6a . the yellow regions near r2, r7, and r6 showed the substituents of the molecule, which proved that these positions were not ideal for sterically favorable functional groups. therefore, compounds 19, 24, and 26 (with pld 50 values of 1.17, 0.84, and 1.82, respectively), which consist of sterically esterified moieties at positions r2 and r7, were less toxic than compounds 6 and 20 (with pld 50 values of 5.00 and 4.95), which were substituted by a small hydroxyl group, and compound 3 (with a pld 50 value of 1.44) has less toxic activity due to the esterified moiety in r6. the green regions, sterically favorable the comfa electrostatic contour map is shown in figure 6b . the blue region near the r2 and r7 substitution revealed that the replacement of electropositive groups is in favor of toxicity. this can be proven by the fact that the compounds with hydroxy in these two positions had higher pld 50 values than the compound with acetoxy or no substituents. the red region surrounding molecular scaffolds was not distinct, which revealed that there was no connection between the electronegative and the toxicity. the comsia hydrophobic contour map is shown in figure 6c . the r2, r6, and r7 around the white region indicated that the hydrophobic groups were unfavorable for the toxicity, so the esterification of hydrophilic hydroxyl or dehydroxylation decreased the toxicity, which is consistent with the steric and electrostatic contour map. the yellow contour map near the r12 manifested that the hydrophilic hydroxy was unfavorable to the toxicity, which can be validated by the fact that aconitine alkaloids with hydroxy substituents in r12 (compound 10, with a pld 50 the ppi network of aconitine alkaloids cardiotoxicity was divided into nine clusters using clusterone. statistical parameters are shown in figure 5 . six clusters, namely clusters 1, 3, 4, 5, 7, and 9, which possess quality scores higher than 0.5, a density higher than 0.45, and a p-value less than 0.05, were selected for further analysis (in figure 7) . clusters 1, 4, and 7 consisted of proteins mainly involved in the effects of various calcium, potassium, and sodium channels. cluster 1 mainly the comsia contour map of hbd is shown in figure 6d . the cyan regions at r2, r6, and r7 represented a favorable condition for the hbd atom, which clearly validated the fact that the compounds with hydroxy in this region show potent toxicity. a purple region was found near r12, which proved that the hbd atom (hydroxyl) in this region has an adverse effect on toxicity. the hba contour map is shown in figure 6 . the magenta region around r1 substitution proved that this substitution was favorable to the hba atom, so compounds 13, 15, 32, and 33 with the hba atom in the r1 substitution exhibit more potent toxicity (with pld 50 values of 3.52, 3.30, 3.16, and 2.84) than compounds with methoxymethyl substituents (compounds 19, 24, and 26 with pld 50 values of 1.17, 0.84, and 1.82). the red contour map where hba atoms are unfavorable for the toxicity was positioned around r2 and r6. these contours were well validated by the lower pld 50 value of compounds with carbonyl in these substitutions. the ppi network of aconitine alkaloids cardiotoxicity was divided into nine clusters using clusterone. statistical parameters are shown in figure 5 . six clusters, namely clusters 1, 3, 4, 5, 7, and 9, which possess quality scores higher than 0.5, a density higher than 0.45, and a p-value less than 0.05, were selected for further analysis (in figure 7) . clusters 1, 4, and 7 consisted of proteins mainly involved in the effects of various calcium, potassium, and sodium channels. cluster 1 mainly consisted of three channel types related to the cardiotoxicity of aconitine alkaloids, cluster 4 contained calcium and sodium channels and some channel exchangers (such as ryr1 and ryr2), and cluster 7 mainly consisted of various potassium channels. all of these findings are consistent with previous research about the arrhythmogenic properties of the toxicity of aconitine alkaloids: the aconitine binds to ion channels and affects their open state, and thus the corresponding ion influx into the cytosol [53] [54] [55] . the channel exchangers play a crucial role in keeping the ion transportation and homeostasis inside and outside of the cell. cluster 9 contained some regulatory proteins that can activate or repress the ion channels through the protein expression level. atp2a1, ryr2, ryr1, cacna1c, cacna1d, and cacna1s mediate the release of calcium, thereby playing a key role in triggering cardiac muscle contraction and maintaining the calcium homeostasis [56, 57] . aconitine may cause aberrant channel activation and lead to cardiac arrhythmia. clusters 3 and 5 consisted of camp-dependent protein kinase (capk), cgmp-dependent protein kinase (cgpk), and guanine nucleotide binding protein (g protein). they have not been fully studied to prove whether the cardiotoxicity induced by aconitine alkaloids is linked to the capk, cgpk, and g proteins; however, some studies have shown that cardiotoxicity-related protein kcnj3 (potassium inwardly-rectifying channel) is controlled by g proteins and the cardiac sodium/calcium exchanger and is said to be regulated by capk and cgpk [58, 59] . the result of clusterone indicated that the constructed network is consistent with existing studies and that the network can be used to screen essential proteins in the cytonca plugin. the protein 2v7o belonging to the camkii (calcium/calmodulin (ca 2+ /cam)-dependent serine/threonine kinases ii) isozyme protein family plays a central role in cellular signaling by transmitting ca 2+ signals. the camkii enzymes transmit calcium ion (ca 2+ ) signals released inside the cell by regulating signal transduction pathways through phosphorylation. ca 2+ first binds to the small regulatory protein cam, and this ca 2+ /cam complex then binds to and activates the kinase, which then phosphorylates other proteins such as ryanodine receptor and sodium/calcium exchanger. thus, these proteins are related to the cardiotoxicity induced by aconitine alkaloids [60] [61] [62] . the excessive activity of camkii has been observed in some structural heart disease and arrhythmias [63] , and past findings demonstrate neuroprotection in neuronal cultures treated with inhibitors of camkii immediately prior to excitotoxic activation of the camkii [64] . the acute cardiotoxicity of the aconitine alkaloids is possibly related to this target. based on the analysis of the ppi network above, camkii was selected as the potential target for further molecular docking and dynamic simulation. the dock result of 2v7o is shown in figure 8a . compound 20 has the highest fit scores, so it was selected as the template for conformational analysis. the mechanisms of camkii activation and inactivation are shown in figure 8b . compound 20 affects the normal energy metabolism of the myocardial cell via binding in the atp-competitive site in figure 8c . the inactive state of the camkii was regulated by cask-mediated t306/t307 phosphorylation, and this state can be inhibited by the binding of compound 20 in the atp-competitive site. such binding moves camkii toward a ca 2+ /cam-dependent activation active state and a ca 2+ /cam-dependent activation through structural rearrangement of the inhibitory helix caused by ca 2+ /cam binding and the subsequent autophosphorylation of t287 [65] , which will induce the excessive activity of camkii and dynamic imbalance of the calcium ions in the myocardial cell, eventually leading to heart disease and arrhythmias. molecules 2018, 23, x for peer review 10 of 24 channel) is controlled by g proteins and the cardiac sodium/calcium exchanger and is said to be regulated by capk and cgpk [58, 59] . the result of clusterone indicated that the constructed network is consistent with existing studies and that the network can be used to screen essential proteins in the cytonca plugin. the protein 2v7o belonging to the camkii (calcium/calmodulin (ca 2+ /cam)-dependent serine/threonine kinases ii) isozyme protein family plays a central role in cellular signaling by transmitting ca 2+ signals. the camkii enzymes transmit calcium ion (ca 2+ ) signals released inside the cell by regulating signal transduction pathways through phosphorylation. ca 2+ first binds to the small regulatory protein cam, and this ca 2+ /cam complex then binds to and activates the kinase, which then phosphorylates other proteins such as ryanodine receptor and sodium/calcium the information of a binding pocket of a receptor for its ligand is very important for drug design, particularly for conducting mutagenesis studies [28] . as has been reported in the past [66] , the binding pocket of a protein receptor to a ligand is usually defined by those residues that have at least one heavy atom within a distance of 5 å from a heavy atom of the ligand. such a criterion was originally used to define the binding pocket of atp in the cdk5-nck5a complex [20] , which was later proved to be very useful in identifying functional domains and stimulating the relevant truncation experiments. a similar approach has also been used to define the binding pockets of many other receptor-ligand interactions important for drug design [30, 31, 33, [67] [68] [69] [70] . the information of a binding pocket of camkii for the aconitine alkaloids will serve as a guideline for designing drugs with similar scaffolds, particularly for conducting mutagenesis studies. in figure 8a , four top fit scores-compounds 1, 6, 12, and 20-generated similar significant interactions with amino acid residues around the atp-competitive binding pocket. four compounds formed with many van der waals interactions within the noncompetitive inhibitor pocket through amino acid residues such as asp157, lys43, glu140, lys22, and leu143. the ligand-receptor interaction showed that the hydroxy in r2 formed a side chain donor interaction with asp157. in addition, the hydroxy in r6 and r7 also formed a side chain acceptor interaction with glu140 and ser26, respectively (the docking result of compounds 6 and 12 in figure 8a ). these results correspond to the comfa and comsia contour maps. however, the small electropositive and hydrophilic group in r2, r6, and r7 possess a certain enhancement function to toxicity. there were aromatic interactions between the phenyl group in r9 and amino acid residues. the phenyl group in r9 formed aromatic interactions with leu20, leu142, and phe90, while the small group hydroxyl did not form any interaction with asp91, which demonstrate that bulky phenyl group is crucial to this binding pattern and toxicity. this was mainly equal to the comfa steric contour map, where r9 was ideal for sterically favorable groups. the methoxymethyl r1 generated backbone acceptor with lys43, which correspond to the comsia hba contour map, where r1 was favorable for the hba atom. compound 20 docked into 2v7o, and the atp-competitive pocket was painted green; the t287, t307, and t308 phosphorylation sites were painted green, orange, and yellow, respectively; the inhibitory helix was painted red. the result of md simulation is shown in figure 9 . the red plot represented the rmsd values of the docked protein. the values of rmsd reached 2.41 å in 1.4 ns and then remained between 2 and 2.5 å throughout the simulation for up to 5 ns. the averaged value of the rmsd was 2.06 å. the md simulation demonstrated that the ligand was stabilized in the active site. finally, we combined the ligand-based 3d-qstr analysis with the structure-based molecular docking study to identify the necessary moiety related to the cardiotoxicity mechanism of the aconitine alkaloids (in figure 10 ). finally, we combined the ligand-based 3d-qstr analysis with the structure-based molecular docking study to identify the necessary moiety related to the cardiotoxicity mechanism of the aconitine alkaloids (in figure 10 ). to build the ppi network of aconitine alkaloids, literature from 1 january 2007 to 31 february 2017 was retrieved from pubmed (http://pubmed.cn/) and web of science (http://www.isiknowledge.com/) with the mesh word "aconitine" and "toxicity" and without language restriction. all documents about cardiotoxicity caused by aconitine alkaloids were collected. the proteins related to the aconitine alkaloids cardiotoxicity of this decade were gathered and taken as the input protein in the string (https://string-db.org/) database [51, 71] , used to search for related proteins or pathways that had been reported. finally, all the proteins and its partners were recorded in excel in order to import information and build a ppi network in cytoscape software. cytoscape is a free, open-source, java application for visualizing molecular networks and integrating them with gene expression profiles [71, 72] . plugins are available for network and molecular profiling analyses, new layouts, additional file format support, making connections with figure 10 . crucial requirement of cardiotoxicity mechanism was obtained from the ligand-based 3d-qstr and structure-based molecular docking study. to build the ppi network of aconitine alkaloids, literature from 1 january 2007 to 31 february 2017 was retrieved from pubmed (http://pubmed.cn/) and web of science (http://www.isiknowledge.com/) with the mesh word "aconitine" and "toxicity" and without language restriction. all documents about cardiotoxicity caused by aconitine alkaloids were collected. the proteins related to the aconitine alkaloids cardiotoxicity of this decade were gathered and taken as the input protein in the string (https://string-db.org/) database [51, 71] , used to search for related proteins or pathways that had been reported. finally, all the proteins and its partners were recorded in excel in order to import information and build a ppi network in cytoscape software. cytoscape is a free, open-source, java application for visualizing molecular networks and integrating them with gene expression profiles [71, 72] . plugins are available for network and molecular profiling analyses, new layouts, additional file format support, making connections with databases, and searching within large networks [71] . clusterone (clustering with overlapping neighborhood expansion) of cytoscape was utilized to cluster the ppi network into overlapping sub-graphs of highly interconnected nodes. clusterone is a plugin for detecting and clustering potentially overlapping protein complexes from ppi data. the quality of a group was assessed by the number of sub-graphs, p-values, and density. the cluster was discarded when the number of sub-graphs was smaller than 3, the density was less than 0.45, the quality was less than 0.5, and the p-value was under 0.05 [73] . the clustering results of the clusterone are instrumental to understanding how the reliability of the ppi network relates to aconitine alkaloids' cardiotoxicity. cytonca is a plugin in cytoscape integrating calculation, evaluation, and visualization analysis for multiple centrality measures. there are eight centrality measurements provided by cytonca: betweenness, closeness, degree, eigenvector, local average connectivity-based, network, subgraph, and information centrality [74] . the primary purpose of the centrality analysis was to confirm the essential proteins in the pre-built ppi network. the three centrality measurements in the cytonca plugin-subgraph centrality, betweenness centrality, and closeness centrality-were used for evaluating and screening the essential protein in the merged target network. the subgraph centrality characterizes the participation of each node in all subgraphs in a network. smaller subgraphs are given more weight than larger ones, which makes this measurement an appropriate one for characterizing network properties. the subgraph centrality of node "u" can be calculated by [75] µ l (u) is the uth diagonal entry of the lth power of the weight adjacency matrix of the network. v 1 , v 2 , . . . , v n is be an orthonormal basis composed of r n composed by eigenvectors of a associated to the eigenvalues λ 1 , λ 2 , . . . , λ n v u v , which is the uth component of v v [75] . the betweenness centrality finds a wide range of applications in network theory. it represents the degree to which nodes stand between each other. betweenness centrality was devised as a general measure of centrality. it is applicable to a wide range of problems in network theory, including problems related to social networks, biology, transport, and scientific cooperation. the betweenness centrality of a node u can be calculated by [76] ρ (s, t) is the total number of shortest paths from node s to node ρ (s, u, t), which is the number of those paths that pass through u. closeness centrality of a node is a measure of centrality in a network, calculated as the sum of the length of the shortest paths between the node and all other nodes in the graph. thus, the more central a node is, the closer it is to all other nodes. the closeness centrality of a node u can be calculated by [77] |nu| is the number of node u's neighbors, and dist (u, v) is the distance of the shortest path from node u to node v. pharmmapper serves as a valuable tool for identifying potential targets for a novel synthetic compound, a newly isolated natural product, a compound with known biological activity, or an existing drug [78] . of all the aconitine alkaloids in this research, compounds 6, 12, and 20 exhibited the most toxic activity and were used for the potential target prediction. the mol2 format of three compounds was submitted to the pharmmapper server. the parameters of generate conformers and maximum generated conformations was set as on and 300, respectively. other parameters used default values. finally, the result of the clusterone and pharmmapper will be combined together to select the potential targets for the following docking study [78] . comparative molecular field analysis (comfa) and comparative molecular similarity index analysis (comsia) are efficient tools in ligand-based drug design and are in use for contour map generation and identification of favorable and unfavorable regions in a moiety [52, 79] . the comfa consists of a steric and electrostatic contour map of molecules that are correlated with toxic activity, while the comsia consists of hydrophobic field, hydrogen bond donor (hbd)/hydrogen bond acceptor (hba) [80] , and steric/electrostatic fields that are correlated with toxic activity. the comfa and comsia have been utilized to generate a 3d-qstr model [81] . all molecule models and the generation of 3d-qstr were performed with sybyl x2.0. alkaloids in mice with ld 50 values listed in table 4 were extracted from recent literature [70] . the ld 50 values of all aconitine alkaloids were converted into pld 50 with a standard tripos force field. these pld50 values were used as a dependent variable, while comfa and comsia descriptors were used as an independent variable. the sketch function of sybyl x2.0 was utilized to illustrate structure and charges, and was calculated by the gasteiger-huckel method. additionally, the tripose force field was utilized for energy minimization of these aconitine alkaloid molecules [81] . the 31 molecules were divided into a ratio of 3:1. the division was done in a way that showed that both datasets are balanced and consist of both active and less active molecules [81] . the reliability of the 3d-qstr model depends on the database molecular alignment. the most toxic aconitine alkaloids (compound 6) was selected as the template molecule, and the tetradecahydro-2h-3,6,12-(epiethane [1,1,2] triyl)-7,9-methanonaphtho [2,3-b] azocine was selected as the common moiety. pls (partial least squares) techniques are associated with field descriptors with activity values such as [80] leave one out (loo) values, the optimal number of components, the standard error of estimation (see), cross-validated coefficients (q 2 ), and the conventional coefficient (r 2 ). these statistical data are pivotal in the evaluation of the 3d-qstr model and can be worked out in the pls method [81] . the model is said to be good when the q 2 value is more than 0.5 and the r 2 value is more than 0.6. the q 2 and r 2 values reflect a model's soundness. the best model has the highest q 2 and r 2 values, the lowest see, and an optimal number of components [80, 82, 83] . in the case of comfa and comsia analysis, the values of the optimal number of components, see, and q 2 can be worked out by loo validation, with use sampls turned on and components set to 5, while in the process of calculating r 2 , the use sampls was turned off and the column filtration was set to 2.0 kcal mol −1 in order to speed up the calculation without the need to sacrifice information content [81] [82] [83] [84] . therefore, components were set to 6 and 4, respectively, which were optimal numbers of components calculated by performing a sampls run. see and r 2 were utilized to assess the non-cross validated model. the applicability domain (ad) of the topomer comfa and comsia model was confirmed by the williams plot of residuals vs. leverage. leverage of a query chemical is proportional to its mahalanobis distance measure from the centroid of the training set [85, 86] . the leverages are calculated for a given dataset x by obtaining the leverage matrix (h) with the equation below: x is the model matrix, while xt is its transpose matrix. the plot of standardized residuals vs. leverage values was drawn, and compounds with standardized residuals greater than three standard deviation units (±3σ) were considered as outliers [85] . the critical leverage value is considered 3 p/n, where p is the number of model variables plus one, and n is the number of objects used to calculate the model. h > 3 p/n mean predicted response is not acceptable [85] [86] [87] . (cadd) software program that incorporates the functions of qsar, molecular docking, molecular dynamics, adme (absorption, distribution, metabolism, and excretion), and homologous modeling. all of these functions are regarded as conducive instruments in the field of drug discovery and biochemistry. the molecular docking and dynamics technology were performed in moe2016 software to detect the stability and affinity between the ligands and predictive targets [88, 89] . the docking process involves the prediction of ligand conformation and orientation within a targeted binding site. docking analysis is an important step in the docking process. it has been widely used to study the reasonable binding mode and obtain information of interactions between amino acids in active protein sites and ligands. the molecular docking analysis was carried out to determine the toxicity-related moiety of aconitine alkaloids through the ligand-amino-acid interaction function in moe2015. the pdb format of 2v7o and 2vz6 was downloaded from the pdb (protein data bank) database (https://www.rcsb.org/), and the mol2 format of compounds was from the sybyl software of qstr research. the structure preparation function in moe software will be carried out to minimize the energy and optimize the structure of the protein skeleton. based on the london dg score and induced fit refinement, all compounds will be docked into the active site of every potential target by taking score values as the scoring function [90] . the dcg (discounted cumulative gain) algorithm was utilized to examine the consistency between the ranking result of pld 50 and our research (fit scores of dock study). they rely on the formula that refers to pld 50 . the idcg (ideal dcg) refers to the ordered pld 50 values. the closer the normalized discounted cumulative gain (ndcg) value is to 1, the better the consistency [91] . preliminary md simulations for the model protein were performed using the program namd (nanoscale molecular dynamics program, v 2.9), and all files were generated using visual molecular dynamics (vmd). namd is a freely available software designed for high-performance simulation of large biomolecular systems [92] . during the md simulation, minimization and equilibration of original and docked proteins occurred in a 15 å3 size water box. a charmm 22 force field file was applied for energy minimization and equilibration with gasteiger-huckel charges using boltzmann initial velocity [93, 94] . integrator parameters also included 2 fs/step for all rigid bonds and nonbonded frequencies were selected for 1 å and full electrostatic evaluations for 2 å were used with 10 steps for each cycle [93] . the particle mesh ewald method was used for electrostatic interactions of the simulation system periodic boundary conditions with grid dimensions of 1.0 å [94] . the pressure was maintained at 101.325 kpa using the langevin piston and the temperature was controlled at 310 k using langevin dynamics. covalent interactions between hydrogen and heavy atoms were constrained using the shake/rattle algorithm. finally, 5 ns md simulations for original and docked protein were carried out for comparing and verifying the binding affinity and stability of the ligand-receptor complex. the method combining network analysis and the in silico method was carried out to illustrate the qstr and toxic mechanisms of aconitine alkaloids. the 3d-qstr was built in sybyl with internal robustness and external high prediction, enabling identification of pivotal molecule moieties related to toxicity in aconitine alkaloids. the comfa model had q 2 , r 2 , optimum component, and correlation coefficient (r 2 ) values of 0.624, 0.966, 6, and 0. 9698, respectively, and the comsia model had q 2 , r 2 , optimum component, and correlation coefficient (r 2 ) values of 0.719, 0.901, 4, and 0.9770. the network was built with cytoscape software and the string database, which demonstrated the reliability of cluster analysis. the 2v7o and 2vz6 proteins were identified as potential targets with the cytonca plugin with pharmmapper server for interactions between the aconitine alkaloids and key amino acids in the dock study. the result of the dock study demonstrates the consistency of the experimental pld 50 . the md simulation indicated that aconitine alkaloids exhibit potent binding affinity and stability to the receptor camk2g. finally, we incorporate pivotal molecule moieties and ligand-receptor interactions to realize the qstr of aconitine alkaloids. this research serves as a guideline for studies of toxicity, including neuro-, reproductive, and embryo-toxicity. with a deep understanding of the relationship between toxicity and structure of aconitine alkaloids, subsequent structural modification of aconitine alkaloids can be carried out to enhance their efficacy and to reduce their toxic side effects. based on such research, aconitine alkaloids can bring us closer to medical and clinical 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this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-001244-qdld7hdc authors: fan, yue-nong; xiao, xuan; min, jian-liang; chou, kuo-chen title: inr-drug: predicting the interaction of drugs with nuclear receptors in cellular networking date: 2014-03-19 journal: int j mol sci doi: 10.3390/ijms15034915 sha: doc_id: 1244 cord_uid: qdld7hdc nuclear receptors (nrs) are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. therefore, nrs have become a frequent target for drug development. during the process of developing drugs against these diseases by targeting nrs, we are often facing a problem: given a nr and chemical compound, can we identify whether they are really in interaction with each other in a cell? to address this problem, a predictor called “inr-drug” was developed. in the predictor, the drug compound concerned was formulated by a 256-d (dimensional) vector derived from its molecular fingerprint, and the nr by a 500-d vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the svm (support vector machine) algorithm. compared with the existing prediction methods in this area, inr-drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at http://www.jci-bioinfo.cn/inr-drug/, which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. it is anticipated that the inr-drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. with the ability to directly bind to dna ( figure 1 ) and regulate the expression of adjacent genes, nuclear receptors (nrs) are a class of ligand-inducible transcription factors. they regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology [1] [2] [3] . the nr superfamily has been classified into seven families: nr0 (knirps or dax like) [4, 5] ; nr1 (thyroid hormone like), nr2 (hnf4-like), nr3 (estrogen like), nr4 (nerve growth factor ib-like), nr5 (fushi tarazu-f1 like), and nr6 (germ cell nuclear factor like). since they are involved in almost all aspects of human physiology and are implicated in many major diseases such as cancer, diabetes and osteoporosis, nuclear receptors have become major drug targets [6, 7] , along with g protein-coupled receptors (gpcrs) [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] , ion channels [18] [19] [20] , and kinase proteins [21] [22] [23] [24] . identification of drug-target interactions is one of the most important steps for the new medicine development [25, 26] . the method usually adopted in this step is molecular docking simulation [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] . however, to make molecular docking study feasible, a reliable 3d (three dimensional) structure of the target protein is the prerequisite condition. although x-ray crystallography is a powerful tool in determining protein 3d structures, it is time-consuming and expensive. particularly, not all proteins can be successfully crystallized. for example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. therefore, so far very few membrane protein 3d structures have been determined. although nmr (nuclear magnetic resonance) is indeed a very powerful tool in determining the 3d structures of membrane proteins as indicated by a series of recent publications (see, e.g., [44] [45] [46] [47] [48] [49] [50] [51] and a review article [20] ), it is also time-consuming and costly. to acquire the 3d structural information in a timely manner, one has to resort to various structural bioinformatics tools (see, e.g., [37] ), particularly the homologous modeling approach as utilized for a series of protein receptors urgently needed during the process of drug development [19, [52] [53] [54] [55] [56] [57] . unfortunately, the number of dependable templates for developing high quality 3d structures by means of homology modeling is very limited [37] . to overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter. the results thus obtained can be used to pre-exclude the compounds identified not in interaction with the nuclear receptors, so as to timely stop wasting time and money on those unpromising compounds [58] . actually, based on the functional groups and biological features, a powerful method was developed recently [59] for this purpose. however, further development in this regard is definitely needed due to the following reasons. (a) he et al. [59] did not provide a publicly accessible web-server for their method, and hence its practical application value is quite limited, particularly for the broad experimental scientists; (b) the prediction quality can be further enhanced by incorporating some key features into the formulation of nr-drug (nuclear receptor and drug) samples via the general form of pseudo amino acid composition [60] . the present study was initiated with an attempt to develop a new method for predicting the interaction of drugs with nuclear receptors by addressing the two points. as demonstrated by a series of recent publications [10, 18, [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] and summarized in a comprehensive review [60] , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: (a) select or construct a valid benchmark dataset to train and test the predictor; (b) represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; (c) introduce or develop a powerful algorithm or engine to operate the prediction; (d) properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; (e) establish a user-friendly web-server for the predictor that is accessible to the public. below, let us elaborate how to deal with these steps. the data used in the current study were collected from kegg (kyoto encyclopedia of genes and genomes) [71] at http://www.kegg.jp/kegg/. kegg is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. here, the benchmark dataset can be formulated as where is the positive subset that consists of the interactive drug-nr pairs only, while the negative subset that contains of the non-interactive drug-nr pairs only, and the symbol represents the union in the set theory. the so-called "interactive" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the kegg database [71] ; while the "non-interactive" pair means that its two counterparts are not interacting with each other in the drug-target networks. the positive dataset contains 86 drug-nr pairs, which were taken from he et al. [59] . the negative dataset contains 172 non-interactive drug-nr pairs, which were derived according to the following procedures: (a) separating each of the pairs in into single drug and nr; (b) re-coupling each of the single drugs with each of the single nrs into pairs in a way that none of them occurred in ; (c) randomly picking the pairs thus formed until reaching the number two times as many as the pairs in . the 86 interactive drug-nr pairs and 172 non-interactive drug-nr pairs are given in supplementary information s1, from which we can see that the 86 + 172 = 258 pairs in the current benchmark dataset are actually formed by 25 different nrs and 53 different compounds. since each of the samples in the current network system contains a drug (compound) and a nr (protein), the following procedures were taken to represent the drug-nr pair sample. first, for the drug part in the current benchmark dataset, we can use a 256-d vector to formulate it as given by where d represents the vector for a drug compound, and d i its i-th (i = 1,2, ,256) component that can be derived by following the "2d molecular fingerprint procedure" as elaborated in [10] . the 53 molecular fingerprint vectors thus obtained for the 53 drugs in are, respectively, given in supplementary information s2. the protein sequences of the 25 different nrs in are listed in supplementary information s3. suppose the sequence of a nuclear receptor protein p with l residues is generally expressed by where 1 r represents the 1st residue of the protein sequence p , 2 r the 2nd residue, and so forth. now the problem is how to effectively represent the sequence of equation (3) with a non-sequential or discrete model [72] . this is because all the existing operation engines, such as covariance discriminant (cd) [17, 65, [73] [74] [75] [76] [77] [78] [79] , neural network [80] [81] [82] , support vector machine (svm) [62] [63] [64] 83] , random forest [84, 85] , conditional random field [66] , nearest neighbor (nn) [86, 87] ; k-nearest neighbor (knn) [88] [89] [90] , oet-knn [91] [92] [93] [94] , and fuzzy k-nearest neighbor [10, 12, 18, 69, 95] , can only handle vector but not sequence samples. however, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction. facing such a dilemma, can we find an approach to partially incorporate the sequence-order effects? actually, one of the most challenging problems in computational biology is how to formulate a biological sequence with a discrete model or a vector, yet still keep considerable sequence order information. to avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition [96, 97] or chou's pseaac [98] was proposed. ever since the concept of pseaac was proposed in 2001 [96] , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides [99] , predicting protein subcellular location [100] [101] [102] [103] [104] [105] [106] , predicting membrane protein types [107, 108] , predicting protein submitochondria locations [109] [110] [111] [112] , predicting gaba(a) receptor proteins [113] , predicting enzyme subfamily classes [114] , predicting antibacterial peptides [115] , predicting supersecondary structure [116] , predicting bacterial virulent proteins [117] , predicting protein structural class [118] , predicting the cofactors of oxidoreductases [119] , predicting metalloproteinase family [120] , identifying cysteine s-nitrosylation sites in proteins [66] , identifying bacterial secreted proteins [121] , identifying antibacterial peptides [115] , identifying allergenic proteins [122] , identifying protein quaternary structural attributes [123, 124] , identifying risk type of human papillomaviruses [125] , identifying cyclin proteins [126] , identifying gpcrs and their types [15, 16] , discriminating outer membrane proteins [127] , classifying amino acids [128] , detecting remote homologous proteins [129] , among many others (see a long list of papers cited in the references section of [60] ). moreover, the concept of pseaac was further extended to represent the feature vectors of nucleotides [65] , as well as other biological samples (see, e.g., [130] [131] [132] ). because it has been widely and increasingly used, recently two powerful soft-wares, called "pseaac-builder" [133] and "propy" [134] , were established for generating various special chou's pseudo-amino acid compositions, in addition to the web-server "pseaac" [135] built in 2008. according to a comprehensive review [60] , the general form of pseaac for a protein sequence p is formulated by where the subscript  is an integer, and its value as well as the components ( 1, 2, , ) u u   will depend on how to extract the desired information from the amino acid sequence of p (cf. equation (3)). below, let us describe how to extract useful information to define the components of pseaac for the nr samples concerned. first, many earlier studies (see, e.g., [136] [137] [138] [139] [140] [141] ) have indicated that the amino acid composition (aac) of a protein plays an important role in determining its attributes. the aac contains 20 components with each representing the occurrence frequency of one of the 20 native amino acids in the protein concerned. thus, such 20 aac components were used here to define the first 20 elements in equation (4); i.e., (1) ( 1, 2, , 20) ii fi   (5) where f i (1) is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned. since aac did not contain any sequence order information, the following steps were taken to make up this shortcoming. to avoid completely losing the local or short-range sequence order information, we considered the approach of dipeptide composition. it contained 20 × 20 = 400 components [142] . such 400 components were used to define the next 400 elements in equation (4); i.e., (2) 20 ( 1, 2, , 400) jj fj where (2) j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned. to incorporate the global or long-range sequence order information, let us consider the following approach. according to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues [143] , gene doubling, and gene fusion. with the accumulation of these changes over a long period of time, many original similarities between initial and resultant amino acid sequences are gradually faded out, but the corresponding proteins may still share many common attributes [37] , such as having basically the same biological function and residing at a same subcellular location [144, 145] . to extract the sequential evolution information and use it to define the components of equation (4), the pssm (position specific scoring matrix) was used as described below. according to schaffer [146] , the sequence evolution information of a nuclear receptor protein p with l amino acid residues can be expressed by a 20 l matrix, as given by where (7) were generated by using psi-blast [147] to search the uniprotkb/swiss-prot database (the universal protein resource (uniprot); http://www.uniprot.org/) through three iterations with 0.001 as the e-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned. in order to make every element in equation (7) be scaled from their original score ranges into the region of [0, 1], we performed a conversion through the standard sigmoid function to make it become now we extract the useful information from equation (8) moreover, we used the grey system model approach as elaborated in [68] to further define the next 60 components of equation (4) ( 1, 2, , 20) in the above equation, w 1 , w 2 , and w 3 are weight factors, which were all set to 1 in the current study; f j (1) has the same meaning as in equation (5) where   and combining equations (5), (6), (10) and (12), we found that the total number of the components obtained via the current approach for the pseaac of equation (4) and each of the 500 components is given by (1) ( since the elements in equations (2) and (4) are well defined, we can now formulate the drug-nr pair by combining the two equations as given by   (19) where g represents the drug-nr pair, å the orthogonal sum, and the 256 + 500 = 756 components are defined by equations (2) and (18) . for the sake of convenience, let us use x i (i = 1, 2, , 756) to represent the 756 components in equation (19); i.e., (20) to optimize the prediction quality with a time-saving approach, similar to the treatment [148] [149] [150] , let us convert equation (20) to where the symbol means taking the average of the quantity therein, and sd means the corresponding standard derivation. in this study, the svm (support vector machine) was used as the operation engine. svm has been widely used in the realm of bioinformatics (see, e.g., [62] [63] [64] [151] [152] [153] [154] ). the basic idea of svm is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function. for a brief formulation of svm and how it works, see the papers [155, 156] ; for more details about svm, see a monograph [157] . in this study, the libsvm package [158] was used as an implementation of svm, which can be downloaded from http://www.csie.ntu.edu.tw/~cjlin/libsvm/, the popular radial basis function (rbf) was taken as the kernel function. for the current svm classifier, there were two uncertain parameters: penalty parameter c and kernel parameter  . the method of how to determine the two parameters will be given later. the predictor obtained via the aforementioned procedure is called inr-drug, where "i" means identify, and "nr-drug" means the interaction between nuclear receptor and drug compound. to provide an intuitive overall picture, a flowchart is provided in figure 2 to show the process of how the predictor works in identifying the interactions between nuclear receptors and drug compounds. to provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by chou [159] [160] [161] in predicting signal peptides was adopted. according to chou's formulation, the sensitivity, specificity, overall accuracy, and matthew's correlation coefficient can be respectively expressed as [62, [65] [66] [67] sn 1 where n  is the total number of the interactive nr-drug pairs investigated while n   the number of the interactive nr-drug pairs incorrectly predicted as the non-interactive nr-drug pairs; n  the total number of the non-interactive nr-drug pairs investigated while n   the number of the non-interactive nr-drug pairs incorrectly predicted as the interactive nr-drug pairs. according to equation (23) we can easily see the following. when 0 n    meaning none of the interactive nr-drug pairs was mispredicted to be a non-interactive nr-drug pair, we have the sensitivity sn = 1; while nn    meaning that all the interactive nr-drug pairs were mispredicted to be the non-interactive nr-drug pairs, we have the sensitivity sn = 0 . likewise, when 0 n    meaning none of the non-interactive nr-drug pairs was mispredicted, we have the specificity sp we have mcc = 0 meaning total disagreement between prediction and observation. as we can see from the above discussion, it is much more intuitive and easier to understand when using equation (23) to examine a predictor for its four metrics, particularly for its mathew's correlation coefficient. it is instructive to point out that the metrics as defined in equation (23) are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in [162] . how to properly test a predictor for its anticipated success rates is very important for its development as well as its potential application value. generally speaking, the following three cross-validation methods are often used to examine the quality of a predictor and its effectiveness in practical application: independent dataset test, subsampling or k-fold (such as five-fold, seven-fold, or 10-fold) crossover test and jackknife test [163] . however, as elaborated by a penetrating analysis in [164] , considerable arbitrariness exists in the independent dataset test. also, as demonstrated in [165] , the subsampling (or k-fold crossover validation) test cannot avoid arbitrariness either. only the jackknife test is the least arbitrary that can always yield a unique result for a given benchmark dataset [73, 74, 156, [166] [167] [168] . therefore, the jackknife test has been widely recognized and increasingly utilized by investigators to examine the quality of various predictors (see, e.g., [14, 15, 68, 99, 106, 107, 124, 169, 170] ). accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. as mentioned above, the svm operation engine contains two uncertain parameters c and  . to find their optimal values, a 2-d grid search was conducted by the jackknife test on the benchmark dataset . the results thus obtained are shown in figure 3 , from which it can be seen that the inr-drug predictor reaches its optimal status when c = 2 3 and 9 2    . the corresponding rates for the four metrics (cf. equation (23)) are given in table 1 , where for facilitating comparison, the overall accuracy acc reported by he et al. [59] on the same benchmark dataset is also given although no results were reported by them for sn, sp and mcc. it can be observed from the table that the overall accuracy obtained by inr-drug is remarkably higher that of he et al. [59] , and that the rates achieved by inr-drug for the other three metrics are also quite higher. these facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates. as mentioned above (section 3.2), the jackknife test is the most objective method for examining the quality of a predictor. however, as a demonstration to show how to practically use the current predictor, we took 41 nr-drug pairs from the study by yamanishi et al. [171] that had been confirmed by experiments as interactive pairs. for such an independent dataset, 34 were correctly identified by inr-drug as interactive pairs, i.e., sn = 34 / 41 = 82.92%, which is quite consistent with the rate of 79.07% achieved by the predictor on the benchmark dataset via the jackknife test as reported in table 1 . it is anticipated that the inr-drug predictor developed in this paper may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. since user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful predictors [98, 172] , a publicly accessible web-server for inr-drug was established. for the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using inr-drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. step 1. open the web server at the site http://www.jci-bioinfo.cn/inr-drug/ and you will see the top page of the predictor on your computer screen, as shown in figure 4 . click on the read me button to see a brief introduction about inr-drug predictor and the caveat when using it. step 2. either type or copy/paste the query nr-drug pairs into the input box at the center of figure 4 . each query pair consists of two parts: one is for the nuclear receptor sequence, and the other for the drug. the nr sequence should be in fasta format, while the drug in the kegg code beginning with the symbol #. examples for the query pairs input and the corresponding output can be seen by clicking on the example button right above the input box. step 3. click on the submit button to see the predicted result. for example, if you use the three query pairs in the example window as the input, after clicking the submit button, you will see on your screen that the "hsa:2099" nr and the "d00066" drug are an interactive pair, and that the "hsa:2908" nr and the "d00088" drug are also an interactive pair, but that the "hsa:5468" nr and the "d00279" drug are not an interactive pair. all these results are fully consistent with the experimental observations. it takes about 3 minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. step 4. click on the citation button to find the relevant paper that documents the detailed development and algorithm of inr-durg. step 5. click on the data button to download the benchmark dataset used to train and test the inr-durg predictor. step 6. the program code is also available by clicking the button download on the lower panel of figure 4 . nuclear receptors in cell life and death 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an integrated framework review: recent advances in developing web-servers for predicting protein attributes the authors would like to express their gratitude to the three anonymous reviewers, whose constructive comments are very helpful for strengthening the presentation of the paper. the authors declare no conflict of interest. key: cord-253987-83h861lp authors: tada, takuya; fan, chen; kaur, ramanjit; stapleford, kenneth a.; gristick, harry; nimigean, crina; landau, nathaniel r. title: a soluble ace2 microbody protein fused to a single immunoglobulin fc domain is a potent inhibitor of sars-cov-2 infection in cell culture date: 2020-09-17 journal: biorxiv doi: 10.1101/2020.09.16.300319 sha: doc_id: 253987 cord_uid: 83h861lp soluble forms of ace2 have recently been shown to inhibit sars-cov-2 infection. we report on an improved soluble ace2, termed a “microbody” in which the ace2 ectodomain is fused to fc domain 3 of the immunoglobulin heavy chain. the protein is smaller than previously described ace2-ig fc fusion proteins and contains an h345a mutation in the ace2 catalytic active site that inactivates the enzyme without reducing its affinity for the sars-cov-2 spike. the disulfide-bonded ace2 microbody protein inhibited entry of lentiviral sars-cov-2 spike protein pseudotyped virus and live sars-cov-2 with a potency 10-fold higher than unmodified soluble ace2 and was active after initial virus binding to the cell. the ace2 microbody inhibited the entry of ace2-specific β coronaviruses and viruses with the high infectivity variant d614g spike. the ace2 microbody may be a valuable therapeutic for covid-19 that is active against sars-cov-2 variants and future coronaviruses that may arise. as the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) continues to spread worldwide, there is an urgent need for preventative vaccine and improved therapeutics for treatment of covid-19. the development of therapeutic agents that block specific steps of the coronavirus replication cycle will be highly valuable both for treatment and prophylaxis. coronavirus replication consists of attachment, uncoating, replication, translation, assembly and release, all of which are potential drug targets. virus entry is particularly advantageous because as the first step in virus replication, it spares target cells from becoming infected and because drugs that block entry do not need to be cell permeable as the targets are externally exposed. in sars-cov-2 entry, the virus attaches to the target cell through the interaction of the spike glycoprotein (s) with its receptor, the angiotensin-converting enzyme 2 (ace2) (li, 2015; li et al., 2005; li et al., 2003) , a plasma membrane protein carboxypeptidase that degrades angiotensin ii to angiotensin-(1-7) [ang-(1-7)] a vasodilator that promotes sodium transport in the regulation of cardiac function and blood pressure (kuba et al., 2010; riordan, 2003; tikellis and thomas, 2012) . ace2 binding triggers s protein-mediated fusion of the viral envelope with the cell plasma membrane or intracellular endosomal membranes. the s protein is synthesized as a single polypeptide that is cleaved by the cellular protease furin into s1 and s2 subunits in the endoplasmic reticulum and then further processed by tmprss2 on target cells (glowacka et al., 2011; hoffmann et al., 2020; matsuyama et al., 2010; shulla et al., 2011) . the s1 subunit contains the receptor binding domain (rbd) which binds to ace2 while s2 mediates virus-cell fusion (belouzard et al., 2012; fehr and perlman, 2015; heald-sargent and gallagher, 2012; li et al., 2006; shang et al., 2020) . cells that express ace2 are potential targets of the virus. these include cells in the lungs, arteries, heart, kidney, and intestines (harmer et al., 2002; ksiazek et al., 2003; leung et al., 2003) . the use of soluble receptors to prevent virus entry by competitively binding to viral envelope glycoproteins was first explored for hiv-1 with soluble cd4. in early studies, a soluble form of cd4 deleted for the transmembrane and cytoplasmic domains was found to block virus entry in vitro (daar et al., 1990; haim et al., 2009; orloff et al., 1993; schenten et al., 1999; sullivan et al., 1998) . fusion of the protein to an immunoglobulin fc region, termed an "immunoadhesin", increased the avidity for gp120 by dimerizing the protein and served to increase the half-life of the protein in vivo. an enhanced soluble cd4-ig containing a peptide derived from the hiv-1 coreceptor ccr5 was found to potently block infection and to protect rhesus macaques from infection (chiang et al., 2012) . the soluble receptor approach to blocking virus entry has been recently applied to sars-cov-2 through the use of recombinant human soluble ace2 protein (hrsace2) (kuba et al., 2005; monteil et al., 2020; wysocki et al., 2010) or hrsace2-igg which encodes soluble ace2 and the fc region of the human immunoglobulin g (igg) (case et al., 2020; lei et al., 2020; qian and hu, 2020) which were shown to inhibit of sars-cov and sars-cov-2 entry in a mouse model. in phase 1 and phase 2 clinical trials (haschke et al., 2013; khan et al., 2017) , the protein showed partial antiviral activity but short halflive. addition of the fc region increased the half-life of the protein in vivo. a potential concern with the addition of the ig fc region is the possibility of enhancement, similar to what occurs with antibody-dependent enhancement in which anti-spike protein antibody attaches to fc receptors on immune cells, facilitating infection rather than preventing it (eroshenko et al., 2020) . we report, here on a soluble human ace2 "microbody" in which the ace2 ectodomain is fused to domain 3 of immunoglobulin g heavy chain fc region (igg-ch3) (maute et al., 2015) . the igg-ch3 fc domain served to dimerize the protein, increasing its affinity for the sars-cov-2 s and decreasing the molecular mass of the protein. the ace2 microbody did not bind to cell surface fc receptor, reducing any possibility of infection enhancement. mutation of the active site h345 to alanine in the ace2 microbody protein, a mutation that has been shown to inactivate ace2 catalytic activity (guy et al., 2005) , did not decrease its affinity for the s protein. the dimeric ace2 microbody had about 10-fold higher antiviral activity than soluble ace2, which was also a dimer, and high a higher affinity for virion binding. the ace2 microbody blocked virus entry into ace2.293t cells that over-expressed ace2 as well as all of the cell-lines tested and was fully active against the d614g variant spike protein and a panel of b coronavirus spike proteins. as a means to study sars-cov-2 entry, we developed an assay based on sars-cov-2 s protein pseudotyped lentiviral reporter viruses. the viruses package a lentiviral vector genome that encodes nanoluciferase and gfp separated by a p2a self-processing peptide, providing a convenient means to titer the virus, and the ability to use two different assays to measure infection. to pseudotype the virions, we constructed expression vectors for the full-length sars-cov-2 s and for a δ19 variant deleted for the carboxyterminal 19 amino acids that removes a reported endoplasmic reticulum retention sequence that blocks transit of the s protein to the cell surface (giroglou et al., 2004) ( figure 1a) . the vectors were constructed with or without a carboxy-terminal hemagglutinin (ha) epitope tag. pseudotyped viruses were produced in 293t cells cotransfected with the dual nanoluciferase/gfp reporter lentiviral vector plenti.gfp.nluc, gag/pol expression vector pmdl and full-length s protein, the δ19 s protein, vesicular stomatitis virus g protein (vsv-g) expression vector or without an envelope glycoprotein expression vector. immunoblot analysis showed that full-length and δ19 s proteins were expressed and processed into the cleaved s2 protein (s1 is not visible as it lacks an epitope tag). analysis of the virions showed that the δ19 s protein was packaged into virions at >20-fold higher levels than the full-length protein ( figure 1b ). this difference was not the result of differences in virion production as similar amounts of virion p24 were present in the cell supernatant. analysis of the transfected 293t cells by flow cytometry showed a minor increase in the amount of cell surface δ19 s protein as compared to full-length ( figure 1c ) suggesting that deletion of the endoplasmic reticulum retention signal was not the primary cause of the increased virion packaging of the δ19 s protein and may result from an inhibitory effect of the s protein cytoplasmic tail on virion incorporation. as a suitable target cell-line, we established a clonal, stably transfected 293t cell-line that expressed high levels of ace2 (figures 1d and s1). a comparison of the infectivity of the viruses on ace2.293t cells showed that the δ19 s protein pseudotype was about 2.5-fold more infectious than the full-length s protein pseudotype ( figure 1e ). the ha-tag had no effect on infectivity and the nevirapine control demonstrated that the luciferase activity was the result of bona fide infection and not carried-over luciferase in the virus-containing supernatant. to determine the cell-type tropism of the pseudotyped virus, we tested several standard laboratory cell-lines for susceptibility to infection to the δ19 s protein pseudotyped virus. the vsv-g pseudotype, which has very high infectivity on most celltypes was tested for comparison and virus lacking a glycoprotein was included to control for potential receptor-independent virus uptake. the results showed high infectivity of the δ19 s protein pseudotyped virus on ace2.293t cells, intermediate infectivity on 293t, vero, vero e6, a549, ace2.a549, caco2 and huh7 and low infectivity on a549, chme3, bhk and u937 ( figure 1f) . analysis by flow cytometry of cell surface ace2 levels showed high level expression on ace2.293t, intermediate levels expression on ace2.a549 and low to undetectable levels on a549, caco2 and huh7 ( figure s1 ). the low level of ace2 expression on cells such as vero and caco2 suggests that virus can use very small amounts of the receptor for entry. moreover, the pseudotyped virus is a highly sensitive means with which to detect virus entry. soluble ace2 and ace2-fc fusions have been shown to inhibit sars-cov-2 infection (case et al., 2020; kuba et al., 2010; lei et al., 2020; monteil et al., 2020; qian and hu, 2020; wysocki et al., 2010) . to increase the effectiveness of soluble ace2 and improve therapeutic potential, we generated an ace2-"microbody" in which the ace2 ectodomain was fused to a single igg ch3 domain of the igg fc region (figure 2a ). this domain contains the disulfide bonding cysteine residues of the igg fc that are required to dimerize the protein, which would serve to increase the ace2 microbody avidity for ace2. to prevent potential unwanted effects of the protein on blood pressure due to the catalytic activity of ace2, we mutated h345, one of the key active site histidine residues of ace2, to alanine, a mutation that has been shown to block catalytic activity (guy et al., 2005) . h345 lies underneath the s protein interaction site so was not predicted to interfere with s protein binding ( figure 2b ). for comparison, we constructed vector encoding soluble ace2 without the igg ch3. the proteins were purified from transfected 293t cells and purified to homogeneity by ni-nta agarose affinity chromatography followed by size exclusion chromatography ( figure s2 ). the oligomerization state of the proteins was analyzed by sds-page under nonreducing and reducing conditions. under reducing conditions, the ace2 and ace2.h345a microbody proteins and soluble ace2 ran at the 130 kda and 120 kda, consistent with their calculated molecular mass (figures 2c and s2 ). under nonreducing conditions, the ace2 microbody and ace2.h345a microbody proteins ran at 250 kda, consistent with dimers while the soluble ace2 ran as a monomer with a mass of 120 kda ( figure 2c) . analysis of the proteins by size-exclusion chromatography coupled with multi-angle light scattering (sec-mals) under nondenaturing conditions showed all three proteins to have a molecular mass consistent with dimers ( figure 2d ). the mass of the ace2 and ace2.h345a microbody proteins was 218 kda and 230 kda, respectively, while soluble ace2 was 180 kda. taken together, the results suggest that the ace2 microbody proteins are disulfide-bonded dimers while soluble ace2 is a nondisulfide-bonded dimer. to compare the relative ability of the soluble ace2 proteins to virions that display the s protein, we established a virion pull-down assay. ni-nta beads were incubated with a serial dilution of the carboxy-terminal his-tagged soluble ace2 proteins. free spike protein was removed and the beads were then incubated with a fixed amount of lentiviral pseudotyped virions. free virions were removed and the bound virions were quantified by immunoblot analysis for virion p24 capsid protein. to confirm that virus binding to the beads was specific for the bead-bound ace2, control virions lacking the spike were tested. the results showed that s protein pseudotyped virions bound to the beads while virions that lacked the s protein failed to bind, confirming that the binding was specific ( figure 3a) . in addition, a high titer human serum from a recovered individual blocked binding of the virions to the bead-bound ace2 microbody ( figure s5 ). analysis of the soluble ace2 and ace2 microbody proteins that had bound to the beads showed that similar amounts of each proteins had bound ( figure 3b ). immunoblot analysis of virion binding to the bead-bound soluble ace2 proteins showed that the wild-type and h345a microbody proteins both bound to virions more efficiently than soluble ace2 ( figure 3c) and that the ace2.h345a microbody bound more virions than the wild-type microbody protein. this was unexpected as h345 does not lie in the interaction surface with the s protein. to determine the relative antiviral activity of soluble ace2 and the ace2 microbody proteins, we tested their ability to block the infection sars-cov-2 δ19 s protein pseudotyped gfp/luciferase reporter virus. a fixed amount of pseudotyped reporter virus was incubated with the ace2 proteins and then used to infect ace2.293t cells. after 2 days, luciferase activity and the number of gfp+ cells in the infected cultures were analyzed. for comparison, a high titered recovered patient serum with a neutralizing titer of 1:330 (figure s5) was also tested. the results showed that soluble ace2 had moderate inhibitory activity with an ec50 of 1.24 µg/ml. the ace2 microbody was significantly more potent, with an ec50 of 0.36 µg/ml and the ace2.h345a microbody protein was somewhat more potent than the wild-type ace2 microbody with an ec50 of 0.15 µg/ml ( figure 4a ). inhibition of infection by the soluble ace2 proteins was comparable to recovered patient serum although it is not possible to directly compare the two inhibitors as the mass amount of anti-s protein antibody in the serum is not known. to confirm the results, we analyzed the infected cells by flow cytometry to determine the number of gfp+ cells. the inhibition curves were similar to the luciferase curves, confirming that the ace2 proteins had decreased the number of cells infected and did not simply reduce expression of the reporter protein ( figure 4b, top) . representative images of the gfp+ cells provide visual confirmation of the results ( figure 4b, below) . the inhibitory activity of the soluble ace2 proteins was specific for the sars-cov-2 s protein as they did not inhibit vsv-g pseudotyped virus ( figure 4c ). the ace2 microbody was somewhat more active when tested on untransfected 293t that express low levels of ace2 ( figure 4d ). to determine the ability of the ace2 microbody proteins to block the replication of live sars-cov-2, we used the replication-competent sars-cov-2, icsars-cov-2mng that encodes an mneongreen reporter gene in orf7 (xie et al., 2020) . serially diluted ace2 microbody proteins were incubated with the virus and the mixture was then used to infect ace2.293t cells. the results showed that 1-0.125 µg of ace2 microbody protein blocked live virus replication ( figure 4e ). soluble ace2 was less active; 1 µg of the protein had a 50% antiviral effect and the activity was lost with 0.5 µg. the antiviral activity of ace2 proteins against live virus was similar to pseudotyped virus, except that in the live virus assay, the wild-type and h345a microbodies were of similar potency. in the experiments described above, the proteins were incubated with virus prior to infection. to determine whether they would be active when at later time points, the ace2 proteins were tested in an "escape from inhibition" assay in which the soluble ace2 and ace2 microbody proteins were added to cells at the same time as virus or up to 6 hours post-infection. the results showed that addition of the microbody together with the virus (t0) blocked the infection by 80%. addition of the microbody 30 minutes postinfection maintained most of the antiviral effect, and even 2 hours post-infection the inhibitor blocked 55% of the infection. at 4 hours post-infection, the ace2 microbody retained its blocking activity at 10 µg/ml but was less active with decreasing amounts of inhibitor ( figure 5a) . these results suggest that the ace2 microbody is highly efficient at neutralizing the virus when present before the virus has had a chance to bind to the cell and that it maintains its ability to block infection when added together with the cells and even 2 hours after the virus has been exposed to cells, a time which most of the virus has not yet bound to the cell. to determine whether the ace2 microbody could prevent virus entry once the virus bound to the cell, the virus was prebound by incubating it with cells for 1 hour at 22°c, the unbound virus was removed and the ace2 microbody was added at increasing time points. the results showed that removal of the unbound virus after 1 hour incubation resulted in less infection as compared to when the virus was incubated with the cells for 4 hours, indicating that only a fraction of the virus had bound to cells. however, virus that was bound could be blocked by the ace2 microbody for another 30 minutes post-binding ( figure 5b) . the ability to block entry of the cell-bound virus suggests that virus binding results from a small number of spike molecules binding to ace2. over the next 30-60 minutes, additional spike:ace2 interactions form, escaping the ability of the ace2 microbody to block virus entry. the results demonstrate that the ace2 microbody is a highly potent inhibitor of free virus and maintains its antiviral activity against virus newlybound to the cell. cov-2 containing a d614g point mutation in the s protein has been found to be circulating in the human population with increasing prevalence (daniloski et al., 2020; eaaswarkhanth et al., 2020; korber et al., 2020; zhang et al., 2020) . the d614g mutation was found to decrease shedding of the spike protein from the virus and to assume a fusion-ready conformation, resulting in increased infectivity and most likely contributing to its increasing prevalence. to determine the ability of the soluble ace2 proteins to block entry of virus with the d614g s protein, we introduced the mutation into the δ19 s protein expression vector and generated pseudotyped reporter viruses ( figure 6a ). analysis of the infectivity of the d614g and wild-type pseudotyped viruses on the panel of cell-lines showed that the mutation increased the infectivity of virus 2-4 fold on 293t, ace2.293t, vero and veroe6 cells, consistent with previous reports (daniloski et al., 2020; yurkovetskiy et al., 2020; zhang et al., 2020) . infectivity of the mutated virus was also increased in a549, ace2.a549 caco2 although the overall infectivity of these cells was low (figure.6b). to determine the ability of the soluble ace2 proteins to neutralize the virus with the variant s protein, serial dilutions of the soluble ace2 proteins were tested for their ability to block wild-type and d614g s pseudotyped virus. the results showed that soluble ace2 had moderate antiviral activity against wild-type virus, while the wildtype and h345a microbody proteins were more potent ( figure 6c ). the ace2.h345a microbody was somewhat more active at low concentrations than the wild-type protein. to test the relative binding affinity of the soluble ace2 proteins for wild-type and d614g mutated spike, we tested the pseudotyped virions in the ace2-virus binding assay ( figure 6d) . the results showed that virus with the d614g s bound efficiently to soluble ace2. the results demonstrate the broad activity of the ace2 microbody. we report the development of a soluble form of ace2 in which the ectodomain of ace2 is fused to a single domain of the igg heavy chain fc. the domain renders the protein smaller than those fused to the full-length fc yet retains the cysteine residues required for dimerization and the ability to increase the in vivo half-life (maute et al., 2015) . the microbody protein was shown to be a disulfide-bonded dimer in contrast to soluble ace2 lacking the fc domain which was dimeric but not nondisulfide-bonded. although both proteins are dimeric, the ace2 microbody had about 10-fold more antiviral activity than soluble ace2 and bound to virions with a >4-fold increased affinity. while high affinity anti-spike rbd monoclonal antibodies that potently inhibit sars-cov-2 infection will be of great value in the treatment of covid-19, the soluble receptor proteins have advantageous features. the ace2 microbody is of fully human origin so should be relatively non-immunogenic. in addition, it is expected to be broadly active against mutated variant spike proteins that may arise in the human population. the microbody was fully active against virus with the d614g s protein, a variant of increasing prevalence with increased infectivity in vitro ( figure 6 ) and was highly active against ace2-specific s proteins from other b coronaviruses. it has been previously shown that a recombinant ace2-fc fusion had a major effect on blood pressure in a mouse model (liu et al., 2018) . it was therefore important to inactivate ace2 carboxypeptidase activity in microbody to decrease unwanted effects on blood pressure associated with its use therapeutically. the h345a mutation alters one of the histidine essential for ace2 catalytic activity yet did not impair antiviral activity against sars cov-2 or other b coronavirus spike proteins. in some of our analyses, the ace2.h345a microbody appeared to be more active than the wild-type protein although the significance of this difference was unclear as the two proteins had similar activity in the live virus replication assay. escape from inhibition studies provided insight into the kinetics of virus infection and the mechanism of inhibition by the soluble receptors. pretreatment of virus with the ace2 microbody potently neutralized the virus as did simultaneous treatment addition of virus and microbody to cells. furthermore, the protein retained its ability to prevent infection even when added to the culture at times after addition of virus, blocking infection by about 50% when added 1 hour after virus addition. the ace2 microbody was partially active even on virus that had already attached to the cell. when virus was pre-bound for 2 hours, a time at which about 10% of the infectious virus had bound the cell, the ace2 microbody retained the ability to prevent infection of about 50% of the bound virus ( figure 5a ). taken together, the experiments suggest a series of events in which the virus binds to cells over a period of about 4 hours. during this time, the ace2 microbody is highly efficient, neutralizing nearly all of the free virus. once the virus binds to the cell, the ace2 microbody retains its ability to block infection for about 30 min, suggesting that binding is initially mediated by a small number of s proteins and that over 2 hours, additional s proteins are recruited to interact with target cell ace2, a period during which the ace2 microbody remains able to block the viral fusion reaction. once a sufficient number of s protein:ace2 interactions have formed, the virus escapes neutralization. it was surprising that the ace2 microbody had more antiviral activity than soluble ace2 as both proteins are dimeric. in addition, the ace2 microbody protein showed somewhat better binding to virions than soluble ace2. the reasons for these differences are not clear. it is possible that the disulfide bonds of the ace2 microbody stabilize the dimer or that they position the individual monomers in a more favorable conformation to bind to the individual subunits of the s protein trimer. it is worth noting that in most of the experiments, we used ace2.293t cells that overexpress ace2 compared to the cell-lines tested. on untransfected 293t cells that express barely detectable levels of ace2, the antiviral activity of the microbody protein was increased, suggesting that the antiviral activity of the ace2 microbody may be under-estimated by the use of ace2 overexpressing cells. recent reports have described similar soluble ace2 proteins. recently soluble ace2-related inhibitor including rhace2 was shown to partially block infection (case et al., 2020; lei et al., 2020; monteil et al., 2020) although the proteins had a short half-life (wysocki et al., 2010 ) (< 2 hours in mice), limiting their clinical usefulness. in contrast a dimeric rhesus ace2-fc fusion protein had a half-life in mice in plasma greater than 1 week (liu et al., 2018) . the half-life of the ace2 microbody in vivo has not yet been tested, but the protein retained antiviral activity for several days in tissue culture, significantly longer than longer than soluble ace2 ( figure s3 ). the phenomenon of antibody-dependent enhancement is caused by the interaction of the fc domain of non-neutralizing antibody with the fc receptor on cells which then serves to promote rather than inhibit virus neutralization. a similar phenomenon is possible with receptor-fc fusion proteins by interaction with fc receptor on cells. because the ace2 microbody contained only a single fc domain, it was not expected to interact with fc receptor. to test whether this was the case, we tested the ace2 microbody in an enhancement assay using u937 cells which express fc receptors. the ace2 microbody protein did not detectably bind to cells that express the fc g receptor and the cells did not become infected, suggesting that this mechanism is not likely to play a role in vivo ( figure s4 and not shown). pseudotyped viruses have been highly useful for studies of sars-cov-2 entry. vectors for producing sars-cov-2 lentiviral pseudotypes have been developed by several laboratories (crawford et al., 2020; nie et al., 2020; ou et al., 2020; schmidt et al., 2020; shang et al., 2020; xia et al., 2020) . the vectors we report here produce pseudotyped lentiviral viruses with very high infectivity. the high infectivity of the pseudotypes produced is due in part to efficient expression of a codon-optimized δ19 s protein and the efficient virion incorporation that results from the cytoplasmic tail truncation. the δ19 s protein was present at only slightly higher levels on the cell surface than the full-length protein, suggesting that this small increase does not fully account for the large increase in virion incorporation. a possible explanation is that the full-length cytoplasmic tail sterically hinders virion incorporation by conflicting with the underlying viral matrix protein and that the deletion removes the conflict. also, contributing to high viral titers, is the use of separate gag/pol packaging vector and lentiviral transfer vector as opposed to a lentiviral proviral dna encoding gag/pol and the reporter gene, a strategy that resulted in higher reporter gene expression as shown in a direct comparison (not shown). moreover, the dual luciferase/gfp reporter allows for measurement of infectious virus titer by flow cytometry and the high sensitivity of nanoluciferase read-out. the lentiviral pseudotypes are highly useful for rapidly titering neutralizing antibody in patient serum. in a study of over 100 sera from recovering patients, we found that the pseudotype assay results to be highly correlated with those of a live virus neutralization assay (submitted). a feature of soluble receptors is that because the virus spike protein needs to conserve receptor binding affinity to maintain transmissibility, they should maintain their ability to neutralize s protein variants. sars-cov-2 s variants have been found to be circulating in the human population and it is likely that others are yet to emerge, some of which may be less sensitive to neutralization by the therapeutic monoclonal antibodies currently under development. the recently identified sars-cov-2 variant encoding the d614g s protein has been found to be spreading with increased frequency in the human population (daniloski et al., 2020; eaaswarkhanth et al., 2020; korber et al., 2020; zhang et al., 2020) . the d614g s protein was found to be more resistant to shedding from the virion and to adopt a conformation that favors ace2-binding and is in a more fusioncompetent state (yurkovetskiy et al., 2020; zhang et al., 2020) . we confirmed the increased infectivity of virions and find that the d614g s protein has a higher affinity for ace2 as measured in a virion binding assay. nevertheless, the ace2 microbody maintained its ability to neutralize d614g s protein pseudotyped virus. the ability of the ace2 microbody to neutralize diverse b coronaviruses suggest that it may also be able to neutralize novel ace2 using coronaviruses that may be transferred to the human population in the future. the microbody protein could serve as an off-the-shelf reagent that could be rapidly deployed. the dual gfp/nanoluciferase lentiviral vector plenti.gfp.nluc was generated by overlap extension pcr. a dna fragment encoding gfp was amplified with a forward primer containing a bamh-i site and a reverse primer encoding the p2a sequence. the nanoluciferase gene (nluc) was amplified with a forward primer encoding the p2a motif and a reverse primer containing a 3'-sal-i site. the amplicons were mixed and amplified with the external primers. the fused amplicon was cleaved with bamh-i and sal-i and cloned into plenti.cmv.gfp.puro (addgene plasmid #17448, provided by eric campeau and paul kaufman) (campeau et al., 2009 ). the sars-cov-2 s expression vector pccov2.s was chemically synthesized as dna fragments a and b encoding codon-optimized 5' and 3' halves, respectively, of the s gene of wuhan-hu-1/2019 sars-cov-2 isolate (table s2 and the amplicon was then cloned into the kpn-i and xho-i sites of pcdna6. the ace2 microbody expression vector pcace2-microbody was generated by overlap extension pcr that fused the extracellular domain of ace2 with human immunoglobulin g heavy chain fc domain 3 using a forward primer containing a kpn-i site and reverse primer containing an 8xhis-tag and xho-1 site. the amplicon was cloned into the kpn-i and xho-i sites of pcdna6. expression vector pcace2.h345a-microbody that expressed the ace2.h345a microbody was generated by overlap extension pcr using primers that overlapped the mutation. full-length cdna sequence, primer sequences and amino acid sequences are shown in tables s1-3. control and recovered patient sera were collected from patients through the nyu vaccine center with written consent under i.r.b. approval (irb 20-00595 and irb 18-02037) and were deidentified. vero e6, caco2, a549, ace2 a549, bhk, huh7 293t, vero and chme3 cells were sars-cov-2 s protein pseudotyped lentiviral stocks were produced by cotransfecting virus stocks were titered on 293t by flow cytometry and for luciferase activity. the p24 concentration was measured and the virus was used at a concentration of 1.0 µg/ml. to test the inhibitory activity of soluble receptors and convalescent sera, 50 µl serially diluted inhibitor or convalescent patient serum was incubated for 30 min at room temperature with 5 µl pseudotyped reporter virus (approximately 5 x 10 6 cps luciferase activity/µl) at a moi of 0.1 in a volume of 100 µl. the mixture was added to ace2.293t cells in a 96 well tissue culture dish containing 1 x 10 4 cells/well. after 2 days, the culture medium was removed and 50 µls nano-glo luciferase substrate (promega) and 50 µls medium was added to each well. the supernatant (70 µls) was transferred to a microtiter plate and the luminescence was read in an envision 2103 microplate luminometer (perkinelmer). alternatively, the gfp+ cells were quantified by flow cytometry with pacific blue viability dye to exclude dead cells (biolegend). 293f cells (thermo fisher) at a density of 2.5 x 10 6 cells/ml were transfected with microbody expression vector plasmid dna using polyethyleneimine (polysciences, inc) at a 1:3 plasmid:pei ratio. the cells were then cultured at 30°c and at 12 hours posttransfection 10 mm sodium butyrate was added. after 4 days, the supernatant culture medium was collected, filtered and adjusted ph to 8.0. the medium was passed over a the absolute molecular masses of the purified protein complexes were determined by sec/mals. the proteins were injected onto a superdex 200 10/300 gl gel-filtration chromatography column equilibrated in sample buffer that was connected to a dawn heleos ii 18-angle light-scattering detector (wyatt technology), a dynamic lightscattering detector (dynapro nanostar; wyatt technology) and an optilab t-rex refractive index detector (wyatt technology). the data were collected at 25°c at a flow rate of 0.5 ml/minute every second. the molecular mass of each protein was determined by analysis with astra 6 software. 293t cells were transfected by lipofection with 4 µg pcace2-microbody. at 72 hours posttransduction, 0.5 ml of culture supernatant was incubated with nickel-nitrilotriacetic acidagarose beads (qiagen). the beads were washed, and bound protein was eluted with laemmle loading buffer. the proteins were analyzed on an immunoblot probed with mouse anti-6xhis antibody (invitrogen) and horseradish peroxidase (hrp)-conjugated goat anti-mouse igg secondary antibody (sigma-aldrich). the proteins were visualized using luminescent substrate and scanned on a li-cor biosciences fc imaging system (li-cor biotechnology). ratios were calculated as the his (spike) signal intensity divided by the p24 signal intensity for an identical exposure of the blot. transfected cells were lysed in buffer containing 50 mm hepes, 150 mm kcl, 2 mm edta, 0.5% np-40, and protease inhibitor cocktail. protein concentration in the lysates was measured by bicinchoninic protein assay and the lysates (40 µg) were separated by sds-page. the proteins were transferred to polyvinylidene difluoride membranes and probed with anti-ha mab (covance), mouse anti-his mab (invitrogen) and anti-gapdh mab (life technologies) followed by goat anti-mouse hrp-conjugated second antibody (sigma). the blots were visualized using luminescent substrate (millipore) on a li-cor bio-sciences fc imaging system. soluble ace2 proteins (10 µg) were mixed with 20 µl nickel beads for 1 hour at 4°c. unbound protein was removed by washing the beads with pbs. the beads were resuspended in pbs and mixed with 40 µl pseudotyped lentiviral virions after 1 h incubation at 4°c, the beads were washed with pbs and resuspended in reducing laemmli loading buffer and heated to 90°c. the eluted proteins were separated by sds-page and analyzed on an immunoblot probed with anti-p24 antibody (ag3.0) followed by goat anti-mouse hrp-conjugated second antibody. mneongreen sars-cov-2 (xie et al., cell host and microbe 2020) was obtained from the world reference center for emerging viruses and arboviruses at the university of texas medical branch. the virus was passaged once on vero e6 cells (atcc crl-1586), clarified by low-speed centrifugation, aliquoted, and stored at -80°c. the infectious virus titer was determined by plaque assay on vero e6 cells after staining with crystal violet. virus neutralization was determined as previously described (xie et al, biorxiv 2020) . ace2.293t cells were seeded in a 96-well plate (1 x 10 4 /well). the next day, mneongreen sars-cov-2 (moi = 0.5) was mixed 1:1 with serially 2-fold diluted soluble ace2 protein in dmem/2% fbs and incubated for 1 hour at 37°c. the virus:protein mixture was then added to the ace2 cells and incubated for 24 hours. at 37°c in 5% co2. the cells were fixed with 4% paraformaldehyde, stained with dapi and the mneongreen+ cells were counted on a cellinsight cx5 platform high content microscope (thermo fisher). all experiments were performed in technical duplicates or triplicates and data were analyzed using graphpad prism (version 7 7.0e). statistical significance was determined by the two-tailed, unpaired t test. significance was based on two-sided testing and attributed to p< 0.05. confidence intervals are shown as the mean ± sd or sem. (*p≤ 0.05, **p≤ 0.01, ***p≤0.001, ****p≤0.0001). mechanisms of coronavirus cell entry mediated by the viral spike protein a versatile viral system for expression and depletion of proteins in mammalian cells neutralizing antibody and soluble ace2 inhibition of a replication-competent vsv-sars-cov-2 and a clinical isolate of sars enhanced recognition and neutralization of hiv-1 by antibody-derived ccr5-mimetic peptide variants protocol and reagents for pseudotyping lentiviral particles with sars-cov-2 spike protein for neutralization assays the d614g mutation in sars-cov spike increases transduction of multiple human cell types. biorxiv could the d614g substitution in the sars-cov-2 spike (s) protein be associated with higher covid-19 mortality? implications of antibody-dependent enhancement of infection for sars-cov-2 countermeasures coronaviruses: an overview of their replication and pathogenesis retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response identification of critical active-site residues in angiotensin-converting enzyme-2 (ace2) by site-directed mutagenesis soluble cd4 and cd4-mimetic compounds inhibit hiv-1 infection by induction of a short-lived activated state quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus a novel coronavirus associated with severe acute respiratory syndrome trilogy of ace2: a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor neutralization of sars-cov-2 spike pseudotyped virus by recombinant ace2-ig functional assessment of cell entry and receptor usage for lineage b beta-coronaviruses enteric involvement of severe acute respiratory syndrome-associated coronavirus infection receptor recognition mechanisms of coronaviruses: a decade of structural studies structure of sars coronavirus spike receptor-binding domain complexed with receptor insights from the association of sars-cov sprotein with its receptor, ace2 angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus novel ace2-fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 engineering high-affinity pd-1 variants for optimized immunotherapy and immuno-pet imaging infections in engineered human tissues using clinical-grade soluble human ace2 establishment and validation of a pseudovirus neutralization assay for sars-cov-2 two mechanisms of soluble cd4 (scd4)-mediated inhibition of human immunodeficiency virus type 1 (hiv-1) infectivity and their relation to primary hiv-1 isolates with reduced sensitivity to scd4 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov ucsf chimera--a visualization system for exploratory research and analysis ig-like ace2 protein therapeutics: a revival in development during the covid-19 pandemic angiotensin-i-converting enzyme and its relatives effects of soluble cd4 on simian immunodeficiency virus infection of cd4-positive and cd4-negative cells measuring sars-cov-2 neutralizing antibody activity using pseudotyped and chimeric viruses cell entry mechanisms of sars-cov-2 a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble cd4 and monoclonal antibodies angiotensin-converting enzyme 2 (ace2) is a key modulator of the renin angiotensin system in health and disease targeting the degradation of angiotensin ii with recombinant angiotensin-converting enzyme 2: prevention of angiotensin ii-dependent hypertension inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion an infectious cdna clone of sars-cov-2 sars-cov-2 spike protein variant d614g increases infectivity and retains sensitivity to antibodies that target the receptor binding domain the d614g mutation in the sars-cov-2 spike protein reduces we thank lin xinhua and hanna nazeeh (nyulh) and benjamin tenoever for cell-lines ace2-mb h345a-mb sace2 a. no serum no serum **** **** **** **** **** tada et al. atgtcaagctcttcctggctccttctcagccttgttgctgtaactgctgctcagtccaccattgaggaacaggccaagacatttt tggacaagtttaaccacgaagccgaagacctgttctatcaaagttcacttgcttcttggaattataacaccaatattactgaaga gaatgtccaaaacatgaataatgctggggacaaatggtctgcctttttaaaggaacagtccacacttgcccaaatgtatccacta caagaaattcagaatctcacagtcaagcttcagctgcaggctcttcagcaaaatgggtcttcagtgctctcagaagacaagagc aaacggttgaacacaattctaaatacaatgagcaccatctacagtactggaaaagtttgtaacccagataatccacaagaatgct tattacttgaaccaggtttgaatgaaataatggcaaacagtttagactacaatgagaggctctgggcttgggaaagctggagat ctgaggtcggcaagcagctgaggccattatatgaagagtatgtggtcttgaaaaatgagatggcaagagcaaatcattatgagg actatggggattattggagaggagactatgaagtaaatggggtagatggctatgactacagccgcggccagttgattgaagat gtggaacatacctttgaagagattaaaccattatatgaacatcttcatgcctatgtgagggcaaagttgatgaatgcctatcctt cctatatcagtccaattggatgcctccctgctcatttgcttggtgatatgtggggtagattttggacaaatctgtactctttgac agttccctttggacagaaaccaaacatagatgttactgatgcaatggtggaccaggcctgggatgcacagagaatattcaagga ggccgagaagttctttgtatctgttggtcttcctaatatgactcaaggattctgggaaaattccatgctaacggacccaggaaat gttcagaaagcagtctgccatcccacagcttgggacctggggaagggcgacttcaggatccttatgtgcacaaaggtgacaat ggacgacttcctgacagctcatcatgagatggggcatatccagtatgatatggcatatgctgcacaaccttttctgctaagaaa tggagctaatgaaggattccatgaagctgttggggaaatcatgtcactttctgcagccacacctaagcatttaaaatccattggt cttctgtcacccgattttcaagaagacaatgaaacagaaataaacttcctgctcaaacaagcactcacgattgttgggactctgc catttacttacatgttagagaagtggaggtggatggtctttaaaggggaaattcccaaagaccagtggatgaaaaagtggtggg agatgaagcgagagatagttggggtggtggaacctgtgccccatgatgaaacatactgtgaccccgcatctctgttccatgttt ctaatgattactcattcattcgatattacacaaggaccctttaccaattccagtttcaagaagcactttgtcaagcagctaaacat gaaggccctctgcacaaatgtgacatctcaaactctacagaagctggacagaaactgttcaatatgctgaggcttggaaaatca gaaccctggaccctagcattggaaaatgttgtaggagcaaagaacatgaatgtaaggccactgctcaactactttgagccctta tttacctggctgaaagaccagaacaagaattcttttgtgggatggagtaccgactggagtccatatgcagaccaaagcatcaaa gtgaggataagcctaaaatcagctcttggagataaagcatatgaatggaacgacaatgaaatgtacctgttccgatcatctgttg catatgctatgaggcagtactttttaaaagtaaaaaatcagatgattctttttggggaggaggatgtgcgagtggctaatttgaa accaagaatctcctttaatttctttgtcactgcacctaaaaatgtgtctgatatcattcctagaactgaagttgaaaaggccatca ggatgtcccggagccgtatcaatgatgctttccgtctgaatgacaacagcctagagtttctggggatacagccaacacttggac ctcctaaccagccccctgtttccatatggctgattgtttttggagttgtgatgggagtgatagtggttggcattgtcatcctgat cttcactgggatcagagatcggaagaagaaaaataaagcaagaagtggagaaaatccttatgcctccatcgatattagcaaagg agaaaataatccaggattccaaaacactgatgatgttcagacctccttttag key: cord-017775-qohf9pxp authors: loa, chien chang; wu, ching ching; lin, tsang long title: recombinant turkey coronavirus nucleocapsid protein expressed in escherichia coli date: 2015-09-10 journal: animal coronaviruses doi: 10.1007/978-1-4939-3414-0_4 sha: doc_id: 17775 cord_uid: qohf9pxp expression and purification of turkey coronavirus (tcov) nucleocapsid (n) protein from a prokaryotic expression system as histidine-tagged fusion protein are presented in this chapter. expression of histidine-tagged fusion n protein with a molecular mass of 57 kda is induced with isopropyl β-d-1-thiogalactopyranoside (iptg). the expressed n protein inclusion body is extracted and purified by chromatography on nickel-agarose column to near homogeneity. the protein recovery can be 10 mg from 100 ml of bacterial culture. the purified n protein is a superior source of tcov antigen for antibody-capture elisa for detection of antibodies to tcov. turkey coronavirus (tcov) is the cause of an acute and highly contagious enteric disease affecting turkeys of all ages. the disease is severe in 1-to 4-week-old turkey poults [ 1 ] . turkey fl ocks that recover from natural or experimental coronaviral enteritis may develop lifelong immunity [ 2 ] . tcov has been recognized as an important pathogen of young turkeys. tcov infection causes signifi cant economic losses in the turkey industry due to poor feed conversion and uneven growth. outbreaks of tcov enteritis in turkey poults remain as a threat to the turkey industry. in order to rapidly diagnose and effectively control turkey coronaviral enteritis , development of an antibody-capture enzymelinked immunosorbent assay ( elisa ) for detecting antibodies to tcov is essential. development of elisa for detection of tcov infection requires large amounts of tcov antigen . molecular cloning and expression of tcov n protein were carried out for preparation of large quantities of highly purifi ed viral proteins. coronavirus is an enveloped and positive-stranded rna virus that possesses three major structural proteins including a predominant phosphorylated nucleocapsid (n) protein, peplomeric glycoprotein, and spike (s) protein that makes up the large surface projections of the virion, and membrane protein (m) [ 3 , 4 ] . the n protein is abundantly produced in coronavirus-infected cells and is highly immunogenic. the n protein binds to the viral genomic rna and composes the structural feature of helical nucleocapsid. the n protein is a preferred choice for developing a groupspecifi c serologic assay because of highly conserved sequence and antigenicity. the nucleocapsid protein s of various rna viruses, such as mumps, rabies, vesicular stomatitis, measles, newcastle disease, and infectious bronchitis (ibv) viruses, have been used as the coating antigens in diagnostic elisa [ 5 -10 ] . prokaryotic expression is an economic and convenient system to prepare large amount of pure recombinant protein. in addition, the antigenic integrity of n protein expressed in prokaryotic system is expected to be maintained due to the lack of glycosylation. this chapter describes expression and purifi cation of tcov n protein with a prokaryotic system for preparation of a large quantity of highly purifi ed viral protein, which can be used as coating antigen for antibody-capture elisa for serologic diagnosis of tcov infection [ 11 , 12 ] . 13. add 2 μl of the above rt mixture to the pcr amplifi cation reaction (100 μl) with primers nf and nr. a mix of taq and pfu at 10:1 is recommended to maintain pcr fi delity (table 1 ) . 2. the nuclease reagent benzonase is added with 1 μl (25 units) for every ml of bugbuster reagent used. 4. ampicillin antibiotic marker is on the expression vector ptriex and chloramphenicol antibiotic marker is on plasmid placi in the expression host strain tuner cells. carbenicillin is recommended to be in place of ampicillin for better stability for ph changes throughout the bacterial cultures. 5. the binding capacity of 1.25 ml of his-bind resin is 10 mg of target protein per column. as for any affi nity chromatography, the best purity of target protein is achieved when the amount of protein extract is near the binding capacity. 6. the purpose of 6 m urea is to improve resolution of the sticky inclusion bodies. the presence of 6 m urea does not affect binding of his-bind resin to target n protein. 7. the suggested ratio of rnapure reagent to sample is 10:1. excess amount of rnapure reagent has no negative impact. the lower ratio (5:1) in this step is intended to obtain higher concentration of viral rna in the fi nal supernatants. if the upper aqueous phase after centrifugation at step 3 is more than half of the total volume, there is not enough rnapure reagent added. the appropriate reagent amount may be adjusted. chloroform is applied at 150 μl for every milliliter of lysate. 8. the sample mixture with chloroform at this step can be stored at −70 °c or even lower temperature before proceeding to the next step. 9. optional: inverting the tube for 5-10 min for air-drying of rna pellet is a helpful tip to completely remove any residual ethanol that may interfere the following rt reaction. 10. it is critical to make sure that the jellylike rna pellet is completely dissolved into solution by repeat pipetting. 11. the synthesized cdna in the rt reaction can be stored at −20 °c or even lower temperature until used. 12. pcr product may be purifi ed. the vector must be gel purifi ed due to the long digested fragment size above 30 bp. 13. the molar ratio between vector and insert is suggested at 1:2 to 1:5. the volumes in this step are illustrated for initial exploration. the concentration of digested vector and insert can be estimated by od 260 or agarose gel electrophoresis with known amount of dna of similar size in adjacent wells. the ligation reaction mixture can be stored at 4 °c until used for transformation or at −20 °c for longer term. 14. after plating, the leftover transformation mix can be stored at 4 °c for further plating in the following days at different amount if needed. 15. the starter culture can be prepared from a fresh colony on a plate or directly from a glycerol storage stock. an od around 0.5 represents a culture at log phase when the cells are at the best condition to expand and for protein expression. 16. this usually takes about 2-3 h to reach the od range. the higher the od of starter culture in the previous step, the shorter the time to reach this od range. 17. the centrifuge tubes should be weighed before and after collection of cell pellet for estimation of wet pellet amount and the volume of bugbuster to be applied in the next step. frozen storage of cell pellets may improve the extraction effi ciency of bugbuster reagents through the freeze/thaw cycle. 18. it is important to completely resuspend the cell pellets for the best results of bugbuster extraction. higher volume of bugbuster reagent does not have adverse effect. roughly 10-20 ml of bugbuster reagent should be enough for cell pellets collected from a 100 ml of culture. bugbuster reagent can be added directly to frozen cell pellets. there is no need to wait for the temperature to return to room temperature. protease inhibitors may be added at this step but usually not necessary. 19. it is critical but somewhat diffi cult to completely dissolve the sticky inclusion bodies. repeat pipetting up and down until the protein solution is homogeneous. any undissolved particles will clot the his-bind column and affect the purifi cation process. it is advisable to centrifuge the dissolved inclusion body protein solution at 5000-10,000 × g for 10-15 min at 4 °c for clarifi cation before application to the column. 20. eluate may be collected in fractions such as 0.5 or 1 ml each fraction. 21. the presence of 6 m urea is compatible with the protein assay reagent. the assay range can be adjusted for protein concentrations from low μg/ml to 1 mg/ml with different assay format. the protein concentration of the target n protein eluate as obtained following this process is about 1-2 mg/ml. the presence of 6 m urea has no adverse effect on plate coating for elisa performance. given the coating concentration of n protein at 20 μg/ml, the eluate is usually diluted in coating buffer for at least 1:10 to reduce the urea content to less than 600 mm and, subsequently, further diminish any possible effect on elisa performance. accordingly, the purifi ed n protein eluate can be directly applied to the elisa assay for detection of antibodies to tcov. coronaviral enteritis of turkeys (blue comb disease) immunity to transmissible coronaviral enteritis of turkeys (blue comb) identifi cation of the structural proteins of turkey enteric coronavirus coronavirus immunogens immunoglobulin class and immunoglobulin g subclass enzyme-linked immunosorbent assays compared with microneutralisation assay for sero-diagnosis of mumps infection and determination of immunity rabies diagnostic reagents prepared from a rabies n gene recombinant expressed in baculovirus baculovirus expression of the nucleocapsid gene of measles virus and utility of the recombinant protein in diagnostic enzyme immunoassays immunological characterization of the vsv nucleocapsid (n) protein expressed by recombinant baculovirus in spodoptera exigua larva: use in differential diagnosis between vaccinated and infected animals a diagnostic immunoassay for newcastle disease virus based on the nucleocapsid protein expressed by a recombinant baculovirus recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus expression and purifi cation of turkey coronavirus nucleocapsid protein in escheria coli recombinant nucleocapsid proteinbased enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus the protocol "recombinant turkey coronavirus nucleocapsid protein expressed in escherichia coli " detailed in this chapter had been successfully carried out in the authors' studies on characterization and immunology of turkey coronaviral enteritis . those studies were in part fi nancially supported by usda, north carolina poultry federation, and/or indiana department of agriculture and technically assisted by drs. tom brien and david hermes, mr. tom hooper, and ms. donna schrader for clinical and diagnostic investigation, virus isolation and propagation, and animal experimentation. key: cord-018647-bveks6t1 authors: butnariu, monica; butu, alina title: plant nanobionics: application of nanobiosensors in plant biology date: 2019-10-01 journal: plant nanobionics doi: 10.1007/978-3-030-16379-2_12 sha: doc_id: 18647 cord_uid: bveks6t1 nanobiosensors (nbss) are a class of chemical sensors which are sensitive to a physical or chemical stimulus (heat, acidity, metabolism transformations) that conveys information about vital processes. nbss detect physiological signals and convert them into standardized signals, often electrical, to be quantified from analog to digital. nbss are classified according to the transducer element (electrochemical, piezoelectric, optical, and thermal) in accordance with biorecognition principle (enzyme recognition, affinity immunoassay, whole sensors, dna). nbss have varied forms, depending on the degree of interpretation of natural processes in plants. plant nanobionics uses mathematical models based on qualitative and less quantitative records. nbss can give information about endogenous concentrations or endogenous fluxes of signaling molecules (phytohormones). the properties of nbss are temporal and spatial resolution, the ability of being used without significantly interfering with the system. nbss with the best properties are the optically genetically coded nbss, but each nbs needs specific development efforts. nbs technologies using antibodies as a recognition domain are generic and tend to be more invasive, and there are examples of their use in plant nanobionics. through opportunities that develop along with technologies, we hope that more and more nbss will become available for plant nanobionics. the main advantages of nbss are short analysis time, low-cost tests and portability, real-time measurements, and remote control. plants are a fascinating research topic if we relate to environmental stress. because they are physically stuck in specific spots, the plants have to handle in that site, regardless of the environmental conditions. moving to another place is not an option. but what plants can do is to modify the internal "environment," and plants are "true masters" of manipulating their metabolism to deal with environmental disturbances. this feature is one of the reasons why plants are useful in various research; we can rely on them as "sensitive indicators" of environmental changes, even in completely new environments. in the absence of normal conditions, plants cannot use the classical pathways of metabolism, so they need to identify other solutions. this is what happens when plants adjust their metabolism for regulating gene activation, thus producing more or less proteins that are useful or not in the new environmental conditions. the different parts of the plant come with their own genetic regulation strategies. a number of genes that are involved in the creation and remodeling of cell walls are activated differently in growing plants. other genes with a role in identifying light, which are normally active on the leaves, are active at the root level. in leaves, many genes associated with transmission of hormonal information are suppressed, and genes associated with insect protection are more active. these trends are also seen in the (higher) number of proteins involved in message transmission, cell wall metabolism, and plant protection. these patterns of gene and protein functioning indicate that in unfavorable conditions, the plants respond by weakening the cell walls and creating new ways to understand the environment. it is possible to monitor changes in the genes in real time by labelling certain proteins with fluorescent elements. plants modified with fluorescent proteins can give useful information about how they respond to the environment. these modified plants act as biological sensors (nbss). specialized cameras and microscopes allow us to monitor how the plant uses these fluorescent proteins (hamers et al. 2014) . chemical or biological nbs functions on the principle of signal emission (voltage or electrical, photonic) in response to a chemical reaction involve a chemical or biological receptor, r (macrocyclic ligand, antibody enzyme), that binds to a specific target molecule of a sample to be studied, the analyte, a. signal transmission is achieved by coupling with a transducer t that interfaces nbs processes with the processing-transform unit in a measurable signal. analysis of signals in plant nanobionics aims at processing signals recorded by measurements in order to extract the maximum of useful information for diagnostics and these devices are mostly used in genetic engineering in agriculture, where it is necessary to know the mechanisms of reaction and the affinity of enzymes and microorganisms for different substrates of interest and signaling molecules. a biochip is a device that contains a structure of individual sensory elements (nbss) interconnected by functions and recognition specifications, integrated on a chip. the number of nbss on a chip may be of the order of 10 6 units. in this degree of integration, a set of distinct tests can be performed extremely quickly and efficiently. in contrast to microchips, biochips are not electronic structures (they contain different electronic structures coupled to nanobiosensory elements). each nbs can be thought of as a "microreactor" that performs a specific chemical reaction with an analyte. nbss from biochips can be designed to detect a wide variety of analytes, including dna, antibodies, proteins, and biomolecules. the advantages are multiple: sensors can be produced in batches or sequences that can be assembled in parallel or serial, providing a high manufacturing yield; sensors can be assembled on very small areas with reduced distances between them; 3d structures can be generated providing high signals besides 2d structures; any type of biochemical reaction may be incorporated; nbss can be produced separately and subsequently assembled according to the specificity and nature of the application. the important features regardless of industry or technology are selectivity, sensitivity, and stability in the design of sensory systems integrated with structures and arrangements of sensory elements (wan salim et al. 2013) . one of the integrated systems including rotational aseptic sampling is a robotic fluid and reusable electrodes formed by ink-jet printing injection system. the system contains an enzyme electrode with immobilized gox in gel, and the detection of hydrogen peroxide is carried out on a rhodinised carbon electrode (rh coating). although the enzyme electrode has stability and efficiency characteristics, the problem of automated sample monitoring and sampling in an integrated system requires multiparameter optimization due to reciprocal interferences. there is a requirement on specific domains (environment and genetic engineering) of highly performing integrated systems that work in in vivo conditions, such as dialysis, the use of biointerfaces, evanescent techniques, and atomic force microscopy to grasp in the depth of the biological phenomena (identifying and understanding the interaction of proteins). the in vivo exploitation of detection systems for both glucose and lactate was confirmed by the efficacy of using phospholipid copolymers and improving hemocompatibility. immunosensors provide an example for the development of integrated systems where microseparations, chromatographic methods, and electrochemical couplings with optical detectors are incorporated, which ultimately lead to a miniaturized system. there are examples where the level of integration and miniaturization becomes more pronounced (dna-nanotube or biochips-biointerfaces). nbss are expected to be widely used in plant nanobionics where physiological/biochemical parameters should be identified. advanced ink-jet technology has developed methods for analyzing nanoliter fractions on a three-dimensional nbs surface at a speed of 6 m/s. it is expected to produce one million nbss/cm 2 areas using photolithography, contact fingerprinting or self-assembling techniques, and adsorption/desorption under the laser beam that allows "writing" proteins on the surface to be analyzed with great precision. laser techniques, maple (matrix assisted pulsed laser evaporation) or dw (direct writing) approached for immobilizing biological materials on substrates, are in the laboratory stage but have prospects for use as molecular imprinting methods (potocký et al. 2014 ). whether nbss are individuals, in integrated systems, or areas of nbss, all are characterized by unique parameters such as sensitivity and detection limit for a range of analytes. trace detection of various analytes (indicators, additives, contaminants) with sufficient sensitivity and safety is the basic criteria of an nbs to be used. the detection limit in the laboratory is pushed to an atom when the atomic force microscopy is used. thus, the enzymatic electrodes, studied and continuously perfected, use palliative such as concentrating the analyte of interest, which leads to major design and miniaturization difficulties. nbss for phenol vapors were identified, where the phenoloxidase was immobilized on a glycerol gel with a range of interdigitated electrodes. phenol vapors are directly partitioned into the gel and oxidized to quinone. signal amplification was improved by redox amplification of the quinone/catechol couple to obtain a reasonable sensitivity resulting in a detection limit of 30 ppb phenol. this principle can be extended to other carbon compounds up to the ppt (parts per trillion) limits. dna structures have been studied as potential receptors. sandwich structures of liquid crystal dispersions and dna-polycation complexes have been studied with relatively good success in identifying different analytes. the polycation with the role to maintain structural integrity of dna and dna-protamine complexes allows detection of hydrolysis of the trypsin enzyme to the detection limit of 10 −14 m. elimination of the polycation leads to an increase in the distance between the two dna strands resulting in the appearance of an intensive band in the circular dichroism spectrum due to the texture modification (espinoza et al. 2014 ). from a practical standpoint, the disadvantage is their inherent instability. different strategies have been approached to improve longevity and preserve the structure of biological receptors. immobilization in matrices by sol-gel technique for glucose nbss is one of the strategies; fluorescence indicators are used: hexahydrate chloride (2,2′-bipyridyl) ruthenium (ii) and 1-hydroxypyrene-3,6,8 trisulfonic acid. in addition to the optical property improvement of the gel, the stability of the gox enzyme has also improved. other examples are the case of monooxygenase used in hydrocarbon detection; detection of organic halides with metalloporphyrins; and detection of carbon tetrachloride, haloalkane (perchloroethylene), and insecticides (ddt) (kazakova et al. 2013 ). improving the selectivity of an nbs can be approached on two levels: through direct transducer-biological receiver interfacing to reduce interference and new receptors with improved affinity or new affinity capabilities. selectivity is a key parameter that requires the performance of an nbs. pyrroloquinoline is used as a mediator in a glucose oxidase enzyme electrode for the measurement of glucose in the raw or elaborated cavity. alternatively, the electrocatalytic detection of the reaction products resulting from the enzymatic reactions can be improved by chemically modified electrodes such as rhodinised electrodes or hexacyanoferrate modified carbon electrodes. prussian blue is used to modify the electrode surface at amperometric detection of oxygenated water at both oxidation and reduction potentials for the enzyme electrode in lactate and glucose detection. one solution is to identify redox centers of the enzyme via a molecular wire to perform the electron transfer to the electrode (enzymes linked by molecular wires), but the concerns have focused on immobilized mediators on different polymer chains. molecular wires are regarded as intermediates in long-range electron transfer, consisting of two pyridine groups linked by thiophenes with different lengths. such wires can be used in conjunction with self-assembly techniques to produce an isolated electrode that transfers electrons to predetermined molecular pathways (jones et al. 2013 ). an ideal nbs is a device that will detect an "analyte," the subject under analysis and which is present in a given sample. most samples also contain other analytes which may interfere with the nbs response. it must have a specific selectivity to identify the target analyte. it is necessary to design nbss with selectivity for an analyte with the ability to discriminate the interferences produced by the other components in the analyzed sample. specific identification and selectivity capacity are the key components of molecular recognition. molecular recognition is accomplished by the sensor component of a host molecule (host-chemoreceptor) that binds selectively to the "guest" target molecule/guest molecule that needs to be identified. for each "host-guest" system, there is a specific chemical reaction from the multitude of possible reaction channels. when the host-guest response was identified, the host molecule is immobilized/incorporated into nbss, typically on a transducer-contacting membrane/ contact electrode. finally, a way to signal that the bindings/recognition event has occurred (transducer to transducer) has to be found (rodríguez-sevilla et al. 2014 ). one of the requirements for molecular recognition is the existence of groups or centers with specific reactivity in the host molecule that can "close" or bind ions, atoms, molecules, and biomolecules. all living organisms use enzymes, which are proteins that contain "pockets," active centers, designed to recognize a specific analyte. this means that only a specific analyte is able to enter the enzyme pocket. enzymes can be used in nbss as host receptors with molecular recognition capability but are unstable. to design host molecules that can be used in an nbs, the following criteria are considered: the host molecule must be stable under the conditions in which it is to be used, must be able to selectively bind the analyte in the sample, must be able to be immobilized in a film/membrane that is in contact with the sample, must signal that a host-host binding response has occurred, and must ideally release the analyte after detection so the host is free to be reusable. biological receptors include antibodies, membrane receptors, signaling molecules, enzymes, ribosomes, lectins, phytohormones, etc. they bind analytes using "lock-and-key" molecular recognition mechanisms (key-lock, identification-immobilization). biological receptors are not practical solutions for many applications because the specificity, sensitivity, and stability cannot be optimized. artificial receivers are immobilization environments that can be optimized by molecular design for any type of application. synthetic receptor design and synthesis are based on tools developed by proteomics and genetic engineering, producing recognition components that can respond to the occurrence and identification of metabolic deficiencies in plant nanobionics. there are platforms and areas of artificial receptors based on combinatorial mathematical techniques, interface biology, and surface chemistry. they have induced the development of various artificial receptor environments with rapid and diversified selection for any target analyte. the current technique for producing synthetic receptors is called cara (combinatorial array of receptor analysis) (fang et al. 2015) . supramolecular chemistry has developed a wide range of synthetic macrocycles. the most common feature for macrocycle classes is that they contain cavities that act as host pockets for guest molecules. the selectivity of the hosts can be done in "read mode" by varying the size of the preformed cavities. 12-crown-4 has a small cavity ideal for the binding of small ions such as li + , while 18-crown-6 has a large cavity that fits better with larger ions such as k + . the size of the cavities is important for the selectivity of the host, but the question remains what attracts an ion or molecule into a preformed cavity and which factors stabilize the host-guest complex. in enzymes, weak noncovalent interactions (hydrogen bonding, electrostatics, dipole-dipole, van der waals, etc.) are used to link the guest into the enzyme pocket (interactions stabilize host-guest interaction). macrocycles contain polar functionalities, capable of interacting with guests via hydrogen bonding, electrostatic interactions, and dipole-dipole interactions. it is desirable that bonding in the cavity is not strong, because it is important that the analyte, the guest, is released from the host after it has been detected and measured. crown ethers and calixarene are ideal for bonding metal cations, based on the size of their cavity, but also on the high density of electrons present on the oxygen atoms in the cavity. the base compound selectively binds li + to other metallic cations; the modified version of the base macrocycle has a good selectivity for na + . by synthetic modification it is possible to increase the capacity of the host cavity, and new functionalities can be introduced that will favor the binding of specific molecules and ions (da silva et al. 2013) . other modified calixarenes, which demonstrate this principle, are the group of tetraphosphine oxide of the calix[4]arene. by changing binding groups on the same template, calix[4]arene from esters in phosphorus hydrogen oxides selectivity changes from na + to ca 2+ . by increasing the number of repeatable units in esters and phosphorus hydrogen oxides at six, the cavity increases, and the selectivity changes in favor of the higher cs + and pb 2+ cations, respectively. some host compounds have been developed for the selective detection of low molecular weight compounds. an example involves the use of the tetra (s-propanol) calix[4]arena containing four lateral chiral halves for selective differentiation of the phenylalanyl enantiomers. other techniques of supramolecular chemistry may be involved in the synthesis of synthetic receptors that simulate the properties of enzymes. the basic structures that can be modified are porphyrins, semiconductor polymers of the tetrathiafulvalene (ttf) class, and ppvs. other patterns can be considered modified polysaccharides. a linear archetype is the polyanilines containing the two types of redox states (meyer et al. 2007 ). among the multiple nbs classifications, bioaffinity has a range of applications, and antigen-antibody interactions (ag-ab) play a role and are considered to be an instrument in the development of molecular recognition principles. in vivo ag-ab interactions are reversible. factors that condition the ag-ab interaction are the structural complementarity between the antigenic determinant and the antibody combining site; this is the exclusive factor of the specificity of the reaction; the structural complementarity involves the conformational adaptation of the two reacting groups and was conceived in structural terms on the key-lock principle; the chemical complementarity of the reaction groups is the consequence of structural complementarity and signifies the entry into action of intermolecular forces that stabilize and consolidate the interaction of the two groups. the formation of intermolecular bonds requires the existence of atomic groups sufficiently close to the two molecules. the distance between them is inversely proportional to the degree of complementarity. although structural complementarity is not strictly obligatory, higher spatial matching is more conducive to interaction. it is expressed by the congruent of contact surfaces that provide intermolecular attraction forces that stabilize the complex. the ag-ab interaction involves the following types of noncovalent bonds: h bonds, electrostatic forces, van der waals linkages, and hydrophobic bonds. all are nonspecific forces of low value and their nature makes the reaction reversible. h bonds form when two atoms share an atomic h nucleus (one proton). the common proton is found between two atoms of n or o or between n and o. the h nucleus is covalently bonded to one of the two atoms (n or o). the h bond has a binding energy of 3-7 kcal/mol. intermolecular forces are involved in ag-ab complex formation. the action of these forces requires close contact between the two reaction groups. the h bonds result from the formation of an h bridge between two nearby atoms. the electrostatic forces are due to the attraction of the ionic groups with opposite charges located at the periphery of the two protein chains. the van der waals forces result from the interaction between different electron clouds, represented in the form of oscillating dipoles. the van der waals relationships, the weakest interaction forces, are active at very small distances between the reaction groups. the binding energy is 1-2 kcal/mol. van der waals' links are not based on a permanent separation of electrical charges but on their fluctuations induced by the proximity of molecules. at intermolecular distance, instantaneous electric fields are formed, with a polarizing effect on neighboring molecules. between the nearby atoms, there is a mutual attraction force induced by fluctuating dipole load, which a dipole induces in the neighboring dipole (dispersion forces). their intensity depends on the distance between the groups involved and is inversely proportional to the seventh power of the distance. their value is optimal at 1-2 å. hydrophobic bonds, which can contribute with half of the ag-ab binding force, are produced by the association of nonpolar and hydrophobic groups, whereby water molecules are excluded. the optimum distance between the reactive groups varies with the type of bond. the electrostatic forces (coulomb or ionic) are the result of the attraction between atoms or groups of atoms with the opposite electric charge located on the two reacting groups: between a cation (na + ) and an anion (cl − ) or between coo − and nh 3+ (agrawal et al. 2012) . the binding energy of these forces is significant at very small (less than 100 å) distances from the reaction groups. exact juxtaposition of ions favors the action of these forces. the binding energy is 5 kcal/mol and varies inversely with the square of the distance between the two reaction groups (1/d 2 ). hydrophobic (or a polar) linkages occur between nonpolar (nonionized) groups in aqueous solutions and are the consequence of the tendency to exclude the ordered molecule of water molecules between the antigen and antibody molecules. these linkages are favored by amino acids with a polar group that tends to associate, reducing the number of water molecules in their vicinity. by removing the water molecules between the reaction groups, the distance between the active sites decreases much, and the value of the stabilizing forces increases. taken each one on their own, space complementarity or intermolecular forces are not sufficient to form stable relationships. for the stability of the ag-ab interaction, both conditions are required. the higher the binding energy of the reactants, the stable the ag-ab complexes. the interaction of the antigen-and antibody-reactive groups is defined by two parameters: the affinity and avidity of the antibodies. measurement of antibody affinity can be achieved by dialysis at equilibrium. the ag-ab interaction is reversible. within the dialysis bag, the hapten is partially free and partially bound to the antibodies, depending on the affinity of the antibodies. through the membrane of the dialysis bag, only the free hapten can be diffused, and its external concentration will equal the concentration of the free hapten inside the bag (kersten and feilner 2007) . measurement of the concentration of the hapten in the dialysis bag allows for the calculation of the amount of antibody-bound hapten. the constant renewal of the buffer results in total dissociation and loss of hapten in the dialysis bag, which indicates the reversible nature of ag-ab binding. affinity of antibodies measures the binding force between an antigenic determinant and the complementary binding site of a specific antibody. affinity is the result of attraction and rejection forces that mediate the interaction of the two reactants. the strength of these interactions is measured in the reaction between a monovalent antigen (hapten) and specific antibodies. a high affinity interaction requires perfect complementary structures, while the imperfect complementarity of the reaction groups causes a low affinity, since the attraction forces are active only at very small distances and are diminished by the rejection forces. complexes formed by antibodies with high affinity are rapidly eliminated from the circulation without adverse effects on renal function. the ag-ab interaction is permanently characterized by the formation and cancellation of various types of intermolecular bonds. in vivo, probably all ag-ab reactions are reversible, but secondary in vitro reactions (agglutination, precipitation) , under the conditions of reagent balance, are irreversible (sakamoto et al. 2018) . it is essential that the host-guest binding event (the receptor-analyte interaction, r-a) is detected. it is thus necessary to have a way of identifying and transducing the signal from the receptor-analyte interaction to the outside to be processed. it is generally defined as a transducer. the transducer must be in contact with the receiver or the diaphragm that immobilized the handset. electrode and interfacial interactions are determinant in capturing the signal from an r-a interaction and transforming it into an electrical or photonic signal. there are ways of identifying the r-a event, collecting the signal and transducing it as an external signal. the way of identifying the signals and their transduction defines the type of nbss. this means immobilizing the receiver on an electrochemical transducer that measures a current (amperometric method) or a voltage (potentiometric) between two electrodes. if r is immobilized on an optical component, then we will define optical nbss (optical fiber, fluorescence, absorption, surface plasmon resonance (spr)). for detection, at most electrochemical nbss, it is necessary for the membranes containing the host molecule to be placed on a surface of an electrode that leads to an electrochemical response to the binding of the guest. the approach works well when target analysts are loaded species such as metal cations. neutral molecules cannot be detected from the point of view of electrochemical transduction, so the optical detection methods have been successfully used. a chiral host in calixarene contains naphthyl, fluorescent units. upon binding to the guest, fluorescence is attenuated as a result of interaction between the naphthyl-phenyl groups in the host or analyte. the fluorescence attenuation is proportional to the concentration of the analyte. optical methods are used because they offer greater sensitivity than electrochemical techniques. in the absence of the guest analyte, this compound does not exhibit fluorescence because the pyrenyl substituent cannot come in contact with the adjacent nitrophenyl substituent (and the fluorescence attenuation occurs due to their interaction). however, in the presence of na + ions, fluorescence is observed, because the na + ion enters the cavity and binds to the oxygen in the phenoxy and carbonyl groups in the host. this binding induces a more rigid conformation by removing the nitrophenyl groups of pyrene to prevent fluorescence attenuation (gaggeri et al. 2012 ). nbs is a bioelectronic analysis system that combines a transducer with a biological component that is in a specific interdependence. nbss use biological systems with different levels of recognition of the substances to be determined. the first step in this interaction is the formation of the specific complex of the biologically active, immobilized substance r (the receptor, the substrate with the sensitive biological component) with the analyte a (often defined as the chemical signal). table 12 .2 summarizes specific patterns of nbss in relation to the nature of the receptor and the chemical/biochemical signal. there are two general classes of nbss that are based on the bioaffinity response between r and a that alters the distribution of electrical charges that can be measured with specific transducers or consuming the substrate by a specific reaction. the biological constituent of the molecular recognition element (r) is represented by various active species that can be enzymes or enzymatic systems, antibodies (ab) or antigens (ag), receptors, populations of bacteria or eukaryotic cells, tissue fragments, and sometimes even signaling molecules. analytes or substances that can be analyzed (a) are glucose or other sugars, amino acids, alcohols, lipids, and nucleotides. they can be identified by their specific interaction, or their concentration can be measured by various methods. both r and a represent distinct molecular species with high macromolecular specialization (antibodies, antigens, enzymes, receptors, etc.) or are complex systems (cells, tissues, etc.) (kersten and feilner 2007) . after the active biological component, they can be grouped as follows: • enzymatic nbss: enzymes are energy proteins characterized by their catalytic function. modified substrate molecules lead to oxidation, reduction, and hydrolysis reactions that can be measured by enzymatic nbss. enzymatic nbss produce a linear response depending on the substrate concentration. • immunosensors: antibodies are glycoproteins produced by the immune system when an external substance, antigen, is involved. it is theoretically possible to produce antibodies without identifying an antigen. an immunosensor is a high sensitivity nbs. the principle of operation is based on the ag-ab interaction of molecular recognition. • nbss with receptors: the regularity of biological processes is ensured by high sensitivity molecular processes based on the specialization of structural proteins (receptors) capable of recognizing a number of physiological signals. this is the case for neurotransmitters, whose action is mediated through the presence of receptors in the plasma membrane, in sites or cell targets. activation of the biologically active site is via the ion channels. the acetylcholine receptor is the first known receptor in neurotransmitting phenomena. • nbss based on cells or tissues: measurement of molecular species is not limited to interaction with the compounds to be analyzed; the transformations that occur can be measured as resulting products. it is desirable to operate with cell populations whose major metabolic pathways are known. relevant is nbss' l-arginine, which associates populations of bacterial cells of streptococcus faecium in combination with an ammonia electrode. arginine is metabolized by microorganisms. it is difficult to obtain complex reactions outside the cellular structures. similar to the use of cell populations as sensitive elements, fragments or parts of plant tissues may be used. the advantage is greater because there is no extra effort to keep the cells viable in a natural arrangement. for adenosine nbss, a tissue biosensitive element has been proposed. for dopamine nbss, specialists have focused on the pulp of banana fruit, considering that it has remarkable biocatalytic properties. • nbss with redox proteins: the redox proteins are involved in biochemical processes such as cellular respiration and photosynthesis reaction (kersten and feilner 2007) . nbs catalysts use enzymes, microorganisms, or cells to catalyze a reaction with a target substance. nbss own an affinity on using antibodies, receptors, and nucleic acids that bind to a target substance. reactions are quantified by electrochemical, optical, evanescence transducers, etc. the main types of known redox proteins are cytochromes, containing iron in the prosthetic group, and cytochrome "c" is involved in the transfer of electrons into mitochondria; ferredoxins contain iron and sulfur ions in dimeric combinations of chloroplasts (2fe-2s) ferredoxin and tetrameric combinations of bacterial ferredoxin 2 (2fe-2s) involved in photosynthesis and transfer of fixed nitrogen ions, respectively; blue proteins contain copper linked to the smallest cysteine residue involved in a tetrahedral structure such as plastocyanin and azurin that mediates electron transfer in photosynthesis and possibly in nitrite reduction; flavoproteins, containing a prosthetic group and an organic conjugate, are involved in the transfer of proteins such as flavotoxins (agrawal et al. 2012) . these proteins play a role in nature, due to the location on their surface of the redox centers. the subtle architecture of molecules offers selectivity and specificity to these molecules in their interaction with other proteins or enzymes, such as the cytochrome c structure. porphyrin iron (heme) is located at the center of the molecule and is well covered or hidden, being exposed to solvents in a small proportion of 0.06% of the total molecular surface. from those presented above and from table 12 .2, we can see that nbss can be classified into two groups according to the biological component. the protein has a positive potential of +9 mv due to excess lysinic base debris. there is a 324 debye dipole moment, which produces an imbalance in the spatial distribution of the acidic chain balance. a number of lysine residues are distributed around the solvent to which the center of the heme that interacts with the redox proteins is exposed (nelsen et al. 1990 ). nbss are classified in three generations. at the first-generation sensors, the biocatalyst is attached to the surface of the membrane, and then this arrangement is fixed to the surface of the transducer. the adsorption or covalent attachment of the biologically active component to the surface of the transducer allows the elimination of the semipermeable membrane, which is the second generation. the direct linking of the biocatalyst to the electronic device that translates and amplifies the signal, such as the compact transistor, is the basis of the third-generation nbs miniaturization. depending on the nature of the immobilization and the interaction between the three components, the a-r membrane contact with the electrodes to the transducer, and the processes in the nbss have evolved over a generation. first, the specificity and selectivity are dominated by the biological component and are directly related to its nature: enzymes, antibodies, and microorganisms. the specificity derives from the binding of the analyte to the biological component used as the receptor. at the base of this dominant sequence is the a-r biochemical reaction and the collision process between a and r. second, the transport of the analyte to and through the surface considered to r is also an important factor. this process is related to the transport of a physical size through typical diffusion, migration, and convection mechanisms. third, the nbs signal is dependent on the a-r reaction assumed to be at a constant speed. transient states and biochemical pseudo-equilibrium conditions are dominated by the reaction kinetics, the nature of the transport, which in turn is coupled with the immobilized a-r interface substrate reactions. even in the case of a real equilibrium, the reaction speed near the steady state will be important in determining the response time. the kinetics of these processes require additional conditions (agrawal et al. 2012) . the new types of nanoscale materials with different levels of biocompatibility, the new generation of biocompound cells based on a better understanding of metabolism, the manipulation of information stored at the molecular level all have led to a generation of nbss with a high level of integration. molecular information initially stored in the base molecular components can be expressed directly to a higher level called "supramolecular" where interactions between molecules are performed by preestablished algorithms, leading to adaptive, functional, and intelligent materials. materials are built on conceptional, supramolecular, and combinatorial principles. separation, storage, and detection techniques are developed using "biomimetic" membranes that function according to biological models or precise physicochemical principles ). electrochemical nbs with dna is the result of medical diagnosis requirements to quickly and accurately determine the segments of a dna sequence. results from genetics, molecular biology, and nanotechnology have led to one of the most accurate detection methods: electrochemical nbss with dna (combining the principle of nbs isfet with molecular wires from nanotubes). the operating principle consists of collecting the signal between two electrodes-one working electrode and another reference electrode. the auxiliary electrodes have a specified role in their turn. the sensing mechanism consists in modifying the i-v characteristic (current-voltage) in the presence of a target molecule. carbon nanotubes are exceptional for the work electrode with high electron transfer velocity and excellent spatial resolution. target in nbs dna is an unknown sequence of dna (or oligonucleotides) attached by functionalization to the carboxyl or amino cnt groups. researchers reported the developing plants' ability to capture 30% more energy by implanting carbon nanotubes into chloroplasts and plant organisms where photosynthesis takes place. they managed to modify plants so as to detect nitric oxide by implanting another type of carbon nanotubes. "the plants are suitable for the role of a technological platform. they heal themselves, they are durable, they resist the harsh environments and they have their own sources of energy and water." the transformation of plants to photon devices, with own energy, such as explosive detectors or chemical weapons, is expected (panchal and upadhyay 2014) . external or surface electrodes are metal electrodes that contact the nbss' bioactive component either directly (dry or solid electrodes) or through an electrolyte solution (liquid electrodes). solid or dry electrodes are made in silver, platinum, gold, and nickel. internal electrodes are made of thin wires made of durable metal: stainless steel, platinum, and tungsten. the active part of the electrode can be covered with a metallic conductor layer (gold, silver) and the inactive one with an insulating layer (polymer/thin film). nbss' active contact surfaces are large in size compared to cell sizes and are used for extracellular recordings. microelectrodes are internal electrodes, but they are built to measure potentials in direct contact with the receiver in nbss. the contact surface with r is micronized. microelectrodes can be solid, compounds which can be achieved by depositing a conductive layer (platinum, gold) on a glass support having a particularly thin peak, and another constructive variant consists of inserting a metallic or carbon fiber conductor into an epoxy resin support mixed with a conductive paste; they are used in cellular samples and consist of a glass pipette having a micronized tip filled with an electrolytic solution containing potassium chloride. in the electrolyte solution, a conducting wire is immersed to pick up the electrical potential (wu et al. 2014 ). it was thought that it is not possible to transfer electrons directly between the electrode and the proteins due to their distortion. several practical considerations have led to the conclusion that the active center of the heme is irreversibly adsorbed when resulting in protein denaturation in contact with the electrode. changing the surface of the gold electrode by surface adsorption of 4,4′-bipyridyl resulted in the modification of the electrode surface configuration for interaction with cytochrome "c." 4,4′ bipyridyl is not an electroactive substance in the potential region and therefore does not play a role as a mediator. this electrochemical addition was possible due to the quasireversible binding of cytochrome "c" to the modified gold electrode with 4.4′ bipyridyl, thereby resulting in the hydrogen linkages in the lysine residues to bound to the nitrogen of the pyridyl which modified the surface of the electrode. transmutation through complex protein electrode rapidly directs the transfer of electrons, which is accomplished by the following scheme: cytochrome diffusion "c" on the electrode, protein binding on the surface, electron transfer, and protein desorption. following this process, more than 60 surface changes were possible for electrochemistry of proteins and the gold electrode. using a bifunctional reagent (x-y), wherein the x group is the n, p or s bonded electrode and the y group, which must be bonded "represent also examples of patterns developed" (agrawal et al. 2012 ). electrochemistry of proteins has been extended to the carbon electrode. pyrolytic carbon-graphite forms, vitreous carbon and mesocarbon, are structures in which graphene plans are arranged in ababa hexagonal mesh or disordered in different turbulent forms. the base graphene plan is hydrophobic, but existing or induced defects lead to free c-c bonds, and there is an increase in c-o linkages by oxidation. the direct electrochemistry of positively charged proteins can therefore be performed on the edges of the graphite plans of the carbon electrode. the direct transfer of electrons to negatively charged proteins, such as plastocyanin with graphite electrode (edges or plane edges), can be aided with mn, ca, cr complex cations complexed with amino compounds, cr (am6) 3+ , used as promoters of reactions. in this context, the promoters are inactive redox species in solutions but allow the transfer of electrons to the redox proteins. microminiaturized electrodes have specific advantages among which we mention the improvement of polarization and contact with biological material at the active sites. specificity and selectivity depend on the nbs receptor biological component and its affinity for the analyte. affinity is a specific feature of enzymes, antibiotics, and receptors being used in functions in living organisms. affinity is based on the chemical coupling between a component and its complementary partner. in the case of high-affinity components, the diffusion process is rapid, leading to the formation of the ag-ab type of complex. the association reaction specific to molecular recognition will be characterized by the first-order response rate constant. in nbs measurements it is essential to consider the concentration of a constant component and other variables. the results of electrochemistry of proteins have been extended to amino acids and peptides (barroso et al. 2015) . the addition of enzymes to solutions containing substrate molecules is the essential condition in enzyme catalysis reactions. extracting the necessary information from the enzyme science to be applied to the development of nbss such as the enzyme electrode is an extremely difficult task. references will be used to outline some of the enzyme properties necessary to describe enzymatic nbss. consider a simple reaction, with a single substrate s, that combines with enzyme e to form the enzyme-substrate intermediate complex, es. this unstable complex undergoes a new reaction resulting in product p. the formation and consumption rates of the complex are equal. as soon as e and s enter the reaction, the system becomes unbalanced, the concentration of the complex will be zero, and the formation speed of the complex is much higher than the rate of its consumption. as the reaction unfolds, es increases and implicitly increases the rate of disappearance of the complex relative to its rate of formation. initially, the excess of the substrate determines the consumption of the enzyme, and during the course of the reaction, the enzyme's constant regeneration begins to reach its steady state. the analysis of these reactions results in two important conclusions: • at a low concentration of the substrate, the rate of the reaction is proportional to the substrate concentration and inversely proportional to the rate of the formation and extinction rate of the complex or the dissociation reaction rate in the initial reactants plus the decomposition reaction rate in products. • at a high concentration of the substrate, maximum speed is limited by enzyme concentration. thus, the two sequences correspond to the two processes that can control the overall reaction rate (stein et al. 2011 ). when the reaction takes place in homogeneous solutions at a uniform rate, the same in the entire environment, it is necessary to consider the change in the concentration of the components over time. three mechanisms of mass transport occur in solution: diffusion, convection, and migration. an electrochemical nbs is an autonomous, self-contained device capable of providing specific quantitative or semiquantitative analytical information using as a molecular recognition element, a biochemical receptor (biological identification element) that is in direct spatial contact with an electrochemical transducer. electrochemical nbss are distinguished only by the nature of the transducer regardless of the nature of the biological component according to the classification in table 12 .3. due to their ability to be calibrated in a repetitive manner, an electrochemical nbs is distinguished by a bioanalytical system requiring additional processing steps, such as the addition of reagent. nbss for a single type of measurement, or unable to continuously monitor concentration analysis or not to be rapidly and reproducibly regenerated, are defined as "disposable." nbss are classified according to their biological specificity-with reference to the mechanism or to the interpretation of the physical-chemical signal (the transducer) (barroso et al. 2015) . the biological recognition element is based on a catalyzed chemical reaction or an equilibrium reaction with macromolecules that have been previously isolated or synthesized in their original biological environment. in the case of reversible reactions, the steady state can be reached if there is no net consumption by the agent of in addition to quantitative determination of analytes, nbss are also used to detect and quantify microorganisms: the receptors are bacteria, yeasts, or oligonucleotides coupled with electrochemical, piezoelectric, optical, or calorimetric transducers the immobilized biocomplex and incorporated into the nbss. electrochemical nbss can be classified according to the analyses and reactions they monitor: direct monitoring of the concentration of the analytes or their production or consumption reactions and, alternatively, an indirect monitoring of the inhibitor or activator of the biological recognition element (the biochemical receptor). criteria include calibration characteristics (sensitivity, linearity, operational range of concentration, quantitative determination limits, and specific detection), selectivity, equilibrium state and response time, reproducibility, lifetime, and stability (fang et al. 2015) . the notion of recognition is used in nbss or in nanobiosensory systems by association with the sensory systems of the plants. sensations such as smell or taste are made up of systems that contain an identification receiver cell coupled with neurotransmitter signal-processing pathways. such phenomena also occur in biochemosensors but at a much-simplified level compared to the complexity of molecular recognition in living systems (barroso et al. 2015) . examples of single or multiple transfer signals, limited to the main biochemosensors, are shown in table 12 .3. for the receptor types shown in table 12 .3, different electrodes and measurement methods can be selected from table 12 .4 to form an electrochemical nbs. nbss are classified by the recognition element (table 12. 3) or by the transduction mode (table 12 .4). nbss irrespective of the type of classification should be treated unitarily as a microsystem, the biological recognition element being in direct contact with the transducer element. an electrochemical nbs is an nbs with electrochemical transducer (table 12 .4). it is considered to be a chemically modified electrode (cme), the electric conductor nbss may use several other types of non-electrochemical transducer: (a) piezoelectric nbss; (b) nbs-saw, measures surface acoustic waves in a resonance circuit (shear and surface acoustic wave); (c) thermometric nbss (the active element is coupled with a thermistor); (d) optical nbss, uses optical phenomena: planar wave guide, optical fiber, surface plasmon resonance) spr nbss use the immobilized analyte-receptor interaction on a metal film deposited on an optical prism measuring the variance of the refractive index due to changes induced in the metal's electrical charge that transmits the electrons from the interaction process to the outside in the electronic measuring system. the electrode may be a metal, a semiconductor, or an ionic conductive material coated with a biochemical or bioactive film. electrochemical nbs is an integrated transducer microsystem capable of providing selective, quantitative, or semiquantitative analytical information using a biological identification element. it can be used to monitor biological and nonbiological elements. chemical nbss that incorporate nonbiological components as receptors, although used to monitor biological processes (ph or nbss of oxygen), are not nbss. the clark electrode is of importance in the nbss' measuring range. similar physical nbss used in biological environments such as those measuring pressure, etc. are not considered nbss (jacoby et al. 2015) . electrochemical nbss according to the terminology set out in tables 12.3 and 12.4 can be classified according to their biological specificity, by mechanism or mode of signal transmission, or alternatively, the combination of two. they can be amperometric, potentiometric, field effect (fet), or nmss' conductometric (electrical conduction measurement) respectively impedance metrics. alternatively, they may be called enzymatic amperometric nbss to specify the nature of the receptor and the transducer. the first nbss that were studied are the enzymes and immunosensors (fang et al. 2015) . nbss are based on a catalyzed reaction of biomacromolecules present in the original biological medium that is preisolated or synthetically produced. the reaction is monitored by an integrated detector (transducer) that measures the stationary or transition states or the final reaction product via the immobilized biocidal product in nbss. types of commonly used biocatalysts are enzymes (simple or enzymatic complexes)-most commonly used as recognition systems, cells, microorganisms (bacteria, fungi, eukaryotic cells, or yeasts), cellular organs, or component (cell walls, mitochondria) sections of plant or animal tissues. nbss with biocatalyst recognition elements are the best known and studied since the beginning of their approach by clark and lyons. one or more analytes, commonly called s and s′ substrates, react in the presence of one or more enzymes, cells, etc., to produce the p and p′ products (fang et al. 2015) . there are four strategies whereby the associated transducer can monitor the consumption of s-analyte through the biocatalytic reaction: • detection of s′ cosubstrate consumption, oxygen depletion through the oxidaseinduced reaction chain, bacteria, or yeast. the measured signal is the decrease in cosubstrate consumption compared to the initial value. • recycling of the reaction product p such as peroxyhydrogen, h + , co 2 , and nh 3 , in oxidoreductase reduction schemes, hydrolysis, lysis, etc. the signal from the transducer will be amplified. • detection of active centers in the biocatalyst: redox, cofactors, prosthetic groups evolving in the presence of substrate s by using an immobilized mediator. it reacts quickly with the biocatalyst and is easily detected in the transduction chain. various ferrocene derivatives, such as tetrathiafulvalene, tetracyanochinodimetane (ttf + tcnq), organic salts, quinones, quinone dyes, ru, or os complexes in polymeric matrices, can be used as mediators. • direct electron transfer is made between the enzymatic redox reactive site and the electrochemical transducer. the third strategy eliminates, partially or totally, the dependence of the nbss' response on the cosubstrate concentration, s′, which decreases the influence of interference between species. the use of mediators leads to the decrease of the substrate concentration together with the reaction chains by using a suitable membrane, whose permeability favors the transport of the cosubstrate. when enzymes are immobilized within the same reaction chains, it can improve the performance and abilities of nbss. three possibilities are commonly used: • some enzymes facilitate biological identification by sequentially converting the products of the enzyme reaction series into an electroactive final form: this way allows for a wide range of nbs analysis. • the enzymatic complex, applied in series, can regenerate the cosubstrate of the first enzyme and amplify the nbs output signal by regenerating another cosubstrate of the first enzyme. • the parallel enzyme complex improves selectivity of nbss by lowering the local concentration of electrochemical interfering substrate: this sequence is an alternative to the use of a permselective membrane or a sequential method (e.g., interpretation of an output signal generated by an nbs and a reference nbs without biorecognition element). the operation of nbss is based on the interaction between the analyte and the macromolecules or organized molecular assemblies. upon reaching the balance, there is no consumption of analyte by the biocomplex agent immobilized in the substrate. the response to the biocomplex analyte-reagent reaction is monitored by an integrator detector. in some cases, the biocomplex reaction is self-monitored by a complementary biocatalytic reaction. the integrator detector monitors stationary or transient states. antibody-antigen interactions, the most relevant examples of nbss using biocomplex receptors, are based on immunochemical reactions, e.g., binding an antigen (ag) to the characteristic antibody (ab). complexes formed by ab-ag can be detected provided that other nonspecific reactions are minimized, for each determination of ag corresponds to a certain ab that must be isolated, purified, etc. some recent studies have analyzed direct monitoring of ag-ab formation using ion-selective field effect transistors (isfets). increasing the sensitivity of immunological nbss is achieved by adding specific enzymes to ag-ab couples, but this requires additional synthesis steps. as the binding strength or affinity constant varies widely, these systems operate irreversibly (disposable nbss) or are coupled to flow injection analysis (fia) systems; then ab can be regenerated from dissociation of the complex with agents such as glycine-hcl to ph 2.5 (kurien et al. 2010 ). recently, they have been used as molecular recognition systems in conductometric analysis, isfet, or optical nbss with receptors with ion channels, membranes, or protein structures. a transporting protein, lactose-permease (lp), can be incorporated into a liposomal bilayer that permits protonic carbohydrate transport with a stoichiometric ratio of 1:1. this mechanism was identified through the ph-dependent fluorescence of a fluorophore immobilized in liposomes. liposomes with lp were incorporated into a lipid bilayer deposited on a ph-sensitive isfet. preliminary results show that this modified isfet is capable of irreversibly detecting lactose from an fia system. protein receptor nbss have been recently discovered. binding of analytes, here called agonists, to immobilized receptor proteins is monitored by changing the flow of ions through these channels. glutamate, as an agonist, can be determined in the presence of other agonists that can interfere with the determination of na + or ca 2+ streams using conductometric method or ion-selective electrodes. due to the dependence of the ionic channel on the nature of the linkages, it produces an independence toward the enzyme nature in order to achieve the desired sensitivity. two methods have been approached. the first refers to oligonucleotide duplex interleaving during the formation of the double helix structure of the dna of a molecule that is electrically active. the second method is the direct detection of guanine which is electroactive. some of these nbss cannot operate through analytical-sensing membrane separation membranes. the sensitive layer often has to be in contact with the biological environment where the analytes are located (fang et al. 2015) . nbss have been developed for indirect monitoring of organic pesticides or inorganic compounds (heavy metals, fluorides, cyanides) that inhibit the biocatalytic properties of enzymes used in the construction of nbss (devices are irreversible). in immunosensors, the initial biological activity can only be regenerated by chemical treatment and therefore is not part of the reconditioned or reusable nbs class. their application potential is to warn and not to accurately monitor a specific analyte (considered as disposable). nbss with cyanide (i.g. inhibition of cytochrome c oxidase) that are used as inhibitor to cytochromoxidase are regenerated by washing with a phosphate buffer at ph 6.3 (armstrong and beckett 2011). with the development of enzymatic glucose nbss, an experiment in which glucose oxidase is immobilized between two membranes, literature has emerged about techniques for immobilizing biological receptors. enzymes, antibodies, cells, or tissues with high biological activity can be immobilized in a thin film on the surface of a transducer through a variety of methods. the following immobilization procedures of biological receptors are used: • immobilization on the membrane on the surface unexposed to the analyte: an enzymatic solution, a cell or tissue suspension, rests between the permeable membrane to the analyte and the measuring electrode (electrochemical detector). • retaining of biological receptors in a polymeric matrix, polyacrylonitrile, agar gel, polyurethane (pu) or polyvinyl alcohol (apv), redox hydrogels with redox centers such as [os (bpy) 2cl] +/2+ . • retaining of biological receptors between self-assembled layers (sam) or in membranes from the double lipid layer (blm). • the covalent binding of membrane surface receptors through bifunctional groups: glutaraldehydes, carbodiimides, sams, avidin-biotin silanized. • modification of the entire electrode structure (modified carbon paste with enzymes or graphite in epoxy resins). receptors are modified either alone or mixed with other proteins, such as bovine serum albumin (bsa), either directly on the surface of the transducer or in the polymer membrane. reactivated membranes can be used directly to immobilize enzymes or antibodies without chemical modification. the covalent binding and cross-linking are more difficult than immobilization or the retaining of receptors on the membrane. in the case of microsensor structures where the membrane is directly deposited on the transducer, the covalent bonding is safer and more stable (muñoz et al. 2008 ). besides reactive layers or membranes with immobilized receptors, many nbss, those for clinical or biological applications, incorporate one or more internal or external separation auxiliary membranes with three important functions: the barrier, the outer diffusion barrier for the substrate, and the biocompatible surfaces. for any nbs built on the principle of molecular recognition, it is important to characterize it by its response, which is related to operating parameters and limiting reaction speeds. accuracy, precision, sensitivity, and reproducibility are basic criteria for estimating nbs performance. these parameters are in direct relation to the reaction mechanisms, the transport phenomena, and the kinetics of the processes in the volume at the interface. most criteria have been developed for enzymatic nbss, being the most studied in the literature. in the case of immunosensors, the key element is the ability to capture the surface, i.e., the number of surface molecules that is active. one method of checking this parameter is to measure the specific activity, meaning the ratio of the number of active molecules to the number of immobilized molecules. this estimation is dependent on the immobilization mode (molecular orientation, number of attachment points or active sites), and the ratio ranges between 0.15 and 0.3, rarely reaching the unit. capture capability becomes important when the surface decreases as in microfluidic applications. a problem encountered in immunosensors is that of regenerating the surface without significant loss of activity. there was a lack of rigor in the performance criteria (affi et al. 2016) . the response signal is corrected for background noise, the reference concentration is usually estimated in mol/l although this high value is never used when measuring ranges refer to 10 −10 mol/l, and currently sensitivities of the order nmol/l and pmol/l have been reached. transient response is important for dynamic assay analysis and sampling techniques but is less significant in continuous monitoring. the transient response is estimated by the slope (dr/dt) max after the addition of the analyte in the measuring cell. one evaluation method is to introduce nbss into an fia system for sequential sample analysis in a specified hydrodynamic regime. the sensitivity and linear range of measurement of stationary concentration are determined through graphical representation. this method is more concise than the current calibration curves used to plot the response corrected to the baseline based on its concentration or logarithm. parameters are estimated in the linear response range of nbss. any electrochemical nbs has a superior linearity of the response. this limit is directly related to the biocatalytic or biocomplex properties of the biochemical or biological receptor. more in the case of nbss with enzymes, this limit is significantly influenced by membranes and immobilized substrates where the diffusion barriers and secondary kinetics play a role. the local concentration of the analyte in the reaction layer may be two orders of magnitude smaller than the volume of the solution (michelini and roda 2012) . enzymatic kinetics are described by michaelis-menten mechanisms and expressed by km and vm parameters. for the kinetics of the enzyme in the solution phase, km is usually determined from the lineweaver-burk graphical representation. for any electrochemical nbss, the number of standards used and how the standard sample matrix can be simulated or duplicated should be set, being required to specify the procedures for each type of nbss related to its application. these are important for the disposable nbss' case using immunoaffinity or inhibition reactions. sensitivity is the slope of the curve and should not be confused with the quantified detection limit (lod) relative to the baseline or noise signals. the range of work concentrations is determined by lower or higher detection limits (fang et al. 2015) . selectivity and safety are determined as any kind of amperometric or potentiometric nbss. they depend on the choice of receiver and transducer. most enzymes are specific, but there are also nonselective enzyme classes, such as alcohol oxidases, the group of oxidases sugars, peroxidases, lactases, tyrosinases, ceruloplasmin, alcohol dehydrogenases, glucose dehydrogenases, nadh dehydrogenases, etc. they have been used to develop nbss to determine environmental phenols or to monitor food quality. on the other hand, oxygen electrodes, ph electrodes, and isfets have a pronounced selectivity, the same as metal electrodes that are sensitive to many substances. their selectivity may be changed when these transducers are associated with receptors. enfet is ph sensitive to the buffer and protonation but its selectivity is not altered. when the transducer interferes with other substances, known as ascorbate or urease, to glucose nbss based on hydrogen peroxide detection, these side effects may be restricted by the use of outer or inner permselective membranes. alternatively, nbss with and without biological receptors that work by differential nbss are designed. safety in operation of nbss depends on the selectivity and reproducibility and accuracy of the measurements (heyl et al. 2012 ). the clark construction principle studied electrochemically oxygen as a reducing gas and platinum as a metal electrode. platinum used for detecting electrochemical oxygen is known as the clark electrode. the electrode has an organic membrane covering the electrolyte layer and two metal electrodes. oxygen diffuses through the membrane and is electrochemically reduced to the cathode. between the cathode and the anode, a fixed voltage is applied, for which the oxygen reduction reaction takes place. temperature greatly influences reaction speed and solubility. this is a polarographic electrode used to measure the concentration of oxygen in body fluids or gases. the sample is in contact with a membrane (polypropylene or teflon) through which the oxygen diffuses into a measuring chamber containing 50% saturated potassium chloride solution. inside the room are two electrodes, one is reference, ag/agcl, and the other is platinum, coated in the glass. the electrical current at the polarization potential of -600 mv is proportional to the oxygen concentration in the solution. for reverse polarization at +600 mv, hydrogen measurements can be made. reactions are very sensitive to temperature and should be maintained at ±0.1 °c. the electrode is calibrated using a mixture of the two gas-oxygen and hydrogen-known concentrations. oxygen electrode or clark electrode has proven to be an analyzer of raw gas or developed gas when performing chemistry analyses in the clinical laboratory and in the field of medical care, ambulatory, or intensive care (on the surface of the platinum electrode an enzyme reacts with oxygen). the enzymes are placed in a closed membrane to the surface, which can be recognized as the simplest model of nbss. the oxygen concentration curve was proportional to the glucose concentration. it was the first nbs built, which helped the progress of laboratory analyses a lot. oxygen diffuses through the membrane and is electrolytically reduced to the cathode. the higher the partial oxygen pressure, the more oxygen diffuses at a time. the temperature nbss attached to the sample allow the membrane to compensate for the diffusion and solubility rate. the measuring instruments record cathode current, sample temperature, membrane temperature, barometric pressure, and salinity. with this information one can calculate the oxygen contained in the sample, either in parts per million (ppm) or in percent of oxygen saturation. the geometric configuration of the clark electrode is of great importance. in particular, the thickness of the electrolytic layer between the cathode and the membrane must have a certain limit, to ensure linearity and decrease of the drift current. calibrating a polarographic system is a must. proportionality between current and oxygen concentration must be ensured, with errors below 1% (biological samples role and air parameters are essential). air, as a gas mixture that has a constant oxygen content of about 20.9%, when in contact with water, the dissolved amount depends on several factors: the optimal time for oxygen dissolution, homogeneity of the water solution, water temperature, air pressure, salts contained in water, and other water-soluble substances that are oxygen-consuming. oxygen contained in water is determinant for biological and chemical processes, so measurement of dissolved oxygen in water is important to find the partial pressure of dissolved oxygen; it must be saturated in pure water at a certain temperature (wolfbeis 2015) . the enzymatic electrode (in some references known as the enzyme electrode) is a combination of an electrochemical probe of any type (amperometric, potentiometric, or conductometric) with a thin layer (10-200 microns) of immobilized enzyme. in these devices the function of the enzyme is to provide selectivity in virtue of its biological affinity for a substrate of molecules. an enzyme can catalyze a reaction of a given substrate for a specific isomer from a plurality of substrates with different isomers. typically, the degree of advancement of an enzymatic reaction (directly related to the concentration of the analyte) is monitored by the rate of product formation or the disappearance of a reagent. if the product or reagent is electrically active, then the response can be directly monitored by amperometry, i.e., the variation of the current for a given applied potential. the main considerations are: does the enzyme contain active redox groups? are the biochemical reaction products electroactive? is one of the substrates or cofactors electrically active? what is the speed and response time? what is the final application of nbss? if the enzyme does not contain redox groups, then nbss are limited to measuring the product or substrate consumption by their reaction to the transduction electrode. the electrical current is directly related to the analyte concentration. nbss are based on electrochemical response due to h 2 o 2 . most common enzymes used in the design of enzyme electrodes are those that contain redox groups that change their redox state during the biochemical reaction. redox enzymes are oxidases and dehydrogenases, pyrroloquinoline quinone (pqq). they act by oxidizing the substrate, accepting electrons during the process, and further transforming in a reduced state. these enzymes return to the oxidized active state by transferring electrons to the oxygen molecule resulting in hydrogen peroxide (h 2 o 2 ). both oxygen and peroxide being electrochemically active, they continue by reducing to cosubstrate (oxygen) or oxidation of peroxide (reaction product). the method based on the reduction of oxygen to the o 2 electrode is one of the simplest methods but suffers from several disadvantages, namely, slow response, miniaturization difficulties, low accuracy, and reproducibility. measurements on peroxide oxidation overcome the above difficulties and are currently the most popular method. mediator systems-a major limitation of the above-described hydrogen permeation system is the high operating potential (about 0.8 v against the ag/ agcl reference electrode) required for oxidation of peroxide which leads to increased interference. the use of mediators (molecules that can carry electrons between the enzyme redox center and the electrode) can minimize this inconvenience. depending on the nature of the mediators, the potential applied may be reduced below the limit of interferences of species such as ascorbate, urate, and paracetamol. a large number of compounds are able to act as mediators in the enzyme electrode. of these, the most popular are the metallic complexes. representatives for organometallic complexes are ferrocenes and their derivatives because they have redox potentials and are independent of ph. bienzymatic systems' recent works have focused on the direct communication of electron transfer between enzymes and electrodes. successes in the field are the peroxidase enzyme hrp (horseradish peroxidase) that catalyzes the reduction of hydrogen peroxide for a number of organic compounds. when the enzyme is immobilized on the electrode, the need for the organic reducer is prevented by the electrode itself providing the reducing equivalences. the coupling of peroxidase with an enzyme oxidase allows for the construction of bienzymatic systems through which the peroxide produced by the oxidase is detected by the electrode-peroxidase system which operates at lower potentials relative to a simple platinum electrode. this is where the minimization of active species interferences results from (frederickson matika and loake 2014). optical fiber as a nanobiosensor can be placed in the surface or inside the plant to directly measure parameters. nbss with optical fiber are proposed to be used in many and rapid medical determinations, and its applications are continuously expanding. it can be attached inside a hollow-like tubular instrument, serving to dilate a hole or channel, and inserted into the tissue, performing a minimal monitoring where it is needed. nbss with optical fiber are nontoxic, chemically inert, and can be successfully used inside the plant. it can be associated with plant monitoring equipment. it's easy to handle with negligible weight. the evolution of fiber-optic nbss is based on multiple performance and biocompatibility. biocompatibility is the first step in the plant's comfort; nbss should not affect the physiological parameters of the plant, but its functionality must not be compromised by plant disassimilation products. fiber-optic nbss can be classified as extrinsic, fiber acts as a way for signal and intrinsic, interactions occur in the fiber itself. there are two types of fobs: minimally invasive nbss that are introduced into the cavities of the plants and invasive nbss that are introduced into the organs or in wood conductive tissue (liu et al. 2015) . in the last decade, optical fiber is a product that is widely used in all the cutting-edge fields of advanced science and technology. given the ease with which it can be manipulated, unlimited sterilization possibilities, and reduced costs, it can be estimated that this product will increasingly gain market. the following applications are known to have used fiber-optic nbss: in epidermis and vascular tissues, for analysis of raw sebaceous elements, saturation in oxygen, raw sewage gas analysis, sap ph; in plant breeding monitoring; easy ph determination with a microabsorbent indicator and ph-modulator, acid-alkaline; in vegetal tissues, when it is intended to monitor the temperature, or to diagnose small and very small injuries that are difficult to reach; in epidermis for can test the quality and integrity of the layers, so, small lesions can be detected, can be used to stimulate tissues, a fobs based on oxygen demand (bod) can be used; in the stem can identify very small injuries that are inappropriate. another possible application is to appreciate the color or integrity of the vascular tissues. optic-fiber nbss can now monitor electrolytes from raw or elaborated sap as well potassium, sodium, and calcium. it takes the form of a tubular instrument, able to expand an optic-fiber channel (0.5 mm diameter) that can be inserted into vascular tissues. it can measure the gas concentration and the ph of the raw or elaborated sap and oxygen saturation also. the materials that make up the chemical transducers are ionophores that can be reversibly attached to the electrolyte by a molecular separator (spacer) and fluorophore, respectively. the degree of fluorescence, through excitation with electroluminescent diodes in purple, is modulated by ionophores proportional to the analyte concentration. nbss are used either extracorporeally in the external raw or elaborated sap gas analysis circuit or intracorporeally for continuous blood gas monitoring in critical situations. the chemical parameter capable of monitoring cell state is ph because lactic acid, formed when cell tissue dies, produces a decrease in ph. any drop in the ph of the raw or elaborated sap from 7.4 indicates cell death (mclamore et al. 2010) . achievements in the domain are invasive ph nbss that determine the state of the cell. nbss are composed of a fluorescent dye encapsulated in a gel matrix (polyacrylamide) attached at the end of the optical fiber. the dye is characterized by the emission of the acidic form centered on 580 nm and the alkaline form centered on 680 nm. the two forms are ph sensitive. excitation occurs at 533 nm for both forms. separation of emission is done directly through optical filters, and sensitivity is 0.05 ph units far below acceptable clinical standards (0.1 ph). a bacterial disorder is a multifactorial condition that is characterized by demineralization of the inorganic portion and an organic destruction of the substance. each bacterial perturbation has as an etiological agent as pathogenic species. the content of the raw or elaborated sap in terms of bacterial load is about 109/ml raw or elaborated sap. therefore, raw or elaborated sap can be considered as a selective medium for bacterial growth. a significant correlation has been demonstrated between the number of pathogens in the raw or elaborated sap and their prevalence in the problems. a simple method has been adapted for detecting and counting the pathogen; it is a device consisting of a special support made with an agar culture medium containing 20% sucrose. it is inoculated with raw or elaborated sap, and the density of growth of the pathogen is assessed after incubation for 48 hours at 37 °c. next comes the morphological identification of distinct colonies on selective and nonselective agar, on distinct cell form, visible in the light of the microscope. the technique also has some disadvantages that it takes time for bacterial growth and also requires additional laboratories. to monitor pathogen activity in the sap, a fobs was developed that monitors the pathogen-mediated sucrose reactions through a photosensitive indicator immobilized in a porous glass coating. the surface of the optical fiber core is treated or coated in a porous glass film using the sol-gel technique (kozan et al. 2007) . spectroscopic analysis showed that there are two phases in light absorption at 597 nm over a duration of 120 min: between 0-60 min and 60-120 min. the investigation shows the potential of nbss in monitoring plant activity. the sol-gel technique is used to immobilize the photosensitive indicator, and it is simpler than compared with the principle of selective medium which takes time and is laborious. criteria at the base of the experiment: pathogens are partially anaerobic with opti-mal growth at 37 °c; glucosyltransferases and fructosyltransferases from pathogens catalyze the synthesis of water-insoluble glucan and fructanic polymers in sucrose to form lactic acid found in acidic sap; pathogenic agents in the sap synthesize both extracellular and intracellular polysaccharides from sucrose; extracellular polysaccharides help the adhesion of bacteria to the surface, while intracellular polysaccharides are stored for bacterial energy; polysaccharides, intracellularly, help the bacterium to continue fermentation even when there is no exogenous form of food. acid tolerance of pathogens causes their activity to continue even at a low ph; a ph indicator, photosensitive, produces a characteristic color, according to a color gradient, depending on the ph of the raw or elaborate sap and is used in the fobs construction (miranda et al. 2011) . on the basis of these considerations, an experimental assembly was designed and performed with a double beam uv spectrometer in which optical fiber was introduced instead of a cuvette. in the reference well, the blue bromophenol buffer solution was used for different ph values, from 4 to 7. the initial experiment helped determine the wavelength and peak characteristic to the buffer indicator and for different ph values in the sap, induced by bacterial activity. uv spectroscopic analysis at a ph of 4 and 7 of the blue bromophenol solution showed slightly prominent at 590 nm wavelength; peak intensity decreases from ph 7 to 4. comparison with literature data showed a good concordance, observing that in the medium with sap, sucrose and bromophenol blue, the absorption was stable at 590 nm wavelength for a time interval of 15 min, 30 min, 1 h, and 24 h. for each set of corroded and processed fibers set, it requires independent calibration due to the in homogeneities resulting from the optic-fiber preparation steps. fobs proves to be a rapid quantity measurement test of pathogen activity in raw or elaborated sap. this test can also be adapted to other plant nanobionics where bacterial activity is involved in cellular or tissue destruction. the method of forming a biosensor from an optical fiber for the observation and detection of the pathogen, the experiments of this study followed the phase of biochemical recognition of the signal and the phase of the spectroscopic analysis. the idea of nanobionic plants has evolved to create solar cells that heal themselves from plant cells. the next step was the desire to try to amplify photosynthesis in isolated chloroplasts in plants to be used in solar cells. chloroplasts host everything they need for two-step photosynthesis. in the first step, pigments such as chlorophyll absorb light, which generates the stimulation of electrons that circulate through the chloroplast tilacloids. the plant captures this electrical energy and uses it to fuel the second stage of photosynthesis, creating glucose. chloroplasts also have these reactions after they have been removed from the plants, but after a few hours, they break down because light and oxygen destroy their photosynthetic proteins. normally, unlike extracted chloroplasts, plants can repair this damaging process. to prolong chloroplast productivity, researchers have attached ceric oxide nanoparticles. these particles are, in fact, powerful antioxidants that remove oxygen radicals and other high reactivity molecules produced by light and oxygen, protecting the decomposition chloroplasts. the researchers introduced nanoparticles into chloroplasts using a new technique called leep (lipid exchange envelope penetration). by wrapping the particles into polyacrylic acid, a heavily charged molecule allowed the particles to penetrate the lipid hydrophobic membranes surrounding the chloroplasts. in these chloroplasts, the level of decomposition of molecules has decreased tremendously. using the same technique, the researchers introduced semiconductor carbon nanotubes embedded in negatively charged dna into chloroplasts. plants use only one tenth of the available sunlight, but carbon nanotubes have functioned as artificial antennas that have allowed chloroplasts to capture unusual light waves such as green, ultraviolet, and near infrared. when carbon nanotubes functioned as prosthetic photoabsorbents, photosynthesis, measured by the activity of electrons in tilacloids, was 49% more intense than in chloroplasts isolated without attached nanotubes. when cerium oxide was joined with carbon nanotubes, the chloroplasts remained active for the next few hours (nikolelis et al. 2008) . researchers went to live plants and used a technique called vascular infusion to attach nanoparticles to arabidopsis thaliana, a small flower plant. using the above method, the researchers applied a nanoparticle solution on the lower side of the leaf, penetrating the stomata that usually allow the carbon dioxide to get in and the oxygen out. in these plants, the nanotubes have penetrated into chloroplasts and have increased the photonic electron circuit by about 30%. it is also to be discovered how these percentages influence the sugar production of plants. scientists have been able to transform the arabidopsis thaliana plant into a chemical nbs by implanting nanotubes that detect nitric oxide, a pollutant produced by combustion (hines et al. 2015) . nbss have been created on the basis of carbon nanotubes for several chemicals, including hydrogen peroxide, trinitrotoluene, and sarin gas. when molecules attach to polymers encased in nanotubes, the fluorescence of these nanotubes is altered. carbon nanotubes can be used to create nbss that detect real-time particle free radicals or signal the presence of molecules with a very low level of concentration and too difficult to detect. this is a tremendous demonstration of how nanotechnology can be combined with synthetic biology to modify and enhance the functions of living organisms of plants. the way that nanoparticles arrange themselves can be used to enhance plant photosynthetic capacity, being used as nbss and stress reducers. by adapting nbss to targets, researchers hope to develop plants that could be used to monitor environmental pollution, pesticides, fungal infections, or exposure to bacterial toxins. attempts to incorporate electron nanomaterials into plants, such as graphene, are currently being made. researchers have long tried to find the best way to transmit information in a timely manner, focusing on electronics and mechanics of nbss for their tasks. nbss used for agricultural purposes are not new. recombinant dna technology now offers the possibility of obtaining new biological insecticides that preserve the benefits of "classic" biological control agents, plus some new features. these technologies are not accessible to all possible users, especially if they are poor, and furthermore, they have also generated a series of public debates about their usefulness and effects on organisms other than the target or the environment. obtaining pest-resistant plants is perhaps the most spectacular field of genetic engineering applied to plants, since it allowed the regeneration of plants containing genes of bacterial origin that provide protection against harmful insects. this ensures, on the one hand, the obtaining of richer harvests and, on the other hand, the reduction of farmers' costs for pesticides (lukács et al. 2006; prasad et al. 2014 prasad et al. , 2017 . a series of new genes for resistance to insect attack, transferable to plants (genes encoding δ-endotoxin production from b. thuringiensis) has been discovered; genes for the synthesis of enzymes or enzyme inhibitors; plant genes encoding the synthesis of specific lectins; genes that cause induction of synthesis of plant compounds such as phytoalexins. the development of cloning methodology was based on the observation that there is a group of gram-positive bacteria belonging to b. thuringiensis species that produce a toxin called δ-endotoxin or crystalline protein capable of killing a wide range of insects (coleoptera, lepidoptera, diptera), depending on the bacterial strain. of great interest is strain b. thuringiensis var. tenebrionis that synthesizes an effective δ-endotoxin against the colorado beetle. the genes involved in the synthesis of this protein are localized, on most bacterial strains, to large plasmids (75 kb), the production of the toxin occurring during sporulation. it has been shown that crystalline protein (δ-endotoxin) is normally expressed as a large inactive protoxin, which undergoes proteolytic processing in the intestine of the sensitive insect, becoming an active toxin. it recognizes the specific receptors in the intestinal cells and blocks the functions of these cells, leading to the death of the insects. studies on genes that encode inhibitory proteins produced by b. thuringiensis led to their grouping into four types based on target species specificity and nucleotide sequence: type i cry genes, encode specific 130 kda proteins for lepidopteran larvae; type ii cry genes, encode active 70 kda proteins on dipterous and lepidopteran larvae; type iii cry genes, encode 70 kda specific activity on coleopteran larvae; and type iv cry genes, encode inhibitory proteins for diptera larvae. the number of genes identified that are coding for δ-endotoxin-like proteins is high: 140 genes specific for lepidopteran, coleopteran, and diphtheria (genfa et al. 2005) . it has been achieved to obtain crystalline protein genes from several strains of b. thuringiensis by genetic amplification (pcr). because the whole gene for the crystal protein was found to be very poor in the transformed plant cells, a modified gene was created, containing only the n-terminal portion of the protein (amino acids 1-645). to increase gene expression in plants, the natural sequence for amino acids 1-415 rich in at was replaced by a synthetic sequence, rich in gc, containing the preferred codons for plant cells. these recombinant genes were introduced into ti plasmid-derived vectors (binary vectors containing the duplicate camv promoter, which increases the transcriptional and fivefold transcription and selection marker genes for antibiotic resistance or phosphinothricin herbicide) transferred to cells by a. tumefaciens containing ti disarmed plasmids. the size of recombinant plasmids obtained ranges between 5000 and 10,000 pb, depending on the size of the bacterial gene and the promoter sequence integrated into the vector. recombinant bacterial strains were then used to infect test plants (potato, tobacco, cotton). selection was first performed according to vector-borne selection markers (antibiotic resistance, gus test, herbicide resistance, etc.), and finally regenerated plants were subjected to insect attack (takakusagi et al. 2013) . regenerated plants have shown resistance to attack by insect pests, the specific character being maintained and expressed in experiments in the field. the first transgenic plant that manages the insect attack resistance belongs to the nicotiana tabacum species, expressing the whole or truncated cry 1a gene, cloned under the control of a constitutive promoter, so that the inhibitory protein represents 0.02% of the total plant protein (leaf). there were obtained cotton plants into which the modified cry 1a (c) gene, cloned under the control of the camv 35s promoter or under the control of a promoter and a sequence for a chloroplast transit peptide isolated from arabidopsis so that the expression level of the gene of interest led to a high level of toxin: 0.1% of total protein, 1%, respectively. another variant of cloning the bacterial toxin gene was that of using genetic elements that ensure expression of the gene of interest exclusively in the green portions of the plant (the promoter derived from the gene for pepc) or pollen (by using a gene derived promoter for a calcium dependent protein kinase (cdpk). a similar methodology has been used to transform rice plants, and by cloning a synthetic cry iii gene, they have obtained tobacco and potato plants resistant to the attack of colorado beetle (vigneux et al. 2007) . a modified 1a (b) modified gene was used for cloning under the control of the camv promoter and obtained sugarcane plants with resistance to diatraea saccharalis larvae. given the practical significance of plant resistance to harmful insects, research has been extended to other plant species, producing eggplant plants resistant to the attack of coleoptera, broccoli with resistance to certain lepidopteran species, maize with resistance to b. fusca, etc., as well as a number of advances in leguminous plants. toxicity studies conducted on plants expressing the gene for δ-endotoxin have shown that the existence of the transgene does not alter the normal features of the plants, except resistance to insect attack. in addition to δ-endotoxin produced by b. thuringiensis strains, other bacterial species have also been identified that produce insecticidal proteins (liu et al. 2011) . this is the case for b. cereus strains producing two vip1 and vip2 proteins with effect on insects, their mode of action being similar to δ-endotoxin. expression by plants of enzymes such as chitinase, cholesterol oxidase, lipoxygenase, phenol oxidase, peroxidase, or isopentenyl transferase (ipt) could be an alternative to using the δ-endotoxin gene. of the enzymes that can provide plant protection against insect attack, a particular place is occupied by chitinases, enzymes that act on chitin, a basic component of insect coatings. tobacco plants expressing genes for chitinase isolated from insects or beans exhibit increased resistance to lepidopterans. it has been observed that by cloning the isolated chitinase gene from the s. marcescens bacterium, a synergistic effect of endocytinase produced by plants containing the endotoxin gene in addition to s. littoralis larvae has been revealed. another enzyme of bacterial origin that exhibits insecticidal action is cholesterol oxidase. the introduction and expression of cho a gene for cholesterol oxidase from streptomyces sp. in tobacco plants have led to increased plant resistance to a. grandis larvae. the use for cloning the gene for the enzyme bacterial isopentenyl transferase (ipt) (involved in cytokine biosynthesis) by fusion with the protease inhibitor ii (pi-iik) gene promoter determined the production of n. plumbaginifolia lepidopteran-resistant plants (m. sexta or m. persicae). another embodiment was that of introducing into the plant cells the cpt2 gene that encodes a trypsin inhibitory protein isolated from vicia faba. this protein has antimetabolic effect, protecting plants from the attack of pests. similarly, other genes encoding protease inhibitors (kunitz trypsin inhibitor, bowman-birk proteinase inhibitor) or lectins have been cloned into different hosts, and encoded proteins may be true "defense guns" for the plants that contain them. it is known that insects, such as lepidopterans, depend on serine proteases (trypsin, chymotrypsin, or elastase), these being the first enzymes to digest (alvarez-fernandez 2010). a series of genes encoding different inhibitors for serine proteases have been isolated from various sources (plants, microorganisms) and cloned into various plant species, such as m. sativa, tobacco, corn, etc.; the plants obtained showed increased resistance to various insect pests compared to normal non-gm plants. in some cases, it has been noted that the insertion of additional genes to plants for protease inhibitors to join endogenous genes causes an increased level of pathogen resistance of transformed plants. however, the use of protease inhibitors for controlling insect pests requires thorough studies of plant and insect interactions because it has been observed that for some inhibitors such as serine proteases, the insecticide effect also manifests on useful insects (e.g., on bees). insect carbohydrate metabolism is another target for inhibitory agents tested in numerous studies. genes for different α-amylase inhibitors (wheat and beans) were isolated and characterized; after cloning the isolated gene from wheat in tobacco plants, an increase in lepidopteran larval mortality of up to 40% was observed. in the case of cloning the α-amylase inhibitor gene from beans in pea plants under the control of the pha 1 gene promoter, an increased expression of the foreign gene in the seeds was obtained, which resulted in a higher resistance to callosobruchus sp. (ramirez and spears 2014) . vegetable lectins are a special group of glycoproteins that have protective functions against a number of harmful organisms. studies on these glycoproteins have shown that they produce strong effects on the development of various types of insects. the first example of plants containing genes for nonspecific lectins that show toxicity to pests is tobacco plants in which the lectin genes from the pea have been cloned. however, many of the vegetable lectins also have a toxic effect on mammals, which limits their use as agents to increase pest resistance. special attention has been given by many specialists to lectin specific for mangosteen from guinea pigs and concanavalin a: the genes for these lectins have been cloned into different plant species (tobacco, tomato, potato, sugarcane, rice, wheat), and the heterologous proteins synthesized by them have determined a reduced sensitivity to the attack of harmful insects (lepidopteran, aphids, coleopteran larvae). the results suggest that the use of plants containing insecticide genes (such as for lecithin) together with integrated management represents promising possibilities for controlling pests from many plant species, including wheat or rice (richard et al. 2014 ). contrary to the remarkable results achieved so far, the genes used to transform crop plants are either too specific or only partially effective on the targets of insect pests. to use plants as true "weapons" for pest control, it would be necessary to have genes at their level to determine the synthesis of compounds with different actions on the same target. the researchers are relatively recent and aim mainly to combine genes for a b. thuringiensis δ-endotoxin with another inhibitory gene in the same host gene: for example, the gene for the v. faba trypsin inhibitor (cpti) or for serine proteases and the protein gene in the potato virus y coating. another interesting approach is that which introduced the cry1a (c) gene into a p. fluorescens strain able to colonize the sugarcane by means of two plasmids, pder405 and pkt240, in which the gene is found in 13 and 28 children, respectively. testing of recombinant bacterial strains on sugarcane-specific insect pests revealed greater resistance of plants treated with the respective bacteria than untreated. also, although pest-resistant plants have been obtained for several plant species, fewer results have been reported for cereals, vegetables, and oleaginous plants (ibrahim et al. 2008) . plant resistance to various diseases (microbial, viral, and nematode phytopathogens) has been a subject for long-term studies, identifying a relatively large number of resistance genes. although it was thought that endogenous resistance genes would provide a sustainable effect for the appropriate plants, this was true in very few cases. in the case of potato, the control program for certain diseases, such as the rot caused by phytophthora infestans, had to be abandoned because the resistance to this disease of potato plants obtained by transferring the 11 resistance genes on the basis of crossbreeds with the solanum demissum species proved to be of short duration. identifying genes of resistance in the genome of different plant species and transferring them to other crop plants are extremely difficult and time-consuming if traditional methods (intra-and interspecific hybridizations) are used. the process is considerably accelerated by the use of molecular markers generated by restriction fragment length polymorphism (rflp) techniques, randomized amplified polymorphic dna (rapd), single sequence repeat (ssr), or single nucleotide polymorphism (snp). the application of molecular markers allowed the isolation of nearly 20 resistance genes (r-genes) from genetically well-characterized plant species, proving that many of them are grouped into specific chromosomal regions (they form clusters). of these resistance genes, some have been transferred to other plant species than to their origin through molecular cloning techniques, with the help of specific vectors that ensure the transfer of large fragments, revealing that in this way, the r-genes act synergistically and provide lasting resistance to some diseases. as it has already been mentioned, few r-genes have been shown to provide a lasting control of plant diseases. this is the case for pepper bs2 genes and rice xa21 which provide resistance to different phytopathogenic strains of x. campestris or x. oryzae in the case of species in which the genes have been cloned (e.g., tomatoes). resistance to these genes is due to the recognition of proteins produced by bacteria or phytopathogenic fungi, resulting in the occurrence of a plant hypersensitivity phenomenon and necrosis of affected tissues. another example of the long-acting r-gene is the barley recessive mlo gene which provides the resistance of the plants containing it to all e. graminis strains, through the accumulation in plant cells of a phenolic antifungal compound named p-coumaroylhydroxyagmatine. it is expected that this gene will be used for suppressing the antisense technique of the dominant mlo gene from wheat or other plant species susceptible to erysiphe sp. in vitro processing of the r-genes and then introducing them into new hosts provide new possibilities for resistance. this is the case for the tomato avrpto gene which, after cloning under the control of a strong promoter such as 35s to camv, determines the resistance of transformed tomato plants to p. syringae pv. strains, tomato, and to unrelated pathogens such as x. campestris and c. fulvum. researchers' efforts to obtain resistance systems applicable to a larger number of plant species are not limited to r-genes but also include systemic acquired resistance mechanisms (sar). several genes involved in sar have been isolated and characterized, of which the npr1 gene encoding a transcriptional regulator is a key gene in this system. overexpression of this gene increases the resistance of arabidopsis thaliana or rice plants to a wide variety of pathogens (knecht et al. 2010 ). an interesting behavior has been observed in the myb1 gene which is induced by vmt infection of resistant tobacco plants and which causes the synthesis of a transcription factor that binds to the promoter of a gene involved in pathogenesis (the pr1a gene). modifying the expression level of the myb1 gene in tobacco plants leads to increased resistance to viral infection (with vmt) as well as to r. solani pathogenic fungus (raymond et al. 2007) . along with this, another recently cloned gene, pad4, isolated from arabidopsis thaliana, proves to be extremely interesting for the development of broad-spectrum resistance: overexpression of the gene in plants increases resistance to phytopathogens. numerous biotech companies and universities have begun to assess the performance of plants that express antifungal proteins through both "in vitro" and field experiments. both plants containing r-genes or genes involved in sars, as well as genes such as those which encode the glucosidase (ago) from aspergillus sp., defensins, h 2 o 2 -generating enzymes, glucanases or chitinases, have been examined. although at the laboratory level potato plants containing the fungal gene for glucose oxidase showed an increased resistance to phytopathogens, the results were inconclusive when they were put into the field. for other genes such as chitinase and intracellular α-1,3-glucanase, overexpression of these in tomato plants resulted in significant resistance to fusarium oxysporum (werner et al. 2016) . companies have created nbss that farmers can use to detect information such as air pollution, soil humidity, and so on. however, given that plants are really good nbss and that they can naturally react to external stimuli and changes, they can be used instead. this is the idea behind the latest nbss initiative called advanced plant technologies. the idea is that, through genetic manipulation, researchers will be able to create self-sustaining plants, which in turn enable them to act as a kind of nbs when it comes to detecting chemical substances, pathogens, etc. this is not the first time this idea with plants as nbss appears, but before that, resources that plants needed to survive were used, which in turn reduced their resistance. this new idea indicates that nbss can be sustained by themselves, which means they can work longer in isolated parts. in the future, plants could be used to detect when a biological attack will take place. in addition, because they are plants, it also means that they can be placed anywhere and nobody will think twice about their presence and that they might be some nbss. through such examples, nature teaches us to optimize by exploring diversity. in this context, integrated nanoscale systems (nbss), energy sources (biocombustible cells) that use plants metabolism, manipulators for nanoscale surgery, and drug reservoirs embedded in intelligent polymers are explored. all of these are virus-sized. the proliferation of these types of nbss leads to a large number of applications, and combinations of these in the future will lead to microminiaturization, versatility, and functionality. plant species present a great diversity of genetics, and wild ones constitute a large genetic reservoir, from which genes that are important from a practical point of view can be obtained. plant genetic engineering research has a great theoretical significance, facilitating the knowledge of how genes of these organisms act, the effects of phytohormones on plant development, genes inactivation mechanisms, etc. by applying molecular biology techniques, useful information can be obtained on the genome of plants used for amelioration, the localization of genes of interest, the degree of relationship between different species, etc. as far as practical applications are concerned, a number of significant results have been obtained so far, some of which have already been applied, such as virus-resistant plants, plants resistant to the attack of pests, herbicide-resistant plants, plants of horticultural interest (ornamental plants with new phenotypes, plants producing softening resistant fruits), plants capable of synthesizing secondary metabolites in increased amounts, and plants producing "edible" antibodies, and enumeration could continue. numerical modeling of the dynamic response of a bioluminescent bacterial biosensor rapid detection of cadmium-resistant plant growth promotory rhizobacteria: a perspective of elisa and qcm-based immunosensor patented applications of gene silencing in plants: manipulation of traits and phytopathogen resistance experimental and modelling data contradict the idea of respiratory down-regulation in plant tissues at an internal [o 2 ] substantially above the critical oxygen pressure for cytochrome oxidase 3d-nanostructured au electrodes for the event-specific detection of mon810 transgenic maize development of biosensor for phenolic compounds containing ppo in β-cyclodextrin modified support and iridium nanoparticles detection of glycoalkaloids using disposable biosensors based on genetically modified enzymes proteomic dissection of plant responses to various pathogens redox regulation in plant immune function chiral flavanones from amygdalus lycioides spach: structural elucidation and identification of tnf alpha inhibitors by bioactivity-guided fractionation the screening and isolation of an effective anti-endotoxin monomer from radix paeoniae rubra using affinity biosensor technology development of fret biosensors for mammalian and plant systems application of genetically engineered microbial whole-cell biosensors for combined chemosensing properties, functions and evolution of cytokinin receptors tracking transience: a method for dynamic monitoring of biological events in arabidopsis thaliana biosensors the influence of different nutrient levels on insect-induced plant volatiles in bt and conventional oilseed rape plants assessment of respiration in isolated plant mitochondria using clark-type electrodes in vivo biochemistry: applications for small molecule biosensors in plant biology chemosensors and biosensors based on polyelectrolyte microcapsules containing fluorescent dyes and enzymes generation of plant protein microarrays and investigation of antigenantibody interactions expression of bvglp-1 encoding a germin-like protein from sugar beet in arabidopsis thaliana leads to resistance against phytopathogenic fungi biosensing hydrogen peroxide utilizing carbon paste electrodes containing peroxidases naturally immobilized on coconut (cocos nucifera l.) fibers heat-solubilized curry spice curcumin inhibits antibody-antigen interaction in in vitro studies: a possible therapy to alleviate autoimmune disorders plant growth enhancement and associated physiological responses are coregulated by ethylene and gibberellin in response to harpin protein hpa1 portable optical aptasensor for rapid detection of mycotoxin with a reversible ligand-grafted biosensing surface effect of bt broccoli and resistant genotype of plutella xylostella (lepidoptera: plutellidae) on development and host acceptance of the parasitoid diadegma insulare (hymenoptera: ichneumonidae) measurement of the optical parameters of purple membrane and plant light-harvesting complex films with optical waveguide lightmode spectroscopy self-referencing optrodes for measuring spatially resolved, real-time metabolic oxygen flux in plant systems redox-sensitive gfp in arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer staying alive: new perspectives on cell immobilization for biosensing purposes colorimetric bacteria sensing using a supramolecular enzyme-nanoparticle biosensor versatile strategy for the synthesis of biotin-labelled glycans, their immobilization to establish a bioactive surface and interaction studies with a lectin on a biochip complex regulation of the immunoglobulin mu heavy-chain gene enhancer: microb, a new determinant of enhancer function development of an electrochemical biosensor for the rapid detection of naphthalene acetic acid in fruits by using air stable lipid films with incorporated auxin-binding protein 1 receptor boron nitride nanotube-based biosensing of various bacterium/ viruses: continuum modelling-based simulation approach live-cell imaging of phosphatidic acid dynamics in pollen tubes visualized by spo20p-derived biosensor nanotechnology in sustainable agriculture: recent developments, challenges, and perspectives. front microbiol 8:1014 nanotechnology in sustainable agriculture: present concerns and future aspects stem nematode counteracts plant resistance of aphids in alfalfa, medicago sativa host plant and population determine the fitness costs of resistance to bacillus thuringiensis fine mapping of co-x, an anthracnose resistance gene to a highly virulent strain of colletotrichum lindemuthianum in common bean electrochemical quantification of the antioxidant capacity of medicinal plants using biosensors enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites kinetic models for detection of toxicity in a microbial fuel cell based biosensor mapping a disordered portion of the brz2001-binding site on a plant monooxygenase, dwarf4, using a quartz-crystal microbalance biosensor-based t7 phage display the xaxab genes encoding a new apoptotic toxin from the insect pathogen xenorhabdus nematophila are present in plant and human pathogens multi-analyte biochip (mab) based on allsolid-state ion-selective electrodes (assise) for physiological research belowground communication: impacts of volatile organic compounds (vocs) from soil fungi on other soil-inhabiting organisms luminescent sensing and imaging of oxygen: fierce competition to the clark electrode comparative quantification of oxygen release by wetland plants: electrode technique and oxygen consumption model key: cord-252147-bvtchcbt authors: domingo-espín, joan; unzueta, ugutz; saccardo, paolo; rodríguez-carmona, escarlata; corchero, josé luís; vázquez, esther; ferrer-miralles, neus title: engineered biological entities for drug delivery and gene therapy: protein nanoparticles date: 2011-11-15 journal: prog mol biol transl sci doi: 10.1016/b978-0-12-416020-0.00006-1 sha: doc_id: 252147 cord_uid: bvtchcbt the development of genetic engineering techniques has speeded up the growth of the biotechnological industry, resulting in a significant increase in the number of recombinant protein products on the market. the deep knowledge of protein function, structure, biological interactions, and the possibility to design new polypeptides with desired biological activities have been the main factors involved in the increase of intensive research and preclinical and clinical approaches. consequently, new biological entities with added value for innovative medicines such as increased stability, improved targeting, and reduced toxicity, among others have been obtained. proteins are complex nanoparticles with sizes ranging from a few nanometers to a few hundred nanometers when complex supramolecular interactions occur, as for example, in viral capsids. however, even though protein production is a delicate process that imposes the use of sophisticated analytical methods and negative secondary effects have been detected in some cases as immune and inflammatory reactions, the great potential of biodegradable and tunable protein nanoparticles indicates that protein-based biotechnological products are expected to increase in the years to come. the development of genetic engineering techniques has speeded up the growth of the biotechnological industry, resulting in a significant increase in the number of recombinant protein products on the market. the deep knowledge of protein function, structure, biological interactions, and the possibility to design new polypeptides with desired biological activities have been the main factors involved in the increase of intensive research and preclinical and clinical approaches. consequently, new biological entities with added value for innovative medicines such as increased stability, improved targeting, and reduced toxicity, among others have been obtained. proteins are complex nanoparticles with sizes ranging from a few nanometers to a few hundred nanometers when complex supramolecular interactions occur, as for example, in viral capsids. however, even though protein production is a delicate process that imposes the use of sophisticated analytical methods and negative secondary effects have been detected in some cases as immune and inflammatory reactions, the great potential of biodegradable and tunable protein nanoparticles indicates that protein-based biotechnological products are expected to increase in the years to come. the design of new chemical entities (nce) for diagnosis and treatment of human diseases has relied on the discovery of active chemical drugs from a diverse library of compounds or from naturally occurring molecules. 1, 2 further chemical modifications improve pharmacokinetic properties to obtain a final product with a known mechanism of action and decreased toxicity. 3 nonetheless, using such approaches, the final products present low specificity for their target molecules, interacting with many other molecules and accumulating in some tissues, disturbing the correct homeostasis of the system. in some cases, the adverse effects of drug administration exceed pharmacological effect and despite the concise mechanism of action of the drug over the target molecule representing an improvement in the patient's state, the treatment has to be prevented or discontinued. 4 in fact, although a maintained steady increase in the number of launched nce has been observed in the last years, the question arises whether this classical approach has already exhausted the discovery of innovative molecules. 5 on the other hand, macromolecular new biological entities (nbe) have been used to supplement cellular deficiencies or to inhibit cellular pathways exploiting their relatively specific mode of action. proteins and peptides have been obtained first from their natural source or produced as recombinant 248 versions after the development of genetic engineering techniques in the late 1970s. however, the delivery of biological entities is sometimes hampered by its low half-life in the bloodstream by unspecific degradation, resulting in an expensive and ineffective process. nevertheless, some solutions have already been explored for biopharmaceuticals to increase solubility and stability and to reduce immunogenicity including postranslational modifications such as glycosylation and covalent conjugation of polyethylene glycol. 6 thus, one of the main objectives in the use of drugs (for either nce or nbe) is the need to optimize the delivery system to reduce the pharmacological dose which would consequently represent a concomitant reduction in toxicity and cost. in that scenario, new delivery approaches have been implemented using biological interactions such as antigen-antibody binding (immunoliposomes) 7 or more sophisticated interactions including the binding between nutrient concentrator sparc (secreted protein acidic and rich in cysteine) and albumin in the treatment of some types of cancer (abraxane ). 8, 9 proteins can be then used for their targeting qualities as molecular delivery vehicles both for the specific delivery of drugs or nucleic acids in gene therapy approaches and by themselves as therapeutic molecules. one of the interesting characteristics of proteins is their ability to form intermolecular driven complexes as sophisticated and structurally perfect as in the case of viral capsids. in addition, through the use of genetic engineering, recombinant proteins can be tuned to include additional properties to optimize drug delivery and nucleic acid delivery in gene therapy. in this chapter, the main available strategies to develop protein-based nanovehicles or biopharmaceuticals will be described. in this context, several parameters will be defined such as proper formulation, stability, immunogenicity, and delivery to the correct cell type and cell compartment. modular protein engineering, virus-like particles (vlps), and other self-assembling entities are envisioned as modulatable novel protein nanoparticles able to include many desirable properties in the correct delivery of drugs and nucleic acids. finally, some successful examples of protein nanoparticles on the market will be described in addition to protein products currently in clinical trials and under preclinical research in order to envision which type of protein nanoparticles will be available soon on the market. with the therapeutic molecule to generate a vehicle capable of being transported in the blood if a systemic administration is needed and retaining a significant stability before reaching the target cell. 10, 11 in addition, the biological system poses specific barriers that have to be overcome such as membranes (cytoplasmic, endocytic, and nuclear), degradation (protease degradation induced by acid denaturalization in lysosomes, cytosolic proteosomes, and nucleases), cytosolic transport, and nuclear entry if necessary. 12, 13 for central nervous system therapies, the blood-brain barrier (bbb) represents the main bottleneck, and for that, a specific strategy has to be designed. 14 furthermore, the therapeutic complex has to be flexible enough in order to release the therapeutic molecule in the specific cell compartment. thus, several protein motifs have been described to overcome each and every process described earlier so that a modular multifunctional protein can be generated including those modules that are necessary to achieve its goal. in order to get a rational construction of the multifunctional vector, each step has to be carefully taken into account so as to overcome every step which is needed to achieve its final goal (table i) . the dna/rna condensation or drug interaction with the protein vector is a critical step in the formulation of protein nanoparticles for gene therapy. they have to remain attached to the vector during the whole transport process through the body and the cell until it can be released in the desired localization within the target cell. highly positively charged peptides containing a large number of arginines or polylysines have been used to promote electrostatic interactions since nucleic acids are highly negatively charged molecules. [15] [16] [17] [18] [19] [20] [21] [22] natural dna-condensing proteins as nuclear histidines or protamines can also be used to bind nucleic acids. [22] [23] [24] [25] protamine, which is the protein that replaces histidines during the spermatogenesis process, is a sperm chromatin component and just as the histidines do, it has very high dna condensation ability to protect nucleic acids form cytosolic endonucleases. 23, 26 in addition, as soon as the complex reaches the cellular nucleus, protamine is degraded by chromatinremodeling proteins, releasing the transported dna allowing its expression. 15, 23 in contrast, polycationic dna condensation modules such as polylysines and polyarginines-even they can present higher dna condensation ability depending on the polycationic chain length-usually present lower dna-releasing ability, interfering negatively with the accessibility of cellular transcription factors and dna expression capacity. 15 all these dna condensation modules described above interact with any dna that is incubated in an unspecific way. however, there are proteins such as gal4 that are able to recognize specific dna sequences [27] [28] [29] and that permit to bind and condensate specific dna sequences in the final vector. 30 in many cases, the multifunctional protein vector is in vivo administrated by the systemic route in order to travel in the blood and reach the target cells. that exposes the vector to all blood components, making it susceptible to be degraded. thus, it is completely necessary that the vector remain in the blood long enough to be able to reach the target cells. it has also been described that naked dna has an estimated half-life in blood of minutes 10 ; so protein nanovehicles in gene therapy, among other properties, are intended to protect nucleic acids from degradation. one important factor when the vector is exposed to the blood is that it can be recognized by the immune system components and produces an immune response against the vector. thus, it is also very important to try to make the vector as less antigenic as possible in order to avoid being degraded or even being toxic to the organism. 32 peptide uptake or internalization involves a step before the protein binding to the cell surface. this attachment can be either specific or unspecific but in all cases the promotion of its internalization is required. 33 positively charged peptides usually bind the cellular surface by unspecific electrostatic interactions with the negatively charged cell surface proteoglicans. this kind of peptides can be used in the multifunctional protein if specific targeting is not required. 33 cell-penetrating peptides (cpps) have been widely described as unspecific cell-binding and internalization peptides [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] (see also the chapter ''peptide nanoparticles for oligonucleotide delivery'' by lehto et al. in this volume). however, specific interactions can be obtained by incorporating cell receptor ligands if cell or tissue targeting is required for the therapeutic action. moreover, some of those ligand-receptor interactions promote the ligand-receptor complex internalization. many peptides have been described in the literature as receptor-specific ligands so any of them can be added to the multifunctional proteins in order to confer them cell specificity. [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] the most natural specific ligands that can also be used for cell targeting are monoclonal antibodies. 32, [63] [64] [65] in addition, if no specific peptides are available for an intended target, new specific binding peptides can be found by using phage display 66 or combinatorial chemistry. 67 2. endosomal escape several internalization pathways are possible depending on the vector properties, 27, 33 including endocytosis (clathrin/caveolae-mediated, clathrin/ caveolae-independent), macropinocytosis, and non-endocytic pathways. 254 it is known that more than one internalization pathway can be performed at the same time but usually the peptide-based vector uses endocytic pathways. 68 moreover, it seems that proteins that interact with a specific cellular receptor are internalized by the clathrin-mediated endocytic pathway. 33 most of the generated endosomal vesicles will converge to late endosomes that eventually will fuse with cellular lysosomes. 15, 33 remaining in the cellular endosomes, the multifunctional protein will be degraded, so it is strictly necessary that the internalized multifunctional proteins be released into the cellular cytoplasm escaping from degradation. several peptides have been described that are able to promote endosomal escape and can be classified into two types depending on their escape mechanism: fusiogenic peptides and histidine-rich peptides. 36 the fusiogenic peptides are small peptides that have hydrophobic amino acids (aa-s) interspersed at constant intervals with negatively charged aa-s. 12, 19, 39, 40, 45, 46, [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] thus, when early endosomes become late endosomes, their low ph induces a conformational change in the peptide, which adopts a alpha-helix structure, in an amphipathic structure able to fuse with the endosomal membrane, leading to pore formation and releasing all the endosomal content into the cell cytoplasm. 36 the histidine-rich peptides are small peptides with a high histidine content whose endosmolytic activity is mediated by a mechanism called ''proton sponge''. 12, 22, [80] [81] [82] [83] when the endosomal ph becomes low in late stages, the imidazole groups of the histidines are protonated and attract endosomal cl à ions, buffering against the proton pump. thus, the endosomes collapse by an osmolytic swelling process and the endosomal content is released to the cell cytoplasm. 36 further details are given in the chapter ''peptide nanoparticles for oligonucleotide delivery'' by lehto et al. in this volume. once the protein has achieved the cellular cytosol, it can be degraded by cellular proteases or by the cellular proteosome system. 84 it is important to avoid this process, especially if the protein has to reach the cellular nucleus. if the final target of the nanoparticle is the cellular cytoplasm, it is necessary that it remain there at least long enough to perform its therapeutical action. several peptide proteosome inhibitors have been described that are able to avoid this type of protein degradation. by adding these peptides to the final protein vector it is possible to protect it and enhance cytoplasmatic stability. epstein-barr virus nuclear antigen 1 (ebna1) contains a proteosome inhibitor consisting of glycine-alanine repeats able to prevent proteosomal proteolysis. it has been shown that a minimum of 4 aa-s gly-ala repeats are necessary to achieve such protective activity. [85] [86] [87] if the protein vector is carrying nucleic acids (dna or rna), degradation by the cytosolic endonucleases has to be taken into account, so it is also very important to protect this nucleic acid in order to maintain its integrity. some dna/rna condensing peptides as protamines also protect the dna against cytoplasmic endonucleases and enhance its stability as has been described above. 15 the cellular cytoplasm is a very crowded and compartmentalized environment where cellular organelles and cytoskeleton make the free diffusion of macromolecules such as protein vectors difficult. however, cytoskeleton elements such as microtubules are used by endosomes and other cytosolic macromolecules for intracytosolic mobility. 33 dyneins have been described as being capable of carrying those macromolecules and endosomes along the microtubules in a retrograde transport toward the nucleus. some small peptides that are able to bind dyneins have been identified. they can be added to the multifunctional protein vector in order to mediate an intracytosolic mobility toward the cellular nucleus. 36 several dynein-binding proteins have been identified in viruses that are able to use this transport system. comparing those protein sequences, a consensus peptide sequence (kstqt) that is able to bind to the dynein lc8 light chain has been identified. 88 molecules lower than 45 kda/10-30 nm are able to enter in the cellular nucleus by passive diffusion. however, macromolecules higher than 45 kda/ 10-30 nm generally require an active transport system through the nuclear pore system. this transport mechanism generally requires a specific targeting signal peptide named nuclear localization signal (nls). these signaling peptides are usually rich in basic aa-s, which are recognized by the cellular importines and actively transported through the nuclear pore. 15, 89 monopartite or bipartite nls sequences which are nls peptides that have one or two nls recognized sequences respectively have been described. 12 thus, these peptidic sequences can be added into the final multifunctional protein if nuclear localization is required in order to express a carried dna. it has been reported that a single nls sequence is sufficient to transport the vector to the nucleus and that a large number of nls sequences can result in inhibition of its activity. 90 one of the most used nls signal peptides are fragments derived from the 111-135 aa-s of the simian virus sv40 large tumor antigen (t-ag). other nls sequences can be found in gal4, protamines, or tat. 23, 36, 37, 77, [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] it is important that when the transported dna reaches the cellular nucleus, it has to be released in order to be accessible to the nuclear transcription factors and achieve the desired expression level. thus, while designing the multifunctional protein vector, this aspect has to be taken into account. once the dna has been released in the cell nucleus, it will be necessary to control its expression level depending on which therapeutic action is being promoted. when the goal is to kill a cell as in cancer therapies, the uncontrolled dna expression levels would not be a problem. however, when a specific protein expression level is required, achieving good control is very important. 13 some expression systems have been developed that can be pharmacologically regulated by oral drug formulation. 103 cell-specific promoters and enhancers can be also used in order to confer high cell specificity to the therapy. 104, 105 d. ways to get over the bbb the bbb is a hermetic barrier that only allows nonlipophilic molecules smaller than 400 da to cross it. however, some human proteins such as insulin, transferrin, insulin-like growth factor, or leptins are able to go across it by receptor-mediated transporters. thus, the most important factor limiting central nervous system-targeting therapeutics is the presence of the bbb. 106 finding the way to cross it will be the main challenge. some peptides have been described that are able to reach the brain crossing the bbb. moreover, it has been seen that they can be associated with another molecule and transported through the barrier. thus, they could be interesting candidates to be included in the multifunctional vectors if central nervous system targeting is required. 14, 56, 107, 108 antibodies have also been described that bind transferrin and insulin receptors and that are able to cross the bbb efficiently. they can be conjugated with large molecules, allowing its translocation to the central nervous system. 63, 64, [109] [110] [111] synthesis, and rational design the development of genetic engineering techniques has increased the natural repertoire of proteins for the design of useful and/or valuable proteins with the aim to obtain new proteins with desired functions. there are three main strategies leading to the construction of engineered proteins: (a) direct evolution, (b) de novo protein design, and (c) rational design. directed evolution has developed quickly to become a method of choice for protein engineers in order to create enzymes having desired properties for all kind of processes. over the past decade, this technique has become a daily part of the molecular toolbox of every biochemist. this is emphasized by the increasing number of publications about the subject. 112 in nature, evolution and creation of new functionalities is achieved by mutagenesis, recombination, and survival of the fittest. directed evolution mimics this and is a process of iterative cycles of producing mutants and finding the mutant with the desired properties. mutations can be introduced at specific places using site-directed mutagenesis or throughout the gene by random mutagenesis. several mutagenesis techniques have been developed in order to avoid codon bias. 113, 114 the first technique used to mimic evolution was dna shuffling. 115 this method is based on the mixing and subsequent joining of different related small dna fragments in order to form a complete new gene. in the process of shuffling, the recombination frequency is dependent on the degree of homology. a high level of recombination is important to get all possible combinations of mutations. since recombination can be biased, several methods to overcome problems arising from the use of shuffling in the early years were tackled by novel strategies, all having their own advantages and disadvantages. 112 the products obtained by these methods have to be screened for desired qualities and not all of them can be easily screened. de novo protein design offers the broadest possibility for new structures. it is based on searches for amino acid sequences that are compatible with a three-dimensional protein backbone template using in silico techniques. several research groups in the field have applied in silico methods to design the hydrophobic cores of proteins, with the novel sequences being validated with experimental data. 116 in silico protein design has allowed novel functions on templates originally lacking those properties, modifying existing functions, and increasing protein stability or specificity. beyond any doubt, intense research activities are ongoing in the field, the potential of which is simply enormous. 117 so far there have been numerous examples of full sequences designed ''from scratch'' that were confirmed to fold into the target three-dimensional structures by experimental data. 118 the zinc-finger protein designed by dahiyat and mayo 119 was the first one to appear by this method. rational design of proteins is based on the modification or insertion of selected amino acids or domains in a polypeptide chain backbone to obtain proteins with new or altered biological functions. when using that strategy, a detailed knowledge of the structure and function of the backbone protein is needed to make desired changes. this generally has the advantage of being inexpensive and technically feasible. however, a major drawback of this approach is that detailed structural knowledge of a protein is often unavailable or it can be extremely difficult to predict the effects of various mutations. modular engineering enables, by using simple dna recombinant techniques, the construction of chimerical polypeptides in which selected domains, potentially from different origins, provide the required activities. an equilibrate combination and spatial distribution of such partner elements has generated promising 258 prototypes, able to deliver expressible dna or molecules to tissue culture but also to specific cell types in whole organisms. 120 modular fusion proteins that combine distinct functions required for cell type-specific uptake and intracellular delivery of dna or drugs present an attractive approach for the development of self-assembling vectors for targeted gene or drug delivery. 121 one of the first examples was described by the group of uherek et al. they combined a cell-specific target module (antibody fragment specific for the tumor-associated erbb2 antigen), a dna-binding domain (gal4), and a translocation domain for endosomal escape. 121 in this context, many strategies for the construction of safer vehicles are being explored and the number of nonviral prototype vectors for gene and drug delivery is noticeably increasing. here, the common steps that an approach like this might explore are presented ( fig. 1) . when designing a new protein for drug or gene delivery there are many critical aspects, namely (a) design of the vehicle itself, required functions, stability, etc.; (b) production of the protein, suitable expression system, purification procedure, scaling up process, etc.; (c) characterization of the vehicle by physicochemical and functional tests; and finally (d) the administration route and regulatory guidance for biological products. although all these aspects belong to different disciplines, they have to be overviewed together. here, the major needs of a modular protein for gene and drug delivery are presented. to enhance the physicochemical stability of the cargo molecules and their resistance to nuclease/protease-mediated degradation, protein vehicles should ideally exhibit, like their natural counterparts (viruses), nucleic-acid binding and condensing properties. 27 such abilities are, in general, conferred by cationic segments of the main scaffold molecules that interact with nucleic acids, mainly through electrostatic interactions. in addition, such complexes need to efficiently release the nucleic acid in the nucleus (if the cargo is a therapeutic gene), for which endosomal escape is required. such functions have been found in some peptides in many natural molecules and they are suitable for functionalizing protein vehicles. the ability to bind a particular cell type with high specificity is especially significant in a systemic delivery in which appropriate biodistribution and tissue targeting are essential. 122 for nuclear targeting, only naked short nucleic acids can freely enter the nucleus of nondividing cells via free diffusion through the nuclear pore. large molecules require active transport mediated by nlss that are often found in viral proteins. because the molecular mass of plasmidic dna varies from to 2 to 10 mda, dna that is to be expressed, and essentially any macromolecular complex for nucleic acid delivery, requires nlss. 123 the role and types of functional modules peptides used for all these purposes will be discussed in depth in the following sections. in vivo experiments finally, which protein or peptide is better for a given cargo is to be determined empirically and only few rules can be taken literally. 38, 124 c. production of protein nanoparticles some steps in the production of a protein-based vehicle after molecular cloning such as protein production and protein purification 125 might be experimentally labor intense with a variable success rate. for that reason, when small proteins are needed, solid-phase peptide synthesis 44 guarantees the process. however, the classical procedure of biological production allows scaling up the process in most of the cases and the production of larger polypeptides and fulllength proteins. generally, in protein nanoparticle approaches, the protein is composed by different modules of natural sources such as the cell-penetrating peptide transactivator of transcription (tat) derived from the tat of the human immunodeficiency virus (hiv) 126 or artificial sequences not present in any organism such as the polylysine dna-condensing sequence. 127 once it has been defined which modules will be part of the protein, it is important to define the order they will have in the final construct. it has been demonstrated by boekle and coworkers using melittin conjugated to polyethylenimine (pei) that depending on the side of the linkage (c-or n-terminus), the lytic activity could be changed. some other modules have the need to be in a determined position for its correct function. 128 when producing a protein for gene or drug delivery, it is important to know the origin of its domains to choose the most suitable expression system for its production. for instance, if any module naturally carries a posttranslational modification that is essential for its biological function, the expression system chosen will have to be able to reproduce the same crucial modification. the main biological production systems for protein drugs are described below. escherichia coli is the most widely used prokaryotic organism for the expression of recombinant proteins. 129 the use of this host is relatively simple and inexpensive. 130 added advantages include its short duplication time, growth to high cell densities, ease of cultivation, and high yields of the recombinant product. however, since it lacks fundamental prerequisites for efficient secretion, recombinant proteins manufactured by e. coli systems are mainly produced as inclusion bodies. 125, 131 moreover, posttranscriptional modifications are not achieved with this system. there are many examples of proteins for gene delivery produced in e. coli with probed efficiency. 132, 133 like e. coli, yeasts can be grown cheaply and rapidly and are amenable to high-cell-density fermentations. besides possessing complex posttranslational modification pathways, they offer the advantage of being neither pyrogenic nor pathogenic and are able to secrete more efficiently. species established in industrial production procedures are saccharomyces cerevisiae, kluyveromyces lactis, pichia pastoris, and hansenulapolymorpha. s. cerevisiae is the best genetically characterized eukaryotic organism among them all and is still the prevalent yeast species in pharmaceutical production processes. 131 in spite of their physiological advantageous properties and natively high expression and secretion capacity, the employability of yeasts in some cases, however, might reach a limit, particularly when the pharmacological activity of the product is impaired by the glycosylation pattern. in such cases, either a postsynthetic chemical modification has to be considered or the employment of more highly developed organisms. most examples of nanoparticles produced in yeast are for vlps. 134 animal cell expression systems show the highest similarity to human cells regarding the pattern and capacity of posttranslational modifications and the codon bias. however, their culture is more complicated and costlier and usually yields lower product titers. among the known systems, insect cells infected by baculovirus vectors have reached popularity since they are considered to be more stress-resistant, easier to handle, and more productive compared with mammalian systems and are thus frequently employed for high-throughput protein expression. for commercial application, scale-up related questions have to be solved. [135] [136] [137] preferably applied in pharmaceutical production processes are mammalian systems like chinese hamster ovary (cho) cells and baby hamster kidney (bhk) cells. these systems are genetically more stable and easier to transform and handle in scale-up processes, to grow faster in adherent and submerged cultures, and to be more similar to human cells and more consistent in their complete spectrum of modification. 138 in some cases, mammalian cell systems can be the only choice for the preparation of correctly modified proteins. peptides, being complex and unique complex molecules with regard to its chemical and physical properties, can be produced synthetically by the solidphase method. 139, 140 this technology can be used to avoid problems related to biological production. general advantages of synthetic peptides are that they are very stable compounds, solid-phase chemistry produces highly standardized peptides, and the crucial polycation component is provided by a ''natural'' polycation, thus minimizing toxicity. 141 however, some disadvantages related to synthetic peptides have been reported such as the difficulty to synthesize long and well-folded oligopeptides, peptides with multiple cysteine, methionine, arginine, and tryptophan residues due to technical limitations or production cost. 141 when working with protein nanoparticles, it is very important to characterize them physically and functionally in order to understand their behavior. the size and charge of protein/cargo particles are crucial properties which influence rates of diffusion, binding to polyanionic components of connective tissues, transversal of anatomical barriers, binding of serum proteins, attachment to cells, and mechanisms of endocytosis, among other factors. stability in physiological salt solutions is a key issue for in vivo delivery, as salt is found everywhere in the body. 141 mixing a multivalent polycation and dna results in electrostatic binding of both molecules, with charge neutralization of dna and a particle formation named conjugate. charge neutralization can be easily seen by retardation gel assays and particle formation by dynamic light scattering (dls). dls is a good method to see particle formation but not to quantify relative number of particles of different sizes. 142 to visualize particles, many groups have used transmission electron microscopy (tem) 15, 143 with good results while others have used fluid particle image analyzer (fpia) to photograph individual particles in physiological solutions. 58 the net charge of protein/cargo particles is an important variable. generally, optimal gene delivery for cell lines requires a net positive charge but, as stated previously, it has to be determined empirically. one of the best techniques to determine the net charge is by calculating the zeta potential that measures the electrophoretic mobility of particles. 144 despite the fact that physical characterization is a key element, understanding and testing the functionality and pharmacokinetics of a gene or drug is the most important part of its development process. most of the initial tests are done using cell lines in in vitro experiments using reporter genes, rna, or drugs. 145, 146 quantifying the percentage of transfected cells or drug-induced changes is a very valuable tool to evaluate nanoparticle performance in both nuclear and cytoplasmic delivery, respectively. in addition, in vitro experiments may be designed to select a candidate for the in vivo experiments from a group of possible therapy vectors. the quantitative kinetics of particle binding, the molecular basis of particle interactions with target cell membranes, the efficiency of particle internalization, and endosomal escape are all poorly understood. 141 interaction of particles with plasma membranes prior to protein internalization can be either unspecific or specific. untargeted delivery normally is the consequence of electrostatic interactions between anionic ligands in the cell surface and cationic components of the vehicle. on the other hand, targeted delivery to specific membrane molecules is a more sophisticated approach. it aims to improve cell specificity and efficiency, by directing to molecules, only expressed or overexpressed in a particular cell type, that initiate internalization by endocytosis. targeting moieties include many types of molecules and is discussed afterwards. internalization of particles, its mechanisms, and kinetics are not well known and most studies about nanoparticle delivery do not focus on this aspect. there are several endocytic pathways each initiated by different ligands. 147 enhancing the delivery by addition of chloroquine, a synthetic molecule used primarily for the prophylaxis and treatment of malaria that disrupts endosomes, 148 is an accepted parameter to demonstrate endosomal localization of particles. endosomal escape is the area most intensively investigated but is poorly understood. an important practical point to note is that some reagents that are used can be toxic. 141 to enhance this step, anionic fusogenic peptides can be used. these peptides fuse to membranes in an acidic-dependent manner causing its disruption. 149 in gene delivery approaches, translocation of dna expression plasmids into the cell nucleus involves an active, energy-dependent process through the nuclear pore complex. 150 directly injected dna into the cytosol is usually, but not always, poorly transferred to the nucleus 150, 151 and because of that, the use of proteins carrying cationic nuclear-localizing sequences (such as that of sv40 large t antigen) has been widely used to overcome this step. 143 iv. natural self-assembling protein nanoparticles: vlps ideal drug delivery and gene therapy vehicles must accomplish some desired features such as appropriate packaging size for its cargo, target cellspecificity, safe and efficient cargo delivery, and protection against immune recognition, or capability to escape immune recognition. moreover, these vehicles must avoid inflammatory toxicity and rapid clearance. 152 in this context, viral vectors have been exploited as one of the vehicles of choice. viruses are nano-sized (15-400 nm) supramolecular nucleoproteinbased entities, covered or not with a lipid bilayer (enveloped/nonenveloped viruses) that satisfy, into relatively simple structures, outstanding properties and functions that are relevant to drug and gene delivery. viruses are able to recognize and interact specifically with cells by receptor-mediated binding, internalize, escape from endosomes, and uncoat and release nucleic acids in different cellular compartments. they are also capable of transcribing and translating their viral proteins to self-assemble into new infectious virus particles and exit the host cell. 120, [153] [154] [155] despite all these relevant properties of viral vectors or some other rising vehicles in drug and gene delivery such as cationic liposomes, their therapeutic use presents some limitations and risks because of the complexity of production, limited packaging capacity, insertional mutagenesis and gene inactivation, low probability of integration, reduced efficacy of repeat administration or reduced expression overtime, unfavorable immunological recognition or strong 264 immune response against vehicle and transgene, inflammatory toxicity, and rapid clearance. 120, 152 in this context, virus capsids or vlps, produced by recombinant capsid proteins but lacking the viral genome, have noticeably emerged as a safer alternative to viral vectors. a. structure of protein self-assembled nanovehicles vlps are classically described as self-assembling, nonreplicative and nonpathogenic, highly organized supramolecular multiprotein nanoparticles (coats) (ranging from 20 to 100 nm) that can be formed from the minimal spontaneous self-assembling of one or more viral structural capsid proteins. it has been described that the self-assembling process of the structural viral proteins for vlp formation involves both spontaneous assembly, under favorable experimental conditions, and the requirement of scaffold proteins as catalysts. 156, 157 therefore, vlps are considered protein ''coats'', ''shells'', or ''boxes'' that lack the viral genome, still conserve the structure, morphology, and some properties of viruses. some of these properties such as cellular tropism and uptake, intracellular trafficking, membrane translocation, and transfer of nucleic acids or molecules across the cytoplasmic, endosomal, and nuclear membranes are important for drug delivery and gene therapy. 120, 153, 155, [158] [159] [160] usually, the degree of similarity of vlps and their viruses depends on the number of proteins incorporated into the constructs. 161, 162 since the first description in 1983 of the viral dna packaging into mouse polyomavirus (mpyv) vlps and its transduction in vitro, 163 vlps of different viruses such as papillomaviruses, [164] [165] [166] hepatitis b, c, and e viruses, [167] [168] [169] polyomaviruses, 163 vlps offer some structure, dynamics, characteristic features, and functions that make them appealing bionanomaterials to be exploited in the biomedicine arena as drug and gene delivery vehicles and are discussed in detail afterward. on the one hand, viral coat proteins have the ability to spontaneously selfassemble, which ensures the formation of highly organized, regular, repetitive structurally stable, and very low morphological polydisperse particles that provide useful properties to be used as scaffolds for bioimaging, synthesis of bionanomaterials, and as nanocarriers in drug and gene therapy. 186 in addition, homogeneity of particle size and composition is a desired production factor when developing therapeutic molecules. the overexpression of structural viral proteins in a convenient expression system renders recombinant proteins capable of being folded and assembled in discrete organized nanoparticles with a defined size corresponding to the natural capsid geometry. [187] [188] [189] moreover, even though vlps are structurally stable particles, some biochemical and structural studies have observed that viral capsids and bacteriophages may show some structurally dynamic properties varying in shape, size, or rearrangements of the coat proteins, in response to different factors such as ph. [190] [191] [192] [193] on the other hand, vlps are considered biologically safe nanostructures since they are not infectious (lack of viral genome) and do not replicate, representing a safer alternative to viral vectors. 160, [194] [195] [196] [197] however, they can elicit immune and inflammatory responses, especially when repeated administration is needed. 152 it has to be also noted that when used in vaccination, vlps could show excellent adjuvant properties and the majority of vlps stimulate strong cellular and humoral immune responses as direct immunogens. 198 it has been suggested that recombinant vlps derived from infection of insect cells with baculovirus or even those derived from prokaryotic systems could be contaminated with different residual components of these host cells, contributing those impurities to the adjuvant properties. 153 one interesting property of vlps is that coat viral proteins present an enormous elasticity and adaptability to be modified chemically and/or by protein genetic engineering 154, 160, 199 to incorporate multiple directed functionalities, in order to be addressed in biomedical applications such as drug delivery or gene therapy. it has been recently reviewed that chemically and/or genetically modified vlps, including cpmv, ccmv, ms2, m13 bacteriophages, and other virus-based nanoparticles, 155,186 could maintain their structural integrity and improve their physical stability 154 and, moreover, these modifications could also confer desired cell-targeting properties to the nanovehicle. [153] [154] [155] 186, 200, 201 vlps can be successfully engineered with spatial precision to incorporate (attached or genetically displayed on the surface) targeting tissue-specific ligands such as epidermal growth factor (egfr) and antibodies, or other molecules such as oligonucleotides, peptides, gold, and other metals, target proteins, carbohydrates, polymers, fluorophores, quantum dots, drugs, or small molecules. 152, 154, 155 moreover, one of the potential benefits of such modifications is that the specific geometric rearrangement confers precise recognition patterns. 200, 201 furthermore, accessibility of the materials carried within the particle and the ability of inclusion and separation of nucleic acids, small molecules, and unusual cargoes with appropriate charge is another outstanding feature and key advantage of vlps that has also made them excellent vessels for gene and drug delivery. 152, 195 as described above, vlps can be used as empty nanocarriers to transport molecules chemically attached on their surface or can be loaded ex vivo with therapeutic small molecules such as drugs, dnas, mrnas, sirnas, oligonucleotides, quantum dots, magnetic nanoparticles, or proteins. 155, 157, 160 vlps of different papillomavirus and polyomavirus have been widely characterized and used for directed delivery in biomedical applications. 132, 165, 173, 174, 194, 202 osmotic shock and in vitro self-assembling of vlp subunits in the presence of 266 the cargo have been the two main strategies used to packaged nucleic acid or other small molecules. it has to be taken into account that some attachment of the cargo on the vlp surface can occur. 195 besides, diversity of natural tropism including liver for hepatitis b vlps, spleen for some papillomavirus and polyomavirus vlps, antigen-presenting cells for certain papillomavirus vlps, and glial cells for human polyomavirus jc (jcv) vlps, among others 152 is one of the key advantages offered by vlps providing a wide spectrum of specific targeting and distribution profiles depending on the directed application. although each vlp has its own characteristic receptors, entry pathway, and intracellular trafficking, it has been demonstrated that tropism of vlps could be customized, modifying the residues identified as ligands of the cellular receptor on vlps' surface or even varying the delivery routes. 155, 189, 203 another key advantage of vlps is that they can be easily produced by using a wide range of hosts and expression systems, each of them with its own conditionings. 162 in the past years, there has been an increasing need to improve and optimize efficient large-scale production systems, process control and monitoring, and up-and down-streaming processes. 153, 157, 159, 204 production of vlps usually involves transfection of the cell host expression system of choice with a plasmid encoding one or more viral structural proteins, further and rigorous purification for the removal of immunogenic cellular contaminants, and quality control of the produced vlp and encapsulation of the cargo ex vivo before administration. 152, 158 the most frequent and convenient expression systems, adaptable to large-scale processes are (1) yeast cells 176 205, 206 , (4) bacteria 204,207 , (5) green plants infected with modified viruses 208, 209 , and (6) cell-free systems. 163, 204 the preparative and large-scale manufacture of vlps in some of these hosts has been reviewed by pattenden et al. and can be classified into two main methods of bioprocessing: in vivo and in vitro systems. 157 in addition, the capability of in vitro dissociation and reassociation of vlps contribute to the application of easy and more accurate purification methods than those of viral vectors. 152, 157 furthermore, depending on the expression system, the resulting vlp might be significantly different even though expressing the same viral proteins. thus, a broad spectrum of vlps could be customized depending on the vlp type, the number of proteins needed for vlp assembling, and the targeted final application. 158, 210 as described above, vlps have great potential as nanocarriers in drug and gene delivery. at the same time, although there is an increasing flow of developments in this area, these vehicles also present some limitations that should be addressed and taken into account, such as residual cellular components, variable yield of functional vlps after disassembly/reassembly process, immunostimulation and unsuitability for repeated administration, tolerance to the transgene, ineffective therapeutic molecule loading, and low transfection rates. 152 protein nanoparticles engineered for drug delivery and gene therapy 267 due to their versatile nanoparticulate structure and morphology, and nonreplicative and noninfecting nature combined with their natural immunogenic properties and ease production, vlps have principally emerged as an excellent alternative tool to attenuate viruses for vaccination. 152 182 and ebola virus. 215, 216 although vlp-based vaccines have been primarily developed for their use against the corresponding virus, in the last decades genetic engineering or chemical modifications have been applied in order to generate chimeric vlps. thus, on the one hand, commonly short heterologous peptide epitopes or full proteins that are unable to form vlps or that are unsafe for vaccination have been presented on surface-exposed loops or fused to n-or c-exposed termini of structural viral capsid proteins on vlps. 154, 161, 210 different hpv, 217-219 hbv, 220,221 parvovirus, 222, 223 and chimeric polyoma vlps have been engineered 170, 175 and tested for different applications including vaccination against viral or bacterial diseases, against virus-induced tumors, and more recently, for immunotherapy of nonviral cancer. 161, 210 on the other hand, chemical bioconjugation for covalent coupling of protein epitopes and small molecules to lysines, cysteines, or tyrosine residues of vlp surfaces has been applied in viral or cancer vaccines. 200 chackerian et al. have demonstrated the efficient induction of protective autoantibodies using self-antigen conjugation to hpv vlps. 224 it is important to point out that vlps can also be engineered to incorporate heterologous cell-specific ligands to cell receptors, thus altering their cellular tropism. 154, 155, 186, 201 this great convertibility and flexibility of vlps to be modified (chemically and/or genetically), their high stability, natural and diverse tropism, their nanocontainer properties, and their ability to enter in the cell and incorporate, bind, and deliver nucleic acids and small molecules have positioned vlps as appealing entities not only for vaccination applications but also for a broad spectrum of other diverse and emerging applications in nanomedicine and nanotechnology such as immunotherapy against cancer, 210,225 gene therapy delivery of therapeutic genes into specific cells, 161, 165, 171, 184, 226, 227 and targeted delivery of drugs and small molecules using vlps as nanocarriers. 174, 196 268 domingo-espín et al. although there is no commercial vlp as vector in gene therapy, since the initial work in 1970 of uncoating polyoma pseudovirus in mouse embryo cells as gene delivery vector 228 and the establishment in 1983 of the viral dna packaging into mpyv vlps and its transduction in vitro, 163 different vlps such as hbv and hepatitis e virus, 229 hpv and polyomavirus nanoparticles 172, 178, 229 have been modified toward the specific delivery of therapeutic genes and proteins in different target cells, organs, and tissues in vitro and in vivo by systemic injection 229 or oral administration. 230 for example, recombinant vp1-based polyomavirus vlps can encapsulate in vitro exogenous dna, and deliver it by cell surface sialic acid residues to human brain cells and fetal kidney epithelial cells. 178 furthermore, vlps have recently emerged as novel nanocarriers or nanocontainers to store unnatural cargos, deliver modified oligonucleotides, 154 synthetic small interfering rnas, and plasmids expressing short hairpin rnas as therapy to downregulate gene expression. 171, 231 in this context, chou et al. have recently described the use of jcv vlps as an efficient vector for delivering rnai in vitro using murine macrophage raw 264.7 cells and in vivo using balb/c mice in silencing the cytokine gene of il-10 without significant cytotoxicity for systemic lupus erythematosus gene therapy. 171 one of the key aspects in targeted gene and drug delivery is cell-specific delivery. it is important to point out that vlps are tunable nanoparticles that can also be chemically or genetically engineered to modify their natural cellular tropism in order to diversify the range of therapeutic applications in targeted gene or drug delivery. 154, 201 some effective approaches to modify the natural cellular tropism include: (1) genetic engineering of vlp chimeras incorporating heterologous cellspecific short peptides that contain recognition sites of target cell receptors. 232 in this context, polyoma and papillomavirus, with solved atomic structures of their major structural capsid proteins, have been extensively used to obtain chimeric vlps as delivery vector systems. 165, 233 however, this approach has some bioprocessing limitations such as low production levels as a consequence of vlp modification, alterations of size and properties of the vlps that could affect the structural interactions and conformations for vlp assembly, disassembly and packaging, and low transduction efficiencies. 157 (2) chemical bioconjugation of purified vlps with epitope-containing peptides 234, 235 or a wide range of small molecules conferring cell-specific targeting such as transferrins, folic acid, or other targeting molecules. as an example, cmpv vlps have been successfully conjugated with tfn using ''click'' chemistry 236 and with nhs-ester-derivatized folic acid, demonstrating both as internalized into hela cells and kb cells, respectively. 183, 184 (3) high-throughput library and directed evolution method is a rational approach that has been recently used to engineer viral vectors with the desired tropism properties. 237 (4) pseudotyping, which consists of replacing the envelope protein of one virus species by the envelope protein of another virus species. 238 (5) modification of the delivery route of the vlps. it has been shown that the levels of expression of b-galactosidase in heart, lung, kidney, spleen, liver, and brain are different depending on the delivery route of polyomavirus vp1 vlps. 203 the great accessibility and reactivity showed by vlps, as well as their ability to serve as nanocarriers, which made them suitable to be exploited in gene therapy, have also been applied to targeted drug delivery. 195 genetic modification and/or chemical functionalization of exposed amino acid residues on the capsid surface in order to attach small molecules, such as markers or bioactives molecules, is one of the most common approaches applied to target drug delivery. 174, 239 as an example, canine parvovirus (cpv) vlps produced in a baculovirus expression system and exhibiting natural tropism to transferrin receptors (tfrs) were chemically modified on accessible lysines of the capsid surface with fluorescent dye molecules and delivered to tumor cells. derivatization of cpv-vlps did not interfere with the binding and internalization into tumor cells. 183, 184 one limitation of vlps in gene therapy is the low efficiency of gene transduction due to inefficient dna packaging. however, a recent study presented a novel in vivo dna packaging of jcv vlps in e. coli that effectively reduced human colon carcinoma volume in a nude mouse model. in this study, the exogenous plasmid dna was transformed into the jcv vp1 expressing e. coli. the packaging of the second plasmid occurs simultaneously as the in vivo assembly of the jcv vlp. even though it is still not clear how the plasmid dna molecules are encapsidated in the vlp, the authors showed that gene transduction efficiency by their in vivo package system was about 80% in contrast to the 1-2% of gene transduction efficiency achieved by the in vitro osmotic shock system. 226 in addition, the administration of exogenous proteins may induce the immune system response, reducing therapy effectiveness or causing undesirable secondary effects, albeit immunological response of protein nanoparticles can be modulated. 240 spontaneous protein self-assembly to form ordered oligomers is a common event in biology. it can prove advantageous in terms of genome-size minimization, formation of large structures, stabilization of complexes, and inclusion of 270 functional features. 241 it has been widely documented that cellular oligomer proteins as well as viral capsids are stabilized by several weak noncovalent interactions as hydrophobic interaction, electrostatic energy, and van der waals forces. [242] [243] [244] these interactions result in a complex quaternary structure described by three symmetry point groups named cyclic (cn), dihedral (dm), and cubic (t, o, i). 245, 246 the development of computational techniques to predict protein-protein interactions using solved 3d protein structures makes it possible to predict and/or strengthen experimental data performing in in silico approaches. 247 furthermore, its use opens up the possibility to design proteins not only displaying specific biological functions but also interesting intermolecular interactions to obtain increased multivalency in the resulting complexes. moreover, it should be considered that not only whole proteins can self-assemble in smart nanoparticles; oligopeptides are also capable of forming organized structures. many applications are possible due to the enormous quantity of different combinations and features that can be exploited with peptides. 248, 249 furthermore, protein-protein interactions are not the unique parameters involved in particle formation, nucleic acid-peptide interactions, salt concentration, order of mix, and ratio between nucleic acid and protein can also strongly influence the condensation process. 250, 251 due to their natural tendency to self-assemble forming highly ordered structures, viruses provide a wide variety of scaffold proteins which are used as gene/drug carriers. among them, vlps have been reviewed in the previous section. however, simple bacterial proteins can be also utilized as carriers for gene delivery. for example, heat shock proteins (hsp) from hyperthermophilic archeaon methanococcus jannaschii can assemble in a small structure of 24 subunits having an octahedral symmetry. these 12 nm structures are stable at high temperature, up to 70 c, and wide range of ph. residue modifications are allowed to elicit specific attachment of small molecules. 186, 252 in bacteria, bacterial microcompartments (bmc) which are intracellular organelles consisting of enzymes encapsulated within polyhedral, protein-only shells, somewhat similar to viral capsids, have been described. bmcs are composed of a few thousand copies of a few repeated protein species (including one or more enzymes involved in specific metabolic pathways), and with sizes of around 100-150 nm in cross section. the general role of bmcs is to confine toxic or volatile metabolic intermediates, while allowing enzyme substrates, products, and cofactors to pass. the first described bmc, the carboxysome, was isolated in the early 1970s 253, 254 and has been found to contain both co 2 -fixing ribulose bisphosphate carboxylase/oxygenase (rubisco) 253, 254 and carbonic anhydrase [255] [256] [257] enzymes. carboxysomes' function is to enhance autotrophic co 2 fixation at low co 2 levels. other bmcs were later identified in cyanobacteria and some chemoautotroph bacteria. among them, bmc proteins have been later found to be encoded in the propanediol utilization operon (pdu) of the heterotroph salmonella 258 and by an operon for metabolizing ethanolamine (eut) in enteric bacterial species, including salmonella and escherichia. 259 salmonella enterica forms a polyhedral organelle during growth on 1,2-propanediol (1,2-pd) as a sole carbon and energy source, but not during growth on other carbon sources. 260, 261 the pdu organelles' function is to minimize the harmful effects of a toxic intermediate of 1,2-pd degradation (propionaldehyde). [261] [262] [263] other studies have shown that a polyhedral organelle is involved in ethanolamine utilization (eut) by s. enterica. 259 the function of the eut microcompartment is to metabolize ethanolamine without allowing the release of acetaldehyde into the cytosol, therefore minimizing the potentially toxic effects of excess aldehyde in the bacterial cytosol [264] [265] [266] and also preventing volatile acetaldehyde from diffusing across cell membrane. 267 so far, about 1700 proteins containing bmc domains have been identified, covering at least 10 different bacterial phyla. the typical bmc protein consists of approximately 90 amino acids, with an alpha/beta fold pattern. 268, 269 some individual bmc proteins self-assemble to form hexamers, which further assemble side by side to form the flat facets of the shell. 268, 270, 271 the formation of icosahedral, closed shells from such flat layers was elucidated in part by structural studies in carboxysomes: some bmc proteins assemble to form pentamers, which are located at and form the vertices of the icosahedral shell. 270 mechanisms directing enzyme encapsulation within protein-based bmcs have been studied during the last years. it has been described that, in some carboxysomes, protein ccmm is used as a scaffold to form interactions between both shell proteins and enzymes, 272,273 through a ccmm c-terminal region with homology to the small subunit of rubisco. 274 other studies revealed that pdu shells can self-assemble without needing interior enzymes 275 and that carboxysomes can self-assemble in vivo when rubisco has been deleted. 276 regarding properties of the encapsulated enzymes, in the pdu bmc some of the internal enzymes are encapsulated by specific n-terminal targeting sequences. 275, 277 in this line, sutter and colleagues 278 described a conserved c-terminal amino acid sequence that mediates the physical interaction of an iron-dependent peroxidase (dyp) or a protein closely related to ferritin (flp) with a specific type of bmc (encapsulins). in another example, an icosahedral enzyme complex, lumazine synthase (aals) from bacillus subtilis and aquifex aeolicus, was engineered to encapsulate target molecules by means of charge complementarity and can also be modified to give different characteristics to the assembled structure. 279 moreover, enzymatic subunits, like e2 of pyruvate dehydrogenase from bacillus stearotermophilus, can be modified to be used in gene delivery. e2 peptides naturally form a dodecahedron of 60 subunits of 24 nm in diameter allowing modification for drug-like accommodation. the assembling/disassembling of these structures can be modulated by changing the operative ph in the experimental environment. these nanoparticles can also be functionalized with antigens for vaccine development. 281, 282 according to these results, specific targeting sequences could be of use in biotechnological applications to package proteins inside the stable selfassembled icosahedral shell of bmcs, offering appealing opportunities to manipulate in the laboratory such nanocages to fill them with therapeutic molecules. the simplicity of this system makes it very attractive for engineering studies to design, mimicking nature, new applications in biotechnology, providing a new, intriguing platform of microbial origin for drug delivery. bovine serum albumin (bsa) is able to form microspheres after sonochemical treatment in aqueous medium. chemical effects of ultrasound radiation and coupling with an anticancer drug such as taxol (paclitaxel) led to the assembling of a spherical carrier with an average diameter of 120 nm. bsa particles resulting from s-s bonds, due to ho 2 radical formation, are able to release the encapsulated taxol in cancer tissue with best results if compared with mere taxol treatment. this drug for breast cancer treatment is commercially available. 283, 284 also little cationic peptides can lead to self-assembling particles. among others, arginine-rich cationic peptides are widely known as good tools for gene delivery. for example, purified r9-tailored gfp in solution is described to form nanodisk particles 20 nm in diameter. this structure is proved to be induced by the 9 arg tails and is able to bind and condense dna. these nanodisks are also able to deliver dna toward the nucleus where the reporter gene is expressed. 285 on the other hand, the expression of recombinant proteins over physiological rates can cause a bad functioning of cellular quality control system, leading to self-organizing, pseudo-spherical, protein aggregates known as inclusion bodies. these mechanically stable nanoparticles, ranging from 50 to 500 nm in diameter, were considered for a long time as undesired bio-products. recently, it became clearer that they are suitable for medical approaches when utilized as scaffold surface to promote cellular proliferation. [286] [287] [288] one of the most difficult goals for a foreign gene delivery is to reach the nucleus. an approach to overpass this obstacle is by fusing an nls in a nonessential position of a dna-binding protein. such type of modification has been described for a tetracycline repressor protein (tetr) fused with an sv40 nls. the tetr-nls affinity and specificity to teto dna sequence is exploited to form spontaneous protein-dna complexes which allow an enhancing of dna transportation into the nucleus and subsequent expression of foreign genes, combining the two peculiar characteristics of each fusion component. 289 there is still a tremendous gap between progresses made in protein-based nanoparticle research for drug delivery and clinical reality. hundreds of publications in basic research describe the combination of two or more functional elements in a single protein nanoparticle, by which the delivery of a carried drug is enhanced. these agents act by improving critical steps in the drug delivery process, such as increasing the systemic stability or tissue specificity, favoring internalization, endosomal escape, and entry into the nucleus, or transporting therapeutic material through the bbb, in in vitro and in vivo studies. besides the human recombinant therapeutic proteins currently on the market (or functional segments of them), there are also some fusion proteins approved for clinical use (most by incorporating an antibody fragment or a ligand to enhance cell specificity). sadly no gene therapy trials have so far used full protein carriers in vivo, but rather peptide-functionalized vehicles. bottlenecking the gap between research and clinical application, the us fda/european medicines agency (emea) only approves human proteins, to avoid the risk of an immune response that could affect not only the effectiveness of the nanoparticle but also challenge patients' health. another critical factor is the administration route, where the protein is degraded before arriving at the target; this problem could be solved or minimized by the use of protein d-isomers, pegylation, or the design of protecting groups for labile sites. despite the current situation mentioned above, there are many good examples of multifunctional modular proteins that, when carrying therapeutic material, can improve the prognosis in vivo in animal models for different diseases. these examples are reviewed below, along with those few protein nanoparticles that are currently on the market or in clinical trials. albumin is a natural protein transporter of hydrophobic molecules throughout plasma that has been approved by the fda to reversibly bind water-insoluble anticancer agents, as is the case of albumin-bound (nab) paclitaxel, abraxane . this albumin-nab technology-based drug is in use in patients with metastatic breast cancer who have failed combination therapy, and it is the first protein nanoparticle approved by the fda. albumin potentiates paclitaxel 274 concentration within the tumor by increasing paclytaxel endothelial transcytosis through caveolae formation. it also contributes to the fact that tumors secrete an albumin-binding protein sparc (also called bm-40) to attract and keep albumin-bound nutrients inside the tumor cell. 290 the albuminpaclitaxel complex was not formally considered a nanoparticle in the united states (due to an average size of 130 nm) but only so in europe. apart from whole recombinant therapeutic proteins being currently commercialized, there are also some examples of vehicles formed by chimerical proteins with target ligands already in the market. dab389il-2 (denileukin diftitox or ontak) is a fusion of diphtheria toxin catalytic and translocation domains for lethal effect and interleukin-2 (il-2) to gain cell specificity in the treatment of persistent or recurrent t-cell lymphoma. belatacept (bms-224818) is a ctla4-ig fusion protein formed by the cytotoxic t-lymphocyteassociated antigen 4 joined to an immunoglobulin g1 fc fragment fusion protein, developed by bristol-miers-squibb. etanercept (enbrel) fusion tumor necrosis factor receptor (tnfr), which binds and inhibits specifically tnf activity, to an immune globulin g1 fc, to prevent inflammation mediated by tnf in autoimmune diseases like arthritis and psoriasis. on the other hand, fusion proteins which include an antihuman epidermal growth factor receptor 2 (her2) monoclonal antibody that binds tumor cell surfaces, among them the so-called ''trastuzumab'' (commercialized as herceptin by roche), associated to dm-1, an antimitotic drug, aimed at improving the treatment of breast cancer. finally, vlps, that is, empty viral entities formed by the self-assembly of a viral capsid protein, are the only truly protein nanoparticles (architectonically speaking) which are currently used in clinical practice. hbsag recombinant protein of hbv expressed in yeast and the capsid l1 recombinant protein of hpv (types 6, 11, 16, and 18) administered currently as vaccines tend to form spontaneously vlps that elicit t and b immune response. recently, there have been preclinical and clinical trials to test the security and efficacy of vlp vaccines against chikungunya 291 and seasonal influenza virus (http://www. medpagetoday.com/meetingcoverage//icaac/22129), respectively. influenza vlp vaccines have proven to provide complete protection against h1n1 2009 flu pandemics, 292 within a record preparation time when compared to 9 months for traditional vaccines. the use of vlps as a delivery system for drugs or nucleic acids in gene therapy is still under investigation. 194 drugs and proteins may be transformed through pegylation, a process that can assist them in overcoming some of the potential problems that delay the adoption of protein nanoparticles for clinical use. the covalent attachment of peg can reduce immunogenicity and antigenicity by hiding the particle from the immune system, can increase the circulating time by reducing renal clearance, and can also improve the water solubility of a hydrophobic particle. the use of pegylation has been approved for commercial use by the fda and emea, and some examples of pegylated protein products are adagen (peg-bovine adenosine deaminase), the first pegylated protein approved by the fda in 1990, pegasys (peg-interferon alpha), and oncaspar (peg-l-asparaginase). the majority of protein nanoparticles studied in clinical trials (http://clinicaltrias.gov) are fusion proteins composed of a therapeutic protein/peptide and a target cell-specific ligand. an example is alt-801, a biologic compound composed of il-2 genetically fused to a humanized soluble t-cell receptor directed against the p53-derived antigen. the clinical trials evaluated whether directing il-2 activity using alt-801 to the patient's tumor sites that overexpress p53 results in clinical benefits (nct01029873, nct00496860). another ligand joined to il-2 is l19, a tumor-targeted immunocytokine constituted of a single chain fragment variable (scfv) directed against the ed-b domain of fibronectin, one of the most important markers for neoangiogenesis. l19-il-2 is in a phase i/ii study for patients with solid tumors and renal cell carcinoma (rcc) (nct01058538). l19 has also been fused to tnfa with the intention to target tnfa directly to tumor tissues resulting in high and sustained intralesional bioactive tnfa concentrations. the l19tnfa is under clinical trial using isolated inferior limb perfusion (ilp) with the standard treatment with melphalan 10 mg/l limb volume in subjects affected by stage iii/iv limb melanoma (nct01213732). ngr-htnf is another bifunctional protein which combines a tumor-homing peptide (ngr) that selectively binds to amino peptidase n/cd13 highly expressed on tumor blood vessels, thus affecting tumor vascular permeability, and htnf, with direct anticancer activity. ngr-htnf is undergoing 14 clinical trials as a single agent to treat different cancers, as well as in combination with chemotherapy agents. another strategy to direct a therapeutic protein to the target cell is through fusion to a growth factor receptor ligand. an example is tp-38, a recombinant chimerical protein composed of the egfr binding ligand (tgf-a) and a genetically engineered form of the pseudomonas exotoxin, pe-38, to treat recurrent grade iv malignant brain tumors (nct00071539). many clinical trials are based on a therapeutic protein fused to a targeting antibody, as is the case of apc8015. this drug stimulates the immune system and stops cancer cells from growing by the combination of biological therapies with bevacizumab , an already approved monoclonal antibody that locates tumor cells and kills them in a specific way (nct00849290). there are also many putative protein drugs against cancer which include antibodies antiintegrins (e.g., cilengitide and imgn388), sometimes in combination with 276 classical therapies. a recently developed tool, the nanobodies or single domain antibodies, 293 have several advantages: small size (only 12-15 kda), which lowers the possibility of triggering immune response, safety in clinical trials (nct01020383), and is easy to be joined to different kinds of compounds. all these features make nanobodies competent drugs against different diseases, and have been tested in vivo as bifunctional proteins associated to a prodrug, very efficient in mice cancer xenografts. 294 even though cpps are very useful tools to deliver drugs and in gene therapy (see the chapter ''peptide nanoparticles for oligonucleotide delivery'' by lehto et al. in this volume), their toxicity and endosomal entrapment slows their inclusion for systemic delivery in clinical trials. nevertheless, there are a few examples of use to prevent undesirable cell proliferation in coronary artery bypass grafts, as is the case of a cpp (r-ahx-r) 4 ahxb-pmo conjugate targeted to human c-myc to be applied ex vivo. the trial, in phase ii, has been completed in 2009 (nct00451256). another case is psorban , a product patented for the treatment of psoriasis based on a cyclosporine-polyarginine conjugate of local application, which circumvents the specificity problem of intravenous (i.v.) application. it is in clinical trial phase iii, but not yet in the market. finally, kai-9803, a pkcd inhibitor peptide conjugated to tat to function as an intravenous drug for the treatment of acute myocardial infarction, is currently in phase 2b clinical trial (nct00785954, kai pharmaceuticals). there are many proteins, often organized as nanoparticles, that when associated to a drug, therapeutic protein, peptide, or nucleic acid increase the therapeutic efficacy of a cargo alone in the treatment of various diseases. some of them proved effective in animal models, which are discussed in more detail in this section, with relevant examples listed in table ii . these nanoparticles may simply be (a) a cpp to promote nonspecific internalization, 295-300 (b) a peptide to confer cargo specificity by joining a receptor distinctive of a cell type, including scfvs or peptides obtained by phage display, 301 and (c) a mixture of both, 302 since as observed in several studies the cpp does not reduce ligand specificity and increases nanoparticle potency. [303] [304] [305] complex and multifunctional vehicles including endosomal escape peptides enhance the therapeutic potency of the complex, or other domains that allow their selective activation in certain contexts. 306, 307 apart from the cases listed in table ii , the spectrum of additional examples of multidomain protein nanoparticles tested in vivo is wide, and a considerable proportion of them include cpps, mainly tat and polyarginines. a classical tat fusion protein is the transducible d-isomer ri-tatp53c 0 ' fusion protein that activates p53 protein in cancer cells, but not in normal cells. ri-tatp53c 0 treatment in terminal peritoneal carcinomatosis and peritoneal lymphoma preclinical models results in significant increases in life span (higher than sixfold) and full recovery from the disease. 308 there are also several studies in vivo using tat-fused therapeutic proteins which have proven effective in treating tumors [309] [310] [311] and cerebral ischemia 312,313 when applied intraperitoneally (i.p.). regarding polyarginines, kumar and colleagues have presented two different models in which a bifunctional peptide formed by nine arginines (9r) and a specific ligand constitute an effective sirna vehicle. in the first model, a chimerical peptide derived from rabies virus glycoprotein (to confer neuronal specificity) fused to 9d-arginines (rvg-9r), was able to transport si-rna across the bbb and silence specific gene expression in the brain when applied intravenously. 56 in the second model, a cd7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scfvcd7-9r) for t cell-specific antiviral sirna delivery in humanized mice reconstituted with human lymphocytes. in hiv-infected humanized mice, this treatment controlled viral replication and prevented the disease-associated cd4 t cell loss. moreover, it effectively suppressed viremia in infected mice. 314 some other examples of polyarginines in tumor models are 9-d-arginines fused to a tumor-suppressor peptide, which stopped tumor growth in hepatocellular carcinoma-bearing mice when applied intraperitoneally, and also colesteryl oligoarginines carrying vegf sirna, which inhibited tumor growth in colon adenocarcinoma after local application. 315 another bbb-crossing peptide is g7, which is able to transport nanoparticles loaded with loperamide. 107 in general, the partner fusion peptide can confer specificity instead of penetrability, as is the case of egfr fab fragment associated to liposomes that contain anticancer drug, which increases efficiency of anticancer effect in egf overexpressing xenograft tumors 316 ; in addition, rgd-4 c-doxorubicin in human breast xenografts increases efficacy and diminishes toxicity. 317 in many conjugates, the therapeutic peptide of the chimerical proteins is a toxin. anthrax lethal toxin has been modified to be activated by methaloproteases, and it has probed to be effective for human xenografted tumors such as melanoma, lung, and colorectal cancer. 318 anthrax toxin has also been associated to antibodies or growth factors for lethal effects specifically on cancer cells. 319 the specific cytotoxicity desired to treat a tumor might derive from a tissue factor, which promotes clotting to restrict blood supply in tumor vessels, fused to peptides that provide specificity, like v-cam antibodies, fibronectin, and integrin ligands. 320 eventually, drug activity may decrease when conjugated to a carrier protein, although if the entry of the drug is favored, the overall balance of activity can be much more efficient. 321 on the other hand, the use of noncovalent bond drug carrier could avoid interfering with the activity of the drug. an important issue in a preclinical study to be considered for a clinical trial is the administration route. in in vivo experiments, most of the protein nanoparticles are administered by local or intraperitoneal injection, avoiding systemic spreading and clearance in the vascular system, in a way very similar to in vitro experiments. the fda and emea, on the other hand, will preferentially approve i.v. and oral administrations rather than intraperitoneal or local injections except for very accessible tissues. another relevant issue is the number of active domains to be included in a therapeutic protein carrier, an issue that seems to be relevant for the functionality of the construct. for example, the cpp neutralization of a ligand may depend on the cpp/ligand ratio that is in the vehicle. 322 it has also been observed that the integrin binding power of rgd-containing motives increases with the number of rgd domains over the monomer until a maxim of four moieties. 323 another example is tat activity empowerment when attached to molecules that form tetramers, such as beta-galactosidase 108 and p-53. 324 some multidomain protein carriers allow the drug entrance only in selected target cells by tailored smart selective mechanisms. 325 for instance, cpps neutralized by polyanions are activated and enter the cells when they are released by metalloproteases 326 or by lowering the ph, 327 both situations being very common in tumors. cpp-morpholino oligomer (pmo) nanoparticles have also shown their effectiveness in treating viral infections by inhibiting viral replication, as demonstrated with the carrier (r-ahx-r) 4ahxb-pmo administered i.v. in animal models infected with picornaviruses, i.p. in mice infected with coronaviruses and flaviviruses, and the carrier r9f2c-pmo administered also i.p. in mice infected with ebola virus. furthermore, it has also been shown in some of these studies that the efficacy of the treatment is dependent on the incorporation of arginine-rich peptides in the nanoparticle. 328 a good example of how a cpp can improve the internalization of a therapeutic protein is the case of insulin. the instability and low absorption in the digestive tract of insulin prevents its oral administration, even though it would be very convenient for a daily administrated drug. in recent studies, noncovalent conjugation of insulin to different cpps enhances its absorption without toxic intestinal effect, l-penetratin being the most efficient as insulin carrier. 329 among the protein nanoparticles tested in vivo, it is worth making special mention of trojan horses generated in pardridge's laboratory to cross the bbb, through a strategy of fusing within a chimerical peptide the therapeutic protein which has to reach the cns to a monoclonal antibody against the human insulin receptor (hirmab). this trojan horse is very potent for humans and primates, and has proven effective to transport b-glucuronidase, a-l-iduronidase, gdnf, abeta amyloid peptides, paroxonase, etc., with potential benefits in diseases like mucopolysaccharidosis type vii, hurler syndrome, parkinson, alzheimer, and organophosphates toxicity, respectively. 330 there are also promising results when protein nanoparticles have been tested as carriers for gene therapy in vivo, some examples being listed in table ii . in this regard, the use of modular proteins generated by insertional mutagenesis of b-galactosidase condensing the sod gene are able to protect neurons against ischemic injury 133 ; a bifunctional galactosylated polylysine is able to conjugate plasmid dna and to differentially promote expression in hepatocytes that display asialoglycoprotein receptor 331 ; a suicide multidomain protein particle formed by herpes simplex virus thymidine kinase (hsv-tk) conjugated to transferrin (tf) by a biotin-streptavidin bridging, which, administered i.v. in k562 massively metastasized nude mice, was able to reduce tumor size and to increase mouse survival. 332 in this chapter, proteins and peptides have been envisioned as potent biotechnological tools for the development of new biocompatible biological entities that can be used as therapeutic agents by themselves or as nanovehicles for the delivery of associated drugs. proteins are nanostructures that can form complex high-order entities such as vlps, resulting in appropriate cages for the internalization of therapeutic molecules. in addition, the design of modular proteins displaying selected functions has been possible by using in silico approximations to the feasibility of recombinant protein production. this approach has demonstrated the versatility of such molecules in the generation of novel delivery nanovehicles opening up the possibility of new functional combinations to enhance the specific interaction with the target tissue. such tunable specificity in the delivery of drugs, nucleic acids, or other proteins is one of the main properties that make multifunctional proteins appealing as more rational delivery vehicles. the presence on the market of such complex entities, which started with the approval of insulin for the treatment of diabetes, has been increasing over the past years, and this tendency is expected to continue. in fact, there are some products in clinical trials that will probably end up being approved and some more are being explored in preclinical experiments which might enter in clinical trials. identifying actives from hts data sets: practical approaches for the selection of an appropriate hts data-processing method and quality control review natural products in the process of finding new drug candidates when analoging is not enough: scaffold discovery in medicinal chemistry dose-toxicity models in oncology is declining innovation in the pharmaceutical industry a myth? the impact of pegylation on biological therapies anticancer activity of celastrol in combination with erbb2-targeted therapeutics for treatment of erbb2-overexpressing breast cancers soon-shiong p. sparc expression correlates with tumor response to albumin-bound paclitaxel in head and neck cancer patients improved effectiveness of nanoparticle albumin-bound (nab) paclitaxel versus polysorbate-based docetaxel in multiple xenografts as a function of her2 and sparc 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peptide with applications in drug and gene delivery design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers new basic membrane-destabilizing peptides for plasmid-based gene delivery in vitro and in vivo melittin enables efficient vesicular escape and enhanced nuclear access of nonviral gene delivery vectors the third helix of the antennapedia homeodomain translocates through biological membranes trojan peptides: the penetratin system for intracellular delivery histidine-rich peptides and polymers for nucleic acids delivery membrane permeabilization and efficient gene transfer by a peptide containing several histidines histidine containing peptides and polypeptides as nucleic acid vectors characterization of the gene transfer process mediated by histidine-rich peptides the proteasome metabolizes peptide-mediated nonviral gene delivery systems inhibition of ubiquitin-dependent proteolysis by a synthetic glycine-alanine repeat 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imported into the nucleus by different pathways nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells the carboxyl 35 amino acids of sv40 vp3 are essential for its nuclear accumulation pentapeptide nuclear localization signal in adenovirus e1a identification of domains involved in nuclear uptake and histone binding of protein n1 of xenopus laevis competition between nuclear localization and secretory signals determines the subcellular fate of a single cug-initiated form of fgf3 the human poly(adpribose) polymerase nuclear localization signal is a bipartite element functionally separate from dna binding and catalytic activity two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence long-term pharmacologically regulated expression of erythropoietin in primates following aav-mediated gene transfer rapid promoter analysis in developing mouse brain and genetic labeling of 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a selfassembling nuclear targeting vector system based on the tetracycline repressor protein unraveling mysteries of the multifunctional protein sparc a virus-like particle vaccine for epidemic chikungunya virus protects nonhuman primates against infection recombinant h1n1 viruslike particle vaccine elicits protective immunity in ferrets against the 2009 pandemic h1n1 influenza virus properties, production, and applications of camelid singledomain antibody fragments efficient cancer therapy with a nanobody-based conjugate effect of cell-based intercellular delivery of transcription factor gata4 on ischemic cardiomyopathy morpholino oligomer-mediated exon skipping averts the onset of dystrophic pathology in the mdx mouse overcoming multidrug resistance of small-molecule therapeutics through conjugation with releasable octaarginine transporters in vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation a non-covalent peptide-based carrier for in vivo delivery of dna mimics targeting cyclin b1 through peptide-based delivery of sirna prevents tumour growth antibody mediated in vivo delivery of small interfering rnas via cell-surface receptors selective inhibition of erbb2-overexpressing breast cancer in vivo by a novel tat-based erbb2-targeting signal transducers and activators of transcription 3-blocking peptide design of a tumor-homing cell-penetrating peptide a tumor-homing peptide with a targeting specificity related to lymphatic vessels penetratin improves tumor retention of single-chain antibodies: a novel step toward optimization of radioimmunotherapy of solid tumors killing hiv-infected cells by transduction with an hiv protease-activated caspase-3 protein development of elastin-like polypeptide for thermally targeted delivery of doxorubicin treatment of terminal peritoneal carcinomatosis by a transducible p53-activating peptide antitumor effect of tat-oxygen-dependent degradation-caspase-3 fusion protein specifically stabilized and activated in hypoxic tumor cells the 104-123 amino acid sequence of the beta-domain of von hippel-lindau gene product is sufficient to inhibit renal tumor growth and invasion dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity protein kinase c delta mediates cerebral reperfusion injury in vivo in vivo delivery of a bcl-xl fusion protein containing the tat protein transduction domain protects against ischemic brain injury and neuronal apoptosis t cell-specific sirna delivery suppresses hiv-1 infection in humanized mice cholesteryl oligoarginine delivering vascular endothelial growth factor sirna effectively inhibits tumor growth in colon adenocarcinoma epidermal growth factor receptor-targeted immunoliposomes significantly enhance the efficacy of multiple anticancer drugs in vivo cancer treatment by targeted drug delivery to tumor vasculature in a mouse model matrix metalloproteinase-activated anthrax lethal toxin demonstrates high potency in targeting tumor vasculature anthrax fusion protein therapy of cancer comparison of three different targeted tissue factor fusion proteins for inducing tumor vessel thrombosis overcoming methotrexate resistance in breast cancer tumour cells by the use of a new cellpenetrating peptide tumor cell retention of antibody fab fragments is enhanced by an attached hiv tat protein-derived peptide improved targeting of the alpha(v)beta (3) integrin by multimerisation of rgd peptides probing the impact of valency on the routing of arginine-rich peptides into eukaryotic cells cell-penetrating and cell-targeting peptides in drug delivery tumor imaging by means of proteolytic activation of cell-penetrating peptides tat peptide-based micelle system for potential active targeting of anti-cancer agents to acidic solid tumors cell penetrating peptide conjugates of steric block oligonucleotides usefulness of cell-penetrating peptides to improve intestinal insulin absorption biopharmaceutical drug targeting to the brain gene transfer in vivo: sustained expression and regulation of genes introduced into the liver by receptor-targeted uptake in vivo gene delivery to tumor cells by transferrin-streptavidin-dna conjugate the authors appreciate the financial support received through grants bfu2010-17450 from micinn, ps0900165 from fiss, and 2009sgr-108 from agaur. the authors also acknowledge the support of the ciber de bioingeniería, biomateriales y nanomedicina (ciber-bbn), an initiative funded by the vi national r&d&i plan 2008-2011, iniciativa ingenio 2010, consolider program, ciber actions and financed by the instituto de salud carlos iii with assistance from the european regional development fund. protein nanoparticles engineered for drug delivery and gene therapy 281 key: cord-002100-dt5zvebj authors: he, yonghua; schmidt, monica a.; erwin, christopher; guo, jun; sun, raphael; pendarvis, ken; warner, brad w.; herman, eliot m. title: transgenic soybean production of bioactive human epidermal growth factor (egf) date: 2016-06-17 journal: plos one doi: 10.1371/journal.pone.0157034 sha: doc_id: 2100 cord_uid: dt5zvebj necrotizing enterocolitis (nec) is a devastating condition of premature infants that results from the gut microbiome invading immature intestinal tissues. this results in a life-threatening disease that is frequently treated with the surgical removal of diseased and dead tissues. epidermal growth factor (egf), typically found in bodily fluids, such as amniotic fluid, salvia and mother’s breast milk, is an intestinotrophic growth factor and may reduce the onset of nec in premature infants. we have produced human egf in soybean seeds to levels biologically relevant and demonstrated its comparable activity to commercially available egf. transgenic soybean seeds expressing a seed-specific codon optimized gene encoding of the human egf protein with an added er signal tag at the n’ terminal were produced. seven independent lines were grown to homozygous and found to accumulate a range of 6.7 +/3.1 to 129.0 +/36.7 μg egf/g of dry soybean seed. proteomic and immunoblot analysis indicates that the inserted egf is the same as the human egf protein. phosphorylation and immunohistochemical assays on the egf receptor in hela cells indicate the egf protein produced in soybean seed is bioactive and comparable to commercially available human egf. this work demonstrates the feasibility of using soybean seeds as a biofactory to produce therapeutic agents in a soymilk delivery platform. each year in the united states, more than 530,000 babies, approximately 12% of total births, are born before 37 full weeks of gestation [1] . as a growing health issue the rate of premature birth has increased by 36 percent since the early 1980s. one of the major problems associated with prematurity is the development of a condition known as neonatal necrotizing enterocolitis (nec) [2] . this is observed clinically as the abrupt development of bloody diarrhea, abdominal swelling, and tenderness in a premature infant who is otherwise doing well [3] . current treatment often requires surgical removal of the damaged and dead intestine, often resulting in mortality (about 40%) or, if the infant survives, to manifest significant resulting lifetime problems [3] [4] [5] . although the direct cause of nec is not known, the most significant contributing factor is premature birth. post-partum establishment of an abnormal gut microbiome creates the opportunity for bacterial invasion into gut due to immature intracellular junctions of the intestinal mucosa [6, 7] . experimental and clinical evidence suggest that prematurity and nec is associated with deficient endogenous production of epidermal growth factor (egf), which is necessary for normal intestinal development and repair [8, 9] . egf is a critical growth factor found in multiple fluids that bathe the developing intestine including amniotic fluid, fetal urine, breast milk, bile, and saliva [2, 10, 11] . in the amniotic fluid, there is an increasing concentration of egf as gestation progresses [12] . egf amounts in mother's milk is highest first days after parturition with mothers of extreme pre-term neonates having 50-80% higher than mother's milk of full term infants [13] . human studies have demonstrated that egf is resistant to proteolytic degradation across a range of gastric ph [14] . while egf is produced to some extent in duodenal brunner's glands and kidney, the vast majority of egf is produced in the salivary glands [15] . exogenous infusion of egf in utero has been shown to accelerate the maturation of intestinal enzyme activity as well as stimulate intestinal growth [16, 17] . the importance of egf to gut development is highlighted by the fact that knockout of the egf receptor in some mice strains results in death due to a bloody diarrhea that is remarkably similar to human nec [18] . transgenic mice directed to intestinally overexpress egf displayed a number of beneficial effects, including increased body weight and villus height, after a small bowel resection compared to nontransgenic mice [19] . conversely, inhibition of egf receptors impairs intestinal adaption following a small bowel resection [20] . a prospective, multi-center trial demonstrated that infants fed regular formula (not containing growth factors) were 6 to 10 times more likely to develop nec than infants fed breast milk [21] . while a large number of biologically active peptides and growth factors have been identified in breast milk, egf is one of the major peptides present in significant concentrations [22] . the concentration of egf in milk is found to be inversely proportional to the gestational age of the infant, therefore, the more premature the infant, the more egf is present in the breast milk [13] . this may be a compensatory response to the premature removal of the fetus from the egf-rich amniotic fluid. it has been demonstrated in several animal models of nec that administration of exogenous egf has been shown to significantly reduce the severity of intestinal injury [23, 24] . the proactive treatment of infants at nec risk with egf supplementation could therefore accelerate intestinal maturation, thus preventing the development of nec. if the proactive egf feeding strategy is effective to induce the maturation of the neonate intestinal tract then this simple approach may mitigate the development of nec with its resulting high costs in medical resources, pain and possible life-long debilitation and for the 40% infants with nec that proves fatal [25] [26] [27] . to accomplish such a proactive therapeutic approach adapting infant formula for egf delivery would be simple and economic and mimic the delivery of egf in mother's milk. the need and potential delivery makes infant formula containing egf a good model for food-sourced plant biotechnology. soybean-derived formula encompasses a significant fraction of the total infant formula market. soybean milk and derived products are a potential food-therapy delivery platform that could include a variety of medically-necessary products including drugs such as egf but might also include oral vaccines [28, 29] . the economy of production and simple conversion into therapeutic materials that has long-shelf life makes soybean biotech products a potentially desirable commodity for use in cost-sensitive scaled applications. addressing the devastating disease of nec through a simple proactive treatment protocol is an excellent platform to explore the potential of soybean-produced therapeutics. here we report the accumulation of human egf (hegf) in geneticallyengineered soybean seeds and show that the recombinant egf is indistinguishable from authentic human egf and is bioactive at stimulating egf receptor (egfr) activity. epidermal growth factor protein from humans was produced in soybean seeds by constructing a plant gene expression cassette that involved a synthetic codon optimized egf nucleotide sequence (protein sequence from genbank accession ccq43157). this 162 bp open reading frame was placed in-frame behind a 20-amino acid endoplasmic reticulum (er) signal sequence from the arabidopsis chitinase gene [30, 31] . the er-directed egf encoding open reading frame was developmentally regulated by the strong seed-specific storage protein glycinin regulatory elements [31] . the entire seed specific cassette to direct egf production was placed in a vector containing the hygromycin resistance gene under the strong constitutive expression of the potato ubiquitin 3 regulatory elements as previously described [31] [32] [33] . the result plasmid pgly::shegf was sequenced using a glycinin promoter primer (5' tcattcac cttcctctcttc 3') to ensure the egf open reading frame was placed correctly between the regulatory elements. somatic soybean (glycine max l. merrill cv jack (wild type)) embryos were transformed via biolistics using 30 mg/l hygromycin b selection and regenerated as previously described [34] . embryos from resistant lines were analyzed by genomic pcr to confirm the presence of inserted hygromycin cassette using primers specific to the hygromycin gene (hygf 5'ctcactattcctttgccctc3' and hygr 5'ctgacctattgcatctcccg3'), cetyl trimethyl ammonium bromide (ctab) extraction genomic dna isolation and the following amplification conditions: 150 ng genomic dna in 25 μl total reaction containing 200 nm primers and 3 u taq polymerase (neb) and the following cycling parameters (initial 95°c 4 min then 45 cycles of 95°c 30 s, 55°c 45 s, 72°c 90s; followed by a final extension of 72°c 7 min). dry seeds from two successive generations of pcr positive plants were analyzed by elisa for the expression of egf protein until all 7 lines were confirmed to be homozygous. egf transgenic soybean plants along with nontransgenic control wild type cultivar plants were grown side by side in a greenhouse at 25°c under 16 h daylight with 1000 μm -2 /s. total soluble protein was extracted from dry seeds of two homozygous egf lines and a nontransgenic control by repeated acetone washes followed by acetone precipitation with the protein pellet dissolved in water. proteins with molecular weight 10 kda and under were isolated by separately passing each extract through an amicon ultra centrifugal filter (merck, kenilworth nj). the samples were each suspended in sample buffer (50mm tris hcl, ph6.8 2% sds (w/v), 0.7 m β-mercaptoethanol, 0.1% (w/v) bromphenol blue and 10% (v/v) glycerol) and then denaturated 5 min 95°c. protein content was determined by bradford assay [35] . a 15% sds-page gel was used to separate 30 μg protein for each of the three samples: negative control wild type, lines 4 and 5 of egf transgenic soybean dry seeds. commercially available human egf (gibco, life technologies,united kingdom) was used at 0.5 μg as positive control. gel was electroblotted onto immobilon p transfer membrane (millipore, bedford ma) and blocked with 3% milk solution in tbs for at least 1 hr. primary antibody was a commercially available anti-egf (calbiochem, san diego ca) and was used in a 1:100 ratio in 3% bsa-tbs buffer overnight at room temperature. after 3 washes of 15 mins each with tbs buffer, the blot was incubated with a 1:10,000 ratio in tbs of secondary antibody anti-rabbit igg fabspecific alkaline phosphatase conjugate (sigma, st. louis mo). after 3 washes, the presence of the egf protein was detected by using a color substrate (bcip/nbt: final concentrations 0.02% (w/v) 5-bromo-4-chloro-3-indoyl phosphate and 0.03% (w/v) nitro blue tetrazolium in 70% (v/v) dementhylformadmide) (kpl, gaithersburg ma). total soluble protein was extracted from dry soybean seeds as described previously [31, 32] from all 7 lines of pgly::shegf transgenic plants along with nontransgenic seeds as a negative control. egf was quantitated by commercially available human egf elisa assay (quantikine elisa kit from r&d systems, minneapolis mn) according to the manufacturer's instructions. the provided positive control was used to create a standard curve in order to determine the amount of egf in each soybean protein extract. each homozygote egf transgenic line was assayed with three biological replicates and results displayed as mean +/-standard error. total soluble proteins were extracted, quantitated and suspended in sample loading buffer as previously described [31, 32] . approximately 30 μg of protein extract from dry seeds of 4 homozygous egf lines were separated on a 4-20% gradient sds-page gel (biorad, hercules ca) along with extract from a nontransgenic seed. the gel was subsequently stained with 0.1% total soluble protein was extracted from 3 biological egf transgenic soybean dry seed samples, lines 4, 5 and 6. as described above, proteins with molecular weights lowers than 10 kda were concentrated using an amicon ultra centrifugal filter (merck, kenilworth nj). non-transgenic seeds were used as a negative control and 5 μg commercially available egf (as above in immunoblot section) was the positive control. protein was precipitated by adjusting the solution to 20% (v/v) trichloroacetic acid and allowed to sit at 4°c overnight. precipitated proteins were pelleted using centrifugation, washed twice with acetone and then dried using vacuum centrifugation. the commercial egf was not filtered or precipitated, only dried. dried pellets were rehydrated with the addition of 10 μl 100 mm dithiothreitol in 100 mm ammonium bicarbonate and placed at 85°c for 5 minutes to reduce disulphide bonds. samples were then alkylated with addition of 10 μl iodacetamide in 100 mm ammonium bromide and placed at room temperature in the dark for 30 minutes. two μg trypsin in 200 μl 100 mm ammonium bromide was added to each samples and placed in 37°c overnight for enzymatic digestion. post trypsin digest samples were desalted using a peptide reverse phase microtrap (michrom bioresources, auburn ca), dried and ultimately resuspended in 2 μl of 2% (v/v) acetonitrile, 0.1% (v/v) formic acid. separation of peptides was performed using a dionex u3000 splitless nanoflow hplc system operated at 333 nl minute using a gradient from 2-50% acetonitrile over 60 minutes, followed by a 15 minute wash with 95% acetonitrile and a 15 minute equilibration with 2% acetonitrile. the c18 column, an in-house prepared 75 μm by 15 cm reverse phase column packed with halo 2.7 μm, 90å c18 material (mac-mod analytical, chadds ford pa) was located in the ion source just before a silica emitter. a potential of 2100 volts was applied using a liquid junction between the column and emitter. a thermo ltq velos pro mass spectrometer using a nanospray flex ion source was used to analyze the eluate from the u3000. scan parameters for the ltq velos pro were one ms scan followed by 10 ms/ms scans of the 5 most intense peaks. ms/ms scans were performed in pairs, a cid fragmentation scan followed a hcd fragmentation scan of the same precursor m/z. dynamic exclusion was enabled with a mass exclusion time of 3 min and a repeat count of 1 within 30 sec of initial m/z measurement. spectra were collected over the entirety of each 90 minute chromatography run. raw mass spectra were converted to mgf format using msconvert, part of the proteowizard software library [36] x!tandem 2013.09.01.1 [37] and omssa [38] algorithms were employed via the university of arizona high performance computing center to perform spectrum matching. precursor and fragment mass tolerance were set to 0.2 daltons for both omssa and x!tandem. trypsin cleavage rules were used for both algorithms with up to 2 missed cleavages. amino acid modifications search consisted of single and double oxidation of methionine, oxidation of proline, n-terminal acetylation, carbamidomethylation of cysteine, deamidation of asparagine and glutamine and phosphorylation of serine, threonine, and tyrosine. x!tandem xml and omssa xml results were filtered using perl to remove any peptide matches with an evalue > 0.05 as well as proteins identified by a single peptide sequence. the protein fasta database for glycine max was downloaded on august 5, 2015 from ncbi refseq with the addition of the egf amino acid sequence. a randomized version of the glycine max fasta was concatenated to the original as a way to assess dataset quality. the mass spectrometry proteomics data have been deposited to the proteomexchange constortium (http://proteomecentral. proteomexchange.org) via the pride partner repository [39] with the dataset identifier pxd003326 and 10.6019/pxd003326. hela cells (obtained from american tissue culture collection) were cultured in minimum essential media (mem) complemented with 10% fetal bovine serum (fbs), 100 units/ml penicillin, and 100 μg/ml streptomycin. for western blotting assay, cells grown in 6-well plate were kept in serum free mem media for 24 hours. cells were then either kept in serum free medium (control) or stimulated with soy milk alone, soy egf or commercial recombined human egf for different time period as indicated. cells were lysed by directly adding 1× sds sample buffer (50 mm tris-hcl, ph 6.8, 10% glycerol, 2% sds and 5% β-me) to the cells after washing 3 times with 1x pbs. egf bio-activity was determined via egfr phosphorylation and downstream akt phosphorylation. total egfr was also measured since egfr is known to undergo internalization when stimulated with egf. antibodies used in western blot are anti-p-egfr (tyr1068) (#2234, cell signaling technology), anti-total egfr (#06-847, millipore), anti-p-akt (#4060, cell signaling technology) and anti-lamin b1 (# 13435, cell signaling technology) [40] . for immunocytochemistry assay, cells were grown on coverslip in 6-well plate and kept in serum free media for 24 hours before stimulation, cells were then either kept in serum free media (control) or stimulated with human or soy egf for 6 hours. cells were washed with pbs and fixed with 4% formalin. egfr was labeled using anti-egfr antibody (#4267, cell signaling technology) and detected with alexa fluor 594 goat anti-rabbit igg (#a11012, life technology). the cell nucleus were shown using mounting medium with dapi (#h-1200, vectorshield). to produce hegf in soybean a strong soybean seed-specific promoter and terminator was used to regulate gene expression of a synthetic soybean codon optimized hegf (shegf) gene that included an n-terminal 60 nucleotide er-signal sequence (fig 1a) . in the engineering strategy for the hegf expression in soybean, the components of the prepro portions of hegf were eliminated in preference to produce only the final recombinant hegf product. to facilitate the co-translational transfer of the egf into the er lumen for disulfide bond formation a plant signal sequence was added so that the hegf synthesized would be as a pre-hegf. the gly::shegf construct was used for biolistic transformation of soybean somatic embryo cells as outlined in [31] [32] [33] [34] . embryos were selected in liquid culture by hygromycin b and individual regenerated lines were separated, propagated, and induced to form cotyledonary embryos. the cotyledonary embryos were evaluated for hegf production using egf-specific elisa that indicated a variation of heterologous protein production (data not shown). the most promising egf expressing lines were moved forward for regeneration by desiccating and subsequent germination. the initial t 0 generation egf transgenic plants were grown in the greenhouse and further selected by genomic pcr for an additional 2-3 generations. additionally, each generation of seeds produced by the selected lines were assayed for hegf content by elisa. the hegf content of each line in seeds representative of the homozygous population is shown in fig 1b. the lines varied in hegf content but seeds within each line had a narrow range of hegf accumulation. the egf transgenic line 5 produced in excess of 100 μg hegf per gm dry seed weight, a level calculated to be much in excess of potential therapeutic requirements. by comparison, yeast stains have been used as an expression system for both human egf [41] and mouse egf [42] with the highest levels produced being from a multicopy insert pichia pastoris clone secreting 49 μg egf/ml. in both the mouse and human egf yeast production systems, truncated versions of the egf were detected. the hegf soybeans and nontransgenic soybeans were evaluated to determine the biochemical authenticity of the soybean-produced egf protein. using 1d sds/page and parallel immunoblots probed with anti-egf, the soluble low molecular weight (<10 kda) seed proteins and the mr of the soybean-produced hegf was evaluated. the total protein polypeptide of the hegf expressing lines appeared to be identical to the standard parental control (fig 2) . immunoblots of the 1d sds/page probed with anti-egf showed a lack of an immunoreactive band in the nontransgenic soybean seed control and recognized a 6 kda mr band in the hegf expressing lines 5 and 4. the soybean-produced hegf has the same apparent mr as authentic recombinant hegf fractioned in an adjacent lane (fig 3) . to further assess the soybean-synthesized hegf the seed lysates were enriched in low mr total proteins and concentrated. the crude low mr proteins were reduced, alkylated, and cleaved with trypsin prior to analysis by mass spectrometry. the resulting data was queried with the hegf sequence and exact matches for peptides encompassing the majority of the sequence of the complete mature hegf protein were obtained (fig 4) . together the data shows that transgenic soybeans successfully produced and accumulated hegf that is the correct mr, is immunoreactive with antibodies directed at authentic egf in both elisa and immunoblot assay, and that a majority mass spectrometry of fragments of the soybean-produced hegf match the human egf sequence. the delivery of any biopharma product in the context of compositionally complex food presents the potential that the components of the food may act to modulate bioactivity. plantsource foods in particular pose problems because plant tissues often possess a wide range of intrinsic biologically active components including proteins and natural products. the natural products of food could mask or enhance the effects of an expressed biopharma product. to evaluate the potential of egf activity in soymilk delivery commercial recombinant human egf (rhegf) was added as a supplement to soymilk and the intrinsic activity of the egf was tested with a hela cell assay. fig 5 shows the effects of soymilk on the display of the egf receptor (egfr) on hela cells and the effect of commercial rhegf supplement to soymilk. soymilk does not modify the display of egfr on hela cells showing that soymilk alone is biologically inactive. the binding of egf to egfr results in the decrease of displayed egfr as it is internalized into the hela cells. hela cells treated with commercially available recombinant rhegfsupplemented soymilk display the same decrease in egfr as cells treated with rhegf in media without soymilk. parallel time-course experiments show that the effect of rhegf binding to efgr is rapid with a reduction of displayed efgr occuring within 5 min of treatment and continuing out to at least 30 min (data not shown). together these assays show that soymilk has no apparent negative bioactivity with respect to both the binding of commercial rhegf to the hela cell egfr or the viability of the hela cells over the course of the assay. to assess the bioactivity of soybean-produced hegf, samples were prepared from both shegf transgenic soybean lines and nontransgenic controls that were used to stimulate hela cells to induce egfr internalization, degradation and phosphorylation. in results shown in fig 5, soybean-produced hegf induces the internalization, degradation and phosphorylation of egfr that is indistinguishable from the bioactivity of commercial rhegf delivered in control samples. in contrast, samples prepared from control nontransgenic soybeans exhibited no apparent bioactivity showing the degradation and phosphorylation of egfr is the result of egf binding of either commercial rhegf added to the media or from the hegf produced by the transgenic soybeans. together these results show that nontransgenic soybean seeds have no intrinsic egf-mimic activity able to induce egfr degradation or phosphorylation, while soybeans producing hegf have identical activity in comparison to commercial rhegf. soybean produced egf displayed comparable bioactivity to commercially available egf. panel a. soybean produced hegf induces a rapid phosphorylation of hela cell egfr. serum free media (sf) and sf media with soymilk alone does not induce egfr phosphorylation and degradation. soymilk from seeds producing shegf added at different concentrations (0.1, 0.05, 0.025 μg/ml) induced concentrationdependent egfr degradation comparable to the effect of rhegf. serum free media and serum free media with nontransgenic soybean soymilk (negative controls) showed no effect on inducing pegfr. in contrast soymilk from shegf soybeans given at different concentrations (0.1, 0.05, 0.025 μg/ml) induced pegfr comparable to control rhegf. pakt indicates the functional activation of egfr. lamin b1 was used as a loading control. panel b. exogenous commercial rhegf and shegf induces an internalization and degradation of egfr in hela cells shown as a decrease in abundance assayed by immunoblot. the results shown demonstrate that soymilk alone has no intrinsic bioactivity with respect to egfr abundance. the rhegf is not degraded in soymilk over 24 hours having the same bioactivity as control recombinant rhegf.-ctrl-sf media alone. soy egf and rhegf are at 0.1 μg/ml. lamin b1 was used as a loading control. panel c. shown is an immunohistochemical assay of hela cells showing that shegf induces internalization of the egfr comparable to that from control rhegf. in c, the cells were first treated with soy/egf or human egf for 6 hours, fixed and then immunostained with egfr antibody overnight. egfr shows red staining while nucleus was stained by dapi and shows blue staining. in developing a food-based delivery platform for biopharma it is important to address the question of whether there are significant collateral consequences in seed composition resulting from the genetic modification. ideally a consumption plant biotechnology platform, such as soymilk, should be fully equivalent to the standard type other than the intended modification. seeds in general, including soybeans, possess an inventory of bioactive proteins and small molecules that will affect the metabolism of consumers in both advantageous and disadvantageous manner. for soybeans some of the relevant molecules are allergens, anti-metabolite proteins, and small molecules especially isoflavones. to test for potential collateral composition in the hegf-producing soybeans, the shegf transgenic and nontransgenic control soybeans were analyzed by non-targeted proteomics and metabolomics. among the significant proteins identified include various well-documented allergens and anti-metabolite proteins. a comparison of standard soybeans with hegf-producing soybean lines showed that there was no significant difference (p = .01) between nontransgenic control and shegf transgenic soybeans aside from the targeted production of hegf for any other proteins of concern. this data is available in pride partner repository with the dataset identifier pxd003326 and 10.6019/pxd003326. non-targeted small molecule metabolomics was used to conduct a parallel analysis of the nontransgenic and hegf soybeans. again there were insignificant differences between nontransgenic soybean seeds and the shegf transgenic seeds (fig 6) with one notable exception. soybean highly regulates sulfur availability and its allocation into protein. from a nutritional perspective soybean is considered a somewhat sulfur deficient crop. there have been a number of biotechnology experiments to increase sulfur content be either modifying assimilation and biosynthesis pathways leading to methionine or over-expressing high-methionine proteins such as maize zeins. modifying sulfur by pathway or competition has an effect on sulfurresponsive proteins including the bowman-birk trypsin inhibitor (bbi) and beta chain of the storage protein conglycinin. egf is a high sulfur content protein that broadly mimics bbi as a small globular protein synthesized by the er and presumptively competing for sulfur amino acid charge trna. expressing hegf in soybean has an effect on metabolites involved in sulfur amino acid metabolism that is consistent with producing a protein of egf's composition. a complete dataset of all metabolite abundance of the standard and hegf-expressing lines is available as an on-line spreadsheet (s1 table) . among the assayed molecules of particular note is the soybean molecule genistein, an isoflavone that has been shown to affect the activity of tyrosine phosphatase in the signal cascade associated with egf signaling [43] [44] [45] [46] . genistein levels were determined to be the same in both the nontransgenic and hegf-expressing soybean lines. this too demonstrates that the expression of hegf in soybeans does not produce any incidental collateral consequences of concern for its potential therapeutic use. since the inception of plant biotechnology its potential use for biopharma applications has been assessed [47] [48] [49] [50] . several different plant organs proposed as production for food/feed delivery systems [51] [52] [53] . for many vaccine applications fruit are a highly advantageous delivery system providing a broadly accepted platform for even the most recalcitrant consumer (for example, [54, 55] ). although fruit are perhaps one of the best delivery systems from the perspective of point of delivery, fruit also has logistical issues with relatively short time that ripened fruit are palatable requiring a tightly coordinated effort to produce, distribute, and use biopharma product that could be challenging for deployment in mass quantities. an alternative fig 6. relative proportion of non-targeted metabolites detected in soybean seeds shown as amount in egf transgenic compared to nontransgenic (wt). complete list of non targeted metabolites quantitated in samples in s1 table. doi:10.1371/journal.pone.0157034.g006 transgenic soybean production of bioactive human epidermal growth factor (egf) is to develop a biopharma platform that is broadly acceptable for food and feed delivery but can be lightly processed to preserve bioactivity and can be massively scaled to maximize the distribution potential of the product. soybean is a potentially useful biopharma platform that could have broad application in both food and feed end uses [29, 30, 56, 57] . soybean has been demonstrated as a platform to produce heterologous proteins at a standard that far exceeds the levels typically needed for biopharma [31] . soybeans can be used to produce both soymilk and formula for potential delivery to human infants or children as well as for production animals such as swine and calves. soybean can also be used to produce protein concentrates for inclusion in industrial food and feed or more simply as protein aggregates as tofu. soybean production is efficient and economic that can be massively scaled if needed. recently developed technology makes it feasible to increase the amount of recombinant protein product by silencing and exchange with a storage protein(s) [31, 58, 59] . as a platform, soybean is an industrial crop with vast majority of its total production being directed toward products including processed food, protein used as animal feed, and its oil for food, feed, fuel, and chemical feedstock uses. many of the goals of further enhancing and modifying soybeans are largely directed at improving its utilization for industry products rather than direct food use. as a biopharma platform to produce soymilk derived products soybean seeds can be stored for years anticipating future needs while retaining the potential to be rapidly processed into formula/milk or tofu using adaptations of traditional technology in use for over a millennium. soybeans like many other seeds produce an array of intrinsic small globular proteins with secondary disulfide bonds accumulated at relatively high levels (>1% of total proteins). soybean in particular accumulates the bowman-birk trypsin inhibitor that is 8.5 kda with 3 intra-chain disulfide bonds [60] . this suggests that soybean seeds are optimized as a potential bioreactor to produce and store proteins like egf, a 6.9 kda protein with 3 intra chain disulfide bonds paralleling intrinsic seed proteins. in a predecessor experiment a mutant inactive bbi was expressed in transgenic soybeans showing that alternate small proteins can be expressed in soybean [60] . expression of a construct encoding shegf regulated by the soybean seed storage protein promoter results in the accumulation of hegf at > 100 μg /gm of dry soybean seed, a level to be many fold over the estimated therapeutic requirements of 50 μg/kg weight of treated individual [61] . soybean-produced hegf appears to be completely comparable to authentic hegf in its mr, immunoreactivity with specific antibodies, correspondence of fragment sequence in mass spectrometry assay, and in bioactivity inducing the internalization, degradation and phosphorylation of efgr. together the results shown demonstrate that soybean seeds will produce hegf at proto-therapeutic levels and the derived hegf from these seeds are bioactive for egf activity in a model hela cell assay. the expression of hegf in soybean has little collateral impact on seed composition soybeans have been used as an expression platform for a wide variety of heterologous proteins with investigative as well potential food/feed and biopharma goals [33, [62] [63] [64] [65] [66] . prior biopharma expressions have included prototype expression of vaccine models [67] as well as proinsulin and fibroblast and human growth factor [68, 69] . in this study potential collateral changes in prototype product mature soybean seeds resulting from hegf expression was evaluated by non-targeted proteomics and metabolomics to assess both large and small molecules. these assessments showed that there was no significant difference in the seed proteome of the egf transgenics compared to nontransgenics. this is a pertinent result as soybeans are regulated in the us under falpa (the 2004 food and allergen labeling protection act) and unintended alterations of any of the known seed allergens or anti-nutritional proteins can be of concern. similarly the non-targeted metabolomics of the soybean seeds showed a significant lack of alteration of the small molecule profile in response to hegf accumulation. among the molecules assessed the lack of change in genistein is among the most significant as this isoflavonoid has been shown to have activity with tyrosine phosphatase that is in the signal cascade of animal and human cells that includes egf/egfr signaling [43, 44, 70] . in the hela cell assessments there was no synergistic effect of standard soybean milk and authentic egf on egfr activity indicating that the identical genistein concentration in the standard and hegf expressing soybeans is below the threshold of effect in the assays conducted. the one significant alteration in the small molecule profile was in methionine-related metabolism. egf is a sulfur rich protein containing three disulfide bonds that has some general resemblance to the soybean bowman-birk inhibitor. soybean is a relatively sulfur deficient crop and much effort has been made to increase its sulfur amino acid content by either the co-expression of sulfurrich proteins such as zeins [71] or by increasing the sulfur flux by altering the sulfur amino acid pathways [72] [73] [74] . these studies have shown that within limits the increase of a sulfur sink such as expressing a high-sulfur content protein will collaterally induce modest increases in sulfur amino acid source. the results of increases in sulfur amino acid metabolites accompanying hegf expression in soybean is in accord with these prior experiments. together the results of the non-targeted proteome and metabolome assessments show that converting soybean into a prototype biopharma delivery platform of hegf does not result in any adverse alterations of the soybean seed's composition. soybeans could be used to produce biopharma products that might be delivered as milk or formula. as a test of this concept human epidermal growth factor (hegf) has been produced in soybeans to potentially address the devastating disease of neonatal necrotizing enterocolitis. this is a disease of premature infants of low birth weight. these infants have underdeveloped organs including the intestinal tract. the resulting gangrenous infection is treated by emergency surgery to remove dead portions of the intestinal tract that even under most optimistic situations has a high mortality rate and high cost of treatment. an alternative approach is to proactively treat infants at risk immediately post-partum to attempt to improve the integrity and maturity of the lining epithelial cells. the bioactivity results with model hela cells shows that hegf can be produced and accumulated in soybean seeds and as crude soy-milk lysate is capable of stimulating a response from the egf receptor (egfr) that occurs on epidermal surfaces such as the intestinal tract. soybean-produced hegf has potential other applications in cosmetics, burn and injury treatment, stimulating improved adaptation of the bowel to massive intestinal loss. supporting information s1 table. non-targeted metabolome set. births: final data for 2010 role of human milk in extremely low birth weight infants' risk of necrotizing enterocolitis or death necrotizing enterocolitis: the search for a unifying pathogenic theory leading to prevention neonatal necrotizing enterocolitis: a nine year experience: ii outcome assessment long-term survival and parenteral nutrition dependence in adult patients with the short bowel syndrome 16s rrna gene-based analysis of fecal microbiota from preterm infants with and without necrotizing enterocolitis fecal microbiota in premature infants prior to necrotizing entercolitis establishment of the gut microbiota in western infants bacterial colonization and gut development in preterm neonates epidermal growth factor: biology and mechanism of action early human milk feeding is associated with a lower risk of necrotizing enterocolitis in very low birth weight infants epidermal growth factor (egf) concentrations in amniotic fluid and maternal urine during pregnancy increased epidermal growth factor levels in human milk of mothers with extremely premature infants human epidermal growth factor: isolation and characterization and biological properties immunocytochemical localization of human epidermal growth factor/urogastrone in several human tissues transforming growth factor alpha and epidermal growth factor in protection and healing of gastric mucosal injury epidermal growth factor enhances intestinal adaptation after massive small bowel resection epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor intestinal overexpression of egf in transgenic mice enhances adaptation after small bowel resection selective inhibition of the epidermal growth factor receptor impairs intestinal adaptation after small bowel resection breast milk and neonatal necrotizing enterocolitis human milk for the premature infant epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model intestinal barrier failure during experimental necrotizing enterocolitis: protective effect of egf treatment short bowel syndrome management of the short bowel syndrome in the pediatric population pediatric shortbowel syndrome: the cost of comprehensive care plant made pharmaceuticals: from edible vaccines to ebola therapeutics towards using biotechnology to modify soybean seeds as protein bioreactors. in, recent advancements in plant expression in crop plants production of escherichia coli heat labile toxin (lt) b subunit in soybean seed and analysis of its immunogenicity as an oral vaccine the collateral protein compensation mechanism can be exploited to enhance foreign protein accumulation in soybean seeds a rnai knockdown of soybean 24 kda oleosin results in the formation of micro-oil bodies that aggregate to form large complexes of oil bodies and er containing caleosin transgenic soybean seeds accumulating β-carotene exhibit the collateral enhancements of high oleate and high protein content traits towards normalization of soybean somatic embryo maturation a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding proteowizard: open source software for rapid proteomics tools development tandem: matching proteins with tandem mass spectra open mass spectrometry search algorithm retinoblastoma protein (prb), but not p107 or p130, is required for maintenance of enterocyte quiescence and differentiation in small intestine the proteomics identifications (pride) database and associated tools: status in 2013 characterization of recombinant human epidermal growth factor produced in yeast production of mouse epidermal growth factor in yeast: high level secretion using pichia pastoris strains containing multiple gene copies genistein, a tyrosine kinase inhibitor, reduces egf-induced egf receptor internalization and degradation in human hepatoma hepg2 cells genistein alters growth factor signaling in transgenic prostate model (tramp) genistein enhances relaxation of the spontaneously hypertensive rat aorta by transactivation of epidermal growth factor receptor following binding to membrane estrogen receptors-α and activation of g protein-coupled, endothelial nitric acid synthase-dependent pathway genistein increases epidermal growth factor receptor signaling and promotes tumor progression in advanced human prostate cancer the production of recombinant pharmaceutical proteins in plants sowing the seeds of success: pharmaceutical proteins from plants recombinant pharmaceuticals from plants: the plant endomembrane system as bioreactor commercialization of biopharmaceutical and bioindustrial proteins from plants efficacy of a food plant-based oral cholera toxin β subunit vaccine oral immunization with hepatitis β surface antigen expressed in transgenic plants immunogenicity of recombinant lt-b delivered orally to humans in transgenic corn expression of hepatitis b surface antigen (hbsag) gene in transgenic banana (musa sp) severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine expression and immunogenicity of an escherichia coli k99 fimbriae subunit antigen in soybean protein expression systems: why soybean seeds?" in soybean-molecular aspects of breeding cosuppression of the α-subunits of β-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies silencing of soybean seed storage proteins results in a rebalanced protein composition preserving seed protein content without major collateral changes in the metabolome and transcriptome reduction of protease inhibitor activity by expression of a mutant bowman-birk gene in soybean seed epidermal growth factor augments adaptation following small bowel resection: optimal dosage, route, and timing of administration expression of functional recombinant human growth hormone in transgenic soybean seeds processing and localization of bovine β-casein expressed in transgenic soybean seeds under control of a soybean lectin expression cassette embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds metabolically engineered oilseed crops with enhanced seed tocopherol an alternative to fish oils: metabolic engineering of oilseed crops to produce omega 3 long chain polyunsaturated fatty acids correct targeting of proinsulin in protein storage vacuoles of transgenic soybean seeds high-level expression of basic fibroblast growth factor in transgenic soybean seeds and characterization of its biological activity expression of correctly processed human growth hormone in seeds of transgenic tobacco plants genistein analogues: effects on epidermal growth factor receptor tyrosine kinase and on stress-activated pathways increased sulfur amino acids in soybean plants overexpressing the maize 15 kd zein protein enhanced levels of methionine and cysteine in transgenic alfalfa (medicago sativa l.) plants over-expressing the arabidopsis cystathionine γ-synthase gene transgenic soybean plants overexpressing o-acetylserine sulfhydrylase accumulate enhanced levels of cysteine and bowman-birk protease inhibitor in seeds effects of proteome rebalancing and sulfur nutrition on the accumulation of methionine rich δ-zein in transgenic soybeans front key: cord-009792-e2vvi8qo authors: pandit, sb; balaji, s.; srinivasan, n. title: structural and functional characterization of gene products encoded in the human genome by homology detection date: 2008-01-03 journal: iubmb life doi: 10.1080/15216540400006105 sha: doc_id: 9792 cord_uid: e2vvi8qo availability of the human genome data has enabled the exploration of a huge amount of biological information encoded in it. there are extensive ongoing experimental efforts to understand the biological functions of the gene products encoded in the human genome. however, computational analysis can aid immensely in the interpretation of biological function by associating known functional/structural domains to the human proteins. in this article we have discussed the implications of such associations. the association of structural domains to human proteins could help in prioritizing the targets for structure determination in the structural genomics initiatives. the protein kinase family is one of the most frequently occurring protein domain families in the human proteome while p‐loop hydrolase, which comprises many gtpases and atpases, is a highly represented superfamily. using the superfamily relationships between families of unknown and known structures we could increase structural information content of the human genome by about 5%. we could also make new associations of domain families to 33 human proteins that are potentially linked to genetically inherited diseases. iubmb life, 56: 317‐331, 2004 an important event in the history of humankind has been made at the turn of the 21st century in the form of the draft version of human genome data (1, 2) . this monumental effort provides us with an opportunity to better understand the various biological processes and possible cognition. the functional characterization of the gene products encoded in the human genome could provide insights into such complex biological process. there are various experimental endeavors to understand the functions of gene products encoded in the human genome. in addition to these experimental efforts, computational analyses of human proteins could form an important step in the functional inference of the genome data. the computational approaches for the prediction of functional features of proteins encoded in genomes relies on establishing relationships to homologues that are experimentally studied. there have been several attempts, using various sophisticated homology search tools, to assign functions to gene products encoded in various proteomes (see for example references 3 -8) . such functional predictions could be used as a guiding tool in order to direct the relatively time consuming, more difficult and expensive experimental methods for exploring protein functions. furthermore, functional inferences of human proteins that are implicated in diseases could provide valuable insights on the molecular basis of human diseases. such an understanding could aid identification of effective drug targets and rational design of lead compounds to combat the diseases. the most commonly used computational approach for genome-wide association of functions to proteins is by identification of well-characterized homologues using sequence-based search procedures such as blast (9) and fasta (10) . but, pairwise sequence alignment based search procedures are unlikely to be able to identify related proteins with low sequence similarity. however, these distantly related proteins could often be identified with the use of threedimensional (3-d) structural information (11) as the structure is conserved better than sequence during evolution (12, 13) thus, use of structural information could potentially enhance the functional assignments (14 -17) . moreover, structure prediction with relevant biochemical motifs can provide more detailed functional insights than sequence comparisons alone (18 -20) . the search methods for such an analysis could be improved by the use of multiple sequence alignment of the homologues in a family, which can indicate structurally/ functionally important positions. the information in these multiple sequence alignments can be converted into position specific scoring matrices (pssm) usually referred as profiles (21) or into a probabilistic model called the hidden markov model (hmm) (22) . the use of profile-based search methods is known to improve sensitivity of detection of remotely related homologues (23 -28) . hence, use of structure and profile-based method would enable detection of remote homologues and thus enrich the functional assignments. some of the commonly used profile-based search methods include psi-blast (25) , impala (29) , rps-blast and hidden markov model (hmmer2)-based (30) procedures. these methods have been shown to detect remote and subtle similarities (31, 32) between proteins that were previously possible only by structure comparison procedures, which obviously demand the knowledge of 3-d structures. although the profile-based annotation methods are among the widely used procedures to detect remote similarities, there are other procedures such as genthreader (33) and environment-based profiles (34) , which are fold recognition methods for assigning the structural domains to amino acid sequences. furthermore, the comparison of proteins across various genomes could also aid in enhancement of the assignment of functions to the proteins (34 -41) . the comparative genomics methods are based on the functional characterization of the gene products by detecting the orthologous proteins in the closely related organisms where the experimental functions of the proteins have been proposed. however, the effectiveness of this approach is dependent on at least two factors: (1) ability to identify homologues of a given protein in other organisms. (2) the extent of divergence of amino acid sequences of the homologues across the organisms and its implication on the similarity of functions between the two proteins. in order to understand the biological function of the human proteins we have associated functional or structural domains using sensitive profile-matching procedures. association of functional domain would provide clues to biochemical role of the protein. the structural domain association could provide enhanced abilities to assign function and also provide the molecular basis of action of proteins. furthermore, we have enhanced the structural information content of human genome by relating families apparently with unknown structures to known structural families, as in the supfam database, which was developed by us earlier (26, 27) . the functional domain information was considered from pfam database (http://www.sanger.ac.uk/software/pfam) (42) and structural domain from pali (http://pauling.mbu.iisc. ernet.in/*pali) (43, 44) databases. pali contains structurebased sequence alignment and phylogeny of proteins in the families derived from the scop database (45) , which is a hierarchical structural classification database. the profiles for pfam and pali families have been generated as described by pandit et al (26) . these profiles were searched using rps-blast. we have also used hmm-based search procedure against the hmm libraries of pfam to associate functional domain to human proteins. profiles of transmembrane sequences have been associated with human proteins in order to predict membrane localisation of the proteins. using sensitive profile-matching procedures, we could make a comprehensive compilation of functional/structural domains to gene protein encoded in human genome. similar attempts have been made in the past by muller et al. (38) . they have used psi-blast for much of their analysis, apart from impala, to assign structural/functional domains. in their approach psi-blast has been scanned against the nonredundant database of protein sequences augmented with scop domain sequences. in the present review we will discuss the current status of large-scale function association, using various computational procedures, of various gene products encoded in the human genome. furthermore, human proteins potentially involved in diseases have been specifically analyzed by associating functional/structural domain to these protein sequences. the amino acid sequences of the open reading frames (orfs) that correspond to the gene products encoded in human genome have been obtained from the ensembl database (46) (release 22.34d.1, http://www.ensembl.org). the total number of gene products predicted in this release is 29,031. the omim database (47) is a comprehensive collection of genes and genetic disorders in humans. in our analysis the protein sequences corresponding to the entries in the omim database have been derived from the swissprot database (48) . it is also possible to obtain details of genes involved in genetic disorders through the genelink table provided at ensembl database (46) . the genelink table indicates the association of human proteins to omim identifiers. we were able to associate 6257 unique protein sequences, which have one or more reference, to the omim database. the number of omim entries referenced in swissprot is 6770, as in march 2002 release of the swissprot database. a possible reason for the difference in the numbers of genes could be that more than one genetic disorder entry in omim database is associated with a given protein. in the data set used by muller et al. (38) there were 5856 proteins linked to omim database entries. we have used profile-matching method rps-blast that matches a sequence to sequence-profile obtained from structural (pali -release 2.2) and functional domain (pfam -version 10.0) families. we used stringent e-value cut off of 3 6 10 75 in our search methods to ensure reliability of the domain association. this e-value cut-off has been extrapolated from the one reported by schaffer et al. (1999) (29) as well as based on the benchmarking (n. s. mhatre, b. anand and n. srinivasan, unpublished results) using the database of structure-based sequence alignments of similarly folded proteins. we have used hmmer based procedure against pfam hmm profiles with an e-value cut off of 10 72 to extract reliable domain association. subsequent to functional/ structural domain identification, all the sequences were subjected to tmhmm2.0 (49) in order to assign transmembrane helical regions. the functional and structural domain assignments for human proteome along with other organisms are made publicly available at: http://hodgkin.mbu.iisc.ernet.in/*human. the following sections discuss some of the interesting results derived by analysing this large dataset of human proteins. we could assign a total of 52,297 functional/structural domains to 21,835 (75%) human proteins out of 29,031 proteins encoded in the human genome. we also surveyed for transmembrane regions in human proteins, since this would suggest putative localization of these proteins to the membrane hence, could aid in function prediction. using tmhmm (49) we could identify transmembrane regions in 6777 gene products. of these, 5424 are found to be present in combination with extracellular or intracellular functional/ structural domains. the total number of residues covered in structural/ functional domain and transmembrane region assignments are about 42% of the proteome. these functional/structural domain assignments would indicate probable biochemical functions for the assigned proteins, which could be useful for biological function prediction. a total of 7196 human proteins with no domain assignment, hence with no function or cellular localization information, could form an interesting set for experimental exploration for their properties and biological roles. the association of gene products with structure can give valuable insights, since structural information provides molecular details of the function of a protein. the structural domain assignment will also help in prioritizing the target for structural genomics consortium by indicating gene products with no structural predictions. with a view to enhance structural information present in human genome, we have used structural information as in pali profiles that is generated using structure-dependent sequence alignments of a large number of protein domain families, since the incorporation of 3-d structural information could aid in effective detection of remotely related proteins. using pali profiles alone, we could associate additional 1191 structural domains to 1076 human proteins that are remotely related. furthermore, we tried relating families with unknown structures to known structural families as in supfam database, which was developed by us earlier (26, 27) in order to enhance information on the structural content of human proteome. the supfam database relates two or more homologous protein families, of either known or unknown structure, using profiles derived from structure-based sequence alignments. integrating the relationships derived in supfam we could provide structural information for an additional *5% of domain families (fig. 1 ). these family assignments would increase known structural content in the genome. a total of 2669 pfam families are assigned in human genome, of which 1195 pfam families, have structural information documented in pfam. out of 1474 pfam families, apparently with no structural information, 129 families could be related to a family of known structure in supfam. there are now 1324 families (*50%) with structural information known directly or indirectly through relationships present in sup-fam (fig. 1 ). these 1324 unique families with structural information are present in 40,947 domains, hence would provide further insights into their functions. a total number of 52,082 functional domains could be assigned to the human proteins. these assigned functional domains belong to 2669 sequence/functional families of the pfam database (42) . we have surveyed for the most commonly occurring pfam families in human genome. fig. 2 shows the most frequently occurring functional domain families in the human genome. the most frequently occurring family is the zf-c2h2 family, which is a classical zinc-finger domain with very short length (typically 25 residues). identification of such a family with short motifs using bioinformatics tools could be unreliable. hence, we did not consider them in our analysis. the other most frequently occurring globular protein family is protein kinase. it was previously shown that protein kinases occur with typical and atypical combination of domain families in the gene products encoded in human genome. these kinase domain-containing proteins are involved in a wide variety of biological roles (50) . among the other most frequently occurring pfam families, the majority are involved in or in part responsible for protein-protein interactions (immunoglobulin, ankyrin repeat, tpr domains), cell attachment adhesion function (fibronectin, collagen, cadherin domains), signalling function (ph, sh3, c2 domains), nucleic acid binding function (zf-c2h2, homoeobox, rrm domains). a considerable number of human proteins are characterized by short lengths, although they match significantly with protein domain families which are typically much longer. there are 129 functional families, associated in 1144 human proteins, apparently with no structural information but could be associated to distantly related families of known structures using relationship described in supfam. these 1144 proteins have 1184 number of domains. most of these 129 families correspond to enzymes. from the structural genomics perspective, structure association for 129 pfam families meant that clues about structure and function could be extended for 1144 proteins. some of the pfam protein families are known to be characteristic of prokaryotic organisms or viruses only. however members of some of these families from human genome could be identified from the current analysis and these families are referred to as atypical families. we could associate 16 bacterial specific families and 18 viral specific families to 41 and 188 human proteins respectively. the list of bacterial and viral specific families, identified in human, along with associated gene products in human genome are listed in table 1a and 1b respectively the complete list of proteins with the region of pfam domain assignment is made available at http:// hodgkin.mbu.iisc.ernet.in/*human. the assigned domain family includes for example cobalamin biosynthesis protein, minor capsid protein, bacteriophage lambda head decoration protein. such functions have not been shown before to be present in humans. there are two possible explanations that could be drawn in the context of occurrence of the bacterial and viral specific families in human genome. first explanation is that the superfamily relationship exists between the assigned bacterial or viral domain families and the corresponding eurkaryotic domain families as the sequence similarity of these domains with human proteins is low, while significant. these regions in the human proteins could have diverged significantly and sequence data corresponding to these families in other eukaryotes is currently lacking. an alternative possibility is horizontal gene transfer of these bacterial/viral specific families to humans. we surveyed the human genome for the occurrence of specific pfam families, which are known to be present only in eukaryotes. such eukaryote specific families are known to be involved in specific functions in eukaryotes. out of 2229 eukaryote specific pfam families, we could not associate 1054 eukaryotic specific families to the human proteome. further, we assessed reasons for the absence of these eukaryotic specific families in human genome. most of these families are organism or lineage specific. some of them have no known functions and other, as mentioned below, are involved in functions not required or present in humans, hence not identified in the human proteome. the probable reasons for absence of eukaryotic specific pfam families in human genome are: (1) class of toxin families (which also includes snake and scorpion toxins) (2) families that are unique to the plant kingdom, like seed storage class of proteins, potato inhibitor and plant disease resistance response protein. the zf-c2h2 has been excluded from this histogram due to low reliability in assignments of short domains. protein srb, c. elegans sre g protein-coupled chemoreceptor and c. elegans srg family integral membrane protein. this analysis showed that human proteome has eukaryotic specific families (1175) which are involved in eukaryotes-like functions. however, absence of some of the eukaryotic specific families could be explained from the observations that such biochemical functions are undesirable for human or they are highly specific to lower eukaryotes. the pfam families, without known 3-d structure, could be clustered into sequence superfamilies and such superfamily relationships are documented in the supfam database. in the current release of supfam, 96 of the 3904 pfam families, with no structural information, could be clustered into 39 new potential superfamilies. it is expected that members of all the families in each new potential superfamily would share the same fold and might have gross similarity in their functional properties. these relationships could help in prioritizing the target for structural genomics, since the 3-d structural determination of one of the representative member in each superfamily would result in 39 structures that can serve as framework models. using these sequence superfamilies information we could identify 18 of the 39 sequence superfamilies in the human genome. the list of these new potential superfamilies with their constituent families identified in human genome is listed in table 2 . the 18 sequence superfamilies identified in human genome consist of 25 pfam families, with no known 3-d structure for any of their members. there are 371 domains belonging to these 18 new potential superfamilies that could be assigned to the 367 unique gene products in human genome. hence, an experimental structure for 18 domains or proteins one each from these superfamilies could provide templates for interpreting the functions of other members in the superfamily. this results in substantial reduction (from 371 to 18) in the number of 3d-structures to be determined experimentally in order to get clues about their functions experimentally. these superfamilies may be considered as priority targets for structural genomics initiatives in order to improve the coverage of structural information for the human proteins. the nature of the superfamily relationships for some of the new potential sequence superfamilies that are identified in human genome is discussed further. this superfamily consists of three families namely patched domain, acr_tran and secd_secf domain families. the acr_tran family is an integral membrane protein family whose members are known to be involved in drug resistance in bacteria (51) . the other family in this superfamily, patched domain, is a receptor for the morphogene sonic hedgehog and transduces hedgehog signals (52) . this secd and secf family consists of various prokaryotic secd and secf protein export membrane proteins (53) . we could identify 16 human proteins with patched family domain assigned. the functional and structural elucidation of other two families could be could be extended to patched domain because of superfamily relationships. this superfamily has four families cluster together viz. sugar_tr, oatp_c, duf791 and duf894. the sugar_tr family is single-polypeptide capable only of transporting small solutes, such as sugar, in response to chemiosmotic ion gradients and lies in uniporter-symporter-antiporter family (54) . oatp_c is eukaryotic organic-anion-transporting polypeptides that vary in tissue distribution and substrate specificity (55) . the functions of dufs (domains of unknown function) are not known. we could associate sugar_tr and oatp_c domains to 85 and 16 gene products respectively. this superfamily constitutes methyltransf_4 and trna_u5-meth_tr pfam families. both families have methyltransferase activity, however, the trna_u5-meth_tr family is involved in methylation of t-rnas (56) . we could identify 1 and 5 human homologues of methyltransf_4 and trna_u5-meth_tr respectively. the gde_c and duf608 pfam families could be clustered in this superfamily. the gde_c family is glycogen branching enzyme and has glucosidase activity (57) . we could identify gde_c and duf608 in 3 and 2 human proteins respectively. from, this relationship it could be suggested that duf608 might have glucosidase-like activity. the 3-d information provides precise molecular details about the function of the protein. the association of gene products encoded in human genome to 3-d structures would assist in providing further insights into their function. the databases of protein structures in which domains with similar 3-d architecture are grouped together could be used for such structural analysis. we have used pali database derived from scop for the present analysis. scop classifies protein domain having high sequence and structural similarity into families. the families are grouped in superfamilies when they share similar functional features and have an evolutionary common ancestor. superfamilies are grouped in fold when major secondary structures are topologically equivalent with similar topological connectivity. the assignment of structural domains to the proteins would aid in the investigation of the preponderance of superfamilies and fold in the human genome. using the various search procedures we could associate 38,017 structural domains to 16,459 human proteins either directly or by using the sequence superfamily relationships as described in supfam. further, we classified these domain assignments at the level of fold or superfamilies to understand the most commonly used function present in human genome. we analyzed the most commonly occurring superfamilies in human proteome. the figure 3 shows the top few superfamilies along with their extent of representation in the human genome. this distribution of superfamilies is similar to the one obtained by muller et al. (38) . the most commonly occurring superfamily is c2h2 zinc finger, followed by immunoglobulin domain. because of the short length of c2h2 zinc-finger domain and associated low complexity region, there is bias in identification of these domains. hence, all the gene products having this domain might not have zinc-finger like function and we excluded them from our present analysis. p-loop containing nucleotide triphosphate hydrolases domain is the next most represented superfamily and it is involved in many different critical biological functions such as cell growth, differentiation, repair and modification of dna, transcription, etc. this superfamily comprises various atpases and gtpases that are essential for cell survival. for example gtpases include elongation factors, ga subunit of the heterotrimeric g-proteins that are absolutely critical in major cellular processes. the other superfamilies among frequently occurring superfamily are involved in various functions in the cell as cellular signalling (protein kinase-like, ph domainlike), cell adhesion (cadherin, fibronectin type iii), nucleic acid binding function (rna-binding domain). interestingly, 'family a-g protein-coupled receptor-like' superfamily that consists of many receptors as other most populous superfamilies. the complete list of structural superfamilies that occur in human genome with their respective frequency of occurrence in human genome is provided at http://hodgkin.mbu.iisc.ernet.in/*human. figure 4 shows population distribution of few most populated folds, which occur in the human proteome. figure 5 shows the 3-d folding patterns in the most populated folds. the c2h2 and c2hc zinc finger is the most frequent occurring fold in human genome. for the reasons mentioned before, we have excluded c2h2 and c2hc zinc finger from this analysis. ferredoxin-like fold has the highest number of superfamilies in the human proteome as well as in scop. however, 16 of the superfamilies occur in the human proteome out of the currently known 36 superfamilies in the ferredoxin fold. ribonuclease h-like motif fold has six out of currently known seven superfamilies in the human proteome. except the superfamily of hypothetical protein mth1175 from methanobacterium, all other superfamilies of ribonuclease h-like motif occur in the human proteome. these superfamilies are actin-like atpase domain, creatinase/ prolidase n-terminal domain, ribonuclease h-like, translational machinery components, dna repair protein muts domain ii and methylated dna-protein cysteine methyltransferase domain. this could be expected as the nucleic acid binding/related superfamilies are highly represented in the human proteome. the complete list of protein structure folds that occur in human genome with their respective frequency of occurrence in human genome is provided at http://hodgkin. mbu.iisc.ernet.in/*human. the sequence to profile matching procedure described in the methods section resulted in the association of at least one functional domain family in pfam database to the 4864 proteins of swissprot database linked to omim entries (77.8% of the total of 6257 proteins in the omim database). the remaining 1393 disease-related proteins could not be associated to any functional or structural domain family. hence these proteins could be high priority targets in structural genomics to provide further insights into the molecular basis of the function of these proteins. these 4864 proteins contain 8431 functional and structural domains from 1288 pfam families. it is important to note that 6491 domains out of 8431 domains could be linked to 802 pfam families with known structural information. in terms of the amino acids coverage in these domains about 51% of the amino acids in the proteins are in the functional or structural domain (58) assigned regions in these 4864 proteins. figure 6 shows the distribution of the domains in the top 15 most populous families in the proteins, these families contain 2551 domains which is about 30% of all assigned domains. protein kinase is the most frequently occurring domain family in the human disease proteins. among the top 15 most populous families 14 have known structural information. the most populous structural superfamily that is assigned to the proteins is p-loop containing nucleotide triphosphate hydrolases and this has 361 domains in it. the largest representations in the p-loop superfamily come from the domain families like ras, helicase_c, and dead. the list of highly populated superfamilies has much in common with the analogous list generated by muller et al. (38) . much of these highly represented superfamilies are associated with regulatory roles in development, differentiation and proliferation. further analysis revealed that there are 33 proteins that have been assigned additional functional domains apart from previously assigned functional domains. these 33 proteins are listed in table 3 . these newly assigned domains may play a significant role in furthering our understanding of overall functions of these proteins. using various methods of domain association we could associate at least one domain to about 75% of gene products in the human genome. interestingly, the assignments of remote homologues to the human proteins revealed the occurrence of some of the viral and bacterial specific proteins in the human genome. among most commonly occurring functional family, protein kinases is one of the most frequently occurring domains, and the p-loop containing nucleotide triphosphate hydrolases is the one of the most represented superfamily. the assignment of 1184 domains to families with apparently no structural information to structural families would aid in the prioritization of targets for structural genomics of human genome. the assignment of new domains in addition to previously identified domains to the proteins possibly linked to genetically inherited human diseases could form a basis for the experimental verification of the roles of these domains as well as the molecular basis of disease. initial sequencing and analysis of the human genome the sequence of the human genome predicting function: from genes to genomes and back structural assignments to the mycoplasma genitalium proteins show extesive gene duplication and domain rearrangements the cath extended protein-family database providing structural annotations for genome sequences trends in protein evolution inferred from sequence and structure analysis studying genomes through the aeons: protein families, pseudogenes and proteome evolution predicting protein function by genomic context: quantitative evaluation and qualitative inferences basic local alignment search tool improved tools for biological sequence comparison distant homology recognition using structural classification of proteins the relation between the divergence of sequence and structure in proteins protein evolution. how far can sequences diverge? how representative are the known structures of the proteins in a complete genome? a comprehensive structural census homology-based fold predictions for mycoplasma genitalium proteins the relationship between protein structure and function a comprehensive survey with application to the yeast genome enhanced genome annotation using structural profiles in the program 3d-pssm predicting structures for genome proteins from protein structure to function genomic-scale comparison of sequence-and structure-based methods of function prediction: does structure provide additional insight? profile analysis: detection of distantly related proteins hidden markov models in computational biology. applications to protein modeling fold and function predictions for mycoplasma genitalium proteins applying motif and profile searches gapped blast and psi-blast: a new generation of protein database search programs supfam-a database of potential protein superfamily relationships derived by comparing sequence-based and structure-based families: implications for structural genomics and function annotation in genomes supfam: a database of sequence superfamilies of protein domains enhanced functional and structural domain assignments using remote similarity detection procedures for proteins encoded in the genome of mycobacterium tuberculosis impala: matching a protein sequence against a collection of psi-blast-constructed position-specific score matrices profile hidden markov models benchmarking psi-blast in genome annotation identification of related proteins on family, superfamily and fold level genthreader: an efficient and reliable protein fold recognition method for genomic sequences a method to identify protein sequences that fold into a known three-dimensional structure comparative genome and proteome analysis of anopheles gambiae and drosophila melanogaster the identification of functional modules from the genomic association of genes genecensus: genome comparisons in terms of metabolic pathway activity and protein family sharing structural characterization of the human proteome protein fold recognition using sequence profiles and its application in structural genomics functional and structural genomics using pedant genome sequences and great expectations the pfam protein families database pali-a database of phylogeny and alignment of homologous protein structures integration of related sequences with protein three-dimensional structural families in an updated version of pali database scop: a structural classification of proteins database for the investigation of sequences and structures the ensembl genome database project omim passes the 1000-disease-gene mark the swiss-prot protein sequence database and its supplement trembl in 2000 a hidden markov model for predicting transmembrane helices in protein sequences the repertoire of protein kinases encoded in the draft version of the human genome: atypical variations and uncommon domain combinations acrab efflux pump plays a major role in the antibiotic resistance phenotype of escherichia coli multiple-antibiotic-resistance (mar) mutants the drosophila patched gene encodes a putative membrane protein required for segmental patterning secd and secf are required for the proton electrochemical gradient stimulation of preprotein translocation major facilitator superfamily. microbiol molecular identification and characterization of novel members of the human organic anion transporter (oatp) family dual function of the trna (m(5)u54) methyltransferase in trna maturation identification of the catalytic residues of bifunctional glycogen debranching enzyme the human serum paraoxonase/arylesterase gene (pon1) is one member of a multigene family setor: hardware lighted three-dimensional solid model representations of macromolecules this research is supported by the award of senior fellowship to n.s. by the wellcome trust, london as well as by the computational genomics initiative supported by the department of biotechnology, new delhi. s.b. and s.b.p. are supported by the wellcome trust, london and csir, new delhi respectively. key: cord-001435-ebl8yc92 authors: hoppe, sebastian; bier, frank f.; von nickisch-rosenegk, markus title: identification of antigenic proteins of the nosocomial pathogen klebsiella pneumoniae date: 2014-10-21 journal: plos one doi: 10.1371/journal.pone.0110703 sha: doc_id: 1435 cord_uid: ebl8yc92 the continuous expansion of nosocomial infections around the globe has become a precarious situation. key challenges include mounting dissemination of multiple resistances to antibiotics, the easy transmission and the growing mortality rates of hospital-acquired bacterial diseases. thus, new ways to rapidly detect these infections are vital. consequently, researchers around the globe pursue innovative approaches for point-of-care devices. in many cases the specific interaction of an antigen and a corresponding antibody is pivotal. however, the knowledge about suitable antigens is lacking. the aim of this study was to identify novel antigens as specific diagnostic markers. additionally, these proteins might be aptly used for the generation of vaccines to improve current treatment options. hence, a cdna-based expression library was constructed and screened via microarrays to detect novel antigens of klebsiella pneumoniae, a prominent agent of nosocomial infections well-known for its extensive antibiotics resistance, especially by extended-spectrum beta-lactamases (esbl). after screening 1536 clones, 14 previously unknown immunogenic proteins were identified. subsequently, each protein was expressed in full-length and its immunodominant character examined by elisa and microarray analyses. consequently, six proteins were selected for epitope mapping and three thereof possessed linear epitopes. after specificity analysis, homology survey and 3d structural modelling, one epitope sequence gavvalsttfa of kpn_00363, an ion channel protein, was identified harboring specificity for k. pneumoniae. the remaining epitopes showed ambiguous results regarding the specificity for k. pneumoniae. the approach adopted herein has been successfully utilized to discover novel antigens of campylobacter jejuni and salmonella enterica antigens before. now, we have transferred this knowledge to the key nosocomial agent, k. pneumoniae. by identifying several novel antigens and their linear epitope sites, we have paved the way for crucial future research and applications including the design of point-of-care devices, vaccine development and serological screenings for a highly relevant nosocomial pathogen. klebsiella pneumoniae is a gram-negative, facultative anaerobic rod-shaped bacterium belonging to the family of enterobacteriaceae. it is a non-motile, lactose fermenting organism, which has been known to cause severe lung damage if aspirated. other clinical symptoms common with klebsiella pneumoniae infections encompass urinary-tract-infections (uti) and wound infection potentially causing bacteremia and septicemia [1] . in recent years it has become one of the most persistent nosocomial agents, especially due to the increasing distribution of multiple resistances to antibiotics. the most prominent group of k. pneumoniae harboring a broad resistance spectrum incorporates the extendedspectrum beta-lactamase (esbl) expressing strains. due to their outstanding clinical relevance and occurrence as agents of nosocomial infections, it is highly desirable to rapidly detect the presence of these organisms and to find suitable measures to effectively counter any infection in the early stages [2] . while numerous dna-based typing methods exist [3] , these are often laborious and time-consuming. in contrast, user-friendly point-of-care devices applying antigen-antibody interactions would allow for a quick and reliable detection [4] . nevertheless, the knowledge about suitable antigens to be incorporated into such a device is scarce. thus, we have utilized a method to quickly assess novel immunogenic proteins of k. pneumoniae, which might serve as potential targets for a diagnostic tool. recently, we have successfully employed this approach to unveil immunogenic proteins for both campylobacter jejuni [5] and salmonella enterica [6] . concisely, prokaryotic cdna libraries are created, fusion proteins expressed and these constructs covalently attached to microarray surfaces via the use of a halotag (promega). subsequently, the microarrays are screened using polyclonal antibodies reactive to the donor species of the cdna. therefore, this approach enables a broad and reliable screening, while reducing cross-reactivity and background to a minimum [7] due to the highly selective and covalent binding of halotag to its specific ligand [8] . moreover, the high specificity of said interaction renders excessive protein purification steps normally encountered in microarray-based screening applications [9] obsolete. thus, it is a faster and more direct approach as spotting combines both the deposition of samples and enables the immediate purification by a simple washing step. in connection with the above screening approach, cdna derived expression libraries were generated to express a vast number of proteins from k. pneumoniae. these libraries offer the advantage of a smaller sample size as compared to genomic libraries. this is mainly due to genomic libraries encompassing highly truncated dna fragments as well as dna representing regions that do not encode proteins in the original organism. contrastingly, cdna libraries generated via the in-fusion smarter directional cdna library construction kit (clontech) have been known to lead to longer fragments and possess a high abundance of full-length clones [10] . thus, the overall number of clones required for screening is substantially reduced. still, prokaryotic cdna libraries display one main disadvantage. as bacterial mrna rarely contains a poly(a)-tail [11, 12] , isolation of the mrna from other rna species is tedious. while some methods exist to isolate the mrna prior to reverse transcription [13, 14] , we rather chose to normalize the cdna afterwards. consequently, the entire rna of k. pneumoniae was used for reverse transcription. next, normalization was performed using a duplex-specific nuclease [15] . this treatment has been shown to effectively reduce the highly abundant rrna derived cdna portions without implementing a bias, thus altering the overall composition of the cdna in favour of the mrna derived molecules [16] . in addition to this, ligation-independent cloning and electroporation were employed to enhance cloning efficiency [17] . in this work, we have screened 1536 clones to detect the presence of previously unknown immunogenic proteins. in summary, we identified 14 proteins that have not been described as immunogenic before. after further analyses and epitope mapping of several promising candidates, three proteins -a channel receptor, a putative transport protein and a hypothetical protein -revealed linear antigenic sites with varying specificity. our results offer the potential to be used for a wide array of applications including the generation of monoclonal antibodies that might be used in diagnostic examination. furthermore, several of the identified antigens or parts thereof might be suitable for vaccine development, either used in passive or active immunization. additionally, many virulence-associated factors harbor some immunogenic potential. thus, identification of novel immunogenic proteins might elucidate proteins involved in the pathogenicity and virulence of k. pneumoniae. consequently, this advances the understanding of this pathogen and illuminates new approaches to counter infections. the mean rin value of rna used for cdna generation and library construction was calculated to 7.361.3 (n = 5). after successful normalization, the cdna was cloned to create an expression library. of this library, 1536 different clones were screened using halolink slides. the known immunogenic proteins [18] , outer membrane protein 1a ompf (uniprot/swiss-prot: a6t721) and outer membrane protein 3a ompa (a6t751), were used as positive references, whereas both dihyroorotase pyrc (a6t7d6) and glyceraldehye-3-phosphate-dehydrogenase gapa (a6t8l2) served as negative references. after screening, the signal intensities of each sample were compared to the references and grouped accordingly. generally, three distinct groups were established. group i represents samples exceeding the intensities of both positive reference proteins, group ii encompasses samples ranging in between the different intensities of ompa and ompf, whereas group iii entails those samples that albeit showing higher intensities than the negative controls, are below both ompa and ompf. here, approximately 25% of samples belong to group i, while ii and iii contain 7% and 14% respectively. the remaining samples, 54%, fall into the same range as the negative protein references. consequently, 192 clones or 12.5% of the entire screening approach were selected for sequencing. these clones were all taken from group i. after sequencing, artificial fragments and known antigens were discarded to reveal potentially novel immunogenic proteins, see table s1 for a list of the initial identification via sequencing. some of the inserts were too heavily truncated to reliably identify the corresponding gene. moreover, in some cases subcloning the initially identified genes in full-length failed even after numerous attempts. therefore, those genes were removed from further characterization as the translated peptide fragments were too short to be significant. despite these limitations, 14 potentially novel immunogenic proteins were expressed in full length and used for further characterization. the 14 antigen candidates are summarized in table 1 including their locus tag, protein name, length and size in kda. the difference in immunodominant behaviour was assessed by ten independent microarray and elisa analyses employing two different antibodies. in summary, table 1 reveals the resulting mean q values and corresponding errors (n = 10). a q value above one represents higher intensity signals than ompa, the used positive reference. the highest mean q value was obtained for kpn_02199, a coa-linked acetaldehyde dehydrogenase and irondependent alcohol dehydrogenase; pyruvate-formate-lyase deactivase with 2.1660.39. however, as this protein is highly conserved within all bacteria, it was not considered for further analyses regarding specific antigenic sites. the same holds true for kpn_03668, the 50s ribosomal protein l11 methyltransferase. although a q value of 1.7060.38 was attained, the highly conserved nature of this protein renders it unsuitable within a diagnostic approach. contrastingly, kpn_00466 and kpn_01584, two hypothetical proteins, were selected for future investigations as little is known about the function of these proteins. their mean q values were 1.1560.22 and 1.9060. 36 respectively. moreover, kpn_01584 shows some homology to a known superantigen of yersinia pseudotuberculosis according to the ncbi protein cluster database [19] . thus, the potential utilization of these proteins within a diagnostic tool seems plausible. in addition, kpn_01100, a histidine triad protein, kpn_02202, glucose-1-phosphate uridylyltransferase, kpn_00 363, a nucleoside channel and receptor of phage t6 and colicin k and kpn_00459, a putative transport protein, were selected for further investigations via epitope mapping. while kpn_01100 and kpn_02202 revealed mean q values above one with 1.3160.27 and 1.6960.56, respectively, kpn_00459 and kpn_00363 failed to reach this level. rather, the q values attained were 0.8360.27 and 0.7760.14. while the q values of the latter two proteins are lower compared to some of the other proteins identified, their functional descriptions and membraneassociation render them highly attractive within a diagnostic question. hence, they might be more easily accessible in a whole cell detection approach than cytoplasmic proteins. epitope mapping revealed the potential presence of linear epitopes within three of the six proteins investigated, namely kpn_00363, kpn_00459 and kpn_00466. for kpn_00363 seven distinct regions were identified with intensities above 1000 a.u., see figure 1 . these comprised the peptides 2 and 3, 16-18, 29-30, 45-47, 50-51, 60-63 and 69-71. the highest mean value for these peptides was obtained for peptide 16 with more than 10000 a.u. the positive reference, i.e. rabbit igg, reached a mean value of more than 30000 a.u., whereas the negative control mbp showed intensities of less than 500 a.u. as the adjacent peptides are identical in all but 4 amino acids in sequence, a consensus can easily be derived from two or more neighboring peptides. as figure 2 reveals, only the first two peptides, llaagavvalsttfa and gavvalsttfaagaa showed some specificity during specificity control assays. here, the arrays were incubated with additional antibodies reactive to different bacterial species, namely campylobacter jejuni, staphylococcus aureus, e. coli and s. enterica. all remaining peptides display similar intensities when these antibodies were used as compared to the original k. pneumoniae antibodies. however, for the first two peptides a significant difference is observed. the mean value for peptide 2 was approximately 6500 a.u. with k. pneumoniae antibodies and dropped to less than 400 a.u. with the other antibodies. a similar trend is discernible for peptide 3, where a drop from 1100 a.u. to less than zero is visible. thus, the consensus sequence gavvalsttfa is likely a suitable linear epitope featuring specificity for k. pneumoniae. for the putative transport protein, kpn_00459, three regions scored intensities above 1000 a.u. including peptides 50 and 51, 59 and 60 as well as 79-81, see figure 3 . the positive reference again reached a mean value above 30000 a.u., while the negative control levelled out at less than 500 a.u. after specificity assays, the peptides 59 and 60 revealed predominantly specific binding by the k. pneumoniae antibody as the mean values dropped from approximately 5800 and 2800 a.u. respectively, to less than zero for all other antibodies tested, see figure 4 . thus, a consensus for this epitope can be derived to giafgavelfd. contrastingly, the remaining peptides revealed equal intensities independent of the antibody used. finally, for the third protein, kpn_00466, one pair of adjacent peptides, namely 11 and 12, achieved mean values of approximately 2000 a.u. within close proximity to the positive reference, as depicted in figure 5 . additionally, peptide 16 displayed the highest overall mean value with more than 7000 a.u., however neither of the two neighboring peptides (15 and 17) attained values of any significance. thus, the presence of a linear epitope in that region is rather unlikely. besides, as specificity assays illustrated, none of the three peptides showed specific interaction to the k. pneumoniae antibodies alone, see figure 6 . rather, signal intensities with antibodies reactive to c. jejuni fell into the same scope. therefore, nonspecific binding to these peptides is probable. the remaining three proteins under investigation failed to disclose any linear peptide region with significant signal intensities to assume linear epitopes to exist. for a better understanding of the suitability of the identified linear epitopes, structural modelling was employed using the swiss model automated mode. for kpn_00363 a model was constructed based on the crystal structure of the bacterial nucleoside transporter tsx of e. coli. however, the derived model only spans residues 31 to 294 and as such does not contain the derived consensus sequence of the linear epitope gav-valsttfa. nevertheless, the model displays a beta barrel structure typical of outer membrane-spanning transport proteins, see figure 7 for details. the first residues of the derived model, starting at position 31, are marked in orange and present a coiled region outside of the beta barrel. therefore, the likelihood of the gavvalsttfa region to reside within the barrel is slim. rather, an extension of the truncated coil seems plausible. consequently, the potential accessibility of the identified linear epitope appears high. furthermore, the predicted 3d structure of the first part of kpn_00459 is displayed in figure 8 . contrary to kpn_00363, two models were devised for kpn_00459. however, only one encompasses the identified linear epitope giafgavelfd, which is highlighted in orange and is situated as part of an alpha helix and an adjacent loop. as the sequence is not enclosed by other residues or structures, good accessibility ought to be provided. in order to predict the potential specificity of the epitopes, homology analyses were performed. whereas giafgavelfd of kpn_00459 exhibits a broad homology throughout with all residues identical in closely related species, see figure s3 : homology of kpn_00459, gavvalsttfa of kpn_00363 features four residues likely specific for k. pneumoniae within this particular sequence. the variance of the latter epitope's sequence considering closely related species to k. pneumoniae is summarized in figure 9 . specifically, the following residues have been replaced: valine by leucine (position four), both threonine residues by serine residues (position 8 and 9) as well as alanine by threonine (position 11). the influence of sequence variations on the binding capacity of the identified epitope gavvalsttfa was subsequently examined by performing an alanine scan. its results are summarized in figure 10 . the original consensus epitope sequence shows a mean intensity of approximately 1000 a.u. alterations of the first, second, fifth, eighth and ninth residue lead to a significant drop of signal intensities. consequently, signals of less than 300 a.u., in close proximity to the negative control mbp with 50 a.u. are obtained. contrastingly, by altering residues three or six of the consensus sequence gavvalsttfa, i.e. replacing the first valine or leucine by alanine, the resulting mean signal intensities are significantly increased to more than 1700 a.u. for replacing valine and surpassing 5600 a.u. after changing leucine to alanine. additionally, specificity assays showed no significant signal intensity for antibodies reactive to closely related species, i.e. e. coli and s. enterica. incubation with antibodies reactive to either of those two bacterial species resulted in signal intensities in the neighbourhood of the negative control independent of sequence alterations, see figure 11 . this was also true for gavlalsssft and saalaltssft. the screening of a cdna based expression library of k. pneumoniae has successfully identified a number of novel, previously unknown immunogenic proteins. this is well in accordance to previous achievements of this method, which we have employed for the identification of both c. jejuni [5] and s. enterica [6] antigens. consequently, in this current study we aimed to detect suitable proteins for the specific identification of k. pneumoniae. furthermore, identification of specific antigens might improve treatment opportunities including the development of suitable vaccines [20] . finally, detecting proteins with yet unknown functional and structural information to be antigenic might be an indication of their potential involvement in the pathogenic nature of an organism. thus, these proteins might be suitable access points for future investigations to improve the understanding of the underlying pathogenicity and virulence of k. pneumoniae. within the 14 proteins only a subset was selected for further analysis using epitope mapping. the rationale for these selections was based on three distinct features: the resulting q value, homology as well as functional and structural properties. while the q value mirrors a normalized intensity compared to a positive reference, it does not fully account for differences in expression levels, misfolding and other factors which might have had an influence on the binding reaction and thus on the overall intensity. still, it facilitates to rank the proteins and increases the likelihood of a given protein to be immunogenic if its q value is significantly high. however, proteins with outstanding q values were not chosen for epitope mapping by default. rather, the known homology and corresponding distribution of each protein was carefully evaluated. thus, proteins like kpn_02199 and kpn_03668 albeit scoring high q values were exempt from epitope mapping as they display a broad spectrum of homologous proteins across bacterial species. finally, the intrinsic properties of each protein, if available, were closely scrutinized. therefore, proteins with hypothetical character were predominantly chosen, as information on them is confined. in addition, all types of membrane-associated proteins deserved a better look, as these type of proteins offer a more direct route and accessibility, which might be of utmost importance in a future rapid point-ofcare device detecting whole organisms. after epitope mapping, three of the six proteins under investigation revealed sites with potential linear epitopes. still, the remaining proteins displayed no such sequences. this is, however, in accordance to the general prevalence of structural epitopes in comparison with linear epitopes in nature. in fact, approximately 90% of all epitopes are conformational rather than sequential [21, 22] . despite these potential shortcomings of the used linear epitope mapping method, a number of intriguing linear epitopes have been identified. notably, three proteins, kpn_00363, kpn_00459 and kpn_00466, harbored sites with potential linear epitopes. further careful examination, however, excluded kpn_00466 from future applications as the identified epitope sequence were nonspecific for k. pneumoniae, which might be due to the conserved character of the protein within the family enterobacteriaceae. still, kpn_00466 is a membrane protein and upcoming investigations might help to elucidate applications using this protein either for prevention of k. pneumoniae infections or detection thereof. in contrast, the other two proteins displaying linear epitopes, kpn_00363 and kpn_00459, indicated some specificity with the antibodies tested and two linear consensus sequences could be derived, gavvalsttfa and giafgavelfd, respectively. while the former sequence is present in kpn_00363, an ion channel protein tsx, which is conserved among enterobacteriaceae, the latter sequence is part of kpn_00459, a hypothetical protein with high similarity to a cation:proton antiport protein and conserved among proteobacteria. consequently, the homology of giafgavelfd (kpn_00459) is high throughout different bacterial species, which renders the sequence inapt for specific k. pneumoniae detection and other applications. in contrast, gavvalsttfa (kpn_00363) displays alterations within this sequence for bacterial species other than k. pneumoniae. as small as these alterations appear, they appear to have a crucial effect on the binding of antibodies to the target sequence and thus benefit specificity. this is well in line with the experimental results that indicate no binding by antibodies reactive to other bacteria to this sequence or any of its alterations. moreover, alanine scanning revealed a number of residues to be paramount for antibody binding. consequently, replacing the glycine (position 1), alanine (position 2), alanine (position 5) or either threonine (positions 7 and 8) leads to a significant loss of antibody binding observable by a dramatic reduction in signal intensity. on the contrary, removing either the first valine or leucine and inserting an alanine residue as a replacement results in a significant increase in signal intensity, potentially hinting at an improved antibody binding. the reason for these effects remains nebulous; however, it is not caused by a simple change in secondary structure. this is apparent as both aavvalsttfa and gavvaasttfa, two sequences at opposite sides regarding signal intensity, are predicted to consist solely of an alpha-helix as compared to the original sequence, which is predicted to contain a beta-strand (position 1 to 7) and a helical part (position 8 to 11) by emboss garnier. furthermore, as the original sequence is fully constructed of uncharged amino acids, alterations via alanine or glycine do not change the overall charge of the peptide. consequently, the differences in signal intensity cannot be deduced to implementation or removal of charged residues. finally, changes in hydrophobicity albeit present are minimal at best and may not suffice to explain the observed characteristics. still, one has to bear in mind that the accuracy of prediction based tools is often lacking, especially for emboss garnier with an accuracy in the range of 65%. consequently, some of the predicted secondary structures might differ. despite some uncertainty as to the mechanistic cause of the altered peptides to behave as they did, one fact remains obvious. none of the peptides showed any significant binding to antibodies not raised against k. pneumoniae. therefore, after alanine scanning, gavvalsttfa remains a suitable candidate for specific k. pneumoniae detection or treatment. still, accessibility of the epitope is paramount to a quick diagnostic tool. thus, modelling of the 3d structure of the protein was performed. unfortunately, the 3d modelling only succeeded for the part of the protein, which does not contain said linear epitope sequence. nevertheless, the pronounced beta barrel structure of the channel protein is visible and the linear epitope sequence, albeit absent, likely to be an extension of the freely accessible n-terminal region outside the barrel structure. thus, accessibility of the epitope by an antibody might be pronounced without the need to enter the cell or channel. membrane proteins harboring a beta barrel like structure have been shown in bacteria to be exclusively found in the outer membrane [23] . moreover, the mobile coils on the extracellular sides of these membrane proteins are pivotal for their function or interaction with other molecules. this renders the identified sequence an intriguing target for antibody-based detection. additionally, channel proteins harboring beta barrel like structures have been shown to be immunodominant in other bacterial species such as salmonella, haemophilus influenzae, e. coli, neisseria meningitides, shigella dysenteriae and chlamydia trachomatis [24] [25] [26] [27] [28] [29] . on the contrary, the model for kpn_00459 encompasses the identified consensus sequence of the linear epitope giafga-velfd, which is part of a loop between two alpha helices. the abundance of alpha helices suggests the protein to span the inner membrane [23] . in this topological design, helices are mostly located within the membrane, notably as transmembrane domains, whereas loops are located either on the cytoplasmic or periplasmic side of the cell. when considering prediction based methods, such as s_tmhmm for topological domains [30, 31] or emboss antigenic [32, 33] , part of giafgavelfd is assumed to be extracellular. combined with the high flexibility and degrees of freedom of random coil structures, the likelihood for good accessibility is high rendering the sequence a potentially attractive target for whole cell detection despite its lack of specificity. furthermore, these findings support the 3d model and underline the accuracy of the specified structure. another key aspect in determining the accuracy of the 3d model prediction is a so-called z-score [34] . the z-score for the model of kpn_00363 is 24.285 and 28.895 for kpn_00459 respectively. although the values are significantly below zero that does not inevitably indicate models of poor accuracy. in fact, low z-scores are often obtained if the protein under investigation is membraneassociated. this is mainly due to the inverse physicochemical properties of membrane proteins in comparison to soluble ones. hence, the low z-scores are more likely induced by this effect than caused by an insufficient accuracy of the models. in conclusion, we have successfully identified several novel antigens of k. pneumoniae and identified three proteins potentially harboring linear epitopes. subsequently, we achieved to identify two sequences displaying specificity during experimental investigations; however, one of these is doubtful as homology analysis has revealed it to be highly conserved among a broad spectrum of bacterial species. still, gavvalsttfa of kpn_00363 was identified to be specific both experimentally and has shown four residues within the eleven amino acid sequence to occur predominantly in k. pneumoniae only. thus, the likelihood for this linear epitope to be specific is high. this assumption was confirmed by alanine scanning revealing a number of pivotal residues for antibody binding. moreover, it was unearthed that neither e. coli nor s. enterica antibodies were able to bind to any of the sequences, original and modified. subsequent investigations might help to further nurture the insight into the suitability of this peptide for diagnostic and therapeutic applications. thus, monoclonal antibodies ought to be devised to be used for affinity investigations via biacore and to determine kinetics. furthermore, monoclonal antibodies could be used within a potential diagnostic tool and after validation ought to be tested with whole bacteria. if these antibodies are able to specifically detect intact k. pneumoniae cells, the resulting antibody might well be suited for integration into a point-of-care device. in a different approach, the identified epitope sequence could easily be produced in large quantity. this peptide might serve some role in serological screenings, especially if it proves to be immunodominant. consequently, antibodies against this epitope might be present in a plethora of patient sera. finally, all proteins identified here might be suitable candidates for vaccine development independent of the existence of a linear epitope, as structural epitopes might well be present and antigenicity ensured. nevertheless, additional in-depth analysis is required to determine a number of the key aspects of vaccine development prior to use. as a donor of rna the fully-sequenced strain k. pneumoniae mgh78578 was grown on solid trypticase-soy-agar (tsa) for 24 h at 37uc under aerobic conditions. for rna isolation a liquid culture was prepared by inoculating 10 ml of brain-heart-infusion broth (bhi) with a single colony and incubated overnight at 37uc, 140 rpm. this overnight culture was used to inoculate a flask containing 100 ml fresh bhi medium. the cells were harvested 6 h after inoculation. for initial screening a rabbit polyclonal igg antibody to k. pneumoniae (acris ap00792pu-n) was used. for further micro-array analyses of a subset of candidate proteins, elisa and epitope mapping this antibody was used as well as rabbit polyclonal igg antibody to k. pneumoniae (abcam ab20947). the antibodies were generated with k. pneumoniae atcc 43816 serving as an immunogen. specificity assays were performed employing rabbit polyclonal igg antibody to c. jejuni (acris ap24002pu-n), s. aureus (fitzgerald 20c-cr1274rp), e. coli (abcam ab137967) and s. enterica (abcam ab35156). detection was achieved by usage of secondary antibodies. goat polyclonal antibody to rabbit igg conjugated with chromeo-546 (abcam ab60317) for fluorescent and antibody conjugated with horseradish peroxidase (abcam ab6721) for a colorimetric readout were applied where appropriate. the cells were harvested by centrifugation (20006g, 10 min) and the resulting supernatant discarded. the pellets were resuspended in fresh bhi medium. for stabilisation of the rna, 1 ml of rnaprotect bacteria reagent (qiagen) was added to 0.5 ml of bacterial suspension and processed according to the manufacturer's instructions. lysis was performed with 200 ml of lysis buffer (30 mm tris-cl, 1 mm edta, 15 mg/ml lysozyme, .12 mau proteinase k) by pipetting and vortexing for 10 s. after incubation, 700 ml buffer rlt and 500 ml 96% ethanol were added and the lysate applied to rneasy bacteria mini kit spin columns (qiagen) for rna isolation following the manufacturer's instructions. during this procedure an on-column dnase digest was performed using rnase-free dnase i solution (qiagen) according to the manufacturer's instructions. the isolated total rna was eluted in 50 ml of rnase-free water and its concentration and purity analyzed by nanodrop (peqlab) measurements. the quality of isolated rna was assessed using the rna 6000 pico kit and bioanalyzer 2100 (agilent). the total rna was diluted to a working concentration of 200-500 pg/ml. the analysis was performed following manufacturer's instructions and the rna integrity number (rin) calculated by the 2100 expert software (agilent). the rin is defined to fall into a range of 0 to 10, with a higher score indicating a more intact rna, whereas lower numbers are associated with degraded rna molecules [35] . in order to use bacterial mrna as a substrate in cdna synthesis, polyadenylation was mandatory. the tailing was achieved using the poly(a) polymerase tailing kit (epicentre) following the alternate protocol offered by the manufacturer. briefly, up to 10 mg of total rna were combined with 2 ml poly(a) polymerase reaction buffer, 2 ml 10 mm atp, 0.5 ml riboguard rnase inhibitor and 1 ml poly(a) polymerase (4 u) in a total reaction volume of 20 ml. the reaction was incubated for 20 min at 37uc, terminated by the addition of 1 ml 0.5 m edta and purified by rneasy mini kit (qiagen) following manufacturer's instructions. yield and purity were determined by nanodrop measurements. for cdna synthesis the in-fusion smarter directional cdna library construction kit (clontech) was used according to manufacturer's instructions with slight modifications. 3.5 ml total, polyadenylated rna were mixed with 1 ml of 39 in-fusion smarter cds primer, heated first for 3 min at 72uc and then incubated for additional 2 min at 42uc. after addition of 5.5 ml mastermix (2 ml 5x first strand buffer, 0.25 ml 100 mm dtt, 1 ml 10 mm dntps, 1 ml 12 mm smarter v oligonucleotide, 0.25 ml rnase inhibitor and 1 ml smartscribe reverse transcriptase) the tubes were incubated for 90 min at 42uc. the reaction was terminated at 68uc for 10 min. for second strand cdna synthesis two 2 ml aliquots of first strand reaction were used in long distance pcr using phusion polymerase (finnzymes). each pcr reaction was comprised as follows: 2 ml first-strand reaction, 70 ml rnase-free water, 20 ml 5x phusion hf buffer and 2 ml each of dntp mix (10 mm), 59 figure 6 . specificity binding analysis of epitope peptides of kpn_00466. bar chart representing the mean relative fluorescence intensities (n = 10) of each peptide potentially harboring a linear epitope site after incubation with polyclonal antibodies reactive to k. pneumoniae (green) and c. jejuni (orange). none of the peptides shows a peculiar specific interaction; rather signal intensities are in the same vicinity for each peptide independent of the antibody used. this indicates mainly non-specific binding to occur. doi:10.1371/journal.pone.0110703.g006 pcr primer ii a (12 mm), 39 in-fusion smarter pcr primer (12 mm) and phusion polymerase with a total reaction volume of 100 ml. the pcr reactions were subjected to the cycling program with 98uc for 1 min as initial denaturation followed by 15 cycles of 10 s denaturation at 98uc, 30 s of primer annealing at 65uc and 6 min extension at 72uc. for improved pcr results optimization was performed as follows; 30 ml of the 15 cycle experimental tube were transferred to a separate pcr tube, cycling commenced and aliquots of 5 ml each were collected after 15, 18, 21, 24 and 27 cycles total. the different cycles were compared by gel electrophoresis and the experimental tubes subjected to additional cycles if necessary. finally, pcr reactions were purified using the qiaquick pcr purification kit (qiagen). the purity and yield of each reaction were analyzed by nanodrop measurements. normalization of double-stranded cdna was achieved with the trimmer-2 cdna normalization kit (evrogen) to reduce the number of cdna molecules derived from rrnas. briefly, 12 ml of cdna (approx. 100 ng/ml) were mixed with 4 ml of 4x hybridization buffer. for the trimming reaction 4 ml of this mixture were distributed to four different pcr tubes and overlaid with a drop of pcr-grade mineral oil. after centrifugation (130006g, 2 min), the tubes were incubated for 2 min at 98uc followed by 5 h at 68uc. next, pre-heated (68uc) duplex-specific nuclease (dsn) master buffer was added to each tube and incubation prolonged for 10 min. dsn was added to the first three tubes in decreasing concentrations -1 u/ml, 0.5 u/ml and 0.25 u/ml -with the fourth tube receiving dsn storage buffer and no enzyme as a control reaction. the incubation prolonged for 25 min at 68uc. after addition of 5 ml dsn stop solution and subsequent incubation for 5 min at 68uc, the tubes were placed on ice. the chilled reaction was diluted by addition of 25 ml sterile, rnase-free water. for amplification of normalized cdna, 1 ml of each reaction was used as template in pcr. each pcr reaction contained 1 ml of template from the normalization reaction, 33 ml nuclease-free water, 10 ml 5x phusion hf buffer, 1 ml 10 mm dntp mix (neb), 2 ml of each primer 59 pcr primer ii a (12 mm), 39 in-fusion smarter pcr primer (12 mm) and 1 ml phusion polymerase. the pcr was performed with initial denaturation at 98uc for 1 min and seven cycles of denaturation at 98uc for 10 s, primer annealing at 65uc for 30 s and extension at 72uc for 3 min, respectively. for optimization, the control tube was subjected to 7, 9, 11, and 13 cycles with 12 ml aliquots taken every two cycles. the optimization samples were analyzed by gel electrophoresis (1% agarose, tae, 100 v) and the optimal cycle number determined. the remaining three tubes were subjected to 9+ x cycles with x being the differential of the optimized cycles to the originally performed seven cycles. after the second pcr, the experimental reactions were compared to the optimal control reaction using gel electrophoresis as above. reactions showing a successful normalization were combined and used in a third pcr reaction. after the final pcr, the reactions were purified by qiaquick pcr purification kit. a major feature of the given model is the prominent beta barrel structure that originates from the abundance of beta strands. this is a typical feature of transport and channel proteins spanning the outer bacterial membrane. contrastingly, the identified linear epitope gavvalsttfa is located at the very beginning of the protein and thus not included in the given model. however, it is likely an extension of the truncated n-terminal region marked in orange. doi:10.1371/journal.pone.0110703.g007 figure 8 . 3d model of predicted structure of kpn_00459. the model was predicted using the automated mode of the swiss model application by expasy (university basel). as a template the crystal structure of a na(+)/h(+) antiporter nhaa of e. coli was used. the resulting model spans residues 12 to 390 of the full-length protein and was subsequently dyed using the chimera 1.7 software. coils are depicted in light green, beta strands in purple and alpha helices in blue. the potential linear epitope giafgavelfd is highlighted in orange. it comprises part of an alpha helix, a connective coil and the start of the next alpha helix. doi:10.1371/journal.pone.0110703.g008 in-fusion cloning and cloning vector for cloning pfn18a (promega) was used as a vector, as it features a n-terminal encoded halotag fusion protein, which allows for specific and covalent binding to a unique ligand, thus reducing background and minimizing cross-reactivity in immunoassays with halolink microarrays harboring the ligand on its surface. first, the vector needed to be linearized to be used with the in-fusion cloning technology. this was achieved by reverse pcr using ifs 18a for (59 ttgataccactgcttttc-catggcgatcgcgttatc 39) and ifs 18a rev (59 tctcatcgtaccccgtgtttaaacgaattcgggctcg 39). each reaction contained 2 ml each of 1:10 diluted pfn18a (10 ng/ml) and the two primers, 10 ml 5x phusion hf buffer, 1 ml 10 mm dntps, 0.5 ml phusion polymerase and 32.5 ml nuclease-free water to reach a total reaction volume of 50 ml. the pcr was run using a 25 cycle two-step program with 98uc denaturation for 10 s and 4 min extension at 72uc. after completion, 2 ml of dpni (20 u/ml) were added to the reaction and incubated at 37uc for 1 h. the presence of a single band was checked by gel electrophoresis and the remaining reaction purified by qiaquick pcr purification kit. cloning of normalized cdna and linearized pfn18a vector was performed following the manufacturer's instructions within the in-fusion smarter directional cdna library construction kit (clontech). electroporation 2 ml of the cloning reaction were mixed with 25 ml of electrocompetent acella e.coli cells (mobitec), a bl21 derivative, figure 9 . homology of linear epitope sequence gavvalstffa of kpn_00363. the sequence derived from k. pneumoniae mgh 78578 was used as a reference. identical residues are marked by dots, gaps by a horizontal dash and differences by the single letter amino acid code. seven of nine k. pneumoniae strains show identical epitope sequences, while two strains display changes in two residues. threonine replaces alanine at position 11, a change observed not only in these two strains but in almost all other bacteria within the list. additionally, threonine at position 9 is substituted by either serine or phenylalanine. bacteria other than k. pneumoniae show an additional number of amino acid substitutions, most prominently leucine for valine at position 4 and serine for threonine at position 8. in s.enterica the changes become more pronounced. glycine at position 1 is replaced by serine, valine at position 3 replaced by alanine and threonine inserted for serine at position 7. in some rare cases, other residues have also been substituted, e.g. valine replaces alanine at position 2 in shigella dysenteriae. doi:10.1371/journal.pone.0110703.g009 figure 10 . alanine scan of gavvalsttfa of kpn_00363. box-whisker plot (n = 12) of gavvalsttfa after alanine/glycine scanning. the box comprises 50% of the data, while the whiskers enclose 98%. the median is represented by a small horizontal line and the mean by a small rectangle. rabbit igg served as a positive reference, whereas mbp was used as a negative control. if alanine was present in the original present it was replaced by glycine, otherwise each amino acid was stepwise replaced by alanine. additionally, gavlalsssft and saalaltssft were included as they resemble sequences present in e. coli and s. enterica. switching glycine (position 1), alanine (positions 2 or 5), or threonine (positions 8 and 9) to alanine or glycine, results in a significant drop in signal intensities to levels below or at the negative control. in contrast, substituting valine (position 3) or leucine (position 6) by alanine, leads to an increase in signal intensities to 1700 a.u. and more than 5600 a.u., respectively. note the different axis scales prior and after axis break at 2000 a.u. doi:10.1371/journal.pone.0110703.g010 and electroporated in 1 mm cuvettes using the easyject plus electroporator (peqlab). conditions for electroporation were as follows: voltage = 1400 v, capacity = 25 mf, resistance = 200 v and a pulse duration of 5 ms. the electroporated cells were added to 970 ml of super optimal broth with catabolite expression (soc) and incubated at 37uc for 1 h with shaking at 250 rpm. afterwards, 150 ml of the transformation reaction were plated on lysogeny broth (lb) agar containing ampicillin. for each reaction at least two plates were prepared and incubated at 37uc for 16 h. a total number of 1536 clones were selected and transferred to 1.3 ml u96 deepwell plates (nunc) containing 0.8 ml lb-amp. the plates were incubated overnight at 37uc, 130 rpm. on the next day, the deepwell plates were centrifuged, the supernatant discarded and the pellets resuspended in 370 ml of lb-amp. a new set of u96 deepwell plates was prepared with 850 ml of fresh lbamp and inoculated with 100 ml each from the resuspended overnight cultures. the remaining 270 ml of resuspended overnight culture were mixed with 30 ml of sterile-filtered dmso and stored at 280uc. the newly inoculated plates were incubated for 6 h at 37uc, 130 rpm. afterwards, the temperature was reduced to 20uc, incubation continued for 1 h and protein expression induced by addition of 2 ml of 0.5 m b-d-1-thiogalactopyranoside (iptg). incubation persisted overnight at 20uc, 130 rpm. the cells were harvested by centrifugation (25006g, 10 min), the supernatant discarded and the pellets frozen at 220uc. after 15 min the pellets were resuspended in 180 ml of easylyse bacterial protein extraction solution (epicentre) and incubated for 5 min at room temperature. dnase i was mixed with dnase reaction buffer (10 mm tris-hcl, 2.5 mm mgcl 2 , 10 mm cacl 2 ), added to the reaction and incubation was carried on for 10 min at 37uc. the plates were centrifuged to collect cell debris for 3 min at 25006g. for each sample 10 ml of lysate were transferred to 384 microtiterplates (genetixx), which were used as reservoirs for the spotting procedure. the samples were spotted onto halolink slides (promega) using the qarray2 microarray spotter (molecular devices). 384 different samples were spotted per slide with three replicate slides per screening. in total 1536 samples were screened on 12 slides (n = 3). each sample was spotted as quadruplicates with controls in two identical sets of eighteen 10610 subarrays each (total number of spots per slide 3600). the controls used included ht-ompa and ht-ompf as positive reference proteins as these have been described as immunodominant before. as specificity controls ht-argc and ht-pyrc were used, representing proteins without known immunodominant behaviour, thus binding of the polyclonal antibodies is not expected. in addition two different e.coli strains -acella electrocompetent cells and krx single-step figure 11 . specificity assay of gavvalsttfa and derivatives. the bar chart represents the mean signal intensities (n = 12) of gavvalsttfa and several modified peptides with single amino acid replacements incubated with antibodies reactive to k. pneumoniae (green), e. coli (orange) or s. enterica (purple). the sequences on the left represent the original epitope and modified versions displaying an increase in signal intensity for k. pneumoniae antibodies. in contrast, sequences on the right harbor modifications causing a significant drop in intensity for k. pneumoniae. rabbit igg is used as a positive reference and mbp serves as a negative control. none of the sequences tested displayed any significant signal intensity above the negative control when incubated with either e. coli or s. enterica antibodies. doi:10.1371/journal.pone.0110703.g011 competent cells (promega) -were spotted as further controls. as those two lack proteins expressed with a halotag, they are used as negative controls. after spotting of the samples, the slides were incubated for 1 h at room temperature in a humidity chamber. next, slides were washed with pbs+0.05% igepal ca-630 (pbsi, sigma aldrich) and dried by nitrogen flow. the 2 well proplate module (grace biolabs) was attached to each slide. the top chamber was filled with 1.5 ml of rabbit-polyclonal antibody to k. pneumoniae (acris, 2 mg/ml) in pbs. the bottom chamber was incubated with pbs only. after 2 h of incubation at room temperature with gentle rocking, both chambers of each slide were washed three times with 2 ml of pbsi. secondary antibody (goat-polyclonal to rabbit igg conjugated with chromeo-546, abcam, 5 mg/ml) was subjected to each chamber in pbs and the slides were incubated at room temperature for 2 h in the dark under gentle rocking. finally, slides were washed three times with pbsi, the proplate modules removed and the slides dried by nitrogen flow. the slides were scanned on an axon genepix 4200a laser scanner (molecular devices) with the following settings: 532 nm laser, pmt gain 400, 40% laser power, lines to average 1, 10 mm resolution and standard green emission filter at 575 nm. the raw data sets of all the microarray analyses in this publication have been deposited in ncbi's gene expression omnibus [36] and are accessible through geo series accession numbers gse52536, gse52537, gse52538, and gse60588. the median fluorescence intensity of each spot corrected by the local background (median f532 -b532) was used. further, relative fluorescence intensity (rfi) was calculated by subtracting the signals of the bottom chamber from the raw data signals of the top chamber to account for non-specific binding of secondary antibodies. for screening of expression libraries we used the contrast method with either argc or pyrc as specificity control to determine the contrast via the formula: with rf f i control the median of all rfis of the control used. clones harboring strong signals in microarray screening were selected to be sequenced. sequencing was performed externally by lgc genomics using ht7f (59 acatcggcccgggtct-gaatc 39) and flxr (59 cttcctttcgggctttgttag 39) primers. after sequencing and identification of potentially immunodominant proteins, primers were designed to generate full-length clones for each identified gene, see table s2 for a list of the primers used. cloning was performed as mentioned above with slight modifications. the pfn18a vector was linearized using the following primer set; 18a if linear for (59 gtttaaac-gaattcgggctc 39) and 18a if linear rev (59 ggcgatcgcgttatcgctctg 39) with pcr conditions as mentioned before. protein expression, lysis, and spotting of fulllength proteins were performed as described above. the slides were incubated for 1 h at room temperature in a humidity chamber. for incubation with antibodies 3 well or 16 well proplate modules (grace biolabs) were attached to the halolink slides. processing of the slides was done similar to the original screening, however several different antibodies were used, see section antibodies. for testing of immunodominant characteristics with elisa, the crude lysate was first purified using halolink magnetic beads (promega) following the manufacturer's instructions. the proteins of interest were subsequently cleaved off by digestion with protev protease (promega) and concentration was determined by nanodrop measurements. the samples were diluted to a total protein content of 20 mg/ml in pbs and 50 ml of each sample was added to maxisorb plates (nunc). each sample was analyzed at least in triplicate. the elisa plate was covered with a lid and incubated overnight at 4uc in a humidity chamber. after five washing steps each with pbs+0.05% tween-20 (pbst), the plates were blocked using 200 ml 5% non fat dried milk in pbs per well for 2 h. afterwards, plates were washed three times with pbst. 100 ml of primary antibody solution (c = 4 mg/ml) in pbs containing 1% non fat dried milk were applied to each well using the respective desired antibody or pbs for controls. the plates were incubated for 2 h at room temperature and washed four times with pbst. next, 100 ml of conjugated secondary antibody (goat polyclonal to rabbit igg conjugated with horseradish peroxidase, abcam ab6721, c = 20 ng/ml) were added to each well and incubation carried on for 1 h. finally, plates were washed once again four times with pbst and 100 ml 3,39,5,59-tetramethylbenzidine (tmb, sigma-aldrich) was added to each well for detection. after 30 min of incubation at room temperature in the dark, the reaction was stopped by applying 100 ml of 2 m h 2 so 4 to each well. the optical density of each well was measured using the omega fluostar (bmg labtech) at a wavelength of 450 nm. primers were designed using primer3 [37] within geneious pro 5.6.5 [38] . the sequenced inserts were identified by blast [39] . peptide sequence secondary structures were predicted using the emboss garnier [40] algorithm and the transmembrane regions predicted by tmhmm2.0 [30, 31] . antigenic sites were predicted by emboss antigenic [32, 33] . data evaluation was performed by originpro 8 g (originlab) and microsoft excel. 3-dimensional structure predictions were performed using the swiss model automated mode [41] [42] [43] [44] [45] and pdb files were visualized and analyzed by the ucsf chimera package [46] .chimera is developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco (supported by nigms p41-gm103311). analysis of full-length proteins was achieved by combining the results from elisa and microarray data. hence, the rfi of each sample was calculated. next, a normalized rfi was generated by dividing the rfi of each sample by the median rfi of all the samples within an area of interest, i.e. incubation compartment. from these normalized rfis a median and standard deviation was calculated. if the median normalized rfi of the positive control was below the median normalized rfi of any of the negative references whilst taking the standard deviations into account, the test was rendered invalid. if the test passed the above criterion, the q values were calculated as follows: with rf f i sample the median of normalized rfis of the sample and rf f i pos:control the median of the normalized rfis of the positive control ompa respectively ompf. the resulting error was calculated by error propagation according to gauss. finally, incorporating all valid tests, the mean q value was determined along with its resulting error following error propagation by gauss, see table 1 . several proteins were chosen for epitope mapping. these were the proteins encoded by kpn_00363, kpn_00459, kpn_00466, kpn_01100, kpn_01584, and kpn_02202. the proteins were divided into 15-mer oligopeptides with an overlap of 11 amino acids in silico. the synthesis and coupling to microarray slides was performed externally by jpt peptide technologies gmbh. each peptide sequence was applied 9 times to one slide. the slides were used with proplate 3-well chamber system (grace) allowing for incubation with different antibodies. first, the slides were blocked with superblock blocking buffer (thermo fischer) for 2 h, washed five times with pbs+0.05% tween-20, primary antibodies applied, incubated overnight at 4uc with mild rocking, washed again, secondary antibodies applied for 2 h in the dark and after a final washing procedure, dried and scanned as above. two different primary antibodies to k.pneumoniae were tested. the bottom chamber was always used as a control chamber, incubated only with secondary antibody. the peptide gavvalsttfa and 11 modifications thereof created by substituting one amino acid by alanine/glycine were synthesized by jpt peptide technologies gmbh. these peptides in combination with two peptides showing closely related sequences, gavlalsssft and saalaltssfte, were applied 9 times to slides. incubation procedure was performed as described above for epitope mapping the expression of the desired halotag fusion proteins was checked by sds-page. after lysis of cells, 2 ml of each protein extract was mixed with 1 ml of 10 mm halotag alexa 488 ligand. after addition of 7 ml 1x tbs (100 mm tris, 150 mm nacl, ph 7.6) the reaction was incubated at room temperature for 30 minutes. 2 ml of each reaction were removed, mixed with 8 ml of 5x loading buffer (fermentas) and 1 ml dtt and heated for 5 min at 70uc. the separation was performed on a mini-protean tgx gel (biorad, any kd, 15 wells) in a protean ii xi cell chamber (biorad) for 30 min at 200 v. as a size reference benchmark fluorescent protein standard (life technologies) was used. fluorescence was measured in a fla-5100 (fujifilm) with excitation at 473 nm. figure s3 homology of giafgavelfd of kpn_00459. the sequence derived from k. pneumoniae mgh78578 was used as a reference. the 100 best matches after blast analysis are shown in the figure with dots indicating identical residues. for differentiation of the sequences the ncbi accession number of the parent protein is given followed by the strain designation, if available. only three e. coli strains in lines 63, 97 and 98 feature a valine residue at the second position instead of the consensus isoleucine. consequently, this sequence is highly conserved within the enterobacteriaceae including e. coli, klebsiella, salmonella, and enterobacter among others. (pdf) table s1 list of 192 sequenced clones after screening the clones were sequenced by lgc genomics using ht7f and flx primers. clones that were not successfully sequenced are indicated by ''-'', clones carrying inserts too short to be reliably mapped to a gene are marked as truncated. additionally, a few inserts were detected deriving from primer concatamers. these are displayed as ''artificial''. the remaining clones are indicated by the corresponding locus tag and protein name. several clones apparently carry identical inserts, especially obvious for kpn_01805 or kpn_02668. these were discarded from further analysis as the mapped inserts are very short and might have an artificial origin. inserts that were highly unlikely to garner new immunogenic proteins, antigens described previously, e.g. ompa, other molecules like trna and rrna were abolished from further analysis. table s2 primers used in this study. each primer is given with a name, its sequence in 59 to 39 direction and the target gene or vector. for each target f represents forward and r the reverse primer. the primers were used for cloning in the in-fusion smarter directional cdna library construction kit. (xls) manual of clinical microbiology clinical epidemiology of the global expansion of klebsiella pneumoniae carbapenemases extended-spectrum beta-lactamase producing klebsiella spp. in chicken meat and humans: a comparison of typing methods modern clinical microbiology: new challenges and solutions rapid identification of novel immunodominant proteins and characterization of a specific linear epitope of campylobacter jejuni application of a microarray-based immunoscreening for rapid identification of novel antigens of salmonella enteritidis microarray-based method for screening of immunogenic proteins from bacteria halotagbased purification of functional human kinases from mammalian cells severe acute respiratory syndrome diagnostics using a coronavirus protein microarray reverse transcriptase template switching: a smart approach for full-length cdna library construction polyadenylic acid sequences in e. coli messenger rna identification of the gene for an escherichia coli poly (a) polymerase a simple method to enrich mrna from total prokaryotic rna magnetic capturehybridization method for purification and probing of mrna for neutral protease of bacillus cereus dsn depletion is a simple method to remove selected transcripts from cdna populations duplex-specific nuclease efficiently removes rrna for prokaryotic rna-seq ligation-independent cloning of pcr products (licpcr) identification of vaccine candidate antigens of an esbl producing klebsiella pneumoniae clinical strain by immunoproteome analysis the national center for biotechnology information's protein clusters database protective efficacy of dna vaccines encoding outer membrane protein a and ompk36 of klebsiella pneumoniae in mice b-cell epitopes: fact and fiction x-ray crystallography of antibodies the structure of bacterial outer membrane proteins identification and characterization of ompl as a potential vaccine candidate for immune-protection against salmonellosis in mice the unique structure of haemophilus influenzae protein e reveals multiple binding sites for host factors directed evaluation of enterotoxigenic escherichia coli autotransporter proteins as putative vaccine candidates structure of the c-terminal domain of neisseria heparin binding antigen (nhba), one of the main antigens of a novel vaccine against neisseria meningitidis in vivo versus in vitro protein abundance analysis of shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism surface expression, singlechannel analysis and membrane topology of recombinant chlamydia trachomatis major outer membrane protein predicting transmembrane protein topology with a hidden markov model: application to complete genomes a hidden markov model for predicting transmembrane helices in protein sequences a semi-empirical method for prediction of antigenic determinants on protein antigens new hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-ray-derived accessible sites toward the estimation of the absolute quality of individual protein structure models the rin: an rna integrity number for assigning integrity values to rna measurements gene expression omnibus: ncbi gene expression and hybridization array data repository primer3 on the www for general users and for biologist programmers basic local alignment search tool analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins the swiss-model workspace: a web-based environment for protein structure homology modelling the swiss-model repository and associated resources swiss-model: an automated protein homology-modeling server swiss-model and the swiss-pdb viewer: an environment for comparative protein modeling protein modeling by email ucsf chimera-a visualization system for exploratory research and analysis the k. pneumoniae strain mgh 78578 was a kind gift of the group of s. bereswill (department of microbiology and hygiene, charitã© -university medicine berlin, berlin, germany). sh is greatly indebted to martina obry for her assistance during expression library construction and clone isolation. the authors would like to thank simone aubele for technical assistance. we also gratefully acknowledge michaela schellhase for microarray printing. conceived and designed the experiments: sh mvnr. performed the experiments: sh. analyzed the data: sh ffb mvnr. contributed reagents/materials/analysis tools: sh. wrote the paper: sh ffb mvnr. key: cord-020757-q4ivezyq authors: saikumar, pothana; kar, rekha title: apoptosis and cell death: relevance to lung date: 2010-05-21 journal: molecular pathology of lung diseases doi: 10.1007/978-0-387-72430-0_4 sha: doc_id: 20757 cord_uid: q4ivezyq in multicellular organisms, cell death plays an important role in development, morphogenesis, control of cell numbers, and removal of infected, mutated, or damaged cells. the term apoptosis was first coined in 1972 by kerr et al.1 to describe the morphologic features of a type of cell death that is distinct from necrosis and is today considered to represent programmed cell death. in fact, the evidence that a genetic program existed for physiologic cell death came from the developmental studies of the nematode caenorhabditis elegans.2 as time has progressed, however, apoptotic cell death has been shown to occur in many cell types under a variety of physiologic and pathologic conditions. cells dying by apoptosis exhibit several characteristic morphologic features that include cell shrinkage, nuclear condensation, membrane blebbing, nuclear and cellular fragmentation into membrane-bound apoptotic bodies, and eventual phagocytosis of the fragmented cell (figure 4.1). in multicellular organisms, cell death plays an important role in development, morphogenesis, control of cell numbers, and removal of infected, mutated, or damaged cells. the term apoptosis was fi rst coined in 1972 by kerr et al. 1 to describe the morphologic features of a type of cell death that is distinct from necrosis and is today considered to represent programmed cell death. in fact, the evidence that a genetic program existed for physiologic cell death came from the developmental studies of the nematode caenorhabditis elegans. 2 as time has progressed, however, apoptotic cell death has been shown to occur in many cell types under a variety of physiologic and pathologic conditions. cells dying by apoptosis exhibit several characteristic morphologic features that include cell shrinkage, nuclear condensation, membrane blebbing, nuclear and cellular fragmentation into membrane-bound apoptotic bodies, and eventual phagocytosis of the fragmented cell (figure 4 .1). cell death is central to the normal development of multicellular organisms during embryogenesis and maintenance of tissue homeostasis in adults. 3 during development, sculpting of body parts is achieved through selective cell death, which imparts appropriate shape and creates required cavities in particular organs. in adults, cell death balances cell division as a homeostatic mechanism regulating constancy of tissue mass. deletion of injured cells because of disease, genetic defects, aging, or exposure to toxins is also achieved by apoptosis. in essence, apoptotic cell death has important biologic roles not only in development and homeostasis but also in the pathogenesis of several disease processes. dysregulation of apoptosis is found in a wide spectrum of human diseases, including cancer, autoimmune diseases, neurodegenerative diseases, ischemic diseases, viral infections, 4 and lung diseases. 5 our knowledge of cell death and the mechanisms of its regulation increased dramatically in the past two decades with the discovery nevertheless, necrosis has been shown to occur in cells having defects in apoptotic machinery or upon inhibition of apoptosis, 7 and this form of cell death is emerging as an important therapeutic tool for cancer treatment. 8 autophagy autophagy, which is also referred to as type ii programmed cell death, is characterized by sequestration of cytoplasm and organelles in double or multimembrane structures called autophagic vesicles, followed by degradation of the contents of these vesicles by the cell's own lysosomal system (see figure 4 .1). the precise role of autophagy in cell death or survival is not clearly understood. autophagy has long been regarded as a cell survival mechanism whereby cells eliminate long-lived proteins and organelles. in this regard, it is argued that autophagy may help cancer cells survive under nutrientlimiting and low-oxygen conditions and against ionizing radiation. 9,10 however, recent observations that there is there is early membrane damage with eventual loss of plasma membrane integrity and leakage of cytosol into extracellular space. despite early clumping, the nuclear chromatin undergoes lysis (karyolysis). apoptosis: cells die by type i programmed cell death (also called apoptosis); they are shrunken and develop blebs containing dense cytoplasm. membrane integrity is not lost until after cell death. nuclear chromatin undergoes striking condensation and fragmentation. the cytoplasm becomes divided to form apoptotic bodies containing organelles and/or nuclear debris. terminally, apoptotic cells and fragments are engulfed by phagocytes or surrounding cells. autophagy: cells die by type ii programmed cell death, which is characterized by the accumulation of autophagic vesicles (autophagosomes and autophagolysosomes). one feature that distinguishes apoptosis from autophagic cell death is the source of the lysosomal enzymes used for most of the dying-cell degradation. apoptotic cells use phagocytic cell lysosomes for this process, whereas cells with autophagic morphology use the endogenous lysosomal machinery of dying cells. paraptosis: cells die by type iii programmed cell death, which is characterized by extensive cytoplasmic vacuolization and swelling and clumping of mitochondria, along with absence of nuclear fragmentation, membrane blebbing, or apoptotic body formation. autoschizis: in this form of cell death, the cell membrane forms cuts or schisms that allow the cytoplasm to leak out. the cell shrinks to about one-third of its original size, and the nucleus and organelles remain surrounded by a tiny ribbon of cytoplasm. after further excisions of cytoplasm, the nuclei exhibit nucleolar segregation and chromatin decondensation followed by nuclear karyorrhexis and karyolysis. decreased autophagy during experimental carcinogenesis and heterologous disruption of an autophagy gene, beclin 1 (bcn1), in cancer cells 11, 12 suggest that breakdown of autophagic machinery may contribute to development of cancer. other interesting studies have shed some light on the relationship between autophagy and apoptosis. these investigations have shown prevention of caspase inhibitor z-vad-induced cell death in mouse l929 cells by rna interference directed against autophagy genes atg7 and bcn1 13 and protection of bax −/− , bak −/− murine embryonic fi broblasts against staurosporine-or etoposide-induced cell death by rna interference against autophagy genes atg5 and bcn1. 14 however, both of these studies were done in cells whose apoptotic pathways had been compromised. thus, it remains to be seen whether cells with intact apoptotic machinery can also die by autophagy and whether apoptotic-competent cells lacking autophagy genes will be resistant to different death stimuli. paraptosis has recently been described as a form of cell death characterized by extensive cytoplasmic vacuolation (see figure 4 .1) caused by swelling of mitochondria and endoplasmic reticulum. this form of cell death does not involve caspase activation, is not inhibited by caspase inhibitors, but is inhibited by the inhibitors of transcription and translation, actinomycin d, and cycloheximide, respectively, 15 suggesting a requirement for new protein synthesis. the tumor necrosis factor receptor family taj/troy and the insulin-like growth factor i receptor have been shown to trigger paraptosis. 16 paraptosis appears to be mediated by mitogen-activated protein kinases and inhibited by aip1/alix, a protein interacting with the calcium-binding death-related protein alg-2. 16 autoschizis autoschizis is a recently described type of cell death that differs from apoptosis and necrosis and is induced by oxidative stress. 17 in this type of death, cells lose cytoplasm by self-morsellation or self-excision (see figure 4 .1). autoschizis usually affects contiguous groups of cells both in vitro and in vivo but can also occasionally affect scattered individual cells trapped in subcapsular sinuses of lymph nodes. 18 the nuclear envelope and pores remain intact while the cytoplasm is reduced to a narrow rim surrounding the nucleus. the chromatin marginates along the nuclear membrane, and mitochondria and other organelles around the nucleus aggregate as a result of cytoskeletal damage and condensation of the cytosol. interestingly, the rough endoplasmic reticulum is preserved until the late stages of autoschizis, in which cells fragment and the nucleolus becomes condensed and breaks into smaller fragments. 19 eventually, the nuclear envelope and the remaining organelles dissipate with cell demise. genetic studies in the nematode worm c. elegans led to the characterization of apoptosis. activation of specifi c death genes during the development of this worm results in death of exactly 131 cells, leaving 959 cells intact. 2 further studies revealed that apoptosis can be divided into three successive stages: (1) commitment phase, in which death is initiated by specifi c extracellular or intracellular signals; (2) execution phase; and (3) clean-up phase, in which dead cells are removed by other cells with eventual degradation of the dead cells in the lysosomes of phagocytic cells. 20 the apoptotic machinery is conserved through evolution from worm to human. 21 in c. elegans, execution of apoptosis is mediated by ced-3 and ced-4 proteins. commitment to a death signal results in the activation of ced-3 by ced-4 binding. the ced-9 protein prevents activation of ced-3 by binding to ced-4. 22, 23 mechanisms of apoptosis caspases studies over the past decade have indicated that two distinct apoptotic pathways are followed in mammalian systems: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. the executioners in both intrinsic and extrinsic pathways of cell death are the caspases, 24 which are cysteine proteases with specifi city to cleave their substrates after aspartic acid residues. the central role of caspases in apoptosis is underscored by the observation that apoptosis and all classic changes associated with apoptosis can be blocked by inhibition of caspase activity. to date, 12 mammalian caspases (caspase-1 to -10, caspase-14, and mouse caspase-12) have been identifi ed. 25 caspase-13 was later found to represent a bovine homolog and caspase-11 appears to be a murine homolog of human caspases-4 and -5, respectively. caspases are normally produced as inactive zymogens containing an n-terminal prodomain followed by a large and a small subunit that constitute the catalytic core of the protease. they have been categorized into two distinct classes: initiator and effector caspases. the upstream initiator caspases contain long n-terminal prodomains and one of the two characteristic protein-protein interaction motifs: the death effector domain (ded; caspase-8 and -10) and the caspase activation and recruitment domain (caspase-1, -2, -4, -5, -9, and -12). the downstream effector caspases (caspase-3, -6, and -7) are characterized by the presence of a short prodomain. apart from the structural differences, a prominent difference between initiator and effector caspases is their basal state. both the zymogen and the activated forms of effector caspases exist as constitutive homodimers, whereas initiator caspase-9 exists predominantly as a monomer both before and after proteolytic processing. 26 initiator caspase-8 has been reported to exist in an equilibrium between monomers and homodimers. 27 although the initiator caspases are capable of autocatalytic activation, the activation of effector caspases requires formation of oligomeric complexes with their adapter proteins and often intrachain cleavage within the initiator caspase. caspases have also been divided into three categories based on substrate specifi city. 28 group i members (caspase-1, -4, and -5) have a substrate specifi city for the wehd sequence with high promiscuity; group ii members (caspase-2, -3, and -7 and ced-3) prefer the dexd sequence and have an absolute requirement for aspartate (d) at p4; and members of group iii (caspase-6, -8, and -9 and the "aspase" granzyme b) have a preference for (i/ l/v)exd sequences. several reports have suggested a role for group i members in infl ammation and that of group ii and iii members in apoptotic signaling events. the extrinsic pathway involves binding of death ligands such as tumor necrosis factor-α (tnf-α), cd95 ligand (fas ligand), and tnf-related apoptosis-inducing ligand (trail) to their cognate cell surface receptors tnfr1, cd95/fas, trail-r1, trail-r2, and the dr series of receptors, 29 resulting in the activation of initiator caspase-8 (also known as fadd-homologous ice/ced-3-like protease or flice) and subsequent activation of effector caspase-3 ( figure 4 .2). 30 the cytoplasmic domains of death receptors contain the "death domain," which plays a crucial role in transmitting the signal from the cell's surface to intracellular signaling molecules. binding of the ligands to their cognate receptors results in receptor trimerization and recruitment of adapter proteins to the cell membrane, which involves homophilic interactions between death domains of the receptors and the adapter proteins. the adapter protein for the receptors tnfr1 and dr3 is tnfr-associated death domain protein (tradd) 31 and that for fas, trail-r1, trail-r2, and dr4 is fas-associated death domain protein (fadd). 32 the receptor/ligand and fadd complex in turn recruits caspase-8 to the activated receptor, resulting in the formation of death-inducing signaling complex (disc) and subsequent activation of caspase-8 through oligomerization and self-cleavage. depending on the cell type and/or apoptotic stimulus, caspase-8 can also be activated by caspase-6. 33 activated caspase-8 then activates effector caspase-3. in some cell types, cleavage of caspase-3 by caspase-8 also requires a mitochondrial amplifi cation loop involving cleavage of proapoptotic protein bid by caspase-8 and its translocation to the mitochondrial membrane, triggering the release of apoptogenic proteins from mitochondria into cytosol (see figure 4 .2). in these cell types, overexpression of bcl-2 and bcl-xl can block cd95-induced apoptosis. 34 tumor necrosis factor-α is produced by t cells and activated macrophages in response to infection. although tnf-α-mediated signaling can be propagated through either tnfr1 or tnfr2 receptors, the majority of biologic functions are initiated by tnfr1. 35 binding of tnf-α to tnfr1 causes release of inhibitory protein silencer of death domain protein (sodd) from tnfr1, which enables recruitment of adapter protein tradd. signaling induced by activation of tnfr1 or dr3 diverges at the level of tradd. in one pathway, nuclear translocation of the transcription factor nuclear factor-κb (nf-κb) and activation of c-jun n-terminal kinase (jnk) are initiated, which results in the induction of a number of proinfl ammatory and immunomodulatory genes. 36 in another pathway, tnf-α signaling is coupled to fas signaling events through interaction of tradd with fadd. 37 the tnfr1-tradd complex can alternatively engage traf2 protein, resulting in activation of transcription factor c-jun, which is involved in survival signaling. furthermore, binding of receptor interaction protein to tnfr1 through tradd results in activation of transcription factor nf-κb, which suppresses apoptosis through transcriptional upregulation of antiapoptotic molecules such as traf1, traf2, ciap1, ciap2, and flip. the flice-associated huge protein was identifi ed to be a ced-4 homolog interacting with the ded of caspase-8 and was shown to modulate fas-mediated activation of caspase-8. 38 another class of protein, flip (flice inhibitory protein), was shown to block fasinduced and tnf-α-induced disc formation and subsequent activation of caspase-8. 39 cytotoxic t cells play a major role in vertebrate defense against viral infection. 40 they induce cell death in infected cells to prevent viral multiplication and spread of infection. 41 cytotoxic t cells can kill their targets either by activating the fas ligand/fas pathway or by injecting granzyme b, a serine protease, into target cells. cytotoxic t cells carry fas ligand on their surface but also carry granules containing the channel-forming protein perforin and granzyme b. upon recognizing the infected cells, the lymphocytes bind and secrete granules onto the surface of infected cells. perforin then assembles into transmembrane channels to allow the entry of granzyme b into the target cell. upon entry, granzyme b, which cleaves after aspartate residues in proteins ("aspase"), activates one or more of the apoptotic proteases (caspase-2, -3, -7, -8, and -10) to trigger the proteolytic death cascade (see figure 4 .2). fas ligand/fas and perforin/granzyme b systems are the main apoptotic machinery that regulates homeostasis in immune cell populations. cells can respond to various stressful stimuli and metabolic disturbances by triggering apoptosis. drugs, toxins, heat, radiation, hypoxia, and viral infections are some of the tnf-α tnfr1 complex can also elicit an antiapoptotic response by recruiting traf2, which results in nf-κbmediated upregulation of antiapoptotic genes. in cytotoxic t lymphocyte-induced death, granzyme b, which enters the cell through membrane channels formed by the protein perforin, activates caspases by cleaving them directly or indirectly. intracellular pathways: lack of survival stimuli (withdrawal of growth factor, hypoxia, genotoxic substances, etc.) is thought to generate apoptotic signals through ill-defi ned mechanisms, which lead to translocation of proapoptotic proteins such as bax to the outer mitochondrial membrane. in some cases, transcription mediated by p53 may be required to induce proteins such as bax. translocated bax undergoes conformational changes in the outer membrane to form oligomeric structures (pores) that leak cytochrome c from mitochondria into the cytosol. formation of a ternary complex of cytochrome c, the adapter protein apaf-1, and the initiator caspase-9 results in the activation of caspase-9 followed by sequential activation of effector caspase(s) such as caspase-3 and others. the action of caspases, endonucleases, and possibly other enzymes leads to cellular disintegration. for example, the endonuclease cad (caspase activated dnase) becomes activated when it is released from its inhibitor icad upon cleavage of icad by an effector caspase. antiapoptotic proteins such as bcl-2 and bcl-xl inhibit the membrane-permeabilizing effects of bax and other proapoptotic proteins. cross-talk between extra-and intracellular pathways occurs through caspase-8-mediated bid cleavage, which yields a 15 kda protein that migrates to mitochondria and releases cytochrome c, thereby setting in motion events that lead to apoptosis via caspase-9. the stimuli known to activate death pathways. cell death, however, is not necessarily inevitable after exposure to these agents, and the mechanisms determining the outcome of the injury are a topic of active interest. the current consensus appears to be that it is the intensity and the duration of the stimulus that determine the outcome. the stimulus must go beyond a threshold to commit cells to apoptosis. although the exact mechanism used by each stimulus may be unique and different, a few broad patterns can be identifi ed. for example, agents that damage dna, such as ionizing radiation and certain xenobiotics, lead to activation of p53-mediated mechanisms that commit cells to apoptosis, at least in part through transcriptional upregulation of proapoptotic proteins. 42 other stresses induce increased activity of stress-activated protein kinases, which result ultimately in apoptotic commitment. 43 these different mechanisms converge in the activation of caspases. a cascade of caspases plays the central executioner role by cleaving various mammalian cytosolic and nuclear proteins that play roles in cell division, maintenance of cytoskeletal structure, dna replication and repair, rna splicing, and other cellular processes. this proteolytic carnage produces the characteristic morphologic changes of apoptosis. once the caspase cascade is initiated, the process of cell death has crossed the point of no return. the roles of various caspases in apoptotic pathways and their relative importance for animal development have been examined in genetic studies involving knockout of different caspase genes. a caspase-1 (interleukin [il]-1b converting enzyme [ice]) knockout study suggested that ice plays an important role in infl ammation by activating cytokines such as il-1b and il-18. however, caspase-1 was not required to mediate apoptosis under normal circumstances and did not have a major role during development. 44 surprisingly, ischemic brain injury was signifi cantly reduced in caspase-1 knockout mice compared with wild-type mice, 45 suggesting that infl ammation may contribute to ischemic injury. caspase-3 deficiency leads to impaired brain development and premature death. also, functional caspase-3 is required for some typical hallmarks of apoptosis such as formation of apoptotic bodies, chromatin condensation, and dna fragmentation in many cell types. 46 lack of caspase-8 results in the death of embryos at day 11 with abnormal formation of the heart, 47 suggesting that caspase-8 is required for cell death during mammalian development. in support of this fi nding, knockout of fadd, which is required for caspase-8 activation, resulted in fetal death with signs of abdominal hemorrhage and cardiac failure. 48 moreover, caspase-8-defi cient cells did not die in response to signals from members of the tnf receptor family. 47 however, cells lacking either fadd or caspase-8, which are resistant to tnf-α-mediated or cd95-mediated death, are susceptible to chemotherapeutic drugs, serum depriva-tion, ceramide, γ-irradiation, and dexamethasone-induced killing. 48 in contrast, caspase-9 has a key role in apoptosis induced by intracellular activators, particularly those that cause dna damage. deletion of caspase-9 resulted in perinatal lethality, apoptotic failure in developing neurons, enlarged brains, and craniofacial abnormalities. 49 in caspase-9-defi cient cells, caspase-3 was not activated, suggesting that caspase-9 is upstream of caspase-3 in the apoptotic cascade. as a consequence, caspase-9-defi cient cells are resistant to dexamethasone or irradiation, whereas they retain their sensitivity to tnf-α-induced or cd95-induced death 49 because of the presence of caspase-8, the initiator caspase involved in death receptor signaling that can also activate caspase-3. overall, these observations support the idea that different death signaling pathways converge on downstream effector caspases (see figure 4 .2). indeed, caspase-3 is regarded as one of the key executioner molecules activated by apoptotic stimuli originating either at receptors for exogenous molecules or within cells through the action of drugs, toxins, or radiation. in c. elegans, biochemical and genetic studies have indicated a role for ced-4 upstream of ced-3. 50 upon receiving death commitment signals, ced-4 binds to pro-ced-3 and releases active ced-3. 50 however, when overexpressed, ced-9 can inhibit the activation of pro-ced-3 by binding to ced-4 and sequestering it away from pro-ced-3. therefore, ced-3 and ced-4 are involved in activation of apoptosis, and ced-9 inhibits apoptosis. after the discovery of caspases as ced-3 homologs, a search for activators and inhibitors analogous to ced-4 and ced-9 led to the discovery of diverse mammalian regulators of apoptosis. the plethora of these molecules and their functional diversity allowed them to be classifi ed into four broad categories: (1) adapter proteins, (2) the bcl-2 family of regulators, (3) inhibitors of apoptosis (iaps), and (4) other regulators. as stated earlier, two major pathways of apoptosis, involving either the initiator caspase-8 or the initiator caspase-9 (see figure 4 .2), have been recognized. signaling by death receptors (cd95, tnfri) occurs through a well-defi ned process of recruitment of caspase-8 to the death receptor by adapter proteins such as fadd. recruitment occurs through interactions between the death domains that are present on both receptor and adapter proteins. receptorbound fadd then recruits caspase-8 through interactions between deds common to both caspase-8 and fadd forming a disc. in the disc, caspase-8 activation occurs through oligomerization and autocatalysis. activated caspase-8 then activates downstream caspase-3, culminating in apoptosis. the inhibitory protein, flip was shown to block fas-induced and tnf-α-induced disc formation and subsequent activation of caspase-8. 39 of particular interest is cellular flip, which stimulates caspase-8 activation at physiologically relevant levels and inhibited apoptosis upon high ectopic expression. 51 cellular flip contains two deds that can compete with caspase-8 for recruitment to the disc. this limits the degree of association of caspase-8 with fadd and thus limits activation of the caspase cascade. it also forms a heterodimer with caspase-8 and caspase-10 through interactions between both the deds and the caspase-like domains of the proteins, thus activating both caspase-8 and caspase-10. 52 apoptotic protease activating factor-1 (apaf-1), a ced-4 homolog in mammalian cells, affects the activation of initiator caspase-9. 53 this factor binds to procaspase-9 in the presence of cytochrome c and 2′deoxyadenosine 5′-triphosphate (datp) or adenosine triphosphate (atp) and activates this protease, which in turn activates a downstream cascade of proteases (see figure 4 .2). 54 by and large, apaf-1 defi ciency is embryonically lethal and the embryos exhibit brain abnormalities similar to those seen in caspase-9 knockout mice. 55 these genetic fi ndings support the idea that apaf-1 is coupled to caspase-9 in the death pathway. unlike ced-4 in nematodes, apaf-1 requires the binding of atp and cytochrome c to activate procaspase-9. the multiple wd40 repeats in the c-terminal end of apaf-1 have a regulatory role in the activation of caspase-9. 56 the ced-9 homolog in mammals is the bcl-2 protein. bcl-2 was fi rst discovered in b-cell lymphoma as a protooncogene. overexpression of bcl-2 was shown to offer protection against a variety of death stimuli. 57 the bcl-2 protein family includes both proapoptotic (bcl-2, bcl-xl, bcl-w, mcl-1, nr13, and a1/bfl -1) and antiapoptotic proteins (bax, bak, bok, diva, bcl-xs, bik, bim, hrk, nip3, nix, bad, and bid). 58 these proteins are characterized by the presence of bcl-2 homology (bh) domains: bh1, bh2, bh3, and bh4 (figure 4.3) . the proapoptotic members have two subfamilies: a multidomain and a bh3-only group (see figure 4 .3). the relative ratio of pro-and antiapoptotic proteins determines the sensitivity of cells to various apoptotic stimuli. the best-studied proapoptotic members are bax and bid. exposure to various apoptotic stimuli leads to translocation of cytosolic bax from the cytosol to the mitochondrial membrane. 59 bax oligomerizes on the mitochondrial membrane along with another proapoptotic protein, bak, leading to the release of cytochrome c from the mitochondrial membrane into the cytosol. 60 other proapoptotic proteins, mainly the bh3-only proteins, are thought to aid in bax-bak oligomerization on the mitochondrial membrane. the antiapoptotic bcl-2 family members are known to block bax-bak oligomerization on the mitochondrial membrane and subsequent release of cytochrome c into the cytosol. 60, 61 after release from the mitochondria, cytochrome c is known to interact with the wd40 repeats of the adaptor protein apaf-1, resulting in the formation of the apoptosome complex. seven molecules of apaf-1, interacting through their n-terminal caspase activation and recruitment domain, form the central hub region of the symmetric wheel-like structure, the apoptosome. binding of atp/datp to apaf-1 triggers the formation of the apoptosome, which subsequently recruits procaspase-9 into the apoptosome complex, resulting in its activation 62 . activated caspase-9 then activates executioner caspases, such as caspase-3 and caspase-7, eventually leading to programmed cell death. the iaps, fi rst discovered in baculoviruses and then in insects and drosophila, inhibit activated caspases by directly binding to the active enzymes. 63 these proteins contain one or more baculovirus inhibitor of apoptosis repeat domains, which are responsible for the caspase inhibitory activity. 64 to date, eight mammalian iaps have been identifi ed. they include x-linked iap (xiap), c-iap1, c-iap2, melanoma iap (ml-iap)/livin, iaplike protein-2 (ilp-2), neuronal apoptosis-inhibitory protein (naip), bruce/apollon, and survivin. in mammals, caspase-3, -7, and -9 are inhibited by iaps. 62 there are reports suggesting aberrant expression of iaps in many cancer tissues. for example, ciap1 is overexpressed in esophageal squamous cell sarcoma 65 ; ciap2 locus is translocated in mucosa-associate lymphoid lymphoma 66 and survivin has been shown to be upregulated in many cancer cells. 67 the caspase inhibitory activity of iaps is inhibited by proteins containing an iap-binding tetrapeptide motif. 62 the founding member of this family is smac/diablo, which is released from the mitochondrial intermembrane space into the cytosol during apoptosis. in the cytosol, it interacts with several iaps and inhibits their function. the other mitochondrial protein, omi/htra2, is also known to antagonize xiap-mediated inhibition of caspase-9 at high concentrations. 68 a serine protease, omi/htra2 can proteolytically cleave and inactivate iap proteins and thus is considered to be a more potent suppressor of iaps than smac. 69 it has been reported that the heat shock proteins hsp90, hsp70, and hsp27 can inhibit caspase activation by cytochrome c either by interacting with apaf-1 or other players in the pathway. [70] [71] [72] a high-throughput screen identifi ed a compound called petcm (α-[trichloromethyl]-4-pyridineethanol) as a caspase-3 activator. further work with petcm revealed its involvement in apoptosome regulation. 73 this pathway also includes oncoprotein prothymosin-α and tumor suppressor putative hla-dr-associated proteins. these proteins were shown to promote caspase-9 activation after apoptosome formation, whereas prothymosin-α inhibited caspase-9 activation by inhibiting apoptosome formation. in an apoptotic cell, the regulatory, structural, and housekeeping proteins are the main targets of the caspases. the regulatory proteins mitogen-activated protein/extracellular signal-regulated kinase kinase-1, p21-activated kinase-2, and mst-1 are activated upon cleavage by caspases. 74 caspase-mediated protein hydrolysis inactivates other proteins, including focal adhesion kinase, phosphatidylinositol-3 kinase, akt, raf-1, iaps, and inhibitors of caspase-activated dnase (icad). caspases also convert the antiapoptotic protein bcl-2 into a proapoptotic protein such as bax upon cleavage. there are many structural protein targets of caspases, which include nuclear lamins, actin, and regulatory proteins such as spectrin, gelsolin, and fodrins. 75 degradation of nuclear dna into internucleosomal chromatin fragments is one of the hallmarks of apoptotic cell death that occurs in response to various apoptotic stimuli in a wide variety of cells. a specifi c dnase, cad (caspase-activated dnase), that cleaves chromosomal dna in a caspase-dependent manner, is synthesized with the help of icad. in proliferating cells, cad is always found to be associated with icad in the cytosol. when cells are undergoing apoptosis, caspases (particularly caspase-3) cleave icad to release cad and allow its translocation to the nucleus to cleave chromosomal dna. thus, cells that are icad defi cient or that express caspase-resistant icad mutant do not exhibit dna fragmentation during apoptosis. apoptosis plays a critical role in the postnatal lung. 76 regulated removal of infl ammatory cells by apoptosis helps in the resolution of infl ammation in the lung. 77 recent evidence also supports a role for apoptosis in the remodeling of lung tissue after acute lung injury 78 and in the pathogenesis of chronic pulmonary hypertension, 79 idiopathic pulmonary fi brosis, and chronic obstructive pulmonary disease. 80, 81 acute lung injury/acute respiratory distress syndrome acute lung injury, which clinically manifests itself as the acute respiratory distress syndrome (ards), involves disruption of the alveolar epithelium and endothelium, increased vascular permeability, and edema. two main hypotheses link the pathogenesis of ards to apoptosis, namely, the "neutrophilic hypothesis" and the "epithelial hypothesis." these two hypotheses are not mutually exclusive, and both could play important roles in the pathogenesis of ards. the neutrophilic hypothesis suggests that neutrophil apoptosis plays an important role in the resolution of infl ammation and that the inhibition of neutrophil apoptosis or the inhibition of clearance of apoptotic neutrophils is deleterious in ards. 82, 83 studies in humans showed that bronchoalveolar lavage fl uids from patients with early ards inhibit the rate at which neutrophils develop apoptosis in vitro. 84 the inhibitory effect of bronchoalveolar lavage fl uids on neutrophil apoptosis is mediated by granulocyte/macrophage colony-stimulating factor, and possibly by il-8 and il-2. 85,86 a membrane surface molecule, cd44, has been shown to play an important role in the clearance of apoptotic cells in vivo and in vitro. 87 in a model of bleomycin-induced lung injury, cd44-defi cient mice failed to clear apoptotic neutrophils, which was associated with worsened infl ammation and increased mortality. 87 activation of phagocytic cells inhibits production of proinfl ammatory cytokines, including il-1β, il-8, il-10, granulocyte/ macrophage colony-stimulating factor, and tnf-α and increases release of anti-infl ammatory mediators such as transforming growth factor-β, prostaglandin e 2 , and platelet-activating factor. 88, 89 the net effects of these changes could favor resolution of infl ammation. the epithelial hypothesis suggests that the apoptotic death of alveolar epithelial cells, in response to soluble mediators such as fas ligand, contributes to the prominent alveolar epithelial injury characteristic of ards. several lines of evidence suggest a role for the fas/fas ligand system in epithelial cell apoptosis. 90 fas is expressed on alveolar and airway epithelial cells, 91, 92 and its expression increases in response to infl ammatory mediators such as lipopolysaccharide. fas-mediated lung cell apoptosis is modulated by surfactant protein a, which inhibits apoptosis in vivo. 93 chronic obstructive pulmonary disease chronic obstructive pulmonary disease, caused primarily by smoking, generally refers to chronic bronchitis and emphysema. several factors, including protease/antiprotease imbalance, oxidative stress, cigarette smokederived toxins, and infl ammation mediated by neutrophils, macrophages, and cd8 + t cells, have been shown to contribute to the disease process. furthermore, matrix metalloproteinase 94 and vascular endothelial growth factor receptor inhibition, 95, 96 but not fas/fas ligand, have been shown to play role in the development of emphysema. asthma allergic asthma is characterized by intermittent or persistent bronchoconstriction and has been linked to airway remodeling and chronic infl ammation, with increased numbers of eosinophils, cd4 + t cells, and mast cells. although at present a role for apoptosis in asthma is not confi rmed, studies ex vivo have shown reduced apoptosis of circulating peripheral cd4 + t cells and eosinophils in asthma, which might contribute to infl ammation. corticosteroids used to reduce infl ammation in asthma have been shown to induce eosinophil apoptosis. 97 pulmonary fi brosis is characterized by epithelial damage, fi broblast proliferation, and deposition of collagen. although the mechanism of alveolar epithelial cell apoptosis in pulmonary fi brosis is not known, several reports have suggested fas pathway, 98 angiotensin pathway, 99 activated t cell-derived perforin, 100 il-13 stimulation, 101 and transforming growth factor-β1 activation 102 to play critical roles. because insuffi cient apoptosis is often associated with tumorigenesis, modulation of apoptotic and antiapoptotic targets seems to be an attractive approach to cancer therapy. lung cancers can be divided into small cell lung cancers (sclcs) and non-small cell lung cancers (nsclcs). 103 the sclcs are relatively more sensitive to anticancer drugs and irradiation than are the nsclcs, 104 but the molecular basis for this difference is not clearly known. evaluation of apoptosis-associated substances has shown that caspase-8, fas, and fas ligand are often downregulated in sclcs but not in nsclcs. 105 an investigation of the basis for these differences revealed that there were no differences in the levels of bax and bcl-xl, but the expression of bcl-2 was found to be signifi cantly higher in sclc than in nsclc cell lines. the observation that in some cases bcl-2 can be converted into a proapoptotic bax-like death molecule may offer an explanation for the paradoxic expression of bcl-2 in sclc. 106 the lack of expression of procaspase-1, -4, -8, and -10 107 reported in sclc suggests that these caspases probably do not contribute to spontaneous apoptosis in these cells. apoptosis regulators apaf-1 and procaspase-3 are overexpressed and are functional in nsclc cell lines. in both types of lung cancer, apoptotic stimuli result in cytochrome c release and activation of caspase-9 and caspase-3, but only sclc cell lines showed a relocalization of caspase-3 into the nucleus 108 ; this suggests that the resistance of nsclc cell lines is probably due to defective relocalization of caspase-3. the expression of caspase-9 and caspase-7 in nsclcs was found to be similar to normal lung tissue. 109 however, these cell lines express the apoptosis inhibitor and splice variant of caspase-9 casp9b. in vitro, chemotherapy-resistant nsclc cell lines exhibit decreased caspase-9 and caspase-3 expression, 110 which suggests an inhibition of apoptosis induction via apoptosome formation in nsclc. additionally, both nsclc and sclc cells express high and almost equal levels of survivin. 107 the resistant nsclc cells showed higher expression of c-iap2, and the radiosensitive sclc cells exhibited increased expression of xiap. 111 these results suggest no correlation between the level of expression of the iaps and the difference in the radiosensitivity between nsclc and sclc cells. cell death has become an area of intense interest and investigation in science and medicine because of the recognition that cell death, in general, and apoptosis, in par-ticular, are important features of many biologic processes. involvement of many genes in the death process suggests that cell death is a complex phenomenon with many redundant mechanisms to ensure defi nitiveness. the realization that defective cell death plays a central role in the pathogenesis of diseases has stimulated work on therapies targeted to these processes, and this work will undoubtedly continue in the future. apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics genetic control of programmed cell death in the nematode c. elegans programmed cell death in animal development apoptosis: defi nition, mechanisms, and relevance to disease apoptosis as a therapeutic target for the treatment of lung disease four deaths and a funeral: from caspases to alternative mechanisms dual signaling of the fas receptor: initiation of both apoptotic and necrotic cell death pathways alkylating dna damage stimulates a regulated form of necrotic cell death a novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles autophagy: in sickness and in health tissue protein turnover during liver carcinogenesis reduced autophagic activity in primary rat hepatocellular carcinoma and ascites hepatoma cells regulation of an atg7-beclin 1 program of autophagic cell death by caspase-8 role of bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes an alternative, nonapoptotic form of programmed cell death paraptosis: mediation by map kinases and inhibition by aip-1/alix autoschizis: a novel cell death inhibition of the development of metastases by dietary vitamin c:k3 combination autoschizis: a new form of cell death for human ovarian carcinoma cells following ascorbate/menadione treatment. nuclear and dna degradation the molecular biology of apoptosis evolutionary conservation of a genetic pathway of programmed cell death interaction between the c. elegans cell-death regulators ced-9 and ced-4 interaction and regulation of the caenorhabditis elegans death protease ced-3 by ced-4 and ced-9 caspases: enemies within vital functions for lethal caspases mechanism of xiapmediated inhibition of caspase-9 insights into the regulatory mechanism for caspase-8 activation a combinatorial approach defi nes specifi cities of members of the caspase family and granzyme b. functional relationships established for key mediators of apoptosis signalling by cd95 and tnf receptors: not only life and death apoptosis control by death and decoy receptors the tnf receptor 1-associated protein tradd signals cell death and nf-kappa b activation fadd, a novel death domain-containing protein, interacts with the death domain of fas and initiates apoptosis caspase-6 is the direct activator of caspase-8 in the cytochrome c-induced apoptosis pathway: absolute requirement for removal of caspase-6 prodomain two cd95 (apo-1/fas) signaling pathways induction of cell death by tumour necrosis factor (tnf) receptor 2, cd40 and cd30: a role for tnf-r1 activation by endogenous membrane-anchored tnf tumor necrosis factor (tnf) receptor 1 signaling downstream of tnf receptor-associated factor 2. nuclear factor kappab (nfkappab)-inducing kinase requirement for activation of activating protein 1 and nfkappab but not of c-jun nterminal kinase/stress-activated protein kinase involvement of mach, a novel mort1/fadd-interacting protease, in fas/apo-1-and tnf receptor-induced cell death the ced-4-homologous protein flash is involved in fas-mediated activation of caspase-8 during apoptosis viral fliceinhibitory proteins (flips) prevent apoptosis induced by death receptors memory and distribution of virus-specifi c cytotoxic t lymphocytes (ctls) and ctl precursors after rotavirus infection fasdependent cd4 + cytotoxic t-cell-mediated pathogenesis during virus infection transcriptional regulation during p21waf1/cip1-induced apoptosis in human ovarian cancer cells activation of c-jun nh2-terminal kinase/stress-activated protein kinase (jnk/ sapk) is critical for hypoxia-induced apoptosis of human malignant melanoma characterization of mice defi cient in interleukin-1 beta converting enzyme reduced ischemic brain injury in interleukin-1 beta converting enzyme-defi cient mice caspase-3 is required for dna fragmentation and morphological changes associated with apoptosis targeted disruption of the mouse caspase 8 gene ablates cell death induction by the tnf receptors, fas/apo1, and dr3 and is lethal prenatally fadd: essential for embryo development and signaling from some, but not all, inducers of apoptosis reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9 the ins and outs of programmed cell death during c. elegans development c-flip(l) is a dual function regulator for caspase-8 activation and cd95-mediated apoptosis the fl ip side of flip apaf-1, a human protein homologous to c. elegans ced-4, participates in cytochrome c-dependent activation of caspase-3 an apaf-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9 apaf1 (ced-4 homolog) regulates programmed cell death in mammalian development autoactivation of procaspase-9 by apaf-1-mediated oligomerization bcl-2 inhibits death of central neural cells induced by multiple agents bcl-2 family proteins role of hypoxia-induced bax translocation and cytochrome c release in reoxygenation injury association of bax and bak homo-oligomers in mitochondria. bax requirement for bak reorganization and cytochrome c release bcl-2 prevents bax oligomerization in the mitochondrial outer membrane mechanisms of caspase activation and inhibition during apoptosis diablo promotes apoptosis by removing miha/xiap from processed caspase 9 iap family proteins-suppressors of apoptosis identifi cation of ciap1 as a candidate target gene within an amplicon at 11q22 in esophageal squamous cell carcinomas the apoptosis inhibitor gene api2 and a novel 18q gene, mlt, are recurrently rearranged in the t(11;18)(q21;q21) associated with mucosa-associated lymphoid tissue lymphomas a novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma a serine protease, htra2, is released from the mitochondria and interacts with xiap, inducing cell death omi/htra2 catalytic cleavage of inhibitor of apoptosis (iap) irreversibly inactivates iaps and facilitates caspase activity in apoptosis hsp27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3 heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the apaf-1 apoptosome negative regulation of cytochrome c-mediated oligomerization of apaf-1 and activation of procaspase-9 by heat shock protein 90 distinctive roles of phap proteins and prothymosin-alpha in a death regulatory pathway caspase-dependent cleavage of signaling proteins during apoptosis. a turn-off mechanism for anti-apoptotic signals caspasemediated proteolysis during apoptosis: insights from apoptotic neutrophils programmed cell death contributes to postnatal lung development granulocyte apoptosis and its role in the resolution and control of lung infl ammation apoptosis is a major pathway responsible for the resolution of type ii pneumocytes in acute lung injury mechanisms of structural remodeling in chronic pulmonary hypertension induction of apoptosis and pulmonary fi brosis in mice in response to ligation of fas antigen essential roles of the fas-fas ligand pathway in the development of pulmonary fi brosis granulocyte apoptosis and the control of infl ammation macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary infl ammation in vivo modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor during the course of acute respiratory distress syndrome g-csf and il-8 but not gm-csf correlate with severity of pulmonary neutrophilia in acute respiratory distress syndrome interleukin-2 involvement in early acute respiratory distress syndrome: relationship with polymorphonuclear neutrophil apoptosis and patient survival resolution of lung infl ammation by cd44 macrophages that have ingested apoptotic cells in vitro inhibit proinfl ammatory cytokine production through autocrine/paracrine mechanisms involving tgf-beta, pge2, and paf phosphatidylserinedependent ingestion of apoptotic cells promotes tgf-beta1 secretion and the resolution of infl ammation recombinant human fas ligand induces alveolar epithelial cell apoptosis and lung injury in rabbits fas expression in pulmonary alveolar type ii cells expression of fas (cd95) and fasl (cd95l) in human airway epithelium natural protection from apoptosis by surfactant protein a in type ii pneumocytes upregulation of gelatinases a and b, collagenases 1 and 2, and increased parenchymal cell death in copd inhibition of vegf receptors causes lung cell apoptosis and emphysema oxidative stress and apoptosis interact and cause emphysema due to vascular endothelial growth factor receptor blockade glucocorticoid-induced apoptosis in human eosinophils: mechanisms of action increased circulating levels of soluble fas ligand are correlated with disease activity in patients with fi brosing lung diseases bleomycin-induced apoptosis of alveolar epithelial cells requires angiotensin synthesis de novo the perforin mediated apoptotic pathway in lung injury and fi brosis interleukin-13 induces tissue fi brosis by selectively stimulating and activating transforming growth factor beta(1) early growth response gene 1-mediated apoptosis is essential for transforming growth factor beta1-induced pulmonary fi brosis united states lung carcinoma incidence trends: declining for most histologic types among males, increasing among females progress in understanding the molecular pathogenesis of human lung cancer loss of expression of death-inducing signaling complex (disc) components in lung cancer cell lines and the infl uence of myc amplifi cation conversion of bcl-2 to a bax-like death effector by caspases differences in expression of pro-caspases in small cell and non-small cell lung carcinoma defective caspase-3 relocalization in non-small cell lung carcinoma increased expression of apaf-1 and procaspase-3 and the functionality of intrinsic apoptosis apparatus in non-small cell lung carcinoma rescue of death receptor and mitochondrial apoptosis signaling in resistant human nsclc in vivo expression of inhibitor of apoptosis proteins in small-and non-small-cell lung carcinoma cells key: cord-002179-v8lpw4r7 authors: viktorovskaya, olga v.; greco, todd m.; cristea, ileana m.; thompson, sunnie r. title: identification of rna binding proteins associated with dengue virus rna in infected cells reveals temporally distinct host factor requirements date: 2016-08-24 journal: plos negl trop dis doi: 10.1371/journal.pntd.0004921 sha: doc_id: 2179 cord_uid: v8lpw4r7 background: there are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. a better understanding of the host pathogen interaction is required to develop effective therapies to treat denv. in particular, very little is known about how cellular rna binding proteins interact with viral rnas. rnas within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication. methodology/principal findings: seventy-nine novel rna binding proteins for dengue virus (denv) were identified by cross-linking proteins to dengue viral rna during a live infection in human cells. these cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in denv amplification. knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. their requirement by denv for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. the protein abundances of these host factors were not significantly altered during denv infection, suggesting their interaction with denv rna was due to specific recruitment mechanisms. however, at the global proteome level, denv altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated. conclusions/significance: the method for identification of host factors described here is robust and broadly applicable to all rna viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral rna within cells. this study significantly increases the number of cellular factors known to interact with denv and reveals how denv modulates and usurps cellular proteins for efficient amplification. dengue is a mosquito-borne viral disease that infects 50-100 million people annually, resulting in dengue fever that is either asymptomatic or flu-like. however, tens-of-thousands of people develop the more severe, and sometimes fatal, dengue hemorrhagic fever/shock syndrome (dhf/dss) [1] . denv is found in most tropical and many subtropical areas with more than 125 countries being endemic for denv [2] . there is no approved vaccine or antiviral therapeutic available for this life-threatening disease. given the seriousness of infection, the expanding geographical range of the denv, and the limitations in the existing measures of control and prevention, there is a pressing need to better understand the biology and pathogenesis of denv. denv is a single-stranded positive-sense rna virus that belongs to the flaviviridae family. it has a 5' cap, no poly(a) tail, highly structured 5'-and 3'-untranslated regions (utr), and a single open reading frame (orf) [reviewed in [3] ]. following virus entry, the viral rna is released into the cytoplasm. viral translation and replication occur in membranous assembly "factories" localized in the perinuclear region of endoplasmic reticulum (er) [4] . the positivestranded rna molecules are encapsidated; virions are further processed as they are transported through the secretory pathway to the cell surface and released extracellularly [reviewed in [3] ]. in addition to the viral proteins, cellular proteins, termed host factors, participate in most, if not all, steps of the denv life cycle, including entry, translation, replication, virion assembly, and release [5] . since viruses require host factors for efficient amplification, targeting host factors can provide an effective antiviral target for which the virus has no genetic control over. therefore, it may be more difficult for viruses to evolve escape mutants that can replicate efficiently in the absence of the host factor [5, 6] . several cellular proteins are known to impact denv infection. for example, the polypyrimidine-tract-binding protein (ptbp1) is relocalized from the nucleus to the cytoplasm following denv infection where it enhances denv amplification by binding to the denv 3'utr and to ns4a, a viral protein required for the formation of the viral replication complex [7] [8] [9] [10] . ptbp1 may also stimulate denv translation [8] , although this is still controversial [7, 9] . while most of the known denv rna binding proteins enhance viral amplification, several reduce denv titers [10] [11] [12] . one such factor, ybx1, inhibits viral translation [12] . although previous studies have laid a foundation for establishing critical interactions between viral rna and cellular proteins [ [13] and reviewed in [14] ], the host factors identified thus far likely represent only a fraction of the total network of denv host factors. previously, we have described a high-throughput mass spectrometry method termed tux-ms (thiouracil cross-linking mass spectrometry) to identify host factors that interact with viral rna during a live infection in cell culture [15] . importantly, tux-ms allows for identification of proteins that are bound directly to the viral rna in living cells. briefly, during a viral infection in cell culture, thiouridine is biosynthetically incorporated into the viral rna to serve as a zero-distance cross-linker upon exposure to ultraviolet (uv) light. thus, proteins that are bound directly to the viral rna during a live infection are cross-linked to the rna prior to disruption of cellular compartmentalization. this is particularly valuable for the identification of denv host proteins, since denv amplification is tightly associated with cellular membrane structures [4] . following cross-linking, the viral rna together with cross-linked proteins are isolated under denaturing conditions and identified by mass spectrometry-based proteomics. using tux-ms, we reported previously the successful identification and validation of host factors for poliovirus, pointing to a low false discovery rate of < 12% [15] . here, we expanded the tux-ms methodology for use with other types of rna viruses, and investigated rna-protein interactions during denv infection. we modified the method to use virus-specific dna oligos to capture the viral rna and cross-linked proteins. furthermore, we used metabolic labelling with stable isotopes to accurately quantify relative protein levels. this quantitative thiouridine cross-linking mass spectrometry (qtux-ms) analysis identified 79 novel host proteins, which were not previously shown to be involved in denv infection. we placed these findings in the context of whole proteome changes upon denv infection, and further validated and functionally analysed a subset of the novel denv host factor candidates. overall, validation of the qtux-ms identified factors using secondary assays indicates a low rate of false positives (17%), suggesting that the majority of the other identified qtux-ms factors may also play significant roles in denv viral amplification. hela uprt cells expressing uracil phosphoribosyltransferase (uprt) were generated previously by transduction of hela cells (atcc, ccl-2) with uprt-gene containing lentivirus [15] . huh7.5 uprt cells were generated by transduction of huh7.5 cells (a kind gift from charles m. rice, rockefeller university) with the same lentiviral construct as in [15] . hela uprt and huh7.5 uprt cells were cultured at 37°c and 5% co 2 in complete dulbecco's modified minimum essential medium (dmem) supplemented with 10% fbs (fetal bovine serum; atlanta biologicals) and penicillin-streptomycin and grown with 1 mg/ml g418 (sigma) to select for the uprt gene; huh7.5 uprt cells were additionally supplemented with non-essential amino acids (cellgro). for silac labelling huh7.5 uprt cells were passed 1:10 at least twice in silac dmem (thermo scientific) with 10% dialyzed heat-inactivated fbs (thermo), l-proline (200 mg/l) and penicillin-streptomycin, and either 50 mg/l 'heavy' (13c6 l-lysine and 13c6-15n4 l-arginine; cambridge isotope laboratories, inc.) or 40 mg/l 'light' l-lysine and l-arginine amino acids [16] . dengue virus serotype 2 (denv2), strain 16681 (genebank accession number u87411) was kindly provided by dr. robert striker (university of wisconsin-madison). denv2 was propagated in the mosquito c6/36 cells at 28°c and 5% co 2 in advanced dmem supplemented with 10% fbs, penicillin-streptomycin (cellgro), l-glutamate and 10% tryptose phosphate broth (20 g/l tryptose; 2 g/l glucose; 5 g/l sodium chloride and 2.5 g/l disodium hydrogen phosphate). titers for denv2 were determined using plaque assays in bhk cells. for infections, cells were incubated with virus containing media for 2 hours, washed twice with the dmem media after removal of the virus and incubated in the serum-free dmem for the indicated time. infections and titer determination of adenovirus 5 (ad5) were performed exactly as in [15] . the denv antisense biotin-labelled dna fragments were generated using pcr and primers listed in (s1 table) from the denv2 complementary dna (cdna) and correspond to positions 4350-4914 and 4740-4914 of denv genome. pcr was followed by removal of the unlabelled dna strand according to the nanolink streptavidin magnetic beads (solulink) manual. the mixture of two biotinylated single-stranded dna fragments of 174 base pairs (bp) and 564 bp long were bound to nanolink streptavidin magnetic beads magnetic beads according to the manufacturer's protocol. 1-3 x 10 7 human hepatoma huh7.5 uprt cells labelled with either 'light' or 'heavy' silac media were infected with denv2 (moi = 10) or mock-treated, respectively. then, virus was replaced with silac media with 1 mm 4-thiouracil and 10% fbs. at 28 hours post-infection (hpi) the cells were washed with pbs and irradiated at 365 nm uv light for 20 min, collected, cell pellets were frozen on dry ice and stored at -80°c. cell pellets were lysed in the lysis buffer (50 mm tris-hcl ph 8, 4 mm magnesium chloride, 150 mm sodium chloride, 0.1% tween-20, 5 mm dithiothreitol, recombinant rnasin ribonuclease inhibitor [500 units/ml; promega], 1× complete protease inhibitor cocktail [roche]), with a half volume of 465-600 μm glass beads (sigma) by shaking at frequency of 30 hz for 1 min using a retsch mm200 mixer mill. an aliquot of 'light' and 'heavy' cell lysates were removed and the remaining lysates were incubated with the streptavidin magnetic beads labelled with denv antisense dna fragments for 15 to 30 min allowing for viral rna to bind. the beads were washed twice with wash buffer i (50 mm tris-hcl ph 8, 500 mm potassium chloride, 0.1% tween-20), once with wash buffer ii (50 mm tris-hcl ph 8, 150 mm sodium chloride, 0.5% sodium deoxycholate) and once with 10 mm tris ph 7.6. the samples were eluted at 65°c for 2 min in 20 μl of 10 mm tris ph 8.0. the eluted 'light' and 'heavy' samples were mixed at a 1:1 ratio, rna was degraded with 0.5 ng/μl bovine rnase a (fisher scientific) at 25°c for 24 hrs and subjected to quantitative mass spectrometry-based proteomic analysis. quantitative mass spectrometry-based proteomic analysis of rnainteracting proteins (qtux-ms) protein eluates and their respective mixed light/heavy input lysates were subjected to in-solution enzymatic digestion using a filter-aided sample preparation approach [17] , then analyzed by nlc-ms/ms, as described in s1 methods and in [18] . light (denv2) and heavy-labelled (mock) cell pellets were lysed in 100 mm abc containing 5% sodium deoxycholate at 95°c to ensure denaturation and virus inactivation. the protein concentrations were determined by the bca assay and mixed in equal protein amounts (100 μg total). proteins were subjected to in-solution digestion with trypsin, then fractionated and analyzed by nlc-ms/ms as described in the s1 methods. proteomic data analysis qtux-ms, its respective mixed input lysate, and the whole cell silac instrument raw data were separately processed using the maxquant software (ver. 1.5.3.8), configured with default settings, except for experiment-specific parameters, which are described in the s1 methods. the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride [19] partner repository with the dataset identifier pxd003593. using the filtered list of protein identifications, unique gene symbols were used for downstream functional ontology analyses. the gene ontology annotations from uniprot were used to generate and assign the denv2 rna interacting candidates into broader functional categories. for the whole cell silac protein expression study, genes and their associated ratios were analyzed by panther gene enrichment (panther database ver 10.0, 2015-05-15) [20] using the panther pathway and protein class ontologies. significant differential protein abundance was determined as a function of ontological classification versus the overall population (bonferroni-corrected p-value < 0.05). for specific functional protein ontologies that were differentially regulated, a subset were selected for analysis by the reactome functional interaction (fi) network cytoscape plugin [21] . the reactome fi plugin was used to visualize candidate host factors identified by qtux-ms. sirna transfections 2 x 10 6 hela uprt cells were transfected with 350 pmol of silencer select negative control (ambion) or the specified sirnas (s1 table) using the xtreamgene sirna transfection reagent system (roche). 24 hrs later, 4 x 10 5 cells/well were plated for infection. 48 hours post transfection cells were infected (moi = 0.1) with denv2 or ad5 (n = 3). at either 30 (ad5) or 40 (denv2) hpi, virus titers were determined by plaque assays using 911 or bhk cells, respectively. knockdown efficiency was measured 48 hrs post transfection by rt-qpcr. experiments were performed in two biological replicates for each host factor. statistical analysis was performed using student's t-test. cell viability and proliferation assay 24 hrs post sirna transfection equal numbers of cells (either 2 x 10 3 or 8 x 10 3 ) were seeded in 96-well plates. cell viability was measured 48 hours after sirna-mediated knockdown of individual host factors using the vybrant mtt [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide] assay kit (invitrogen) according to the manufacturer's protocol and reported relative to the negative-control sirna (set to 100%; n = 3). statistical analysis was performed using student's t-test. cdna was generated from 1μg trizol (ambion) purified total rna using moloney murine leukemia virus (mmlv) reverse transcriptase (promega) as described by the manufacturer using random primers (invitrogen). qpcr was performed using iq sybr green supermix (bio-rad) with the primers listed in the s1 table. the amplification efficiency for each primer set was 100±10% as determined using a standard curve. development of a quantitative thiouridine cross-linking mass spectrometry (qtux-ms) method for identification of proteins associated with the denv rna tux-ms can be used to identify host factors by incorporating 4-thiouridine (4su), a zero-distance cross-linker, into the viral rna (vrna) to enable cross-linking of proteins bound to vrna during a live infection in cell culture [15] . cross-linking is carried out under physiological conditions prior to cell lysis to ensure specificity and reduce false-positives from non-specific rna protein interactions that occur upon loss of compartmentalization. vrna is isolated under denaturing conditions and cross-linked proteins are identified using liquid chromatography-tandem mass spectrometry (lc-ms/ms). to improve quantification of the tux-ms identified host proteins (qtux-ms), a silac (stable isotope-labelled amino acids in cell culture) approach [16] was used to label the uninfected (mock) and infected cells with either 'heavy' or 'light' amino acids, respectively ( fig 1a) . when 4-thiouracil (4tu) is present in the medium, huh7.5 uprt human hepatoma cell lines stably expressing uprt (uracil phosphoribosyltransferase) convert 4tu to ump. then, the ump is converted to thiouridine triphosphate (4sutp) by cellular kinases [22] . both cellular and viral rna polymerases use 4sutp as a substrate during rna synthesis, which serves as a zero-distance cross-linker, covalently binding proteins to rnas upon exposure to long wave uv-light. importantly, protein-protein crosslinking is very inefficient at long uv wavelengths, ensuring that only proteins in direct contact with the reactive thiol group of the 4su-containing rna will be cross-linked [23] . we have shown previously that immunoisolation of candidate vrna-binding proteins identified by tux-ms (and confirmed by western) could be specifically co-isolated with viral rna [15] . together, this study established that tux-ms can identify bona fide interactions between host proteins and viral rna. the tux-ms method was originally developed to capture polyadenylated rna using oligo(dt) beads [15] . however, as denv rna is not polyadenylated, we modified the method to use sequence specific capture of the vrna using magnetic beads. following crosslinking in huh7.5 uprt cells infected with denv at 28 hpi and affinity capture of the vrna, the ribonucleoprotein complexes were eluted from the beads, 'heavy' and 'light' eluates were mixed, and rnase a was used to degrade the rna. the proteins were digested insolution with trypsin and subjected to quantitative ms-based proteomics ( fig 1b) . the median 'light' to 'heavy' peptide and protein ratios were calculated, reflecting the specificity of vrnaprotein capture. we identified several classes of proteins, including denv proteins, known denv host factors, and putative rna-interacting host proteins, but most of the qtux-ms identified factors have not been previously identified through interactions with denv (s2 table) . consistent with previous knowledge of flaviviral rna [24] [25] [26] [27] , our qtux-ms analysis identified several viral proteins-c, e, ns3, ns4a and ns5-as associated with vrna. in addition, qtux-ms identified six known denv host factors: polypyrimidine tract-binding protein 1 (ptbp1), interleukin enhancer-binding factor 3 (ilf3), calreticulin (calr), calnexin and heterogeneous nuclear ribonucleoproteins hnrnp h1 and hnrnp k, as well as a known denv anti-viral protein-eukaryotic initiation factor 4a (eif4a)-and 12 other proteins previously shown to associate with denv rna or proteins (s2 table) . altogether, since several known host factors were identified using qtux-ms, this suggests that the adapted method is effective at identifying host factors for denv. for identification of novel host factors, cellular proteins with a denv/mock silac ratio of 1.5-fold (n 3 quantified peptides) were considered putative denv vrna interactions. this threshold was selected when considering the median variance in the silac ratio (for (blue) amino acids are infected with denv or treated with mock, respectively, in the presence of 4tu. 4su is incorporated into cellular and denv rnas and proteins are uv cross-linked to the contacting thio-containing rna (represented as either balls to indicate native conformation or curved lines to indicate denatured proteins) in living cells at 28 hpi prior to cell lysis under denaturing conditions. viral ribonucleoprotein complexes were isolated using dna molecules complementary to denv rna bound to magnetic beads, the rna was degraded with rnase a and the proteins were identified by mass spectrometry. (b) workflow for quantitative proteomic analysis of rna-bound host factors isolated in (a). isolated proteins were mixed between mock and virus-infected samples, digested into peptides, and analysed by mass spectrometry. relative 'light' and 'heavy' peptide abundances were quantified to determine the specificity of interaction. host factor candidates were identified and subjected to functional validation. doi:10.1371/journal.pntd.0004921.g001 proteins with > 3 quantified peptides), which was approximately 25%. therefore, we opted for a conservative cut-off at 50%, representing twice this median value or 1.5-fold. common environmental and non-human cell culture contaminants were excluded, since they existed in only the 'light' silac state. in addition, our qtux-ms samples also contained histones: h3, h4, h2a, h2b, h1.5 and macroh2a.1. in a previous study, histones were shown to play roles in dengue infection [28] . however, their functions were mediated through an interaction with a viral capsid protein and were shown to be independent of rna. in addition, histones are primarily nuclear and highly abundant proteins commonly detected (> 50%) in control affinity purifications compiled across diverse protein-protein interaction studies [29] . for these reasons, histones likely represent non-specific associations rather than denv rna binding factors, and thus were excluded from further analysis. in total, 93 cellular proteins passed these selection criteria, 79 of which have not been previously shown to associate with denv (s2 table, s1 dataset). several of the known denv host factors were enriched but did not meet the stringent inclusion criteria (s2 table) , suggesting that there may be additional host factors below our enrichment threshold (see s1 dataset). importantly, the subset of 79 host factors represents a significant potential expansion in the number of known denv host factors, providing a valuable resource to test for pro-viral or antiviral activities during denv infection. it is well recognized that viral infections can induce significant changes in cellular proteomes [30] [31] [32] and an increase in protein levels during denv infection may contribute to the increased protein capture measured by qtux-ms. to address this, we used silac-ms to quantify proteome (i.e., total protein abundance) changes following denv infection. comparison of qtux-ms and proteome silac ratios showed that the protein abundances for the 93 qtux-ms-identified vrna-binding factors remained largely unchanged (fig 2a, s1 fig and s1 dataset). on average, for these proteins, the denv-induced changes in whole cell abundance were ±1.1-fold, suggesting that their identification by qtux-ms was not due to an increase in their abundance in the cell following denv infection. noteworthy, a retrospective qualitative comparison of the qtux-ms identified factors for denv with those identified in the tux-ms analysis on poliovirus revealed less than 10% were identified for both viruses [15] ( fig 2b) . since the identified proteins are largely denv specific, qtux-ms is not biased towards identifying a sub-set of cellular rna binding proteins. taken together, our results indicate that the enrichment ratios measured by qtux-ms is predominantly due to their specific association with the denv rna. while proteins that bound denv rna did not show significant changes in abundance upon infection, we performed bioinformatics analysis on the complete proteome dataset of whole cell abundance to determine the global proteome effects of denv infection under these conditions. in total, 4,907 host proteins were quantified by silac ms in biological duplicates (s1 dataset). the abundance ratios between biological duplicates were reproducible, with only~2% of the ratios varying by > 50% (fig 3a) . from these duplicates, an average abundance ratio was calculated and the respective proteins were analyzed by panther gene enrichment analysis (s2 fig) [20] . this analysis found systematic up regulation of proteins in the ubiquitin proteasome pathway (upp), comprising 18 members of the 26s proteasome as well as various ubiquitinconjugating enzymes (s3 fig). many of these enzymes are linked to ubiquitin-dependent proteasome degradation, consistent with the current knowledge that the upp is important for production of infectious virions [31, 33, 34 ]. yet, other enzymes, such ube2n and ube2v2, catalyze polyubiquitination at lys-63, which does not lead to proteasome degradation but rather participates in transcriptional activation of target genes and may promote innate immune signaling [35, 36] . in contrast, proteins in the transporter protein class were on average down regulated (fig 3b and s2 fig). assembly of the annotated proteins into reactome protein networks identified several subnetworks with various transporter activities (s4 fig). while the abundances of mitochondrial transporters and nucleoporins were the most consistently decreased, not all transporters were down regulated; for example, the lipoprotein (apo) transporters were increased in expression (fig 3c) . interestingly, the rna binding protein class was significantly down regulated (fig 3b and s2 fig) , though the effect was not as pronounced as the transporter class (fig 3b) . the overall down-regulation of rna binding proteins appears to be driven by changes in cytoplasmic and mitochondrial ribosomal subunits, and proteins involved in rna degradation and processing (s5 fig). nevertheless, the relative protein abundance for the set of 93 (known and putative) denv binding factors identified by qtux-ms was largely unchanged, despite being enriched in rna processing and translation factors (fig 2a) . overall, the quantitative proteome analysis suggests that denv selectively alters the abundance of proteins, and reveals several pathways that could be directly or indirectly modulated in the host response to denv infection. to gain insight into potential molecular mechanisms and biological processes of the 93 qtux-ms identified factors, we performed a functional network-based analysis using the curated human pathway relationships from the reactome database. this analysis revealed a high degree of connectivity, with 62 proteins forming a large interconnected network (fig 4) . the densest network connectivity included proteins involved in rna processing/translation (orange nodes) and dna binding/transcription (blue nodes). several additional proteins with rna and/or translational activities were also identified, but lacked annotation in reactome (orange single nodes). overall, our bioinformatic evaluation further supports the ability of qtux-ms to capture vrna-bound host factors and points to their possible function in denv amplification. since most of the factors were associated with rna processing in the reactome analysis (fig 4) , we focused on these factors for functional analysis of their roles in dengue infection. we have randomly selected six qtux-ms identified host proteins with functions in rna processing/translation, which were enhanced in the denv sample ranging from 1.32-to 2.26-fold (denv/mock). thus, by validating factors that are only modestly enhanced in the qtux-ms analysis this will indicate if the qtux-ms identified factors that are near the cut-off of 1.5-fold are bona fide host factors or not. we assessed the effect of sirna knockdown of these factors (fig 5a) on viral production. hela uprt cells were used for sirna silencing experiments due to higher sirna transfection efficiency compared with huh7.5 uprt cells. knockdown of five out of six qtux-ms identified candidates: non-pou domain-containing octamer-binding protein (nono), embryonic stem cell-specific 5-hydroxymethylcytosine-binding protein (hmces), rbmx (rna-binding motif protein, x chromosome), hnrnp m and hnrnp f significantly decreased denv production, while knockdown of hnrnp l had no effect on denv titer ( fig 5b; s5 fig) . knockdown of ptbp1, a positive control [7, 8] , also resulted in decreased viral titers (fig 5) . for negative controls, we selected two rna binding proteins (ddx39 and hnrnp a0) that were not identified by qtux-ms. denv titers were not altered following silencing of these two proteins, suggesting that only specific rna binding proteins are used by denv. altogether, our data suggests that rbmx, nono, hmces, hnrnp m, hnrnp f are required for viral production. these results demonstrate that qtux-ms is a robust method with a low false discovery rate for high-throughput identification of viral host factors. reduced viral amplification could be due to compromised cell fitness rather than a specific requirement of the virus for a particular host factor. using an mtt assay, we confirmed that knockdown of these factors did not impact cell viability (fig 5c) . as a positive control, knockdown of g3bp2 did reduce cell fitness, as previously shown (s6 fig) [37] . to more rigorously rule out any potential effects of host factor knockdown on cell fitness that would affect viral amplification, an unrelated virus (adenovirus 5), was amplified following knockdowns of the candidate factors. adenovirus 5 replication was not significantly decreased by rbmx, nono, hnrnp m, hnrnp f or hmces sirna knockdown (fig 5b and s6c fig) demonstrating that knockdown of these factors does not affect cell fitness for viral amplification. given that these proteins bind directly to viral rna and are required for viral amplification, rbmx, nono, hnrnp m, hnrnp f and hmces are novel denv host factors. to determine if the host factor is required for a step prior or subsequent to viral replication, vrna was quantified by qrt-pcr in the denv infected cells knocked down for rbmx, nono, hnrnp m, hnrnp f or hmces (fig 6) . as a positive control, knockdown of ptbp1, which is required for denv replication [7] , reduced denv rna levels. similarly, the intracellular vrna levels were reduced in cells knocked down for either hnrnp f, rbmx or hmces. the decrease in dengue rna levels (fig 6) is consistent with the decrease in viral titers ( fig 5b) . thus, hnrnp f, rbmx or hmces are required for the early steps in the viral replication cycle, such as translation, replication or rna stability. in contrast, knockdown of hnrnp m and nono did not change intracellular viral rna levels despite the dramatic decrease in viral titers, suggesting they may play a role downstream of replication. altogether, we have identified and validated five novel host factors for denv, demonstrating that qtux-ms can identify factors that function at different stages of the virus life cycle. an estimated 40% of the world's population is at risk from dengue for which vaccines or antivirals are not yet available. since diagnostic tests can detect denv infection at early stages, administration of antivirals could significantly improve survival rates as viral load is correlated with symptom severity [38] . using antivirals that target host factors may limit the appearance of drug-resistant viruses and may be effective for all denv serotypes and possibly for multiple flaviviruses [5, 6, 39, 40] . the qtux-ms analysis identified 79 novel cellular proteins, for which the majority are distinct from those previously identified for poliovirus using a similar approach [15] . this suggests that unrelated rna viruses have evolved to utilize distinct host rna binding proteins. interestingly, ptbp1 and nono, which were identified in both the poliovirus and denv tux-ms analyses, were shown to be required for production of both viruses (this study, figs 5 and 6) [15, 41, 42] . further analysis of virus-specific and shared host factors will reveal whether unrelated viruses utilize similar or diverse mechanisms to control viral rna replication, processing and packaging within cells. the host factors that enhance amplification of both poliovirus and dengue, (fig 2b) could serve as attractive targets for the development of broad-spectrum antivirals. the novel denv rna interactions identified in our study reveal a large network of cellular proteins which belong to different functional classes primarily associated with the nucleic acid metabolism, including numerous components of splicing, rna processing and translation machineries. these factors likely play direct roles in denv translation, replication or packaging. in addition, we have detected multiple components of cell signaling and stress response, such as several members of 14-3-3 adapter proteins, heat shock proteins and β-catenin. these factors are known to regulate diverse pathways, including host innate immune and cellular homeostasis [43] [44] [45] suggesting their possible role in host antiviral response or viral strategies to subvert the innate immune response. we demonstrated that the majority of the qtux-ms factors that we selected for validation were required for efficient denv amplification (fig 5) . specifically, we found that hnrnp f, hmces and rbmx are required for the early steps in the viral life cycle. in contrast, hnrnp m and nono appear to act downstream of viral rna replication (fig 6) , which may be significant given that both have been shown to be in a complex together [46] . nono and its binding partners are predominantly nuclear, bind rna, and are involved in pre-mrna processing, splicing, and rna transport, as well as in transcriptional activation and repression [47] [48] [49] . interestingly, other nono binding partners: psf/sfpq (polypyrimidine tract-binding protein (ptb)-associated splicing factor) and matrin3 were identified by qtux-ms as well. many of the qtux-ms identified cellular proteins are hnrnps, which encompass a large class of rna binding proteins that either localize to the nucleus or shuttle between the nucleus and the cytoplasm in order to perform multiple functions in rna metabolism, from transcription to rna turnover [50] [51] [52] . importantly, the vast majority of these factors have established roles in viral infections or in modulating the antiviral host response to various viruses [53] [54] [55] [56] , including denv (s2 table and references within). our study establishes that rbmx (hnrnp g), hnrnp f and hnrnp m are required for efficient denv amplification (fig 5b) . since several hnrnps, such as ptbp1 (hnrnp i), hnrnp a1 and hnrnp k, were previously shown to re-localize from the nucleus to the viral replication sites during denv infection [8, 57, 58] , rbmx, hnrnp f, hnrnp m and possibly other nuclear qtux-ms identified factors are also likely to be either actively recruited to the viral replication sites or retained in the cytoplasm upon denv infection. interestingly, the qtux-ms identified hnrnps affect different steps in the denv life cycle (fig 6) , suggesting that they have distinct functions during infection. this is consistent with studies that have suggested that denv rna structures are dynamic during the viral life cycle [59] and may suggest that host factors play an important role in these structural changes. furthermore, we showed that knockdown of some hnrnps (hnrnp a0 and hnrnp l) did not affect dengue viral titers significantly demonstrating that only certain hnrnps are required for denv amplification. since some of hnrnps are known to modulate cellular gene expression in response to dengue infection [60, 61] , we can not rule out that some of the observed effects on virus titers derive from their roles in regulating host mrnas. among the numerous qtux-ms identified factors of interest, our study is the first to demonstrate the involvement of hmces (or c3orf37) in viral infection. while the cellular role of human hmces is currently unknown, the mouse homologue was suggested to be an rnabinding protein and predicted to contain a putative peptidase domain [62, 63] . interestingly, it is possible that the nucleic acid binding domain enhances the protease activity or visa versa as has been shown for other such proteins [64] [65] [66] . for example, adenovirus uses a nucleic acid binding protease to localize the protease activity to the viral substrates [64] . it has been suggested that the protease is recruited to the empty capsid as an inactive protease, then it becomes fully activated once bound to the viral dna inside the virion. using the dna as a guide wire, it moves along the nucleic acid, searching for capsid and core proteins to cleave, which is required to render the viral particle infectious [64, 67, 68] . however, since we observed that knockdown of hmces resulted in a decrease in viral rna, it seems more likely that it might participate in translation, replication or the switch from translation to replication as has been shown for other nucleic acid binding proteases [65, 69] . only one of the qtux-ms identified factors was increased at the protein level in whole cells following denv infection, suggesting that qtux-ms identifications derived from the specific associations to vrna rather than changes in protein abundances. our analysis of the host cell proteome upon infection also revealed interesting changes, including up regulation of proteins in the ubiquitin pathway and down regulation of transporter proteins. the ubiquitin-proteasome pathway is one of two major cellular pathways used to degrade 80 to 90% of proteins. previous studies on denv infected cell lines and patient samples showed that the ubiquitin pathway was upregulated [31, 39, 70] . many groups have consistently shown that the ubiquitin proteasome pathway is critical for amplification of a number of flaviruses, including denv and west nile virus [31, 34, 71, 72] . however, it remains controversial as to which step in viral amplification is affected by ubiquitination, but it appears to be early during internalization or viral genome release [33, 39, 73] . further studies will be required to understand how denv up-regulates the pathway and the mechanism that ubiquitination has in denv amplification. altogether, our study has significantly increased the number of cellular proteins known to interact with the denv rna during a live infection in cells. we have also placed these interactions in the context of proteome abundance changes in the infected cells. a recent study by phillips and colleagues [13] exploited a cross-linking label-free ms approach to identify denv rna associating proteins in cell culture by cross-linking the rna to the proteins using short wavelength uv light and isolating denv rna bound proteins by anti-sense dna affinity capture [74] . while their method identified several denv host factors [12, [75] [76] [77] , the qtux-ms method reported here resulted in improved identification of known dengue host factors and putative denv rna interacting proteins. there could be several reasons for these results, such as, the qtux-ms approach achieves greater cross-linking efficiency by using long wave uv light to form crosslinks to 4-thio-uridines compared to short-wavelength uv light, which is inherently inefficient [78] . moreover, since thio-uridine is a zero distance cross-linker for rna-bound proteins at long uv wavelengths and protein-protein cross-links are not formed at long uv wavelengths, qtux-ms may also have achieved improved specificity, as only proteins in direct contact with the viral rna would be captured [23] . additionally, qtux-ms used an ms-based silac approach to determine which host proteins were specifically enriched in the vrna isolations versus mock. though isotope-labelling is not applicable in all model systems, it does afford greater quantitative accuracy compared to label-free ms strategies [16] . overall, the qtux-ms method identified 93 cellular proteins that bind to denv rna, which include 14 previously known or putative interactions. importantly, five out of the six qtux-ms identified novel factors that were tested were shown to be bona fide host factors. we used robust assays to show that the identified host factors were specifically required for denv amplification and did not merely result in a decrease in cell fitness for viral amplification. future studies will reveal whether the identified factors may also be required for other flavivirus infections that cause life-threatening illnesses, such as yellow fever, west nile, zika, japanese and tick-borne encephalitis. therefore, our data demonstrates that qtux-ms is an effective technique for identifying novel virus host factors that can be used for a broad spectrum of rna viruses by simply designing antisense dna oligonucleotides to allow for efficient sequence-specific isolation of the vrna. supporting information s1 table containing all proteins quantified by qtux-ms, including proteins enriched in denv infection (red highlighted rows) and those that did not meet the specificity threshold. for each protein group, the following are provided (from left to right), uniprot accession number, gene name, protein name, the linear and log2 denv/ mock qtux-ms enrichment ratios (columns d and e), the qtux-ms ratio variability, the number of qtux-ms quantified peptides, the average log2 denv/mock whole cell relative abundance silac ratio, whether this ratio was up (u) or down (d) regulated by more than ± 1.75-fold, the average silac ratio variability, the average number of quantified peptides, the number of razor+unique peptides and sequence coverage for qtux-ms, the protein's molecular weight and sequence length, and the complete fasta header entry for the primary protein group member. columns h-k are cross-referenced from the respective whole cell data (b). nd = not detected. (b) table containing all proteins quantified in whole cell lysates by silac. for each protein group, the following are provided (from left to right), uniprot accession number, gene name, protein name, the log2 denv/mock relative abundance ratios for replicates 1, 2, and the average (columns d and e), whether this ratio was up (u) or down (d) regulated by more than ± 1.75-fold. for each replicate, the following are provide: the silac ratio variability, the number of quantified peptides, the number of razor+unique peptides, and the number of unique peptides, total protein intensity for light (denv) and heavy (mock) conditions, and the overall sequence coverage. the complete fasta header entry is listed for the primary protein group accession number. (xlsx) s1 (a) hela uprt cells were transfected with either control or specific sirnas. 24 hours post transfection cells were counted and seeded (4 x 10 5 cells/well) in 6-well plates in triplicates. 48 hours post transfection cells were infected with denv2 at moi 0.1 and virus released to the media collected 40 hours post infection. denv2 titers were measured using plaque assays. the bars represent average values from triplicate, a standard error is reported. the representative data from one of at least two independent experiments is shown. (b) to verify sensitivity of the mtt assay, we performed sirna knockdown of g3bp2, which is known to bind denv and was previously shown to affect cell viability [37, 75] . relative viability of non-infected cells was measured using an mtt assay (invitrogen) and represented exactly as described in fig 5c. cell viability or proliferation is decreased by knockdown of g3bp2 compared with control sirna (p<0.01). sirna transfection was performed as described in the methods section using previously published sirna sequence [37] . in cells treated with g3bp2-specific sirna but not control sirna, g3bp2 protein was knocked down to the levels undetectable by western analysis using antibodies against g3bp2 (abcam, ab86135). (c). hela uprt cells were seeded 4.0 x 10 5 cells in 60 mm plates and transfected with either control or specific sirnas. 24 hours post transfection cells were infected with ad5 at moi 0.1 and collected 30 hours post infection. sirna knockdown efficiency was determined by measuring respective mrna levels in comparison to β-actin mrna abundance as described in the experimental section and reported relative to control sirna transfection (upper panel). ad5 titers were determined using plaque assays on 911 cells. the bars represent average values from triplicate, a standard error is reported. the representative data from one of at least three independent experiments is shown. (tif) the global distribution and burden of dengue perspectives for the treatment of infections with flaviviridae dengue virus life cycle: viral and host factors modulating infectivity. cellular and molecular life sciences: cmls composition and three-dimensional architecture of the dengue virus replication and assembly sites targeting a host process as an antiviral approach against dengue virus targeting host factors to treat west nile and dengue viral infections the polypyrimidine tract-binding protein is required for efficient dengue virus propagation and associates with the viral replication machinery polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in vero cells polypyrimidine tract-binding protein influences negative strand rna synthesis of dengue virus translation elongation factor-1alpha, la, and ptb interact with the 3' untranslated region of dengue 4 virus rna eukaryotic initiation factor 4ai interacts with ns4a 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ribosomal entry is identical to the nuclear pyrimidine tract-binding protein translation of polioviral mrna is inhibited by cleavage of polypyrimidine tract-binding proteins executed by polioviral 3c(pro) genome-wide rnai screen reveals a new role of a wnt/ctnnb1 signaling pathway as negative regulator of virus-induced innate immune responses the role of the 14-3-3 protein family in health, disease, and drug development. drug discovery today virus-heat shock protein interaction and a novel axis for innate antiviral immunity hnrnp m interacts with psf and p54(nrb) and co-localizes within defined nuclear structures psf and p54(nrb)/nono-multi-functional nuclear proteins kinesin transports rna: isolation and characterization of an rnatransporting granule the multifunctional protein p54nrb/psf recruits the exonuclease xrn2 to facilitate pre-mrna 3' processing and transcription termination advances in experimental medicine and biology hnrnp complexes: composition, structure, and function. 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requires the functional activities of the proteasome appraising the roles of cbll1 and the ubiquitin/proteasome system for flavivirus entry and replication antisense-mediated affinity purification of dengue virus ribonucleoprotein complexes from infected cells quantitative mass spectrometry of denv-2 rna-interacting proteins reveals that the dead-box rna helicase ddx6 binds the db1 and db2 3' utr structures a)-binding protein binds to the non-polyadenylated 3' untranslated region of dengue virus and modulates translation efficiency. the journal of general virology role of human heterogeneous nuclear ribonucleoprotein c1/c2 in dengue virus replication detecting rna-protein interactions by photocross-linking using rna molecules containing uridine analogs we would like to thank those who generously supplied reagents: lncx uprt-myc, tat, and vsvg plasmids (edward mocarski), denv2 (robert striker), and huh 7.5 cells (charlie rice). we would like to thank quynh-mai trinh for technical assistance. conceptualization: ovv tmg imc srt. key: cord-026012-r0w0jbpg authors: tennant, bud c.; hornbuckle, william e. title: gastrointestinal function date: 2014-06-27 journal: clinical biochemistry of domestic animals doi: 10.1016/b978-0-12-396350-5.50013-9 sha: doc_id: 26012 cord_uid: r0w0jbpg this chapter discusses the functions of gastrointestinal tract. the principal functions of the gastrointestinal tract are assimilation of nutrients and excretion of the waste products of digestion. within the gastrointestinal tract, these substances are solubilized and degraded enzymatically to simple molecules, sufficiently small in size and in a form that permits absorption across the mucosal epithelium. the distribution of the different types of secretory cells in the salivary glands varies among species. the mandibular and sublingual glands are mixed salivary glands containing both mucous and serous types of cells, and produce a viscous secretion that contains large amounts of mucus. the cytoplasm of the secretory cells contains numerous zymogen granules that vary in size and number depending on the activity of the gland. these granules contain the precursors of the hydrolytic enzymes responsible for digestion of the major dietary components. the cells of the terminal ducts probably secrete the bicarbonate ion responsible for neutralizing hydrochloric acid that enters the duodenum from the stomach. the digestive system is composed of the gastrointestinal tract or alimentary canal, salivary glands, liver, and exocrine pancreas. the principal functions of the gastrointesti nal tract are assimilation of nutrients and excretion of the waste products of digestion. most nutrients are ingested in a form which is either too complex or insoluble for absorp tion. within the gastrointestinal tract, these substances are solubilized and degraded enzymatically to simple molecules, sufficiently small in size and in a form which permits absorption across the mucosal epithelium. in the following section, the normal biochemi-283 cal processes of intestinal secretion, digestion, and absorption are described. with these in perspective, we then discuss the mechanisms involved in the pathogenesis of the most important gastrointestinal diseases and the biochemical basis for diagnosis and treatment. a. saliva saliva is produced by three major pairs of salivary glands and by small glands distrib uted throughout the buccal mucosa and submucosa. two types of secretory cells are found in the acinar portions of the salivary glands: (1) the mucous cells, which contain droplets of mucus, and (2) the serous cells, which contain multiple secretory granules. in those species which produce salivary amylase, the secretory granules are the zymögen precur sors of this enzyme. a third cell type is found lining the striated ducts. the striations along the basal borders of these cells are caused by vertical infoldings of the cell membrane, a characteristic of epithelial cells involved in rapid movement of water and electrolytes. the primary secretion of the acinar cells is modified by active transport processes of the ductal epithelium. the distribution of the different types of secretory cells in the salivary glands varies among species. the parotid glands of most animals are serous glands which produce a secretion of low specific gravity and osmolarity, containing electrolytes and proteins including certain hydrolytic enzymes. the mandibular (submaxillary) and sublingual glands are mixed salivary glands containing both mucous and serous types of cells and produce a viscous secretion which contains large amounts of mucus (dukes, 1955) . a. mucus. mucus is an aqueous mixture of protein-poly saccharide complexes and glycoproteins (gottschalk, 1972) , which have relatively large amounts of carbohydrate bound to protein. the protein-poly saccharide complexes have long polysaccharide chains containing repeating units bound to a protein core. the glycoproteins contain numerous oligosaccharide residues distributed along the polypeptide chain. one of the most completely studied glycoproteins is mucin from the submaxillary glands of ruminants. the carbohydrate portion is a disaccharide of yv-acetylneuraminic acid (a sialic acid) and 7v-acetylgalactosamine. approximately 800 such disaccharide molecules are present per molecule of mucin (bhavanandan et al., 1964; bertolini and pigman, 1967 ). an enzyme capable of linking protein with hexosamine was demonstrated in sheep submaxillary glands (mcguire and roseman, 1967) . the physiological functions of mucin are closely related to its high viscosity. n-acetylneuraminic acid is the component responsible for the formation of viscous aque ous solutions. at physiological ph, it causes expansion and stiffening of the mucin molecule (gottschalk and thomas, 1961) . the resistance of mucin to enzymatic break down is also due to the presence of disaccharide residues. removal of the terminal yv-acetylneuraminic acid residues by neuraminidase significantly increases the susceptibil ity of peptide bonds to trypsin (gottschalk and fazekas de st. groth, 1960) . b. amylase. the saliva of most species contains the α-amylase ptyalin. this enzyme is said to be absent, however, in the saliva of dogs, cats, and horses (dukes, 1955) . salivary amylase splits the a-1,4-glucosidic bonds of various polysaccharides. the sali vary enzyme is similar in all major respects to pancreatic α-amylase, which is described below (section ii,d). salivary amylase initiates digestion of starch and glycogen in the mouth of those species which secrete the enzyme. the optimal ph for amylase activity is approximately 7, and activity therefore terminates when the enzyme mixes with acidic gastric contents. saliva bathes the oral cavity continuously, serving to protect the surface epithelium. ingested food is moistened and lubricated by saliva, facilitating mastication and swallow ing. the teeth also are protected from decay by saliva, which washes food particles from the surfaces of the teeth and, because of its buffering capacity, neutralizes the organic acids produced by bacteria normally present in the mouth. ruminants produce much greater quantities of saliva than simple-stomached animals, and the saliva has a higher ph and bicarbonate ion concentration. in ruminants, saliva serves several unique functions (phillipson, 1977) . it is required for maintenance of the composition of the contents of the rumen. the great buffering capacity is necessary to neutralize the large amounts of short-chain fatty acids which are the major end products of rumen fermentation. the urea in saliva can be utilized by rumen bacteria for protein synthesis. protein synthesized in the rumen is then used to meet dietary protein require ments. in this way, urea nitrogen can be "recycled" through the amino acid pool of the body and in ruminants need not be considered an end stage in protein catabolism. the ability to reutilize urea has also been demonstrated in the horse and may be of particular benefit during periods of protein deficiency (houpt and houpt, 1971; prior et al., 1974) . the stomach is divided into two main regions on the basis of secretory function (grossman, 1958) . the oxyntic gland area corresponds approximately to the body of the stomach in most species of domestic animals and also to the fundus in the dog and cat. the oxyntic glands contain (1) oxyntic or parietal cells, which are responsible for hydro chloric acid production, (b) peptic (zymogenic, chief) cells, which produce pepsinogen, and (c) mucous cells. the pyloric gland area contains the pyloric glands, which are slightly alkaline, and, in addition to mucus, contains the polypeptide hormone gastrin. a variety of stimuli can initiate gastric secretion. the sight or smell of food or the presence of food within the mouth causes gastric secretion by a reflex mechanism involv ing the vagus nerve. the presence of certain foods within the stomach or distention of the stomach alone also can initiate both intrinsic and vagai nerve reflexes which cause secre tion of gastric juice. in addition to neural reflexes, these stimuli cause release of the polypeptide hormone gastrin from the pyloric gland area, which enters the bloodstream, stimulating gastric secretion. the release of gastrin from the specific g cells responsible for synthesis is inhibited by excess hydrogen ion, and this negative feedback mechanism is 1 fig. 1. amino acid sequence of porcine gastrin i (gregory, 1966) . gastrin ii differs from gastrin i by the presence of a sulfate ester group on the single tyrosyl residue. believed to be of physiological importance in the control of hydrochloric acid production. gastrin has been isolated in pure form from the antral mucosa of swine gregory and tracy, 1964; . when administered intraven ously, the purified hormone causes the secretion of hydrochloric acid and pepsin. it also stimulates gastrointestinal motility and causes pancreatic secretion. two separate peptides have been obtained from porcine gastric mucosa and have been designated gastrin i and gastrin ii. the structure of gastrin has been determined and has been confirmed by synthesis (anderson et al., 1964) . it is a heptadecapeptide amide, with a pyroglutamyl n-terminal residue and the amide of phenylalanine as the c-terminal residue (fig. 1 ). in the center of the molecule is a sequence of five glutamyl residues, which give the molecule its acidic properties. gastrin ii differs from gastrin i only in the presence of a sulfate ester group linked to the single tyrosyl residue. the c-terminal tetrapeptide amide, trp-met-asp-phe-nh 2 , is identical in all species so far studied (gregory, 1967) . the tetrapeptide has all of the activities of the natural hormone. it is not as potent as the parent molecule, but activity can be increased by lengthening the peptide chain. gastrin is the only hormone known to stimulate hc1 secretion (walsh and grossman, 1975) . as indicated above, gastrin is released in response to vagai stimulation by distention of the pyloric antrum and by direct luminal contact with food, particularly partially hydrolyzed protein (walsh and grossman, 1975) . the exact mechanism of action is not known, but studies using isolated preparations of isolated parietal cells suggest that the effects of gastrin are not mediated by cyclic amp (soil, 1977) . some of the other factors which are important in regulation of hc1 secretion are summarized in fig. 2 after dousa and dozois (1977) . there is little doubt that histamine secreted locally within the mucosa has a major effect on the function of parietal cells (soil dousa and dozois, 1977.) and grossman, 1978) . histamine has been recognized as a potent stimulant of hc1 production for many years (code, 1965) . this effect, however, was not inhibited by traditional antihistaminic drugs (hj antagonists), and, until the demonstration of h 2 recep tors in the stomach (the atrium and uterus) by black et al. (1972) , the physiological role of histamine in hc1 secretion was controversial. specific h 2 antagonists (burimamide, cimetidine, metiamide) now have been shown to inhibit the secretory response not only to histamine, but also to gastrin, to cholinergic stimuli, and to food (grossman and konturek, 1974) . although there has been significant conflict in the published literature, current evidence suggests that histamine activates the adenylate cyclase of parietal cells (dousa and dozois, 1977) , resulting in synthesis of cyclic amp and ultimately in hc1 secretion (fig. 2 ). the controversy with regard to the role of cyclic amp as a mediator of histamine action has come from observations that prostaglandins and secretin, both potent inhibitors of gastric hc1 secretion, also stimulate adenylate cyclase (thompson et al., 1977) . it is now believed that prostaglandins, in addition to inhibiting hc1 secretion, act on a mucosal cell population which is different from parietal cells and that these cells secrete cytoprotective substances (mucin, glycosaminoglycans). the ulcerogenic effects of prostaglandin inhibitors (indomethacin, acetylsalicylic acid) apparently result from inhibition of this protective effect of endogenous prostaglandins. a. basal versus stimulated secretion. gastric juice is composed of two compo nents. one is secreted continuously by the surface epithelial cells and other mucusproducing cells. the other component is produced by the oxyntic glands in response to various stimuli. the basal component is neutral or slightly alkaline. the electrolyte composition is similar to that of an ultrafiltrate of plasma (table i) and contains large amounts of mucus, which protects the epithelium. the secretory component produced by the oxyntic glands in response to stimulation contains free hydrochloric acid and pepsinogen, the principal enzyme of gastric digestion. the composition of gastric juice depends on the relative amounts of the two secretory components present, which in turn is a function of flow rate. in the dog, gastric juice is produced in the resting state at a rate of approximately 5 ml/hour (gray and bûcher, 1941) , and the composition is similar to that of the basal component, containing practi cally no peptic activity or hydrochloric acid. when the flow of gastric juice is stimulated maximally, the dog may produce 80 ml or more per hour (gray and bûcher, 1941) , and this secretion contains large amounts of peptic activity and hydrochloric acid. sodium, which is the principal cation in the basal secretion, is replaced to a large extent by hydrogen ion. the concentration of potassium is similar in both basal and stimulated secretions and therefore remains relatively constant at various rates of flow. hydrochloric acid and pepsinogen are secreted by separate mechanisms, but these appear to be closely linked under physiological conditions. stimulation of the vagus nerve (bachrach, 1953; hirschowitz and sachs, 1965) or intravenous injection of gastrin (hirschowitz, 1966) increases pepsinogen and hydrochloric acid levels together. other stimuli may affect the two processes differently. in the dog, for example, histamine infusion stimulates hydrochloric acid production maximally but inhibits pepsinogen secre tion (abrams and brooks, 1960; hirschowitz, 1966; ernas and grossman, 1967) . inhibitor 6 ( n · leu · leu · · · leu · glu (3200 mol. wt.) pepsinogen is the zymögen, or inactive precursor, of pepsin, the principal proteolytic enzyme of gastric juice. pepsinogen was first crystallized from the gastric mucosa of swine (herriott, 1938) , and several pepsinogens have been separated by ryle (1965) , ryle and porter (1959) , and ryle and hamilton (1966) . porcine pepsinogen has a molecular weight of approximately 43,000 and is composed of the pepsin molecule and several smaller peptides (fig. 3) . one of these peptides has a molecular weight of 3200 and is an inhibitor of peptic activity (herriott, 1962) . activation of pepsin from pepsinogen occurs by selective cleav age of this small basic peptide from the parent pepsinogen (neurath and walsh, 1976) . autocatalytic conversion begins below ph 6.o. at ph 5.4, the inhibitor peptide dis sociates from the parent molecule, and, at ph 3.5-4.0, the inhibitor is completely digested by pepsin (taylor, 1968) . pepsin has a very acidic isoelectric point, being stable in acidic solution below ph 6.0 but irreversibly denatured at ph 7.0 or above. in contrast, pepsinogen is stable in neutral or slightly alkaline solution. the optimal ph for peptic activity is generally between 1.6 and 2.5, but the effect of ph may vary with the substrate. pepsin is capable of hydrolyzing peptide bonds of most proteins, mucin being one important exception. pepsin splits bonds involving phenylalanine, tyrosine, and leucine most readily but can hydrolyze almost all other peptide bonds. c. rennin. rennin is another proteolytic enzyme produced by the gastric mucosa and has some characteristics which are similar to those of pepsin. it has been separated from pepsin in preparations from the stomachs of newborn calves. rennin splits a mucopeptide from casein to form paracasein, which then reacts with calcium ion to form an insoluble coagulum. the coagulated milk protein probably delays gastric emptying and increases the efficiency of protein digestion in young calves. d. hydrochloric acid. hydrochloric acid is produced by the oxyntic cells. when the normal mucosa is stimulated, both chloride and hydrogen ions are secreted together, but current evidence suggests that h + and cl" are secreted by separate, closely coupled pump mechanisms. small amounts of cl~ are secreted continuously by the unstimulated parietal cells in the absence of h + secretion, and this mechanism is responsible for the relative negative charge of the resting mucosal surface. hydrogen ion and clsecretory systems may also be differentiated in vitro by the demonstration of hydrogen ion secretion in the absence of cl~. a scheme for the secretion of hydrochloric acid is presented in fig. 4 . for every h + secreted, an electron is removed. the electron ultimately is accepted by oxygen to form oh -, which is neutralized within the cell by h + from carbonic acid. the bicar bonate ion produced enters the venous blood, and this explains why the ph of gastric venous blood frequently is greater than that of arterial blood during hydrochloric acid secretion (davenport, 1966) . conversion of carbon dioxide and water to carbonic acid is catalyzed by carbonic anhydrase, which is present in high concentration within parietal cells. when the rate of acid secretion is high, this enzyme contributes to the secretory mechanism by maintaining normal intracellular ph. carbonic anhydrase inhibitors, such as acetazolamide, interfere with hydrochloric acid production in high concentrations and when the rate of acid secretion is high (janowitz et al., 1952) . bile is secreted continuously by the hepatocytes into the bile canaliculi and is trans ported through a system of ducts to the gallbladder, where it is modified, concentrated, and stored. during digestion, bile is discharged into the lumen of the duodenum, where it aids in emulsification, hydrolysis, and solubilization of dietary lipids. the digestive functions of bile are accomplished almost exclusively by the detergent action of its major components, the bile salts and phospholipids. the primary bile acids are c 24 carboxylic acids synthesized by the liver from choles terol. bile acid formation represents the major pathway for cholesterol metabolism (danielsson, 1963) . cholic acid (3a,7a,12a-trihydroxy-5/3-cholanoic acid) and chenodeoxycholic acid (3a,7a-dihydroxy-5/3-cholanoic acid) are the primary bile acids formed by most species of domestic animals. in swine, chenodeoxycholic acid is hydroxylated at the 6a position by the liver to yield hyocholic acid, which is a major primary bile acid in this species (haslewood, 1964) . bile acids are secreted as amino acid conjugates of either glycine or taurine. taurine conjugates predominate in the dog, cat, and rat. in the rabbit, the conjugating enzyme system appears to be almost completely specific for glycine (bremer, 1956) . both taurine and glycine conjugates are present in ruminants. in the newborn lamb, 90% of the bile acids are conjugated with taurine. as the lamb matures, glycine conjugates increase, accounting for one-third of the total in mature sheep (peric-golia and socie, 1968) . under normal conditions, only conjugated bile acids are present in the bile and in the contents of the proximal small intestine. in the large intestine, the conjugated bile acids are hydrolyzed rapidly by bacterial enzymes so that, in the contents of the large intestine and in the feces, free or unconjugated bile acids predominate. several genera of intestinal bacteria, including clostridium, enterococcus, bacteroides, and lactobacillus (midtvedt and norman, 1967) , are capable of splitting the amide bonds of conjugated bile acids. intestinal bacteria also modify the basic structure of the bile acids. one such reaction is the removal of the a-hydroxyl group at the 7 position of cholic acid or chenodeoxycholic acid. these bacterial reactions yield the secondary bile acids, deoxycholic acid, and lithocholic acid, respectively (gustafsson et al., 1957) . lithocholic acid is relatively insoluble and is not reabsorbed to any great extent (gustafsson and norman, 1962) . deoxycholic acid is reabsorbed from the large intestine in significant quantities and is either rehydroxylated by the liver to cholic acid and excreted (lindstedt and samuelsson, 1959) or excreted as conjugated deoxycholic acid. the extent to which bacteria transform the primary bile acids depends on the nature of the diet, the composition of the intestinal microflora, and the influences which these and other factors have on intestinal motility (gustafsson et al., 1966; gustafsson and norman, 1969a,b) . the carboxyl group of the bile acids is completely ionized at the ph of bile and is neutralized by sodium ion, resulting in the formation of bile salts. the bile salts are effective detergents. they are amphipathic molecules, which have both hydrophobic and hydrophilic regions. in low concentrations, bile salts form molecular or ideal solutions, but, when their concentration increases above a certain critical level, they form polymolecular aggregates known as micelles. the concentration at which these molecules aggregate is called the critical micellar concentration (cmc). bile salt micelles are spherical and consist of a central nonpolar core and an external polar region. fatty acids, monoglycerides, and other lipids are solubilized when they enter the central core of the micelle and are covered by the outside polar coat. solubilization occurs only when the cmc is reached. for the bile salt-monoglyceride-fatty acid-water system present during normal fat digestion, the cmc is approximately 2 mm, which is ordinarily exceeded both in bile and in the contents of the upper small intestine (hofmann, 1963) . phospholipids, principally lecithin, are also major components of bile. in the lumen of the small intestine, pancreatic phospholipase catalyzes the hydrolysis of lecithin, forming free fatty acid and lysolecithin. the latter compound also is a potent detergent which acts with the bile salts to disperse and solubilize lipids in the aqueous micellar phase. the enterohepatic circulation begins as conjugated bile acids near the duodenum and mix with the intestinal contents, forming emulsions and micellar solutions. the bile acids are not absorbed in significant amounts from the lumen of the proximal small intestine. absorption occurs primarily in the ileum (lack and weiner, 1961 weiner, , 1966 weiner and lack, 1962) , where an active transport process has been demonstrated . the conjugated bile acids pass unaltered into the portal circulation (playoust and isselbacher, 1964) and return to the liver, where the cycle begins again. this arrangement provides optimal concentrations of bile acids in the proximal small intestine, where fat digestion and absorption occur, and then efficient absorption after these functions have been accomplished. absorption of unconjugated bile acids from the large intestine ac counts for 3-15% of the total enterohepatic circulation (weiner and lack, 1968) . in dogs, the total bile acid pool was estimated to be 1.1-1.2 gm. the half-life of the bile acids in the pool ranged between 1.3 and 2.3 days, and the rate of hepatic synthesis was 0.3-0.7 gm/day (wollenweber et al., 1965) . the daily requirement for bile acids greatly exceeds the normal synthetic rate. this necessitates repeated reutilization of the bile acids, which is accomplished by means of the enterohepatic circulation. under steady-state conditions, the entire bile acid pool passes through the enterohepatic circulation approxi mately ten times each day (hofmann, 1966) . the size of the bile acid pool is dependent upon diet, the rate of hepatic synthesis, and the efficiency of the enterohepatic circulation. surgical removal of the ileum in dogs interrupts the enterohepatic circulation, causing an increase in bile acid turnover rate and a reduction in the size of the bile acid pool (playoust et al., 1965) . in diseases of the ileum, there may be defective bile salt absorption and bile salt deficiency. if severe, impaired utilization of dietary fat may occur, resulting in steatorrhea and impaired absorption of the fat-soluble vitamins. the exocrine pancreas is an acinous gland with the same general structure as the salivary glands. the cytoplasm of the secretory cells contains numerous zymogen granules, which vary in size and number depending on the activity of the gland. these granules contain the precursors of the hydrolytic enzymes responsible for digestion of the major dietary components. the cells of the terminal ducts probably secrete the bicarbonate ion responsible for neutralizing hydrochloric acid which enters the duodenum from the stomach. /. composition a. electrolyte composition. the cation content of pancreatic secretion is similar to that of plasma. sodium is the predominant cation, with smaller concentrations of potas sium and calcium being present. a unique characteristic of pancreatic juice is its high bicarbonate ion concentration and alkaline ph. in the dog, the ph ranges from 7.4 to 8.3, depending on hc0 3~ content. the volume of pancreatic juice is directly related to hc0 3~ content and ph increase and the cl~ concentration decreases. the sodium and potassium ion concentrations and osmolarity appear to be independent of secretory rate (fig. 5) . b. a-amy läse. the amylase produced by the pancreas catalyzes the specific hy drolysis of a-l,4-glucosidic bonds, which are present in starch and glycogen (a-1,4glycan-4-glycan hydrolase). pancreatic amylase appears to be essentially identical to the amylase of saliva. it is a calcium-containing metalloenzyme (vallee et al., 1959) . re moval of calcium by dialysis inactivates the enzyme and markedly reduces the stability of the apoenzyme. pancreatic amylase has an optimal ph for activity of 6.7-7.2 and is activated by chloride ion. synthesis of pancreatic α-amylase occurs in the ribosomes. the enzyme is transferred -rasmussen et al., 1956.) from the endoplasmic reticulum to cytoplasmic zymogen granules for storage (redman et al., 1966) . it is secreted in active form upon stimulation of the acinar cells. newborn calves (huber et al., 1961) and pigs (walker, 1959 ) secrete amylase at a significantly lower rate than mature animals. the rate of synthesis is also influenced by diet. animals fed a high-carbohydrate diet synthesize amylase at several times the rate of animals on a high-protein diet (ben abdeljlil and desnuelle, 1974) . unbranched a-1,4-glucosidic chains, such as those found in amylase, are hydrolyzed in two steps. the first is rapid and results in formation of the disaccaride maltose and maltotriose. the second step is slower and involves hydrolysis of maltotriose with forma tion of glucose and maltose. polysaccharides such as amylopectin and glycogen contain branched chains with both a-\ ,4-and a-\ ,6-glucosidic linkages. when α-amylase attacks these compounds, the principal products are maltose (a-l,4-glycosidic bond), isomaltose (a-l,6-glucosidic bond), and small amounts of glucose. final hydrolysis of the maltose and isomaltose occurs at the surface of the mucosal cell, where the enzymes maltase and isomaltase are integral parts of the microvillous membrane. c. proteolytic enzymes. the proteolytic enzymes of the pancreas are responsible for the major portion of protein hydrolysis, which occurs within the lumen of the gastrointes tinal tract. two types of peptidases are secreted by the pancreas. trypsin, chymotrypsin, and elastase are endopeptidases, which attack peptide bonds along the polypeptide chain, producing smaller peptides. the exopeptidases attack either the carboxy-terminal or amino-terminal peptide bonds, releasing single amino acids. the principal exopeptidases secreted by the pancreas are carboxypeptidases a and b. the endopeptidases and exopep(table ii) , producing free amino acids, which are absorbed directly, or small peptides, which are further hydrolyzed by the aminopeptidases of the intestinal mucosa (see section iii,c). the pancreatic peptidases are secreted as inactive proenzymes or zymogens termed trypsinogen, chymotrypsinogen, and procarboxypeptidase a and b. trypsinogen is con verted to active trypsin in two ways. at alkaline ph, trypsinogen can be converted autocatalytically to trypsin, the activated enzyme converting more zymögen to active enzyme. trypsinogen can also be activated by the enzyme enter okinase, which is pro duced by the duodenal mucosa. the latter reaction appears to be highly specific in that enterokinase will not activate chymotrypsinogen. chymotrypsinogen, proelastase, and the procarboxypeptidases a and b are converted to active enzymes by the action of trypsin. the amino acid sequences and other structural characteristics of bovine trypsinogen and chymotrypsinogen have been determined (hartley et al., 1965; hartley and kauffman, 1966; brown and hartley, 1966) . the polypeptide chain of trypsinogen contains 229 amino acid residues. activation of the proenzyme occurs with hydrolysis of a single peptide bond located in the 6 position between lysine and isoleucine. the c-terminal hexapeptide is released as enzyme activity appears. there is also substantial change in the helical structure of the parent molecule (davie and neurath, 1955; neurath et al., 1956) . chymotrypsinogen a is composed of 245 amino acid residues and has numerous structural similarities to trypsinogen. activation of the chymotrypsinogen also occurs with cleavage of a single peptide bond. for a complete discussion of this subject, see the review by keller (1968) . d. lipase. the pancreas produces several lipolytic enzymes with different substrate specificities. the most important of these from a nutritional viewpoint is the lipase re sponsible for hydrolysis of dietary triglycéride. this enzyme has the unique property of requiring an oil-water interface for activity so that only emulsions can be effectively at tacked (sarda and desnuelle, 1958) . the principal products of lipolysis are glycerol, monoglycerides, and fatty acids. the monoglycerides and fatty acids accumulate at the oil-water interface and can inhibit enzyme activity. their transfer from the interface to the aqueous phase is favored by the presence of sodium bicarbonate also secreted by the pancreas and by bile salts. mattson and volpenhein (1966) described two other carboxylic ester hydrolases in pancreatic juice. both enzymes have an absolute requirement for bile salts, in contrast to glycerol ester hydrolase, which is actually inhibited by bile salts at ph 8. one of these enzymes is a sterol ester hydrolase responsible for hydrolysis of cholesterol esters. the other enzyme hydrolyzes various water-soluble esters. the two enzyme activities have been differentiated on the basis of stability and optimal ph. the pancreas secretes a third lipolytic enzyme which hydrolyzes phospholids. phospholipase a converts lecithin which is present in bile to lysolecithin, an effective deter gent which aids in emulsification of dietary fat. pancreatic secretion is controlled and coordinated by neural and endocrine mechanisms. when ingesta or hydrochloric acid enters the duodenum, the hormone secretin is released into the circulation by the duodenal mucosa. secretin increases the volume, ph, and hc0 3~ concentration of the pancreatic secretion. secretin is a polypeptide hormone which contains 27 amino acid residues. all 27 amino acids are required to maintain the helical structure of the molecule and its activity (bodanszky et al., 1969) . the c-terminal amide is a property of other polypeptide hormones, such as gastrin and vasopressin, which act on the flow of water in biological systems (mutt and jorpes, 1967) . in addition to its effects on the pancreas, secretin increases the rate of bile formation (wheeler and mancusi-ungaro, 1966) . the pancreatic juice which results from stimulation by secretin is large in volume and has high bicarbonate concentration but is low in enzyme activity. stimulation of the vagus nerve causes a significant rise in enzyme concentration. this type of response also is produced by pancreozymin, another polypeptide hormone secreted by the duodenal mu cosa. pancreozymin is now believed to be identical to cholecystokinin, an intestinal hormone which causes contraction of the gallbladder (thompson, 1969) . the c-terminal pentapeptide of pancreozymin-cholecystokinin is exactly the same as that of gastrin. this fascinating relationship suggests that gastrin and pancreozymin-cholecystokinin may par ticipate in some unified but as yet poorly understood system of digestive control (thompson, 1969) . during the past several years, a large number of papers have been published on the endocrine function of the gastrointestinal mucosa and on several new polypeptides which are being classified as gut hormones (table iii) . many of these new substances have not met the rigid physiological requirements for true hormone status, including (1) biological action in very small concentration, (2) release into the bloodstream, and (3) normal serum levels comparable to those provided experimentally by exogenous administration. these criteria probably will be modified, particularly with regard to requirements for transport in the vascular system. a large class of peptides are under investigation which have paracrine rather than endocrine activities; that is, their actions are on cells and tissues in the immediate vicinity of the cells of origin. motilin is a polypeptide containing 22 amino acids that was originally isolated from porcine duodenal mucosa (brown et al., 1971) . the amino acid composition and se quence have been described (brown et al., 1972 (brown et al., , 1973 . immunoreactive motilin has been found in the enterochromaffin cells of the duodenum and jejunum of several species (polak et al., 1975) and, by means of radioimmunoassay, motilin has been identified in the plasma of dogs (dryburgh and brown, 1975) . motilin has been shown to stimulate pepsin output and motor activity of the stomach (brown et al., 1971) and to induce lower esophageal sphincter contractions (jennewein et al., 1975) . studies by itoh et al., (1978) suggest that motilin plays an important role in initiating interdigestive gastrointestinal contractions. somatostatin, which is named for its growth hormone release-inhibiting activity, was first purified from bovine hypothalamus (brazlua and guilleman, 1974) . somatostatin also has been demonstrated in the stomach, pancreas, and intestinal mucosa in concen trations higher than in the brain (pearse et al., 1977) . somatostatin is a potent inhibitor of insulin and glucagon release. it also inhibits gastrin release and gastric acid secretion (barros d'sa et al., 1975; bloom et al., 1974) , apparently acting independently on parietal cells and on g cells. these and a variety of other physiological effects suggest that somatostatin has important gastrointestinal regulatory functions (pearse et al., 1977) . enteroglucagon is the hyperglycémie, glycogenolytic factor isolated from the intestinal mucosa. it occurs primarily in the distal small intestine and colon in at least two forms, one with a molecular mass of 3500 dal tons and the other somewhat larger (val verde et al., 1970) . enteroglucagon differs from pancreatic glucagon biochemically, immunologically, and in its mode of release. the physiological function of enteroglucagon is not known, but its release from the mucosa following a meal and the associated increase in circulating blood levels have suggested a regulatory role on bowel function (pearse et al., 1977) . enteroglucagon also differs significantly from glucagon produced by the a cells of the gastric mucosa of the dog (sasaki et al., 1975) . canine gastric glucagon is biologi cally and immunochemically identical to pancreatic glucagon. gastric glucagon appears to be unique to the dog, similar activity not being observed in the stomach of the pig or the abomasum of cattle and sheep (sutherland and de du ve, 1948) . the microvillous membrane of the intestinal mucosa, like other cell membranes, is a lipid structure which acts as a barrier to water and water-soluble substances. water and polar solutes penetrate in one of two ways. (1) they may pass through pores in the membrane, which are believed to be aqueous channels connecting the luminal surface of the cell with the apical cytoplasm. the "effective" diameter of jejunal pores has been estimated to be approximately 0.4 nm (lindemann and solomon, 1962) . (2) they may attach to membrane carriers, which facilitate passage through the lipid phase of the membrane. transport of water and water-soluble compounds is influenced by the permeability characteristics of the limiting membrane and by the nature of the driving forces which provide energy for transport. passive movement occurs either by simple diffusion or as a result of gradients in concentration (activity), ph, osmotic pressure, or electrical potential which may be present across the membrane. the passive movement of an ion in the direction of an electrochemical gradient is referred to as single-file diffusion (hladky, 1965) . when a substance moves in a direction opposite that of an established electrochem ical gradient, an active transport process is said to be responsible. most water-soluble compounds, such as monosaccharides and amino acids, cannot diffuse across the intestinal mucosal membrane at rates which are adequate to meet nutritional requirements. transport of these substances is believed to be by means of membrane carriers. the nature of thse carriers is not well understood, but they are believed to be an integral part of the membrane and responsible for binding the transported substance in a rather specific way. their existence is based primarily on kinetic evidence. carrier-mediated transport systems can be saturated and are competitively inhibited by related compounds. three types of carrier transport mechanisms are recognized (curran and schultz, 1968 ). (1) active transport, as stated previously, involves movement of electrolytes against an electrochemical gradient. in the case of nonelectrolytes, such as glucose, active transport is defined as movement against a concentration gradient. active transport requires metabolic energy and is inhibited by various metabolic blocking agents or by low tempera ture. (2) facilitated diffusion occurs when the passive movement of a substance is more rapid then can be accounted for by simple diffusion. facilitated diffusion systems can increase the rate of movement across the membrane by two or three orders of magnitude. the carrier mechanism is similar to that involved in active transport in that it displays saturation kinetics, may be inhibited competitively, and is temperature dependent. how ever, transport does not occur against concentration or electrochemical gradients, and direct expenditure of energy is not required. (3) exchange diffusion is a transfer mechanism similar to facilitated diffusion. it was postulated originally by ussing (1947) to explain the rapid transfer of radioactive na + across cell membranes. the mechanism does not give rise to net transport but contributes in a major way to unidirectional flux rates, which are measured with isotopie tracers. in the intestine, net water absorption is the result of bulk flow through pores in the membrane. diffusion in the usual sense plays no important role in a water movement (section iii,a,4). when bulk flow occurs, it is possible for solutes to move across the membrane in the direction of flow by a phenomenon called solvent drag. the effect of solvent drag on the transport of a given solute depends on the rate of volume flow and upon the reflection coefficient, which is an expression of the relationship between the pore radius and the radius of solute molecule being transported. a solute such as urea can be transported by the intestine against a concentration gradient by means of solvent drag (hakim and lifson, 1964) . studies with isotopie tracers have shown that transport of water and electrolytes by the intestinal mucosa is a dynamic process, with rapid unidirectional fluxes of the substances occurring continuously in both directions. net absorption occurs when the flow from lumen to plasma exceeds that in the opposite direction (code et al., 1960; berger et al., 1959; hindle and code, 1962) . active transport of na + can occur along the entire length of the intestine, but the rate of absorption is greatest in the ileum and colon, where most net sodium and water absorption occurs. sodium transport is believed to be accomplished by an energy-requiring "sodium pump. " the characteristics of this pump are not completely understood, but skou (1965) presented evidence that the pump is intimately related to the activity of a na + -in dependent adenosine triphosphatase located within the cell membrane. this enzyme is inhibited by cardiac glycosides, such as oubain, which also are effective inhibitors of na + transport, and it has been suggested that this enzyme system may actually be the pump. in the jejunum, net absorption of sodium occurs slowly unless nonelectrolytes, such as glucose or amino acids, are absorbed simultaneously. in in vivo studies by fordtran et al. (1968) , jejunal absorption of sodium appeared to be explained, in part, by solvent drag which was associated with active glucose transport. in the ileum, na + absorption was independent of glucose absorption. water absorption in the jejunum also appears to be almost entirely dependent upon the absorption of glucose, while absorption from the ileum is unaffected by glucose (barry et al., 1961) . the differential effect of glucose on absorption from the jejunum and ileum appears to be the result of fundamental metabolic differences between these two areas of the intestine (curran, 1960; gilman and koelle, 1960) . as sodium is transported across the mucosal membrane, an equivalent amount of anion must be transported simultaneously to maintain electrical neutrality. a significant amount of chloride ion absorption can be accounted for on this basis. it is generally agreed that chloride transport in the intestine is a passive process (clarkson et al., 1961) , although active secretion by the gastric mucosa seems well established. the intestinal mucosa can, under certain circumstances, absorb cl" independently of cation absorption and maintain electrical neutrality by exchange secretion of bicarbonate into the lumen (ingraham and visscher, 1936). dietary potassium is absorbed almost entirely in the proximal small intestine. absorp tion appears to be a passive process since movement across the mucosa occurs down a concentration gradient (high luminal concentration to a low concentration in plasma). the fluid which reaches the ileum from the jejunum has a potassium concentration and a sodium/potassium ratio which is similar to that of plasma. in the ileum and colon, the rate of sodium absorption is much greater than that of potassium so that, under normal conditions, the sodium/potassium ratio in the feces is much lower than that of plasma, approaching a ratio of 1. the absorption of water has been one of the most extensively studied aspects of intestinal transport. it is now generally agreed that water movement is the result of bulk flow through membranous pores and that simple diffusion plays only a minor role. the question of whether water is actively or passively transported has been the subject of considerable controversy, and the controversy itself points to the fundamental difficulties which arise in trying to establish a definition of active transport. hypertonie saline so lutions can be absorbed from canine intestine in vivo (grim, 1962) and from canine and rat (parsons and wingate, 1961) intestine in vitro. these observations indicate that water absorption can occur against an activity gradient and that the process is dependent upon metabolic energy. this would suggest that an active transport process is involved. curran (1965) , however, presents an alternate interpretation which is now generally accepted. this view is that water transport occurs secondarily to active solute transport and is the result of local gradients established within the mucosal membrane. water transport is then coupled to the energy-dependent process responsible for solute transport but is one step removed from it. in the dog and probably other carnivores, the ileum is the main site of net sodium and water absorption. the colon accounts for no more than perhaps 20% of the total. in the case of herbivorous animals in which the large intestine is developed extensively, net secretion of water may occur in the ileum so that all net absorption of water must take place in the cecum and colon (powell et al., 1968; argenzio, 1975) . carbohydrate is present in the diet primarily in the form of polysaccharides of glucose. the most common polysaccharides are starch, glycogen, and cellulose. starch and glycogen are composed of long chains of glucose molecules linked together by repeating a-1,4-glucosidic bonds. branching chains are linked by a-1,6-glucosidic bonds. in those species which secrete salivary amylase, digestion of starch and glycogen begins in the mouth when this enzyme mixes with food. the action of salivary amylase is interrupted in the stomach, however, because of the low ph of the gastric secretion. starch digestion begins again in the proximal small intestine with the action of pancrea tic amylase. this enzyme catalyzes a series of stepwise hydrolytic reactions, resulting in formation of the principle end products of starch digestion, the disaccharides maltose and isomaltose, and small amounts of glucose. glucose is absorbed directly by the intestinal mucosa and transported to the portal vein. the disaccharides are broken down further by hydrolytic enzymes of the brush border. b. cellulose. cellulose, like starch, is a polysaccharide of glucose but differs from starch in that the glucose molecules are linked by ß-1,4-glucosidic bonds. starch can be utilized by all species, but cellulose is utilized as a source of energy only by animals which have extensive bacterial fermentation within the gastrointestinal tract. ruminant species digest cellulose most efficiently, but other animals in which the large intestine is well developed also can utilized cellulose to some degree. in ruminants, hydrolysis of cellulose is accomplished by cellulytic bacteria, which are part of the complex rumen microflora. the end products of cellulose fermentation are short-chain fatty acids-acetic, propionic, and butyric acids. these are absorbed directly from the rumen and serve as the major source of energy for ruminants. propionic acid is the major precursor for synthesis of carbohydrate. maltose and isomaltose are the disaccharides (glucose-glucose) produced as end prod ucts of starch digestion. the diet also contains lactose (galactose-glucose) and sucrose (fructose-glucose). it once was believed that disaccharides were hydrolyzed within the (1968) malathi (1967) malathi (1967) eichholz (1967), forstner et al. (1968) eichholz (1967), forstner et al. (1968) lumen of the intestine by enzymes secreted by the mucosa. there is now general agree ment, however, that disaccharide digestion is completed at the surface of the cell by disaccharidases (gray, 1975) , which are components of the brush border (table iv) . this is considered a form of intracellular digestion (ugolev, 1965) . the disaccharidases have been solubilized from the brush border and partially purified. two separate maltases have been isolated (auricchio et al., 1965) . isomaltase and sucrase have been separated and purified together as a two-enzyme complex (kolinskâ and semenza, 1967) . the mucosa also contains two enzymes with lactase activity. one of these is a nonspecific /3-galactosidase which hydrolyzes synthetic /3-galactosides effec tively but which hydrolyzes lactose at a slow rate. this enzyme has an optimal ph of 3 and is associated with the lysozomal fraction of the cell. the other lactase hydrolyzes lactose readily. it is associated with the brush border fraction of the cell and is the enzyme which is important in the digestive process (alpers, 1969) . maltase, isomaltase, and sucrase are almost completely absent from the intestine in newborn pigs dahlqvist, 1961) and calves (huber et al., 1961) . the activity of these disaccharidases increases after birth and reaches adult levels during the first months of life. lactase activity is highest at birth and decreases gradually during the neonatal period. the relatively high lactose activity seems to be an advantage to the newborn in utilizing the large quantities of lactose present in the diet. by water and demonstrated lactase deficiency following acute enteric infections and suggested that lactose utilization may be decreased in such cases. a. specificity of monosaccharide transport. regardless of whether monosaccharides originate in the lumen of the intestine or are formed at the surface of the mucosal cell, transport across the mucosa involves processes which have a high degree of chemical specificity. glucose and galactose are absorbed from the intestine more rapidly than other monosaccharides. fructose is absorbed at approximately one-half of the rate of glucose, and mannose is absorbed at less than one-tenth the rate of glucose (kohn et al., 1965) . glucose and galactose can be absorbed against concentration gradients and are said, by definition, to be actively transported. active absorption requires metabolic energy and can be inhibited by a variety of substances which block oxidative phosphorylation. the monosaccharides that are transported most efficiently against concentration gradients have certain common structural characteristics, which were summarized by wilson (1962) . these include (1) the presence of a pyranose ring, (2) a carbon atom attached to c-5, and (3) a hydroxyl group at c-2 with the same stereoconfiguration as d-glucose. these features once were believed to be necessary for active monosaccharide transport, but recent observations suggest that they are not absolute requirements. both d-xylose, which has no substituted carbon atom at c-5, and d-mannose, which lacks the appropriate hydroxyl configuration at c-2, can be transported against concentration gradients under proper experimental conditions (csâky and lassen, 1964; csâky and ho, 1966; alvarado, 1966b) . most current concepts imply that, during the initial phase of monosaccharide absorption, the monosaccharide molecule attaches to a mobile carrier located within the cell membrane . the evidence for such membrane carriers comes from kinetic studies of the overall transport process. the rate of glucose absorption is independent of luminal concentration over a rather wide range, but a maximal rate of absorption can be demonstrated at very high concentrations. this limitation of transport is believed to be due to saturation of binding sites on the membrane carrier. glucose transport is competitively inhibited by galactose (cori, 1925; fisher and par sons, 1953) and by a variety of substituted hexoses, which compete with glucose for carrier binding sites. the glucoside phlorizin is a very potent inhibitor (parsons et al., 1958; alvarado and crane, 1962) . phlorizin also competes for binding sites but has a much higher affinity for these sites than does glucose. the absorptive surface of the mucosal cell is the microvillous membrane, or brush border (figs. 6a and 6b). it is through this part of the plasma membrane that glucose must pass during the initial phase of mucosal transport. techniques have been developed for isolating highly purified preparations of microvillous membranes from mucosal homogenates forstner et al., 1968) . faust et al. (1967) studied the binding of various sugars to these isolated membrane fractions. they found that d-glucose was bound by the membrane preferentially to l-glucose or to d-mannose and that glucose binding was completely inhibited by 0.1 mm phlorizin. the specificity of their observa tions suggested that binding represented an initial step in glucose transport, namely, attachment to a membrane carrier. c. sodium requirement. the absorption of glucose and other monosaccharides is influenced significantly by sodium ion (schultz and curran, 1970; kimmich, 1973) . when sodium is present in the solution bathing the intestinal mucosa, glucose is absorbed rapidly, but, when sodium is removed and replaced by equimolar amounts of other cations, glucose absorption virtually stops (riklis and quastel, 1958; csâky, 1961; bihler and crane, 1962; bihler et al., 1962) . glucose abosrption is inhibited by oubain, digitalis, and other cardiac glycosides which are also inhibitors of na-k-dependent adenosine triphosphatase activity and sodium transport (csâky and hara, 1965; schultz and zalusky, 1964) . these observations suggest a close relationship between the transport of glucose and sodium. on the basis of their own observations, crane and co-workers (1965) sug gested that sodium ion acts directly upon the membrane carrier to increase affinity of the carrier for glucose. csâky (1963) interprets the apparent coupling of sodium transport to the transport of various nonelectrolytes as being due to the need to maintain a critical intracellular sodium concentration, which, in turn, is essential for conversion of metabolic energy (atp, etc.) to energy for transport. the initial step in protein digestion is enzymatic hydrolysis of peptide bonds with formation of smaller peptides and amino acids. the endopeptidases (proteases) hydrolyze peptide bonds within the protein molecule and also hydrolyze certain model peptides. exopeptidases hydrolyze either the carboxy-terminal (carboxypeptidase) or the aminoterminal (aminopeptidase) amino acids of peptides and certain proteins. dietary proteins first come in contact with proteolytic enzymes in the stomach. the best known of the gastric proteases is the family of pepsins (samloff, 1971) , which attack most proteins with the exception of keratins, protamines, and mucins. pepsins are relatively nonspecific endopeptidases and split peptide bonds involving many amino acids. the most readily hydrolyzed peptide bonds are those of leucine, phenylalanine, tyrosine, and glutamic acid (ryle, 1965; ryle and hamilton, 1966; meyer and kelly, 1977) . the extent of proteolysis in the stomach depends on the nature of the dietary protein and the length of time spent in the stomach. the food bolus mixed with saliva has a neutral or slightly alkaline ph as it enters the stomach, and a certain period of time is necessary for it to mix with gastric secretions and become acidified. proteolytic digestion begins when the ph of the gastric contents approaches 4 and occurs optimally in two ph ranges, 1.6-2.4 and 3.3-4.0 (taylor, 1959a,b) . because of the relative lack of specificity of the pepsins, some peptide bonds of almost all dietary proteins are split during passage through the stomach. the gastric phase of protein digestion appears to have only a minor and probably dispensable role in overall protein assimilation (freeman and kim, 1978) . the reservoir function of the stomach, however, contributes to the gradual release of nutrients, insuring more efficient utilization in the small intestine. partially digested protein passes from the stomach to the duodenum, where the acidic contents are neutralized by sodium bicarbonate secreted in the bile and pancreatic juices. peptic activity persists in the duodenum only during the period required to raise the ph above 4.0. the major peptidase activity in the lumen of the small intestine comes from the pancreatic enzymes trypsin, chymotrypsin, elastase, and carboxypeptidases a and b. the action of these enzymes is integrated so that the endopeptidases produce peptides with c-terminal amino acids which are appropriate substrates for the exopeptidases. trypsin produces peptides with basic c-terminal amino acids which are particularly suited for the action of carboxypeptidase b. chymotrypsin produces peptides with aromatic amino acids in the c-terminal position, and elastase produces peptides with c-terminal amino acids which are nonpolar. carboxypeptidase a hydrolyze both types of c-terminal peptide bonds (table ii) . the intestinal mucosa contains a broad range of aminopeptidases which complete the process of protein digestion (heizer and laster, 1969) . most of the aminopeptidase activity is found in the soluble fraction of the cell (newey and smyth, 1960), but a small fraction is tightly bound to the microvillous membrane and appears to serve a digestive function at the cell surface similar to that described for the disaccaridases (rhodes et al., 1967 ). an endopeptidase from the intestinal mucosa was studied by hsu and tappel (1965) using hemoglobin as substrate. over 95% of the activity was located in the particulate fraction of the cell. the association with other acid hydrolases suggests that this is a lysozomal enzyme, and its relationship to the normal process of protein digestion is not known. despite the long interest in and controversy regarding the subject of this section, the relative amounts of the various types of protein digestion products, i.e., peptides and amino acids, which are actually absorbed by intestinal mucosal cells during normal digestion are still not known. it is a difficult process to investigate from a kinetic standpoint because the products of proteolysis are absorbed rapidly after they are formed. studies of luminal contents, therefore, give only an estimate of the overall rate of protein digestion. in addition, dietary protein is continually mixed with endogenous protein in the form of digestive secretions and extruded mucosal cells. endogenous protein is hydrolyzed and the amino acids absorbed in a manner similar to that of dietary protein, and the two processes occur simultaneously. endogenous protein accounts for a significant part of the amino acids of the intestinal contents (nässet and ju, 1961) . even when the dietary protein is labeled with a radioactive tracer, there is such rapid utilization that the tracer soon reenters the lumen in the form of endogenous protein secretion. in adult mammals, protein is not absorbed from the intestine in quantities of nutritional significance without previous hydrolysis. most neonatal animals absorb significant amounts of immunoglubin and other colostral protein, but this capacity is lost soon after birth (see section iii,a,4 below). the intestinal mucosa is not totally impermeable to large polypeptide molecules, however. the absorption of insulin (mw 5700) (laskowski et al., 1958; danforth and moore, 1959) , ribonuclease (mw 13,700) (alpers and isselbacher, 1967), territin (bockman and winborn, 1966) , and horseradish peroxidase (cor nell et al., 1971 ) has been demonstrated. the intestine produces a part of the plasma /3-globulin. this is believed to be the result of de novo synthesis of protein, however, presumably from individual amino acid precursors. during the digestion of protein, the amino acid content of the portal blood increases rapidly. attempts to demonstrate parallel increases in the level of peptides in the portal blood have not been successful (levenson et al., 1959) . this has sometimes been taken as evidence that only amino acids can be absorbed by the intestinal mucosa and that the absorption of peptides does not occur. while it seems clear that a significant part of the dietary protein is absorbed in the form of free amino acids, peptides also may be taken up by the mucosal cell. evidence of the mucosal uptake of peptides came originally from experiments with isolated loops of intestine (wiggans and johnston, 1959; newey and smyth, 1959) . various peptides were placed in solutions bathing the mucosa and analyses made sub sequently of the serosal fluid. with the exception of small amounts of glycylglycine, peptides were never found on the serosal side, but free amino acids were found in significant quantities. the final steps to peptide digestion appear to be associated with mucosal epithelial cells. almost all of the aminopeptidase activity is associated with the mucosa, and very little activity is present in luminal contents (lindberg, 1966) . as described above, mucosal aminopeptidase activity is located in the cytosol and in the brush border mem brane fractions of the epithelial cell (heizer and laster, 1969; kim et al., 1972) . these physically separate enzymes have remarkably different substrate specificities (kim et al., 1974) . the brush border enzyme has more than 50% of the activity for tripeptides, yet less than 10% of the total activity for dipeptides relative to the cytosolic enzyme(s) (peters, 1970; kim et al., 1972) . almost all activity for tetrapeptides is present in the brush border (freeman and kim, 1978) . proline-containing peptides are hydrolyzed almost exclusively by cytosolic peptidases, whereas leucine aminopeptidase activity is located primarily in the brush border. from these studies, it appears that, in the intact animal, peptides are absorbed in physiologically important quantities by intestinal mucosal cells and hydrolyzed either at the cell surface or intracellularly to constituent amino acids. the individual amino acids then are transported to the apical part of the cell and finally enter the portal circulation. amino acids, like glucose and certain other monosaccharides, are absorbed and trans ferred to the portal circulation by active transport processes. the same type of saturation kinetics observed in studies of monosaccharide absorption are observed with amino acids, suggesting carrier transport mechanisms. certain monosaccharides inhibit amino acid transport (saunders and isselbacher, 1965; newey and smyth, 1964) . inhibition generally has been of the noncompetitive type, but alvarado (1966a) demonstrated competitive inhibition between galactose and cycloleucine, suggesting that some form of common carrier may be involved. most amino acids are transported against concentration and electrochemical gradients, and the overall transport process requires metabolic energy. the chemical specificity of these transport mechanisms is demonstrated by the observation that the natural / forms of various amino acids are absorbed more rapidly than the corresponding d forms, and only the /-amino acids appear to be actively transported. sodium ion is necessary for absorp tion of amino acids as it is for a variety of other nonelectrolyte substances (schultz and curran, 1970; gray and cooper, 1971) . separate transport systems appear to exist for different groups of amino acids. each member of a group inhibits the transport of other members competitively, suggesting that they share the same binding site. there is some overlap between groups, indicating that, in the overall transport process, certain steps may be common to all amino acids and other steps more specific (saunders and isselbacher, 1966; matthews and laster, 1965; wise man, 1968) . these groups are the following: 1. monoaminomonocarboxylic (neutral) amino acids, including histidine. these amino acids show mutual competition for transport and have the greatest requirement for na + . 2. monoaminodicarboxylic amino acids. aspartic and glutamin acids are not trans ported against concentration gradients. following uptake, they are transaminated, and, under physiological conditions, almost all of the aspartic and glutamic acid enters the portal blood as alanine. 3. dibasic amino acids, including lysine, arginine, ornithine, and the neutral amino acid cystine. these amino acids are apparently transported by the same transport system. 4. proline, hydroxyproline, the n-substituted glycine derivatives n-methylglycine (sarcosine), and n-dimethylglycine, and betaine. proline and hydroxyproline also can be transported by the first mechanism but the affinity of both amino acids for the nadependent pathway is low. the γ-glutamyl cycle has been proposed as a possible transport system for amino acids (meister and tate, 1976) . γ-glutamyltransferase (ggt) is a membrane-bound en zyme which is present in a number of mammalian tissues and catalyzes the initial step in glutathione degradation. the γ-glutamyl moiety of glutathione is transferred to amino acid (or peptide) receptors with the production of cysteinylglycine: glutathione + amino acid ^τ γ-glutamyl-amino acid + cys-gly the highest ggt activity is present in tissues which are known to transport amino acids actively, e.g., the jejunal villus and the proximal convoluted tubule of the kidney. meister and his colleagues (1976) have suggested that ggt may function in translocation by interaction with extracellular amino acids and with intracellular glutathione. the hypothet ical mechanism involves the noncovalent binding of extracellular amino acids to the plasma membrane, while intracellular glutathione interacts with ggt to yield a γ-glutamyl enzyme. when the γ-glutamyl moiety is transferred to the membrane-bound amino acid, a γ-glutamyl-amino acid complex is formed and, when released from the membrane binding site, moves into the cell. the γ-glutamyl-amino acid complex is split by the action of γ-glutamylcyclotransferase, an enzyme appropriately located in the cytosol. glutathione is regenerated by means of the γ-glutamyl cycle, which are good substrates for ggt (thompson and meister, 1975) . the γ-glutamyl cycle does not require sodium, and the previously demonstrated sodium dependence for amino acid transport would not be explained by the cycle. the cycle is not considered to be the only amino acid transport system, and its quantitative significance in individual tissues is unknown. certain nutrient cell types which are deficient in ggt have been shown to transport amino acids normally. at birth most domestic species, including the calf, foal, lamb, pig, kitten, pup, and infant, absorb significant quantities of colostral protein from the small intestine (brambell, 1958; walker and isselbacher, 1974) . γ-globulin either is absent in the serum of these species at birth, or is at a low level. within a few hours after ingestion of colostrum, the serum γ-globulin level rises. this is the principal mechanism by which the young of the above-listed species acquire maternal immunity. under normal environmental con ditions, ingestion of colostrum is an absolute requirement for the health of these species during the neonatal period (fig. 7) . in the neonatal calf, immunoglobulin deficiency has a role in the pathogenesis of gram-negative bacterial septicemia (smith, 1962; gay, 1965; roberts et al, 1954) . most calves deprived of colostrum develop septicemia early in life but may develop diarrhea before death (smith, 1962; roberts et al., 1954; wood, 1955; tennant et al, 1975) . hypogammaglobulinemia is almost always demonstrable in calves dying of gramnegative bacterial septicemia (fey, 1971) , and hypogammaglobulinemia is believed to be due to insufficient immunoglobulin intake or to insufficient intestinal absorption. the factor in colostrum that protects against systemic infections is the igm fraction (penhaie et al, 1971) . serum immunoglobulin values of neonatal calves vary, and a 10% incidence of hypogammaglobulinemia may occur in clinically normal calves (tennant et al, 1969a; house and baker, 1968; smith et al, 1967; thornton et al, 1972; braun et al, 1973) . most hypogammaglobulinemic individuals probably had insufficient colostrum intake. even when calves were given the opportunity to ingest colostrum, a surprising number were hypogammaglobulinemic. some of the reasons for varying gammaglobulinemia values are recognized, but the relative importance of each reason is not known. the concentration of lactoglobulin, the volume consumed (bush et al, 1971; selman et al, 1971 ) , the time elapsed from birth to ingestion of colostrum , and the method of ingestion (natural suckling versus bucket feeding) may have an important influence on the serum γ-globulin (smith et al, 1967; mcbeath et al, 1971) . calves that suckle their dams usually attain serum γ-globulin concentrations that are higher than those attained by calves given colostrum from a bucket. the frequency of hypogamma globulinemia may be influenced by seasons (gay et al., 1965b; mcewan et al., 1970a) , although this relationship has not always been observed (smith et al., 1961; thornton et al., 1972) . familial factors also influence hypogammaglobulinemia (tennant et al., 1969a) . regardless of cause, the mortality of hypogammaglobulinemic calves is higher than that of calves with normal serum γ-globulin values (gay, 1965; house and baker, 1968; thornton et al., 1972; mcewan et al., 1970a; boyd, 1972; naylor et al., 1977) . in addition to having more septicémie infections (smith, 1962; gay, 1965a; roberts et al., 1954; wood, 1955; fey, 1971; mcewan et al., 1970a) , hypogammaglobulinemic calves have a greater prevalence of acute diarrheal disease (boyd, 1972; naylor et al., 1977; penhale et al., 1970; gay et al., 1965) ; the local protective effects of immunoglobulin in the intestine apparently are important (fisher et al., 1975; . the prevalence of hypogammaglobulinemia and the high mortality associated with it has led to the development of several rapid tests for identification of hypogamma globulinemic calves (mcbeath et al., 1971; aschaffenburg, 1949; fisher and mcewan, 1967a; patterson, 1967; stone and gitter, 1969) . the zinc sulfate turbidity test (kunkel, 1947) was the first to be used for determination of serum immunoglobulin concentrations of neonatal calves (mcewan et al., 1970) . a close correlation has been established between test results and the amount of serum igg and igm (fisher and mcewan, 1967a,b; mcewan et al., 1970b; penhale et al., 1967) . the sodium sulfite turbidity test is similar to the zinc sulfate test and also has been used to identify hypogammaglobulinemic calves (stone et al., 1969; pfeiffer and mcguire, 1977) . failure of turbidity to develop when serum is added to a saturated solution of sodium sulfite indicates immunoglobulin deficiency, and semiquantitative assessment of the immunoglobulin concentration may be made by grading the degree of turbidity (stone and gitter, 1969) . the refractometer is used as a rapid test for immunoglobulin deficiency (mcbeath et al., 1971; boyd, 1972) . the close relationship between the concentration of γ-globulin and that of total serum protein in neonatal calves was described previously (tennant et al., 1969a) , and the wide variation in total protein concentration was due to differences in γ-globulin concentration. direct linear correlation between the serum protein concentra tion (refractive index) and the immunoglobulin concentration also has been described (mcbeath et al., 1971) . the equation for the regression line in that report was virtually identical to that observed recently (tennant, et al., 1978) . the y intercepts in our study and in that previously reported were identical (4 gm/dl). the refractometer has value as a rapid field instrument for the assessment of immunoglobulin status, but in cases of hemoconcentration it has limitations (boyd, 1972) . the glutaraldehyde coagulation test was used originally for the detection of hypergammaglobulinemia in cattle, using whole blood (sandholm, 1974) . glutaraldehyde reagent also has been used in a semiquantitative test to evaluate γ-globulin in canine (sandholm and kivisto, 1975) and human serum (sandholm, 1976) . we modified this procedure to detect hypogammaglobulinemic calves ( table v) . calves that had a negative test result (serum γ-globulin ^0.4 gm/dl) had markedly higher mortality than did calves with posi tive results (table vi) (tennant, et al., 1979) , findings similar to those obtained by using the zinc sulfate turbidity test (gay et al., 1965a; mcewan et al., 1970a) . many tests can be initiated at one time using the glutaraldehyde coagulation test, and all results can be evaluated rapidly without instrumentation (tables v and vi) . protein enters the absorptive cell by pinocytosis and passes across the cell to the lymphatics. the process is not selective because many proteins other than the immune globulins can be absorbed (payne and marsh, 1962a,b) . the ability to absorb intact protein is lost by domestic species within 1 or 2 days following birth. in rodents, protein absorption normally continues for approximately 3 weeks. the mechanism of intestinal "closure" was studied by lecce and co-workers (1964; lecce, 1966 ; lecce and morgan, a samples of serum were obtained at birth, but no follow-up of calves was made. 0 the death rate of calves that were test negative was significantly (p < 0.01) greater than that of testpositive calves, using t test for significance of differences between two percentages. 1962). they found that complete starvation of pigs lengthened the period of protein absorption to 4-5 days, whereas early feeding shortened the period. feeding different fractions of colostrum including lactose and galactose resulted in loss of protein absorptive capacity. the route of feeding may not be the critical factor, however. calves prevented from eating but which receive nutrients parenterally lose the ability to absorb protein at the same time as control calves (deutsch and smith, 1957). a. luminal phase. the fat present in the diet is primarily in the form of triglycérides of long-chain fatty acids. the initial step in utilization of triglycérides occurs in the lumen of the proximal small intestine, where hydrolysis is catalyzed by pancreatic lipase. this enzyme, which is secreted in active form, requires an oil-water interface for activity so that only emulsions are attacked (sarda and desnuelle, 1958) . enzyme activity is directly related to the surface area of the emulsion. the smaller the emulsion particle, the greater the total surface area of a given quantity of triglycéride and the greater the rate of hydrolysis (benzonana and desnuelle, 1965) . bile salts are not an absolute requirement, but they favor hydrolysis (1) by their detergent action, which causes formation of emul sions with small particle sizes, and (2) by stimulating lipase activity within the physiologi cal ph range of the duodenum (borgström, 1954 (borgström, , 1964a . a colipase is present in the pancreatic secretion which facilitates the interaction of lipase with its triglycéride substrate and protects lipase from inactivation (borgström and erlanson, 1971) . pancreatic lipase splits the ester bonds of triglycérides preferentially at the 1 and 3 positions (sari et al., 1966) , so that the major end products of hydrolysis are 2-monoglycerides and nonesterified fatty acids (mattson et al., 1952; volpenhein, 1962, 1964) . both compounds are relatively insoluble in water but are brought rapidly into micellar solution by the detergent action of bile salts. the mixed micelles so formed have a diameter of approximately 2.0 nm (borgström, 1964b; laurent and persson, 1965) and are believed to be the form in which the products of fat digestion are actually taken up by the mucosal cell (hofmann and small, 1967) . the intraluminal events which occur in fat absorption are schematically summarized in fig. 8. b. mucosal phase. the initial step in fat transport is the uptake of fatty acids and monoglycerides by the mucosal cell from micellar solution. just how this occurs is not completely clear, but present evidence suggests that the lipid contents of the micelle are somehow discharged at the cell surface so that they enter the cell in molecular rather than micellar form . the net effect is the absorption of the end products of lipolysis with the exclusion of bile salts, which are absorbed farther down the intestine, primarily in the ileum (lack and weiner, 1963) . uptake of fatty acids appears to be a passive process having no requirement for metabolic energy (johnston and borgström, 1964; strauss, 1966) . within the mucosal cell, the fatty acids are transported by a soluble binding protein to the endoplasmic reticulum, where the fatty acids and monoglycerides are rapidly reesterified to triglycéride (ockner and manning, 1974; ockner and isselbacher, 1974) . the two biochemical pathways for triglycéride biosynthesis in the intestine are summarized in fig. 9 . direct acylation of monoglyceride occurs in the intestine (senior and isselbacher, 1962) and probably is the major pathway for lipogenesis in the intestine during normal fat absorption (kern and borgström, 1965; mattson and volpenhein, 1964) . the initial step in this series of reactions involves activation of fatty acids by acyl-coa synthetase, a reaction which requires mg 2+ , atp, and coa (dawson and isselbacher, 1960; clark and hübscher, 1960, 1961; brindley and hübscher, 1965 ) and which has a marked specificity for long-chain fatty acids (dawson and isselbacher, 1960; brindley and hübscher, 1965) . this specificity appears to explain the observation by bloom et al. (1951) that mediumand short-chain fatty acids are not incorporated into triglycérides during intestinal trans port but enter the portal circulation as nonesterified fatty acids. the activated fatty acids (from isselbacher, 1966.) then react sequentially with mono-and diglycerides to form triglycérides in steps catalyzed by mono-and diglyceride transacylases (ailhaud et al., 1964) . the enzymes responsible for this series of reactions were partially purified by rao and johnston (1966) from the microsomal fraction of the cell. they observed that purification of the separate enzyme activities occurred simultaneously, suggesting that these enzymes occur together in the endoplasmic reticulum as a "triglyceride-synthetase" complex. an alternate route which is available for fatty acid esterification involves la-glycerophosphate, which may be derived from glucose or from dietary glycerol by the action of intestinal glycerokinase (haessler and isselbacher, 1963; clark and hübscher, 1962) . activated fatty acid coa derivatives react with l-a-glycerophosphate to form lysophosphatidic acid (monoglyceride phosphate), which by a second acylation forms phosphatidic acid (diglyceride phosphate). phosphatidic acid phosphatase then hydrolyzes the phosphate ester bond, forming diglyceride, and by means of a transacylase step similar to that described in the previous paragraph, triglycéride can then be formed. although this pathway appears to be one of minor importance for triglycéride synthesis in the intestine, johnston (1968) pointed out the importance of certain of the intermediates in this sequence of reactions in the synthesis of phospholipids which are necessary for stabilization of the chylomicron. the next step in fat transport is formation of chylomicrons within the endoplasmic reticulum. the chylomicron is composed primarily of triglycéride and has an outer mem branous coating of cholesterol, phospholipid, and protein (zilversmit, 1965) . the js-lipoprotein component of the chylomicron is synthesized by the intestinal mucosal cell (isselbacher and budz, 1963; hatch et al., 1966; windmueller and levy, 1968) . inhibi tion of protein synthesis by puromycin or acetoxycycloheximide interferes with chylomic ron formation and significantly reduces fat transport (sabesin and isselbacher, 1965) . the final step in fat absorption is extrusion of the chylomicron into the intercellular space opposite the basal lateral portion of the absorptive cell. this is accomplished by a process which is essentially the reverse of pinocytosis (palay and karlin, 1959) . from the intercellular space the chylomicron passes through the basement membrane and enters the lacteals through small pores. the chylomicron passes from the lacteal into lymph ducts and ultimately reaches the general circulation, having bypassed the liver completely during the initial phase of absorption. a. cholesterol. dietary cholesterol is present in both free and esterified forms, but only nonesterified cholesterol is absorbed (vahouny and treadwell, 1964) . cholesterol esters are hydrolyzed within the lumen of the intestine by sterol esterase secreted by the pancreas. bile salts are required both for the action of this enzyme (vahouny et al., 1965) and for the absorption of nonesterified cholesterol. in the mucosal cell, cholesterol is reesterified and transferred by way of the lymph to the general circulation. the type of triglycéride present in the diet significantly affects the absorption of cholesterol and its distribution in lymph lipids . b. vitamin a. the diet contains vitamin a activity in two principal forms: (1) as esters of preformed vitamin a alcohol (retinol) and fatty acids and (2) as provitamin a, primarily in the form of jö-carotene. vitamin a ester is hydrolyzed by a pancreatic esterase within the lumen (murthy and ganguly, 1962) , and the free alcohol is absorbed in the upper small intestine by a process which apparently requires metabolic energy (skala and hrubâ, 1964) . vitamin a alcohol is reesterified in the mucosa utilizing primarily palmitic acid (mahadevan et al., 1963) . the vitamin a ester is absorbed by way of the lymph. after reaching the general circulation, it is rapidly cleared from the plasma and stored in the liver. in the postabsorptive state, vitamin a circulates as the free alcohol. this is also the form released from the liver as needed by the action of a specific hepatic retinylpalmitase esterase (mahadevan et al., 1966) . the blood level of vitamin a is independent of the liver reserve, and, as long as a small amount of vitamin a is present in the liver, the blood level remains normal (dowling and wald, 1958) . in diets which lack animal fat, the carotenes, mainly /3-carotene, serve as the major vitamin a precursors. the intestinal mucosa plays the primary role in conversion of provitamin a to the active vitamin, although conversion can occur to a limited degree in other tissues (bieri and pollard, 1954; zachman and olson, 1963) . the exact mechanism involved in the conversion of /3-carotene to vitamin a is not completely established, but studies by olson (1961) suggest that there is central cleavage of/3-carotene into two active vitamin a alcohol molecules, which are subsequently esterified and transported by the lymphatics as with the preformed vitamin. bile salts are required for the mucosal uptake of /3-carotene and for the conversion of ß-carotene to vitamin a. uptake of carotene and release of vitamin a ester into the lymph appear to be rate-limiting steps. cattle also absorb substantial amounts of carotene without prior conversion to vitamin a, and these pigments are responsible for much of the yellow color of the plasma. most other species have no carotene in the plasma, and it has been suggested that extraintestinal conversion may be more efficient in these species than in cattle . c. vitamin d. vitamin d, like cholesterol, is a sterol which is absorbed from the intestine by way of the lymph (schachter et al., 1964) . intestinal absorption differs, however, in that vitamin d is transported to the lymph in nonesterified form (bell, 1966) . the uptake of vitamin d by the mucosal cell is favored by the presence of bile salts. simultaneous absorption of fat from micellar solutions increases transport out of the cell into the lymph, a step which appears to be rate limiting (thompson et al., 1969) . one of the major actions of vitamin d is to enhance the intestinal absorption of calcium ion. the mechanism of action of vitamin d has been described by wasserman and co-workers (1968; wasserman and taylor, 1966, 1968) . they have shown that vitamin d causes synthesis of a calcium-binding protein present in the soluble fraction of the intesti nal mucosal cell. they have accumulated a substantial amount of evidence which suggests that this protein plays a central role in the active transport of calcium. vomiting is a coordinated reflex act which results in rapid, forceful expulsion of gastric contents through the mouth. the reflex may be initiated by (1) local gastric irritation caused by a variety of toxic irritants or infectious agents, (2) foreign bodies, (3) gastric tumors, (4) obstruction of the pyloric canal or of the small intestine, or (5) drugs, such as apomorphine, or other toxic substances which act centrally on the "vomiting center" located in the medulla. severe vomiting produces loss of large quantities of water and of h + and cl~ ions. these losses cause dehydration, metabolic alkalosis with elevated plasma bicarbonate concentration, and hypochloremia. chronic vomiting may also be associated with loss of tissue k + and hypokalemia. the k + deficit is caused primarily by increased urinary excretion, which is the result of the existing alkalosis (leaf and santos, 1961) . gastric secretions contain significant quantities of k + (section ii,b), and losses in the vomitus also contribute to the k + deficiency. potassium deficiency, which develops initially because of alkalosis, ultimately may perpetuate the alkalotic state by interfering with the ability of the kidney to conserve h + (koch et al., 1956; darrow, 1964) . both potassium deficiency and the hypovolemia caused by dehydration may result in renal tubular damage and ultimately in renal failure (haden and orr, 1923, 1924) . vomiting occurs frequently in the dog, cat, and pig but is an unusual sign in the horse, which has anatomical restrictions of the esophagus that interfere with expulsion of gastric contents. in cattle, sheep, and goats, the physiological process of rumination utilizes neuromuscular mechanisms similar to those involved in vomiting. uncontrolled expulsion of ruminai contents is, however, an uncommon sign, occurring most frequently after ingestion of toxic materials. the contents of the abomasum are not expelled directly even when the pyloric canal is obstructed. a syndrome does occur in cattle with pyloric obstruction, however, which is similar metabolically to that observed in nonruminants. the syndrome has been observed in right-sided displacement of the abomasum with torsion (espersen, 1961; boucher and abt, 1968) . we have also observed the syndrome in cows with functional pyloric obstruction, the result of reticuloperitonitis (a variety of "vagai indigestion"). when the pylorus is obstructed, abomasal contents are retained, causing distention of the abomasum, which in turn stimulates further secretion and reten tion. retained abomasal contents may be regurgitated into the large reservoir of the rumen and there are sequestered from other fluid compartments of the body. the net result is loss of h + and cl" ions and development of metabolic alkalosis, hypochloremia, and hypokalernia (espersen and simesen, 1961; svendsen, 1969) . chronic hypertrophie gastritis has been demonstrated in the dog (van der gagg et al., 1976; happe and van der gagg, 1977; kipnis, 1978) which resembles menetrier's disease in man. van kruiningen's series of cases were basenjis which had concomitant lymphocytic-plasmocytic enteritis. three unpublished cases were studied at the new york state college of veterinary medicine. signs of illness usually involved chronic vomiting, weight loss, and occasionally diarrhea. hypoalbuminemia was documented in most of these cases. in man, hyperchlorhydria or achlorhydria can occur. the morphological changes in the stomach wall (hypertrophie rugae) as well as some of the clinical features help to differentiate this disease from gastric neoplasia and canine zollinger-ellison syndrome. canine zollinger-ellison syndrome was reported in four dogs (straus et al., 1977; van der gagg et al., 1978) . vomiting, diarrhea, inappetance, and weight loss were reported. all of the dogs had pancreatic non-js islet cell tumors, resulting in hypergastrinemia, hyperchlorhydria, hypertrophie gastritis, peptic esophagitis, and duodenal ulcers. the term "diarrhea" is used loosely to describe the passage of abnormally fluid feces with increased frequency and/or with increased volume. the significance of diarrhea depends primarily on the underlying cause and on the secondary nutritional and metabolic disturbances which are caused by excessive fecal losses. there are theoretically three factors which could act independently or in combination to produce diarrhea: (1) increased rate of intestinal transit, (2) decreased intestinal absorptive capacity, and (3) increased secretion into the intestinal lumen. an increase in the rate of intestinal transit has been considered to be important in various functional disorders of the gastrointestinal tract in which "hypermotility" has been considered the primary cause. although increased intestinal motility may be a factor in certain types of diarrheal disease when the direction of motility has been investigated, diarrhea has actually been associated with decreased motility (christiansen, 1972) . decreased intestinal assimilation of nutrients may result from either (1) decreased intraluminal hydrolysis of nutrients, e.g., maldigestion (kaiser, 1964) , due to pancreatic exocrine insufficiency or to bile salt deficiency or (2) defective mucosal transport of nutrients, malabsorption, which may be the result of various types of inflammatory bowel disease, intestinal lymphoma, or intrinsic biochemical defects in the mucosal cell which interfere with normal digestion and absorption. the role of increased intestinal secretion in the pathogenesis of certain types of acute diarrhea is now recognized. enteropathogenic strains of escherichia coli have been shown to produce soluble enterotoxins (smith and halls, 1967; köhler, 1968; moon, 1978) , which alter bidirec tional sodium and water flux (fig. 10) . rapid advances in understanding the pathogenesis of enterotoxin-induced diarrhea and the molecular basis of enterotoxin action have been made. the most extensively studied enterotoxin is that produced by vibrio cholerae. this bacterium produces a large molecular weight heat-labile toxin (ct), one subunit of which has properties similar to those of heat-labile (lt) enterotoxin produced by certain strains of e. coli (richards and douglas, 1978) . the mechanism of action of ct is believed to moon, 1978.) involve the activation of adenylate cyclase. this membrane-bound enzyme converts atp to cyclic 3',5'-adenosine monophosphate (camp), which is then responsible for the greatly increased secretion of water and electrolytes by the intestinal mucosa (moon, 1978) . although species differences have been observed (hamilton et al., 1978a,b; forsyth et al., 1978) , this mechanism appears to be important in the mode of action of e. coli lt as well (richards and douglas, 1978) . additional extensive studies have centered on the molecular mechanism of action of ct. under physiological conditions, adenylate cyclase is activated by the binding of guanosine triphosphate to the inactive enzyme. an associated gtpase inactivates the enzyme by converting enzyme-bound gtp to gdp and inorganic phosphate. this gtp-gdp system apparently plays a critical role in the regulation of adenylate cyclase. cholera toxin is believed to bind to the adenyl cyclase in a way which inhibits hydrolysis of gtp, thereby maintaining the enzyme in an activated state (levinson and blume, 1977; johnson et al., 1978; cassel and pfeuffer, 1978) (fig. 11) . certain enteropathogenic strains of e. coli produce a low molecular weight heat-stable toxin (st) alone or in addition to lt (richards and douglas, 1978; moon, 1978; hamil ton et al., 1978a) . in most epidemiological studies of neonatal diarrheal diseases of calves, isolated strains of e. coli produce only st (moon et al., 1976; braaten and myer, 1977; lari vier et al., 1979) . in contrast to lt and ct, which induce intestinal sodium and . proposed mechanism of action of cholera toxin, which inhibits hydrolysis of gtp, thereby increas ing adenylate cyclase activity. (after cassel and selinger, 1978.) water secretion only after a lag phase of several hours, st increases intestinal secretion at once. recent evidence suggests that st induces intestinal secretion by activating guanylate cyclase and that the mediator of intestinal secretion induced by st is cyclic 3',5'guanosine monophosphate field et al., 1978) . such advances in our fundamental knowledge of the pathogenesis of enterotoxininduced diarrheal disease have opened several avenues of investigation which may lead to pharmacological modification of intestinal secretion as a mode of therapy or prophylaxis. enterotoxin-induced intestinal secretion has been shown to be effectively blocked by cycloheximide, inhibitor of protein synthesis (serebro et al., 1969) . the lack of speci ficity and the toxicity of cycloheximide precluded its clinical use, but acetazolamide has been shown to inhibit intestinal fluid secretion (norris et al., 1969; moore et al., 1971) , and ethacrynic acid, another potent diuretic, has been shown to inhibit enterotoxininduced fluid secretion (carpenter et al., 1969) . unfortunately, the diuretic effects of these drugs preclude their clinical use, but an "intestinal-specific" derivative would have significant therapeutic potential. adenosine analogues also have been shown in prelimi nary studies to inhibit cholera toxin-stimulated intestinal adenylate cyclase, but their potential as prophylactic or therapeutic agents is not known. prostaglandin e t and ct have a similar effect on electrolyte transport in rabbit ileum. application of either to the mucosa inhibits sodium absorption and stimulates chloride secretion. one possible explanation for the effects of ct is that it stimulated release of prostaglandin, which then acted on adenylate cyclase, producing camp. to test this hypothesis, the effects of inhibitors of prostaglandin release on enterotoxin-stimulated intestinal secretion were investigated. both indomethacin (gots et al., 1974) and acetylsalicylic acid (farris et al., 1976) were shown to be potent inhibitors of enterotoxininduced intestinal secretion using laboratory animal models. current evidence does not support the hypothesis that prostaglandins play a primary role in the pathogenesis of cholera or other enterotoxin-induced diarrheal diseases (schwartz et al., 1975) , but the effects of these known prostaglandin inhibitors and other drugs on the intestinal secretory process warrant their evaluation as possible prophylactic and therapeutic agents. in pre liminary studies, jones and his colleagues demonstrated a positive therapeutic response to a new prostaglandin inhibitor (jones et al., 1977) . the autonomie nervous system has important effects on intestinal ion transport and water absorption (tapper et al., 1978) . catecholamines stimulate formation of camp in a variety of mammalian cells (sutherland and rail, 1960; schultz et al., 1975) , apparently by activating the gtp-gdp system described above (cassel and selinger, 1978; ciment and devellis, 1978) . adrenergic blocking agents, such as chlorpromazine (holmgren et al., 1978) and propranolol (donowitz et al., 1979) , have been shown to have significant inhibitory effects on enterotoxin-induced intestinal secretion. although the mechanism of action of these two adrenergic blockers is not known, they represent still another class of drugs which may be of therapeutic benefit. the intestinal "adsorbent" drug pepto bismol, a patented medication containing bis muth subsalicylate, and attapulgite, a heat-treated silicate, have been shown to have antienterotoxic effects (drucker et al., 1977; ericsson et al., 1977; gyles and zigler, 1978) . controlled therapeutic trials with bismuth subsalicylate have demonstrated signifi cant therapeutic benefit in certain large-volume diarrheal diseases in man suspected of being enterotoxigenic in origin (portnoy et al., 1976; dupont et al., 1977; dupont, 1978) . the mechanism of action in inhibiting intestinal secretion has not been determined, but the chemical relation of bismuth subsalicylate to other known prostaglandin inhibitors is recognized. it is possible that such drugs, by decreasing endogenous production of prostaglandin, decrease the basal level of cyclic nucleotides, which in turn causes an increase in the threshold of response to enterotoxin. recent evidence suggests that salicylates also may stimulate sodium chloride absorption (powell et al., 1979) . these observa tions taken collectively suggest that new, innovative methods for therapy and control of acute clinical diarrheal disease may be developed in the not too distant future. acute diarrhea represents the leading cause of morbidity and mortality in neonatal calves and pigs. the pathogenesis of the neonatal enteric infection is complex, often involving nutritional or environmental factors as well as infectious agents, such as enteropathogenic strains of e. coli, the transmissible gastroenteritis virus (tge), rota viruses, and other bacterial and viral pathogens. the severe clinical signs and frequently fatal outcome of acute diarrheal disease are often directly related to dehydration and to associated hydrogen ion and electrolyte disturbances (dalton et al., 1965; fisher and mcewan, 1967b; tennant et al, 1972 tennant et al, , 1978 . in acute diarrhea with watery stools of large volume, the fecal fluid originates primarily in the small intestine. the electrolyte composition of the stool in such cases is similar to that of the fluid found normally in the lumen of the small intestine, which in turn is similar to that of an ultrafiltrate of the plasma. the rapid dehydration which accompanies acute enteritis in the newborn soon produces hemoconcentration and ultimately hypovolemic shock. such cases are characterized by metabolic acidosis (dalton et al., 1965; phillips and knox, 1969) caused by decreased excretion of h + due to renal failure and by in creased production of organic acids, the result of decreased tissue oxygénation, which leads to excessive anaerobic glycolysis. hyperkalemia also is observed characteristically in young, severely dehydrated animals. hyperkalemia in such cases is the result of increased movement of cellular potassium into the extracellular fluid and to decreased renal excretion. cardiac irregularities caused by hyperkalemia can be demonstrated with the electrocardiogram, and cardiac arrest related to hyperkalemia is believed to be a direct cause of death in calves with acute diarrhea fisher and mcewan, 1967b) . marked hypoglycemia also has been observed occasionally prior to death in calves with acute enteric infections. hypoglycemia is believed to be due to decreased gluconeogenesis and increased anaerobic glycolysis, the result of hypovolemic shock (tennant et al., 1968) . the sequence of metabolic changes which occur during acute neonatal diarrhea is summarized in fig. 12 . in chronic forms of diarrheal disease, excessive fecal losses of electrolyte and fluid may be compensated in part by renal conservation mechanisms and by oral ingestion. if water is consumed without adequate ingestion of electrolytes, hyponatremia and hypokalemia may develop (tasker, 1967; patterson et al., 1968) . in such cases, the osmolarity of the plasma is significantly decreased and hypotonie dehydration occurs. in longer-standing cases of chronic diarrhea, the plasma k + concentration may become dangerously low. it is imperative, in this situation, that intravenous fluids contain sufficient k + to prevent further reduction in plasma concentration. if they do not, additional cardiac irregularities or cardiac arrest may result. decreased assimilation of nutrients may occur either as a result of defective intraluminal digestion (maldigestion) (kaiser, 1964) , or because of defects in mucosal transport (jeffries, et al., 1969; floch, 1969; wilson and dietchy, 1971 (from tennant et al., 1972.) or the malabsorption syndrome is observed in several types of intestinal disease, including chronic intestinal granulomatous diseases such as johne's disease, intestinal parasitic infections, and lymphoma of the intestine. primary clinical signs include persistent or recurrent diarrhea, nutrient loss in the feces (e.g., steatorrhea), and weight loss. mucosal cell-enzymatic defects may be accompanied by chronic inflammation, villous atrophy, or cellular infiltrations of the lamina propria of the intestine. early reports of primary or idiopathic intestinal malabsorption in dogs (miller, 1960; vernon, 1962; kaneko et al., 1965) were compared to nontropical sprue (adult celiac disease, gluten induced enteropathy) of man, but no convincing association to with gluten sensitivity was demonstrated. subsequent reports of malabsorption syndromes in the dog have described a variety of causes (van kruiningen, 1968; ewing, 1971; van kruiningen andhayden, 1972; hill, 1972; hill and kelly, 1974; schall, 1974; anderson, 1975 anderson, , 1977 burrows et al., 1979) , which must be distinguished from the maldigestion caused by pancreatic insufficiency (anderson and low, 1965a,b) (juvenile pancreatic atrophy, chronic pancreatitis) and from certain forms of hepatic or gastric disease. intestinal malab sorption can occur with protozoal enteritis (giardiasis, coccidiosis), lactase deficiency, eosinophilic gastroenteritis, lymphangiectasis, villus atrophy, lymphocytic-plasmacytic enteritis, histoplasmosis, chronic "bacterial" enteritis, malignant lymphoma, and intesti nal amyloidosis of the bowel. some authors (anderson, 1977; hay den and van kruiningen, 1973; arrick and kleine, 1978) described malabsorption and pseudoobstruc tion secondary to hypoplasia of the tunica muscularis of the jejunum in a dog. intestinal malabsorption is reported less frequently in the cat than in the dog (theran and carpenter, 1968; wilkinson, 1969) .malabsorption syndromes similar to those recog nized in dogs are being recognized with increased frequency in farm animals (blood et al., 1979) . meuten et al. (1978) , cimprich (1974), and merritt et al., (1976) have reported malabsorption in the horse secondary to chronic granulomatous enteritis and specific amino acid malabsorption has been reported in johne's disease (patterson and berret, 1969) . steatorrhea, the presence of excessive amounts of fat in the feces, is a prominent sign of intestinal malabsorption in dogs. the stools are bulky, gray or tan, and, grossly, may have an oily appearance. the normal dog excretes 3-5 gm of fat in the stool each day. this level of fecal fat is quite constant and is independent of dietary fat intake over a wide range of 15 to 48 gm/day (heersma and annegers, 1948) . in intestinal malabsorption, the ability to absorb fat is decreased and fecal fat excretion increases significantly. under these conditions, the amount of fecal fat excreted becomes proportional to dietary intake. merritt et al. (1979) reported that body weight is an important factor in fat output. small dogs (i.e., less than 10-15 kg body weight) with intestinal malabsorption had fecal fat outputs lower than or equal to published normal values. fecal fat excretion for normal dogs was 0.24 ± 0.01 gm/kg body weight per day. steatorrhea can be documented qualitatively by staining the fresh stool with a lipophilic stain, such as sudan iii, and observing increased numbers of oil droplets under the light microscope. in experienced hands, this method is a reliable diagnostic procedure (drummey et al., 1961) . the following methods can be used to demonstrate neutral and split fats. for neutral fat, two drops of water are added to a stool sample on a glass slide and mixed. two drops of 95% ethanol are then added and mixed followed by several drops of a saturated solution of sudan iii in 95% ethanol. a coverslip is applied to the mixture, which is then examined for yellow or pale orange refractile globules of fat, particularly at the edges of the coverslip. normally, two or three fat droplets per high-power field are present. a large number of neutral fat droplets suggests a lack of pancreatic lipase activity, i.e., exocrine pancreatic insufficiency. ¥ or free fatty acids, several drops of 36% acetic acid are added to a stool sample on a glass slide and mixed. several drops of sudan iii solution are then added and mixed. a coverslip is applied, and the slide gently heated over an alcohol burner until it begins to boil. the slide is air-cooled and then quickly heated again, this procedure is repeated two or three times. the warm slide is examined for stained free fatty acid droplets, which, when warm, appear as deep orange fat droplets from which spicules and soaps, resem-bling the pinna of the ear, form as the preparation cools. normal stools may contain many tiny droplets of fatty acids (up to 100 per high-power field). with increasing amounts of split fats, the droplets become larger and more numerous, which suggests an abnormality in fat absorption. quantitation of fecal fat is the most accurate method of assessing steatorrhea (burrows et al., 1979) with dietary fat balance being determined for a period of 48-72 hours. fecal fat is analyzed using a modification of the technique of van de kamer et al. (1949) , which employs ether extraction of fecal lipid and titration of fatty acids. the results are ex pressed as grams of neutral fat excreted per 24 hours. merritt et al. (1979) have suggested that dogs be fed 50 gm fat per kilogram per day for two to three days prior to fecal collection. analysis of a 24-hour collection of stool when this is done is believed to be as accurate as a 72-hour stool collection. results are expressed as fat excretion in grams per kilogram body weight. in addition to mal absorption of fat, the canine malabsorption syndrome is associated with decreased absorption of other nutrients. these defects in absorption are responsible for the progressive malnutrition which is a cardinal feature of the disease. there may be malabsorption of vitamin d and/or calcium, resulting in osteomalacia. the anemia some times observed may be the result of malabsorption of iron or of the b vitamins, which are required for normal erythropoiesis. malabsorption of vitamin k can result in hypoprothrombinemia. glucose malabsorption has been clearly documented by kaneko et al. (1965) , and it is likely that amino acids, which are absorbed at a similar level of the small intestine, are also malabsorbed. carbohydrate and fat malabsorption unquestionably con tributes to the calorie deficit which results in weight loss. amino acid malabsorption may contribute to the development of hypoproteinemia, although this is thought to be due primarily to increased intestinal loss of plasma protein (see section iv,c). the diagnosis of idiopathic canine malabsorption can be made only after appropriate diagnostic procedures have ruled out the presence of (1) other primary inflammatory, neoplastic, or parasitic diseases of the intestine and (2) the diseases of the pancreas, liver, or stomach which result in defective intraluminal digestion. the presence of parasitic infection is determined by examining the feces for parasite ova. other inflammatory or neoplastic diseases of the intestine may be suggested on the basis of clinical or radiologi cal examination, but a definitive diagnosis usually depends on histopathological examina tion of an intestinal biopsy specimen. both primary and secondary intestinal malabsorption must be differentiated from those diseases in which there is decreased intraluminal hydrolysis of nutrients. the latter are due most frequently to pancreatic exocrine insufficiency, the result of such diseases as chronic pancreatitis or juvenile atrophy. in these diseases, degradation of the major dietary con stituents is reduced because of a primary lack of pancreatic enzymes. intraluminal hy drolysis of fat may also be decreased because of a deficiency of bile salts caused either by decreased hepatic secretion or by bile duct obstruction. under certain experimental condi tions, diversion of bile flow in the dog actually has a quantitatively small effect on fat absorption (wells et al., has a quantitatively small effect on fat absorption (wells et al., 1955; hill and kidder, 1972a ). the problems of pancreatic exocrine deficiency are discussed in detail elsewhere in this text (chapter 7). the most simple and perhaps most widely used test to differentiate intestinal malabsorption from pancreatic exocrine insufficiency is that described by jasper (1954) . the test is employed to detect reduction in trypsin-like activity in the feces of dogs with decreased pancreatic exocrine secretion (grossman, 1962) . there is wide variation in normal activity, making interpretation of the test difficult (frankland, 1969; hill and kidder, 1970; burrows et al., 1979) . the test reveals only the presence or absence of hydrolysis of gelatin and does not differentiate between gelatinase activity produced by intestinal bacteria from that secreted by the pancreas. there is evidence in some species that trypsin is almost completely destroyed by bacteria during its passage through the intestine and that the proteinase activity of the feces is primarily of bacterial origin (borgström et al., 1959) . despite these theoretical objections, the test has been of clinical diagnostic value in our hands. fecal gelatinase activity has been detected consistently in cases of intestinal malabsorption and is almost always absent when severe pancreatic exocrine insufficiency is present. burrows et al. (1979) reported that the mean 24-hour trypsin output in dogs with pancreatic insufficiency was significantly lower, and in dogs with malabsorption significantly higher, than clinically normal dogs. an indirect method to test chymotrypsin activity has been described (strombeck, 1978) . a synthetic peptide, rc-benzoyltyrosine//?-aminobenzoic acid, is administered to test dogs orally. if chymotrypsin is present in the duodenum, hydrolysis of this peptide occurs and p-aminobenzoic acid (paba) is released in a free form, which is absorbed and subsequently excreted in the urine within 6 hours. the urine is analyzed for paba. less than 43% paba excretion identifies dogs with suspected pancreatic exocrine insuffi ciency. a. oleic acid and triolein absorption. several tests have been developed for the clinical evaluation of intestinal absorptive capacity. the absorption of 131 i-labeled oleic acid and 131 i-labeled triolein has been studied extensively in normal dogs (turner, 1958; michaelson et al., 1960) , and kaneko et al. (1965) used this test to study dogs with in testinal malabsorption. the day before administration of the 13l i-labeled compound, a small amount of lugol 's iodine solution is administered to block thyroidal uptake of the isotope. tracer amounts of the test substances are mixed with nonradioactive carrier and are administered orally. absorption is determined by measuring the radioactivity of the plasma at intervals following administration can calculating the percentage of the dose absorbed based on plasma volume. it is possible to use the results of these two tests, when performed in sequence, to differentiate between steatorrhea caused by a deficiency of pancreatic enzymes and that caused by a primary defect in absorption (kallfelz et al., 1968) . if steatorrhea is caused by a lack of pancreatic lipase, oleic acid absorption will be normal, whereas that of triolein, which requires lipolysis for absorption, will be significantly reduced. the absorption of both compounds is reduced in intestinal malabsorption (fig. 13a,b) . the results of this test also may vary depending on the rate of intestinal motility (tennant et al., 1969b) . kaneko et al., 1965.) of vitamin a, mean serum vitamin a concentrations peak at 6-8 hours, with values ranging between three and five times fasting serum levels in normal dogs. breed dif ferences and delayed gastric emptying will alter results. c. glucose absorption. the absorption of glucose can be measured by means of an oral glucose tolerance test in which a test dose of glucose is given by mouth and the blood glucose level measured at intervals for 3-4 hours following administration. the test has been used in canine malabsorption in which the normal rise in blood glucose level is reduced (kaneko et al., 1965) . the test also has been reported for use in the horse (roberts and hill, 1973) . dogs with pancreatic exocrine deficiency may, however, have "diabetic" tolerance curves (hill and kidder, 1972b) . the major disadvantage of relying on this test alone is that it does not differentiate between decreased intestinal absorption and increased tissue uptake following absorption. this problem can be minimized by comparing results of the oral glucose tolerance test with those obtained with the intraven ous tolerance test. the results of this test, however, must be interpreted carefully and in relation to other clinical and laboratory findings. hill and kidder (1972) reported that dogs on low-carbohydrate diets can have "diabetic" tolerance curves; test dogs should be on a high-carbohydrate diet 3-5 days before testing. the absorption of d-xylose also can be used to evaluate intestinal function. d-xylose is not metabolized by the body to any significant degree, and the problems of evaluating tissue utilization which occur with glucose are eliminated. because of the large amounts of d-xylose used in the test, absorption is independent of active transport processes, and the rate of absorption is proportional to luminal concentra tion. a d-xylose absorption test for dogs has been described by van kruiningen (1968) . in this procedure, a standard 25-gm dose of d-xylose is administered by stomach tube. during the 5-hour period following administration, the dog is confined in a metabolism cage, and urine is collected quantitatively. at the end of the 5-hour test period, the urine remaining in the bladder is removed by catheter, and the total quantity excreted in 5 hours is determined. normal dogs excreted an average of 12.2 gm during the test period, with a range of 9.1-16.5 gm. the results obtained by this method are dependent not only on the rate of intestinal absorption, but also on the rate of renal excretion, and it is necessary, therefore, to know that kidney function is normal. the oral xylose tolerance test has received most clinical use (hill et al., 1970; hayden and van kruiningen, 1973) . dogs are fasted overnight, a blood sample is obtained, and d-xylose is administered by stomach tube at the rate of 0.5 gm/kg. a control test is performed on a normal dog simultaneously with each dog with signs of intestinal malab sorption. the first blood sample is obtained one-half hour after administration. the second sample is obtained 1 hour following administration, and additional samples are taken at hourly intervals for 5 hours. the xylose concentration in the blood is determined by the method of roe and rice (1948) . maximal blood levels almost always are reached at 1 hour after administration of the test dose; hill expects a xylose level of at least 45 mg/dl within 60-90 minutes in a normal dog. in preliminary studies of four dogs with the malabsorption syndrome, maximal blood xylose levels averaged 58% of corresponding control values. in dogs with pancreatic exocrine insufficiency with normal intestinal mucosa, there should be a normal xylose response test. the d-xylose absorption test also has been described for use in differential diagnosis of equine diarrheal diseases (roberts, 1974) . bolton et al. (1976) reported that a dosage of 0.5 gm xylose per kilogram body weight was useful in detecting horses that absorbed the pentose abnormally. gastrointesti nal lesions associated with abnormal results were classified as (1) villous atrophy, (2) edema of the lamina propria, or (3) necrosis of the lamina propria. at this dosage in normal horses, the mean peak plasma concentration is less than one-third that seen in normal dogs given xylose (normal dogs: 60-70 mg % at 60 minutes). albumin, γ-globulin, and other plasma proteins are present in normal gastrointestinal secretions. because protein usually undergoes complete degradation within the intestinal lumen, it has been suggested that the gastrointestinal tract must have a physiological role in the catabolism of plasma proteins. the relative significance of this pathway, however, has been the subject of considerable controversy. some investigators have concluded that as much as 50% or more of the normal catabolism of albumin (glenert et al., 1961 campbell et al., 1961; wetterfors, 1964 wetterfors, , 1965 wetterfors et al., 1965) and γ-globulin occurs in the gastrointestinal tract. others believed that the physiological role of the intestine in plasma protein catabolism is far less significant, accounting for only about 10% of the total catabolism (waldmann et al., 1967 (waldmann et al., , 1969 katz et al., 1960; franks et al., 1963a,b) . regardless of the questions concerning the relative importance of the gastrointestinal tract in plasma catabolism, it is well established that normal intestinal losses are increased significantly in a variety of gastrointestinal diseases, which are referred to collectively as protein-losing enteropathies. the increased loss causes hypoproteinemia (especially hypoalbuminemia), which may be observed in various types of chronic enteric diseases. the excessive losses are produced by ulcérations or other mucosal changes which alter permeability or by obstruction of lymphatic drainage from the intestine. if severe, hypoalbuminemia may result in retention of fluid with development of ascites and subcutaneous edema of pendant areas. excessive plasma protein loss has now been demonstrated in swine with chronic ileitis (nielsen, 1966) , in calves with acute enteric infections (marsh et al., 1969) , in cattle with parasitic or other inflammatory abomasal disease (nielsen and nansen, 1967; halliday et al., 1968; murray, 1969) , and in johne's disease (patterson et al., 1967; nielsen and andersen, 1967; patterson and berret, 1969) . in addition to the classic mucosal and submucosal lesions of johne's disease, nielsen and andersen (1967) demonstrated the presence of secondary intestinal lymphangiectasia. meuten et al., (1978) described protein-losing granulomatous enteritis in two horses and discussed a comparative over view of diseases causing mal absorption in the horse, cow, dog, pig, and man. protein-losing enteropathy has been seen with some frequency in the dog (campbell et al., 1968; farrow and penny, 1969; hill, 1972; fineo et al, 1973; hayden and van kruiningen, 1973; mattheeus, et al; hill and kelly, 1974; milstein and sanford, 1977; barton et al, 1978; olson and zimmer, 1978) . intestinal lymphangiectasia was com monly reported. the dog described by milstein and sanford (1977) was not hypoproteinemic because the rate of albumin synthesis by the liver was greater than protein loss into the intestine. protein loss has also been documented in dogs with chronic hypertrophic gastritis (section iv,a). increased intestinal protein loss is the most likely explanation for the hypoalbuminemia associated with certain other enteric diseases, including intestinal mal absorption and lymphoma of the intestine. munro (1974) demonstrated that protein loss in dogs with experimentally induced protein-losing gastropathy occurs by an intercellular route. isotope-labeled polyvinylpyrolidine ( 131 i-pvp), 67 cr-labeled ceruloplasmin, and 51 crlabeled albumin have been used to evaluate enteric protein loss in the dog (fineo et al, 1973; barton et al, 1978; hill and kelly, 1974; van der gagg et al, 1976; olson and zimmer, 1978) . canine ulcerative colitis was described originally in the report of cello (1964). since that time, ulcerative colitis and its variant form, granulomatous colitis of boxer dogs, has been reported by several investigators kennedy and cello, 1966; koch and skelley, 1967; sander and langham, 1968; ewing and gomez, 1973; gomez et al, 1977; russell et al, 1971 ). the etiology is generally unknown. ewing and aldrete (1973) reported a case of canine giardiasis presenting as chronic ulcerative colitis and cases of ulcerative colitis in dogs have been attributed to trichuriasis, balantidiasis, protothecosis, histoplasmosis, eosinophilic ulcerative colitis, or neoplasia (lorenz, 1975) . rarely, severe ulcerative colitis is seen in the cat. in some of these cases, feline leukemia virus is demonstrated. shindel et al (1978) described colonie lesions in cats caused by feline panleukopenia. histopathologically, periodic acid-schiff-positive macrophages are pathognomonic for the granulomatous colitis of boxer dogs. the disease causes chronic, intractable diarrhea, which is often hemorrhagic. in addition, afflicted dogs may vomit and are often emaciated. fever is usually not present. biochemical manifestations of ulcerative colitis depend upon duration and severity of ewing and gomez (1973) . 0 thirty-six observations on 29 affected dogs. illness, degree of colorectal involvement, and the presence of systemic complications. in severe cases of long duration with extensive colorectal involvement, hypoalbuminemia and hypergammaglobulinemia (table vii) are sometimes observed. the pathogenesis of hypoalbuminemia probably involves increased loss of plasma through the denuded and inflammed colorectal mucosa. hypergammaglobulinemia is probably an associated re sponse to chronic inflammation. the digestive process of ruminants differs from that of other animals because of microbial digestion and metabolism in the rumen which occurs prior to other normal digestive processes. the short-chain fatty acids (acetic, propionic, and butyric acids) are the pri mary end products of rumen fermentation and represent the chief dietary source of energy for ruminants (hungate et al., 1961) . the polysaccharide cellulose, which undergoes only very limited digestion in most simple-stomached animals, is readily utilized by ruminants because of the activity of cellulytic bacteria. significant quantities of nonprotein nitrogen also can be utilized by ruminai bacteria for protein synthesis, and this bacterial protein subsequently can be utilized to meet the protein requirements of the animal. bacterial production of vitamins may also meet essentially all the requirements of ruminants. maintenance of bacterial fermentation within the rumen also presents certain unusual hazards to ruminant animals. when rapid changes in dietary intake occur, the products of fermentation can be released more rapidly than they can be removed. acute rumen tympany, acute indigestion of d-lacticacidosis, and urea poisoning are diseases which result from such abrupt changes in diet (hungate, 1966 (hungate, , 1968 . acute rumen indigestion occurs in sheep or cattle on a high-roughage diet when they inadvertently are allowed access to large amounts of readily fermentable carbohydrate, e.g. grain and apples (dunlop, 1972) . streptococcus bovis is the rumen microorganism believed to be chiefly responsible for rapid fermentation and for production of large quantities of lactic acid (hungate et al., 1952; krogh, 1963a,b) . as lactic acid accumulates more rapidly than absorption, the rumen ph falls and rumen atony results. rumen bacteria produce a racemic mixture of lactic acid. some l-lactate may be metabolized by the liver and other tissues, but d-lactate cannot be and contributes significantly to the acid load of the body. the excessive lactic acid production results in metabolic acidosis, which is characterized by reduced blood ph and bicarbonate concen tration and by a fall in urine ph from a normal value of 8.01 -8.0 to as low as 5.0. fluid accumulates in the rumen because of increased osmolarity of its contents, causing hemoconcentration, which may lead to hypovolemic shock and death (hyldgaard-jensen and simesen, 1966) . if affected animals survive the initial period of explosive fermenta tion, a chemical rumenitis, caused by lactic acid, may develop. secondary mycotic rumenitis may also occur and be fatal. hepatic abscesses also may result from severe rumenitis. the rumen of mature cattle can produce 1.2-2.0 liters gas per minute (hungate et al., 1965) . the gas is composed primarily of carbon dioxide and methane, which are products of rumen fermentation. carbon dioxide is also released when salivary bicarbonate is acted upon by organic acids within the rumen. under normal conditions, these large amounts of gas are continually removed by eructation. any factor which interferes with eructation can produce acute tympany of the rumen (bloat), leading to rapid death. interruption of the normal eructation reflex or mechanical obstruction of the esophagus typically results in free-gas bloat. the most important form of bloat, however, is seen in cattle consuming large quantities of legumes or in feedlot cattle on high-concentrate diets. the primary factor in these more common types of bloat is a change in the ruminai contents to a foamy or frothy character. because of altered surface tension, gas is trapped in small bubbles with the rumen and cannot be eliminated by eructation (clarke and reid, 1974) . the chemical changes which cause foam to form within the rumen are not completely clear. some reports (nichols, 1966; nichols and deese, 1966) suggest that plant pectin and pectin methyl esterase, an enzyme system also from plants, are critical factors. the enzyme acts on pectin to release pectic and galacturonic acids, which greatly increase the viscosity of the rumen fluid, resulting in formation of a highly stable foam. slimeproducing bacteria also have been incriminated in the pathogenesis of frothy bloat. these microorganisms produce an extracellular polysaccharide, which results in stable foam formation. effective medical treatment and control are directed toward decreasing or preventing foam formation. this has been accomplished with certain nonionic detergents with surfac tant properties which break up or prevent formation of foam within the rumen (bartley, 1965) . another approach has been the prophylactic administration of sodium alkyl sulfonate, which inhibits pectin methyl esterase activity, preventing foam formation by eliminating the products of this enzyme reaction (nichols, 1963) . much effort is now being directed toward genetic selection of cattle which are less susceptible to rumen tympany and to varieties of legumes which are less likely to produce bloat (howarm, 1975) . unlike monogastric species, ruminants can effectively use nonprotein nitrogen to meet dietary protein requirements. urea, biuret (oltjen et al., 1969) and ammonium salts (webb et al., 1972) all can serve as dietary supplements. urea, which is the most frequently used, is hydrolyzed by bacterial urease within the rumen and the free ammonia formed is incorporated into amino acids by microorganisms within the rumen. the bacte rial protein so produced is digested and absorbed in the small intestine along with protein from the diet. signs of urea poisoning typically develop within minutes after consumption of food containing toxic amounts of urea. clinical manifestations are the result of excessive ammonia production (word et al., 1969; elmer and barclay, 1971) and are due to the encephalotoxic effects of free ammonia absorbed from the rumen. tolerance to urea may be significantly increased by gradually elevating the amounts of urea in the diet or by adding readily fermentable carbohydrate. it has actually been possible for ruminants to adapt and thrive on a diet in which urea was the sole source of dietary nitrogen. however, if urea is fed at a level of more than 3% in the diet of unadapted animals, toxic effects are likely. poisoning may occur when, by accident, animals obtain access to large amounts of urea-containing dietary supplement or in animals receiving bulk feed when there has been an error in formulation or when the urea-containing additive is incompletely mixed. oral administration of acetic acid has been shown to reduce acute urea toxicity, apparently by decreasing absorption of free ammonia from the rumen. acetic acid also has been used clinically for the treatment of urea poisoning but under experimental conditions it has more value prophylactically than in animals with frank signs of poisoning (word et al., 1969) . textbook of veterinary internal medicine-diseases of the dog and cat current veterinary therapy veterinary medicine cornell vet. 66, 183 vet. ree. 90, 645 nature (london) 185, 35 handbook of physiology a guide to learning fluid therapy physiology of the digestive tract proc nati. acad. sci. u.s.a. 44, 648. drucker the physiology of domestic animals current veterinary therapy proc. nati. acad. sci the vitamins vet. ree. 77, 994 glycoproteins: composition, structure and function handbook of physiology the rumen and its microbes handbook of physiology nord. veterinaermed. 18, 73 handbook of physiology handbook of physiology acta vet. scand. 4, 27. krogh, n proc. soc. exp. biol. med. 66, 217. lack textbook of veterinary internal medicine-diseases of the dog and cat nature (london) 202, 400 vet. ree. 81, 416. penhale current veterinary therapy handbook of physiology proc. nati. acad. sci nature (london) 160, 262 diabetes 19, 614. van de kamer current veterinary therapy gastroenterology 67, 531 handbook of physiology intestinal absorption handbook of physiology key: cord-006331-s2qf98lj authors: spiridonova, v. a. title: molecular recognition elements: dna/rna-aptamers to proteins date: 2010-05-23 journal: biochem mosc suppl b biomed chem doi: 10.1134/s1990750810020046 sha: doc_id: 6331 cord_uid: s2qf98lj the review summarizes data on dna/rna aptamers, a novel class of molecular recognition elements. special attention is paid to the aptamers to proteins involved into pathogenesis of wide spread human diseases. these include aptamers to serine proteases, cytokines, influenza viral proteins, immune deficiency virus protein and nucleic acid binding proteins. high affinity and specific binding of aptamers to particular protein targets make them attractive as direct protein inhibitors. they can inhibit pathogenic proteins and data presented here demonstrate that the idea that nucleic acid aptamers can regulate (inhibit) activity of protein targets has been transformed from the stage of basic developments into the stage of realization of practical tasks. during the last decade a significant break has been achieved in the use of basic knowledge on dna in applied studies. the development of highly technolog ical analytic methods employing immobilized dna is one of rapidly developing directions. the major achievement of microarray technology (i.e. dna chips) consists in possibility of the use of various dna libraries amplified by polymerase chain reaction (pcr) for the development of sets of dna sequences. using hybridization these sets can rapidly analyze and compare sequences of thousands genes, their mutant forms, dna polymorphism and to discover new genes. the second direction consists in the develop ment of irrational design of nucleic acids for studies of nucleic acid protein recognition. in 1990, two labs (the gold and szoztak laboratories; usa) indepen dently developed the selex method (systematic evolution of ligands by exponential enrichment) [1, 2] . using this method it is possible to isolate targeted nucleic acid molecules (aptamers) from the large set of individual molecules (more than 10 18 ) known as the combinatorial library. aptamers are small single stranded dna or rna molecules of 40⎯100 nucle otide residues in length with rather complex three dimensional structure. such complex structure deter mines aptamer ability to bind various molecules including proteins. thus, such complex process of biosynthesis of protein recognizing elements, antibod ies, which nature has been naturally creating for thou sands years, is now modeled in vitro. selection begins with generation of a large rna library with fixed 5' and 3' ends and a degenerated region of 30-60 nucleotides in length (fig. 1) . such library contains 10 14 -10 15 variants of rna molecules, which are folded in complex 3d structure. the library is incubated with a protein and rna molecules bound to the protein target are separated from unbound rna molecules. the bound rna molecules are separated from proteins and then amplified by means of reverse transcriptase and pcr to obtain a new pool of mole cules with increased affinity. the procedure is usually repeated 10-15 times until maximal number of aptamers exhibiting affinity to the target will be clearly detectable in the enriched fraction. aptamers are then cloned (usually into a bacterial vector) and sequenced. in the case of dna the selection also begins with dna library in which the randomized region is flanked by fixed sequences at 5' and 3' ends. to pro duce single stranded dna molecules either asymmet ric pcr or one of primers carries a biotinylated tag, which helps to separate one dna strand from another on streptavidin columns are used. many reviews on detailed description of all steps of this method have been published to date [3] [4] [5] [6] [7] [8] [9] [10] . now, the idea that nucleic acid aptamers can regulate (inhibit) activity of protein targets has been transformed from the stage of basic developments into the stage of realization of practical tasks. in this review the major attention is paid to the aptamers to proteins involved into patho genesis of wide spread human diseases. tuer and gold proposed to use combinatorial rna libraries for creation of rna ligands selectively bound to t4 dna polymerase [1] . rna ligands were named as aptamers by ellington and szoztak [2] . they also introduced a name of this method, selex. now convincing evidence exists that aptamers are a new effective group of therapeutics, which may represent the scheme illustrating the selex method for preparation of dna and rna aptamers. an initial randomized dna library is transformed into single stranded dna (ssdna) and then introduced into binding reaction with a protein, pre immo bilized onto a column. rna is obtained by transcription of the initial library; the latter carries introduced promoter of t7 rna polymerase. rna molecules are also exposed for binding with the protein immobilized onto the column. after removal of unbound molecules, dna/rna molecules that bind to the immobilized protein are separated from this protein by phenol chlo roform treatment and then subjected to alcohol sedimentation. rna molecules are used for cdna preparation by means of reverse transcriptase. all resultant molecules are then transformed into double stranded dna (dsdna). in the case of dna ssdna prepared using asymmetric pcr is used again for repeated binding. rna molecules are subjected for transcription and resultant molecules are used for binding with the immobilized protein. the selection includes 10⎯15 rounds and this yields an enriched fraction of aptamers. the next step includes cloning into a plasmid vector and sequencing of resultant sequences. an important tool against many diseases. the drug macugen has already been approved by us fda (food and drug administration) for the treatment of age related macular degeneration (amd). some aptamers are now in different stages of pre and clini cal trials [11, 12] . highly specific (antibody like) recognition and binding of aptamers to their protein targets make them attractive therapeutics. aptamers (as well as antibod ies) are folded into complex three dimensional struc tures and form hairpins and loops. the range of disso ciation constants characterizing binding of dna and rna aptamers to their protein targets varies from nanomolar to subnanomolar levels. aptamers can dis criminate related proteins consisting of the same structural domains [13] [14] [15] [16] . it should be noted that the use of 1000 fold excess of aptamer doses in animal models employed in preclinical trials and in therapeu tic applications in humans did not cause allergic reac tions [11, 17] . studies on biocompatibility and phar macokinetics of aptamers and investigations of various modifications of aptamer structures have been per formed for their further applications as drugs [12] . nuclease degradation is the major problem that complicates manipulations with oligonucleotides. protection against nonspecific action of nucleases during selection includes modification of pyrimidine nucleotides at ribose c2' (amino and fluoro deriva tives) [18] [19] [20] , use of liposomes as carriers [21] , post selection ribose c2' hydroxyl modifications by intro ducing methyl, allyl, amino groups, etc. [22] [23] [24] . the molecular mass of short polynucleotides is 8-14 kda; this corresponds to 25-40 nucleotides. such a small size facilitates rapid renal filtration within a few minutes. aptamer modification by conjugation to polyethylene glycol or other agents and their attach ment to the liposome surface prolonged the period of aptamer action [21, 25] . usually, aptamers exhibit high specificity towards their targets and this should be taken into consider ation in preclinical trials on animal models. however, protein orthologs may decrease efficacy of such com pounds. nevertheless, an improved selection process named as "toggle selex" seems to overcome this problem. toggle selex has been proposed for rna libraries incubated with the same protein from human plasma. during the follwing rounds of selection it was also exposed to binding with the animal ortholog used in subsequent preclinical trials [26] . studies on pharmacokinetics of aptamers subjected to modifications should be accompanied by analysis of their excretion from the body. this is important because in addition to the main disease patients may have multiple dysfunctions including renal insuffi ciency. for regulation of aptamer activity rusconi pro posed the antidote proof of concept (the method of rational design) [27] . using watson crick base pairing he designed an antidote structure as a complementary sequence that bound to the aptamer, altered its struc the mechanism of antidote action in the pair aptamer antidote (modified from [26] ). the antidote is an oligonucleotide of 15 nucleotides in length; it forms an inactive complex with the aptamer to factor ixa involved into the blood coagulation cas cade. possessing a sequence complementary to the aptamer, the antidote forms a complex with the aptamer and thus alters its 3d structure. antidote inactive form ture and, thus, prevented its complex formation with the protein target (fig. 2) . the proposed concept gave a unique possibility of aptamer regulation because it allows to control administration of an aptamer based drug in any clinical use. considering aptamers as direct protein inhibitors it is very attractive to use them for studies of inhibitory mechanisms and also for therapeutic application. pos sibility of aptamer preparation for any class of biomol ecules allows to evaluate importance of their use and increases areas of their applications. advantage of aptamers maintaining their activity in multicellular organisms significantly facilitates preclinical trials, saves time and reduces costs required for antibody preparation on animal models. finally, design of an antidote that control aptamer activity possibly repre sents the most important contribution to the develop ment of nuclei acid therapy because it can control drug dosage and, thus, determine required safety. it should be noted that in addition to biomedical studies aptam ers are also used as a recognizing elements in microar ray based biosensors; this is another important and logic continuation of the development of the dna chip technology. this review summarizes current knowledge on aptamers to various proteins, their affinity to protein targets; it describes inhibitory properties of aptamers and information about preclinical and clinical trials. 2.1. thrombin thrombin is a key protein in blood clotting process. this serine protease is generated during a cascade of proteolytic reactions initiated by epithelial damage. thrombin is produced from prothrombin by factor xa. active thrombin catalyzes the reaction of fibrinogen conversion into fibrin, which forms a fibrin matrix for the thrombus by "capturing" blood cells [28] . throm bin also activates platelets via interaction with their par receptors and regulates expression of some sub strates and activation molecules such as p selectin [29, 30] . problem of hemostasis requires creation of such thrombin inhibitor, which would be specific for blood clotting process, does not cause allergic reaction and effectively regulates this process. an anti thrombin aptamer was one of the first therapeutic aptamers obtained by the selex method. the single stranded dna aptamer was isolated from a pool of ~10 13 oligo nucleotide sequences containing a 60 nucleotide ran domized region [31] . the 5 round selection resulted in identification of aptamers forming a complex with thrombin, which was characterized by the k d values ranged from 25 to 200 nm. these aptamers were based on the 15 nucleotide sequence: dggttggtgtg gttgg (15tba). the aptamer increased time required for clot formation from 25 to 170 s in vitro and from 25 to 43 s in human blood plasma. the 15tba structure was investigated by means of nmr analysis, which included the study of 15tba alone, in the complex with thrombin and the study of aptamer binding with the anion binding site of thrombin, exosite 1 [32] [33] [34] [35] . 15tba forms a complex compact tertiary structure known as g quadruplex (fig. 3 ). other laboratories also used the selex method to perform aptamer selection to thrombin [36, 37] . 15tba also influenced platelet aggregation stimulated by thrombin. thrombin also caused proteolytic activa tion of platelet par 1 receptor and its aptamer inhib ited this activation in a dose dependent manner [38] . the anticoagulant activity of the aptamer was tested on monkeys. the prothrombin time (pt) increased by 1.7 fold in 10 min and returned to base line 10 min after aptamer administration. 15tba also inhibited platelet aggregation and prolonged platelet activation induced by thrombin [39] . the aptamer was also investigated using an anticoagulation model of extracorporeal circulation in sheep. the pt values reached 40⎯45 s (versus 21.7 s of the baseline level), whereas control pt remained close to the baseline. in the other experiment 15tba was investigated using a the structure of the 15tba aptamer to thrombin, which inhibits fibrinogen hydrolyzing activity. 15tba has the oligonucleotide sequence dggttggtgtggt tgg. the g quadruplex structure is a structure forming element for dna. eight of nine guanines form two planar g quartets with three loops; the loop tgt located in the center and two symmetrical loops tt. the presence of octa coordinating calcium ion and stacking interaction between g quartets of the duplex determine maintenance of the g quadruplex. calcium ion is located between par allel planes of the g quadruplex. cardiopulmonary bypass (cpb) model. the study included examination of anticoagulation activity, pharmacokinetics and renal clearance of the aptamer [40] . animals were subdivided into two groups: one group received injections of heparin (300 u/kg) and protamine in boluses and was used as control of activ ity by cpb. the second group of animals received aptamer infusion (0.3-0.5 mg/kg per 1 min). these animals were characterized by increased pt, activated partial thromboplastin time (aptt), and activated clotting time (act), which then returned to the base line after infusion [39] [40] [41] . in the pharmacokinetic studies using the cpb model, the elimination half life of the aptamer was 1.9 min; however, during the 60 min infusion this parameter increased up to 7.7 min. these results sug gested that the aptamer would function in the animal model and that unmodified dna aptamers were rap idly eliminated from the body. now this dna aptamer is under preclinical trials by archemix corp. for subsequent trials in humans. anti thrombin rna aptamers were obtained using the library with a 30 nucleotide randomized sequence [42] . the anti thrombin aptamers were iso lated after 12 rounds of selection. the enriched frac tion was then cloned in the plasmid puc18 and sequenced; this yielded two classes of aptamers. the conservative motif in 22 clones of the class i rnas was represented by the sequence uccggaucgaag uuaguaggcgga inside a variable zone. one of the best anti thrombin aptamers was characterized by the k d value of 9.3 ± 1.0 nm. members of the second class exhibited lower affinity (the k d value of 155 ± 9.0 nm). competitive analysis with heparin and hirudin dem onstrated that heparin but not hirudin displaced the rna aptamer from its complex with thrombin. this suggests affinity of this aptamer to thrombin exosite ii. however, tests on functional activity in animal models have not been performed. in preclinical trials highly specific aptamers to human proteins may demonstrate lowered affinity to protein orthologs in animal models. to overcome this problem so called "toggle" approach has been pro posed: 2' fluoro rna aptamers were incubated with a mixture of human and porcine thrombin during the first round and then porcine and human thrombin were alternatively used in subsequent rounds of selection [43] . after 13 rounds of selection clones with the con servative sequence gggaacaaagcugaaguac uuaccc have been found; they exhibited cross reac tivity with porcine and human thrombin. the complex with human thrombin was characterized by the disso ciation constant k d of 2.8 ± 0.7 nm and the complex with porcine thrombin had the k d value of 83 ± 3 pm. the aptamer increased clotting time (thrombin con centration was 10 nm) of blood plasma from 11.6 ± 0.2 to 22.6 ± 1.4 s. in porcine plasma tog 25 increase clot ting time from 15.7 ± 0.7 to 61.9 ± 1.2 s. improvement of thrombin dependent platelet aggregation by the aptamer occurred in the dose dependent manner. the higher effect was achieved using porcine platelets: a 10 fold excess of tog 25 inhibited thrombin activity by 90% [26, 43] . factor viia (fviia) is a trypsin like protease involved into the coagulation cascade. in combination with the tissue factor (tf) fviia plays a critical role in thrombin formation and thus promotes active clot for mation. aptamers to fviia have been isolated from an rna library using the selex method. these aptam ers inhibited activation of factor x (inactive precursor in the coagulation cascade) by fviia [15] . after 16 rounds of selection from the 2' amino modified rna library the isolated aptamers formed a complex with fviia characterized by the k d value of 11.3 ± 1.3 nm. specificity of some aptamers was investigated in bind ing reactions with other protein factors (fxia and fxa). the micromolar range of k d values determined for these complexes suggested nonspecific binding of these aptamers with protein factors. addition of the anti fviia aptamer inhibited an initial rate of fx acti vation by about 95%. experiments on dilution of the reaction mixture revealed a dose dependent mode of inhibition. the aptamer prolonged clotting time up to 175% in the pt test. factor ixa (fixa) is a serine protease that plays an important role in formation of critical mass of throm bin required for coagulation. the complex tf/fviia performs proteolytic cleavage of the protein factor fix into its active form fixa; the latter binds to fviiia on the platelet surface and activates factor fx to fxa, which catalyzes conversion of prothrombin into thrombin [28] . rusconi et al. performed rna selection to fixa; after eight rounds of selection they found an aptamer, which bound to fixa with the k d value of 0.65 ± 0.2 nm and exhibited 5000 fold higher affinity to fixa compared with fviia, fxa, fxia and activated protein c [20] . a truncated version of this aptamer (9.3t) maintained high affinity to fixa (k d of 0.58 ± 0.1 nm) and totally inhibited fx hydrolysis by the enzyme complex. the anticoagulation activity of 9.3t was evaluated using activated partial thromboplastin time (aptt). the aptamer increased clotting time in the dose dependent manner and caused a several fold increase in aptt. in continuation of the antidote theory rus coni obtained an rna antidote, which caused revers ible inhibition of 9.3 t thus creating a drug/antidote pair for the anticoagulation therapy. using the com plementary base pairing principle the second rna oligonucleotide complementary to the 9.3t aptamer was created. after administration of the antidote nucleotide the anticoagulation activity of the anti fixa aptamer changed in 10 min and this effect per sisted for over 5 hours [16] . almost in 5% of 12 million people receiving heparin therapy heparin induced thrombocytopenia (hit) is developed after one year [28] and this is the reason for cessation of the heparin therapy. patients who need repeated anticoagulant therapy receive hemodialysis, which complicates patient's life. to prolong the effect of the anti fixa aptamer in vivo rusconi c.p. et al. prepared a cholesterol deriva tive (ch 9.3t), which exhibited high affinity (k d = 5.3 ± 1.1 nm) and anticoagulant activity [27] . tests on por cine and mouse plasma have shown the same efficacy of the animal models as in the case of human plasma. using the porcine anticoagulant system the aptamer increased pt and aptt comparable with its effects on pt and aptt in human plasma samples. there was significant difference between the modified and initial aptamers: the cholesterol moiety increased half life of ch 9.3t to 60-90 min (versus 5-10 min for 5-10 min). the antidote 5 2 c neutralized more than 95% of the aptamer effect within 10 min in animal models [27] . the antithrombotic effect of the aptamer was investigated in mouse thrombosis model, which was induced by administration of ferric chloride to the carotid artery; mice were pretreated with ch 9.3t or a functionally inactive aptamer with scrambled nucle otide sequence (negative control). all the mice in the negative control group developed an occlusive throm bus in 8.1 ± 0.1 min. in the aptamer treated group 80% of mice maintained clear normal carotid artery blood flow during 30 min (time required for the occlu sive thrombus formation ≥24.4 min) [27] . the effect of the 5 2c antidote was accessed using the model of active bleeding (tail transection). mice were pre treated with ch 9.3t or an aptamer with scrambled sequence and the tail was cut 1 h after the treatment. blood losses were measured for 15 min after tail transection. animals treated with ch 9.3t exhibited significantly more blood loss (176 ± 23.7 μl) compared with controls (48 ± 17.8 μl). administration of the 5 2c immediately after tail transection prevented hem orrhage in the aptamer treated animals (blood loss was 54.5 ± 13.6 μl) [27] . the biopharmaceutical company regado bio sciences continues studies on the fixa aptamer anti dote pair named as reg1, which is under first stage of clinical trials. hepatitis c virus (hcv) is a major cause of both sporadic and viral hepatites differed from hepatitis a or b [44] . the nonstructural protein 3 (ns3) is a serine pro tease that exhibits protease and helicase activity and represents a good target for inhibition of hcv. aptamer selection was performed using a library car rying a 120 nucleotide randomized region and after 6 rounds of selection two aptamers inhibiting protease and helicase activities were obtained [45] . for identi fication of the aptamer demonstrating affinity to the active site of ns3 subsequent selection was performed using a truncated polypeptide δns3. using an rna library with a 30 randomized region authors per formed 9 rounds of selection and identified 45 clones, which bound δns3 [46] . according to their nucleotide sequences aptamers were subdivided into three families. they all contained a conservative region ga(a/u)ugggac. these aptamers formed a complex with δns3 with the k d value of 10 nm, caused 90% inhibition of protease activity of the δns3 peptide and full sized ns3 bound to a maltose binding protein (mbp ns3). in vivo hcv proteins are processed by ns3 and ns4a cofac tor. for modeling of physiological conditions the aptamer effect on ns3 activity was tested in the pres ence of the p41 peptide, which caused a sevenfold increase of mbp ns3 activity. under these conditions the aptamer inhibited mbp ns3 activity by 70% [46] . human neutrophil elastase (hne) is involved in various inflammatory diseases, including acute respi ratory distress syndrome (ards), septic shock, arthritis, and ischemia reperfusion injury [47] . a covalent inhibitor of hne, a diphenyl phosphate derivative of valine (valp), was coupled to an rna library to enhance the binding of the inhibitor with hne [47] . ten rounds of selection yielded an rna aptamer conjugated to the dna:valp substrate (rna 10.11: dna:valp). the aptamer demonstrated bind ing to hne (k d = 71 nm) and enzyme inhibition (k i = 5 nm) in vitro. in contrast to the rna aptamer 10.11 or the substrate dna:valp administered separately the aptamer modified with the substrate inhibited hne ex vivo in the rat model of ards [48] . the same group also performed a valyl phosphonate: dna library selection to find more potent hne inhibitors. authors used a single enantiomer form of the valyl phospho nate, which was compared with a racemic mixture. inhibitor selection was performed using purified elastase and also secreted elastase in the presence of neutrophils [49] . after 18 rounds of selection the aptamer ed45, which inhibited hne, was found. the aptamer was truncated to a 42 mer, named nx21909, and tested in a rat model of lung inflammation. a 40 nmol dose of nx2109 inhibited neutrophil infiltration by 53% in the lung of rat in vivo [49] . 3.1. vegf angiogenesis plays a central role in various physio logical and pathological processes. vegf (vascular endothelial growth factor) is one of the best character ized growth factors; it is involved into initial steps of angiogenesis and represents one of the most promising targets for anticancer therapy [50] . an increased vegf level associated with angio genesis was observed during tumor growth and metastases, premature aging and age related degener ation of tissues [50] [51] [52] . using the selex method ruckman performed 12 rounds of selection cycles and isolated aptamers to human vegf 165 with k d of 50 pm in a 2' f pyrimi dine rna library [19, 20, 53⎯55] . for increased sta bility against nucleases two aptamers were additionally modified by the 2' o position [55] . these aptamers were characterized by the k d values of 49 and 130 pm; they were specific to vegf 165 and did not bind to related proteins: vegf 121 and placenta growth factor plgf 129 . the aptamers to vegf 165 inhibited the bind ing of vegf 165 to its receptors, flt 1 and kdr (kinase domain receptor). using 125 i labeled vegf 165 inhibition of receptor binding was evaluated: theic 50 values for aptamer competition with the flt 1 receptor and kdr were ranged from 50-300 and 2-60 pm, respectively [55] . therapeutic potential of the aptamers to vegf was evaluated by the miles assay representing simple and rapid means of monitoring the ability of aptamers to inhibit the activity of vegf 165 in vivo. it is assessed as vascular wall permeability in animal models. the test was performed using adult guinea pigs and the most effective aptamer inhibited vascular permeability by 58% at 1 μm [55] . pharmacokinetics of the 2 fluoro pyrimidine and 2' o methyl purine aptamer to vegf called as nx 1838 has been investigated in monkeys. during intravenous administration of this aptamer as a conjugate with a 40 kda polyethylene glycol was characterized by half life of 9.3 h and a clearance rate of 6.2 ml/h. subcuta neous administration resulted in 80% absorption into the tissues within 8-12 h [25] . preclinical and clinical trial of nx 1838 also called as macugen was performed by eyetech pharmaceuti cals inc for the treatment of age related macular degeneration in diabetic patients [11, 12] . the synthetic aptamer nx 1838 was also investi gated in the rat model of angiogenesis. these studies confirmed a significant inhibition by 80% of angio genesis by means of vegf in the presence of this aptamer. the 1a phase of clinical trials did not reveal any significant complications after a single adminis tration of the drug. in addition 80% of patients dem onstrated stable improvement during observation for 3 months after injections, 27% of patients demon strated a threefold improvement of vision among dia betic patients (etdrs) [10] . clinical trials (phase 2) have shown that multiple macugen administration with or without photody namic therapy (pdt) did not cause any serious impairments; moreover 87.5% of patients demon strated stable vision improvement and 25% of patients demonstrated significant improvement evaluated using the etdrs system (early treatment for dia betic retinopathy study). during the third phase of clinical trials macugen (under the commercial name pegaptanib) was used as the only drug every 6 weeks over a period of 48 weeks at a dose of 0.3, 1 and 3 mg intravitreously [56] . all three groups of patients demonstrated significant improvement of vision. the severe loss of visual activ ity determined as the loss of 30 letters of visual acuity reduced from 22 to 10% in the group receiving 0.3 mg of macugen. in addition, 33% of patients receiving this dose maintained their visual acuity or gained acu ity (versus 23% of control group). no antibodies against macugen were found. eytech in cooperation with pfizer obtained fda approval for the use of macugen for the treatment of amd. these first results demonstrate that aptamers may be effective drug prep arations. it is known that the intensive tumor development is accompanied by neovascularization and therefore an aptamer to vegf was used for inhibition of tumor vascularization (and tumor growth). the aptamer isolated and optimized by ruckman et al. was tested in a mouse model of wilmis tumor (the most common malignant tumor of the kidneys in chil dren). tumor was implanted into a mouse kidney and its growth was maintained for one week, and then the aptamer to vegf (200 μg) or vehicle (phosphate buff ered saline) were administered for experimental and control mice respectively, daily for 5 weeks [57] . after decapitation of animals authors observed that tumors weighed 84% less in treated versus control animals. lung metastases were seen only in 20% of the aptamer treated animals (versus 60% of animals from the control group). the aptamers tested in the murine nephroblastoma exhibited the decrease in tumor growth by 53% compared with control [57] . the increase in bfgf correlates with appearance of various diseases including retinopathy, rheumatoid arthritis, leukemia [58] . jellinek and co authors used a 2' amino pyrimi dine derivative rna library and performed 11 rounds of aptamer selection [58] . they found an aptamer named as m21a, which exhibited binding to bfgf with k d of 0.35 nm; a competitive binding study revealed that it competed for bfgf binding with unfractionated heparin and low molecular weight hep arin. the inhibitory activity of m21a was also investi gated using chinese hamster ovary (cho) cells: the aptamer bound to its target with the k d values of 1-3 nm [59] . the effect of m21a on the endothelial cell motility was also investigated using the migration of endothelial cells to a denuded area in bovine aortic cells where endogenous bfgf is essential for activity. at concentrations > 50 nm the aptamer inhibited cell migration in a dose dependent manner (as compared with control). the rna aptamer inhibited bfgf bind ing to its cell receptor [20] . platelet derived growth factor (pdgf) is a mito gen composed of two homologous (a and/or b) chains linked by three disulfide bonds; this dimeric protein is involved into wound healing and progression of vari ous diseases including atherosclerosis and glomerulo nephritis. many tumor cell lines produce and secrete pdgf [60] . a dna selection in vitro against human recombi nant pdgf ab was performed and after 12 rounds dna aptamers characterized by k d of 50 pm were iso lated. three aptamers effectively inhibited pdgf bb binding to pdgf α and β receptors with the k i value of 1 nm. the anti pdgf aptamers also inhibited mitogenic effects of pdgf on cells expressing pdgf β receptors with k i of 2.5 nm [61] . one aptamer termed 36t was truncated, 2'o methyl, 2' fluoro modified and capped at the 3' end (to increase resistance to nucleases) and conjugated to 40 kda peg (to increase its lifetime in blood circula tion) [62] . the modified aptamer exhibited high affin ity binding to the human protein (k d = 100 pm); it was tested using a rat glomerulonephritis. in this model intravenous administration of this aptamer (2.2 mg/kg) twice a day decreased mitoses by 64% on day 6 and by 78% on day 9. animals treated with this aptamer were characterized by a decreased mono cyte/macrophage index and glomerular matrix over production. control animals received a scrambled sequenced oligonucleotide or peg for 6 days [63] . interferon γ (ifn γ)exhibits various immunoregu latory effects. although its antiproliferative effect is less pronounced than in ifn α and ifn β, ifn γ is the most potent activator of macrophages and the inducer of expression of mhc class ii molecules [58] . in healthy nervous tissue ifn γ is almost absent, how ever, during inflammatory processes in the nervous system and in multiple sclerosis it is overproduced. ifn γ secretion can result in inflammatory and autoim mune diseases. rna selection using 2 fluoropyrimi dine and 2 aminopyrimidine rna or a mixture of these two modifications were screened for aptamers that inhibited receptor binding of ifn γ [64] . the resultant aptamer, 2' amino 30 had a k d value for its complex with receptor of 2.7 nm. in the culture of a549 cells it inhibited receptor binding of ifn γ with k i value of 10 nm. this aptamer also inhibited induction of the mhc complex regulated by ifn γ and icam 1 expression with ic 50 values of 700 and 200 nm, respectively [64] . endothelial receptor tyrosine kinase tie2 plays an important role in vascular wall stability. angiopoietin 2 (ang 2) is a natural antagonist, which is obviously expressed only during active angiogenesis (e.g. tumor growth) [65] . for investigation of ang 2 by aptamers 11 rounds of rna selection were performed and rna molecules exhibiting specific binding to ang 2 were iso lated. one aptamer demonstrated high affinity to ang 2 (k d = 3.1 nm); it did not bind to ang 1 (k d > 1 μm). this aptamer was truncated to 41 mer (k d = 2.2 nm) and the truncated aptamer inhibited ang 2 function in a cell culture and in a rat model, where it significantly inhibited neovascularization by 40% [65] . influenza is one of the most widespread disease in the world. control of this disease includes active cam paigns of vaccination, use of drugs blocking neuro minidase action. the use of aptamers helps to block virus binding to cell receptors. binding of isolated dna aptamers to viral hemagglutinins blocked virus penetration into cells [66, 67] . extracellular domains of influenza hemagglutinin cause agglutination of blood cells (mainly erythro cytes). hemagglutinin determines virus binding to cells. neuraminidase is responsible for : 1) ability of a viral particle to penetrate into the host cell; 2) ability of viral particles to leave host cells after reproduction. two dna aptamers were obtained to the hemaggluti nin peptide (residues 91-261), which is responsible for binding of an oligosaccharide component of cell receptors. the aptamer a22 exhibited high binding activity and blocked agglutination of chicken red blood cells [66] . the effect of a22 was confirmed by microscopic studies; they revealed preservation of cell structure compared with control preparations, in which damage of cell structure in the presence of influenza virus was observed. the same authors iso lated two rna aptamers to hemagglutinin using an rna library containing a 30 nucleotide randomized region. a predicted secondary structure in 73 bases long included nucleotides of the randomized region and also constant sequences of the flanking region. one of the aptamers exhibited binding to hemaggluti nin with the k d value of 2.9 nm. the affinity of this aptamer was 15 fold higher than that of a monoclonal antibody to this protein. moreover, the rna aptamer allowed to discriminate hemagglutinins isolated from two various strains [67] . binding proteins 5.1. tat protein therapeutic applicability of aptamers has been undertaken during studies of hiv replication. human immunodeficiency viruses hiv 1 and hiv 2 that belong to a lentivirus class selectively affect t helpers. a regulatory tat protein activates viral replication. the tar element (of 60 nucleotides long) presented in all predicted viral transcripts is required for func tioning of tat protein. it was proposed to express the 60 nucleotide tar sequence to capture tat protein into an rna decoy [68, 69] . tar rna, hiv tran script, was expressed in cem ss cells. the rna decoy inhibited hiv 1 replication over 99% in vitro. inhibition of viral replication in cd4+ cells proceeded at the high level tar aptamer expression from the trna promoter. changes in the nucleotide sequence of hair pins or loop in the structure of the tar aptamer abol ished the ability of the tar aptamer to inhibit hiv rep lication [68, 70] . the selex library consisted of a randomized sequence of 120 nucleotides was used for selection of aptamers to tat proteins. after 11 rounds of selection a truncated variant of an rna aptamer named as rna tat was obtained; it formed a complex with the tat protein with k d of 120 pm. this 37 mer rna aptamer inhibited hiv 1 in vitro and decreased viral replica tion by 70% in a cell culture [71] . other groups also found rna decoys, which bound tat protein and inhibited hiv 1 [72, 73] . no significant incompatibility between tar and tat interactions of hiv 1 and hiv 2 belonging to var ious subfamilies were found. however, although tat 1 could transactivate hiv 2 through tar 2, tat 2 did not interact with tar 1 in hiv 1 [74] . the viral protein rev promotes transportation of partially spliced rna molecules to cytoplasm, where they provide synthesis of usual retroviral products. the rre (rev responsive element) element composed of about 234 nucleotides forms a complex three dimen sional structure, to which rev protein binds. using a trna promoter for expression of the rre element, the major rev binding site in hiv 1, overexpression of this construct has been estimated in the cells. expres sion of the chimeric trna rre aptamer caused inhi bition of viral replication by more than 90% [75] . the aptamers passed phase 1 clinical trials, in which this construct was transduced in vitro to cd34+ cells obtained from bone marrow of a hiv 1 infected sub jects followed by subsequent reinfusion into these sub jects. aptamer administration did not cause adverse effects, however, a rather low level of rre gene expression was observed possibly due to inappropriate conditions for gene transfer [76] . reverse transcriptase (rt) was the first target for the development of the selex method for hiv ther apy. tuerk and gold published the pioneer paper on selex. they used rt as the target for isolation of rna ligands, inhibiting hiv replication [1] . after 9 rounds selection from rna populations random ized at 32 positions authors isolated rna that specifi cally bound to hiv rt and inhibited activity of this enzyme [77] . the sructure of the rna aptamer was subsequently characterized and experiments per formed on cells indicated inhibition of hiv 1 replica tion by 90-99% [78] . in addition, aptamer expressing t cells completely blocked the spread of hiv in cul ture [79] . proliferation of myocardial and vascular cells is a central problem in the development of such cardiovas cular diseases as hyperplasia, atherosclerosis, malig nant tumors [80, 81] . e2f plays a central role in regu lation of cell proliferation. this factor exhibits highly specific binding to a double stranded dna containing eight base pairs tttcgcgc. the constructed 14 mer dna aptamer containing a sequences for e2f binding was tested for inhibition of e2f activity [82] . in vascular smooth muscle cells (vsmc) stimulated by e2f the 14 mer oligonucleotide (odn) inhibited vsmc proliferation and expression of the genes c myc and cdc2, controlling cell cycle, and proliferating cell nuclear antigen (pcna). in vivo the 14 mer odn was transfected to rats with experimental carotid injury and this markedly suppressed the fibrosis for mation compared with nontransfected arterial seg ments. furthermore, this inhibition continued up to 8 weeks after a single transfection [81] . transfer of an e2f decoy can therefore modulate gene expression and inhibit smooth muscle proliferation and vascular lesion formation in vivo. using the selex method other aptamers to e2f were also prepared; they inhib ited dna binding activity of this protein [82] . these interesting results prompted dzau's group to test the e2f aptamer in humans in order to determine whether it can limit intimal hyperplasia during intra venous administration [83, 84] . the e2f aptamer was delivered to the infra inguinal vein by transfection. cell transfection efficiency was 89%, expression of c myc and pcna reduced by 73 and 70%, respectively, com pared with the control group. after 12 months in the group of patients treated with the e2f aptamers fewer occlusions were registered compared with control. the e2f dna aptamer is now evaluated by cor genetch inc., in a phase iii study to estimate its effi cacy at limiting coronary and peripheral vascular damages. this aptamer is very close to clinical appli cation. the e2f aptamer was also used for evaluation of long term protection from neointimal hyperplasia and atherosclerosis [85] . hypercholesterolemic rabbits were treated with intravenous injections of e2f aptamer or scrambled oligonucleotide. after 6 months (when animals were put on a cholesterol containing diet) the e2f aptamer treated group of animals was free of plaque whereas animals treated with the scram bled oligonucleotide and also control animals had extensive plaque formation [85] . finally, selex was used to obtain an rna aptamer that would bind and inhibit e2f. insertion of the e2f aptamer into a trna expression cassette yielded rnas exhibited effective inhibition of e2f1 binding to dna [86] . to test the ability of the e1 rna aptamer to block proliferation, human fibro blasts were treated with e1 rna aptamer and prolifer ation was then induced. the rna aptamer inhibited s phase by 80% compared with control [86] . thus, natural and in vitro selected aptamer can act as prolif eration inhibitors. transcription factor nf κb activates genes involved into inflammatory processes and synthesis of cytokines, interferons, mhc proteins, growth factors, and cell adhesion molecules, which play a central role in infarctions and various ischemic pathologies [87] . it is also required for hiv 1 gene expression and regula tion of cell tumors. a double stranded dna aptamer exhibiting high affinity binding to nf κb and named as a "natural decoy" was investigated in vivo using a cardiac ischemic/reperfusion model and a significant effect in inhibiting this injury was observed [88] . in a rat model of thrombosis animals transfected with the nf κb aptamer showed improved recovery of coro nary flow (97% versus 61% in control) 3 days after transfection [89] . the aptamer treated group demon strated a lower percentage of neutrophil adhesion to endothelial cells (38% versus 81%) and a lower level of interleukin 8 (109 versus 210 ng/mg) as compared with control [89] . a fluorescent labeled aptamer to nf κb was investigated in a murine model of nephritis, where it blocked glomerular inflammation and expression of the inflammatory markers il 1α, il 1β, il 6, icam 2, vcam 1 [90] . using the selex method an rna aptamer was also genetade against the p50 subunit of nf kb. four teen rounds of selection yielded the rna aptamer, exhibiting high affinity binding to p50 and inhibition of nf kb binding to dna by preventing protein dimerization [91] . work with aptamers has important advantages over antibodies: ⎯in clinical practice aptamers may be applicable in the same fields where antibodies are already used for treatment, but in contrast to antibodies aptamers are non immunogenic; ⎯aptamers exhibit the same high affinity to their protein targets as antibodies; ⎯aptamers can bind and penetrate to a pathologi cal nidus faster than antibodies; ⎯aptamer antidotes may be developed and they can control activity of the administered aptamer. the selex method originally developed for nucleic acid binding proteins is now actively used for studies of proteins, which lack natural complexes. the method is applicable for manipulations with individ ual proteins and for work with cell cultures. proc. natl. acad. sci. usa trombozy v kardiologii. mekhanismy razvitiya i vozmozhnosti terapii (thromboses in cardiology. mechanisms of develop ment and therapeutic capacities) proc. natl. acad. sci. usa proc. natl. acad. sci. usa proc. natl. acad. sci. usa proc. natl. acad. sci. usa proc. natl. acad. sci. usa proc. natl. acad. sci. usa, 1992 proc. natl. acad. sci. usa proc. natl. acad. sci. usa key: cord-000257-ampip7od authors: bagowski, christoph p; bruins, wouter; te velthuis, aartjan j.w title: the nature of protein domain evolution: shaping the interaction network date: 2010-08-17 journal: curr genomics doi: 10.2174/138920210791616725 sha: doc_id: 257 cord_uid: ampip7od the proteomes that make up the collection of proteins in contemporary organisms evolved through recombination and duplication of a limited set of domains. these protein domains are essentially the main components of globular proteins and are the most principal level at which protein function and protein interactions can be understood. an important aspect of domain evolution is their atomic structure and biochemical function, which are both specified by the information in the amino acid sequence. changes in this information may bring about new folds, functions and protein architectures. with the present and still increasing wealth of sequences and annotation data brought about by genomics, new evolutionary relationships are constantly being revealed, unknown structures modeled and phylogenies inferred. such investigations not only help predict the function of newly discovered proteins, but also assist in mapping unforeseen pathways of evolution and reveal crucial, co-evolving interand intra-molecular interactions. in turn this will help us describe how protein domains shaped cellular interaction networks and the dynamics with which they are regulated in the cell. additionally, these studies can be used for the design of new and optimized protein domains for therapy. in this review, we aim to describe the basic concepts of protein domain evolution and illustrate recent developments in molecular evolution that have provided valuable new insights in the field of comparative genomics and protein interaction networks. the protein universe is the collection of proteins of all biological species that exist or have once existed on earth [1] . our sampling and understanding of it began over half a century ago, when the first peptide and protein sequences were determined by sanger [2, 3] and, subsequently, the sequencing of rna and dna [4] [5] [6] . in the meantime, the genome projects of the last decade have uncovered an overwhelming amount of sequence data and researchers are now starting to address a series of fundamental questions that should shed light onto protein evolution processes [7] [8] [9] [10] . for instance, how many gene encoding sequences are present in one genome? how many sequences are repetitive and are these sequences similar in the various organisms on earth? which genes were involved in the large scale genome duplications that we see in animals? a comparison of sequences for evolutionary insight is best achieved by looking at the structural and functional (sub)units of proteins, the protein domains. by convention, domains are defined as conserved, functionally independent protein sequences, which bind or process ligands using a core structural motif [11] [12] [13] . examples of domain modes of actions in signaling cascades for instance, are to connect different components into a larger complex or to bind signaling-molecules [14, 15] . protein domains can usually fold independently, likely due to their relatively limited size, and are well known to behave as independent genetic elements within genomes [16, 17] . the sum of these features makes protein domains readily identifiable from raw nucleotide and amino acid sequences and many protein family resources (e.g., superfamily and smart [see table 1 ]) indeed fully rely on such sequence similarity and motif identifications [18, 19] . the algorithms that are used for domain identification are built around a set of simple assumptions that describe the process of evolution. in general, evolution is believed to form and mold genomes largely via three mechanisms, namely i) chemical changes through the incorporation of base analogs, the effects of radiation or random enzymatic errors by polymerases, ii) cellular repair processes that counter mutations, and iii) selection pressures that manifest themselves as the positive or negative influence that determines whether the mutation will be present in subsequent generations [20, 21] . by definition, each of these phenomena styles, reproductive strategies, or the lack of apparent polymerase-dependent proofreading such as in positivestranded rna viruses [22] [23] [24] [25] . consequently, substitution rates need therefore be calculated to correctly compare two or more sequences and hunt uncharted genomes for comparable domains. particularly this last strategy, using general rate matrices like blosom and pam, is an elegant example of how new protein functions can be discovered [26] [27] [28] [29] [30] . fast algorithms for pair-wise alignments can be found in the basic local alignment search tool (blast), whereas multiple sequence alignments (msas, fig. 1a) in which multiple sequences are compared simultaneously are commonly created with for example clustalx and muscle (see table 1 ) [31] [32] [33] [34] . close relatives, sharing an overall sequence identity above for example 50% and a set of functional properties, can also be grouped into families and subfamilies. in turn, these families share also evolutionary relationships with other domains and form together so-called domain superfamilies [18, 35] . evolutionary distances between related domain sequences can easily be estimated from sequence alignments, provided that the correct rate assumptions are made. subsequently, these can be used to compute the phylogenies of the domain that share an evolutionary history. these, often tree-like graphs (fig. 1b) , depend heavily on rate variation models, such as molecular clocks or relaxed molecular clocks (e.g., maximum likelyhood and bayesian estimation), which are calibrated with additional evidence fig. (1a) . it was computed using bayesian estimation and presents the best-supported topology for the alignment. numbers indicate % support by the two methods used, while # indicates gene duplication events in the common ancestor and * marks a species-specific duplication event. for computational details, please see [42] . such as fossils and may therefore also provide valuable information on aspects like divergence times and ancestral sequences [36] [37] [38] . commonly used phylogenetic analysis strategies are listed in table 1 . a limitation of all inferred phylogenetic data is that it is directly dependent on the alignment and less so on the programs used to build the phylogenetic tree [39] . one of the shortcomings of automated alignments may thus derive from the fact that they commonly employ a scoring and penalty procedure to find the best possible alignment, since these parameters vary from species to species [22, 23] , as mentioned above. careful inspection of alignments is therefore advisable, even though software has been developed that combines the alignment procedure and phylogenetic analysis iteratively in one single program [40] . although sequence and phylogenetic analysis provide a relatively straightforward way for looking at domain divergence, comparison of solved protein structures has shown that protein tertiary organizations are much more conserved (>50%) than their primary sequence (>5%) [41] . for this reason, protein structures and their models provide significantly more insight into the relations of protein domains and how domain families diverged [16] . for example, the inactive guanylate kinase (gk) domain present in the maguk family was shown to originate from an active form of the gk domain residing in ca2+ channel beta-subunits (cacnbs) through both sequence and structural comparison [42] . furthermore, identification of functionally or structurally related amino acid sites in a fold sheds light on the complex, co-evolutionary dynamics that took place during selection [43] . as described above, the evolution of a protein domain is generally the result of a combination of a series of random mutations and a selection constraint imposed on function, i.e., the interaction with a ligand. the interaction between protein and ligand can be imagined as disturbances of the protein's energy landscape, which in turn bring about specific, three-dimensional changes in the protein structure [44, 45] . binding energies however, need not be smoothly distributed over the protein's binding pocket as a limited number of amino acids may account for most of the free-energy change that occurs upon binding [45] [46] [47] . in these cases, new binding specificities (including loss of binding) may therefore arise through mutations at these hot spots. an example is a recent study of the pdz domain in which it was shown that only a selected set of residues, and in particular the first residue of -helix 2 ( b1), directly confers binding to a set of c-terminal peptides [48] . the folding of a domain is essentially based on a complex network of sequential inter-molecular interactions in time [49] . this has of course significant implications for domain integrity, particularly if one assumes that the core of a protein domain is and has to be largely structurally conserved. indeed, even single mutations that arise in this area may easily derail the folding process, either because their free energy contribution influences residues in the direct vicinity or disturbs connections higher up in the intermolecular network [49] . it is therefore hypothesized that protein evolution took place at the periphery of the protein domain core, and that gradual changes via point mutations, insertions and deletions in surface loops brought about the evolutionary distance we see among proteins to date [21, [50] [51] [52] . however, distant sites also contribute to the thermodynamics of catalytic residues. this is achieved through a mechanism called energetic coupling, which is shaped by a continuous pathway of van der waals interactions that ultimately influences residues at the binding site with similar efficiency as the thermodynamic hotspots [53, 54] . indeed in such cases, evolutionary constraints are not placed on merely one amino acid in the binding pocket, but on two or more residues that can be shown to be statistically coupled in msas [54, 55] . in addition to contributions to binding, these principles also explain why the core of a domain structure will remain largely conserved, while at functionally related places residues can (rapidly) co-evolve with an overall neutral effect [56] . of course, these aspects of co-evolution are also of practical consequence for structure prediction and rational drug design [43] . through selective mutation, protein domains have been the tools of evolution to create an enormous and diverse assembly of proteins from likely an initially relatively limited set of domains. the combined data in genbank and other databases now covers over 200.000 species with at least 50 complete genomes and this greatly facilitates genome comparisons [57] [58] [59] . following such extensive comparisons, currently > 1700 domain superfamilies are recognized in the recent release of the structural classification of proteins (scop) [60] and it has become clear that many proteins consist of more than one domain [17, 61, 62] . indeed, it has been estimated that at least 70% of the domains is duplicated in prokaryotes, whereas this number may even be higher in eukaryotes, likely reaching up to 90% [35] . there are various mechanisms through which protein domain or whole proteins may have been duplicated. on the largest scale, whole genome duplication such as those seen in the vertebrate genomes duplicated whole gene families, including postsynaptic proteins, hormone receptors and muscle proteins, and thereby dramatically increased the domain content and expanded networks [42, 63, 64] . on the other end of the scale, domains and proteins have been duplicated through genetic mechanisms like exon-shuffling, retrotranspositions, recombination and horizontal gene transfer [65] [66] [67] . since the genetic forces, like exon-shuffling and genome duplication vary among species, the total number of domains and the types of domains present fluctuate per genome. interestingly, comparative analyses of genomes have shown that the number of unique domains encoded in organisms is generally proportional to its genome size [60, 68] . within genomes, the number of domains per gene, the socalled modularity, is related to genome size via a power-law, which is essentially the relation between the frequency f and an occurrence x raised by a scaling constant k (i.e., f (x) x k ) [69, 70] . a similar correlation is found when the multi-domain architecture is compared to the number of cell types that is present in an organism, i.e., the organism complexity or when the number of domains in a abundant superfamily is plotted against genome size (fig. 2) [71, 72] . given the amount of domain duplication and apparent selection for specific multi-domain encoding genes in, for example, vertebrates, it may come as little surprise that not all domains have had the same tendency to recombine and distribute themselves over the genomes [68, 73] . in fact, some are highly abundant and can be found in many different multi-domain architectures, whereas others are abundant yet confined to a small sample of architectures or not abundant at all [68, 70] . is there any significant correlation between the propensity to distribute and the functional roles domains have in cellular pathways? some of the most abundant domains can be found in association with cellular signaling cascades and have been shown to accumulate non-linearly in relation to the overall number of domains encoded or the genome size [70] . additionally, the on-set of the exponential expansion of the number of abundant and highly recombining domains has been linked to the appearance of multicellularity [70] . a reoccurring theme among these abundant domains is the function of protein-protein interaction and it appears that particularly these, usually globular domains, have been particularly selected for in more complex organisms [70] . this positive relation is underlined by the association of these abundant domains with disease such as cancer and gene essentiality as the highly interacting proteins that they are part of have central places in cascades and need to orchestrate a high number of molecular connections [74, 75] . their shape and coding regions, which usually lie within the boundaries of one or two exons, make them ideally suited for such a selection, since domains are most frequently gained through insertions at the n-or c-terminus and through exon shuffling [76] [77] [78] . from a mutational point of view, protein-protein interaction domains are different from other domains as well and this appears to be particularly true for the group of small, relatively promiscuous domains like sh3 and pdz. these domains are promiscuous in the sense that they both tend to physically interact with a large number of ligands [79, 80] and are prone to move through the genome to recombine with many other domains. it has been found that particularly these domains evolve more slowly than non-promiscuous domains [70] . this likely stems from the fact that they are required to participate in many different interactions, which makes selection pressures more stringent and the appearance of the branches on phylogenetic trees relatively short and more difficult to assess when co-evolutionary data in terms of other domains in the same gene family or expression patterns is limited [42, 63] . non-promiscuous domains on the other hand can quite easily evade the selection pressure by obtaining compensatory mutations either within themselves or their specific binding partner [70] . the overall phenomenon that the number of protein domains and their modularity increases as the genome expands has not been linked to a conclusive biological explanation yet. a rationale for the increase in interactions and functional subunits, however, may derive from the paradoxical absence of correlation between the number of genes encoded and organism complexity, the so-called g-value paradox [81] . there is indeed evidence that domains involved in the same functional pathway tend to converge in a single protein sequence, which would make pathways more controllable and reliable without the need for supplementary genes [73] . additionally, the number of different arrangements found in higher eukaryotes is, given the vast scale of unique domains present, relatively limited. this in turn implies that evolutionary constraints have played an important role in selecting the right domain combinations and the right order from n-to c-terminus in multi-domain proteins [13, 82] . in fact, the ordering and co-occurrence of domains was demonstrated to hold enough evolutionary information to construct a tree of life similar to those based on canonical sequence data [70] . furthermore, the increased use of alternative splicing and exon skipping in higher eukaryotes likely supplied a novel way of proteome diversification by restricting gene duplication and stimulating the formation of multi-domain proteins [83, 84] . in plants, however, the latter notion is not supported since both mono-and dicots show limited alternative splicing and a more extensive polyploidy [85] [86] [87] . it is clear that some of the above characteristics are underappreciated in the phylogenetic analysis of linear amino acid sequences. moreover, the effects of evolution extend even further than these aspects and entail transcriptional and translational regulation, intramolecular domain-domain interactions, gene modifications and post-translational protein modifications [88] [89] [90] [91] [92] [93] [94] [95] [96] . new methods are thus being developed to take into account that when sequences evolve, their close and distant functional relationships evolve in parallel. correlations of mutations have already been found between residues of different proteins [97, 98] and compensating mutational changes at an interaction interface were shown to recover the instability of a complex [99] . these observations are evidence for the current evolutionary models for the protein-protein interaction (ppis) networks that are being constructed through large-scale screens [100] [101] [102] . in these, a gene duplication or domain duplication (depending on the resolution of the network) implies the addition of a node, while the deletion of a gene or domain reduces the amount of links in the network (fig. 3) . in the next step, extensive network rewiring may take place, driven by the effect of node addition or node loss in the network (i.e., the duplicability or essentiality of a domain/protein) and mutations in the domain-interaction interface [67, 74, [103] [104] [105] . beyond mutations at the domain and protein level, regulation of protein expression provides another vital mechanism through which protein networks can evolve. microarray studies are now well under way to map genome-wide ex-pression levels of related and non-related genes under a variety of conditions [91, [94] [95] [96] . for example, transcriptional comparisons have investigated aging [106] and pathogenicity [107] . unfortunately, given the highly variable nature of gene expression and the fact that different species may respond different to external stimuli, such comparisons can only be performed under strictly controlled research conditions. to date most studies have therefore focused on the embryogenesis, metamorphosis, sex-dependency and mutation rates of subspecies [94, [108] [109] [110] [111] . other studies have revealed valuable information on promoter types and duplication events [91] [92] [93] [94] . to overcome the limitations mentioned in the previous paragraph, the analysis of co-expression data has been developed to supplement the direct comparison of individual gene expression changes [95] . in this procedure, a coexpression analysis of gene pairs within each species precedes the cross-comparison of the different organisms in the study. this approach thus primarily focuses on the similarity and differences of the orthologous genes within network, and is therefore ideally suited for the study of protein domain evolution and has already revealed that species-specific parts fig. (3) . evolutionary models for protein-protein interactions. the evolution of protein networks is tightly coupled to the addition or deletion of nodes. additionally, events that introduce mutations in binding interfaces of proteins may result in the addition or loss of links in the network. node addition may take place through e.g., domain duplication or horizontal gene transfer, while rewiring of the network is mediated by point mutations, alternative splice variants and changes in gene expression patterns. of an expression network resulted via a merge of conserved and newly evolved modules [95, 112, 113] . finding evolutionary relationships protein domains is mostly based on orthology and thus commonly performed on best sequence matches. identifying these and categorizing them depends largely on multiple sequence alignments and this will in most cases give good indications for function, fold and ultimately evolution. however, this approach usually discards apparent ambiguities that arise from speciesspecific variations (e.g., due to population size, metabolism or species-specific domain duplications or losses) and may therefore introduce significant biases [114] . biases may also derive from the method of alignment, the rate variation model used to infer the phylogeny, and the sample size used to build the alignment [39, 40, 115] . care should therefore be taken to not regard orthology as a one-to-one relationship, but as a family of homologous relations [91] , to select for appropriate analysis methods [39, 115] and extend comparative data to protein interactions and expression profiles [91] . indeed, as our wealth of biological information expands, our systems perspective will improve and provide us with an opportunity to reveal protein domain evolution at the level network organization and dynamics. large-scale expression studies are beginning to show us evolutionary correlations between gene expression levels and timings [94, 106, 107, 112, 116] , while 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lim kinases during zebrafish development towards cellular systems in 4d gene expression map of the arabidopsis shoot apical meristem stem cell niche a gene expression map of arabidopsis thaliana development key: cord-011012-5mev3otu authors: rathore, abhishek singh; sarker, animesh; gupta, rinkoo devi title: production and immunogenicity of fubc subunit protein redesigned from denv envelope protein date: 2020-03-30 journal: appl microbiol biotechnol doi: 10.1007/s00253-020-10541-y sha: doc_id: 11012 cord_uid: 5mev3otu dengue virus (denv) is a vector-borne human pathogen that usually causes dengue fever; however, sometime it leads to deadly complications such as dengue with warning signs (dws+) and severe dengue (sd). several studies have shown that fusion (fu) and bc loop of denv envelope domain ii are highly conserved and consist some of the most dominant antigenic epitopes. therefore, in this study, fu and bc loops were joined together to develop a short recombinant protein as an alternative of whole denv envelope protein, and its immunogenic potential as fusion peptide was estimated. for de novo designing of the antigen, fu and bc peptides were linked with an optimised linker so that the three dimensional conformation was maintained as it is in denv envelope protein. the redesigned fubc protein was expressed in e. coli and purified. subsequently, structural integrity of the purified protein was verified by cd spectroscopy. to characterise immune responses against recombinant fubc protein, balb/c mice were subcutaneously injected with emulsified antigen preparation. it was observed by elisa that fubc fusion protein elicited higher serum igg antibody response either in the presence or in absence of freund’s adjuvant in comparison to the immune response of fu and bc peptides separately. furthermore, the binding of fubc protein with mice antisera was validated by spr analysis. these results suggest that fu and bc epitope-based recombinant fusion protein could be a potential candidate towards the development of the effective subunit vaccine against denv. electronic supplementary material: the online version of this article (10.1007/s00253-020-10541-y) contains supplementary material, which is available to authorized users. in the recent decades, dengue has emerged as one of the world's most dominant tropical diseases (guzman et al. 2016) with around 390 million (95% credible interval 284 to 528 million) cases recorded per year, of which only 96 million (95% credible interval 67 to 136 million) cases were manifested clinically (giraldo-garcía and castaño-osorio, 2019; bhatt et al. 2013 ). according to world health organization (who), an estimate of 3.9 billion people in 128 countries are living in areas with a high risk of dengue infection (brady et al. 2012 ). the recently estimated annual 390 million dengue cases reveals that the dengue disease burden has tripled as compared to previous predictions of 50 to 100 million reported cases without dengue warning signs. nonetheless, 250,000 to 500,000 patients were hospitalised due to dengue with warning signs (dws+) and severe dengue (sd), and the total annual cost of dengue burden was estimated globally around us$ 8.9 billion (giraldo-garcía and castaño-osorio, 2019; guzman et al. 2016) . in spite of having paucity of effective vaccines and drugs to expel dengue comprehensively, still it's enduring a big challenge to human (martina et al. 2009 ). therefore, next-generation vaccine strategies such as inactivated purified virus, dna or protein-based subunit vaccines, and their fusion chimeras are now going under investigation and some of them even under clinical trials (coller et al. 2011; danko et al. 2011; liu et al. 2015) . recently, dengvaxia (cyd-tdv), a tetravalent live attenuated vaccine has been approved for use in some of the highly dengue endemic areas where the sero-prevalence is higher than 70% (guy et al. 2015; pang et al. 2018 ). according to abhishek singh rathore and animesh sarker contributed equally to this work. electronic supplementary material the online version of this article (https://doi.org/10.1007/s00253-020-10541-y) contains supplementary material, which is available to authorized users. who, it has shown significant efficacy and acceptable safety profile during clinical trials in seropositive individuals; however, it carries a risk of severe dengue infection in seronegative individuals, and also failed to confer subsequent protection in denv-2 positive people in areas with less exposure of dengue (arredondo-garcía et al. 2018; durbin et al. 2011; guy et al. 2011) . several other vaccine candidates were also formulated based on live-attenuated dengue viruses (e.g. tv003, tv005) and inactivated purified virus (e.g. dpiv), of which some have already completed clinical trials and some are currently going under phase ii to iii clinical trials (whitehead et al. 2017; diaz et al. 2018 ). on the other hand, dna or proteinbased alternative subunit vaccines (e.g. tvdv, den-80) and dengue-flavivirus chimeras (e.g. ediii-p64k, 80e-stf2, ediii-hbsag) are going either under preclinical study or phase i clinical test (govindarajan et al. 2015; danko et al. 2018; castaño-osorio et al., 2019) . although, still most of them are seemed to be slow immunity booster and require a strong adjuvant and longer immunisation to achieve full protection against each of the dengue serotype, which all make them ill-suited for universal vaccine licence . the dengue envelope (e) protein is composed of three ecto-domains, a membrane-proximal stem and a transmembrane anchor (klein et al. 2013 ). various crystal structures have shown that the ecto-domains are arranged into three antiparallel dimer on virus surface in a icosahedral symmetry, where ecto-domain i (ei) is located at the centre holding domain ii (edii) and iii (ediii) in two opposite sites in each of the monomer of a dimeric unit (fibriansah et al. 2015; kuhn et al. 2002; modis et al. 2004 ). most of the protective antibodies against dengue virus are identified to domain iii, and initially were thought to be a potential subunit vaccine target (murphy and whitehead 2011; sukupolvi-petty et al. 2010) . although, recombinant ediii domain injected by plasmid dna or purified from bacterial expression systems was found to be poorly immunogenic, and has shown low protective efficacy in animal model (guzman et al. 2010 ). on the other hand, edii has been reported as the dimerisation domain and consists of the most conserved fusion (fu) and bc loop (li et al. 2019; zhang et al. 2004 ). several studies have also shown that the conserved fu loop is highly immunogenic (lai et al. 2008; cherrier et al. 2009; smith et al. 2013) , and induces cross-reactive antibodies, of which some are crosstalk with adjacent bc loop (gallichotte et al., 2015; cherrier et al. 2009 ). moreover, during endocytosis, both of the loops are found to involve in tertiary conformation of membrane fusion (nayak et al. 2009; sukupolvi-petty et al. 2010) . therefore, it has been anticipated that both of the loops might have integrated role for boosting cross-neutralizing immunity. in this study, we aim to develop an alternative vaccine by combining fu and bc loop together in a single orf for production of a fusion antigen. although, the recombinant antigen in isolation tends to be poorly immunogenic in vivo; the use of potent immunomodulating compounds, fusion partner or suitable delivery systems improve specific immune response (higgins et al. 2007; garçon et al. 2011) . herein, we have expressed the recombinant fubc antigenic protein, optimised its large-scale purification protocol and finally evaluated its protein-specific immune response in balb/c mice. prior to the animal challenge, its secondary structure was checked by cd spectroscopy, and binding specificity was cross-checked with a characterised anti-fusion loop scfv antibody ). all of the male and female balb/c mice were obtained from national institute of nutrition, telangana, hyderabad, india and were maintained in standard light: dark (12:12) cycle with the supplement of adequate standard food, and water was provided from ad libitum. all of the animals were acclimated and randomly distributed into different experimental groups. furthermore, all the in vivo experiments were performed in accordance with the committee for the purpose of control and supervision on experiments on animals (cpcsea) guidelines and were approved by the south asian university institutional animal ethics committee (iaec) that was responsible for the care and use of laboratory animals. the dna sequence of fubc gene was retrieved from fusion (fu) and bc loop of denv serotype 2 envelope protein deposited in the protein data bank (pdb: 1oan). in order to construct stable and immunogenic protein, highly conserved fusion (fu) and bc loops and their neighbouring residues (amino acid residues 62 to 122) were selected by using antigen variability analyser (avana), pblast and multiple sequence alignment tool of ncbi. for convenient expression in bacteria, all of the oligos were codon optimised for e. coli, using codon optimisation tool of integrated dna technology. complete fubc gene was constructed by assembly pcr reactions using four 60 nucleotide long overlapping oligonucleotides. for cloning into pet28a expression vector, two unique restriction sites, ecori and xhoi, were also incorporated at the 5′ and 3′ ends respectively during the final amplification of fubc full-length gene using forward and reverse primers. initially, the full-length fubc gene was cloned into a ta cloning vector using instaclone kit (thermo scientific). positive clones were screened using x-gal bluewhite screening method and digested with ecori and xhoi restriction enzymes. the digested fubc gene was further sub-cloned into a pet28a expression vector. the recombinant plasmid (fubc + pet28a) was transformed into e. coli xl-10 gold for cloning, and subsequently into e. coli bl-21 rosetta (de3) for protein expression. the e. coli bl-21 (rosetta) cells carrying fubc gene in pet28a vector were grown overnight at 37°c in 10 ml lb broth (luria-bertani medium) containing 50 μg/ml kanamycin (sigma, usa). overnight grown 1 ml primary culture was used to inoculate kanamycin containing 100 ml secondary culture and was further incubated at 37°c. the incubated secondary culture was induced by 0.5 mm isopropyl β-d-1thiogalactopyranoside (iptg) when its od 600 reached at around 0.5, and the culture was grown for another 4 h at 37°c. the cells were harvested by centrifugation at 4000 rpm for 10 min. the resulting cell pellet was resuspended in lysis buffer containing 50 mm tris-hcl ph 8.0, 1 mm cacl 2 , with 0.5% triton x-100, lysozyme 0.1 mg/ml, 1 mm edta and 1 mm pmsf. then, the resuspended cells were kept on a rocker for an hour at room temperature, and sonication was done using 30% amplitude for five times 30 s on/off pulse. the lysed sample was separated into supernatant and pellet by centrifugation at 10,000 rpm for 10 min at 4°c. finally, fubc expression level in supernatant and pellet were checked on 15% sds-page. a major fraction of the fubc protein was observed in pellet after a number of efforts made to recover it in a soluble form. then, it was decided to recover soluble protein from pellet fraction. the resulting pellet protein was washed 4 to 5 times with te 50/20 (50 mm tris ph 8.0 and 20 mm edta 20) buffer to remove impurities and extra salts. the remaining pellet was re-solubilised using mild denaturing agent such as 4.0 m urea and 5% n-propanol along with pbs buffer (ph 8.1) and was centrifuged at 20,000 rpm for 10 min. the soluble protein fraction was initially purified by using ni-nta agarose beads and was confirmed by western blot using anti-his antibody. however, the purity and yield were not sufficient. therefore, soluble protein fraction was subjected to gel filtration in superose 6 10/300 column by using fast protein liquid chromatography (fplc) for largescale good quality protein production. the column was preequilibrated with pbs (50 mm phosphate buffer ph 7.4 and 150 mm nacl), and the protein sample was eluted with the same pbs buffer. 0.5 ml protein sample was injected in each run by using 0.5 ml loop at 0.4 ml/min flow rate. the elution profile of injected protein was followed by monitoring uv absorbance at 280 nm on the akta fplc system with u9-l uv monitor. different peaks greater than 20 mau were collected in fraction collector and checked using a 15% sds page. multiple runs of fplc were carried out, and the fraction containing fubc protein was pooled together. finally, fubc was concentrated and desalted by using 0.5 ml millipore-amicone filter with a cut-off of 3 kda. concentration of the purified fubc was also measured by using bca protein assay kit. in order to assure the proper folding of purified (> 95%) fubc protein, secondary structure was analysed by cd spectroscopy at far uv wavelength ranging from 180 to 260 nm. to achieve the best conformational reading, cd spectrum was obtained at the different protein and buffer concentrations, because the cd spectrum of a protein needs to be adequately intense for interpreting the data as the intensity of a cd spectrum directly relies on the protein and buffer concentration (kelly et al. 2005; miles and wallace 2006) . the cd spectra of pbs buffer and fubc protein samples at 0.1 mg/ml and 0.2 mg/ml concentrations (diluted in 50 mm and 10 mm pbs buffer, ph 7.4) were recorded at far uv spectra ranging from 200 to 280 nm with a step size of 1 nm to bandwidth 1 nm. the measurement was performed at room temperature (22°c), and the uv spectra were recorded for each sample with five scans. the baseline cd spectrum of the buffer was deducted from the spectrum containing the protein to yield the actual fubc protein cd spectrum. the mean residue ellipticity [θ] mrw at wavelength λ was quoted in units of degree cm 2 / dmol, and was calculated as [θ] mrw, λ = mrw × θλ/10 × d × c, where θ is the observed ellipticity (degrees) at wavelength λ, d is the path length (cm) and c is the concentration (g/ml). mrw is the mean residual weight for the peptide bond which is given as mrw = m/(n − 1); where m is the molecular mass of the polypeptide chain (in da), and n is the number of amino acids in the chain; the number of peptide bonds is n − 1. complete and incomplete freund's adjuvant (sigma, usa) was used for primary (day 0) and booster immunisation (days 7, 14 and 21) respectively. to prepare a primary dose of the immunogen, complete freund's adjuvant (fa) was mixed with equal volume of purified fubc (25 μg) in pbs and emulsified by vigorous vortex. similarly, three booster doses of immunogen were prepared with an equal volume of fubc protein (25 μg) and incomplete freund's adjuvant. all of the immunogens were prepared according to the protocol "immunization of mice" by maira-litrán, 2017. finally, emulsified fubc immunogens were checked by observing stable droplet on the water surface. healthy balb/c mice (10 weeks old, male and female) were randomly distributed into four different groups. each group was immunised with different antigen preparations. out of the four, two groups were immunised with fubc antigen: one group was injected with fubc protein with adjuvant and other was with fubc protein without adjuvant. rest of the two groups were immunised with fu and bc peptide: one group was injected with fu and bc peptides with adjuvant, and other was fu and bc peptide without adjuvant. each of the groups was also subdivided into male and female sets, and with each set, one adjuvant control was used replacing adjuvant with pbs buffer. the mice of each group were subcutaneously immunised with emulsified antigen preparations, first dose (day 0) with cfa and three subsequent booster doses (at days 7, 14 and 21) with ifa (according to protocol maira-litrán 2017). after 5 days of the final booster dose, blood samples were collected by retro-orbital cavity, and all the antisera isolated from blood were stored at − 80°c for further analysis. recombinant fubc protein-specific igg was measured by indirect elisa. for this, 96 well elisa plates were coated by incubating overnight at 4°c with fubc protein (1 μg/ well) in 50 mm sodium carbonate-bicarbonate buffer ph 9.5. blocking was done for 2 h with 2% bsa in pbst (pbs plus 0.05% tween 20). serum collected from immunised and control mice were incubated for 1 h at 37°c after making 7 double dilutions in pbst starting from 1/500 μl. subsequently, the elisa plate was washed once with pbst, and 100 μl of anti-mouse hrp-conjugated secondary antibody (1:1000 dilution in pbst) was added and incubated for 30 min at room temperature. then, the plate was washed three more times with pbst, and the binding reaction was developed by adding 200 μl/ well opd substrate (prepared in a phosphate-citrate buffer, 0.05 m, ph − 5.0 plus 0.03% h 2 o 2 ). the reaction was stopped by adding 50 μl of 2.5 m h 2 so 4 just after observing an optimum colour, and the absorbance was measured at 492-nm wavelength using biotek synergy ht microplate reader (winooski, vt). mean absorbance was plotted against anti-sera dilutions, and two-way anova was performed to find a statistically significant difference between two groups. comparison of the immune response in different groups of antisera was made by converting each elisa curve to linear equations (y = mx + c), and computing serum dilutions for a fixed elisa absorbance. the data are expressed as mean ± standard deviation (sd). p values of < 0.05 were considered statistically significant. to validate the immune response generated by recombinant fubc, collected mice anti-sera were allowed for spr interaction experiment with a autolab esprit instrument. similar to elisa, here also recombinant fubc protein of denv envelope was immobilised on a gold plate of spr device. for coupling of fubc protein on gold disc, 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (edac) and nhydroxysuccinimide (nhs) were applied for activation. nhs activates the carboxymethyl groups by creating a highly reactive succinimide ester on the disc surface, which reacts with amine and other nucleophilic groups on proteins that subsequently help to bind the target protein on activated disc surface. ethanolamine was added to block the remaining activated carboxymethyl group. for the qualitative assay, all of the serum samples (diluted in running buffer) were applied at a flow rate of 20 μl/min over 2 min. in between injections, the surface of the sensor chip was regenerated by injecting 2 m nacl at the same flow rate for 15 s. in addition, surface coupling was done by using buffer (150 mm nacl, 3 mm edta, 0.005% surfactant p20 and 10 mm hepes-naoh) at ph 7.4, and the buffer containing 50 mm phosphate and 150 mm nacl at ph 7.1 was used for sample running. initially, a series of serum dilutions were applied as analytes to find an optimum refractive index. it was observed that the refractive index at 1:500 dilution was within the detection limit among the series of serial dilutions (fig. 6a) . therefore, 1:500 dilution of different experimental samples was applied as standard for further comparative analysis between fubc immune response with and without freund's adjuvant. serum control (the serum collected before immunisation) sample with the identical dilution was also applied to find the basal (non-specific) immune response of serum. finally, refractive index was then analysed from association and dissociation curve of the spr sensorgram. the fubc synthetic gene sequence was deposited in genbank database with accession number mn781186. generally, dengue envelope (e) folded into three distinct domains (designated by domain i, ii and iii), membrane proximal stem and a transmembrane anchor (klein et al. 2013) . throughout these structural element, four highly conserved regions have been identified by in silico sequence analysis, and two of them were found in the domain ii with a very less informational entropy . further studies have revealed that these conserved regions are the part of previously characterised flavivirus fusion (fu) and bc loop (kuhn et al. 2002) . during the process of endocytosis, this hydrophobic fusion loop remains buried at the dimer interface in the prefusion state and forms cluster into larger hydrophobic surface at one end to form trimer at later state that finally initiates membrane fusion (klein et al. 2013 ). in addition to this structural property, fu and bc regions consist some of the most potent antigenic epitopes that were identified by denv neutralising conformation sensitive anti-denv hmabs (goncalvez et al. 2007; costin et al. 2013; yamanaka et al. 2013) . therefore, these two conserved fu and bc loops have been selected to design a recombinant fubc antigenic protein for the development of dengue subunit vaccine. in this study, an alignment of the domain ii amino acid sequences of denv1-4 envelope proteins spanning residues from 61 to 120 was used to find optimum conserved sequence for the development of fubc fusion protein (fig. 1a) . the structural details of truncated fubc protein compare to whole envelope in dengue and fu, and bc peptides were also analysed by modelling each of their three dimensional structure. it reveals that the presence of three anti-parallel β-strands and one disulphide linkage play a crucial role in preserving the three dimensional conformations of truncated fubc protein same as in original denv envelope protein (fig. 1b, c) . ironically, due to the absence of anti-parallel β-strands and a single protein frame of fu and bc loops, these two separately expressed peptides fail to form similar three-dimensional conformation as in original denv envelop (fig. 1c) . the electrostatic and solvent accessible areas of fubc truncated protein are also comparable with original dengue envelope. hence, it is speculated that the recombinant fubc protein would be an alternative vaccine target of whole dengue envelope. fu loop is shown in orange and bc loop is shown in yellow. b in denv envelope, the fu and bc loops are present at the end of domain ii and are linked by the di-sulphide linkage that holds both loops together to form a stable structure that can also work as an epitope. c when the structure of fubc is compared to original denv envelope, it is plausible that the presence of three anti-parallel β-strands present in fubc protein (same as denv protein) plays a crucial role in maintaining the three dimensional conformation of fubc protein as it is in denv protein. another major factor that plays in conformation of fu and bc loops is the disulphide linkage between the two highly conserved loop in the fubc protein that ensures the same conformation of denv envelope as it is shown in b and c, and d due to the lack of anti-parallel β-strands and same protein frame while these peptides are expressed separately, it is unlike to form di-sulphide linkage and fold in the same conformation as reside in denv envelope and fubc proteins for in vitro synthesis of short antigenic protein, a recombinant fubc gene was constructed by assembly pcr using overlapping oligonucleotides, designed from fu and bc loop encoding dna sequences of dengue envelope. therefore, full length of fubc gene was amplified by using 5 and 3 end primers flanked by ecori and xhoi restriction sites. the fubc gene was then cloned into a ta cloning vector, and the clones were screened by colony pcr using fubc gene specific end primers. two positive clones were then confirmed by restriction digestion using ecori and xhoi enzymes (fig. s1a-d) . the digested and purified fubc insert gene was further sub-cloned into pet28a expression vector, and the positive clones were confirmed by restriction digestion (fig. s2b) and sanger sequencing. primarily, the recombinant fubc protein expression was observed only in pellet fraction; there was no such fubc equivalent protein band in supernatant fraction, while they were separated on sds page (fig. s2c) . therefore, fubc expression level was checked further by lowering growth temperature and iptg concentration, but no significant change was noticed in supernatant fraction. chaperone-assisted folding system did not help remarkably to express the fubc recombinant protein in the soluble form. therefore, the insoluble pellet fraction of fubc protein was further utilised in mild solubilisation process to recover soluble fubc protein. initially, the recovered soluble fubc protein fraction was purified by using ni-nta agarose beads, and the purified protein band was confirmed by western blot analysis using anti-his tag monoclonal antibody (fig. s3) . although, ni-nta affinity chromatography has not revealed good purification quality and the yield was also not sufficient. furthermore, gel filtration chromatography was used to recover good quality large-scale recombinant protein, and single distinct peak greater than 20 mau was observed at approximately 20.57 ml position for all of injected fubc protein samples (fig. 2a) . after pooling together, the peak fractions were separated on 15% sds-page, and single bright band was observed at molecular weight 11.5 kda (fig. 2b) . originally, the fu and bc loop of dengue envelope is composed of three anti-parallel β-sheets. and the formation of recombinant fubc secondary structure was characterised here by negative bending at 208 nm and 222 nm wavelength (kumagai et al. 2017) . the cd spectrum of recombinant fubc protein has showed significantly decreased spectral peaks around 222 and 208 nm that signify the formation of β-sheet (fig. 3 ). in addition, it was noticed that the acquired cd spectrum of fubc at a protein concentration of 0.1 mg/ml in 10 mm pbs buffer was adequately intense (fig. 2) . therefore, it can be inferred from fig. 3 (lower panel) that fubc at concentrations of 0.2 mg/ml and 0.1 mg/ml in 10 mm pbs retained the best globular folded state as compared to other conditions. immune response to the purified fubc protein in balb/c mice from indirect elisa, it was observed that the serum igg levels in both male and female groups treated with fubc proteins were significantly higher than those treated with only adjuvant and pbs control (fig. 4a, b) . in addition, the response with only fubc recombinant protein (without adjuvant) in the female group was also observed higher than their male counterpart group (fig. 4b) . all of the elisa data were also found statistically significant by two-way anova (table s1) . similarly, by spr assay, high level immune response was also observed for serum injected with recombinant fubc protein along with adjuvant, and without any adjuvant, the response was also higher than the control (serum with pbs only) (fig. 6b ). in post-dengue infection, most of the circulating antibodies are non-neutralizing and found to be raised against dengue envelope e protein and the prm protein (wahala and de silva 2011) . due to the absence of highly specific neutralising antibodies in secondary infections, cross-reactive nonneutralising antibodies usually enhance the dengue severity (de alwis et al. 2014 ). according to the revised dengue case classification (denco), 2 to 3% of secondary infections with another serotype causes life threating dengue with warning signs (dws+) and severe dengue (sd). it was reported that serotype-cross-reactive non-neutralising antibodies enhance the entry of dengue genome into fc receptor-bearing monocyte cells and promote disease severity by a process known as antibody-depended enhancement (ade) flipse et al. 2013) . that means nature of the antibody response to denv is most likely to play a major role in defining disease outcome. therefore, it is predictable that antibodies that recognise specific neutralizing epitopes help in virus clearance and reduce symptoms; however, antibodies that recognise non-neutralising epitopes lead to more severe forms of disease like dws+/sd. hence, there is an urgent need for an advanced vaccine which could generate highly specific and cross-neutralising antibody (costin et al. 2013 ). recently, several attempts have been made towards the development of a potent dengue vaccine. the most advanced candidate, dengvaxia (cyd-tdv), was licenced in some of the dengue endemic countries (imai and ferguson 2018) . however, it was revealed risky in children or naïve dengue patient with severe infection as they were vaccinated (arredondo-garcía et al. 2018) . therefore, considering safety issues, production of recombinant subunit vaccine with efficient immune protective properties is looking attractive (govindarajan et al. 2015) . meanwhile, an admixture of four live attenuated recombinant dengue vaccine tv003/tv005 have completed phase iii clinical trial and licenced to several manufacturers including butantan, vabiotech and merk (whitehead et al. 2017) . moreover, some recombinant tetravalent vaccines (e.g. den-80e, tvdv) expressing the prm and e genes of each of the four denv serotypes from plasmid dna, have already completed phase i clinical trial (govindarajan et al. 2015; danko et al. 2018 ). it has also been shown that the recombinant dengue envelope domain iii can inhibit dengue infectivity, and induce dengue-neutralising immunoglobulin in mice (hermida et al. 2006) . in addition, a number of antibodies which were raised in mice and fig. 2 purification of recombinant fubc protein by size exclusion chromatography. a fplc chromatogram of fubc protein expressed in e. coli inclusion bodies. three distinct peaks at 19.88 ml, 20.57 ml and 22.57 ml position were collected separately. b gel image of unpurified and purified fubc protein sample separated on 15% sds-page. lanes 1 and 2 represent the un-purified sample and lanes 3 and 4 represent purified protein sample collected from peak at position 20.57 ml fraction chimpanzees against dengue domain ii fusion loop were found cross-reactive to other flaviviruses (goncalvez et al. 2004; stiasny et al. 2006) . although, the hmabs specific to dengue domain ii fusion loop were not found equally effective on other flaviviruses including wnv, yfv and other denv strain (costin et al. 2013) . therefore, it was conjectured that adjacent to the fusion loop, additional contact residues of original domain ii surface structure might be involved to raise cross-neutralizing antibodies. several studies have identified that adjacent to the fusion loop, another similar loop exists in most of the flaviviral (e.g. wnv, tbe, jev, yfv) envelope (fibriansah and lok, 2016; li et al. 2019) . our previous bioinformatics studies have also shown that the envelope of all denv serotype consists of four conserved regions (> 90%), and two of them were found in domain ii, in which one is fusion loop (fu) with more than 99% conservation and another is its nearby bc loop (fig. 1a ) . therefore, these two highly conserved loops were targeted in this study for the development of a fusion protein. by using reverse dna technology, we have previously shown the structural integrity ( fig. 1b-d) and binding specificity of fubc protein with an anti-fusion loop scfv antibody, derived from dengue and wnv-specific mab e53 (rodenhuis-zybert et al. 2011; rathore et al. 2019) . the very early challenge of subunit vaccine design is to produce large quantities of functional protein. hereby, we have successfully optimised the expression and purification methods for the production of large-scale high-quality recombinant fubc protein. the e. coli expression system was used here, and the recombinant protein was found to be expressed in higher quantity, though most of the protein was extracted initially in pellet fraction. no significant change was noticed by altering regular growth temperature and inducer concentration. therefore, we have utilised the mild-solubilisation methodology to recover soluble fubc protein from insoluble pellet protein. for purification, firstly, we have used convenient ninta affinity purification method, and by adjusting the lysis and elution buffer, we were able to purify recombinant fubc protein to some extent but the quality and quantity was not sufficient. finally, to scale up the quality protein production, size exclusion chromatography was used in akta fplc system. to confirm the recombinant protein expression, simple western blot was performed by using anti-his monoclonal antibody as primary. furthermore, in vitro structural integrity and functional authenticity of the experimental fubc protein were also verified by cd spectroscopy (fig. 3) and indirect elisa (fig. 4) . however, most of the hmabs identified earlier from dengue patient serum were predominantly cross-reactive and recognise epitopes containing highly conserved residues at the fu loop of domain ii (lai et al. 2008 ). however, having such sequence homology in fu loop of all flaviviruses, these hmabs are non-neutralizing against heterologous serotypes (lai et al. 2008; smith et al. 2013) and thus was found to be responsible for ade in animals (goncalvez et al. 2007 ). previously, it has also been stated that this scenario might happen due to the cryptic nature and poor accessibility of the fusion loop epitope on the surface of the mature virion (lai et al. 2008; cherrier et al. 2009 ). however, in addition to the partially exposed fusion loop on immature flaviviruses, some neutralizing hmabs were also found to bind with bc loop (costin et al. 2013) . therefore, it is suggestive that along with conserved fusion loop, adjacent less conserved linker sequence and bc loop might be required to be exposed as a whole to generate effective cross-neutralizing immunity. our current mouse immunisation experiment also supports this idea as it was observed that the antibody response to full length fubc protein was stronger than the response elicited by the fu and bc peptides in separate (figs. 4 and 5) . moreover, without freund's adjuvant, recombinant fubc protein was found immunogenic, and the response in female mice was stronger than in male (figs. 4 and 5) . these observations were also symmetrical with other studies where it was stated that female mice have a better immune response due to the higher number of leukocytes occupying the naive peritoneal and pleural cavities, and also have a number of t-and blymphocytes as well as macrophages (terres et al. 1968; weinstein et al. 1984; klein et al. 2015) . therefore, we have used female mice serum samples for further qualitative elisa and spr test to quantify immune response of recombinant fubc protein. it was noticed that igg antibody response to fu, and bc peptides were significantly lower than the response observed to fubc recombinant protein, and the immune response achieved to fu and bc peptide with freund's adjuvant was significantly higher as compared to the peptides without adjuvant and serum control (fig. 5) . two-way anova was also performed to analyse the significance of igg immune responses between fubc + fa and fubc without fa; fu, bc peptide + fa and fu, bc peptide without fa. from f values and p values obtained by anova test, it can be interpreted that the difference between fubc protein with freund's adjuvant and fu, bc peptide with freund's adjuvant were significantly higher than protein and peptides injected without freund's adjuvant (table s2) . also, the igg response to recombinant fubc protein was recorded better than fu and bc peptides in both cases either with or without adjuvant. in addition, the p value of igg immune response between fubc protein with fa and without fa was found insignificant (p = 0.1740 > 0.05) that suggests that fubc without any adjuvant is sufficient to elicit significant immune response (table s2) . consistently, it was observed by spr experiment that the refractive index of fubc + fa was significantly higher than fubc + without fa and serum control (fig. 6b) . since, the newly developed fubc recombinant protein expresses vastly in e. coli and induces significant immune response in mice, it might be a good agent of dengue subunit vaccine development. due to the presence of highly conserved fusion loop epitope, overlapping less conserved linker sequences and also bc epitope, this fusion protein might induce cross-neutralisation immunity against heterologous dengue serotypes. though, still a lot of investigations, like the evaluation of a proper adjuvant to induce robust immune response, the memory immune response generated against the fubc protein both by humoral and cell-mediated immunity, and whether this memory response will provide protection against a secondary encounter with denv, are required before going clinical trial. nevertheless, the production process and immune response of this fusion protein would provide new insight for the development of dengue subunit vaccine. four-year safety 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in a randomized trial, the live attenuated tetravalent dengue vaccine tv003 is well-tolerated and highly immunogenic insubjects with flavivirus exposure prior to vaccination a mouse monoclonal antibody against dengue virus type 1 mochizuki strain targeting envelope protein domain ii and displaying strongly neutralizing but not enhancing activity publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-000884-zq8kqf6h authors: shen, hsin-hui; lithgow, trevor; martin, lisandra l. title: reconstitution of membrane proteins into model membranes: seeking better ways to retain protein activities date: 2013-01-14 journal: int j mol sci doi: 10.3390/ijms14011589 sha: doc_id: 884 cord_uid: zq8kqf6h the function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. the activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. the reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. however, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. this review will discuss the reconstitution of membrane protein activities in four different types of model membrane—monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. we also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. it is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro. the cell membrane separates intracellular components from the outside environment and is constituted by various phospholipids, cholesterol, glycolipids and proteins. integral membrane proteins have at least one polypeptide segment spanning the membrane bilayer whereas peripheral membrane proteins are temporarily attached to the lipid bilayer or to integral membrane proteins by various interactions such as hydrophobic, electrostatic and other types of non-covalent interactions. membrane proteins work as a selective filter to regulate molecules entering cells and also serve in communicating with the surrounding environment. thus, membrane proteins play an essential role in the physiological functions needed for cell survival. the functional activities of membrane proteins are modulated by the structure of the surrounding lipids molecules in the membrane [1, 2] ; thus the composition of the lipid bilayer can affect the inter-or intra-molecular interactions between the lipid bilayer and membrane proteins [3] . investigating membrane proteins in vivo is difficult because the membrane proteins are associated with a complex mixture of other proteins, and are prone to aggregation in solution [4] . it is still a major challenge at this stage to extract information needed in vivo to address specific questions in the function of the cell membrane. to simplify cell membrane systems, model membranes such as monolayers, bilayers, liposomes and nanodiscs have been developed, enabling detailed investigation of membrane protein structure in lipid membranes. model membrane environments more closely resemble the natural lipid bilayer than alternatives such as detergents. however, many features of phospholipid structure need to be considered and optimized in the creation of a suitable model membrane. for example, the hydrophobicity of the lipid chain defined by the lengths of the fatty acid chains, is an important parameter for retaining protein activity. other factors affecting the reconstituted membrane protein activity are the chemical properties of the lipid head groups which control membrane hydrophilicity. both parameters are crucial in stabilizing membrane protein structure. there are a number of approaches used to create a model membrane in order to mimic properties of the native cell membrane, and we will review these various approaches for reconstituting membrane proteins into different types of model membrane-monolayers [5] , supported planar lipid bilayer [6] and liposomes [7] as shown in figure 1a -c. we will also discuss the emerging technology of nanodiscs [8] ( figure 1d ). nanodiscs are a new class of model membrane, with attractive properties that address shortcomings of other approaches in the study of membrane proteins. the first section gives a brief summary of each method and a comparison of their strengths and weaknesses. in the following section, we describe four case studies and will compare the protein activity changes when the membrane proteins are reconstituted into different model membranes. in these case studies, we demonstrate how protein activities are modulated by the lipid environment and discuss how this environment helps to retain protein activities. and black in b represent water and a substrate respectively. nanodiscs contain membrane scaffold proteins, shown in green. one of the most common approaches to study the membrane protein structure and activity uses a langmuir monolayer at the air-water interface. this method has been extensively used for more than a century [9, 10] . reconstitution of membrane proteins helps obtain further information on their organization and structure in the langmuir membrane [11, 12] . it is a simple method to create a phospholipid monolayer at an air-water interface. basically, a desired amount of lipid or lipid mixtures are dissolved in organic solvents such as chloroform or chloroform/ethanol mixtures, followed by spreading the lipid/solvent mixtures on the water surface. by evaporating out the solvent, the phospholipid molecules self-assemble vertically as a monolayer film at the air-water interface, with their hydrophilic head groups immersed in the water and their hydrophobic tail pointed to the air as shown in figure 1a [13] . a major advantage of using the langmuir monolayer system is that parameters such as thickness, surface pressure, molecular area and subphase thickness can be well controlled [10] . more advanced characterization techniques, such as π-a isotherm uv-vis adsorption, x-ray reflectivity, ellipsometry and rheology, have been developed to gain detailed information on the binding of proteins onto the phospholipid monolayer and to monitor enzyme activities when binding to the monolayer [14] . however, a limitation of langmuir monolayers is that the lack of a layer comparing to the natural cell structure (bilayer) and the high surface tension of water that can cause protein denaturation. despite this limitation, there are several successful studies using this approach. two types of membrane proteins in monolayer model membrane system will be briefly described below: rhodopsin [15, 16] , bacteriorhodopsin [17, 18] and the aliphatic peptide gramicidin [19, 20] have been successfully reconstituted and studied in monolayers at the air-water interface. in order to obtain information on the secondary structure and orientation, the protein layer can be investigated in situ at the air-water interface by either polarization modulation infrared reflection absorption spectroscopy (pm-irras) or x-ray reflectivity in combination with surface pressure-area isotherms [21] . the study of gramicidin is an example of such an approach, and while gramicidin is unfolded at high molecular area (low pressure), it is refolded upon compression and retains its precise structure and orientation. likewise, for both rhodopsin and bacteriorhodopsin, the secondary structures measured in monolayers are indistinguishable from that in native membranes when appropriate conditions are used. while some experiments have suggested that spreading of rhodopsin in certain conditions (>5 m/n) leads to denaturation [21] , bacteriorhodopsin, in contrast, is very stable in most testing conditions (compression and temperature change). the different properties of the protein are probably due to the ability of baceriorhodopsin to form a stable two-dimensional crystalline structure at the air-water interface [21] . phospholipid monolayers are simple model membrane systems that are perfectly suited to study the binding of peripheral proteins onto a membrane surface. peripheral membrane proteins spontaneously bind onto phospholipid monolayers at the air-water interface by injecting themselves into the subphase underneath the lipid monolayer. in most cases, useful information can be obtained by measuring the binding of peripheral proteins onto the monolayer. for example, the kinetics and dynamics of adsorption of myristoylated and nonmyristoylated recoverin onto phospholipid monolayers have been investigated using surface pressure isotherm described in figure 2 [21] . the curve can be fitted with stretched exponential which can convert into the rate of adsorption of myristoylated and nonmyristoylated which is 0.028 s −1 and 0.0048 s −1 , respectively. this indicates that the adsorption of myristoylated recoverin is six times faster than nonmyristoylated recoverin. reconstituting enzymes into the langmuir monolayers at the air-water interface has been found to be a very useful approach to understand the hydrolysis of membrane phospholipids. for example, the interfacial recognition and adsorption of phospholipases a2 (pla2) and phospholipases c (plc) to the phospholipid membrane interface are poorly understood. by using this approach, it appears that both pla2 and plc are active at the monolayer model membrane, indicating that the kinetics of phospholipid hydrolysis at the air-water interface can be monitored by biophysical characterization techniques in situ such as pm-irras and infrared reflection adsorption spectroscopy [22] . moreover, it has been found that in the presence of calcium, phospholipid hydrolysis by pla2 resulted in the production of calcium-palmitate complexes. this suggests that calcium is necessary for pla2 secretion. the formation of a supported lipid bilayer on a solid substrate was reported by tamm and mcconnell in 1985 as a new model membrane system to study the physical properties of biological membranes and their constituent lipid and protein molecules [23, 24] . supported planar lipid bilayers are prepared by several methods [25, 26] . vesicle fusion is the simplest method for supported bilayer formation and the fusion mechanism on a hydrophilic support is well understood [27, 28] . essentially, the bilayer is prepared by the fusion of small unilamellar vesicles on solid supports such as sio 2 , glass and modified gold surface by van der waals, electrostatic, hydration and steric forces. the supported lipid bilayer has polar hydrophilic headgroups facing the aqueous surroundings and two hydrophobic tails that face the interior of the membrane which more closely resembles biological membranes than the langmuir monolayer. the supported lipid bilayer can confer many key functions to biological membranes. however, one side of the hydrophilic head group is still tightly attached to the solid support and this may, in some cases, affect the fluidity of the model membrane. this matters, since integral membrane proteins may not diffuse in the plane of the membrane. furthermore the orientation of membrane proteins cannot be controlled in the supported planer lipid bilayer. to alleviate some of these problems, a new tethered polymer-supported planar lipid bilayer system was developed to investigate the reconstitution of integral membrane proteins in a laterally mobile form into the supported lipid bilayer [29] . wagner and tamm [30] have successfully designed a supported lipid bilayer on a polyethyleneglycol cushion shown in figure 3 . the polymer cushion minimizes the interactions of the proteins with the substrate and the polymer. it also provides a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. in low polyethyleneglycol concentration regimes, the bilayers were assembled with high lateral lipid diffusion coefficients (0.8-1.2 × 10 −8 cm 2 /s). cytochrome b5 and annexin v were used to test the polyethyleneglycol cushion system. two populations of laterally mobile proteins were observed in the polyethyleneglycol cushion-supported bilayers. approximately a quarter of cytochrome b5 diffused with a diffusion coefficient of 0.8-1.2 × 10 −8 cm 2 /s, and more than half of the cytochrome b5 diffused with a diffusion coefficient of ~2 × 10 −10 cm 2 /s. similar results were found in the annexin v system. annexin v diffused with two populations with diffusion coefficients of 3 × 10 −9 cm 2 /s and 4 × 10 −10 cm 2 /s. the new polymer-supported lipid bilayer system has increased the mobile fraction and retained the full lateral mobility of both cytochromes b5 and annexin when integrated or bound to the supported lipid. although polymer cushions allow for successful integration of small membrane proteins into bilayers, further challenges stem from studies with large transmembrane proteins. polymer cushions cannot provide large transmembrane proteins with good solvent accessibility, or enough space for the motion; required for the activity. while several types of polymer cushions have been developed, including polymethyl methacrylate diblock polymer cushions [31] , poly(ethylene imine) [32, 33] cushions and poly(ethylene glycol) tethered lipopolymers [30] , these cushions are mostly limited to a thickness of up to 10 nm. a recent development of a maleic anhydride copolymer thin film has film thickness up to 60 nm [34] . the hydrophilic polymer-cushioned supported lipid bilayers provide a higher mobility and homogeneous distribution of the incorporated beta-amyloid precursor protein cleaving enzyme (bace) on the bilayer surface, and enhances the enzymatic activity of bace (increased from 8% to 16%). even so, the activity of the incorporated bace remains significantly lower (16%) than that of the native enzyme (100%). another important classic category of membrane proteins are the transporters of ions and small molecules. studies of how ion channels regulate the transport of substrates [7] are important for fundamental biology. however, it is challenging to incorporate ion channels in supported lipid bilayers due to leakage or instability issues. detailed studies of ion channel conduction or gating require considerable period of time (possibly >1 h), and it is difficult to set up a stable and electrically quiet environment for the ion channel in planar lipid bilayer. a better alternative has proven to be reconstitution of ion channels into proteoliposomes. lipid vesicles, also known as liposomes, consist of a self-closed lipid bilayer. they have been widely used for more than 30 years to reconstitute the membrane proteins in unilamellar phospholipid vesicles. liposomes are relatively easy to construct by procedures such as extrusion method or ultrasonication, with reverse-phase evaporation. furthermore, giant vesicles of unilamellar or multilamellar nature can be "micro-manipulated" under an optical microscope. reconstitution of membrane proteins in liposomes usually requires detergents wherein purified membrane proteins are solubilized in detergent, then mixed with the desired phospholipid vesicles forming an isotropic solution of mixed phospholipid-protein-detergent micelles. the detergent can then be removed slowly by dialysis, gel filtration or biobead adsorption. when the detergent concentration reaches a critical level, the protein will spontaneously associate with the phospholipid membrane to form biologically active liposomes, called proteoliposomes. however, it has been a hard feat to control the final orientation of protein in the proteoliposomes [35] , as well as the amount of protein inserted due to the limited area available. in many cases, disorientation of the protein causes aggregation. despite these difficulties, there have been many successful cases of membrane proteins reconstituted in the proteoliposomes, and we describe two examples below. several integral membrane proteins have been successfully reconstituted into proteoliposomes such as rhodopsin [36] , g proteins [37, 38] , proapoptotic bcl-2 proteins and t-bid [39] , phosphocholine cytidylyltransferase (ct) [40] and p protein kinase c (pkc) [41] . however, these studies also found that the resulting protein activities are sensitive to the membrane curvature of the liposomes. this indicates that different phospholipids can cause considerable curvature stress changes in the liposomes [42] . specifically, the curvature stress has been suggested to modulate the free energy and folding of the integral membrane proteins [43] . sometimes the activity of different enzymes is modulated by the same driving force of the membrane curvature, but there may also be variation of activity through different mechanisms. for example, the activities of both ct [40] and pkc [41] are enhanced by increasing the negative curvature strain of the membrane. the activity of ct appears to be directly coupled with the membrane curvature, in contrast, the activity of pkc does not have a direct relationship with the curvature strain and enzymatic activity [41] . the activity of pkc is instead modulated by nonlamellar-forming lipids via a less direct mechanism. liposomes have been commonly used for reconstituting different types of transporters to allow for the free diffusion of solution or catalysis of obligatory co-transporters. a large number of functional membrane proteins have been successfully reconstituted into liposomes but only a few examples will be discussed here. the reconstitution of colicin ia and e1 in either soybean phospholipids or e. coli phospholipids show that there is channel formation in the liposomes but there are unspecific channels allowing passage of ions, such as rubidium, sodium, chlorine, potassium or phosphate but not of sugars [7, 44] . an example of the reconstitution of selective transport comes from the d-glucose transporter, purified from human erythrocytes and extracted from detergents followed by incorporation into proteoliposomes. with incorporation of the d-glucose transporter, the proteoliposomes become permeable to d-glucose but not to l-glucose. the transport was inhibited by cytochalasin b which is a potent inhibitor of d-glucose transporter [45, 46] . several types of atp-dependent ion transporters such as ca 2+ /mg 2+ -atpase, na + /k + -atpase, and h + /k + -atpase have been reconstituted into proteoliposomes [47] . upon addition of atp, ions are observed to be transported inwards and can form a complex. the single-channel property of channels incorporated into proteoliposomes can be investigated using the well-known patch-clamp method [48] . channel activity is monitored following excision of the patch from the proteoliposomes. ion-channel reconstitution makes possible the investigation of the influence of membrane lipid composition on channel function. the kinetic investigation of these channels under physiological conditions has been discussed elsewhere [47] . another up-to-date method is using organic solvent or oil mixed with water that creates water-in-oil (w/o) microdroplets coated by phospholipid. the hydrophilic head group immerses in the water and the hydrophobic tail locates in the oil/organic solvent phase. the application of the water-in-oil system could cover a wide range of applications from monolayer, planer lipid bilayer and liposomes. funakoshi et al. [49] and maglia et al. [50] used a planer lipid bilayer formed by two microdroplets driven to come in contact to reconstitute ion channels in the bilayer. this method is extremely simple and reproducible. recently, the water-in-oil microdroplets are extended to form liposomes by using droplet-transfer method invented by yoshikawa [51] . by using this approach, it is possible to modulate the lipid compositions of outer and inner leaflets and furthermore to orient a reconstituted membrane protein in liposomes [52] . nanodiscs offer a solution to some of the challenges described in the previous sections. the first attempt to reconstitute membrane proteins in the phospholipid bilayers using nanodisc technology was initiated by sligar's group a decade ago [8] . the nanodisc is a self-assembly of phospholipids and a membrane scaffold protein derived from human serum apolipoprotein a1. the detergent, cholate, can be used to solubilize phospholipids and membrane scaffold proteins into a micelle mixture. following detergent removal with dialysis or bio-beads adsorbent, a nanodisc self-assembles. the phospholipid associates as a bilayer domain while the membrane scaffold protein wraps around the edges of the discoidal structure in a belt-like configuration ( figure 1d ). it is possible to modify the diameter of the bilayer disc by genetically engineering the apolipoprotein a1 by changing the number of amphipathic helices. by this approach, the diameter of nanodiscs can be made anywhere from 9.8 to 17 nm, and therefore accommodate a range of membrane proteins. the ratio of phospholipid: membrane scaffold protein is precisely defined which helps engineer the different size of membrane proteins in the nanodiscs. detailed formation of different types of nanodiscs has been described elsewhere [53, 54] . the great advantage of using nanodiscs is keeping the membrane proteins in aqueous solution, in native-like phospholipid bilayer environment that is soluble, stable, monodisperse and detergent-free. most important, it isolates proteins or complexes as individual particles in monomeric or oligomeric states for analysis by techniques that range from activity assays to electron microscopy. since 2003, there have been more than 100 membrane proteins reconstituted into nanodiscs [54] , ranging from signaling receptors to transport machines. we will discuss the applications separately below. nanodiscs have been used to analyze the influence of binding substrate on monodisperse receptors which are isolated from the cell-surface membrane. those receptors include g protein-coupled receptors (gpcr) [55, 56] , cholera toxin receptor ganglioside g m1 , bacterial chemoreceptor [57] and epidermal growth factor receptor. introduced into nanodiscs, the receptors stay in monodispersed, controllable, predefined oligomeric states in which it is possible to characterize the oligomeric status. for example, two different gpcr proteins, the beta-adrenergic receptor (β 2 ar) and rhodopsin [58] have been extensively studied using nanodiscs. β 2 ar was one of the first receptors assembled into nanodiscs which was found to be functionally active (54% of starting activity recovered) and shown coupling to its g-protein. rhodopsin is a light-activated gpcr present in the photoreceptor cells of the retina and transducin is an important g-protein naturally expressed in retina rods and cones. assembly of functional rhodopsin into nanodiscs was found to activate transducin with high efficiency and to isolate the high affinity of transducin-metharhodopsin ii complex. this provides strong evidence that the monomeric state of rhodopsin can activate and interact with the transducin. a dimeric rhodopsin nanodisc was separated for monomeric forms using sucrose density gradients. even with two rhodopsins in the nanodiscs, interaction with a single transducin molecule was observed and found to activate the transductin with high efficiency [56] . numerous membrane associated enzymes have been incorporated into nanodiscs. cytochrome p450 (cyp) enzymes have been extensively studied, including cyp2b4 [59] , cyp6b1 [8] , cyp73a5 [60] and cyp19 [61, 62] . this system has provided a means for studying the extensive collection of membrane bound cytochromes p450 with the same biochemical and biophysical tools that have been previously limited to use with the soluble p450s. for example, the cytochrome p450 3a4 (cyp3a4) is a membrane-bound protein which is a human hepatic drug-metabolizing enzyme. most studies of the ligand binding by cyp34a are carried out in the presence of detergents below their critical micelle concentrations [63, 64] but are compared by the propensity of cyp34a to aggregate. even in studies attempting to use liposomes, cyp3a4 is unlikely to exist in its native state because the detergent concentrations are much higher than the phospholipid concentrations. as a result, the understanding of the structure and composition of cyp3a4 in the lipid phase was limited and the membrane effect on cyp3a4 ligand binding behavior is unclear. nanodiscs have been utilized to study cyp3a4 which displays monophasic reduction kinetics. with a high lipid-protein ratio, cyp3a4 is captured as a monomer. however, at lower lipid ratio, cy3a4 self-associates and heterogeneous behaviors are induced. the nanodiscs prohibit self-association in this case as there is only one cyp3a4 per nanodisc and show significant improvement in homogeneity and stability. this opens up new possibilities for detailed analysis of equilibrium and steady-state kinetic characteristics of catalytic mechanisms of cyp enzymes [63] . the sec translocon is a membrane-embedded protein assembly that drives protein translocation into or across membranes. the core translocon is formed from a trimeric arrangement of secy, sece and secg [65] . the secyeg promoter has 15 transmembrane helices sitting in the phospholipid membrane. the oligomerization of secyeg has been proposed to be necessary to proper function. researchers were successful in reconstituting sec into membrane vesicles in 1990 and have had great success in characterizing several partial reactions of secyeg functions [65] . reconstituting a single secyeg into a nanodisc with different types of lipids [66] suggests that the acidic lipids can stabilize the secyeg channel in the nanodisc bilayer and trigger dissociation of the seca dimer. a model has been proposed by alami et al. [66] , suggesting that the dissociation of the seca dimer provoked by the secyeg complex is followed by activation of the seca atpase. furthermore, dalal et al. [67] , using the nanodisc technology, have also shown that only the secy dimer together with acidic lipids supports the activation of the seca translocation atpase. recently, a high resolution single-particle cryo-em structure of single secyeg complexes in nanodiscs, bound to translating ribosomes was first solved at subnanometer resolution [68] . it allows the secyeg complex to be investigated in a natural lipid bilayer environment and identifies the ribosome-lipid interactions. wu et al. [69] also used surface plasmon resonance to investigate the competitive binding of ribosomes and seca. the data suggest that both ribosomes and seca can interact simultaneously with secyeg complex during membrane protein insertion, but seca competes with ribosome when it binds to the secyeg complex. in the previous section, we have shown that membrane proteins can be assembled into four different types of model membrane and the activities of some of the membrane proteins can be retained, allowing their physicochemical properties to be studied. but is there a model membrane system that is the best for membrane-protein reconstitution? the reconstitution of the same membrane protein into different model membranes has been compared and, in this section, we list four membrane proteins with varying activities in different model membranes. ganglioside g m1 is a naturally occurring native receptor that binds to cholera toxin via hydrogen bonds [70] . it is an excellent receptor for studying lipid-receptor interaction. several different approaches to reconstituting the glycolipid receptor g m1 in model membranes have enabled the measurement of binding of its interaction partner cholera toxin. in liposomes and supported lipid bilayer systems, the ganglioside g m1 is free to diffuse across long distances and exhibits a non-uniform lateral distribution, i.e., self-aggregation, even at low incorporation ratios. therefore the binding activity of ganglioside g m1 with cholera toxin b is restricted [71] . investigations of ganglioside g m1 incorporated into nanodiscs found reduced protein aggregation. bricarello et al. [72] found that the reconstitution of a low concentration of ganglioside g m1 in nanodiscs, shows binding of cholera toxin with a significantly higher affinity than in liposomes or supported lipid bilayers. this is due to the interaction of ganglioside g m1 with the headgroup region of the disc which reduces the oligomerization, thereby causing a potential effect on the affinity of toxin binding. thus, nanodisc technology restricts the ganglioside g m1 oligomerization by controlling the number of ganglioside g m1 monomer isolated by each nanodisc. borch et al. [73] have also used sensor chip-based surface plasmon resonance (spr) technology to measure the detailed kinetic binding of the interaction between soluble molecules and membrane receptors inserted in the bilayer of nanodiscs. the corresponding spr sensorgrams are displayed in figure 4 . overall, the change of the sensorgram indicates that the spr sensorchip is immobilized with histidine-modified nanodisc or the cholera toxin b bound to the nanodiscs. the sensorgrams in both figure 4a ,b shows the binding of nanodiscs (576 ru) on the antibody immobilization surface on the sensor chip. by injecting the cholera toxin b over two flow cells presented in figure 4a and 4b, the spr sensorgrams can detect the interaction of the cholera toxin b with nanodisc with or without the existence of g m1 . it has been revealed that the captured 2% g m1 -nanodiscs bound 238 ru of the cholera toxin b without binding to the capturing nanodiscs without g m1 ( figure 4b ). the measured kinetic values of the interaction are in agreement with those reported by previous studies on the interaction of the cholera toxin with the g m1 receptor embedded in different membrane systems. this, therefore, serves as a proof of concept that nanodiscs can be employed in kinetic spr studies. the nucleus envelope is composed of two bilayers (the outer nuclear membrane and inner nuclear membrane) and contains abundant ion channels, through which ions and small molecules diffuse between the cytoplasm, nucleoplasm and perinuclear (i.e., intermembrane) space. the nuclear ionic channels represent a ubiquitous structure in the nuclei in a wide range of cells, although little is known about its functional properties. to characterize nuclear ionic channels, guihard et al. [74] attempted to reconstitute nuclear envelope vesicles derived from the canine liver nuclei into a planar lipid bilayer and giant proteoliposomes. they found that the success rate of nuclear envelope fusion into planar lipid bilayers was extremely low although cardiac nuclear ionic channels were successfully incorporated into planar lipid bilayers. the detection of the nuclear ionic channels activity was not possible. such a low efficiency can be explained by the clustering of nuclear envelope vesicles, and the low density of single vesicles, as well as the presence of residual chromatin and/or nuclear proteins (histones or lamins) which would prevent fusion events with the bilayer. another approach is reconstituting nuclear envelope vesicles into giant proteolipsosmes and detecting the single ion channel by the patch-clamp technique [49] . large conductance, voltage-gated, k + and cl − selective nuclear ionic channels are characterized and plotted as a current-voltage relationship presented in figure 5a ,b respectively. it has been found that under asymmetrical 150/50 mm kcl conditions, the zero current potential for unitary currents is at 322 mv ( figure 5a ). calculated from the goldman-hodgkin-katz (ghk) flux equation, a p k +/p cl − ratio is 9.4. this value indicates the k + selectivity for this channel. in figure 5b , the cl − selective nuclear ionic channel yields a positive zero current potential of +27.3 mv, with a p cl −/p k + ratio of 80, indicative of a high cl − selectivity over k + . this suggests super fusion of the channel under asymmetrical (150/50 mm) kcl conditions. the current-voltage relationship curves indicate that the nuclear ion channels can be functionally characterized by incorporating the proteins into the giant proteoliposomes where it is possible to retain their channel activity. furthermore, the measured activities are consistent with those described for native nuclear ion channels. p-glycoprotein, the most extensively studied atp-binding cassette transporter, has been implicated in the phenomenon of multidrug-resistance in tumor cells and has been suggested to play a significant role in drug absorption and deposition. how p-glycoprotein interacts with its substrates is still unknown. functional studies are limited because of the difficulty of obtaining large quantities of stable p-glycoprotein. besides that, no atpase activity of p-glycoprotein solubilized in detergent could be detected. when p-glycoprotein is reconstituted into proteolipsomes, it has detectable atpase activity; however, the whole complex is very unstable. heikal et al. [75] have further found that p-glycoprotein reconstituted in the proteoliposomes has a half-life of less than one day. in 2009, ritchie et al. [76] performed a detailed study of drug-stimulated atpase kinase activity of p-glycoprotein using the nanodisc technology. the p-glycoprotein protein was reconstituted into both msp1e3d1 disc and liposomes in order to compare its atpase kinase activities. the results described in figure 6 demonstrate that p-glycoprotein is functionally active when reconstituted into the nanodiscs (close squares). comparing to the atpase kinase activity of p-glycoprotein reconstitution in lipsosomes (close circles), there is a twofold increase in the maximum atpase activity in the nanodiscs. this could be due to the uniform orientations of p-glycoprotein in the nanodiscs while there are two possible orientations in liposomes. these data not only show that p-glycoprotein is functionally active when reconstituted into the nanodiscs, but that it also exhibits higher specific activity than the current standard reconstitution system. figure 6 . comparsions of the atpase activity of p-glycoprotein in nanodiscs (square) and proteoliposomes (circle). open symbols: basal activity in the absence of drug; filled-in symbols: activity in the presence of nicardipine [76] . atp-binding cassette transporters utilize the energy of atp hydrolysis to transport a wide range of substrates across cellular membranes and for non-transport-related processes such as translation of rna and dna repair [77] . a member of the atp-binding cassette super family, the maltose transporter malfgk2 from e. coli, together with the substrate-binding protein male, is one of the best-characterized atp-binding cassette binding cassette transporters suitable for various reconstitution techniques. bao and fuong have reported the reconstitution of the maltose transporter in nanodiscs, in detergent and in proteoliposomes. the atpase activity of the malfgk2 complex in various environments is shown in figure 7 . the data presented in the first column of figure 7 show that the basal atpase activity for assembly in the nanodiscs and detergent (~700 nmol/min/mg) is 10-fold higher than in proteoliposomes because of the decrease in the activation energy barrier of the transporter [78] in detergent micelles and nanodiscs. however, in the presence of male, the rate of atp hydrolysis increases in all assembly conditions. this is because male captures maltose and delivers the sugar to the transporter. note that the basal atpase activity assembly in the nanodiscs dramatically increases from 700 to 2300 nmol/min/mg. the maltose alone has no effect on the basal atpase activity in the nanodiscs and detergent. however, in nanodisc and detergent, an inhibition of the atpase activity was observed in the presence of both maltose and male in the nanodiscs. this is because that maltose reduces the binding affinity of the male-malfgk 2 complex, which therefore has reduced the atpase activity. in proteoliposomes, the atpase activity (~40 nmol/min/mg) shows a further 10-fold increase in the presence of both maltose and mele in the figure. the author used another type of male mutant which binds maltose with higher affinity. this male mutant, in contrast, shows a reduction of the atpase activity in proteoliposomes which has the same effect as the nanodiscs and detergents. overall, proteoliposomes have shown a low basal atpase activity because the lipid stabilized the transporter. however, the nanodiscs have been shown to be a better medium than proteoliposomes for studying the atp hydrolysis ability of atp-binding cassette transporters. this review summarizes and compares the most up-to-date methods for reconstituting membrane proteins into model membranes. there is no superior method for reconstituting membrane proteins in the model membrane; instead two or more model membranes should be considered, depending on the particular needs of the system and the proteins of interest. in general, systems based on lipid bilayers supported on a solid substrate are still the most favored and well-developed of the methods to study membrane proteins in the bilayer. this approach allows detailed study of the fundamental properties of biological membranes and is practical to reproduce the bilayer system. on the other hand, the proteoliposome is more suitable for ion channel reconstitution in the bilayer, as well as for combination with the patch-clamp method to detect the ionic selectivity of the channel. finally the self-assembled nanodiscs system provides a robust and common means for rendering these targets soluble in aqueous media while providing a native-like bilayer environment that maintains functional activities. nanodisc technology offers another way to prepare monodisperse samples of membrane proteins in the bilayer environment, and it is emerging as the favored approach in studies concerning membrane protein complexes. biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein lipid-protein interactions in human erythrocyte-membrane acetylcholinesterase. modulation of enzyme activity by lipids correlation between the effect of the anti-neoplastic ether lipid 1-o-octadecyl-2-o-methyl-glycero-3-phosphocholine on the membrane and the activity of protein kinase calpha weak dependence of mobility of membrane protein aggregates on aggregate size supports a viscous model of retardation of diffusion structure and phase transitions in langmuir monolayers allogeneic stimulation of cytotoxic t cells by supported planar membranes effect of colicins ia and e1 on ion permeability of liposomes direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers surface enhanced raman scattering of a lipid langmuir monolayer at the air-water interface lipid monolayers: why use half a membrane to characterize protein-membrane interactions? langmuir monolayer of artificial pulmonary surfactant mixtures with an amphiphilic peptide at the air/water interface: comparison of new preparations with surfactant polymyxin b-lipid interactions in langmuir-blodgett monolayers of escherichia coli lipids: a thermodynamic and atomic force microscopy study langmuir balance investigation of superoxide dismutase interactions with mixed-lipid monolayers modern physicochemical research on langmuir monolayers structure of rhodopsin in monolayers at the air-water interface: a pm-irras and x-ray reflectivity study formation, structure, and spectrophotometry of air-water interface films containing rhodopsin proton transport by bacteriorhodopsin in planar membranes assembled from air-water interface films structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films spectroscopic and structural properties of valine gramicidin a in monolayers at the air-water interface effects of gramicidin-a on the adsorption of phospholipids to the air-water interface organization, structure and activity of proteins in monolayers monitoring of phospholipid monolayer hydrolysis by phospholipase a2 by use of polarization-modulated fourier transform infrared spectroscopy supported planar membranes in studies of cell-cell recognition in the immune system supported phospholipid bilayers formation of high-resistance supported lipid bilayer on the surface of a silicon substrate with microelectrodes supported lipid bilayer self-spreading on a nanostructured silicon surface simulations of temperature dependence of the formation of a supported lipid bilayer via vesicle adsorption simulations of lipid vesicle rupture induced by an adjacent supported lipid bilayer patch membrane lateral mobility obstructed by polymer-tethered lipids studied at the single molecule level tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker reversible activation of diblock copolymer monolayers at the interface by ph modulation, 1: lateral chain density and conformation polymer-cushioned bilayers. i. a structural study of various preparation methods using neutron reflectometry polymer-cushioned bilayers. ii. an investigation of interaction forces and fusion using the surface forces apparatus controlled enhancement of transmembrane enzyme activity in polymer cushioned supported bilayer membranes orientation and reactivity of nadh kinase in proteoliposomes modulation of rhodopsin function by properties of the membrane bilayer role of lipid polymorphism in g protein-membrane interactions: nonlamellar-prone phospholipids and peripheral protein binding to membranes influence of the membrane lipid structure on signal processing via g protein-coupled receptors the apoptotic protein tbid promotes leakage by altering membrane curvature modulation of ctp:phosphocholine cytidylyltransferase by membrane curvature elastic stress the role of membrane biophysical properties in the regulation of protein kinase c activity membrane lipid polymorphism: relationship to bilayer properties and protein function elastic coupling of integral membrane protein stability to lipid bilayer forces reconstitution of colicin e1 into dimyristoylphosphatidylcholine membrane vesicles the permeability of bilayer lipid membranes on the incorporation of erythrocyte membrane extracts and the identification of the monosaccharide transport proteins binding of cytochalasin b to human erythrocyte glucose transporter conformational dynamics of na+/k+-and h+/k+-atpase probed by voltage clamp fluorometry the extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes lipid bilayer formation by contacting monolayers in a microfluidic device for membrane protein analysis droplet networks with incorporated protein diodes show collective properties cell-sized liposomes and droplets: real-world modeling of living cells oriented reconstitution of a membrane protein in a giant unilamellar vesicle: experimental verification with the potassium channel kcsa phospholipid phase transitions in homogeneous nanometer scale bilayer discs membrane protein assembly into nanodiscs functional reconstitution of beta2-adrenergic receptors utilizing self-assembling nanodisc technology transducin activation by nanoscale lipid bilayers containing one and two rhodopsins using nanodiscs to create water-soluble transmembrane chemoreceptors inserted in lipid bilayers atomic-force microscopy: rhodopsin dimers in native disc membranes single-molecule height measurements on microsomal cytochrome p450 in nanometer-scale phospholipid bilayer disks co-incorporation of heterologously expressed arabidopsis cytochrome p450 and p450 reductase into soluble nanoscale lipid bilayers the critical iron-oxygen intermediate in human aromatase the ferrous-oxy complex of human aromatase kinetics of dithionite-dependent reduction of cytochrome p450 3a4: heterogeneity of the enzyme caused by its oligomerization ligand binding to cytochrome p450 3a4 in phospholipid bilayer nanodiscs the effect of model membranes the atpase activity of seca is regulated by acidic phospholipids, secy, and the leader and mature domains of precursor proteins nanodiscs unravel the interaction between the secyeg channel and its cytosolic partner seca two copies of the secy channel and acidic lipids are necessary to activate the seca translocation atpase cryo-em structure of the ribosome-secye complex in the membrane environment competitive binding of the seca atpase and ribosomes to the secyeg translocon crystal structure of cholera toxin b-pentamer bound to receptor gm1 pentasaccharide self-aggregation-an intrinsic property of g(m1) in lipid bilayers ganglioside embedded in reconstituted lipoprotein binds cholera toxin with elevated affinity nanodiscs for immobilization of lipid bilayers and membrane receptors: kinetic analysis of cholera toxin binding to a glycolipid receptor patch-clamp study of liver nuclear ionic channels reconstituted into giant proteoliposomes the stabilisation of purified, reconstituted p-glycoprotein by freeze drying with disaccharides chapter 11-reconstitution of membrane proteins in phospholipid bilayer nanodiscs abc transporters, mechanisms and biology: an overview discovery of an auto-regulation mechanism for the maltose abc transporter malfgk2 the authors gratefully acknowledge financial support from the australian research council (arc). hhs is an arc super science fellow and tl is an arc federation fellow. tl and lm were awarded the arc super science fellowships and grant (fs110200015). we thank victoria hewitt for her critical reading of the manuscript. key: cord-005145-1l87fdmi authors: marquet-blouin, e.; bouche, f.b.; steinmetz, a.; muller, c.p. title: neutralizing immunogenicity of transgenic carrot (daucus carota l.)-derived measles virus hemagglutinin date: 2003 journal: plant mol biol doi: 10.1023/a:1022354322226 sha: doc_id: 5145 cord_uid: 1l87fdmi although edible vaccines seem to be feasible, antigens of human pathogens have mostly been expressed in plants that are not attractive for human consumption (such as potatoes) unless they are cooked. boiling may reduce the immunogenicity of many antigens. more recently, the technology to transform fruit and vegetable plants have become perfected. we transformed carrot plants with agrobacterium tumefaciens to generate plants (which can be eaten raw) transgenic for an immunodominant antigen of the measles virus, a major pathogen in man. the hemagglutinin (h) glycoprotein is the principle target of neutralizing and protective antibodies against measles. copy numbers of the h transgene were verified by southern blot and specific transcription was confirmed by rt-pcr. the h protein was detected by western blot in the membrane fraction of transformed carrot plants. the recombinant protein seemed to have a 8% lower molecular weight than the viral protein. although this suggests a different glycosylation pattern, proper folding of the transgenic protein was confirmed by conformational-dependent monoclonal antibodies. immunization of mice with leaf or root extracts induced high titres of igg1 and igg2a antibodies that cross-reacted strongly with the measles virus and neutralized the virus in vitro. these results demonstrate that transgenic carrot plants can be used as an efficient expression system to produce highly immunogenic viral antigens. our study may pave the way towards an edible vaccine against measles which could be complementary to the current live-attenuated vaccine. the development of genetic transformation technology has allowed the expression of foreign genes in an increasing number of plant species. the use of plants for the production of foreign antigen proteins that could serve as experimental immunogens was first reported in the early 1990s (cardineau and curtis, 1990; mason et al., 1992) . since then, a number of viral and bacterial antigens have been expressed in a variety of plant species (mcgarvey et al., 1995; thanavala et al., 1995; carrillo et al., 1998; gomez et al., 1998; modelska et al., 1998; tacket et al., 1998; wigdorovitz et al., 1999) . despite differences in post-translational processing viral and bacterial antigens preserved their immunogenic properties when produced in plants and induced cross-reactive and sometimes neutralizing and protective antibodies. plants could therefore be an inexpensive source of antigens that could be easily purified for parenteral inoculation (thanavala et al., 1995; gomez et al., 1998) . moreover, oral ingestion of plants expressing high levels of antigens bear the potential of edible vaccines (kong et al., 2001) . strategies based on potent mucosal adjuvants such as cholera toxin and heat-labile enterotoxin of escherichia coli (haq et al., 1995; arakawa et al., 1998a, b) may pave the road for oral immunization. research on edible plant vaccines has been carried out mostly in plant species largely inappropriate for human consumption (e.g. tobacco, nicotiana tabacum, thanavala et al., 1995; mason et al., 1996; huang et al., 2001; nicotiana benthamiana, modelska et al., 1998; arabidopsis thaliana, carrillo et al., 1998; gomez et al., 1998) . for most proteins of human pathogens expressed in edible plants, potatoes were used, which are normally boiled before consumption (mason et al., 1996; arakawa et al., 1998a; richter et al., 2000; kong et al., 2001) . it can be anticipated that many antigens would not resist cooking without being denatured and that cooked plant material is less immunogenic than raw plants (kong et al., 2001) . therefore, there is a need to develop other and more appropriate transgenic plant species that can serve as edible vaccines. the technology for creating other transgenic edible plants, including fruits and vegetables has been further perfected (schenk et al., 2001; brodzik et al. 2000) . we chose to transform transgenic carrots, which can be grown in most parts of the world, which can be eaten both raw and cooked, and are part of the early diet of infants. current life-attenuated measles vaccines are given routinely at 9 to 15 months of age. after a single injection, seroconversion rates are high, complications are rare and protection is long-lasting. these advantages are difficult to match by other experimental measles vaccine. however, after 25 years 50% of vaccinees are thought to have lost protective levels of antibodies (mossong et al., 1999) . revaccination with an oral vaccine which would boost the residual immunity would be a preferred strategy since it can be selfadministered, requires less training of health workers and avoids the risks associated with needle injections. the potential of a parenteral/oral prime-booster schedule has been demonstrated for a number of pathogens (kong et al., 2001; mantis et al., 2001) . such a schedule is probably less liable to problems of weak and variable responses after oral vaccination. a strategy based on oral vaccination could be a particularly useful for large-scale booster immunization in developing countries where the need to deliver parenteral vaccines may hamper eradication efforts. measles is caused by a paramyxovirus (mv) which projects two glycoproteins, the hemagglutinin (h) and the fusion protein, from the outer viral envelope. the h protein is responsible for the attachment of the virus to the host cell (naniche et al., 1993) , whereas the fusion protein is directly involved in the fusion of viral and target cell membranes required for the penetration of the virus (wild et al., 1991) . virus-neutralizing and protective antibodies are mainly directed against the hemagglutinin and, to a lesser extent, the fusion protein (mcfalin et al., 1980; giraudon and wild, 1985) . the aim of this study was (1) to explore the potential of carrots as an expression system for antigens that is suitable for human consumption, and (2) to test whether the measles virus hemagglutinin glycoprotein would preserve its neutralizing immunogenicity in this system. although some work has been done with transgenic carrot callus cells (brodzik et al., 2000) , this is one of the first reports of the expression of a transgenic antigen in mature carrots, showing that high levels of virus-neutralizing antibodies can be induced with a glycoprotein produced in this plant. the coding sequence corresponding to the measles virus hemagglutinin (mv-h) protein (bouche et al., 1998a) was subcloned into the expression cassette of the prtl2 vector at the ncoi-bamhi sites (restrepo et al., 1990) . in this vector, the mv-h sequence was under the control of the constitutively expressed cauliflower mosaic virus (camv) 35s promoter fused to the tobacco etch virus (tev) 5 -untranslated region, a translational enhancer, and the camv 35s terminator (odell et al., 1985; pietrzak et al., 1986) . these control sequences were flanked with hindiii restriction sites that allowed their transfer, together with the h sequence, into the t-dna region presents in binary vector pbin19 (bevan, 1984) , creating the recombinant plasmid pbin19-mvh. the t-dna region, delimited by the right and left border sequences, also contains the neomycin phosphotransferase ii gene (nptii) that provides neomycin and kanamycin resistance to transformed plants ( figure 1 ). after subcloning of the expression cassette in pbin19 and transformation of xl1-blue cells kanamycin-resistant colonies were picked and checked for the presence of the expression cassette by pcr and automated sequencing (373 a model, perkin elmer, netherlands). plasmid dna was then isolated from a positive clone and introduced in agrobacterium tumefaciens strain lba4404 by electroporation (25 µf, 2500 v, 400 ). transformed bacteria were selected on yeb-agar solid medium containing 50 µg/ml kanamycin (28 • c, 48 h) and were used for subsequent carrot transformation. the protocols of a. tumefaciens-mediated transformation of hypocotyls were modified for the production of transgenic carrot plants (hardegger and sturm, 1998; tokuji and fukuda, 1999; brodzik et al., 2000) . sterilized carrot seeds (herrera-estrella and simpson, 1988; daucus carota cv. senkou-gosun, kindly given by dr tokuji, japan) were sown in 0.8% agar in the dark at 25 • c. after 14 days hypocotyl segments were harvested in gamborg b5 liquid medium (b5) supplemented with 3% sucrose and with the phytohormone 2,4-dichlorophenoxyacetic acid (1 mg/l) for 48 h at 25 • c. after washing, the segments were placed for 5 days on phytohormone-free b5 solid medium (0.8% agar) containing 3% sucrose. the hypocotyl fragments were then transformed by immersion (2 h) in the bacterial suspension of a. tumefaciens containing the recombinant pbin19-mvh binary plasmid. the segments were further co-cultured in the dark at 25 • c with the bacteria on b5 solid medium with 3% sucrose. five days later, the explants were washed with b5 medium containing cefotaxime (250 mg/l) to kill remaining agrobacteria. explants were grown in darkness at 25 • c on b5 solid medium containing cefotaxime (250 mg/l) and geneticin (10 mg/l) for the selective growth of transgenic cells. after 4 weeks of selection, somatic embryos resistant to geneticin appeared on the hypocotyl segments. each hypocotyl usually gave rise to a few embryos. embryos were subcultured and rooted on selective medium at 25 • c under light. plantlets with adequate roots were transferred to potting soil and grown in a greenhouse under normal light and humidity conditions. several lines of transgenic carrots were obtained and further studied. genomic dna was isolated from both untransformed and transformed plants by macerating frozen leaves (ca. 3 g) in liquid nitrogen. the resulting powder extract was re-suspended in the extraction buffer (100 mm tris-hcl, 20 mm edta, 1.4 m nacl, 100 mm 2-mercaptoethanol and 2% n-cetyl-n,n,n,trimethylammonium bromide, ph 8.0) and incubated at 60 • c for 30 min. after a chloroform extraction, nucleic acids were precipitated with 0.7 volume of isopropanol and the pellet obtained after centrifugation was resuspended in tris-hcl (10 mm, ph 8.0)/ edta (1 mm) buffer. after rnase treatment the dna was stored at −20 • c until being used. a fragment of the mv-h expression cassette (promotor-mvh-terminator) was specifically amplified with a forward primer (5 -gcaagacccttcctctatat-3 ) from the 35s promoter region and a reverse primer (5 -atctgggaactactcacac-3 ) from the 35s terminator region. the presence of the nptii gene was detected by pcr amplification with a pair of specific primers within the nptii gene (5 -tgctcctgccgagaaagtatc-3 and 5 -tcctgtatcgcaaccgatgggc-3 ). genomic dna of untransformed plants was used as negative control. southern blotting with genomic dna was performed following conventional protocols. briefly, 30 µg of extracted dna was digested overnight at 37 • c with ecori which cuts the recombinant t-dna at a single position (between the 35s promoter and the tev leader). after agarose gel (0.8%) electrophoresis, digestion products were transferred overnight onto a nylon membrane (hybond+, amersham-pharmacia biotech, uk). the membrane was equilibrated with 20× sspe prior to immobilization of dna by uvcross-linking (uv stratalinker, stratagene, netherlands). for hybridization, a 32 p-labelled h-specific cdna probe, generated by nick translation according to feinberg and volgelstein (1983) , was incubated with the membrane for 4 h at 65 • c. the membrane was then washed for 20 min at 65 • c successively with 0.1% sds in 5× ssc, 2× ssc and finally in 0.2× ssc. hybridized complexes were detected by autoradiography. total rna from leaves (1 g) of transformed plants was isolated as described by hughes and galau (1988) . rna from untransformed plants was used as a negative control template. reverse transcription (rt) was carried out for 2 h at 40 • c in 50 µl final reaction volume containing 5 µg rna, 500 ng random hexamers and a mixture of dntps (20 mm each), 10 mm dtt, 10 units of rnasin (promega, netherlands) and 200 units of m-mlv reverse transcriptase (superscript ii, gibco life sciences, belgium). after adding 0.1 volume of 10 mm atp, ligation was carried out by incubation for 45 min at 37 • c in the presence of 2 units of t4 dna ligase. pcr amplification was performed with two h-specific primers including the first 18 and the last 12 nucleotides of the coding sequence. the pbin19-mvh vector was used as positive control template. a pcr reaction was also performed directly on the total rna to confirm the absence of specific dna in the extract. plant tissues were homogenized on ice in pbs containing 5 mm edta and protease inhibitors (10 µg/ml aprotinin, 1.8 mg/ml iodoacetamide, 10 µg/ml leupeptin, 1 mm pmsf). the mixture was centrifuged at 1700 × g (10 min, 4 • c) to remove insoluble debris. the membrane fraction was sedimented by ultracentrifugation at 100 000 × g (45 min, 4 • c), and re-suspended in pbs containing 0.5% np-40. protein concentration was determined with the dc protein assay kit (biorad, belgium). one gram of wet weight of carrot plant gave about 100 µg of membrane protein. proteins of the membrane fraction were separated by 12% sds-page under reducing and denaturing conditions (100 mm dtt, 4 m urea, 2% sds). proteins were blotted onto nitrocellulose membrane in tris-glycine buffer for 2 h at 250 ma. the membrane was blocked for 1 h at room temperature with 5% non-fat dehydrated milk in pbs containing 0.1% tween-20. for detection of the mv-h protein two specific monoclonal antibodies (mabs; bh47 and bh195, 1:1000 dilution) and goat anti-mouse iggconjugated horseradish peroxidase secondary antibodies (1:5000 dilution) were used. bound antibodies were detected by enhanced chemiluminescence (ecl kit, amersham-pharmacia biotech). to characterize recombinant h protein in leaf and root extracts microtiter plates (maxisorb, nunc, denmark) were coated with increasing concentrations of plant extract (in pbs) and revealed with h-specific mabs (dilution 1:1000). bh47 recognizes the sequential helix-forming epitope h236-350 (fournier et al., 1997; deroo et al., 1998) ; bh67 and bh81 are conformation-dependent antibodies (unpublished); bh1 binds only denatured protein (ziegler et al., 1996) ; bh216 binds to the hemagglutinin noose epitope (hne) h386-400 (ziegler et al., 1996) . mouse sera (1:250 to 1:32000) were titrated against immobilized purified h protein (bhk-h) produced in bhk-21 cells (50 ng/well; bouche et al., 1998b) . the coated antigens were washed and blocked with 1% bsa in tris-buffered saline (15 mm, ph 7.4). after addition of mabs or mouse serum, alkaline phosphatase-conjugated goat anti-mouse igg (1:750; southern biotechnology association, usa) and pnitrophenylphosphate (sigma, usa) were used for detection. optical density (od) was measured after 90 min at 405 nm. data were expressed as net od values after subtracting either the absorbance of the negative control antigen (e.g. extract of wild-type roots or leaves, or bhk-0 antigen; figure 4 ) or the absorbance of the conjugate without mouse serum (<0.150 od). h-specific antibody isotypes and subclasses were determined in mouse sera (1:500) by elisa with specific conjugates and substrate of a commercial kit (biorad). data were expressed as od at 415 nm after 30 min as recommended. groups of four spf balb/c mice were primed by intraperitoneal injection of with 500 µg of leaf or root extracts from transgenic or wild-type (wt) plants or 100 µg of bhk-h or bhk-0 antigens emulsified (1:1) in freund's complete adjuvant (sigma). by comparison with the elisa signal of mammalianexpressed purified h protein, the plant extract and the bhk extract was estimated to contain about 10 and 20 µg of the specific protein, respectively. mice were boosted on days 14, 28 and 42 with the same antigen preparation emulsified in freund's incomplete adjuvant (sigma). sera were drawn 7 days after boosting and were tested as pooled sera (after the second boost) or as individual sera (after the third boost). the reactivity of immune sera (1:50) with native h protein or mv was tested by flow cytometry as described before using permanently h-transfected mel-juso cells expressing the recombinant protein at their surface (mel-juso/h, gift of r. de swart, rotterdam, netherands; de swart et al., 1997) or mvsuperinfected ebv-transformed human b cell line (mv-wmpt; gift of b. chain, london, uk; muller et al., 1995) . for both assays wt or uninfected cells (mel-juso/wt or wmpt) were used as negative control cells. cells were incubated on ice (30 min) with diluted serum of individual mice. after washing, fitc-conjugated goat anti-mouse fc-specific antibody (1:200, sigma) was used for detection. fitcconjugate alone, naïve serum on positive and negative cells or test serum on negative cells served as negative controls. bh26 (1:500; ziegler et al., 1996) served as an antibody positive control. dead cells were excluded by propidium iodide staining (1 µg/ml). to test mv-neutralizing activity, mouse sera were heated for 30 min at 56 • c to inactivate complement. duplicates of two-fold serial dilutions (1:64 to 1:32768) of heat-inactivated serum were mixed with an equal volume of medium containing 35 plaqueforming units of edmonston strain mv. after 2.5 h of pre-incubation at 37 • c, the mixture was added to a subconfluent vero cell culture in 24-well plates (2.5 × 10 5 cells per well). after one hour unbound virus was removed and cells were covered with a carboxymethylcellulose (4%) overlay. after four days of incubation under tissue culture conditions, a 0.2% neutral red solution was added. on day 5, the overlay was removed and cells were fixed with a 10% formaline solution. the 50% neutralization titre, defined as the reciprocal of the dilution that reduces the number of plaques by 50%, was calculated accord-ing the spearman-kärber method. titres <64 were considered to be negative. the transformation of carrot plants was mediated by recombinant agrobacterium infection using the pbin19-mvh plasmid (figure 1 ). the regenerated transgenic plants showed no morphological changes in comparison to wt carrot plants (data not shown). about 30 plants resulting from independent transformation events were selected and grown in the greenhouse. ten of them were analysed further. the presence of the mv-h expression cassette in transgenic plants was confirmed by pcr followed by gel electrophoresis of the amplified fragments (figure 2a) . from all transformed plants tested a product of the expected size (2.2 kb) was amplified (lanes 3-12). the same size was obtained with pbin19-mvh vector as a positive control template (lane 1). this product was absent in dna of untransformed plants (lane 2). similarly, the presence of the nptii gene was confirmed as a specific product of 360 bp in all geneticin-resistant plants ( figure 2b, lanes 3-12) . this product was absent from untransformed plants (lane 2). copy numbers of the transgene and the integration pattern were determined by southern blot by digesting genomic dna of the 10 transformed plants ( figure 2c ) with ecori (which has a unique restriction site in the t-dna, see figure 1 ). hybridization with a mv-h-specific probe showed in every transgenic plant a unique restriction pattern (lanes 3-12) indicating that insertions occurred at random sites throughout the genome. all plants appeared to have integrated a single copy of the transgene, as illustrated by the presence of a single hybridization band. the insert corresponding to the radioactive probe was used as positive control template (lane 1). no signal appeared when genomic dna of wt plants was used as a template (lane 2). the transcription of the mv-h gene was analysed by rt-pcr on total rna extracted from transgenic plants ( figure 3a ). untransformed plants served as a negative control (lane 3). all tested plants produced a specific major transcript of the expected size lanes 4-13) . the minor unspecific, smaller band was also found when other transgenes were tested. no amplified dna was detected when the pcr was directly performed on the rna preparations, confirming the rna-specificity of the reaction (lane 2). crude membrane preparations of leaves (500 ng protein) and of bhk-h cells (50 ng protein) gave specific bands of similar intensities in western blots ( figure 3b ). based on elisa with purified h protein the content of specific protein was estimated at about 2% and 20% of specific protein in membrane fraction. the h-specific monoclonal antibodies bh47 and bh195 that bind to two non-overlapping epitopes revealed a strong under reducing conditions with an estimated molecular mass of about 68 kda (lane 3). under the same conditions, h protein produced in mammalian cells (bhk-h) migrated with an apparent size of ca. 74 kda as reported earlier (lane 1; bouche et al., 1998b) . this difference in mass may correspond to different levels of glycosylation of the monomer, but this was not further explored (vialard et al., 1990) . negative controls included an irrelevant mab (lanes 4-6) as well as a crude membrane preparation of wt leaves (lanes 2). the western blot suggested that there may be differences in glycosylation. glycosylation is well known to be important for the proper folding of the h protein (hu et al., 1994) . several monoclonal antibodies were used to investigate the conformational integrity of the transgenic protein ( figure 4a ). microtitre plates were coated with increasing concentrations (62.5-1000 ng/well) of membrane fractions from both transgenic leaves and roots. high net signals were obtained with two conformational-dependent mabs (bh67 and bh81), and bh47 which recognizes the sequential epitope h236-250 (fournier et al., 1997) with a putative helical conformation (deroo et al., 1998) . in contrast, bh1, which binds denatured protein only, showed essentially no reactivity above the background of wt plants. bh216 binds to the hemagglutinin noose epitope (hne, h386-400) with its oxidized cysteine bridge (ziegler et al., 1996) . the antibody binding pattern was essentially the same in root and leaf extracts and resembled that of purified h protein of mammalian origin ( figure 4b ). in general, signals tended to be higher in younger than in older tissues (data not shown). since the transgenic h protein seemed to be antigenically conserved, its immunogenicity was tested in mice. after 2 or 3 boosts with transgenic plant extracts all animals showed reactivity with purified h protein produced in mammalian cells ( figure 5a ). after the third boost, average antibody levels obtained with leaf extracts were about 4 times higher than after immunization with root extracts. similar levels were found after boost 2 for leaves, whereas antibodies still increased between boost 2 and 3 with root extracts. sera of control mice immunized with wt plant extract showed no cross-reactivity with the h protein (net od <0.03; data not shown). interestingly, antibodies generated with the transgenic leaves were of both the igg1 and igg2a subclass, whereas the immune response against the h protein produced in bhk-21 cells was essentially restricted to igg1, suggesting a difference in the th1/th2 balance ( figure 5b ). no h-specific igg2b, igg3, iga and igm were detected. to exclude that the reactivity may be due to partially denatured recombinant protein, the sera were further tested for antibodies against the intact native protein expressed on mv-superinfected wmpt cells ( figure 6a ) and h-transfected mel-juso cells (figure 6b ). the flow cytometry data showed that all mice vaccinated with transgenic leaf or root extracts produced high levels of antibodies cross-reacting with the native protein independently whether virus-infected or h-protein transfected cells were used. antibody levels were similar to those of mice immunized with h protein produced in mammalian cells, while wt plants induced no cross-reactive antibodies. in addition to a strong virus cross-reactivity, all sera showed high levels of neutralizing antibodies in a standard plaque reduction neutralization assay. in the group immunized with leaf extracts, mean neutralizing titres of 10 700 (range 5500-22 500) were observed, while root extracts induced significantly lower titres (3000; range 800-7400). all sera from mice given extracts of untransformed plants had titres <64 and were considered negative. in general, both cross-reactive and neutralizing titres were higher in leaves than in roots. this could reflect a difference in expression or in the yield of the extraction procedure. although edible vaccines seem to be feasible, very few antigens have been expressed in plants fit for human consumption (modelska et al., 1998; kapusta et al., 1999; sandhu et al., 2000) . for instance, potatoes have been used to express antigens of human pathogens (mason et al., 1996; arakawa et al., 1998a, b; richter et al., 2000; kong et al., 2001) . however, raw potatoes are not very appealing, and cooking can drastically reduce the immunogenicity of the vaccine (kong et al., 2001) . this is the first report of the expression of an antigenic protein as a transgene in mature carrots (which can be eaten raw by human beings) and of the antigenic and immunogenic properties of the heterologous protein in mice. genomic integration of the mv-h gene under the camv 35s double promoter was obtained using agrobacterium tumefaciens. southern blot analysis showed that all regenerated transgenic plants analysed integrated a single copy of the transgene and that integration occurred randomly in the carrot genome. in all clones a specific transcript of the expected size could be amplified by rt-pcr. under the fluorescence microscope, a fusion protein of mv-h with the green fluorescence protein (gfp) expressed in tobacco protoplasts showed a predominant association with the plasma membrane (data not shown). the membrane fraction of carrot cells produced a strong signal in western blot correspond-ing to an estimated 2 µg of specific protein per gram of wet weight. interestingly, the transgenic protein migrated faster in sds-page than the same protein produced in mammalian cells, suggesting a size difference of about 6 kda. a similar observation was made when the rabies virus glycoprotein g was expressed in plants (mcgarvey et al., 1995) . the expression of the c-terminally fused gfp demonstrates that the h protein is fully translated and that the difference in apparent size is most probably due to post-translational modifications. in the virus, the edmonston strain h protein undergoes glycosylation at 4 of the 5 predicted n-glycosylation sites (hu et al., 1994) . it has been shown that glycosylation of mammalian proteins is efficient in plants, but normally different carbohydrate side chains are utilized (bardor et al., 1999) . the complex glycans of plants are often smaller than those of animals, partially because they lack sialic acid (faye et al., 1993) . in insect cells, which also lack sialic acid, the reduced size of the recombinant h protein was explained by a difference in glycosylation (vialard et al., 1990) . authentic post-translational modifications such as glycosylation and cystine-bridge formation are thought to be important for intracellular trafficking (hu and norrby, 1995) as well as the antigenic conformation of the h protein (hu et al., 1994; hu and norrby, 1995) . recently, huang et al. (2001) reported the expression of the h protein in tobacco leaves. in this system, the native h protein was undetectable. after adding a retention signal for the endoplasmic reticulum low levels of protein became detectable by elisa with polyclonal sera from rabbits and man; the reactivity with mabs was even weaker or negative suggesting that the conformation may have been less than optimal. the immunogenic integrity of the h protein is critical for the induction of antibodies that protect against virus infection and disease. conformational dependent mabs confirmed the proper antigenic structure of the transgenic protein produced in carrots; while mab bh1 which recognizes only denatured protein showed no reactivity. after immunization with extracts of transgenic carrots high levels of virus-neutralizing antibodies were found, using the who recommended plaque neutralization assay (miller et al., 1995) . these titres were comparable to those obtained with the mammalian cell-derived h protein extract. the antibody isotype subclasses suggested that in contrast to the h protein of mammalian origin, which produced primarily a th2 (or antibody-dominated) response, the plant protein induced a th1/th2-balanced response, indicative of both a humoral and cellular response. this may be important to safeguard against atypical measles when such a vaccine is used in unprimed individuals. in many countries carrots are components of the diet of both adults and children and a stable antigenic transgene in an edible plant that can be consumed raw without further processing could bring a vaccine within reach of the most destitute. although plants can be used as efficient bioreactors for producing antigens, the full potential of plant-based vaccines becomes apparent when immunogenicity can be demonstrated after oral ingestion. a frequent problem of plant-based vaccines is that the response after oral delivery is inconsistent and variable. sometimes even low levels of antigen can give an effective response after oral administration in mice (mason et al., 1996; wigdorovitz et al., 1999) and man (kapusta et al., 1999) . in other cases mucosal immunity was improved by mucosal adjuvants (arakawa et al., 1998b; kong et al., 2001) . in the study by huang et al. 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co-immunization with measles virus human membrane cofactor protein (cd46) acts as a cellular receptor for measles virus identification of dna sequences required for activity of a plant promoter: the camv 35s promoter expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector nuclear transport of plant potyviral proteins production of hepatitis b surface antigen in transgenic plants for oral immunization oral immunization in mice with transgenic tomato fruit expressing respiratory syncytial virus-f protein induces a systemic immune response promoters for pregenomic rna of banana streak badnavirus are active for transgene expression in monocot and dicot plants immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes immunogenicity of transgenic plant-derived hepatitis b surface antigen a rapid method for transformation of carrot (daucus carota l.) by using direct somatic embryogenesis synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the β-galactosidase gene induction of a protective antibody response to foot and mouth disease virus in mice following oral or parental immunization with alfalfa transgenic plants expressing the viral structural protein vp1 measles virus: both hemagglutinin and fusion glycoproteins are requiered for fusion protection against measles virus encephalitis by monoclonal antibodies binding to a cystein loop domain of the h protein mimicked by peptides which are not recognized by maternal antibodies we are grateful to richard wagner and his team (ibmp) for taking care of the transgenic carrots. we also thank b. jérouville, s. willieme and w. ammerlaan for technical assistance. this research was supported by a grant from the ministère de l'education nationale et de la formation professionnelle du grand-duché de luxembourg, the european union 4th framework programme (project pl970242) and the crp-santé, luxembourg. key: cord-004400-li1sc47z authors: ma, jingjiao; wu, rujuan; xu, guanlong; cheng, yuqiang; wang, zhaofei; wang, heng’an; yan, yaxian; li, jinxiang; sun, jianhe title: acetylation at k108 of the ns1 protein is important for the replication and virulence of influenza virus date: 2020-02-24 journal: vet res doi: 10.1186/s13567-020-00747-3 sha: doc_id: 4400 cord_uid: li1sc47z non-structural protein 1 (ns1) of influenza virus is a multifunctional protein that plays an important role in virus replication and virulence. in this study, an acetylation modification was identified at the k108 residue of the ns1 protein of h1n1 influenza virus. to further explore the function of the k108 acetylation modification of the ns1 protein, a deacetylation-mimic mutation (k108r) and a constant acetylation-mimic mutation (k108q) were introduced into the ns1 protein in the background of a/wsn/1933 h1n1 (wsn), resulting in two mutant viruses (wsn-ns1-108r and wsn-ns1-108q). in vitro and mouse studies showed that the deacetylation-mimic mutation k108r in the ns1 protein attenuated the replication and virulence of wsn-ns1-108r, while the constant acetylation-mimic mutant virus wsn-ns1-108q showed similar replication and pathogenicity as the wild-type wsn virus (wsn-wt). the results indicated that acetylation at k108 of the ns1 protein has an important role in the replication and virulence of influenza virus. to further explore the potential mechanism, the type i interferon (ifn-i) antagonistic activity of the three ns1 proteins (ns1-108q, ns1-108r, and ns1-wt) was compared in cells, which showed that the k108r mutation significantly attenuated the ifn-β antagonistic activity of the ns1 protein compared with ns1-wt and ns1-108q. both ns1-wt and ns1-108q inhibited the ifn-β response activated by rig-i card domain, mavs, tbk1, and irf3 more efficiently than the ns1-108r protein in cells. taken together, the results indicated that acetylation at ns1 k108 is important for the ifn antagonistic activity of the ns1 protein and virulence of the influenza virus. influenza virus non-structural protein 1 (ns1) is a multifunctional protein that is responsible for interacting with cellular factors to antagonize the host antiviral response during viral infection [1] . the major role of the ns1 protein is inhibition of both interferon (ifn) and ifn-stimulated proteins by different mechanisms. ns1 inhibits the transcription of type i ifn by binding the 5′ triphosphate viral double-stranded rnas generated during viral replication to prevent the recognition of viral genomic material by host pattern recognition receptors (prrs), including rig-i, dsrna-dependent protein kinase r (pkr), and 2′5′-oligoadenylate synthetase (oas)/rnase l [2] [3] [4] . ns1 can also interact directly with rig-i in the absence of rna binding to inhibit the conformational change of rig-i required for mavs activation [5] . moreover, ns1 is able to interrupt mrna maturation by inhibiting the nuclear export of host mrnas by binding to host poly(a)-binding protein ii (pabpii) and cleavage and polyadenylation specific factor 30 (cpsf30), which are required for host mrna processing, resulting in the accumulation of ifn pre-mrnas in the nucleus of infected cells [6] . in addition, ns1 also antagonizes the open access *correspondence: lijinxiang@caas.cn; sunjhe@sjtu.edu.cn 1 shanghai key laboratory of veterinary biotechnology, key laboratory of urban agriculture (south), ministry of agriculture, school of agriculture and biology, shanghai jiao tong university, shanghai 200240, china 2 chengdu national agricultural science and technology center, sichuan, china full list of author information is available at the end of the article ifn signalling response by regulating other host factors, such as phosphoinositide 3-kinase (pi3k) activity, crklike protein (crkl), and the jak-stat signalling pathway [7] [8] [9] [10] [11] . the ns1 protein, typically 202-237 aa in length depending on the strain, contains four functional regions: an rna binding domain (rbd, 1-73 aa), linker region (lr, 74-88 aa), effector domain (ed, 89-202 aa), and c-terminal "tail" (ctt, 207-237 aa) [12] . multiple basic amino acids (e.g., 35r, 38r, 41k, and 46r) in the rbd are important for rna binding activity and suppressing the activation of pkr [12, 13] . the ed plays an important role by targeting multiple host factors, such as pkr, cpsf30, and p85β (pi3k), to inhibit antiviral responses and enhance viral replication [1] . the residue 187w in the ed domain is important for the dimerization of the ns1 protein, and the w187r substitution impaired ns1 dimerization and attenuated the virus in vivo [14] . in addition, residues 186e, 189d, and 194v play important roles in the binding of ns1 to cleavage and polyadenylation specificity factor 30 (cpsf30), and mutations in those residues weaken the binding of ns1 to cpsf30 and impair the ability of the ns1 protein to shut off host gene expression [15, 16] . post-translational modifications, such as phosphorylation, sumoylation, and acetylation, are important for protein function. phosphorylation at 49t, 80t, and 215t of the ns1 protein are important for interferon antagonistic activity and replication of human influenza virus [17, 18] . sumoylation at positions 219 and 221 of ns1 are crucial for host protein expression shutoff and replication of h5n1 influenza virus [19] . acetylation is an important post-translational modification that occurs in two forms [20] . one is the co/post-translational acetylation at the n α -termini of the nascent polypeptide chains [21] . the other form is acetylation of the ε-amino group of lysine, which was first recognized in histones regulating gene translation [22] and later was found in non-histone proteins [23] . the acetylation status is reversible and well balanced by lysine (k) acetyltransferases (kats) and lysine deacetylases (kdacs), which are tightly regulated to perform many cellular functions [24] . dysfunction of the acetylation machinery can inhibit protein functions and consequently lead to severe diseases [25, 26] . acetylation has been found in multiple proteins of influenza viruses. acetylation was identified in the np protein of influenza virus, and deacetylation of the np protein prevented the virus from assembling functional virus particles [27] . a histone-like sequence (histone mimic) was identified in the ns1 protein of influenza a h3n2, which contributes to suppression of the antiviral response [28] . the n-terminal acetylation of pa-x is required for the host shutoff activity of pa-x and for viral polymerase activity [29, 30] . acetylation of pa has been reported to be crucial for polymerase activity, and deacetylated pa protein restricts iav rna transcription and replication. the influenza virus haemagglutinin (ha) has three conserved cysteine residues (551, 559, and 562) at its c terminus serving as acylation sites that are essential for the formation of fusion pores and infectivity [31] . in the present study, an acetylation modification at k108 of the ns1 protein was identified and characterized. the results showed that the deacetylation-mimic mutation k108r in the ns1 protein attenuated the replication and virulence of the virus in vitro and in vivo. ifn-β antagonist assays indicated that the k108r mutation attenuated ifn antagonistic ability compared with the ns1-wt or ns1-108q (constant acetylation-mimic) proteins. overall, this study indicated that acetylation at k108 of the ns1 protein plays an important role in the replication and virulence of influenza virus. madin-darby canine kidney (mdck) cells were maintained in eagle's minimal essential medium (emem, hyclone, grand island, usa) with 5% foetal bovine serum (fbs, gibco, grand island, usa), l-glutamine (gibco), and 1% antibiotic (gibco). human embryonic kidney (hek) 293t cells and adenocarcinomic human alveolar basal epithelial cells (a549 cells) were maintained in dulbecco's modified eagle's medium (dmem, hyclone) supplemented with 10% fbs (gibco), l-glutamine (gibco), and 1% antibiotic (gibco). the virus strain a/wsn/1933 h1n1 (wsn), a mouse-adapted human influenza virus, was propagated and titrated in mdck cells. to identify the putative acetylation sites in influenza viral proteins, mass spectrometry was conducted with concentrated influenza virus. briefly, the h1n1 virus was propagated in mdck cells, and then a total of 100 ml of virus stock was prepared. to concentrate the virus, the collected virus was pelleted by centrifugation at 2000 g for 10 min to remove the cell debris. clarified virus supernatants were layered on a 30% (w/v) sucrose cushion and centrifuged at 200 000 g for 3 h. the virus pellet was suspended in water and subjected to mass spectrometry analysis performed by ptm biolabs llc (hangzhou, china). to generate mutant viruses, the site mutations k108r (deacetylation-mimic mutation) and k108q (constant acetylation-mimic) were introduced into the reverse genetic plasmid phw2000-wsn-ns by a commercial site-directed mutagenesis kit (invitrogen, grand island, usa). the mutant viruses were rescued in the background of wsn-h1n1 virus as described previously [32] , resulting in wsn-ns1-108r and wsn-ns1-108q viruses, and all the mutant viruses were verified by sequencing. then, the ns1-wt, ns1-108r, and ns1-108q genes were amplified and cloned into the pcdna3.0-flag expression vector (flag-ns1-wt, flag-ns1-108r, and flag-ns1-108q). the three viruses were inoculated on monolayer mdck and a549 cells cultured in 12-well plates with multiplicities of infection (mois) of 0.001 and 0.01 for each virus, respectively. each time point was set up in triplicate, and then the samples were collected at 12, 24, 36, and 48 hours post-inoculation (hpi). the supernatants were titrated on mdck cells cultured in 96-well plates following the reed and muench method to calculate tcid 50 /ml. the cells were collected and subjected to western blotting. briefly, the cell lysates were separated on a sodium dodecyl sulfate (sds)-polyacrylamide gel and transferred to a pvdf membrane. the membrane was blocked in pbs containing 5% skim milk and then incubated with rabbit anti-ns1 polyclonal antibody (genscript, piscataway, usa) and then with horseradish peroxidase-conjugated secondary anti-mouse antibody (thermo fisher, grand island, usa). the proteins were visualized by using an ecl kit (yeasen, shanghai, china). additionally, to determine the protein expression levels of the three ns1 expression plasmids, the flag-ns1-wt, flag-ns1-108q, and flag-ns1-108r plasmids were transfected into 293t cells. forty-eight hours post-transfection, the cells were collected and subjected to western blotting. forty-eight 4-week-old female balb/c mice were randomly allocated into four groups, and each group contained 12 mice. three groups were challenged with the indicated viruses, and one group was challenged with pbs as a control. the mice were inoculated with virus intranasally with 10 5.5 tcid 50 of virus in 50 µl solution under slight anaesthesia with co 2 . the mice were monitored for body weight, clinical signs, and survival rate each day until 14 days post-infection (dpi), and they were euthanized if they lost more than 25% of their original body weight. three mice from each group were euthanized at 3 and 5 dpi. the mouse lungs were collected for viral titration and cytokine analysis. to quantify the cytokine levels of il-1β, ifn-β, and tnf-α in mouse lungs, total rna was extracted from lung tissues, reverse-transcribed and subjected to quantitative realtime polymerase chain reaction as described previously [33] . to detect the ifn-β antagonistic ability of ns1 proteins, 293t cells were transfected with the indicated ns1 expression plasmids (0.2 μg/well) together with a plasmid expressing firefly luciferase under the control of the ifn-β promoter (pgl-ifn-β-luc, 0.2 μg/well), the renilla luciferase expressing plasmid prl-tk (0.07 μg/ well), and the ifn-β stimulator poly(i:c) (0.2 μg/well) or a plasmid expressing the active caspase recruitment domain (card) of rig-i (pcdna-rig-i 0.2 μg/well), pcdna-mavs (0.2 μg/well), pcdna-tbk1 (0.2 μg/well) or pcdna-irf3 (0.2 μg/well) as described previously [34] . twenty-four hours post-transfection, the cells were lysed and subjected to a dual-luciferase reporter assay kit (promega, madison, usa). mdck cells cultured on glass slides were infected with wsn-ns1-k108r, wsn-ns1-k108q, or wsn-wt at an moi of 1. all cells were fixed with 4% paraformaldehyde (pfa) and permeabilized with 0.1% triton x-100 in pbs at 12, 24, and 36 hpi. to detect the ns1 proteins, the fixed cells were incubated with rabbit anti-ns1 polyclonal antibody (genscript), followed by fitc-conjugated antirabbit igg antibody (yeasen), and then the cells were stained with dapi. all images were obtained on a leica tcs sp2 confocal microscope (leica microsystems inc., buffalo grove, usa). the animal study was conducted in accordance with the guidelines of the animal care and use committee of shanghai jiao tong university, and the animal study protocols were approved by shanghai jiao tong university (approval no. 20181012). all data were analysed using analysis of variance (two-way anova) in graph-pad prism version 5.0 (graphpad software inc., la jolla, usa); a p-value of 0.05 or less was considered significant. the mass spectrometry results showed that one acetylation modification was identified at position k108 of the ns1 protein of the wsn-wt virus (figure 1 ). to further explore whether k108 is subtype-specific, we compared the ns1 amino acid sequences of 1000 randomly selected influenza virus strains of each endemic subtype in birds and humans from genbank. most avian h9n2 (100%), h5n1 (99.9%), h7n9 (100%), human h3n2 (99.4%), and human h1n1 (99.9%, isolated before 2009) viruses contained k108 in the ns1 genes, whereas 99.9% of 2009 pandemic h1n1 viruses contained 108r in the ns1 protein. these data demonstrated that the k108 residue is relatively conserved in influenza viruses except the 2009 pandemic h1n1. since the ns1 protein is an important virulence marker and antagonist of host innate immunity, we chose to further explore the influence of acetylated k108 on virus replication and virulence in this study. to mimic deacetylated lysine, a k108r substitution was introduced into the ns1 protein, since an r substitution prevents acetylation but preserves the positive charge, and a mutant virus containing the ns1-k108r substitution was generated in the background of wsn virus (wsn-ns1-108r). moreover, to mimic constantly acetylated lysine at k108 of the ns1 protein, a k108q substitution that is a known acetylation mimic was introduced into ns1, resulting in a mutant virus containing ns1-k108q (wsn-ns1-108q). to determine the effect of acetylated k108 on virus replication, mdck and a549 cells were infected with wsn-wt or the two mutant virus wsn-ns1-108r (deacetylation mimic) or wsn-ns1-108q (constant acetylation mimic) at the indicated mois to obtain multicycle growth curves. all three viruses replicated efficiently in mdck and a549 cells, and wsn-ns1-108q and wsn-wt replicated to similar levels at each time point. however, the growth of the deacetylated mutant virus wsn-ns1-108r was significantly impaired compared with that of the other two viruses in mdck and a549 cells at 36 and 48 hpi, which indicated that the acetylated k108 of ns1 is important for virus replication in vitro at the late stage of infection (figures 2a and b) . similarly, ns1 protein levels were detected by western blotting. the results showed that the ns1 expression levels of wsn-ns1-108r were lower than those of wsn-wt and wsn-ns1-108q at different time points in mdck and a549 cells (figures 2c and d) . all the mice infected with viruses showed clinical signs such as ruffled fur, depression, and inappetence. the mice infected with constantly acetylated wsn-ns1-108q displayed more severe clinical signs and started to show mortality earlier than wsn-wt-infected mice. both wsn-ns1-108q and wsn-wt caused 100% mortality in infected mice. however, the deacetylated wsn-ns1-108r virus infection resulted in 80% mortality, and the mortality was delayed by 2 days compared with other viruses, which indicated that the deacetylation-mimic k108r substitution attenuated the wsn-ns1-108r virus in mice ( figures 3a and b) . virus titers were slightly lower in the lungs of mice infected with wsn-ns1-108r than in the other two groups ( figure 3c) . notably, significantly higher levels of ifn-β, il-1β, and tnf-α mrna were detected in wsn-ns1-108r-infected mice than in the other two groups at 3 dpi but not at 5 dpi ( figures 3d-f) , which indicated that wsn-ns1-108r was less efficient at inhibiting the innate immune response than wsn-ns1-108q and wsn-wt at 3 dpi in mice. to determine whether the k108r and k108q mutations affect the expression of the ns1 protein, the expression levels of flag-ns1-wt, flag-ns1-108q, and flag-ns1-108r in 293t cells were compared. the three proteins were expressed at similar levels in transfected 293t cells, which indicated that the k108r and k108q mutations did not influence protein expression ( figure 4e) . the major function of the ns1 protein is inhibition of type i ifn induction, and the acetylated k108 residue is located in the effector domain of ns1, which is important for its ifn antagonistic ability. to determine the effect of the acetylated k108 residue on ifn suppression by the ns1 protein, the inhibition of ifn-β promoter activity by flag-ns1-wt, flag-ns1-108q, and flag-ns1-108r was evaluated. the results showed that flag-ns1-wt and acetylation-mimic flag-ns1-108q suppressed the ifn-β promoter activity stimulated by poly(i:c) ( figure 4a) ; however, the deacetylation-mimic flag-ns1-108r protein was significantly less capable of inhibiting the activation of the ifn-β promoter compared with the acetylated ns1 proteins ( figure 4a ). this result indicated that the impaired ifn-β antagonistic ability might be responsible for the attenuation of the wsn-ns1-108q virus in vitro and in vivo. influenza virus infection stimulates type i ifn production by signal transduction from rig-i to tbk1 to irf3. to further explore how the k108r mutation attenuated the ifn-β antagonistic ability of the ns1 protein, we co-transfected ns1 expression plasmids, an ifn-β reporter plasmid and different type i interferon pathway components, including rig-i card, tbk1, and the active form of irf3, into 293t cells. the results showed that ns1-108q and ns1-wt inhibited the ifn-β response stimulated by each component more efficiently than ns1-108r, which suggested that the acetylated k108 residue is important for inhibiting the ifn-β response of ns1 that targets factors downstream of irf3 or other proteins (figures 4b-d) . two nuclear localization signals (35-41 and 216-221) have been identified in the ns1 protein, which drive ns1 to the nucleus during the early stage of infection. to determine whether k108r changes the subcellular localization of ns1 during infection, mdck cells were infected with the three viruses. the ns1 protein of wsnwt was located in the nucleus and cytoplasm of infected cells at 12 and 24 hpi ( figures 5a and b) . at the late stage of infection (36 hpi), the ns1 protein was mainly located in the nucleus and perinuclear area of infected cells (figure 5c ). the ns1-108q protein was located in both the nucleus and cytoplasm at 12 hpi and 24 hpi, while it was mainly located in the perinuclear area of infected cells at 36 hpi. in contrast, the ns1-108r protein accumulated mostly in the cytoplasm during the whole infection course. this result indicated that the deacetylation-mimic k108r substitution retained ns1 protein in the cytoplasm of infected cells, suggesting that the acetylated k108 residue is important for the nuclear localization of the ns1 protein ( figures 5a-c ). post-translational modification is important for protein function, stability, cellular localization, and protein-protein interactions. recent studies have shown that posttranslational modifications of viral proteins modulate the virus life cycle, e.g., phosphorylation of influenza viral proteins (ns1, m1, and np) plays important roles in virus replication [35] [36] [37] [38] . the ubiquitination of np and m2 proteins is crucial for viral rna replication and the production of infectious virus particles [37] . acetylation is an important post-translational modification in eukaryotes, but the occurrence and function of acetylation in influenza viral proteins remain largely unclear. giese et al. reported that acetylation of 77 k, 113 k, and 229 k of np proteins is important for virus polymerase activity and replication [27] . in the present study, we identified and characterized the acetylation of k108 in the ns1 protein, and the deacetylation of k108q affected viral replication in cells at 36 and 48 hpi. the expression levels of ns1-k108q and ns1-k108r in transfected 293t cells were similar ( figure 4e ), while the ns1 levels in infected mdck and a549 cells were different ( figures 2c and d) . this result could be attributed to the deacetylation affecting virus replication, which resulted in low expression of ns1 in infected cells. moreover, the acetylation of 108q contributes to the ifn antagonistic ability of the ns1 protein. the mrna levels of ifn-β, il-1β, and tnf-α in mouse lungs of the wsn-ns1-k108r-infected group were significantly higher than those in the other two groups at 3 dpi, which indicated that ns1-108r was less efficient at inhibiting the production of innate antiviral cytokines at 3 dpi in mice. the ns1 protein of influenza virus is a virulence factor that inhibits the antiviral immunity of the infected host, and c-terminal truncation has been widely used as a strategy to generate attenuated virus vaccine candidates [39] . one mechanism used by ns1 to inhibit the ifn response is through direct binding and sequestration of rna as well as direct interaction with trim25 and complex formation with the rna sensor rig-i, resulting in inhibition of the activation of the rig-i card and hence inhibition of irf3 activation [12] . the rna binding, rig-i and trim25 interacting domains are located in the n-terminus (1-73 aa) of the ns1 protein. however, the acetylated k108 is located in the ed domain, and acetylation of k108 may not affect the rna binding capability of the ns1 protein. the ns1-k108r substitution impaired the suppression of ifn promoter activation by poly(i:c), rig-1 card, tbk-1, and irf3, which suggested that the ns1-k108r substitution affected the ifn antagonism of ns1 either through targeting downstream of irf3 or a general mechanism that ns1 uses to inhibit ifn, such as interaction with cpsf30, resulting in inhibition of the processing of mrna, including ifn mrna [6] . notably, the cpsf30 protein is mainly located in the nucleus and is required for the 3′ end processing of all host pre-mrnas. interestingly, the deacetylation-mimic k108r substitution retained ns1 protein in the cytoplasm of infected cells, resulting in a possible impaired interaction between cpsf30 and the ns1 protein, subsequently leading to attenuated ifn antagonism. benjamin hale and colleagues found that the a/california/04/2009 (h1n1) virus has 108r in ns1, and ns1 was unable to suppress general host gene expression. nevertheless, the rk108 substitution in the ns1/2009 protein restored its ability to block general gene expression and bind cpsf30 [40] . this could explain why attenuation of the ifn antagonistic ability of ns1-k108r is independent of rig-i card, tbk-1, and irf3 activation. in addition, anastasina et al. [41] reported that the ns1 protein binds to cellular dna to block the cellular transcription of ifns and isgs; thus, ns1 proteins retained in the cytoplasm lose their cellular dna binding function, resulting in impaired ifn-β antagonistic ability. two known nuclear localization sequences (nlss) of ns1 proteins are located at the 35-41 and 219-232 positions. the 35-41 nls of the ns1 protein is highly conserved among influenza a virus strains [42] . the second nls (219-232) is virus strain specific, and the 2009 pandemic h1n1 lacks the second nls in the ns1 protein [43] . single point mutations, either r35a, r38a, or k41a, completely eliminated importin protein binding, which transports target proteins to the nucleus [42] . notably, in this study, acetylation of k108 located outside of the nls affected the cellular localization of ns1 protein, and the deacetylation-mimic k108r substitution blocked the nuclear localization of the ns1 protein in infected cells; however, the underlying mechanism remains unknown. interestingly, the ns1-k108 residue is relatively conserved in most influenza viruses, except for the 2009 pandemic h1n1. the 2009 pandemic h1n1 has 108r in ns1, which causes inefficient general host gene expression shutoff, while r108k restores its ability to block general host genes and bind cpsf30 [40] . potentially, the 2009 pandemic h1n1 virus might use different strategies to overcome the ifn response compared with the other influenza viruses. overall, we identified an acetylation of k108 of the ns1 protein of influenza virus, and the acetylation of k108 plays an important role in the cellular localization, ifn antagonistic ability, replication, and virulence of influenza virus. conformational plasticity of the influenza a virus ns1 protein inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns1 protein of influenza a virus rig-i-mediated antiviral responses to single-stranded rna bearing 5′-phosphates 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nuclear and nucleolar targeting of influenza a virus ns1 protein: striking differences between different virus subtypes influenza a h3n2 subtype virus ns1 protein targets into the nucleus and binds primarily via its c-terminal nls2/nols to nucleolin and fibrillarin this study was supported by the national natural science authors' contributions jm, jl, and js designed the study; jm was involved in the acquisition of data, analysis, and figure preparation; rw, gx, yc, and zw contributed to some of the laboratory experiments and data analysis; hw and yy helped revise the manuscript; jl and js supervised the study; jm drafted the original paper. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-103320-2rpr7aph authors: bhandari, bikash k.; gardner, paul p.; lim, chun shen title: solubility-weighted index: fast and accurate prediction of protein solubility date: 2020-03-26 journal: biorxiv doi: 10.1101/2020.02.15.951012 sha: doc_id: 103320 cord_uid: 2rpr7aph motivation recombinant protein production is a widely used technique in the biotechnology and biomedical industries, yet only a quarter of target proteins are soluble and can therefore be purified. results we have discovered that global structural flexibility, which can be modeled by normalised b-factors, accurately predicts the solubility of 12,216 recombinant proteins expressed in escherichia coli. we have optimised b-factors, and derived a new set of values for solubility scoring that further improves prediction accuracy. we call this new predictor the ‘solubility-weighted index’ (swi). importantly, swi outperforms many existing protein solubility prediction tools. furthermore, we have developed ‘sodope’ (soluble domain for protein expression), a web interface that allows users to choose a protein region of interest for predicting and maximising both protein expression and solubility. availability the sodope web server and source code are freely available at https://tisigner.com/sodope and https://github.com/gardner-binflab/tisigner-reactjs, respectively. the code and data for reproducing our analysis can be found at https://github.com/gardner-binflab/sodope_paper2020. high levels of protein expression and solubility are two major requirements of successful recombinant protein production (esposito and chatterjee 2006) . however, recombinant protein production is a challenging process. almost half of recombinant proteins fail to be expressed and half of the successfully expressed proteins are insoluble ( http://targetdb.rcsb.org/metrics/ ). these failures hamper protein research, with particular implications for structural, functional and pharmaceutical studies that require soluble and concentrated protein solutions (kramer et al. 2012; hou et al. 2018) . therefore, solubility prediction and protein engineering for enhanced solubility is an active area of research. notable protein engineering approaches include mutagenesis, truncation (i.e., expression of partial protein sequences), or fusion with a solubility-enhancing tag (waldo 2003; esposito and chatterjee 2006; trevino, martin scholtz, and nick pace 2007; chan et al. 2010; kramer et al. 2012; costa et al. 2014 ) . protein solubility, at least in part, depends upon extrinsic factors such as ionic strength, temperature and ph, as well as intrinsic factors-the physicochemical properties of the protein sequence and structure, including molecular weight, amino acid composition, hydrophobicity, aromaticity, isoelectric point, structural propensities and the polarity of surface residues (wilkinson and harrison 1991; chiti et al. 2003; tartaglia et al. 2004; diaz et al. 2010) . many solubility prediction tools have been developed around these features using statistical models (e.g., linear and logistic regression) or other machine learning models (e.g., support vector machines and neural networks) (hirose and noguchi 2013; habibi et al. 2014; hebditch et al. 2017; sormanni et al. 2017; heckmann et al. 2018; z. wu et al. 2019; yang, wu, and arnold 2019) . in this study, we investigated the experimental outcomes of 12,216 recombinant proteins expressed in escherichia coli from the 'protein structure initiative:biology' (psi:biology) (chen et al. 2004; acton et al. 2005) . we showed that protein structural flexibility is more accurate than other protein sequence properties in predicting solubility (craveur et al. 2015; m. vihinen, torkkila, and riikonen 1994) . flexibility is a standard feature that appears to have been overlooked in previous solubility prediction attempts. on this basis, we derived a set of 20 values for the standard amino acid residues and used them to predict solubility. we call this new predictor the 'solubility-weighted index' (swi). swi is a powerful predictor of solubility, and a good proxy for global structural flexibility. in addition, swi outperforms many existing de novo protein solubility prediction tools. we sought to understand what makes a protein soluble, and develop a fast and accurate approach for solubility prediction. to determine which protein sequence properties accurately predict protein solubility, we analysed 12,216 target proteins from over 196 species that were expressed in e. coli (the psi:biology dataset; see supplementary fig s1 and table s1a ) (chen et al. 2004; acton et al. 2005) . these proteins were expressed either with a c-terminal or n-terminal polyhistidine fusion tag (pet21_nesg and pet15_nesg expression vectors, n=8,780 and 3,436, respectively). they were previously curated and labeled as 'protein_soluble' or 'tested_not_soluble' (seiler et al. 2014) , based on the soluble analysis of cell lysate using sds-page (r. xiao et al. 2010) . a total of 8,238 recombinant proteins were found to be soluble, in which 6,432 of them belong to the pet21_nesg dataset. both the expression system and solubility analysis method are commonly used (costa et al. 2014) . therefore, this collection of data captures a broad range of protein solubility issues. protein structural flexibility, in particular, the flexibility of local regions, is often associated with function (craveur et al. 2015) . the calculation of flexibility is usually performed by assigning a set of 20 normalised b-factors-a measure of vibration of c-alpha atoms (see supplementary notes)-to a protein sequence and averaging the values by a sliding window approach (ragone et al. 1989; karplus and schulz 1985; m. vihinen, torkkila, and riikonen 1994; smith et al. 2003) . we reasoned that such sliding window approach can be approximated by a more straightforward arithmetic mean for calculating global structural flexibility (see supplementary notes). we determined the correlation between flexibility (vihinen et al. 's sliding window approach as implemented in biopython) and solubility scores calculated as follows: where is the normalised b-factor of the amino acid residue at the position , and is the b i i l sequence length. we obtained a strong correlation for the psi:biology dataset (spearman's rho = 0.98, p-value below machine's underflow level). therefore, we reasoned that the sliding window approach is not necessary for our purpose. we applied this arithmetic mean approach (i.e., sequence composition scoring) to the psi:biology dataset and compared four sets of previously published, normalised b-factors (bhaskaran and ponnuswamy 1988; ragone et al. 1989; m. vihinen, torkkila, and riikonen 1994; smith et al. 2003 ) among these sets of b-factors, sequence composition scoring using the most recently published set of normalised b-factors produced the highest auc score ( to improve the prediction accuracy of solubility, we iteratively refined the weights of amino acid residues using the nelder-mead optimisation algorithm (nelder and mead 1965) . to avoid testing and training on similar sequences, we generated 10 cross-validation sets with a maximised heterogeneity between these subsets (i.e. no similar sequences between subsets). we first clustered all 12,216 psi:biology protein sequences using a 40% similarity threshold using usearch to produce 5,050 clusters with remote similarity (see methods and supplementary fig s4) . the clusters were grouped into 10 cross-validation sets of approximately 1,200 sequences each manually. we did not select a representative sequence for each cluster as about 12% of clusters contain a mix of soluble and insoluble proteins (supplementary fig s4c) . more importantly, to address the issues of sequence similarity and imbalanced classes, we performed 1,000 bootstrap resamplings for each cross-validation step (fig 2a and supplementary fig s5) . we calculated the solubility scores using the optimised weights as equation 1 and the auc scores for each cross-validation step. our training and test auc scores were 0.72 ± 0.00 and 0.71 ± 0.01, respectively, showing an improvement over flexibility in solubility prediction (mean ± standard deviation; fig 2b and supplementary table s3 ). the final weights were derived from the arithmetic means of the weights for individual amino acid residues obtained cross-validation (supplementary table s4) . we observed over a 20% change on the weights for cysteine (c) and histidine (h) residues (fig 2c and supplementary table s4 ). these results are in agreement with the contributions of cysteine and histidine residues as shown in supplementary fig s2b. we call the solubility score of a protein sequence calculated using the final weights the solubility-weighted index (swi). flow chart shows an iterative refinement of the most recently published set of normalised b-factors for solubility prediction (smith et al. 2003) . the solubility score of a protein sequence was calculated using a sequence composition scoring approach (equation 1, using optimised weights , w instead of normalised b-factors ). these scores were used to compute the auc scores for b training and test datasets. (b) training and test performance of solubility prediction using optimised weights for 20 amino acid residues in a 10-fold cross-validation (mean auc ± standard deviation). related data and figures are available as supplementary table s3 and supplementary fig s4 and s5 . (c) comparison between the 20 initial and final weights for amino acid residues. the final weights are derived from the arithmetic mean of the optimised weights from cross-validation. these weights are used to calculate swi, the solubility score of a protein sequence, in the subsequent analyses. filled circles, which represent amino acid residues, are colored by hydrophobicity (kyte and doolittle 1982) . solid black circles denote aromatic amino acid residues phenylalanine (f), tyrosine (y), tryptophan (w). dotted diagonal line represents no change in weight. see also supplementary table s4 and fig s4. auc, area under the roc curve; roc, receiver operating characteristic; , arithmetic w mean of the weights of an amino acid residue optimised from 1,000 bootstrap samples in a cross-validation step. to validate the cross-validation results, we used a dataset independent of the psi:biology data known as esol (niwa et al. 2009 ) . this dataset consists of the solubility percentages of e. coli proteins determined using an e. coli cell-free system (n = 3,198) . our solubility scoring using the final weights showed a significant improved correlation with e. coli protein solubility over the initial weights (smith et al. 's normalised b-factors) [spearman's rho of 0.50 (p = 9.46 ✕ 10 -206 ) versus 0.40 (p = 4.57 ✕ 10 -120 )]. we repeated the correlation analysis by removing extra amino acid residues including his-tags from the esol sequences (mrgshhhhhhtdpalra and glcgr at the n-and c-termini, respectively). this artificial dataset was created based on the assumption that his-tags have little effect on solubility. we observed a slight decrease in correlation for this artificial dataset (spearman's rho = 0.47, p= 3.67 ✕ 10-176), which may be due to the effects of his-tag in solubility and/or the limitation(s) of our approach that may overfit to his-tag fusion proteins. we performed spearman's correlation analysis for both the psi:biology and esol datasets. swi shows the strongest correlation with solubility compared to the standard and 9,920 protein sequence properties (fig 3 and supplementary fig s2, respectively) . swi also strongly correlates with flexibility, suggesting that swi is also a good proxy for global structural flexibility. we asked whether protein solubility can be predicted by surface amino acid residues. to address this question, we examined a previously published dataset for the protein surface 'stickiness' of 397 e. coli proteins (levy, de, and teichmann 2012) . this dataset has the annotation for surface residues based on previously solved protein crystal structures. we observed little correlation between the protein surface 'stickiness' and the solubility data from esol (spearman's rho = 0.05, p = 0.34, n = 348; supplementary fig s6a) . next, we evaluated if amino acid composition scoring using surface residues is sufficient, optimising only the weights of surface residues should achieve similar or better results than swi. as above, we iteratively refined the weights of surface residues using the nelder-mead optimisation algorithm. the method was initialised with smith et al. 's normalised b-factors and a maximised correlation coefficient was the target. however, a low correlation was obtained upon convergence (spearman's rho = 0.18, p = 7.20 ✕ 10 -4 ; supplementary fig s6b) . in contrast, the swi of the full-length sequences has a much stronger correlation with solubility (spearman's rho = 0.46, p = 2.97 ✕ 10 -19 ; supplementary fig s6c) . these results suggest that the full-length of sequences contributes to protein solubility, not just surface residues, in which solubility is modulated by cotranslational folding (natan et al. 2018 ) . to understand the properties of soluble and insoluble proteins, we determined the enrichment of amino acid residues in the psi:biology targets relative to the esol sequences (see methods). we observed that the psi:biology targets are enriched in charged residues lysine (k), glutamate (e) and aspartate (d), and depleted in aromatic residues tryptophan (w), albeit to a lesser extend for insoluble proteins (supplementary fig s7a) . as expected, cysteine residues (c) are enriched in the psi:biology insoluble proteins, supporting previous findings that cysteine residues contribute to poor solubility in the e. coli expression system (diaz et al. 2010; wilkinson and harrison 1991) . in addition, we compared the swi of random sequences with the psi:biology and esol sequences. we included an analysis of random sequences to confirm whether swi can distinguish between biological and random sequences. we found that the swi scores of soluble proteins are higher than those of insoluble proteins (supplementary fig s7b) , and that true biological sequences also tend to have higher swi scores than random sequences, highlighting a potential evolutionary selection for solubility. to confirm the usefulness of swi in solubility prediction, we compared it with the existing tools protein-sol (hebditch et al. 2017 ) , camsol v2.1 (sormanni, aprile, and vendruscolo 2015; sormanni et al. 2017) , parsnip ) , deepsol v0.3 (khurana et al. 2018) , the wilkinson-harrison model (davis et al. 1999; harrison 2000; wilkinson and harrison 1991) , and ccsol omics (agostini et al. 2014 ) . we did not include the specialised tools that model protein structural information such as surface geometry, surface charges and solvent accessibility because these tools require prior knowledge of protein tertiary structure. for example, aggrescan3d and solart accept only pdb files that can be downloaded from the protein data bank or produced using a homology modeling program (kuriata et al. 2019; hou et al. 2019) . swi outperforms other tools except for protein-sol in predicting e. coli protein solubility (table 1, fig 4a) . our swi c program is also the fastest solubility prediction algorithm (table 1, fig 4b and supplementary table s7 ). prediction accuracy of solubility prediction tools using the above cross-validation sets (fig 2a) . for swi, the test auc scores were calculated from a 10-fold cross-validation (i.e., a boxplot representation of fig 2b) protein structural flexibility has been associated with conformal variations, functions, thermal stability, ligand binding and disordered regions (mauno vihinen 1987; teague 2003; ma 2005; radivojac 2004; schlessinger and rost 2005; yuan, bailey, and teasdale 2005; yin, li, and li 2011) . however, the use of flexibility in solubility prediction has been overlooked although their relationship has previously been noted (tsumoto et al. 2003) . in this study, we have shown that flexibility strongly correlates with solubility (fig 3) . based on the normalised b-factors used to compute flexibility, we have derived a new position and length independent weights to score the solubility of a given protein sequence (i.e., sequence composition based score). we call this protein solubility score as swi. upon further inspection, we observe some interesting properties in swi. swi anti-correlates with helix propensity, gravy, aromaticity and isoelectric point (fig 2c and 3) , suggesting that swi incorporates the key propensities affecting solubility. amino acid residues with a lower aromaticity or hydrophilic are known to improve protein solubility (trevino, martin scholtz, and nick pace 2007; niwa et al. 2009; kramer et al. 2012; warwicker, charonis, and curtis 2014; han et al. 2019; wilkinson and harrison 1991) . consistent with previous studies, the charged residues aspartate (d), glutamate (e) and lysine (k) are associated with high solubility, whereas the aromatic residues phenylalanine (f), tryptophan (w) and tyrosine (y) are associated with low solubility (fig 2c and supplementary fig s7a) . cysteine residue (c) has the lowest weight probably because disulfide bonds couldn't be properly formed in the e. coli expression hosts (stewart, aslund, and beckwith 1998; rosano and ceccarelli 2014; jia and jeon 2016; aslund and beckwith 1999) . the weights are likely different if the solubility analysis was done using the reductase-deficient, e. coli origami host strains, or eukaryotic hosts. higher helix propensity has been reported to increase solubility (idicula-thomas and balaji 2005; huang et al. 2012 ) . however, our analysis has shown that helical and turn propensities anti-correlate with solubility, whereas sheet propensity lacks correlation with solubility, suggesting that disordered regions may tend to be more soluble (fig 3) . in accordance with these, swi has stronger negative correlations with helix and turn propensities. these findings also suggest that protein solubility can be largely explained by overall amino acid composition, not just the surface amino acid residues. this idea aligns with our understanding that protein solubility and folding are closely linked, and folding occurs cotranscriptionally, a complex process that is driven various intrinsic and extrinsic factors (wilkinson and harrison 1991; chiti et al. 2003; tartaglia et al. 2004; diaz et al. 2010) . however, it is unclear why sheet propensity has little contribution to solubility because β-sheets have been shown to link closely with protein aggregation (idicula-thomas and balaji 2005) . we conclude that swi is a well-balanced index that is derived from a simple sequence composition scoring method. to demonstrate the usefulness of swi, we developed a web server called sodope (soluble domain for protein expression; https://tisigner.com/sodope ). sodope calculates the probability of solubility of a user-selected region based on swi, which can either be a full-length or a partial sequence (see methods and supplementary table s8 ). this implementation is based on our observation that some protein domains tend 345 to be more soluble than the others. to demonstrate this point, we have analysed three commercial monoclonal antibodies and the severe acute respiratory syndrome coronavirus proteomes (sars-cov and sars-cov-2) (wang et al. 2009; marra et al. 2003; f. wu et al. 2020 ) ( supplementary fig s8 and s9 ). these soluble domains may enhance protein solubility as a whole. sodope also provides options for solubility prediction at the presence of solubility fusion tags. similarly, solubility tags may act as soluble 'protein domains' that can outweigh the aggregation propensity of insoluble proteins. however, some soluble fusion proteins may become insoluble after proteolytic cleavage of solubility tags (lebendiker and danieli 2014) . in addition, sodope is integrated with tisigner, a gene optimisation web service for protein expression. this pipeline provides a holistic approach to improve the outcome of recombinant protein expression. the standard protein sequence properties were calculated using the bio.sequtils.protparam module of biopython v1.73 (cock et al. 2009 ) . all miscellaneous protein sequence properties were computed using the r package protr v1.6-2 (n. xiao et al. 2015) . we used the standard and miscellaneous protein sequence properties to predict the solubility of the psi:biology and esol targets (n=12,216 and 3,198 , respectively) (seiler et al. 2014; niwa et al. 2009 ) . for method comparison, we chose the protein solubility prediction tools that are scalable (table 1) . default configurations were used for running the command line tools. to benchmark the wall time of solubility prediction tools, we selected 10 sequences that span a large range of lengths from the psi:biology and esol datasets (from 36 to 2389 residues). all the tools were run and timed using a single process without using gpus on a high performance computer [ /usr/bin/time -f '%e' <command> ; centos linux 7 (core) operating system, 72 cores in 2× broadwell nodes (e5-2695v4, 2.1 ghz, dual socket 18 cores per socket), 528 gib memory]. single sequence fasta files were used as input files. to improve protein solubility prediction, we optimised the most recently published set of normalised b-factors using the psi:biology dataset (smith et al. 2003 ) (fig 2) . to avoid including homologous sequences in the test and training sets, we clustered the psi:biology targets using usearch v11.0.667, 32-bit (edgar 2010) . his-tag sequences were removed from all sequences before clustering to avoid false cluster inclusions. we obtained 5,050 clusters using the parameters: -cluster_fast <input_file> -id 0.4 -msaout <output_file> -threads 4 . these clusters were divided into 10 subsets with approximately 1,200 sequences per subsets manually . the subsequent steps were done with his-tag sequences. we used smith et al. 's normalised b-factors as the initial weights to 391 maximise auc using these 10 subsets with a 10-fold cross-validation. since auc is non-differentiable, we used the nelder-mead optimisation method (implemented in scipy v1.2.0), which is a derivative-free, heuristic, simplex-based optimisation (oliphant 2007; millman and aivazis 2011; nelder and mead 1965) . for each step in cross-validation, we used 1,000 bootstrap resamplings containing 1,000 soluble and 1,000 insoluble proteins. optimisation was carried out for each sample, giving 1,000 sets of weights. the arithmetic mean of these weights was used to determine the training and test auc for the cross-validation step (fig 2a) . to examine the enrichment of amino acid residues in soluble and insoluble proteins, we compute the bit scores for each amino acid residue in the psi:biology soluble and insoluble groups ( supplementary fig s7a) , we normalised the count of each residue in each x) ( group by the total number of residues in that group. we used the normalised count of amino acid residues using the esol e. coli sequences as the background. the bit score of residue for soluble or insoluble group is then given by the following equation: where is the normalised count of residue in the psi:biology soluble or insoluble (x) f i x) ( group and is the normalised count in the esol sequences. (x) f esol for a control, random protein sequences were generated by incrementing the length of sequence, starting from a length of 50 residues to 6,000 residues with a step size of 50 residues. a hundred random sequences were generated for each length, giving a total of 12,000 unique random sequences. to estimate the probability of solubility using swi, we fitted the following logistic regression to the psi:biology dataset: (3) robability of solubility where, is the swi of a given protein sequence, and . the x 81.05812 a = − 2.7775 b = 6 p-value of log-likelihood ratio test was less than machine precision. equation 3 can be used to predict the solubility of a protein sequence given that the protein is successfully expressed in e. coli ( supplementary table s8 ). on this basis, we developed a solubility prediction webservice called the soluble domain for protein expression (sodope). our web server accepts either a nucleotide or amino acid sequence. upon sequence submission, a query is sent to the hmmer web server to annotate protein domains ( https://www.ebi.ac.uk/tools/hmmer/ ) (potter et al. 2018) . once the protein domains are identified, users can choose a domain or any custom region (including full-length sequence) to examine the probability of solubility, flexibility and gravy. this functionality enables protein biochemists to plan their experiments and opt for the domains or regions with high probability of solubility. furthermore, we implemented a simulated annealing algorithm that maximised the probability of solubility for a given region by generating a list of regions with extended boundaries. users can also predict the improvement in solubility by selecting a commonly used solubility tag or a custom tag. we linked sodope with tisigner, which is our existing web server for maximising the accessibility of translation initiation sites (bhandari, lim, and gardner 2019) . this pipeline allows users to predict and optimise both protein expression and solubility for a gene of interest. the sodope web server is freely available at https://tisigner.com/sodope . jupyter notebook of our analysis can be found at https://github.com/gardner-binflab/sodope_paper_2020 . the source code for our solubility prediction server (sodope) can be found at https://github.com/gardner-binflab/tisigner-reactjs . 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 robotic cloning and protein production platform of the northeast structural genomics consortium ccsol omics: a webserver for solubility prediction of endogenous and heterologous expression in escherichia coli the thioredoxin superfamily: 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thank new zealand escience infrastructure for providing a high performance computing platform. we are grateful to harry biggs for proofreading our manuscript and providing feedback for the web server. this work was supported by the ministry of business, innovation and employment, new zealand (mbie grant: uoox1709). key: cord-018479-mvnm98hv authors: rehm, fabian b. h.; grage, katrin; rehm, bernd h. a. title: applications of microbial biopolymers in display technology date: 2017-11-16 journal: consequences of microbial interactions with hydrocarbons, oils, and lipids: production of fuels and chemicals doi: 10.1007/978-3-319-50436-0_377 sha: doc_id: 18479 cord_uid: mvnm98hv microorganisms produce a variety of different polymers such as polyamides, polysaccharides, and polyesters. the polyesters, the polyhydroxyalkanoates (phas), are the most extensively studied polymers in regard to their use in display technology. the material properties of bacterial phas in combination with their biocompatibility and biodegradability make them attractive substrates for use in display technology applications. by translationally fusing bioactive molecules to a gene encoding a pha-binding domain, the appropriate functionalization for a given application can be achieved such that the need for chemical immobilization is circumvented. by separately extracting and processing the biopolymer, using it to coat a surface, and then treating this surface with the fusion proteins, surface functionalization for immunodiagnostic microarray or tissue engineering applications can be accomplished. conversely, by expressing the fusion protein directly in the pha-producing organisms, one-step production of functionalized beads can be achieved. such beads have been demonstrated in diverse applications, including fluorescence-activated cell sorting, enzyme-linked immunosorbent assays, microarrays, diagnostic skin test for tuberculosis, vaccines, protein purification, and affinity bioseparation. the display of biologically active molecules is utilized for a range of applications such as diagnostics, biosensing, and microarray technologies. the substrate on which such display takes place is greatly deterministic of functionality and applicability. common, well-established techniques include display on cell surfaces, ribosomes, and phage particles (lee et al. 2003; zahnd et al. 2007; rakonjac et al. 2011) . the use of microbial biopolymers as substrates has more recently been revealed in a variety of contexts. although bacteria can produce a range of polymers, only a few of them have been considered for display technologies (fig. 1 ). bacterial cellulose, which exhibits various properties superior to plant-based cellulose with respect to display technology applications, has been limited to enzyme, bacterial cell and fungi immobilization (wu and lia 2008; ullah et al. 2016) . in contrast, bacterial polyhydroxyalkanoates (phas) have been extensively investigated as substrates for display technology applications, and thus will be the focus of this chapter. phas are biopolyesters which serve as carbon and energy storage materials in a range of bacteria and archaea (lenz and marchessault 2005; rehm 2010 ). during excess carbon availability, they are stockpiled as the amorphous core of pha inclusions, surrounded by structural proteins (phasins), pha metabolism-associated enzymes, and regulator proteins jendrossek 2009 ). critical enzymes for pha synthesis and inclusion assembly are the pha synthases (such as phac) which catalyze the stereoselective conversion of the activated precursor (r)-3-hydroxyacyl-coa (rehm 2003) . these coa thioesters, depending on their carbon chain length, are synthesized from intermediates of fatty acid metabolism or directly from acetyl-coa to polyoxoesters with the simultaneous release of coenzyme a (steinbuchel et al. 1993; rehm 2006) (fig. 2) . unlike the other hydrophobically interacting pha inclusion surface proteins, the pha synthase remains covalently linked to the pha inclusion core (hezayen et al. 2002; peters and rehm 2006 although phas are all hydrophobic and water insoluble, they can drastically vary in composition and thus physical properties. melting points can range from 50 c to 180 c and crystallinity can range from 30% to 70%, which is largely determined by monomer composition (rehm 2010) . as such, phas have been classified on the basis of monomer chain length. medium-chain-length phas (mcl, c6-c14) are naturally produced primarily by pseudomonads, whereas short-chain-length phas (scl, c3-c5) production is more widespread throughout bacteria and archaea (anderson et al. 1990) (fig. 2) . while the common laboratory bacterium escherichia coli does not naturally accumulate phas, it becomes a competent pha producer upon introduction of the appropriate pha biosynthesis genes (schubert et al. 1988; lee et al. 1994) . the intracellular pha inclusions may be isolated for polymer extraction and purification for processing into various materials, or maintained as functional shell-core beads (fig. 3) . the latter requires engineering of proteins attached to the pha core in order to obtain functionality. the pha material properties enable film coating of solid surfaces suitable for microarray applications. indeed, by exploiting the ability of pha-associated proteins to specifically bind phas, and thereby overcoming specificity-or orientation-associated issues, pha substrates for immobilization have been demonstrated as attractive for such applications. the first relevant example of pha as a protein micropatterning substrate was described in a study by park and colleagues (2005) . poly(3-hydroxybutyrate) (p(3hb)) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) were independently produced, purified, and used to spin-coat glass substrate, producing pha films. subsequently, enhanced green fluorescent protein and red fluorescent protein, fused with the hydrophobic side chain-interacting substrate binding domain (sbd) of a pha depolymerase, were micropatterned onto the film using microcontact printing. confocal fluorescent microscopy clearly revealed the printed patterns after several wash steps and surface plasmon resonance spectroscopy using anti-green fluorescent protein polyclonal antibody further confirmed this specific fusion protein immobilization, additionally demonstrating the system as capable for examining proteinprotein interactions. spurred on by these encouraging results, a further study aimed to demonstrate the possibilities of this system for protein microarray development centered on immunodiagnostic applications (park et al. 2006) . pha depolymerase sbd fusions with the single-chain antibody variable region (scfv) against hepatitis b virus (hbv) pres2 surface protein as well as the severe acute respiratory syndrome coronavirus envelope protein (scve) were produced and microspotted onto p(3hb) films. fluorescence-labelled hbv antigen and anti-scve antibody were then used to detect the interactions on the films. fluorescence signals were detected only at the corresponding microspotted regions, indicating high affinity and selectivity and thus indicating the technology as appropriate for use in immunodiagnostics. subsequent investigation into whether such a platform could be applicable for clinical pathogen detection via immobilized dna-protein complexes was undertaken by park et al. (2009) (fig. 4) . pathogen-specific (acinetobacter baumannii, e. coli, klebsiella pneuomoniae, and pseudomonas aeruginosa) biotin-labelled 15-mer dna probes immobilized to core streptavidin fused to the pha depolymerase sbd were microspotted onto polyhdroxybutyrate (phb)-coated slides. by hybridizing differentially fluorescently labelled target dnas to each pathogen-specific probe simultaneous detection of the corresponding pathogens added to the slide was demonstrated, further evidencing the potential of phas for use in immunodiagnostic microarrays. the diverse, adjustable material properties of phas in concert with their biodegradability, biocompatibility, noncarcinogenicity, and low cytotoxicity have driven their development in the area of biomaterials research (ali and jamil 2016) . on the basis of pha surface functionalization by addition of pha-binding protein fusions, several approaches to enhancing tissue engineering and culturing techniques have been undertaken. by translationally fusing the pha-binding phasin, phap, to the cell adhesion motif arg-gly-asp (rgd) and applying this fusion protein to coat pha surfaces, the effect on fibroblast growth was investigated (dong et al. 2010) . under serum-free conditions, confocal laser scanning microscopy in combination with cell counting assays revealed a significant cell attachment increase on the phap-rgd-coated pha films relative to the phap-(nonfused) and noncoated films, in a manner that is unlikely to be attributable to changes in pha surface topology. further cell proliferation assays revealed increased fibroblast proliferation levels on phap-rgdcoated pha films. thus, pha surface functionalization, using a well-known cell adhesion motif, was demonstrated using aqueous solutions, avoiding toxicity of chemical immobilization techniques. continuing this pattern of investigation, similar increases in cell adhesion and cell proliferation of human vascular smooth muscle cells (hvsmcs) were demonstrated using the pha repressor protein (phar) fused to the specific integrin ligand peptide (kqagdv) (dong et al. 2012) , and, more recently, phap fused to rgd or the laminin-derived ikvav peptides used to coat pha films demonstrated enhanced neural stem cell (nsc) attachment, proliferation, and better neurite outgrowths, without effecting nsc differentiation (xie et al. 2013 ). fluorescence activated cell sorting (facs) is a qualitative and quantitative technique used in biomolecule detection for in vitro diagnostics. generally, antigen-displaying beads are used to bind antibodies which are then detected via fluorescent-signaling secondary antibodies when the bead suspension passes through the facs machine flow cell. current bead preparation techniques involve tedious antigen purification and chemical crosslinking. therefore, more cost-effective, reliable means of producing antigen-displaying beads with comparable detection efficacy could greatly impact the accessibility of facs. a series of studies have shown that one path towards such improvement may be via one-step production of surface protein engineered pha beads. the first demonstration of in vivo-produced pha inclusions for facs applications was described by bäckström and coworkers (bäckström et al. 2007 ). the authors generated fusions between either interleukin-2 (il2) or the myelin oligodendrocyte glycoprotein (mog) and the c-or n-terminus of phap via an enterokinase (specific protease) recognition site providing linker. subsequently, the hybrid genes were expressed in e. coli and the pha granules extracted. the resultant antigen-displaying pha beads were then used for facs using the corresponding fluorescently labelled monoclonal antibody which showed significant and specific antibody binding. enterokinase treatment reversed this recognition indicating removal of the fusion partners/antigens. finally, sera from mice immunized with mog or ovalbumin (as a negative control) were analyzed using the mog-displaying beads and facs. again, high specificity and sensitivity (antibody detection in sera diluted 1:100,000) were demonstrated, providing strong support for application of engineered pha beads in facs (fig. 5) . a subsequent investigation explored c-terminal phac-streptavidin fusions (peters and rehm 2008) . the remarkably high streptavidin-biotin binding affinity has given rise to its use in range of biotechnological applications, thus making it a great candidate for further demonstrating the applicability of pha beads in facs. the study revealed a biotin binding capacity of 61 ng per μg of bead protein and demonstrated the detection of goat polyclonal biotinylated igg in facs using a secondary conjugated antibody. most recently, pha inclusions with gfp and mog fused to phap or phac, such that each granule is simultaneously displaying two protein-based functionalities, were examined in the context of facs, providing proof-of-concept for the biotechnological application of bifunctional pha beads which may extend beyond facs ). the enzyme-linked immunosorbent assay (elisa) is based on antigen-antibody binding on solid support either detecting antigen or antibody in samples. in vivo assembled engineered pha inclusions displaying antigens or specific binding domains as described above were directly used to coat elisa plates. respective elisas showed specific and sensitive detection of the corresponding antibody or in vivo manufactured antigen displaying pha beads used in fluorescence activated cell sorting for specific antibody detection. pha beads were incubated in sera of mice either immunized with mog or ova. after washing, bound antibodies were detected using a fluorescently labelled secondary anti-igg antibodies combined with facs. anti-mog antibodies were still detectable at a serum dilution 1:100,000. mog, myeline oligodendrocyte glycoprotein; phap, phasin (structural pha inclusion surface protein); ova ovalbumin (facs data representation was adapted from bäckström et al. (2007)) antigen confirming the applicability of bioengineered pha beads in elisa (peters and rehm 2008; atwood and rehm 2009; parlane et al. 2009 ). one in vivo-produced pha bead-based approach to in vivo diagnosis is a skin test for the detection of bovine tuberculosis (tb) (chen et al. 2014 ). the major disadvantage of the currently widespread tuberculin skin test is the lack of detection specificity for pathogenic mycobacterium tuberculosis complex members, that is, cattle exposed to nonpathogenic, environmental mycobacteria may result in falsepositives. thus, with the aims of improving specificity and lowering production cost, an alternative test was developed. three immunodominant tb antigens, which are absent in most environmental mycobacteria, were fused to phac mediating production of triple-antigen-displaying pha beads. these granules showed in vitro increased reactivity with antigen-specific tb antibodies when compared with granules displaying only one antigen. assessment of triple-antigen-displaying pha beads in the skin test (in vivo) showed specificity as all cattle experimentally infected with mycobacterium bovis were detected while no false-positive reactions in cattle previously exposed to environmental mycobacteria were observed. a fourth antigen was added to the triple-antigen-displaying pha beads to boost skin test sensitivity (parlane et al. 2016) . dose response studies showed that very low amounts of mycobacterial antigens (0.1 μg) were already sufficient for the skin test suggesting greatly increased immunogenicity of antigen-displaying beads versus soluble antigens. hence, it was demonstrated that antigen-displaying pha beads mediate an antigen-specific immune reaction upon injection into the skin, that is, serve as specific immune response stimulating antigen delivery systems. the concept of in vivo assembly of nano-/microsized pha beads displaying diseasespecific antigen as particulate vaccines was investigated. since phas are considered as biomaterials, when produced coated with pathogen-specific antigens they might serve as safe and efficient particulate vaccine. in general, the nano-/microsized particulate nature of a vaccine mimics the dimensions of a pathogen which boosts immunogenicity via enhanced uptake by antigen presenting cells (apc) (shah et al. 2014) . immunodominant antigens from pathogens such as hcv and m. tuberculosis were displayed on pha beads using pha synthase engineering (parlane et al. , 2011 . purified antigen-coated pha beads were injected into mice which resulted in strong and specific immune responses mediating protective immunity as assessed by challenge of vaccinated animals with the pathogen (parlane et al. 2012; martinez-donato et al. 2016) (fig. 6) . it is noteworthy that these pha bead-based vaccines stimulated both a humoral antibody (th2) and cell-mediated (th1) immune response, the latter being particularly relevant to protect against intracellular pathogens and is more challenging to achieve. purification of recombinantly produced proteins, generally with the target protein fused to an affinity tag, can be a time-consuming and costly process since it often requires multiple chromatography steps which all need to be individually optimized, and additionally require cleavage and removal of the tag. over the past 10 years, various approaches have been undertaken to make use of pha and pha-associated proteins for the development of alternative, simple and cost-effective protein purification systems. the general principle that most of these studies have followed is to produce the target protein as a translational fusion to a pha inclusion-associated protein in cells that also make pha and to then copurify the protein with the pha beads. this can be done in a host that naturally produces pha or in an organism that has been genetically engineered to make pha (e.g., e. coli). different methods have then been applied to release the target protein from the beads. early examples used the phasin protein phap as an affinity tag and inducible self-cleaving inteins inserted between phap and the target protein to release the protein of interest. this method was applied by barnard and coworkers to purify green-fluorescent protein (gfp) and β-galactosidase (lacz) from ralstonia eutropha, a natural phb producer (barnard . gfp and lacz could be successfully purified fused to either end of phap using a thiol-inducible intein. similarly, several proteins (maltose-binding protein (mbp), lacz, chloramphenicol acetyltransferase (cat), and nusa) were tagged with phap and purified from recombinant e. coli with the help of a ph-inducible intein (banki et al. 2005) . to strengthen the binding of the fusion protein to the bead surface to the point where significant leakage during the purification process could be avoided, multiple (two or three) phasins were used as a tag. geng and coworkers chose a protein which is generally difficult to produce in bacteria due to the presence of several disulfide bonds (geng et al. 2010) . they fused recombinant human tissue plasminogen activator (rpa), a truncated version of tissue plasminogen activator, to phap and inserted a thrombin cleavage site as a linker. active rpa could be released from isolated beads by thrombin treatment. in another study which focused on diagnostic applications (aforementioned) the mog and interleukin-2 were produced as phap-fusions containing an enterokinase recognition site (bäckström et al. 2007 ). the successful removal of mog or il2, respectively, after enterokinase treatment was monitored by facs analysis. a slightly different approach to pha-based protein purification is based on production of fusion proteins separate to pha extraction and processing into beads. various target proteins were fused to different granule-associated proteins such as phap or the regulatory protein phar, the fusion proteins recombinantly produced in e. coli and crude cell lysates incubated with beads processed from extracted pha (wang et al. 2008 , zhang et al. 2010 . release of the target protein was also achieved via intein-mediated self-cleavage. while this method is more labor-intensive than producing pha and proteins in the same cell, advantageous might be the possibility to produce the tagged target protein in any host organism including eukaryotes. a simplified approach used the n-terminal part of phaf to anchor a target protein to pha beads in vivo, followed by bead isolation and release of the target protein (i.e., the entire fusion of target protein and phap) from the beads by detergent treatment (moldes et al. 2004 ). however, a general drawback of using phap (or phar) as a tag for protein purification is that these proteins are only attached to the pha inclusion surface via hydrophobic interactions, so there is a risk that the fusion protein could detach during either the bead purification process or the tag cleavage process resulting in loss of target protein. in the natural system, phasins have the advantage of being the predominant protein on the surface of pha inclusions (wieczorek et al. 1995) ; however, in a recombinant system, proteins such as the pha synthase can be overproduced to achieve a high density at the inclusion surface (brockelbank et al. 2006; mifune et al. 2009 ). grage et al. harnessed the covalent attachment of the pha synthase to pha inclusions by translationally fusing hcred or an anti-β-galactosidase single-chain antibody fragment (scfv) (martineau et al. 1998) , separated by an enterokinase recognition site . both target proteins were successfully released from purified beads by enterokinase treatment; however, cleavage efficiency was relatively low. in an attempt to find a robust and inexpensive auto-processing module, hay and coworkers used a modified soluble form of the cell surface sortase transpeptidase a (srta) from staphylococcus aureus which had been engineered to self-cleave in the presence of ca 2+ (mao 2004; hay et al. 2015a) . srta and the target protein were fused to the c-terminus of phac using an extended linker . using this technique, gfp, mbp, and antigen rv1626 from mycobacterium tuberculosis could be released from isolated pha beads at a high yield and purity (e.g., 6 mg/l of soluble gfp at a purity of about 98%) (hay et al. 2015a ). it may not always be feasible or desirable to produce the target protein fused to a bead-associated protein (and to coproduce it with the beads/resin). hence pha beads have also been engineered to serve as affinity resins for bioseparation, generally exploiting the possibility of densely displaying binding domains on nano-/microbeads. these resins can be produced in one step by fusing the binding domain of choice to the bead-associated protein and then isolating these functionalized beads from the production host (similar to the protein purification approach described above). the first example of this was the immunoglobulin g (igg) binding zz domain of staphylococcus aureus protein a translationally fused to phac (brockelbank et al. 2006 ). the resulting zz domain displaying beads were isolated from e. coli and successfully purified igg from human serum and mouse hybridoma supernatants, with purity and yield comparable to commercially available protein a sepharose (brockelbank et al. 2006; lewis and rehm 2009 ). further pha bead-based resins assembled by engineering phac displayed streptavidin which bound various biotinylated compounds such as enzymes, antibodies, and dna (peters and rehm 2008 ). grage and rehm were able to purify β-galactosidase from a mixture of proteins using anti-lacz scfv immobilized to pha beads (grage and rehm 2008 ). an endotoxin-removing resin was developed by producing human lipopolysaccharide-binding protein (hlbp) fused to phap in pichia pastoris (li et al. 2011) . after secretion and recovery from the culture supernatant, the hlbp-phap fusion was incubated with beads processed from extracted pha, and the resulting hlbp-beads were tested for their endotoxin removal abilities under a variety of conditions. according to the authors, the beads performed better than commercially available endotoxin-removing gels (li et al. 2011) . recently, hay and coworkers published a more extensive study that aimed at broadening the applicability of pha bead affinity resins by identifying and testing several easily customizable affinity binding domains which they translationally fused to phac (hay et al. 2015b ). this study demonstrated that v hh domains from camelid antibodies, designed ankyrin repeat proteins (darpins) and ob-folds (obodies), could be densely displayed on pha beads resulting in high affinity binding resins for purification of various target proteins. these binding domains were used to establish extensive libraries of variants enabling screening for binders specific for the target compound of interest (binz et al. 2004; harmsen and de haard 2007; stumpp et al. 2008; steemson et al. 2014) . pha-based affinity resins showed a purification performance at least equal to current commercial offerings (hay et al. 2015b) . a recent surface topology study of the r. eutropha phac attached to pha inclusions suggested new engineering strategies towards the development of pha-based affinity resins with increased binding capacity via improved display (hooks and rehm 2015) . this study identified several surface-exposed flexible regions of phac, which tolerated the insertion of the igg-binding zz domain. one of the double zz domain insertions (i.e., zz inserted in two of the surface-exposed regions) showed greatly improved igg binding capacity with some of the single insertions also showing improved igg binding capacity when compared with terminal fusions. overall, phac engineering such as n-or c-terminal fusions and/or insertions enabled efficient display of binding domains for interaction with target compounds resulting in purification performance suitable for application as bioseparation resin. research needs microbial phas show great promise as polymers providing a support structure for display of a range of protein functions. since phas can be composed of various constituent resulting in a diversity of material properties, it currently remains unexplored how these different phas perform in the context of anchoring binding domains for display. additionally, research needs to address bioprocessing challenges to obtain phas of consistent structure and shape for improved implementation in various protein display applications. although microorganisms are capable of producing a variety of polymers, the hydrophobic thermoplastic phas currently hold the greatest promise as support material to display protein functions. the pha material properties allow processing into nano-/microbeads, films, and 3d structures, while pha-related proteins provide specific pha binding domains to anchor protein functions of interest. these approaches enabled implementation in microarray-based diagnostics as well as tissue engineering. besides the binding of protein functions to isolated and processed pha, recent research elucidated the concept of producing pha inclusions within the bacterial cell already coated with desired protein functions. the applicability of these pha beads as vaccines, in diagnostics and as bioseparation resin was demonstrated. polyhydroxyalkanoates: current applications in the medical field biosynthesis and composition of bacterial poly (hydroxyalkanoates) protein engineering towards biotechnological production of bifunctional polyester beads recombinant escherichia coli produces tailormade biopolyester granules for applications in fluorescence activated cell sorting: functional display of the mouse interleukin-2 and myelin oligodendrocyte glycoprotein novel and economical purification of recombinant proteins: intein-mediated protein purification using in vivo polyhydroxybutyrate (phb) matrix association integrated recombinant protein expression and purification platform based on ralstonia eutropha high-affinity binders selected from designed ankyrin repeat protein libraries recombinant escherichia coli strain produces a zz domain displaying biopolyester granules suitable for immunoglobulin g purification new skin test for detection of bovine tuberculosis on the basis of antigen-displaying polyester inclusions produced by recombinant escherichia coli the improvement of fibroblast growth on hydrophobic biopolyesters by coating with polyhydroxyalkanoate granule binding protein phap fused with cell adhesion motif rgd the cytocompatability of polyhydroxyalkanoates coated with a fusion protein of pha repressor protein (phar) and lys-gln-ala-gly-asp-val (kqagdv) polypeptide expression of active recombinant human tissue-type plasminogen activator by using in vivo polyhydroxybutyrate granule display in vivo production of scfv-displaying biopolymer beads using a selfassembly-promoting fusion partner bacterial polyhydroxyalkanoate granules: biogenesis, structure, and potential use as nano-/micro-beads in biotechnological and biomedical applications recombinant protein production by in vivo polymer inclusion display properties, production, and applications of camelid singledomain antibody fragments in vivo polyester immobilized sortase for tagless protein purification bioengineering of bacteria to assemble custom-made polyester affinity resins biochemical and enzymological properties of the polyhydroxybutyrate synthase from the extremely halophilic archaeon strain 56 insights into the surface topology of polyhydroxyalkanoate synthase: self-assembly of functionalized inclusions tolerance of the ralstonia eutropha class i polyhydroxyalkanoate synthase for translational fusions to its c terminus reveals a new mode of functional display polyhydroxyalkanoate granules are complex subcellular organelles (carbonosomes) microbial cell-surface display construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) by recombinant escherichia coli bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology zz polyester beads: an efficient and simple method for purifying igg from mouse hybridoma supernatants endotoxin removing method based on lipopolysaccharide binding protein and polyhydroxyalkanoate binding protein a self-cleavable sortase fusion for one-step purification of free recombinant proteins expression of an antibody fragment at high levels in the bacterial cytoplasm protective t cell and antibody immune responses against hepatitis c virus achieved using a biopolyester-bead-based vaccine delivery system production of functionalized biopolyester granules by recombinant lactococcus lactis vivo immobilization of fusion proteins on bioplastics by the novel tag biof micropatterning proteins on polyhydroxyalkanoate substrates by using the substrate binding domain as a fusion partner polyhydroxyalkanoate chip for the specific immobilization of recombinant proteins and its applications in immunodiagnostics microarray of dna-protein complexes on poly-3-hydroxybutyrate surface for pathogen detection bacterial polyester inclusions engineered to display vaccine candidate antigens for use as a novel class of safe and efficient vaccine delivery agents production of a particulate hepatitis c vaccine candidate by an engineered lactococcus lactis strain vaccines displaying mycobacterial proteins on biopolyester beads stimulate cellular immunity and induce protection against tuberculosis display of antigens on polyester inclusions lowers the antigen concentration required for a bovine tuberculosis skin test in vivo enzyme immobilization by use of engineered polyhydroxyalkanoate synthase protein engineering of streptavidin for in vivo assembly of streptavidin beads filamentous bacteriophage: biology, phage display and nanotechnology applications polyester synthases: natural catalysts for plastics genetics and biochemistry of polyhydroxyalkanoate granule self-assembly: the key role of polyester synthases bacterial polymers: biosynthesis, modifications and applications cloning of the alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (phb) and synthesis of phb in escherichia coli the impact of size on particulate vaccine adjuvants tracking molecular recognition at the atomic level with a new protein scaffold based on the ob-fold molecular basis for biosynthesis and accumulation of polyhydroxyalkanoic acids in bacteria darpins: a new generation of protein therapeutics advances in biomedical and pharmaceutical applications of functional bacterial cellulose-based nanocomposites a novel self-cleaving phasin tag for purification of recombinant proteins based on hydrophobic polyhydroxyalkanoate nanoparticles analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in alcaligenes eutrophus application of bacterial cellulose pellets in enzyme immobilization enhanced proliferation and differentiation of neural stem cells grown on pha films coated with recombinant fusion proteins ribosome display: selecting and evolving proteins in vitro that specifically bind to a target microbial polyhydroxyalkanote synthesis repression protein phar as an affinity tag for recombinant protein purification key: cord-005034-wyipzwo4 authors: gleeson, paul a.; teasdale, rohan d.; burke, jo title: targeting of proteins to the golgi apparatus date: 1994 journal: glycoconj j doi: 10.1007/bf00731273 sha: doc_id: 5034 cord_uid: wyipzwo4 the golgi apparatus maintains a highly organized structure in spite of the intense membrane traffic which flows into and out of this organelle. resident golgi proteins must have localization signals to ensure that they are targeted to the correct golgi compartment and not swept further along the secretory pathway. there are a number of distinct groups of golgi membrane proteins, including glycosyltransferases, recyclingtrans-golgi network proteins, peripheral membrane proteins, receptors and viral glycoproteins. recent studies indicate that there are a number of different golgi localization signals and mechanisms for retaining proteins to the golgi apparatus. this review focuses on the current knowledge in this field. the survival of a cell depends on maintaining the integrity of the intracellular organelles. this feat is achieved by highly selective sorting and accurate transport of proteins to their correct destinations. over the past few years the dissection of the molecular machinery for targeting and localization of proteins, in particular proteins of the secretory pathway, has been studied vigorously. defined sequence motifs have been identified on proteins which can act as 'address labels'. the golgi apparatus represents the 'hub' of the secretory pathway where intense membrane traffic is controlled. this organelle not only co-ordinates the sorting of newly synthesized proteins but is also responsible for the control of posttranslational modifications, in particular glycosylation. a fundamental question currently being addressed in cell biology is how the golgi apparatus is organized to achieve these demanding functions and how it maintains its structural integrity in spite of the intense membrane traffic which enters and leaves this organelle. this review will focus primarily on our understanding of the molecular signals and mechanisms for the retention of resident golgi proteins. the golgi apparatus is a highly complex and dynamic organelle, which has been difficult to define in three-dimensional terms [1] . it consists of a number of (reflecting in part the cholesterol content), ph, and most importantly in the populations of resident proteins which they contain. however, detailed biochemical characterization of the individual cisternae is lacking as the current methods are inappropriate to allow their purification. newly synthesized proteins are transported sequentially from the er to the golgi and then to their final destination. the transport of newly synthesized proteins from the endoplasmic reticulum to golgi cisternae, between adjacent cisternae within the golgi stack, and from golgi cisternae to various destinations is mediated by vesicles shuttling between donor and recipient compartments. vesicles bud from one compartment and then target and fuse with the next compartment [7, 8] . an increasing number of structural and regulatory components have been identified which are involved in the orchestration of the complex and intriguing processes of budding, specific targeting, docking and fusion [9] [10] [11] . some of the components of this machinery are localized only to golgi membranes and are thought to be specific for membrane transport through and from the golgi apparatus. the restricted location of these components indicates the presence of specific localization signals. until recently it was widely believed that, if correctly folded, newly synthesized proteins are transported from the endoplasmic reticulum, through the golgi stack to the tgn without the requirement for a specific transport signal [7, 12] . however, the concept of 'bulk flow' of membrane proteins from the er has recently been challenged by the finding that vesicular stomatitis virus g glycoprotein is significantly concentrated during export from the er [13] . nonetheless forward transport, at least from the golgi apparatus to the cell surface, appears to constitute a signal-independent or default pathway. despite this extensive flux of proteins, it is imperative that the golgi apparatus maintains the set of resident proteins which define its unique structural and functional properties. thus, it would appear that newly synthesized golgi membrane proteins must stop at the correct cisterna, or subcompartment, and be prevented from being swept into transport vesicles that bud from the dilated rims of the cisternae. clearly, specific localization signals are required for retention of proteins which reside in the golgi apparatus. what do we know about the sorting signals and mechanisms for the localization of non-golgi proteins within the secretory and endocytic pathways? a number of sorting signals have been found associated with the cytoplasmic domains of membrane proteins [14] . for example, short tyrosine-containing peptide motifs found on cytoplasmic domains direct the sorting of proteins from the plasma membrane via the receptor mediated endocytosis pathway [15] and the transcytosis of basolateral proteins to the apical surface of certain polarized cells [16] , while a dileucine-containing peptide motif directs the transport of man-6-p receptors from the tgn to the late endosomes [17, 18] . these cytoplasmic domain sorting signals mediate interactions with coat structures of budding vesicles and thereby allow the selective vesicular transport of these membrane proteins between a variety of compartments [ 19] . much progress has been made in defining the retention signals for resident er proteins. targeting signals have been identified for both soluble and membrane-bound proteins residing in the er. a specific retention signal, comprising the carboxy terminal sequence kdel/hdel, has been identified for a number of resident soluble er proteins [20, 21] , and a receptor for this retention sequence has been identified [22] [23] [24] . retention of these soluble er proteins is mediated by a receptor-based salvaging mechanism, whereby escaped kdel-bearing proteins are retrieved from a post-er compartment by a recycling kdel receptor [24] . for membrane proteins of the e r a double lysine motif (kkxx) at the cytoplasmically exposed carboxy terminus of certain type i membrane proteins has been shown to specify er residence [25] . for type li membrane er proteins, a related double arginine motif at the cytoplasmically orientated amino terminus has been identified [26] . interestingly, the localization of ergic-53 (p53), a type i membrane protein of the intermediate compartment or cgn, requires a kkxx er retention motif, again suggesting that the cgn may be an extension overall, the localization signals of non-golgi proteins are hydrophilic motifs located on either the cytoplasmic or luminal domains of the protein, and some of these signals have been shown to interact specifically with receptor molecules or with protein coats of budding vesicles. it is now clear from recent studies that there are a number of distinct types of golgi localization signals. based on these localization signals and other biochemical features the resident proteins of the golgi apparatus can be divided into five groups (table 1) . they are all membrane proteins. interestingly no soluble resident golgi proteins have been identified within the lumen of the golgi apparatus, which probably indicates that the mechanisms for retaining proteins to this organelle are restricted to membraneassociated proteins. the five groups are described individually as the mechanism of golgi localization may be unique for each group. the golgi apparatus plays a key role in the glycosylation of newly synthesized membrane and secreted proteins [29, 30] . based on the exquisite specificities of the currently defined glycosyltransferases [29, 31] , the synthesis of all the known carbohydrate chains of glycoconjugates must require in the region of 100-200 different glycosyltransferase enzymes distributed throughout the golgi stack. however, very little is known about the structural organization of these integral membrane enzymes within the membranes of the golgi cisternae and the signals which define their localization within the golgi are only now beginning to be defined. a number of glycosyltransferases have restricted distributions within the golgi apparatus, notably /~1,4 galactosyltransferase (/? 1,4galt) (trans-golgi) [32] [33] [34] , :~2,6 sialyltransferase (e2,6st) (trans-golgi and trans-golgi network) [35, 36] and n-acetylglucosaminyltransferase i (glcnacti) (medial-golgi) [34, 37, 38] . furthermore, simultaneous immunogold localization of /~i,4galt and gtcnacti in the same golgi apparatus, confirms that these enzymes have distinct, though overlapping, distributions [34] . from the purification tables ofgolgi glycosyltransferases, it is clear that individual transferases constitute only a minor percentage of the proteins of the cisternae in which they reside. for example, glcnacti constitutes about 1~ of medial-specific golgi membrane protein in rabbit liver [39] . however, in view of the estimated number of golgi glycosyltransferases, collectively the glycosyttransferases of each golgi cisterna may represent a very significant proportion, if not the bulk, of the resident membrane proteins. numerous mammalian glycosyltransferases have been cloned and sequenced (see reviews [40] [41] [42] [43] ). individual glycosyltransferases are highly conserved across species, for example rabbit glcnacti shares 92~o and 93~ amino acid sequence identity with human and mouse glcnacti, respectively [4447] . but comparison of the amino acid sequences between the glycosyltransferases has revealed only isolated cases of sequence similarity. for example, there is a high degree of sequence similarity between blood group a and b glycosyltransferases [48] , which are products of two alleles, and between a number of e3(4) fucosyltransferases [49] . furthermore, a conserved motif has been identified in the catalytic domain of cloned sialyltransferases (the 'sialylmotif') [50] . however, these examples of sequence similarity are the exceptions and, overall, little sequence similarity has been detected between different glycosyltransferases. this is dramatically illustrated by a lack of obvious amino acid similarity between the sequences of four different glcnac transferases involved in the synthesis of the outer antennae of complex n-glycans, namely glcnacti [44, 45] , glcnactii [51] , glcnactiii [52] , and glcnactv [53] . one would expect there to be structural similarity between the catalytic sites of these glcnac transferases but this has not been detected from their amino acid sequences. thus, comparison of the primary structures of golgi glycosyltransferases has not revealed a potential golgi localization motif. there is, however, a striking similarity in the domain structure of these golgi enzymes. all golgi transferases cloned to date are nin/cou t (type ii) membrane proteins containing a single hydrophobic membrane-spanning domain (16-25 amino acids) which also serves as a non-cleavable signal sequence. each has a short n-terminal cytoplasmic domain (many have less than 10 amino acids), and a large carboxyl-terminal catalytic domain situated in the lumen of the golgi apparatus. the catalytic domain is linked to the transmembrane domain by a loosely defined 'stem' region which may play a role in positioning the catalytic domain away from the lipid bilayer facilitating access to the substrate. over the past 4 years a number of groups have attempted to identify the targeting signal responsible for the localization of gtycosyltransferases. three glycosyltransferases have been extensively examined, namely cd,6st, /~i,4galt, and glcnacti. these enzymes are residents of the tgn, tgn/trans-gotgi, and medial-golgi respectively. a common strategy has been employed by all groups to identify a putative golgi retention signal(s) by analysing the localization, in transfected mammalian cells, of hybrid molecules containing limited sequences derived from golgi glycosyltransferases. in all cases, the membrane-spanning domains of the golgi glycosyltransferases have been shown to direct, at least partial localization of hybrid molecules to the golgi apparatus [54] [55] [56] [57] [58] [59] [60] [61] . indeed it has been further demonstrated that the transmembrane domain of/?i,4galt and glcnacti can specifically localize hybrid proteins to the trans and medial cisternae, respectively [55, 61] . however, a number of studies have shown that sequences flanking the transmembrane domain also play auxiliary roles in mediating golgi localization [54, [61] [62] [63] . these studies all agreed that the transmembrane domain plays a central role in the targeting and localization of resident golgi glycosyltransferases. this was an unexpected finding as localization signals up until then were hydrophilic glycopinion mini-review regions of the cytoplasmic or luminal domains. the involvement of a hydrophobic stretch of amino acids in targeting indicated a unique mechanism for the localization of these resident golgi proteins. these initial studies were based to a large extent on the premise that a discrete region, or motif, was responsible for the golgi localization signal. further studies have indicated that, although the transmembrane domain is relevant, the situation is far more complex than at first appreciated. figs 2-4 summarize graphically the regions of glycosyltransferases that have been examined and their ability to direct reporter molecules to the golgi apparatus. collectively, a large number of constructs have now been analysed. figs 2-4 include many of these constructs, however, it is by no means all-inclusive. those selected are the most instructive and highlight the complexity of the situation. there is considerable variability in the results obtained between groups, even when comparing the same glycosyltransferase. for example, wong et al. [60] reported that the transmembrane domain of e2,6st resulted in very efficient golgi localization of a hybrid molecule, whereas a hybrid construct of munro [54] , containing the equivalent c~2,6st domain, resulted in considerable leakage to the cell surface ( fig. 2) . site-directed mutagenesis of residues of the transmembrahe domain of //1,4gait, in the context of hybrid molecules, suggested that uncharged polar residues are critical for the ability of these hydrophobic domains to mediate golgi retention [56] . however, a number of other studies have indicated that considerable alterations can be made to the transmembrane domain of the native enzymes without abolishing gotgi retention. for example, colley et al [62] showed that sequential replacement of 4-5 amino acid blocks of the transmembrane domain of e2,6st had no effect on golgi localization. further, munro [54] made the striking demonstration that the transmembrane domain of an e2,6st hybrid protein (containing the stem and tail of e2,6st) can be totally replaced by a poly-leucine sequence of similar length without adversely affecting golgi retention. munro [54] also reported that the length of the polyteucine segment appeared to be important in maintaining efficient golgi localization as a transmembrane domain of 23 leucine residues showed leakage to the cell surface. in contrast to this apparent length requirement, dahdat and colley [63] replaced the 17 amino acid transmembrane domain of e2,6st with the long 29 amino acid transmembrane domain from influenza neuraminidase without any apparent disruption of the retention signal. the difficulty in defining the structural elements associated with transmembrane domains in golgi localization has been highlighted by a recent study by low et al. [64] who demonstrated that swapping the transmembrane domains of two cell surface proteins resulted in hybrid molecules which either accumulated in the golgi or were retarded in transport through the golgi apparatus. gol0jeell smc~ace (m) 6o~0~ ce~ surface (n,w) -_ ~. t 17aa i stem37aa 6 gotgi 0~w) ce~ surface (lv~ go~ce~ surface (d) thus, although both native proteins are transported efficiently to the cell surface, swapping the transmembrane domains of these two proteins altered golgi to cell surface transport. these investigators concluded that the hydrophobic transmembrane domain in relation to its charged flanking sequences is important in transport from the golgi apparatus to the cell surface. for both ct2,6st and glcnacti it is clear that regions flanking the transmembrane domain can augment the efficiency of golgi localization. for example, additional sequences from the stem region and the cytoplasmic tail increase the efficiency of golgi localization of ~2,6st and glcnacti [54, 58, 61] , although the tail and/or stem of ~2,6st and glcnacti alone is not capable of retaining a reporter molecule to the golgi 1-54, 60, 61] , and removal of the stem region from wild type c~2,6st [62] or /31,4gait [561 does not disrupt golgi localization. although the potential of the stem region has been addressed in a number of studies, the potential role of the catalytic domain has been overlooked in most studies. yet, removal of the cytoplasmic tail and stem, and considerable alteration of the signal/anchor domain, still allowed hybrid ~2,6st molecules containing the catalytic domain to be golgi localized [-62, 63] . it should be noted that the membrane flanking sequences, comprising short stretches of charged residues, were maintained in the latter constructs which may also be an important factor in localization. the contribution of the membrane flanking sequences of /~i,4galt to golgi localization is not known. comparison of the results of all these studies is not straightforward and there are a number of factors which may account for the apparent lack of agreement between them. first, as yet there is no direct evidence that glycosyltransferases localized to different golgi subcompartments are retained by identical mechanisms. there may be subtle differences between the localization signals which specify residency in medialand trans-cisternae and in the tgn. second, in the majority of studies sequences involved in golgi localization have been identified by their ability a. third, the definition of the transmembrane domain has varied from group to group; in some cases the transmembrane domain has been defined as a stretch of hydrophobic residues, excluding charged residues necessary for anchoring membrane proteins within the lipid bilayer [38, 54] , whereas in other studies, two or three charged amino acids on either side of the hydrophobic stretch have been included in the sequence defined as the transmembrane domain [55] . fourth, the appearance of hybrid or mutant glycosyltransferases at the cell surface has been frequently used as a measure of disruption of golgi localization. however, the majority of groups have only used fluorescence microscopy to compare levels of cell surface expression. fluorescence microscopy is relatively insensitive and comparisons are at best qualitative. only a few studies have employed the more sensitive and quantitative technique of flow cytometry to compare levels of cell surface expression between constructs. fifth, the expression levels of the hybrid constructs vary between and within studies. we believe this to be a critical factor in assessing these results. whereas many groups have shown that the native gtycosyltransferases can be expressed at very high levels without saturation of the golgi retention mechanism [38, 54, 55, 59] [44] . of expression have been observed between different constructs. in addition, for any one construct there is a wide range of expression within one transfection experiment, and indeed dahdal and colley [63] noted that surface expression of some hybrid proteins appeared to be related to the level of expression in the cells. in our studies on fll,4galt, we have observed that a construct expressed transiently in cos cells showed a different intraceltular distribution to that of the same construct stably expressed in mouse l cells. replacement of the 20 amino acid transmembrane domain of /?i,4galt with the 27 amino acids from the transferrin receptor resulted in abundant cell surface expression in cos cells, and with very little detected in the golgi region, whereas in stable l cells, which expressed the hybrid molecules at a 50 to 100-fold lower level, substantial amounts of the hybrid molecules were specifically retained within the golgi apparatus [66] . clearly, stable clones expressing low levels of the hybrid molecules are likely to be more informative. sixth, several groups have identified glycosyltransferase sequences which are capable of conferring golgi localization upon reporter proteins, but have neglected to assess the role played by these sequences within the context of the full length enzyme. strategies involving reporter proteins are useful for determining the minimum sequence requirements for golgi localization of hybrid molecules. however, it does not necessarily follow that a sequence which is sufficient to confer golgi localization upon a reporter molecule is the only sequence involved in retention of the native enzyme. this point was illustrated earlier with the golgi localization of glycosyltransferases bearing substituted transmembrane domains (figs 2-4) . furthermore, most of the studies which have made substitutions of the native enzyme have not assessed the effect of those substitutions on enzyme activity, thus it is unclear whether the structure of the luminal catalytic domain has been perturbed in these studies. in our recent study on glcnacti [61] we have attempted to address many of these problems and have assessed the relative contribution of the cytoplasmic tail, transmembrane domain, and catalytically active luminal domain in medial-golgi localization. stable l cell clones expressing hybrid molecules were generated, and clones which expressed equivalent amounts of hybrid proteins were selected for analyses. all hybrid molecules expressing the luminal domains of glcnacti were catalytically active, inferring a native structure for this domain. cellular localization was assessed by fluorescence microscopy, immuno-electron microscopy and flow cytometry. overall our study showed that each of the three gicnacti domains contributes significantly to medial-golgi localization. soluble, catalytically active forms of /?i,4galt and ~2,6st, which lack the cytoplasmic tail, transmembrane domain, and luminal stem region, have been detected in body fluids and are thought to be derived from the membrane-bound forms by proteolytic cleavage [67-693. when the cytoplasmic tail, transmembrane domain and luminal stem region of either /~2,6st or /~i,4galt are replaced by a cleavable signal sequence, the resulting truncated enzymes are also rapidly secreted from transfected cells [59, 70] . from these data it has been argued that the catalytic domains of glycosyltransferases do not contain golgi retention signals. however, it is entirely possible that the luminal catalytic domain can only function in golgi retention if it is anchored to the membrane. at this stage the relative contribution of the stem and catalytic domains in the localization of glycosyltransferases is unresolved. overall, it is most unlikely that golgi retention is determined by a discrete and continuous sequence motif or peptide segment, but rather localization of golgi glycosyltransferases could be mediated by interactions spanning the entire length of the molecule. what are the possible mechanisms for the compartmentspecific localization of the membrane-bound glycosyltransferases? it is unlikely that localization of glycosyltransferases involves a simple receptor-ligand interaction where the receptor is fixed in the golgi cisternae, as over-expression of wild-type transferases does not result in saturation of the retention mechanism [38, 54-56, 59, 62]. an alternative glycopinion mini-review possibility is that escaped golgi glycosyltransferases are retrieved from post-golgi compartments, as with soluble er proteins. however, experimental evidence seems to argue against a retrieval system for golgi glycosyltransferases. wong et al. [60] have demonstrated that ~2,6st leaked to the cell surface is not retrieved back to the golgi apparatus. also we [66] have demonstrated that/~i,4galt which has escaped golgi retention undergoes a post-translational modification, probably in the tgn, before appearance at the cell surface; as the golgi-localized /?1,4gait does not accumulate this modification, a retrieval system would appear unlikely to play a dominant role. what could be the basis of an active golgi retention mechanism? it has been suggested by a number of investigators that retention of golgi glycosyltransferases could be mediated by the formation of protein aggregates within the membranes of the correct golgi cisternae [71] [72] [73] . this model proposes that such oligomers or aggregates of glycosyltransferases would then be excluded from entry into vesicles for forward transport. although an attractive hypothesis, the evidence for aggregation remains largely indirect. recent elegant experiments performed by nilsson et al. [74] have shown that the addition of an er retention motif to the glcnacti cytoplasmic tail not only causes glcnacti to localize to the er but also partially retains another medial-golgi enzyme, namely ~mannosidase ii, within the er. furthermore, burke [75] has demonstrated co-precipitation of glcnactii activity, another medial-golgi enzyme, using antibodies specific to glcnacti. as the amino acid sequences of the glcnacti and glcnactii transferases are not related, a likely explanation is the association of enzymes which occupy the same golgi cisternae. warren and colleagues have coined the term 'kin recognition' to denote this self-association of golgi enzymes [72] . the proposed aggregation of golgi glycosyltransferases is also consistent with earlier observations that the majority of glcnacti and/~i,4galt exist as high molecular weight material following detergent extraction of tissue [39, 76] . how could the three domains of a glycosyltransferase play a role in aggregation? the fact that each domain of glcnacti is required for complete golgi retention implies that all three domains may be involved in the lateral interactions which lead to aggregate formation. for example, the transmembrane domains of resident golgi proteins may mediate homo-or hetero-dimerization via protein protein interactions along uncharged polar faces of c~-helixes, predicted for some of the glycosyltransferases [71] , or along one face of the c~-helix containing a leucine zipper, as predicted for glcnacti [44] . such dimers may form prior to their arrival in the golgi, as indicated by the results of er retention of glcnacti/manii [74] . these homo-or hetero-dimers may then be induced to interact, within the correct golgi microenvironment, through their large luminal domains, resulting in a two-dimensional aggregate ( fig. 5) . aggregation may be induced by differences within the golgi cisternae, such as ph and calcium concentration. this model differs somewhat from that of warren's group which proposes that the golgi enzymes form homo-dimers (via their stem regions) and interact via their transmembrane domains with different neighbours to generate linear hetero-oligomers. ~-mannosidase ii has been shown to be a disulphide-bonded dimer, but there is no evidence of stable covalent dimer formation for any of the golgi glycosyltransferases. the first report of a purified membrane-bound form of glycosyltransferase, namely /~i,4galt, indicates that no disulphide bonded dimers exist [76] , contrary to an earlier suggestion [77] . in addition, warren's model of linear heteroaggregates cannot readily explain the efficient golgi retention of hybrid molecules containing a transmembrane domain of a glycosyltransferase and a reporter molecule known to be a monomer in the native state, such as ovalbumin [38, 59, 61] . retention of such hybrid molecules could only occur at the ends of the iinear aggregates, via their transmembrane domains, and would effectively cap these linear structures resulting in only a very minimal number of hybrid molecules retained in the golgi apparatus. finally, the cytoplasmic tail of the glycosyltransferases may be necessary for either transmembrane-mediated dimerization or, as proposed by slusarewicz et al. [78] , may interact in a salt-dependent manner with a putative intercisternat matrix. consistent with this proposal, the differences in solubility of wild-type gicnacti and the glcnacti hybrid proteins indicate that golgi localized molecules may exist in a different physical state from their cell surface counterparts [61] . an interaction of the glycosyltransferases with the intercisternal matrix (the "golgi glue'), either directly or indirectly, would ensure that the aggregates are immobilized within the golgi membranes and so are excluded from transport vesicles. clearly an aggregation model of retention may involve many additional components and further biochemical analysis is now required. a model of golgi localization also needs to account for the presence of soluble catalytically active forms of fil,4galt and ~2,6st which have been detected in body fluids. the retention model proposed above could allow the release of soluble catalytic oligomers from the golgi aggregate by proteolytic cleavage, with the subsequent dissociation of the oligomers to monomers. gotgi membranes differ in lipid composition from the er and plasma membranes. such lipid differences may be important in mediating interactions between the transmembrane domains of glycosyltransferases. a lipid mediated mechanism of protein sorting has been proposed by bretscher and munro [79] who have suggested that the typically shorter transmembrane domains of golgi proteins may interact selectively with the low cholesterol bilayers of golgi membranes and be excluded from the thicker, cholesterol-sphingotipid enriched bitayers of post-golgi membranes. a protein-lipid interaction is compatible with the observations that the length of the hydrophobic stretch coupled with the adjacent flanking residues is important in gotgi retention, rather than the actual amino acid sequence. the models discussed above are by no means mutually exclusive and it is possible that the golgi retention mechanism includes both a protein-lipid interaction (via the transmembrane domains of the proteins) as well as protein-protein aggregation. based on the aggregation model of golgi retention outlined above, wild type glycosyltransferases expressed in transfected cells may be retained within golgi cisternae as a consequence of self aggregation, or by virtue of their ability to interact or 'dock' to existing aggregates within the golgi apparatus of the mouse cells. self aggregation, as opposed to docking, represents a potentially non-saturable means of retention, consistent with the many reports that glycosyltransferases expressed in heterologous cell lines do not leak to the plasma membrane even when expressed at vastly elevated levels [38, 54-56, 59, 62] . on the other hand, hybrid constructs would have a reduced capacity to self-aggregate, due to insufficient domains, and may be retained by interacting or 'docking' to existing aggregates within goigi membranes, either through their luminal domain or transmembrane domain (fig. 6) . this would be a more readily saturable means of retention, with only a finite number of exposed, endogenous molecules available as 'docking' sites. the fact that golgi localized glcnacti hybrid proteins, including those which lack the glcnacti cytoplasmic tail, are predominantly localized to medial-golgi cisternae is in agreement with this proposal [61] . thus, golgi localization of hybrid molecules probably reflects the minimum structure(s) required to 'dock' with endogenous golgi aggregates. this model would also help to explain the discrepancies between studies as the expression levels of the hybrid molecules would be an important factor in the efficiency of golgi localization. the high level of conservation of individual glycosyltransferases across species is also consistent with structural constraints imposed by such an aggregation model. a conserved structure would be required in order to preserve the many interactions with neighbouring enzyme molecules of a heteroaggregate within the golgi compartment. if golgi glycosyltransferases have evolved with a fundamental requirement for such inter-molecular interactions, it would also explain the conservation of the retention mechanism across species and also the ability of an animal glycosyltransferase to be apparently correctly housed in the golgi apparatus of plant cells [80] . while most viruses mature at the plasma membrane, a limited number of viruses acquire their envelopes by budding into intracellular compartments. viruses which assemble from golgi membranes include, coronavirus, bunyavirus and pox virus [81, 82] . viral budding from the golgi apparatus is probably determined by the targeting of one or more viral glycoproteins to the golgi membranes. indeed, a number of viral proteins have been shown to be independently targeted to the golgi apparatus, including the m glycoproteins of an avian coronavirus [83] and a related murine coronavirus [84, 85] , the e1 and e2 spike glycoproteins of rubella virus [86] and the g1 glycoprotein of punta tora virus [87] . as a consequence of the specific localization of these viral glycoproteins they represent useful tools for the study of protein targeting to the golgi apparatus. the m (formerly called el) glycoprotein of the avian coronavirus, infectious bronchitis virus (ibv), has been shown to be localized specifically to the cis-golgi cisternae [83] . in contrast to the type ii membrane orientation of the glycosyltransferases, the ibv m gtycoprotein contains a short glycosylated amino-terminal domain, three membrane spanning domains and a carboxy-terminat cytoplasmic tail. only the first of the three membrane spanning domains of m glycoprotein of ibv is required to retain this protein in the golgi [88] . furthermore, this membrane spanning domain is sufficient to confer golgi localization upon a plasma membrane localized protein [89] . thus, as for the gtycosyltransferases of the medial and trans-cisternae and the tgn, the transmembrane domain of a resident protein of the cis-cisternae has also been implicated in retention. extensive mutagenesis showed that four polar residues in the first m transmembrane domain were critical for golgi retention of a hybrid protein with the vsv g glycoprotein [90] . these four polar residues are predicted to form an uncharged polar face along one side of an c~-helix, which has potential to be involved in protein-protein interactions and mediate oligomer formation. indeed, aggregation has been shown to correlate with retention of this m hybrid protein in the golgi apparatus [91] . these investigators demonstrated that the appearance of sds-resistant aggregates of the m hybrid protein correlated with golgi localization, whereas mislocatized transmembrane domain mutants do not form oligomers. the aggregates have not been biochemically characterized but it is possible that they include endogenous golgi proteins. however, sds-resistant oligomers of the native m glycoprotein were not detected in this study [91] , thus the relationship between aggregate formation of the m hybrid molecule and golgi retention of the native m protein remains unclear. in contrast to the findings for the m glycoprotein of ibv, machamer et al. [90] in the past few years it has become apparent that there is a distinct set of resident gotgi proteins in the tgn of mammalian cells, and the late golgi of yeast, that have features associated with their localization which are distinct from the golgi glycosyltransferases [93] [94] [95] . these differences are associated with the structure of the proteins. the group includes the mammalian tgn38/41 [95] and furin [96] , and the yeast proteolytic enzymes kexlp, kex2p, and dipeptidyl aminopeptidase a (dpap a) (for review see [97] ). in contrast to the golgi glycosyltransferases, tgn38/41, furin, kextp and kex2p are type i membrane proteins, however, membrane orientation is not a distinguishing characteristic of the group as dpap a is a type ii membrane protein. in contrast to the golgi glycosyltransferases, the cytoplasmic tail of all these proteins is essential for golgi localization and, in addition, a retrieval signal plays a role in defining residence of these proteins to the tgn or late golgi. tgn38/41 is a heterodimeric membrane protein complex which cycles between the tgn and the cell surface [95, [98] [99] [100] . tgn38/41 has been shown to interact with cytosolic proteins and may be involved in the formation of exocytic vesicles from the tgn [95, [98] [99] [100] . a number of groups have demonstrated that the tetrapeptide sequence yqrl, within the 33 amino acid cytoplasmic tail of tgn 38, is both necessary and sufficient to target this membrane protein to the tgn [101] [102] [103] . this tyrosine-containing motif also acts as an internalization motif from the plasma membrane, via interaction with clathrin-coated pits. recently this tyrosine motif has been shown to lie within an ~-helix, and not a tight /~-turn conformation which is typical of other tyrosine-containing internalization motifs [104] . there is evidence that individual amino acids around the tyrosine of the tgn 38/41 internalization motif could signal different intracellular locations. for example, mutation of the yqrl sequence to yqdl abrogated tgn localization of tgn 38 but did not affect internalization [101] . recently furin, a membrane associated subtilisin-like protease, has been shown to be concentrated in the tgn [96] . like tgn38/42, furin also cycles between the cell surface and tgn. sequences of the cytoplasmic tail of furin are required for tgn targeting, and a potential tyrosine motif has been identified [96, 105] . on the other hand, potential tyrosine motifs for internalization appear to be absent in the cytoplasmic tails of gotgi glycosyltransferases. the yeast proteins dpap a, kex2p and kexlp are all integral membrane proteins with cytoplasmic tails of about 100 amino acids. these cytoplasmic tails are required for retention of these enzymes in the late golgi since deletions in the tail reduce the efficiency of retention [106] [107] [108] . the retention signals within the cytoplamic tails of these proteins have been identified and are very similar to the proposed general motif for clustering into clathrin-coated pits of animal cells (see [94, 97] ). deletion of the golgi retention signal, or over-expression of these proteins, results in mislocalization to the vacuolar compartment. this initially surprising finding has led to the conclusion that the default destination for membrane proteins in the yeast secretory pathway is the vacuolar compartment and not the plasma membrane. studies on the yeast vps mutants suggest that dpap a may leak from the late golgi and is transported, via the default pathway, to a post-golgi/pre-vacuolar compartment [-97] . the cytoplasmic localization signals of these escaped dpap a moiecules then mediate retrieval back to the late golgi; in the absence of the cytoplasmic tail golgi localization signals these membrane proteins would continue to be transported along this default pathway to vacuoles. thus there are clear similarities in the mechanism of golgi localization of these yeast proteolytic enzymes and mammalian tgn38/41 and furin. the kdel receptor resides in the cgn and possibly throughout the entire golgi stack [22, 24] . the kdel receptor is predicted to have six or seven transmembrane domains [109] . empty receptors do not recycle back to the er, however, after binding to ligand the ligand-receptor complex is then returned by retrograde transport to the er [24] . thus, this receptor is likely to have signals for goigi localization. however, mutational analysis of the kdel receptor, although defining structural features associated with ligand binding and retrograde transport, revealed very little about the nature of the putative golgi localization signal [t09]. there are a number of structural membrane proteins and proteins associated with the machinery of vesicular transport that are localized specifically to the golgi apparatus, for example/~-cop [110], rab6 and tab12 [111] , p230 [112] , p200 [113] , heterotrimeric g proteins [114, 115] , sec 7 [116] and the actin binding protein, comitin [117] (table 1) . these are not integral membrane proteins as they do not have transmembrane domains, but rather are peripheral membrane proteins associated with the cytosolic face of golgi membranes. some of these components recycle between a cytosolic pool and golgi membranes. in general very little is known about the gotgi localization signals for these peripheral membrane proteins. there is evidence that the carboxy-terminat region of the gtp binding protein g,n is required for golgi membrane binding [118] . membrane association of rab proteins requires the geranylgeranylation of one or two c-terminal cysteines [119] as well as a localization signal to define the organelle-specificity. the hypervariable c-terminus of rab proteins has been implicated in localization [120] , although a recent study on rab6 indicates that efficient localization of this rab protein to golgi membranes requires both n-terminal and c-terminal domains [121] . the identification of the precise nature of the targeting signals and the mechanism of localization of these peripheral membrane proteins will be important to the understanding of the organization of the golgi apparatus and vesicular transport. it is now apparent that the localization of resident golgi proteins includes more than one mechanism. for some late golgi membrane proteins a retrieval system operates to recycle proteins from post-golgi compartments. on the other hand, golgi glycosyltransferases appear to be actively retained within golgi membranes; there is no evidence that glycosyltransferase molecules which have leaked from the golgi apparatus can be retrieved. from many 'cut and paste' experiments it is apparent that the localization of glycosyltransferases does not involve a discrete retention signal but may be dependent on many interactions spanning the length of the molecule. furthermore, there is increasing evidence to suggest that retention of glycosyltransferases involves the formation of aggregates within the golgi apparatus. the challenge now is to biochemically characterize these aggregates, to identify any associated molecules that may be important in mediating retention, and to identify the conditions which induce aggregation. this will require the development of novel strategies to allow the isolation and biochemical analyses of individual golgi compartments, in particular the lipid composition, the organization of the resident proteins within golgi membranes, and the nature of interactions with the intercisternal matrix. thus, the problem now is understanding the biogenesis of golgi membranes themselves. the enzymes of biological membranes (martonosi an this work was supported by grants from the national health and medical research council of australia and the australian research council. key: cord-031937-qhlatg84 authors: verma, anukriti; sharda, shivani; rathi, bhawna; somvanshi, pallavi; pandey, bimlesh dhar title: elucidating potential molecular signatures through host-microbe interactions for reactive arthritis and inflammatory bowel disease using combinatorial approach date: 2020-09-15 journal: sci rep doi: 10.1038/s41598-020-71674-8 sha: doc_id: 31937 cord_uid: qhlatg84 reactive arthritis (rea), a rare seronegative inflammatory arthritis, lacks exquisite classification under rheumatic autoimmunity. rea is solely established using differential clinical diagnosis of the patient cohorts, where pathogenic triggers linked to enteric and urogenital microorganisms e.g. salmonella, shigella, yersinia, campylobacter, chlamydia have been reported. inflammatory bowel disease (ibd), an idiopathic enteric disorder co-evolved and attuned to present gut microbiome dysbiosis, can be correlated to the genesis of enteropathic arthropathies like rea. gut microbes symbolically modulate immune system homeostasis and are elementary for varied disease patterns in autoimmune disorders. the gut-microbiota axis structured on the core host-microbe interactions execute an imperative role in discerning the etiopathogenesis of rea and ibd. this study predicts the molecular signatures for rea with co-evolved ibd through the enveloped host-microbe interactions and microbe-microbe ‘interspecies communication’, using synonymous gene expression data for selective microbes. we have utilized a combinatorial approach that have concomitant in-silico work-pipeline and experimental validation to corroborate the findings. in-silico analysis involving text mining, metabolic network reconstruction, simulation, filtering, host-microbe interaction, docking and molecular mimicry studies results in robust drug target/s and biomarker/s for co-evolved ibd and rea. cross validation of the target/s or biomarker/s was done by targeted gene expression analysis following a non-probabilistic convenience sampling. studies were performed to substantiate the host-microbe disease network consisting of protein-marker-symptom/disease-pathway-drug associations resulting in possible identification of vital drug targets, biomarkers, pathways and inhibitors for ibd and rea. our study identified na((+))/h((+)) anti-porter (nhaa) and kynureninase (kynu) to be robust early and essential host-microbe interacting targets for ibd co-evolved rea. other vital host-microbe interacting genes, proteins, pathways and drugs include adenosine deaminase (ada), superoxide dismutase 2 (sod2), catalase (cat), angiotensin i converting enzyme (ace), carbon metabolism (folate biosynthesis) and methotrexate. these can serve as potential prognostic/theranostic biomarkers and signatures that can be extrapolated to stratify rea and related autoimmunity patient cohorts for further pilot studies. www.nature.com/scientificreports/ approach has advantages over the traditional approach for network analysis that can help to simultaneously characterize several protein interaction modules and has the potential to study complex diseases. the vital information obtained in our study from in-silico analysis is cross-validated through targeted gene expression experimental analysis on patient cohorts. this study will help us to obtain clinico-molecular informatics-based outcomes and expand our knowledge regarding the understanding of biological functions for ibd co-existent rea. text mining: data screening and selection. systematic data search and organization was carried out incorporating data identification, data screening and data selection to find target microorganisms involved in inflammatory bowel disease (ibd) and reactive arthritis (rea). data identification was carried out to obtain records through data sources utilising keywords (e.g. "microorganism and inflammatory bowel disease and reactive arthritis") incorporating boolean operators (and/or/not). data screening and selection were carried as part of the manual curation through primary and secondary screening scrutinizing collected data records to obtain organized records relevant for the autoimmune and enteric disorders triggered by microorganisms, especially ibd and rea and the microbial triggers implicated in ibd and rea that were utilised for further metabolic network reconstruction. bottom-up approach consisting of draft reconstruction and manual reconstruction refinement was followed to create metabolic networks of obtained target microorganisms. genome-scale metabolic models simulation, reconstruction and visualization (gemsirv) software 51 that includes reciprocal basic local alignment search tool (blast) of target microorganisms against a template metabolic network of its phylogenetic neighbour and incorporates information from national center for biotechnology information (ncbi), kyoto encyclopedia of genes and genomes (kegg) and transport db was used for creating draft reconstructs. the manual curation of missing links or gaps in the draft reconstruct was done by mapping the incomplete information to other databases such as expert protein analysis system (expasy) 52 and integrated relational enzyme database (intenz) 53 . this fully connected and annotated network was used for further simulation studies 54 . the metabolic networks thus obtained were visualized using celldesigner, a tool for modelling and editing biochemical and gene-regulatory networks. simulation analysis was carried by converting the metabolic networks obtained into a mathematical model and performing the gene deletion analysis to retrieve essential genes. model conversion was through generation of stoichiometric based matrixes consisting of reactions (columns) and metabolites (rows) corresponding to respective genes. upper boundary and lower boundary fluxes i.e. movement of matter across a system were generated for the gene associated reactions and metabolites that was extracted in systems biology markup language (sbml) format. the next step was gene deletion analysis done using the constraint based reconstruction and analysis toolbox (cobra) that runs in matrix laboratory (matlab) 55 for finding the essential genes based upon the gene-reaction matrix and boolean relationship between genes and reactions 56 . the purpose of data filtering is to remove repeats and homologs from essential genes of target microorganisms associated with ibd co-existent rea. the non-homologous protein sequences corresponding to the essential genes of target microorganisms were extracted from pathosystems resource integration (patric) database 57 . refinement of protein sequences was further done using cluster database at high identity with tolerance (cd-hit) 58 suite so as to have 60% identity non-repeat sequence tolerance stringency. blast-p was further used to remove the homologs from such non-repeats against human database at e-value of 10 -4 to obtain nonhomologous protein sequences used for further in-silico analysis. essential host-microbe and microbe-microbe interactions. the host-microbe interactions of the non-homologous proteins for the selected target microorganisms were obtained using host-pathogen interaction database (hpidb) 59, 60 . the host-microbe interactions were visualised using cytoscape. simulation analysis (gene essentiality) was done to obtain the essential host proteins interacting with common microbe proteins of microorganisms triggering ibd and rea utilising the human metabolic model hmr 2, a cobra compliant metabolic model of human consisting of around 3,765 genes, 8,000 reactions and 3,000 metabolites 61 . this led to profiling of the common host-microbe and microbe-microbe interactions comprehending the complex 'interspecies communication' as complex interaction maps, executed using search tool for the retrieval of interacting genes/proteins (string) 62,63 . host-microbe disease network and molecular mimicry studies. the host-microbe disease network is a multilayered archetype that connects the protein-marker-symptom/disease-drug-pathway associations. the contributions of the microorganisms in the co-evolved ibd and rea as part of the disease network was created through the interactive maps of the essential host interaction proteins (verified using literature survey) and the information processed through gene expression data analysis 64 . the information patronised here is mostly scored through the available non-specific protein diagnostic markers of both ibd and rea e.g. c-reactive protein (crp), interleukin 6 (il6) and toll like receptor 4 (tlr4), major histocompatibility complex, class i, b (hla-b) and major histocompatibility complex, class ii, dr beta 1 (hla-drb1) with the essential host proteins determined using string 65 . database genecards 66 was used to assess the role of these interacting partners aka proteins further with symptoms/diseases associated with ibd and rea. the pathways of the above host interacting proteins were found out using kegg database that provides ontologies for proteins related to biological processes 67 www.nature.com/scientificreports/ subsequently, the role of drugs or inhibitors used to suppress the effect of ibd and rea such as indomethacin, prednisone, ciprofloxacin, sulfasalazine, azathioprine, methotrexate and hydroxychloroquine was scored in the disease network through their docking studies against the potential targets (both host as well microbial targets) as per published methodologies 68, 69 . the host-microbe disease network which is an amalgamation of all the above patterned associations was visualized using cytoscape software 70 . molecular mimicry analysis between the vital targets triggering ibd co-evolved rea, essential human proteins including hla-b27, hla-b51 and hla-drb1 was done using data repository expasy. this led to retrieval of microbe relayed protein sequences that have been implicated in disease development after sequence alignment performed using emboss 71 . experimental evidences to identify the signature molecules in patient samples. the cross-validation of vital in-silico targets was done in rea patient cohort cases via targeted gene expression analysis. scientific and ethical clearance was taken from amity university ethics committee and institutional ethics committee, fortis noida for handling the patient samples. all experiments were performed in accordance with indian council of medical research (icmr) guidelines constituting the ethics committees. the study was carried out for 6 months on the rare disorder rea patients, with the inclusion criteria as patients having rea according to european spondyloarthropathy study group (essg) 72 and exclusion criteria as patients undergoing treatment from last 3-6 months and healthy controls (hc). the participants were inducted in the study design with an informed consent form along with a questionnaire containing information regarding symptomatic and diagnostic history of patient and linked disorders. blood (5 ml) was drawn from participants in ethylenediaminetetraacetic acid (edta) vacutainers. these were transported to the laboratory for further analysis. the processing of the samples was done within 2-4 h of procurement 73 . peripheral blood mononuclear cells (pbmc's) were isolated from blood using density gradient centrifugation 74 . rna was isolated from pbmc's using trizol method 75 . the quantification of rna was done using nano-drop 76 . the high capacity cdna reverse transcription kit (applied biosystems™) was used for conversion of rna to single-stranded cdna as per the standard protocol 77 . quantitative pcr analysis of target gene was executed using biorad cfx96 real time-pcr taking human housekeeping gene, gapdh as a reference. previously reported primers for qpcr analysis of target and reference gene were selected for this study 78, 79 following the standard protocol 80 . relative gene expression analysis from qpcr data was performed using the relative expression software tool (rest® 2009) 81 that utilises the expression of reference genes to normalize expression of target genes in different samples. the schematic representation of methodology involved in our combinatorial analysis is provided in fig. 1 . text mining: data screening and selection. a systematic literature mining and curation for our thematic connecting autoimmune disorders, inflammatory bowel disease (ibd) and reactive arthritis (rea) was carried out. data identification extracted 1,071 records (articles in journals, book chapters, conference papers etc.) corresponding to autoimmune and enteric disorders. data screening extracted 426 records of autoimmune and enteric disorders triggered by microorganisms that belong to class of bacteria, fungi, protozoan, mites, virus, yeast and nematode. data selection yielded 48 ibd, 32 rea and 5 ibd co-evolved rea records. data selection was directed towards the microbial contenders implicated here resulting in 6 target microorganisms namely campylobacter jejuni, escherichia coli o157:h7, klebsiella oxytoca, salmonella typhimurium, shigella dysenteriae and yersinia enterocolitica, whose genome information was available. the etiopathogenesis in the co-evolved disorders have been documented through gut microbiome associated host-pathogen interactions studies, perpetuating where pathogen microorganisms involve in dysbiosis leading to autoimmunity. the results of text mining are provided in fig. 2 . the list of microorganisms is provided in supplementary table s1 online. ing of genes along with their corresponding proteins, reactions and metabolites for the selected microorganisms serve as primary set of partial metabolic network information. the missing data persistent in the draft reconstruct obtained through genome-scale metabolic models simulation, reconstruction and visualization (gem-sirv) was manually refined. entirely associated metabolic networks of target microorganisms were obtained (genes, proteins and reactions). the essential genes of microorganisms (vital for survival. sustenance and growth) were obtained after performing simulation on mathematical models consisting of gene associated reactions and metabolites (metabolites, inner cell reactions, exchange reactions and essential genes). due to lack of availability of exchange reactions for campylobacter jejuni, simulation analysis on the partial metabolic network could not be carried out and essential genes could not be retrieved. an alternative approach for finding essential genes of campylobacter jejuni was carried out. the essential genes of campylobacter jejuni were taken from our previous published report and were found out to be 228 69 . table 1 portrays the results of metabolic network reconstruction and simulation of target microorganisms. the metabolic network and simulation analysis data of target microorganisms is provided in supplementary table s2 online. the proteins corresponding to essential genes, non-repeats and non-homologs were obtained as stated below according to the parenthesis {proteins corresponding to essential genes, non-repeats, non-homologs}. the essential genes, their corresponding proteins, reactions and metabolites from the curated dataset were refined to create a list of most relevant molecular indicators to assess their coveted role in disease establishment. the non-redundant filtered proteins were utilised further in the computational work-pipeline canvassing the drug targets and signatures in the interspecies communication. essential host-microbe and microbe-microbe interactions. the central mechanism of hostmicrobe/microbe interface conferred through gut microbiome was correlated for the selected microbial species and processed to obtain the common signatures so as to follow the core system of metabolic changes affecting the host harbouring them as either commensal or pathogenic loads. the interactors between human and target microorganisms were obtained. the interactors of escherichia coli o157:h7 were 136; klebsiella oxytoca were 141; salmonella typhimurium were 136; shigella dysenteriae were 117 and yersinia enterocolitica were 133. there were no interactors for campylobacter jejuni (supplementary table s3 -s7 online). table 2 shows the results of filtering and host-microbe interactions of protein sequences corresponding to essential genes of target microorganisms. www.nature.com/scientificreports/ the host-microbe interactors were analysed for all the target microbial species and processed to obtain the common signatures. 43 proteins were found between all target microorganisms having interaction among themselves and with 130 human proteins. the essential host correlative targets to the microbial gene targets were followed by obtaining host essential genes and corresponding proteins from human metabolic model hmr 2. there were 1,401 essential proteins (supplementary table s8 online) the essential human protein was found out to be kynu having interaction with essential microbial protein nhaa (fig. 3) . nhaa was also having interactions with non-essential hcls1 associated protein x-1 (hax1), prolyl endopeptidase-like (ppcel), biogenesis of lysosomal organelles complex 3 subunit 1 (hps1) and eukaryotic translation initiation factor 2 alpha kinase 1 (e2ak1) proteins of human host. kynu was further mapped with host proteins (direct and indirect) resulting in 1994 interactions. out of these the single connected essential protein interactions were 988 and protein interactors were 412 ( fig. 4 and see supplementary table s9 online). the research design here followed to assess the interaction map of essential proteins in human host to indicate the clinical insights in pathophysiological trends in the autoimmune development. host-microbe disease network and molecular mimicry. the human essential proteome complement with its interacting proteins were analysed further as part of the disease network. 394 human essential protein interactors were found to be associated with ibd and similarly 3 essential protein interactors namely adenosine supplementary table s10 online) . these 397 proteins can be postulated as probable contenders transcending their role in the simulated network as important regulators in the co-existent disorders. the composite associations of the above 397 proteins with non-specific protein diagnostic markers of ibd and rea were obtained (see supplementary table s11 online) . this gave rise to a single connected protein network consisting of 402 proteins and 13,350 interactions. the association of above 402 with symptoms and diseases linked with ibd and rea were obtained (see supplementary table s12 online) . apart from non-specific diagnostic markers, the major protein linked with majority of symptoms/diseases is angiotensin i converting enzyme (ace). 78 pathways of the 402 proteins were obtained (see supplementary table s13 online) in total out of which the pathway associated with majority of proteins was carbon metabolism. another layer of disease network substantiates the role of therapeutic regime followed in the studied autoimmune diseases, so the docking analysis of drugs used to suppress the effect of ibd and rea against nhaa of target microorganisms and kynu of human host was done. the docking analysis resulted in docking scores that represent binding of drugs with host kynu and microbial nhaa of all 5 microorganisms selected in our study. higher the negative docking score more is the binding 68 . escherichia coli o157:h7 nhaa shows highest and lowest docking score with methotrexate (− 7.362) and azathioprine (− 3.491); klebsiella oxytoca nhaa with methotrexate (− 5.083) and azathioprine (− 3.459); salmonella typhimurium nhaa with ciprofloxacin (− 5.135) and hydroxychloroquine (− 2.597); shigella dysenteriae nhaa with methotrexate (− 8.059) and azathioprine (− 3.847); yersinia enterocolitica nhaa with hydroxychloroquine (− 7.47) and azathioprine (− 3.451) and human kynu with hydroxychloroquine (− 5.357) and indomethacin (1.113). our results portray methotrexate to have highest docking scores with maximum proteins and therefore can be considered as a vital drug for ibd associated rea. the resultant docking scores are provided in fig. 5 . the extensive interaction pattern of nhaa with kynu along with 396 proteins, 5 markers, 66 symptoms/ diseases, 78 pathways and 7 drugs give rise to a host-microbe disease network of ibd co-existent rea (fig. 6 and see supplementary table s14 online) . the final league of information processed in this study design was to accommodate the concept of molecular mimicry between the essential host proteins and selected microorganisms. nhaa protein of target microorganisms shows homology with human hla-b27, hla-b51 and hla-drb1 (fig. 7) . peptides homologous to hla-b27: peptides homologous to hla-drb1: experimental evidences to identify the signature molecules in patients. the in-silico analysis followed for the molecular signature identification till far through gene expression datasets and curated metabolic reconstructs strongly indicate the host protein, kynu being the singular common predictive markers for all pathogenic microbes. kynu has also been indicated in the expression data of inflammatory linked disorder, www.nature.com/scientificreports/ ibd. there is lack of data available regarding kynu differential expression in rea, therefore the experimental evaluation of kynu through targeted expression analysis in rea patients was carried out. a non-probabilistic convenience sampling was followed for our single blind study. this study encompassed 15 individuals: 60% male with mean age of 45.7 and 40% female with mean age of 38 (9 males and 6 females). out of these cases were: 10 with rea and controls were: 3 currently undergoing treatment, 1 with poncet's disease (pd) and 1 healthy control (hc). the clinical characteristics of the patients recruited in the study included inflammatory back pain in 33%, fatigue in 60%, fever in 27%, swollen joint in 47%, ankylosing spondylitis (as) that affects spine in 7%, dactylitis that is inflammation in finger or toe in 7% and poncet's disease (pd) in 7% of participants. the clinical characteristics of the recruits are provided in table 3 . the expression of kynu in peripheral blood mononuclear cells (pbmc's) of rea cases vs controls was evaluated using relative expression software tool (rest) software that estimated a sample's relative expression ratio in relation to the control housekeeping gene (here gapdh) by calculating an intermediate absolute concentration value: where cp = point at which fluorescence escalates considerably above the background fluorescence. here the cp values for reference and target genes are collectively redistributed to control and sample groups and the expression ratios are calculated based on the mean value. a pair wise fixed reallocation randomisation test is followed for normalisation of the target genes with a reference gene and for calculating the statistical difference of variation between 2 groups 81 . it utilises a bootstrapping technique providing a 95% confidence interval for expression ratios. it uses a p(h1) test for testing the significance between the samples and controls. according to our analysis, kynu sample group is different to control group where p(h1) = 0.025. kynu was found to be downregulated in sample group (in comparison to control group) by a mean factor of 0.115 (standard error range is 0.018-0.837) as depicted in the whisker-box plot (fig. 8) . kynu expression showed a ~ ninefold decline in rea cases as compared to controls. gut microbiome is pitched to be the central theme housing enormous diversity of microbial species, characterizing the fine balance between healthy and diseased states. the physiological drifts from healthy to diseased and vice-versa is tuned to sophisticated interactive networks of human host and the microbial flora residing the gut. the autoimmune conditions reactive arthritis (rea) and inflammatory bowel disease (ibd) have been linked to prevalent dysbiosis of the gut, where disease development occurs as a perceptive reaction due invading population of microbes. to find out the basal networks of interactions at the host-microbe interface, common microbes affecting the co-evolved diseases with shared characteristics were studied. these involved comprehensive analysis of the bimolecular functional networks including the gene, protein, metabolite molecular signatures engraved at the host-microbe and microbe-microbe interface. this 'interspecies communication' have been linked now with immuno-pathogenesis of most human autoimmune disorders 82, 83 . www.nature.com/scientificreports/ the etiopathology of these interactions have remained elusive leading to non-specific diagnostic criteria and therapeutic regimes. it is suggested that microbial dysbiosis, pathogenic infection and host-microbe interactions cause incidence of rea. in this study, utilising the combinatorial approach we have compiled a repertoire of microorganisms, biomolecules and pathways that are possibly involved in triggering co-evolved autoimmune disorders ibd and rea. in our study, text mining results convey the presence of microorganisms namely campylobacter jejuni, escherichia coli o157:h7, klebsiella oxytoca, salmonella typhimurium, shigella dysenteriae and yersinia enterocolitica implicated in both the disorders. the thematic concepts for microbe contribution in host immunity have been explored in our previous analysis of metabolic reconstruction and simulation of campylobacter jejuni and salmonella enterica 69, 84 . in our current study, we used a designated work-pipeline for metabolic network reconstruction and simulation of target microorganisms. the analysis conducted extracted the information via constraint-based bottom-up approach that was filtered and utilised for further computational analysis. the essential genes, proteins and metabolites of microorganisms represent the promising drug targets as these are speculated to contribute towards infection triggered host physiological drifts leading to development of the co-evolved pattern of autoimmunity in ibd and rea. a thorough curation pattern followed led to provide robust molecular cues in terms of essential proteins and biological networks that are correlated to the 'interspecies communication' using the host-microbe and microbemicrobe interaction profiling. the most closely associated common protein observed in all the selected common microbial species involved in both ibd and rea is na (+) /h (+) antiporter (nhaa), microbial integral membrane protein, catalyzing the exchange of 2 h (+) per na (+)85 and involved in processes crucial for cell viability. similarly, the common host interacting protein with nhaa is kynureninase (kynu), involved in tryptophan metabolism and whose differential expression (upregulation and downregulation based on the control samples) have been followed in ibd patient cohorts [86] [87] [88] . as per the scientific discourse presented in the studied disorders, the pathological mechanism hypothesizes that after bacterial infection, antigen-presenting cells transport bacterial antigens/peptides into the synovial membrane, where the bacterial components persist causing inflammation. it is suggested that in host-microbe interactions, bacterial proteins entering host cells interact with host proteins and inject their effector components, but has not been proven in rea and ibd. so, this formed a basis of one of the parameters in our study design where we found the physical interactions between nhaa and kynu and predicted that these might be the early host-microbe interactors for establishing pathogenesis in ibd associated rea. this could assist to comprehend the very few reports indicated in the rare autoimmune rea, where gene expression datasets of the co-evolved disorder ibd can serve to incorporate the larger theme of gut-microbiome associations. the theme of gut-microbiome paradigm shifts thus contemplates the vital cues in triggering autoimmunity with indirect linkages to diet and environmental triggers. this is indicative of the identified target molecular signature, kynu, found to be differentially regulated in the patient cohorts with history of infection triggered or ibd co-evolved rea. kynu and nhaa could serve as the robust early and essential host-microbe interacting targets and molecular indicators involved in interspecies communication in ibd associated rea. the investigations further were targeted for parallel analysis of other host-essential protein partners enmeshed to have interaction with host protein kynu indicating the intricate details of host-microbe interaction information. the disease network constructed through our approach consists of 412 single connected essential protein interactors of kynu, where 394 human essential protein interactors are found to be associated with ibd, while 3 of them (adenosine deaminase (ada), catalase (cat) and superoxide dismutase 2 (sod2)) are associated with both ibd and rea. ada protein has been reported in juvenile idiopathic arthritis and rea patient cohorts in serum samples 89 . similarly, cat and manganese superoxide dismutase (sod) genes polymorphisms were observed in rea patient cohorts 90, 91 . these become part of the host-microbe disease network where such molecular elements and co-regulatory pathways represent the intricate biological cross-talk followed during disease development. pathological conditions can also trigger immune cells such as il's and tlr's and various cytokines leading to immune cell infiltration in host and higher levels of inflammation. genetic factors such as hla alleles encode susceptibility, contribute to bacterial persistence and increase risk in rea cases. based on this we also found the interactions of important targets in our study with immunogenic and genetic factors. the host harboured assorted essential proteins were further probed for their association with non-specific protein diagnostic markers as well as with symptoms/diseases linked with ibd and rea, accruing towards a single connected network consisting of 402 interdependent proteins. the reciprocation of these integrated protein indicators to the disease development is conveyed through metabolite monitoring as in the study, angiotensin i converting enzyme (ace) was found to be linked with maximum symptoms/diseases. ace is involved in catalyzing the conversion of angiotensin i into angiotensin ii that is a potent vasopressor and aldosterone-stimulating peptide that controls blood pressure and fluid-electrolyte balance 92 . this could be the indicator of involvement of microbe triggered host physiological drifts. subsequently, the pathways associated with the proteins ramified into 78 pathways of human host speculated to give details of metabolic regulatory checkpoints where carbon metabolism is found to be associated with majority of deduced proteins. carbon metabolism pathway implicated here as the vitally generic pathway for ibd co-related rea confers how diet, balance of gut microbiome, antibiotic exposures can have layered impact on autoimmune disease progression and remissions. kynu is found to be downregulated in rea patients as compared to controls through our targeted gene expression analysis. collectively, the disease network followed here confers interaction of microbial nhaa with host kynu, that is further correlated to 396 proteins, 5 markers, 66 symptoms/diseases, 78 pathways and 7 drugs. docking analysis of drugs used to suppress the effect of ibd and rea predicts methotrexate as an important drug that could be useful for early treatment of ibd co-evolved rea. www.nature.com/scientificreports/ genetic factors found common in both rea and ibd are hla-b27, hla-b51 and hla-drb1. the most important mechanism of susceptibility of hla in rea is molecular mimicry that is microbial peptides mimicking hla autopeptides of human host leading to autoimmunity. this mechanism has been observed in rea where reports have predicted microorganism peptides such as chlamydial proteins (clpc, nqra and dnap) and yersinia pseudotuberculosis peptides (yoph) showing homology with human hla-b27 via bioinformatic analysis 14 . similarly, molecular mimicry has also been observed in ibd cases having extraintestinal manifestations. we performed targeted molecular mimicry analysis in our study using our robust microbial protein (nhaa) with hla-b27, hla-b51 and hla-drb1, enhancing the importance of nhaa acting as a trigger for generating ibd associated rea. we generate a putative hypothesis amalgamating key findings with literature. we state that the initial hostmicrobe triggers for ibd associated rea is when pathogenic microbial protein nhaa interacts with host protein kynu that further interacts with human proteins ada, sod2, cat and ace and carbon metabolism involving the above host proteins is hampered. methotrexate regulates carbon metabolism and the associated host-microbe proteins reducing effect of ibd associated rea. since carbon metabolism is the most basic aspect of life and therefore an extensive network consisting of sub-pathways, we narrowed down our findings towards a consequentially central and a significant pathway that embrace the carbon metabolism pathway involving the molecular signatures kynu, ada, sod2, cat and ace, further is also effectuated by potential drug methotrexate and is associated with ibd/ rea/ ibd and rea cohorts. it is reported that methotrexate is incorporated intracellularly interfering with adenosine concentrations and affecting proinflammatory cytokines in ibd reducing inflammation 93 . in inflammatory arthritis, the mechanisms reported by which methotrexate reduces inflammation include enhanced adenosine release, de novo synthesis of purines and pyrimidines, inhibition of transmethylation reactions, diminished accumulation of polyamines and nitric oxide synthase uncoupling. most of the mechanisms are associated with folate biosynthesis, a type of carbon metabolism 94 . kynu, ada, sod2, cat and ace are also found to be involved in folate biosynthesis and metabolism from genecards. apart from the above targets, parallel interactors, pathways and drugs for ibd co-evolved rea obtained in our host-microbe disease network can be utilised further as disease determinants. the experimental validation of these targets in patient cohorts need to be performed on a pilot scale in future to increase the robustness of this network. the intertwined information processed through the knowledge-base created for the linked disorders have given the most elaborate layout of patterns observed in disease diagnosis and analysis. the major information after processing the gene expression profiles, protein markers, molecular networks and metabolic networks involved here have led to chalk out as well as connect the strings for robust gut microbiome paradigm shifts. the current work on host-microbe interactions provides a starting point for researchers and clinicians to investigate inflammatory bowel disease (ibd) associated reactive arthritis (rea). in this study a combinatorial approach is utilised to reveal the interactions of gut microbes with human host extensively sketched through the work-pipeline providing the vital insights for the drug targets, biomarkers, pathways and inhibitors for etiology, prognosis, diagnosis and treatment attributes of pathogenic rheumatic autoimmunity. the information sorted through the combinatorial study will be useful in deciphering the etiopathogenesis of the co-linked disorders especially for the rare rea, from synonymous analyses of ibd datasets, conferred through common microbial triggers. these predictions substantially furnish the intricate details of the cross-talk between post-infectious inflammatory reactions with shared patho-immunogenesis as the starting point for researchers and clinicians for detailed and newer experimental analysis. future studies are required on larger cohort of patients having rea due to ibd in 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superoxide dismutase genes polymorphisms in reactive arthritis coronavirus disease 2019 (covid-19): do angiotensin-converting enzyme inhibitors/angiotensin receptor blockers have a biphasic effect the current role of methotrexate in patients with inflammatory bowel disease methotrexate and its mechanisms of action in inflammatory arthritis the authors are grateful to amity institute of biotechnology, amity university uttar pradesh, noida and department of biotechnology, teri school of advanced studies, new delhi for providing the facility and technical support during the preparation of the manuscript. we also thank fortis hospital, noida for providing the patient samples. s.s. and b.r. conceived the study concept; s.s., b.r. and p.s. jointly designed and supervised the work; b.d.p. supervised the clinical setting and recruitment of participants; b.d.p. and a.v. recruited the participants and contributed to the sample collection and preparation; a.v. performed the experiments; s.s., b.r., p.s. and a.v. contributed to the analysis and interpretation of data; a.v. generated all figures and tables; a.v. wrote the first draft of the manuscript; s.s., b.r., p.s. and b.d.p. critically reviewed and edited the manuscript; all authors reviewed and approved the final version of the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-71674 -8.correspondence and requests for materials should be addressed to s.s.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-001898-ntqyjqqk authors: huang, chih-wei; lin, hui-chen; chou, chi-yuan; kao, wei-chuo; chou, wei-yuan; lee, hwei-jen title: lys-315 at the interfaces of diagonal subunits of δ-crystallin plays a critical role in the reversibility of folding and subunit assembly date: 2016-01-05 journal: plos one doi: 10.1371/journal.pone.0145957 sha: doc_id: 1898 cord_uid: ntqyjqqk δ-crystallin is the major structural protein in avian eye lenses and is homologous to the urea cycle enzyme argininosuccinate lyase. this protein is structurally assembled as double dimers. lys-315 is the only residue which is arranged symmetrically at the diagonal subunit interfaces to interact with each other. this study found that wild-type protein had both dimers and monomers present in 2–4 m urea whilst only monomers of the k315a mutant were observed under the same conditions, as judged by sedimentation velocity analysis. the assembly of monomeric k315a mutant was reversible in contrast to wild-type protein. molecular dynamics simulations showed that the dissociation of primary dimers is prior to the diagonal dimers in wild-type protein. these results suggest the critical role of lys-315 in stabilization of the diagonal dimer structure. guanidinium hydrochloride (gdmcl) denatured wild-type or k315a mutant protein did not fold into functional protein. however, the urea dissociated monomers of k315a mutant protein in gdmcl were reversible folding through a multiple steps mechanism as measured by tryptophan and ans fluorescence. two partly unfolded intermediates were detected in the pathway. refolding of the intermediates resulted in a conformation with greater amounts of hydrophobic regions exposed which was prone to the formation of protein aggregates. the formation of aggregates was not prevented by the addition of α-crystallin. these results highlight that the conformational status of the monomers is critical for determining whether reversible oligomerization or aggregate formation occurs. introduction δ-crystallin is a taxon-specific eye lens protein. it is the major soluble protein in the eye lens of reptiles and birds and functions as a structural protein to maintain the refraction properties of the lens [1, 2] . δ-crystallin and argininosuccinate lyase (asl) are homologous proteins. asl is in this study, the effects of this interaction on the folding pathway of wild-type and mutant proteins were investigated using urea as a denaturant. the different distributions of dissociated component from wild-type and mutant proteins, as measured by sedimentation velocity experiment, suggests the quaternary structure dissociates in different ways for wild-type and mutant proteins. structural simulation supports different dissociation processes for the two proteins. these results highlight the critical role of k315 in stabilizing the quaternary structure of δ-crystallin. the residue appears to control both the dissociation of dimers into monomers and the stability of the produced monomers. the monomers dissociated from the k315a mutant protein with a stable and compact conformation provided a good model for studying the folding mechanism of the δ-crystallin. this study reveals the conformational status of the monomers, which determines whether functional protein or aggregates are formed. the recombinant wild-type and the k315a mutant δ-crystallin or αa-crystallin plasmid were transformed and over-expressed in e. coli bl21 (de3) with induction by isopropyl-β-d-thiogalactopyranoside (iptg). proteins were purified as previously described [8, 17] . the supernatants of crude cell extracts were loaded onto a q-sepharose anion exchange column (hiprep 16/10 q xl, ge healthcare) pre-equilibrated in buffer a (50 mm tris-hcl buffer, ph 7.5) and eluted with a linear gradient of 0 to 0.4 m nacl in buffer a. recombinant protein was eluted at approximately 0.15 m nacl. the eluted protein was pooled and treated with ammonium sulfate to 1.2 m. after filtration, the sample was loaded onto a hydrophobic interaction column (source™ 15phe) pre-equilibrated in buffer a containing 10% (v/v) glycerol and 1.2 m ammonium sulfate and eluted with a linear gradient to the same buffer lacking ammonium sulfate. the retained proteins were eluted at~0.3 m ammonium sulfate. fractions were pooled and loaded onto s-300 sephacryl column (26 mm x 85 cm) pre-equilibrated in 50 mm tris-acetic acid buffer, ph 7.5. fractions were analyzed by sds-page and protein concentrations determined by the method of bradford [18] . proteins possessing a c-terminal his 6 tag were purified on ni affinity column (chelating sepharose ff, ge healthcare) then desalted using a sephadex g-25 column (26 mm x 12 cm) as previously reported [11] . equilibrium unfolding experiments were carried out by overnight incubation of δ-crystallin with various concentrations of urea or gdmcl in 50 mm tris-acetic acid buffer, ph 7.5 at 25°c. the refolding experiments were undertaken by dilution of equilibrium-denatured δ-crystallin to a series of urea or gdmcl concentrations in the same buffer. the experiments for equilibrium unfolding of monomeric δ-crystallin were undertaken by overnight incubation of δ-crystallin in 1.5 m urea at 25°c followed by addition of various concentrations of gdmcl to the solution followed by incubation for 2 hrs. the refolding experiments were carried out by dilution of the denatured δ-crystallin into a solution containing 1.5 m urea and 0.8, 3 or 5 m gdmcl in 50 mm tris-acetic acid buffer, ph 7.5. to analyze the conformation and quaternary structure of the refolded δ-crystallin, the refolding experiments were undertaken by 20-fold dilution of the denatured monomeric δ-crystallin with buffer. the transition region in fig 2b for the reversible dissociation of k315a mutant δ-crystallin in urea solution was analyzed using the following method. the unfolding process was described as a two-state transition for the conversion of the tetramer (t) into monomers (m). the thermodynamic parameters were obtained by global fitting of the tryptophan fluorescence signal to eq 1 [19] : where y is the observed signal from tryptophan fluorescence, y n and y u are the signals in the folded and unfolded states, and m f and m u are the slopes of the baselines preceding and following the transition region. δg u 0 is the free energy difference in the absence of urea and m is the variation in the free energy of unfolding with the urea concentration. reversible unfolding of monomeric k315a mutant δ-crystallin were described as a four state process (m$i 1 $i 2 $u). the unfolding curve from tryptophan fluorescence was analyzed using a four-state unfolding model described as the transition from n to u with two intermediates, i 1 and i 2 , in the process. the thermodynamic parameters were calculated by fitting the data to eq 2 [8, 20] : where y i1 and y i2 are the signals in the i 1 and i 2 states. δg 1 0 , δg 2 0 and δg 3 0 are the free energy differences in the absence of gdmcl for n to i 1 , i 1 to i 2 and i 2 to u transition, respectively, and m 1 , m 2 and m 3 are the variation in the free energy of unfolding with the gdmcl concentration. enzymatic activity assay δ-crystallin was assayed for asl activity by monitoring the absorption of fumarate at 240 nm in a perkin-elmer lambda 40 spectrophotometer. assays were performed at least in triplicate in 50 mm tris-hcl buffer (ph 7.5) with 1 mm sodium argininosuccinate as substrate. a molar absorption coefficient of 2.44 x 10 3 m -1 cm -1 was used for all calculations [21] . the fluorescence spectra were measured on a perkin-elmer ls-50 luminescence spectrophotometer at 25°c. intrinsic tryptophan fluorescence spectra of the protein were recorded with excitation wavelength set at 295 nm and using 5 nm band-width for both excitation and emission wavelength. all spectra were corrected for buffer or denaturant absorption. the average emission wavelength was utilized for data analysis [22] . the ans (1-anilinonaphthalene-8-sulfonic acid) (molecular probes; eugene, oregon) was used as probe to bind with proteins and the fluorescence was recorded from 450 to 550 nm the circular dichroism (cd) spectra were measured on a jasco j-810 spectropolarimeter at 25°c. experiments were performed in 20 mm tris-acetic acid buffer (ph 7.5) with a 1 mm path-length over a wavelength range from 200 to 250 nm. all spectra were averaged from three accumulations and were buffer corrected. the observed ellipticity (θ) (degrees) was converted into the mean residue ellipticity [θ] by the equation: [θ] = θ×m mrw /10×d×c [23] , where m mrw is the mean residue weight, d is the light path (cm), and c is the concentration of protein in g/ml. the protein sedimentation were performed on a beckman-coulter (palo alto, ca) xl-a analytical ultracentrifuge (auc) with an an50 ti rotor at 20°c with 130 000 g in standard double sectors aluminum centerpieces. the radial scans were recorded with a time interval of 7-min and a step size of 0.003 cm. all data were fitted to the continuous c(s) distribution model and a continuous size-distribution with respect to frictional ratio (f/f o ) model using the sedfit program [24, 25] . the partial specific volume of the protein, solvent density and viscosity were calculated using the sednterp program [26] . the solvent density and viscosity of varied urea concentrations were calculated and included in the fitting. electrophoresis was performed using a phastsystem (ge healthcare). the samples were subjected a phastgel 4-15% gradient gel which contains the native buffer strip (0.88 m l-alanine, 0.25 m tris/hcl ph 8.8) attached to the surface of the gel and both electrodes. the electrophoresis was carried out at 10 ma and 15°c for 400 vh. after electrophoresis, the gel was fixed in 20% (w/v) trichloroacetic acid and stained with coomassie brilliant blue r250. the turbidity of protein solution was measured using a perkinelmer lambda 40 spectrophotometer equipped with a peltier temperature control accessory to monitor the light scattering at 360 nm. all measurements were carried out at 25°c. spectra were corrected using measurements recorded for native protein in the absence of denaturants. the aggregation rate was calculated by fitting the data to the single or double exponential equation: where y t is the signal at time t, y 0 is the signal of the final state, y i is the change in amplitude, and k i is the rate constant for aggregation. data for the linear increase in signals was fitted to a linear equation using sigmaplot 10. the assay was performed by setting the excitation wavelength at 440 nm and measuring the emission spectrum from 460 to 600 nm. proteins unfolded in denaturant were diluted into 50 mm tris-acetic acid buffer (ph 7.5) to give 0.05 mg/ml of protein in the presence of thioflavin t (tht) (50 μm). the spectrum of tht alone was used as a correction for each assay. the crystal structure of goose δ-crystallin (pdb code: 1xwo) with all water molecule removed was subjected to the charmm force field and energy minimized with the smart minimization algorithm to satisfy (rms gradient~0.1 kcal/mol/å). the implicit solvent model of generalized born was included in the calculation. the structural model was used as a template to build the k315a mutant model using the build mutant protocol (accelrys discovery studio 3.5, accelrys inc.). the best scoring model conformation was selected for energy minimization. molecular dynamics (md) simulations were performed using the standard dynamics cascade protocol. the structures of wild-type and mutant δ-crystallin in the charmm force field were subjected to initial energy minimization for 500 steps by steepest descent followed by a conjugate gradient for 500 steps. the minimized models were then heated from 50 to 300 k in 2 ps md simulations followed by equilibration for 2 ps at 300 k in the absence of any structural restraint. finally, the equilibrated models were submitted to md simulations for 100 ps at nvt (constant number of particles, volume, and temperature) using the berendsen coupling algorithm [27] . wild-type and k315a mutant δ-crystallin purified to near homogeneity were used for all analysis. equilibrium unfolding experiments were conducted by incubation of proteins in buffer supplemented with different gdmcl or urea concentrations overnight. tryptophan fluorescence was used to monitor the conformational changes during the unfolding process in the microenvironment around the tryptophan residues (fig 2a and 2b ). there are two tryptophan residues, w74 and w169, in the structure of δ-crystallin. they are located in the solvent accessible domain 1 and the helix bundle of domain 2, respectively ( fig 1a and 1b) . unfolding of the wild-type and k315a mutant protein follows a multistep process in gdmcl solution and was not reversible after 20-fold dilution (fig 2a) . as shown in fig 2a, the dramatic changes in the signal for the first transition were due to subunit dissociation as reported previously, with the gdmcl concentrations for half transition ([d] 1/2 ) at 1 ± 0.05 and 0.5 ± 0.01 m for wildtype and k315a mutant protein, respectively [28] . unfolding of the two proteins in urea solution followed a three-state process, with the [d] 1/2 values in the first transition at 3.6 ± 0.1 and 1.7 ± 0.1 m urea, respectively ( fig 2b) [8, 12] . the differences in the denaturant concentration required for the half transition clearly show the potency of gdmcl when disrupting of the conformation of δ-crystallin [11] . in the presence of urea, the k315a mutant protein was dissociated and stayed in a stable conformation between 2 and 5 m urea. the conformation was more stable than for wild-type protein at urea concentrations exceeding 4 m. when the urea was removed by 20-fold dilution into buffer, the denatured mutant protein was able to refold into a conformation similar to the original state (fig 2b) . in contrast, dilution of 3.5 or 4 m urea denatured wild-type δ-crystallin or 6 m urea denatured k315a mutant protein did not result in the restoration of the properly folded conformation. these results suggest that the reversible assembly of the quaternary structure of δ-crystallin is correlated with the conformation of the dissociated monomers. the exposure of hydrophobic surfaces in the presence of urea was investigated using ans titration. a dramatic increase in fluorescence was observed at around 3 m and 1 m urea, for wild-type and the k315a mutant, respectively, indicating that subunit dissociation had occurred due to the exposure of hydrophobic areas (figs 2c and 3) . the result is consistent with the observed changes in the tryptophan micro-environment as probed by tryptophan fluorescence ( fig 2b) . the highest signals occurred around 2 and 5 m urea for the mutant and wildtype protein, respectively. dilution of the 2.5 m urea denatured k315a mutant protein resulted in restoration of a native-like conformation. however, dilution of 4 or 6 m urea denatured wildtype or mutant protein, respectively, resulted in higher levels exposure of hydrophobic areas. since α-helices are the major secondary structure in δ-crystallin, the ellipticity at 222 nm was used to analyze the structural changes induced by the presence of urea (fig 2b) . the results showed both proteins retaining relatively stable α-helical structure at concentrations of urea below 2 m. there was about 13% and 30% loss of the structure at 3 m and 4 m urea, respectively. effect of urea on the size-and-shape changes of δ-crystallin variants the size and size-and-shape changes of wild-type and k315a mutant protein in different urea concentrations were determined by sedimentation velocity measurements and using continuous c(s) distribution and c(s, f/f 0 ) distribution analysis, respectively (figs 3 and 4) (s1 and s2 figs) [29] . in the absence of urea, the two proteins appeared as one major component with sedimentation coefficients about 8.5 and 8.4 s, respectively (fig 3) . this peak corresponds to tetrameric δ-crystallin [11] . they possessed the native conformation as judged from the friction ratio (f/f 0 ) distribution profile (with the centre region below 1.5 as shown by red in the contour) (fig 4a and 4b ). at 2.5 m urea two components were observed for wild-type protein with sedimentation coefficients about 6.6 and 3.2 s, and these are assumed to be the dissociated dimeric and monomeric forms. the s values of the two peaks decreased with increasing urea concentration. the proportion of the second (monomeric) peak increased from 6% to 27% to 60% in the presence of 2.5, 3.0 to 3.5 m urea, respectively. dilution of the denatured wild-type protein at 3.5 m urea resulted in refolding into one major component with an s value of 8.1 (fig 3a) . measurement of asl activity showed that around 25% activity was recovered following refolding (table 1) . subunit dissociation was observed at about 1.2 m urea for the k315a mutant protein, with s values for the major peak of 6.8 and a shoulder at about 4.5 s (fig 3b) . a single peak with an s value of 4.5 was observed for the mutant protein at~1.5 m urea. this species is thought to be dissociated monomers possessing the native conformation as judged from the friction ratio (f/ f 0 ) distribution profile (fig 4c) . the monomers were reassembled into a similar quaternary structure of wild-type protein after removing the urea (fig 4d) . when the urea concentration was increased to 4.5 m, the s values for the single component were gradually decreased to about 2.4 ( fig 3b) . dilution of the protein denatured with 2.5 m urea resulted in refolding into one major peak with a s value of 8.1 s. the refolded protein showed around 80% asl activity was recovered (table 1) . since k315a mutant protein was reversible dissociated into stable monomers at 1.5 m urea, the conformational reversibility of monomers was investigated. monomeric k315a mutant protein that had been produced by equilibrium incubation of the native protein in 1.5 m urea was treated with various gdmcl concentrations. unfolding of monomeric k315a mutant proteins followed a multistep pathway as measured by both tryptophan and ans fluorescence ( fig 5a and 5b ). stable intermediates were identified from the unfolding curve of ans fluorescence which included the highest ans fluorescence region at around 1 m gdmcl and a steady state at~3 m gdmcl (fig 5b) . the monomeric protein had lost about 30 and 55% of its secondary structures at 1 and 3 m gdmcl, respectively. the changes in tryptophan fluorescence were dilution of monomeric k315a mutant protein denatured in 5 m gdmcl resulted in refolding to a similar conformation as the original monomeric state (fig 5a and 5b) . however, dilution of 1 and 3 m gdmcl denatured monomeric protein resulted in the increasing of the ans fluorescence, indicating higher exposure of hydrophobic area (fig 5b) . the results suggest the conformation of the partly unfolded intermediate could affect the folding reversibility of the monomeric k315a δ-crystallin mutant. k315a mutant δ-crystallin that denatured in 3 m urea was reversible folded back to the original conformation after dilution (fig 2b) . signal changes in the tryptophan fluorescence with different urea concentration were used to calculate the thermodynamic parameters by directly fitted to the two-state mechanism (eq 1) [19] . the free energy difference in the absence urea (δg 0 ) for the transition was determined to be 6.5 ± 0.3 kcal/mol ( table 2) . the changes of tryptophan fluorescence as a function of gdmcl concentration were used to calculate the thermodynamic parameters for the reversible unfolding of the monomeric k315a δ-crystallin mutant (fig 5a) . the unfolding curve was best fitted into a four-state model [8, 20] . the [gdmcl] 1/2 for the transitions from the m to i 1 , i 1 to i 2 and i 2 to u (denaturation) were about 0.6 ± 0.04 m, 2.1 ± 0.2 m and 3.9 ± 0.1 m, respectively. the thermodynamic parameters determined are summarized in table 2 . the total free energy difference (δg 0 ) for folding of monomeric k315a δ-crystallin mutant was determined to be 12.8 ± 0.7 kcal/mol. to determine the ability about refolding of denatured monomeric k315a mutant followed by reassembly to a tetrameric protein, the monomeric proteins that denatured by gdmcl were diluted with buffer to remove both of the urea and gdmcl. the results showed that the quaternary structure of k315a mutant protein was recovered from the denatured monomers instantly after dilution. the amount of the assembled protein was increased with the incubation time possessing about 60% of the activity recovered ( fig 5c and table 1 ). in contrast, without denaturant treatment, respectively. samples from refolding of 5 m gdmcl denatured wild-type and mutant protein for time period of zero or overnight are shown in lanes 3-4 and 5-6, respectively. the protein concentrations used in the assays were 0.03, 0.1 and 0.1 mg/ml for (a), (b) and (c), respectively. doi:10.1371/journal.pone.0145957.g005 is the concentration of denaturant at which the transition is half completed. the data was calculated by global fitting to eq 1. b the data were fitted to a 4-state unfolding model (eq 2) described as the transition from m to u. two intermediates, i 1 and i 2 , were assumed in the process before denatured species (u). these data are the mean ± sd of at least three independent experiments. doi:10.1371/journal.pone.0145957.t002 dilution of 5 m gdmcl denatured wild-type δ-crystallin, the quaternary structure was recovered after overnight incubation with no detectable activity ( table 1 ). the similar result was also shown for 5 m gdmcl denatured k315a mutant protein. these results suggested the different pathway for protein folding that seems due to the distinct conformation of the denatured protein caused by different means of denaturation. since refolding of partly unfolded monomeric mutant δ-crystallin resulted in a conformation with high exposure of hydrophobic regions, the occurrence of protein aggregation in the process was determined using light scattering measurement. no protein aggregation was detected upon dilution of 0.84, 3 and 5 m gdmcl denatured monomeric mutant protein into buffer containing 1.5 m urea. however, when 0.84 and 3 m gdmcl denatured monomeric mutant protein was diluted into buffer, protein aggregation was occurred. the rates for aggregate formation were calculated to be ca. 0.14 and 0.0004 min -1 , respectively (fig 6a) . when αa-crystallin, the chaperone protein, was added in a 5:1 ratio to 0.84 m gdmcl denatured monomeric mutant protein in folding buffer, no change in the rate of protein aggregation was observed. formation of aggregates by αa-crystallin alone did not occur under the same conditions. it is notable that upon dilution of 5 m gdmcl denatured monomeric mutant protein into buffer, no aggregation occurred. the structural features of the protein aggregate were investigated using the thioflavin t assay [30] . an increase in fluorescence intensity resulting from binding of tht with the aggregates over time was observed following dilution of 0.84 and 3 m gdmcl denatured monomeric mutant δ-crystallin into buffer (fig 6b) . the results suggest the possible formation of ordered aggregates. no changes in the signal were observed during the incubation period upon refolding of 5 m gdmcl denatured monomeric mutant protein. to determine the effect of the interactions provided by k315 at the diagonal subunits in disassembly of the quaternary structure, a md simulation were run for 100 ps for wild-type and mutant δ-crystallin in the absence of any structural restraints. from the simulation trajectory, the dynamic motion for disassembly of the quaternary structure and conformational changes in the tertiary structure were elucidated. the distances between the c α of d237 and r182, r302 and e330 and the two k315 or a315 residues were measured to evaluate the extent for subunit dissociation between the a-c, a-d and a-b dimeric pairs, respectively (fig 7a) . these residues interact with each other by hydrogen bonding or salt bridges at the dimeric pair interface in the native structure. these interactions are lost on replacement of k315 with a315. the results showed that the distances between d237 and r182, r302 and e330 and the two a315 residues increased linearly at a similar rate, except that the rate of change for the r302-e330 interaction in wild-type protein was about half of that for the mutant protein. in contrast, no changes in the distance between the k315 residues were observed before 80 ps. inspection of the timecourse at 20 ps in wild-type protein showed that the primary dimers of subunit a and c or b and d showed were separated, while the diagonal dimers of subunit a and b or c and d were connected by the interactions of residue k315 (fig 7b) . however, the subunits for both of the primary and diagonal dimers were separated from each other in the mutant protein. the results suggest a different disassembly process for tetrameric wild-type and mutant proteins. the quaternary structure of δ-crystallin is assembled as two pairs of closely associated dimers. previous mutagenesis studies have used to investigate the interactions at the interfaces of double dimers to elucidate their role in the stabilization of the quaternary structure [11] . the unique stable conformation from unfolding of k315a mutant protein in the presence of urea suggests that the interactions provided by this residue at the interfaces may play a critical role in stabilization of the quaternary structure of δ-crystallin. lys-315 is the only residue which is arranged symmetrically at the diagonal subunit interfaces (fig 1b) . the ε-amino group in the side-chain of this residue forms hydrogen bonds with the carbonyl groups of m312, v313 and k315 within the symmetric subunit (from pisa analysis: http://www.ebi.ac.uk/msd-srv/prpt_int/cgi-bin/piserver) (fig 1d) . substitution of this residue by alanine reduces the structural stability of the protein. the results from the previous study showed about a 9°c reduction in the thermal stability of the secondary structure and the changes in the micro-environment surrounding the tryptophan residues [11] . this mutant protein was also more susceptible to chemical denaturation, since about half of the concentration of denaturant was required to disrupt its quaternary structure compared to wild-type protein. both of the wild-type and mutant protein showed similar and not reversible denaturation in the presence of gdmcl. however, differences in the denaturation pathway were observed when urea was used as the denaturant. the results suggest that the non-covalent interactions between the intra and inter-subunits might be disrupted by the ionic character of gdmcl, with subunit dissociation and denaturation occurring simultaneously for both proteins [31] . the previous studies showing the presence of only a monomeric molten globule intermediate in the dissociation pathway of wild-type δ-crystallin in gdmcl supports this assumption [12, 13] . thus, the role of k315 in the folding process of δ-crystallin cannot be distinguished under the strong denaturant. around 2~5 m urea, the k315a mutant δ-crystallin is in a stable conformation, as judged by tryptophan fluorescence measurements (fig 2b) . subunit dissociation occurs under these conditions, resulting in the exposure of hydrophobic regions (figs 2c and 3b ). only monomers were identified in this state, as measured by sedimentation velocity analysis. the monomers that dissociated from the tetramers at~1.5 m urea possessed a nearly identical content of secondary structure as native protein and native-like conformation (figs 2b and 4c ). monomers in this conformation were able to refold and re-associate into tetramers with a similar conformation as the native protein, and possessed significant asl activity. when the urea concentration was increased to 2.5 m, the conformational changes led to further exposure of hydrophobic regions. however, following this conformational change at higher urea concentrations, protein folding was not reversible. the contrasting result found for wild-type protein was that the dissociation and denaturation were concurrent under the effect of urea (fig 3a) . in this condition, the conformation of the dissociated monomers was partly unfolded, as judged from the reduced level of α-helix (fig 2b) . the dissociated monomers seem to refold into alternative conformations then re-associate into tetramers with only part of the catalytic activity recovered (table 1 ). these results indicated that the conformation of the monomers seems related to the assembly pattern for functional protein. it is interesting that the interactions provided by k315 at the interfaces seem to affect the disassembly pathway of the quaternary structure of wild-type protein. it was found that both of the dimers and monomers were dissociated from the wild-type protein at around 2 to 4 m urea, as measured by sedimentation velocity while only monomers were dissociated from the mutant protein (fig 3) . these results suggest that the interactions provided by k315 at the interfaces seem to increase the energy barrier for dimeric dissociation. when these interactions were disrupted by mutation, the monomers could be isolated with intact conformation from the tetramers earlier than for the wild-type protein in urea. thus, the steps for dissociation and denaturation could be distinguished in the unfolding process of the k315a mutant δ-crystallin. in this study, the dynamic motion of protein structure in the process was simulated for elucidation the dissociation mechanism of δ-crystallin. from the trajectory, the tetramers were found to disassemble at the early stage. due to the interactions by k315, the interfaces between the diagonal dimers remain connected while distances increase between the subunits of the primary dimers in wild-type protein. in contrast, the subunits between both of the diagonal and primary dimers were dissociated in the k315a mutant protein (fig 7) . as δ-crystallin was assembled by two close contact dimers as transthyretin, dissociation at the two primary dimer interfaces would be expected to occur at the initial stage [4, 32] . the simulation result provides a novel pattern for dissociation of the double dimeric protein consistent with the results of sedimentation velocity experiments. a possible explanation for this earlier dissociation of the subunit from the primary dimers compared to diagonal dimers is differences in solvent accessibility. unlike the location of the interfaces between the subunits of the primary dimer, the position of k315 is buried at the interior interfaces away from solvent. thus, the interactions of k315 at the interfaces of the protein seem to elevate the stability of the quaternary structure. for wild-type protein, two diagonal dimers were presumed to disassemble initially from the tetramers followed by subunit dissociation of the diagonal dimers. however, dissociation at the interfaces of two primary dimers would assume to be the first step in the unfolding process of the k315a mutant protein (fig 8) . the detail mechanism for folding of monomeric protein remains elusive due to the monomers dissociated from wild-type δ-crystallin were in a molten globule conformation. thus, the monomers that reversible dissociated from k315a mutant δ-crystallin with a stable conformation and possessing similar level of secondary structure as the original state, and this would be a good model for studying the folding process. the monomeric protein was reversible denatured in a four-state mechanism in the presence of gdmcl and two intermediates were detected in the process (fig 5) . refolding of the partly unfolded intermediate was not reversible which in turn resulted in a conformation with more exposure of hydrophobic regions. only denatured δ-crystallin was reversible folded into the monomers with a similar conformation to the original state. it is interesting that the refolded monomers were able to reassemble into tetramer instantly upon dilutions, with substantial recovery of activity (fig 5 and table 1 ). this contrasts with the slow refolding of gdmcl denatured wild-type protein into its tetrameric form with no detectable activity. the slow recovery of the quaternary structure for the latter protein is due to an energy barrier for the appropriate assembly of double dimers, as reported previously [14] . the results suggest that the conformation of the denatured monomers which the tetrameric wild-type (t) and k315a mutant (t*) δ-crystallin was dissociated through the diagonal dimer (d) and primary dimer (d*) to monomers with partial unfolded (m) and stable (m*) conformation, respectively. monomers of the wild-type or the mutant protein were then denatured through intermediate (i or i*) into respective unfolded form (u or u*). the monomers (m) of wild-type protein in partial unfolded conformation was associated in alternative pathway to form dimers (d 1 ), and then assembled into tetramers (t 1 ) or aggregates (a). the aggregation was prevented by αa-crystallin. refolding followed by assembly of the intermediates (i 1 * and i 2 *) of the mutant protein resulted in the aggregates (a 1 and a 2 ) formation and the chaperone function of αa-crystallin was invalid in this process. doi:10.1371/journal.pone.0145957.g008 was unfolded by stepwise dissociation or directly unfolded with 5 m gdmcl could be different. the consequence of this might be that protein folding occurs via different pathways leading to the refolded monomers with different conformations to associate into native structure or alternative structures without function. protein aggregates are prone to form during the reassembly process from refolding of partly unfolded monomeric intermediates of δ-crystallin. the intermediate with the highest exposure of hydrophobic conformation is particularly prone to aggregate formation (fig 5) . aggregate formation by monomeric intermediates with defined conformations was also reported for transthyretin under mildly acidic conditions [33] . the result implies that the conformational status of the monomers influences subunit association. it is interesting that the presence of αcrystallin seems to increase the formation of aggregates from the monomeric intermediates of δ-crystallin with partly unfolded conformation, while α-crystallin alone was not affected under these conditions (fig 6) . the studies for αa peptide which induces the aggregation of soluble α-crystallin suggested that the mechanism for aggregate formation might due to the changes in the hydrophobicity of α-crystallin induced by the peptide [34, 35] . our previous study reported that aggregate formation during refolding of gdmcl denatured wild-type δ-crystallin was due to the improper assembling of double dimers and was prevented by the presence of α-crystallin [14] . in this study, the aggregate formation was caused by assembly of the refolded monomeric intermediate which facilitated the aggregate formation of α-crystallin. it thus postulated that the electrostatic interaction with the substrate seems to be key factors to determine the chaperon-like or anti-chaperone activity of the α-crystallin [34, 36] . the underlying mechanism requires further investigation. nonetheless, the result highlights the conformational status of the monomers which play a critical role in the folding pathway for reversible oligomerization or aggregate formation. in conclusion, the folding pathways of wild-type and mutant δ-crystallin are summarized as the working models shown in fig 8. this model depicts the key interactions from k315 at the interfaces of diagonal subunits not only to stabilize the quaternary structure of δ-crystallin but also to act as the energy barrier for dissociation of stable monomers. the stability might be one of the reasons for recruitment of the metabolic enzyme asl into the lens as a crystallin protein [37] . the single polypeptide chain of δ-crystallin after translation would be assumed to fold into functional tetramers as the proposed refolding pathway for k315a mutant. however, due to the interactions by k315, the tetrameric protein would be assumed to dissemble in an alternative manner to form the diagonal dimers, followed by simultaneous subunit dissociation and denaturation. monomers in this status might associate into dimers via a different pathway which then assemble slowly into a non-native tetrameric form or self-associate into aggregates which can be prevented by the presence of α-crystallin. the reversible folding of the monomers that dissociated from the k315a mutant protein with near native conformation provided the folding mechanism of the δ-crystallin. in this process, the ordered aggregate formation from re-association of the partly unfolded intermediate reveals a specific status of the protein to avoid the chaperone function of α-crystallin. this model proposes a possible mechanism about the aggregate formation for lens protein under stress effect and their interaction with α-crystallin. this study reveals the key role of monomers that dissociated from the oligomeric crystallin; their conformational status determines the levels of aggregate formation. , the panels show the raw sedimentation and theoretical fitted data (solid lines), and the fitting residual, respectively. (c) grayscale of residual bitmap. the raw sedimentation data were fitted to the continuous size distribution model using the sedfit program [25] . (tif) lens crystallins: gene recruitment and evolutionary dynamism the recruitment of crystallins: new functions precede gene duplication lens crystallins. innovation associated with changes in gene regulation the structure of avian eye lens δ-crystallin reveals a new fold for a superfamily of oligomeric enzymes human argininosuccinate lyase: a structural basis for intragenic complementation evidence for neutral and selective processes in the recruitment of enzyme-crystallins in avian lenses structural comparison of the enzymatically active and inactive forms of δ crystallin and the role of histidine 91 the effect of n-terminal truncation on double-dimer assembly of goose δ-crystallin structural studies of duck δ1 and δ2 crystallin suggest conformational changes occur during catalysis crystal structure of an inactive duck δii crystallin mutant with bound argininosuccinate substitution of residues at the double dimer interface affects the stability and oligomerization of goose δ-crystallin guanidine hydrochloride induced reversible dissociation and denaturation of duck δ2-crystallin monomeric molten globule intermediate involved in the equilibrium unfolding of tetrameric duck δ2-crystallin kinetic refolding barrier of guanidinium chloride denatured goose δ-crystallin leads to regular aggregate formation disruption of a salt bridge dramatically accelerates subunit exchange in duck δ2 crystallin guanidine hydrochloride-induced denaturation and refolding of transthyretin exhibits a marked hysteresis: equilibria with high kinetic barriers distinct interactions of αa-crystallin with homologous substrate proteins, δ-crystallin and argininosuccinate lyase, under thermal stress. biochimie a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding determination and analysis of urea and guanidine hydrochloride denaturation curves equilibrium unfolding of bombyx mori glycyl-trna synthetase metabolism of amino acids and amines resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants the application of circular dichroism to studies of protein folding and unfolding macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation determination of the sedimentation coefficient distribution by least-squares boundary modeling analytical ultracentrifugation in biochemistry and polymer science molecular dynamics with coupling to an external bath d18g transthyretin is monomeric, aggregation prone, and not detectable in plasma and cerebrospinal fluid: a prescription for central nervous system amyloidosis? reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride mechanism of thioflavin t binding to amyloid fibrils protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis the acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid mechanism of the chaperone-like and antichaperone activities of amyloid fibrils of peptides from αa-crystallin the αa66-80 peptide interacts with soluble αcrystallin and induces its aggregation and precipitation: a contribution to age-related cataract formation amyloid-induced aggregation and precipitation of soluble proteins: an electrostatic contribution of the alzheimer's beta(25-35) amyloid fibril gene sharing by δcrystallin and argininosuccinate lyase we thank dr m. d. lloyd (university of bath, u. k.) for reading of this manuscript before publication. conceived and designed the experiments: hjl wyc. performed the experiments: cwh hcl wck cyc. analyzed the data: cwh cyc. wrote the paper: cwh hjl. key: cord-003761-ikni2acz authors: li, zengbin; zou, zixiao; jiang, zeju; huang, xiaotian; liu, qiong title: biological function and application of picornaviral 2b protein: a new target for antiviral drug development date: 2019-06-04 journal: viruses doi: 10.3390/v11060510 sha: doc_id: 3761 cord_uid: ikni2acz picornaviruses are associated with acute and chronic diseases. the clinical manifestations of infections are often mild, but infections may also lead to respiratory symptoms, gastroenteritis, myocarditis, meningitis, hepatitis, and poliomyelitis, with serious impacts on human health and economic losses in animal husbandry. thus far, research on picornaviruses has mainly focused on structural proteins such as vp1, whereas the non-structural protein 2b, which plays vital roles in the life cycle of the viruses and exhibits a viroporin or viroporin-like activity, has been overlooked. viroporins are viral proteins containing at least one amphipathic α-helical structure, which oligomerizes to form transmembrane hydrophilic pores. in this review, we mainly summarize recent research data on the viroporin or viroporin-like activity of 2b proteins, which affects the biological function of the membrane, regulates cell death, and affects the host immune response. considering these mechanisms, the potential application of the 2b protein as a candidate target for antiviral drug development is discussed, along with research challenges and prospects toward realizing a novel treatment strategy for picornavirus infections. the picornaviridae family consists of 35 genera and 80 species, mainly including enterovirus, hepatovirus, cardiovirus, aphthovirus, and rhinovirus [1] . to date, research on picornaviruses has mainly focused on enterovirus (ev) 71, coxsackievirus (cv), poliovirus (pv), encephalomyocarditis virus (emcv), foot-and-mouth disease virus (fmdv), human rhinovirus (hrv), and hepatitis a virus (hav). picornavirus infections can cause enormous damage in humans and animals. the ev71, cva16, and cva10 cause hand, foot, and mouth disease in millions of children in asia-pacific region each year and can cause more serious clinical symptoms such as aseptic meningitis, acute flaccid paralysis, and neurological respiratory syndrome [1] [2] [3] . picornaviruses are non-enveloped spherical viruses with an icosahedral-structured viral capsid. the picornaviral genome consists of a single-stranded positive-sense rna, which is approximately 6.7-10.1 kilobases in length, with a highly conserved structure, including a 5 -noncoding region , an open reading frame, a 3 -ncr, and a 3 -end polya tail [1] . the 5 -ncr contains multiple rna secondary structural elements, including the internal ribosome entry site. the open reading frame of the viral genome consists of three regions: p1, p2, and p3. the p1 region is translated and processed to form the structural proteins vp1, vp2, vp3, and vp4, which compose the capsid structure of a picornavirus. the p2 and p3 regions are separately translated to the non-structural proteins 2a, 2b, and 2c and 3a, 3b, 3c, and 3d, respectively. the majority of related research has focused on structural proteins of picornaviruses, such as vp1, whereas the importance of the non-structural protein 2b has been relatively overlooked. viroporins are proteins found in a variety of viruses and are generally comprised of 50 to 120 amino acids. each viroporin contains a highly hydrophobic domain capable of forming at least one amphipathic α-helical structure, which oligomerizes to form transmembrane hydrophilic pores [4, 5] . the 2b protein is a crucial component of picornaviruses that exhibits viroporin or viroporin-like activity, plays a key role in the picornavirus life cycle by inducing a series of cytotoxic reactions to promote picornaviral replication and release [6] [7] [8] [9] [10] [11] [12] [13] [14] . the 2b protein has a highly conserved sequence, which can be exploited for viral detection [15] [16] [17] , vaccine development [18] [19] [20] , and rna interference [21] [22] [23] [24] [25] . in addition, the 2b protein exhibits a viroporin or viroporin-like activity, and thus, targeted drugs against viroporin could potentially target 2b protein as a novel strategy to treat or prevent picornavirus infections. however, the detailed mechanism of action of the 2b protein has not been elucidated to date. therefore, here, we review the recent research data on the role of the 2b protein in the picornaviral life cycle and discuss its possible application in antiviral therapy. the picornaviral 2b protein is a relatively short molecule, containing a maximum of two predicted putative transmembrane hydrophobic helices, along with n-and c-terminal domains, which are connected by a short stretch of amino acid residues. the α-helix-turn-α-helix sequence of the 2b protein is the basis for forming a transmembrane pore through homo-multimerization and the major determinant of the 2b protein function [4, 11, [26] [27] [28] [29] [30] . a computational approach has demonstrated that the ev 2b protein is a tetramer, and 2b proteins with different orientations have different activities [31, 32] . the 2b protein belongs to the type ii family of viroporins, which can be further divided into different types according to the number and orientation of the membrane-spanning domains ( figure 1 ). in type iia viroporins, the n-and c-termini stretch to the organelle lumen, such as in the 2b protein of cvb3 [6, 9, 26, 33] , whereas the n-and c-termini of type iib viroporins face the cytoplasmic matrix, such as in pv [9, 27, 34] and fmdv [7, 14, 28] . in addition, the c-terminus of the hav 2b protein has a viroporin-like activity [11] . picornaviral 2b proteins target the membrane and form pores mainly through their transmembrane regions. the protein molecules are first inserted into the membrane individually and then self-interact and homo-oligomerize to form higher-order structures, which are important for the pore-forming activity, determined by the specific sequence and structure [4, 34, 35] . the majority of 2b proteins are localized to organelles, with predominant co-localization with the golgi apparatus and the endoplasmic reticulum (er) in cvb3, pv, and hrv14 ( figure 1 ) [36] . the hrv16 and fmdv 2b proteins are mainly localized to the er, whereas the emcv 2b protein is not localized to either the golgi complex or the er [28, 36, 37] . seggewiss et al. [38] found that the hav 2b protein was not localized to the er either but was involved in the amendment of the er-golgi apparatus intermediate compartment. since the protein structure determines its ultimate function and 2b proteins belonging to different viroporin species show both similarities and differences in their functions, much insight can be gained from research on the same viroporin 2b protein and on different viroporins. picornavirus infects the host cell, and then viral gene encodes 2b proteins. the 2b protein belongs to the type ii family of viroporins, including two transmembrane hydrophobic helices which are the basis for forming a transmembrane pore that results in the changes in cell membrane permeability. in type iia viroporins, the n-and c-termini stretch to the organelle lumen, whereas the n-and c-termini of type ii b viroporins face the cytoplasmic matrix. the majority of 2b proteins are localized to organelles, with predominant co-localization with the golgi apparatus and the endoplasmic reticulum (er), resulting in an obvious decrease in ca 2+ of the er and golgi complex, along with a decrease in calcium uptake by the mitochondrion, and causing an influx of extracellular ca 2+ . the 2b protein can induce many cellular reactions, such as changing membrane permeability, regulating apoptosis and autophagy, and affecting host immune responses. these functions are all related to changes in ion concentrations, especially of calcium ions (ca 2+ ). therefore, here, we mostly focus on the role of ca 2+ in these 2b protein activities. a common feature of infection by animal viruses is the damage to the ion balance in host cells. the picornaviral 2b protein may change the membrane permeability of target cells, disturbing the ion balance, especially that of ca 2+ , in organelles, such as the er and the golgi apparatus ( figure 1 ) [6, 36, 39] . the changes in membrane permeability, caused by the 2b protein, have also been suggested to be regulated by the content of specific membrane phospholipids [11, 40] . the ca 2+ are involved in the activation of enzymes in cells and play a crucial role in viral replication and other viral biological processes [41] [42] [43] . however, the role of the 2b protein in ca 2+ homeostasis remains unclear. initial studies only indicated that host cells had elevated the ca 2+ levels, owing to the expression of the 2b protein [9, 28, 29, 36, [44] [45] [46] , but the mechanism was not clarified. since then, some researchers have proposed that the decrease in the concentration of ca 2+ stored in organelles triggers the opening of specific calcium ion channels on the plasma membrane of cells, causing an influx of extracellular ca 2+ [29] . this idea was supported by the findings that expression of the cvb3 and pv 2b proteins resulted in an obvious decrease in the ca 2+ concentration in the er and golgi complex, along with a decrease in calcium uptake by the mitochondria. meanwhile, the increased ca 2+ level in the cytoplasm was suggested to be mainly due to the influx of extracellular ca 2+ [36, 44, 45] . similarly, the expression of the hrv 2b protein was shown to decrease the ca 2+ concentrations in the er and golgi apparatus, whereas the emcv 2b protein only significantly reduced the ca 2+ concentration in the er [36] . in contrast, other studies showed that expression of the hav and fmdv 2b proteins elevated the cytoplasmic ca 2+ level but did not alter the level of stored ca 2+ in organelles, such as the er and golgi complex [28, 36] . taken together, these studies suggest that there are different mechanisms by which 2b proteins affect the ca 2+ concentrations, depending on the virus type. furthermore, it is unknown whether ca 2+ directly pass through the channel formed by the 2b protein. pham et al. [47] demonstrated, using a planar lipid bilayer and liposome patch-clamp electrophysiological technique, that the rotavirus non-structural protein 4 (nsp4) viroporin region acts as a ca 2+ conduction channel. although there is currently no direct evidence that the 2b protein can directly induce the observed changes in the ca 2+ concentration in host cells upon infection, the above-reviewed studies suggest an association, and the mechanism requires further investigation. given the importance of ca 2+ signaling for numerous cellular processes, further studies on picornaviral 2b protein function should include determination of the ca 2+ concentration, which may provide more insight into the detailed function of the 2b protein. in particular, the 2b protein may change the ca 2+ concentration to regulate autophagy and apoptosiswhich are distinct cell death mechanisms controlled by the virus to effectively evade the host immunity, thereby promoting viral replication and release [48] [49] [50] [51] [52] . picornaviruses can form new cytoplasmic vesicles by inducing membrane remodeling, thereby promoting their own proliferation [39, 53, 54] . the 2b protein is capable of binding to the membrane and inducing target membrane remodeling to form a unique membrane structure that can serve as a viral replication site. this site, known as the viroplasm, is generated from the er to accumulate all of the cellular components required for viral replication ( figure 2 ) [13, 39, [54] [55] [56] . the viroplasm is also the main membrane source of autophagy [54, 57] . the cvb3 2b protein is dependent on its transmembrane hydrophobic region to induce autophagy [8] , which may be related to alterations in membrane permeability, especially with regard to the ca 2+ concentration. moreover, at an early stage of fmdv cell infection, the virus specifically recognizes and binds to the cell surface receptors, and the 2b protein rapidly upregulates the autophagy pathway, leading to punctate aggregation of a large number of autophagy marker proteins, such as the microtubule-associated protein 1 light chain 3 (map1-lc3) [28, 58] . in addition, rotavirus encodes the nsp4 viroporin, which releases the er-stored ca 2+ into the cytoplasm, thereby activating the ca 2+ /calmodulin-dependent kinase kinase-β (camkk-β) signaling pathway, leading to autophagy ( figure 2 ) [59] . further, cvb4 induces autophagy in a calpain-dependent manner, causing an accumulation of lc3 lipids and autophagosomes [60] . considering the ability of the 2b protein to alter cellular calcium homeostasis, along with its viroporin-like activity, it is feasible that the 2b protein may regulate autophagy mainly by changing the ca 2+ concentration. the 2b protein has also been shown to regulate apoptosis through the endogenous pathway, which can be divided into er stress and the mitochondrial pathway, providing another potential mechanism of bypassing the host immune response to facilitate infection [32, 37, 44, 45, 48] . the ca 2+ plays a pivotal role in er stress-dependent apoptosis by regulating the flow between the er and the mitochondria [45, 61] . excessive mitochondrial uptake of ca 2+ exerts a cytotoxic effect because a high ca 2+ concentration can open numerous mitochondrial transition pores, increase mitochondrial permeability, and destroy the mitochondrial outer membrane; consequently, cytochrome c and other proapoptotic factors are released, leading to apoptosis ( figure 2 ) [9, 32, 44, 45, 62] . the cvb3 2b protein was shown to inhibit caspase activation and cell death induced by actinomycin d and cycloheximide by regulating the intracellular ca 2+ concentration [44, 45] . additionally, the 2b protein of hrv16 induced an er stress response, accompanied by an increased expression of cleaved caspase-3 and ccaat-enhancer-binding protein homologous protein (chop), which might have also involved a change in the ca 2+ level [37] . collectively, these results suggest that the 2b protein may regulate apoptosis by altering calcium homeostasis. furthermore, the 2b protein can regulate apoptosis through the mitochondrial pathway. madan et al. [32] showed that the pv 2b protein interacted with the mitochondria and altered the mitochondrial morphology, in addition to the release of cytochrome c after the pv 2b gene expression. cong et al. [63] reported that the ev71 2b protein was localized to the mitochondria and induced apoptosis by directly activating the proapoptotic b-cell lymphoma 2-associated x (bax) protein, without a significant uptake of ca 2+ by the mitochondria. therefore, the activation of the mitochondrial apoptotic pathway and subsequent apoptosis, induced by the ev71 2b protein, may not involve ca 2+ signaling. collectively, picornaviral 2b proteins can induce cell death in a variety of ways, with ca 2+ playing an important role in most of these mechanisms. figure 2 . 2b protein regulates autophagy and apoptosis. the 2b protein induces target membrane remodeling to form the viroplasm, which is generated from the endoplasmic reticulum (er). the isolation membrane is produced by the viroplasm. activation of the ca 2+ /calmodulin-dependent kinase kinase-β (camkk-β) signal pathway is due to an increased intracellular calcium concentration. furthermore, mitochondrion takes up ca 2+ from the er, thereby cytochrome cis released, leading to apoptosis. the host immune system is an important line of defense against pathogens, and pathogens can affect the immune system in a variety of ways. the 2b protein mainly affects the host immune response through inflammasome activation and by direct antagonism of the host immune response. recognition of pathogens by the immune system is mainly mediated by pathogen-associated molecular pattern receptors, known as pattern recognition receptors, including nucleotide-binding oligomerization domain (nod)-like receptors (nlrs), retinoic acid-inducible gene-i (rig-i)-like helicases, and pyrin domain-containing 3 (nlrp3) [64] [65] [66] . activation of nlrp3 inflammasome occurs during a period of changes in ion concentrations [67, 68] . the nlrp3 belongs to the nlr family of inflammasomes and causes interleukin (il)-1β and il-18 secretion via caspase-1 activation [67] . the emcv, pv, ev71, and hrv 2b proteins all activate the nlrp3 inflammasome but use distinct mechanisms [12, 69] . the hrv and emcv 2b proteins can stimulate the nlrp3 inflammasome pathway to activate caspase-1, which catalyzes the proteolysis of pro-il-1β to il-1β, leading to its secretion from across the plasma membrane by inducing a ca 2+ efflux from intracellular storage ( figure 3 ) [12, 69] . wang et al. [70] found that cvb3-infected cells induced the nlrp3 activation in association with a k + efflux. the influenza virus m2 protein, which is also a viroporin, is capable of transporting na + and k + , resulting in activation of the nlrp3 inflammasome [71, 72] . since the cvb3 2b protein acts as a viroporin and can disrupt the intracellular ion balance [36, 46] , it has been speculated that the induction of nlrp3 activation in cvb3-infected cells may be related to the 2b protein. in addition to activating the inflammasome, the 2b protein also antagonizes the host immune response. both in vitro and in vivo studies have suggested that inhibition of protein trafficking would effectively allow viral evasion of the host immune response (figure 3 ) [73] [74] [75] . moreover, inhibition of protein transport may be related to changes in the ca 2+ concentration [46] . similar to the cvb3 2b protein, the 2b proteins of pv, hrv16 and hrv14, were shown to significantly inhibit the protein transport through the golgi complex, whereas the hav, fmdv, and emcv 2b proteins did not inhibit the protein transport [36, 46, 76] . the fmdv 2b and 2c proteins did not block protein secretion, whereas the transport of proteins from the er to the golgi complex were blocked by the fmdv 2bc protein, and this effect was reproduced upon co-expression of the 2c and 2b genes [7, 77] . collectively, these findings suggest that the 2b protein may participate in a viral evasion of the host immune response, mainly by inhibiting protein transport. figure 3 . 2b protein affects the host immune response. the 2b protein can stimulate the nlrp3 inflammasome pathway to activate caspase-1, which catalyzes the proteolysis of pro-il-1β to il-1β, which leads to their secretion across the plasma membrane by inducing a ca 2+ efflux from intracellular storage. moreover, the 2b protein inhibits protein transport through the golgi protein which may be effective to evade the host immune response. the 2b protein has also been suggested to facilitate the viral evasion of the host immune response through other means. thus, the 2b protein antagonizes rig-i-mediated antiviral responses by inhibiting the expression of rig-i as an fmdv-specific reaction [14] . the rna helicase lgp2 (also known as dexh-box helicase 58, dhx58) is a crucial factor involved in the host antiviral immune response [78] . the fmdv leader protein (lpro), 3c protein, and the 2b protein have the ability to induce a decrease in lgp2 protein expression [79] . in addition, pv 2b variants were shown to inhibit the antiviral interferon (ifn) system [80] , whereas the hav 2b protein inhibited the synthesis of ifn-β by affecting the mitochondrial antiviral signaling protein activity, thereby antagonizing the host immune response [81] . collectively, these evidences indicate that picornaviral 2b proteins can affect the host immune response, thereby promoting viral amplification or the release of viral particles. as discussed above, picornaviral 2b proteins have a viroporin or viroporin-like activity and play an important role in the picornaviral life cycle. therefore, many common applications targeting viroporins may be translatable to those targeting 2b proteins. in addition, the 2b protein may serve as a new target for the development of antiviral drugs. thus, further studies on the structure and function of the 2b protein might open up new avenues for the prevention and control of picornaviruses. owing to its highly conserved sequence, the use of the 2b gene as a marker could effectively improve the accuracy of virus detection. li et al. [15] designed primers and taqman probes, based on the 2b and 3d regions, which were successfully used in real-time polymerase chain reaction to accurately detect and quantify fmdv during infection and replication. in addition, wang et al. [16] developed a lateral-flow detection system, which could rapidly and easily detect fmdv using the 2b gene. in addition to gene-based detection, the virus could be detected using a 2b antibody. biswal et al. [17] used an indirect enzyme-linked immunoassay based on a recombinant 2b protein to detect antibodies specific for fmdv. this method can be applied not only to fmdv but also to other picornaviruses, including cvb3 and ev71. given the significant threat that picornaviruses pose to humans and animals, resulting in enormous economic damage to the livestock industry, development of picornavirus vaccines is of great significance. although inactivated virus vaccines can offer effective prevention, there are associated residual risk issues, including incomplete virus inactivation and escape during the vaccine production process [82] [83] [84] . therefore, genetically engineered vaccines are considered more suitable options to overcome these shortcomings of inactivated viral vaccines. the ev vp1 protein is located outside the viral membrane and is thus exposed to the greatest amount of immune stress. accordingly, vp1 shows an extreme serological variability, thus providing the most reliable molecular epidemiological information. consequently, the vp1 region of ev71 has become a focus of vaccine research for picornavirus infections [84] . however, dna constructs containing the vp1 gene of ev71 showed low levels of antigenicity. therefore, there is still a need to develop an effective adjuvant strategy to increase the antigenicity. one possibility in this regard is the use of recombinant vaccines incorporating the 2b gene to enhance the efficacy of vaccines. at present, applications of the 2b gene in recombinant vaccines have mainly concentrated on fmdv. the addition of a 2b fragment to a vaccine designed with vp1 as the core has been shown to effectively enhance the vaccine efficacy [18] [19] [20] and reduce the dose and side effects [20] . these effects may be similar to those leading to a greater efficacy of the adenoviral vector vaccine fused to the fmdv 2b protein against serotype o, which is associated with the induction of specific cd4 + and cd8 + protective t cell responses [85] . therefore, future designs of other picornaviral genetically engineered vaccines would benefit from the addition of the 2b gene to increase the vaccine efficacy, including the addition of the 2b gene to a genetically engineered ev vaccine with the vp1 gene as the core. since viroporin plays an important role in all life stages of the virus, it is an attractive antiviral therapeutic target, and there have been great breakthroughs in this regard. by contrast, research and development of drugs targeting the 2b protein are relatively delayed. since 2b proteins have a viroporin or viroporin-like activity, screening for anti-picornavirus drugs among existing viroporin-targeting drugs may be a viable approach. there are four main types of inhibitors of viroporin activity, including adamantane, amiloride, alkyl iminosugar, and spirane amine [86] . adamantane (amantadine and rimantadine) inhibits the m2 channel of influenza a virus by destroying the transmembrane network of hydrogen-bonded water molecules, thereby inhibiting the viral amplification [87] . in bhk-21 cells infected with fmdv, the virus titer gradually decreased with an increase in amantadine concentration, which may have been due to abrogation of the pore-forming activity of the 2b protein and ultimate inhibition of fmdv replication [28] . however, clinical trials showed that amantadine was not only selective for specific resistance mutations in hepatitis c virus (hcv) p7 [88] , but also caused a rapid emergence of amantadine-resistant variants of influenza a virus during monotherapy for influenza [89] . amiloride is a composite of two drugs, 5-(n,n-hexamethylene) amiloride and a novel inhibitor, bit225, targeting hcv p7 and hiv-1 vpu, which can together block the viroporin ion channel activity or prevent ion channel formation, resulting in a potent antiviral effect [90] [91] [92] [93] . the alkyl iminosugar inhibits the formation of ion channels by targeting the hcv p7 viroporin [94] . finally, spirane amines, such as bl-1743, also inhibit the influenza a virus m2 protein, with an antiviral mechanism similar to that of amantadine [95] . there are also other drugs that act as viroporin inhibitors, including 1,3-dibenzyl-5(2h-1,2,3,4tetraazol-5-yl) hexahydropyrimidine (cd), n-(1-phenylethyl)-2-[4-(phenylsulfonyl)-1-piperazinyl]-4quinazolinamine (lds25), and 6-methyl-1,3,8-trihydroxyanth-raquinone (emodin), among others [88, 96, 97] . the mechanism of action of these viroporin inhibitors is based on the inhibition of the viroporin channel activity. therefore, these drugs may have the potential to be applied for the treatment of picornavirus infections by targeting the 2b gene. however, this application will require further detailed investigations and drug screening. nevertheless, the 2b protein has the potential to widen the range of antiviral treatment strategies. furthermore, specific degradation of complementary mrna can be triggered by small interfering rnas (sirnas) or folded short hairpin rnas (shrnas) [98] , which can be explored as an rna interference strategy, a relatively novel technology that has already been applied to treat many important pathogens, including hiv-1, hepatitis b virus, and herpes simplex virus [99] [100] [101] . currently, shrnas targeting the highly conserved 2b gene sequence are widely used in picornavirus research, including fmdv [21, 25] , emcv [23] , and cvb3 [24] , and significant experimental viral suppression has been achieved. basically, rna interference against 2b gene affects the stability and integrity of the whole viral genome. the high nucleotide sequence conservation makes the 2b gene an attractive target for rna interference, which may potentially be effective against multiple picornavirus types, and open the door for additional sirna drugs. to date, there have been few studies specifically focusing on inhibitors of the 2b protein. xie et al. [102] found that 4,4 -diisothiocyano-2,2 -stilbenedisulfonic acid (dids) blocked a chloridedependent current, mediated by the ev71 2b protein, and suppressed viral amplification. however, further research is needed to uncover the underlying mechanism. despite the many challenges in drug development, new technologies such as fourier-transform infrared spectroscopy and design of molecular dynamics analogs, as well as cryo-electron microscopy and spectroscopy, are expected to greatly contribute to the development of antiviral drugs. recent studies have gradually clarified the function and the potential of the 2b protein, along with increasingly recognizing its importance in the viral life cycle. however, there are still some challenges to overcome in investigations of the picornaviral 2b protein. in particular, its strong hydrophobicity makes it difficult to achieve soluble expression. ao et al. [28] conjugated the small ubiquitin-like modifier (sumo) protein to the n-terminus of the fmdv 2b protein and successfully achieved soluble expression. therefore, this method can be tested for other picornaviral 2b proteins. moreover, the detailed molecular mechanism of the action of the 2b protein requires further study, along with the identification of interactions of 2b protein with host proteins, to better understand the role of the 2b protein in the pathogenesis of picornaviruses. in murine cells, the 2b protein was suggested to react with host proteins to promote rhinovirus proliferation [103] . using a yeast two-hybrid system, the fmdv 2b protein was found to interact with the host elongation factor 1γ (eef1g), and mislocalization of eef1g demonstrated that the eef1g deletion affected the synthesis of membrane proteins [104, 105] . although a yeast two-hybrid system is a common laboratory protein-screening technique, it has a low success rate and is time-consuming. alternatively, affinity purification-mass spectrometry can be used to overcome these shortcomings, which has already been widely used in studies on dengue, zika, and ebola viruses [106, 107] . at present, the development of antiviral drugs against viroporins is focused on three aspects, including viroporin and membrane fusion inhibitors, ion channel inhibitors, and targeted viroporin antibodies [9] . with respect to the biological function of the 2b protein, antiviral drugs targeting the 2b protein could be designed based on the following three approaches: broad-spectrum screening for anti-picornavirus drugs among existing viroporin inhibitors, screening for 2b protein and membrane fusion inhibitors, and screening for 2b protein pore activity inhibitors. as discussed herein, the most important basis for the function of the 2b protein is that it can be polymerized into pores, thereby changing the permeability of the membrane. therefore, the design of drugs targeting 2b protein should be based on inhibiting polymerization of the 2b protein into pores, thereby reducing its effects on cellular ion homeostasis. however, these designs first require detailed determination of the refined atomic structure of the 2b protein, along with the expansion of screening techniques and applications of meticulous medicinal chemistry. furthermore, to develop better antiviral drugs, it will be necessary to elucidate the exact role of the 2b protein channel in the viral life cycle. thus, the main points of focus for research on the structure and function of the 2b protein toward ultimate drug development are: (1) mechanism of increasing membrane permeability to disturb the ion balance, (2) regulation of autophagy and apoptosis, (3) inhibition of the host immune response, and (4) promotion of viral replication and release. taken together, as research aimed at further elucidation of the role of the 2b protein progresses, along with the adoption of new technologies, it is expected that more strategies will come to light for antiviral drug development and disease control. author contributions: z.l., z.z. and z.j. wrote the manuscript; q.l. and x.h. revised for its integrity and accuracy; q.l. approved the final version of this manuscript and takes responsibility for its contents. the authors declare no conflicts of interest. human parechoviruses-biology and clinical significance examples of the two-stage membrane protein folding model. viruses viroporins: structure and biological functions membrane-active peptides derived from picornavirus 2b viroporin molecular characterization of the viroporin function of foot-and-mouth disease virus nonstructural protein 2b protein 2b of coxsackievirus b3 induces autophagy relying on its transmembrane hydrophobic sequences. viruses topology and biological function of enterovirus non-structural protein 2b as a member of the viroporin family single point mutation in the rhinovirus 2b protein reduces the requirement for 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foot-and-mouth disease virus empty capsid subunit antigen with nonstructural protein 2b improves protection of swine versatility of the adenovirus-vectored foot-and-mouth disease vaccine platform across multiple foot-and-mouth disease virus serotypes and topotypes using a vaccine dose representative of the adta24 conditionally licensed vaccine poly iclc increases the potency of a replication-defective human adenovirus vectored foot-and-mouth disease vaccine transgenically mediated shrnas targeting conserved regions of foot-and-mouth disease virus provide heritable resistance in porcine cell lines and suckling mice cross-inhibition to heterologous foot-and-mouth disease virus infection induced by rna interference targeting the conserved regions of viral genome specific small interfering rnas-mediated inhibition of replication of porcine encephalomyocarditis virus in bhk-21 cells short hairpin rna targeting 2b gene of coxsackievirus b3 exhibits potential antiviral effects both in vitro 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calpain-dependent necrosis are essential for release of coxsackievirus b from polarized intestinal epithelial cells a dual role for ca(2+) in autophagy regulation the coxsackievirus 2b protein suppresses apoptotic host cell responses by manipulating intracellular ca 2+ homeostasis enterovirus protein 2b po(u)res out the calcium: a viral strategy to survive? the coxsackievirus 2b protein increases efflux of ions from the endoplasmic reticulum and golgi, thereby inhibiting protein trafficking through the golgi the rotavirus nsp4 viroporin domain is a calcium-conducting ion channel picornaviruses and apoptosis: subversion of cell death regulation of apoptosis during flavivirus infection virus infection and death receptor-mediated apoptosis autophagy promotes the replication of encephalomyocarditis virus in host cells coxsackievirus b3 infection induces autophagic flux, and autophagosomes are critical for efficient viral replication viral reorganization of the secretory pathway generates distinct organelles for rna replication remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles a cytopathic and a cell culture adapted hepatitis a virus strain differ in cell killing but not in intracellular membrane rearrangements induction of intracellular membrane rearrangements by hav proteins 2c and 2bc subversion of the cellular autophagy pathway by viruses foot-and-mouth disease virus induces autophagosomes during cell entry via a class iii phosphatidylinositol 3-kinase-independent pathway autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-beta signaling is required for rotavirus replication coxsackievirus b4 uses autophagy for replication after calpain activation in rat primary neurons cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis viral calciomics: interplays between ca 2+ and virus 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novel ev71 anti-therapeutics and vaccines. viruses increased efficacy of an adenovirus-vectored foot-and-mouth disease capsid subunit vaccine expressing nonstructural protein 2b is associated with a specific t cell response viroporins: structure, function and potential as antiviral targets inhibitors of the m2 proton channel engage and disrupt transmembrane networks of hydrogen-bonded waters resistance mutations define specific antiviral effects for inhibitors of the hepatitis c virus p7 ion channel combination chemotherapy for influenza a phase 1b/2a study of the safety, pharmacokinetics and antiviral activity of bit225 in patients with hiv-1 infection antiviral efficacy of the novel compound bit225 against hiv-1 release from human macrophages understanding the inhibitory mechanism of bit225 drug against p7 viroporin using computational study a novel hepatitis c virus p7 ion channel inhibitor, bit225, inhibits bovine viral diarrhea virus in vitro and shows synergism with recombinant interferon-alpha-2b and nucleoside analogues the hepatitis c virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives growth impairment resulting from expression of influenza virus m2 protein in saccharomyces cerevisiae: identification of a novel inhibitor of influenza virus =structure-guided design affirms inhibitors of hepatitis c virus p7 as a viable class of antivirals targeting virion release emodin inhibits current through sars-associated coronavirus 3a protein specific inhibition of gene expression by small double-stranded rnas in invertebrate and vertebrate systems rna interference approaches for treatment of hiv-1 infection fatality in mice due to oversaturation of cellular microrna/short hairpin rna pathways an sirna-based microbicide protects mice from lethal herpes simplex virus 2 infection dids blocks a chloride-dependent current that is mediated by the 2b protein of enterovirus 71 amino acid changes in proteins 2b and 3a mediate rhinovirus type 39 growth in mouse cells kinectin-dependent assembly of translation elongation factor-1 complex on endoplasmic reticulum regulates protein synthesis eef1g interaction with foot-and-mouth disease virus nonstructural protein 2b: identification by yeast two-hybrid system protein interaction mapping identifies rbbp6 as a negative regulator of ebola virus replication comparative flavivirus-host protein interaction mapping reveals mechanisms of dengue and zika virus pathogenesis this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-009614-lbjesv8y authors: durmuş tekir, saliha d.; ülgen, kutlu ö. title: systems biology of pathogen‐host interaction: networks of protein‐protein interaction within pathogens and pathogen‐human interactions in the post‐genomic era date: 2012-11-29 journal: biotechnol j doi: 10.1002/biot.201200110 sha: doc_id: 9614 cord_uid: lbjesv8y infectious diseases comprise some of the leading causes of death and disability worldwide. interactions between pathogen and host proteins underlie the process of infection. improved understanding of pathogen‐host molecular interactions will increase our knowledge of the mechanisms involved in infection, and allow novel therapeutic solutions to be devised. complete genome sequences for a number of pathogenic microorganisms, as well as the human host, has led to the revelation of their protein‐protein interaction (ppi) networks. in this post‐genomic era, pathogen‐host interactions (phis) operating during infection can also be mapped. detailed systematic analyses of ppi and phi data together are required for a complete understanding of pathogenesis of infections. here we review the striking results recently obtained during the construction and investigation of these networks. emphasis is placed on studies producing large‐scale interaction data by high‐throughput experimental techniques. despite immense technological advances in medicine, pathogenic organisms remain the source of much human morbidity and mortality. hiv/aids, acute lower respiratory tract infections, hemorrhagic fever, diarrheal diseases, tuberculosis and malaria are particularly notorious for high mortality rates [1] [2] [3] . the continuous emergence of new diseases and drug-resistant pathogens has heightened the global burden of infectious diseases in the 21 st century [1, 4] . to tackle such biological threats, an improved understanding of pathogenic microorganisms and their interactions with host organisms is needed since pathogen-host molecular interactions have crucial roles in initiating, sustaining, or preventing infection. pathogenic microorganisms communicate with human cells through interactions with human proteins both on the surface of the cell and within the interior of the cell. these interactions allow the microorganisms to enter the host cell and manipulate cellular mechanisms in order to use the host cell's capabilities to their own advantage, resulting in infection in the host organism. detailed knowledge of pathogen-host protein interactions may enable us to comprehend the mechanisms of infection and to identify better strategies to prevent or cure infection [5, 6] . however, the identification of new drug and vaccine targets for infectious diseases is only possible when the molecular machinery within individual pathogenic and host organisms is understood. for instance, anti-infection therapeutics should target essential genes in the pathogens which have no homology with human genes [7] . the very first genome sequencing was published in 1977 with the dna sequence for the genome of a virus, bacteriophage phix174 [8] . following the sequencing of the bacterial pathogen haemophilus influenzae in 1995 [9] and the human genome in 2000 [10] , sequence data for prokaryotic and eukaryotic genomes have appeared at an accelerated rate. today, genomic data for most of the pathogen and host organisms are available [11] . these data are used to study individual genes and corresponding proteins as well as to identify intra-and interspecies connections between proteins. in the light of these advances, the initial steps towards complete understanding of infection mechanisms through protein interactions have been recently published. in this review, the efforts to systematic determination and analysis of protein interaction networks underlying infection pathogenesis are summarized (mainly in a chronological order) to present the current picture of the research on infectious diseases. from a classical perspective, a protein is a functional unit that specifies a small, but discrete, part of the cellular physiology of an organism. in the post-genomic era, a protein is seen to function as an element within network of its interaction, and its role should be evaluated within this network together with its interacting partners [12] . advances in genomics and proteomics have been followed by the first large-scale efforts to identify functional networks of interacting proteins using the two-hybrid method [13] [14] [15] , pull-down assays [16, 17] , and protein chips [18] . to increase our understanding of the mechanisms of infection, protein-protein interaction (ppi) networks of pathogenic organisms should be determined in order to capture their functional and structural organizations. pathogenic ppi maps reveal biological pathways and processes, allowing prediction of protein functions and discovery of new drug and vaccine targets. the first genome-wide protein interaction networks were determined for viruses [19] [20] [21] . the first large-scale bacterial networks [22] [23] [24] followed successes in eukaryote mapping [15, [25] [26] [27] . today, the genome-wide ppi maps for a number of pathogens and hosts are available in public databases: bind [28] , biogrid [29] , dip [30] , hprd [31] , intact [32] , mint [33] , mips [34] , reactome [35] and string [36] . primarily due to their small genome size, whole genome ppi maps were first constructed for viruses. the first interaction map of whole proteome was determined for escherichia coli bacteriophage t7, mapping 25 interactions among viral proteins [19] . subsequently, genomewide analyses of important human pathogens, hepatitis c virus [20, 37] , vaccinia virus [21] , herpesviruses [38, 39] , and sars coronavirus [40, 41] were performed through intraviral ppi maps. hepatitis c virus (hcv), a flaviviridae family member causing severe liver disease, is a positive-sense singlestranded rna virus. it encodes only a single polyprotein which is co-or post-translationally processed into at least 10 viral proteins [42] . a controlled two-hybrid strategy based on a random genomic hcv library screen was used by flajolet et al. [20] , resulting in the identification of known and novel ppis. interactions among structural and non-structural proteins were revealed in the study, leading to the conclusion that almost all of the viral proteins encoded by the genome function in the hcv life-cycle, as in the cases of other members of the flaviviridae [43] . the roles of these functional interactions were discussed within the framework of the constructed genome-wide interaction map. interacting domains of the viral polyprotein were also identified to shed light on the development of anti-viral agents [20] . another genome-wide ppi map of hcv was then generated for the viral non-structural proteins [37] . vaccinia virus, well-known as a smallpox vaccine and also the source of potential recombinant vaccines against cancer and infectious diseases, is a member of poxviridae family. it is a large, double-stranded dna virus. poxviruses replicate themselves in the cytoplasm of the host cells without depending on the host's transcriptional machinery. the large genome of vaccinia virus can potentially express more than 200 proteins [44, 45] . mccraith et al. [21] performed a comprehensive two-hybrid analysis of full-length vaccinia virus proteins and detected 37 ppis (including 28 novel interactions) among both characterized and uncharacterized proteins. many of the ppis mapped involved one partner which was known to function in a specific process, coupled with another of unknown function, allowing functions to be assigned to previously unannotated proteins within dna replication, transcription, virion structure, or host evasion. another double-stranded dna virus family is herpesviridae whose members encode 70-170 proteins. herpesviruses cause human diseases such as kaposi sarcoma, b-cell lymphomas, chickenpox, shingles, and nasopharyngeal carcinoma [46] [47] [48] . the genome-wide intraviral protein interaction maps for three members of this family, kaposi sarcoma-associated herpesvirus (kshv), varicella-zoster virus (vzv), and epstein-barr virus (ebv) were generated by two-hybrid and analyzed comprehensively to reveal viral network properties [38, 39] . in the work of uetz et al. [38] , 123 ppis for kshv and 173 ppis for vzv were identified, the largest dataset published to date, allowing the construction of the first viral networks. topological network analyses of these interactome maps indicated that the viral networks appear as a single, highly coupled module ( fig. 1 ) with relatively many hubs and few peripheral nodes [38] in contrast to scale-free cellular networks with well-separated functional modules [49, 50] . just after this study was published, calderwood et al. [39] reported the detection of 43 ppis among ebv proteins. the construction of a ppi map for ebv by merging these interactions with already published ones resulted in a network of 52 proteins with 60 interactions. this large-scale network allowed the prediction of functions of uncharacterized proteins, further defining viral mechanisms. in these consecutive studies [38, 39] , core proteins common to all herpesviruses and noncore ones specific to each strain were investigated thoroughly. the severe acute respiratory syndrome coronavirus (sars-cov) is a positive-sense single-stranded rna virus belonging to the family of the largest rna viruses known, coronaviridae. its genome encodes 14 open reading frames expressing up to 30 structural and non-structural proteins that have roles in viral replication, assembly, and other functions for viral amplification in host cells [40, 41, 51] . for a genome-wide analysis of ppis of sars-cov, interactions between all sars-cov proteins were determined [40, 41] by two-hybrid producing 65 and 40 interactions, respectively. intraviral ppis were analyzed to elucidate the functions of the proteins as well as to identify the essential proteins in viral replication. von brunn et al. [40] compared the intraviral network topology of sars-cov with a previously defined viral network [38] and cellular networks [52] [53] [54] , concluding sars-cov network contained similarities to the kshv network [38] . insights gained into molecular mechanisms and topological network properties provided by the genome-wide analyses of intraviral ppi maps (table 1 ) may be used as a basis for further characterization of the functions and mechanisms of viral proteins, especially for other members of the same virus families. having successfully built genome-wide ppi maps for viruses, similar two-hybrid methodology was applied to construct ppi networks for the larger, more complex genomes of pathogenic bacteria. the first prokaryotic ppi map was built for helicobacter pylori [22] . other large-scale prokaryotic networks eventually emerged for campylobacter jejuni [55] , treponema pallidum [56] mycobacterium tuberculosis [57] , and bacillus subtilis [58] . genome-scale analysis of interacting proteins that assemble into protein complexes were performed for e. coli [23, 24] and mycoplasma pneumoniae [59] . the first large-scale intrabacterial ppi map was constructed for the human gastric pathogen, and gram-negative bacterium h. pylori, identifying 1280 interactions between 46.6% of all 261 bacterial proteins using the twohybrid method [22] . the comparison of these h. pylori ppis with previously described interactions between orthologous e. coli proteins resulted in prediction of protein functions within biological pathways such as chemotaxis and urease activity, essential for h. pylori pathogenicity. in this study, the interacting domains of h. pylori proteins were also identified and used in protein function predictions. interacting domains may serve in mapping new functional domains, providing crucial information for antibacterial drug design studies. gram-negative bacterium e. coli, the main cause of urinary tract infections and a model bacterial system, is one of the best characterized and early studied organisms [60] [61] [62] . however, any large-scale analysis of protein complexes in e. coli was not performed until the studies of butland et al. [23] and arifuzzaman et al. [24] . first, 716 binary interactions involving 83 essential and 152 nonessential proteins, were identified by pull-down assay using tandem affinity purification-mass spectrometry, targeting 1000 orfs (about one-quarter of the e. coli genome) [23] . a small number (15%) of these interactions were already available in dip, bind, and string. ten newly described e. coli ppis were found as orthologous to the interactions reported for h. pylori [22] . the novel interactions were analyzed for functional annotations of uncharacterized proteins, allocating them within ribosome function, rna processing, rna binding, and so on. the graph theoretical analysis of the ppi map of e. coli revealed scale-free behavior and a high correlation between connectivity and the degree of conservation. the genome-wide ppi map of e. coli k-12 strain with 11 511 interactions among 2667 proteins was then constructed by a similar method [24] . the comprehensive analysis of this large-scale network also validated the scale-free nature and the connectivity-conservation correlation found previously [23] . arifuzzaman et al. [24] identified 107 functional units which have roles in metabolic pathways, transcriptional and translational machinery, recombination and flagella assembly. analysis of ppis based on this functional unit categorization provided further functional annotations. the gram-negative, food-borne pathogen c. jejuni is the major cause of gastroenteritis. the proteome-level analysis revealed 11687 interactions involving 80% of 1654 c. jejuni proteins [55] , the most comprehensive bacterial ppi map determined by two-hybrid. a scale-free network was obtained, removing low confidence-scored interactions. this ppi map of c. jejuni was used to identify evolutionarily conserved subnetworks through comparison with protein networks of h. pylori [22] , e. coli [23] and saccharomyces cerevisiae in dip. further analyses of the identified conserved sub-networks allowed the prediction of new c. jejuni interactions using orthologous interactions. this comparative analysis also enabled the identification of essential c jejuni genes based on their orthology to essential genes in other organisms. this comprehensive interactome data were next used to predict protein roles and to map functional pathways such as chemotaxis. the causative agent of syphilis, t. pallidum, has one of the smallest genomes known in extracellular bacteria, encoding 1039 proteins [63] . the global ppi network of t. pallidum, involving 3649 interactions connecting 726 bacterial proteins, was identified by two-hybrid [56] . the high-confidence subset connects 576 proteins by 991 interactions. in that study, an integrated network of dna-metabolism related processes was constructed and 18 proteins were functionally annotated within this network. additionally, various orthologous interactions were predicted for completely sequenced genomes, allowing the description of phylogenetically conserved interaction patterns. atypical pneumonia causing human pathogen, m. pneumoniae also has one of the smallest genomes in self-replicating organisms with 689 protein-encoding genes, making it a good model organism to study proteome organization in prokaryotes [64] . a proteome-wide analysis was performed by tandem affinity purificationmass spectrometry, identifying 62 homo-multimeric and 116 hetero-multimeric protein complexes [59] . about a third of the found hetero-multimeric complexes were observed to interact with proteins forming 35 larger, multiprotein complexes implying higher level of proteome organization and protein multifunctionality, allowing functional annotations of assemblies as well as prediction of biological roles of individual proteins within the complexes. m. tuberculosis causes millions of deaths each year with tuberculosis infection [65] . after computational efforts to construct large-scale ppi maps of m. tuberculosis [66, 67] , its genome-wide network was identified experimentally by two-hybrid [57] . this global network is composed of 8 042 interactions among 2907 proteins which represent 74.1% of the whole proteome. the topological properties of the undirected network of these interactions were calculated and compared with those of the previously defined prokaryotic ppi networks [22-24, 55, 56] . similar scale-free behavior following a power-law distribution was observed. in fact, the networks obtained by pull-down assay [23, 24] differ in values of clustering coefficient from the networks obtained by two-hybrid analysis [22, 55, 56] . moreover, wang et al. [57] performed a cross-species network comparison analysis of m. tuberculosis interactions with the available large-scale ppi data [22-24, 55,56] and identified conserved sub-networks. additionally, the highly connected critical proteins and mechanisms of the protein secretion pathways which have roles in its pathogenesis were revealed. a large-scale ppi network was recently constructed for the gram-positive bacterium b. subtilis (which is rarely pathogenic) by two-hybrid [58] . this network of 793 interactions involves 287 bacterial proteins. due to its role as a model organism, many studies were performed to characterize the biological functions of its ppis in cellular processes [68] [69] [70] . however, many processes remained uncharacterized. hence marchadier et al. [58] performed a comprehensive analysis with the integration of transcriptomic data focusing on cell division, cell responses to stresses, the bacterial cytoskeleton, dna replication and chromosome maintenance. these sequential efforts on construction of large-scale ppi networks for prokaryotes (table 1) constitute the first comprehensive description of the intraspecies mechanisms of the bacterial pathogens. the protozoan pathogen plasmodium falciparum causes malaria which results in deaths of nearly a million of people each year [71] . a comprehensive protein interaction map of this pathogen was generated by two-hybrid, identifying a highly interconnected, scale-free network of 2823 interactions within 1267 proteins (~25% of the predicted p. falciparum proteins) [72] . in this network, 33% of the interactions are between two uncharacterized proteins whereas 49% of the interactions include one such protein. bioinformatic analysis of this network yielded functional annotations of the proteins within the processes; chromatin modification, transcription, messenger rna stability, ubiquitination, and invasion of host cells. more detailed studies of ppis within p. falciparum are required in order to unravel its pathogenesis mechanisms thoroughly. despite the increasing rate in the identification of genome-wide ppi networks, they remain unconstructed for most pathogens. in the light of accelerating advances in genomics, proteomics, and interactomics, large-scale maps for many more organisms are expected to be built in the near future. increasing numbers of ppi networks will allow the comparison of networks across diverse organisms, resulting in generalized conclusions about pathogenic molecular mechanisms. the first examples of such comparative studies have been highlighted in the sections 2.1 and 2.2 above. integration of several highthroughput interaction datasets to generate more detailed networks is also possible, as indicated by recent examples for the e. coli system [73, 74] . the frequency of such integrated networks is expected to increase, owing to the large number of diverse data sets. these will be invaluable in defining whole proteomic maps of the pathogens. one of the most striking results of bioinformatic analyses on the constructed ppi maps is the identification of essential proteins functioning within pathogens. these proteins should be examined thoroughly to test their potential as novel therapeutic targets. the exploration of genome-wide ppi maps of the pathogens permits the assignment of unannotated proteins to biological pathways with function prediction. the proteins annotated to the host invasion processes may provide a launching point for pathogen-host interaction studies. biochemical interactions of pathogens with their hosts are necessary to invade the host organism. these connections between pathogens and hosts include interactions between proteins, nucleotide sequences, and small ligands [75, 76] . however, the protein interactions of pathogen-host systems have been identified as the most important, and therefore the most studied, type of pathogen-host interactions (phis) [76, 77] . since these interspecies crosstalks determine the pathogenesis, focusing on the whole phi system, instead of investigating a pathogen or host individually, may allow us to capture critical mechanisms (i.e. strategies used by pathogens and host immune responses) during infection that cannot be provided by traditional methods. due to a lack of sufficient experimental phi data until recent years, many computational phi prediction methods have been developed [78] [79] [80] [81] [82] [83] [84] . these studies focused mainly on interactions of p. falciparum and human immunodeficiency virus (hiv), as these are some of the most threatening pathogens to humans. very recently experiments have been carried out to determine the first large-scale molecular interactions between human and viruses [39, 85, 86] and bacteria [87, 88] . as a result of an increase in data available for pathogen-host systems, phispecific databases have been introduced such as phibase [89] , virusmint [90] , virhostnet [91] , patric [92] , and phisto [93] . although these advances in data archiving are promising, most data relevant to phi are still buried in the biomedical literature. some rare efforts have been performed to obtain hidden phis from the literature by text mining [94] [95] [96] . as in the case of intraspecies pathogen ppis, large-scale phi data were generated for viral systems before bacterial systems ( table 2 ). the first examples are for commonly observed human pathogens, ebv [39] , hcv [85] and influenza a virus (h1n1 and h3n2) [86] and then recently for hiv [97] . in calderwood et al. [39] , protein interactions between herpesvirus, ebv and human were mapped by twohybrid in conjunction with ebv intraviral ppi mapping, providing 173 phis between 40 ebv proteins and 112 human proteins. a systematic analysis of these interactome maps of ppis and phis enabled hypotheses of the roles of ebv proteins in pathogenesis to be generated. furthermore, intraspecies protein interaction data for human were integrated from databases (bind, dip, hprd, mips) and from the literature [52, 53] to analyze the organization of the human proteins targeted by ebv within human molecular machinery. it was found that ebv proteins tend to target human proteins which are highly connected (hubs) and central to many paths (bottlenecks) in the human ppi network. on the other hand, the degree distribution of the ebv-human protein interaction network could not be fitted to any model because of its incompleteness (fig. 2) . attempts to analyze incomplete maps of ppis and phis are still able to supply a partial understanding of mechanisms underlying infection. a similar thorough analysis was earlier performed with herpesviral protein networks of kshv and vzv and their interaction with the human proteome [38] . in that study, protein interactions between herpesviruses and human were predicted using the interacting orthologs of both proteins in other organisms [54] . combined virus-human networks were constructed by starting with the viral networks, adding their human protein targets, and then adding the cellular interactions among the targeted human proteins. the topological analyses of the combined herpesviruses-human networks revealed distinct properties from both viral and human interactomes providing insights into the impact of the two organisms on each other [38] . a proteome-wide phi map for the flavivirus hcv was mapped by two-hybrid and then by literature mining of previously found interactions between hcv and human [85] . a map of 481 interactions between 11 hcv proteins and 421 human proteins was generated (314 phis by twohybrid). 65% of this phi network included novel interactions. the integrated human network of 44 223 ppis among 9520 proteins [98] was used to evaluate the interplay between hcv and human. very similar behavior to ebv [39] was observed for hcv in terms of attacking hub and bottleneck proteins in the human network. to assess the human pathways targeted by hcv, kegg functional annotation pathways [99] were used. four pathways were detected to be enriched in hcv-targeted human proteins. three of them were associated already with hcv clinical www.biotechnology-journal.com syndromes as insulin, tgf-β and jak/stat pathways. the last enriched pathway, focal adhesion, is a novel observation as a human pathway affected during hcv infection [85] . influenza a is a member of negative-sense singlestranded viruses of orthomyxoviridae family. it is the sources of all flu pandemics infecting multiple species. for h1n1 a/pr/8/34 strain of influenza virus, 31 intraviral ppis among 10 viral proteins and 135 phis between 10 viral and 87 human proteins, most of which are expressed in primary human bronchial cells, were detected by twohybrid [86] . some of the phis constructed had been published previously [100] . the topology of the constructed intraviral network revealed a highly interconnected nature, as observed previously for other viral networks [38, 101] . in the case of the influenza a-human interaction network, important properties about connectivity of proteins were observed. first, viral proteins interact with significant number of human proteins, reflecting the multifunctionality of the small number proteins encoded in rna viruses. second, each of 24 human proteins connects with two or more viral proteins forming virus-human multiprotein complexes. additionally, it was observed that viral proteins generally target human proteins which are highly connected within their own network, as it was the case in herpesviruses-human system [39] . in shapira et al. [86] another phi network was identified for strain of influenza virus, h3n2 a/udorn/72 by the same experimental approach. this phi network consists of 81 interactions between 10 viral and 66 human proteins, reflecting a similar nature to the network for h1n1 strain-human system. this confirms the conserved functions of influenza virus proteins through strains. besides direct physical interactions between viral and human proteins, host responses in bronchial cells to influenza infection was identified by expression profiling, generating a regulatory map of interactions between influenza proteins and their human targets. comprehensive analysis of the physical and regulatory maps of the phi system elucidated human mechanisms involved in infection. for example, nf-κb, mitogen-activated protein kinase, apoptosis, and wnt signaling pathways are regulated through transcriptional and/or physical interactions during influenza a infection. one of the most dangerous human pathogens, hiv, belongs to positive-sense single-stranded rna virus family retroviridae. acquired immunodeficiency syndromecausing hiv has been extensively studied since its first observation near the end of the 20 th century [102] [103] [104] [105] . similar to other rna viruses, hiv has a small genome and depends largely on human cellular machinery to be replicated. identifying the physical contacts between hiv and human proteins during hiv replication is critically important for a full understanding of hiv infection. being one of the most studied pathogens, there are many phi data for hiv-1 in virusmint and phisto. the current phi data have been produced mainly by small-scale experiments [106] [107] [108] . very recently, a global phi network was generated for hiv-human protein complexes by affinity tagging and purification mass spectrometry, producing 497 phis between 16 hiv-1 proteins and 435 human proteins [97] . it was observed that hiv-targeted human proteins are highly conserved across primates. the novel interactions identified in that study requires further work to detail their biological significance in terms of hiv infection. besides whole proteins, domains of the interacting proteins were investigated and the enriched domain types in targeted human proteins were indicated for facilitating future structural modeling studies regarding hivhuman system. the first large-scale interaction networks between viruses and humans [39, 85, 86, 97] provide crucial clues about the viral infections, verifying the critical importance of phi analyses in infection researches. until very recent years, the phi data were scarce for bacterial systems because of lack of any large-scale experiments. the first extensive bacterial phi networks were identified for important human pathogens, bacillus anthracis, francisella tularensis, and yersinia pestis [87] , then another high-throughput experimental study generating phi data of y. pestis was reported [88] . gram-positive bacteria b. anthracis and y. pestis and gram-negative bacterium f. tularensis are respiratory pathogens causing anthrax, bubonic plague, and acute pneumonic disease, respectively. using a two-hybrid assay, large-scale interaction data were generated between these bacteria and human producing 3073 phis between 943 b. anthracis proteins and 1748 human proteins, 4059 phis between 1218 y. pestis proteins and 2108 human proteins, and 1383 phis between 349 f. tularensis proteins and 999 human proteins [87] . the first conclusion of computational analyses of these comprehensive bacteria-human networks, in combination with the integrated human ppi network from databases bind, dip, hprd, intact, mint, mips, and reactome, was that bacterial proteins tend to target hubs and bottlenecks in the human network. secondly, the roles of human proteins targeted by these bacteria were investigated using their gene ontology annotations [109] . the tendency of all three pathogens to target human proteins involved in immune responses was observed as previously reported [110] [111] [112] . besides being effectors of immune signaling, the bacteria-targeted human proteins also have crucial roles in apoptosis [87] . thirdly, the conserved protein interaction modules of the three phi networks were computed [113, 114] for a more systematic comparative analysis. conserved modules revealed common attacks by the bacterial pathogens to same human pathways. subsequently, another phi map was generated for plague causing y. pestis by a different two-hybrid strategy by choosing only potential virulence factors as bait pro-teins [88] . 204 phis were yielded between 66 y. pestis proteins and 109 human proteins and then 23 previously reported phis were integrated to construct a comprehensive network between y. pestis and human. a graph theoretic analysis confirms that y. pestis preferentially targets hub and bottleneck proteins in the human intranetwork as concluded previously for viruses [39, 86] and bacteria [87] . signaling pathways, crucial for human immune system, were found to be enriched in human proteins targeted by y. pestis. these pathways include mitogen-activated protein kinase signaling and toll-like receptor signaling and also pathways functioning in focal adhesion, regulation of cytoskeleton, and leukocyte transendoepithelial migration. finally, y. pestis-targeted human proteins were compared with those targeted by viruses whose phi networks were identified previously. 16 of 109 y. pestis-targeted human proteins are included in phi networks of ebv [39] and hcv [85] indicating the common infection strategies of both viruses and bacteria. the recent detected first large-scale phi networks of bacteria-human systems [87, 88] contribute largely to the understanding of bacterial infection mechanisms with immune evasion. as phi data available for various pathogens increase, a need to analyze comprehensive phi data for all pathogen types together arises in order to draw a generalized picture. although infection mechanisms of individual pathogens have been studied through intraspecies pathogenic ppi maps and interspecies phi maps, a general overview of infection mechanisms was missing until analyses of phi data from different infection agents were attempted [6, 93] . in the absence of large-scale phi networks for bacterial, protozoan and fungal systems, dyer et al. [6] performed the first global analysis of 10 477 protein interactions between 190 pathogen strains of viruses, bacteria, protozoa, and human through properties of targeted 1233 human proteins. diversity of the available phi data was not rich, 98.3% of 10 477 phis belonged to the virushuman systems with 77.9% of the interaction data drawn from hiv -human interaction systems. the importance of the pathogen-targeted proteins was evaluated within the intraspecies human ppi network of 75 457 interactions. these phi and ppi data were integrated from public databases; mint, intact, dip, hprd, reactome, bind and mips. firstly, targeting hub and bottleneck proteins was concluded to be global behavior for all pathogens, as reported for individual pathogen strains previously [39, [86] [87] [88] . gene ontology [109] functions enriched in the targeted human proteins by different pathogens revealed common infection mechanisms. attack of human transcription factors and key proteins that control the cell cycle and regulate apoptosis and transport of genetic material across the nuclear membrane were found to be among the common viral strategies. despite its scarcity (174 interactions in the datset), bacterial phi data allowed identification of specific human proteins that function in the host immune response (via toll-like receptors and i-κb kinase/nf-κb signaling cascade) as a target of bacterial infection strategy [6] . recently we performed another study with comprehensive phi data to explore common and special infection strategies for viruses and bacteria [93] . a significant amount of bacterial phis, constituting 36.5% of all data, was avaiable thanks to dyer et al. [87] . we analyzed 23 435 interactions between 3419 proteins of viral, bacterial, protozoan and fungal pathogens (totally 257 strains) and 5210 proteins of human obtained from phisto (www.phisto.org). to generate the intra species human protein network, 194006 ppis were integrated from biogrid, dip, intact, mint and reactome. the significant amount of bacterial and viral phi data allowed us to focus on comparisons between their specific infection mechanisms. firstly, attacking hub and bottleneck proteins in the human ppi network was verified as a common infection strategy of both bacteria and viruses. furthermore, viruses were observed to target human proteins of much higher connectivity and centrality values in comparison to bacteria. secondly, gene ontology enrichment analysis of the targeted human proteins verified the special mechanisms of bacteria and viruses use to manipulate of human immune defense mechanisms and cellular processes, respectively (as reported in dyer et al. [6] but relying on lower amounts of phi data). a first attempt at the investigation of the human proteins targeted by both bacteria and viruses revealed that attacking human metabolic processes is a common strategy used by both pathogens during infections [93] . global analysis of phi data provides insights into the strategies adapted by bacteria and viruses to subvert human cellular processes and immune system for the infection. however, large-scale phi networks for pathogens other than bacteria and viruses are still undetermined, leaving their pathogenesis mechanisms to be relatively uncharacterized. research on infectious diseases through phis has accelerated within the post-genomic era (fig. 3) . however, large-scale phi networks have been infrequently studied. efforts to identify and analyze large-scale phis for diverse pathogen types would be expected to parallel the acceleration of biotechnology and bioinformatics research. increasing amounts of data available will allow more complete data sets to be compiled, resulting in characterization of topological properties of phi networks. the first attempt to fit the degree distribution of ebv-human interaction network failed due to scarcity of data [39] . on the other hand, bioinformatic analyses of the pathogen-tar-geted human proteins succeeded in unraveling some infection strategies such as targeting human hubs and bottlenecks, subverting cellular processes for the usage of pathogens' own advantages and evasion of immune defenses [6, 39, 85, 87, 88, 93] . the huge amount of data expected to be generated for phi systems will enable us to capture all details of infection processes. potentially leading to the development of new and more efficient therapeutics. conventional treatments for infectious diseases often aim to kill pathogens by targeting their essential proteins. this approach unfortunately forces the pathogens to evolve for survival and consequently selects resistant strains (especially in the case of rna viruses with a high mutation rate). to fight drug-resistant patho gens, novel alternative therapeutics are emerging which target host proteins required by pathogens to replicate and persist within the host organism. if these host factors are indispensable for pathogens, but not essential for host cells, their silencing may inactivate pathogenic activity, allowing them to serve as therapeutic targets [4, 115] . in the light of phi studies, some human factors required by viral and bacterial pathogens have been determined for hiv [115] [116] [117] [118] [119] , hcv [120] , west nile virus [121] , influenza virus [122, 123] , and m. tuberculosis [124] in recent years. despite the efforts reviewed here, the use of systems biology approaches to investigate phi is still considered relatively undeveloped. the availability of new phi network data, together with further topological and functional analyses of pathogen-host systems, are expected to shed more light on infection mechanisms and novel therapeutic targets for infectious diseases in the near future. we particularly thank dr. tunahan çakır for critical reading of the manuscript and for his contributions to figure 3 . the financial support was provided by the research funds of bogaziçi university, through project 5554d. the doctoral scholarship for saliha durmuş tekir is sponsored by tübitak, is gratefully acknowledged. the authors declare no conflict of interest. figure 3 . the number of scientific publications including phi-related terms in pubmed in the post genomic era. the searched phi-related terms: "pathogen host interactions", "host pathogen interactions", "pathogen host interaction", "host pathogen interaction", "pathogen-host interactions", "pathogen-host interaction", "host-pathogen interactions", "host-pathogen interaction". infectious diseases: for considerations the 21st century host-pathogen systems biology filoviruses are ancient and integrated into mammalian genomes new strategies to fight 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switch recombination through a cd40-independent mechanism involving baff and c-type lectin receptors gene ontology: tool for the unification of biology francisella tularensis induces cytopathogenicity and apoptosis in murine macrophages via a mechanism that requires intracellular bacterial multiplication macrophage apoptosis by anthrax lethal factor through p38 map kinase inhibition inhibition of mapk and nf-kb pathways is necessary for rapid apoptosis in macrophages infected with yersinia graemlin: general and robust alignment of multiple large interaction networks conserved patterns of protein interaction in multiple species network-based prediction and analysis of hiv dependency factors identification of host proteins required for hiv infection through a functional genomic screen global analysis of hostpathogen interactions that regulate early-stage hiv-1 replication genome-scale rnai screen for host factors required for hiv replication host cell factors in hiv replication: meta-analysis of genome-wide studies a genome-wide genetic screen for host factors required for hepatitis c virus propagation rna interference screen for human genes associated with west nile virus infection genome-wide rnai screen identifies human host factors crucial for influenza virus replication human host factors required for influenza virus replication genome-wide analysis of the host intracellular network that regulates survival of mycobacterium tuberculosis key: cord-007211-prygoc0q authors: segawa, hiroaki; kato, masahiko; yamashita, tetsuro; taira, hideharu title: the roles of individual cysteine residues of sendai virus fusion protein in intracellular transport(1) date: 1998-06-17 journal: j biochem doi: 10.1093/oxfordjournals.jbchem.a022044 sha: doc_id: 7211 cord_uid: prygoc0q the role of intramolecular disulfide bonds in the fusion (f) protein of sendai virus was studied. the 10 cysteine residues were changed to serine residues using site-directed mutagenesis. none of the cysteine mutant f proteins reacted with a monoclonal antibody specific for the mature conformation of the f protein, but eight of ten mutants reacted with an immature conformation-specific monoclonal antibody. the transport of these mutant proteins to the cell surface was drastically reduced. all of the cysteine mutant f proteins remained sensitive to endoglycosidase h (endo h) for 3 h after their synthesis. moreover, cell surface transport of the hemagglutinin-neuraminidase (hn) protein co-expressed with each of these cysteine mutant f proteins was also reduced. these results suggest that all cysteine residues participate in the formation of intramolecular disulfide bonds, that co-translational disulfide bond formation is crucial to the correct folding and intracellular transport of the f protein, and that interaction of the f and hn proteins takes place intracellulary. the two glycoproteins of paramyxoviruses, the fusion (f) and hemagglutinin-neuraminidase (hn) proteins, are present as integral proteins which form spike-like projections on the outer surface of the viral envelope. the hn protein exhibits both the hemagglutinating and the neuraminidase activities, while the f protein has been shown to be involved in virus penetration, hemolysis and cell fusion (for reviews, see refs. 1 and 2). the f protein is synthesized as an inactive precursor, f o , which is cleaved by proteases to form the biologically active protein consisting of the disulfide-linked subunits fi and f 2 . the n-terminal portion of the fi subunit is very hydrophobic, a feature which is highly conserved among paramyxovirus f proteins, and is suggested to mediate fusion of the virus envelope with the target membrane as well as cell fusion resulting in the syncytium formation. the sequences of paramyxovirus f proteins reveal the conservation not only of amino acid residues of the fusioninducing domain, cleavage site, and transmembrane domain, but also of cysteine residues at specific positions. as shown in fig. 1 , mature sendai virus f o protein has 10 cysteine residues, designated cl to c10, and the relative position of cysteine residues is highly conserved among paramyxovirus f proteins (2, 3) . this suggests that these cysteine residues play important structural and functional roles. two cysteine residues are present in the signal peptide and are not likely to participate in intramolecular disulfide bonds of the mature protein. the cl cysteine is in the f 2 subunit, and the remaining 9 cysteine residues are in the fi subunit. eight of the 9 cysteine residues are clustered in a narrow region near the transmembrane domain. the formation of disulfide bonds takes place in the er and is co-translationally catalyzed by protein disulfide isomerase. it is suggested that only native disulfide bonds are found in the principal folding intermediates and that disulfide bond formation plays an integral role in the folding. the mechanisms of folding, oligomeric assembly, and sorting of viral membrane proteins have been characterized in recent years (for review, see ref. 4) . to date, the roles of individual cysteine residues in the folding of newcastle disease virus (ndv) (5) and measles virus (6) have been reported. on the other hand, the roles of individual cysteine residues of paramyxovirus f proteins have not been characterized and remain largely unknown. intramolecular and intermolecular disulfide bonds are essential components of the structure of the majority of proteins, and it is of great interest to understand their functions. roles of disulfide bonds that have been suggested to include (i) aiding in protein folding and maturation and (ii) maintenance of stability and solubility. two general approaches, in vivo reduction using reducing reagents (7) (8) (9) (10) and site-directed mutagenesis to substitute each of the cysteine residues with other amino acids (5, 6, (11) (12) (13) (14) , have been used to study the functional role of disulfide bond formation within cells. recently, the sites of the disulfide bonds of sendai virus f protein were determined using protein sequencing analysis, and all 10 cysteine residues were shown to participate in disulfide bond formation {15). thus, mutagenesis to substitute each cysteine residue should provide additional tgc-tcc tgt-»tct tgt-uct tgotcc tgt-»tct tgt-uct tgotcc tgt-»tct tgc-»tcc tgt-»tct a findings regarding the functional role of each disulfide bond in the folding, intracellular transport and antigenicity. previously, we described the efficient expression of sendai virus f gene cdna with mutations at its cleavage site and hn gene cdna to induce cell fusion (16) . in this study, we employed site-directed mutagenesis to prepare mutant f protein genes at each of the 10 cysteines and analyzed the role of each cysteine residue in f protein intracellular processing, cell surface expression, and immunoreactivity with different monoclonal antibodies (mabs) after transient expression of the cysteine mutants in cos cells. plasmids-mutagenesis of pcr fragments by use of mismatched primers was performed in a three-step pcr (17) . to replace each cysteine, except for c4, we used the sets of four primers listed in table i . in the first pcr, two separate pcr reactions were run using the 5'-mutagenic and 3'-outer primers and the 3'-mutagenic and 5'-outer primers to amplify separate, overlapping sequences of the template plasmid puc-f (18). the 5'-mutagenic and 3'-mutagenic primers have overlapping homologous regions containing a mutation of interest. following amplification, the pcr products were purified, mixed, then denatured and re-annealed. an overlapping duplex was formed and extended by the second pcr reaction without primer to give the full-length target sequence containing the mutation. this mutated pcr fragment was amplified by the third pcr using the 5'-outer primer and 3'-outer primer used in the first pcr. plasmid psrd-fcls was constructed as follows: the pcr product containing a mutation at its cl site was digested with clal and ligated to the fc fragment, which was excised from puc-f by digestion with clal and hindlll. the ligated whole f gene was purified by gel extraction, treated with klenow fragment of dna polymerase i (klenow fragment) to create blunt ends, then treated with t4 polynucleotide kinase and ligated into the expression vector pcdl-srd (psrd) (19) , which had been plasmid psrd-fc2s was constructed as follows: the pcr product containing a mutation at its c2 site was digested with bglll and pstl and used to replace the corresponding fragment of puc-f. subsequently, the fc2s gene was excised by digestion with hindlll, blunt-ended by treatment with klenow fragment, and inserted into psrd, which had been linearized by digesting with £cori and treated with klenow fragment. to construct the expression plasmids psrd-fc3s, psrd-fc5s, psrd-fc6s, psrd-fc7s, psrd-fc8s, psrd-fc9s, and psrd-fclos, we used the following procedure: a cdna fragment containing the carboxyl terminal portion of f protein from 235 to 565 amino acid residues was subcloned into pbluescript ii sk+ (stratagene cloning systems, la jolla, ca) at the pstl site to yield pbs-fc. mutagenesis was performed as described above, and the resulting pcr products containing a mutation at each cysteine site were digested with xmnl and ndel, then inserted into pbs-fc, which had been digested with smal and ndel. the resulting plasmids were digested with pstl, and the mutated fc fragment was ligated with 4.1 kb pstldigested psrd-f. plasmid psrd-fc4s was constructed as follows: pcr was performed using n primer and mutagenic primer 3'-flcs3. the pcr product was digested with bamkl and inserted into pbs-fc, which had been digested with the same restriction enzyme. the resulting plasmid was digested with psti, and the mutated fc fragment was ligated with 4.1kb psti-digested psrd-f as described above. the mutations at the desired sites of all the mutant dnas were confirmed by dna sequencing (20) . as shown in fig. 1 , a total of 10 cysteine mutants were prepared and designated fcls to fc10s from the amino terminus of the f o protein to the carboxyl terminus, corresponding to the cysteine residues at amino acid positions 70, 199, 338, 347, 362, 370, 394, 399, 401, and 424, respectively. the expression plasmid psrd-hn was constructed by inserting the whole hn gene into psrd as described previously (16) . cell culture and dna transfection-monkey cos-1 cells (21) were grown in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal calf serum at 37°c in 5% co 2 . for transient expression of the f protein, cos cells were grown to 80% confluency in 35-mm tissue culture dishes, then transfected with 5 jug of expression plasmid per dish by calcium phosphate precipitation (22) . cells incubated for 48 h after transfection were analyzed by immunoprecipitation (18) . antibodies-anti-sendai virus antiserum was prepared from sendai virus-infected rabbits. the antiserum used for immunoprecipitation was obtained from rabbits immunized with f proteins, which were expressed in escherichia coli as maltose-binding fusion proteins and purified by amylose resin as described by the manufacturer (new england biolabs, beverly, massachusetts). anti-f serum used to detect cell surface or intracellular expression of the f protein was obtained from rabbits immunized with the fj protein, which was purified from virions by sds-polyacrylamide gel electrophoresis (sds-page) (23) . anti-hn serum used to detect hn protein by western blotting was obtained from rabbits immunized with the hn protein, which was purified from virions by sds-page. monoclonal antibodies (mabs) f-49, f-236, f-921, and yl-111 were the generous gift of dr. h. tozawa (kitasato university). mab f-49 reacts with the mature form of f protein (24) , and f-236 recognizes both f o and f! in immunoprecipitation but reacts specifically with f o in western blot analysis. mab f-921 reacts with the immature form of f protein. in a competitive binding assay, the mabs were divided into two groups, f-i and f-ii. mabs f-236 and f-921 belonged to the f-i group and f-49 belonged to the f-ii group (25) . mab yl-111 recognizes the hn protein. indirect immunofluorescence staining-indirect immunofluorescence staining was performed as described previously (26) . in general, cos cells were grown on glass coverslips and transfected with plasmid dna as described above. forty-eight hours after transfection, cells were washed with ice-cold phosphate buffered saline (pbs) and fixed with acetone for 10 min at -20°c or with 3.7% paraformaldehyde in 100 mm sodium phosphate buffer (ph 6.5) for 30 min at room temperature. the cells were washed twice with pbs for 10 min, then incubated for 1 h at 37°c in pbs containing 3% bsa, 0.02% nan 3 , and anti-f rabbit serum or yl-111 (diluted 1:100). after incubation with the antiserum, cells were washed with pbs twice, then incubated for 1 h at 37°c in pbs containing nan 3 , bsa, and fluorescein isothiocyanate (fitc)-conjugated anti-rabbit igg or anti-mouse igg (wako pure chemical industries), each diluted 1:100, as secondary antibody to anti-f rabbit serum or yl-111, respectively. immunofluorescence was examined by fluorescence microscopy. western blot analysis-cos-1 cells (in 12-well dishes) were transfected with plasmid dna as described above. forty-eight hours after transfection, cells were washed with ice-cold pbs containing 20 mm iodoacetamide (iaa), lysed in 100 a l of ripa buffer [0.01 m tris-hcl (ph 7.5), 0.15 m nacl, 1% triton x-100, 1% sodium deoxycholate, 0.1% sds] containing 2 mm pmsf and 20 mm iaa, and sonicated for 30 s. twenty-five microliters of 5xsds sample buffer [312.5 mm tris-hcl (ph 6.8), 25% 2-mercaptoethanol (2-me), 50% glycerol, 10% sds, 0.05% bromophenol blue] was added to the cell lysates and boiled for 3 min. fifteen microliters of each sample was electrophoresed on a 9% polyacrylamide gel. proteins were transferred to immobilon-p membrane (millipore) by semi-dry electroblotting. the membrane was preincubated in trisbuffered saline (tbs) [0.02 m tris-hcl (ph 7.6), 0.137 m nacl] containing 0.05% tween20, 5% low fat milk for 1 h at room temperature. the membrane was then washed in tbs containing 0.05% tween20, incubated for 1 h at room temperature in tbs-tween buffer containing a 1:1,000 each dilution of the anti-f and anti-hn antisera, washed twice for 30 min in tbs-tween, and incubated for 1 h at room temperature in tbs-tween containing a 1:4,000 dilution of horseradish peroxidase (hrp)-conjugated protein a (e. y laboratories). after extensive washing of the membrane, bound antibodies were detected using the ecl western blotting detection reagent system (amersham). immunoprecipitation-cos-1 cells (in 35-mm dishes) were transfected with plasmid dna as described above. forty-eight hours after transfection, the medium was replaced with methionine-and cysteine-free minimum essential medium (mem). twenty minutes later, cells were labeled with 100 ix\ of methionine-and cysteine-free mem supplemented with [ 35 s] methionine and [ 35 s] cysteine (200/^ci/ml, 1,000 ci/mmol; du pont/nen), and labeling was continued for 10 min. the cells were chased with dmem supplemented with 5% fbs, 5 mm methionine, and 5 mm cysteine for the time indicated in each figure legend. then the cells were washed twice with ice-cold pbs containing 20 mm iaa, lysed in 300/^1 of ripa buffer containing 20 mm iaa, sonicated for 30 s, and centrifuged for 10 min at 15,000 rpm. fifty microliters of the supernatant (cellular extract) was incubated with 5 //i of antibody raised against recombinant f protein expressed in e. coli or mabs reacting with various epitopes (25) on ice for 1 h. twenty microliters of a suspension of protein-a sepharose fast flow (pharmacia biotech, uppsala, sweden) was added to the lysates. the mixture was incubated at 4°c for 1 h with gentle mixing, and immune complexes adsorbed on the beads were washed three times in 50 mm tris-hcl (ph 7.5), 150 mm nacl, 1 mm edta, 0.25% (w/v) gelatin, 0.1% (v/v) np-40, 0.02% (w/v) nan 3 , then denatured in sds sample buffer [6.25 mm tris-hcl (ph 6.8), 2% sds, 10% glycerol] in the presence or absence of 5% 2-me by boiling for 3 min. the samples were analyzed by 9% sds-page followed by fluorography. glycosidase treatment-the immune complexes adsorbed on protein-a sepharose were resuspended in 100 fi\ of 50 mm sodium acetate buffer (ph5.5), then samples were incubated with 2 mu of endo-y3-at-acetylglucosaminidase h (endo h: boehringer mannheim biochemica) for 16 h at 37°c. after digestion, the immune complexes on the beads were recovered by centrifugation and denatured in sds sample buffer containing 5% 2-me by boiling for 3 min. the immune complexes were analyzed by sds-page followed by fluorography. expression of the cysteine mutant f proteins-the f protein of the z strain of sendai virus possesses 12 cysteine residues. the first and second cysteine residues are present in the signal peptide while the rest are distributed across the entire ectodomain of the protein. the signal peptide is cleaved in the er, and thus the remaining 10 cysteine residues are present in the mature f protein. the relative locations of these cysteine residues are highly conserved among paramyxovirus f proteins, and these cysteines are likely to participate in intramolecular disulfide bonds in the mature protein. to examine the processing of the intramolecular disulfide bonds and their contribution to the structure of the f protein, site-directed mutagenesis using pcr was used to substitute each cysteine residue by a serine residue to prevent the formation of disulfide bonds. ten expression plasmids of the cysteine mutant f proteins, named fc1s to fc10s, were prepared as shown in fig. 1 . recombinant plasmids were introduced into cos cells by the calcium phosphate precipitation method (22) , and the cells were incubated for 48 h. transfected cells were labeled with [ 35 s]methionine and [ 35 s] cysteine for 10 min, chased for 1 h, then lysed in ripa buffer. the lysates were immunoprecipitated with anti-f antiserum and analyzed by 9% sds-page under reducing ( fig. 2a) or nonreducing (fig. 2b) conditions. in fig. 2a , psrd-f transfected cells gave one main band (lane 3) with mobility corresponding to the uncleaved form of f, or f o , synthesized in sendai virusinfected cos cells as reported previously (16) . the 10 cysteine mutant-transfected cells also gave one main band corresponding to the uncleaved f o protein. a minor band of about 60 kda was also detected in all cells transfected with recombinant plasmids (lanes 3-13), and this seems to be nonspecifically degraded f proteins or unglycosylated f proteins, because the unglycosylated f proteins and immature f proteins could be detected by the antiserum used here, which was raised against the unglycosylated f proteins expressed in e. coli. the cysteine mutant psrd-fc1s ( fig. 2a, lane 4) transfected cells gave three bands, one main band corresponding to the f o protein, the common minor band (60 kda), and another higher molecular weight species of f protein. the alteration of the cysteine residue of the f 2 subunit to a serine residue introduced a new glycosylation site in this protein sequence. thus, the higher molecular weight species seems to be the f protein utilizing the additional new glycosylation site. approximately equal levels of most of the f proteins in the cells transfected with the above plasmids were detected by western blot analysis (data not shown). this showed that these 10 cysteine mutant f proteins were efficiently expressed at almost the same level as wild-type f protein in psrd-f transfected cells and were relatively stable. under nonreducing conditions, formation of disulfide bonds in a protein should allow it to assume a more compact form and to migrate faster on sds-page than unfolded forms without disulfide bonds, as reported by machamer et al. (27) . as shown in fig. 2b , the compact and unfolded forms of monomer f proteins and aggregates were detected with the mutants as well as the wild-type f protein. the compact form migrated as a 60 kda band and the unfolded form migrated as a 66 kda band, which is similar to the molecular mass of the reduced form of f protein as shown in fig. 2a . most of the wild-type f protein migrated as the compact form (lane 3). on the other hand, the relative amount of the unfolded form of the each 10 cysteine mutant f proteins was increased, and the ratio of the compact form to unfolded form was roughly 1 as determined by densitometry (lanes [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] . this indicated that all cysteine residues contributed to make the compact form. no obvious oligomeric form of f proteins was detected in the mutant or wild-type f protein-expressing cells. this implied that f protein does not form disulfide-linked oligomers. although the aggregates disappeared from the top of the gel when the radiolabeled cell lysates were reduced by boiling in 5% 2-mercaptoethanol before sds-page, treatment with thiol alkylation reagents such as iodoacetamide (iaa) or iv-ethylmaleimide (nem) had little effect on the formation of the aggregates. in preliminary experiments, we did not detect such aggregates in sendai virus-infected cells analyzed under nonreducing conditions. these observations indicated that these aggregates were formed before sample preparation and that other viral factors might be required for efficient folding of f protein. antibodies-to examine the antigenicity of the cysteine mutant proteins, the cell lysates were immunoprecipitated with mabs f-49, f-236, and f-921 and analyzed by sds-page under reducing conditions. in preliminary experiments, f-921 detected intracellular f proteins but not cell surface-transported f proteins. mab f-236 recognized both f o and fj in the immunoprecipitation, but specifically recognized f o in western blot analysis (unpublished data). mab f-49 recognized the mature form of f protein (24) . thus, these mabs bind independent epitopes. as shown in fig. 3 , only the wild-type f protein was precipitated with anti-mature conformation mab f-49 (panel a, lane 3), the wild-type f protein and a small amount of fc2s protein were precipitated with mab f-236 (panel b, lanes 3 and 5), and the wild-type f protein and most of the cysteine mutant f proteins, except for the fc5s and fc6s proteins, were precipitated with mab f-921 (panel c, lanes 3-7 and 10-13). these data indicated that loss of any one of the cysteine residues of the f protein had dramatic effects on the immunoreactivity to anti mature mabs and that all of the cysteine mutant f proteins were blocked at some stage in their maturation. cell surface transport of cysteine mutant f proteins-to examine the expression of cysteine mutant f proteins in the cells and their transport to the cell surface, the cells were fixed with acetone or paraformaldehyde and examined by indirect immunofluorescence staining using anti-f antiserum. to detect subcellular localization, the cells expressing wild-type and cysteine mutant f proteins were fixed with acetone, then examined by indirect immunofluorescence staining (fig. 4 , panels a, c, e, g, i, k, and m). we expected that a pair of cysteine mutants which had altered cysteine residues involving the same disulfide bond might show the same expression phenotype. recent results by iwata et al. (15) identified specific disulfide bonds between cl and c2, c3 and c4, and c5 and c6, while cio appears to be linked to c7, c8, or c9. thus, only the 5 cysteine mutants fc1s, fc3s, fc5s, fc7s, and fc8s were shown in fig. 4 . the cos cells transfected with vector plasmid psrd showed no fluorescence (panel a), whereas the cells transfected with psrd-f (panel c), psrd-fcls (panel e), psrd-fc3s (panel g), psrd-fc5s (panel i), psrd-fc7s (panel k), and psrd-fc8s (panel m) displayed a bright staining pattern, and the frequency of positive cells was estimated to be 5-10% of total cells in each case. as shown in panel c, the cells expressing wild-type f protein displayed internal staining throughout the cytoplasmic reticulum as well as in the juxtanuclear region. on the other hand, the cysteine mutant-expressing cells showed an intracellular staining pattern limited to a reticular perinuclear structure (panels e, g, i, k, and m). this indicates that cysteine mutant f proteins were efficiently expressed in these cells. to detect whether the expressed proteins were transported to the cell surface, the cells were fixed with paraformaldehyde instead of acetone, then examined by indirect immunofluorescence staining. the cells transfected with psrd-f displayed a staining pattern on the cell surface (panel b), whereas the surface fluorescence of the cells transfected with the cysteine mutant f plasmids was clearly much less intense than that of cells expressing wild-type f protein (panels d, f, h, j, and l). this indicates that the wild-type f protein was properly transported to the cell surface, but these cysteine mutant f proteins were not transported to the cell surface. in sendai virus-infected cells, both f protein and hn protein are transported to the cell surface. to examine the effect of hn on cell surface expression of wild-type and cysteine mutant f proteins, cells co-transfected with the hn gene and wild-type f or one of the cysteine mutant f genes were fixed with paraformaldehyde, then cell surface expression of f or hn proteins was tested with anti-f antiserum or anti-hn mab yl-111. the cell surface expression of cysteine mutant f proteins was not detected (data not shown). furthermore, as shown in fig. 5a , the cell surface expression of the hn protein was drastically reduced in the cells co-expressing hn and cysteine mutant f proteins. in panels (b) and (c), hn protein was efficiently detected at the cell surface of psrd-hn-or psrd-f and psrd-hn-transfected cells. on the other hand, the cell surface expression of the hn protein was drastically reduced in the cells co-transfected with psrd-hn and one of the cysteine mutant f genes (examples shown in fig. 5a panels d, e, and f). as shown in fig. 5b , in western blot analysis, both hn and wild-type or the cysteine mutant f proteins were detected in the cells co-transfected with hn and wild-type or cysteine mutant f genes (lanes [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] . the expression levels of hn proteins in these cells and in the cells transfected with hn gene alone were similar (lanes 3-14) . these results indicated that association of f and hn proteins takes place intracellulary and that the transport deficiency of the cysteine mutant f proteins affected the intracellular transport of the hn protein. intracellular transport of f proteins-the f protein has three iv-glycosylation motifs and contains complextype sugar chain(s) (28) . the f protein is synthesized on the rough er, then glycosylated in a well-defined sequence in the er and golgi apparatus in the process of transport to the cell surface. high mannose-type sugar chains of the glycoprotein in the er and the cis-golgi compartments are sensitive to digestion with endoglycosidase h (endo h), while sugar chains once trimmed and further processed in the medial and trans-golgi compartments are not. we therefore determined the critical state in the transport process of the cysteine mutant f proteins by testing endo h sensitivity of the sugar chains. to follow the process of maturation of glycoproteins, the transfected cells were labeled with [ 35 s]methionine and [ 35 s] cysteine for 10 min at 48 h after transfection. after being chased for 3 h, cells were lysed and the lysates were analyzed after immunoprecipitation with anti-f followed by endo h digestion, as described in "materials and methods." as shown in psrd-fcls-transfected cells was partially glycosylated f protein modified at a novel glycosylation motif generated by the mutagenesis. because the antiserum used in this study reacts efficiently with immature f protein, relatively large amounts of endo h-sensitive species of f proteins were detected even in the cells expressing wild-type f protein. these data showed that cysteine mutant f proteins were trapped in the er or the cis-golgi region, which prevented their transport to the cell surface. we used mutagenesis of the cysteine residue of the f protein to examine the effect of the disruption and rearrangement of the disulfide bonds on the folding of the f protein and its transport to the cell surface. we found that all cysteine mutant f proteins were efficiently expressed in cos cells ( fig. 2a) , but failed to fold into the compact form and to achieve proper conformation (fig. 3) . they were retained in the er to cis-golgi compartments (fig. 6) , formed aggregates (fig. 2b) , and failed to be transported to the cell surface (fig. 4) . this indicated that disulfide bond formation involving all cysteine residues is required for the f protein to exit the er. although our cysteine mutant f proteins were glycosylated, none of them acquired native structure. assignment of disulfide bonds of the f protein has been reported (15); thus, we expected that we would be able to detect some specific folding intermediates corresponding to the lack of individual disulfide bonds by analyzing individual cysteine mutant f proteins under nonreducing conditions. when mutations are made in either one of a pair of cysteines that are normally linked in a mature structure, the phenotype of the mutant proteins is often similar. in our study, two species of f proteins were detected in the cysteine mutant f proteins under nonreducing conditions: folded f protein and unfolded f protein. this indicated that formation of incorrect disulfide bonds led to rapid unfolding or rearrangement of disulfide bonds. as shown in fig. 3 , almost all cysteine mutants exhibited a drastic decrease in reactivity to the mature conformation specific mab f-49. this result showed that cysteine residues contribute to the whole protein structure through disulfide bridging, even though they are fairly distant from each other in the primary amino acid sequence. mab f-921 could not immunoprecipitate fc5s and fc6s proteins but could immunoprecipitate the rest of the mutants. this implied that the epitope of f-921 is present in the loop formed by the c5 and c6 disulfide bond, and the antigenic site is exposed when the f protein is inside the cells, but buried when the f protein is expressed on the cell surface. fc2s showed only slight reactivity to mab f-236. these data indicated that f-236 and f-921 may recognize cysteine-rich domains and supported the idea that the cysteine-rich domain in the bunched structure of paramyxovirus f proteins is recognized by mabs (29) . since the formation of the correct intramolecular disulfide bonds seems to be a complex process involving disruption and rearrangement of disulfide bonds during intracellular transport, the three-dimensional structure of these cysteine mutants was changed, and the immunoreactivity of mabs with these mutant proteins decreased dramatically. these data suggested that co-translational disulfide bond formation is an absolute requirement for subsequent folding. because of the incorrect folding of cysteine mutant f proteins, their transport to the cell surface was reduced drastically and they contained only high mannose-type oligosaccharides. these results indicated that the mutant proteins were retained in the er. viral glycoprotein mutants that had a temperature-sensitive phenotype and folded correctly at 32°c but misfolded at 37°c have been reported (4). in hsv-1, the cysteine mutant gd proteins showed temperature sensitive cell surface expression (12) . we examined whether cysteine mutant sendai virus f proteins show temperature sensitivity, but we could not detect cell surface expression of the cysteine mutant f proteins by incubating mutant gene-expressing cells at 33°c or at 31.5°c (data not shown). these results suggest that the inhibition of the expression of the cysteine mutant f proteins on the cell surface occurred at a stage involved in the intracellular transport. though many reports have shown that cysteine mutant proteins are improperly folded and found as disulfide -linked aggregates in the er, there are some reports that cysteine mutant proteins could be transported to the cell surface, e.g., newcastle disease virus (ndv) hn, measles virus h, and asialoglycoprotein receptor h2b subunit, all of which have type ii topology (5, 6, 14) . it seems that type i transmembrane proteins more strictly require disulfide bond formation than do type ii proteins in order to exit from the er. we previously found that cleavage of an intrinsically protease-sensitive mutant f, fmut, was enhanced by coexpression with hn protein (16) , indicating that f protein associates with hn protein intracellulary. thus, we examined the effect of co-expression of hn on cell surface expression of cysteine mutant f proteins. although the expression of both proteins was detected at roughly equal levels by western blot analysis (fig. 5b) , cell surface expression of cysteine mutant f proteins was not detected, and a decrease of cell surface expression of hn protein was observed (fig. 5a) . these observations are in accord with the down-regulation of the human parainfiuenza type 3 (hpiv3) hn protein cell surface expression by the mutant f protein containing an intracellular retention signal (30) . recently, many reports have shown a direct interaction of paramyxovirus f and hn proteins (31) (32) (33) (34) . however, the cell surface expression of f and hn proteins is reported to occur at different rates because of the different degrees of association with bip (35) . thus, decrease of cell surface expression of hn protein in the cysteine mutants coexpressing cells may be caused not only by direct interactions of the f and hn proteins but also by indirect interaction affected by binding of molecular chaperons such as bip or calnexin. morrison et al. reported that ndv f protein undergoes conformational change with disruption and rearrangement of disulfide bonds during intracellular transport, and that the conformational change occurs before the cleavage reaction (36) . because of the intracellular transport deficiency of the cysteine mutant f proteins, we could not identify the cysteine residues involved in such disulfide bond rearrangements. to study sequential disulfide bond formation and bond rearrangements, in vivo reduction experiments using well-characterized mabs are also now under way. paramyxovirus fusion: a hypothesis of changes the paramyxoviruses determination of the complete nucleotide sequence of the sendai virus genome rna and the predicted amino acid sequences of the f, hn and l protein folding and assembly of viral membrane proteins the role of the individual cysteine residues in the formation of the mature, antigenic hn protein of newcastle disease virus role of individual cysteine residues in the processing and antigenicity of the measles virus haemagglutinin protein ing disulfide bond formation and protein folding in the endoplasmic reticulum quality control in the endoplasmic reticulum folding and misfolding of vesicular stomatitis virus g protein in cells in vitro role of cotranslational disulfide bond formation in the folding of the hemagglutinin-neuraminidase protein of newcastle disease virus disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins cysteine mutants of herpes simplex virus type 1 glycoprotein d exhibit temperature-sensitive properties in structure and function disulfide bond structure of glycoprotein d of herpes simplex virus types 1 and 2 the contribution of cysteine residues to antigenicity and extent of processing of herpes simplex virus type 1 glycoprotein d enhanced folding and processing of a disulfide mutant of the human asialoglycoprotein receptor h2b subunit assignment of disulfide bridges in the fusion glycoprotein of sendai virus transfection of sendai virus f gene cdna with mutations at its cleavage site and hn gene cdna into cos cells induces cell fusion a general method for rapid site-directed mutagenesis using the polymerase chain reaction construction of expression plasmids for the fusion protein of sendai virus, and their expression in e. coli and eucaryotic cells sra promoter: an efficient and versatile mammalian cdna expression system composed of the simian virus 40 early promoter and the r-u5 segment of the human t-cell leukemia virus type 1 long terminal repeat dna sequencing with chain-terminating inhibitors sv40-transformed simian cells support the replication of early sv40 mutants a new technique for the assay of infectivity of human adenovirus 5 dna cleavage of structural proteins during the assembly of the head of bacteriophage t4 conformational aberrance of sendai virus f0 protein in thapsigargin-treated cells allowing exit from the endoplasmic reticulum but causing arrest at the golgi complex neutralizing activity of the antibodies against two kinds of envelope glycoproteins of sendai virus expression of the sendai virus fusion protein and the hemagglutinin-neuraminidase protein using a baculovirus vector heavy chain binding protein recognizes incompletely disulfide-bonded forms of vesicular stomatitis virus g protein carbohydrate structures of hvj (sendai virus) glycoproteins perturbation of cellular calcium blocks exit of secretory proteins from the rough endoplasmic reticulum downregulation of paramyxovirus hemagglutinin-neuraminidase glycoprotein surface expression by a mutant fusion protein containing a retention signal for the endoplasmic reticulum identification of regions on the hemagglutinin-neuraminidase protein of human parainfluenza virus type 2 important for promoting cell fusion functional interaction of paramyxovirus glycoproteins: identification of a domain in sendai virus hn which promotes cell fusion association of the parainfluenza virus fusion and hemagglutinin-neuraminidase glycoproteins on cell surfaces detection of an interaction between the hn and f proteins in newcastle disease virus-infected cells selective and transient association of sendai virus hn glycoprotein with bip conformational change in a viral glycoprotein during maturation due to disulfide bond disruption key: cord-020788-a33vcapl authors: gottardi, cara j.; caplan, michael j. title: signals and mechanisms of sorting in epithelial polarity date: 2008-05-22 journal: nan doi: 10.1016/s1569-2558(08)60020-x sha: doc_id: 20788 cord_uid: a33vcapl this chapter discusses epithelial-membrane polarity, sorting pathways in polarized cells, and the sorting-signal paradigm. polarized epithelial cells have long captured the attention of cell biologists and cell physiologists. at the electron-microscopic level, one of the most apparent and fundamental features of this cell type is its polarized organization of intracellular organelles and its structurally and compositionally distinct lumenal (apical) and serosal (basolateral) plasma-membrane domains. the polarized epithelial phenotype is an absolute necessity for organ-system function. in the most general sense, these cells organize to form a continuous, single layer of cells, or epithelium, which serves as a semi-permeable barrier between apposing and biologically distinct compartments. within the tubules of the nephron, these cells orchestrate complex ion-transporting processes that ultimately control the overall fluid balance of the organism. at the surface of the gastrointestinal tract, specialized versions of this cell type control the digestion, absorption, and immuno-protection of the organism. polarized epithelial cells have long captured the attention of cell biologists and cell physiologists. this is largely because the architecture of these cells so tellingly bespeaks their function. at the electron microscopic level, one of the most apparent and fundamental features of this cell type is its polarized organization of intracellular organelles and its structually and compositionally distinct lumenal (apical) and serosal (basolateral) plasma membrane domains (figures 1 a, b) . through the eyes of the physiologist, the polarized epithelial phenotype is an absolute necessity for organ system function. in the most general sense, these cells organize to form a continuous, single layer of cells, or epithelium, which serves as a semi-permeable barrier between apposing and biologically distinct compartments. within the tubules of the nephron, these cells orchestrate complex ion-transporting processes that ultimately control the overall fluid balance of the organism. at the surface of the gastrointestinal tract, specialized versions of this cell type control the digestion, absorption and immuno-protection of the organism. thus while polarized epithelial cells can carry out myriad functions, they share one defining feature: a structural polarity which serves their underlying functional polarity. the differential distribution of membrane proteins between the plasmalemmal surfaces of polarized epithelial cells enables these cells to both respond to and effect changes upon their environment in a directed fashion. the gastric parietal cell of the stomach, for example, contains a population of h,k-atpase-rich vesicles. upon stimulation, these vesicles fuse selectively with the lumenal membrane, resulting in the massive apical secretion of hcl which initiates digestion. without two important elements of the polarized phenotype, that is, junctional integrity and the precision of this membrane insertion, proton pumps might be delivered to a compartment which would be adversely affected by the secretion of acid. another illustration of the utility of the polarized phenotype is provided by the principal cells of the kidney, which carry out net sodium absorption through a mechanism which is entirely dependent upon the polarized distribution of two membrane proteins. sodium absorption is stimulated by the hormone aldosterone, which increases the amount or activity of na,k-atpase at the basolateral surface, while increasing the number or activity of apical sodium channels and thus the sodium conductance of the lumenal membrane (doucet and barlet-bas, 1989) . because the na,k-atpase generates low intracellular { na+}, sodium is these morphologically distinct apical and basolateral membrane domains are separated by a unique ultrastructure known as the tight junction (tj). this structure is just visible as an area of close, uniform membrane apposition located at the apices between adjacent epithelial cells. (photo courtesyof dr. marian neutra, children's hospital, boston, ma). able to pass from the lumen of the kidney tubule through apical sodium channels and into the cytoplasm down its electrochemical gradient. the na+ is then pumped across the basolateral membrane and into the interstitum by the sodium pump and is ultimately prevented from leaking back into the lumen by impermeable tight junctions. therefore, it is the differential assignment of na' channels to the apical surface and na,k-atpase molecules to the basolateral domain that ensures the vectoriality of this transport process. how the polarized cell assigns these two proteins (and apical and basolateral membrane proteins in general) to their respective surface domains has been the subject of much investigation and is the general focus of this review. it is perhaps important to point out that the fundamental questions of plasma membrane protein aniosotropy are not unique to surface membrane proteins or even to the study of epithelial polarity. the golgi apparatus, for example, is a polarized organelle whose cis-and trans-most cisternae are structurally and biochemically distinct. this organization is thought to enable the ordered addition and trimming of glycoprotein sugar residues as they traverse the stacked cisternae. as is clearly represented in the breadth of topics covered in this book, numerous cell types adopt a polarized state for some functional purpose. the propagation of a nervous impulse from dendrite to axon requires compositionally different membrane proteins in each of these domains, while the localization of determinants to specific parts of an egg's cytoplasm gives rise to cells with different growth potentials and the necessary assymetries required for embryo development . what we hope will become clear in this chapter and related chapters in this book is that we are beginning to appreciate the universality of polarity. the mechanisms involved in establishing and maintaining the polarized state appear to be so fundamental that some of the schemes through which a cell is able to localize a particular protein to a given cellular domain are turning out to be conserved between epithelia and neurons, and even between epithelia and yeast. while the need for protein asymmetries in development, or membrane polarity in epithelial transport is clear, the means through which it is achieved are only beginning to be elucidated. before we embark upon our review of the field, we first introduce the conceptual framework onto which the results in this field are organized and interpreted. first, a protein destined to accumulate with a polarized distribution needs to be recognized as different from other proteins. we presume that what is recognized is some structural aspect of the protein itself. we refer to that part of the protein that is recognized for polarized localization as a sorting signal or localization determinant. these two terms are often used interchangeably, but in fact there is a subtle difference between the two. "sorting signal" is often taken to imply a signal that is recognized and acted upon before the protein is delivered to its ultimate residence. sorting signals are thought to be those signals that enable a cohort of similar proteins with similar destinations to be sorted and sifted away from all of the other molecules traversing the biosynthetic pathway at the same time. a "localization determinant" is perhaps a more general term that carries fewer mechanistic implications. it is defined here as the determinant that specifies a protein's polarized distribution, but it does not make a distinction between recognition that takes place before the protein has reached its final destination or after (e.g., through a selective retention mechanism). the proteins which serve to recognize a particular signal and act upon it are generally referred to as sorting machinery. often, a distinction is made in the literature between "sorting" and "targeting machinery." in these cases, the sorting machinery is exclusively those elements which recognize the sorting signal. any downstream effectors of this sorter that orchestrate the vectorial directing of a vesicle to its final destination are referred to as targetting machinery. a simple schematic of these elements is presented in figure 2 . as is discussed in the second half of this review, we know much more about general targeting machinery than the sorters themselves. one of many possible ways to think about how a secretory or membrane protein could be sorted into a vesicle. it is presumed that the "sorters" will recognize a sorting signal ("1 embedded within the protein structure. it seems likely that this recognition event would need to take place in the lumen of the golgi for a secretory protein, but this might not be necessary for a membrane protein, which could interact with a sorter from either a lumenal-or cytoplasmic-facing signal domain. ultimately, the sorted protein(s) could be contained within a "domain-specific vesicle," which would then be targetted (with the help of protein targetting machinery x, y, and z) to the appropriate apical or basolateral surface domain. it is thought that proteins destined for either the apical or basolateral domain of a polarized cell occupy the same golgi cisternae during their biosynthesis ( m a t h and simons, 1984; misek et al., 1984; rindler et al., 1984; fuller et al., 1985; pfeffer et al., 1985) . immunoelectron microscopic studies performed on nonpolarized endocrine cells which manifest two biochemically and kinetically different secretory pathways suggested that the process of sorting components away from one another takes place at the tgn (orci et al., 1987; tooze et al., 1987) . however, recent studies have demonstrated that sorting may not take place exclusively at the tgn. sorting mechanisms have been suggested to take effect as early along the biosynthetic pathway as the er (balch et al., 1994) as well as at the recycling endosome (matter et al., 1993; . in hepatocytes, sorting appears to occur after all newly synthesized membrane proteins are delivered to the basolateral plasmamembrane (bartles et al., 1987) . similar delivery routes have been detected in polarized intestinal epithelial cell lines (matter et al., 1990) . finally, in at least one subclone of the canine renal mdck cell line, sorting may take place both at the golgi as well as at the level of the plasma membrane. while most proteins in this cell line are sorted in the tgn, the na,k-atpase can be preferentially localized to the basolateral membrane through domain-specific stabilization mechanisms after random insertion into both plasmamembrane domains (hammerton et al., 1991; siemers et al., 1993) . apically and basolaterally sorted proteins have been shown to be packaged into distinct classes of golgi-derived vesicles which are ultimately targeted to their appropriate domains. recently it has been shown that membrane and secretory proteins are segregated into distinct vesicular carriers upon transport from the golgi to the basolateral surface of hepatocytes (saucan and palade, 1994) the extent to whch distinct basolateral (or apical) proteins are cosorted and incorporated within the same vesicle either due to common localization signals or the ability to co-aggregate has not yet been determined. after proteins are sorted, the targeting of a vesicle to a particular surface domain can occur directly (vectorially) from the tgn to the apical domain (matlin and simons, 1984; rindler et al., 1984; fuller et al., 1985) , basolateral domain (caplan et al., 1986) or indirectly as has been shown for the poly-immunoglobulin receptor (pigr) (mostov and deitcher, 1986) . in the latter case, the protein is first targeted to the basolateral surface where the receptor can bind its ligand and is then transported to the apical surface via a process known as transcytosis (reviewed in mostov and simister, 1985) . as noted above, in hepatocytes all apical proteins studied to date make use of this indirect pathway for apical delivery (bartles et al., 1987) , while cell lines derived from intestine and kidney can employ both routes for surface delivery (matter et al., 1990; casanovaet al., 1991; low et al., 1991) while the details of the routes have been determined for a number of sorting pathways, the molecular signals and recognition components which control each of them are not well understood. the search for these molecular signals and recognition components has been the focus of much study over the last 15 years. during this period, the subjects of protein sorting and epithelial polarity have been extensively reviewed. several of these reviews are listed here for those seeking more background on specific aspects of this field: for general reviews on protein sorting pathways (burgess and kelly, 1987) ; general concepts of sorting and targeting (caplan and matlin, 1989 ); a discussion of the mechanisms required for the establishment and maintenance of epithelial polarity (rodriguez-boulan and nelson, 1989) ; polarized transport of surface porteins and lipids in epithelial cells ; comparative epithelial and neuronal polarity (rodriguez-boulan and powell, 1992) ; the generality of the polarized phenotype (nelson, 1992) ; cytoskeleton as a component of the protein sorting machinery (mays et al., 1994) ; summary of the few known sorting signals in polarized epithelial cells ; common signals involved in sorting from the tgn and endosomes . perhaps now more than ever before, it is becoming a rather daunting task to provide a synthesis of the observations relevant to the study of epithelial polarity. this is in part due to the fact that important insights into the mechanisms of sorting are being contributed by fields that are not exclusively focussed on epithelial biology. as we discussed in this review, some important contributions are emerging from studies of endocytosis, secretion in yeast and neurons, and the sorting of yeast lyso-soma1 enzymes (see chapter i of this volume), in addition to more "classical" approaches to epithelial polarity. in this review, we explore the current paradigm that the generation and maintainance of distinct membraneous compartments requires "sorting signals," the recognition domains embedded within the amino acid sequence or polypeptide structure of the protein, and "sorting machinery," the proteins which interpret and act upon these signals. in the first half, we review and categorize the signals that have begun to be elucidated, as well as discuss the approaches and difficulties associated with finding and interpreting sorting signals. while the polarity field itself has not yet succeeded in characterizing the definitive sorting machinery, numerous components of the membrane budding and fusion apparatus are rapidly being elucidated. we have chosen to review some of the important findings in the field of membrane transport, and in particular examine the potential roles that gtp-binding proteins of the rab, arf and heterotrimeric classes may play. we also discuss a class of proteins referred to as adaptins as well as the implications that the snare hypothesis may have for epithelial polarity. although these components have not been shown to be directly involved in sorting per se, it is becoming increasingly clear that in a general sense, the composition of the membrane vesicle budding and fusion machinery may be part of the overall apparatus which "acts upon" the sorted species and contributes to domain specific surface targeting. as stated above, the paradigm for conceptualizing the mechanisms responsible for biosynthetic sorting requires that each protein contains signal information embedded within its polypeptide sequence/structure (sorting signal) which is interpreted and acted upon by components referred to as sorting machinery. this scheme takes its cue from the process through which ribosomes translating secretory and membrane proteins are targeted to the endoplasmic reticulum to initiate cotranslational protein translocation (blobel, 1980) . prior to the elucidation of this process, it was suggested that protein targeting might require cellular sorting machinery to recognize certain signals which would be shared by proteins with common destinations (blobel, 1980) . shortly after this suggestion, it became clear that targeting to the rer, mitochondria and chloroplasts required short, contiguous, n-terminal signal peptides (reviewed in burgess and kelly, 1987) . in the case of the former, the signal was recognized by a receptor, srp (lingappa et al., 1978; von heijne, 1984; kurzchalia et al., 1986; walter and lingappa, 1986 ). subsequently, a number of short, contiguous amino acid domains have been shown to play a role in later stages of post-synthetic targeting. these include: (1) the kdel and adenovirus e l 9 signals which ensure the retention or recapture of resident er proteins (munro and pelham, 1987; nilsson et al., 1989); ( 2 ) a transmembrane domain signal responsible for golgi retention (swift & machamer, 1991; machamer, 1993) ; (3) the cluster of positively charged lysine residues (sv40-nls) sufficient for nuclear targeting (richardson et al., 1986) ; (4) the critical tyrosine/ "tight-turn'' structural motif which can mediate localization to clathrin coated-pits (goldstein et al., 1985; pearse and robinson, 1990; collawn et al., 1991) ; and (5) the discovery that lysosomal hydrolases were targeted to lysosomes through the recognition of a phosphorylated sugar residue (mannose-6-phosphate; reviewed by kornfeld and mellman, 1989) . in several of these cases receptors for these signals have been well-characterized: the signal recognition particle (srp) for secretory and membrane proteins (walter and lingappa, 1986) , the mannose-6-phosphate receptor (m6pr) for the targeting of lysosomal hydrolases to the lysosome (sly and fischer, 1982; vonfiguraandhasilik, 1986) , the kdelreceptor (tanget al., 1993) and the adaptins which couple coated pit localization sequences to clathrin cages (pearse and robinson, 1990; robinson, 1994) . the search for definitive signals which mediate the delivery of proteins to a particular epithelial surface domain has proven to be quite difficult. this is due in part to general limitations imposed by certain molecular biological approaches, as well as to some inherent difficulties specific to the investigation of epithelial polarity. our goal in this section is to outline reasonable criteria for the identification of a sorting signal. the observation that the influenza and vesicular stomatitis viruses bud from opposite surface domains of polarized mdck cells (madin darby canine kidney) (rodriguez-boulan and sabatini, 1978) spawned an extensive search in which chimeric and deletion analyses were applied to the problem of identifying the underlying apical and basolateral sorting signals (reviewed in caplan and matlin, 1989) . these efforts to characterize sorting signals have generally involved the generation of chimeric or truncated contructs prepared from portions of apical and basolateral membrane proteins. through analysis of the subcellular distributions of the resulting proteins, sorting information can, at least in theory, be assigned to particular portions of the parent molecules. while a large number of chimeric and truncated viral glycoproteins have been generated and analyzed, it has been difficult to interpret many of the resultant observations. with the benefit of hindsight, we now know that these difficulties can be attributed to a number issues that we discuss in more detail below (including the tertiary stuctures of the experimental constructs, the confounding possibilities introduced by uncharacterized default pathways, and the potential for multiple and hierarchical signals to be embodied within the structures of the studied proteins). until recently (thomas and roth, 1994) , the analysis of viral spike glycoproteins did not produce a definitive sorting signal. much of the uncertainty associated with this work is likely attributable to the fact that these studies engineered chimeras from portions of structurally dissimilar molecules. the tertiary structures of the resultant chimeras may thus differ substantially from those of either parent molecule, which may in turn exert unpredictable effects upon sorting behavior. clearly, if sorting signals are formed from domains arising from noncontiguous regions of a polypeptide, for example, in much the same manner that heterotrimeric g proteins are thought to "see" their effectors (berlot and bourne, 1992) , or in the way that the human growth hormone receptor (hghbp) is thought to interact with its ligand (cunningham and wells, 1989) , it is easy to imagine how the structural integrity of the putative sorting signal could become compromised in a chimeric construct. while producing a rough map of the signal-bearing domain of a protein can be relatively straight forward, determining the exact residues which constitute the signal is turning out to require a collaboration between many different types of mutagenesis approaches. often, contradicting results can arise from alanine scanning, truncation and point mutation/deletion mutagenesis, since a mutated protein can manifest impaired sorting behavior even though the altered residues are not part of the actual sorting signal (aroeti et al., 1993) . it is becoming clear that a judicious and thorough comparison of many different types of mutagenesis approaches may be necessary to determine definitively the key residues necessary for sorting. perhaps another difficulty in looking for apical or basolateral sorting signals is that the default pathway for "signal-less" membrane proteins is still not known. a protein that is sorted "by default" is, by definition, unable to interact with and be acted upon by any sorting machinery whatsoever. in theory, at least, such "unsorted'' proteins may be distributed with polarity, depending on the nature and characteristics of the membrane vesicular traffic arising from the golgi complex in a particular cell type. obviously, if the localization of a protein construct under study is identical to that produced by the cell's default pathway, elucidation of a signal will be difficult, since elimination of the signal will not alter the protein's distribution. thus, one can appreciate the difficulty in assigning localization information to a particular domain in the context of an undefined default pathway. this caveat accounts for at least some of the reasons which explain why a definitive basolateral sorting signal in the c-terminal domain of vsv-g protein took so long to discern. in the following example we summarize the ha-vsvg spike glycoprotein chimera literature as a means to illustrate the difficulties in interpretating these types of studies. when acdna encoding the influenza ha was expressed in mdck cells, the encoded protein localized to the apical membrane (roth et al., 1983) , while a cdna encoding the vsvg polypeptide produced a protein that is localized to the basolateral domain (gottlieb et al., 1986b; stephens and compans, 1986) . when truncation mutants were expressed in which soluble ectodomain versions of these proteins were synthesized, the vsvg ectodomain was secreted from both apical and basolateral domains gonzalez et al., 1987) while the ha ectodomain was predominantly secreted from the apical domain (gonzalez, et al., 1987; roth et al., 1987b) . based on evidence that the default pathway for secreted proteins leads to nonpolarized secretion from both surface domains (kondor-koch et al., 1985; gottlieb et al., 1986a; caplan et al., 1987) , it was reasoned that the ectodomain of ha encodes an apical sorting signal while the vsvg ectodomain lacks signal information. this was further confirmed by the observation that a hybrid ha-vsvg protein comprising the ha ectodomain fused to the vsvg transmembrane and cytoplasmic tail region was targeted to the apical membrane (mcqueen et al., 1986; roth et al., 1987a) . but if the vsvg ectodomain is randomly secreted and the vsvg tail domain fused to ha is apical, which domain of vsvg encodes basolateral sorting information? the complementary hybrid comprised of the ectodomain of vsvg (presumably signal-less) tethered to the ha transmembrane and tail region (perhaps also signal-less) was targeted either to the basolateral membrane or to both surface domains (mcqueen et al., 1986; puddington et a] ., 1987; roth et a]., 1987a; compton et al., 1989) . the interpretation of the behavior of this chimera was clearly complicated; it was suggested that this protein could be pursuing its distribution by default. (as discussed above, the default pathway for membrane proteins is still not defined in polarized cells). an alternative interpretation was that the vsvg ectodomain indeed contains basolateral sorting information, but that perhaps this domain needs to be tethered to the plasmamembrane with a transmembrane anchor in order to interact with its presumptive sorting machinery. this interpretation, however, was proved incorrect by the observation that the anchoring of this ectodomain to the membrane through a lipid-linkage resulted in apical targeting (brown et al., 1989) . interestingly, when the ectodomain of the normally apical placental alkaline phosphatase (plap) was attached to the vsvg transmembrane and cytosolic tail domains (which were though to lack a dominant signal), the resulting chimeric protein was targeted basolaterally. it is difficult to reconcile the ha-vsvg and plap-vsvg chimeras without invoking hierarchical and competing signals. recently, a basolateral targeting signal has been precisely localized to the cytoplasmic domain of the vsvg protein (thomas and roth, 1994) . in light of the vicissitudes which attended the interpretation of each round of chimeric constructs discussed above, it was certainly unexpected that definitive sorting information would be localized to the cytoplasmic tail of vsvg. the nature and function of this signal will be discussed in depth below. the preceding discussion was presented simply to reinforce the caveat that the default pathway, protein structural considerations and the possible interactions between "dominant" and "recessive" sorting signals can considerably cloud the interpretation of chimera experiments. recent studies of the polymeric immunoglobulin receptor (pigr), the low density lipoprotein receptor (ldlr) and polytopic hetero-oligomeric proteins (h,k-atpase and na,k-atpase) suggest that individual proteins can interact in multiple and complex fashions with the machinery responsible for surface targeting. it is becoming increasingly clear that there can be an array of signals encoded within an individual protein, and the sorting problem is becoming evermore complicated by the apparent redundancy, multiplicity and hierarchical nature of these signals (matter et al., 1992; mostov et al., 1992) . for example, brewer and roth's (1991) demonstration that they could completely overwhelm the apical signal present in the ha ectodomain and redirect it to the basolateral surface by changing a single amino acid in this protein's cytoplasmic tail strongly suggests that multiple signals present in a single protein can interact in a heirarchical fashion. the newly created cytoplasmic signal is dominant over the presumed apical sorting signal present in the ectodomain of ha. as discussed below, the ldl receptor has been shown to encode redundant, basolateral sorting information, since either of two cytoplasmic determinants could independently mediate basolateral delivery (matter et a]., 1992) . moreover, the protein may also contain acryptic apical sorting signal in its ectodomain, since a cytoplasmic tail-minus construct of this protein (ct12) is sorted with great efficiency to the apical membrane in mdck cells (matter et al., 1992 ). an ectodomain apical localization signal has also been found within the pigr, whose initial surface delivery is to the basolateral plasmalemma. why do these proteins need multiple signals? what does the ldlr gain by expressing two basolateral localization signals? recent studies (discussed in greater detail in the following section) have more finely decoded these two signals and are revealing functional differences. for ex-ample, the "membrane proximal determinant" encodes coated-pit internalization information, while the "membrane distal determinant" appears to ensure efficient sorting from a basolateral endosome back to the basolateral surface (matter et al., 1993) . analysis of the sorting behavior of multisubunit ion pumps provides further insight into the possible utility of multiple signals (reviewed in the gastric h,k-atpase and the na,k-atpase are close cousins in the large family of p-type ion transporting atpases. both are composed of 100 kda a-subunits and heavily glycosylated 55 kda p-subunits. they share similar reaction mechanisms and catalytic properties and, not surprisingly, are highly homologous at the amino acid sequence level. the a-subunits are -65% identical, whereas the p-polypeptides manifest roughly 40% identity. while the na,k-atpase is a basolateral protein in most polarized epithelial cell types (with the exception of neural epithelia such as choroid plexus and retinal pigment epithelium), the h,k-atpase occupies the apical membrane and a pre-apical storage compartment in gastric parietal cells. hormonal stimulation of gastric acid secretion induces fusion of the membrane vesicles which comprise the intracellular reservoir with the plasma membrane, resulting in delivery of the h,k-atpase to the apical cell surface. during the interdigestive period, the h,k-atpase is re-endocytosed and returned to its storage compartment. chimera studies reveal that each subunit of the h,k-atpase possesses a sorting signal which participates in regulating this complex traffic . the a-subunit is endowed with a dominant apical targeting signal, which can drive the apical sorting of chimeric pumps expressed in both mdck and llc-pkl renal epithelial cells. the p-subunit of the h,k-atpase possesses a tyrosine-based endocytosis signal (roush et al., manuscript submitted). this signal causes the protein to be sorted basolaterally when it is expressed in mdck cells and apically when it is expressed in llc-pk1 cells. the na,k-atpase p-subunit does not possess a similar sequence domain. it seems likely that the two h,k-atpase signals participate in distinct stages of pump sorting in the gastric parietal cells. the apical signal in the a-subunit probably mediates the sorting of the entire complex to the apical membrane or the pre-apical storage compartment, whereas the p-subunit signal is responsible for ensuring the re-internalization of the pump following the cessation of secretagogue stimulation (courtois-coutry et al., 1997) . it remains to be determined why the p-subunit's tyrosine-based signal is differentially interpreted by mdck and llc-pk1 cells. investigation of this phenomenon may shed light on the nature and function of the epithelial sorting machinery. this apparent trend towards a multiplicity of signals is not entirely surprising, since many proteins are required to perform highly sophisticated feats of membrane targeting during the course of their transits throughout the endomembranous networks of the cell. for example, the pigr receptor expressed in its native hepatocytes or by transfection in mdck cells travels first to the basolateral membrane to pick up ligand and is then transported to the apical surface domain. it appears that an apical sorting signal in this protein's ectodomain might be required for basolateral to apical transcytosis, while a basolateral signal in the cytoplasmic domain ensures the initial basolateral delivery. unlike proteins that are constitutively expressed at one surface domain, a number of distinct and individually acting signals are necessary to orchestrate the more complicated surface targeting events displayed by pigr receptor, and other molecules like it. obviously, the hierarchical (both temporal and spatial) regulation of each signal will be of utmost importance in ensuring that a protein follows a physiologically relevent trafficking pathway. recent evidence, for example, demonstrates that the pig receptor undergoes phosphorylation on a cytosolic serine residue around the time that it is delivered to the basolateral surface (larkin et al., 1986) . this phosphorylation event appears to inactivate the protein's basolateral signal and thus permit its transcytosis to the apical membrane (casanova et al., 1990 ). perhaps not surprisingly, the greatest advances in the elucidation of sorting signals have been made with single membrane-spanning monomeric or homooligomeric proteins (e.g., pigr, ldl-r, tfr). with these molecules the requirements for surface expression are easily met and the effects of mutagenesis on tertiary structure can be assessed through well-characterized functional assays, such as receptor-ligand or antibody binding. through deletion analysis and heterologous expression in mdck cells, it was determined that the pigr (casanova et al., 1991) and the ldlr (hunziker et al., 1991) each contained basolateral targeting determinants which mapped to short, contiguous regions of their cytoplasmic domains (table 1). both signals could be grafted onto heterologous proteins and cause them to be targeted to the basolateral surface, supporting the idea that each determinant was truly an autonomous basolateral sorting signal. exhaustive mutagenesis studies have more finely mapped each of these determinants. the ldlr possesses two distinct basolateral targeting determinants, one that is "coated-pit related" (proximal determinant) and another which is tyrosine-dependent but not capable of mediating localization into coated-pits (distal determinant) (matter et al., 1992; . interestingly, the polymeric immunoglobulin receptor (pigr) signal may constitute yet another class of basolateral targetting determinant, since it shares little in the way of sequence homology with either determinant of the ldlr and shows weak tyrosine dependence (aroeti et al., 1993) . the general characteristics of these three determinants and the degree to which they are related are only beginning to be eluciated thomas and roth, 1994 ). an attempt to categorize these basolateral sorting determinants has been made by and is summarized in table 1 . before discussing the nature of the "coated-pit related" basolateral targeting determinant, it is necessary to be familiar with the signals that are known to mediate the accumulation of plasma membrane receptors into clathrin-coated pits (goldstein et al., 1985) . it is now generally accepted that tyrosine-and dileucinecontaining sequence motifs present in the cytoplasmic tails of a number of coated-pit clustering proteins serve as the critical recognition elements for the adaptor components of clathrin coats (pearse and robinson, 1990; trowbridge, 1991) . more recently, numerous studies have demonstrated a strong relationship between the signals which mediate localization into coated pits and a subset of those involved in basolateral targeting (brewer and roth, 1991; hunziker et al., 1991; lebivic et al., 1991) . for example, brewer and roth (1991) found that the apically targeted ha molecule could be completely rerouted to the basolateral membrane by replacing a strategically localized cysteine residue (cys 543) with a tyrosine in the cytoplasmic domain. this tyrosine was also sufficient to localize this protein into coated-pits and direct the protein's incorporation into endosomes. this observation that an endocytosis signal might also double as a basolateral targeting signal led to the suggestion that the recognition determinants for endocytosis and for tgn-to-basolateral targeting might be similar or identical to one another. thorough mutagenesis studies on the coated-pit localization and basolateral sorting determinants of ha-y543 (thomas and roth, 1994; lin et al., 1997) , vsvg protein (thomas et al., 1993) , and the ldlr , however, have led to a revision of this initial interpretation. it turns out that the "endocytosis signal" of both the ha-ys43 and the ldlr (proximal signal) can be resolved into two overlapping but distinct signal components. in other words, there is information recognized for endocytosis that is distinct from that recognized for basolateral sorting, even though the sequences are in part superimposed and share marked similarity. table 2 shows the systematic mutagenesis that ultimately unraveled this relationship. brewer and roth (1991) found that ha-y543 is capable of both basolateral sorting and endocytosis. the second generation mutant ha-y543,rs46, however, behaved as aprotein that was capable of endocytosis, but whose basolateral localization was inhibited (lin et al., 1997) . similar results were found with the ldlr proximal determinant. matter and colleagues (1994) showed that the truncation mutant ct27 was basolaterally targetted and rapidly endocytosed, while the removal of terminal acidic residues in ct22 produced a protein that was not capable of basolateral targetting, but could nonetheless be endocytosed. thus, the initial correlation between endocytosis signals and basolateral targeting has now resolved into two distinct but overlapping signals that can share common residues for their respective activities. the implications of this result are very exciting for the field of epithelial polarity. first, they suggest that the signals for basolateral sortingkargetting may be structurally similar to signals for clathrin-coated pit localization and endocytosis. the involvement of similar signals suggests that the sortinghecognition molecules themselves may be related. at least for endocytosis signals, there is evidence in favor of clathrin "adaptins" (of the ap2 plasma membrane class) playing a role in recognizing these sequences (pearse, 1988; glickman et al., 1989; beltzer and spiess, 1991; sorkin and carpenter, 1993; sosa et al., 1993) . in light of the recent characterization of adaptin related molecules (cops, discussed in section 111, below), it has been suggested that a family of structurally and functionally similar sorting adaptors may serve as the sorting machinery which interacts with these basolateral sorting signals . the findings support the more general contention that sorting at the level of the tgn may be mechanistically similar to that at the level of the endosome (matter et al., 1993 . taken altogether, there now appear to be two general classes of basolateral targeting determinants. one of these is biochemically related to the signals that mediate sorting into coated pits. this type of signal can be colinear with an endocytosis determinant and may share the critical tyrosine residue required for the activity of both, but it is nonetheless distinct and dissociable from an endocytosis signal. the second class of basolateral targeting determinants appears to be unrelated to clathrin-coated pit localization signals, although it may also strongly depend on a tyrosine for activity. this second type of determinant appears to be unique to the ldlr, pigr (casanovaet al., 1991) and the tw (dargement et al., 1993) , although these signals share no primary sequence homology with one another. it is possible however, that this second determinant present in these three proteins may be mutually similar in three-dimensional structure but not in primary sequence. in this context it is important to note that adaptor proteins are thought to recognize tyrosine residues in the context of a tight turn, which can be achieved by many different primary sequences (glickman et al., 1989; collawn et al., 1990 collawn et al., , 1991 bansal and gierasch, 1991) . more detailed analyses are revealing that while the dependency on tyrosine is crucial, other residues which are acidic and c-terminal to the tyrosine are also important. demonstrated that the clusters of two or more acidic amino acids downstream from a tyrosine, phenylalanine or di-leucine are important for signal function (see table 1 ). while the authors of this study have argued that it is premature to propose a common motif characteristic of all basolateral targeting determinants, they have found that this critical aromatic amino acid followed by acidic residues can be discerned in the cytoplasmic domains of many known proteins which are targeted to the basolateral membrane of mdck cells, including ecadherin, transferrin receptor, cation-independent and dependent mannose-6-phosphate receptors, lap, pigr and fcriib2. (see discussion of . as these authors have suggested, it will be exciting to define mutations that will prevent the recognition of these sequences so that the identification and characterization of the molecules which serve to interact with and interpret these signals can be facilitated. an ever growing list of proteins are anchored to membranes through a covalent attachment to glycosylphosphatidylinositol or gpi. proteins of this class are initially synthesized on bound polysomes as transmembrane polypeptides and, while still resident within the er, are cleaved from their transmembrane portions and transferred covalently to lumenally facing glycosyl-phosphatidylinositol molecules (cross, 1990) . gpi-anchored proteins are widely distributed with respect to both cell type and function. members of this class of proteins include protozoal surface coat proteins (e.g, the variant surface glycoproteins of trypanosomes), differentiation antigens (e.g., thy-i ) , adhesion molecules (e.g., the gpi-linked isoform of n-cam), hydrolases (e.g., alkaline phosphatase and snucleotidase), and receptors (folate receptor). the functional advantages that this membrane linkage confers upon a particular protein is presently unclear, and has been the focus of a great deal of attention (reviewed in brown, 1992) . in general, the gpi-linkage has been suggested to be important for enabling proteins to "c1uster"at a surface density much higher than is possible for single-pass transmembrane proteins (hooper, 1992) . studies have also shown that these clusters of gpi-anchored proteins may be important for certain cell surface signal transducing events (reviewed in anderson, 1993) . gpi-linked proteins captured the attention of epithelial biologists because of their polarized distribution in mdck cells (lisanti et al., 1988) and other cultured epithelial cell lines (lisanti et al., 1990) . the nearly exclusive correlation of membrane anchoring via gpi with apical localization raised the question as to whether or not the gpi membrane anchor was itself a signal for apical targeting. chimeric analyses showed clearly that the gpi-linkage is sufficient for apical targeting in mdck cells (brown et al., 1989; lisanti et al., 1989a,b) . of course, in the absence of a known default pathway for membrane proteins, it remains formally possible that the gpi-anchor prevents a protein from gaining entry into the basolateral sorting pathway. moreover, the fact that the cytoplasmic tail-minus versions of the ldl and pig receptors are directly targetted to the apical membrane is consistent with the possibility that apical sorting occurs by default (discussed in . nonetheless, the gpi-linkage is the field's best accepted apical localization signal characterized to date. interestingly, glycosphingolipids (gsls) share the apical preference of gpi-linked proteins and are generally found exclusively in the outer leaflets of the apical membranes of mdck cells. the means through which gpi-anchored proteins and glycosphingolipids (gsls) are sorted and subsequently targetted to the apical membrane are poorly un-derstood. it has been shown that gsls manifest biophysical properties which enable them to self-associate or form clusters in the plane of the membrane (thompson and tillack, 1985) . these properties have been invoked to support the proposal that gsl clustering occurs at the level of the tgn, and that newly synthesized gpi-linked proteins might co-cluster with these lipids (simons and van meer, 1988) . it has been further suggested that apically-destined transmembrane proteins could similarly be sorted through an ability to co-cluster with gsls and gpi-linked proteins . according to this model, apical sorting could take place through selective inclusion within these gsl microdomains, while certain basolateral membrane protein components would be sorted by selective exclusion. however, it should be pointed out that there is still no experimental evidence showing that the gsl clusters are important for apical sorting. one cell line in particular suggests that the role of gsls in sorting of gpi-anchored proteins may be more complex. a rat thyroid epithelial cell line (frt) distributes its gsls and gpi-anchored proteins to the basolateral surface while the polarized distribution of a number of transmembrane proteins is identical to that of mdckcells (zurzolo et al., 1993) . this suggests that at least some of the apical proteins analyzed (e.g., ha) do not partition with basolaterally directed gsls. the frt cell line will serve as an excellent tool for furthering our understanding about the role of glycolipid clustering in the sorting of proteins and lipids in polarized epithelial cells. most of the early studies in epithelial polarity used the kidney-derived mdck cell line as their workbench. however, the last six years has seen the introduction of a number of new cell culture models into the field: caco2 (pinto et al., 1983; matter et al., 1990; costa de beauregard et al., 1995) ; ht-29 and t-84 (human intestinal epithelial), (madara et al., 1987; polak-charcon et al., 1989; mikogami et al., 1994) ; llc-pk1 (pig kidney proximal tubule) (hull et al., 1976; gstrauthaler et al., 1985; gottardi et al., 1995) ; mdbk (madin-darby bovine kidney) (furuse et al., 1994) , frt (fischer rat thyroid) (zurzolo et al., 1993) , as well as primary cultures of choroid plexus and retinal pigmented epithelium (marrs et al., 1993) . as we have discussed in the first half of this review, we arejust beginning to elucidate the nature of certain "apical" and "basolateral" sorting signals. however, the "nonstandard" sorting of gpi-link proteins in frt cells mentioned above, and the fact that a number of proteins display tissue and cell-type specific membrane localizations (see table 3 ), calls into question the ways in which we think about polarized sorting signals and the mechanisms of sorting. as shown in table 3 , there are notable differences in the localization of certain membrane proteins expressed in different tissue cell-types. the na,k-atpase, nearly ubiquitously expressed at the basolateral domain of most polarized cell types, is localized to the lumenal (apical) domain of both retinal pigmented epithelial and chorid plexus cells (wright, 1972; steinberg and miller, 1979; spector and shiel and caplan, 1995a,b; (m) schwartz et al., 1985; (n) brown et al., 1988 . johanson, 1989 gundersen et al., 1991) . when the cdna encoding the ldl receptor was placed under the control of a metallothionein promoter and employed in the generation of a transgenic mouse, the receptor was expressed at the basolateral domains of liver and intestinal epithelial cells, but unexpectedly localized to the apical domains of proximal kidney tubule cells (pathak et al., 1990) . the polarized budding of certain viruses and the localization of their respective spike glycoproteins was shown to vary considerably between kidney derived mdck and thyroidderived frt cells (zurzolo et al., 1992a) . in some instances, ashift in the type of targeting pathway used by a protein can depend on the differentiated state of the cell culture (zurzolo et al., 1992b) . furthermore, the polarized localization of a particular gpi-linked protein was found to be developmentally regulated in drosophila embryos (shiel and caplan, 1995a) . finally, a remarkable flexibility and "plastic-ity" of protein sorting has been suggested to be present in kidney intercalated cells, which appear to direct the vacuolar proton pump to either surface domain, depending on particular environmental cues (schwartz et al., 1985; brown et al., 1988a) at the present time, we have little understanding of the signals or sorting mechanisms that mediate the differential sorting of the same protein in distinct cellular types. are different signals recognized by the different epithelial cells or is the same signal interpreted differently? is the sorting machinery itself different between polarized cells, or is the sorting machinery basically conserved between different cell-types while its regulation, adaptation, or wiring to the targeting machinery is different? evidence discussed in the second half of this review on the rab family of proteins suggests that elements of the targeting machinery are in fact highly conserved between different cell types, and it is the cell-type specific adaptation of this machinery which accounts for differences. nonetheless, it is becoming clear that the sorting of a particular protein can be a highly idiosyncratic feature of each polarized cell model. the observation that different epithelial cell lines may handle the same protein (or the same signal) differently has to reflect more than a mere capriciousness of epithelial cells in culture. each of the cultured cell models employed in polarity studies derive from and reflect some of the differentiated features of a tissue or organ system. accordingly, the sorting behavior observed in a particular cell type needs to be evaluated in the context of this cell's functional history. for example, is this cell derived from a tissue specialized for apical secretion or apical endocytosis? studies of the sorting of ion-transporting atpase molecules expressed in distal tubule-derived mdck and proximal tubule derived-llc-pk1 kidney cells suggest that the distinct cell surface distributions which an atpase subunit achieve in these two lines are consistent with established physiologic differences between the distal and proximal tubule epithelial cells (roush et al., manuscript submitted). these observations have led to the suggestion that sorting mediates delivery to functionally defined rather than topographically defined domains (gottardi and caplan, 1993a) . it is becoming quite clear that the findings in the field of intracellular protein transport (reviewed by rothman, 1994 and by mellman, 1994) will prove to be extremely valuable to the discipline of epithelial polarity. in this field, the convergence of studies on synaptic vesicle (regulated) secretion in neurons, constitutive secretion in yeast, and intra-golgi transport have led to the rapid identifcation and characterization of the basic components necessary for vesicle formation, docking and fusion. clearly, the general components of the bud-ding and docking machinery lie at the heart of any transport process, whether we are considering the transport of a membrane protein from er to golgi, or a secretory protein from the tgn to a particular cell surface. in the following sections we touch upon some of the key discoveries in the field of intracellular transport and focus on the relevant molecules that may contribute to polarized sorting and delivery processes. one of the recent paradigms in intracellularprotein transport is based on the concept that vesicle shuttling between different organellar compartments is regulated through the coordinated efforts of different gtp-binding proteins. there are two broad classes of gtp-binding proteins which have been shown to regulate membrane trafficking events; the small g proteins (rabs and arf) reviewed by (donaldson and klausner, 1994; pfeffer, 1992 pfeffer, ,1994 and the trimeric g proteins (reviewed by bomsel and mostov, 1992) . the role of a gtp-binding protein in regulating vesicular transport was first realized with the analysis of one of the temperature sensitive sec (secretory) mutants in yeast (salminen and novick, 1987) . sec4 mutants display a rather striking accumulation of secretory vesicles when cultured at the restrictive temperature. the cloning, sequencing, and characterization of the sec4 gene revealed that it encoded a ras-like or 'small' gtp-binding protein which was present on the surfaces of the vesicles and could bind and hydrolyze gtp (salminen and novick, 1987; goud et al., 1988; kabcenell et al., 1990) . since the phenotype of cells bearing mutant sec4 is the accumulation of transport vesicles, it was apparent that sec4 is necessary for the targeting and/or fusion of secretory vesicles with the plasma membrane. similar results were found with another yeast protein yptl(48% identical to sec4), which in its mutant form inhibited vesicular transport between the er and golgi complex (gallwitz et al., 1983; segev et al., 1988) . the suggestion that two small gtpases were important in the regulation of two different vesicular transport events in yeast led to the hypothesis that each step in vesicular traffic was regulated by a specific gtpase (bourne et al., 1990) . these ras-like gtpases are known to zdopt either of two distinct conformations, depending upon whether or not they are complexed with gtp or gdp. consequently, these gtpases have been postulated to serve as key regulators or "molecular switches" for membrane fission and fusion events. the apparent generality of ras-like gtpase in yeast, as revealed by sec4 and yptl, inspired asearch for these proteins' mammalian counterparts. to date, 30 yptl/seccrelated proteins have been identified and are often referred to as rab proteins ("ras-like" proteins from rat brain) reviewed in (balch, 1990; hall, 1990; goud and mccaffrey, 1991; zerial and stenmark, 1994) . a number of rabs have been localized to specific organelles within the cell and through the combined efforts of in vitro and in vivo approaches have been shown to regulate membrane traffic between these organelles (reviewed in zerial and stenmark, 1994) . how this class of molecules contributes to the overall fidelity of membrane trafficking events is still unclear (rothman, 1994) . the idea that specific rab proteins regulate distinct steps along the transport pathway (e.g., rabl always regulates er to golgi traffic, whether in a kidney cell or neuron) led to the hypothesis that cells which contain unique, cell-type specific transport processes might be regulated by disinct rabs. indeed, the best example of this is the family of rab3 isofoms which have been found to be localized within cells which are well-adapted for regulated secretory events. rab3a has been suggested to be important in the regulation of caz+ dependent secretion in neuronal (fischer von mollard et al., 1991) , neuroendocrine (darchen et al., 1990) and endocrine cell types (mizoguchi et al., 1989) . interestingly, an isoform of rab3a, rab3d, has been localized to the glucose transporter-containing vesicles of adipocytes, which are known to undergo regulated exocytosis after insulin stimulation (baldini et al., 1992) . thus, despite cell-specific differences, or vesicle-content differences, these regulated pathways rely on similar rabs (rab3). thus, distinct regulated exocytic events in a variety of cell types make use of similar molecular machinery (lutcke et al., 1993) . in this context, it has been speculated that polarized epithelial cells, with their distinct apical and basolateral targeting pathways, may employ epithelia-specific rab molecules. recent data suggest that this may be true. there are four rabs which have been implicated in polarized epithelial-specific functions: rab 17, rab 3b, rabl3, and rab8. of the four, only rabl7 is truly specific to polarized epitheiial cells. in the developing kidney, rabl7 mrna is detected only after mesenchyme is induced to differentiate into polarized epithelial structures (lutcke et al., 1993) . interestingly, rabl7 induction was shown to occur just prior to the appearance of apical markers and has therefore been suggested to be involved in the generation of apicalhasolateral polarity in these cells. rab 17 localizes to the basolateral membrane and to electron dense tubules near the apical membrane. since rab proteins have been shown to regulate transport between the subcellular compartments with which they associate, it has been suggested that rabl7 regulates epithelial transcytosis. as we stated previously, two isoforms of rab3 (3a and 3d) have been implicated in the regulated exocytosis events shared by neuronal, endocrine and adipocyte cell types. interestingly, another isoform of rab3,3b, has been shown to be specific for polarized epithelial cells and is exclusively localized to the apical pole of cells, near the tight junctions (weber et al., 1994) . rabl3, like rab3b, also accumulates at the apical poles of polarized cells and co-localizes with the tight junction associated protein, zo-1 (zahraoui et al., 1994) . it has been suggested that these two rabs could regulate events necessary for the establishment of polarity. for example, since the localization of both rabs are completely dependent on the presence the of cell-cell contacts, it is possible that these mole-cules control the recruitment of membrane protein-containing vesicles required for establishing the tight junction "fence," a structure thought to maintain the distinct protein and lipid compositions of apical and basolateral membranes (dragsten et al., 1981) . it has also been proposed that these rabs control general vesicle targetting to the apical membrane (zahraoui et al., 1994) . this hypothesis was based on two independent observations. it has been shown that an apical membrane protein (aminopeptidase) inserts preferentially into the apical membrane at regions of cell-cell contact in mdck cells (louvard, 1980) . furthermore, under conditions in which mdck cells are denied intercellular contacts, apical proteins appear to be sorted and retained within a large subapical vacuolar compartment (vacuolar apical compartment, or vac) which, after initiation of cell-cell contact, is inserted preferentially at regions of cell-cell contact (vega-salas et al., 1988) . taken together, the localization of r a b l 3 and rab3b at this region of cell contact places these monomeric gtpases in a position to regulate the delivery of apical proteins to the cell surface (zahraoui et al., 1994) . moreover, the localization of a regulated, exocytic compartmentspecific rab (rab3) to a subdomain of the apical membrane of polarized cells is intriguing and suggests possible functional relationships between these subcellular compartments. the last rab worth exploring in the context of epithelial polarity is rab8. while rab8 is not solely expressed in polarized cells, it is the only rab that has been functionally implicated in vectorial targeting. a peptide derived from the c-terminal region of rab8 can inhibit basolateral but not apical transport of membrane proteins in a permeabilized-mdck cell assay (huber et al., 1993a) . interestingly, rab8 can also regulate membrane transport to the dendritic plasma membranes of neurons in culture; antisense rab8 oligonucleotides decrease the level of viral glycoprotein transported to this domain (huber et al., 1993b) . this observation is consistent with the model which suggests that the mechanisms which produce axoddendrite polarity in neurons may be similar to those involved in apicallbasolateral polarity in epithelia (simons et al., 1992) . taken together, the identification of a polarized epithelia-specific rab (rab 17), and the localization of other rabs to specific polarized epithelial domains (rab 13 and rab3b, apical; rab8, basolateral) suggests that rabs may regulate specific pathways in polarized epithelial cells. for the epithelial cell biologist, the obvious question is, "what brings about the pathway-specific localizations of rab proteins in polarized epithelial cells?" it has been demonstrated that the carboxy-terminal regions of rab proteins are responsible for their unique cellular localizations (chavrier et al., 1991) . it has been suggested that organelle-specific receptors exist which recognize the c-terminal domains of these molecules. at least in terms of polarized cells, it would be tempting to speculate that identification of such receptors for rabl3,3b and rab8 will bring us one step closer to an understanding of the overall machinery that orchestrates domain-specific vesicle formation and targeting. recent evidence, however, suggests that rabs may not provide the primary level of specificity in membrane targeting events (brennwald and novick, 1993; reviewed by rothman and warren, 1994) . as we discuss below, a new class of proteins, the snares, may provide the necessary specificity for vesicle-membrane targeting events throughout the cell. the snare hypothesis for vesicle targeting arose from research in three related fields: synaptic vesicle release in neurons, transport between cisternae of the golgi, and secretion in yeast. briefly, a number of synaptic proteins were discovered to be important for the regulated fusion of synaptic vesicles with their targets on the pre-synaptic plasma membranes (reviewed by pevsner and scheller, 1994) . homologues of these proteins were found in yeast and shown to be required for constitutive vesicle transport (aalto et al., 1993) . at the same time, key elements of the general machinery for intracellular membrane fusion were being elucidated. in all three .cases, membrane fusion requires an nem-sensitive factor (nsf), adaptors that link nsf to membrane proteins (snaps: soluble nsf attachment proteins) and the membrane receptors for the nsf-snap complexes (snares: snap receptors) (reviewed in rothman and warren, 1994) . distinct snare proteins are present in the membranes of the vesicle and the target. the snare hypothesis stipulates that each transport vesicle is endowed with its own vesicle-(v-) snare (or vamp-like molecule) that can specifically interact with its cognate target-( t -) snare (or syntaxin/snap25-like protein). this 'pairing' could ensure vesicleharget membrane specificity, while a general fusion apparatus consisting of nsf and snaps could be used throught the cell (sollner et al., 1993) . in the context of epithelial polarity, this hypothesis suggests that vectorial targeting of apical and basolateral proteins will require distinct v-snares. interesting recent data suggest that the situation in at least one epithelial cell type may be somewhat more complicated. when the surface delivery of membrane proteins is examined in mdck cells permeabilized at their apical or basolateral surfaces with streptolysin 0, it appears that basolateral transport involves all of the machinery discussed above. toxins which cleave snares inhibit basolateral delivery, as do antibodies directed against snaps. in contrast, apical protein insertion is unaffected by these reagents. isolation of apically-bound vesicles from mdck cells reveals the presence of high concentrations of an adducin homologue in their surface membranes. adducins are calcium-dependent phospholipid binding proteins thought to be involved in a number of membrane fusion events (ilkonen et al., 1995) . it would appear, therefore, that completely distinct classes of vesicular targeting and fusion machinery may operate in the two membrane delivery pathways present in polarized epithelial cells. in the absence of a readily available genetic system with which to identify the genes and gene products necessary for such higher eukarotic functions as transcytosis or polarized targeting, epithelial cell biologists have been resigned to the prospect of "poking" at the epithelial cell with various reagents and watching how it responds. reagents which prevent the polymerization of actin (gottlieb et al., 1993; jackmon et al., 1994) and tubulin (achler et al., 1989; parczyk et al., 1989) , toxins which modify a particular class of g proteins (stow et al., 1991; pimplikar and simons, 1993b) , or toxins that inactivate the vamp, syntaxin and snap-25 molecules described above, second messanger stimulators, analogues of the messangers themselves (apodaca et al., 1994; cardone et al., 1994; hansen and casanova, 1994) and the remarkable fungal metabolite brefeldin a (bfa) are all being incorporated into the repetoire of tools which we hope will enable us to gleen more information from a particular transport pathway. those interested in polarized and nonpolarized cell functions alike have made use of such cell-perturbing reagents. since the focus of this review is epithelial polarity, we have chosen to summarize some of the studies which are providing insights about the mechanisms of polarized sorting and targeting. brefeldin a is a fungal metabolite that endeared itself to cell biologists because of its dramatic effect on the protein secretory pathway (reviewed in . protein secretion is inhibited by bfa: membrane trafficking out of the er is blocked and the golgi appears to breakdown and become redistributed into the er (lippincott-schwartz et a]., 1989). before golgi redistribution, bfa causes this organelle to form tubular extensions which are devoid of any cytoplasmic (nonclathrin) "coat" material (lippincottschwartz et al., 1990) . it has been shown that these morphological changes are not restricted to the golgi but rather are observed in a number of organelles of the endomembranous network such as endosomes, lysosomes and the tgn (hunziker et al., 1991; lippincott-schwartz et al., 1991; wood et al., 1991) , suggesting that the bfa "effector" might play a role in membrane transport events all over the cell. perhaps surprisingly, while membrane transport phenomena are remarkably altered in the presence of bfa, several processes are clearly unaffected, including receptor mediated endocytosis and endocytic recycling . from the standpoint of sorting and polarized delivery, bfa's most interesting property is its ability to differentially affect polarized cell surface targeting events. for example, low and colleagues ( i 991,1992 ) determined a concentration of bfa where er-golgi trafficking was not inhibited, so that delivery from the tgn to the surface could be assayed for bfa sensitivity. interestingly, bfa inhibited the apical delivery of both endogenous, mdck secretory proteins (199 1) and the membrane protein dppiv (1992) while also enhancing their mis-delivery to the basolateral surface. basolateral targeting of the endogenous mdck protein, uvomorulin, was not affected under these conditions. taken together, it would seem that a target molecule for bfa action exists that is exclusively involved in directing apical vesicles or which is simply more sensitive to the effects of bfa than similar molecules participating in the basolateral pathway. either way, these results provide a hint that there are indeed molecular differences between these two targeting pathways. it is important to add that in addition to inhibiting the exocytic apical pathway in mdck cells, basolateral to apical transcytosis is also inhibited by this drug (hunziker et al., 1991; low et al., 1992) . these findings have led to the suggestion that sorting mechanisms for apically destined proteins, whether along the exocytic or the transcytotic pathway may be functionally and biochemically similar (hunziker et al., 1991) . the fact that the loss of the structural integrity of the golgi induced by bfa correlates with a striking absence of its characteristic "coat" (observed at the em level) led to the idea that coat proteins might be rendered non-functional due to bfa action. through a number of studies (reviewed by donaldson et al., 1992; helms and rothman, 1992; rothman & orci, 1992 ) molecules which make up this "coat" were identified and characterized (e.g., pcop and a m ) . an "order of events" necessary for vesicle budding emerged from these studies and is outlined below. arf is a gtp-binding protein loosely related to ras and distinct from the family of rabs. in its gtp-bound state, it is capable of associating with the membrane by virtue of its myristoyl group, while its gdp-bound form is soluble and not membrane bound. arf binding to membranes appears to be the signal for coatomer binding, that is, the binding of pcop in addition to other as yet uncharacterized coat proteins. coatomer binding is believed to be absolutely necessary for vesicle budding. therefore proper coatomer assembly would be required for any event downstream of budding, such as targeting. recently, it has been determined that bfa inhibits coatomer assembly and vesicle formation through arf, by essentially allowing it to remain in its gdp-bound or inactive form. there exists a class of proteins which are able to catalyze the exchange of gdp for gtp called guanine nucleotide exchange factors (gne). bfa has been proposed to antagonize the action of a gne on arf, thus preventing coatomer assembly and membrane budding helms and rothman, 1992) . with the recent identification of an ever-growing family of new arf-related genes (kahn et al., 1991) and the speculation that different cops may exist in the control of membrane budding events from different organelles , there is growing excitement that arfs and cops will turn-out to be essential components for regulating a particular level of specificity inherent to membrane targetting events. in the context of bfas affect on apical sorting and targeting in polarized mdck cells (low et al., 1991 (low et al., , 1992 , it is likely that distinct arfkoatomer complexes regulate the budding of apical and basolaterallydestined vesicles from the tgn. moreover, the fact that significant missorting into the basolateral pathway was observed in the presence of bfa (low et al., 1992) suggests that coatomer assembly may be inextricably linked to proper secretory and membrane protein sorting. it has been known for some time that members of the heterotrimeric family of g proteins are associated not only with the plasma membrane but also with intracellular membranes (reviewed by bomsel and mostov, 1992) . a number of toxins (cholera, pertussis and mastoparan) known to activate or inhibit various classes of g proteins have been applied to studies of polarized sorting and targeting. stow et al. (1991) found that overexpression of gai-3 in polarized llc-pk1 cells significantly reduced the level of constitutive basolateral secretion of an extracellular matrix component, heparan sulfate proteoglycan. pertussis toxin, which adp-ribosylates and inactivates the a-subunits of the g a i/o class of heterotrimeric g proteins, relieved this inhibition. similarly, pimplikar and simons (1993) suggested that gi and gs may differentially regulate the trafficking of apical and basolateral vesicles in slo-permeabilized mdck cells, while leyte et al. (1992) found that gi/o and gs associated with the tgn could oppositely regulate constitutive secretory vesicle formation. it should be noted that in no case did the g protein related inhibition or stimulation appear to affect the actual sorting or missorting of apical or basolaterally destined proteins (in contrast to the bfa results discussed above (low et al., 1992) , but rather may only affect the rate or "efficiency" of sortingkargetting . a possible link between heterotrimeric g proteins and coatomer formatiodvesicle budding was provided by ktistakis et al., (1992) . this group found that activation of a g a protein with mastoparan promoted pcop binding and prevented bfainduced effects. pretreatment of cells with pertussis toxin, which is known to specifically affect g a i subclass of heterotrimerics, prevented mastoparan's antagonizing effects on bfa. stated more simply, these results showed that activation of a pertussis-toxin-sensitive ga promotes the binding of pcop to golgi membranes and thus antagonizes the action of bfa. the authors of this study suggest further that different subclasses or isoforms of ga could be responsible for some of the differences in bfa-sensitivities observed between cell types and organellar membranes. these key observations have led to the idea that heterotrimeric g proteins, by virtue of their membrane topology would be ideal candidates for coordinating the transfer of sorting information to the cytoplasmic surface of the tgn necessary for vesicle budding (bomsel and mostov, 1992; ktistakis et al., 1992) . the outer surface of a fruit fly embryo is composed of a monolayer of polarized epithelial cells. the apical membranes of these epithelial cells face the outer shell, or chorion, while their basolateral surfaces face the embryonic interior and yolk space. invaginations of this surface epithelium give rise rise to all of the embryo's internal tissue structures (for review see shiel and caplan, 1995b) . recent investigations have examined the mechanisms through which proteins are sorted in these epithelial cells. human placental alkaline phosphatase (plap) is a gpi-linked protein which has been shown to be sorted to the apical plasma membrane when it is expressed by transfection in mdck cells. a chimeric construct of plap, in which the gpilinkage domain is replaced by the transmembrane and cytoplasmic domains of the vsv g protein (plapg), is sorted to the basolateral surfaces of mdck cells (brown et al., 1989) . these two proteins have been expressed under the control of heat shock promoters in transgenic flies and their distributions have been examined in embryonic epithelia throughout embryogenesis (shiel and caplan, 1995a) . as would be expected, the plapg protein is restricted to basolateral surfaces throughout ontogeny in the surface epithelial cells as well as in the internal epithelia which derive from invaginations of the surface cells. surprisingly, plap was also restricted to a basolateral distribution in the surface epithelial cells in both early and late stage embryos. biochemical experiments demonstrated that this mis-sorting of the plap protein can not be attributed to problems with the addition of the gpi-linkage, since at all embryonic stages plap is correctly glipiated. internal epithelial cells sorted plap exclusively to their apical surfaces. since in many cases internal epithelia form from surface epithelia without undergoing any mitosis (e.g., salivary gland), essentially the same epithelial cell is capable of differentially sorting plap depending on that cell's physical position within the embryo. examination of epithelia undergoing invagination (e.g., ventral furrow, tracheal placode) demonstrate that the transition in plap sorting occurs in the early stages of the invagination process. while the mechanism responsible for this switch remains unclear, the power of drosophilu genetics will hopefully allow the cellular components responsible for this transition to be readily identified. it is likely that the isolation of the proteins responsible for this phenomenon will shed light on the drosophilu as well as on the mammalian epithelial sorting machinery. a drosophilu mutation whose phenotype includes peturbations of the polarized organization of the surface epithelial cells has recently been identified and characterized at the molecular level wodarz et al., 1993) . the crumbs gene encodes a transmembrane protein which is normally expressed in the apical membranes of surface and internal epithelial cells. mutation of the crumbs gene results in a loss of crumbs polarity and markedly alters embryonic morphology. genetic studies have demonstrated that the crumbs gene product is necessary not only for its own apical sorting, but for the apical delivery of other proteins as well. furthermore, the product of the stardust gene appears to interact with the crumbs protein and also appears to participate in apical sorting. understanding these proteins' biochemical functions and their intermolecular associations will undoubtedly provide enormous insight into the cellular components responsible for generating and maintaining the polarized phenotype. hopefully, the development of genetic approaches such as these, in concert with the continuing refinement of in vitro and model systems, will allow us to develop a clear and fundamental understanding of how epithelial cells produce their remarkable asymmetry. yeast syntaxins ssolp and sso2p belong to a family of related membrane proteins that function in vesicular transport role ofmicrotubules in polarized delivery of apical membrane proteins to the brush border ofthe intestinal epithelium distribution of transport proteins over animal cell membranes podocytosis of small molecules and ions by caveolae the calmodulin antagonist, w-13, alters transcytosis, recyclingand morphology ofthe endocyticpathway in madine-darby canine kidney cells mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum cloning of a rab3 isotype predominately expressed in adipocytes the npxy internalization signal of the ldl receptor adopts a reverse turn conformation biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation. 1 in vifro binding oftheasiatoglycoproteinreceptor to the betaadaptin of plasma membrane coated vesicles identificationofeffector-activatingresidues ofgsa lntracellular protein topogenesis. proc. natl. acad. sci. usa 77 role of heterotrimeric g proteins in membrane traffic the gtpase superfamily: aconserved switch sorting of gpi-anchored proteins to glycolipid-enriched membrane an h+-atpase in opposite plasma membrane 1317-1328. for diverse cell functions 413421. subdomains during transport to the cell surface interactions between gpi-anchored proteins and membrane lipids mechanism of membrane anchoring affects polarized expression oftwo proteins in mdck cells constitutive and regulated secretion of proteins intracellular sorting and polarized cell surface delivery of nqk-atpase, an endogenous component of mdck cell basolateral plasma membranes dependence on ph of polarized sorting of secreted proteins sortingofmembrane and secretoryproteins in polarizedepithelial cells phorbol myristate acetate-mediated stimulation of transcytosis and apical recycling in mdck cells phosphorylation of the polymeric immunoglobulin receptor required for its efficient transcytosis an autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor hypervariable c-terminal domain of rab proteins acts as a targeting signal transferrin receptor internalization sequence yxrf implicates a tight turn as the structural recognition motif for endocytosis transplantedldl and mannose-6-phosphate receptor internalization signals promote high-efficiency endocytosis of the transferrin receptor asortingsignal for the basolateral delivery of the vesicular stomatitis virus (vsv) g protein lies in its luminal domain: analysisofthe targetingofvsv g-influenzahemagglutininchimeras suppression of villin expression by antisense rna impairs brush border assembly in polarized epithelial intestinal cells a tyrosine-based signal targets h,k-atpase to a regulated compartment and is required for the cessation of gastric acid secretion high-resolution epitope mapping of hgh-receptor interactions by alanine-scanning mutagenesis association of the gtp-binding protein rab3a with bovine adrenal chromaffin granules the internalization signal and the phosphorylation site of transferrin receptor are distinct from the main basolateral sorting information arf: a key regulatory switch in membrane traffic and organelle structure brefeldin a inhibits golgi membrane-catalysed exchange of guanine nucleotide onto arf protein polarized sorting ofglypiated proteins in hippocampal neurons involvement of na,k-atpase in antinatriuretic action of mineralocorticoids in mammalian kidney membrane assymetry in epithelia: is the tight junction a barrier to diffusion in the plasma membrane? a small gtp-binding protein dissociates from synaptic vesicles during exocytosis an enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late golgi compartments direct association of occludin with zo-1 and its possible involvement in the localization of occludin at tightjunctions a yeast gene encoding a protein homologous to the human c-hashas proto-oncogene product specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor 11 receptor receptor-mediated endocytosis: concepts emerging h m the ldl receptor system nonpolarized secretion of truncated forms of the influenza hemagglutinin and the vesicular stomatitis virus g protein from mdck cells an ion transporting atpase encodes multiple localization signals biotinylation and assessment of membrane polarity: caveats and methodological conerns sorting of ion transport proteins in polarized cells secretion of endogenous and exogenous proteins from polarized mdck monolayers sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface ofpolarized epithelial cells small gtp-binding proteins and their role in transport a gtp-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast biochemical characterization ofrenal epithelial cell cultures (llc-pk1 and mdck) apical polarity ofna,k-atpase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton mechanism for regulatingcell surfacedistributionofna,k-atpase inpolarizedepithelial cells gs alphastimulates transcytosis and apical secretion in mdck cells through camp and protein kinase a inhibition by brefeldin a of a golgi membrane enzyme that catalyzes exchange of guanine nucleotide bound onto arf protein more than just a membrane anchor rab 8, a small gtpase involved in vesicular traffic between the tgn and the basolateral plasma membrane protein transport to the dentritic plasma membrane of cultured neurons is regulated by rab8p the origin and characteristics of a pig cell strain, llc-pk 1 basolateral sorting in mdck cells requires a distinct cytoplasmic domain determinant different requirements for nsf, snap, andrab proteins in apical and basolateral transport inmdck cells inhibition of apical but not basolateral endocytosisofricinandfolateincaco-2 cells by cytochalasind binding and hydrolysis of guanine nucleotides by sec4p, ayeast protein involved in the regulation of vesiculartraffic human adp-ribosylationfactors: a functionally conserved family of gtp-binding proteins crumbs and stardust, two genes of drosophila required for the development of epithelial cell polarity. development suppl exocytotic pathways exist to both the apical and the basolateral cell surface of the polarized epithelial cell mdck the biogenesis of lysosomes action of brefeldin a blocked by activation of a pertussis-toxin-sensitive g protein the signal sequence of nascent preprolactin interacts with the 54k polypeptide of the signal recognition particle phosphorylationofthe rat hepaticpolymericiga receptor an internal deletion in the cytoplasmic tail reverses the apical localization of human ngf receptor in transfected mdck cells multiple trimeric g-proteins on the trans-golgi network exert stimulatory and inhibitory effects on secretory vesicle formation tyrosine-dependent basolateral sortingsignals are distinct from tyrosine-dependent internalization signals a signal sequence for the insertion of a transmembrane glycoprotein rapid redistribution of golgi proteins into the er in cells treated with brefeldin a: evidence for membrane cycling from golgi to er microtubule-dependentretrograde transport of proteins into the er in the presence of brefeldin a suggests an er recycling pathway brefeldin a's effects on endosomes, lysosomes, and the tgn suggest a general mechanism for regulating organelle structure and membrane traffic polarized apical distribution of glycosyl-phoshatidylinositol-anchoredproteins in a renal epithelial cell line steady-state distribution and biogenesis of endogenous mdck-glycoproteins: evidence for intracellular sorting and polarized cell surface delivery preferred apical distribution of glycosyl-phosphatidylinositol (gpi) anchored proteins: a highly conserved feature of the polarized epithelial cell phenotype apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells selective inhibition ofprotein targeting to the apical domain of mdck cells by brefeldin a inhibition by brefeldina ofprotein secretion from the apical cell surfaceofmadin-darby caninekidney cells rabl7, a novel small gtpase, is specific for epithelial cells and is induced during cell polarization targeting and retentioon ofgolgi membrane proteins structural analysis o f a human intestinal epithelial cell line distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different na,k-atpase distributions in polarized epithelia sortingofan apical plasmamembraneglycoproteinoccurs before it reaches the cell surface in cultured epithelial cells sortingofendogenous plasmamembrane proteins occurs from two sites in cultured human intestinal epithelial cells (caco-2) basolateral sorting of ldl receptor in mdck cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants mechanisms ofcell polarity: sorting and transport in epithelial cells structural requirements and sequence motifs for polarized sorting and endocytosis of ldl and fc receptors in mdck cells polarizedexpressionofa chimeric protein in which the transmembrane and cytoplasmic doamins of influenza hemagglutinin have beenreplaced by those of the vesicular stomatitis g protein membranes and sorting. c u r apical-to-basal transepithelial transport biogenesis of epithelial cell polarity: of human lactoferrin in the intestinal cell line ht-29c1.19a intracellular sorting and vectorial exocytosis of an apical plasmamembrane glycoprotein tissue distribution of smg p25a, a ras p21-like gtp-binding protein, studied by use of a specific monoclonal antibody polymeric immunoglobulin receptor expressed in mdck cells transcytoses iga plasma membrane protein sorting in polarized epithelial short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum the trans-most cisternae of the golgi complex: a compartment for sorting of secretory and plasma membrane proteins microtubules are involved in the secretion of proteins at the apical cell surface of the polarized epithelial cell, madin-darby canine kidney tissue-specific sorting of the human ldl receptor in polarized epithelia of transgenic mice clathrin, adaptors, andsorting receptors compete for adaptors found in plasmamembranecoated pits mechanisms of vesicle docking and fusion: insights from the gtp-bindingproteins inintracellulartransport. trendsincell biology2 intracellular sorting and basolateral appearance of the g j. 7,333 1-3336. nervous system isoformsofthena,k-atpase are presentin both axons and dendrites of hippocampal neurons in culture regulation of apical transport in epithelial cells by a gs class of heterotrimeric g protein role of heterotrimeric g proteins in polarized membrane transport the effect ofmodifying the culture medium on cell polarity in a human colon cell line replacement of the cytoplasmic domain alters sorting of a viral glycoprotein in polarized cells localization of sodium pumps in the choroid plexus epithelium nuclear localization signals in polyomavirus large-t viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same golgi apparatus during their intracellular transport in doubly infected madin-darby canine kidney cells the distribution of na,k-atpase in the retinal pigmented epithelium from chicken embryo is polarized in vivo but not in primary cell culture the roleofclathrin, adaptors and dynamin inendocytosis morphogenesis of the polarized epithelial cell phenotype polarity of epithelial and neuronal cells asymmetric budding of viruses in epithelial monolayers: a model system for study of epithelial polarity influenza virus hemagglutinin expression is polarized in cells infected with recombinant sv40 viruses carrying cloned hemagglutinin dna the large extracellular domain is sufficient for the correct sorting of secreted or chimeric influenza virus hemagglutinins in polarized monkey kidney cells the large external domain is sufficient for the correct sorting of secreted or chimeric influenza virus hemagglutinins in polarized monkey kidney cells mechanisms of intracellular protein transport molecular dissectionofthesecretory pathway implications ofthe snare hypothesis for intracellularmembrane topology and dynamics a ras-like protein is required for a postgolgi event in yeast secretion membrane and secretory proteins are transported from the golgi complex to the sinusoidal plasmalemmaofhepatocytes by distinct vesicular carriers plasticity of functional epithelial polarity developmental regulation of membrane protein sorting in the generation of epithelial polarity in mammalian and lipid sorting in epithelia polarized sorting in epithelia biogenesis of cell-surface polarity in epithelial cells and neurons the phosphomannosyl recognition system for intracellular transport of lysosomal enzymes snap receptors implicated in vesicle targeting and fusion interaction of activated egf receptor with coated pit adaptins in vitro binding of plasma-coated vesicle adaptors to the cytoplasmic domain of lysosomal acid phosphatase 38), c207<216. drosophila embryos atpase in polarized epithelial cells the mammalian choroid plexus transport and membrane properties of the retinal pigment epithelium nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells a heterotrimeric g protein, gai-3, on golgi membranes regulates the secretion o f a heparan sulfate proteoglycan in llc-pki epithelial cells a golgi retention signal in a membrane-spanning domain of coronavirus el protein molecularcloning,characterization, subcellular localization and dynamics of p23, the mammalian kdel receptor vesicular stomatitis virus glycoprotein contains a dominant cytoplasmic basolateral sorting signal critically dependent on tyrosine the basolateral targetingsignal inthe cytoplasmicdomainofvsv g protein resembles a variety of intracellular targeting motifs related by primary sequence but having diverse targeting activities organization of glycosphingolipids in bilayers and plasmamembranes of mammalian cells sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans golgi network of att 20 cells endocytosis and signals for internalization exocytosis of vacuolar apical compartment (vac): a c e l k e l l contact controlled mechanism for the establishment ofthe apical plasma membrane domain in epithelial cells analysisofthedistributionofchargedresidues inthen-terminal regionofsignal sequences: implications for protein export in prokaryotic and eukaryotic cells mechanisms of protein translocation across the endoplasmic reticulum distinct transport vesicles mediate the delivery of plasmamembrane proteins to the apical and basolateral domains of mdck cells expression and polarized targeting of a rab3 isoform in epithelial cells crumbs is involved in the control of apical protein targeting during drosophrlu epithelial development brefeldin a causes amicrotubule-mediatedfusionofthe mechanismsofiontransportacrossthe choroidplexus a small rab gtpase is distributed in cytoplasmic vesicles in non-polarized cells but colocalizes with the tightjunction marker 20-1 in polarized epithelial cells opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in fischer rat thyroid epithelial cells key: cord-013315-plptulfb authors: tilocca, bruno; soggiu, alessio; greco, viviana; piras, cristian; arrigoni, norma; ricchi, matteo; britti, domenico; urbani, andrea; roncada, paola title: immunoinformatic-based prediction of candidate epitopes for the diagnosis and control of paratuberculosis (johne’s disease) date: 2020-08-27 journal: pathogens doi: 10.3390/pathogens9090705 sha: doc_id: 13315 cord_uid: plptulfb paratuberculosis is an infectious disease of ruminants caused by mycobacterium avium subsp. paratuberculosis (map). map is an intracellular pathogen with a possible zoonotic potential since it has been successfully isolated from the intestine and blood of crohn’s disease patients.since no cure is available, after the detection of the disease, animal culling is the sole applicable containment strategy. however, the difficult detection of the disease in its subclinical form, facilitates its spread raising the need for the development of effective diagnosis and vaccination strategies. the prompt identification and isolation of the infected animals in the subclinical stage would prevent the spread of the infection.in the present study, an immunoinformatic approach has been used to investigate the immunogenic properties of 10 map proteins. these proteins were chosen according to a previously published immunoproteomics approach. for each previously-described immunoreactive protein, we predicted the epitopes capable of eliciting an immune response by binding both b-cells and/or class i mhc antigens. the retrieved peptide sequences were analyzed for their specificity and cross-reactivity. the final aim is to employ the discovered peptides sequences as a filtered library useful for early-stage diagnosis and/or to be used in novel multi-subunit or recombinant vaccine formulations. bovine paratuberculosis, also known as johne's disease (jd) is an infectious disease of ruminants caused by mycobacterium avium subsp. paratuberculosis (map). it is characterized by chronic and progressive granulomatous enteritis. the infected animals initially show normal appetite and food consumption, but the intestinal wall thickening and the impaired nutrient absorption cause a reduced feed-conversion rate and a progressive weight loss. milk yield is also negatively affected by the progression of the infection. nevertheless, clinical manifestations do not involve all infected animals [1] [2] [3] ; the subclinical stageof infection can last from 2 to 15 years [4] and, despite the absence of visible symptoms, animals in this stage can shed map and spread the disease [3, [5] [6] [7] . for these aforementioned reasons, this pathology leads to significantly increased veterinary costs worldwide [3, 8, 9] . the causal agent of jd is map. it is considered a zoonotic pathogen [10] because of its possible link with crohn's disease. map infection affects animals and there is considerable evidence that might be a co-cause of human crohn's disease [11] . map isolation from the intestine and blood of crohn's disease patients has extensively documented. more precisely, map presence was found to be seven times higher in crohn's disease patients than what has been found in patients with any other bowel inflammation [12, 13] . map also infected animals and crohn's disease patients show similar alterations of the immune system response reinforcing the hypothesis about the analogy between the two [14] [15] [16] [17] . map is a slow-growing bacterium, commonly acquired via the fecal-oral route by both animals and humans [18] . despite the pathogenetic mechanism of map, infection has not been fully understood, it has been demonstrated that its acid resistance enables it to survive in the gastric environment, allowing its entrance in the intestinal tract. at the ileal level, map invades the lymphatic system overlying peyer's patches. this stimulates the host's immune response that, besides activating the humoral response, promptly phagocytizes map into macrophages [8] . as an intracellular pathogen, map can either survive into macrophagic cells or being killed and disassembled to present its antigens to t-lymphocytes [3] . evidence from multibacillary jd revealed a massive humoral antibody response along with a tendency to suppress the cell-mediated immune response [3, 19, 20] . whereas, a recent comparative study between two groups of cows, one in the sub-clinical and the other in the clinical stage, highlighted an increased t-cell activity in the first group of animals compared to the second one [21] . studies on cattle at the early stage of map infection revealed an upregulation of class i mhc molecules, suggesting a pivotal role of these molecules in the very beginning of the infective process [22] ; this is of great interest for both diagnosis and prophylaxis-oriented studies. figure 1 provides an overview of the major immunological mechanisms triggered bymap infections. to date, jd diagnosis relies on both direct (map culture, pcr, microarrays etc.) and indirect (elisa) detection of map from feces, milk and necroscopy-derived tissues. however, all the available diagnostic methods suffer from sensitivity (especially in the sub-clinical phase) that strongly reduce their robustness and efficient applicability on large-scale control programs. the failure to detect the subclinical infection makes it difficult to timely apply the control measures necessary to block the spread of the infection within the same, and to other, herds. a thorough comprehension of the etiopathogenetic mechanisms of map infection and host response would be beneficial for diverse research scenarios, providing guidance for the design of map-specific diagnostic tools capable of jd diagnosis in the subclinical phase. from this perspective, a previous study from our research group [18] employed an immunoproteomic approach to investigate and select map-specific immunoreactive proteins. here, incubation of map proteome with sera from infected bovines highlighted several possible candidate immunoreactive proteins. these candidates represent a good starting point for an immunoinformatic analysis of their sequences in order to find the best immunoreactive sub-sequences and epitopes. this would provide a library of peptides that might be useful for novel prophylactic strategies and/or for their potential application in the immune-based detection of map. the rapid development of the bioinformatics tools and databases provides the possibility to detect the antigenic/epitopic regions of given protein sequences. this innovative strategy for the in-silicoanalysis is time-and cost-effective compared to the "old-fashioned" laboratory-based approach. recently, carlos et al. [23] and rana et al. [24] applied immunoinformatics-based studies to detect class ii mhc epitopes possibly useful for the control of jd in a rapid and cost-effective manner. over the last decade, these computational approaches lead to the achievement of successful epitopes prediction in several research fields as virology [25, 26] , bacteriology and cancer research [27] . in this study, previously-selected immunogenic proteins [18] were studied via several immunoinformatics approaches aiming at the detection of the most promising peptide sequences useful for diagnostic purposes. the parameters taken into account were affinity for both the humoral antibody binding and the class i mhc molecule binding. we predicted the most suitable peptide sequences and discuss their potential employment in the design of innovative control measures against jd, with a specific focus on the early diagnosis of jd and/or potential use in novel specific vaccine formulations. pathogens 2020, 9, x for peer review 3 of 16 over the last decade, these computational approaches lead to the achievement of successful epitopes prediction in several research fields as virology [25, 26] , bacteriology and cancer research [27] . in this study, previously-selected immunogenic proteins [18] were studied via several immunoinformatics approaches aiming at the detection of the most promising peptide sequences useful for diagnostic purposes. the parameters taken into account were affinity for both the humoral antibody binding and the class i mhc molecule binding. we predicted the most suitable peptide sequences and discuss their potential employment in the design of innovative control measures against jd, with a specific focus on the early diagnosis of jd and/or potential use in novel specific vaccine formulations. the peptide sequences of the previously-identified immunoreactive proteins [18] were used to recall the novel protein identifiers in the ncbinr protein database. because of the continuous evolution of the data repositories and the increasing knowledge on their entries, some protein accession numbers were re-classified into other identifiers. table 1 summarizes the blast-based alignments of the peptides performed to line up the selected proteins to the current identification system. all pblast alignments matched at 100% with the reference protein kept. the low e-value of each alignment supports the attribution of the immunoreactive proteins to the novel identifiers. the peptide sequences of the previously-identified immunoreactive proteins [18] were used to recall the novel protein identifiers in the ncbinr protein database. because of the continuous evolution of the data repositories and the increasing knowledge on their entries, some protein accession numbers were re-classified into other identifiers. table 1 summarizes the blast-based alignments of the peptides performed to line up the selected proteins to the current identification system. all pblast alignments matched at 100% with the reference protein kept. the low e-value of each alignment supports the attribution of the immunoreactive proteins to the novel identifiers. once the updated protein identifiers are inferred, the major immunogenic domains of the selected proteins were predicted through an immunoinformatic approach. prediction of the linear b-epitopes provided a list of epitopes capable of eliciting antibody production (supplementary table s1 ). all the selected proteins showed relevant epitopes from an immunogenic point of view. a large number of short epitope sequences is predicted for each immunogenic protein; whereas, an average of six candidate epitopes (min 4-max 8) meeting the threshold of a minimal length of 10 aminoacids is predicted for each of the selected immunogenic proteins. figure 2 depicts the ten immunogenic proteins of map along with the relative distribution of the predicted b-epitopes. whole protein calculated immunogenic potential based on the type-b epitopes prediction indicates the "hypothetical protein map_1386c" (aas03703) as the most immunogenic one. this evidence is supported by its highest number of predicted epitopes and the highest average epitope length ( figure 2 ). on the other hand, the fructose-bisphosphate aldolase (eta93906), reported the lowest number of predicted epitopes along with the lowest epitope sequence length. regardless of the number of predictions, candidate epitopes are evenly mapped over the full sequence length of the immunogenic proteins, suggesting a good versatility of the predicted sequences ( figure 2 ). pathogens 2020, 9, x for peer review 5 of 16 prediction of binding affinity for the diverse class i bolas histocompatibility antigens predicted a high number of peptide sites. the full list of class i mhc epitope prediction is provided in the supplementary table s2 . epitope prediction from the previously selected immunogenic proteins yielded a total of 7044 peptides, each of which scoring a peculiar binding affinity. peptides scoring a binding affinity among the top 0.5% are considered as strong binders (sb); whereas, peptides with a percentile rank comprised between 0.6% and 2% were labelled as weak binders (wb). sorting all the entries using the "sole" wb and sb resulted in a total of 818 candidate epitopes when considering all the mhc haplotypes for the ten immunogenic proteins (supplementary table s2) . for a better evaluation of the most suitable map epitopes, we focused our attention towards the sequences that are most commonly recognized by the immune system effectors (i.e., bola haplotypes and, in turn, cd8 + t-cells). figure 3 lists, for each of the tested immunogenic protein, the shared epitopes among the mhc haplotypes. pathogens 2020, 9, x for peer review 6 of 16 prediction of binding affinity for the diverse class i bolas histocompatibility antigens predicted a high number of peptide sites. the full list of class i mhc epitope prediction is provided in the supplementary table s2 . epitope prediction from the previously selected immunogenic proteins yielded a total of 7044 peptides, each of which scoring a peculiar binding affinity. peptides scoring a binding affinity among the top 0.5% are considered as strong binders (sb); whereas, peptides with a percentile rank comprised between 0.6% and 2% were labelled as weak binders (wb). sorting all the entries using the "sole" wb and sb resulted in a total of 818 candidate epitopes when considering all the mhc haplotypes for the ten immunogenic proteins (supplementary table s2) . for a better evaluation of the most suitable map epitopes, we focused our attention towards the sequences that are most commonly recognized by the immune system effectors (i.e., bola haplotypes and, in turn, cd8 + t-cells). figure 3 lists, for each of the tested immunogenic protein, the shared epitopes among the mhc haplotypes. summarizes bolas haplotypes predicted to bind each sequence as means of a color scale that depends on the binding affinity. peptides "amlqdmail" belongs to heat shock protein 65 (caa52630.1); "aqldlsnal" and "hmlrhqqir" belong to hypothetical protein map_0323c (aas02640.1); "arleppgpl" belong to hypothetical protein map 1386c (aas03703.1); "gvfnleatl" and "neraveeal" belong to the protein fixa (aas05609.1); "rlleiepal", "aagqigysl" and "alegvvmel" belong to the malate dehydrogenase protein (p61976.1); "amqtlpqvl" and "rmaqtgspl" belong to phosphoglucosamine mutase (q73s29.1); "nqielhpll", "teravsaal", "amdaceasl" and "tqsytplal" belong to uncharacterized oxidoreductase map_3007 (q73vk6.1); "emfdlihqm" and "amrkwessm" belong to fructose-bisphosphate aldolase (eta93906.1); "glyefftpl", "gmigltqal" and "tqmtaaipl" belong to 3-ketoacyl-acp reductase (ajk73649.1); "fitwrgipl" and "nqsaiatyl" are peptides of the hypothetical protein ega31_12440 (azp81686.1). the vast majority of the listed epitopes are classified as sb; while eight of them, belonging to the proteins malate dehydrogenase (p61976), uncharacterized oxidoreductase map_3007 (q73vk6) and hypothetical protein ega31_12440 (azp81686), are to be considered as wb on the basis of their affinity rank. regardless of the binding affinity, all these sequences are predicted to be commonly bound by a plurality of mhc haplotypes. an average of 2 (min 1-max 4) suitable epitopes are selected for each of the tested protein. such epitopes are predicted to be recognized by five diverse bolas out of the six mhc haplotypes used for the computer-based prediction; except for the immunogenic proteins fixa (aas05609) whose epitopes can be bound by four bolas out of six. the bola-hd6, bola-jsp.1 and bola-t2c are able to recognize all the selected epitopes sequences . selected class i mhc binding peptides. the heat map displays the selected t-epitopes and summarizes bolas haplotypes predicted to bind each sequence as means of a color scale that depends on the binding affinity. peptides "amlqdmail" belongs to heat shock protein 65 (caa52630.1); "aqldlsnal" and "hmlrhqqir" belong to hypothetical protein map_0323c (aas02640.1); "arleppgpl" belong to hypothetical protein map 1386c (aas03703.1); "gvfnleatl" and "neraveeal" belong to the protein fixa (aas05609.1); "rlleiepal", "aagqigysl" and "alegvvmel" belong to the malate dehydrogenase protein (p61976.1); "amqtlpqvl" and "rmaqtgspl" belong to phosphoglucosamine mutase (q73s29.1); "nqielhpll", "teravsaal", "amdaceasl" and "tqsytplal" belong to uncharacterized oxidoreductase map_3007 (q73vk6.1); "emfdlihqm" and "amrkwessm" belong to fructose-bisphosphate aldolase (eta93906.1); "glyefftpl", "gmigltqal" and "tqmtaaipl" belong to 3-ketoacyl-acp reductase (ajk73649.1); "fitwrgipl" and "nqsaiatyl" are peptides of the hypothetical protein ega31_12440 (azp81686.1). the vast majority of the listed epitopes are classified as sb; while eight of them, belonging to the proteins malate dehydrogenase (p61976), uncharacterized oxidoreductase map_3007 (q73vk6) and hypothetical protein ega31_12440 (azp81686), are to be considered as wb on the basis of their affinity rank. regardless of the binding affinity, all these sequences are predicted to be commonly bound by a plurality of mhc haplotypes. an average of 2 (min 1-max 4) suitable epitopes are selected for each of the tested protein. such epitopes are predicted to be recognized by five diverse bolas out of the six mhc haplotypes used for the computer-based prediction; except for the immunogenic proteins fixa (aas05609) whose epitopes can be bound by four bolas out of six. the bola-hd6, bola-jsp.1 and bola-t2c are able to recognize all the selected epitopes sequences among the immunogenic proteins. on the other hand, the bola-t2a is not showing any binding affinity to the epitope sequences; while bola-d18.4 and bola-t2b fail to bind the epitopes of aas05609 protein (figure 3 ). the class i mhc epitopes as of figure 3 are further aligned against both the mycobacteria and cow databases to assess the specificity of the predicted epitope sequences for map. complete list of alignments is available in the supplementary table s3 . sequence alignment highlighted that the peptide amdaceasl and amrkwessm respectively of the "uncharacterized oxidoreductase map_3007" (q73vk6) and "fructose-bisphosphate aldolase" (eta93906) proteins are the most specific for map. specifically, amdaceasl scores 100% identity with the map and the mycobacterium aviumcomplex (mac); whereas, hits with other mycobacteria specimens are featured by a lower sequence identity (below 89%) and a far higher e-value when compared with map and mac hits (0.16 vs. 5.3, supplementary table s3) . similarly, the peptide amrkwessm scores 100% sequence identity with map and mac and only less than 73% of sequence similarity is scored by the alignments with other mycobacteria. the e-value supports the robust alignment against the map and mac (e-value 0.01) in spite of the other alignments (e-value > 86) further supporting the hypothesis on the specificity of this peptide sequence (supplementary table s3 ). concerning the alignment of the peptides against the cow database, both amdaceasl and amrkwessm did not score relevant matches with any of the cow proteins. several hits were matching with discontinuous sequences of the cow proteins database with high e-values (supplementary table s3 , topic better commented in the discussion section). the host's immune response to map infection is complex and heterogeneous. debates on the sequelae of immunological events following map infection are currently ongoing. nevertheless, it seems widely accepted that the early stage of the infection elicits an important cell-mediated response. once map is phagocytized, its antigen presentation is accomplished through the loading of the processed antigen onto mhc molecules. the bovine mhc genes complex (i.e., bovine leukocyte antigen, bola) is carried in the chromosome 23 and represent a fundamental component of the bovine immune system that allows the recognition and presentation of a virtually infinite number of antigens [28] (figure 1 ). such a high versatility relies on diverse factors, including the polygenetic origin of the mhc genes, the codominance of the parental alleles, the polymorphism of the genetic variants and the peptides/proteins splicing [29] . class i mhc molecules recognize, bind and present peptide antigens from intracellular pathogens to cd8 + t-lymphocytes [28] . in this view, class i mhc molecules and cytotoxic t-lymphocytes (ctl) are likely to play a pivotal role in the early stage of the map infection [30] . thus, of potential interest for early diagnosis-oriented studies and the design of efficient vaccine formulations. class i mhc peptide antigens are to be considered among the main triggers of the cell-mediated responseand their specific immunostimulation would lead to a more efficient prophylactic outcome. nevertheless, a study from rana etal. highlighted an important involvement also of the humoral response to map infection, other than the adaptive immunity mediated by the class ii mhc molecules [24] . huge efforts have been made to optimize diagnostics for the efficient detection of map by means of both direct and indirect methods [4, 31, 32] . the slow-growing rate of map along with the reduced sensitivity of the culture-based methods raised the need to develop alternative diagnostic strategies. pcr-based diagnosis targeting the multicopy insertion sequence is900 held the promise of fast detection of map in both environmental and clinical samples. however, the presence of is900-like sequences in other bacterial specimens resulted in a reduction of the pcr specificity. this, along with the elevated costs of the reagents, equipment and procedures, precludes the pcr applicability in large-scale programs [33] . among the indirect methods, elisa-based detection of anti-map antibodies enables faster diagnosis time but still suffer from drawbacks related to sensitivity and specificity. although great improvements have been made in optimizing elisa kits to reduce cross-reactions with environmental mycobacterium strains [18, 34] . still, this method suffers from a lack of sensitivity. moreover, the high antigen similarity between map and mycobacterium bovis obstacles the discrimination between bovines infected with tuberculosis and inoculated with live or attenuated paratuberculosis vaccines [35] . this promotes the seek of molecular target(s) capable of offering a more robust diagnosis. the present work describes a companion study that relies on previously-obtained datasets of our research group [18] . employing an immunoproteomic approach, we experimentally validated the whole map proteome for its capability of being complexed by the antibodies naturally occurring in sera of infected bovines. ms-based identification of the immunogenic proteins enabled the detection of 10 protein candidates whose protein sequences have been now further investigated for their immunogenic features. we employed an immunoinformatic approach for further focusing on the peptide sequences, potentially involved in the immunostimulation. a key point of the immunoinformatic approach is the prediction of the protein epitope sequences. epitopes prediction can be based on several features such as physical-chemical properties and structural folding of the primary protein sequence [36] [37] [38] . the present investigation is mainly focused on linear epitopes because protein-antibody complexes were selected through two-dimensional electrophoresis (2-de) and western blotting; thus, on linearized proteins [18, 39] . however, the application of other mass spectrometry technologies is quickly developing in the field on immunoproteomics [40] there are still significant limitation to map on a large scale conformational epitopes. as expected from the previous experimental data, all the screened protein sequences showed the capability of being recognized by both b-cells and class i bolas. the comprehension of recognition and binding of map by the host immune cells is still controversial. some studies document a relevant humoral response to map infections. on the other hand, other pieces of evidence describe the importance of the cell-mediated response to control map growth [41] [42] [43] [44] [45] . from our perspective and, according to the collected evidence, map-targeted antibodies could play a key role in the specific and sensitive detection of this pathogen in the subclinical stage of the infection. b-cell epitopes prediction highlighted the "hypothetical protein map_1386c" (aas03703) protein as the most active in stimulating antibody production. this finding is in agreement with our previous study [18] , where this protein showed a high level of immunoreactivity exclusively against the serum of the map infected animals. to the best of our knowledge, this protein was not described before as an antigen and, according to our dataset on its functional domains, it is possible to hypothesize that it is part of a surface-associated dehydrogenase with oxidoreductase activity involved in pentose phosphate pathway [18, 46] . the fructose-bisphosphate aldolase (eta93906), instead, is described as less prone to elicit antibody production. this is consistent with its intracytoplasmic localization and with its major role in the central metabolism. despite its cellular localization, several moonlighting properties have been described as part of its multiple functions [47] [48] [49] . interestingly, b-cell epitopes prediction highlighted a homogenous distribution of multiple peptide sequences throughout all the proteins primary sequences (figure 2 ). this suggests the potential usefulness of the selected proteins for a variety of implications where two or more epitopes are needed in a single protein molecule (e.g., sandwich elisa, and other indirect diagnostic tests ensuring higher sensibility) [50, 51] . prediction of the class i bolas binding peptides confirmed the immunogenicity of the previously studied proteins. similarly to hlas, bolas are highly polymorphic proteins; thus, including a plurality of bolas while computing the peptide binding affinity would benefit the robustness and reliability of the prediction [52, 53] . among the class i mhc epitopes predicted in the present study, the hypothetical protein ega31_12440 (azp81686) differs by only one amino-acidic residue from the map membrane protein 2121c (v7kre0), whose immunogenic properties have been already demonstrated by both our previous investigation and other studies [18, 31, 54] . it is, indeed, a surface-exposed protein involved in the mechanism of invasion of the epithelial cells [55, 56] . its expression is upregulated when the map is exposed to the physicochemical conditions similar to the intestine environment and the specific block of this protein reduces the virulence up to 60% [34] . interestingly, this protein is among the entries classified as wb suggesting that more immunogenic properties can also be exploited by the other wb protein besides the others predicted as being sbs. moreover, we specifically focused on the sole peptide sequences whose binding affinity is shared among multiple bolas. in this manner, the most suitable epitope sequences are likely to have a broad recognition in a higher portion of the bovine population [57, 58] . epitopes identified with this approach are of potential interest for diverse purposes and studies, including the investigations aimed at elucidating the order of immunological events following the map infection, and shedding light on the controversial aspect of suppression, or not, of the cellular-mediate immune response following map infection [3] . to prove selected epitopes as suitable candidates for the unbiased diagnosis of map infection, we aligned the peptides sequences against a database comprising the closest taxonomically-related bacteria. such alignment generated a steep reduction of the number of input sequences and returned two peptides suitable for a specific diagnosis of map. these two candidates were not overlapping with other mycobacteria other than mycobacterium avium complex (mac). the described approach resumes the pipeline of an in-silico method, therefore, empirical tests will be required for the definitive assessment of differential diagnosis capability of the selected sequences. the sequence alignment against the host-specific protein (i.e., the publicly available cow proteome) fails to identify significant sequence identities. the only alignment hits observed (supplementary table s3 ) were not continuously overlapping and showed a low percentage of identity and a high e-value. acknowledged the prediction of linear epitopes, the matching of our candidates with gapped sequences of cow is likely to be of a negligible relevance since regarding amino acid residues that are not laying in a concatenate order. thus, we speculate that the candidate epitopes suggested in this study are of potential value for the design of either multi-subunit or recombinant vaccines to confer protection against the first-time infection of the calves by map. nevertheless, confirmatory experimental trials are warmly encouraged especially to assess the specificity between map infected animals and bovine with tuberculosis [59, 60] . although less significant, a certain level of identity hasbeen registered with other mycobacterium strains. however, the discrepancy observed in the sequence alignment might be used as the driving force for the differential diagnosis. at this purpose, application of optimized laboratory protocols expecting high stringency condition might be the key to improve the specificity of the diagnostic methods. finally, empirical evaluation of the synergistic effect of both b-cell epitopes and the class i mhc epitopes are desirable. this will aim at the evaluation of the successful diagnosis of map infection at the subclinical stage and at the potential in elicitation of protective immunity. to conclude, the present study describes an innovative pipeline based on the in-silico survey of selected immunoreactive proteins capable to uncover the immunogenic features of each protein. this pipeline was applied to the detection of a restricted number of peptides potentially useful for the diagnosis of jd at the early subclinical stage. obtained results are as well useful for the implementation of innovative vaccination strategies. the obtained results confirmed the immunogenicity observed experimentally through the immunoproteomic approach applied to the map proteome. this evidence demonstrates, once again, reciprocal support between immunoproteomics and immunoinformatics. nevertheless, empirical confirmations are warmly required to test the provided epitope sequences both in-vitro and ex-vivofor the possible detection of the subclinical phase of the infection and for the efficacy of the eventual vaccinal formulations. such experimental tests might also help with the comprehension of the controversial role of the host immune cell-response underlying behind jd. complementation of the linear epitopes array with other conformational ones is also of importance for befitting efficacy and safety of the deliverables empirical confirmation may serve as further proof of the robustness of the immunoinformatics approaches as key contributors in the study of diverse infectious diseases. this would provide reliable scientific support in a safe, rapid and cost-effective approach. the current study focuses on ten proteins whose immunoreactivity has been experimentally investigated by means of an immunoproteomic approach [18] . brielfy, the map proteome was incubated with sera from infected animals to screen for proteins with immunoreactive potential. the most promising entries were then subjected to ms-based identification. identifiers of the candidate immunoreactive proteins were queried in the ncbi non-redundant (ncbinr) protein database to retrieve the whole protein sequences and export them as a fasta file. update of the protein accession numbers operated by the reference data repository (i.e., ncbi) required the run of a protein sequence alignment for the attribution of the novel protein identifiers (gi numbers). selected peptide sequences of the immunoreactive proteins were searched against the ncbinr database restricted to mycobacterium avium subsp. paratuberculosis (taxid 1770) and the best hit was used to transform the former protein accession numbers into the novel ncbi protein gis. the list of proteins employed in this study along with their current gi number is provided in table 1 and supplementary table s4 . prediction of the protein sequences that are likely to elicit antibody production and/or bind class i mhc proteins was performed through two tools that are commonly employed for the epitope prediction [27] , namely iedb (http://tools.iedb.org/bcell/) and netmhc (http://www.cbs.dtu.dk/ services/netmhc/), respectively for b-and class i mhc epitopes prediction. bepipred algorithm was chosen for the prediction of linear protein epitopes capable of binding b-cells. this employs a combination of a hidden markov model and a propensity scale method [61] . each protein residue is scored for its epitope behavior and the sole aminoacid with a score greater than or equal to 0.35 was considered as a potential epitope. linear peptide epitopes of the least length of 10 aminoacids were selected for this study. prediction of epitopes binding class i mhc molecules was performed through netmhc prediction tool, using the artificial neural network (ann) algorithm [62] . the algorithm was set for the prediction of nine-aminoacids long peptides capable of binding the following bola alleles: bola-d18.4; bola-hd6; bola-jsp.1; bola-t2a; bola-t2b; bola-t2c. the binding affinity of the peptides wasscored, and a percentile rank wasprovided by computationally comparing the score of each queried peptide sequence against 400,000 natural peptides of the same length. peptides scoring a binding affinity up to 0.5% were considered as strong binders (sb); whereas, peptides with a percentile rank comprised between 0.6% and 2% were labelled as weak binders (wb). all other peptides were discarded [3, 62, 63] . the resulting list of selected peptide epitopes was further quality-checked and filtered. for each of the selected proteins, the epitopes shared among the major number of bola haplotypes were kept (i.e., the most commonly recognized in the bovine population), resulting in a consensus list of epitopes to be further used in the study. a summary of the experimental worklow employed in this study is provided in figure 4 . . schematic representation of the immunoinformatic approach. blue and orange arrows refer to the input and output of the data, respectively. data arising from our previous immunoproteomic study [18] were used to retrieve the protein sequences of the immunogenic proteins, selected on the basis of their capability of being complexed by the immunoglobulins in the sera of the infected cows. the sequence of the immunogenic proteins is subjected to the epitope prediction through dedicated tools and algorithms. the b-epitope prediction was performed via the iedb prediction tool, that provides a list of candidate b-epitopes. the class i mhc epitopes waspredicted via netmhc. this makes use of the bola haplotypes from the data repository (ncbi) as references for computing the linear peptides capable of being recognized and presented by the diverse bolas. the list of potential t-epitopes is further refined by selecting the most commonly recognized epitopes with a relatively high binding affinity. the refined list of epitopes is further tested for sequence specificity and cross-reactivity by pblast alignment versus the mycobacteria and cow protein database which, in turn, arise from the publicly available data repository (ncbi). the list of epitope sequences was further analyzed through the basic local alignment search tool for protein sequences (pblast) [64] . this tool implements the pam30 algorithm to compare protein sequences and calculates the robustness of matches as means of expected values (e-value). . schematic representation of the immunoinformatic approach. blue and orange arrows refer to the input and output of the data, respectively. data arising from our previous immunoproteomic study [18] were used to retrieve the protein sequences of the immunogenic proteins, selected on the basis of their capability of being complexed by the immunoglobulins in the sera of the infected cows. the sequence of the immunogenic proteins is subjected to the epitope prediction through dedicated tools and algorithms. the b-epitope prediction was performed via the iedb prediction tool, that provides a list of candidate b-epitopes. the class i mhc epitopes waspredicted via netmhc. this makes use of the bola haplotypes from the data repository (ncbi) as references for computing the linear peptides capable of being recognized and presented by the diverse bolas. the list of potential t-epitopes is further refined by selecting the most commonly recognized epitopes with a relatively high binding affinity. the refined list of epitopes is further tested for sequence specificity and cross-reactivity by pblast alignment versus the mycobacteria and cow protein database which, in turn, arise from the publicly available data repository (ncbi). the list of epitope sequences was further analyzed through the basic local alignment search tool for protein sequences (pblast) [64] . this tool implements the pam30 algorithm to compare protein sequences and calculates the robustness of matches as means of expected values (e-value). this value describes the statistic of matches occurring "by chance"; thus, it decreases exponentially as the score of the match increases. in the pblast, each epitope sequence has been aligned against both mycobacteria (ncbi taxid 85007) and cow (ncbi taxid 9913) protein repertoires to evaluate sequence specificity and cross-reactivity (figure 4) , of importance while selecting candidate epitopes to be employed for the effective diagnosis and/or prophylaxis of map. supplementary materials: the following are available online at http://www.mdpi.com/2076-0817/9/9/705/s1. table s1 . b-cell binding protein epitope prediction. the file summarizes the b-epitope prediction results. each sheet of the xls file is relative to one of the ten selected proteins. "position" column is relative to the position of each aminoachid along the protein sequence; "residue" indicates the type of aminoacid; "score" is relative to the epitope propensity attributed by the algorithm and "assignment" rank each aminoacid residue as epitope or not depending on the prefixed settings. table s2 . class i mhc binding protein epitope prediction. the xls file summarizes the results in a table reporting: the predicted affinity (nm); the percentile rank, and the predicted binding core for all the selected alleles. two additional columns summarize the predictions across alleles: harmonic mean of the %rank calculated over all specified alleles (h_avg_ranks); the number of alleles covered by a given peptide (n_binders). table s3 . selected peptide epitopes alignment against the mycobacteria and cow databases. the xls file provides a summary of the pblast alignment search. full description of the table columns and the alignment criteria is available at https://blast.ncbi.nlm.nih.gov/blast.cgi?page=proteins. table s4 . full list of the prototypic peptides alignment. the file summarizes the p-blast alignment of the two selected epitopes against both the mycobacteria and the cow database. full description of the table columns and the alignment criteria is available at https://blast.ncbi.nlm.nih.gov/blast.cgi?page=proteins. the authors declare no conflict of interest. defining resilience to mycobacterial disease: characteristics of survivors of ovine paratuberculosis experimental infection model for johne's disease using a lyophilised, pure culture, seedstock of mycobacterium avium subspecies paratuberculosis gene expression profiles during subclinical mycobacterium avium subspecies paratuberculosis infection in sheep can predict disease outcome control of paratuberculosis: who, why and how. a review of 48 countries paratuberculosis in sheep and goats johne's disease) in cattle and other susceptible species temporal patterns and 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epitope mapping approaches new latex bead agglutination assay for differential diagnosis of cattle infected with mycobacterium bovis and mycobacterium avium subsp. paratuberculosis duplex pcr for differential identification of mycobacterium bovis, m. avium, and m. avium subsp. paratuberculosis in formalin-fixed paraffin-embedded tissues from cattle improved method for predicting linear b-cell epitopes gapped sequence alignment using artificial neural networks: application to the mhc class i system in silico identification of epitopes in mycobacterium avium subsp. paratuberculosis proteins that were upregulated under stress conditions protein database searches using compositionally adjusted substitution matrices key: cord-001726-d7iwkatn authors: henry, kevin a.; arbabi-ghahroudi, mehdi; scott, jamie k. title: beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold date: 2015-08-04 journal: front microbiol doi: 10.3389/fmicb.2015.00755 sha: doc_id: 1726 cord_uid: d7iwkatn for the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. however, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. this review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. the filamentous bacteriophage (genera inovirus and plectrovirus) are non-enveloped, rod-shaped viruses of escherichia coli whose long helical capsids encapsulate a single-stranded circular dna genome. subsequent to the independent discovery of bacteriophage by twort (1915) and d 'hérelle (1917) , the first filamentous phage, f1, was isolated in loeb (1960) and later characterized as a member of a larger group of phage (ff, including f1, m13, and fd phage) specific for the e. coli conjugative f pilus (hofschneider and mueller-jensen, 1963; marvin and hoffmann-berling, 1963; zinder et al., 1963; salivar et al., 1964) . soon thereafter, filamentous phage were discovered that do not use f-pili for entry (if and ike; meynell and lawn, 1968; khatoon et al., 1972) , and over time the list of known filamentous phage has expanded to over 60 members (fauquet et al., 2005) , including temperate and gram-positivetropic species. work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle (reviewed in marvin, 1998; rakonjac et al., 2011; rakonjac, 2012) . in the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by smith and colleagues (smith, 1985; parmley and smith, 1988) . based on the ideas described in parmley and smith (1988) , groups in california, germany, and the uk developed phage-display platforms to create and screen libraries of peptide and folded-protein variants (bass et al., 1990; devlin et al., 1990; mccafferty et al., 1990; scott and smith, 1990; breitling et al., 1991; kang et al., 1991) . this technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. many excellent reviews are available on phage-display libraries and their applications (kehoe and kay, 2005; bratkovic, 2010; pande et al., 2010) . however, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. these tend to fall into four major categories of use: (i) filamentous phage as a vaccine carrier; (ii) engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; (iii) filamentous phage as a scaffold for bioconjugation and surface chemistry; and (iv) filamentous phage as an engine for evolving variants of displayed proteins with novel functions. a final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins ( table 1) . the display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. display may be achieved by fusing dna encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type 8 display on pviii, type 3 display on piii, etc.), resulting in fully recombinant phage. much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy (e.g., type 88 display if both recombinant and wild-type pviii genes are on the phage genome, type 8+8 display if the parmley and smith (1988), mcconnell et al. (1994) , rondot et al. (2001) hybrid (type 33 and 3+3 systems) type 3+3 system <1 2 smith and scott (1993) , smith and petrenko (1997) pvi hybrid (type 6+6 system) yes <1 2 >25 kda hufton et al. (1999) pvii fully recombinant (type 7 system) no ∼5 >25 kda kwasnikowski et al. (2005) hybrid (type 7+7 system) yes <1 2 gao et al. (1999) pviii fully recombinant (landscape phage; type 8 system) no 2700 3 ∼5-8 residues kishchenko et al. (1994) , petrenko et al. (1996) hybrid (type 88 and 8+8 systems) type 8+8 system ∼1-300 2 >50 kda scott and smith (1990) , greenwood et al. (1991) , smith and fernandez (2004) pix fully recombinant (type 9+9 * system) yes ∼5 >25 kda gao et al. (2002) hybrid (type 9+9 system) no <1 2 gao et al. (1999) , shi et al. (2010) , tornetta et al. (2010) 1 asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 the copy number depends on polypeptide size; typically <1 copy per phage particle but for pviii peptide display can be up to ∼15% of pviii molecules in hybrid virions. 3 the total number of pviii molecules depends on the phage genome size; one pviii molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication) resulting in a hybrid virion bearing two different types of a given coat protein. multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene (e.g., type 3 * +3 display). by far the most commonly used coat proteins for display are the major coat protein, pviii, and the minor coat protein, piii, with the major advantage of the former being higher copy number display (up to ∼15% of recombinant pviii molecules in a hybrid virion, at least for short peptide fusions), and of the latter being the ability to display some folded proteins at an appreciable copy number (1-5 per phage particle). while pviii display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion (sidhu et al., 2000) . for the purposes of this review, we use the term "phage display" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface (or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins), and the term "phage-displayed library" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants (e.g., antibody fragments; peptides). such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest (e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries) followed by amplification of the bound phage in e. coli cells. early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants (meynell and lawn, 1968 ) and that only the major coat protein, pviii, and the minor coat protein, piii, are targeted by antibodies (pratt et al., 1969; woolford et al., 1977) . thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la cruz et al. (1988) . the phage particle's low cost of production, high stability and potential for high valency display of foreign antigen (via pviii display) also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pviii or piii (de la cruz et al., 1988) . as library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies (selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; geysen et al., 1986; knittelfelder et al., 2009) were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. some of the pioneering work in this area used peptides derived from infectious disease antigens (or peptide ligands of antibodies against these antigens; table 2) , including malaria and human immunodeficiency virus type 1 (hiv-1). when displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits (de la cruz et al., 1988; greenwood et al., 1991; willis et al., 1993; demangel et al., 1996) , and antibodies raised against the latter cross-reacted with the full-length protein. various peptide determinants (or mimics thereof) of hiv-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins (minenkova et al., 1993; di marzo veronese et al., 1994; de berardinis et al., 1999; scala et al., 1999; chen et al., 2001; van houten et al., 2006 van houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses (di marzo veronese et al., 1994; scala et al., 1999) . the list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens ( table 2) . while in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: (i) in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins (irving et al., 2010 ; see below); (ii) antibodies against a single epitope may be of limited utility, especially for highly variable pathogens (van regenmortel, 2012); and (iii) for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. more recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. immunization with phage displaying alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs (frenkel et al., 2000 (frenkel et al., , 2003 esposito et al., 2008; tanaka et al., 2011) , possibly reduced amyloid plaque formation in mice (frenkel et al., 2003; solomon, 2005; esposito et al., 2008) , and may have helped maintain cognitive abilities in a transgenic mouse model of alzheimer's disease (lavie et al., 2004) ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. yip et al. (2001) found that antibodies raised in mice against an erbb2/her2 peptide could inhibit breast-cancer cell proliferation. phage displaying peptide ligands of an anti-ige antibody elicited antibodies that bound purified ige molecules (rudolf et al., 1998) , which may be useful in allergy immunotherapy. several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. for example, immunization with phage displaying follicle-stimulating hormone peptides on pviii elicited antibodies that impaired the fertility of mice and ewes (abdennebi et al., 1999) . phage displaying or chemically rubinchik and chow (2000) conjugated to sperm antigen peptides or peptide mimics (samoylova et al., 2012a,b) and gonadotropin-releasing hormone (samoylov et al., 2012) are also in development. for the most part, peptides displayed on phage elicit antibodies in experimental animals ( table 2) , although this depends on characteristics of the peptide and the method of its display: piii fusions tend toward lower immunogenicity than pviii fusions (greenwood et al., 1991) possibly due to copy number differences (piii: 1-5 copies vs. pviii: estimated at several hundred copies; malik et al., 1996) . in fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin (bsa) and keyhole limpet hemocyanin (klh; melzer et al., 2003; su et al., 2007) , and has comparatively few endogenous b-cell epitopes to divert the antibody response from its intended target (henry et al., 2011) . excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. the overall picture is considerably bleaker than that painted by table 2 , since in several studies either: (i) peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or (ii) the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. irving et al. (2010) describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. while the peptide may mimic the chemistry of a given epitope on a folded protein (allowing it to crossreact with a targeted antibody), being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins (henry et al., 2011) . this may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity (see immunological mechanisms of vaccination with filamentous phage below). the filamentous phage has been used to a lesser extent as a carrier for t-cell peptide epitopes, primarily as fusion proteins with pviii ( table 3) . early work, showing that immunization with phage elicited t-cell help (kölsch et al., 1971; willis et al., 1993) , was confirmed by several subsequent studies (de berardinis et al., 1999; ulivieri et al., 2008) . from the perspective of vaccination against infectious disease, de berardinis et al. (2000) showed that a cytotoxic t-cell (ctl) epitope from hiv-1 reverse transcriptase could elicit antigen-specific ctls in vitro and in vivo without addition of exogenous helper t-cell epitopes, presumably since these are already present in the phage coat proteins (mascolo et al., 2007) . similarly, efficient priming of ctls was observed against phage-displayed t-cell epitopes from hepatitis b virus (wan et al., 2001) and candida albicans (yang et al., 2005a; wang et al., 2006 wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. vaccination with a combination of phagedisplayed peptides elicited antigen-specific ctls that proved effective in reducing porcine cysticercosis in a randomized controlled trial (manoutcharian et al., 2004; morales et al., 2008) . while the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies (plotkin, 2010) , in certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production (gram et al., 1993) . e. coli f17a-g adhesin (van gerven et al., 2008) , hepatitis b core antigen (bahadir et al., 2011) , and hepatitis b surface antigen (balcioglu et al., 2014) all elicited antibody responses when displayed on piii, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. phage displaying schistosoma mansoni glutathione s-transferase on piii elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone (rao et al., 2003) . two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. immunization with phage displaying the 1e10 idiotype scfv (mimicking a vibrio anguillarum surface epitope) elicited antibodies that protected flounder fish from vibrio anguillarum challenge (xia et al., 2005) . a chemically linked phage-bcl1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a b-cell lymphoma model (roehnisch et al., 2013) , and was welltolerated and immunogenic in patients with multiple myeloma (roehnisch et al., 2014) . one study of dna vaccination with an anti-laminarin scfv found that dna encoding a piii-scfv fusion protein elicited stronger humoral and cell-mediated immune responses than dna encoding the scfv alone (cuesta et al., 2006) , suggesting that under some circumstances, endogenous phage t-cell epitopes can enhance the immunogenicity of associated proteins. taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional t-cell help to displayed or conjugated proteins. however, the low copy number of piii-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination (figure 1) . the phage particle is immunogenic without adjuvant in all species tested to date, including mice (willis et al., 1993) , rats (dente et al., 1994) , rabbits (de la cruz et al., 1988) , guinea pigs (frenkel et al., 2000; kim et al., 2004) , fish (coull et al., 1996; xia et al., 2005) , non-human primates (chen et al., 2001) , and humans (roehnisch et al., 2014) . various routes of immunization have been employed, including oral administration (delmastro et al., 1997) as well as subcutaneous (grabowska et al., 2000) , intraperitoneal (van houten et al., 2006) , intramuscular (samoylova et al., 2012a) , intravenous (vaks and benhar, 2011) , and intradermal injection (roehnisch et al., 2013) ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. antibodies are generated against only three major sites on the virion: (i) the surface-exposed n-terminal ∼12 residues of the pviii monomer lattice (terry et al., 1997; kneissel et al., 1999) ; (ii) the n-terminal n1 and n2 domains of piii (van houten et al., 2010) ; and (iii) bacterial lipopolysaccharide (lps) embedded in the phage coat (henry et al., 2011) . in mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization (frenkel et al., 2000) . primary antibody responses against the phage appear to be composed of a mixture of igm and igg2b isotypes in c57bl/6 mice, while secondary antibody responses are composed primarily of igg1 and igg2b isotypes, with a lesser contribution of igg2c and igg3 isotypes (hashiguchi et al., 2010) . deletion of the surface-exposed n1 and n2 domains of piii produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity (van houten et al., 2010) . figure 1 | types of immune responses elicited in response to immunization with filamentous bacteriophage. as a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity (macrophages, neutrophils, and possibly natural killer cells), which are recruited to tumor sites by phage displaying tumor-targeting moieties. the phage likely activates t-cell independent antibody responses, either via phage-associated tlr ligands or cross-linking by the pviii lattice. after processing by antigen-presenting cells, phage-derived peptides are presented on mhc class ii and cross-presented on mhc class i, resulting in activation of short-lived ctls and an array of helper t-cell types, which help prime memory ctl and high-affinity b-cell responses. frontiers in microbiology | www.frontiersin.org although serum anti-phage antibody titers appear to be at least partially t-cell dependent (kölsch et al., 1971; willis et al., 1993; de berardinis et al., 1999; van houten et al., 2010) , many circulating pviii-specific b cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates t-cell-independent b-cell responses in addition to highaffinity t-cell-dependent responses (murira, 2014) . filamentous phage particles can be processed by antigen-presenting cells and presented on mhc class ii molecules (gaubin et al., 2003; ulivieri et al., 2008) and can activate t h 1, t h 2, and t h 17 helper t cells (yang et al., 2005a; wang et al., 2014d) . anti-phage t h 2 responses were enhanced through display of ctla-4 peptides fused to piii (kajihara et al., 2000) . phage proteins can also be cross-presented on mhc class i molecules (wan et al., 2005) and can prime two waves of ctl responses, consisting first of short-lived ctls and later of long-lived memory ctls that require cd4 + t-cell help (del pozzo et al., 2010) . the latter ctls mediate a delayed-type hypersensitivity reaction (fang et al., 2005; del pozzo et al., 2010) . the phage particle is self-adjuvanting through multiple mechanisms. host cell wall-derived lps enhances the virion's immunogenicity, and its removal by polymyxin b chromatography reduces antibody titers against phage coat proteins (grabowska et al., 2000) . the phage's singlestranded dna genome contains cpg motifs and may also have an adjuvant effect. the antibody response against the phage is entirely dependent on myd88 signaling and is modulated by stimulation of several toll-like receptors (hashiguchi et al., 2010) , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system (molenaar et al., 2002) , particularly of the liver and spleen, where it is retained for days (zou et al., 2004) , potentially activating marginal-zone b-cell responses. thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former soviet union and eastern europe (reviewed in sulakvelidze et al., 2001) . the filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of inovirus and plectrovirus includes many pathogens of medical importance, including salmonella, e. coli, shigella, pseudomonas, clostridium, and mycoplasma species. in an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a "trojan horse" to introduce various antibacterial agents into cells. m13 and pf3 phage engineered to express either bglii restriction endonuclease (hagens and blasi, 2003; hagens et al., 2004) , lambda phage s holin (hagens and blasi, 2003) or a lethal catabolite gene activator protein (moradpour et al., 2009) effectively killed e. coli and pseudomonas aeruginosa cells, respectively, with no concomitant release of lps (hagens and blasi, 2003; hagens et al., 2004) . unfortunately, the rapid emergence of resistant bacteria with modified f pili represents a major and possibly insurmountable obstacle to this approach. however, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage (hagens et al., 2006) or phage engineered to repress the cellular sos response (lu and collins, 2009) . filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in e. coli (may et al., 2011) . thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. more advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity (figure 2) . the first work in this area showed as proof-of-concept that phage encoding a gfp expression cassette and displaying a her2specific scfv on all copies of piii were internalized into breast tumor cells, resulting in gfp expression (poul and marks, 1999) . m13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of staphylococcus aureus growth than high-concentration free chloramphenicol (yacoby et al., 2006; vaks and benhar, 2011) . m13 phage loaded with doxorubicin and displaying a targeting peptide on piii specifically killed prostate cancer cells in vitro (ghosh et al., 2012a) . tumorspecific peptide:pviii fusion proteins selected from "landscape" phage (romanov et al., 2001; abbineni et al., 2010; fagbohun et al., 2012 fagbohun et al., , 2013 lang et al., 2014; wang et al., 2014a) were able to target and deliver sirna-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells (jayanna et al., 2010a; wang et al., 2010a wang et al., ,b,c, 2014b bedi et al., 2011 bedi et al., , 2013 bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models (jayanna et al., 2010b; wang et al., 2014b,c) . using the b16-ova tumor model, eriksson et al. (2007) showed that phage displaying peptides and/or fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site (eriksson et al., 2009) . phage displaying an scfv against β-amyloid fibrils showed promise as a diagnostic (frenkel and solomon, 2002) and therapeutic (solomon, 2008) reagent for alzheimer's disease and parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue (ksendzovsky et al., 2012) . similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis (rakover et al., 2010) . the advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are (i) its ability to carry very high amounts of drug or peptide, and (ii) its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. first and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating lps, in the range of ∼10 2 -10 4 endotoxin units (eu)/ml (boratynski et al., 2004; branston et al., 2015) , which have the potential to cause severe adverse reactions. lps is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation (smith and gingrich, 2005; branston et al., 2015) , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography (boratynski et al., 2004; zakharova et al., 2005) , polymyxin b chromatography (grabowska et al., 2000) , and treatment with detergents such as triton x-100 or triton x-114 (roehnisch et al., 2014; branston et al., 2015) . these strategies routinely achieve endotoxin levels of <1 eu/ml as measured by the limulus amebocyte lysate (lal) assay, well below the fda limit for parenteral administration of 5 eu/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated lps which may be undetectable. a second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline (reviewed in dalmasso et al., 2014) or for detection of foodborne pathogens post-production (reviewed in schmelcher and loessner, 2014) . filamentous phage displaying a tetracysteine tag on piii were used to detect e. coli cells through staining with biarsenical dye . m13 phage functionalized with metallic silver were highly bactericidal against e. coli and staphylococcus epidermidis . biosensors based on surface plasmon resonance (nanduri et al., 2007) , piezoelectric transducers (olsen et al., 2006) , linear dichroism (pacheco-gomez et al., 2012) , and magnetoelastic sensor technology (lakshmanan et al., 2007; huang et al., 2009) were devised using filamentous phage displaying scfv or conjugated to whole igg against e. coli, listeria monocytogenes, salmonella typhimurium, and bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/ml. proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce (li et al., 2010b) and eggs (chai et al., 2012) . the filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pviii monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups (henry et al., 2011) . the regularity of the phage pviii lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation (figure 3) . the most commonly used approach is functionalization of amine groups with nhs esters (van houten et al., 2006 (van houten et al., , 2010 yacoby et al., 2006) , although this can result in unwanted acylation of piii and any displayed biomolecules. carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively (li et al., 2010a) . carrico et al. (2012) developed methods to specifically label pviii n-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides (ng et al., 2012) or enzymes (chen et al., 2007; hess et al., 2012) , but this can be cumbersome and is less general in application. for more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. it has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions (welsh et al., 1996) . lee et al. (2002) engineered m13 phage to display a zns-binding peptide on piii and showed that, in the presence of zns nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament hess et al., 2012) , this pioneering figure 3 | chemically addressable groups of the filamentous bacteriophage major coat protein lattice. the filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pviii, arranged in a shingle-type lattice. each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups (shown in red), three carboxyl groups (show in blue) and two hydroxyl groups (show in green). the 12 n-terminal residues generally exposed to the immune system for antibody binding are in bold underline. figure adapted from structural data of marvin, 1990 , freely available in pdb and scope databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires (mao et al., 2003 (mao et al., , 2004 , nanoparticles , and nanocomposites (oh et al., 2012; chen et al., 2014) . using hybrid m13 phage displaying co 3 o 4 -and gold-binding peptides on pviii as a scaffold to assemble nanowires on polyelectrolyte multilayers, nam et al. (2006) produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors (nam et al., 2008) . the electrochemical properties of such batteries were further improved through piii-display of single-walled carbon nanotube-binding peptides (lee et al., 2009) , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. phagebased nanomaterials have found applications in cancer imaging (ghosh et al., 2012b; yi et al., 2012) , photocatalytic water splitting (nam et al., 2010a; neltner et al., 2010) , light harvesting (nam et al., 2010b; chen et al., 2013) , photoresponsive technologies (murugesan et al., 2013) , neural electrodes (kim et al., 2014) , and piezoelectric energy generation (murugesan et al., 2013) . thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. it is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. magnetic alignment of high-concentration filamentous phage in solution can partially order dna, rna, proteins, and other biomolecules for measurement of dipolar coupling interactions (hansen et al., 1998 (hansen et al., , 2000 dahlke ojennus et al., 1999) in nmr spectroscopy. because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution (reviewed in husimi, 1989; wichman and brown, 2010; kawecki et al., 2012; hall et al., 2013) . the filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. first and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on piii. libraries of variant fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system (marks et al., 1992; bradbury et al., 2011) . however, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation (although each display technology has complementary strengths; koide and koide, 2012) , and regardless of the display method, selection of "improved" variants can be slow and cumbersome. iterative methods have been developed to combine computationally designed mutations (lippow et al., 2007) and circumvent the screening of combinatorial libraries, but these have had limited success to date. recently, esvelt et al. (2011) developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution (pace), which allows multiple rounds of evolution per day with little experimental intervention. the authors engineered m13 phage to encode an exogenous protein (the subject for directed evolution), whose functional activity triggers gene iii expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone dna polymerases. by supplying limiting amounts of receptive e. coli cells to the engineered phage variants, esvelt et al. (2011) elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. carlson et al. (2014) later showed that pace selection stringency could be modulated by providing small amounts of piii independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated piii variant that impairs infectivity in a dominant negative fashion. pace is currently limited to protein functions that can be linked in some way to the expression of a gene iii reporter, such as protein-protein interaction, recombination, dna or rna binding, and enzymatic catalysis (meyer and ellington, 2011) . this approach represents a promising avenue for both basic research in molecular evolution (dickinson et al., 2013) and synthetic biology, including antibody engineering. filamentous bacteriophage have been recovered from diverse environmental sources, including soil (murugaiyan et al., 2011) , coastal fresh water (xue et al., 2012) , alpine lakes (hofer and sommaruga, 2001) and deep sea bacteria (jian et al., 2012) , but not, perhaps surprisingly, the human gut (kim et al., 2011) . the environmental "phageome" in soil and water represent the largest source of replicating dna on the planet, and is estimated to contain upward of 10 30 viral particles (ashelford et al., 2003; chibani-chennoufi et al., 2004; suttle, 2005) . the few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques (typically direct observation by electron microscopy) found that filamentous phage made up anywhere from 0 to 100% of all viral particles (demuth et al., 1993; pina et al., 1998; hofer and sommaruga, 2001) . there was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria (hofer and sommaruga, 2001) . environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. the existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater (roux et al., 2012) and reclaimed and potable water (rosario et al., 2009) but have much higher frequencies in wastewater and sewage (cantalupo et al., 2011; alhamlan et al., 2013) , with the caveat that biases inherent to the methodologies for ascertaining these data (purification of viral particles, sequencing biases) have not been not well validated. there are no data describing the population dynamics of filamentous phage and their host species in the natural environment. at the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. this can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. temperate filamentous phage may also play a role in genome evolution (reviewed in canchaya et al., 2003) . perhaps the best-studied example of virulence modulation by filamentous phage is that of vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding ctxφ phage (waldor and mekalanos, 1996) . integration of ctxφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, tlc-knφ1 (hassan et al., 2010) . thus, filamentous phage species interact and coevolve with each other in addition to their hosts. infection by filamentous phage has been implicated in the virulence of yersinia pestis (derbise et al., 2007) , neisseria meningitidis (bille et al., 2005 (bille et al., , 2008 , vibrio parahaemolyticus (iida et al., 2001) , e. coli 018:k1:h7 (gonzalez et al., 2002) , xanthomonas campestris (kamiunten and wakimoto, 1982) , and p. aeruginosa (webb et al., 2004) , although in most of these cases, the specific mechanisms modulating virulence are unclear. phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen ralstonia solanacearum (yamada, 2013) . since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes (lin et al., 2011) . finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants (petrenko and makowski, 1993) , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings (curtis et al., 2011 (curtis et al., , 2013 . engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies (curtis et al., 2009 ). the filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. while novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. kh and js conceived and wrote the manuscript. ma-g read the manuscript and commented on the text. evolutionary selection of new breast cancer cell-targeting peptides and phages with the cell-targeting peptides fully displayed on the major coat and their effects on actin dynamics during cell internalization generating fsh antagonists and agonists through immunization against fsh receptor n-terminal decapeptides metagenomics-based analysis of viral communities in dairy lagoon wastewater elevated abundance of bacteriophage infecting bacteria in soil phage displayed hbv core antigen with immunogenic activity cost effective filamentous phage based immunization nanoparticles displaying a full-length hepatitis b virus surface antigen hormone phage: an enrichment method for variant proteins with altered binding properties protective immune responses induced by the immunization of mice with a recombinant bacteriophage displaying an epitope of the human respiratory syncytial virus selection of pancreatic cancer cell-binding landscape phages and their use in development of anticancer nanomedicines targeted delivery of sirna into breast cancer cells via phage fusion proteins delivery of sirna into breast cancer cells via phage fusion protein-targeted liposomes association of a bacteriophage with meningococcal disease in young adults a chromosomally integrated bacteriophage in invasive meningococci preparation of endotoxin-free bacteriophages beyond natural antibodies: the power of in vitro display technologies a nonchromatographic method for the removal of endotoxins from bacteriophages progress in phage display: evolution of the technique and its application a surface expression vector for antibody screening raw sewage harbors diverse viral populations negative selection and stringency modulation in phage-assisted continuous evolution n-terminal labeling of filamentous phage to create cancer marker imaging agents rapid and sensitive detection of salmonella typhimurium on eggshells by using wireless biosensors phage display evolution of a peptide substrate for yeast biotin ligase and application to two-color quantum dot labeling of cell surface proteins versatile three-dimensional virus-based template for dye-sensitized solar cells with improved electron transport and light harvesting assembly of viral hydrogels for three-dimensional conducting nanocomposites protection of rhesus macaques against disease progression from pathogenic shiv-89.6pd by vaccination with phage-displayed hiv-1 epitopes phage-host interaction: an ecological perspective evaluation of filamentous bacteriophage as immunogens in atlantic salmon enhancement of dna vaccine potency through linkage of antigen to filamentous bacteriophage coat protein iii domain i bacteriophageinduced aggregation of oil sands tailings biomining with bacteriophage: selectivity of displayed peptides for naturally occurring sphalerite and chalcopyrite effects of bacteriophage on the surface properties of chalcopyrite (cufes(2)), and phage-induced flocculation of chalcopyrite, glacial till, and oil sands tailings induced alignment and measurement of dipolar couplings of an sh2 domain through direct binding with filamentous phage exploiting gut bacteriophages for human health recognition of hiv-derived b and t cell epitopes displayed on filamentous phages phage display of peptide epitopes from hiv-1 elicits strong cytolytic responses immunogenicity and epitope mapping of foreign sequences via genetically engineered filamentous phage triggering dth and ctl activity by fd filamentous bacteriophages: role of cd4+ t cells in memory responses immunogenicity of filamentous phage displaying peptide mimotopes after oral administration reproducing the immune response against the plasmodium vivax merozoite surface protein 1 with mimotopes selected from a phage-displayed peptide library direct electron microscopy study on the morphological diversity of bacteriophage populations in lake pluβsee monoclonal antibodies that recognise filamentous phage: tools for phage display technology a horizontally acquired filamentous phage contributes to the pathogenicity of the plague bacillus random peptide libraries: a source of specific protein binding molecules experimental interrogation of the path dependence and stochasticity of protein evolution using phage-assisted continuous evolution structural mimicry and enhanced immunogenicity of peptide epitopes displayed on filamentous bacteriophage. the v3 loop of hiv-1 gp120 sur un microbe invisible antagoniste des bacilles dysentérique tumor specific phage particles promote tumor regression in a mouse melanoma model tumor-specific bacteriophages induce tumor destruction through activation of tumor-associated macrophages immunogenicity and therapeutic efficacy of phage-displayed beta-amyloid epitopes a system for the continuous directed evolution of biomolecules landscape phages and their fusion proteins targeted to breast cancer cells metastatic prostate cancer cell-specific phage-like particles as a targeted genedelivery system the potential of phage display virions expressing malignant tumor specific antigen mage-a1 epitope in murine model virus taxonomy. 8th report of the international committee on the taxonomy of viruses mimicking of discontinuous epitopes by phage-displayed peptides, ii. selection of clones recognized by a protective monoclonal antibody against the bordetella pertussis toxin from phage peptide libraries a general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera reduction of β-amyloid plaques in brain of transgenic mouse model of alzheimer's disease by efrh-phage immunization immunization against alzheimer's β-amyloid plaques via efrh phage administration filamentous phage as vector-mediated antibody delivery to the brain a method for the generation of combinatorial antibody libraries using pix phage display making artificial antibodies: a format for phage display of combinatorial heterodimeric arrays processing of filamentous bacteriophage virions in antigenpresenting cells targets both hla class i and class ii peptide loading compartments a priori delineation of a peptide which mimics a discontinuous antigenic determinant refactored m13 bacteriophage as a platform for tumor cell imaging and drug delivery m13-templated magnetic nanoparticles for targeted in vivo imaging of prostate cancer conserved filamentous prophage in escherichia coli o18:k1:h7 and yersinia pestis biovar orientalis immunisation with phage displaying peptides representing single epitopes of the glycoprotein g can give rise to partial protective immunity to hsv-2 phage display as a rapid gene expression system: production of bioactive cytokinephage and generation of neutralizing monoclonal antibodies multiple display of foreign peptides on a filamentous bacteriophage. peptides from plasmodium falciparum circumsporozoite protein as antigens genetically modified filamentous phage as bactericidal agents: a pilot study augmentation of the antimicrobial efficacy of antibiotics by filamentous phage therapy of experimental pseudomonas infections with a nonreplicating genetically modified phage viral host-adaptation: insights from evolution experiments with phages pf1 filamentous phage as an alignment tool for generating local and global structural information in nucleic acids tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions immunological basis of m13 phage vaccine: regulation under myd88 and tlr9 signaling satellite phage tlcφ enables toxigenic conversion by ctx phage through dif site alteration developing strategies to enhance and focus humoral immune responses using filamentous phage as a model antigen m13 bacteriophage display framework that allows sortasemediated modification of surface-accessible phage proteins seasonal dynamics of viruses in an alpine lake: importance of filamentous forms on infectious substructures from e. coli bacteriophages. 3. demonstration and properties of "ht2" particles peptide mimotopes of rabies virus glycoprotein with immunogenic activity sequential detection of salmonella typhimurium and bacillus anthracis spores using magnetoelastic biosensors programmable assembly of nanoarchitectures using genetically engineered viruses phage display of cdna repertoires: the pvi display system and its applications for the selection of immunogenic ligands selection and evolution of bacteriophages in cellstat filamentous phage associated with recent pandemic strains of vibrio parahaemolyticus exploring peptide mimics for the production of antibodies against discontinuous protein epitopes landscape phage ligands for pc3 prostate carcinoma cells landscape phage fusion protein-mediated targeting of nanomedicines enhances their prostate tumor cell association and cytotoxic efficiency dynamic modulation of dna replication and gene transcription in deep-sea filamentous phage sw1 in response to changes of host growth and temperature th2-type immune response induced by a phage clone displaying a ctla4-binding domain mimicmotif effect of infection with filamentous phage xf on the growth, ultrastructure and virulence of xanthomonas campestris pv. oryzae n 5850 linkage of recognition and replication functions by assembling combinatorial antibody fab libraries along phage surfaces experimental evolution filamentous phage display in the new millennium a new filamentous bacteriophage with sex-factor specificity diversity and abundance of single-stranded dna viruses in human feces genetically engineered bacteriophage delivers a tumor necrosis factor alpha antagonist coating on neural electrodes expression of a foot-and-mouth disease virus immunodominant epitope by a filamentous bacteriophage vector structure of a foreign peptide displayed on the surface of bacteriophage m13 epitope structures recognised by antibodies against the major coat protein (g8p) of filamentous bacteriophage fd (inoviridae) mimotope vaccination-from allergy to cancer affinity maturation of single-domain antibodies by yeast surface display genetics of the immune response. i. the immune response to the phage fd in high and low responding inbred strains of mice convection-enhanced delivery of m13 bacteriophage to the brain multivalent display system on filamentous bacteriophage pvii minor coat protein phage immobilized magnetoelastic sensor for the detection of salmonella typhimurium specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen efrh-phage immunization of alzheimer's disease animal model improves behavioral performance in morris water maze trials ordering of quantum dots using genetically engineered viruses fabricating genetically engineered high-power lithium-ion batteries using multiple virus genes chemical modification of m13 bacteriophage and its application in cancer cell imaging direct detection of salmonella typhimurium on fresh produce using phagebased magnetoelastic biosensors mimotopes selected with a neutralizing antibody against urease b from helicobacter pylori induce enzyme inhibitory antibodies in mice upon vaccination phage display for site-specific immunization and characterization of high-risk human papillomavirus specific e7 monoclonal antibodies inhibition of bacterial conjugation by phage m13 and its protein g3p: quantitative analysis and model computational design of antibody-affinity improvement beyond in vivo maturation isolation of a bacteriophage specific for the f plus and hfr mating types of escherichia coli k-12 engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy role of capsid structure and membrane protein processing in determining the size and copy number of peptides displayed on the major coat protein of filamentous bacteriophage recombinant bacteriophage-based multiepitope vaccine against taenia solium pig cysticercosis viral assembly of oriented quantum dot nanowires virus-based toolkit for the directed synthesis of magnetic and semiconducting nanowires genetically engineered phage fibers and coatings for antibacterial applications molecular evolution of proteins on filamentous phage. mimicking the strategy of the immune system model-building studies of inovirus: genetic variations on a geometric theme filamentous phage structure, infection and assembly a fibrous dna phage (fd) and a spherical rna phage (fr) specific for male strains of e. coli. ii. physical characteristics phage display of a ctl epitope elicits a long-term in vivo cytotoxic response exposure of conjugative plasmid carrying escherichia coli biofilms to male-specific bacteriophages phage antibodies: filamentous phage displaying antibody variable domains constrained peptide libraries as a tool for finding mimotopes humoral immune response against proteophosphoglycan surface antigens of entamoeba histolytica elicited by immunization with synthetic mimotope peptides antigenicity and immunogenicity of phage library-selected peptide mimics of the major surface proteophosphoglycan antigens of entamoeba histolytica immunisation with phage-displayed variable region 2 from meningococcal pora outer membrane protein induces bactericidal antibodies against neisseria meningitidis derivation of vaccines from mimotopes. immunologic properties of human hepatitis b virus surface antigen mimotopes displayed on filamentous phage molecular evolution picks up the pace filamentous phages specific for the i sex factor design of specific immunogens using filamentous phage as the carrier uptake and processing of modified bacteriophage m13 in mice: implications for phage display genetically engineered phage harbouring the lethal catabolite gene activator protein gene with an inducer-independent promoter for biocontrol of escherichia coli inexpensive anti-cysticercosis vaccine: s3pvac expressed in heat inactivated m13 filamentous phage proves effective against naturally acquired taenia solium porcine cysticercosis recognition by human sera and immunogenicity of hbsag mimotopes selected from an m13 phage display library characterization of molecular correlates of the chronic humoral immune response: clues towards eliciting broadly neutralizing antibodies against hiv characterization of filamentous bacteriophage pe226 infecting ralstonia solanacearum strains virusbased photo-responsive nanowires formed by linking site-directed mutagenesis and chemical reaction virus-enabled synthesis and assembly of nanowires for lithium ion battery electrodes stamped microbattery electrodes based on self-assembled m13 viruses biologically templated photocatalytic nanostructures for sustained light-driven water oxidation virus-templated assembly of porphyrins into light-harvesting nanoantennae spr biosensor for the detection of l. monocytogenes using phage-displayed antibody production of hydrogen using nanocrystalline protein-templated catalysts on m13 phage quantitative synthesis of genetically encoded glycopeptide libraries displayed on m13 phage graphene sheets stabilized on genetically engineered m13 viral templates as conducting frameworks for hybrid energy-storage materials affinity-selected filamentous bacteriophage as a probe for acoustic wave biodetectors of salmonella typhimurium detection of pathogenic bacteria using a homogeneous immunoassay based on shear alignment of virus particles and linear dichroism phage display: concept, innovations, applications and future antibody-selectable filamentous fd phage vectors: affinity purification of target genes potential applications of phage display to bioremediation a library of organic landscapes on filamentous phage abundance, morphology and distribution of planktonic viruslike particles in two high-mountain lakes correlates of protection induced by vaccination targeted gene delivery to mammalian cells by filamentous bacteriophage conditional lethal mutants of the small filamentous coliphage m13. ii. two genes for coat proteins selection of antigenic and immunogenic mimics of hepatitis c virus using sera from patients towards a solution for hepatitis c virus hypervariability: mimotopes of the hypervariable region 1 can induce antibodies crossreacting with a large number of viral variants filamentous bacteriophages: biology and applications, " in els (the encyclopaedia of life sciences) filamentous bacteriophage: biology, phage display and nanotechnology applications antigen-specific therapy of eae via intranasal delivery of filamentous phage displaying a myelin immunodominant epitope expression of a 28-kilodalton glutathione s-transferase antigen of schistosoma mansoni on the surface of filamentous phages and evaluation of its vaccine potential structural requirements for the activity of the mirb ferrisiderophore transporter of aspergillus fumigatus chemically linked phage idiotype vaccination in the murine b cell lymphoma 1 model phage idiotype vaccination: first phase i/ii clinical trial in patients with multiple myeloma phage display selection of peptides that affect prostate carcinoma cells attachment and invasion a helper phage to improve single-chain antibody presentation in phage display metagenomic analysis of viruses in reclaimed water assessing the diversity and specificity of two freshwater viral communities through metagenomics recombinant expression and neutralizing activity of an mhc class ii binding epitope of toxic shock syndrome toxin-1 epitope-specific antibody response to ige by mimotope immunization some physical-chemical and biological properties of the rod-shaped coliphage m13 generation and characterization of phage-gnrh chemical conjugates for potential use in cat and dog immunocontraception phage display allows identification of zona pellucida-binding peptides with species-specific properties: novel approach for development of contraceptive vaccines for wildlife infective and inactivated filamentous phage as carriers for immunogenic peptides vaccination with filamentous bacteriophages targeting dec-205 induces dc maturation and potent anti-tumor t-cell responses in the absence of adjuvants the use of filamentous bacteriophage fd to deliver hla-a2-restricted peptides and to induce strong antitumor ctl responses selection of hiv-specific immunogenic epitopes by screening random peptide libraries with hiv-1-positive sera application of bacteriophages for detection of foodborne pathogens searching for peptide ligands with an epitope library de novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pix fusion proteins high copy display of large proteins on phage for functional selections filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface effect of dna copy number on genetic stability of phage-displayed peptides hydroxyapatite chromatography of phage-display virions phage display libraries of peptides and proteins displayed on filamentous phage generation of anti-β-amyloid antibodies via phage display technology towards alzheimer's disease vaccination filamentous bacteriophage as a novel therapeutic tool for alzheimer's disease treatment comparison of phage pviii and klh as vector in inducing the production of cytokines in c57bl/6j mice bacteriophage therapy viruses in the sea a mimotope peptide of aβ42 fibril-specific antibodies with aβ42 fibrillation inhibitory activity induces anti-aβ42 conformer antibody response by a displayed form on an m13 phage in mice accessibility of peptides displayed on filamentous bacteriophage virions: susceptibility to proteinases antibody fab display and selection through fusion to the pix coat protein of filamentous phage an investigation on the nature of ultra-microscopic viruses antigenic properties of hcmv peptides displayed by filamentous bacteriophages vs. synthetic peptides in vivo characteristics of targeted drug-carrying filamentous bacteriophage nanomedicines presentation of the functional receptor-binding domain of the bacterial adhesin f17a-g on bacteriophage m13 engineering filamentous phage carriers to improve focusing of antibody responses against peptides filamentous phage as an immunogenic carrier to elicit focused antibody responses against a synthetic peptide basic research in hiv vaccinology is hampered by reductionist thinking lysogenic conversion by a filamentous phage encoding cholera toxin induction of hepatitis b virus-specific cytotoxic t lymphocytes response in vivo by filamentous phage display vaccine crosspresentation of phage particle antigen in mhc class ii and endoplasmic reticulum marker-positive compartments bio-mimetic nanostructure self-assembled from au@ag heterogeneous nanorods and phage fusion proteins for targeted tumor optical detection and photothermal therapy enhanced tumor delivery and antitumor activity in vivo of liposomal doxorubicin modified with mcf-7-specific phage fusion protein paclitaxel-loaded peg-pe-based micellar nanopreparations targeted with tumor specific landscape phage fusion protein enhance apoptosis and efficiently reduce tumors hybrid phage displaying slaqvkytsassi induces protection against candida albicans challenge in balb/c mice protective immune responses against systemic candidiasis mediated by phage-displayed specific epitope of candida albicans heat shock protein 90 in c57bl/6j mice enhanced binding and killing of target tumor cells by drug-loaded liposomes modified with tumor-specific phage fusion coat protein paclitaxel-loaded polymeric micelles modified with mcf-7 cell-specific phage protein: enhanced binding to target cancer cells and increased cytotoxicity cytoplasmic delivery of liposomes into mcf-7 breast cancer cells mediated by cell-specific phage fusion coat protein bacteriophage and phenotypic variation in pseudomonas aeruginosa biofilm development evidence for tilted smectic liquid crystalline packing of fd inovirus from x-ray fiber diffraction experimental evolution of viruses: microviridae as a model system immunological properties of foreign peptides in multiple display on a filamentous bacteriophage adsorption protein of bacteriophage fl: solubilization in deoxycholate and localization in the fl virion sensitive and selective bacterial detection using tetracysteine-tagged phages in conjunction with biarsenical dye phage display particles expressing tumor-specific antigens induce preventive and therapeutic anti-tumor immunity in murine p815 model development of a phage displayed disulfide-stabilized fv fragment vaccine against vibrio anguillarum high frequency of a novel filamentous phage, vcyφ, within an environmental vibrio cholerae population targeting antibacterial agents by using drug-carrying filamentous bacteriophages filamentous phages of ralstonia solanacearum: doubleedged swords for pathogenic bacteria prophylactic vaccination with phage-displayed epitope of c. albicans elicits protective immune responses against systemic candidiasis in c57bl/6 mice epitope mapping of mycoplasma hyopneumoniae using phage displayed peptide libraries and the immune responses of the selected phagotopes m13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors comparison of phage piii, pviii and gst as carrier proteins for peptide immunisation in balb/c mice spontaneous assembly of viruses on multilayered polymer surfaces characterization of murine coronavirus neutralization epitopes with phage-displayed peptides purification of filamentous bacteriophage for phage display using size-exclusion chromatography conformational mimicry of a chlamydial neutralization epitope on filamentous phage f1, a rodshaped male-specific bacteriophage that contains dna biodistribution of filamentous phage peptide libraries in mice this work was supported by funding from the national research council of canada (kh, ma-g) and the canada research chair program (js). we thank jyothi kumaran and roger mackenzie for critical appraisal of the manuscript, and jasna rakonjac for inviting us to contribute it. this is national research council canada publication number 53282. key: cord-020235-stcrozdw authors: nan title: abstracts of papers presented at the 38th meeting of the deutsche gesellschaft für hygiene und mikrobiologie, virology section, göttingen, 5.–8.10.1981 date: 2012-03-15 journal: zentralbl bakteriol mikrobiol hyg a doi: 10.1016/s0174-3031(82)80128-5 sha: doc_id: 20235 cord_uid: stcrozdw nan schaefer, a., zibirre, r., kabus, p., kohne, jutta, and koch. g. na +, k + -atpase activity was studied in a plasma membrane rich fraction isolated from control and poliovirus infected he la cells and compared to membrane potential and amino acid uptake in parallel cultures of intact cells. na + , k + -at pase activity in membranes from infected hela cells increased relative to control with a maximum at 90 minutes post infection (+ 30 %) and decreased again (180 rnin.: -30 %). similar but slighter changes were observed in the membrane potential dependent tetraphenylphosphonium (t pp+ ) uptake, indicating a correlation between membrane porential in intact cells and our measurment of plasma membrane na + , k +-at pase. at approximately 1 h post infection we observed a decrease in the uptake of amino acid (methionine, leucine) in infected cells relative to controls. these results suggest that the decline in amino acid upt ake is not mediated by virus-induced changes in the na + , k + -atpase activity or membrane potential. mpi f. biochemic, abt. virologic, sequence homology in different strains of fmdv and other picoma viruses marquardt, o. restriction enzyme generated subgenomic fragments of cloned cdna prepared from rna of the strain 01k of fmdv were compared quantitatively for sequence complementarity with radioactive rna from strains cobb and a2s or with rna from mengo or polio virus in hybridization experiments by use of the southern technique. nucleic acid sequences neighbouring or including the 3' end of viral genomes are demonstrated to be 80 ofo homologous in fmdv. in contrast, sequences coding for the capsid proteins vpl and vp3 were remarkably heterologous (less than 20010) in fmdv. sequences coding for non-structural proteins showed 35-50010 homology. thus no highly conserved coding sequence was detectable in fmdv by this technique. no hybridization could .be detected between 01k specific dna and polio rna, while weak hybridization was observed with mengo rna at areas including the 3' end and with a part of the gene for precursor protein p52. abr, molekularbiologie, physiol-chern. inst., grindelallee 117, d-2000 hamburg 13 modification of poliovirus capsid proteins scharli, claudia and koch, g. poliovirus type 1, strain mohoney contains a protein kinase activity. a highly purified (two cycles of csci gradient centrifugation) poliovirus preparation is able to transfer the y-phosphoryl-group of [32p]_atp to acid precipitable material. when the reaction product is anal yzed by sds page, all structural proteins of polio are phoshorylated. most of the label is incorporated in the minor capsid protein vpo and to a lesser extend in vp1, vp2 and vp3. hepatitis a virus (hav) was isolated directly from human stool in diploid human fibroplasrs, viral antigen was expressed only after 210 days of incubation of the infected cultures. in contrast, hav first adapted to growth in human hepatocellular carcinoma cells caused antigen expression in fibroplast cultures already 90 days after infection. during further passage the time of first appearance of antigen in infected fibroblasts decreased to about 14 days in both passage series (i. e. cultures inoculated with virus recovered directly from human stool or after adaption to human hepatoma cells). antigen was predominantly cell-associated and was shown by immunofluorescence to be located exclusively in the cytoplasm. -biophysical properties of hav particles extracted from infected cells were comparable to those described for hav extracted from human stool. those findings are of importance for preparation of large amounts of hav for vaccine. production. max von pettenkofer-inst. u. inst. f. biochemie d. univ. d-8000 miinchen 2 cloning of hepatitis a virus genomes helm, k. von der, winnacker, e. l., and deinhardt, f. subgenomic fragments of the genomic rna of hepatitis a virus (hav) were cloned via edna into pst 1 site of pbr 322. by restriction enzyme pattern analysis and hybridization of the cloned hav dna with selected fragments of the ha v rna it was determined that the major part of the cloned hav dna fragments are located at the 3' end of the genome but a few clones are distributed over the entire genome. we have now identified subtype specific sites in hbv-restriction map s. the maps were aligned to the eco ri site. only subtype ay revealed one subtype specific site of each xbai (2000 bp), h ineli (2200 bp) and bam hi (2900 bp). they are situ ated outside the s-gene. employing rhis system the subty pes of three hbv-ona sequences that ha ve been published so far , could be determined . thereupon the first 54 triplets of a hbv (ad)-dna sequence of the s-gene could be com pared with pub lished data . nucleotide exchanges in triplets 12 and 13 did not cau se an amino-acid (aa)-exchange, bu t cha nges in the triplets 45 and 46 do cause such exchange. thus, in subtype ay, aa 45 is thr, and aa 46 is thr, wh ile in subtype ad, aa 45 is ser and aa 46 is pro. these exchanges may produce different conf ormation of th e peptides. dept. m ed. mic robio l., univ. 0 -3400 gottingen characterization of the hepatitis b virus (hbv) associated proteinkinase gerlich, w. h. purified hbv-core preparations were shown to contain a pr ot eink inase which phosphory lates the major core protein (albin and robinson, 1980) . in this study it was found th at th is enzyme copurifies with the light (d = 1.31) but not with the heavy subfraction (d = 1.35) of the core-particles. the enzyme has a high affinity for atp and it transfers approx. one ph osphat e per particle within minutes. the 32p-phosphate introd uced in vitro cannot be remove d from core-particles by digestion with alkaline ph osph at ase. after lysis of the cores with sos the 32p can be cleaved from rhe protein. t his suggests th at th e enzyme and its ph osphate acceptor site are located with in the pa rticle. after acid hydrolysis the incor porated 3!p co-migrates with ph osph oserin but not with phosphor hreonin or phos pho tryosin. m ax von petrenk ofer-inst., univ. 0 -8000 miin chen 2 a phosphokinase activity associated with the hepatitis b virus (hbv) core helm, k. von der, roggendorf, m., and siegert, w. prepar ation s of core antigen positive (hbcag) fractions obta ined from hbv positive hu man liver tissue are associated with a ph osphokinase wh ich copurifies with the hbcag in cscl density-and sucrose velocity grad ient centrifugations. the acceptor protein for this kina se is the 19000 d hbcag protein; casein as an exogenous acceptor is also phospho rylate d to a minor extend. the ph osphorylated amino acid is serine or threonine and not tyrosi ne, as it is frequently the case with tumor virus specific prot ein kinases. dept. med . microbiol., univ. 0 -3400 gott ingen effect of glycosidases on the proteins of hepatitis b surface antigen (hbsag) stibbe, w. and gerlich, w. h . the protein composition of purified hbsag was stud ied by sos·gc1e1ectrophoresis and staining with silver. in additio n to p25 and gp28 of hbsag two furt her proteins, gp33 and gp36, were found consistently in preparations from 9 different blood donors. the glycoprotein nature of gp 33 and gp 36 was shown by the increase of electrophoretic mobility after treatment with endoglycosidase h or neuraminidase. immune precipitation with anti-hbs in ripa-buffer confirmed the specifity of the glycoproteins, the change in apparent mol. weight after treatment with neuraminidase was larger for gp36 (900) than for gp33 (600) or gp28 (300). the data suggest that gp 33 and gp36 are multiply glycosylated proteins with n-glycosidic linked carbohydrate side chains of the mixed type. inst, f. virologie, univ., hb s antigen screening was done in the blood samples of 1,790 pregnant women. 33 blood samples were found to be hb s antigen positive. 32 women were asymptomic carriers, 1 woman suffered from chronic hepatitis. 31 children have been born until now. the examination of cord blood showed hb s antigen in 6 samples; the venous blood samples taken thereupon demonstrated hb s antigen only in two samples. the hb s antigen negative children were treated with hbig. the follow-up examination of the treated children showed no signs of infection up to now. -one child born to an hb e antigen positive mother was already infected at the time of birth. another child born to an anti hb e positive mother became hbs antigen positive at the age of 4 months. unfortunately, this child had not been treated with hbig at the time of birth. willers, h., ponnath, h., sipos, s., and moller, r. sera of 5,847 pregnant women living in the hannover area were investigated for the presence of hbsag. 56 healthy hbsag carriers were found: 17 (0.340/0) among 4,962 women of german origin and 39 (4.280/0) among 912 foreign-borne women from southern european and non-european countries. 10 out of the 56 hbsag carriers (114,962 german women and 9/912 foreign-borne women) were recognized to be hbeag-positive. -the risk of perinatal transmission of hepatitis b virus in infants of hbeag-positive mothers is suggested to be 90 % and of hbeag-negative mothers 12 0/0. -thus in the hannover area 6 out of 10,000 infants born to german mothers and 129 out of 10,000 infants born ro foreign mothers become hbsag carriers due to perinatal hepatitis b virus-infection. max von pettenkofer insr., univ. d-8000 munich 2 a radioimmunoassay for anti-hbc igm using higher concentration for the dilution of serum (5 mg/ml) and hbcag (10 mg/ml) was compared with an elisa test ]. din. microb iol. 13 (1981) , 618. onl y 10 of 157 (6 0 /0) hbsag positive blood don ors were positive (40 in elisa) and no correl ation with the total anti -hbc titer could be found . after fraction ation of a serum with discrepant results a positi ve result was found in the elisa only in the igg fractions. in a collaborative stu dy with a/torfer et al. (presented also at lisbo n, easl 1981 (1981) , who show ed a 10 6 fold reduction in hepatitis b titer by means of !i-plluv treatme nt. the effectiveness of the cold sterilization procedure as regards reducing virus infectivity is considera bly greater than that of pasteurization (10 h 60 dc) for which shikata et al. (1977) sho wed a 10 4 fold reduction of hep atitis b virus infectivity. it has also been found that factor ix conce ntrate and stab ilized serum (biseko®) der ived from pooled. cold-sterilization hu man plasma transmitted neither hepatitis b nor hepatit is no n-a, non-b in chimpa nzees. max von pettenk ofer-insr., 0 -8000 miinchen 2 comparison of different evaluation systems for determination of viral antibodie s of the igg and igm class in the enzymeimmunoassay r o g g endorf, m. , zachoval, r ., zoulek, g., a nd deinharot, f. t he enzymeimmunoassays used to demonstrate antibodies of the igg and igm class against viral antigens reveal a high sensitivity. at onset of acute viral infections igm antibodies can be demonstrated up to serum dilutions of 10-7 • due to th is high sensitivity, the determin ation of antibody tite rs is not accurate and reproducible, because titer end points are determ ined as that dilution of sera giving an 0.0. sample/o .o. negative control equal or greater than 2.1. -for the dete rmination of antibody con centr ations an evaluation method is proposed which correlates, the measured 0 .0. of sera at one dilut ion step to the 0 .0. of a reference serum which is defined arbitrarily to contain 100 anti body units. using an elisa for detection of antibodies to adenovirus, a significant increase in antibod y units of acute phase sera over that of convalescent pha se sera is observed. low day to da y varia tions are seen in tests car ried out on different days. in an elisa which is designed to detect anti bodies to tick-born e encephalitis virus of th e igm class, the day to day variatio n using antibody units was significantly lower th an using pin ratios. inst. f. virolog ie, jusrus-liebi g-un iv., 0 -6300 gieben; max-planck-insr, f. m olekulare genet ik, d -looo berlin structure and function of the core protein of alphaviruses boege, ulrike and witt man n-liebold, brigitte the com plete primary struc tures of the core proteins of sindbis and semliki forest virus have been established by pr ote inchemical methods. both proteins contain clusters of basic amino acids and proline in th e n-terminal part, suggesting th at its function is to bind to the nucle ic acid. the c-termi nal pa rts have ext ensive iden tical sequence regions, pr ob abl y p roviding the recognition sites for pr otein-protein interactions . both proteins contain within these highl y conserved portion s the sequence gly-asp-ser-gly wh ich is typical for serine proteases and most likely is related to the cata lytic function of the core prot ein as a protease wh en cleaving its own peptide chai n from the nascent protein precursor. experimental studies using peptides which contain certai n sequence regions will help to elucidate the relationships of struc ture and functions. an ac ute and a persistent infectio n with rab ies virus (h ep flury) was established in th e cn s-derive d cell 1ine 108-cc-i5 (ng 108-15). these cells possess specific membrane recept ors to many hormon es and neurotran smitters including opiate receptors. in both cases we found increases in th e dissociation constant (kn) for the agonist 3h-etorp hine as estimated by scatcha rd plot ana lysis. h owe ver, in both cases th ere was no change in th e nu mb er of op iat e receptors per cell compa red to uninfected cells. these studies complete our previou s published observations of the impai rme nt o f receptor func tions in rabies virus infe cted cells (1) . (1) kosch el, k. an d m. halb ach: j. gen. virol. 42 (1979) , 627-632. insr, f. virologic u. immunobi olo gie d. univ., 0-8700 wiirzburg effect of measles (sspe) antiserum on viral surface proteins and hormone receptor activity in c6/sspe persistently infected cells barrett, p. n. and koschel , k. it has been reported (1) th at measles antis erum ca n modulate the expression of certain viru s prot eins in acu tely measles infected cells. we have exam ined th e effect of measles (sspe) antiserum on the exp ressio n of vira l surface proteins in c6isspe infected cells. we have shown an over 50 % redu ction in the am ount of viral antigen present on the memb ranes of these cells following incuba tion with sspe antiserum. this was demon-strated both by immunofluorescence and rad ioimmunoassay. however this loss of antigen had no effect on memb rane receptor linked camp synthesis. this is discussed with respect to the effect of virus antigen insertion in cell membranes on specialised membrane bound functions. (1) we have analyzed the effect of phosphorylation and dephosphorylation on the structure and dna binding of d2-t antigen. on non-denaturing pore-gradient gels the purified protein migrated with an apparent size of 135,000 daltons, in vitro phosphorylation by the protein kinase associated with the purified protein resulted in a shift of most of the protein to a size of 740,000 daltons, and it was this form that contained most of the phosphate incorporated. this aggregation was completely reversible by treatment of phosphorylated d2-t antigen with alkaline phosphatase. partial tryptic digestion indicated that phosphorylation of sites in the nvrerminal part of the protein is responsible for the observed aggregation. -as show n by protein blotting onto nitrocellulose filters predominantly the 740,000 dalton form bound to sv40 dna. however, onl y a fraction of the in vitro phosphorylated protein did bind to dna, suggesting that aggregation alone is not sufficient for dna bind ing. subclasses of sv40 large t antigen were separated by zone velocity sedimentation. three major forms, which sedirnented at about 5-6s, 14-16s and 23-255, have been shown to differ biologically and biochemically (1) . each subclass was tested for specific binding to restriction fragments of sv40 dna using an immunoprecipitation assay. -all three forms of t antigen bound specifically to a restriction fragment containing the sv40 origin of replication. however, the 5-6s form bound more origin fragment per unit t antigen than the other forms . the 5-6s form bound equally well to origin dna in the presence and absence of excess cellul ar dna , whereas the binding of the 14-16s form was reduced in the presence of cellular dna . all forms of t antigen from sv40-transformed sv80 cells bound much less origin dna than that from infected cells. (1) fanning, nowak , burger: j. virol37 (1981) wart scrapings from several small skin regions of an epidermodysplasia verruciformis patient were tested for papilomavirus-specific sequences by means of a 32p-labelled hpv8 dna. uncleaved wart dna contained uniforme full length hpv genomes. cleavage with bam hi revealed six different patterns and a surprising heterogeneity even within small biopsies. at least two virus types showed only limited cross-hybridization with hpv 8a. one of these closely resembles hpv 5b (bam hi fragments 2,9 and 2,1; eco ri fragments 3,6 and 1,1). hind ii fragments a and c of his virus perfectly hybridize with hpv 8a; b, d and 6 anneal only partially and f, g show no detectable hybridization. the dnas of four subtypes were partially characterized and mapped. only dna of the hpv 5b-like virus was detected in a probe from the center of a carcinoma at the patient's forehead. this dna persists extrachromosomally with more than 100 genome equivalents per cell. inst, f. virologie, zentrum hygiene, univ., hermann-herder-str. 11, d-7800 freiburg gene expression of papillomaviruses in hamster tumors freese, u. k.,schulte, p., and pfister, h. bpv 1 includes fibrosarcomas in hamsters. the tumors contain large amounts of complete virus genomes which persist extrachromosomally but there is no evidence for capsid protein synthesis or virus particle production. we used this system to study early viral gene expression. rna from the tumors contained a single virus-specific rna with about 1300 nucleotides which was shown to be polyadenylated by affinity chromatography on poly-uesepharose. the transcribed dna region of bpv 1 was mapped within the two hpa ii fragments, which are next to the bam hi cleavage site within the 1.4 x 10 6 d bam hiieco ri fragment. cross-hybridization studies under low stringency revealed some sequence relationship of hpv 1 and hpv 4 dna to the transcribed region of bpv 1. inst. of genetics, univ., d-5000 cologne the adenovirus type 12-mouse cell system: permissivity and analysis of viral dna in tumor cells starzinski-powitz, a., schulz, m., and doerfler, w. interactions between viruses and eucaryotic cells can be studied in a genetically well defined system like the mouse system. we have investigated whether ad 12 dna is able to replicate in primary mouse kidney cells or in the mouse cell line l929. it was shown by southern blot experiments that ad12 dna was not able to replicate in l929 cells, w hereas in prima ry mouse kidne y cells of (balb/c x cs7/b16) fl origin, viral dna repl icated. moreover , we subcutaneously injected ad12 into mice of various genetic origins. in ab out 100 mice injected, one rumor emerged in a balblc mouse almost 8 months after injection. restriction pattern anal ysis of the rumor or dna indicated that abou t 2-3 copies of ad12 dn a were integrated and covalently bound to cellular dna. with th e techniques available no deletion or rearrangement of viral dna could be found. t he sites of jun ction betw een viral and cellular dna will now be cloned and sequenced. inst, of genetics, univ., 0-5000 co logne virus-cell dna recombinants in human cells lytically infected by ad2 neumann, r., weyer, u., and doerfler, w. there is ample evidence for the notion that virus-cell dna recombinants are formed in human cells productively infected with adenovirus type 2 (ad2). these high molecular weight forms were detected at 1-2 h postinfection and were generated at high frequency, a limited set of rather specific recombinants was produced (neumann and doerfler, j. virol. (1981) 887 suggesting th at recomb ination exhibited a cert ain specificity. we now succeeded in molecularly cloning dna frag ments excised from the high mole cular weight dn a of ad2-infected hum an cells by th e restri ction endonuclease ecori in agtwes . ab or in acharon 4b. some of these clon ed frag ments had sequence ho mology to both viral and cellular d na. th is result provided proof fo r the occurrence of virus-cell dna recombi na nts . inst, of genetics, univ., 0-5000 cologne unmethylated dna sequences in the promoter regions of integrated adenovirus genes correlate with gene expression kruczek, 1. and doerfler, w. an inverse correlation was established between the levels of dn a met hylation at 5'-ccg g-3' sites in specific segments of integrated adenovirus dna and the extent to wh ich the se segments were expressed (sutt er and doerfler, pnas 77 (1980) 253. similar correlatio ns were reported in other viral and non-viral systems. more recently, the results of in vitro experiments prov ided direct evidence for the notion th at dna methylation at specific sites led to gene inactivation (vardimon et al., pnas, in press ). -in the present stud y a detai led methylation map at 5'-cc gg-3' (h paiilm spi) sites in the expressed early and the silent late genes of ad12 dn a was determined in three adl2-tr ansformed hamster cell lines. the early region s of inte grated ad12 dna were unmethylated; in particular their promot er regions were unrnethylated ar the hpall sites in all three lines investigated. t he late regio ns including the promot er sites were completely meth ylated. gahlmann, r., deuring, r., stabel, s., winterhoff, u. vardimon,1., and doerfler, w. the sites of junction between viral and cellular dna were sequenced to investigate two problems: 1. are the sites of insertion of viral dna specific? 2. what type of recombinatorial events occur in viral dna integration? we have studied junction sites from the ad2-transformed hamster line hes, and from the ad12-induced hamster tumor lines claci and clac3. the virus-cell dna junctions were cloned in the dna of bacteriophage 2gt· j.b. appropriate restriction fragments were sequenced by the maxam-gilbert technique. there was no direct sequence identity apparent at the viral and cellular junction sequences. deletions at the termini of the viral genome were seen involving 5 (he5), 45 (clac3) or 174 (clacl) nucleotides. peculiar patch type homologies between the cellular and viral sequence adjacent to the site of junction and also in remote areas were observed. these patches may have an important function in integrational recombination. buttner, w., veres-malnar,s., and block, j. as a first step to understand the relationship between structure and function of the adenovirus type 2 dna binding protein (ad2 dbp) we have begun to determine the primary structure of the dbp gene. the isolation and characterization of tupaia adenovirus (tav) has been reported. in order to construct a genetic map of the tav genome the use of temperature-sensitive mutants (ts) of tav was necessary. -a variety of ts mutants of t av, which were generated by treatment of t av virions (1 x 10 10 pfu) with hydroxylamine, were isolated and preliminary characterized. six of these mutants its: 1, 4, 11, 12, 13 , and 54) did not replicate in tupaia embryonic kidney cells at 39.5 dc, but did replicate well at 32°c. the characterization of these mutants was carried out using complementation tests, host range studies and dna anal ysis using different restriction enzymes. according to complementation analysis four groups were determined : group i = ts 1, group ii = ts 12, group iii = ts 13, and group iv = ts 4, 11, and 54. the host rage study revealed that ts 1 and 54 also had properties of host range mutants. these mutants did not replicate in tupaia bab y fibroblasts in contrast to wild-type virus. genome analysis of these mutants revealed that the mutated region is located between 69.9 to 74.5 map units. in addition, in ts 1 and 54 mutants a deletion from 0.77 kb to 1.39 kb (map unit 76.5 to 100) was detected. interaction of viral substructures with serum and serumcomponents resp. during the final stage in the course of many viral infections complete virus particles as well as viral substructures enter the intercellular space, e. g. hbsag, hbcag in hepatitis b virus infections . the present study deals on the interaction of the potentially infectious adenovirus cores with dna-specific antibodies and immunoglobulin solutions which had been absorbed by dna-antigens. cores were prepared by sarcosyl treatment of purified adenovirus typ 5. by electron microscopy immunocomplexes could be demonstrated which are composed of several individual core units. by buoyant density centrifugation in metrizamide gradients a drastic rise in the density of cores could be shown too. with immunoglobulin solutions, absorbed with native as well as with denatured dna, so far no complexes could be assessed. a cell line designated sbl-h12578 and several cell clones were established from skin tumours of the sporadic leukosis form. the cells were proved to be free of bovine leukemia (blv), and some other bovine viruses. by indirect immunofluorescence macroschizonts of a theileria species were seen in the cytoplasm. the cells originated from a blvantibody negative animal and carried a female karyotype with some morphological aberrations. evidence for a possible t cell origin of sbl-h12578 cells was obtained. after inoculation of 8 x loa cells into 'nude mice' transplantable sbl-h12578 tumours developed. by incorporarion of 3h-uridine and electron microscopy, the production of retrovirus particles by the cultured cells was detected . in the simultaneous detection test a highmolecular weight rna co-migrating with felv 70 s rna was demonstrated. no antigenic or genetic relationship between th e skin tumour virus isolate and blv or other major mammalian retrov iruses has been found. -(supported by stiftung volkswagenwerk ). abt. f. pathologie, gesellschafl: f. strahlen-u. umweldorschung, d-8042 neuherberg insectpathogenic baculoviruses: studies of the activation of endogenous c-type retroviruses in mammalian cell cultures schmidt, j. and erfle, v. activation of endogenous c-type retroviruses by baculoviruses was studied in "in vitro" cell culture systems of four mammalian species: mouse, rat, monkey and man. cells were treated with baculoviruses (from larvae and insect cell cultures), baculovirus-dna, c-rype retrovirus-activating chemicals and chemical insecticides alone and in combination. the activation of retroviral genomes was tested by the determination of reverse transcriptase activiry in concentrated cell culture supernatants and by the demonstration of the intracellular localisation of retrovirus structural protein p30 applying the indirect immunoperoxydase technique. -c-type retroviruses were activated in mouse cells only by the halogenated pyrimidine analogue iododeoxyuridine. in baculovirus-treated cell cultures no c-type retrovirus activation was detectable. in simultaneous treatments of the cells with baculoviruses and chemicals no potentiating effects could be detected. virions of baculoviruses in mammalian cell cultures showed unaltered morphology and upon reisolation their infectivity in homologous insect cell cultures was lowered by approximately one log. no influence upon growth or morphology of the cells could be observed. the gene products of replication-defective oncornaviruses are high molecular weight proteins which comprise a gag related and one gene product. they are probably not cleaved because the p15 protease gene is lacking. in vitro, however, the gag-specific peptide sequences are cleaved off upon addition of the purified viral pis protease; in the case of the replication-defective, transforming avian sarcoma virus prc ii the cleaved nongag part has a ryrosine-phosphorylaring kinase activity similar to that described for the rsv src-gene product pp60 src . processing of pr92 gp , the precursor to the viral glycoproteins of rous sarcoma virus bosch, v., schwarz, r. t., ziemiecki, a., and friis, r. r. the viral glycoproteins of rous sarcoma virus gp85 and gp35 are synthesized via a precursor polyprotein pr92". this precursor is already glycosylated and contains the polypeptides of both gp85 and gp35. we have studied the nature and site of processing of pr92'~to mature disulfide-linked gp85 and gp35 (vgp). we could show that in addition to proteolytic cleavage, processing involves conversion of the high-mannose oligosac-charides found in pr92' " to the complex, sialidated oligosaccharides found in vgp. experiments pertaining to the site of processing indicate that processing does not occur extracellularly as has been proposed by others iklemenz and diggelmann, j. virol. 29 (1979) 285-292. we have determined that the small amount of mature vgp found in infected cell lysates is localized chiefly within the cell, not at the cell surface. we favour the view that further glycosylation and proteolytic cleavage occur concomitantly on smooth membranes within the cell and that subsequent to this, export in virus is rapid. 1 paul-ehrlich-inst., d-6000 frankfurt; 2 lnst. f. virologie, univ., d·6300 giefen, 3 a protein of a molecular weight of about 38.000 d has been found to be phosphorylated 1 h after the onset of cell transformation by rous sarcoma virus (rsv). it is assumed to be a physiological target of the pp60'" kinase, since, apart from being phosphorylated in the transformed cell, it can be phosphorylated in vitro by the pp60"· kinase in tyrosine (1, 2) . -using 6 different chromatographic procedures (chromatography on deae-sephacel, poly (a)-sepharose, blue sepharose, and hydroxylapatite, isoelectric focusing and gel filtration ) the 38,000 d protein could not be separated from cytosolic malic dehydrogenase activity (c-m dh ). antiserum against the 38,000 d protein inhibited c-mdh. the transformation-specific protein "v-myc" of the acute avian leukemia virus mc29 donner, p., greiser-wilke, irene, and moelling, karin avian acute leukemia viruses transform cells through a virus-coded oncogene. in the case of the rnyelocytornatosis virus, mc29, which transforms fibroblasts as well as bone marrow cells in vitro , this oncogene is fused to a viral structural component, p19. the fusion protein, v-rnyc, was characterized by using monoclonal antibodies against p19. mc29 transformed quail fibroblasts which do not produce any virus, mc29-q8, were analyzed b y immunofluorescence. a nuclear fluorescence was observed which was not detected in normal cells or virus-producing cells. the monoclonal antibodies were used to purify the v-myc protein by immuno-affinity chromatography. the purification achieved by this single-step purification was 3,500 fold . the eluted protein was precipitable by anti-sera and will be further characterized for its biological properties. med. poliklinik, univ., d-8000 miinchen; abr, f. pathologie d. gsf, neuherberg biochemical characterization of antigens in human leukemic sera that crossreact with sisv p30 and baevp30 leib, c., schetters, h., erfle, v., and hehlmann, r. antigens crossreacting with the core proteins p30 of baboon endogenous virus (baev) and/or of simian sarcoma associated virus (sisv) have been isolated from human leukemic sera by immunoaffinity chromatography. the antigens have an apparent molecular weight of 70,000 in sds-polyacrylamide gel electrophoresis. peptide maps performed with the antigens from two different leukemic sera show that the two antigens are identical. peptide analyses of sisv p30 and of baev p30 and simultaneous mapping of p30 proteins mixed with human antigens show that 11 out of 21 major peptides of sisv p30 and 10 out of 20 major peptides of baev p30 have identical mobility with peptides of the human antigens. human serum albumin, transferrin, fibrinogen, igg and igm share clearly less peptides of identical mobility with the human antigens. the isoglycoproteins gp69 and gp71 were purified from f-mulv particles (propagated in eveline cells) by solubilization (freezing and thawing), ion exchange chromatography (phosphocellulose) and preparative sds-page. prior to amino acid and nh 2-terminal amino acid sequence analysis, the purified glycoproteins were subjected to high performance liquid chromatography (hplc) to remove contaminants. -the nh 2-terminal amino acid sequences (23 residues) of gp71 and gp69 were found to be different (in 10 positions) but highly related. f-mulv gp69 shows 410f0 homology to gp71 but lacks the potential glycosylation site (sequon) at position 12 in both f-mulv gp71 and r-mulv gp70. et al., 1980) . in our laborato ry 99 breast can cers, 60 normal breast tissues, 44 benign breast lesions and 10 other carcin om as from south german patients were tested for crossrea ctivit y w ith mmtv-gp52. the tests were done by indirect immunoperoxidase staining on paraffin-sections using antise ra against gp52 prov ided by dr. spiegelman, n. y. specificity of positive reactions wa s controlled by preabsorption with purified mmtv-gp52. 86 breast cancers (87 0 /0) and 13 benign breast lesions (30 0 /0) gave positive reactions, whereas normal bre ast tissue and other carcinomas were negative. -the test might be an additional useful diagnostic too l for the earl y detection of micrometastases, for the assignment of metastases from unknown pr imary tumors and in doubtful cases of breast cancer. it can possibly serve as an add itional criterion for the classification of mastopath ies. viruses wer e found in 3,691 patients. nearly all illnesses were caused by mumps-and entero-viruses , other viru ses were found in less than 5 % of all cases. the mumpsmeningitides were ascertained constantly over all these year s. in 1967 in , 1974 in and 1980 an increased incide nce of meningitides caused by echo-virus type 30 wa s seen. -there was no seaso na l dep endence on the occurrence of mumps virus meningitides. meningitides, caused by ent ero-viruses was found mor e often in the autumn. male patients fell ill twice as ofte n as female patients. mumps-meningitides were rarel y found in the first year of life. in contras t, enterovirus meningitides could be dete cted during the first year and caused men ingitides to age of 14. virus meningitides among adults were rarel y found. we suspected that a number of peripheral facial pareses (p.f.p.) considered "idiopathic" might, in fact, have a viral aetiology . 71 patients of the cologne university e.n.t. clinic with so-called idiopathic p.f.p. were examined under the aspect of a viral aetiology. in 32 cases conditions for virological investigations were optimal (paired sera, first serum within the first week after onset of disease). in 10 of these 32 cases a viral infection could be proven (varicella zoster virus: 7, herpes simplex virus: 2, coxsackie b 4 :1). in 5 of 7 vzv cases minimal zoster lesions were observed, 4 facial and 1 thoracic. among the 39 oth er patients (with late sera) no viral infection could be proven. in conclusion, by means of alert clinical and virological examinat ions, a considerable fract ion of idiopathic p.f.p. could be associated with a virus infection. 152 neonates were screened from the delivery to the time of discharge for rotavirus infections. daily faecal specimens were examined by an enzyme-linked immunosorbent assay (elisa) and a subgroup of positive specimens were also tested by a negative staining electromicro scopic method. -22 babies (14.5 0/0) were found to excrete rotavirus. 20 of them were asymptomatic infected and 2 showed mild gastrointestinal symptoms. with one exception viruses were not detected in babies less than 24 h old, but 10.9 % of them excreted virus during the second day of life and after 10 days 30 % of the neonates were positive for rotavirus. -excretion persisted for 1 to 5 days. according to our study most babies (40 0 /0) excreted roravirus for 3 days. -a great number of the stools (29%) from the first da y which were tested by elisa were found to have nonspecific activity in the absence of rotaviral antigen. theref ore such stool specimens should only be examined by electron microscopy. the single radial hemolysis test (srh-test = hemolysis-in-gel test) is a suitable technique for detection of rubella and influenza antibodies in a large number of sera. in our stud y we looked for the effect of att achment of the viral antigens to the erythrocytes by different coupling reagent s. chrom ic chloride, cyanogen chloride, glutaraldehyd and tetraazotized -0 -dianisid ine (tod) were used for the sensitization of the erythrocytes. -in the rubella srh-test no improvement on regard to sensitivity of the test was seen. moreover it is not possible to detect igm specific antibodies after different kinds of attachment in the srh-test. -in the influenza srh-test with allantoic fluid of eggs infected with h 3n2 virus it was possible to increase the sensitivity with tod, chromic chloride and potassium periodate. if tween-ether treated hemagglutinin was used only after sensitization with tod, chromic chloride and periodate hemolytic zones were detectable . univ.-kinderkiinik, mathildenstr. 1, d-7800 freiburg rna-electropherotyping of human rotaviruses 1978 . and pastor, s. rotaviruses contain a double-stranded ribonucleic acid genome consisting of 11 segments. this can easily be extracted from crude stool suspension (method by rodger et al., j. clin. microbial. 1981) . 55 out of 210 rota antigen positive samples contained sufficient rna to produce a satisfying pattern in the sds-acrylamide-electrophoresis. we demonstrate six electropherotypes with differences in the relative mobility of segments 2, 3, 4, 5, 7, 8, 9, 11 . of these diarrhea producing strains at least two different electropherotypes were found during every outbreak of rotavirus gastroenteritis. our findings are in good agreement with the results reported by rodger ), espejo et al. (1980 ), and kalica et al. (1978 , 1976 . the so-called m-type of emc virus is capable of inducing diabetes mellitus in mice by a selective b cell damage. the m-emc strain used in our laboratory has partially lost this capacity. we attempted to reisolate a diabetogenic variant and to elucidate the causes of the change. seven serial heart passages in mice did not enrich such a variant. cloning of the virus stock yielded one clone diabetogenic in two of ten animals (5 clones tested so far), -in a different substrain of m-ecm virus (the highly diabetogenic dvariant, obtained from dr. petersen) we found both diabetogenic and non-diabetogenic clones. incidence and severity of diabetes has been shown to be age-related. t his safety study was to demonstrate whether or not granulosis virus (gv) of laspeyresia pomonella can rep licate in vertebrates. after feeding gv to nmri-mice, no virus induced antibodies could be detected within eighty days by radioimmunoassay (ria) and no vertical virus transmission was observed. gv did not replicate in mice. human sera, as well as sera from horses, cattle, sheep and swine reacted with gv in the ria. by characterization the positive reacting human immunoglobulin c1ass(es), igg showed the strongest positive reaction, less positive reactions were shown by ige and igm and no reaction by iga and igd. by concentrating the immunoglobulins of the negative reacting sera to 250 pg igg/ml, all sera could be recognized as positive. thus a non-immunological reaction has been suggested. infection of human fibroblasts with cytomegalovirus induces typical cytopathic alterations. cell rounding within 5-6 h postinfection (p.i.) is followed by an increase in cell size, appearance of cytoplasmic and nuclear inclusions and a morphological change to an epitheloid cell shape by 48 h p.i. in order to elucidate the participation of the cytoskeleton in this alteration of cell morphology experiments were initiated in serum-starved human fibroblasts to visualize changes in actin arrangement by indirect immunofluorescence. a early as 3 h p.i. cytoplasmic microfilamenrs had shortened and were rearranged to a more irregular pattern. at 12 h p.i. actin fibers were absent from rounded cells. the same was observed in epitheloid cells at 48 h p.i. cultivation of infected cells with phosphonoacetic acid resulted in a partial preservation of the normal actin fiber distribution. in contrast, infected cells did not exhibit major changes in actin synthesis as estimated from the specific radioactivity of cytoplasmic actin isolated from 3h-leucine pulse labelled infected cells by dnaase i-sepharose affinity chromatography and sdspolyacrylamide electrophoresis with fluorography. chicken erythrocytes were coated with glycine-extracted cmv antigen and negative control antigen by treatment with formaldehyde and crcl s and were used for detection of cmv specific serum antibodies in pha tests. their sensitivity was proven to be in good agreement and their specificity superior to results seen in cft. igm-specific cmv antibody detection was performed either after a simple and rapid deae-cellulose exchange chromatography of serum samples or by igg immunoprecipitation combined with me-reduction controls. the main advantage of the modified cmv-phat was seen in the stability of sensitized erythrocytes, which can be lyophilized. lanvers, a., mertens, th., and eggers, h. j. there are reports that herpes simplex virus (hsv) could be isolated from the genitourinary tract of up to 15 ofo of males without manifest herpes. in order to confirm these results we first wanted to establish a method for typing possible hsv isolates. we used 3 published methods: a plaque test in chick embryo cells, a neutralisation test, and the analysis of the early hsv-proteins (sds-page). all 3 methods yielded identical results. we then tried to isolate hsv from 192 materials of 181 asymptomatic males (89 urethral swabs, 55 seminal fluids, 48 fresh tissue probes). all materials were immediately inoculated into tube cultures of two cell lines shown to be highly hsv-susceptible. additionally, the tissue probes were co-cultivated with permissive cells. we also induced cell fusion (peg) in such cocultures. despite all efforts we did not isolate any virus from all these materials. for an experimental approach to answer the question as to which viral genes may control pathogenesis after peripheral infection of inbred mice with hsv, we have isolated a variant strain (ang-path) that proved highly neuropathogenic both upon i.p. -or intravaginal infection from the apathogenic strain hsv-1 ang. two alterations of the ang genome have been detected by restriction enzyme analysis: the loss of the amplifiable 500 b.p. nucleotide sequence typical for ang and a 400 b.p, deletion approximately at the position mapped for the structural viral glyco-protein d. despite the induction of interferon and nk-cells both variants multiply to a similar extent, probably in the same target cells of the peritoneum. the spread from the peritoneum, spleen, liver, thymus and local lymph nodes to the ens seems, however, to be controlled by the action of gene products coded for in only one of the variants. div, of expo virology, insr, for med. microbiology, univ., d-6500 mainz knoblich, a., friedrich, d., goertz,] ., and falke, d. herpesviruses are known to cause infections in men and animals. strong differences in resistance to herpes simplex virus (hsv-1) are noted among mice of various strains. anti-hsv-activity of t-iymphocytes, macrophages and nk cells has been demonstrated in the last years. our interest was focused on neutralizing antibodies in primary hsv-1 abstracts of the 38 th meeting of the oghm infections of mice. antibodies become detectable by day 5 after infection and reach a plateau level at day 21, the day we chose to test the sera. comparison of antibody titers in 18 strains of mice revealed titers always to be higher in female mice, whereas no clear influence of either h-2-haplotype or background genome could be detected. sexual steroids produced in ovaries and testes were identified to exert influence on antibody formation by castration experiments. treatment of mice with silica once between day 1 before and day 12 after infection resulted in a strong increase of antibody titers both in females and males at the same time abolishing the difference in antibody titers between the sexes. silica could enhance antibody levels also after immunisation of mice with a formol-inactivated hsv-vaccine. bestatin is a small peptide known-selectively to stimulate dna metabolism in t-lymphocytes and to enhance hsv-antibody -titers maximally when given at day 5 after infection . after pretreatment of mice with silica the antibody augmenting effect is already achieved at day 1 after infection . in secondary hsv-l infection bestatin acts best 1 day after infection, too. insr. f. med. virologie d. univ., 0-6900 heidelberg induction and characterization of herpes simplex virus reisolates, isolated after intertypic superinfection of latent infected tupaias darai, g. and scholz, j. the susceptibility of juvenile and adult tupaias to herpes simplex virus type 1 and 2 had been reported. the intertypic recombination of herpes simplex virus using temperature-sensitive mutants of hsv-l and 2 and superinfection with wild-type hsv-l and 2 was studied. it was found that animals survived an infection of 1 x 10 7 to 1 x l()8 pfu of rs mutants of hsv-l and /or 2 which were inoculated intravenously (l0 50 for hsv-l = 10-3 • 75 and for hsv-2 = 10-2 • 25 ) . the inoculated animals were protected against a superinfection of hsv-l or 2 (5.0 x 10 6 pfu/animals). the state of viral latency in surviving animals was investigated. it was found that infectious virus was recovered from ganglia of those animals which had initially been infected with wild-type hsv-l or 2 and /or superinfected with wild-type viruses. in contrast, the infectious viruses were recovered only from spleens of those animals which had initially been infected with ts mutants of hsv-l or 2 and superinfected with wild-type hsv-2 and /or 1. it was found that recovered viruses from the spleen of the animals lost their pathogenicity and their natural tissue tropism . significant changes in the genome of the recovered viruses from the spleen were detected. recombination between ts mutants of hsv-l and 2 and challenged wild-type viruses was observed. thus, the pathogenicity and genomic properties of recovered viruses were altered . this observation is the first description of generation of inrerrypic recombinants of hsv-l and 2 in in vivo. oiv. of expo virology, inst. for med. microbiology, univ., 0-6500 mainz the functions of the hsv-coded dpyk-complex enzyme labenz, j., brauer, d., moller, w. e. g., and falke, d. analysing the phosphorylating capacity of the hsv-coded dpyk of hsv-l and 2 by glycerol gradient centrifugation we detected each three peaks differing in molecular weights using atp, aop or amp as phosphate donors. also by page peaks with differ-ing rj-values could be seen. indeed, 32p_amp and 32p_adp were used for phosphorylation of dthd. the amp-dependent activiry could be purified boo-fold. two antisera against hsv-coded pdyk neutralized all three activities. the tk-mutant mdk (b2006) did not induce in'i'kr cells the respective activities, only the 3 cellular tk's were detected and identified by their rj-values. further experiments have shown that only the hsv-l-dpyk has thymidylate kinase activity, but not that of hsv-2. the hsv-l thymidylate kinase activity could be neutralized by a tk-antiserum. -the ph-optimum, sensitivity to mg'", fe'", zn'", co" and mri'" ions differed. the susceptibility to thiol reagents was different, the amp-dependent activity proved to be susceptible to phenanthroline. also the k m and vrnax-values were detected. a diagram summarizes the biochemical function of the hsv-coded dpyk-complex. finally there is some indication that the enzyme phosphorylates ado by using dtmp or atp as a phosphate donor. the importance of the enzyme complex in hsv-infected cells is discussed. dkfz, inst. f. virusforschung, 1m neuenheimer feld 280, deletion of n uc1eotide-sequences in cloned hsv-1 fragments during propagation in the rec a e. coli, hb 101 gray, c. p., jellinghaus, u., and kaerner, h. c. passage of hsv at high mol results in the generation of defective genomes which are of full length, consisting of a relatively short region of the wild type genome that is repeated. they are packaged into mature virus particles and thus leave the cell in a state that is capable of entering a new host. such defective molecules are of interest as they represent a simpler model in which to study replication, recombination and packaging of hsv. -such a defective molecule arising from the serial passage of hsvtype l-ang has been mapped, and restriction fragments have been cloned into pbr 322 using the rec a-e.coh, hb 101, as host. -all the resulting clones were found to be unstable and to contain deletions, one of which has been characterised in more detail. the isolation and characterization of tupaia herpesviruses (thy 1 to 4) has been reported. the analysis of dna of these viruses showed the absence of submolar dna fragments, when the dna was cleaved with different restriction enzymes, as well as of a stem loop structure, when analysed by electron microscopy. with respect to this genomic structure it was of interest to study he recombination events between thv 1 to 4. recombinants were generated between these viruses using a co-infection technique in vitro on tupaia fibroblasts. recombinants were selected after the stocks of new progency virus were treated with specific antiserum against each parental virus. different recombinants were isolated, plaque-purified and characterized. results for one recombinant thv-r-26 between thv-2 and 3 were as follows: (i) the in vitro host range and the in vivo pathogenicity in juvenile tupaia was altered compared to parental viruses. phosphorylation of proteins is a posttranslational modification which is regulatory for the activity of several enzymes. most animal viruses code for phosphoproreins and the degree of their phosphorylation is thought to be a controlling factor during viral macromolecular synthesis. recent studies on protein kinases from a number of tumor viruses have raised the possibility that the phosphorylation of cell proteins is involved in the processes leading to cell transformation. -incubation of purified tree shrew (tupaia) herpesvirus (thv) particles with y.32p atp resulted in the incorporation of 32p labelled phosphate into proteins. a nonionic detergent such as np-40 was necessary for the detection of protein kinase activity. the incorporation of 32phosphate was proportional to the quantity of th viral proteins, indicating that the viral proteins can serve as substrates for the viral enzyme. a_ 32p datp or a_ 32p atp did not function as phosphate donors. a divalent cation such as mg2+ or mn'" is essential for the enzymatic activity. a product analysis revealed that six viral polypeptides are phosphorylated. the predominant sites of phosphorylation are the (f-oh groups of serine and threonine. in 1972 a herpesvirus was isolated from young goats with a severe generalized infection in california. this isolate has been characterized in some detail and named caprine herpesvirus 1. recently a herpesvirus was isolated in switzerland from goats with a similar infection. we report a further characterization of both isolates and propose their classification as bovid herpesvirus 6 (bhv-6). -bhv-6 multiplies rapidly and shows a broad hostcell range. crossneutralization onl y could be observed with bhv-1 (ibr/ipv-virus), in a one way reaction. in gel immunoelectrophoresis the serologic relationship envolved the major antigenic components of bhv-1. we have evaluated two methods for the analysis of antibodies directed against ebvspecified proteins: 1. indirect immunoprecipitation (ip) and 2. radioimmunoassay of electroblots of sds page separated ebv-specified proteins (riab). using ip we have identified 20 proteins against which antibodies are made during infection, some of these proteins have also been found using the latter technique. many of the proteins are only reactive with ea'vca+ sera and not ractive with ea-vca + sera and may therefore be candidates for the ea specifying proteins. it seems likely that the failure to identify all proteins using the riab technique is probably due to the strong denaturating conditions used during the sds page step. only those antibodies directed towards the primary sequence of the protein will react with the blotted proteins, whereas with ip analysis the native proteins are available for interaction with the sera. previousl y we demonstrated that the synthesis of ebv-induced proteins in superinfected raji cells (raji 51) could be divided into 3 phases: primary, secondary and tertiary (bay-liss and wolf, j. gen. virol., 1981, in press) . recent experiments show that incubation of raji si in the presence of canavanine and the absence of arginine allows only a limited expression of the viral genome. if the cells are released from a canavanine block (applied from 0 to 8 h post infection) then between 2 and 4 h later several new proteins are synthesized, however, if m-rna synthesis is inhibited (with actinomycin d) after removal of the canavanine then these new proteins fail to appear. amongst these proteins are members of the ebv-specified early antigen complex (ea). these results indicate that an arginine-containing protein is synthesized soon after infection and that this protein is required in an active form in order to synthesize m-rna for the secondary group of proteins. amongst these proteins are the major components of the early antigen (ea) complex. max von pettenkofer inst., univ., d-8000 miinchen 2; ent-klinik, univ., d-8700 wiirzburg; dept. tumor virology, chinese acad. med. sci., beijing, china seibl, r., richter, w., zeng, y., gu,s.-y., and wolf, h. antibody titers of iga antivirus capsid antigen (vca) can be used for screening early tumor cases, confirming histological diagnoses and longtime surveillance of therapy. however, in high risk areas (s. china, incidence of npc: 20/10 5 ) 2 % of the population have iga anti vca antibody indicating a need for methods which allow monitoring of additional parameters for deciding on the need for therapy. the same need exists in case of long term (1-2 years) survivors of npc with constant iga anti vca titers. -we have developed a system to collect cell specimens by application of buffer to the tumor site and collection of cells directly on to nitrocellulose filters. these cells can be examined cytologically or for ebv dna in nucleic acid hybridization. for the latter tests a modification of the grunstein hogness colony hybridization test and cloned ebv dna have been used. in reconstruction experiments 25 virus-producing cells could be detected. inst. f. klin. virologie, univ., d-8520 erlangen-niimberg keil, g. and fleckenstein, b. after infection of permissive cell cultures with overlapping restriction-fragments of viral dna derived from different strains of herpesvirus saimiri (h. saimiri) a number of recombinants could be isolated. the analysis of the viral proteins of the wt-strains 11, omi and s295c led to the identification of four proteins that differed within these strains with respect to molecular weight. we are able to localize these proteins in the internal region of the m-dna by comparing the protein patterns of recombinant and parent strains. the exact localization was so far not possible because the recombination events occured mainly in regions near the ends of the l-dna. -furthermore recombinants were constructed to identify the genomic region responsible for oncogenicity. the wt-strains of h. saimiri are oncogenic while attenuated strain 11-att, that was obtained from strain 11 has lost this property. this may be due to a deletion of 1,1 md at the left end of the l-dna. recombinants between this attenuated strain and wt-strains were constructed. the test for oncogenicity in vivo is in progress. the t-iymphoid cells can contain up to 300 genome copies per cell as episomes. we have begun to study translation and transcription in transformed cells in comparison to lytically infected omk-cells. about 25 new proteins can be detected in lyrically infected cells having apparent molecular weights between 12-200 kd. -at early and late stages of infection the right part of the genome is preferentially transcribed and each dna-fragment encodes a series of specific rnas. -in contrast, we do not find any virus-specific proteins in the transformed cells after labeling with 3ss-methionine and subsequent immunoprecipitation. preliminary results may suggest that the only virus-specific rna found in the transformed cells are small rnas with molecular weights of about 0.13 kb. -a more detailed analysis has to be performed in order to confirm these hybridization data. max von pettenkofer inst., d-8000 mi.inchen 2 characterization of herpesvirus saimiri glycoproteins modrow, s. and wolf, h. for the identification of herpesvirus saimiri glycoproteins we seperated h. saimiri 11 induced cell proteins on sds-polyacrylamide gels and transfered the polypeptides by elec-trophoretic blotting (2 h, 3,7 ma/cm 2 ) to nitrocellulose paper using carbon electrodes and buffer soaked sponge to cover the gel/filter layer. lectins, which are known to bind very specifically to certain sugar residues (concanavalin a to d-glucose and d-mannose derivates, soy bean agglutinin to n-acetyl-d-galactosamine and d-galactose, dolichos biflorus agglutinin to n-acetyl-d-galactosamine, ulex europaeus agglutinin to l-fucose) were iodinated using the nen-iactoperoxidase system. the glycoproteins bound to the nitrocellulose sheets were detected by incubation with the 1z5j-labelled lectins. by this, eight viral glycoproteins could be identified, two of them were synthesized in the presence of canavanine, and characterized according to the type of glycosylation. lymphocystis disease (ld) is a virus disease of marine fish with an almost world-wide geographical distribution. this disease is characterised by papilloma-like tumour lesions. lymphocystis disease virus (fdlv) has been tentatively classified as belonging to the family of iridoviridae. this report describes the anatomy of fdlv: the fdlv virions were isolated from a total of 22 fish with ld lesions, caught near the doggerbank, including 12 flounders, 6 dabs, and 4 plaice, which were analysed individually. the purity of the virus preparations was examined by a negative-staining technique and the virion diameters were determined: flounder 227.5 ± 12.5 nm, ldv-plaice 198.8 ± 12.9 nm, and ldv-dab 200.5 ± 12 nm. dnas of these different ldv isolates were cleaved with different restriction endonucleases and the resulting dna fragments were separated electrophoretically on agarose slab gels. the fragment patterns demonstrate that ldv dna of flonders and of plaice are indistinguishable, but clearly different from those of dab. the determination of the molecular weights of fdlv dnas using contour length measurements by electron microscopy resulted in a value ranging from 60 to 150 x 10 6 daltons. in contrast the molecular weight estimations by restriction enzyme analysis resulted in lower values ranging from 60 to 90 x 10 6 daltons. this discrepancy is probably due to a restricted, permutated structure of the ldv genome similar to the genome of frog virus 3 as reported by granoff. the structure of fldv constituents and its interaction with host cells remains obscure. in an effort to clarify the viral components and their functions we have studied the proteins and purified fldv and searched for virion-associated enzymes. -at least 33 distinct viral polypeptides were detected by polyacrylamide gel electrophoresis under denaturing conditions. the polypeptide patterns are remarkably specific for a given fish species (flounder, plaice, and dab) although some heterogeneity was found when proteins of individual fish of the same species were analysed. -it was found that a nucleoside triphosphate phosphohydrolase activity is closely associated with fldv particles. this activity hydrolyses atp with a high preference. the reaction requires a non-ionic detergent and a divalent cation, such as mg2+. reaction rates and substrate specifities were determined. the products of the reaction are nucleosides disphophares and inorganic phosphate. characterization of a poxvirus isolated from white rhinoceros (ceratotherium s. simum) pilaski,] ., schaller, k., olberding, p., and finke, hannelore in september 1977 an outbreak of poxdisease occurred in white rhinoceroses (ceratotherium s. simum) in the munster zoo. at the same time a similar outbreak was observed in elephants (elephas maximus, loxodonta africana) of the zoo in frankfurt (airline distance about 230 km). in both cases orthopoxvirus strains could be isolated which were similar but not identical in their biological properties (small efflorescences on the cam with hemorrhagic center, inclusion bodies of type a v+, high pathogenicity for rabbit skin, characteristic skin lesions in adult mice) and their dna restriction patterns (xho i, eco r i, hin d iii). both virus strains were incorporated into the group of "cowpoxlike viruses" (baxby and ghaboosi, 1977) . the results indicate that both outbreaks had occurred independently from each other. characterization of a 37k protein specifically associated with released extracellular vaccinia particles hiller g. and aulbach, h. infectious vaccinia virus can be isolated from infected cells after experimentally induced lysis (intracellular virus) or from the growth medium of infected cultures (extracellular virus). we have characterized a 37k protein only present on extracellular particles by its amino acid composition and its behaviour on isoelectric focusing. in addition we have used a 37k-specific antiserum to detect its distribution within infected cells. -37k protein is a late viral protein appearing 5-6 h p.i. it is predominantly found associated with the cellular golgi complex but never with structures representing pox virus "factories". later in infection 37k protein is incorporated into single viral particles preferentially found in the cell periphery. upon electron microscopy approximately 3040 % of morphologically mature virions inside the cell are enwrapped by a double-membranate vesicular structure. thus 37k viral protein is probably a component of this vesicular structure and only vesiculated virions can be released by the cell before lysis occurs. epidemiology of influenza in lower saxony willers, h. and hopken, w. the influenza surveillance in lower saxony is mainly based on laboratory investigations especially on the attempts to isolate influenza viruses throughout the year. -in the winter 1977-78 and in the winter 1980-81 the two influenza subtypes h3n2 and hini circulated at the same time. the h3n2 subtype affected during the last years persons of all age-groups whereas the hini subtype affected only persons younger than 30 years. -in the winter 1978-79 influenza b was found in an epidemiological extent. the disease caused outbreaks mostly in schools and kindergartens but also affected adults. -in 1980-81 from mid-january to mid-february scattered outbreaks were caused by the a subtype hinl, which particularly affected schoolchildren contrarily to 1978 where the hini subtype mostly infected young adults. influenza of the h3n2 subtype circulated from january to march. inst. f. med. mikrobiologie. abr, virologie d. tu, biedersteiner str. 29, antibodies against influenza c virus in the population of germany, kenia and australia pfeil-putzien, c. and meier-ewert, h. over one hundred human sera from each of the three countries germany, kenia and australia were tested for antibodies against influenza c virus, using the conventional hemagglutination inhibition test (hi). the rate of positive sera, showing a hi titer 8 amounted to 59 ofo for germany, 93 ofo for kenia and 96 ofo for australia. in the age group up to 5 years, 34 ofo of german and 94 ofo of kenian sera had already antibody titers against influenza c virus. the australian sera were tested for the age group of 16-25 years and showed 95 ofo seropositivity. the results show that influenza c virus is circulating to a higher extend in the populations of countries with subtropical climate, as compared to the more temperate middle european zones. inst, f. virologie, justus-liebig-univ., d-6300 giessen the proteolytic activation of influenza hemagglutinin, structure of the cleavage site and the mechanism of cleavage garten, w. and bosch, f. x. the hemagglutinin precursor ha is posttranslationally cleaved by proteases to the complex h i , 2' in vitro cleavage of ha by trypsin and trysin-like proteases yields infectious virus. cleavage by thermo lysin or chymotrypsin yields non-infectious virus. we have analyzed the cleavage sites of hi, h3 and hlo hemagglutinins. the amino acid sequences at the cleavage site, i. e. c-terminus of hal and the n-terminus of ha 2 are identical when virus is activated in vivo and in vitro. under both conditions, an arginine residue connecting hal and ha 2 in the precursor is eliminated. the elimination of argmme results in a shift of the isoelectric point of the hemagglutinin as demonstrated by isoelectric focusing. non-activating enzymes cleave only one peptide bond in the ha 2-nterminal region of activated ha and thus do not affect the isoelectric point. we have also analyzed the cleavage site of the hemagglutinin of fowl plaque virus (h7). a connecting peptide containing several basic amino acids is eliminated. -the data show that activation of the influenza hemagglutinin involves the action of a cellular protease with trypsin-like specificity followed by the action of an exopeptidase of the carboxy-peptidase b-type. the latter enzyme activity is associated with purified virus and can be analyzed by an assay employing peptides bearing 3h-arginine at the c-terminus. the carbohydrates of the glycoproteins of 21 influenza a strains containing hemagglutinin and neuraminidase of all serotypes known to date have been compared by analysis of glycopeptides labeled with radioactive sugars. analysis of incompletely glycosylated glycoproteins synthesized in the presence of glycosylation inhibitors allowed the determination of the number of oligosaccharide side chains on ha 2 • with all strains, the neuraminidase contains side chains of both the complex type i and the mannose-rich type ii. there are distinct quantitative and qualitative differences between the strains in the distribution of type i and type ii side chains on the hemagglutinin fragments hal and ha 2 • the majority of the hemagglutinin oligosaccharides is located on hal' these side chains are usually of type 1. only the hem agglutinins of serotype h3 have, in addition, a substantial amount of type ii side chains on hal' most strains have on ha 2 a single side chain which is usually of type i. with serotype hs this side chain is free of fucose, and with serotype h8 it appears to be missing completely. serotypes h7 and h10 have, in addition to the type i, a type ii side chain on ha 2 • these observations strengthen the concept that the primary structure of the polypeptide chain is an important determinant for the carbohydrate moiety of the hemagglutinin. inst. f. virologie, ]ustus-liebig-univ., d-6300 giessen acylation of viral glycoproteins schmidt, m. f. g. covalent binding of fatty acids to viral glycoproteins was first detected with sindbis virus and vesicular stomatitis virus (1, 2) . studies on acylation in other enveloped viruses revealed that covalent addition of fatty acid to spike glycoproteins is a more general feature. while in sindbis and in semliki forest virus both species of spike glycoprotein (el and e2) carry fatty acid chains, acylation with the other viruses studied (corona-, influenza a-and paramyxovirus family) seems restricted to those glycoproteins that are known to carry fusion activity. this new type of modification of viral glycoproteins occurs in a wide variety of host cells including those of human, bovine, mouse, hamster, avian and insect origin (3) . -with the aid of controlled digestion of 3h-palmitic acid labelled virus particles and by the analysis of cyanogen bromide fragments of fatty acid labelled glycoprotein the fatty acid bind ing site in influenza hemagglutinin (hat), vsv g-protein and sindbis virus e1 and e2 could be located to the membrane spanning portion of the respective proteins (3). heinrich-pette-institut f. exp. virologie u. immunologie an d. univ., martinistr. 52, d-2000 hamburg 20 mannweiler, k., bohn, w., rutter, g., and hohenberg, h. in replica-immunocytochemical (ric) -and ultrathin section (us) preparations the ultrastructures of the specific alterations and of virus antigens, which appear at the plasma membrane of he la cell coverslip cultures after infection with an adapted measles virus strain were investigated. as immunomarker protein a-coated gold particles were used (1) . this method is sufficiently sensitive to enable labeling of even small altered areas of the plasma membrane (70-100 nm cb). due to the high atomic number contrast in the tem and the small size of the marker (-<:: 10 nm) the ultrastructure of characteristic alterations morphologically still remains visible with ease in a three-dimensional aspect. data obtained by ric and us preparations after labeling with antimeasles immune serum or with monoclonal antibodies against ha (2) are demonstrated, compared and discussed. bohn, w., rutter, g., and mannweiler, k. by use of the mouse hybridoma technique, monoclonal antibodies were obtained with specificity for the ha (79k), p (72k) and m (36k) polypeptides of measles virus. balblc mice were immunized with native measles virus and measles virus treated with detergents and heat. clones obtained after immunization of mice with native measles virus showed specificity for the ha polypeptide only. after immunization with measles virus, treated with 1 0/0 sodium sarkosyl sulfate (sss) at 20°c a clone was obtained producing antibodies to the m polypeptide. heating of measles virus in the presence of 1 0/0 sds under reducing conditions elicited a selective immune response to the p and np polypeptides. thus, clones producing antibodies to the p polypeptides were isolated. contains a 50s rna, but it does not show any infectivity even after trypsin treatment. an activation of the 6/94 cl virus can be obtained by i) cocultivation of the cl-e-8 cells with standard cells (e. g. bhk-21) and ii) serial passaging of the 6/94 cl virus in several cell lines (e. g. bsc-1). infectious 6/94 cl virus can be detected in the cell supernatant after a period of 5-10 days and 25-30 days respectively. this virus cannot be propagated in chicken eggs, but it can replicate in serial cell cultures without trypsin treatment; moreover trypsin treatment does not influence the viral replicaiton. in comparison 6/94 virus released from an in-vitro generated persistent infection (bsc-1 cells infected with egggrown 6/94 standard virus) can be propagated in chicken eggs. this virus termed 6/94 pi virus also can grow on serial cell passages without trypsin treatment, whereas the serial replication of 6/94 standard virus on cell cultures depends on the trypsin-activation. inst, f. virologie u. immunbiologie, versbacher str. 7, d-8700 wiirzburg monzel, p. and koschel, k. the paramyxvviruses measles (sspe) virus and canine distemper virus (cdv) cause an impairment of the catecholamine induced p-adrenergic receptor dependent c-amp generation in persistently infected c6 rat glioma cells. in cdv persistently infected c6 cells the number of receptors is greatly reduced. hirata and axelrod have shown that the number of ii-adrenergic receptors could be regulated by methylation of phosphatidyl ethanolamine (pe) resulting in lecithin synthesis (1) . we have therefore studied the methylation of pe in persistently infected cells by the incorporation of (3h) methyl groups from (3h-methyl)-methionine into pe. in both infected systems, c6/sspe and c6/cdv, we observed a total loss of catecholamine stimulated p-adrenergic receptor dependent methylation whereas the p-receptor independent methylation of phospholipids is unchanged. inst. f. med. virologie d. univ., d-6900 heidelberg; t robert-koch-institut, d-1000 berlin 65; 2 inst. f. virusforschung, dkfz, d-6900 heidelberg kurz, w., gelderblom, h.t, flogel, r. m.2, and darai, g. a paramyxovirus was isolated from a kidney biopsy of a tupaia (three shrew) and termed (tpv). the detailed host range study revealed that only tupaia embryonic fibroblasts and tupaia kidney cells are the cells of choice for the efficient propagation of tpv. tpv can be plaque-assayed on tupaia embryonic fibroblasts and this cell line was used for the continued propagation of tpv. electron microscopy of purified tpv revealed the presence of typical paramyxovirus particles. -the hemagglutination test was performed with erythrocytes with a variety of different species. it was found that guinea pig erythrocytes were agglutinated with tpv. the buoyant density of purified virions was determined in sucrose gradient and found to be 1.19 g x ml", -the biological chara cterization of tpv which was perfo rmed by host range study in vivo revealed that tpv is highly pathogenic for new born mice and hamsters. -the characterization of viral rna and proteins of th is new member of paramyxo viridae is now in progress. coronaviruses contain two glycoprotein species £2 (180 k) and £1 (23 k) which are both synthesized in the r£r of the infected host cell. glycosylation of £2 is initiated at the cotranslational level and it can be inhibited by 2-deoxyglucose and tunicamycin indicating the presence of n-glycosidic carbohydra te protein linkages. particles formed in the presen ce of these inh ibit ors are non infectious and lack detect able amounts of £2. -cell fractionation experiments show that glycosylation of glycoprotein £1 occur s posttranslationally in smooth memb ranes. the carb ohydrate protein linkages in £1 are susceptible to mild alkaline reductive conditions and n-acetylgalactosamine was determined to be the reduc ing sugar of the released oligo saccharides . this together with the finding that glycosylation of £1 is not sensitive to inhibito rs of n-glycosylation suggests that glycoprotein in £1 of coronaviruses is the first structural virus glycoprotein containing o-glycosidic side chains exclusively. insr, £. virologie, jusrus-liebig-univ., d-6300 giessen target cells of infectious bursal disease virus (ibdv) of chickens moller, h. and becht, h. infectious bursal disease virus (ibdv), the causative agent of a highl y contagious disease of young chickens result ing in severe necrotic lesions in the bur sa of fabricius (gumboro disease), is a non-enveloped icosahedral particle with a diameter of about 60 nm. its genome con sists of 2 segments of double-stranded rna with mol ecular weight s of 2.2 x 10· and 2.5 x 10· daltons. th e virion is composed of 5 structural pol ypeptides with mole cular weights of 90 kd, 48 kd. 40 kd, 32 kd and 28 kd. the 40 kd pol ypeptide, one of the two main structural proteins, is derived from the 48 kd pol ypeptide, perh aps by proteolytic modification (1) . with immunofluorescence and "i nfectious center assays" we were able to show th at 1. after infection of isolated lymphoid cells in vitro only 20 ofo of bur sa cells, 2 % of thymus cells and 5 ofo of spleen cells produce infective virus (i, e. plaques in infectious center assays) alth ough the donor chickens were in the most susceptible age of 4 to 5 weeks. 2. the number of virus producing cells is not cor related with the appe arance of slgm or sigg. 3. virus yields seem to be influenced by the cell cycle: the number of chick emb ryo fibro blasts producing plaques in "infectious center assays" is increased after synchro nisa tion of the cells before infection. 4. cells that produce infective virus show an increased uptake of 3h-thymidine. 5. isolated lymphoid cells from thymus or spleen as well as blood lymphocytes can be stimulated by mitogens to produce higher virus yields. infection of borna virus without any influence of the immunoresponse. the antigens first appeared in the nucleus of neurons and sometimes fibroblasts, then filled the cytoplasm, most brilliantly 7 days post infection. thereafter they disappeared from the cytoplasm, but remained persistently only in the nucleus in point shape. no morphological changes were seen in the infected cells during 60 days post infection. we can say that the virus does not kill the cell. for the destruction of nerve cells in in vivo conditions it can be pointed to the importance of the immunological events, which might cause the clinical pictures. abr, molekularbiologie, physiol.-chem. lnst., grindelallee 117, d-2000 hamburg 13; heinrich pette-inst., martinistr. 52, d-2000 hamburg 20 large scale production of biologically active vsv in eat-cells mack, d., kruppa, j., and breindl, m.i ehrlich ascites tumor cells maintained in mice were used to prepare milligram quantities of biologically active vsv. at the 6th day after passage when the cell number reached approx. 7 x 10 8 cells/mouse, mice were infected by intraperitoneal injection of appropriate concentrations of vsv. ascites fluid was harvested after 20 h. virus production was exponential for at least 16 h and continued for at least 20 h p.i. -approximately 3-4 mg of viral protein/mouse and 2 x 1011 pfulmouse were routinely obtained. the specific infectivity of vsv isolated from eat-cells reached nearly 1.5 x 10 8 pfui,ug protein. the endogenous transcriptase activity of vsv produced in eat, bhk, and hela cells showed no significant differences. large amounts of biologically active vsv may be produced rapidly and much less costly with the described procedure than using tissue culture cells. it should be possible to adapt the procedure for the production of other viruses. lonza a.g., ch-4000 basel, schweiz the quantitative determination of bardac-22, formaldehyde, glyoxal and glutardialdehyde in disinfectants weinreis, p. and goller, s. several institutes of hygiene have been provided with two disinfected formulations, a and b, with the intention to analyse these mixtures, containing different amounts of quaternary ammonium compounds, formaldehyde, glyoxal and glutardialdehyde, -the quantitative analysis of the components of the two mixtures depends on well known volumetric and spectrophotometric methods. -the results of this analysis have shown that it is possible to use the described procedure for routine check ups of disinfectants without using microbiological tests. proc . nat. acad. sci virolog y (1982) previous studies have shown, that mouse-neurovirulent recombinants can be obtained from mixtures of avirulent influenza a-viruses provided one of the parents had previously been adapted to that host. this study shows that prior adaptation of parental strains is not necessary and that generation of neurovirulent recombinants is frequent. the gene constellation for neurovirulence was predictible for recombinants derived from a particular pair of parental strains in influenza c virus -infected cells a virus-specific protein of a molecular weight of 65,000 dalton can be detected when glycosylation is inhibited by tunicamycin. since this protein can not be found in untreated control cells, it probably constitutes the unglycosylated precursor of the viral glycoprotein gp 88 since the petide pattern is identical for the doublet bands, it remains to be established whether this reflects a differential glycosylation or dissimilar proteolytic cleavage sites. the coding capacity of the viral genome rna-segment no abstracts of the 38th meeting of the oghm 1 augenklinik d. justus-liebig-univ., 0-6300 giessen; 2 inst. f. neuropathologie u. virologie, freie univ., 0-1000 berlin retino-cerebral manifestation of experimental borna disease in rhesus monkeys krey, h.t, roggendorf, w.2, and ludwig, h.3 inflammation of the uveal tract, the retina meninges and brain represent uveo-meningoencephalitic syndromes of unknown origin. one of these, the vogt-koyanagi-harada syndrome is primarily manifested by inflammation of the retinal pigment epithelium, the uveal tract and meninges. severe visual and neurological impairment can occur. in our experimental studies 14 rhesus monkeys were experimentally infected with borna disease virus. after a 4-7 week incubation period a progressive retino-cerebral syndrome was observed. focal inflammatory lesions in the retinal pigmentepithelium and the uveal tract were accompanied by encephalitic and meningeal infiltrates. infectious virus could be demonstrated in the retina and in the brain. experimental borna disease can serve as an appropriate model for uveo-meningo-encephalitic syndromes in men. borna disease (bo) virus induced encephalitis in horses, sheep, rodents and primates shows similarities in many aspects with other so-called slow virus diseases. the eeg has shown to be a specific tool in studying encephalitides of different types in man. this is a report on the eeg of the bo virus specific encephalitis. twenty-eight rabbits inoculated by different routes and with different virus preparations were screened for eeg changes. the basic frequency was measured optically and an analysis of the eeg was performed. the following conclusions were made: 1. a significant slowing down of the basic frequency was observed in the eegs of bo virus infected rabbits. 2. spikes and spike-waves were present rather regularly and correlated with epileptic seizures at the end of the disease when they appeared rhythmically in intervalls of twenty seconds. rademakercomplexes appeared from the third week on. virological and serological data collected from all animals demonstrated a strong correlation of bo virus specific reactions with the eeg alterations. the patterns of eeg changes are reminiscent of those in sspe. eeg features in this kind of slow virus diseases may have rather similar characteristics which could suggest that they may underlie common pathophysiological mechanism. we established the primary cultures of neural retina and pigment epithelium of rabbit and of the brain of chicken embryo to study the sensitivity of each kind of cells to the key: cord-021626-ck2kybtp authors: walker-smith, john title: dietary protein intolerance date: 2013-10-21 journal: diseases of the small intestine in childhood doi: 10.1016/b978-0-407-01320-9.50011-x sha: doc_id: 21626 cord_uid: ck2kybtp this chapter discusses dietary protein intolerance. clinical food intolerance has many causes and many manifestations, including psychological aversion to the sight, smell, or taste of food as well as psychological intolerance to one or more of the many constituents of food. dietary protein intolerance is the clinical syndrome resulting from the sensitization of an individual to one or more proteins that have been absorbed via a permeable mucosa in the small intestine. intolerance to various food proteins, especially to cows' milk, has been recognized in children for many years. such food intolerance may be the result of a variety of causes—for example, a congenital digestive enzyme defect such as sucrase–isomaltase deficiency or an acquired lactase deficiency secondary to small-intestinal mucosal damage, which in turn can be the result of a food allergy. the incidence of gastrointestinal food allergy diseases is greatest in the first few months and years of an infant's life and decreases with age. the acute syndrome is usually characterized by the sudden onset of vomiting, after cows' milk ingestion, occasionally followed by pallor and a shock-like state; however, acute anaphylaxis is rare. acute abdominal pain seems to be a particular feature of fish hypersensitivity, while peanuts often produce immediate reactions in the oral mucosa as well as abdominal pain. clinical food 'intolerance' has many causes and many manifestations, including psychological aversion to the sight, smell or taste of food as well as psychological intolerance to one or more of the many constituents of food. intolerance to various food proteins especially the protein of cows' milk, has been recognized in children for many years. such food intolerance may be a result of a variety of causes; for example, congenital digestive enzyme defect such as sucrase-isomaltase deficiency, or acquired lactase deficiency secondary to small intestinal mucosal damage, which in turn can be the result of a food allergy. bleumink (1974) has classified adverse reactions after food ingestion as follows: • toxic effects, including those due to bacterial contamination and food additives. • intolerance phenomena due to enzyme deficiencies, e.g. lactose intolerance as a sequel to lactase deficiency. • allergic reactions. • symptoms resembling allergic reactions but not elicited by immunological phenomena. to this category belong symptoms caused by histamine releasers, e.g. strawberries, where histamine release is not the consequence of an immunological reaction. in recent publications the term 'food idiosyncrasy' has been used in the sense of a non-immunological abnormal response to food. there is, however, increasing evidence that dietary protein intolerance may be mediated via an allergic reaction. in this chapter, the varieties of food protein intolerance in which there is some evidence for such an allergic reaction or reactions affecting the small intestine will be discussed. von pirquet of vienna in 1906 introduced the term allergy. he used it to describe a deviation from the original state or normal behaviour of the individual. his contribution and its relevance to current concepts of immunity and allergy have been very clearly reviewed by turk (1987) . today, when the term allergy is used, it implies a heterogeneous group of conditions which have in common a state of altered reactivity to foreign proteins (antigens) (gell and coombs, 1968) . these antigens are called allergens when they produce symptoms in an allergic person. a child who has an allergy is distinguished from other children by an abnormal response on contact with an allergen or allergens, a response that does not occur when a non-allergenic child is exposed to the same allergen. the typical features of an allergic reaction are: first, the lack of any untoward reaction on the child's first exposure to the allergen; and second, that subsequent exposure to the allergen produces a hypersensitivity reaction. indeed, ferguson (1976) regards the term 'hypersensitivity' as preferable to the term 'allergy' when used to describe tissue damage resulting from the immune reaction to a further dose of antigen occurring in a previously immunized host. gell and coombs (1968) have classified the allergic or hypersensitivity reactions that may produce tissue damage of some kind into four types, as follows. this is initiated by an allergen reacting with mast cells that have been passively sensitized by ige (reaginic) antibody with release of vasoactive agents such as histamine. the reaction occurs within minutes of exposure. in the modern usage of the term atopy, this is the state where an individual is prone to develop antibodies of the igê class. the presence of such antibodies, however, does not necessarily mean that the child is intolerant to the antigen producing the antibody, in a clinical sense. this reaction is initiated by antibody reacting with an antigenic component of a cell or tissue element or one that is intimately associated with these. complement is usually necessary to affect cellular damage. in this type of reaction, antigen and antibody (igg or igm) react in the presence of antigen excess with the subsequent fixation of complement and consequent local inflammatory response. this reaction is maximal a few hours after exposure to antigen. this reaction is mediated by t-lymphocytes and macrophages and manifests by infiltration of lymphocytes and macrophages at the site where antigen is present, due to release of lymphokines. these are soluble factors secreted by lymphocytes on contact with antigen. this reaction takes 1-2 days after antigen exposure. evidence of such an allergic reaction, with tissue damage, in children who show clinical intolerance to dietary protein is not always available. therefore, in clinical practice, the descriptive term 'food protein intolerance' is often used rather than the more precise term 'food allergy'. until more is known about the pathogenesis of food allergy terminological difficulties will continue to arise . the first case report of food allergy (cows' milk allergy) was made by hamburger in 1901 . then finkelstein in 1905 described cows' milk as a cause of acute death in an infant. schloss, in 1911 , related gastrointestinal symptoms to food allergy. he made the diagnosis of egg allergy on the basis of a positive skin test with a protein fractionated from ovomucoid. gastrointestinal food allergy has since come to be recognized as an important cause of gastrointestinal symptoms in infancy. many paediatricians in the past, however, have been sceptical about this diagnosis because of the absence of precise and objective diagnostic criteria, nevertheless most paediatricians now accept the existence of the condition. on the other hand, there are those who undoubtedly have exaggerated its true importance. there is still debate, however, concerning its frequency and importance in different parts of the world. gastrointestinal symptoms may be the only manifestation of clinical intolerance to food protein but there may also be respiratory symptoms, skin reactions and other clinical features. only gastrointestinal effects will be discussed in any detail here. dietary protein intolerance is the clinical syndrome resulting from the sensitization of an individual to one or more dietary proteins that have been absorbed via a permeable small intestinal mucosa. clinically, it appears to be a transient phenomenon of variable duration in children. gastrointestinal food allergies may be defined as clinical syndromes which are characterized by the onset of gastrointestinal symptoms following food ingestion where the underlying mechanism is an immunologically mediated reaction within the gastrointestinal tract. a food-sensitive enteropathy is a disorder characterized by an abnormal small intestinal mucosa whilst having the offending food in the diet; the abnormality is reversed by an elimination diet only to recur once more on challenge with the relevant food. clinical intolerance to a variety of food proteins has been described. the most common are cows' milk, eggs and fish; but intolerance to tomatoes, oranges, bananas, meat, nuts, chocolates and cereals, including soy protein, have been described (bleumink, 1974) . there is no consistent association between a particular food and specific syndromes. in fact the clinical manifestations that may occur in cows' milk protein intolerance are large in number and diverse in nature (table 5 .1) (bahna and heiner, 1980; hill et al, 1986; hutchins and walker-smith, 1982) . chemically, allergens are usually glycoproteins with a molecular weight of between 20 000 and 40 000. broadly, gastrointestinal reactions to food in children with gastrointestinal food allergy may be divided into those that manifest quickly, i.e. within minutes to an iv secondary general effects iron deficiency anaemia hypoproteinaemia thrombocytopenia eosinophilia angioedema hour of food ingestion, and those in which the onset is slow, taking hours or days after food ingestion. both types of reaction may occur individually or together in different children. yet there are clear immunological differences between these groups. for example, it has been shown by fallstrom et al. (1986) that children with slow onset reactions to cows' milk feedings have significantly elevated titres of igg antibodies against both native and digested beta-lactoglobulin, when compared with both controls and those children who develop symptoms quickly after milk ingestion. these children also tended to have higher levels of antibody of ig a class to both native and processed milk. seven out of nine children with quick onset cows' milk allergy had ige antibodies to cows' milk but these did not occur at all in the slow onset group. the incidence of gastrointestinal food allergic diseases is greatest in the first months and years of life and decreases with age. this is especially true for late onset reactions to food with food induced small intestinal mucosal damage (dannaeus and johansson, 1979) . it remains uncertain as to whether gastrointestinal syndromes of allergic origin causing small intestinal mucosal damage exist in adult life. these gastrointestinal food syndromes of early childhood appear to be temporary in duration, although it does seem possible-as in the case of cows' milk protein intolerance-that gastrointestinal syndromes may be replaced with the passage of time by syndromes involving other systems. these syndromes are usually easy to diagnose on historical grounds. levels of foodspecific ige antibodies are typically elevated and skin prick tests are also often positive. children with such problems often present to allergy clinics rather than to gastroenterology clinics. thus diagnosis is usually simple and specific diagnostic tests are available. a good example of this is egg hypersensitivity. the most dramatic deleterious response is acute possibly life threatening anaphylaxis to food. the peculiar attribute some proteins possess, when injected, to diminish instead of to increase the defences of the body against their harmful action is described as anaphylaxis (the reverse of a guard or protection). anaphylaxis was first observed at the beginning of this century. charles richet and paul portier in 1901 on the yacht of prince albert of monaco discovered anaphylaxis by injecting dogs with an extract of the sea anemone la physalie without their becoming ill after the first injection. a second injection lead to acute vomiting and diarrhoea with their rapid death. only 4 years later schlossman in 1905 documented similar symptoms of acute shock not after injection but after ingestion of a foreign protein namely cows' milk in infants. in the same year finkelstein described a death due to cows' milk ingestion in infancy. it is now known that anaphylaxis usually results from a generalized immediate ige mediated reaction following the introduction of sufficient antigen into a previously sensitized individual, releasing histamine and other biologically active mediators from sensitized mast cells. reactions with the clinical features of anaphylaxis have also been described without evidence of ige mediation. thus their precise causation is not clear. the term anaphylaxis in clinical practice remains a term used to describe a severe collapse-like reaction, not necessarily ige mediated. this phenomenon of an acute anaphylactic reaction to an ingested food represents the most severe example or one extreme of the clinical spectrum of gastrointestinal food allergy, but fortunately does not usually result in death. the acute syndrome is usually characterized by the sudden onset of vomiting, after cows' milk ingestion, occasionally followed by pallor and a shock-like state, but acute anaphylaxis is rare. when it occurs, acute anaphylaxis is a dramatic syndrome ( figure 5 .1) (de peyer and walker-smith, 1977) and can be fatal. a breast-fed infant given cows' milk feeding may react in this dramatic way. in these circumstances, acute vomiting with or without diarrhoea can be clearly related to the ingestion of cows' milk by taking a careful history. other clinical features usually accompany these gastrointestinal symptoms, such as swelling of the lips and tongue, oedema, and urticaria. all these symptoms disappear in a few hours if cows' milk is stopped. the amount of cows' milk responsible for this can be extremely small. it has been proven that infants can be sensitized to cows' milk via its presence in maternal breast milk when mother is herself drinking cows' milk lake, whitington and hamilton, 1982) . investigations show a high serum ige level and elevated milk specific rasts. the milk antibodies of the other immunoglobulin classes (igg, ig a and igm) are present but usually at a low titre (firer, hosking and hill, 1981) . skin prick tests are also positive (ford et al., 1983) . in a series of 100 children with cows' milk allergy (hill et al., 1986) 27 children fell into the quick onset group. the role of cows' milk hypersensitivity in the genesis of sudden unexpected death or 'cot death' was raised by parish et al. (1960) . the postulated mode of death was an acute anaphylactic reaction to cows' milk. devey et al. (1976) , in cambridge, went on to report that guinea-pigs given cows' milk to drink, instead of water, soon became anaphylactically sensitized to the proteins of cows' milk. coombs, devey and anderson (1978) then found that when the drinking of cows' milk was continued by the guinea-pigs for more than 70 days they became refractory to the effect. anderson et al. (1979) then showed that there were differences in the anaphylactic sensitizing capacities of different milks in their animal model. evaporated whole cows' milk was practically without sensitizing capacity to beta lactoglobulin, and a formula in a liquid concentrate form had extremely low sensitizing capacity to both casein and beta lactoglobulin. in both cases this only occurred when given to the guinea-pigs by mouth, sensitizing capacity being retained when given parenterally. this suggests that these formula are handled differently in the small intestine from other milk feeds. these observations have far-reaching implications if they are true for human infants, because they suggest that modification of artificial feeding formulae may profoundly influence their allergenic or sensitizing capacity. this aspect is discussed further on page 154. rudd, manuel and walker-smith (1981) have described an acute anaphylactic reaction after feeding an infant with a wheat rusk. this infant did not have rast antibody to wheat and his ige was only marginally elevated to 17 iu ml" 1 . egg hypersensitivity was first described by schloss in 1911. vomiting within a few minutes to an hour of egg ingestion is characteristic of egg hypersensitivity. diarrhoea, abdominal pain and nausea may also occur. typically, skin and respiratory manifestations also occur and may be a more important part of the clinical presentation than gastrointestinal symptoms (ford and taylor, 1982) . ovomucoid is the most important egg protein capable of producing this syndrome (bleumink and young, 1969) . rast and skin prick responses to egg are usually positive and helpful diagnostically. they provide a useful guide to resolution or persistence of egg allergy ( figure 5 .2). acute abdominal pain seems to be a particular feature of fish hypersensitivity (niziami, lewin and baloo, 1977) , whilst peanuts often produce immediate reactions of the oral mucosa (wraith et al., 1979) as well as abdominal pain. where one or a number of foods produces quick onset symptoms skin prick and rasts responses are usually positive and give helpful information. the total serum ige is usually elevated (dannaeus and johansson, 1979) . one unfortunate aspect of recent times has been the appearance of a number of commercial laboratories offering diagnostic tests for allergy directly to the public. the ability of such laboratories to accurately diagnose nine fish-allergic patients and nine controls who provided specimens of blood and hair for testing was assessed by sethi et al. (1986) . all five laboratories were not only unable to diagnose fish allergy but reported many allergies in apparently non-allergenic subjects and also provided inconsistent results on duplicate samples from the same subject. thus laboratory investigations appear to be most helpful when they are clinically least necessary, as history should give a clear indication of the diagnosis in most cases of quick onset syndromes. sodium cromoglycate as an oral preparation (nalcrom, fisons ltd.) has been reported to be an effective treatment for some cases of immediate food allergic disease. symptoms provoked by ingestion of one or more foods may be prevented by sodium cromoglycate if it is taken before taking the food. the literature contains many anecdotal reports of the beneficial effect of this treatment of small numbers of patients at various ages (freier and berger, 1973; watson and timmins, 1979) , but larger studies are still awaited. scepticism about the value of this drug has been based upon its failure to act on mucosal mast cells in the animal model (pearce et al., 1982) . however, recent evidence that it has an effect on improving abnormal gastrointestinal sugar permeability in patients suggests its mode of action may be other than on the mast cell (scotto et al., 1987) . in england and wales there has been a sharp decline in the number of childhood deaths reported to be due to choking on food. the number of deaths from this cause fell from 144 in 1974 to 46 in 1984. this was especially due to a fall in mortality for those under 3 months of age. roper and david (1987) ford.) attention to this, relate the fall to the change in infant feeding practice, namely that early introduction of solid food should be avoided. this appears to be yet another beneficial consequence of the dhss present day practice in infant feeding publication which recommended that solids should not be given before the age of 3 months. some individuals have gastrointestinal and other symptoms related to a wide variety of foods. such patients characteristically have a number of immediate symptoms such as vomiting, urticaria or wheezing upon exposure to multiple foods. they often have an individual and family history of atopy, peripheral eosiniphilia, elevated total serum ige and positive rast and skin tests to specific foods. diets involving the elimination of a number of foods may be impractical or ineffective on their own. however, the addition of disodium cromoglycate may be highly effective as in the group of children described by syme (1979) . the therapeutic dose is empiric at present (kocoshis and gryboski, 1979) . it is usually 100 mg twice daily. curiously if oral disodium cromoglycate alleviates symptoms these may not relapse when the drug is discontinued. these patients need to be distinguished from cases of eosinophilic gastroenteritis. whereas quick onset syndromes often present to allergy clinics, by contrast the slow onset syndromes usually present as a gastroenterological problem to paediatric or paediatric gastroenterology clinics. such children may often have failure to thrive. in these cases there is often no clear history of food being related to the onset of symptoms. diagnosis may be difficult. accurate diagnosis centres upon the following three groups of tests: • investigation of gastrointestinal structure, e.g. proximal small intestinal mucosal biopsy. • investigation of gastrointestinal function, e.g. intestinal sugar permeability. • investigation of immunological function: (a) systemic, e.g. specific antibody production. (b) gut associated, e.g. studies of local antibody producing cells. once these initial investigations have been performed, dietary elimination and challenge continue to have an important diagnostic role. this approach is of best value when such elimination and challenge is related to gastrointestinal structure and function, i.e. serial observations. at present there are no simple laboratory tests available for diagnostic screening of children with these slow onset gastrointestinal symptoms. in individual patients cows' milk antibody estimation is not diagnostically useful {see figure 1.1) . in children such problems often overlap with gastrointestinal infection which may coexist thus making diagnosis difficult. unless full microbiological study of the stools is done, i.e. stool electron microscopy for viruses as well as stool bacterial culture, infection of the gastrointestinal tract can be easily overlooked. food allergy and infection often coexist. changes in the structure of the small intestinal mucosa in response to the ingestion of particular foods provide clear objective evidence for the existence of food sensitive disorders affecting the small intestinal mucosa. this approach of using serial small intestinal mucosal biopsies related to dietary elimination and challenge was first used for the diagnosis of coeliac disease in the interlaken or espgan diagnostic criteria (see chapter 5). coeliac disease is a state of permanent food sensitivity, but there also exists a group of temporary food sensitive enteropathies, presenting in infancy. indeed ingestion of a number of foods apart from gluten have now been shown to produce food sensitive enteropathies in infancy. from studies in the experimental animal (macdonald and ferguson, 1977; ferguson, 1980) it seems likely that such enteropathies may result from a type iv or tcell mediated reaction within the mucosa. in such animal studies a type i reaction in the small gut mucosa is associated with only minimal morphological changes (mast cell degranulation and some oedema), while mucosal type iii reactions which are associated with polymorph infiltration do not cause crypt hyperplasia. of course more than one type of allergic response may coexist within the mucosa at any one time and it is possible, for example, that a type i reaction may precede a type iv reaction. abnormalities of the small intestinal mucosa have been reported in children suffering temporary intolerances to cows' milk protein, soy protein, gluten, eggs, chicken, ground rice and fish. the evidence that the enteropathy is directly related to ingestion of a particular food is based upon serial small intestinal biopsy studies related to dietary elimination and challenge, as in coeliac disease. the enteropathy is not usually as severe as that seen in coeliac disease, although a flat mucosa may occasionally be seen. these disorders usually resolve by the age of 18 months to 2 years. in some cases the children appear to develop food intolerance after an acute episode of gastroenteritis. the underlying causes of these temporary food intolerances of infancy probably relate to a transient sensitization of the child to dietary antigens, which may be a result of a breach of the mucosal barrier. the precise mechanisms which cause the enteropathy are unclear although the application of the gell and coombs classification of hypersensitivity reaction provides a basis for investigation. for the reactions to occur the offending food antigen must enter the mucosa in appropriate amounts to cause sensitization. there are two hypotheses regarding this process: one suggests sensitization caused from an overstimulation of the immune system by excess antigen entry, the other proposes a minimal entry of antigen sufficient to stimulate a reaginic response, which in turn leads to increased antigen entry leading to mucosal damage. in the experimental animal it has been shown that intestinal anaphylaxis can lead to increased uptake of intestinal luminal antigens (walker et al., 1975) . both hypotheses may be correct. post-enteritis food sensitive enteropathies may result from excess local food antigen entry in susceptible individuals following gut damage induced by viral or bacterial pathogens. an hypothesis relating acute gastroenteritis and cows' milk sensitive enteropathy is illustrated in figure 5 .3. it is known from the observations of gruskay and cook (1955) that excess antigen absorption (in their studies egg albumin) occurs in infants with acute gastroenteritis. this has been well documented in animal studies of viral enteritis (keljo, butter and hamilton, 1985) . clinical studies have also shown increased entry of both small molecular weight sugars in acute gastroenteritis ( figure 5 .4) (ford et al., 1985) , and a larger molecular weight protein, horse radish peroxidase, in post-enteritis food sensitive enteropathies as observed using organ culture (jackson, walker-smith and phillips, 1983) . thus direct and indirect evidence exists that damage to the small intestinal mucosa may result in a local increase in antigen entry. however, most children in whom this happens are not sensitized to food. thus for this excessive antigen entry to be of pathogenic importance it must occur in susceptible individuals. the nature of such susceptibility remains to be established but clearly relates in part to impaired immunoregulation by the local defence mechanisms in the small intestinal mucosa. the local effect of excess antigen absorption may be also determined by the allergenicity of the antigen entering the mucosa, as suggested by the guinea-pig studies of coombs and colleagues in cambridge referred to earlier and supported by the clinical studies of manuel, walker-smith and france (1979) . the role of cows' milk sensitive enteropathy as a cause of the post-enteritis syndrome is discussed further in chapter 6. it remains yet to be established whether small intestinal mucosal damage due to food ingestion occurs in adults, other than with gluten ingestion in patients with coeliac disease. cows' milk proteins cows' milk and human breast milk have different protein compositions, as table 5 .2 illustrates. human milk does not contain beta-lactoglobulin, which represents the major protein in cows' milk whey proteins. most observers, including visakorpi and immonen (1967) and freier and his colleagues (1969) , have noticed that this protein is often the factor responsible for cows' milk allergy, although the other proteins may also be allergenic in children. cows' milk contains three times more protein than human milk (due to its higher content of casein), but has the same (table 5 .2). it is perhaps unfortunate that proteins in breast milk and cows' milk such as casein are not distinguished by special names to describe human casein and cows' casein respectively. both these proteins, despite the same name, are biologically and chemically (e.g. their amino acid composition) quite distinct. in modern adapted milks, the so-called 'humanized' milks, the total protein content is reduced to about the level of human milk and the proportion of soluble proteins to casein is corrected by the addition of whey proteins (i.e. all the soluble proteins in milk after precipitation of casein either by the action of rennin or by acidification to ph 4.6). thus these milks contain more beta-lactoglobulin. despite this there is both clinical and experimental evidence that they are less sensitizing (walker-smith, 1986 ). the immunoglobulins present in breast milk are chiefly of the iga class. table 5 .3 indicates the difference in the immunoglobulin composition of breast and cows' milk. there are probably two syndromes, a primary disorder of immunological origin and a secondary disorder, a sequel of mucosal damage. abnormal handling of dietary antigens across the intestinal mucosa probably occurs in infants with this disorder. this may be related to a temporary immunodeficiency state such as transient iga deficiency (taylor et al., 1973) , or to non-specific small intestinal mucosal damage from any cause permitting excess antigen entry as referred to above. there is indeed clinical evidence that acute enteritis may be followed by not only lactose intolerance but by more persistent and longer lasting cows' milk sensitive enteropathy (harrison, wood and walker-smith, 1976; harrison et al., 1976; walker-smith, 1982 ) (see chapter 6). in the experimental animal increased protein antigen uptake occurs when the mucosa is damaged by parasitic infection (bloch et al., 1979) . the pathogenetic role of circulating antibodies to cows' milk remains to be established. lippard et al. (1936) showed that at whatever age a child first begins to drink cows' milk, cows' milk antigen and then cows' milk antibody can be detected in his blood. délire, cambiaso and masson (1978) have found that neonates fed on cows' milk have in their blood immune complexes containing cows' milk protein antigens, and igg antibodies of maternal origin. despite these findings, only a few children go on to develop cows' milk protein intolerance. how such a state of clinical intolerance develops is unknown. even the presence of a high level of serum anti-milk antibodies is not necessarily associated with damage, e.g. there is a high incidence of elevated titres of cows' milk antibodies in children with both coeliac disease and kwashiorkor (chandra, 1976) yet, as a rule, these children improve clinically on cows' milk diet. as stated earlier the local reaction in the small intestine may be mediated via one of the allergic reactions as classified by gell and coombs (1968) , namely type i, type iii, and type iv. evidence that these three types of reaction may occur in children with cows' milk protein intolerance include the following observations. first, in relation to type i reaction, elevated titres of ige antibodies to cows' milk protein, have been observed in some children with enteropathy but are more typical of quick reactors. however, such elevated titres can also be found in milk tolerant children. positive skin prick tests may be found in milk intolerant children but, again, correlation with symptoms is variable. the involvement of ige in the immunological response of the lamina propria to milk challenge in children with cows' milk protein intolerance has been described by shiner and her colleagues (1975) , also by kilby, walker-smith and wood (1976) , who showed an increase in ige cell numbers in the small intestinal mucosa after a milk challenge in a child with cows' milk sensitive enteropathy. however, whether ige mediated small intestinal disease due to sensitized mast cells releasing mediators at the site of reaction between cows' milk protein, and reaginic ige antibody fixing to mast cells really occurs, has not yet been proved. if this does happen, disodium cromoglycate may help. second, in relation to type iii reaction, elevated titres of igg and igm milk antibodies have been described in cows' milk protein intolerance but again with a poor clinical correlation. increased numbers of ig a cells, but not in general igm plasma cells after a positive milk challenge, may occur. matthews and soothill (1970) have observed the effect of milk feeding on complement activation, and reports of circulating immune complexes in children fed with cows' milk has already been referred to. whether such complexes are implicated in the genesis of the mucosal lesion of cows' milk protein intolerance is unclear. third, abnormalities of cell mediated immunity leading to abnormal lymphocyte transformation tests (fontaine and navarro, 1975) have been reported. studies by macdonald and ferguson (1977) and ferguson (1980) in the animal show that type iv hypersensitivity produce an appearance similar to cows' milk sensitive enteropathy. the importance of the type i reaction in the gut would be in allowing increased amounts of antigen to cross a damaged mucosa, and by causing capillary dilatation and increased permeability, allowing large amounts of antigen into the systemic circulation to initiate secondary immunization. if this antigen meets tissue fixed ige on mast cells then type i reaction would occur, e.g. in the skin (rash), in the gut (mucosal damage) and in bronchial mucosa (wheeze). thus the very variable clinical reactions encountered can be accounted for by differences in antigen reaching ige on mast cells in different sites in the body. involvement of systemic immunity, and the local immune system, could explain the transient nature of the illness. the illness could disappear after a period on a milk-free diet when the small intestinal mucosa local immune system was mature enough to prevent much antigen getting through. the exact role of cell-mediated immunity in this hypothesis is not certain but it is clearly important. the allergenicity or antigenicity of the cows' milk formula may be of critical importance in pathogenesis, as clearly most individuals who have acute gastroenteritis associated with increased gut permeability do not become sensitized to food. manuel et al. (1981) showed a remarkable difference in the incidence of delayed recovery between infants fed with different formulae immediately after an acute attack of gastroenteritis. old formula pregestimil (high osmolality and based on a casein hydrolysate) and al 110 (based on casein) and standard sma (a conventional adapted formula) were compared for infants aged under 6 months. it is likely that delayed recovery in these circumstances is related to cows' milk sensitive enteropathy; 26% of infants fed with the casein formula al 110 developed delayed recovery compared with only 5% with pregestimil and the very low figure of 2.5% with sma. this later figure may have been by chance unusually low. nevertheless, when these milks are tested in an animal model (guinea-pigs) then similar results are found: al 110 is sensitizing, pregestimil not sensitizing at all and sma sensitizing significantly less often (mclaughlan and coombs, 1983; manuel et al., 1979) . thus adapted feeding formulae appear to be much less sensitizing than the older infant feeding formula still routinely used in much of the developing world. this is consistent with the decline in the severity of cows' milk sensitive enteropathy in societies where such milks are now universally used. thus the allergenicity of the milk formula fed at the time of an acute attack of gastroenteritis may be a central factor in the development of cows' milk sensitive enteropathy. boys and girls appear to be equally affected (table 5 .4). although an atopic family history is very common, no definite genetic factor has been identified. kuitunen et al. (1975) have shown an hla status identical to that of the community. however, swarbrick, stokes and soothill (1979) have shown, in animals, a genetic variation in the control of antigen absorption by the gut, which suggests certain individuals may be predisposed to develop dietary protein intolerance. in the vast majority of patients with slow onset of symptoms related to cows' milk ingestion the architecture of the proximal small intestinal mucosa is abnormal but the severity of the enteropathy is variable (kuitunen et al., 1975; fontaine and navarro, 1975; harrison et al., 1976) . however, in some early reports the mucosa was flat and indistinguishable from that seen in coeliac disease (kuitunen et al., 1975) . more recently from the same centre in finland less severe mucosal damage has been characteristic and now very few children are seen in finland with severe cows' milk sensitive enteropathy (verkasalo et al., 1981) . when present, the enteropathy can be shown to be cows' milk sensitive by serial biopsies related to withdrawal and challenge with cows' milk ( figure 5 .5). unlike the gluten sensitive enteropathy of untreated coeliac disease, this cows' milk sensitive enteropathy is of variable severity on proximal mucosal biopsy, and is patchy in distribution, while a flat mucosa, indistinguishable from the mucosal appearances found in coeliac disease, may occasionally occur {figure 5.5). typically the mucosa in untreated cows' milk sensitive enteropathy is thin (maluenda et al., 1984) (figure 5.6) . the pathological changes are often patchy (manuel et al., 1979) . the intra-epithelial lymphocytes count is increased although not to the level found in untreated coeliac disease (figure 5.7) . there is also often a dense accumulation of fat in the epithelium which is a particular feature of this disorder and the post-enteritis syndrome (variend et al., 1984) (figure 5.8) . the mucosa rapidly returns towards normal or near normal on withdrawal of milk, only to relapse following challenge with cows' milk albeit to a variable and inconsistent degree (walker-smith, 1975) . however, unlike coeliac disease the mucosa remains thin throughout (maluenda etal., 1984) (see figure 5 .6) and the intra-epithelial lymphocytes fall to levels below normal on a milk-free diet rising to levels within normal limits after cows' milk challenge (see figure 5 .7). cows' milk sensitive enteropathy only affects children less than 3 years of age as a general rule. one remarkable exception is the case report of watt, pincott and harries in 1983 of a child with coeliac disease whose small intestinal mucosa was responsive to cows' milk until at least the age of 7 years. after a positive milk challenge, alteration in microvilli of the enterocyte may be seen (figure 5 .9) with a reduction in microvillous surface area (phillips et al., 1980) in parallel with a fall in disaccharidase activity (figure 5.10) . figure 5 .11 shows the relationship between milk challenge and lactose tolerance. the onset of symptoms may be acute with the sudden onset of vomiting and diarrhoea; the diarrhoea persisting and becoming chronic. alternatively in some infants the onset is insidious and the presentation is very similar to coeliac disease. in fact in most cases in the early reports the onset with acute symptoms commenced before the age of 6 months. in those earlier reports the majority of infants were less than 3 months of age at the onset of symptoms (harrison et al., 1976; walker-smith, kilby and france, 1978) . in the more recent study of digeon et al. (1986) the mean age of onset was 7 months and some patients were over 1 year at the age of presentation. this change may relate to the fact that for the first 6 months of life most infants were fed modern adapted formulae, whereas in the previous reports infants were fed old-fashioned partly modified milks. the onset of symptoms usually occurred at a time when infants were having ordinary pasteurized (doorstep) milk. as referred to earlier, it seems likely that modern adapted milks are less sensitizing, and there is both animal model (coombs and mclaughlin, 1985) and clinical evidence to support this view. there is usually a latent interval between the introduction of cows' milk and the onset of symptoms. such an onset may be clinically indistinguishable from acute gastroenteritis. indeed the illness may begin with acute gastroenteritis and then after return to a cows' milk feeding the diarrhoea becomes persistent. lactose intolerance may also be present. whatever the mode of onset of symptoms most often at the time of diagnosis these infants have chronic diarrhoea and failure to thrive. cows' milk sensitive enteropathy is a most important cause of the syndrome of chronic diarrhoea and failure to thrive in infancy. the mode of presentation of 18 infants at time of diagnosis seen at queen elizabeth hospital for children is illustrated in table 5 .4. cows' milk protein intolerance may also be associated with protein losing enteropathy and with iron deficiency anaemia due to intestinal blood loss (either occult or overt). this is related to an endoscopie colitis, which is not discussed here (gryboski, 1967; jenkins et al., 1984) . usually cows' milk sensitive enteropathy and cows' milk induced colitis do not coexist in the same patient. cows' milk protein intolerance has also been described as producing severe gastritis with antral erosions accompanied in some cases by duodenitis, diagnosis being made by upper endoscopy. these infants presented between 2 and 4 months with vomiting and failure to thrive as the principal symptoms. all had hypochromic anaemia and occult faecal blood, responded favourably to cows'-milk-free diet, and relapsed on milk challenge. neither small intestinal biopsy nor colonoscopy was performed so the state of the rest of the gastrointestinal tract in these infants remains unknown (coello-ramirez and larrosa-haro, 1984). until recently, the only satisfactory way to make the diagnosis of cows' milk protein intolerance has been based purely on clinical observations of repeated withdrawal of milk and challenge with milk, associated with clinical remission and relapse as formulated by goldman and colleagues in 1963, and since spoken of as the goldman criteria (table 5 .5). these criteria have obvious drawbacks. most mothers are reluctant to submit their infants to three potentially hazardous challenges, especially after one positive challenge. diagnosis of clinical relapse can be misleading, e.g. intercurrent illness may cause vomiting and diarrhoea and lead to error in interpretation. it is also now clear that children may take longer than 48 hours to relapse after a milk challenge. finally, positive challenges do not always have a similar onset, duration and clinical features. the use of these rigorous criteria has probably led to under-diagnosis of this syndrome. serial small intestinal biopsies taken first at the time of initial presentation, and second after the return of symptoms following a milk challenge, now permit a firm diagnosis to be made on the basis of one diagnostic milk challenge. because of its transient nature it may be difficult to fulfil all the diagnostic criteria for cows' milk sensitive enteropathy. nevertheless, accurate diagnosis is important. it is important to exclude an infective cause of the enteropathy in any infant with chronic diarrhoea. as with all dietary protein intolerances, diagnosis is based upon the response to withdrawal and subsequent reintroduction of the offending protein. there is no specific laboratory test apart from serial biopsy related to elimination and challenge. the first stage in making the diagnosis is the suspicion that the child's symptoms relate to milk ingestion. if the small intestinal mucosa is shown to be abnormal on biopsy the finding of a patchy enteropathy with a thin mucosa in a cows' milk fed infant, when the infant responds rapidly to a cows' milk elimination diet provides firm presumptive evidence for this diagnosis. such children may then be described as having a milk elimination responsive enteropathy. the transient nature of this disorder as well as the desire to avoid early milk challenge (because of the potential risk of acute anaphylaxis) (de peyer and walker-smith, 1979) leads in practice to a late milk challenge at the age of 9 months to 1 year or even later. these children may then no longer be milk intolerant at the time of milk challenge (i.e. at the age of 9-12 months). milk provocation in such circumstances merely establishes the safe return of cows' milk into the diet. it does not confirm the diagnosis of a cows' milk sensitive enteropathy which must remain forever unproven, at least on present-day diagnostic criteria. it is important that a challenge with cows' milk should be carried out in hospital so that it may be critically evaluated and because of the occasional risk of an acute anaphylactic reaction. the infant who is having a milk-free diet (almost the same as a lactose-free diet) is admitted to hospital. first, he has a control pre-challenge small intestinal biopsy to show that his mucosa has returned to normal or near normal. if it has not improved, challenge should be deferred and the diagnosis reconsidered, the diet being carefully checked. in these circumstances if the child is truly on a milk-free diet but continues to have gluten in his diet coeliac disease must be considered. when the mucosa has been shown on biopsy to have healed, the following day an oral lactose load of 2 g lactose/kg is given. this is followed by a lactose mixture containing 7% lactose for 24 hours. during this period the infant should be observed carefully and any loose stools tested for reducing substances. if watery diarrhoea with excess reducing substances occurs, the infant is clearly lactose intolerant and goes back to his milk-free diet, and milk challenge is deferred. if there is no diarrhoea the milk challenge takes place the next day. the amount given in the challenge will depend upon the previous severity of symptoms. if the history suggests the possibility of a previous anaphylactic reaction an intravenous infusion should be set up before the challenge and the initial amount of milk given should be small; for example 0.2 ml. if there has been a previous anaphylactic reaction before challenge, resuscitation equipment should be ready, and 1:10 000 adrenaline 0.1 ml kg -1 for injection, hydrocortisone 100 mg for intravenous use, intramuscular chlorpheniramine (piriton) 2.5 mg for intravenous use, plasma infusion, oxygen and equipment for intubation. all children who have had a previous anaphylactic reaction should be admitted to hospital. it is suggested that at least a year should elapse before reintroduction is attempted. in clinical practice an anaphylactic reaction may be defined as collapse, hypotension, impairment of consciousness or significant upper airway obstruction (swelling of structures in mouth or throat). when this has occurred previously the following method is used to determine if the child is still allergic. a drop of full-strength cows' milk is placed on the skin of the forearm. if no skin reaction occurs, cautiously administer 1 ml of a 1 in 20 dilution of milk or 15 ml teaspoon on the tongue. if a reaction such as swelling, itching, redness or pain occurs, no further milk is given. if no reaction occurs within 20 minutes, 1 ml full-strength milk may be given. in most children who have no history of previous reaction, 5 ml of milk should be given, followed after an hour by a further 10 ml if no symptoms occur. if this is tolerated the child is then regraded in quarters back onto cows' milk or his normal milk feeding. should symptoms recur, such as vomiting and diarrhoea, evidence of an intercurrent infection, e.g. hospital acquired rotavirus gastroenteritis, should be sought, i.e. stools should be sent for rapid viral diagnosis. if viral gastroenteritis is excluded, it is only a biopsy that can establish whether a relapse has occurred and should be done as soon as possible. if the previously normal mucosa is now abnormal, then this is regarded as a positive challenge, i.e. a cows' milk sensitive enteropathy has been shown and a firm diagnosis of cows' milk protein intolerance is made. if the mucosa is still normal, he continues on milk, and other causes for the symptoms are sought, such as an intercurrent illness, e.g. urinary tract infection or other infection. an occasional child may clinically relapse after a milk challenge despite a normal biopsy in the absence of any other explanation. this poses a difficult problem in diagnosis and may be due to patchiness of the cows' milk sensitive enteropathy. the symptoms that occur after challenge vary considerably and range from an alarming anaphylactic reaction that comes on rapidly after the child has ingested cows' milk, to the development of diarrhoea, with stools obviously blood-stained 24 or more hours after exposure to milk protein (table 5 .6). vomiting is usually a striking symptom and is often the first to appear. however, there is still no unanimity concerning the diagnostic criteria for cows' milk protein intolerance; for example, there is no consensus as to how a milk challenge should be performed; some use whole milk for the challenge and others use milk protein fractions such as lactoglobulin. goldman and his colleagues in 1963 reported investigations of 89 children with cows' milk protein intolerance and found the following frequencies of reactions to challenge with various milk protein fractions: beta-lactoglobulin, 66%; casein, 57%; alpha-lactalbumin, 54%; bovine serum albumin, 51%. in addition to the above challenge procedure, related to small intestinal biopsy, various laboratory tests have been studied to help to diagnose cows' milk protein intolerance. anderson and schloss in the usa in 1923 found antibodies to cows' milk protein in the serum of infants exposed to cows' milk. since then the occurrence of circulating milk precipitins in the serum of children has been reported in a wide variety of disorders, including chronic respiratory disease and ig a deficiency. however, most authorities believe that the presence of circulating milk antibodies cannot be correlated with clinical intolerance to this protein. a good example of this is provided by some children with coeliac disease who still have cows' milk antibodies in their sera whilst in remission on a gluten-free diet with no symptoms related to milk ingestion. some observers, e.g. matthews and soothill (1970) , have studied complement activation after milk challenge. five children with gastrointestinal symptoms due to milk protein intolerance had evidence of complement activation after milk challenge with 5 ml of milk. however, assay of complement activity after milk challenge has not been adopted as a useful laboratory confirmation of the diagnosis of cows' milk allergy. however, the above authors were not considering cows' milk sensitive enteropathy specifically but were looking at cows' milk allergy as a whole. it may not be possible to distinguish cows' milk sensitivity from lactose malabsorption at the time of initial presentation as both cows' milk sensitive enteropathy and lactose intolerance may coexist. however, when a child is on a cows' milk-free diet it is possible by cows' milk challenge to make the distinction. in a classic paper, liu, tsao and moore, in 1967 , showed that challenge with cows' milk protein could induce intestinal malabsorption of lactose in children who were intolerant to cows' milk protein. it is now clear that lactase deficiency with secondary lactose intolerance may be produced by cows' milk challenge in children who have cows' milk sensitive enteropathy, particularly when this has occurred as a sequel to acute gastroenteritis. this secondary lactase deficiency is a consequence of mucosal damage produced by cows' milk protein per se {see figure 5 .1) (harrison, 1974; harrison et al., 1976) . while it may be impossible at the time of initial presentation to make the differential diagnosis, at the time of milk challenge it is practicable and important that the distinction should be made. the therapy for this disorder is to eliminate cows' milk from the child's diet and also all foods based upon cows' milk. this latter point is most important as dietetic failure may sometimes relate to neglect of restriction of foods such as ice-cream, which are based on cows' milk, despite strict adherence to avoidance of cows' milk per se. treatment involves substituting cows' milk feeds with the commercially available cows' milk protein-free formulae (francis, 1987) . in practice five categories of cows' milk substitutes have been used, namely those based on: • casein hydrolysate, pregestimil, and for infants over 6 months, nutramigen. • lactalbumin hydrolysate, alfare. • soya protein (cow and gate formula s, prosobee, wysoy). • a formula based upon comminuted chicken which requires supplements with the complete range of vitamins and minerals. • boiled goats' milk for children over 6 months plus vitamins a, d, c, b 12 and folic acid tablets (these contain lactose). as children may become sensitized both to soy and goats' milk feeds, the author prefers a hydrolysate formulation in most circumstances, with the occasional use of comminuted chicken formulae. this is used when the child is intolerant to one of the hydrolysates. a casein hydrolysate has most often been used but there are some children who are intolerant to it. this sometimes is due to glucose polymer intolerance (see chapter 7). in a study comparing a casein hydrolysate and a whey hydrolysate, whilst both were effective, weight gain and healing of the mucosa was better in some cases fed a whey hydrolysate. however, the latter formula was more unpalatable walker-smith, digeon and phillips, 1987) . only those formulae that are nutritionally complete (if necessary with vitamin supplementation) are to be recommended. those with a low osmolality should be chosen for young infants or infants with small intestinal disease. it is important to ensure that both liquid and solid feeds are free of cows' milk proteins. lactose intolerance may accompany the protein intolerance, and in such circumstances lactose should also be withdrawn from the diet. the necessity for dietary treatment is always temporary, and reintroduction of a normal diet is normally nearly always possible between the age of 1 and 2 years. this may be done in the home, but a history of previous severe reactions, such as urticaria or anaphylactoid shock, is an absolute indication for reintroduction of a normal diet under very close medical supervision usually in hospital. when a repeat biopsy is done to assess mucosal healing before challenge, clearly this will be done in hospital, which the author recommends on most occasions. usually this shows significant improvement. because of the risk of anaphylaxis milk challenge is usually delayed to the age of 9-12 months. soy bean was first proposed as a substitute for cows' milk in infants by ruhrah in 1909 . however, it was not until the recommendation by hill and stuart in 1929 that a soy bean food prepared to resemble milk began to be used in many countries for infants with milk allergy. glaser and johnstone went on in 1953 to suggest that soy bean milk when used as a substitute for cows' milk could play an important role in the prevention of allergy to cows' milk in those who were at risk. soy bean is rich both in protein (40%) and fat (20%). it belongs to the leguminaceae family as does the pea. it is principally produced in the usa. there were some difficulties with the first-generation soy formulae. these included carbohydrate intolerance because of indigestible carbohydrates and vitamin deficiencies. from the mid-1960s second-generation soy bean formulae have been used, based on a soy-protein isolate. the commercial formulae available in britain-formula s, isomil, prosobee and wysoy-are based on soy protein isolate. soy beans contain trypsin inhibitors, and in the experimental animal soy bean diets may cause growth retardation, pancreatic hypertrophy and even adenocarcinoma (mcguiness et al., 1980) . none of these serious effects has been described in infants probably because heat-labile trypsin inhibitors are inactivated in the manufacture of the formulae. however, the kunitz soy bean trypsin inhibitor has been reported to be the antigenic stimulant for an acute anaphylactic reaction in an adult woman (moroz and yang, 1980) . some problems with the second-generation formulae do remain. amongst these are mineral bioavailability. this is probably due to its 1% phytate content and there is a report of its use exacerbating acrodermatitis enteropathica (glasgow and elmes, 1975) . one other immunological concern about the use of soy formulae is the report from verona that healthy non-atopic infants fed soy formulae have lower immunoglobulin levels and more infections than do similar infants fed cows' milk (zoppi et a/., 1979 (zoppi et a/., , 1983 . the globulin fraction of soy beans is the major protein component. it consists of four main components (shibaski et al., 1980) . the 2s globulin has the highest allergenic potency. heat treatment increases this potency. haemagglutinating titres to heat treated soy were higher when injected into rabbits than crude soy powder extract (eastham et al., 1982) . this is in contrast to cows' milk formulae which became less antigenic after heat treatment (mclaughlin et al., 1981) . soy-based formulae have been shown to be at least as antigenic as milk-based formulae in a study by eastham et al. (1978 eastham et al. ( , 1982 . circulating antibodies developed rapidly for up to 3 months with little further rise in normal infants fed these formulae. high levels of haemagglutinating antibody have been shown in amniotic fluid, suggesting in-utero sensitization (kuruome et al., 1976) . although it has been shown that soy bean has low antigenicity in guinea-pig studies (ratner and crawford, 1955) , over recent years there has been an increasing number of clinical reports of intolerance to soy protein. such reactions have varied from a dramatic anaphylactic response, the onset of respiratory symptoms and the appearance of gastrointestinal symptoms. these observations are in accord with the concept that soy protein is in fact not a weak antigen in man. the first report of soy allergy was as long ago as 1934 (duke) . acute anaphylaxis has been described in infancy (david, 1984) . it has been shown that soy protein can produce a small intestinal enteropathy which resolves with soy elimination and which reappears when soy protein is reintroduced into the diet, i.e. a soy-sensitive enteropathy (ament and rubin, 1972) . these workers described a flat mucosal lesion indistinguishable to that found in coeliac disease. this observation has been confirmed by others, but more usually the small intestinal damage is less severe and is similar to cows' milk sensitive enteropathy (perkkio et al., 1981) . as with cows' milk protein intolerance the colon may also be affected and there are reports both of enterocolitis (mcdonald et al., 1984) and colitis (halpin et al., 1977) . soy formulae have been recommended in three situations: • when the small intestinal mucosa is normal as a prophylaxis against cows' milk allergy. • in those already who are cows' milk protein intolerant, some of whom may have small intestinal mucosal damage. • for the management of gastroenteritis by virtue of the formulae being lactose free. here too the small intestinal mucosa is likely to be abnormal. it is particularly in these last two situations when the mucosa is already damaged that the use of soy formulae may lead to intolerance by sensitizing the mucosa to this protein. there is clear evidence that the incidence of soy protein intolerance is lower when used for prophylaxis than as treatment for cows' milk allergy (0.5% versus 15-50% or even higher). the very high figure of 80% reported by wong in 1965 (wong, 1985 for children with severe cows' milk protein intolerance contrasts to the figure of 11% reported by kuitunen et al. referred to earlier. however, the former report concerned first-generation isolate. in between is the figure of 15% reported by perkkio, savilahti and kuitunen (1981) who described 16 cases of soy intolerance from 108 children with cows' milk sensitive enteropathy treated with soy formula. it is because of such data that the author recommends a protein hydrolysate rather than a soy formula for the management of children with cows' milk sensitive enteropathy. the pathogenesis of soy protein intolerance is probably very similar to that of cows' milk sensitive enteropathy. it frequently appears to occur as its sequel. butler et al. (1981) studied neutrophil chemotaxis and neutrophil random migration in infants with milk and/or soy protein intolerance. chemotaxis is the ability of certain cells to move or to turn towards other cells or substances that exert a chemical influence (positive chemotaxis) or away from them (negative chemotaxis). all infants showed a significant decrease in chemotaxis at a time when the disease was active. however, this depression did not relate to the severity of the protein intolerance as judged by symptoms or the nutritional status. random migration of neutrophils was increased in patients with active protein intolerance as compared with controls. the relationship of this observation to pathogenesis is unclear at present. kuitunen et al. (1975) who described the clinical findings in 54 children with cows' milk intolerance reported that 35 of these children were given soya protein as a cows' milk substitute. four of these developed soya protein intolerance. the symptoms were vomiting, diarrhoea and weight loss. three had partial villous atrophy on biopsy and the fourth had a flat mucosa. it is clear that soy sensitive enteropathy may occur as a sequel to cows' milk sensitive enteropathy. treatment is with a protein hydrolysate formula such as pregestimil. strict avoidance of soy may be difficult as so many modern prepared foods contain some soy. like cows' milk protein intolerance, the need for dietary avoidance is temporary. definition transient gluten intolerance may be defined as the syndrome seen when a child with gastrointestinal symptoms and an abnormal small intestinal mucosa who responds clinically and histologically to a gluten-free diet, subsequently thrives and continues to have a normal mucosa despite returning to a normal gluten-containing diet. dicke in holland, in 1952, described a transient wheat sensitivity in pre-school children following gastroenteritis. visakorpi and immonen in finland, in 1967 , described a state of transient gluten intolerance in 28 children associated in some cases with temporary cows' milk protein intolerance. these reports did not include serial small intestinal biopsies. in 1970, a child with transient gluten intolerance was described in australia, and this report included such biopsies (walker-smith, 1970) . this child had an abnormal small intestinal mucosa (a severe degree of partial villous atrophy) and he responded clinically to a gluten-free diet. after 1 year, while he was still on a gluten-free diet, a further biopsy revealed a normal mucosa. he was put back onto a normal gluten-containing diet, and 16 months later a further biopsy demonstrated a persistently normal mucosa. he has subsequently remained in excellent health. despite these case reports there has been considerable scepticism expressed that transient gluten intolerance does actually exist. in particular scepticism has been expressed as to whether those children reported to have had transient gluten intolerance had really been gluten intolerant ab initio. hence stricter criteria were laid down for diagnosis. these were first, the need to provide evidence that gluten toxicity was in fact present and that the apparent clinical response to gluten restriction was not fortuitous and second, the need to demonstrate the presence of a normal small intestinal mucosa 2 years or more after the return to a normal diet (i.e. the 2 years rule) as laid down by the european society for paediatric gastroenterology in interlaken in 1970 to exclude coeliac disease {see chapter 4) (meeuwisse, 1970) . the precise criteria necessary to establish the existence of any form of transient intolerance to a dietary substance are indicated in diagrammatic form in figure 5 . 12. mcneish et al. (1976) , in fact, have demonstrated such early evidence of gluten toxicity by serial xylose absorption studies at the time of an early gluten challenge in an infant with an enteropathy who had previously responded to a gluten-free diet. serial biopsies were not used to establish gluten toxicity at that time in their infant, but the mucosa has been shown to be normal 2 years after return to a normal gluten-containing diet. at the queen elizabeth hospital, a child has been observed who completely fulfils both the espgan and mcneish criteria for the diagnosis of transient gluten intolerance (see table 5 .8) . this child had an early gluten challenge at age of 1 year 2 months, having previously had a flat mucosa and a clinical response to a gluten-free diet. she relapsed clinically and histologically with gluten. subsequently, after a second challenge with gluten, she has remained clinically well and her mucosa 2 years 3 months after return to a normal glutencontaining diet has remained normal. she has now left the paediatric age group symptom free. it thus seems clear that this child had transient gluten intolerance from which she has now recovered (see table 5 .7). thus application of these very strict criteria has established evidence that transient gluten intolerance does in fact exist in this very small number of patients. in routine clinical practice these criteria are impossible to fulfil as early gluten challenge is no longer performed. thus the diagnosis of transient gluten intolerance is usually retrospective and presumptive, i.e. a child provisionally diagnosed as coeliac disease fails to relapse clinically and histologically after 2 years or more back on a gluten-containing diet. thus the child fails to fulfil the espgan criteria for the diagnosis of coeliac disease (see chapter 4). thus in current clinical practice the term transient gluten intolerance is reserved for the small group of children provisionally diagnosed as coeliac disease who fail to relapse after 2 years or more back on a gluten-containing diet. this retrospective diagnosis is then based upon the following diagnostic criteria. • initial illness associated with a severe small intestinal enteropathy. • complete clinical remission on a gluten-free diet. gluten started 2\ months of age gluten-free gluten 10 g powder daily for 3 months gluten-free 3 years 2 months gluten 5 g in diet daily for 3 months gluten 5-10 g in diet, daily for 2 years 2 months • healing of the enteropathy on a gluten-free diet. • normal intestinal mucosa 2 years or more after return to a gluten-containing diet (the two years rule'). in a review of the espgan criteria, mcneish et al. in 1979 commented upon the paucity of published evidence, at that time, to endorse the concept that all coeliac children relapse within 2 years of gluten challenge and therefore that patients not relapsing after this time had transient gluten intolerance. in fact the literature is now more extensive. several published studies of gluten challenge in children have established that relapse most often occurs within 2 years. there are, however, a few reports to suggest that it may take more than 2 years for a relapse in some exceptional cases of coeliac disease. mcnicholl et al. (1974) described two children who took more than 2 years to relapse after return to a normal gluten-containing diet. only one case has had the full clinical details published (egan-mitchell, fottrell and mcnicholl, 1978) . in that case the intra-epithelial lymphocyte count rose and disaccharidase activities fell before frank mucosal relapse. a survey by the european society of paediatric gastroenterology and nutrition suggested that others had a similar experience (shmerling, 1978) . in a study of 65 children, originally diagnosed as having coeliac disease, at the queen elizabeth hospital (see chapter 4), 15 proved on reinvestigation to have a normal mucosa 2 or more years after having gluten in their diet (walker-smith, kilby and france, 1978) . nine of these had a documented abnormal initial biopsy at the time of the original presentation on a normal gluten-containing diet. they responded clinically to a gluten-free diet, but despite this they had a final biopsy which was normal or near normal after 2 or more years on a gluten-containing diet. all have now left the paediatric age group symptom free. figure 5 .13 demonstrates the histological findings in one of these children. seven of these children had serial disaccharidase assay after start of challenge. on two occasions, disaccharidase activity fell despite normal morphology as reported in such children by mcnicholl, egan-mitchel and fottrell (1974) . their ages ranged from 8 weeks to 1 year 5 months at the time of the initial biopsy, i.e. all were less than 2 years of age at that time. a critical review of the early history in three children reveals evidence of a preceding episode of acute enteritis and evidence of other food intolerances, e.g. cows' milk protein intolerance, but in the remainder there was no such evidence (table 5. within this group there were some children who met all the classical criteria for the initial diagnosis of coeliac disease as they had a completely flat mucosa ( figure 5 .13) and evidence of malabsorption, and responded dramatically to a gluten-free diet alone without any evidence of other food intolerances. it is impossible now to convince the mothers of these children that a gluten-free diet at that time did not account for their clinical improvement. figure 5 .14 shows the weight progress over the years of one of these children. these cases with two others making a total of 11 were further reviewed (walker-smith, 1987) . this represents the total number of children diagnosed at queen elizabeth hospital between 1972 and 1986. they have now been followed up for a period of 8-10 years in most cases but in one for 20 years. many of these children had gluten introduced into their diet at an early age as did children with coeliac disease diagnosed at the same time. all had normal biopsies 2 years or more after a return to a normal gluten containing diet. four had further biopsies. one was abnormal and thus this child has coeliac disease. he had in fact been symptom free, the indication for biopsy being the development of serum gliadin antibodies. nusslé et al. (1978) have described a similar group of six children, initially diagnosed as having coeliac disease, who have had a normal small intestinal mucosa 2h-^h years after reintroduction of gluten to their diet. all had normal iel levels, and all except one, normal disaccharidases. thus, again, these children appear to have had transient gluten intolerance from which they have recovered. schmitz, jos and rey (1978) , from paris, in a very important paper have described three children who had earlier responded to a gluten-free diet but who then developed flat mucosa on a gluten-containing diet at the ages of 10 years, 62 years and 4 years 8 months respectively. however, they did not return to a gluten-free diet but continued on a gluten-containing diet. further biopsies after more than 9 years, 12 years and 5 years respectively on a normal gluten containing diet, showed surprisingly normal or near normal mucosae. the third case was having a low gluten intake but had relapsed earlier on a similar low gluten diet. thus these three children appear to have recovered spontaneously on a gluten-containing diet. a further seven children have since been described (schmitz et al., 1984) . the author would regard this as good evidence that these children had transient gluten intolerance which was longer lasting than other children described from which they had recovered, but schmitz, jos and rey have raised the possibility that the mucosal lesion of coeliac disease may disappear during adolescence only to reappear in adulthood. further prospective studies will establish whether this is so. it must be acknowledged that the ages of the cases described by schmitz et al. are clearly different to those reported by walker-smith et al. (1975) and nusslé et al. (1978) as they are much older than those cases which were all under 2 years at time of initial diagnosis. further evidence comes from the study of shmerling and franckx (1986) who described three patterns of response after a gluten challenge: • a group of 24 coeliac patients who fulfilled the espgan criteria; 21 relapsed within 2 years of commencement of the gluten-challenge. three took up to nearly 5 years to relapse. • six children who after a gluten challenge of 2.42-6.92 years have not relapsed. these children are being followed carefully and would appear to have had transient gluten intolerance. • a group of 11 children whose mucosa after gluten challenge deteriorated without becoming flat. all were symptom-free and have continued on gluten. they resemble the cases described by schmitz et al. likewise their future is uncertain. thus the 2-year rule appears to be valid for the majority of children with coeliac disease who have a gluten challenge although there are occasional exceptions. thus most children previously diagnosed on firm criteria as suffering from coeliac disease but who fail to relapse by 2 years after a return to a gluten-containing diet can be provisionally labelled as transient gluten intolerance. in each individual child, however, follow-up must be close in order to detect the occasional child who eventually has a late relapse and thus proves in the end to have coeliac disease, i.e. permanent gluten intolerance. from all these studies it is clear that there is thus a particular need for detailed and long-term follow-up into adult life of these children retrospectively diagnosed as transient gluten intolerance. it is therefore difficult to regard the diagnosis of transient gluten intolerance in our present state of knowledge as ever being any more than provisional and it cannot be regarded as final. it remains possible that any or all of the children referred to above ultimately may relapse after many years in adult life. similar uncertainty must remain concerning the fate of the children described by schmitz et al. and shmerling and franckx. a schematic model of the present scene is outlined in figure 5 .15. two explanations have been proposed to explain the development of this syndrome. firstly, it has been suggested that there may be a temporary depression of dipeptidase activity occurring in the small intestinal mucosa, secondary to nonspecific mucosal damage, such as may occur as a sequel to gastroenteritis. such a suggestion at present is only speculative and is based on reports of this syndrome following clinical episodes of gastroenteritis where there has been the demonstration of an abnormal small intestinal mucosa on biopsy. secondly, it is possible that a transient 'allergy' to gluten may occur in a similar and equally unknown manner to that suggested earlier in this chapter in relation to cows' milk protein. there is little evidence available so far to support either theory. all the cases described by walker-smith 1985 had gluten introduced very early in to their diet (under 2 months). this may account for the fact that no new case has been diagnosed at queen elizabeth hospital presenting since 1974. from 1975 mothers have been encouraged in britain to introduce gluten at a later time and to use rice rather than wheat cereal as a weaning food. thus the early age of introduction of gluten into the diet of these children may be an important factor in the pathogenesis of transient gluten intolerance and could account for its importance in the early 1970s. the small intestinal mucosa is by definition abnormal, i.e. thickened ridged mucosa characterized histologically by partial villous atrophy, or sometimes a flat mucosa. the demonstration of a flat mucosa, however, should not ordinarily suggest this diagnosis, as it is more characteristic of coeliac disease. the mucosal abnormality is therefore typically less severe than that found in coeliac disease. in the child referred to in table 5 .9 it is notable that there was an increase in the intra-epithelial lymphocyte count in the initial diagnostic biopsy, a fall in the count after a gluten-free diet, followed by a rise on mucosal relapse following a gluten challenge and, finally, a return to normal level which has remained normal on a gluten-containing diet. thus at the time when the child was gluten sensitive, the intra-epithelial lymphocyte count appeared to be as responsive to gluten in the diet as in children with coeliac disease (i.e. permanent gluten intolerance). transient gluten intolerance shibuld be considered as part of the differential diagnosis of the infant who develops gastrointestinal symptoms when he first encounters wheat protein, especially when he appears to be intolerant to other food proteins such as milk and egg. it should also be considered as a possibility in a child who fails to thrive following gastroenteritis, e.g. salmonellosis (walker-smith, 1970) in the presence of an abnormal small intestinal mucosa and the absence of other explanations, such as secondary lactose intolerance, particularly when such an infant has not responded to several other dietary measures but is having cereal in his diet. there appear to be two clinical syndromes associated with transient gluten intolerance: first, when gluten intolerance accompanies other forms of food intolerance; and secondly, when there is gluten intolerance alone producing a clinical picture identical with coeliac disease (walker-smith, kilby and france, 1978; nussle et al., 1978) . gliadin antibodies may be detected in the serum of such children even though their mucosa is now normal. table 5 .9 histological grading, lactase activity and intra-epithelial lymphocyte counts/100 epithelial cells (iel) in serial biopsies from a child s.s. related to age and gluten-containing (g) or gluten-free diet (gf) the dietary management is identical with that prescribed for children with coeliac disease but the need for such dietary restriction is, of course, by definition, a temporary one. the duration of the need for such dietary restriction will vary from child to child. clearly, at present this is a confusing field and in need of much clarification. it has been discussed here at some length, not because of the intrinsic importance of transient gluten intolerance itself, but rather because of the importance of distinguishing the condition from coeliac disease. if it were not for the existence of a permanent gluten intolerance this disorder would be looked upon much the same as cows' milk and soy intolerance. finally, it is most important that those children diagnosed as transient gluten intolerant, but who have completely recovered, should have long-term follow-up. these children could eventually relapse. it is the author's practice to follow-up such children throughout their childhood and then refer them to adult gastroenterologists for indefinite follow-up. it has now been established by serial biopsy and dietary elimination and challenge that egg protein (lyngkaran, 1982) , ground rice, chicken meat and fish (vitoria et al., 1982) may all temporarily damage the small intestinal mucosa in infancy. in this latter study all infants also were cows' milk intolerant and were under the age of 6 months at the time of diagnosis. these findings are of more theoretical importance than practical as the author would not advocate in clinical practice serial biopsy and challenge if a child with an enteropathy who responds to milk elimination develops diarrhoea in the absence of infection when given one of these foods. rather, the offending food would be removed from the diet. what is important is that there is now firm evidence that these food sensitive enteropathies do exist in infancy. soy protein-another cause of the flat intestinal lesion allergy to cow's milk in infants with nutritional disorders anaphylactic sensitivity of guinea-pigs drinking different preparations of cow's milk and infant 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of cow's milk from mother's diet. british paediatric association meeting evaluation of a caesin and a whey hydrolysate in the management of cow's milk sensitive enteropathy in infancy food allergy. response to treatment with sodium cromoglycate combined cow's milk protein and gluten induced enteropathy: common or rare rice gruel in management of infantile diarrhoea recognition of food allergic patients and their allergens by the rast technique and clinical investigation gammaglobulin level and soy protein intake in early infancy diet and antibody response to vaccinations in healthy infants key: cord-000479-u87eaaj8 authors: stolf, beatriz s.; smyrnias, ioannis; lopes, lucia r.; vendramin, alcione; goto, hiro; laurindo, francisco r. m.; shah, ajay m.; santos, celio x. c. title: protein disulfide isomerase and host-pathogen interaction date: 2011-10-11 journal: scientificworldjournal doi: 10.1100/2011/289182 sha: doc_id: 479 cord_uid: u87eaaj8 reactive oxygen species (ros) production by immunological cells is known to cause damage to pathogens. increasing evidence accumulated in the last decade has shown, however, that ros (and redox signals) functionally regulate different cellular pathways in the host-pathogen interaction. these especially affect (i) pathogen entry through protein redox switches and redox modification (i.e., intraand interdisulfide and cysteine oxidation) and (ii) phagocytic ros production via nox family nadph oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. the protein disulfide isomerase (pdi) family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (er) and towards the cytosol, a thiol-based redox locus for antigen processing. here, we summarise examples of the cellular association of host pdi with different pathogens and explore the possible roles of pathogen pdis in infection. a better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections. host cells have the ability to cope with the progression and severity of infection in response to different types of pathogen. on the other hand, numerous mechanisms have evolved that support the use of the host cell machinery to facilitate pathogen survival and multiplication. such co-evolutionary processes are directly affected by different physicochemical factors within different cell compartments, both in the host and in pathogen. for instance, ph critically affects antigen stability of the influenza virus which modulates endosome acidity that attenuates its own infection [1] . ros (and reactive nitrogen species) production and the redox state of different cell compartments are also critically involved in cellular hostparasite interaction. among the many redox sensitive proteins that are altered during the course of different infections, protein disulfide isomerase (pdi-) mediated redox switches have been associated with pathogen attachment-internalization, antigen processing in the er/phagosome, and the regulation of ros production by nox family enzymes. thus, pdi emerges as a ubiquitous redox protein that regulates different steps of diverse infection processes. several pathogens also have their own pdi that act as an important virulence factor (table 1 ). other redox modifications directly mediated by ros and especially via nitric oxide (no) generated by inducible nitric oxide synthase (inos), which is abundant in phagocytic cells, have been reviewed elsewhere [2, 3] and are not considered in this article. below, the main cellular redox aspects of host and pathogen pdi will be discussed. the ancient pdi is a ubiquitous redox chaperone belonging to the thioredoxin oxireductase super family and can reduce (reaction 1), oxidize (reaction 2), and catalyse dithiol-disulfide exchange reactions (i.e., isomerase activities, reaction 3, figure 1 ). such broad range of activities overlaps with the chaperone role of pdi that overall performs a housekeeping function in helping to maintain proteins in a more stable conformation. there are around 20 pdi homologues, and the detailed structure and function of eukaryotic pdis have been covered in recent excellent reviews [4, 5] . the classic mammalian pdi (55 kda) has several domains ordered as a-b-b -a -c with 2 thioredoxin-like motifs (trp-cys-gly-his-cys) displayed in the a and a domain [4] [5] [6] (figure 1 ). pdi is abundant in the er (∼ 0.5 mm) where the relatively oxidizing conditions at basal level (i.e., gsh/gssg ratios ∼ 2-3 : 1) favours pdi isomerase/oxidase activity, which is primarily involved in client protein redox folding (reaction 2-3, figure 1 ). the oxidizing equivalents for this process are driven mainly by the er thiol-containing oxidase, ero1 (endoplasmic reticulum oxidase-1), which binds fad and is in turn re-oxidized via electron transfer to oxygen, generating h 2 o 2 in the process [7] [8] [9] [10] . the h 2 o 2 destiny is elusive, but it can oxidize er-located peroxiredoxin iv (prxiv) that is further reduced by pdi that is oxidized in the process [11] . this redox circuit is thought to increase total protein folding and thiol oxidation via ero1 [11] . however, even in the absence of ero1, protein folding still occurs, and it is suggested that other oxidases may compensate for redox demand in the er in some circumstances [12, 13] . nevertheless, the pdi-ero1-dependent oxidative activity is balanced to cytosolic glutathione levels suggesting a functional redox interplay between these compartments [12] . pdi reductase activity has been primarily associated to more reducing compartments (i.e., gsh/gssg ratios ∼ 30-100 : 1), such as those in the vicinity of the plasma membrane [6, 10] . pdi redox versatility is mainly governed by the low pka of the proximal cysteine on the active n-terminal a domain. indeed, the lower pka of 4.5 renders pdi a much better oxidase than thioredoxin, which has a pka of 7.1 and is mainly a reductase in most neutral ph cell compartments. it should also be noted that pdi functions as a chaperone independently of its redox-active domains as especially required for its atpase and ca 2+ activity, although pdi redox motifs still stabilize binding interaction [4, 5, 10] . in the er, pdi is tightly associated with prolyl-4 hydroxylase (the rate-limiting enzyme for collagen biosynthesis), the sec61 translocon, and the mhc class i complex (see later). it can be also found as a heterodimer with microsomal triglyceride transfer protein [6, 10] . pdi is a soluble homodimer and does not have a transmembrane domain and, similarly to other er chaperones, carries the kdel c-terminal sequence which binds to respective receptors in the cop vesicles that circulate in the er-golgi vicinity, recycling proteins back to er. pdi also undergoes intense intracellular trafficking and is found on the surface of diverse prokaryotic and eukaryotic cells [14] [15] [16] . despite limited knowledge about this traffic, it is possible that pdi exits the er through the translocon sec61 pore and/or via secretory vesicles [17] . pdi is thought to attach to lipids, glycans, and integral membrane proteins via electrostatic interactions at the cell plasma membrane [14, 15] , where its reductive activity mediates the infection of different pathogens ( figure 2 , discussed later). pdi along with erp57 have been found in the nuclei in association to dna and affecting transcriptional activity of nf-kb, ap-1, and stat3 [15] . these transcriptional regulators are key elements in many inflammatory processes, but their functional association to pdi and to different pathogen still elusive. in contrast to many other members of its family, such as thioredoxin itself and erp57, pdi is not normally found in the cytosol, where it is likely cleaved by caspase-3 and -7 [18] . protozoans and bacteria have their own pdis ( table 1 ). the function of these pdis in protein folding is poorly understood; however, there are intriguing data correlating pdi expression and the pathogenicity of several parasites, especially obligatory intracellular protozoans. leishmania that leads to distinct types [20] . if l. chagasi is a subgroup of l. infantum brought to america by european colonist or other specie in its own still a matter of controversy (see discussion in [21] ). the infection cycle in the vertebrate host and is initiated when leishmania promastigote is injected into the skin by the insect vector. in the host, the promastigote is phagocytised especially by macrophages, and further it is converted into intracellular amastigote. amastigote replicates inside the phagosome within the cell and is liberated after the cell lyses, subsequently infecting other cells resulting in the progression to disease [21, 22] . l. amazonensis has at least four pdis, and the use of specific pdi inhibitors substantially affected parasite growth [23] . in l. major, the increased levels of leishmania pdi (lmpdi) expression and secretion at the parasite surface reflects optimal protein folding balanced to parasite multiplication. importantly, this is correlated to high virulence of the parasite strains [16] . more recently, the use of lmpdi antigens to generate a vaccine for l. major partially protected balb/c animals and accelerated the cure of different strains of mice [24] . similarly to leishmania species, other parasites of the trypanosomatid group such as trypanosoma contain several genes predicted to encode for pdis, that can execute n-glycosylation and protein folding in the er [25, 26] . although pdi was considered essential for t. brucei survival, pdi activity was not essential for the growth of trypanosomes in vitro [26] . pdi is also expressed in different species of plasmodium protozoans (table 1) , the parasites that cause malaria [27, 28] . p. falciparum expresses at least nine different pdis and the pfpdi-8 has great similarity to the prototype pdi and is expressed during all stages of parasite life cycle. this pdi facilitates the disulfide-dependent conformational folding of eba-175 protein, an emerging candidate for the development of malaria vaccines [28] . this is intriguing given that malaria parasites express proteins with high content of cysteine, which are associated to parasite invasion and sequestration in the vertebrate host and transmission into mosquito host [28] . finally, toxoplasma gondii pdi was identified in host tears, suggesting an extracellular location and adhesion to host cells during the initial phase of infection [29] . antigen presentation occur through two distinct pathways. antigen presenting cells (apcs; especially macrophages and dendritic cells; dcs) are long-lived cells that capture antigens and subsequently process and present them at the cell surface, where they are recognized by t-lymphocytes. this process provides a long-term adaptive immune response to fungi, bacteria, and parasite. after internalization by the apc, antigens pass through phagosome/lysosome vesicles, where they form complexes with mhc class ii (figure 2 ), which are recognized by helper cd4+ t lymphocytes (exogenous pathway). in contrast, self cell antigens and virus synthesized within cells (mostly non-apcs) are degraded by the proteasome in the cytosol and nucleus. in successive steps, the antigen is processed, folded, and incorporated into the mhc class i ( figure 2 ) and the complex exposed on the cell surface, and recognized by cytotoxic cd8+ t lymphocytes (endogenous pathway). these two pathways overlap and some antigens are presented by both mhc class i and ii, in a process called cross-presentation. this has been described in dcs responding to viral infection, transplant rejection, and some autoimmune diseases and cancer. moreover, a wide range of pathogens passing or living in the phagosome such as mycobacterium tuberculosis, salmonella typhimurium, toxoplasma gondii, and especially leishmania spp and trypanosoma cruzi are all crosspresented in association to high levels of cd8 + t cells [31] . pdi as part of the er protein folding machinery directly regulates antigen processing of the mhc class i complex [32] [33] [34] [35] . antigens that are degraded by peptidases and proteasome to shorter peptides in the cytosol and nucleus can be further transported to the er through the tap system, a transmembrane er type of atp-binding cassette (abc) peptide transporter family [36] . er-located pdi interacts with the peptide-loading complex (pcl) that efficiently promotes peptide assembly with mhc class i molecules and supporting the exit of the peptide-antigen complex from the er [32] [33] [34] . other pcl components include calreticulin, tapasin, erp57 (another pdi family member), and the tap transporter itself. cells lacking pdi present much less peptide loading to mhc class i and the disulfide bridge between the peptide and mhc groove remains in a reduced redox state [32] . normally, this interaction is affected by the redox exchange between pdi (predominantly oxidized) and erp57 (predominantly reduced) [32] , a condition in which pdi favours the release of peptide-mhc class i from the pcl and the antigen-mhci complex is exited from er [34] . in fact, pdi-bound peptide facilitates the disassembly of the tapasin-erp57 complex while the pdi unbound to the complex is unable to interact with tapasin-erp57, retaining mhc i molecules in the er [34] . overall, pdi redox activity modulates the stability of the antigen peptide-mhc class i complex and further determines the transport of the complex to the plasma membrane [32] [33] [34] [35] . these redox effects may vary according to the type of antigen and some pathogens interfere with this pathway to escape antigen process and evading cd8 + t-cells recognition. this is the case for us3 protein from human cytomegalovirus, which enhances pdi degradation via the proteasome [32] . pdi participation in immune response, however, goes beyond its role in the er protein folding machinery and it acts at other cellular steps of host-pathogen interaction. pdi in the er is also thought to play a role in parasite phagocytosis, and the pdi displayed on the cell surface can mediate the entry of some viral, bacterial, and protozoan. pdi is also implicated in protein unfolding and trafficking of some pathogenic antigens across the endoplasmic reticulum and towards the cytosol by the endoplasmic reticulum-associated degradation system (erad). this is the main pathway where proteins are retrotranslocated from the er to cytosol and further degradated by the proteasome. next, we discuss some examples of the cellular association between host pdi and different pathogens. phagocytosis is the main gate for large microbes to enter into apcs. after binding and attaching to the pathogen, these cells can internalize organisms and large particles even bigger then their own size, which are then phagocytosed in an active process that involves intense membrane remodelling [37] . proteomics studies accumulated over the last decade revealed the presence of er chaperones in the isolated phagosome, uncovering a process called er-mediated phagocytosis [38] [39] [40] [41] [42] [43] [44] [45] . er chaperones were detected in phagosomes of macrophages exposed to different particulate material and pathogens, including latexbeads opsonised or not with immunoglobulin g (igg) or mouse serum (to facilitate entry through the fcr or complement receptors), igg-opsonized erythrocytes, promastigotes of leishmania donovani derived from wild-type cells or cell-surface lpg knockout, among other parasites [38] . a mix of er and endocytic vesicles in the formation of the parasitophorous vacuoles (pvs) during the uptake of different leishmania spp. was recently shown in macrophages overexpressing er-tagged green fluorescent protein [41] . the presence of the er proteins sec61, bip/grp78, and pdi in the phagosome of apcs [38, 41] support the idea that the er provides the necessary machinery for antigen translocation from the phagosome to the cytoplasm and thus, possibly converges mhc class i and class ii antigen cross-presentation [42] (figure 2 ). there are several other complementary hypotheses on how peptides cross from phagosomes to cytoplasm during the cross-presentation process [43, 44] . neutrophils are short-lived cells (half-life of 4-8 h in the human circulation) and very active in the phagocytosis of large microbes such as bacteria, parasites, and fungus. contrary to apcs, neutrophils only contain restricted amounts of er machinery and are thought to lack the er-mediated phagocytosis process [38] . whether er proteins functionally operate on phagocytosis-mediated infection has not been well characterised yet. an important work has shown that dictyostelium lacking both er calreticulin and calnexin present altered phagocytic cup formation and substantial decline in phagocytosis [45] . these two proteins utilise ca, and their disruption per se affects actin filaments and plasma membrane remodelling during phagocytosis [45] . we recently showed that pdi is critically involved in leishmania parasite infection in vitro [22] . we showed that phagocytosis of promastigotes (but not amastigotes) of leishmania chagasi was significantly inhibited by macrophage incubation with the thiol/pdi inhibitors dtnb, bacitracin, phenylarsine oxide, and neutralizing pdi antibody in a parasite redox-dependent way [22] . the phenylarsine response is of particular interest, since this arsenic compound may act similarly to antimonials, widely used in leishmaniasis chemotherapy [46, 47] . pdi preferentially affects parasite internalization and the phagocytosis of the promastigote forms is increased when wild-type pdi is overexpressed in macrophages, an effect opposed by pdi knockdown. at later stages of infection (i.e., after 4 h), pdi from promastigote-infected j774 macrophages was immunoprecipitated and subsequently blotted with an anti-leishmania antibody revealing a parasite band at ∼ 94 kda [16, figure 10 (b), lane 5]. subsequent removal and analysis of this band by mass fingerprint spectrometry showed a 58% match with elongation factor 2 (ef2) of l. major (q4q259; data not shown). the incubation of purified bovine pdi (sigma, p3818) and parasites did not yield any detectable protein complexes, suggesting that the macrophage milieu may be important to sustain pdi-ef2 association [22] . interestingly, leishmania ef2 has important virulent features and acts as a soluble antigen in lymphocyte stimulation in vitro [48] and in vivo [49] . moreover, proteomics studies revealed that ef2 is secreted during promastigote differentiation into the amastigote stage with potential immunomodulatory proprieties in animal models [50] . leishmania ef2 is therefore of particular interest for leishmania therapeutic interventions such as vaccines. although our studies did not address the role of the er in mediating phagocytosis, these data provide compelling evidence for a functional role of er-pdi in a host-parasite interaction. other mechanisms underlining pdi-mediated l. chagasi promastigote phagocytosis involves its association to ros production by phagocyte nadph oxidase and this is discussed next. the nadph oxidase (nox) family of enzymes uses nadph as an electron donor to convert oxygen to superoxide anion (o 2 •− ), a precursor of h 2 o 2 and other powerful oxidants such as hydroxyl radical and peroxynitrite (in the presence of nitric oxide), collectively called ros [3, 51, 52] . each of the seven oxidase family members is characterized by a distinct catalytic subunit (i.e., nox1-5 and duox1-2), and has differing requirements for additional protein subunits [51, 52] . the prototypic member of the nox family, nox2 oxidase (or gp91 phox oxidase), is best known for its role in neutrophil and macrophage phagocytosis. genetic defects in the enzyme are related to chronic granulomatous disease, a condition in which affected children suffer from recurrent severe fungal and bacterial infections due to defective phagocyte function [51] . each nox isoform forms heterodimers with a lower molecular weight p22 phox subunit and is predicted to be membrane-bound. nox2 is normally quiescent and acutely activated by agonists such as pma, lpg, and cytokines in a tightly regulated process in which cytosolic subunits (p47 phox , p67 phox , p40 phox , and rac1 in the case of macrophages and dendritic cells, or rac2 in neutrophil) associate with the nox2-p22 phox heterodimer to initiate enzyme activity [51, 52] . nox2 also has electrogenic features [53] and in apc cells is linked to the regulation of phagosome/lysosome ph and antigen processing [54, 55] . usually, phagosome acidity is maintained by a vacuolar atpase (v-atpase) that transports protons from the cytosol into the phagosome lumen, therefore regulating the function of lysosome proteases in the fused phagolysosomes. savina et al. [56] [57] [58] have shown that nox2-derived superoxide in the phagosomal vesicle promptly consumes protons maintaining a higher ph ambient in dendritic cells during particle internalization, which favours antigen processing and presentation [56] [57] [58] . opposite results were found in macrophages [56] [57] [58] . the selective role of nox2 in different phagocytic cells remains to be defined. the jury is out on whether the results shown in macrophages (association of nox2 to rab27a; a member of rab family of gtpases) are related to vesicle traffic molecule assembly and quality, or rather associated to degradation processes [59] . nox complex protein expression and function is greatly affected by redox compounds, and it is especially regulated by pdi with implications for cell signalling [60] . the association of pdi to p22 phox and other nox isoforms in different cell types, especially in vascular cells, has been previously described [60] [61] [62] . a functional and spatial/physical interaction between pdi and the p22 phox oxidase subunit was shown in macrophages [22] and more recently between pdi and p47 phox in neutrophils [62] . in macrophages, pdi-nox association was correlated to leishmania infection in vitro [22] . it is well known that during phagocytosis of leishmania, nox2 is activated and parasite uptake is inhibited by antioxidants such as catalase [22] . intriguingly, in the course of promastigote infection, some parasites evade that stressful condition and convert themselves into intracellular amastigotes, multiplying and resulting in progression to a disease process. overall, our studies support the view that parasite phagocytosis/infection by macrophages is a redox process mediated by pdi in at least two ways. initially, pdi-nadph oxidase increases ros production generating an oxidizing milieu, which seems to favour promastigote infection. the downstream role of ros generated by pdi-nadph oxidase remains unknown but can be related to the unfolded protein response signalling [63] or, similar to pdi-ero1, to protein folding in the macrophage er compartment with key implications for antigen processing. nevertheless, at later stages of infection, macrophage pdi physically associates with leishmania elongation factor-2 (as discussed earlier). some viruses envelop their genetic material within a protein-coated capsid in a further lipid membrane layout, for example, influenza virus, baculovirus, hepatitis-c, hiv, and herpes virus. these enveloped particles require successive steps to successfully entry and infect host cells. they usually first attach onto host receptors (and attachment factors), and their membranes fuse to interact with endosome vesicles that traffic the virus toward the endoplasmic reticulum, where it is uncoated. the proteins are finally transported to the cytosol and nucleus [64] (figure 2 ). there is convincing evidence showing that most viral infections are strongly influenced by changes in the redox environment and that host pdi mediates infection of enveloped viruses [65] [66] [67] [68] [69] [70] . in the course of hiv infection, the virus first binds to attachment factors, for example, mannose binding c-type lectin receptor and intracellular adhesion molecule (icam-3) on the surface of host cd4 + t cells. the glycoprotein 120 (gp120) subunit of the virus envelope binds to immunoglobulin g of cd4 + and undergoes conformational changes, allowing the virus to interact with its coreceptors, cxcr4 or ccr5. these interactions favour downstream conversions of gp41 envelope subunit to a competent fusion conformation. initial studies showed that membrane-impermeable pdi inhibitors and monoclonal antibodies against pdi prevent hiv-1 infection [65] . it was then revealed that the domain d2 of the cd4 has redox-active disulfide bonds and is regulated by thioredoxin [66] . using membrane-impermeable reducing agents (especially arsenical-derived compounds) and labelling thiol reagents, it was demonstrated that cd4 + reactive thiols critically drive hiv entry into cells [66] . work from another group also revealed that pdi, on the surface of hiv-1 target cells, reduces disulfide bonds of the recombinant envelope glycoprotein gp120 (reaction 1, figure 1 ), a reaction prevented by the usual pdi inhibitors [67] . intriguingly, pdi silencing in u373 and hela cells had little impact on hiv infection itself as compared to the effect mediated by general thiol inhibitors [68] . the reasons for this discrepancy remain to be elucidated and raise the question whether the reductive effect of pdi is coupled to other redox proteins (e.g., thioredoxin or nox's) that could amplify virus-cd4 redox association in some cells. it is noteworthy that in these later studies, pdi knockdown on the cell surface was not evident as compared to massive loss of most pdis within the er; an observation that supports the idea that pdi in the er has little impact in hiv-mediated infection [68] . thiol inhibitors also affect viral fusion as that mediated by the fusion (f) protein from the paramyxovirus newcastle disease virus [69] . the overexpression of pdi family members pfdi and erdj5 has also been shown to significantly catalyze the reduction of thiols in f protein, facilitating membrane fusion [70] . there is evidence suggesting a possible association between pdi and infection mediated by some members of the of herpesviridae viruses family [71] . pdi is also implicated in the attachment of some bacteria from different species of chlamydia [72] [73] [74] . chlamydia is an obligatory intracellular pathogen that causes diverse diseases in humans. the most common species are chlamydia trachomatis, which is sexually transmitted and can cause blindness and infertility, and c. pneumoniae, which affects the respiratory tract. cho cells have impaired endogenous pdi expression due to a defect in truncated mrna processing, thus providing a valuable model to understand the effect of pdi-mediated cell-cell interaction and infection. these cells are very resistant to chlamydia infection showing impaired attachment, an effect restored by ectopic expression of pdi [73] . similar to hiv infection, the molecular mechanisms most likely include the reductive activity of pdi (reaction 1, figure 1 ) on the surface of cho cells [72] . crossing the endoplasmic reticulum (er) membrane is an irreversible process for most proteins. in some cases, however, this flow is reversed and misfolded proteins retained in the er are retrotranslocated to the cytosol via erad to be degraded by the proteasome. this pathway is also exploited by small pathogens, especially non-enveloped viruses and some bacterial toxins, to gain access to the cytosol. in these cases, antigenic particles that reach the er by different means suffer molecular redox rearrangements and binding to pdi allowing them to be transported back to the cytosol or nucleus. well-known examples are infections mediated by polyomaviruses (py) and simian virus 40 (sv40) extensively studied in the field of carcinogenesis. after sv40 interaction with the gm1 receptor on the cell surface, the particle enters the host cell through endocytosis and traffics via the caveosome (a particular caveolin containing endosome with neutral ph) towards the er compartment [75, 76] . sv40-coated pentamers are linked to each other by disulfide bonds between cysteine 104 (c104). further isomerisation in the er is crucial for the viral uncoating process. in vitro cell screening shows that among all er-resident proteins, pdi and erp57 more specifically regulate sv40 infection [75] . pdi silencing substantially decreases sv40 infection that is also dependent on some redox sensitive cysteines on the viral particle [75] . pdi cooperation with erassociated erad proteins derlin-1 and sel1l is ca dependent and facilitates sv40 traffic through erad [75] . a similar pathway is used by some nonobligatory intracellular bacteria that exert their effect through production of potent endotoxins, such as diphtheria toxin (dt) and cholera toxin (ct). these proteins function similarly to some plant toxins, such as ricin and abrin. conversion into toxic proteins involves cleavage of their interchain disulfide bond, allowing them to traffic into the endocytic pathway within the host cell [77, 78] . in humans, ct is derived from the bacterium vibrio cholerae that causes cholera disease and has 2 subunits (a1 and a2). the protein first attaches to the host cell surface via gm1 and the subunit a2, which contains a kdel sequence, and is transported back to the er (see earlier discussion). there, pdi reduces and unfolds a2 and a1 that exit the er via the sec61 channel into the cytosol. pdi in the reduced state (reaction 1, figure 1 ) binds to the toxin and subsequent oxidation of pdi, probably via ero1α, enables the release of ct toxin [79, 80] . the active polypeptide a1 efficiently modifies a heterotrimeric g protein in the cytosol that leads to massive loss of chlorine and water secretion by intestinal epithelial cells in mammals, resulting in severe diarrhoea. in this article we have reviewed the main cellular aspects of pdi-mediated host pathogen interactions and the pathways that are involved in viral, bacterial (including bacterial toxins), and parasitic infections. a number of cellular mechanisms through which pdi modulates some specific cellular pathways in immune cells have been described, such as redox-sensitive attachment, antigen presentation in the er and exit from it, and association to phagosome and ros production by nadph oxidase (figure 2 ). many of these responses are antigen-specific and the precise mechanisms of action remain to be fully elucidated, especially in the context of redox changes in cross-presentation phenomena. moreover, little is known about the role of pdi in infection per se, as well as how pdi signals to a more integrated cellular response to stress [63] . pdi global knockout mice are only viable until birth, but partial gene-modified mice and also modified pathogens will help to reveal the significant redox role of pdi and its redox partners. overall, pdi is a key regulator that may propagate or limit the severity of the infection processes, depending on the infectious organism involved. a better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections. endoplasmic reticulum mhc: major histocompatibility complex h 2 o 2 : hydrogen peroxide erp57: a member of pdi family also know as glucose-regulated protein or 58-kd (grp58) nf-kb: factor nuclear kappa b ap-1: activator protein 1 stat-3: signal transducer and activator of transcription 3. cell entry by enveloped viruses: redox considerations for hiv and sars-coronavirus role of nitric oxide synthesis in macrophage 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reticulum oxidoreductases, mitochondrial electron transport, and nadph oxidase how viruses enter animal cells inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction disulfide exchange in domain 2 of cd4 is required for entry of hiv-i inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent hiv-1 entry role of protein disulfide isomerase and other thiol-reactive proteins in hiv-1 envelope protein-mediated fusion thiol/disulfide exchange is required for membrane fusion directed by the newcastle disease virus fusion protein overexpression of thiol/disulfide isomerases enhances membrane fusion directed by the newcastle disease virus fusion protein the human cytomegalovirus ul37 immediate-early regulatory protein is an integral membrane n-glycoprotein which traffics through the endoplasmic reticulum and golgi apparatus attachment and entry of chlamydia have distinct requirements for host protein disulfide isomerase chlamydia attachment to mammalian cells requires protein disulfide isomerase protein disulfide isomerase, a component of the estrogen receptor complex, is associated with chlamydia trachomatis serovar e attached to human endometrial epithelial cells simian virus 40 depends on er protein folding and quality control factors for entry into host cells downregulation of protein disulfide isomerase inhibits infection by the mouse polyomavirus cleavage of disulfide bonds in endocytosed macromolecules. a processing not associated with lysosomes or endosomes cell surface sulfhydryls are required for the cytotoxicity of diphtheria toxin but not of ricin in chinese hamster ovary cells unfolded cholera toxin is transferred to the er membrane and released from protein disulfide isomerase upon oxidation by ero1 the ero1α-pdi redox cycle regulates retro-translocation of cholera toxin protein disulfide isomerase and host-pathogen interaction the authors thank daniel c. pimenta for mass spectrometry experiments (centre for applied toxinology cat/cepid, butantan institute). they also thank dr. joão wosniak jr. from vascular biology laboratory, heart institute (incor) for helpful discussion. this paper was supported by auxílio fundação de amparò a pesquisa do estdado de são paulo (fapesp) and conselho nacional de pesquisa (cnpq), instituto do milênio redoxoma; fundação ej zerbini (incor). a. m. shah, s. ioannis and c. x. c. santos are also supported by the british heart foundation and a leducq fondation transatlantic network of excellence award. key: cord-001567-3bw7jbzq authors: borlak, jürgen; singh, prashant; gazzana, giuseppe title: proteome mapping of epidermal growth factor induced hepatocellular carcinomas identifies novel cell metabolism targets and mitogen activated protein kinase signalling events date: 2015-02-25 journal: bmc genomics doi: 10.1186/s12864-015-1312-z sha: doc_id: 1567 cord_uid: 3bw7jbzq background: hepatocellular carcinoma (hcc) is on the rise and the sixth most common cancer worldwide. to combat hcc effectively research is directed towards its early detection and the development of targeted therapies. given the fact that epidermal growth factor (egf) is an important mitogen for hepatocytes we searched for disease regulated proteins to improve an understanding of the molecular pathogenesis of egf induced hcc. disease regulated proteins were studied by 2de maldi-tof/tof and a transcriptomic approach, by immunohistochemistry and advanced bioinformatics. results: mapping of egf induced liver cancer in a transgenic mouse model identified n = 96 (p < 0.05) significantly regulated proteins of which n = 54 were tumour-specific. to unravel molecular circuits linked to aberrant egfr signalling diverse computational approaches were employed and this defined n = 7 key nodes using n = 82 disease regulated proteins for network construction. string analysis revealed protein-protein interactions of > 70% disease regulated proteins with individual proteins being validated by immunohistochemistry. the disease regulated network proteins were mapped to distinct pathways and bioinformatics provided novel insight into molecular circuits associated with significant changes in either glycolysis and gluconeogenesis, argine and proline metabolism, protein processing in endoplasmic reticulum, hifand mapk signalling, lipoprotein metabolism, platelet activation and hemostatic control as a result of aberrant egf signalling. the biological significance of the findings was corroborated with gene expression data derived from tumour tissues to evntually define a rationale by which tumours embark on intriguing changes in metabolism that is of utility for an understanding of tumour growth. moreover, among the egf tumour specific proteins n = 11 were likewise uniquely expressed in human hcc and for n = 49 proteins regulation in human hcc was confirmed using the publically available human protein atlas depository, therefore demonstrating clinical significance. conclusion: novel insight into the molecular pathogenesis of egf induced liver cancer was obtained and among the 37 newly identified proteins several are likely candidates for the development of molecularly targeted therapies and include the nucleoside diphosphate kinase a, bifunctional atp-dependent dihydroyacetone kinase and phosphatidylethanolamine-binding protein1, the latter being an inhibitor of the raf-1 kinase. electronic supplementary material: the online version of this article (doi:10.1186/s12864-015-1312-z) contains supplementary material, which is available to authorized users. liver malignancies are common cancers worldwide and are responsible for approximately one million deaths each year with most hcc patients having poor prognosis as a result of rapid disease progression. the relative 5-year survival rate is about 15% and can be attributed to an advanced stage of disease at the time of diagnosis, the occurrence of cirrhosis and of other co-morbidites [1] . detection of early stages of disease is essential for an improved prognosis and overall survival. however, apart from alpha-fetoprotein (afp) only a few serological markers are available in clinical practice (such as glypican-3, mir-21, fucosylated gp73, α-fucosidase) with afp diagnostics remaining unsatisfactory because of its low sensitivity and the non-specific correlation between the clinical behavior of hcc and afp blood levels. for this reason, new biomarkers are in strong demand [2, 3] and more selective markers, such as soluble interleukin-2 receptor levels, are evaluated [4] . importantly, research into the molecular pathogenesis of hcc identified several signalling pathways as deregulated. this inspired the development of molecularly targeted therapies such as the multikinase inhibitor sorafenib that inhibits signalling of c-raf-1, mek, erk, vegfr, pdgfr and other kinases, effectively [5] . given that epidermal growth factor is an important mitogen for hepatocytes we were particularly interested in an understanding of the consequences of its targeted overexpression in liver. in our initial study we reported the oncogenomics and pathology of egf induced hepatocarcinogenesis and an identification of molecular circuitries linked to exaggerated egfr signalling [6] . furthermore, we investigated the serum proteome of egf-tumourbearing mice to obtain information on disease-regulated proteins and to search for novel biomarkers at different stages of disease [7] . note, a regulatory loop was proposed whereby egf induces transcriptional activation of hdac2 by ck2α/akt activation in liver cancer cells [8] . additionally, inhibition of egfr in different animal models by erlotinib was shown to attenuate liver fibrosis and the development of hepatocellular carcinoma [9] thus suggesting new therapeutic intervention strategies in the prevention of hcc. indeed, the egf receptor tyrosin kinase plays a much wider role in the immortalization of different cell types as originally anticipated [10] , and is highly expressed in a number of solid tumours and egfr over-expression correlates well with tumour progression, resistance to chemotherapy and poor prognosis. the present study aimed at an identification of disease regulated proteins to facilitate an improved understanding of its complex signalling networks and to search for cross-talk amongst other pathways while an identification of disease regulated proteins would aid the development of molecularly targeted therapies. for this purpose, a two-dimensional electrophoresis and maldi-tof/tof ms strategy was employed to identify disease regulated proteins in an egf transgenic mouse model of hcc. this resulted in an identification of 96 statistically significant regulated proteins of which 54 are uniquely expressed in liver cancer. importantly, 11 out of 54 mouse tumour specific proteins were likewise uniquely expressed in human hcc and 49 disease regulated proteins identified in egf induced liver cancer were similarly regulated in human hcc, as determined by immunhistochemistry using different antibodies and the information given in the publically available human protein atlas depository. clinical significance of the identified proteins could be demonstrated and a total of 37 so far unkown proteins could now be related to egf induced liver cancer, several of which are likely candidates for the development of molecularly targeted therapies. this includes the nucleoside diphosphate kinase a, bifunctional atp-dependent dihydroxyacetone kinase, phosphatidylethanolamine binding protein1, i.e. an inhibitor of the raf-1 kinase as well as aldo-keto reductase family 1 proteins, members c14 and c6, interleukin 25 and the v-crk sarcoma virus ct10 oncogene homolog. finally, to gain insight into the molecular circuitries of egfr induced hepatocarcinogenesis diverse computational approaches were employed. this revealed master regulatory proteins and permitted network constructions of 82 disease regulated proteins with protein-protein interactions being confirmed for > 70% of regulated proteins in string analysis. their regulations were also studied by immunohistochemistry in egf transgenic hcc. we also compared the serum and liver proteomes of hcc bearing mice and found 10 proteins to be similarly regulated, thus evidencing leakage of tumour proteins that can be detected in serum. obviously, these are highly interesting biomarker candidates, 6 of which were also regulated in human hcc as determined by immunohistochemistry. all animal work followed strictly the public health service (phs) policy on humane care and use of laboratory animals of the national institutes of health, usa. formal approval to carry out animal studies was granted by the animal welfare ethics committee of the state of lower saxony, germany ('lower saxony state office for consumer protection and food safety' (laves)). the approval id is az: 33.9-42502-04-06/1204. a total of n = 12 c57/bl6 non-transgenic and n = 12 egf transgenic mice (aged 6-8 months), weighing 25-33 g, were housed in makrolon® type iii cages. water and food (v1124-000, ssniff, the netherlands) was given ad libitum. the temperature and relative humidity was set to 22 ± 2°c and 40-70%, respectively and a 12-h day and night cycle was used. tris, urea, thiourea, chaps, dithiothreitol, bromophenol blue, glycerin, sodium dodecyl sulfate, glycine, temed, ammoniumperoxodisulfate, ammonium sulfate, ammonium bicarbonate, colloidal coomassie blue, and acrylamide were purchased from roth (karlsruhe, germany). iodacetamide was obtained from serva (heidelberg, germany) and benzonase was purchased from novagen (darmstadt, germany). ampholytes (biolyte 3-10) were purchased from bio-rad laboratories (münchen, germany) and destreak was obtained from amersham bioscience (freiburg, germany). mice were anesthetized with ketamine 10% 100 μl/100 g and xylazine 2% 50 μl/100 g, and after surgical removal the liver was perfused and rinsed with ice cold ringer solution until free of blood. approximately 0.1 g of the liver sample was ground in a mortar under liquid nitrogen flow. then, the samples were processed with 0.5 ml of a buffer containing 40 mm tris base, 7 m urea, 4% chaps, 100 mm dtt, and 0.5% (v/v) biolyte 3-10 first (lb2). the suspensions were homogenized by sonication (3 × 20 s) and after addition of 3 μl of benzonase (endonuclease that degrades dna and rna) were left at room temperature for 20 min. the samples were then centrifuged at 12,000 g for 20 min. the pellets were washed and sonicated for 5 min with a further 0.5 ml of lb2 and centrifuged at 12,000 g for another 20 min, and the resulting two fractions of supernatant were collected (extract a). finally, the pellets were redissolved with 0.5 ml of buffer containing 40 mm tris base, 5 m urea, 2 m thiourea, 4% chaps, 100 mm dtt, and 0.5% (v/v) biolyte 3-10 (lb3), sonicated, and centrifuged at 12,000 g for 20 min. the pellet was collected, and the supernatant was marked as extract b. from the same animals, a further 0.1-g portion was ground in a mortar, but was now treated with 0.5 ml of lb3. the suspensions were sonicated, incubated with benzonase, and centrifuged. the pellets were then washed with another 0.5 ml of lb3, sonicated and centrifuged, and the supernatants were collected (extract c). proteome mapping was done under a variety of conditions, e.g. extraction with lysis buffers 2 and 3. in addition, proteins were separated at two different ph ranges [5] [6] [7] [8] [9] [10] . a total of 4 independent experiments were carried out, and duplicate measurements were run for each experiment. the protein concentration of all extracts was determined using the bradford assay. liquid-phase ief pre-fractionation was performed in the rotofor cell system (bio-rad) following the supplier's instructions. ion exchange membranes were equilibrated overnight in the appropriate electrolyte (anion exchange membranes in naoh 0.1 m and cation exchange membranes in h 3 po 4 0.1 m). after four runs ion exchange membranes were always discarded and new membranes were replaced for the other samples. for each run, the electrode chambers were filled with appropriate fresh electrolytes (30 ml) . initially, the cell was filled with pure water and run for 5 min at 5 watts constant power to remove residual ionic contaminants from the membrane core and ion exchange membranes. approximately 32 ml of lb2 were used to fill the cell. a total of 60 mg of total proteins in approximately 2 ml of lb2 were added to the cell to reach the maximum loadable volume (40 ml) . focusing started at 12 watts constant power. after approximately 4 hours the voltage increased to 3000 v and the wattage decreased to 3 w. the focused proteins were harvested in 20~1.5 ml fractions, and ph values were checked. fractions having ph values between 3 and 7.0 were collected and denoted "a-a" (acid). fractions having ph values > 7.0 were collected and denoted "a-b" (basic). again, the protein concentration was determined for both fractions (a-a and a-b) by the bradford method. approximately 30 mg of protein were recovered at the end of the liquid-phase ief pre-fractionation from an initial 60-mg load. the losses are accounted for by the multi-step pre-fractionation procedure, but are not the result of a precipitate that could not be dissolved in our lysis buffer. after each run the membrane core was cleaned with naoh 0.1 m overnight and sonicated for 5 min in water before the new focusing. isoelectric focusing (ief) -first dimension ief was performed using precast linear ipg strips. the 17-cm ipg strips 7-10 and 5-8 were loaded with 1.5 mg of proteins by active rehydration (12 h, 50 v) . samples destined to be separated by ipg strips 7-10 received an excess of hydroxyethyldisulphide (hed) (destreak™) prior to the focusing run. focusing began at 250 v for 20 min in rapid mode, 10,000 v for 5 h in linear mode and 10,000 v for 50,000 vh in rapid mode (for the ipg strips 5-8). ief for the strips 7-10 was carried out at 250 v for 60 min in rapid mode, 10,000 v for 3 h in linear mode and 10,000 v for 50,000 vh in rapid mode. each sample was analysed in duplicate. control and hcc samples were run always at the same time (6 control and 6 hcc samples). after ief, the ipg strips were either stored at −80°c or transferred to 10 ml of equilibration buffer (6 m urea, 30% w/v glycerin, 2% w/v sds, 50 mm tris-hcl ph 8.8) with 2% w/v dtt and 0.5% v/v bromophenol blue solution (0.25% w/v bromophenol blue, 1.5 m tris-hcl ph 8.8, 0.4% w/v sds) and incubated for 20 min at room temperature. strips were removed and incubated in equilibration buffer with 4% w/v iodoacetamide and 0.5% v/v bromophenol blue solution for further 20 min at room temperature. finally, the strips and 10 μl sds-page molecular weight standard on filter paper were placed on top of the 20 cm x 20.5 cm 12% second-dimension gel (12% v/ v acrylamide/bis solution, 375 mm tris, ph 8.8, 0.1% v/v sds, 1/2000 temed, 0.05% v/v aps). both were fixed in place with a 0.5% w/v agarose overlay. gels were run in protean plus dodeca cell (bio-rad) at 70 v for approximately 14 h, followed by 200 v until the bromophenol blue dye reached the bottom of the gel. the running buffer (25 mm tris, 0.2 m glycin, 0.1% sds) was cooled externally to 16°c. gels/proteins were fixed overnight in 30% ethanol, 2% phosphoric acid, and washed 3 x 20 min with 2% phosphoric acid. the gels were equilibrated with 15% ammoniumsulfate, 18% ethanol, 2% phosphoric acid for 15 min and finally stained with colloidal coomassie blue for 48 h. after staining, gels were washed 10 min with pure water and scanned on a molecular fx scanner (bio-rad) at 100 μm resolution. protein spots were imaged first automatically and then manually and analysed using the pdquest™ software (bio-rad). the normalization was carried out in total density in gel mode according to the manufacturer's recommendation. gels were excised using the spot cutter of bio-rad and placed into 96-well microtiter plates. excised gel spots were washed manually with 20 μl of water for 10 min and destained twice, first with 15 μl ammonium bicarbonate 50 mm for 5 min and then with 15 μl 50% ammonium bicarbonate 50 mm -50% acetonitrile for 5 min. finally, the gel particles were covered by acetonitrile until gel pieces shrunk and left to dry for 10 min. all gels/proteins were digested manually in situ with 4 μl of ammonium bicarbonate 50 mm containing 20 ng trypsin (sequencing grade modified trypsin, promega, germany). after 15 min each gel piece was re-swelled with 10 μl of ammonium bicarbonate 50 mm and incubated for 4 h at 37°c. after 4 h the reaction was stopped by adding 10 μl of trifluoroacetic acid 1% containing 1.5% (w/v) n-octyl-beta-d-glucopyranoside (ogp) (applichem). for the application of the samples, 4 μl of peptide solution were loaded onto an mtp anchor chip target 600/384 (bruker daltonics) previously prepared with a saturated solution of matrix, alpha-cyano-4-hydroxy-cinnamic acid (alpha-hcca) (bruker daltonics, bremen, germany). maldi-ms was performed on an ultraflex ii maldi-tof/tof (bruker daltonics) mass spectrometer equipped with a smartbeam™ laser and a lift-ms/ms facility. the instrument was operated in positive ion reflectron mode and an acceleration voltage of 25 kev for the peptide mass fingerprint (pmf) mode. typically, 600 spectra, acquired at 100 hz, were summed and externally calibrated. in the case of ms/ms-cid the lift device was used for selection and fragmentation of the ions; the acceleration voltage in the ion source 8 kv, the timed ion selector was set to 0.4% (relative to parent mass), and argon was used as collision gas (~4-6 × 10-6 mbar). resulting fragments were further accelerated in a second source by 19 kv and analysed by a two-stage gridless reflectron. typically, 400 shots were accumulated for the parent ion signal and 1000 shots for the fragments. flexcontrol™ 3.0, and flexanalysis™ 3.0 were used as instrument control and processing software (bruker daltonics, bremen, germany). a calibration standard was used for the external calibration of spectra (peptide calibration standard for mass spectrometry, which covered the mass range~1000-4000 da internal calibration was achieved using trypsin autolysis products (m/z's 1045.564, 2211.108 and 2225.119) resulting in a mass accuracy of ≤ 50 ppm. spectra were collected by the flexcontrol software without smoothing or baseline subtraction and a peak resolution higher than 6000 or 7000 a.u. in case of dhb and chca matrixsample preparation, respectively. the spectra were sent to the flexanalysis software which labeled the peaks for protein identification by proteinscape 1.3 or biotools 3.1 (bruker daltonics). trypsin autolysis products, tryptic peptides of human keratin and matrix ions were automatically discarded by proteinscape (mass control list). proteinscape score booster feature was used to improve database search results by automatic iterative recalibrations and background eliminations. protein scores greater than 53 were considered significant (p <0.05, mascot) and an annotation as mouse protein as the top candidates was requested in the search when no restriction was applied to the species of origin. identified proteins were checked individually for further considerations. for pmf peak picking the snap peak detection algorithm, a signal to noise threshold of 6, maximal number of peaks 100, a quality factor threshold 50 and baseline subtraction tophat was applied. peptide masses were searched against the swiss-prot database (download 2005-197 228 sequences, 71 501 181 residues) employing the mascot server (in-house mascot-server, matrix sciences ltd., http://www.matrixscience.com/, revision 2.0.0), taking into account carbamidomethyl of cysteines -carbamidomethyl (c)-as fixed modification and possible oxidation of methionine -oxidation (m)-as a variable modification but allowing one missed cleavage. based on initial data, ion precursors were selected by proteinscape for tandem ms data acquisition (by lift-tof/tof, bruker daltonics, bremen, germany). in the mascot ms/ms ions search, the restriction mammalia was applied with peptide tolerance of (70 ppm and ms/ms tolerance of (0.9 da (fixed and variable modifications as pmf). the acceptance criteria for pmf-based identification were an individual ions score >27, at least five matching peptides and 10% peptide coverage of the theoretical sequences. livers, dissected from egf-overexpressing mice aged between 7-9 months, were fixed in 4% buffered paraformaldehyd and embedded in paraffin. 5 μm thick sections were deparaffinized and rehydrated through a descending alcohol series followed by a 4 min washing step in destilled h 2 o. subsequently, antigen retrieval was performed in citrate buffer (ph 6) by autoclaving the sections 15 min at 121°c. the envision kit (dako, hamburg, germany) was used for immunohistochemistry. the slides were rinsed with destilled h 2 o and after a 5 min incubation step in tris-buffered saline (washing buffer), endogenous peroxidase activity was blocked with dako peroxidase blocking reagent for 5 min followed by a second washing step. thereafter, the sections were blocked for 10 min with protein-block serum free (dako) and incubated with primary antibodies for 45 min. details of antibody dilutions with washing buffer are given in additional file 1: table s1. in the case of goat primary antibodies a rabbit-anti-goat bridging antibody (dako) was employed. specifically, the bound primary antibodies or bridging antibodies were detected by use of labelled polymer hrp anti-rabbit secondary antibody (envision kit; dako) and the immunoreactivity was visualized by dako liquid dab substrate chromogen system in a 5 min incubation. finally, the sections were counterstained with harris haematoxylin for 2 min, dehydrated in an ascending alcohol series, coverslipped and examined under a light microscope (leica, jülich, germany). a total of n = 122 disease regulated proteins were filtered for statistical significance at p < 0.05 (table 1 ). this yielded n = 96 statistically significantly regulated proteins two of which had identical accession number, i.e. aah81431 = atp synthase h+ transporting mitochondrial f0 complex, subunit d and bac36241 = apoa1 but differed in their spot ids as a result of posttranslational modifications. the statistically significantly regulated proteins were grouped into four different categories to yield 54 tumour specific (to), 9 up-regulated (ur), 19 down-regulated (dr) and 14 proteins only expressed in healthy non-transgenic control livers (co). categorization of tumour regulated proteins based on ontology terms 82 non-redundant tumour proteins covering to, ur and dr categories were considered and analysed for ontologies using the genexplain software (v.2.4.1), the biological pathways tools reactome (http://www.reactome.org) and kegg (http://www.genome.jp/kegg) and wikipathways (http://wikipathways.org). the tumour regulated proteins (to + ur + dr) were subjected to functional classification based on ontology terms and a p-value of <0.01 was considered to be significant. moreover, disease regulated proteins were analysed with the cytoscape software version 3.0.2 using the function go-tree levels and number or % of proteins for a given term (see additional file 2: table s2 ). master regulatory proteins were searched based on the designated workflow of the genexplain software. it is designed to find master regulatory molecules upstream of an input list of regulated tumour proteins. after annotation of the input datasets the tool for master regulator finding over geneways network (http://www.genexplain.com) was applied. specifically, the geneways software is used to automatically extract, analyse, visualise and integrate molecular pathway data from the published peer reviewed literature. it is based on document sorting, term identification, term meaning disambiguation, information extraction, ontology, visualization and system integration [61] . the following filtering threshold was used, i.e. score cutoff (0.2), search collection (geneways hub), maximum radius [4, 10] , fdr cutoff (0.05), z-score cutoff (1.0), penalty (0.1) and decay factor (0.1) (additional file 3: table s3 ). protein network for disease regulated proteins were also constructed using the string software (http:// string-db.org/). the underlying database informs on known and predicted protein-protein interaction and the constructed networks are based on active prediction methods of neighborhood, gene fusion, co-occurrence, co-expression, databases and textmining. eventually, confidence scores were calculated for each interaction pair and only those above default cutoff scores (0.4) were selected. finally, mapping of pathways information from reac-tome, kegg and wikipathways have been implemented the proteins are sorted in alphabetical order, and the ncbi annotation is given in the accession number column. molecular weight, pi, and mascot scores are also given. the column "gels", "c" (c = control) and "t" (t = tumour) indicate the frequency of positive identification of proteins in a total of 48 independent gels, whereas "lb2" and "lb3" (lb = lysis buffer) refers to the different lysis buffers employed. furthermore, references are given for those proteins which have already been described as hcc-associated whereas those marked with a star (*) are so far unknown as egfr disease regulated in hepatocellular carcinoma. over protein networks using information of known pathways and sustained proteins connecting these pathways in a given network. the histopathology and oncogenomics of egf induced liver cancers was previously reported [6] and an important finding of the study was the 100% incidence of malignant tumour formation in less than one year after birth. notably, a sequence of events was observed that initially consisted of diffuse large cell dysplasia followed by multiple dysplastic foci and nodules and growth of hcc. figure 1 a and b depict the histopathology of healthy non-transgenic control liver and egf induced tumours, respectively. after protein extraction 2de was performed. subsequently, the gels were scanned on a bio-rad molecular fx scanner at a 100 μm resolution. image analysis was done with the pdquesttm software and spots were detected automatically. a total of 122 proteins differed in expression or were de novo expressed when 2de gels of non-transgenic controls and hcc mice were compared (see table 1 for detailed information on the proteins identified and figure 1e -g depicting examples of zoomin-gels of some regulated proteins). among them are 96 statistically significantly regulated proteins (p ≤ 0.05) of which 63 were significantly up-regulated (ratio hcc/control ≥ 2) and included fibrinogen and subunits of it, vimentin, cu/zn superoxide dismutase, and apolipoprotein e ( figure 1f (i-iv), while 33 proteins were repressed in expression (ratio hcc/control ≤ 0.6) and included arginase 1, dhdh protein, glutathione peroxidase 1 and agmatine ureohydrolase ( figure 1g (i-iv) and table 1 ). a reference 2-de map of mouse liver and serum proteins was constructed that consists of more than n = 500 proteins [2, 7] . note, in our previous efforts we identified n = 25 serum proteins as regulated in the egf transgenic disease model. among them were alpha-fetoprotein, clusterin, fibrinogen-α and -γ, serum amyloid component p and several apolipoproteins all of which were significantly up-regulated. based on the combined use of 2de and maldi-ms a total of n = 122 differentially expressed proteins were identified (table 1 ) and included isoforms as well as post translational modifications of albumin (5 up-regulated spots), alpha enolase (4 down-regulated spots), apoliproptein a-i (2 up-regulated spots), atp synthase h+ transporting mitochondrial (2 down-regulated spots), fibrinogen beta (2 up-regulated spots), glycine n-methyltransferase (3 spots, in controls only), hsp60 (2 down-regulated spots), nit protein 2 (2 down-regulated spots), peroxiredoxin 6 (1 up-regulated spot and 1 down-regulated spot), and 4931406c07rik (2 up-regulated spots) (see table 1 ). importantly, a total of n = 37 so far unknown disease regulated proteins were identified that can now be related to egf induced liver cancer. these are marked with an asterisk in table 1 . furthermore, a comparison of serum and liver proteoms revealed n = 10 proteins to be regulated in common, thus evidencing leakage of tumour proteins into systemic circulation (table 2) . among them was serum afp; it's up-regulation and that of others was confirmed by western blot analysis (figure 2a-e) . likewise, apolipoprotein e was up-regulated both in serum and tumour samples, the ratio hcc/control being 2.2 and 3.9, respectively. in a previous study on human hcc increased expression of apoe was observed in 88% of study cases; however, gene apoe expression and serum levels were unchanged to suggest its accumulation and impaired secretion [21] . two isoforms of alpha-2-macroglobulin were up-regulated in serum of hcc-bearing mice (spot 1: ratio hcc/control = 1.8; spot 2: ratio hcc/control = 3.2). its expression was exclusively associated with tumours. finally, serum amyloid component p was up to 10-fold up-regulated in serum and its tissue expression was tumour specific ( table 2) . to further evidence disease regulated proteins and to provide information on their subcellular localization a total of n = 8 proteins were studied by immunohistochemistry. five of them were selected for their novelty (see table 1 ) while amphiregulin and epiregulin were chosen for their importance in the egf-signalling pathway. furthermore, hnf4α was studied for its pivotal role in liver cancer [62] . depicted in figure 3 are immunohistochemistry stainings performed with egf transgenic livers to confirm regulation and predominant cytoplasmic expression of arginase ii. note, arg2 is only expressed in hcc and recent evidence suggest modulation of arginine levels in the extracellular milieu to be part of an immune escape mechanism whereby lack of local arginine weakens tumour-infiltrating lymphocytes as t cells require adequate argine levels [63] . likewise, the tumour specific and cytoplasmic expression of the f-actin capping protein α1 subunit (capza1) and the predominant nuclear expression of tubulin β that was particularly visible beneath the liver capsule may possible promote microtubule stability and interactions of microtubules with endogenous proteins. furthermore, the induced and predominat cytoplasmic expression of the gdp dissociation inhibitor 2 (gdi2) protein is part of the control of vesicular trafficking. this protein is known to regulate gdp-gtp exchange amongst members of the rab family of proteins. the tumour specific and cytoplasmic expression of amphiregulin supports the notion of a switch in autocrine signalling and it has been reported that amphiregulin is a prognostic marker for poor outcome of a variety of malignancies including colorectal liver metastasis [64] . finally, the repressed nuclear expression of hnf4a was not unexpected and confirms earlier findings [62] . based on the information given in table 1 the human protein atlas depository (www.proteinatlas.org, version 12) was interrogated. as shown in additional file 4: table s4 48 out of 96 mouse liver cancer regulated proteins were likewise regulated in human hcc. it should be noted that for some proteins several antibodies were used to study their expression; only representative data were considered. importantly, out of the 54 proteins uniquely expressed in mouse liver tumours n = 11 were likewise uniquely expressed in human hcc thus evidencing clinical significance of our findings. we compared our previously published transcriptomic data of egf induced liver cancers with the proteomic data obtained in the present study. such comparisons revealed n = 22 genes to be significantly regulated of which n = 17 are in common regulated whereas for n = 5 genes transcript expression was opposite to that of the coded proteins (see additional file 5: table s5 ). 82 of the 96 significantly regulated proteins were mapped to 40 different biological processes (see figure 4a ) of which prominent examples are regulation of arginine metabolism and amino acid import, regulation of cdc42 protein signal transduction', cellular response to oxidative stress, hydrogen peroxide and superoxide, glycolysis and gluconeogenesis, regulation of cholesterol transport, protein-lipid complex and plasma lipoprotein particle remodeling, positive regulation of steroid metabolic process, negative regulation of calcium ion transmembrane transporter activity and release of sequestered calcium ion into cytosol by sarcoplasmic reticulum, (see additional file 6: table s6 ). in figure 4a -c the go biological process, cellular components and molecular functions are depicted. note, some of the ontology terms could be grouped, i.e. chaperone-mediated protein complex assembly and folding, endoplasmic reticulum unfolded protein response, er-nucleus signalling pathway and response to er oxidative stress as well as hypoxia, blood coagulation, developmental growth and regulation of programmed cell death. as depicted in figure 4b 76 significantly regulated proteins were mapped to 21 cellular components (see additional file 7: table s7 ), i.e. mitochondrial crista, matrix and inner membrane, endoplasmic reticulum lumen, early endosome and cytoplasmic membrane-bounded vesicle, chylomicron, very-low and high density lipoprotein particle, proteasome accessory complex, peroxisome, extracellular vesicular exosome and extracellular membrane-bounded organelle. furthermore, 75 significantly regulated proteins were mapped to 21 molecular functions (see figure 4c ) and included arginase activity, fructose-bisphosphate aldolase activity, hydrolase and oxidoreductase activity, acting on carbon-nitrogen (but not peptide) bonds, acting on aldehyde, ch-oh group or oxo group of donors, nad or nadp as acceptor as well as steroid dehydrogenase activity. in addition, phosphatidylcholine-sterol o-acyltransferase activator activity, cholesterol transporter activity, sterol transporter, antioxidant and lipid transporter activity as well as electron carrier and serine-type endopeptidase inhibitor activity were prominent functions. finally, proteins functioning in metal ion and purine ribonuleoside triphosphate binding, lipoprotein particle receptor binding, chaperone and oxygen binding, binding of magnesium ion and nad, protease and single-stranded dna binding were observed as disease regulated (additional file 8: table s8 ). in all, 96 significantly regulated proteins were classified by the reactome, kegg and wikipathway databases, respectively. the different databases provided similar information with the majority of tumour proteins acting in 4 major metabolic pathways (see figure 5 and information derived from cluego and cluepedia). for example, the proteins aldoa, aldoc, fbp1 and pkm function in glycolysis and gluconeogenesis whereas akr1c6, aldoa, aldoc and fbp1 are part of the fructose and mannose metabolic pathway. likewise, atp5h and ndufv1 are part of the oxidative phosphorylation pathway and mdh1 and pkm contribute to pyruvate metabolism. similarly, the proteins akr1c14, akr1c18, akr1c6, alb, apoa1, apoa4, apoe, fdps, gpx1, hacl1 and plg take part in the metabolism of lipids, arachidonic acid and lipoproteins whereas the proteins agmat, arg1, arg2, bckdha, ckb, cps1, haao and phgdh are specified for arginine and proline metabolism. in the same manner the proteins gpx1, itpa and nme1 contribute to the metabolism of nucleotides and related to this are the proteins itpa, pkm and psmc5 which are part of the purine metabolic pathway. apart from these pathways a highly significant regulation of the blood coagulation cascade, figure 2 western blotting of serum proteins in control and egf transgenic mice. for the commonly regulated proteins in serum and tumours their regulation in liver tissue was confirmed by 2de and maldi-tof/ms (see table 1 ). depicted are western blots for serum proteins. note, with the exception of egf the regulated serum proteins were already reported in our earlier publication [7] . platelet activation and fibrinolysis was observed as defined by the proteins crk, fga, fgb, fgg, plg and sod1 all of which were highly significantly regulated. furthermore, trna aminoacylation (aars, gars and sars), advanced glycosylation endproduct receptor signalling (alb, capza1 and lgals3), peroxisome (ech1, hacl1 and sod1), protein processing in endoplasmic reticulum (ganab, hyou1 and pdia4), proteasome (psmc5 and psmd11) and activation of chaperone genes by xbp1(s) and 'unfolded protein response' (hyou1 and lmna) are pathways significantly perturbed in liver cancer induced by egf (see additional file 9: table s9 and additional file 10: table s10 ). using the designated workflow of the genexplain platform (see methods section) we searched for master regulatory proteins. the software is designed to identify molecules upstream of regulated tumour proteins to assist in the construction of molecular circuitries. after annotation of the input datasets the tool "find master regulators in networks (geneways)" was used to identify key nodes amongst 54 proteins exclusively expressed in tumours (to). this revealed 24 upstream regulatory molecules. among them five were selected for their link to the egfr signalling pathway, i.e. plaur, fgfr1, ptbp1 and agtrap while the protein s100a1 was chosen for its importance in the plaur/egfr network, (see additional file 11: figure s1a -e). in additional file 3: table s3 and additional file 12: table s11 , the tumour regulated proteins distributed amongst the selected master regulatory molecules are summarized. in support of its biological significance the constructed networks were enriched with gene expression data from transgenic non-tumour and tumour tissues. thus, the gene and protein data were merged and hybrid networks for each master regulatory protein were constructed. subsequently, these were merged into one (see figure 6 ) and the integrated hybrid network consisted of n = 82 network proteins of which n = 20 were tumour specific. in support, the genes coding for lmna, i.e. a component of the nuclear lamina that is frequently up-regulated in cancers and mvp that codes for multidrug resistance were up-regulated (ur-t) whereas nme, a suppressor of metastasis was repressed in expression (dr-t). likewise, the genes coding for igals3, i.e. a beta-galactoside-binding protein frequently overexpressed in cancers and pcbp1 that is involved in transcription and functions as an inhibitor of invasion [65] were up-regulated in transgenic nontumour livers (ur-tr-nt) whereas transcript expression of aars, a member of trna synthases and anaxa6, a calcium-dependent, phospholipid-binding protein with important roles in the tumour microenvironment and metastasis were repressed (dr-tr-nt). finally, the entire network was enriched with expression data of 16 and 17 genes, respectively that were significantly regulated in tumour and non-tumour transgenic livers. next, we searched for master regulatory molecules by considering 82 regulated tumour proteins obtained from the comparison tumour specific or up-and down regulated as compared to healthy non-transgenic controls (to + ur + dr). this revealed 29 filtered (threshold radius of 10) upstream regulators. among these 7 were selected as candidates because of their regulation in liver tumours and their link to egfr signalling. notably, in the constructed network all master regulators were significantly up-regulated and included pdia4, apeh, pebp1 and apoe while the protein expression of arg1, fbp1 and haao was repressed (see additional file 13: figure s2a-g) . note, in the case of arg1 transcript expression was equally repressed. in additional file 3: table s3 and additional file 12: table s11 the tumour regulated proteins distributed amongst the selected master regulatory molecules are summarized. in support of its biological significance the fused hybrid network was enriched for gene expression data derived from transgenic non-tumour and tumour tissues. thus, the integrated hybrid network consisted of 34 out of 82 regulated proteins and gene expression calls evidenced 6 of the 27 up-regulated tumour (to + ur) proteins to be regulated at the transcript level as well whereas among the 7 down-regulated tumour proteins (dr) the gene arg1 was repressed in expression. likewise, gene expression data from non-tumour transgenic livers evidenced 5 genes out of 27 networks partners to be increased in expression (ur-tr-nt) and among the 7 down-regulated networks proteins the gene phb was repressed (dr-tr-nt). thus, when the tumour gene expression data of the entire network was considered a total of 22 genes were regulated, of which 13 were up-regulated and 9 were repressed in expression, (see figure 7) . based on the information of the hybrid master regulatory network and in addition to other disease regulated proteins summarized in table 1 (note, some of the proteins were not part of the networks) a total of n = 122 disease regulated proteins were considered for network construction. after filtering for non-connected proteins the string database informed on n = 151 protein-interactions of which n = 76 were disease regulated as identified in the present study. among these 45, 24 and 7 were either up-, down-or not statistically significantly regulated. furthermore, gene expression calls for 45 up-regulated proteins were supported by 5 up-and 4 down-regulated genes identified in tumours and 4 up-and 6 down-regulated genes in transgenic non-tumour livers. likewise, gene expression calls for 24 down-regulated proteins were supported by 8 and 5 downregulated genes in tumours and transgenic non-tumour livers, respectively. therefore, the entire network was supported by 14 induced and 17 repressed tumour specific gene expression changes and 16 up-regulated and 13 downregulated genes observed in transgenic non-tumour livers. as depicted in figure 8 the proteins of the fusion network displayed functional associations via the egf/egfr network and included 69 out of 96 (72%) significantly regulated tumour proteins with 6 out of 7 master regulators being connected to egfr through the network's proteins (see additional file 14: table s12 for possible protein-protein interactions and related scores). of the 151 network proteins 109 could be mapped to distinct pathways. after removal of non-relevant terms such as alzheimer disease a total of 94 proteins were mapped to 6 pathways with meaningful associations (see figure 9 ) and consisted of 'platelet activation, signalling and aggregation (platelet degranulation)' , 'lipoprotein metabolism' , 'mapk signalling pathway' , 'glycolysis and gluconeogenesis', 'metabolism of amino acids and derivatives (arginine and proline metabolism)', 'apoptosis' and 'egfr1 signalling pathway'. additionally, a total of 2 and 3 tumour regulated proteins were mapped to the hif-1 signalling and protein processing in endoplasmic reticulum pathways, respectively. the pathway mapping was also supported by gene expression data with 10 up-and 9 down-regulated genes in tumours and 9 up-and 6 downregulated genes in transgenic non-tumour livers. note, two of the significantly regulated tumour proteins, i.e. crk and pebp1 are members of the egfr1 signalling pathway with pebp1 also functioning as a master regulator while the other regulated proteins are connected to egfr signalling through cross-talk among the pathways (see additional file 15: table s13 ). figure 6 integrated master regulatory network for proteins uniquely expressed in tumours. based on network information obtained for the 5 different master regulators an integrated hybrid network was constructed. the network contained 82 proteins including 20 with connectivity to egfr signalling (yellow coloured inner node). the master regulator, the connecting proteins (network elements) and regulated proteins are given as red, green and blue coloured inner node, respectively. furthermore, each node is partitioned into four segments whereas the first segment seen from left refers to tumour specific proteins and is red-coloured. the second, third and fourth segments refer to either up-and down-regulated proteins, tumour specific gene expression changes and gene regulations in transgenic non-tumour liver tissue, respectively. increased expression of either proteins or genes is given in red, whereas the blue colour denotes repressed expression. recent research into the molecular pathogenesis of hcc evidenced significant alterations in signalling pathways. given the fact that the epidermal growth factor is an important mitogen for hepatocytes we were particularly interested in investigating the consequences of its targeted overexpression in the liver. in our previous study we employed chromatin immunoprecipitation followed by cloning and sequencing of dna to search for tumour associated gene regulations targeted by novel hnf4alpha p1 and p2 promoter-driven isoforms. this identified egf-receptor substrate (eps15r) and eps15 as regulated by the p2 promoter-driven hnf4alpha splice variant in mouse and human hcc. a molecular circuitry was proposed whereby eps15 and eps15r mediate internalization of activated egfr to stimulate receptor recycling, therefore responding to mitogenic signalling of egf [66] . in the present study disease proteomics was performed to further investigate the role of egf in liver cancer. this identified 122 regulated proteins of which 37 are novel and have not been reported so far. a total of 63 proteins were significantly up-regulated (table 1) . among these 18 were extra-cellular or secreted proteins and included albumin and isoforms of it, apolipoproteins (apoe, apoa4 and apoai), α-, β-, γfibrinogen, plasminogen as well as interleukin 1 receptor antagonist (il-1ra). note, an isoform of apoa1 was already proposed as serum marker of hcc [67] and based on ihc staining il-1ra expression was confirmed in about 70% of mouse liver adenoma and carcinoma cases; however preneoplastic foci as well as normal hepatocytes surrounding the lesions were negative. furthermore, rt-pcr analysis confirmed mouse hepatic tumours to contain both secreted and intracellular forms of il-1ra [51] and figure 7 integrated master regulatory network for hcc regulated proteins. based on network information obtained for 7 different master regulators an integrated hybrid network was constructed. the network contained 114 proteins including 34 with connectivity to egf/egfr signalling (yellow coloured inner node). the master regulator, the connecting proteins (network elements) and regulated proteins are given as red, green and blue coloured inner node, respectively. furthermore, each node is partitioned into four segments whereas the first segment seen from left refers to tumour specific proteins and is red-coloured. the second, third and fourth segments refer to either up-and down-regulated proteins, tumour specific gene expression changes and gene regulations in transgenic non-tumour liver tissue, respectively. increased expression of either proteins or genes is given in red, whereas the blue colour denotes repressed expression. serum levels of il-1ra were monitored to assess therapeutic efficacy of radiofrequency ablation in hcc patients [68] . an important finding of the present study is the statistically significant regulation of 20 mitochondria associated proteins of which 13 were repressed while 7 were upregulated. similar results were reported by chignard and wei sun with mitochondrial proteins being the second largest proportion of regulated proteins in human viral hcc [15, 69] . among the repressed proteins were nadh dehydrogenase (ubiquinone) 1 alpha subcomplex 8 and prohibitin, a mitochondrial chaperone. this protein, when deleted (prohibitin ko mice) induced fibrosis, bile duct metaplasia, liver dysplasia and eventually multifocal hcc. however, its overexpression in tumour cell lines inhibited cell proliferation to demonstrate tumour suppressor function [70] . likewise, glutathione peroxidase 1 (response to oxidative stress) and argininosuccinate synthetase 1 (ass, urea cycle) were repressed. note, ass is the first of two enzymes to convert citrulline to arginine and this pathway allows cells to synthesize arginine from citrulline to function in no production, ammonia detoxification and synthesis of polyamines. several reports suggest ass deficiency to be common in tumour cell lines [25] [26] [27] [28] [29] [30] , and the present study confirms ass expression to be confined to healthy non-transgenic control liver, but ass was absent in tumour tissue extracts (see table 1 ). ablation of ass in diverse tumours suggests a tumour suppressor function and the fact that forced expression of ass in osteosarcoma cell lines suppresses growth adds weight to this notion [71] . another example of tumour specific ablation of proteins refers to glycine n-methyltransferase (gnmt). the enzyme catalyzes the transfer of a methyl group from s-adenosylmethionine (sam) to glycine thereby generating s-adenosylhomocysteine and n-methylglycine. this protein was completely downregulated in liver tumours. gnmt is known to play a role in the maintenance of genetic stability [44, 72] , and a novel tumour suppressor function was recently reported that is independent of its catalytic activity but does require its nuclear localization [73] . several of the proteins listed in table 1 were already reported for their tumour specific regulation while proteins so far unknown for their regulations in hcc, are marked with an asterisk (table 1) . these function in diverse biological processes including metabolism, translation and signalling. specifically, changes in carbohydrate metabolism are commonly observed in tumours where energy production relies on glycolysis rather than mitochondrial oxidative phosphorylation. in the present study induced expression of several glycolytic enzymes was observed, most notable [1] pyruvate kinase 3 that catalyzes the transfer of a phosphate group from phosphoenolpyruvate to adp and was shown to be a target of mi-rna122 in hcc [2, 74] aldolase, an enzyme that converts fructose 1,6-bisphosphate into dihydroxyacetone phosphate (dhap) and glyceraldehyde 3-phosphate and was reported to be a sensitive marker for benign and malignant liver disease [75] and [3] alpha glucosidase 2, a hydrolase that cleaves glycosidic bonds with the release of alpha glucose from carbohydrates. further important findings include the tumour specific expression of alanyl-, glycyl-and seryl-trna synthetases which catalyze the transfer of specific amino acids to trna, as well as regulation of eukaryotic translation elongation factor 2 and poly(rc) protein 2 that binds to oligo dc. note, knowledge on the role of aminoacyl-trna synthetases in cancer is just emerging [76] and through the use of a lentiviral mediated shrna vector, a link between aminoacyl-trna synthetases [aars]-interacting multifunctional protein 2 (aimp2) and repressed egfr signalling was established that resulted in repressed glucose uptake [77] . we also observed induced expression of heterogeneous ribonucleoprotein (hnrnp) that takes on diverse functions in the processing of mrna. its expression was reported to be increased in serum of hcc patients . in contrast, proteins involved in the synthesis and degradation of cholesterol, lipids, steroids and fatty acid were in part oppositely regulated and included induced expression of the aldo-keto reductase family 1. regulation of this protein has been reported for lung and pancreatic cancers [78] , and gene silencing of aldo-keto reductase family 1 b10 resulted in growth inhibition of colorectal cancer cells that might be of therapeutic utility [79] . the repressed expression of figure 9 pathways mapping of fussed network proteins. cytoscape 3.0.2 with plugins (see methods section) are used to generate functionally grouped network of pathways. grouping of significant pathway terms (p ≤ 0.05) were based on kappa score threshold of 0.4, initial group size of 2 and sharing group percentage of 50. the pathway network consisted of 35 significantly and 7 non-significantly regulated proteins involved in distinct pathways which are colour-coded. note, the two individual terms are grey-coloured. up and down-regulated proteins are coded as orange and green small discs, respectively. up-and down-regulated as well as non-significantly regulated proteins and connecting proteins of the network are given as orange, green, yellow and blue coloured discs, respectively. the network depicts protein-protein interactions in liver tumours of egfr transgenic mice and their relation to various pathways under the influence of egfr signalling. egfr is highlighted as blue triangle in this network. certain proteins may also be considered as an adaptive response and includes the enzyme enoyl coenzyme a hydratase 1. its activity was shown to contribute to lymphatic spread of liver tumours as was evidenced in gene silencing studies [80] . likewise, we observed repressed expression of dihydrodiol dehydrogenase in tumours. this enzyme plays an important role in the metabolism of steroids that leads to inactivation of circulating androgens, progestins and glucocorticoids and was repeatedly reported to be overexpressed in non-small cell lung cancer. amongst patients with high dhd expression the incidence of early tumour recurrence and distant metastasis is significantly higher and patients are highly resistant to chemo and radiotherapy [81] . intriguingly, complete ablation of mitochondrial butyryl coenzyme a synthetase 1, a gtp-dependent lipoateactivating enzyme was observed in tumours of egf transgenic mice. little is known about the possible link between butyrate metabolism and liver cancer. however, butyrate is well known to inhibit proliferation of human colon carcinoma cells in an epigenetic manner that involves histone acetylation [82] . note, it was recently reported that due to the warburg effect butyrate-mediated histone acetylation and cell proliferation is dictated [83] . several lines of evidence therefore suggest butyrate to act as a cytosolic sensor for histone acteylation and when transformed to intermediates by butyryl coenzyme a synthetase is unable to escape the mitochondria. moreover, we observed a highly significant repression of 2-hydroxyphytanoyl-coa-lyase. this peroxisomal thiamine pyrophosphate-dependent enzyme is rate limiting in the breakdown of 2-hydroxy fatty acids. the biological role of 2-hydroxy fatty acids has only recently become apparent [84] and cumulative evidence suggests intermediates of energy metabolism to specifically activate g-protein coupled receptors which are now classified as hydroxy carboxylic acid receptors (hca1-3). the hca2 receptor is involved in a complex negative feed-back loop whereby ketone bodies derived from fatty acid oxidation are sensed by hca2 via the activity of 3-hydroxybutyrate that leads to inhibition of lipolysis and to restriction of further fatty acid supply. in this way triglyceride use is diverted and energy demands for tumour growth are met more efficiently. specifically, during rapid tumour growth and the herewith associated ischemia the yield of high energy bonds (atp) from glucose oxidation is about twice that of fatty acid oxidation. our observation that proteins involved in the ß-oxidation of fatty acids were either repressed or unchanged agrees well with this principle (see also discussion below). the reduced expression of lysophosphopholipase signifies an adaptive response; it catalyses the production of lysophosphatidic acid, i.e. a second messenger known to contribute to tumour cell motility, survival and proliferation [85] . additionally, the repressed expression of mitochondrial acyl-coa thioesterase 1 in liver tumours which hydrolyzes acyl-coas to free fatty acids and coenzyme a, will influence the supply of ligands for nuclear receptors and the regulation of fatty acid oxidation in mitochondria and peroxisomes. equally, the regulation of farnesyl diphosphate synthetase, i.e. a key enzyme in the isoprenoid biosynthetic pathway is highly interesting and this enzyme is explored as a drug target of bisphosphonates to treat tumour growth [86] . it's up-regulation in colon cancers was reported [39] . in the present study repressed expression of the ribosom-compononent rps12 and enzymes of amino acid metabolism like branched chain ketoacid dehydrogenase e1 as well as dimethyl glycine dehydrogease was observed. conversely, expression of the proteasome 26s atpase subunit 5 (p45/sug) and its non-atpase regulatory subunit 11 (psmd11) was confined to tumour tissues (see table 1 ); the latter subunit is known to display high activity in embryonic stem cells. this multicomplex molecular machinery degrades intracellular proteins marked up by ubiquitin chains. psmd11 was reported to be up-regulated in breast cancer cells [87] . enhanced expression of cytoskeletal proteins such as tubulin β 5 and capza1 was also confirmed by ihc staining (see figure 3 ). differences in the localization of these proteins were obvious with tubulin ß 5 expression being primarily associated with cells proximal to the liver capsule, whereas expression of capping protein zline α1 (capza1) was strongly associated with tumour foci and this protein is known to play a pivotal role in cytoskeletal networks to support cell mobility, invasion and metastasis. additionally, gdi2, a protein functioning in the cycling of rab gtpases and arginase ii, i.e. a non-liver isoform of the urea cycle were up-regulated in tumours of egf transgenic mice (see figure 3 ). regulation of arginase ii was observed in various malignancies including lung cancer [88] . besides, the actin-binding protein lasp1 was uniquely expressed in tumours and is also up-regulated in breast cancer [89] to possibly support migration of cancer cells [90] . furthermore, pdia4, a disulfide bond isomerase and master regulator of the constructed networks (see below) was up-regulated as was kininogen that is part of the blood coagulation system and functions as a precursor of kinin. conversely, the serinproteinase inhibitor serpinb1a was repressed in expression to possible limited immunological responses in tumour growth and to influence inflammatory cytokine production by infiltrating monocytes [91] . the significant regulation of the calcium binding protein sorcin and nucleobindin 1 are further highly interesting results. sorcin is associated with multidrug-resistance in human leukemia cells [92] and nucleobindin 1 is evaluated as a biomarker of colon cancer [93] . in egf induced liver tumours transthyretin was also up-regulated. this protein is involved in the transport of thyroid hormones and was reported to be aberrantly regulated in thyroid cancer [94] . among the newly identified proteins is v-crk sarcoma virus ct10. this oncoprotein interacts with several tyrosinephosphorylated proteins and is part of the intracellular signalling cascades notably the phosphoinositide 3-kinase (pi3k)/akt pathway [95] . likewise, regulation of the 170 kda glucose-regulated protein grp170 is of great importance. this lumenal endoplasmic reticulum plays a role in immunoglobulin folding as was confirmed by coimmunoprecipitation in four different b cell hybridoma cell lines [11] . in our previous study several immunoglobulins were found to be either repressed or absent in serum of egf tumour bearing mice and this was particularly obvious for the ig k and l classes [7] . it remains to be determined whether repression of immunoglobulins can be attributed to aberrant grp170 activity. a summary of the biological functions in addition to their previous reported tumour association is given in additional file 16: table s14 while the regulation of genes coding for newly identified proteins and of genes coding for commonly regulated proteins in liver tumours and serum of egf2b-transgenic mice is given in additional file 17: table s15 and additional file 18: table s16 . initially the network construction was based on proteins exclusively expressed in tumours and by selecting master regulatory proteins linked to egfr signalling. thereafter, a fused hybrid network was developed in which tumour specific proteins were part of it. subsequently, the search was extended to all significantly regulated proteins (table 1) . this revealed 7 master regulatory proteins and its associated networks and encompassed 114 proteins of which 34 were disease regulated. eventually a fused network was developed; however not all disease regulated proteins are part of it. the performed pathway mapping over fused networks (see string analysis) defined protein interactions and grouped 76 disease regulated proteins into 6 distinct pathways of which platelet activation, signalling and aggregation is a major one (see figure 8) . specifically, the glycoprotein fibrinogen is a multimeric protein and consists of α, ß and y subunits. it is synthesized by hepatocytes and an essential blood coagulation factor with all polypeptide chains being highly regulated in tumours of egf transgenic mice. note, an association between coagulation factors and malignancies was established whereby fibrinogen functions as an extracellular matrix protein to interact with integrin receptors in the control of cell proliferation and cell migration [96] . accordingly, induced gene expression of the integrin receptors itgb1, itga3 and itgav was observed in egf induced liver tumours. in cancer progression a regulatory loop between fibrinogen, platelets and tumour cells has been determined that is activated by platelet cytosolic ca2+. this second messenger induces integrin receptor complex formation through an association of platelet glycoprotein chains iib and iiia (cd41/cd61) thereby creating an active binding site for fibrinogen. an association of tumour regulated proteins with the regulatory loop was confirmed in string analysis ( figure 8 ) and fibrinogen was reported to be an important determinant for metastasis of circulating tumour cells [97] . it is therefore of no surprise that elevated blood fibrinogen is a poor prognostic factor. haemostatic complications are commonly observed in cancer patients and future therapeutic strategies may focus on the hemostatic system by targeting tumour stroma. in this regard the tumour specific induction of plasminogen is of great importance. this zymogen [98] is converted to plasmin by urokinase (upa), a serine protease which itself was unchanged; however, gene expression of its receptor was significantly up-regulated in transgenic non-tumour livers. one report suggests the urokinase receptor to prime cells for proliferation in response to egf by promoting tyr845 phosphorylation and stat5b activation; nonetheless, this depended on intracellular c-src levels [99] . further studies established a link between induced expression of plasminogen activator, upa receptor and plasminogen activator inhibitor type-1 (pai-1) and invasiveness and metastasis of hcc [100, 101] . indeed, a fine balance exists between the plasminogen activating system and its inhibition by pai-1 and pai-2. based on transcriptomic data a highly significant induction of pai-i (up to 12-fold) in large tumours of egf transgenic mice was observed [6] ; consequently, the regulation of components of the plasminogen activating system may be considered as part of a strategy to degrade extracellular matrix thereby facilitating invasion and metastasis [102, 103] . to meet energy demands efficiently different sources are utilized and the induction of the proteins aldoa, aldoc, eno1, pkm and fbp1 is testimony to an altered glycolytic and pentose phosphate pathway. however, with the exception of acyl-coa thioesterase 2 that was below the limit of detection and functions in the hydrolysis of myristoyl-palmitoyl-, stearoyl-and arachidoyl-coa esters the regulation of enzymes linked to fatty acid metabolism in mitochondria and peroxisomes was hardly observed. in pursue of tumour growth and to sustain organelle and membrane biogenesis lipids are de novo synthesized and mobilized from stores and while the complex interaction of hepatic lipid and glucose metabolism in liver disease is the subject of intense research [104] the present study evidences significant regulation of several apolipoproteins, i.e. apoe, apoa1, apoa4 and isoforms of albumin. apart from lipid transport apolipoproteins play a wider role in cancers and are known to interact with diverse receptors to elicit cellular events as demonstrated for apoe to cause sustained proliferation and survival of cancer cells [105] . a further group of highly regulated proteins are aldoketo reductases. their quantitative evaluation in different hepatocellular carcinoma (hcc) cell lines was recently reported [106] . this superfamily of proteins comprises nad (p)(h)-dependent enzymes which catalyze oxidoreduction of a variety of prostaglandins, steroids and toxic aldehydes. their involvement in tumorigenesis is supported by several studies and they are explored as drug targets to overcome chemoresistance. in the present study the aldo-keto reductases akr1c14, akr1c18 and akr1c6 were uniquely expressed in tumours, however glutathione peroxidase 1 was repressed to 30% of healthy control livers to possibly support hif-1 signalling. indeed, the redox state and therefore glutathione participates in the hypoxic induction of hif-1 [107] , and two proteins of the glycolytic pathway, i.e. aldoa1 and eno1, which respond to hif-1 signalling, were regulated. moreover, glutathione peroxidase 1 was shifted in the gel as shown in figure 1 panel g iii as a result of post translational modifications that most likely involved c-abl and arg kinase activity at tyr 96 of gpx1 [108] . likewise, the genes coding for aldo1 and eno3 were significantly up-regulated in egf induced liver tumours. a complex interaction exists between egfr and rage signalling. this receptor for advanced glycation end-products is a member of the immunoglobulin family of cell surface molecules and was reported to significantly influence hepatic tumour growth in murine models of colorectal carcinoma [109] . there is strong evidence for rage to promote cancer growth upon ligand dependent activation and several proteins of the s100 family bind to the extracellular domain of rage [110, 111] . it is of considerable importance that gene expression of s100a4 and s100a11 was up to 34-fold induced in tumours of egf transgenic mice, however expression of s100a1 was repressed. likewise the tumour specific expression of the rage binding proteins lectin, galactoside-binding, soluble, 3 and capza1 in tumours of egf transgenic mice is highly suggestive for a sustained crosstalk between rage and egfr [112] . although the precise mechanism by which s100 proteins stimulate egfr signalling remains to be elucidated binding of s100a4 to egf and to other egfr ligands was reported to possibly facilitate interaction with the receptor [113] . similarly, the binding of s100a8/a9 to rage was shown to promote migration and invasion of human breast cancer cells through actin polymerization and epithelial-mesenchymal transition [114] . conversely, advanced glycation endproduct (age) receptor 1 suppressed oxidant stress-dependent signalling via the egfr and shc/grb2/ras pathway [115] . as depicted in figure 5 the amino acid metabolism was another distinct pathway to which several of the regulated proteins could be mapped to. note, the tumour specific regulations of arginine 1 and 2 as well as the regulation of subunits of the proteasome 26s atpase (psmc5 and psmd11) were already discussed (see above). in the following additional proteins regulated in this pathway are briefly summarized. specifically, 3-hydroxyanthranilate-3,4-dioxygenase (haao) catalyzes oxidation of 3-hydroxyanthranilate to quinolinate and this intermediate functions as a precursor in nad and pyridine biosynthetic pathways. expression of haao was significantly repressed in tumours of egf transgenic mice and hypermethylation of the coding gene was observed in ovarian cancer [116] . due to the fact that haao is significantly repressed at the gene and protein level in at least two different tumour entities (ovarian and liver cancer) the protein may function as a tumour suppressor that appears to be repressed by an epigenetic mechanism. a significant finding is the tumour specific expression of 3-phosphoglycerate-dehydrogenase which catalyses the production of 3-phosphoglycerate. this intermediate of glycolysis is an essential precursor of the serine biosynthetic pathway. importantly, a recent metabolomic study evidenced 3-phosphoglycerate to be diverted into serine and glycine metabolism and repressed expression of 3-phosphoglyceratedehydrogenase resulted in impaired tumour cell proliferation [117] . in support of tumour growth the diversion of intermediate of glycolysis affects protein, membrane lipid and nucleotide synthesis. moreover, the observed induction of creatine kinase in tumours of egf transgenic mice creates a circuitry for cellular energy homeostasis in conditions of high metabolic demands [118] . the enzyme catalyses the reversible transfer of phosphate from phosphocreatine to adp to yield atp and creatine. its induction has been observed in many cancers including liver cancer cell lines [119, 120] and a further study suggested a possible interplay between p53 mutations, hcc, ck expression with growth-inhibitory effects of cyclocreatine in hcc [121] . while the rationale of tumour cells in embarking on abnormal metabolism had already been discussed (see above) the finding that agmatine ureohydrolase was strongly repressed in egf induced liver tumours to about 10% of non-transgenic healthy livers is of great importance. this enzyme hydrolyzes agmatine (= decarboxylated arginine) to form putrescine and urea and repression of the enzyme will significantly increase agmatine tissue concentration to influence diverse cellular control mechanisms. importantly, in the study of battaglia and coworkers [122] 1 mm agmatine induced large amounts of superoxide production in rat liver mitochondria; however, it did not affect mitochondrial respiration or redox levels of thiols and glutathione. furthermore, atp synthesis remained normal and prevented ca(2+)-induced mitochondrial permeability transition in the presence of phosphate to suggest an intriguing regulatory loop whereby h2o2 induces hypoxia signalling that is linked to abberant metabolism, nonetheless by selecting interconnected physiological pathways tumour cells are equipped to avoid programmed cell death [122, 123] . thus, arginine deprivation is evaluated for its utility in cancer therapy [124] . a further enzyme repressed to 20% of healthy nontransgenic liver is carbamoyl phosphate synthetase 1 (cps1), i.e. a liver specific ligase to function in ammonia detoxification. it is perplexing that tumour cells disable such an important pathway of the urea cycle. however, a recent study demonstrated dna hypermethylation as a key mechanism of silencing cps1 gene expression in human hcc. note, forced expression of cps1 induced cell proliferation and the observed repression in human hcc may simply be the result of genomic instability as was observed in tumour cells [125] . the present study identified novel disease regulated proteins induced by overexpression of egf to provide new insight into the complex signalling events in hcc. six major pathways perturbed by egfr hyperactivity were identified and several of the regulated proteins are interesting drug target candidates and this includes tumour specific expression of kinases as well as proteins involved in aberrant metabolism. an identification of commonly regulated proteins in tumour and sera will be of great utility in the development of biomarkers to monitor disease progression and responses to therapy. the following additional data are available with the online version of this paper. aminoacyl-trna synthetases interacting multifunctional protein 2; alpha-hcca: alpha-cyano-4-hydroxycinnamic acid; aps: ammonium persulfate; ass: argininosuccinate synthetase 1; atp: adenosine triphosphate; ck: creatine kinase; ck2α: casein kinase ii subunit alpha; co: control specific protein dr: down-regulated protein; dr-t: down-regulated tumour; dr-tr-nt: down-regulated transgenic non-tumour; egf: epidermal growth factor egfr: epidermal growth factor receptor; eps15: epidermal growth factor receptor substrate 15; eps15r: epidermal growth factor receptor substrate 15r; er: endoplasmic reticulum; erk: extracellular signalling regulated kinase; fdr: false discovery rate; fgfr1: fibroblast growth factor receptor 1 go: gene ontology; grp170: 170 kda glucose-regulated protein; gtp: guanosine triphosphate h 3 po 4 : orthophosphoric acid; hca: hydroxy carboxylic acid; hcc: hepatocellular carcinoma; hdac2: histone deacetylase 2; hed: bis(2-hydroxyethyl) disulfide; hrp: horseradish peroxidase ihc: immunohistochemistry; il-1ra: interleukin-1 receptor antagonist ko: knockout mouse mek: mitogen-activated protein kinase kinase; nad: nicotinamide adenine dinucleotide; nad(p)(h): nicotinamide adenine dinucleotide phosphate nadp: nicotinamide adenine dinucleotide phosphate; no: nitric oxide; ogp: n-octyl β-d-glucopyranoside pai-1: plasminogen activator inhibitor type-1 pdgfr: platelet-derived growth factor receptors; plaur: plasminogen activator, urokinase receptor; pmf: peptide mass fingerprinting; ptbp1: polypyrimidine tract binding protein 1; rage: receptor for advanced glycation endproduct s100a1: s100 calcium binding protein a1; sam: s-adenosylmethionine short hairpin rna; stat5b: signal transducer and activator of transcription 5b; temed: tetramethylethylenediamine; to: tumour specific protein; trna: transfer rna; tyr845: tyrosine residue 845; upa: urokinase-type plasminogen activator; ur: up-regulated protein up-regulated transgenic non-tumour; vegfr: vascular endothelial growth factor receptor; xbp1(s): x-box binding protein 1 isoform surgery and ablative therapy for hepatocellular carcinoma improved method for proteome mapping of the liver by 2-de maldi-tof ms identification of specific 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mir-122 targets pyruvate kinase m2 and affects metabolism of hepatocellular carcinoma serum aldolase isoenzymes in benign and malignant liver disease aminoacyl trna synthetases and their connections to disease lentiviral vector-mediated shrna against aimp2-dx2 suppresses lung cancer cell growth through blocking glucose uptake overexpression and oncogenic function of aldo-keto reductase family 1b10 (akr1b10) in pancreatic carcinoma aldo-keto reductase family 1 b10 gene silencing results in growth inhibition of colorectal cancer cells: implication for cancer intervention enoyl coenzyme a hydratase 1 is an important factor in the lymphatic metastasis of tumors expression of dihydrodiol dehydrogenase and resistance to chemotherapy and radiotherapy in adenocarcinoma cells of lung butyrate metabolism in human colon carcinoma cells: implications concerning its growth-inhibitory effect the warburg effect dictates the mechanism of butyrate-mediated histone acetylation and cell proliferation biological roles and therapeutic potential of hydroxy-carboxylic acid receptors autotaxin has lysophospholipase d activity leading to tumor cell growth and motility by lysophosphatidic acid production farnesyl pyrophosphate synthase: a key enzyme in isoprenoid biosynthetic pathway and potential molecular target for drug development over-expression of genes and proteins of ubiquitin specific peptidases (usps) and proteasome subunits (pss) in breast cancer tissue observed by the methods of rfdd-pcr and proteomics arginase 2 is expressed by human lung cancer, but it neither induces immune suppression, nor affects disease progression nuclear localization and cytosolic overexpression of lasp-1 correlates with tumor size and nodal-positivity of human breast carcinoma regulation of cell migration and survival by focal adhesion targeting of lasp-1 serpinb1 regulates homeostatic expansion of il-17+ gammadelta and cd4+ th17 cells sorcin, an important gene associated with multidrug-resistance in human leukemia cells autoantibodies to ca2+ binding protein calnuc is a potential marker in colon cancer detection fine-needle aspiration of thyroid nodules: proteomic analysis to identify cancer biomarkers v-crk activates the phosphoinositide 3-kinase/akt pathway by utilizing focal adhesion kinase and h-ras tumors and fibrinogen. the role of fibrinogen as an extracellular matrix protein fibrinogen is an important determinant of the metastatic potential of circulating tumor cells plasminogen receptors and their role in the pathogenesis of inflammatory, autoimmune and malignant disease urokinase receptor primes cells to proliferate in response to epidermal growth factor invasion and metastasis of hepatocellular carcinoma in relation to urokinase-type plasminogen activator, its receptor and inhibitor urokinase-type plasminogen activator receptor transcriptionally controlled adenoviruses eradicate pancreatic tumors and liver metastasis in mouse models plasminogen activator system and its clinical significance in patients with a malignant disease the urokinase plasminogen activator system: role in malignancy the interaction of hepatic lipid and glucose metabolism in liver diseases apolipoprotein e is required for cell proliferation and survival in ovarian cancer quantitative evaluation of aldo-keto reductase expression in hepatocellular carcinoma (hcc) cell lines the redox state of glutathione regulates the hypoxic induction of hif-1 controlled elimination of intracellular h(2)o(2): regulation of peroxiredoxin, catalase, and glutathione peroxidase via post-translational modification rage signaling significantly impacts tumorigenesis and hepatic tumor growth in murine models of colorectal carcinoma binding of s100 proteins to rage: an update rage and rage ligands in cancer identification of galectin-3 as a high-affinity binding protein for advanced glycation end products (age): a new member of the age-receptor complex epidermal growth factor receptor ligands as new extracellular targets for the metastasis-promoting s100a4 protein rage-binding s100a8/a9 promotes the migration and invasion of human breast cancer cells through actin polymerization and epithelial-mesenchymal transition advanced glycation end product (age) receptor 1 suppresses cell oxidant stress and activation signaling via egf receptor identification of candidate epigenetic biomarkers for ovarian cancer detection phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the'phosphocreatine circuit' for cellular energy homeostasis creatine and creatinine metabolism mitochondrial creatine kinase as a tumor-associated marker elevated creatine kinase activity in primary hepatocellular carcinoma different behavior of agmatine in liver mitochondria: inducer of oxidative stress or scavenger of reactive oxygen species? hypoxia signaling pathways in cancer metabolism: the importance of co-selecting interconnected physiological pathways arginine deprivation as a targeted therapy for cancer submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we gratefully acknowledge support from the virtual liver network (grant 031 6154) of the german federal ministry of education and research (bmbf) to jb. additional file 1: table s1 . antibodies and dilutions used to study disease regulation by immunohistochemistry.additional file 2: table s2 . statistics and selection criteria for functional grouping of pathways terms of disease regulated proteins. a hypergeometric test followed by bonferroni correction was used with p-value ≤ 0.05. for grouping of pathway terms, the kappa score threshold was set to 0.4. additional file 3: table s3 . different master regulators and associated network for proteins expressed in tumours only (to) or significantly regulated when compared to healthy liver of non-transgenic control animals (t + ur + dr).additional file 4: table s4 . comparison of disease regulated proteins in mouse and human hcc. data were taken from 'the human protein atlas' database.additional file 5: table s5 . comparison of transcriptomic and proteomic data. this comparison revealed 19 significantly regulated genes of which 15 are regulated in common whereas for 4 genes transcript expression was opposite to that of the coded proteins.additional file 6: table s6 . biological processes ontology for significantly regulated proteins. additional file 7: table s7 . cellular component ontology for significantly regulated proteins.additional file 8: table s8 . molecular function ontology for significantly regulated proteins.additional file 9: table s9 . biological pathways and their cluster. cytoscape 3.0.2 with plugins (cluego and cluepedia) were used to generate functionally grouped network of pathways based on reactome, kegg and wikipathways databases. the grouping of significant pathway terms (p ≤ 0.05) is based on a kappa score of 0.4, initial group size of 2 and sharing group percentage of 50.additional file 10: table s10 . biological pathways information based each significantly regulated protein (to + ur + dr). this table depicts pathway terms of nearly 80% of regulated proteins (to + ur + dr) and are taken from reactome and kegg databases.additional file 11: figure s1 . master regulatory networks for proteins uniquely expressed in tumours with link to egfr signalling: (a) the plaur network consists of 44 proteins of which 20 are tumour-specifically regulated, (b) the fgfr1 consists of 41 proteins of which 18 are tumour-specifically regulated, (c) the ptbp1 network consists of 44 proteins of which 18 are tumour-specifically regulated, (d) the agtrap network consists of 43 proteins of which 19 are tumour-specifically regulated and (e) the s100a1 network consists of 38 protein of which 16 are tumour-specifically regulated. note all networks display connectivity to egfr signalling (yellow coloured inner node) and in the case of the s100a1 master regulatory protein egfr s ignalling is via the plaur/egfr network.the master regulator, the connecting proteins (network elements) and regulated proteins are given as red, green and blue coloured inner node, respectively. furthermore, each node is partioned into four segments whereas the first segment seen from left refers to tumour specific proteins and is red-coloured. the second, third and fourth segments refer to either up-and down-regulated proteins, tumour specific gene expression changes and gene regulations in transgenic non-tumour liver tissue, respectively. increased expression of either proteins or genes is given in red, whereas the blue colour denotes repressed expression.additional file 12: table s11 . integrated hybrid network with master regulator information for proteins expressed in tumours only (to) and significantly regulated proteins when compared to heathy liver of non-transgenic control animals (t + ur + dr).additional file 13: figure s2 . master regulatory networks for regulated proteins with link to egfr signalling: (a) the pdia4 network consists of 68 proteins including 32 significantly regulated proteins, (b) the apeh network consists of 68 proteins including 32 significantly regulated proteins, (c) the pebp1 network consists of 63 proteins including 31 significantly regulated proteins, (d) the apoe network consists of 66 proteins including 31 significantly regulated proteins, (e) the arg1 network consists of 67 proteins including 31 significantly regulated proteins, (f) the fbp1 network consists of 69 proteins including 31 significantly regulated proteins, (g) the haao network consists of 66 proteins including 32 significantly regulated proteins. note, all networks display connectivity to egf protein (yellow coloured inner node). the master regulator, the connecting proteins (network elements) and regulated proteins are given as red, green and blue coloured inner node, respectively. furthermore, each node is partioned into four segments whereas the first segment seen from left refers to tumour specific proteins and is red-coloured. the second, third and fourth segments refer to either up-and down-regulated proteins, tumour specific gene expression changes and gene regulations in transgenic non-tumour liver tissue, respectively. increased expression of either proteins or genes is given in red, whereas the blue colour denotes repressed expression.additional file 14: table s12 . protein interaction information of the fused network. given are interacting proteins with association score from different prediction methods, i.e. neighborhood, gene fusion, co-occurrence, co-expression, databases and textmining.additional file 15: table s13 . biological pathways and their cluster for fused network proteins. reactome, kegg and wikipathways database information was used as input data file for cytoscape 3.0.2. the table shows grouping of significant pathway terms (p ≤ 0.01) and is based on a kappa score of 0.4, initial group size of 2 and sharing group percentage of 50.additional file 16: table s14 . biological function of newly identified proteins and their previously reported tumour association.additional file 17: table s15 . regulation of genes coding for newly identified proteins in egf2b-transgenic liver tumours.additional file 18: table s16 . regulation of genes coding for common proteins in tumour tissue and serum of egf transgenic mice. the authors declare that they have no competing interests.authors' contributions jb conceived the study and contributed the reagents, gg performed the experiments, ps performed the bioinformatics analysis. jb, gg and ps analysed the data, jb wrote the manuscript and all authors read and approved the final manuscript. key: cord-007208-wnkjdg6y authors: li, sheng-hsiang; lee, robert kuo-kuang; hsiao, ya-ling; chen, yee-hsiung title: demonstration of a glycoprotein derived from the ceacam10 gene in mouse seminal vesicle secretions(1) date: 2005-09-01 journal: biol reprod doi: 10.1095/biolreprod.105.039651 sha: doc_id: 7208 cord_uid: wnkjdg6y ceacam10 was purified from mouse seminal vesicle secretions by a series of purification steps that included ion exchange chromatography on a deae-sephacel column and ion exchange high-performance liquid chromatography on a sulfopropyl column. it was shown to be a 36-kda glycoprotein with an n-linked carbohydrate moiety. the circular dichromoism spectrum of ceacam10 in 50 mm phosphate buffer at ph 7.4 appeared as one negative band arising from the β form at 217 nm. ceacam10 was expressed predominantly in seminal vesicles of adult mice. both ceacam10 and its mrna were demonstrated on the luminal epithelium of the mucosal folds in the seminal vesicle. the amount of ceacam10 mrna in the seminal vesicle was correlated with the stage of animal maturation. castration of adult mice resulted in cessation of ceacam10 expression, while treatment of castrated mice with testosterone propionate in corn oil restored ceacam10 expression in the seminal vesicle. during the entire course of pregnancy, ceacam10 might be silent in the embryo. a cytochemical study illustrated the presence of the ceacam10 binding region on the entire surface of mouse sperm. ceacam10-sperm binding greatly enhanced sperm motility in vitro. mammalian sperm display an intriguing sense of timing in undergoing some modification during their transit in the reproductive tract before encountering an egg. studying how the lumen of the reproductive tract affects sperm function is a prerequisite to unraveling the molecular mechanisms underlying the complex modification of sperm. factors that affect sperm motility have been reported in the seminal plasma of several mammals including the pig [1] [2] [3] , bovine [4] , mouse [5] , and human [6] . the seminal vesicle is a male accessory sexual gland found in many species of more than 4000 mammalian species alive on the earth today. after puberty, the gland secretes a fluid called seminal vesicle secretion (svs), which accumulates in its lumen. svs contains both protein and nonprotein components. when ejaculated, svs squirts into the urethra, contributing the major part of the liquid portion of seminal plasma, which is the complex biological fluid formed from mixing of various fluid in the male reproductive tract. it has been found that extirpation of the seminal vesicle from mice and rats greatly reduces fertility [7, 8] , demonstrating the importance of svs to sperm modification under natural circumstances. svs differs extensively in terms of volume and composition in various species of mammals. however, rodents have proven to be good experimental animals for the molecular study of mammalian reproduction, so attempts have been made to isolate the proteins involved in sperm modification by mouse svs, which contains several minor proteins and seven well-defined major proteins designated svs i-vii, named in decreasing order of molecular mass according to their mobility in sds-page [9] . previously, we demonstrated that svs vii enhances sperm motility [10] , and two of the minor proteins modulate sperm activity. one is a caltrin-like trypsin inhibitor/p12, which suppresses ca 2ϩ uptake by sperm [11] , and the other is a seminal vesicle autoantigen, which serves as a decapacitation factor [12, 13] . here we report the purification and identification of an androgen-stimulated 36-kda glycoprotein, a minor protein component of mouse svs that is able to enhance sperm motility in vitro. we have demonstrated that its core protein is derived from the ceacam10 gene [14] , which is a member of the cell adhesion molecule (cam) subgroup belonging to the carcinoembryonic antigen (cea) family. the following materials were obtained from commercial sources: deae-sephacel (amersham pharmacia biotech, uppsala, sweden); protein pak sp 5pw column (waters, milford, ma); vydac 218tp54 c 18 column (separations group, hesperia, ca); aminolink coupling gel, bicinchoninic protein assay kit (pierce, rockford, il); testosterone propionate, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate (bcip), pmsf, periodic acid schiff reagent, and silanated glass slides (sigma chemical co., st louis, mo); cdna integrity kit, alkaline phosphataseconjugated streptavidin, and biotin-conjugated goat anti-rabbit immunoglobulin g (igg; kirkegaard & perry laboratories, gaithersburg, md); rhodamine-conjugated goat anti-rabbit igg (zymed laboratories, san francisco, ca); nuclear fast red (vector laboratories, burlingame, ca); enhanced chemiluminescent substrate and [␣32 a) fractionation of soluble mouse svs proteins by ion exchange chromatography on a deae-sephacel column. b) resolution of fraction iii sample from a by ion-exchange hplc on an sp column. c) demonstration of the glycoprotein nature. each of the a-to-d peaks from b were digested with n-glycosidase f. the parent proteins (lane 1, peak a; lane 3, peak b; lane 5, peak c; lane 7, peak d) and their deglycosylated forms (lanes 2, 4, 6, and 8) were identified by sds-page on a 12% polyacrylamide gel slab. the proteins in the gel were stained with coomassie brilliant blue. pgem-t-easy vector (promega, madison, wi); ultraspec-ii rna isolation kit (biotecx laboratories, inc., houston, tx); thermoscript reverse transcriptase (invitrogen life technologies, carlsbad, ca); n-glycosidase f, taq dna polymerase (takara, shiga, japan); and mouse embryo-stage blots designed to observe gene expression during pregnancy (seegene, inc., korea). all other chemicals were reagent grade. outbred icr mice were purchased from charles river laboratories (wilmington, ma) and were maintained and bred in the animal center at the college of medicine, national taiwan university. animals were treated according to institutional guidelines for the care and use of experimental animals. they were housed under controlled lighting (14l:10d) at 21-22њc and were provided with water and nih-31 laboratory mouse chow ad libitum. for investigation of androgenic effects, 8-wk-old adult male mice, which had been castrated 3 wk earlier, received a daily s.c. injection of testosterone propionate in corn oil (5 mg/kg body weight) for 8 consecutive days. control animals received corn oil only. seminal vesicles were removed from the animals 12 h after the last injection. normal adult mice (8 to 12 wk old) were killed by cervical dislocation. the seminal vesicles of 30 mice were carefully dissected to free them from the adjacent coagulating glands, and the secretions were squeezed directly into 50 ml of ice-cold 10 mm tris-hcl in the presence of 1 mm pmsf at ph 8.0. after centrifugation at 10 000 ϫ g for 15 min, the supernatant was resolved by ion exchange chromatography on a deae-sephacel column (12 ϫ 2.6 cm) that had been pre-equilibrated with 10 mm tris-hcl at ph 8.0. after the nonretarded fractions were washed out, the column was eluted with 0.5 m nacl in the same buffer at a flow rate of 18 ml/h. fractions (4 ml) were collected, and their absorbance at 280 nm was recorded (fig. 1a) . fraction iii was further subjected to ionexchange high-performance liquid chromatography (hplc) on a sulfopropyl (sp) column (7.5 cm ϫ 7.5 mm). the column was sequentially eluted with three linear gradients, including 0%-15%, 15%-40%, and 40%-80% of 0.6 m nacl in 20 mm sodium acetate at ph 6.0 at a flow rate of 1.0 ml/min for 60 min (see fig. 1b ). thirty micrograms of each peak (a-d) in 50 l of 100 mm tris-hcl ph 8.6 were digested with 0.6 g of trypsin-tpck at 37њc for 18 h. the reaction was stopped by adding 100 l of 0.1% trifluoroacetic acid and the reaction mixture was resolved by a reverse-phase c 18 column (4.6 ϫ 250 mm). the column was eluted with a linear gradient of 0%-80% acetonitrile at a flow rate of 1.0 ml/min for 60 min (see fig. 2a ). the n-glycoconjugate was removed from a glycoprotein by following the method described by tarentino and plummer [15] . the protein was boiled in 1.0% sds and incubated with n-glycosidase f (40 u/mg of protein) in 20 mm sodium phosphate at ph 7.2 in the presence of 50 mm edta, 0.5% nonidet p-40, and 10 mm sodium azide for 16 h at 37њc. the concentration of ceacam10 was determined using the bicinchoninic acid protein assay [16] according to the manufacturer's instructions. the amino acid sequence was determined using automated edman degradation with a 492 protein sequencer with an online 140 c analyzer (applied biosystems, foster city, ca). the circular dichromoism (cd) spectra were measured with a jasco j-700 spectropolarimeter under constant flushing with n 2 at room temperature. the mean residue elipticity () was estimated from the mean residue weight, which was calculated from the primary structure. antisera against ceacam10 were raised in new zealand white rabbits. the anti-ceacam10 antibody was purified from the antiserum on a column (2.5 ϫ 1.0 cm) of aminolink gel coupled with the purified antigen according to our previously described method [17] . proteins were resolved using sds-page on a 12% gel slab (8.2 ϫ 7.3 ϫ 0.075 cm) according to the method described by laemmli [18] . the proteins on the gel were stained with coomassie brilliant blue or transferred to a nitrocellulose membrane using an electroblotting method, which was conducted at 35 v at 4њc for 18 h in a solution of 25 mm tris-hcl, 197 mm glycine, and 13.3% methanol. membranes were blocked with 10% normal goat serum in pbs for 2 h, and then incubated with anti-ceacam10 antibody (0.5 g/ml) in the blocking solution for 1 h at room temperature. after gently agitating in four changes of pbs for 15 min each, they were immunoreacted with horseradish peroxidase-conjugated goat anti-rabbit igg diluted to 1:15 000 in the blocking solution for 1 h. immunoreactive bands were revealed using an enhanced chemiluminescent substrate according to the manufacturer's instructions. mouse seminal vesicles were fixed in bouin solution, embedded in paraffin, and 8-m serial cross-sections were mounted on silanated glass slides. deparaffinized sections were blocked in blocking solution for 1 h at room temperature and then incubated with affinity-purified anti-cea-cam10 antibody at a concentration of 1 g/ml in the blocking solution for 1 h. the slides were gently agitated in three changes of washing solution for 10 min each and then treated with biotin-conjugated goat antirabbit igg (1 g/ml) in the blocking solution for 1 h at room temperature. the slides were washed again as described above and then incubated with alkaline phosphatase-conjugated streptavidin (1 g/ml) in blocking solution for 1 h at room temperature. protein signals were observed after the slides were incubated for 10 min with 0.0375% nitroblue tetrazolium and 0.0188% bcip in a solution of 100 mm tris-hcl, 100 mm nacl, and 5 mm mgcl 2 at ph 9.5. the slides were washed in three changes of water for 3 min each and then counterstained with nuclear fast red for 3 min. fig. 2. identification of the 36-kda glycoprotein derived from ceacam10 and its circular dichroism. a) the trypsin-digested sample of peak c from figure 1b was resolved by reverse-phase hplc on a c 18 column (see materials and methods). b) the protein sequence was deduced from the reading frame of ceacam10 cdna (genbank accession number nm 007675). the initial and stop codons are underlined. the potential n-linked glycosylation sites are denoted by open boxes. the deduced protein sequence and the amino acid sequences determined directly from protein analysis for peaks a to d in figure 1b and peaks 9 and 18 in figure 2a agree in all positions except that asn 11 from the cdna-deduced protein was not identified in protein sequencing. the cleavage points for the generation of mature protein are indicated by an arrow. c) circular dichroism of ceacam10 in 50 mm phosphate buffer at ph 7.4 at room temperature. finally, the slides were briefly washed with water and photographed using a brightfield microscope (ah3-rfca; olympus, tokyo, japan). seminal vesicle tissues were oriented in a small aluminum foil cup and frozen immediately in tissue-tek oct medium. embedded samples were then stored at ϫ80њc before further processing. serial 8-m cryostat sections were mounted on uncoated glass slides and stored at ϫ80њc. before use, sections were fixed in 70% ethanol and stained with a histogene lcm frozen section-staining kit following the supplier's protocol. slides were dipped in xylene twice for 5 min each time and then air-dried. mucosal folds or smooth muscle cells were harvested using a pixcell ii lcm system (arcturus engineering, mountain view, ca). captured tissues from three sections were collected on capsure hs lcm caps containing transfer film. tissue samples from three different animals were pooled for subsequent analysis. total rna was extracted from the captured cells by using the picopure rna isolation kit, followed by treatment with dnase i before cdna synthesis. the total rna was reverse-transcribed using thermoscript reverse transcriptase for first-strand cdna synthesis according to the manufacturer's instructions. a cdna integrity kit was employed to examine the quality of the cdna. the qualified cdna samples were used as templates for pcr. the primer pair for amplification of a 237-base pair (bp) ceacam10 gene fragment was a forward primer (5ј-tatgctattt-caaaacttcgatcat-3ј), which corresponds to sequence 822-846, and a reverse primer (5ј-gttatgcggactttattg-3ј), which corresponds to sequence 1058-1041 (genbank accession number nm 007675). the primer pair for amplification of a 557-bp mouse glyceraldehyde-3-phosphate dehydrogenase (gapd) dna fragment was a forward primer (5ј-cggcaaattcaacggcacagt-3ј), which corresponds to sequence 199-219, and a reverse primer (5ј-tgggggtaggaacacggaagg-3ј), which corresponds to sequence 755-735 (genbank accession number xm 194302). pcr reaction mixtures consisted of 2 l of template, 1. for 30 sec at 53њc, extension for 30 sec at 72њc, and a final extension for 7 min at 72њc. the polymerase chain reaction (pcr) products were analyzed by electrophoresis on a 2.0% agarose gel. the identity of pcr products was confirmed by cloning and sequencing. dna sequencing was carried out with an abi prism 377-96 dna sequencer using the abi prism bigdye terminator cycle sequencing ready reaction kit (applied biosystems). total rna was extracted from tissue homogenates using an ultraspec-ii rna isolation kit. a pcr-amplified fragment of ceacam10 cdna (237 bp), which was inserted in pgem-t-easy, and a cdna fragment of the mouse gapd gene (1233 bp), which was inserted in pgem3 vector, were used as a template to prepare a 32 p-labeled cdna probe using a promega random-priming kit. rna samples (20 g) were subjected to denaturing by 1.0% agarose-formaldehyde gel electrophoresis and then blotted onto nylon membranes by capillary transfer as previously described [19] . after incubation with the prehybridization buffer (50% deionized formamide, 6ϫ ssc, 5ϫ denhardt solution, 1.0% sds, and 100 g/ml of sheared salmon sperm dna) for 2 h at 50њc, the membranes were hybridized with one labeled probe overnight at 50њc. following hybridization, the membranes were washed using standard procedures. rna messages on one filter membrane were observed after autoradiography and the probes were removed from the membranes as previously described [19] . the same membrane was then hybridized with another labeled probe. thus, hybridization with ceacam10 or gapd cdna probe was performed on the same filter membrane. in accordance with a method previously used [20] , a modified tyrode buffer, which consisted of 124.7 mm nacl, 2.7 mm kcl, 0.5 mm mgcl 2 , 0.4 mm nah 2 po 4 , 5.6 mm glucose, 0.5 mm sodium pyruvate, 15 mm nahco 3 , 10 mm hepes, 100 iu/ml penicillin, and 100 g/ml streptomycin was adjusted to ph 7.3-7.4 by aeration with humidified air/co 2 (19:1) in an incubator for 48 h at 37њc before use. mouse epididymides were removed and immersed in the medium. after careful dissection from the connective tissue, spermatozoa were extruded from the distal portion of the tissues for 10 min at 37њc. the cells were gently filtered through two layers of nylon gauze, layered on top of a linear gradient of 20%-80% percoll (v/v), and centrifuged at 275 ϫ g for 30 min at room temperature [21, 22] . three distinct cell layers formed. the lowest layer, which contained cells with progressive motility, was washed with three volumes of the medium and collected using centrifugation at 60 ϫ g for 10 min at room temperature. the sperm were resuspended and centrifuged two more times in a similar manner. the cell pellets were resuspended, and cacl 2 was added to the culture medium at a final concentration of 1.8 mm before the sperm were assayed. freshly prepared epididymal spermatozoa (10 6 cells/ml) were blocked in pbs containing 10% normal goat serum for 30 min at room temperature. the cells were further incubated with 1 m ceacam10 for 1 h. at the end of incubation the cells were centrifuged and the cell pellets were washed with pbs to remove the unbound ligands. the cells were air-dried on a glass slide and washed twice with pbs. the slides were incubated with the affinity-purified anti-ceacam10 antibody at a concentration of 1 g/ml in blocking solution for 1 h. the slides were washed three times with pbs to remove excess antibody before they were incubated with rhodamine-conjugated goat anti-rabbit igg diluted to 1:500 in blocking solution for 40 min. all slides were then washed with pbs, covered with 50% (v/v) glycerol in pbs, and photographed with a fluorescence microscope (axioplan 2 imaging; carl zeiss, oberkochen, germany). ejaculated sperm were collected from semen that existed in the uterine cavity of three vagina-plugged female mice. after extensive washing with pbs the ejaculated sperm without incubation with the exogenous cea-cam10 were smeared onto slides for immunolocalization of ceacam10 as mentioned above. sperm motility was determined using a computer-assisted sperm assay (casa) with a sperm motility analyzer (ivos version 10; hamilton-thorne research, beverly, ma). a 10-l sample was placed in a 10-m-deep makler chamber at 37њc. the analyzer was set as follows: negative phasecontrast optics and recording at 60 frames/sec; minimum contrast, 40; minimum cell size, 4 pixels; low-size gate, 0.2; high-size gate, 1.5; lowintensity gate, 0.5; high-intensity gate, 1.5; nonmotile head size, 29; nonmotile head intensity, 76; medium average path velocity, 50 m/sec; low path velocity, 7.0 m/sec; slow motile cells, yes; and threshold straightness, greater than 80%. ten fields were assessed for each sample. the fresh preparation of soluble svs was divided into fractions. i to iv by ion exchange chromatography on a deae-sephacel column (fig. 1a) . the fraction iii sample was further resolved into peaks a-e (fig. 1b) by ion exchange hplc on an sp column. peak e was a fad-dependent sulfhydryl oxidase (unpublished observation). on reducing sds-page gel, each of the a-to-d peak samples gave one rather broad 36-kda band that could be stained with either coomassie brilliant blue or periodic acid-schiff reagent, demonstrating their glycoprotein nature (fig. 1c, lanes 1, 3, 5, and 7) . each protein sample could be deglycosylated either by trifluoromethane sulfonic acid or exhaustive digestion with n-glycosidase f to a core protein that was identified as a sharp band between 26 and 28 kda by sds-page (fig. 1c, lanes 2, 4, 6 and 8) , indicating a similar molecular mass of the protein cores. apparently, peaks a-d were glycoproteins with an n-linked carbohydrate moiety. they were purified to homogeneity. automated edman degradation for each a-to-d peak samples for 14 cycles gave reliable data, which were assembled to the n-terminal sequences. avppxvtadnnvll was determined from the peak a sample. two amino acids were detected in each cycle during protein analysis for each peak (b to d). the actual yield of the two sequences in an individual cycle was such that the ratio of the major sequence to the minor one was estimated to be 2.5-3.0:1. assembly of the major and minor sequences gave a peptide sequence of aqvtveavppxvta and qvtveavppxvta, respectively. the three n-terminal peptides were completely confirmed in the ceacam10-deduced protein consisting of 265 amino acid residues in all positions except that x (asparagine), one potential site for an n-linked carbohydrate in ceacam10, was not identified in the protein sequencing (fig. 2b) . the post-translational cleavage at the peptide bond between glu and ala, thr and ala, or ala and gln in the signal peptide of the putative ceacam10 sequence gives rise to a peak a protein or to peak b-to-d proteins. as a result, peaks a to d share a very similar protein core with a slight difference in their n-terminal sequences. thereafter, we combined them for further study. among the svs protein components on sds-page gel, antibody against cea-cam10 immunoreacted only to a 36-kda protein band corresponding to the antigen, showing high specificity of the antibody. taken together, these data indicate that peaks a to d are translational products of the ceacam10 gene. each peak (a to d) was digested with trypsin, and the digests were subjected to hplc on a c 18 column. the chromatographic patterns of the four trypsin-digested samples were very similar. one representative chromatogram is shown in figure 2a . three amino acids were identified in each cycle of automated edman degradation of peak 9 on figure 2a . these data could be assembled to three peptide sequences of vfywyk, etiysn, and aiywyr in cea-cam10. the peptide sequence of ndegayaldmlfqnf in ceacam10 was completely confirmed by automated edman degradation of peak 18 (see fig. 2b ). ceacam10 was stable in 10 mm tris-hcl at ph 8.0, but it was degraded to an 18-kda protein component in 5% acetic acid (not shown). the cd spectrum of this protein at ph 7.4 shows a negative band with a minimum mean residue ellipticity of 12 000 deg cm 2 dmol ϫ1 at 217 nm (fig. 2c ). in addition, a positive band appeared as the cd profile extending below 200 nm. the spectral profile in the uv region shows some resemblance to that of the ␤ form of protein conformation [23] [24] [25] [26] , suggesting the presence of a considerable amount of ␤ form, a ␤ turn, or both in the protein molecule. we examined the distribution of ceacam10 and its rna message in the tissue homogenates of reproductive glands, including the seminal vesicle, epididymis, testis, coagulating gland, vas deferens, prostate, uterus, and ovary. the rna message was predominantly detected in the seminal vesicles (fig. 3a) . this was confirmed by the results of western blot analysis showing that ceacam10 was abundant in the seminal vesicle; a trace appeared in the epididymis and prostate only after over autoradiography (fig. 3b) . when equal amounts of total rna from the homogenate of a nonreproductive organ were compared with those of the seminal vesicle, very litter to no ceacam10 mrna was found in brain, heart, lung, liver, spleen, kidney, stomach, small intestine, muscle, skin, and thymus (not shown). ceacam10 was immunolocalized primarily to the luminal epithelium of the mucosal folds in the seminal vesicle slides of adult mice (fig. 4a) . the smooth muscle layer contained almost none. the strong immunochemical staining in the lumen supports the view that ceacam10 accumulates in the lumen as a result of its secretion from the luminal epithelium. further, we separated mucosal epithe-lial cells and smooth muscle cells from the tissue slices by lcm. ceacam10 transcripts were relatively abundant in the mucosal cells, but only trace amounts of the rna message appeared in the smooth muscle cells (fig. 4b) . the amounts of ceacam10 mrna in the seminal vesicles of mice at different ages were compared. the rna message first appeared at a considerable level in 3-wk-old mice. thereafter, the amount of transcript began increasing rapidly at 4 wk and reached a maximum in 7-wk-old mice (fig. 5a) . we analyzed mouse embryos from 4.5 to 18.5 days postcoitus (d.p.c.). the 4.5-6.5 d.p.c. samples included early stage embryos, extraembryonic tissue, and maternal uterus; the 7.5-9.5 d.p.c. samples included embryos and extraembryonic tissues, and the 10.5-18.5 d.p.c. samples were solely embryos. the rna message in the embryo samples was present in trace amounts on 5.5 d.p.c, increased remarkably from 6.5 d.p.c. to a maximum on 9.5 d.p.c., and rapidly declined thereafter to an almost undetectable level until delivery (fig. 5b) . because seminal vesicle growth is known to be androgen-dependent, we examined how androgen influenced ceacam10 expression in the seminal vesicles of adult mice that had been castrated 3 wk earlier (fig. 6) . ceacam10 mrna was undetectable in the total rna prepared from the control castrates that had received a daily injection of corn oil only compared with that of normal adults. induction of ceacam10 mrna was observed in the castrates treated with testosterone (5 mg/kg per day) for 8 consecutive days. figure 7a shows micrographs of epididymal spermatozoa with indirect fluorescence staining. no fluorescence was observed on the cells after they were treated successively with the ceacam10 antibody and rhodamine-conjugated anti-rabbit igg, demonstrating a lack of cea-cam10 on the cell surface. when spermatozoa were preincubated with 1 m ceacam10 in a blocking solution at room temperature for 45 min, rhodamine fluorescence was prominent on the middle piece, relatively weak on the tail, and faint on the head (fig. 7a, d) . apparently, sperm have ceacam10-binding sites that cover the entire cell surface. to access the binding of ceacam10 to epididymal sperm upon ejaculation, the ejaculated sperm were directly stained with the antibody. ceacam10 was immunodetected on the surface of the ejaculated sperm, in spite of high fluorescence background due to the free cea-cam10 that was difficult to removed completely during cell preparation (fig. 7b, d) . most spermatozoa freshly retrieved from the caudal epididymis of mice in modified tyrode buffer were mobile with visible tail beating. the result of casa for the cell incubation at specified conditions revealed that 90.0 m ceacam10 in cell culture greatly enhanced sperm motility relative to the motility of control cells at any incubation time (fig. 7c) . this work is the first to purify ceacam10 from mouse svs. we demonstrated it to be a 36-kda protein with an n-linked glycoconjugate. asn 11 , asn 54 , asn 71 , and asn 191 , each being part of consensus asn-xaa-(ser/thr) [27, 28] in the protein molecule, are the potential acceptor sites for the attachment of the carbohydrate moieties. our results of edman degradation support asn 11 as being one n-glycosylated site but rule out asn 71 in that role. removal of the hydrophobic leader sequence from the putative form of this protein gives a protein core consisting 226, 231, or 232 amino acid residues that sum to have a molecular mass of 25 270, 25 827 or 25 898 daltons, which is close to the molecular size of the deglycosylated proteins as determined by sds-page (fig. 1c) . the cea family consists of ceacam and pregnancyspecific glycoprotein subfamilies. these evolutionarily and structurally divergent glycoproteins of mammals share many common structural features [29] . they are characterized by the assembly of immunoglobulin variable (igv)like domain and immunoglobulin constant (igc)-like domains in each member of the family. according to the molecular model established by watt et al. [30] and tan et al. [31] , there are nine ␤ strands in one immunoglobulin-like domain involved in the maintenance of the three-dimensional architecture. in the ceacam10 molecule, residues 2-96 form one igv-like domain and residues 122-216 form another [14] . this may account for the characteristic cd shown in figure 2c . many members of the murine and human ceacam family contain either a transmembrane domain or a glycosyl ptdins moiety, but no such structural element is present in the ceacam10-deduced protein sequence, suggesting that ceacam10 is not a membrane-bound protein [32] . this is substantiated by our demonstration of its secretion from the luminal epithelium of seminal vesicle (fig. 4) . in the sexual glands of adult mice, the ceacam10 gene is predominantly transcribed and translated in the seminal vesicle. on ejaculation, the ceacam10-sperm binding may take place in the semen to enhance sperm motility. finkenzeller et al. [33] demonstrated that ceacam10 ϫ/ϫ male and female mice developed indistinguishably from wild-type litter mates with respect to sex ratio, weight gain, and fertility, but a significant reduction by 23% of litter size was observed in ceacam10 ϫ/ϫ mating. although this may be partially attributed to the lack of ceacam10 in the semen of ceacam10-inactivated males, it remains arguable whether in vitro ceacam10-enhanced sperm motility may play a significant role after coitus under natural circumstances. the maternal decidua surrounding the implantation site was not removed from the mouse conceptus that was used to prepare the commercially available embryo-stage blot for the observation of gene expression during pregnancy. as a result, the appearance of ceacam10 mrna in embryo samples at the blastula or gastrula stage (6.5-9.5 d.p.c.) (fig. 5b ) may arise from the maternal decidua as suggested in the study by finkenzeller et al. [33] . this finding, together with the lack of an rna message in embryo samples at 4.5 and 10.5-18.5 d.p.c. (fig. 5b) implies that ceacam10 might be weakly expressed if not silent during the entire course of embryonic development. in fact, we were unable to immunodetect the presence of ceacam10 in the em(a and b) or the ceacam10 antibody (c and d) and followed by incubation with rhodamine-conjugated anti-rabbit igg. the slides were observed via light microscopy (a and c) or fluorescence microscopy (b and d). bar ϭ 10 m. c) freshly prepared mouse spermatozoa in modified tyrode solution (10 5 cells/ml) containing 1.8 mm cacl 2 were incubated alone (⅙) or in the presence of 90 m ceacam10 (•) at 37њc for 0 to 60 min. cell motility determined at each specified incubation time was expressed as a percentage of control cell motility at time zero. points are mean ϯ sd for three determinations. *p ͻ 0.01 in a paired statistical comparison with the corresponding control. values were evaluated using one-way analysis of variance. bryo samples obtained by carefully microdissecting the extraembryonic tissues from the conceptus at 8.5 to 18.5 d.p.c. cytochemical observations shown in figure 7 suggest the presence of ceacam10-binding sites on the entire sperm surface. considering the presence of ceacam1 on human sperm cells [34] , it raises a possibility that heterophilic adhesion exists between ceacam10 and other ceacam molecules on the mouse sperm surface. this is unlike the action of other sperm motility effectors in the mouse svs, such as svs vii, which binds neutral phospholipid to enhance sperm motility [10] , and sva, which predominantly binds membrane phosphatidylcholine to suppress sperm motility [12] . motility of spermatozoa in hydrosalpingeal and follicular fluid of pigs purification and characterization of a sperm motility-dynein atpase inhibitor from boar seminal plasma. mol reprod purification and characterization of reversible sperm motility inhibitors from porcine seminal plasma low molecular weight components in bovine semen diffusate and their effects on motility of bull sperm effects of seminal vesicle fluid components on sperm motility in the house mouse purification and characterization of the active precursor of a human sperm motility inhibitor secreted by the seminal vesicles: identity with semenogelin the role of the seminal vesicles, coagulating glands and prostate glands on the fertility and fecundity of mice effects of seminal vesicle removal on fertility and uterine sperm motility in the house mouse the androgendependent mouse seminal vesicle secretory protein iv: characterization and complementary deoxyribonucleic acid cloning a novel heat-labile phospholipid-binding protein, svs vii, in mouse seminal vesicle as a sperm motility enhancer developmental profile of a caltrin-like protease inhibitor, p12, in mouse seminal vesicle and characterization of its binding sites on sperm surface seminal vesicle autoantigen, a novel phospholipid-binding protein secreted from luminal epithelium of mouse seminal vesicle, exhibits the ability to suppress mouse sperm motility a seminal vesicle autoantigen of mouse is able to suppress sperm capacitation-related events stimulated by serum albumin the cea10 gene encodes a secreted member of the murine carcinoembryonic antigen family and is expressed in the placenta, gastrointestinal tract and bone marrow enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from flavobacterium meningosepticum measurement of protein using bicinchoninic acid various forms of mouse lactoferrins: purification and characterization cleavage of structural proteins during the assembly of the head of bacteriophage t4 molecular cloning. cold spring harbor recovery, capacitation, acrosome reaction, and fractionation of sperm choosing among different technical variations of percoll centrifugation for sperm selection motility and fertilizing ability of rat epididymal spermatozoa washed by a continuous gradient of percoll determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion determination of the helix and beta form of proteins in aqueous solution by circular dichroism circular dichroic analysis of protein conformation: inclusion of the beta-turns analysis of the circular dichroism spectrum of proteins using the convex constraint algorithm: a practical guide sequence differences between glycosylated and non-glycosylated asn-x-thr/ser acceptor sites: implications for protein engineering the carcinoembryonic antigen (cea) family: structures, suggested functions and expression in normal and malignant tissues homophilic adhesion of human ceacam1 involves n-terminal domain interactions: structural analysis of the binding site crystal structure of murine sceacam1a[1,4]: a coronavirus receptor in the cea family redefined nomenclature for members of the carcinoembryonic antigen family zimmermann w. carcinoembryonic antigen-related cell adhesion molecule 10 expressed specifically early in pregnancy in the decidua is dispensable for normal murine development soluble isoforms of ceacam1 containing the a2 domain: increased serum levels in patients with obstructive jaundice and differences in 3-fucosyl-n-acetyl-lactosamine moiety key: cord-022499-7d58f1k3 authors: mall, sanjay; malcolm east, j.; lee, anthony g. title: transmembrane α helices date: 2004-01-07 journal: curr top membr doi: 10.1016/s1063-5823(02)52014-7 sha: doc_id: 22499 cord_uid: 7d58f1k3 this chapter discusses effects of intrinsic membrane proteins on lipid bilayers and model transmembrane α helices. incorporation of a protein into a lipid bilayer has significant effects on the properties of the bilayer. the rough surface presented by a protein to the surrounding lipid bilayer tends to produce poor packing unless the lipid fatty acyl chains distort to match the surface of the protein. in a liquid crystalline bilayer the lipid fatty acyl chains are disordered, because the chains undergo extensive wobbling fluctuations. the presence of a rigid protein surface reduces the extent of these motional fluctuations. however, the chains tilt and become conformationally disordered to maximize contact with the rough surface of the protein. the net result is that the presence of a protein leads to decreased order for the chains, with a wide range of chain orientations relative to the bilayer normal, but with reduced extent and rate of motion. because of the reduced motion, lipids adjacent to membrane proteins are often referred to as being motionally restricted. it is clear that the reasons for the disorder of the bulk lipids and the disorder of the lipids adjacent to the protein are different; for the bulk phospholipids, the disorder is dynamic, whereas, for the boundary lipids the disorder is static. bilayer of 30/~, about 20 residues will be required to span the core of the bilayer. the residues in the transmembrane region will be predominantly hydrophobic. however, for membrane proteins such as transporters and ion channels the transmembrane ot helices will also have to contain polar groups; the transmembrane helices will then be amphipathic rather than just hydrophobic. an extreme case could be a transmembrane a helix totally surrounded by other t~ helices in the center of a helical bundle: such a helix would not need to be hydrophobic at all, because it is not in direct contact with the lipid bilayer. however, although small in number, the available high-resolution structures for membrane proteins show no evidence for the presence of such purely hydrophilic transmembrane ot helices. it may be that the process of insertion of membrane proteins into the membrane during biogenesis requires all the transmembrane c~ helices to be relatively hydrophobic. analysis of the compositions of a large number of membrane proteins predicted to contain single transmembrane ot helices has shown that the amino acid composition of a transmembrane ot helix is distinctly different from that of hydrophobic a helices in water-soluble proteins. as expected, hydrophobic residues make up the bulk of the residues, the most common being leu (landolt-marticorena et al., 1993; wallin et al., 1997) . amino acids essentially excluded are the basic (arg and lys) and acidic (asp and glu) amino acids and their amide counterparts (asn and gin). transmembrane c~ helices are, however, relatively rich in bulky residues, such as ile, val, and thr, which, in water-soluble proteins, are classed as membrane destabilizers (their bulky side chains interfere sterically with the carbonyl oxygen in the preceding turn of the ot helix and thus destabilize the helical conformation). thus, factors such as residue volume and packing, which are important in determining helix stability in water-soluble proteins, are not so important for transmembrane ct helices, at least for membrane proteins containing single transmembrane c~ helices; effects of large residue volume will, in the membrane, be balanced by the favorable hydrophobic interactions of a large side chain with the fatty acyl chains. in water-soluble proteins, the conformationally flexible gly residue is also classed as a helix breaker, because it is an intrinsically flexible residue with the potential to adopt most of the dihedral angles available in a ramachandran plot. the observation that gly is quite common in transmembrane ot helices suggests that its potential flexibility is constrained in the bilayer environment. because gly possesses the smallest of all the side chains, it may play a role in mediating helix-helix interactions and packing in the membrane. the polar amino acids most commonly found within transmembrane ot helices are cys, thr, and ser. these residues can be stabilized within a hydrophobic environment by hydrogen bonding between their polar side chains and the peptide backbone at positions i -3 and i -4 (eilers et al., 2000) . figure 1 shows the positional preferences of the residues in the transmembrane ot helices of type i membrane proteins with a single transmembrane c~ helix oriented figure 1 positional preferences for amino acids in the transmembrane domains of human type i membrane proteins with single transmembrane a helices. modified from landolt-marticorena et al. (1993) . with its c-terminus on the cytoplasmic side of the membrane (landolt-marticoreno et al., 1993) . the amino-terminal end of the transmembrane domain contains an lie-rich region followed by a val-enriched region. the carboxyl-terminal half of the transmembrane a helix is leu-rich. ala is found randomly distributed throughout the transmembrane domain. aromatic residues are found located preferentially in the boundary regions, with trp at either end of the transmembrane domain, but with tyr and phe only at the carboxyl-terminal boundary. unlike the other aromatic amino acids, phe, is also found in the hydrophobic segment as well as in the boundary region. the polar regions flanking the transmembrane domain are enriched in arg and lys on the c-terminal side; asn, ser, and pro are enriched in the n-terminal flanking region. the presence of a positively charged c-terminus (cytoplasmic) could play a role in the process of insertion into the membrane, according to the inside positive rule of von heijne (1996) . the presence of particular residues at the n-and c-terminal ends of the helices could also be important in meeting the requirement to "cap" the ends of the ot helices; the initial four --nh and final four --c=o groups of an ot helix have no hydrogen-bonding partners provided by the peptide backbone of the a helix itself, and so suitable hydrogen-bonding partners have to be provided in some other way. one way is to extend the helix by three or four residues at each end with polar residues containing suitable hydrogen-bonding partners such as pro and ash. alternatively, if the hydrophobic, nonpolar residues in the transmembrane c~ helix extend into the headgroup region, hydrogen bonds could form with suitable groups in the glycerol backbone and headgroup regions of the lipid bilayer. either way, if about 20 residues are required to span the hydrophobic core of the bilayer, the total helix length could be up to 28 residues. the result is that there is a degree of indeterminacy in where the ends of transmembrane helices should be drawn; the precise ends of transmembrane ~ helices are often not known. the observed preference for trp and tyr residues for the ends of transmembrane ct helices agrees with measurements of the binding of small peptides at the lipid-water interface, which show that aromatic residues have a preference for the interface (wimley and white, 1996) . further, a number of small tryptophan analogues have been shown to bind in the glycerol backbone and lipid headgroup region of the bilayer, stabilized partly by location of the aromatic ring in the electrostatically complex environment provided by this region of the bilayer, and partly by exclusion of the fiat, rigid ring system from the hydrocarbon core of the bilayer for entropic reasons (yau et al., 1998) . thus, although it is agreed that aromatic residues at the ends of transmembrane a helices probably act as "floats" at the interface serving to fix the helix within the lipid bilayer, it is unclear whether the aromatic rings are located in the hydrocarbon or the headgroup region of the bilayer. this uncertainty is also apparent in the crystal structures of a number of membrane proteins. for example, the trp residues in the bacterial potassium channel kcsa (doyle et al., 1998) are found clustered at the ends of the transmembrane ot helices, forming clear bands on the two sides of the membrane, as shown in fig. 2 . however, the tyr residues clearly form a band on the periplasmic side of the membrane above the band formed by the trp residues. similarly, in the bacterial photosynthetic reaction center (rees etal., 1994) the majority of the trp residues are found near the periplasmic side of the protein near the ends of the transmembrane c¢ helices, as shown in fig. 3 . however, the band of trp residues is more diffuse than in kcsa, and some trp residues are likely to be located in the hydrocarbon core and some in the headgroup region. the average number of figure 2 the crystal structure of the potassium channel kcsa. a cross section with just two of the four identical subunits is shown. trl o residues are shown in space-fill representation and tyr residues are shown in ball-and-stick representation. two potassium ions in space-fill representation are shown moving through the channel. the separation between the two planes representing the outer edges of the trp residues is 35/~. (protein data bank [pdb] file ib18.) p ¢ figure 3 the structure of the l and m subunits of the photosynthetic reaction center of rhodobacter sphaeroides. trp residues are shown in ball-and-stick representation. an approximate location for the hydrophobic core of the bilayer of thickness 30 a is shown, as defined by the surface covered by detergent. (pdb file 1 aij.) residues in the transmembrane c~ helices of the bacterial photoreaction center is 26, corresponding to a length of about 39/~. the stretch of hydrophobic residues in these helices is, however, only about 19 amino acids or about 28.5/~ long (ermler et al., 1994; michel and deisenhofer, 1990) . this matches the thickness of the nonpolar region of the complex (about 30/~) as defined experimentally as the part covered by detergent in the crystal (roth et al., 1989 (roth et al., , 1991 . detergent is seen to cover some of the trp residues on the periplasmic side of the membrane, but not others (roth et al., 1991) . the distribution of trp residues on the cytoplasmic side of the complex is much less distinct than on the periplasmic side (fig. 3 ). if the hydrocarbon core of the bilayer around the complex does have a thickness of 30 ]~, then again the trp residues on the cytoplasmic side of the membrane will be located in both the hydrocarbon core and the headgroup regions of the bilayer (fig. 3) . in the ca2+-atpase of skeletal muscle sarcoplasmic reticulum the situation is more complex, as shown in fig. 4 (toyoshima et al., 2000) . many of the transmembrane o~ helices extend above the membrane surface to form a central stalk linking the transmembrane region to the cytoplasmic head of the protein. as a consequence some of the helices are very long; helix m5, for example, contains 41 residues. a ring of trp residues can be seen on the cytoplasmic side of the membrane helping to define the location of the membrane surface (fig. 4) . a lys residue (lys-262) in transmembrane c~ helix m3 can be seen pointing up from the hydrophobic core of the bilayer like a snorkel. because the cost of burying a charged residue in the hydrophobic core of a bilayer is very high (about 37 kj mo1-1 for a lys residue ; engelman et al., 1986) , it is likely that the amino group on lys-262 will be located at the interface; the trp residues in the ca 2+-atpase will then be located in the headgroup region of the bilayer. the structure of the ca2+-atpase is also unusual in that the first transmembrane ot helix contains two polar residues, asp-59 and arg-63, pointing out into the hydrocarbon core; presumably, stacking of asp-59 against arg-63 allows formation of an ion pair. the distribution of trp residues on the lumenal face of the ca2+-atpase is much more diffuse than on the cytoplasmic side. the hydrophobic thickness of the ca2+-atpase could be expected to be about 30/~, because that is the hydrophobic thickness of a bilayer of di(c18:1)pc, 1 the phospholipid that supports highest activity for the atpase . however, as shown in fig. 4 , this definition locates the lumenal loops between transmembrane ~ helices m5 and m6 i phospholipid designations are pc, ps, and pa for phosphatidylcholine, phosphatidylserine, and phosphatidic acid, respectively. fatty acyl chains are given in the format m:n, where m is the number of carbon atoms and n is the number of double bonds. thus, for example, dioleoylphosphatidylcholine is di(c 18:1)pc. and between m9 and m10 within the hydrocarbon core. further, it locates a lys residue (lys-972) totally within the hydrocarbon core, which seems unlikely. the hydrophobic thickness of the bilayer would have to be about 21 a to locate the two interhelical loops and lys-972 at the lumenal surface (fig. 4) ; this is close to the thickness of a bilayer of di(c14:i)pc (22 a). the crystal structure shown in fig. 4 corresponds to the ca2+-bound, e 1 conformation of the atpase. it has been shown that the e1 conformation of the atpase is favored by di(c14:1)pc, whereas di(c 18:1)pc favors the other major conformation of the atpase, e2 (starling et al., 1994) . thus, it is possible that conformational changes within the transmembrane region of the ca2+-atpase lead to changes in the interhelical loops and thus to changes in the effective hydrophobic thickness of the atpase (lee and east, 2001) . incorporation of a protein into a lipid bilayer can be expected to have significant effects on the properties of the bilayer. the rough surface presented by a protein to the surrounding lipid bilayer will tend to produce poor packing unless the lipid fatty acyl chains distort to match the surface of the protein. in a liquid crystalline bilayer the lipid fatty acyl chains are disordered, because the chains undergo extensive wobbling fluctuations. the presence of a rigid protein surface would be expected to reduce the extent of these motional fluctuations. however, the chains will have to tilt and become conformationally disordered to maximize contact with the rough surface of the protein. the net result is that the presence of a protein will lead to decreased order for the chains, with a wide range of chain orientations relative to the bilayer normal, but with reduced extent and rate of motion. because of the reduced motion, lipids adjacent to membrane proteins are often referred to as being motionally restricted. it is clear, therefore, that the reasons for the disorder of the bulk lipids and the disorder of the lipids adjacent to the protein (the boundary or annular lipids) are different; for the bulk phospholipids the disorder is dynamic, whereas for the boundary lipids the disorder is static. an example is provided by the bacteriorhodopsin trimer, whose crystal structure is unusual in showing a few well-defined lipid molecules (belrhali et al., 1999; luecke et al., 1999) . figure 5 shows some of the lipids located at the surface of the trimer. the electron densities for the chains are well defined, but the headgroups are disordered, so that the headgroups could not be identified; the lipids were therefore modeled simply as 2,3-di-o-phytanylsn-propane (belrhali et al., 1999) . the considerable static disorder of the chains is clear in fig. 5 , the rotational disorder of the chains being necessary to obtain good van der waals contacts with the molecularly rough surface of the bacteriorhodopsin trimer. lipids on the extracellular side of the membrane are better resolved than 10~ figure 5 structures of four phospholipid molecules identified in x-ray crystallographic studies of bacteriorhodopsin (belrhali et al., 1999) . the lipids have been modeled as 2,3-.di-o-phytanyl-snpropane. (pdb file lqhj.) those on the cytoplasmic side; the degree of order of the lipids parallels that of the protein, which is also greater on the extracellular side (grigorieff et al., 1996) . the average distance between the glycerol backbone oxygens for phospholipids on the two sides of the membrane was 31.6/~ (mitsuoka et al., 1999) . as expected, this closely matches the hydrophobic length of the transmembrane helices of bacteriorhodopsin; the mean helix length is 23 residues, corresponding to a length of about 35 a. the crystal structure also makes clear the very different conformations adopted by the various lipid molecules located on the surface of the tfimer. for example, one lipid molecule forms a hydrogen bond from its ether oxygens to a tyrosine --oh group at the end of a transmembrane a helix (fig. 6; belrhali et al., 1999; essen et al., 1998) . the result is that the strength of the interactions of individual boundary lipid molecules with the protein will be different. the disorder of the chains seen in fig. 6 is consistent with the results of molecular dynamics simulations of the bacteriorhodopsin trimer in a bilayer of diphytanyl phosphatidylglycerophosphate (edholm et al., 1995) . the molecular dynamics simulation agrees with experiment in predicting higher order for both the lipids and the protein on the extracellular side of the membrane; fluctuations in the loops and the ends of helices on the cytoplasmic side of the membrane are greater than on the extracellular side. this is also seen in fluctuations of the lipids, with lipids on the cytoplasmic side of the membrane fluctuating more strongly than those on the extracellular side (edholm et al., 1995) . the calculated order parameters for the chains are low, mainly due to a static tilt of the chains necessary to allow them to nestle against the rough surface of the protein. the chains in the purple membrane behave more like parts of the protein than parts of a fluid lipid phase, consistent with the idea of boundary lipids (edholm et al., 1995) . the boundary lipids and the bulk lipids in a membrane can be distinguished experimentally in many systems, because of the static disorder of the boundary lipids and the dynamic disorder of the bulk lipids. static and dynamic disorder give rise to very different electron spin resonance (esr) spectra for spin-labeled lipids, and esr spectra for membrane protein systems usually show two components, one "immobilized," corresponding to boundary lipid, and one relatively mobile, corresponding to bulk lipid (devaux and seigneuret, 1985; marsh, 1995) . studies with oriented samples have confirmed a wide range of orientational distributions for the boundary lipid, in contrast to the bulk lipid phase, where motion of the lipid long axis is about the bilayer normal (jost et al., 1973; pates and marsh, 1987) . a particularly important feature of a membrane protein as far as the lipid bilayer is concerned is the thickness of the transmembrane region of the protein. the cost of exposing hydrophobic fatty acyl chains or protein residues to water is such that the hydrophobic thickness of the protein should match that of the bilayer. the question then is how the system responds when these do not match. most models of hydrophobic mismatch assume that the lipid chains in the vicinity of the protein adjust their length to that of the protein, with the protein acting as a rigid body. when the thickness of the bilayer is less than the hydrophobic length of the peptide the lipid chains must be stretched. conversely, when the thickness of the bilayer is greater than the hydrophobic length of the peptide the lipid chains must be compressed (fig. 7) . stretching the fatty acyl chains will effectively decrease the surface area they occupy in the membrane surface, and, conversely, compressing the chains will increase the effective area occupied in the surface (fig. 7) . thus, figure 7 the result of a mismatch between the hydrophobic length of a peptide and the hydrophobic thickness of a lipid bilayer. left: positive hydrophobic mismatch (dp > dl). right: negative mismatch (dp< de). the top shows a "side" view of the chain packing around the peptide and the bottom shows a top view, illustrating the variation in chain cross-sectional area with distance from the peptide. figure based on fattal and ben-shaul (1993) . changes in fatty acyl chain order are linked to changes in average interfacial areas per lipid molecule. a number of terms have been suggested to contribute to the total free energy cost of deforming a lipid bilayer around a protein molecule (fattal and ben-shaul, 1993; nielsen et al., 1998): 1. loss of conformational entropy of the chains imposed by the presence of the rigid protein wall 2. bilayer compression/expansion energy due to changes in the membrane thickness 3. surface energy changes due to changes in the area of the bilayer-water interface 4. splay energy due to changes in the cross-sectional energy available to the chains along their length, resulting from curvature of the monolayer surface near the protein a number of models have been proposed to estimate these terms. fattal and ben-shaul (1993) calculated the total lipid-protein interaction free energy as the sum of chain and headgroup terms. for the chains the loss of conformational entropy imposed by the rigid protein wall was positive (unfavorable) even for perfect hydrophobic matching. the other contribution to the chain term arose from the requirement for hydrophobic matching and the consequent stretching or compression of the chain. the term due to the headgroup region was treated as an interracial free energy, including an attractive term associated with exposure of the hydrocarbon core to the aqueous medium and a repulsive term due to electrostatic and excluded-volume interactions between the headgroups (excluded-volume interactions signify that no two atoms can occupy the same position in space). the resulting profile of energy of interaction as a function of hydrophobic mismatch was fairly symmetrical about the point of zero mismatch. the lipid perturbation energy f (in units of kt per angstrom of protein circumference) calculated by ben-shaul (1995) fits to the equation where dp and dl are the hydrophobic thicknesses of the protein and lipid bilayer, respectively, and the unperturbed bilayer thickness is 24.5/~. the hydrophobic thickness of a bilayer of phosphatidylcholine in the liquid crystalline phase is given by where n is the number of carbon atoms in the fatty acyl chain (lewis and engelman, 1983; sperotto and mouritsen, 1993) . a simple, but crude calculation gives an idea of the size of the effect that can be expected from hydrophobic mismatch. it is assumed that the protein is very large and so appears fiat to a lipid molecule. it is also assumed that all the lipid perturbation energy is concentrated in the first shell of lipids around the protein. equation (1) then shows that if a lipid occupies 6 a of the protein circumference, the lipid-protein interaction energy would change by 3.6 kj tool -1 for a hydrophobic mismatch of 7 a, corresponding to an increase in fatty acyl chain length of 4 carbons, and by 22.8 kj mol -z for a hydrophobic mismatch of 17.5 a, corresponding to an increase in acyl chain length of 10 carbons. changes in interaction energies of 3.6 and 22.8 kj mo1-1 correspond to decreases in the lipid-protein binding constant by factors of 4.3 and 10 n, respectively. if the change in lipid-protein interaction energy were to propagate out from the protein surface to affect more than the first shell of lipids, effects of hydrophobic mismatch would be reduced. for example, if effects were averaged over three shells of lipids, changes in fatty acyl chain lengths by 4 and 10 carbons from that giving optimal interaction would decrease lipid-protein binding constants by factors of 1.6 and 21, respectively. energies of the magnitude calculated by ben-shaul (1995) are easily sufficient to result in conformational changes on a protein. if a protein conformational change results in a change in the hydrophobic thickness of the protein, the change will result in a deformation of the adjacent bilayer. because the equilibrium constant describing the equilibrium between two conformational states of a protein is determined by the total free energy difference between the two states, the energetic cost of the membrane deformation will contribute toward the equilibrium constant. the approach adopted by nielsen et al. (1998) came to rather similar conclusions. the most important energy terms were found to be the splay energy and the compression-expansion term, the splay energy term being most important close to the protein, with the compression-expansion term being more import'ant further from the protein. even though the bilayer deformation was calculated to extend some 30 a from the protein, most of the deformation was found concentrated in the component immediately adjacent to the protein. an alternative model for mismatch is the mattress model of bloom (1984, 1993) . this again expresses mismatch as two terms. the first is an excess hydrophobic free energy associated with exposing either lipid chains or the protein surface to the aqueous medium. the second is proportional to the contact area between the lipid chains and the hydrophobic surface of the protein. the calculations showed that, for a protein of hydrophobic thickness 20 a, which matches a bilayer ofdi(c 14:0)pc in the liquid crystalline state, the binding constant for di(c14:0)pc is about a factor of 2.5 greater than for a bilayer of di(c18:0)pc, which will give a bilayer too thick by 7 a (sperotto and mouritsen, 1993) . thus, effects of mismatch calculated in this way are similar to those calculated using the approach of fattal and ben-shaul (1993) . the importance of hydrophobic matching has been confirmed in a number of experimental studies (dumas et al., 1999; killian, 1998) . for example, although bacteriorhodopsin has relatively little effect on the phase transition temperatures of di(c14:0)pc or di(c16:0)pc (alonso et al., 1982) , it increases the transition temperature of di(c12:0)pc and decreases that of di(c18:0)pc (piknova etal., 1993) . this is consistent with hydrophobic matching models; because di(c 12:0)pc gives a too thin bilayer in the liquid crystalline phase, bacteriorhodopsin favors the gel phase, whereas di(c18:0)pc gives a too thick bilayer in the gel phase, so that bacteriorhodopsin favors the liquid crystalline phase. hydrophobic matching could also be the explanation for the unexpected observation that, in mixtures of di(c14:0)pc and di(c18:0)pc at temperatures where the mixture contains both gel and liquid crystalline phases, bacteriorhodopsin partitions equally between the two phases (piknova et al., 1997; schram and thompson, 1997) . this contrasts with the observed exclusion of bacteriorhodopsin from gel-phase lipid in mixtures containing single species of phospholipid (alonso et al., 1982; cherry et al., 1978) . it has been suggested that this shows that the requirements of hydrophobic matching are of prime importance; the hydrophobic thickness of bacteriorhodopsin is intermediate between the hydrophobic thickness of di(c14:0)pc in the gel phase and di(c18:0)pc in the liquid crystalline phase, so that bacteriorhodopsin shows little preference between the two. in mixtures of di(c 12:0)pc and di(c 18:0)pc, where, at low temperatures, two separate gel phases are formed, one enriched in di(c 12:0)pc and one enriched in di(c 18:0)pc, bacteriorhodopsin partitions very strongly into the di(c12:0)pc-enriched domains; this could be because the hydrophobic thickness of bacteriorhodopsin is better matched to gelstate di(c12:0)pc than to gel-state di(c18:0)pc (dumas et al., 1997) . however, in studies of the ca2+-atpase of sarcoplasmic reticulum using either spin-labeled (london and feigenson, 1981) or brominated phospholipids , strengths of binding of liquid crystalline-phase phospholipids to the atpase were found to be independent of fatty acyl chain length. these results are not consistent with the expectations of hydrophobic matching theory and suggest that, in liquid crystalline bilayers, u-helical membrane proteins are not rigid, but, in fact, can distort to match the thickness of the bilayer. such a distortion could explain why bilayer thickness affects the activity of membrane proteins such as ca2+-atpase (lee, 1998) . the structural distortion could take the form of a change in the tilt of the transmembrane ~ helices with respect to the bilayer normal or could be a change in the packing of the transmembrane c~ helices. an alternative approach to these questions, avoiding the complexity of real membrane proteins, is to use simple model transmembrane ~ helices, which can be synthesized chemically in large quantity. a number of studies have used peptides of the type ac-k2-g-ln-k2-a-amide (pn) consisting of a long sequence of hydrophobic leu residues capped at both the n-and c-terminal ends with a pair of charged lysine residues. the poly(leu) region forms a maximally stable u helix, particularly in the hydrophobic environment of the lipid bilayer. the charged lys caps were chosen both to anchor the ends of the peptides in the lipid headgroup region and to inhibit the aggregation of the peptides in the membrane. the peptide has been shown to adopt the expected u-helical structure in both liquid crystalline-and gel-phase bilayers (davis et al., 1983; huschilt et al., 1989; zhang et al., 1992a) . rates of hydrogen/deuterium exchange for the peptide p16 in lipid bilayers suggest that at least 80% of the peptide is in an u-helical conformation in the bilayer, meaning that the whole of the poly(leu) core must be u-helical (zhang et al., 1992b) . rates of hydrogen/deuterium exchange were greater at the n-and c-termini of the peptides than in the middle, suggesting some unraveling of the peptide at its ends (zhang et al, 1992b) . experiments with the peptides of this type suggest that about 15 lipid molecules are required for complete incorporation of the peptide into a bilayer of the appropriate thickness (webb et al., 1998) . this agrees with estimates from molecular modeling that about 16-18 lipid molecules will be required to form a complete bilayer shell around the peptide. at molar ratios of lipid less than this, non-bilayer phases can be induced, particularly when the hydrophobic length of the peptide is less than the hydrophobic thickness of the bilayer and when the peptide contains interfacial aromatic groups (de planque et al., 1999; killian et al., 1996; morein et al., 1997) . effects of single transmembrane u helices on lipid bilayers are likely to be less than those of a protein containing a bundle of transmembrane u helices. the cross-sectional area of a single transmembrane u helix is not much greater than that of a phospholipid molecule in the liquid crystalline phase, so that the hydrophobic surface presented to the lipid molecules is rather small. the structure of the lipid bound to bacteriorhodopsin shown in fig. 6 shows the two chains interacting predominantly with two different transmembrane u helices. this kind of interaction will obviously not be possible with a single transmembrane u helix. less extensive interactions between lipids and single transmembrane u helices than between lipids and membrane proteins is suggested by esr experiments. whereas esr spectra of spin-labeled lipids in the presence of membrane proteins typically show two-component spectra, as described above, esr spectra for lipid bilayers containing the peptide l24 and for a tryptophan-containing peptide of the type aw2(la)nw2a are single-component (de planque et al, 1998; subczynski et al., 1998) . this means either that the lipid fatty acyl chains are not "immobilized" on the peptide surface or that the rate of exchange between bulk and boundary lipid is fast on the esr time scale (i.e., exchange is faster than 107 s-l). effects of peptides on chain order in di(c14:0)pc or di(c16:0)pc measured using deuterium nuclear magnetic resonance (nmr) methods are small in both the liquid crystalline and the gel phases (davis et al., 1983; de planque et al., 1998 de planque et al., , 1999 roux et al., 1989) . thus, addition of a peptide aw2(la)tw2a to bilayers of di(c12:0)pc in the liquid crystalline phase resulted in only a 1.4-/~ increase in thickness (de planque et al., 1998) , whereas about an 11-/~ increase would be necessary for the bilayer thickness to match the hydrophobic length of the peptide. similarly, nezil and bloom (1992) estimated that the peptide p24 increased the thickness of a bilayer of (c16:0,c18:1)pc by just 0.6/~, despite the hydrophobic mismatch between the peptide and the lipid bilayer being ca. 10 a. increases in chain order caused by p24 in (c 16:0,c 18:1)pc in the liquid crystalline phase were detected using esr, but again the effects were small (subczynski et al., 1998) . effects of peptides on chain order will depend on the relative hydrophobic length of the peptide compared to the hydrophobic thickness of the bilayer, with long peptides decreasing order and short peptides increasing order, and such effects have been detected using infrared (ir) spectroscopy, but again effects were small (zhang et al., 1992a) . thus it appears that lipids will distort slightly to improve the match between the hydrophobic length of the peptide and the hydrophobic thickness of the bilayer, but the extent of these modifications is very limited and much less than required to produce full matching. a number of studies have been published on the effects of these peptides on the phase transition properties of lipid bilayers. addition of the peptide pi6 to bilayers of di(ci6:0)pc both broadens the main gel-to-liquid crystalline phase transition and decreases the enthalpy of the transition (morrow et al., 1985) . similar effects have been seen on incorporation of membrane proteins such as bacteriorhodopsin and ca2+-atpase (alonso et al., 1982; gomez-fernandez et al., 1980) . the decrease in enthalpy of the transition has often been taken to mean that the lipids adjacent to the protein (the boundary lipids) are very strongly perturbed by the peptide and so are unable to take part in the normal phase transition: they are effectively withdrawn from the transition. however, deuterium nmr spectra of mixtures of p24 and di(c 16:0)pc above and below the phase transition are typical of liquid crystalline-and gel-phase lipids, respectively, with no evidence for any "special" lipid unable to take part in the phase transition . similarly, as already described, esr spectra of spin-labeled lipids show the presence of a single type of lipid in the system, not separate bulk and boundary lipids in slow exchange (subczynski et al., 1998) . thus the peptides (or proteins) do not remove lipid from the main transition, but, rather, perturb the whole lipid bilayer. the peptide decreases the enthalpy difference between the liquid crystalline and gel phases, whereas the lipids in the bilayer remain recognizably liquid crystalline or gel (morrow et al., 1985) . morrow et al. (1985) showed that mixtures of lipids and peptides can be modeled in terms of regular solution theory (lee, 1978) . unfortunately, the number of free parameters in fitting to regular solution theory is high, so that little useful information is obtained from the analysis, apart from showing that the data are consistent with regular solution theory. effects of peptides or proteins on phase transition properties have therefore been interpreted qualitatively in terms of a twocomponent model in which one component is more or less unperturbed bulk lipid and the other is highly perturbed boundary lipid, which undergoes a broad phase transition of low enthalpy; this approach works for peptides of the poly(leu) type, but, for some reason, does not work with peptides of the k2(la)nk2 type (zhang et al., 1992a (zhang et al., , 1995 . differential scanning calorimetry (dsc) thermograms for mixtures with the poly(leu) peptides have been fitted to two components, attributed to the phase transitions of peptide-free and boundary lipid, respectively. the phase transition temperature for the peptide-free component is slightly less than that for pure lipid; this is probably due to the normal colligative effects that will follow from mixing the "pure" lipid phase with the boundary lipid, the latter acting as an "impurity." the phase transition temperature for the boundary lipid is higher than the bulk transition temperature for short-chain lipids, but is lower for long-chain lipids (zhang et al, 1992a) . the same observation has been made for membrane proteins (dumas et al., 1999; piknova et al., 1993) . this is consistent with the idea that short fatty acyl chains have to stretch to match the hydrophobic thickness of the membrane protein, whereas long fatty acyl chains have to compress. however, the experimental changes are much smaller than expected from models ofhydrophobic matching. further insights into how transmembrane ot helices might interact with lipid molecules in a bilayer have come from molecular dynamics simulations. one study was of a transmembrane ot helix of 32 alanine residues in a bilayer of di(c 14:0)pc in the liquid crystalline state (shen et al., 1997) . the peptide is not an ideal model for a transmembrane ot helix, because it lacks charged groups at each end to interact with the polar headgroups of the phospholipids. nevertheless many features of the simulation are informative. the simulation was started with the peptide as a pure ot helix. the central 15 residues (ala-12-26), which interacted just with the lipid fatty acyl chains, remained as a stable 0t helix. the n-and c-terminal regions of the ot helix, located in the lipid headgroup region, were less stable and fluctuated more, because of transient hydrogen bonding between the peptide bond amide hydrogen and the phosphate or fatty acyl ester oxygen atoms and the water; as a result, the ends of the helices become frayed. the length of the central helical region oscillated slightly about a 22-a average expected for an ot helix, varying between 20 and 23 a. the helix was tilted up to 30 ° with respect to the bilayer normal. because the helix contains no resides that would make strong contacts in the headgroup region or with water, there is no reason for it not to tilt (shen et al., 1997) . tilting in fact allows more hydrophobic contact by allowing more of the ala residues to be located in the core of the bilayer. the presence of the peptide had little effect on the calculated properties of the bilayer. the average bilayer thickness was not significantly changed, although the average order parameter for the ch2 groups in the chains decreased in the presence of the peptide. many different lipid molecules contributed to the immediate surroundings of the peptide (shen et al., 1997) . even if the fatty acyl chain of a particular lipid was immediately adjacent to the peptide, the headgroup of the lipid could be a substantial distance away. given the diameter of the helix and the size of the phosphate group, a phosphate immediately adjacent to the helix would be between 8 and 10 a away from the center of the helix (shen et al., 1997) . on average, five lipid molecules having their chains adjacent to the helix also had their headgroups adjacent, using this definition. however, the headgroup, as given by the position of the phosphorus atom, could be up to 16-18 a away. since the average distance between lipid headgroups is 8 a, this puts these lipids in the "second" shell around the peptide. other lipids existed between these extremes, suggesting a very diffuse environment around the protein rather than a discrete set of well-ordered shells of lipid. only rarely did an entire lipid molecule pack tightly around the helix. the shell around the helix contains contributions from a large number of lipid molecules, each contributing a small number of atoms (shen et al., 1997) . thus there is no evidence for a distinct shell of lipids around the peptide and any perturbation of the lipids extends out just a few angstroms. the lack of a clear shell of lipids around the poly(ala) peptide contrasts with the boundary lipids observed for membrane proteins and illustrated for bacteriorhodopsin in figs. 5 and 6. in part this could be an intrinsic feature of single transmembrane a helices, which will not be able to present a large surface area on which fatty acyl chains could be immobilized. the regular structure of a poly(ala) peptide compared to the rough surface of a typical or-helical peptide might also contribute to the lack of an immobilization shell of lipids. however, a significant factor is also likely to be the lack of polar groups at the ends of the peptide able to interact with the phospholipid headgroups. the importance of polar groups at the ends of the peptides has been shown in a simulation of isolated helices from bacteriorhodopsin (woolf, 1998) . the simulations show that a small number of the lipids surrounding a helix interact with it much more strongly than other lipids, due to a combination of van der waals (mediated by chains) and charge interactions (mediated by beadgroups) (woolf, 1998) . a molecular dynamics simulation of the peptide el6 in a bilayer of di(c 14:0)pc in the liquid crystalline phase showed that the peptide tilted with an average angle of 15.3 ° with respect to the bilayer normal, even though the thickness of the hydrophobic region of the bilayer (22 a) was a good match to the length of the ~ helix, 24 a (belohorcova et al., 1997) . a molecular dynamics simulation has been reported for pf1 coat protein in di(c16:0)pc (roux and woolf, 1996) . the coat protein contains an amphipathic a helix on the bilayer surface and a hydrophobic transmembrane a helix. fatty acyl chains next to the transmembrane helix were slightly more ordered than bulk lipids (roux and woolf, 1996) . similarly, in a simulation of the seventh transmembrane c~ helix of the serotonin 5ht receptor in di(c 14:0)pc, lipids in contact with the peptide had slightly higher order parameters than bulk lipids (duong et al., 1999) . the theories described above show that there will be an energetic cost associated with any change in the thickness of a bilayer. this would be reflected in values for the equilibrium constant describing the binding of lipids to the protein. a lipid that can bind to a protein without a change in bilayer thickness would bind more strongly to the protein than one for which binding required a change in bilayer thickness. the strength of interaction between a peptide and a phospholipid in a bilayer can be measured using a fluorescence quenching method (webb et al, 1998) . peptides used are of the type kkgl7wl9kka (l16) and kkgl10wl12kka (l22) containing a central trp residue as a fluorescence reporter group. the peptide is incorporated into bilayers containing the brominated phospholipid dibromostearoylphosphatidylcholine (di(br2c 18:0)pc); di(br2c 18:0)pc behaves much like a conventional phospholipid with unsaturated fatty acyl chains, because the bulky bromine atoms have effects on lipid packing similar to those of a cis double bond . contact between the bromine atoms in the lipid and the trp residue in the peptide leads to fluorescence quenching. in mixtures of brominated and nonbrominated phospholipids, the degree of quenching of the fluorescence of the tryptophan residue is related to the fraction of the surrounding (boundary) phospholipid molecules that are brominated, and thus to the strength of binding of the nonbrominated lipid to the peptide. an example of the method is shown in fig. 8 . the fluorescence intensity for the peptide l16 incorporated into bilayers of di(br2c 18:0)pc at a molar ratio of lipid to peptide of 100:1 is 5% of that in di(c18:1)pc, demonstrating highly efficient quenching of the tryptophan by the bromine-containing fatty acyl chains (fig. 8) . the fluorescence intensity in mixtures of di(br2c 18:0)pc and di(c18:1)pc decreases with increasing content of di(br2c 18:0)pc, reflecting the increasing number of boundary lipids that will be di(br2c 18:0)pc. as shown in fig. 8 , fluorescence quenching curves for l16 in mixtures of di(br2c 18:0)pc and (c 14:1)pc show more fluorescence quenching at intermediate mole fractions of di(br2c 18:0)pc than in mixtures with di(c 18:1)pc. this shows that di(c 18: i)pc binds more strongly to the peptide than does di(c14:1)pc. the results can be analyzed to give relative lipid-binding constants, as described in webb et al. (1998) . these lipid-binding constants for l16 and l22 are given in table i. for l22 strongest binding is seen with di(c22:1)pc, for which the relative binding constant is about double that for di(c 18:1)pc (table i ). the hydrophobic length of the peptide l22 is about 36 a, calculated for a stretch of 24 hydrophobic residues in total, with a helix translation of 1.5 a per residue. thus, strongest binding is seen when the hydrophobic length of the peptide matches the hydrophobic thickness of the bilayer, as expected from theories of hydrophobic mismatch. however, relative binding constants do not continue to decrease with decreasing chain length from di(c 18:1)pc to di(c 14:1 )pc as would have been predicted (table i ). an even larger deviation from theoretical predictions is observed with the short peptide l16 (table i ). in this case strongest binding is observed with di(c 18:1)pc, with binding decreasing with decreasing chain length to di(c14:i)pc, as expected. however, the peptide was found not to incorporate at all into bilayers of di(c24:l)pc, instead forming aggregates of peptide separate from the bilayer. ren et al. (1999) obtained very similar results, except that under their conditions, unincorporated peptide bound to the surface of the lipid bilayer, with the long axis of the peptide parallel to the surface. similarly, l16 was found to be only partly incorporated into ~bilayer hydrophobic thickness d calculated from d = 1.75(n -1), where n is the number of carbon atoms in the fatty acyl chain (sperotto and mouritsen, 1988) . bestimated hydrophobic length is 27 a for li6 and 36 a for l22. bilayers of di(c22:1)pc (webb et al., 1998) . thus, a short peptide cannot incorporate into a too-thick bilayer. it is suggested that a too-thin bilayer can match to a too-long peptide both by a slight stretching of the lipid and by tilting of the long axis of the helix with respect to the bilayer normal so that its effective length across the bilayer is reduced. however, a too-thick bilayer can only match a too-thin peptide by compression of the lipid, which becomes energetically unfavorable when the difference between the bilayer thickness and the peptide length exceeds about 6/~ (webb et al., 1998) . possible effects of aromatic residues at the ends of transmembrane ~ helices have been studied using peptides k2gfl6wl8fk2a (f2l14) and k2gyl6wl8yk2a (y2li4), in which one leu residue at each end of the poly(leu) stretch is replaced by either a phe or a tyr (mall et al., 2000) . in contrast to the results with l16, peptide f2l14 incorporated fully into bilayers of di(c24:1)pc, and y2li4 partitioned partially into di(c24:1)pc. the fluorescence quenching method was again used to obtain binding constants for phosphatidylcholines to the peptides, measured relative to the binding constant for di(c 18:1)pc (table ii) . the effective hydrophobic length of the peptide y2li4 might be expected to be somewhat greater than that of l16; if the peptide is modeled as an c~ helix with the two tyr residues oriented to be roughly parallel to the long axis of the c~ helix, the distance between the two tyr--oh groups is ca. 33 a, about 6/~ greater than the hydrophobic length of l16. the hydrophobic length of y2l14 calculated in this way matches the hydrophobic thickness of a bilayer of di(c20:1)pc, whereas the relative lipid-binding constants increase from di(c14:1)pc to di(c22:1)pc (table ii) . similarly, relative binding constants for f2li4 increase with increasing chain length from di(c14:i)pc to di(c24: i)pc. the results with y2l14 and f2li4 show that introduction of aromatic "hydrophobic thickness of the bilayer calculated from d = 1.75(n -1), where n is the number of carbon atoms in the fatty acyl chain (sperotto and mouritsen, 1988 ). residues at the two ends of the hydrophobic sequence increases the ability of the short peptides to partition into thick lipid bilayers. the observation that the highest relative binding constant is obtained with bilayers considerably thicker than the calculated hydrophobic length of the peptides suggests that the presence of aromatic residues at the ends of the helices could lead to marked thinning of the bilayer around the peptides (mall et al., 2000) . the chain length dependence of lipid binding to y2l20 is much less marked than for the shorter peptides; the relative binding constant increases from di(c14:1)pc to di(c18:i)pc, but then hardly changes with increasing chain length between di(c18:i)pc and di(c24:)pc (table ii) . this contrasts with l22, which shows markedly stronger interaction with di(c22:l)pc than with phospholipids with shorter or longer chains. this again suggests that the introduction of the two tyr residues leads to an increase in the thickness of the bilayer with which optimal interaction of the peptide is observed. interactions between transmembrane c~ helices and the phospholipid headgroups also have to be considered. using the fluorescence quenching method, it was shown that a small number of anionic phospholipid molecules (possibly just one) bound strongly to the peptide l16 , the remaining molecules binding with an affinity equal to that of phosphatidylcholine. the binding constant for the strongly bound phosphatidic acid molecule relative to phosphatidylcholine in a medium of low ionic strength was 8.6 (in mole fraction units), corresponding to a difference in unitary binding energies of -5.3 kj mo1-1 . at ph 7.2, phosphatidic acid bears a single negative charge (cevc, 1990) . the binding constant for phosphatidic acid changed little with ionic strength, suggesting that the interaction with the positively charged peptide did not follow simply from a high positive potential in the vicinity of the positively charged lys residues on the peptide, increasing the local concentration of anionic phospholipid. the energy of interaction between two ions u is given by where zl and z2 are the charges on the two ions eo is the permittivity of a vacuum, er is the relative permittivity (dielectric constant) of the medium, and r is the distance between the two ions. assuming a dielectric constant of 78.5 (water), we find that an energy of interaction of 5.3 kj tool -1 corresponds to a distance of separation between two monovalent ions of 3.3/~. this therefore suggests that strong interaction requires the anionic headgroup of phosphatidic acid to be in close contact with one of the lys residues on the peptide. once this strong interaction with a single phosphatidic acid molecule has been made, other phosphatidic acid molecules will then interact with el6 relatively nonspecifically, with a binding constant relative to phosphatidylcholine close to 1. the relative binding constants for phosphatidylserine were less than for phosphatidic acid and are more sensitive to ionic strength . for phosphatidylserine, the presence of the positively charged ammonium group as well as the negatively charged carboxyl group in the headgroup region may reduce interaction with the positively charged peptide. in contrast to l16, the binding constants for anionic phospholipids to l22 are very similar to those for zwitterionic phospholipids, with a relative binding constant close to 1. it could be that tilting of l22 in the bilayer, necessary to match the hydrophobic length of l22 to the hydrophobic thickness of a bilayer of di(c18:1)pc, locates the lys residues on the peptide too far from the lipid headgroup region to allow a strong interaction between the anionic phospholipid and the peptide. both phosphatidylserine and phosphatidic acid bind more strongly to the peptides yell4 and yel20 than does phosphatidylcholine, the effect of anionic phospholipid decreasing slightly with increasing ionic strength. however, in this case the experiments are consistent with a model in which the binding constants for all the anionic phospholipid molecules binding to the peptide are increased slightly (mall et al., 2000) . this suggests that the presence of the tyr residues prevents close association of the anionic phospholipid group with the cationic lys residues. these results suggest that the effects of charge on the interactions between phospholipids and transmembrane ot helices will often be rather small and will be strongly dependent on the detailed structure of the peptide and its orientation in the membrane. this picture is consistent with the results of the molecular dynamics simulations of individual ~ helices of bacteriorhodopsin in bilayers of di(ci4:0)pc, which showed that a small proportion of the lipid molecules interacted with the o~ helices much more strongly than the others, and that these strong interactions were dominated by electrostatic terms rather than van der waals terms (woolf, 1998) . in general, binding constants for phospholipids to membrane proteins also show relatively little selectivity for anionic phospholipids. for example, binding constants for phosphatidic acid and phosphatidylserine relative to phosphatidylcholine are close to 1 for the caz+-atpase (dalton et al., 1998) , and binding constants for phosphatidic acid and phosphatidylserine for the (na+-k+)-atpase are about twice those for phosphatidylcholine (esmann and marsh, 1985) . however, there is evidence for the presence of a small number of "special" anionic phospholipids binding to some membrane proteins, acting as "cofactors." an example is provided by cytochrome c oxidase, whose crystal structure shows the presence of a lipid molecule bound between the transmembrane a helices (iwata et al., 1995) . interaction between an anionic phospholipid and a binding site on a membrane protein would be specific if strong binding requires close interaction between the anionic headgroup and a positively charged residue on the protein, as suggested by the results presented here. the phase of the phospholipid is important in determining interactions with transmembrane ot helices. as shown in fig. 8 , fluorescence quenching is much more marked in mixtures of di(br2c18:0)pc and di(c16:0)pc at temperatures where both liquid crystalline and gel phases are present than in mixtures of di(brac18:0)pc and di(c18:1)pc (mall et al., 2000) . thus, l16 is excluded from regions of lipid in the gel phase and accumulates in regions in the liquid crystalline phase. the binding constants of l|6 and l2z for di(c16:0)pc in the gel phase relative to di(c18:i)pc in the liquid crystalline phase are ca. 0.15 (mall et al., 2000) . this is consistent with the expectation that van der waals contacts between an all-trans fatty acyl chain and the molecularly rough surface of a peptide will be poor; one way that this poor packing can be overcome is by exclusion of the peptide from the gel phase. quenching plots for y2l14 are very similar to those of l16 (fig. 8) , showing that the presence of bulky aromatic residues does not have any significant effect on the selectivity for liquid crystalline-over gel-phase lipid (mall et al., 2000) . further, since y2li4 shows a preference for longer chain phospholipids than l16, y2li4 might have been expected to show a greater preference for gel-phase lipid than l16, because phospholipid in the gel phase gives a thicker bilayer than the corresponding lipid in the liquid crystalline phase. because y2l14 and l16 show equal preferences for liquid crystalline-over gel-phase lipid, any effects of hydrophobic matching between the peptide and the bilayer must be small compared to effects of lipid phase on the interaction energies between the peptides and the lipid (mall et al., 2000) . preferential partitioning of proteins from domains in the gel phase into domains of liquid crystalline lipid has been demonstrated for a variety of membrane proteins, including bacteriorhodopsin (cherry et al., 1978) and ca2+-atpase kleeman and mcconnell, 1976) . effects of sphingomyelin at 25°c are very similar to the effects of gel-phase di(c16:0)pc ( fig. 8 ; mall et al, 2000) . mixtures of bovine brain sphingomyelin and di(c 18:1)pc are in a two-phase region at 25°c, with gel-phase domains enriched in sphingomyelin (untracht and shipley, 1977) . thus, partitioning of the peptides between gel-and liquid crystalline-phase lipid shows little dependence on the structure of the phospholipid. it has been suggested that plasma membranes of mammalian cells contain domains or "rafts" enriched in sphingomyelin and that particular enzymes, particularly those associated with cell signaling, are concentrated within the rafts (simons and ikonen, 1997) . the results presented here suggest that membrane proteins containing transmembrane ot helices will tend to be excluded from these rafts, and it may therefore be significant that many of the signaling proteins suggested to be contained within the rafts are anchored to the membrane by glycosylphosphatidylinositol anchors (harder and simons, 1997) . the presence of cholesterol has a marked effect on incorporation of the peptides into phospholipid bilayers (webb et al., 1998) . incorporation of cholesterol at a 1 : 1 molar ratio to phospholipid leads to a general reduction in incorporation of the peptides l16 and l22, but superimposed on this effect is a chain length effect. in the presence of cholesterol, the binding constant of p16 for di(c 14:1)pc relative to di(c 18 : 1)pc increased from 0.4 to about 1, as expected if the presence of cholesterol increases the effective chain length of the c14 chain so that it more nearly matches the hydrophobic length of the peptide (fig. 8) . consistent with this interpretation, the presence of high molar ratios of cholesterol prevented the incorporation of p16 into bilayers of di(c18:i)pc. nezil and bloom (1992) showed that incorporation of cholesterol at 33 mol% increases bilayer thickness by about 4 ,~. studies with brominated analogues of cholesterol showed that cholesterol binds to the peptides with a binding constant only a factor of about two less strongly than di(c 18:1)pc . this is rather surprising, given the relatively rigid structure of the steroid ring of cholesterol and the molecularly rough surface of the peptide. in other studies, it has been shown that cholesterol binds relatively weakly at the lipid-protein interface of the atpase (simmonds et al., 1982 (simmonds et al., , 1984 ; comparison with the peptide studies reported here suggests that weak binding of cholesterol to the atpase involves interactions in the lipid headgroup region rather than interactions between the sterol ring and the hydrophobic transmembrane helices. the requirement to match the hydrophobic thickness of a membrane protein to that of the surrounding lipid bilayer could be important in a number of ways. targeting of proteins to their correct final destinations in a cell is essential in maintaining cell integrity. in the bulk flow model, the vast majority of proteins synthesized in the endoplasmic reticulum (er) are believed to leave the er by default and flow along the exocytic pathway until they reach the plasma membrane (nilsson and warren, 1994) . some proteins, however, have to be retained at particular points along the exocytic pathway. compartmental localization could be achieved in one of two ways. the first involves a retention signal in the protein, which, at the appropriate point in the exocytic pathway, prevents forward movement of the protein by denying it access to budding transport vesicles of the onward pathway. the second involves a retrieval signal, leading to recapture of the protein after it has left the compartment in which it resides. the classical retrieval signal is the kdel sequence found in many er-resident proteins; the situation appears to be different for golgi-resident proteins, where membrane-spanning domains act as retention signals (nilsson and warren, 1994) . despite the extensive flux of proteins through the golgi, the golgi maintains its own distinctive population of resident proteins. furthermore, the distribution of enzymes within the golgi is organized according to function, so that, for example, the distributions of glycosyltransferases and glycosidases, although overlapping, are distinct (colley, 1997; roth, 1987) . many of the proteins in the golgi membrane are predicted to contain a single transmembrane ot helix, oriented with the n-and c-termini on the inner and outer faces of the membrane, respectively. the golgi retention signal in such proteins has been shown to involve the membrane spanning domain (munro, 1991 (munro, , 1995 nilsson et al., 1991; swift and machamer, 1991) . however, the membrane-spanning domains show no sequence homology, and it has not been possible to identify any particular motif leading to retention (bretscher and munro, 1993; colley et al., 1992; munro, 1991) . thus, sialyltransferase remains localized in the golgi even when its 17-amino-acid transmembrane domain is replaced by 17 leu residues (munro, 1991) . however, a longer stretch of 23 leu residues did not provide an efficient retention signal (munro, 1991) . similarly, a 4-residue insertion into the transmembrane domain of galactosyltransferase reduced its retention in the golgi (masibay et al., 1993) . the reverse effect has been shown with the influenza virus neuraminidase, which shifted from the plasma membrane to the golgi and er when the number of residues in the transmembrane domain was reduced (sivasubramanian and nayak, 1987) . the lack of a clear retention motif, together with the inability to saturate the mechanism for golgi retention by overexpression, suggests that retention is not a receptor-mediated event (nilsson and warren, 1994) . one possible model is then retention by preferential interaction with membranes of optimal thickness (nilsson and warren, 1994) . both bretscher and munro (1993) and masibay et al. (1993 ) showed that transmembrane domains of golgi proteins are shorter (average 15 residues) than transmembrane domains of plasma membrane proteins (average 20 residues). it has therefore been suggested that if the golgi membrane is thinner than the plasma membrane, membrane proteins with short transmembrane domains will interact "more strongly" with the lipid bilayer of the golgi than with that of the plasma membrane, leading to retention in the golgi (bretscher and munro, 1993; masibay et al., 1993) . the studies with model peptides described above show that a protein containing a transmembrane ot helix with a hydrophobic length greater than the hydrophobic thickness of the golgi membrane will be able to move out of the golgi into the plasma membrane. however, a protein whose transmembrane ot helix has a hydrophobic length less than the hydrophobic thickness of a particular membrane will not be able to enter that membrane, and such a protein would then be retained in the golgi (webb et al., 1998) . studies of targeting of proteins in yeast are also consistent with a lipid-based model (rayner and pelham, 1997) . the length of the transmembrane domain is important in targeting with long helices (24 residues), ensuring transport to the plasma membrane. however, for proteins with shorter transmembrane domains, the relative hydrophobicity of the transmembrane domain has been suggested to be important as well as its length, this determining targeting to the golgi and the vacuole (rayner and pelham, 1997) . retention of some membrane proteins in the er could also depend on the length of the transmembrane domain of the protein. an important class of er membrane proteins are those with an n-terminal catalytic domain exposed to the cytoplasm and a c-terminal membrane anchor. such proteins are inserted into the er membrane post-translationally by a signal-recognition-particle-independent pathway. no er retrieval signals have been identified in these proteins. instead, it has been observed that the hydrophobic domain is rather short. for example, cytochrome bs, a protein of this type, has a transmembrane domain containing just 17 hydrophobic amino acid residues (pedrazzini et al., 1996) . if the length of the hydrophobic stretch is increased to 22 residues, the protein is transported out of the er along the secretory pathway (pedrazzini et al., 1996) . it could therefore be that matching of the thickness of the lipid bilayer and the transmembrane length of the protein is important in retention in er, as was suggested for the golgi complex. although the length of the transmembrane domain appears to be the most important factor, the structure of the c-terminal, lumenal, region has also been shown to contribute to retention (honsho et al., 1998) . experiments with another c-terminalanchored protein, the ubiquitin-conjugating enzyme ubc6 from yeast, suggest that the thickness requirements of the er and golgi membranes may be different, explaining targeting between these two organelles (yang et al., 1997) . whereas ubc6 containing the wild-type 17-residue transmembrane domain targets to the er, increasing the length of the transmembrane domain to 21 residues results in movement to the golgi, and increasing the length further to 26 residues allows movement to the plasma membrane. these experiments show that the length of the transmembrane ot helix is often an important factor in targeting, although it is likely to be only one of a number of important factors. the lengths of the transmembrane ot helices are also likely to be important in the proper function of membrane proteins containing multiple transmembrane ot helices. an example already described is that of the ca2+-atpase, which shows highest atpase activity in di(c 18:1)pc and lower activities in bilayers of phospholipids with longer or shorter fatty acyl chains (lee, 1998) . changes in the atpase underlying these changes in atpase activity are complex (lee, 1998) , but all must be mediated by the transmembrane ot helices, because these are the parts of the atpase that can "sense" the change in bilayer thickness. in the case of the ca2+-atpase it seems that, as described above, the two major conformational states of the atpase (el and e2) have different preferences for bilayer thickness, the e1 conformation favoring thin bilayers and the e2 conformation favoring thick bilayers (lee, 1998) . changing the bilayer thickness could change the tilt of the transmembrane u helices in the ca2+-atpase, it could change the packing of the helices, and, possibly, it could lead to changes in the structures of the loops connecting the helices, changing the effective lengths of the helices. all these changes could be linked to changes in the phosphorylation domain of the ca2+-atpase, located well above the surface of the membrane. if, as seems likely, the various membranes in a cell have different thicknesses because of their different lipid compositions, the structure of each 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dimyristoylphosphatidylcholine. ii. interaction energy analysis the transmembrane domain of a carboxyl-terminal anchored protein determines localization to the endoplasmic reticulum the preference of tryptophan for membrane interfaces interaction of a peptide model of a hydrophobic transmembrane a-helical segment of a membrane protein with phosphatidylcholine bilayers: differential scanning calorimetric and ftir spectroscopic studies ftir spectroscopic studies of the conformation and amide hydrogen exchange of a peptide model of the hydrophobic transmembrane a-helices of membrane proteins peptide models of helical hydrophobic transmembrane segments of membrane proteins. 2. differential scanning calorimetric and ftir spectrooscopic studies of the interaction of ac-k2-(la)12-k2-amide with phosphatidylcholine bilayers we thank the biotechnology and biological sciences research council (bbsrc) for financial support of the original studies reported here. key: cord-002835-qaogpxy9 authors: too, issac horng khit; bonne, isabelle; tan, eng lee; chu, justin jang hann; alonso, sylvie title: prohibitin plays a critical role in enterovirus 71 neuropathogenesis date: 2018-01-11 journal: plos pathog doi: 10.1371/journal.ppat.1006778 sha: doc_id: 2835 cord_uid: qaogpxy9 a close relative of poliovirus, enterovirus 71 (ev71) is regarded as an important neurotropic virus of serious public health concern. ev71 causes hand, foot and mouth disease and has been associated with neurological complications in young children. our limited understanding of the mechanisms involved in its neuropathogenesis has hampered the development of effective therapeutic options. here, using a two-dimensional proteomics approach combined with mass spectrometry, we have identified a unique panel of host proteins that were differentially and dynamically modulated during ev71 infection of motor-neuron nsc-34 cells, which are found at the neuromuscular junctions where ev71 is believed to enter the central nervous system. meta-analysis with previously published proteomics studies in neuroblastoma or muscle cell lines revealed minimal overlapping which suggests unique host-pathogen interactions in nsc-34 cells. among the candidate proteins, we focused our attention on prohibitin (phb), a protein that is involved in multiple cellular functions and the target of anti-cancer drug rocaglamide (roc-a). we demonstrated that cell surface-expressed phb is involved in ev71 entry into neuronal cells specifically, while membrane-bound mitochondrial phb associates with the virus replication complex and facilitates viral replication. furthermore, roc-a treatment of ev71-infected neuronal cells reduced significantly virus yields. however, the inhibitory effect of roc-a on phb in nsc-34 cells was not through blocking the craf/mek/erk pathway as previously reported. instead, roc-a treated nsc-34 cells had lower mitochondria-associated phb and lower atp levels that correlated with impaired mitochondria integrity. in vivo, ev71-infected mice treated with roc-a survived longer than the vehicle-treated animals and had significantly lower virus loads in their spinal cord and brain, whereas virus titers in their limb muscles were comparable to controls. together, this study uncovers phb as the first host factor that is specifically involved in ev71 neuropathogenesis and a potential drug target to limit neurological complications. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 enterovirus 71 (ev71) is a non-enveloped, positive-sense, single-stranded rna virus, and causes hand, foot and mouth disease (hfmd) in humans. being a close relative of poliovirus, ev71 is deemed as an important neurotropic virus worldwide [1] . since its first isolation in california in 1969, several major outbreaks have been reported in china, singapore, korea, and japan [2] [3] [4] [5] . although the clinical manifestations are generally mild and self-limiting, including hfmd and herpangina, severe neurological complications have been consistently reported with ev71-associated infections, causing brainstem encephalitis, acute flaccid paralysis, pulmonary edema and cardiopulmonary failure [6, 7] . in addition, some patients who have recovered from severe disease have been reported to develop long term neurologic and psychiatric disorders [8] . there are currently no effective prophylactic or therapeutic agents against ev71. although several vaccines have completed phase iii clinical trials [9] , regulatory issues may limit their widespread utilization. in addition, as these vaccine candidates consist of inactivated virus from a single ev71 genotype (c4), cross-protection against other genotypes may be limited [10, 11] . the increasing awareness of life-threatening ev71 infections has boosted research in recent years to further understand virus-host interactions and develop effective antiviral strategies [12] [13] [14] [15] [16] [17] . however, the neuropathogenesis of ev71 is still poorly understood. infection occurs when the virus enters the body upon ingestion and/or inhalation. the virus multiplies initially in the alimentary tract mucosa and rapidly reaches the deep cervical and mesenteric lymph nodes via the tonsils and peyer's patches [18] . after a short transient systemic dissemination phase, the virus accumulates and actively replicates in muscles where it is believed to infect motor neurons at the neuromuscular junctions. experimental evidence supports that ev71 migrates to the brainstem via retrograde axonal transport as previously described for its close relative poliovirus [1, 2, [19] [20] [21] . however, the molecular mechanisms involved in ev71 infection of motor neurons to access the central nervous system (cns) have not been studied. indeed in vitro studies aiming at studying ev71 neurovirulence have employed neuroblastoma cell lines that may not reflect accurately infection in motor neurons. to address this gap, we have recently reported a novel in vitro model of ev71 infection in the murine motor neuron cell line nsc-34 [22] . nsc-34 cells originate from the fusion between murine neuroblastoma and spinal cord cells, and possess motor neuron-like properties, such as generation of action potentials and production of acetylcholine [23] , therefore making it a relevant model to study the mechanism of ev71 neuropathogenesis. we demonstrated that nsc-34 cells are permissive to ev71 clinical isolates and found that, unlike any other mammalian cell types so far reported, ev71-infected nsc-34 cells do not undergo apoptosis and lysis. instead we showed that the virus exits the cells via a non-lytic mode, a phenomenon that has also been previously described for poliovirus [21, 24, 25] . these unique features thus suggested that the infection cycle of ev71 in nsc-34 cells involves host pathways and partners that are likely to be different from those previously identified in other mammalian cell types such as muscle cells and neuroblastoma cells. in this work, using a proteomics approach coupled with mass spectrometry, we have identified a panel of cellular proteins that were dynamically regulated during ev71 infection of nsc-34 cells. among the host protein candidates that were up-regulated, we focused our attention on prohibitin (phb) and characterized its role during ev71 infection in nsc-34 cells. we also demonstrated the importance of phb during ev71 infection in a symptomatic mouse model of ev71 infection. to identify the host proteins involved in ev71 infection cycle in nsc-34 cells, a 2de proteomic approach was undertaken. nsc-34 cells were infected with ev71 at m.o.i. 10 , and the cell lysates were harvested at 6, 24, 48 and 72 hours for downstream proteomic analysis in which a range of 350-800 spots were resolved. by using pdquest 2-d analysis software (biorad), a total of 81 protein spots (fig 1a) that displayed at least 0.5-fold differential expression (p<0.05, two-tailed student's t-test) compared to uninfected controls, were excised for in-gel digestion and maldi-tof ms analysis. the peptide fingerprints were then searched against ncbinr mouse genome database for protein identification using mascot program (http://www.matrixscience.com/). the protein candidates were then categorized based on their primary functional class indicated in uni-protkb database (s1 table) . to illustrate the dynamic regulation of host proteins during the viral infection, a heat map was generated using multiexperiment viewer (mev), with the distance between proteins represented by euclidean average linkage clustering (fig 1b) . this clustering analysis revealed that proteins that were up-regulated (fig 1c) during infection are mainly involved in motility (23%) and catalytic processes (20%), while proteins that participate in rna processing (32%) and energy biosynthesis (20%) generally displayed a down-regulation trend during the course of infection (fig 1d) . functional interactions among the selected host proteins were analyzed by string (search tool for the retrieval of interacting genes/proteins). this platform allows establish proteinprotein interactions based on published literature, online databases, predicted functional associations using genomic information or observations made with other organisms [26] . the protein network obtained was significantly enriched with the p value of less than 0.05, suggesting that the interactions are highly associated and unbiased (fig 2; s2 table) . furthermore, some of the selected host proteins appear to have strong associations among each other as indicated by the thickness of connecting lines which reflects the confidence level of the interactions [26] . using go annotations for biological processes, molecular functions, cellular compartments and protein classes, the protein candidates were localized within the cytoplasm (29.2%), organelles (20.8%) and macromolecular complexes (13.9%) (s1a fig). in addition, they were found to be involved in various biological processes including mitochondrial biogenesis, proteolytic activity, cytoskeletal machinery and rna processing (s1a fig) , consistent with the protein clusters observed in the string network (fig 2) . finally, molecular function analysis indicates that majority of these proteins contribute to nucleic acid binding transcription factor activity (34.7%), structural molecule activity (25%) or binding (22.2%) (s1a fig). a meta-analysis with other selected proteomic studies of ev71-infected muscle and neuronal cells [13] [14] [15] 17, [27] [28] [29] revealed minimal overlap between nsc-34, rd and other neuronal cell types with 4 protein candidates only, namely actb, tubb, pdia3 and eno1, suggesting that the host-pathogen interactions in nsc-34 cells are unique (s1b fig). the limited overlap may also be partly explained by differential proteomics approaches. act and tubb are involved in maintaining cytoskeletal structure, and they are found highly modulated during viral infection to facilitate virus internalization and transportation [30] [31] [32] . eno1 has been shown previously to interact with cytoskeletal proteins in intermediate filaments framework rearrangement [33] . on the other hand, pdi functions in catalyzing reduction and oxidation processes and protein folding [34] . it has also been demonstrated to be involved in humoral immune response [35] or viral replication (denv) [36] and entry (hiv) [37] . not surprisingly, greater overlap was seen between motor-neuron nsc-34 and other neuronal cells (26 shared hits) than between nsc-34 and rd cells (5 shared hits). importantly, neuro-specific proteins such as prph and uchl1 were only identified from profiling studies in nsc-34 and other neuronal cells, thus validating our 2de proteomic approach. seven protein candidates namely, alpha-enolase (eno1), dep domain-containing mtorinteracting protein (deptor), peripherin (prph), phosphatidylethanolamine-binding protein 1 (pebp1), prohibitin (phb), stomatin-like protein 2 (stoml2) and protein disulfideisomerase a3 (pdia3) were selected for validation of the proteomic findings by gene knockdown. these host proteins have been previously shown to be associated with various steps in the life cycle of viruses, such as entry [37] [38] [39] [40] and replication [15, 41, 42] , or to be involved in autophagy [43] [44] [45] and axonal transport [46] [47] [48] . silencing of each selected gene target was achieved by reverse transfecting the on-targetplus sirna smartpool into nsc-34 cells, prior to viral infection. sirna smartpools consist of four highly potent gene-specific sirna molecules which have been modified to minimize off-target activity and enhance gene specificity [49] . cytotoxicity of the sirnas smartpools was first established. apart from the sirna pool targeting pdia3, no significant cytotoxicity was observed with the other sirna pools at 25 and 50 nm with cell viabilities greater than the 70% viability threshold (fig 3a) . the pdia3-specific sirna pool concentrations were lowered to 5 and 10nm to avoid cytotoxicity (fig 3a) . virus titers in the culture supernatants of sirna-transfected cells were then determined at 48 h.p.i. results indicated that silencing of stoml2, prph, phb and deptor led to significantly lower virus titers, whereas pdia-, pebp1-and eno1-knocked down resulted in increased viral titers in the supernatants of ev71-infected nsc-34 cells compared to control (fig 3b) . importantly, both trends were dose-dependent. therefore, these results validate the 2d-proteomics approach as a powerful way to identify host proteins that play a role during ev71 infection in nsc-34 cells. prohibitins belong to a highly conserved protein family present in unicellular and multicellular eukaryotes [50] . prohibitin (phb; bap-32) and prohibitin 2 (phb2, rea, bap-37) are two highly homologous members of this family and are ubiquitously expressed in multiple cellular compartments including the mitochondria, nucleus, and the plasma membrane. prohibitins have been involved in multiple cellular functions including cell proliferation and maintenance of the functional integrity of the mitochondria [50] . in addition, phb specifically has been previously reported to be involved in the entry step of alphavirus chikungunya (chikv) [40] , and flaviviruses dengue (denv) [38] and hepatitis c (hcv) [39] , and to interact with envelope proteins from white spot syndrome virus to prevent infection [51] . however, there has been no report so far on the role of phb during ev71 infection. first, modulation of phb expression during ev71 infection in nsc-34 cells was confirmed by western blot and showed an overall up-regulation of phb during the course of infection compared to uninfected control (s2 fig). next, the impact of phb gene silencing on virus production was further analyzed using a wider range of sirna pool concentrations including 5, 10, 25 and 50 nm. efficacy of the gene silencing was assessed by western blot and showed a dose-dependent decrease of phb expression in nsc-34 cell lysates (fig 3c) . this dose-dependent phb knockdown correlated well with a dose-dependent reduction in the viral titers measured in the culture supernatant (fig 3d) , therefore supporting the role for phb in ev71 infection cycle. to address the possibility of false positive or off-target effects of the sirna pool, phb gene silencing was performed with the individual sirnas species from the pool. western blot showed that each individual sirna was capable of silencing phb expression significantly (s3a 10 . the viral titers in the culture supernatants were determined by plaque assay at 48 h.p.i.. cell viability of the transfected cells was assessed using alamarblue assay. statistical analysis was performed using two-tailed student's t-test ( ãã p<0.005, ããã p<0.005). relative band quantification (below western blot) was determined by imagej, by normalizing to loading control, β-actin. error bars represent mean ± standard deviation. one representative of two biological repeats is shown. to determine if phb mediates entry of ev71 into nsc-34 cells, a competition assay was performed using commercially available anti-phb antibody. incubation of nsc-34 cells with anti-phb antibody prior to infection led to significant reduction of the viral titer in a dosedependent manner (fig 4a) . to demonstrate a physical interaction between cell surfaceexpressed phb and ev71, a proximity ligation assay (pla) was performed. in this assay, phb and ev71 are recognized by specific primary antibodies raised in two different species, which are in turn recognized by species-specific secondary antibodies conjugated to a probe and a target, respectively. should ev71 and phb be in close proximity, the probe and the target ligate, amplify and result in emission of a fluorescent signal. scarb-2 which has been previously demonstrated as the main receptor for ev71 in rd cells [52] was used as positive control and a red fluorescent signal was readily detected in ev71-infected rd cells (fig 4b) . a positive signal was also detected with nsc-34 cells incubated with anti-phb and anti-ev71 antibodies thus supporting the close proximity between ev71 virus particles and surfaceexpressed phb (fig 4b) . in contrast, and expectedly, no signal was detected with mouse scarb-2 (mscarb2) and ev71 antibodies (fig 4b) , since we have shown previously that entry of ev71 into nsc-34 cells is not mediated by mscarb-2 [22] . the physical interaction between ev71 and cell surface-expressed phb was further assessed by performing a co-immunoprecipitation experiment. nsc-34 cells were incubated with ev71 for 2 hours at 4˚c to allow viral adsorption onto the cell surface but no internalization. the total cell lysate was obtained and a pulldown was carried out using antibody specific to phb or an isotype igg antibody control. the immunoprecipitates were then analyzed by western blot using anti-ev71 primary antibody. a discrete band at the expected size was obtained when pulldown was performed with the anti-phb antibody whereas no band was seen when pulldown was done with the igg isotype (fig 4c) . these findings thus support that ev71 physically interacts with cell surface-expressed phb, and suggest that phb may serve as a receptor for ev71 entry into nsc-34 cells. in addition to its association to the plasma membrane at the cell surface, phb is also present intracellularly [50] . to study the role of intracellular phb in ev71 infection cycle, phbknocked down nsc-34 cells were transfected with ev71 rna genome and the viral titers were determined at 6, 12, 18 and 24 hours post-transfection, in order to assess virus production within a single cell infection cycle. transfection of viral genome was meant to bypass the virus entry step which we have shown involves cell surface-expressed phb. no viral titer was obtained at 6 h.p.t. in the phb-knocked down and control cells (fig 5a) . from 12 h.p.t onwards the viral titers detected in the culture supernatant from phb-knocked down cells were consistently lower than those measured in sintc or non-treated cells (fig 5a) . this result thus demonstrates that phb plays a role in the intracellular virus infection cycle. to further confirm this hypothesis, a luciferase ev71 (lucev71) replicon transfection assay was performed. in this replicon, the viral structural genes have been replaced with a luciferaseencoding gene while the other parts of the viral genome are retained (fig 5b) . upon transfection, the replicon undergoes a single replication cycle with no production of virus progeny since it is deficient in viral structural proteins. the luminescence measured from the cell culture is proportional to the amount of luciferase produced inside the cell thereby reflecting the replication activity of the replicon. here, a significant reduction in the luminescence signal was observed in phb-knocked down nsc-34 cells compared to sintc-treated and nontreated cells (fig 5c) . thus, together the data support that intracellular phb is involved in ev71 viral replication. ) were incubated with ev71 at 4˚c for 2 hours to allow viral adsorption, prior to pla staining. scarb2-stained nsc-34 and rd cells served as negative and positive controls, respectively. scale bar represents 20μm. a representative experiment of two independent repeats is shown. (c) co-immunoprecipitation of ev71 and surface-expressed phb. ev71 and nsc-34 cells were co-incubated for 2 hours at 4˚c. the cell lysate was pulled down with anti-phb or igg isotype control antibodies prior to immunoblotting using anti-vp1 primary antibody. mock-infected cells and igg isotype served as control. a representative experiment of two independent repeats is shown. ip, immunoprecipitation; ib, immunoblot. to further investigate the role of intracellular phb during ev71 infection cycle, immunostaining was performed on ev71-infected nsc-34 cells probing for phb, ev71 capsid proteins vp0/vp1 and the viral replication intermediate dsrna. co-localization between phb and dsrna, and between phb and ev71 capsid proteins was readily observed (fig 6a) , supporting that intracellular phb could be involved in the viral replication and/or assembly processes. to further study the role of intracellular phb in viral replication, co-immunoprecipitation was carried out using antibody against phb. pull down with anti-phb antibody followed by western blot using anti-ev71 3d/3cd antibody led to the detection of a 72kd band that corresponds to the ev71 3cd protein complex and a 53 kda band (3d polymerase) which comigrated with igg heavy chain (fig 6b) . taken together, these data support a physical interaction between intracellular phb and ev71 non-structural proteins 3d and 3cd, indicating that intracellular phb is likely involved in viral replication. previous studies have shown that the main replication sites of picornavirus are located at the golgi apparatus and endoplasmic reticulum (er) [53] . we have demonstrated that intracellular phb co-localizes with dsrna and is closely associated with the ev71 3d polymerase. given that intracellular phb is abundantly and mainly expressed on mitochondria [54] , we speculated that in nsc-34 cells mitochondria may be exploited by ev71 as replication site. consistently, co-localization of phb and ev71 with mitochondria was observed by ifa (fig 7a and 7b) . furthermore, co-localization of phb, dsrna and mitochondria was also readily detected, thus indicating that mitochondrial phb is associated with the viral replication (fig 7c) . to exclude the possibility that the replication complexes detected were actually associated to the er, which is in close proximity to mitochondria, the mitochondrial fraction was prepared from ev71-infected nsc-34 cells and western blot analysis revealed the presence of viral capsid protein vp1 (38 kd), 3d (53 kd) and 3cd (72 kd) proteins (fig 7d) . furthermore, the mitochondrial fraction was shown to be free of cytoplasmic contamination, as evidenced by the presence of mitochondrial marker (atpb, 52 kd) and lack of er marker (calreticulin, 48 kd). similar observation was made with the mitochondrial fraction prepared from ev71-infected rd cells (fig 7d) , suggesting that ev71 is able to exploit various cellular organelles as replication scaffolds in various mammalian cell types from different species. this finding is consistent with a previous study where ev71 vp1 was found to be associated with mitochondria in hela cells [55] . to further support the close proximity and likely interactions between the viral replication complexes and mitochondria, transmission electron microscopy was performed on ev71-infected nsc-34 cells. clustering of mitochondria surrounding viral replication complexes could be seen (electron-dense like structures) in the infected cells, and examination at a higher magnification indicated a close association between mitochondria membrane and virus complex/virus particle (fig 7e) . collectively, the data strongly indicate that mitochondria in nsc-34 cells are exploited by ev71 as a replication scaffold and that mitochondria-associated phb plays a role in this process. recent studies have shown that phb activity is inhibited by a group of phytochemicals called rocaglamides, which are derived from the traditional chinese medicinal plants aglaia [39, [56] [57] [58] . we therefore investigated whether roc-a could interfere with ev71 infection cycle by blocking phb activity in nsc-34 cells. incubation of roc-a with virus prior to nsc-34 cell infection (co-treatment) did not result in any significant reduction in viral titer (s4a fig) . when cells were pre-treated with roc-a prior to ev71 infection (pre-treatment), less than 1 log pfu/ml of decrease in viral titer was observed at the highest drug concentration only (500 nm) (s4b fig). in contrast, a dose-dependent decrease in the viral titer was seen when roc-a treatment was applied after infection (post-treatment) at concentrations ranging between 10-100 nm (fig 8a) . western blot analysis of the cell lysates further confirmed the dose-dependent reduction of intracellular viral capsid protein and phb (fig 8b) . taken together, the data suggest that the antiviral effect of roc-a on ev71-infected nsc-34 cells targets the viral replication step and not the entry step, in contrast to a previous study with hcv [39] . prior studies focusing on cancer have reported that the mechanism by which roc-a targets and inhibits phb activity involves blocking the craf/mek/erk pathway [56, 57] , and this was also described in the hcv study [39] . to investigate whether the craf/mek/erk to decipher the mode of action of roc-a in nsc-34 cells, immunostaining of roc-a treated nsc-34 cells was performed. decreased signals for phb and mitochondria were observed with increasing concentrations of roc-a (fig 8c) , suggesting that roc-a might affect mitochondrial integrity. we thus assessed the mitotoxicity and cytotoxicity of roc-a using the mitochondrial toxglo assay (promega). while cytotoxicity remained generally minimal over the range of roc-a concentrations tested, the intracellular atp levels were significantly reduced in a dose-dependent manner, thus indicating functional impairment of the mitochondria in roc-a-treated nsc-34 cells (fig 8d) . consistently, using the membrane-permeant jc-1 dye as an indicator of mitochondrial membrane potential, roc-a-treated nsc-34 cells displayed marked and dose-dependent mitochondrial depolarization as evidenced by the decrease of red fluorescent j-aggregates, compared to untreated cells (s6 fig). together, these observations suggest that roc-a treatment in nsc-34 cells results in reduced levels of phb, leading to mitochondrial destabilization and lower atp production. one could thus speculate that the lack of intact mitochondria and reduced intracellular atp levels might eventually impact negatively on ev71 replication efficacy. the role of phb in ev71 infection cycle was also studied in human muscle (rd) and neuronal (sk-n-sh) cell lines. as human (gi246483) and murine (gi6679299) phb display high similarity in their amino acid composition, most of the anti-phb antibodies commercially available demonstrate good cross reactivity with cell lines of both species. we first showed by flow cytometry comparable levels of surface expression of phb on rd, sk-n-sh and nsc-34 cells (s7 fig). however, both phb gene silencing and phb receptor blocking experiments performed in human muscle rd cells did not impact the viral titer (s8a and s8b fig) . on the contrary, phb silencing in the human neuroblastoma cells sk-n-sh led to a significant dosedependent reduction in viral titer in the culture supernatant (fig 9a) . in addition, reduced virus titers were observed with sk-n-sh cells pre-treated with anti-phb antibodies, thus supporting that cell surface-expressed phb is involved in ev71 entry into this human neuroblastoma cell line (fig 9b) . to investigate if intracellular phb is also involved in viral replication in sk-n-sh cells, transfection of lucev71 replicon into phb-silenced sk-n-sh cells was performed. results showed a significant reduction in the luminescence signal compared to controls (fig 9c) . finally, the effectiveness of roc-a treatment in ev71-infected sk-n-sh cells was also assessed. similar to our observations with nsc-34 cells, a significant dose-dependent decline in viral titers was observed (fig 9d) . taken together, these findings thus strongly indicate the specific involvement of phb in both viral entry and replication of ev71 in neuronal cells from both human and murine origins. concentrations of roc-a for 48 hours before assessment of cytotoxicity (fluorescence) and mitotoxicity (luminescence) using mitochondrial toxglo assay. statistical analysis was performed using one-way anova with dunnett's post-test ( ã , p<0.05; ãã , p<0.005; ããã , p<0.001; ãããã , p<0.0001). one representative from two independent experiments is shown. https://doi.org/10.1371/journal.ppat.1006778.g008 i. the culture supernatant was collected for viral titer determination by plaque assay, and the cell lysate was harvested for western blot analysis. statistical analysis was performed using one-way anova with dunnett's post-test ( ãã , p<0.005). relative band quantification (below western blot) was determined by imagej, by normalizing to loading control, β-actin. error bars represent mean ± standard deviation. cellular cytotoxicity was assessed using alamarblue assay. one representative from two independent experiments is shown. https://doi.org/10.1371/journal.ppat.1006778.g009 the role of phb was further investigated in vivo, using the mouse model of ev71 infection that we established previously where 2-week old ag129 mice (deficient in type i&ii ifn pathways) infected with ev71 display progressive limb paralysis and spatio-temporal virus accumulation in the limb muscles, spinal cord and brainstem [59] . here, immunohistochemical analysis showed that phb was readily detected in the limb muscles, brainstem and spinal cord at day 4 p.i. (fig 10a) . furthermore, some co-localization with ev71 was observed (fig 10a) . next, the in vivo anti-ev71 efficacy of roc-a was assessed by treating therapeutically ev71-infected mice with roc-a at day 1 and 3 p.i. the development of clinical manifestations was clearly delayed in the roc-a-treated mouse group which resulted in increased survival time compared to the untreated or vehicle-treated control groups (fig 10b) . in addition, the viral loads in limb muscles, spinal cord and brain in both roc-a-treated and vehicle-treated mice were determined. comparable viral loads were detected in the limb muscles from both groups (fig 10c) . in contrast, viral titers in the spinal cord and brain from the roc-a-treated animals were significantly lower compared to the vehicle-treated group (fig 10c) , thus supporting that roc-a treatment specifically impairs ev71 neuropathogenesis. these findings correlate well with our in vitro data showing that the role of phb during ev71 infection cycle is specific to neuronal cells. together, the in vivo data support that phb plays a critical role in ev71 neurovirulence, and that roc-a represents a potential therapeutic strategy to limit ev71 neuropathogenesis, thereby minimizing neurological manifestations and complications. the re-emergence of neurotropic enteroviruses in recent years has motivated investigations into ev71 transmission in the neuronal system. understanding the interplay between virus and host proteins is likely to result in the identification of potential novel drug targets and development of novel antiviral strategies. here, using a proteomics approach, we have identified a panel of host factors that displayed dynamic regulation during the course of ev71 infection in the motor neuron nsc-34 cells. the host protein candidates are mainly involved in cytoskeletal structure maintenance, rna processing and mitochondrial biogenesis. by employing a sirna gene silencing approach, we have shown that some of these host factors either facilitate or limit ev71 productive infection. among these host factors, phb was found to exert a pro-viral effect as evidenced by the reduced viral titers measured in the culture supernatant of nsc-34 cells when the expression of phb was down-regulated, and by an increased viral titer in phb over-expressing cells. phb is mainly localized on plasma membrane, mitochondria and nucleus, and has been involved in multiple signaling pathways regulated by growth factors, immune response, mitochondrial biogenesis, cell migration, proliferation and survival [50, 58] . interestingly, knockdown in nsc-34 cells of stoml2, which was shown to interact with phb and participate to mitochondria biogenesis [60] , resulted in significant reduction of viral titer, similar to that seen with phb-knocked down cells. this further supports the involvement of mitochondrial proteins during ev71 infection cycle in nsc-34 cells. in addition, previous studies have reported the association of phb with internalization of several viruses, including hcv [39] , chikv [40] , denv [38] , and coronavirus (sars-cov) [61] . furthermore, phb was shown to promote hiv replication by interacting with the hiv-1 glycoprotein [62] . using various experimental approaches, we have demonstrated that cell surface-expressed phb is physically associated with ev71 and is involved in the entry of the virus into nsc-34 cells. on the other hand, by employing a lucev71 replicon, we have shown that intracellular (mitochondrial) phb plays a role in ev71 replication activity. this was further supported by (a) two week-old ag129 mice (n = 3) were infected i.p. with ev71 (10 7 pfu) and the limb muscles, spinal cord and brainstem were harvested for immunohistochemical analysis at day 4 p.i. scale bars denote 100 μm (20× magnification) and 50 μm (40× magnification). (b) ev71-infected ag129 mice (n = 8) were treated i.p. with roc-a at 0.25 mg/kg at day 1 and 3 p.i. and were monitored for survival and clinical manifestations. clinical scores were defined as follows: 0, healthy; 1, ruffled hair and hunched back; 2, limb weakness; 3, one limb paralysis; 4, both limbs paralysis at which point the animals were euthanized. statistical analysis of survival curve was performed using logrank (mantel-cox) test ( ãã , p<0.005). (c) limb muscles, spinal cord and brain were harvested at day 4 p.i. for viral load determination by plaque assay (n = 6/7). dotted line represents the limit of detection. statistical analysis was performed using mann-whitney u test ( ã , p<0.05). error bars represent mean ± sem. one representative of two biological repeats is shown. https://doi.org/10.1371/journal.ppat.1006778.g010 role of prohibitin in ev71 neuropathogenesis the observation that mitochondrial phb co-localizes with the replicating viral genome (dsrna) and the non-structural proteins 3d polymerase and 3cd complex. co-immunoprecipitation and tem approaches also confirmed the physical proximity and likely interactions between viral complexes/viral particles and mitochondria. these data thus led us to propose that mitochondria could serve as replication site for ev71 in nsc-34 cells. the association of ev71 with mitochondria was reported in a previous study where it was proposed that ev71 could potentially interact with some mitochondrial signaling proteins to evade host anti-viral innate immunity [55] . previous studies have reported that membrane-bound phb binds to ras in a gtp-dependent manner, which in turn activates craf kinase and eventually triggers the mapk pathway [58] . similar to other flavaglines, rocaglamide (roc-a) is a natural product that displays insecticidal, anti-fungal, anti-inflammatory and anti-cancer activities [63] . mechanistically, roc-a was found to inhibit craf-phb interactions in tumor cells [64] [65] [66] , and in an in vitro model of hcv infection [39] . in ev71-infected nsc-34 cells, incubation with nm concentrations of roc-a resulted in a dose-dependent reduction in virus titers. however, the mechanism by which roc-a exerts its antiviral effect against ev71 in nsc-34 cells does not seem to be mediated by blocking the craf/mek/erk pathway, given that phosphorylated erk proteins could not be detected in uninfected nsc-34 cells, suggesting that this pathway is not functional in these cells. instead, we found that roc-a-treated cells displayed reduced expression of mitochondrial phb and lower levels of intracellular atp, which suggests that mitochondria integrity/functionality is impaired in roc-a treated nsc-34 cells. since we showed that mitochondrial phb is involved in ev71 replication and that mitochondria serve as replication site for this virus, the impact of roc-a on phb expression and mitochondria integrity could represent the basis of its antiviral activity. further investigation is necessary to decipher the molecular mechanisms by which roc-a affects the expression of mitochondrial phb. interestingly, we found that the role of phb in ev71 entry and replication was limited to cells of neuronal origin, thus supporting a role of phb specifically in ev71 neuropathogenesis. this neuro-specific phenotype was also observed in vivo where roc-a-treatment resulted in reduced virus loads in the cns (spinal cord and brain) only but not in the limb muscles from infected mice, although phb was readily detected in the muscle cells as well. the cell typedependent involvement of phb during ev71 infection likely reflects differential intracellular events with different host factors being engaged during ev71 intracellular life cycle. since ev71 is known to be able to use multiple receptors to enter host cells, one could speculate that the host factors that are engaged during ev71 infection depend on the entry receptor that is being used by the virus. further study is necessary to explore this idea. in conclusion, our work has uncovered a novel host factor that is specifically involved in ev71 neurovirulence. in addition, our data support that roc-a, a previously established anticancer drug that targets phb, could represent a therapeutic approach to limit ev71 neuropathogenesis, and thus prevent or limit associated neurological complications. given the current attrition in effective antiviral drugs against ev71, roc-a repurposing is worth considering seriously. all the animal experiments were carried out under the guidelines of the national advisory committee for laboratory animal research (naclar) in the aaalac-accredited nus animal facilities. the animal experiments described in this work were approved under the nus institutional animal care and use committee (iacuc) protocol number 16-0136. non-terminal procedures were performed under anesthesia, and all efforts were made to minimize suffering. murine motor neuron nsc-34 cells (cellutions biosystems, clu140), human rhabdomyosarcoma (rd) cells (atcc ccl-136) and human neuroblastoma sk-n-sh cells (atcc htb-11) were used in this study. all cell lines were cultured in dulbecco's modified eagle's medium (dmem) (gibco) containing 10% fetal bovine serum (fbs) (gibco) at 37˚c with 5% co 2 . non-mouse-adapted ev71 s41 (5865/sin/00009, accession no.: af316321), kindly provided by prof. chow v. t. k. at national university of singapore, was isolated from the lymph node of a ev71-infected patient who died of encephalitis and pulmonary edema in singapore [67] . the virus stocks were made in rd cells and the viral titers were determined by plaque assay on rd cells. total proteins extract from infected cells was prepared using proteoextract complete mammalian proteome extraction kit (millipore). briefly, the cell pellet was thawed by resuspension in ice-cold resuspension buffer and the proteins were extracted with extraction buffer at room temperature (rt). benzonase and reducing agent were added during protein extraction to minimize nucleic acid contamination and to remove disulphide bonds, respectively. the solubilized protein suspension was subjected to centrifugation at 25,000 ×g for 30 minutes at 4˚c to remove the remaining insoluble material. the recovered cell extract was stored at -20˚c until further analysis. the protein samples (200 μg, quantified using rc dc bradford assay, biorad) were loaded onto individual lanes of the isoelectronic focusing (ief) tray with pre-wetted electrode wicks. passive rehydration was performed for each protein sample using 11cm ph4-7 readystrip ipg strips (biorad) for 12 hours at rt with gel side down configuration. after rehydration, the protein sample was subjected to ief on protean ief cell i11 (biorad) according to the following conditions: 250 v for 20 minutes with linear ramp, 8,000 v for 2.5 hours with linear ramp and 8,000 v for 30,000 v-hours with rapid ramping. ipg strips equilibration was achieved by incubating the strips with pre-warmed dtt equilibration buffer i (biorad) followed by iodoacetamide-supplemented equilibration buffer ii (biorad) for 10 minutes each on orbital shaker. equilibrated ipg strips were then transferred onto 12.5% tris-hcl criterion gel (biorad) and overlaid with readyprep overlay agarose (biorad). electrophoresis was run at 200 v for 65 minutes. after electrophoresis, the gels were stained with instantblue (expedeon) for 1 hour and submerged in milliq water overnight to remove background signal. gels were scanned using gs-800 calibrated densitometer (biorad). gel images were further processed using pdquest 2-d analysis software (biorad), whereby the different gel images from three independent experiments were matched and the intensities of detected spots were measured. protein spots that showed at least 0.5-fold change in spot intensity (p<0.05, two-tailed student's t-test), compared to the uninfected control sample, were excised for maldi-tof ms. the fold change was calculated using the equation: mean of spot expression in infected samples for each time point. in-gel digestion and maldi-tof ms of the excised protein spots were done by protein and proteomics centre, national university of singapore (singapore). the data was search against the murine and viruses national centre for biotechnology information non-redundant (ncbinr) database using a mascot program (http://www.matrixscience.com). no threshold was applied to the ms/ms fragment ions intensities. data mining of the identified proteins was done by searching in panther (http://www. pantherdb.org/) and swiss-prot/trembl (http://www.uniprot.org/) databases. the enrichment analysis of protein-protein interactions was performed using string network analysis version 10 (http://string-db.org/). hierarchical clustering and classification were performed using multiexperiment viewer version 4.9 (http://mev.tm4.org/#/welcome). on rd cells (10 5 cells/well) were seeded onto 24 wells plate. culture supernatant from ev71-infected samples was serially diluted (10-fold) with dmem containing 2% fbs prior to infection. the cell monolayer was incubated with 100 μl of the diluted viral suspension for 1 hour at 37˚c. the cells were then washed twice with pbs and replaced with 1 ml dmem containing 2% fbs and 1% carboxymethyl cellulose (cmc, sigma aldrich). after 3 days incubation at 37˚c, the infected monolayers were fixed and stained with 4% paraformaldehyde/ 0.1% crystal violet solution (sigma aldrich). the number of plaques was scored visually and viral titers were expressed as plaque-forming units (pfu) per milliliter (pfu/ml). drug treated or sirna-transfected cells (2.5×10 4 nsc-34 or 5×10 4 sk-n-sh cells/well) were washed twice with pbs and 1× alamarblue reagent (invitrogen) diluted with dmem containing 2% fbs was added. after 4 hours incubation at 37˚c, the fluorescence signals were determined using microplate reader (infinite 2000, tecan) at ex 570nm and em 585nm . percentage of viable cells was calculated using non-treated cells as control. table) for 20 mins at rt, the protein complexes were pulled down and subjected to western blot analysis (s6 table) . briefly, the protein extracts were incubated with the conjugated magnetic beads and further incubated for 3 hour at 4˚c with constant rotating, before eluting using laemmli buffer. isotype antibody pull-down and uninfected cells were used as controls. table) . ) . briefly, 20 μl of 5× cytotoxicity reagent were added into each well and incubated at 37˚c for 30 minutes. cytotoxicity was measured using fluorescence at ex 485nm and em 525nm . after equilibrating the assay plate to rt for 10 minutes, 100 μl of atp detection reagent were added into each well and mitotoxicity was measured by luminescence. all readings were normalized against non-treated cells. sodium azide (s2002, sigma aldrich) and staurosporine (s6942, sigma aldrich) were used as mitochondrial toxin and cytotoxin positive controls, respectively. nsc-34 (10 5 cells/well) cells were seeded onto 8-wells chamber slides (ibidi) and incubated overnight. the cells were treated with various concentrations of roc-a (diluted with 2% dmem) for 48 hours. jc-1 dye (invitrogen) (10 μg/ml in 2% dmem) was added into each well and incubated for another 15 mins, prior to imaging. nsc-34 cells incubated with sodium azide nan3 at 1 μm in 2% dmem (s2002, sigma aldrich) was used as positive control. cell lysate was prepared using m-per mammalian protein extraction reagent containing 1% of halt protease inhibitor cocktail and 1% of 0.5 m edta (thermoscientific). protein quantification was performed using quick start bradford protein assay (biorad). denatured proteins (5 μg) were resolved in 10% sds-page gel and transferred electrophoretically onto nitrocellulose membrane using trans-blot turbo transfer system (biorad). after blocking with 5% (w/v) milk in tbst (tbs buffer with 0.01% tween 20) for 1 hour at rt, the membrane was probed using specific primary antibodies and relevant secondary antibodies (s6 table) . the chemiluminescence signal was visualized using clarity western ecl substrate (biorad) on x-ray films. densitometric quantification was performed using imagej and the relative band intensity was normalized against β-actin. nsc table) . the nucleus was revealed using nucblue live readyprobes reagent (molecular probes). fluorescence images were captured using olympus ix81 microscope and further processed using imagej. limb muscles, spinal cord and brainstem from ev71-infected ag129 mice were harvested at day 4 p.i. after systemic perfusion. the organs were fixed in 4% pfa overnight prior immersing in 15% and 30% sucrose solution, and embedded in tissue-tek oct (vwr) solution. the organ samples were then frozen at -80˚, sectioned (10 μm) using a cryostat (leica) and mounted on a glass slide prior to blocking and staining as described above. samples were fixed for 1h at rt with 2.5% glutaraldehyde containing 1% tannic acid in 0.1m cacodylate buffer (ph 7.2), then washed three times for 5 min (each time) in 0.1m cacodylate buffer and post-fixed for 1h at rt with 1% osmium tetroxide in the same buffer. samples were then dehydrated in a graded series of ethanol and embedded in spurr. thin sections were stained with 2% uranyl acetate and lead citrate and observations were performed by transmission electron microscopy using a fei tecnai spirit g2 at 100kv. image were taken using a fei eagle 4k ccd camera. nsc-34, sk-n-sh (8 × 10 6 cells) and rd (4 × 10 6 cells) cells were seeded onto 6-well plate overnight. the cells were dislodged by incubating them in 10 mm edta/pbs in 4˚c for 10 mins and spun down to collect the cell pellet. cells were then blocked with either human or murine fc-blocker (564200 or 553142, bd pharmigen; 1:400) for 30 min at rt, and subsequently stained with anti-phb antibody (ab75766, abcam; 1:200) and/or anti-rabbit af488 antibody (a27034, thermoscientific; 1:500) for 30 min at 4˚c. stained cells were then fixed with 4% pfa. flow cytometry analysis was carried out using the becton-dickinson fortessa flow cytometer and analysed using flowjo v10. two-week old ag129 mice (b&k universal) were bred and housed under specific pathogenfree conditions in individual ventilated cages. infection was performed by injecting intraperitoneally (i.p.) 10 7 pfu of s41 (in 100 μl) per mouse. at day 1 and 3 post-infection (p.i.), mice were injected i.p. with 0.25 mg/kg of roc-a (in 0.25% dmso in sterile olive oil). the control group was inoculated with 0.25% dmso in olive oil. clinical manifestations were observed for a period of 20 days. clinical score was graded as follows: 0, healthy; 1, ruffled hair and hunched back; 2, limb weakness; 3, one limb paralysis; 4, two limbs paralysis; and 5, death. two limbs paralysis was used as criterion for early euthanasia. for virus titer determination, infected mice were euthanized at day 4 p.i. and perfused with 50 ml of sterile pbs systemically. the fore and hind limb muscles, spinal cord and brain were harvested and weighed before mechanical homogenization in 1 ml of serum-free dmem. the homogenates were spun down at 10,000 rpm for 10 minutes at 4˚c, and clarified using a 0.22 μm filter before serial dilution was carried out for plaque assay. viral titers were expressed as pfu per gram of tissue. supporting information s1 relative band quantification (below western blot) was determined by imagej, by normalizing to loading control, β-actin. statistical analysis was performed using two-tailed student's t-test ( ãã , p<0.005). one representative from two independent experiments is shown. (pdf) (a) for co-treatment assay, virus was incubated with roc-a for 1 hour before adding the mixture onto the cells. after one hour of incubation on cell monolayer, the mixture was then removed and replaced with fresh 2% dmem. (b) for pre-treatment assay, the cells were pre-treated with roc-a for 3 hours, prior to viral infection. culture supernatants were harvested at 48 h.p.i. for viral titer determination by plaque assay. cell viability was assessed using alamarblue viability assay. statistical analysis was performed using one-way anova with dunnett's post-test ( ãã , p<0.005). one representative from two independent experiments is shown. (pdf) . viral titer in the culture supernatant was determined by plaque assay at 12 h.p.i. statistical analysis was performed using two-tailed student's t-test ( ã p<0.05, ãã p<0.005, ããã p<0.005, ãããã p<0.0001). error bars represent mean ± standard deviation. relative band quantification (below western blot) was determined by imagej, by normalizing to loading control, β-actin. non-targeting sirna (ntc) served as control. (b) rd cells were pre-incubated with anti-phb antibody or isotype control antibody for 1 hour before infection. culture supernatant was harvested at 12 h.p.i. for viral titer determination. cell viability was determined using alamarblue cytotoxicity assay. one representative of two biological repeats is shown. 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prohibitin from the ap2 gene promoter leads to obesity-linked tumor development in insulin resistance-dependent manner & chellappan s. prohibitin induces the transcriptional activity of p53 and is exported from the nucleus upon apoptotic signaling prohibitin: a potential target for new therapeutics complete sequence analyses of enterovirus 71 strains from fatal and non-fatal cases of the hand, foot and mouth disease outbreak in we would like to thank a/p vincent chow from the department of microbiology & immunology, nus, for sharing the s41 strain and the electron microscopy unit core facility from yong loo lin school of medicine, nus. conceptualization: issac horng khit too, sylvie alonso. formal analysis: issac horng khit too, isabelle bonne. key: cord-006636-xgikbdns authors: ühlein, e. title: übersicht über neue ernährungswissenschaftliche publikationen date: 1964-02-01 journal: z ernahrungswiss doi: 10.1007/bf02021334 sha: doc_id: 6636 cord_uid: xgikbdns nan shoe~taxer, w. c., 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(4 s.) . lowrey, r. s., pond, w. g., lo0sli, j. k. u. barnes, r. h.: effect of dietary protein and fat on growth, protein utilization, and carcass composition of pigs fed purified diets. j. animal sci. 22 [1963] nr. 1, s. 109ft. (6 s.) . lvnd, c. c., u. ) [1963] nr. 1, s. 32ff. (5 8.) . nr~cleod, l. b. : effect of liming and potassium fertilization on soil solution and on yield and composition of alfalfa and orchard grass mixtures. dlss. abs~r. 23 [1962] nr. 6, 62--5826 (322 s.) . ~ds~n, k. 0., u. edmonds, e. j. : prolonged effect on caries of short-term feeding of rice hulls to cotton rats. j. dental res. 42 [1963] nr. 1, s. 137ff. (9 s.) . m~a~.~, a. c. : biological responses of young rats fed diets containing genistin and genistein. j. nutrition 89 [1963] nr. 2, s, 151] 156. ~_ajaj, a.s., dnc~g, j.s., azzam, s.a., u. da_~by, w. j.: vitamin e responsive megaloblastic anemia in infants with protein-calorie malnutrition. amcr. j. clinical nutrition 12 [1963] nr. 5, s. 374 ft. (6 s.) . 1v~jcm~owicz, e., u. quxstel, j. h. : effects of aliphatic alcohols on the metabolism of glucose and fructose in rat liver slices. canad. j. bioehem. physiol. 41 [1963] nr. 3, s. 793ff. (12 s.) . m_al~otra, o. p., n~a-wl)ov, a.v., r~ber, e. f., u. norton, h . w., effects of rat strain, stilbestrol, and testosterone on the occurrence of hemorrhagic diathesis in rats fed a ration containing irradiated beef. j. nutrition 79 [1963] nr. 3, s. 381ff. (8 8,) . --, u. r~ber, e. f. : effect of methionine and age of rat on the occurrence of hemorrhagic diathcsis in rats fed a ration containing irradiated beef. j. nutrition 80 [1963] nr. 1, *misga, u. k. : effect of corn oil feeding on the lipids of dog bile. indian j. exper. biol. 1 [1963] nr. 2, s. 87/90. * miyao, m., tsvn•isnz, m., nagano, k., u. ttosogi, t.: experimental studies on the digestibility and absorbability of milk proteins. 4. effects of carbohydrate addition on the digestibility and absorbability of cow's milk proteins. tokushima j. exper. med. 9 [1962] nr 1963, nr. 5334, s. 846ff. (4 s.) . nagra, c. l., brerrenbach, r. p., u. meyer, 1~. k. "-influence of hormones on food intake and lipid deposition in castrated pheasants. poultry sci. 42 [1963] nr. 3, s. 770ff. (6 s.) . *+na~:ler, w. g.: the significance of calcium ions in cardiac excitation and concentration. amer. heart j. 65 [1963] nr l~, b. l., u. re(~a~, w. s., u. dobozy, a.: effect of vitamin a on the nucleic acid metabolism of rats. acts biologics acad. sci. hungariae 1963, suppl. 5, s. 46 . 01~itz, k., u. loeser, a. : uber den einflufl appetithemmender substanzen auf das fettgewebe. klin. wschr. 41 [1963] nr. 4, s. 1931196. *0rstadius, k., nordstrom, g., u. lannek, n. : combined therapy with vitamin e and selenite in experimental nutritional muscular dystrophy of pigs. cornell veterinarian 53 [1963] nr. 1, s. 60ft. (9 s.) . ern~hrung und therapie. med. u. ern~hrung 4 [1963] nr. 6, s. 146/150 . *--pflanzliche polysaccharide zur steigerung der kiirpereigenen abwehr. ivied. welt 1963, nr. 6, s. pxrkrnson, t. m., u. 0lso~r, j. a. : inhibitory effects of bile acids on the uptake, metabolism, and transpor~ of water-soluble substances in the small intestine of the rat. life sci. 1963, nr. 6, s. 393] 398. *pxl~o% j.-l., u. mord~l~t-dx~rin~, )i..' action du potassium et du calcium sur l'histaminopexie s~rique. recherches sur le cobaye, le rat et l'homme. j. physiol. 54 [1962] nr. 4, s. 579ff. (12 s.) . * patek, a. j., de fr1tsc] t, n. m., kendall, f. e., n. hirsch, r. l.: corn and coconut effects in dietary cirrhosis of rats. arch. pathology 75 [1963] nr. 3, s. 264ff. (7 s.) . patil, s. s.: the relation of ehlorogenic acid and total free phenols in potato plants to resistance to infection by verticillum alboatrum. diss. abstr. 23 [1962] nr. 6, 65--5602 (92 s.) . petuet,y, f.: diskussionsbemcrkungen zu rcferaten fiber sauermilchprodukte auf einer vortragsveranstaltung der gesellsehaft fiir erniihrungsbiologie e.v., miinchen, 22. juni 1961 . milchwissensehaft 18 [1963 nr. 5, s. 236] 237. pfei~ter, (3. j., gass, g. h., u. schw~rz, (3. s.: reduction by chlorpromazine of ulcem due to acute starvation in mice. nature 197 [ ] nr. 4871, s. 1014 1015. prrrllros, a. w., newc0m~, tl r., u. sha.wklrs, d. r.: long-term rat feeding studies on irradiated chicken stew and irradiated cabbage. toxicol. appl. pharmacol. 5 [1963] nr. 3, s. 273]297. prrmt~s, p. h., sutti~, j. w., u. ze~rowski, e. j. : effects of dietary sodium fluoride on dairy cows. vii.: recovery from fluoride ingestion. j. dairy sei. 46 [1963] l~r. 6, s. 513ff. (4 s. browi~, t. h., u. levee, r. v.: the effect of milk intake on nematode infestation of the lamb. prec. nutrition 8oc. 22 [1963] nr. 1, s. 32/41. * srnwrvasan, m., n~o~bhusha_~a~, a., u. sm~rrvas~, k. s.: changes in serum inorganic phosphate following ingestion of protein. curr. 8ci. 32 [1963] nr. 1, s. 21. stallwoth, h. : some effects of 2.4-dichlorophen-oxyacetie acid on sweet corn (zea mays rugosa l.) with emphasis on yield, tillering, root development, and exudation of electrolytes from roots and stems. diss. abstr. 23 [1963] nr. 8, 62---6233 (79 s.) . starcher, b., u. i~.ratzer, f~ h.: effect of zinc on bone alkaline phosphatase in turkey poults. j. nutrition 79 [1963] nr. 1, s. 18/22. stei~-~off, d., u. ~rqu~lrdt, p. : kombination yon kaliumpyrosulfit und ~xthy]alkohol ira tr~nkungsversuch an ratten. arzneimittelforschung 13 [1963] nr. 3, s. 237/238. stevermer, e. j. : influences of level of nutrition of the boar and of ionic environment of the spermatozoa on the properties of boar semen. diss. abstr. 23 [1962] nr. 6, 63--619 (104 s.) . stn~p~., f., u. 8c~warz, k.: incorporation of valine-l-x*c into serum and tissue proteins of rats fed torula yeast diets. j. nutrition 79 [1963] nr. 2, s. 151/160. 8ton~., d. b., u. co~or, w. e. : the prolonged effects of a low cholesterol, high carbohydrate diet upon the serum lipids in diabetic patients. diabetes 12 [1963] nr. 2, 8.127 ft. (6 s.) . *stormont, j. •., u. waterhouse, c. : effect of variations in previous diet on fasting plasma lipids. j. labor. clinical med. 61 [1963] nr. 5, s. 826ff. (6 s.) . * stuart, a. e., u vity of the heart extract of rats. medlcina exporimentalis 7 [1962] nr. 6, s. 363ff. (5 s.) . szepsenwol, j.: carcinogenic effect of egg white egg yolk and lipids in mice. prec. soc. exper. biol. med. 112 [1963] nr. 4, s. 1073ff. (3 s.) . *t~cs, l, u. ny~i, i.: effect of saline infusion, acth infusion, and blood transfusion on the hormone excretion of patients with hypereme~is. act~ medics aead. sei. hun. garicae 18 [1962] nr. 4, s. 385ff. (14 s.) . tanner, j. w.: an external effect of inorganic nitrogen on nodulation. diss. abstr. 23 [1963] nr. 10, 63--3007 (50 s.) . --, u [1963] nr. 4, s. 308ff. (13 s.) . *t~i~er, l., m~az, m., u. cs:menafiov4, m. : the effect of glucose and glucose together with insulin on the resistance of fasted rats to trauma in the noble-collip drum. physiologia bohemoslovenica 12 [1963] nr. 2, s. 136ff. (9 8.) . ude~iend, s. : factors in amino acid metabolism which can influence the central nervous systems. amer. j. clinical nutrition 12 [1963] nr. 4, s. 287ff. (4. 8 .) vahouny, g. v., moede, a., silver, b., n . treadw~ll, c. r. : nutrition studies in the cold. iv. effect of cold environment on experimental atherosclerosis in the rabbit. j. nutrition 79 [1963] nr. 1, s. 45/52. van p1lsum, j. f., olsen, b., taylor, d., rozyc'ki, t., u voss, r. d.: yield and foliar composition of corn as affected by fertilizer rates and environmental factors. i)iss. abstr. 23 [1963] nr. 10, 63--3008 (293 s.) . *vyval,ro, i. g. : the effect of gibberellin on the transformation of substances in germinating corn seeds. i)oldady akad. nauk sssr [russ.] 149 [1963] nr. 4, s. 979ff. (3 s.) . wao~ei~, g. r., cl~mk, a. j., hays, v. w., u amer. j. clinical nutrition 12 [1963] lgr. 3, s. 235ff. (6 s.) . the assessment of marginal protein malnutrition. prec. nutrition soc. 22 [1963] mr. 1, s. 66]72. --, u. stnp~, j. m. l.: the free ly~ine and amino nitrogen content of liver, muscle, and serum in normal and protein-depleted rats. prec. nutrition soc. 22 [1963] mr. i, s . viii/ix. *watson, w. c.: the morphology and lipid composition of the erythrocytes in normal and essential-fatty-acid-deflcient rats. bri6. j. haematology 9 [1963] lgr. 1, s. 32ff. (7 s.) . waite, r., u. : blackburn, p. s. : the relationship between milk yield, composition and tissue damage in a case of subclinical masticis. j. dairy res. 30 [1963] 1963, nr. 5330, s. 561ff. (1 s.) . *n. n.: paraty1~hoid fever from frozen chinese eggs. brit. reed. j. . (1 s.). *n. n. : radioactivity and human diet. chem. ind. 1963, nr. 18, 8. 721 . ~q. ~.: 8eh~digungen dutch konservlerungsmittel bei zitrusfrfichten? dr. reed. wschr. 88 [1963] nr. 17, s. 927. *n. n. : salt-poisoning in infancy. lancet 1 [1963] l~r. 7293, s. 1251. n.n.: kouoquium des arbeitskrcises hamburg der gdch-fachgruppe lebensmittelchemic und gerichtliche chemic am 7. januar 1963 . lebensmittelchem. u. gerichtl. chem. 17 [1963 nr. 6, s. 1161118. *n. n. : smoking and heath disease. new england j. med. 268 [1963] nr. 15, s. 903. +hi. n. : toxic components of lathyrus peas. nutrition rev. 21 [1963] nr. 1, s. 28/30. +n. n. : cariogenic ability of different diets. nutrition roy. 21 [1963] nr. 2, s. 50]52. +n. 1~.: aminoaeiduria in lead intoxication. nutrition rev. 21 [1963] nr. 3, s. 75/76. +n. n. : pyridoxine and dental caries. human studies. nutrition rev. 21 [1963] nr. 5, s. 1431145. +n. n.: pyridoxine and dental caries. animal studies. i~utrition rev. 21 [1963] nr. 5, s. 1451147. +hi. n. : rcporb: the prophylactic requirement and the toxicity of vitamin d. pediatrics 31 [1963] nr. 3, s. 512ff. (? s.). axrkroa, a.: caesium-137 from fall-out in human milk. nature 197 [1963] nr. 4868, s. 667ff. (1 s.) . *allcroft, r., u. car~agha~, r. b. a. : groundnut toxicity: an examination for toxin in human food products from animals fed toxic groundnut meal. veteri. rec. 75 [1963] nr. 10, s. 259ff. (5 s.) . *--, l~wis, g., u. hill, k. 1~.: groundnut toxicity in cattle: experimental poisoning of calves and a report on clinical effects in older ca~tle. ve~er. rcc, 75 [1963] nr, 19, s. 487ff. (6 s. nr. 1, 2, 3 nr. 1, 2, 3, 4, 5 nr. 1, 2 nr. 1, 2 influence of calcium and ouabai bain upon potassium influx in human erythrocytes the enzymatic assimilation of nitrate in the tomato plant translocation of '~p, i~n, and ~c in plants einige neue gesiehtspunktr zum caleiumstoffwechsel dis~ibution of water, sodium, and potassium in resting and stimulated mammalian muscle. canad the influence of vitamin bi, on calcium ('sca) metabolism of maxillodental tissues kidney, water, and electrolyte metabolism intermediiirer elektroly~toffwechsel und zellgrenzfl~chenphysiologie im theoretischen zusammenhang mit der krebsentstehtmg. tell i dcr einflul~ yon vollkornbrot auf den calcium-stoffwechsel bei schulkindern recherches sur le m6tabolisme du soufre. x. : la non-6quivalence de la eystine et de la eyst4ine dans la couvcrture des besoins sufr~s du rat adulte potassium-magnesium antagonism in soils and crops low serum iron levels in obese adolescents metal content of human organs studies on the requirements and interaction of copper and iron in broad breasted bronze turkeys to 4 weeks of age iron absorption and excretion in experimental iron deficiency the measurement of exchangeable magnesium in dogs the copper metabolism of warmblooded animals with special reference to the rabbit and the sheep comparative studies of the metabolism of strontium and barium in the rat the utilization of iron in erythropoiesis binding of strontium in blood ~iolybdenum, copper, and zinc contents of mouse liver and sarcoma 180 treated with molybdenum compounds biochemical effects of zinc deficiency in tomato plants excretion of sodium, potassium, magnesium, and iron in human sweat and the relation of each to balance and requirements turnover rate of zinc in the body as determined by the study of 6szn in rats a study of the iron absorption in mice as modified by various agents funktionsteste des radiojodstoffwechselstudiums und ihre bedeutung in der diagnostik der sehilddrfisenerkrankungen. ~rztl. laboratorium 9 the fate of radioiodine after parcnteral administration a possible humeral regulator of iron absorption beitrag zur kl~rung der ursachen der anreicherung yon caesinm-137 im 0rganismus blood-and serum-level of watersoluble vitamins in man and animals significados metabslicos do ~cido ~-lip6ico. 3. o ~cido r162 e o metabolismo do ferro observations on a magnesium-fluoride interrelationsip in chicks prevention of ,meat anemia" in mice by copper and calcium iron metabolism in experimental pyridoxine deficiency aluminium in soils and plants on the coastlands of british guinea physiology of adolescence. ii. : nutrition -basal oxygen consumption -energy expenditure and balance -nitrogen metabolism -calcium metabolism -iron metabolism -red cell mass and hemoglobin dietary strontium and calcium, and deposition of 89sr and asca in the bones of rats -~iengenelementansatz wachsender sehweine bei unterschicdiichen cuso4-zulagen differences in copper retention in two strains of chickens untersuchungen fiber anreicherung und verteilung yon rubidium in gerstenkcimpflanzen 112 [1963] nr. 3, s. 631/636. *+lv~ointyr~, i.: an outline of magnesium metabolism in health and disease. a review uptake by the root and subsequent distribution within the potato plant of strontium-89 leached from the foliage nor zinkstoffwechsel in der schwangerschaft foetal metabolism of caesium-137 in the rat magnesium metabolism of chickens zinc metabolism in patients with the syndrome of iron deficiency anemia hepatospenomegaly dwarfism, and hypogonadism. labor. clinical mcd c~isium-137 trod kalium in menschlichen organen und in der milch 1959/60 l~ole of the genotype in controlling accumulation of strontium-89 by plants copper and zinc interrelationships in the pig effect of chromium, cadmium, and other trace metals on the growth and survival of mice studies in the metabolism of zinc. iv. some observations on the urinary zine-porphyrin relationship in non-porphyries and in a patient with aeutm intermittent porphyria aastrontium balances in man studies on zinc metabolism. ii.: effect of the diabetic state on zinc metabolism: an experimental aspect effect of diabetic state on zinc metabolism: a clini. cal aspect copper metabolism and the liver iron metabolism and the liver with particular reference to the pathogcnesis of haemachromatosis studies on iron metabolism uber den eisenstoffwechsel. (bemerkung zu t. su~di~, miinchener reed yifinchener reed. wschr. 105 [1963] nr. 19, s. 99911000. 2c -7 wirkstoffe -biocatalysts +n. n. : vitamin a in human livers. nutrition biological half.llfe of vitamin b~ in plasma hypervitaminosis a and mast cells. a study of the interrelationship of mast cells and vitamin a in vivo and in vitro evidence concerning the human requirement for vitamin bi~. use of the whole body counter for determination of absorption of vitamin blz vitamin c in plasma and leucocy~s of smokers and non-smokers some factors affecting the absorption of vitamins the determination of vitamin a in animal tissues and its presence in the liver of the vitamin a deficient rat metabolic activities of vitamin a and related compounds in animals. i.: role of vitamin a in intestinal muscular contraction zum vitamin-b--haushalt dcr ratte bei sorbitfiitterung contribution a l'~tude do la relation entre la vitamine big et la glande thyrolde effects of deficiencies of certain b vitamins and ascorbic acid on absorption of vitamin blv amer ascorbic acid metabolism in plants. ii. : biosynthesis dietary and thyroid interrelationships affecting vitamin a status of feedlot beef cattle die wirkung gesteigerter kupferzufuhr auf den vitamin-c-haushalt vom meerschweinchen bei parental zugeffihrter ascorbins~ure kritische auswe~ung der naeh 1949 erschienenen arbeiten fiber gebundene ascorbins~ure im tierischen gewebe metabolism and biological activity of vitamin a acid in the chick biochemical studies of vitamin metabolism in poultry urine. ii. : on the excretion of thiamine in poultry urine after subcutaneous and oral administrations of some thiamine derivatives untersuchungen fiber die speiehcrung und fiber die ansscheidung yon vitamin a nach ungeniigender vitamin-a-versorgung bei legenden hfihnern studies on metabolism of vitamin a. 1.: the biological acticity of vitamin a acid in rats vitamin a and cholesterol absorption in the chicken studies on the interaction of vitamin bi~, intrinsic factor, and receptors. il the possible absorption of intrinsic factor human growth hormone in infant malnutrition macro-and micromethods for the determination of serum vitamin a using trifluoroacetic acid the activation of sulphate by extracts of cornea and colonic mucosa from normal and vitamin a-deficient animals the importance of blood as a pool of vitamin d studies on metabolism of vitamin a. 2.: enzymic synthesis ~nd hydrolysis of phenolic sulphates in vitamin-a-deficient rats human metabolism of l-ascorbie acid and erythorbic acid tissue distribution and storage forms of vitamin b~, injected and orally administered to the dog relation between vitamin a, tocopherol, and cholesterol serum levels in the elderly interrelationships of vitamin bi~, folio held, and ascorbie acid in the megaloblastie anemias zum wirkungsmechanismns des vitamins e. helvetica physiologica et pharmacologica effect of some physiologic factors on the absorption of vitamin bi~ in rats transport of dietary nitrogen mitochondrial fatty acids of fish and fish-eating birds the biosynthesis of fatty acids influence of age and dietary protein on cerbain free amino acids in chick blood plasma nitrogen metabolism in coldexposed rats ~ber des vermehrte auftreten yon fettsiiuren mit 10 bis 14 c-atomen in den depotfetten siiugender ratteu und den ubergang der linolsi~ure yon den mfittern auf die jungen die bildung yon antiksrpcrn gegen verschicdene kuhmilehproteine bei neugeborenen, kindern, erwachsenen und graviden the myocardial arterio-venous differences of free amino acids and of free fatty acids in healthy individuals, patients with diabetes and with essential hypercholesterolemia urinary amino acids on phenylalanine-tyrosine-supplemented diets difference in the metabolic fate of acetate and ethanol fed to higher plant tissues iiemodynamic relationships of anaerobic metabolism and plasma free fatty acids during prolonged, strenuous exercise in trained and untrained subjects effects of palmitate on the metabolism of leukooytes from-guinea pig exudate the dynamics of plasma free fatty acid metabolism during exercise ~ber die retention, den abbau und die ausseheidung yon 2-thion-tetrahydro-l.3.5-thiadiazinen transport systems for amino acids effects of protein intake and cold exposure on selected liver enzymes associated with amino acid metabolism quantitative studies on tryptophan metabolism in the pyridoxine-deficient rat effect of desoxypyridoxine-induced vitamin b~ deficiency on polyunsaturated fatty acid metabolism in human beings studies on the wheat plants using carbon-14 compounds. xix.: observations on the metabolism of lysine-14c. canad genetic defects of amino acid metabolism. pediatric clinics north america metabolism of tryptophan in diabetes mellitus the two-carbon chain in metabolism der einfluss yon vitamin a auf den citronens~urestoffwechsel ~)ber phenolspeicherung und phenolabbau in wasserpflanzen. naturwissensehaften 50 effect of physical exercise on nitrogen balance in obese subjects metabolism of nitrogen compounds in the rumen of ruminants. izvest. acad der intermedli~r-stoffwechsel s~ffweehsel der carotine im hiihnerembryo nucleic acid metabolism of germinating corn seedlings carbon metabolism of ~4c-labelled amino acids in wheat leaves. ii.: serine and its role in glycine metabolism bovine metabolism of insecticides. the metabolism of ~evin in dairy cows stoffweehsel der carotine. wiss. veroff. dr. oes. ern~hrnng 9 metabolism of labelled linolcic-l-tac acid in the sheep rnmen nonessential nitrogen supplements and essential amino acid requirements. nutrition rev vitamin c requirements of man re-examined. new values based on previously unrecognized exhalatory excretory pathway of ascorbie acid studies on the requirements and interaction of copper and iron in broad breasted bronze turkeys to 4 weeks of age water requirements of men as related to salt intake evidence for a high zinc requirement at the onset of egg production effect of lysine and glyeine upon arginine requirement of guinea-pigs the cobalt requirement of subterranean clover in the field fluid and electrolyte, requirements of newborn infants with intestinal obstruction the requirement and availability of dietary iron for young pigs studies on the protein and methionine requirements of young bobwhite quail and young ringnecked pheasants phenylalanine requirement of women consuming a minimal tyrosine diet and the sparing effect of tyrosine on the phenylalanine requirement effects of starvation on the cardiovascular system of the chicken calcium and phosphorus requiremeats of finishing broilers using phosphorus sources of low and high availability water intake of normal children sex differences in the ~4oeopherol requirement of rats as shown by the haemolysis test further studies on protein and energy requirements of chicks selected for high and iow body weight smoking and blood dotting dental caries and trace elements a statement approved by the board of directors of the canadian heart foundation the question of fats. il : fats and disease moldy peanuts and liver cancers vitamin c and healing of wounds berieht fiber die vortragstagung des fachverbandes lehensmittelchemie der chemisehen gesellschaft in der d])r veto 19. his 21 diet and human depot fat ethanol and plasma free fatty acid in man dietary water and protein efficiency in rats. nutrition rev. 21 [1963] nr. 1, s. 16/17. +n. n. : nature of the coagulation defect in rats fed diets producing thrombosis or experimental atheroselerosis factors affecting growth depression by raw soybeans bones in undernourished animals. nutrition rev. 21 [1963] nr. 1, s. 21123. +n. n. : vitamin e and the etiology of muscular dystrophy in the rabbit riboflavin eoenzymes and congenital malformations the thyroid gland in infant malnutrition. nutrition rev. 21 [1963] nr. 1, s. 32. +n. n. : a proposed mechanism for the effect of fats on serum cholesterol effect of varying levels of dietary protein on synthesis and excretion of urea dietary phosphates and dental caries folic acid restriction and cancer inhibition changes induced by lipoie acid in normal rat liver vitamin b6 deficiency and tryptophan metabolism effect of ubichromenol on development of encephmomalacia in vitamin e deficient chicks leucine-induced hypoglycemia nutritional muscular dystrophy in lambs. nutrition rev. 21 [1963] nr. 4, s. 120/122. +n. n.: amino acid imbalance in cold-exposed rats. nutrition rev. 21 [1963] nr. 4, s. 1221124. +n. n.: milk and athletic performance effect of vitamin a deficiency on the ubiquinone content of rat liver idiopatjaie stea~orrhea gastrointestinal protein loss in iron-deficiency anemia nutritional cirrhosis of the liver. nutrition rev. 21 [1963] nr. 6, s. 175/178. +n. n. : exercise and heart disease calcium deficiency in the etiology of osteoporosis the relation of dietary fat to the fatty acids in the intestinal wall clot-strength and elot-lysis in rats fed hyperlipemic diets effect of potassium iodide and duodenal powder on the growth and organ weights of goitrogen-fed rats ver~nderungen im stoffwechsel und wachsturn junger tomatenpflanzen nach giberillins~,urebehandiung effect of restricted feeding during the growing period on reproductive performance of large type white turkeys keys, a. l glucose, sucrose, and lactose in the diet and blood lipids in man ambient temperature and survival on a protein-deficient diet some considerations of changes in total body composition in relation to nutritional status the effect of variations in the energy and protein levels of the ration upon performance in the pig studies in choline deficiency. fate of injected 1-1~c-pal. mitio acid and fatty acid spectra in fasting and refed rats bartou i~ vitamin a requirements of chicks at moderately elevated temperature influence of age and dietary protein on eer~ain free amino acids in chick blood plasma the effects of nicotine on weight increment, activity, food intake, and water intake in weanllng albino rats effect of pyridoxine deficiency upon delayed hypersensitivity in guinea-pigs vitamin a deficiency in chickens ccrebellar encephalomalacia produced by diets deficient in toeopherol vitamin b12 deficiency in indian infantm plasma liplds in scurvy: effect of ascorblc acid supplement and insulin treatment effect of gibberellin on the variations of the growth-point in winter wheat uptake of dinitrophenol and its effect on transpiration. calcium accumulation in barley seedlings l~[ethylmalonate excretion in vitamin b~ deficiency reduction of plama cholesterol levels in atherosclerosis by diet and drug treatment. australasian ann effects of reduced dietary intake on the activities of various enzymes in the livers and kidneys of growing male rats the effect of feeding d-methionine on the d-amino acid oxidase activity of chick tissues l~etabolic effects of dietary protein level in cold-exposed rats relative effects of rapeseed oil and corn oil on rats subjected to aclrenalectomy, cold, or pyridoxine deprivation metabolic effects of dietary protein level with caloric restriction in coldexposed rats. canad metabolic effects of three dietary protein levels fed isocalorically to coldexposed rats. caned die nolle versehiedener fette im eiweis des organismus. nahrung the bursa of fabricius and xanthine oxidase activity of liver and kidney following dietary supplementation of iodina~d casein to chickens effects of linolsate and dietary fat level on plasma and liver cholesterol and vascular lesions of the cholesterol-fed rat a comparative study of the effect of bile acids and cholesterol on cholesterol metabolism in the mouse, rat, hamster, and guineapig the effects of ruminal and plasma sodium concentrations on the sodium appetite of sheep effect of level and sequence of feeding and breed on ovulation rate, embryo survival, and fetal growth in the mature ewe inhibitory effects of carbohydrates on histamine release and mast cell disruption by dextran fett-s~urestoffwechsel bci the effect of calcium infusions, parathyroid hormone, and vitamin i) on renal clearance of calcium nutritional supplementation during pregnancy effect of level of dietary protein with and without added cholesterol on plasma cholesterol levels in man die schilddrfisenfunktion bei enteralem eiweiflverlust effects of pelleting and varying grain intakes on milk yield and composition fatty acid composition of lipids of serum and aorta in the chicken on different diets rat intestinal suerase. ii.: the effects of rat age and sex and of diet on suerase activity the effect of selenium administration on the growth and health of sheep on scottish farns die wirkung yon vitamin b 6 bei leukopenien effect of energy source and level of alfalfa pellets on growth and tissue hpids of beef calves effect of magnesium deficiency on mast cells and urinary histamine in rats histamine-liberating effect of magnesium deficiency in the rat zur wirkung des wassers bei der seitenwurzelbildung an luftwurzeln influence of the aqueous potato extract and its fractions on growth and spore formation of the b. pumilus and the production of the antibiotic tetaine influence of mineral nutrition on the resistance of peach tree to fusicoceum amygdali de la croix the effect of dietary protein on the course of various infections in the chick bifidus intestinal flora in infants fed on mamysan b. acta paediatriea 52 effect of food fats on concentration of ketone bodies and citric acid level in blood and tissues is there a hemostatie effect of peanuts in hemophilioid disorders? milk allergy in infancy dental effects of fluoridation of water with particular reference to a study in the united kingdom influence of previous feeding with a high-fat diet on liver steatosis produced by acute starvation of growth hormone in mice effect of amino acid imbalance on nitrogen retention. ii.-interrelationships between methionine, valine, isoleucine, and threonine as supplements to corn protein for dogs supplementation of cereal proteins with amino acids. v. : effect of supplementation lime-treated corn with diffe. rent levels of lysine, tryptophane, and isoleueine of the nitrogen retention of young children the interrelation of nutrient supply, leaf nutrient content, and vegetative growth of ilex crenata gastric content of fasted primates. a survey serum cholesterol in vitamin c deficiency in man the effect of carbohydrates on the production of staphylococcal pigment effect of a low dietary level of three types of fat on reproductive performance and tissue lipid content of the vitamin b6-defieient female rat effect of magnesium deficiency on synthesis of hear~ and liver mit~chondria phospholipids ~ber den einflub l~nger dauernder ksrperlicher inaktivit~t auf die blutzuckerkurve nach oraler glucosebelasttmg. helvetica medica acts 30 action of trace elements on the metabolism of fluoride zur frage des einflusses yon kondensmilch und einer protein-valerina-polyphosphat-komplexverbindung auf die kreislaufwirktmg des kaffees beim menschen modifications de la croissance de la plantule de lapin blanc (lupinns albns l.) provoqu6es par une diminution exp6rimentale des r6serves influence of the dietary protein level on the magnesium requirement effects of high levels of copper and chlorotetracycline on performance of pigs effect of dietary calcium lactate and lactitic acid on faecal escherichia coli counts in pigs die entwickhing von calcium-mangelsymptomen. z. pflanzenern~hrung, diingung, bodenkde. 100 [1963] nr. 1, s. 53/58. --calcium-mangelsymptome an hsheren pfianzen copper deficiency in relation to swayback in sheep. i. : effect of molybdate and sulphate supplements during pregnancy long-term, low fat, low protein diets and their effect on normal trappist subjects further studies of the influence of diet on radiosensitivity of guinea-pigs, with special reference to broccoli and alfalfa effects of the infusions of ammonia, amides, and amino acids on excretion of ammonia answirlmngen langfristig fettreicher ern~ihrung auf das plasma-cholesterin the relationship of dietary energy level and density to the growth response of chicks to fats influences of dietary carbohydrate-fat combinations on various functions associated with glycosis and lipogenesis in rats. i. effects of substituting sucrose for rice starch with unsaturated and with saturated fat compensatory carcass growth in steers following protein and energy restriction fatty acid composition and glyceride structure in rats fed rapeseed oil or corn oil. canad influence of selective and nonselective hydrogenation of rapeseed oil on carcass fat of rats. canad studies in serum lipids. with special reference to spontaneous variations and the effect of short-term dietary changes experimentelle untersuchungen zur wirkung yon kaffeefett evaluation of the effect of breed on vitamin be requirements of chicks 1-iigh salt content of western infant's diet: possible relationship to hypertension in the adult the effect on the serum cholesterol levels of the consumption of a special dietary fat with a high content of unsaturated fatty acids in elderly people effect of dilution of the diet with an indigestible filler on feed intake in the mouse effect of tea and its tannins upon capillary resistance of guinea-pigs food input and energy extraction efficiency in carassins auratns effect of calcium and magnesium upon digestibility of a ration containing corn oil by lambs effects of calorie restriction during the growing period on the performance of egg-type replacement stock effects of insulin on hepatic glucose metabolism and glucose utilization by tissues cellulase and polygalacturonase in tomato fruits and the effect of calcium on fruit cracking effect of nutritional muscular dystrophy and of starvation on amino acid penetration in rabbit tissues plasma protein synthesis in nutritional muscular dystrophy inulin and sucrose distribution in tissues of vitamin e-deficient and control rabbits protein-bound dyes in the serum and liver of rats fed aminoazo dyes vanadium. excretion, toxicity, lipid effect in man the influence of vitamin a status on the proteoly~ic activity of lysosomes from the livers and kidneys of rats disauxie metainfettive e da malnutrizione induced drinking in dogs: comparative effects of hypertonic sodium chloride and sorbitol the influence of early nutrition on brain cholesterol accumulation during growth changes in composition of the saliva of cows on grazing heavily fertilized grass. res. veter changes in composition of the saliva of sheep on feeding heavily fertilized grass efect of varying alfalfa: barley ratios on energy intake and volatile fatty acid production by sheep the influence of calcium on the secretory response of the submaxillary gland to acetyi-choline or to noradrenaline clinical dentistry and fluoride food allergy as a cause of abdominal pain the effect of various dietary levels of ddt on liver function, cell morphology, and ddt storage in the rhesus monkey effect of natural and purified diets on survival of x-irradiated mice effect of autoclaving and ])'sine supplementation of skimmilk-powder diets on growth and caries in rats effects of alcohol intake on subjective and objective variables over a five-hour period nitrogen metabolism of african cattle fed diets with an adequate energy feeding value of fl-caroteno following treatment with n~o~ the relationship of the quantity and quality of dietary fats to serum cholesterol levels in men of different ages and weights effects on girls of greater intake of milk, fruits, and vegetables effect of antioxidant, protein, and energy on vitamin a and feed utilization in steers the growth-maintaining activity of ascorbic acid the effects of an induced pyridoxine and pantothenie acid deficiency on excretions of oxalic and xanthurenic acids in the urine influence of lactose and dried skim milk upon the magnesium deficiency syndrome in the dog. i.: growth and biochem chronic malnutrition in turkey. v. study on serum fatty acids in malnourished children the effect of nutrition conditions on the growth of and nitrogen accumalation by fodder beans when sown together with indian corn effects of a diet high in polyunsaturated fat on the plasmalipids of normal young females citrate and action of vitamin d on calcium and phosphorus metabolism beeinflussung der sportliehen lelstungsf/~higkeit durch eine geeignete er-nehrung effect of dietary erotic acid on liver proteins effect of barbiturie acid and ehlortetraeyeline upon growth, ammonia concentration, and urease activity in the gastrointestinal tract of chicks effects of feeding low levels of dimethoate on milk and whole blood eholinesterase activity of dairy cattle changes in serum lipoproteins after a large fat meal in normal individuals and in patients with isehemic hear~ disease the relationship of specific nutrient deficiencies ~) antibody response in swine. i. : vitamin a. ii. : pantothenie acid, pyridoxine or riboflavin. i)iss. abstr relationship of specific nutrient deficiencies to antibody production on swine. i. : vitamin a relationship of specific nutrient deficiencies to antibody production in swine. il : pantothenic acid, pyridoxine or riboflavin histechemistry of dietary cardiac lesions effect of various levels of fluorine, stilbestrol, and oxytetraeycline, in the fattening ration of lambs uptake of copper and its physiological effects on chlorella vulgarls the effects of a small dose of ethyl alcohol on certain basic components of human physical performance. i. the effect on cardiac rate during muscular work. arch. internat some effects of feeding stilbestrol, chlortetracycline and penicillin with alfalfa soilage on steer performance and carcass quality experimental induction of ciguatera toxicity in fish $hrough diet effects of potassium fertilizer, age of ewe, and small magnesium supplementation on blood magnesium and calcium levels of lactating ewes the influence of higher volatile fatty acids on the intake of urea-supplemented low quality cereal hay by sheep un~emuchungen fiber den umsatz wachsender schweine ab geburt. 2. mitt eczema and cow's milk. brit. med. j. 1963, nr. 5332, s. 753. isaacso~, a. : the effects of zinc on responses of frog skeletal muscle effects of zinc on responses of skeletal muscle effect of diet on work metabolism carbohydrate-phosphorus metabolism in the skeletal muscles of epinephrectomized animals durlng treatment with cortisone and vitamins c and p. ukrainskii biokhimichnii zhurnal composition of dietary fat and the accumulation of liver lipid in the choline-deficlent rat nutrition and palatability the incidence of protein-calorie malnutrition of early childhood theories on the mode of action of fluoride in reducing dental decay saccharase deficiency, familial entailing intolerance ~ cane sugar. acts paediatrica 52 raw and heat-treated soybeans for growing-finishing swine and their effect on fat firmness effect of the administration of isoniazid and a diet low in vitamin be on urinary excretion of oxalic acid dietary and thyroid interrelationships affecting vitamin a status of feedlot beef cattle ration effects on rumen acids, ketogenesis, and milk composition. i.: unrestricted roughage feeding the effect of supplements of groundnut flour or groundnut protein isolate fortifed with calcium salts and vitamins or of sklmmilk powder on the digestibility coefficient, biological value and net utilization of the proteins of poor indian diets given to undernourished children a comparison of skin-testing with natural foods and commercial extracts die wirkung yon hungern auf den ammoniakgehalt und das ph der pansenfi(issigkeit sowic auf die harnstoff-, cholesterin-und zuckerkonzentration im blu~. acts veterinaria acad increase of plant virus infection by magnesium in the presence of phosphate effect of intramuscular injection of magnesium sulphate solution on the growth rate and serum composition in rats diskussionsbemerkungen zu referaten fiber sauermilchprodukte auf einer vortragsveranstaltung der gesellschaft fiir eru~ effect of massive sodium bicarbonate infusion on renal function excessive insulin response to glucose in obese subjects as measured by immunoehemical assay die wirkung gesteigerter kupferzufuhr auf den vitamin-c-hanshalt yon meerschweinehen bci paren~ral zugef'dhrter ascorbinsrure the influence of growth hormone on fat and protein metabolism. dies. abs~r. 23 [1962] nr phenolearbons~iuren in mensehlichen nahrungsprodukten. zum vorkommen yon phenolcarbons~uren in mensehlichen nahrungsprodukten und ihr einflul~ auf den intermedit~ren stoffwechsel influence of pregnancy and an oxidized lipid diet on the fatty acid composition of blood and tissue an experimental approach to the mechanisms of weight loss. ii. 9 a comparison of effects of thyroxine, fat-mobilizing substance (fms) and food deprivation in achieving weight loss in mice fat accumulation in the regenerating media of arteries in rats given an atherogenic diet the effect of nutrition on the growth of faseiola hepatica in its snail host the effects of specific viruses, virus complexes, and nitrogen nutrition on the growth, flowering, and mineral composition os strawberry plants body weight and food intake as initiating factors for puberty in the rat 9 the role of catecholamines in the free fatty acid response to cigarette smoking die renale steroidausseheidung bei experimentellem vitamin-e-mangel 9 peculiar features of respiration and redox processes in the rice plants grown under different nutritional conditions der bohnenkaffee und die migrrne repeatability of litter size and weight in the laboratory rat as affected by selection and plane of nutrition wirkungen yon muskelextrakt auf den stoffwechsel. arzneimittelforschung is [1963] nr. 3, s. 238/239 einflul] der silageftitterung auf die qualit~t der butter einflul] der silageftitterung auf die qualit~t yon milch und milchproduk. ten. 3. mitt.: einflul] von silagefiitterung auf die organoleptischen eigenschaften dcr milch effect of protein intake and cold exposure on selected liver enzymes associated with amino acid metabolism body iron levels and hematologic fin. dings during excess methionine feeding der einttul~ des kulturmediums auf die bildung von streptolysis s durch ruhcnde zellen influence of polyphosphates in chilling water on quality of poultry meat influence of breed-type, feed level, and sex on characteristics of the lamb carcass, and some relationships among live animal and carcass measurements the toxic action of phenothiazine and some disturbances of intermediary metabolism in undernourished sheep efficiency of feed use in beef cattle uber die wirkung der nalmmgsfette auf die blutlipoide, teil i. ernahrungs-umsehau 10 [1963] nr. 8, s. 174/176. --~ber die wirkung der nahrungsfette auf die blutlipide fette und blutgerinnung. bibliothcca hacmatologiea effect of heavy cigarette smoking on postprandial triglycerides, free fatty acids, and cholesterol role of calcium in fibrin formation glucose-6-phosphate dehydrogcnase and aldolase in lenses of lactose-fed rats -effect of riboflavin, choline, pantothenic acid and vitamin a on the excretion of sodium in urine of rats the effect of waterwashed rice in the diet on the growth, excretion of sodium in urine and blood pressure of rats effect of aseorbic acid, vitamin b, and milk on the dark adaption effect of single deficiency of vitamin a, thiamine der einflub yon vitaminen auf die psyehomotorisehe leistungsi'~higkcit beim menschen. naunyn-schmiederberg's arch. exper feeding response of adult tribolium to carbohydrates in relation to their utilization nutritional secondary hyper hieronymi: influence of nutritional conditions on the cellular rna metabolism in rive and in vitro diffusible auxin increase in a rosette plant treated with gibberellin transamination in muscular dystrophy and the effect of exogenous glutamate: a study on vitamin e deficient rabbits, and mice with hereditary dystrophy. canad effect of auxin on the emergence of lateral roots in p. mungo seedlings the effect of nutritive status on oestrus, ovulation, and graafian follicles in merino ewes biologische wirkungcn autoxydierter, epoxydierter und bestrahlter fette s~ure-basen-gleichgewicht mad chronische acidogene und alkalogene eruehrung effect of protein level in milk replacers on growth and protein metabolism of dairy calves effect of sodium bicarbonate in the drinking water of ruminants on the digestibility of a pelleted complete ration sucrose diet and bfliary chelate excretion in rats: with note on procedure for chelate determination eirdlub sauerer milcherzeugnisse auf die darmflora untersuchungen fiber die speicherung und die ausscheidung yon vitamin a nach ungenfigender vitamin-a-versorgung bei legenden hiihnern the effects of low magnesium intake on lactating ewes effect of vitamin a on the content of pyridinnucleotides, pyrovic, and lactic acid and on anaerobic phosphorylation. ukrainskii biokkimichnii zhurnal the pyridine nucteotides. a study of a method of measurement. a study of the alterations in rat fiver under the conditions of diabetes and starvation. a preliminary study of various marine fish tissues with the emphasis on the islet of langerhans iron uptake-transport of soybeans as influenced by other cations hshe und zeitpm~kt der dfingung yon sommerweizen mit chlorcholinchlorid zur vcrkiirzung der halmlange nutritional significance of soluble nitrogen in dietary proteins for ruminants effects of long-term feeding of vegetable fats on atherosclerosis effects of feeding various mile, corn, and protein levels on laying performance of egg production stock some observations on the influence of a magnesiumdeficient diet of rats, with special reference to renal concentrating ability effect of gibberellic acid on flowering of apple trees the effects of dietary fat and energy levels on the performance of caged laying birds motivational producing properties of the feeding system of the rat hypothalamus the influence of diet and age on lipid metabolism of chickens effect of age on the response of chickens to dietary protein and fat absorption and excretion of biotin after feeding minced liver in achlorhydria and after partial gastrectomy variations de la calcsmie du chien normal apr~s ingestion de cholesterol eristallis~ dans l'6thanol ou dans rhexane changes in activity of rat epididymal adipose tissue in vitro due to time elapsed since last feeding differences in rat strain response to three diets of different compositions l'alcoolisme ~ l'hspital psychiatrique de colsou (martinique). ann. m~dico-psychologiques 1 nitrogen, lipid, glycogen, and deoxyribonu. eleie acid content of human liver. the effect of brief starvation and intravenous administration of glucose some metabolic and nutritional factors affecting the survival of erythrocytes erythrocyte survival of rats deficient in vitamin e or vitamin b 6. j. nutrition 80 [1963] nr. 2, s. 185/190 nutrition and lactation the effect of administering sodium chloride, sodium bicarbonate, and potassium bicarbonate to newly born piglets strontinm-90 and calcium in milk of miniature swine further studies on cariostatic effect of organic and inorganic phosphates urinary excretion of magnesium in man following the ingestion of ethanol the effects of magnesium compounds and of fertilizers on the mineral composition of herbage and on the incidence of hypomagnesaemia in dairy cows behavioral, dietary, and autonomic effects of ehlordiazepoxide in the rat the effect of a high and low sodium diet in a patient with familial periodic paralysis the effect of quaternary ammonium anion exchange resin on plasma and egg yolk cholesterol in the laying hen vitamin e deficiency and ion transpor~ in rat liver slices effect of level of nitrogen on growth and reproductive physiology of young buus and rams influence of low protein rations on growth and semen characteristics of young beef bulls, if treatment of nutritional cirrhosis in rats with choline and methionine; with special reference to fibrogenesis and fibroelasia probleme der beurteilung yon sauermilcherzeugnissen. milchwissen. schaft 18 [1963] nr. 5, s. 228/231. --antwort auf die diskussionsbemerkungen auf einer vortragstagung der gesellschaft ffir ern~hrungsbiologie e. v., miinehen, am 22 response of early-weaned pigs to variations in dietary calcium level with and without lactose effect of low calcium diet on bone crysta]linity and skeletal uptake of 4sca in rats response of rural guatemalan indian children with hypocholesterolemia to increased crystalline cholesterol intake source of plasma free fatty acids in dogs receiving fat emulsion and heparin alcoholic intoxication in the newborn infant. bril mcd dental caries of rats fed a rice diet and modifications a study of zinc deficiency in the dairy calf effects of different levels of zinc and phosphorus on the growth of subterranean clover (trifolium subterraneum l) lrber den einflu8 yon fluor auf den wassergehalt des knochens gegevens ovvr vitamine b,-deficientie, -behoefto en -voorziening the liquefying action of pancreatic, cereal, fungal, and bacterial co-amylases ern~ihrung und endokrines system 1. mitt.: der einfiul3 der erniihrung auf die schilddriise the effect of saline water on kidney tubular function and electrolyte excretion in sheep zinc and iron deficiencies in male subjects with dwarfism and hypogonadism but without ancylostomiasis, schistosomiasis or severe anemia die yextriiglichkeit yon xyli~ beim diabetiker einflni3 der laevulose auf die fu~ionsbrelte. sport~tl. praxis der einflul~ yon vitamin a auf den zitronensi~urestoffwechsel studies on the growth-promoting value and digestibility of passion fruit seed oil ration effects on drylot steer feeding patterns dextrothyroxine on metabolism of cholesterol some effects of feeding lactates to dairy cows copper deficiency problems in south-east scotland bone changes in iron deficiency anaemia a preliminary report on nucleic acid levels in mineral deficient plants metabolism of histidine in protein malnutrition ttypoglycemie effect of l-leucine during periods of endogenous hyperinsulinism nutritional studies with the guinea-pig. viii. : effect of different proteins, with and without amino acid supplements, on growth some effects of sulphur-containing amino acids on the growth and composition of wool effects of hunger and vi value on vi pacing potency of conditioned reinforcem based on food and on food and punishment sfibwaren und karies in theorie und praxis effect of magnesium on the changes of myocardial potassium confent untersuchungen fiber den einflub oral verabreiehten oxytetraeyclins auf leberlipide und serumeholesterin der weil~en ratte further studies on manganese nutrition of tobacco in relation to virus infection and synthesis aminobutyric acid (),-aba)-vitamin be relationships in the brain serum lipids and diet: a comparison between three population groups with low, medium, and high fat intake effects of light and gibberellin on elongation of intact wheat coleoptiles experimental magnesium deficiency in the cow thyroid function in chickens and rats: effect of iodine content of the diet and hypophysectomy on iodine metabolism in white leghorns cockerels and long-evans rats kuhmilehallergie beim sgugling und a rapid method for assessing drug inhibition of feeding behavior variation in, and the effects of vitamins on vertieillinm albo-atrnm influence of high level vitamin a supplement on semen characteristics and blood composition of breeding bulls influence of diet on viral hepatitis der einflul] der stiekstoffdfingung auf die zusammcnsetzung yon karf~ffeleiweib insulin response to fructose and galactose the effect of excess vitamin a on the oxygen consumption of young female rats effect of dietary amino acid pattern on plasma ~mino acid pattern and food intake gibbere/lln: effect on diffusible auxin in fruit development effect of intravenous versus oral fat administration in fat-deiicient dogs plasma vitamin bi~ levels in some nutritional deficiency states nutritional factors influencing the conversion of tryptophan to niacin pancreatic hypertrophy and chick growth inhibition by soybean fractions devoid of trypsin inhibitor production, interior egg quality, and some physiological effects of feeding raw soybean meal to laying hens effect of palm jaggeries on the growth and blood and liver composition of albino rats kept on rice and jowa effects of polyphosphates on water uptake, moisture retention, and cooking loss in broilers untersuchungen fiber den ern~hrungsphysiologischen wer~ yon kasein entgegnung zu diskussionsbemerkungen auf einer vortragsveranstaltung der gesellschaft for ern~ihrungsbiologie e. v., mfinchen, am 22. juni die erg~nzungswirkung yon dl-methionin allein oder in kombination mit l-lysin beim wachsenden schwein der einflul~ der nahnmg auf den kauapparat der einflu6 der nahrung auf den kauapparat. teil il changes in bone mass and density in living rats during the manipulation of calcium intake effect of chromium, cadmium, and other trace metals on the growth and survival of mice a study of fermentation in the production of mahewu, an indigenous sour maize beverage of southern africa the vitamin b~ deficiency syndrome in human infancy, biochemical and clinical observations lipids in chick urine: the influence of dietary rapeseed oil effects of dietary nitri~ on the chick" growth, liver vitamin a stores, and thyroid weight influence of radioactive sodium-24 on higher nervous activity of dogs, when chronically administered into the organism in comparatively small doses 9 the change of erythroeyte blood composition in persons with prolonged complete alimentary starvation (without limiting the water intake) and subsequent feeding dental abnormalities in rats attributable to protein deficiency during reproduction the effect of environment, and nutrition of pathogen and host, in the damping off of seedlings by rhizoctonia solani effect of dietary protein on fructose, citric acid, and 5-nucleotldase activity in the semen of bulls the effect of fluorine on dairy cattle. v. : fluorine in the urine as an estimator of fluorine intake some effects of diet and therapy on the survival and metabolism of adrenalectomized rats effect of methonine and choline deficiency on liver choline oxidase activity in young rats untersuchungen fiber die wasserlssliehen hemmstoffe aus dem 8chnittholz der weinrebe (vi~is vinifera l.). i. zur wirkung der hemmstoffe auf die keimlmg mad entwicklung yon rebs~mlingen nutrition and palatability. lancek 11 the effect of feeding fluoride on some enzymes of bovine tissues diet and histamine in the rmninant effect of food reflexes on cholinestera~e activity of cortical tissue. federation essential fatty acid deficiency and rat liver homogenate oxidations the effect of vitamin and antibiotic injections on early turkey poult growth and mortality alimentary production of gallstones in hamsters. 12. studies with rice starch diets with and without antibiotics nitrogen studies with apple and cranberry the influence of diet on the quality of faecal fat in patients with and without steatorrhoea uber die unterschiediiche beeinflussung des tryptophansteffwechsels dutch vitamin b6-mangel in der ratte. hoppe-seyler's influence of vitamin b12 and its coenzyme upon incorporation in rive of amino acids into tissue proteins in rats relativer vitamin b6-mangel hei erkrankungen der schilddriise strukturanomalien der z/ihne bei vitamin d-mangel-raehitis und der vitamin d-resistenten renalen rachitis the effect of fluoride on bone effects of insulin on hepatic glucose production and utilization prevention by hydrocortisone of changes in connective tissue induced by an excess of vitamin a acid in amphibia acute hypervitaminosis a in guinea-pigs. i. : effect on acid hydrolases der einfiu~ yon vollkornbrot auf den calciumstoffwechsel bei schulkindern effect of feeding milk replacers with varying amounts of f~ for hothonsc lamb production egg yolk and serum cholesterol levels: importance of dietary cholesterol intake effect of protein intake on glutamic dehydrogenase and amino acid desruination in rive observations in experimental magnesium depletion effect of gibbercluc acid on growth, gibberellin content, and chlorophyll content of leaves of potato ~ physiological factors influencing growth, reproduction, and production of well-fed dairy heifers. i. age at first breeding. ii. feeding of diethytstilbestrol tm~ influence of diet on the development of parotid salivation and the rumen of the lamb bericht fiber den wissenschaftlichen kongreb 1963 der deutschen geseuschaft fiir erniihrung influence of variations in dietary calcium: phosphorus ratio on performance and blood constituents of calves the lack of a consistent chick growth response to norwegian kelp meal some effects of kinctin on the growth and flowering of intact green plants incorporation of [,2p] orthophosphate into phospholipids of frog tissues during feeding and stmrvation growth-modifying and antimetabolite effects of amino acids in chrysanthemum a study of techniques used by advertisers in dealing with weight control. a national health problem lipid excretion. 3.: examination of faecal lipids of rats injected intravenously with serum lipoprotcin containing ~ac-labelled cholesterol effect of diet and diabetes on plasma glucose, fatty acid, and insulin effect of cigaret smoking during pregnancy: study of 2000 cases. obstetrics gynecology 21 respiration and phosphorylation in live homogenates from rats exposed to hypoxia and food restriction the influence of mi]l~ fat depressing rations on the yield and composition of bovine milk phosphatides and cholesterol in the rat bed: effects of growth, diet, and age the effect of plant nutrients and antagonistic microorganisms on the damp. ing-off of cotton seedlings caused by rhizoctonia solani kurus l~utrition of gram-negative anaerobic bacilli. nutrition rev. 21 effects of glucose on the production by escherichia coli of hydrogen sulphide from cysteine. j. general iylierobiol. 30 enumeration of psyehrophilie microorganisms vitamin requirements of three pathogenic fungi calorie requirements of rat intestinal microorganisms specificity of the salt requirement of halobacterium cutirubrum influence of the aqueous potato extract and its fractions on growth and spore formation of the b. pumilus and the production of the antibiotic tetaine the relationship between hormonal activity and sugar metabolism in protoparce scxta (joka~sen) and blabcrus craniifer bur~ieister apparent incorporation of ammonia and amino acid carbon during growth of selected species of ruminal bacteria l~ber die wirkung anorganischer st~ube auf das wachstum yon mikroorganismen effect of dietary calcium lactate and lactic acid of faecal eseherichia coli counts in pigs uber den einflul3 des n~ihrsubstrats auf die hemmung des bakterienwachstums durch cyanid autoradiographic studies of the differential incorporation of glycine, and purine and pyrimidine ribosides by paramecium aurelia correlation between the essential amino-acid requirements of staphylococcus aureus, their phage types, and antibiotic patterns nutrition and metabolism of marine bacteria. xii.: ion activation of adenosine triphosphat~se in membranes of marine bacterial cells carbon dioxide fixation in bacillus anthracis bacterial growth under conditions of limited nutrition interrelationship between temperature and sodium chloride on growth of lactic acid bacteria isolated from meat-curing brines morphological variation and nutrition of a new monoeentric marine fungns feeding stimulants required by a polyphagons insect, schlstocera gregaria vitamin requirements of root nodule bacteria phcnotypic, genotypic, and chemical changes in starving populations of aerobacter aerogenes studies on the d-amino acids. ii.: utilization of d-amino acids by lactic acid bacteria role and formation of the acid phosphatase in yeast der einflub yon co~-partia]druck und glucose-konzentration auf wachsturn und stiekstoffbindung yon azotobacter chroococcum bei~ inorganic polyphosphate metabolism in chlorobium thiosulfatophilum effects of molybdenum, vanadium, tungsten, and cobalt on growth of rhizobia and their hosts nutrition of leptospira pomona. ii.: fatty acid requirements 9 sterilization by beta-propiolactone of solid nutrient media for eultivation of moulds the digestion of natural food protein by the elearnose skate raja eglanteria (bose.) utilization of amino acids during metabolism in escheriehia coil the effect of nutrition on the growth of fasciola hepatica in its snail host cobamide coenzyme contents of soybean nodules and nitrogen fixing bacteria in relation to physio]ogical conditions determination of carbohydrate metabolism of marine bacteria the amino acid requirements of various types of shigella mushroom culture. factors affecting the growth of morel mushroom myecelium in submerged culture lebensmittelzusatzstoffe und mutagene wirkung. vii. : priifung einiger xanthen-farbs~offe auf mutagene wirkung an escherichia coll the biological control of glycogen metabolism in agrobaeterium tumefaeiens the maintenance requirement of escheriehia coll methionine requirement for growth of a species of mieroeoecus ~iorphogenesis and nutrition in the memnionella-stachybotrys group of fungi viable organisms from feces and food-s~uffs from early antarctic expeditions the metabolism of yeas~ sporulation. v. : stimulation and inhibition of sporulation and growth by nitrogen compounds the effect of lipids on citric acid production by an aspergillns niger mutant relationship between deuterium inhibition of growth and glucose catabolism in saecharomyees cerevisiae function of trehalose in baker's yeast (saccharomyces cerevisiae). arch. biochem preparation and lyophilization of colicine suspensions. i. production of eolicines in liquid nutrient media lvber den einflub der kulturbedingungen auf die stramenempfmdliehkeit der glueoseoxydation in baeterium cadaveris nutritional requirements and metabolism of myeoplasma laid-lawil j. gen. microbiol. 30 die wirkung subletaler konzentrationen yon sorbinsi~ure auf escherichia coli und aspergillus niger the genetic analysis of carbohydrate utilization in aspergillus nidulans the amino acid nutrition of respiration deficient and respiration competent saecharomyces. a. van leewenhoek nutritional studies of some membem of mucorales. iv.: 1. sugars, amino-, and organic acids of the myceaium selektivn~hrboden fiir staphylokokken effects of certain amino acids in anthranilate production in neurospora crassa studies on the polysaccharide-fermenting lactic acid bacteria. in. : nutritional requirements and the existence of fermentation promoting factors for sucrose and inulin the catabolism of proteins and nucleic acids in starved aerobacter aerogenes aerobic fermentation and the depletion of the amino acid pool in yeast cells influence of hydrogen ion concentrations on the utilization of sodium nitrite by fungi oxidative metabolism of glucose in leaf tissues infected with tobacco mosaic virus differential effects of amino acid deficiencies on bacterial cytochemistry nutritional requirements of an aspergiuus niger mutant for citric acid production culture de tissus d'insectes ~, l'aide d'extrait d'embryon de poulet en l'absenee d' h6molymphe. c. r. acad utilization of some carbon and nitrogen sources by pseudomorms fluorescens on the selection of microorganisms for use in bacterial fertilizers in vitro and -rive uptake of carbon-14 labelled alanine and glucose by ascaridia galli, parasitic nematode of chickens growth of psychrophiles. i. : lipid changes in relation to growgh-temperature reductions vitamin requirements of listeria monoeytogenes parasitism and nutrition of gonatobo~rys simplex the effect of alkali metals on the growth of staphylococcus pyogenes the uptake of potassium and rubidium by staphylococcus pyogenes metabolism of nucleic acids and of nucleotides in the course of synchronous development of azotobacter vihelandii studies on the biotin-oleie acid requirements of a lactobaeillus plantarum variant isolated from chick feces unusual response to iron-dextran. brit. *ned. j. 1963, nr. 5331, s. 630. +in'. n.: tissue trypsin and trypsinogen levels in pancreatitis skeletal development of suckling kittens rate of liver regeneration atherogenesis in the monkey the significance of serum triglyeerides anaemia and parasitism in man physiological mechanisms in nutritionally-induced differences in ovarian activity of mature ewes bone development in suckled pigs production performance of artificiauy and non~r~ifically sired herd-mates in wisconsin search for an unidentified nutrient in mammalian liver. part i.: growth studies with various ox liver preparations proline control of the feeding reaction of cordylo-phora relationship between longevity and production in holstein-friesian cattle circadian adrenal cycle in c mice kept without food and water for a day and a half metabohc pmduc~s form labelled ethanol. iv. : disappearance of ethanol-carbon from morphological fractions and lipids of rat tissues acetate utilization by maize roots vajda, ]3. : i~c~sll-rcs of body fat and hydration in adolescent boys some characteristics of a proteolytic enzyme system of pseudomonas fluorescens some metabolic relationships between host and parasite with particular reference to the eimcriae of domestic poultry nucleotide degradation in the muscle of iced haddock (gadns aeglefinns), lemon sole (pleuronectes microcephalus), and plaice (pleuronectes platessa) effect of starvation and 6-mcthylprednisolone (m_edrol) on the acid phosphatase of rat liver and muscle metabolic patterns in preadolescent children. vii. : intake of niacin and tryptophan and excretion of niacin or tryptophan metabolites biochemical changes in fish muscle during rigor morris studies on ornithine synthesis in relation to benzoic acid excretion in the domestic fowl effect of manual total collection of feces upon nutrient digestibilities polarographie studies on storage of meats. xxii. : influence of proteolytic enzymes on the polarographie wave of beef protein solutions post-mortem changes in chilled and frozen muscle genetic-nutritional interactiions as affecting the early growth rate of chickens effect of unequal milking intervals on lactation milk, milk fat, and total solids production of cows changes with age in glutamic oxalacetic transaminase activity of sonically oscillated tureen juice compared to total steam volatile fatty acids in calves fed different roughages catecholamine metabolism and some functions of the nervous system a study of some of the conditions affecting the rate of excretion and stability of creatinine in sheep urine changes in feeding behavior after intracerebral injections in the rat kanzcrogene substanzen in wasser und boden food additives and contaminants and cancer milk allergy in infancy food poisoning due to salmonella cnteritidis vat the mineral element content of spring pasture in relation to the occurrence of grass tetany and hypomagnesaemia in dairy cows insecticide residues in meat. residues in body tissues of livestock sprayed with sevin or given sevin in the diet over de giftigheid van solanum-alkaloidcn toxic products in groundnuts zur beziehung zwischen lipidcn hepatotoxicity of foods: a consideration of the hepatotoxicity of a few phanerogams and eryptogams. their possible influence in the pathogenesis of cirrhosis and hepatoma food-poisoning potential of the enterococei vanadium. excretion, toxicity, lipid effect in man an institutional food-poisoning outbreak examination of market milk of novokuznctsk for brucellosis an outbreak of food poisoning in a mental hospital food allergy as a cause of abdominal pain radioactivity in the diet the effect of microbial contamination on the requirement of chicks for certain nutrients the acute oral toxicity of cottonseed pigment glands and intraglandular pigments sur l'absorption du edsium radioactif par rorge. c. r. hebd. s6ances aead die experimentelle alimentiire lebernekrose a]s empfindlicher indikator bei thermiseher belastung der milch. uber die magermilehtroekntmg the comparative toxicity of ethylene dibromide when fed as fumigated grain and when administered in single daily doses repository polyvalent insect antigen treatment for patients sensitive to hymenoptera. a clinical evaluation precursors of carcinogenic hydrocarbons in tobacco smoke toxin production in naturally separated liquid and solid components in preparations of heated surface-ripened cheese inoculated with clostridium botulinum allergieversuehe am tier zur ~tiologie der sogenannten margarinekrankheit. dr. reed. wschr. 14 [1963] nr. 1, s. 9/12. --, allergenwirkung oder immunologisohe adjuvanswirkung in der ~tiologie der sogenarmten margarinekrankheit radium-226 in human diet and bone miodine in the thyroids of north american deer experimental groundnut poisoning in pigs cholesterol as carcinogen safety factors in water fluoridation based on toxicology of fluorides entelo epidemiologische gegevens over her ,planta-exantheen" te rotterdam, verkregen door enquetc-onderzoek planta-~x~ntheem" epidemie te rotterdam in de m~nden ~ugnstu~ en september 1960 salmonella-verontreiniging van plantaardige grondstoffen veer voedingsmiddelen van mens en dier increase of strontium-90 and caesium-137 sodium fluoride intoxication salmonellosis in the netherlands zwei seltene salmonellenfunde untersuehungen fiber die chronisehe toxizit~t der ascorbins~ure bei der ratte captan in green vegetables rfickst~nde yon pflanzenschutzmitteln, insektiziden mid dergleichen in der nahrung und ihre bedeutung ffir die gesundheit der gehalt der milch an 181j, 1,~co ' u0ba _{_ 140la in der deutschen )iileh in der zeit yon langfristige nutritive anwendung yon antibiotika in der tierern~hrung im hinblick auf die menschliche gesundheit mi~ besonderer beriicksichtigung yon chlortetrazyklin a milk-borne outbreak of food poisoning due to salmonella heidelberg ergebnisse yon schwebversuehen an farbstoffen zur farbmattierung yon tabakwaren nutritional secondary hyperparathyroidism of the cat insecticide residues in fat. a screening method for chlorinated pesticide residues in fat without cleanup untersuehungen fiber die quantitative verteilung radioaktiver falloutprodukte in milch too many vitamins radios~ron$ium removal from milk. determination of apparent equilibrium constants of the exchange reactions of sodium, potassium, calcium, and magnesium with amberlite ir-120 ern~hrungsphysiologische eigenschaften der margarine. fette, seffen, anstrichmittel 65 smoking and cancer: retrospective studies and epidemlologieal evalution beobachtungen fiber den verlauf der alkoholkrankheit am krankengut einer heilanstalt die verschmutzung yon trinkwasser dutch i)etergentien grain fumigant residues. occurrence of bromides in the milk of cows fed sodium bromide and grain fumigated with methylbromide insecticide persistence. the disappearance of endrin residues on cabbage lebensm ittelchem u. gerichtl reproductive performance of female miniature swine ingesting strontium-90 daily toxicity of nitrate nitrogen to cattle methods of residues of phosphated insecticides and miticides in foods on bacillary excretion in food toxinfections of salmonella etiology relationships between the deposition of strontium-90 and the contamination of milk in the united kingdom staphylococci in cottage cheese is~cs and potassium in people and diet. -a study of finnish lapps effect of treatment of seeds with 2-chloroethanol on the resistance to boron toxicity in wheat seedlings desferrioxamin, eine neue das eisen bindende und eliminierende substanz zur behandlung der 9rim~rcn und sckund~ren i-i~mochromatose akuter eisenvergiftungen toxic products in groundnuts smoking, arteriosclerosis, and age the incidence of milk sensitivity and the development of allergy in infants einflul] yon fluor und jod auf den stoffwechsel, insbesondere auf die schilddrtise quelques exemples illnstrant la valour et l'utllit6 des m~thodes de lysotypie clans certaines salmonelloses humaines d'origine alimentaire food poisoning caused by panthogenic halophilic bacteria (pseudomonas enteritis txkikawa): 1%ep0rt of four autopsy cases procaine penicillin g in milk following intramuscular injections comparative subacute toxicity for rabbits of citric, fumaric, and tartaric acids distribution of pesticides in fermentation products obtained from artificially fortified grape musts mercurial fungicidal seed protectant toxic for sheep and chickens the problem of salmonella food poisoning dietary factors in the pathogenesis and treatment of cirrhosis of the liver. med. clinics of north america 47 an outbreak of salmonella food poisoning in l~ehmadabad town, kaira i)istxiet, gnjaxat 8~a~e la tossinfezionl alimentarl da salmonella nell' 0spedale maggiore di milano dal 1954 al 1961 water intoxication due to oxytocin: reporb of a case c~sium-137 und kalium in menschlichen organen und in der milch 1959/60 caesium-137 in dried milk produets in relation to phytoellmatic zones smoking and oral cancer occurrence of hepatomas in rats fed diets containing peanut meal as a major source of protein nachweis yon mangan-54 und kobalt-57 in pflanzen als fo]ge russischer kernwaffenvcrsuche e-ruhr-epidemie durch speiseeis bcricht fiber eine arbeitstagung bei der internationalen atomenergic-behsrde in wien vom 12. bis 14. i)ezember 1962. i)t. lebensmittel-rdsch the development of microbiological standards for foods. j. milk food technol a case of breslau salmoneliosis caused by eating chicken internationales rundgespr~ch fiber lebensmittelchemische probleme in wiesbaden und eltvllle a. rh. (4 vortragsreferate) staphylococcal infection of raw milk as a cause of food poisoning. monthly bull. ministry health pub carcinogenic effect of egg white, egg yolk, and lipids in mice eczema and cow's milk exitus letalis nach lebensmittelvergiftung dutch bacillus cereus repression of staphylococcus aureus by food bacteria. ii. : causes of inhibition a further report on the radioactive contamination of milk and milk products in japan. determination of strontlum-90 and cesium-137 concentrations in milk powder in japan concerning sporadic salmonelloses insecticide residues. extraction and cleanup studies for parathion residues on leafy vegetables salmonellosis epidemiology zur frage der deponierung yon nutrltiven allergenen im organismus. allergic, _~sthma 9 allergic children with various symptoms caused by cows' milk messungen der umweltsradioaktivit~t und der radioak-tivit~t yon lebensmitteln im jahre ein cxperimenteller bcitrag zur tabakrauehkanzerogenese. dr. reed. wschr. 88 [1963] nr. 13. u n. : contamination of leaves by radio active fall-out insecticide residues in milk and meat. residues in butterfat and body fat of dairy cows fed at two levels of kelthane (1.0 and 2 insecticide residues in milk. residues in milk from dairy cows fed low levels of toxaphene in their daily rations tier-und pfhnzenerniihrnug _anlrnal and plant nutrition summary of ,tropical crops soil, analysis, and its relation to plant composition and growth fertilisers and plant nutrients ulcers in swine tnfluence of chelating agents on the concentration of some nutrients for plants growing in soil under acid and under alkaline conditions nutritional evaluation of permanent pastures with dairy cattle in louisiana the herbage intake of eattle grazing lucerne and cocksfoot pastures terminology and methods for feeding and weighing animals the effect of feeding on evaporative heat loss and body temperature in zebu and jersey heifers studies on the requirements and interaction of copper and iron in broad breasted bronze turkeys to 4 weeks of age some factors affecting iron uptake by strawberry plants feed consumption during lactation and involution in sprague-dawley-rolfsmeycr rats the effect of variations in the energy and protein levels of the ration upon performance in the pig use of barley in high-efficiency broiler rations. 6. poultry sci tierarzncimittcl und aufzuchtmittel in der landwirtschaftlichen praxis. gesund. heitliche erwggungen zum schutze des konsumenten bei der anwendung yon tierarzneimitteln und aufzuchtmitteln in der landwirtschaftlichcn praxis, tell ii mechanism for movement of plant nutrients from soil and fertilizer to plant root growth rate of the tea leaf as determined by shade and nutrients. empire j. exper note on induction of flowering in ~railing shoots of clones of saccharum spontaneum effect of level and sequence of feeding and breed on ovulation rate, embryo survival, and fetal growth in the mature ewe evidence for a high zinc requirement at the onset of egg production aufnahme und wirkung des mikronghrstoffs knpfer in ionogencr und ehelatisierter form bei gerste effects of pelleting and varying grain intakes on milk yield and composition the relationship of gibberellic acid to flower initiation in column stock, math~ela incana the effect of selenium administration on the growth and health of sheep on scottish farms the horsebean (vicia faba l.) as a vegetable protein concentrate in chick diets size and feeding of different types of fishes the influence of age of chicks on their sensitivity to raw soybean oil meal influence of the mineral nutrition on the resistance of peach trees to fusicoceum amygdali de la croix granular fertilizer. influence of associated salts on plant response to dicalcium phosphate a comparison of feeding growing pigs once or twice daily the interrelation of nutrient supply, leaf nutrient content, and vegetative growth of ilcx crenata 'green island' effect of rationing grass on the growth rate of dairy heifers and on output per acre, with a note on its significance in experimental design experiments on the nutrition of the dairy heifer. iv.: protein requirements of 2-year-old heifers grass silage vs. hay for lactating dairy cows hay vs. silage for two to six months old dairy calves weaned at 25 or 60 days effects of high levels of copper and ehlortetracycline on performance of pigs seedlings resistance of corn to leaf feeding of the european corn borer ostrina nubflalis ease of hydrolysis of the hemieeiluloses of forage plants in relation to digestibility bodenkde. 100 [1963] mr. 1, s. 53/58. --caleium-mangelsymptome an h6heren pflanzen effects of frequency of feeding on urea utilization and growth charae%oristics in dairy heifers factors affecting the voluntary intake of foods by cows. 6. : a preliminary experiment with ground, pelleted hay the relationship of dietary energy level and density to the growth response of chicks to fats salt tolerances of pinus thunbergii compensatory carcass growth in steers following protein and energy restriction a guide to production, care, and use of laboratory animals. federation prec. 22 estimation of essential fatty acid intake in swine automatic dispensing at frequent regular intervals of liquid diet for piglets chelation in nutrition. chelates and the trace element nutrition of corn der einflul3 yon humuss~ure auf wachstum und ver~inderungen des freien zuekergehaltes bci winterweizenpflanzen, die im dtmkeln kultiviert wurden a comparison of the growth of chicks in the gustafsson germ-free apparatus and in a conventional environment, with and without dietary supplements of penicillin an evaluation of weed competition and the effects of weed extracts and leachates on the development of field corn (zea mays l.) and oats (arena sativa l digestibility of rations containing different sources of supplementary protein by young pigs the effects of urea supplements on the utillzation of straw plus molasses diets by sheep production performance of artificially and nonartifieiallysired herd-mates inwiseonsin dietary phosphorus for laying hens tolerance to acid soil conditions in barley effect of calcium and magnesium upon digestibility of a ration containing corn oil by lambs effects of caloric restriction during the growing period on the performance of egg-type replacement stock untersuchungen fiber die verwertung yon calcium-und phosphorsalzen aus fisehgr~itenmehl einigcr frischfische und fischkonserven bei der verffitt~rung a nut~rient requirement for optimum water absorption by intact root systems preparation of feed for animal nutrition experiments responses of winter wheat to nitrogen and soil nitrogen status studies on calcium requirements of broilers znm problem dcr nahrungspflanzenwabl der aphiden some factors affecting leaf development and longevity and the subsequent yield of corn grain efficiency of energy utilization by young cattle ingesting diets of hay, silage, and hay supplemented with lactic acid the effects of a plant steroid on body weight and feed efficiency of broilers feeding troughs for guinea-pigs beitrag zur ]~ackfruchtmast mit schweinen unter besondercr beriicksichtigung des n~hrstoffgehaltes der beifuttermischungen und der the feeding of thyroprotein to lactating sows zur planung, durchfiihrnng und auswertung yon schweinemastvcrsuchen bei gruppenfiitterung the influence of barbituric acid derivatives on the development of plant roots and root hairs factors affecting the voluntary intake of food by cows. 5.: the relationship between the voluntary intake of food, the amount of digesta in the retieulo-rumen, and the rate of disappearance of digesta from the alimentary tract with diets of hay, dried grass or concentrates artificial food for oak-silkworm raising the comparative toxicity of ethylene dibromide when fed as fumigated grain and when administered in 8ingle daily do~0~ gibberellin at the vineyard oak wilt development and its reduction by growth regulators. i. production and activity of oak wilt fungus pectinase, ecmulase, and auxin. ii. effect of halogenated benzoic acids on oak trees, the oak wilt diseases, and the oak wilt fungus regulating nutrient intake in laying hens with diets fed ad libitum some effects of different soils on composition and growth of sugar beet production of fodder yeast from barley. i. preliminary studies on the use of the waldhof fermenter development and nutrition of new species of thranstochytrium studies on the effect of frequency of feeding upon the biology of a rabbit-adapted strain of pediculns humanus the influence of previous vitamin k nutrition on thromboplastie activity of brain extract the effect of nutrition conditions on the growth of and nitrogen accumulation by fodder beans when sown together with indian corn. i)oklady akad. nauk sssr dutch phenylbors~ure induzierte fragen der resistenzsteigerung in der modernen gefliigelhaltung chelation in nutrition. metal chelates in plant nutrition beziehungen zwischen dcm kupfergehalt und dem zeitlichen auftreten yon kupfermangelsymptomen an hafer in wasserkultur mit kleincn bodenmengen increased tolerance of bean plants to soil drought by means of growth-retarding substances effect of monocaleium and diammonium phosphates on crop yield, and their influence on soil solution movement and characteristics when associated with different salts effect of barbituric acid and ehlortetraeyeline upon growth, ammonia concentration, and urease activity in the gastrointestinal tract of chicks effects of feeding low levels of dimethoate on milk and on whole blood cholinesterase activity of dairy cattle die ziichtung von fleischschweinen und die folgeerscheinungen, die sich besenders im hinblick auf die qualit~t yon fleiseh und fett ergeben the relationship of specific nutrient deficiencies to antibody response in swine. i.: vitamin a. ii.: pantothenie acid, pyridoxine or riboflavin further studies of diet composition on egg weight effect of various levels of fluorine, stilbestrol, and oxytetraeycline, in the fattening ration of lambs studies on the properties of l~ew zealand butterfat. viii. the fatty acid composition of the milk fat of cows grazing on ryegrass at two stages of maturity and the composition of the ryegrass lipids soil potassium and the growth of vegetable seedlings artificial feed for silkworm, bombyx mori some effects of feeding stilbestrol, ehlortetracyeline, and penicillin with alfalfa soflage on steer performance and carcass quality food agric. 14 [1963] nr. 2, s. 66/75. --, mineral supplements for sheep the influence of higher volatile fatty acids on the intake of urea-supplemented low quality cereal hay by sheep untersuehungen fiber den umsatz waehsender sehweine ab geburt. 2. mitt growth of edible emorophyllous plant tissues in vitro chelates in agriculture. metal chelation by glucose-ammonia derivatives economic analysis of high-level grain feeding for dairy cows evaluation of the dacron bag technique as a method for measuring cellulose digestibility and rate of forage digestion n~ihrlssungen fiir zuckerriiben in wasser-und sandkultur activity of gibbereuin:'d' on the germination of photosensitive lettuce seeds raw and heat-treated soybeans for growing-finishing swine, and their effect on fat firmness ration effects on tureen acids, ketogenesis, and milk composition. i.: unrestricted roughage feeding a new growth stimulant, ~ growth hormone the effects of specific viruses, virus complexes, and nitrogen nutrition on the growth, flowering, and mineral composition of strawberry plants peculiar feature of respiration and redox processes in the rice plants grown under different nutritional conditions einflul] der silagefiitterung auf die qualit~t yon milch und milchprodukten. 3. mitt. : einflul] der silagcfiitterung auf die organoleptischen eigenschaften dcr milch effect of grinding and pelleting on the utilization of coastel bermuda grass hay by dairy heifers langfristige nutritive anwendung yon antibiotika in der tierernlihrung im hinblick auf die menschliche gemmdheit mit besonderer beriicksichtigung yon chlor-te~azyklin mode of action of growth retarding chemicals yield of sugarcane in louis-ana as influenced by soil moisture status and climate diss effect of auxin on the emergence of lateral roots in p. mungo seedlings compound mouse diets a semipurified caries-test diet for rats present status of feeding antibiotics to htctating dairy cows effect of sodium bicarbonate in the drinking water of ruminants on the digestibility of a pelleted complete ration semi-purified diets for sheep effect of vacuum-drying, freeze-drying, and storage environment on the viability of pea pollen. ii. : effect of boron, sucrose, and agar on the germination of pea pollen hshe und zeitpunkt der diingung yon sommerweizen mit chlorcholinchlorid zur verkiirzung der halml~nge nutritional signifieance of soluble nitrogen in dietary proteins for ruminants primary signs of nutritional deficiencies of laboratory animals ]~ffe[~b of dlctary pro~in and fat on growth, protein utilization, and carcass composition of pigs fed purified diets trans-fetts~iuregehalt yon schweineschmalz nach fiitterung yon sehweinen mit rindertalghaltigem kraftfutter. (ein beitrag zur quantitativen infrarotspektroskopischen bestimmung yon trans-fetts~uren in fetten effect of liming and potassium fertilization on soil solution and on yield and composition of alfaffa and orchard grass mixtures effects of feeding various milo, corn, and protein levels on laying performance of egg production stock effect of gibberellie acid on flowering of apple trees the effects of dietary fat and energy ]evels on the performance of caged laying birds effect of age on the response of chickens to dietary protein and fat chemical control of flowering. concentration of a floral-inducing entity from plant extracts strontium-90 and calcium in milk of miniature swine studies on the properties of newzealand butterfat. vii. effect of the stage of maturity of ryegrass fed to cows on the characteristics of butterfat and its carotene and vitamin a contents new radioactive tests show how termites feed mechanisms regulating the feeding rate of daphni~ magna straus influence of low protein rations on growth and semen characteristics of young beef bulls a study of zinc deficiency in the dairy call effects of different levels of zinc and phosphorus on the growth of subterranean clover (trifolium subterraneum l.). australian j. agrie absorption, translocation, exudation, and metabolism of plant growth-regulating substances in relation to residues the effect of the performance of growing pigs of the level of meal fed in conjunction with an unrestricted supply of whey increase in yield of legumes by fer~iliser mixture with lime chemically defined medium for growth of animal cells in suspension dis sieherung der eiweiljwrsotgung in dor l~ndwirt influences of previous calcium and phosphorns intake and plant phosphorus on the requirement of developing turkeys for calcium and phosphorus relationship between isotopicauy exchangeable calcium and absorption by plants effect of adding buffers to all-concentrate rations on fcedlot performance of steers, ration digestibility, and intrarumen environment lysine supplementation of corn -and barley-base diets for growing-finishing swine the effect of gibbereliin on the germination of seeds of arboreal plants effect of physical state of coastal bermuda grass hay on passage through digestive tract of dairy heifers nitrate reduction and carotene stability. effect of nitrate and some of its reduction products on carotene stability, d. agric. food chem chemical preparations for plant protection untersuchungen fiber die ksrperzusammensetzung und den stoffansatz waehsender mastschweine und ihre beeinflussung dutch die erniihrung. 3. mitt the cobalt requirement of sub-~erranean clover in the field comparing mile and corn in broiler diets on an equivalent nutrient intake basis effect of mineral nutrition on the invasion and response of turnip tissue to plasmodiophora brassicae wor the relation of chlorogenic acid and total free phenols in potato plants to resistance to infection by verfieillium alboafxum nitrogen and potassium as variables influencing soluble nitrogen and organic acid accumulation in soybean (glyoine max). di~s. abstr. 23 bett~r british beef and barley feed. veter effect of nitrogen fertilization upon yield and digestibility of aftermath timothy forages fed to dairy heifers ration effects on dltlot steer feeding patterns effects on zea mays seedlings of a strontium replacement for calcium in nutrient media evaluation of albumen quality in a poultry breeding program nutritional studies with the guinea-pig. viii. : effect of different proteins, with and without amino acid supplements, on growth some effects of heredity and environment on appetite in dairy animals further studics on manganese nutrition of tobacco in relation to virus infection and synthesis amillo acid supplementation of pig diets chelation as a basic biological mechanism der einflu~ der stiekstoffdiingung auf die znsammensetzung yon kartoffeleiweiil z studies on photosynthesis. i. : biosynthesis of sucrose from glycolate. par~ ii. : bicarbonate utilization by washed algae production, interior egg quality, and some physiological effects of feeding raw soybean meal to laying hens alteration of post-mortem changes in porcine muscle by preslaughter heat treatment and diet modification chelation in nutrition. soft microorganisms and soil chelation. the pedogenie action of lichens and lichen acids die ergiinzungswirkung yon dl-•ethionin allein oder in kombination mit l-lysin beim wachsenden schwein untersuchungen iibcr den einflub unterschiedlicher wasservemorgung auf ertr~ge, gehalte an ~therischem 01, trenspirationsquotienten, biattgrsl~en und relative ~)ldriisendichtsn bei einigen arten aus der familie der labiaten. 2. teil gehalte an iitherischem 01, transpirationsquotienten, blattgrsflen und relative 01-driisendichten bei einigen arten aus dcr familie der labiaten. iii.: blattgrsflen, relative 01driisendichten, anzahl am haupttricb inseriertcr blattpaare und internodien. liingen cadmium: uptake by vegetables from supcrphosphate in soil studies on the protein and methionine requirements of young bobwhite quail and young ringnecked pheasants chelation in nutrition. evidence for natural chelates which aid in the utilization of zinc by chicks selective fertilization of apple-trees some soils and fertilizer relationships of the cavendish banana (muss cavendlshl lambert) on three different soils in costa rice soil organic phosphorus and the phosphorus nutrition of plants the effect of heat treatment on the nutritive value of milk for the young calf. 5. : a comparison of spray-dried skim milks prepared with different preheating treatments and roller.dried skim milk, and the effect of chlortetracyclinc supplementation of the spray-dried skim milks the effect of heat, ~reatmen~ on the nutritive value of milk for the young calf. 6. :the effect of the addition of calcium a biological assay for metabolizable energy in poultry feed ingredients together with findings which demonstrate some of the problems associated with the evaluation of fats feed additives in livestock rations: part i. : urea in dairy rations. part.i/: use of thyroprotein in cattle nutrition diet and histamine in the ruminant synthetic ion-exchange resins as a medium for plant growth nutrition of vibrio fetus theoretical basis of unicellula algae cultivation amino acid supplementation of barley diets for growing swine some effects of 2.4-dichiorophen-oxyacetic acid on swect corn (zea ]~ays rugosa l.) with emphasis on yield, tillering, root development, and exudation of electrolytes from roots and stems. i)iss. abstr feeding and management of broiler strain breeder hens relationships among seven elements in the nutrition of corn in sand culture an external effect of inorganic nitrogen on nodulation influence of enzyme supplements in lamb fattening rations gravenstein and jonathan apples produced with giberellle acid the role of carotene in the dairy cow. wiss. ver6ff. dr. ges. ern~hrung 9 vitamin a-wirksamkeit der carotine bei versehiedenen tierarten. wiss. ver-5ff. dt. ges. eru~hrung 9 upgrading the indigenous poultry of uganda. i. : the growth rates and feed conversion from hatching to maturity of indigenous poultry crossed with four imported breeds effect of different kinds of litter on growth and feed efficiency in chick rearing investigation of the mineral nutrition of datura innoxia the effect of flooding in the availability of phosphorus and on the growth of rice nutrition of the boll weevil larva ascorbic acid in the nutrition of plant-feeding insects effects on the s~maeh worm, i-iaemonehus contortus, of feeding lambs natural versus semipuriiicd diets yield and foliar composition of corn as affected by fertilizer rates and environmental factors. i)iss. abstr. 2~ [1963] nr praktische erfahrm]gen in der carotinoidversorgung yon vsgeln effect of protein-energy relationship on the performance and carcass quality of growing swine the use of quarter samples in the assessment of the effects of feeding treatments on milk composition * calcium and phosphorus requirements of finishing broilers using phosphorus sources of low and high availability amino acid supplementation of peanut meal diets for broiler chicks the effects of feeding various levels of vitamin a on chicks with cecal coccidiosis chelation in nutrition. review of chelation in plant nutrition water use by irrigated arabia coffee in the failure of certain dietary ingredients to affect the incidence of blood spots in chicken eggs dcr einflul~ der anbauverh~ltnisse auf die eigensehaften der kartoffelknolle und der st~rke effect of feeding milk replacers with varying amounts of fat for hothouse lamb production l~hysiologieal factors influeneelng growth, reproduction, and production of wcll-fed dairy heifers. i. ago at first breeding. ii. feeding of diethylstflbestrol results of an experiment ot rothamsted testing farmyard manure and n, p, and k fertilizers on five arable crops. i. : yields results of an experiment at rothamsted testing farmyard manure and n, p, and k fertilizers on five arable crops. ii.: nutrients removed by crops 9 the utilization of carotenoids by the hen and chick some effects of potassium and lime on the relation between phosphorus in soil and plant, with particular reference to glasshouse tomatoes, carnations, and winter lettuce the lack of a consistent chick growth response to norwegian kelp meal further studies on protein and energy requirements of chicks selected for high and low body weight some effects of kinetin on the growth and flowering of intact green plants individual feed consumption of turkey breeder hens and the correlation of feed intake, bocly weight, and egg production crop analysis technique for studying the food habits and preferences of chickens on range supplemental value of turkey protein for wheat herbicides and plant growth regulators preparation of purified ration for chick. parg iv. : preparation of crystalline amino acid diet evaluation of algae as a food for human diets the influence of milk fat depressing rations on the yield and composition of bovine milk the effect of plant nutrients and antagonistic microorganisms on the damping-off of cotton seedlings caused by rhizoetonia solani kukn ki;-;sehe ern~ihrung und di~tetlk clinical nutrition and dietetics a statement approved by the board of directors of the canadian heath foundation radioactivity and human diet probleme der ern~hrung durch gefrierkost. sympomum der d0utschen gcgell~ehaft ffir ernghrung veto 14. bis 15. mgrz klinische ernghrungslehre" und wissenschaftlicher kongreb der deutschen gesellschaft flit ern~hrung an der johannes-gutenberg-universit~t mainz veto 17. bis 19 wissenschaftlicher kongreb der deutsehen gesellschaft ftir ern~hrung an der johannes-gutenberg-universit~t mainz am 18. und 19 ernghrung nnd digit" 12. deutseher kongreb ffir ~irztliehe fortbildung in berlin veto 5. bis 9 arbeitstagung fiber klinisebe ernghrungslehre. ern~ foods of the future (forts.). problems in space foods and nutrition. foods for extended space travel and habitation the question of fats. ii.: fats and disease behandlung fettbedingter gerinnungsstgrungen mit lipostabil sugar and dental caries obesity and sugar addiction hunger and malnutrition lancet 2 nutrition and general practice bericht fiber die vortragstagung des fachverbandes lebensmittelchemie der chemischen gesellschaft in der ddr vom 19 arbeitstagung fiber kommission ffir volksern~hrung, lebensmittelgesetzgebung und -kontrolle (eek) zu yi~inden des eidg the national diet-heart study low fat diet in familial mediterranean fever the thyroid gland in infant malnutrition evaluation of fao amino acid reference pattern studies on the physiology of nutrition in surinam rickets in southern israel diet and heart disease maiskeims1 in ernt~hrung und ditltetik apha conference report safe and nutritious food supply malnutrition and disease expert committee on medical assessment of nutritional status protein malnutrition the fat tolerance curves of patients with hyperllpidcmia and athcrosclcrosis die lactose im rahmen der ernt~hrung effect of environment on nutritional status zur theorie und praxis der zuckerkrankheit. wiener z dietetically induced experimental flous of rats physikalisch-diiitetische therapie yon hautkrankheiten. arch. phys. therapie 15 err~hrungsforschung 7 [1962/63] :nr. 4, s. 598/ 612. +bo rtiw~l, p. w. : milk-borne disease consumers' reactions to instand foods de voeding van woonwagenbcwoners experimental investigations on nutrition and human behavior. a post-script. amer di~tetische therapie der chronischen herzinsuffizienz construction and validation of the food attitude scale why we have a safe and wholesome food supply use of food in a psychiatric setting stature and nutrition in cystinuria and hartnup disease ])as endokrinologisehe syndrom des proteinmangels urinary excretion of 3.4-dlhydroxyphenylalanine (dopa) in two children of short stature with malnutrition current problems affecting consumption of milk and indnstry's response to them preparedness for emergency feeding fluoridation and public relations dieetprodukten in vlaanderen incorporation of labelled glycine into erythrocyto glutathione of rabbits; effect of nutritional muscular dystrophy hot wcreldvoedselvraagstuk sniker -glycogcen -tandbederf dietary in take in patients with arthritis and other chronic diseases a clinical trial of iron-fortified bread effects of freater intake of milk, fruits, and vegetables joint fao/w/to expert committee on nutrition" fiber eine sitzung in genf vom 18. his 25 serum cholesterol in a military population. its relation to obesity and the military diet ). ~z~, a. c. 9 some nutritional problems of older age groups neuere biochemisehe untersuchungen zur diagnostik und therapie yon b-vitamin-mangelzust~nden call-harvard nutrition project. iii.: the erythroid atrophy of severe protein deficiency in monkeys cali-harvard nutrition projeet. ii. : the erythroid a~rophy of kwashiorkor and marasmus zur hshe des erwiinsehten fottverbrauehs ern~hrung des sportlers voeding 24 moderne ern~hrungsbedarfsnormen. i. mitt. z. ges. hyg. 9 [1963] nr. 1, s. 11122. *--~ioderne ern~hrungsbedarfsnormen. 2. mit~.: z. ges. hyg. 9 a comprehensive home-care program for the chronically ill fat-modified foods for serum cholesterol reduction besonderheiten der ern~hrung alter menschen chronic malnutrition in turkey. v. studies on serum fatty acids in malnourished children prevention of ,meat anemia" in mice by copper and calcium beeinflussung der sportlichen leistungsfdhigkeit dutch eine geeignete er-n~ihrung physiology of adolescence. ii, l~u-trition -basal oxygen consumption -energy expenditure and balance -nitrogen metabolism -calcium metabolism -iron metabolism -red cell mass and hemoglobin ern~hrungsproblemc bei chirurgisehcn kranken. wiss. versff. dr. ges. ern~hrung 11 moderne vitamin b~-therapie: oral, rektal oder parenteral ? a palatable diet for producing experimental folate deficiency in man smoking in hospital was ist hungern und was heibt the cultivation of tflapia. this prolific fish as a fine source of proteinrich food in underdeveloped areas pyridoxine supplementation during pregnancy. clinical and laboratory observations on japanese foods nutritional sequelae of ga~trio surgery koehsalzarme kost und nierenerkrankungen theoretische und praktische grundiagen der ernilhrung in der fett in der diabeteskost foods or supplements? g zur hygiene und ,di~tetik des rauehens zur dis behandlung der uremic anaphylactie shock of the lungs triggered by mieroaspiration of cows' milk: a form of sudden unexpected death in early infancy the food service industry and its relation to the control of foodborne illness die konservative therapie des peptischen gesehwiirs gezondheid op reis addictive aspeeta in heavy cigarette smoking die ern~hrung im rahmen de~ heilvers im kurort the diet in renal faiiure is the rationale for gaetrointestinal diet therapy sound? familie-beruf-ern~hrung die dfiitbehandiung der leberkrankheiten. i)t. reed vom hunger his zum ~beritul3-weltweite ern~hrungsprobleme die bedeutung der vitamine in der t~tglichen erniihrung s~ure-und baseniibersehiissige naln'ung. therapiewoehe 13 [1963] nr. 13, s. 563/565. --ss und chronische acidogene und alkalogene erns z. em~hrungswiss industrial lunches and public health the assessment of nutritional status in man: chairman's opening remarks therapie der essentiellen hypertonie speeifieke voedings-en voorlichtingsproblemen in tropische landen wie kann man unsere kos~ und unsere kostgewohnheiten beeinflussen? neue konzeptionen in der wasser-und salzsubstitution some aspects of the relation of nutrition and pregnancy is coronary heart disease preventable? world hunger demineralization of whey. use of its protein in infant feeding elaidinized olive oil and cholesterol atherosclerosis soziatrischo aspekte dee genul)-und arz~aeimittelkonsums verwendung yon htilsenfriichten in der diabetiker-di~t sanitation and dishes sanitation and dishes. aspects old and new. part ii smoking, arteriosclerosis, and age neue weg~ zur erni~hrungsphysiologi~chen aufwertung yon getreide-erzeugnissen diphyliobothrium latum and human nutrition, with particular reference to vitamin bli deficiency milk and diverticulosis dietary factors in the pathogenesis and treatment of cirrhosis of the liver. ivied. clinies north america 47 zur frage der quanti~tiven charakteristik der ern~hrung der berufst probleme der gemeinschaftsverpflegung aus der sieht des ern~hrungsphysio-logen gegevens over vitamine b,-deficientie, -behoefte en -voorziening use of government-donated foods in a rural community fluoride, teeth, and the analyst eiweil3bedarfsnormen im rahmen unserer ern~hrungsrichts~tze physiologic discomforts in 1962 navy protective shelter tests di~tvorschl~ige : akute nierenentziindtmg siii]waren und karies in theorie und praxis ernehrtmgsberatung im krankenhaus use of a low-sodium formula as an improved karell diet, with emphasis upon the outpatient management of heart failure and lymphedema pflanzliches eiweil~ fiir die erniihrung des menschen influence of diet on viral hepatitis influence of siblings on student smoking patterns voeding yon leerlingen van een lagere technische school. ii. calorie~n-en nutri~ntenwaarde early use of circulating blood volume, weights, and normal diet in acute renal failure fette in tier nahrung. dr. recd. wschr. 14 [1963] nr. 9, s. 247/250. *scm~-idt-b~bach, a.: ~j~ber die di~tetik der sauermilch begriff und anfgabe di~tetischer lebensmittel zur ursache yon geschmackskalamit~ten in trinkwassertalsperren psychologische motive im wandel des brotverzehrs improving levels of nutrition through better food practices the vitamin b~ deficiency syndrome in hun~n infancy. biochemical and clinical observations treatmen~ of ,refractory obesry" with ,formula die~ nr. 1, s. 66ff. (45 s.). 3elwzea, c. c.: morphologie constitution and smoking prediction the outcome for obese dieters symposium fiber probleme der ern~hrung durch gefrierkost in karlsruhe yore 18 coordination of long-term care of :pku children die di~t bei diabetes meuitus a nutritional supplement (nutrament) for elderly patients dietary intake of five groups of subjects. 24-hr. recall diets vs. dietary patterns the prolonged effects of a low cholesterol, high carbohydrate diet upon the serum lipids in diabetic patients stand und perspektiven der eiwei~versorgung. zur zielsetzung des verhandiungsthemas de voeding van rijswerkers signification des standards calorieo-azotes utilists en france erg~nzungen veto standpunkt des lebensmittelehemikers zu (dem beitrag) official acceptance of homogenized milk in the united states advances in nutrition and dietetics nutrition of 96 naval recruits during a shelter habitability study hot ombuigen van voedingsgewoonten de voeding van schippemkinderen san boord en in de internaten voeding 24 [1963] iqr. 5, s. 291/308. --le traitement de rinsuffisance rtnale her zoutarme-eiwitarme dicer ein beitrag zur allgemeinen el3problematik, ausgehend yon einer anorexia nervosa rroblem~ in nutrltlon~l supplementation an4 ~mri~hnl~ut detection of nutritional imbalances theorie und praxis der schwangerenern~hrung die pharmakologisehe beeinflussung yon hunger mad s~ttigung problems in the evalutaion of nutritional status in chronic illness europ~ische di~ttagung in amstercl~m (17. bls 19 entwieklung des brot-und gctreidevcrzehrs in der neuercn zeit nutrition and palatability coehac dis~tse. -biochemical and technological aspects die di~itetik der :fettsucht kinderemll}~mg nutrition of infants and children report on infant feeding childhood nutrition in lapland. :nutrition rev the thyroid gland in infant malnutrition. nutrition rev. 21 [1963] nr. 1, s. 32. +n. n.: physical activity of obese girls appraisal of nutritional adequacy of infant formulas used as cow milk substitutes isomaltose intolerance causing decreased ability to utilize dietary starch passagere hypereh]or~mische azidose bei zwei ausgetragenen s~uglingen wghrend s~uremflch-em~hrung ober erfahrungen in der frfihgeborenonaufzucht mit einer neuen bedarfsangepa6ten friilmahrung nutritional defects in adolescence g p 27 intravenous glucose tolerance in the normal newborn infant: the effects of a double dose of glucose and insulin ! attitudes towards physical ac. tivity, food, and family in obese and nonobese adolescent girls urinary excretion of 3.4-dihydroxyphenylalanine (dopa) in two children of short stature with malnutrition high salt content of western infant's diet: possible relationship ~o hypertension in the adult vitamin e to premature infants des enzym yon ~'leming (lysozym) und seine bedeutung flit die si~ugllngsern~ihrung. ann investigation on the relation of between-meal eating and dental caries of sixth-year molars in school children studies in infantile malnutrition. i.: nature of the problem in peru chronic malnutrition in turkey. v. studies on serum fatty acids in malnourished children emi~hrnng mad faekalc lysozymaktivitiit beim s~iugiing role of linoleio acid in infant nutrition factors related to the eating behavior and dietary adequacy of girls 12 to 14 years of age. dies. abstr. 23 des vitamin c im jugendalter. ii. mitt.: uber die wirkung yon natiirlichem und synthctisehemvitamin c bei l~ngeren zugaben the incidence of protein-calorie malnutrition of early childhood ein besonderer znsammenhang zwischen dem bedarf an nahrungsfett und dem stoffwechsel in den ersten lebensjahren the effect of supplements of groundnut flour or groundnut prorein isolate fortified with calcium salts and vitamins or of skim-milk powder on the digestibility coefficient, biological value, and net utilization of the proteins of poor indian diets given to undernourished children lysine fortifications of wheat bread fed to haitian school children lrber den 24-stunden-rhythmus der kalorienerzeugung bei friihgeborenen beitriige zur frage der spezifisch-dyrmmi~ehen wlrkung auf grund yon glykokoll-belastungen bei friihgeborenen. acta paediatriea acad carotine in tier stiuglingserni~hrung. wiss. versff. d$. ges. ernkhrung 9 liver and depot lipids in children on normal and high carbohydrate diets response of rural guatemalan indian children with hypocholesterolemia to increased crystalline cholesterol intake feeding value of soy milks for premature infants early feeding and birth difficulties in childhood schizophrenia. a brief study zusammenh~nge zwischen stoffwechsel und fl~issigkeitsbedarf beim s~ug-ling kuhmilchauergie beim s~ugling und ,cot death". die unspezifische kumulative sensibilisicrung malnutrition and the health of children practical aspects of infant feeding breast-feeding, weaning, ~nd acculturation appetithemmer in der ]~ehandlung der fettsucht bei kindern. miinchener med the effect of different amounts of vitamin d on growth and serum levels of calcium, inorganic phosphorus, and alkaline phosphatase in premature infants partition of urinary nitrogen in children with kwashiorkor treated with animal and vegetable proteins der einflud yon voukornbrot auf den caleiumstoffweehsel bei schulkindern ist eine rektale vitamin blz-behandlung vertretbar? dr ethyl alcohol in the pathogenesis of gout c|e~rance of infused fat emulsion in diabetic dogs praktische durehffihrung der parentcralon ern~hrung die parcntcrale ern~hrung chirurgischor paticntcn. wiss. ver6ff. dr. ges. em~hrung 11 verwertung intravenss verabfolgter aminos~,urengemische. wiss. versff. dr. ges die erkcnnung yon fe~-transportstsrungen und ihre bedeutung ftir die intra-ven6se fettzufuhr intravenous glucose tolerance in the normal newborn infant: the effect of a double dose of glucose and insulin new intravenous fat emulsion indikationen und kontraindikationen der intraven5sen fettzufuhr in der chirurgie anwendung intraven6s gegebener aminos~urengemische in dcr p~diatrie ymtravensse fettinfusionen. wiss. versff. dt. ges. ern~rung ~qotwendigkcit und erfolge der parenteralen mad sonder-ern~hrung moderne vitamin blz-therapie: oral, rektal oder p~renteral? mcd anwendung intravenss gegebener aminos~urengemische in der gyn~kologie und geburt~hilfe aminos~ureninfusionen. schweiz. reed. wsehr. 93 klinische anwendung mad erfahrungen bei der verabreiehung intravensser fettemulsionen an ehirurgischen patienten diskussionsbemerkung zum thema: die parenteralo ern~hrung ~tude exp~rimentale de la tolerance d'une solution de graisse vsgstale d'administration intraveineuse ern~hrungsphysiologische grundlagen der parenteralen ern~hrung erfahrungen mit der parenteralen ern~hrung mittels fettinfusionen. helvetica chirurgica aeta 30 nitrogen, lipid, glycogen, and deoxyribonucleic acid content of human liver. the effec~ of brief starvation and intravenous administration of glucose techn~ und indikationen der parenteralen ern~hrung des neugeborenen die praktische organisation der klinisehen infusionstherapie mit zuckerund elektrolytlt)sungen. l ~ed untersuchungen und bcobachtungen fiber intravensse fettinfusionen in der inneren klinik. wiss. versff. d$. ges. ern~hrung 11 l'alimentation parenttrale, 6mulsions lipidiques. (a suivre) ann intravcnsse ern~hrungstherapie mit fettemulsionen parenteral-und sondeneru~hrte patienten zur rekt~len kaliumsubstitufion parenteraie ern~hrtmg mit fettemulsionen konservierung mad zubereitung yon lebens-und futtermitteln nutritional hygiene, preservation and preparation of foodstuffs and feeds n. n.: vortragsveranstaltung ,fleischhygieno" der th seminar on the use of radioisotopes in nutrition science and of ionising radiation in food technology. strasbourg, 1st -6th october progress of food irradia~on work and programmes in o.e.c.d. member countries (16 berichte) safe heat processing of canned cured meats with regard to bacterial spores the role of food science i and technology on the freeze dehydration of foods public health aspects of handling animal products in the txopics fack)rs affecting bacfcrial spoilage of animal products at elevated temperatures. food technol sterilized concentrated millr. food teehnol. 17 [1963] nr. 6, s. 43/44, 49. n.n.: foods of the future. now opportunities for flavor modification unrestricted approval for irradiated bacon 3-a sanitary standards for multiple-use rubber and rubber-like materials used as product contact surfaces in dairy equipment 3-a sanitary standards for batch and continuous freezers for ice cream, ices, and similarly-frozen dairy foods bericht fiber die vortrag~tagung des fachverbandes lebensmittelchemio der chemischen gesellsch~ft in der ddr vom 19. bis 21 lebensmittelchem. u. geriehtl. chem. 17 fluoridation in great britain die enzymatische phycinspaltung in geschrotetem getreide in abh~ingigkeit yon der relativen lufffeuchtigkeit the effect of certain antioxidants during freezer storage of pork chops and sausage the mechanics of treating hatching eggs for disease prevention beitrag zur sfil~gerinnung yon kakaotrunk jodophore als desinfektionsmittel in der milchwirtschaft. milchwissenschaft 17 [1962] nr. 9, s. 513ff. (? s.). zitat: dr. lebensmittel tierarzneimittel und anfzuchtmittcl in der landwirtschaftllehen praxis. gesundheitliche erwrgungen znm sehutze des konsumenten bei der anwendung yon tierarzneimitteln und aufzuchtmitteln in der landwirtschaftlichen praxis n~ihrwertminderung dureh zubereitung dcr nahrung suue modifieazioni della flora mierobiea dei mollusohi eduli par effetto di eonservazione impropria verlinderungen des inhaltes yon dosenkonserven w~hrend lgngerer lagerung digtbrote aus der sieht ihrer praktischen gestmtung. wiss. versff. dr. ges. ern~hrung l0 erniihrungshygienische untersuchungen in kindergarten an budapester kindern im alter yon 1 bis 3 jahren. z. ges. hyg. 9 die eignung der bakteriologischen untersuehung yon kannenmilchproben als grundlage eines eutergesundheitsdienstes adequacy of cooking procedures for the destruction of salmonellae zur revitaminierung des mehles bzw. brotes. wiss. versff. dr. ges. er-n~hrung 10 conventionele verwarningsmethoden beitrag zur kenntnis der wechselwirkungen zwischen proteinen und poly. phenolcn der kakaobohnen wtihrend der fermentation nihydrazone feed medication ag~ins~ ar~iiieiaily induced escheriehia eoli air-sac infection foam-mat dried orange juice. i. time-temperature drying studies lebensmittelhygicnische probleme bei der herstellung yon gemeinschaftsverflpegung. 5. mitt. : z. ges. hyg. 9 lebensmittelhygienische probleme bci der herstellung yon gemeinschaftsverpflegung. 6. mitt.: z. ges. ttyg. 9 untersuchungen fiber die temperaturvorg~nge im innern yon lebensmittein w~hrend ihrer thermischen zubereitung, erl~uter~ am kochen yon kartoffelklsl~en. z. ges. hyg. 9 zur gewinnung yon niederverestertem pektin aus toehnisehen apfelpektinextrakten mit ammoniak: einflul] der entesterungsbedingungen auf das geliervermsgen hygienisohe beurteilung einer dutch clostridlum verursaehten massen. lebensmittelvergiftung [ungar neuartige teehnik der lebensmlttelverpaekung filr ge. schmaeks-aromastoffe und andere artikel bei ~berdruek studies with a natural source of xanthophylls for the pigmentation of egg yolks and skin of poultry die problematik der tuberkulosebeurteilung in der sehlachttier-und fleischuntersuchung freezing rate of beef as affected by moisture, fat, and wrapping materials ~ber mit komblnier~en konservierungsmitteln hergestellte konfitiiren und consumers' reactions to instant foods. food teclmol effect of supplementing lime-~reated corn with different levels of lysine, tryptophane, and isoleueine on the nitrogen retention of young children effect of freezing on autoxidation of oxymyoglobin solutions the control of gloecsporium album rot of stored apples by orchard sprays which reduce sporulation of wood infections bakteriologische befunde bei der spelseeisuntersuchung im sommer the microbiology of vacuum packed sliced bacon the stability of canned foods in long4erm storage the effect of proofing and baling on concentrations of organic acids, carbonyl compounds, and alcohols in bread doughs prepared from pre-ferments nutrients in raw vs. cooked globe artichokes effect of gamma-radiation, chemical, and packaging treatments on refrigerated life of strawberries and sweet cherries. food teehnol beeinflussung der wirkung yon kaffeeinhaltsstoffen dutch be-s~immte behandiungsverfahren der l~hbohne. (eine tierexperimentell-toxikologische studie influence of surface pasteurization and ehlortetraeycline on bacterial incidence on fryers the hydrolysis of grass hemicelluloses during ensilage post-harvest storage studies with selected fruits the science of food technology in venezuela beitrag zur aufbewahrung von sti~rkesirup in verzinkten ge-f~ben inactivation-rate studies on a radiation.resistant spoilage microorganism. ii. : thermal inactivation rates in beef the oceurrence and growth of staphylococci on packed bacon, with special reference to staphylococcus aureus zur verhiitung yon lebensmittelinfektionen in grobklichenanlagen dutch desinfektionsmabnahmen. ~rztl the influence of selected bacteria upon the flavor of a precooked frozen poultry product flour maturing and bleaching with aeyclie acetone peroxides effect of processing conditions on dry-heat expansion of bulgar wheat zur vcrwendung von pentachlornitrobenzol bei der lagerung yon kohl. dr. lebensmittel-rdsch. 59 [1963] nr. 1, s. 14115 los mati~res f6cales des pores et les selles des ouvriers d'aba~toir constituent une source permanente de diss6mination des salmonella studies on cooking fats and oils aspect sanitaire et l~gal aetuel des aliments conserv&s. rev. d'hyg over de betekenis van postduiven als besmettingsbron van levensmiddelen met salmonella-kiemen. ti]dschr. v. diergeneesk 87 over bet voorkomen van salmonella-kiemen bij slagerijen. tijdschr. v. diergeneesk 88 ursache und entstehung yon brotfehlern les salmonella des oeufs et ovoproduite frangais eg 6trangers the microstructure of baked products and doughs. food technol tomsto powder by foam-mat drying zitat: dt. lebensmittel-rdsch. 59 [1963] nr. 4, s. 123. --die verwendung yon gefrosteter saline zur butterherstellung. teil iv. die ergebnisse der in d~nemark, frankreich und in den l~iederlanden durchgefiihrten praktischen versuche und ihre bedeutung ffir die in der deutschen molkereiwirtschaft erfolgendo verwendung yon gefrosteter sahne bei der butterherstellung tenderne~ of the turkey meat as influenced by pre-cooling before proce~ing and hand masv~ging vortragsmaterialien flit die ern~hrungsproplldeutik. (erlruterungen zu insgesamt 6 groben sehautafeln.) 2. mitt. : behandlung der tafeln iv bis vi. ernahrungsforschung 7 die unterschiedliehe problematik und ihre konsequcnzen bei der bekrmplung der rinder-und schwefnefinnen salmonellae from flies in a mexican slaughterhouse factors affecting quality of pies prepared from frozen bulkpack red sour pitted cherries zur bedeutung antimikrobiellcr stoffc in der nahrung modified equipment for pasteurizing and deodorizing market milk and for pasteurizing, deodorizing, and slightly concentrating cheese milk adhesion of coatings on frozen fried chicken oob~age eheeae problems in production and sanitation. publle health aspects ~ber den einflub yon licht, 8auerstoff und tempera~ur auf die hal~barkoib yon verpaektem emmentaler ks in scheiben aktuelle notwendigkeiten -gesetzliche m6glichkeiten zur fischkfihlung in eis und seewasser techniques de recherche des salmonella dans les oeufs frais et de conserve food hygiene on board ship safety factors in water fluoridation based on toxicology of fluorides the effect of oiling before and after cleaning in maintaining the albumen condition of shell eggs lebensmi~t~l-aerosole. fette preservation of the natural color in processed sweetpotato products. i.: flakes. food technol temporary inhibition of fermentation in apple juice preservatives and artificial sweeteners the mechanism of the development of rancidity in frozen fresh pork sausage and practicable methods for its inhibition die entwicldung der trinkwasseriinoridierung in den usa microbiological principles in prcpaeking meats st~ndard-kapazit~tstest ffir die bestimmung der desinfektionswirkung yon desinfektionsmitteln in der milchwirtsehaft. internationaler standard fil/ii)f 18-1962 die anwendung einiger arteu, bzw. st~mme, yon propions~urebakterien zur herstellung bestimmter k~scsorten mit hohem vitamin b12-gehalt the extraction of pectins from apple marc preparations zur hygiene und ,di~tetik des rauchens studies on control of respiration of mcintosh apples by packaging methods. food teehnol effects of ingredients used in condermed and frozen dairy products on thermal resistance of potentially pathogenic staphylococci der frisehkllse und seine verpackung studies on the viscosity of mayonnaise. ii.: the influence of addition of vinegar on the vi~co~isy of mayonnaise e~'ec~ of chemical v~lditives on the spreading quality of butter. ii. laboratory and plant churnings studies on browning mechanisms of fruit juice products. i.: changes in chemical composition which accompany browning of commercial concentrated lemon juice during storage ergebnisse der dlg-qualit~tspriifung 1963 fiir speiseeis. dr. molkerei-ztg beeinttnssung versehiedenartig verpaekter lebensmittel dureh desinfektion mit formaldehyd grunds~itzliches zur stabilisierung und solubilisierung yon carotin und carotinoidpr~paraten riickst~nde yon pflanzensehutzmitteln, insektiziden und dergleichen in der nahrung und ihre bedeutung fiir die gesundheit absehliebende stellungnahme lebensmittel-rdsch langfristige nutritive anwendung yon antibiotilm in der tierern~hrung im hinbliek auf die menschliehe gesundheit mit besonderer beriieksiehtigung yon chiortetrazyklin nutritional studies on the utilization of distiller's stillage. part l: insolubles of me]lasses-butanol distiller's stillage auswertung der dlg-priifung fiir frischk~ise in verbraueherpaekungen zu den fermentativen eigensehaften der milchs~urebakterien (,laetobacillus meijerinek"), zugleieh ein beitrag zur vermeidung yon fehlfabrikaten bei roh-nnd briihwurst. arch. lebensmittel-hyg microbiological aspects of one-trip glass bottles as used by the carbonated beverage industry de bereiding van bouillon aktnelle milchhygienische aufgaben und zicle des organisation der ~berwachung der umweltradioaktivit~t unter besonderer beriieksichtigung der l~berwaehung des gehaltes yon lebensmitteln an radioaktiven stoffen. dr. lebensmittel bakteriologie der 8auermilcherzeugnisse l~ber die italtbarkeit yon lebensmittelkonserven preparation of aeid-modifid flour for tub sizing radiostrontium removal from milk. determination of apparent equilibrium constants of the exchange reactions of sodium, potassium, calcium, and magnesium wish amberlite ir-120 probleme der vitaminierung yon brot the effect of several operational variables on the rate of freeze-drying of beef studies on beef quality. x. effect of temperature, freezing, frozen s~orage, thawing, and p~ on the rate of hypoxanthine production. div. food preservation techn retardation of gelation in high temperature-short-time sterile milk concentrates with polyphosphates nonenzymatic bread browning and flavor. changes in amino acids and formation of earbonyl compounds during baking ttinweise fiir konservierende wirkungen synthetiseher senfslbildner naeh versuehen an fisehen neuo wege zur herstellung haltbarer fisch-pr~iserven behavior of ethylene dibromide, methyl bromide, and their mixtures. i. : in columns of grains and milled materials der einflui~ des wksserns auf die kartoffel irradiation of fruits and vegetables in india effect of storage in nitrogen on the soluble sugar and dry matter contents of ryegrass drying of seaweeds and other plants. v. throughcirculation drying of asophyllum nodosnm in a semi-continuous dryer niacin, thiamin, and riboflavin in fresh and cooked pale, soft, watery versus dark, firm, dry pork muscle nouvelles observations concernant la survie des salmonellae clans les fromages pyroearhonie acid diethyl ester as a potential food preservative the effect of phosphates on moisture absorption, retention, and cooking losses of broiler carcasses gur hygienisehen beur~ilung der trinkwasserverh~ltnisse des oberon vogtlandes -eine hydrobiologische s~udie. z. ges. hyg. 9 studies on preserving quality in market eggs rapid detection of faecal coliform bacteria in the food processing plant. j. milk food technol the relationship between the loss of water and carbon dioxide from eggs and the effect upon albumen quality plastic pacckaging of eggs. 2 study on improvement of digestibility of milk protein. i.: the effect of heating, adjustment of activity of calcium ion, addition of whey protein, homogenization, and elimination of coarse casein micelle from milk by ultracentrifuge on the digestibility of milk especially on the coagulability of it part iii.; the digebtibility of slightly hydrolized milk with proteinase and the preparation of rnill~ which has same eoagulability as human milk. g. agrie, chem. see study on improvement of digestibility of milk protein. part iv. : the nature of coagulation of casein of milk preparation which has same coagulability as human milk the influence of added microorganism on the quality of margarine. i. : the influence of mold inoculation die 8ilberung yon tafelw/~ssern. dr. lebermmittel-rdsch technological aspects of the radiation pasteurization of foods rapid hydration of dried fruits. food technol untersuchungen fiber polygalakturonase-enzyme aus sehimmelpilzen. 6. mitt.: eigenschaften der polygalakturonasen aus schimmclpilzen role of individual phospholipids as antioxidants association of veterinary food hygienists symposium on the marketing, transport, and slaughter of calves. iil: scientific aspects. ve~cr ricerche sulla resistenza della brucella abor~us helle salsicce. riv pr6senee des salmonelles dans les viandes. donn6es frangaises et 6tran-gbres biochemisehe vorg~nge inl fleisch bei der lagerung einflul3 des r6stgrades yon kaffee auf die extinktion w~briger extrakte und die menge der trockensubstanz die herstellung yon quark und weillk~e unter ansnfitzung ss eiweibstoffe der milch vcrluste yon vitamin b~ und c beim kochen und turmkoehen yon gemfise effect of chilled storage on the frozen storage life of whiting salmonellenfunde in einer importsendung amerikanischer tiefgefrierhiihner. arch. lebensmittel-hyg iron sulfide blackening in canned protein foods: oxidation and reduction mechanisms in relation to sulfur and iron raft research on food preservation by irradiation in poland no~: gas chromatography of chicken and turkey volatiles: the effect of temperature, oxygen, and type of tissue on composition of the volatile fraction l'inaetivation dens l'eau de meret l'eau d'alimentation de eertains entdrovirus de voedingswaarde van aardappelen van versehiuende re, sen en de invloed daarop van bemesting en bewaring effecb of l-arab]nose and d-xylose on dough fermentation and crust browning gelation of egg yolk corn carotenoids: effects of temperature and moisture on losses during storage salmonellenfunde in einer importsendung amerikanischer tiefgefriorhiihner. arch. lebensmittel-ttyg bacteriological examination of unbottled soft drink ~berbliek fiber kunststoff-folien und -kombinatlonen ale verpackungsmaterial in der mflchwirtschaft pigmentierung des eidottem bei gettiigel. wiss. versff. dr. ges. eruiilu'ung 9 s~ beltrag zur bedeutung wasserlsslieher hochmolekularer kohlenhydrate f'tir die verkleisterung der st~irke einfiul3 ehemischer verbindungen auf die antimikrobielle konservierungs-~toffwirkung. 1. mitt.: einflub verschiedener stoffgruppen auf die konservierungswirkung gegen aspergillus niger a quantitative s~udy of changes in dried skim-milk and lactose cnscin in the 'dry' state during storage the role of the major sugars of potatoes in ~he browning roa0tion during chipping probleme der zuverl~sigkeit yon kunststoffen zur lebensmittelverpackung in europi~ischer sicht bakteriologiseh-hygienisehe beur~ilung yon speiseeis weizenkeime ale wertvoller rohstoff-einige ~ragestellungen und probleme probleme der frischhaltung und haltbarmachung yon brot end backwaren the effect of selected polymers upon the albumen quality of eggs after storage for short periods preparation and quality evaluation of processed fruits and fruit products with sucrose and synthetic sweeteners the microflora within the tissue of fruits and vegetables changes in carbohydrate and phosphorus content of potato tubers during storage in nitrogen preparation of "natural" cow-milk fat globules; preliminary investigation of materials adsorbed at their surfaces lethal doses of gamma radiation of some fruit spoilage microorganisms alteration of post-mortem changes in porcine muscle by preslaughter heat treatment and diet modification ober die msglichkeiten end grenzen eines effects of polyphosphates on water uptake, moisture retention, and cooking loss in broilers flavors imparted to dairy products by phenol deriva aromatisehe crackproduktc yon sterinen. (i). z. ern~hrungs-wiss dose requirements for the radiation sterilization of food berichb fiber eine arbeitstagung bei der internationalen atomenergie-beh6rde in wien vom 12 zur bok~mpfung d6r rinderfinne zum einfiu]~ handelsfiblicher, in lebensmittelbetrieben gebr~uch-]icher desinfektionsmittel auf lactobakterien; zugleich ein beitrag zur desinfektion in der marinadenindustrie einflu~ chemischer umsetzungen bei trockenen lebensmittelgemischen in hinsich~ auf die lagerfestigkeit. vi. mitt. : lebensmittelgemische mit troekenmagermileh als hauptkomponente. z. lebensmittel-untersuchung u die technologie yon sauren milcherzeugnissen, insbesondere der sauermilcharten und sauerrahmarten effects of several edible coatings on poultry meat quality how to control insects in stored foods. part 2 die antibiotika und die ans ihrer anwendung fiir die ~iilehwirtsehaft sich ergebenden probleme zur haltbarkeitsverli~ngerung empfindlicher l~ahrungs-und genuflmittel dureh abpaeken unter vakuum. fette, seifcn the diffusion of hydrogen through tinplate containers packed with grapefruit juice effect of ice cream stabilizem on the freezing characteristics of various aqueous systems ist die infektion mit trichinen aus amtlich untersuchtem schweinefieisch im liehte der mathematisehen analyse der bestimmungen der fleischbcschau yon schweinefleisch msglieh? hygiene in milk production, processing, and distribution effect of pre-eooling eggs and cartons upon quality after storage a biological after-effect in radiation-processed chicken muscle accounting for farm tank milk factors related to the flavor stability during storage of foam-dried whole milk. iii. effect of antioxidants untersuchungen zur hygienischen beurteihing yon ~ietallverunreinlgungen in lvben~mitteln the effect of bleed time prior to scald and refrigerated storage upon bacterial counts in the axillary diverticula of the interclavicular air sac of chickens techniques de recherche des salmonella darts les viandes the serotypes of salmonella isolated from foods carotinverluste beider zubereitung der nahrung. wiss. vcrsff. dr. gcs. er-n~hrung 9 stability of ascorbie acid in a liquid multivitamin emulsion containing sodium fluoride the effect of storage time and holding temperature on egg interior quality in uganda uber den einflul3 versehiedener fangverfahren auf die qualit~t und lagerreserve der fische zerst~ubungstrocknung yon tomatenkonzentraten. dr. lebensmittel radiation pasteurization of fresh fruits and vegetables a bacteriological survey of certain processed meat~. part l population studies at packet and retail levels association of veterinary food hygienists symposium on the marketing, transport, and slaughter of calves. l : marketing and slaughter die kombinierte verarbeitung yon kartoffeln auf st~rke und alkohol (fortschrittsbericht) usaec program in radiation research preservation of certain fish and fruits biochemical and quality changes in chicken meat during storage at above-freezing temperatures inleidend onderzoek naar strnctuurveranderingen die ontstaan bi] verhitten van plantaardige produkten veranderingen van hot vetgehalte bij de bereiding van vlces a study on the relationship between the factors influencing the time of cheese salting maple sirup. xxi. : the effec~ of temperature and formaldehyde on the growth of pseudomonas geniculata in maple sap vacuum-tempering corn for dry. milling organoleptische eigenschappen, thiamine-en ascorbinezuurgehalte van enige week-en diepvriesgroenten growth of psyehrophiles. il : growth of poultry meat spoilage bacteria and some effects of chlortctracycline tests of corn stored four years in a commercial bin association of veterinary food hygienists symposium on the marketing, transport and slaughter of calves. ii. : the slaughter and inspection of calves. veter the effect of processing conditions upon the nutritional quality of vegetable oils berieht fiber den wisscnsehaftlichen kongrel3 1963 der dcutschen gesellschaft ftir ern~hrung (5 vortragsrcferate) an objective measurement of the freshness of ready-to-cook broilers the fieldman's responsibilities in milk quality and procurement studies on the bacteria found in the wine during its making. i.: multiplication of bacteria in wine and male-lactic fermentation hydrophilic colloids as additives in white layer cakes the acceptability of cooked poultry protected by an edible acetylated monoglyeeride coating during fresh and frozen storage istes zu vertreten, dal3 das fleiseh sehwachfinniger rinder auch in gebriitetem zust~nd eingcfrorcn wird? arch. lebensmittel-hyg =[efenlnfit.ierte kondensmilch als ursache yon fehlfabrikation bei schokolade. arch. lebensmittel.hyg. 14 [1963] nr. 1, s. 6110. m, ober die bedeutung aerober sporenbildner als bombageerreger yon wfimtehenkonscreen. arch. lebensmittel-hyg aluminiumfolio zur verpaekung tiefgekiihlter und gefriergetrockneter lebcnsmittel. fette fiber die milcldtuorierung. bull. schwciz. akad. reed. wiss vitamin stability in diets sterilized for germfree animal~ die trinkwasserversorgung ernliln-ungsstatistlk nutritional statistics african nutrition problems der vcrbrauch yon alkoholisehen getr~,nken in 0sterreich childhood nutrition in lapland overweight children in stockholm iron deficiency in the finnish population vitamin b12 deficiency in indian infants the indices of nutritional change in great britain dietary values from a 24 h recall compared to a 7-day survey on elderly people her vaststellen van de voedingstoestand van sen bevolldng in de tropen en subtropen dental effects of fluoridation of water with particular reference to a study in the united kingdom de voedlng van woonwagenbewoncrs nutritional attitudes of some london housewives de vocdingsgewoonten van bcjaarden in amsterdam community studies of drinking behavior fats and carbohydrates as factors on atherosclerosis and diabetes in yemenite jews nutritional beliefs among a low-income urban population algemene gezondimidsaspccten van de vocding in de ontwikkelingsgcbieden a comparative study of the nutritional adequacy of the morning intake of women clerical workers and women factory workers serum cholesterol in a military population. its relation to obesity and the military diet nutrient intakes of healthy older women analysis of the structures of food consumption by groups in japan thiamin (vitamin b,)-untercru~hrung in deutschland? lvied vortragsmaterialien ftir die ern~hrungsprops (erli~uterungen zu insgesamt 6 groben sehautafeln.) 2. mitt.: behandlung der tafein iv bis vi. erns 7 [1962163] nr. 4, s. 6131634 zur ern~ihrungssituation in arbeitexfamilien aus verschiedcnen bezirken der ddr. 2. mitt. : ern~hrungssoziologischc answertung der lebensmittelverzehrungen in 6 itaushaltungen mit 2 erwachscnen und 2 kindern~ stand 1950 studies in infantile malnutrition. i. : nature of the problem in peru zur ~ethodik yon ern~ihrungserhebungen bei der gemeinschaftsverpflegung weight changes in relation to birthweight of papuan, indonesian, and chinese children during the first two weeks of life numbers of tasters required to determine consumer preferences for fruit drinks onderzoek naar de menupatronen in de noordoostpolder smoking habits of medical and non-medical university staff i**cs and potassium in people and diet. -a study of finnish lapps. ann. acad. scientiarum fennicae a ~berbliek fiber die radioaktivit~t der in 0sterreich im jahre 1961 konsumierten lebensmittel diet and plasma cholesterol in 99 bank men der mengenmi~bige getr~inkeverbrauch je einwohner im bundesgebiet predictors of human food consumption use of goverument.donated foods in a rural community bericht fiber die durum-und teigwarentagung der arbcitsgemeinschaft der oetreldeforschung e. v. vorn 12. bis 13. miirz voeding van leerlingen van con lagere teehni~eho school. ii. calorie~in-en nutriiintenwaarde nutritional deficiencies in developing countries dietary survey in surinam vitamine a-tekor~en op de aarde schwierigkeiten beim erreichen einer vollwertigcn ern~ihrung in ausgew~hl-ten vcrbrauchergruppen die entwicklung des brot-und getreideverzehrs in der neueren zeit. wiss. ver6ff. dr. ges. ern~hrung 10 n~rwert und zusammensetzung yon lebens. und futtermltteln nutritive values, composition of food~tuffe and f 9 physical chemistry of ice cream vitamin e in human nutrition appraisal of nutritional adequacy of infant formulas used as cow milk substitutes enkele gegevens betreffende de calorisehe waarde van klsine hepjes, gerechtcn en maaltijden the public health aspects of the use of antibiotics in food and foodstuffs. report of an expert committee niihrwertminderung dutch zubereitung der nahrung metals and other elements in foods die farbe der nahrungsmittel in anthropologischer sicht the enzymatic destruction of carotene and carotenoids foodstuff flavors. some factors affecting the flavor of sodium caseinate chemical and radiochemical composition of the rongelapese diet the organic constituents of food. i. : lettuce the influence of dehydration of foods on the digestibility and the biological value of the protein a stfidy of two methods of assessing vitamin b6 nutriture mincraisalze mad spurenelemente in der nahrung distribution of the bound form of nicotinic ac|d in natural material~ the distribution of c~rotenoids in nature and their biological significance zur bedeutung antimikrobieller stoffe in der mahrung die konsistenz yon margarine mad fetten an improved nutrient solution for diploid chinese hamster and human cell lines extraneous materials in foods and drugs the composition of food flavors studies on pantothenic acid intake. i. pantothenic acid content in japanese foods haben wir mangel an essentiellen fettsiiurcn? phonolcarbons~uren in menschlichen nahrungsprodukten. zum vorkommen yon phenolcarbons~uren in menschlichen nahrungsprodukten und ihr einttud auf den intermedi~ren sboffwechscl drugs in feeds relationship between the sulphur/nitrogen ratio and the protein value of diets gums in foods zur definition der begriffe ,aroma" und begriff und aufgabe dii~tetischer lebensmittel valettr vitaminique des carot6nes pour rhomme. wiss. versff. dr. ges. ern~hrung 9 internationales rundgespriich fiber lebensmittelchemische probleme in wiesbaden und eltville a. rh. (4 vortragsreferate) consumer awareness of texture and other food attributes organisehe und organisierte substanz in der lebensmittelchemie frost resistivity of fruit plants californ'ia association of chemistry teachers : inorganic nutrients in the sea fluoride in food antibiotics in feeds and other products planktorm as foods relation between color of cranberries and color and stability of sauce berieht fiber die vortragstagung des fachverbandes lebensmittelchemie der chemischen gesellsehaf$ in der ddr vom 19. bis 21 bulletin on tobacco evaluation of algae as a food for human diet~ digestibility of high-amylose corn starch. nutrition red. 21 [1963] nr. 1, s. 27/28. 1~. n. : comparative evaluation of corn mesa and steam-processed whole corn flours the major anthocyanin pigments of vitis vinifera varieties flame tokay, emperor, and red ~alaga peonidin-3-monoglucoside in vinifera grapes formation and distribution of amylosc and amylopectin in the starch granule nutrient in seeds. amino composition of some seeds sugar levels in fruits of the lowbush blueberry estimated at four physiological ages the relation of pectic substances to firmness of processed sweet potatoes (ipomoea berates) processed vegetable produc~s the protein composition of different flours and its relationship to nitrogen content and baking performance relationship between 'antitryptic factors' of some plant protein feeds and products of proteolysis precipitable by trichloroacetic acid. g. sci. food agric a thermostable haemolytic factor in soybeans foam-mat dried orange juice. i. time-temperature drying studies bound" growth inhibitor in raw soybean meal leaf analysis as a guide to the nutrition of fruit crops. ii. : distribution of total n, p, k, ca and mg in the laminae and petioles of raspberry (rubns idaeus l.) as influenced by soil treatments nutritive value of pumpkin seed. essential amino acid content and protein value of pumpkin seed (cueur bi~a farinoaa) effect of cooking and of amino acid supplementation on the nutritive value of black beans (phaseolns vulgaris l.) supplementation of cereal proteins with amino acids. iv.: lysine supplementation of wheat flour fed to young children at different levels of protein intake in the presence and absence of other amino acids uber den chemischen total ascorbic acid in potatoes. raw, fresh, mashed, and reeonstituted flakes moisture contents of hard red winter wheat as determined by meters and by oven drying, and influence of small differences in moisture content upon subsequent deterioration of the grain in storage rheological studies with canned tomato juice the chemical composition of maple sugar sand soluble carbohydrate content of varieties of te~raploid ryegrass natiirlicher gehalt und stabilit~t yon carotine11 und carotinoiden in citrnss~ften ~ber wein und weinuntersuchungen fatty acids and other lipids in mayonnaise cyclic fatty acid yields from linseed oil factors ~ffecting enzymatic solubilization of beef proteins weibulls original ring, en ny medeltidig varvetesort. (a new medium early variety of spring wheat, weibull's ring.) agri hortique genetiea 21 ober die askorbins~uresynthese in zerschnittene11 kartoffeln die bestimmung der amylaseaktivit~t und einige studicn fiber amylaseaktivit~t in gekeimtem roggen effect of processing conditions on dry-heat expansion of ~ulgar wheat oils, fats, and waxes ~.~ber das 01 der johannisbro~.kerne. fetto, seifen fruit and fruit products uber die variabili~it einiger eigcnschaften der kartoffelstilrke in abhiingigkcit yon witterung a short.term effect of weather on malie acid in pineapple fruit the specific surface of flour and starch granules in a hard winter wheat flour and in its five subsieve-size fractions oranges and lemons factors affecting quality of pies prepared from frozen bulk-pack red sour pitted cherries studies on the consish~ncy of thiamin and protein contents of pure.bred strains of rice a comparison of the nutritional value of protein from several soybean frac4ions zum vitamin-und aminos~uregehal~ yon maisquellwasser dark discoloration of canned all-green asparagus. i. chemistry and related factors enzymatic enhancement of flavor peetinestcrase in normal and abnormal tomato fruit storage effects on winter squashes. varietal differences and storage changes in the ascorbie acid content of six varieties of winter squashes grape pigments. concord grape pigments corn meal as a source of ribonuclease banana odor components. volatile components of bananas. part i. isolation of an odor concentrate. part il separation and identification lipids of algae. ill. : the components of unsaponifable matter of the algae chlore]la volatile esters of bartlett pear. ii studies on the nutritive value of raw and cooked soybeans for growing rats and swine and their effect on fat firmness characterization of fruit juices by acid profiles nitrate content of beets, collards, turnip greens lysine fortifications of wheat bread fed to haitian school children studies on the flavor of green tea. par~ iv. : dimethyl sulfide and its preeursor funktionelle eigenschaften yon lehens-mittclst~rken ~)ber das vorkommen yon xylit im speisepi]z champignon ein beitrag zur znsammensetzung yon apfeisinens~ften aus spanischcn und l~iarokko-apfelsinen an examination of the free amino acids of the common onion (allium cepa) a trypsin inhibitor in wheat flour the protein quality, digestibility, and composition of algae, chlorella 71105 chemical and color changes in canned apple sauce digestibility of the a-cellulose and pentosan components of the cellulosic mieelle of rescue and alfalfa safeness and serva. bility of meringued pie alcoholic beverages diurnal-nocturnal changes in the starch of tobacco leaves sugar and sugar products modification of flour proteins by dough mixing: effects of suffhydryl-blocking and oxidizing agents ~ber das haferprotein. z. lebensmittel-untersuchung u sodium and potassium in wines and distilled spirits non-volatile organic acids of the dwarf cavendish (chinese) variety of banana~ biological evaluation of soybean meal and cottonseed meal by amino acid digestibility and protein efficiency ratio studies. oiss. abstr. 23 [1963] l~r oxidation-reduction potentials of sak~f and synthetic sak6. xi.: on the relationship of the various fermenting processes of sak~ to oxidereduction potentials and indicator time test (i.t.t.) values of sak6 mash neue wege zur ern~hrungsphysiologischen aufwcrtung von getreideerzeugnissen cereal products ascorbie acid in dehydrated po~es die eiweibqualit~t yon ,getoastetem" (dampferhitztem) und ungetoastetem sojaextraktionssehrot color studies on processed dried fruits studies on the basic amino acid of the soy sauces and the seasoning liquids. ii.: the quantitative changes of l-arginine in the process of soy sauces brewing studies on the flavorons substances in soy sauce. xxii. : the differences between the soy sauce made from soy bean and wheat and that made from defatted soy bean and wheat feeding value of soy milks for premature infants flavors and non-alcoholic beverages fat content and fatty acids in some commercial mixes for baked products sensory examination of four organic acids added f~ wine 13ber die inhaltsstoffe der robktmtanie und versuehe zu ihrcr gehaltsbestimmung. diss. univ. hamburg, 3.2 malt beverages, sirups, extracts, and brewing materials vitamin b 6 and niacin in potatoes. retention after storage and cooking chemical investigation of some wild indian legumes zusatzstoffe der margarine. forte componen~ glyeerides of an indian fresh-water fish fat einflub des rsstgrades yon kaffce auf die extinktion w~$riger extrakte und die 1v[enge der trockensubstanz gaschromatographische untersuehungen yon fusclslen aus vcrsehiedchen g~irprodukten. 1. mitt.: problemsteilung und literaturfibemieht de voedingswaarde van aardappelen van versehillende rassen en de invloed daarop van bcmesting en bewaring eleetrophoretic separation of beet pigments studies on the growth-promoting value and digestibility of passion fruit seed oil polycyclisehe und aliphatisehe kohlenwasserstoffe dc~ tabakrauehes carotenoid, oil, and tocopherol content of corn inbreds location and possible role of esterified phosphorus in starch fractions untersuchungen fiber die chemischen u beim a]tern yon r6stkaffee. 1. mitt.: gaschromatographische analyse der leichtflfichtigen aromabestandteile nature, origin, and prevention of hydrogen sulphide aroma in wines the magnesium contents of soil and crops versuehe zur ehemischen differenzierung der eiweibst~ffe des weizens und roggens lemon juice composition. iii.: characterization of california-arizona lemon juice by use of a multiple regression analysis weizenkeime als wertvoller rohstoff -einige fragestellungen und probleme isolation of gram quantities of a rhamnoglucoside of apigenin from grapefruit determination of distribution of water in wheat grains by interference microscopy preparation and quality evaluation of processed fruits and fruit products with sucrose and synthetic sweeteners changes in carbohydrate and phosphorus content of potato tubers during storage in nitrogen chemical composition of some natural and processed orange juices effects of various factors in the candy test zum stand der kenntnlsse fiber die v{eclmelwir-kung zwischen nativer sts und wasser some volatile compounds from cooked potatoes firmness of canned apple slices as affected by maturity and steam-blanch temperature nutritive values of ten samples of western canadian grains proteins of wheat and flour. the separation and purification of the pyrophosphate-soluble proteins of wheat flour by chromatography on dear-cellulose changes in quality and composition produced in wine by s~ gamma irradiation citrus essential oils. iii. evaluation of silician natural lemon oils mineral analysis of plant tissues a. : nature of colloids in clarified cane juices studies on amino acid content of rice. i. : amino acid composition of polished rice glutelln estimated by beckman amino acid analyzer yliichtige carbonylverbindungen in honig neue aminos~iuren in h6heren pflanzen studium der wirkung der gammastrahlung auf die l-ascorbins~iure flour liplds and oxidation of sulfhydryl groups in dough zur kenntnls eincr weiteren in der sti~rke vorkommenden kohlenhydrat-komponente. ern~ihrungsforschung 8 lemon juice composition. ii. : characterization of california.arizona lemon juice by its polyphenolic content lemon juice composition. i.: characterization of california-arizona lemon juice by its total amino acid and 1-malic acid content safflower amino acids amino acid composition of safflower kernels, kernel protein, and hulls, and solubility of kernel nitrogen citrus fruit enzymes. i. : ascorbie acid oxidase in oranges untersuehungen fiber die verf~rbung gekochter kartoffeln an den sorten des kulturlrartoffelsortiments ides instituts ffir pflanzenziiehtung groi~-liisewitz nutritive value of red kidney beans (phaseolns vulgaris) for chicks instability in potable spirits. ii.: rum and brandy effect of size classification and maturity on the protein content of alaska and perfection peas ma~tr~, d. c.: ascorbie acid retention and color of strawberries as related to low-level irradiation and storage time historical aoac data on four fema samples of vanilla extract the acid-extracted pentosan content of wheat as a measure of milling quality of pacific northwest wheats petroleum ether extraetables in tobacco isolation, origin, and synthesis of a bread flavor constituent the protein composition of airclassifled flour fractions * studies on the flavor of green tea. v. : examination of the essential oil of the tea-leaves by gas lipid chromatography nutritive value of starches. iv.; comparison of digestibility of 15 natural starches estimated by a new procedure 1o c lebensmittel tierischen ursprungs _foodstu~$ o/animal origin n.n.: gdch-faehgruppe ,lebensmib~el. und gerichtliche chemie bericht fiber die vortragstagung des fachverbandes lebensmittelchemie der chemischen gesellsehaft in der ddr yore 19. bis 21 a taxometric study of the propionic acid bacteria of dairy origin comparisons of the caseins of buffalo's and cow's milk matiirlicher gehalt und stabilit~t der carotinoide in fetten und milchprodukten. wiss. versff. dr. ges. ernhhrung 9 antibiotics in milk vitamin a and d enrichment of nonfat dry milk some characteristics of yolk solids affecting their performance in cake doughnuts. i. effects of yolk type, level, and contamination with white proteine des eidotters post-mortem changes in the muscles oflandrace pigs t~ber den fettgehalt yon flcischkonserven. dr. lebensmittel der einflul~ der verfahrenstechnik auf die ks163 jahreszeitliche einfliisse alff den gehalt der fischmuskulatur an freien ami-nos~iuren u. deren bedeutung fiir die qualitiit fangtechnik und fisch-qualit~t. fette spcei~c distribution of fatty acids in marine lipids a comparison of pigs slaughtered at three diffcrcn~ weights. i. : carcass quality and performance a comparison of pigs slaughtered at three different~ weights. ii. : association between dissection results, various measuremen~ and visual assessments a note on the effect of heat on the colour of goat's milk chemical and nutritional changes in stored herring meal. 4.: nutritional significance of oxidation of the oil relation of pork muscle quality factors to zinc con~ent and other properties the chemical nature of the characteristic flavor of cultured buttermilk occurence of vaniuin in hea~ed milks the electrophoretie properties of the proteins in cottage cheese curd the influence of post-mortem glycolysis on poultry tenderness seasonal variations in cod liver oil isolation and characterization of the flavor components of rancid pork some problems in the evaluation of egg albumen quality quality evaluation studies of fish and shellfish from certain northern european waters indices for lamb carcass composition subjective and objective evaluations of prefabricated cuts of beef 0ils, fats, and waxes die bildung yon eiskristallen in diinnen milchschichten. milchwissensehaft 18 the incidence of bacteria in cheese milk and cheddar cheese and their association with flavour body composition of market weight pigs some factors affecting tenderness of turkey meat ovine bioenergetics and nutritional efficiency, with special reference go forage utilization die ziichtung yon fleisehsehweinen und die folgeerseheinungen, die sich insbesondere im hinbliek auf die qualit~t yon fleisch und fett ergeben. arch. lebensmitgel-ityg 0n the structure of highly unsaturated fatty acids of fish oils by high resolution nuclear magnetic resonance spectral analysis the fatty acid composition of the milk fat of cows grazing on ryegrass at two stages of maturity and the composition of the ryegrass hpids effect of intrauterine infusion of penicillin-streptomycin and furacin and vaginal deposition of furacin on chemical residues tevei~ in millr beziehungen zwisehen l-aseorbinsaure und milch comparison of chemical and organoleptic data obtained on thawed and unthawed frozen cod, haddock, and perch fillets die ~4m~uosfiurenzusammensetzung der ziegenmflch und des ziegenmileh-caseins food flavors and odors. meat flavor: lamb dairy products de ehemische samenstelling van visen visprodukten chemical studies on the herring (clupen harengus). vii.: collagen and cohesiveness in heat-processed herring and observations on a seasonal variation in collagen content growth and pro~eolycie activity of pseudomonas fluoresccns in eggs and egg products the fatty acid composition of some perirenal and subcutaneous beef depot fats inactivation of peroxidase in milk by homogenization a study of the "cured meat" color producing reaction and the effects of some curing adjuncts t~yoer den fettgchalt yon br'tihwiirsten stress effects, eareass composition, and carcass quality in lambs effect of chemical additives on the spreading quality of butter. ii. laboratory and plant ehurnings preliminary studies on protein and moisture relationship in fresh and proeessed hams die zusammensetzung des kuhmilchfettes in abh~ngig-keit yon der ffitterung. fette chemical characterization of off-flavors in concentrated and nonfat dry milk zur ausseheidung yon xanthindehydrase mid molybdiin in der kuhmilch copper distribution in milk during early lactation die physikaliseh-ehemischen ursaehen der hitzestabilitet yon mileheiweil]stoffen. milehwissenscha sobn~a: ~oer einige verenderungen in den caseinfraktionen normaler und anomaler milch (colostralmilch und milch an lviastitis erkrankter kiihe). milchwissensehaft 18 some characteristics of yolk solids affecting their performance in cake doughnuts. ii. variability in commercial yolk solids zum problem des znsammenhanges zwisehen der konsistenz und der physikalischen struktur der butter. fette relationships between milk fat acidity, short-chain fatty acids, and rancid flavors in milk induced and natural inhibitory behavior of milk and significance to antibiotics disc assay testing. j. dairy sei. 46 [1963] nr. 2, s. 95ff. (7 s.). --some distribution patterns of cottage cheese particles and conditions contributing to curd shattering natural inhibitory eharacteristies of some irish manufacturing milks thermophylie aetinomycetes in milk and dairy products. mikrobiologiya the free fatty acids of purdue swiss-type eheese die zusammensetzung yon siilzen lind ihre beurteilung im 1regierungsbezirk diisseldorf not~ on tyrosine production in frozen stored liver studies on the muscles of meat animals. hi. : comparative composition of various muscles in pigs of the three weight groups studies on beef quality. x. effect of temperature, freezing, frozen storage, thawing, and pe on the rate of hypoxanthine production. die. food preservation t~chn increased iodine in milk as a countermeasure for ~liodine time-temperature studies of baked, loaves. meat, fish, and poultry vergleichende untersuehungon an butter und einem butter~hnli-chen umgeesterten fort. forte, seifen, anstrichmittel 65 zur beziehung zwisehen fettgehalt und wassergehalt bei ungewa~ohener s/il~rahmbutter (fritzbutter) variation of ovine fat composition within the carcass studies on the properties of new zealand butterfat. vii. effect of the stage of maturity of ryegrass fed to cows on the characteristics of butterfat and its carotene and vitamin a contents studies on turkey body composition. 2. poultry sci thermal conductivity of beef studies on turkey body composition. 1 free fatty acid, tyrosine, and 1~ changes during ripening of blue cheese made from variously treated milks l : factors influencing the nutritional value of fish flour. il: availability of lysine and sulphur amino acids. canad characterization of flavor compounds isolated from evaporated milk effect of egg yolk size on yolk cholesterol concentration ~tudo immuno~lectrophor~tiquo du lair dans los divers types de mammites. i. milchwissenschaft 17 rdsultats hemmstoffe in der anlieferungsmflch und methoden zu ihrem nachweia eiweibverenderungen gefriergetroekneter muskulatur meat and meat products effect of high doses of vitamin a palmitate on vitamin a aldehyde, esters, and alcohol and carotenoid contents of hen's eggs. brig. j. nutrition 17 [1963] nr. 2, s. 235/242. --the amounts of vitamin a aldehyde, esters, and alcohol and of earotenoids in hen's eggs and in day-old chicks nutritive value of marine oils i. : ]~lenhaden oil at varying oxidation levels, with and without antioxidants in rat diets a quantitative study of changes in dried skim-milk and lactose-casein in the 'dry' state during storage physico chemical characteristics of canadian milk fat. unsaturated fatty acids collagen content and its relation t~ tenderness of connective tissue in two beef muscles ~ber eine braune verf~rbung yon mariniertem hering. dr. lebensmitbel versuche fiber den sonnenlichtgesehmaek in mit ass markierter milch effect of unequal milking intervals on lactation milk, milk fat, and total solids production of cows wei~re untersuehungen fiber die nitratreduktase verschiedener an der reifung yon rohwurst beteiligter mlkroorganismen nutritive value of leg of lamb roasts. l~oisture, energy, protein, fat, and iodine values ~ber einige gul~sigkai~s~renzen bei konsistenzfehiern yon butter influence of linoleic acid content of milk lipids on oxidation of milk and milk fat fish hydrolysates-iii.: influence of degree of hydrolysis on nutritive value einige beobaehtungen fiber die bildung des durch lieht verursaehten oxydationsgesehmackes s.). zitat: dt. lebensmittel comminuted meat emulsions: factors affecting meat proteins as emulsion stabilizers factors related to the flavor stability during storage of foam-dried whole milk. iil effect of antioxidants upgrading the indigenous poultry of uganda. hi. : shell and egg interior quality relation between carcass composition and live weight of sheep diethylstilbestrol occurence in eggs of subcutaneously injected hens biochemical and quality changes in chicken meat during storage a~ above-freezing temperatures investigations on the allerged goitrogenie properties of milk fish and other marine products bioehemieal properties of pork muscle in relation to curing potassium content of dried milk vergleiehende histometrische untersuehungen fiber den kollagengehalt yon brfihwfirsten a comparison of the volatile compounds of fresh and decomposed cream by gas chromatography studies on the volatile carbonyl compounds in ladino clover and their influence on the flavor of milk aut~xidation of fish oils. ii.: changes in the carbonyl distribution of autoxidizing salmon oils the public health aspects of the use of antibiotics in food and feedstuffs. repor~ of an expert committee composition and digestibility of corn silage as affected by fertilizer rate and plant population factors influencing the nitrate content of forage the component sugars and rate of hydrolysis of forage hemicelhflose as related to digestibility digestibility trials on forages in trinidad and their use in the prediction of nutritive value acetyl-(para-nitrophenyl)-sulfanilamide in feeds distribution of major and trace elements in some common pasture species 0-dimcthyl 0-(2,4,5-trichlorophcnyl)phosphorothioate) in feeds feeding value of low-moisture alfalfa silage from conventional silos ovine bioenergeties and nutritional efficiency, with special reference to forage utilization a system for naming and describing feeds, energy terminology, and the use of such information in calculating diets supplemental methionine in a sixteen percent protein diet for laying chickens l : organic arsenicals in feeds cellulose degradation by enzymes added to ensiled forages influence of corn distillers dried grains with sohbles on the feeding value of wheat silage zinc content of certain feeds, associated materials, and water der natiirliche gehalt und die stabilit~it yon carotin und carotinoiden in lieu und silage forage digestibility. benzene-ethanol extracts of forage and faeces as indicators of digestibility the prediction of the metabolizable energy content of poultry fcedingstuffs from a knowledge of their chemical composition factors affecting the metabolizable energy content of poultry feeds. 11 facters affeeting the metsbohzablo energy content of poultry feeds unidentified chick growth factor in fish solubles diet and histamine in the ruminant. occurrence of histamine in silage ethopabate in feeds die isolierung yon apocarotinalen aus luzernemehl isoflavone contents of red and subterranean clovers metabolizable energy of some oil seed meals and some unusual feedstuffs ii methoden der untersuchung yon lebens-und futtermitteln techniques analysis of foodstuffs and feeds n.n.: gdch-faehgruppe analysis of foods by neutronaetivation techniques continuous measurement of dissolved solids in food processes by critical-angle refractometry berieht fiber die vortragstagung des fachverbandes lebensmittelchemie der chemischen gesellschaft in der ddr vom 19. bis 21 a rapid test for anionic detergents in drinking water fortschritte in der lebensmittelehemio dureh moderne analysenmethoden direct potcntiometrio determination of chloride in cheese modification of the polarimetrie starch determination on hig-hamyloso corn ~iethoden der ]~berwachung des wassers auf radioaktiviuit. btmdes prediction of quality in protein concentrates by laboratory procedures involving determination of soluble nitrogen direct chromatographic analysis of milk die eignung der bakteriologischen untersuehung yon kannenmilchproben als grundlage eines eu~ergesundheitsdienstes. arch. lebensmittel-i~yg nachweis fremder carotinoide in 0rangensiiften mittels diinnschichtchromatographie. i)t. lebensmit~el-l~dseh determination of phosphate composition of stock food calcium phosphate. 5. assoe. off. agric. chemists 46 molybdenum in plants and animals. determination of molybdenum in biological materims with dithiol control of copper interference the determination of dissolved oxygen in canned drinks using a vibrating mercury-plated platinum electrode determination of chromium and lead in periodic acid solution and dialdehyde starch the utilization of infrared and ultraviolet spectrometric procedures for assay of pesticide residues ultramicro determination of potassium and sodium in biologic fluids gum quautativen naehweis des pektins und der alginsiiure ein schliffapparat zur abtrennung yon flfichtigcn stoffen mittel~ wasscrdampfdcstillation ~tude par chromatographic on phase gazeuse des acidea gras du beurre fabriqu6 en italic et clans d'autres pays. application ~, la recherche des falsifications clans le bcurre commercim. ann. falsiticatiorm expertise chim note on the determination of caffeine in coffee a comparison of the press method with taste-panel and shear measurements of tenderness in beef and lamb muscles zur diehtebestimmung der milch mit der neuen milehspindel. lebensmittelchem, u. geriehtl. chem. 17 [1963] nr. 6, s. 113/116. --aufschlu6-.i~thercxtrakt und titrationswert bei der teigwarenunbersuehung als beispiel einer dynamisehen lebensmittelanalyse quantitative measures of carcass composition and qualitative evaluations fruit preservatives analysis. determination of calcium in cherry brines by versenat~ titration: elimination of anthocyanin interference by means of carbonyl reagents, g. agric. food chem allgemeine prinzipien der analytik yon carotinen und carotinoiden zur bestimmung yon ethoxyquin kolorimctrische bestimmung yon dipterex-riickst~nden yon lebensmittein the direct determination of shear stress-shear rate behavior of foods on the presence of a yield stress methods for evaluating bhe feeding quality of meat-andbone meals a paper chromatographic method for determination of vanillin and ethyl vanillin in vanilla flavorings ~drber die anwendung der papierchromatographisehen analyse auf dem fettgebiet. 4. mitt.: uber 5ls~iurereiehe samenole. ermittlung der konsti~uierenden fettsiiuren der samensie yon margosa (azadiraehta indies), cashewkern (anacardicum oecidentale) und putranjiva roxburghli. nahrung 7 die jodzahl des riickenspeeks im verhiiltnis zu der qualit~t des futterfettes, dem alter der schweine und dem fettungsgrad bei sehweinen der ditnisehen landrasse determination of parathion, methyl parathion, epn, and their oxons in some fruit and vegetable crops an improved chromatographic method for determining trace elements in foodstuffs determination of guthion residues on fruits techniques used in meat flavor research the analysis of edible oils contaminated with synthetic ester lubricants colorimetrische bestimmtmg yon nitrat und nitrit in biologisehem material confrontation de quelques proe~d6s de dosage iodometrique de l'anhydride sulfureux dans les vins zur anwendung der massenspektroskopie zur strukturermittlung yon naturstoffen, mit besonderer berfieksiehtigung der lebensmittelanalytik z lebensmittel cellulose solubility as an estimate of cellulose digestibillty and nutritive value of grasses the 2-thiobarbiturie acid reagent for determination of oxidative rancidity in fish oils bet die eignung yon daphnia magna zur ermittlung yon riieksti~nden auf frisehem 0bst und gemfise bemcrkung zum diinnschiehtchromatographischen kakaoschalennaehweis nach the determination of vitamin a in animal tissues and its presence in the liver of the vitamin a-deficient rat elution column preparation of leaf sample for flame photometry. ii. : determination of calcium in tobacco enzymatic-ultraviolet method for determination of uric acid in flour die bestimmung der amyiaseaktivit~t und einige studien fiber amylaseaktivit~it in gekeimtem roggen identification and determination of ascorbic acid (vitamin c) with janus green and its localisation in mitochondria cho]estehnbestimmung im kleinen laboratorlum chick edema factor. iii.: application of mieroeoulometric gas chromatography to detection of chick edema factor in fats or fatty acids bioassay of chick edema factor. 1962 collaborative study determination of nih4te and nitrate in meat products the measurement of the surface areas of milk powders by a permeability procedure sedimentbeurteilung und sehaim-~iastitis-test als sortierverfahren zur ermittlung yon sekretionsstsrungen bei der 1~iassenuntersuchung yon milchproben aoac methods for nutritional adjuncts die ermittlung der ribonucleins~ure im pflanzenmaterial beitrag zur analytischen beurteilung des frischezustandes der pharmazeutisch verwendeten 01e und fe~te sehnellbestimmung yon kupfer in fe~n applications of oscillographic polarography to the determination of organophosphorus pesticides. ii. : a rapid screening procedure for the determination of parathion in some t~its and vegetables zur standardisierung der vitaminb~-bestimmung in getreide und getreideprodukten eine mikromethode zur bestimmung des fettgehaltes der milch kleiner laboratoriumstiere the determination of organophosphato pesticides and their residues by paper chromatography die l~fikrochemie beim studium yon nahrung und erni~hrung aearicide residues. an improved method for kelthane residue analysis with applications for determination of residues in milk collaborative study of the determination of ethoxyquin in feeds ~rber den naehweis yon quellstoffcn in fleischwaren und m6gliche stsrungen durch andere polysaccharide ersatz manueller labormethoden der l6sungsspektralanalyse durch den automaten determination of total acids in wines: american society of enologists determination of aldehydes in wines and spirit~ by the direct bisulilte method ~fber einen vereinfachten nachweis des vitamin b12 mit poteriochromonas slipitata extraction of nys~tin in animal feeds for microbiological analysis zur quantitativen bestimmung der sorbinsi~ure mit dem thiobarbitur-s~urereagenz cottage cheese problems in production and sanitation. quality control in cottage cheese sitzung des arbeitskreises berlin der gdch-fachgruppe lebensmittelchemie und gerichtliche chemie am 23. 11. 1962 in ]~erlin-dahlem (2 vortragsreferate) ein neues elektronisches sctmeuverfahren zur ermittlung der ~risehe yon seefischen fatty acids of lard. a. identification by gas.liquid chromatography oxydative abbauprodukte der l-aseorbins~ure. 1. mitt. : papierchromatographischer l~achweis analysis of orange juice for total earotenoids, carotenes, and added betacarotene. food technol. 17 [1963] nr. 3, s. 95/98. u. r~ck~, a. a. : refractometrio measurement of soluble solids in orange juice analysis of iron chelates in plant extracts. il: ferric ethylenediamine'bis'(~176 acid) determination of n-aeetylglucosamine-1-phosphate and n-aeetylglucosamine in milk arsenic in foods: collaborative comparison of the aminemolybdenum blue and the silver diethyldithiocarbamate methods improved method for testing macaroni products neuere beitri~ge zur chemic der st~rkefraktionen. 13. mitt.: die mikro-ameisens~urebestimmung bei der perjodat-0xydatonsbestimmungsmethode yon stiirke recherche des falsifications dans les extraits de vanille. ann. falsifications expertise chim bestimmung des gesamtstckstoffgehaltes yon milch nach der kjwld~t:l-methode. internationaler standard fil vorteile und grenzen des einsatzes yon markiertem phosphat bei untersuchungen am hiihnerei paper chromatography of carotene and carotenoids determinaton of sevin insecticide residues in fruits and vegetables insecticide residues in meat and eggs. determinaton of sevin insecticide and its metabolites in poultry tissues and eggs bcstimmung der jodzahl yon fetten und 01en mittels n-bromsuccinimid. 2. mitt.: l~ber eine !~i6gliehkeit zur bestimmung der gesamtjodzahl yon el~iostearins~ure und holz61. nahrung 7 [1963] nr. 5, s. 375/381. kxrr:~e~r, m. a. : ~3ber die anwendbarkeit des thiobarbi~urs~iuretestes boi der untersuchung yon milchprodukten application of gas chromatography to the measurement of gas permeability of packaging materials a sensitive method for quantitative microdetermination of lipids comparison of chemical and microbiological methods for the determination of procaine penicillin in prcmixes and mixed feeds electrophoretc analysis of flour proteins from various varieties of wheat katalytische methode zur bestmmung kleinster manganmengen in lebensmitteln am beispiel der milch eine methode zur papierchromatographischen qualits yon silagen comparison of methods of measuring potassium in pork and lamb and prediction of their composition from sodium and potassium electron capture gas chromatography for determination of ddt in butter and some vegetable oils eine methode zum schnelinachweis yon nitriten in yleiseh-und wurstwaren. arch. lebensmittel-hyg determination ofglyodin residues on pears and peaches eleetrophoretic analysis of flour proteins erstes internationales symposium fiber methoden zur analyse yon lebensmitteln in bordeaux-tatence (frankreich) veto 8. bis 12. oktober on the problem of luminescence technique of protein definition in milk beitr~ge zur aminos/iuren-bestimmung in biologischem material aktuelle fragen der lebensmitteluntersuehung, insbesondere der histologischen wurstanalyse untersuchungen yon importiertem tiefgefrorenem tiasenfleisch argentinischer herkunft fish hydrolysates. iv.: microbiological evaluation untersuehungon zum naehweis yon emulgatoren in lebensmit~eln. 3. mitteilung. forte use of a slice-tenderness evaluation device with pork studies on improvements in quantitative paper chromatography of amino acids in foods. i. : research on procedure of development studies on improvements in quantitative paper chromatography of amino acids in foods. ii.: selection of solvent systems determination of the equilibrium relative humidity of foods untersuchungen fiber die methodik der quantitativen bestimmung yon heizolen und flfissigen treibstoffen im wasser determination of ~-lactalbumin in complex systems hydrogen sulphide in cheddar cheese; its estimation and possible contribution to flavour ascorbic acid measurement. polarographic determination of total ascorbic acid in foods trans-fettsguregehalt yon sehweineschmalz nach fiitterung von schweinen mit rindertalghaltigem kraftfutter. (ein beitrag zur quantitativen infrarotspektroskopischen bestimmung yon trans-fetts~uren in fetten the importance of starch on the microscopic identification of cereal grains in feeds insecticide residues. chromatographic identification of some organophosphate insecticides in the presence of plant extracts chemical and biological estimation of the carotene content in fresh and processed italian apricots sialir acid as an index of the u-casein content of bovine skimmilk foodstuffs analysis. nonvolatile acids of blueberries le dosage de la matibre grasse dans les fromages. ]~tude critique de la m6thode en usage au laboratoire municipal la d6termination de residus d'insecticides et de fongieides par la m6thode polarographique fusel oil determination by gas.liquid chromatography * die brabanter mastitis-reaktion, ein neues verfahren zur ermittlung yon sekretionsstsrungen des euters dutch die kannenmilchuntersuchung uber eine enzymatisehe _&pfels~urebestimmung in wein und traubensaft direct microscopic technique to detect viable yeast cells in pasteurized orange drink die enzymatisehe bestimmung der glucose und saecharose und ibre anwendung in der lebensmittelanalyse a modified zirconium-alizarin method for determining fluoride in natural waters paper chromatography of some cholesterol derivatives determination of moisture by nuclear magnetic resonance and oven methods in wheat, flour, doughs, and dried fruits dye binding by soybean and fish meal as an index of quality the absorptiometrie determination of silicon in water: part l: formation, stability, and reduction of cr and fl-molybdosilicie acids column chromatography of soybean whey proteins enzymatic determination of carbon dioxide in lightly carbonated wines. 1962 collaborative study nachweis yon biiffelmilch als verf/flschungsmittel in kuhmilch durch serologisehe methoden photometric determination of phosphate in wines pertinent references to analytical lipid methods published recently, ft spectrophotometric estimation of nucleic acid of plant leaves hemmstoffe in der aalieferungsmilch und methoden zu ihrem nachweis determination of potassium in tobacco determination of chlorides in tebacco phospholipase c determination by egg yolk turbidimetry oher die inhaltsstoffe der rol]kastanie und versuche zu ihrer gehaltsbestimmung mierobiologlcal method for assaying nystatin in animal feeds /63] nr. 5, s. 401/415. --gaschromatographische untersuchungen yon fuselslen aus versehiedenen g~rproduk-ten. 2. mitt.: methodik der fusel61bestimmung lactose activity measurements. evaluation of laetase preparations for use in breadmaking comparison of methods for determination of lysino in cereals direct determination of calcium in plants, soils, and milk by means of a flame photometer colorimetrie determination of urea in feeds kolorimetrische bestimmung des dihydrox-yacetons fluoride, teeth, and the analyst the quantitative micro-determination of biphenyl in citrus fruit kolorimetrisehes verfahren zur gleichzcitigen bestimmung der weinsiiure und milchsiiurc in wein und most estimation of extra-cellular starch of dehydrated potatoes versuehe zur chromatographischen trennung yon kohlenhydra~en und eiweil3en aus dem w~ibrigen extrakt yon roggenvollkornmehl quantitative determination of the amino acid content of rumen fluid from twin steers fed soybean oil meal or urea vemnche zur chemischen differenzierung der eiwei•stoffe des weizens und roggens aromastoffe des brotes. versuch einer auswertung chemiseher gesehacksanalysen mit hilfe des schwellenwertes zur trennung yon sacchariden an kohle]celit~-s~ulen. ern~hrungs-fomchung 7 untersuehungen zur bestimmung der lsslichkeit yon milchpulver * selection of a medium for the isolation and enumeration of enterocoeei in dairy products colorimetric determination of amino nitrogen in corn syrups 0stxogene und versuche zu ihrem nachweis in gefliigelflelsch. dt. lebensmittel ein beitr~g bert. die verwendung der anatysen-q~arzlampe zu friihzeitiger erkennung der l~anzigkeit colorimetrlsche methode zur bestimmung yon 1-monoglyceriden in eiskrem determination of fusel oil in distilled spirits zu den m6glichen fehlerquellcn bei der histometrischen ermittlung des kollagen-, bzw. gelatine-gehaltes bei briihwiirsten studium der uv-spektren der auf hshere tempcraturen erhitzten 01e measuring of oil-binding characteristics of flour naehweis der konservierungsmittel mit hilfe cl~r papierehromatographie identification of ch]orogenic acid in castor bean and oranges. canad evaluation of forages in the laboratory. iii.: comparison of various methods for predicting silage digestibility feed microscopy essential oils. determination of botanical and geographical origin of spearmint oils by gas chromatographic and ultraviolet analysis fluorometric determination of chlortetracycline in premixes dfinnschicht-chromatographie yon carotin-und carotinoidgemischen measurement of the sub-sieve particle size distribution of flour chemisehe bestimmung der riickst~nde yon parathion, malathion und diazinon auf blumenkohl, kohlrabi, bohnen und gurken determination of cadmium anthranilate in feeds nachweis yon penicillin und anderen antibiotika in milch microbiological evaluation of protein quality with tetrahymenapyriformis w. 3.: a simplified assay procedure peanut lipoprotein. ii. : analysis in foods by gas chromatography beitrag zum papierchromatographisehen und spektrophotometrisehen i~achweis fettlsslieher synthetiseher farbstoffe in lebensmitteln und kosmetika elektroehemische sauerstoffbestimmung in olefinischen fetich bestimmung der gesamten sehwefligen s~ure in getr~nken the modified whiteside test. recommended procedures for bulk or blended milk deliveries die bestimmung yon mileheiweib in fleiseherzeugnisscn. dr. lebensmittel zur bestimmung des veresterungsgrades yon pektin a quantitative fluorometrie method for the determination of serpasil (reserpine) in feeds at the micro level fluorimetric mierodetermination of carbohydrates zur l~iethodik der klebrigkeitsbestimmung yon brot frage der papierchromatographischen untersuchung von amylopektin und amylose chemical determination of diethylstilbestrol residues in the tissues of treated chickens gas chromatographic identification of components in m~ple sirup fl~vor extract dark discoloration of canned all-green asparagus. ii. development of a new tin plate for its control thermal conductivity and density of chicken breast mnsele and skim beitrag zur bestimmung der fluoreszenz in spriten microbiological determination of alanine in proteins and foods collaborative study of the method for counting microorganisms in maple sirup glass fiber paper strip charring. a rapid and simple method for monitoring column chromatography of lipids verbesserte mcthode zum serienm~bigcn quantitativen nachweis yon insektizidrficksti~nden bei 0bst und gemfise use of the shear press in determining fibronsness of raw and canned green asparagus betrachtungen fiber verschiedene methodcn zur bestimmnng des bindegewebeanteiles in rohem fleisch und fleischwaren. arch. lebensmittel-hyg the determination of citric acid in milk and milk sera formaldehyde in maple sirup: an adaption of the nash method polarographische bestimmung dcr ascorbinsiiure und des gesamt-vitamin c untersuchungcn fiber die mbglichkeit zur objektiven beurteilung der organoleptischen eigensehaften yon kokosraspeln analysis of the flavor and aroma constituents of florida orange juices by gas chromatography assay for cyzine in finished feeds dfinnsehicht-ehromatographische trennnngen yon synthetischen lebensmitteffarbstoffen auf ceuulose-schichten evaluation of quantitative methods of determining peroxidase in vegetables. i.: the indophenol and the o-phenylenediamine methods ein beitrag zur untersuchung thermisch oxydierter fette. dt. lehensmittel a more accurate method for determination of caffeine in decaffeinated coffee bestimmung yon vitamin c in friichten, fruchtsiiften, gemiise und konserven naeh der methode naeh tillma~s unter ausschaltung reduzierender stoffe lebensmittelrecht und lebensmitteliiberwaehung nutritional laws and nutritional control briihwiirst~ einfaeher qualit~t, die zu einem kaliber unt~r 32 mm in den verkehr gebracht werden, sind i.s. des w 4 nr. 3 lmg irreffihrend aufgemaeht new regulations under the food and drugs act lebcnsmittel-rdseh orb der kenntlichmaehung fremder stoffe probleme des geltenden lebensmittelrechts glasurmittel ftir r6stkaffee beschr~nkt zugelassen food technol. 17 [1963] nr. 6, s. 96. n. iv. : revised u. k. preservatives regulations. food teehnol ausschub fiir lebeusmittelrechtliche fragen der fachgruppe lebensmittelehemio und gerichtliche chemic in der geseuschaft dcutscher chemiker vi. t~tigkeitsberieht der eidg. kommission fiir volksern~hrung, lebensmittelgesetzgebung und -kontrolle (eek) zu h~nden des eidg. departementes des i_nnern, umfassend die jahre federal food, drug, and cosmetic act, as amended. selected u. s. government publ. 1963 nr. 8; 50 h. catalog i~o verkauf yon frikadellen mit brotzusatz in gastwirtsehaften tierarzneimittel und aufzuchtmittel in der landwirtschaftlichen praxis. gesundheitliche erw~gungen zum schutze des konsumenten bei der anwendung yon tierarzneimitteln und aufzuchtmit~ln in der landwirtschaftliehen praxis. teil hi der amtsarzt und das neue lebensmitt~lgesetz. off ist der beschlub des bundesgerichtshofes vom 18. 5. 1962 -1 stl~ 546161 -geeignet, den vertrieb yon hackfleiseh befriedigend zu regeln ? arch. lebensmittel-hyg zur beurteilung des saccharingehaltes yon meerrettiehzubereitungen. dt. lebensmittel zum begriff ,mai]gebend" in w 4a abs. 2 des lebensmittelgesetzes aspect sanitaire et ldgal actuel des aliments conservds verbrauchererwaxtung und lebensmitte]kontrolle bei fleiseherzeugnissen bei ger~ueherten fleischwaren. sehwarz-w~lder speck, schwarzw~lder sehinkenspeck, sehwarzw~lder sehinken. arch. lebensmittel-ttyg kampf der wasserverseuchung. aktuelle notwendigkeiten -gesetzliehe ~/isgliehkeiten. ~iiinchener reed. wschr. 105 lebensmittelreehtliehe stellung yon carotin und carotinoiden in der schweiz die lebensmittelgesetzgebung 0sterreiehs, der sehweiz und der bundesrepublik deutschland. eine vergleiehende untersuchung arzneimittel-lebensmittel dr. lebensmittel-rdseh erwiderung des autors (zur stellungnahme yon f. nitzsc~, d~. lebensmittel-rdseh. 59 [1963] nr. 5, s. 146). i)t. lebensmittel das lebensmittelgesetz und seine auswirkung auf die gummiindtlstrie codex alimentarius austriacus absehliel]ende stellungnahme der ort der kenntlichmachtmg fremder stoffe. dr. lebensmittel-rdsch _rreffihrende bezeiehnung und angaben bei limonade aus mineralamem tafelwasser. dr. lebensmittel zur herkunftsbezeichnung yon lebensmitteln. dt. lebensmlttel aktuelles zur lebensmitteliiberwachung einige beispiele fiir die auswirkung der neuen lebensmittelrechtliehen vorschriften auf erniihrungs-und landwirtschaft auf dem wege zum einheitliehen lebensmittel-rech~ zur ~r yon b. rsssl~: welehe anforderungen sind an alkoholhaltige siigwaren zu stellen? dr die silbertmg yon tafelwiissern mokka-kaffee" auch im handel mit kaffeebohnen keine tterkunftsbezeiehnung 59 [1963] nr. 1, s. 29131. --zuliissigkeit trod grenzen bildlicher darstellungen yon fleiseh und ylelscherzeugnissen auf paclmngen yon suppen in trockener form. db. lebensmit~el-l~lsch bedeutung und beur~eitung yon galtstreptokokken in vorzugsmileh. arch. lebensmittel-hyg auf clipverschliissen zul~ssig ist die infektion mit trichinen aus amtlich untersuehtem schwelnefleiseh im lichte der mathematischen analyse der bestimmungen der fleischbesehau yon schweinefleiseh msglich? arch. lebensmittel-hyg intema~ionales rundgespr~ch fiber lebensmitte]ehemische probleme in wiesbaden und eltville a. rh. z. lebensmittel-untersuch. u. -forschung. 129 [1963] nr. 2, s. 1271130. --kommission zur priifung fremder stoffe bei lebensmitteln (fremdstoff-kommission) der deutsehen forsehungsgemeinsehaft entwicklung des lebensmittelrechts im nationalen und internationalen bereich entwicklung des lebensmittelrechts im nationalen und inter~ationalen bereich entwicklung des lebensmittelrechts im nationalen und internationalen bereich de warenwet en de hierop berustendc besluiten zum entwurf einer harmonisierung der lebensmittelrechtlichen bestimmungen fiber konservierungsstoffe im gebiet der europaischen wirtschaftsgemeinschaft. er lebensmittelrechtliche stellung yon carotinen und carotinoiden in der bundesrepublik deutschland die instrumente der lebensmittelfiberwachung in osterreich 0sterreichischer standpunkt zur l~rage der f~rbung yon lebensmitteln mit carotinen und carotinolden. wiss. ver6ff. dr. ges. ernkhrung 9 lst es zu vcrtreten, dab das fleisch schwacbfinniger rinder auch in gebr~tetem zustand eingefroren wird? z lrch. lebensmittel-iiyg richtlinien fiber die zulassung yon gegeusachverst~ndigen zur untersuchung yon lebensmittel-gegenproben die ]ebensmittelrechtliche bedeutung yon bildiichen darstellmlgen auf verpackungen diverse problems (education, documentation associations, terminology etc the nutritional education of the food technologist proposed formation of a food engineering panel within the food group studium der hauswirtschafts-und ern~hrungswissenschaften. ern~hrungswirt-sehaft l0 an information service for the american food industry 1st international congress of food science and technology. symposium on education and training technically trained people for developing countries lft paces the food frontier un committee on food additives international standardization of fruit and vegetables. food technol terminology and methods for feeding and weighing animals food additives and food standards beitriige zur durehffihrung der umweltsradloaktivitiits-0berwaehung training dairy personnel in denmark training opportunities for the sanitarian-specialized in-service training r die benennungen honigkueheniihnlicher oebiieke als lobkuehen und lebzelten sowie als biber und bibenzelten. dt. lebensmittel nutrition and dietetics for the medical student problem of keeping dairy plants supplied with the foreman-type employee literaturdokumentation fiir hochschulassistenf~n a system for naming and describing feeds, energy terminology, and the use of such information in calculating diets a brief sketch of veterinary periodical literature in great britain before the foundation of the veterinary record a conference on nutrition teaching in medical schools the food service industry and its relation to the control of foodborne illness research and educational progress in nutrition informationsm6gliehkeiten fiir tieriirzte auf dem measuring readability of health education literature the dai~y literature problem the central food technological research institute the education and function of the nutritionist training in food service for nursing homes. l: tools for evaluation training in food service for nursing homes. iii.: observations on management of twelve units pennsylvania takes a look at nutrition in the orthopedic program zur definition der begriffe ,aroma" und l~ber die notwendigkeit einer priifung for beamtete sehlaehthofleiter. arch. lebensmittel-hyg organisation der dokumentation in einem forschungsinstitut (bundes. forsehungsanstalt f'tir lebensmittelfrischhaltung in karlsruhe). dt. lebensmittel-rdsch on changing the name of our assoziation. for a name change 0pporbunities in nutrition education questionnaires to identify nursing homes most in need of dietary counsel. publ. health irep lebensmittelwissenschafttiche institute in bulgarien zur stellung des psychiaters in der alkoholfrage naas nutrition chemists. vcter on changing the name of our association. against a name changing begriffe) in 10 sprachen: deutseh, russiseh, polnisch, tsehechisch, slowakisch, ungariseh, bulgariseh, serbokroatiseh, rum~nisch, englisch the role of nutrition in the teaching of medicine key: cord-021500-sy6lnt7b authors: jean harry, g.; toews, arrel d. title: myelination, dysmyelination, and demyelination date: 2007-05-09 journal: handbook of developmental neurotoxicology doi: 10.1016/b978-012648860-9.50007-8 sha: doc_id: 21500 cord_uid: sy6lnt7b nan normal functioning of the nervous system involves the transmission, processing, and integration of information as nervous impulses. impulse transmission along axons is greatly facilitated by the presence of myelin, the compact multilamellar extension of the plasma membrane of specialized glial cells that spirals around larger axons. in the central nervous system (cns), oligodendroglial cells are responsible for the synthesis and maintenance of myelin, whereas schwann cells subserve this role in the peripheral nervous system (pns). schwann cells produce a single segment of myelin (called an internode), whereas oligodendroglial cells furnish multiple myelin segments around different axons, although only one segment for a given axon. there are periodic interruptions along the axons between adjacent myelin internodes; termed nodes of ranvier, these short intervals where axons are not enveloped by myelin are vital for normal nervous system function (see later this chapter). myelin is an electrical insulator, and the periodic interruptions at the nodes allow for rapid and efficient transmission of nervous impulses. in unmyelinated axons, impulse transmission involves a wave of membrane depolarization that moves down the axon in a continuous sequential manner. however, in myelinated axons, the myelin internodes function as high-resistance insulators, so the excitable axonal membrane, containing a high concentration of voltage-sensitive sodium channels, is exposed only at the nodes of ranvier. impulse conduction thus involves excitation at the nodes only, and the impulse jumps from node to node (saltatory conduction) (see funch and faber, 1984; waxman et al., 1989; and morell et al., 1994, for details) . saltatory conduction is much more rapid and requires much less energy for membrane repolarization than conduction in unmyelinated axons. myelin thus greatly increases the efficiency of the nervous system, facilitating conduction while conserving metabolic energy and space. it is not difficult to imagine how even minor loss of myelin or perturbations in its structure and/or function could have deleterious effects on normal nervous system function. structural aspects of the process of myelination are most easily illustrated in the pns. each myelin-forming schwann cell produces an elaborate specialized extension of its plasma membrane, which is wrapped spirally around a segment of one axon (fig. 1 ). schwann cells, the glial cells of the pns, are derived from the portion of the neural epithelium that gives rise to the neural crest (le douarin, 1982) . during development, schwann cells invade the developing nerves, where they migrate along bundles of axons, proliferate (probably in response to an axonal mitogen; webster and favilla, 1984) , and segregate the axons individually within invaginations on the surface of schwann cells. as they cease migrating, they synthesize a basal lamina (billings-gagliardi et al., 1974) , composed of laminin, merosin, type iv collagen, fibronectin, nidogen/entactin, and heparan sulfate proteoglycan (sanes and cheney, 1982; tohyama and ide, 1984; bannerman et al., 1986; leivo and engvall, 1989; sanes et al., 1990) . as the axons continue to enlarge, the larger axons become further segregated so that a single schwann cell envelops a single axon. after the plasma membrane of the schwann cell has completely enclosed the axon, the external surfaces of the plasma membrane fuse to form a structure known as the mesaxon. the mesaxon then elongates and spirals around the axon, eventually resulting in a "jelly-roll" structure consisting of double layers of the schwann cell plasma membrane. myelin internodes can be as much as 2 mm long and contain 5 mm of myelin spiral (friede and bischhausen, 1980) . myelin compaction occurs as cytoplasm is extruded and the cytoplasmic faces are condensed to produce the dark major period line visible in electron micrographs (fig. 2) . the juncture of what was originally the outer faces of the apposing plasma membranes form the lighter appearing intraperiod line. mature myelin thus has a characteristic compact multilamellar structure, but cytoplasmic inclusions continu-figure 2 electron micrograph of compact myelin from the mammalian cns. although there are minor ultrastructural differences, compact pns myelin has a similar ultrastructural appearance. note the alternating pattern of darker major dense lines and paler intraperiod lines, originally formed by fusion of apposing surfaces of the inner and outer leaflets, respectively, of the oligodendroglial plasma membrane. cytoplasm-containing internal mesaxons can be seen on two of the myelinated axons. ous with the perikarya cytoplasm of the schwann cells are also present ( figs. 1 and 2 ). in addition, the myelin internode contains several ultrastructurally and biochemically distinct membrane domains, including the outer plasma membrane of the myelin-forming cell and the compact myelin itself, as well as the cytoplasmcontaining schmidt-lanterman incisures, paranodal loops, nodal microvilli, and outer and inner mesaxons. the latter cytoplasm-containing structures provide connections with the perikaryal cytoplasm, and are vital for myelin maintenance. the process of myelination in the cns is similar, except that a single oligodendroglial cell extends a number of processes from its cell body; each process then envelopes and myelinates a single segment of a given axon (fig. 3) . much of the local membrane assembly to give mature, compact myelin occurs within the oligodendroglial cytoplasmic processes (waxman and sims, 1984) . the size of the fibers and the thickness of the sheaths are very different in the pns and the cns, but the overall surface area of myelin generated by an oligodendrocyte around multiple axons may be no larger than that formed by a schwann cell around a single internode. the term oligodendrocyte, meaning "few processes," is actually somewhat of a misnomer, as a given oligodendrocyte may myelinate anywhere from less than five axons up to dozens of axons (butt and ransom, 1989; bjartmar et al., 1994) . as in the pns, the onset of myelination is preceded by proliferation of oligodendroglia. as development continues, both the diameter and length of the axons increase, and this is associated with a corresponding increase in nodal length as well as increases in myelin thickness. thus, despite its compact highly ordered appearance, myelin continues to expand in all planes during growth and development. in general, myelination follows the order of phylogenetic development, with the peripheral nerves myelinating first, then the spinal cord, and finally the brainstem, cerebellum, and cerebrum. there is, however, considerable overlap in this progression. in addition, each fiber tract may have its own spatiotemporal pattern of myelination, so that the degree of myelination may differ in different fiber tracts at a given developmental stage. for example, myelination in the spinal cord proceeds in a rostral-caudal gradient, whereas in the optic nerve, myelination progresses with a retinal to chiasmal gradient. although not necessarily an absolute prerequisite for function, in general, fiber tracts are myelinated before they become fully functional. myelination is a major metabolic and structural event that occurs during a relatively brief but precisely defined period in the normal progression of events involved in nervous system development. in both the cns and pns, an enormous amount of myelin membrane is formed (some pns axons may have as many as 100 layers) and this membrane must be maintained at a considerable distance from the supporting glial cell body. the surface area of myelin per adult oligodendrocyte in the rat brain has been calculated to be 1-20 x 105/zm 2, several orders of magnitude greater than the perikaryal membrane surface area of about 100 /zm 2 (pfeiffer et al., 1993) . myelinating glial cells are thus maximally stressed in terms of their metabolic and synthetic capacity during this time, with each cell synthesizing myelin equivalent to up to 3 times the weight of its perikarya each day . because of these very high levels of synthetic and metabolic activity, these myelinating cells are especially vulnerable to nutritional deficits and/ or to toxic insults or injuries during this period. the tightly programmed sequence of events eventually resulting in the formation of mature compact myelin and the consequent initiation of impulse transmission is regulated by interactions between axons and glial cells at numerous stages (see waxman and black, 1995, for detailed discussion) . during the early stages of myelination, the axon is loosely ensheathed by processes arising from immature, relatively undifferentiated glial cells. loose glial ensheathment of axons in the optic nerve is seen in the rat beginning at postnatal day 6. this is followed by spiral wrapping of the axon by oligodendroglial processes that form compact myelin. in some tracts, the immature myelin sheath is initially close to the oligodendroglial cell body (remahl and hildebrand, 1990) , but with maturation the sheath is displaced radially and often is connected to the cell body by only a thin cytoplasmic bridge. a single oligodendrocyte can myelinate axons of various diameters in their vicinity and can form myelin sheaths of different thicknesses around axons of differing diameter. the development, maturation, and maintenance of the myelin sheath is dependent on both the normal functioning of the myelinating glial cells and the integrity of its relationship to the axon it ensheaths. during development, physical features of the myelin sheath, such as thickness and number of lamellae, are not preprogrammed within the myelin-forming cells, but rather depend on local regulation by the axon, with larger axons having thicker myelin sheaths (waxman and sims, 1984) . myelin internodal distance is also matched to fiber diameter (hess and young, 1952) and the internodal distance:diameter ratio is different for fibers in different tracts. there is some evidence that myelination is initiated when a developing axon reaches a "critical diameter." however, myelination occurs over a range of axonal diameters (fraher, 1972) and at various times along a single axon (waxman et al., 1972; waxman, 1985) . the signal for initiation of myelination thus appears to be specific for particular axons, or for specific domains along axons. the axonal membrane contains molecules that trigger mitogenesis in schwann cells and oligodendrocytes (salzer et al., 1980a; 1980b; devries et al., 1983; chen and devries, 1989) and regulate the rate and degree of myelin formation (black et al., 1986; waxman 1987a,b) . although some schwann cells go on to myelinate axons, others only ensheath bundles of unmyelinated axons. these nonmyelinated schwann cells express distinct molecular markers such as the low-affinity nerve growth factor receptor (ngf-r), neural cell adhesion molecules (n-cam and l1), and growth associated protein-43 (gap-43), but none of the myelin specific proteins (see following sections, as well as mirsky and jessen, 1990; curtis et al., 1992) . transplantation studies have shown that the axon determines the phenotype of the schwann cell (aguayo et al., 1976; weinberg and spencer, 1976) . thus, there are a number of potentially distinct physical interactions between schwann cells and axons as schwann cells proliferate, migrate, ensheath axons, and form myelin sheaths during development of the pns. although the axon influences and helps direct the formation of myelin, the myelin sheath also significantly defines features of the axon. the premyelinated axon is electrically excitable (foster et al., 1982; waxman et al., 1989) and the loss of excitability in the internodal axonal membrane that occurs with myelination involves an active suppression of na + channels by the overlying oligodendrocyte or myelin sheath (black et al., 1985 (black et al., , 1986 . proliferation of oligodendrocyte precursors in the optic nerve is dependent on axonal electrical activity in that the blockage of optic nerve electrical activity by transection or exposure to tetrodotoxin resulted in a dramatic loss of oligodendrocyte precursor cells (barres and raft, 1993) . potassium channels may also participate in myelin formation; the potassium channel blocker, tea + was shown to be effective in eliminating myelination in spinal cord explants while leaving axonal conduction and synapse formation intact. the ontogenic development of the myelinating schwann cell lineage has been relatively wellcharacterized, particularly in rodents. most of our knowledge derives from cell-culture studies, but in vivo developmental studies in normal as well as in transgenic and gene knock-out mice have also proved useful. understanding of the events and stages involved is of potential clinical relevance, not only with respect to various toxic neuropathies and to pns nerve regeneration, but also because schwann cell precursors may be attractive schwann cells of the pns originate from primitive neural crest cells, proliferative multipotential cells also capable of differentiating into neurons or melanocytes. the first step along the schwann cell lineage gives the schwann cell precursor, a proliferative cell that becomes associated with many axons and expresses the lowaffinity nerve growth factor receptor (ngf-r), growth-associated protein 43 (gap-43), and the neural cell adhesion molecules n-cam and l1. the subsequent "committed" schwann cell becomes associated with progressively fewer axons and expresses, in addition to the previously noted markers, s-100 protein (from this stage onward, all schwann cells express s-100). committed schwann cells develop into either nonmyelinating schwann cells, which remain associated with several axons and express galactocerebroside (galc) in addition to the previous markers, or into myelinating schwann cells. myelinating schwann cells progress through a proliferative "premyelinating" stage, characterized by transient expression of suppressed camp-inducible pou-domain transcription factor (scip), followed by a "promyelinating" galc-positive stage, becoming associated with a single axon in the process. the final differentiation into a mature myelinating schwann cell involves candidates for transplantation to facilitate remyelination and repair in injured cns. a detailed discussion of this subject is beyond the scope of this chapter, but the reader is referred to several comprehensive reviews on this subject if more details are desired (jessen and mirsky, 1991; gould et al., 1992; mirsky and jessen, 1996; zorick and lemke, 1996) . schwann cells develop from neural crest cells and most of the key developmental stages in schwann cell maturation seem to depend on axon-associated signals. schwann cell precursors give rise to immature schwann cells, which have a distinct phenotype (fig. 4a ). these cells then develop into either myelin-forming or nonmyelin-forming schwann cells, under the control of reversible processes regulated by axon-associated signals. even mature schwann cells show a high degree of plasticity. following a demyelinating insult, schwann cells dedifferentiate into more primitive precursor cells, but generally do not die. when appropriate conditions present themselves (such as the regrowth of axons that occurs following a nerve-crush injury), these cells can proliferate, reestablish contact with axons, redifferentiate to the myelinforming phenotype, and remyelinate the axons, thereby restoring normal function. it is possible that primitive neural crest cells enter the schwann cell lineage only when they first encounter axons in the developing nerves, and that a signal related to axonal contact guides them down the path to mature schwann cells. such signals may involve members of the neu-differentiation factor (ndf) family. members of the ndf growth factor family, including glial growth factor (ggf), heregulin, acetylcholine-inducing activity (aria), and neuregulin, are alternatively spliced products of a single gene, and these molecules are emerging as important regulators of schwann cell lineage development (dong et al, 1995; zorick and lemke, 1996) . alternatively, it is possible that selected neural crest cells have already entered the schwann cell lineage before they encounter axons (see mirsky and jessen, 1996, for discussion) . investigation of which transcription factors regulate schwann cell development is a current area of active study. among factors likely to play significant roles are the zinc-finger transcription factors krox-20 (topilko, 1994) , scip (see zorick and lemke, 1996) , pax3 (kioussi et al, 1995) , and possibly c-jun (stewart, 1995) . a number of signalling molecules also regulate schwann cell proliferation and myelination, including insulin-like growth factor-1 (igf-1), which promotes expression of a myelinating phenotype in cell culture, and transforming growth factor/3s (tgf-/3), which inhibit myelin formation. the latter may be involved in generating the nonmyelinating schwann cells, which ensheath smaller pns axons. the developmental lineage of the oligodendrocyte, the myelin-producing cell of the cns, is also relatively well characterized, particularly in rodent in vitro systems (fig. 4b ). oligodendrocytes originate as neuroectodermal cells of the subventricular zones and then migrate, proliferate, and further differentiate into mature, postmitotic myelin-forming oligodendroglial cells. their development is discussed only briefly here, but a more comprehensive review is available (warrington and pfeiffer, 1992) . panels of cell-and stage-specific antibodies have proved especially useful in characterizing the sequential expression of various developmental markers, and this has allowed identification of distinct phenotypic stages, each characterized by its proliferative capacity, migratory ability, and distinct morphologies. primitive precursor cells differentiate into proliferative, migratory bipolar o2a progenitor cells. these cells are bipotential, being capable of differentiating into either astrocytes or oligodendrocytes. the oligodendrocyte lineage progresses through several additional stages, including an immature oligodendrocyte expressing galactocerebroside, sulfatide, and cyclic nucleotide phosphodownregulation of ngf-r, gap-43, n-cam, and l1 expression, with upregulation of expression of galc and myelin proteins, and in vivo, the synthesis and elaboration of myelin. because of the high degree of plasticity of schwann cells, most of the developmental steps shown are reversible. (modified from mirsky and jessen [1996] and zorick and lemke [1996] ). (b) myelinating oligodendroglial cells of the cns originate from neuroectodermal cells of the subventricular zones of the developing brain. the earliest precursor cells recognized to date (pre-gd3 stage) are proliferative, unipolar cells that express the embryonic neural cell adhesion molecule (e-ncam). these cells develop into gd3 ganglioside-expressing proliferative bipolar cells, termed o-2a progenitor cells because they are capable (in culture, at least) of developing into either "type 2" astrocytes or oligodendrocytes. development continues through a postmigratory but proliferative multipolar pro-oligodendroblast (pro-ol) and a pre-galc stage, characterized by lack of expression of galc. the onset of terminal oligodendroglial cell differentiation (immature ol stage) is identified by the surface appearance of a subset of "myelin components," consisting of the lipids galc and sulfatide, as well as the enzyme cyclic nucleotide phosphodiesterase (cnp). immature ols then undergo final differentiation into mature oligodendrocytes (mature ol), characterized by regulated expression of myelin components such as mbp and plp and by the synthesis and elaboration of sheets of myelin membrane. diesterase (all myelin components), finally arriving several days later at the mature oligodendrocyte stage. mature oligodendrocytes express all of the myelinspecific proteins and are capable of myelin synthesis in vitro in the absence of axons. it is worth noting that oligodendroglial cells show some developmental plasticity, and this may be of clinical relevance with respect to cns remyelination. in addition, a small population of oligodendrocyte progenitors persist in the adult rat brain (see wolswijk and noble, 1995, for details) , and these constitute a potential source of myelin-forming cells for cns remyelination. immature, cycling oligodendroglial progenitor cells endogenous to adult white matter are capable of remyelinating cns axons following lysolecithin-induced demyelination (gensert and goldman, 1997) . also, 02-a progenitor cells from mice subjected to coronavirusinduced demyelination show increased phenotypic plasticity and enhanced mitotic potential, properties that may be linked to the efficient remyelination that occurs following the demyelinating phase of this disease (armstrong et al., 1990) . manipulation of these progenitor cells by various factors (see later this chapter) thus may also be useful in promoting remyelination in a clinical context. as is the case for schwann cells in the pns, development of oligodendrocytes is governed by a number of growth factors, including platelet-derived growth factor (pdgf), basic fibroblast growth factor (bfgf), igf-1, tgf-/3, and nerve growth factor (ngf), as well as several cytokines (see pfeiffer et al., 1993) . oligodendrocytes differ from schwann cells in that they can be induced to produce myelin in culture in the absence of axons. this raises the question of the extent to which neurons and their axons influence oligodendrocytes with respect to development and myelination in vivo. it is, however, difficult to imagine the lack of significant interaction between these two cells in the developing nervous system, and in fact, many such interactions are known. neurons produce many of the growth factors involved, and they are known to modulate steady-state levels of myelin components as well as mrna levels for these components (singh and pfeiffer, 1985; macklin et al., 1986; kidd et al., 1990; barres and raft, 1993,) . as in the pns, production of myelin by oligodendrocytes requires the coordinated synthesis of massive amounts of myelin components. the marked upregulation of myelin-specific proteins, as well as of enzymes involved in synthesis of myelin lipids (see later this chapter), reflects corresponding increases in abundance of the respective mrna transcripts, suggesting that most regulation of the program for myelination occurs at the level of transcription (see hudson, 1990 , for review). a possible candidate for the coordinate control of cns myelination is myelin transcription factor 1 (myti), a zinc-finger dna-binding protein first identified by its ability to recognize the myelin proteolipid protein (plp) gene (kim and hudson, 1992) . myti mrna transcripts are most abundant in oligodendrocyte progenitor cells, suggesting that this factor acts at a very early stage in the regulation of transcription for myelinogenesis (armstrong et al, 1995) . myelin of both the cns and pns has a distinctive composition that differs somewhat from that of most cellular membranes. it is the major component of white matter of the cns, accounting for about half the dry weight of this tissue, and is responsible for its glistening white appearance. the same is true for larger nerves of the pns, such as the sciatic nerve. myelin in situ has a water content of about 40%, and is characterized by its high lipid content (70-85% of its dry mass) and its correspondingly low protein content (15-30%). most biologic membranes have a much higher protein:lipid ratio, usually somewhere around unity. the insulating properties of myelin, vital to its physiological function, are related to this high lipid content. the high lipid content of myelin also results in a buoyant density less than that of other biologic membranes, and advantage can be taken of this to isolate myelin with a high yield and a high degree of purity (norton and poduslo, 1973a) . the lipid content of cns myelin (table 1) is characterized by high levels of galactolipids (about 32% of lipid dry weight), including galactocerebroside (gal-c) and its sulfated derivative, sulfatide, and cholesterol (about 26% of total lipid weight), with phospholipids accounting for most of the remainder (norton and cammer, 1984; dewille and horrocks, 1992; morell et al., 1994) . plasmalogens, phospholipids having a fatty aldehyde linked to the c1 of glycerol instead of a fatty acid, are especially prominent in myelin. ethanolamine plasmalogens and phosphatidylcholine (lecithin) are the major phospholipid species. gangliosides are also present in minor amounts. pns myelin has a similar composition, although there are quantitative differences (see smith, 1983) . pns myelin has less cerebroside and sulfatide and more sphingomyelin than cns myelin. these differences are minor, however, relative to the larger differences in protein composition discussed later. the protein composition of myelin is relatively simple in that a few major structural proteins account for the bulk of the total protein ( table 2 ). the plp and the myelin basic proteins (mbp) together account for about 80% of the protein content of cns myelin (nornorton and cammer, 1984; morell et al., 1994; and morell and toews, 1996b , for references and additional details. bother lipids are also present in myelin, including gangliosides, galactosyl diglycerides, and fatty acid esters of cerebroside; although not shown in the table, polyphosphoinositides may account for up to 7% of total myelin lipid phosphorus (see morell et al., 1994) . ccalculated from data in norton and cammer (1984) , using 800 and 750 as average molecular weights for phosphoglycerides and sphingomyelin, respectively. aabbreviations: epg, ethanolamine phosphoglycerides; cpg, choline phosphoglycerides; spg, serine phosphoglycerides; ipg, inositol phosphoglycerides. eprimarily ethanolamine plasmalogens. fvalue includes both spg and ipg. ton and cammer, 1984; morell et al, 1994) . in contrast, p0 protein, a protein not found in the cns, accounts for more than half the total protein of pns myelin. aonly major proteins are shown; see text for discussion of other myelin proteins, and morell et al (1994) and newman et al. (1995) for additional references and details. babbreviations: plp, proteolipid protein; mbp, myelin basic protein; cnp, cyclic nucleotide phosphodiesterase; mag, myelin-associated glycoprotein; pmp-22, peripheral myelin protein-22. c composite values representative of adult mammalian myelin. dalthough mrna for this protein has been detected, the protein itself, if present at all, is present in myelin at only low to undetectable levels. mbp, p2-protein, and pmp-22 account for most of the remainder of pns myelin proteins. plp, the major protein component of cns myelin, is present in pns myelin at only very low levels, if at all (see later). in both cns and pns myelin, there are a number of other minor but integral protein components, and the list will continue to grow as research continues. these include structural proteins and proteins involved in cell-cell interactions, as well as a large number of enzymes, receptors, and second messenger-related proteins. all of these have vital roles in maintaining the complex structure of myelin and/or in its function. characteristics of some myelin proteins, including selected aspects of their gene structure and expression, follows, but it is necessarily brief. for a more detailed discussion of individual myelin proteins, see lemke (1988) , morell et al. (1994) , campagnoni (1995) , and newman et al. (1995) . it is worth noting at this point that the composition of myelin changes during development, with the first myelin deposited having a somewhat different composition than that present in adults (norton and cammer, 1984) . in the rat brain, myelin galactolipids increase by about 50%, and phosphatidylcholine decreases by a similar amount. similar changes have been noted in human myelin as well. other minor changes in lipids and gangliosides also occur. the protein portion of myelin also changes somewhat during development; both mbp and plp increase during development, whereas the amount of higher molecular weight proteins decreases. myelin basic proteins (mbps) are highly basic proteins of related isoforms derived from alternative splicing of a single gene. the mbp gene consists of seven exons distributed over about 32 kb of chromosome 18 in the mouse (roach et al, 1985) and human (sparkes et al, 1987) and chromosome 1 in the rat (koizumi et al., 1991) ; at least six transcripts are expressed via alternative splicing of rna (table 3 ). the mbp gene is actually a "gene within a gene," being part of a much larger (approx. 105 kb in mice and 179 kb in humans) transcriptional unit, called the golli-mbp gene pribyl et al, 1993) . portions of the golli-mbp gene are expressed outside the nervous system, including the immune system, although the exact function of these gene products remains unknown. this may be of relevance to clinical disorders related to autoimmunity against mbp. expression of the various mbp protein products is also very complex; in addition to alternative splicing of a number of exons, there is also considerable transcriptional and posttranscriptional control (see campagnoni, 1995, for details) . this complexity of gene expression is augmented by posttranslational protein modifications, including loss of c-terminal arginine, n-acylation, glycosylation, phosphorylation, methylation, deamination, and substitution of some arginine residues with citrulline (toews and morell, 1987; smith, 1992; morell et al, 1994) . mbps are extrinsic membrane proteins localized to the cytoplasmic membrane surface (major dense line) of myelin of both the cns and pns. in the cns this protein accounts for approximately 30% of the total myelin protein, whereas in the pns it accounts for only 18% (greenfield et al, 1980) . myelin lipids can promote mbp self-association, suggesting it may exist as oligomers on the cytoplasmic surface of the myelin membrane (smith, 1992) . it has been suggested that stabilization and maintenance of the myelin structure may be due to specific associations between mbps and sulfatides and gangliosides (ong and yu, 1984; yu, 1989, 1992; mendz, 1992; smith, 1992) . proteolipid protein (plp) and dm20 integral membrane proteins with several transmembrane domains. plp is the most abundant protein in cns myelin (50%), and although mrna for this protein is present in the pns, the protein itself is present at only very low levels in pns myelin and its function in pns myelin is unknown kamholz et al., 1992) . a report (garbern et al, 1997 ) of a human plp null mutation phenotype characterized by a demyelinating peripheral neuropathy suggests that plp/dm20 is necessary for proper myelin formation in the pns as well as the cns. this report also demonstrates by immunoelectron microscopy the presence of plp in compact pns myelin (however, see also puckett et al., 1987) . like mbp, plp is one of the products of alternative splicing of a single gene, having a molecular weight of approximately 25kda. dm20, a second isoform that migrates as a 20kda band on sds gel electrophoresis, is identical to plp except for the deletion of amino acid residues 116-150 (macklin, 1992) . the plp-dm20 gene, located on chromosome x in the mouse, rat, and human, is approximately 17 kb in length and consists of seven exons. the alternative splicing of this gene to give plp and/or dm20 is developmentally regulated, with the dm20 splice product predominating during early myelination. some mutations of the plp/dm20 gene (e.g., jimpy mice; see later this chapter) result in developmental abnormalities prior to any myelination, suggesting that gene may be involved in other functions besides myelination. in addition, expression is not confined to myelin-producing oligodendrocytes in the cns. the role of protein products of this gene outside the nervous system remain unknown, however. missense mutations of the plp/dm20 gene give rise to a host of cns pathologies, the most devastating being pelizaeus-merzbacher disease (pmd) (see seitelberger, 1995) . in some forms of pmd, there is complete deletion of the plp/dm20 gene (raskind et al, 1991) . the described single base pair deletion in humans leads to the absence of plp/dm20 expression; this produces a disease similar to, but less severe than, classic pmd, but also involving a progressive demyelinating peripheral neuropathy (garbern et al, 1997) . a number of both positive and negative cis-acting elements, as well as some trans-acting factors, have been identified for this gene (see campagnoni, 1995, for details) . in its orientation in the myelin membrane, the extracellular domains of plp may be instrumental in stabilizing the intraperiod line of myelin . in addition, plp may play an active role as an ion channel (toews and morell, 1987; lees and bizzozero, 1992) . as noted previously, dm20 is generally a relatively minor product of the plp gene but it shows a pattern of developmental regulation distinct from plp. it is expressed earlier in development than plp and is the major plp gene product in the developing embryo (ikenaka et al., 1992; macklin, 1992; timsit et al., 1992) . its presence in "premyelinating" glial cells and in cells outside the glial cell lineage suggest a possible functional role unrelated to myelination. myelin-associated glycoprotein (mag) is the principal glycoprotein of central nervous system myelin (for review, see milner et al, 1990; quarles et al, 1992) . mag is heavily glycosylated and is specific to myelin sheaths, with an especially high concentration in the periaxonal regions of both cns and pns myelin. it is a member of the immunoglobulin gene superfamily (sutcliffe et al, 1983; lai et al., 1987; salzer et al, 1987) . examination of the extracellular, n-terminal domains suggest that mag is most closely related to the cell adhesion molecules n-cam, l1, and contactin. in the pns, mag immunostaining is seen in glial membranes of the schmidt-lantermann incisures, paranodal loops, and mesaxons (trapp, 1990) and is distinctly absent from the compact myelin sheath. it is thought that mag plays a major role in membrane-membrane interactions during myelin formation and maintenance quarles et al, 1992; morell et al., 1994) . it is presumed to be involved in the adhesion of the myelin sheath to the axonal plasmalemma and in membrane spacing (trapp, 1990) , and it has been implicated in various peripheral neuropathies (mendell et al, 1985; tatum, 1993) . mag exists as two isoforms (l-mag and s-mag) that are derived by alternative splicing from a single gene. l-mag is produced almost exclusively in the cns and is the predominant variant during early development and active myelination (campagnoni, 1988; trapp, 1990) . s-mag is the major isoform in the adult cns and in the pns at all ages. it is thought that the differences in distribution within the cns and pns may be associated with either the phosphorylation or other posttranslational modifications (e.g., sulfation of oligosaccharide moieties, acylation of the transmembrane domain) altering interactions with the cytoskeleton (trapp, 1990; quarles et al, 1992) . homotypic interaction may be operational in schmidt-lantermann incisures, paranodal loops, and mesaxon membranes in pns myelin, whereas heterotypic interactions with axolemmal constituents may mediate glia-axon adhesion trapp, 1990; quarles et al., 1992) . 2'-3'-cyclic nucleotide-'-phosphodiesterase (cnp) is localized within oligodendrocytes in the cns and within schwann cells in the pns. one of the earliest markers for cells of the oligodendroglial lineage, cnp is an enzyme that hydrolyzes 2',3'-cyclic nucleotides to form 2'-nucleotides exculsively. because no physiologically relevant substrate molecules have been found in myelin, however, this enzymatic activity may actually be vestigial and unrelated to its function in myelin. the current view of cnp is that it may be a key component of an interactive protein network within oligodendroglial cells, possibly involved in extension of processes (e.g., see braun et al, 1990) . it is isoprenylated, suggesting possible involvement in signal transduction processes (braun et al., 1991) . furthermore, the presence of potential nucleotide-binding domains on cnp (sprinkle, 1989) suggest it might exert regulatory influence on various cellular processes such as growth and differentiation by serving as a link between extracellular signals and intracellular effector molecules. its exact function within myelin and oligodendroglial cells, however, remains unknown. glycoprotein (mog) mylein/oligodendrocyte glycoprotein (mog) is localized primarily at the external surfaces of the myelin sheath and oligodendrocytes. it is developmentally regulated, appearing with the onset of myelination as one of the last myelin protein genes expressed (scolding et al., 1989) . features of the protein structure suggest it may be a member of the immunoglobulin gene superfamily (gardinier et al., 1992) . because anti-mog antibodies can cause demyelination in vivo (schluesener et al., 1987) , it has received attention for a potential role in autoimmune-mediated demyelination such as experimental autoimmune encephalomyelitis (eae) and multiple sclerosis (gunn et al., 1989; bernard and kerlero de rosbo, 1991) . oligodendrocyte-myelin glycoprotein (omgp) is one of the minor protein components of myelin that appears in the cns during the period of myelination. it is highly glycosylated and appears to be specific to oligodendrocytes and myelin membranes in the cns (mikol and stefansson, 1988) . the protein is anchored to the membrane through a glycosylphosphatidylinositol intermediate. a subpopulation of omgp molecules contain the human natural killer cell antigen-1 (hnk-1) carbohydrate (mikol et al, 1990b) . the presence of a tandem leucine repeat domain in the predicted polypeptide sequence and the apparent presence of omgp at the paranodal regions of the myelin sheath have lead to speculation that its major role is as an adhesion molecule that mediates axon-glial cell interactions (mikol et al., 1990a) . p0 protein (peripheral myelin protein zero) accounts for more than 50% of the protein in peripheral nerve myelin (ishaque et al., 1980) . this has lead to the suggestion that p0 is the pns equivalent of plp in the cns, although the properties of these two proteins are very different. p0 is a transmembrane protein with a glycosylated extracellular domain, a single membrane spanning region, and a highly basic intracellular domain (lemke, (lemke and axel, 1985) . it is thought to play an important role in the compaction of myelin through the homotypic interaction of molecules on adjacent myelin lamellae (lemke, 1988) . like mag, it is a member of the immunoglobulin superfamily. unlike many of the other myelin protein genes, expression of the p0 gene is highly restricted to schwann cells. the p0 gene contains six exons distributed over about 7 kb in both rats and mice (lemke, 1988; you et al, 1991) . based on transgenic experiments, elements regulating its expression appear to reside in first 1.1 kb of the 5'-flanking region (messing et al, 1992) . mutation of the p0 gene in humans is associated with charcot-marie-tooth disease, type 1b, an inherited peripheral neuropathy (hayasaka et al., 1993; kulkens et al., 1993) . peripheral nerve protein p2 is a basic protein distinct from mbp. interest in p2 arose from its ability to induce experimental allergic neuritis, a demyelinating disease of the pns (kadlubowski and hughes, 1980) . it has sequence homology to cellular retinol and retinoic acidbinding proteins (crabb and saari,1981; eriksson et al., 1981) and fatty acid-binding proteins (fabps) (veerkamp et al., 1991) , and has high affinity for oleic acid, retinoic acid, and retinol (uyemura et al., 1984) . the p2 protein gene belongs to an ancient family of fabps that diverged into two major subfamilies (medzihradszky et al., 1992) . p2 mrna levels parallel myelination during development as well as the levels of microsomal enzymes involved in fatty acid elongation. this suggests the p2 protein may be involved in fatty acid elongation or in the transport of very long-chain fatty acids to myelin (narayanan et al, 1988) . peripheral myelin protein-22 (pmp-22) is a glycoprotein with an apparent molecular weight of about 22 kda (kitamura et al., 1976; smith and perret, 1986 ). the rat and human genes have been cloned (spreyer et al., 1991; welcher et al, 1991; hayasaka et al., 1992) ; although these have a high homology to the growth arrestspecific mrnas for gas 3 and pasii-glycoprotein, the function of pmp-22 in pns myelin remains unknown. the pmp-22 gene maps to mouse chromosome 11 and a point mutation in this gene is apparently responsible for the autosomal dominant mutation in the trembler (tr) mutant mouse (suter et al, 1992a,b) . in humans, the gene maps to chromosome 17; this gene is duplicated in patients with charcot-marie-tooth disease, type 1a, and this alteration is presumably related to the pathology (patel et al., 1992; valentijn et al., 1992; see campagnoni, 1995, and newman et al., 1995 for additional references and discussion). the rat pmp-22 gene, the expression of which is largely confined to the pns, is developmentally regulated in schwann cells, where its expression coincides with myelination (spreyer et al, 1991; snipes et al., 1992) . mrna expression is coordinately down-regulated along with other myelin proteins during tellurium-induced primary demyelination and during degeneration induced by nerve transection or crush; message levels are consequently up-regulated along with other myelin genes during remyelination if it occurs (spreyer et al., 1991; toews et al, 1997) . although myelin initially was believed to be metabolically inert (due largely to the very slow metabolic turnover of some of its components) and to function exclusively as an electrical insulator, it is now known that the picture is considerably more complex and interesting. all protein and lipid components of myelin turn over with measurable turnover rates (see benjamins and smith, 1984; morell et al, 1994) . although some structural components of myelin are indeed relatively stable metabolically, with half-lives of several months, there is also a very rapid turnover of some myelin components. the phosphate groups modifying myelin basic protein in compact cns myelin turn over with half-lives of minutes or less (desjardins and , and the phosphate groups on myelin polyphosphoinositides also show a very rapid turnover rate. at least 40 different enzyme activities have been documented in cns myelin (newman et al, 1995) . in addition to cnp described previously, these include enzymes related to second messenger signalling, as well as associated receptors and g-proteins (larocca et al., 1990) . a number of enzymes involved in myelin lipid metabolism are also present, including those for phospholipid synthesis and catabolism. noteworthy among the latter are phospholipase c activities for polyphosphoinositides, and these may have important roles in signal transduction mechanisms in myelin (ledeen, 1992) . also of note are a cholesterol ester hydrolase, and udp-galactose : ceramide galactosyltransferase, the terminal enzyme in biosynthesis of galactocerebroside, the most "myelinspecific" lipid. various proteases, protein kinases and phosphatases, and transport-related enzymes are also present; of particular interest with respect to the transport-related enzymes are carbonic anhydrase (cammer et al, 1976) and na+,k+-atpase (zimmerman and cammer, 1982) . these enzymes may be involved in controlling k + levels at nodes of ranvier (lees and sapirstein, 1983) and/or in removal of carbonic acid from metabolically active axons. the cell surface area of myelin-forming cells is so large as to suggest the need for specialized structures and mechanisms for transporting components between the perikaryon and the remote extensions. there is indeed a great deal of protein and lipid transport and targeting within the myelinating glial cell, and cytoskele-tal elements play important roles in these processes. p0, mag, and laminin (a secreted extracellular matrix component ; cornbrooks et al, 1983) are synthesized and modified in the rough endoplasmic reticulum (rer) and golgi membranes of the perinuclear cytoplasm and then sorted into different carrier vesicles upon exit from the trans golgi network (trapp et al, 1993) . these proteins must be transported over millimeter distances prior to insertion into their proper surface membrane locations. various proteins enriched in compact myelin reach their proper destinations via different mechanisms. for example, p0 reaches compact myelin by vesicular transport, whereas it is the mrna for mbp that is translocated, with synthesis of mbp occurring close to the site of its insertion into the forming myelin (colman et al, 1982; trapp et al, 1987; griffiths et al, 1989) . as noted previously, the cytoskeleton plays a major role in transport and assembly of myelin. in myelinating schwann cells, microfilaments are enriched beneath the membranes of the schmidt-lanterman incisures, the outer and inner mesaxons, and portions of the outermost compact myelin lamellae and schwann cell plasma membrane (trapp et al., 1989; zimmerman and vogt, 1989; kordeli et al, 1990) . it is thought that interactions between mag and microfilaments play a role in membrane motility during myelination (trapp and quarles, 1982; trapp et al, 1984; martini and schachner, 1986; salzer et al, 1987) . mag colocalizes with microfilaments at membranes that move during internodal growth (trapp et al, 1989) . a role for mag in myelin wrapping and spacing is supported by studies showing precocious spiral wrapping by myelinating schwann cells transfected with additional copies of mag (owens et al, 1990) , and impaired or prevented wrapping when mag expression is reduced or eliminated in schwann cells (owens and bunge, 1991; mendell et al., 1985) . microfilaments also help to define and maintain organelle-rich channel regions and organelle-free nonchannel regions of the myelin internode. the channel regions are important to formation and maintenance of the myelin internode and to intracellular transport of myelin components. they include the external cytoplasmic channels, schmidt-lanterman incisures, para nodal loops, and periaxonal cytoplasm. microfilaments associated with the abaxonal plasma membrane and the adaxonal periaxonal membrane may have multiple functions in dealing with the external environment, including endocytosis (blok et al., 1982) , pinocytosis (phaire-washington et al, 1980) , and exocytosis ( john et al, 1983; koffer et al., 1990) , as well as stress resistance. just as they do in axons, microtubules may also subserve a role in the directional movement of organelles within glial cells. post-golgi vesicles transported in this way could be involved in the growth, turnover, or modification of compact myelin at distant sites. microtubules are the largest of the cytoskeletal filaments and provide a dynamic substrate for organelle trafficking and structural organization (for review see dustin, 1984; kirschner and mitchison, 1986; schroer and sheetz, 1991) . they are present in all major cytoplasmic compartments of the myelin internode, but are excluded from compact myelin (peters et al., 1991) . in myelinating schwann cells, microtubules are crucial to the transport of myelin proteins and organelles (trappet al, 1995) . this function is determined by the orientation and organization of microtubules, which in turn are influenced by axons (kidd et al., 1994) . during the formation of the myelin sheath, contact with a myelin-inducing axon results in a more complex microtubule organization (kidd et al., 1994) . as in axons, microtubules are inherently unstable and oscillate between phases of elongation and collapse (dustin, 1984; kirschner and mitchison, 1986 ). the extent of depolymerization and repolymerization is determined by complex assembly/ disassembly kinetics and can be influenced by modifications such as binding of maps (sloboda et al., 1976; pryer et al., 1992) . microtubule disassembly causes marked accumulation of p0, mag, and laminin in the perinuclear cytoplasm of myelinating schwann cells (trapp et al., 1995) . because of this, chemically induced neurotoxicity involving microtubules may lead to alterations not only in axons, but also in myelin and myelinating cells as well. in myelinating schwann cells, the major intermediate filament is vimentin (dahl et al., 1982; schachner et al., 1984; kobayashi and suzuki, 1990) . in most cells, intermediate filaments are considered to have a structural role in mechanically maintaining cell shape against external forces (klymkowsky et al., 1989; skalli and goldman, 1991) it has been proposed that vimentin intermediate filaments interact with microfilament-associated molecules and with microtubules in resisting stress (wang et al., 1993) . it is thought that intermediate filaments play a role in the process of myelination in the pns because myelinating schwann cells contain abundant intermediate filaments and the content of these filaments between myelinating and nonmyelinating schwann cells can vary substantially. because the structure and composition of myelin is unique, its formation involves activation of a set of unique genes (see lemke, 1988 , for details). these genes include those related to induction of myelination (e.g., glial-specific receptors for differentiation signals), those involved in controlling and directing the initial deposition of myelin (e.g., axon-glial cell-adhesion molecules), and those involved in actual production of compact myelin (e.g., structural proteins of myelin). genes for enzymes involved in synthesis of lipids enriched in myelin are also preferentially activated as well. the process of myelination is a highly regulated event that begins postnatally during the first few weeks of life in the rodent brain and within the third fetal trimester in the human spinal cord. in the cns of rodents, maximum levels of synthesis of myelin components and actual accumulation of myelin occurs at about 3 weeks of age (norton and poduslo, 1973b; norton and cammer, 1984; morell et al., 1994) , and although myelin accumulation continues for an extended time, the rate of synthesis declines considerably by about 6 weeks of age. this time course is similar to the profile of expression of myelin protein genes (see campagnoni and macklin, 1988) . in the rodent pns, myelination begins at about birth, peaks at about 2 weeks, and then decreases to a low basal level by the end of the first month (webster, 1971) . as is the case in the cns, the pattern of myelin synthesis and accumulation is closely paralleled by expression of mrna for pns myelin protein components (lemke and axel, 1985; stahl et al, 1990 ) and for enzymes involved in synthesis of major myelin lipids (hydroxymethylglutaryl-coenzyme a [hmg-coa] reductase, the rate-limiting enzyme in cholesterol biosynthesis; and ceramide galactosyltransferase, the ratelimiting enzyme in cerebroside biosynthesis) (lemke and axel, 1985) . regulation of the expression of myelin genes occurs at a number of different levels including promotor choice, transcription, mrna splicing and stability, translation, and posttranslational processing (for reviews see campagnoni, 1988; campagnoni and macklin, 1988; lemke, 1988; nave and milner, 1989; ikenaka et al, 1991; mikoshiba et al, 1991; campagnoni, 1995) . the synthesis and assembly of myelin has been examined by measuring incorporation of radioactive precursors into myelin components both in vivo and in tissue slices, by measuring the in vitro activities of enzymes involved in synthesis of myelin components, by examining levels of expression of mrna species for myelinrelated genes, and by actual isolation and analysis of myelin (for reviews see benjamins and smith, 1984; dewille and horrocks, 1992; morell et al., 1994) . after individual myelin components have been synthesized, they must be assembled to form mature compact myelin. some components, such as plp, which is synthesized on bound polyribosomes in the oligodendroglial perikaryon, show a time lag of about 45 min between their synthesis and their appearance in myelin, reflecting the time required for transport from their site of synthesis to the forming myelin. other components, such as mbp, show only a short lag, in keeping with their synthesis of free polyribosomes in oligodendroglial processes, near the actual site of myelin assembly. in keeping with this difference, studies have shown that myelinating cells spatially segregate mrna species for myelin-specific proteins (see trapp et al., 1987) . mrna for mbp is transported to near the sites of myelin assembly before the protein is synthesized gillespie et al., 1990) , whereas plp mrna is present in a perinuclear location. individual lipids also show different kinetics of entry into myelin following synthesis, and some of this may be due to synthesis in, or movement through, different intracellular pools benjamins and iwata, 1979 ; for review, see benjamins and smith, 1984) . the mature myelin internode contains several ultrastructurally and biochemically distinct membrane domains that include the outer plasma membrane of the myelin-forming cell and its attached compact myelin, as well as the schmidt-lanterman incisures, paranodal loops, nodal microvilli, the periaxonal membrane, and the membranes of the outer and inner mesaxons (see figs. 1 and 3) . some of these membrane domains are compositionally distinct, containing different structural proteins and differing in lipid composition as well. with respect to the pns, p0, mbp, and pmp-22 are enriched in compact pns myelin (trapp et al., 1981; omlin et al., 1982) , whereas plp and mbp predominate in compact cns myelin. the exact mechanisms by which individual components are targeted to their respective membrane domains and other molecular aspects of actual myelin membrane assembly are not well understood, but this continues to be an active area of investigation. it seems clear that cytoskeletal elements are closely involved in the intracellular sorting and transport of myelin components, as discussed in a previous section, and they presumably also function in the actual process of myelin assembly. in the mouse, terminal differentiation of myelinforming cells occurs mostly after birth, following establishment of the basic wiring of the nervous system. many neurologic mutations result in dysmyelination, the inability of myelin-forming glial cells to assemble qualitatively and/or quantitatively normal myelin (see quarles et al., 1994; nave, 1995) . because myelination is a postnatal event in rodents, this inability to assembe normal myelin is not immediately lethal, and the animals usually survive for at least several weeks. mutations that affect myelin are characterized behaviorally by abnormalities such as shivering, ataxia, and frequently including seizures; these signs of abnormal nervous system function begin at about the time when myelin accumulation becomes significant, probably an indication of the importance of myelin for motor control and normal brain function. in general, myelin-deficient mice possess a mutant gene for some structural myelin protein (see lemke, 1988) . the major known "myelin-deficient" mutations in mice are described as follows, as are some related transgenic and "knockout" mouse models. the shiverer mouse (shi, mouse chromosome 18) was one of the first neurologic mouse mutants examined at the molecular-genetic level (roach et al., 1983) . affected homozygotes lack any detectable mbp and fail to make normal cns myelin. the behavioral phenotype of this mouse is first observed within the second postnatal week, when a general body tremor, which becomes more pronounced with intentional movements, develops (biddle et al., 1973; chernoff, 1981) . the shivering behavior derives from a loss of spinal motor and reflex control and increases with age, often progressing to include seizures. the life span of the shiverer mouse is limited to approximately 6 months of age. histologic examination shows severe dysmyelination throughout the entire cns, but with normal-appearing pns myelin (privat et al., 1979; kirschner and ganser, 1980; rosenbluth, 1980) . at the ultrastructural level, the pattern of dysmyelination is dominated by a severe lack of myelin. the myelin-like structures that are occasionally present are loosely wrapped around the axon and the intracellular adhesion zone of the extended cell process that normally forms the "major dense line" of myelin cannot be discerned . the lack of proper myelin sheath formation may be the result of a defect in myelin compaction or a related process, because oligodendroglia appear to be normally differentiated. histologic evidence of dysmyelination is supported biochemically by a dramatic reduction of all major myelin proteins, and more specifically by the complete lack of mbp. this is the result of a large (20 kb) genomic deletion encompassing exons 3-7 of the mbp gene (or exons 7-11 of the larger golli-mbp gene) resulting in no coding capacity for any of the mbp isoforms (roach et al., 1985; molineaux et al., 1986) . the tremoring phenotype can be cured by a number of manipulations associated with restoration of mbp expression, indicating that the amount of mbp available to the oligodendrocytes is a rate-limiting step in the assembly of cns myelin. successful approaches include reintroduction of the entire wild-type mbp gene into the germ line of the shiverer mouse , increasing the transgene copy number shine et al., 1990) , and the reintroduction of a mbp minigene encoding only the smallest (14 kda) mbp isoform (kimura et al., 1989) . a shiverer-like phenotype can also be generated in normal mice by specifically down-regulating the amount of mbp mrna available for protein synthesis via transgenic expression of the mbp gene in "anti-sense" orientation under the control of its cognate mbp promotor (katsuki et al., 1988) . similarly, the formation of antisense mbp mrna is the presumed primary defect of the myelin-deficient (shi-mld) mouse mutant, an allele of the shiverer mutation on chromosome 18 (doolittle and schweikart, 1977; popko et al., 1988) . the presence of this antisense rna is thought to reduce and dysregulate the amount of normal mbp mrna functionally available, thereby resulting in a level insufficient for normal myelin formation (fremeau and popko, 1990; tosic et al., 1990) . the dysmyelination is less severe in the myelin deficient mouse when compared to the shiverer mouse. isolated white matter tracts in cns have a 3-4-fold increase in sodium channel density and it has been suggested that some myelin-associated molecule absent from the shiverer white matter tracts could cause a down-regulation of either the synthesis or accumulation of sodium channels in myelinating axons (noebels et al., 1991) . the lack of dysmyelination in the pns is thought to be due to the substitution of p0, the major integral protein of pns myelin, for the structural function of mbp (lemke and axel, 1985) . the mammalian plp gene is linked to the x chromosome and defects in this gene are associated with neurologic abnormalities in the mouse and with pelizaeus-merzbacher disease in humans. in the mouse, three mutations have been characterized: the jimpy (jp), myelin synthesis-deficient (jp msd), and rumpshaker (rsh). each derives from a point mutation that alters the structure of the encoded protein. in the jimpy mouse, the mutation is a single nucleotide change in the plp gene that inactivates the splice-acceptor site of intron 4. the last a-helical transmembrane domain is replaced by an aberrant carboxy terminus, and the resulting abnormally folded protein is degraded in the endoplasmic reticulum shortly after synthesis, failing to reach the golgi apparatus for further processing and transport (roussel et al, 1987) . the cns is nearly completely devoid of myelin, with less than 1% of the axons ensheathed; pns myelin, however, is ultrastructurally intact (sidman 1964; her-schkowitz et al, 1971) . there are only a few layers of abnormally thin myelin around cns myelin, consisting of either uncompacted membrane whorls or compacted myelin with abnormal ultrastructure (duncan et al, 1989) . the major cause of the dysmyelination in the jimpy mouse seems to be a lack of differentiated oligodendrocytes. the proliferation rate of oligodendrocyte precursor cells is increased but an abnormally high rate of apoptotic cell death eliminates most of these maturating oligodendrocytes (skoff, 1982; knapp et al, 1986; barres et al., 1992) . islands of myelinated fibers are formed by the few oligodendrocytes that escape degeneration and developmental arrest. the behavioral phenotype is evident at 2 weeks of age and consists of general body tremor and ataxia; the animals die with seizures and convulsions by about 4 weeks of age. heterozygous jimpy females, which are mosaics with respect to the x-linked plp gene, display normal behavior. in the allelic mouse mutant, jimpy msd, there are similar ultrastructural alterations in myelin as seen in the jimpy mouse, but about twice as many glial cells escape premature degeneration (billings-gagliardi et al, 1980) . the rumpshaker mutant is the result of a novel mutation of the plp gene (schneider et al, 1992) and displays a phenotype very different from the jimpy and the jimpy msd. rumpshaker mice have more myelin than other dysmyelinated mutants and the degree of dysmyelination varies among cns regions, with early myelinated regions appearing normal whereas late myelinating regions are severely hypomyelinated. the oligodendrocytes appear differentiated and most escape apoptotic cell death resulting in a normal complement of mature oligodendrocytes (griffiths et al, 1990) . the rumpshaker mutation appears to allow the oligodendrocyte to survive but somehow interferes with its ability to normally deposit plp in the myelin membrane (schneider et al, 1992) . although sparse, some myelin sheaths subsist in the rumpshaker mutants and these show selective immunostaining for dm20 (schneider et al., 1992) . these findings suggest that dm20 may serve a critical purpose in glial cell development that is distinct from any function in myelin formation and maintenance. p0-deficient mice have been generated by homologous recombination of the p0 gene in mouse embryonic stem cells with the cloned gene and subsequent generation of germline chimeric mice (giese et al, 1992) . animals lacking one functional copy of the p0 gene are phenotypically normal, but the homozygotes develop a behavioral phenotype by the third week of life. these mice show a body tremor and dragging movements of the hindlimbs. there is no evidence of paralysis or sei-zures and the mutants have a normal life span. histologically, the deficit is characterized by the inability of the schwann cell to assemble a compacted multilamellar pns myelin sheath. the high degree of variability in the pathology is thought to be due to the intervening actions of other proteins such as cell adhesion molecules (mag; n-cam), and perhaps other myelin proteins. using promoter and regulatory regions of the p0 gene in a fusion gene construct, schwann cells were destroyed when they began to express p0, after associating in a 1 : 1 ratio with axons (messing et al, 1992) . the behavioral phenotype of the schwann cell-ablated mice was similar to the phenotype displayed in the homogygous p0 mutants. a proliferation of nonmyelin-forming schwann cells was induced along with skeletal muscle atrophy. the trembler mouse (tr; mouse chromosome 11) contains a mutation of the pmp-22 gene, which results in pns dysmyelination. the pmp-22 gene encodes an integral membrane protein specific to schwann cells (22 kda peripheral myelin protein) believed to be important for normal schwann cell development (spreyer et al, 1991; welcher et al., 1991) . histologically, the majority of large caliber axons in the sciatic nerve are devoid of a myelin sheath and, if present at all, these are abnormally thin and relatively uncompacted (henry and sidman, 1988) . the total number of schwann cells is dramatically increased at the time of segregation of axons, and myelin deposition is arrested. in the absence of pns myelin, the mice display a behavioral phenotype characterized by a coarse-action tremor that begins at the end of the second postnatal week and results in moderate quadriparesis and a waddling gate. under controlled conditions, these animals can experience a normal life span. the quaking mouse (qk; mouse chromosome 17) is the result of an autosomal recessive mutation (sidman et al., 1964) . homozygous mice carrying the viable quaking allele (qkv/qkv) show the typical motor coordination signs of dysmyelination in the absence of seizures and have a normal life span. the myelin deficiency, characterized by fewer than normal myelin lamellae, is predominately in the brain and spinal cord, although a lack of normal compaction and enlarged intraperiod lines of some myelinated fibers has been noted in the pns as well (trapp, 1988) . interestingly, the distribution of mag is shifted from the innermost myelin layer facing the axon to throughout the compact myelin sheath. myelin mutants in a number of other species besides humans and mice have also been described (see duncan, 1995 , for detailed discussion). these include x-linked mutations in the dog (shaking pup, griffiths et al., 1981) , pig (type a iii hypomyelinogenesis congenita, blakemore et al., 1974) , rat (myelin-deficient; dentinger et al., 1982; jackson and duncan, 1988) , and rabbit (paralytic tremor, taraszewska, 1988) , as well as the autosomal recessive taiep mutant rat . as noted previously, myelination is a critical process in the maturation of the nervous system. it involves the synthesis of an enormous amount of specialized membrane within a relatively short period of time. much of the myelin in both the cns and the pns is formed during a relatively short "developmental window" (first few years in humans; first 30 days of age in rodents), and this period is preceded by a burst of myelinatingcell proliferation. during these time periods, a large portion of the nervous system's metabolic capacity is devoted to myelinogenesis. during these "vulnerable periods," the process of myelination is especially susceptible to perturbations such as toxic insults, nutritional deficiencies, genetic disorders of metabolism, viral infections, substances of abuse, and other environmental factors (for review, see wiggins, 1986) . insults occurring during the period of proliferation of myelinating cells may be especially disruptive, as this may lead to an irreversible deficit of myelin-forming cells and consequent permanent hypomyelination. perturbation of myelination at a later stage may result in a myelin deficit that can be reversed. depending on the timing of the insult, a myelin deficiency can result from alterations related to several different developmental events, including failure of myelin-forming cells to proliferate, reduction of axonal development resulting in fewer and/ or smaller axons to myelinate, and decreased formation of myelin at time of maximal synthesis. the morphologic term myelinopathy describes damage to white matter or myelin, and disorders of myelin can be classified by a number of different factors. such factors include the preferential effects on either the cns or on the pns or an involvement of both systems. in addition, effects on myelin can sometimes be delineated as the result of a primary effect on myelin itself or the myelinating glial cell. myelin loss due to a primary insult to myelin or the myelinating cell are termed primary demyelination. there are a number of factors relevant to selective targeting of various toxic or metabolic in-suits to myelin (for discussion, see morell et al, 1994; morell and toews, 1996a ). an intact axon is a prerequisite for maintenance of normal myelin; alterations in myelin due to an effect on the neuron or the underlying axon is termed secondary demyelination. secondary demyelination is an inevitable consequence of serious damage to neurons supporting myelinating axons or to axonal transcetion or crush (wallerian degeneration). however, the distinction between primary and secondary demyelination is often somewhat vague; the basis for this distinction usually involves morphologic evidence of the initial target site. the term hypomyelination is used to describe developmental alterations of myelination in which an insufficient amount of myelin accumulates. hypomyleination can be the result of disease processes, undernutrition, or toxic insult. the term dysmyelination, when used in its strictest sense, refers to certain inborn errors of metabolism in which a block in the breakdown of a myelin lipid causes accumulation of myelin of an abnormal composition (which eventually leads to a collapse and degeneration of myelin), but it is also in wide use as a general descriptor of situations characterized by any abnormalities in myelin. some specific myelinopathies that are preferential to developing organisms are discussed later in this chapter. additional toxicants have been demonstrated to disturb myelin in the adult animal and the morphologic descriptions and mechanistic processes involved have been previously discussed (morell, 1994; morell and toews, 1997) . these include tellurium, diphtheria toxin, 2'3'dideoxycytidine, vigabatrin, carbon monoxide, triethyltin, lead, hexachlorophene, cuprizone, and isoniazid. in the human infant, several studies have provided evidence supporting the concept of a critical period from birth to about 2 years of age, during which time the nervous system is most vulnerable to malnutrition. the production of neurons is virtually completed by about the midpoint of gestation, but glial cell production continues through the end of gestation into the second postnatal year. the vulnerability of the developing nervous system to various factors is determined by the developmental stage of the cellular activities targeted by a specific insult. the effects of an agent or condition may vary depending on the agent, the time of insult during development, and the species under study (dobbing, et al, 1971) . general factors such as undernutrition can have maximal effects on processes that are most active during what has been called the "brain growth spurt" (dobbing and sands, 1979) . depending on the developmental process ongoing at the time of exposure, alterations can be produced in either the number of neurons and extent of axonal arborizations, the number of glial cells, or the degree of myelination. myelination in both the cns and pns is sensitive to nutritional factors (see wiggins, 1986; blass, 1994) . brains of rats undernourished from birth contain a lower amount (20% deficit) of total lipids, cholesterol, and phospholipids and a 50% deficit in cerebrosides (benton et al, 1966) . following severe nutritional deprivation during lactation and post-weaning, total brain galactolipids, cholesterol, and lipid phosphorus showed a slower rate of accumulation (krigman and hogan, 1976) . lipid phosphorus and cholesterol levels recovered by adulthood, whereas galactolipids remained at a 60% decreased level. myelin recovered from undernourished rats was normal in lipid composition but siginificantly reduced in total amount (fishman et al., 1971) . a reduced proportion of basic and proteolipid protein was seen in myelin isolated from undernourished rats at postnatal days 15 and 20, but the composition was similar to normals by postnatal day 30. at all time points examined, myelin yield was 25% less than normal levels (wiggins et al., 1976) . these studies suggested that undernutrition produced a delay in myelin maturation. morphologic examination of animals undernourished from birth showed a decreased number of mature oligodendroglia and poorly stained myelin (bass et al., 1970; krigman and hogan, 1976) . the number of myelin lamellae per axon and the number of myelin lamellae for a given axon diameter were both lower (krigman and hogan, 1976) . some studies suggest that in some cases, myelination is able to catch-up and achieve normal levels once unrestricted feeding is initiated. rats deprived by an increased litter size rapidly gained body and brain weight and normal brain lipid composition within 3 weeks after weaning to an unrestricted diet (benton et al, 1966) . however, nutritional deprivation during the first 21 days of life resulted in reduced levels of total brain lipids, cerebrosides, cholesterol, and plp, and this deficit persisted through 120 days of age (bass et al., 1970) . similar persistent myelin deficits were found in brains of rats subjected to either moderate or severe food deprivation during the first 30 days of life (toews et al., 1983) . although metabolic studies showed that after 6 days of free feeding following 20 days of postnatal starvation, incorporation of labeled precursors into myelin proteins was higher than in animals starved for the entire 26 days, and it was still depressed relative to controls (wiggins et al., 1976) . severe underfeeding in rats from 1 to 14 days of age resulted in a lasting significant deficit in myelin, even with rehabilitation (wiggins and fuller, 1978) . overall, these studies point to the possibility of irreversible deficits in myelin resulting from nutritional deficiencies during development. additional studies suggest the most vulnerable period for myelin may be the time of oligodendroglial proliferation. animals deprived during this period are left with a permanent deficit of myelinforming cells, resulting in irreversible hypomyelination (wiggins and fuller, 1978) . apparently, once normal numbers of oligodendroglia have been formed, the process of myelin formation itself is somewhat more capable of nutritional rehabilitation. similar effects can be found with a specific nutritional manipulation of depleting protein in the diet. in rats subjected to a protein and calorie deficiency during gestation and lactation, glial numbers were greatly reduced, and by postnatal day 19 the majority of cells in the corpus callosum appeared to be glioblasts rather than differentiated oligodendroglia (robain and ponsot, 1978 ). an early postnatal protein deficiency resulted in reduced levels of brain myelin and an altered myelin composition in rats (nakhasi et al., 1975) . in myelin from offspring of rats maintained on a 4% protein diet during lactation, an excess of high molecular weight proteins and a deficiency of plp in heavy myelin was found at postnatal day 17, with normal protein composition seen at 53 days (figlewicz et al., 1978) . the mag persisted in its higher molecular weight form longer than normal, suggesting that protein deficiency results in a delay in development and maturation of the myelination process (druse and krett, 1979) . animals raised on a fat-deficient diet are able to synthesize all fatty acids except the essential fatty acids (linoleic and linolenic acid families). essential fatty acid deficiency induced prenatally in the mothers and postnatally in the offspring resulted in lower brain weights (white et al., 1971) , and a low level of galactolipid and plp (mckenna and campagnoni, 1979) . in the optic nerve of essential fatty acid-deficient rats, vacuolation, intramyelinic splitting, and wallerian degeneration were present (trapp and bernsohn, 1978) . several studies have demonstrated hypomyelination in the offspring of copper-deficient mothers (dipaolo et al., 1974; prohaska and wells, 1974) . in the third generation of mice maintained on a copper-deficient diet, the offspring showed approximately 60% decrease in myelin yield with the major glycoprotein shifted to a higher molecular weight (zimmerman et al., 1976) . thyroid hormones influence the temporal onset of myelination and its compositional maturation. neonatal thyroidectomy in rats results in a lasting reduction of total cerebroside in the brain and a 30% reduction in myelin yield (balazs et al., 1969) . it is thought, however, that hypothyroidism does not exert a specific effect on myelin but rather delays myelin development and matu-ration (dalal et al, 1971; walters and morell, 1981) . whereas hypothyroidism resulted in a 1-2 day delay of myelinogenesis with prolonged immature myelin formation, it eventually attained a normal composition, although the myelin deficit persisted. a classic example of differential susceptibility of the developing organism to the effects of an environmental chemical is that of inorganic lead exposure. children are more vulnerable to lead in terms of external exposure sources, internal levels of lead, and timing of exposures during development. at high exposure levels, lead induces encephalopathy in children and can be life threatening. experimental animal studies have allowed examination of various specific target sites and processes of development susceptible to lead toxicity . during development, the process of cns myelination shows an increased vulnerability to lead exposure. the amount of lead that accumulates in the brain of the developing animal during lactation can be as much as 4 times higher than brain levels in the lactating dam receiving lead in the drinking water. under these conditions, myelin was significantly reduced; however, the relationship between the axon diameter and myelin lamellae remained normal, suggesting that the hypomyelination was the result of altered axonal growth (krigman et al., 1974) . direct administration of lead via to pups intubation from 2 to 30 days of age resulted in a reduction of myelin accumulation in the forebrain and optic nerve. these effects were not due to undernutrition, as the accumulation of brain myelin was decreased significantly relative to controls undergoing a similar degree of malnourishment (toews et al., 1980) . in developing rats, there is a synergistic interaction between lead exposure and mild malnutrution induced by milk deprivation with respect to decreasing the normal developmental accumulation of myelin (harry et al., 1985) . this interaction appeared to be more prevalent in females as compared to males. the decrement in myelin induced by development exposure to inorganic lead is a long-lasting effect that persists into adulthood (toews et al., 1980 (toews et al., , 1983 . myelination is not necessarily the most sensitive target for lead as low doses sufficient to produce some microscopically discernible hemorrhagic encephalopathy in the cerebellum of young rats did not depress myelination (sundstrom and karlsson, 1987) ; this hemorrhagic encephalopathy may be related to concentration of lead in brain capillaries (toews et al., 1978) . the basic cns change induced by exposure to tet is a massive cerebral edema, restricted primarily to the white matter (magee et al., 1957; torak et al, 1970) , with the formation of intramyelinic vacuoles (jacobs et al., 1977) . the pathologic effect varies with the age of the animal (suzuki, 1971) . young rats exposed to tet develop severe spongious white matter similar to that seen in the adult, but with the absence of major clinical signs seen in the adult (suzuki, 1971; blaker et al., 1981) . it is thought that the severe paralysis seen in the adult animal is due in part to the intracranial pressure developed during severe edema, whereas the open cranial sutures in the young rat may allow for edema in the absence of increased pressure. when newborn rats are exposed to tet, brains became swollen and petechial hemorrhages are observed, particularly in the cerebellum. necrotic cells were found diffusely throughout the brain (watanabe, 1977) . when older (postnatal day 8) animals were exposed, both the hemorrhagic and necrotic changes occurred, but damage was also seen in the myelinated fibers of the brain stem and cerebellum. although the morphologic alterations in myelin dissipate with time, biochemical evidence suggests that the amount of myelin produced is decreased and that this myelin deficit persists through adulthood (blaker et al., 1981; toews et al., 1983) . chronic exposure to tet from 2 to 30 days after birth decreases myelin yield and cerebroside content (55%) and 2',3'-cyclic nucleotide 3'-phosphohydrolase activity (20%) (blaker et al, 1981) . in studies using radioactive tracer, smith (1973) demonstrated that it is the newly forming cns myelin that is preferentially susceptible to degradation by tet. interestingly, administration of tet to quaking mice did not produce intramyelinic edema (nagara et al, 1981) . when young animals are exposed to trialkyllead, the process of myelination is inhibited (konat and clausen, 1976) . unlike triethyltin, this impairment in myelinogenesis is not accompanied by edema of white matter. the impairment appears to be primarily in the deposition of myelin rather than in the program for myelination, as the protein composition of forebrain myelin isolated from triethyllead-intoxicated young rats was normal (konat and clausen, 1978) . in vitro studies suggest that the alteration involves posttranslational processing and transport of integral membrane proteins, processes particularly important for myelin proteins during development (konat and clausen, 1980; konat and offner, 1982) . hexachlorophene (2,2'-methylenebis-3,4,6-trichlorophenol) is an antimicrobial agent that has been used previously in soaps and detergents, as well as in the bathing of newborn babies to prevent bacterial infections (herter, 1959; powell et al., 1973; for review, see towfighi, 1980) . both cns and pns myelin show a severe white matter edema following exposure, and young rats are more vulnerable than adults (towfighi et al, 1974) . in young rats, edema of the myelin sheath becomes evident after postnatal day 15, probably because the myelin membrane provides a hydrophobic reservoir for accumulation of this toxic compound and thereby becomes a significant site for fluid accumulation (nieminen et al., 1973) . developmental exposure results in a decrease of the normal accumulation of myelin during development (matthieu et al., 1974) . in 22-dayold rats nursed by mothers fed hexachlorophene, there was a decrease in myelin yield, yet the myelin composition remained normal. abnormal "dissociated" myelin accounted for about 10% of the total myelin and contained the typical myelin constituents with the exception of mag, which was absent (matthieu et al., 1974) . degeneration caused by this antimetabolite involves myelin, neurons, astrocytes, and oligodendroglia. in young animals injected with 6-aminonicotinamide, the pns shows a selective swelling of schwann cell cytoplasm at the inner surface of the myelin sheath. the nerve is compressed by the swelling and results in an overgrowth of the myelin sheath (friede and bischhausen, 1978) . young ducklings fed a diet containing ihn developed a wobbling gait and head tremor after 2 weeks, progressing to ataxia and inability to stand (lampert and schochet, 1968) . examination of the cns showed spongy degeneration of the myelin-containing white matter. cuprizone (bis-cyclohexanoneoxalyhydrazone) is a copper chelator that results in cns demyelination following dietary exposure to weanling mice. the loss of myelin can reach as much as 70% in white matter regions of the cerebrum (carey and freeman, 1983) . deficits in adenosine triphosphate (atp) production secondary to reduced activity of cytochrome oxidase (a copperrequiring enzyme) may lead to alterations in energyrequiring ion transport mechanisms, but the underlying reason for targeting of this compound to cns myelin is not clear. interestingly, cuprizone inhibits carbonic anhydrase, an enzyme present in myelin, and this inhibition takes place well before any demyelination is observed (komoly et al., 1987) . in experimental studies of cuprizone neurotoxicity, mrna for mag, a protein located at the myelin-axonal interface, is downregulated during demyelination and returns to normal levels following cessation of exposure (fujita et al, 1990) . the mrna for this glycoprotein exists in two major splice variants that are both severely downregulated. on recovery, one splice variant returns to normal levels whereas the other shows an accumulation above control levels. prolonged exposure to cuprizone (9 weeks or longer in mice) results in irreversible demyelination (tansey et al., 1996) , possibly due to death of oligodendrocytes and/or oligodendrogilal precursor cells. exposure of weanling rats to a diet containing tellurium (element 52) leads to a highly synchronous demyelinating peripheral neuropathy (lampert and garrett, 1971; duckett et al., 1979; said and duckett, 1981; takahashi, 1981; bouldin et al, 1988) . when tellurium exposure is discontinued, there is rapid and synchronous remyelination. although tellurium toxicity in humans is rare, this model is of considerable interest as a system for studying the manner in which a specific metabolic insult can lead to demyelination. because there is little or no associated axonal degeneration, it has also proved useful for examining events and processes related to pns remyelination, independently of processes related to axonal regeneration. inclusion of 1-1.5% elemental tellurium in the diet of weanling rats leads to a primary segmental demyelination of about 20-25% of myelinating internodes in the sciatic nerve, but with sparing of axons (bouldin et al, 1988; harry et al, 1989) . this demyelination results in a peripheral neuropathy characterized by hindlimb paresis and paralysis. older rats are much more resistant to tellurium, and the cns is generally not affected, although some pathologic alterations can be induced with prolonged exposure periods. the nature of the underlying metabolic insult has been delineated. tellurium blocks cholesterol synthesis, specifically by inhibiting the enzyme squalene epoxidase, an obligate step in the cholesterol biosynthesis pathway (harry et al, 1989; wagner-recio et al, 1991; wagner et al, 1995) . tellurite, a water-soluble oxidized metabolite of the administered insoluble element, is the active species in vitro, effective at micromolar concentrations in a cell-free system (wagner et al., 1995) . the organotellurium compound dimethyltelluronium dichloride, (ch3)zteci2, is also effective in inhibiting squalene epoxidase in cul-tured schwann cells and in inducing demyelination when administered intraperitoneally (goodrum, 1997) . presumably, the resulting cholesterol deficit in schwann cells eventually leads to an inability to maintain preexisting myelin and to assemble new myelin; this in turn leads to the observed demyelination. although the telluriuminduced inhibition of cholesterol biosynthesis is systemic, deleterious effects are confined largely to the sciatic nerve. in the liver, which supplies cholesterol for most body tissues, the resulting intracellular cholesterol deficit results in a marked upregulation of the cholesterol biosynthetic pathway (toews et al, 1991b; wagner-recio et al, 1991; wagner et al, 1995) , presumably via well-characterized feedback mechanisms (see goldstein and brown, 1990, for review) . this allows normal levels of cholesterol synthesis in this tissue despite considerable inhibition of one of the steps in the synthesis pathway, and normal levels of lipoprotein-associated circulating cholesterol are maintained. however, unlike many other tissues, the sciatic nerve cannot use circulating cholesterol; all cholesterol required for myelin in the sciatic nerve must be synthesized locally (jurevics and morell, 1994) . this fact, coupled with the great demand for cholesterol in the rapidly myelinating pns at the time of tellurium exposure, may account for the specificity of toxicity observed. expression of mrna for myelin proteins is markedly down-regulated during the demyelinating phase of tellurium neuropathy, as is gene expression for enzymes involved in synthesis of lipids enriched in myelin (toews et al, 1990 (toews et al, , 1991a (toews et al, ,b, 1997 . the latter include hmg-coa reductase, the rate-limiting enzyme in cholesterol biosynthesis. although this enzyme is markedly upregulated in the liver (as expected from the telluriuminduced intracellular sterol deficit), it is down-regulated in the sciatic nerve in concert with other myelin-related genes (toews et al., 1991b) . failure to up-regulate the cholesterol biosynthesis pathway in the sciatic nerve is, in fact, probably the major underlying reason for the preferential susceptibility of this tissue. the co-ordinate down-regulation for myelin-related genes seen following exposure to tellurium suggests that gene expression of all proteins involved in myelin synthesis and assembly may be under the co-ordinate control of the overall program for myelination (see morell and toews, 1996a; toews et al, 1997 , for discussion). the coordinate downregulation of myelin gene expression takes place in all myelinating schwann cells and not just in those undergoing demyelination (toews et al., 1992) . thus, this downregulation is not just a secondary response to injury but rather reflects the co-ordinate control of myelin gene expression. when tellurium exposure is discontinued, there is co-ordinate up-regulation of these messages during the remyelinating period. thus, tellurium toxicity specifically leads to pns demyelination because (1) synthesis of cholesterol, a major myelin lipid, is severely inhibited; (2) unlike other tissues, peripheral nerve cannot up-regulate the synthesis of cholesterol in response to the tellurium-induced cholesterol deficit; (3) because the pns is isolated from the circulation by barriers, it cannot use circulating cholesterol derived from the diet or from synthesis in the liver; and (4) there is a particularly high demand for cholesterol in the myelinating pns at the time of tellurium exposure. the process of myelination by oligodendroglia in the cns and by schwann cells in the pns represents a complex series of metabolic and cell-biologic events involving intercellular recognition and interaction, adhesion, synthesis, sorting and assembly of specialized myelin membranes, compaction of myelin lamellae, and axonal (and possibly glial) ion channel reorganization. this entire process must be completed during an intense burst of metabolic activity at a specific predetermined interval during development, and failure to complete this program of myelination within the proper "developmental window" may have permanent deleterious effects. because myelin-forming cells are operating at near their metabolic capacity, they are especially sensitive to toxic or other types of insults during this "vulnerable period" of nervous system development, and deficiencies in myelin, either qualitative or quantitative, may result. these myelin deficiencies can result from underlying alterations of various developmental events, including failure of myelin-forming cells to proliferate in normal numbers, reduction of axonal development, and/ or decreased or altered formation of myelin at its normal time of maximal synthesis. the timing of any toxicant exposure or other insult can also differentially effect one or more of these events necessary for normal myelination. although these processes are best examined in the developing nervous system, a clearer understanding of the biochemistry, molecular biology, and cell biology of these events is also of particular relevance with regards to remyelination in injured adult nervous tissue. further delineation of the underlying nature of insults that result from toxic, genetic, nutritional, or other perturbations will also be useful in better understanding these vital processes. multipotentiality of schwann cells in cross-anastomosed and grafted unmyelinated nervesmquantitative microscopy and radioautography 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surface expression patterns in transfectants myelin deficity produced by early postnatal exposure to inorganic lead or triethyltin are persistent primary demyelination induced by exposure to tellurium alters mrna levels for nerve growth factor receptor, scip, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin proteolipid protein in rat sciatic nerve tellurium-induced alterations in hmg-coa reductase gene expression and enzyme activity: differential effects in sciatic nerve and liver suggests tissue-specific regulation of cholesterol synthesis primary demyelination induced by exposure to tellurium alters schwanncell gene expression: a model for intracellular targeting of ngfreceptor alterations in gene expression associated with primary demyelination and remyelination in the peripheral nervous system experimental lead encephalopathy in the suckling rat: concentration of lead in cellular fractions enriched in brain capillaries effect of inorganic lead exposure on myelination in the 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thickness morphological correlates of functional differentiation of nodes of ranvier along single fibers in the neurogenic electric organ of the knife fish sternarchus low density of sodium channels supports action potential conduction in axons of neonatal rat optic nerve the geometry of peripheral myelin sheaths during their formation and growth in rat sciatic nerves development of peripheral nerve fibers studies on the control of myelinogenesis. ii. evidence for neuronal regulation of myelin production a myelin protein is encoded by the homologue of a growth arrest-specific gene brain recovery from essential fatty acid deficiency in developing rats myelination: a critical stage in development early postnatal starvation causes lasting brain hypomyelination myelin synthesis during postnatal nutritional deprivation and subsequent rehabilitation in vitro studies of the development, maintenance and regeneration of the oligodendrocyte-type-2 astrocyte (o-2a) lineage in the adult central nervous system dna sequence, genomic organization, and chromosomal localization of the mouse peripheral myelin protein zero gene: identification of polymorphic alleles hypomyelination in copper-deficient rats atpase activities in myelin and oligodendrocytes isolated from the brains of developing rats and from bovine brain white matter membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of ranvier in electric ray and rat schwann cell differentiation key: cord-018276-elb93kp6 authors: li, shitao title: proteomics defines protein interaction network of signaling pathways date: 2012-12-27 journal: bioinformatics of human proteomics doi: 10.1007/978-94-007-5811-7_2 sha: doc_id: 18276 cord_uid: elb93kp6 protein interactions play fundamental roles in signaling transduction. analysis of protein–protein interaction (ppi) has contributed numerous insights to the understanding of the regulation of signal pathways. different approaches have been used to discover ppi and characterize protein complexes. in addition to conventional ppi methods, such as yeast two-hybrid (yth), affinity purification coupled with mass spectrometry (ap-ms) is emerging as an important and popular tool to unravel protein complex and elucidate protein function through the interaction partners. with the ap-ms method, protein complexes are prepared first by affinity purification directly from cell lysates, followed by characterization of their components by mass spectrometry. in contrast to most ppi methods, ap-ms reflects ppi under near physiological conditions in the relevant organism and cell type. ap-ms is also able to probe dynamic ppi dependent on protein posttranslational modifications, which is common for signal transduction. ap-ms mapping protein interaction network of various signal pathways has dramatically increased in recent years. here, i’ll present the strategies toward obtaining an interactome map of signal pathway and the methodology, detailed protocols, and perspectives of ap-ms. protein interaction plays essential role in cell structure and function. in a simpli fi ed diagram of a signaling pathway, upon interaction of a ligand, the receptor alters its conformation, such as dimerization, phosphorylation, and ubiquitination, leading to recruitment of intracellular molecules and subsequent activation of downstream signal cascades. each level of the signaling cascades requires protein interaction to work as a well-assembled, multifunctional protein complex essential for signal transduction. the functionality of proteins relies on their ability to interact with one another, whereas pathogenic conditions can re fl ect the perturbations of these protein interactions. numerous protein-protein interaction (ppi) methods have been developed, but only a few of them are used for large-scale ppi detection, including yeast twohybrid (yth), protein fragment complementation assay (pca), luciferase-mediated interactome (lumier), mammalian protein-protein interaction trap (mappit), protein array, and af fi nity puri fi cation coupled with tandem mass spectrometry (ap-ms). the yth system is the fi rst assay for analysis of large-scale protein-protein interactions and widely accepted method (fields and song 1989 ) . in yth system, interested gene (bait, x) is fused to the dna-binding (db) domain of a transcription factor such as gal4 (db-x), while the interacting protein (prey, y) is fused to an activation domain (ad) such as gal4-ad (ad-y). physical interaction between x and y brings ad and db together, which reconstitutes the transcription factor and subsequently activates the downstream reporter genes (fields and song 1989 ) . like the yth, pca requires that bait and prey are each fused with incomplete fragments of a third protein, which acts as a reporter. interaction between bait and prey proteins brings the fragments of reporter protein in close enough proximity to allow them to form a functional reporter protein (rossi et al. 1997 ) . when fl uorescent proteins are reconstituted, the pca is called bimolecular fl uorescence complementation assay (kerppola 2009 ) . lumier is basically a co-immunoprecipitation assay, in which bait is linked to an epitope for puri fi cation and prey protein is fused to renilla or fi re fl y luciferase for detection (barrios-rodiles et al. 2005 ) . in the mappit, bait and prey proteins are linked to signaling de fi cient cytokine receptor chimeras. interaction of bait and prey restores jak-stat cascade after the receptor has been stimulated with ligand, which leads to stat3-dependent reporter gene activation (eyckerman et al. 2001 ) . protein microarray is a microscopic array glass slide on which interested proteins have been af fi xed at separate locations in an ordered manner using a variety of available chemical linkers (macbeath 2002 ) . protein microarrays are typically high-density arrays that are used to identify novel proteins or protein-protein interactions. antibody microarrays are the most common analytical microarray. ap-ms is biochemical puri fi cation of protein complexes followed by characterization of their components by mass spectrometry. however, unlike the methods discussed above, ap-ms is not designed for one-to-one protein interaction (i.e., binary interaction). instead, ap-ms detects multi-protein complexes. as with 2 proteomics de fi nes protein interaction network of signaling pathways ap-ms, gene of interests is tagged with desirable epitope for af fi nity puri fi cation. various tags have been developed, such as flag tag, ha tag, glutathione s-transferase (gst) tags, the calmodulin-binding peptide, the streptavidin-binding peptide, or the in vivo biotinylation of the target tagged peptide using coexpression of the bira ligase (waugh 2005 ) . with af fi nity tag, protein complexes are enriched fi rst by af fi nity puri fi cation. one early developed ap-ms is to use the tandem af fi nity puri fi cation (tap) tag (puig et al. 2001 ) . the original tap tag is composed of a protein a tag and a calmodulin-binding peptide for two sequential enrichment puri fi cations. in the fi rst puri fi cation step, the protein complex is isolated from the cell lysate using immunoglobulin gamma (igg) resin with high protein a af fi nity. after protein complex is cleaved from the protein a tag with tev protease, the eluate undergoes second puri fi cation on an immobilized calmodulin column. to date, ap-ms has been performed in combination with other techniques, such as biochemical fractionation and chemical cross-linking, for characterization of protein complex. combining biochemical fractionations, like size fractionation, with ap-ms can provide a more precise characterization of multi-protein complexes according to the factions. for example, a combination of tap puri fi cation with standard gel fi ltration has allowed for a better characterization of rna polymerase ii complex (mueller and jaehning 2002 ) . crosslinker is used for detecting weak interactions, such as membrane complex, which may be interrupted by detergents in lysis buffer. a combination of tap with in vivo cross-linking with formaldehyde was used to identify novel proteasome interactors (tagwerker et al. 2006 ) . ap-ms can also be combined with quantitative proteomics approaches, such as silac and icat, to better understand the dynamics of protein complex assembly. stable isotope labeling by amino acids in cell culture (silac) is an approach for in vivo incorporation of a label into proteins for mass spectrometry (ms)-based quantitative proteomics (ong et al. 2002 ) . isotope-coded af fi nity tags (icat) are complementary to silac and measure dynamic changes in complexes isolated from tissues or organisms that cannot be metabolically labeled (gygi et al. 1999 ) . both entail labeling the samples with isotope labels that allow the mass spectrometer to distinguish between identical proteins in separate samples. differentially labeled samples are combined and analyzed together, and the differences in the peak intensities of the isotope pairs accurately re fl ect difference in the abundance of the corresponding proteins. given the fundamental importance of protein interactions, systematically mapping protein-protein interaction (ppi) in various species has dramatically increased in recent years. using high-throughput yth, proteome-wide physical interaction maps have been generated for several organisms: saccharomyces cerevisiae (fromont-racine et al. 1997 ; uetz et al. 2000 ; ito et al. 2001 ) , caenorhabditis elegans (walhout et al. 2000 ; reboul et al. 2003 ; li et al. 2004 ) , drosophila melanogaster (giot et al. 2003 ; guruharsha et al. 2011 ) , and human (guruharsha et al. 2011 ; rual et al. 2005 ) . virus-host protein interactomes were also explored, such as severe acute respiratory syndrome (sars)-coronavirus (pfefferle et al. 2011 ) , kaposi sarcoma herpesvirus (kshv), and varicella zoster virus (vzv) (uetz et al. 2006 ; rozen et al. 2008 ) . in addition to global mapping, protein interaction networks of several important signal pathways, such as mapk (bandyopadhyay et al. 2010 ) , tgf b ( tewari et al. 2004 , smad (colland et al. 2004 ) , and pi3k-mtor (pilot-storck et al. 2010 ) , have been investigated. in addition to yth, ap-ms is another widely used ppi tool to map protein interactomes. due to many advantages that will be discussed later, ap-ms mapping protein interaction network of various signal pathways has dramatically increased in recent years. global-wide interactomes have been established in escherichia coli (hu et al. 2009 ) and mycoplasma pneumonia (kuhner et al. 2009 ) , saccharomyces cerevisiae (krogan et al. 2006 ; gavin et al. 2006 ; ho et al. 2002 ) , drosophila melanogaster (guruharsha et al. 2011 ) , and hiv-host interactome (jager et al. 2012 ) . in vertebrate, this approach has so far been used to de fi ne proteomic subspaces or speci fi c signal pathways: antiviral innate immunity pathway (li et al. 2011 ) , autophagy pathway (behrends et al. 2010 ) , deubiquitinase interactome (sowa et al. 2009 ) , endoplasmic reticulum-associated protein degradation network (erad) (christianson et al. 2012 ) , tnf pathway (bouwmeester et al. 2004 ) , proteasome interaction network (guerrero et al. 2008 ) , and disease-related protein network (ewing et al. 2007 ) . systematic identi fi cation of protein interactions within an organism will facilitate systems-level studies of biological processes. current binary ppi networks are mainly generated by high-throughput yeast two-hybrid. due to the small overlap of these maps, it has been assumed that these maps are of low quality containing many false positives (parrish et al. 2006 ) . recent efforts to map interactions using ap-ms illustrate the promise to measure speci fi c protein interactions in vivo (instead of in yeast) and provide a more powerful tool to model the in vivo interactome. first, i discuss the advantages of ap-ms versus yth, and then focus the details of the methodology, applications, and perspectives of ap-ms. despite the wide acceptance of yth system for protein-protein interaction analysis and discovery, high-throughput yth for protein interaction network bears several major limitations: (1) reporter analysis method indirectly re fl ects protein-protein interaction which usually leads to high false positives. for example, proteins with transcriptional activity can lead to autoactivation of the reporter genes. (2) some heterologous protein expressions are incompatible or toxic to yeast, i.e., membrane proteins which are unlikely to be appropriately assayed as a fusion with a reconstituted transcription factor in yth. (3) yth cannot re fl ect the endogenous protein interactions in the relevant organism. (4) lots of signaling pathways in vertebrates do not exist in yeast. thus, interactions triggered by posttranslational modi fi cations do not occur in yeast, resulting in many intrinsic false negatives. (5) the coverage of prey library usually is not completed. in addition, in high-throughput yth, the bait expression is not monitored. heterologous full-length protein expression, especially high-molecular-weight protein, expects to have low expression level in yeast. although both yth and ap-ms detect protein-protein interaction, they have several distinct differences (table 2 .1 ). ap-ms couples af fi nity puri fi cation with mass spectrometry and requires more labor works and sophisticated equipments. basically, baits can be expressed in any cell line, which investigator is interested in. after antibiotic selection, bait expression levels are monitored in stable cell lines by western blot, and cell line expressing low bait protein level (close to endogenous level) is usually chosen for following af fi nity puri fi cation. since the bait expression is close to the counterpart endogenous protein level, we expect the puri fi ed complex re fl ects the endogenous protein interactions under physiological conditions. ap-ms also can be used to detect dynamic protein interactions dependent on protein posttranslational modi fi cation by signal stimulation. unlike yth detecting one-to-one interaction (aka binary interaction), ap-ms analyzes the entire bait complex and provides all prey information in one run. however, the puri fi ed complex represents a mix of direct and indirect binding partners since the nature of the interactions identi fi ed in ap-ms data cannot be determined to be either direct or indirect. last, protein abundance and speci fi city in different cell lines also limits the detection of protein complex. for example, mib1 and mib2 have comparable af fi nity with tbk1, but we did not detect mib2 in tbk1 complex in 293t cells by ap-ms. using real-time pcr, we found mib1 predominantly expressed in 293t cell line (li et al. 2011 ) . taken all together, ap-ms overcomes the limitations of yth discussed above except several disadvantages over yth: high cost, indirect interaction, and cell type speci fi city. the pipeline of ap-ms from gene construction to interaction network mapping is shown in fig. 2 .1 (li et al. 2011 ) . in brief, interested gene is tagged with desirable epitopes such as flag, gst, his, and biotin. depending on the puri fi cation strategy, one or two tags (usually tandem tags) are adopted. these vectors should carry one antibiotic resistance gene for mammalian cell stable line selection. after transfection or infection into the desirable mammalian cell line, cells are selected by designated antibiotics to obtain stably and close to endogenous protein expression. protein complexes are precipitated from lysates of bulk cells by using various immobilized matrixes, such as resin conjugated with antibody. protein complexes are then eluated from the matrixes after several washing steps to remove nonspeci fi c interactors. protein complex is either separated on gel following silver staining or precipitated. sliced gel bands or solution samples are analyzed by mass spectrometry. after data collection and statistical analysis, protein interaction network is generated and ready for validation and further function analysis. to purify protein complex closing to physiological level, cell line stably expressing tagged bait is a prerequisite. therefore, antibiotic resistance gene should be included in the vector for stable cell line selection. genes of interest also needs to be tagged in-frame with an epitope (at either the n or c terminus), which is used to af fi nity purify the tagged protein (aka bait) along with its interacting partners (aka prey). any af fi nity tag can be used for ap-ms in theory, and most successful tags developed to date are flag, ha, s-tag, and tandem af fi nity puri fi cation (tap) tag. each puri fi cation tag has advantages and disadvantages, and the appropriate technique should be selected depending on the goals of the experiment. for example, a single flag or ha epitope only adds 8-11 amino acids (li et al. 2011 ) , while the tap tag adds a >20-kda tag (krogan et al. 2006 ) which may cause more nonspeci fi c binding. because tag may interfere with protein expression or interaction, both n-terminal and c-terminal fusion could be tested for optimal ap-ms. for example, membrane protein may need to put the tag on the c-terminal or after signal peptide on the n-terminus. furthermore, two kinds of puri fi cation methods (single and tandem puri fi cation) are used for ap-ms, which requires bait fused with single or double epitopes, respectively. depending on the number of tags on the vector, there are one-step and two-step puri fi cation methods for speci fi c protein complex, cell line, or organism. originally developed for yeast, the fi rst tap tag consists of calmodulin-binding peptide (cbp), followed by tobacco etch virus protease (tev protease) cleavage site and protein a with high af fi nity to immunoglobulin gamma (igg). protein complex is fi rst puri fi ed from the cell lysate on an igg af fi nity resin and cleaved from the protein a tag with tev protease. the eluate is then enriched in a second af fi nity puri fi cation step on an immobilized calmodulin column. several variants of tap with different combinations of tags, such as flag-ha double tags, are developed. usually, one-step puri fi cations on average preserve weaker or more transient protein-protein interactions in the price of a higher number of nonspeci fi c binding proteins. conversely, the tandem procedure tends to yield cleaner results, but weak interactions can be lost. flag and ha double tags are most commonly applied for tandem puri fi cation of protein complexes. we compared the effect of tandem tag versus single tag puri fi cation on the yield of total prey and hcip by examining four protein complexes puri fi ed by single puri fi cation with flag versus a two-step puri fi cation with flag followed by ha (li and dorf 2013 ) . ms analysis revealed that the number of total interactors was dramatically reduced in all protein complexes (tbk1, nap1, irf3, and sintbad) isolated by tap puri fi cation. however, the ratio of hcip to total prey did not increase. consistently, more hcip were detected by single-step af fi nity puri fi cation ( fig. 2. 2 ). in brief, tandem puri fi cation reduces the nsbp at the price of hcip loss. due to on average more than 90% of proteins as nonspeci fi c binding protein in one-step puri fi cation, researchers prefer to tandem af fi nity puri fi cation to get a cleaner background if they only study on a few protein complexes. however, if the study is to map the protein interaction network of a speci fi c signaling pathway, nsbp from one-step puri fi cation can be excluded by statistical analysis of the whole database. in most proteomics experiments, the puri fi ed proteins are separated by onedimensional sds-page and stained with a mass spectrometry-compatible dye such as silver, sypro ruby, or coomassie. sds-page separation removes unwanted contaminants such as buffer components from the protein sample, and the sample complexity is decreased by separating the proteins according to molecular weight. moreover, it also can be used to compare bands distribution with and without stimulation. in some cases, like irf3 complexes shown in fig. 2 .1 , unique bands are only found in the bait complex with stimulation, indicating these interacting proteins are dependent on ligand stimulation. individual protein bands of interest are excised, or the entire lane is cut into approximately 1-mm 3 pieces. gel pieces were then subjected to an in-gel trypsin digestion procedure to produce peptides for mass spectrometry analysis. but the extraction ef fi ciency of peptides from a gel is low and dependent on the primary structure of the peptide. as an alternative approach to in-gel digestion, protein mixtures can be digested in solution without prior separation (behrends et al. 2010 ) . because buffer components, such as detergents, interfere with the mass spectrometry ionization process, protein samples need to be precipitated with trichloroacetic acid (tca), washed, and redissolved in a digestion buffer. the main advantages of solution digestion are the reduction of the time and a higher recovery of peptides compared to in-gel digestion. however, bear in mind that some proteins like membrane proteins are resistant to be redissolved. the peptide mixture can be directly introduced into the mass spectrometer or separated by hplc before mass spectrometric analysis (lc-ms). the two primary mass spectrometry methods developed for identi fi cation of proteins are electrospray ionization (esi) (fenn et al. 1989 ) and matrix-assisted laser desorption/ionization (maldi) (hillenkamp et al. 1991 ) . electrospray ionization mass spectrometry is a desorption ionization method. a sample solution is sprayed from a small tube into a strong electric fi eld in the presence of a fl ow of warm nitrogen to assist desolvation. the droplets formed evaporate in a region maintained at a vacuum of several torr causing the charge to increase on the droplets. the multiply charged ions then enter the analyzer. the most obvious feature of an esi spectrum is that the ions carry multiple charges, which reduces their mass-to-charge ratio compared to a singly charged species. this advantage allows mass spectra to be obtained for large molecules. a major disadvantage is that this technique cannot analyze mixtures very well. the other most used technique, maldi, is a two-step process. first, desorption is triggered by a uv laser beam. matrix material heavily absorbs uv laser light, leading to the ablation of upper layer (~micron) of the matrix material. a hot plume produced during the ablation contains many species: neutral and ionized matrix molecules, protonated and deprotonated matrix molecules, matrix clusters, and nanodroplets. the second step is ionization (more accurately protonation or deprotonation). in the most common instrumental designs, esi and maldi are performed with mass spectrometers capable of tandem mass spectrometry (ms/ ms) experiments. ion traps, quadrupole time-of-fl ight instruments (q-tof), fourier transform ion cyclotron resonance (ft-icr) mass spectrometers (ftms), and the orbitrap are the most common types of instrumentation now used in high-end protein analysis. most protein interactomes only represent as static entities, which however only poorly captures the dynamics of complex composition. there has been increasing efforts to detect dynamic views of interactomes using various modi fi ed ap-ms. systematic methods to map dynamic changes include semi-quanti fi cation based on total spectral counts or ion intensities of precursor peptide (ms1) or fragment ions (ms2) and use of isotopic labeling approaches to obtain more accurate relative quanti fi cation. relative quanti fi cation methods such as the stable isotope labeling by amino acids in cell culture (silac) detect differences in protein abundance among samples using nonradioactive isotopic labeling. although relative quantitation is more costly and time-consuming, and less sensitive to experimental bias than label-free quantitation, it entails labeling the samples with stable isotope labels that allow the mass spectrometer to distinguish between identical proteins in separate samples. differentially labeled samples are combined and analyzed together, and the differences in the peak intensities of the isotope pairs accurately re fl ect difference in the abundance of the corresponding proteins. thus, relative quantitation may discover the dynamic interactions by comparing the change of identical protein abundances from same bait cells with and without extracellular stimulation. absolute quantitation of proteins is also developed by using isotopic peptides entails spiking known concentrations of synthetic, heavy isotopologues of target peptides into an experimental sample (mirgorodskaya et al. 2012 ) . however, the cost of absolute quantitation is too high and not realistic for large-scale interactome mapping. as quantitative methods become more robust, there will be increasing demand for detection of dynamic protein interaction upon extracellular stimulation. for example, we revealed that ~20% protein interactions are dependent on ligand stimulation, such as viral dsrna mimics poly(di:dc), in the h uman i nnate i mmunity i nteractome for type i i nterferon (hi5) (li et al. 2011 ) . another example in insulin pathway, glatter et al. de fi ned the interaction network of insulin receptor/target of rapamycin pathway in drosophila (glatter et al. 2011 ) . they found that 22% of the detected interactions were regulated by insulin. in addition to the quantitative power of mass spectrometry, it is also crucial to establish a stable cell line sensitive to stimulations. when overexpressed in cells, bait protein may not respond to stimuli as sensitive as the corres ponding endogenous protein. in most cases, the raw data fi les are fi rst processed by the software controlling the respective mass spectrometry instrument. the generated data sets are then searched against a protein database using search engines such as mascot (hirosawa et al. 1993 ) or sequest (maccoss et al. 2002 ) . a valid approach for validation of the chosen parameters is to search the obtained data sets against a decoy protein database. the data also need to be further fi ltered by setting speci fi c thresholds such as a minimum peptide length or a speci fi c number of peptides to consider a protein identi fi cation. mass spectrometry has some intrinsic problems, such as the common problem of carryovers between mass spectrometry runs. to circumvent the carryover problem in mass spectrometry, we usually analyze the repeated sample in different batch. the carryovers in two independent ap-ms of the same bait will not be possible to show up twice. the record of each batch of ms runs will also help to discriminate the carryovers. in addition to mass spectrometry, af fi nity puri fi cation also has its own inherent false positives and false negatives, which is critical general limitation encountered in the interpretation of the ap-ms due to lack of binary interaction information. false positives are nonspeci fi c binding proteins and contaminants found in puri fi ed bait complex. several types of false positives are present in typical af fi nity puri fi ed protein samples. the most common ones are from researchers' hands when they perform puri fi cation and handle samples. these contaminants usually are keratin proteins and easy to remove from the dataset. there are also other various kinds of nonspeci fi c binding proteins: (1) proteins binding to af fi nity matrices, like stk38 and prmt5; (2) proteins bind to af fi nity tag, like kif11 binding to flag tag; (3) abundant proteins (e.g., actin, tubulin); (4) proteins prefer binding to speci fi c domain, like ribosomal proteins binding to baits with nucleic acid-binding domain; (5) and heat-shock proteins for protein folding. therefore, it is important to use cell line stably expressing baits at near physiological levels to avoid nsbps, as transient overexpression may probably result in protein aggregation and improper intracellular localization. to discriminate nsbp from the protein complex, repetition of ap-ms is mandatory. in our experiences, nsbps are dramatically different in two independent ap-ms of the same bait. proper controls including cells expressing gfp with the same epitope will be also useful to exclude nsbps. last, large database with the same af fi nity tag and the same cell line background from high-throughput study will be a good resource for identi fi cation of nsbps and hcips. if a protein is often isolated with many unrelated bait proteins, it is easily recognized through analysis of the high-throughput data. however, systematic large-scale experiment does not allow for the subjective and individual evaluation of their results, which means the removal of potential contaminating proteins cannot be based on judging individual puri fi cations. therefore, statistic tools for analysis of database are required to fi lter out nonspeci fi c proteins and yield high-con fi dence interacting proteins. for statistical analysis of ap-ms data, three main parameters are protein abundance, uniqueness (the frequency of observed protein in database), and reproduci bility. total spectral counts (tsc) have gained acceptance as a practical, label-free, semiquantitative measure of protein abundance in proteomics study. several computational tools have been developed for the processing of ap-ms data, like comppass (sowa et al. 2009 ) , saint (breitkreutz et al. 2010 ) , and mist (jager et al. 2012 ) . we designed a simpli fi ed method for analysis of ap-ms data, combining three main parameters: protein abundance, uniqueness (the frequency of observed protein in the database), and reproducibility. total spectral counts (tsc) have gained acceptance as a practical, label-free, semiquantitative measure of protein abundance for proteomics studies. we adopted the z -score statistic to compare protein abundance because z -score calculates the probability of tsc occurring within a normal distribution. however, z -score does not re fl ect reproducibility. in our protocol, each protein complex is tested in 4 ms runs, so reproducibility can be readily factored into the analysis. z -score also does not analyze information about prey occurrence (i.e., prey uniqueness). to explore the likelihood that an interaction is speci fi c, we set a value of prey occurrence at <5%. we now propose a simple 3-stage scoring system to identify hcip. this algorithm combines z -score plus prey occurrence and reproducibility (zspore) (li and dorf 2013 ) . in the zspore scoring system, each interaction must pass all three criteria to merit classi fi cation as hcip. the fl owchart of zspore is shown as in fig. 2.3 , and a detailed description is provided in sect. 2.4.6 . taken together, the zspore method combines three parameters ( z -score based on tsc, prey occurrence, and reproducibility) and is a simple, ef fi cient, and robust way to analyze ap-ms data. as with any large screening database, ap-ms also has false negatives, like lacking many known protein-protein interactions documented previously. there are several reasons why a known interaction fail to be found in ap-ms. first, statistical analysis tool may fi lter out the known interaction as a nonspeci fi c binding. second, the nature and location of the tag might interfere bait protein function and disrupt its interactions. third, to parallel comparison, all ap-ms experiments are performed in a same single condition. the generic conditions of af fi nity puri fi cation may be too harsh to preserve some protein interactions, such as the buffer for membrane proteins should be different from other ones. fourth, the known protein interaction depends on different stimulation. some proteins may be involved in several pathways and have different interactors in response to the relevant stimulation. last, the absence of detection is often due to the protein expression level in the speci fi c cell type, especially when the cells have relative low abundances of the protein. to visualize the protein interaction network formed by hcips and baits, graphic representation of two protein interactions basically consists of drawing two circles (nodes) linked by a line (edge). all interactions are combined to generate a map of (smoot et al. 2011 ) . for comprehensive and dynamic visualization of the network, various kinds of attributes can be applied to the node and the edge by representation of different color and line thickness. in addition, the functional classi fi cations of hcips can be analyzed by a few online programs. for example, hcip list can be uploaded to panther (thomas et al. 2003 ) or david (da huang et al. 2009 ) via a web interface. these programs group these proteins by protein domains, molecular functions, biological processes, and signal pathways. the functional classi fi cations may help discover common threads underlying the proteins of interest. another approach is to obtain clues from known protein interactions to discover regulation mechanisms. several protein-protein interaction databases are available for online search, repository, and free download, such as biogrid, string, intact, and mint. the biogrid database is an online protein interaction repository with data compiled through comprehensive curation efforts. the latest version searches 31,739 publications for 510,188 raw protein and genetic interactions from major model organism species . the string is a database of known and predicted protein interactions. the interactions include direct (physical) and indirect (functional) associations. string quantitatively integrates interaction data from these sources for a large number of organisms and transfers information between these organisms where applicable (szklarczyk et al. 2011 ) . the intact database provides a freely available, open-source database system and analysis tools for molecular interaction data (kerrien et al. 2012 ) . all interactions are derived from literature curation or direct user submissions and are freely available. the mint database focuses on experimentally veri fi ed protein-protein interactions mined from the scienti fi c literature by expert curators (licata et al. 2012 ) . ap-ms raw data also can be deposited in the tranche repository (smith et al. 2011 ) , which is a distributed fi le system into which any sort of proteomics data may be uploaded. the data then are distributed on the internet and downloaded by anyone who has access to the hash key identi fi ers for the data, which may be kept private or publicly released. in summary, all these free online programs are useful and convenient research tools for mapping, analysis, and repository of ap-ms data. ap-ms has applied for mapping of protein interactome of various cellular signaling pathways in mammalian cells. our lab has established an ef fi cient ap-ms pipeline for de fi ning protein interaction network and successfully applied in several pathways including h uman i nnate i mmunity i nteractome for type i i nterferon (hi5) (li et al. 2011 ) , mi rna pathway i nteractome (mii), and in fl uenza-host (ihost) protein interaction network (li and dorf, unpublished data) . detailed pipeline of our ap-ms is provided in this section, and how this applies on different pathways in mammalian cells will be discussed. genes known to regulate the studied signaling pathway are usually selected as primary baits. baits cover from extracellular signals like ligands to cognate receptors on cell membrane and to signaling intermediates, kinases, and transcription factors involved in these signaling pathways and their family members. after analysis of primary bait ap-ms, some new and important hcips with primary baits are also chosen to be as secondary baits. secondary baits will validate the association with primary baits but also expand the protein interaction network, provide new insights into this signaling pathway, and cross talk with other pathways. bait cdnas can be tagged with various epitopes, such as flag or ha epitope. as we discussed earlier, commercially available anti-flag beads have much higher af fi nity than anti-ha beads. we use two mammalian expression vectors, pcmv-3tag8 (stratagene) and viral expression vector, plpcx (clontech), for transfection and infection, respectively. vector pcmv-3tag8 harbors a hygromycin resistance gene, while plpcx confers cells' resistance to puromycin. transfection and transduction are two common dna delivery methods into mammalian cells. for cell lines easy to be transfected like hek293 cells, bait constructs are directly transfected into cells. for cell lines with low transfection ef fi ciency, such as thp-1 cell line, bait gene needs to be fi rst packaged into retroviral virion. the following infection will allow bait gene to integrate into cell genome dna and subsequent expression in cells. two days after transfection and infection, cells are treated with puromycin or hygromycin for 14 days. single colonies are picked and expanded in 6-well plates. protein expression levels in each colony are determined by immunoblotting. colony with protein expression close to endogenous level is picked up for ap-ms. most protein interactomes are descriptions of homeostasis of a speci fi c signaling pathway, such as dub network (sowa et al. 2009 ) , autophagy interaction network (behrends et al. 2010 ) , and erad interactome (christianson et al. 2012 ) . however, many protein interactions depend on protein posttranslational modi fi cations induced by different stimuli. for example, we found that about 20% interactions were ligand dependent in hi5 protein interaction network (li et al. 2011 ) . we also noticed many new interactions between in fl uenza virus protein and human host after viral infection (li and dorf, unpublished data) . therefore, in our pipeline for ap-ms, each stable cell line is divided into two groups, and cells are treated with ligand speci fi c for the signaling pathway or infected with virus for studying virus-host interactome. each group of cells is cultured in four or fi ve 15-cm 2 culture dishes (about 5 × 10 7 cells) to scale up for af fi nity puri fi cation. cells are lysed in 10 ml tap buffer (50 mm tris hcl [ph 7.5], 10 mm mgcl 2 , 100 mm nacl, 0.5% nonidet p40, 10% glycerol, phosphatase inhibitors, and protease inhibitors). after shaking on ice for 30 min, cell lysates were centrifuged for 30 min at 15,000 rpm. supernatants are collected and precleared with 50 m l of protein a/g resin. after shaking for 1 h at 4°c, resin is removed by centrifugation. cell lysates are added to 20 m l anti-flag m2 resin (sigma) and incubated on a shaker for 12 h. then the anti-flag resin is 3× washed (15 min/time) with 10 ml tap buffer. after removing the wash buffer, the resin is transferred to a spin column (sigma) and incubated with 40 m l 3× flag peptide (sigma) for 1 h at 4°c in a shaker. eluates are collected by centrifugation and stored at −80°c. puri fi ed complexes are loaded on 4-15% nupage gels (invitrogen) and run about 1 cm 2 distance for 8 min at 200 v. gels were stained using the silverquest staining kit (invitrogen). each entire stained lane was excised and rinsed twice with 50% acetonitrile. the taplin biological mass spectrometry facility (harvard medical school) performs ms analysis for our samples. excised gel bands were cut into approximately 1-mm 3 pieces. gel pieces are then subjected to a modi fi ed in-gel trypsin digestion procedure. gel pieces were washed and dehydrated with acetonitrile for 10 min followed by removal of acetonitrile. pieces were then completely dried in a speed-vac. gel pieces were rehydrated with 50 mm ammonium bicarbonate solution containing 12.5 ng/ m l modi fi ed sequencing grade trypsin (promega, madison, wi) at 4°c. after 45 min, the excess trypsin solution was removed and replaced with 50 mm ammonium bicarbonate solution to just cover the gel pieces. peptides were later extracted by removing the ammonium bicarbonate solution, followed by one wash with a solution containing 50% acetonitrile and 1% formic acid. the extracts were then dried in a speed-vac (~1 h) and stored at 4°c until analysis. on the day of analysis, the samples were reconstituted in 5-10 m l of hplc solvent a (2.5% acetonitrile, 0.1% formic acid). a nanoscale reverse-phase hplc capillary column was created by packing 5 m m c18 spherical silica beads into a fused silica capillary (100-m m inner diameter x ~12-cm length) with a fl ame-drawn tip. after equilibrating the column, each sample was loaded via a famos auto sampler (lc packings, san francisco, ca) onto the column. a gradient was formed and peptides were eluted with increasing concentrations of solvent b (97.5% acetonitrile, 0.1% formic acid). as peptides eluted, they were subjected to electrospray ionization and then entered into an ltq velos ion trap mass spectrometer (thermo fisher, san jose, ca). peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of speci fi c fragment ions for each peptide. dynamic exclusion was enabled such that ions were excluded from reanalysis for 30 s. peptide sequences (and hence protein identity) were determined by matching protein databases with the acquired fragmentation pattern by the software program sequest (thermo fisher, san jose, ca). the human ipi database (ver. 3.6) was used for searching. precursor mass tolerance was set to ±2.0 da, and ms/ms tolerance was set to 1.0 da. a reversed-sequence database was used to set the false discovery rate at 1%. filtering was performed using the sequest primary score, xcorr, and delta-corr. spectral matches were further manually examined, and multiple identi fi ed peptides (>1) per protein were required. as with many screening methods, un fi ltered ap-ms data contain many nonspeci fi c binding proteins due to some intrinsic characteristics, such as nonspeci fi c binding to bead or tag, protein aggregation, and carryover during ms runs. we now describe a simple ef fi cient statistic method, z -score plus prey occurrence and reproducibility (zspore) scoring system, for identi fi cation of hcip. using this pipeline, we achieve a higher ef fi ciency of ap-ms and better identi fi cation of high-con fi dence interacting proteins. the methods and criteria used to remove nonspeci fi c binding proteins and identify high-con fi dence interacting proteins include: (a) gfp and controls. ap-ms of gfp-flag and various controls, such as non-flag igg conjugated resin for ap-ms, were used to identify nonspeci fi c binding proteins in the database. (b) z -score. a z -score (aka a standard score) indicates how many standard deviations an element is from the mean. to calculate z -score, mass spectrometry data were transformed into a "stats table," where the columns are total spectral counts (tsc) from 4 ms runs, the rows are bait-associated proteins (table 2. 2 ). then we calculated z -score of each x i,j (i prey interacts with j bait) based on the maximum total spectral counts (tsc) of 4 ms runs. for hi5 database analysis, we set the cutoff of z -score as 2. z is the z-score, x is the value of the element, m is the population mean, and s is the standard deviation. (c) prey occurrence. we considered any prey associated with a single bait as an hcip while preys associated with all baits as nsbp. generally, we set the bar of prey occurrence as <5%, which means one speci fi c prey interacts less than 5% of total baits in the entire database. in hi5, we showed that preys that interact with less than 5 baits represented statistically signi fi cant interactions in hi5 dataset. so the threshold for prey occurrence in hi5 is set as 4. due to known high interconnectivity among selected baits, bait-to-bait interactions were considered as hcip. (d) reproducibility. each prey must appear in at least 2 out of 4 ms runs. (e) batch reproducibility. to account for possible variations in the list of background contaminants observed in our dataset that were not identi fi ed by other statistical approaches, we intentionally sequenced each duplicate puri fi ed complex in different experiments. any protein that did not appear in different puri fi cations was considered an nsbp and manually removed from hcip list. after statistical analysis of dataset, all pairwise interactions are collected and analyzed by cytoscape. several important attributes, such as z -score and tsc, can be integrated into the interaction map. except generating interaction map, the functional classi fi cations of hcips also need to be analyzed. interactors can be grouped by protein domains, molecular functions, biological processes, and signal pathways, which may help discover common mechanism underlying the proteins of interest. to fi gure out the new interactions in database, several protein-protein interaction databases such as biogrid, string, intact, and mint can be used to identify the known interaction. however, protein interactions in new publication will not be included in these databases. the interaction information is also not completed, and many known interactions may not be found in these database. therefore, it is important to dig out protein interaction information in curated literature. take together, all ap-ms data must be interpreted with care and validated with additional experiments. as with any screening approach, the database does not represent a fi nal or complete interaction network. understanding how proteins interact in complex and dynamic networks is the key to dissect the complexity of many genotype-to-phenotype relationships. the systematic mapping of physical interactions is therefore critical for post-genomic research. comprehensive analysis of protein-protein interactions is still a challenging endeavor of functional proteomics. since intrinsic negatives are inherent to every technique, the physical interaction data generated by ap-ms may carry many false positives and negatives. thus, ap-ms is unlikely to grasp the entire interactome. it is also still a challenge to develop optimal computational tools to visually and computationally represent the multiple layers of data and integrate existing biological knowledge and functional data in literature with the interactome data. since most ap-ms data represent static graph of ppi map, advanced methods have to be developed and focused on dynamic and spatial changes in ppi. we have presented the general principles of the ap-ms approach and highlighted some recent developed technologies and successful applications on various signaling pathways. despite of the increasing ap-ms data and analysis tools, there are still many major challenges. it includes (1) the speci fi city of protein complex in different cells and tissues, (2) the dynamics of protein complex with different stimulations or posttranslational modi fi cations, (3) the absolute and relative quantitation of proteins, (4) mapping of transient or weak ppi and endogenous ppi from native cells and tissues, (5) the integration of ppi data sets with the other functional data sets, (6) the standardization and benchmarking for interactome mapping, and (7) the challenges for primary cells like neuronal cells and the detection of weak endogenous interaction. given the different types of mass spectrometric instrumentation, ionization processes, and software platforms, the assessment of published data becomes increasingly dif fi cult. to facilitate sharing experimental data, common standards in data acquisition, data interpretation, and data storage are required. many processes in a cell depend on ppi, and perturbations of these interactions can lead to diseases. comprehensive knowledge of ppi network of signaling pathways will not only give us insights on how the cells respond to stimulation but will also provide new drug targets for therapeutic application. moreover, many viral and bacterial pathogens rely on host ppis to survive in host cells and tissues and exert their damaging effects. ultimately, such high-quality ppi networks will become invaluable resources for better understanding the mechanisms underlying major human diseases and will enable the better de fi nition of drug targets. shitao li , ph.d., usa shitao li is a research fellow in department of microbiology and immunobiology, harvard medical school. he obtained his ph.d. from wuhan university, china. he is a recipient of kaneb fellowship and aai abstract award (2010). dr. li studies on protein interaction network using proteomics approach. he has mapped a dynamic antiviral innate immunity protein interaction network and currently is working on virus-host protein interaction network. by examining the protein network, he is investigating the signaling mechanisms controlling innate antiviral immunity and new drug targets for host defense to viral infection. he published his research on several prestigious journals such as nature, immunity, and molecular cell. a human map kinase interactome high-throughput mapping of a dynamic signaling network in mammalian cells network organization of the human autophagy system a physical and functional map of the human tnfalpha/nf-kappa b signal transduction pathway a global protein kinase and phosphatase interaction network in yeast de fi ning human erad networks through an integrative mapping strategy functional proteomics mapping of a human signaling pathway systematic and integrative analysis of large gene lists using david bioinformatics resources large-scale mapping of human protein-protein interactions by mass spectrometry design and application of a cytokine-receptor-based interaction trap electrospray ionization for mass spectrometry of large biomolecules a novel genetic system to detect protein-protein interactions toward a functional analysis of the yeast genome through exhaustive two-hybrid screens proteome survey reveals modularity of the yeast cell machinery a protein interaction map of drosophila melanogaster modularity and hormone sensitivity of the drosophila melanogaster insulin receptor/target of rapamycin interaction proteome characterization of the proteasome interaction network using a qtax-based tag-team strategy and protein interaction network analysis a protein complex network of drosophila melanogaster quantitative analysis of complex protein mixtures using isotope-coded af fi nity tags matrix-assisted laser desorption/ionization mass spectrometry of biopolymers mascot: multiple alignment system for protein sequences based on three-way dynamic programming systematic identi fi cation of protein complexes in saccharomyces cerevisiae by mass spectrometry global functional atlas of escherichia coli encompassing previously uncharacterized proteins a comprehensive two-hybrid analysis to explore the yeast protein interactome global landscape of hiv-human protein complexes visualization of molecular interactions using bimolecular fl uorescence complementation analysis: characteristics of protein fragment complementation the intact molecular interaction database in 2012 global landscape of protein complexes in the yeast saccharomyces cerevisiae proteome organization in a genome-reduced bacterium a map of the interactome network of the metazoan c. elegans optimization and zspore analysis of af fi nity auri fi cation aoupled with tandem mass spectrometry in mammalian cells mapping a dynamic innate immunity protein interaction network regulating type i interferon production mint, the molecular interaction database: 2012 update protein microarrays and proteomics yates 3rd jr. probability-based validation of protein identi fi cations using a modi fi ed sequest algorithm absolute quantitation of proteins by acid hydrolysis combined with amino acid detection by mass spectrometry ctr9, rtf1, and leo1 are components of the paf1/rna polymerase ii complex stable isotope labeling by amino acids in cell culture, silac, as a simple and accurate approach to expression proteomics yeast two-hybrid contributions to interactome mapping the sars-coronavirus-host interactome: identi fi cation of cyclophilins as target for pan-coronavirus inhibitors interactome mapping of the phosphatidylinositol 3-kinasemammalian target of rapamycin pathway identi fi es deformed epidermal autoregulatory factor-1 as a new glycogen synthase kinase-3 interactor the tandem af fi nity puri fi cation (tap) method: a general procedure of protein complex puri fi cation elegans orfeome version 1.1: experimental veri fi cation of the genome annotation and resource for proteome-scale protein expression monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation virion-wide protein interactions of kaposi's sarcoma-associated herpesvirus towards a proteome-scale map of the human proteinprotein interaction network tranche distributed repository and proteomecommons. org cytoscape 2.8: new features for data integration and network visualization de fi ning the human deubiquitinating enzyme interaction landscape the biogrid interaction database: 2011 update the string database in 2011: functional interaction networks of proteins, globally integrated and scored a tandem af fi nity tag for two-step puri fi cation under fully denaturing conditions: application in ubiquitin pro fi ling and protein complex identi fi cation combined with in vivo cross-linking systematic interactome mapping and genetic perturbation analysis of a c. elegans tgf-β signaling network panther: a browsable database of gene products organized by biological function, using curated protein family and subfamily classi fi cation a comprehensive analysis of protein-protein interactions in saccharomyces cerevisiae herpesviral protein networks and their interaction with the human proteome protein interaction mapping in c. elegans using proteins involved in vulval development making the most of af fi nity tags key: cord-007648-tm0hn0hz authors: mockett, a.p.adrian title: envelope proteins of avian infectious bronchitis virus: purification and biological properties date: 2002-12-20 journal: j virol methods doi: 10.1016/0166-0934(85)90138-7 sha: doc_id: 7648 cord_uid: tm0hn0hz immunoadsorbents, made with monoclonal antibodies, were used to purify the spike and membrane proteins of infectious bronchitis virus (ibv). the purified proteins were inoculated into rabbits to produce antisera. the rabbit anti-spike sera neutralized the infectivity of the virus whereas the anti-membrane sera did not. ibv-infected chickens produced antibodies to both the spike and membrane proteins. both these antibodies were at their highest concentration about 9–11 days after inoculation, whereas neutralizing antibodies were present only at very low concentrations at that time. neutralizing antibodies were at their highest concentration 21 days after inoculation. a second inoculation of virus at 42 days induced an anamnestic antibody response to the spike and membrane proteins and also for the neutralizing antibodies. the neutralizing, anti-spike and anti-membrane antibodies all reached highest concentrations 7–11 days after this inoculation. the advantages of purifying viral proteins using affinity chromatography with monoclonal antibodies are discussed. avian infectious bronchitis virus (ibv) is a coronavirus whose principal site of replication is the ciliated epithelial cells of the respiratory tract mucosa of chickens. viral replication occurs in the cytoplasm of the cell and virions are formed by budding from the endoplasmic reticulum. there are three viral structural proteins: spike (s; peplomers), membrane (m) and nucleocapsid (n). s and m proteins are both glycosylated and parts of them are exposed at the surface of the virion. the spike protein consists of two glycopolypeptides, s 1 and $2, which have molecular weights of 90 kda and 84 kda, respectively (cavanagh, 1981) . the membrane protein is present as a number of distinct species which have molecular weights ranging from 23 kda to 34 kda; the molecular weight differences are associated with the various degrees of glycosylation. n protein (54 kda) is associated with the viral rna. the ibv spike protein, associated with the outer projections, plays an important part in the infection of cells. chicken antisera to this protein and spike-specific monoclonal antibodies (mockett et al.. 1984) can neutralize the infectivity of the virus. a similar function has been found for the spike protein of murine coronavirus mhv-4 (collins et al., 1982; fleming et al., 1983) and the porcine coronavirus tgev (garwes et al., 1978) . the spike protein of ibv also contains strain-specific determinants (mockett et al., 1984) . the membrane protein appears to be a more highly conserved antigen and it is possible that only a small amount (approx. 1 kda) is exposed at the viral surface (boursnell et al., 1984) . the nucleocapsid protcin interacts with the viral rna to form a helical nucleocapsid. the objectives of this work were to produce immunoadsorbents using monoclonal antibodies which have been prepared previously (mockett et al., 1984) and to purify the virus-coded proteins of the viral envelope in a single step in relatively large amounts. this allowed hyperimmune rabbit antisera to the proteins to be produced and tested for neutralizing antibodies. in addition the sequential humoral antibody response of chickens after ibv infection has been studied using the purified viral proteins and whole virus in elisas and compared to the results using the neutralization test. the massachusetts m41 strain of ibv was grown in the allantoic cavities of i 1-day-old embryonated chicken eggs and purified on isopycnic sucrose gradients as described by cavanagh (1981) . purified virus was pelleted in a 6 x 14 ml rotor at 70,000 x g for 3 h at 4°c and resuspended in phosphate-buffered saline (pbs). an equal volume of pbs containing 4% (wt./vol.) np40 was added, mixed using a dounce homogeniser and incubated for 2 h at 25°c. the material was centrifuged for 5 min in an eppendorf microcentrifuge and the resulting supernatant, containing soluble viral components, was used for the affinity chromatography purification. monoclonal antibodies (designated a38 and c!24) to the spike and membrane proteins respectively of ibv strain m41 were prepared (mockett et al., 1984) . the gammaglobulin fraction of ascitic fluids containing either anti-spike or anti-membrane monoclonal antibodies was isolated by salt precipitation using a final concentration of 18% (wt./vol.) na2so4. for the spike immunoadsorbent 5.6 mg of gammaglobulin was coupled to 0.75 mg of cnbr-sepharose 4b (pharmacia) according to the manufacturers' instructions and for the membranc immunoadsorbent 5.5 mg was coupled to the same amount of gel. unreactive groups on the gel were blocked using 1 m ethanolamine, pit 8.0, and any non-covalently bound proteins wcre removed by repeated washings with 0.1 m nahco~ buffer, ph 8.3, containing0.5 m naci and 0.1 m acetic acid buffer, ph 4.4, containing0.5 m naci. the immunoadsorbent was stored in pbs containing 0.2% nan~ at 4°c until used. it was washed twice with 3 m nh4scn in pbs containing 0.1% octylglucoside, four times with pbs and twice with pbs containing 2% np40 before use. all wash volumes were 10 ml. the solubilised virus preparation was mixed with the immunoadsorbent for 16 h at 4°c using a rotary stirrer. the gel was poured into a chromatography column and washed with pbs containing 0.1% np40 (40 ml) and pbs containing 0.1% octylglucoside (10 ml). 3 m nh,scn in pbs containing 0.1% octylglucoside was added and 10 fractions of 1 ml collected. the absorbance at 280 nm of each of the fractions was read using a sp1800 pyeunicam spectrophotometer. the fractions in the absorbance peak were dialysed against pbs. a sample of each fraction was then subjected to electrophoresis in a polyacrylamide gel. those fractions containing detectable viral protein were pooled and constituted the purified protein prepar~,tion. ten per cent polyacrylamide slab gels containing sds were used (laemmli, 1970) and after electrophoresis samples were stained first with coomassie brilliant blue r-250 and then silver (morrisey, 1981) . new zealand white rabbits were used. samples of purified proteins were mixed with an equal volume of freund's complete adjuvant and inoculated intramuscularly into the rabbits. a similar inoculation was given 1 wk later. after 5 wk the same antigen in incomplete freund's adjuvant was inoculated subcutaneously. five months later blood was collected from the ear vein and the resulting serum stored at -20°c until used. the houghton poultry research station line of rhode island red chickens was used. ibv (m41) was inoculated intratracheally (500 ciliostatic dose fifty (cd 50); darbyshire et al., 1979) into 8 chickens and sequential blood samples were taken from the wing vein (see fig. 2 for times after inoculation). sera from the blood samples were stored at -20°c until used. a serum pool for each time of sampling was made by mixing an equal volume of serum from each of the eight samples. three different antigens were used for the ei,isas: spike protein, membrane protein and ib virus. the purified spike and membrane proteins were used at a dilution of 1 : 20 in carbonate buffer whilst the purified ib virus was used at a 1 : 100 dilution. antigens were adsorbed for i h at 37°c. in the second step chicken sera were serially diluted in 0.5 m nacl containing 0.5~7c np40 (saline/np40) from an initial 6.64 iog2 dilution. any specifically bound antibody was detected using a rabbit anti-chicken igg serum ( 1 : 300) and a goat anti-rabbit igg alkaline phosphatase conjugate ( 1 : 1,000) (sigma), both diluted in saline/np40. the substrate was p-nitrophenyl phosphate (1 mg/ml)in diethanolamine buffer and the reaction stopped using 3 m naoh. each step was for 30 min at 37°c, the reaction volumes 50 pl and the plate was washed three times with pbs containing 0.1c~ tween 20 between each step. titres were calculated graphically (mockett and darbyshire, 1981) using an absorbance value of 0.1 as the cut off. the chicken antisera were tested for neutralizing antibody to ibv as described by darbyshire et al. (1979) . rabbit sera were precipitated with 18°~ (wt./vol.) nazso4 and the resulting gammaglobulin tested because rabbit sera have high concentrations of non-specific inhibitors of ib virus replication. the viral proteins purified by affinity chromatography are shown in fig. 1 . the spike protein, which is composed of two polypeptides, was the only protein detected in the two fractions shown using the sensitive silver staining procedure. similarly the membrane protein was not contaminated with other proteins, although this protein did not stain as well as the spike. there were other stained bands present, but these are artifacts sometimes observed, even in the absence of protein, with this staining procedure. the purification was highly reproducible. the purified viral proteins were inoculated into rabbits and the gammaglobulin fraction of the antisera tested for neutralizing activity. only the anti-spike gammaglobulin neutralized the virus. however the membrane protein was a good immunogen because the rabbit anti-membrane sera tested in the elisa had high activity against the whole virus: in fact the titres were higher than those of the rabbit anti-spike sera (see table 1 ). the results of testing sera from ibv-infected chickens for antibody to spike and membrane proteins showed that both anti-spike and anti-membrane antibodies were produced early after infection (see fig. 2 ). peak titres were between 9 and 11 days after infection. the antibody response to the whole virus had a similar profile. however, the the second inoculation of virus induced an anamnestic antibody response. the elisa detected a similar increase in antibody titres using spike, membrane and whole virus -the peak was at 10-11 days after infection. the neutralizing antibody response was also similar and the peak titres were at 11 days after infection which contrasted to the slow rise to the peak titres after the primary inoculation. this paper describes the application of affinity chromatography using monoclonal antibodies for the purification of the two viral structural proteins present at the surface of the ib virion -spike and membrane. a previous report has described procedures for the purification of these viral proteins and also nucleocapsid protein, the only other major structural protein (cavanagh, 1983) . ibv was solubilised in np40 detergent and centrifuged in a sucrose gradient containing this detergent in order to purify the nucleocapsid protein. the addition of 1 m naci to the sucrose solutions was required for the purification of the spike and membrane proteins, as they co-migrated in gradients containing low salt concentrations. however, the nucleocapsid protein could not be purified in gradients containing high salt concentrations. the yield of material from these gradients was relatively low, due to the limited number of fractions which contained purified viral components. in other studies purified spike material contained some nucleocapsid protein and the membrane preparation contained other proteins which were thought to be of cellular origin. there are a number of advantages in using affinity chromatography. by making use of the specificity of the antibody pure material can be isolated, even from a crude mixture of proteins. the method is very quick and easy and the immunoadsorbent can be used several times. thus, relatively large amounts of purificd material can be obtained. the availability of spike and membrane proteins in a highly purified form will allow more biochemical, structural and immunological studies to be done. the conditions used to solubilise the virus did not dissociate the two spike polypeptides, therefore, both s i and $2 were detected in thc material eluted from the anti-spike immunoadsorbent. the results of experiments using the rabbit antisera to the viral proteins confirmed the biological importance of the spike protein as only antibodies to this protein neutralized the infectivity of the virus. thc chicken, about 10 days after an ibv infection, has antibodies to both the spike and membrane proteins in its serum but only very low concentrations of neutralizing antibodies. thc profile of neutralizing antibodies shown in this paper agrees with previous published findings (holmes, 1977; mockett and darbyshire, 1981; hawkes et al., 1983) . the results show that anti-spike antibodies produced early after infection are non-neutralizing, as assessed by our in vitro technique. this raises the question as to the function of these antibodies in thc chicken. previous evidence has shown only the spike protein to be capable ofeliciting neutralizing antibodies. there is a possibility that the anti-spike antibodies could be neutralizing in vivo and the function of the anti-membrane antibodies could be similar. the possible role of these antibodies in protection remains to be resolved. purified viral proteins can be used to determine which is responsible for protecting thc chicken from infection. protection tcsts such as thc ciliostasis (darbyshire, 1980) and the mixed infcction (escherichia coil and ibv) (smith et al., 1985) tcsts are available. it is only by using methods such as affinity chromatography that stflficientl~, large amounts of pure viral proteins can be made available to enable such tests to bc done. 1984. \.'iru~ rc~ i 11. pov, cll virolog, 119, 35~. darbyshire artwrlght, lg":'bl vet ph. i). i hcsis, i. tnl,ct~,it', oi ]'he author wishes to thank ms. ,i.k.a. ('ook for her help with the neutralilatton tests, ms. debra southee for her excellent technical assistance and i)r. i'.i).k. br()wn for useful discussions. key: cord-000979-cav9n18w authors: hoppe, sebastian; bier, frank f.; nickisch-rosenegk, markus v. title: rapid identification of novel immunodominant proteins and characterization of a specific linear epitope of campylobacter jejuni date: 2013-05-29 journal: plos one doi: 10.1371/journal.pone.0065837 sha: doc_id: 979 cord_uid: cav9n18w campylobacter jejuni remains one of the major gut pathogens of our time. its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. antibody-based detection presents a suitable means to identify pathogenic bacteria. however, the knowledge about immunodominant targets is limited. thus, an approach is presented, which allows for the rapid screening of numerous cdna derived expression clones to identify novel antigens. the deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium’s pathogenicity as well as revealing potential candidates for vaccination. we have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. after subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and elisa. subsequently, seven proteins were selected for epitope mapping. for cj0669 and cj0920c linear epitopes were identified. for cj0669, specificity assays revealed a specific linear epitope site. consequently, an eleven amino acid residue sequence tlikelkrlgi was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. the innovative approach presented herein of generating cdnas of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of c.jejuni. the findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. consequently, it is desirable to simplify the identification of structural epitopes, as this would extend the spectrum of novel epitopes to be detected. c. jejuni is a gram-negative, microaerophilic bacterium possessing a helical-shaped morphology [1] . in industrialized countries, c. jejuni has been one of the primary causal agents of gastroenteritis. in 2012, in germany alone 62626 cases have been reported [2] . campylobacteriosis predominantly induces mild, self-limiting diarrhoea, however severe cases have been reported [3] . several studies have shown the potential contribution of campylobacteriosis in the development of neuropathies such as the guillain-barré syndrome [4, 5] . the prominent route of infection is the improper handling and insufficient cooking of poultry [6] . the broad distribution of this pathogen in combination with a high clinical relevance necessitates fast and reliable diagnosis. although several genomic typing methods exist [7, 8] , these are often timeconsuming and inappropriate for a point-of-care application. instead, a direct approach detecting the whole bacterium is beneficial. this can be achieved by using specific antibodies to membrane-associated antigens similar to the latex-agglutination-test that is already available for several bacterial pathogens including campylobacter [9] . in order to achieve this, copious knowledge of potentially suitable targets, i.e. immunodominant proteins, is indispensable. in the past, screening for immunogenic proteins has been carried out on nitrocellulose membranes [10] or using microarray library screening with extensive protein purification [11] . however, these methods have some major drawbacks as the former is prone to non-specific binding and cross-reactivity when using polyclonal sera for screening of bacterial libraries [12] , while the latter method is time-consuming and laborious due to the purification steps needed prior to microarray printing. as we have shown elsewhere, an approach using halotagh and specifically coated halolink tm slides is better suited to detect immunodominant proteins while reducing cross-reactivity to a minimum [13] . the halotagh provides several advantages to other commonly used tags as it enhances the amount of soluble proteins expressed [14] reducing the formation of inclusion bodies. in addition, the interaction of tag and its specific ligand is based on covalent binding [15] . this negates the need for additional purification steps as the crude lysate can simply be spotted onto coated microarrays. only the target proteins presented as fusion constructs bearing a halotagh will bind to the surface, whereas the remaining proteins are washed off. combining the described screening method with expression libraries derived from c. jejuni cdna allows for the fast analysis of hundreds of different proteins. thus, suitable immunodominant proteins can be detected, isolated and identified via sequencing the encoding cdna sequence. the generation of a cdna derived expression library offers advantages in contrast to genomic libraries. the latter demand excessive screenings as the genetic information is mostly truncated or of little relevance representing areas within the genome that do not encode for proteins, whereas the former focuses on the genes transcribed [16] . this reduces the amount of clones to be screened. nevertheless, for effective cdna library screening normalization is needed, as rrna is mainly overrepresented due to its extreme abundance within a total rna extraction prior to reverse transcription [17] . bacteria only posses a poly(a)-tail on their mrna in rare cases [18, 19] . although methods exist to isolate mrna from bacteria [20, 21] , it is generally considered to be more challenging as compared to eukaryotic rna, where oligo(dt) primers are sufficient [22] . therefore, we refrained from isolating the mrna prior to reverse transcription. instead, the generated cdna was normalized, i.e. trimmed down, afterwards by the use of a duplex-specific nuclease [23] . this approach has been shown to effectively reduce the amount of rrna-derived molecules, thereby altering the overall composition in favour of the mrnaderived cdna without including a bias [24] . further optimization of library construction was achieved by using a ligationindependent cloning as well as electroporation, which have been shown to enhance overall cloning efficiency [25, 26] . using this approach, a relatively small number of clones can be screened to illuminate immunodominant proteins effectively. the identification of previously unknown or incompletely described antigens offers several benefits and potential applications. first, these proteins might serve in a diagnostic tool to rapidly identify c. jejuni in biological samples, e.g. in food manufacturing industry or hospitals. secondly, proteins eliciting an immune response might be suitable candidates for vaccination. finally, gaining insight into the structure and function of novel immunodominant proteins might improve the overall understanding of a bacterium's pathogenicity as it could accelerate the elucidation of novel virulence-associated factors. in this paper, we show the successful screening of an expression library of c. jejuni identifying several potentially immunodominant proteins. after further investigations, we selected a subset of these proteins for epitope mapping and succeeded in identifying linear epitopes for two proteins, namely cj0669 and cj0920c not described before. furthermore, assays were performed to assess specificity of the binding as well as investigating the relevance of the amino acid residues involved via alanine scanning. additionally, the structure and antigenicity of the proteins and epitopes were modelled to analyze the suitability of the identified sequences for future applications like diagnostic tools or vaccine development. the rin values for the c. jejuni rnas isolated were above 8.5 in all cases (see s1: rin). after polyadenylation of the rna, it was reverse transcribed and subsequently normalized using a duplexspecific nuclease (dsn). assessing the performance of normalization, we sequenced a sample set of 96 individual clones after transformation with trimming and without trimming. without dsn treatment 28% of clones contained 23s rrna derived cdna and 8% other rrna derived cdna (16s and 5s). in comparison, after incubation with dsn only one clone in 96 showed a 23s rrna derived cdna. a total number of 1536 different clones were screened using halolink tm slides. we used hisj (uniprot/swiss-prot: q46125), cjaa (q0p9s0) and peb1a (q0p9x8) as positive reference markers, as they have been described previously to be immunodominant [27] [28] [29] . in contrast, argc (q9pis0) and pyrc (q0pbp6) were used as negative reference proteins. after comparing the median value and the standard deviation of each sample to the values of the positive references, a selection of 192 clones were picked to be sequenced. generally, we grouped clones into three categories after screening. group i included samples showing a higher median contrast value than all the positive references used, group ii encompassed the samples with median contrast values in between those of different positive references and group iii the remaining samples which albeit below the lowest positive reference were still above all the negative reference signals. after screening 1536 clones, 32% fell into group i, 15% encompassed group ii and 4% made up group iii while the remaining 50% were below the negative controls. the sequenced clones were ''blasted'' against the genome of c. jejuni nctc 11168 (refseq: nc_002163) to identify the corresponding genes and proteins. for further experiments, known antigens of c. jejuni found within the sequenced clones were discarded and we focused solely on 22 novel potentially immunogenic proteins. several of the identified clones carried only truncated fragments of the corresponding genes. in addition, some of the sequenced inserts possessed a frame shift. in order to overcome these limitations fulllength inserts were prepared and subcloned to express the fulllength proteins. the 22 genes and their corresponding proteins are summarized in table 1 including the length as well as the size of the protein without the attached halotagh. recombinant expression of the full-length fusion proteins was assessed by sds-page using a halotagh 488 alexa ligand, see figure 1 . the correct-sized proteins are highlighted as they are expressed fused to the 34 kda halotagh. bands at roughly 34 kda in size are visible throughout, likely representing early-termination transcripts comprised only of the halotagh. evaluating immunodominance of these proteins, we performed 32 microarray and elisa analyses using three different primary antibodies against c. jejuni. summing up these results, table 1 shows the respective mean q values and errors (n = 25) for each of the proteins identified. the highest scores were attained by cj0926 and cj0927 with 1.7760.75 and 2.2360.58 respectively, indicating a potentially stronger immunogenicity as compared to the known immunodominant proteins hisj and cjaa. however, we restrained from further characterizing the former two proteins via epitope mapping as both are highly conserved and show multiple homologies in cellular organisms (cj0927) or epsilonproteobacteria (cj0926) according to the ncbi protein cluster database [30] . the same rationale was applied for cj1576, which is conserved in cellular organisms and peaks at 1.5560.43. instead, we focused on cj0623, cj1575 and cj0669, which are highly conserved in campylobacter only as well as cj0920c and cj1380, which in turn are conserved in campylobacteraceae. cj1380, cj0623 and . for almost all proteins, bands were visible with the respective sizes of their molecular weight and the halotag tm . the halotag tm alone at 34 kda is visible throughout. the proteins were as follows: 1 -cj0476, 2 -cj1723, 3 -cj1208, 4 -cj1619, 5 -cj0920c, 6 -cj1380, 7 -cj1381, 8 -cj0669, 9 -cj1624, 10 -cj1320, 11 -cj1486, 12 -cj0130, 13 -cj1366, 14 -cj0016, 15 -cj1575, 16 -1576, 17 -cj1729, 18 -cj0571, 19 -cj0926, 20 -cj0927, 21 -cj0623 and 22 -cj1138. the pictures of the two gels as well as the righthand marker were fused by coreldraw to minimize space. for all proteins except cj0920c (5) and cj1320 (10) the overlapping peptide sequences corresponding to each protein were mapped with three different primary antibodies to c. jejuni. each peptide contains 15 amino acid residues with an overlap of 11 to each adjacent peptide. cj0669, an abc-transporter atp-binding protein, showed significant intensities in two adjacent peptide spots as shown in figure 2 . the consensus sequence derived from these spots is tlikelkrlgi, which is highly conserved in c. jejuni (see s2: tlikelkrlgi is conserved in c. jejuni). next, we assessed specificity of these signals by applying antibodies reactive to other bacteria then c. jejuni in the epitope mapping to assess whether the binding occurs via the paratope of the antibody or in a nonspecific manner. figure 3 shows the results of these investigations. the mean rfis (n = 12) of peptide 44 and 45 are at 8000 and 6000 a.u. respectively after incubation with polyclonal antibody to c. jejuni, whereas these intensities drop below 50 a.u. if an e.coli antibody is used. the remaining proteins painted a different picture. for cj1575 (s3:epitope mapping of cj1575) -a fragment with only 75 residues -no linear epitope was identified. the same was true for cj0623 (s4:epitope mapping of cj0623), cj0476 (s5:epitope mapping of cj0476) as well as cj1723 (s6:epitope mapping of cj1723) which harboured no linear epitopes. for the protein cj1380 (s7:epitope mapping of cj1380) only one peptide showed signal intensities above the positive control. another abc-transporter protein, cj0920c, possesses several regions, where signal intensities were above those of the positive controls, namely peptides 6 to 8 (aa 21 -43), 17 to 19 (aa 65 -87) as well as 56 and 57 (aa 221 -239), see s8: epitope mapping of cj0920c. the three potential antigenic regions were further assessed using transmembrane prediction tool tmhmm2.0 and the antigenic prediction tool by emboss. here, peptides 6 -8 showed a consensus sequence, namely spfavwkfldal, which ought to be presented extracellular as well as being antigenic according to the prediction tools. for the other two sites, either no antigenicity was predicted or most of the amino acids lie within a transmembrane region. this is true for amino acids 47 -69, 90 -112 and 214 -236. for a summary of the predicted characteristics, see s9: transmembrane and antigenic potential of three potential epitope sites for cj0920c.further, specificity control assays revealed that these signals do not drop significantly when using antibodies to salmonella enterica indicative of nonspecific binding to occur, see s10: specific vs. non-specific binding to potential linear epitopes of cj0920c. the influence of each amino acid within the consensus sequence tlikelkrlgi was assessed by performing an alanine scan. as figure 4 shows, the majority of amino acids in the sequence do not confer a change in binding intensity if replaced by alanine. however, in case of glycine a substantial drop in rfi values to less than 10% of the original value is observed. this indicates an important role of the glycine residue in the binding of the antibody. further, these results were highly reproducible in a second set of experiments and the specificity of the binding was evaluated using antibodies reactive to helicobacter pylori as summarized in figure 5 . the green boxes on the left represent the values of original sequence tlikelkrlgi and altered sequence tlikelkrlai after incubation with polyclonal antibodies to c. jejuni. as was observed in the previous figure, the drop in intensity is significant from a mean value of approximately 38000 a.u. to less than 7900 equal to roughly 80% decrease in the signal. in comparison, the adjacent boxes represent the data of the original sequence as well as tlikelkrlai after incubation with antibodies reactive to helicobacter pylori (orange).here, no significant difference between unaltered and altered sequence could be observed. furthermore, the mean intensities were below 800 a.u. thus, binding of an anti-helicobacter pylori antibody to the sequence is approximately 2% as compared to the original binding of polyclonal antibodies to campylobacter jejuni against this linear epitope. the same trend was observed when using antibodies reactive to s. enterica (s11: binding specificity assay of tli-kelkrlgi with anti-salmonella antibodies). this indicates low non-specific binding. modelling of cj0669's structure was performed by swiss-model using automated mode. the template chosen during automatic identification was pdb1ji0a from the rcsb protein data bank [35] , which referred to the crystal structure analysis of the abc transporter from thermotoga maritima [36] . the model spans amino acids 3 to 236 of the molecule incorporating more than 90% of the residues. figure 6 shows a graphical display of the structure with the distinct secondary structures highlighted. in addition, the epitope sequence tli-kelkrlgi is marked in orange. the first nine residues comprise parts of an alpha helix, whereas glycine and isoleucine form a loop to attach the alpha helix with a beta strand further down the primary sequence of the protein. the modelling was supported by statistical analyses including determination of a z-value [37] based on published data in the protein data bank. figure 7 shows the accuracy of the model based on the z-value calculations. we analyzed the consensus peptide sequence tlikelkrlgi by geneious pro 5.6.5. notably, when predicting secondary structure and antigenic regions by emboss garnier and antigenic tool respectively, the results matched the previous observations. an alpha helix precedes a loop that is connected to a beta-sheet. the only difference to the structural modelling by swiss model is the number of residues within the loop as emboss only includes the glycine residue in the loop. for antigenic potential, emboss antigenic predicts all amino acids within the consensus sequence to be part of antigenic sites except for lysine. the development of a cdna derived expression library and subsequent screening of the expressed fusion proteins has revealed several novel immunodominant proteins. optimization during library construction by reducing the amount of rrna molecules via dsn treatment has proven to greatly reduce the amount of rrna clones as was shown for other applications elsewhere [38, 39] . this enhances the number of clones carrying mrnaderived cdna, thus increasing the number of clones potentially expressing proteins of interest. for screening purposes antibodies were used, which were generated by immunizing rabbits with whole and partly lysed cells of c. jejuni. therefore, both membrane-associated as well as soluble proteins were available during immune response. thus, it was to be expected that the antibodies used in screening target both membrane and cytosolic proteins, which could be observed in the proteins identified. the screening not only detected the 22 proteins listed here but included other already known immunogenic proteins. these were excluded from further analyses as the main goal of this work was to identify novel proteins and their linear epitopes. the screening and analysis of immunodominant behaviour by microarrays was highly reproducible. some of the proteins showing the strongest signals during screening and microarray experiments were discarded, as they possess strong homologies within a wide spectrum of organisms. however, we focused on proteins specific for campylobacter due to the potential application in a diagnostic tool. thus, linear epitope mapping was performed on a subset of seven proteins. this revealed linear epitopes for two proteins, cj0669 and cj0920c, both abc-type transporters, which were expected to be immunogenic [40] . the identified region tlikelkrlgi of cj0669 proved to be specific showing little to none non-specific interactions with other antibodies. most notably, h. pylori antibodies did not reveal any binding to the linear epitope. in the wake of the other antibodies tested, i.e. anti-e.coli and anti-s. enterica, this strongly suggests non-specific binding to be minute. furthermore, the applicability of tlikelkrlgi as a specific target for campylobacter identification, antibody or vaccine production seems plausible. alanine scan has further identified the glycine residue to be of utmost importance for the antibody binding as replacing it by alanine reduces the intensity by at least 80%. this has been somewhat surprising as glycine is a non-charged, achiral residue. however, the presence of glycine might cause the protein to turn more easily provided by the glycine's flexibility and consequently leading to a better accessibility. thus, immunogenic ability might be rendered by the presence of glycine. in fact, epitopes containing glycine as an important residue have been reported before [41] [42] [43] . next, we modelled the 3-dimensional structure of cj0669 in order to evaluate the accessibility of the potential epitope. this is an important feature when bearing a diagnostic application in mind. from the model, we could conclude that the glycine residue and the linear epitope are most likely presented as part of a loop structure adjacent to the end of an alpha helix. these are located on the outer rim of the protein, hence easily accessible for antibody binding to occur. although, the structural information is merely based on modelling, the z-values of this model fall within close proximity to zero (-0.88) commonly associated with x-ray crystallographic results [37] . consequently, the probability of this model to match the real structure is high. these findings are supported by further calculations performed using the emboss garnier and emboss antigenic algorithms. these results underline the antigenic potential of the consensus sequence tlikelkrlgi as well as predicting almost identical secondary figure 5 . binding specificity assay of epitope sequence in alanine scan. box-whisker-plot (n = 12) of the eleven residue long consensus peptide sequence tlikelkrlgi and its derivative tlikelkrlai of cj0669 after incubation with antibodies reactive to c. jejuni (green) and h. pylori (blue). each box represents 50% of the values, while 98% fall within the whiskers. the median is represented by a horizontal line within each box and the small rectangle corresponds to the mean for each sample. for references the rfi values of the positive control, rabbit igg, and negative control, mbp, are indicated on the right for both antibody incubations. for the original sequence a mean value of 38000 a.u. can be observed after incubation with antibody to c. jejuni. this dropped to less than 8000 a.u. after alanine substitution. this represents a drop of roughly 80% in overall intensity. in contrast, neither original nor substituted peptide showed any significant mean values after incubation with the anti-h. pylori antibodies indicating specific binding to the peptide by the anti-campylobacter antibody. doi:10.1371/journal.pone.0065837.g005 structures as the automated swiss model. moreover, the secondary structure prediction changes when glycine is replaced by alanine, which further underlines the important role glycine most likely plays in antibody binding due to its ability to create a loop structure which would otherwise be absent with high probability. in conclusion, tlikelkrlgi seems a suitable candidate to be studied further and shows high potential for applications in a diagnostic tool or vaccination due to its good accessibility and antigenicity. this gains further momentum as tlikelkrlgi is highly conserved in c. jejuni, whereas other species of campylobacter, e.g. c. coli, c. upsaliensis or c. lari possess other amino acid residues within this region. for helicobacter pylori, this effect is even more pronounced. specifically, the glycine residue at the tenth position that was revealed paramount for the binding is not present in helicobacter pylori, see s2. thus, tlikelkrgli seems a suitable candidate for specific diagnostic applications targeting c. jejuni. the analysis of the remaining six proteins revealed linear epitopes only in the case of cj0920c, another abc-type transporter. the region encompassing amino acids 27 -38 spfavwkfldal seems to be the most promising as its rfi values are high in microarray experiments. furthermore, the prediction tools determined this sequence to be antigenic and located extracellularly, thus easily accessible. this is not true for the other two regions detected from cj0920c, which are either lacking antigenic potential or are located within the cell or in transmembrane regions. still, the results indicate non-specific binding to contribute mainly to the positive signal. this exempts spfavwkfldal from a suitable diagnostic application; however, further analysis might be helpful to investigate the full potential of this sequence as a specific epitope. although the analyses of full-length proteins on microarrays hint at the presence of antigenic sites within each protein, a lack of linear epitopes was observed. however, this was expected, as most naturally occurring epitopes are conformational and not linear [44] [45] [46] [47] . the current method used for epitope mapping cannot detect conformational epitopes. still, the presence of linear epitopes and their detection are important features in serological applications. this is particularly true for the high-throughput analysis of sera on microarrays as described elsewhere [48, 49] . the present work has accomplished two main goals. first, we have been able to identify a previously unknown antigenic site tlikelkrlgi of cj0669 from c. jejuni and were able to determine the important residue involved in antibody binding as well as modelling the epitope's accessibility within the full-length protein. for c. jejuni the generation of monoclonal antibodies against cj0669 is mandatory to further investigate the affinity and kinetics of the observed binding event by biacore. this might grant further insight whether or not this sequence is a suitable candidate for specific detection in a biosensor or if cj0669 is a suitable vaccine candidate. mutagenesis of cj0669 might help to illuminate the function of this protein and its potential role if any in pathogenicity. once this is determined, a monoclonal antibody might be used to cocrystallize the antigen for x-ray crystallography to assign a measured structure to the predicted model. on top of that, antibody validation by elisa with a wide array of c. jejuni isolates is needed to evaluate the applicability of the derived antibody for future clinical point-of-care devices. additionally, next-generation sequencing of transcriptomes of a broad spectrum of c. jejuni strains ought to be beneficial in analyzing the presence and expression of the protein. this might provide further insight into the suitability of the protein for clinical applications and vaccine development. consequently, it could help to identify potential sequence homologies or discrepancies, which as of now are limited to already published datasets. thus, nextgeneration sequencing might be an attractive procedure to complement the approach presented herein. secondly, we have established and applied a quick and easy method for the screening of cdna expression libraries in order to rapidly detect novel immunodominant proteins of bacteria, see figure 8 for a summary. this approach is easily transferable to other bacterial pathogens such as methicillin-resistent staphylococcus aureus, klebsiella pneumonia, neisseria gonorrhoea, pseudomonas aeruginosa, salmonella enterica and others. the information needed prior to analysis is minute, yet using fully sequenced strains is advantageous as it speeds up the identification of genes and proteins. still, even unsequenced strains such as clinical isolates can easily be analyzed and should not pose any hindrance, as homologous proteins ought to be identified via blast. the more important prerequisite for the screening is the availability of polyclonal antibodies or patient sera. in the context of a diagnostic tool to be used in hospitals, patient sera are beneficial, as they resemble the desired immune response better, thus narrowing down the results to the clinically most relevant proteins. finally, suitable references are useful, yet in most cases, some immunodominant antigens have already been described. regardless, even without a suitable known antigen, this method could identify novel immunoreactive proteins and their linear epitopes with some minor adjustments to the overall calculations. in conclusion, we have successfully shown the application of this method while gaining insight into some novel immunodominant proteins of an important gut pathogen, c. jejuni. the current research emphasizes the need for future investigations in two distinct areas. first, more insight into the newly identified proteins of c. jejuni might foster the understanding of this enigmatic pathogen and help to illuminate its pathogenicity while providing suitable means for rapid detection and combating its spreading. second, transferring this method to other bacterial pathogens will help to discover other immunodominant proteins, potentially leading to a broad spectrum of clinical applications and serological assays. additionally, it might be preferable to simplify the identification of conformational epitopes as this could greatly enhance the retrieval rate of suitable antigenic sites. the state-of-the-art technique involves the cocrystallization of an antigen with a monoclonal antibody [50, 51] . besides, mutation analysis [52] , mass-spectrometry [53] or clips technology [54] are other methods to identify structural epitopes. however, the existing methods are extremely costly, time-consuming and demand high material inputs. therefore, advances to simplify structural epitope detection are essential and would be ideally suited to be combined with our current approach. nevertheless, linear epitopes provide an important tool for clinical applications. while their low abundance poses a problem, their simplicity grants major benefits. they are rapidly synthesized and modified. this enables them to be used in a broad spectrum of assays. in a clinical context where antibody titer determination of patient sera is necessary, short peptides are extremely valuable. production costs using chemical synthesis are relatively low and easier than recombinant expression of full-length proteins. furthermore, specific peptides of pathogenic bacteria remove the need to use the entire pathogen, thus reducing the associated risk. although prediction-based strategies for antigens and linear epitopes have been published [55] [56] [57] [58] , their accuracy is often lacking [59] . in contrast, our approach offers an attractive dual procedure as it rests on experimental data that are complemented by a widespread support of bioanalytical tools. thus, such a thorough approach facilitates to focus on relevant epitopes quickly, while rapidly evaluating their suitability for prospective diagnostic applications as compared to prediction-based or experimental methods alone. consequently, we believe our present findings to be of outstanding interest for diagnostic applications and to pave the way for future implementation. moreover, the ability to quickly generate cdna libraries and identify novel immunodominant figure 8 . schematic summary of the methods involved for library construction, screening and characterization. a bacterial pathogen is selected and its rna isolated. after cdna generation, normalization is performed to minimize the number of rrna derived clones. next, screening is performed using polyclonal antibodies and potentially immunogenic proteins are selected. the corresponding clones are sequenced and the genes and proteins identified, either by exact match (sequenced strain) or homology (clinical isolate). a set of candidate proteins is selected focusing only on previously unknown antigens, while known antigens are discarded. immunodominant nature of each full-length protein is assessed by elisa and microarray analyses to further narrow down the number of proteins to be tested via epitope mapping, if desired. linear epitope mapping reveals potential antigenic sites, which are tested for specificity and in alanine scan. finally, bioinformatic tools are used to model the 3-dimensional structure, accessibility and antigenicity of each protein. afterwards, the most promising candidates can be used in future applications including but not limited to monoclonal antibody generation, kinetic measurements, structural and functional analyses and diagnostic applications. doi:10.1371/journal.pone.0065837.g008 proteins independent of the bacterium used should lay the foundations for future research with highly relevant pathogens. creating a method to extend the detection to structural epitopes would add another tier to the current approach and greatly enhance the knowledge about antigenic sites. further, we are expanding our focus to other pathogens to help elucidate novel antigens within a wide array of clinically relevant bacteria. the strain c. jejuni nctc 11168 was grown on solid karmali media for 48 h at 37 uc under microaerophilic conditions (85% n 2 , 5% o 2 and 10% co 2 ) within a hypoxic workstation (coylabs). for rna isolation, a liquid culture was prepared by inoculating 10 ml of brain-heart-infusion broth (bhi) with a single colony and incubated overnight at 37uc, 140 rpm under microaerophilic conditions. this overnight culture was used to inoculate a flask containing 100 ml fresh bhi medium and incubated for 16 h prior to harvesting. for initial screening, a rabbit polyclonal igg antibody to c. jejuni (acris ap24002pu-n) was used. for further microarray analyses of a subset of candidate proteins, elisa, epitope mapping and alanine scanning this antibody was used as well as two other rabbit polyclonal igg antibody to c. jejuni (abcam ab22542 and abd serotec 1744-9035). the immunogen used for generation of antibodies to c. jejuni was c. jejuni atcc 29428. for specificity assays rabbit polyclonal igg antibody to e.coli bl21 (micromol #322), s.enterica (abcam ab35156), s. aureus (fitzgerald 20c-cr1274rp) and h. pylori (abcam ab20459) were used respectively. detection was achieved by usage of secondary antibodies. a goat polyclonal to rabbit igg conjugated with chromeo tm -546 (abcam ab60317) for fluorescent and conjugated with horseradish peroxidase (abcam ab6721) for a colorimetric readout was applied. the cells were harvested by centrifugation (2000 x g, 10 min). the supernatant was discarded and the pellets resuspended in fresh bhi medium. subsequently, 0.5 ml of the bacterial suspension were added to 1 ml of rnaprotect bacteria reagent (qiagen) in a 2 ml tube, vigorously vortexed for 5 s and incubated for 5 min at room temperature. after centrifugation (5000 x g, 10 min) the pellets were resuspended in 200 ml of lysis buffer (30 mm tris-cl, 1 mm edta, 15 mg/ml lysozyme, .12 mau proteinase k) by pipetting and vortexing for 10 s. the solution was incubated for 10 min using a thriller thermomixer (peqlab) at room temperature and 1000 rpm. after addition of 700 ml buffer rlt and 500 ml 96% ethanol, the lysate was applied to rneasy bacteria mini kit spin columns (qiagen) for rna isolation following the manufacturer's instructions. after loading of the lysate, an on-column dnase digest was performed using rnasefree dnase i solution (qiagen) according to manufacturer's instructions. the isolated total rna was eluted in 50 ml of rnasefree water and its concentration and purity analyzed by nanodrop (peqlab) measurements. the quality of isolated rna was assessed using the rna 6000 pico kit and bioanalyzer 2100 (agilent). the total rna was diluted to a working concentration of 200 -500 pg/ml. the analysis was performed following manufacturer's instructions and the rna integrity number (rin) calculated by the 2100 expert software (agilent). the rin is defined to fall into a range of 0 to 10, with a higher score indicating an intact rna, whereas lower numbers are associated with degraded rna molecules [60] . in order to use bacterial mrna as a substrate in cdna synthesis, polyadenylation was mandatory. the tailing was achieved using the poly(a) polymerase tailing kit (epicentre) following the alternate protocol offered by the manufacturer. briefly, up to 10 mg of total rna were combined with 2 ml poly(a) polymerase reaction buffer, 2 ml 10 mm atp, 0.5 ml riboguard rnase inhibitor and 1 ml poly(a) polymerase (4 u) in a total reaction volume of 20 ml. the reaction was incubated for 20 min at 37uc, terminated by the addition of 1 ml 0.5 m edta and purified by rneasy mini kit (qiagen) following manufacturer's instructions. yield and purity were determined by nanodrop measurements. for cdna synthesis, the in-fusionh smarter tm directional cdna library construction kit (clontech) was used according to manufacturer's instructions with slight modifications. 3.5 ml total, polyadenylated rna were mixed with 1 ml of 39 in-fusion smarter cds primer, heated first for 3 min at 72uc and then incubated for additional 2 min at 42uc. after addition of 5.5 ml mastermix (2 ml 5x first strand buffer, 0.25 ml 100 mm dtt, 1 ml 10 mm dntps, 1 ml 12 mm smarter v oligonucleotide, 0.25 ml rnase inhibitor and 1 ml smartscribe tm reverse transcriptase) the tubes were incubated for 90 min at 42uc. the reaction was terminated at 68uc for 10 min. for second strand cdna synthesis two 2 ml aliquots of first strand reaction were used in long distance pcr using phusion polymerase (finnzymes). each pcr reaction was comprised as follows: 2 ml first-strand reaction, 70 ml rnase-free water, 20 ml 5x phusion hf buffer and 2 ml each of dntp mix (10 mm), 59 pcr primer ii a (12 mm), 39 in-fusion smarter pcr primer (12 mm) and phusion polymerase with a total reaction volume of 100 ml. the pcr reactions were subjected to the cycling program with 98uc for 1 min as initial denaturation followed by 15 cycles of 10 s denaturation at 98uc, 30 s of primer annealing at 65uc and 6 min extension at 72uc. for improved pcr results optimization was performed as follows; 30 ml of the 15 cycle experimental tube were transferred to a separate pcr tube, cycling commenced and aliquots of 5 ml each were collected after 15, 18, 21, 24 and 27 cycles total. the different cycles were compared by gel electrophoresis and the experimental tubes subjected to additional cycles if necessary. finally, pcr reactions were purified using the qiaquick pcr purification kit (qiagen). the purity and yield of each reaction were analyzed by nanodrop measurements. normalization of double-stranded cdna was achieved with the trimmer-2 cdna normalization kit (evrogen) to reduce the number of cdna molecules derived from rrnas. briefly, 12 ml of cdna (approx. 100 ng/ml) were mixed with 4 ml of 4x hybridization buffer. for the trimming reaction 4 ml of this mixture were distributed to four different pcr tubes and overlaid with a drop of pcr-grade mineral oil. after centrifugation (13000 x g, 2 min), the tubes were incubated for 2 min at 98uc followed by 5 h at 68uc. next, pre-heated (68uc) duplex-specific nuclease (dsn) master buffer was added to each tube and incubation prolonged for 10 min. dsn was added to the first three tubes in decreasing concentrations -1 u/ml, 0.5 u/ml and 0.25 u/ml -with the fourth tube receiving dsn storage buffer and no enzyme as a control reaction. the incubation was continued for 25 min at 68uc. after addition of 5 ml dsn stop solution and subsequent incubation for 5 min at 68uc, the tubes were placed on ice. the chilled reaction was diluted by addition of 25 ml sterile, rnase-free water. for amplification of normalized cdna, 1 ml of each reaction was used as template in pcr. each pcr reaction contained 1 ml of template from the normalization reaction, 33 ml nuclease-free water, 10 ml 5x phusion hf buffer, 1 ml 10 mm dntp mix (neb), 2 ml of each primer 59 pcr primer ii a (12 mm), 39 in-fusion smarter pcr primer (12 mm) and 1 ml phusion polymerase. the pcr was performed with initial denaturation at 98uc for 1 min and seven cycles of denaturation at 98uc for 10 s, primer annealing at 65uc for 30 s and extension at 72uc for 3 min, respectively. for optimization, the control tube was subjected to 7, 9, 11, and 13 cycles with 12 ml aliquots taken every two cycles. the optimization samples were analyzed by gel electrophoresis (1% agarose, tae, 100 v) and the optimal cycle number determined. the remaining three tubes were subjected to 9 + x cycles with x being the differential of the optimized cycles to the originally performed seven cycles. after the second pcr, the experimental reactions were compared to the optimal control reaction using gel electrophoresis as above. reactions showing a successful normalization were combined and used in a third pcr reaction. after the final pcr, the reactions were purified by qiaquick pcr purification kit. for cloning pfn18a (promega) was used as a vector, as it features a n-terminal encoded halotagh fusion protein, which allows for specific and covalent binding to a unique ligand, thus reducing background and minimizing cross-reactivity in immunoassays with halolink tm microarrays harbouring the ligand on its surface. first, the vector needed to be linearized to be used with the in-fusion cloning technology. this was achieved by reverse pcr using ifs 18a for (59 ttgataccactgcttttc-catggcgatcgcgttatc 39) and ifs 18a rev (59 tctcatcgtaccccgtgtttaaacgaattcgggctcg 39) . each reaction contained 2 ml each of 1:10 diluted pfn18a (10 ng/ml) and the two primers, 10 ml 5x phusion hf buffer, 1 ml 10 mm dntps, 0.5 ml phusion polymerase and 32.5 ml nucleasefree water to reach a total reaction volume of 50 ml. the pcr was run using a 25 cycle two-step program with 98uc denaturation for 10 s and 4 min extension at 72uc. after completion, 2 ml of dpni (20 u/ml) were added to the reaction and incubated at 37uc for 1 h. the presence of a single band was checked by gel electrophoresis and the remaining reaction purified by qiaquick pcr purification kit. cloning of normalized cdna and linearized pfn18a vector was performed following the manufacturer's instructions within the in-fusion smarter directional cdna library construction kit (clontech). electroporation 2 ml of the cloning reaction were mixed with 25 ml of electrocompetent acella tm e.coli cells (mobitec), a bl21 derivative, and electroporated in 1 mm cuvettes using the easyject plus electroporator (peqlab). conditions for electroporation were as follows: voltage = 1400 v, capacity = 25 mf, resistance = 200 v and pulse duration of 5 ms. the electroporated cells were added to 970 ml of super optimal broth with catabolite expression (soc) and incubated at 37uc for 1 h with shaking at 250 rpm. afterwards, 150 ml of the transformation reaction were plated on lysogeny broth (lb) agar containing ampicillin. for each reaction, at least two plates were prepared and incubated at 37uc for 16 h. a total number of 1536 clones were selected and transferred to 1.3 ml u96 deepwell tm plates (nunc) containing 0.8 ml lbamp. the plates were incubated overnight at 37uc, 130 rpm. on the next day, the deepwell tm plates were centrifuged, the supernatant discarded and the pellets resuspended in 370 ml of lbamp. a new set of u96 deepwell tm plates was prepared with 850 ml of fresh lb-amp and inoculated with 100 ml each from the resuspended overnight cultures. the remaining 270 ml of resuspended overnight culture were mixed with 30 ml of sterile-filtered dmso and stored at -80uc. the newly inoculated plates were incubated for 6 h at 37uc, 130 rpm. afterwards, the temperature was reduced to 20uc, incubation continued for 1 h and protein expression induced by addition of 2 ml of 0.5 m b-d-1thiogalactopyranoside (iptg). incubation persisted overnight at 20uc, 130 rpm. the cells were harvested by centrifugation (2500 x g, 10 min), the supernatant discarded and the pellets frozen at -20uc. after 15 min the pellets were resuspended in 180 ml of easylyse tm bacterial protein extraction solution (epicentre) and incubated for 5 min at room temperature. dnase i was mixed with dnase reaction buffer (10 mm tris-hcl, 2.5 mm mgcl 2 , 10 mm cacl 2 ), added to the reaction and incubation was carried on for 10 min at 37uc. the plates were centrifuged to collect cell debris for 3 min at 2500 x g. for each sample 10 ml of lysate were transferred to 384 microtiter plates (genetixx), which were used as reservoirs for the spotting procedure. the samples were spotted onto halolink tm slides (promega) using the qarray2 microarray spotter (molecular devices). 384 different samples were spotted per slide with three replicate slides per screening. in total 1536 samples were screened on 12 slides (n = 3). each sample was spotted as quadruplicates with controls in two identical sets of eighteen 10610 subarrays each (total number of spots per slide 3600). the controls used included ht-hisj, ht-cjaa and ht-peb1a as positive reference proteins as these have been described as immunodominant before. as specificity controls ht-argc and ht-pyrc were used, representing proteins without known immunodominant behaviour, thus binding of the polyclonal antibodies is not expected. in addition, two different e.coli strains -acella tm electrocompetent cells and krx single-step competent cells (promega) -were spotted as further controls. as those two lack proteins expressed with a halotagh, they are used as negative controls. after spotting of the samples, the slides were incubated for 1 h at room temperature in a humidity chamber. next, slides were washed with pbs + 0.05% igepalh ca-630 (pbsi, sigma aldrich) and dried by nitrogen flow. the 2 well proplate tm module (grace biolabs) was attached to each slide. the top chamber was filled with 1.5 ml of rabbit-polyclonal antibody to c. jejuni (acris, 2 mg/ml) in pbs. the bottom chamber was incubated with pbs only. after 2 h of incubation at room temperature with gentle rocking, both chambers of each slide were washed three times with 2 ml of pbsi. secondary antibody (goat-polyclonal to rabbit igg conjugated with chromeo tm -546, abcam, 5 mg/ml) was subjected to each chamber in pbs and the slides were incubated at room temperature for 2 h in the dark under gentle rocking. finally, slides were washed three times with pbsi, the proplate tm modules removed and the slides dried by nitrogen flow. the slides were scanned on an axon genepix 4200a laser scanner (molecular devices) with the following settings: 532 nm laser, pmt gain 400, 40% laser power, lines to average 1, 10 mm resolution and standard green emission filter at 575 nm. the raw data sets of all the microarray analyses in this publication have been deposited in ncbi's gene expression omnibus [61] and are accessible through geo series accession number gse44717. the median fluorescence intensity of each spot corrected by the local background (median f532 -b532) was used. further, relative fluorescence intensity (rfi) was calculated by subtracting the signals of the bottom chamber from the raw data signals of the top chamber to account for non-specific binding of secondary antibodies. for screening of expression libraries we used the contrast method with either argc or pyrc as specificity control to determine the contrast via the formula: with rf f i control the median of all rfis of the control used: clones harbouring strong signals in microarray screening were selected to be sequenced. sequencing was performed externally by lgc genomics using ht7f (59 acatcggcccgggtct-gaatc 39) and flxr (59 cttcctttcgggctttgttag 39) primers. after sequencing and identification of potentially immunodominant proteins, primers were designed to generate full-length clones for each identified gene, see s12 for a list of the primers used. cloning was performed as mentioned above with slight modifications. the pfn18a vector was linearized using the following primer set; 18a if linear for (59 gtttaaac-gaattcgggctc 39) and 18a if linear rev (59 ggcgatcgcgttatcgctctg 39) with pcr conditions as mentioned before. protein expression, lysis and spotting of fulllength proteins were performed as described above. the slides were incubated for 1 h at room temperature in a humidity chamber. for incubation with antibodies 3 well or 16 well proplate tm modules (grace biolabs) were attached to the halolink tm slides. processing of the slides was done similar to the original screening, however several different antibodies were used, see section antibodies. for testing of immunodominant characteristics with elisa, the crude lysate was first purified using halolink tm magnetic beads (promega) following the manufacturer's instructions. the proteins of interest were subsequently cleaved off by digestion with protev protease (promega) and concentration was determined by nanodrop measurements. the samples were diluted to a total protein content of 20 mg/ml in pbs and 50 ml of each sample was added to maxisorb plates (nunc). each sample was analyzed at least in triplicate. the elisa plate was covered with a lid and incubated overnight at 4uc in a humidity chamber. after five washing steps each with pbs + 0.05% tween-20 (pbst), the plates were blocked using 200 ml 5% non fat dried milk in pbs per well for 2 h. afterwards, plates were washed three times with pbst. 100 ml of primary antibody solution (c = 4 mg/ml) in pbs containing 1% non fat dried milk were applied to each well using the respective desired antibody or pbs for controls. the plates were incubated for 2 h at room temperature and washed four times with pbst. next, 100 ml of conjugated secondary antibody (goat polyclonal to rabbit igg conjugated with horseradish peroxidase, abcam ab6721, c = 20 ng/ml) were added to each well and incubation carried on for 1 h. finally, plates were washed once again four times with pbst and 100 ml 3,39,5,59-tetramethylbenzidine (tmb, sigma-aldrich) was added to each well for detection. after 30 min of incubation at room temperature in the dark, the reaction was stopped by applying 100 ml of 2 m h 2 so 4 to each well. the optical density of each well was measured using the omega fluostar (bmg labtech) at a wavelength of 450 nm. primers were designed using primer3 [62] within geneious pro 5.6.5 [63] . the sequenced inserts were identified by blast [64] .peptide sequence secondary structures were predicted using the emboss garnier [65] algorithm and the transmembrane regions predicted by tmhmm2.0 [66, 67] . antigenic sites were predicted by emboss antigenic [68, 69] . data evaluation was performed by originpro 8 g (originlab) and microsoft excel. 3dimensional structure predictions were performed using the swiss model automated mode [70] [71] [72] [73] [74] and pdb files were visualized and analyzed by the ucsf chimera package [75] . chimera is developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco (supported by nigms p41-gm103311). analysis of full-length proteins was achieved by combining the results from elisa and microarray data. hence, the rfi of each sample was calculated. next, a normalized rfi was generated by dividing the rfi of each sample by the median rfi of all the samples within an area of interest, i.e. incubation compartment. from these normalized rfis a median and standard deviation was calculated. if the median normalized rfi of the positive control was below the median normalized rfi of any of the negative references whilst considering the standard deviations, the test was rendered invalid. if the test passed the above criterion, the q values were calculated as follows: with rf f i sample the median of normalized rfis of the sample and rf f i pos:control the median of the normalized rfis of the positive control hisj respectively cjaa. the resulting error was calculated by error propagation according to gauss. finally, incorporating all valid tests, the mean q value was determined along with its resulting error following error propagation by gauss, see table 1 . several proteins were chosen for epitope mapping. these were the proteins encoded by cj0467, cj1723, cj0669, cj1380, cj0920, cj1575 and cj0623. the proteins were divided into 15-mer oligopeptides with an overlap of 11 amino acids in silico. the synthesis and coupling to microarray slides was performed externally by jpt peptide technologies gmbh. each peptide sequence was applied 9 times to one slide. the slides were used with proplate 3-well chamber system (grace) allowing for incubation with different antibodies. first, the slides were blocked with superblock blocking buffer (thermo fischer) for 2 h, washed five times with pbs + 0.05% tween-20, primary antibodies applied, incubated overnight at 4uc with mild rocking, washed again, secondary antibodies applied for 2 h in the dark and after a final washing procedure, dried and scanned as above. three different primary antibodies to c.jejuni were tested. the bottom chamber was always used as a control chamber, incubated only with secondary antibody. the peptide tlikelkrlgi and 11 derivatives thereof substituting one amino acid for alanine were synthesized and coupled to microarray slides by jpt peptide technologies gmbh. each peptide was applied 9 times to one slide. incubation procedure was performed as described above for epitope mapping. the expression of the desired halotagh fusion proteins was checked by sds-page. after lysis of cells, 2 ml of each protein extract was mixed with 1 ml of 10 mm halotagh alexa 488 ligand. after addition of 7 ml 1x tbs (100 mm tris, 150 mm nacl, ph 7.6) the reaction was incubated at room temperature for 30 minutes. 2 ml of each reaction were removed, mixed with 8 ml of 5x loading buffer (fermentas) and 1 ml dtt and heated for 5 min at 70uc. the separation was performed on a mini-protean tgx gel (biorad, any kd, 15 wells) in a protean ii xi cell chamber (biorad) for 30 min at 200 v. as a size reference benchmark fluorescent protein standard (life technologies) was used. fluorescence was measured in a fla-5100 (fujifilm) with excitation at 473 nm. figure s1 rin. electropherogramm and rna integrity number (rin) for sample 5, a total rna isolated from c. jejuni nctc 11168, after analysis using the rna 6000 pico kit and the agilent bioanalyzer 2100. the rin equals 9 and the ratio of 23s to 16s rrna is 1.8. on the right hand, a virtual gel picture is presented as calculated by the agilent expert 2100 software. (tif) figure s2 likelkrlgi is conserved in c. jejuni. the protein sequence of cj0669 was blasted and subsequently aligned according to a blosum62 matrix. as a reference sequence tlikelkrlgi of q0pak5, the protein encoded by cj0669 from c. jejuni nctc 11168 was used. the dots indicate an agreement to the reference, while differences are given by the one letter amino acid code. the first 13 sequences including the references are 100% identical and are all derived from c. jejuni proteins. for other species of campylobacter such as c. coli or c. upsaliensis several of the residues are replaced. for other organisms, especially helicobacter pylori the degree of replaced residues increases. the positions showing the most conservation throughout are residues 3, 6, 9 and 11. however, residue 10, the glycine which was revealed to be paramount for the binding of the antibody is not found in helicobacter pylori proteins. (pdf) figure s10 specific vs. non-specific binding to potential linear epitopes of cj0920c. the bars represent the mean values (n = 15) of rfi values after incubation with polyclonal antibody to c. jejuni (green) and salmonella enterica (orange). the mean values for each peptide fall within the same range or possess overlapping standard deviations. thus, no specific interaction of the antibody to the epitope is present; rather a non-specific binding seems likely. (tif) figure s11 binding specificity assay of tlikelkrlgi with anti-salmonella antibodies. the different sequences tested in alanine scanning are shown in the box-whisker-plot (n = 15) with each box representing 50% of the values. the whiskers encompass 98% of the values, the median is indicated by a horizontal line and the mean represented by a small rectangle. the 12 boxes in green on the left represent the results after incubation with polyclonal antibody to s. enterica. for comparison, the two red boxes show the original signals from fig. 4 for the sequence tlikelkrlgi as well as tlikelkrlai, after incubation with polyclonal antibodies to c. jejuni. all the green boxes fall into the same range as the altered sequence tlikelkrlai, where alanine replaced the glycine residue, which possessed only 10% intensity of the original sequence. thus, no specific interaction of the antibody to the epitope is present; rather a non-specific binding seems likely. (tif) figure s12 primers used in this study. the name of each primer, the corresponding target gene or vector and its sequence in 59 to 39 direction is given. for each gene, f represents the forward primer and r the reverse. the primers were used for cloning in the in-fusion smarter tm directional cdna library construction kit. (xls) proposal for a new family epidemiologisches bulletin 03 campylobacter jejuni and related species campylobacter and guillain-barré syndrome genetic basis for the variation in the lipooligosaccharide outer core of campylobacter jejuni and possible association of glycotransferase genes with post-infectious neuropathies risk factors for sporadic campylobacter infection in the united states. a case-control study in foodnet sites typing of campylobacter jejuni and campylobacter coli isolated from live broilers and retail broiler meat by flaa-rflp, mlst, pfge and rep-pcr rapid pulsed-field gel electrophoresis protocol for subtyping of campylobacter jejuni evaluation of three commercial latex agglutination tests for identification of campylobacter spp molecular cloning: a laboratory manual, third edition severe acute respiratory syndrome diagnostics using a coronavirus protein microarray immunogenic cross-reaction among outer membrane proteins of gram-negative bacteria microarray-based method for screening of immunogenic proteins from bacteria proteinprotein interactions: analysis of a false positive gst pulldown result halotag: a novel protein labeling technology for cell imaging and protein analysis validation of two ribosomal rna removal methods for microbial metatranscriptomics polyadenylic acid sequences in e. coli messenger rna identification of the gene for an escherichia coli poly(a) polymerase a simple method to enrich mrna from prokaryotic rna magnetic capturehybridization method for purification and probing of mrna for neutral protease of bacillus cereus direct detection of recombinant gene expression by two genetically engineered yeasts in soil on the transcriptional and translational level normalization of full-length-enriched cdna duplex-specific nuclease efficiently removes rrna for prokaryotic rna-seq ligation-independent cloning of pcr products (lic-pcr) high efficiency transformation of e. coli by high voltage electroporation the campylobacter jejuni/coli cjaa (cj0982c) gene encodes an n-glycosylated lipoprotein localized in the inner membrane genetic diversity of the campylobacter genes coding immunodominant proteins immunogenicity and immunoprotection of recombinant peb1 in campylobacter-jejuni-infected mice the national center for biotechnology information's protein clusters database characterization of genetically matched isolates of campylobacter jejuni reveals that mutations in genes involved in flagellar biosynthesis alter the organism's virulence potential nutrient acquisition and metabolism by campylobacter jejuni identification of campylobacter jejuni genes contributing to acid adaptation by transcriptional profiling and genome-wide mutagenesis structure, function, and evolution of bacterial atp-binding cassette systems the protein data bank: a computer-based archival file for macromolecular structures the 2.0 crystal structure of abc transporter from thermatoga maritima toward the estimation of the absolute quality of individual protein structure models construction and evaluation of normalized cdna libraries enriched with full-length sequences for rapid discovery of new genes from sisal (agava sisalana perr.) different development stages construction of normalized rna-seq libraries for next-generation sequencing using the crab duplex-specific nuclease atp-binding cassette transporters are targets for the development of antibacterial vaccines and therapies a glycine-rich bovine herpesvirus 5 (bhv-5) ge-specific epitope within the ectodomain is important for bhv-5 neurovirulence identical igm antibodies recognizing a glycine-alanine epitope are induced during acute infection with epstein-barr virus and cytomegalovirus glycine-rich cell wall proteins act as specific antigen targets in autoimmune and food allergic disorders epitope mapping x-ray crystallography of antibodies b-cell epitopes: fact and fiction antigenic diversity among helicobacter pylori vacuolating toxins functional peptide microarrays for specific and sensitive antibody diagnostics serodiagnosis of echinococcus spp. infection: explorative selection of diagnostic antigens by peptide microarray three-dimensional structure of an antibody-antigen-complex epitope mapping using the x-ray crystallographic structure of complement receptor type 2 (cr2)/cd21: identification of a highly inhibitory monoclonal antibody that directly recognizes the cr2-c3d interface high-resolution epitope mapping of hghreceptor interactions by alanine-scanning mutations characterization of an anti-borrelia burgdorferi ospa conformational epitope by limited proteolysis of monoclonal antibody-bound antigen and mass spectrometric peptide mapping functional reconstruction and synthetic mimicry of a conformational epitope using clips technology diagnostic peptide discovery: prioritization of pathogen diagnostic markers using multiple features best: improved prediction of b-cell epitopes from antigen sequences an introduction to epitope prediction methods and software immunoinformatics and the in silico prediction of immunogenicity. an introduction benchmarking b cell epitope prediction: underperformance of existing methods the rin: an rna integrity number for assigning integrity values to rna measurements gene expression omnibus: ncbi gene expression and hybridization array data repository primer3 on the www for general users and for biologist programmers geneious v5 basic local alignment search tool analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins predicting transmembrane protein topology with a hidden markov model: application to complete genomes a hidden markov model for predicting transmembrane helices in protein sequences a semi-empirical method for prediction of antigenic determinants on protein antigens new hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-ray derived accessible sites the swiss-model workspace: a web-based environment for protein structure homology modeling the swiss-model repository and associated resources swiss-model: an automated protein homology-modeling server swiss-model and the swiss-pdbviewer: an environment for comparative protein modeling protein modeling by e-mail ucsf chimera -a visualization system for exploratory research and analysis the c. jejuni strain nctc 11168 was a kind gift of the group of s. bereswill (department of microbiology and hygiene, charité -university medicine berlin, berlin, germany). sh is greatly indebted to martina obry for her assistance during expression library construction and clone isolation. the authors would like to thank simone aubele for technical assistance. we also gratefully acknowledge michaela schellhase for microarray printing. conceived and designed the experiments: sh mvnr. performed the experiments: sh. analyzed the data: sh ffb mvnr. contributed reagents/materials/analysis tools: sh. wrote the paper: sh ffb mvnr. key: cord-010260-8lnpujip authors: anthonsen, henrik w.; baptista, antónio; drabløs, finn; martel, paulo; petersen, steffen b. title: the blind watchmaker and rational protein engineering date: 1994-08-31 journal: j biotechnol doi: 10.1016/0168-1656(94)90152-x sha: doc_id: 10260 cord_uid: 8lnpujip in the present review some scientific areas of key importance for protein engineering are discussed, such as problems involved in deducting protein sequence from dna sequence (due to posttranscriptional editing, splicing and posttranslational modifications), modelling of protein structures by homology, nmr of large proteins (including probing the molecular surface with relaxation agents), simulation of protein structures by molecular dynamics and simulation of electrostatic effects in proteins (including ph-dependent effects). it is argued that all of these areas could be of key importance in most protein engineering projects, because they give access to increased and often unique information. in the last part of the review some potential areas for future applications of protein engineering approaches are discussed, such as non-conventional media, de novo design and nanotechnology. nature has evolved using several types of random mutations in the genetic material as a fundamental mechanism, thereby creating new versions of existing proteins. by natural selection nature has given a preference to organisms with proteins which directly or indirectly made them better adapted to their environment. thus nature works like a blind watchmaker, trying out an endless number of combinations. this may seem to be an inefficient approach by industrial standards, but nevertheless nature has been able to develop some highly complex and sophisticated designs, simply by the power of natural selection over millions of years, occurring in a large number of parallel processes. by virtue of reproduction several copies of each organism have been able to test the effect of different mutations in parallel. it is quite probable that the mutation frequency was higher in ancient species (doolittle, 1992) , although it is still possible to find highly mutable loci in genes involved in adaptation to the environment (moxon et al., 1994) . enzymes have been used by man for thousands of years for modification of biological molecules. the use of rennin (chymosin) in rennet for cheese production is a relevant example. and with increased knowledge about proteins, genes and other biological macromolecules scientists started starting with a protein with known sequence and properties, we make a 3-d model of the protein from experimental structure data or by homology. by modelling and simulation we identify mutations that will modify selected properties of the protein (the design part of the process), these mutations are implemented at the dna level and expressed in a suitable organism (the production part of the process), and the success of the design is verified by experimental methods. to look at methods for making modified proteins with new or improved properties. at first this was done by speeding up nature's own approach, by increasing the number of mutations (e.g., by using chemicals or radiation) and by using a very strong selection based on tests for specific properties. with the introduction of new and powerful techniques for structure determination and site directed mutagenesis, it is now possible to do rational protein modification. rather than testing out a large number of random mutations, it has become feasible to identify key residues within the protein structure, to predict the effect of changing these residues, to implement these changes in the genetic material, and finally to produce large amounts of modified proteins. this is protein engineering. there are several reviews describing the fundamental ideas in protein engineering, see fersht and winter (1992) for a recent one. the basic protein engineering process is shown in fig. 1 (see also petersen and martel (1994) ). in most cases it starts out with an unmodified protein with well-characterised properties. for some reason we want to modify this protein. in the case of an enzyme we may want to make it more stable, alter the specificity or increase the catalytic activity. first we enter the design part of the protein engineering process. based on structural data we create a computer model of the protein. by a combination of molecular modelling and experimental methods the correlation between relevant properties and structural features is established, and changes affecting these properties can be identified and evaluated for implementation. in more and more cases the effect of these changes can be simulated, and the modifications can be optimised with respect to these simulations. as soon as a new design has been es~tablished we may enter the production part of the process. the necessary mutations must be implemented in the genetic material, this genetic material is introduced into a production organism, and the resulting modified protein can (in most cases) be extracted from a bioprocess. this protein can be tested with respect to relevant properties, and if necessary it may be used as a basis for re-entering the design part of the protein engineering process. after a few iterations we may reach an optimal design. there are several examples of successful protein engineering projects. protein engineering may be used to improve protein stability (kaarsholm et al., 1993) , enhance or modify specificity (getzoff et al., 1992; witkowski et al., 1994) , adapt proteins to new environments (arnold, 1993; gupta, 1992) , or to engineer novel regulation into enzymes (higaki et al., 1992) . in some cases even de novo design of new proteins may be relevant, using knowledge gained from existing structures (kamtekar et al., 1993; shakhnovich and gutin, 1993; ghadiri et al., 1993; ball, 1994) . in a truly multidisciplinary project chymosin mutants with optimal activity at increased ph values compared to wild-type chymosin was designed and produced (pitts et al., 1992) . point mutations changing the charge distribution of superoxide dismutase have been used to increase reaction rate by improved electrostatic guidance (getzoff et al., 1992) . a project on converting trypsin into chymotrypsin has been important for understanding the role of chymotrypsin surface loops (hedstrom et al., 1992) , a serine active site hydrolase has been converted into a transferase by point mutations (witkowski et al., 1994) , and mutations in insulin aiming at increased folding stability have given an insulin with enhanced biological activity (kaarsholm et al., 1993) . an example of a rational de novo project (as opposed to the random approach used, e.g., in generation of catalytic antibodies) is the design of an enzymatic peptide catalysing the decarboxylation of oxaloacetate via an imine intermediate, in which a very simple design gave a three to four orders of magnitude faster formation of imine compared to simple amine catalysts . in some cases it may also be an interesting approach to incorporate nonpeptidic residues into otherwise normal proteins (baca et al., 1993) , or to build de novo proteins by assembling peptidic building blocks on to a nonpeptidic template (tuchscherer et al., 1992) . it has been shown that incorporation of nonpeptidic residues into e-turns of hiv-1 protease gives a more stable enzyme (baca et al., 1993) . the main problem with this approach is how to incorporate the non-standard residues. in the hiv-1 protease case solid-phase peptide synthesis combined with traditional organic synthesis was used, others have suggested that the degeneracy of the genetic code may be used to incorporate novel residues via the standard protein synthesis machinery of the cell (fahy, 1993) . in the present review we will look at the design part of the protein engineering process, with emphasis on some of the more difficult steps, especially homology based modelling in cases with very low sequence similarity, nuclear magnetic resonance (nmr) of very large proteins and modelling of electrostatic interactions. in the last part of the review we will discuss some possible future directions for protein engineering and protein design. any protein engineering project is based on information about the protein sequence. this information may stem from either direct protein sequencing or a deducted translation of the dna/rna sequence. the amount of information on protein and nucleic acid sequences, as well as on relevant data like 3-d structures and disease-related mutations, is growing at a very rapid pace, and novel databases and computer tools give increased access to these data (coulson, 1993) . it is very reasonable to expect that projects like the human genome project will succeed in providing us with sequence information about every single gene in our chromosomes within the next decade. this information will be after transcription, the mrna may be edited, a process that now has been reported in man, plants and primitive organisms (trypanosoma brucei). the mrna is then translated into a protein sequence. this protein sequence can subsequently be modified, leading to n-or o-glycosylation, phosphorylation, sulfation or the covalent attachment of fatty acid moieties to the protein. at this stage the protein is ready for transport to its final destination -which may be right where it is at the time of synthesis, but the destination may also be extracellular or in secluded compartments such as the mitochondria or lysosomes. in this case the protein is equipped with a signal sequence. after arrival to its destination the protein is processed, often involving proteolytic cleavage of the signal sequence. shorter routes to the functional protein with fewer steps undoubtedly exist as well as routes with interchanged steps of processing. finally, the catabolism of the protein is also part of the process, but has been left out in the figure. of key importance for our understanding of the biology, development and evolution of man. it should, however, be kept in mind that the sequence itself may give us little information about regulation of gene expression, i.e., under what conditions genes are expressed, if they are expressed at all. most protein sequences have been deducted from gene sequences. it is in most cases a priori assumed that a trivial mapping exists between the two sets of information. however, this may not necessarily be the case. in fig. 2 , the various steps currently recognised as being of importance for the production of the mature enzyme are shown, and several of these steps may affect the mapping from gene to protein. posttranscriptional editing is modifications at the mrna level affecting the mapping of information from gene to protein, often involving modification, insertion or deletion of individual nucleotides at specific positions (cattaneo, 1994) . currently only speculative models exist for the underlying molecular mechanism(s) for posttranscriptional editing. in the case of mammalian apolipoprotein b two forms exist, both originating from a single gene. the shorter form, apo b48, arises by a posttranscriptional mrna editing whereby cytidine deamination produces an uaa termination codon (teng et al., 1993) . in the ampa receptor subunit glur-b mrna editing is responsible for changing a glutamine codon (cag) into a arginine codon (cgg) (higuchi et al., 1993) . this editing has a pronounced effect on the ca 2+ permeability of the ampa receptor channel, and it seems to be controlled by the intron-exon structure of the rna. similar mrna editing has been reported in the related kainate receptor subunits giur-5 and giur-6, where two additional codons in the first trans-membrane region are altered (sommer et al., 1991; k6hler et al., 1993) . it is also interesting in this context that certain human genetic diseases have been related to reiteration of the codon cag (green, 1993) . mrna editing in plant mitochondria and chloroplasts has also been reported (gray and covello, 1993) . here the posttranscriptional mrna editing consists almost exclusively of c to u substitutions. editing occurs predominantly inside coding regions, mostly at isolated c residues, and usually at the first or second position of the codons, thus almost always changing the amino acid compared to that specified by the unedited codon. in trypanosoma brucei some extensive and well-documented posttranscriptional cases of editing have been reported (read et al., 1992; harris et al., 1992; adler et al., 1991) . the editing takes place at the mitochondrial transcript level where a large number of uridine nucleic acid bases are added or deleted from the mrna, which then subsequently is translated. several non-editing processes affecting the transcription/translation steps are also known. although the ribosomes in an almost perfect manner translate the message provided by the mrna (with error rates less than 5 x 10 -4 per amino acid incorporated), it appears as if the mrna in certain cases contain information, that forces the ribosome to read the nucleic acid information in a non-canonical fashion (farabaugh, 1993) . a special case, that may deserve some attention as well, is the seleno proteins, were seleno cystein is introduced into the protein by an alternative interpretation of selected codons (b6ck et al., 1991; yoshimura et al., 1990; farabaugh, 1993) . translational frameshifting has been found in retroviruses, coronaviruses, transposons and a prokaryotic gene, leading to different translations of the same gene. two cases of translational 'hops' have been reported, where a segment of the mrna is being skipped by all ribosomes, in the two cases 50 and 500 nucleotides were skipped, respectively (farabaugh, 1993) . to our knowledge posttranscriptional editing and related processes are uncommon but definitely present in humans. it is, therefore, important to understand precisely how these mechanisms work, in order to correctly deduct the protein sequence from the gene sequence. the most common posttranslational modifications are side chain modifications like phosphorylations, glycosylations and farnesylations, as well as others. however, some modifications may also affect the (apparent) gene to protein mapping. posttranslational processing may involve removal of both terminal and internal protein sequence fragments. in the latter case an internal protein region is removed from a protein precursor, and the external domains are joined to form a mature protein (hodges et al., 1992; xu et al., 1993) . interestingly, all intervening protein sequences reported so far have sequence similarity to homing endonucleases (doolittle, 1993) , which also can be found in coding regions of group i introns (grivell, 1994) . posttranslational modifications like phosphorylation, glycosylation, sulfation, methylation, farnesylation, prenylation, myristylation and hydroxylation should also be considered in this context. they modify properties of individual residues and of the protein, and may thus make surface prediction, dynamics simulations and structural modelling in general more complex. the residues that are specifically prone to such modifications are tyrosines (phosphorylation and sulfation), serine and threonine (o-glycosylation), asparagine (nglycosylation), proline and lysine (hydroxylation) and lysine (methylation). in addition glutamic acid residues can become y-carboxylated leading to high affinity towards calcium ions (alberts, 1983) . specific transferases are involved in the modification, e.g., tyrosylprotein sulfotransferases (suiko et al., 1992) and farnesyl-protein transferases (omer et al., 1993) . phosphorylation of amino acid residues is an important way of controlling the enzymatic function of key enzymes in the metabolic and signalling pathways. tyrosine kinases phosphorylate tyrosine residues -thus introducing an electrostatic charge at a residue, which under normal physiological ph is uncharged. phosphorylation is central to the function of many receptors, such as the insulin and insulin-like growth factor i receptors. given the possibility that several modifications may be introduced in the sequence when we move from gene to mature protein, the task of deducting a protein sequence from the gene sequence may be more complex than we normally assume. although the protein sequence itself is a valuable starting point, the optimal basis for a rational protein engineering project will be a full structure determination of the protein. in many cases this turns out to be an expensive and timeconsuming part of the project. most structure determinations are based on x-ray crystallography. this approach may give structures of atomic resolution, but is limited by the fact that stable high quality crystals are needed. many proteins are very difficult to crystallise, in particular many structural and membrane-associated proteins. a large number of important x-ray structures have been published over the last few years, and the structures of the hhai methylase (klimasauskas et al., 1994) , the tbp/tata-box complex (kim et al., 1993a; kim et al., 1993b) and the porcine ribonuclease inhibitor (kobe and deisenhofer, 1993) are mentioned as examples only. nmr may be an alternative in many cases, as the proteins can be studied in solution, and for some experiments they can even be membrane associated. however, nmr is limited to relatively small molecules, and even with incorporation of labelling in the protein the upper limit for a full structure determination using current state of the art methods seems to be close to 30 kda. some novel techniques for studying structural aspects of larger proteins will be discussed (vide infra). representative examples of important nmr structures may be interleukin 1/3 (clore et al., 1991a) , the glucose permease iia domain (fairbrother et al., 1992) and the human retionic acid receptor-/3 dna-binding domain (knegtel et al., 1993) . cryo electron microscopy (cem) is a relatively new approach to protein structure determination. the resolution of the structures are still lower than the corresponding x-ray structures, and a 2-dimensional crystal is a prerequisite. however, despite this cem appears to be a very promising approach to structure determination of membrane associated proteins that can form 2-dimensional crystals. cem has been used to study the nicotinic acetylcholine receptor at 9 .~ resolution (unwin, 1993) and the atp-driven calcium pump at 14 a resolution (toyoshima et al., 1993) , and in a combined approach using high resolution x-ray data superimposed on cem data the structure of the actin-myosin complex (rayment et al., 1993) and of the adenovirus capsid (stewart et al., 1993) has been studied. the recent structure by kiihlbrandt et al. (1994) of the chlorophyll a/b-protein complex at 3.4 a resolution shows that the resolution of cem rapidly is approaching the resolution of most x-ray protein data. scanning tunnelling microscopy (stm) is another new approach for studying protein structures (amrein and gross, 1992; lewerenz et al., 1992; haggerty and lenhoff, 1993) . the method is interesting because of a very high sensitivity, as individual molecules may be examined. the method will give a representation of the surface of the molecule, rather than a full structure determination. however, it is possible that both cem and stm can be used for identification of protein similarity. if data from these methods show that the overall shape of a protein is similar to some other known high resolution protein structure, then the known structure may be evaluated as a potential template for homology based modelling. we believe that such a model can either be used as an improved starting point for a full structure determination (i.e., for doing molecular replacement on x-ray data), or as a low resolution structure determination by itself. in homology based modelling a known structure is used as a template for modelling the structure of an homologous sequence, based on the assumption that the structures are similar. this is a very simple and rapid process, compared to a full structure determination. the sequences may be homologous in the strict sense, meaning that there is an evolutionary relationship between protein data banks the sequences. the same approach may obviously also be used for sequences that are similar, but not necessarily evolutionary related, and in that case we probably should talk about similarity based modelling. however, in this paper we will use homology based modelling as a general term, especially since the distinction between homology and similarity may be difficult in many cases. homology based modelling may turn out to be essential for the future of protein engineering. in fig. 3 , the number of entries in the swissprot protein sequence database (bairoch and boeckmann, 1992 ) and the brookhaven protein structure database (bernstein et al., 1977; abola et al., 1987) are shown as a function of time. as we can see, there is a very significant gap between the number of sequences and the number of structures. this gap is in fact even larger than shown in fig. 3 , as not all entries in the brookhaven database are unique structures. a large number of entries are mutants of other structures or identical proteins with different substrates or inhibitors. there has been an exceptional growth in the number of protein structures over the last 2-3 years. however, it is unrealistic to assume that we will be able to get high resolution experimental structures of all known proteins. the structure determination process is too time consuming, and the sequence databases are growing at a far faster pace, as shown in fig. 3 , especially as a consequence of several large-scale genome sequencing projects. on the other hand, it may not really be necessary to do experimental structure determination of all proteins (ring and cohen, 1993) . the assumption that similar sequences have similar structures (see fig. 4 ) has been proved valid several times and it seems to be true even for short peptide sequences as long as they come from proteins within the same general folding class ). an interesting case which is to some degree an exception to this rule is the structure of hiv-1 reverse transcriptase (kohlstaedt et al., 1992) . two units with identical sequence have similar secondary structure, but very different tertiary structure. however, this seems to be a rather exceptional case. new approaches to general structure alignment (orengo structure distance sequence distance fig. 4 . sequence and structure similarity. in most cases similar sequences have similar structures (region 1), and dissimilar sequences (i.e., measured by a standard mutation matrix) have dissimilar structures (region 2). in several cases quite dissimilar sequences have been shown to have very similar structures, at least with respect to individual domains (region 3). in very special cases we may have similar sequences with different structures (region 4), at least with respect to tertiary structure, showing that environment and binding to other proteins may be essential for the final conformation in some cases. however, in most cases it seems to be safe to assume that structures can be found in the lower grey triangle of this graph, indicating that structure is better conserved than sequence. holm et al., 1992; alexandrov and go, 1993; lessel and schomburg, 1993) have made it possible to search for structurally conserved domains in proteins with very low sequence similarity (swindells, 1994) . this is an important approach, as structure normally is better conserved than sequence (doolittle, 1992) . several cases have been identified where the sequences are very different (at least by traditional similarity measures), whereas the three-dimensional structures are surprisingly similar. the identification of a globin fold in a bacterial toxin (holm and sander, 1993) , and the similarity between the dsba protein and thioredoxin (martin et al., 1993) are relevant examples. recently the structure of the human serum amyloid p component was shown to be similar to concanavalin a and pea lectin, despite only 11% sequence identity (emsley et al., 1994) , and the similarity between hen egg-white lysozyme and a lysozyme-like domain in bacterial muramidase "is remarkable in view of the absence of any significant sequence homology", as noted by thunnissen et al. (1994) . this shows that there probably is a limited number of protein folds, and this number must be lower than the number of sequence classes, defined as groups of similar protein sequences. recent estimates show that this number probably is close to 1000 different protein folds (chothia, 1992) , and approx. 160 of these folds are known so far (burley, 1994; orengo et al., 1993) . this means that rather than full structure determination of a very large number of proteins, it may be sufficient to do structure determination of only a few selected examples of each protein fold, and use this as a basis for homology based modelling of other proteins shown to have the same fold. homology based modelling of the 3-d structure of a novel sequence can be divided into several steps. first, one or more templates must be identified, defined as known protein structures assumed to have the same fold as the trial sequence. then a sequence alignment between trial sequence and template is defined, and based upon this alignment an initial trial model can be built. this initial model must be refined in several steps, taking care of gap splicing, loops, side chain packing etc. the final model can be evaluated by several quality criteria for protein structures. an example of homology based modelling is the modelling of cinnamyl alcohol dehydrogenase based on the structure of alcohol dehydrogenase (mckie et al., 1993) . the protein folding problem is a fundamental problem in structural biology. this problem can be defined as the ab initio computation of a protein's tertiary structure starting from the protein sequence. this problem has not been solved and appears to be extremely difficult. if we want to solve the problem by computing an energy term for all conformations of a protein, defined by rotation around the ~b and ~o backbone angles of n residues in 10 degree steps, we have to evaluate 36 2(n-d alternatives, even without considering the side chains. for a peptide with 15 residues this corresponds to 10 44 conformations. a hypothetical computer with 106 processors, each processor running at 1015 hz (the frequency of uv light) and completing the energy evaluation of one conformation per cycle would need 3 x 1015 years in order to test all conformations. the estimated age of the universe is 14 x 10 ~2 years. a more realistic approach is the use of molecular dynamics or monte carlo methods for simulation of protein folding. however, it is still very difficult to use this as an ab initio approach, both because folding is a very slow process compared to a realistic simulation time scale, and also because it is very difficult to distinguish between correctly and incorrectly folded structures using standard molecular mechanics force fields (novotny et al., 1984) . a possible alternative approach may be to generate potential folds on a simplified lattice representation of possible residue positions (covell and jernigan, 1990; crippen, 1991) . however, this approach is still very experimental. some progress has been achieved in the area of secondary (rather than tertiary) structure prediction (benner and gerloff, 1993) . studies of local information content indicate that 65% match may be an upper limit for single-sequence prediction methods (rao et al., 1993) , whereas methods taking homology data into account may probably raise this limit to approx. 85%. methods based on neural networks and combinations of several prediction schemes seem to give good predictions, and especially methods using homology data from multiple alignments may give predictions at 70% match or better in many cases (salzberg and cost, 1992; boscott et al., 1993; rost and sander, 1993a; rost et al., 1993; levin et al., 1993) . also methods taking potential residue-residue interactions into account, like the hydrophobic cluster analysis (hca), may be used for identification of potential secondary structure elements (woodcock et al., 1992) . it has been shown that by restricting the prediction to a consensus region with stable conformation it is possible to make very reliable predictions (rooman and wodak, 1992) . in one case, neural networks were shown to be capable of returning a limited amount of information on the tertiary structure (bohr et al., 1993) . . structure retrieval by secondary structure. a flow chart for structure retrieval by secondary structure (right side) compared to retrieval by sequence (left side). please see the text for details. in this example the secondary structure library was generated using dssp (kabsch and sander, 1983) , the secondary structure was predicted with the phd program (rest et al., 1994), and fasta (pearson, 1990; pearson and lipman, 1988 ) was used to search the secondary structure library and the nrl-3-d databases (namboodiri et al., 1988; george et al., 1986) . only the secondary structure based method was able to identify the hla class i structure as similar to the class ii structure. the ribbon representation of the hla class i antigen binding region used in this figure was generated with molscript (kraulis, 1991) . be inconsistent, compared to the more sophisticated classification which can be achieved by a trained expert. recent studies show that the average agreement between alternative assignment methods used on identical structures is close to 65% for three methods (colloc'h et al., 1993) , or 79% if only two methods are compared (woodcock et al., 1992) . vadar is a new classification method which is aiming at a better agreement between manual and automatic assignment (wishart et al., 1994) , to what degree this may have influence on prediction systems remains to be seen. over the last few years it has been realised that the inverse folding problem is much easier to solve (bowie et al., 1991; blundell and johnson, 1993; bowie and eisenberg, 1993) . the inverse folding problem can be defined as follows: given a known protein structure, identify all protein sequences which can be assumed to fold in the same way. a large number of protein structures must be available in order to use this as a general approach, as the relevant protein fold has to be represented in the database in order to be identified. however, with a limited number of possible folds actually used by nature, a complete database of all folds appears to be possible. some information about possible folding classes can be derived from experimental data. circular dichroism can be used as a crude way of measuring the relative amounts of secondary structure in a protein. classification methods based on amino acid composition can be used for classification of proteins into broad structural classes zhou et al., 1992; chou and zhang, 1992; dubchak et ai., 1993) . this information may limit the number of different folds which have to be evaluated. it is also possible that such information may be used to improve the performance of other methods, although data on secondary structure prediction of all-helical proteins seems to indicate that the gain may be small (rost and sander, 1993b) . however, for a unique identification of folding class more sensitive methods are needed, and the most useful one is probably some kind of protein sequence library search. in order to identify the folding class we have to search a database of known protein structures with our trial sequence. the problem is that standard methods for sequence retrieval may not be sensitive enough in all cases. if the sequences are similar, then retrieval is trivial. however, we know that there are cases where structures are known to be similar despite very different sequences. how can these cases be identified in a reliable way? the most promising approaches are based on methods for describing the environment of each residue (bowie et al., 1991; eisenberg et al., 1992; overington et al., 1992; ouzounis et al., 1993; wilmanns and eisenberg, 1993; liithy et al., 1994) . this description can be used for generating a profile, showing to what degree each residue is found in a similar environment in other structures, and this profile can be used as a basis for sequence alignment and library searches. similar property profiles can also be used for searching database systems of protein structures (vriend et al., 1994) . a very simple approach can be used if we accept the hypothesis that protein sequences representing structures with a similar linear distribution of secondary structure elements may fold in a similar way. we can then create a sequence type library of known structures where the residues are coded by secondary structure codes rather than residue codes (see fig. 5 ). given the sequence of a protein with unknown 3-d structure, we can use a secondary structure prediction method and translate the sequence into a secondary structure description. if we define a suitable 'mutation' matrix describing the probability of inter conversion between different secondary structure elements, then a standard library search program like fasta (pearson and lipman, 1988; pearson, 1990) can be used in order to identify potential template structures. the example shown in fig. 5 is the identification of hla class i as a suitable candidate for homology modelling of hla class ii. the sequence similarity is very low, 11% sequence identity in the antigen binding region (based on alignment of the structures), and especially for this region most sequence based methods will retrieve a large number of alternative sequences before any of the class i molecules. bly improve the performance to use a positiondependent gap penalty, where most gaps are placed in loop regions rather than in helices or strands. however, the method is very simple to implement and test, as necessary tools and data already are available in most labs. however, for the secondary structure based approach the hla class i sequences are retrieved as top candidates. the structure prediction did not include any information about the hla class ii structure, which recently has been published (brown et al., 1993) . it should be mentioned that the 11% sequence identity score is not significantly higher than the score from a random alignment of sequences. if we, for each sequence in the swissprot protein sequence library, align it against a sequence selected at random from the same library (alignment without gaps, using the full length of the shortest sequence, and start the alignment at a random position within the longest sequence), then the average percentage of identical residues is (6 _+ 6)% at 3 standard deviations. the identification method using secondary structure is based on an assumption which has to be examined more closely, and the implementation of it is very crude. much work can be done on the secondary structure prediction, the 'mutation' matrix and the search method. it will proba-4.2. sequence alignment pip4_rat as described in the introduction a crucial feature in molecular evolution has been the parallel exploration of several different mutations. and although mechanisms like horizontal gene transfer and intragenic recombination may have been important as key steps in the evolution of new proteins, the most common mechanism seems to have been gene duplication followed by mutational modification (doolittle, 1992) . this means that especially multiple sequence alignment can give essential information about the mutation studies already performed by nature. conserved residues are normally conserved because they . multim alignment. alignment of inositol triphosphate specific phospholipase c /3 1 from rat (pip1 rat) against three other pip sequences from rat. each horizontal bar represents a sequence, marked in 50 residue intervals. black lines connecting the bars represent well conserved motifs found in all sequences, in this case subsequences of 8 residues where at least 4 residues are completely conserved in all 4 sequences. it is very easy to identify two well conserved regions, annotated as region x and y in the swissprot entries, despite a 400 residue insertion in two of the sequences. this insertion contains sh2 and sh3 domains (pawson, 1992) . it is an interesting observation that the extra c-terminal domain of the pipi_rat sequence shows a weak similarity to myosin and tropomyosin sequences. have an important structural or functional role in the protein, and identification of such residues will thus give vital information about structure and activity of a protein. several tools have been developed for multiple alignment. a very attractive one is macaw (schuler et al., 1991) , which will generate several alternative alignments of a given set of regions, and in a very visual way help the user to identify a reasonable combination of (sub)alignments. an even more general tool is muitim (drablcs and . here all possible alignments, based on short motifs, are shown simultaneously, and the user is free to identify potential similarities even in cases with low sequence identity and very disperse motifs. this is possible because of the superior classification potential of the human brain compared to most automatic approaches. the method includes an option for probability based filtering of motifs, and an example of a multim alignment is shown in fig. 6 . however, it is important to realise that in standard sequence alignment we are trying to solve a three-dimensional problem (residue interactions) by using an essentially one-dimensional method (alignment of linear protein sequences). as a consequence important conserved throughspace interactions may not be evident from a standard sequence alignment. a good example can be found in the alignment of lipases (schrag et al., 1992) . in fig. 7 , the sequence alignment of residues in a structurally conserved core of three lipases (rhizomucor miehei lipase (derewenda et al., 1992) , candida antarctica b lipase (a. jones, personal communication) and human pancreatic lipase (winkler et al., 1990 ) is shown. the active site residues, ser (s), asp (d) and his (h), are shown as black boxes. the ser and his residues are at identical positions. however, the asp residue of the pancreas lipase is at a very different sequence position compared to the other two lipases. it would be very difficult to identify this as the active site asp from a sequence alignment. if we look at the structural alignment in fig. 8 , we see that the positions are structurally equivalent, it is possible for all three lipases to have highly similar relative orientation of the active site atoms, despite the fact that the alternative asp positions are located at the end of two different /3-strands. an improved alignment may be generated if we can incorporate 3-d data for at least one of the sequences in the linear alignment (gracy et al., 1993) . however, in order to get a reliable alignment of sequences with low sequence similarity, we have to take true three-dimensional effects into account. this means that if we are able to identify a known 3-d structure as a potential basis for modelling, then the sequence alignment should be done in 3-d using this structure as a template. this can be done by threading the sequence through the structure and calculating pairwise interactions (jones et al., 1992; bryant and lawrence, 1993) . as soon as a template has been identified, and an alignment between this template and a sequence has been defined, a 3-d model of the protein can be generated. we can either use the template coordinates directly, combined with diffig. 7 . sequence alignment of lipases. alignment of structurally conserved regions of three lipases. for each lipase the solvent accessible surface in % compared to the gxg standard state (grey scale, white is buried and black is exposed), the secondary structure as defined by the dssp program, and the sequence is shown. the position of each subsequence in the full sequence is also shown. the active site residues are shown in white on black. please observe the shift of the active site asp (d) between two very different positions. the alignment was generated using alscript (barton, 1993) . ferent modelling approaches for the ill-defined regions, or the template can be used as a more general basis for folding the protein by distance geometry (srinivasan et al., 1993) or general molecular dynamics methods. loop regions are often highly variable, and must be treated with special approaches (topham et al., 1993) . it is also necessary to consider the orientation of side chains. although the backbone may be well conserved, many residues especially at the protein surface will be mutated, as shown in fig. 9 . the stability of a protein depends upon an optimal packing of residues, and it is important to optimise side chain conformation if we want to study protein stability and complex formation. a very common approach is the use of rotamer libraries combined with molecular dynamics refinement. recent studies show that this step of the modelling in fact may be less difficult than has been assumed . and important interactions, and exposed regions may to some degree be identified by using antibodies. however, in many cases the rational for modelling by homology is the very lack of experimental data related to structure, and we have to use other more general methods for evaluation of models. some of the approaches we already have described for sequence alignment can obviously also be used for evaluation of models. in general, model evaluation can be based on 3-d profiles (lfithy et al., 1992) , contact profiles (ouzounis et al., 1993) or more general energy potentials (hendlich et al., 1990; jones et al., 1992; nishikawa and matsuo, 1993) . some of these approaches have been implemented as programs for evaluation of structures or models, like procheck (laskowski et al., 1993) and prosa (sippl, 1993a, b) . however, in general no model (or even experimental structure) should be trusted beyond what can be verified by experimental methods. a protein model based on homology (or similarity) has to be verified in as many ways as possible, and experimental methods should always be preferred. mutation studies may give valuable information about active site residues a prerequisite for rational protein engineering is 3-d structure information about the protein. in fig. 6 , including parts of the sequences connecting the core regions. the active site asp is able to maintain a similar relative orientation, despite very different sequence positions. the alignment was generated using insight (biosym technologies). adddition to x-ray crystallography, nmr is the most important method for protein structure determination. x-ray crystallography has several advantages when compared to nmr. solving the crystal structure by x-ray crystallography is usually fast as soon as good crystals of the protein are obtained (even if it may not be so easy to obtain these crystals). it is also possible to determine the structure of very big proteins. the major disadvantage of x-ray crystallography is that it is the crystal structure that is determined. this implies that crystal contacts may distort the structure (chazin et al., 1988; wagner et al., 1987) . since active sites and other binding sites usually are located on the surface of the proteins, very important regions of the protein may be distorted. some structures even show large differences between nmr and x-ray structure (frey et al., 1985; klevit and waygood, 1986) the advantage of nmr is that it is dealing with protein molecules in solution, usually in an environment not too different from its natural one. it is possible to study the protein and the dynamical aspects of its interaction with other molecules like substrates, inhibitors, etc. it is also possible to obtain information about apparent pk a values, hydrogen exchange rates, hydrogen binding and conformational changes. all nuclei contain protons, and therefore they carry charge. some nuclei also possess a nuclear spin. this creates a magnetic dipole, and the nuclei will be oriented with respect to an external magnetic field. the most commonly studied nuclei in protein nmr (1h, 13c and 15n) have two possible orientations, representing high and low energy states. the frequency of the transition between the two orientations is proportional to the magnetic field. at a magnetic field of 11.7 tesla the energy difference corresponds to about 500 mhz for protons. in an undisturbed system there will be an equilibrium population of the possible orientations, with a small difference in spin population between the high and low energy orientation. the equilibrium population can be perturbed by a radio frequency pulse of a frequency at or close to the transition frequency. in addition, the spins will be brought into phase coherence (concerted motion) and a detectable magnetisation will be created. the intensity of the nmr signal is proportional to the population difference between the levels the nuclei can possess. nuclei of the same type in different chemical and structural environments will experience different magnetic fields due to shielding from electrons. the shielding effect leads to different resonance frequencies for nuclei of the same type. the effect is measured as a difference in resonance frequency (in parts per million, ppm) between the nuclei of interest and a reference substance, and this is called the chemical shift. in molecules with low internal symmetry most atoms will experience different amounts of shielding, the resonance signals will be distributed over a well-defined range, and we get a typical nmr spectrum. the process that brings the magnetisation back to equilibrium may be divided into two parts, longitudinal and transverse relaxation. the longitudinal or t 1 relaxation describes the time it takes to reach the equilibrium population. the transverse or t 2 relaxation describes the time it takes before the induced phase coherence is lost. for macromolecules the t 2 relaxation is always shorter than the t a relaxation. short t 2 relaxation leads to broad signals because of poor definition of the chemical shift. most molecules have dipoles with magnetic moment, and the most important cause of relaxation is fluctuation of the magnetic field caused by the brownian motion of molecular dipoles in the solution. how effective a dipole may relax the signal depends upon the size of the magnetic moment, the distance to the dipole, and the frequency distribution of the fluctuating dipoles. a nucleus may also detect the presence of nearby nuclei (less than three bonds apart), and this will split the nmr signal from the nucleus into more components. several nuclei in a coupling network is called a spin system. by applying radio frequency pulses it is possible to create and transfer magnetisation to different nuclei. it is, as an example, possible to create magnetisation at one nucleus, and transfer the magnetisation through bonds to other nuclei where it may be detected. the pulses are applied in a so-called pulse sequence (ernst, 1992; kessler et al., 1988) . the methodology for determination of protein structure by two-dimensional nmr is described in several textbooks and review papers (wagner, 1990; wiithrich, 1986; wider et al., 1984) . the standard method is based on two steps, sequential assignment: assignment of resonances from individual amino acids, and distance information: assignment of distance correlated peaks between different amino acids. the first step involves acquiring coupling correlated spectra (cosy, tocsy) in deuterium oxide to determine the spin system of correlated resonances. some amino acids have spin systems that in most cases make them easy to identify (gly, ala, thr, ile, val, leu). the other amino acids have to be grouped into several classes, due to identical spin systems, even though they are chemically different. the spin systems can be correlated to the nh proton by acquiring cosy and tocsy spectra in water. the assigned nh resonance is then used in distance correlated spectra (noesy) to assign correlations to protons (nh, h,, h~) at the previous amino acid residue (fig. 10) . by combining the knowledge of the primary sequence (which gives the spin system order) with the nmr data collected it is possible to complete the sequential assignment. when the sequential assignment is done the assignment of short range noe (up to four residues) will give information about secondary structure (a-helix, fl-strand). long-range correlations will serve as constraints (together with scalar couplings) to determine the tertiary structure of the protein. excellent procedures describing these steps are available (roberts, 1993; wiithrich, 1986) . with large proteins there will be spectral overlap of resonance lines. the problem is partially solved by labelling the protein with 13c and 15n isotopes. triple resonance multidimensional nmr methods (griesinger et al., 1989; kay et al., 1990 ) may then be applied. the resonances will then be spread out in two more dimensions (13c and ~sn) and the problem with overlap is reduced. these methods depend upon the use of scalar couplings to perform the sequential assignment, the sequential assignment procedure will then be less prone to error. the noesy spectra of such large proteins are often very crowded, but four-dimensional experiments like the 13c-~3c edited noesy spectrum (clore et al., 1991b) have been designed. such experiments will spread the proton-proton distance correlated peaks by the chemical shift of its corresponding 13c neighbour and reduce the spectral overlap. secondary structure elements may also be predicted from the chemical shift of 1h and 13c (spera and bax, 1991; williamson and asakura, 1991; wishart et al., 1992) . obtaining nmr-spectra of proteins has some aspects that should be considered. spectral overlap. as we move to larger proteins the probability of overlap of resonance lines increases. at some point it will become impossible to do sequential assignment due to this overlap. application of 3-d and 4-d multiresonance nmr has made it possible to assign proteins in the 30 kda range (foght et al., 1994; stockman et al., 1992) . fast relaxation. as the size of the protein is increased the rate of tumbling in solution is re-duced. this leads to a reduced transverse relaxation time (t2), and broadening of the resonance lines in the nmr spectra. the intensities of the peaks are reduced and they may be difficult to detect. the short transverse relaxation time will also limit the length of the pulse sequences it is possible to apply (because there will be no phase coherence left), and multidimensional methods become difficult. it is possible to determine a 3-d structure by nmr or x-ray crystallography are probably a subset of all proteins (wagner, 1993) . proteins may have regions with mobility and few cross peaks. the effective size of a protein is often increased by aggregation. the amount of aggregation can often be reduced by reducing the protein concentration. thus, very often the degree of aggregation will determine whether it is possible to assign and solve a protein structure by nmr, by limiting the maximum concentration that may be used. the stability of the proteins is also a major issue. a sample may be left in solution for days, often at elevated temperatures, so denaturation may become a problem. photo-cidnp (chemically induced nuclear polarisation) is an interesting technique for the study of surface positioned aromatic residues in proteins (broadhurst et al., 1991; cassels et al., 1978; hore and kaptain, 1983; scheffier et al., 1985) . by introducing a dye and exciting it with a laser, it is possible to transfer magnetisation to aromatic residues, where it can be observed. in addition to high-resolution nmr, solid state nmr has also been applied to studies of proteins. studies of active sites and conformation of bound inhibitors yields interesting information. the stability of proteins may be monitored under different conditions by detecting signals from transition intermediates bound to the active site (burke et al., 1992; gregory et al., 1993) . structural constraints on transition state conformation of bound inhibitors can be obtained (auger et al., 1993; christensen and schaefer, 1993) . structural constraints of the fold and conformation of the amino sequence may be gathered by setting upper and lower distances for lengths between specific amino acids (mcdowell et al., 1993) . using solid-state nmr it is also possible to study membrane proteins and their orientation with respect to their membrane (killian et al., 1992; ulrich et al., 1992) . we expect such studies to give insight into ion channels in membranes (woolley and wallace, 1992). an important mechanism for relaxation in high-resolution nmr is dipolar relaxation. usually this is induced by the spin of nuclei in the immediate vicinity, and it is a function of the size of the dipole. the electron is also a magnetic dipole, and the magnitude of this dipole is about 700-times that of a proton. paramagnetic compounds have an electron that will interact with 11 . the paramagnetic relaxation method. outline of the paramagnetic relaxation method. the protons located at the protein surface will be closer to the dissolved paramagnetic relaxation agent than the protons located inside the protein core, hence the resonance lines from protons at the surface will be broadened more than resonance lines stemming from protons located inside the protein. nearby protons and increase the relaxation rate of these protons. the widest use of paramagnetic compounds has been of gd 3÷ bound to specific sites in a protein , but also other compounds have been used (chang et al., 1990; hernandez et al., 1990a, b ). this will make it possible to identify resonance lines from residues in the vicinity of the binding site. it is also possible to calculate distances from the paramagnetic atom as the relaxation effect is distance dependent. the paramagnetic broadening effect can also be used with a compound moving freely in solution (drayney and kingsbury, 1981; esposito et al., 1992; petros et al., 1990) . in this way residues located on or close to the protein surface will give broadened resonance lines compared to residues in the interior of the protein. this method can be used to measure important noe and chemical shifts inside the protein directly, or it can be used as a difference method to identify resonances at the surface by comparing spectra acquired with and without the paramagnetic relaxation agent (fig. 11) . we have used the paramagnetic compound gadolinium diethylenetriamine pentaacetic acid (gd-dtpa) as a relaxation agent. gd-dtpa will increase both the longitudinal and the transverse relaxation rates of protons within the influence sphere. suitable nmr experiments to highlight the relaxation effect may be noesy, roesy and tocsy (bax and davis, 1985; braunschweiler and ernst, 1983) gd-dtpa is widely used in magnetic resonance imaging (mri) to enhance tissue contrast. it is assumed to be non-toxic and we do not expect it to bind to proteins. we used the wellstudied protein hen egg-white lysozyme as a test protein. both the structure and the nmr spectra of this protein are known (diamond, 1974; redfield and dobson, 1988) , and the protein is extremely well suited for nmr experiments. in fig. 12 , the 1-d ih-nmr spectrum recorded in the presence and absence of gd-dtpa is shown. although it is evident that there is a selective broadening in the 1-d spectrum, it is also clear that there are problems with overlapping spectral lines. we therefore applied two-dimensional nmr methods, and shown in fig. 13 is the low field region of a noesy spectrum of lysozyme. the region corresponds to the same region as shown in fig. 12 . from fig. 13 we see that the signals from w63, w 63 and w123 disappear with addition of gd-dtpa, while the signals from w28, w108 and wlll still are observable. by examination of the solvent accessible surface of lysozyme it is evident that the indole nh of w62, w63 and w123 is exposed to solvent, while the indole nh of w28, w108 and wlll is not exposed. this shows that the changes in the spectrum are as expected from the structure data. the appearance of the nh-nh region of the spectrum (fig. 14) also shows the reduction in the number of signals in the gd-dtpa exposed spectrum. this shows that the paramagnetic broadening effect can be used for selective identification of signals from solvent exposed residues in a protein. one of the fundamental steps in the protein engineering process shown in fig. 1 is the design step, where a correlation between structure and properties is established in order to select potential structural candidates that match new functional profiles. the understanding of this correlation implies a realistic modelling of the physical chemical properties involved in the functional features to be engineered. these features are basically of two types: diffusional and catalytic. any ligand binding to a protein, whether ligandreceptor or substrate-enzyme, is essentially a diffusional encounter of two molecules. electrostatic interactions are the strongest long-range forces at the molecular scale and, thus, it is not surprising that they are one of the determinant effects in the final part of the encounter (berg and von hippel, 1985) . in the case of substrateenzyme interactions the catalytic step that follows the binding of the substrate seems to be possible mainly by the presence of electrostatic forces that stabilise the reaction intermediates in the binding site (warshel et al., 1989) , from which the product formation may proceed. another and much more basic necessary condition for a successfully engineered protein is that a functional folded conformation is maintained. solvation of charged groups is one of the determinants in protein folding (dill, 1990) , so that even the conformation of the protein is electrostatically driven. given the ubiquitous role of electrostatic interactions, it is then obvious that their accurate modelling is an essential prerequisite in the design of engineered proteins. several good reviews exist on protein electrostatics (warshel and russel, 1984; matthew, 1985; rogers, 1986; harvey, 1989; davies and mccammon, 1990; sharp and honig, 1990) . this section intends to give a brief overview of the subject. we start by presenting the methods one can use to model electrostatic interactions. the most familiar methodology in biomolecular modelling is certainly molecular mechanics (mm) (either through energy minimisations or molecular dynamics (md)). we point out some of the limitations of mm in the treatment of electrostatic interactions, and the need to use alternative ways of describing the system, such as continuum methods. the computation of ph-dependent properties and some potential extensions of mm are also discussed. finally, we refer some applications of electrostatic methods relevant to protein engineering. in mm simulations, electrostatic interactions are usually described with a pairwise coulombic term of the form qlq2/dr12,were ql and q2 are the charges of the pair of atoms, r12 their distance, and d the dielectric constant. d is usually set equal to 1 when the solvent is included. a complete simulation in a sufficiently big box with water molecules should, in principle, give a realistic description of the protein molecule (harvey, 1989) . this would be specially true if a force field including electronic polarizability effects (see 6.3.) was available for use with biomolecular systems, which unfortunately is not the case (harvey, 1989; davis and mccammon, 1990) . we use the term force field in this context as including both the functional form and parameters describing the energetics of the system, from which the forces are derived. simulations where solvent molecules are not treated explicitly are naturally appealing, since the computation time increases with the square of the number of atoms. several methods have been proposed that attempt to account for solvent effects. the more popular approach is an ad hoc dielectric 'constant' proportional to the distance (e.g., mccammon and harvey, 1987) but different distance dependencies can be used (e.g., solmajer and mehler, 1991) . a variety of more elaborated methods were also suggested (northrup et al., 1981; still et al., 1990; gilson and honig, 1991) . all these methods should be viewed as attempts of including solvent screening effects in a simplified way. they can be useful when inclusion of water is computationally prohibitive, but they cannot substitute for an explicit inclusion of solvent since, e.g., the existence of hydrogen bonding with the solvent is not properly described by these approaches. mm of biomolecules has, in general, heavy computation needs. the number of water molecules that should be included in order to simulate a typical protein in a realistic way is quite large, especially if one wants to perform md. also, each pair of atoms has its own electrostatic interaction and the number of pairs cannot be lowered by a short cut-off distance (e.g., 7 a) as in van der waals interactions, since electrostatic interactions are very long range, typically up to 10 ~,. mm simulations have also some limitations on the description of the system, since ph and ionic strength effects usually are difficult or impossible to include. the only way to include ph effects is through the protonation state of the residues. each titrable group (in asp, glu, his, tyr, lys, arg, c-and n-terminal) in the protein have two states, protonated or unprotonated. thus, a protein with n titrable groups will have 2 n possible protonation charge sets. the best we can do is to choose the set corresponding to the protonation states of model compounds at the desired ph. free ions can be included in md simulations of proteins (levitt, 1989; mark et al., 1991) , but it is not clear if the simulated time intervals are long enough to realistically reflect ionic strength effects. another problem with mm is that the understanding it provides of the system (through energy minimisation or md) does not include entropic aspects explicitly, i.e., it does not give free energies directly. there are methods to calculate free energies based on mm potentials (beveridge and dicapua, 1989) , but even though several applications have been made on biomolecular systems (for a review see beveridge and dicapua, 1989) , they are still too demanding for routine use in systems of this size. then, when the properties under study are related to free energies rather than energies (which is often the case), mm by itself can only be seen as a first approach. in summary, although mm simulations can provide some unique information on the structural and dynamical behaviour of biomolecular systems, some limitations exist due to both conceptual and practical reasons, in particular regarding the treatment of electrostatic interactions. fortunately, other methods exist that can provide insight on aspects whose modelling is poor or absent in mm simulations, although at the cost of the atomic detail in the description. there is no 'best' modelling method and we should resort to the several methods available in order to gain an understanding of the system that is as complete as possible. the so-called continuum or macroscopic models assume that electrostatic laws are valid at the protein molecular level and that macroscopic concepts such as dielectric properties are applicable. protein and solvent are treated as dielectric materials where charges are located. these charges may be titrable groups (whose protonation state may vary), permanent ions (structural and bound ions, etc.) or, more recently, permanent partial charges of polar groups. given the dielectric description of the system and the placement of the charges, the problem can be reduced to the solution of the poisson equation (or any equivalent formulation), as can any problem of electrostatics (e.g., jackson, 1975) . the electrostatic potential thus obtained can be used to study diffusional processes or visually compare different molecules (see 6.6.). the simplest continuum model assumes the same dielectric constant inside and outside the protein. typically, a value somewhere between the protein and solvent dielectric constants has been used (sheridan and allen, 1980; koppenol and margoliash, 1982; hol, 1985) . this approach completely ignores the effects of having two very different dielectric regions, but can be used for a first qualitative computation. the more common continuum models treat the protein as a low dielectric cavity immersed in a high dielectric medium, the solvent. the way the charges are placed in this cavity and the way the electrostatic problem is solved vary with the particular method. analytical solutions can be obtained for the simplest shapes, such as spheres, but in general the more complex shapes require numerical techniques. in the first cavity model the protein was assumed to be a sphere with the charge uniformly distributed over its surface (linderstr~m-lang, 1924) . tanford and kirkwood (1957) proposed a more detailed model in which each charge has a fixed position below the surface. assuming a spherical geometry allows for a simple solution to the electrostatic problem. it is even possible to include an ionic atmosphere that accounts for ionic strength effects (leading to the poisson-boltzman equation). the effect of ph occurs naturally in the formalism. the energy cost of burying a charge inside the low-dielectric protein (self-energy) is taken to be the same as in small model compounds, since at the time when this method was developed (before protein crystallography) charges were believed to be restricted to the protein surface. this limits the method to proteins without buried charges, unless we have some estimate on the self-energy. there are, obviously, some problems in fitting real, irregularshaped proteins to a spherical model. some solutions to this problem were proposed, including an ad hoc scaling of interactions based on solvent accessibility (shire et al., 1974) , and the placing of more exposed charges in the solvent region (states and karplus, 1987) . the inclusion of non-spherical geometries im-plies the use of numerical techniques, as referred above. warwicker and watson (1982) and used the finite differences technique to solve, respectively, the poisson and poisson-boltzman equations. self-energies can be included (gilson and honig, 1988) , such that the method is fully applicable when buried charges exist. the intrinsic discretization of the system in the finite differences technique, makes these methods readily applicable to any kind of spatial dependency on any of the properties involved. the inclusion of a spatially-dependent dielectric constant, for instance, will be relatively simple. other extensions such as additional dielectric regions (ligands, membranes, etc.), eventually with charges, should also be possible. alternative numerical techniques for solving the poisson or poisson-boltzman equations have also been used, including finite elements (orttung, 1977) and boundary elements (zauhar and morgan, 1985) . the dielectric constant in a region comes from the existence of dipoles in that region, permanent or induced. permanent dipoles are due to atomic partial charges (e.g., water dipole, peptide bond dipole). induced dipoles are due to the polarizability of electron clouds. warshel and levitt (1976) represented this electronic polarizability by using point dipoles in the atoms. as pointed out by davies and mccammon (1990) this representation is roughly equivalent to a spatially-dependent dielectric constant. this approach is usually combined with a simplified representation ot water by a grid of dipoles (warshel and russel, 1984) . ionic strength and ph effects are not considered. all the above methods deal with a particular charge set (see 6.1.), even when ph effects are considered. however, a protein in solution does not exist in a single charge set. we are usually interested in the properties of a protein at a given ph and ionic strength, not at a particular charge set. moreover, if we want to test the available methods, we have to test them against experimental results which usually do not correspond to a specific charge set. a common test on the accuracy of electrostatic models is their ability in predicting pk a values of titrable groups in a protein (see 6.6.), obtained via titrations, nmr, etc. these values can be quite different from the ones of model compounds, due to environment of the groups in the protein. this difference (pk a shift) can be of several pk units. the experimentally determined apparent pka (pkap p) is determined as the ph value at which half of the groups of that residue are protonated in the protein solution, i.e., when its mean charge is 1/2 (thus, the equivalent notation pk1/2). then, if we can devise a method to compute the mean charge of the titrable groups at several ph values, we can predict their pkap p values. as mentioned above (see 6.1.), we have 2 n possible charge sets. any structural property can, in principle, be computed through a boltzman sum over all those sets, with each one contributing according to its free energy (taken as the electrostatic energy) (tanford and kirkwood, 1957; bashford and karplus, 1990) . the property thus computed is characteristic of the chosen ph value (and ionic strength, if considered) instead of a specific charge set. we are particularly interested in computing the mean charges at a given ph (see last paragraph). a sum with 2 n terms is not, however, a trivial calculation in terms of computer time. tanford and roxby (1972) avoided the boltzman sum by placing the mean charges directly on the titrable groups, instead of using one of the integer sets. this corresponds to considering the titration of the different groups as independent (a mean field approximation; bashford and karplus, 1991) . other alternatives to the boltzman sum are the monte carlo method (beroza et al., 1991) , less drastic mean field approximations (yang et al., 1993; gilson, 1993) , the 'reduced site' approximation (bashford and karplus, 1991) , or even assume that the predominant charge set is enough to describe the system (gilson, 1993) . since electrostatic interactions in proteins are typically dominated by titrable groups whose charge is affected by ph, no electrostatic treat-ment can be complete without taking this effect into account. a simple, although effective, way of doing this is to: (i) compute the electrostatic free energies (e.g., by a continuum method); (ii) compute the mean charge of each titrable group at a given ph (e.g., by a mean field approximation); (iii) use those charges to compute the electrostatic potential (e.g., by a continuum method), which can be displayed together with the protein structure (see the human pancreatic lipase example in section 6.6.). in this way a ph-dependent electrostatic model of the protein can be obtained, which is not possible with usual mm-based modelling techniques. as stated above (see 6.1.), electronic polarizability is not explicitly considered in common force fields. van belle et al. (1987) included the induced dipole formalism (warshel and levitt, 1976) in mm calculations. the electrostatic interactions in the applied force field were simply 'corrected' with additional terms due to inducible dipoles. however, it should be noted that a force field fitted to experimental data without polarizability terms, should be fitted again if those terms are included. the protein conformation used in molecular modelling is usually an experimentally based (xray, nmr) mean conformation, characteristic of those particular experimental conditions. that conformation may, however, be inadequate for modelling the protein properties at different conditions. in particular, proteins are known to denaturate at extreme ph conditions. thus, ph-dependent methods such as the continuum methods may give incorrect results when using one single conformation over the whole ph range. actually, md simulations have shown that the results can be highly dependent on side chain conformation (wendoloski and matthew, 1989) . although overall properties like titration curves did not seem to be very sensitive, individual pka's showed variations up to 2.0 pk units. as mentioned in section 6.1, mm has the problem of what charge set to use in simulations. instead of using a charge set corresponding to model compounds at the intended ph, one may use the predominant charge set of the protein, determined, e.g., by a continuum method, as suggested by gilson (1993) . a different approach to this problem would be to devise a way of including the averaged effect of all charge sets in the mm simulation. we have recently developed a method where a force field is derived which includes the proper averaged effect of all charge sets (a potential of mean force) (to be published). the method depends on the calculation of electrostatic free energies obtained from, e.g., a continuum method. the electrostatic potential, computed in some of the referred methods, can help to understand the contribution of electrostatic interactions in the diffusional encounters of proteins with ligands (substrates or not). the diffusional process driven by the electrostatic field can be simulated through brownian dynamics (bd) and diffusion rates may be computed (for references see, e.g., davies and mccammon, 1990) . the effect of mutations on the diffusion of superoxide ion into the active site of superoxide dismutase has been studied by this technique (sines et al., 1990) and faster mutants showing 2-3-fold increase in reaction rate could be designed (getzoff et al., 1992) , although this enzyme usually is considered to be 'perfect'. electrostatically driven bd simulations can help to reveal steric 'bottlenecks' (reynolds et al., 1990) and orientational effects (luty et al., 1993) . this method can also be applied to study the encounter of two proteins (northrup et al., 1988) . visual comparison of electrostatic fields can also provide useful information. soman et al. (1989) showed that rat and cow trypsins have similar electrostatic potentials near the active site, despite a total charge difference of 12.5 units. as an illustration of such type of comparisons, using ph-dependent electrostatics, we have applied the solvent accessibility-modified tanford-kirkwood method (see 6.2.) to the human pancreatic lipase structures with both closed (van tilbeurgh et al., 1992) and open lid (van tilbeurgh et al., 1993) , as shown in fig. 15a and b. fig. 15c-f shows surfaces corresponding to an electrostatic potential equal to + 1.0 kt/e (where k is the boltzman constant, t the absolute temperature and e the proton charge). these surfaces correspond to regions were the electrostatic interactions on a charge are roughly of the same magnitude as the thermal effects due to the surrounding solvent, i.e., where charged molecules in solution start to feel electrostatic steering or repulsion. at ph 7 clear differences exist between the closed and open forms, the latter showing a dipolar groove in the presumed binding site region. at pi-i 4 the molecule is strongly positively charged and most electrostatically differentiated regions have disappeared. given the role of electrostatic interactions on molecular orientation and association (see the beginning of this section (6)), this is expected to markedly affect the interaction with the lipid-water interface. for enzymes the catalytic activity involving a charged residue can be modulated by shifting the pk a of that residue. the pk a shifts of the active site histidine has been successfully predicted for a number of mutants of subtilisin loewenthal et al., 1993) . one of the main reasons why enzymes are good catalysts is because they stabilise the transition state intermediate (fersht, 1985) . for enzymatic reactions that are not diffusion limited, engineering leading to an enhanced stabilisation of the intermediate will result in an increased activity. the induced dipole method was used to compute the activation free energy for different mutants of trypsin and subtilisin (warshel et al., 1989) , with some qualitative agreement with the experimental results. the prediction of changes introduced by mutations on redox potentials could also be of interest to protein engineering. prediction of redox potentials has been made with some success (rogers et al., 1985; durell et al., 1990) . in plastocyanin the effect of chemically modifying charged groups was also considered (durell et al., 1990) . the effect of mutations could also be analysed, as has been done for pk a shift calculations (see above). the above examples clearly show that, whatever the particular method used, the modelling of 15a c e 13 d t electrostatic interactions in proteins has an important role to play in protein engineering. a highly relevant example is the design of a faster 'perfect' enzyme (getzoff et al., 1992) , which also illustrates the combination of different methods (bd and electrostatic continuum methods) that can sometimes be determinant in a modelling study. the science of protein engineering is advancing rapidly, and is emerging in many new contexts, such as metabolic engineering. rational protein engineering is a complex undertakingand only the groups with sufficient understanding of sequences and 3-d structures can handle the complex underlying problems. predicting protein structure may be difficult -but predicting future developments in a very active branch of science can be hazardous at the best. however, we will review a few of the more recent research aspects that we are convinced will be of key importance in the future development of protein engineering. often the substrates or products in an enzymatic process are poorly soluble in an aqueous medium. this may lead to poor yields and difficult or expensive purification steps. the potential of using other solvents, either pure or in mixture, where substrates and/or products may be soluble has attracted a great deal of attention (tramper et al., 1992; arnold, 1993) . dissolving the protein in organic solvents will alter the macroscopic dielectric constant and lead to a much less pronounced difference between the interior and exterior static dielectric behaviour. protein function in such media may be altered and is poorly understood; we can expect a significant development in the future. despite the often dramatic change in dielectric constant when changing the solvent from, e.g., water to an organic substance, the protein 3-d structure can remain virtually intact, as has been documented in the case of subtilisin carlsberg dissolved in anhydrous acetonitrile (fitzpatrick et al., 1993) . the hydrogen bonding pattern of the active site environment is unchanged, and 99 of the 119 enzyme-bound structural water molecules are still in place. one-third of the 12 enzymebound acetonitrile molecules reside in the active site. many enzymes remain active in organic solvents and in the case of enzyme reactions where the substrate has very poor water solubility, a change to organic solvent can be of major importance (gupta, 1992 ). an extreme case of a non-conventional medium for enzymatic action is the gas phase. certain enzymes, immobilised on a solid bed, have been shown to be active at elevated temperatures towards selected substrates in the gas phase (lamare and legoy, 1993) . obviously the range of substrates that potentially can be used is limited to those that actually can be brought into the gas phase under conditions where the enzyme is still active. enzymes for which such reactions have been studied include hydrogenase, alcohol oxidase and lipases. the fact that even interfacially activated lipases (such as the porcine pancreatic and the candida rugosa lipases) function with gas phase carried substrate molecules opens up the interesting possibility of studying the role of water in this reaction. protein engineering may be used to enhance enzyme activity in organic solvents (arnold, 1993; fig. 15 . electrostatic maps of hpl with closed and open lid. ribbon models of human pancreatic lipase with colipase are shown with closed (left: a,c,e) and open (right: b,d,f) lid. the colipase is shown in blue and the mainly a-helical 'lid' region is highlighted in cyan. the residues of the active site are shown in green. access to the active site pocket seems to be controlled by the conformational st'ate of the lid. electrostatic isopotential contours of + 1.0 kt/e are shown at ph 4 (c,d) and ph 7 (e,f). the negative surfaces are represented in red and the positive surfaces in blue. the models and isopotentiai contours were produced with insight h and delphi (biosym technologies, san diego). the ph-dependent charge sets were computed with titra (to be published). chen and arnold, 1993) . when dissolving subtilisin e in 60% dimethylformamide (dmf) the kcat/k m for the model substrate suc-ala-ala-pro-met-p-nitroanilide drops 333-fold. after ten mutations were introduced, the activity in dmf was restored almost to the level of the native enzyme in water. all metabolic conversions in micro-organisms are carried out directly or indirectly by proteins. our ability to manipulate single genes has opened up for the actual control of such processes. we may alter the efficacy of a certain pathway or we may introduce totally new pathways. thus, escherichia coli can be modified in such a way that one can use i>glucose in the e. coli based manufacture of hydroquinone, benzoquinone, catechol and adipic acid (dell and frost, 1993; draths and frost, 1990; frost, 1993) . presently such compounds are produced through organic chemical synthesis using aromatics as one of the reactants. the prospect of producing the same compounds using only microbes and glucose thus has some obvious environmental benefits. we expect to see a virtual surge in the engineering of microorganisms towards the production of rare chemical or biochemical compounds or compounds for which the current synthetic route is costly either economically or from an environmental perspective. the perspective of designing and producing functional protein molecules from scratch is extremely attractive to many visionary scientists. some central questions arise: do we know enough to undertake such tasks, and what goals can we define? screening mutation studies of protein interfaces show that the majority of mutations reduce activity or binding affinity (cunningham and wells, 1993) , indicating that most proteins already represent highly optimised designs. the groups active in this area have aimed at constructing certain 3-dimensional folds such as the four helix bundle (felix) (hecht et al., 1990 ) and histidine-based metal binding sites (arnold, 1993) and even the observation of limited enzymatic activity is regarded as a successful result . protein de novo design of helix bundles may even follow a very simple binary pattern of polar and nonpolar amino acids as was concluded in a study of four-helix bundle proteins (kamtekar et al., 1993) . the helix-helix contact surfaces are mainly hydrophobic, whereas the solvent exposed regions are hydrophilic. many variants conforming to this hydrophobic pattern were generated and two of these proteins were stabilised with 3.7 and 4.4 kcal tool -~ relatively to the unfolded form, thus approaching what is found for many natural proteins. the authors suggest that such a binary pattern may have been important in the early stages of evolution. in our laboratory we have results supporting this conclusion for the trypsin family of proteins, which is predominantly in a /3-strand based fold . fusion and hybrid proteins may be produced by fusing the genes or gene fragments including a proper linking region between the two genes (argos, 1990) . this in principle may allow for combining properties from two different proteins. thus artificial bifunctional enzymes have been produced by fusing the genes for the proteins, e.g., /3-galactosidase and galactokinase (bulow, 1990 ). in a recent paper an elegant hybrid protein concept is described. a hybrid antibody fragment was designed to consist of a heavy-chain variable domain from one antibody connected through a linker region of 5-15 residues to a short lightchain variable domain from another antibody (holliger et al., 1993) . the antibody fragments displayed similar binding characteristics as the parent antibodies. the prospect of engineering multifunctional antibodies for medical applications is imminent. a hybrid protein between the glucose transporter and the n-acetylglucosamine transporter of e. coli have been produced. the two proteins displayed 40% residue identity. the hybrid protein consisted of the putative transmembrane do-main from the glucose transporter and the two hydrophilic domains from the n-acetylglucosamine transporter. the hybrid protein was, somewhat surprisingly, still specific for glucose (hummel et al., 1992) . interestingly, several naturally occurring proteins themselves seem to have originated through gene fusion. in the case of hexokinase it is proposed that it originated from a duplication of the glucokinase gene maintaining even the gene organisation (kogure et al., 1993) . several other proteins such as receptor proteins of the insulin family can best be understood as gene fusion products of a kinase domain onto the rest of the receptor (which in itself may consist of several fragments). with potential medical applications, proteinnucleic acid hybrids have been constructed, where the nucleic acid fragment complemented the sequence of a fragment of mrna that the rnase should be targeted towards. the results obtained confirmed that this approach indeed worked (kanaya et al., 1992) . the potentials for generating anti-viral agents against, e.g., hiv are obvious. as a consequence of the enormous growth in our understanding of molecular biology and material technology, a new technological sector is emerging which takes aim at exploring the possible advantages in creating micro-machines and switchable molecular entities. this concept is currently known as nano technology (birge, 1992) . two concepts that we find particularly interesting are described briefly below. rhodopsin is a very ancient molecular construct -we find rhodopsin like molecules in a range of roles, all of them associated with its membrane location. proton transport and receptor functions are particularly interesting. bacteriorhodopsin from halobacterium halobium maintains a large ph gradient across the bacterial membrane. this protein complex is coloured, and its colour can be changed by exposing the protein to light of an appropriate frequency. the lifetime of the excited state can be adjusted by adjusting the physical chemical parameters of the medium the rhodopsin is embedded in (birge, 1992) . this protein can be used as a molecular switch in a very broad sense, e.g., as part of a high density memory device. however, changing the colour of a protein molecule is just one example that could be considered. another molecular based switch concept involves the transfer of a molecular ring (paraquat-derived rotaxane ring) between two binding sites (bradley, 1993) . currently the transfer is induced by a solvent change, but it is believed that an electrochemical transfer mechanism can be developed as well. similar concepts can probably also be developed for proteins. the present paper reviews some of many new developments in protein engineering. the review is not exhaustive -it is simply not possible to do this properly within the limits of this paper. we have tried to review some selected scientific areas of key importance for protein engineering, such as the validity of protein sequence information as well as structural information. sometimes the translation of a gene sequence to amino acid sequence is not trivial -a range of posttranscriptional editing and splicing events may occur, leading to a functional protein, where the amino acid sequence cannot be directly deducted from the gene sequence. in addition, posttranslational modification may provide triggers for other parts of the cells molecular machinery. we are thus in a situation where the full benefits and profits from projects such as the human genome project may escape us for a while. we have covered some of the recent developments in the modelling of protein structure by homology, which we regard as one of the most strategic areas of development. we will be flooded with sequence information deducted from gene sequences, and in the cases where the deducted amino acid sequences are assumed valid, we have to use homology based structure prediction in most cases. given that the number of protein structure families is expected to be limited the task is durable. here we should again caution the reader. we have no a priori reason to assume that non-soluble proteins, such as structural proteins, have structures that can be predicted from our limited library of mostly globular, soluble proteins. some structural proteins are gigantic, the cuticle collagen in the riftia worms from deep sea hydrothermal vents have a molecular mass of 2.600 kda (gaill et al., 1991) . it is extremely unlikely that a 3-d structure at atomic resolution of such a protein will ever be determined using methods we have available today. nmr has emerged with surprising speed as a structure determination tool. many excellent reviews have been written on this topic. we have decided to direct the readers attention to some recent developments that we believe will be of significant importance to the usage of nmr in protein engineering projects. the potential of using nmr to study the solvent exposed outer shell of larger proteins, that by far exceed the 30 kda limit mentioned earlier is intriguing. this is particularly so, since most functionality of a protein is a feature of exactly the residues in the outer shell. thus, we can 'peel' the protein, and thereby isolate the spectral information that pertains to the surface only. this simplifies the spectra, and in some cases even allows for a partial assignment of specific residues. recent developments in ph-dependent protein electrostatics have been given special attention here. the similarities and differences within a family of structurally related proteins can only be understood if we are capable of interpreting the consequences of the substitutions, insertions and deletions that mostly occur at the surface of the proteins. when such changes are found and they involve charged residues, this will effect the extent or polarity of the electrostatic fields that the protein molecule is embedded in. we believe that the consequences of charge mutations to a large extent can be predicted through the use of ph-dependent electrostatics although practical examples are still lacking. to our knowledge the results on the electrostatic consequences of the lid motion in the human pancreatic lipase (vide 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cloning of cdna coding for rat plasma glutathione peroxidase a new method for computing the macromolecular electric potential an optimization approach to predicting protein structural class from amino acid composition a weighting method for predicting protein structural class from amino acid composition we want to thank christian cambillau, cnrs, marseille, for kindly providing us with pre-release 3-d data of human pancreatic lipase, jerry h. brown, harvard university, for sending us a prerelease dataset for the hla ii structure, alwyn jones, uppsala university, for pre-release 3-d data of candida antarctica b lipase, and johnmccarthy, brookhaven national laboratory, for helping us with data on previous pdb releases. the french norwegian foundation (fns 27958) and the norwegian research council (bp 29345) have contributed with financial support to some of the research activities described in this paper. a.b. and p.m. thank junta nacional de investi-ga~o cientlfica, portugal, for their grants. key: cord-010604-3d37o05y authors: rein, theo title: post-translational modifications and stress adaptation: the paradigm of fkbp51 date: 2020-04-29 journal: biochem soc trans doi: 10.1042/bst20190332 sha: doc_id: 10604 cord_uid: 3d37o05y adaptation to stress is a fundamental requirement to cope with changing environmental conditions that pose a threat to the homeostasis of cells and organisms. post-translational modifications (ptms) of proteins represent a possibility to quickly produce proteins with new features demanding relatively little cellular resources. fk506 binding protein (fkbp) 51 is a pivotal stress protein that is involved in the regulation of several executers of ptms. this mini-review discusses the role of fkbp51 in the function of proteins responsible for setting the phosphorylation, ubiquitination and lipidation of other proteins. examples include the kinases akt1, cdk5 and gsk3β, the phosphatases calcineurin, pp2a and phlpp, and the ubiquitin e3-ligase skp2. the impact of fkbp51 on ptms of signal transduction proteins significantly extends the functional versatility of this protein. as a stress-induced protein, fkbp51 uses re-setting of ptms to relay the effect of stress on various signaling pathways. the physiological stress response is elicited whenever a change in the environment is sensed and interpreted as threat to homeostasis. effector systems comprise the autonomic nervous system and the hypothalamic-pituitary-adrenocortical (hpa) axis [1] . the effector molecules of these systems are noradrenaline and adrenaline for the autonomic nervous system, and cortisol for the hpa axis (corticosterone in rodents). adrenaline and noradrenaline act through g protein-coupled receptors that are hooked to various intracellular pathways involving determinants of post-translational modifications (ptms) such as kinases and phosphatases [2] . these determinants are also referred to as 'writers' and 'erases' of ptms [3, 4] . using ptms for signal transduction comes with the obvious advantage of allowing for quickly changing the mode of action of pre-synthesized proteins in a vast functional space defined by the huge number of possible combinations of ptms at different amino acids [4, 5] . moreover, ptms are reversible at a small time scale, which is another important feature for the stress response, in particular when stress exposure is short [5] . the hpa axis also activates g protein-coupled receptors, namely the receptors for the hormones corticotropin-releasing factor and adenocorticotropic hormone [1] . however, its final effector cortisol operates through the steroid receptors glucocorticoid receptor (gr) and mineralocorticoid receptor [6] . these receptors are transcription factors, and thus this part of the overall stress response typically takes longer than the time required to redefine the function of proteins through re-setting their ptms. nevertheless, the action of these receptors also is intertwined with ptms: they are subject to regulation by ptms and they impact writers and erasers of ptms [7] [8] [9] . in part, this is achieved through the synthesis of specific proteins that take part in the orchestration of proteome function through ptms. fk506 binding protein (fkbp) 51 turned out to be one of these proteins which is subject of this review. the stress protein fkbp51, promoted through translational research fkbp51 is a show-case of translational research where clinical and basic science approaches stimulated each other. the background of fkbp51 is laid out here only shortly, and the reader is referred to the numerous recent reviews for more detailed information [10] [11] [12] [13] [14] [15] . originally discovered as part of steroid receptor-heat shock protein 90 hetero-complexes, fkbp51 was shown to be a potent inhibitor of gr by several laboratories [16] [17] [18] [19] . by virtue of its binding to immune suppressive drugs such as fk506, fkbp51 also has been classified as 'immunophilin' [15] . biochemically, fkbp51 is able to isomerize peptidyl-prolyl bonds [20] ; the physiological relevance, if any, of this function is not clear [11, 12, 21] . however, the peptidylprolyl isomerase domain of fkbp51 is engaged in protein interactions, and drug binding to this domain likely affects several functions of this protein [13] . the inducibility of fkbp51 gene (named fkbp5) expression by the activated gr [22] [23] [24] [25] [26] [27] gives rise to an intracellular ultra-short negative feedback loop, as one of the hallmarks of adaptive molecular circuits [11] . of particular interest for neuropsychiatric research was the observation that fkbp51 was overexpressed in squirrel monkeys featuring altered set-points of the hpa axis [28] . thus, the molecular settings in these animals were considered a model for gr-resistance [29] . this was related to the situation in patients suffering from depression where malfunction of gr was hypothesized to be causal for the development of the disease [30] . based on these considerations, fkbp5 was included as candidate gene in an early gene association study in depression that found this gene linked to the response to antidepressant treatment [31] . later, fkbp5 could be linked to additional stress-related diseases such as post-traumatic stress disorder [12, 14] . these findings strongly amplified the interest in fkbp51 and stimulated research on its function and regulation in several laboratories; this greatly expanded the knowledge base on fkbp51's ( patho)physiological role, regulation on several levels and involvement in multiple molecular pathways, going beyond stress regulation [12, 13] . the variety of its physiological functions goes along with its association with several proteins, including proteins involved in writing and erasing ptms, as detailed in the subsequent sections. more than 200 ptms are known with a major impact in the configuration of protein networks [32] [33] [34] ; the vast majority of these modifications are reversible with prominent examples being the attachment of chemical groups (e.g. phosphorylation, acetylation, methylation, nitrosylation, sulfonation), the conjugation with polypeptides (e.g. ubiquitination, nedd8 [neural-precursor-cell-expressed developmentally down-regulated 8], and ubiquitin-like peptides such as sumo [small ubiquitin-like modifier] or atg8 [autophagy-related gene 8]) and the addition of a complex group of molecules including, e.g. prenylation, farnesylation, glycosylation, palmitoylation, myristoylation, glutamylation, adp-ribosylation and ampylation [4, 35] . protein phosphorylation is one of the first known ptm [36, 37] and probably the best studied one affecting almost all biological processes [38] . in eukaryotes, proteins are phosphorylated primarily through phosphor-ester bonds formed at the residues serine, threonine and tyrosine, and up to 2% of the protein-coding genes produce the enzymatic machinery governing this process [39] . while this review focusses on the effect of fkbp51 on ptms of other proteins, it should be noted that fkbp51 also is subject to ptms itself. this has been reviewed very recently [40] , and thus is mentioned here only briefly: not surprisingly, the first reported ptm of fkbp51 was phosphorylation. originally, it was inferred from the pattern of this protein in 2d gel electrophoresis and the modulation of this pattern by the use of kinase inhibitors or phosphatases [40] [41] [42] . more recently, pten-induced putative kinase 1 (pink1) was found to phosphorylate fkbp51 at yet to be mapped serine residues [43] . thereby, pink1 regulates the interaction of fkbp51 with the kinase akt1 and the phosphatase phlpp [43] . this interaction further is controlled by acetylation of fkbp51 at lysines 28 and 155 [44] . the sirtuin sirt7 has been identified as deacetylase acting at these sites [44] . sumoylation of fkbp51 was detected and mapped to lysine 422 [45] . it regulates the inhibitory action of fkbp51 on gr [45] . the first evidence for the involvement of fkbp51 in the regulation of the phosphoproteome was provided by the observation that fk506-bound fkbp51 inhibits the serine/threonine-phosphatase calcineurin (also known as protein phosphatase 2b), thereby inhibiting nuclear factor of activated t cells (nfat) [46, 47] . it also has been reported that fkbp51 interacts with calcineurin in the absence of fk506 [48] , which was not observed by others [47] . nevertheless, impacting ptms through re-arranging protein association of kinases and phosphatases as in the case of calcineurin/nfat is the mode of action also revealed for the effect of fkbp51 on many other signaling pathways. the inhibition of calcineurin by fkbp51 is reported to also affect the nuclear factor (nf)κb pathway [49] . in this pathway, phosphorylation of the inhibitor of κb (iκb) by the kinase of iκb (ikk) leads to activation of nfκb [50] . therefore, the protein associations of fkbp51 with calcineurin as well as with ikkα and other kinases of the nfκb pathway [51] support a model where fkbp51 impacts nfκb activity through re-setting phosphorylation at multiple levels with variable outcome [11] . for example, a direct association between fkbp51 and both tnf receptor-associated factor 2 (traf2) and ikkγ was found [52] . traf2 catalyzes k63-linked poly-ubiquitination of receptor-interacting protein 1 (rip1) in response to tnfα, thereby facilitating the recruitment of the ikk complex to its upstream activating kinase tak1 (transforming growth factorbeta activated kinase 1) [53, 54] . fkbp51 enhances and shapes this polyubiquitin-mediated interaction, thereby changing the phosphorylation of ikk and iκb and thus the activity of nfκb [52] . the serine/threonine kinase akt (also known as protein kinase b) is another well-examined example of fkbp51's impact on ptms through organizing protein complexes. akt is activated by step-wise phosphorylation in response to extracellular signals that involves its translocation from the cytoplasm to the cell membrane [55, 56] . fkbp51 employs its ability to interact with various proteins, frequently referred to as scaffolding, to recruit the phosphatase phlpp that de-phosphorylates and thereby inactivates akt [57, 58] (figure 1a ). akt is a central pathway regulator that inhibits apoptosis and promotes cell growth [59] . accordingly, fkbp51 expression is enhanced in most cancer types and is linked to resistance to chemotherapy [57, [60] [61] [62] . evidence has also been provided that fkbp51 mediates the inactivation of akt induced by stress [58] . thus, fkbp51 may also relay the effect of stress on the phosphoproteome [11] . fkbp51's effect on akt also alters downstream ptm-dependent pathways. for example, through akt1 fkbp51 impacts p38 mapk, thereby differentially regulating the transcription factors gr and peroxisome proliferator-activated receptor-γ [63] . fkbp51 also governs the effects of akt1 on its targets beclin 1 (becn1) and s-phase kinase-associated protein 2 (skp2), constituting a link to autophagy as detailed below [58, 64, 65] . [58, 64, 65] . the recruitment of phlpp leads to lower akt phosphorylation and activity, entailing less phosphorylation of becn1 and of the e3-ligase skp2. thereby, skp2 is less active resulting in lower ubiquitination of becn1. whether or not fkbp51 associates with all these proteins in one complex remains to be elucidated. another downstream target of akt is glycogen synthase kinase 3β (gsk3β) [66] . consistent with the inhibitory effect of fkbp51 on akt1, it has been reported that overexpression of fkbp51 decreased the phosphorylation of gsk3β at serine 9 [57] . however, it also has been found that fkbp51 associates with gsk3β, and increases its phosphorylation [67] . this appears to be accomplished through rearrangement of the protein heterocomplex governing phosphorylation and thus the activity of gsk3β. more specifically, fkbp51 recruits cyclin-dependent kinase 5 (cdk5) and furthermore associates with the three subunits of the phosphatase pp2a [67] (figure 1b) , which acts in concert with cdk5 to regulate gsk3β affecting downstream targets [68] . thus, fkbp51 redefines signaling pathway connections through protein associations. fkbp51's impact on protein phosphorylation furthermore provides a link to epigenetic regulation as well as to metabolic function. the link to epigenetics is evidenced by its impact on phosphorylation, and thus the activity of dna methyltransferase 1 (dnmt1) [69] . mechanistically, this effect appears to be achieved through the differential association of fkbp51 and its close homolog fkbp52 with cdk5 and its regulatory protein p35 [69] . metabolic function is impacted by fkbp51 through protein associations that dephosphorylate and thus inhibit akt2, leading to dephosphorylation and thus inhibition of as160 and reduced glucose uptake [70] . ptms are also involved in the effect of fkbp51 on microtubule dynamics. it is assumed that phosphorylation of tau leads to its dissociation from microtubules and adoption of the trans configuration of specific peptidylprolyl bonds, while the association of phosphorylated tau with fkbp51 promotes its dephosphorylation and cis configuration that is required for microtubule association [71] [72] [73] . another mass spectrometry-based screen for fkbp51-associated proteins revealed the rho (ras homologous) gtpase-activating proteins deleted in liver cancer (dlc) 1 and dlc2 as novel interaction partners [74] . accordingly, fkbp51 enhances rhoa activity and signaling through the serine/threonine kinase rock (rho-associated coiled-coil containing protein kinase) along with the linked processes cell migration and invasion [74] . the exact mechanism remains to be elucidated. it appears that this is an example for a more indirect effect of fkbp51 on the phosphoproteome: it inhibits a protein, dlc, that serves as a gtpase activator for members of the rho family of gtpases that regulate downstream kinases [74, 75] . ubiquitination is an essential ptm in all eukaryotes [3, 76] . it determines protein stability as well as protein function through changing protein-protein interaction. biochemically, ubiquitination is a process where the 76 amino acid protein ubiquitin is covalently linked to other proteins, in most cases through the formation of an amide bond between its carboxy terminus and the ε amino group of lysine residues in the substrate proteins [34, 76] . in addition to this isopeptide bond, ubiquitin forms other links to target diverse protein residues such as cysteines, serines or threonines [3] . ubiquitination comes in the form of mono-ubiquitination and polyubiquitination, where distinct lysines of one ubiquitin serve as attachment sites for additional ubiquitin moieties. the site of linkage destines the modified protein to different functions. for example, poly-ubiquitination through the lysines at position 11 and 48 typically lead to degradation of the protein through the 26s proteasome [77, 78] . the first indication that fkbp51 influences the ubiquitination of other proteins came from the observation that it stabilizes tau and protects it from becoming ubiquitinated [71] . the mode of action appears to be an indirect mechanism where fkbp51 influences the conformation and/or phosphorylation of tau to prevent the access of ubiquitinating enzymes [71, 73, 79] . for the differential effect of fkbp51 and fkbp52 on nfκb, it has been proposed that a mechanism is involved that is similar to pin1's promotion of the ubiquitin-mediated proteolysis of the nfκb subunit p65/rela [80] . further experimental evidence is awaited; in any case, this effect also would be indirect. the reported effect of fkbp51 on nfκb signaling through the association with traf2 and ikk is dependent on the k63-poly-ubiquitination of rip1, but does not appear to impact ubiquitination in this context [52] . the e3 ubiquitin-protein ligase complex members traf3 and traf6 also were discovered as fkbp51 associating proteins, with currently unknown consequences for their enzymatic activity [81] . more recently, a direct association of fkbp51 with glomulin, a regulator of the scf (skp1-cul1-f-box protein) e3 ubiquitin-protein ligase complex, has been described in detail and in comparison with other fkbps [82] . it is likely that this interaction affects the ubiquitination activity of scf; experimental investigation of this potential impact of fkbp51 on the ubiquitination machinery has not been reported yet. a yeast two-hybrid screen indicated protein associations of fkbp51 with the ubiquitin-specific peptidases (usps) 18, 36 and 49 as well as with the e3 ubiquitin ligases ring finger protein 219 and skp2 [83] . while the functional consequences were not explored in this report, another study revealed that usp49 stabilizes fkbp51 by the removal of ubiquitin chains [84] . thus, in this case, the association with a ptm executer results in fkbp51 being a target rather than a modifier. conversely, fkbp51 regulates the ubiquitination activity of skp2 through phosphorylation, probably by functioning as protein scaffolder for the association with phlpp and akt1 [65] . this novel fkbp51 function on protein ubiquitination has far-reaching consequences: the study further discovered the autophagy regulator becn1 as novel target of skp2 executing k48-linked poly-ubiquitination at this autophagy regulator [65] . thus, fkbp51 drives autophagy involving at least two types of ptms of becn1, ubiquitination and phosphorylation [58, 64, 65] (figure 1c ). given the multiple targets of skp2 [85] [86] [87] [88] , it is assumed that the ubiquitination of more proteins will be changed by fkbp51 with diverse functional consequences. autophagy is another fundamental cellular process essential for protein, organelle and energy homeostasis [89] . it involves several atg products that through several steps form autophagosomes, vesicles that engulf material destined for degradation which is accomplished upon the fusion with lysosomes [89, 90] . an important step in this process is the lipidation of atg8 (also known as microtubule associated protein 1 light chain 3 beta), a ubiquitin-like protein that is integrated into the autophagosomal membrane upon formation of an amide bond the stress protein fkbp51 is intertwined with gr as both inhibitor and target. as target, it has the potential to relay the stress response to downstream pathways through the association with writers and erasers of ptms. known associations include de-ubiquitinases, ubiquitinases, protein kinases and protein phosphatases (first box, writers and erasers of ptms are grouped according to the type of their biochemical activity). some of the associated proteins are changed in their activity, others are redirected to certain targets. examples of target proteins affected by the altered activities of ptm writers and erasers are provided in the box below. '+' and '−' after the protein names indicate the overall effect of fkbp51 on the activity of associating proteins or downstream proteins. not all potential interactions are displayed. becn1 is a downstream protein that also forms a complex with fkbp51. however, most of the downstream target proteins are indirectly affected in the sense that they were not shown to associate with fkbp51. with phosphatidylethanolamine [91, 92] . similar to the process of ubiquitination, the formation of this amide bond is executed by a set of ligases (e1-e3) that use cysteine linked thioesters as intermediates. the last step, conjugation to phosphatidylethanolamine, is mediated by the e3-like atg12-atg5:atg16 complex [93] . the effect of fkbp51 on this protein lipidation probably is indirect through driving autophagy by regulating becn1. however, fkbp51 also leads to enhanced levels of atg12, a member of the conjugation system [58] , pointing to different pathways used by fkbp51 to enhance atg8 lipidation. • fkbp51 is receiving increased attention for its pivotal role in research on stress and stressrelated diseases, but also several additional fields such as immunology, metabolism, oncology, neurology, etc. • the stress protein fkbp51 is engaged in various signaling pathways through diverse protein interactions. it both affects the stress response and is affected by the stress response, and furthermore relays stress to multiple pathways. likewise, it is the subject of ptms and affects the activity of ptm writers and erasers ( figure 2 ). • protein associations of fkbp51 with ptm writers and erasers could affect ptms of fkbp51 itself or of other proteins, and deciphering these scenarios will significantly contribute to our mechanistic understanding of this versatile protein. furthermore, it will be of high interest to elucidate which of the ptm-mediated downstream effects of fkbp51 contribute to the physiological stress reaction. the author declares that there are no competing interests associated with this manuscript. atg, autophagy-related gene; becn1, beclin 1; cdk, cyclin-dependent kinase; dlc, deleted in liver cancer; dnmt1, dna methyltransferase 1; fkbp, fk506 binding protein; gr, glucocorticoid receptor; gsk, glycogen synthase kinase; hpa axis, hypothalamic-pituitary-adrenocortical axis; iκb, inhibitor of κb; ikk, kinase of iκb; mapk, mitogen-activated protein kinase; nfκb, nuclear factor kappa b; nfat, nuclear factor of activated t cells; phlpp, pleckstrin homology domain leucine-rich repeat protein phosphatase; pp2a, protein phosphatase 2a; pparγ, peroxisome proliferator-activated receptor-γ; ptm, post-translational modification; rip1, receptor-interacting protein 1; scf, skp1-cul1-f-box protein; skp2, s-phase kinase-associated protein 2; tnf, tumor necrosis factor; traf, tnf receptor-associated factor; usp, ubiquitin-specific peptidase. the corticotropin-releasing factor family: physiology of the stress response molecular pathways: 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downstream role of fk506-binding protein 51 in the control of apoptosis of irradiated melanoma cells hsp90-binding immunophilin fkbp51 forms complexes with htert enhancing telomerase activity fkbp51 immunohistochemical expression: a new prognostic biomarker for oscc? fkbp51 controls cellular adipogenesis through p38 kinase-mediated phosphorylation of grα and pparν fkbp5/fkbp51 enhances autophagy to synergize with antidepressant action skp2 attenuates autophagy through beclin1-ubiquitination and its inhibition reduces mers-coronavirus infection inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase b fkbp51 inhibits gsk3β and augments the effects of distinct psychotropic medications mice lacking phosphatase pp2a subunit pr61/b 0 δ (ppp2r5d) develop spatially restricted tauopathy by deregulation of cdk5 and gsk3β chaperoning epigenetics: fkbp51 decreases the activity of dnmt1 and mediates epigenetic effects of the antidepressant paroxetine stress-responsive fkbp51 regulates akt2-as160 signaling and metabolic function the hsp90 cochaperone, fkbp51, increases tau stability and polymerizes microtubules organization and function of the fkbp52 and fkbp51 genes bending tau into shape: the emerging role of peptidyl-prolyl isomerases in tauopathies fkbp51 regulates cell motility and invasion via rhoa signaling rhoa-rock signaling as a therapeutic target in traumatic brain injury the ubiquitin system protein modifications: beyond the usual suspects' review series cellular quality control by the ubiquitin-proteasome system and autophagy accelerated neurodegeneration through chaperone-mediated oligomerization of tau nf-κb transcriptional activity is modulated by fk506-binding proteins fkbp51 and fkbp52: a role for peptidyl-prolyl isomerase activity mitochondria-nucleus shuttling fk506-binding protein 51 interacts with traf proteins and facilitates the rig-i-like receptor-mediated expression of type i ifn fkbp51 and fkbp12.6-novel and tight interactors of glomulin a quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways usp49 negatively regulates tumorigenesis and chemoresistance through fkbp51-akt signaling skp2 is required for ubiquitin-mediated degradation of the cdk inhibitor p27 expression of skp2, a p27(kip1) ubiquitin ligase, in malignant lymphoma: correlation with p27(kip1) and proliferation index kip1 meets skp2: new links in cell-cycle control dysregulated expression of skp2 and its role in hematological malignancies autophagy: renovation of cells and tissues dynamics and diversity in autophagy mechanisms: lessons from yeast the atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice atg8 controls phagophore expansion during autophagosome formation atg systems from the protein structural point of view key: cord-013046-r6dtiu97 authors: liu, bin; zhang, deyuan; xu, ruifeng; xu, jinghao; wang, xiaolong; chen, qingcai; dong, qiwen; chou, kuo-chen title: combining evolutionary information extracted from frequency profiles with sequence-based kernels for protein remote homology detection date: 2014-02-15 journal: bioinformatics doi: 10.1093/bioinformatics/btt709 sha: doc_id: 13046 cord_uid: r6dtiu97 motivation: owing to its importance in both basic research (such as molecular evolution and protein attribute prediction) and practical application (such as timely modeling the 3d structures of proteins targeted for drug development), protein remote homology detection has attracted a great deal of interest. it is intriguing to note that the profile-based approach is promising and holds high potential in this regard. to further improve protein remote homology detection, a key step is how to find an optimal means to extract the evolutionary information into the profiles. results: here, we propose a novel approach, the so-called profile-based protein representation, to extract the evolutionary information via the frequency profiles. the latter can be calculated from the multiple sequence alignments generated by psi-blast. three top performing sequence-based kernels (svm-ngram, svm-pairwise and svm-la) were combined with the profile-based protein representation. various tests were conducted on a scop benchmark dataset that contains 54 families and 23 superfamilies. the results showed that the new approach is promising, and can obviously improve the performance of the three kernels. furthermore, our approach can also provide useful insights for studying the features of proteins in various families. it has not escaped our notice that the current approach can be easily combined with the existing sequence-based methods so as to improve their performance as well. availability and implementation: for users’ convenience, the source code of generating the profile-based proteins and the multiple kernel learning was also provided at http://bioinformatics.hitsz.edu.cn/main/∼binliu/remote/ contact: bliu@insun.hit.edu.cn or bliu@gordonlifescience.org supplementary information: supplementary data are available at bioinformatics online. by march 2013, 89 003 experimentally determined protein structures were deposited in the protein data bank (berman et al., 2007) . however, this number is only about one-sixth of 539 616, the number of protein sequences held in the uniprotkb/swiss-prot database (wu et al., 2006) . to timely use such vast amount of structure-unknown protein sequences for basic research and drug development, it is highly desired to determine their 3d structures and functions by means of homology approaches (chou, 2004) . unfortunately, protein remote homology detection is still a challenging problem in bioinformatics. the early methods in dealing with this problem were based on the pairwise sequence comparison approaches, such as blast (altschul et al., 1990) and smith-waterman local alignment algorithm (smith and waterman, 1981) . however, in many cases, this kind of sequence alignment method failed to detect remote homologies due to the low sequence similarities. later methods to challenge this problem were based on the generative models to induce a probability distribution over the protein family, and then to generate the unknown proteins as new members of the family from the stochastic model. for example, the hidden markov model (hmm) (karplus et al., 1998) can be trained iteratively in a semi-supervised manner by using both positively labeled and unlabeled samples of a particular family to generate the positive set (qian and goldstein, 2004) . recently, the discriminative methods, such as support vector machine (svm) (vapnik, 1998) , were used to address this problem by focusing on the differences between protein families. the key of the svm methods is the kernel function by which to compute the inner product between two samples in the feature space. the most straightforward approach to generate the kernels was based on the features extracted from protein sequences. svm-ngram (dong et al., 2006) , svm-pairwise (liao and noble, 2003) and svm-la (saigo et al., 2004) were three of the most successful sequencebased kernels. svm-ngram (dong et al., 2006) was based on the feature space that contains all short subsequence of length n. in svm-pairwise (liao and noble, 2003) , a protein sequence was represented as a vector of pairwise similarities to all protein *to whom correspondence should be addressed. sequences in the training set, and then inner product between these vector-space representations was taken as the kernel. svm-la (saigo et al., 2004) measured the similarity between a pair of proteins by taking all the optimal local alignment scores with gaps between all possible subsequences into account. besides these kernels, several other sequence-based kernels were also proposed, such as mismatch (leslie et al., 2004) and svm-balsa (webb-robertson et al., 2005) . the profile-based kernels could further improve the performance by using the evolutional information extracted from the profiles. for example, top-n-grams (liu et al., 2008) extracted the profile-based patterns by considering the most frequent elements in the profiles; profile kernel (kuang et al., 2005) extracted the short substrings according to the profile-based ungapped alignment scores; some profile-based methods improved the predictive performance by developing more sensitive profiles. hhsearch method (so¨ding, 2005) was based on a novel profile using the hmm. in compass (sadreyev et al., 2009) , numerical profiles were generated to construct optimal profile-profile alignments and to estimate the statistical significance of the corresponding alignment scores. in the meantime, some other features and techniques have been applied to this field to further improve the predictive performance. for instance, the kernel combination methodology (vbkc) (damoulas and girolami, 2008 ) used a single multiclass kernel machine to combine various kernels based on different feature spaces; svm-physicochemical distance transformation (pdt) (liu et al., 2012) combined the amino acid physicochemical properties and the profile features via pdt to incorporate the local sequence-order information of the entire protein sequences. also, based on the similarities between protein sequences and natural languages, the natural language processing techniques were applied to this field. it was shown that the performance of building-block-based methods could be improved by using the latent semantic analysis (lsa) (dong et al., 2006) . moreover, p rot e mbed (melvin et al., 2011) detected protein remote homology by embedding protein sequences into a low-dimensional semantic space. as we can see from the aforementioned introduction, most of the top-performing methods were developed based on the features extracted from profiles. this is consistent with the fact that a profile is much richer than an individual sequence in encoding information. also, biology is a natural science with historic dimension. all biological species have developed beginning from a limited number of ancestral species. it is true for protein sequence as well (chou, 2004) . their evolution involves changes of single residues, insertions and deletions of several residues, gene doubling and gene fusion (chou, 1995) . with these changes accumulated for a long period, many similarities between initial and resultant amino acid sequences are gradually eliminated, but the corresponding proteins may still share many common features, such as having basically the same biological function (loewenstein et al., 2009) , folding topology, subcellular location and other attributes (chou, 2013) . accordingly, the key to improve the performance of these methods is to find a suitable approach to extract the evolutionary information from the profiles. in view of this, the current study was initiated in an attempt to propose a profile-based protein representation by extracting the evolutionary information from the frequency profiles. as shown by a series of publications liu et al., 2009; xiao et al., 2013; xu et al., 2013) and summarized in a comprehensive review , to develop a useful statistical prediction method or model for a biological system, one needs to engage the following procedures: (i) construct or select a valid benchmark dataset to train and test the predictor; (ii) formulate the samples with an effective mathematical expression that can truly reflect their intrinsic correlation with the target to be predicted; (iii) introduce or develop a powerful algorithm (or engine) to operate the prediction; (iv) properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; and (v) provide the downloadable source code or a web-server for the prediction method. below, let us describe how to deal these procedures. suppose s is a remote homology system investigated in the current study that contains 4352 protein sequences, which were taken from (liao and noble, 2003) at http://noble.gs.washington.edu/proj/svm-pairwise/. these proteins were selected from scop version 1.53 and extracted from the astral database (brenner et al., 2000) . none of the 4352 proteins has sequence pairwise similarity to any other with higher than 10 à25 in the e-value [for more about the e-value and its implication in reducing homology bias and redundancy, see (brenner et al., 2000) ]. these proteins were also used by others (dong et al., 2006; lingner and meinicke, 2006; saigo et al., 2004) to study remote homology detection. the 4352 proteins in s can be classified into 853 superfamilies and 1356 families; i.e. where s f i ði ¼ 1, 2, . . . , 853þ is the ith superfamily, s f k ðk ¼ 1, 2, . . . , 1356þ is the kth family and the symbol [ represents the 'union' in the set theory. for readers' convenience, the codes of the 4352 proteins and their sequence as well as the attributes of their families and superfamilies are given in supplementary material s1. because some families and superfamilies in s do not contain significant number of protein sequences, and also because the negative dataset for each protein family can be any proteins except those belonging to its own superfamily, it is not so straightforward but a little more complicated and subtle for how to select protein samples from s to define the training and testing datasets. to provide a clear description, let us consider a different manner to address this. as demonstrated by many previous studies on a series of important biological topics, such as enzymecatalyzed reactions (zhou and deng, 1984) , inhibition of human immunodeficiency virus-1 reverse transcriptase (althaus et al., 1993) , drug metabolism systems (chou, 2010) and applying wenxiang diagram or graph to study protein-protein interactions (zhou, 2011; zhou and huang, 2013) , using graphical approaches to study complicated problems can provide an intuitive picture and useful insights for in-depth studying and analyzing various complicated relations in these systems (lin and lapointe, 2013) . in view of this, let us also use graphic approach to describe the feature and relation of the families and superfamilies in s, as shown in figure 1 , where the open circles denote the families or superfamilies that have significant number of protein sequences and the gray circles denote those that do not. of the 1356 families in s (cf. equation 1), 54 contain significant number of proteins (see the third row of fig. 1 ) and form a target family set s f target ; i.e. of the 853 superfamilies in s, 23 contain at least one target family (see the open circles in the second row of fig. 1 ) and form a target superfamily set s f target ; i.e. thus, we have meaning that s f target is the subset of s f target , and s f target is the subset of s; each of the three contains 857, 1508 and 4352 proteins, respectively. now, for each of the 54 families in the target family set s f target , we can define a training dataset and testing dataset given by where the positive training dataset s þ train ðkþ contains at least 10 of its superfamily members, none of which belongs to the kth family, and the positive testing dataset s þ test ðkþ contains at least five protein domains within the family. the proteins in the negative training and testing datasets, s à train ðkþ and s à test ðkþ, were picked from s by excluding the superfamily of the kth family and randomly split between the two in the same ratio as the positive ones. the 54 training and testing datasets thus obtained are given in the supplementary materials s2 and s3, respectively. the frequency profile m for protein p with l amino acids can be represented by where 20 is the total number of standard amino acids; m i,j (0 m i,j 1) is the target frequency, which reflects the probability of amino acid i (i ¼ 1,2, . . . ,20) occurring at the sequence position j (j ¼ 1,2, . . . ,l) in protein p during evolutionary processes. for each column in m, the elements add up to 1. the target frequency is calculated from the multiple sequence alignments generated by running psi-blast (altschul et al., 1997) against the ncbi's nr with default parameters except that the number of iterations was not set at 1 but was set at 10 in the current study. the target frequency of amino acid i in sequence position j is calculated as: where f ij is the observed frequency of amino acid i in column j; is a free parameter set to a constant value of 10, which is initially used by psi-blast; is the number of different amino acids in column j à 1; and g ij is the pseudo-count for amino acid i in protein sequence position j, which can be calculated as: where p k is the background frequency of amino acid k, and q ik is the score of amino acid i being aligned to amino acid k in blosum62 substitution matrix, which is the default score matrix of psi-blast (altschul et al., 1997) . although the methods by using amino acid sequence information achieve certain degree of success, only using sequence information cannot accurately detect protein remote homology. recent studies demonstrated that the profile-based methods would show better performance because a profile is richer than an individual sequence as far as the encoding information is concerned. however, a profile is a 2d matrix, whereas a protein sequence is an amino acid string. therefore, the 2d evolutionary profile information cannot be directly incorporated into the sequence-based methods for prediction. to deal with this problem, we propose an approach to convert the frequency profiles into a series of profile-based proteins. thus, the existing sequence-based methods can be directly performed on these proteins for the prediction. the target frequencies in the frequency profiles reflect the probabilities of the corresponding amino acids appearing in the specific sequence positions. the higher the frequency is, the more likely the corresponding amino acid occurs. it is reasonable to use the nth most frequent amino acids in the frequency profiles to represent the protein sequences. below is the description on how to convert frequency profiles into profile-based proteins. given the frequency profile m for protein p (equation 6), we can sort the amino acids in each column according to a descending order. the frequency profile thus obtained by the sorting operation is called the sorted frequency profile and denoted by m 0 . for each row in m 0 , the amino acids are combined to produce the profile-based protein. by following this approach, the frequency profile m is converted into 20 profile-based proteins p 1 , p 2 , . . . , p 20 ( supplementary fig. s1 in supplementary material s4), which contain the evolutionary information in the frequency profile. these 20 proteins have different importance. during evolutionary process, protein p is preferred to transform into p1, but not preferred to transform into p20. for reader's convenience, the source code for generating the profile-based proteins is accessible by clicking the link at http://bioinformatics.hitsz.edu.cn/main/ *binliu/ remote/. three state-of-the-art sequence-based kernels [svm-ngram (dong et al., 2006) , svm-pairwise (liao and noble, 2003) and svm-la (saigo et al., 2004) ] were used to validate whether the proposed approach could improve their performance. in svm-ngram (dong et al., 2006) method, the ngrams were the set of all possible subsequences of amino acids of a fixed length. a protein sequence was mapped to a feature vector by the occurrence frequency of each ngram. the value of n was set at 3 as suggested by the authors (dong et al., 2006) , and therefore the dimension of the vector is 8000. at the heart of the svm is a kernel function that acts as a similarity score between pairs of vectors. the kernel was normalized so that each vector had length 1 in the feature space: where x and y are two proteins in the dataset. this normalized step was also used by svm-pairwise (liao and noble, 2003) and svm-la (saigo et al., 2004) . the normalized kernel thus obtained was then transformed into a radial basis kernel. in the svm-pairwise (liao and noble, 2003) method, the feature vector was a list of pairwise sequence similarity scores, computed with respect to all of the sequences in the training set. the radial basis function was used as the kernel. the rest steps were the same as the ones used in svm-ngram (dong et al., 2006) . in the svm-la (saigo et al., 2004) , the kernel was calculated by summing up scores obtained from the local alignments with gaps between the two sequences, computed by smith-waterman dynamic programming algorithm. such kernel might not be a positive definite kernel and the authors (saigo et al., 2004) provided two solutions for this problem. owing to its performance and simplicity, we implemented one of the two methods, namely, the la-ekm kernel. the parameters of la-ekm kernel took the optimal values ( ¼ 0.5, d ¼ à11, e ¼ à4). the kernel described in the previous section can be used by kernel methods to train the svm classifier. each kernel contains different discriminative information, and therefore combining the kernels automatically is a promising way to improve the performance. in machine learning field, this approach is called multiple kernel learning (mkl) (cortes et al., 2010; varma and babu, 2009) , which has attracted a lot of attention recently. the mkl technique aimed to combine different kernels to improve the performance, and showed the state-of-the-art results on image classification field (varma and babu, 2009) . in this article, we focused on the weighted linear combination of kernels. the weight of each kernel can be optimized based on different criterion, which can be categorized by two groups. one group is the one-stage kernel learning methods, which optimize the weight and the svm objective function simultaneously (varma and babu, 2009 ). these methods suffer from the high training complexity. the other group is two-stage kernel learning methods, which optimize the weight by using a criterion first and then train the svm classifier using the kernel combined by the learned weight of each kernel. compared with one-stage learning methods, the two-stage kernel learning methods showed better performance with reduced training cost. therefore, in this study, we adopted the two-stage kernel learning method. specifically, the kernel target alignment (kta) objective function was used to optimize the weight of each kernel, which showed theoretical guarantees and can improve the performance in practice (cortes et al., 2010; varma and babu, 2009) . given m training samples x 1 , x 2 , . . . , x m and their corresponding labels y 1 , y 2 , . . . , y m , the ideal kernel matrix can be formulated as k y ¼ y t y, where y is the vector of labels [y 1 , y 2 , . . . , y m ]. for the given n kernels k 1, k 2 , . . . k n , the aim is to learn the weight of each kernel. to avoid the kernel scaling problem, we center kernel k k and the corresponding ideal kernel k y in feature space by the following equation: where k ck is normalized by: following the above steps, each kernel is normalized, and then these n kernels are linearly combined by the following equation: where w k (0 w k 1, p n k¼1 w k ¼ 1) is the weight of kernel k 0 k . the weight is learned by kta objective function, which maximizes the alignment between k comb and the centered ideal kernel k cy . this leads to a quadratic program problem and can be solved quite efficiently. for implementation details, please refer to (cortes et al., 2010) . in this study, three kernels (k p , k p1 and k p2 ) for each selected sequence-based method were linearly combined by using the above kta approach to further improve the performance. for reader's convenience, the source code of the mkl is accessible by clicking the link at http://bioinformatics.hitsz.edu.cn/main/ *binliu/remote/. svm is a class of supervised learning algorithms first introduced by vapnik (1998) . svm-based machine learning algorithm has been successfully used to investigate various problems in molecular biology, such as identifying dna recombination spots , membrane protein types (cai et al., 2003) and heat-shock protein functions (feng et al., 2013) , among many others. in this study, the publicly available gist svm package (http://www.chibi.ubc.ca/gist/) was used. because the test sets have many more negative than positive samples, simply measuring error-rates will not give a good evaluation of performance. for the cases in which the positive and negative samples are not evenly distributed, the best way to evaluate the trade-off between the specificity and sensitivity is to use a receiver operating characteristic (roc) score (gribskov and robinson, 1996) . an roc score is the normalized area under a curve that plots true positives against false positives for different classification thresholds. a score of 1 means perfect separation of positive samples from negative ones, whereas a score of 0 means that none of the sequences selected by the algorithm is positive. another performance measure is roc50 score, which is the area under the roc curve up to the first 50 false positives. the frequency profile of a protein p can be converted into 20 profile-based proteins (p1, p2, . . . , p20) by using the proposed approach (see section 2 for details). these 20 proteins have different importance. p1 is the most important protein, as it is the combination of the top frequent amino acids in frequency profile, whereas p20 is the profile-based protein to which protein p is the most unlikely to convert because it is the combination of the amino acids with lowest frequencies in frequency profile. if all the 20 profile-based proteins are used in the prediction, the computational cost is relatively high. in this study, only the top n most important profile-based proteins (p1, . . . , pn) were used in the prediction. to select the value of n, the following experiment was conducted. the frequencies of 20 standard amino acids in each column of a frequency profiles add up to 1. therefore, the average frequency is 0.05 (1/20 ¼ 0.05). if an amino acid with frequency 40.05, it is likely to occur during evolutionary process; otherwise, it is not likely to occur. the percentage of the amino acids with frequencies40.05 in each profile-based protein on the scop benchmark was calculated, and the results are shown in figure 2 . as we can see from the figure, such amino acids are abundant in profile-based proteins p1, p2 and p3 (99.99%, 99.60% and 98.13%, respectively), but for the other 17 profile-based proteins, the percentage decreases significantly (from 89.28 to 0%). therefore, in this study, only the top three profile-based proteins were used in the prediction. these profile-based proteins were combined with three state-ofthe-art methods based on sequence composition, including svm-ngram (dong et al., 2006) , svm-pairwise (liao and noble, 2003) and svm-la (saigo et al., 2004) , and the results are shown in supplementary table s1 of supplementary material s4. for each of the three methods, the best performance was achieved for the top important protein p1. compared with the methods performed on the raw protein sequence p, the performance of the proposed methods can be improved by 3.7$7.5% and 9.6$13.7% in terms of average roc and roc50 scores, respectively, indicating that the proposed profile-based protein representation is useful for protein remote homology detection. the performance of the methods performed on p2 is similar as that of the methods performed on the raw protein p. the predictive results of the methods performed on p3 were the lowest. these results are consistent with the different importance of the three profile-based proteins p1, p2 and p3. besides the current method, there are some other methods for predicting protein remote homologies based on profiles, such as svm-top-n-gram-combine-lsa (liu et al., 2008) , svm-pdt-profile (liu et al., 2012) , profile (kuang et al., 2005) , biosvm-2l (muda et al., 2011) and hhsearch (so¨ding, 2005) . svm-top-n-gram-combine-lsa (liu et al., 2008) extracted the building blocks of proteins from the frequency profiles, which could be treated as the 'words' of protein language. the lsa (dong et al., 2006) was applied to further improve the performance of this method. svm-pdt-profile (liu et al., 2012) combined the amino acid physicochemical properties in the amino acid index (aaindex) (kawashima et al., 2008) with the frequency profiles for the prediction. the feature vector of profile method (kuang et al., 2005) was constructed by the short subsequences whose pssm-based ungapped alignment score was above a predefined threshold. biosvm-2l constructed two-layer svm classifiers with profile-based kernels (muda et al., 2011) . all the above three methods were based on svm, and the difference among them was in the extracted features. hhsearch (so¨ding, 2005) was one of the best protein remote homology detection methods, which used a novel profile-based hmm. the results obtained by these four methods on the scop benchmark are listed in supplementary table s1 of supplementary material s4, from which we can see that the current method outperforms svm-top-n-gram-combine-lsa (liu et al., 2008) , svm-pdt-profile (liu et al., 2012) and biosvm-2l (muda et al., 2011) and is highly comparable with profile (kuang et al., 2005) and hhsearch (so¨ding, 2005) , indicating that the profile-based protein representation is a promising approach to extract the evolutionary information from frequency profiles for protein remote homology detection. as mentioned above, the approaches based on the top two profile-based proteins p1, p2 and the raw protein p are among the top performing methods. it is interesting to investigate whether these methods can be combined to further improve the performance. in this study, the mkl framework was used to combine these methods. the kta method was used to automatically optimize the weight of each kernel on the training set, and then these kernels are combined with weights into a single kernel for the svm-based prediction. the results are shown in supplementary table s2 of supplementary material s4 as well as supplementary materials s5-s7. the mkl approach can improve the performance of svm-ngram (dong et al., 2006) , but only has minor impact on the svm-pairwise (liao and noble, 2003) and svm-la (saigo et al., 2004) . to uncover the reason, the weight of each kernel was analyzed. for each kernel, the average weight on all the 54 protein families is shown in supplementary table s2 of supplementary material s4. for these three methods, the p1-based kernel was weighted most heavily. for svm-pairwise (liao and noble, 2003) and svm-la (saigo et al., 2004) , the weight values of their corresponding p and p2 kernels are 50.1, indicating these kernels only have minor impact on the final results, and hence the performance improvement is modest. vbkc (damoulas and girolami, 2008) is another method based on the mkl, which combined four string kernels: svm-pairwise (liao and noble, 2003) , svm-la (saigo et al., 2004) , svm-mm (leslie et al., 2004) and svm-mono (lingner and meinicke, 2006) . our proposed svm-pairwise-kta and svm-la-kta outperform vbkc (damoulas and girolami, 2008 ) by 1.2 $ 2.2% and 29.9 $ 31.3% according to the average roc and roc50 scores, respectively. the obvious performance improvement is mainly due to the proposed profile-based protein representation and mkl approach. the svm-ngram (dong et al., 2006) method is based on the explicit feature space representation, which provides the possibility to measure the correlations between ngrams and protein families. the sequence-specific weight learnt from the svm training process can be used to calculate the discriminant weight for each ngram, which indicates the importance of the corresponding ngram. by following lingner and meinicke's approach (lingner and meinicke, 2008) , given the weight vector of a set of m sequences obtained from the kernel-based training process ¼ [ 1 , 2 , 3 , . . . , m ], the discriminant weight vector w in the feature space can be calculated by the following equation: where f is the matrix of sequence representatives. the magnitude of the element in w represents the discriminative power of the corresponding feature. in most protein families, kernel p1 is weighted more heavily than kernel p and kernel p1. two such protein families (scop id: 2.1.1.4 and 3.2.1.5) were selected from the scop benchmark for further study, and the results are shown in supplementary tables s3 and s4 of supplementary material s4, respectively. for each kernel, the top 10 most discriminative ngram features calculated by equation 14 are shown in the tables too. for protein family 2.1.1.4, kernel p and kernel p1 share some common most discriminative ngrams, such as 'mtm', 'yty', 'mtf' and 'wwf', indicating these ngrams remain stable during evolutionary process and therefore these ngrams would be the important sequence patterns for maintaining the structure and function of this protein family (supplementary table s3 of supplementary material s4). however, there are a few common most discriminative ngrams between kernel p and kernel p1 in protein family 3.2.1.5. the top 10 most discriminative ngrams of kernel p1 are all different from those in kernel p (supplementary table s4 of supplementary material s4). these ngrams would contribute to the higher discriminative power of kernel p1 for this protein family. although in most cases, kernel p1 was weighted most heavily, some exceptions were observed. for example, for protein family 7.3.6.1, kernel p2 is the most discriminative kernel with weight value of nearly 1, while the other two kernels only have little contribution to the mkl (supplementary table s5 of supplementary material s4). the top 10 most discriminative ngrams for each kernel were investigated, and the results are shown in supplementary table s5 of supplementary material s4, from which some interesting patterns can be observed. the ngrams containing amino acids 'n' and 'f' tended to show strong discriminative power in both kernel p and kernel p1, whereas amino acid 'a' was abundant in the top discriminative ngrams in kernel p2, indicating the ngrams with amino acid 'a' could better describe the prosperities of protein family 7.3.6.1 in the evolutionary process. 3.5 application of the proposed remote homology detection methods for studying the 3d structure of nck5a in addition to provide useful insights for evolution study, protein remote homology detection is useful for drug development as well. as is well known, many drug-targeted proteins are still without x-ray or nuclear magnetic resonance structure. pharmaceutical scientists have to resort to the homology modeling technique or structural bioinformatics tools (chou, 2004) to timely develop their 3d structures, so as to be able to conduct molecular docking study (chou et al., 2003; wang et al., 2009) , one of the key steps in structure-based drug design. however, a reliable template, or a structure-known protein homologous to the target protein, is the necessary prerequisite in this regard (chou, 2004) . unfortunately, many target proteins did not have significant sequence similarity with any structure-known proteins, and hence it was hard to find a proper template to develop their 3d structures. actually, many of them did have structure-known homologous proteins, but the problem was how to detect them. for example, the sequence similarity between nck5a and cyclina was 520% (chou et al., 1999) and hence their homologous relationship could not be detected by the simple sequence alignment technique (mohabatkar, 2010) . now let us see what will happen if the current remote homology detection technique is applied. to realize this, a dataset was constructed based on scop, from which 11 proteins were selected as the positive samples in the cyclin family (scop id: a.74.1.1), while 3605 negative samples were selected from the scop version 1.67 by excluding all the proteins within the cyclin-like superfamily. none of these proteins shares 495% sequence similarity. trained with such 11 positive proteins and 3605 negative proteins, the proposed best performing method svm-la (p1) was used to predict nck5a. it was found that nck5a is homologous to cyclina, fully consistent with the experimental results obtained by the site-directed mutagenesis studies (tang et al., 1997) . actually, chou et al. (1999) did use cyclina as a template to construct the 3d structure of activation domain of nck5a, one of the important parts of tau protein kinase ii, an important therapeutic target against alzheimer's disease. furthermore, based on the computed structure thus obtained, the molecular truncation experiments (zhang et al., 2002) were conducted with an outcome that confirmed and validated the structure computed by using such a remote homologous protein as a template. therefore, it is anticipated that the proposed method for detecting remote homology proteins will certainly enhance the power of homology modeling, and hence have impacts on drug development as well. discriminative methods based on svm are the most effective and accurate methods for protein remote homology detection. the performance of the svm-based methods depends on the kernel function, which measures the similarity between the samples in any pair. varieties of kernels based on sequence composition have been proposed. however, these methods often fail to accurately predict the proteins sharing low sequence similarity. recently, methods using the evolutionary information extracted from profiles achieved great success, such as profile (kuang et al., 2005) , sw-pssm (rangwala and karypis, 2005) , svm-top-ngram (liu et al., 2008) and svm-acc (liu et al., 2011) . a key step to improve the performance of these methods is in how to find a suitable approach to incorporate the evolutionary information extracted from the profiles for prediction. in this article, we proposed a method that can convert the frequency profile into a series of profile-based proteins. three state-of-the-art sequence-based kernels, i.e. svm-ngram (dong et al., 2006) , svm-pairwise (liao and noble, 2003) and svm-la (saigo et al., 2004) , were selected for demonstration on a well-known benchmark. it was shown that the methods based on the profile-based proteins p1 and p2 achieved the best performance, outperforming the original three string kernels by 3.7 $ 7.5% and 9.6 $ 13.7%, respectively, according to the average roc and roc50 scores. these results are fully consistent with our previous findings that the top two most frequent amino acids show stronger discriminative power than the other low frequent amino acids in the frequency profiles (liu et al., 2008) , further confirming that the proposed profile-based protein representation is a promising approach in extracting the evolutionary information from frequency profiles for protein remote homology detection. it has not escaped our notice that the current approach can be easily combined with sequence-based methods, and hence, with the development of the sequence-based kernels, the currently proposed method can be further improved accordingly. it is instructive to point out that since the concept of pseudo amino acid composition, or chou's pseaac (lin and lapointe, 2013) , was introduced in 2001 (chou, 2001) , it has been successfully used to predict various attributes of proteins (e.g. chen and li, 2013; chou, 2005; georgiou et al., 2009; huang and yuan, 2013; khosravian et al., 2013; mohabatkar, 2010; mohabatkar et al., 2011 mohabatkar et al., , 2013 mohammad beigi et al., 2011; nanni et al., 2012; sahu and panda, 2010; zhang et al., 2008; zhou et al., 2007; liu et al., 2013) . accordingly, the potential would be high to develop a powerful method for protein remote homology detection by combing pseaac with profile-based protein representation. in the original pseaac, it only uses three indices, including the hydrophobicity index, hydrophilicity index and side-chain mass index. because protein remote homology detection is a more difficult problem, proteins in the dataset only share low sequence similarity. only these three indices would not be enough to capture the different properties of various proteins. therefore, our further research will focus on incorporating new amino acid indices into pseaac and applying it to protein remote homology detection. conflict of interest: none declared steady-state kinetic studies with the non-nucleoside hiv-1 reverse transcriptase inhibitor u-87201e basic local alignment search tool gapped blast and psi-blast: a new generation of protein database search programs the worldwide protein data bank (wwpdb): ensuring a single, uniform archive of pdb data the astral compendium for sequence and structure analysis support vector machines for predicting membrane protein types by using functional domain composition irspot-psednc: identify recombination spots with pseudo dinucleotide composition predicting membrane protein types by incorporating protein topology, domains, signal peptides, and physicochemical properties into the general form of chou's pseudo amino acid composition the convergence-divergence duality in lectin domains of the selectin family and its implications prediction of protein cellular attributes using pseudo amino acid composition review: structural bioinformatics and its impact to biomedical science using amphiphilic pseudo amino acid composition to predict enzyme subfamily classes graphic rule for drug metabolism systems some remarks on protein attribute prediction and pseudo amino acid composition (50th anniversary year review) some remarks on predicting multi-label attributes in molecular biosystems a model of the complex between cyclin-dependent kinase 5 (cdk5) and the activation domain of neuronal cdk5 activator binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against sars wenxiang: a web-server for drawing wenxiang diagrams two-stage learning kernel algorithms probabilistic multi-class multi-kernel learning: on protein fold recognition and remote homology detection application of latent semantic analysis to protein remote homology detection ihsp-pseraaac: identifying the heat shock protein families using pseudo reduced amino acid alphabet composition use of fuzzy clustering technique and 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homology detection a discriminative method for protein remote homology detection and fold recognition combining top-n-grams and latent semantic analysis prediction of protein binding sites in protein structures using hidden markov support machine using amino acid physicochemical distance transformation for fast protein remote homology detection protein remote homology detection by combining chou's pseudo amino acid composition and profile-based protein representation protein remote homology detection based on auto-cross covariance transformation protein function annotation by homology-based inference detecting remote evolutionary relationships among proteins by large-scale semantic embedding prediction of cyclin proteins using chou's pseudo amino acid composition prediction of gaba(a) receptor proteins using the concept of chou's pseudo-amino acid composition and support vector machine prediction of allergenic proteins by means of the concept of chou's pseudo amino acid composition and a machine learning approach prediction of metalloproteinase family based on the concept of chou's pseudo amino acid composition using a machine learning approach remote protein homology detection and fold recognition using two-layer support vector machine classifiers identifying bacterial virulent proteins by fusing a set of classifiers based on variants of chou's pseudo amino acid composition and on evolutionary information performance of an iterated t-hmm for homology detection profile-based direct kernels for remote homology detection and fold recognition compass server for homology detection: improved statistical accuracy, speed and functionality a novel feature representation method based on chou's pseudo amino acid composition for protein structural class prediction protein homology detection using string alignment kernels identification of common molecular subsequences protein homology detection by hmm-hmm comparison cyclin-dependent kinase 5 (cdk5) activation domain of neuronal cdk5 activator. evidence of the existence of cyclin fold in neuronal cdk5a activator statistical learning theory more generality in efficient multiple kernel learning insights from investigating the interactions of adamantanebased drugs with the m2 proton channel from the h1n1 swine virus svm-balsa: remote homology detection based on bayesian sequence alignment the universal protein resource (uniprot): an expanding universe of protein information icdi-psefpt: identify the channel-drug interaction in cellular networking with pseaac and molecular fingerprints isno-aapair: incorporating amino acid pairwise coupling into pseaac for predicting cysteine s-nitrosylation sites in proteins identification of the n-terminal functional domains of cdk5 by molecular truncation and computer modeling using the concept of chou's pseudo amino acid composition to predict protein subcellular localization: an approach by incorporating evolutionary information and von neumann entropies an extension of chou's graphic rules for deriving enzyme kinetic equations to systems involving parallel reaction pathways the disposition of the lzcc protein residues in wenxiang diagram provides new insights into the protein-protein interaction mechanism using chou's amphiphilic pseudo-amino acid composition and support vector machine for prediction of enzyme subfamily classes the ph-triggered conversion of the prp(c) to prp(sc.) key: cord-004719-3stcx0dd authors: mushegian, a. r.; koonin, e. v. title: cell-to-cell movement of plant viruses: insights from amino acid sequence comparisons of movement proteins and from analogies with cellular transport systems date: 1993 journal: arch virol doi: 10.1007/bf01313766 sha: doc_id: 4719 cord_uid: 3stcx0dd cell-to-cell movement is a crucial step in plant virus infection. in many viruses, the movement function is secured by specific virus-encoded proteins. amino acid sequence comparisons of these proteins revealed a vast superfamily containing a conserved sequence motif that may comprise a hydrophobic interaction domain. this superfamily combines proteins of viruses belonging to all principal groups of positive-strand rna viruses, as well as single-stranded dna containing geminiviruses, double-stranded dna-containing pararetroviruses (caulimoviruses and badnaviruses), and tospoviruses that have negative-strand rna genomes with two ambisense segments. in several groups of positive-strand rna viruses, the movement function is provided by the proteins encoded by the so-called triple gene block including two putative small membrane-associated proteins and a putative rna helicase. a distinct type of movement proteins with very high content of proline is found in tymoviruses. it is concluded that classification of movement proteins based on comparison of their amino acid sequences does not correlate with the type of genome nucleic acid or with grouping of viruses based on phylogenetic analysis of replicative proteins or with the virus host range. recombination between unrelated or distantly related viruses could have played a major role in the evolution of the movement function. limited sequence similarities were observed between i) movement proteins of dianthoviruses and the mip family of cellular integral membrane proteins, and ii) between movement proteins of bromoviruses and cucumoviruses and m1 protein of influenza viruses which is involved in nuclear export of viral ribonucleoproteins. it is hypothesized that all movement proteins of plant viruses may mediate hydrophobic interactions between viral and cellular macromolecules. it is generally accepted that plant viruses exploit plasmodesmata to move from initially infected cells, which are usually negligible in amount, into neighbouring healthy cells. without cell-to-cell movement productive virus infection is not established. also it is known that specific movement proteins encoded by virus genomes are essential for cell-to-cell spread. frequently, a plant virus protein is referred to as movement protein if i) it is not a capsid protein and ii) disruption of the coding sequence of this protein abolishes infection in whole plants but has no effect on virus replication in protoplasts. putative movement proteins have been identified in this manner in about 15 different plant virus groups (see [4, 19, 40] for recent reviews). in some cases, additional virus proteins are also required for movement, e.g. coat protein [13, 52, 58] or a specific segment in a replication protein [55] , but these phenomena are not universal. on the other hand, a specific movement protein or at least movement domain is thought to be present in all viruses that are able to spread from cell to cell [4] . apart from this genetic evidence for the movement function, the current knowledge of mechanisms which facilitate intercellular spread of plant virus genomes is insufficient. recently certain properties of some movement proteins, notably of the 30 kda movement protein of tobacco mosaic virus (tmv), were characterized in vitro and in vivo and models for movement of plant viruses have been prompted by these studies [ 16, 19] . additionally, movement proteins were subjects of comparative sequence analysis, which allowed to delineate common motifs in movement proteins from otherwise unrelated groups implying existence of common features among many movement proteins [33, 43] . in this work, we sought to critically analyze currently available biochemical and ultrastructural data on movement proteins. we then investigated whether sequences of movement proteins could provide information on their function based on similarities with other viral or cellular proteins. fluorescent probes injected into a plant cell are either transported into neighbouring cells via plasmodesmata or retained in the initially injected cell. the crucial determinant for the exclusion of a particle from plasmodesmatal movement is its hydrodynamic (stokes) radius [51] . studies of transgenic tobacco plants constitutively expressing tmv 30 kda movement protein (mp + plants) revealed that certain molecules, which were too large to be transported in wild type plants, gained such an ability in mp + tobaccos. specifically, fitc-labelled dextran (mr 9.4 kda) moved from injected to neighbouring cells only in plants transformed with and expressing tmv 30 kda protein but not in control mp-plant virus movement proteins 241 plants [57] . interestingly, plasmodesmata in mp + and mp-plants appeared to be structurally indistinguishable [19, 57] . these data indicate that movement protein of tmv functionally modifies plasmodesmata. it is not understood yet whether these modifications and the changes required for moving virus genome through plasmodesmata are the same or different. virus nucleic acid would not be expected to exist in naked form in the cell; rather, it seems reasonable that, like all nucleic acids in eukaryotic cells, it is associated with cellular and/or virus proteins throughout its lifetime. while stokes radius of a 10 kda dextran molecule is estimated to be about 3.1 nm [20, 57] , ribonucleoproteins (rnps) are clearly larger, many of them having sizes within the range of 10-50 nm [41] , which would exclude them from movement even in mp + plants described in [57] . recently, plasmodesmatal permeability in nicotiana clevelandii leaves infected with tobacco rattle virus (trv) has been investigated [20] . movement protein of trv, the 29 kda protein, shares high sequence similarity with tmv movement protein, and both proteins are considered to be functionally very similar. simultaneous injection of virus and fluorescent tracers into cells of leaf trichomes allowed to observe movement of the labels under the conditions when the virus itself was known to move in the same cells. it has been shown that fitc-labelled dextran (mr 4.4kda) moved from cell to cell only in trvinfected cells and synchronously with the virus. however, lucifer yellow-labelled dextran (mr 10 kda) was excluded from plasmodesmatal movement even in infected cells. thus, certain molecules with the dimensions resembling the smallest of the known rnps did not move under conditions when virus rnps readily moved. apparently, either an extremely thin virus-specific rnp should be postulated (see below), or it might be concluded that slight increase in plasmodesmatal permeability induced by movement proteins hints at some important circumstances but is not sufficient to explain celt-to-cell movement of the virus genome. binding of movement proteins to single-stranded nucleic acids tmv 30 kd movement protein has been overexpressed in e. coli, and purified protein has been shown to bind single-stranded (ss) dnas and rnas nonspecifically and cooperatively [14] . a model has been proposed, in which movement protein binds genomic rna stoichiometrically to unfold and shape it into a thin complex [ 14, 16, 17] . electron microscopy of such complexes formed in vitro revealed thin structures at the limit of microscope resolution; it was concluded that, as it was barely seen under em, the complex was thin enough to fit in plasmodesmata with size exclusion limit of 3 nm [17] . additionally, ss nucleic acid-binding properties have been reported for movement proteins of cauliflower mosaic caulimovirus (camv) [ 15] , red clover necrotic mosaic dianthovirus (rcnmv) ( [47] , d. cookmeyer, pers. comm.) and alfalfa mosaic virus (aimv) [-40] . interestingly, p1, the movement protein of cauliflower mosaic virus, has been shown to preferentially bind rna rather than dna 242 a.r. mushegian and e. v. koonin [15] ; it was speculated that 35 s transcript, a genome length virus replication intermediate, might be the form which is transported from cell to cell. these in vitro observations offer new insights into a mechanism of virus cell-to-cell movement. however, is should not be forgotten that virus rnas in vivo are associated with proteins, as revealed for tmv-specific rnas [22] ; it is thought that such an association is maintained throughout the whole life of the rnps in the cell; a mechanism of stripping virus rna from these proteins has not yet been proposed. also, as binding of movement proteins to rna is thought not to be sequence-specific [14, 17] , it could be anticipated that the majority of movement protein will bind to cellular rnas, which in the case of tmv infection are in excess over virus rnas at the time when 30 kd protein is transiently expressed [18] . the "ss nucleic acid-binding" model, therefore, has to be modified to deal with these difficulties. biochemical fractionation of infected cells revealed that in many virus-plant interactions movement proteins are found in cell wall-enriched fraction. immunogold labelling has confirmed these observations and demonstrated localization of tmv, a1mv, camv, and rcmv movement proteins in plasmodesmatal channels or in cell wall near plasmodesmata [36, 40] . movement protein of cowpea mosaic comovirus (cpmv), the 58 kd/48 kda protein, has been found to form tubular structures perpendicular to and intruding into plasma membrane and cell wall. these tubules have been found both in whole leaves and in protoplasts [-56] . virions have been observed inside the tubules [56] . it has been speculated that the tubular inclusions are the structures required for movement. cucumber mosaic virus (cmv) movement requires the 32 kda movement protein [7] . this protein has not been found to associate with the cell wall or from tubules; instead, it was located in nucleoli of infected cells [-37] . observations on movement proteins of tmv and cpmv have given rise to the idea of two different types of virus movement, "tobamo-type" and "comotype". it has been proposed that the tobamo-type is characterized by movement protein localization in the plasmodesmatal area of the cell wall and by the virus infectious entity capable of cell-to-cell movement in the absence of the viral capsid protein. como-type of movement was thought to require both movement protein and coat protein and to involve formation of tubular structures protruding from cell wall into cytoplasm and formed by the movement protein [19, 56] . complementation data argued, however, that there is certain compatibility among both types of movement as tobamoviruses were able to complement comovirus movement [4] . when all available ultrastructural data are considered, the picture becomes not so clear-cut. it should be expected that rcmv, a comovirus closely related to cpmv, moves according to the como-type; instead, movement protein of rcmv was found exclusively in plasmodesmata of the infected plants [-40] which had been proposed to be a tobamo-type feature. camv movement protein is found in cell wall fraction and, more specifically, in plasmodesmata [2, 36] ; this seemingly supports the attribution of caulimovirus movement to the tobamo-type. however, tubular structures of unknown composition have been observed in the cytoplasm of camv-infected cells [36] and recently it has been shown that p1, the movement protein of camv, forms tubular structures on the surface of inoculated protoplasts [49] . these findings seem to be more consistent with the como-type action of the caulimovirus movement protein. the above discussion shows that it may be premature to draw too sharp a distinction between different types of movement based on ultrastructural observations. it also should be remembered that we see movement proteins at the sites where they are most easily observed and/or are retained for longer periods, but not necessarily at the sites of their essential activity. with full-length dna copies of numerous virus genomes available, attempts have been made to genetically dissect movement protein coding sequences. in most cases, lengthy deletions have been introduced, and effect of these deletions on the overall infectivity or individual properties of the movement proteins has been investigated. it has been shown that even small changes near the n-terminus of tmv 30 kd movement protein abolish virus infectivity [6] ; at the c-terminus, up to 33 of the 268 amino acid residues of this protein could be deleted without apparent effect on infectivity or cell wall localization of the movement protein. when 55 c-terminal amino acids were deleted, virus moved slowly though movement protein was still found in plasmodesmata [-6] . deletion of 74 cterminal residues (195 to 268) was lethal, and, at the same time, movement protein was no longer found in plasmodesmata. it has been noted that essential residues from 195 to 214 could comprise a domain with a specific function, or their deletion might simply cause misfolding of the protein i-6]. in another study, c-terminal and internal deletions have been introduced into tmv movement protein to determine location of ss nucleic acid-binding site(s) [-17] . initially, it was thought that residues 65 to 86 constitute rnabinding domain [14] . later, this domain has been shown to rather decrease protein solubility [17] . up to 84 amino acids from the c-terminus of tmv movement protein could be eliminated without effect on ss nucleic acid binding capacity of the protein [-17]. in the remaining part, deletion of amino acids 111-125 abolished ss nucleic acid binding, but when the gene segment encoding these amino acids was fused to an unrelated sequence, the resulting himeric protein did not bind to ss nucleic acids [17] . movement protein of a1mv associates with cell wall upon infection and in transgenic tobacco plants; however, this did not happen when n-proximal amino acids (12-77) have been deleted [-24] . strains of tmv have been characterized that are temperature sensitive in 244 a.r. mushegian and e. v. koonin: plant virus movement proteins cell-to-cell movement or break the host resistance which is based on plant genes restricting virus movement. involvement of mutations in 30 kd movement protein cistron could be implied, and, indeed, in many cases nucleotide changes responsible tbr the altered phenotype were shown to be confined to the movement protein gene [11] . many of these mutations were scattered around the middle one-third of 30 kd movement protein and resulted in a change in polarity of the encoded amino acid [11] . as no known function could be mapped to the area, these data are difficult to interpret. with representative sequences of plant virus genomes from many groups available, efforts were made to identify regions of similarity between known movement proteins and to predict movement functions for uncharacterized orfs. early observations made by this approach have been reviewed previously (e.g. [4] ) and included strong similarity between tobamovirus and tobravirus movement proteins [29] as well as less pronounced but significant local similarity between segments of tobamovirus and caulimovirus movement proteins [30] . later, similarities between movement proteins of tricornaviruses and dianthoviruses were observed [59] . in fact, the movement function for the respective proteins of tobraviruses, caulimoviruses and dianthoviruses has been implied by these observations and only subsequently confirmed by site-directed mutagenesis [54, 59, 60] . more detailed analyses performed by melcher [43] and by ourselves [33] revealed longer (ca. 150 amino acids) segments of sequence similarity between movement proteins from different virus groups. interestingly, one family of movement proteins included proteins encoded by viruses with positive-strand rna genomes, single-strand dna geminiviruses and by pararetroviruses with virion dna, namely caulimoviruses [33] . in the time past after the publication of these analyses, further significant increase in the number of sequenced virus genomes was achieved and it has been argued that the current collection of such sequences may represent the majority of the existing virus groups [32, 34] . with this in mind, we undertook a systematic comparison of the sequences of the movement proteins with each other and with the current sequence databases. using the blast program [3] , statistically highly significant similarities were observed only between proteins of viruses belonging to a single group or two closely related groups. further comparisons were done using programs ma-caw [53 a] and optal [27] to generate multiple alignments according to the progressive alignment strategy [21] , i.e. starting with pairs of most closely related sequences and proceeding to incorporate more and more distant ones. it was demonstrated that movement proteins of viruses belonging to 17 groups contain a weak but confidently identified conserved motif consisting of approximately 30 amino acid residues (fig. 1) . this motif includes a nearly invariant aspartic acid residue (replaced by asparagine in dianthoviruses and ilarviruses) preceded by a region enriched in hydrophobic and nonpolar residues. in some of the movement proteins this region was predicted to form a transmembrane helix using the algorithm of rao and argos ( [50] , data not shown; see also discussion below). remarkably, this conserved motif unites viruses with single-stranded dna genomes (geminiviruses of group ii), pararetroviruses with double-stranded dna genomes (caulimoviruses and badnaviruses), negative-strand rna viruses with ambisense genome segments (tospoviruses), and numerous groups of positive-strand rna viruses. among the latter, all the three major phylogenetically defined divisions [31, 34] are represented, namely run-like viruses (tobamoviruses, tobraviruses, cucumoviruses, bromoviruses, ilarviruses, capilloviruses, furoviruses, raspberry bushy dwarf virus), picornalike viruses (comoviruses, parsnip yellow fleck virus, pea enation mosaic virus), and flavi-like viruses (dianthoviruses and tombusviruses). several groups of movement proteins showed significant similarity on longer sequence stretches. in fig. 2 we show the amino acid sequence alignments for four of these groups, delineation of which included identification of new putative movement proteins. such new identifications are: i) 38 kda protein of apple stem grooving virus, which is related to the putative movement proteins of the other capilloviruses, namely apple chlorotic leaf spot virus and potato virus t (fig. 2 a) ; ii) 37 kda protein of soil-borne wheat mosaic furovirus related to the movement proteins ofdianthoviruses (fig. 2 b) ; iii) 27 kda protein of pea enation mosaic virus and 21 kda protein of tombusviruses related to the movement proteins of bromoviruses and cucumoviruses (fig. 2 c) ; and iv) nsm protein of fig. 2 . amino acid sequence alignments for groups of proteins including newly identified putative movement proteins. for each group of boundaries of the conserved segments were determined using the macaw program and the alignment itself was constructed hierarchically using the optal program. in the consensus u indicates a bulky aliphatic residue (i, l, v, or m), a indicates an aromatic residue (f, i7, or w) & indicates a bulky hydrophobic residue (aliphatic or aromatic) and x indicates any residue. the conserved motif of the "30 k superfamily" is highlighted by bold typing, a putative movement proteins of capilloviruses. aclsv apple chlorotic leaf spot virus; pvt potato virus t; asgv apple stem grooving virus; b movement protein of red clover necrotic mosaic dianthovirus (rcnmv), putative movement proteins of raspberry bushy dwarf virus (rbd v) and soil-borne wheat mosaic furovirus (sb wmv); e movement proteins of cucumber mosaic cucumovirus (cmv) and brome mosaic bromovirus (bmv), putative movement proteins of pea enation mosaic virus (pemv) and cucumber necrosis tombusvirus (cnv); the similarity between 21 kda proteins of tombusviruses and movement proteins of bromoviruses and cucumoviruses has been independently reported by u. melcher (pers. comm.); d putative movement proteins of caulimoviruses, badnaviruses, parsnip yellow fleck virus, and tomato spotted wilt tospovirus; asterisks show identical residues and colons show similar residues between the sequences of pyfv and fmv, and coymv and tswv tospoviruses and the n-terminal domain of the parsnip yellow fleck virus polyprotein related to the putative movement proteins of caulimoviruses and badnaviruses (fig. 2 d; the similarity between caulimovirus movement proteins and n-terminal domains of badnavirus polyproteins was noted by u. melcher and cited in [8] ). even among these smaller groups, the latter three brought together proteins of very different viruses. group iv) is particularly notable in that it compiles proteins of a positive-strand rna virus, negative-strand (ambisense) rna viruses, and dna-containing pararetroviruses. curiously, the array of domains, namely n-movement domain-capsid protein(s)-replicative domains-c in the polyproteins of such seemingly unrelated viruses as parsnip yellow fleck virus and the badnaviruses is very similar. a relevant observation from our previous work is the relationship between movement proteins of two component geminiviruses (bl1/bc1) with those of tobamoviruses and tobraviruses [33] . all the proteins containing the conserved motifs shown in fig. 1 should be considered a single vast, and highly diverged superfamily of plant virus movement proteins. we would like to coin the nickname "30 k superfamily", after the most thoroughly studied movement protein of tobamoviruses, and also because most of the proteins of this superfamily have the size around this value (fig. 1 a) . several groups of positive-strand rna viruses, namely potexviruses, carlaviruses, hordeiviruses, and a group including beet necrotic yellow vein virus, nicotiana velutina mosaic virus, and peanut clump virus, encompass the triple gene block that consists of two small membrane proteins and a putative rna helicase [34, 44] . it has been demonstrated that disruption of any of the triple block genes abolished the cell-to-cell movement of hordeiviruses, potexviruses and bnyvv [5 a, 25 a, 49 a]. the putative helicases in the triple block comprise a distinct group within superfamily i of dna and rna helicases and contain several unusual amino acid substitutions in the conserved helicase motifs [26, 34] . unlike the genes coding for the proteins of the "30 k superfamily", the triple block is found only in positive-strand rna viruses. carmoviruses and necroviruses encode a pair of small proteins, one of which is predicted to be an integral membrane protein. it has been shown that disruption of either of these genes precluded cell-to-cell movement of turnip crinkle carmovirus [28] . these two genes perhaps may be considered a "truncated triple block" although it was hard to demonstrate this at the level of sequence comparison due to the small size of the hydrophobic proteins. a third type of movement protein has been revealed in tymoviruses. a 75 kda proline-rich protein that is encoded in the 5' portion of the genomic rna and overlaps with the replicative polyprotein gene is essential for the movement of tymv in infected plants [10] . this protein may have specific secondary and tertiary structure similar to that of other proline-rich filamentous proteins. interestingly, upon screening of the amino acid sequence databases with the sequence of the tymovirus movement protein the highest (albeit moderate) similarity was observed with the plant cell wall-associated protein extensin (data not shown). recently it has been shown that cell-to-cell movement of geminivirus msv is dependent on the small protein v1 [9] . this protein and the related proteins of other one component geminiviruses did not show appreciable sequence similarity to movement proteins of other viruses. interestingly, however, alignment of their amino acid sequences revealed the conservation of a highly hydrophobic central domain terminating at an invariant aspartic acid residue and thus distantly resembling the conserved motif of the 30 k superfamily (fig. 3) . this domain was flanked by two proline-rich domains showing some similarity to extensins (fig. 3) . thus, in a sense the movement proteins of one component geminiviruses combined the 30 k superfamily and the tymovirus structural themes. for several virus groups, in which at least one member has been completely sequenced, the movement function has not been genetically mapped and identification of a putative movement proteins by amino acid sequence comparison was very uncertain if possible at all. among these, uncharacterized proteins of nepoviruses and closteroviruses showed a very limited sequence similarities to some members of the 30 k superfamily, in particular to caulimovirus movement proteins [33] . the observations discussed in the previous sections show that classification of movement proteins based on comparison of their amino acid sequences does not correlate with the type of genome nucleic acid or with grouping of viruses based on phylogenetic analysis of replicative proteins 1-31, 34, 61] . also, we were unable to notice any correlation between grouping of movement proteins and biological properties of the viruses encoding these proteins (e.g. host ranges). recombination between unrelated or distantly related viruses could have played a major role in the evolution of the movement function. this is clearly the preferential explanation for situations when related movement proteins are found in viruses with different type of genome nucleic acid (e.g. the striking similarity between the putative movement proteins of caulimo/badnaviruses fig. 2 d) . in some cases recombinational transfer of movement protein genes appears very likely also between remote groups of positive-strand rna viruses (e.g. dianthoviruses and soil borne wheat mosaic furovirus; fig. 2 c) . another equally important trend in the evolution of movement protein genes is the apparent high rate of mutational change leading to the very limited level of sequence conservation as featured above. even within compact groups of viruses that share common genome organization and functional properties (e.g. tobarnoviruses), the divergence between the movement protein sequences is much higher than that between the principal repticative domains although comparable to that between capsid proteins. it has been proposed that plasmodesmata are plant analogues for certain animal cell-cell contacts, namely gap junctions, based mainly on studies in permeability of both to fluorescent dye-labelled molecules [5, 51] . details of protein organization of plasmodesmata are poorly understood, although a protein serologically related to a major gap junctions protein, connexin, was reportedly found in plasmodesmata and gene encoding this plant protein was cloned [42] . in animal kingdom, gap junctions are important for electrical coupling of cells in various tissues [51] . interestingly, permeability of both plasmodesmata and gap junctions for some low molecular weight tracer dyes is down-regulated by activation of protein kinase c [5] . however, movement of macromolecules, in particular nucleoproteins, through gap junctions, has not been described. a case of movement of a nucleoprotein through a channel in a membrane is represented by nucleocytoplasmic trafficking of rnps (reviewed in [38, 45] ). among the features of the nucleocytoplasmic export of cellular rnas revealed thus far, several might be relevant to the mechanisms of movement of plant virus genomes. in particular, it has been shown that at all stages of nucleocytoplasmic export, mrna is attached to specific protein frameworks, being transferred from nucleoskeleton to nuclear pore complex to cytoplasm [12] ; specific proteins in mrnp are thought to chaperone association-dissociation of the mrna to and from these frameworks [1, 53] . components of mrnp activate enzymes within the nuclear pore complex which are essential for energydependent translocation of rnp through nuclear pore; two of these enzymatic activities are atpase and rna helicase [53] . it is tempting to speculate that mechanistic analogies may exist between the nucleocytoplasmic transport of cellular rnps and intracellular and/or intercellular movement of viral rnps. more specifically, the parallel between the rna helicase in the nuclear pore and the putative viral rna helicase in the triple block that is involved in virus movement (see above) certainly is provocative. it should be noted that plant viruses, which undergo some stages in the nucleus of the infected cells (i.e. pararetroviruses and geminiviruses), should be able to perform both nucleocytoplasmic movement and cell-to-cell movement of their genomes; one may wonder to what extent these two processes are similar and whether movement proteins are involved in both of them. comparison of the amino acid sequences of dianthovirus movement proteins with amino acid sequence databases revealed a marginally significant but provocative similarity with membrane proteins of the mip family [48] . with the blast score of 67 and probability of random matching of 0.23, the similarity of the movement protein of rcnmv with tonoplast membrane protein tip 1 from arabidopsis was the highest in the entire database except for the similarities with the homologous proteins of the other dianthoviruses. analysis of the multiple alignment of the dianthovirus movement proteins with a representative set of the mip proteins (fig. 4) showed that the region of similarity included the conserved motif of the "30 k superfamily" described above and the characteristic conserved motif of the mip family [48] . each of these motifs consisted of a putative transmembrane helix succeeded by a loop containing conserved amino acid residues (fig. 4) . the drastic difference between the two types of proteins is that mip proteins contain six transmembrane helices whereas in the virus movement proteins the region shown in figs. 1 and 4 is the only such predicted helix. nevertheless, we believe that the observed sequence similarity may be functionally relevant indicating that the conserved motif of the "30 k superfamily" may mediate membrane interaction. for many proteins of this superfamily, membrane interaction is compatible with the observed localization in the cell wall [40] . however, caution is due in the interpretation of these observations as the hydrophobicity of the conserved region in different movement proteins differed significantly and not for all of them a membrane-spanning helix was confidently predicted ( fig. 1 b and fig. 4 . a conserved sequence motif in the movement proteins of dianthoviruses and the mip family of membrane proteins. asterisks show identical residues and colons show similar residues in the sequences of the movement protein of rcnmv and tip1. the consensus (for symbols see legend to fig. 2) includes the conserved residues, with one possible exception in the mip-related proteins. the hydrophobic regions predicted to form transmembrane helices are underlined. the motif was extracted from an alignment generated using the macaw program in some of these proteins the conserved motif may be involved in hydrophobic interactions other than membrane spanning. as mentioned above, membrane localization also has been proposed for the triple block proteins and strongly predicted for the movement proteins of monopartite geminiviruses. the observations on the involvement of influenza virus m 1 protein in nuclear export of virus ribonucleoproteins [-39] prompted us to directly compare the m 1 sequence to those of plant virus movement proteins. moderate similarity was revealed between m 1 and the movement proteins of bromoviruses and cucumoviruses (fig. 5) . the region of similarity included the conserved motif of the "30 k superfamily", with the invariant aspartic acid residue and the preceding hydrophobic stretch. m 1 protein chaperones newly formed influenza virus rnps from the nucleus to the cytoplasm; upon formation of virions, this protein is thought to provide the link between the rnp and the virion membrane (reviewed in [35] ). both of these functions are apparently based on the ability of m 1 to interact both with rnp and the membrane providing a provocative analogy with the plant virus movement proteins. very recently, a mutation in m 1 protein gene resulting in impaired nucleocytoplasmic movement was mapped within the region of similarity to bromovirus and cucumovirus movement proteins [23] . finally, our previous observations indicated a weak sequence similarity between a domain of cellular 90 kda heat shock proteins with some of the movement proteins, specifically those of caulimoviruses [33] . a common denominator for all these observations may be that the function of plant virus movement proteins is determined by domains mediating hydrophobic interactions. taking into account the limited sequence conservation and the absence of strictly invariant amino acid residues, it is very unlikely that these proteins (with the exception for the putative helicase in the triple block) have any enzymatic activity. among the known groups of plant viruses [25] , movement proteins so far have been identified genetically or by sequence comparison for about 75 percent. the variety of these proteins could be reduced to only three types, namely the "30k superfamily", with the distantly similar proteins of monopartite geminiviruses; the triple block; and the proline-rich proteins of tymoviruses. obviously, future studies may lead to characterization of movement proteins unrelated to any of these groups. different types of movement proteins may affect different aspects in viruscell interaction leading to virus spread in planta. however, it appears likely that in many cases these proteins mediate hydrophobic interactions between viral and cellular components, in a general analogy with molecular chaperone action [16, 33] . as discussed in the first part of this review, a large fraction of experimental data on plant virus movement proteins is very hard to be interpreted unequivocally. conceivably, this reflects the highly complex nature of virus-plant interaction. in this situation, it appears reasonable to focus further experiments on probing the specific functions fo conserved domains revealed by amino acid sequence comparison. hopefully, this approach will highlight new important aspects of plant virus lifestyle. recently, it has been shown that tmv movement protein expressed by transgenic tobacco behaves as an integral membrane protein [moore pj, fenczik ca, deom cm, beachy rn (1992) developmental changes in plasmodesmata in transgenic tobacco expressing the movement protein of tobacco mosaic virus. protoplasma 170:115-127]. between nucleus and cytoplasm cauliflower mosaic virus gene i product detected in cell wall-enriched fraction basic local alignment search tool expression of a plant virus-coded transport function by different viral genomes dynamic continuity of cytoplasmic and membrane compartments between plant cells triple gene block proteins of white clover mosaic potexviurs are required for transport the tmv movement protein: role of the c-terminal 73 amino acids in subcellular localization and function mutational analysis of cis-acting sequences and gene function in rna 3 of cucumber mosaic virus an analysis of the complete nucleotide sequence of a sugarcane bacilliform virus genome infectious to banana and rice replication of maize streak virus mutants in maize protoplasts: evidence for a movement protein expression of orf-69 of turnip yellow mosaic virus is necessary for virus spread in plants nucleotide sequence analysis of the movement genes of resistance breaking strains of tomato mosaic virus a three-dimensional view of precursor messenger rna metabolism within the mammalian nucleus potato virus x as a vector for gene expression in plants the p 30 movement protein of tobacco mosaic virus is a single-stranded nucleic acid binding protein gene i, a potential cell-to-cell movement locus of cauliflower mosaic virus, encodes an rna-binding protein how do plant virus nucleic acids move through intercellular connections visualization and characterization of tobacco mosaic virus movement protein binding to single-stranded nucleic acids relationship of tobacco mosaic virus gene expression to movement within plant plant virus movement proteins increase in plasmodesmatal permeability during cell-to-cell spread of tobacco rattle virus from individually inoculated cells of urfs and orfs. a primer on how to analyze derived amino acid sequences the plant virus movement proteins 255 informosome-like virus-specific ribonucleoprotein (vrnp) may be involved in the transport of tobacco mosaic virus infection an influenza virus temperature-sensitive mutant defective in the nuclear-cytoplasmic transport of the negative-sense viral rnas an n-proximal sequence of alfalfa mosaic virus movement protein is necessary for association with cell walls in transgenic plants classification and nomenclature of viruses. fifth report of the international committee on taxonomy of viruses efficient cell-tocell movement of beet necrotic yellow vein virus requires 3' proximal genes located on rna 2 a novel superfamily of nucleoside triphosphate-binding motif-containing proteins which are probably involved in duplex unwinding in dna and rna replication and recombination an atp-binding motif is the most conserved sequence in a highly diverged group of proteins involved in positive strand rna viral replication turnip crinkle virus genes required for rna replication and virus movement the complete nucleotide sequence of tobacco rattle virus rna-1 the sequence of carnation etched ring virus dna: comparison with cauliflower mosaic virus and retroviruses the phylogeny of rna-dependent rna polymerases of positivestrand rna viruses virus evolution: time for sturm und drang diverse groups of plant dna and rna viruses share related movement proteins that may possess chaperonelike activity evolution and taxonomy of positive-strand rna viruses: implications of comparative analysis of amino acid sequences genes and proteins of the influenza viruses the subcellular localization of gene i product of cauliflower mosaic virus is consistent with a function associated with virus spread ultrastructural location of non-structural protein 3a of cucumber mosaic virus in infected tissue using monoclonal antibodies to a cloned chimeric fusion protein nuclear mrna export nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (m1) promotes export and inhibits import virus movement in infected plants translocation of a specific premessenger ribonucleoprotein particle through the nuclear pore studied with electron microscope tomography gap junction protein homologue in arabidopsis thaliana: evidence for connexins in plants similarities between putative transport proteins of plant viruses probable reassortment of genomic elements among elongated rna-containing plant viruses nuclear import-export: in search of signals and mechanisms detection of the movement protein of red clover necrotic mosaic virus in a cell wall fraction from infected nicotiana clevetandii plants cooperative binding ofthe red clover necrotic mosaic movement protein to single stranded nucleic acids evolution of the mip family of integral membrane transport proteins cauliflower mosaic virus gene i product (p 1) forms tubular structures which extend from the surface of infected protoplasts identification of barley stripe mosaic virus genes involved in viral rna replication and systemic spread a conformation preference parameter to predict helices in integral membrane proteins spray dc (eds) (1990) parallels in cell-to-cell junctions in plants and animals effects of deletions in the n-terminal basic arm of brome mosaic virus coat protein on rna packaging and systemic infection evidence for involvement of a nuclear envelope-associated rna helicase activity in nucleocytoplasmic rna transport a workbench for multiple alignments construction and analysis a mutation in cauliflower mosaic virus gene i interferes with virus movement but not virus replication deletion analysis of brome mosaic virus 2 a protein: effects on rna replication and virus spread tubular structures involved in movement of cowpea mosaic virus are also formed in infected cowpea protoplasts plasmodesmatal function is probed using transgenic tobacco plants that express a virus movement protein cell-to-cell transport of cowpea mosaic virus requires both the 58/48 k proteins and the capsid proteins the roles of the red clover necrotic mosaic virus capsid and cell-to-cell movement proteins in systemic infection tobacco rattle virus rna-1 29 k gene product potentiates viral movement and also affects symptom production in tobacco evolution of rna viruses we appreciate helpful discussions with dr. v. v. dolja and dr. u. melcher. we would like to thank drs. d. cookmeyer, s. a. demler, r. kormelink, u. k. melcher, n. olszewski and yu. shirako for communicating data prior to publication. a. m. is grateful to dr. r. j. shepherd for constant support and encouragement. received june 9, 1993 key: cord-048471-7jszm1nd authors: salim, omar; clarke, ian n.; lambden, paul r. title: functional analysis of the 5′ genomic sequence of a bovine norovirus date: 2008-05-14 journal: plos one doi: 10.1371/journal.pone.0002169 sha: doc_id: 48471 cord_uid: 7jszm1nd background: jena virus (jv), a bovine norovirus, causes enteric disease in cattle and represents a potential model for the study of enteric norovirus infection and pathogenesis. the positive sense rna genome of jv is organised into orf1 (non-structural proteins), orf2 (major capsid protein) and orf3 (minor capsid protein). the lack of a cell culture system for studying jv replication has meant that work to date has relied upon in vitro systems to study non-structural protein synthesis and processing. principal findings: only two of the three major orf1 proteins were identified (p110 and 2c) following in vitro translation of jv rna, the n-term protein was not detected. the n-term encoding genomic sequence (5′gs) was tested for ires-like function in a bi-cistronic system and displayed no evidence of ires-like activity. the site of translation initiation in jv was determined to be at the predicted nucleotide 22. following the insertion of an epitope within the 5′gs the jv n-term protein was identified in vitro and within rna transfected cells. conclusions: the in vitro transcription/translation system is currently the best system for analysing protein synthesis and processing in jv. unlike similarly studied human noroviruses jv initially did not appear to express the n-terminal protein, presenting the possibility that the encoding rna sequence had a regulatory function, most likely involved in translation initiation in an ires-like manner. this was not the case and, following determination of the site of translation initiation the n-term protein was detected using an epitope tag, both in vitro and in vivo. although slightly larger than predicted the n-term protein was detected in a processed form in vivo, thus not only demonstrating initial translation of the orf1 polyprotein but also activity of the viral protease. these findings indicate that the block to noroviral replication in cultured cells lies elsewhere. jena virus, a bovine norovirus, is a member of the caliciviridae family of positive sense rna viruses and was first isolated from the diarrhoeic stools of newborn calves [1, 2] . jv is a type i genogroup iii (giii) norovirus which is closely related to the type ii giii bovine noroviruses newbury agent 2 and dumfries [3, 4] . the giii noroviruses are responsible for causing enteric disease in cattle [2, 5] and, thus, likely share a similar tissue tropism to the human-associated enteric noroviruses. like human noroviruses [6] bovine noroviruses have a high seroprevalence [4] . jv is therefore a potentially useful model for studying the molecular biology of enteric norovirus pathogenesis and replication. the 7.3 kb polyadenylated rna genome of jv has been characterised previously [7] and, like other noroviruses, is organised into 3 open reading frames (orfs). orf1 encodes the non-structural proteins in the form of a large 185 kda polyprotein, which is subsequently cleaved into functional replication proteins by the viral encoded 3c-like protease. orf2 encodes the structural capsid protein (56 kda) and orf3 encodes a small basic protein, which has been shown to function as a minor capsid component [8] . jv orf1 is consistent with other caliciviruses in that it encodes a 39 kda 2c-like nucleoside triphosphatase (ntpase), a 3c-like protease and a 56 kda 3d-like rna-dependent rna polymerase [7, [9] [10] [11] [12] [13] . however, the genomic sequence within the 59 region of jv orf1 (59gs) displays a high level of divergence. this divergence is mainly attributed to the presence of several proline-encoding polypyrimidine tracts within the region predicted to encode a 35 kda nterminal protein [7] . the predicted size of n-terminal proteins relative to the size of the respective 2c proteins differs within the norovirus genus. within the gi noroviruses, such as southampton virus, the n-terminal protein (44.8 kda) is larger in size compared to the 2c protein (39.6 kda). this is in contrast to the gii noroviruses, such as lordsdale virus and camberwell virus, in that the n-terminal protein is smaller in size compared to the 2c protein [11, 14] . this is also the case for jena virus in which the predicted jv n-terminal protein (35 kda) is smaller than the jv 2c protein (39 kda) [7] . the norovirus n-terminal protein varies in relative size across the genus, and the encoding sequence bears no similarity to other cellular or viral proteins. alignment of the n-term protein sequences of various noroviruses indicates little similarity between genogroups within the first 180 residues, however towards the cterminal end of the protein similarity between the amino acid residues increases. recent studies investigating the functions of the norwalk virus n-terminal protein have successfully demonstrated association with the golgi apparatus in transfected cells [15] . in addition this study also identified a picornaviral 2b like region within the n-terminal protein, suggesting that the protein is involved with host cell membrane interactions, reinforcing other findings that have suggested that the norwalk virus n-terminal protein disrupts intracellular protein trafficking, including proteins destined for the host cell membrane [16] . a 3c protease-mediated cleavage event within the n-terminal protein (37 kda) was described for camberwell virus, a genogroup 2 norovirus, yielding proteins of 22 kda and 15 kda [17] . based on these observations and location within the genome it was hypothesised that the nterminal protein of noroviruses corresponds to the 2ab region in picornaviruses. another possibility is that the n-term encoding rna itself serves to function as a translational enhancer by interacting with cellular proteins involved in translation. indeed, this phenomenon has been previously reported for norwalk virus, within which a double stem loop structure has been predicted at the 59 end of the genomic rna [18] . it was subsequently demonstrated that elements within the 59 end of norwalk virus bind specifically with cellular proteins such as la, ptb and pcbp2 [19] which have all been implicated in ires-mediated cap-independent translation in the closely related picornaviruses [20] [21] [22] [23] . in this study the role of the jv 59gs was investigated, including its potential to direct capindependent translation initiation. the precise location of translation initiation in jv was also investigated. previous studies of norovirus polyprotein processing have yielded three major products following in vitro transcription and translation, representing the uncleaved 3abcd, n-term and 2c proteins. however, initial analysis of jv polyprotein processing indicated that only two major proteins are synthesised initially which, based on molecular weight predictions, are the 3abcd (110 kda) and the 2c (39 kda). the lack of an n-terminal protein encoded by the jv 59gs, predicted to be 35.3 kda, is unique among the noroviruses that have been studied in this way. the in vitro transcription and translation profile for jv was therefore studied in more detail. as initial experiments had analysed tnth reactions following a 1 hr incubation, reaction aliquots were harvested at time points before and after the recommended 1 hr incubation. the results in figure 1 show that there are no major reaction products synthesised prior to the 1 hr time point, at which time the 3abcd/p110 and 2c/p39 proteins are clearly visible. extended incubation past the 1 hr point resulted in further proteolytic cleavage of p110 that coincided with the appearance of proteins of the following sizes: 86 kda, 55 kda, and 51 kda. in addition proteins of 29 kda, 22 kda and 20 kda were also visible at the 24 hr time point (figure 1, lane 7) . the only protein that was consistently visible following the 1 hr time point was the 2c/p39 protein. despite prolonged incubation there was no indication that the n-terminal/p35 protein was synthesised. a comprehensive study of polyprotein processing within the murine norovirus (mnv) suggests likely identities for the equivalent proteins in the similar profile for jv [12] . using region specific antisera the authors were able to identify p110 as the 3abcd uncleaved precursor, p90 as the 3bcd, p57.5 as the 3d-like polymerase, p52 as a 3abc precursor and p40 as the 2clike ntpase, which was determined by mutagenesis and microsequencing experiments. the 19 kda protein was identified as the 3c-like protease. the antisera used to detect the mnv nterm protein recognised 3 products; one was the predicted molecular weight at 39 kda and the other two bands migrated as a 45 kda doublet. the 59gs region of jv is highly divergent compared to other noroviruses, mainly due to the relatively high cytosine content (32%), which contributes to an overall g/c content of 58%. there are many polypyrimidine tracts within the sequence, potentially yielding a relatively high degree of rna secondary structure. previous studies have described potential secondary rna structure and interaction with proteins involved with iresmediated translation within the 59 genomic region of norwalk virus [18, 24] . it was of interest therefore, based on these findings, to ascertain whether or not the 59gs of jv possessed ires-like properties within the context of a 'bi-cistronic' expression system, independently of other viral proteins, including the vpg which, in other caliciviruses, has been shown to be associated with translation initiation factors [25, 26] . traditionally the bi-cistronic vector system has been used to define potential ires-like sequences from a variety of viral and cellular mrnas, and is recognized as being the standard test for this function [27] . a bi-cistronic vector is comprised of a 59 and 39 cistron; translation of the 59 cistron being cap-dependent and translation of the 39 cistron regulated by the putative ires-like sequence. thus, if the 39 cistron is translated in addition to the 59 cistron then the sequence of interest is said to have ires-like properties, as translation is initiating internally. to test for ires-like function in jv, bi-cistronic constructs were made with a cap dependent 59 egfp cistron and a 39 lacz cistron under the translational control of either the jv 59gs (pegfp-c1/ jv59gs/lacz) or an authentic emcv ires (pegfp-c1/ires/ lacz). crfk cells were transfected with the bi-cistronic constructs and, following incubation, were assayed for egfp and lacz expression. both constructs were able to direct translation of the egfp cistron effectively as expected (figure 2a and figure2d). the use of an authentic emcv ires to direct translation of the lacz cistron was also effective (figure 2e), with levels of b-galactosidase activity comparable to those of the b-galactosidase reporter ( figure 2f ). however, no b-galactosidase activity was detected from cells transfected with the pegfp-c1/jv59gs/lacz construct (figure 2b ), demonstrating that the jv 59gs was unable to initiate translation, and therefore, in this context, did not possess any ires-like functions. as it was clear that the jv 59gs did not posses any ires-like functions it was necessary to determine the location of translation initiation within orf1. this was predicted be the atg encoding methionine at nucleotide position 22, as it is situated in a favourable context for translation initiation [7] . to investigate this multiple translation termination codons (polystop) were inserted into the jv genome within the 3b-encoding region, downstream of the 59gs, to halt translation at a defined point. in vitro transcription and translation of this construct would, in theory, yield a product whose size would relate to the initiation codon used within the 59gs (figure 3 ). to address the unlikely event of translation read-through or re-initiation downstream of the polystop, which would result in subsequent translation of the 3c protease and cleavage of the truncated orf1 polyprotein, a mutation was made within the active site encoding region of the 3c protease within jv orf1, to prevent any viral mediated cleavage of orf1 translation products (jv 3c mut /polystop). a point mutation of the critical cysteine residue within the highly conserved gdcg motif to a glycine residue was performed, and this approach has been described for the successful inactivation of other norovirus' 3c activity [28] . in vitro transcription and translation analysis was performed on jv wild type ( figure 4 within the 3c region of jv successfully inactivated the 3c protease, thus a large, .200kda uncleaved polyprotein is yielded following tnth. the major product generated by jv 3c mut / polystop was calculated to be 103kda in size. based on computer predictions this is in agreement with the initiation of translation occurring at nucleotide 22, which demonstrates that the jv n-term protein is translated in full in vitro. at this time it is not possible to determine whether translation of intracellular vpgbound viral rna initiates at nucleotide 22, although it is likely given the favourable context in which the initiation codon is situated. as the jv n-term was found to be translated in vitro attempts were made to express and purify the protein in bacteria for immunisation so that the protein could be identified by radioimmune precipitation assay (ripa), as it was possible that the nterm protein was migrating on gels aberrantly and possibly comigrating with 2c. attempts to express the protein in bacteria were unsuccessful due to toxicity. therefore, the 14aa v5 epitope encoding sequence was cloned in frame into the jv cdna construct at nucleotide position 123 (jv v5). the v5 epitope originates from the p and v proteins of the sv5 paramyxovirus [29] , for which a commercially available monoclonal antibody is used for detection. following in vitro transcription and translation of jv v5 a new product, approximately 42 kda in size, was visible ( figure 5 , lane 2). this product was not observed in any prior analyses of jv. to confirm that this protein was v5/n-term associated the tnth reaction was subjected to ripa using the anti-v5 antibody ( figure 5, lane 3) . this confirmed expression of the n-term protein in vitro. to confirm expression of the v5/n-term protein in cell culture capped rna was synthesised from the jv v5 t7 cdna construct, which was used to transfect crfk cells. as there is currently no host cell line in which to propagate jv the crfk cell line was used as it has been shown to support the replication of feline calicivirus [30] . confocal immunofluorescence of transfected cells using the anti-v5 antibody demonstrated expression of the v5/nterm protein in cultured cells ( figure 6 ). expression of the v5/nterm protein was diffuse and did not co-localise with the golgi/ er/plasma membrane marker wheat germ agglutinin (wga) and therefore displays a different pattern of cellular expression compared to norwalk virus [15] . cells transfected with the wild type full length jv rna were negative for fluorescence (data not shown). lysates of cells transfected with wild type jv and jv/v5 rna were subjected to western blot using the anti-v5 antibody ( figure 7) . no product was present for cells transfected with wild type jv rna, but a protein of approximately 42 kda in size was visible in cells that had been transfected with jv/v5 rna, confirming n-term expression and size as seen in the in vitro system. in addition, this important observation also confirms for the first time that the jv 3c protease was active in cells transfected with capped rna as the size of the v5/n-term indicated successful cleavage of the protein from the orf1 polyprotein. to address the issue of potential rapid degradation of the jv nterm protein crfk cells were transfected with jv v5 rna and were harvested at designated time points following the addition of the protein synthesis inhibitor cycloheximide. cell lysates were analysed by western blot using the anti-v5 antibody (figure 8 ). the consistent appearance of the n-term/v5 protein suggested that it is stable and insensitive to degradation by viral and host cell proteases. the predicted molecular weight of the jv n-term is 35.3 kda, based on the site of initiation of translation and location of conserved cleavage sites. the appearance, therefore, of a previously unseen 42 kda protein in the in vitro transcription and translation profile was unexpected but this protein does represent a translation product for the jv 59gs. to date, it has not been possible to explain the difference in the predicted and observed sizes for the jv n-term, and the addition of the 14 amino acid v5 epitope within jv n-term does not account for this apparent large shift in molecular weight. however, a recent study described a similar anomaly when investigating proteolytic processing in the murine norovirus mnv-1 [12] . the predicted molecular weight for the mnv-1 n-term protein was 38.3 kda. the authors successfully generated antisera against the mnv-1 n-term and used it to immunoprecipitate the protein from in vitro transcription and translation reactions and observed that the n-term existed as a 45 kda doublet, in addition to the predicted size of 38 kda. however, when mnv-1 n-term antisera was used to probe mnv-1-infected cell lysates only the 43-45 kda doublet and a large 115 kda precursor could be detected, suggesting that the predicted 38 kda form of the n-term is not generated in cell culture. again, it was not possible to conclusively determine the cause of this discrepancy, but it was speculated that the n-term protein may migrate abnormally in sds-page, or may be proteolytically processed at a previously unknown cleavage site downstream of the protein's predicted c-terminus. it is also possible that the n-term protein might be modified in some way leading to a shift in observed molecular weight. at this time the same conclusions would seem appropriate for the jv n-term. in addition, it is not known why the jv n-term was previously not detected in in vitro transcription and translation studies prior to the insertion of the v5 epitope. it cannot be ruled out, however, that the wild type jv n-term aberrantly co-migrates with the 39 kda jv 2c protein in sds-page. indeed, the appearance of the v5/ n-term product from transfected cell lysates would appear to be one of a doublet (figure 8) , also analogous to the observed appearance of the mnv n-term protein in infected cells, suggesting the likelihood of a further cleavage site within the jv n-term protein which has yet to be elucidated. nevertheless, these studies clearly demonstrate that a protein representative of the 59gs of jv is translated both in vitro and in vivo and is proteolytically processed from the orf1 polyprotein following translation initiation at nucleotide 22. human norovirus infection has been shown to be the leading cause of non-bacterial gastroenteritis [31] , however there is currently no cell culture system available to facilitate viral replication and ethical considerations have hindered progress in establishing a permissive human organ culture system. the study of jena virus offers a potential animal model of enteric noroviral infection. however, until a permissive bovine cell and/or organ culture systems is established analysis of the molecular mechanisms underpinning viral replication and pathogenesis rely upon in vitro systems, most notably polyprotein synthesis and processing. unlike similarly studied human noroviruses jv initially did not appear to express the n-terminal protein, presenting the possibility that the encoding rna sequence had a regulatory function itself, most likely involved in translation initiation in an ires-like manner. this was shown not to be the case and, following determination of the site of translation initiation at the predicted nucleotide 22 the n-term protein was detected following the insertion of an epitope tag, both in vitro and in vivo. although slightly larger than predicted the n-term protein was detected in a processed form in vivo, thus not only demonstrating initial translation of the orf1 polyprotein but also activity of the viral encoded protease. these important findings indicate that the block to replication of enteric norovirus in cultured cells cannot be attributed to a failure to synthesise and process the non-structural proteins. the detection of processed and active orf1 proteins in transfected cultured cells, however, highlights the potential for the development of cell and bovine organ based systems to facilitate the replication of jena virus. the pegfp-c1 vector (clontech) comprises of an egfp coding sequence under the control of a cmv promoter and a kozak translation initiation site. downstream of the egfp sequence is the multiple cloning site containing unique bglii, saci, hindiii and apai restriction sites. contruction of pegfp-c1/jv 59 gs/lacz was as follows; the jv 59 gs sequence was amplified from the jv full length cdna clone [7] using bio-x-act dna polymerase (bioline) with the primers 59 gs f (59-aactgca-gatcttaataagtgaatgaagactttgacgat-39), containing the bglii restriction site (bold) and two in-frame translation termination codons (underlined) to ensure that translation of the egfp sequence did not carry over to the 59 gs, and 59 gs r (59-aactgcaagcttctgcaggacacaatgagg-39), containing thehindiii restriction site. the jv 59 gs amplicon was ligated to the pegfp-c1 vector, following restriction enzyme digestion of both amplicon and vector with bglii and hindiii restriction enzymes, and the ligated dna used to transform e.coli top10 (invitrogen). this intermediate construct was named pegfp-c1/ jv 59 gs. the lacz coding sequence was amplified from the psvb-gal reporter vector (promega) using bio-x-act dna polymerase and the primers lacz f (59-aactgcaagcttga-tatgggggatcccgtcgttttacaacg-39), containing the hindiii restriction site (bold) and a kozak translation initiation site (underlined), and lacz r (59-aactgcgggcccttat-tatttttgacaccagacca-39) containing the apai restriction site (bold) and translation termination codons (underlined). the lacz amplicon was ligated to the pegfp-c1/jv 59 gs vector following restriction enzyme digest of both amplicon and vector with hindiii and apai restriction enzymes, and the ligated dna used to transform e.coli top10. the construct was verified by sequencing. construction of pegfp-c1/ires/lacz was as follows; the emcv ires sequence was amplified from the pires2-egfp vector (clontech) using bio-x-act dna polymerase and the primers ires bgl f (59-actcgaagatcttaa-tagagcttcgaattctgcagtcga-39), containing the bglii restriction site (bold) and translation termination codons (underlined) to prevent carry over translation as before, and ires sac r (59-actcgagagctctgtggccatattatcatc-gtg-39), containing the saci restriction site (bold). the ires amplicon was ligated to the pegfp-c1 vector follwing restriction whole cell lysate was collected at the following time points following chx treatment: 0 hr, 1 hr, 3 hr, 6 hr, 12 hr, 24 hr. bradford analysis was performed on the lysates to ensure equal loading. following western analysis the ecl treated membrane was exposed to film for 1 min. molecular weight marker is represented in lane 1. doi:10.1371/journal.pone.0002169.g008 enzyme digest of both amplicon and vector with bgiii and saci restriction enzymes, and the ligated dna used to transform e.coli top10. the lacz amplicon described previously was ligated to the intermediate pegfp-c1/ires vector following restriction enzyme digest of both amplicon and vector with hindiii and apai restriction enzymes, and the ligated dna used to transform e.coli top10. the jv 3c protease mutant was created by point mutation of the critical tgt encoded cysteine residue, within the gdcg active site motif, to a ggt encoded glycine residue by mutagenic overlap pcr using bio-x-act dna polymerase. three rounds of amplification using the jv full length cdna clone as template were used to generate the final mutant protease cassette. round 1 used the primers jv f1 (59-cgtctcagggttgatact-39) and jv mut 1 (59-gcaaccaccgtcaccag-39), yielding a 222 bp amplicon (point mutation nucleotide shown in bold). round 2 used the primers jv mut 2 (59-ctggtgacggt-ggttgc-39) and jv r2 (59-ttcctgggaggaacaagtt-39), yielding a 651 bp amplicon. amplicons generated in rounds 1 and 2 were pooled to serve as template for round 3 using the primers jv nf (59-atgtcaaccaccaccagc -39) and jv nr (59-aagggctccggtgaagg-39). this cassette contained two bcli restriction sites flanking the 3cprotease active site, as also found in the wild-type full length clone. restriction digest using bcli was used to remove the appropriate wild-type cassette from the jv full length clone. the mutant cassette was also digested with bcli prior to ligation to the bcli-digested jv full length clone. the ligated dna was used to transform e.coli top10, and was designated jv 3c mut . construction of jv 3c mut /polystop was as follows: complementary oligonucleotides with three translation termination codons (underlined) in each reading frame in sense and anti-sense orientations were desgined in such a way that upon annealing the duplex would contain blunt termini. the oligonucleotides were termed pstop top (59-ctaggtaagtaaacgcgtctact-cactcac-39) and pstop comp (59-gtgagtgagta-gacgcgtttacttcaatag-39). each oligo (1 mg) was incubated with t4 polynucleotide kinase and atp to phosphorylate the 59 termini, pooled and heated to 75uc for 15 min, and left to cool to room temperature to anneal the oligos. following purification the polystop duplex was ligated to eco47iii digested jv 3c mut , and ligated dna was used to transform e.coli top10. the duplex contained the unique restriction site mlui (shown in bold) to assist screening of recombinant clones. the v5 epitope (n-gly-lys-pro-ile-pro-asn-pro-leu-leu-gly-leu-asp-ser-thr-c) is recognized by the anti-v5 monoclonal antibody (invitrogen). complementary oligonucleotides encoding the v5 epitope were designed in such a way as to generate sacii compatible termini following annealing (bold), and to preserve the reading frame when inserted into the sacii restriction site at nucleotide 123 within the 59 gs of the jv genome (underlined). the oligos were termed v5 top (59-ggtaagcctatccc-taaccctctcctcggtctcgattctacgagc-39) and v5 comp (59-tcgtagaatcgagaccgaggagagggt-tagggataggcttaccgc-39). the oligos were phosphorylated and annealed as described previously and the duplex ligated to the sacii digested jv full length clone. ligated dna was used to transform e.coli top10. in vitro coupled transcription and translation was performed using the tnth coupled reticulocyte lysate system (promega) as per the manufacturer's instructions. reactions were incubated at 30uc for 1-2 hr. for non-radiolabelled reactions the 35 s-methionine was replaced with 1 mm unlabelled methionine (2 ml). reaction products (1-2 ml) were analysed by sds-page. gels were stained and prepared for autoradiography by incubating for 30 min in a solution containing 32 g sodium salicylate, 100 ml methanol and 100 ml dh 2 o. gels were dried under vacuum and the reaction products were detected by exposure to kodak x-omat scientific imaging film (sigma) at 270uc for 16 hr followed by developing using a kodak automated developer. specific v5-tagged proteins synthesised by tnth were precipitated from 5-10 ml of reaction product using the anti-v5 monoclonal antibody (invitrogen) at the recommended dilution in 600 ml of 16 ripa buffer (diluted from 10x stock: 10 mm tris-hcl (ph 7.5), 1 mm edta, 0.15 mm nacl, 0.1% sds, 0.5% empigen bb, 0.1 mm phenylmethylsulphonylfluoride) for 1 hr at 37uc. this was followed by a second incubation of tube for 2 hr rotating at room temperature with goat anti-mouse immunoglobulin g agarose beads (sigma) to absorb the immune complexes. the beads were washed three times with 500 ml 16 ripa buffer and once with 500 ml pbs. the beads were resuspended in sample buffer for analysis by sds-page and autoradiography as before. endotoxin-free preparations of plasmid dna were prepared using the genelute tm endotoxin free plasmid midi prep kit (sigma). crandall-reese feline kidney cells (crfks) were seeded into a 12 well tray at approximately 40-50% confluence. crfk cells were transfected with no dna (negative control), psv-b-gal (control for b-galactosidase activity), pegfp-c1/jv 59 gs/lacz and pegfp-c1/ires/lacz (control for ires activity) using the superfect tm transfection reagent (qiagen) as per the manufacturer's recommendations. following a 16 hour incubation the cells were observed for egfp expression using a leica leitz dmrb fluorescence microscope. the cells were washed in pbs and fixed using a 0.5% solution of glutaraldehyde for 30 min at room temperature. the cells were incubated with an x-gal stain solution: 5 mm k 3 fe(cn) 6 , 5 mm k 4 fe(cn) 6 , 2 mm mgcl 2 , 1x x-gal (sigma) for 4 hours at 37uc and were observed for b-glactosidase activity by light microscopy. the experiment was performed more than once to confirm the results. jv v5 and jv flc t7 cdna plasmid constructs were linearised using ndei (invitrogen). capped rna was synthesised using the mmessage mmachineh capped rna transcription kit (ambion) according to the manufacturer's instructions. crfk cells were seeded into 6 well trays at approximately 50% confluence and were transfected with 2 mg purified rna per well using transmessenger transfection reagent (qiagen) according to the manufacturer's instructions. for immunofluorescence crfk cells were seeded onto 19 mm coverslips in 6 well trays and were transfected with rna as described. following a 24 hr incubation the coverslips were washed with pbs and fixed in 4% formaldehyde for 15 min at room temperature. cells were permeabilised and blocked in saponin buffer, also used as staining buffer, (0.1% saponin, 10% foetal calf serum, 0.1% sodium azide) for 1 hr at 4uc. cells were stained using an anti-v5 monoclonal antibody (invitrogen) followed by an anti-mouse alexafluor 488 conjugated secondary antibody (molecular probes) at the recommended dilution in staining buffer for 30 min in the dark. cells were then stained for 30 min in the dark with a wheat germ agglutinin alexafluor 594 nm conjugate (molcular probes) to allow identification of plasma and golgi membranes. coverslips were washed and mounted onto slides using vectashield containing dapi (vector labs). microscopy was performed using an inverted leica tcs-nt confocal laser scanning microscope. the anti-v5 antibody was also used to detect v5-tagged protein by western blot. cell lysates were prepared following transfection using lysis buffer (0.15 m sodium chloride, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) sds, 50 mm tris-cl ph 8.0) and protease inhibitor cocktail (sigma). lysates were incubated for 15 min on ice followed by sonication to shear genomic dna. following bradford analysis equal protein content from jv v5 and jv flc lysates were run on a 10% sds-page gel and subsequently transferred onto immobilon-p pvdf membrane (millipore) according to the manufacturer's recommendations. the membrane was probed using the anti-v5 monoclonal antibody at the manufacturer's recommended dilution, followed by an anti-mouse hrp-copnjugated secondary antibody (santa cruz) at the recommended diltution. the ecl western blotting reagents kit (g.e. healthcare) was used to detect antibody bound protein, which was visualised by exposure to biomax light film (kodak). crfk cells were seeded into 6 well trays and transfected with capped jv v5 rna as described. following a 24 hr incubation cycloheximide (sigma) was added to the cells at a final concentration of 50 mg/ml. cells were harvested at indicated times for the preparation of lysates for v5 western analysis as described above. bradford reagent (sigma) was used to ensure equal loading of lysates according to the manufacturer's recommendations. studies into diarrhoea of young calves. sixth communication: detection and determination of pathogenicity of a bovine corona virus and an undefined icosahedric virus. archives of experimental veterinary medicine studies into diarrhoea of young calves-seventh communication: ''zackenvirus'' (jena-agens 117/80)-a new diarrhoea pathogen to calf. archives of experimental veterinary medicine complete genomic characterization and antigenic relatedness of genogroup iii, genotype 2 bovine noroviruses genotype 1 and genotype 2 bovine noroviruses are antigenically distinct but share a cross-reactive epitope with human noroviruses characterization of a calici-like virus (newbury agent) found in association with astrovirus in bovine diarrhea the seroepidemiology of genogroup 1 and genogroup 2 norwalk-like viruses in italy molecular characterization of a bovine enteric calicivirus: relationship to the norwalk-like viruses two nonoverlapping domains on the norwalk virus open reading frame 3 (orf3) protein are involved in the formation of the phosphorylated 35k protein and in orf3-capsid protein interactions processing map and essential cleavage sites of the nonstructural polyprotein encoded by orf1 of the feline calicivirus genome rabbit hemorrhagic disease virus: genome organisation and polyprotein processing of a calicivirus studied after transient expression of cdna constructs organisation and expression of calicivirus genes cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells norovirus protein structure and function open reading frame 1 of the norwalklike virus camberwell: completion of sequence and expression in mammalian cells norwalk virus n-terminal nonstructural protein is associated with disassembly of the golgi complex in transfected cells norwalk virus nonstructural protein p48 forms a complex with the snare regulator vap-a and prevents cell surface expression of vesicular stomatitis virus g protein activity of the norovirus camberwell proteinase and cleavage of the n-terminal protein encoded by orf1 sequence and genomic organization of norwalk virus la, ptb, and pab proteins bind to the 39 untranslated region of norwalk virus genomic rna requirement of poly(rc) binding protein 2 for translation of poliovirus rna stem-loop structure synergy in binding cellular proteins to the 59 noncoding region of poliovirus rna direct evidence that polypyrimidine tract binding protein (ptb) is essential for internal initiation of translation of encephalomyocarditis virus rna sonenberg n (1993) la autoantigen enhances and corrects aberrant translation of poliovirus rna in reticulocyte lysate interaction of cellular proteins with the 59 end of norwalk virus genomic rna the genomelinked protein vpg of the norwalk virus binds eif3, suggesting its role in translation initiation complex recruitment vpg of murine norovirus binds translation initiation factors in infected cells new ways of initiating translation in eukaryotes? polyprotein processing in southampton virus: cleavage in trans by the 3c-like protease identification of an epitope on the p-proteins and v-proteins of simian-virus 5 that distinguishes between 2 isolates with different biological characteristics electron-microscopic observation of feline kidney-cells infected with a feline calicivirus viral gastroenteritis outbreaks in europe key: cord-017817-ztp7w9yh authors: land, walter gottlieb title: cell-autonomous (cell-intrinsic) stress responses date: 2018-03-28 journal: damage-associated molecular patterns in human diseases doi: 10.1007/978-3-319-78655-1_18 sha: doc_id: 17817 cord_uid: ztp7w9yh in this chapter, the role of cell-intrinsic stress responses is examined which include autophagic processes, the oxidative stress response, the heat shock response, the unfolded proteins response, and the dna damage response. autophagy (macroautophagy, microautophagy, and chaperone-mediated autophagy) is a self-digestive process in response to environmental stress to eukaryotic cells, by which cytoplasmic components are delivered to the lysosome for recycling and degradation. the oxidative stress response is directed against any oxidative stress and is mediated by antioxidative defense systems including antioxidant enzymes such as superoxide dismutase, detoxifying enzymes such as glutathione peroxidase, and energy-dependent efflux pumps. the heat shock response is induced upon exposure of cells to any stress condition and characterized by emission of heat shock proteins which operate as damps to maintain and restore homeostasis. the unfolded protein response is induced by any stress of the endoplasmic reticulum that is perceived by three sensor molecules. under remediable endoplasmic reticulum stress conditions, the sensors trigger signalling pathways to resolve this stress. however, in severe irremediable endoplasmic reticulum stress, the unfolded protein response may lead to pro-inflammatory and pro-apoptotic responses resulting in regulated cell death. finally, the dna damage response is induced by any dna damage that occurs in a variety of exogenous and endogenous conditions. when successful, this stress response leads to dna repair and is associated with the emission of various damps which contribute to restoration of homeostasis. when unsuccessful, the dna damage response, like the unsuccessful unfolded protein response, can result in regulated cell death, either in form of apoptosis or necrosis. together, the ultimate goal of all the stress responses is to maintain cellular homeostasis and ensure cell integrity. when they fail, the incidence of regulated cell death is frequently observed. as comprehensively described in part ii, prms are specifically involved in the recognition of mamps and damps. as will be discussed in part vi, each of these recognition receptors can trigger distinct signalling cascades in innate immune cells that modify their gene expression to create and execute efferent innate immune responses that involve (1) production of inflammatory mediator substances such as cytokines and chemokines, (2) phagocytosis, and (3) cytotoxicity, as well as, as described in part viii, may elicit and shape antigen-specific adaptive immune responses. beyond this well-characterized mamp/damp engagement of prms leading to a variety of downstream efferent cellular and humoral responses, the innate immune defense program also depends on cell-autonomous, that is, cell-intrinsic, responses which counteract any stressful insult [1] . constitutive cell-autonomous immunity mobilizes pre-existing molecules and processes in order to primarily and quickly defend the cell and the host against infectious and sterile injury. hence it can be considered as the very first line of innate immune defense. here, the role of constitutive cell-autonomous responses will be examined, whose involvement in the innate immune defense to stress and injury has only been appreciated within the last few years. the focus of this brief overview will be mainly directed toward cellular stress responses. the term autophagy comes from the greek words "phagy" meaning eat and "auto" meaning self. autophagy is an evolutionarily highly conserved self-digestive process in response to environmental stress to eukaryotic cells, by which cytoplasmic components such as defective/damaged or redundant organelles or protein aggregates are delivered to the lysosome for recycling and degradation. there is convincing evidence indicating that activation of the autophagic process is promoted by mamps and/or damps [2, 3] . in more simple words, autophagy is a classical cellprotective and cell-autonomous process of the innate immune system aimed at maintaining and restoring homeostasis at both the cellular (cell-intrinsic) and organismal (cell-extrinsic) level [4] . although autophagy was initially identified in mammals, a significant breakthrough in our understanding of how autophagy is controlled came from the analysis in the genetically tractable yeast system. pioneering work from ohsumi's group showed that the morphology of autophagy in yeast was similar to that documented in mammals [5] . (as known, ohsumi received the nobel prize in physiology or medicine 2016.) in fact, the discovery of the autophagyrelated genes in yeast has significantly advanced the understanding of the molecular mechanisms participating in autophagy and the genes involved in regulating the autophagic pathway. many yeast genes have mammalian homologues, confirming that the basic machinery for autophagy has been evolutionarily conserved along the eukaryotic phylum [6] [7] [8] [9] . notably, a panel of leading experts in the field of autophagy has recently published a new definition of several autophagy-related terms based on specific biochemical features [10] . accordingly, in the following, three types of autophagy are briefly sketched including macroautophagy, microautophagy, and, in mammals, chaperone-mediated autophagy. each of them fulfils very specific tasks in intracellular degradation. there is general agreement on two main features that characterize bona fide, functional autophagic responses, irrespective of type: (1) they involve cytoplasmic material; and (2) they culminate with (and strictly depend on) lysosomal degradation [10] . thus, although autophagy substrates can be endogenous such as damaged cellular organelles or exogenous such as viruses or bacteria escaping phagosomes, autophagy acts on entities that are freely accessible to cytosolic proteins. this property is essential in order to distinguish between autophagic responses and branches of vesicular trafficking that originate at the plasma membrane, which also culminates in lysosomal degradation. such endocytic processes include phagocytosis, receptor-mediated endocytosis, and macropinocytosis, that is, processes which will be dealt with in part vi, sect. 22.6. of note, however, some forms of autophagy and the endocytic pathway interact at multiple levels, and the molecular machinery responsible for the fusion of late endosomes (also known as mvbs) or autophagosomes with lysosomes is essentially the same [11] . as stressed [10] , the strict dependency of autophagic responses on lysosomal activity is necessary to discriminate them from other catabolic pathways that also involve cytoplasmic material, such as proteasomal degradation [12] . thus, the 26s proteasome (box 18.1) degrades a large number of misfolded cytoplasmic proteins that have been ubiquitinated (for (poly)ubiquitination, see box 18.2) as well as properly folded proteins that expose specific degradation signals, such as the socalled n-degrons [13] . on the other hand, the proteasome system shares some substrates with different forms of autophagy whereby these two catabolic pathways differ drastically in their final products. thus, proteasomal degradation results in short peptides that are not necessarily degraded further but may flow into additional processes including but not limited to antigen presentation/cross-presentation at the plasma membrane, thereby generating mhc-ii and mhc-i epitopes (compare part viii, chap. 31). by contrast, lysosomal proteases fully catabolize polypeptides to their constituting amino acids which eventually become available for metabolic reactions or repair processes. together, as summarized [10] , bona fide functional autophagic responses navigate cytoplasmic material of endogenous or exogenous origin to degradation within lysosomes (or late endosomes, in specific cases). the binding of many ubiquitin molecules to the same target protein. in its simplest form, ubiquitin can be attached to the target protein as a single moiety resulting in monoubiquitination. ubiquitin itself can be ubiquitinated, resulting in the formation of ubiquitin chains attached to the target protein: polyubiquitination. polyubiquitination of proteins is the triggering signal that leads to subsequent degradation of the protein in the proteasome. ligases play a central role in polyubiquitination. ligases are enzymes that catalyze the synthesis of polyubiquitin chains. ubiquitin conjugation requires typically box the proteasome is a common complex for all living cells, needed to recycle and eliminate unwanted proteins. in analogy, it resembles a chaff-cutter. this molecular machine provides a pathway that is involved in many cellular levels such as protein degradation, antigen processing, cell cycle, apoptosis, and dna repair. the 26s proteasome that is present in the cytoplasm and nucleus is usually formed by one 20s proteasome complex and two 19s proteasome complexes, which are composed of proteases and structural units. the 26s proteasome is a giant protease responsible for the regulated degradation of polyubiquitylated proteins (see box 18.2) . it consists of at least 33 distinct subunits and is arranged into two modules: core particle containing catalytic sites and regulatory particles. the cylinder-shaped proteolytic core is the 20s core particle, which is capped at one or both ends by 19s regulatory particles. further reading: wehmer m, sakata e. recent advances in the structural biology of the 26s proteasome. int j biochem cell biol 2016;79:437-442. basically, the term macroautophagy is often used when describing autophagy in general. the phenomenon is characterized by its typical morphological features which involve dedicated vesicles that can occupy a considerable part of the cytoplasm. typically, macroautophagy is one type of autophagic processes in which the substrates are sequestered within cytosolic double-membrane vesicles termed autophagosomes. the substrates of macroautophagy include superfluous and damaged organelles, cytosolic proteins, and invasive microbes. mechanism of formation and regulation of macroautophagy are very complex and complicated processes that are outlined here in a considerably simplified way. macroautophagy involves the sequestration of cytoplasm via a double-membrane intermediate structure termed the phagophore which matures into an autophagosome; the latter compartment fuses with a lysosome allowing degradation and recycling of the cargo [14] . in more detail, the process begins with the formation of a membrane of unknown origin, the initial phagophore or isolation membrane. the phagophore then expands, surrounds proteins or organelles, sequesters cytoplasm, and, on completion, develops into a large double-membrane transport vesicle, the autophagosome. subsequently, the autophagosome fuses with a lysosome containing acid hydrolases and releases its contents into the lytic acid hydrolases-containing compartment as part of single-membrane vesicles, termed autophagic bodies. the fused compartment where the autophagic body and its contents are degraded is called an autophagolysosome or autolysosome ( fig. 18.1) . notably, the process of phagophore expansion three classes of enzymes. e1 (ubiquitin-activating enzyme) hydrolyzes atp and forms a thioester-linked complex between itself and ubiquitin. e2 (ubiquitin-conjugating enzyme) receives ubiquitin from e1 and forms a similar thioester-linked intermediate with ubiquitin. e3 (ubiquitin ligase) finally binds both the e2 and a substrate and catalyzes the transfer of ubiquitin to the substrate. ubiquitin itself is often a substrate for further ubiquitylation, which results in the formation of so-called polyubiquitin chains. ubiquitin has seven lysine residues, and depending on the lysine residue used for ubiquitin-ubiquitin chain formation, the polyubiquitin chain can signal different functions. proteins modified by lysine-48 (k48)-or lysine-29 (k29)-linked chains are usually degraded by the proteasome. in contrast, modification by k63-linked chains or by a single ubiquitin moiety (monoubiquitylation) seems to trigger other functions, e.g., protein sorting, gene expression, and dna repair. further reading: callis j. the ubiquitination machinery of the ubiquitin system. arabidopsis book 2014;12:e0174. provides tremendous flexibility and capacity with regard to cargo, allowing entire organelles to be deleted via autophagy; however, this flexibility also means that autophagy must be tightly controlled in order to prevent inappropriate degradation, which could lead to cell death (for relevant papers, see [6] [7] [8] [9] [14] [15] [16] . intensive studies have been carried out in the past two decades to understand the mechanism and regulation of autophagy. the biogenesis of autophagosomes needs the ordered intervention of autophagy-regulated (atg) proteins that act on different modules. thus, more than 30 atg genes have been identified in human that orchestrate the complex membrane dynamics involved in autophagic sequestration. these atg proteins act sequentially in three macromolecular complexes involved in the three successive stages of autophagy. initiation of autophagy requires the unc-51like kinase 1 (ulk1)-atg13-fip200 (also known as rb1-inducible coiled-coil 1) complex, whereby the kinase activity of ulk1 is controlled by the kinase mammalian target of rapamycin (mtor) in mtor complex 1 (mtorc1), which is sensitive to rapamycin [9] . the next process, membrane nucleation, requires the beclin1/ class iii pi3k complex, which also plays a major role in membrane trafficking and restructuring involved in autophagy [15, 17] ; the final process refers to the elongation, expansion, and closure of the phagophore membrane/autophagosome which mainly relies on atg8/microtubule-associated protein 1 light chain 3 (lc3) lipidation. in fact, atg8/lc3 lipidation is regarded as a hallmark of autophagy and is established by a covalent linkage of cytosolic lc3 to the lipid phosphatidylethanolamine on the surface of the autophagosome [7, 9] . of note, in addition to the cytoplasmic ptm of various atg proteins, recent studies have explored the transcriptional and epigenetic control of autophagy [18] . notably, in human cells, tfeb (for transcription factor eb) and zkscan3 (for zinc finger with krab and scan domains 3) were shown to be implicated in playing a crucial role in autophagy regulation [19, 20] . also, there is growing evidence in support of the notion that histone modification/dna methylation acts as an alternative approach for long-term autophagy control [21] (for histone modification, see part vi, sect. 24.2.3) . also recently, a new ampk→skp2→carm1 (for: ampactivated protein kinase; s-phase kinase-associated protein 2 (p45); coactivatorassociated arginine methyltransferase 1) regulatory axis was reported that incorporated cellular nutrient sensing with transcriptional as well as epigenetic control of autophagy [22] . as concluded by xu and klionsky [14] , "…this ampk-skp2-carm1 signalling axis integrates the various levels of autophagy regulation including cell signalling, and transcriptional regulation as well as epigenetic modification. epigenetic and transcriptional regulation provides an energy-saving approach for control and also create an enduring memory in preparation for future adverse events. thus, this study has deepened our understanding of how autophagy can be controlled in a holistic manner by pathways linking a multitude of regulation mechanisms. given the extensive involvement of autophagy in human diseases, this work also presents potential directions for novel therapeutic intervention." indeed, besides its beneficial function in controlling cellular homeostasis, macroautophagic pathways when disrupted can have severe consequences leading to major diseases such as cancer, metabolic and neurodegenerative disorders, and cardiovascular and pulmonary diseases [23] . of note, macroautophagy can be divided into two subtypes depending on the organelle that is targeted for autophagic degradation; thus, the process of mitophagy corresponds to autophagy of mitochondria, whereas the term er-phagy refers to autophagy of the endoplasmic reticulum (er). both processes deserve a few more words in the following subsection. the term mitophagy corresponds to cargo-specific autophagy of mitochondria, a process which mediates the selective removal of mitochondria [24, 25] . the aim of mitophagy is to eliminate mitochondria, either to regulate their number to adjust to metabolic demand or to explicitly remove those that are damaged in terms of a quality control. mechanistically, mitochondria are selectively recruited into isolation membranes, which seal and then fuse with lysosomes to eliminate the trapped mitochondria. as discussed [24] , mitophagy is preceded by so-called mitochondrial fission that divides elongated mitochondria into pieces of manageable size for encapsulation and also controls segregation of damaged mitochondrial material for selective removal by mitophagy. the term er-phagy (also called micro-er-phagy) refers to a process of distinct selective degradation of er membranes and proteins in the lysosome under stress, and this is independent of the core autophagy machinery [26] [27] [28] (for er stress, see sect. 18.5) . studies on yeast showed that er-phagy is characterized by the fact that stress-induced er whorls are selectively taken up into the vacuole, the yeast lysosome. import into the vacuole was found not to involve autophagosomes but occurs through invagination of the vacuolar membrane, indicating that er-phagy is topologically equivalent to microautophagy [27] . recent studies on yeast provide evidence suggesting that the atg proteins atg39 and atg40 are specific receptors for this pathway of er-phagy [28] . at this point, it also appears worthwhile to mention that a more recent study on yeast revealed a novel er quality-control pathway, namely, the so-called macro-er-phagy. first results from this study suggest that this pathway delivers an excess of integral-membrane proteins from the er to the lysosome for degradation and, typically, requires the core autophagy machinery [29] . the brief overview about macroautophagy provides another typical example of innate immune responses which, when controlled, operate in a beneficial homeostatic way but, when uncontrolled, may lead to severe pathologies. for other forms of phagocytic responses, this phenomenon has not been investigated sufficiently. clearly, mitophagy also plays a key homeostatic role in mitochondrial quality control. upregulation of mitophagy has been shown to mitigate excessive mitochondrial accumulation and toxicity to safeguard mitochondrial fitness. hence, mitophagy is a viable target to promote longevity and prevent age-related pathologies [25] . concerning the two types of er-phagy (micro-and macro-er-phagy), one has to state that research in this exciting field has just begun. several questions remain to be addressed, for example, what is the purpose of er-phagy and what are the underlying mechanisms. future studies will probably provide a clue to elucidating the molecular mechanisms and physiologic roles of er-phagy in other organisms. of note, besides mitophagy and er-phagy, other specific forms of phagocytic pathways have been described. they include -pexophagy as a macroautophagic response preferentially targeting peroxisomes -nucleophagy as an autophagic response selectively targeting portions of the nucleus -ribophagy as a specific autophagic response targeting ribosomes -aggrephagy as an autophagic response specific for protein aggregates -lipophagy in terms of selective autophagic degradation of neutral lipid droplets -bacterial xenophagy as a macroautophagic removal of cytoplasmic bacteria which have escaped the phagosomal compartment upon phagocytosis -viral xenophagy as a macroautophagic response targeting fully formed cytoplasmic virions or components thereof -proteaphagy in terms of macroautophagic responses specific for inactive proteasomes -lysophagy as a specific macroautophagic disposal of damaged lysosomes in mammalian cells for details of these specific forms of phagocytic responses, the reader is referred to the excellent comprehensive review article of galluzzi et al. [10] . in addition to macroautophagy, two other types of autophagy have been described called microautophagy and chaperone-mediated autophagy (cma). microautophagy together with macroautophagy plays, for example, a role in nutrient recycling under starvation. on the other hand, cma is known to contribute to the maintenance of cellular homeostasis by facilitating recycling of amino acids of the degraded proteins and by eliminating abnormal or damaged proteins, thereby exerting major regulatory functions in different pathophysiological scenarios such as metabolic regulation. here, a few aspects of these two types of autophagic responses are skimpily touched. by contrast to macroautophagy, the process of microautophagy is much less defined in mammals since most studies have been performed in yeast and plants. according to current models, the term refers to a collection of diverse processes. unlike autophagy, microautophagy does not involve the autophagosome-dependent degradation of cytoplasmic components but rather and characteristically relies on the direct engulfment of small portions of cytoplasm into lysosomes or late endosomes by invagination and inward budding of the lysosomal/endosomal membrane, a process that leads to their degradation [30] [31] [32] . though microautophagy is the least studied form of autophagy, a molecular signature of the process has begun to emerge and has led to the definition of microautophagy as a type of autophagy in which the cargo is directly internalized in small vesicles that form at the surface of the lysosome/vacuole or late endosomes (multivesicular bodies), respectively [10] . in addition to macroautophagy and microautophagy, there is another type of autophagy experiencing increased attention, the cma. characteristically, in cma, cargo delivery also occurs directly at lysosomes, but it does not require formation of vesicles nor membrane invagination. instead, the substrate proteins for this autophagic pathway cross the lysosomal membrane through a protein-translocation complex, that is, a process that requires protein interaction with the chaperone hspa8 (also known as hsc70) and association of hspa8 with a specific splicing isoform of lamp-2, that is, the lysosomal protein lamp-2a. thus, chaperone-bound autophagy substrates bind lamp-2a monomers on the cytosolic side of the lysosome, which stimulate the formation of an oligomeric lamp-2a translocation complex [10, [33] [34] [35] . essential functions that cma fulfils in cells include a contribution to amino acid recycling during prolonged starvation as well as quality control, directly linked to the ability of this pathway to selectively remove single proteins from the cytosol. for example, cma is up-regulated during oxidative stress where it contributes to the degradation of oxidized proteins (reviewed in [36] ) (see next sect. 18.3). of note, growing evidence demonstrates that malfunction of cma plays a vital role in the pathogenesis of severe human disorders. often, the mechanisms underlying the alterations of cma in these pathologies involve perturbations in the functioning of the cma translocation complex. both diminished and enhanced cma activities have been shown to associate with diseases, an observation that emphasizes the importance of a tight regulation of cma activity (highlighted in [36] ). as argued above, current knowledge about mechanism and physiological relevance of microautophagy in mammalian cells is hard to judge since most findings derive from studies in yeast. however, future studies aimed at identifying proteins controlling microautophagy-related vacuolar membrane changes in yeast will probably allow to search for homologues in mammals and then investigate their contribution to mammalian microautophagy. by contrast, research on cma has already made substantial progress. for example, the recent identification of a plethora of new cma substrates and deficiencies in cma associated with diverse human pathologies has expanded our understanding of the importance of cma in multiple cellular functions. in fact, the growing number of connections between cma and human diseases has already generated interest in modulating cma activity for therapeutic purposes. there is a close relationship between autophagy and mamps and/or damps in the cellular response to injury. in fact, autophagy cannot be restricted to an innate immune mechanism that controls intracellular homeostasis alone but has to be extended in terms of "immunological autophagy" to a process that is committed to control and regulate efferent innate and potential adaptive immune responses. the "medium" for achieving this goal is the mamps and/or damps which operate as a link between intracellular and extracellular events. this scenario is briefly touched in the following. growing evidence indicates that autophagy regulates release and degradation of damps-here in terms of inducible damps-including hmgb1, atp, and dna in several cell types [37] . for example, autophagic mechanisms reportedly promote and regulate the release and secretion of hmgb1 in a ros-dependent manner in fibroblasts, macrophages, and cancer as well as net-mediated release of hmgb1 in neutrophils [37, 38] . moreover, autophagy has been shown to be required for the liberation/active secretion of atp by dying cancer cells [39, 40] . in addition, autophagy was found to contribute to the regulation of the ddr at multiple levels, that is, a process associated with the emission of damps [37, 41] (for ddr, see sect. 18.6). via emission of damps, eventually, together with mamps, autophagy can amplify or even instigate mamp/damp-prm signalling leading to efferent innate immune responses. on the other hand, autophagy can inhibit pro-inflammatory signalling cascades. for example, the atg5-atg12 complex, a key regulator of the autophagic process, was shown to negatively regulate rlr signalling by direct binding to card domains of rig-i and interferon promoter-stimulating factor-1 (ips-1) [42] (compare part vi, sect. 22.3.6). moreover, as reviewed elsewhere [1] , autophagy has been found to inhibit both nlrp3 and aim2 inflammasome activation and subsequent production of pro-inflammatory cytokines il-1β and il-18 (for inflammasomes, see part vi, sects. 22.4.2 and 22.4.4). as a possible mechanism, the authors propose that inflammasome components and pro-il-1β are subjected to ubiquitination and subsequent degradation by autophagy, thereby leading to functional inactivation of inflammasomes. conversely, an increasing number of studies suggest that damps, including hmgb1, atp, and dna, are powerful stimuli and regulators to elicit autophagic responses [3, [43] [44] [45] [46] [47] [48] [49] [50] . for example, hmgb1 was demonstrated to be an important regulator of autophagy in various types of cancer cells and keratinocytes. mechanistically, the reduced form of the hmgb1 protein was proposed to be responsible for the promotion of autophagy in an ager/rage-dependent fashion [47, 48] . also, and of high interest, in a clinical study on patients with chronic hepatitis b, hmgb1-induced autophagy was found to maintain treg function during chronic viral infection [49] . moreover, in studies on a mini pig lung iri model, evidence was provided indicating that autophagy, when triggered by damps such as hmgb1 and hsp60 during iri, amplifies the inflammatory response through enhancing k63-linked ubiquitination of traf6 and activation of the downstream mapk and nf-κb signalling (for traf6, mapk, and nf-κb signalling, see part moreover, there is already first evidence suggesting a role of atp in the regulation of autophagy [50] . in addition, there are accumulating data indicating that cytosolic dna, dislocated as a result of dna damage, may contribute to the regulation of autophagy whereby the dna damage-regulated autophagy modulator 1 (dram1) appears to play a mechanistically crucial role [51] [52] [53] . the mechanisms involved in mamp/damp-activated autophagic responses have only partially been elucidated. in fact, there is convincing evidence suggesting that many mamp/damp-recognizing prms including tlrs (in particular endosomal tlrs), nlrs, and anti-dna receptors can activate autophagic responses by triggering specific signalling pathways (reviewed or discussed in [1, 2, 54, 55] ). the crosstalk between autophagic responses and damps represents a powerful instrument of the innate immune system to integrate and unify various tools for the promotion and regulation of injury-induced inflammation and, in the presence of nonself-or altered self-antigens, injury-induced adaptive immunity. thus, on the one hand, autophagy is known to promote and regulate the release of damps (though the exact mechanisms are still elusive); subsequently, damps via prmtriggered pathways participate in the regulation of inflammation. on the other hand, activation of prms by mamps and/or damps promotes autophagy activation through a mechanism that has been partially elucidated. in fact, an increasing number of findings suggest that this activation process is triggered by prms following recognition of mamps and/or damps. nevertheless, the precise molecular mechanisms by which prms modulate autophagy remain largely unknown. there is increasing evidence in support of the notion that mamp/damp-activated autophagic responses promote emission of damps which in turn support cellular homeostasis in the course of adaptive stress responses in healthy cells. notably, this cellular homeostatic effect may spread out and affect the whole organism via emission of autophagy-dependent damps. in other words, via damps, autophagy as a cell-intrinsic stress response can fortify its defending capability by providing a link to promotion and regulation of cell-extrinsic efferent innate immune and eventually subsequent adaptive immune responses. however, despite the fact that autophagy is one of the best-known cell-autonomous responses in innate immunity and has clearly been shown to counteract dangerous infectious and sterile cell stress, much is left unclear. one such issue concerns the definition of autophagy-dependent cell death. as discussed and summarized [10] , autophagy-dependent cell death can be defined as a form of rcd (see next chapter) that can be retarded by pharmacological or genetic inhibition of macroautophagy. in this context, as stressed by galluzzi et al. [10] , it is important to note that (1) specificity issues affect most, if not all, pharmacological agents employed so far for suppressing macroautophagic responses and (2) multiple components of the macroautophagy machinery have autophagy-independent functions. in view of these facts and findings, these authors recommend to favor genetic approaches and to test the involvement of at least two different proteins of the macroautophagy apparatus in a specific instance of rcd before etiologically attributing it to macroautophagy. other unclear issues refer to the specific modulation of autophagy by mamps and/or damps, the precise interaction of autophagy with innate immune signalling cascades, and the cooperation between autophagy and other physiologic cell-intrinsic and cell-extrinsic processes during scenarios of cell stress and tissue injury. efforts to solve these problems are of utmost importance in view of the fact that autophagy-when induced by excessive, chronic, or acute-repetitive emission of damps-can contribute to the pathogenesis of many human diseases, that is, acute and chronic, infectious, or sterile inflammatory disorders. at the respective places, they will often be mentioned in the following chapters as well as in volume 2. oxidative cell stress and tissue injury reflect most potent and omnipresent threats an organism is exposed to. though there is a robust defense response continuously operating, this kind of injury is known to contribute to the pathogenesis of many human diseases. how can this be? oxidative stress is caused by an imbalance between the production of oxidants such as ros on one side and the biological antioxidative defense system's ability on the other side to counter the oxidant levels with antioxidants, that is, to readily detoxify the toxic reactive species or easily repair the resulting damage. thus, it is the excessive production of ros-overriding the antioxidative capacities-that is pathophysiological and contributes to dysfunction, damage, and even death of cells. by contrast, generation of ros in physiological low/ moderate concentrations-operating as second messenger molecules and causing so-called oxidative "eustress" [56] -assists in intracellular signalling pathways and, thus, is essential for optimal cell functions and homeostasis of an organism. in other words, the biological effects of ros-beneficial or deleterious-considerably depend on the amounts of ros present and, in action, a phenomenon that is in agreement with the idea that cellular ros generation has characteristics of hormesis implying a dose-response phenomenon that is characterized by beneficial effects at low doses and deleterious effectivity at high toxic doses [57] . to guarantee this homeostatic function of ros, to keep these molecules within physiological limits, and to prevent their deleterious effects, that is, to maintain hormesis, a smooth running of the oxidative stress response is of utmost importance. accordingly, a few aspects of this critical stress response are addressed in the following. reactive oxygen species are produced from molecular oxygen as a result of normal cellular metabolism. to understand any discussion on a role of ros in host defense or human diseases, one should define free radicals. according to halliwell and gutteridge [58] , "a free radical is any species capable of independent existence that contains 1 or more unpaired electrons." an unpaired electron is one that occupies an atomic or molecular orbital by itself. radicals can be formed by the loss of a single electron from a non-radical, or by the gain of a single electron by a non-radical. in this sense, superoxide anions (o 2 ·− ), hydroxyl radicals ( · oh), peroxyl radicals (ro 2 · ), and alkoxyl radicals (ro˙) are oxygen radicals. of note, ros is a collective term often used by scientists to include not only the oxygen radicals but also some non-radical derivatives of oxygen such as h 2 o 2 , hypochlorous acid (hocl), ozone (o 3 ), and singlet oxygen ( 1 o 2 ). nitrogen-containing oxidants, such as no · are called rns. generation of ros is generally a cascade of reactions that starts with the production of superoxide anions. superoxide rapidly dismutases to h 2 o 2 either spontaneously (especially at the low ph) or catalyzed by sod. other elements in the cascade of ros generation include the reaction of superoxide with no to form the very toxic peroxynitrite, the peroxidase-catalyzed formation of hocl from h 2 o 2 , and the iron-catalyzed fenton reaction, leading to the generation of hydroxyl radical. the oxidants are produced endogenously as by-products or metabolites of various metabolic processes. multiple enzyme systems produce superoxide radicals and their derivatives including xanthine oxidoreductase (xor), the reduced form of nadph oxidases (noxes), and mitochondrial electron transport chain (etc)associated molecular complexes ( fig. 18 [61, 62, [64] [65] [66] [67] [68] [69] [70] mitochondrial etc is believed to be the main source of ros. in the following, some aspects of these three systems (other systems not mentioned here) are briefly touched, exemplified by and focused on their role as vascular sources of ros production as investigated on models of iri. xanthine oxidoreductase (xor), as a housekeeping enzyme, is probably expressed in all cells but primarily in surface epithelia such as capillary endothelial tissue of various organs. xanthine oxidoreductase, a complex molybdoflavoprotein, is the ratelimiting step in the catabolism of purines, where it catalyzes the last steps of purine metabolism: the conversion of hypoxanthine to xanthine and of xanthine to uric acid, with superoxide/h 2 o 2 generated as by-products. there is a definite role of xor in reperfusion of tissue and organs [59, 60] . for example, experiments performed in isolated rat hearts have demonstrated that radical generation and functional injury are decreased by inhibition of xor with oxypurinol. similarly, in human aortic or venous ecs, xor-mediated ros generation has been shown to be a central mechanism of oxygen radical generation upon postischemic reoxygenation [61, 62] . the nadph oxidases were initially considered as enzymes expressed only in phagocytic cells involved in host defense and innate immunity; however, recent evidence indicates that there is an entire family of noxes based on the discovery of gp91phox homologues. the family comprises seven members, including nox1, nox2 (formerly termed gp91phox), nox3, nox4, nox5, duox1, and duox2 [63] . three members out of the enzyme family are important sources of ros in the vasculature, namely, nox1, nox2, and nox4 [64] [65] [66] . today, noxes are perhaps the best-studied enzymes involved in ros production in the blood vessels. remarkably, different members of the nox/duox family engaged in iri are localized in various cells, that is, in vascular cells and phagocytes. this may lead to the notion that noxes in vascular cells are responsible for the first wave of ros production because vascular cells are first confronted with reintroduced molecular oxygen. as generally believed, there is, in fact, no vascular specific nox isoform but rather a complex expression of various nox isoforms in different cells and regions of the vascular system. nevertheless, in arteries from humans and animals, nox2, nox4, and a shallow level of nox1 have been consistently found to be present both as messenger rna and as protein [63] . altogether, the findings and data briefly described here make clear that vascular cells are equipped with efficient machinery able to efficiently produce ros. regarding the different enzymatic sources, superoxide radicals appear to be predominantly generated compared, for example, to hydroxyl radicals. mitochondria have been implicated as potential oxygen sensors by increasing the generation of ros which regulate a variety of hypoxic responses [67] [68] [69] [70] . in fact, mitochondria are increasingly recognized as lynchpins in the evolution of tissue injury during postischemic reperfusion. it is generally acknowledged that the majority of intracellular ros production is generated in the mitochondrial etc and its associated metabolic enzymes. however, very little is known about which mitochondrial sites are involved in physiological or pathological ros production under native conditions. of note, using inhibitors to manipulate the redox states of particular sites and prevent superoxide generation from others, at least ten different locations of superoxide/h 2 o 2 production in the etc and associated enzymes (krebs cycle, β-oxidation, etc.) have been identified in mammalian mitochondria. in fact, the relative and absolute contributions of specific sites to the production of ros in isolated mitochondria depend very strongly on the substrates being oxidized, and the same is likely valid in cells and in vivo [71] . for example, superoxide formation occurs on the outer mitochondrial membrane, in the matrix, and on both sides of the inner mitochondrial membrane (fig. 18.3) . complex i (nadh-ubiquinone oxidoreductase) accepts electrons from nadh; these electrons are carried to complex ii (the succinate dehydrogenase-coq oxidoreductase), where they are used to oxidize succinate to fumarate. afterward, electrons continue to travel down their electrochemical gradient to complex iii (the cytochrome bc1 complex (ubiquinol-cytochrome c oxidoreductase)), and subsequently to complex iv (cytochrome c oxidase); finally, the electrons are used to reduce molecular oxygen to water. thus, complex i and complex ii oxidize the energy-rich molecules nadh and flavin adenine dinucleotide h 2 , respectively, and then transfer the resulting electrons to ubiquinol that carries it up to complex iii (for competent articles, see [72, 73] notably, complexes i and ii generate superoxide within the mitochondrial matrix, whereas complex iii produces superoxide at the qo site, resulting in the release of superoxide into either the intermembrane space or the matrix. regarding complex i, it was recently demonstrated that inhibition of nd5, a subunit of complex i, suppresses the activity of this complex and thus ros production [74] . furthermore, data from another set of studies on complex i showed that stable down-modulation of its subunits grim-19 and ndufs3 decreased complex i activity that was associated with a significant reduction in the overall nadh oxidation rate but with an increased production of ros by the target cells [75] . similar results have been found in studies on complex ii: there is evidence suggesting that inhibition of complex ii on the level of subunits even leads to an increase in ros production. the phenomena can be explained by assuming that, if electrons provided in the course of the etc cannot efficiently be transferred to the next complex, they would leak out from the inhibited complex and generate ros [76] . the complex iii subunits rieske iron-sulfur protein (risp) encoded by ubiquinol-cytochrome c reductase, rieske iron-sulfur polypeptide 1 (uqcrfs1), and ubiquinol-cytochrome c reductase binding (uqcrb) protein appear to play a crucial role in hypoxia-triggered mitochondrial ros generation (for rieske, see box 18.3). thus, it was shown that risp promotes the hypoxic stabilization of the transcription factor hif-1α protein [77] and uqcrb was found to mediate hypoxia-induced tumor angiogenesis via mitochondrial ros-mediated signalling [78, 79] . also, and of note, a mouse model to permit conditional deletion of the nuclear-encoded risp gene was recently developed to assess its role in hypoxiainduced ros signalling in the pulmonary circulation [80] . it was found that depletion of risp abolishes the ros response to hypoxia in isolated pulmonary the rieske protein is an iron-sulfur protein (isp) component of the cytochrome bc1 complex that was first discovered and isolated by john s. rieske and coworkers in 1964. the rieske iron-sulfur protein is an essential subunit of mitochondrial cytochrome bc1 complexes and, like the majority of mitochondrial proteins, is encoded by a nuclear gene and synthesized on cytoplasmic ribosomes as a precursor with a 32-residue amino-terminal extension. the iron-sulfur protein is then post-translationally imported into the mitochondria where it is inserted into the bc1 complex in the inner mitochondrial membrane. at first, the precursor is translocated via translocation contact sites into the matrix. there, cleavage to an intermediate containing an 8-residue extension occurs. the intermediate is then redirected across the inner membrane, processed to the mature subunit, and assembled into complex iii. arterial smcs and isolated pulmonary artery segments. further, in this article, it was discussed that mitochondria are not the only source of ros during hypoxia. thus, studies using a genetic knockout of p47phox suggested that cytosolic nadph oxidase systems may also contribute to a hypoxic pulmonary vasoconstriction response during acute hypoxia [81, 82] . according to the authors' conclusion, the blockade of hypoxia-induced ros responses (in these studies observed with depletion of risp) suggests that the mitochondria may act as the initiators of ros production, which could be amplified by engagement of nadph oxidase systems elsewhere in the cell. such "ros-induced ros release" might permit small ros signals generated by mitochondria to activate ros signalling throughout the cell, thereby avoiding mitochondrial damage that might arise if the entire cellular oxidant signal originated from that organelle [83] (or even leading to excessive ros production?). indeed, the substantial advances in oxidative stress research of recent times, in particular, the specification of hypoxia-sensing ros-producing enzyme systems, will contribute to new therapeutic strategies to be applied in acute and chronic human diseases known to be influenced by oxidative stress. for example, discrimination of oxidative eustress, a fundamental process in maintaining health, from oxidative damage will improve clarity in developing "redox medicine" [56] . when the redox equilibrium of a cell is upset by pro-oxidant environmental stimuli, that is, when oxidative stress exists, an adaptive stress response takes place which can result in upregulation of antioxidant proteins and detoxification enzymes. these antioxidative defense molecules comprise the following [58] : (1) agents that catalytically remove free oxygen radicals and other reactive species, for example, sod, catalase, peroxidase, and thiol-specific antioxidants; (2) proteins that minimize the availability of pro-oxidants such as iron ions, copper ions, and heme, for example, transferrins, haptoglobins, hemopexin, and metallothionein; (3) proteins that protect biomolecules against damage (including oxidative damage) by other mechanisms, for example, hsps; and (4) low-molecular-mass agents that scavenge ros and rns, for example, glutathione, α-tocopherol, and (possibly) bilirubin and uric acid. in the past, it was fashionable to divide the oxidative stress response into three main tiers: (1) antioxidant enzymes including sod, catalase, glutathione peroxidase, and glutathione; (2) detoxifying enzymes such as glutathione peroxidase, glutathione s-transferase, aldo-keto reductase, and aldehyde dehydrogenase; and (3) energy-dependent efflux pumps. as a fourth defense system, the antioxidant nutrients such as vitamins e and c as well as carotenoids were appreciated [84] . it was generally accepted that the first line of enzymes is of enormous importance in limiting ros-mediated damage to intracellular macromolecules. for example, among the most important regulators of ros levels were the sod enzymes: cu/ znsod in the cytoplasm and outer mitochondrial space and mnsod exclusively in the inner mitochondrial space. mechanistically, superoxide is converted to h 2 o 2 and oxygen (o 2 ·− + o 2 ·− + 2h + → h 2 o 2 + o 2 ) by sod. peroxiredoxins and abundant catalase enzyme then scavenge h 2 o 2 , converting it to molecular oxygen and water. another example of a first-line defense molecule is trx. thioredoxin contains two adjacent -sh groups in its reduced form which are converted to a disulfide in oxidized trx. notably, it can undergo redox reactions with multiple proteins using the reaction trx however, it then turned out that these antioxidative principles were clearly not 100% effective at performing this task, as under normal physiological conditions, lipid and dna oxidation products can be detected in blood and urine. because certain compounds of the chemicals generated after an interaction of ros with macromolecules are highly reactive, there must be an equal necessity to detoxify these secondary oxidation products to prevent them from also damaging dna, proteins, and lipids. without the adequate detoxification of such products, an extended chain reaction will occur resulting in the degradation of cellular components and the ultimate death of the cell. this second line of defense against ros is provided by those detoxifying enzymes. finally, detoxified metabolites produced by these enzymes are eliminated from the cell by energy-dependent efflux pumps such as the glutathione s-conjugate transporter, also called the multidrug resistance-associated protein (mrp) [58] . today, one must state that these previous notions are incomplete. at first, it became apparent that members of the so-called cnc (for cap 'n' collar)-basic region-leucine zipper (bzip) family of transcription factors are principal mediators of defense responses to redox stress. in mammals, the cnc family members nrf1 and nrf2 were shown to be involved in the transcriptional upregulation of cytoprotective genes encoding a large number of diverse detoxification, antioxidant, and anti-inflammatory proteins (e.g., glutamate cysteine ligase, nadph-quinone oxidoreductase, glutathione s-transferases, and aldo-keto reductases) as well as enzymes with essential roles in cell metabolism [85] . more recent studies then revealed that these transcription factors, notably nrf2, are activated by keap1 as the primary negative regulator of nrf2, that is, a molecule that simultaneously operates as a sensor protein able to perceive dyshomeostatic subclass iic-4 damps, for example, in terms of redox changes reflecting electrophilic stress. it is worth to add here that six critical domains have been defined in nfr2 (neh1-neh6), and it is neh2, located at the n terminus of nrf2 that acts as the regulatory domain for the cellular stress response. actually, neh2 interacts with the cytoplasmic protein keap1 [86] . in the following, this very important damp-induced and gene-based antioxidative and cytoprotective system is addressed a bit more in detail. increasing evidence indicates that several redox-regulated gene products serve to protect cells from ros damage. the antioxidant response element (are), a cis-acting dna regulatory element or enhancer sequence is known to be activated by oxidative stress and to be responsible for the transcriptional regulation of several redox-regulated gene products. both nrf 1 and 2 bind to are and regulate are-mediated gene expression and induction. the molecule nrf2 is more potent than nrf1 in activation of are-regulated gene expression and is regarded as the principal transcription factor that binds to the are. this transcription factor is ubiquitously expressed and present in various organs and tissues including the kidney, muscle, lung, heart, liver, and brain (for reviews, see [87] [88] [89] [90] [91] ). as touched in part ii, sect. 5.3.2 and part iv, sect. 13.4.5, the nrf2-triggered antioxidant response is initiated by activation of keap1 that functions as a substrate adaptor protein for the degradation of nrf2 and serves as an intracellular sensor for redox changes reflecting the presence of subclass iic-4 damps (for reviews see [92] [93] [94] ). earlier studies had already shown that nrf2 is a bzip transcription factor that translocates to the nucleus after liberation under oxidative stress conditions from its cytosolic inhibitor keap1 [86] . in the nucleus, nrf2 was found to form dimers with the proteins maf, jun, fos, activating transcription factors 4 (atf4), and/or creb binding protein (cbp) and, in addition, regulates transcription by binding to the are upstream of a variety of cytoprotective and detoxification target genes to combat the oxidative stress [95] . thus, established nrf2-regulated genes reportedly included cu/zn sod, catalase, trx, trx reductase, glutathione reductase (gr), glutathione peroxidase (gpx), and ferritin l (ftl) [96] (ftl and ferritin h (fth) subunits are responsible for intracellular iron storage). all of these genes are involved in the response to oxidative stress. there are several other genes also known to be engaged in the response to oxidative stress that are not described here. recently, the molecular signalling mechanism involved in the keap1 ↔ nrf2 pathway has been further elucidated and specified. the core can be seen in an axis consisting of redox change (subclass iic-4 damps)-initiated → keap1-induced → nrf2-triggered → are-driven expression of antioxidant and detoxifying genes ( fig. 18.4 ) (discussed in [92-94, 97, 98] ). the complex and complicated sequelae of the pathway are simplified in the following text. under homeostatic and stress-free conditions, binding of keap1 to the nrf2 molecule leads to its polyubiquitination and subsequent degradation by the proteasomal pathway, thereby maintaining a consistent generation of nrf2 and retaining its very low levels in the cytoplasm. in this scenario, keap1 homodimer binds to a single nrf2 protein via a high-affinity so-called "etge" motif and low-affinity so-called "dlg" motif. the twosite recognition of nrf2 by the keap1 dimer is essential for polyubiquitination of nrf2 (also see part ii, sect. 5.3.2) (for polyubiquitination, see box 18.2) . in this sense, the keap1 ↔ nrf2 system can be regarded as a vital part of regulating cells under a homeostatic environment. however, in case of dangerous and threatening oxidative (and xenobiotic) stress, the system instigates a stress response that is characterized by a rapid and dramatic cessation of the keap1-dependent polyubiquitination process resulting in a rapid increase of nrf2 abundance. in fact, exposure to ros-mediated stress (or electrophilic stress) is thought to modify the reactive "iic-4 damps-sensing" cysteine residues in keap1, which is associated with a conformational change of the protein resulting in a loss of keap1 ubiquitination activity (fig. 18.4) . notably, as a cysteine-rich protein, the human keap1 possesses 27 cysteine residues, and they are all reactive to stress to varying degrees. among these sensor cysteines of keap1, c151 is best characterized. evidence from a number of studies has suggested that c151 is the most reactive and critical to the keap1 ↔ nrf2 stress-sensing response. even, there is already evidence from a first atomic-level view suggesting that the unique environment of cys 151 (besides cys171, cys273, and cys288) appears to be the critical residue of keap1 responsible for detecting increased levels of oxidative stress [91, 92, 97] . oxidative modification of cysteine sensors of keap1 leads to a loss of its polyubiquitination and degradation activity thereby stabilizing nrf2. consequently, fig. 18.4 the oxidative stress-induced keap1 ↔ nrf2 pathway. under non-oxidative homeostatic conditions, the sensor keap1is bound to the nrf2 molecule resulting in its polyubiquitination and subsequent degradation via the proteasomal pathway. oxidative stress modifies the ros-sensing cysteine residues of keap1 leading to loss of its polyubiquitination and degradation activity, dissociation of nrf2 that becomes stabilized and accumulates. then, nrf2 translocates to the nucleus and forms a heterodimer with the smaf transcription factor. the nrf2/smaf heterodimer binds to are and induces transcription of numerous cytoprotective antioxidant and detoxification genes. are antioxidant response element, keap1 kelch-like erythroid cell-derived protein with cnc homology (ech)-associated protein 1, nrf2 nuclear factor-erythroid 2 p45-related factors 2, smaf small musculoaponeurotic fibrosarcoma, ub-ub-ub poly-ubiquitin chain. sources: refs. [92-94, 97, 98] stabilized nrf2 accumulates in the cytoplasm, translocates into the nucleus, and forms a heterodimer with a smaf transcription factor (smaf, small musculoaponeurotic fibrosarcoma). thus, this accumulation of nrf2 in response to ros (and electrophiles) cannot be regarded as an induction in a strict sense but instead is a mechanism referred to as derepression, that is, from the rapid degradation-based repression. as highlighted and discussed [93] , there are two models of how to explain the cytoplasmic accumulation process of nrf2. the "hinge-and-latch" model holds that the modification of the sensing cysteine residues of keap1 reduces its affinity for nrf2 but does not result in release. instead, newly synthesized nrf2 is translocated to the nucleus to trigger the transcription of nrf2-dependent genes. the other model denoted as the "conformation cycling" model claims that keap1 uses a cyclic mechanism to target nrf2 for polyubiquitination and proteasomal degradation. an important feature of this cyclic mechanism is that it ensures regeneration of keap1 which allows the cycle to proceed. modification of specific reactive cysteine residues of keap1 may block the cycle of keap1-dependent nrf2 degradation allowing de novo synthesized nrf2 to accumulate. the subsequent transcriptional process in the nucleus has been specified as well. as partially mentioned above, the nrf2-smaf heterodimer binds to are or electrophile-responsive element (epre) and induces transcription of numerous cytoprotective genes. of note, recently, an extensive genome-wide analysis of the nrf2-smaf-binding sequence, that is, the are/epre, and the maf homodimerbinding sequence (the so-called maf responsive element or "mare") was conducted, and the differences between these elements were clarified. as a result, it was proposed that are, epre, and the nf-e2 binding sequence be collectively named cnc-smaf-binding elements (csmbe) [99] . interestingly, prms appear to be co-players in this scenario. thus, tlrs have been observed to induce nrf2 activation. remarkably, in a recent study, tlr agonists were shown to activate nrf2 signalling via reduction of keap1 [100] . the authors could demonstrate that tlr signalling-induced keap1 reduction promotes nrf2 translocation from the cytoplasm to the nucleus, where it activated transcription of its target genes. further, tlr agonists were found to modulate keap1 at the protein post-translation level through autophagy. in fact, tlr signalling increased the expression of autophagy protein p62 and lc3-ii and induced their association with keap1 in the autophagosome-like structures. the keap1 ↔ nrf2 system as briefly described here is a robust oxidative stress response. its regulatory mechanisms, for example, stress-sensing mechanism, proteasome-based regulation of nrf2 activity, and selection of target genes have been elucidated mainly in mammals (for proteasome, see box 18.1). nevertheless, the pathway is now regarded as an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress across the tree of life. thus, the keap1 ↔ nrf2 system has been found to be also present in zebrafish, fruit fly, and caenorhabditis elegans indicating that its roles in cellular defense are conserved throughout evolution among vertebrates and suggesting that analogous defense systems are widely conserved throughout the animal kingdom [101, 102] . as briefly demonstrated in this subchapter, aerobic organisms have integrated antioxidant systems, which include damp-promoted generation of enzymatic and nonenzymatic antioxidants that are usually effective in blocking harmful effects of ros. however, when ros is produced in excess causing pathological conditions, the stress response against oxidative damage can be overridden. thus, oxidative stress is known to contribute to many pathological conditions, including cancer, neurological disorders, atherosclerosis, hypertension, ards, and chronic obstructive pulmonary disease, just to mention a few of them. plausibly, these disorders are motivation enough to search for new effective therapeutic options by harnessing the new insights into mechanisms of the keap1 ↔ nrf2 system. certainly, intense research is essential for a detailed understanding of the precise consequences of targeting keap1 for disease prevention and treatment. all the more so as the oxidative stress response is integrated into other forms of innate stress responses that will be further outlined in the following subchapters. the heat shock response-one of the most ancient and evolutionarily conserved cytoprotective mechanisms found in nature-is induced upon exposure of living cells to acute, subacute, or chronic stress conditions. this defense response is characterized by the expression of a group of phylogenetically conserved intracellular hsps, which possess the capacity to recognize structures commonly found in the interior of proteins and to bind such structures. thanks to this property, they form a chaperone network involved in correct protein folding, trafficking, and complex assembly (for reviews, see [103] [104] [105] [106] [107] [108] [109] ). in order to be released from engagement with proteins after folding and take part in further rounds of activity, hsp70 and other chaperones utilize an intrinsic atpase domain to hydrolyze atp and assume a free conformation [110] . once released, hsps mediate protective cellular defense mechanisms including regulation of apoptosis, the aim being to maintain and restore cellular protein homeostasis (proteostasis). exposure of almost any cell to heat shock most often leads to the rapid transcription, translation, and accumulation of a variety of hsps that increase to quite considerable levels when the stress is pronounced. in fact, these molecules are induced by various environmental insults that can cause protein denaturation and unfolding within the cells, leading to the formation of nonnative proteins and protein aggregates, thereby emitting dyshomeostatic damps. of note, besides their protective role in different intracellular compartments, hsps in terms of inducible damps can translocate to the cell surface to get exposed or are actively secreted via non-canonical pathways. in case of necrotic cell death, they are passively released in large amounts into the extracellular space to act as constitutively expressed damps (compare part iv, sect. 12.2.3 and sects 12.3.2.3 and 14.2.2.2). once emitted, hsps are sensed by classical recognition receptors and/or non-classical receptors (e.g., cd 91) [111] [112] [113] [114] [115] [116] [117] . interestingly, recent knowledge about similarities between allograft and tumor rejection has visualized that the processes of both iri to allografts [108, 118, 119] and therapy-mediated injury to tumors [120] [121] [122] [123] are characteristically associated with emission of hsps. in all eukaryotes, the hsr is primarily regulated and controlled by the hsfs, in particular, hsf1, a sequence-specific factor that binds upstream to heat shock elements in the promoters of target genes [124] (fig.18.5 ). for example, hsf1 is activated by environmental stress including oxidative and tumor-associated stress [125, 126] . notably, in all eukaryotes, hsf1 responds to such stress conditions by undergoing a monomer to trimer transition and becomes massively phosphorylated, leading to its acquiring ability to bind to dna rapidly and activate transcription [127] . there is at least preliminary evidence suggesting that intracellular perturbations reflecting dyshomeostatic damps may activate hsfs [128] [129] [130] [131] . such molecular alterations reportedly include changes in cytosolic ca 2+ concentration, for example, caused by increase of fluidity in specific membrane domains [128, 129] . interestingly, a recent study in support of these earlier findings provided evidence of the existence of a plasma membrane-dependent mechanism of hsf1 activation in animal cells, which is initiated by specific membrane-dependent trpv calcium channel-like receptors [130] . these findings lend support to the notion that heat sensing and signalling in mammalian cells are dependent on trpv channels, suggesting that these receptors may act as a major hsr sensor in different epithelial non-cancerous and cancerous cells, capable of triggering the cellular hsr [130] . in another line of experiments, trpv2 was demonstrated to mediate the effects of transient heat shock on endocytosis of human monocyte-derived dcs, suggesting a central role of trpv2 in mediating the cellular action of heat shock on these important cells of the innate immune system [131] (for trpv channel receptors, also compare part ii, sect. 5.3.6). induction of an hsr is not only mediated by sterile stress conditions but is also believed to be promoted by cell-invading viruses or bacteria. consequent emission of hsps in their role as damps may reflect a mechanism by which pathogens may contribute to sterile inflammation. for example, exacerbation of hepatitis b virus (hbv)-associated liver injury is reportedly characterized by an abnormal immune response that not only mobilizes specific antiviral effects but also poses a potentially lethal non-specific sterile inflammation to the host [132] . heat shock proteins may be the most extensively studied damps in the context of hbv infection. a number of hsps such as hsp70 and hsp90 have been reported to be supportive factors in the process of hbv replication, and selective inhibition of these hsps was proposed to be host-based anti-hbv strategies [133] [134] [135] . thus, as argued [132] , both infective and sterile inflammation may synergistically contribute to the exaggeration of chronic hepatitis, if the hbv cannot be cleared entirely. likewise, bacterial infections were also reported to promote induction of the hsr [136, 137] . for example, in studies on h. pylori and e. coli infection models, the initiation of an hsp70 stress response could be demonstrated [138, 139] . the hsr is recognized and accepted as a classical stress response in nearly all species across the tree of life. it reflects the desperate efforts of a cell to restore homeostasis and survive upon both infectious and sterile insults. its products, the hsps, operate as damps in commission of the innate immune system to reach this goal. notably, via this mechanism, an hsr, induced by infectious damage to a cell, can promote sterile inflammation. whereas a successful hsr upon stress leads to restoration of cellular homeostasis and cell survival, an unsuccessful hsr may result in rcd such as apoptosis [109] . this phenomenon will be resumed in the following subchapters. the er is a continuous membrane system that forms a series of flattened sacs within the cytoplasm of eukaryotic cells. as a subcellular organelle in the control of proteostasis, it is responsible for calcium storage and lipid biosynthesis as well as the synthesis, correct folding, processing, and maturation of proteins as well as for the orchestration of their transport along the classical/conventional secretory pathway. the er delivers these components to their destination compartments which include the er itself, the golgi apparatus, the plasma membrane, and the extracellular milieu or the endocytic and autophagic pathways. plausibly, the multifunctional nature of this organelle requires a myriad of proteins, unique physical structures, and coordination with and response to perturbations in the intracellular environment. a series of chaperones, folding enzymes, glucosidases, and carbohydrate transferases support and execute these processes. perturbation of er-associated functions such as accumulation of unfolded/misfolded proteins, excessive ros production, hypoxia, calcium and glucose depletion, or viral and bacterial infections reflect stress of the organelle and result in activation of an er stress-coping response, the evolutionary conserved upr [140] [141] [142] [143] . for example, the processes of both iri-mediated cell damage/cell death [144] [145] [146] and induction of the icd of cancer cells [147] [148] [149] are characterized by demonstration of er stress that is almost always associated with oxidative stress and vice versa [150] . to cope with er stress, cells have the unique possibility to activate the upr, a dynamic signalling network that orchestrates the recovery of homeostasis or triggers rcd modalities, depending on the level of damage. perception of any perturbation of the er is provided by three sensor molecules of the upr embedded in the er membrane: the perk, ire-1, and atf6. the upr-mediated recovery of er homeostasis mainly occurs through the perk-eif2α-mediated temporary shutdown of protein translation and the activation of a complex genetic program that aims to improve er quality control and adaptive responses [140] [141] [142] [143] (fig. 18.6) . accordingly, perk, devoted to perceiving dyshomeostatic damp-emitting er perturbations (so far, no clear evidence for ire-1 and atf6 in this respect), may be regarded as a new family of non-classical prms, at least in a broader sense. in nonstressed conditions, the three sensors of the er homeostasis are kept in an inactive state by the er-luminal binding immunoglobulin protein (bip). the protein bip, also known as glucose regulated protein 78 (grp78), is a member of the hsp70 family of proteins (specifically hspa5). it functions as a chaperone to selectively bind unfolded proteins in the er lumen by interacting with exposed hydrophobic 18 .6 simplified scenario model illustrating the er stress-induced three arms of the unfolded protein response. perception of any perturbation of the er is provided by three sensor molecules of the unfolded protein response embedded in the er membrane: the perk, ire-1, and atf6. perk perceives dyshomeostatic damp (subclass iic-4 damp)-emitting er perturbations (ire1 and atf6 still questionable). perk phosphorylates eif2a to up-regulate transcription factor atf4 that induces the expression of transcription factor chop. ire1a signals through its rnase via the splicing of xbp1 mrna. the active transcription factor xbp1s translocates to the nucleus. atf6 is exported from the er to the golgi complex to enter the nucleus as a potent transcription factor. together, these transcription factors of the unfolded protein response determine the cell fate by the regulation of distinct subsets of target genes toward recovery of er homeostasis and cell survival or the induction of regulated cell death in form of apoptosis and ferroptosis. in addition, the transcription factor atf4 is differentially translated, up-regulating genes participating in autophagy and other homeostatic pathways. atf activating transcription factor, chop cytidine-cytidine-adenosine-adenosine thymidine-enhancer-binding homologous protein, eif2α eukaryotic translational initiation factor 2α, er endoplasmic reticulum, ire1α inositol-requiring transmembrane kinase/endoribonuclease 1α, perk protein kinase-like eukaryotic initiation factor 2α kinase, upr unfolded protein response, xbp1 x-box binding protein 1. xbp1s, x-box binding protein 1 whereby the "s" stands for the spliced form of xbp1. sources: refs. [142, [153] [154] [155] [156] [157] [158] [159] [160] [161] residues on nascent peptides [151] . however, in conditions of er stress, bip is detached from these sensors allowing their activation to trigger pathways, collectively included in the term of upr. this stress response acts as a corrective path, capable of both increasing the er folding capacity and decreasing the incoming polypeptide load. of note, this downstream pathway of each of the three upr sensors appears to have an innate preference for a particular type of er stress. moreover, as reviewed [152] , upon dissociating from bip, each of the three sensors modifies the er to mitigate stress in its own unique way. for example, atf6 is often the first sensor to respond to er stress. once atf6 dissociates from bip, it is translocated to the golgi apparatus for cleavage. the cytosolic domain of atf6 is then free to move to the nucleus, where it moderates increased expression of several proteins involved in lipid biosynthesis and chaperones. this allows an increase in the volume of the er and provides more chaperone proteins to aid in folding, thus relieving some of the er stress. the other two sensors, ire-1 and perk, remain as integral er proteins but oligomerize and autophosphorylate following bip disassociation (autophosphorylation, a type of post-translational modification of proteins (see part vi, sect. 24.3); typical for this biochemical process, a phosphate is added to a protein kinase by itself). under remediable er stress conditions, the three sensors trigger signalling pathways to resolve er stress aiming at maintaining cellular integrity (fig. 18.6) . they are briefly touched in the following (for reviews and original articles, see refs [142, [153] [154] [155] [156] [157] [158] [159] [160] [161] [162] .). for example, misfolded proteins are dislocated in the cytosol where degradation processes such as the er-associated degradation (erad) and autophagy will clear them, thereby reducing their potential toxicity. characteristically, erad is a protein quality control mechanism conserved in all eukaryotic cells and represents a critical arm of the upr, necessary to alleviate er stress. the erad mechanism results in the selective dislocation of unfolded and misfolded proteins from the er to the cytosol via specific membrane machinery. the erad targets are subsequently degraded by the cytosolic ubiquitin proteasome system (ups). the whole process includes transcriptional activation of a variety of er-associated chaperones and folding enzymes which include but are not limited to bip and the lectins calr, calmodulin (cam), and calnexin (cnx). a pathway that represents the most conserved branch of the upr is mediated by ire-1, a multifunctional protein that possesses kinase and endonuclease activities. upon activation, ire-1 aggregates and autophosphorylates, thereby activating its endonuclease activity to catalyze the unconventional splicing of x-box binding protein 1 (xbp1) via removal of a 26-nucleotide intron. this processing event changes the open reading frame of the mrna, resulting in the translation of the transcription factor now termed xbp1s ("s" stands for the spliced form of xbp1) (for splicing, see box 18.4) . production of xbp1s leads to upregulation of several genes involved in the upr's adaptive phase, for example, expression of er-resident molecular chaperones and protein folding enzymes. activation of perk leads to the phosphorylation of eif2α that is required for the initiation of translation. this factor inhibits global protein synthesis by inhibiting the assembly of the 80s ribosome, thereby reducing er load and promoting cellular survival. at the same time and under these conditions, the transcription factor atf4 is differentially translated, up-regulating genes participating in protein folding, amino acid metabolism and transport, autophagy, and oxidative stress resistance/redox homeostasis. under conditions of prolonged or severe er stress that the upr cannot resolve (see below), atf4 also contributes to apoptosis through the induction of the transcription factor chop (for cytidine-cytidine-adenosine-adenosine-thymidine-enhancer-binding homologous protein) and by enhancing oxidative stress and protein synthesis. finally, the third branch of the upr is initiated by atf6. the sensor atf6 is retained at the er under homeostatic conditions but translocates to the golgi apparatus under er stress where it is cleaved by the golgi-resident proteases site-1 protease (sp1) and sp2. this event leads to the release of atf6 n-terminal fragment, a potent transcription factor that moves to the nucleus, where it binds the er stress response element upstream of a subset of upr genes to activate their transcription. together with xbp1, this fragment regulates the expression of several genes with functions in protein folding, protein transport, and lipid biosynthesis, that is, genes involved in re-establishing er homeostasis. strikingly, there is a considerable interconnectedness of the er stress-promoted upr with other innate immune processes. for example, an increasing number of studies support the view that oxidative stress has a strong connection with er stress. during the protein folding process, ros are produced as by-products, leading to impaired redox balance conferring oxidative stress. as the protein in molecular biology, splicing is a modification of an rna after transcription in which introns are removed and exons are joined. thus, an intron is any noncoding nucleotide sequence within a gene that is removed by rna splicing during maturation of the final rna product; an exon is any part of a gene that encodes a part of the final mature rna produced by that gene after introns have been removed by rna splicing. this process is needed for the typical eukaryotic messenger rna before it can be used to produce a correct protein through translation. for many eukaryotic introns, splicing is done in a series of reactions catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snrnps), but there are also self-splicing introns. folding process is dependent on redox homeostasis, the oxidative stress can disrupt the protein folding mechanism and facilitate the production of misfolded proteins, causing further er stress [163] . moreover, this stress response functions as a productive source of damps. typically, hsps and calr, like other er chaperones, can translocate to the cytosol and eventually to the surface of cells. once exposed, these molecules can operate as subclass ib-1 damps to facilitate engulfment of antigens as mostly described in the context of induction of icd [148, [164] [165] [166] [167] [168] . via these mechanisms and by crosstalk with other molecular machines of the innate immune system including nlrp3 inflammasome activation via txnip (see part iv, sect. 13.4.6.3 and part vi, sect. 22.4.2.2) , the upr may contribute to sterile inflammation and immunity (for articles, see refs [153] [154] [155] [156] [157] [169] [170] [171] [172] ). also, several signalling cascades triggered by the three sensors are apparently potent inducers of autophagy at a cellwide level that-as mentioned above-normally has an adaptive/protective function and consists of the three major types: chaperone-mediated, macro-and microautophagy. interestingly, recent evidence has indicated that upr-induced autophagic processes are capable of alleviating the upr pointing to a crosstalk between these two innate immune defense mechanisms [173, 174] . strikingly, a complex relationship reportedly exists between autophagy and damps in cellular adaption to stress and injury and cell death characterized by a crosstalk between autophagy induction and secretion or release of damps. in fact, growing evidence indicates that autophagic mechanisms are involved in regulating release and degradation of damps including calr, hmgb1, atp, and dna in several cell types [37, 148, 175] . this scenario may contribute to the observation that autophagy is also able to shape a supportive cellular immune response [176, 177] . this kind of innate immune interrelationships is, for example, involved in mechanisms of both reperfusion-mediated cell injury and icd of cancer cells (see refs [40, [178] [179] [180] [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] ). overall, depending on the duration and intensity of the stress, the upr engages different cellular pathways to restore and maintain cell survival, on the one hand, or trigger apoptosis, on the other hand. in cases of severe irremediable er stress, however, the balance is tipped in favor of pro-death signalling; that is, the upr, now mediated by different biochemical pathways, may lead to pro-inflammatory and pro-apoptotic responses resulting in catastrophic rcd (fig. 18.6) . while the precise pathways of apoptosis induced by er stress are not known, the up-regulated perk → eif2α → atf4 → chop pathway plays an essential role by reversing translational arrest, increasing generation of ros, and promoting calcium efflux from the er. together, these signals lead to cytochrome c release from mitochondria and loss of membrane potential, resulting in apoptosis [191] . in this context, it is interesting that one pathway of rn (ferroptosis; see sect. 19.3.3) apparently shares a partially overlapping machinery with er stress, suggesting a molecular interconnectivity between these two events [192] . the issue of er-stress-promoted upr is a parade example of an injury-induced innate immune response that can decide besides life and death of a cell. placed at the very beginning of defense processes of multicellular organisms upon injury, this stress response nicely reflects a hierarchy of damp emission (see part iv, chap. 16) . striking is also the existence of an inter-organelle communication, for example, between upr, inflammasome activation, and autophagic pathways which emerge as a homeostatic network determining the switch from adaptive life-saving programs to cell death under stress conditions, where specialized sentinels are localized at organelle membranes to induce the core apoptosis pathway. as briefly sketched in the next section, this innate immune defense response is not only directed against sterile stress but also against pathogen-mediated stress. in the previous section, induction of a upr was mainly exemplified by referring to reperfusion-mediated cell damage and icd of cancer cells, that is, instances of sterile stress. however, growing evidence is coming to light clearly indicating that viruses and bacteria also induce er stress, thereby activating a robust upr. in fact, the constitution of cellular stress responses is meanwhile regarded as the first line of defense against both viral and bacterial infection. however, the outcome is not always in favor of the host; the pathogen may also profit from this stress response, at least under certain circumstances. an increasing number of reports have recently been published on this emerging topic (such as refs [192] [193] [194] [195] [196] [197] ), the quintessence briefly being addressed here. there are several mechanisms described of how a virus can induce er stress. the central mechanisms of perturbation of the er during virus infection can be seen in the production of large amounts of viral proteins by the virus concerned. such accumulation of viral proteins in the er implies a challenge to the protein folding machinery which may cause er stress and, in turn, activate the upr resulting in restoration of the er homeostasis or apoptosis. so far, at least 36 viruses have been found to be able to induce er stress and activate the three upr stress signalling pathways [198] . moreover, er stress can be caused by viruses via other mechanisms, for example, as a result of er membrane exploitation, imbalance of calcium concentration, or sabotage/depletion of the er membrane during virion release. viral infections may activate these pathways resulting in the inhibition or promotion of viral replication. for example, the perk-mediated global translation shutdown is a very efficient antiviral mechanism, and a similar shutdown by pkr has been used in the interferon pathway to defend against viral infection [199] . also, the virus-related upr was found to trigger host inflammatory signalling cascade through innate immune signalling pathways that activate nf-κb and ap-1 transcription factors as a result of chronic er stress. in fact, overexpression of viral proteins in the er has long been known to activate these transcription factors which induce expression of pro-inflammatory cytokines such as il-6 [200, 201] . increasing evidence supports the notion that the upr signalling synergistically interacts with virus-induced signalling to produce inflammatory cytokines and type i ifns. in addition, other lines of studies have shed light on a role of nod1 and nod2 receptors in transducing virus-related er stress signals to elicit inflammation [197] . so far, however, a possible contribution of damps to the promotion of these signalling pathways has not been investigated. importantly, however, the effects of virus-induced upr have been observed not only to inhibit but also to potentiate viral infection. in fact, manipulation of the upr response has become an asset for many viruses to promote their translation, thereby leading to chronic er stress. in other words, during infection, viruses are capable of hijacking the host translational machinery and fill the er with viral proteins. for example, this is the case for many positive-strand rna viruses, which house the virus replication machinery in the protective er-membrane. in fact, viruses need host er to produce increased quantities of viral proteins to continue replication. intriguingly, as discussed [194] , many viruses have evolved strategies aimed at continuing the replication cycle. thus, viruses were shown to manipulate the host upr in various ways to stimulate protein synthesis capacity and to improve cell survival by inhibiting cellular apoptosis. in particular, the link between the upr and autophagy are intensely discussed to be involved in this scenario. these two systems may act dependently, or the induction of one system may interfere with the other [202] . thus, experimental studies could demonstrate that different viruses modulate these mechanisms to allow them to circumvent and bypass the host immune response or, worse, to exploit the host's defense to their advantage. according to current knowledge, rna viruses including influenza virus, poliovirus, coxsackievirus, enterovirus, japanese encephalitis virus, hcv, and dengue virus were shown to regulate these processes. for example, recent studies on hcv-infected hepatocytes confirmed the evidence that virus-associated er stress and upr are linked to cellular autophagy. thus, induction of the cellular autophagic response is reportedly required to improve survival of infected cells by inhibition of cellular apoptosis. moreover, the autophagic response was demonstrated to inhibit the cellular innate antiviral program that usually inhibits virus replication. nevertheless, as argued by the authors [194] , though hcv induces er stress and autophagy, their cause-effect relationship is not clear. notably, the upr is not only modulated by viruses! recent evidence indicates that this stress response plays multiple roles during bacterial infections as well. thus, as comprehensively reviewed by celli and tsolis [203] , the upr has been shown to be induced in murine lungs by m. tuberculosis (associated with apoptotic events) and also be correlated with helicobacter-induced gastric carcinogenesis. similarly, in vitro infectious models have revealed upr induction in macrophages and epithelial cells infected with either brucella melitensis and b. abortus or listeria monocytogenes. on the other hand, there is evidence suggesting that bacteria can subvert the upr for their own advantage. nevertheless, indications that bacteria can modulate this response are still somewhat sparse. one example refers to l. pneumophila that recruits components of the erad on its vacuole to mediate turnover of bacterial effectors on the vacuolar surface and uses the proteasome to generate amino acids necessary for its intracellular growth. interestingly, like with virus-induced upr, a bacteria-initiated upr may turn out to be of advantage for the host or in favor of the bacteria, but it is not clear in each case whether this response benefits the host or the pathogen. thus, as argued by celli and tsolis [203] , "modulation of er function during infection by intracellular bacteria can promote bacterial infection by providing a replicative niche, but at the same time the resulting disruption of the secretory pathway can provide a pattern of pathogenesis that aids the innate immune system in recognizing intracellular infection and in mounting an appropriate defence. however, considering the more rapid evolution of bacterial pathogens compared to their hosts, it is likely that bacteria have evolved to modulate the upr to their advantage during infection." the function of the er stress-induced upr appears to provide another impressive evidence in support of the notion that immunity is induced by both sterile and infectious stressful injury and not primarily by invading nonself. in fact, this stress response is at the forefront of any stressful injury and is dedicated to initiating involvement of further damp-promoted defense mechanisms. however, like all the other innate immune processes, the upr may become uncontrolled. together, this may be reason enough that this stress response is involved in the pathogenesis of many human systemic and organ-specific disorders. in fact, the list of human diseases that are pathogenetically associated with er stress and activation of the upr is steadily growing [204] [205] [206] . they include neurodegenerative and metabolic diseases, autoimmune disorders, and atherosclerosis. given such an ever-increasing list of diseases, it is not a surprise that chemical compounds and inhibitors targeting the upr signalling pathways with a high degree of specificity have been and will be further developed [207] [208] [209] . maintaining genome integrity and transmission of intact genomes is a condition sine qua non for cellular, organismal, and species survival [210] . this homeostatic integrity is threatened by dna damage that occurs in a variety of conditions including but not limited to ionizing radiation, chemical reactions, and viral infections, two of the most dominant conditions being oxidative stress and replication stress. these exogenous and endogenous factors induce diverse lesions in the dna such as nucleotide alterations (substitution, deletion, and insertion), bulky adducts, collapsed dna replication forks, ssbs, and dsbs (see refs [57, [211] [212] [213] [214] [215] [216] [217] [218] [219] ). in response to dna damage, cells initiate and activate a complex network of cellular signalling cascades that cooperate to sense and repair lesions in dna, denoted as the ddr. this stress response plays an important role in fighting against detrimental effects of cell stress and injury. it orchestrates many processes, including not only dna repair but also regulation of cell-cycle checkpoints, transcription of ddr genes, and autophagy ( fig. 18.7 ). the ddr is controlled by three pi3k-related kinases (pikks): the atm, the dna-pk, and the atr kinases (mostly nuclear proteins). all three pikks are enormous polypeptides with similar domain organizations and various common structural features. equipped with the capability to autophosphorylate (see above however, at the beginning of this dance, sensors have recently been identified which, as prms in a wider sense or even as a new group of recognition receptors, are capable of detecting dna damage-as manifested by the toxic dsbs or generation of ssdna to activate these three pikks. as already briefly touched in part ii, sect. 5.2.6.4, two highly conserved multiprotein complexes, mrn and ku, are considered the primary sensors of dsbs to subsequently activate atm and dna-pk [221, 227, 228] . in addition, ssdna is sensed by the recognition molecule rpa that then, analogous to ku, acts as a signalling and repair platform for downstream factors such as the prp19, an e3 ubiquitin ligase involved in pre-mrna splicing, and atr kinase [229] . other lines of studies have provided evidence suggesting that the protein prp19 itself may act as the primary sensor of rpa-ssdna to subsequently activate atr [224] (fig. 18.7) . in brief, upon dna damage, dsbs are recognized by the mrn complex. following recognition, mrn recruits atm to this dna lesion where it binds to the c-terminus of nbs1 as a component of mrn [230] . following binding, atm kinase is activated. however, the exact mechanism whereby mrn activates atm is still not fully understood (discussed in [226] ). recognition of dsbs is also realized by ku70/80 heterodimer that has been shown to bind broken dsdna ends preferentially [231] . it is the ku70/80 then that recruits the catalytic subunit of dna-pk (dna-pkcs) at the site of dsbs to form the dna-pk holoenzyme. the major role of activated dna-pk is to promote a peculiar dna repair mechanism called non-homologous end joining (nhej), a pathway that repairs double-strand breaks in dna [231] . in fact, nhej repairs most dsbs in mammalian cells. as its name implies, nhej involves ligation of two broken dna ends without needing a repair template [232] . in contrast to atm and dna-pkcs which respond primarily to dsbs, atr is activated by a much wider range of genotoxic stresses, for example, reflected by exposure of increasing amounts of ssdna as a consequence of compromised activity of replication proteins or nucleolytic processing of various forms of damaged dna. in fact, first evidence suggests that ssdna is initially sensed by rpa which then recruits and activates prp19 [229] . in turn, prp19 facilitates the accumulation of atr-interacting protein (atrip, the regulatory partner of the atr kinase) at dna damage sites, thereby activating atr [233, 234] . plausibly, the recent discovery of dna damage-sensing molecules such as mrn, ku70/80, and rpa calls for the definition of those molecules they recognize, that is, broken dna ends at the site of dsbs and ssdna. as outlined in part iv, sect. 13.4.2, they have been tentatively sorted into a subclass of cell-intrinsically emitted damps (subclass iic-1). future studies will have to assign their exact place in the world of damps. apart from those damps emitted in the nucleus, other lines of studies lend support to the suggestion that dna damage or dna replication stress, in case of unsuccessful dna repair, promotes release of aberrant dna structures into the cytosol in the form of ssdna and dsdna breaks/fragments which operate as cell-intrinsically emitted dislocated damps. they are sensed by the dna receptor cgas and probably other as-yet-not-identified dna receptors to activate sting-dependent pathways to promote defense pathways [235, 236] (compare part ii, sect. 5.2.6; part iv, sect. 13.4.3; as well as part vi, sect. 22.3.7). as discussed by the authors [236] , it is conceivable that cytosolic dna is released by dysfunctional mitochondria upon dna damage or generated during repair of damaged genomic dna. moreover, as shown in studies on tumor models, the ddr-through the activation of sting-mediated pathways-induces the expression of another class of constitutive damps which are exposed at the cell surface, namely, the mics and different ulbps [236] [237] [238] [239] [240] (compare part iv, sect. 12.3.3). of note, however, the ddr does not always result in a happy end. in fact, when unsuccessful in repairing dna damage, the ddr can lead to cellular senescence or-like a upr in case of irremediable er stress-ultimately induces an rcd, most often in the form of apoptosis, less frequently in the form of necrosis. such subroutines of rcd are presumably aimed at mitigating the propagation of potentially mutated cells leading to cancer or other age-related pathologies (fig. 18.7) [222, 223, 241]. the dna damage response must be regarded as an efficient cell-intrinsic defense process, which is sophistically connected with other stress responses such as autophagy that also plays a significant role in maintaining genome stability [242] and the er stress-induced upr [243] . also, of particular interest is the observation that three tiers of damps are involved in this pathway, starting with broken dna ends at the site of dsbs and ssdna in the nucleus immediately generated upon dna damage; followed by dna fragments dislocated in the cytosol and operating as class iic-2 damps to promote intracellular innate immune signalling; and finishing with the exposure of subclass ib-2 damps (e.g., mics) able to activate nk, nkt, and γδ t cells (compare part vii, sects. 27.2.2, 28.2.2 and 28.4.2). this phenomenon may again reflect a particular hierarchy in the work of damps to maintain homeostasis. in fact, genomic integrity is of utmost importance and key to human health, and this is retained by the ddr. this stress response, however, may fail in maintaining and restoring homeostasis. accordingly, dna damage has been observed to play a causal role in numerous human pathologies associated with genome instability or aberrant pikk function such as cancer, leukemia, premature ageing, and certain chronic inflammatory conditions. furthermore, there is growing evidence suggesting that uncontrolled ddr signalling is associated with various neurodegenerative diseases, as documented by recent work showing that atm inhibition alleviates pathologies in models of huntington's disease [244] . this is reason enough that ddr pathways have been and are being explored therapeutically to induce freedom from these diseases. in particular, small molecule inhibitors of atm, dna-pk, and atr are regarded as potential therapeutic agents, for example, as innovative drugs in cancer treatment [245, 246] . an increasing amount of publications in the international literature provides convincing evidence that cell-intrinsic, cell-autonomous stress responses are initiated by any damage to a cell, regardless of being of sterile or infectious in nature. the ultimate goal of those responses, which are all highly conserved among eukaryotic species, is to maintain cellular homeostasis and ensure cell integrity. again this knowledge is in support of the concept that any form of host defense is primarily directed against injury and not against microbes. autophagy in terms of "immunological autophagy" appears to act as the center of all stress responses that is committed to control and regulate efferent innate and potential adaptive immune responses. the 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the cytochrome bc1 complex key: cord-022354-aqtceqqo authors: hunter, eric title: membrane insertion and transport of viral glycoproteins: a mutational analysis date: 2012-12-02 journal: protein transfer and organelle biogenesis doi: 10.1016/b978-0-12-203460-2.50007-x sha: doc_id: 22354 cord_uid: aqtceqqo nan the eukaryotic cell faces a continual problem of partitioning a variety of enzyme activities into different subcellular organelles where specific macromolecular reactions can occur. this subcellular organization requires specific intracellular targeting of macromolecules through the cytoplasm, by as yet ill-defined mechanisms, and through the secretory pathway via a more clearly defined vesicular transport mechanism (sabatini et al. y 1982) . as obligate intracellular parasites with only limited genetic complexity, viruses must utilize the existing cellular transport mechanisms to colocalize their virion components in a part of the cell where assembly can take place. this problem of intracellular targeting is compounded for the enveloped viruses because they must transport capsid polypeptides through the cytoplasm and envelope components through the secretory pathway to a common point of assembly. since these viruses depend on the preexisting host cell processes and possess lipid envelopes that are biochemically similar to cellular membranes, they can provide ideal systems for probing the cellular mechanisms involved in glycoprotein biosynthesis and transport. the relatively simple structure of virions, the high level of expression of viral genes, the ease of molecularly cloning and manipulating those genes, together with the availability of conditional lethal mutants with defects in viral protein transport, confer several additional advantages for such studies. we have chosen an enveloped, rna-containing virus, rous sarcoma virus (rsv), for an analysis of viral glycoprotein biosynthesis and transport, because in addition to the aspects delineated above, the availability of molecularly cloned, infectious dna copies of the genome of this retrovirus has also allowed us to pose questions about the role of the glycoproteins in virus assembly and virus infectivity. like most simple enveloped viruses, the retroviruses consist of a host membrane-derived, lipid bilayer that surrounds (envelopes) a protein capsid structure (fig. 1) . the icosahedral capsid of rsv assembles as the virus particle buds from the plasma membrane of the cell, and so these two events are linked both temporally and spacially. glycoprotein knobbed spikes extend from the surface of the virion, and it is generally thought that during virus assembly a specific interaction between the glycoproteins and capsid (and/or "matrix" protein) is required, since cellderived polypeptides are for the most part excluded from the budding structure. the envelope glycoproteins span the lipid bilayer and are thereby divided into three distinct domains: an external, hydrophilic receptor-binding domain that functions in virus-cell attachment; a hydrophobic membrane-spanning domain; and a hydrophilic cytoplasmic domain. in rsv two polypeptides, gp85 and gp37, make up this structure (fig. 2) ; the external domain is primarily made up of the 341 amino acid long gp85 which contains regions that define the host-range and neutralization properties of the virus. the 198 amino acid long gp37 polypeptide, on the other hand, is a bitopic protein that anchors the envelope glycoprotein complex into the virion via disulfide linkages to gp85. it is less heavily glycosylated than gp85 (having only 2 versus 14 potential glycosylation sites) and contains two apolar regions in addition to the hydrophilic cytoplasmic domain. one apolar region is located near the amino terminus of gp37 and may be analogous to the fusion peptide of the hemagglutinin ha2 polypeptide of influenza virus that mediates viral entry into the cell. the hatched regions represent the highly hydrophobic signal and anchor sequences found at the n and c terminus, respectively. the location of a nonpolar region that may be analogous to the fusion peptide of ortho-and paramyxoviruses is shown by the stippled box. the aug at the start of the env orf was used to initiate translation of transcripts expressed in an sv40 vector (wills et ai, 1984) , but during a virus infection the genomic length transcript is spliced such that the aug and first 5 codons from gag (black box) are spliced into the env orf . in both cases the signal peptide is removed during translation, and cleavage of the polyprotein precursor to gp85 and gp37 occurs in the golgi at the basic tetrapeptide, -arg-arg-lys-arg-. branched structures denote potential n-linked oligosaccharide addition sites (asn-x-ser or asn-x-thr). (b) orientations of gp85 and pg37 in the viral membrane are depicted schematically. in the electron microscope this structure is seen as a spiked knob, where gp37 is the spike and gp85 is the knob. second apolar region in gp 37 consists of a 27 amino acid long stretch of hydrophobic residues near the carboxy terminus that functions during translation to stop the movement of the protein into the lumen of the rough endoplasmic reticulum (rer) and to anchor the complex in the membrane. the general orientation and structure of the rsv glycoprotein is thus similar to that of the influenza virus hemagglutinin (ha) (porter et al, 1979; gething et al, 1980) , the vesicular stomatitis virus (vsv) g protein (gallione and rose, 1983) , and several membrane-spanning cellencoded glycoproteins. the two viral glycoproteins of rsv are encoded by a single viral gene, env, and are translated in the form of a heavily glycosylated precursor polypeptide, pr95 e/ii; . since several processing and maturation events occur during the transport of the env gene products to the plasma membrane, they provide excellent markers for the subcellular compartments of the cell. a long (62 amino acid) amino-terminal signal peptide, which mediates translocation of the env gene product across the rough endoplasmic reticulum (rer), is removed cotranslationally from the precursor protein, and in the lumen of the rer 15-16 high-mannose core glycosylation units are added to the nascent pr95. the marked increase in molecular weight (mw) (approximately 40k) that results from the addition of this endo-ß-tv-acetylglucosaminidase h (endo h) sensitive carbohydrate provides a clear indicator for the translocation event. removal of glucose residues and some mannose moities appears to occur prior to transport of the protein to the golgi, where further trimming of the mannose residues and addition of glucosamine, galactose, and fucose are observed. cleavage of pr95 to gp85 and gp37 takes place after galactose and prior to fucose addition; it thus provides an excellent marker for trans-golgi locations. while the transit time of pr95 from the rer to golgi is quite long (t = 90 min) compared to other viral glycoproteins, movement through the golgi appears rapid and precludes any dissection of individual compartments within this organelle. these major biochemical modifications to the env glycoprotein coupled with sensitive immunological probes allow a fairly accurate mapping of the cell's secretory pathway. several viruses assemble at points within the secretory pathway, and since in many cases the location of the viral glycoproteins defines the virus maturation point (roth et al., 1983b; jones et al., 1985; gottlieb et al., 1986; gahmberg, 1984; kabcenell and atkinson, 1985) these systems are proving useful in investigating the signals that target proteins to specific subcellular locations. figure 3 is a schematic summary of the assembly points for the major groups of enveloped viruses. herpes simplex virus (hsv), a complex enveloped dna virus that encodes several glycoproteins (gb, gc, gd, ge) , buds into the nuclear envelope (spear, 1985) . virion glycoproteins synthesized on the rer appear to be transported to the nuclear membrane in an endo h-sensitive form where they are incorporated into nascent virions (compton and courtney, 1984) . it has been postulated that intact hsv virions traverse the secretory pathway thereby exposing the glycoproteins to the entire array of carbohydrate-modifying enzymes such that the mature virion contains glycoproteins with complex carbohydrate side chains (compton and courtney, 1984; spear, 1985) . a majority of the hsv glycoproteins can be found on the surface of infected cells, suggesting that targeting to the nuclear membrane is not absolute. expression of the cloned hsv gd gene in the absence of other viral components resulted in more rapid alphaviruses ortho-, paramyxoviruses transport to the plasma membrane and reduced accumulation on the nuclear membrane, indicating that interactions with other viral-encoded proteins may be required for normal nuclear membrane localization (johnson and smiley, 1985) . the corona-, flavi-, and rotaviruses have been reported to undergo assembly at the rer (dubois-dalcq et al, 1984) , but the rotaviruses appear to target this organelle most specifically. while the mature rotavirus is not enveloped, it contains a glycosylated capsid protein in its outer shell that is derived from a transient membrane during the assembly process. the inner and outer protein shells of these viruses form sequentially and by very different mechanisms. the inner capsids, containing the genomic rna segments, are the equivalent of nucleocapsids of other viruses and assemble in the cytoplasm at the edge of electron-dense inclusions called "viroplasm" (petrie et al, 1982) . they acquire a transient envelope, or pseudoenvelope, by budding at rer membranes adjacent to the viroplasm. further maturation of rotaviruses occurs within the cisternae of the rer where enveloped particles are converted to mature double-shelled virions and the lipid bilayer is removed by a process that remains to be elucidated (dubois-dalcq et al, 1984) . vp7, the glycosylated protein found in the outer capsid, has been shown to have carbohydrate structures consistent with its rer location and to target specifically to this organelle when expressed in the absence of other viral proteins from a recombinant expression vector (kabcenell and atkinson, 1985; poruchynsky et al, 1985) . the golgi body is the site of assembly for several viruses. coronaviruses, for example, mature by budding into the lumina of rer or golgi cisternae; virions form as the intracytoplasmic, helical neucleocapsids align under regions of intracellular membranes containing viral proteins. two glycoproteins, el and e2, comprise these membrane-associated polypeptides. e2 forms the large peplomers or spikes characteristic of coronaviruses and is a multifunctional molecule, being responsible for virus-induced cell fusion, binding of the virion to receptors on the plasma membrane of susceptible cells, and for inducing neutralizing antibody (dubois-dalcq et al, 1984) . el, in contrast, is an unusual polypeptide; it has only a short amino-terminal domain, which contains the glycosylation sites of the protein, and two long stretches of hydrophobic amino acids, suggesting that it may traverse the membrane more than once (dubois-dalcq et al, 1984; boursnell et al, 1984) . during infection, e2 can be transported through the secretory pathway to the plasma membrane, whereas el is transported only as far as the golgi apparatus, where it accumulates during the infection cycle (sturman and holmes, 1983) ; it has been suggested that this restricted intracellular movement of el ac-counts for the intracellular budding site of coronaviruses (sturman and holmes, 1983) . members of the bunyavirus family also mature intracellularly, by budding at the golgi complex (bishop and shope, 1979; dubois-dalcq et al., 1984) . by immunofluorescent microscopy, kuismanen and colleagues showed that in cells infected with uukuniemi virus the golgi region underwent an expansion and became vacuolized . both glycoproteins, gl and g2, accumulated in the golgi region during virus infection; neither polypeptide could be chased out of the golgi even after a 6-hr treatment with cycloheximide (gahmberg et al., 1986) , conditions that would allow complete transport of the semliki forest virus membrane proteins from the golgi (green et al., 1981a) . furthermore, the glycoproteins of a temperature-sensitive strain of uukuniemi virus were retained in the golgi even under conditions where no virus maturation took place and no nucleocapsids accumulated in the golgi region (gahmberg et al., 1986) . thus intracellular targeting of these viral components appears to be independent of other viral components and of the assembly process itself. moreover, it supports the concept that it is the glycoproteins themselves that dictate the cellular site of virus maturation. for several virus groups, virion assembly does not occur until the envelope components have traversed the entire secretory pathway. thus the ortho-and paramyxoviruses, rhabdoviruses, alphaviruses, and retroviruses mature at the plasma membrane. even these proteins, however, possess additional membrane-targeting information such that in polarized epithelial cells, where different cell proteins are inserted in the apical and basolateral membranes, the different viruses assemble from distinct membranes; for example, the ortho-and paramyxoviruses bud from apical membranes, rhabdo-and retroviruses from basolateral membranes (rodriguez-boulan and sabatini, 1978; herrler et al., 1981; roth et al., 1983a; rindler et al., 1985) . as with those viruses that mature at points within the secretory pathway, it is the glycoproteins themselves that appear to specify the specific plasma membrane location for virus assembly, since glycoproteins expressed from recombinant expression vectors are transported in a polarized fashion (roth et al., 1983b; jones et al., 1985; gottlieb et al., 1986; . the problem of polarized expression will be dealt with in more detail later in this chapter (see section ii, b, 4) . from the brief outline presented above it is clear that viral envelope components provide a plethora of systems for studying intracellular protein targeting. recombinant dna approaches, described below, are already providing information on the role of the different glycoprotein domains in this important aspect of viral and cell biology. in addition to utilizing the secretory pathway of vertebrate cells for transporting viral components to the point of virus maturation, several enveloped viruses take advantage of a second vesicle-mediated transport system, the endocytic pathway, to gain entry into susceptible cells. these viruses, such as orthomyxoviruses, rhabdoviruses, and togaviruses, in contrast to paramyxoviruses, such as sendai virus, which bind and fuse with the plasma membrane of the host cell, bind to the host cell surface and are subsequently internalized by endocytosis. this latter process serves an important role in the normal uptake of nutrients and in the internalization of receptor-bound ligands such as hormones, growth factors, lipoproteins, and antibodies (mellman et al., 1986; hopkins, 1983) . bound virions are carried into clathrin-coated pits, which form continually on the surface of the cell, and which fold inward and pinch off into the cytoplasm to form "coated vesicles." as the coated vesicle moves into the cytoplasm, it loses its clathrin and fuses with an endosome, a large acidic vacuole with a smooth outer surface. for viruses entering by this pathway, membrane fusion occurs in the endosomal compartment (marsh, 1984; yoshimura and ohnishi, 1984) . fusion is triggered by the mildly acidic endosomal ph and is catalyzed by the virally encoded glycoproteins which undergo a low ph-dependent configurational change (skehel et al., 1982; kielian and helenius, 1985) . the ph dependence of fusion varies among virus types, with the optimal ph for fusion generally falling within the range of ph 5.0-6.2 for endocytosed viruses (white et al., 1983) . in order to obtain an understanding of the molecular mechanisms involved in these low ph-induced fusion reactions, virus mutants have been isolated which fuse with ph optima different from those of their respective parents. through the use of an elegant selection scheme, in which mutagenized virus was allowed to fuse with nuclease-filled liposomes at a ph below 6.0, kielian et al. (1984) isolated the first such fusion mutant of semliki forest virus. this virus, fus-1, fused at a ph optimum 0.7 ph units lower than that of the wild type (ph 5.5 versus 6.2). the mutant was, nevertheless, fully capable of infecting cells under standard infection conditions and even under conditions that prevent fusion of endosomes with lysosomes. on the other hand, the fus-1 mutant showed increased sensitivity to lysosomatropic agents that increase the ph in acidic vacuoles of the endocytic pathway. in addition to proving that alterations within viral structural components can significantly affect the ph at which virus-induced fusion can occur, these results showed that a ph below 5.5 exists within the endosomal compartment and thereby demonstrated the usefulness of mutant viruses as biological ph probes of this pathway. in parallel studies, rott and co-workers (1984) have shown that variants of the x31 strain of influenza virus, selected for their ability to undergo activation cleavage and growth in madin-darby canine kidney (mdck) cells, have an elevated fusion ph threshold (approximately 0.7 ph units higher than the wild type). similar virus variants have been selected by growth of influenza virus in the presence of amantadine, a compound that raises endosomal ph (daniels et al., 1985) . in this latter study, viruses were obtained that fused at ph values 0.1-0.7 units higher than the parental strain. analogous mutants have been reported to occur naturally within stocks of the x31 strain of influenza virus (doms et al., 1986) . such mutants should provide useful probes for elucidating the endocytic pathway. the mechanisms by which cells send membrane-bound and secreted proteins to their proper subcellular locations remain a central problem in cell biology. it has been postulated to involve the specific interaction of "sorting signals," located within the structure of the newly synthesized proteins, with membrane-bound receptors in the rer and golgi apparatus of the cell (for review, see sabatini et al., 1982; silhavy et al., 1983) . this concept is supported by the facts that cell, as well as viral, glycoproteins can be retained at or targeted to specific points in the secretory pathway and that cells can transport and secrete a variety of glycosylated and nonglycosylated proteins at distinctly different rates (strous and lodish, 1980; fitting and kabat, 1982; gumbiner and kelly, 1982; ledford and davis, 1983; , kelly, 1985 . very little is known about the composition(s) or indeed the exact role of sorting signals, but it is generally thought that they are composed of protein. clearly, the initial step that introduces polypeptides into the secretory pathway is mediated by the interaction of a sequence of amino acids (the signal sequence) within the polypeptide and the signal recognition particle (srp)/docking protein (dp) complex (blobel and dobberstein, 1975; blobel, 1980, 1981a,b; walter et al., 1981; meyer and dobberstein, 1980; meyer et al., 1982; gilmore et al., 1982a,b; . mutants of secreted proteins that are defective in later stages of transport have been identified that differ from the wild-type forms by one (mosmann and williamson, 1980; wu et al., 1983; shida and matsumoto, 1983) or two (yoshida et al., 1976; hercz et al., 1978) amino acid substitutions, supporting the concept that sorting signals are composed of protein. also, several conditional transport-defective mutants of membrane-bound viral glycoproteins have been identified (for example, knipe et al, 1977b; zilberstein et al, 1980; pesonen et al, 1981) . studies using tunicamycin, an inhibitor of glycosylation, suggest that carbohydrate moieties are not recognized directly by the sorting machinery but may be important for maintaining the proper secondary or tertiary structures of (protein-composed) sorting signals (struck et al., 1978; gibson et al, 1978 gibson et al, , 1979 leavitt et al, 1977; hickman et al, 1977; roth et al, 1979; strous et al, 1983; green etal, 1981b) . the lack of a direct role for carbohydrate moieties in the sorting process is perhaps to be expected in view of the fact that many secreted proteins are not glycosylated at all (for example, strous and lodish, 1980; underdown et al, 1971) . nevertheless, addition of carbohydrate to molecules that are unable to be transported through the secretory pathway can release the block to their transport (guan et al, 1985; machamer et al, 1985) . in addition, the transport of certain hydrolases to the lysosome (and away from the secretory pathway) does appear to require the addition of a carbohydrate moiety (mannose 6-phosphate) (hasilik and neufeld, 1980; sly and fisher, 1982; creek and sly, 1984) , but these additions in turn must require the recognition of signals within the polypeptide chains. even less is known about the intramolecular location(s) of sorting signals. in the case of the membrane-spanning glycoproteins, three protein domains exist which together or separately may harbor sorting signals: (1) the internal or cytoplasmic domain, (2) the hydrophobic or transmembrane domain, and (3) the extracytoplasmic or external domain. since most secreted proteins (which may be cotransported with membranebound glycoproteins, strous et al, 1983) possess only external domains, it might be reasonable to expect the transmembrane and cytoplasmic domains to be unimportant to the sorting process. we have tested this hypothesis by introducing genetic lesions into the gene encoding the envelope glycoproteins of rsv (wills et al, 1983 (wills et al, , 1984 hardwick et al, 1986; , as have others for the vsv g protein bergmann, 1982, 1983; rose et al, 1984; adams and rose, 1985a,b) , the influenza virus hemagglutinin protein (sveda et al, 1982 gething and sambrook, 1982; doyle et al, 1985 doyle et al, , 1986 gething et al, 1986) , and the glycoproteins of semliki forest virus (garoff et al, 1983; garoff, 1985; . genetic analyses of protein transport in prokaryotic systems have provided both support for the role of the signal peptide in protein translocation and valuable insights into the polypeptide interactions that are required for the intracellular targeting of bacterial secreted and membrane proteins (reviewed by michaelis and beckwith, 1982; silhavy et al, 1983; benson et al, 1985; oliver, 1985) . while similar experiments are more difficult to perform in eukaryotic cells with the enveloped virus systems described here, both the classic and molecular genetic approaches outlined below are providing information on the role of different protein domains in the transport process. during the genetic analysis of enveloped virus replication through the isolation and biochemical characterization of spontaneous and mutageninduced variants, complementation groups were established for several viruses that contained mutants defective in normal transport of the viral glycoproteins (knipe et al., 1977a,b; zilberstein et al., 1980; pesonen et al, 1981; gahmberg, 1984; ueda and kilbourne, 1976) . the existence of conditional lethal mutants that were blocked at different stages of virus glycoprotein maturation suggested that the viral polypeptides themselves might contain the signals necessary for normal sorting by the cells' transport machinery and raised the possibility that such mutants could be used tq dissect the maturation pathway of a glycoprotein. since it is impossible in this chapter to provide a detailed review of the characterization of mutants in each of these systems, and since the transport of the influenza virus glycoproteins has recently been discussed in detail by , this section will concentrate primarily on mutants of the vsv g protein gene as an example of these approaches. temperature-sensitive (ts) mutants in complementation group v of this vsv have defects in the structural gene for the viral glycoprotein, g, and cells infected at the nonpermissive temperature with such mutants produce markedly reduced yields of virus like particles which are noninfectious and specifically deficient in g protein. at the nonpermissive temperature the mutant g polypeptide is synthesized normally; however, it does not accumulate on the cell surface, nor is it incorporated into virions (knipe et al., 1977b; zilberstein et al., 1980) . the ts(v) mutants can be subdivided into two subclasses with respect to the stage of posttranslational processing at which the block occurs (zilberstein et al., 1980) . three mutants, tsl5\3, tsm50l, and ts045, encode g proteins that at the nonpermissive temperature are blocked at an early, pre-golgi step of the secretory pathway. while insertion into the er membrane, removal of the amino-terminal hydrophobic signal sequence, and addition of the two n-linked high-mannose core oligosaccharides occur in a way that is indistinguishable from the wild type, all subsequent golgi-mediated carbohydrate processing reactions are blocked (zilberstein et al., 1980) . these results are consistent with subcellular fractionation and immunoelectron microscopy studies which indicated that the g protein in tam501 or ta045-infected cells was arrested in its transport from the rer to the golgi complex at the nonpermissive temperature (zilberstein et al., 1980; bergmann et al., 1981; bergmann and singer, 1983) . the defect in transport in these mutants is a reversible phenomenon, thereby excluding irreversible denaturation as the basis for lack of movement; proteins synthesized at the nonpermissive temperature rapidly move by stages to the plasma membrane upon shift to the permissive temperature bergmann and singer, 1983) . within 3 min after shift to 32°c, g protein of tsoa5 could be seen by immunoelectron microscopy at high density in saccules at one face of the golgi complex and by 3 min later was uniformly distributed through the complex (bergmann and singer, 1983) . movement of the mutant proteins to the cell surface occurred rapidly and was accompanied by incorporation into virions . the second class of ts mutants of vsv is represented by tal511. g protein encoded by this mutant is transported normally through most of the golgi-mediated functions involved in the processing of carbohydrate side chains, including addition of the terminal sialic acid residues. however, this molecule does not undergo two posttranslation modification reactions that take place with the wild-type g protein. the first is the addition of fucose to the tel511 oligosaccharide chains, which is reduced at both permissive and nonpermissive temperatures (zilberstein et al., 1980) . in the second, a molecule of palmitic acid (a 12-carbon fatty acid) is covalently attached to the wild-type g polypeptide near the membranespanning region rose et al., 1984) . attachment occurs at a late stage of maturation, just before oligosaccharide processing is completed (schmidt and schlesinger, 1980) , probably in the eis compartment of the golgi complex (dunphy et al., 1981) . this modification of the g protein does not occur at the nonpermissive temperature in cells infected with the tsl5ll mutant. taken together with the almost complete processing of the mutant's oligosaccharide side chains, this suggests that at the nonpermissive temperature the asl511 glycoprotein accumulates at a specific region within the golgi complex. the mutants of vsv, together with equivalent mutants in other viral systems that were blocked at different stages of the secretory pathway (pesonen et al., 1981; gahmberg, 1984; kuismanen et al., 1984) , raised the possibility of identifying and understanding the nature of sorting signals in secreted polypeptides. with the advent of recombinant dna and rapid nucleotide sequence techniques, it has been possible to determine at the amino acid level the basis for these defects (gallione and rose, 1985; arias et al., 1983) , but the interpretation of this information with regard to protein transport has been less than straightforward. gallione and rose (1985) determined the nucleotide sequence of the ts045 mutant of vsv and compared it to that of the parent and a wildtype revertant. the mutant and revertant differed in three amino acid residues, and through the construction and expression of hybrid genes it was possible for these investigators to demonstrate that the basis of the temperature-sensitive phenotype was a single amino acid change of phenylalanine to serine. since this polar substitution occurred within a very hydrophobic region of the g protein, it was suggested that it might significantly affect protein folding in this region such that reversible denaturation of the protein might occur at the nonpermissive temperature. this denaturation could prevent further transport to the golgi apparatus and the cell surface. alternatively, gallione and rose (1985) pointed out that the conformational change at the nonpermissive temperature might be more subtle, perhaps preventing recognition by a component of the protein transport machinery. however, since hydrophobic residues are generally buried within a protein (kyte and doolittle, 1982) , it is unlikely that the mutated sequence itself would play a direct role in such an interaction. nothing is known about the 3-dimensional structure of the g protein or whether accessory proteins are involved at this stage of protein transport, thus both suggestions remains viable possibilities. a similar analysis has been carried out by arias et al. (1983) , who sequenced the genes encoding the viral glycoproteins of tslo and ts23, mutants of sindbis virus defective in the intracellular transport of their glycoproteins, and of revertants of these mutants. these investigators found ts23 to have a double mutation in glycoprotein el, while ts was a single mutant in the same glycoprotein. in each case reversion to temperature insensitivity occurred by changes at the same site as the mutation, in two cases restoring the original amino acid and in the third case substituting a homologous amino acid (arginine in place of lysine). since the three mutations were far apart from each other in the protein, these authors concluded that the 3-dimensional conformation of el was very important for the correct migration of the glycoproteins from the er to the plasma membrane. similarly, two ts mutants in the ha gene of influenza virus that result in ha protein transport being arrested in the rer are also caused by single point mutations that probably disrupt the tertiary structure of the molecule (nakajima et al., 1986) . in summary, several conditional mutants have been isolated by classic genetic approaches that at the nonpermissive temperature disrupt the normal transport of viral glycoproteins through the secretory pathway. while these mutants carried the promise of defining specific protein domains that might interact with components of the transport machinery, the evidence from the nucleotide sequencing experiments outlined above suggests that many or all of the mutations may exert their phenotype through distortion of the 3-dimensional shape of the molecule. while this mechanism does not preclude a role for specific protein-protein interactions in the secretory pathway, it provides no direct evidence for it at this time. the recent development of cdna cloning, gene sequence manipulation, and gene expression technologies has opened up new approaches for localizing and characterizing those structural features of a protein that act as sorting signals. these new methodologies have allowed investigators to delete or modify potentially important structural regions of a protein at the nucleotide level and then to determine the effect of such changes by expressing modified genes in suitable eukaryotic expression vectors. furthermore, in some instances it has been possible to test directly the functional role of a particular peptide region by fusing it to another protein and analyzing the behavior of the chimera. since these general approaches to protein sorting have been reviewed recently (garoff, 1985; gething, 1985) , this chapter will describe our recent recombinant dna analyses of the biosynthesis and transport of the rsv envelope glycoproteins, within the context of similar analyses in other viral systems. as we have discussed earlier, the rsv env gene encodes two viral glycoproteins, gp85 and gp37, that mediate recognition of, attachment to, and penetration of the susceptible target cell. these proteins are synthesized as a glycosylated precursor protein, pr95, that is proteolytically cleaved in the golgi complex. the coding sequences for gp85 and gp37 have been placed in an open reading frame that extends from nucleotide 5054 to nucleotide 6863, and predict sizes of 341 amino acids (40,000 mw) for gp85 and 198 amino acids (21,500 mw) for gp37 ( fig. 2) . carbohydrate makes up a significant contribution to the observed molecular weights of these polypeptides-the predicted amino acid sequence contains 14 potential glycosylation sites (asn-x-ser/thr) in gp85 and 2 in gp37. experiments aimed at determining the number of carbohydrate side chains yielded results consistent with most or all of the sites being occupied . although an initiation codon is located early (codon 4) in the open reading frame, during a viral infection splicing yields an mrna on which translation initiates at the same aug as the gag gene to produce a nascent polypeptide in which gp85 is preceded by a 62 amino acid long leader (signal) peptide (fig. 2) . this peptide contains a hydrophobic sequence that we have shown (see below) is necessary for translocation across the rer and is completely removed from the env gene product during translation . it represented one of the longest signal peptides described to date, and we were therefore interested in determining the signal peptide requirements for normal biosynthesis of gp85 and gp37. for these studies the env open reading frame was excised from the rsv genome and inserted into an sv40 expression vector under the control of the late-region promoter (wills et al., 1983; . in this construction translation is initiated at the aug present at the start of the open reading frame (at nucleotide 5054 of the rsv genome) and results in the synthesis of an even longer (64 amino acid) signal peptide; nevertheless, biosynthesis of pr95 and signal peptide cleavage occur normally. furthermore, expression of the rsv env gene in african green monkey (cv-1) cells parallels that seen in a normal virus infection in avian cells (wills et al., 1984; hardwick et al., 1986) , making it an excellent system for the analysis of mutant env genes. a. deletion/substitution of the signal peptide. in order to examine the role of the signal peptide in rsv glycoprotein biosynthesis we constructed a series of deletion mutations within the 5' coding region of the env gene using the double-stranded exonuclease babl. oligonucleotide linkers of the sequence catcgatg were ligated to the ends of the truncated molecules to introduce a unique restriction endonuclease cleavage site and to replace the deleted in-frame aug. the mutants were then sized and their nucleotide sequence determined to find those with a suitable deletion and an in-frame aug. one such mutant, al, contained a deletion of 171 nucleotides within the env coding sequences and encoded an env product that completely lacked an aminoterminal hydrophobic sequence (fig. 4) . expression of this gene from the sv40 vector resulted in the synthesis of a nonglycosylated, 58 kda cytoplasmic protein that was similar in size to the nonglycosylated wild-type env gene product produced in the presence of the glycosylation inhibitor, tunicamycin. in contrast to the tunicamycin product, however, the al protein was not associated with membrane vesicles and was rapidly degraded (half-life < 5 min; e. hunter, k. shaw, and j. wills, unpublished) . thus the signals for initiating translocation of the rsv env gene product must reside within the cotranslationally removed amino-terminal sequence, and in their absence the molecule is synthesized as an unstable cytoplasmic protein. this result is similar to those obtained by gething and sambrook (1982) and by sekikawa and lai (1983) with the influenza virus ha gene product. influenza hemagglutinin sequences are depicted by italicized text. underlined text denotes amino acid residues encoded by oligonucleotide linkers. arrows depict the signal peptidase cleavage site at which the signal is removed cotranslationally from each of the constructs. plus symbols indicate that translocation, glycosylation, or transport to a plasma membrane location is observed; minus symbols mean that the properties above are not observed. since the signal peptide of the rsv env gene product is exceptionally long, it was of interest to determine whether another signal peptide could substitute for it. for these experiments we have utilized the signal sequences of the influenza virus ha gene (a/jap/305/57; gething et al., 1980) . two constructions were made: in the first of these the al deletion mutant coding sequence was fused in-frame to the ha signal coding sequence at the signal peptidase cleavage site of the latter (fig. 4) and in the second, we made use of a sail restriction enzyme site in the ha gene and an xhol site in the rsv env gene, so that the signal sequence and 16 amino acids of ha1 were fused with env 6 amino acids into gp85 (fig. 4) . expression of these hybrid genes in cv-1 cells resulted in the biosynthesis of a glycosylated pr95 protein that was transported to the golgi complex, cleaved to gp85 and gp37, and displayed on the cell surface (e. hunter, k. shaw, and j. wills, unpublished) . to demonstrate that the pr95 molecules expressed from the ha-a1 fusion gene had undergone signal peptide cleavage, pr95 was immunoprecipitated from [ 3 h]leucine, pulse-labeled cells and analyzed by sequential edman degradation, in order to determine the amino-terminal sequence. to our surprise the signal peptidase had cleaved at the ha cleavage site, despite the fact that according to the analyses of von heijne (1983) only the rsv cleavage sequence should have been recognized. thus the h a -al fusion protein contains two potential signal peptidase cleavage sites (that from the ha and that remaining in the env sequences), but only the first of these is utilized. both gene fusions, therefore, result in the synthesis of aberrant gp85 proteins-that from the ha-a1 fusion having an 8 amino acid amino-terminal extension, and that from the sallxho fusion having lost 6 amino-terminal amino acids and gained 16 from ha1which nevertheless can be transported to the plasma membrane (wills et al., unpublished data) . reciprocal gene fusions, in which the env gene signal peptide was fused to the structural sequences of ha (fig. 4) , also resulted in translocation of the ha molecule across the rer membrane, supporting the concept that this transient sorting sequence is not polypeptide specific. however, only in the construction where the signal sequence of env was precisely fused to the amino terminus of ha1 was transport beyond the rer observed (wills et al., unpublished data) . in constructions where the amino-terminal sequence of ha1 was perturbed, the recombinant protein was apparently prevented from assembling into trimers and its transport was blocked in the rer . thus, while signal peptides may be capable of mediating the translocation of foreign polypeptides across the rer, other sorting "signals" must be active for transport of the molecule to continue. site. the experiments described above have been extended to determine the following: (a) whether the hydrophobic region of the signal peptide carries all the information required for transfer of the env gene product into the rer; (b) what the structural specificities of the signal peptide are; and (c) where the specificity for signal peptidase cleavage is located. more than 200 prokaryotic and eukaryotic signal peptides have been sequenced (watson, 1984) . comparison shows that most extensions comprise 20-40 amino acid residues; one of the longest being that of the rsv envelope glycoprotein. there is no homology between sequences, but a characteristic distribution of amino acid chains is observed. three structurally distinct regions have been observed so far: a positively charged aminoterminal region, a central region of 9 or more hydrophobic residues, and a more polar carboxy-terminal region that appears to define the cleavage site (von heijne, 1983 (von heijne, , 1984 (von heijne, , 1985 perlman and halvorson, 1983) . the importance of these general features has been supported by the genetic studies in prokaryotic systems (reviewed by silhavy et al., 1983; benson et al, 1985) . to investigate these questions we initially constructed a series of internal deletion mutants that initiated within the amino-terminus of gp85 and extended into the signal peptide. the deletion mutations were introduced into the coding region for the envelope glycoprotein by digestion of a plasmid containing the env gene at a unique xhol site located 13 base pairs (bp) from the 5' end of the coding sequence for gp85, followed by digestion with the double-stranded exonuclease bal3l. potential mutants were identified by restriction enzyme analysis and dna sequencing, and those of interest were engineered into the sv40 expression vector. these are depicted in figs. 5 and 6. mutants x4-a -b, and -c were derived from a single out-of-frame parent, and they represent a nested set of mutants in which the hydrophobic sequence varies from the wild-type length of 11 to only 9 amino acids. expression of these mutant genes in cv-1 cells gave the results summarized in fig. 5 . mutant polypeptides with the shortest hydrophobic domain (x4-c) resembled the al mutant polypeptides in that they had a cytoplasmic location, were nonglycosylated, and were rapidly degraded. they differed from the al mutant in length (65 versus 58 kda) confirming that the mutated signal was not removed. mutant x4-a polypeptides, on the other hand, were translocated and glycosylated with an efficiency equivalent to wild type, despite the substitution of serine and isoleucine for leucine and cysteine residues within the hydrophobic domain. mutant x4-b expressed a phenotype intermediate between that of x4-a and x4-c, approximately 50% of the polypeptides being translocated and glycosylated. none of the mutants contain the sequences that specify the signal peptidase cleavage site, and molecules of x4-a/x4-b that were translocated across the rer retained an uncleaved signal peptide. the data from these mutations suggest the following: (1) that the length, rather than the amino acid composition, of the hydrophobic domain of the env signal peptide is critical for translocation across the rer and (2) that signal peptide cleavage is not a requirement for translocation. the first of these conclusions is supported by genetic experiments in prokaryotic systems, where a requirement for secondary structure in the signal peptide was suggested bankaitis et al., 1984) . the second is consistent with the presence of permanent insertion sequences in secreted and membrane-spanning proteins that are translocated across the rer membrane without removal of an amino-terminal signal peptide (palmiter et al., 1978; bos et al., 1984; markoff et al., 1984; zerial et al., 1986; spiess and lodish, 1986) . several membrane-spanning proteins are anchored in the membrane by an amino-terminal anchor/signal domain and display what is termed group ii protein topology (garoff, 1985; wickner and lodish, 1985) , where the amino terminus of the protein is cytoplasmic and the carboxy terminus is luminal. after translocation of a nascent chain across the endoplasmic reticulum has been initiated, the signal peptide is removed. this cleavage is carried out by signal peptidase, a cellular gene product. two classes of signal peptidases have been described. a signal peptidase of escherichia coli (spase i) has been cloned into pbr322 (date and wickner, 1981) , and has been shown to accurately cleave eukaryotic precursor proteins as well as bacterial protein precursors (talmadge et al., 1980) . conversely, the eukaryotic signal peptidase will accurately cleave prokaryotic proteins (watts et al., 1983) . the latter enzyme has been studied, using detergentsolubilized dog pancreas signal peptidase (jackson and white, 1981) and hen oviduct signal peptidase (lively and walsh, 1983) , and been demonstrated to be an integral membrane protein that can be solubilized only when the lipid bilayer is dissolved. a second prokaryotic signal peptidase (e. coli spase ii) has been described that is specific for prolipoproteins (hussain et al., 1982; tokunaga et al., 1982) and membrane-bound penicillinases (nielsen and lampen, 1982) . this enzyme maps to a different locus on the e. coli genome and requires a glyceride-modified cysteine for cleavage. perlman and halvorson (1983) and von heijne (1983) have examined sequences of a number of membrane proteins and have described amino acid sequence patterns that allow prediction of signal peptidase cleavage sites with greater than 90% accuracy. the most striking feature of signal peptidase cleavage sites is the presence of an amino acid with a small, uncharged side chain at the carboxy terminus of the signal peptide. the most common amino acids found at this position are alanine and glycine. from statistical analyses, the peptidase cleavage site appears to be determined by sequences within the signal peptide and not by sequences beyond the cleavage site. this is in contrast to the observations that mutations within the structural protein itself prevent signal peptidase cleavage of the lamb gene product and the m13 coat protein (emr and bassford, 1982; benson and silhavy, 1983; rüssel and model, 1981) . to investigate this question with regard to the rsv env gene product, the deletion mutants shown in fig. 6 were constructed as described above. mutant xi has a 14 amino acid deletion encompassing residues 4-17 of gp85, which results in the loss of one potential glycosylation site. this deletion resulted in the synthesis of a slightly smaller precursor polypeptide that lacked one carbohydrate side chain but was otherwise glycosylated normally. based on size estimations in pulse-labeling experiments, in the presence and absence of tunicamycin, the precursor polypeptide lacked the long, signal-containing leader peptide. thus, although the mutation in xi significantly alters the sequences near the signal peptidase site, the signal peptidase still recognized and removed the signal peptide. in mutants x2 and x3 the amino-terminal nine and six amino acids, respectively, of gp85 are deleted. they therefore encode gp85 polyxhol site in dna r\ 5 | 10 15 °| asp val his leu leu glu gin pro gly asn leu trp ile thr trp ala asn arg . 1 fig. 6 . amino acid sequence deduced from dna sequence of mutants xi, x2, and x3. the rsv glycoprotein is schematically represented: the location of the hydrophobic signal sequence within the long (64 amino acid) leader peptide is denoted by a stippled bar, and the mature gp85 glycoprotein by a hatched bar. the signal peptidase cleavage site in both the cartoon and the numbered amino acid sequence is denoted by a long arrow. the potential glycosylation site in the amino terminus of gp85 is shown as a cho. the amino acid sequence of the last 7 amino acids of the signal peptide and the amino-terminal 18 amino acids of gp85 are shown for the wild-type gene product. the solid black bars show the lengths and positions of the deletions in mutants xi, x2, and x3. peptides with novel amino termini that alter the signal peptidase cleavage site from ala/asp-val-his to ala/asn-leu-trp and ala/gln-pro-gly, respectively. thus both the charge and secondary structure of the cleavage site would be predicted to be altered by the loss of asp and his (x2) and by the relocation of proline near the cutting site (x3). nevertheless, the signal peptidase efficiently cleaved the leader peptide from the nascent polypeptide, and its specificity of cleavage was unaffected by these alterations (hardwick et al, 1986) . these experiments then support the generalized conclusion from statistical analysis, that the sequence to the right of the signal peptidase site is not critical for signal peptidase cleavage. none of the deletions generated in the rsv glycoprotein gene resulted in a loss of recognition and cleavage by the signal peptidase. this result contrasts with previously described prokaryotic mutants. a 12 amino acid deletion starting at the fifth residue beyond the signal peptidase site of the lamb gene product blocked cleavage of the signal (emr and bassford, 1982; emr et al, 1981) , and a deletion of 130 amino acids beginning 70 amino acids downstream from the signal abolished signal cleavage although the shortened protein was localized correctly . in addition, the substitution of a leucine, in place of the glutamic acid, at residue 2 of the mature m13 coat protein also inhibited signal peptidase cleavage; however, in this latter instance the procoat protein was transported inefficiently across the inner membrane (boeke et al., 1980; rüssel and model, 1981) . although more mutants will be required to properly define these systems, the prokaryotic cleavage site appears to be more sensitive to manipulation than that of eukaryotes. there is accumulating evidence that transported prokaryotic proteins, unlike those of eukaryotes, may not be transferred across membranes in a strictly cotranslational manner (randall and hardy, 1984) . thus, altered regions within the structural protein portion of a molecule would have the opportunity to interact and interfere with signal peptidase cleavage; such as interaction would not be possible in the cotranslational system described for eukaryotes. although the mutant xi polypeptides were translocated across the rer membrane in a normal fashion, immunofluorescence experiments and posttranslational modification probes indicated that the transport and maturation of the xi glycoprotein was halted shortly after exiting the rer, perhaps within pre-or cis-golgi vesicles. cells synthesizing the mutant protein showed no surface immunofluorescence, no cleavage of the pr95 to gp85/gp37, and no terminal sugar additions (hardwich et al., 1986) . the basis for this block appears to be the altered amino acid sequence rather than the loss of the carbohydrate side chain since by using a mutagenic oligonucleotide we have modified the amino terminus of the xi gp85 from asp-val-his-arg-thr-to asp-val-asn-arg-thr-, thereby reinserting the glycosylation site missing from this mutant. the derivative mutant, x1a, is glycosylated at this site but remains blocked at the same intracellular point in the secretory pathway (k. shaw, k. kervin, and e. hunter, unpublished) . a second deletion mutant of the rsv env gene is also blocked in intracellular transport. this mutant, c3, has an engineered deletion at the carboxy terminus of gp37 that removed the cytoplasmic tail and transmembrane region (see below and wills et al., 1984) . its transport is clearly blocked at an earlier stage than that of the xi mutant since it was localized to the er and never reached the golgi apparatus, whereas by immunofluorescent staining of fixed cells the xi protein appeared to colocalize with the golgi complex (see fig. 7 ; hardwick et al., 1986) . although the xi and c3 mutants contain alterations at opposite ends of the env gene product, they both appear to lack an element that normally signals their transport to and beyond the cis-golgi. while there may be a specific amino acid sequence (analogous to the amino-terminal signal sequence) that is required for these transport steps, it is as likely that a correctly aligned tertiary structure is the critical factor. just as small changes as the amino terminus of ha1 can disrupt assembly and transport un rhodamine fig. 7 . intracellular immunofluorescence of cells expressing wild-type and mutant polypeptides. fixed infected cv-1 cells were stained to detect the intracellular localization of wild-type and mutant glycoproteins. rabbit anti-glycoprotein antibodies were tagged with fluorescein-conjugated goat anti-rabbit antibodies which in wild-type and xi-infected cells could be localized on the nuclear membrane (nm), endoplasmic reticulum (er), and golgi apparatus (g). the golgi was localized by staining the same cells with rhodamine-conjugated wheat germ agglutinin. in mutant c3-infected cells neither the nuclear membrane nor the golgi apparatus stained with the antiglycoprotein antiserum. magnification is 500. 131 of the ha trimer, the deleted amino acids unique to the xi mutation may similarly play a critical role in the tertiary structure of the env complex, such that sorting signals required for transport through the golgi complex are lost. mutants x2 and x3 have deletions that begin at the amino terminus of gp85 and extend nine and six amino acids into this structural protein; thus these mutations overlap with the deletion in xi. nevertheless, both mutant proteins were transported to the cell surface and were indistinguishable from the wild type. mutants x2 and x3 thus indicate that the terminal nine amino acids of pr95 are not required for normal intracellular transport and define the critical region in gp85 as the seven amino acids that are uniquely deleted in the xi mutant. a. truncation of the cytoplasmic sequences. dna and protein sequence studies demonstrated the presence of a 27 amino acid long hydrophobic (and presumably membrane-spanning) domain and a 22 amino acid long cytoplasmic domain at the carboxy terminus of gp37 ; see fig. 1 ). comparison of these domains with those of other exogenous and endogenous strains of rsv has revealed that the sequence within the hydrophobic domain is highly conserved and that within the cytoplasmic domain the sequence of the first 18 amino acids (adjacent to the hydrophobic domain) is also highly conserved while those at the carboxy termini diverge greatly (hughes, 1982; hunter et al., 1983) . these results raised the possibility that the conserved region of the cytoplasmic domain might play a functional role in either transport of the env gene product through the secretory pathway or in virus assembly. to investigate this question we initially altered the cytoplasmic domain by introducing deletion mutations into the molecularly cloned sequences of the proviral env gene and examined the effects of the mutations on transport and subcellular localization in cv-1 cells. we found that replacement of the nonconserved region of the cytoplasmic domain with a longer unrelated sequence of amino acids from sv40 vector sequences (mutant cl) did not alter the rate of transport to the golgi apparatus nor the appearance of the glycoprotein on the cell surface. larger deletions, extending into the conserved region of the cytoplasmic domain (mutant c2), however, resulted in a 3-fold slower rate of transport to the golgi complex, but did not prevent transport to the cell surface (wills et al., 1983 (wills et al., , 1984 . these results were thus consistent with the cytoplasmic domain of the rsv env gene product playing some role in transport to the golgi complex. similar results were obtained by rose and bergmann (1983) who introduced into the cdna clone encoding the vsv g protein a series of deletions that affected the cytoplasmic domain. these mutants fell into two classes; the first was completely arrested in their transport at a stage prior to the addition of complex oligosaccharides (presumably the rer) and the second showed severely reduced rates of transport to the golgi complex although the proteins were ultimately transported to and expressed on the cell surface. the method by which these mutants were constructed (as with the rsv env mutants) meant that the truncated g proteins terminated in sv40 sequences, and in at least one case the block to transport could be alleviated by substitution of a termination codon for these "poison" sequences. even in these constructions, however, three foreign amino acids were translated prior to termination (rose and bergmann, 1983) . the concept that the cytoplasmic domain might influence or govern the rate at which membrane-spanning proteins were transported to the golgi complex was supported by similar studies of doyle et al. (1985) on the ha polypeptide. while the ha cytoplasmic domain could be replaced by the equivalent region from the rsv env gene product without affecting the rate of transport of the hybrid ha from the rer to the golgi complex, truncation of the ha cytoplasmic domain or addition of the 21 amino acid long cytoplasmic domain from gp37 slowed transport significantly, and addition of 16 amino acids encoded by pbr322 sequences blocked transport of the ha from the rer. on the other hand, studies on the class i histocompatibility antigens (zuniga et al., 1983; murre et al., 1984) , the p62 of semliki forest virus (garoff et al., 1983) , and additional studies on the ha of influenza indicated that the cytoplasmic domains of these proteins could be truncated without affecting transport to the cell surface, although the kinetics of transport were not determined in every case. b. substitution mutations. it should be noted that in most of the mutant constructions described above, the carboxy-terminal region contained one or several aberrant amino acids as a result of the recombinant dna approach. thus in order to determine more directly the role of the cytoplasmic domain of gp37, we have used oligonucleotide-directed mutagenesis to introduce an early termination codon in the coding sequences of gp37 such that the arginine residue that represents the first amino acid of the cytoplasmic domain is changed to an opal terminator. this mutation creates a truncated viral glycoprotein lacking specifically the cytoplasmic domain of gp37. the biosynthesis and transport of the products of this mutant viral glycoprotein gene were analyzed by expression from an sv40 late-region replacement vector, and its ability to be active in viral assembly was investigated by substitution of the mutated gene for the wild-type gene in an infectious avian retrovirus vector. in contrast to our previous results, deletion of the entire cytoplasmic domain alone had no effect on the biosynthesis or rate of intracellular transport of the env glycoprotein. thus it seems unlikely that the conserved amino acids present in this region play a role in intracellular transport. although the cytoplasmic domain contains several charged, hydrophilic residues, it does not appear, by itself, to be required for anchoring the complex in the membrane, since molecules lacking the cytoplasmic domain were expressed stably on the plasma membrane and were not shed into the cell culture medium . a recent study by gething et al. (1986) has demonstrated that mutations within the cytoplasmic domain of the influenza virus ha can affect the conformation of the extracellular domain by preventing assembly and trimerization of the ha molecule, thereby resulting in a failure of those mutants to be efficiently transported. a similar requirement for the assembly of oligomeric forms of the vs v g protein prior to its transport to the golgi complex has also been reported (kreis and lodish, 1986) . the inconsistency of our previous results with the ones obtained with the opal mutant could be explained in a similar way. we cannot rule out the possibility that in our earlier experiments the extra amino acids, added as a consequence of the loss of the env termination codon, created a conformational change in the extracellular domain of pr95 and slowed its transport from the rer. our present results indicate that the cytoplasmic domain of gp37 is neither a recognition signal for transport to the plasma membrane nor a requirement for anchoring the molecule to it. these findings also support the idea that the charged amino acids present in most of the cytoplasmic domains of many transmembrane proteins (garoff et al., 1983; sabatini et al., 1982) are dispensable for anchor function (davis etal., 1984) . this latter question has also been addressed by cutler and co-workers , who mutated the cytoplasmic domain of the p62 polypeptide of semliki forest virus. this region, which normally contains a charge cluster (arg-ser-lys) flanking the hydrophobic domain, was changed to a neutral (met-ser-gly) or an acidic (met-ser-glu) one using oligonucleotide mutagenesis. expression analyses of these mutant proteins confirmed that the basic amino acids were not required for cell surface transport since they reached the surface in a biologically active form. nevertheless, both mutant polypeptides showed reduced stability when membranes containing them were extracted with high-ph buffer . charged residues within the cytoplasmic domain may thus provide an additional measure of stability to the membrane-bound complex. since the conserved residues in the cytoplasmic domain of gp37 were not required for protein transport it seemed possible that this region might play a role in the process of infectious virus assembly. the fact that the mutant protein was efficiently transported to the cell surface allowed us to analyze this potential role for the cytoplasmic domain in the process of virus budding. chemical cross-linking experiments have demonstrated an interaction between gp37 and pl9, one of the gag gene products that structure the viral core of rsv (gebhardt et al, 1984) . while it is clear that virus assembly can occur in the absence of glycoproteins, it was suggested that the pl9/gp37 interaction may be part of the driving force for the process of viral assembly and budding. furthermore, since host membrane glycoproteins are excluded from the viral membrane there must be some positive signal for inclusion of the viral env gene products in the budding virion. to determine whether the cytoplasmic domain is involved in this interaction and required for infectious virus assembly, we reconstructed a retrovirus genome carrying the "tail(-)" env gene mutation. surprisingly, such mutant viruses were infectious on avian cells and spread through the culture with similar efficiency to those containing a native env glycoprotein complex. furthermore, this truncated env gene complex was incorporated as efficiently into virus particles as the wildtype complex . this fact suggests that if an interaction between gp37 and pl9 is required to mediate the incorporation of the glycoproteins into the envelope of the budding viral particle, it must occur within the lipid bilayer, presumably with the hydrophobic anchor domain. it is thus unlikely that interactions between viral capsid proteins and the cytoplasmic domain of the env complex constitute a driving force for preferential incorporation of the viral glycoproteins in the avian retroviral envelope. what then is the function of the cytoplasmic domain of the env glycoprotein? since this segment of the viral polypeptide does show a region of conserved sequence, it is possible that it has evolved to facilitate transport to the plasma membrane without being a requirement for it; clearly, randomly inserted alterations within this domain can exert a negative effect on the transport process. while we have observed normal assembly and infection by virus encoding a "tail(-)" env product, it will be of interest to determine whether continued growth of the virus results in the dominant appearance of revertants that encode a functional cytoplasmic domain. many cell surface and membrane proteins of animal viruses are bound to the lipid bilayer by a membrane-spanning hydrophobic peptide close to the carboxy terminus of the polypeptide (reviewed by warren, 1981; armstrong et al, 1981) . experimental evidence for this first came from deletion mutants of the influenza virus ha (gething and sambrook, 1982; sveda et al, 1982) , the vsv g protein (rose and bergmann, 1982) , and the minor coat protein of phage fl (boeke and model, 1982) in which removal of sequences that encoded the cytoplasmic and membrane-spanning domains resulted in secretion of the protein. the hydrophobic membrane-spanning peptide of these polypeptides is thought to be an essential component of the cotranslational signal that results in the arrest of chain transfer across the rer membrane during synthesis. these stop-translocation sequences have been proposed to be a region of the nascent protein molecule which halts insertion through the membrane by disassembling the translocation apparatus and thereby creates proteins with three topological domains (blobel, 1980) . they appear to be inseparable from the anchor sequences (yost et al, 1983; rettenmier et al., 1985) since the transfer of intact transmembrane domains to normally secreted proteins has caused translocation of the constructed hybrid molecules to stop at the added sequences (yost et al., 1983; guan and rose, 1984) . however, the precise structural and physical properties of the stop-translocation sequences have not been defined. wold et al. (1985) have suggested that the cytoplasmic domain of membrane-spanning proteins might act to interrupt translocation; however, this seems unlikely since deletion mutants lacking this domain are found to be associated with the membrane in a normal manner (see above; garoff et al., 1983; zuniga et al, 1983; murre et al, 1984; doyle et al, 1986; . while length and sequence vary widely among regions described as transmembrane anchors, they do have characteristics in common. most often, they are long stretches (19-30 residues) of predominantly nonpolar and hydrophobic amino acids, bounded by charged residues, at the carboxy terminus of membrane proteins. membrane-spanning sequences have also been described, however, at the amino terminus of some viral proteins (blok etal.,a9s2; palmiter et al, 1978; bos et al, 1984; markoff et al, 1984; zerial et al., 1986; spiess and lodish, 1986) and in the middle of other proteins (rettenmier et al, 1985; kopito and lodish, 1985; finer-moore and stroud, 1984) . we have investigated the structural requirements for a functional anchor/stop-translocation sequence in the rsv env system by constructing both deletion and point mutations in this region. a. deletion of the anchor domain. during our studies on the role of the cytoplasmic domain in env product transport, we characterized a mutant (c3) in which the entire cytoplasmic and transmembrane domains were deleted. this mutant, in contrast to those described for the influenza virus ha and the vsv g protein, was arrested in its transport at the rer and thus was not secreted from the cell (wills et al., 1984) . pulse-chase experiments coupled with oligosaccharide precursor labeling experiments showed that the c3 polypeptide was not transported to the golgi complex, even though it accumulated in a soluble, nonanchored form in the lumen of the rer; the mutant thus appeared to lack a functional sorting signal. surprisingly, immunofluorescent labeling studies showed that the c3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm (fig. 7) , suggesting that movement to the nuclear membrane, blocked in c3, may require a specific transport event, even though the rer and nuclear membranes appear to be continuous. this hypothesis is supported by studies on the vsv g protein that indicate that transitional vesicles (for the transport of glycoproteins to the golgi apparatus) may be derived from "blebs" in the nuclear membrane (bergmann and singer, 1983) . although these studies raised the possibility that sorting signals might exist within the deleted region of c3, the 95 amino acid deletion in this mutant, which extends into the external domain of gp37, would be expected to prevent normal folding of the glycoprotein. to determine whether the transmembrane domain was required for intracellular transport, we have modified the env gene by oligonucleotide-directed mutagenesis, changing the lysine (aaa) codon, which precedes the hydrophobic domain of gp37, to an ochre nonsense codon (taa). this modified gene thus encodes a protein consisting of the entire external domain of pr95 and lacking precisely the hydrophobic membrane-spanning and hydrophilic cytoplasmic domain. the biosynthesis and intracellular transport of the truncated protein in cv-1 cells was not significantly different from that of the wild-type glycoprotein, suggesting that any protein signals for biosynthesis and intracellular transport of this viral glycoprotein complex must reside in its extracellular domain. in contrast to the case of the c3 mutant, this complex lacking just the transmembrane and cytoplasmic domains is secreted as a soluble molecule into the culture medium . since the glycoprotein complex lacking only the cytoplasmic domain of gp37 is stably expressed on the cell surface, in a manner similar to the wild-type complex, it can be concluded that the transmembrane domain alone is required for anchoring the rsv env complex in the cell membrane. b. requirements for a functional stop-translocation/anchor sequence. we have approached the question of the compositional requirements for membrane anchoring and orientation (stop-translocation) of a membrane-spanning protein by substituting an arginine for a centrally positioned leucine in the hydrophobic anchor region of the rsv env gene product (fig. 8) . the arginine substitution is one of the most drastic ^ membrane-spanning mutant « domain * wt his leu leu lysjgly leu leu leu gly leu val val ile leu leu leu leu val cys leu pro cys leu leu gin phe val ser ser ser ile|arg lys met μά^ his leu leu lys|gly leu leu leu gly leu val val ile leu leu leu leu val cys|arg|pro cys leu leu gin phe val ser ser ser ile) arg lys met t24 his leu leu lys|gly leu leu leu gly leu val val ile leu leu leu leu val cysj ;;;-::;;;: : :;|i|leu leu gin phe val ser ser ser ile|arg lys met t18 his leu leu lys|gly leu leu leu gly leu val val ile leu leu leu leu val| |ser ser se7üe|arg lys met t16 his leu leu lys|gly leu leu leu gly leu val val ile leu leu[ jval ser ser ser ile| arg lys met t11 his leu leu lys|gly leu leu leu gly leu val| |ser ser ser ile|arg lys met t5 his leu leu lys|gj7leu||| |ser ser ile|arg lys met compositional point mutations that could be made since it is only rarely found buried in hydrophobic environments (kyte and doolittle, 1982) and has a high predicted potential for terminating membrane-buried helices (rao and argos, 1986) . the substitutions we have made fall within the conserved leucine-rich " i c " region proposed by patarca and haseltine (1984) and near the two cysteine residues where palmitate may be covalently added (gebhardt et al, 1984; kaufman et al., 1984) . by changing the anchor's hydrophobic integrity through the insertion of point mutations we hoped to define better what constituted a functional anchor sequence. placing a highly charged basic side chain into the hydrophobic core of the membrane might be expected to either (1) terminate the membrane-spanning helix, thereby partitioning the charged residue to one side or the other of the membrane, or (2) destroy the stop-translocation signal, causing the protein to be secreted. the results of these experiments showed that a single amino acid substitution in the transmembrane anchor did not affect membrane association or its orientation in the membrane; unexpectedly, however, it affected targeting of the protein at a stage late in the transport pathway, such that the mutant protein was rapidly degraded in lysosmes . the early translation products of both the arginine-mutant and wild-type genes behaved normally: they were synthesized with equal efficiency, had normal bitopic symmetry, and were glycosylated. the kinetics for the turnover of these precursors were nearly identical to those previously reported in infected chicken embryo fibroblasts (bosch and schwarz, 1984) and in sv40 expression vectors (wills et al., 1984) . furthermore, in the golgi, palmitate was added to the precursors, they were cleaved to gp85-gp37, and they received terminal sugars. only after this last stage did the presence of the charged side chain of the substituted arginine alter expression. at the level of the trans golgi, a post-golgi compartment (saraste and kuismanen, 1984) , or at the cell surface, the gp85-gp37 complex was rapidly shunted to lysosomes and degraded-as shown by the protection afforded the terminally glycosylated env proteins by the lysosomatropic agent, chloroquine . the exact pathway that the molecules take to the lysosome is not known. they may be transported directly from the trans golgi or first to the surface where they are rapidly endocytosed. discriminating between the alternate pathways has not been possible from current data. why the insertion of an arginine into the anchor should result in targeting to lysosomes is not obvious. a possible explanation is that we have introduced a specific sorting signal into the molecule; however, this is unlikely since other polypeptides with charged residues in the membranespanning domain are not so targeted (kabcenell and atkinson, 1985; saito et al., 1984; hayday et al., 1985) . the arginine's charge is incompatible with the hydrophobic environment of the lipid bilayer; to achieve stability, the charged guanidinium group needs to be neutralized, and how this is done inside the bilayer is not clear. parsegian (1969) has postulated that a lone charge sequestered in a membrane must form a pore or tunnel along with localized membrane thinning to acheive the lowest energy state. if the charged residue in the gp37 anchor causes the mutant molecules to aggregrate and form channels in the membrane in an analogous manner, it would likely kill the cell unless there was a mechanism to remove it rapidly. alternatively, since the env protein is not an isolated entity in the membrane, it is conceivable that it aggregates with other components of the membrane to reduce net charge cooperatively, and thereby triggers the endocytotic machinery (mellman and plutner, 1984) . charged residues are found in several proposed membrane-spanning helices (kabcenell and atkinson, 1985; saito et al., 1984; hayday et al., 1985; reviewed by rao and argos, 1986) . the charged residues in bacteriorhodopsin membrane-spanning a helices have been suggested to be neutralized by forming ion pairs (engelman et al., 1980) . this is likely a special case, however, since the energy required to bury an ion pair in the membrane is not much different from that required to bury the free charged group itself (parsegian, 1969) . neutralization of strong charges, particularly of lysine and arginine, may occur through the formation of strong hydrogen bonds with tyrosine (kyte and doolittle, 1982) ; however, no tyrosine residues are present in the anchor domain of the env gene product which could participate with the arginine. the t cell a, ß, and y gene products and the rotavirus vp7 protein have a putative structure similar to that of the arginine mutant, with a lysine centered within the transmembrane anchor; however, unlike the molecule we created, they invariably have tyrosine residues adjacent to the lysine (saito et al., 1984; kabcenell and atkinson, 1985; hayday et al., 1985) which could stabilize the charge through hydrogen bonds (kyte and doolittle, 1982) . adams and rose (1985a) have described the similar insertion of an arginine (and glutamine) residue at the center of the transmembrane domain of the vsv g protein. their mutant protein, like the env mutant we have characterized, was bitopic, could be seen localizing in the golgi, and did not accumulate on the cell surface. since these investigators observed a "lower level of protein expression" with their mutant g protein, it is possible that it also was rapidly degraded in lysosomes following terminal glycosylation. in contrast, observed no alteration in the biosynthesis and transport of a mutant p62 polypeptide of semliki forest virus in which the hydrophobic domain was interrupted by insertion of a glutamic acid residue. in this protein, however, the outer boundary of the hydrophobic domain is not delineated by a charged residue and so it is possible that additional uncharged residues from the external domain were pulled into the membrane. since the insertion of a charged polar residue into the transmembrane region of the rsv env gene product did not interfere with its anchor/stoptranslocation function, we have investigated the requirement for the long (27 amino acid) hydrophobic domain in arresting translocation and anchoring the env complex. a series of deletion mutations was generated by progressively removing base pairs to either side of a unique sph\ restriction site that had been previously engineered into the center of the anchor coding region. this produced env proteins with truncated transmembrane anchors that ranged in length from 24 (t24) to a single apolar amino acid (tl) summarized in fig. 8 ; . while the effects of the deletions on the transport and subcellular localization of the env gene product appeared to be a complex function of the length and composition of the remaining anchor, the mutants appeared to fall into three broad phenotypic classes (summarized in table i ). even the smallest deletion (t24), which removed only three amino acids, greatly reduced the surface expression of the mature env proteins. t24 and mutants t18, t17, and t16 had a normal bitopic orientation in the membrane but appeared to be cleared from the cell surface and degraded in lysosomes, since they accumulated only in the presence of choroquine, an inhibitor of lysosomal degradation. the reduced surface expression of the as determined by the distance between charged residues. b hydrophobicity score as determined using the hydropathicity values of kyte and doolittle (1982) . mean hydrophobicity is hydrophobicity score divided by the apparent anchor length. c symbols: + denotes either a wild-type or positive response; ± denotes an intermediate response; -denotes a negative response. d sec, secreted. e nd/na, not determined/not applicable. f with chloroquine treatment, pr95 appears in the medium with gp85 and gp37. « anchorless and tailess deletion mutant . largest deletion mutant (t24) was surprising since the effective hydrophobic peptide remaining in this construct was as long or longer than functional anchors reportedly present in other integral membrane proteins [e.g., 19 amino acids: m 2 protein of influenza (lamb, 1985) ; 20 amino acids: vsv g protein (rose et al., 1980) and adenovirus e3 protein (wold et al., 1985) ]. deletions which reduced the size of the transmembrane anchor to seven amino acids (t7) or less resulted in the secretion of mature glycoproteins into the medium. a third class of mutants with hydrophobic regions of 14 (t14) and 11 (til) amino acids, respectively, while remaining membrane associated, no longer appeared to span the rer as bitopic proteins. neither mutant could be found at the surface of cells, nor could their degradation be arrested by chloroquine treatment. from the sum of the data obtained with these mutants, it would appear that bitopic insertion of the env gene product is possible with effective anchor domains of at least 16 amino acids; if additional amino acids are removed from the domain, the protein can no longer exist bitopically, and it either partitions monotopically to the luminal side of the rer membrane or withdraws amino acids from the cytoplasmic side of the membrane into the bilayer. nevertheless, such sequences from the cytoplasmic domain are not able to stabilize the shortest anchors (tl, t5, and t7) since these are secreted from the cell. davis and model (1985) have investigated the requirements of a functional anchor domain by inserting artificial hydrophobic peptides of varying length into the membrane-associated pill protein of the bacteriophage fl. their results show that 17 hydrophobic amino acids are sufficient to maintain the protein in a bitopic configuration; however, the 17 amino acid anchor was "deleterious to the cell" presumably because it was too short to assume a stable conformation compatible with existence in the bilayer and thereby destabilized the membrane. a construct with an anchor of 12 hydrophobic amino acids was membrane associated but showed an intermediate phenotype in its sensitivity to solubilization by alkali. in contrast, a construct with only 8 hydrophobic amino acids-also membrane associated-was completely released into the supernatant at high ph. adams and rose (1985b) reduced the anchor domain of vsv g by precisely deleting amino acids from within the hydrophobic core. when the length of the anchor was reduced from 20 amino acids to as few as 14, the protein was normally membrane associated and expressed in a bitopic fashion on the cell surface. on the other hand, proteins with an anchor domain of 12 or 8 amino acids, while spanning the membrane, appeared to be transported only as far as the golgi where they accumulated; the surface expression of these proteins was greatly reduced (12 amino acid anchor) or undetectable (8 amino acids). doyle et al. (1986) have characterized a series of carboxy-terminal deletion mutants of the ha polypeptide in which the 27 amino acid anchor domain was truncated to 17, 14, and 9 amino acids, respectively. in this case, molecules with a 17 amino acid long transmembrane domain were stably anchored but were transported less efficiently to the plasma membrane. truncation of the hydrophobic anchor to 9 or 14 residues resulted in ha proteins that were unstable and whose transport appeared to be blocked in the rer or in a pre-golgi compartment-resembling the til mutant of the rsv env gene described above. from the results of these different systems and approaches it would seem that in strictly physical terms, anchors may be significantly reduced in their length without any consequence to the membrane association. the limits for this length ap-pear to be about 8-12 amino acids, but it must be appreciated that the mere presence of a stretch of hydrophobic amino acids within a protein does not serve to constitute an anchor. several mammalian virus envelope proteins contain in their external domain long hydrophobic amino acid regions that are equivalent to the truncated bitopic anchors have described here (gething et al, 1978; white et al, 1981) . indeed, gp85 contains a strongly hydrophobic 11 amino acid region that clearly does not act as a stop-translocation sequence or play a role in membrane association . the work of davis and model (1985) , on the other hand, implies that the length of a hydrophobic region is the major determinant as to whether or not it will confer membrane association properties to a protein, although they point out that the position of such sequences within the molecule may play a role. most eukaryotic membrane-spanning polypeptides have a complex tertiary structure that is stabilized by multiple disulfide linkages, and it is possible that the entropy of a correctly folded molecule is sufficient to pull potential stop-transfer regions through the membrane. a corollary of this hypothesis, therefore, is that, once folding is complete, short hydrophobic regions that can potentially span the membrane as an a helix would stop translocation. the length requirements for such a region could be shorter than the 20 residues predicted from a-helix dimensions if the region were flanked by arginines or lysines. since the latter have long side chains, equivalent in length to a single turn of an a helix, a stretch of hydrophobic amino acids 13-14 amino acids long might be sufficient. such a prediction fits well with the data we have obtained and with those of adams and rose (1985b) and davis and model (1985) . it will be of interest to determine what effect inserting the truncated anchors of mutants t18 and t16 into the middle of the env precursor has on translocation; if the above speculations are correct they should be extruded into the external domain. finally, it should be reemphasized that merely providing a bitopic membrane anchor/stop-translocation is not sufficient to confer wild-type biological activity on a polypeptide. mutant t24 of the rsv env gene has a hydrophobic domain which might be expected to be sufficiently long and hydrophobic in character to span the membrane stably; it is modified normally by palmitic acid and can clearly be transported to its targeted cellular location. nevertheless, it is degraded rapidly by the cell. these results imply that hydrophobic transmembrane domains contain additional (and perhaps subtle) signals that remain to be deciphered, a conclusion that is supported by the finding that deletion of the anchor domain of the rotavirus vp7 protein abolishes its specific targeting and retention in the rer (poruchynsky et al, 1985) . polarized epithelial cells exhibit apical and basolateral membrane domains that are separated by well-defined tight junctions. each membrane domain has a unique protein composition (louvard, 1980; reggio et al., 1982) , indicating that mechanisms must exist to specifically target membrane proteins to different surfaces. the sorting process occurs during or shortly after passage of the glycoproteins through the golgi complex pfeiffer et al., 1985; rindler et al., 1984 rindler et al., , 1985 . however, the mechanisms determining this directed transport to either the apical or basolateral membranes are not understood. their study has been facilitated by the use of cultured epithelial cell lines, such as the mdck cell line, and by the observation that certain rna viruses bud exclusively from apical or basolateral domains of these polarized cells in culture (rodriguez-boulan and sabatini, 1978; herrler et al., 1981; roth et al., 1983a; rindler et al., 1985) . avian and mammalian retroviruses together with rhabdoviruses such as vsv mature from the basolateral surface, while ortho-and paramyxoviruses bud from the apical surface. the carbohydrate residues present on the different proteins do not appear to play a role in this sorting process, since tunicamycin does not interfere with the polarized release of the viruses (roth et al., 1979; green et al., 1981b) . as with the maturation of viruses that assemble at intracellular locations within the secretory pathway, polarized budding of enveloped viruses is dependent on the site to which viral glycoproteins are transported. expression of cloned viral glycoprotein genes from both sv40-based and vaccinia expression vectors in polarized cells has demonstrated that the ha and neuraminidase polypeptides of influenza virus are targeted to the apical surface (roth et al., 1983b; jones et al., 1985; gottlieb et al., 1986) while the g protein of vsv and the gp70/pl5e complex of murine leukemia virus (mulv) are transported exclusively to the basolateral membranes . in an attempt to locate the signals which direct these glycoproteins to the apical or basolateral domains, recombinant dna techniques have been employed to construct chimeric proteins and express these in polarized cells in culture. in experiments where sequences encoding the external domain of ha were fused to those encoding the transmembrane and cytoplasmic domains of the vsv g protein, the hybrid glycoprotein behaved in the same manner as wild-type ha and was transported to the apical domain of polarized cells (mcqueen et al., 1986; roth et al., 1986) . conversely, fusing the external domain of g protein to the anchor/cytoplasmic domain of ha results in basolateral transport (mcqueen et al., 1987) . these experiments thus suggest that the ectodomains of ha and g protein contain signals for apical and basolateral transport, respectively. while expression of a secreted form of the ha glycoprotein in an apical polarized manner supports this conclusion (roth et al. y 1986) , the unanchored ectodomain of the mulv gp70/pl5e complex, which is normally targeted to the basolateral membrane, is secreted in a nonpolarized fashion . it is possible that this soluble protein is improperly folded and thus is unable to interact with the sorting machinery, alternatively it also raises the possibility that targeting signals may be located in more than one domain of these molecules. further analyses should shed light on this problem. the studies described in this chapter demonstrate the breadth of information that has been and can be obtained from studies on enveloped virus glycoprotein biosynthesis. many of the studies were performed at a time when the cloned genes and molecular probes for cellular glycoproteins were unavailable and thus provided valuable insights into the manner in which cells compartmentalized and transported membrane proteins. the exciting possibility of utilizing viral glycoprotein genes for genetic analyses of the transport pathway, in a way analogous to that pursued in prokaryotic systems (michaelis and beckwith, 1982; silhavy et al. y 1983; oliver, 1985) , has led to a plethora of studies that utilized both classic and recombinant dna genetic approaches. these investigations have resulted in great progress in our understanding of the general processes involved in intracellular transport of proteins through the secretory pathway but at the same time have raised difficult questions about the molecular interactions required for protein sorting. the initial observation that proteins destined for secretion contain an amino-terminal sorting sequence provided a precedent on which to build models for protein targeting based on topogenic sequences (blobel, 1980) . to a large extent the identification of signal sequences was facilitated by their transient nature, not by a conserved primary sequence. indeed, while some common characteristics of signal peptides can be recognized (von heijne, 1985) , the identification of signal peptides in proteins where they are not removed has proved difficult and has required the use of sophisticated recombinant dna technology [e.g., ovalbumin (tabe et ah, 1984) ]. the requirement for a signal sequence to initiate translocation across the er membrane has been clearly confirmed through the isolation and construction of mutants which lack this functional region (as discussed above) and by fusion of this sequence to proteins that are not normally translocated (lingappa et al., 1984) . these approaches have also been facilitated by the transient, nonstructural nature of many aminoterminal, translocation signals. recent experiments by friedlander and blobel (1985) and kaiser et al. (1987) , however, raise questions about the informational content of signal sequences. in particular kaiser and colleagues showed that several random amino acid sequences derived from human genomic dna fragments could act as signal sequences for translocation of the yeast invertase enzyme. thus even in this well-defined situation, where an amino acid sequence is known to play a functional role in the sorting process, it can be impossible to predict with confidence its location in the protein; how then might we expect to identify additional sorting sequences that may or may not exist within the structural domain of a transported protein? the possibility that additional sorting sequences might be involved in steering the transport of a membrane-spanning or secreted protein through the vesicular maze of the secretory pathway remains open. at the present time the necessity for a native tertiary structure cannot be separated from the possibility of such additional sorting sequences. it is clear that disruption of a poly peptide's normal folding can completely prevent its transport from the er kreis and lodish, 1986) , and the simplest explanation for the phenotypes of a variety of conditional and nonconditional transport mutants would be that they alter the tertiary structure of the mature protein (see section ii, a, above). a 3-dimensional structure has been determined for only a few molecules that traverse the secretory pathway, and even with these proteins the current, predictive algorithms are insufficiently accurate to model potential changes in molecular shape in response to mutations. the question of a direct role for tertiary structure in protein transport thus represents a major challenge to molecular biologists. furthermore, one might argue that a change in protein shape could also mask or distort a necessary (peptide) sorting sequence. this possibility is supported by the observation that for a majority of eukaryotic proteins the amino-terminal signal peptide is unable to initiate translocation across the er if translation is allowed to proceed to completion, presumably because the tertiary structure of the nascent polypeptide precludes the interaction of the signal peptide with the translocation machinery. it is quite feasible that sorting signals and targeting signals could be represented by different entities within a single polypeptide, particularly if the latter were required to fix the intracellular location of a protein. for example, the rotavirus vp7 polypeptide accumulates within the er un-less its amino-terminal hydrophobic anchor region is deleted, whereupon it is transported to the cell surface and secreted (poruchynsky et al., 1985) ; in this instance the deleted region presumably contains a sequence that can fix the intracellular location of the protein despite the fact that the molecule has the potential to be exported from the cell. since most translocated polypeptides appear to follow a common pathway to a late compartment of the golgi (kelly, 1985) , it might be argued that a native conformation is the sole requirement for transport to this organelle and that the observed differences in the transport rates of proteins to the golgi merely reflect the time necessary for completion of the folding process. nevertheless, proteins leaving the golgi appear to be sorted into specific pathways; for example, in secretory cells proteins may follow either the constitutive pathway or be sequestered in secretory granules (moore and kelly, 1985; reviewed by kelly, 1985) , and in epithelial cells specific proteins appear to be transported directly to either the apical or basolateral membranes (see above). thus, it would seem likely that some form of sorting signal must be present in the polypeptide at this point in the secretory pathway in order to correctly direct its transport; initial results from viral glycoprotein expression studies indicate that at least in polarized cells the ectodomain of the sorted protein plays a dominant role (mcqueen et al., 1986; . additional studies should provide a clearer picture of this complex process. in summary, studies on the biosynthesis and transport of enveloped virus glycoproteins have provided important insights into the general processes involved in the intracellular movement of these membrane-associated molecules. the specific questions that remain to be answered are many and difficult, but it is likely that these viral systems will continue to play a vital role by providing clues and direction in this important area of cell biology. incorporation of a charged amino acid into the membrane spanning domain blocks cell surface transport but not membrane anchoring of a viral protein structural requirements of a membrane-spanning domain for protein anchoring and cell surface transport sequence analysis of two mutants of sindbis virus defective in the intracellular transport of their glycoproteins domain structure of bacteriophage fed adsorption protein intragenic suppressor mutations that restore export of maltose binding protein with a truncated signal peptide information within the mature lamb protein necessary for localization to the outer membrane of eseherichia coli k-12 genetic analysis of protein export in escherichia coli k-12 immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (g) of vesicular stomatitis virus in infected chinese hamster ovary cells passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the golgi apparatus en route to the plasma membrane bunyaviridae. in "comprehensive virology intracellular protein topogenesis transfer of proteins across membranes. i. presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma studies on the size, chemical composition and partial sequence of the neuraminidase (na) from type a influenza viruses show the nterminal region of na is not processed and serves to anchor na in the viral membrane a prokaryotic membrane anchor sequence: carboxyl terminus of bacteriophage fl gene iii protein retains it in the membrane processing of filamentous phage precoat protein: effect of sequence variations near the signal peptidase cleavage site nh 2 -terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation processing of gpr92env, the precursor to the glycoproteins of rous sarcoma virus: use of inhibitors of oligosaccharide trimming and glycoprotein transport sequence of the membrane protein gene from avian coronavirus ibv virus-specific glycoproteins associated with the nuclear fraction of herpes simplex virus type 1-infected cells mutants of the membrane-binding region of semliki forest virus e2 protein. i. cell surface transport and fusgenic activity mutants of the membrane-binding region of semliki forest virus e2 protein. ii. topology and membrane binding fusion mutants of the influenza virus hemagglutinin glycoprotein isolation of the escherichia coli leader peptidase gene and effects of leader peptidase overproduction in vivo a charged amino acid substitution within the transmembrane anchor of the rous sarcoma virus envelope glycoprotein affects surface expression but not intracellular transport altered surface expression, membrane association and intracellular transport result from deletions within the transmembrane anchor of the rous sarcoma virus envelope glycoprotein an artificial anchor domain: hydrophobicity suffices to stop transfer fine structure of a membrane anchor domain variant influenza virus hemagglutinin that induces fusion at elevated ph mutations in the cytoplasmic domain of influenza virus hemagglutinin affect different stages of intracellular transport analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin assembly of enveloped rna viruses early and late functions associated with the golgi apparatus reside in distinct compartments localization and processing of outer membrane and periplasmic proteins in escherichia coli strains harboring export-specific suppressor mutations suppressor mutations that restore export of a protein with a defective signal sequence importance of secondary structure in the signal sequence for protein secretion path of the polypeptide in bacteriohodopsin amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor evidence for a glycoprotein "signal" involved in transport between subcellular organelles bovine opsin has more than one signal sequence characterization of two recombinant-complementation groups of uukuniemi virus temperature-sensitive mutants uukuniemi virus glycoproteins accumulate in and cause morphological changes of the golgi complex in the absence of virus maturation nucleotide sequence of a cdna clone encoding the entire glycoprotein from the new jersey serotype of vesicular stomatitis virus a single amino acid substitution in a hydrophobic domain causes temperature-sensitive cell-surface transport of a mutant viral glycoprotein using recombinant dna techniques to study protein targeting in the eucaryotic cell expression of semliki forest virus proteins from cloned complementary dna. ii. the membrane-spanning glycoprotein e2 is transported to the cell surface without its normal cytoplasmic domain rous sarcoma virus pl9 and gp35 can be chemically crosslinked to high molecular weight complexes. an insight into viral association cold spring harbor laboratory, cold spring habor construction of influenza haemagglutinin genes that code for intracellular and secreted forms of the protein purification of the fusion protein of sendai virus: analysis of the nh 2 -terminal sequence generated during precursor activation cloning and dna sequence of double-stranded copies of haemagglutinin genes from h2 and h3 strains elucidates antigenic shift and drift in human influenza virus expression of wild-type and mutant forms of influenza hamagglutinin: the role of folding in intracellular transport synthesis and infectivity of vesicular stomatitis viruses containing nonglycosylated g protein the nonglycosylated glycoprotein of vesicular stomatitis virus is temperature-sensitive and undergoes intracellular aggregation at elevated temperatures protein translocation across the endoplasmic reticulum. i. detection in the microsomal membrane of a receptor for the signal recognition particle protein translocation across the endoplasmic reticulum. ii. isolation and characterization of the signal recognition particle receptor the mechanism of protein translocation across the endoplasmic reticulum membrane sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells passage of viral membrane proteins through the golgi complex glycosylation does not determine segregation of viral envelope proteins in the plasma membrane of epithelial cells conversion of a secretory protein into a transmembrane protein results in its transport to the golgi complex but not to the cell surface glycosylation allows cell-surface transport of an anchored secretory protein two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells amino-terminal deletion mutants of the rous sarcoma virus glycoprotein do not block signal peptide cleavage but block intracellular transport biosynthesis of lysosomal enzymes in fibroblasts: phosphorylation of mannose residues structure, organization, and somatic rearrangement of t cell a genes antitrypsin: the presence of excess mannose in the z variant isolated from liver isolation and structural analysis of influenza virus c virion glycoproteins studies on the mechanisms of tunicamycin inhibition of iga and ige secretion by plasma cells the importance of the endosome in intracellular traffic sequence of the long terminal repeat and adjacent segments of the endogenous avian virus rous-associated virus complete sequence of the rous sarcoma virus env gene: identification of structural and functional regions of its product mechanism of signal peptide cleavage in the biosynthesis of the major lipoprotein of the escherichia coli outer membrane phospholipid is required for the processing of presecretory proteins by detergent-solubilized canine pancreatic signal peptidase intracellular transport of herpes simplex virus gd occurs more rapidly in uninfected cells than in infected cells surface expression of influenza virus neuraminidase an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells processing of the rough endoplasmic reticulum membrane glycoproteins of rotavirus sah many random sequences functionally replace the secretion signal sequence of yeast invertase cysteines in the transmembrane region of major histocompatability complex antigens are fatty acylated via thioester bonds pathway of protein secretion in eukaryotes ph-induced alterations in the fusogenic spike protein of semlilci forest virus membrane fusion mutants of semliki forest virus separate pathways of maturation of the major structural proteins of vesicular stomatitis virus maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus primary structure and transmembrane orientation of the murine anion exchange protein oligomerization is essential for transport of the vesicular stomatitis virus glycoprotein to the cell surface uukuniemi virus maturation: an immune fluorescence microscopy study using monoclonal glycoprotein-specific antibodies a simple method for displaying the hydropathic character of a protein influenza m2 protein is an integral membrane protein expressed on the infected-cell surface impaired intracelluiar migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and sindbis virus kinetics of serum protein secretion by cultured hepatoma cells: evidence for multiple secretory pathways determinants for protein localization: /3-lactamase signal sequence directs globin across microsomal membranes hen oviduct signal peptidase is an integral membrane protein reversible block in intracellular transport and budding of mutant vesicular stomatitis virus glycoprotein hepatoma secretory proteins migrate from rough endoplasmic reticulum to golgi at characteristic rates apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells l985). a single n-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus g protein to the cell surface polarized expression of a chimeric protein in which the transmembrane and cytoplasmic domains of the influenza virus hemagglutinin have been replaced by those of the vesicular stomatitis virus g protein basolateral expression of a chimeric protein in which the transmembrane and cytoplasmic domains of vesicular stomatitis virus g protein have been replaced by those of the influenza virus hemagglutinin glycosylation and surface expression of the influenza virus neuraminidase requires the n-terminal hydrophobic region the entry of enveloped viruses into cells by endocytosis internalization and degradation of macrophage fc receptors bound to polyvalent immune complexes acidification of the endocytic and exocytic pathways identification and characterization of a membrane component essential for the translocation of nascent proteins across the membrane of the endoplasmic reticulum secretory protein translocation across membranes-the role of the "docking protein mechanism of incorporation of cell envelope proteins in escherichia coli secretory protein targeting in a pituitary cell line: differential transport of foreign secretory proteins to distinct secretory pathways structural mutations in a mouse immunoglobulin light chain resulting in failure to be secreted construction, expression and recognition of an h-2 molecule lacking its carboxyl terminus identification of the defects in the hemagglutinin gene of two temperature-sensitive mutants of a/wsn/33 influenza virus membrane-bound penicillinases in grampositive bacteria carbohydrate moieties of glycoproteins, a reevaluation of their function protein secretion in escherichia coli ovalbumin: a secreted protein without a transient hydrophobic leader sequence energy of an ion crossing a low dielectric membrane: solutions to four relevant problems similarities among retro virus proteins mutations within the proteolytic cleavage site of the rous sarcoma virus glycoprotein precursor block processing to gp85 and gp37 mutants of the rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and/or cytoplasmic domains: analysis of intracellular transport and assembly into virions a putative signal peptidase recognition site and sequence in eucaryotic and procaryotic signal peptides reversible defect in the glycosylation of the membrane proteins of semliki forest virus tsl mutant localization of rotavirus antigens in infected cells by ultrastructural immunocytochemistry intracellular sorting and basolateral appearance of the g protein of vesicular stomatitis virus in mdck cells complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned dna deletions into an nh 2 -terminal hydrophobic domain result in secretion of rotavirus vp7, a resident endoplasmic reticulum glycoprotein export of protein in bacteria a conformation preference parameter to predict helices in integral membrane proteins surface and cytoplasmic domains in polarized epithelial cells transmembrane orientation of glycoproteins encoded by the w-fms oncogene viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same golgi apparatus during their intracellular transport in doubly infected madin-darby canine kidney cells polarized delivery of viral glycoproteins to the apical and basolateral plasma membranes of madin-darby canine kidney cells infected with temperature-sensitive viruses asymmetric budding of viruses in epithelial monolayers: a model system for study of epithelial polarity intracellular transport of influenza virus hemagglutinin to the apical surface of madin-darby canine kidney cells expression from cloned cdna of cell-surface secreted forms of the glycoprotein of vesicular stomatitis virus in eukaryotic cells altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein vesicular stomatitis virus is anchored in the viral membrane by a hydrophobic domain near the cooh terminus the presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition polarity of influenza and vesicular stomatitis virus in mdck cells; lack of a requirement for glycosylation of viral glycoproteins influenza virus hemagglutinin expression is polarized in cells infected with recombinant sv40 viruses carrying cloned hemagglutinin dna basolateral maturation of retroviruses in polarized epithelial cells heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway membrane insertion and intracellular transport of influenza virus glycoproteins the large external domain is sufficient for the correct sorting of secreted or chimeric influenza virus hemagglutinins in polarized monkey kidney cells studies on the adaption of influenza viruses to mdck cells a mutation downstream from the signal peptidase cleavage site affects cleavage but not membrane insertion of phage coat protein mechanisms for the incorporation of proteins in membranes and organelles complete primary structure of a heterodimeric t-cell receptor deduced from cdna sequences pre-and post-golgi vacuoles operate in the transport of semliki forest virsus membrane glycoproteins to the cell surface fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of posttranslational modification of the viral glycoprotein relation of fatty acid attachment to the translation and maturation of vesicular stomatitis and sindbis virus membrane glycoproteins evidence for covalent attachment of fatty acids to sindbis virus glycoproteins defects in functional expression of an influenza virus hemagglutinin lacking the signal peptide sequences analysis of the hemagglutinin glycoprotein from mutants of vaccinia virus that accumulates on the nuclear envelope mechanisms of protein localization changes in the conformation of influenza virus hemagglutinin at the ph optimum of virus-mediated membrane fusion the phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes glycoproteins specified by herpes simplex viruses an internal signal sequence: the asialoglycoprotein receptor membrane anchor nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells polarized transport of the vsv g surface expression of viral glycoproteins is polarized in epithelial cells infected with recombinant vaccinia viral vectors intracellular transport of secretory and membrane proteins in hepatoma cells infected by vesicular stomatitis virus vesicular stomatis virus glycoprotein, albumin, and transferrin are transported to the cell surface via the same golgi vesicles effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chicken hepatocytes the molecular biology of coronaviruses cell surface expression of the influenza virus hemagglutinin requires the hydrophobic carboxy-terminal sequences influenza virus hemagglutinin containing an altered hydrophobic carboxy terminus accumulates intracellularly segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in xenopus oocytes: identification of an ovalbumin signal sequence eukaryotic signal sequence transports insulin antigen in escherichia coli prolipoprotein signal peptidase in escherichia coli is distinct from the m13 procoat protein signal peptidase temperature-sensitive mutants of influenza virus: a mutation in the hemagglutinin gene structural studies of iga myeloma proteins having anti-dnp antibody activity patterns of amino acids near signal-sequence cleavage sites how signal sequences maintain cleavage specificity signal sequences, the limits of variation purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum signal recognition protein (srp) mediates the selective binding to microsomal membranes of m-i>//ro-assembled poly somes synthesizing secretory protein translocation of proteins across the endoplasmic reticulum iii. signal recognition protein (srp) causes signal sequence-dependent and sitespecific arrest of chain elongation that is released by microsomal membranes translocation of proteins across the endoplasmic reticulum i. signal recognition protein (srp) binds to m-wvro-assembled polysomes synthesizing secretory protein membrane proteins: structure and assembly compilation of published signal sequences m13 procoat and a preimmunoglobulin share processing specificity but use different membrane receptor mechanisms cell fusion by semliki forest, influenza, and vesicular stomatitis viruses membrane fusion proteins of enveloped animal viruses multiple mechanisms of protein insertion into and across membranes alterations in the transport and processing of rous sarcoma virus envelope glycoproteins mutuated in the signal and anchor regions mutations of the rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products the 19-kda glycoprotein precursor coded by region e3 of adenovirus secretion of a x2 immunoglobulin chain is prevented by a single amino acid substitution in its variable region molecular abnormality of human a r antitrypsin variant (pi-zz) associated with plasma activity deficiency uncoating of influenza virus in endosomes a stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems the transmembrane segment of the human transferrin receptor functions as a signal peptide mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein expression and function of transplantation antigens with altered or deleted cytoplasmic domains key: cord-014685-ihh30q6f authors: nan title: posters p788 p999 date: 2005-09-21 journal: eur biophys j doi: 10.1007/s00249-005-0504-x sha: doc_id: 14685 cord_uid: ihh30q6f nan in order to understand mrna stability, we proceed to the studie of life time model system composed of the regb ribonuclease and the ribosomal protein s1. the t4 bacteriophage life cycle is modulated by regb which mediate speci c mrna degradations. regb cleaved the mrna at the middle of the translation initiation region (shine-dalgarno sd). studies had shown that regb activity is enhanced with addition of s1 which is normaly required for the translation of mrna in case of unusual sd regions. s1 is considered as a key factor of translation's inititiation. composed of six similar domains (f1-f6), it interacts with the ribosome by his f1-f2 domains and with the mrna by f3-f6. we had shown that the f345 fragments got the same properties than the whole protein. many global architectures had been published (linear or globular conformation). then we try to understand what is the catalysis way of s1 in the regb activation and in rna recognition. we study the global conformation of f345 fragment. for that, we use nmr to determinate the domains interfaces. at rst we made the nmr backbone assignement of 15n/13c/2h labeled fragments (f34-f45). then we analysed hsqc overlapping of each fragments in order to identify interfaces residues. the residues identi cation on s1 homologous model allowed us to determinate interaction region between domains. currently we study of s1-rna interactions to determinate if the ligand conformation could change the interaction region and involve conformationals changes. translocation of amino acids from the membrane interface to the interior: theory and experimiment c. aisenbrey 1 , e. goormaghtigh 2 , j.-m. ruysshaert 2 , b. bechinger 1 1 ulp/cnrs, chemistry, 4 rue blaise pascal, 67070 strasbourg, 2 université libre, campus plaine cp206/2, 1050 brussels the interactions of a series of histidine-containing peptides with biological model membranes have been investigated by attenuated total re ection fourier transform infra red (atr-ftir) and oriented solid-state nmr spectroscopies. related peptides have previously been shown to exhibit antibiotic and dna transfection activities. the 26-residue lah 4 x 4 and lah 4 x 6 peptides were designed in such a manner to form amphipathic helical structures in membrane environments. four histidines and four/six variable amino acids x constitute one face of the helix whereas leucines and alanines characterize the opposite hydrophobic surface. the dichroic ratio of atr-ftir spectra, or the orientation-dependent 15 n solid-state nmr chemical shift have been used to follow the ph-dependent transition from in-plane to transmembrane alignments upon increase in ph. a theoretical model of the topological modulations is presented and the experimental transition curves analysed in order to reveal the gibbs free energy of transition. the novel concept provides access to the free energy changes associated with the amino acids x incorporated into an extended -helix and in the context of phospholipid bilayers. for the peptides of the lah 4 x 4 series the gibbs free energies associated with the transition from the membrane interface to the bilayer interior follow the sequence of amino acids: l < a i < s f < t g < v w << y. here we present a novel solid-state nmr approach which allows for the accurate determination of the tilt and rotational pitch angles of peptides reconstituted into uniaxially oriented membranes. the method works with transmembrane or in-plane oriented peptides that have been labelled with 3,3,3-2 h 3 -alanine and 15 n-leucine at two selected sites. proton-decoupled 15 n and 2 h solid-state nmr spectroscopy at sample orientations of the membrane normal parallel to the magnetic eld direction have been used to characterize the tilt and rotational pitch angle of these peptides in considerable detail. the same samples when inserted into the magnetic eld at 90 degrees tilted alignments provide valuable information on the rotational diffusion constants in membranes and thereby of the association and size of peptide complexes within the membrane environments. whereas monomeric transmembrane peptides exhibit spectral averaging and well-de ned resonances, larger complexes are characterized by broad spectral line shapes. in particular the deuterium line shape is sensitive to association of a few transmembrane helices. in contrast, the formation of much larger complexes affects the 15 n chemical shift spectrum. aisenbrey ch. & bechinger, b. biochemistry 43, 10502-10512 (2004) a meccano set approach of joining trpzip a water soluble -hairpin peptide with a didehydrophenylalanine containing hydrophobic helical peptide p. chetal, v. chauhan, d. sahal international centre for genetic engineering & biotechnology,new delhi 110067,india a 16 residues long, water soluble, monomeric -hairpin peptide "trpzip" [cochran et al (2001) pnas 98, 5578-5583], stabilized by tryptophan zipper has been linked via a tetraglycyl linker to a hydrophobic didehydrophenylalnine ( f) containing helical octapeptide. circular dichroism studies of this 28 residues long peptide, "trpzipalpha" (ac-gewtwddatktwtwte-gggg-fal fal fa-nh 2 ) in water have revealed the presence of both the -hairpin and the helical conformations. this is the rst instance where a f containing peptide has been found to display a helical fold in water. the uorescence emission wavelengths of tryptophan in ac-g-w-g-nh 2 , trpzip and trpzipalpha were 341.5, 332.8 and 332.6 nm respectively. the uorescence quantum yield of trpzip was 2.6 fold higher than trpzipalpha suggesting that proximal interactions between the -hairpin and the helix caused the quenching of tryptophan uorescence in the former by the fs in the latter. the molar ellipticity of the far uv couplet characteristic of trpzip was reduced in trpzipalpha and the cd based thermal melting temperatures at 228 nm were 62 c (trpzip) and 57 c (trpzipalpha). a concentration dependent variable temperature cd study in water showed that in trpzipalpha, increasing temperature is detrimental to the -hairpin, but it augments the helical motif by intermolecular oligomerization. our results show that in water, trpzipalpha exhibits long-range interactions between two different secondary structures. structural-based differential stability in the yoeb-yefm toxin-antitoxin module i. cherny, e. gazit department of molecular microbiology and biotechnology, faculty of life sciences, tel aviv university, tel aviv 69978, israel the speci c physiological role of natively unfolded proteins is only recently beginning to be explored. a notable case in which natively unfolded state appears to have physiological signi cance is the e. coli yoeb-yefm toxin-antitoxin (ta) module. a crucial element in proper functioning of ta systems requires physiological instability of the antitoxin in contrast to the stable pro le of its toxin partner. we have shown that yefm antitoxin is a natively unfolded protein, lacking secondary structure even at low temperatures. in contrast, its toxin partner has a well-folded conformation at physiological temperatures. we suggest that the structural-based differential thermodynamic stability between the two components is the cause for their differential physiological stability, since structural instability of the antitoxin exposes it to cellular quality-control machinery. we further revealed that yefm and yoeb interact and form tight complex and determined it stoichiometry. a potential use of ta systems is as novel antibacterial targets. indeed, we identi ed homologous yefm-yoeb systems in a large number of bacteria including major pathogens. we aim to design peptides capable of interfering with the yefm-yoeb interaction, thus releasing the toxin to execute its detrimental function. for this purpose, we identi ed a short linear determinant within yefm that is involved in yoeb interaction. this peptide motif will be optimized for development of antibacterial lead molecules. a. chatterjee, a. prabhu, a. ghosh-roy, r. v. hosur tata institute of fundamental research, mumbai, india-400005 dynein light chain (ddlc1), a member of the cytoplasmic motor assembly exists as a monomer or a dimer (functional form) under different experimental conditions. here we report the unfolding characteristics of the monomeric ddlc1 at ph 3, due to urea and guanidine hydrochloride, by various biophysical techniques. it is observed that the unfolding pathways due to the two denaturants have many differences. urea unfolding seems to be two state, while guanidine unfolding is more complex. the nmr experiments carried out at low denaturant concentrations have enabled detailed characterization of the structure and dynamics of the near native excited states of the protein. these are similar to the native state in structure, except for the small extensions of the helices in the nterminal half of the protein. however, the local stabilities of the and -strands are perturbed and this occurs differently in the two denaturants. in the guanidine case the entire multi-stranded -sheet in the c-terminal half is destabilized. in either case the motional characteristics, seem to suggest the presence of a nite population of the dimer in the excited state ensemble. these states are suggested to be likely intermediates in the momoner-dimer transition, and their characterization here thus provides clues to the molecular mechanism of the transition. it is also envisaged that the near native excited states could play regulatory roles in the functioning of the protein. kinetic bottlenecks identi cation in different folding models f. cecconi 1 , c. guardiani 2 , r. livi 3 1 infm -istituto di sistemi complessi -cnr, italy, 2 centro interdipartimentale per lo studio delle dinamich complesse, università di firenze, italy, 3 dipartimento di sica, università di firenze, italy the ww domains are a family of fast folding, compact, modular domains featuring a triple-stranded, antiparallel beta-sheet. the ww domain of the pin1 protein, due to the availability of a complete picture of the residues involved in thermodynamic stability and in the formation of the transition state, in particular, represents an excellent benchmark to test computational methods. the objective of the present work is to identify the kinetic bottlenecks in the folding process through md unfolding simulations at increasing temperatures. the kinetic bottlenecks are related to the establishment of contacts requiring the overcoming of a large entropy barrier and acting as a nucleus for the creation of further contacts. the key sites are therefore those involved in contacts showing a dramatic decrease in fractional occupation near the speci c heat peak. the technique was applied to the go model and to a model based on the knowledge of secondary structure, providing in both cases a picture of the folding process consistent with the experimental data. evidence is also shown that while the go model allows a more accurate prediction of the native structure, the folding pathway is better described by the other model. the protein talin plays a key role in coupling the integrin cell adhesion molecules to the actin cytoskeleton and in integrin activation. the globular head of talin, which binds -integrins, is linked to a rod containing an actin-binding site and binding sites for the protein vinculin, which regulates the dynamic properties of cell-matrix junctions. we have determined the structure of three domains which contain vinculin binding sites (vbss) and shown that each of these are made up of helical bundles. the structures of complexes between vinculin and peptides corresponding to the vbss show that the residues which interact with vinculin are buried in the hydrophobic core of the helical bundles of the talin domains. nmr studies of the interaction of one of these domains with vinculin shows that it involves a major structural change in the talin fragment, including unfolding of one of its four helices, to make the vbs accessible. while the observation of folding of unstructured regions of a protein on interaction with a 'partner' is quite common, this kind of major unfolding to permit a protein-protein interaction is much less common. ways in which it may be regulated will be discussed. conformational changes of eye lens proteins studied by combined saxs and high pressure s. finet 1 , f. skouri-panet 2 , a. tardieu 3 1 european synchrotron radiation facility, 6 rue horowitz, bp220, 38043 grenoble france, 2 institut de minéralogie et de physique des milieux condensés, 140 rue de lourmel, 75015 paris france, 3 protéines: biochimie structurale et fonctionnelle, case 29, 7 quai st-bernard, 75252 paris france -, -and -crystallins are the main components of vertebrate eye lenses, with exceptional structural and associative properties. the crystallins are known to be exceptionally stable in vivo since they have to last the lifetime of the organism. they therefore represent an extreme case of stability versus unfolding and aggregation. these proteins are mainly beta strands. -crystallins are 21 kda monomers (from 50 to 80% sequence identity), and -crystallins are large hetero-oligomers of about 800kda. -crystallins are molecular chaperone; they belong to the ubiquitous superfamily of small heat shock proteins, shsps. here, the conformation and the stability of -and -crystallins were investigated by small angle x-ray scattering (saxs) and high pressure, depending upon temperature and ph. at room temperature, -crystallins have shown a partially reversible change in size from 2 to 3kb, and this effect was enhanced by the combination of temperature and pressure. in the case of -crystallins and in the pressure range up to 2kb, the pressure was combined with temperature and ph. the results depend upon the different itself. structural studies on pore-forming peptides s. m. ennaceur 1 , d. bown 2 , j. m. sanderson 1 1 chemistry department, university of durham, 2 biology department, university of durham the resistance of pathological microorganisms to conventional antibiotic drugs has created a need for new antibacterial agents. biologically active antimicrobial peptides that act as primary defense agents in a large variety of species are thought to have the potential as precursors for a new range of drugs from antibiotics to cancer treatments. this study has attempted to analyse the structural properties of membrane peptides and proteins through the use of model systems that have been designed to mimic their natural counterparts: we have successfully synthesised model membrane peptides with a beta-sheet structural motif and have used a wide range of techniques to analyse their interactions with phospholipid (pc) membranes. the synthetic peptides were very hydrophobic and only soluble in uorinated alcohols such as hfip and to a lesser extent tfe. we found hfip to have a very strong af nity for pc membranes and carried out a series of experiments to investigate this af nity. 1 binding of hfip to pc membranes was found to be reversible and we exploited this property in 2d crystal trials of our synthetic peptides. we over expressed the c-terminal domain of brka, a gram negative autotransporter protein, which forms a beta-barrel channel in the outer membrane (omp), for comparison with our model peptides. we performed 2d crystal trials on the omp and imaged the resulting protein arrays by stem and afm. a protein spontaneously folds into a unique native structure in physiological conditions. this process accompanies a huge loss of the conformational entropy (ce). our major concern is to specify the factor that can compete with the ce loss. the previous discussions concerning protein folding have been focused on contributions to the free energy of folding from the interaction potentials in a system. a view lacking in earlier studies is that the folding is critically in uenced by the translational movement of water molecules. when solute particles contact each other in a solvent, the excluded volumes for the solvent molecules overlap, and the total volume available to their translational movement increases. this leads to a gain in the translational entropy (te) of the solvent. this type of te effect should be much stronger in protein folding where the tight packing of the side chains occurs. an elaborate statistical-mechanical theory is employed to analyze the te of water in which a peptide or a protein molecule is immersed. it is shown that the te gain upon folding is large enough to compete with the ce loss. when water is replaced by another solvent whose molecular size is larger, the te gain decreases to a remarkable extent. we suggest that the entropic loss accompanying the self-assembly and the formation of ordered structures in a living system is compensated mostly by the te gain of water, highlighting an aspect of the crucial importance of water in sustaining life. domain ii of ribosome recycling factor is required for disassembly of the post-termination complex p. guo, l. zhang, y. feng, g. jing institute of biophysics, chinese academy of sciences, national laboratory of biomacromolecules, beijing, china ribosome recycling factor (rrf) consists of two domains and, in concert with elongation factor ef-g, triggers dissociation of the post-termination ribosomal complex. however, the exact function of the individual domains of rrf remains unclear. to clarify this, two rrf chimeras, ecodi/ttedii and ttedi/ecodii, were created by exchanging the rrf domains between the proteins from escherichia coli and thermoanaerobacter tengcongensis. the ribosome recycling activity of the rrf chimeras was compared with their wild-type rrfs by using in vivo and in vitro activity assays. the experiments show that like wild-type tterrf, the ecodi/ttedii chimera fails to complement the rrf ts phenotype of e.coli lj14 (frr ts ) strain and has no polysome breakdown activity. however, under the same conditions, the ttedi/ecodii chimera complements the rrf ts phenotype and has polysome breakdown activity equivalent to that of wild-type ecorrf. the results indicate that domain ii of rrf is the functional domain that is mainly responsible for the disassembly of the post-termination ribosomal complex, and the speci c interaction between rrf and ef-g on the ribosomes mainly depends on the interaction between domain ii of rrf and ef-g; while domain i of rrf is the main contributor for binding ribosomes and maintaining the stability of the rrf molecule. this study provides direct genetic and biochemical evidence for the assignment of individual functions of rrf domains. self-assembly of natural somatostatin into liquid crystalline nano brils w the natural neuropeptide somatostatin-14 is a cyclic tetradecapeptide hormone, with broad inhibitory effects on both endocrine and exocrine secretions. we report the self-assembly of somatostatin in solution, into stable liquid crystalline nano brils, based on the neuropeptide bioactive backbone conformation. the system was studied as a function of peptide concentration, milieu composition and time, using optical and electron microscopy, x-ray scattering, vibrational spectroscopy and sec/rp-hplc. in pure water, the formation of twisted nano brils (around 10nm wide and a few microns long) was characterized. their structure relies on the native somatostatin -hairpin and on intermolecular antiparallel -sheets networks. the nano brils were observed to laterally associate further with increased concentration and time, as well as to generate hexagonal phases. increase in ionic strength (sodium chloride, phosphate) was found to signi cantly favor the self-association process. the soft conditions of formation of the somatostatin nano brils support biological relevance, for instance to the biological mechanism of storage of the neuropeptide hormone. unraveling the physical origin of the structure of fully denatured ubiquitin s. golic grdadolnik, f. avbelj national institute of chemistry, hajdrihova 19, 1001 ljubljana, slovenia the structure and dynamics characterization of non-native states of proteins is crucial for understanding the mechanism of protein folding. recently many experimental studies have shown variations of conformational propensity and exibility along the backbone chain of fully denatured proteins. it has been supposed that areas of residual structure may serve as initiation sites of protein folding. however, the physical origin of these variations is still unclear. we analyze the structure of fully urea-denatured ubiquitin. the experimental veri cation of conformational propensities of protein backbone is obtained through structurally dependent nmr parameters. although the secondary structure of ubiquitin under strong denatured conditions is not detectable and no correlation with the native overall topology is found, the variations of nmr parameters along the backbone follow the secondary structure elements of its native state. we show that these variations are in accord with the recently developed electrostatic screening model of denatured proteins (1). in this model, the backbone conformations of residues in unfolded protein are determined by local backbone electrostatic interactions and their screening by backbone solvation. many virulence factors from gram-negative bacteria are autotransporter proteins. the nal step of autotransporter secretion is c nterminal threading through the outer membrane (om), followed by folding. this process requires neither atp hydrolysis nor a proton gradient. pertactin, an autotransporter from bordetella pertussis and the largest -helix structure solved to date, folds much more slowly than expected based on size and native state topology, yet folding intermediates are not aggregation-prone. equilibrium denaturation results in the formation of a partially folded structure, a stable core comprising the c-terminal half of the protein. examination of the pertactin crystal structure does not reveal the origin of the enhanced c-terminal stability. yet sequence analysis reveals that, despite size and sequence diversity, all autotransporters are predicted to fold into parallel -helices, suggesting this structure may be important for secretion. for example, slow folding in vivo could prevent premature folding of in the periplasm prior to the assembly of the om porin. moreover, extra stability in the c-terminal rungs of the -helix may serve as a template for the formation of the native protein during secretion, and formation of the growing template may contribute to the energy-independent translocation mechanism. coupled with the sequence analysis, these results suggest a general mechanism for autotransporter secretion. thermal and functional properties of e.coli outer membrane protein-receptor fhua fhua is e.coli outer membrane protein, which transports iron into the bacterial cell and also serves as receptor for several phages. in order to get more deep information about structural properties of fhua, we've studied its thermal properties by means of calorimetry. we've also investigated the interaction between t5, ira phages and directly fhua by means of viscometry. the calorimetric result of heat denaturation of membrane protein fhua and next deconvolution of the recorded calorimetric curve with two transitions has shown that in a chosen conditions the structure of fhua consists of 4 domains. though both t5 and ira phages grow on the common bacterial strain (e.coli k12 ho830), expressing fhua the results of viscometric investigation show that under direct interaction of phages with fhua the receptor activity of protein revealed only for t5 phage. therefore, we conclude that other than fhua protein serves as receptor for ira phage. it should be mentioned that the phage dna ejection process induced by receptor was observed for the rst time by us in an incessant regime. electron spray ionization mass spectroscopy (esi-ms) is a powerful tool for the investigation of the protein folding or proteins non-covalent interactions in solution since charge state distributions (csds) in esi-ms are affected by the conformational state and mass relates on the association state. we used this tecnique to inquire at different ph and different conditions the dimerization process of the porcine and bovine -lactoglobulins that share a high sequences similarity and close 3d structures. dimerization oflactoglobulins is reversible and involves both electrostatic and hydrophobic interactions. it was possible to detect simultaneously both the monomeric and dimeric form of the proteins in solution, pointing out the different dimerization behaviour of the two isoforms. we assessed the maximal stability of the dimeric structure at ph 4 for the porcine protein and ph 6 for the bovine one. moreover we showed that bovine lactoglobulin has a stronger dissociation costant than the porcine protein. further we showed that it is possible to modulate the dimerization equilibrium of the bovine isoform at ph 6 both increasing temperature and adding methanol without inducing denaturation of the protein. a possible novel method of protein structure prediction; a. ikehata division of structural bioinformatics science of biological spramolecular systems graduate school of integrated sciences mechanism of protein folding has been mysterious since an nsen's dogma was sugested.here i would like to propose a possible novel method of protein structure prediction; origami method. the method comes from a protein backbone property of uctuation and the residue hydrophobicity. l. marsagishvili 1 , m. shpagina 1 , z. podlubnaya 2 1 institute of theoretical and experimental biophysics ras, 2 pushchino state university amyloid brils are formed by proteins or their peptides in the result of a conformational transition from alpha helix into beta-sheet structure. despite the different nature of proteins-precursors their amyloids have common properties: beta-pleated sheet structure with individual beta-sheets oriented parallel to the main axis; insolubility in vivo; speci c binding to congo red and thyo avin-t. amyloid deposits are observed in different diseases such as myositis, myocarditis, cardiomyopathies and others. we showed that sarcomeric cytoskeletal proteins of titin family (x-, c-, h-proteins) of rabbit skeletal muscles are capable to form amyloid brils in vitro. these proteins already contain 90% of beta-sheet structure necessary for formation of amyloids. the amyloid nature of their brils was conrmed by electron, polarization and uorescence microscopy. as x-, c-, h-proteins form amyloid brils easily in vitro, there is a danger of fast growth their amyloid deposits in vivo. taking into account common properties of amyloids formed by different proteins, our results clear the ways for conducting by amyloidogenesis in human organs and tissues. work is supported by rfbr grants 03-04-48487, "universities of russia" 11.01.462 and program of the presidium ras "fundamental sciences for medicine". probing the folding capacity and residual structures in 79-and 110-residue fragments of staphylococcal nuclease d. liu, x. wang, y. feng, l. shan, j. wang national laboratory of biomacromolecules, institute of biophysics, chinese academy of sciences n-terminal fragments of staphylococcal nuclease (snase) with different chain lengths were used as a model system in the folding study. the detailed characterization of conformational states of 1-79 and 1-110 residues snase fragments (snase79 and snase110) and their v66w and g88w mutants can provide valuable information on the development of conformations in the folding of snase fragments of increasing chain lengths in vitro. in this study, the presence of retained capacity for folding and residual structures in snase79 and snase110 is detected by cd, uorescence, ftir, and nmr spectroscopy. snase79 is represented as an ensemble of interconverting conformations. the uctuating nascent helix-and âsheet-like structures, localized in regions of a58-a69 and t13-v39, respectively are transiently populated in snase79. the native-like tertiary conformations are obtained for g88w110 and v66w110 and for snase110 in the presence of 2.0 m tmao. analysis of the results of such studies indicate that folding of snase fragments is dominated by developing the local and non-local nucleation sites from native-like secondary structures and by intensifying the longrange interactions of residues at nucleation sites with residues further removed in sequence. thermal disruption of a spanning network of hydration water and conformational changes of elastin a. krukau 1 , i. brovchenko 2 , a. oleinikova 2 , a. geiger 2 1 international max-planck research school in chemical biology, otto-hahn str. 11, d-44227, germany, 2 physical chemistry, university of dortmund, otto-hahn str. 6, d-44227, germany hydration water strongly in uences the structural and dynamical properties of biomolecules. the existence of a spanning hydrogenbonded network formed by the hydration water enables the function of biomolecules at low hydration levels. we can expect that the formation/disruption of the spanning network formed by the hydration water in solution also affects crucially the protein properties. we present the rst computer simulation study of the thermal disruption of a spanning network formed by the hydration water of a biomolecule (elastin-like peptide). this process obeys the laws of 2d percolation transition, similarly to the formation of a spanning water network with increasing hydration level [1]. the spanning water network transforms into an ensemble of small water clusters with increasing temperature: it is still permanent at 280 k and exists with probability 50% at 320 k. in the same temperature interval, the conformation of the peptide changes noticeably: its radius of gyration increases sharply (by 15%) at about 295 k. these two phenomena may be related to the "inverse temperature transition" at about 310 k, where an elastin solution separates into two phases. in our simulations, the displacement of hydration water by the addition of a denaturant (urea) or by other peptide molecules causes an even stronger increase of the radius of gyration (up to 25% lipidic cubic phases formed by distinct water and lipid volumes provide bicontinuous 3d bilayer matrices that have speci c and controllable water channel sizes and large surface areas. these systems have proven to be also valuable as membrane mimetic structures, as promising matrices for controlled-release and delivery of proteins, vitamins and small drugs in pharmacological applications, and they offer a 3d lipid matrix for successful crystallization of membrane proteins which do not easily crystallize in bulk solution. the present study is directed towards a better understanding of the interplay between curved cubic lipid phases and the protein entrapped within their aqueous channel structures. as model systems, we have chosen a cubic ia3d phase formed by an uncharged lipid, monoolein, and incorporated different proteins, such as cytochrome c, -chymotrypsin and insulin, in its narrow water channels. we show that the protein secondary structure and unfolding behaviour may be in uenced by the con nement and, vice versa, the topology of the lipid matrix may change as a function of protein size and concentration. in fact, even new cubic lipid structures may be formed that are not known in pure lipid systems. furthermore, we compare the aggregation scenario of insulin in bulk solution and in the narrow water channels of the cubic lipid matrix and discuss the differences found in terms of the geometrical limitations imposed by the con nement. after endocytosis, i.e. at acidic ph, the t domain inserts in the membrane of the target cell and helps the translocation of the catalytic domain into the cytoplasm. therefore, the t domain has a key role in the strategy of internalization of the toxin. the study of the interaction of the t domain with membranes and its ph dependence is important for a better understanding of the diphtheria toxin translocation mechanism. at least, two steps can be distinguished during the membrane insertion of the t domain. the rst step involves hydrophobic interactions with the membrane and is related to the ph-induced stabilization in a molten-globule state. in the second step, electrostatic interactions are preponderant and the ph-sensitivity comes from changes of the balance between repulsive and attractive electrostatic interactions. the role of the n-terminal part of the t domain in the second step has been investigated by studying peptides corresponding to the amphiphilic helices found in this part of the domain. the results are correlated with those obtained with a single trp mutant probing the n-terminal region of the whole domain. the translocation mechanism will be discussed in view of the physico-chemical properties of the peptides. in complex systems with many degrees of freedom such as peptides and proteins there exist a huge number of local-minimumenergy state. one way to overcome this multiple-minima problem is to perform a simulation in a generalized ensemble where each state is weighted by a non-boltzmann probability weight factor so that a random walk in potential energy space may be realized (for reviews, see refs. [1] [2] [3] xas spectroscopy results show that there are two different structures of the metal binding site in the a 1 40 peptide according to whether they are complexed with cu 2 or zn 2 ions. while the geometry around copper is suggestive of an intra-peptide binding with three histidine residues bound to the metal, the zinc site geometry is compatible with an inter-peptide aggregation mode. this result reinforces the hypothesis that assigns opposite physiological roles to the two metals, with zinc favouring and copper blocking peptides aggregation and consequent plaque formation. effect of pressure on the conformation of proteins. a molecular dynamics simulation of lysozyme a. n. mccarthy, r. grigera instituto de física de líquidos y sistemas biológicos (iflysib), conicet-unlp-cic, university of la plata, argentina the effect of pressure on the structure and dynamics of lisozyme was studied by md computer simulation at 1bar (101 325 pa) and 3 kbar using gromacs package. all-atoms (ff2gmx) force eld were used for the minimization process and for all the md simulation and kept all protein bond lengths constrained (lincs algorithm). water molecules (spc/e model) were constrained using the settle algorithm. for the electrostatic forces we applied the reaction field method. lennard-jones interactions were calculated within a cut-off radius of 1.4nm. the results have good agreement with the available experimental data, allowing the analysis of other features of the effect of pressure on the protein solution. the studies of mobility show that although the general mobility is restricted under pressure this is not true for some particular residues. from the analysis of secondary structures along the trajectories it is observed that the conformation under pressure is more stable, suggesting that pressure acts as a 'conformer selector' on the protein. the difference in solvent accessed surface (sas) with pressure shows a clear inversion of the hydrophilic/hydrophobic sas ratio, which consequently shows that the hydrophobic interaction is considerably weaker under high hydrostatic pressure conditions. direct observation of mini-protein folding using uorescence correlation spectroscopy h. neuweiler, s. doose, m. sauer applied laser physics & laser spectroscopy, university of bielefeld, 33615 bielefeld, germany the "trp-cage" motif represents the smallest and one of the fastest folding mini-proteins known to date. the globular fold is characterized by a hydrophobic core burying a single tryptophan (trp) residue. here, we report on the direct observation of trp-cage folding kinetics using uorescence correlation spectroscopy (fcs). our method is based on the selective uorescence quenching of oxazine dyes by trp which becomes ef cient only upon contact formation between the dye and the indole moiety of trp. by sitespeci cally labeling the dye to trp-cage, temporal uorescence uctuations of the dye-peptide conjugate, caused by intramolecular contact formation between dye and trp, directly report on conformational dynamics and folding transitions of the peptide chain. in order to measure uorescence uctuations directly in solution we used fcs on a confocal uorescence microscope setup. fcs allows us to reveal conformational dynamics with nanosecond timeresolution, under thermodynamic equilibrium conditions, and in highly dilute solutions (i.e. at nano-molar sample concentrations). our method con rms microsecond folding kinetics of the trp-cage motif, previously estimated with non-equilibrium temperature-jump techniques. we further investigated stability and folding rates under denaturing conditions and at various temperatures, giving further insight into structural transitions during the folding process. identi cation and mutagenesis of a region of tnt required for the stability of tnt-tni coiled-coil evolutionarily conserved heptad hydrophobic repeat (hr) domains present in troponin subunits tnt and tni are involved in alpha helical coiled-coil formation. using recombinant peptides from fast-skeletal tnt and tni, we examined the contributions of amino acid residues within these hr domains as well as anking these domains, to the stability of the coiled-coil interaction. a series of tnt fragments were tested for their ability to form coiledcoil with tni hr domain. we show that the tnt region 166-178, although remains outside of the coiled-coil domain, is absolutely required for the stability of the coiled-coil. interestingly, the region tnt 166-178 contains few absolutely conserved residues that are potential candidate for ionic interaction, as predicted by molecular modeling. using single point mutants we show that among all the conserved residues, residue lysine 175 is most important in stabilizing the coiled-coil interaction, whereas others play accessory role. we propose that the lysine 175 initiates the stabilization of the coiled-coil interaction and then the other residues acts in a zipper like fashion. magnesium promotes conformational switching of ca 2 sensor s. mukherjee, k. v. r. chary tata institute of fundamental research the importance of mg 2 , one of the most abundant metal ion in the cell cytoplasm, relating to the calcium sensor mechanism is demonstrated. in this study it has been shown that the 134 amino acid long calcium binding protein from entamoeba histolytica (ehcabp) can exist in three different forms namely, the calcium-free (apo) form, the magnesium bound (apo-mg) form and the calcium bound (holo) form. these three forms have been characterized using chromatographic, calorimetric as well as various spectroscopic techniques. there is a radical difference in the stability between the calcium free and ca 2 bound forms. the calcium free form has molten globule like characteristics. mg 2 stabilizes the closed conformation of the apo form, where the hydrophobic core remains buried. the presence of mg 2 signi cantly alters the calcium binding cooperativity thereby increasing the cooperativity of the conformational switching between the open and closed conformation which is an important aspect of such regulatory proteins. a structural model for the molten-globular form of apo-ehcabp and its equilibrium folding towards completely folded holo state in presence and absence of mg 2 will be presented. intermediate states of formin binding protein ww domain: explored by replica-exchange simulation y. mu school of biological science, nanyang technological university, singapore ww domain of formin-binding protein (fbp) is a model system for beta strand folding study. although it is small, only 37 amino residues in total, the folding kinetics of fbp ww domain proved to be biphasic. an extensive molecular dynamics-monte carlo hybrid method, called replica-exchange simulation strategy is employed to study the folding/unfolding of the fbp ww domain in explicit water model. begin with randomly chosen conformations from high temperature unfolding trajectory distributed in 88 replicas (88 different temperatures covering from 290k to 570k), the simulation lasts 30 nanoseconds in each replica. in the end an interesting distribution of conformations shapes up. we nd that there are three distinctive subgroups, one being the unfolded conformations with rmsd averaging around 10å , one being native conformations with rmsd 2.5å, more interestingly, the third group having rmsd 5å, an intermediate folding ensemble. by checking the intermediate ensemble in detail, we nd that it is quite heterogeneous and the heterogeneity mainly comes from the exibility of the c-terminal loop region. our ndings provide a microscopic picture of folding kinetics of the ww domain: the stable intermediate states with mis-registered hydrogen bonds on the c-terminal beta strand make this peptide folding as a three-state folding model rather than a usual two-state model. h. rezaei, f. eghiaian, j. perez, y. quenet, y. choiset, t. haertle, j. grosclaude institut national de la recherche agronomique, france in pathologies due to protein misassembly, low oligomeric states of the misfolded proteins rather than large aggregates play an important biological role. to get better insight into the molecular mechanisms of prpc/prpsc conversion, we studied the kinetic pathway of heat induced amyloidogenesis of the full length recombinant ovine prp (arq) at ph 4.0. according to the size exclusion chromatography experiments, three sets of oligomers were generated from the partial unfolding of the monomer. the effect of concentration on the oligomerization kinetics was different for the three species obsreved suggesting that they are generated from distinct kinetic pathways. limited proteolysis and peptide analysis of the two best separated peaks showed a difference in the accessibility of the c-terminal domain of these two oligomers, and allowed the identi cation of regions undergoing a structural change during the conversion process. the analysis of correlations between oligomer populations as well as numerical kinetic modeling led us to propose a multi-step kinetic pathway describing the evolution of each species as a function of time. the existence of at least three distinct oligomerization pathways on one hand and the differences in the accessibility of the two puri ed oligomers on the other hand re ect the structural plasticity of the prp protein. studying the mechanism of retention of ovine prion protein in soils will tackle the environmental aspect of potential dissemination of scrapie infectious agent. the conformational transition from the monomeric cellular prion protein prp c in -helical structure into the aggregated -sheet-rich multimer prp sc is supposed to be responsible for the so-called prion diseases. it is commonly admitted that the recombinant prp could serve as model of conversion of the normal prion protein prp c . fundamentally, the interaction of proteins with surfaces either uid or solid involves both protein binding and unfolding. our goal in studying protein adsorption is to determine the nature and the amplitude of the structural changes occurring during non-speci c adsorption. the protein-clay interaction depends on several parameters such as protein hydration, net charge, charge distribution on the protein surface. ftir spectroscopy is well-suited to probe structural changes of proteins at a molecular level at aqueous/solid interfaces. the conformational states of the full-length ovine prp adsorbed on the electronegative clay surface are compared to its solvated state in deuterated buffer in the pd range 3.5-9, using ftir spectroscopy. during the intoxication of a cell, the diphtheria toxin binds to a cell surface receptor, is internalized and reaches the endosome. the translocation (t) domain from the toxin interacts with the membrane of the endosome in response to the acidic ph found in this compartment. this process drives then the passage of the catalytic domain of the toxin through the membrane into the cytoplasm. the interaction of the t domain with the membrane involves at least two steps. in the rst step occurring at ph 6, t adopts a molten globule conformation, which is able to bind super cially to the membrane through hydrophobic interactions by the c-terminal region (helices th8 and th9). in a second step occurring between ph 6 and 4, penetration into the membrane involves electrostatic interactions. this step leads to a functional inserted state. trp 281 was mutated to phe in order to use trp 206 located in helix th1 as a probe of its behavior during the interaction with the membrane as a function of ph. we found that the second step is correlated with the reorganization of the n terminal region in the membrane and is controlled by electrostatic interactions. peptide conformational search using generalized simulated annealing method p. g. pascutti 1 , f. p. agostini 2 , c. osthoff 2 , k. c. mundim 3 , m. a. moret 4 1 ibccf -ufrj -brazil, 2 lncc -brazil, 3 iq-unb -brazil, 4 ceppev -fundação visconde de cairú / df-uefs -brazil the three-dimensional structure of proteins is mainly determined by the sequence of amino acids, making possible the development of ab initio methods for peptides and protein structure prediction. we proposed a stochastic method based on a classical force field and the generalized simulated annealing (gsa), which utilizes tsallis generalization of boltzmann-gibbs statistics. we have applied this method for peptide conformational search and as a complement for comparative modeling of proteins, searching the conformation for loops and mismatched sequence alignments. the gsa ef ciency depends on the right choice of the parameters involved in the conformational calculation: the qv parameter which de nes a function for visiting the molecular energy surface; and the qa parameter de ning an acceptance probability, both according with tsallis statistics. to avoid the conformational trapping in local energy minima we introduced a new parameter in this work, qt, to control the temperature decreasing. to investigate the qv, qa and qt best parameters set, we used the 18-alanine and 26-alanine peptides, which have a known alpha-helical structure in low dielectric environment. the global minimum energy occurs for the alpha-helix folded structure, and was found for qv ranging from 1.1 to 1.9, qa from 1.6 to 2.9, and qt from 1.1 to 2.4. we observed also that convergence values for qv decrease while for qa and qt increase for folded structures. p. s. santiago, é. v. de almeida, m. tabak instituto de quimica de são carlos-usp cp780 13560 são carlos sp brasil extracellular hbgp is a giant hemoglobin, similar to other annelid hemoglobins, having a molecular weigth of 3.1 mda. the effect of ctac in the oligomeric protein structure was assessed by optical absorption and emission spectroscopies. optical absorption spectra of hbgp 0.075 mg/ml as a function of ctac concentration from 0 up to 10.5 mm evidentiate changes both in soret band at 414 nm, and at 490 nm associated to light scattering which appears at low ctac concentration. below 1 mm of surfactant extensive light scattering occurs together with signi cant shifts of max of soret band from 414 nm to around 400 nm, which is probably due to oxidation of the original oxyhbgp. light scattering reaches a maximum value disappearing for higher ctac concentrations. fluorescence spectra show a signi cant increase in intensity (30fold) upon titration with ctac. this is consistent with the dissociation of the oligomer with signi cant reduction of intrinsic quenching of tryptophan uorescence due to the heme groups. similar data were obtained at protein concentration of 3 mg/ml. in this case a signi cant increase of light scattering is observed with protein precipitation at a narrow ctac range followed by re-disolution at higher ctac concentration. differently from anionic sds surfactant, cationic one induces protein aggregation. support: cnpq and fapesp. in situ formation of silk protein nano-particles studied by small angle x-ray scattering m. w. roessle 1 , c. riekel 2 , d. i. svergun 1 1 european molecular biology laboratory; outstation hamburg/germany, 2 european synchrotron radiation facility; grenoble/france silk threads from the mulberry silkworm bombyx mori are used for the production of textiles since centuries. in modern applications silk proteins are chosen because of their good tensile strength, their high biocompatibility as well as of the resorbability for the human body. however, the processing of silk by the silkworm is barely understood. the silk proteins are stored as a solution in the glands of the silkworm and processed during the spinning into a co-block polymer like ber. in a rst step the random coil silk proteins are transformed into molecules with beta-sheet subdomains, which provide a protein-protein interface for the ber assembly. this transformation can be mimicked by a rheometer applying a shear force to the silk protein solution. applying combined small/wide angle x-ray scattering (saxs/waxs) transient formed silk nanoparticles upon increasing shear force were found. further details of these macromolecules were derived by xing the transient state with chemicals such as polyethylene oxide (peo). the resulting data can be analyzed in detail by saxs data evaluation software and low resolution models of the found nanoparticles were derived. moreover, the internal structure of the particles was explored as well as suggestions for the silk processing of the silkworm could be made. on the three-dimensional information of a protein sequence v. a. risso, l. g. gebhard, r. g. ferreyra, j. santos, m. noguera, m. r. ermacora universidad nacional de quilmes conicet in this work, lactamase of b. licheniformis (es l) was used as an experimental model to (a) study the conversion of sequential information into 3d structure and (b) to investigate the distribution of conformational information in the polypeptide chain. by a novel approach, over thirty connectivity variants of the polypeptide chain were prepared; witch were also nterminally truncated to a variable degree. the variants produced in e. clil were puri ed to homogeneity, refolded, and its structure content analyzed by circular dichroism, hydrodynamic behaviour and aggregation state. several variants were dimeric in solution, suggesting a possible general inespeci c stabilization mechanism. most variants were compact and had different degrees of secondary and tertiary structure. a strikingly large number of variants showed native like spectroscopic signatures and signi cant enzymatic activity, which means that the very elaborate active site of beta lactamase is formed, at least in fractions of the molecules, despite the absence of long stretches of sequence. these ndings are discussed in the light of the current knowledge of the protein folding process. var and lgg contributed equally to this work. on the three-dimensional information of a protein sequence v. a. risso, l. g. gebhard, r. g. ferreyra, j. santos, m. e. noguera, m. r. ermacora universidad nacional de quilmes conicet in this work, lactamase of b. licheniformis (es l) was used as an experimental model to (a) study the conversion of sequential information into 3d structure and (b) to investigate the distribution of conformational information in the polypeptide chain. by a novel approach, over thirty connectivity variants of the polypeptide chain were prepared; witch were also nterminally truncated to a variable degree. the variants produced in e. clil were puri ed to homogeneity, refolded, and its structure content analyzed by circular dichroism, hydrodynamic behaviour and aggregation state. several variants were dimeric in solution, suggesting a possible general inespeci c stabilization mechanism. most variants were compact and had different degrees of secondary and tertiary structure. a strikingly large number of variants showed native like spectroscopic signatures and signi cant enzymatic activity, which means that the very elaborate active site of beta lactamase is formed, at least in fractions of the molecules, despite the absence of long stretches of sequence. these ndings are discussed in the light of the current knowledge of the protein folding process. var and lgg contributed equally to this work. unfolding of the extrinsic proteins of photosystem ii (33kda, 23kda and 17 kda) induced by pressure unfolding of the three extrinsic proteins of spinach photosystem ii induced by pressure has been systematically investigated. thermodynamic equilibrium studies indicated that these proteins are very sensitive to pressure. at 20 o c all the proteins show a reversible unfolding transition by about 180 mpa for 33kda, 200mpa for 23kda and 270 mpa for 17kda. the ph and temperature dependence of pressure unfolding of these proteins were explored. the stabilization effect of reagents sucrose etc on the proteins was found to associate not only with the increase in the unfolding free energy, but also with the reduction of the absolute value of v u . pressure-jump studies of unfolding of 23kda protein revealed a negative activation volume for unfolding and a positive activation volume for refolding, indicating that in terms of system volume, the transition state lies between the folded and unfolded states. comparison of temperature dependence of v u # , v f # and v u indicated that the thermal expansivities of the transition state and the unfolded state are similar and larger than that of the folded state. aggregation-prone intermediate protein structures on the refolding pathway l. smeller 1 , j. fidy 1 , k. heremans 2 1 semmelweis university dept. biophysics and radiation biology, budapest, hungary, 2 department of chemistry, katholieke universiteit leuven, belgium the folding of the polypeptide chain into a native conformation can be studied by experimental systems, where the environmental parameters causing the denatured state can be easily and fast eliminated. one of such parameters is the high hydrostatic pressure. refolding of the unfolded protein can be studied after decompression. the refolding pathway can contain several intermediate states. on the other hand, the destabilization of the native proteins can populate conformations, where the polypeptide chain is not completely folded. these metastable conformations can easily aggregate. deposition of insoluble protein aggregates plays a crucial role in the conformational diseases (parkinson, alzheimer's disease, amyloidozis, etc.) stability and conformation of the above mentioned metastable conformations were investigated in case of myoglobin, apo-horseradish peroxidase, lipoxygenase. fluorescence and infrared spectroscopy and light scattering experiments were used to explore not only the structure but also the aggregation af nity of the intermediates in case of all the above mentioned proteins a well de ned temperature range could be determined, where the metastable not completely folded structures were populated considerably during the refolding process. these intermediate conformations were significantly more aggregation prone, than the native conformers existing in the same temperature range. aggregation processes in beta-amyloid peptides: effects of molecular chaperons a. sgarbossa, d. buselli, f. lenci cnr istituto bio sica, pisa, italy several neurodegenerative pathologies, like parkinson's, hungtington's and alzheimer's diseases, are related to the formation of small peptides aggregates, which amyloid brils originate from. understanding the molecular mechanisms responsible for these processes can, therefore, contribute to clarify the origin and, hopefully, to control the development of the afore mentioned diseases. here we report the results of an in vitro study aiming to affect the aggregation kinetic of 1-42 and 1-40 beta-amyloid peptides by means of an endogenous chaperone-like protein (alpha-crystallin) and an exogenous polycyclic aromatic pigment (hypericin) that can perturb the aggregation process through stacking interactions with the peptides aromatic residues. because of the well known problems in getting reproducible and reliable results, particular attention has been devoted to carefully check the preparation procedures of the samples. the effects of both alpha-crystallin and hypericin on the self-assembly process have been examined at different times of the aggregation kinetics. the results are discussed in relation with the involvement of different molecular structures in the amyloid brillation phenomenon. remodelling the folding of thioredoxin by removal of the c-terminal helix j. santos, j. m. del no iquifib and departamento de química biológica, facultad de farmacia y bioquímica, uba, junín 956, c1113aad buenos aires, argentina e. coli thioredoxin (trx) is a monomeric / protein of 108 amino acids with a fold characterized by a central beta sheet surrounded by alpha helices. two subdomains are topologically noticeable, but it is unclear whether their folding occurs in a concerted fashion. subdomain trx1-73 has been extensively studied as a model of a partially folded, with no tertiary or persistent secondary structure. this work describes the expression and characterization -by circular dichroism (cd), uorescence emission, size exclusion chromatography, chemical cross-linking and light scattering-of a novel engineered fragment (trx1-93) lacking the last stretch of 15 amino acids. after refolding from inclusion bodies, trx1-93 shows a strong propensity to form soluble oligomers endowed with distinctive optical properties unlike those observed for the full protein. although trx1-93 also shows signi cant changes in secondary structure, trp residues appear to occupy rigid and apolar environments. these ndings support the existence of an alternatively folded form for trx1-93. in addition, the secondary structure content of chemically synthesized peptide trx94-108 and its ability to complement fragment trx 1-93 upon refolding were also evaluated by cd. taken together, the data herein presented shed light upon issues such as the distribution of information content relevant for folding along the polypeptide chain in regard to conformational stability. with grants from anpcyt, ubacyt and conicet. thermal aggregation of two "beta-protein" models at different ph values v. vetri, f. librizzi, v. militello, m. leone physical and astronomical sciences, univ. of palermo, italy & infm the structural stability of proteins strongly depends on the environment and the lost of this stability may trigger a partial unfolding, leading in turn to the formation of aggregates. such processes have been extensively studied also in view of their biotechnological and medical implications. in fact a large number of diseases is associated with protein misfolding and aggregation. conformational changes play a keyrole in the aggregation processes and have their onset under particular external conditions. the aggregation pathways and the topology of the obtained supramolecular structures sizeably depend on the details of the involved conformational changes, which are determined by the details of the external conditions. here we present an experimental study on thermal aggregation processes of two model proteins mainly composed from structures: -lactoglobulin and concanavalin a, at different ph values. the conformational changes of the proteins (whose association state depends by ph) and the aggregation pathways were monitored by intrinsic and suitable external dyes uorescence. at the same time, the growth of supramolecular structures was followed by measuring the rayleigh scattering of the excitation light. secondary structure changes were followed by circular dichroism measurements. the results show that at different ph values the aggregation processes of both proteins follow different pathways determined by the variations in the native structure and by the details of the involved conformational changes. a. verma 1 , t. herges 2 , w. wenzel 2 1 iwr, forschungszentrum karlsruhe, po box 3640, 76021, karlsruhe, germany, 2 int, forschungszentrum karlsruhe, po box 3640, 76021, karlsruhe, germany ab-initio protein structure prediction and the elucidation of the mechanism of the folding process are among the most important problems of biophysical chemistry. investigations of the protein landscape may offer insights into the folding funnel and help elucidate folding mechanism and kinetics. we investigate the landscape of the internal free-energy of the 36 amino acid villin headpiece with a modi ed basin hopping method in the all atom force eld pff01, which was previously used to fold several helical proteins with atomic resolution. we identify near native conformations of the protein as the global optimum of the force eld. more than half of the twenty best simulations started from random initial conditions converge to the folding funnel of the native conformation, but several competing low-energy metastable conformations were observed. from 76,000 independently generated conformations we derived a decoy tree which illustrates the topological structure of the entire low-energy part of the free energy landscape and characterizes the ensemble of metastable conformations. these emerge as similar in secondary structure content, but differ in the tertiary arrangement. exploring the free energy landscape of a folded protein by means of afm stretching experiments m. vassalli 2 , b. tiribilli 1 , l. casetti 2 , a. torcini 1 , a. pacini 3 , a. toscano 3 1 isc -cnr, florence -italy, 2 csdc, univ. of florence -italy, 3 anatomy dept, univ. florence -italy the aim of our work is to study the mechanically induced folding and refolding of single proteins by means of an atomic force microscope (afm). the resulting data will be analyzed with theoretical methods, both to determine the folding pathway and to gain information on the energy landscape of real systems. recently, experiments employing atomic force microscopy have shown that mechanical and thermal unfolding share several common features. we are using an afm to perform mechanical stretching experiments on single biomolecules. the experiments that can be performed are particularly well-suited to reconstruct the folding-unfolding pathways as well as the free energy landscape of the examined protein. in particular we are interested in the free energy pro le associated to titin and elastin, by considering a periodic loading of the afm cantilever, instead of the usual linear ramp, and measuring the force as a function of displacement. these experiments will be complemented by theoretical and numerical studies. molecular dynamics simulation of simple models but including the experimental geometry, will allow to examine in detail the effect of different experimental procedures (periodic loading versus linear ramp) proposed to reproduce equilibrium energy landscapes. moreover, we will investigate the limit of applicability of the jarzynski's equality which has been claimed to be able to be used to extract equilibrium results from non equilibrium measurements. association of subunits is a prerequisite for formation of the native structure of the dimeric ipmdh a. varga, é. gráczer, i. hajdú, p. závodszky, m. vas institute of enzymology, biological research center, hungarian academy of sciences, budapest, hungary to answer the question whether subunits are autonomous folding units, or their association at an early stage of folding is required for formation of the native protein structure, denaturation-renaturation experiments were carried out with the dimeric isopropylmalatedehydrogenase (ipmdh). denaturation was induced by guanidine hydrochloride, renaturation was initiated by dilution and followed by activity measurements and uorimetry. reactivation is a complex process with an initial lag phase, indicating the presence of an inactive intermediate. the kinetics of the process is independent of protein concentration, suggesting that association of the two polypeptide chains takes place much faster than the rate limiting rst order isomerisation step(s). restoration of protein uorescence during renaturation is also protein concentration independent, biphasic process, however the initial lag phase is replaced by an even faster burst increase of uorescence. the rst step leads to formation of an intermediate with a native-like uorescence spectrum. based on our experiments the following mechanism is proposed for refolding of ipmdh: d+d i 2 i 2 n 2 , where d is denaturated monomer, i 2 and i 2 are inactive dimer intermediates, n 2 is native dimer, that means initial association of the polypeptide chains during refolding is a prerequisite for formation of the native 3 dimensional structure of ipmdh. we describe a new beamline for optical biophysic in construction on the synchrotron soleil. the high briliance of the synchrotron beam, associated with its tunability on a broad part of the electromagnetic spectrum make it an excitation source of choice for several biophysical optical techniques. the disco beamline we present will consist of three endstations : 1. the circular dichroism (cd) endstation will bene t from the inclusion of the energies accessible in the vuv wavelength range (350-165nm) and from the natural polarization of the synchrotron beam. cd spectra of proteins covering a broad range of wavelenghts will enable better and ner structural analysis. moreover, new biological chromophores such as sugars which absorb in the deep uv will be accessible in cd. 2. the mass spectrometry endstation,will bene t from an ionisation beam with even greater energies (down to 60 nm) comprising nebulisation at atmospheric pressure. photoionisation of macromolecular bio-structures without any solvent restriction will produce perfect analytes for mass spectrometry, 3. the multiparametric imaging endstation, build on a confocal microscope, will use the great tunability of the synchrotron radiation (200-700 nm) to excite samples at many wavelenghts simultaneously. the temporal component of the beam will allow natural lifetime imaging by phase modulation -demodulation. predictive all-atom protein folding with stochastic optimization methods the prediction of protein tertiary structure, in particular based on sequence information alone, remains one of the outstanding problems in biophysical chemistry. according to the thermodynamic hypothesis, the native conformation of a protein can be predicted as the global optimum of its free energy surface with stochastic optimization methods orders of magnitude faster than by direct simulation of the folding process. we have recently developed an all-atom free energy force eld (pff01) which implements a minimal thermodynamic model based on physical interactions and an implict solvent model. we demonstrated that pff01 stabilizes the native conformation of several helical proteins as the global optimum of its free energy surface. in addition we were able to reproducibly fold several helical proteins (the 20 amino acid (aa) trp-cage protein, the villin headpiece (36 aa), the conserved headpiece of the hiv accessory protein (40 aa), the headpiece of protein a (40 aa) and the 4-helix bacterial ribolsomal protein l20 (60 aa), as well as several beta-sheet peptides. we used several stochastic optimization methods: the stochastic tunneling method, an adapted version of parallel tempering, basin hopping techniques and distributed evolutionary optimization strategies. we discuss advantages and limitations of this approach to de-novo allatom protein structure prediction. the 9 independent thermal unfolding simulations of gb1 have been performed. 12 physical property parameters of protein structure were chosen to construct a 12-dimension physical property space. then the 12-dimension property space was reduced to 3dimensions principle component property space. under the property space, the unfolding pathway ensemble of gb1 was obtained. the pathway ensemble likes a funnel that was gradually emanative from the native state ensemble to the unfolded state ensemble. the unfolding trajectories have the similar variable trend during the native state and the transition state ensemble. during the unfolded state, the 9 unfolding trajectohries were divided into two types that one includes only one trajectory and the other include 8 trajectories. the rst type of unfolded state was a discontinuity step distribution model, which is not random distribution. the second type of unfolded state was a near ellipsoid distribution model and a near random. there were substantial overlaps of unfolded state, indicating that thermal unfolded state consists of a con ned set of property values that makes the number of unfolded state of protein to be much smaller than that was believed before. the protein circular dichroism data bank (pcddb): a bioinformatics and spectroscopic resource b. a. wallace 1 , l. whitmore 1 , r. w. janes 2 1 dept. of crystallography, birkbeck college, university of london, 2 school of biological sciences, queen mary, university of london we describe the development and creation of the protein circular dichroism data bank (pcddb), a deposition data bank for validated circular dichroism spectra of biomacromolecules. its aim is to provide a resource for the biological and bioinformatics communities, by providing open access and archiving facilities for circular dichroism spectra. it is named by parallel with the protein data bank (pdb), a long-existing valuable reference data bank for protein crystal structures. it will permits spectral deposition via userfriendly web forms and will include automatic reading of a range of data formats and data mining from le headers to facilitate the process. it will be linked, in the case of proteins whose crystal structures and sequences are known, to the appropriate pdb and sequence data band les, respectively. a series of validation tools that will provide reports on data quality are included (and are accessible as stand-alone software). it is anticipated that this data bank will provide readily-accessible biophysical catalogue of information on folded proteins that may be of value in structural genomics programs, for quality assurance and archiving in industrial and academic labs, as a resource for programs developing spectroscopic structural analysis methods, and in bioinformatics studies. the relation of n-terminal residues and structural stability of l-chain apoferritin k. yoshizawa 1 , k. iwahori 3 , y. mishima 1 , i. yamashita 2 1 crest/jst, 2 atrl-matsushita, 3 nara institute of science and technology the denaturation of apoferritin by acidic solution was studied. ferritin, the ubiquitous iron storage protein, represents a well known polymeric assembly that is highly resistant to chemical and physical denaturants. it is a cage-shaped protein which is composed of 24 subunits. natural vertebrate ferritins are copolymers of two different subunits, l-and h-chains. in the recombinant h-chain apoferritin (rhf), the structural stability is decreased by deletion of the n-terminal residues. we studied the effect of n-terminal residues of recombinant l-chain apoferritin (rlf) on the acidic ph denaturation and re-assembly. we constructed rlf and mutant rlfs which are lack of 4 (fer4) or 8 (fer8) amino acid residues from the n-terminus and investigated their stability by cd spectra. among three, fer8 has the least endurance against ph decrease. in the case of fer8, the re-assembly of subunits into apoferritin can be performed by increasing solution ph without causing the by-product while huge aggregations are caused in fer0 and fer4. the structural comparison of three mutants indicates that the hydrogen bonds of inter-and intra-subunits decrease by the loss of the n-terminal residues. therefore, it is elucidated that the hydrogen bonds of inter-and intra-subunits from n-terminal residues affect the molecule stability and re-assembly of l-chain apoferritin. pressure perturbation calorimetic studies of solvation, unfolding and aggregation of proteins r. winter university of dortmund, physical chemistry, otto-hahn-str. 6, d-44227 dortmund pressure perturbation calorimetry (ppc) was used to study the solvation and volumetric properties of various proteins in their native and unfolded state. in ppc, the coef cient of thermal expansion of the partial speci c volume of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps, which strongly depends on the interaction of the protein with the solvent or cosolvent at the protein-solvent interface. the effects of ph and various chaotropic and kosmotropic cosolvents (glycerol, sucrose, urea, guhcl, salts, etc.) on the solvation and unfolding behavior of the proteins was also investigated, and the observed volume and expansivity changes are correlated with further thermodynamic and spectroscopic properties of the systems. depending on the type of cosolvent and its concentration, speci c differences are found for the solvation properties of the proteins, and the volume change upon unfolding may even change sign. taken together, the data obtained lead to a deeper understanding of the solvation process of proteins in different cosolvents in their native and unfolded states. in addition, the effects of con nement and crowding on the solvational properties of the proteins were studied. finally, the use of ppc for studying intermolecular interactions and aggregation (amyloidogenesis) phenomena of proteins (e.g., insulin, prp) will be discussed. the in uence of semisynthetic derivatives of phenolic lipids on activity of yeast abc pumps phenolic lipids are the natural amphiphilic long-chain homologues of orcinol (1,3-dihydroksy-5-methylbenzene). they occur in numerous plants and microorganisms. resorcinolic lipids exhibit high af nity for lipid bilayer and biological membranes and are able to modify the activity of membrane enzymes (e.g. pla 2 , ache). the in uence of semisynthetic derivatives of phenolic lipids on yeast pdr protein activity was studied by spectro uorimetric method using the potentiometric uorescence probe dis-c 3 (3). the probe is expelled from s. cerevisiae by abc pumps and can conveniently be used for studying their performance. two pump-competent s. cerevisiae strains and different pump-free mutant strains were used to experiment to check the effect of the semisynthetic derivatives of phenolic lipids on activity of abc transporters. two of these derivatives, named 23.1 and 23.2, seem to affect the activity of pdr pumps. their in uence on activity of yeast plasma membrane multidrug resistance abc pumps is concentration-dependent. in uence of lipid membrane composition on pglycoprotein activity k. bucher, s. d. krämer, h. wunderli-allenspach department chemistry and applied biosciencies, eth zurich, switzerland p-glycoprotein (p-gp), a membrane atpase expelling many structurally unrelated compounds, is one of the major contributors to multidrug resistance. it is proposed that substrates bind to it within the membrane and are exported from there out of the cell. p-gp substrates are generally hydrophobic and their binding to the transporter is governed by their ability to partition into the membrane. the intimate association of both p-gp and its substrates with the membrane suggests that p-gp function may be regulated by the composition of the lipid bilayer. as detergents in uence the membrane properties and have been shown to affect p-gp atpase activity, we developed virtually detergent-free proteoliposomes to investigate the in uence of the membrane environment on the atpase activity of p-gp. the basal and substrate induced atpase activity was dependent on the cholesterol level of egg phosphatidylcholine (pc) membranes. the compound concentration at half maximal activation of p-gp (k m ) in proteoliposomes correlated with the af nity of the respective compound to liposomes consisting of the same lipids as the proteoliposomes tested. in conclusion, the basal and drug-induced atpase activity of p-gp is strongly dependent on the cholesterol content in detergent-free p-gp/egg pc/cholesterol proteoliposomes. m. berchel 1 , j. jeftic 1 , t. benvegnu 1 , j.-y. thepot 2 , d. plusquellec 1 1 enscr umr cnrs 6052, campus de beaulieu, 35700 rennes, 2 université de rennes 1, umr cnrs 6509, 35042 rennes bipolar lipids found in archaebacterial membranes, generally termed bolaamphiphiles, induce increased stability in membranes exposed to environments such as acidic conditions, high temperatures, high salt concentrations and/or absence of oxygen. we have synthesized a spin labeled unsymmetrical bolaamphiphile that selforganises in water solutions in multilamellar vesicles and shows slow ip-op phenomenon in comparison to conventional liposomes. generally, the ip-op from the exovesicular to the endovesicular membrane surface is a relatively slow process, which is due to the high energy barrier in transferring the polar amphiphilic heads through the lipophilic membrane. it can be involved in membrane transport mechanisms and in facilitating the transport, cells have evolved to use various supramolecular strategies. the half-life of the ip-op is estimated to more than twelve hours. we are now modulating the ip-op rate by incorporating chemical modi cations such as addition of cyclopentanes, double or triple bonds into the bridging chain of the molecule, in order to control the membrane transport via the ip-op mechanism. transport activity of the monocarboxylate transporter 1 is increased by carbonic anhydrase h. m. becker, j. w. deitmer abt. allgem. zoologie, tu kaiserslautern, 67663 kaiserslautern the enzyme carbonic anhydrase (ca), which catalyses the conversion of co 2 and h 2 o to bicarbonate and protons, is present in nearly all animal cells, and is highly expressed in astrocytes. it is known that ca can bind to several membrane transporters, forming a transport metabolon and thereby enhancing the transport activity of the protein. in this project we have studied the functional interaction of the enzyme with the monocarboxylate transporter 1 (mct1), which transports lactate and other monocarboxylates together with protons and is believed to play a pivotal role in the metabolite shuttling between astrocytes and neurons. therefore we expressed mct1 and then injected ca into xenopus oocytes. our results indicate a direct binding of ca to the mct1, leading to a ca-induced increase in acid/base ux mediated by the transporter. interestingly, the effect was insensitive to the ca inhibitor ethoxyzolamide and to the nominal absence of co 2 /hco 3 , but disappeared when binding of ca to the mct1 was hindered. it seems, that ca, bound to the mct1, mediates local buffer capacity by removing protons transported into the cell via the mct1. this helps to stabilise the proton gradient close to the cell membrane, and thereby enhances the transport activity of the mct1. these ndings suggest that ca can enhance metabolite-acid/base transport, by forming a transport metabolon with the mct1. melibiose permease of e.coli (mel b) is a membrane bound ioncoupled sugar symporter that uses the favorable na , li , or h electrochemical potential gradient to drive cell accumulation ofor -galactosides. cysteine scanning mutagenesis, electrophysiological (ssm -solid supported membranes) and uorometric measurements, were used in order to better understand the role of speci c parts of the protein in the function of this symporter. the ssm technique combines a rapid solution exchange with the high sensitivity of planar lipid membranes. it employs a solid supported membrane as a capacitive electrode and allows the time resolved investigation of charge translocation during the catalytic cycle of such transporters as na /solute symporters. in order to obtain some more precise information about the function of mel b symporter, starting from the c-less melb, the mutant g117c was constructed and from electrical, spectro uorimetric and fret measurements carried out on this mutant, in the absence and in the presence of speci c inhibitors, conclusions were drawn about the possible role of the helix iv in the function of the symporter. two-dimensional crystallization of co-reconstituted ca2+-atpase, phospholamban and sarcolipin the sarcoplasmic reticulum ca 2 -atpase and its regulators phospholamban (plb) and sarcolipin (sln) form a primary control mechanism in the recovery of resting state calcium levels in the myocardium. defects in the regulation of ca 2 -atpase by plb and sln are central determinants in cardiac contractility and disease states such as cardiomyopathy. given the signi cance of these proteins, the structural details of their regulatory mechanisms remain an important future goal for the clinical improvement of heart disease. using co-reconstitution into proteoliposomes at low lipidto-protein ratios, we have examined the effects of mutation on the functional properties of plb and sln, revealing novel insights into calcium pump regulation. in addition, these same co-reconstituted proteoliposomes have been used for structural studies by electron cryo-microscopy. in an attempt to better de ne the structural interactions between plb, sln and ca 2 -atpase, we have sought methods for the production of large two-dimensional crystals suitable for high resolution electron crystallography. we previously utilized the co-reconstituted proteoliposomes to produce long, tubular crystals suitable for helical reconstruction. our new procedure comprises three steps -co-reconstitution, membrane fusion, and crystallization -producing large two-dimensional crystals suitable for high resolution structural studies. herein, we will present our latest results characterizing the structural interaction between plb, sln and ca 2 -atpase. t. v. demina 1 , n. s. melik -nubarov 1 , h. frey 2 , e. e. pohl 3 1 state university, chemistry department, moscow, russia, 2 institute of organic chemistry, johannes-gutenberg-university, mainz, germany, 3 humboldt university, charite, institute of celland neurobiology, berlin, germany multidrug resistance (mdr) of tumours is associated with overexpression of the p-glycoprotein responsible for an active drug ef ux from cells. block copolymers of ethylene oxide and propylene oxide (pluronics) are known to cause a pronounced chemosensitization of tumour cells. the effect may be due either to speci c polymer -protein interactions or to unspeci c lipid bilayer disturbance. we have shown recently that amphiphilic copolymers with various hydrophilic and hydrophobic blocks can disturb lipid bilayers. importantly, that block copolymers of propylene oxide and glycerol (ppo-pg) with hyperbranched "corona" induced larger effects then pluronics with linear polyethylene oxide chains. in the present work we have shown that ppo-pg copolymers increase dox cytotoxicity towards human erythroleukemia (k562is9, k562/dox) and breast carcinoma (mcf7/dox) resistant cell lines. using confocal and two-photon microscopy, we demonstrated that these copolymers accelerated dox penetration into resistant cells, inhibited efux and caused drug redistribution into nuclei. a clear correlation between the ability of the polymers to disturb lipid bilayers and favour drug accumulation in mdr cells was disclosed. this nding points to an unspeci c mode of the copolymers' chemosensitizing activity. voltage dependence of processes related to electrogenic membrane transporters electrogenic membrane transporters, such as the sodiumbicarbonate cotransporter (nbce1), may induce dependence on membrane potential upon processes which are innately voltageindependent. we tested this hypothesis by heterologously coexpressing electrogenic nbce1 from human kidney in oocytes of the frog xenopus laevis with the electroneutral rat monocarboxylate transporter mct1. the apparent intracellular buffer capacity was increased by nbce1 expression and became voltage-dependent by 7 mm/10 mv membrane depolarisation. lactate transport via the mct1 not only became enhanced after co-expression with nbce1, but also dependent upon membrane potential. injection of carbonic anhydrase caii from bovine erythrocytes into oocytes enhanced the ef cacy of nbce1 activity, identifying an additional, ca-sensitive, membrane current via nbce1. our results show that nbce1 adds voltage-dependent buffer capacity to the cytosol; this is suggested to be the prime cause for enhancing acid/base-coupled transport and conferring membrane potential dependence on transporters which are stoichiometrically electroneutral. these interactions may have functional consequences for cells and tissues, where electrogenic and electroneutral processes interact, such as in brain, heart and muscle. . by using carboxy-snarf-1 as ph-sensitive uoroprobe and microspectro uorimetry, we now show that nhe1 activation is due to jun kinase (jnk) activation, resulting from reactive oxygen species (ros) produced during metabolism of b(a)p and might involve lipid raft. when analysing b(a)p-induced apoptosis, we have found that cariporide signi cantly reduces both nuclear fragmentation and caspase-3 like activity. we further show that nhe1 activation and/or alkalinization affects the mitochondrial ros production detected during the apoptotic cascade, likely via an effect on the complex iii of the electron transport chain. altogether, our results suggest that apoptotic xenobiotics, such as benzo[a]pyrene, induce an early activation of nhe1 that might play a signi cant role in the subsequent mitochondria-dependent apoptosis. conformational dynamics of a lactose proton symporter j. c. holyoake, m. s. sansom biochemistry department, university of oxford, south parks road, oxford, ox1 3qu, u.k. transporter proteins are an essential class of proteins, catalysing the transfer of molecules across membranes. increasing numbers of transporter structures are becoming available, opening the way to study their dynamic properties using computational techniques. we present a study on the conformational dynamics of the lactose proton symporter lactose permease using molecular dynamics simulations. these simulations exhibit large scale conformational changes from the initial intracellularly open conformation to a more closed conformation that may be signi cant to the transport mechanism. the conformational change is analysed to identify the contributing motions. effects of nortriptyline and chlorpromazine on anthroylouabain-labeled na,k-atpase e. a. guevara 1 , m. l. barriviera 1 , a. hassón-voloch 2 , s. r. louro 1 1 departamento de física, puc-rio, rio de janeiro, brazil, 2 instituto de biofísica carlos chagas filho, ufrj, rio de janeiro, brazil the effects of nortriptyline and chlorpromazine (cpz) on the uorescence properties of anthroylouabain (ao)-treated na ,k -atpase of electrocyte membranes from e. electricus are studied. na ,k -atpase oscillates between two major conformations e 1 and e 2 during ion transport cycle. the cardiotonic steroid ouabain speci cally inhibits this enzyme binding to the e 2 conformation. the uorescent label ao presents increased uorescence when binding to the ouabain site of na ,k -atpase. tricyclic drugs such as the antipsychotic cpz and the antidepressant nortriptyline inhibit na ,k -atpase activity in the micromolar range. for the e 2 enzyme, but not e 1 , nortriptyline was found to increase the uorescence in a concentration dependent manner, suggesting a further stabilization of e 2 . for both conformations, cpz induces negligible uorescence change up to 10 µm. the uorescence of atpasebound ao, however, strongly increases upon ultraviolet exposure after cpz treatment at concentrations around 20 µm. fluorescent products of cpz-photodegradation were studied in pure buffer and in the presence of membranes. the results suggest that cpz binds to na ,k -atpase and photolabels amino-acid residues near the ouabain binding site. how important is protein exibility for transport through ion channels? a. a. gray-weale university of sydney certain ion channels are selective for k+ over other ions, but the geometry of the pore does not explain selectivity because thermal uctuations are too large. i extend the usual treatment of ion channels with molecular dynamics simulation by calculating the static and dynamic pair correlations between monovalent ions and ion channels (gramicidin-a and kcsa), and also between certain small, complex cations and the gramicidin channel. this means not only the radial-distribution functions or the density pro les, but also correlations between the ion and the mass and charge densities of various regions of the protein. the advantage of this approach is that it systematically identi es the elements of the protein and modes of motion that contribute to selectivity, and illustrates the decay of correlations. recently, noskov et al. [1] showed that thermal uctuations protect selectivity. my results on the interaction of ions with carbonyl groups agree with theirs, but take the analysis further to higher correlations. the key new element is the study of the time-correlation functions that describe the motion of the ions through the channel, borrowing methods originally developed for the study of dense or even supercooled liquids. the surface-enhanced infrared absorption spectroscopy (seiras) is used for the investigation of two membrane proteins, the cytochrome c oxidase (cco) and the bacteriorhodopsin (br). of central interest are the transport-mechanisms of electrons (cco) and ions (br). the main parts of the setup are an infrared light source, a hemispherical si-crystal, in which the beam is internal re ected and a plexiglas cell with buffer solution (see schematic scetch). the beam is totally re ected on the inner at surface of the crystal, but the evanescent wave excites surface plasmons in a chemically adsorbed gold-layer on the crystal (attenuated total re ection spectroscopy-atr). the protein to be analysed is attached at the gold surface and can absorb certain wavelengths. the gold is need for the surfaceenhancing effect. cco plays a major role in the respiratory chain, the retinal protein br is a photosynthetic protein. the cco is immobilized on the gold surface via the af nity of its histidine-tag to a nickel-chelating nitrilo-triacetic acid (nta) surface. for the br we incorporate the protein in a lipid membrane, which is attached on the gold surface by the sulfuric bindings of 2,3-di-o-phytanyl-sn-glycerol-1-tetraethylene glycol-d,l-lipoic acid ester lipid (dptl). the sensitivity of this method is further enhanced by modulation of an external parameter, like the electric potential. effects of copper ions on the escherichia coli growth and proton-potassium exchange copper ions are required for the function of many important enzymes in escherichia coli but can cause a number of toxic cellular effects also. it's interesting to reveal the in uence of copper ions on the growth of bacteria and proton-coupled membrane systems. upon transition of e. coli mc 4100 wild-type culture to stationary growth phase a decrease in redox potential (e h ) from the positive values ( +140 mv) to the negative ones (of -380 to -550 mv), resulting h 2 production by formate hydrogenlyase (fhl) have been studied. copper increased a latent growth phase duration as well as delayed a logarithmic growth phase in concentration-dependent manner. during the anaerobic growth, the production of h 2 was strongly inhibited in the presence of cucl 2 (2 mm). 0.1 mm cucl 2 was inhibited h 2 production under experimental conditions with glucose. the inhibitory effect of copper ions (0.1 mm) on n',n'dicyclohexylcarbodiimide (dcc)-sensitive h /k exchange was also observed: k uptake was decreased and the stoichiometry of dcc-inhibited ion uxes varied. interestingly, these effects on h and k uxes were absent for the mutant hd700 (hyc-operon for hydrogenase was deleted). we suggest that copper ions, inhibiting the activity of fhl, have an effect on h /k -exchanging mechanism which is the proton f 0 f 1 -atpase associated with k uptake trk system. this effect may be due to the relationship of fhl with the ion-exchanging mechanism above under fermentation at alkaline ph. lateral diffusion in tethered bilayer membranes m. jung, v. atanasov, w. knoll, i. koeper max planck institute for polymer research, mainz, germany tethered bilayer membranes (t'blm) provide a useful platform for the investigation of bilayer membranes as well as embedded membrane proteins. we have developed a modular system, which is suitable for surfaces providing gold and oxide surface coatings. these systems serve as a quasi natural environment for the study of membrane proteins, being functionally incorporated into a lipid bilayer, which is covalently bound (tethered) to the substrates. functionality could be shown using electrochemical methods. here we present a study of these systems, basically the membrane itself, using uorescence recovery after photobleaching (frap) studies in order to investigate lateral motion in the lipid bilayers. lateral mobility is essential for successful incorporation of large membrane protein complexes. we will present rst results of experiments that try to differentiate motions hindered due to the tethering from diffusion in free oating or suspended bilayers. the information gained in this study will serve for improvements in the chemical structure of the tethered molecules. we will develop the system as a basis for bio sensing applications, where embedded proteins will serve as actual sensing elements. a. d. ivetac, j. campbell, m. s. p. sansom biochemistry department, university of oxford, south parks road, oxford, ox1 3qu, u.k. atp-binding cassette (abc) transporters form an important superfamily of membrane proteins which couple atp hydrolysis to the active transport of diverse compounds across the cell membrane. their biomedical relevance is highlighted in examples such as multidrug resistance to antibacterial and anticancer agents, and cystic brosis. the availability of crystal structures of three complete bacterial abc transporters provides an opportunity to study structurefunction relationships at the atomic level. in this work, we carry out multi-nanosecond molecular dynamics simulations of the vitamin b 12 importer from e. coli (btucd), with both the complete multimeric transporter embedded in a phospholipid bilayer and the soluble subunits in a membrane-free environment, in an attempt to elucidate some of the conformational changes which arise during the transport event. atp-bound and atp-free structures are used to investigate the effect of nucleotide on the system. a range of analytical techniques have been applied to assess the dynamic behaviour of the protein during the simulations, which includes measurements of: conformational drift, residue exibility, transmembrane domain (tmd) movement, concerted protein motions, nucleotide-binding and translocation pathway changes. an in-vitro method was designed to measure transmembrane transport rates. liposomes were prepared by extrusion with dipalmitoyl phosphatidylcholine (dppc) and optionally cholesterol, and loaded with a peptide (zinc-insulin tagged with a uorescent group or bsa). after removal of the non-encapsulated peptide from the liposome solution by gel ltration, the release of the peptide from the liposomes was monitored by uorescence as a function of time at various temperatures. the transport was greatly accelerated by the presence of a speci c proprietary excipient molecule (cyclopentadecanolide -cpe215™), which effectively triggered the release of the peptide. a mathematical model was developed to quantify these results. a semi-empirical nonlinear equation involving four parameters ts the protein release pro les. then a neural network predictions model was used to correlate the different release condition parameters and the four semi-empirical tting parameters based on the experimental data sets. most release data t well with the mathematical model, further supporting our theory of a two step release mechanism. phenyltin the well known group of organotin compounds that exhibit toxic properties in relation to the biological systems are phenyltins. no studies have been performed as yet to establish directly whether organotins such as diphenyltin dichloride ( dpht) and triphenyltin chloride (tpht) cross the lipid bilayer. we have performed experiments that showed transfer of those compounds across the lipid bilayer using the stopped-ow technique and desorption of those compounds from a monolayer using the langmuire technique. obtained results demonstrate that dpht and tpht rst adsorb onto the lipid bilayer surface, in diffusion controlled manner, within a very short time (0.05 s), whereas the membrane passing was observed in a minute's time range. the long time kinetics show a complex dependence on the kind of compound, its concentration and the presence of cholesterol in the membrane. the desorption of both compounds from the monolayer to water subphase occurs in a minute's time range. these observations may explain the known fact, that the in uence of organic, amphiphilic tin (and also lead) compounds is more toxic than that of inorganic ones.the phenyltins much easier (compared with tin or lead ions ) penetrate e.g. blood -brain barrier. [ under the resting conditions ca 2 concentration in agonistsensitive ca 2 stores re ects a balance between active uptake mediated by a ca 2 -atpase (serca) and passive ef ux of ca 2 . this ca 2 leak appears to be a common property of ca 2 -storing organelles, but the nature of the leak in submandibular acinar cells remains unclear. we have studied the ca 2 leak pathways in the endoplasmic reticulum (er) of acinar cells of rat submandibular salivary gland by directly measuring concentration ca 2 in the er ([ca 2 ] er ) in mag-fura 2/am preloaded cells while [ca 2 ] i was clamped at a resting level with a egta/ca 2 mixture. we have shown that thapsigargin (tg) or ca 2 -free buffer treatment completely blocked ca 2 uptake by serca after the rst minute of superfusion and caused a ca 2 leak represented by continuous decline in [ca 2 ] er . this ca 2 leak from the er was not sensitive to tg, heparin and ruthenium red and therefore appears to be independent of the serca, the insp 3 receptor and the ryanodine receptors. however, treatment with puromycin (0.1-1 mm) to remove nascent polypeptides from er-ribosome translocon pores increased ca 2 leak from the er by a mechanism independent of the serca, insp 3 or ryanodine receptors. thus we conclude that basal ca 2 leak from the er of submandibular acinar cells occurs through translocon pores in the er membrane. r. b. kishore, j. reiner, e. edgu-fry, a. jofre, k. helmerson physics laboratory, national institute of standards and technology we have developed a procedure to make lipid and polymer nanotubes of up to one cm long and 50 nm in diameter, from the surface of giant liposomes and polymersomes, using micro uidics and optical tweezers. the liposomes and polymersomes were formed, using elcetroformation method, from phospholipids and amphiphilic diblock copolymers, respectively. the polymer tubes were made extremely robust by cross-linking them using chemical reactions. we are currently studying the transport of molecules in the crosslinked nanotubes for use in nano uidic networks. p-glycoprotein (p-gp) is an active membrane transporter capable of expelling out of the cell a large number of potentially cytotoxic amphiphilic molecules with unrelated chemical structures. as a consequence, p-gp may be responsible for multidrug resistance of tumors against chemotherapy (mdr); it also plays a key role in absorption, biodisposition and elimination of many pharmaceuticals. to understand the molecular mechanisms of the transmembrane drug transport mediated by pgp, it is highly desirable to design a convenient assay for measuring both p-gp atpase activity and p-gp transport function. to do so, we used inside-out native membrane vesicles, prepared from mdr cells and containing high amounts of p-gp. we took advantage of the speci c property of a uorescent dye, the carbocyanin jc-1, known to be expelled out of mdr cells: above a critical concentration (the "cjc"), this dye forms j-aggregates which emit a uorescence at a wavelength very different from that emitted by the monomer. in the presence of mgatp, the p-gp-containing vesicles accumulated jc-1, which exceeded locally the cjc and thus formed intraluminal j-aggregates; these aggregates allowed accumulated jc-1 both to be sequestered inside the vesicles, by dramatically slowing down its passive backdiffusion, and to be speci cally detected. kinetic characterization of this transport suggests that jc-1 is rst translocated to the exoplasmic lea et of the vesicle membrane before its internalization into the aqueous phase of the vesicle lumen. interaction between the energy metabolism and externally applied electric elds in yeast cells electric elds are often used for biophysical or biomedical treatment of biological cells, e.g. cell fusion or killing of cells. however, only a few data about the possible mechanisms of electrosensitivity of biological cells are available. since electrostimulation always induces depolarization of biomembranes, an impact of the energy metabolism is obvious due to the regeneration of electrochemical gradients by the expenditure of cellular energy. our aim is to investigate the interactions between externally applied electric elds and the aerobic/anaerobic energy metabolism of yeast cells. for this, we have constructed a new electrical interface for local stimulation of biological cells with variable duration and amplitude. when applying short lasting electrical pulses to yeast cells, we nd a direct response of the energy metabolism (measured by nadh-uorescence) to these pulses. a sudden and fast decrease in nadh is followed by a slower recovery of the uorescence signal. these nadh-signals are abolished in the presence of antimycin a or kcn, demonstrating the importance of mitochondrial energy production for this phenomenon. we attribute these changes to the immediate break down of atp as a consequence of the regeneration of the membrane potential (atpases) and the slower regeneration of atp by mitochondrial respiration. new insights of hypericin blood transport and its incorporation into the plasma membrane b. m. macri 1 , g. stoian 2 , m. l. flonta 1 1 department of animal physiology and biophysics, faculty of biology, university of bucharest, 2 department of biochemistry, faculty of biology, university of bucharest, bucharest, romania hypericin (hyp) is one of the active compounds from hypericum perforatum (an herb usually prescribed as antidepressant). the bioavailability of this hydrophobic molecule is a very important issue for medical applications. the goal of our work was the study of hyp blood transport mechanisms. techniques of absorbtion spectroscopy, electrophoresis and uorescence microscopy were used in order to de ne the properties of hyp-albumin and hyp-lipoproteins complexes and to explain the action of hyp at the plasma membrane level. hyp bind to several electrophoretic (sds-page) bands evidenced by mice plasma migration. both albumin and lipoproteins bind to hyp, forming complexes, during the blood transport process. different types of lipoproteins from males and females plasma mice were evidenced by gradient electrophoresis to bind hyp. hyp-albumin complex was also identi ed by absorbtion spectra, and the ratio a 594 /a 550 has a ph-dependence. hyp interaction with plasma membranes was also examined on cell culture by uorescence microscopy, and hyp plasma incorporation is a dose-and incubating time-dependent process. our results partially elucidate the plasma fractions that bind hyp, contributing to its blood transport. this study proposes a new mechanism of hyp cellular insertion, discussing its plasmatic membrane penetration due to its high hydrophobicity. the overexpression of p-glyoprotein (p-gp) is one of the major causes of multidrug resistance (mdr) in cancer chemotherapies. many p-gp inhibitors have been designed to reverse the mdr effect, but the structure-activity relationships of the p-gp inhibitors and substrates still remain largely unknown. until now, it is still very challenging to obtain the high-resolution p-gp structures and currently only low-resolution electron microscopy structure is available. this has caused the structure-based design of "p-gp-ignoring" therapeutic agents a remote goal. however, the recent determination of x-ray crystal structures of bacterial lipid a transporter, msba, has provided eligible structure templates for homology modeling of p-gp. we have therefore conducted explicit solvent molecular dynamics simulations of the fulllength ef ux pump, human p-gp, in an excessively hydrated popc bilayer to re ne the homology model. both free and atp-bound forms of p-gp have been simulated. the entire system consists of more than 365,000 atoms. our molecular dynamics simulations have shown that the overall architecture of p-gp remained very stable for tens of nanoseconds, while the observed membrane undulation was rather large. the simulation results have allowed us to investigate the conformational changes of p-gp upon atp binding in the ef ux process and to predict the possible binding site of various known substrates and inhibitors. the re ned structure models of p-gp by our simulations could be used as the basis for further drug design. electroporation (ep) is a phenomenon where increased permeability of cells exposed to an external electric eld is observed. the induced transmembrane voltage presumably leads to the formation of aqueous pores in the phospholipid bilayer, which increases permeability of the membrane for molecules and ions. ep is currently used in many biomedical applications including transfer of genes and electrochemotherapy of tumors. still, the molecular mechanisms of the process are not fully explained. recently it was proposed that ep could be monitored in real-time by measuring electric conductivity of tissue. so far the studies focused mostly on a single pulse, however in biomedical applications usually several pulses are used. in our study we used a train of electric pulses to analyse the relationship between electric conductivity and cell permeabilization. current-voltage measurements during and after pulse application were performed in dense suspension of cells. conductivity changes were analysed numerically using nite elements method and compared with the percentage of permeabilized cells. we obtained a transient increase in conductivity above a certain voltage with complete relaxation in < 1s. substantial changes in conductivity are also due to the diffusion of ions through membrane pores and osmotic swelling. we further show that relation between conductivity and permeabilization level is indirect. interaction of quinolones with bacterial porin ompf: uorescence quenching studies quinolones are widely used antibiotics witch develop their antibacterial action by inhibition of important bacterial enzymes. consequence of the internal location of their target of action, the translocation of this drugs trough the outer membrane is an essential step for their antibacterial action. in vivo studies have been showing that ompf is important for the entrance of some of these antibiotics, but the exact degree of involvement of this protein in the transport of the different members of this group of antibiotics, remains unknown. in this study, the quenching of the intrinsic tryptophan uorescence of ompf, in presence and in the absence of the drugs and by two distinct quenchers, was used as a rst approach, to elucidate ligandinduced structural changes and consequently prove the differential involvement of ompf in the entrance of these antibiotics in the bacterial cell. the results obtained reveal that the degree of interaction with the protein is related with the hydrophobicity of the different antibiotics. this kind of evidence suggests that the entry by the porin channel is not the only path used by these antibiotics and that it is more important for the latest generations of this group because of their increased hydrophilic characteristics. inhibition of multidrug resistance-associated protein mrp1 and kv channels by natural polyphenols k. michalak, a. teisseyre, b. ania-pietrzak department of biophysics, wroclaw medical university, poland resistance to cytotoxic agents remains a major obstacle to successful chemotherapy in cancer. best-characterized form of drug resistance is caused by the overexpression of genes encoding membrane drug pumps, like p-gp or mrp1. in present study, activity of several plant polyphenols ( avonoids and stilbene) against mrp1 has been studied using functional assay based on ef ux of mrp1 uorescent substrate. very recently, a role of kv1.3 potassium channels in proliferation of various cancer cells was suggested. in our study the effect of the plant polyphenols on voltage-gated potassium channels kv1.3 was investigated by patch-clamp electrophysiological method. some of studied compounds were found to be active inhibitors of multidrug resistance-associated protein mrp1 and voltage-gated potassium channels, and their properties are promising for further research in the eld of anticancer activity of natural products. the transport mechanism of melibiose permease: a study using electrical measurements and uorescence techniques k. meyer-lipp 1 , c. ganea 2 , t. pourcher 3 , g. leblanc 3 , k. fendler 1 1 max planck institut für biophysik, frankfurt, germany, 2 c. davila medical university, bucharest, romania, 3 cea, université de nice so a antipolis, nice, france the melibiose permease (melb) of escherichia coli is a membrane bound carrier that uses the favorable na+, li+, or h+ electrochemical potential gradient to drive cell accumulation of alfa-galactosides (melibiose, raf nose) or beta-galactosides (methyl-1-thio-beta-dgalactopyranoside). electrophysiological techniques have proved to be extremely useful tools to investigate the mechanism of ion transfer across the membrane by ion-coupled transporters. using a solid supported membrane (ssm) as a capacitive electrode a rapid solution exchange can be combined with the high sensitivity of planar lipid membranes and allows time resolved investigation of the charge translocation during the catalytic cycle of na+/solute symporters. this technique has been combined with uorescence measurements, which report on structural changes during the substrate transport process of the carrier. we have used time resolved tryptophane uorescence, uorescence energy transfer with a uorescent sugar substrate and site speci c uorescence of a label attached to a cysteine residue on the protein. this allowed us to identify conformational transitions during the reaction cycle of the melibiose permease. we could assess their electrogenicity and determine rate constants. a kinetic model for na+ and melibiose binding and transport is presented. electrophysiological characterization of the vast number of annotated channel and transport proteins in the postgenomic era would be greatly facilitated by the introduction of rapid and robust methods for the functional incorporation of membrane proteins into dened lipid bilayers. we present an automated method for reconstitution of membrane proteins into lipid bilayer membranes, that substantially reduces both the reconstitution time and the amount of protein required. we have applied this well-de ned system to the characterization of a novel mitochondrial uncoupling protein, ucp2 and demonstrated that ucp2 exhibits protonophoric function exclusively in the presence of fatty acids, similar to that previously shown for its homologue ucp1. the membrane conductance was proportional to the concentration of the reconstituted ucp2 in presence of oleic acid or eicosatrienoic acid, and was inhibited by atp. amphipols are amphipathic polymers designed to replace or supplement detergents in membrane protein solution studies. for the study of the ca 2 -atpase from sarcoplasmic reticulum, previous experiments have revealed both advantages and disadvantages to the use of a polyacrylate-based amphipol, a8-35. these issues have been reinvestigated using four different amphipols. size exclusion chromatography showed that, although a8-35 aggregates in the presence of millimolar concentrations of calcium -an effect that probably accounts for most of the aggregation of atpase/a8-35 complexes observed in our previous work-, aggregation can be avoided by resorting to a sulfonated version of a8-35. we also found that all amphipols tested slowed down the rate of calcium dissociation from its binding sites and reduced atpase activity, while protecting the solubilized protein against denaturation. this suggests that association with the polymer may damp the protein's dynamics, perhaps due to the multipoint attachment of the polymer to its hydrophobic transmembrane surface. such a "gulliver" effect could contribute both to the protection of membrane proteins against denaturation and to the reversible inhibition of serca1a. clc proteins are found from prokaryotes to mammals. they function as plasma membrane chloride channels or provide neutralizing anion currents for v-type h -atpases that acidify compartments of the endosomal/lysosomal pathway. vesicular clcs have been thought to be cl -channels, in particular because clc-4 and clc-5 mediate plasma membrane cl -currents upon heterologous expression. we have shown, however, that these two mainly endosomal clc proteins rather function as electrogenic cl /h exchangers, resembling the transport activity of the bacterial clc-e1 that has been crystallized. neutralization of a critical glutamate residue not only abolished the steep voltage-dependence of transport, but also eliminated the coupling of anion ux to proton counter-transport. clc-4 and clc-5 may still compensate the charge accumulation by endosomal proton pumps, but are expected to tightly couple vesicular ph-gradients to cl -gradients. calorimetry and mechanics of ca 2 transporting systems in rat myocardial bigeminies a series of new n-oxides of tertiary amines (nta) was checked for its biological activity. individual compounds differed in the length of substituted alkyl chain. the primary goal was to nd if they can be used as effective antioxidants and, to what degree they modify used model (liposomes) and biological (erythrocytes, algae and cucumber) membranes. various methods were used in order to do that. a mechanism of the interaction between nta and membranes was studied by measuring their potency to hemolyse erythrocytes, to in uence a phase transition temperature in dppc liposomes, to change a membrane potential of algae cells. the measure of the interaction of nta with cucumber cells were potassium leakage, chlorophyll content and inhibition of growth of hypocotyls. antioxidative abilities of nta were determined by measuring their ef ciency to protect erythrocytes against membrane lipid oxidation induced by uv irradiation and by comparising their antioxidative ef ciencies with that of trolox (vitamin e analogue) in chromogen experiments. the mostly widely employed mechanism of drug extrusion in bacteria is via membrane transport proteins called ef ux pumps. in gram-negative bacteria, multidrug resistance is conferred by tripartite complexes, rather than by a single transport protein. through these systems, a wide range of substrates is expelled from the cytoplasm, through the periplasmic region, to the exterior of the cell. among these complexes, the acrab/tolc system in escherichia coli is formed by an inner membrane ef ux pump, acrb, an outer membrane protein, tolc, and a periplasmic protein known as an adaptor, acra. the components of this complex are studied, in order to provide insights into drug transport in bacteria. here we present a dynamics study on mexa, homologue of acra from pseudomonas aeruginosa. the protein has been studied by molecular dynamics simulations in bulk water. a structural adjustment by the periplasmic protein is required in order to engage both the bottom part of the om protein and the top region of im protein. the dynamics on mexa reveals a exible behaviour of the protein in water. the major concerted motions observed are the hinge-bending of the two domains, and the rotation of the -barrel domain. these can be related to the adaptation of mexa (and acra) to the om and im proteins during the process of assembly in forming the complex, and during the opening of the channels. electrophysiologic study of ap in chara corallina -indication of its biochemical nature the speci c conductance's of aqueous solution of electrolytes (viz.naf, nacl, nano 3 , na2so 4 , kf, kcl, kno 3 , k 2 so 4 , mgcl 2 , cacl 2 , fecl 3 , mncl 2 ,crcl 3 ,cucl 2 , cocl 2 , )have been measured across peritoneum at temperatures between(15-35) c.conductance attains a maximum limiting value at higher concentrations for each electrolyte due to a progressive accumulation of ionic species within the transmembrane region. the membrane becomes more and more conductive to incoming ions and attaining a limiting value due to the fact that an electrically neutral pore, which is speci c for a particular ion, is unlikely to contain more than one type of ion. consequently, at high electrolyte concentration, the pore saturates and the conductance's approaches a limiting value. the values of speci c conductance measured follow the sequence for anions; so 4 2 > cl > no 3 > f . whereas for the cations the sequence is k > na ; ca 2 > mn 2 > co 2 > cu 2 > mg 2 ; cr 3 > fe 3 . the energy of activation for the cations as well as for the anions follows the sequence (for cations): the low temperature (77k) chlorophyll uorescence, photochemical activity, oxygen ash yield and oxygen burst decay of thylakoid membranes with different organization of the light-harvesting chlorophyll a/b complex of photosysytem ii (lhcii) were investigated after freeze-thaw cycle in criotoxic and cryoprotective medium. the increase of lhcii oligomerization, which is associate with signi cant reduction of the surface charge density of the thylakoid membrane, correlates with lower extent of freezing damage of the photosynthetic apparatus, when the procedure is carried out in cryotoxic medium (nacl). in the presence of the cryoprotective compound (sucrose) freezing damage is less pronounced and is not affected by the degree of the lhcii oligomerization. the mechanisms of damage and protection of photosynthetic apparatus in the process of freeze-thaw treatment are discussed. spectral and redox characterization of the novel heme ci in the cytochrome b6f complex . this is an indication of different spin delocalization in the primary donor, for the mutant being typical of a monomeric oxidized bchl. considering the fact that the properties of both isolated and membrane-associated mutant rcs were similar, we conclude that missing bchl molecule from the mutant rc was the result of the introduced mutation but not of the protein puri cation procedure. authors acknowledge the support by the russian brf. we created two site-directed mutants, a249s and l267i in the d2 protein of photosystem ii in thermosynechococcus elongatus. both mutations are within the binding pocket of the primary quinone acceptor (q a ). we investigated the effects of the mutations in vivo and in isolated psii. while the l267i mutant exhibits characteristics similar to the wild type, the a249s mutation effects q a charge recombination measured by thermoluminescence and uorescencedecay. these results strongly indicate that the a249s mutation induce a shift in the redox potential of q a . the a249s accelerates the rate of photoinhibition, an effect consistent with the negative shift in the redox potential. epr was used to measure the temperature dependence of the electron transfer from q a to q b in the a249s mutant. it was found to be indistinguishable from the wild type despite the difference in the midpoint potential of q a . this is taken as an indication as a gating mechanism on the acceptor side of psii similar to that in bacterial reaction centers. protochlorophyllide oxidoreductase takes an abnormal reaction pathway below the glass transition g. durin 1 , d. j. heyes 2 , c. n. hunter 2 , d. bourgeois 1 1 ibs and esrf, grenoble, france, 2 krebs institute and r hill institute for photosynthesis, shef eld university, shef eld, uk motions through the energy landscape of proteins lead to biological function. at temperatures below a dynamical transition (150-250 k), the activity of some proteins cease. in this work, we describe an enzyme that, instead, engages into a non-productive pathway below 160k. protochlorophyllide oxidoreductase (por) catalyzes the reduction of protochlorophyllide (pchlide) into chlorophyllide (chlide), a key step in chlorophyll biosynthesis. por is one of the two enzymes known to require light for catalysis. when illuminated with gentle light at 165 k, the complex of t. elongatus por with pchlide and nadph transforms into a nonuorescent intermediate. upon warming, several uorescent intermediates develop, and at 290k chlide is released. when illuminated at temperatures below 155k, por behaves differently. if gentle light is used, the reaction can not start. instead, if a blue laser source is used, the initial complex disappears, like at 165k. however, upon warming, a new intermediate develops that uoresces at 694nm and leads to a dead-end product. by using uorescence microspectrophotometry, we have measured the solvent glass transition temperature of the system to be 158k. the solvent glass transition, possibly controlling a por dynamical transition, may be the determinant that switches the enzyme reaction pathway from a non productive to a productive one. the nonproductive pathway results from a two-photons absorption mechanism, whereas the productive pathway is a one-photon mechanism. sensory rhodopsin ii from n. pharaonis (npsrii) forms a complex with its cognate transducer nphtrii in a 2:2 stoichiometry 1 . light activation of npsrii leads to a movement of helix f which triggers a rotation of tm2 in nphtrii 2 3 . the mechanism of signal transduction through the hamp region to the cytoplasmic domain of the transducer is still unknown. structural information exists for the transmembrane and cytoplasmic regions, however the hamp domain is not yet characterized. in order to obtain structural information on this domain, twenty-four residues in the membrane adjacent region (78-101), and six residues in the following region were spin labeled and investigated by cw and pulsed x-band epr. to analyze the overall architecture of the complex, doubly spin labeled variants between the transducer and the receptor were also engineered. depending on their function, the absorption spectra of rhodopsins can be tuned by the protein over a wide range. a major determinant for spectral shifts between different rhodopsins are electrostatic interactions between the chromophore retinal and the protein. we compute and compare the classical electrostatic potential at the retinal of three archaeal rhodopsins: bacteriorhodopsin (br), halorhodopsin (hr), and sensory rhodopsin ii (srii). these proteins are an excellent test case for understanding the spectral tuning of retinal. the absorption maxima of br and hr are very similar, while the spectrum of srii is considerably blue shifted. we nd that the electrostatic potential is similar in br and hr, but differs signi cantly in srii. a quantum mechanical model of a particle in a box with a step potential can qualitatively relate the differences between the electrostatic potentials of the proteins to the relative shifts of their absorption maxima. by decomposing the electrostatic potential into contributions of individual residues, we could identify six residues that are responsible for the differences in electrostatic potential between the proteins. three of these residues are close to the retinal, while the other three residues are more then 8 angstroem away from the retinal. the counterion of the schiff base, which is frequently discussed to be involved in the spectral tuning, does not contribute to the dissimilarities between the electrostatic potentials. effect of uv-a radiation on thylakoid membranes with different organization p. ivanova, a. dobrikova, t. markova, s. taneva, e. apostolova institute of biophysics, bulgarian academy of sciences, 1113 so a, bulgaria the effect of uv-a (320-400 nm) radiation on the energy transfer and the photosynthetic oxygen evolution of thylakoid membranes from pea mutants was investigated. the membranes have different pigment composition, stoichiometry and organization of pigment-protein complexes. the aim of our work was to nd out whether uv-a induced damage is affected by the altered content and/or oligomerization of the main light-harvesting chlorophyllprotein complex (lhcii) in thylakoid membranes. the data for the effect of uv-a radiation on the oxygen evolution demonstrate that: (i) the inhibition of photosystem ii (psii)-mediated electron transport and ash-induced oxygen yields strongly depend on the amount of lhcii; (ii) the increase of the s o populations of psii centers in darkness is more pronounced in thylakoid membranes with smaller amount of lhcii; (iii) the inhibition of the oxygen evolution is related to the reduced number of the functionally active psii centres; (iv) the degree of impairing of active psii centres depend on the amount and oligomerization of lhcii. the results also show that the altered content and organization of lhcii in uence the uv-a light-induced changes in the energy transfer between psii and psi and within the supramolecular lhcii-psii complex. the effects of uv-a radiation on leaves and isolated thylakoid membranes are compared. sudden polarisation -a large change in the electric dipole moment between the excited and the ground state -is a well-known phenomenon for retinal chromophore. some early models of the energy transduction mechanism in bacteriorhodopsin (br) even attribute a primary functional role of that. however, it was apparently unrecognized that the maxwell theory intuitively predicts the appearance of an ultrafast transient electromagnetic radiation due to this dipole moment change. here we show that the existence of this type of radiation can be derived from semiclassical quantum electrodynamics as a second order phenomenon. in optical terms it corresponds to the previously unstudied resonant case of optical recti cation. recently we experimentally observed a major component in the fs coherent infrared emission of oriented purple membranes of br corresponding well to this effect (groma et. al, proc. natl. acad. sci. 101, 7971, 2004). our theory predicts that such a signal holds detailed information on the dynamics of excited state polarization, opening a new branch of impulsive spectroscopy on asymmetric systems. beyond optical recti cation we found a complex phase a coherent oscillation living for a few ps, i.e. much longer than the excited state of br. fitting analysis resulted in at least seven vibrating modes in the 700-1500 cm 1 region, while windowed fourier transform indicated time-dependent frequency distribution. a. ghignoli, g. cercignani, s. lucia, g. colombetti istituto di bio sica, cnr -pisa, dipartimento di fisiologia e biochimica, università di pisa, italy the life cycle of ophryoglena ava, a histophagous ciliate dwelling in fresh waters, reportedly includes several stages that feature morphology changes and different phototactic responses. previous studies on the phototactic responses in o. ava during its phase of maximal positive phototaxis led to an action spectrum with two main peaks at 420 and 590 nm, and a minor peak at 540 nm. starting from those results, we analyzed the phototactic response at various cell ages, using three broad-band interferential lters (fwhm = 50 nm) centred respectively at 420, 550 and 600 nm, and constructed dose-effect curves for each band. a higher photosensitivity at 420 nm, and lower photosensitivies with the other two lters (550 and 600 nm) have been observed at any cell age. however, the photosensitivities in the blue and orange regions show a different time course vs. cell age with respect to the photosensitivity in the green region. measures were also carried out on cells whose feeding cycle was altered by a 4-day starvation (a double time with respect to the standard protocol) before being fed at t = 0. the maximal photoresponse values reached by starved cells are lower than the highest values reached with standard cultures; in other words, a general reduction of the phototactic response is observed. these results suggest that, while feeding optimally induces cell division, it does not generally reset all cellular functions. a. quaranta 1 , f. lachaud 2 , y. pellegrin 2 , p. dorlet 2 , m.-f. charlot 2 , s. un 1 , a. aukauloo 2 , w. leibl 1 1 service de bioénergétique, cea-saclay, bât. 532, 91191 gif-sur-yvette cedex (france), 2 laboratoire de chimie inorganique, bât. 420, université paris-sud, 91405 orsay (france) coordination complexes based on a photoactive rutheniumpolypyridyl moiety linked to simple, rigid ligands with binding sites for transition metals, are developed to mimic the light induced charge separation and water oxidation processes taking place in the photosynthetic apparatus. inspired by the structure around the donor side of photosystem ii a family of phenanthroline based ligands holding an imidazole, a phenol or an indole unit simulating the amino acids histidine, tyrosine and tryptophan in the oxygen evolving complex, were developed as models for proton-coupled electron transfer. in some of the molecules investigated the hydrogen bonding interaction present in the natural system is reproduced. combined data from photophysical, spectroelectrochemical studies and dft calculations evidenced the photogeneration of a phenoxyl or a tryptophan radical upon excitation of the chromophore in presence of an external electron acceptor, therefore mimicking the electron trade between p 680 and tyrz-hist190. finite element model to predict the electric potential distribution in ps i containing vesicles c. p. a. pennisi 1 , e. chemineau 1 , e. greenbaum 2 , k. yoshida 1 1 center for sensory motor interaction, aalborg university, denmark, 2 chemical sciences division, oak ridge national laboratories, usa, 3 facultad de ingeniería, uner, argentina photosynthetic reaction centers (rc) are integral membrane proteins and molecular photovoltaic structures. recently, it was suggested their use as triggers of voltage-gated ion channels in excitable cells, where a certain voltage threshold has to be reached to evoke a response (kuritz et al., ieee trans. nanobiosci. in press 2005). experimental studies with rc's reconstituted in lipid vesicles have shown different values of transmembrane voltage, depending on parameters like light intensity, rc concentration and membrane passive properties. ultimately, the purpose of this work is to have a tool to estimate the proximity, number and density of rc's required near a voltage-gated channel to activate an excitable cell. as a starting point, we aim to predict the spatial distribution of the membrane potential in vesicles. a nite element model was realized using a commercial package (femlab, comsol a/s). the three-dimensional distribution of the electrical potential near a single rc in the surface of a spherical vesicle was calculated. in terms of density, in conditions of saturating light, a minimum of 1,8e 12 rcs/cm 2 is needed to develop a potential of 20 mv, capable to activate voltage-gated sodium channels. microsecond time-resolved x-ray diffraction study of purple membrane t. oka 1 , k. inoue 2 , m. kataoka 3 , n. yagi 2 1 faculty of science and technology, keio university, japan, 2 japan synchrotron radiation research institute (jasri), japan, 3 nara institute of science and technology, japan the structural changes in the photoreaction cycle of bacteriorhodopsin, a light-driven proton pump, was investigated at a resolution of 7 å by time-resolved x-ray diffraction experiment utilizing synchrotron x-rays from an undulator of spring-8. the xray diffraction measurement system, used in coupling with a pulsed yag laser, enabled to record diffraction pattern from purple membrane lm at a time-resolution of 6 µsec over the time domain of 5 µsec to 500 msec. the low temperature (77k) chlorophyll uorescence, photochemical activity, oxygen ash yield and oxygen burst decay of thylakoid membranes with different organization of the light-harvesting chlorophyll a/b complex of photosystem ii (lhcii) were investigated after freeze-thaw cycle in cryotoxic and cryoprotective medium. the increase of lhcii oligomerization, which is associate with signi cant reduction of the surface charge density of the thylakoid membrane, correlates with lower extent of freezing damage of the photosynthetic apparatus, when the procedure is carried out in a cryotoxic medium (nacl). in the presence of a cryoprotective compound (sucrose) freezing damage is less pronounced and independent of the degree of the lhcii oligomerization. the mechanisms of damage and protection of photosynthetic apparatus during the freeze-thaw process are discussed. we have studied the effect of a cytokinin meta-topolin (mt, 10 4 m) on senescence-induced changes in the photosynthetic apparatus of detached primary leaves of wheat (triticum aestivum l. cv. hereward). the senescing leaves were kept under continuous light conditions. mt signi cantly slowed down the senescenceinduced decrease in chlorophyll content and markedly stimulated violaxanthin zeaxanthin (z) conversion. the high z content was maintained even after an hour in darkness. mt treatment caused also the appearance of an emission band f699 peaking at 698-700 nm. this emission band is attributed to aggregates of lightharvesting chlorophyll a/b-binding proteins (lhc), the production of which is associated with a higher z content. the presence of lhc aggregates in mt treated leaves was documented also by electron microscopy imagines. besides the lhc aggregation, mt induced also a decrease in photosystem i content which was documented by electrophoresis and 77k-uorescent spectra. supported by grants frvs 3190/2005 and msm 6198959215. recently high resolution images of bacterial photosynthetic membranes have revealed the organization of membrane proteins in these native membranes. the organization revealed is remarkable, and all the more so when we realize that these specialized, protein rich, membranes differentiate from the cytoplasmic membrane which has a more complex composition and is richer in lipids. analysis of the protein organization in these specialized membranes from several different bacteria suggest that the organization results from a phase separation of several different contiguous phases. in order to better understand our observations we have undertaken an examination of the different phase behaviors that are possible for membrane proteins considered as a two dimensional colloid. monte-carlo modeling of the phase diagram of this system shows the importance of interaction distance in the determination of system behavior. transcription of our observations on the model systems to the photosynthetic membranes suggests that electrostatic and elastic forces in the membrane are of particular importance in determining the high level order of membrane proteins. the recent crystal structure of photosystem i (psi) from synechococcus elongatus shows two quasi-symmetric branches of potential electron transfer cofactors including primary donor (dimer of chlorophylls p700), monomeric chlorophylls a and a 0 and quinone a 1 , bound to the psaa/psab heterodimer. so far, it is not clear if both potential electron transfer pathways are active in this process or only one of them. to solve this issue, we studied a set of 6 mutants with methionine coordinating the primary electron acceptor, a 0 , replaced by histidine, leucine, or serine in either of two branches. our results obtained with a technique of femtosecond transient absorption spectroscopy show that both branches are equally active in electron transfer. mutation in either branch slows the forward electron transfer between a 0 and a 1 from 20 ps in wilde type psi to 1-2 ns in all these mutants. this strong effect is explained by signi cant change in the redox midpoint potential and change in the position of a 0 by the mutations. i. s. zaharieva department of biophysics and radiobiology, faculty of biology, university of so a an approach to the investigation of structural and functional properties of the photosystem ii supramolecular complex in native photosynthesizing objects based on the registration of delayed chlorophyll a uorescence is developed. using a disc phosphoroscope, we register simultaneously: i) changes of the intensity of millisecond delayed uorescence (decayed in 0.35 -5.5 ms time range) during the transition of the photosynthetic apparatus from dark to lightadapted state; ii) changes of the intensity of delayed uorescence decaying in different subintervals of the investigated time range; iii) dark relaxation curves at different moments of the transition; iv) changes of the intensity of prompt chlorophyll a uorescence. the analysis of these data allows the correlation of the delayed uorescence characteristics to particular processes occurring in the photosystem ii complex -proton or electrical gradient accumulation, changes in the redox state of quinone acceptors, changes in the pigment-protein complexes caused by different stress factors, for example temperature. three-dimensional structure of major light-harvesting antenna of photosystem ii from cucumber h. yan 1 , z. liu 1 , k. wang 2 , t. kuang 2 , j. zhang 1 , l. gui 1 , x. an 1 , w. chang 1 1 national lab of biomacromolecules, institute of biophysics, chinese academy of sciences(cas), 15 datun road, chaoyang distr., beijing 100101, china, 2 lab of photosynthesis and environmental molecular physiology, institute of botany, cas, 20 nanxincun, xiangshan, beijing 100093, china the major light-harvesting antenna complex of photosystem ii (lhc-ii), the most abundant integral membrane protein, functions in light capture, energy transfer/distribution and photoprotection. lhc-ii from different species or conditions shows different spectral properties and variation in polypeptide and pigment components. this indicates some speci c function-related alterations in the organization of lhc-ii. here we report a 2.66-å crystal structure of cucumber homo-trimeric lhc-ii, organized in a perfect virus-like icosahedral particle. the electron-density map shows the reasonable existence of a chlorophyll (chl) a/b mixed binding site in the complex. the occurrence and locus of lactucaxanthin (lac) was seen directly for the rst time. based on the credible structure information, a mechanism of the energy transfer, regulation and excess excited energy dissipation under high light condition was proposed. coherent anti-stokes raman scattering microscopy (cars) is a new approach for chemical imaging of molecular systems within cells and tissues, with high sensitivity, high spatial resolution, and three dimensional sectioning capabilities, without using uorophores that are prone to photobleaching. this technique permits to map selectively molecular species, by using vibrational properties of their chemical bounds. the epi detected (e-cars) and forward detected (f-cars) intensities depends on the shape, the size of the sample, as well as the index of the solvent. in this presentation, after introducing the cars microscopy technique, we show the rst cars studies of the refractive effect of the sample, comparing the e-cars and f-cars signals for different diameters of polystyrene beads, in different refractive index solvents. we present several simulations, comparing forward-detected and backward-detected signals in different sized polystyrene beads, embedded in different index solvents, and we show that, the backwardre ected f-cars dominates the experimentally epi-detected signals. furthermore, we demonstrate experimentally and theoretically that the maxima of forward and epi-detected signals are generated at different positions along the z axis in the sample. we nally discuss how index mismatch in cells can alter cars images. the effects of static magnetic elds on humans have been the subject of continuous investigations. since one of the major static magnetic eld sources is nuclear magnetic resonance imaging (mri), the present study aimed to investigate the effects of 1.5 t magnetic eld that is produced by mri on humans. the study is carried out with 33 voluntary and healthy young men from 20 to 25 years of age. the subjects informed about the purpose of the study at the beginning. the subjects exposed to 30 minutes of 1.5 t static magnetic eld by means of putting the subjects into the magnetic resonance unit. 5 ml blood was taken from each subject one minute before and one minute after exposure. t1 and t2 relaxation times and trace elements were measured in of pre and post exposure plasma of the subjects. the obtained post exposure values were compared with pre-exposure values of the subjects. pre and post exposure results were analyzed by means of student t-test. evaluation of tumor response of breast cancer patients by diffusion weighted mri k. a. danishad 1 , v. seenu 2 , u. sharma 1 , p. k. julka 3 , g. k. rath 3 , n. r. jagannathan 1 1 department of nmr, 2 department of surgery, 3 department of radiotherapy, all india institute of medical sciences, new delhi, india diffusion weighted mr imaging (dwi) measures the diffusion of water molecules in tissues and is quanti ed by apparent diffusion coef cient (adc). dwi can be used to differentiate tumors from normal tissue and also can be used to monitor the response of tumor to chemotherapy. thirteen healthy volunteers and twelve patients were recruited for the study. dw images were obtained prior to therapy (n=10) and after three cycles of therapy (n=3). the mean adc value of tumors (0.83 x 10 3 0.05 mm 2 /s) was signi cantly less (p < 0.05) compared to the normal tissue (1.80 x 10 3 0.2 mm 2 /s). decrease in adc in tumor is due to an increase in the cellularity which restricts the diffusion of water molecules. in patients receiving neo-adjuvant chemotherapy, the adc values were higher (1.36 x 10 3 0.86 mm 2 /s) and were closer to that of the normal tissue (p <0.05), indicating response of the tumor to chemotherapy. the post-therapy increase in adc is due to the cell damage caused by the therapeutic agents which increases the fractional volume of the interstitial space, causing an increase in the mobility of water. the study showed that dwi can be used non-invasively to assess the response of breast cancer patients to neo-adjuvant chemotherapy. quanti cation by optical imaging of gene electrotransfer in mouse muscle and knee optical imaging was evaluated for monitoring and quanti cation of the mouse knee joint and tibial cranial muscle electrotransfer (et) of a luciferase encoding plasmid. the substrate of luciferase (luciferin) was injected i.p or locally in the muscle or the knee joint. luminescence resulting from the luciferase-luciferin reaction was measured with a cooled ccd camera. luminescence of the knee joint and muscle were higher after local than after i.p injection of luciferin, but both measurements were highly correlated. local injection procedure was adopted. a signi cant correlation was observed between measurements in vivo and in vitro on the same muscle. reproducibility of individual luminescence measurements was also veri ed, and the luminescence levels were clearly dependant of the amount of plasmid injected. in vivo luciferase in the electrotransfered knee joint was detected for two weeks. intramuscular electrotransfer of 0.3 or 3 µg of plasmid led to stable luciferase expression for 62 days, whereas injecting 30 µg plasmid resulted in a luminescence fall two weeks after electrotransfer. these decreases were, at least partly, related to the production of antibodies against luciferase. thus, optical imaging was shown to be a relevant technique to quantify variations of luciferase activity in vivo in one given tissue. furthermore, evaluating the effective amount of luciferase in tissues from in vivo luminescence levels requires calibration since it relies on conditions of the enzymatic reaction and light absorption. acute effect of corticosterone on nmda receptormediated ca2+ elevation in mouse hippocampal slices m. saito 1 , s. sato 1 , h. osanai 1 , a. hirano 1 , y. komatsuzaki 1 , s. kawato 2 1 department of physics and applied physics, college of humanities and sciences, nihon university, 2 department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo corticosterone (cort) is a principal glucocorticoid synthesized in the rodent adrenal cortex and secreted in response to stress. we examined the rapid effects of cort on n-methyl-d-aspartate (nmda) receptor-mediated ca 2 signals in adult mouse hippocampal slices by using ca 2 imaging technique. application of nmda caused a transient elevation of intracellular ca 2 concentration followed by a decay to a plateau within 150 sec. the 30 min preincubation of cort induced a signi cant decrease of the peak amplitude of nmda-induced ca 2 elevation in the ca1 region. the rapid effect of cort was induced at a stress-induced level (0.4-10 µm). because the membrane non-permeable bovine serum albuminconjugated cort also induced a similar rapid effect, the rapid effect of cort might be induced via putative surface cort receptors. in contrast, cort induced no signi cant effects on nmdainduced ca 2 elevation in the dentate gyrus. in the ca3 region, cort effects were not evaluated, because the marked elevation of nmda-induced ca 2 signals was not observed there. in vivo subcellular structures recognized with phase k. nagayama 1 , r. danev 1 , n. usuda 2 , y. kaneko 3 , k. nitta 1 , a. nakazawa 2 , k. atsuzawa 2 , m. tanaka 4 , m. setou 1 1 okazaki institute for integrative bioscience, okazaki, japan, 2 fujita health university, school of medicine, toyoake, aichi, japan, 3 saitama university, saitama, japan, 4 tokyo metropolitan institute of gerontology, itabashi-ku, tokyo, japan phase contrast transmission electron microscopy has been developed to enable a high contrast and a high resolution observation for unstained ice-embedded samples. to enhance the image contrast, two methodologies have already been developed; i) scattering contrast for stained samples with small aperture diaphrams and ii) defocus contrast for unstained or stained samples with deep defocusing. the former prevails in histochemical sciences and the latter is popular in electron crystallography. both methods, however, have a common drawback that the contrast is only improved by impairing the image quality. this drawback can be removed with use of the phase contrast method using phase plates, which has traditionally been used in visible light microscopy. due to the severe obstacle of the charging of phase plates, however, the idea has not yet been materialized. we have solved the phase-plate charging problem. an experiment 300kv with tem for a whole cell from cyanobacterium unstained and ice-embed ful lled the expectation. only weak and vague contrast was obtained for the conventional image of the cell even with a very deep defocus. contralily a high-contrasted image has appeared for phase contrast images, where various ne structures are clearly recognized. this may be a rst example to observe nanometer scale structures in details in the intact cell. other examples including intact state intravesicular structures will be shown. j. lichtenberger, p. fromherz max-planck-institute for biochemistry we cultured bovine chromaf n cells on an array of electrolyteoxide-silicon eld-effect transistors (eos fet) and monitored granule secretion. by stimulation with barium chloride, vesicles are released into the narrow sheet of electrolyte between the chip surface and the plasma membrane. the interaction of released protons with the silicon dioxide surface of the chip alters the threshold voltage of the transistor and gives rise to a measurable signal. simultaneously performed measurements with a carbon bre showed a correlation of the transistor signals and amperometric current traces. we conclude that the transistors are able to monitor exocytosis on a single vesicle level. to elucidate the role of protons, we destroyed the proton gradient across the vesicle membrane by nigericin and valinomycin. as a result a massive reduction of the transistor signals was induced, whereas there was only little change of the amperometric records. we conclude that released protons are responsible for the detection of vesicles with transistors. the individual transistor records of vesicle exocytosis can be explained by combining the dynamics of the exocytotic event with the diffusion in the cell-chip junction. transistor recording of exocytosis does not depend on the electrochemistry of transmitters. as many kinds of exocytotic vesicles contain a large amount of buffered protons it can be applied to numerous kinds of exocytotic events, independent on the nature of the transmitter. we tried various solvents for the solubility of the uorescent product, and found that the product was insoluble in water and most organic solvents. a quite bright uorescence emitted by the particles was observed by uorescent microscope when emitted by uv365 nm. sem indicated that the size of the particles was 1µm 20µm, depending on the reaction time and phospholipid concentration in hexane solution. endothelial cells from human vein grew better on the surface prepared from the particles than the culture plate, implies a possible application as a new type of biomaterial as a coating material for medical devices, and as a uorescent tracer for human bodies. confocal microscopy of the phototactic ciliate f. salina. fabrea salina is a marine heterotrich ciliate, which dwells in salt ponds. in previous works we have described the phototaxis and the uorescence properties of a hypericin like endogenous pigment in an albino strain. we have recently obtained a heavily pigmented strain from the saline of torre colimena (taranto, italy). we have used confocal microscopy to characterize the uorescent properties of this strain and to compare them with those of the albino strain. the results obtained by one and two photon confocal microscopy show that, as in the albino strain, the uorescence intensity of the pigment is higher in dead cells than in the living cells. the excitation and emission spectra are quite similar in the two strains and this is also true for the uorescence lifetime, which is about 2 ns. all together, these measurements indicate that the pigment of the new strain belongs to the family of hypericin-like chromophores. the analysis of different confocal planes shows that the pigment is localized not only in granules under the somatic membrane in the cellular body, as currently thought, but also in the cilia. some experiments of fotobleaching "in situ" con rm this result, that might have important implications in the understanding of the mechanisms of the photomotile responses of f. salina and probably of other heterotrich ciliates. there is no doubt that modern physician should have the knowledge of basic sciences as physics, chemistry and biology. furthermore, the biophysics is incorporated into the curriculum of most european medical schools. at medical school in zagreb, the course of physics and biophysics is positioned in rst and forth year of study. the students learn basic physics phenomena of structure of matter, mechanics, thermodynamics, electromagnetism, optics and acoustics applied on the human body. additionally, the interactions of the body with the surrounding are thoroughly discussed as the basis for different diagnostic methods. the arguing at our school is still going on where to include the content of this course. should it be the autonomous course or the part of physiology and radiology courses in the problem based learning approach?! so far at zagreb medical school the biophysical courses are autonomous structured according to the biophysics programs at other european universities. the highlighting is on seminar work and lab, encouraging the students for individual learning. the seminars are made more vivid and instructive for students by inclusion of different model devices constructed in our department learning on science is also learning how scienti c knowledge is produced. in this sense, issues related to the dynamics of science should be brought into focus in science education. the idea has support in the 1999 "declaration on science and the use of scienti c knowledge", particularly in the statement that "science curricula should include science ethics, as well as training in the history and philosophy of science and its cultural impact". thinking on rst year undergraduate students, we are interested in an integrated approach of that kind of themes. this presentation describes a practical teaching module included in the basic biophysics course for biochemistry majors. organized in case studies, it deals with stories of biophysics (and biochemistry), and addresses the role of the biophysical approach in the progress of the life sciences. the module follows the whole course, and consists in small exercises on the way a given understanding has been constructed explored within the practice trend of contemporary science studies. examples of the chosen stories -anchored in the subjects covered in the lectures of the course -relate to the search for the mechanism of energy production through atp-synthase, the development of radioactive labelling techniques, and the discovery of protein water channels. beyond their value as cultural legacy and as motivating tools, the insight they might provide is vast. axiomatic theory of biophysics q. zhao china rehabilitation research centre, beijing, china. until now, all approaches to interpret biology by applying principles of physics have been announced failure. the achievement of biophysics is limited in area to provide essential tools for biological research and biophysics is far from to be the basic theory of biology. to resolve it requires the fundamental research about the logic features of biophysical processes of biology. we promoted system logic and then protein thermodynamics structure theory. based upon it, the axiomatic theory of biophysics and biology could be developed. our result shows that the real understanding of biology or biophysics must be constructed based upon new thinking methods. a successful ligand-receptor docking methodology depends strongly in the ef ciency of the global optimization algorithm used to explore the ligand conformational space. in this work we have implemented and analyzed the performance of a new exible ligand-receptor docking methodology. this methodology uses as optimization method a multisolution version of the generalized simulated annealing algorithm adapted to problems with box constraints. a grid-based methodology, considering the receptor rigid, and the gromos96 classical force eld are used to evaluate the ligand-receptor scoring function. the methodology was tested in redocking (ligand within it own protein conformation) and cross-docking (ligand within another protein conformation) experiments for ve hiv1 protease-ligand complexes with known threedimensional structures. all ligands tested are highly exible, having 12 to 20 conformational degrees of freedom. the implemented docking methodology was able to redock successfully all exible ligands with a success ratio 95% and a mean rmsd lower than 1.52 å with respect to the corresponding experimental structures. in the cross-docking experiments we observed a strong dependence of the mean success ratio with respect to the protein structure used as reference. in 4 situations we observed a mean success ratio 40% and 70% in 13 cases among the 20 possible ones. s. aida-hyugaji 1 , h. nakagawa 2 , j. nomura 2 , m. sakurai 2 , d. tokushima 3 , t. takada 3 , u. nagashima 4 , t. ishikawa 2 1 tokai university, japan, 2 tokyo institute of technology, japan, 3 nec corporation, japan, 4 national institute of advanced industrial science and technology, japan irinotecan is a widely-used antitumor drug that inhibits mammalian dna topoisomerase i. however, overexpression of abcg2 can confer cancer cells resistance to sn-38, that is, the active form of cpt-11. in the present study to develop a platform for the molecular modeling to circumvent drug resistance associated with abcg2, we have characterized a total of fourteen new sn-38 analogues by some typical properties, which were evaluated by molecular orbital (mo) calculations and neural network (nn) analysis. the nn was rst applied to estimate hydrophobic properties (logp) of the analogues. thereafter, the electrostatic potential (esp) and the solvation free energy ( g) were evaluated by mo calculation. these indexes were found to be well correlated with the drug resistance ratio experimentally observed in abcg2-overexpressing cells. it is suggested that hydrophilic analogues carrying oh-or nh 2 -groups are good substrates for abcg2 and therefore exported from cancer cells. in contrast, sn-38 analogues with cl or br atom at those positions have similar logp values and high af nities toward the putative active site of abcg2, however they were not substrates of abcg2. from these results, it is strongly suggested that hydrogen bond formation with oh-or nh 2 -groups are critically involved in the transport mechanism of abcg2. a. agopian 1 , j. depollier 1 , e. gros 1 , g. aldrian-herrada 1 , p. clayette 2 , n. bosquet 2 , g. divita 1 1 crbm-cnrs, 1919 route de mende, 34293 montpellier, france., 2 spi-bio-cea fontenais aux roses, france reverse transcriptase (rt) plays an essential role in the replication of hiv and constitutes the main target for the development of aids therapies. the biologically active form of hiv rt is a heterodimer of two subunits, p51 and p66, each consisting of distinct subdomains: the ngers, the palm, the thumb, the connection and the rnase h subdomain, the latter only present in p66. we have demonstrated that formation of fully active rt is a two-step process involving rapid association of the two subunits (dimerization) followed by a conformational change (maturation). thanks to the crystal structure of rt we have identi ed a new class of inhibitors based on short peptide motifs derived from the dimer interface. we rst identi ed a short 9mer peptide (pep-7) derived from the tryptophanrich motif of the connection subdomain that blocks dimerization of rt and ef ciently abolishes hiv-1 replication. pep-7 interacts preferentially in a pocket involving residues trp 24 and phe 61 on p51. we then designed 15mer peptides derived from the thumb domain which inhibit rt maturation as well as viral replication when delivered into cells. taking into account these results we propose that dimerization of rt constitutes a potential target for the design of more speci c new antiviral drugs. . the success of gene therapy largely relies on the availability of vectors that would deliver the genetic material ef ciently to the target cells with a minimal toxicity. in this context, our purpose was to evaluate as possible vectors a series of newly synthesized low molecular weight (5 kda) chitosan derivatives grafted with dodecenoyl (ddc) groups at different percentages (3, 9, 16 and 25 %). in the absence of dna, the critical micellar concentration (cmc) of these derivatives in 20 mm mes buffer ph 6.5 was found to be strongly dependent on the percentage of ddc but not on ph or salt concentrations. this indicates that the ddc groups confer to the chitosan derivatives the potency to self-assemble probably in micellar structures: a property that may dictate the formation and the structure of their complexes with dna. next, we investigated by quasielastic light scattering the size and the surface charge of complexes of plasmid dna with these derivatives at different ph, salt concentrations and n/p ratios (expressed in charged units of chitosan amines to dna phosphates). we found the smallest and more positively charged complexes were obtained at ph 5.8 and n/p=5 in the absence of salt: a condition where the chitosan derivatives were fully protonated and in excess over the dna phosphate groups. biophysical and biological examination of dna/lipids complexes particles of virus-like structure designed for in vivo gene transfer d. durand 1 , m. schmutz 2 , b. lebleu 3 , a. r. thierry 3 1 lure, centre universitaire paris sud, orsay, france, 2 institut henri sadron, strasbourg, france, 3 laboratoire des défenses antivirales et antitumorale, umr 5124, montpellier, france the structure of complexes made from dna and suitable lipids (lipoplexes lx) was examined by cryo electron microscopy. we observed a distinct concentric ring-like pattern with striated shells when using plasmid dna. these spherical multilamellar particles have a mean diameter of 254 nm with repetitive spacing of 7.5 nm with striation of 5.3 nm width. small angle x-ray scattering (saxs) con rmed cryoem data and revealed repetitive ordering of 6.9 nm, suggesting a lamellar structure containing at least a dozen layers. this concentric and lamellar structure with different packing regimes was also observed by cryoem with linear dsdna, ssdna and oligodeoxynucleotides. for the rst time, dna chains could be visualized in dna/lipid complexes. such speci c supramolecular organization is the result of thermodynamic forces, which cause compaction to occur through concentric winding of dna in a liquid crystalline phase. cryoem of t4 phage dna packed either in t4 capsides or in lipidic particles showed similar patterns. saxs suggested an hexagonal phase in lx-t4 dna. thus, both lamellar and hexagonal phases may coexist in the same lx preparation or particle and transition between both phases may depend upon equilibrium in uenced by type and length of the dna used. organization of such nucleotidic supramolecular assemblies is relevant for prebiotic chemistry. engineering self-assembly peptides for targeted delivery of therapeutics and imaging agents s. s. dhadwar, m. sung, k. kawamura, j. gariépy department of medical biophysics, university of toronto, canada peptide-mediated delivery systems have recently emerged as a means to substitute or augment conventional drug and gene delivery technologies. these approaches are versatile and easily designed to incorporate a number of speci c attributes required for ef cient delivery of therapeutic and imaging agents. in particular, self-associating peptide domains can be utilized to construct stable and structurally well-de ned protein-like assemblies displaying a series of cell-routing functions. more speci cally, a peptide-based self-assembling intercellular delivery vehicle was designed by incorporating the 30-residue long tetramerization domain of the human tumor suppressor protein p53 (hp53tet). the resulting peptide tetramer displays 8 termini within its structure that allows for the simultaneous presentation of distinct cell targeting signal or functional domains. the fusion of polycationic sequences to the hp53tet domain promotes the cellular import of the resulting constructs into eukaryotic cells. this internalization event was dramatically enhanced for such multivalent peptides in relation to their monomeric counterparts. peptides containing a nuclear localization sequence along with a polycationic sequence were found to shuttle reporter plasmids ef ciently to the nucleus of cells. these results have important implications in the design and construction of novel targeted delivery vehicles. mechanisms of non-covalent peptide mediated cellular delivery of therapeutics: a biophysical study s. deshayes 1 , m. c. morris 1 , a. heitz 2 , p. charnet 1 , g. divita 1 , f. heitz 1 1 crbm -cnrs fre 2593 montpellier france, 2 cbs -cnrs umr 5048 -inserm u554 montpellier france two different cell-penetrating peptides mpg and pep-1 were shown to promote non-endosomal intracellular delivery of non-covalent bound cargos, namely nucleic acids and proteins; respectively. in order to identify the peptide mediated internalization pathway, we undertook conformational investigations of both peptides with and without associated cargos and checked the conformational consequences of the presence of phospholipids. from the conformational point of view, pep-1 behaves differently from mpg. cd analysis revealed a transition from a non-structured to a helical conformation upon increase of the concentration while mpg remained nonstructured. determination of the structure by nmr showed that in water, it's a-helical domain extends from residue 4 to 14. cd and ftir indicated that pep-1 adopts a helical conformation in the presence of phospholipids while mpg is in a -sheet form. adsorption measurements performed at the air-water interface were consistent with the helical form. pep-1 did not undergo conformational changes upon formation of a particle with a cargo peptide. in contrast, we observed a partial conformational transition when the complex encountered phospholipids. for mpg, interactions with nucleic acids generated a partial folding into -sheet which was more pronounced in the presence of lipids. electrophysiological measurements showed that both peptides, whether associated or not with their cargo, can induce transmembrane pore-like structures. self-assembly of hydrolysed alpha-lactalbumin into nanotubes j. f. graveland-bikker 1 , k. g. de kruif 2 1 nizo food reseach, ede, netherlands, 2 van´t hoff laboratory, netherlands nanotubes are formed by self-assembly of partially hydrolysedlactalbumin, a 14 kda milk protein. there are several promising applications of these -lactalbumin tubes, in food, pharmacy and nanotechnology. we studied the mechanism of self-assembly, the structure and the properties of the nanotubes. limited proteolysis of the -lactalbumin (by a serine protease) makes the molecule prone to self-assembly. in the presence of ca 2 tubular structures are formed. other divalent ions like mn 2 and zn 2 can also induce tubular self-assembly, while mg 2 leads to random aggregation. light scattering showed that the self-assembly is reversible, which is of relevance for controlled release applications. on the other hand, we could also make stable tubes by cross linking, which would be a requisite for several other applications. from afm and saxs measurements, we obtained values for the outer diameter: 21 nm; and the inner diameter: 8 nm. afm and cryo-em revealed the helical structure of the tube wall; it is a right-handed helix. by performing nano indentations with afm we determined mechanical properties of the tubes. the tubes were shown to be relatively resilient upon small deformations; the elastic modulus is of the order of 0.1 gpa. targeted delivery of photosensitizers into the cancer cell nuclei enhances their cytotoxic ef cacy the search for new pharmaceuticals has raised interest in locallyacting drugs which act over short distances within the cell, and for which different cell compartments have different sensitivities, e.g. photosensitizers used in anticancer therapy should be transported to the most sensitive subcellular compartments where their action is most pronounced. earlier we have produced a number of modular recombinant transporters for locally-acting drugs comprising several functional modules for cell-speci c targeting, internalization, escape from intracellular acidic vesicle, and targeting to the nuclei of melanoma cells overexpressing melanocortin receptors. here we describe new transporters on the basis of epidermal growth factor which are speci c for a wide variety of cancers. these transporters possess all necessary functional activities and deliver photosensitizers into the nuclei of human carcinoma cells to result in photocytotoxic effects almost 3 orders of magnitude greater than those of nonmodi ed photosensitizers. characterization of mixtures of dna and nonionic polymeric agents for gene delivery in muscle j. m. gau 1 , j. lal 2 , l. auvray 2 1 laboratoire mpi -lrp, umr cnrs 7581, université d´évry val d´essonne, 91025 évry cedex, france, 2 argonne national laboratory, ipns, 9700 south cass avenue, illinois 60439, usa a strategy to cure muscle disease is to introduce genes (dna) into the muscle cell to correct or to add genes. nonionic polymeric agents have emerged as an ef cient vector to deliver dna in the muscle. these polymers protect dna from extracellular nuclease degradation by allowing the dna diffusion throughout the muscle tissue. there is at present no understanding about how nonionic polymers enhance transfection in the muscle. the kind of interactions between these nonionic agents and dna, dna-nonionic polymeric agent mixtures and cell membrane are currently unknown. also the structure of dna-nonionic polymeric agent mixtures is not yet well de ned. more information is needed to improve this delivery system. neutron scattering (contrast variation) and light scattering were used to investigate the interaction between: dna and nonionic polymers (pvp, di-and triblock copolymers). furthermore, electrical measurements with the same polymer complexes and black lipid membrane were also performed. depending on the polymer type there is either direct interaction with dna or in other cases polymers exhibit strong interaction with the lipid membrane. an explanation for transfection ef ciency of these nonionic agents in gene delivery to muscle will be given. high throughput in-silico screening against exible protein receptors b virtual screening of chemical databases to targets of known threedimensional structure is developing into an increasingly reliable method for nding new lead candidates in drug development. based on the stochastic tunneling method (stun) we have developed flexscreen, a novel strategy for high-throughput in-silico screening of large ligand databases. each ligand of the database is docked against the receptor using an all-atom representation of both ligand and receptor. in the docking process both ligand and receptor can change their conformation. the ligands with the best evaluated af nity are selected as lead candidates for drug development. using the thymidine kinase inhibitors as a prototypical example we documented the shortcomings of rigid receptor screens in a realistic system. we demonstrate a gain in both overall binding energy and overall rank of the known substrates when two screens with a rigid and exible (up to 15 sidechain dihedral angles) receptor are compared. we note that the stun suffers only a comparatively small loss of ef ciency when an increasing number of receptor degrees of freedom is considered. flexscreen thus offers a viable compromise between docking exibility and computational ef ciency to perform fully automated database screens on hundreds of thousands of ligands. maturation and inhibitor design of sars-cov 3cl protease based on a product-bound crystal structure severe acute respiratory syndrome (sars) is an emerging infectious disease caused by a novel human coronavirus. here we report that the 3cl pro containing n-and/or c-terminal additional in-frame sequences underwent autoactivation to cleave the tags and yielded the mature protease in vitro. the 3-d structure of the c145a mutant protease shows that the active site of one protomer of the dimeric protease is bound with the c-terminal six amino acids of the protomer in another asymmetric unit, suggesting a possible mechanism for maturation. the crystal structure of this product-bound form shows that the active site has a p1 pocket that binds the gln side chain speci cally. in addition, the p2 and p4 sites are clustered together to accommodate large hydrophobic side chains. the tagged c145a mutant protein served as a substrate for the wildtype protease and the n-terminus was rst digested (55-fold faster) followed by the c-terminal cleavage as shown by the sds-page analysis. the analysis of t analytical ultracentrifuge experiments reveals the remarkably tighter dimer formation for the mature enzyme (k d = 0.35 nm) than for the mutant (c145a) containing the n-terminal (k d = 17.2 nm) or the c-terminal 10 extra amino acids (k d = 5.6 nm). taken together, the study here provides insights to the design of our new structure-based inhibitors. nevertheless, a signi cant proportion of patients do not respond to this therapy, and adverse effects are common. here we report the delivery and expression of recombinant mycobacterial dna vaccines in vivo and demonstrate the ability of multicomponent dna vaccines to enhance th1-polarized immune responses. splenocytes from immunized groups of mice were re-stimulated in vitro and examined for cytotoxicity against bladder tumour cells. we used four combined recombinant bcg dna vaccines (multi-rbcg) for electroporative immunotherapy in vivo, and found that tumour growth was signi cantly inhibited and mouse survival was prolonged. increased immune cell in ltration and induction of apoptosis were noted after treatment with multi-rbcg alone, with the interleukin-23 (il-23) vaccine alone, and-most signi cantly-with their combinations. thus, electroporation immunogene therapy using multi-rbcg plus il-23 may be an attractive regimen for the treatment of bladder cancer. this approach presents new possibilities for the treatment of bladder cancer using recombinant bcg dna vaccines and il-23 dna vaccine. the cell-penetrating peptide (cpp) pep-1 is capable of introducing large proteins into different cell lines, maintaining their biological activity. two mechanisms have been proposed to explain the entrance of other cpps in cells, endosomal-dependent and independent. we evaluated the molecular mechanisms of pep-1mediated cellular uptake of -galactosidase ( -gal) from e. coli, in large unilamellar vesicles (luv) and hela cells. fluorescence spectroscopy and immuno uorescence microscopy were used to study the translocation. internalization of -gal into luv and protein functionality in hela cells were detected by enzymatic activity. -gal translocated into luv in a transmembrane potentialdependent manner. likewise, -gal incorporation was extensively decreased in depolarized cells. furthermore, -gal uptake efciency and kinetics were temperature-independent and -gal did not co-localize with endosomes, lysosomes or caveosomes. therefore, -gal translocation was not associated with the endosomal pathway moreover transmembrane pores were not detected. these results indicated that the protein uptake in vitro and in vivo was mainly, if not solely, dependent on a physical mechanism governed by electrostatic interactions between pep-1 (positively-charged) and membranes (negatively-charged). peptide the dramatic acceleration in the identi cation of new nucleic acidbased therapeutic molecules has provided new perspectives in pharmaceutical research. however, the development of nucleic acidand peptide-based therapeutics is limited by their poor cellular uptake and traf cking. with the aim of addressing these issues, we have designed a family of short amphipatic peptides for delivery of nucleic acids (mpg) and of peptide/pna (pep). these carriers consist of a hydrophobic moiety and a nls-derived hydrophilic domain. they form stable non-covalent complexes with peptides, proteins, sirna or pna without any requirement for prior covalent cross-linking. both mpg and pep carriers enter cells rapidly, in a process involving membrane disorganization, independently of the endosomal pathway. mpg ef ciently delivers short odns and sirna into a wide variety of mammalian cell lines, without interfering with their biological function. pep signi cantly improves delivery of pna and peptides. both carriers were used for the delivery of sirna or antisense pna targeting the cell cycle regulatory protein cyclin b1 in an animal model and were found to block tumor growth upon intravenous injection. we believe that mpg and pepbased technologies will contribute signi cantly to the development of basic and therapeutic applications. probing the bound conformation of acetylcholinesterase (ache) inhibitor at the binding site c. g. kim, x. zhao, s. goodall, a. watts department of biochemistry, university of oxford, south parks road, oxford, ox1 3qu, uk acetylcholinesterases are the enzymes which preferentially hydrolyze acetyl esters (such as ach or acetyl-â-methylcholine), containing 543 amino acid residues in the eeache form and arranged as a 12-stranded â-sheet surrounded by 14 á-helices. the protein is ellipsoidal in shape, with approximate dimensions of 45 å by 60 å by 65 å. inhibitors of acetylcholinesterase are of commercial and medical interest as pesticides and as therapeutics in the treatment of alzheimer's disease. an understanding of the conformation of inhibitors in the binding site enables the rational design of novel inhibitors with increased potency and speci city. interaction between the ligand, amino-2-methyl-3-(3tri uoroacetylbenzyl-oxymethyl)quinoline (r414983), and ache inhibitor has been studied by advanced solid-state nmr through double-quantum chemical shift and distance measurements. combining solid-state nmr data and docking simulations, conformation of the ache inhibitor at the active site has been predicted. in vivo, heat shock proteins (hsps) being stress-inducible chaperones can attenuate detrimental consequences of ischemic insults, inammation, neurodegenerative diseases, etc. also, intracellular accumulation and chaperone activities of some hsps may contribute to improved cell survival following uv or ionizing radiation. in models of pathological states and their treatment, we used special virusbased vectors for overexpression of hsp70 or hsp27 in cell cultures to confer cytoprotection under simulated ischemia/reperfusion. in parallel, similar cytoprotection was achieved after pretreatments of the cells with a pharmacological hsp inducer, geranylgeranylacetone. the cytoprotective effects were manifested in the lesser extent of oxidative modi cation and aggregation of cellular proteins, better preservation of the cytoskeleton, faster restoration of energy metabolism and the improved post-stress cell survival. in the other model, we treated normal and tumor cells with an inhibitor of the chaperone activity of hsp90, geldanamycin. only the drug-treated tumor cells became more sensitive to gamma-irradiation; such results characterize this drug as a potentially selective radiosensitizer of tumors. taken together our data demonstrate promising approaches to clinically bene cial manipulating the levels of expression and/or chaperone activity of hsp(s) by means of gene therapy or pharmacotherapy. characterizaton of proteins from human pleural uid r. jain, s. kumar, n. singh, s. sharma, t. p. singh all india institute of medical sciences, new delhi ,india 110029 the samples of human pleural uid were obtained from both healthy subjects and patients infected by tuberculosis. after the preliminary processing these samples were run in independent lanes of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the two lanes indicated variations in the intensities of a few bands and some new bands were also observed in the infected samples. these were characterized by determining their nterminal sequences. the new bands which had low density were carefully identi ed and cloned. some of the common bands that showed intensity variations were characterized. these were matrix metalloproteins, secretory phospholipasea 2 , transferrin and ceruloplasmin. they were also studied with maldi-tof and their molecular weights have been determined. some of these proteins have been crystallized and their detailed crystal structure determinations are in progress. biophysical study of non-lethal stress response of cultured dc3f cells stress factors may induce two kinds of responses in living cells: either cell death or adapting mechanisms. our aim was to search for non-lethal effects of various stress conditions on cultured hamster lung broblasts (dc3f cells) as well as to assess the recovery time after stress removal. dc3f cells were cultured in standard conditions and were submitted to stress, either by incubation with chemical substances (sodium arsenate, sodium nitroprusiate) and drugs (bleomicine and statins) either by irradiation (uv, he-ne laser). the doses and exposure times were chosen as to avoid cell death. after stress removal, cells were allowed to recover and the recovery time period was measured. structural and functional parameters were evaluated before and after stress, as well as during recovery. by now, experimental models for the in vitro study of non-lethal stress inducing factors have been set up. severe acute respiratory syndrome (sars) is an emerging infectious disease caused by a novel human coronavirus. the viral maturation requires a main protease (3cl pro ) to cleave the virus-encoded polyproteins. we report here that the 3cl pro containing n-and/or c-terminal additional in-frame sequences underwent autoactivation to cleave the tags and yielded the mature protease in vitro. the 3-d structure of the c145a mutant protease shows that the active site of one protomer of the dimeric protease is bound with the cterminal six amino acids of the protomer in another asymmetric unit, suggesting a possible mechanism for maturation. the tagged c145a mutant protein served as a substrate for the wild-type protease and the n-terminus was rst digested (55-fold faster) followed by the c-terminal cleavage as shown by the sds-page analysis. the analysis of the quaternary structures for the tagged and mature proteases by analytical ultracentrifuge experiments reveals the remarkably tighter dimer formation for the mature enzyme than for the mutant (c145a) containing the n-terminal or the c-terminal 10 extra amino acids. taken together, the study here provides insights to the design of our new structure-based inhibitors. characterisation of macromolecular transport in physiologically relevant mixed ecm based gels s. lelu, a. pluen school of pharmacy and pharmaceutical sciences, university of manchester (uk) the extracellular matrix (ecm) a complex gel made of hyaluronic acid, collagen and proteoglycans (pg) impedes the penetration of macromolecules especially in tumours, and may compromise the success of novel therapies. though recent in vivo investigations pointed out that, not only ha but brillar collagen its content and organisation and its interactions with pg were involved in macromolecular transport hindrance, transport mechanisms relating the macromolecular drug and these ecm components are unknown. in this study we seek to evaluate the determinants of passive transport mechanisms of biomacromolecules in complex gels made of ha, collagen using uorescence techniques (frap and confocal re ection microscopy (crm)) and rheology. focus was on conditions relevant to tumours and, initially, on low collagen and relatively high ha content. rheology experiments showed that mixed systems containing less than 10mg/ml of ha present higher elastic modulus ge than pure ha network or pure collagen gels. interestingly crm and frap studies revealed similarities for collagen and mixed gels: the organisation and spacing of the collagen bres did not change and the ratio of the diffusivities (d/d 0 ) of dextran 2m and igg were not different but higher than those in ha networks. systems with higher collagen content are under investigation to complete the characterisation of transport. encapsulation of clone vector dna by cationic diblock copolymer vesicles for gene delivery a. v. korobko 1 , j. r. van der maarel 2 1 leiden university, the netherlands, 2 national university of singapore, singapore we will discuss the design, control, and structural characterization of cationic copolymer vesicles loaded with dna. these vesicles serve as a model system for diverse applications such as gene delivery, micro-arraying techniques and packaging of dna in congested states. encapsulation of dna was achieved with a single emulsion technique. for this purpose, an aqueous puc18 or pegfp-n1 plasmid solution is emulsi ed in an organic solvent and stabilized by an amphiphilic diblock copolymer. the neutral block of the copolymer forms an interfacial brush, whereas the cationic block complexes with dna. a subsequent change of the quality of the organic solvent results in a collapse of the brush and the formation of a capsule. the capsules are subsequently dispersed in aqueous medium to form vesicles and stabilized with an osmotic agent in the external phase. inside the vesicles, the dna is compacted in a liquid-crystalline fashion as shown by the appearance of birefringent textures under crossed polarisers and the increase in uorescence of labeled dna. the compaction ef ciency and the size distribution of the vesicles were determined by light and electron microscopy, respectively, and the integrity of the dna after encapsulation and subsequent release was con rmed by gel electrophoresis. we demonstrate the gene transfer ability of this new carrier system by the transfection of encapsulated pegfp plasmid into hela cancer cells. cellular transduction of nucleotide kinases to improve the activation of nucleoside analog prodrugs m. konrad 1 , c. monnerjahn 1 , s. ort 1 , a. lavie 2 1 max-planck-institute for biophysical chemistry, goettingen, germany, 2 university of illinois at chicago, chicago, usa the objective of our study is to improve therapeutic enzymeprodrug systems by generating catalytically superior nucleoside and nucleotide kinases that are essential for activation of nucleoside analogs. compounds, such as azt for the treatment of hiv infections, acv and gcv used against herpes virus, or the anticancer compounds arac and gemcitabine, can enter cells only in the unphosphorylated state (prodrug) and need to be transformed by different kinases to their pharmacologically active triphosphate state that interferes with dna replication. we have rst designed mutants of the human tmp kinase (htmpk) that phosphorylate aztmp up to 200-fold faster than wildtype. expression of this enzyme in human cells leads to 10-fold higher intracellular concentrations of azttp and to enhanced hiv inhibition. second, the prodrugs acv and gcv are not phosphorylated by human kinases, but are converted to their monophosphate forms by hsv1-tk which is used in enzyme/prodrug-dependent cancer suicide gene therapy. we generated enzyme variants which show selective and ef cient phosphorylation of gcv. third, an engineered human dck variant catalyzes more ef ciently the activation of the prodrugs arac and gemcitabine. thus, the concept of a gene (or enzyme) therapeutic treatment involving expression (or direct intracellular transduction) of a catalytically improved human enzyme may pave the way to the development of novel strategies in nucleoside prodrug-dependent cancer chemotherapy. docking-molecular dynamics studies on the peroxidase site of prostaglandin endoperoxide h2 synthase prostaglandin endoperoxide h 2 synthases-1 and 2 (pghs-1 and 2) catalyze the rst step in the biosynthesis of prostaglandins, prostacyclins and thromboxanes. arachidonic acid is transformed into prostaglandin g 2 (pgg 2 ) at the cyclooxygenase site of the enzyme and the 15-hydroperoxide oxygen-oxygen bond of pgg 2 is subsequently cleaved by reaction with haem at the distinct peroxidase site (pox) to produce prostaglandin h 2 (pgh 2 ). herein we present a plausible productive conformation obtained by docking calculations for the binding of pgg 2 to the pox site of pghs-1. the enzyme-substrate complex stability was veri ed by a 500-ps molecular dynamics simulation. structural analysis unveils the requirements for enzyme-substrate recognition and binding: the pgg 2 15-hydroperoxide group is in the proximity of the haem iron and participates in a hydrogen bond network with the invariant his207 and gln203 and a water molecule, whereas the carboxylate group establishes salt bridges with the remote lysines 215 and 222. the interaction of the peptide lah4 with anionic lipids during dna/rna delivery to eukaryotic cells a. j. mason 1 , a. martinez 2 , c. leborgne 2 , a. kichler 2 , b. bechinger 1 1 faculté de chimie, université louis pasteur, strasbourg, france, 2 généthon, evry, france the histidine rich amphipathic peptide lah4 has antibiotic and dna delivery capabilities. the peptide has a strong af nity for anionic lipids found in the outer membrane of bacterial membranes and has shown evidence of higher transfection activity against transformed over healthy tissue in culture. it has been proposed that anionic lipids can ip-op to reach the cytoplasmic monolayer. here they neutralise the cationic transfection complexes thereby causing release of oligonucleotides into the cytoplasm. we were, therefore, particularly interested to test for the role of the acidic lipid phosphatidylserine (ps) in mediating lah4-mediated delivery of dna ef ciency. to understand the potential peptide-lipid interactions in more detail, solid-state nmr experiments on model membranes have been performed. 31 p mas nmr on mixed phosphatidylcholine (pc)/ps and pc/phosphatidylglycerol (pg) membranes has been used to investigate speci c lah4 interactions with anionic lipids. by using deuterated lipids and wide-line 2 h nmr when probing lipid chain order, it is demonstrated that lah4 preferentially interacts with ps over pc. lah4 thereby effectively disorders the anionic ps lipid fatty acyl chains. the lipid chain destabilising effect of lah4 and also lah4 analogues can then be compared with their transfection ef ciency for dna or sirna in cell culture to aid in rational peptide vector design. virtual screening is now widely accepted as a basis for drug discovery thanks to signi cant improvement and good hit rates [1]. however, it is still highly cpu-consuming. at the same time, the number of protein-ligand complexes described at the atomic level is rising and the sequence similarity is used for structure and function predictions. new approaches are being developed to take advantage of the available structural data and the huge number of protein sequences in order to allow better tuned virtual screening. new web servers are being built to ease and to speed up the whole process (http://abcis.cbs.cnrs.fr/kindock/). integrating these servers into a pipeline dedicated to molecular modelling (http://abcis.cbs.cnrs.fr/atome/) shall allow both the re ned validation of modelled active sites as well as the oriented screening for the primary caracterization of potential ligands. an ideal drug delivery system should own the following characteristics, the rst is the targeting of therapeutic agent to the speci c site of their action. the second is the controlled delivery of a therapeutic molecule or protein in a pulsatile or staggered fashion. the third is the achieving sustained zero-order release of a therapeutic agent over a prolonged period of time. in this study, a new drug delivery system combined these characteristics was provided, which contains azobenzene derivatives (ab lipid) as an on-off switch incorporated into liposomes. the drastic release of calcein was observed on the rst uv irradiation of ab lipid to the cis isomer, while a suppressed release was observed when irradiated with the rst visible light. after that, the slope of release pro le became coincident. furthermore, calcein release was greatly increased after uv irradiation of ab lipid to the cis isomer and the drug release was greatly suppressed after vis irradiation of ab lipid to the trans isomer. we can control the release rate of calcein from ab lipid/egg pc mixed liposomes by uv or vis light irradiation. tryptophanase (trpase), a bacterial enzyme with no counterpart in eukaryotic cells, produces l-trp pyruvate ammonia and indole. it was suggested that indole is essential for bacteria multiplication and bio lm formation. bio lms destroy equipment and food and cause many illnesses. most synthesized quasi-substrates inhibit trpase at mm range. an optimal and speci c inhibitor of trpase may eliminate indole production and prevent bio lm formation. x-ray crystallography of the holo-wt e. coli trpase soaked with l-trp and the known mechanism of trpase activity should provide the information for the design and synthesis of active-sitespeci c quasi-analogs. utilizing the chromogenic substrate s-(onitrophenyl)-l-cysteine, the following michaelis-menten kinetics analyses determined the mode of trpase inhibition by trp and quinone based quasi-analogues and the corresponding ki values (in µm): dl-2-alanyl-2,10 anthraquinone, noncompetitively, 110; trypthophan ethylester, competitively, 24; acetyltryptophan, uncompetitively, 61.5; s-phenylbenzoquinone-l-tryptophan, uncompetitively, 92. plp-l-trp, inhibited irreversibly only the apo form of trpase and may serve for structure-determination purposes. further attempts are being made to synthesize improved trpase inhibitors, i.e., in the nm range. polyelectrolyte multilayer lms (pem) adsorbed on biomaterial surfaces are a new way to create a controlled release system. using biodegradable polymers, the lms can be degraded in vivo and release active molecules. in this work, we demonstrate the possibility of tuning the degradability of polysaccharide pem in vitro and in vivo. chitosan and hyaluronan pem (chi/ha) were either native or cross-linked (cl) using a water soluble carbodiimide (edc) at various concentrations in combination with nhydroxysulfosuccinimide. the in vitro degradation of the lms in contact with enzymes was followed by quartz crystal microbalance measurements and confocal laser scanning microscopy after lm labeling with chi fitc . whereas the native lms were subjected to degradation, the cl lms were more resistant to enzymatic degradation. films made of chitosan of medium molecular weight were indeed more resistant than lms made of chitosan-oligosaccharides. in addition, macrophages could degrade all types of lms and internalize the chitosan in vitro. the native lms implanted in vivo in mouse peritoneal cavity for a week showed an almost complete degradation whereas the cl lms were only partially degraded. these results suggests that the polysaccharides pem are of potential interest for in vivo applications as biodegradable coatings and that degradation can be tuned by controlling lm cross-linking. membrane electroporation -tool for therapeutic electrotransfer of drugs and gene dna e. neumann, s. kakorin physical and biophysical chemistry, faculty of chemistry, university of bielefeld, germany membrane electroporation (mep) is a new electrical high voltage scalpel, transiently opening the cell membranes of tissue for the penetration of foreign substances. due to the enormous complexity of cellular membranes, many fundamental problems of mep have to be studied at rst on model systems, such as the curved bilayer membranes of unilamellar lipid vesicles. electrooptical and conductometrical data of unilamellar liposomes indicate that electric eld pulses cause not only the formation of membrane electropores but also shape deformation of the liposomes, both processes mutually affecting each other. the primary eld effects of mep and cell deformation can trigger a cascade of numerous secondary phenomena, such as pore percolation and transport of small and large molecules across the electroporated membrane. the chemical mep theory represents a molecular physico-chemical approach to electrochemomechanical pore formation, yielding transport parameters, such as permeation coef cients, pore fractions and pore sizes. the pore concept is successfully applied to rationalize optimization strategies for biotechnological and medical applications of mep. in silico elucidation of xenobiotic processing loops k. nakata 1 , y. tanaka 2 , t. nakano 1 , t. ishikawa 3 , h. tanaka 2 , t. kaminuma 4 1 national institute of health sciences, tokyo, japan, 2 tokyo medical and dental university, tokyo, japan, 3 tokyo institute of technology, yokohama, japan, 4 hiroshima university, hiroshima, japan one of the important challenges for drug designers is to predict and analyze how drugs are absorbed, distributed, metabolized and excreted (adme) in the body. these processes highly correlated with toxicity of drugs and are actively studied in pharmacology. two classes of proteins, the drug metabolizing enzymes such as cytochrome p450s (cyps) and transporters, are the target of such adme/tox research. it was relatively recent that these two classes of proteins are synthesized by the genes that are the target genes of the nuclear receptors. nuclear receptors are ligand-activated transcription factors that form a superfamily. in case of humans there are 48 nuclear receptors almost half of whose ligands are identied, leaving some as true orphans. thus it was now recognized that these nuclear receptors play the role of sensors of drugs and other xenobiotic substances including environmental chemical pollutants and nutritional ingredients, while the drug metabolizing enzymes and the transporters are the processors which carry the actual cleaning jobs. we have started to elucidate the feedback loops that are formed by the xenobiotic ligands, nuclear receptors, their target genes, their product proteins, and their feedback actions on the ligands. the work is being carried out on our background database on the ligands and their receptors called kibank, and search programs for target genes of nuclear receptors algorithmically. the most recent results will be presented at the presentation. the major route for drug entry into cells is permeation across lipid bilayers. due to methodological limitations there are only few studies on permeation of drug-like molecules across lipid bilayers. an assay developed in our lab allows the direct measurement of lipid bilayer permeation of aromatic carboxylic acids (acas). tb 3 , which forms a uorescent complex with acas, is entrapped in liposomes and aca entry is determined from luminescence increase. lipid bilayer permeation was ph-dependent, following a henderson-hasselbalch function with a plateau for the neutral and the anionic species, respectively. in contrast to the expectations of the ph-partition hypothesis, permeation of the anionic species was only 1 to 3 magnitudes lower than that of the neutral species, leading to anion-controlled permeation at ph 7.4, independently of bilayer state and lipid composition. permeation across bilayers with a biologically relevant lipid composition was signi cantly slower than across egg-phosphatidylcholine membranes. the in uence of single lipids, such as cholesterol, was dependent on the structure and ionization state of the permeant. permeation coef cients of the neutral species correlated better with the polar surface area (psa) than logp oct , therefore psa is a better predictor for bilayer permeation of the neutral species of small acas than logp oct . interplay between polymerized liposomes physicochemical properties and composition and citotoxicity this study was aimed at investigating whether there is an interplay between diacetylenic polymerized liposomes physicochemical properties and lipid composition affecting citotoxicity in vitro. unsaturated 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine with saturated 1,2-dimiristoyl-sn-glycero-3phosphocholine in molar ratio 1:1, were combined to give a chemically modi ed membrane by uv-polymerization. biophysical characterization was carried out determining the hydrophobic factor and hydrodynamic radius. citotoxicity was evaluated through haemolytic capacity on bovine red blood cells and indirectly by capacity of induction of lipid peroxidation on microsomes or mitochondrial membranes. the haemolysis percentage in presence of dc8,9pc/dmpc is less than that induce by polymers used in dentistry. the data obtain suggests that the polymerized lipids can not induce lipid peroxidation on natural membranes. the polymerized diacetylenic liposomes showed less interaction with serum proteins than non polymerized and lower citotoxicity as compared with natural lipids. also cell viability was determined in cell line nih3t3 after exposure to lipids systems under study. the hydrophobic factor showed further augmentation for polymerized liposomes and is discussed in relation to in vitro stability. the above results suggest that polymerized and non-polymerized liposomes would serve as an effective delivery vehicle. s. sonar, s. d´souza, k. p. mishra radiation biology and health sciences division,bhabha atomic research centre,mumbai-400085, india liposomes offer new approaches for drug delivery through their encapsulation to alter pharmacodynamic properties of loaded drug leading to reduction in toxicities and/ or improved ef cacy. for prolonged systemic circulation, the liposomes size has been shown to be limited to 200 nm or less. the ethanol injection method is an excellent technique for the formation of liposomes of < 200nm without the need of sonication or extrusion. the present study was aimed to produce liposomes encapsulating doxorubicin in minimum procedural steps. liposomes were prepared using distearoyl phosphatidylcholine and cholesterol, distearoyl phosphatidylcholine, cholesterol and oleic acid. the effects of different operational conditions for vesicle production and drug encapsulation were evaluated, with a view to achieve process cost to a minimum, suitable size and high encapsulation ef ciency. although high ef ciency of doxorubicin encapsulation was obtained by 'active' or 'remote' loading process in dspc/chol system, it was poor in one-step injection method. oleic acid was included to cut down the active loading by ph-gradient. dspc/chol/oa systems spontaneously loaded doxorubicin with encapsulation ef ciency of 50 % and nal drug to lipid molar ratio upto 0.172. the mean diameter of the vesicles was 175 + 5 nm. the method offers liposomes of small size with high loading. design of peptides with consecutive dehydro phenylalanine residues r in order to develop general rules for the design of peptide conformations with consecutive alpha, beta-dehydro phenylalanine-residues, peptides were synthesized, crystallized and crystal structures and molecular conformations were determined. following conclusions were drawn based on the structural data: -peptide unit sequences with two consecutive dehydro-phe residues at (i+1) and (i+2) positions adopt an unfolded s -shaped structure with dihedral angles phi-psi centred at 60 , 30 . -the peptides containing two consecutive dehydro-phe residues at (i+2) and (i+3) positions • form two overlapping type iii beta -turns (incipient 3 10 -helix). • with branched beta -carbon residue only at (i+1) position adopt a conformation with two overlapping types ii and iii beta -turns. • with branched beta-carbon residues such as val and ile at both (i+1) and (i+4) positions form two overlapping types ii and i beta -turns. the consistency in the formations of these conformations makes the design of peptides with alpha,beta ?-dehydro -residues a useful and highly predictable method for developing speci c ligands for various biological applications including drug design. binding of cationic porphyrin to isolated double-stranded dna and nucleoprotein complex k. zupán 1 , l. herényi 1 , k. tóth 2 , z. majer 3 , g. csík 1 1 institute of biophysics and radiation biology, semmelweis univ., budapest, hungary" 2 biophysics of macromolecules, german cancer research center, heidelberg, germany, 3 department of organic chemistry, eötvös loránd univ., budapest hungary the complexation of tetrakis(4-n-methylpyridyl)porphyrin (tmpyp) with free and encapsidated dna of t7 bacteriophage was investigated. to identify binding modes and relative concentrations of bound tmpyp forms, the porphyrin absorption spectra at various base pair/porphyrin ratios were analyzed. spectral decomposition, uorescent lifetime, and circular dichroism measurements proved the presence of two main binding types of tmpyp, e. g., external binding and intercalation both in free and in encapsidated dna. tmpyp binding does not in uence the protein structure and/or the protein -dna interaction. concentrations of tmpyp species were determined by comprehensive spectroscopic methods. our results facilitate a qualitative analysis of tmpyp binding process at various experimental conditions. we analyzed the effect of base pair composition of dna, the presence of protein capsid and the composition of buffer solution on the binding process. protein crystallization and structural study of upa protease domain with active site serine mutation the urokinase (upa) system is composed of upa, its receptor (upar), and inhibitor (pai). it plays important role in various physiologic processes, including brinolysis, cell adhesion, and signal transduction, and has been recognized as a target for intervention in tumor growth and tumor metastasis. we constructed an active site mutant of upa protease domain (159-405) with three mutations (c279a, n302q, and s356a) and expressed it as secreted protein in pichia pastoria with ppicza vector. the secreted mutant was captured from culture medium by a cation exchange column and then further puri ed on a gel ltration column. the puri ed mutant was then crystallized by sitting drop vapor diffusion method with several precipitant conditions: (1) 1.5-2.1m ammonium sulphate, 5-8% peg400, 50mm sodium citrate ph 4.6 or 50mm sodium phosphate ph 7.5, 0.05% sodium azide. (2) 1.8m ammonium sulphate, 0.14m lithium sulphate, 50mm sodium acetate at ph 5.2, (3) 2.5-2.8m sodium formate, 50mm sodium acetate at ph 5.2. (4) 3.2-3.6m sodium chloride, 50mm sodium acetate at ph 5.2. the crystals were of varying quality but generally diffracted from 1.7å-2.1å with inhouse x-ray source. the structure of this upa mutant and its complex with various inhibitors will provide a platform for rational upa inhibitor design. abstract in this work, the linear interaction energy (lie) method was used to calculate the binding free energies of hiv-1 integrase (in) and a series of dicaffeoyl -or digalloyl pyrroliding and furan derivatives inhibitors. the model of binding free energy prediction for homogeneous inhibitors of hiv-1 in has been obtained with a root-mean-square deviation (rmsd) of 1.39 kj/mol and estimated to be a precise model with good prediction capability. in addition, the probable binding mode of this series of inhibitors with hiv-1 in was proposed by using molecular docking and molecular dynamics (md) simulation methods. our results indicate that caffeoyl -or galloyl group of inhibitors have close interaction with a hiv-1 in conservative dde motif. a speci c non-competitive inhibitor of a small g protein/gef complex on the protective role of selenium and catechin in cadmium toxicity s. özdemir 1 , s. dursun 1 , s. toplan 1 , n. dariyerli 2 , m. c. akyolcu 1 1 istanbul university, cerrahpasa medical faculty, department of biophysics, turkey, 2 istanbul university cerrahpasa medical faculty, department of physiology, turkey cadmium as heavy metal is toxic and carcinogenic for organisms. cadmium perform their effects on living organisms by accumulation in blood and various tissues. due to their accumulation in various tissues and in blood, tissue antioxidant enzyme systems are affected. the present study was planned to determine the possible protective roles of selenium and catechin against the toxic effects of considered heavy metals. the study has been performed in wistar albino type rats which divided into four groups as control and cadmium, cadmium+selenium, cadmium+catechin received groups. besides cadmium as heavy metal, selenium concentration determinations were performed in blood, liver and kidney tissues of each group of rats. in the same tissue samples besides lipid peroxidation measurements, glutathione, glutathione peroxidase and superokside dismutase enzyme activity determinations were also performed. the accumulation of heavy metals was determined in blood, liver and kidneys after cadmium administration during experimental period. in the tissue of experimental group animals there was an increased lipid peroxidation but decreased antioxidant enzyme activities were observed. while effects of selenium in decreased toxicity of cadmium have been detected, there was no statistically signicant effect of catechin observed. proposed that motors could dynamically cluster at the tip of tubes when they are individually attached to the membrane. we demonstrate, in a recently designed experimental system, the existence of an accumulation of motors allowing tube extraction. we determine the motor density along a tube by using uorescence intensity measurements. we also perform a theoretical analysis describing the dynamics of motors and tube growth. the only adjustable parameter is the motor binding rate onto microtubules, which we measure to be 4.7 +/-2.4 s 1 . in addition, we quantitatively determine, for a given membrane tension, the existence of a threshold in motor density on the vesicle above which nanotubes can be formed. we nd that the number of motors pulling a tube can range from four at threshold to a few tens away from it. the threshold in motor density (or in membrane tension at constant motor density) could be important for the understanding of membrane traf c regulation in cells. kinesin and dynein move a peroxisome in vivo: a tug-ofwar or coordinated movement? c. kural, p. r. selvin, k. hwajin, g. goshima, v. i. gelfand university of illinois at urbana-champaign, usa we have used fluorescence imaging with one nanometer accuracy (fiona) for analysis of organelle movement by conventional kinesin and cytoplasmic dynein in a cell. we can locate a green uorescence protein (gfp)-tagged peroxisome in cultured drosophila s2 cells to within 1.5 nanometer in 1.1 milliseconds, a 400-fold improvement in temporal resolution, suf cient to determine the average step size to be 8 nanometers for both dynein and kinesin. furthermore, we nd that dynein and kinesin do not work against each other in vivo during peroxisome transport. rather, we nd that multiple kinesins or multiple dyneins work together, producing up to 10 times the in vitro speed. engineering a bio-molecular walker h. jankevics, j. e. molloy division of physical biochemistry, mrc national institute of medical research, the ridgeway, mill hill nw7 1aa, london, uk in this work we describe the design and development of a biomolecular walker based on the motile system found in certain ciliated protists. the motile system is driven by the binding of ca 2 ions and in contrast to other commonly studied motor proteins is independent of atpase (amos et al., 1975) . the motor protein is a 20kda ca-binding protein called spasmin (maciejewski et al.,1999; itabashi et al., 2003) which belongs to the ef-hand family of calcium binding proteins called calmodulins. upon calcium binding, spasmin is thought to undergo a large conformational change as it binds its own target peptide and we wish to exploit this in order to create our own novel molecular walker.we have created a recombinant spasmin with sequence tags to enable speci c immobilization and various conjugate chemistries. cystein mutants have been introduced at speci c points in the protein to enable attachment of other small molecules, for example uorophores. we are now optimising protein expression and puri cation to maximise the yield of active protein on which we can perform the conjugate chemistries. we will characterize the structural changes using biophysical methods such as circular dichroism, analytical ultracentrifugation, electron microscopy, afm and total internal re ection uorescence spectroscopy on single molecules. the force generation in muscle arises from direct interaction of the two main protein components of the muscle, myosin and actin. the process is driven by the energy liberated from the hydrolysis of atp by myosin. the interaction is performed by cyclic interaction of myosin with atp and actin, and at least six intermediates are proposed for actomyosin atpase in solution. the powerful dsc technique allows the derivation of heat capacity of proteins as a function of temperature. from the deconvolution of the thermal unfolding patterns it is possible to characterize the structural domains of the motor protein. in this work we tried to approach the temperature-induced unfolding processes in different intermediate state of atp hydrolysis in striated muscle bres. we have extended the experiments to study the ber system prepared from psoas muscle of rabbit in rigor, strongly binding and weakly binding states of myosin to actin where the inorganic phosphate (p i ) was substituted by the phosphate analogue orthovanadate. the dsc transitions were analyzed in different buffer solutions (tris and mops) to get information about the temperature dependence of ph on the conformational changes. single kinesin motor proteins walking through the searchlight s. verbrugge, l. c. kapitein, e. j. peterman vrije universiteit, de boelelaan 1081, 1081 hv, amsterdam, the netherlands the dimeric motor protein kinesin steps by a hand-over-hand mechanism. this means that the centre of mass moves with 8 nm steps, while the two motor domains, the one after the other, move 16 nm to the next binding site on the microtubule. the molecular details of what happens during a step are not fully understood, partly because of lack of time resolution in wide-eld, single-molecule uorescence experiments. we set out to develop an approach to study the motility of kinesin with a time resolution below a millisecond (a single step takes on the order of 10 milliseconds). this approach allows us to look into the mechanochemistry and coupling of the two kinesin motor domains while they are stepping. our method is based on confocal microscopy and we study the uorescent properties of single labeled motors while they walk through the confocal laser spot. we present the experimental details of our approach and show our results on human kinesin constructs that are speci cally labeled in the tail. we show that our approach enables us to study the mechanism of kinesin with a much higher time resolution than what was achieved before with single-molecule uorescence experiments. movement of coupled single-headed kinesins analysed by a brownian-ratchet model . the analysis of this system is expected to provide insights into the mechanism underlying the motility of conventional double-headed kinesin, espetially the roles played by individual heads. we would like to clarify whether the experimentally observed behaviors that are supposed to be caused by a pair of single-headed kinesins can be explained by a simple brownianratchet model, which is successful in describing the motion of an unconventional single-headed kinesin kif1a. our model consists of two brownian motors (ratchets) separated by a xed distance r. the velocity and other quantities of the coupled motors are calculated by solving the fokker-planck equation with various choices of r and other parameters. then, assuming a certain probability distribution of r associated with random attachment of kinesin heads on a bead in the experiment, the statistical properties of the motion of the coupled brownian motors are analysed. the force-velocity relation observed experimentally is found to be consistent with the present model with appropriate choices of the model parameters. the adequacy of the parameter choice needs to be con rmed by other experiments. optical trap with fast programmable feedback loop to study rotary molecular motors t. pilizota, f. bai, r. m. berry clarendon laboratory, univeristy of oxford an optical trap with back-focal plane detection and fast programmable feedback has been developed for the study of rotary molecular motors. a helium-neon laser (632 nm) is used for position detection and a solid state bre laser (1064 nm, 3w cw) forms the trap. acousto-optic de ectors (aods) controlled by a digital signalling processing board are used to achive programmable feedback loops with exible control options and speeds up to 8 khz. several modes of feedback are demonstrated, controlling both bead position (x,y) and angle (r, ). polystyrene beads or bead pairs can be held at set (x,y) or , and the set-point can be changed while the program is running. for example, feedback can be used to move a bead or a bead pair in a circle. results of using the system to study the bacterial agellar motor are presented. a dimeric 1-d lattice gas as model for molecular motors collective dynamics p. pierobon 1 , t. franosch 2 , e. frey 3 1 ludwing-maximilian universitaet, münchen, germany, 2 hahn meitner institut, berlin, germany, 3 arnold sommerfeld center for theoretical physics, münchen, germany the transport of molecular motors along microtubules closely resemble the dynamics of a driven lattice gas of dimers without conservation of particles. the unidirectionality, asymmetry and stochasticity of the motion are encoded in the well studied totally asymmetric simple exclusion process (tasep). we extend the model to a more realistic one, including attachment and detachment kinetics and extended (dimeric) particles. we study the stationary phase diagram by means of monte carlo simulations combined with a continuum description (based on an extended mean eld theory). we also evaluate the domain wall theory nding out the effective potential con ning the phase interface into the bulk. a. e. wallin 1 , j. lisal 2 , r. tuma 2 1 department of physical sciences, pobox 64, fin-00014, university of helsinki, finland, 2 institute of biotechnology, university of helsinki, finland molecular motors often consist of two or more subunits that cooperate to convert chemical energy into mechanical motion. hexameric helicases and viral packaging atpases constitute a special class of molecular motors that translocate along nucleic acids. recent structural and spectroscopic characterization of these motors revealed that their enzymatic cooperativity does not result from cooperative binding [1-2]. in order to understand this new type of cooperativity we simulated the kinetics of a single hexameric motor by multiple coupled stochastic reactions using the gillespie algorithm [3] . in contrast to analytical methods, our direct simulation allowed us to investigate the kinetics with an arbitrary model for cooperativity between the subunits. simulations on the kinetics of the hexameric rna packaging motor p4 from dsrna bacteriophage [1] with different cooperativity mechanisms provided insight into the rnamediated cooperativity and yielded a sound theoretical basis for the interpretation of experimental results [2]. the viral infectivity factor (vif) encoded by hiv-1 is a small basic protein that strongly modulates the viral replication and is required for pathogenicity. vif is packaged into hiv-1 particles through a strong interaction with genomic rna and is associated with viral nucleoprotein complexes. moreover vif acts during the early stages of the viral infection (capsid disassembly, reverse transcription) as well as during the late stages of virus replication (virus assembly and maturation of the virion). however the effect on early stages is probably a consequence of a defective assembly and / or virion maturation. understanding the rna-binding properties of vif would contribute to elucidate the role played by vif in the regulation of the genomic rna traf cking in the cytoplasm to unable ef cient packaging, and the prevention of cellular inhibitors from altering hiv-1 rna. in this context, we have characterised the interactions of recombinant vif with hiv-1 genomic rna by uorescence spectroscopy, and determined the af nity of the protein for synthetic rnas corresponding to various regions of hiv-1 genome. taken together our results demonstrate cooperative and speci c binding. in particular, we showed that vif has a high af nity for the 5'untranslated region of hiv-1 genomic rna. s. bernacchi 2 , e. ennifar 1 , k. toth 2 , p. walter 1 , j. langowski 2 , p. dumas 1 1 cnrs upr 9002 -strasbourg (france), 2 dkfz -heidelberg (germany) we have used the dimerization initiation site (dis) of hiv-1 genomic rna as a model to investigate hairpin-duplex interconversion by using a combination of uorescence, uv-melting, gel electrophoresis and x-ray crystallographic techniques. fluorescence studies with molecular beacons and crystallization experiments with 23-nucleotide dis fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on rna concentration, and not on cooling kinetics. with natural sequences able to form a loop-loop complex (or 'kissing complex'), concentrations as low as 3 µm in strands are necessary to obtain a majority of the hairpin form. on the contrary, when kissing-complex formation was made impossible by mutation in the loop, a majority of hairpins was obtained even at 80 µm in strands. this mutated sequence also showed that kissing-complex formation is not a prerequisite for an ef cient conversion to duplex in presence of salts. we proved that this happens through hairpins engaged in a cruciform intermediate, but not from free strands after hairpin melting. supporting this view, the very rst step of formation of such a cruciform intermediate could be trapped in a crystal structure. such a mechanism might be biologically signi cant beyond the strict eld of hiv-1 rna dynamics. generation of rna dimeric form of the human immunode ciency virus type 1 (hiv-1) genome is important for the viral replication. the dimerization initiation site (dis) has been identi ed as a short sequence that can form a stem-loop structure with a selfcomplementary sequence in the loop and a bulge in the stem. a 39mer dis rna fragment, dis39, spontaneously formed "loosedimer" and was converted into "tight-dimer" by supplement of nucleocapsid protein ncp7. nmr chemical shift analysis for dis39 in the kissing-loop and extended-duplex dimers revealed that three dimensional structures of the stem-bulge-stem region were similar between the two types of dimers. therefore, we determined the solution structures of two shorter rna molecules corresponding to the loop-stem region and the stem-bulge-stem region of dis39, and the solution structures of dis39 in the kissing-loop and extendedduplex dimers were determined by combining the parts of structures. the mechanism of conformational conversion will be discussed based on the solution structures and the molecular dynamics analysis. nmr and molecular modelling studies of an rna hairpin containing a g-rich hexaloop the mrna of the pgy1/mdr1 gene encoding the transmembrane p-glycoprotein (p-gp) contains a hairpin that is the target of antisense oligonucleotides, suppressing the p-gp function of multidrug resistance. the solution conformation of this hairpin constituted by the 5'(gggaug)3' loop closed by a g-u mismatch containing stem is studied by nmr and molecular dynamics in explicit solvent. special attention is given on the sugar and the backbone conformations and the hexaloop intrinsic properties of these two components are carefully investigated. the stem structures obtained by molecular dynamics with and without nmr constraints converge to the same a-type double helix. the wobble g-u mismatch moderately perturbs the overall conformation, despite of c2'-endo sugars and unusual backbone conformations located between the mismatch and the loop. in the hexaloop part, the sugar puckers are in majority in c2'-endo conformations, probably to extend the strand with the help of unusual backbone angles conformations. the loop appears stabilized by one hydrogen bond and stacking interactions. thus, from the 5' to the 3'-ends, the four purine bases ggga are stacked together, then a u-turn like is observed, and nally, u stacks on the last g that remains rather far from the stem. nmr and molecular modelling studies of an rna hairpin containing a g-rich hexaloop the mrna of the pgy1/mdr1 gene encoding the transmembrane p-glycoprotein (p-gp) contains a hairpin that is the target of antisense oligonucleotides, suppressing the p-gp function of multidrug resistance. the solution conformation of this hairpin constituted by the 5'(gggaug)3' loop closed by a g-u mismatch containing stem is studied by nmr and molecular dynamics in explicit solvent. special attention is given on the sugar and the backbone conformations and the hexaloop intrinsic properties of these two components are carefully investigated. the stem structures obtained by molecular dynamics with and without nmr constraints converge to the same a-type double helix. the wobble g-u mismatch moderately perturbs the overall conformation, despite of c2'-endo sugars and unusual backbone conformations located between the mismatch and the loop. in the hexaloop part, the sugar puckers are in majority in c2'-endo conformations, probably to extend the strand with the help of unusual backbone angles conformations. the loop appears stabilized by one hydrogen bond and stacking interactions. thus, from the 5' to the 3'-ends, the four purine bases ggga are stacked together, then a u-turn like is observed, and nally, u stacks on the last g that remains rather far from the stem. aminoglycoside binding to hiv-1 dis kissing-loop complex: from crystals to cells e. ennifar 1 , j.-c. paillart 1 , a. bodlenner 2 , p. pale 2 , r. marquet 1 , p. dumas 1 1 cnrs upr 9002, strasbourg -france, 2 cnrs/université louis pasteur strasbourg umr 7123, strasbourg -france all retroviral genomes consist in two homologous single stranded rnas. dimerization is an essential step for viral replication. hiv-1 dimerization initiation site (dis) is a strongly conserved stem-loop in the 5' leader region of the genomic rna. it was shown in vivo that alteration of the dis dramatically reduces viral infectivity. we have previously solved crystal structures of the dis kissing-loop complex. analysis of these structures revealed an unexpected resemblance between the dis kissing-loop and the 16 s ribosomal aminoacyl-trna site (a-site), which is the target of aminoglycoside antibiotics. we have shown that some aminoglycosides specifically bind to the dis kissing-loop complex with an af nity and geometry similar to that observed in the a-site. in agreement with these previous results, we have now solved highresolution crystal structures of the dis kissing-loop complex bound to four aminoglycosides. these structures show that, as expected, two aminoglycosides are bound per kissing-loop complex. importantly, the binding is observed not only in vitro on large hiv-1 genomic rna fragments, but also on infected cells. moreover, we showed that some of these aminoglycosides stabilize the kissingloop rna dimer, which is consistent with the observation in crystal structures of numerous direct and water-mediated drug-rna contacts. these structures are currently used as starting points for designing potential new drugs targeted against the viral rna. modeling the long range entropy of rna: w. k. dawson 1 , k. fujiwara 1 , k. yamamoto 2 , g. kawai 1 1 chiba institute of technology, 2-17-1 tsudanuma, narashino, chiba, japan, 2 international medical center of japan, 1-21-1 toyama, shinjuku-ku, tokyo, japan non-coding rna appears to make up a large part of the human genome. a reliable rna structure prediction program is needed to understand the structure of this non-coding rna. we recently developed a new way to model the long range entropy in rna and applied it to rna secondary structure prediction. in some of instances, the new approach is able to achieve far better predictions than the state of the art secondary structure programs even given exactly the same parameters. predictions using this method tend to show distributions that are funnel shaped. a new and important parameter in these calculations is the persistence length (a measure of the correlation and exibility of the rna). (url: http://www.rna.it-chiba.ac.jp/ vsfold/vsfold4/) this new approach has now been extended to prediction of pseudoknots. the method is a heuristic wherein the hierarchical folding hypothesis is used to nd the pseudoknots as the rna secondary structure is folding, and corrections to that secondary structure are made to accommodate the pseudoknot. it is able to do these searches in roughly n^4 time. the model is consistent with the hierarchical hypothesis and it is possible to estimate rna folding times that are of the correct order of magnitude using this model. with further adaptations to account for the size, shape and variablity of amino acid residues (hydrophobicity etc.), the model also appears to be transferable to protein folding problems. a. v. melkikh ural state technical university, ekaterinburg, russia a model of the genome as a gene network capable of receiving information about the environment and performing some operations on genes has been considered. the evolution rate of replicators for the mechanism of random mutations has been estimated. it was shown that the evolution rate under random actions is negligibly small for real dimensions of genomes of replicators [1]. it was inferred that only a deterministic mechanism of the evolution can explain the known evolution rate of replicators. a deterministic model of the evolution has been proposed. the basic principles of this model include: 1) information about the replicators evolution is encoded in the conformational states of proteins; 2) the conformational language of proteins is translated into the language of nucleotide sequences during the evolution; 3) the structure of genes is controlled such that the transition to a nearest free ecological niche takes a minimum time (at a preset restriction on the control). transfer rnas are synthesized as part of longer primary transcripts that require processing of both their 3' and 5' extremities in every living organism known. the 5' side is matured by the quasiuniversally conserved endonucleolytic ribozyme, rnase p, while removal of the 3' tails can be either exonucleolytic or endonucleolytic. the endonucleolytic pathway is catalysed by an enzyme known as rnase z. rnase z cleaves precursor trnas immediately after the discriminator base in most cases, yielding a trna primed for addition of the cca motif by nucleotidyl transferase. rnase z is found in the vast majority eukaryotes and archaea and in about half of the sequenced bacteria. it is often essential for growth and mutations in one of the two genes encoding rnase z (elac2) have been linked with prostate cancer in man. in this poster we present the crystal structure of bacillus subtilis rnase z at 2.1å resolution (1) resolved by mad method and propose a model for trna recognition and cleavage. the structure explains the allosteric properties of the enzyme and also sheds light on the mechanisms of inhibition.it also highlights the extraordinary adaptability of the metallohydrolase domain of the b-lactamase family for the hydrolysis of covalent bonds. (1)i. most rnas undergo several steps of post-transcriptional modi cation before carrying out their assigned functions. one of the major modi cations is the splicing process, by which non-coding introns are removed from the coding exons. splicing can be performed by autocatalytic, self-splicing introns (e.g. group ii introns), i.e. catalytic rna or ribozymes. group ii introns, which occur in bacterial genomes and in organellar genes of plants, funghi and lower eukaryotes, consist of a conserved set of six domains. domain 1 recognizes the 5'-exon through a 10-15 base pairing interaction formed by two regions within the intron (exon binding sites, ebs1 and ebs2) and the last 10-15 nucleotides of the 5'-exon (intron binding sites, ibs1 and ibs2). as the correct recognition of ibs1 by ebs1 is crucial for a successful splicing event we are investigating the structural and metal ion requirements of this part by various spectroscopic techniques, e.g. nmr. our data shows that the hairpin including ebs1 consists of a helical region followed by an unstructured single stranded part, which is ready for splice site recognition. the results of the structure analysis will be presented. financial support by boehringer ingelheim fonds (fellowship to d. k.) and the swiss national science foundation (snf-förderungsprofessur to r. k. o. s.) is gratefully acknowledged. structural basis for the antigene and antisense properties of modi ed dna:dna and rna:dna duplexes e. c. m. juan 1 , t. kurihara 1 , j. kondo 1 , t. ito 2 , y. ueno 3 , a. matsuda 2 , a. takenaka 1 1 graduate school of bioscience and biotechnology, tokyo institute of technology, 2 graduate school of pharmaceutical sciences, hokkaido university, 3 faculty of engineering, gifu university oligonucleotides containing polyamines are currently being evaluated as potential antigene and antisense compounds. those with 5-(n-aminohexyl)carbamoyl-2'-deoxyuridine ( n u) and its 2'-omethyl derivative ( n u m ) exhibit improved nuclease resistance and form stable duplexes with their dna and rna targets. x-ray structures of these duplexes have shown good correlation between the conformational changes and the observed chemotherapeutic properties. the amide groups of the modi ed uracil bases form six-membered rings through the intra-molecular nh-o4 hydrogen bonds, so that the aminohexyl chains protrude into the major grooves. some of the terminal ammonium groups are involved in intra-duplex interactions with phosphate oxygen anions, whereas the others interact with those of the adjacent duplex. such interactions contribute to the stability of duplex formation. the 2'-o-methyl modi cation in n u m shifts the ribose ring toward the c3'-endo conformation and in uences duplex stability. observed changes in the dimensions of the minor grooves and in the hydration structures are also well correlated to nuclease resistance and duplex stability. group ii intron ribozymes catalyze selfsplicing in bacterial genomes as well as in organellar genes of lower eucaryotes. for correct structure and function these ribozymes need speci c concentrations of monovalent and divalent cations such as k and mg 2 . most of these ions are used for charge screening, but some are also bound to distinct sites ful lling various speci c tasks. the conserved secondary structure of group ii intron ribozymes consists of six domains grouped around a central wheel. here the focus is set on domain 5 (d5) of the yeast mitochondrial intron ai5 , a hairpin of 34 nucleotides, which is crucial for catalytic activity. the 3-nucleotide bulge in d5 is known to be exible and acts as a metal ion binding platform. we have investigated the binding of different metal ions (mg 2 , ca 2 , mn 2 , cd 2 ) to this platform by uorescence spectroscopy. for this the bulge site adenosine in d5 was replaced by the uorescent nucleotide base analogue 2aminopurine (2ap). the binding data ts to an equation describing a binding to a single class of sites. the titration experiments not only reveal different dissociation constants for the tested metal ions but also indicate different effects on the bulge structure. financial support by the swiss national science foundation (snf-förderungsprofessur to r.k.o.s) is gratefully acknowledged. modi ed nucleosides and across the anticodon loop interactions in trna u. b. sonavane, k. d. sonawane, r. p. tiwari national chemical laboratory, pune 411008, india in several interesting trna molecules, the (34 th ) as well as the (37 th ) nucleoside are hyper modi ed. as an example, unique hypermodi ed nucleosides mcm 5 s 2 u 34 and ms 2 t 6 a 37 are crucial in human trna lys , which acts as a primer in hiv replication. modi ed nucleosides may facilitate or hinder across the loop interactions. large substituents in 34 th and 37 th modi ed nucleosides if oriented suitably may also interact with each other. across the loop interactions may lead to unconventional anticodon loop structures also affecting exibility of the anticodon loop. this may restrict or enlarge synonymous codon choice and decoding during protein biosynthesis. except for trna asn (with interacting q 34 and t 6 a 37 ), our studies show conventional 'open' loop structure -free of across the loop interactions, for a number of interesting trna anticodon loops with diverse hyper modi ed nucleosides at both of these locations. molecular dynamics simulations of hydrated anticodon arm of trna asn show persisting interaction involving the diol group of q 34 and carbonyl group of ureido linkage in t 6 a 37 . additionally, the hoogsteen edge of 37 th adenine base participates in hydrogen bonding with watson -crick edge of 33 rd base and thus contributes to unique loop structure of trna asn . resulting suboptimal q:c base pairing leads to unbiased reading of u or c as the third codon letter. absence of queuosine modi cation, q 34 happens to be also associated with uncontrolled rapid proliferation of cells and malignant growth. structural properties of ctg/cag repeats, and preliminary x-ray analysis of cug repeats y. sato, k. kimura, a. takénaka graduate school of bioscience and biotechnology, tokyo institute of technology, yokohama, japan. the human genome contains so many different types of repetitive sequences. some of them are tandem repeats of trinucleotides. their unusual expansions cause genetic diseases such as type 1 myotonic dystrophy (dm1) and huntington's disease (hd), the unit sequences being ctg and cag, respectively. the numbers of repeats of the two complementary sequences change independently during dna replication or repair. the direct origin of dm1 is, however, the transcribed rna fragments with cug repeats, which forms a speci c structure and inhibits other protein syntheses. in the present study, structural versatilities of such dna and rna fragments has been examined. native pages of (cug) n show that the hairpin structure with even number is more stable than that with odd number. this difference might be ascribed to the structural difference at the hairpin head. the pages also show that duplex formation is dependent on coexisting cationic species and their concentration. crystal data of (cug) 6 (a=b=39.6, c=141.0 å, and the space group r32) suggest that the asymmetric unit contains the rna fragment. an approximate crystal structure solved by molecular replacement techniques at 1.95 å resolution shows that the rna fragments form a duplex similar to an a-form rna. context-dependent selection of promoter in a natural selection-type evolution reactor h. nagayasu, y. ageno, x. t. ma, y. husimi saitama university, saitama, japan using an isothermal ampli cation of hairpin dna/rna (developed by g.joyce), we drove a natural selection-type evolution reactor taking the speci c growth rate as the tness. we used hiv-1 rt and thermot7 rnap at 50 c. starting from the random promoter pool, we selected the strongest promoter at 50 c, which was separated by hamming distance 2 from the strongest promoter at 37 c. the latter was found to be identical to the natural t7 promoter. when we used a simple random pool, the selection process showed one-step convergence. when we used a random pool with a speci c short sequence at the upstream anking region of the random region, we observed an evolution process as convergencedivergence-convergence of the promoter sequence, driven by deletion of the speci c short sequence. this context-dependent selection was found to come through a neutral path, judged from the tness measurement. intronic sirna and mirna, and dna methylation gif sur yvette, france. abnormal activation of small g proteins is involved in several human diseases. small g proteins are activated by gdp/gtp exchange, which is stimulated by their guanine nucleotide exchange factors (gefs). thus, small g protein/gef complexes appear as emerging targets for interrupting signalling networks regulated by small g proteins in pathological contexts. the activation of small g proteins of the arf family is initiated by exchange factors which carry a sec7 catalytic domain stimulating the dissociation of the bound gdp nucleotide. the structure of the arf1-gdp-arno reaction intermediate, trapped by a mutation of the catalytic glutamate, was recently solved by x-ray crystallography (pdb code: 1r8s) acknowledgements: the work was supported by contract kj. q. yin, f. chen, y. tan, t. gu institute of biophysics, chinese academic sciences, beijing, china sirna/mirna can ef ciently induce mrna cleavage or translational repression at the posttranscriptional level in a sequencespeci c manner. recently, it has been shown that these small rnas guide genome modi cation in mammalian cells. however, their ability to direct cognate dna methylation has been con rmed so far only in plants, and their biogenesis, functions, and modes of silencing genes are yet elusive. here, we report that small rnas derived from intron regions of some genes can target homologous dna sequence in promoter, 5'utr or 3'utr regions of genes in different human tumor cells. surprisingly, we also discovered that endogenous sirnas from introns of genes possessed a large number of target mrnas by using bioinformatics, and con rmed their existence in human cells with northern blot analysis. intronic small rnas generated by sliceosomes can form mature mirnas or sirnas through the processing of drosha and /or dicer. rt-pcr analysis indicated that vector-based small rna repressed expression of homologous genes at the transcriptional and/or translational levels. western blotting demonstrated that the expression of some proteins was greatly reduced or completely inhibited owing to promoter methylation. these ndings reveal that the expression of some genes can incredibly control cell activities at both protein and rna levels. our results also suggest that these small rnas may regulate gene expression in different modes of action. role of stacking in speci c recognition of capped rna by the cbc protein the stacking interaction involving 7-methylguanine moiety of mrna 5' terminal cap (m 7 g) and aromatic amino acid side chains is a common feature of all known cap-binding proteins. the crystal structures of the human cap binding complex (cbc) showed its induced folding upon m 7 gpppg cap analogue binding. stabilization of the cbc-m 7 gpppg complex by sandwich stacking of m 7 g in between y20 and y43 is additionally enhanced by stacking of the second base of capped mrna with y138. gibbs free energy of the association of various cbc mutants with the synthetic cap analogues, m 7 gpppg, m 7 gpppu, and m 7 gtp, has been determined by uorimetric titration. preference of the wild type (wt) cbc for the dinucleotide analogues is also observed for the y43a mutant, with the energy loss of 0.8-1.5 kcal/mol. however, all proteins with the mutated second stacking partner y20 prefer to bind m 7 gtp compared with m 7 gpppg. the binding of m 7 gtp to the y20a mutant is only 0.3 kcal/mol less favourable compared with the wt cbc. these divergences may be ascribed to smaller entropic costs of conformational rearrangement of cbc in the case of a smaller ligand, which can nd more favourable contacts when its second part is not ef ciently 'constrained' by stacking with y138. supported by kbn 3 p04a 021 25 annealing of the tar dna hairpin to a complementary tar rna hairpin, resulting in the formation of an extended duplex, is an essential step in the minus-strand transfer process of hiv-1 reverse transcription. in this work, we use gel-mobility-shift analysis to follow the kinetics of this reaction in the absence or presence of hiv-1 nc prepared by solid-phase peptide synthesis. to elucidate the reaction pathway, we use either the complete 59-nt tar hairpins or truncated 27-to 32-nt minihelices (mini-tar) derived from the top part (i.e. hairpin loop) of tar. assays were also carried out with mutant tar constructs. the annealing kinetics were studied systematically as a function of dna concentration and temperature. we show that the annealing initiates through a weak loop-loop kissing interaction, followed by a much slower conversion step, which results in formation of the extended duplex. nc facilitates both reaction steps, resulting in the overall 10 4 -fold and 10 6 -fold rate enhancement for mini-tar and tar annealing, respectively. we show that the kissing step is facilitated by the nc-induced nucleic acid aggregation, which is more pronounced for the longer tar hairpins. at the same time, the conversion steps in tar and mini-tar appear to be very similar and are similarly facilitated by nc 10-100-fold. the later effect relays on the ability of nc to destabilize nucleic acid duplexes, and is equivalent to destabilization of a few base pairs required for the conversion initiation. linking of the n-terminus of a peptide to its encoding mrna s. ueno, h. arai, y. husimi department of functional materials science, saitama university, saitama, japanin evolutionary protein engineering, in vitro selection using a cellfree translation system has advantages of large library size and also of applicability to cytotoxic protein. although many in vitro protein selection techniques such as in vitro virus, ribosome display are developed, most of these are the techniques that link the genotype molecule to the peptide at its cterminus. we developed a method to link the genotype molecules to the nterminus of its encoding peptide. the mrna has dna-linker hybridize region, translation enhancer, initiation codon, single codon, amber stop codon, n-terminus sequence in gfp gene, his-tag and ochre stop codon. the mrna is also linked at 5'-terminus to sup trna via spacer and the speci c amino acid. the mrna is translated in the cell-free translation system. thus, c-terminus of the protein becomes free. moreover, in this system, the free protein of the full length is never generated. for this reason, the problem in the conventional technique, that is, the competition between the protein displayed on the genotype molecule and the free protein is eliminated. we succeeded to turn one round of the " life cycle " of the in vitro virus, judged by his-tag selection. key: cord-014661-mrh2pbi6 authors: dumitrascu, georgiana r.; bucur, octavian title: critical physiological and pathological functions of forkhead box o tumor suppressors date: 2013-12-31 journal: nan doi: 10.15190/d.2013.5 sha: doc_id: 14661 cord_uid: mrh2pbi6 the forkhead box, subclass o (foxo) proteins are critical transcription factors, ubiquitously expressed in the human body. these proteins are characterized by a remarkable functional diversity, being involved in cell cycle arrest, apoptosis, oxidative detoxification, dna damage repair, stem cell maintenance, cell differentiation, cell metabolism, angiogenesis, cardiac development, aging and others. in addition, foxo have critical implications in both normal and cancer stem cell biology. new strategies to modulate foxo expression and activity may now be developed since the discovery of novel foxo regulators and non-coding rnas (such as micrornas) targeting foxo transcription factors. this review focuses on physiological and pathological functions of foxo proteins and on their action as fine regulators of cell fate and context-dependent cell decisions. a better understanding of the structure and critical functions of foxo transcription factors and tumor suppressors may contribute to the development of novel therapies for cancer and other diseases. the forkhead box (fox) proteins represent a wide family of transcription factors that display an extraordinary functional diversity, regulating a variety of critical biological processes 1, 2 . fox proteins are well known to control the following physiological procceses: apoptosis, cell-cycle, cellular metabolism, immune response, differentiation, development (such as cardiac development) or aging. fox proteins are also involved in various pathologies, such as cancer, diabetes and neurodegenerative diseases 3, 4 . the first forkhead transcription factor was first identified in 1989, and its function was related to the development of the anterior and posterior gut of the drosophila embryo. the "forkhead" name was given because of the two spiked-head structures found in the embryos of the drosophila melanogaster forkhead mutant presenting modifications of the gut formation 5 . one year later, the sequence comparison between forkhead and mammalian hepatocyte-enriched transcription factor hnf-3a showed a conserved 110-aminoacid dna binding domain, bringing evidence that forkhead proteins are a new class of transcription factors 6 . hence, forkhead transcription factors are defined by their winged-helix dna binding domain, a conserved structure called "forkhead box", the symbol fox being assigned to all vertebrate forkhead transcription factors, according to the revised nomenclature 7 . since its discovery, numerous forkhead genes have been identified in a broad range of organisms, from yeast and worms to humans 2 . interestingly, up to now, fox genes have not been identified in plants 8 . however, an update published in 2010 reported that are 50 fox genes identified and classified in the human genome and 44 in the mouse genome 9 . therefore, a standard nomenclature system was required for this extended number of discovered factors. in 2000, daniel e. martinez and his team established fox nomenclature committee, the first step towards an unified nomenclature for the winged-helix / forkhead transcription factors. the committee has changed the initial terms for the forkhead proteins (e.g. freac -forkhead related activator; fkh -drosophila gene fork head) 7 . currently, based on phylogenetic analysis, foxo genes are classified in subclasses that range from foxa to foxs to yield 23 subclasses in total 10, 11, 12, 13 . each subclass has several members noted with an arabic number. while for human forkhead proteins abbreviations have all letters in uppercase (e.g., foxd3), for mouse only the first letter is capitalized (e.g., foxd3) and for all other chordates the first and subclass letter are in uppercase (e.g., foxd3) 7 . one of the largest and the most important subclass of fox family is represented by foxo (forkhead box, subclass o) transcription factors. foxo transcription factors (foxos) are characterized by the same conserved dna binding domain that define the family of forkhead proteins 1 . however, foxos share an additional unique 5 amino acid sequence insertion within the dna binding domain that is not present in other forkhead proteins 14 . only one foxo transcription factor is known in invertebrates (named abnormal dauer formation protein 16/daf-16 in the nematode worm caenorhabditis elegans and dfoxo in the fruit fly drosophila melanogaster), while in mammals four foxo proteins encoded by four different genes were identified: foxo1, foxo3, foxo4 and foxo6 15 . initially, foxo1 transcription factor (previously known as fkhr-forkhead in rhabdomyosarcoma) was identified through its involvement in chromosomal translocations t(2;13) and t(1;13) in alveolar rhabdomyosarcoma tumors due to pax3/7-fkhr fusion transcript 16, 17 . a few years later, foxo3 (previously known as fkhrl1 -forkhead in rhabdomyosarcoma like protein 1) was characterized and named, based on similarities to fkhr 18 . the gene for foxo4 was described as being fused to mll transcription factor as a result of the t(x; 11) chromosomal translocation in acute lymphoblastic leukemia, therefore, the initial term for foxo4 was afx (the acute leukemia fusion gene located on chromosome x) 19 . foxo proteins are considered unique cellular targets, regulating a wide variety of critical cellular processes, such as: apoptosis, oxidative stress, dna damage repair, cell cycle, stem cell proliferation and maintenance, metabolism, angiogenesis, vascular tone, cardiovascular development, fertility, immune response and neuronal survival 4, 20 . the aim of this review is to discuss the most recent advances in elucidating the functions, structure and transcriptional regulation of forkhead box o transcription factors, both in physiological and pathological conditions. the advances in understating the mechanism of foxos regulation of stem cells, cancer stem cells and how non-coding rnas are regulated and regulate the function of foxo genes/protein are presented. in humans, the primary structure of the foxo proteins is characterized by a length of approximately 655-675 amino acid residues (aa) for foxo1 (655 aa) and foxo3 (673 aa), and a shorter sequence of approximately 500 amino acids for foxo4 (505 aa) and foxo6 (492 aa) 21,22,23,24 . all members of the foxo family consist of four domains: a forkhead dna-binding domain (dbd), a nuclear localization signal (nls), a nuclear export sequence (nes) and a c-terminal transactivation domain 25 (figure 1) . the forkhead dna-binding domain (dbd), also called the forkhead box, is described as a "winged helix" due to the butterfly-like aspect on x-ray crystallography and nuclear magnetic resonance 26 . analysis of the amino acid sequences alignment of foxo proteins revealed a highly conserved dna binding domain among all the members of the broader group of forkhead transcription factors, but also among species, in eukaryotic organisms, from yeast to humans 3, 27 . moreover, several studies point out that forkhead dbd is not the only region in the foxo molecule that is highly conserved. similarities were also observed in the n-terminal region near the first akt/protein kinase b (pkb) phosphorylation site (thr24 for foxo1 and thr32 for foxo3), the region containing nls, and sequences from c-terminal transactivation domain 25 . as mentioned above, the forkhead domains in foxo subclass of fox family are similar to the forkhead regions of other subclasses. for example, structural similarities have been proven in the core region of foxo3 compared to foxa3, foxk1 and foxp2 28 . the forkhead/winged helix motif is a shared sequence between subclasses of foxo, of around 100 amino acids that folds into three alphahelices (h1, h2 and h3), three beta-strands (s1, s2 and s3) and two wing-like loops (w1 and w2). the structure has a h1 -s1 -h2 -h3 -s2 -w1 -s3 -w2 topology and the strands s1, s2 and s3 interact with each other forming a beta-sheet. while the n-terminal part of the domain is formed by a cluster of three alpha-helices, the c-terminal half consists of beta strands s2 and s3 and two large loops (wings w1 and w2) 25 . the main dna recognition site is represented by the highly conserved helix h3 of forkhead dna-binding domain. the stability of forkhead dbd -dna complexes is increased by the more variable regions of the dbd, including h1, the region between h2 and h3, wing w1 and c-terminal segment 25 . although the foxo forkhead box is clearly related to those found in other forkhead genes, all foxo members contain an aditionally segment of five amino acid (198-gdsns-202) between helices h2 and h3, that creates a small extra loop, forming a coil structure in the foxo3 dbd, and a short helix in the foxo4 dbd 28, 29 . this is important in sequence-specific interactions with dna-binding sites 30 . foxo proteins mediate transcriptional activation through binding to the conserved consensus core motif ttgtttac in the dna 31 . they bind dna through a foxo-recognized element with a t/c-g/a-a-a-a-c-a-a consensus sequence in the forkhead dbd (c-terminal) 26 . there are fourteen protein-dna contacts described, which mediate the activation/inhibition of foxo's target genes, such as bim, trail, p27, p21 and catalase. the main recognition site is the α-helix h3 32 . forkhead proteins function as transcription factors that bind to dna through their forkhead domain in order to upregulate or downregulate the expression of a tremendous number of target genes 20 . hence, the foxo transcription factors control the expression of a wide spectrum of genes that regulate essential physiological cellular processes, such as cell death/cell survival, cell cycle, cell proliferation, cell differentiation/development, angiogenesis, cell metabolism, stress response and stem cell maintenance 33,34,35,36,37,38 (figure 2) . moreover, foxos are also involved in a wide range of pathological cellular processes, such as cancer, neurodegenerative diseases (parkinson's disease, alzheimer), diabetus mellitus, cardiac failure, atherosclerosis, hypertension and anovulation 39, 40, 41, 42, 43, 44, 45, 46 . differences between functions and regulatory mechanisms of foxo1 and foxo3 proteins are considered to be partially redundant, with several exceptions. for example, akt phosphorylates all foxo family members, inducing their translocation into the cytoplasm and inactivation, while pp2a dephosphorylates part of the akt phosphorylation sites in foxo1 and foxo3, reactivating them 47 . several particular differences regarding the regulatory kinases, e3 ligases or other important enzymes and regulators exist. thus, their activity is in part controlled by different mechanisms 31 . foxo transcription factors regulate the expression of multiple pro-apoptotic and anti-apoptotic proteins, and they have the ability to induce apoptosis by activating either intrinsic or extrinsic pathways of apoptosis 48,49,50,51,52 . moreover, the consensus foxo recognition element (fre) -(g/c)(t/a)aa(c/t)aa -which differs from that of other forkhead proteins, seems to have a very important role in both apoptotic pathways, since matching functional fre sites have been identified in the promoters of foxo target genes encoding fas ligand (fasl), insulin like growth factorbinding protein 1 (igfbp1), the apoptotic regulator bcl-2 interacting mediator of cell death (bim) and others 30 . foxo triggers the mitochondria-dependent intrinsec apoptotic pathway through upregulation of multiple pro-apoptotic bcl-2 family members, such as puma, bim, noxa, bnip3, and downregulation of the anti-apoptotic bcl-2 family member bcl-xl 48,49,50,51,52 . the expression of the anti-apoptotic protein bcl-xl is suppressed by foxo proteins, such as foxo4, after increasing the expression of the transcriptional repressor bcl-6, in order to trigger apoptosis 52 . both foxo-induced expression of the proapoptotic bcl-2 family members and foxodownmodulated anti-apoptotic bcl-2 family members lead to apoptosis due to mitochondrial outer membrane permeabilization (momp), as a response to intracellular stress, growth factor deprivation or other factors 53 . these mitochondrial modifications result in release of cytochrome c, smac/diablo and omi/htra2 from mitochondria, subsequently triggerring downstream caspases activation 54,55 . apaf 1 binds cytochrome c forming a complex named apoptosome, required for caspase 9 activation (initiator caspase) 54 . caspase 9 activation further activates the effector caspases, such 3, 6 or 7, triggering caspase cascade 54 . foxo proteins are also able to induce apoptosis by activating the receptor-dependent extrinsic pathway of apoptosis. they induce the upregulation of the death receptor ligands fasl and trail, promoting an autocrine and/or paracrine action of these death ligands on the death receptors 56 . fas signaling pathway is important in apoptosis induction through the extrinsic pathway, since jurkat cells lacking several critical components of fas pathway fail to induce foxodependent cell death 57 . upregulation of tumor necrosis factor related apoptosis inducing ligand (trail) by increased levels of foxo1 or foxo3 in cancer cells, such as prostate carcinoma cells, leads to apoptosis 58 . thus, binding of fas ligand (fasl) and trail to their receptors (fas/cd95/apo-1 for fasl and dr4, dr5 for trail) triggers a death-inducing signaling complex (disc) with a subsequent activation of caspases, mainly initiator caspase 8 and effector caspase 3, leading to apoptosis 59 . moreover, foxo transcription factors directly regulate the expression of tumor necrosis factor receptorassociated death domain (tradd). activation of foxo proteins by pi3k-akt pathway inhibitors results in increased expression of tradd protein 60 . tradd is an important adaptor protein interacting with tnfr1 and fas receptors, mediating apoptosis and nfkb pathway activation 61 . under normal physiological conditions, cyclins and their associate cyclin dependent kinases (cdks) are very important for the progression of the cell cycle 62 . in response to dna damage, foxo transcription factors increase the expression levels of the cdk inhibitors binding to cyclin/cdk complexes, such as p21 (also known as cdk inhibitor 1) and p27 (kip1) 63,64 . cdk inhibitors together with foxo-induced inhibition of cyclins expression act by stopping the cell cycle at different checkpoints in order to repair the dna damage or to remove the damaged cell 65 . for example, a study performed on 32d murine myeloid cells and on baf3 murine pre-b-cell lines brings evidence that endogenous foxo proteins are required to enforce cell cycle checkpoints after the cell cycle arrest at g1/s transition is induced by foxo by upregulating the negative regulators of the g1/s phase, such as the cdk inhibitors: p27 kip1 , p57 kip2 , p21 cip1/waf1 (cip/kip family), p15 ink , p19 ink (ink family) and the retinoblastoma protein family member p130 27 .also, foxo decreases the positive regulators, such as cyclin d1, or cyclin d2, blocking the g1/s transition 66 . additionally, foxo increases the expression of negative regulators such as gadd45alpha and cyclin g2, resulting in cell cycle arrest at g2/m 66 . surprisingly, foxo proteins act as transcription factors for plk1 expression during cell cycle, plk1 being critical in promoting g2-m phase transition, m phase progression and end of mitosis 67 . a complete knock-down of foxo protein levels induces an arrest in cell cycle, suggesting that a basal, low levels of foxos activity is required for the cell cycle progression 67 . surprisingly, in specific settings, foxo may actually serve as a promoter of proliferation. for example, neutrophils isolated from foxo3 deficient mice show high levels of fasl expression and apoptosis, revealing that foxo3 may represses fasl expression in neutrophils, leading to proliferation in this type of cells 68 . foxo1 was also shown to positively influence proliferation induction in pancreatic β cells in vitro, under low nutritional circumstances. the mechanisms implicated in this process are not completely understood. however, the induction of ccnd1 gene transcription, which encodes cyclin d1, may at least partially be involved. cyclin d1 represents one of the earliest cell cycle-related events, being critical for the g1 to s phase progression during the cell cycle 34 . thus, foxo proteins coordinate the expression of multiple important cell cycle regulators, in order to block the g0/g1, g1/s or the g2/m transitions during cell cycle when needed, such as after dna damage. however, a basal level of foxo proteins is required for g2-m cell cycle transition, when foxo-dependent plk1 expression seems to be necessary. these results suggest that foxo proteins have to be tightly regulated and are important and sensitive regulators of cell cycle, proliferation and other critical cellular processes. foxo proteins play an important role in regulating differentiation of a wide variety of tissues. it is well known that foxos can control the differentiation of precursor cells into muscle, adipose tissue or blood cells. interestingly, foxos effect on differentiation is context dependent and sometimes foxo isoform dependent. while foxo3 promotes differentiation of erythroid cells, foxo1 suppresses the differentiation of precursor cells in adipose and muscle tissues 69 . foxos can also suppress bone formation by inhibiting wnt-β catenin-tcf signaling. wnt signaling is known to stimulate bone formation 70 . moreover, foxo proteins are required for maintenance of somatic/adult stem cells, such as hematopoietic stem cells, and they also regulate cancer stem cells 71 (see details in chapter 5). metabolic signaling mediated by forkhead transcription factors is conserved among multiple species, including but no limited to mammals, drosophila melanogaster and caenorhabditis elegans 20 . it was first noticed for the foxo homolog named dauer formation-16 (daf-16), in the caenorhabditis elegans worm 72 . foxo proteins are involved in critical physiological processes that regulate cellular metabolic activity in many organs, such as liver, pancreas, adipose tissue and hypothalamus 73, 74, 75, 76 . in the liver, foxo1 forms a complex with another transcription factor, the liver specific pgc1alpha, in order to induce gluconeogenesis by upregulating g6pase and pepck genes. this is important for maintaining glucose homeostasis 73 . confirming these results, loss of hepatic foxo genes in mice induces a downmodulation of gluconeogenesis and an upregulation of glycolysis 77 . in addition, ectopic expression of foxo1 in rat primary hepatocytes increases apociii, an inhibitor of lipoprotein lipase, suggesting that foxo1 is involved in regulating the lipid metabolism 78 . recently, nicotinamide phosphoribosyltransferase (nampt) gene was described to be a new transcriptional target gene of foxos for regulating hepatic triglyceride levels 79 . in pancreas, foxo1 plays an important role in the maintenance of beta cell function, but also in the development of the pancreas, through repression of the pancreatic transcription factor pdx1 74 . aditionally, foxo1 increases the food intake by upregulating agrp and npy, and by downregulating pomc in hypothalamus, antagonizing the anorexigenic hormone leptin 76, 80 . recently, g-protein-coupled receptor gpr17 was described to be a foxo1 target that activates agrp, suggesting a new mechanism for foxo1-agrp mediated food intake that might provide a new treatment for obesity 81 . all these results establish foxo proteins as important regulators of cellular metabolism and potential target in metabolic related diseases. foxo transcription factors function as sensors for reactive oxygen species (ros) and play an important role in cellular resistance to stress, increasing cellular survival. foxo proteins are able to protect the cell from oxidative damage, decreasing the availability of ros. they stimulate the expression of certain genes responsible for the ros inactivation, such as the antioxidant enzymes manganese superoxide dismutase (mnsod), that catalyzes the conversion of superoxide to hydrogen peroxide, and catalase, that converts hydrogen peroxide into water and oxygen 27 . studies on human cardiac fibroblasts revealed that foxo also modulate the antioxidant enzyme peroxiredoxin iii, fighting against cell damage induced by oxidative stress. concomitant peroxiredoxin iii knockdown and foxo3 knockdown resulted in higher levels of hydrogen peroxide in response to serum starvation, as compared to peroxiredoxin iii knockdown only 82 . other foxo transcriptional targets (figure 3 ), such as sterrole carrier proteins (scps), are now known to play an important role during the defence against oxidative stress. scps are implicated in protecting fatty acids from peroxidation 83 . foxo proteins are also able to increase dna repair, inducing a cell cylce arrest at g2/m checkpoint, in order to provide time for repairing the dna damage. one of the transcriptional target of foxo, implicated not only in cell cycle arrest but also in dna repair and cell survival in response to cellular stress, is gadd45 84 . similarly, ddb1 gene protein product is able to repair the dna damage through upregulation by foxo 69 . foxos regulate the longevity of the cell mainly by increasing resistance to stress, modulating the dna repair process and maintaining the stem cells 27 . the lifespan extension is an important function of foxo that is conserved among several species. a well studied example is the role of foxo homologous daf-16 (caenorhabditis elegans) in aging 85 . loss of foxo3 in human skin fibroblasts results in aging specific morphological changes, suggesting that foxo3 is necessary for maintenance or promotion of cellular longevity 86 . moreover, loss of foxo3 activity leads to decreased mnsod and enhanced cell injury in vascular smooth muscle explanted from aged mice, due to limied ros inactivation 87 . thus, cellular lifespan maintenance requires a certain level of foxos activity and this is mainly achieved by inactivation of akt activity 88 . foxo proteins do not only play an important protective role during senescence/aging, but also during exercise. it was previously suggested that foxos may at least partially be involved in the exerciseinduced beneficial cardiac effects. interestingly, exercise induces an upregulation of foxo3 and sirt1 in the heart, with subsequent increased mnsod, catalase and gad45alpha, and decreased cyclin d2, suggesting the protective role of foxo proteins 89 . interestingly, other studies revealed that, in conditions of oxidative stress, foxo3 is also able to induce apoptosis by triggering a fas-mediated death pathway in cultured motoneurons, by activating trail, bh3-only proteins noxa and bim, and by promoting pro-apoptotic activity of p53 20 . additional reports suggest that suppression of foxo proteins expression during oxidative stress can be protective to some extent for cells, since protein inhibition or gene knockdown of foxo1 and foxo3 decreases the ischemic infarct size in the brain, protects the metabotropic glutamate receptors during vascular injury, enhances pancreatic β-cell or neuronal survival through nad + precursors and provide trophic factor protection with erythropoietin (epo) and neurotrophins 20 . this suggests that while a certain level of foxo3 activation is required in cellular stress resistance, sustained activation or activation over a threshold of foxo3 is detrimental and may induce apoptosis. thus, foxo3 plays an important role in cellular decision during stress, helping the cells to survive by multiple mechanisms (such as dna repair, ros inactivation etc) and inducing apoptosis when dna or cellular damage can't be repaired. foxo proteins play a central role in maintaining the immune response of the human body. cell type-specific deletions of foxo1 and/or foxo3 in mice revealed an important role of foxo proteins in regulating immunological homeostasis and tolerance, by controlling the function and development of b and t lymphocytes. thus, foxo1 and foxo3 are essential transcription factors involved in early b cell development and peripheral b cell function, since early deletion of foxo1 blocked b cell differentiation at the pro-b cell stage. this is due to a defect in interleukin 7 receptor alpha (il-7ralpha) expression. deletion of foxo1 in peripheral b cells was associated with defective expression of both cd62l and aid, with subsequent failure in class-switch recombination and reduced igg production upon immunization. moreover, it is known that the pi3k-akt-mediated inactivation of foxo1 is essential for the optimal proliferation of b cells, while ectopic expression of pi3k-independent variants of foxo1 or foxo3 (active triple mutants at the akt phosphorylation sites) resulted in cell cycle arrest and increased cell death in b cells 90 . another study on mice with t cell-specific deletion of foxo1 showed a decreased expression of il-7 receptor on mature t-cells, suggesting that il7-r is a transcriptional target of foxo1, which is mediating through binding with il-7 the survival and homeostatic proliferation of peripheral t cell 91 . excesive inflammatory cells may become harmful for the human body through the generation of ros and through the production of cytokines. studies performed on mice deficient of foxo3 ilustrated lymphoproliferation, inflammation of the salivary glands, lung, and kidney, and increased activity of helper t cells. these observations demonstrate the beneficial role of foxo proteins in human body, by preventing t cell hyperactivity 20 . also, it was demonstrated that mir-182 has a central role in the late phase of clonal expansion of the helper t lymphocytes, by inducing il-2 which is able to inactivate foxo1. foxo1 was found to be a suppressor of proliferation in resting helper t lymphocytes and, in order to allow proliferation, t cell activation via tcr/cd28 and il-2r signaling must inhibit foxo1 90 . other studies reported that t cells derived from bim and puma deficient mice were resistant to apoptosis after il-2 deprivation, demonstrating that foxo3, through upregulation of bim, puma and p27 kip1 is important for the induction of cell cycle arrest and apoptosis of t cells, in the absence of cytokines 48 . foxo proteins may be benefical for autoimmune disorders by inducing a fas mediated apoptosis that target activated t cells, followed by a decrease in cytokine stimulation in patients with autoimmune lymphoproliferative syndromes 20 . foxo proteins play an important role not only during physiological cellular processes, but also in few pathologies, such as cancer. foxos are well known tumor suppressor proteins. although foxo proteins protect the human body by playing a central role in a wide range of mainly physiological functions (as described earlier), under some circumstances, foxo's roles can become harmful for the human cells. this is because foxo is also involved in a number of pathological functions, such as inflammation and muscle atrophy. for example, apoptosis places foxo proteins on the good side when it leads to tumor suppression. however, cellular apoptosis can become itself a significant component for pathology in diseases such as neurodegenerative disease, diabetes mellitus (dm), and cardiovascular injury 40,41,92,93 . cancer: foxo proteins are tumor suppressors foxo proteins are inactivated in a wide variety of malignancies, eiher posttranslational (mainly by pi3k-akt mediated phosphorlation) or by fusion mutations (such as pax3-foxo1; mll-foxo3, mll-foxo4) 94, 95 . inactivation of foxo by pi3k-akt pathway activation is a common feature of many malignancies, such as prostate cancer, breast cancer, leukemia and glioblastoma 69 . conditional deletion of foxo family members in mice leads to the lymphomas and hemangiomas, which suggests that loss of foxos maintains or promotes survival of tumor cells 96 . foxos loss or inactivation is known to play an important role in cancer tumorigenesis or progression, in vivo 97 . for example, a recent study described a significant correlation between low expression of foxo3 and a poor prognosis for gastric cancer patients, bringing evidence that foxo3 could be a valuable prognostic biomarker for patients with gastric cancer 98 . in addition, foxo3 overexpression was shown to reduce motility, invasiveness, and aggressiveness in estrogen receptor α-positive (erα+) breast cancer cells 39 . foxo proteins exert their tumor suppressor functions predominantly by promoting cell cycle arrest, apoptosis, ros inactivation and dna repair, through expression of their target genes 47,99 . for instance, foxo3-induced expression of bim, a pro-apoptotic member of the bcl-2 family of proteins induced a caspase-dependent cell death in several types of cancers, such as in chronic leukemias, breast and gastric cancers 27 . similarly, foxo-induced trail and noxa expression induces apoptosis in many malignancies, including leukemias 100 . interestingly, foxo3 can also inhibit the protooncogene c-myc, indirectly controlling the transcription of a wide set of target genes implicated in cell survival, cell cycle, apoptosis and tumorigenesis. myc is a transcription factor and a well known promoter of survival, proliferation and tumorigenesis, being found upregulated in a large variety of malignancies. noteworthy, activation of foxo proteins not only induces cell cycle arrest or apoptosis, but also a differentiation program. in chronic myeloid leukemia (cml), foxo3 can induce cml leukemic cells differentiation, by inhibiting the expression of id1 (inhibitor of dna binding 1). cml is characterized by the presence of the bcr-abl fusion protein, which is a constitutive active kinase, controlling many crucial downstream pathways implicated in cell cycle, proliferation, apoptosis and cell adhesion. bcr-abl activates pi3k-akt pathway, inactivating foxo proteins and their pro-apopotic and cell cycle arrest signals 27 . the use of the tyrosine kinase inhibitors (tkis) and of the proteasome inhibitor bortezomib results in inhibition of bcr-abl activity and its downstream pathways, with a subsequent activation of foxo proteins 102 . tkis treatment induces a foxo-dependent suppression of id1 expression, leading to k562 bcr-abl positive cell line differentiation. thus, bcr-abl can maintain the leukemic state not only by promoting proliferation, cell cycle progression and inhibiting apoptosis, but by also inhibiting foxo-mediated differentiation 27 . inflammation (rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus) it was shown that loss of functional foxo proteins lead to inflammatory cell activation in several disorders, with the subsequent cellular damage, through oxidative stress and excess of cytokines. inactivation of foxo3 in t lymphocytes, as well as inactivation of foxo1 and foxo4 in synovial macrophages in patients with rheumatoid arthritis and osteoarthritis result in inflammatory cell activation. in addition, loss of foxo proteins may be a potential etiology for systemic lupus erythematosus (sle) and rheumatoid arthritis, since foxo1 gene transcript levels are downregulated in peripheral blood mononuclear cells of these patients 20 . a link between inflammation, insulin resistance and foxo transcription factors was previously suggested when downregulated foxo1 decreased the levels of c/ebp beta transcription factor in adipocytes, with the subsequent reduction in expression of the pro-inflammatory cytokines ccl2 (chemokine ligand 2) and il-6 103 . thus, foxo1 indirectly might induce an inflammatory status of the adipose tissue, responsible for the insulin resistance in type 2 diabetes. yet, there are studies that describe the ability of foxo proteins to directly induce inflammation through upregulation of the inflammatory cytokine il-1b 104 . however, further experiments are required in order to clearly understand the context related mechanisms of foxo-dependent modulation of inflammation. muscle atrophy appears in a variety of diseases, including cancer, diabetes and sepsis, and is characterized by accelerated proteolysis that can be induced through two pathways: the ubiquitinproteasome pathway and through lysosomal pathway, as a consequence of autophagy 27 . studies revealed that foxo proteins can induce muscle atrophy, characterized by decreased muscle function 105 . thus, foxo proteins have been shown to increase the transcription of key regulators of both lysosomal and proteasomal proteolysis: the autophagy followed by the lysosomal proteolysis is stimulated by upregulation of bnip3, lc3, and gabarapl1, while the proteasomal proteolysis is induced by increasing the ubiquitin ligase atrogin1. it was also shown that foxo3 activity is both required and sufficient for induction of autophagy in muscle cells, since studies performed on adult muscle fibers from mice show that the ectopic expression of an active foxo3 mutant leads to lysosomal proteolysis after formation of autophagosomes, while the knockdown of foxo3 in these muscles fibers blocked autophagosome formation after starvation 106, 107 . autophagy has a critical role in maintaining the cellular and metabolic homeostasis. it seems that the metabolic status of the cell strongly influences this process in both normal cells and cancer cells, despite the profound differences in their metabolism 108 . in cancer cells, atp is predominantly produced through the constitutive activation of aerobic glycolysis, process that is modulated by the transcription factor hif1α 108 . since p38α is required to maintain the levels of hif1α target genes, researchers demonstrated that in colorectal cancer cells, the inhibition of p38α causes a rapid drop in atp levels, with an acute energy need which activates foxo3 in an ampkdependent manner, in order to induce autophagy, cell cycle arrest and cell death in these cells 96, 108 . moreover, the knockdown of foxo3 was sufficient to induce hypertrophy in cultured neonatal rat cardiomyocytes. in these cells, stimulation with insulin inhibits foxo3 function, with subsequent downregulation of the antioxidant enzyme catalase and increased levels of ros, that in low levels may act as second messengers for intracellular signaling, making possible the increase in cell size 109 . atrogin 1, upregulated by foxo1 and foxo3, plays important roles in cardiovascular system, since mice lacking atrogin-1 are susceptible to cardiac hypertrophy 20 . foxo proteins are inducing atrophy of differentiated cardiac and skeletal muscle cells through protein synthesis inhibition, which leads to a decrease in cell size 69 . in skeletal muscle, this mechanism involves myostatin, a foxo transcriptional target and a secreted molecule that can induce atrophy by protein synthesis inhibition 110 . foxo1 might be implicated in the development or progression of type 2 diabetes, since increased foxo1 expression in diabetic mouse liver is associated with increased expression of pepck and g6pase, and inhibition of foxo1 activity downregulated both pepck and g6pase expression and normalized the blood glucose levels. thus, foxo1-mediated expression of g6pase and pepck is critical for gluconeogenesis in the liver during fasting, but its deregulation may be involved in diabetes etiology 111 . physiological functions of foxo take place under certain circumstances, since sometimes the ability to maintain the proper control is overwhelmed 20 . experiments on insulin-producing mouse pancreatic beta cells (betatc-6) show that chronic exposure to high glucose activates foxo transcription factors and leads to upregulation of endogenous inflammatory cytokines interleukin-1beta (il-1beta) and suppressors of cytokine signalling (socs). these events trigger the activation of caspase-3 with subsequent apoptosis, suggesting a new mechanism that leads to the destruction of endocrine pancreas in type 2 diabetes 92 . also, exposure to high glucose of the cardiac microvascular endothelial cells (cmecs) isolated from hearts of adult rats show that foxo transcription factors leads to reactive oxygen species (ros) accumulation and apoptosis, suggesting that foxos might be involved in microvascular complications of diabetes 93 . foxo is also involved in insulin resistance and metabolic syndrome, since the activation of foxo1 in cardiomyocytes leads to increased akt activity and attenuated cellular response to insulin, followed by decreased glucose uptake 112 . interestingly, clinical studies regarding metabolic status profile on age-related diseases, fertility, fecundity and mortality revealed higher hba1c levels and increased mortality risk associated with specific haplotypes of foxo1 113 . foxo proteins are also activated in an attempt to protect the human body against the oxidative stress resulted due to hyperglycemia that leads to increased production of ros in endothelial cells, liver cells, and pancreatic β-cells. this hyperglycemia-dependent ros increase leads to a subsequent development of insulin resistance and significant neurodegenerative and cardiovascular diseases in the patients with diabetus mellitus 20 . foxos are necessary for endothelial cell development and angiogenesis, since mice that are deficient in foxo1 lack development of the vascular system and die by embryonic day eleven 114 . in addition, foxo1 and foxo3 were shown to be the most abundant foxo isoforms in mature endothelial cells, having also an important role in the regulation of postnatal vessel formation, not only in the embryogenesis 36 . unfortunately, angiogenesis is not only involved in critical physiological processes, such as embryogenesis and postnatal vessel formation, but it is also involved in pathological events, such as chronic inflammation and tumor growth 115 . thus, it is fascinating how the angiogenesis mediated by foxo proteins may become a negative element for the organism, antagonizing the tumor suppresor's main function through new vessel formation, that can lead to tumor cell growth 116 . foxo3 was associated with both cardiomyocyte survival after oxidative stress and heart muscle loss with subsequent ventricular dysfunction 43, 117 . foxos are activated after akt inhibition by insulin or other factors, leading to atrogin-1 induction, which results in a suppression of heart muscle cell size 43 . foxo3 proteins seem to inhibit the vascular smooth muscle cell proliferation and growth in a rat balloon carotid arterial injury model, suggesting a role of foxos in the regulation of vascular tone and systemic arterial blood pressure, preventing or at least lessening the effects of atherosclerosis and hypertension 118 . also, decreased foxo1 expression due to high flow states in vessels leads to proliferation of vascular smooth muscle cells, vascular neointimal hyperplasia, and subsequent hypertension 45 . moreover, experiments on lowdensity lipoprotein (ldl) receptor knockout mice resulted in the prevention of atherosclerosis when the triple ablation of foxo1, foxo3 and foxo4 was induced in endothelial cells 44 . interestingly, the same experiment on myeloid cells lead to more severe atherosclerosis compared to the controls, explained by authors through increased proliferation of granulocyte-monocyte progenitors and high levels of inducible nitric oxide synthase (inos) and oxidative stress, which predispose to atherosclerosis 119 . noteworthy, analysis of mouse oocytes revealed overexpressed foxo3 transcription factors in primordial and early primary follicles, but downregulated foxo in primary and more developed follicles, suggesting that foxo proteins also have reproductive functions, modulating oocyte and follicular cell maturation. to confirm the hypothesis, constitutively active foxo3 was induced in transgenic mouse oocytes in primary and more developed follicles, which affected the oocyte growth and follicular development, leading to anovulation and luteinization of unruptured follicles, with subsequent infertility 46 . in addition, foxo3 and foxo1 mutations were detected in a small percentage of womens with premature ovarian failure 120 . involvement of foxo family of transcription factors in stem cells self-renewal, survival, proliferation and differentiation is currently under investigation. foxos have been shown to play critical functions in maintaining self-renewal potential and quiescence of hematopoietic stem cells, however, the mechanisms of these processes are not yet well understood 1, 69 . recent reports show that foxo-mediated regulation of cell cycle, oxidative stress and apoptosis plays an important role in these processes 69 . stem cells are characterized by the capacity of self-renewing and the ability to differentiate 121 . they are necessary in maintenance and propagation of several adult tissues, including but not limited to blood, skin and gastro-intestinal epithelium. adult stem cells or cells with stem cell properties were also found in other critical organs/systems, such as the central nervous system and the lung 69 . previous studies suggested that hematopoietic stem cells are sensitive to reactive oxygen species (ros) levels. foxo family members are known to play a central role in ros detection and in inducing an adaptative response after ros exposure, by inducing the expression of critical enzymes that neutralise ros, such as catalase and manganase superoxide dismutase (mnsod). interestingly, foxo1, foxo3, and foxo4 inactivation leads to an upregulation of ros levels in hematopoietic stem cells and their death 121 . deletion of these three foxo family members in mice revealed their importance in controlling ros in stem cells, in vivo 27 . while the number of hematopoietic stem cells in bone marrow of foxo-deficient mice is low, an increase in myeloid progenitor cells in blood is observed. moreover, the repopulation ability is decreased in the absence of foxos and the treatment of foxo-deficient mice with n-acetylcysteine (nac) at least partially rescued these effects 27 . thus, persistent akt activation, which induces inactivation of foxo transcription factors by akt-mediated phosphorylation, results in ros-induced cell death. this is due to the fact that the cells can't synthesize the foxo-dependent ros neutralizing factors catalase and mnsod 121 . between foxo family members, the most important regulator of hematopoietc stem cells survival and self-renewal is foxo3, since foxo3 knockdown induces hematopoietic stem cells depletion 121 . ros neutralizing agent n-acetylcysteine (nac) can rescue hematopoietic stem cells quiescence and at least partially resque their ros-induced loss 121 . all these results suggest that foxo1, foxo3 and foxo4-induced resistance to ros is critical in maintaining homeostasis of hematopoietic stem cells in bone marrow 27 . pten tumor suppressor was revealed as an important modulator of hematopoietic stem cells self-renewal and survival. pten is a major inhibitor of akt activation. depletion of pten, induces akt activation and subsequent foxo inactivation. it is likely that foxos mediate at least a part of the pten effects on hematopoietic stem cells 121 . although foxo3 was established as the most important foxo family member regulator of hematopoietic stem cell's self-renewal, foxo1 is critical for the human embryonic stem cells (hesc) pluripotency maintenance. foxo1induced sox2 and oct4 expression is one of the mechanisms responsible for this process. interestingly, in embryonic stem cells akt is not the major regulator of foxo1 71 . fascinating, a recent study brings evidence that foxo is a critical regulator of stem cell maintenance in hydra vulgaris, a member of the phylogenetically old animal phyla cnidaria, which has been suggested to be biologically immortal due to the unlimited self-renewal capacity of their stem cells 38, 122 . foxo transcription factors are not only required for maintenance of somatic stem cells, but they also play an important role in cancer stem cells 71 . recent reports provided evidence that in many types of malignancies, such as leukemia, colon, or gastro-intestinal malignanciens, a small population of cells similar to stem cells exists 121 . these cells, called cancer stem cells, have the potential of forming new tumors. notably, most of the time, the cancer stem cells are resistant to current cancer treatments, and these therapies may result in cancer stem cells enrichment. these cells serve as a starting point in cancer recurrence 121 . similarities between stem cells and cancer stem cells were best described in the hematopoietic system, where similar surface markes and signal transduction patterns were described between the hematopoietic stem cells and leukemia-initiating cells 121 . presented results have not only implications in uncovering the stem cells/cancer stem cells regulation, self-renewal and differentiation, but also for the development of novel therapeutic strategies in cancer, degenerative diseases and many other pathologies 71 . foxo family members are expressed in almost every tissue of the human body, including the nervous system, cardiovascular system, reproductive system of males and females, lung, liver, spleen, pancreas, thymus, and skeletal muscle. however, each member of the foxo family has its own expression pattern, since they are not equally expressed in all tissues 20 . foxo1 is better represented in adipose tissue 27 . foxo3 has the highest expression in liver, but it is also being predominantly expressed in heart, brain, kidneys, and ovaries, while foxo4 is found mainly in the muscle and heart 20,27 . the newest member of the transcription family, foxo6, is present in the brain. the association of this member with other tissues is still a matter of study 123 . cell lines (immortalized and/or cancer cells) are some of the most used and useful tools for studying the structure, function, regulation and expression of proteins in general, and foxo family members in particular. foxo members expression in different cell lines (including nci 60 group of cell lines) is partially known 124, 125 . foxo1 is found to be expressed at high levels in igrov 1 (human ovarian carcinoma cells), astrocyte cells, rl 7 (human follicular lymphoma cells), ht29 (human colon carcinoma cells) and hek 293 (human embryonic kidney cells). knowing the qualitative and quantitative expression pattern of foxo family members is important in elucidating their functions and regulation. thus, databases summarizing these patterns in various tissues and cell lines are very helpful and necessary. for example, biogps presents experimental results showing the mrna expression levels of a wide number of genes in most of the human tissues and many (mostly human) cell lines 126, 127, 128 . regulation of foxos expression is not yet well understood. p300 was shown to control foxo1 gene expression by binding to the proximal region of the foxo1 promoter (cre tandem sites) 129 . non-coding rnas were shown to suppress translation of foxo family members in various tissues and contexts. in particular, the micrornas-dependent inhibition of foxo1 and foxo3 expression is better studied and is summariez below, in chapter 7. however, the main transcription factors implicated in foxos expression are not well understood. interestingly, methylation of fox promotors induces suppression of their expression. as an example, promoter methylation induced by braf results in inactivation of fox genes expression 130 . the mirnas are 18-28 nucleotide-long noncoding rna molecules with an important role in post-transcriptional regulation of protein expression that regulate a variety of cellular processes, including cell differentiation, cell cycle progression and apoptosis. these mirnas can function as oncogenes or tumor suppressors, and oncogenic mirnas (oncomirs) are upregulated in cancer cells. in cancer, mirnas were found to be situated both upstream and downstream of the carcinogenesis process and modified expression of some mirnas is the outcome of carcinogenic transformation or progression, revealing that mirnas may be potential diagnostic or prognostic tools in cancer. 131 for instance, micrornas gained a special attention in melanoma studies and the altered pattern of mirna in melanoma seems to be related to apoptosis (mir-15b), cell cycle (mir-193b) and invasion/metastasis (mir-182) 132 . the microrna (mirna)-mediated regulation of foxo transcription factors was demonstrated by several groups within the last three years. mir-182 has been shown to specifically target foxo transcription factors irrespective of cell type, since mir-182 seems to target foxo3 in melanoma cells, foxo1 in breast cancer cells and foxo1 in activated helper t (th) lymphocytes 90 . thus, in melanoma cells, mir-182 modulate the expression of both foxo3 and microphthalmia-associated transcription factor (mitf). the inhibition of mir-182 by anti-mirs (blocking antisense oligonucleotides) hindered melanoma cell migration and triggered their apoptosis 90 . in breast cancer cells, foxo1 was coordinately targeted by mir-27a, mir-96, and mir-182, while the inhibition of each mirna resulted in induced levels of foxo1 and reduced breast cancer cell survival 90 . mir-182 also targets foxo1 in osteoblasts lineage cells in order to inhibit osteoblast proliferation and differentiation, repressing the osteogenesis 133 . many other micrornas were shown to regulate foxos activity and functions, including apoptosis or cell cycle. mir-96 has been shown to regulate foxo1-induced apoptosis in transitional cell carcinoma 134 . also, increased expression of mir-96 in breast cancer cells have been shown to downregulate foxo3 transcription factor with consequent induction of cell proliferation 135 . also, increased mir-96, mir-182 and mir-183 downregulate the expression of foxo1 transcription factor in classical hodgkin lymphoma (chl) cell lines, suggesting that decreased foxo1 expression is involved in lymphomagenesis 136 . moreover, a recent study performed on du145 and lncap human prostate cancer cells show that upregulated microrna-370 induces proliferation due to downregulation of the foxo1 transcription factor 137 . in addition, mir-155, which is highly induced in mature activated t and b cells and in treg cells, has recently been shown to target foxo3 in t cells, but it remains to be shown whether foxo3 via mir-155 contributes to the observed phenotypes in b and t lymphocytes 90 . new strategies to modulate foxo expression may now be developed since the discovery of mirnas targeting foxo transcription factors 90 . however, some difficulties may appear in the mirna-based therapeutic manipulation of foxo transcription factors. for example, a single mirna may target hundreds of genes among foxos, and the therapeutic manipulation of a specific mirna could have unanticipated adverse effects by influencing whole gene networks, while having only moderate effects on foxo genes 90 . since delivery of mir-182 mimics could induce lymphoproliferative disease or other forms of cancer by systemic repression of foxo transcription factors, another challenge in the mirna-foxo therapeutic manipulation is the specific delivery of the mirna into the target cell, in order to avoid adverse effects in other cell types or tissues 90 . several micrornas were identified as being targets of foxo transcription factors. an akt -foxo -mir-30d signaling pathway was recently identified. after inhibition of akt, activated foxo3 leads to upregulation of mir-30d. mir-30d acts as a tumor suppressor in renal cell carcinoma, further inhibiting the oncoprotein metadherin (mtdh) 138 . another recent study shows that foxo1 stimulates the expression of a microrna cluster located on a x chromosome, in a direct manner, dependent on rna polymerase ii, but not on the de novo protein synthesis. thus, foxo1 upregulates mir-506, mir-508, mir-513c, mir-513a-1, mir-513a-2. also, the same study shows that inhibition of pi3k-akt axis in lncap and mcf7 human carcinoma cell lines is followed by increased mir-506. as suggested by authors, mirnas could be valuable biomarkers of foxo activity 139 . a study performed on primary cultures of neural stem/progenitor cells (nspcs) from adult mice show that the expression levels of the mir-106b~25 cluster members (mir-106b, mir-93, and mir-25) is modulated by foxo3 transcription factors in a complex manner 140 . the precursors of the mir-106b~25 cluster members are located on mcm7 gene. foxo3 directly binds to the first intron of this gene, modulating the expression of the micrornas. thus, foxo3 transcription factors could be an important tool in preventing the loss of neurogenesis during aging 140 . further studies are needed to completely understand the regulation of micrornas expression by activated foxo proteins. foxo transcription factors and tumor suppressors are ubiquitously expressed in the human body, with some specific differences between its members. as resumed above, foxo proteins are characterized by a remarkable functional diversity, being implicated in regulation of many critical cellular functions, such as cell cycle arrest, apoptosis, oxidative detoxification, dna damage repair, stem cell maintenance, cell differentiation, cell metabolism, angiogenesis, cardiac development, aging and others. foxo proteins play an important role not only during physiological cellular processes, but also in few pathologies, such as cancer. they are well known tumor suppressors proteins. although foxo proteins protect the human body by playing a central role in a wide range of mainly physiological functions, under some circumstances, foxo's roles can become harmful for the human cells. this is because foxo is also involved in a number of pathological functions, such as inflammation, muscle atropy, and a number of physiological functions that become harmful for the organism. for example, apoptosis places foxo proteins on the good side when it leads to tumor suppression, but in some cases cellular apoptosis can become itself a significant component for pathology in diseases such as neurodegenerative disease, diabetes mellitus (dm), and cardiovascular injury. interestingly, while excessive foxo levels induce cell cycle arrest and cell death, complete knock-down of foxos leads to cell cycle progression impairment, suggesting that certain levels of foxo activation are required for cell cycle progression. moreover, foxo proteins play a variety of roles in the cells dependending on the context. in deciphering the role of forkhead transcription factors in cancer therapy foxo tumor suppressors and bcr-abl-induced leukemia: a matter of evasion of apoptosis snapshot: forkhead transcription factors i sly as a foxo": new paths with forkhead signaling in the brain the homeotic gene fork head encodes a nuclear protein and is expressed in the terminal regions of dynamic foxo transcription factors forkhead transcription factors contribute to execution of the mitotic programme in mammals inflammatory arthritis requires foxo3a to prevent fas ligandinduced neutrophil apoptosis foxo transcription factors and stem cell homeostasis: insights from the hematopoietic system foxos attenuate bone formation by suppressing wnt signaling foxo1 is an essential regulator of pluripotency in human embryonic stem cells the fork head transcription factor daf-16 transduces insulin-like metabolic and longevity signals in c. elegans insulin regulated hepatic gluconeogenesis through foxo1-pgc-1alpha interaction the forkhead transcription factor foxo1 links insulin signaling to pdx1 regulation of pancreatic beta cell growth forkhead transcription factor foxo1 in adipose tissue tissue regulates energy storage and expenditure forkhead protein foxo1 mediates agrpdependent effects of leptin on food intake deletion of hepatic foxo1/3/4 genes in mice significantly impacts on glucose metabolism through downregulation of gluconeogenesis and upregulation of glycolysis foxo1 mediates insulin action on apoc-iii and triglyceride metabolism hepatic foxos regulate lipid metabolism via modulation of expression of the nicotinamide phosphoribosyltransferase gene minibrain/dyrk1a regulates food intake through the sir2-foxo-snpf/npy pathway in drosophila and mammals foxo3 regulates peroxiredoxin iii expression in human cardiac fibro blasts regulation of sterol carrier protein gene expression by the forkhead transcription factor foxo3a dna repair pathway stimulated by the forkhead transcription factor foxo3a through the gadd45 protein daf-16/foxo directly regulates an atypical amp-activated protein kinase gamma isoform to mediate the effects of insulin/igf-1 signaling on aging in caenorhabditis elegans down-regulation of a forkhead transcription factor, foxo3a, accelerate s cellular senescence in human dermal fibroblasts down-regulation of manganese-superoxide dismutase through phosphorylation of foxo3a by akt in explanted vascular smooth muscle cells from old rats akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway exercise training promotes sirt1 activity in aged rats lymphocyte signaling: regulation of foxo transcription factors by micrornas an essential role of the forkhead-box transcription factor foxo1 in control of t cellhomeostasis and tol erance high glucose induces suppression of insulin signalling and apoptosis via upregulation of endogenous il-1beta and suppressor of cytokine signalling-1 in mouse pancreatic beta cells high glucose induced oxidative stress and apoptosis in cardiac microvascular endothelial cells are regulated by foxo3a apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand in prostate cancer therapy common mechanism for oncogenic activation of mll by forkhead family proteins applications of post-translational modifications of foxo family proteins in biological functions foxos are lineage-restricted redundant tumor suppressors and regulate endothelial cell homeostasis decreased expression of the foxo3a gene is associated with poor prognosis in primary gastric adenocarcinoma patients the dna damage repair protein ku70 interacts with foxo4 to coordinate a conserved cellular stress response therapy-resistant acute lymphoblastic leukemia (all) cells inactivate foxo3 to escape apoptosis induction by trail and noxa induction of mxi1-sr alpha by foxo3a contributes to repression of myc-dependent gene expression combination of bortezomib and mitotic inhibitors down-modulate bcr-abl and efficiently eliminates tyrosine-kinase inhibitor sensitive and resistant bcr-abl-positive leukemic cells foxo1 increased pro-inflammatory gene expression by inducing c/ ebpbeta in tnf-alpha-treated adipocytes foxo1 links insulin resistance to proinflammatory cytokine il 1beta production in macrophages inhibition of foxo transcriptional activity prevents muscle fiber atrophy during cachexia and induces hypertrophy foxo3 controls autophagy in skeletal muscle in vivo foxo3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells inhibition of p38alpha unveils an ampk-foxo3a axis linking autophagy to cancer-specific metabolism foxo3a inhibits cardiomyocyte hypertrophy through transactivating catalase regulation of myostatin expression and myoblast differentiation by foxo and smadtranscription factors inhibition of foxo1 function is associated with improved fasting glycemia in diabet ic mice foxo transcription factors activate akt and attenuate insulin signaling in heart by inhibiting protein phosphatases haplotypes in the human foxo1a and foxo3a genes; impact on disease and mortality at old age abnormal angiogenesis in foxo1 (fkhr)-deficient mice multifaceted link between cancer and inflammation constitutive phosphorylation of the foxo1 transcription factor in gastric cancer cells correlates with microvessel area and the expressions of angiogenesis -related molecules foxo transcription factors promote cardiomyocyte survival upon induction of oxidative stress forkhead transcription factors inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia expanded granulocyte/monocyte compartment in myeloid-specific triple foxo knockout increases oxidative stress and accelerates atherosclerosis in mice mutational screening of foxo3a and foxo1a in women with premature ovarian failure the pi-3kinase pathway in hematopoietic stem cells and leukemia-initiating cells: a mechanistic difference between normal and cancer stem cells mortality pattern suggest lack of senescence in hydra foxos: signalling integrators for homeostasis maintenance modulators of sensitivity and resistance to inhibition of pi3k identified in a pharmacogenomic screen of the nci-60 human tumor cell line collection global proteome analysis of the nci-60 cell line panel biogps: an extensible and customizable portal for querying and organizing gene annotation resources biogps and mygene.info: organizing online, gene-centric information a gene atlas of the mouse and human protein-encoding transcriptomes control of foxo1 gene expression by co-activator p300 braf mutation-specific promoter methylation of fox genes in colorectal cancer tissular and soluble mirnas for diagnostic and therapy improvement in digestive tract cancers immune-related biomarkers for diagnosis/prognosis and therapy monitoring of cutaneous melanoma mir-182 is a negative regulator of osteoblast proliferation, differentiation, and skeletogenesis through targeting foxo1 mir-96 regulates foxo1-mediated cell apoptosis in bladder cancer unregulated mir-96 induces cell proliferation in human breast cancer by downregulating transcriptional factor foxo3a foxo1 is a tumor suppressor in classical hodgkin lymphoma upregulation of mircorna-370 induces proliferation in human prostate cancer cells by downregulating the transcription factor foxo1 akt/foxo pathway in renal cell carcinoma foxo1 regulates expression of a microrna cluster on x chromosome the microrna cluster mir-106b~25 regulates adult neural stem/progenitor cell proliferation and neuronal differentiation key: cord-022779-himray6q authors: nan title: abstracts of oral presentations date: 2005-06-10 journal: biopolymers doi: 10.1002/bip.20321 sha: doc_id: 22779 cord_uid: himray6q nan s. tchertchian, f. oplinger, m. paolini, s. manganiello, s. raimondi, b. depresle, n. dafflon, h. gaertner, and p. botti geneprot inc., geneva branch, 2, pré de-la-fontaine, 1217 meyrin, switzerland the last decade has provided extensive demonstration of the key role played by native chemical ligation (ncl) for the preparation of small and medium size proteins [1] . yet the requirement for cysteine at the site of ligation in standard ncl has limited its flexibility. recently, different types of auxiliary groups [2, 3] have been developed to extend the application of ncl to other ligation sites. however, the generally slower ligation rates especially with large fragments and the additional step required to cleave the auxiliary post-ligation have reduced their utility. here we present a novel strategy to synthesize proteins through a chemical ligation using unprotected peptide segments. our scheme does not make use of auxiliary groups [2, 3] , instead originally exploits the features of some side chain removable functionalities. ligation rates are high, comparable to ncl and the residues available for ligation are more frequent than cysteine. furthermore the whole process is "one pot" and at the end a native polypeptide is obtained directly in the ligation mixture. the total chemical syntheses of c5a (1-74) using both ncl and our method will be presented and compared. [ during the biosynthesis of glycopeptide antibiotics of the vancomycin family, several oxidative phenol coupling reactions take place. the enzymes catalyzing these reactions are of interest from structural and mechanistic viewpoints. in this work [1, 2] , it is shown that the oxygenase oxyb from the vancomycin producer only catalyzes a phenol coupling reaction when the putative peptide substrate is linked as a thioester to a peptide carrier domain (pcd) derived from the nonribosomal peptide synthetase. an efficient access is described to representative free linear peptide substrates, which makes use of alloc-solid phase peptide chemistry, but largely avoids the use of amino acid side chain protecting groups. in this way, the target linear peptides can be released from the resin under very mild conditions, and then be activated as thioesters, prior to loading onto the pcd. [ we have recently discovered a new nonenzymatically-formed product from n-(3-oxododecanoyl)-l-homoserine lactone. interestingly, both the n-acylhomoserine and its novel tetramic acid degradation product 1 are potent antibacterial agents. bactericidal activity was observed against all tested gram-positive bacterial strains, while no toxicity was seen against gram-negative bacteria. we propose that p. aeruginosa utilizes this tetramic acid as an interference strategy to preclude encroachment by competing bacteria. additionally, we have discovered that this tetramic acid binds iron with comparable affinity to known bacterial siderophores, possibly providing an unrecognized mechanism for iron solubilization. using short portions (7-11 amino acid) rich in positively charged residues from either human lactoferricin or the marcks protein as templates, a panel of 70 peptides each possessing a specific chemical structure was synthesized. these included amino acid omissions, substitutions, and insertions in the aim to modify the peptides overall charge, hydrophobic core, and/or amphipathicity. the peptides antimicrobial activities against a large panel of bacteria were assessed using both conventional tests (mic, mbc) and non-conventional assays (mic quantified by an automated turbidimetry-based system and mbc measured on resting cells suspended in low-ionic strength medium-"survival assay"). furthermore, the membrane permeabilizing activity of the peptides on strains of several gram negative bacterial species was quantitated by measuring their ability both to decrease the mic of novobiocine and to promote the uptake of the hydrophobic fluorescent probe npn. while the mic determined by turbidimetry or by the conventional method did not significantly differ, bactericidal activity of the peptides measured by the survival assay was 1 to 2 orders of magnitude higher than that measured by the conventional mbc test. on the other hand, the two assays used to measure the permeabilizing activity of the peptides rendered similar results. interestingly, the most potent permeabilizers did not correspond with the peptides exhibiting the highest bactericidal activity thus indicating that these two activities have different structural bases. protein farnesyl transferase (pftase) catalyzes the attachment of farnesyl diphosphate (fpp) to proteins that contain a caax-box sequence at their c-termini [1] . several analogues of fpp that incorporate azide functional groups have been synthesized and shown to be incorporated into peptides using pftase as a catalyst. in particular, it has been shown that the prenyl azide moiety from 1 or related analogues can be transferred to the peptide substrate, n-dansyl-gcvia to yield the corresponding thioether-linked products. the resulting azide-containing peptides have been derivatized with a triphenylphosphine-based reagent to generate o -alkyl imidate-linked products rather than the amide-linked material expected via a staudinger reaction [2] . since caax-box sequences can be appended to the c-termini of many different proteins, these analogues provide a simple and general method for incorporating orthogonal azides into proteins at unique sites. subsequent functionalization of such azide groups via staudinger or "click" chemistry should provide a convenient method for linking proteins with a diverse array of probes, biomolecules, surfaces and other materials under mild conditions. chemoselective glycosylation, acylation, and alkylation of completely unprotected peptides can be accomplished by incorporating n-alkylaminooxy amino acids into the peptide sequence. the n-alkylaminooxy side chains react selectively with reducing sugars, activated alkyl halides, and various acylating agents in mildly-acidic aqueous buffers (ph 4) to furnish neoglyco-and neolipopeptides. a key feature of the approach is that a single parent peptide can be quickly reacted with a variety of agents to provide a large number of "post-translationally"modified peptides. the ability to easily synthesize arrays of modified peptides allows comprehensive studies of the effects that glycosylation and lipidation have on peptide structure and function. here we present an overview of the methodology and initial results on its application to studying problems of biological interest. this presentation describes cyclic peptides that fold into well-defined ␤-sheet structures in aqueous solution and can dimerize through ␤-sheet interactions. the cyclic peptides contain the unnatural amino acid hao, which mimics the hydrogen-bonding pattern of one edge of a peptide ␤-strand, and ␦-linked ornithine, which mimics a ␤-turn and provides enhanced water solubility or a linkage point for creating multivalent structures. institute for molecular bioscience, the university of queensland, queensland 4072, australia the human genome project and other major sequencing projects have rapidly provided a vast array of new protein/peptide sequences. in contrast, many other new proteins/peptides are also being uncovered from plant and animal sources whose genomes are yet to be tapped. in the post-genomic era, the physical form of many of these gene-encoded sequences will be vital for biomedical research and drug development. moreover, the advantages of peptide and protein chemical synthesis over recombinant-dna methods are increasingly being used to provide rapid structure-activity information of complex bioactive peptides, small proteins and functional receptor domains. in a program designed to exploit the potential of australian conus species we have isolated, characterised and chemically synthesised a wide range of novel conotoxins. these cysteine rich microproteins have well-defined tertiary structures with considerable rigidity and stability and contain many elements of protein secondary structure. of particular interest are the two disulfide bond containing conotoxins (examples below) which target transporters, ion channels and receptors at nanomolar potencies. in this presentation i will describe some of our research on controlling the shape and potency/selectivity of these microproteins through intramolecular native ligation chemical approaches. it appears that there is considerable scope to control the properties of these native sequences which in some cases may prove useful in the development of these molecules as therapeutic candidates. despite identical amino acid composition, differences in the properties of class a amphipathic helical peptides due to differences on the hydrophobic face results in substantial differences in anti-inflammatory properties. one of these peptides is an apolipoprotein a-i mimetic, d-4f. when given orally to mice and monkeys, d-4f caused the formation of pre-␤ hdl, improved hdl-mediated cholesterol efflux, reduced lipoprotein lipid hydroperoxides, increased paraoxonase activity and converted hdl from pro-inflammatory to anti-inflammatory. in apoe null mice d-4f increased reverse cholesterol transport from macrophages. oral d-4f reduced atherosclerosis in apoe null and ldl receptor null mice. in vitro d-4f caused the formation of pre-␤ hdl, reduced lipoprotein lipid hydroperoxides and converted hdl from pro-inflammatory to anti-inflammatory. physical properties and the ability of various class a amphipathic helical peptides to activate enzyme lcat in vitro did not predict biologic activity in vivo. in contrast, the use of cultured human artery wall cells in evaluating these peptides was more predictive of their efficacy in vivo. thus, anti-inflammatory properties of different class a amphipathic helical peptides depends on subtle differences in the configuration of the hydrophobic face of the peptides. physical-chemical properties provide an explanation for the mechanism of action of the active peptides. peptides to ameliorate atherosclerosis and other inflammatory diseases can be designed using this strategy. inflammatory diseases. this chemokine belongs to the family of cxc chemokines, its response is mediated through binding to seven transmembrane helical g-protein coupled receptors cxcr1 and cxcr2. in order to investigate the relevance of selected protein segments for biological activity we synthesized chemically modified and biologically active analogues of the 77-mer of hil-8 by expressed protein ligation (epl). for ligation naturally occurring cysteine at position 55 was chosen. c-terminal peptides carrying an n-terminal cysteine were synthesized by solid phase peptide synthesis (spps) applying the fmocstrategy and used to introduce modifications. ligation of the recombinantly produced thioester with synthetic peptides yielded in full length hil-8 that finally was correctly folded and stabilised by two disulfide bridges as in the native protein. in addition to fluorescent and photoactivatable analogues, we produced variants that contain a ␤-peptide helix instead of the naturally occurring ␣-helix. thus, for the first time, we received a protein containing a whole ␤-peptide segment and still showing high biological activity. depending on the linker between ␤-sheet and ␤-peptide helix of interleukin 8 we could discriminate between active and inactive proteins suggesting that the overall orientation of the c-terminal segment is highly relevant for the folding of the protein and subsequently for the signalling of interleukin 8. a peptide based on residues 109 -122 of the syrian hamster prion protein (h1) forms ␤-sheet aggregates in solution, which grow to form large fibers. isotope-edited infrared spectroscopy has shown that the initial antiparallel ␤-sheet formed by this peptide is disordered. a slow rearrangement occurs to form a structure in which the hydrophobic core of the strands (residues 112 -122) pack together, resulting in the alignment of residue 117 across the sheet. the kinetics of the realignment have been monitored for h1 and for peptides with mutations at residue 117 (a117i, a117l and a117b where b is aminobutyric acid). h1 and a117i align with non-exponential kinetics. at low concentrations h1 aligns via the repeated detachment and annealing of strands, whereas at higher concentrations a reptation mechanism is observed. a117b aligns instantaneously within the dead-time of our experiments. a117l does not align at residue 117 but some undefined reordering can still be observed as a shift of the 13 c band. these data are the first experimental probes of the types of intersheet rearrangements which are required for the nucleation of fibrous peptide aggregates, and the evidence for strand reptation within the ␤-sheet confirms observations in molecular dynamics simulations. [1, 2] . we have since established the microfluidic peptide chip as a miniaturizing platform. the challenging issues in making peptide chips a practical tool for understanding biology, drug discovery, and diagnostics are quality of synthesis, specificity in reported activities, and ability for quantitative measurements. we will present the results of the work which involves our intensive effort in: • development of the method for monitoring and analyzing quality of peptide chip synthesis • improvement in peptide chip synthesis • development of the methods for quantitative analysis of (a) the specific binding of antibodies/proteins to peptides on chip and (b) kinase enzymatic activities against substrate peptides on chip. our presentation should demonstrate novel applications of peptide chips that can be implemented as routine laboratorial processes. [1] gao, x., pellois, j. p., kim, k., na, y. , gulari, e., and zhou, x. to prototype this approach we developed a protein array consisting of the ras-binding domain of craf-1 (rbd) that was c-terminally modified with a 24mer oligonucleotide and immobilized via hybridization with the complementary oligonucleotide on a wafer [1] . the rbd-dna conjugate was generated by native chemical ligation using a recombinantly produced rbd-thioester and an oligonucleotide carrying a 5'-cystein residue [2] . incubation of the immobilized rbd with ras(gtp), the activated rbd-binding form, retains sufficient amounts of ras on the protein array to allow detection by mass spectrometry with high sensitivity. controls carried out with inactive ras(gdp) did not produce any signal, demonstrating a sufficient selectivity for biotechnological applications. protein microarrays in which proteins are immobilized to a solid surface are ideal reagents for high-throughput experiments that require very small amounts of analyte. such protein microarrays ('protein chips') can be used very efficiently to analyze all kind of protein interactions en masse. the present work describes a general method for the selective attachment of proteins to solid surfaces through its c-termini that can be used for the creation of protein chips. our method is based in the chemoselective reaction between a protein c-terminal ␣-thioester and a modified surface containing n-terminal cys residues. ␣-thioester proteins can be obtained using standard recombinant techniques by using expression vectors containing modified inteins. we also present an efficient solid-phase approach for the rapid synthesis of cys-containing linkers that can be used for the modification of au-and si-based surfaces. this new method was used to immobilize two fluorescent proteins and a functional sh3 domain. a series of glycopeptides based on the leu-enkephalin analogue yt-gfs*-conh 2 led to greatly enhanced stability in vivo and effective penetration of the bbb. transport through the bbb hinges on the biousian nature of the glycopeptides-the glycopeptides have two conflicting conformational manifolds, a h 2 o soluble state, and an amphipathic state at h 2 o-membrane phase boundaries. multiple lines of evidence suggest that the bbb transport mechanism is absorptive endocytosis. mixed /␦-agonists showed antinociceptive potencies greater than morphine, and lacked many of the side effects generally associated with classical -selective opiate analgesics. the biousian design was extended to larger glycopeptides (16 residues) related to ␤-endorphin, which also penetrated the bbb and produced antinociception in mice. plasmon waveguide resonance (pwr) studies showed that the amphipathic helices bound to membrane bilayers with m to low nm k d 's. the presence of diverse endogenous neuropeptide transmitters and neuromodulators in the human brain is potentially applicable to the treatment of a wide range of behavioral disorders. clemencia pinilla, 1 mireia sospedra, 2 yindong zhao, 3 we have recently demonstrated the feasibility of utilizing the ligase activity of inteins for the in vivo backbone cyclization of peptidic chains. this procedure -called siclopps for split intein circular ligation of peptides and proteins-provides a biosynthetic pathway for peptides that are metabolically stable, and can be produced with spatial and temporal control [1] . to screen for bacteriotoxic peptides, a sic-lopps library was introduced into an escherichia coli population, such that each bacterium encodes a different peptide sequence. siclopps library over-expression afforded six distinct bacteriostatic peptides that reduce cell growth. one of these peptides (ln05) also caused cell aggregation. an e. coli genomic library was introduced into cells encoding ln05. co-expression of the genomic library and ln05 peptide rescues growth only in cells expressing genomic fragments able to counteract peptide toxicity. genomic library and ln05 co-expression resulted in enrichment of a single genomic construct, a fragment of the narz gene. narz is part of a nitrate reductase complex and has a role in tuberculosis persistence [2] . ln05 production in mycobacterium smegmatis resulted in a slow-growth phenotype. [1] abel-santos, e., scott, c. p., and benkovic, s. j., methods in the special feature of proteins involved in alzheimer's or prion diseases is their ability to adopt at least two different (meta) stable conformations. thus, amyloid-forming proteins that mainly contain ␣-helical structures in their native conformation must undergo an ␣-helix3␤strand conversion before or during fibril formation. the conformational transition that shifts the equilibrium from the functional to the pathological isoform can happen sporadically. it can also be triggered by mutations in the primary structure, changes of the environmental conditions such as ph, ionic strength, metal ions, protein concentration, oxidative stress, free radicals, action of physiological, or pathological chaperones. alternatively, the introduction of a small quantity of protein polymer may act as a structural template and initiate the disease. therefore, the development of model systems which allow the investigation of the complex folding mechanisms that lead to ␤-sheet aggregation appears to be one of the main challenges in the detailed understanding of the pathways from incubation to mortality. in order to create an ␣-␤ switch system we designed ideal ␣-helical parallel coiled coil peptides, introduced trigger functions by mutations in the primary structure, and studied the consequences that these mutations have on the secondary structure properties of the resulting peptides under certain environmental conditions. based on these results we continued to change the primary structure of the coiled coil system subsequently by mutations in the heptad repeat untill we ended up with soluble ␤-sheet peptides. the most important feature of these new ␤-sheet peptides is that they still follow the characteristic hydrophobic heptad repeat of an ␣-helical coiled coil and that all of the positions which are part of the dimerization domains of the coiled coil remained untouched. thus, these new peptides bear all of the requirements which are necessary for formation of cooperatively interacting helical structures and, furthermore, contain domains for cooperative sheet aggregation as well. the folded structure will now strongly depend on the environment. this peptidic model system allows a systematic study of the subtle influences that environmental conditions may have on protein folding stepwise, which means changing these conditions one after one or in all of the possible combinations that nature applies in vivo. plasmon waveguide resonance (pwr) spectroscopy is a powerful new biophysical method which allows us to examine structural changes, kinetics and thermodynamics of anisotropic biological systems and processes such as proteolipid membranes. this method has probed the mechanisms of g-protein coupled receptor (gpcr) signal transduction, and has obtained new insights into specific signaling pathways of agonists and antagonists with gpcrs. we now have extended these studies to examine the effects of lipid microdomains (rafts) on the binding, signaling and transduction pathways. using a 1:1 mixture of palmitoyloleoylphosphatidyl-choline (popc) and sphingomyelin (sm), we have directly observed the formation of two lipid bilayer microdomains, and the preferred segregation of the delta opioid receptor (hdor) into sm lipid rafts when the agonist ligand was bound, but not for the unoccupied receptor which preferentially incorporated into the popc-rich domain. furthermore, we can demonstrate directly that, g-proteins bind much more strongly to the hdor receptor in the lipid raft (sm-rich) environment than in the fluid non-raft (popc-rich) domain of the lipid bilayer. the implications of these findings for novel design of drugs, and drug screening will be emphasized. † supported by grants from the u.s.p.h.s., national institute of drug abuse and national science foundation. their measurement in high resolution nmr requires partial molecular alignment. for proteins in aqueous solution a number of standard methods exist to achieve such small alignment. we found that swelling of cross-linked polymers inside the nmr tube results in anisotropic gels. peptides in such a gel phase exhibit well resolved spectra of the partially aligned molecules. this allows to scale the orientation depending on the cross-linking of the polymer, the thickness of the unswollen stick or the temperature. rdcs can now easily be measured in natural abundance in peptides 3] all common nmr solvents can be used. with the chiral gel gelatin it is possible to discriminate d-and l-alanine to determine enantiomeric purity. the procedure is demonstrated to refine the solution structures of peptides such as cyclosporin a, somatostatin analogues and others galia blum, 1 georges von degenfeld, 2 kinneret keren, 3 misregulation of cysteine protease activity is associated with numerous pathologies ranging from cancer to autoimmune disease. protease activity is controlled by a delicate balance of many factors such as levels of natural inhibitors and posttranslational modifications. thus developing a detection method for monitoring protease activity rather than abundance is desirable. here we describe the design and synthesis of a novel class of chemical tools, activity based probes (abps), that detect protease activity. these probes are composed of a fluorescent tag and its cognate quenching group, a peptide recognition scaffold, and a reactive "warhead". these fluorescently quenched "smart probes" covalently modify protease active sites in a fashion that is dependent on activity of the protease. this results in loss of the quenching group, producing a fluorescent signal. we report the production of selective, cell permeable activity based probes for the study of papain family cysteine proteases in cells and whole animals. these probes are used to monitor real time protease activity in living cells using fluorescence microscopy techniques as well as standard biochemical methods. key: cord-012878-j9vndxgg authors: tremblay, reynald; feng, mary; menassa, rima; huner, norman p. a.; jevnikar, anthony m.; ma, shengwu title: high-yield expression of recombinant soybean agglutinin in plants using transient and stable systems date: 2010-06-18 journal: transgenic res doi: 10.1007/s11248-010-9419-0 sha: doc_id: 12878 cord_uid: j9vndxgg soybean agglutinin (sba) is a specific n-acetylgalactosamine-binding plant lectin that can agglutinate a wide variety of cells. sba has great potential for medical and biotechnology-focused applications, including screening and treatment of breast cancer, isolation of fetal cells from maternal blood for genetic screening, the possibility as a carrier system for oral drug delivery, and utilization as an affinity tag for high-quality purification of tagged proteins. the success of these applications, to a large degree, critically depends on the development of a highly efficient expression system for a source of recombinant sba (rsba). here, we demonstrate the utility of transient and stable expression systems in nicotiana benthamiana and potato, respectively, for the production of rsba, with the transgenic protein accumulated to 4% of total soluble protein (tsp) in nicotiana benthamiana leaves and 0.3% of tsp in potato tubers. furthermore, we show that both plant-derived rsbas retain their ability to induce the agglutination of red blood cells, are similarly glycosylated when compared with native sba, retained their binding specificity for n-acetylgalactosamine, and were highly resistant to degradation in simulated gastric and intestinal fluids. affinity column purification using n-acetylgalactosamine as a specific ligand resulted in high recovery and purity of rsba. this work is the first step toward use of rsba for various new applications, including the development of rsba as a novel affinity tag for simplified purification of tagged proteins and as a new carrier molecule for delivery of oral drugs. electronic supplementary material: the online version of this article (doi:10.1007/s11248-010-9419-0) contains supplementary material, which is available to authorized users. soybean agglutinin (sba) is a legume lectin glycoprotein that binds non-covalently to specific cell-surface carbohydrates, provoking agglutination of the bound cells when in solution. sba has been used to fractionate different cell types for use in clinical and biomedical applications. one such application is the separation of pluripotent stem cells from human bone marrow. cells fractionated by sba do not produce graft versus host disease (gvhd) and can be used in bone marrow transplantation across histocompatibility barriers (yura et al. 2008) . another application is the enrichment and isolation of fetal cells from the blood of pregnant women as a means of detecting fetal genetic and chromosomal abnormalities. it has been shown that the number of fetal erythroblasts recovered by a soybean agglutinin galactose-specific lectin method was approximately eightfold higher than the number obtained by a standardized magnetic cell-sorting (macs) procedure (babochkina et al. 2005 ). in addition, sba binds very effectively to some tumor cells and has been used to detect and treat several cancers including breast cancer (pusztai et al. 2008) . sba and other lectins, which are carbohydrate-binding proteins that bind sugar residues reversibly and specifically, have also been exploited as a ligand for targeted oral drug delivery, because their glycoprotein and glycolipid targets are integral parts of the enterocyte membrane (smart 2004) . procedures for high recovery and purity of sba have been developed, with extraction of sba from soybean flour resulting in high-quality purification with over 90% yield (percin et al. 2009 ), opening the door for the use of recombinant sba as a potential novel affinity tag for purification of genetically fused proteins. the potential production of a variety of recombinant proteins genetically fused to sba, however, necessitates the generation of an efficient recombinant production system. native sba is produced in the developing seeds of glycine max, with maximum production in seeds reaching over 2% tsp (lindstrom et al. 1990 ). it is relatively simple to obtain large amounts of purified native sba given that soybean seeds express high levels of native sba together with the establishment of efficient purification methods. however, many potential applications of sba in biotechnology and medicine could not be achieved or are difficult to achieve by use of unmodified native sba. for example, it is difficult to use native sba as an affinity tag for protein purification unless the target protein is genetically fused to sba and produced as a recombinant fusion protein. sba has previously been expressed in transgenic tobacco seeds in order to study the upstream and downstream cis-regulatory sequences mediating the expression of native sba (lindstrom et al. 1990 ). although a protein band with a molecular mass expected for the monomer of sba protein was seen on western blot, no analysis or characterization of the recombinant protein itself was performed; it was merely used as a marker for gene expression. others have successfully expressed sba in both e. coli and monkey bs-c-1 cells in an attempt to generate recombinant sba in order to analyze the relationship between glycosylation and tetramer assembly (adar et al. 1997) . they demonstrated that the recombinant sba retained its agglutination ability and specificity for n-acetylgalactosamine. the sba generated in bacteria, however, lacks glycosylation that, while not required for agglutination or assembly, is important to the stability of the bioactive tetramer (sinha and surolia 2005) . production in bs-c-1 cells resulted in a similarly glycosylated protein to native sba with an identical sugar binding profile but for some unknown reason had a lower agglutination ability. the minimum amount of bs-c-1 cell-derived sba required to induce hemagglutination is 10-20 lg/ml, which is 4-5 times larger than that of native sba. in addition, for production of a therapeutic protein, animal cells carry an inherent risk of pathogen contamination and have a high production cost due in large part to media costs. therefore, both bacteria and mammalian cells are not ideal bioreactors for sba production for therapeutic uses. plants are a suitable alternative expression system for recombinant sba production. as bioreactors, plants enable unlimited scalability, elimination of product contamination by mammalian pathogens, and reduced production costs compared with microbial or animal cell-based systems (boehm 2007; ma et al. 2008; tremblay et al. 2010 ). because of their eukaryotic nature, plants can perform the complex post-translational modification and processing required by many transgenic therapeutic proteins for biological and/or immunological function. furthermore, plant bioreactors have the short turn-around time needed to obtain gram quantities of a recombinant protein in a matter of weeks when expressed transiently. this is not only economically advantageous, but is also critical to meeting challenges related to quick access to life-saving biotechnology drugs and therapies. in addition, stable edible transgenic plant tissue might enable direct oral delivery of plant-derived therapeutic proteins and peptides, eliminating the need for expensive downstream protein purification and processing. our long-term objective is to generate recombinant sba for use as a carrier molecule for oral delivery of protein and peptide drugs and as an affinity tag for simplified and high-yield, high purity isolation of recombinant proteins. as a first step toward this objective, we report here the transient production of recombinant sba (rsba) in nicotiana benthamiana (nb), a close relative of tobacco (nicotiana tabacum), and stable production in solanum tuberosum (st) under the control of a ubiquitous camv35s promoter. we demonstrate transient expression of sba in nb plants at levels as high as 4% of total soluble protein (tsp), whereas its stable expression in st tubers reaches 0.3% tsp. furthermore, nbrsba and strsba are similarly glycosylated compared with native sba, retain their ability to induce hemagglutination, bind specifically to n-acetylgalactosamine, are stable in simulated gastric fluid (sgf) containing pepsin at acidic ph and are rapidly isolated in high purity from total soluble protein. the cdna of soybean agglutinin (sba) was cloned from glycine max cdna derived from 1 to 5 mm developing seeds. in brief, total messenger rna was extracted from the seeds using the rneasy plant mini kit (qiagen #74903) and converted to cdna by following the superscript ii procedure (invitrogen #18064). the primers f 5 0 aatccatggctacttc aaagttgaaaacc 3 0 and r 5 0 tctagattaa tgatgatgatgatgatggatggcctcatgca acacaaaacttg 3 0 (with addition of a 6xhis-tag to the c-terminus underlined, restriction sites in bold, and a silent mutation to remove an internal hindiii site italicized) were constructed using the previously published sequence for sba (genbank accession #k00821.1). the generated sba cdna was then inserted into puc-19. after confirmation by sequencing, the sba cdna was inserted into prtl-2 via digestion with ncoi and xbai, replacing the gus gene, providing a 35s promoter, 5 0 and 3 0 utr (carrington and freed 1990) . the resulting expression cassette was digested with hindiii and inserted into pbi-101 to create pbi-rsba. tri-parental mating was used to transfer pbi-rsba to agrobacterium tumifaciens strain lba4404 and confirmed via pcr using specific primers. transient expression of sba in n. benthamiana transient expression of pbi-rsba was accomplished by infection of 6-8-week-old leaves of nicotiana benthamiana as described by sparkes et al. (2006) . briefly, overnight cultures of agrobacterium were grown until density reached 0.5-1.0 a 600 , at which time the cells were centrifuged at 800g for 10 min, rinsed four times with infiltration medium (0.5% d-glucose, 50 mm mes, 2 mm na 3 po 4 , 0.0001 m acetosyringone), and then resuspended to the desired cell density, between 0.01 to 0.75 a 600 . the cells were then infiltrated into the abaxial side of the leaves using a 1-ml syringe and infected tissue was harvested each morning at day 1 through 9 postinfection. for co-infiltration with a second agrobacterium harboring the t-dna vector encoding the p19 suppressor gene of tomato bushy stunt virus (lakatos et al. 2004) , the two agrobacterial cultures were mixed at equal concentration before infiltration. stable expression of sba in s. tuberosum solanum tuberosum mini-tubers were generated as described previously (bourque et al. 1987) . overnight cultures of agrobacterium with pbi-rsba were used to transform tubers as previously described (ma et al. 2005) . regenerating plantlets were transferred to magenta boxes containing ms media supplemented with 50 lg/l kanamycin. the presence of the transgene in transgenic plants was confirmed by pcr, and pcr-positive plants were then selected to produce mini tubers for protein expression analysis. for mini tuber induction, a stem section (1 cm) with one resting auxiliary bud and one fully developed leaf was excised from a sterile magenta-grown transgenic plant. the leaf was removed and the stem was transferred to tuber-inducing medium as described above and placed in darkness at 20°c. after 4 weeks, the tubers formed (3 mm in diameter) were harvested and used for protein analysis. total soluble protein was extracted from infected n. benthamiana leaf tissue or potato tuber and then quantified as described previously . briefly, approximately 100 mg plant material, either solanum tuberosum tuber or nicotiana benthamiana leaf, was ground in a 1.5-ml tube with a plastic pestle and mixed with protein extraction buffer (200 mm tris ph 8.0, 100 mm nacl, 400 mm sucrose, 1 mm phenylmethylsulfonyl fluoride, 10 mm edta, 14 mm b-mercaptoethanol, 0.05% tween-20, 2 lg/ml leupeptin, 2 lg/ml aprotinin). the mixture was incubated on ice for 30 min and clarified by centrifugation at top speed for 10 min at 4°c. the supernatant was transferred to a new 1.5-ml tube and used for all subsequent assays. protein samples, boiled or unboiled, were loaded on to a 12.5% sds-page gel and resolved. gels were then either stained with coomassie blue to visualize the protein profile or transferred to a pvdf membrane as described previously ). the blot was then blocked for 1 h at room temperature with 5% w/v milk in tbs-t and washed 3 times, each time for 5 min, with tbs-t. the blots were incubated overnight at 4°c in 1:2000 rabbit anti-sba (cedarlane al-1301-2) in 1/3 blocking buffer 2/3 wash buffer. the blots were washed 3 times, each time for 10 min, in wash buffer and incubated in 1:5,000 goat anti-rabbit-hrp (g-7641, sigma) for 1 h at room temperature. the blots were washed 3 times, each time for 10 min, in wash buffer and then incubated with supersignal west pico chemiluminescent substrate (34080, pierce, rockford, il, usa). blots were exposed and then developed using a film processor. the amount of accumulated rsba in nicotiana benthamiana leaves or solanum tuberosum tubers was quantified by enzyme linked immunosorbent assay (elisa). briefly, commercial native sba standard (sigma l-1395) and triplicate ntrsba or strsba samples were bound to 96 well plates by incubation in phosphate buffer overnight at 4°c. the plates were then washed with pbs-t and blocked with 3% bsa in pbs-t for 1 h at room temperature. the plates were washed 3 times and then incubated overnight at 4°c in 1:2,000 rabbit anti-sba in 1/2 blocking:1/2 wash buffer. the plates were washed 3 times and incubated for 1 h at room temperature with 1:5,000 goat anti-rabbit-hrp in 1/2 blocking:1/2 wash buffer and then rinsed 3 times. the plates were incubated with substrate reagent pack (dy999, r&d systems) according to the manufacturer's instructions. the color was developed for 20 min and then stopped by addition of an equal volume of 2 m h 2 so 4 . the plate was then read using a thermomax microplate reader (molecular devices, usa). standard curves were calculated and used to determine protein concentrations of the individual samples. negative control was protein extract prepared from wild-type plant tissue. purification of rsba from nicotiana benthamiana or potato tubers was carried out according to the procedure for ge healthcare histrap hp column (#17-5248-01) or using an n-acetylgalactosamineagarose column. for purification with the n-acetylgalactosamine-agarose column, agarose columns with pre-bound n-acetylgalactosamine (sigma a2787-5 ml) were washed and equilibrated with 0.1 m nacl. the tsp containing rsba was then applied to the column and rinsed with excess 0.1 m nacl. samples were taken throughout rinsing and total protein in rinse was determined by spectrophotometry at a 280 . once protein levels were negligible, rsba was eluted with 0.5 m galactose/0.1 m nacl. the purified protein was confirmed via western blot and then desalted via dialysis against excess 0.59 pbs buffer. hemagglutination assay was performed using 2% rabbit red blood cells (rbc) suspended in saline buffer. commercial native sba standard and purified ntrsba and strsba were used for the assay. the proteins were diluted to different concentrations with saline in order to determine the effective unit for each sample, with one unit defined as the amount of sba required to induce agglutination (lin et al. 2008) . all agglutination assays were repeated in triplicate. rbc (2%, 50 ll) was added to round-bottomed 96-well plates and then mixed with 50 ll protein dilutions. the mixture was then allowed to settle for 1 h and the results were recorded. competitive sugar-binding assay sugar-binding assay was carried out as described above for hemagglutination, with the following modifications. one unit of native sba or nbrsba or strsba was mixed with saline containing 40 lm or 400 lm concentrations of one of the sugars n-acetylgalactosamine, n-acetylglucosamine, arabinose, lactose, or raffinose. the mixture (50 ll) was then added to 50 ll 2% rbc and incubated for 1 h. the results were then recorded as positive or negative for hemagglutination. deglycosylation of nbrsba and strsba was carried out with pngase f according to the manufacturer's procedure (sigma p-9120-1set). the samples were then loaded on to an sds-page gel followed by western blot analysis using anti-sba antibody. n-linked glycan removal was confirmed via a band shift on the western blot. to confirm the presence of mannose-type glycans on plant-derived rsba, blots containing both pngase f-treated and untreated samples were incubated with 20 lg/ml concanavalin a (c-2010, sigma) at room temperature for 1 h. after several washes with 0.5% tween-20 in pbs (ph 7.5), the blot was incubated with hrp-conjugated anti-cona antibodies (hal-1104-1, e.y. laboratories) at 1,000-fold dilution for 1 h. after the same wash step, the blot was incubated with supersignal west pico chemiluminescent substrate. the signals were then developed using a film processor. analysis of in-vitro digestion of nbrsba was carried out using either simulated gastric fluid (sgf) or simulated intestinal fluid (sif). for sgf (0.2 g nacl, 0.32 g pepsin, 700 ll hcl, in 100 ml h 2 o, final ph 2.5) and sif (0.68 g monobasic kh 2 po 4 , 7.7 ml 0.2 m naoh, 1.0 g pancreatin, in 100 ml h 2 o, final ph *6.8), purified protein was incubated in a 37°c water bath with samples taken and mixed in neutralization buffer (1.7 g na 2 co 3 in 100 ml h 2 o) at time 0, 15 s, 30 s, 1 min, 5 min, 15 min, and 30 min. samples were boiled for 10 min, separated on a 12.5% sds-page gel and subjected to western blot analysis as described above. control native sba and a non-glycoprotein, human gad65 made in e. coli (plantigen), were also tested. isolation and cloning of cdna encoding sba to obtain a cdna clone encoding sba, rna was extracted from wild-type soybean seeds that were approximately 1-5 mm in size, then converted into cdna that was then used as a template for the pcr cloning of the sba coding sequence. the primers included the addition of a 5 0 ncoi and 3 0 xbai restriction sites to facilitate sub-cloning of the pcr products, and addition of the 69 histidine tag to the c-terminus. the cloned sba gene was confirmed by sequencing. for the convenience of sub-cloning, the internal hindiii site within the native sba coding sequence was removed by converting a g to an a in the 2nd codon position encoding serine, resulting in no change of the amino acid. the complete sba expression cassette was released from the prtl-rsba via hindiii digestion and inserted into pbi101.1 to form pbi-rsba (supplemental fig. 1) . the plasmid pbi-rsba was then transferred into a. tumifaciens strain lba4404 for plant transformations. six-week-old n. benthamiana plants were infiltrated with the a. tumefaciens clone harboring pbi-rsba or mixed with cultures of a. tumefaciens containing the p19 expression cassette, a viral inhibitor of the rna silencing pathway in plants that has proven effective in increasing the yield of transiently expressed proteins (lakatos et al. 2004) (fig. 1a) . leaf samples were collected from three independent plants on different days post-infiltration with different concentrations of a. tumefaciens cultures, with an equal amount of p19 added for each assay, in order to determine the peak of rsba expression in the agroinfiltrated n. benthamiana plants. the results of immunoblot analysis showed that the highest expression using the gene-silencing suppressor p19 was reached at day 5, with an optimum concentration of agrobacterium of 0.25 a 600 (fig. 1b,c) . as calculated by elisa, the expression level of rsba at day 5 reached 4% of tsp (fig. 1d) . examination of unboiled samples on sds-page gels followed by western blotting showed the presence of two bands, one corresponding in size to the monomer and the other equivalent to the size of the tetramer of sba (supplemental fig. 2) , suggesting that n. benthamiana made rsba was assembled into a tetrameric protein complex, essential for its biological activity. stably transformed potato plants were also tested for expression of rsba. thirteen individual transgenic potato lines were generated after agrobacteriummediated transformation and transferred to minituber-inducing magenta boxes in order to generate consistently sized tubers for further analysis. immunoblot analysis of rsba expression in unboiled samples of tuber extract showed two major bands of 32 kda and just over 100 kda (supplemental fig. 2) , expected for the monomer and tetramer of sba, respectively. immunoblot analysis of boiled samples of tuber extract showed only the small band of 32 kda (data not shown). the level of strsba accumulation in potato mini-tubers was variable among individual transgenic lines, ranging from no detectable signal to 0.31% tsp (supplemental fig. 3) . soybean agglutinin (sba) specifically binds to n-acetylgalactosamine, enabling the purification of this protein via an n-acetylgalactosamine-agarose column to a high degree of purity. purification through this column also serves as confirmation of authentic sba production. figure 2a shows that sugar-specific purification of rsba from nicotiana benthamiana leaf extracts resulted in efficient protein retention during purification and elution. in addition, the sugar-purified sample was of high-quality when examined on sds-page gel and coomassie blue staining, with negligible contamination of non-specific proteins (fig. 2b) . sugar-specific purification of rsba from potato mini tubers also resulted in high-quality, purified protein (data not shown). as a control, the isolation of nicotiana benthamiana-derived rsba using a traditional histrap column was also performed (fig. 2b) . hemagglutinating activity of plant-derived rsba soybean agglutinin is known to agglutinate human and animal red blood cells (hemagglutination). rsba was therefore assessed for its ability to agglutinate rabbit erythrocytes. purified nbrsba was found to c accumulation of rsba at day 5 with different a 600 concentrations of agrobacterium. ? is native sba, nb is nicotiana benthamiana wild-type tsp. d elisa data for accumulation at days 1-9 after infection at optimized concentration. values are expressed as a percentage of total soluble protein (tsp), with each bar representing the mean value of the three collected samples repeated in triplicate from each day, with standard error. all western blots were loaded with approximately 30 lg tsp loaded per lane, 150 ng native sba control induce the agglutination (clumping) of rabbit erythrocytes within 1 h of treatment (fig. 3a) . similar results were obtained with strsba (data not shown). the minimum amount of native sba required to induce agglutination in the assay was 2.5 lg, whereas nbrsba and strsba required approximately 2 and 3.5 lg, respectively. as agglutination of erythrocytes by sba is because of its ability to bind to specific sugar residues on the cell surface, it is therefore speculated that the agglutinating activity of sba could be inhibited by addition of specific sugars. indeed, addition of n-acetylgalactosamine even at low concentrations was able to prevent the agglutinating ability of nbrsba, similarly to the native sba control (fig. 3b) . in contrast, the agglutinating ability of nbrsba or native sba was unaffected by high concentrations of non-specific sugars (supplemental table 1 ; see also fig. 3b ). native sba is a glycosylated protein with a single high-mannose glycan (adar et al. 1997) . the presence of glycosylation, while not required for assembly or function, is important in maintaining the stability of sba (sinha and surolia 2007) . to determine the glycosylation status of plant-derived rsba, nbrsba, strsba, and sba standard were digested with pngase f and analyzed by immunoblotting following separation by sds-page gels. both sba standard and plant-derived rsba had a decrease in molecular size of approximately 2-3 kda, which is an expected size for a single glycan (fig. 4a) . to determine the type of glycans of plant-derived rsba, blots containing both pngase f-treated and untreated nbrsba, strsba, and sba standard were incubated with cona, which binds to n-linked mannose, and then with anti-cona antibody. figure 4b shows that binding of cona occurs only in the untreated samples, with no binding to any of the pngase f treated sba proteins, suggesting that plant-derived rsba contains high-mannose type glycans, similar to native sba. plant-derived rsba was further investigated in simulated gastric and simulated intestinal fluid (sgf and sif) to assess its stability in the digestive system. sgf (3.2 g/l pepsin, ph 2.5) has been used to mimic the acidic stomach environment in animals, and sif (10 g/l pancreatin, ph 6.8) is used for proximal small bowel conditions. to this end, purified nbrsba and native sba were used in simulated digestion experiments. the effect of sgf and sif on the degradation of nbrsba, as evaluated by immunoblot analysis, is shown in fig. 5a ,b. as can be seen, nbrsba remains relatively intact after digestion even for 30 min in either solution. native sba showed similar resistance to sgf and sif digestion (data not shown). to ensure proper activity of the simulated fluids, e. coli-derived recombinant gad65 (glutamic acid decarboxylase) previously produced in our laboratory was tested as a control. the protein lasts less than 1 min in sgf and 5 min in sif (fig 5c,d) . we have demonstrated the successful recombinant production of sba through both stable and transient expression in solanum tuberosum and nicotiana benthamiana, respectively. sba has served as an excellent system of choice to study the fundamentals of protein-carbohydrate interaction (loris et al. 1998; sharon and lis 1990) . with a vast increase in knowledge of sba and other lectins, it has become apparent that sba has many additional applications, especially in medical and biotechnology-focused industrial areas such as the utilization of sba to selectively remove cancerous cells from whole blood without removing normal t cells, to prevent graftversus-host disease following transplant, or to enrich hematopoietic stem cells (bakalova and ohba 2003; kernan et al. 1987; yura et al. 2008) . moreover, recombinant sba has the potential to serve as a carrier system for oral drug delivery or to be used as an affinity tag for isolation of high-purity fusion proteins. to make these applications feasible, however, a more affordable source of large quantities of high-quality rsba is required. previous recombinant production of sba in tobacco was incidental, because it was simply used as a marker for studying the promoter activity and, as such, no assays were performed to confirm that the protein made retained authentic sba activity (lindstrom et al. 1990) . similarly, recombinant production in both bacterial and mammalian cell culture was for the purpose of elucidating the functional role of glycosylation and the produced recombinant sba was either lacking glycosylation (e. coli-derived) or had reduced agglutinating ability (mammalian cell-derived) (adar et al. 1997) . our objective was to develop a robust expression system that can generate large quantities of low-cost authentic rsba, which retains its binding specificity to facilitate high-purity simplified protein isolation and maintains its stability in the gastrointestinal tract (gi) needed for future use as a carrier molecule for oral drug delivery. authenticity of both nbrsba and strsba was confirmed by several results. the foremost is the binding and inhibition studies using rabbit red blood cells. sba binds to different surface proteins bearing n-acetylgalactosamine, and, in the case of red blood cells, results in hemagglutination (gordon and marquard 1974) . both of the plant-derived rsbas were able to induce hemagglutination of rabbit red in addition, a similar minimum amount of sba, whether native or from n. benthamiana or s. tuberosum, was able to induce agglutination. this suggests that, unlike monkey cell-made sba, nb/strsba is functionally equivalent to native sba. many lectins can induce hemagglutination, however, so it is important to confirm the specificity of rsba for its ligand. for example, the chinese black soybean variety is able to cause agglutination of rabbit red blood cells but is inhibited by a different sugar, in that case melibiose (lin et al. 2008) . the inhibition of hemagglutination by addition of n-acetylgalactosamine but not other sugars confirms the rsba retains the authentic binding profile of native sba. in addition to this, the ability to specifically purify high-quality rsba via n-acetylgalactosamine bound to agarose further confirms the authenticity of the recombinant protein, as there was no detectable level of rsba in either the flow-through or wash steps, only in the elution step. the capture rate for this technique has been reported to be over 90% efficient, which is supported by the lack of rsba signal in the flowthrough steps seen in fig. 2b (percin et al. 2009 ). the ability to purify high-quality rsba also enables potential use as an affinity tag. through genetic fusion with a gene of interest (goi), it may be possible to first purify the fusion protein on the sugar columns, followed by subsequent cleavage of the desired goi via specific endoproteolytic cleavage, such as with tobacco etch virus (tev) protease (tubb et al. 2009) . a second possibility would be in combination with a linker intein, an intervening protein sequence that can be induced to undergo n-terminal, c-terminal or both termini cleavage under specific conditions (evans et al. 2005) . this could enable cleavage of the goi from rsba without any expensive proteases and result in high-purity protein. it should be mentioned that because of the requirement of the tetrameric formation for proper sba activity, addition of a fusion partner may affect the formation of a tetrameric sba. however, several groups, including our own, have previously demonstrated that other multimer forming proteins, for example the non-toxic b subunit of cholera toxin, retain their ability to form pentamers and bind to their target ligand when fused with other proteins (arakawa et al. 1998; li et al. 2006; ruhlman et al. 2007; tremblay et al. 2008 ). the production of rsba was accomplished using both transient and stable transformation of nicotiana benthamiana and potato, respectively. transient gene expression in plants has a number of advantages over stable genomic transformation in plants or other recombinant expression platforms. first and foremost is that new recombinant proteins can be generated in sufficient quantities for in-vitro analysis and in-vivo studies at a speed not possible with traditional bioreactors. this is best demonstrated in the generation of idiotpe-specific scfvs to treat non-hodgkins lymphoma patients. researchers were able to generate patient-specific scfvs by first cloning the antibody fragment sequences from the patient's biopsy and then generated sufficient quantities in plants to inject the plant-made scfvs back into the patient in less than 16 weeks, something unachievable with any other biological system (mccormick et al. 2008) . transient expression can also be used to generate and validate new vaccines against future pandemics, for example h1n1 or sars, for which large quantities of vaccine are needed in a short-period of time in order to meet world-wide demand. an additional benefit of transient expression in plants is the level of accumulation. in our work, we achieved almost 4% accumulation of rsba in less than a week. this is a tenfold increase over the accumulation seen in our stably transformed potato lines. based on our data, the transient system can generate an approximate yield of between 1.5 and 3 g of rsba per kg fresh weight in 1 week, meaning that a greenhouse setting capable of producing 1,000 kg of tissue per week could generate up to 3 kg of purified rsba every week. the use of stable lines expressing rsba can likewise prove advantageous. the production of a stable expression platform provides long-term, lowcost therapeutic proteins, typically required where the therapeutic agent is commonly used in multiple individuals over a long period of time, for example in the potential prevention of type 1 diabetes using glutamic acid decarboxylase 65 (gad65) (ma et al. 2004) . we were able to accumulate rsba to over 0.3% of tsp in transgenic potato tubers. a major benefit of tuber accumulation is the stability of recombinant proteins during long periods of storage. in the production of monoclonal antibodies, for example, the recombinant antibodies were stable for over 6 months when stored in the dark at room temperature, with little to no loss in either quantity or activity (de wilde et al. 2002) . this makes potatoes an excellent system for recombinant protein production, because one of the major disadvantages of field/ greenhouse grown plants for recombinant production is losses in yield owing to protein degradation during transit and processing. an additional benefit of the use of tubers is that they can be used to orally deliver the target protein with minimal processing, especially given that potato tubers form a staple part of many cultures' diets and can be processed for consumption via freeze-drying that enables ingestion without cooking and thus could help minimize protein denaturation/loss during food preparation. in-vitro digestion of n. benthamiana-derived rsba under simulated gastric and intestinal conditions proved it to be stable. there was no significant loss or degradation of the protein after incubation of nbrsba in sgf for 30 min, as seen in fig. 5a ,c. these results suggest that the plant-derived rsba may hold great promise for use as a carrier system for oral delivery of protein and peptide drugs. the oral route for drug delivery is the most preferred route and has considerable advantages: requiring neither sterile needles nor trained personnel, lower cost and increased quality of life, increased access to a large population, reduced side-effects often seen with systemic delivery, and greater patient compliance and acceptability. however, administration of therapeutic peptide or protein drugs by the oral route is a major challenge. orally administered peptide or protein drugs are readily degraded because of their exposure to the harsh environment of the gi (low ph and various proteinases and peptidases). therefore, development of suitable delivery systems is crucial to the success of oral administration of protein drugs. plant-derived rsba may provide an ideal vehicle for oral drug delivery. moreover, sba undergoes endocytosis through the epithelial lining of the intestine, adding to its potential as an adjuvant for therapeutic protein delivery (benjamin et al. 1997) . a potential concern with the use of rsba as a carrier system for targeted drug delivery is its antinutritional property. indeed, sba is one of the predominant anti-nutritional factors found in the raw soybean and accounts for approximately 50% of the growth inhibition in rats fed unheated soybean (liener 1996) . however, its anti-nutritional activity is strictly dose-dependent. a negative effect of sba on growth and immune function in rats was observed only when very high levels of this protein was given in diets (14 mg/per rat daily or equivalent to 0.2% of its body weight). in our previous work on oral tolerance induction in mice using plant-derived autoantigenic protein, we showed that only microgram quantities (or .005% of the animal's body weight) of the plant-derived antigen is required to induce a response (ma et al. 2004 ). this suggests that when used as a carrier system, the minimum effective dose of rsba-antigen fusion protein required to be delivered is in the range of micrograms, not milligrams. delivery of microgram quantities of sba to animals is safe (buttle et al. 2001; tang et al. 2006) . however, as with all therapeutics destined for human use, thorough studies would be required to determine the overall bio-safety. in conclusion, our work has demonstrated the feasibility of high accumulation, high-purity recombinant production of sba. this is the first molecular characterization of rsba in plants. the availability of low-cost, high-quality rsba may make many important applications of this protein possible, especially in medicine. synthesis of soybean agglutinin in bacterial and mammalian cells a plant-based cholera toxin b 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plants: from transgene containment to protein purification factors affecting hemagglutination by concanavalin a and soybean agglutinin prevention of gvhd in hlaidentical marrow grafts by removal of t-cells with soybean agglutinin and srbcs molecular mechanism of rna silencing suppression mediated by p19 protein of tombusviruses expression of cholera toxin b subunit and the b chain of human insulin as a fusion protein in transgenic tobacco plants effects of processing on antinutritional factors in legumes: the soybean case purification of melibiose-binding lectins from two cultivars of chinese black soybeans expression of soybean lectin gene deletions in tobacco legume lectin structure induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (gad) and il-4 production of biologically active human interleukin-4 in transgenic tobacco and potato plant-based pharmaceuticals and its application in oral tolerance. in: pontell eb (ed) immune tolerance research development plant-produced idiotype vaccines for the treatment of non-hodgkin's lymphoma: safety and immunogenicity in a phase i clinical study n-acetyl-dgalactosamine-specific lectin isolation from soyflour with poly(hpma-gma) beads uses of plant lectins in bioscience and biomedicine expression of cholera toxin b-proinsulin fusion protein in lettuce and tobacco chloroplasts-oral administration protects against development of insulitis in nonobese diabetic mice carbohydrate-protein interactions oligomerization endows enormous stability to soybean agglutinin: a comparison of the stability of monomer and tetramer of soybean agglutinin attributes of glycosylation in the establishment of the unfolding pathway of soybean agglutinin lectin-mediated drug delivery in the oral cavity rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants effects of purified soybean agglutinin on growth and immune function in rats expression of a fusion protein consisting of cholera toxin b subunit and anti-diabetic peptide (p277) from human heat shock protein in transgenic tobacco plants tobacco, a highly efficient green bioreactor for production of therapeutic proteins purification of recombinant apolipoproteins a-i and a-iv and efficient affinity tag cleavage by tobacco etch virus protease selection of hematopoietic stem cells with a combination of galactose-bound vinyl polymer and soybean agglutinin, a galactose-specific lectin key: cord-003435-ke0az7nf authors: schlake, thomas; thess, andreas; thran, moritz; jordan, ingo title: mrna as novel technology for passive immunotherapy date: 2018-10-17 journal: cell mol life sci doi: 10.1007/s00018-018-2935-4 sha: doc_id: 3435 cord_uid: ke0az7nf while active immunization elicits a lasting immune response by the body, passive immunotherapy transiently equips the body with exogenously generated immunological effectors in the form of either target-specific antibodies or lymphocytes functionalized with target-specific receptors. in either case, administration or expression of recombinant proteins plays a fundamental role. mrna prepared by in vitro transcription (ivt) is increasingly appreciated as a drug substance for delivery of recombinant proteins. with its biological role as transient carrier of genetic information translated into protein in the cytoplasm, therapeutic application of mrna combines several advantages. for example, compared to transfected dna, mrna harbors inherent safety features. it is not associated with the risk of inducing genomic changes and potential adverse effects are only temporary due to its transient nature. compared to the administration of recombinant proteins produced in bioreactors, mrna allows supplying proteins that are difficult to manufacture and offers extended pharmacokinetics for short-lived proteins. based on great progress in understanding and manipulating mrna properties, efficacy data in various models have now demonstrated that ivt mrna constitutes a potent and flexible platform technology. starting with an introduction into passive immunotherapy, this review summarizes the current status of ivt mrna technology and its application to such immunological interventions. our bodies are continuously exposed to molecules that may indicate disease or parasitic invasion. the immune system is responsible for the detection and clearance of such molecules and for control of the underlying causes. immediate discrimination between self and foreign upon first exposure is mediated by innate immunity. signals that induce innate immunity are called pathogen-associated and damageassociated molecular patterns (pamps and damps) [1, 2] . the innate immune system is characterized by induction of generic defenses against a broad spectrum of infectious agents and is mainly aimed at clearance of tissue damage at the site of infection and interruption of further pathogen replication. responses against specific targets following prolonged or repeated exposures are processed by the adaptive immune system [3] . important effectors of adaptive immunity are highly specialized cells: b cells secrete antibodies against soluble or cell-associated antigens [4] . cytotoxic cd8 + t cells (ctls) recognize and kill infected or neoplastic cells [5] . regulatory cd4 + t cells augment b cell maturation or inhibit auto-reactive immune cells [6] . dendritic cells (dcs) process and present antigen for the transition from innate to adaptive immunity [7] . some b and t cells progress towards persisting memory cells that react faster and with greater affinity to the foreign molecules upon re-exposure. compared to innate immunity, adaptive immunity usually requires weeks (as opposed to minutes) until an effective response against novel targets is achieved. advantages of the adaptive immune system include the ability to launch specific immune responses also against antigens of endogenous (not only microbial) origin that may be associated with degenerative or neoplastic disease. manipulation of the immune system is an important component of many prophylactic and therapeutic applications against infectious, degenerative and neoplastic diseases. the diverse repertoire of methods can be roughly divided into four approaches: active immunization (vaccination) prepares adaptive memory responses, usually prior to first exposure, and constitutes one of the most efficacious and cost-effective medical interventions. immunization with antigens generally only leads to the induction of antibody production, whereas for instance inoculation with attenuated viruses also elicits cytotoxic effector cells [8] . in addition, active immunization can be accomplished by adoptive cell transfer. in a typical setting, dendritic cells are loaded with tumor antigens ex vivo and subsequently infused into patients to induce an adoptive immune response against cancer cells [9] . in contrast, passive immunotherapy circumvents the initial steps required for immune responses to be launched and directs the immune system efficiently to the desired medical targets. for cellular approaches, ctls are equipped with recombinant receptors ex vivo. these cells are designed to attack neoplastic cells that express the cognate tumorassociated antigen immediately after infusion [5] . passive immunization by administration of processed antibodies derived from human or animal donors is a well-established emergency procedure for treatment of snake-bite envenomation or post exposure prophylaxis against, for example, rabies [10] . the advantage of passive immunization is that protective antibodies can be provided in a very short time. application of recombinant antibodies further expands the number of available targets and is increasingly important for augmentation of conventional therapies against cancer [10, 11] . passive immunotherapies either require or can exploit modern nucleic acid-based methods, among which mrna is the latest technology. the present review is dedicated to provide an overview on mrna in passive immunotherapy after introducing the immunological approach as well as mrna technology as such. it is well known that protection raised by most of today's licensed vaccines is primarily antibody-dependent (fig. 1a ) [8, 12] . basically, this explains the long and successful history of passive immunotherapy (fig. 1b) . the protective capacity of serum against bacterial toxins was discovered in the early 1890s [13] . the avoidance or control of infection by such passive immunization is based on the transfer of serum and later polyclonal immune globulin (= antibody) preparations from convalescent or vaccinated humans or animals [14, 15] . prior to the discovery of antibiotics, serum was the only antidote for bacterial diseases [16] . thus, following successful passive immunization against diphtheria toxin [13] , a whole plethora of serum or immune globulin therapies for viral and bacterial diseases as well as to neutralize snake toxins was developed [17] . clinical benefits of serum and immune globulin therapy were demonstrated for viral diseases such as influenza, measles, and polio and bacterial infections with meningococcus or pneumococcus [18] [19] [20] . with the advent of antibiotics, the use of serum or polyclonal immune globulin preparations as antibacterial agent was largely discontinued in late 1940. however, such therapies retained a niche as treatment for venoms, toxins, and certain viral infections. in the second half of the twentieth century, polyclonal antibody preparations were developed and in part licensed for the prophylaxis and treatment of hepatitis a and b, cytomegalovirus, varicella-zoster virus, vaccinia virus, rabies, respiratory syncytial virus, west nile virus, and various hemorrhagic fevers [21] . for instance, passive immunization against the argentine hemorrhagic fever shows beneficial effects when applied within 1 week after the emergence of symptoms, and post-exposure treatment with human or equine immunoglobulins are recommended for rabies [22] [23] [24] . moreover, botulism is treated with equine antitoxin [25, 26] . such immune globulin preparations of non-human origin are particularly prone to elicit an immune response that obstructs their therapeutic use or efficacy [27] . further disadvantages or difficulties associated with the use of serum or polyclonal globulin preparations are the often high content of non-neutralizing antibodies, batch-to-batch variations, and in case of human sources the availability of appropriate immune donors [21, 28] . in 1975, groundbreaking work described the production of monoclonal antibodies (mab) by immortalization of b cells [29] . the resulting hybridoma technology was then rapidly exploited for clinical use, for instance, to produce a mab to cd3 for preventing organ rejection [30] . recombinant technologies further expanded the available therapies based on mabs. in vitro antibody selection technologies like phage or ribosome display were developed to enable the generation of highly specific human mabs out of libraries that may even be naïve for the specific antigens [31] [32] [33] [34] [35] [36] 1 schematic illustration comparing active immunization and passive antibody immunotherapies. a during active immunization triggered by natural infection or vaccination, antigenic patterns are presented by antigen-presenting cells in the lymph nodes. this leads to t cell mediated activation of antigen-specific b cells. as a consequence, b lymphocytes differentiate into plasma cells which produce and secrete antigen-specific antibodies that bind to cognate structures, finally leading to their clearance. b instead of being produced by plasma b cells, antibodies can be manufactured recombinantly and administered for instance by subcutaneous injection for passive immunization. after injection, antibodies enter circulation by diffusion and act like endogenous antibodies. c for dna-based passive immunization, dna is often packaged in nanoparticles, e.g., virus capsids, which for instance can be injected intramuscularly. after uptake by muscle cells, dna is released into the cytosol. for transcription into mrna, the dna has to enter the cell nucleus first. mrna is then translated into antibodies which are secreted to bind their cognate targets a high throughput technique to amplify and clone antibody genes from single human b cells was described [37, 38] . although recombinant mab technology is exploiting a large variety of different antibody formats (fig. 2) , the prevalent type is still a full-size antibody of the igg class. in addition to the variable domains essential for antigen binding, they contain constant domains including the fc-region. the latter is important for antibody function and can mediate antibody-dependent cellular cytotoxicity (adcc), complement-dependent cytotoxicity (cdc), and antibodydependent cellular phagocytosis (adcp) [39] . furthermore, binding to the neonatal fc receptor (fcrn) plays a role in controlling the antibody half-life which is 21-28 days for human igg [40] [41] [42] . fcrn rescues bound antibody from degradation by transporting it back to the cell surface where it is released into the extracellular space [43, 44] . specific mutations in the fc region that increased the affinity to fcrn have been shown to prolong antibody half-life up to fivefold [45] . in addition to the impact of fcrn binding, half-life also benefits from the large size of iggs. it obstructs antibody clearance by the kidney as well as metabolization by cytochrome p450 [42, 46] . the downside of the large size is the correspondingly low access to and penetration of tissue which can affect therapeutic efficacy [47] . full-size antibodies are often posttranslationally glycosylated, modulating fc function. although aglycosylated iggs can be produced in bacteria [48] , modern production processes usually take advantage of the cellular machinery for advanced posttranslational maturation and secretion of eukaryotic cells [48] [49] [50] [51] . the vast majority of approved therapeutic antibodies are currently produced in mammalian cells [52, 53] . production processes have been optimized especially for the predominant production system, the continuous chinese hamster ovary (cho) cell line. cho cells secrete antibodies with negligible non-human glycoforms [54] and are also amenable to glycoengineering [55] . differences in glycoforms depend on the production system and affect distribution and stability of the antibody, fc effector function and immunogenicity in recipients [54] . since traditional expression hosts such as e. coli do not allow efficient production of full-size antibodies, smaller proteins consisting of fragments derived from the variable domains were developed as promising alternatives. such single-chain variable fragments (scfv) and various derivatives thereof preserve antigen binding while facilitating manufacturing (fig. 2b, c) [56] . another type of antibody fragment is derived from camelids or cartilaginous fish. these animals produce single-domain antibodies devoid of light chains (fig. 2e) [57, 58] . since antigens are recognized by a heavychain-only v h domain (v h h) in camelids [59] , the variable v h h fragment can be easily engineered into nanobodies that offer additional advantages such as improved heat and ph stability [60] . moreover, they can also be assembled into v h h-based neutralizing agents (vnas) (fig. 2e) [61] . various studies demonstrated that multivalent formats were more effective than monovalent single-domain antibodies [62, 63] . notably, all formats based on antibody fragments can be relatively efficiently produced with less expensive bacterial expression systems, typically employing e. coli [64, 65] . the antibody fragments produced in this system are often targeted to the oxidative environment of the periplasm using specific signal peptides to foster disulfide bond formation and proper folding [64, 65] . moreover, enhanced expression of chaperones and cytoplasmic oxidases has been demonstrated to increase the yield of antibody fragments [48, 66] . small antibody fragments were also the basis for developing the concept of bispecific antibodies more than 20 years ago. initially, single chain antibodies with a different binding specificity were fused to the c-terminal ends of heavy chains of iggs [67] . generation of first bispecific igg molecules benefited from the knob-into-hole technology [68] . today, many different bispecific antibody formats combining two different antigen binding domains in one molecule are available ( fig. 2d ) [69] [70] [71] [72] . among them, bispecific diabodies (bi-(scfv) 2 ) and bite antibodies are prominent examples [73, 74] . in general, bispecific antibodies can be deployed to target therapeutic substances such as toxins, radionuclides, and drugs as well as effector cells like ctls to the site of cognate antigen expression [75] . associated with their small size, many formats using antibody fragments are cleared by renal elimination [76, 77] . moreover, in the absence of an fc region, recycling by the fcrn rescue mechanism cannot take place [77] . as a consequence, these formats usually reveal short plasma half-lives [77] . for instance, bi-(scfv) 2 antibodies have a serum half-life of less than 2 h which usually requires continuous infusion [78] . in case of the bite blinatumomab, the antibody is usually administered daily due to its short halflife [79] . possible strategies to extend serum half-lives are site-specific pegylation and fusion to an fc region [80, 81] . however, the latter approach would negate various advantages of antibody fragments including their better and faster tissue penetration [41, 82] . it has been shown that small single-domain antibodies could even cross the blood-brain barrier [83] . in case of an anti-rabies antibody, this allowed partial rescue of mice challenged with virus injection into the brain in contrast to full-size immunoglobulins [84, 85] . today, monoclonal antibodies play an important role in the therapeutic armamentarium. dozens of antibodies have been licensed to treat cancer, rheumatoid arthritis, multiple sclerosis, psoriasis, allergy, systemic lupus, and other diseases. in addition, mabs have shown promise in protecting against various microorganisms, viruses, and fungal infections as well as in treating neurodegenerative diseases [86] [87] [88] [89] . however, mabs for infectious disease indications are still scarce among licensed products. palivizumab for respiratory syncytial virus prophylaxis in high-risk infants was the first antiviral mab approved by the fda [90] . since then, antibodies against anthrax and rabies in india became available. in addition, bezlotoxumab binding to clostridium difficile toxin is used to prevent recurrent bacterial infections [91] . mabs for treating cancer are still the largest group of licensed products. here, rituximab, directed against the transmembrane protein cd20 on the surface of b lymphocytes, was the first mab in clinical use [92] . however, as for many other mabs in antitumor therapy, high doses are needed to obtain clinical efficacy [93] . a successful example for a bispecific antibody is the first-in-class bite against cd19/cd3, blinatumomab, which is approved for the treatment of acute lymphoblastic leukemia [94] [95] [96] . in addition to the administration of immunoglobulins, passive immunity can also be conferred by transferring functionalized immune cells. adoptive transfer of ctls was shown to be a potent therapeutic means to treat both viral infections and cancers [97] [98] [99] . to this end, t cells can be equipped with an additional t-cell receptor (tcr) or a chimeric antigen receptor (car) [100] . while cars are limited to the binding of surface antigens, tcrs recognize mhc-presented peptides derived also from intracellular proteins. the first successful clinical trial with an engineered tcr on ctls demonstrating tumor regression was reported in 2006 [99] . subsequently, passive immunotherapies with tcr-engineered t cells became an important approach for antitumor treatments [101] . efficient targeting and killing of cancer cells expressing the respective antigen in patients with various forms of cancer including metastatic melanoma, synovial sarcoma, and colorectal carcinoma have been demonstrated [99, [102] [103] [104] . a possible problem specific to the use of tcr-engineered t cells is the presence of an endogenous tcr. mispairing of endogenous and introduced α-and β-chains may create new specificities with potential reactivity to host molecules [105, 106] . to avoid such mispairing, the use of γ/δ t cells has been suggested, since γ/δ-chains do not pair with α-or β-chains [107] [108] [109] . alternatively, gene editing is now being explored to disable endogenous tcr expression [110, 111] . the concept of cars was developed in 1989 [112] and then refined using a scfv fragment to obtain antibody-like receptor specificity without the need to transfer multiple genes [113] . first generation cars consisted of an antigenspecific scfv, fused to a transmembrane and intracellular cd3ζ tcr signaling domain, conferring transient activation and cytotoxicity to t cells [114] . upon target binding through the scfv domain, the engineered t cell is activated in an mhc-independent manner [115] . subsequent generations of cars were improved with respect to cytotoxicity and persistence by including additional co-stimulatory domains such as cd28, ox-40 or 4-1bb [114, [116] [117] [118] . t cells engineered with such cars targeting specific tumor antigens are remarkably successful in treating hematological malignancies like leukemia and lymphoma [119, 120] . for instance, cd19-directed car t cells repeatedly revealed complete and durable remissions in patients with b-cell acute lymphoblastic leukemia (b-all) [121] [122] [123] . in contrast, car t cell therapies face various challenges for solid tumors [124] . at present, the most common techniques for generating tcr-or car-engineered t cells utilize viral gene transduction with retro-or lentiviral vectors [125] . however, permanent expression of the transgenic receptor mediated by this efficient technology can be disadvantageous in case of therapy-related severe toxicities due to accidental cross reactivity [126] [127] [128] . on-target/off-tissue and off-target toxicities by engineered t lymphocytes attacking healthy host cells as well as cytokine release syndrome are feared side-effects which were repeatedly reported when virus vector transduced cells were applied [129] [130] [131] [132] . another concern of using retroviral vectors is the potential to induce insertional mutagenesis and genotoxicity in effector cells [133] [134] [135] [136] . hence, more precise cell manipulations are currently under investigation. among them, gene editing of primary human t cells was recently demonstrated to be an efficient approach [137] . while t cell engineering for adoptive transfer inevitably requires the use of nucleic acids encoding a target-specific receptor, antibody immunotherapy can deploy recombinant proteins. however, as pointed out above, maintaining therapeutically effective levels may require frequent administrations dependent on clearance and indication [138] . thus, dna-mediated antibody expression directly in the body may represent an attractive alternative to administration of recombinant proteins (fig. 1c ). both, plasmids as well as viral vectors have been used for passive immunization. although efficient in small animal models, strong expression of recombinant genes using unformulated dna does not scale well to larger animals (including primates) [139] . consequently, application of recombinant adeno-associated viruses (aavs) is currently the preferred method for transduction of the antibody gene of interest [140] . early work reached single digit µg/ml serum titers [141] . later studies with advanced vector designs reported expression levels in the high µg/ml or even in the single digit mg/ml range [142] [143] [144] . much work has been done in the field of hiv prophylaxis. here, a single intramuscular injection of recombinant aav was demonstrated to elicit peak antibody titers above 100 µg/ml in mice [142] . treated mice revealed substantial anti-hiv antibody levels for more than a year and were protected from hiv-1 challenge. although aav usually maintains an episomal state, this vector still harbors an inherent risk of insertional mutagenesis. in patients with hepatocellular carcinomas integration of aav2 into known cancer genes was observed [145] . moreover, aav-based immunotherapy faces various issues regarding immunogenicity [146] [147] [148] . a substantial percentage of the population has already been in contact with the used virus and consequently shows pre-existing immunity limiting the efficacy of treatment [149, 150] . the induction of anti-viral responses during immunotherapy may have similar consequences if a single virus serotype is used in repeated treatments [148] . pre-existing or induced immunity could lead to clearance of the viral vector and/or aavtransduced cells. finally, gene delivery by aav has been reported to induce immune responses against the encoded protein. even when using an endogenous gene such as erythropoietin (epo), some macaques receiving epo-encoding aav intramuscularly developed severe autoimmunity against the protein [151] . when non-human primates were treated with aav vectors expressing antibody or antibody-like proteins, titers dropped rapidly in some animals [152] . one reason is the possibility of inducing anti-idiotype antibodies which requires immune suppression to obtain sustained expression [153] . further studies indicated that primates may be more prone to develop a robust t cell response to the aavencoded protein compared to mice [154] . in summary, the risks of insertional mutagenesis and genotoxicity, long-lasting expression without control in case of adverse events, and various potential issues regarding immunogenicity associated with viral vectors that may limit efficacy emphasize the high demand for other vectors for passive immunotherapy than dna. how mrna offers a viable option to meet the demands is reviewed in the following sections. the cellular machinery uses mrna as a transient carrier of genetic information for the synthesis of proteins. based on this fundamental biological concept administering exogenous mrna represents an alternative to dna-mediated protein delivery in vitro and in vivo. using mrna instead of dna as therapeutic substance is attractive due to the absent risk of insertional mutagenesis. moreover, efficient expression is even obtained in non-dividing cells, since mrna does not require a nuclear phase for activity. compared to the delivery of proteins and peptides, mrna may prolong the availability of effector molecules, however, not as much as dna. in contrast to the latter, mrna therapy, therefore, has to cope with the short half-life in vivo of exogenously delivered mrna as for instance indicated by mrna-mediated vegf expression in myocardium returning to baseline within 72 h [155] . while this may be a therapeutic disadvantage in various instances, it can be considered advantageous from a safety perspective, particularly in case of adverse events. mrna was first employed for the expression of a protein of interest in the early 1970s when rna preparations were microinjected into xenopus oocytes and synthesis of the encoded protein was demonstrated [156, 157] . in 1989, the group of inder verma presented a reliable method to efficiently introduce rna into a variety of cells using a cationic lipid [158] . almost at the same time, mrna-mediated protein expression in vivo was demonstrated after direct injection into mouse muscle [159] . much of the early work on a potential therapeutic use of mrna focused on the development of active vaccination approaches, in part since low amounts of antigen suffice due to the amplifying nature of the immune response. subcutaneous injection of liposomeencapsulated, antigen-encoding mrna was the first example of eliciting an antigen-specific ctl response in mice [160] . gene gun delivery of mrna into mouse epidermis provided the first evidence of an antigen-specific antibody response [161] . in addition, mrna turned out to be a potent means to load dendritic cells with antigens to convert them to tailored antigen-presenting cells in vitro and in vivo [162] . later, a new vaccination protocol was introduced which elicited a complete adaptive immune response consisting of antigenspecific antibodies and t cells with lytic activity without requiring any transfection reagents, special equipment or heterologous boost [163] . from a retrospective view, this event marked the starting point of the commercial development of mrna vaccines. in the minimal structure, mrna contains a protein-encoding open reading frame (orf) flanked at the 5′-and 3′-end by two elements essential for the function of eukaryotic mrna: the "cap", a 7-methyl-guanosine residue (m7g) bound to the 5′-end of the rna via a 5′-5′ triphosphate bond, and, with the exception of histone mrnas, a poly(a) tail at the 3′-end [164] [165] [166] . synthetic mrna is transcribed in vitro from a plasmid dna template that contains at least a bacteriophage promoter, the orf, and a unique restriction site for linearization of the plasmid to ensure defined termination of transcription. typically, the template also contains a poly(d[a/t]) sequence transcribed into poly(a). alternatively, the poly(a) tail can be generated by enzymatic in vitro polyadenylation of transcribed rna. the cap may be incorporated into the transcript during transcription by including an m7gpppg dinucleotide as a structural homolog of the endogenous cap structure in the transcription reaction. different options for how to derive ivt mrna are summarized in fig. 3 . various elements of an mrna molecule contribute to the level and duration of expression of the encoded protein. the cap structure is required for efficient translation and stabilizes mrna towards exonucleolytic decay [167] [168] [169] . various structures have been repeatedly used for ivt mrna (fig. 4) . the basic m7gpppg cap analog is incorporated in both orientations into the rna by the bacteriophage rna polymerase [170] . however, reverse incorporation of the cap analog results in mrna molecules that lack the m7 methylation at the cap and are not recognized by the translational machinery [171] . substitution of the hydroxyl group in c2′ or c3′ position of the m7g with a methoxy group prevents reverse incorporation of the cap analog by inhibiting elongation at the m7g. the dinucleotide is, therefore, called 'antireverse cap analog' or arca [171, 172] . arca-capped mrna revealed increased as well as prolonged protein expression in cultured cells and enhanced reporter protein expression in mouse dendritic cells up to 20-fold [173, 174] . for further optimization of the cap structure, modifications were introduced within the triphosphate linkage to inhibit decapping. substitution of a non-bridging oxygen in the β-phosphate moiety of an arca by sulfur results in the β-s-arca dinucleotide. while β-s-arca maintains recognition by the translational machinery, protein expression from mrna capped with β-s-arca was extended in hc11 cells and immature dcs, but not in mature dcs [175, 176] . enzymatic capping represents an alternative to the cotranscriptional approach, avoiding a n7-unmethylated cap as does arca. to this end, mrna is transcribed without cap analog and subsequently capped using the vaccinia virus capping complex [177] . this complex with triphosphatase, guanylyltransferase and (guanine-7)-methyltransferase activity adds a natural cap to the 5′-triphosphate of an rna molecule. such a cap0 structure can be further converted into a cap1 group by o-methylation of the ribose of the cap-proximal nucleotide using the vaccinia virus 2′o-methyltransferase [178] . cap1 (and cap2 harboring a further o-methyl-ribose at the subsequent nucleotide) structures are typical of eukaryotic mrna and are recognized less by cytosolic rna sensors of the innate immune system, thereby rendering the mrna less immunostimulatory. for instance, rig-i is activated by cap0 but not cap1 mrna and ifit1 binds cap0 mrna more tightly than cap1 molecules [179, 180] . such sensor-mediated immune stimulation may in turn negatively impact mrna translation [181] . it is interesting to note that since recently a new cap analog, cleancap, is available which enables the cotranscriptional incorporation of a cap1 structure into ivt mrna. like the cap structure at the 5′-end, details of the poly(a) tail at the 3′-end influence translation and stability of mrna [167, 182] . while numerous studies demonstrated a positive effect of a poly(a) tail and some correlation of effectiveness and length of the element, details of observations were remarkably variable. the majority of studies indicate an enhancement of translation when extending the poly(a) length from approximately 60-70 to 100-150 nucleotides or by tail extension using enzymatic polyadenylation [174, [183] [184] [185] . however, effects were mostly moderate, but reached a maximum of a 35-fold increase in one particular setting. in contrast, another study reported an optimum of approximately 60 nucleotides; protein expression declined with further increasing poly(a) length [186] . in addition to poly(a) length, one report suggests a positive role of using a type iis restriction enzyme for dna template linearization to obtain a free poly(a) end rather than one extended with unrelated nucleotides [183] . further elements that can affect mrna translation and/or half-life are untranslated region (utr) sequences flanking fig. 4 schematic representation of different cap structures. a the typical 5′ cap of eukaryotic mrnas. a guanosine is methylated at position 7 and linked to the first nucleotide of the mrna by an unusual 5′ to 5′ triphosphate bridge. depending on the degree of methylation of the first two bases of the mrna, the full 5′ terminal structure is referred to as cap0, cap1 or cap2. the cleancap™ analog, a trinucleotide introducing a cap1 structure during ivt, is indicated in blue. b a plain cap0 analog (orange) is incorporated in two orientations during ivt. c inverse orientation can be avoided using anti-reverse cap analogs (arcas, highlighted in orange). such analogs are characterized by the presence of a methoxy group at either c2′ or c3′ of m 7 g. to improve resistance to decapping, a phosphorothioate was positioned in the 5′-5′ bridge of arca (β-s-arca) the orf sequence. trans-acting regulatory rna-binding proteins (rbps) interact with distinct rna sequence elements, thereby affecting ribosome recruitment and transit [187] . for instance, β globin 5′-and 3′-utrs, duplicating the β globin 3′-utr, the 5′-utr of tobacco etch virus, and a structure of the 5′-utr of human heat shock protein 70 all enhanced mrna translation in mammalian cells [158, 183, 188, 189] . according to a very recent survey of a combinatorial utr library, 5′-utr sequences appear to be most critical for protein expression [190] . in contrast, 3′-utrs seem to be the key driver for mrna half-life as exemplified by the stabilizing effects of the α globin 3′-utr as well as a duplication of the β globin 3′-utr [183, 191] . codon usage of the orf sequence also affects translation efficacy in many species. although in humans codon usage bias does not correlate with trna levels and gene expression [192, 193] , still increased protein expression from mrna upon codon usage optimization has been reported. for instance, codon usage adaptation of hiv-1 gag improved protein yield from mrna approximately 1.6-fold in a human t lymphocyte cell line [194] . a more pronounced increase in protein expression as a result of codon optimization was reported upon transfection of mrna encoding angiotensinconverting enzyme 2 into a549 and hepg2 cells [195] . however, alternatively to a direct effect of codon usage, the enhancement may be an indirect result of the use of modified nucleotides, the content of which was altered by codon optimization. furthermore, coding sequence engineering exploiting more advanced concepts like codon optimality may prove valuable in designing therapeutic mrnas of high efficacy. according to recent insights, codon usage may also affect fidelity of translation or the stability of transcripts [196] . in addition, orf as well as utr sequences can have an effect on immunostimulation and thus on translational activity. initially, researchers applied mrna containing only the four unmodified bases a, u, c, and g [158] [159] [160] 197] . in this context, for instance, u-rich sequences, as well as several rna structural features were described as immunostimulatory due to interactions with various rna sensors such as toll-like receptors (tlr), rig-i, and protein kinase r (pkr) [198] [199] [200] [201] [202] [203] [204] [205] . as a consequence, development of mrna therapies was hampered by the immunogenicity of in vitro transcribed mrna. the consideration of mrna for therapeutic purposes gained momentum by the finding that incorporation of modified bases into in vitro transcribed mrna reduced immunostimulation of such preparations. various modified bases found in natural rnas suppressed recognition by tlrs in vitro [206] . among these, particularly pseudouridine increased translation and stability of mrna [207] . in addition to the effect on tlr binding, replacement of uridine by pseudouridine affected binding to and activation of further sensors such as pkr and 2′-5′-oligoadenylate synthetase, contributing to higher and longer protein expression [208, 209] . however, the effects of mrna modifications on translation, immunostimulation and resulting protein expression appear to be variable, for instance dependent on the type of target cells. in vitro testing of various modifications and combinations thereof revealed decreased protein yield for any of them in a context dependent manner, while most of them reduced immunostimulation in raw124.7 macrophages [210] . as found in vitro, pseudouridine modification of mrnas reduced immunostimulation and increased level and duration of protein expression after intravenous (iv) or intraperitoneal administration of formulated mrna in mice [188, 207] . in contrast, another study on systemic administration of nanoparticle-complexed mrna concluded that neither immunostimulation nor protein expression benefited from pseudouridine modification [211] . after intradermal or intramuscular injection of formulated mrna, only n1-methyl-pseudouridine, but not pseudouridine, substantially enhanced expression [212] . in line, a different study applying lipid nanoparticle (lnp)-formulated mrna intradermally found that replacement of uridine with n1-methylpseudouridine resulted in much increased and longer lasting protein expression [213] . notably, recent studies revealed that also endogenous eukaryotic mrna harbors various modified nucleotides [214] [215] [216] [217] . however, the total level of modification is rather low which is in strong contrast to the usually 100% replacement of an unmodified nucleotide in ivt mrna. moreover, heavy nucleotide modification appears to interfere with the function of translation-enhancing rna elements such as utrs and internal ribosomal entry sites (ireses) [218] . at the time when modified nucleotides were introduced to minimize immunostimulation of ivt mrna, the importance of stringent purification of such preparations was recognized. chromatographic purification, particularly hplc, can separate mrna according to size, thereby removing smaller or larger by-products such as abortive transcripts, mrna from traces of non-linearized dna template or double-stranded rna (fig. 5) [219, 220] . such purification enhanced protein yield, most probably by enriching functional transcripts and depleting contaminants causing detrimental immunostimulation [220, 221] . the latter is corroborated by the finding that stringent purification reduced the beneficial effect of chemical modification or even made it dispensable, particularly in combination with specific sequence-engineering of the mrna [218, 220] . importantly, the specifics of mrna formulation are another layer that can influence activation of the innate immune system by masking mrna from recognition by sensors, particularly tlrs. for example, immune responses after mrna administration into the central nervous system were effectively suppressed by the use of a nanomicelle formulation compared to naked mrna [222] . after in vivo administration of mrna was proven to be feasible, the concept of using mrna as a basis for therapeutics was pursued almost immediately. the very first report on a therapeutic effect with exogenous mrna was already published in 1992 and described a temporary reversion of diabetes insipidus in rats by intrahypothalamic injection of vasopressin mrna [197] . thereafter, it took almost two decades until further studies started to demonstrate the broad potential of mrna-based protein therapies. meanwhile, there is a plethora of publications on a huge variety of indications comprising anemia [188, 218] , hemophilia [223, 224] , myocardial infarction [155, 225] , cancer [226, 227] , lung disease such as surfactant b deficiency and asthma [228] [229] [230] , metabolic disorders [231] [232] [233] [234] [235] , fibrosis [195] , skeletal degeneration [236] , tendon impairment [237] , and neurological disorders such as sensory nerve dysfunction, friedreich's ataxia and alzheimer's disease [238] [239] [240] . whereas evidence for the therapeutic potential of mrna is mostly restricted to mouse models, first data in swine indicate that mrna-based protein therapies are feasible also in large animals [218, 225] . in view of the various indications, it is hardly surprising that this diversity goes along with different routes of administration and various formulations. only very few studies looking at local administration used uncomplexed and thus unprotected mrna [225, 228, 230, 237] . the majority of investigations built on lipid-based formulations with a clear tendency to the application of lnps [223, 224, [231] [232] [233] 239] . most if not all groups purified their ivt mrna before in vivo administration. while some simply precipitated the mrna [195, 227, 236] , most used commercial purification kits. only a few researchers applied hplc purification [188, 218, 232] . with respect to the mrna, the vast majority of studies used long poly(a) tails of at least 100 nucleotides. likewise, there is a clear prevalence to chemically modified mrna, although various examples suggest that this is not mandatory [218, 223, 239, 240] . while most mrnas harbored 5-methyl-cytosine and/or pseudouridine initially [155, 188, 226] , there appears to be a trend towards the use of n1-methyl-pseudouridine at present [224, 225] . regarding the cap structure, almost all early studies cotranscriptionally generated cap0 mrnas using arca [226, 228, 229] . however, since about 2 years, research groups prefer to apply mrnas with a cap1 5′-end [223, 227, 231] . initial attempts to apply mrna to passive immunotherapy focused on cellular approaches for various reasons. adoptive transfer of ctls equipped with either an additional tcr or a car had shown great promise in cancers and viral infections. in contrast to typical scenarios of antibody therapy, receptor expression usually requires much lower protein levels. furthermore, t cells are loaded with receptor-encoding nucleic acid (dna or mrna) ex vivo. hence, passive cellular immunotherapy does not require sophisticated and highly efficient formulations for in vivo delivery but can build on the armamentarium of cell transfection methods. previous work on active cellular vaccination with antigen-presenting cells that had been transfected with antigen-encoding mrna revealed electroporation as easy and efficient means to load cells [241] . comparison to passive cell pulsing and lipofection demonstrated that electroporation was also most efficient for transfection of t lymphocytes [242] . rna electroporation had up to 90% efficiency without eliciting any critical toxicity [243] . onset of transgene expression was very rapid and lasted about 7 days [243] . receptor transfer into t cells by mrna electroporation has now been well established for many years [243, 244] . moreover, gmpcompliant protocols for manufacturing receptor-expressing retention time t cell preparations via mrna electroporation are available today [245, 246] . electroporation of human t lymphocytes with various antigen-specific tcrs redirected them to recognize cancer cells in an mhc-dependent manner in vitro [243] . mrnamediated tcr expression conferred in vitro cytotoxicity to t cells for at least 72 h [244] . the lytic efficacy of such cells was comparable to retrovirally transduced lymphocytes [244, 247] . likewise, transfection of car-encoding mrnas generated cells that were lytically active in vitro. using an optimized ivt mrna for a cd19-specific car, surface expression and cytotoxic function were detectable for up to 10 days [248] . to avoid as many manipulation steps as possible in generating t cells for adoptive transfer, it was demonstrated that human peripheral blood lymphocytes instead of purified t lymphocytes could be used as well to elicit strong cytotoxicity in vitro upon electroporation of a car mrna [249] . currently, most car approaches deploy αβ t cells. however, γδ t lymphocytes are an attractive target as well due to their antitumor effector function which is not mhc restricted and does not require co-stimulation. accordingly, mrna-mediated tcr and car expression in such cells was investigated very recently and shown to kill target cells in an antigen-specific manner [246] . to the best of our knowledge, there is so far just one study that started to systematically analyze the role of different mrna elements for receptor expression in t cells. to this end, the group of carl june built on previous findings in dendritic cells which revealed the superiority of a duplicated β globin 3′-utr over a single copy of the same element and of a long (120 nt) over a short (16-51 nt) poly(a) tail [183] . with respect to receptor expression, 150 as enhanced expression compared to 64 as [184] . a tandem repeat of the β globin 3′-utr had also a beneficial effect, particularly in combination with a long poly(a). in contrast, the vegf translational enhancer as 5′-utr element had even detrimental consequences. the authors speculated that this may be due to reduced capping efficacy but did not provide data corroborating their hypothesis. however, they demonstrated the important role of the cap structure. co-transcriptional cap0 using arca and an enzymatically generated cap1 structure were equivalent and outperformed the basic cap analog as well as an enzymatic cap0. besides expression level, capping also appeared to have an effect on the persistence of expression. modification of the orf sequence by removing all internal orfs had no effect on receptor production. t lymphocytes transfected with tcr-or car-encoding mrna proved to be functional also in vivo. robust antitumor effects were observed in various preclinical models [184, 249] . they were mrna-specific, since mock-transfected t cells had no or very little unspecific impact. although mrna-mediated receptor expression is transient, a single injection of car t cells against cd19 was sufficient to prolong survival of mice [248] . using peripheral blood lymphocytes instead of purified t cells for car mrna transfection (see above) also enabled a strong antitumor response in vivo although those cells could not persist long-term in vitro [249] . very recently it was shown that mrna cannot only be used to drive receptor expression, but can support the generation of t cells for adoptive immunotherapy. by expressing a chimeric membrane protein against cd3, cells could be efficiently stimulated and expanded in vitro [250] . after transfection with an mrna for an anti-cd19 bite, those cells mediated sustained reduction in tumor burden upon intraperitoneal injection. based on encouraging preclinical data, adoptive t cell therapies using mrna were already subjected to first clinical testing. in a phase 1 trial on solid tumors addressing the safety and feasibility of using such cells, car transfected lymphocytes migrated to tumor sites after iv injection [251] . in addition, the study appeared to provide initial evidence of antitumor activity. due to the transient nature of mrna expression, subjects received repeated infusions of t cells. this led to an anaphylactic response in one patient who developed antibodies specific to the scfv domain of the car [252] . however, this could be a consequence of the murine origin of this domain. in another trial, mrnatransfected car t cells were injected intratumorally in metastatic breast cancer patients [253] . treatment was well tolerated and elicited an inflammatory response within tumors. as discussed above, the use of viral vectors for adoptive t cell therapy has potential safety issues. in various studies, authors ascribed toxicities particularly to the persistence of receptor-expressing t cells. regarding such concerns, mrna-mediated tcr or car expression offers at least two advantages. first, mrna does not integrate into a cell's genome, thus excluding genotoxicity. second, due to its transient nature, any potential toxicities accompanying treatment are temporary as well [254] . however, the increased level of safety has a substantial drawback. apparently, clinical efficacy correlates with long-term persistence of receptor-engineered t cells [255, 256] . as a consequence, mrna-transfected cells are expected to have limited antitumor activity because of rapidly declining receptor expression. substantial mrna translation only lasts about 1 day which translates into efficacious receptor levels on the cell surface for several days [183, 184] . importantly, clinical studies demonstrated that iv infused t lymphocytes reached tumor sites only 2-3 days after administration [257, 258] . as a consequence, intratumoral or intraarterial administration was suggested to counteract the delayed cell arrival. the problem of transience of mrna is further enhanced by the well-known ligand-induced receptor internalization. upon target recognition, tcrs as well as cars are rapidly internalized, a mechanism which is important for proper signal transduction [259, 260] . this explains why lentiviral vectors generated a more robust treatment effect than mrna [184] . mrna transfection can give rise to high receptor expression, equaling lentiviral vectors [261] . however, mrna was only similarly effective during the first hours after electroporation. later, contact to target cells strongly down-regulated receptor on the cell surface while lentiviral expression remained constant [261] . in comparison to a single transfer of cells with retroviral car expression, an mrna-encoded receptor required three consecutive lymphocyte infusions to obtain a comparable antitumor effect [262] . these observations and considerations may be put into perspective at least in part by the finding that transferred t cells can become tolerized rather rapidly, thereby losing their ability to function in the tumor microenvironment [263] . thus, frequent injection of t cells may be desired even for viral vector transduced t cells. notably, mrna was also considered to be of use in settings that require long-term expression for therapeutic efficacy and preclude repeated cell administration. the greater safety of mrna-transfected t cells may make the initial testing of novel antigens and receptors with unknown on-target/off-tissue toxicity less hazardous [248] . passive immunization with antibodies often requires considerable amounts of polypeptide to obtain therapeutically active concentrations after systemic administration. this poses a substantial challenge to the broad applicability of any nucleic acid mediated passive immunization strategy. thus, compared to the very first attempts regarding in vivo protein expression with mrna in the 1990s, the optimization of ivt mrna to enhance and extend expression is a prerequisite for many mrna-based passive immunotherapies. as reviewed above, great progress towards this goal was made during the last almost three decades. another potential hurdle for passive mrna immunization is related to delivery. to obtain high levels of in vivo antibody expression, the mrna should be targeted to as many "producer" cells as possible which in turn should be transfected with high efficiency. to this end, the mrna which is prone to degradation by rnases should also be protected against these ubiquitous nucleases in an appropriate manner. moreover, a viable mrna complexation reagent needs to be well tolerated. notably, various commercial transfection reagents can be used to formulate mrna and suffice research purposes. among them, transit has been repeatedly used for in vivo studies [188, 218, 264] . with respect to potential therapeutic applications, the class of lipid nanoparticles (lnps) became the most widely deployed means of complexation [223, 224, [231] [232] [233] . after iv delivery, lnps mainly route to the liver on the basis of an apolipoprotein e (apoe)-dependent mechanism [265] . however, such nanoparticles were demonstrated to be also applicable to intramuscular and subcutaneous administration [266] . advances in mrna and formulation technology led to a couple of recent intriguing studies as to passive immunization with mrna (fig. 6) . while the group of drew weissman described successful passive mrna immunization for prophylaxis of viral infections [267] , stadler et al. demonstrated the applicability of mrna-mediated antibody expression for cancer immunotherapy [264] . the feasibility of using mrna for such indications was confirmed by thran et al. who applied different mrnaencoded antibody formats to diverse biological threats, viruses, toxins, and tumors [268] . finally, sabnis and colleagues presented first antibody expression data in nonhuman primates (nhps) in a publication dealing with the development of novel lnp formulations [269] . 6 schematic illustration of mrna-mediated passive antibody immunotherapy. for in vivo administration, mrna is usually formulated in nanoparticles which for instance can be administered by iv injection. for many formulations, liver is the main target organ. upon uptake of nanoparticles by hepatocytes and release of the mrna into the cytosol, it is translated into antibodies that are typically secreted into circulation and finally bind their cognate antigens mrna design and formulation fundamental designs of antibody-encoding mrnas reveal only a few common features but several differences (table 1) . among the latter, the exclusive use of chemically unmodified nucleotides by thran et al. as in a previous publication from the same group [218] may be the most prominent one, since it contrasts to all other reports. other differences are much more diverse among these studies. hence, they do not provide an unequivocal guidance for future work, but at least commonalities may be taken as a recommendation. although obviously not mandatory, mrna with cap1 structure was clearly preferred. in addition, all mrnas harbored a poly(a) tail. the use of bipartite poly(a) elements by some groups may be owed to the experience that maintenance of long poly(d[a/t]) vector sequences is challenging and strongly dependent on bacterial strains [186] . beyond these common rna elements, the publications suggest that mrna should be subjected to further optimizations to exploit its full potential for antibody expression. however, different strategies appear to be applicable, but little is known about the interchangeability of individual elements. finally, chromatographic purification of ivt mrna appears to be generally recommended as well (table 1) . whether pardi et al. actually used fplc as stated throughout their report instead of hplc applied by other groups is not fully clear, since they referred to earlier publications describing the use of hplc [220, 270] . pardi et al. encoded a well-known, broadly neutralizing antibody against hiv-1, vrc01 [267] . to this end, heavy and light chains of the full igg antibody were represented on separate mrna molecules. for delivery, heavy and light chain mrnas were mixed in a molar ratio of 1:1. likewise, thran et al. used separate mrnas to encode heavy and light chain of various full igg antibodies [268] . titration of heavy and light chain mrna found a molar ratio of approximately 1.5:1 to be optimal for co-delivery. neither report provides a rationale for encoding chains on separate molecules. in principle, a bicistronic construct separating heavy and light chain by an ires sequence or an mrna for a polypeptide where a 2a sequence between heavy and light chain would lead to separate antibody chains by ribosome skipping could have been used [271] . for pardi et al. the observation that modified nucleotides can hamper the function of ires elements may have affected the selection [218] . sabnis et al. also worked with a full igg antibody, directed against influenza a, but did not provide any details on how heavy and light chain were represented [269] . in contrast, stadler et al. chose bite antibodies directed against tcr-associated cd3 and one of three different tumor-associated antigens (taas) [264] . they displayed the bites as fab(scfv) 2 or scfv 2 molecules but focused on the latter format. their findings on single-chain antibodies are complemented by thran et al. whose work covers single domain-derived vnas in addition to igg antibodies [268] . for iv administration of antibodyencoding mrna all but the group of ugur sahin used lnp formulations which, however, may differ from each other in composition ( table 1 ). the latter team exploited transit but switched the route of administration which had been intraperitoneal in previous studies [188, 218] . as with lnps, nanoparticles were shown to mainly target the liver upon iv injection [264] . drew weissman's group administered 30 µg of vrc01encoding mrna in most in vivo studies [267] . this corresponded to doses between 1 and 1.4 mg/kg due to differences in mouse weight among experiments. antibody serum titers 24 h after administration, the earliest time of analysis, ranged between approximately 80 and 200 µg/ml in various mouse strains. obviously, slight differences in dosage, as well as the respective strain contributed to varying peak levels. notably, increasing the administered mrna dose in steps of two enhanced serum titers by more than twofold with each step. moreover, 30 µg of mrna in lnps generated higher serum titers than 600 µg of recombinant vrc01 protein. the kinetics of antibody serum titers revealed an accelerated decline after about a week in balb/c mice. the kinetics in nsg mice appeared to be basically the same, since the level at 1 week after single administration was largely the same as in balb/c at this time. the observed kinetics may also explain why weekly injections of mrna-lnps in nsg mice did not show additive effects on serum titers at the times of analyses. since measurements were conducted 7 days after each treatment, antibodies from the preceding injection probably dropped to background levels within this 2-week period as observed in balb/c animals. such accelerated decline of protein titers after a few days is often indicative of the induction of an anti-drug antibody (ada) response [139] . the likelihood of such a response may be particularly high for the reported experiments, since the authors expressed a human antibody in mice. the emergence of adas cannot be fully ruled out because animals were not analyzed accordingly. however, the apparently similar kinetics in immunocompromised nsg mice which are unable to develop adas suggests a different explanation for the pharmacokinetics. possibly, the mrna continues to express antibody for a few days which would inevitably lead to a seemingly extended antibody serum half-life during that period. only after expression ceases, the actual shorter antibody half-life becomes evident. while this could also easily explain the serum profile of repeated treatment of nsg mice, it remains hypothetical due to the lack of respective analyses. stadler et al. first characterized their mrna in vitro demonstrating expression and secretion of functional antibodies [264] . in a pbmc-mediated killing assay, mrna-derived bite antibodies targeted ctls to tumor cells via binding to cd3 on pbmcs and to the cognate taa on tumor cells, thereby inducing t cell activation and tumor cell lysis. these antibodies were equally potent as the corresponding recombinant protein. in immunodeficient nsg mice, antibody plasma levels peaked within 6 h, but rapidly declined by more than 50% within the next 18 h. subsequently, the decrease of bite titers became much slower. the authors did not provide an explanation of this striking kinetics. a pharmacokinetic analysis in non-tumor-bearing mice could have elucidated whether the initial kinetics reflects the trapping of antibody in the engrafted tumor until saturation of binding sites. bite plasma levels above background for a few days were in accordance with the sustained ex vivo cytotoxicity of plasma from mrna-treated mice. in contrast to the antibody plasma kinetics, cytotoxicity showed a steady and slow decline during the observation period. 0.05 µg of mrna were already sufficient to obtain strong plasma activity in the ex vivo killing assay. 5 µg of mrna (approx. 0.25 mg/kg) were comparable to 4-7 µg of recombinant antibody with respect to peak plasma concentrations that were in the range of 6.5 µg/ml in nsg mice. as opposed to this modified and hplc-purified mrna, antibody plasma levels were almost undetectable with mrna preparations without modification and chromatographic purification. plasma titers declined much faster for recombinant protein compared to mrna, thereby demonstrating the substantial impact of mrna on bite pharmacokinetics. consequently, only mrna was able to maintain a sustained cytotoxic activity of plasma by weekly administrations. the various mrna-encoded antibodies of thran et al. included vrc01 which had been used in drew weissman's work [267, 268] . however, analyses were limited to in vitro characterization, preventing a direct comparison between studies. as observed for bites, igg and vna antibodies produced from mrna in vitro revealed potencies comparable to that of the respective recombinant proteins. iv administration of 40 µg of unmodified mrna (approx. 2 mg/kg) gave rise to antibody serum titers between 15 and 400 µg/ ml in immunocompetent mice. this contrasts strongly with the finding of stadler et al. who found unmodified mrna to be basically inactive. differences in purification and mrna design may be responsible for this striking discrepancy. similar to the weissman work, thran and colleagues observed a disproportionate increase of antibody serum titers with elevated mrna doses. onset of antibody expression was rather rapid, being already substantial 2 h after injection and reaching peak levels after approximately 4 h. this confirms findings on other mrna-mediated protein therapies showing that mrna starts accumulating in hepatocytes within minutes after administration and leads to substantial protein levels within a couple of hours [223, 231] . serum half-life of igg antibodies appeared to be in the range of 1 week and thus slightly longer compared to pardi et al. [267] . as in the latter study, one of two iggs showed an accelerated decline after about 1 week, however, only in approximately half of the animals. here, the expedited clearance could be assigned to the development of an ada response against the mrna-encoded antibody. importantly, this response was antibody-dependent and not intimately linked with the use of mrna. as expected, vnas revealed a much shorter serum half-life of about 1-2 days. compared to published kinetics data on recombinant vnas, mrna appeared to contribute to extended antibody availability during the first days after administration as it has been observed for bite-expressing mrna by ugur sahin's group. however, the lack of a headto-head comparison hampers a detailed analysis. while previous in vivo studies on mrna-mediated antibody expression were limited to mice, sabnis et al. presented expression results in nhps using a proprietary lnp formulation [269] . a 0.3 mg/kg dose of mrna gave rise to antibody serum titers of about 4 µg/ml 24 h after iv administration which is at least at the lower end of the range of efficacy observed in mouse studies. however, data on a different protein suggest that efficacy of the formulation may be slightly lower in nhp than in mouse. whereas a 0.5 mg/ kg dose induced protein levels of approximately 7 µg/ml in mice, a 0.2 mg/kg dose generated protein titers between 200 and 800 ng/ml in nhps. all mouse studies on mrna-encoded antibodies investigated their therapeutic efficacy. pardi et al. used two different humanized mouse models to demonstrate that mrnaderived vrc01 protects from hiv-1 challenge [267] . mrna encoding a reporter protein was utilized as control. mrna-lnps were administered 24 h prior to challenge with one of two hiv-1 isolates. in the authors' first model, a vrc01 mrna dose of 0.35 mg/kg was ineffective, but 0.7 mg/kg already reduced viral rna copies in the plasma to undetectable levels as assessed by quantitative real-time polymerase chain reaction (qrt-pcr). the latter dose is well below the 10-20 mg/kg doses that are typically used for prophylactic immunization with recombinant antibody in humanized mice to reach therapeutic concentrations [272, 273] . however, the authors did not titrate the dose of recombinant vrc01 but used a 28 mg/kg dose as control which was sufficient to completely eradicate viral rna copies in the plasma. mrna efficacy could be also demonstrated in the second mouse challenge model. to show in vivo efficacy of mrna-mediated bite expression, stadler et al. implanted tumor cells subcutaneously in immunodeficient nsg mice [264] . about 1 week before mrna treatment, human pbmcs were engrafted into these animals. 3 µg of bite mrna (approx. 0.15 mg/ kg) given three times with an interval of 1 week could eliminate tumors entirely. in contrast, tumors progressed in control animals that received mrna encoding a reporter protein. the recombinant bite required three injections per week and a total of ten injections of 4-7 µg each to obtain a comparable antitumor effect as with bite mrna. the need for a more frequent administration corroborated the previous finding that mrna substantially improved antibody plasma half-life. due to the diversity of antibodies included in their study, thran et al. utilized various disease models for demonstrating therapeutic efficacy [268] . in contrast to all other studies, the authors applied mrna encoding irrelevant antibodies instead of a reporter protein as control. a 40 µg dose (approx. 2 mg/kg) of antibody mrna could protect mice from challenges with either rabies virus or botulinum toxin. in the intoxication model, mrna was proven to be equally protective as recombinant antibody. however, mice received approximately 0.1 mg/kg of recombinant vna compared to approximately 2 mg/kg of mrna. based on protein expression levels from mrna dose titrations, lower doses than 2 mg/kg may still confer full protection but this remains hypothetical, since the authors did not conduct an mrna dose titration in their challenge model. notably, mrna was effective in pre-as well as in post-exposure settings. the latter is important for some indications of passive immunization and confirms the aforementioned rapid onset of antibody expression. the post-exposure scenario for botulinum toxin requires very rapid availability of neutralizing antibodies. whereas recombinant protein can act immediately after administration, mrna needs more time to provide the antibody. hence, it may well be that in such instances higher doses of mrna than of protein are required, not for obtaining the same peak level but for reaching meaningful titers in a timely manner. in a further model, thran et al. evaluated their mrna approach with respect to anti-tumor efficacy. using a disseminated tumor model for rituximab, they showed efficient tumor growth control with injections of 50 µg (approx. 2.5 mg/kg) of rituximab mrna twice a week. higher doses (200 µg, approx. 10 mg/kg) of recombinant rituximab were less potent. this finding is reminiscent of results of drew weissman's group and contrasts those of ugur sahin and colleagues who required similar doses of mrna and recombinant protein (but less frequent dosing with mrna) to obtain equivalent therapeutic effects [264, 267] . notably, the difference among studies may be related to the use of igg antibodies on the one hand and a scfv 2 protein on the other hand. moreover, the irrelevant antibody control used by thran et al. appeared to have a slight unspecific anti-tumor effect. it may have contributed to the superiority of mrna compared to recombinant protein regarding dosing. amongst other explanations, the potential unspecific effect may be due to an mrna-lnp-independent response to repeated treatment or may be the consequence of a weak and transient cytokine response observed after mrna-lnp administration. however, the phenomenon was not investigated further. in line with previous reports, pardi et al. confirmed the importance of highly purified ivt mrna. in combination with lnps, only modification with n1-methyl-pseudouridine plus chromatographic purification was sufficient to avoid cytokine release by innate immune activation [267] . for this analysis, however, the authors deployed an mrna encoding a different protein than the vrc01 antibody which had been used for in vivo expression and efficacy experiments. tolerability of mrna-lnps was also addressed by repeated mrna treatments. translation of vrc01 mrna was not compromised over time, but the analysis was conducted in immunodeficient nsg mice. to overcome this caveat, the authors complemented their study by repetitive treatment of immune competent balb/c mice. to this end, they switched to an endogenous protein, since human vrc01 may be recognized as foreign and thus elicit an immune response. again, mrna injections did not lose efficacy over time. however, the authors also changed the formulation (transit instead of lnp) as well as the route of administration (intraperitoneal instead of iv) compared to the use of vrc01 mrna. hence, evidence for immune silence and overall tolerability of vrc01 mrna in lnps is just circumstantial yet. stadler et al. did not observe any liver toxicity upon treatment with mrna in transit according to liver enzyme analyses [264] . moreover, bite mrna administration did not elevate murine cytokines such as ifnα and tnfα above background in plasma. likewise, analysis of systemic human cytokine release from engrafted pbmcs did not show any unspecific t cell activation. as opposed to modified and hplc-purified mrna, preparations without nucleotide modification and chromatographic purification induced detectable levels of murine cytokines. similar to the weissman group, the authors also assessed the tolerability of formulated mrna by repeated injections. administrations did not lose efficacy over time, but as in the corresponding weissman experiment immunodeficient nsg mice were used. using chemically unmodified mrna formulated in lnps, thran et al. did not observe any liver toxicity in histopathological analyses [268] . only a few animals developed an ada response which was dependent on encoded antibody and was, thus, no intrinsic consequence of treatment with mrna-lnp. in addition, treatment appeared to elicit a transient weak cytokine release which, however, neither suppressed antibody expression nor induced adverse effects. since there are ample differences among studies on mrna-mediated antibody expression and no detailed analyses of the issue, the role of mrna, lnp, and/or encoded antibody/protein in cytokine induction remains elusive. an earlier study on erythropoietin showing the absence of any appreciable immunostimulation suggests that the use of chemically unmodified instead of modified mrna is not the decisive parameter [218] . quite a few in vivo studies provided compelling evidence for the principle feasibility of mrna-based immunotherapies. as discussed above, challenges and open questions regarding adoptive t cell transfer are less related to the mrna and its formulation or transfection but more of fundamental character. in contrast to ex vivo loading of cells, mrnamediated antibody expression is strongly affected by body size. thus, while there are now convincing efficacy data in diverse small rodent models, the translation to larger animals and finally humans has still to be demonstrated. first data suggest that substantial expression can be obtained in small nhps. however, the utilized lnps appeared to lose some efficacy when switching from mouse to nhp. hence, the development of human therapies may perhaps require further advancements of the mrna technology as well as primatespecific formulations with improved efficacy. in addition, tolerability of formulations has to be analyzed further and in more depth in the future. for instance, repeated dosing of nanoparticles can induce complement activation-related pseudoallergy (carpa) [274] . however, this can in principle be counteracted by optimization towards better biocompatibility. in case of lnps, fast degradation was shown to be particularly important [231, 232, 269] . while antibodies for cancer treatment were initially developed for iv administration, there is a trend towards subcutaneous injections today. for instance, rituximab was initially formulated for iv infusion which is typically administered over a period of 1.5-6 h [275] . this treatment schedule poses a substantial burden to patients as well as the healthcare system. thus, a formulation which reduces the time and required resources would be advantageous. to meet these goals by subcutaneous administration, the antibody solution was concentrated 12-fold [276, 277] . since this volume was still too large for subcutaneous injection, rituximab was co-formulated with human hyaluronidase which limits swelling and associated pain by increasing the dispersion and absorption of co-administered substances [276, 278] . now, median administration time for rituximab using the subcutaneous route is 6 min. as a consequence, antibody immunotherapy with mrna does not only require competitive efficacy and costs but also routes of administration to become a viable alternative to recombinant proteins. although other routes than iv have been shown to be possible for mrna, there are still a few open questions to be addressed by future studies. where are the advantages of using mrna for antibody immunotherapies? compared to dna it may be primarily the safety aspect. concerning recombinant proteins various points matter. as reviewed above, mrna provides benefits as to the pharmacokinetics when short-lived antibodies such as scfv, (bi)-scfv 2 or vna are used. moreover, solving the challenges of antibody cocktails may be easier using mrna. different mrna sequences are much more similar with respect to their physicochemical characteristics than different proteins are. hence, producing a 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acids how the messenger got its tail: addition of poly(a) in the nucleus formation of the 3′ end of histone mrna the cap and poly(a) tail function synergistically to regulate mrna translational efficiency the enzymes and control of eukaryotic mrna turnover concerted action of poly(a) nucleases and decapping enzyme in mammalian mrna turnover reverse 5′ caps in rnas made in vitro by phage rna polymerases synthesis and properties of mrnas containing the novel "anti-reverse" cap analogs 7-methyl(3′-o-methyl)gpppg and 7-methyl (3′-deoxy)gpppg novel "anti-reverse" cap analogs with superior translational properties effective delivery with enhanced translational activity synergistically accelerates mrna-based transfection mrna transfection of dendritic cells: synergistic effect of arca mrna capping with poly(a) chains in cis and in trans for a high protein expression level phosphorothioate cap analogs stabilize mrna and increase translational efficiency in mammalian cells phosphorothioate cap analogs increase stability and translational efficiency of rna vaccines in immature dendritic cells and induce superior immune responses in vivo modification of the 5′ end of mrna. association of rna triphosphatase with the rna guanylyltransferase-rna (guanine-7-)methyltransferase complex from vaccinia virus cap-specific mrna (nucleoside-o2′-)-methyltransferase and poly(a) polymerase stimulatory activities of vaccinia virus are mediated by a single protein a conserved histidine in the rna sensor rig-i controls immune tolerance to n1-2′o-methylated self rna inhibition of translation by ifit family members is determined by their ability to interact selectively with the 5′-terminal regions of cap0-, cap1-and 5′ppp-mrnas viral stressinducible protein p56 inhibits translation by blocking the interaction of eif3 with the ternary complex eif2.gtp.met-trnai mrna poly(a) tail, a 3′ enhancer of translational initiation modification of antigen-encoding rna increases stability, translational efficacy, and t-cell stimulatory capacity of dendritic cells multiple injections of electroporated autologous t cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor mrna with a < 20-nt poly(a) tail imparted by the poly(a)-limiting element is translated as efficiently in vivo as long poly(a) mrna optimized transfection of mrna transcribed from a d(a/t)100 tail-containing vector trans-acting translational regulatory rna binding proteins increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mrna encoding erythropoietin an element within the 5′ untranslated region of human hsp70 mrna which acts as a general enhancer of mrna translation optimization of mrna untranslated regions for improved expression of therapeutic mrna an mrna stability complex functions with poly(a)-binding protein to stabilize mrna in vitro codon usage and trna genes in eukaryotes: correlation of codon usage diversity with translation efficiency and with cgdinucleotide usage as assessed by multivariate analysis evolution of synonymous codon usage in metazoans quantitative effect of suboptimal codon usage on translational efficiency of mrna encoding hiv-1 gag in intact t cells translation of angiotensin-converting enzyme 2 upon liver-and lung-targeted delivery of optimized chemically modified mrna codon optimality, bias and usage in translation and mrna decay reversal of diabetes insipidus in brattleboro rats: intrahypothalamic injection of vasopressin mrna species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 innate antiviral responses by means of tlr7-mediated recognition of single-stranded rna 5′-triphosphate rna is the ligand for rig-i differential roles of mda5 and rig-i helicases in the recognition of rna viruses rig-i-mediated antiviral responses to single-stranded rna bearing 5′-phosphates rig-i detects viral genomic rna during negative-strand rna virus infection 5′-triphosphate-dependent activation of pkr by rnas with short stem-loops suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna incorporation of pseudouridine into mrna yields superior nonimmunogenic vector with increased translational capacity and biological stability incorporation of pseudouridine into mrna enhances translation by diminishing pkr activation nucleoside modifications in rna limit activation of 2′-5′-oligoadenylate synthetase and increase resistance to cleavage by rnase l screening of mrna chemical modification to maximize protein expression with reduced immunogenicity efficacy and immunogenicity of unmodified and pseudouridine-modified mrna delivered systemically with lipid nanoparticles in vivo n(1)-methylpseudouridine-incorporated mrna outperforms pseudouridine-incorporated mrna by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice nucleoside-modified mrna vaccines induce potent t follicular helper and germinal center b cell responses chemical and structural effects of base modifications in messenger rna chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome a mettl3-mettl14 complex mediates mammalian nuclear rna n6-adenosine methylation transcriptome-wide mapping reveals reversible and dynamic n(1)-methyladenosine methylome sequence-engineered mrna without chemical nucleoside modifications enables an effective protein therapy in large animals messenger rna-based vaccines generating the optimal mrna for therapy: hplc purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mrna spontaneous cellular uptake of exogenous messenger rna in vivo is nucleic acid-specific, saturable and ion dependent in vivo messenger rna introduction into the central nervous system using polyplex nanomicelle therapeutic efficacy in a hemophilia b model using a biosynthetic mrna liver depot system systemic delivery of factor ix messenger rna for protein replacement therapy biocompatible, purified vegf-a mrna improves cardiac function after intracardiac injection 1 week post-myocardial infarction in swine systemic delivery of modified mrna encoding herpes simplex virus 1 thymidine kinase for targeted cancer gene therapy exploring cytotoxic mrnas as a novel class of anti-cancer biotherapeutics expression of therapeutic proteins after delivery of chemically modified mrna in mice in vivo genome editing using nuclease-encoding mrna corrects sp-b deficiency modified foxp3 mrna protects against asthma through an il-10-dependent mechanism systemic messenger rna therapy as a treatment for methylmalonic acidemia targeted mrna therapy for ornithine transcarbamylase deficiency g6pc mrna therapy positively regulates fasting blood glucose and decreases liver abnormalities in a mouse model of glycogen storage disease 1a quantitative systems pharmacology model of hugt1a1-modrna encoding for the ugt1a1 enzyme to treat crigler-najjar syndrome type 1 mrna treatment produces sustained expression of enzymatically active human adamts13 in mice chemically modified rna induces osteogenesis of stem cells and human tissue explants as well as accelerates bone healing in rats tendon healing induced by chemically modified mrnas treatment of neurological disorders by introducing mrna in vivo using polyplex nanomicelles intrathecal delivery of frataxin mrna encapsulated in lipid nanoparticles to dorsal root ganglia as a potential therapeutic for friedreich's ataxia messenger rna-based therapeutics for brain diseases: an animal study for augmenting clearance of beta-amyloid by intracerebral administration of neprilysin mrna loaded in polyplex nanomicelles efficient genetic modification of murine dendritic cells by electroporation with mrna highly efficient gene delivery by mrna electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mrna and to electroporation of plasmid cdna for tumor antigen loading of dendritic cells high-efficiency transfection of primary human and mouse t lymphocytes using rna electroporation a new way to generate cytolytic tumor-specific t cells: electroporation of rna coding for a t cell receptor into t lymphocytes a gmp-compliant protocol to expand and transfect cancer patient t cells with mrna encoding a tumor-specific chimeric antigen receptor rna-transfection of gamma/delta t cells with a chimeric antigen receptor or an alpha/beta t-cell receptor: a safer alternative to genetically engineered alpha/beta t cells for the immunotherapy of melanoma transfer of mrna encoding recombinant immunoreceptors reprograms cd4 + and cd8 + t cells for use in the adoptive immunotherapy of cancer treatment of advanced leukemia in mice with mrna engineered t cells adoptive immunotherapy using human peripheral blood lymphocytes transferred with rna encoding her-2/neu-specific chimeric immune receptor in ovarian cancer xenograft model novel t cells with improved in vivo anti-tumor activity generated by rna electroporation mesothelin-specific chimeric antigen receptor mrna-engineered t cells induce anti-tumor activity in solid malignancies t cells expressing chimeric antigen receptors can cause anaphylaxis in humans ) safety and efficacy of intratumoral injections of chimeric antigen receptor (car) t cells in metastatic breast cancer nonviral rna transfection to transiently modify t cells with chimeric antigen receptors for adoptive therapy adoptive t cell therapy for cancer in the clinic adoptive cell transfer: a clinical path to effective cancer immunotherapy survival and tumor localization of adoptively transferred melan-a-specific t cells in melanoma patients phase i trial of adoptive immunotherapy with cytolytic t lymphocytes immunized against a tyrosinase epitope signal transduction and endocytosis: close encounters of many kinds endosomes: a legitimate platform for the signaling train redirecting t cells to ewing's sarcoma family of tumors by a chimeric nkg2d receptor expressed by lentiviral transduction or mrna transfection immunological quality and performance of tumor vessel-targeting car-t cells prepared by mrna-ep for clinical research interleukin-15 rescues tolerant cd8 + t cells for use in adoptive immunotherapy of established tumors elimination of large tumors in mice by mrna-encoded bispecific antibodies structure, activity and uptake mechanism of sirna-lipid nanoparticles with an asymmetric ionizable lipid expression kinetics of nucleoside-modified mrna delivered in lipid nanoparticles to mice by various routes administration of nucleoside-modified mrna encoding broadly neutralizing antibody protects humanized mice from hiv-1 challenge ) mrna mediates passive vaccination against infectious agents, toxins, and tumors a novel amino lipid series for mrna delivery: improved endosomal escape and sustained pharmacology and safety in non-human primates hplc purification of in vitro transcribed long rna comparison of ires and f2a-based locus-specific multicistronic expression in stable mouse lines hiv therapy by a combination of broadly neutralizing antibodies in humanized mice passive immunization with a human monoclonal antibody protects hu-pbl-scid mice against challenge by primary isolates of hiv-1 complement activation-related pseudoallergy: a stress reaction in blood triggered by nanomedicines and biologicals phase iii safety study of rituximab administered as a 90-minute infusion in patients with previously untreated diffuse large b-cell and follicular lymphoma subcutaneous administration of rituximab (mabthera) and trastuzumab (herceptin) using hyaluronidase non-clinical pharmacokinetic/pharmacodynamic and early clinical studies supporting development of a novel subcutaneous formulation for the monoclonal antibody rituximab a recombinant human enzyme for enhanced interstitial transport of therapeutics delivery of antibodies to the cytosol: debunking the myths intracellular and cell surface displayed single-chain diabodies phenotypic lentivirus screens to identify functional single domain antibodies construction and expression of a bispecific single-chain antibody that penetrates mutant p53 colon cancer cells and binds p53 barbas cf 3rd (2000) functional deletion of the ccr5 receptor by intracellular immunization produces cells that are refractory to ccr5-dependent hiv-1 infection and cell fusion a novel intracellular antibody against the e6 oncoprotein impairs growth of human papillomavirus 16-positive tumor cells in mouse models a human singlechain fv intrabody blocks aberrant cellular effects of overexpressed alpha-synuclein trapping prion protein in the endoplasmic reticulum impairs prpc maturation and prevents prpsc accumulation a cell-penetrating whole molecule antibody targeting intracellular hbx suppresses hepatitis b virus via trim21-dependent pathway delivery of macromolecules using arginine-rich cell-penetrating peptides: ways to overcome endosomal entrapment acknowledgements we thank mariola fotin-mleczek for discussion on the manuscript. we are grateful to nigel horscroft and michael stolz for critical reading of the review. we also thank bettina danker for her graphical illustrations. finally, we apologize to those authors whose work was not cited owing to space limitations. key: cord-222664-4qyrtzhu authors: coban, mathew; morrison, juliet; freeman, william d.; radisky, evette; roch, karine g. le; caulfield, thomas r. title: attacking covid-19 progression using multi-drug therapy for synergetic target engagement date: 2020-07-06 journal: nan doi: nan sha: doc_id: 222664 cord_uid: 4qyrtzhu covid-19 is a devastating respiratory and inflammatory illness caused by a new coronavirus that is rapidly spreading throughout the human population. over the past 6 months, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the virus responsible for covid-19, has already infected over 11.6 million (25% located in united states) and killed more than 540k people around the world. as we face one of the most challenging times in our recent history, there is an urgent need to identify drug candidates that can attack sars-cov-2 on multiple fronts. we have therefore initiated a computational dynamics drug pipeline using molecular modeling, structure simulation, docking and machine learning models to predict the inhibitory activity of several million compounds against two essential sars-cov-2 viral proteins and their host protein interactors; s/ace2, tmprss2, cathepsins l and k, and mpro to prevent binding, membrane fusion and replication of the virus, respectively. all together we generated an ensemble of structural conformations that increase high quality docking outcomes to screen over>6 million compounds including all fda-approved drugs, drugs under clinical trial (>3000) and an additional>30 million selected chemotypes from fragment libraries. our results yielded an initial set of 350 high value compounds from both new and fda-approved compounds that can now be tested experimentally in appropriate biological model systems. we anticipate that our results will initiate screening campaigns and accelerate the discovery of covid-19 treatments. covid-19 is a disease cause by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). it was identified in wuhan city, in the hubei province of china in december 2019 (chen et al., 2020; huang et al., 2020; zhu et al., 2020) . the virus is spread between people via small droplets produce by talking, sneezing and coughing. the disease was declared a global pandemic by the world health organization (who) on march 11th, 2020. while a large proportion of the cases results in mild symptoms such as fever, cough, fatigues, loss of smell and taste, as well as shortness of breath, some cases progress into more acute respiratory symptoms such as pneumonia, multiple-organ failure, septic shock and blood clots. these more severe symptoms can lead to death and are likely to be precipitated by a cytokine storm after infection and multiplication of the virus in humans. indeed, recent data indicate that the levels of il-6 correlate with respiratory and organ failures (gubernatorova et al., 2020) . so far, the estimated death rate of sars-cov-2 is above 1.3%, which is more than 10 times higher than the death rate of seasonal influenza (abdollahi et al., 2020) . older patients and patients who have serious underlying medical conditions such as hypertension, diabetes, and asthma are at higher risk for severe disease outcomes . a clear understanding of the genetics and molecular mechanisms controlling severe illness remains to be determined. sars-cov-2 is a positive-sense, single-stranded rna betacoronavirus, closely related to sars-cov-1, which caused severe acute respiratory syndrome (sars) in 2003, and middle east respiratory syndrome coronavirus (mers-cov), which caused mers in 2012. positive-strand rna viruses are a large fraction of known viruses including common pathogens such as rhinoviruses that cause common colds, as well as dengue virus, hepatitis c virus (hcv), west nile virus. the first genome sequence of sars-cov-2 was released in early january on the open access virological website (http://virological.org/) (zhou et al., 2020) . its genome is ~29.8 kb and possesses 14 open reading frames (orfs), encoding 27 proteins (wu et al., 2020a) . the genome contains four structural proteins: spike (s) glycoprotein, envelope (e) protein, membrane (m) protein, and nucleocapsid (n) protein. the e and m proteins form the viral envelope, while the n protein binds to the virus's rna genome. the spike glycoprotein is a key surface protein that interacts with cell surface receptor, angiotensinconverting enzyme 2 (ace2) mediating entrance of the virus into host cells (zhu et al., 2018) . in addition to its dependence on the binding of s to ace2, cell entry also requires priming of s by the host serine protease, transmembrane serine protease 2 (tmprss2). tmprss2 proteolytically processes s, promoting membrane fusion, cell invasion and viral uptake (heurich et al., 2014; hoffmann et al., 2020) . blocking viral entry by targeting s/ace2 interaction or tmprss2-mediated priming may constitute an effective treatment strategy for covid-19. the non-structural proteins, which include the main viral protease (nsp5 or m pro ) and rna polymerase (nsp12), regulate virus replication and assembly. they are expressed as two long polypeptides, pp1a and pp1ab, which are proteolytically processed by m pro . the key role of m pro in viral replication makes it a good therapeutic target as well. a third group of proteins are described as accessory proteins. this group is the least understood, but its members are thought to counteract host innate immunity (kim et al., 2020, cell 181, 914-921) (fig. 1a) . there is currently no treatment or vaccine available to prevent or treat covid-19 (baden and rubin, 2020; lurie et al., 2020) (https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19update-daily-roundup-june. while the fda has granted emergency use authorization (eua) for the 65-year-old antimalarial drug, hydroxychloroquine, covid-19 treatment based on early results from clinical trial in china and france gautret et al., 2020a; gautret et al., 2020b; million et al., 2020) , more recent results reported that hydroxychloroquine does not decrease viral replication, pneumonia or hospital mortality, and may in fact increase cardiac arrest in patients infected with covid-19 rosenberg et al., 2020) . the accuracy of the statistical analyses in these studies raised serious concerns in the scientific community. more accurate data are needed to reach a conclusion about the effect of hydroxychloroquine in covid-19 patients. in another recent study published in the new england journal of medicine, the antiviral remdesivir, an unapproved drug that was originally developed to fight ebola, seemed to improve patients with severe breathing problems (beigel et al., 2020) and has also recently been granted eua by the fda. repurposing drugs that are designed to treat other diseases is one of the quickest ways to find therapeutics to control the current pandemic. such drugs have already been tested for toxicity issues and can be granted eua by the fda to help doctors to treat covid-19 patients. another efficient way to attack the virus is to use drug cocktails to target multiple enzymes/pathways used by the virus. combination therapy has the advantage of being less likely to select for treatmentresistant viral mutants. such a strategy has been successfully used to treat hepatitis c virus (hcv) and human-immunodeficiency virus (hiv) infections. in the case of hcv, the treatment, enpclusa, combines sofosbuvir, which inhibits the viral rna-dependent rna polymerase (ns5b), and velpatasvir, a defective substrate that inhibits ns5a. antiretroviral therapy (art) against hiv combines drugs from different drug classes to target disparate aspects of the hiv replication cycle. these drug classes include nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors, ccr5 antagonists, post-attachment inhibitors, and integrase inhibitors. one example from hiv-aids literature is the randomized comparison of 4 groups of patients comparing monotherapy to combination therapies: zidovudine (zdv) monotherapy; zdv zidovudine and didanosine; zdv plus zalcitabine; or didanosine monotherapy. this randomized trial showed positive results when zdt was combined with didanosine or zalcitabine, and for didanosine compared to zdt monotherapy in raising cd4 counts greater than 50% (hammer et al., 1996) . combination therapy has become standard of care initial treatment in other infectious diseases such as mycobacterium tuberculosis and failure to cure with monotherapy and requires multidrug therapy (mdt) (collaborative group for the meta-analysis of individual patient data in et al., 2018) . similar mdt is also found effective in hepatitis c virus infection using glecaprevir and pibrentasivr combination therapies which lead to sustained virological response rates as far out as 12 weeks' post-treatment (wang et al., 2019) . we propose an effective combination therapy for covid-19 could target the sars-cov-2 replication cycle at multiple levels to synergistically inhibit viral spread and dissemination. using a computational pipeline that aimed to expeditiously identify lead compounds against covid-19, we combined compound library preparation, molecular modeling, and structure simulations to generate an ensemble of conformations and increase high quality docking outcomes against two essential sars-cov-2 viral proteins and their host protein interactions; s/ace2, tmprss2, cathepsin l and k, and m pro that are known to control both viral binding, entry and virus replication (fig. 1a) . our in silico approach (fig. 1b) , which will most likely lead into experimental virus screening, structural characterization of binding interactions by x-ray crystallography, and compound safety profiling. virtual screening (vs) is a rational driven controller for identification of new hits from compound libraries (willett, 2006) using either ligandbased (lbvs) or structure-based (sbvs) virtual screening (dror et al., 2004) . lbvs tactics use structural and biological data of known active compounds to select favorable candidates with biological activity from experiments (jahn et al., 2009; maldonado et al., 2006) . sbvs approaches, on the other hand, examine quantitative structure-activity relationships (qsar), clustering, pharmacophore and 3d shape matching (villoutreix et al., 2007) . the utility of vs is evident in the growth of our knowledge base of new compounds and existing drugs as well as the expansion of our structural databases. sbvs is generally the preferred approach when access to the target 3d-information derived from nmr, x-ray crystallography or homology models (jahn et al., 2009; maldonado et al., 2006) is possible. molecular docking (docking) is the most common sbvs approach used today (bottegoni et al., 2009; corbeil et al., 2012; fernandez-recio et al., 2005; friesner et al., 2006; mcgann, 2012; morris et al., 2009 ) and searches for the ideal position and orientation (called "pose") of the small molecule within a target's binding site, which gives a score for the pose. when including knowledge of experimentally known compounds ("actives") from a 3d target, lbvs and sbvs can be combined to increase likelihood of obtaining new actives from searches (kruger and evers, 2010) . hit identification in vs also requires careful selection of the methods used based on the goal of the project (e.g. compound databases and libraries can be either proprietary, commercial or public) (bender, 2010) . zinc is one such large public database often used in vs (irwin and shoichet, 2005) , which contains millions of compounds. by contrast, other libraries have structure-activity relationships (sar) databases (scior et al., 2007) that integrate information about compound interactions with their known targets. drugbank, chem-space are other attractive sources of compounds for drug repurposing (or repositioning) (ashburn and thor, 2004; duenas-gonzalez et al., 2008; o'connor and roth, 2005) (wishart et al., 2008) , and maintain drug diversity that is useful for scaffold development (gozalbes et al., 2008; schreiber, 2000) . advances in computing power have increased utility of in silico screening capabilities and balanced the need for accuracy with virtual high-throughput screening approximations and assumptions (anthony, 2009; lee et al., 2008; mcgaughey et al., 2007; plewczynski et al., 2009) , while recent techniques have improved accuracy without sacrificing cpu time (caulfield and devkota, 2012; caulfield et al., 2011; jiang et al., 2014; mackerell et al., 1998; phillips et al., 2005) (fig. 1b) . further innovations in docking methods have improved the exactness of empirical docking equations (corbeil et al., 2012; fernandez-recio et al., 2005; friesner et al., 2006; kalid et al., 2012; kruger and evers, 2010; mcgann, 2012) . accuracy is improved by incorporating molecular flexibility with simulations (caulfield, 2012; caulfield et al., 2019; caulfield and medina-franco, 2011; caulfield et al., 2011; caulfield et al., 2014; kayode et al., 2016) , thus capturing conformational information on structural changes that directly impact compound docking results. to target the covid19 problem on multiple fronts (e.g. ace2:s protein, tmprss2, m pro , and cathepsin l and k), as well as improve our screening accuracy using our selected repurposing libraries and new chemical entity libraries (zinc database), we implemented a novel method that integrates protein flexibility/shape, adaptive biasing algorithms, machine learning from drug data, and final z-score matrix weighting to our drug modeling. we matched all fda compounds with our realistic (x-ray derived) protein structures over a dynamic range of protein conformations with accelerated dynamics using our algorithms, such as maxwell's demon molecular dynamics (mdmd); this approach combines docking with simulations for exploration of both ligand and protein flexibility (caulfield, 2012; caulfield et al., 2019; caulfield and devkota, 2012; caulfield and medina-franco, 2011; caulfield et al., 2014; kayode et al., 2016; von roemeling et al., 2018) . we then refined the drug-target interface our specific leaderlike hit compounds using the quantum mechanics (qm)-based scoring within our mdmd matrix (caulfield, 2012) to make our go/no-go assessment, which is particularly useful with nces and de novo compound design (dcds). the protocol for library, structural modeling, dynamics, refinement, and hit identification as part of a pipeline is given (fig. 1b) . to improve our docking outcome, we constructed x-ray structure-based models of ace2 bound to sprotein, m pro , and tmprss2 in our molecular dynamics simulations (mds) and virtual screening (fig. 1b,s1 ). as s-protein interfaces with ace2 at a distinct region from the active site ( fig. s1a-d) , inhibition of the binding site by ligands may disrupt the ace2/s-protein interaction. canonical inhibitors of ace2 bind at the active site where angiotensin interacts, whereas drugs directed at the structural region for s-protein binding are not overlapping with the binding site. the modulation of ace2/s-protein interaction by canonical ace2 inhibitors is likely allosteric and suboptimal. therefore, directly targeting the interface of the interaction should increase efficacy of the approach and block covid viral binding, precluding entry (fig. s1) . additional investigation into the glycosylation sites of the s-protein demonstrated that the ace2 binding site is mostly unaffected by these additions (fig. s2) . a. s-protein:ace2 interaction (protein-protein inhibitor, ppi) requires dynamics to reveal binding site to get the optimal interface for drug screening, we used our grid searching algorithms, as well as site mapping and protein-protein docking, to examine the protein-protein interactions surface using mds ( fig. 2-3 ,s1) (bhachoo and beuming, 2017; caulfield and devkota, 2012; caulfield et al., 2011; caulfield and harvey, 2007; fernandez-recio et al., 2005; kozakov et al., 2006) . the protein-protein inhibitor (ppi) interaction complex did not identify any immediate binding site on the surface of the ppi interfaces. nevertheless, a small pore around one single beta-sheet in the center of the ppi interaction area could be exploited as a weak point that may perturb the interface equilibrium. using uniprot, which contains information about a number of confirmed mutations, we determined the relative potencies of ppi binding residues, identifying those that would likely affect the integrity of the complex (fig. 2) . residues k353 and y41, which interact with d155 at the center of the ppi, are likely stabilizing its surface, potentially forming a useful "hot spot" for targeted druggability ( fig. 2-3,s2) . to check whether this is true and to understand how ace2:s-protein cooperation functions, we performed two md simulations, one with and one without the mutation of y41a. this mutation causes strict inability to form the s-protein:ace2 complex. analysis of the trajectory of the wild-type protein, which possessed an intact complex, revealed the three most stable conformations of the "hot spot" region with expanded pores inside the triangle of residues k353, d155, y41. since it is impossible to determine which of these three conformations is the most stable, we ran three high-throughput screenings based on the donor-acceptor atoms and hydrophobic areas of the region. we then performed three md simulations with top pose ligands. as demonstrated in figure 3k , ligands failed binding within 10 ns, while docked ligands became leaders, as determined by energetic stability, during md and interaction energy values (electrostatic -red, van der waals -blue) ( fig. 3j/l) . to identify inhibitors of the s-protein:ace2 interaction via docking, we used the best scoring compounds obtained after combination of molecular docking and molecular dynamics simulations, which feeds into the pipeline for constraint-based screening. the high-throughput screening (hts) of a ppi library did not produce any results, since the ppi binding sites were weakly identified shallow regions (fig. 2,4a -d,s1). compounds that made good insertion into the sites situated between ace2 and s-protein were able to perturb the association of s-protein with ace2 via steric hindrance of s-protein association (fig. 3) . from the mds, we detected compounds that decreased energy of stability between the ace2:sprotein complex, which is desired in an inhibitor of protein-protein interaction. as a whole, this approach identified a deep and narrow binding site to disturb the s-protein interaction with ace2 (fig. 3,4a-d) . c. tmprss2 and m pro modeling requires dynamics to reveal optimal inhibitor binding to optimize the binding site of our inhibitors, we constructed a full-length (zymogen) model of tmprss2 (epitheliasinogen), as well as a mature version of the protease (epitheliasin), as described in our method section ( fig. 4e-g) . the mature protease model was used for mds studies to generate a reference dynamical profile that can be used to assist in silico screening of tmprss2 inhibitors. a control experiment was also completed with the uncleaved (non-catalytic) form of tmprss2 to demonstrate the pocket's instability and poor ligand binding capacity ( fig. s3 ) (ko et al., 2015; lucas et al., 2014; wilson et al., 2005) . a full-length model of monomeric m pro was also constructed, as well as a homodimer ( fig. 4h-k,s1 ). the structure derived from pdb code 6y2f with its ligand was used for a consensus virtual screen . in addition, we used the dimer to generate a reference dynamical profile to assist with in silico screening and study its interdomain behavior. we acquired the dimer protein sequence from the uniprot database. blast search showed the highest identification values against factor xi, prothrombin, kallikrein proteases (~41-42%). however, we focused on ligands that could be active against active form of tmprss2 protein. thus, we found the ligand: (2s)-1-[(2r)-2-(benzylsulfonylamino)-5-guanidino-pentanoyl]-n-[(4-carbamimidoylphenyl)methyl]pyrrolidine-2-carboxamide, contained within the chembldb repository (chembl1229259) and active against tmprss2, prothrombin, and factor xi. likewise, another docked model was recovered with macrocyclic ligand (chembl3699198), called: ethyl14-[[(e)-3[5-chloro-2-(tetrazol-1-yl) phenyl]prop-2-enoyl]amino] -5-(methoxycarbonylamino)-17-oxo-8,16 diazatricyclo[13.3.1.02,7] nonadeca-1(18),2(7), 3,5,15(19) -pentaene-9-carboxylate. we launched several molecular dynamics simulations (up to 75 ns of duration) to understand the interaction with the target protein-binding site. figure s3 shows the initial and stable/final states of our various models ( fig. 4e-g) . the md analysis provided useful results for selecting the appropriate model. after 15 ns md, the putative binding site collapsed (fig. s3,4e-g) . although the active form of thrombin was used for tmprss2 modeling, as a negative control we also examined the region with prothrombin-based binding site for completeness of the docking study (fig. s3) . the overlay of the average homology model structure from md and structure 3f68 (pdb code) was used as a template to compare protein-ligand interaction map and assign docking constraints (baum et al., 2009) . two optimal inhibitors for tmprss2 were selected for demonstration purpose in figure 5 . we also modeled cathepsins l and k for preliminary work, since these can be implicated in late-endosomal entry of the virus (fig. s4 ). for the viral main proteinase, m pro , a key enzyme for coronavirus replication (sars-cov-2), and a potential target for anti-sars drug development, several peptidomimetics synthetized in early 2012 against sars-cov-1 proteases were identified as selective. there is a high degree of sequence identity between the sars-cov-1 and sars-cov-2 m pro . this means that sars-focused ligands could form similar interaction map with m pro protein and offers good launching points for 3d-qsar/machine learning-drive based drug design for future iterations. to perform the virtual screening, protein structure was taken from the pdb code 7bqy complex and significant attention was paid to the interaction between the crystallized ligand from the complex and protein-binding site (jin et al., 2020) (fig. 6) . as the binding site is quite large (fig. 6a) we used a set of additional crystal structures (pdb code 6y2f and fragment-like compounds from https://www.diamond.ac.uk/) to narrow the source of possible conformations. the binding of the compounds inserted into this region demonstrated a very canonic and recurring interacting motif, represented with î±-keto amide group flanked with aliphatic or saturated rings. we then performed molecular dynamics of 75 ns for the ligand-free dimer structure of the m pro to evaluate and "catch" the most flexible elements of the binding site. our simulation revealed that the extended binding pocket was not very stable, unlike its individual sub-pocket, which contains active cysteine (c145) residue (fig. 6b,6c) . we began our molecular docking after assigning several combinations of constraints that should define specific interactions with the protein-binding site. we performed several high-throughput screening procedures using the same set of features in different combinations of constraints by partial matching algorithm ( fig. 6d-e) . we then ranged docking scores and compared obtained conformations inside the binding site with the co-crystalized ligands from 7bqy, 6y2f structures to select the most potent compounds. by disrupting the sars-cov-2 viral process in three different critical routes: binding, entry, and replication with our virtual screening approaches against dynamic structures, we were able to identify 350 compounds (dataset s1) and compile data reflecting physiochemical and chemoinformatic properties. an exemplar top hit from each target is summarized for docking score in table 1 . to classify the compounds and their chemical space, we completed various regression, k-means analyses and fingerprint measurements, and provide further details about their structures and properties, including commonly evaluated traits: mw, hba, hbd, docking score, rule of three (jorgensen), rule of five (lipinksi), logp o/w , and logs (dataset s2). we focused on new compound searches. the mw for these initial screening compounds ranges from large fragment (~250 da) to mature drug sized molecules (~500 da) with only 10 of the 310 top scoring compounds being over 500 da in size and the smallest fragment-based compound measured 178 da. overall the docking scores were very good with median around -7 kcal/mol using the glide xp calculations. we also generated a list of most commonly related drugs and discuss some of our best hits to known and clinical trial drugs (dataset s3). the general process for pruning the >30 million total chemical fragments and compounds from commercially available compounds for the initial round of virtual screening is described (fig. 1b) , which reduces the primary large set to 3 million per conformation of target. as an example, when examining some prototype compounds from our selected dataset of >300 nces screened from >10 million total compounds, we find the predicted interactions between drug and protein ( table s2 ) have some common binding modalities. when looking at the dynamical data for the drugs binding to the protein-protein site on ace2, we find the rmsd, rmsf, and h-bond occupancy evidence strong binding capability, as calculated from three separate simulations of ace2 with different ligands, referred to as 300, 392, and 488 ( fig. 4,s1) . these observations can be applied to generate constraints for additional virtual screening to improve the performance at higher throughput. based on these results, ligand 392 reduced the overall rmsd and per residue rmsf, while maintaining strong hydrogen bonds, as demonstrated by its greater occupancy during the simulation (table s1 ). this information, particularly h-bond occupancy and modulation of interface residue rmsfs, can be used in conjunction with docking and other data to profile the compounds more thoroughly (fig. 4) . in some cases, where constraints were utilized, the docking score underrepresents the compound and testing is needed to get important single-point data to clarify actives from non-actives, as well as determine the real ic50s for the selected active compounds. we will enrich our dataset with the top compounds for future rounds of parallel chemical screening and eventual de novo chemical design for novel chemical entities. current results of our approach are presented on all three targets (ace2, tmprss2, m pro ). for each of our targets, we screened for hits from a library of fda-approved compounds alongside the more extensive library of nces. our final result across all three targets identified a total of 350 specific compounds, with 167 against ace2, 40 against tmprss2, and 103 against m pro . among these are fdaapproved drugs that could be repurposed: 21 against ace2, 11 against tmprss2, and 8 against m pro (supplemental dataset tables1_topnce-fda-hits.xlsx). isoprenaline hydrochloride (isoprotenerol) is an adrenoreceptor agonist that can be repurposed as a vasopressor to augment cardiovascular function with a beta-receptor side benefit of bronchodilation to improve breathing function. metaraminol bitartrate, a stereoisomer of meta-hydroxynorephedrine, is a potent sympathomimetic amine to raise blood pressure. atenolol and nadolol are beta-receptor blocking agents used in chronic hypertension, a comorbid risk factor in covid-19 patients. propafenone is an anti-arrhythmic agent approved for patients with life-threatening ventricular tachycardia. levosulpiride is an atypical antipsychotic medication with prokinetic function that can be used in patients with agitated delirium, and gut immotility. valganciclovir hydrochloride is an antiviral agent used for cytomegalovirus (cmv), varicella zoster virus (vzv), and preventative medication in hiv patients (wu et al., 2020b) . recent data shows covid-19 deplete cd8 t helper cells similar to hiv (zheng et al., 2020) . amikacin sulfate and cephalexin are antibiotic anti-bacterial drugs that can treat bacterial super-infection. prochlorperazine dimaleate is a phenothiazine derivative prescribed in medicine for nausea. isoetharine mesylate is a selective adrenergic beta-2 agonist and fast-acting aerosolized bronchodilator for covid-19 respiratory distress. benserazide hydrochloride is an aromatic l-amino acid decarboxylase (dopa decarboxylase inhibitor) used with levodopa for the treatment of parkinsonism. glucosamine hydrochloride is constituent found in cartilage and used for osteoarthritis joint pains. s4701 or 2-deoxy-d-glucose (2d-dg) compound can induce ketogenic state, a powerful pathway involved in reducing systemic inflammation. inulin is a natural prebiotic agent that enhances gi function and digestion by increasing prebiotic gi homeostasis critical to stabilize downstream anti-inflammatory effects and prevent overgrowth of harmful bacteria. metaproterenol is a bronchodilator (beta-2 receptor agonist) that is commonly used to treat a variety of respiratory disorders including asthma, copd, bronchitis and wheezing associated with viral pneumonias in clinical practice. the novelty of this drug is that is aerosolized and can be given as a breathing treatment and similar reach the lungs, which have a tremendous surface area and enter the blood rapidly. by inhalation this drug acts rapidly and potentially with or in combination with other aerosolized drugs or oral or iv combination drugs. its inhalational route of delivery also can reach alveolar type ii cells which express ace2 for dual synergism. metaraminol bitartrate, a stereoisomer of meta-hydroxynorephedrine, is a potent sympathomimetic amine. this drug is used in patients with hypotension or low blood pressure. covid-19 hospitalized patients in the intensive care unit (icu) setting often need vasopressor agents to raise blood pressure in a condition called shock (dangerously low blood pressure) from covid-19 disease or sepsis. therefore, metaraminol has dual purpose of antiviral function at ace2 docking site /entry as well as helping with systemic blood pressure in those acutely ill covid-19 patients. this drug has immediate repurposing use in this patient population. atorvastatin is a statin drug with anti-inflammatory, immunomodulatory (diamantis et al., 2017) and endothelial benefits (ackermann et al., varga et al., 2020) . carbenicillin disodium is a penicillin derivative antibacterial antimicrobial agent. catechins are derived from plants with many beneficial properties in human health including anticancer, anti-obesity, antidiabetic, anti-cardiovascular, antiinfectious, hepatoprotective, and neuroprotective effects (isemura, 2019) . these substances fall outside fda purview since supplements and generally have a wide safety margin that will be tested on the multidrug platform. epicatechine s5105 is a naturally occurring flavonoid found in chocolate with anti-sarcopenic effects on skeletal muscle (gutierrez-salmean et al., 2014) . ivosidenib is an experimental drug for treatment of several forms of cancer. bezafibrate is a fibrate lipid-lowering drug, which creates a favorable anti-inflammatory ratio against cardiovascular diseases. pf299804 or dacomitinib is an egfr inhibitor used in cancer therapeutics. metaproterenol is a bronchodilator (beta-2 receptor agonist) that is commonly used to treat a variety of respiratory disorders with viral pneumonias in clinical practice. carbenicillin disodium is a penicillin derivative antibacterial antimicrobial agent that as mentioned above can be used in conjunction with other anti-sars-cov-2 agents to shut down antiviral effects and used in combination with those covid-19 patients with secondary super-infection with bacterial infection of lung, blood, or skin. bumetanide is a loop-diuretic used to remove extra fluid in the body (edema) such as pulmonary edema. aloin is an anthraquinone glycoside found naturally in aloe vera plants, a natural cathartic, and decreases 16s rrna sequencing of dysbiosis-producing butyrate producing bacterial species via an emodin breakdown product (gokulan et al., 2019) . emodin blocks ace2 and viral docking (ho et al., 2007) . salbutamol sulfate (albuterol) is a bronchodilator used in various breathing disorders. s4953 usnic acid is a naturally occurring dibenzofuran derivative found in lichen plant species, in some kombucha teas, with adrenergic function to raise blood pressure and potential bronchodilator. usnic acid is an active ingredient in some and a preservative in others and has a wide array of antimicrobial action against human and plant pathogens with antiviral, antiprotozoal, antiproliferative, antiinflammatory, and analgesic activity (ingolfsdottir, 2002) . avanafil is a class of medications called phosphodiesterase (pde) inhibitors, which are pulmonary artery and circulation dilators. s3612 rosmarinic acid is a naturally occurring compound found in plants (rosemary and sage), which has broad range of antimicrobial activity including antiviral activity including hiv (shekarchi et al., 2012) . ractopamine is a beta-agonist function used for bronchodilatation. neohesperidin dihydrochalcone (nhdc) is a naturally derived plant sweetener (bitter orange) with anti-tmprss2 effects. cidofovir and zidovudine (zdv) are both antiviral drugs used in hiv patients. there is a critical unmet patient need for therapeutics to treat the acute phase of covid-19 disease now and for the future. efforts to create and trial a vaccine are underway, but 11.6 million patients are confirmed infected globally (>540k deaths) with 25% infected within the united states and we are just at the midpoint of 2020. therefore, there is an urgent need to rapidly speed drug discovery from the bench to the bedside. in order to accelerate drug discovery, translation and human application, a design funnel using high-powered artificial intelligence is needed to screen millions of compounds against macromolecular mechanistic targets against the virus. at the back end of this funnel 40 drug candidates emerged, many of which may represent repurposing candidates for use in humans due to known safety and tolerability profiles. however, the approach with the highest probability of overall clinical therapeutic success may be not a single drug therapy for this viral rna disease but rather a multi-pronged drug approach gleaned from decades of hiv-aids epidemic research. a multidrug approach for hiv has improved survival, markedly reduced viral loads, and vastly improved management of the disease by preventing aids end-stage fatal complications. we therefore suggest that a multifaceted drug approach for sars-cov-2 may prove superior by attacking 3 viral entry and replication cycle sites simultaneously: ace2 receptor docking site and entry, tmprss2 endosomal packaging, and m pro viral replication. multiple drug targets for each of the 3 sites also allow permutations and optimization for combinatorial success. a recent study that screened commercially available >10,000 clinical-staged and fda-approved small molecules against sars-cov-2 in a cell-based assay (riva et al., 2020) identified interesting compounds for alternative targets that complement our results. these fda approved compounds included mdl-28170, a selective cathepsin b inhibitor; vby-825, a non-specific cathepsin b, l, s, v inhibitor; apilimod, an inhibitor of production of the interleukins il-12 and il-23; z-lvg-chn2, a tripeptide derivative inhibitor for cysteine proteinases; ono 5334, a selective cathepsin k inhibitor; and sl-11128, a polyamine analogs designed against e. cuniculi, a antimicrobial agents used as an adjuvant treatment for opportunistic aids-associated infections. overall these compounds are cathepsin-centric or antibiotic in nature, with little to no effect on our intended targets (tmprss2, ace2, m pro ). additional top hits identified by riva et al. include: amg-2674, an amgen compound inhibitor of trpv-1 (vanilloid receptor); sb-616234-a that possesses high affinity for human 5-ht1b receptors; sdz 62-434 that strongly inhibited various inflammatory responses induced by lipopolysaccharide (lps) or function-activating antibody to cd29; hafangchin a (also called "tetrandrine"), a bisbenzylisoquinoline alkaloid, which acts as a calcium channel blocker; elopiprazole an antipsychotic drug of the phenylpiperazine class (antagonist at dopamine d2 and d3 receptors and an agonist at serotonin1a receptors) that was never marketed; yh-1238, which inhibits dipeptidyl peptidase iv (dpp-iv) enzyme prolonging the action of the incretin hormones, glucagon-like peptide-1 (glp-1) and glucose-dependent insulinotropic polypeptide (gip); kw-8232, an anti-osteoporotic agent that can reduce the biosynthesis of pge2; astemizole, an antihistamine; n-tert-butyl isoquine (also called "gsk369796"), an antimalarial drug candidate; and remdesivir, a broad-spectrum antiviral medication developed by the biopharmaceutical company gilead sciences. again, none of these compounds were geared toward targeting tmprss2 or mpro, and are also not specific to ace2. while the lack of overlap may be surprising, results generated by riva and colleagues are not in opposition to our findings and both approaches can complement each other. most importantly, these approved fda compounds can be combined with our set of identified nce (310 compounds) that have been demonstrated to have low toxicity issues based on our chemoinformatics filtering (fig. 1b) . all nce compounds identified were chemical moieties that do not overlap any fda drugs. altogether, the data presented here complements previously generated data and should help prioritize and rapidly identify safe treatments for covid-19. future work will rely on advanced 3d-qsar, fragment-based drug design principles for de novo drug optimization. among millions of potential covid-19 drugs screened the majority of the final 40 drug candidates have known medical use and/or fda approval for a primary indication (e.g., hypertension, cardiac indication, hyperlipidemia) with well-established patient safety and tolerability profiles from large phase iii human trials and post-market (phase iv) analyses. these large human data provide both a clinically significant and scientifically innovative window of opportunity to test 40 compounds on the multidrug platform, and, in conjunction, observe longitudinal human survival outcomes of covid-19 patients on these drugs for comparative effectiveness within established and ongoing patient registries. an emerging example of this important parallel is ace2 pathway drugs (ace inhibitors [acei] and angiotensin receptor blocking drugs [arb]), which are increasingly observed in humans with covid-19 to be associated with improved survival advantage (jarcho et al., 2020; mancia et al., 2020; mehta et al., 2020; patel and verma, 2020; vaduganathan et al., 2020) . however, there is a scientific knowledge gap within human registries data regarding a scientifically robust and testable translational platform to test mechanistic effects of these different molecular compounds. therefore, creation of a "pandemic platform" using newer technology of compound ai drug throughput screening combined with animal multi-drug screening models creates an early phase i/ii safety, tolerability and early efficacy platform which is rapidly needed to expedite bedside human use for covid-19 pandemic, and as a platform that can be used in future pandemics. a flurry of activity to identify compounds for sars cov-2 targets has been underway by academic labs globally. here in our approach we introduce our novel maxwell's demon molecular dynamics method for screening flexibility required to get rare and essential conformational transitions and pathways to find the most likely druggable state. we also used our quantum docking technique (qm-driven adaptive molecular dynamics scanning docking) (caulfield, 2012) to identify compounds effective for targeting ace2, tmprss2 and m pro . the compounds identified by our large-scale in silico platform can next be experimentally validated as binders for intended targets and for efficacy in models of the disease, evaluated for ec50/safety-toxicity data, and carried into hit-to-lead and lead optimization in a drug development pipeline. structural studies such as x-ray crystallography will also be important to generate structural sar data for these efforts. in sum, our leading edge in silico methods incorporating structural dynamics have produced a set of 350 candidate compounds suitable for screening in biological disease models. among these, 40 fdaapproved compounds are eligible for rapid clinical trial testing. additionally, our results bring forward 310 nces predicted to possess potency and specificity for viral or human accessory target proteins to lower the viral load. moreover, this resource offers the community a set of chemical tools to probe the behavior of these enzymes essential for sars-cov-2 progression, namely, binding, entry and replication. as sars-cov-2 is already endemic, the rapid identification of effective antivirals remains a paramount focus until we have an efficient vaccine to provide long-lasting protection. in general, coot was used for building in missing residues and regularizing geometry (emsley and cowtan, 2004; emsley et al., 2010) . more details for the preparation of each model are given in the respective subsections. since these structures were all used in downstream computational studies, a uniform structural preparation was implemented. the full-length structures are comprised of all residues and side chains. we added missing atoms in rotamers and de-clashed atoms, added missing residues for chain continuity, and removed extraneous molecules/atoms (e.g. artifacts of crystallography or alternative conformations of residues were removed (keeping the highest occupancy)), and the bfactors were set to isotropic. the pdbepisa server was used to data mine the interface between ace2 and s-protein (krissinel and henrick, 2007) . surface interactions data is provided (supplemental). calculations on molecular dynamics trajectories including rmsd, rmsf, and h-bonds were performed using vmd and internal tools thereof (rmsd trajectory tool and tk console). prior to calculations, the backbone (concî±) atoms of each frame of the trajectories were aligned to the first frame as a reference, to remove the effect of random rotation/translation. after alignment, the per residue average of rmsf or rmsd per frame in ã� across the entire mds trajectory is given. for the ace2-ligand simulations, the number of hydrogen bonds between the protein and ligand were recorded for each frame, and the occupancy of each specific h-bond is defined as the percentage of frames the bond is present. rmsd, rmsf, and h-bond data were plotted in 2d format in excel. the rmsf was also appended to the beta column of the pdb and heat-mapped to the structure using a custom tcl/tk script and pymol. all molecular graphics were generated in pymol (mooers, 2016) . molecular dynamics and monte carlo simulations were performed on the protein to allow local regional changes for full-length structure for all acids of each structure. the x-ray refinement for monte carlo was built using yasara ssp/pssm method (altschul et al., 1997; hooft et al., 1996a; hooft et al., 1996b; king and sternberg, 1996; krieger et al., 2009; qiu and elber, 2006) . the structure was relaxed to the yasara/amber force field using knowledge-based potentials within yasara. the side chains and rotamers were adjusted with knowledge-based potentials, simulated annealing with explicit solvent, and small equilibration simulations using yasara's refinement protocol (laskowski ra, 1993) . the entire full-length structure was modeled, filling in any gaps or unresolved portions from the x-ray. refinement of the finalized models was completed using either schrodinger's lc-mod monte carlo-based module or namd2 protocols. these refinements started with yasara generated initial refinement of tmprss2 (altschul et al., 1997; hooft et al., 1996a; hooft et al., 1996b; krieger et al., 2009) . the superposition and subsequent refinement of each protein regions yields a complete model. the final structures were subjected to energy optimization with pr conjugate gradient with an rdependent dielectric. atom consistency was checked for all amino acids of the full-length wild-type structure, verifying correctness of chain name, dihedrals, angles, torsions, non-bonds, electrostatics, atom typing, and parameters. model was exported to the following formats: maestro (mae), yasara (pdb). model manipulation was done with maestro (macromodel, version 9.8, schrodinger, llc, new york, ny, 2010), or visual molecular dynamics (vmd) (humphrey et al., 1996) . mds and mc searching were completed on each model for conformational sampling, using methods previously described in the literature (caulfield and devkota, 2012; caulfield and medina-franco, 2011; caulfield, 2011; caulfield et al., 2011) . briefly, each protein system was minimized with relaxed restraints using either steepest descent or conjugate gradient pr, then allowed to undergo the mc search criteria, as shown in the literature (caulfield and devkota, 2012; caulfield and medina-franco, 2011; caulfield, 2011; caulfield et al., 2011) . the primary purpose of mc, in this scenario, is examining any conformational variability that may occur with each protein. for ace2/s-protein, pdb code 6vw1 was used to construct the model (shang et al., 2020) . while the structure was mostly complete, chain f (s-protein) was missing more residues, though it had residue ala522. chain e (s-protein) was only missing residue 522. residue ala522 was built into chain e using coot and where the extraneous molecules (solvent/cryoprotectant) and chains were deleted to leave only the heterodimer ace2/s-protein, which was processed to be used for computational studies, not to generate a de novo model or complete structure with missing atoms and sections. all information about the protein was found on the corresponding uniprot page. after identifying the hot spot residues using sitemap or protein-protein interfaces, we used md to find out how the y41a mutation can affect of ppi inhibition. we performed md for wild type and mutated protein. residual mutation was also performed using pymol's built-in tools. gromacs 2018 and amber99 force field were used to conduct md and further analysis of the results (baugh et al., 2011; dilip et al., 2016; janson et al., 2017; kazmierkiewicz, 2013, 2016; mooers, 2016) . visual inspection of every 10 frames allowed us to determine some tendency of structural deformation in a certain place on the protein surface. according to the literature data and our finding, we focused on the predicted binding site. then, each trajectory was analyzed via the built-in clustering tool based on the rmsd distribution. three the most stable conformations of the binding site were chosen for the docking studies. all received docking poses from each docking study were evaluated based on the docking scores, interaction diagrams and solvent exposure. to make some prediction regarding the binding method, we carried out another molecular dynamics simulation for the upper poses of each docking. after such a confirmation of our assumptions, we selected the most powerful and accurate compounds from the results of docking. a homology model was constructed on the basis of prothrombin crystal structure in complex with the ligand analog (pdb code 3f68) (baum et al., 2009) . we modeled the 492 amino acid tmprss2 protein two different ways: yasara based and swissmodel server based (krieger et al., 2002; waterhouse et al., 2018; zoete et al., 2011) . first, the yasara based model begins with the fasta sequence: malnsgsppaigpyyenhgyqpenpypaqptvvptvyevhpaqyypspvpqyaprvltqasnpvvct qpkspsgtvctsktkkalcitltlgtflvgaalaagllwkfmgskcsnsgiecdssgtcinpsnwcdg vshcpggedenrcvrlygpnfilqvyssqrkswhpvcqddwnenygraacrdmgyknnfyssqgi vddsgstsfmklntsagnvdiykklyhsdacsskavvslrciacgvnlnssrqsrivggesalpgawp wqvslhvqnvhvcggsiitpewivtaahcvekplnnpwhwtafagilrqsfmfygagyqvekvishpn ydsktknndialmklqkpltfndlvkpvclpnpgmmlqpeqlcwisgwgateekgktsevlnaakvll ietqrcnsryvydnlitpamicagflqgnvdscqgdsggplvtsknniwwligdtswgsgcakayrp gvygnvmvftdwiyrqmradg. topological domains have the following characteristics: residues 1 -84 forms the cytoplasmic sequence; residues 85 -105 form the transmembrane domain region (helical 21 aa); and residues 106 -492 form the signal-anchor for type ii membrane protein (extracellular), where the protein as two main chains: non-catalytic chain (met1-arg225) and catalytic chain (ile256-gly492), where each domain modeled as a separate unit built together in composite. disulfide bonds exist between several residues (113 â�� 126), (120 â�� 139), (133 â�� 148), (172 â�� 231), (185 â�� 241), (244 â�� 365), (281 â�� 297), (410 â�� 426), (437 â�� 465), which can be informative for building the structure. glycosylation sites are also possible at residues n213 and n249. cleavage site (active) exists between arg255 and ile256 (see refinement section). the second method, homological modeling was performed using the swissmodel server after performing a blast search on available protein structures in the rcsb database. molecular dynamics simulations of 100 ns of both, suggested and re-modeled protein structures, was performed with gromacs 2018 kazmierkiewicz, 2013, 2016) . based on the structural analysis and the generated connolly surfaces, we identified critical changes in the binding site of the proposed model and began creating a mesh for the binding site of the new homology model. since our model was based on the structure of thrombin, we used its co-crystallized ligand as a template for assigning constraints and ensured we built the catalytically active state. for m pro (pdb 6y2f) co-crystallization with tert-butyl (1-((s)-1-(((s)-4-(benzylamino)-3,4-dioxo-1-((s)-2oxopyrrolidin-3-yl)butan-2-yl)amino)-3-cyclopropyl-1-oxopropan-2-yl)-2-oxo-1,2-dihydropyridin-3yl)carbamate (also referred to as alpha-ketoamide 13b) was used; the structure was also mostly complete. residues e47 and d48 were built in using coot, where the other preparations previously described were also performed. to build the missing residues, the coordinates and structure factors were downloaded, generated 2mfo-dfc and fem maps, and real space refine zone/regularize zone were used to fit to electron density and optimize local geometry. the ligand (alpha-ketoamide 13b) was left for usage as a cognate ligand for virtual screening. the protein structure was initially studied using md to find out if the binding site is cruel enough or can break down without a ligand molecule during the simulation. simulation of the dimeric complex for 100 ns was sufficient to compare conformational changes from different md states. a set of positional and hydrogen bonds were assigned based on the available peptidomimetic structure. thus, two screenings were conducted with an emphasis on positional constraints or interactions of hydrogen bonds. using mds and mc refinement with schrodinger and/or yasara ssp/pssm methods (altschul et al., 1997; hooft et al., 1996a; hooft et al., 1996b; king and sternberg, 1996; krieger et al., 2009; qiu and elber, 2006) , each structure was relaxed to the yasara/amber force field using knowledge-based potentials within yasara. the side chains and rotamers were adjusted with knowledge-based potentials, simulated annealing with explicit solvent, and small equilibration simulations using yasara's refinement protocol (laskowski ra, 1993) . the entire full-length structure was modeled, filling in any gaps or unresolved portions from the x-ray structure. refinement of the finalized models was completed using either schrodinger's monte carlobased module or in-house protocols. these refinements started with generated initial refinement for each independent structure (altschul et al., 1997; hooft et al., 1996a; hooft et al., 1996b; krieger et al., 2009) . the superposition and subsequent refinement of the overlapping regions yields a complete model for all four proteins. the final structures were subjected to energy optimization with pr conjugate gradient with an r-dependent dielectric. atom consistency was checked for all amino acids (and atoms) of the full-length wild-type model, verifying correctness of chain name, dihedrals, angles, torsions, non-bonds, electrostatics, atom typing, and parameters. a multimeric-complex model is predicted, including cofactors and ions. all of the models were exported in the following formats maestro (mae), yasara (pdb). model manipulation was done with maestro (macromodel, version 9.8, schrodinger, llc, new york, ny, 2010), or visual molecular dynamics (vmd) (humphrey et al., 1996) . analyses were emphasized on the protein-protein interaction regions containing. monte carlo dynamics searching (mc-search) was completed on each model for additional conformational sampling, using methods previously described in the literature (caulfield and devkota, 2012; caulfield, 2011; caulfield et al., 2011) . briefly, each protein system was minimized with relaxed restraints using either steepest descent or conjugate gradient pr, then allowed to undergo the mc search criteria, as shown in the literature (caulfield and devkota, 2012; caulfield, 2011; caulfield et al., 2011) . the primary purpose of mc, in this scenario, is examining any conformational variability that may occur with different orientations in the region near to protein-protein interfaces. the total atomic force field was used to minimize the energy of the system, namely, the descent algorithm for 20,000 steps with an iteration interval of 2 fs. the equilibrium of the solvent was carried out using positional restrictions imposed on the atoms of protein structures, while the solvent molecules remained mobile for all 100 ps. each system was placed in a box in which the layer of the tip3p water molecule was 10 ã�. the final systems were neutralized by the addition of na + and cl-ions to a concentration of 150 mm. all simulations were performed under periodic boundary conditions using the v-rescale thermostat algorithm to maintain temperature (310 k) and the parrinello-rahman barostat algorithm for constant pressure (1 bar) (bussi et al., 2007; parrinello and rahman, 1981) . long-range unrelated interactions were calculated using the particle-mesh-ewald (pme) method (abraham and gready, 2011). all molecules were relaxed with a molecular dynamics simulation of 100 ns. ligand topologies were created using the antechamber module from the ambertools18 package (case et al., 2005) . we used sitemapper (bhachoo and beuming, 2017) to identify possible binding sites for docking affinity with the proteins ace2 (allosteric site), tmprss2, and m pro . we also used our novel mds biasing technique algorithm, maxwell's demon md, for searching within these sites for potential flexible zones that would have beneficial peptide interactions, which served as a reductive filter limiting the total number of possible sites screened on the proteins to those with adequately deep binding grooves (caulfield, 2011; kayode et al., 2016) or interesting insertion sites (ace2). prior to the docking with the ace2 (allosteric site), tmprss2, and m pro , we had completed rigorous molecular dynamics simulations (mds) and monte carlo (mc) conformational searching for each model for additional conformational sampling, using methods previously described in the literature (caulfield and devkota, 2012; caulfield, 2011; caulfield et al., 2011) . the primary purpose of mc, in this scenario, is examining any conformational variability that may occur with different orientations in the region near to protein-protein interfaces. over three million compounds were docked to each site using the glide xp docking program (bhachoo and beuming, 2017). all compounds were accounted for using opls3 within maestro program (maestro-9.4, 2014). using our published docking protocols on each identified site, we reductively scanned from 100s to the top 10 poses from each docking and then did cross-comparisons of docking scores to retain only the top binding pose of each compound from each site in a winnertakes-all strategy. each compound has been converted into a set of energy minimized three-dimensional shapes with the ligprep module. without protein preparation, it was used for the correct distribution of protonation and post-minimization in the opls3 force field. in the case of assigning restrictions based on ligands (m pro , tmprss2), we tried to cover the most important and strong interactions. in the case of ace2, a set of constraints was generated in sufficient quantities to generate combinations of possible interactions. positional constrains (1.8 a radius) and h-bond constraints were generated in the schrodinger glide module, namely in the mesh generation tool. aromatic and hydrophobic features were represented with short smarts. a partial matching protocol for applying constraints has also been used to improve process accuracy. a high throughput screening protocol with regulated ligand flexibility was applied. each compound has been converted into a set of energy minimized three-dimensional shapes with the ligprep module. without protein preparation, it was used for the correct distribution of protonation and post-minimization in the opls3 force field. in the case of assigning restrictions based on ligands (m pro , tmprss2), we tried to cover the most important and strong interactions. in the case of ace2, a set of constraints was generated in sufficient quantities to generate combinations of possible interactions. positional constrains (1.8 a radius) and h-bond constraints were generated in the schrodinger glide module, namely in the mesh generation tool. aromatic and hydrophobic features were represented with short smarts. a partial matching protocol for applying constraints has also been used to improve process accuracy. a high throughput screening protocol with regulated ligand flexibility was applied. conformations of compound orientations were generated using our standard protocols (bhachoo and beuming, 2017; kalid et al., 2012; unger et al., 2015) . the starting conformation of relaxed protein structures was first obtained by the method of polak-ribiã¨re conjugate gradient (prcg) energy minimization with the optimized potentials for liquid simulations (opls) 2005 force field (jorgensen, 2004; jorgensen and tiradorives, 1988) for 5000 steps, or until the energy difference between subsequent structures was less than 0.001 kj/mol-ã� units. our docking methodology has been described previously (caulfield and devkota, 2012; friesner et al., 2006; loving et al., 2009; vivoli et al., 2012) . briefly, compounds were docked within the schrã¶dinger software suite (mohamadi et al., 1990 ) using a virtual screening workflow (vsw) (bhachoo and beuming, 2017; friesner et al., 2006; jacobson et al., 2002; kalid et al., 2012; kozakov et al., 2006) . alternative docking methods were also employed, including in-house software techniques for top leads for sar elucidation. the top seeded poses were ranked and unfavorable scoring poses were discarded. top favorable scores from initial dockings yielded hundreds of poses with the top five poses retained. molecular interactions of the ligand-protein interfaces were used to help determine the optimal binding set, which included descriptors were used to obtain atomic energy terms like hydrogen bond interaction, electrostatic interaction, hydrophobic enclosure and ï�-ï� stacking interaction that result during the docking run. molecular modeling for importing and refining the proteins was completed (maestro-9.4, 2014). examinations of structure stability were examined for all proteins investigated, s-protein:ace2, tmprss2, and m pro , respectively (caulfield and devkota, 2012; caulfield and medina-franco, 2011; caulfield, 2011; reumers et al., 2005; schymkowitz et al., 2005; zhang et al., 2013) . object stability was used to determine if any changes in structure that were deleterious to function from immediate inspection, which the foldx algorithm can provide, prior to docking studies. thus, we examined the local residues around the docking site and determined an electrostatic calculation may be useful to explain the change in function. the molecular model for the full structure and its truncated form are given (fig. s1 ) using our state of the art methods, which have been established (abdul-hay et al., 2013; ando et al., 2017; caulfield and devkota, 2012; caulfield and medina-franco, 2011; caulfield, 2011; caulfield et al., 2011; caulfield et al., 2014; caulfield et al., 2015; fiesel et al., 2015a; fiesel et al., 2015b; puschmann et al., 2017; zhang et al., 2013) . local residues within the 12ã� cutoff near docking sites were analyzed ( fig. s1-s2) . any interactions requiring inducible fit, or threonine/serine hydroxyl rotation or other docking parameter (ï�stacking/halogen-directionality) were also included. mapping electrostatics was accomplished using the poisson-boltzmann calculation for solvation on all amino acids for each docked structure (caulfield and devkota, 2012; caulfield and medina-franco, 2011; caulfield, 2011; reumers et al., 2005; schymkowitz et al., 2005; zhang et al., 2013) e. libraries used compounds were derived from either a set of all fda approved and clinical tested compounds, bioactive set of compounds, or a large multi-million compound set from zinc database. in the all cases the libraries were prepared using ligprep described above. the zinc database was pruned using parameters for better drug-like profile and removal of reactive functional groups and poor chemoinformatics properties delivering a large set suitable for screening on all targets across dynamic time points from mds. supplemental information can be found online at: xxx acknowledgements author contributions t.c. designed and conducted most experiments, analyzed data, and wrote the manuscript with inputs from j.m., k.l., m.c. and e.r.; t.c. and m.c. performed mds, docking, and generated analyses; t.c. and m.c. performed post simulation analyses; c.b. and m.c. performed bioinformatics analysis; t.c. supervised m.c.; t.c. provided expertise on data analysis; e.r. provided expertise and insight interpreting experimental structures and homology models; and t.c. proposed the project to k.l., whom helped with formatting, detailing analysis and edited the manuscript. the authors declare no competing interest. zhu, z., zhang, z., chen, w., cai, z., ge, x., zhu, h., jiang, t., tan, w., and peng, y. (2018) . predicting the receptor-binding domain usage of the coronavirus based on kmer frequency on spike protein. infect genet evol 61, 183-184. zoete, v., cuendet ma fau -grosdidier, a., grosdidier a fau -michielin, o., and michielin, o. (2011) . swissparam: a fast force field generation tool for small organic molecules. (g) ligand interaction diagram rendered with maestro for ace2 with ligand 300 at the allosteric site impacting s-protein binding from sar-cov2. this 2d "flat" representation shows the interactions at this particular compounds interface on ace2 that would interfere with s protein binding. in particular, extending from deeply inserted to superficial, the interactions are described in the subsequent sentences. d382 and d350 are hydrogen bond acceptors (side chains) from the opposite nh+ on the piperazine-like ring deeply inserted into the binding pocket. r393 is a hydrogen bond donor (side chain) to the alcohol group connecting the piperazine-like ring to the fused ring. e37 is a hydrogen bond acceptor (side chain) to one of the nh on the fused ring. the fluorocyclohexane group is entirely solvent-exposed at the mouth of the binding pocket. although glycosylation sites at residues n165, n234, n343 from s-protein (pdb code 6vsb), are nearby the ace2:s-protein binding interface, they do not overlap and interfere with the protein-protein interface, offering an adjacent site is readily available for ppi docking (s-prot glycosylation analysis: doi: 10.1126/science.abb9983; 10.1101/2020.04.29.069054}. the majority of glycosylation sites are not on the rbd (fig. s2) , the glycosylation site that is actually present on the rbd, n343, is not in 3d proximity to the binding interface. recently, a variant of the s-protein, d614g, was identified to possess enhanced transmissibility and resistance to contemporary interventions and this site is not present on the rbd. neither the glycosylation sites, nor the enhanced transmissibility variant d614g, are within the 3d proximity to the drug binding site for our targeted protein-protein interface disrupting therapeutics for ace2. 3-methyl-7-{2-methyl-3-[(1-phenyl-1h-1,2,3,4tetrazol-5-yl) sulfanyl]propyl}-8-(4-methylpiperazin-1-yl)-2,3,6,7-tetrahydro-1h-purine-2,6-dione ace2 -7.596339 [ (2-chloro-1-benzofuran-3-yl) rac-(3r,4s)-1-[ (3-cyclopentyl-1,2,4-oxadiazol-5yl) methyl]-4(1-methyl-1h-imidazol-5-yl) 2-(decahydroisoquinolin-2-yl)-n(2-oxo-2,3-dihydro-1h-1,3-benzodiazol-5-yl) predicting molecular interactions in silico: i. a guide to pharmacophore identification and its applications to drug design the prince and the pauper. a tale of anticancer targeted agents a common feature pharmacophore for fdaapproved drugs inhibiting the ebola virus coot: model-building tools for molecular graphics features and development of coot optimal docking area: a new method for predicting protein-protein interaction sites patho-)physiological relevance of pink1-dependent ubiquitin phosphorylation structural and functional impact of parkinson disease-associated mutations in the e3 ubiquitin ligase parkin extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in 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composition and divergence of the novel coronavirus (2019-ncov) originating in china analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods crystal structure of sars-cov-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors the dual functions of the extreme n-terminus of tdp-43 in regulating its biological activity and inclusion formation functional exhaustion of antiviral lymphocytes in covid-19 patients a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china fc1c(f)c(f)c(cc(=o)nc2cccc(c2)c(=o) nc2cc2)c(f) coc1ccc(ccc(=o)nc2ccccc2c(=o)occ (=o)nc(c)c2ccc(f)cc2f) -methoxyphenyl)methyl]-4h-1,2,4-triazol-3-yl}sulfanyl)-n-(3,5-dimethylphenyl =o)nc3cc(c)cc( c)c3)c3ccccc3)n2n ccoc(=o)nc1ccc2c(coc(=o)cns(=o)( =o)c3cccc(br)c3)cc(=o) cc1ccc(c(c1)c(=o)nc1cccc(occ(=o)nc 2ccccc2)c1)-n1cnnc1 o=c(coc(=o)cc1ccsc1)nc1cccc2c(=o) c3ccccc3c(=o) cn(c1ccccc1)s(=o)(=o)c1cccc(c1)c(=o )oc(c(=o)nc1cccc(c1)c(c)=o) =o)c(c1)c(f)(f)f nc(=o)cnc(=o)nc1cccc(nc(=o) ccoc(=o)c1c(nc(=o)c(c)sc2nc3ccc(o cc)cc3[nh]2)sc2cc(c) -fluorophenyl)-3-methyl-1h-pyrazol-5-yl cc1cc(nc(=o)csc2nnc3ccccn23)n(n1)-c1ccc(f) fc(f)(f)c1cccc cn1cc(nc1c1cccn(c1)c(=o)cc1ccc(o) c(f)(f)f 4-tetrahydropyrimidin-1-yl)-nmethyl-n-{[(2r,3s)-1-methyl-2-(1-methyl-1h-pyrazol-5-yl) c(=o)cn1ccc(=o)[nh]c1=o 3-carbamoylphenyl)-4-(2-fluorophenyl)-1,4-diazepane-1-carboxamide mpro -7.652268 nc(=o)c1cccc(nc(=o)n2cccn(cc2)c2 ccccc2f -(difluoromethyl)-5-methyl-1h-pyrazol-4-yl )f)c1nc(=o)c1cnc2[nh] c(=o) coc(=o)cc(nc(=o)c1cc(=o)nc2cc(f) ccc12) c=cc(=o)c2ccc3[nh]c(=o)[nh] c3c2)cc1 6-dichlorophenyl)prop-2-enoyl clc1cccc(cl)c1c=cc(=o)c1ccc2[nh]c(= o) 2-yl)methyl]sulfamoyl}phenyl)-3-(2-oxo-1,2,3,4-tetrahydroquinolin-3-yl)propanamide s(=o)(=o) -fluorophenyl)-1-(2-methoxyacetyl)-4,5-dihydro-1h-pyrazol-3 fc1ccc(cc1)s(=o)(=o)nc1ccccc1c(=o) nc1cccc(c1) coc1ccc(cc1)n(c)s(=o)(=o)c1cccc(c1) c(=o)nc1cccc(c1)c#n 4-dihydrophthalazine-1-carboxamide mpro -7.71278 cc(nc(=o)c1nn(cc2ccccc2)c(=o)c2ccc cc12)c1ccc(cc1)s(n)(=o)=o -methyl-2,3-dihydro-1h-indol-1-yl cc1cc2ccccc2n1c(=o)csc1cc(c(n)=o) 2-dimethyl-3-oxo-1,2,3,4-tetrahydroquinoxalin-1-yl coc1ccc(cc1ncc(=o)n1c2ccccc2nc(= o)c1(c)c)s(=o)(=o) c(=o)ncc2ccccc2)c1 cn1cnnc1scc(=o)nc1cccc(c1)c(=o)n cn(c)c1ccc(cn(c)c(=o)coc2ccc3nc(= o)ccc3c2) benzothiazol-2-yl)piperidine-1-carbonyl cc1ccccc1nc(=o)cc1nc(coc(=o) )=o)c2ccccc2)cs1 nc(=o)nc(c)c1cccc(nc(c)=o)c1)c1 cccc(f) coc1ccc(oc)c(cnc(=o)nc(c)c2cccc(n c(c)=o)c2) -ethoxypyridin-3-yl)methyl ccoc1ncccc1cnc(=o)cc1sc(c)nc1-c1ccc(c) 3-dihydro-1-benzofuran-2-yl)methyl]-2-oxo-1,2,3,4-tetrahydroquinoline-6-carboxamide mpro -7.632513 o=c(ncc1cc2ccccc2o1)c1ccc2nc(=o) yl)acetyl]pyrrolidin-2-yl}-4-methyl-4,5-dihydro-1h-1,2,4-triazol-5-one 4-dichlorophenyl)methyl]-n-({4-oxo-4h-pyrido[1,2-a]pyrimidin-2-yl}methyl clc1ccc(cn2ccc3(c2)cccn(c3)c(=o)n cc2cc(=o)n3ccccc3n2) ccc(=o)nc1ccc(cc1)c(c)nc(=o) ccc(=o)nc1ccc(cc1)c(c)nc(=o) nc(=o)c1ccn(n1)-c1cccc(nc(=o)c2ccn(cc2)s(=o)(=o)c =cc2ccccc2) cc(n1ccn(cc=cc2ccccc2)cc1)c(=o)n c1cccc(c1)c#n 4,5-tetrahydro-1h-1 cccn1c(scc(=o)n2c(c)cc(=o)nc3ccc cc23 -oxo-1,2-dihydropyridin-1-yl)acetamide mpro -7.623712 fc(f)(f)c1cc(nc(=o)cn2ccccc2=o)cc(c c(f)(f)f 3-dihydro-1,4-benzodioxin-6-yl)ethyl fc1cccc(c1)n(ccn1c(=o)c2cccc3cccc( c1=o)c23)c(=o) 10-dihydroanthracen-1-yl)-2-(thiophen-2-yl)quinoline-4-carboxamide mpro -7.800731 o=c(nc1cccc2c(=o)c3ccccc3c(=o)c12 ) c(=o)c3ccccc3c(=o) coc1ccc(ns(=o)(=o)c2cccc(c2) coc1ccc(cc1)s(=o)(=o)nn=cc1ccc(oc (=o)nc2ccccc2)c(oc) 3-dimethylcyclohexyl)carbamoyl]methyl 5-(benzylsulfamoyl s(=o)(=o)ncc2ccccc2) key: cord-004672-0lf5j8lo authors: anderson, kevin; bond, clifford w. title: structural and physiological properties of mengovirus: avirulent, hemagglutination-defective mutants express altered alpha (1 d) proteins and are adsorption-defective date: 1987 journal: arch virol doi: 10.1007/bf01313891 sha: doc_id: 4672 cord_uid: 0lf5j8lo structural and physiological properties of two mutants of mengovirus, 205 and 280, were compared to those of wild-type virus to understand the molecular basis of changes exhibited in their biological function. two dimensional gel electrophoresis of wild-type and mutant structural proteins revealed alterations in the isoelectric character of the alpha (1 d) protein of both mutant 205 and 280. these data suggest that alterations in the alpha (1 d) protein may be responsible for the phenotypic changes by the mutants. a delay in detectable virus-specified protein synthesis was exhibited in mutant-infected cells in comparison to wild-type. the amount of rna synthesized in mutantand revertant-infected cells was less than that synthesized in wild-type infected cells. changes in virus-specified macro-molecular synthesis in mutant and revertant-infected cells reflected a decrease in the ability of the viruses to attach to cells. biological properties of two mengovirus mutants, 205 and 280, were compared with those of wild-type virus (2) . these mutants were defective in their ability to agglutinate erythroeytes, produced smmler plaques in cell culture, were avirulent in mice, but were not temperature-sensitive. these data suggested that changes in the structure of tile mutant viruses may be responsible for the expression of altered phenotypie traits. in this communication, the structurm proteins of the wfld-ty]pe and mutant, viruses were compared by sds-page, peptide mapping, and twodimensional gel electrophoresis to determine which of the mutant structural proteins differed from that of wild-type and the extent of homology among analogous proteins. the structural proteins of two revertants of mutant 205 were also examined by two-dimensional gel electrophoresis to relate changes in biological function to structural differences in the analogous proteins of the mutants and wild-type mengovirus. in addition, the progression of events associated with wild-type, mutant, and revertant virus infection were examined to identify-factors responsible for physiological changes associated with mutant and revertant virus-specified macromolecular s:ymthesis. the growth and assay of wild-type, mutant and revertant viruses using bhk-21 cells have been described previously (2) . mutant and revertant virus stocks used in the experiments described below were from the second passage of virus isolated by two successive rounds of plaque purification. purified virions were radiolabeled in vivo with [hc(u)]-l-amino acid mixture (6 ~ci/ ml) [new england nuclear (nen), nec-445] were prepared and analyzed by sds-polyacrylamide gel electrophoresis as described previously (2) . surface tyrosine residues of purified virions were labeled with [12~i]-sodium iodide [(nen), nez-033 hi using a modification of the method described by millar and smith (15) . pellets containing purified virions were resuspended in tn buffer [50 mm tris-hc1 (ph 7.5), 150 m~ nac1] and quantitated by absorbanee at 260 nm as described (19) . suspensions of purified virus in tn buffer containing 0.3 absorbance units (260 nm), 200 ~ci of lesi, and 100 ~g of iodogen (pierce) were mixed in a total volume of 0.3 ml and agitated for l0 minutes. an equal volume of 0.4 ~m nai, 5.0 ~m 2-mercaptoethanol was added to the mixture and layered onto sephadex g-25 columns (60 × 7 mm) equilibrated with tn buffer. the excluded volumes were collected and centrifuged in an sw 41 rotor at 37,000 rpm for 90 minutes at 6°c. pellets were prepared for preparative sds-polyacrylamide gel eleetrophoresis (sds-page). virus-specified intracellular proteins were labeled with l-[35s]-methionine, immunoprecipitated and analyzed by sds-page. cells were mock-infected or infected with virus in suspension (1 × 107 eells/ml) at an moi of 3, adsorbed, plated into plastic 35 mm dishes (2.5 × 106 ceils/dish), and incubated at 33 ° c. cells were pulse-labeled for 15 minutes with 100 ~ci/ml 3~s-methionine in 0.3 ml methionine-free medium. the cells were washed twice with serum free medium and lysed with 0.1 ml b 10 [10 mm tris-hcl (ph 7.4), 5 mm mgci~, 0.5 percent np 40, 0.1 percent sds, 1 percent aprotinin, 50 txg/ml ribonuelease a, 50 ~g/ ml deoxyribonuelease] for 5 minutes on ice. cellular lysates were centrifuged at 6500 × g for 1 minute at 4 ° c and the supernatant fluid was colleeted and stored at -2 0 ° c. b 11 [50 m~ tris (ph 7.4), 150 mm nacl, 5 m~i edta, 0.02 percent sodium azide, 0.05 percent np 40, 1 percent aprotinin, 0.1 percent bovine sernm albumin]. twelve txl of mouse anti-mengovirus hyperimmune aseitic fluid (2) was added to the diluted lysates (0.5 ml volume) and incubated at 0 ° c for 60 minutes. immune complexes were precipitated with 100 t~l of 10 percent fixed staphylococcus aureus (cowan strain) (13) by incubation at 0 ° c for 60 minutes and pelleted by centrifugation for 1 minute at 6500 × g. pellets were washed four times with b 11 buffer at 0 ° c and resuspended in 40 t~l 20 mm dithiothreitol (dtt), 1 percent sds. after 15 minutes of incubation at room temperature, the immunoprecipitated proteins were eluted and reduced by heating at 60 ° c for 5 minutes. bacteria were removed by eentrifugation and the supernatant fluids were prepared for sds-page. virus-specified intracellular rna was labeled with [5, 6-'~h]-uridine [(nen), net-367]. cells were infected in suspension with virus (1 × 107 ceus/ml) at an moi of 3, plated in plastic 35 mm dishes (2.5 × 106 cells/dish) following adsorption at 33 ° c for 30 minutes, and incubated at 33 ° c. actinomyein d (5 l~g/ml final concentration) was added to each dish at various time points and the cells were incubated at 33 ° c for 20 minutes. the medium was aspirated, replaced with 0.3 ml dme 2 containing 10 ~ci/mt '~h-uridine, and the monolayers were incubated at 33 ° c for 60 minutes. at the end of the labeling period, the cells were lysed at 4 ° c for 5 minutes with net buffer [10 mm tris-hc1 (ph 7.4), 100 mm nac1, 1 mm edta] supplemented with 1 percent np 40. the lysate was centrifuged at 6500 × g for i minute. duplicate samples containing 25 i~t of the supernatant fluid were spotted onto whatman gfc glass fiber filters and precipitated in ice cold 10 percent trichloroacetic acid (tca). filters were placed in vials with scintillation fluid [5 g / l 2, 5diphenyloxazoie (pp0) in xylene], and counted in a packard lsc 460 cd liquid scintillation counter using the pre-set tritium channel (4). to prepare virus particles containing radiolabeled i~na, cells were infected with virus at an m0i of 3 and were labeled at 4.5 hpi with 20 ~ci/ml [5, 6-3h]-uridine [(nen), net-367] or 500 ~ci/m132p-orthophosphate [(nen), nex-054] in medium containing 12.5 rag/ l monosodium phosphate. virus-infected cells were allowed to incubate at, 33 ° c until lysis and virions were purified as described above. a~s-methionine labeled virions were suspended in sample buffer containing 9.95 m urea (bio-l~ad), 4 percent. np-40 (particle data laboratories), 2 percent bio-lyte 3/10 (bio-rad), and 100 m~[ dtt as described (8) and incubated at 25 ° c for 30 minutes. isoelectrie focusing (ief) was performed as described (17). following equilibration, the tube gels were placed onto 10 percent polyaerylamide slab gels and subjected to sds-page. the ph gradients for ief and nephge were generated from 1 cm serial gel sections equilibrated in 50 mm nac1 and the ph values were lowered by 0.5 ph unit~s to correct for measurement in the presence of urea (22) . the isoclectric point (pi) value for each protein species was estimated from these gradients. bands corresponding to the structural proteins of mcngovirus were identified by exposure of dried preparative sds-page gels to x-ray film, excised, rehydrated in 10 mm ammonium bicarbonate, 0.1 percent sds, and electroeluted as described (7, 21) . protein samples were lyophilized and sds was removed as described (10) . the proteins were dried, oxidized for 90 minutes at 4 ° c with 0.2 mt of fresh performie acid and lyophitized three times in distilled water. proteins were anmyzed for purity by sds-page prior to digestion. purified, oxidized proteins were resuspended in 0.2 ml of 1 percent ammonium bicarbonate (ph 7.8) for digestion with n-mpha-p-tosyl-l-lysine ehloromethyl ketonetreated chymotrypsin (tlck-chymotrypsin) at 37 ° c as described (7), enzyme treated samples were frozen and lyophilized before peptide analysis by thin layer chromatography (tlc). two-dimensional peptide mapping was performed as previously described (ll). lyophilized samples were dissolved in electrophoresis buffer [butanol-pyridine-acetie acid-water (2 : 1 : 1 : 36)] and 2--5 pl was spotted onto cellulose tlc plates (e. merck). separation of peptides in the first dimension was perfomed by eleetrophoresis for 45 minutes at 1000v (40 to 50 ma). the tlc plates were air-dried and the peptides were separated in the second dimension by ascending chromatography in n-butanol-pyridineacetic acid-water (393 : 304 : 61 : 243) until the solvent front reached 2 cm from the top. dried plates were sprayed with en3hanee (nen) and exposed to preflashed kodak nag-2 x-ray film at -70 ° c. the number and percentage of wild-type, mutant, and revertant virus particles adsorbing to bhk-21 cells were determined as follows. cells were infected with purified ~ep_ labeled virus at a multiplicity of 2000 particles/cell. the 32p-labeled virus suspensions were allowed to adsorb to bhk-21 cell monolayers in plastic dishes (60 mm diameter) for 60 minutes at 33 ° c. the cells were lysed with 0.1 ml b 10 for 5 minutes on ice. duplicate 100 t~1 samples were spotted onto whatman glass fiber filters and precipitated in ice-cold 10 percent tca. the filters were placed in vials with scintillation fluid and counted in a packard 460 cd liquid scintillation counter using the pre-set ~2p channel (4) . the fraction of cell-associated virus particles was calculated by dividing the number of cell associated counts per minute (cpm) by the cpm of the input virus. the average number of virus particles adsorbed per cell was calculated by multiplying the fraction of cell-associated virus by the multiplicity of infection (2000 particles/cell). the structural proteins of purified 14c-labeled wild-type and m u t a n t mengovhnases were analyzed by s d s -p a g e on 8, 10 a n d 15 p e r c e n t polya e r y l a m i d e slab gels. a n a l y s e s of the structural proteins resolved on 15 percent gels are shown (fig. 1) . five structural proteins were resolved for each of the viruses: epsilon (lab), 40 kd; alpha (1 d), 37 kd; b e t a (1b), 33 kd; g a m m a (1 c), 25 kd; and delta (t a), 7.8 kd. no changes in the migration of the m u t a n t structurm proteins were o b s e r v e d in c o m p a r i s o n to those of the wild-type virus. in addition, the migration of the structural proteins of several h a + r e v e r t a n t s of m u t a n t 205 was also similar to t h a t of wild-type (data n o t shown). although some v a r i a t i o n in the a m o u n t of delta (1 a) p r o t e i n of the viruses w a s o b s e r v e d (fig. 1) , this was not a result found consistently t h r o u g h o u t our analyses. 35s-methionine-labeled structural proteins of wild-type and m u t a n t viruses 205 and 280 were analyzed following digestion with t p c k -t r y p s i n b y r e v e r s e -p h a s e h p l c to further e x a m i n e the structure of the proteins by separating peptides in a gradient on the basis of charge, however, the spectra of methionine-eontmning peptides of the delta (1a), beta (1b), gamma (1 c), and alpha (1 d) proteins from the different viruses were identical (data not shown). although not all of the peptides generated by tpck-trypsin digestion were likely to contain methionine, the results suggested that the peptide compositions of the four strueturm proteins of the wild-type and mutant viruses were remarkably similar. hplc analyses of the tryptie peptide compositions of the four structural proteins of the wildtype and mutant viruses uniformly labeled with 14c-amino acid mixture by labeled strueturm proteins was inconclusive because the resulting ehromatograms contained many unresolvable, overlapping peaks (data not shown). to determine whether the arrangement of the structural proteins on the surface of mutants 205 and 280 was similar to that of wild-type, purified virions were labeled with r25i and analyzed by sds-page (fig. 2) . most of the tyrosine residues on the surface of the wild-type and mutant viruses were on the alpha (1 d) protein as demonstrated by the extensive labeling of this protein. the beta (1 b) protein was labeled to a much lesser extent. there was no detectable difference in the pattern of surface protein labeling among the three viruses suggesting that the arrangement of the structural proteins on the surfaces of the virions of the wild-type and mutant viruses was similar. to examine the structure of the surface proteins of the three viruses in greater detail, two-dimensional tlck-ehymotryptie peptide ehromatoalthough not all of the surface peptides generated by tlck-chymotrypsin are likely to be labeled, numerous peptides were labeled. these data suggest that the arrangement of the structural proteins on the surface of the three viruses was remarkably similar. the structural proteins of the wfld-ts~e, mutant, and revertant strains were analyzed by ph gradient gel electrophoresis to determine whether any changes were apparent in migration of the mutant and revertant structural proteins relative to those of the wild-type virus. the proteins were analyzed by ief and nephge two-dimensional gel electrophoresis in order to resolve both acidic and basic proteins. the two-dinlensional electrophoretie migration of the structural proteins of wild-type mengovirus is shown in fig. 4 , panel a. the epsflon (1 ab) protein had a pi of 5.32. the alpha (1 d) protein was separated into four major protein species, each with a distinct pi (5.35, 5.52, 5.61, and 5.72). the beta (1 b) protein was separated into two major protein species with pi values of 5.55 and 5.62 and a heterogenous species ranging from 6.3 to 6.6 the gamma (1 c) protein was not well resolved by this technique and appears to be slightly acidic or neutral in charge. the delta (1 a) protein does not appear in any of the two-dimensional ief gels. the two-dimensional electrophoretic migration of the structural proteins of mutant 205 is shown in fig. 4, panel b . the etectrophoretic migration of the proteins was identical to that of the wild-type strain with one exception. only three alpha (1 d) protein species were resolved. one of the protein species resolved in separation of the wild-type proteins, p i = 5.72, was absent. however, this protein species was resolved in the wild-type/205 mixed sample separation (fig. 4, panel d) . these data suggest that the absence of this particular protein species represents a phenotypie change or mutation in the alpha (1 d) protein of mutant 205 relative to the wild-type virus. in addition, the two-dimensional electrophoretic patterns of the structural proteins of the revertent viruses, 205-a 7 and 205-d 2, were identical to those of mutant 205 (data not shown). the two-dimensional electrophoretie migration of the structural proteins of mutant 280 is shown in fig. 4 , panel c. the eleetrophoretic migration of the proteins was identiem to t h a t of the wild-type strain with one exception. the pi of one of the four alpha (1 d) protein species (pi = 5.58) was slightly more acidic t h a n tile analogous wild-type isoelectric species ( p i = 5.61). this species a p p e a r e d b r o a d e r and slightly lower in molecular weight t h a n the corresponding wild-type protein species. the wild-type/280 mixed sample separation (fig. 4, panel e) shows a broad protein species in the region of the gel corresponding to tile apparent, change in the pis of the wild-type and 280 alpha (1 d) protein species. the altered migration of the m u t a n t 280 isoeleetrie species was found consistently in two dimensional profiles of independently derived virion protein samples. these data suggest that the change in form and migration of this protein species represents a phenotypic change or mutation in the alpha (1 d) protein of mutant 280 relative to the wild-type virus. two-dimensionm nephge analyses confirmed the results obtained by ief analysis of the wild-type, mutant, and revertant structural proteins. the delta (1 a) proteins of the wild-type and mutant viruses were resolved by this technique and migrated similarly as a single acidic protein species at ph 2.5 (data not, shown). the specific activities (cpm/particle) of the wild-type and mutants differed when bhk-21 cells were intbcted and labeled with 35s-methionine or 14c-amino acids under similar conditions (ani)e~son and bo~-d, unpublished data). therefore, to determine whether differences in the kinetics of virus-specified macromotecular synthesis exist among the viruses, virusspecified intraeellular protein and t~na synthesis were examined. virusinfected cells were pulse-labeled at 1 hour intervals from 3 to ll hpi with 35s-methionine for 15 minutes. cytoplasmic lysates were immunoprecipitated and analyzed by sds-page (fig. 5) . eight virus-specified intracellular proteins were resolved in wild-type and mutant strain-infected cells: a (1-2 a), b (1), c (3), d (3 cd), 1 abc, d 2 (1 cd), alpha (1 d), and gamma (1 c). the number and molecular weights of the proteins specified by the wild-type and mutant strains were identical. however, virus-specified protein synthesis was detected initimly at 5 hpi for the wild-type strain and at 6 hpi for mutant strains 205 and 280. to determine whether changes in the kinetics of lgna synthesis could explain the delay in detectable protein synthesis observed in mutantinfected cells, virus-infected cells were treated with aetinomyein d, pulselabeled with 3h-uridine, lysed, and counted. the results are shown in fig. 6 . the peak of rna synthesis for each virus was from 10 to 11 hpi. however, the amount of rna synthesized by the wild-type virus was 10-fold greater than that of the mutants and 2-fold greater than that of the revertants. since the magnitude of virus-specified rna synthesized in the mutant virusinfected cells was 10-fold less than that of wild-type virus, the beginning of virus-specified protein synthesis, as detected by immunoprecipitation (fig. 5) , would appear to be delayed due to the fact that less rna would be available for translation. since the magnitude of virus-specified i%na synthesis in mutant virusinfected cells was 10-fold less than that of the wild-type virus, mterations in uncoating or adsorption of the m u t a n t viruses to cells m a y account for this difference. however, surface peptide analysis suggested t h a t the arrangem e n t of the strueturm proteins of the wild-type and m u t a n t virions was r e m a r k a b l y similar. therefore, it is possible t h a t uncoating of the virions would occur at a similar rate in infected ceils due to their similarity in structure. the average n u m b e r of wild-type, mutant, and revertaa~t virus particles adsorbing to bhk-21 cells was examined tbllowing incubation at 33 ° c for 60 minutes. u n d e r these conditions, the a m o u n t of virus adsorption to cells would approximate saturation. the results are shown in table 1 . the average n u m b e r of virus particles adsorbing per cell clearly differed among the wild-type, mutant,, and r e v e r t a n t viruses. therefore, it is a p p a r e n t t h a t the mechanisms of adsorption of the m u t a n t and r e v e r t a n t viruses to cells is modified from t h a t of wild-type. the diftbrenee in the cellular binding affinities among the wild-type, mutant, and revei'~ant viruses would be an i m p o r t a n t factor contributing to the differences in the magnitude of viral rna and protein synthesis observed fbr these viruses. these data suggest t h a t fewer cells would be infected by the m u t a n t and r e v e r t a n t viruses and therefore, fewer virus-specified macromoleeules would be synthesized. kevln anderson and clifford w. bond: cells were infected at an moi of 2000 particles/cell and incubated at 33 ° c for 60 minutes b the average number of particles adsorbed/cell ° the percentage of adsorption of the mutants and revertants in comparison to wild-type mengovirus biologiem and structural properties of two mengo~drus :mutants have been compared to those of the parental wild-type strain. ~e s e mutants, 205 and 280, exhibited alterations in agglutination of erythrocytes, virulence in mice, and plaque morphology (2) . in addition, biological characterization of several ha + revertants of mutant 205 isolated from the brains of mice infected intraeranieally indicated that agglutination and virulence may be linked traits and that the size of plaques produced by a pa~ieular mengovirus isolate may reflect its bindhag affinity to cells and virulence in mice. analysis of 35s-methionine-labeled structural proteins of wild-type and mutant viruses by sds-page and hplc revealed that extensive homology exists among these viruses. to further examine these viruses for structural differences, surface labeling of intact wild-type and mutant virus particles was done to compare the arrangement of the structural proteins which form the xdral capsids. previous studies have shown that surface iodination of intact potiovirus and mengovirus labels primarily the 1 d (vp 1 or alpha) protein of the respective virus eapsids (3, 12) and the 1 b (vp 2 or beta) protein is also labeled, but to a much lesser extent than the 1 d protein. we obtained similar data tbr the surface iodination of intact wild-type and mutant virus particles. in addition, no apparent differences were evident from the analysis of ehymotryptie peptides of 125i-labeled wild-type and mutant alpha (1 d) proteins. these data suggest that arrangement of structural proteins on the surface of mutant eapsids did not differ from that of wild-type. therefore, the mutations may result in the expression of altered determinants that reflect differences in biologicm function and may not result in the masking of otherwise funetionm determinants due to altered arrangement of the capsid proteins. we compared the isoeleetrie character of the viral capsid proteins by two-dimensional gel electrophoresis. multiple species of the alpha (1 d) and beta (1 b) proteins were reprodueibly deteeted by this technique in both the presence (fig. 4 ) and absence (data not shown) of sds prior to isoeleetrie focusing. previous studies have demonstrated multiple isoelectrie species for the major eapsid proteins of poliovirus [vp 1 (1 d), vp 2 (1 b), and vp 3 (1 c)] (9, 23) and emc virus [alpha (1 d) and beta (1 b)] (6). vriasen et al. (23) also excluded the possibility that the multiple species are derived from differential binding of sds or the accumulation of mutants in the original virus stoek. these authors suggested that the multiple species may result from heterogeneity in the cleavage of structural protein precursors; although the sequenees of the amino-and earboxyl-termini of the major eapsid polypeptides of mengovirus were determined without mention of sequenee variations (24) . we suggest that the charge heterogeneity of these molecules may reflect differences in the interaction of these proteins with the ampholines. the different protein species observed may represent different forms of the viral proteins associated with either intraeellular partieles or free extraeellular particles or extraeellular particles bound to or eluted from membranes, assuming that these particles share the same buoyant density in equilibrium gradients. therefore, the heterogeneity observed may reflect differenees in the manner of isolating virus partieles, whether intraeellularly or from lysed cells. differenees were deteeted among the alpha (1 d) structural proteins of the wild-type and mutant viruses. since the mutants exhibited different alterations in the alpha (1 d) protein, yet similar ehanges in biological activity, it is possible that alteration of this protein resulted in the observed phenotypie ehanges. the differenees in the number and migration of the alpha (1 d) protein isoelectrie species are likely to be due to changes in uneharged amino acid residues affeeting the interaction of the altered species with the ampholines. changes in eharged amino acid residues would result in altered migration of all four isoeleetrie species. since the ehromatograms of surface-and metabolieally-labeled peptides were similar, we speculate that the alterations in the mutant alpha (1 d) proteins may be assoeiated with a particular determinant on this protein. this determinant may serve as or be part of a funetional attachment site on the surface of virus partieles that determines their affinity for various cells. atomic resolution of the structure of another pieornavirus, human rhinovirus 14, revealed a large cleft on the ieosahedral faee that has been postulated to serve as the host eell receptor binding site (18) . the large cleft separates the major part of five 1 d (alpha) subunits from the other viral protein subunits. sinee the pieornaviruses share similar struetural eharaeteristies, by analogy it seems reasonable to speculate that mutations in the 1 d (alpha) protein could affect the strueture of the deft, and thus, alter the binding affinities of the mutant and revertant viruses. alteration of the mengovirus host cell receptor binding site may lead to changes in affinity for erythrocytes as well as other cell types which may explain the lack of virulence of the mutants in mice. previous work by morishima et al. (16) demonstrated that differences in binding affinities of closely related strains of emc and mengovirus to various cells reflects their difference in pathogenicity for mice. ha + revertants were isolated from the brains of mice infected intracranially with mutant 205 (2) . in addition to regaining agglutination activity, the revertants were also virulent for mice and exhibited a slight increase in plaque size. however, revertants required 103. to 104-fold more p f u to kill mice than the wild-type virus. since the isoelectric character of t/he proteins of the ha + revertants were identicm to that of mutant 205, this partial phenotypic reversion may have resulted from a change in an uncharged amino acid associated with a determinant, located on the alpha (1d) protein, which partially restores its biological activities. the partial reversion of this mutation may involve modification of the altered surface determinant, increasing the binding affinity of the virus for cells and restoring virulence, but would not result in reappearance of the isoelectric species absent in mutant 205 and revertant alpha (1 d) capsid proteins. alternatively, mutant 205 may express more than one mutation since complete reversion to w i l d -t~e virulence and plaque size did not occur and the isoelectric profile of the alpha (1 d) protein was the same as that of mutant 205. however, revertants were not isolated from mice infected intracranially with mutant 280, which shared biological properties as well as alpha (1 d) protein alterations with mutant 205. therefore, the mutation observed in the alpha (1 d) structural protein of mutant 280 is apparently more stable than that of mutant 205. however, unlike mutant 205, expression of the mutation of mutant 280 resulted in a more subtle, yet reproducible change in the isoelectric point of one of the four alpha (1 d) protein species. changes in virus-specified macromolecular synthesis in mutant and revertant virus-infected cells can be explained by a decrease in the ability of these viruses to attach to cells. these data suggest that a smaller proportion of cells were productively infected with the mutant and revertant viruses in comparison to wild-type. therefore, the effective moi for the mutant and revertant viruses would be less than that of wild-type. this difference can be explained by changes in the cellular binding affinities; and the higher particle : p f u ratios exhibited by the mutant and revertants (2), assuming that a greater proportion of noninfectious particles would lower the probability of these viruses to infect cells. this interference phenomenon would explain why p f u values obtained under dilute conditions could not be used to accurately predict the fraction of infected cells in high particle : cell infections. since fewer cells would be productively infected with mutant and revertant viruses, less virus-specified rna would be synthesized, although the peak hour of rna synthesis would be the same as that of wild-type. therefore, less rna would be available for translation; and protein synthesis, as detected by immunoprecipitation, would appear to be delayed in ceils infected with the mutant viruses. changes in the metabolic rates of mutant, virus-specified gna synthesis would predict different results than those presented here if tile fraction of infected cells were similar to ~t d -t y p e infections. collectively, our data suggest that the phenotypie changes expressed by the mutants may be due to mutations toeated exclusively within the alpha (1 d) coding region of the genome. previous work by a(~ol et al. (1) has indicated that the neurovirulenee of poliovirus maps to the 5' end of the genome which includes the coding region of the eapsid proteins. consistent with these results, we have identified two different mutations in the alpha (1 d) capsid protein, which maps to the 3' end of tiffs region, of two avirulent mengovirus mutants. in addition, ko~tara et al. (14) have stated that a molecular recombinant of the sabin vaccine strain of poliovirus containing vp 1 (1 d) and most of vp 3 (1 c) of the neurovirulent mahoney strain is virulent, but not as virulent as the mahoney strain and retain the small plaque morphology of the sabin strain. our data also indicate that changes in the alpha (1 d) protein result in altered virulence of a pieornavirus. since revertants of mutant 205 shared similar characteristics with the poliovirus recombinant, the plaque-forming ability of a particular virus isolate may be a characteristic that reflects as well as contributes to its virulence. future experiments which compare the nueleotide sequences of the nmtants and revertant structural proteins to that of the parental wild-type virus will be useful in determining the genetic basis for the altered biological properties exhibited by the mutant and revertant viruses. construction and properties of intertypie poliovirus reeombinants: first approximation mapping of the major determinants of neurovirulence biological properties ofmengovirus: characterization of avirulent, hemagglutination-defective mutants iodination of poliovirus capsid proteins liquid scintillation counting: elimination of spurious results due to static electricity relatedness of virion and intracellular proteins of the murine coronaviruses jhm and a 59 two-dimensional electrophoretic analysis of encephalomyocarditis viral proteins identification of the initiation site of poliovirus protein synthesis two-dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines isoelectric points of polypeptides of standard poliovirus particles of different serological types and of empty capsids and dense particles of poliovirus type 1 a micromethod for complete removal of dodecyl sulfate from proteins by ion-pair extraction characterization of t antigens in polyoma-infected and transformed cells structure of the mengo virion. distribution of the capsid polypeptides with respect to the surface of the virus particle rapid isolation of antigens from cells with a staphylococcal protein a-antibody absorbent: parameters of the interaction of antigen-antibody complexes with protein a in vitro phenotypic markers of a poliovirus recombinant constructed from infectious cdna clones of the neurovirulent mahoney strain and the attenuated sabin 1 strain protein iodination using iodogen genomic and receptor attachment differences between mengovirus and encephalomyocarditis virus two-dimensional polyacrylamide gel electrophoretic fraetionation structure of a human common cold virus and functional relationship to other picornaviruses on the structure and morphogenesis of picornaviruses systematic nomenclature for picornavirus proteins poliovirus replication proteins: rna sequence encoding p 3-1 b and the sites of proteolytie processing isoelectric points and conformations of proteins. i. effect of urea on the behavior of some proteins in isoelectric focusing gesolution of the major poliovirus eapsid proteins into doublets structure of the mengo virion. iv. amino-and carboxylterminal analysis of the major capsid polypeptides we thank drs. sandra ewald and andreas luder for their hetpfhl discussions and interest in our work and dr. andrew king for helpful comments on the manuscript. support for this work was obtained from three geseareh creativity development grants awarded to k. a. by the department of graduate studies, montana state universi~-and a grant from the montana heart association, inc. awarded to c.w.b. p~eceived march 20, 1986 key: cord-193133-puqcbf8t authors: piplani, sakshi; singh, puneet kumar; winkler, david a.; petrovsky, nikolai title: in silico comparison of spike protein-ace2 binding affinities across species; significance for the possible origin of the sars-cov-2 virus date: 2020-05-13 journal: nan doi: nan sha: doc_id: 193133 cord_uid: puqcbf8t the devastating impact of the covid19 pandemic caused by sars coronavirus 2 (sarscov2) has raised important questions on the origins of this virus, the mechanisms of any zoonotic transfer from exotic animals to humans, whether companion animals or those used for commercial purposes can act as reservoirs for infection, and the reasons for the large variations in susceptibilities across animal species. traditional lab-based methods will ultimately answer many of these questions but take considerable time. in silico modeling methods provide the opportunity to rapidly generate information on newly emerged pathogens to aid countermeasure development and also to predict potential future behaviors. we used a structural homology modeling approach to characterize the sarscov2 spike protein and predict its binding strength to the human ace2 receptor. we then explored the possible transmission path by which sarscov2 might have crossed to humans by constructing models of ace2 receptors of relevant species, and calculating the binding energy of sarscov2 spike protein to each. notably, sarscov2 spike protein had the highest overall binding energy for human ace2, greater than all the other tested species including bat, the postulated source of the virus. this indicates that sarscov2 is a highly adapted human pathogen. of the species studied, the next highest binding affinity after human was pangolin, which is most likely explained by a process of convergent evolution. binding of sarscov2 for dog and cat ace2 was similar to affinity for bat ace2, all being lower than for human ace2, and is consistent with only occasional observations of infections of these domestic animals. overall, the data indicates that sarscov2 is uniquely adapted to infect humans, raising questions as to whether it arose in nature by a rare chance event or whether its origins lie elsewhere. the devastating impact of covid-19 infections caused by sars-coronavirus 2 (sars-cov-2) has stimulated unprecedented international activity to discover effective vaccines and drugs for this and other pathogenic coronaviruses. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] it has also raised important questions on the mechanisms of zoonotic transfer of viruses from animals to humans, questions as to whether companion animals or those used for commercial purposes can act as reservoirs for infection, and the reasons for the large variations in sars-cov-2 susceptibility across animal species. [17] [18] [19] understanding how viruses move between species may help us prevent or minimize these pathways in the future. elucidating the molecular basis for the different susceptibilities of species may also shed light on the differences in susceptibilities in different sub-groups of humans. very recently, shi et al. published the results of experiments to determine the susceptibility to sars-cov-2 of ferrets, cats, dogs, and other domesticated animals. 20 they showed that sars-cov-2 virus replicates poorly in dogs, pigs, chickens, and ducks, but ferrets and cats are permissive to infection. other studies have reported the susceptibility of other animal species to sars-cov-2. 17, 20, 21 susceptible species such as macaques, hamsters and ferrets are used as animal models of sars-cov-2 infection. [22] [23] [24] in the absence of purified, isolated ace2 from all the relevant animal species that could be used to measure the molecular affinities to spike protein experimentally, computational methods offer considerable promise for determining the rank order of affinities across species, as a method to impute which species may be permissive to sars-cov-2. here we show how computational chemistry methods from structure-based drug design can be used to determine the relative binding affinities of the sars-cov-2 spike protein for its receptor, angiotensin converting enzyme (ace)-2, a critical initiating event for sars-cov-2 infection, across multiple common and exotic animal species. [25] [26] [27] the aim of these studies was to better understand the species-specific nature of this interaction and see if this could help elucidate the origin of sars-cov-2 and the mechanisms for its zoonotic transmission. to construct the three-dimensional structure of the sars-cov-2 spike protein, the sequence was retrieved from ncbi genbank database (accession number yp_009724390.1). a psi-blast search against the pdb database for template selection was performed and the x-ray structure of sars coronavirus spike template (refcode 6acc) was selected with 76.4% sequence similarity to sars-cov-2 spike protein. the protein sequences of the ace2 proteins for different species is summarized in table 11 and full sequence alignment in supplementary figure 1 . the phylogenetic tree for ace2 proteins from selected animal species is illustrated in supplementary figure 2 . the 3d-structures were built using modeller 9.21 (https://salilab.org/modeller/). 28 the quality of the generated models was evaluated using the ga341 score and dope scores, and the models assessed using swiss-model structure assessment server (https://swissmodel.expasy.org/assess). 29 the x-ray crystal structures of human ace2 (recode 3sci) and human sars spike protein (refcode 5xlr) were retrieved from protein data bank. protein preparation and removal of nonessential and non-bridging water molecules for docking studies were performed using the ucsf chimera package (https://www.cgl.ucsf.edu/chimera/). 30 these modelled structures were docked against sars-cov-2 spike protein structure using the hdock server (http://hdock.phys.hust.edu.cn/). 31, 32 molecular docking was performed on the homology modelled sars-cov-2 spike protein with human and animal ace2 proteins. the sars-cov spike protein was also docked with human ace2 protein to obtain the docking pose for binding energy calculations. the docking poses were ranked using an energy-based scoring function. the docked structures were analyzed using ucsf chimera software. the final models were optimized using the amber99sb-ildn force field in gromacs2020 (http://www.gromacs.org/). 33 docked complexes (sars-cov-2 spike with human ace2, human sars-cov spike with human ace2, sars-cov-2 spike with bat ace2 etc) were used as starting geometries for md simulations. simulations were carried out using the gpu accelerated version of the program with the amber99sb-ildn force field i periodic boundary conditions on an oracle cloud server. docked complexes were immersed in a truncated octahedron box of tip3p water molecules. the solvated box was further neutralized with na+ or cl− counter ions using the tleap program. particle mesh ewald (pme) was employed to calculate the long-range electrostatic interactions. the cutoff distance for the long-range van der waals (vdw) energy term was 12.0 å. the whole system was minimized without any restraint. the above steps applied 2500 cycles of steepest descent minimization followed by 5000 cycles of conjugate gradient minimization. after system optimization, the md simulations was initiated by gradually heating each system in the nvt ensemble from 0 to 300 k for 50 ps using a langevin thermostat with a coupling coefficient of 1.0/ps and with a force constant of 2.0 kcal/mol·å2 on the complex. finally, a production run of 100 ns of md simulation was performed under a constant temperature of 300 k in the npt ensemble with periodic boundary conditions for each system. during the md procedure, the shake algorithm was applied for the constraint of all covalent bonds involving hydrogen atoms. the time step was set to 2 fs. the structural stability of the complex was monitored by the rmsd and rmsf values of the backbone atoms of the entire protein. finally, the free energies of binding were calculated for all the simulated docked structures. calculations were also performed for up to 500 ns on human ace2 to ensure that 100ns is sufficiently long for convergence. duplicate production runs starting with different random seeds were also run to allow estimates of binding energy uncertainties to be determined for the strongest binding ace2 structures. the binding free energies of the protein-protein complexes were evaluated in two ways. the traditional method is to calculate the energies of solvated sars-cov-2 spike and ace2 proteins and that of the bound complex proteins and derive the binding energy by subtraction. δg (binding, aq) = δg (complex, aq) -(δg (spike, aq) + δg (ace2, aq) we also calculated binding energies using the molecular mechanics poisson boltzmann surface area (mm-pbsa) tool in gromacs that is derived from the nonbonded interaction energies of the complex. 34, 35 the method is also widely used method for binding free energy calculations. the binding free energies of the protein complexes were analyzed during equilibrium phase from the output files of 100 ns md simulations. the g_mmpbsa tool in gromacs was used after molecular dynamics simulations, the output files obtained were used to post-process binding free free energy decomposition analyses were also performed by mm-pbsa decomposition to get a detailed insight into the interactions between the ligand and each residue in the binding site. the binding interaction of each ligand-residue pair includes three terms: the van der waals contribution, the electrostatic contribution, and the solvation contribution. another estimate of the strength of the interaction between protein-protein complex can be obtained from the non-bonded interaction energy between the complex. gromacs has the ability to decompose the short-range nonbonded energies between any number of defined groups. to compute the interaction energies as a part of our analysis, we reran the trajectory files obtained during simulation to recompute energies using -rerun command. the interaction energy is the combination of short range coulombic interaction energy (coul-sr:protein-protein) and the short-range lennard-jones energy (lj-sr:protein-protein (see table 3 ). while this paper was being prepared, a paper by guterres and im described a substantial improvement in protein-ligand docking results using high-throughput md simulations. 36 they employed docking using autodock vina, followed by md simulation using charmm. the parameters they advocated were very similar to those used in our study. proteins were solvated in a box of tip3p water molecules extending 10 å beyond the proteins and the particle-mesh ewald method was used for electrostatic interactions. nonbonded interactions over 10 and 12 å were truncated. their systems were minimized for 5000 steps using the steepest descent method followed by 1 ns equilibration with an nvt setting. for each protein-ligand complex, they ran 3 × 100 ns production runs from the same initial structure using different initial velocity random seeds and an integration step size of 2 fs. the ancestry of sars-cov-2 traces back to the human, civet and bat sars-cov strains, which all use the same ace2 proteins for cellular entry. [37] [38] [39] the similarities and variations in sequences for both the sars-cov-2 spike protein and the sars spike protein were determined from sequences retrieved from ncbi genbank databank and aligned using clustalw. the spike protein receptor binding domain (rbd) region showed a 72% identity between the two viruses ( figure 1 ). conserved regions in black and non-conserved residues in red and blue. the three-dimensional structure of sars-cov-2 spike protein ( figure the ramachandran scores of the modeled ace2 structures for selected species are summarized in table 1 with the actual predicted ace2 structures and ramachandran plots for each selected species shown in supplementary figure 3 . figure 3) . the modelled structures were further assessed for quality control using ramachandran plot and molprobity scores in swissmodel structure assessment. the ramachandran plot checks the stereochemical quality of a protein by analyzing residue-by-residue geometry and overall structure geometry, is also a way to visualize energetically allowed regions for backbone dihedral angles ψ against φ of amino acid residues in protein structure. the ramachandran score of sars-cov-2 spike protein was 90% in the binding region and molprobity scores that provide an evaluation of model quality at both the global and local level was 3.17. further, the ace2 modelled structures from selected species were also assessed using swiss model structure assessment. ramachandran score or the percentage of amino acid residues falling into the energetically favored region for all species ranged from 96-99% (table 1) . these ramachandran and molprobity scores show that all the built structures were of good quality and were suitable for use in further studies. the ramachandran graphs are presented in supplementary figure 3 . the receptor binding domain (rbd) of sars-cov-2 spike protein was docked against ace2 receptor of various species using hdock server. the interacting residues of ace2 and sars-cov-2 spike protein are depicted in table 3 . we found certain key amino acids in the receptor binding motif (rbm) that were in accordance with previous studies 40 . certain amino acids including phe28, asn330, asp355 and arg357 were conserved in ace2 of most of the selected species and were observed to take part in the interaction with spike protein. tyr41, lys353, ala386 and arg393 also interacted with spike protein residues and were highly conserved across all species except bat, mouse, ferret and pangolin, respectively. spike protein interacting residues with ophiophagus hannah (king cobra) were least common amongst all the ace2 species included in the study, consistent with its low sequence similarity to human ace2. homo sapiens (human) the molecular dynamics simulation of complexes of sars-cov-2 spike protein and ace2 receptors of various species were performed for 100ns. all complexes became stable during simulation with rmsd fluctuations converging to a range of 0.5 to 0.8 nm from the original position. the calculated binding energies for the interactions of sars-cov-2 with ace2 from the species studied are presented in table 4 . the table 4 also includes observational in vivo data on sars-cov-2 infectivity and disease symptoms in the species where this has been reported. although bats carry many coronaviruses including sars-cov, a relative of sars-cov-2, direct evidence for existence of sars-cov-2 in bats has not been found. as highlighted by our data, the binding strength of sars-cov-2 for bat ace2 is considerably lower than for human ace2, suggesting that even if sars-cov-2 did originally arise from a bat precursor it must later have adapted its spike protein to optimise its binding to human ace2. there is no current explanation for how, when or where this might have happened. instances of direct human infection by coronaviruses or other bat viruses is rare with transmission typically involving an intermediate host. for example, lyssaviruses such as hendra are periodically transmitted from bats to horses and then to humans who contact the infected horse. similarly, sars-cov was shown to be transmitted from bats to civet cats and from them to humans. to date, a virus identical to sars-cov-2 has not been identified in bats or any other non-human species, making its origins unclear. to date, the most closely related coronavirus to sars-cov-2, is the bat coronavirus, batcov ratg1, which has 96% whole-genome identity to sars-cov-2. 50 hence other genetic factors could underlie the apparent lack of susceptibility of dogs to covid-19 clinical infection. it is known that gain of function (gof) mutations occur in viruses that can lead to pandemics. gof means viruses gain a new property e.g. in influenza virus gof has been associated with the acquisition of a new function, such as mammalian transmissibility, increased virulence for humans, or evasion of existing host immunity. 49 the conditioning of viruses to humans as pandemics progress is well recognized. however, the sars-cov-2 structures and sequences that we employed were from viruses collected very early in the pandemic. it is therefore not clear how sars-cov-2 could have developed such a high affinity for human ace2, notably higher than for those of putative zoonotic sources for sars-cov-2, unless it has been previously selected on human ace2 or an ace2 of another species bearing a closely homologous spike protein binding domain. interestingly, pangolin ace2 bears some similarities in its sbd to human ace2. this marries with the fact that pangolin-cov shares a highly similar rbd to sars-cov-2, although their remaining sequence has only 90% similarity. this could be consistent with a process of convergent evolution whereby human and pangolin coronaviruses infecting via ace2, have come to the same solution in respect of evolving an optimal spike rbd for binding of either human or pangolin ace2, respectively. our data does indicate that humans might be permissive to pangolin covs that use ace2 for cell entry, a fact that needs to be borne in mind in respect of future potential coronavirus pandemic sources. however, this does not mean that pangolin ace2 was the receptor on which the sars-cov-2 spike protein rbd was initially selected, with the strength of binding to pangolin ace2 lower than binding to human ace2. this makes it unlikely that pangolins are the missing intermediate host. if sars-cov-2 spike was selected on pangolin ace2, then given the higher affinity of sars-cov-2 for human ace2 than for bat ace2, sars-cov-2 would have to have circulated in pangolins for a long period of time for this evolution and selection to occur and to date there is no evidence of a sars-cov-2 like virus circulating in pangolins. another possibility would be a short term evolutionary step where a pangolin was recently coinfected with a bat ancestor to sars-cov-2 at the same time as it was infected by a pangolin cov allowing a recombination event to occur whereby the spike rbd of the pangolin virus was inserted into the bat cov, thereby conferring the bat cov with high binding for both pangolin and human ace2. such recombination events are known to occur with other rna viruses and can explain creation of some pandemic influenza strains 49 . nevertheless, such events are by necessity rare as they require coinfection of the one host at exactly the same time. most importantly, if such a recombination event had occurred in pangolins it might have been expected to have similarly triggered an epidemic spread of the new highly permissive sars-cov-2 like virus among pangolin populations, such as we now see occurring across the human population. currently there is no evidence of such a pangolin sars-cov-2 like outbreak, making this whole scenario less likely. indeed, pangolins might be protected from sars-cov-2 infection due to the existence of crossprotective spike rbd neutralising antibodies induced by exposure to pangolin cov, given the rbd similarity of these two viruses. another possibility which still cannot be excluded is that sars-cov-2 was created by a recombination event that occurred inadvertently or consciously in a laboratory handling coronaviruses, with the new virus then accidentally released into the local human population. given the seriousness of the ongoing sars-cov-2 pandemic, it is imperative that all efforts be made to identify the original source of the sars-cov-2 virus. in particular, it will be important to establish whether covid-19 is due to a completely natural chance occurrence where a presumed bat virus was transmitted to humans via an intermediate animal host or whether covid-19 has alternative origins. this information will be of paramount importance to help prevent any similar human coronavirus outbreak in the future. clinical trials for the treatment of coronavirus disease 2019 (covid-19): a rapid response to urgent need progress and prospects on vaccine development against sars-cov-2 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zoonotic aspects susceptibility of ferrets, cats, dogs, and other domesticated animals to sarscoronavirus 2 absence of sars-cov-2 infection in cats and dogs in close contact with a cluster of covid-19 patients in a veterinary campus infection and rapid transmission of sars-cov-2 in ferrets age-related rhesus macaque models of covid-19 simulation of the clinical and pathological manifestations of coronavirus disease 2019 (covid-19) in golden syrian hamster model: implications for disease pathogenesis and transmissibility structural basis for the recognition of sars-cov-2 by full-length human ace2 ace2 the janus-faced protein -from cardiovascular protection to severe acute respiratory syndrome-coronavirus and covid-19 structural basis of receptor recognition by sars-cov-2 comparative protein modelling by satisfaction of spatial restraints toward the estimation of the absolute quality of individual protein structure models ucsf chimera--a visualization system for exploratory research and analysis hdock: a web server for proteinprotein and protein-dna/rna docking based on a hybrid strategy the hdock server for integrated protein-protein docking high performance molecular simulations through multilevel parallelism from laptops to supercomputers electrostatics of nanosystems: application to microtubules and the ribosome open source drug discovery, c. & lynn, a. g_mmpbsa--a gromacs tool for high-throughput mm-pbsa calculations improving protein-ligand docking results with high-throughput molecular dynamics simulations isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov predicting the angiotensin converting enzyme 2 (ace2) utilizing capability as the receptor of sars-cov-2 silico analysis of intermediate hosts and susceptible animals of sars-cov-2. chemrxiv identifying sars-cov-2 related coronaviruses in malayan pangolins an overview of sars-cov-2 and animal infection sars-cov-2 spike protein favors ace2 from bovidae and cricetidae the pathogenicity of 2019 novel coronavirus in hace2 transgenic mice comparative pathogenesis of covid-19, mers and sars in a nonhuman primate model risks and benefits of gain-of-function experiments with pathogens of pandemic potential, such as influenza virus: a call for a science-based discussion a pneumonia outbreak associated with a new coronavirus of probable bat origin alignment of ace2 amino acid sequence from selected species. the sar-cov-2 spike protein binding region is highlighted in red we would like to thank harinda rajapaksha for assistance to optimise gromacs for this project. we would like to thank oracle corporation for providing their cloud computing resources through the oracle for research program for the modelling studies described herein. in particular, we wish to supplementary figure 4 . predicted and confirmed utilization of ace2 receptors by the sars-cov-2 spike protein based on sequence homology from qui et al. 41 key: cord-010938-12igesqw authors: patra, prasanta; mondal, niladri; patra, bidhan chandra; bhattacharya, manojit title: epitope-based vaccine designing of nocardia asteroides targeting the virulence factor mce-family protein by immunoinformatics approach date: 2019-08-21 journal: int j pept res ther doi: 10.1007/s10989-019-09921-4 sha: doc_id: 10938 cord_uid: 12igesqw nocardia asteroides is the main causative agent responsible for nocardiosis disease in immunocompromised patient viz. acquired immunodeficiency syndrome (aids), malignancy, diabetic, organ recipient and genetic disorders. the virulence factor and outer membrane protein pertains immense contribution towards the designing of epitopic vaccine and limiting the robust outbreak of diseases. while epitopic based vaccine element carrying b and t cell epitope along with adjuvant is highly immunoprophylactic in nature. present research equips immunoinformatics to figure out the suitable epitopes for effective vaccine designing. the selected epitopes vlgssvqta, vnielkpef and vvpsnlfav amino acids sequence are identified by hla-drb alleles of both mhc class (mhc-i and ii) molecules. simultaneously, these also accessible to b-cell, confirmed through the abcpred server. antigenic property expression is validated by the vaxijen antigenic prediction web portal. molecular docking between the epitopes and t cell receptor delta chain authenticate the accurate interaction between epitope and receptor with significantly low binding energy. easy access of epitopes to immune system also be concluded as transmembrane nature of the protein verified by using of tmhmm server. appropriate structural identity of the virulence factor mce-family protein generated through phyre2 server and subsequently validated by prosa and procheck program suite. the structural configuration of theses epitopes also shaped using distill web server. both the structure of epitopes and protein will contribute a significant step in designing of epitopic vaccine against n. asteroides. therefore, such immunoinformatics based computational drive definitely provides a conspicuous impel towards the development of epitopic vaccine as a promising remedy of nocardiosis the gram positive bacillus bacteria, nocardia asteroides causing nocardiosis targeting to immunocompromised patients in direct state (wilson 2012) . a total of 225 species have been estimated till now under the genera of nocardia (bennett et al. 2014) . it is an opportunistic pathogen responsible for several diseases in human as well as other vertebrate animal (moylett et al. 2003) . practically these bacteria serve as predominant causing factors for pulmonary infection. consequently it shows acute necrotizing pneumonia that also reflects in the inflammation of cutaneous and subcutaneous tissue. while the brain is recognized as most favored site for secondary infection in 25% patients amongst all victims (ellis and beaman 2002; quinones-hinojosa 2012) . subsequently n. asteroides is the prime cause of serious cerebral abscess as reported by researcher (fleetwood et al. 2000) . nocardiosis is distributed worldwide but it is most prevalence through the united states (patil et al. 2012; saubolle and sussland 2003) . infection of nocardiasis is influences by the sex and age of the patient. as noted the male female patient ratio 3:1 reflects that men are prone to nocardiasis rather than the female. recently, around 0.375 nocardia infection for every 100 000 individuals have been reported throughout the world (palmieri et al. 2014) . exclusively 500-1000 individuals are affected in united states every year in a random mode (sethy et al. 2016) . numerous endeavors are taken to overcome the fatal disease but till now the disease is prevalence as research data reported. the nocardiosis disease is predominant to the alcoholic, diabetic, cancer, aids and organ transplanted patient so, its need immediate control and monitoring supports. in present scenario records of these patients are quiet in frequent numbers. therefore, this disease already occupied great concern and requires immediate remedy by promising, cost effective ways. vaccination against the disease will be the finest and reliable option for controlling the disease outbreak (nichol et al. 2003) . a vaccine would have more effective if it can elicit both b and t-cell dependent immune responses. vaccine designing against any disease can be performed using two specific approaches, conventional approach and reverse vaccinological approach. within the present research plan the reverse vaccinological approach has been applied to design epitopic vaccine against n. asteroides as the conventional technique has some limitations. the conventional technique requires much time and also very expensive to formulate. furthermore, the technique applied to killed, attenuated or indolent pathogens for vaccine development which will prove deadly if the pathogen alters to its pathogenic appearance (khan et al. 2015) . besides from such conventional approach, the reverse vaccinology harnesses the immunoinformatics for developing vaccine against pathogenic diseases (vivona et al. 2008) . the advance approach involves several web portals and computational tools to predict the potential vaccine candidate. the fundamentality of reverse vaccinology showing it targets of protein derived epitopic sequence for novel vaccine generation. the reverse vaccinology provides easy and reliable techniques to find out the accurate b and t-cell epitopes (srivastava et al. 2019) . while the exomembrane and secreted protein of pathogens are the ideal for vaccine generation as these can easily interact with immune system (gourlay et al. 2013) . considering the bacterial proteins are foreign to host body, it will be further recognized as antigenic determinants. expression of both b and t-cell epitopes will enhance the immunogenicity driven by b and t-cell proliferation. the virulence factor of mce family protein found to be responsible for the virulency of n. asteroides and pathogenicity expression. the current research focuses on the computational analysis and identification of potential epitopes present within the bacterial mce family protein candidate. therefore, identified epitopes will be served as ideal component for future vaccine development against the infection of n. asteroides. in order to execute the subsequent research performance the supreme computational aided techniques has been employed. consequently the targeted protein sequence has been crucially analyzed through several authentic web prediction portals focus to extract the functional epitopes leading to narrative vaccine generation against nocardiosis disease. the amino acid sequences of a protein is the preliminary requirement for development of computer aided epitopic vaccine component. amino acid sequence of the virulence factor mce-family protein has been retrieve from ncbi protein database (coordinators 2017) . identified amino acids sequence was downloaded and carried out further computer based analysis particularly supported in fasta file format. b-cell epitope identification is one of the most crucial steps intended to development of epitopic vaccine (jamal et al. 2017) . potential b-cell epitope has been predicted employing the abcpred web server (saha and raghava 2006) . the server utilizes recurrent neural network technique to locate the specific b-cell epitopes present in targeted protein sequence. the server also uses five parameters to validate the predicted outputs. these factors are: q ppv (positive prediction value) = tp tp + fp tp, fn, tn and fp represents true positive, false negative, true negative and false positive output respectively (saha and raghava 2006) . the collective fasta sequence of targeted protein virulence factor mce-family protein has been submitted to the abcpred server. further, for the b-cell epitope identification, threshold score is set to 0.75 and window length is set to 20 in order to clarify the result. moreover an overlapping filter has been used to eliminate the overlapped epitopic prediction. in order to generate the immense immune response, an antigenic epitope necessarily to be accessible for the both mhc class type molecules (naz et al. 2015) along with the b-cell (barh et al. 2010; bhattacharya et al. 2019) . in support to the prediction of potent t-cell epitopes, the propred (singh and raghava 2001) and propred-i (singh and raghava 2003) servers are appointed for mhc-ii and mhc-i molecules correspondingly. the propred and propred-i web servers predict epitopes that can be recognized by 51 mhc-ii and 47 mhc-i alleles (mustafa and shaban 2006) . both the server applied matrices prediction algorithm method (lafuente and reche 2009; lin et al. 2008) to find out the potent t-cell epitope. current research work predicted epitopes for b-cell, are submitted to the propred and propred-i server with default parameters to identify the common epitopes anticipate by both b and t-cell. predicted common epitopes for both the b and t-cell are further investigated for their antigenic property through the vaxijen (v. 2.0) server (doytchinova and flower 2007) . the server requires a query sequence of amino acids to evaluate the antigenic propensity with 70-97% accuracy level (dimitrov et al. 2016) . the server also specifies the field of antigenic prediction in five subsequent target group like-bacteria, virus, tumor, parasite and fungal (zaharieva et al. 2017 ). the auto cross covariance (acc) method has been also incorporated to convert the submitted sequence into uniform length of amino acids chain (doytchinova and flower 2007) . the acc is estimated by the formulas (1) where 'j' is the z-scale (j = 1, 2, 3), 'n' is the amino acids present in sequence (i = 1, 2,…n) and 'l' is the lag (l = 1, 2,…l). the eq. (2) is used when there are two different zscales. the query sequence has been submitted to the server by selecting bacteria as target organism. in order to obtain more precise result, a threshold value of 1.0 has been set instead of default threshold value (0.4). the common epitopes of b and t-cell having antigenic property are promising candidate for the epitopic vaccine designing. the epitopes predicted by abcpred, propred and propred-i server also having vaxijen predicted antigenic nature are selected for the vaccine development. three dimensional conformation of the epitope have its significant role leading to vaccine development. it is the most vibrant object for investigation of epitope-antibody docking system (alam et al. 2016 ). due to short amino acid length of the epitope conventional modeling servers are not in use so, distill 2.0 (baú et al. 2006 ) server have been introduced for the process. the server uses two sets of bidirectional recurrent neural networks technique (pollastri et al. 2002) . for the result output, filtering is applied in two successive stages. in first stage network input has been amplified through prophecies and averaged over several adjacent windows. if σj = (αj, βj, ϒj) signifies output of j, subsequent to helix, strand and coil prediction reflects the inputs. in second stage network, i j is the input instead of j. in which k f = j + f(2ω + 1), 2ω +1 represents window size (ω = 7) and 2p + 1 represents window numbers (p = 7) taken into account (pollastri and mclysaght 2004) . pbd file of the experimental epitopic sequence is provided by the server as result element. the three dimensional (3d) architecture of a protein plays essential role in protein function and stability (roy et al. 2012; willard et al. 2003) . so, unrevealing the protein structural configuration is quiet necessary and purposeful for the study. the virulence factor mce-family protein structure of n. asteroides is unavailable in pdb database (berman et al. 2000) . because of the unavailability of pdb structure, the phyre2 (kelley et al. 2015) web server has been introduced to generate the protein structural data. the phyre2 server applies the hidden markov model alignment to detect structure of target protein through hhsearch, open-source software package (kumar and jena 2014; nema and pal 2013) . this server also uses poing folding simulation to figure out the non allied part of the protein sequence (nema and pal 2013) . amino acid sequence of virulence factor mce-family protein has been submitted to the server and a zip file containing predictions is comes out as systematic result component. justification of a predicted protein model is very much fundamental aspect for establishing the specified protein 3d configuration (laskowski et al. 2006; rodriguez et al. 1998) . to properly justify the model quality, two web portals are methodologically assigned. the procheck web server (laskowski et al. 1993) , to analyze stereochemical properties of the protein model emphasizing on the torsion angle of c α atoms of amino acids. a pdb file of the targeted protein model has to provide to the online web server. the server offered a ramachandran plot of all available amino acids, that also vital for the validation of targeted protein model (laskowski et al. 1993 ). the prosa-web is introduce to calculate the z score of the protein model and furnishes a plot of the protein model within all known protein structure (wiederstein and sippl 2007) . this server also furnishes energy plot for perfect assessment of the model quality. while the negative energy value of the amino acid residues indicates a good model quality of examined protein (belkina et al. 2001; wiederstein and sippl 2007) . transmembrane helix prediction of the protein model was performed through tmhmm (ver. 2.0) server, the standalone software package (sonnhammer et al. 1998 ). the server predicts transmembrane helices based on a hidden markov model method with 97-98% accuracy of result (krogh et al. 2001) . fasta sequence of the protein has been submitted to the server and the server provides lists of transmembrane prediction along with an apparent graphical interpretation. molecular docking between epitope and antibody component is the critical for proper functioning of vaccine candidate. in order to carry out molecular docking the patchdock server have been appointed in present research (duhovny et al. 2002) . the geometric contour complementarity method has been applied for docking the peptide and protein sequences (schneidmanduhovny et al. 2003) . hence, the pdb file or pdb code of the protein and epitope has to be submitted to the server for molecular docking analysis. the accurate docked complexes ranked according to geometric shape complimentary score and presented along with ace value, interface area and pdb data (schneidmanduhovny et al. 2005) . in molecular docking the aggregated desolvation energy of atom pairs termed as ace. atom set inside s 1 and s 2 with threshold distance d, the ace is: here |s − t| signifies euclidean distance amid s and t, t [s,t] signifies the prearranged score of s and t atom pair. t [s,t] is estimated using the subsequent formula: here 0 signifies the solvent. (n s,t ) is number of s,t connection and the number of s-0 connection (n t,0 ) are suitable connection numbers of recognized complexes. moreover, cs,t and cs,0 are signifies as the possible numbers of s,t connection and s-0 connection (guo et al. 2012) . the amino acid sequence of the protein virulence factor mce-family protein has been retrieved from the ncbi protein database. the virulence factor mce-family protein consisting of 493 amino acids and the sequence was downloaded in fasta format (genbank: sfl64340.1) (benson et al. 2012) . afterwards, this sequence processed through several web servers to find out the potential epitopes for promising vaccine development. abcpred server predicts 20 linear epitopes (table 1) within the virulence factor mce-family protein of 20 amino acids length (window length). the sensitivity, specificity and accuracy are 57.14%, 71.57% and 64.26% correspondingly at window length of 20 (saha and raghava 2006) . a score is also assigned against predicted epitopes and are ranked accordingly (han et al. 2015) . predicted sequence with the higher score secures better chance to be an potent epitope (jones and carter 2014) . the predicted epitopes are superior because of high threshold score of 0.75. the identified b-cell epitope were further investigated for detection of prospective t-cell epitope through propred and propred-i to improve immunogenicity (oprea and antohe 2013). the b-cell epitopes those are recognized by both mhc molecules are taken under additional consideration. the epitopes common to both mhc classes and b-cell are listed in the table 2 which may be recognized for ideal vaccine candidate. following mhc alleles are also enlisted in the supplementary table. the propred server predicts epitopes of only nine amino acids moreover, the common epitopes comprised of 9mer sequence. manifestation of antigenicity is the prime criterion of a novel epitope in immunobiological aspect (chen et al. 2007 ). the common b and t-cell epitopes are successively validated against antigenic propensity via vaxijen server; table 3 presents the epitopes along with relevant antigenic score. the 9mer epitopes availing antigenic score beyond the threshold value 1.0 secures antigenic characteristics. in that consequence 10 out of 13 9meric epitopes proved futile to express antigenicity. three 9mers vlgssvqta, vnielkpef and vvpsnlfav having antigenic score 1.1110, 2.4569 and 1.0810 respectively proved to secure antigenic propensity. the three 9mer b and t-cell epitopes vlgssvqta, vnielkpef and vvpsnlfav having antigenicity (table 4 ) will be easily accessible to the immune system (comerford et al. 1991) . these epitopes can be utilized for the future prospective of epitopic vaccine development. the epitopes will elicit strong immune response when administrated in the body as potent vaccine element. the selected epitopes vlgssvqta, vnielkpef and vvpsnlfav are submitted to distill server (2.0). the distill server contributes five models of each of the epitope in pdb format. only the top ranked model of epitopes is taken under consideration. ucsf chimera (ver. 1.13.1) program has been implemented for visualization of the selected pdb files of the targeted epitopes (pettersen et al. 2004 ). subsequently the generated images are retrieved and presented in the fig. 1a , b, c in order of vlgssvqta, vnielkpef and vvpsnlfav protein. the virulence factor mce-family protein lacking pdb entry, for that reason the structure of protein has been depicted by homology modeling. modeling of virulence factor mce-family protein has been performed via hidden markov model in phyre2 server. amongst 493 amino acids of virulence factor mce-family protein 41%, 11%, and 4% amino acid construct α-helix, β-strand and transmembrane helix respectively. phyre2 predicted pdb data of the protein processed through ucsf chimera program in order to generate structural image. figure 2 showing the three dimensional conformation of virulence factor mce-family protein in n. asteroids. the topology of virulence factor mce-family protein is justified using prosa and procheck web servers. according to the prediction of procheck generated ramachandran plot (fig. 3) , 79.3% of residues exist in most favoured region and only 1.7% exists in disallowed region (table 5 ). existence of only two non-glycine and non-proline residues in disallowed region established the model quality. prosa estimated z score (− 2.38) of the model resides within the range of experimentally proved protein structures (fig. 4 ) (sharma and jaiswal 2009) . as presented in fig. 5 , most of the residues of the model possess negative energy value only few have positive value then the model is justified (bodade et al. 2010 ). pdb file of vlgssvqta, vnielkpef and vvpsnl-fav epitopes and variable domain of t cell receptor delta chain (pdb id:1tvd) are submitted to patchdock algorithm based server (li et al. 1998) . the server provides 20 docking complex for each epitope ranking them against geometric shape complimentary score. only the top ranked complex of each epitopes is picked for computational analysis. epitope along with score, area of interface and ace value are listed in table 6 . the significantly low ace value of docking complexes indicated elevated reactivity between epitope and (guo et al. 2012; lavi et al. 2013 ). the docking of selected epitopes and t cell receptor confirms that the epitopes will accessible to immune system and generate specific immunogenicity. rational identification, authentication and in sillico analysis of epitopic components facilitates the successful generation of novel vaccines. as noted the vaccine element performs a key role not only recovering from the infections but also controlling the future disease outbreak. hence, the nocardiosis has carried a dreadful influence over the immunocompromised patient as they manifest high level of disease susceptibility. particularly the organ transplant patients encounter the greater chances of nocardiosis. the casualty of organ recipient patients for nocardia infection often reflects in higher degree (husain et al. 2002) . consequently, the causing pathogen also highly resistance to several well known, market available antibiotics (husain et al. 2002) . greater survivability, infections and antibiotic resistance nature of the bacterial pathogen is one of the greatest apprehensions for medical biotechnologist. present advanced, computational analysis assisted research emphasizes much on the discovery of effective vaccine against to n. asteroides. the virulence factor mce family protein of n. asteroides is responsible for the pathogenesis of the bacteria to other organism. hence this protein served as the supreme component for designing epitopic vaccine against nocardiosis. the virulence factor mce family protein is processed through several bioinformatic tools to execute the suitable epitopes having antigenicity. the transmembrane localization of the protein permits it to intermingle with exact immune system. the transmembrane helix prediction server tmhmm server (ver. 2.0) attests the exomembrane localization of the protein component. both the b and t-cell epitopes are considerate for obtaining the maximum immune response through humoral and cell mediated immunity. the epitopes were identified through sequence based prediction method using the several web prediction servers. after retrieving the protein sequence of virulence factor mce family protein (genbank: sfl64340.1) from ncbi database, it is processed through the abcpred server for b-cell epitope motif. the 20 numbers of b-cell epitopes having considering threshold value are again submitted to propred and propred-i servers simultaneously for mhc-ii and mhc-i binding allele (barh et al. 2010) . the common epitopes are shortened to only 9mers because of propred prediction module provides only 9meric epitopes. the epitopes vlgss-vqta, vnielkpef and vvpsnlfav were selected for vaccine designing after accurate validation against antigenic property through vaxijen server. these are highly antigenic (vlgssvqta = 1.1110, vnielkpef = 2.4569 and vvpsnlfav = 1.0810) being laid over the customized threshold antigenic score. this research design reveled that, three epitopes are the finest vaccine components as identified by b-cell and mhc molecules. the motif of the particular epitopes was mapped out through distill 2.0 for conformational uniqueness and molecular docking. intend to proper binding of epitopes with the t cell receptor delta chain reflects through the significantly lower ace values. the structural profile of virulence factor mce family protein also been mapped out via phyre2 server that will be fundamental for therapeutics to design the vaccine. the epitopes vlgssvqta, vnielkpef and vvpsnlfav will be much more effectual for perfect designing of epitopic vaccine against n. asteroides limiting nocardiosis and subsequent casualties linked with it. specialized immunoinformatic studies focuses the virulence factor mce-family protein of n. asteroides established its significances lead to expression of bacterial pathogenicity. existing research will be surely valuable in modern therapeutics purposes, to resist the nocardiosis outbreak. particular manifestation of antigenicity by the common b and t-cell epitopes (vlgssvqta, vnielk-pef and vvpsnlfav) substantiates the critical aptitude to generate humoral and cell mediated immunity. consequently, the targeted epitopes assist for easy interaction with the immune receptors favor to transmembrane localization of protein element. literal structural signature of considerable protein along with its epitopes served as decisive factor for novel vaccine development. however, the epitopes requires substantial in vivo and in vitro justification for accurate refinement to generate finest vaccine component restricting the nocardia infection. such computer aided research techniques are also highly influential and efficient for designing of desired epitopic vaccine against several associated diseases in light of immunoinformatics. from zikv genome to vaccine: in silico approach for the epitope-based peptide vaccine against zika virus envelope glycoprotein a novel strategy of epitope design in neisseria gonorrhoeae distill: a suite of web servers for the prediction of one-, two-and three-dimensional structural features of proteins modelling of three-dimensional structures of cytochromes p450 11b1 and 11b2 mandell, douglas, and bennett's principles and practice of infectious diseases: 2-volume set the protein data bank computational characterization of epitopic region within the outer membrane protein candidate in flavobacterium columnare for vaccine development homology modeling and docking study of xanthine oxidase of arthrobacter sp. xl26 prediction of linear b-cell epitopes using amino acid pair antigenicity scale identification of t-and b-cell epitopes of the e7 protein of human papillomavirus type 16 database resources of the national center for biotechnology information pymol: an open-source molecular graphics tool a cohesive and integrated platform for immunogenicity prediction. vaccine design vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines efficient unbound docking of rigid molecules murine polymorphonuclear neutrophils produce interferon-γ in response to pulmonary infection with nocardia asteroides nocardia asteroides cerebral abscess in immunocompetent hosts: report of three cases and review of surgical recommendations exploiting the burkholderia pseudomallei acute phase antigen bpsl2765 for structure-based epitope discovery/design in structural vaccinology protein-protein binding site identification by enumerating the configurations identification of immunodominant b-cell epitope regions of reticulocyte binding proteins in plasmodium vivax by protein microarray based immunoscreening nocardia infection in lung transplant recipients identification of b-cell epitope of leishmania donovani and its application in diagnosis of visceral leishmaniasis prediction of b-cell epitopes in listeriolysin o, a cholesterol dependent cytolysin secreted by listeria monocytogenes the phyre2 web portal for protein modeling, prediction and analysis epitopebased peptide vaccine design and target site depiction against ebola viruses: an immunoinformatics study predicting transmembrane protein topology with a hidden markov model: application to complete genomes understanding rifampicin resistance in tuberculosis through a computational approach prediction of mhc-peptide binding: a systematic and comprehensive overview pro-check: a program to check the stereochemical quality of protein structures procheck: validation of protein-structure coordinates detection of peptide-binding sites on protein surfaces: the first step toward the modeling and targeting of peptidemediated interactions structure of the vδ domain of a human γδ t-cell antigen receptor evaluation of mhc class i peptide binding prediction servers: applications for vaccine research clinical experience with linezolid for the treatment of nocardia infection propred analysis and experimental evaluation of promiscuous t-cell epitopes of three major secreted antigens of mycobacterium tuberculosis identification of putative vaccine candidates against helicobacter pylori exploiting exoproteome and secretome: a reverse vaccinology based approach exploration of freely available web-interfaces for comparative homology modelling of microbial proteins influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly reverse-vaccinology strategy for designing t-cell epitope candidates for staphylococcus aureus endocarditis vaccine soil-acquired cutaneous nocardiosis on the forearm of a healthy male contracted in a swamp in rural eastern virginia ucsf chimera-a visualization system for exploratory research and analysis porter: a new, accurate server for protein secondary structure prediction improving the prediction of protein secondary structure in three and eight classes using recurrent neural networks and profiles schmidek and sweet: operative neurosurgical techniques 2-volume set: indications, methods and results in silico identification of catalytic residues in azobenzene reductase from bacillus subtilis and its docking studies with azo dyes homology modeling, model and software evaluation: three related resources cofactor: an accurate comparative algorithm for structure-based protein function annotation prediction of continuous b-cell epitopes in an antigen using recurrent neural network membrane association and epitope recognition by hiv-1 neutralizing anti-gp41 2f5 and 4e10 antibodies nocardiosis: review of clinical and laboratory experience taking geometry to its edge: fast unbound rigid (and hinge-bent) docking patchdock and symmdock: servers for rigid and symmetric docking pleuroplumonary nocardiosis in an immunocompetent host egf domain ii of protein pb28 from plasmodium berghei interacts with monoclonal transmission blocking antibody 13.1 propred: prediction of hla-dr binding sites propred1: prediction of promiscuous mhc class-i binding sites a hidden markov model for predicting transmembrane helices in protein sequences design of novel multi-epitope vaccines against severe acute respiratory syndrome validated through multistage molecular interaction and dynamics computer-aided biotechnology: from immuno-informatics to reverse vaccinology prosa-web: interactive web service for the recognition of errors in three-dimensional structures of proteins vadar: a web server for quantitative evaluation of protein structure quality nocardiosis: updates and clinical overview epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies immunogenicity prediction by vaxijen: a ten year overview key: cord-023726-2fduzqyb authors: strauss, james h.; strauss, ellen g. title: the structure of viruses date: 2012-07-27 journal: viruses and human disease doi: 10.1016/b978-0-12-373741-0.50005-2 sha: doc_id: 23726 cord_uid: 2fduzqyb nan virus particles, called virions, contain the viral genome encapsidated in a protein coat. the function of the coat is to protect the genome of the virus in the extracellular environment as well as to bind to a new host cell and introduce the genome into it. viral genomes are small and limited in their coding capacity, which requires that three-dimensional virions be formed using a limited number of different proteins. for the smallest viruses, only one protein may be used to construct the virion, whereas the largest viruses may use 30 or more proteins. to form a three-dimensional structure using only a few proteins requires that the structure must be regular, with each protein subunit occupying a position at least approximately equivalent to that occupied by all other proteins of its class in the final structure (the principle of quasi-equivalence), although some viruses are now known to violate the principle of quasi-equivalence. a regular three-dimensional structure can be formed from repeating subunits using either helical symmetry or icosahedral symmetry principles. in the case of the smallest viruses, the final structure is simple and quite regular. larger viruses with more proteins at their disposal can build more elaborate structures. enveloped viruses may be quite regular in construction or may have irregular features, because the use of lipid envelopes allows irregularities in construction. selected families of vertebrate viruses are listed in table 2 .1 grouped by the morphologies of the virions. also shown for each family is the presence or absence of an envelope in the virion, the triangulation number (defined later) if the virus is icosahedral, the morphology of the nucleocapsid or core, and figure numbers where the structures of members of a family are illustrated. electron micrographs of five dna viruses belonging to different families and of five rna viruses belonging to different families are shown in fig. 2 .1. the viruses chosen represent viruses that are among the largest known and the smallest known, and are all shown to the same scale for comparison. for each virus, the top micrograph is of a virus that has been negatively stained, the middle micrograph is of a section of infected cells, and the bottom panel shows a schematic representation of the virus. the structures of these and other viruses are described next. helical viruses appear rod shaped in the electron microscope. the rod can be flexible or stiff. the best studied example of a simple helical virus is tobacco mosaic virus (tmv). the tmv virion is a rigid rod 18 nm in diameter and 300 nm long ( fig. 2.2b ). it contains 2130 copies of a single capsid protein of 17.5 kda. in the right-hand helix, each protein subunit has six nearest neighbors and each subunit occupies a position equivalent to every other capsid protein subunit in the resulting network ( fig. 2.2a) , except for those subunits at the very ends of the helix. each capsid molecule binds three nucleotides of rna within a groove in the protein. the helix has a pitch of 23 å and there are 16 1 /3 subunits per turn of the helix. the length of the tmv virion (300 nm) is determined by the size of the rna (6.4 kb). many viruses are constructed with helical symmetry and often contain only one protein or a very few proteins. the popularity of the helix may be due in part to the fact that the length of the particle is not fixed and rnas or dnas of different sizes can be readily accommodated. thus the genome size is not fixed, unlike that of icosahedral viruses. virions can be approximately spherical in shape, based on icosahedral symmetry. since the time of euclid, there have been known to exist only five regular solids in which each face of the solid is a regular polygon: the tetrahedron, the cube, the octahedron, the dodecahedron, and the icosahedron. the icosahedron has 20 faces, each of which is a regular triangle, and thus each face has threefold rotational symmetry ( fig. 2.3a ). there are 12 vertices where 5 faces meet, and thus each vertex has fivefold rotational symmetry. there are 30 edges in which 2 faces meet, and each edge possesses twofold rotational symmetry. thus the icosahedron is characterized by twofold, threefold, and fivefold symmetry axes. the dodecahedron, the next simpler regular solid, has the same symmetry axes as the icosahedron and is therefore isomorphous with it in symmetry: the dodecahedron has 12 faces which are regular pentagons, 20 vertices where three faces meet, and 30 edges with twofold symmetry. the three remaining regular solids have different symmetry axes. the vast majority of regular viruses that appear spherical have icosahedral symmetry. in an icosahedron, the smallest number of subunits that can form the three-dimensional structure is 60 (5 subunits at each of the 12 vertices, or viewed slightly differently, 3 units on each of the 20 triangular faces). some viruses do in fact use 60 subunits, but most use more subunits in order to provide a larger shell capable of holding more nucleic acid. the number of subunits in an icosahedral structure is 60t, where the permissible values of t are given by t = h 2 + hk + k 2 , where h and k are integers and t is called the triangulation number. permissible triangulation numbers are 1, 3, 4, 7, 9, 12, 13, 16 , and so forth. a subunit defined in this way is not necessarily formed by one protein molecule, although in most cases this is how a structural subunit is in fact formed. some viruses that form regular structures that are constructed using icosahedral symmetry principles do not possess true icosahedral symmetry. in such cases they are said to have pseudo-triangulation numbers. examples are described later. a protein subunit, shown as blue trapezoids labeled "a." the twofold, threefold, and fivefold axes of symmetry are shown in yellow. this is the largest assembly in which every subunit is in an identical environment. (b) schematic representation of the subunit building block found in many rna viruses, known as the eightfold β barrel or β sandwich. the β sheets, labeled b through i from the n terminus of the protein, are shown as yellow and red arrows; two possible α helices joining these sheets are shown in green. some proteins have insertions in the c-d, e-f, and g-h loops, but insertions are uncommon at the narrow end of the wedge (at the fivefold axis). from granoff and webster (1999) vol. 3, color plate 31. [originally from j. johnson (1996) ]. structural studies of viruses have shown that the capsid proteins that form the virions of many plant and animal icosahedral viruses have a common fold. this fold, an eightstranded antiparallel β sandwich, is illustrated in fig. 2.3b . the presence of a common fold suggests that these capsid proteins have a common origin even if no sequence identity is detectable. the divergence in sequence while maintaining this basic fold is illustrated in fig. 2 .4, where capsid proteins of three viruses are shown. sv40 (family polyomaviridae), poliovirus (family picornaviridae), and bluetongue virus (family reoviridae) are a dna virus, a single-strand rna virus, and a double-strand rna virus, respectively. their capsid proteins have insertions into the basic eight-stranded antiparallel β-sandwich structure that serve important functions in virus assembly. however, they all possess a region exhibiting the common β-sandwich fold and may have originated from a common ancestral protein. thus, once a suitable capsid protein arose that could be used to construct simple icosahedral particles, it may ultimately have been acquired by many viruses. the viruses that possess capsid proteins with this fold may be related by descent from common ancestral viruses, or recombination may have resulted in the incorporation of this successful ancestral capsid protein into many lines of viruses. because the size of the icosahedral shell is fixed by geometric constraints, it is difficult for a change in the size of a viral genome to occur. a change in size will require a change in the triangulation number or changes in the capsid proteins sufficient to produce a larger or smaller internal volume. in either case, the changes in the capsid proteins required are relatively slow to occur on an evolutionary timescale and the size of an icosahedral virus is "frozen" for long periods of evolutionary time. for this reason, as well as for other reasons, most viruses have optimized the information content in their genomes, as will be clear when individual viruses are discussed in the following chapters. cryoelectron microscopy has been used to determine the structure of numerous icosahedral viruses to a resolution of 7 to 25 å. for this, a virus-containing solution on an electron microscope grid is frozen very rapidly so that the sample is embedded in amorphous frozen water. the sample must be maintained at liquid nitrogen temperatures so that ice crystals do not form and interfere with imaging. unstained, slightly out-of-focus images of the virus are captured on bluetongue virus vp7 t = 13 figure 2.4 structure of three vertebrate virus protein subunits that assemble into icosahedral shells. the n termini and c termini are labeled with the residue number in parenthesis. the β barrels are shown as red arrows, α helices are gray coils, and the subunit regions involved in quasi-symmetric interactions that are critical for assembly are colored green. sv40 and pv have triangulation numbers of "pseudo-t=7" or p = 7 and "pseudo-t=3" or p = 3, respectively. adapted from granoff and webster (1999), vol. 3, plate 32. film, or more recently captured electronically, using a low dose of electrons. these images are digitized and the density measured. mathematical algorithms that take advantage of the symmetry of the particle are used to reconstruct the structure of the particle. a gallery of structures of viruses determined by cryoelectron microscopy is shown in fig. 2 .5. all of the images are to scale so that the relative sizes of the virions are apparent. the largest particle is the nucleocapsid of herpes simplex virus, which is 1250 å in diameter and has t=16 symmetry (the virion is enveloped but only the nucleocapsid is regular figure 2.5 gallery of three-dimensional reconstructions of icosahedral viruses from cryoelectron micrographs. all virus structures are surface shaded and are viewed along a twofold axis of symmetry except ross river, which is viewed along a three-fold axis. all of the images are of intact virus particles except for the herpes simplex structure, which is of the nucleocapsid of the virus. most of the images are taken from baker et al. (1999) , except the images of ross river virus and of dengue virus, which were kindly provided by drs. r. j. kuhn and t. s. baker. rest are not enveloped). b19 parvovirus has t=1. the general correlation is that larger particles are constructed using higher triangulation numbers, which allows the use of larger numbers of protein subunits. larger particles accommodate larger genomes. because the simplest viruses are regular structures, they will often crystallize, and such crystals may be suitable for x-ray diffraction. many viruses formed using icosahedral symmetry principles have been solved to atomic resolution, and a discussion of representative viruses that illustrate the principles used in construction of various viruses is presented here. among t=3 viruses, the structures of several plant viruses, including tomato bushy stunt virus (tbsv) (genus tombusvirus, family tombusviridae), turnip crinkle virus (tcv) (genus carmovirus, family tombusviridae), and southern bean mosaic virus (sbmv) (genus sobemovirus, not yet assigned to family), have been solved. all three of these viruses have capsid proteins possessing the eight-stranded antiparallel β sandwich. t=3 means that 180 identical molecules of capsid protein are utilized to construct the shell. the structures of two insect viruses that are also simple t=3 structures have also been solved. as an example of these simple structures, the t=3 capsid of the insect virus, flock house virus (family nodaviridae), is illustrated in fig. 2 .6. the 180 subunits in these t=3 structures interact with one another in one of two different ways, such that the protein shell can be thought of as being composed of an assembly of 60 ab dimers and 30 cc dimers (fig. 2.6a ). the bond angle between the two subunits of the dimer is more acute in the ab dimers than in the cc dimers (figs. 2.6b and c). for the plant viruses, there are n-terminal and c-terminal extensions from the capsid proteins that are involved in interactions between the subunits and with the rna. the n-terminal extensions have a positively charged, disordered domain for interacting with and neutralizing the charge on the rna and a connecting arm that interacts with other subunits. in the case of the cc dimers, the connecting arms interdigitate with two others around the icosahedral threefold axis to form an interconnected internal framework. in the case of the ab conformational dimer, the arms are disordered, allowing sharper curvature. for flock house virus, the rna plays a role in controlling the curvature of the cc dimers, as illustrated in fig. 2 .6. b. proteins that make up a triangular face are only quasi-equivalent. the angle between the a and b 5 units (shown with a red oval and in diagram (c) is more acute than that along the c-c 2 edge, shown with a blue oval, and diagram (b). this difference in the angles is due to the presence of an rna molecule located under the c-c 2 edge. from johnson (1996) , with permission. the structures of several picornaviruses and of a plant comovirus (cowpea mosaic virus) have also been solved to atomic resolution. the structures of these viruses are similar to those of the plant t=3 viruses, but the 180 subunits that form the virion are not all identical. a comparison of the structure of a t=3 virus with those of poliovirus and of cowpea mosaic virus is shown in fig. 2 .7. poliovirus has 60 copies of each of three different proteins, whereas the comovirus has 60 copies of an l protein (each of which fills the niche of two units) and 60 copies of an s protein. all three poliovirus capsid proteins have the eight-stranded antiparallel β-sandwich fold. in the comoviruses, the l protein has two β-sandwich structures fused to form one large protein, and the s protein is formed from one sandwich. the structures of the picornavirus and comovirus virions are called pseudo-t=3 or p=3, since they are not true t=3 structures. the picornavirus virion is 300 å in diameter. the 60 molecules of each of the three different proteins have different roles in the final structure, as illustrated in fig. 2 .8, in which the structure of a rhinovirus is shown. notice that five copies of vp1 are found at each fivefold axis (compare fig. 2 .7 with fig. 2.8) . vp1, vp2, and vp3 are structurally related to one another, as stated, all possessing the common βsandwich fold. there exists a depression around each fivefold axis of rhinoviruses that has been termed a "canyon." this depression is believed to be the site at which the virus interacts with the cellular receptor during entry, as illustrated in fig. 2 .9. this interaction is thought to lead to conformational changes that open a channel at the fivefold axis, through which vp4 is extruded, followed by the viral rna. the structures of both mouse polyomavirus and of sv40 virus, two members of the family polyomaviridae, have been solved to atomic resolution. both viruses possess pseudo-t=7 icosahedral symmetry. although t=7 l vp1 vp2 vp3 vp1 vp2 vp1 vp2 vp3 vp1 vp2 vp3 vp1 vp2 vp3 vp1 vp2 vp3 vp1 vp2 vp3 vp1 vp2 vp3 vp1 vp2 vp3 vp1 vp2 vp3 vp1 vp2 vp3 vp1 vp2 vp3 vp3 figure 2.7 arrangement of the coat protein subunits of comoviruses compared with those of simple t=3 viruses and picornaviruses. in simple viruses, the asymmetric unit contains three copies of a single protein β sandwich, labeled a, b, and c in order to distinguish them. in picornaviruses such as poliovirus the asymmetric unit is made up of three similar but not identical proteins, all of which have the β-sandwich structure. in comoviruses such as cowpea mosaic virus, two of the β-sandwich subunits are fused to give the l protein. adapted from granoff and webster (1999) , p. 287. symmetry would require 420 subunits, these viruses contain only 360 copies of a major structural protein known as vp1. these 360 copies are assembled as 72 pentamers. twelve of the 72 pentamers lie on the fivefold axes and the remaining 60 fill the intervening surface in a closely packed array ( fig. 2 .10). these latter pentamers are thus sixfold coordinated and the proteins in the shell are not all in quasi-equivalent positions, a surprising finding for our understanding of the principles by which viruses can be constructed. the pentamers are stabilized by interactions of the β sheets between adjacent monomers in a pentamer ( fig. 2.10c ). the pentamers are then tied together by c-terminal arms of vp1 that invade monomers in an adjacent pentamer (figs. 2.10b and c). because each pentamer that is sixfold coordinated has five c-terminal arms to interact with six neighboring pentamers, the interactions between monomers in different pentamers are not all identical ( fig. 2.10b ). flexibility in the c-terminal arm allows it to form contacts in different ways. members of the reovirus family are regular t=13 icosahedral particles. they are composed of two or three concentric protein shells. cryoelectron microscopy has been used to solve the structure of one or more members of three genera within the reoviridae, namely reovirus, rotavirus, and orbivirus, to about 25-å resolution. structures of a reovirus and of a rotavirus are shown in fig. 2 .5. the complete structure of virions has not been determined because of their large size, but in a remarkable feat the atomic structure of the core of bluetongue virus (genus orbivirus) has now been solved. this is the largest structure determined to atomic resolution to date. solution of the structure was possible because the virus particle had been solved to 25 å by cryoelectron microscopy, and the structures of a number of virion proteins had been solved to atomic resolution by x-ray diffraction. fitting the atomic structure of the proteins into the 25-å structure gave a preliminary reconstruction at high resolution, which allowed the interpretation of the x-ray data to atomic resolution. core particles are formed following infection, when the outer layer is proteolytically cleaved (described in more detail in chapter 5). the structure of the inner surface of the bluetongue virus core is shown in fig. 2 .11a and of the outer surface in fig. 2 .11b. the outer surface is formed by 780 copies of a single protein, called vp7, in a regular t=13 icosahedral lattice. the inner surface is surprising, however. it is formed by 120 copies of a single protein, called vp3. these 120 copies have been described as forming a t=2 five-fold axis lattice. because t=2 is not a permitted triangulation number, these 120 copies, strictly speaking, form a t=1 lattice in which each unit of the lattice is composed of two copies of vp3. however, the interactions are not symmetrical, leading to the suggested terminology of t=2. it has been suggested that the inner core furnishes a template for the assembly of the t=13 outer surface. the reasoning is that a t=13 structure may have difficulty in forming, whereas the t=2 (or t=1) structure could form readily. in this model, the threefold symmetry axis of the inner surface could serve to nucleate vp7 trimers and organize the t=13 structure. cryoelectron microscopy has also been applied to adenoviruses, which have a triangulation number of 25 or pseudo-25. various interpretations of the structure of adenoviruses, both schematic and as determined by microscopy or crystallography, are shown in fig. 2 .12. three copies of a protein called the hexon protein associate to form a structure called a hexon ( fig. 2.12c ). the hexon is the basic building block of adenoviruses. five hexons, called peripentonal hexons, surround each of the 12 vertices of the icosahedron (which, as has been stated, have fivefold rotational symmetry). between the groups of peripentonal hexons are found groups of 9 hexons, which are sixfold coordinated. each group of 9 hexons forms the surface of one of the triangular faces. thus, there are 60 peripentonal hexons and 180 hexons in groups of nine. the structure of the hexon trimer has been solved to atomic resolution by x-ray crystallography ( fig. 2.12c ). the hexon protein has two eight-strand β sandwiches to give the trimer an approximately sixfold symmetry (and each hexon protein fills the role of two symmetry units). there are long loops that intertwine to form a triangular top. these structures can be fitted uniquely into the envelope of density determined by cryoelectron microscopy, which produces a structure refined to atomic resolution for most of the capsid. the minor proteins can be fitted into this structure. from the 12 vertices of the icosahedron project long fibers that are anchored in the surface by a unit called the penton base. each fiber terminates in a spherical extension that forms an organ of attachment to a host cell (fig. 2.12a ). the length of the fiber differs in the different adenoviruses. in addition to the nonenveloped viruses that possess relatively straightforward icosahedral symmetry or helical symmetry, many viruses possess more complicated symmetries made possible by the utilization of a large number of structural proteins to form the virion. the tailed bacteriophages are prominent examples of this ( fig. 2.13) . some of the tailed bacteriophages possess a head that is a regular icosahedron (or, in at least one case, an octahedron) connected to a tail that possesses helical symmetry. other appendages, such as baseplates, collars, and tail fibers, may be connected to the tail. other tailed bacteriophages have heads that are assembled using more complicated patterns. for example, the t-even bacteriophages have a large head, which can be thought of as being formed of two hemi-icosahedrons possessing regular icosahedral symmetry, which are elongated in the form of a prolate ellipsoid by subunits arranged in a regular net connecting the two icosahedral ends of the head of the virus. many animal viruses and some plant viruses are enveloped; that is, they have a lipid-containing envelope surrounding a nucleocapsid. the lipids are derived from the host cell. although there is some selectivity and reorganization of lipids during virus formation, the lipid composition in general mirrors the composition of the cellular membrane from which the envelope was derived. however, the proteins in the nucleocapsid, which may possess either helical or icosahedral symmetry, and the proteins in the envelope are encoded in the virus. the protein-protein interactions that are responsible for assembly of the mature enveloped virions differ among the different families and the structures of the resulting virions differ. the virions of alphaviruses, and of flaviviruses, are uniform the essential features of the orbivirus native core particle. the asymmetric unit is indicated by the white lines forming a triangle and the fivefold, threefold, and twofold axes are marked. (a) the inner capsid layer of the bluetongue virus (btv) core is composed of 120 molecules of vp3, arranged in what has been called t=2 symmetry. note the green subunit a and the red subunit b which fill the asymmetric unit. (b) the core surface layer is composed of 780 copies of vp7 arranged as 260 trimers, with t=13 symmetry. the asymmetric unit contains 13 copies of vp7, arranged as five trimers, labeled p, q, r, s, and t, with each trimer a different color. trimer "t" in blue sits on the icosahedral threefold axis and thus contributes only a monomer to the asymmetric unit. from granoff and webster (1999) , color plate 17. structures that possess icosahedral symmetry. poxviruses, rhabdoviruses, and retroviruses also appear to have a regular structure, but there is flexibility in the composition of the particle and the mature virions do not possess icosahedral symmetry. the herpesvirus nucleocapsid is a regular icosahedral structure ( fig. 2.5 ), but the enveloped herpesvirions are not regular. other enveloped viruses are irregular, often pleiomorphic, and are heterogeneous in composition to a greater or lesser extent. the structures of different enveloped viruses that illustrate these various points are described next. the nucleocapsids of enveloped rna viruses are fairly simple structures that contain only one major structural protein, often referred to as the nucleocapsid protein or core protein. this protein is usually quite basic or has a basic domain. it binds to the viral rna and encapsidates it to form the nucleocapsid. for most rna viruses, nucleocapsids can be recognized as distinct structures within the infected cell and can be isolated from virions by treatment with detergents that dissolve the envelope. the nucleocapsids of alphaviruses, and probably flaviviruses and arteriviruses as well, are regular icosahedral structures, and there are no other proteins within the nucleocapsid other than the nucleocapsid protein. in contrast, the nucleocapsids of all minus-strand viruses are helical and contain, in addition to the major nucleocapsid protein, two or more minor proteins that possess enzymatic activity. as described, the nucleocapsids of minus-strand rna viruses remain intact within the cell during the entire infection cycle and serve as machines that make viral rna. the coronaviruses also have helical nucleocapsids, but being plus-strand rna viruses they do not need to carry enzymes in the virion to initiate infection. the helical nucleocapsids of (−) rna viruses appear disordered within the envelope of all viruses except the rhabdoviruses, in which they are coiled in a regular fashion (see later). the nucleocapsids of retroviruses also appear to be fairly simple structures. they are formed from one major precursor protein, the gag polyprotein, that is cleaved during maturation into four or five components. the precursor nucleocapsid is spherically symmetric but lacks icosahedral symmetry. the mature nucleocapsid produced by cleavage of gag may or may not be spherical symmetric. the nucleocapsid also contains minor proteins, produced by cleavage of gag-pro-pol, as described in chapter 1. these minor proteins include the protease, rt, rnase h, and integrase that are required to cleave the polyprotein precursors, to make a cdna copy of the viral rna, and to integrate this cdna copy into the host chromosome. the two families of enveloped dna viruses that we consider here, the poxviruses and the herpesviruses, contain large genomes and complicated virus structures. the nucleocapsids of herpesviruses are regular icosahedrons but those of poxviruses are complicated structures containing a core and associated lateral bodies. the external proteins of enveloped virions are virusencoded proteins that are anchored in the lipid bilayer of the virus or whose precursors are anchored in the lipid bilayer. in the vast majority of cases these proteins are glycoproteins, although examples are known that do not contain bound carbohydrate. these proteins are translated from viral mrnas and transported by the usual cellular processes to reach the membrane at which budding will occur. when budding is at the cell plasma membrane, the glycoproteins are transported via the golgi apparatus to the cell surface. some enveloped viruses mature at intracellular membranes, and in these cases the glycoproteins are directed to the appropriate place in the cell. both type i integral membrane proteins, in which the n terminus of the protein is outside the lipid bilayer and the c terminus is inside the bilayer, and type ii integral membrane proteins, which have the inverse orientation with the c terminus outside, are known for different viruses. many viral glycoproteins are produced as precursor molecules that are cleaved by cellular proteases during the maturation process. following synthesis of viral glycoproteins, during which they are transported into the lumen of the endoplasmic reticulum (er) in an unfolded state, they must fold to assume their proper conformation, and assume their proper oxidation state by formation of the correct disulfide bonds. this process often occurs very quickly, but for some viral glycoproteins it can take hours. folding is often assisted by chaperonins present in the endoplasmic reticulum. it is believed that at least one function of the carbohydrate chains attached to the protein is to increase the solubility of the unfolded glycoproteins in the lumen of the er so that they do not aggregate prior to folding. during folding, the solubility of the proteins is increased by hiding hydrophobic domains within the interior of the protein and leaving hydrophilic domains at the surface. the glycoproteins possess a number of important functions in addition to their structural functions. they carry the attachment domains by which the virus binds to a susceptible cell. this activity is thought to be related to the ability of many viruses, nonenveloped as well as enveloped, to bind to and agglutinate red blood cells, a process called hemagglutination. the protein possessing hemagglutinating activity is often called the hemagglutinin or ha. the viral glycoproteins also possess a fusion activity that promotes the fusion of the membrane of the virus with a membrane of the cell. the protein possessing this activity is sometimes called the fusion protein, or f. the glycoproteins, being external on the virus, are also primary targets of the humoral immune system, in which circulating antibodies are directed against viruses; many of these are neutralizing antibodies that inactivate the virus. the glycoproteins of some enveloped viruses also contain enzymatic activities. many orthomyxoviruses and paramyxoviruses possess a neuraminidase that will remove sialic acid from glycoproteins. the primary receptor for these viruses is sialic acid. the neuraminidase may allow the virus to penetrate through mucus to reach a susceptible cell. it also removes sialic acid from the viral glycoproteins so that these glycoproteins or the mature virions do not aggregate, and from the surface of an infected cell, thereby preventing released virions from binding to it. the viral protein possessing neuraminidase activity may be called na, or in the case of a protein that is both a neuraminidase and hemagglutinin, hn. the structure of most enveloped viruses is not as rigorously constrained as that of icosahedral virus particles. the glycoproteins are not required to form an impenetrable shell, which is instead a function of the lipid bilayer. they appear to tolerate mutations more readily than do proteins that must form a tight icosahedral shell and appear to evolve rapidly in response to immune pressure. however, the integrity of the lipid bilayer is essential for virus infectivity, and enveloped viruses are very sensitive to detergents. in some enveloped viruses, there is a structural protein that underlies the lipid envelope but which does not form part of the nucleocapsid. several families of minus-strand rna viruses possess such a protein, called the matrix protein. this protein may serve as an adapter between the nucleocapsid and the envelope. it may also have regulatory functions in viral rna replication. the herpesviruses also have proteins underlying the envelope that form a thick layer called the tegument. the thickness of the tegument is not uniform within a virion, giving rise to some irregularity in its structure. the tegument proteins perform important functions early after infection of a cell by a herpesvirus (see chapter 7). the alphaviruses, a genus in the family togaviridae, are exceptional among enveloped rna viruses in the regularity of their virions, which are uniform icosahedral particles. virions of two alphaviruses have been crystallized and the crystals are regular enough to diffract to 30-40-å resolution. higher resolution has been obtained from cryoelectron microscopy, which has been used to determine the structures of several alphaviruses to 7-25 å (fig. 2.5) . more detailed reconstructions of sindbis virus and ross river virus (rrv) have been derived from a combination of cryoelectron microscopy of the intact virion and x-ray crystallography of alphavirus structural proteins. a cutaway view of rrv at about 25-å resolution is shown in fig. 2.14a . the nucleocapsid, shown in red and yellow, has a diameter of 400 å, and is a regular icosahedron with t=4 symmetry. it is formed from 240 copies of a single species of capsid protein of size 30 kda. note the fivefold and sixfold coordinated pedestals in yellow that rise above the red background of rna and unstructured parts of the protein. each of these pedestals is formed by the ordered domains of one capsid protein molecule. the lipid bilayer is shown in green and is positioned between the capsid and the external shell of glycoproteins, shown in blue. the glycoproteins are also icosahedrally arranged with t=4 symmetry. the complete structure is therefore quite regular and the virion has been described as composed of two interacting protein shells with a lipid bilayer sandwiched between. the structure of the ordered part of the capsid protein of sindbis virus has been solved to atomic resolution by conventional x-ray crystallography and this structure is shown in fig. 2. 14b. the first 113 residues are disordered and the structure is formed by residues 114-264. this ordered domain has a structure that is very different from the eightfold β sandwich described earlier (compare fig. 2.14b with figs. 2.3b and 2.4). instead, its fold resembles that of chymotrypsin, and it has an active site that consists of a catalytic triad whose geometry is identical to that of chymotrypsin. the capsid protein is an active protease that cleaves itself from a polyprotein precursor. after cleavage, the c-terminal tryptophan-264 remains in the active site and the enzymatic activity of the protein is lost. the interactions between the capsid protein subunits that lead to formation of the t=4 icosahedral lattice have been deduced by fitting the electron density of the capsid protein at 2.5-å resolution into the electron density of the nucleocapsid found by cryoelectron microscopy. such a reconstruction, based on a cryoem structure of sindbis virus at a resolution of better than 10 å, is shown in fig. 2.14c . the fit of the capsid protein is unique and the combined approaches of x-ray crystallography and cryoelectron microscopy thus define the structure of the shell of the nucleocapsid to atomic resolution. the envelopes of alphaviruses contain 240 copies of each of two virus-encoded glycoproteins, called e1 and e2. e2 is first produced as a precursor called pe2. e1 and pe2 form a heterodimer shortly after synthesis, and both span the lipid bilayer as type i integral membrane proteins (having a membrane-spanning anchor at or near the c terminus). the c-terminal cytoplasmic extension of pe2 interacts in a specific fashion with a nucleocapsid protein so that there is a one-to-one correspondance between a capsid protein and a glycoprotein heterodimer. the 240 glycoprotein heterodimers form a t=4 icosahedral lattice on the surface of the particle by interacting with one another and with the capsid proteins. because of the glycoprotein-capsid protein interactions, the icosahedral lattices of the nucleocapsid and the glycoproteins are coordinated. at some time during transport of the glycoprotein heterodimers to the cell surface, pe2 is cleaved by a cellular protease called furin to form e2. e1 and e2 remain associated as a heterodimer. if cleavage is prevented, noninfectious particles are produced that contain pe2 and e1. in the virion, three glycoprotein heterodimers associate to form a trimeric structure called a spike, easily seen in figs. 2.5 and 2.14a. it is not known if the spike assembles during virus assembly or if heterodimers trimerize during their transport to the cell surface. a reconstruction of a spike of sindbis virus at a resolution better than 10 å is shown in fig. 2 .15a. in this reconstruction, the electron density of e1 has been replaced by the e1 structure of the related semliki forest virus determined to atomic resolution by x-ray crystallography. the three copies of e1 project upwards at an angle of about 45° and are shown in three colors because they have slightly difb. dengue virus figure 2.15 comparison of (a) the spike structure of mature sindbis virus (an alphavirus) with (b) the spike of immature dengue virus (a flavivirus). the cα backbones of the three e1 (sindbis) and e (dengue) glycoprotein ectodomains are shown in red, green, and blue, as they were fitted into the cryoelectron density envelope. the e1 and e densities have been zeroed out, leaving the gray envelope that corresponds to e2 for sindbis and prm for dengue. the density corresponding to the lipid bilayer is shown in bright green. adapted from figure 5 in y. with permission. ferent environments. the electron density in gray that remains after subtracting the density due to e1 is thus the electron density of e2. e2 projects further upward than does e1 and covers the apex of e1, which has the fusion peptide. thus, e2 covers the fusion peptide with a hydrophobic pocket so that it does not interact with the hydrophilic environment. the apex of the e2 spike contains the domains that attach to receptors on a susceptible cell. both e1 and e2 have c-terminal membrane-spanning anchors that traverse the lipid bilayer shown in green. the c-terminal domain of e1 is not present in the protein whose structure has been determined because hydrophobic domains do not easily crystallize. thus, the electron density shown traversing the lipid bilayer arises from both e1 and e2 and shows that the two membrane spanning anchors go through as paired α helical structures (fig. 2.16) . upon entry of an alphavirus into a cell, the acidic ph of endosomal vesicles causes disassembly of e2/e1 heterodim-ers and trimerization of e1 to form homotrimers. the fusion peptide is exposed and penetrates the target bilayer of the host endosomal membrane. fusion follows by methods discussed in chapter 1. flaviviruses also possess a regular icosahedral structure (fig. 2.5 ) that has been solved by methods similar to those used to determine the structure of alphaviruses. the structures of alphaviruses and flaviviruses are related and have descended from a common ancestral structure. like alphaviruses, flaviviruses produce two structural glycoproteins, called e and prm (for precursor to m). e is homologous to e1 of alphaviruses. although no sequence identity is detectable, the structures of the two proteins are virtually identical and are formed with a similar fold (fig. 2.17 ). prm and e form a heterodimer and immature particles can be formed if cleavage of prm is prevented. the glycoprotein heterodimers in these immature particles trimerize to form spikes whose structure is very similar to the spikes of alphaviruses (fig. 2.15b ). the arrangement of the glycoproteins on the immature virus particle is illustrated in fig. 2.18a and a cryoem reconstruction that illustrates the surface of the immature dengue virus particle is shown in fig. 2.18d . the major differences between the immature flavivirus particle and the alphavirus particle are that there are 180 copies of the heterodimer in the flavivirus particle arranged in a t=3 icosahedral structure rather than 240 heterodimers arranged in a t=4 structure in alphaviruses; that prm is a smaller molecule than pe2 so that in the flavivirus spike there is but a thin trace of density that projects downward, parallel to e, from the cap that shields the fusion peptide ( fig. 2.15b ) rather than a substantial trace of density in the alphavirus spike ( fig. 2.15a) ; and that the c-terminal regions of prm and e that enter the membrane do so independently and do not emerge from the internal side of the membrane (illustrated in fig. 2.19b ), unlike the alphavirus membrane spanning regions (fig. 2.16 ). there is no evidence that the membrane glycoproteins interact with the nucleocapsid in flaviviruses, and the flavivirus nucleocapsid, assuming it is a regular icosahedral structure, is not coordinated with the icosahedral structure formed by the spikes, which is again different from alphaviruses where the c-terminal domain of pe2 interacts with the nucleocapsid. following cleavage of prm by furin to form m, there is a dramatic rearrangement of the flavivirus glycoproteins such that the final virion structure is very different from the alphavirus structure. the heterodimers dissociate and e-e homodimers are formed that collapse over the lipid bilayer (fig. 2.19) . the e-e homodimers occur in two totally different environments, either perpendicular to the twofold axis where they interact with homodimers in sideto-side interactions, forming a herringbone pattern, or parallel to a twofold axis where they interact at fivefold and threefold axes (fig. 2.18b) . a comparison of the immature flavivirus particle, t398 figure 2.16 the e1 and e2 transmembrane helices of sindbis (an alphavirus) determined from a 9å resolution cryoelectron microscopy reconstruction. shown are e1 residues from 409 to 439 and e2 residues 363 to 398 fitted into the transmembrane density. this is figure 6 from mukhopadhyay et al. (2006) , reprinted with permission. the mature flavivirus particle, and the alphavirus particle is shown in fig. 2.18 . the mature flavivirus is smooth, with no surface projections, and is 50 nm in diameter (fig. 2.18e ). the immature particle is ragged in appearance and is 60 nm in diameter (fig. 2.18d ). the alphavirus virion shows conspicuous spikes and is 70 nm in diameter (fig. 2.18f ). the arrangement of e or e1 in the three particles is also shown (figs. 2.18a, b, c) , illustrating the differences in their arrangement. entry of flaviviruses follows pathways similar to those used by alphaviruses. the acidic ph of the endosome causes the e-e homodimers to reorganize to form e homotrimers. these trimers must reorient so that the exposed fusion peptide is projected upwards where it penetrates the host membrane. the arteriviruses possess icosahedral nucleocapsids, but the mature virion does not appear to be regular in structure. detailed structures of these particles are not available. the herpesviruses are large dna viruses that have a t=16 icosahedral nucleocapsid (fig. 2.5) . a schematic diagram of an intact herpesvirion is shown in fig. 2.20a . underneath the envelope is a protein layer called the tegument. the tegument does not have a uniform thickness, and thus the virion is not uniform. two electron micrographs of herpesvirions are shown in figs. 2.20b and 2.20c that illustrate the irregularity of the particle and the differing thickness of the tegument in different particles. the retroviruses have a nucleocapsid that forms initially using spherical symmetry principles. cleavage of gag during virus maturation results in a nucleocapsid that is not icosahedral and that is often eccentrically located in the virion. fig. 2 .21a presents a schematic of a retrovirus particle that illustrates the current model for the location of the various proteins after cleavage of gag and gag-pol. figs. 2.21b, c, and d show electron micrographs of budding virus particles and of mature extracellular virions for three genera of retroviruses. betaretrovirus particles usually mature by the formation of a nucleocapsid within the cytoplasm that then buds through the plasma membrane. this process is shown in fig. 2 .21b for mouse mammary tumor virus. in the top micrograph in fig. 2 .21b, preassembled capsids are seen in the cytoplasm. in the middle micrograph, budding of the capsid through the plasma membrane is illustrated. in the bottom micrograph, a mature virion with an eccentrically located capsid is shown. in gammaretroviruses, the capsid forms during budding, and the nucleocapsid is round and centrally located in the mature virion. this process is illustrated in fig. 2 .21c for murine leukemia virus. the top micrograph shows a budding particle with a partially assembled capsid. the bottom micrograph shows a mature virion. in the lentiviruses, of which hiv is a member, the capsid also forms as a distinct structure only during budding. in the top panel of fig. 2 .21d is shown a budding particle of bovine immunodeficiency virus. after cleavage of gag to form the mature virion, the capsid usually appears cone shaped or bar shaped (bottom panel of fig. 2.21d) . mason-pfizer monkey virus is a betaretrovirus whose capsid is cone shaped and centrally located in the mature virion. a single amino acid change in the matrix protein ma determines whether the capsid preassembles and then buds, or whether the capsids assemble during budding. thus, the point at which capsids assemble does not reflect a fundamental difference in retroviruses. preassembly of capsids or assembly during budding appears to depend on the stability of the capsid in the cell. stable capsids can preassemble. unstable capsids require interactions with other viral components to form as a recognizable structure. the coronaviruses and the minus-strand rna viruses have nucleocapsids with helical symmetry. the structures of the mature virions are irregular, with the exception of the rhabdoviruses, and the glycoprotein composition is not invariant. because of the lack of regularity in these viruses, as well as the lack of symmetry, detailed structural studies of virions have not been possible. the lack of regularity arises in part because in these viruses there is no direct interaction between the nucleocapsid and the glycoproteins. the lack of such interactions permits these viruses to form pseudotypes, in which glycoproteins from other viruses substitute for those of the virus in question. pseudotypes are also formed by retroviruses. the structures of paramyxoviruses and orthomyxoviruses are illustrated schematically in fig. 2.22 . the helical nucleocapsids contain a major nucleocapsid protein called n or np, and the minor proteins p (ns1) and l (pb1, pb2, pa), as shown. there is a matrix protein m (m1) lining the inside of the lipid bilayer and also two glycoproteins anchored in the bilayer that form external spikes. the two glycoproteins, called f and hn in paramyxoviruses and ha and na in orthomyxoviruses, do not form heterodimers but rather form homooligomers so that there are two different kinds of spikes on the surface of the virions. ha in the orthomyxoviruses forms homotrimers whereas na forms homotetramers, and the two types of spikes can be distinguished in the electron microscope if the resolution is high enough. as occurs in many enveloped rna viruses, f and ha are produced as precursors that are cleaved by furin during transport of the proteins. cleavage is required to activate the fusion peptide of the virus, which is found at the n terminus of the c-terminal product (see fig. 1.6 ). electron micrographs of virions are shown in figs. 2.22c and d. the particles in the preparations shown are round and reasonably uniform, but in other preparations the virions are pleomorphic baglike structures that are not uniform in appearance. in fact, clinical specimens of some orthomyxoviruses and paramyxoviruses are often filamentous rather than round, illustrating the flexible nature of the structure of the virion. the micrograph of the paramyxovirus measles virus shown in fig. 2 .22c is a thin section and illustrates the lack of higher order structure in the internal helical nucleocapsid. the micrograph of the orthomyxovirus influenza a virus shown in fig. 2 .22d is of a negative-stained preparation and illustrates the spikes that decorate the virus particle. the structures of rhabdoviruses and filoviruses are illustrated in fig. 2 .23. the rhabdoviruses assemble into bullet-shaped or bacilliform particles in which the helical nucleocapsid is wound in a regular elongated spiral conformation (figs. 2.23b and c). the virus encodes only five proteins (fig. 2.23a) , all of which occur in the virion (fig. 2.23b ). the nucleocapsid contains the major nucleocapsid protein n and the two minor proteins l and ns. the matrix protein m lines the inner surface of the envelope, and g is an external glycoprotein that is anchored in the lipid bilayer of the envelope. budding is from the plasma membrane ( fig. 2.23d) . the filoviruses are so named because the virion is filamentous. a schematic diagram of a filovirus is shown in fig. 2 the structure of viruses the poxviruses, large dna-containing viruses, also have lipid envelopes. in fact, they may have two lipid-containing envelopes. the structures of poxviruses belonging to two different genera, orthopox and parapox, are illustrated in fig. 2 .24. electron micrographs of the orthopox virus vaccinia virus and of a parapox virus are also shown. vaccinia virus has been described as brick shaped. the interior of the virion consists of a nucleoprotein core and two (in vertebrate viruses) proteinaceous lateral bodies. surrounding these is a lipid-containing surface membrane, outside of which are several virus-encoded proteins present in structures referred to as tubules. this particle is called an intracellular infectious virion. as its name implies, it is present inside an infected cell, and if freed from the cell it is infectious. a second form of the virion is found outside the cell and is called an extracellular enveloped virion. this second form has a second, external lipid-containing envelope with which is associated five additional vaccinia proteins. this form of the virion is also infectious. parapox virions are similar to orthopox virions. however, their morphology is detectably different, as illustrated in fig. 2 .24. virions self-assemble within the infected cell. in most cases, assembly appears to begin with the interaction of one or more of the structural proteins with an encapsidation signal in the viral genome, which ensures that viral genomes are preferentially packaged. after initiation, encapsidation continues by recruitment of additional structural protein molecules until the complete helical or icosahedral structure has been assembled. thus, packaging of the viral genome is coincident with assembly of the virion, or of the nucleocapsid in the case of enveloped viruses. the requirement for a packaging signal may not be absolute. in many viruses that contain an encapsidation signal, rnas or dnas lacking such a signal may be encapsidated, but with much lower efficiency. for some viruses, there is no evidence for an encapsidation signal. assembly of the tmv rod ( fig. 2. 2) has been well studied. several coat protein molecules, perhaps in the form of a disk, bind to a specific nucleation site within tmv rna to initiate encapsidation. once the nucleation event occurs, additional protein subunits are recruited into the structure and assembly proceeds in both directions until the rna is completely encapsidated. the length of the virion is thus determined by the size of the rna. the assembly of the icosahedral turnip crinkle virion has also been well studied. assembly of this t=3 structure is initiated by formation of a stable complex that consists of six capsid protein molecules bound to a specific encapsidation signal in the viral rna. additional capsid protein dimers are then recruited into the complex until the structure is complete. it is probable that most other viruses assemble in a manner similar to these two well-studied examples. at least some viruses deviate from this model, however, and assemble an empty particle into which the viral genome is later recruited. it is also known that many viruses will assemble empty particles if the structural proteins are expressed in large amounts in the absence of viral genomes, even if assembly is normally coincident with encapsidation of the viral genome in infected cells. the nucleocapsids of most enveloped viruses form within the cell by pathways assumed to be similar to those described above. they can often be isolated from infected cells, and for many viruses the assembly of nucleocapsids does not require viral budding or even the expression of viral surface glycoproteins. after assembly, the nucleocapsids bud through a cellular membrane, which contains viral glycoproteins, to acquire their envelope. budding retroviruses were illustrated in fig. 2 .21 and budding rhabdoviruses in fig. 2 .23. a gallery of budding viruses belonging to other families is shown in fig. 2 .25. the membrane chosen for budding depends on the virus and depends, in part if not entirely, on the membrane to which the viral glycoproteins are directed by signals within those glycoproteins. many viruses bud through the cell plasma membrane (figs. 2.25b-f); in polarized cells, only one side of the cell may be used. other viruses, such as the coronaviruses and the bunyaviruses, use the endoplasmic reticulum or other internal membranes. the herpesviruses replicate in the nucleus and the nucleocapsid assembles in the nucleus; in this case, the first budding event is through the nuclear membrane (fig. 2.25a) . although the nucleocapsid of most enveloped viruses assembles independently within the cell and then buds to acquire an envelope, exceptions are known. the example of retroviruses, some of which assemble a nucleocapsid during virus budding, was discussed earlier. in these viruses, morphogenesis is a coordinated event. the forces that result in virus budding are not well understood for most enveloped viruses. in the case of the alphaviruses, there is evidence for specific interactions between the cytoplasmic domains of the glycoproteins and binding sites on the nucleocapsid proteins. the model for budding of these viruses is that the nucleocapsid first binds to one or a few glycoprotein heterodimers at the plasma membrane. by a process of lateral diffusion, additional glycoprotein heterodimers move in and are bound until a full complement is achieved and the virus is now outside the cell. additional free energy for budding is furnished by lateral interactions between the glycoproteins, which form a contiguous layer on the surface (fig. 2.18c ). this model accounts for the regularity of the virion, the one-to-one ratio of the structural proteins in the virion, and the requirement of the virus for its own glycoproteins in order to bud. in other enveloped viruses, however, there is little evidence for nucleocapsid-glycoprotein interactions. the protein composition of the virion is usually not fixed, but can vary within limits. in fact, glycoproteins from unrelated viruses can often be substituted. in the extreme case of retroviruses, noninfectious virus particles will form that are completely devoid of glycoprotein. the matrix proteins appear to play a key role in the budding process, as do other protein-protein interactions that are yet to be determined. for most animal viruses, there are one or more cleavages in structural protein precursors during assembly of virions that are required to activate the infectivity of the virion. interestingly, these cleavages may either stabilize or destabilize the virion in the extracellular environment, depending on the virus. many of these cleavages are effected by viral proteases, whereas others are performed by cellular proteases present in subcellular organelles. virions are formed by the spontaneous assembly of components in the infected cell, sometimes with the aid of assembly factors ("scaffolds") that do not form components of the mature virion. for most nonenveloped viruses, complete assembly occurs within the cell cytoplasm or nucleoplasm. for enveloped viruses, final assembly of the virus occurs by budding through a cellular membrane. in either case, the virion must subsequently disassemble spontaneously on infection of a new cell. the cleavages that occur during assembly of the virus potentiate penetration of a susceptible cell after binding of the virus to it, and the subsequent disassembly of the virion on entry into the cell. a few examples will be described that illustrate the range of cleavage events that occur in different virus families. in the picornaviruses, a provirion is first formed that is composed of the viral rna complexed with three viral proteins, called vp0, vp1, and vp3. during maturation to form the virion, vp0 is cleaved to vp2 and vp4. no protease has been found that performs this cleavage, and it has been postulated that the virion rna may catalyze it. cleavage to produce vp4, which is found within the interior of the capsid shell, as illustrated schematically in fig. 2.9 , is required for the virus to be infectious. as described in chapter 1, vp4 appears to be required for entry of the virus into the cell. this maturation cleavage has another important consequence. whereas the provirion is quite unstable, the mature virion is very stable. the poliovirus virion will survive treatment with proteolytic enzymes and detergents, and survives exposure to the acidic ph of less than 2 that is present in the stomach. only on binding to its receptor (figs. 1.5 and 2.9) is poliovirus destabilized such that vp4 can be released for entry of the viral rna into the host cell. similarly, the insect nodaviruses first assemble as a procapsid containing the rna and 180 copies of a single protein species called α (44 kda). over a period of many hours, spontaneous cleavage of α occurs to form β (40 kda) and γ (4 kda). this cleavage is required for the particle to be infectious. these events in nodaviruses have been well studied because it has been possible to assemble particles in vitro, and the structures of both cleaved and uncleaved particles have been solved to atomic resolution. rotaviruses, which form a genus in the family reoviridae, must be activated by cleavage with trypsin after release from an infected cell in order to be infectious. trypsin is present in the gut, where the viruses replicate, and activation occurs normally during the infection cycle of the virus in animals. when the viruses are grown in cultured cells, however, trypsin must be supplied exogenously. a different type of cleavage event occurs during assembly of retroviruses and adenoviruses, as well as of a number of other viruses. during assembly of retroviruses, the gag and gag-pol precursor polyproteins are incorporated, together with the viral rna, into a precursor nucleocapsid. these polyproteins must be cleaved into several pieces by a protease present in the polyprotein if the virus is to be infectious. these cleavages often visibly alter the structure of the particle as seen in the electron microscope (fig. 2.21 ). an analogous situation occurs in adenoviruses, where a viral protease processes a protein precursor in the core of the immature virion. in most enveloped viruses, one of the envelope proteins is produced as a precursor whose cleavage is required to activate the infectivity of the virus. this cleavage may occur prior to budding, catalyzed by a host enzyme called furin, or may occur after release of the virus, catalyzed by other host enzymes. the example of the hemagglutinin of influenza virus was described in chapter 1. this protein is produced as a precursor called ha0, which is cleaved to ha1 and ha2 ( fig. 1.6 ). cleavage is required to potentiate the fusion activity present at the n terminus of ha2. as a second example, alphaviruses produce two envelope glycoproteins that form a heterodimer and one of the glycoproteins is produced as a precursor, as described before. the heterodimer containing the uncleaved precursor is quite stable, so that a particle containing uncleaved heterodimer is not infectious. the cleaved heterodimer, which is required for virus entry, is much less stable and dissociates readily during infection. thus, in contrast to the poliovirus maturation cleavage, the alphavirus cleavage makes the virion less stable rather than more stable. maturation cleavages also occur in the envelope glycoproteins of retroviruses, paramyxoviruses, flaviviruses, and coronaviruses. dna or rna has a high net negative charge, and there is a need for counterions to neutralize this charge in order to form a virion. in many viruses, positively charged polymers are incorporated that neutralize half or so of the nucleic acid charge. the dna in the virions of the polyomaviruses is complexed with cellular histones. the viral genomes in these viruses have been referred to as minichromosomes. in contrast, the adenoviruses encode their own basic proteins that complex with the genome in the core of the virion. another strategy is used by the herpesviruses, which incorporate polyamines into the virion. herpes simplex virus has been estimated to incorporate 70,000 molecules of spermidine and 40,000 molecules of spermine, which would be sufficient to neutralize about 40% of the dna charge. among rna viruses, the nucleocapsid proteins are often quite basic and neutralize part of the charge on the rna. as one example, the n-terminal 110 amino acids of the capsid protein of sindbis virus have a net positive charge of 29. the positive charges within this domain of the 240 capsid proteins in a nucleocapsid would be sufficient to neutralize about 60% of the charge on the rna genome. this charged domain is thought to penetrate into the interior of the nucleocapsid and complex with the viral rna. virions differ greatly in stability, and these differences are often correlated with the means by which viruses infect new hosts. viruses that must persist in the extracellular environment for considerable periods, for example, must be more stable than viruses that pass quickly from one host to the next. as an example of such requirements, consider the closely related polioviruses and rhinoviruses, members of two different genera of the family picornaviridae. these viruses shared a common ancestor in the not too distant past and have structures that are very similar. the polioviruses are spread by an oral-fecal route and have the ability to persist in a hostile extracellular environment for some time where they may contaminate drinking water or food. furthermore, they must pass through the stomach, where the ph is less than 2, to reach the intestinal tract where they begin the infection cycle. it is not surprising, therefore, that the poliovirion is stable to storage and to treatments such as exposure to mild detergents or to ph < 2. in contrast, rhinoviruses are spread by aerosols or contaminated mucus, and spread normally requires close contact. the rhinovirion is less stable than the poliovirion. it survives for only a limited period of time in the external environment and is sensitive to treatment with detergents or exposure to ph 3. general structure of viruses thin section of a herpes simplex virion (herpesviridae) in an infected hep-2 cell. the particle is apparently coated with an inner envelope, and is in the process of acquiring its outer envelope from the nuclear membrane. from roizman (1969). (b) machupo virus (arenaviridae) budding from a raji cell. from murphy et al. (1969). (magnification: 120,000×). (c) sindbis virus (togaviridae) budding from the plasma membrane of an infected chicken cell 000×). (e) a portion of the cell surface with sv5 filaments (paramyxoviridae) in the process of budding. from compans and choppin (1973). (45,000 ×). (f) a row of sv5 virions budding from the surface of a monkey kidney cell. cross sections of the nucleocapsid can be seen within several of the particles principles of virus structure functional implications of protein-protein interactions in icosahedral viruses principles of virus structure structural biology of viruses ultrastructure of animal viruses and bacteriophages: an atlas. ultrastructure in biological systems cryoelectron microscopy adding the third dimension to virus life cycles: three-dimensional reconstruction of icosahedral viruses from cryo-electron micrographs. microbiol membrane proteins organize a symmetrical virus mapping the structure and function of the e1 and e2 glycoproteins of alphaviruses structures of immature flavivirus particles conformational changes of the flavivirus e glycoprotein visualization of membrane protein domains by cryo-electron microscopy of dengue virus the refined crystal structure of hexon, the major coat protein of adenovirus type 2, at 2.9å resolution nucleocapsid and glycoprotein organization in an enveloped virus three-dimensional structure of poliovirus at 2.9å resolution the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph a ligand-binding pocket in the dengue virus envelope glycoprotein x-ray crystallographic structure of the norwalk virus capsid structure of a human common cold virus and functional relationship to other picornaviruses the structure of simian virus 40 refined at 3.1å resolution the canyon hypothesis in a nutshell: structure and assembly of the vaccinia virion structure of dengue virus: implications for flavivirus organization, maturation, and fusion a structural perspective of the flavivirus life cycle rotavirus proteins: structure and assembly bluetongue virus assembly and morphogenesis budding of alphaviruses key: cord-001093-5l0fthw3 authors: jin, fan; yu, chen; lai, luhua; liu, zhirong title: ligand clouds around protein clouds: a scenario of ligand binding with intrinsically disordered proteins date: 2013-10-03 journal: plos comput biol doi: 10.1371/journal.pcbi.1003249 sha: doc_id: 1093 cord_uid: 5l0fthw3 intrinsically disordered proteins (idps) were found to be widely associated with human diseases and may serve as potential drug design targets. however, drug design targeting idps is still in the very early stages. progress in drug design is usually achieved using experimental screening; however, the structural disorder of idps makes it difficult to characterize their interaction with ligands using experiments alone. to better understand the structure of idps and their interactions with small molecule ligands, we performed extensive simulations on the c-myc(370–409) peptide and its binding to a reported small molecule inhibitor, ligand 10074-a4. we found that the conformational space of the apo c-myc(370–409) peptide was rather dispersed and that the conformations of the peptide were stabilized mainly by charge interactions and hydrogen bonds. under the binding of the ligand, c-myc(370–409) remained disordered. the ligand was found to bind to c-myc(370–409) at different sites along the chain and behaved like a ‘ligand cloud’. in contrast to ligand binding to more rigid target proteins that usually results in a dominant bound structure, ligand binding to idps may better be described as ligand clouds around protein clouds. nevertheless, the binding of the ligand and a non-ligand to the c-myc(370–409) target could be clearly distinguished. the present study provides insights that will help improve rational drug design that targets idps. intrinsically disordered proteins (idps), discovered in the 1990s, are proteins that lack a stable three-dimensional native structure under physiological conditions [1] [2] [3] [4] [5] . idps are sometimes described as ''protein clouds'' because of their structural flexibility and dynamic conformation ensemble [6] . various bioinformatics methods have been developed to predict idps based on their sequences [7, 8] . it was revealed that idps are abundant in all kingdoms of life; for example, more than 40% of the proteins in eukaryotic cells possess disordered regions longer than 50 residues [9, 10] . because of the flexibility of the chain and the resulting advantages in protein-protein interactions [1, 11, 12] , idps play important roles in various critical physiological processes such as the regulation of transcription and translation [2] , cellular signal transmission, protein phosphorylation and molecular assemblies [3, 13, 14] . on the other hand, idps also have some adverse effects. it was revealed that many idps are associated with human diseases such as cancer, cardiovascular disease, amyloidosis, neurodegenerative diseases, and diabetes [15] . it was also reported that the swiss-prot keywords for eleven severe diseases are strongly correlated with idps [16] . given their abundance and their biological importance, idps are regarded as promising and potential drug targets [15, [17] [18] [19] . compared with rational drug design targeting ordered proteins [20] [21] [22] , drug design targeting idps is still in its infancy. though some general strategies have been proposed [23] , most of the studies [24] [25] [26] [27] [28] [29] [30] have been limited to only a few systems, namely, p53-mdm2, ews-fli1 and c-myc-max. among them, the oncoprotein c-myc is an encouraging example. c-myc is a transcription factor with a basic helix-loop-helix leucine zipper (bhlhzip) domain which becomes active by forming a dimer with its partner protein max [31] . in their unbound forms, both c-myc and max are disordered. however, in the dimerized forms, they undergo coupled folding and binding. in most cancers cells, c-myc protein is expressed persistently by a mutated myc gene, causing its unregulated expression in cell proliferation and signal transmission. therefore, inhibiting either the overexpression of c-myc and/or its dimerization with max may provide a therapy for cancer. yin et al. [30] have used high-throughput experimental screening to successfully identify seven compounds that inhibit dimerization between c-myc and max. further biophysical studies using nuclear magnetic resonance (nmr), circular dichroism (cd) and fluorescence assays have verified three different binding sites (residues 366-375, 374-385, and 402-409) in the bhlhzip domain of c-myc [28] . these binding sites contain several successive residues that can independently bind different small molecules [28] [29] [30] . it should be noted that, after binding with the small molecule inhibitors, the c-myc sequence remains disordered, making the detailed experimental characterization of the molecular interactions almost impossible. therefore, the inhibition mechanism is still unclear. for example, a recent study using drifttime ion mobility mass spectrometry suggested that the binding between c-myc and these inhibitors is not as specific as previously thought [32] . the lack of conformation data also hampers the application of the well-developed structure-based drug design approach to optimize the inhibition. molecular simulations are useful in understanding the characteristics of idps because they can provide an atomic description of molecular interactions. coarse-grained models [11, [33] [34] [35] and allatom simulation [36] [37] [38] [39] [40] [41] [42] have both been used to investigate idps. recently, knott and best [40] used large-scale replica exchange molecular dynamics (remd) simulations with a well-parameterized force field to obtain a conformational ensemble of the nuclear coactivator binding domain of the transcriptional coactivator cbp. their simulation results were in good agreement with nmr and small-angle x-ray scattering measurements, validating the efficacy of all-atom simulations in exploring the highly dynamic conformations of idps. for the c-myc/inhibitor complex described above, michel and cuchillo [43] built a structural ensemble using all-atom simulations for c-myc 402-412 with and without an inhibitor (10058-f4) and found that 10058-f4 bound to multiple distinct binding sites and interacted with c-myc 402-412 . however, because the c-myc segment used in their simulation contained only the 11 residues that covered the binding sites of 10058-f4 (residues 402-409), it is unclear how the inhibitors would interact with longer segments of c-myc and how specific the interaction would be. in the present study, we conducted extensive all-atom molecular dynamic (md) simulations to investigate the c-myc 370-409 conformational ensemble and its interactions with a smallmolecule inhibitor (10074-a4). first, we performed implicitsolvent remd simulations to clarify the conformational features of the unbound c-myc 370-409 . next, we performed md simulations with an explicit water model to explore in detail the interactions between c-myc 370-409 and 10074-a4. finally, a negative control using a different peptide segment (c-myc 410-437 ) was simulated to address the issue of interaction specificity. the conformational ensemble that we obtained will be useful not only in clarifying the structural features of c-myc and the binding mechanism with inhibitors, but also in providing reference structures for drug design targeting c-myc via structure-based approaches. conformational sampling of idps for molecular modeling is challenging because the energy landscapes of idps are relatively flat [44, 45] . in the present study, extensive remd simulations using an implicit solvent model were performed to explore the conformational characteristics of c-myc 370-409 . the accumulative total of simulation time reached 34.5 ms (see methods). c-myc 370-409 is a 40-residue truncated construct of a full-length c-myc. the conformational properties of c-myc 370-409 in its bound state (with 10074-a4) and more dynamic unbound state, have been studied experimentally using cd and nmr spectroscopy, and a likely average conformation was built based on chemical shift data which is not meant to (and cannot) define detailed structural features [28] . we compared our simulation results with the available experimental results. to assess the sampling quality of the remd simulations, we computed 1 h and 13 c chemical shifts from the simulated conformational ensemble using shifts [46] and compared the computed values with the experiment values ( figure 1 ). the agreement is reasonable, though not excellent. deviations between the average chemical shift values for a simulated ensemble and experimental values have been observed previously in several studies on idps [40, 47, 48] . the chemical shift calculation performed using several other software (shiftx [49] , camshift [50] , sparta+ [51] ) also showed deviations between the computed and experimental values ( figure s1 ). a possible reason is that chemical shifts are difficult to calculate accurately and the underlying parameterizations applied in current software for the calculation of chemical shifts have been optimized for ordered proteins but not for idps [47] . interestingly, when we backcalculated chemical shifts from the nmr-refined structure using either the shifts [46] or shiftx [49] software, the resulting values also deviated from the experimental ones ( figure s2 ). in addition, the ensemble nature of idp conformations suggests that the chemical shifts of idps should be described as a distribution, and not merely as average values. the calculated distributions of the h a chemical shifts obtained from our simulations are summarized in figure 2 . all the h a chemical shifts are distributed over a broad range. the experimental values, indicated by arrows in figure 2 , are located close to the centers of the distributions, indicating the validity of the conformational sampling. data for the h n , c a and c b chemical shifts are given in figure s3 , showing similar behaviors as the h a chemical shifts. we also computed the distribution of the backbone dihedral angles (ramachandran (w,y) plot) for the simulations and the dihedral angles of the nmrrefined apo structure lie well within the simulation distributions ( figure s4 ). the secondary structure content of the simulated structures was also calculated [43, [52] [53] [54] and compared with that estimated from the experimental chemical shifts (figure 3 ). the helix and polyproline ii content of the simulated structure were consistent with the experimental structures ( figure 3a ). however, the sheet content of the simulated structures was much lower than the sheet content of the experimental structures. in a previous study [43] on a shorter c-myc segment, c-myc 402-412 , a similar underestimation of sheet content was observed in the simulated structures. the deficiency of sheet content in the simulated structures might be caused by a bias in the force fields. although c-myc 370-409 is intrinsically disordered, it possesses a high content of residual helical structure (.25%). the simulated helix propensity ( figure 3b ) showed three helical regions separated by proline residues, pro382 and pro391. intrinsically disordered proteins (idps) exist as conformational ensembles that change rapidly. they are an important and common class of proteins in all kingdoms of life. idps are widely associated with human diseases and may serve as potential drug design targets. however, drug design targeting idps is difficult and only limited examples have been reported. one example is the oncoprotein, c-myc, for which seven inhibitors were discovered by experimental screening. understanding how small inhibitor molecules bind to c-myc may help in understanding the binding mechanism of idps with ligands. in the present study, we conducted extensive molecular dynamics simulations to explore the binding mechanism for the c-myc peptide with an inhibitor 10074-a4. we found that 10074-a4 could bind to c-myc 370-409 at different sites along the peptide chain and its binding behavior could be described as a 'ligand cloud'. even in the bound state, the structure of the c-myc 370-409 peptide remained a dynamic ensemble. compared to c-myc peptides that do not bind to 10074-a4, c-myc 370-409 binds selectively with 10074-a4, but the specificity of binding was not high. the interactions of idps with ligands can perhaps be described as a scenario in which ligand clouds around protein clouds. to clarify the conformational features of c-myc 370-409 , backbone-rmsd clustering with a cutoff of 2.0 å of the conformations was performed. representative structures (the central structure of each group) of the first eight groups were depicted in figure 4 . they are all somewhat collapsed compared to the fully extended structure and possess a rich residual helical structure. these states with considerable population will be useful references for rational drug design targeting c-myc. the existence of residual structure may be related to the functional misfolding that prevents idps from unwanted interactions with non-native partners [55] . a quantitative analysis on the distributions of dimension and helix content was provided in figure s5 . the mean radius of gyration is around 10.360.6 å , which is much smaller than the expected value of random coils (18.5 å ) under the same chain length. the mean helix content of the conformational ensemble is 27.7611.1%, showing a broad distribution. these results indicated that c-myc 370-409 is disordered in nature and interconversions between dispersed structures occur. to reveal how the conformations of apo c-myc 370-409 were stabilized, we analyzed the lennard-jones and electrostatic residue-residue interactions among all the residues ( figure s6 ). the lennard-jones interaction matrix was rather weak ( figure s6a ), indicating that the conformations were disordered and that the packing in the collapsed structures was poor. this finding is consistent with the contact map, which showed that residueresidue contacts were dispersed and low in magnitude ( figure s6b ). the electrostatic interactions, on the other hand, were comparatively strong ( figure s6c ), probably because nearly onethird of the residues in c-myc 370-409 (12 out of the 40) are charged residues. the favorable electrostatic interactions of the arg372, arg378, lys389, lys392 and lys398 residues with the asp379, glu383, glu385 and glu409 residues ( figure s6c ) are the result of the electrostatic attraction between residues with opposite charges. residues like ser373 and gln380 also contributed to the electrostatic interactions by forming hydrogen bonds ( figure s6d ). therefore, charge-pair interactions and hydrogen bonds were the main stabilized factors for the c-myc 370-409 conformations. we conducted md simulations with an explicit solvent model to investigate the interactions between c-myc 370-409 and the inhibitor 10074-a4. 10074-a4 is the only inhibitor (among seven inhibitors of c-myc) that binds to the 375-385 sites in loop region of the bhlhzip domain of c-myc and we wanted to see whether or not stable local structures were induced when 10074-a4 interacted with the flexible loop region. in the experimental study, 10074-a4 is a mixture of two chiral forms, the s and r forms ( figure 5 ). in the simulations, both chiral forms were tested. for comparison, the apo c-myc 370-409 was simulated with the same explicit solvent model. the accumulative simulation time for each group was 7 ms (see methods). we calculated and compared the simulated chemical shifts with experimental chemical shifts for both implicit solvent remd and explicit solvent simulations ( figures s7, s8 , s9, s10). reasonable agreements were found. for example, the average discrepancy between the simulated and experimental chemical shifts for ha atoms of apo c-myc370-409 is 0.14 and 0.16 in the md simulations with explicit solvent model and remd simulations, respectively (see table s1 ). the relative binding free energy of c-myc 370-409 with the two chiral 10074-a4 forms was analyzed from the md trajectories for the remd simulations (red), the helix and sheet content was computed using the dssp [52] method; the polyproline ii content was computed with the pross software [53] . for the experimental data (black), the secondary structure content was estimated from the chemical shifts using d2d [54] . b helix propensity from the remd simulations using the dssp method. doi:10.1371/journal.pcbi.1003249.g003 using the molecular mechanic/poisson-boltzmann surface area (mm/pbsa) method [56] . the results of this analysis, together with the average non-bonded interactions u non-bonded (lennard-jones and electrostatic potentials) between c-myc 370-409 and 10074-a4, are given in table 1 . we found that the interaction between c-myc 370-409 and the s form of 10074-a4 was much stronger than the interaction with the r form. the difference of u non-bonded between the s and r forms (23.7 kcal/mol) was close to the difference of dh from mm/pbsa (23.2 kcal/mol). the difference of binding free energy between the s and r forms was 22.2 kcal/mol, resulting in a binding-affinity ratio of for the s and r forms. therefore, compared with the binding of the s form to c-myc 370-409 , the binding of the r form can be ignored. thus, only the holo system with the s form of 10074-a4 is discussed further. hammoudeh et al. [28] reported an induced circular dichroism (icd) effect on c-myc 370-409 by the binding of a racemate (1:1 mixture of the s and r forms) of 10074-a4. there were two possible reasons for the observed icd effect [28] ; either the chiral surroundings affected the absorption transition of the compound, or the enantiomer-specific effect (the different binding affinity of the s and r forms) led to the icd effect. we have shown above that the s form of 10074-a4 bound much stronger with c-myc 370-409 than the r form. therefore, we suggest that it was the enantiomer-specific effect that was responsible for the observed icd effect. further experiments using single chiral forms of 10074-a4 would be helpful in clarifying this observation. we clustered the conformations from md simulations with the explicit solvent model for both the apo and holo c-myc 370-409 peptide based on rmsd of the backbone atoms. figure 6 and 7 showed the representative conformations for the top eight clusters of the apo and holo peptides. it is clear that both the apo and the holo peptides have a rather broad conformation distribution, which is typical of disordered proteins. upon binding to the ligand 10074-a4, the conformational distribution became more condensed. the top eight conformation clusters of the holo peptide were more highly populated compared to that of the apo peptide, with a total of about 77% occupancy compared to 50%. similar to the apo c-myc 370-409 structure, the holo c-myc 370-409 structure is rich in helical structures. a quantitative analysis indicated that the helix and polyproline ii content was almost unaffected by the binding of 10074-a4 ( figure s11 ), while the sheet content was enhanced (see also in figure 7 ). the electrostatic interactions (from both charged residues and hydrogen bonding) dominated the intramolecular stabilizing force for holo c-myc 370-409 ( figure s12 ). the binding free energy was calculated using the mm/pbsa method [56] . all quantities are in kcal/mol. *u non-bonded is averaged non-bonded potential, which is composed of lennard-jones and electrostatic potential. 10074-a4 usually binds simultaneously to two or more regions that are flanked by several residues. the binding was highly dynamic and could switch between different modes within a trajectory. the time percentage of binding for each residue was calculated and is shown in figure 9 . three binding sites were detected, which included site i (residues 372 to 384), site ii (387 to 395), and site iii (398 to 408). site i was near the n-terminal and showed stronger potency than that of the other two sites. this result was supported by the intermolecular interaction analysis (figure 10 ), which showed that both the electrostatic and lennard-jones interactions for site i were much stronger than those of the other two sites. in fact, in the latter cases, hydrogen bonds hardly formed and the electrostatic interactions were weak. site i was similar to the experimentally determined binding site of 10074-a4 on c-myc at residues 374-385 [28] . binding at all the other sites generated in our simulations was much weaker, which would make them difficult to be observed experimentally. the low residue interaction specificity that we observed in the simulations is consistent with a recent simulation on an 11 residue peptide of c-myc 402-412 that suggested that ligand binding was driven by weak and nonspecific interactions [43] . the mass spectrometry experiment on c-myc reported by harvey et al. [32] also supported this conclusion. to further investigate the inherent specificity features of idps, we conducted a negative control study in which we chose another segment of c-myc (residues 410-437) that does not bind with 10074-a4 [28] . the simulated binding between c-myc 410-437 and 10074-a4 is shown in figure 11 the specificities of idps in molecular recognition are complicated [57] . our simulation results showed that the specificity of c-myc in binding the small-molecule ligand 10074-a4 was not high. c-myc is a typical example of idps. it is sticky and binds the ligands at different regions with different interaction strengths. because of the lack of coupled folding and binding, after binding, c-myc is still in an ensemble with diverse conformations and the distinct conformations are all capable of binding the ligand. furthermore, for a given c-myc structure, the binding of ligand occurred at disperse sites ( figure 12 ). we named this phenomenon ligand clouds. ligand clouds are remarkably different from the type of binding that is found in ordered proteins where a dominant binding structure is formed. we expect that ligand clouds may be a general feature for idps binding with small-molecule ligands. for idps binding with macromolecule partners, it was reported that some idps remain disordered in the holo state [57] ; for example, b-catenin/tcf4, b-catenin/apc peptide, b-catenin/apc phosphorylated, vif/elob/eloc, and errclbd/pgc-1a. these idp complexes assume dynamic structures upon binding, suggesting that idps may interact with their partners in a similar manner to the ligand clouds. the ligand clouds concept supports the idea that there is no definite binding mode in the interactions between idps and small-molecule inhibitor [43] . it suggests that the interactions could be described as protein clouds interacting with ligand clouds. the ligand cloud concept describes a scenario for the interactions between idps and small-molecule ligands and may provide a basis for drug design targeting idps. a straightforward strategy for rational drug design on idps is to extract metastable structures from simulations and then to conduct a virtual screen on them to identify potential inhibitors. a similar strategy was applied successfully in designing an inhibitor for ab fibrillation [58] . however, the ligand clouds concept for small molecules binding with idps implies that different strategies from those used for ordered proteins should be developed for better rational drug design on idps. for example, because ligand binding on idps occurs in disperse locations and in different orientations, multimode interactions should be considered in the scoring functions instead of the single-mode interaction that is commonly used for other proteins. therefore, schemes that can consider binding energy landscapes [59] might be expected to perform better when designing small molecule ligands for idps. on the other hand, in contrast to the conventional ordered proteins that are in either ''binding'' or ''non-binding'' states with small molecules, idps are ''sticky'' and would be either in ''strong binding'' or ''weak binding'' with small molecules. so more cares should be paid to the problem of specificity in drug design targeting idps. for conventional ordered proteins, the binding conformation is unique which could be selected from pre-existing conformations (the conformational selection mechanism) or be induced (the induced fit mechanism) by particular ligands. the scenario of ligand clouds around protein clouds for idps indicates that multiple protein conformations are selected and/or induced by the binding of a ligand on idps. this may extend the conformational selection-induced fit continuum in a new dimension. in conclusion, we conducted extensive simulations to explore the conformational ensemble of c-myc 370-409 and its complex with a small-molecule inhibitor 10074-a4. the conformational space was found to be rather dispersed. in contrast to conventional structured proteins, the conformations of c-myc 370-409 were mainly stabilized by charge interactions and hydrogen bonds. upon binding to 10074-a4, c-myc 370-409 remained disordered. the 10074-a4 ligand bound at different sites throughout the c-myc 370-409 chain with different strength. accordingly, a ligand cloud concept was proposed, that is, the interactions between small molecule ligands and idps were like ligand clouds around protein clouds. the different binding probabilities between the protein clouds and ligand clouds indicated that the ligand could be selective and thus specific. though the specificity of the binding was not high, the binding of ligand and non-ligand to the target idp could be clearly distinguished. hammoudeh et al. [28] measured chemical shifts and several noe signals of c-myc 370-409 and predicted dihedral angle distributions and atomic contacts. to build the c-myc 370-409 peptide, we first built a completely extended conformation with the following sequence: 370 lkrsffalrdqipelenne-kapkvvilkkatayilsvqae 409 (accession number: p01106). we then built the initial structures from the reported dihedral angles [28] using pymol [60] . the apo and holo structures for c-myc 370-409 were refined further using the gromacs 4.5.4 software package [61] and the amber99sb force field, with the nmr data [28] as the dihedral angle and distance restraints in the simulation. each initial structure was minimized in vacuum. then, it was solvated, minimized, and equilibrated as described below. the time step was set to 0.5 fs. finally, a 5 ns production simulation was performed and the final structure was adopted as the refined structure. the conformations of the c-myc 370-409 peptide were sampled by remd simulations with a generalized born/surface area (gb/sa) implicit solvent model. the amber molecular simulations package was used with amber99sb force fields [62] . a total of 30 replicas were adopted with temperatures ranging between 284.6 k and 608.8 k. all adjacent replicas attempted to exchange temperature every 10 ps with the average exchange rate between 35% and 40%. to produce the 30 starting conformations for an remd simulation, an initial structure (described below) was minimized using steepest descent for 500 steps and then switched to conjugate gradient for another 500 steps. the minimized conformation was then heated to the defined temperature over a time of 200 ps for each replica. the obtained conformations were adopted as starting conformations in the remd simulations, which were run with a time step of 2 fs. replica temperature was controlled with a coupling time constant of 2 ps. bonds involving hydrogen atoms were constrained with shake. chirality restraints on the backbone were employed to prevent non-physical chiralities. ionic strength was set to 0.2 m. the cutoff for non-bonded interactions and for the gb pairwise summations involved in calculating born radii was 999 å to consider all probable interactions entirely. snapshots from each trajectory were stored every 10 ps. we conducted four groups of remd simulations with different initial structures: (a) the extended structure of the peptide; (b) apo nmr refined structure; (c) the structure after a 80-ns md simulation at 300 k starting from the extended conformation; and (d) the most occupied representative conformation generated previously from the remd simulations of the extended structure in (a). the simulation time for the four groups of remds was 150 ns, 270 ns, 210 ns and 520 ns, respectively. the total simulation time was 34.5 ms (1.15 ms per replica). the trajectories of 292.2 k, 300 k and 308 k were used in the further analyses except that only the trajectory of 300 k was used in the chemical shifts calculations. to investigate the interactions between c-myc 370-409 and 10074-a4, md simulations for the complex structure were carried out with an explicit solvent model [63] . the apo c-myc 370-409 was also simulated with the same explicit solvent model for comparison. three groups of simulations were performed, one for the apo and two for the holo (with the two chiral 10074-a4 forms (see figure 5) ). each group contained seven trajectories of 1 ms, therefore, the total simulation time was 21 ms. one of the seven initial structures was the nmr refined structures (apo and holo); the other six initial structures were adopted from representative conformations generated previously in the 150-ns remd simulations (for the holo structures, the 10074-a4 isomers were docked using the autodock 4.2 program [64] ). md simulations with the explicit solvent model were performed with the gromacs 4.5.4 software package [61] and am-ber99sb force field under particle mesh ewald periodic boundary conditions. the tip4p-ew water model [63] was used with amber99sb force field because of its previously reported good performance in other simulations of idps [36, 47, 65] . in the holo simulations, the small molecule 10074-a4 ligand involved was parameterized using a general amber force field (gaff) with acpype software [66] . an am1-bcc charge model [67] was used to assign charges to the ligand. each initial structure was immersed in an explicit tip4p-ew truncated octahedral water box. the dimensions of the box, defined as the distance between the farthest atoms of the peptide and the edge of the box, was set to 10 å . the system was neutralized by adding ions, and extra nacl was added to represent a solution with an ionic strength of 0.15 m. the system was minimized using the steepest descent minimization approach. after the minimization, the system was equilibrated in the nvt ensemble with all-heavy atom restrained with a force constant of 239 kcal/mol. the temperature was maintained at 300 k using a v-rescale thermostat with a coupling constant of 0.1 ps. further equilibration was carried on in the npt ensemble without strains, and where the pressure was maintained at 1 atmosphere using a parrinello-rahamn barostat with the coupling constant set to 2.0 ps. both equilibrations were performed for 200 ps with a time step of 1 fs. for the production run, the thermostat and barostat settings were the same as for the npt run. to enable 2 fs time steps, bonds involving hydrogen atoms were constrained to equilibration length using the lincs algorithm [68] . a realspace cutoff of 10 å was used for the electrostatic and lennard-jones forces. snapshots from each trajectory were stored every 20 ps. to further investigate the inherent specificity features of idps, we conducted a negative control study using the c-myc 410-437 truncated peptide ( 410 eqkliseedllrkrreqlkhk-leqlrns 437 ), which did not bind to 10074-a4. the extended structure of the peptide was used as the initial structure in an 80 ns implicit solvent md simulation and the final structure that was generated was applied in all-atom explicit simulations. two groups of simulations were performed for each of the two chiral 10074-a4 isomers. each group contained one trajectory of 1 ms; the other parameters were the same as the parameters used for the holo c-myc 370-409 simulations described above. all the simulations were analyzed using the gromacs utilities [61] with either pymol [60] or in-house scripts. dsasa was used in determinations of the binding sites. upon small molecule binding, for each residue in the peptide there would be a clear decrease of sasa related to the difference between the sasa of the bound and unbound states. backbone rmsd clustering of peptide conformations was performed to identify distinct structural clusters and to estimate their populations. the relative binding free energy was calculated every 200 ps using mm/pbsa [56] methods. figure s1 comparisons of the computed and experimental chemical shifts for apo c-myc 370-409 . the computed values using shiftx (red circles), camshift (blue square) and sparta+ (green squares) are from the remd simulations and the experimental values are from hammoudeh et al. [28] (black triangle). note that the experimental values for some residues were not available. chemical shifts are for the atoms : a h a , b h n natively unfolded proteins: a point where biology waits for physics intrinsic disorder and protein function intrinsically unstructured proteins and their functions intrinsically unstructured proteins intrinsically disordered proteins: the new sequencestructure-function relations drugs for 'protein clouds': targeting intrinsically disordered transcription factors predicting intrinsic disorder in proteins: an overview inherent relationships among different biophysical prediction methods for intrinsically disordered proteins prediction and functional analysis of native disorder in proteins from the three kingdoms of life comparing and combining predictors of mostly disordered proteins kinetic advantage of intrinsically disordered proteins in coupled folding-binding process: a critical assessment of the ''fly-casting'' mechanism smoothing molecular interactions: the ''kinetic buffer'' effect of intrinsically disordered proteins malleable machines take shape in eukaryotic transcriptional regulation exploring the binding diversity of intrinsically disordered proteins involved in one-to-many binding intrinsically disordered proteins in human diseases: introducing the d 2 concept functional anthology of intrinsic disorder. 3. ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins intrinsically disordered proteins are potential drug targets rational drug design via intrinsically disordered protein novel strategies for drug discovery based on intrinsically disordered proteins (idps) dynamic modeling of human 5-lipoxygenase-inhibitor interactions helps to discover novel inhibitors discovery of multitarget inhibitors by combining molecular docking with common pharmacophore matching virtual screening of novel noncovalent inhibitors for sars-cov 3c-like proteinase rational drug design via intrinsically disordered protein inhibition of the p53-mdm2 interaction: targeting a proteinprotein interface in vivo activation of the p53 pathway by small-molecule antagonists of mdm2 a small molecule blocking oncogenic protein ews-fli1 interaction with rna helicase a inhibits growth of ewing's sarcoma improved low molecular weight myc-max inhibitors multiple independent binding sites for small-molecule inhibitors on the oncoprotein c-myc structural rationale for the coupled binding and unfolding of the c-myc oncoprotein by small molecules low molecular weight inhibitors of myc-max interaction and function x-ray structures of myc-max and mad-max recognizing dna: molecular bases of regulation by proto-oncogenic transcription factors smallmolecule inhibition of c-myc:max leucine zipper formation is revealed by ion mobility mass spectrometry polyelectrostatic interactions of disordered ligands suggest a physical basis for ultrasensitivity electrostatically accelerated coupled binding and folding of intrinsically disordered proteins binding-induced folding of a natively unstructured transcription factor optimizing protein-solvent force fields to feproduce intrinsic conformational preferences of model peptides anchoring intrinsically disordered proteins to multiple targets: lessons from n-terminus of the p53 protein energy landscape analyses of disordered histone tails reveal special organization of their conformational dynamics a free-energy landscape for coupled folding and binding of an intrinsically disordered protein in explicit solvent from detailed all-atom computations a preformed binding interface in the unbound ensemble of an intrinsically disordered protein: evidence from molecular simulations residual structures, conformational fluctuations, and electrostatic interactions in the synergistic folding of two intrinsically disordered proteins binding of two intrinsically disordered peptides to a multi-specific protein: a combined monte carlo and molecular dynamics study the impact of small molecule binding on the energy landscape of the intrinsically disordered protein c-myc ensemble modeling of protein disordered states: experimental restraint contributions and validation constructing ensembles for intrinsically disordered proteins automated prediction of 15n, 13ca, 13cb and 13c' chemical shifts in proteins using a density functional database structure and dynamics of the ab(21-30) peptide from the interplay of nmr experiments and molecular simulations homogeneous and heterogeneous tertiary structure ensembles of amyloid-b peptides rapid and accurate calculation of protein 1h, 13c and 15n chemical shifts fast and accurate predictions of protein nmr chemical shifts from interatomic distances sparta+: a modest improvement in empirical nmr chemical shift prediction by means dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features a physical basis for protein secondary structure determination of secondary structure populations in disordered states of proteins using nuclear magnetic resonance chemical shifts intrinsically disordered proteins may escape unwanted interactions via functional misfolding mmpbsa.py: an efficient program for end-state free energy calculations do intrinsically disordered proteins possess high specificity in protein-protein interactions inhibitor discovery targeting the intermediate structure of beta-amyloid peptide on the conformational transition pathway: implications in the aggregation mechanism of beta-amyloid peptide binding energy landscape analysis helps to discriminate true hits from high-scoring decoys in virtual screening the pymol molecular graphics system algorithms for highly efficient, load-balanced, and scalable molecular simulation development of an improved four-site water model for biomolecular simulations: tip4p-ew autodock4 and autodocktools4: automated docking with selective receptor flexibility atomic-level characterization of the ensemble of the ab(1-42) monomer in water using unbiased molecular dynamics simulations and spectral slgorithms acpype -antechamber python parser interface fast, efficient generation of highquality atomic charges. am1-bcc model: i. method lincs: a linear constraint solver for molecular simulations the authors thank daqi yu, dr. changsheng zhang and dr. fangjin chen for helpful discussions. f.j. gratefully acknowledges the help of prof. tsun-mei chang and dr. shuangyu bi in preparing the manuscript. key: cord-103528-3tib5o1m authors: ahmed, asad; mam, bhavika; sowdhamini, ramanathan title: deelig: a deep learning-based approach to predict protein-ligand binding affinity date: 2020-09-28 journal: biorxiv doi: 10.1101/2020.09.28.316224 sha: doc_id: 103528 cord_uid: 3tib5o1m protein-ligand binding prediction has extensive biological significance. binding affinity helps in understanding the degree of protein-ligand interactions and has wide protein applications. protein-ligand docking using virtual screening and molecular dynamic simulations are required to predict the binding affinity of a ligand to its cognate receptor. in order to perform such analyses, it requires intense computational power and it becomes impossible to cover the entire chemical space of small molecules. it has been aided by a shift towards using machine learning-based methodologies that aids in binding prediction using regression. recent developments using deep learning has enabled us to make sense of massive amounts of complex datasets. herein, the ability of the model to “learn” intrinsic patterns in a complex plane of data is the strength of the approach. here, we have incorporated convolutional neural networks that find spatial relationships among data to help us predict affinity of binding of proteins in whole superfamilies towards a diverse set of ligands. the models were trained and validated using a detailed methodology for feature extraction. we have also tested deelig on protein complexes relevant to the current public health scenario. our approach to network construction and training on protein-ligand dataset prepared in-house has provided significantly better results than previously existing methods in the field. proteins are a diverse class of dynamic macromolecular structures in living organisms and are essential for the biochemistry and physiology of the organism. depending on their functional role (s), proteins may bind to other proteins, peptides, nucleic acids and non-peptide ligands with varying affinities. determining protein-ligand affinity helps in understanding the reaction mechanism and kinetics of the reaction and has applications in drug development and pharmacology (1) . protein-ligand interaction is measured in terms of binding affinity. the stronger the readout for binding affinity, the stronger the interaction between protein and ligand may be inferred. it is quantified in terms of inhibition constant (ki), dissociation constant (kd), changes in free energy measures (delta g, delta h) and (ic50) (2) . predicting binding affinity between a protein and ligand complements experimental approaches and is usually used as a start-point for the latter. prediction-based approaches are also useful in cases where experimental determination of binding affinity may not be feasible. classical prediction methods to score free binding energies of small ligands to biological macromolecules such as mm/gbsa and mm/pbsa typically rely on molecular dynamic simulations for calculations and aid in silico docking and virtual screening as well as experimental approaches. however, there is a trade-off between computational resources and accuracy (3) . with a recent shift towards the use of machine learning and deep-learning based methods in the field of structural biology, making biologically significant predictions using regression and 'learning' intrinsic patterns in a complex plane of available data has led to resourceoptimal predictions without compromising on accuracy. deep learning has been known to learn representations and patterns in complex data forms. our aim was to apply deep learning to predict binding affinity of protein-ligand interaction. convolutional neural networks (cnn) are deep neural networks that use an input layer, output later as well as convolutional hidden layer(s). the first cnn was incorporated by lecunn in 1998 (4) the connectivity pattern of which was inspired by the elegant experiments of hubert and weisel on the mammalian visual cortex in the 1960s (5) . with the growing technical advancements and massive amounts of data, cnns have emerged popular in biological fields in the recent decade with various applications (6) . in our study, we have used cnns to provide a quantitative estimate of protein-ligand binding using various sets of features corresponding to protein and ligand respectively by finding spatial relationships amongst the data. our approach was validated using ligand-bound complexes from kinases superfamily in the pdb. kinases belong to a class of enzymes required for substrate dependent phosphorylation. they are represented across diverse cellular functions like signaling, differentiation, glycolysis (7) . we have also tested our model on covid-19 main protease (8) of the novel coronavirus strain complexed with various inhibitors of which binding affinities have not been predicted or experimentally determined so far. the raw data for our novel database was obtained from rcsb pdb (9) database, where following were selected as the query parameters. these criteria resulted in a list of 5464 protein pdb ids, 2568 complexed ligand (s) and corresponding binding affinity values. the search results include the structures present in pdbdatabase, pdbbind (10, 11, 12) , pdbmoad (13, 14) and scpdb (15) for its results. initial raw data database created contained protein structures in pdb format, protein sequences in fasta format, ligand in sdf format and binding affinity values of corresponding protein-ligand pairs for 5464 complexes. the pdb, fasta and sdf files filtered were further processed to refine our novel dataset, as shown in figure 1 . protein-ligand complexes were 5,464 in number and corresponded to 29,650 complex unique chain-ligand pairs. binding affinity values were obtained from the rcsb database and protein chain-ligand pairs with corresponding binding affinity as 0 were discarded to reduce statistical errors. this narrowed down the total complexes to 4,750 protein-ligand pairs. pocket information was extracted from the protein using ghecom (16) and converted to mol2 format using chimera (17) , which narrowed our results to 4699 pocket-ligand pairs. it narrowed down the size of the dataset to 4286 pocket-ligand pairs. we discarded other protein-ligand pairs with missing pssm profiles, secondary structure or dihedral angle information. it resulted in a total of 4041 pocket-ligand pairs, which corresponds to 7414 pocket-ligand pairs containing unique chains. training the deep learning network on raw information is known to result in longer time for convergence and less accuracy. we followed a conventional methodology for feature extraction and used the deep learning framework to learn the interaction between the proteinpocket and ligand for their affinity prediction. a comprehensive two-level feature extraction methodology, one at the atomic level and the other at the level of amino acids utilizing structural information and protein sequence respectively. • 9 bit 1 hot or all null hot encoding for atom types: b, c, n, o, p, s, se, halogen and metal. • 1 integer for hybridization • 1 integer representing the number of bonds with heavy atoms • 1 integer representing the number of bonds with hetero atoms • 5 bits (1 if present) encoding properties defined with smarts patterns: hydrophobic, aromatic, acceptor, donor and ring • 1 float for partial charges • 1 integer to distinguish between ligand as -1 and protein as 1 we utilized the sequence information of protein to get more features about the protein pocket-ligand interaction. • position-specific scoring matrix (pssm): pssm is a matrix that represents the probability of mutation at each point of the sequence. it gives a 20 bit-probability for each amino acid at each location. pssm profiles were obtained using psi-blast (18) with swissprot as subject database and e-value threshold as 0.001. chains with less than 50 amino acids were removed from the input dataset. • relative solvent accessibility (rsa): it is encoded by 1 bit of information for each amino acid that provides whether it is buried or exposed to the solvent. we set a threshold of 25% in rsa values. rsa was obtained using naccess (19) . • secondary structure: it is encoded by 1 bit of information about the structure as coil, helix or plate and was predicted using the dssp (20, 21). • dihedral angles: it is encoded by 2 bits of information with phi / psi angles of each of the amino acids and was predicted using dssp (20, 21) for obtaining dihedral angles. standard ligand features were calculated for ligands in our dataset using padel (22) and 1d, 2d and chemical fingerprints, which includes hybridisation, atom pair interaction, counts of various functional group. we also used qikprop (34) and canvas (23) to derive admet (absorption, distribution, metabolism, excretion, and toxicity) properties, which includes the physical properties, solubility and partition coefficients. the exhaustive list of every property calculated is given in the appendix. it results in a 1d array of 14,716 dimensions containing the various properties of a given ligand. this is used as a feature vector representing the ligand represented in mol2 format. the three-dimensional co-ordinates of atoms were converted into a 3d grid of resolution 10å with 1å spacing between the two axes centered along the centroid of the ligand. atoms outside each such grid were discarded. the atoms lying inside the grid were rounded up to the nearest coordinate of the grid where features of corresponding atoms that lay in the same coordinates were added up. this resulted in projecting ligand-interacting residues into a three-dimensional cube with features representing the atomic as well as protein-based properties of each atom of the protein pocket. features were calculated at the atomic level (section 4.1.1) corresponding to each atom of an amino acid and ligand. a 19-bit vector was calculated that uniquely identified each of the atoms in the 3d co-ordinates of a given protein-pocket and ligand complex. a 4d tensor each of size m x m x m x 19, i.e. the 3 coordinates (x, y, z) and the features, where m represents the number of atoms present in a complex was constructed as the feature vector representing the given protein pocket-ligand. the 4d vector contains the protein-pocket features and was converted to a 3d grid using grid featurization (section 4.3). the 3d-featurized grid is essentially a 4d tensor, where the coordinates are approximated to the points on the grid. the dataset is converted to vectors and is divided into training:validation:test sets in ratio 80:10:10. convolutional neural networks (24) have been used to capture spatial features in an image. we use cnns to capture the interaction between ligand and protein atoms in threedimensional space. a network was constructed ( figure 2 ) with a 3d cnn of varying channel sizes of [64, 128, 256] with non-linear activation relu after each layer, each 3d cnn had a filter of 5å cube which was used to perform convolution operations. maxpool (25) layer that acts in three dimensions to lower the dimension with a pool size of 2å cube and batch normalization (26) layer is added after each cnn layer, this in turn decreases the training time and helps in faster convergence. the latent features learnt from the above cnn layers were then flattened and used for calculating the binding affinity of the protein pocket-ligand pair. the cnn derives the relation among the 3d coordinates and their features, which would correspond well to the binding affinities of complexes. the features from the last cnn layer are then flattened out, and passed through a fully connected neural network having the number of neurons as [1000, 500, 250] with relu as non-linearity after each layer. dropout (25) is added after each layer to prevent overfitting by forcing the neural network to learn various other pathways by randomly assigning neurons to zero, 0.50 as dropout threshold. dense network predicts a regressive value of binding affinity, corresponding to a single neuron output. training framework is shown in figure 2 and a detailed layer network is shown in figure 4 (a). the featurized protein-pocket grid formed was rotated to all 24 combinations possible, such that the network is able learn in an orientation invariant form. the network was trained by taking mean square error between the predicted and actual values as a loss function. the network was optimized using adam (27) as the optimizer with a learning rate of 1e-5 and weight decay of 0.001 for 20 epochs. network was trained on an nvidia pascal gpu using pytorch (28) as the framework. features were calculated at the amino acid level (section 4.1.2) and were concatenated alongside the atomic level features (section 4.1.1) to each atom of amino acid. it results in a 44-bit vector uniquely identifying each of the atoms in the 3d co-ordinates of a given protein. a 4d tensor each of sizes m x m x m x 44, i.e. the 3 coordinates (x, y, z) and the features, where m represents the number of atoms present in a complex is constructed as the feature vector of protein pocket. the 4d vector contains the protein-pocket features, it was converted to a 3d grid using grid featurization (section 4.3). the 3d featurized grid is essentially a 4d tensor, where the coordinates are approximated to the points on the grid. the ligands were separately featurized by calculating the ligand properties (section 4.2), which results in a 1d tensor. the dataset is converted to vectors and is divided into training:validation:test sets in ratio 80:10:10. a multi-input network was constructed (29) training framework is shown in figure 3 and a detailed layer network is shown in figure 4 (b) the featurized protein-pocket grid formed was rotated to all 24 combinations possible, such that the network is able to learn in an orientation invariant form. the featurized protein pocket-ligand pair of training set was passed through corresponding the network and trained by taking mean square error between the predicted and actual values as a loss function. the network was optimized using adam (27) as the optimizer with a learning rate of 1e-5 and weight decay of 0.001. the network was trained on an nvidia pascal gpu using pytorch (28) as the framework. the performance of the models was quantified using mean absolute error (mae) and root mean square error (rmse). it was tested on validation and testing sets which were initially divided from our dataset as mentioned in the training section. lower error corresponds to better learning capacity of the model. standard deviation among the real and predicted values was also calculated. the mae, rmse and sd values are shown in table 1 . for the purpose of training and testing models, one nvidia tesla p100 gpu cluster was used. computational time taken for featurization of dataset, training and testing were 52 hours, 22 hours and 8 minutes respectively. two modules were trained. the first module was trained using a small set of features for protein and ligand, which were represented together in a 3d grid space. this approach has also been part of a previous study (29) . however, the previous study uses a restricted ligand set that does not involve larger ligands. here we have used a diverse set of ligands as one of our inputs. with training of atomic model for 35 epochs, mae score of 2.84 was achieved (table 1) . we constructed another module that enabled us to improve on the ligand and protein based information. to this purpose, we used an increased feature vector size which amounted to 14716 bits in size for ligand and 44 bits for each atom of protein. with training of composite model for only 4 epochs, mae score of 2.27 was achieved ( table 1) . the performance of our model was further evaluated using ligand-bound complexes from the kinase superfamily from pdb. the composite model outperformed the atomic model significantly and with lower standard deviation. in light of the ongoing coronavirus pandemic, we tested protein-ligand complexes from the coronavirus (cov) family. the covid-19 main protease is a key enzyme for the novel strain of coronavirus that is being implicated in the pandemic. a recent study involved testing of invitro binding efficacy of coronavirus covid-19 virus main protease (mpro) with a potent reversible synthetic inhibitor, n3 (31) . however, the highly potent inhibition by n3 rendered the experimental determination of binding affinity not achievable. using the structure of mpro at high resolution (7bqy: 1.7 angstrom), we have been able to predict the binding affinity of n3 to 3.1e+4 nanomolar (table 3) . this value agrees with the observed high affinity in the course of recent experiments (31) . another study has deposited the complex of the covid-19 main protease with a broadspectrum inhibitor x77 (n-(4-tert-butylphenyl)-n-[ (1r)-2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl]-1h-imidazole-4-carboxamide) (2020; unpublished). we used these complexes to predict their respective binding affinities as they have not been made available. based on our model-based predictions, broad spectrum inhibitor x77 scores for highest affinity followed by ligands z45617795, n3, z31792168, z1220452176 and z219104216 in the order of decreasing binding affinity ( we propose a deep-learning based approach to predict ligand (eg., drug)-target binding affinity using only structures of target protein (pdb format) and ligand (sdf format) as inputs. convolutional neural networks (cnn) were used to learn representations from the features extracted from these inputs and hidden layers in the affinity prediction task. we used two approaches to feature extraction-atomic level as well as composite level and compared their performance using the same network. deep-learning based approaches have been implemented for prediction of binding affinity. one of the studies used atomic level features of complex in a cnn based framework for binding affinity prediction (32), while another study used protein sequence level features in a cnn based framework for prediction (33) . another approach used as been to use feature learning along with gradient boosting algorithms to predict binding affinity (34) . here, we provide a composite model that incorporates tripartite structural, sequence and atomic level features with those of the atomic and other chemical features of the ligand to predict binding affinity of a putative complex. we have trained two models to predict the binding affinity between protein and ligand in a given complex. this would help existing databases like rscb pdb, pdbbind in filling missing binding affinity data for complexes. we have constructed a novel dataset that represents a diverse set of ligands and using a novel deep learning based approach we have achieved significant improvement in prediction of binding affinity of protein-ligand complexes. interestingly, our approach performed better without ligand coordinates as input. to counter filtering or noise reduction in our dataset, our dataset constructed is smaller than pdbbind (35) but we have overcome the constraints on ligand selection part of a previous study (29) . although our dataset contains 5464 complexes compared to 16,151 complexes found in pdbbind, the ligands used as part of our training include 452 unique ligands absent in pdbbind. this helps in achieving ligand diversity during training the cnn model. the similarity matrix constructed from the binary fingerprints of ligands used in the dataset supports our claim of improved ligand diversity in our dataset (supplementary file s1). we have also eliminated the need of providing ligands in a complex form with protein. thus a given protein pocket may be tested for the degree of binding for any given ligand. this can be extended to predicting potential binding partners for proteins in other superfamilies as well. it is also important to consider that docking score and pose is not a reliable correlation with mm/gbsa poses (36). deelig can be used for a member of any protein superfamily and a non-peptide ligand, the docking pose of which may or may not be known. the code repository for the project is publicly available at : https://github.com/asadahmedtech/deelig binding affinity predictions through deelig can be extended to protein-ligand complexes of protein superfamilies where the affinity is quantitatively unknown due to experimental limitations or where the potential for binding is yet to be explored in vitro. a webserver to implement deelig for easy online access would be useful for the general scientific community and this will also be in the pipeline. a later version of deelig which is trained on peptide ligand dataset will also be worked on. following properties of ligand were calculated using padel (22) • ip (ev) ( ionization potential) • ea (ev) (electron affinity) • #metab (likely metabolic reactions) • psa (van der waals sa of polar n and o atoms) • #nando, #ringatoms (number of atoms in rings) • #in34 (number of atoms in 3 or 4 membered rings) • #in56 (number of atoms in 5 or 6 membered rings) • #noncon (ring atoms cannot form conjugated aromatic bonds) • #nonhatm (heavy atoms-nonhydrogen atoms) • ruleofthree • ruleoffive (lipinski violations) • qplogkhsa (binding to human serum albumin) • percenthuman-oralabsorption recent improvements to binding moad: a resource for protein ligand binding affinities and structures gapped blast and psi-blast: a new generation of protein database search programs analysis and comparison of 2d fingerprints: insights into database screening performance using eight fingerprint methods the mm/pbsa and mm/gbsa methods to estimate ligandbinding affinities expert opinion on drug discovery simboost: a read-across approach for predicting drug-target binding affinities using gradient boosting machines receptive fields, binocular interaction and functional architecture in the cat's visual cortex batch normalization: accelerating deep network training by reducing internal covariate shift structure of m pro from sars-cov-2 and discovery of its inhibitors a series of pdb related databases for everyday needs sc-pdb: a 3d-database of ligandable binding sites-10 years on dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features on the binding affinity of macromolecular interactions: daring to ask why proteins interact detection of multiscale pockets on protein surfaces using mathematical morphology adam: a method for stochastic optimization imagenet classification with deep convolutional neural networks binding moad (mother of all databases gradient-based learning applied to document recognition deepatom: a framework for protein-ligand binding affinity prediction deep learning in bioinformatics bindingmoad, a high-quality protein-ligand database deepdta: deep drug-target binding affinity prediction pytorch: tensors and dynamic neural networks in python with strong gpu acceleration. pytorch: tensors and dynamic neural networks in python with strong gpu acceleration ucsf chimera-a visualization system for exploratory research and analysis the rcsb protein data bank: redesigned website and web services very deep convolutional networks for largescale image recognition development and evaluation of a deep learning model for protein-ligand binding affinity prediction the pdbbind database: collection of binding affinities for protein-ligand complexes with known three-dimensional structures the pdbbind database: methodologies and updates the pdbbind database: methodologies and updates padel-descriptor: an open source software to calculate molecular descriptors and fingerprints pdb-wide collection of binding data: current status of the pdbbind database binding of nicotinoids and the related compounds to the insect nicotinic acetyicholine receptor schrödinger release 2020-3: qikprop, schrödinger, llc supplementary files a. similarity matrix of ligands used in dataset (supplementary file s1) b. dataset details (supplementary file s2) c. dataset distribution aa acknowledges funding awarded by the indian academy of sciences, bangalore (2019).bm would like to acknowledge tata trusts-tdu fellowship for phd awarded to her from 2017 to 2019. all authors acknowledge ncbs for infrastructural support. the authors declare no conflict of interest. key: cord-022200-hqc8r31t authors: hyatt, alex d. title: protein a–gold: nonspecific binding and cross-contamination date: 2012-12-02 journal: colloidal gold doi: 10.1016/b978-0-12-333928-7.50007-1 sha: doc_id: 22200 cord_uid: hqc8r31t nan colloidal gold has been a popular electron-dense marker in immunoelectron microscopy for the past decade. over this period protein a-gold has been used extensively as a reagent for binding immunoglobulins from many mammalian species. due to the broad reactivity of the probe and the ease of preparation, protein α-gold remains a popular choice as an immunolabel. the aim of this chapter is to discuss the potential problems associated with protein α-gold as stated in the literature and as recog nized in this laboratory. to understand the potential difficulties, it is nec essary first to discuss the techniques involved with the preparation of colloidal gold and protein α-colloidal gold conjugates. colloidal gold is generally prepared by the reduction of tetrachloroauric acid, h(aucl 4 )-4h 2 0, with a range of chemicals including phosphorus (zsigmondy, 1905; zsigmondy and thiessen, 1925) , trisodium citrate (frens, 1973) , ascorbic acid (stathis and fabrikanos, 1958) , sodium borohydride (tschopp et al, 1982) , and trisodium citrate and tannic acid (slot and geuze, 1985) . the gold colloid is formed from micromolecular units (nuclei) and the increase in particle size is dependent on the rate of forma tion of nuclei and the rate of crystal growth. thus, by controlling the reduction of tetrachloroauric acid, gold probes of varying diameters can be produced. reduction with white phosphorus, ascorbic acid, or triso dium citrate, for example, produces gold particles with average diameters of 5, 12, and 16 nm, respectively (slot and geuze, 1981) . for further de tails, see handley (1989) . an important surface property of colloidal gold is its negative charge. in an aqueous phase, colloidal gold will stay in suspension by electro static repulsion. if electrolytes are added, the ion layers of the particles are compressed and so the critical "cohesion" distance is reduced, thereby facilitating flocculation. it has been recognized since 1901 (zsig mondy, 1901) that gold particles can be stabilized in solution by the addi tion of proteins. one of the more successful of such proteins is staphylo coccus aureus coat protein a. the complexing of protein a (as with other proteins) to colloidal gold involves electrostatic interaction between the negatively charged surface of gold particles and positively charged groups of the proteins. it is generally accepted that the protein is attracted into the action radius of van der waals-london attractive forces and there firmly binds to the surface of the gold particles. the important parameters for successful preparation of stable and ac tive protein α-gold complexes are the ph of the colloidal gold suspension (roth, 1983) and the determination of the minimum amount of protein required for stabilization. although the isoelectric point for protein a is 5.1, complexes are generally produced at a ph range of 5.9 to 6.2. how ever, adequate protein binding can be obtained at a ph between 5 and 6 (slot and geuze, 1981) ; the overall bioactivity and stabilization do not appear to be compromised in such complexes. the amount of protein a required for stabilization of colloidal gold is generally determined by the technique described by zsigmondy and thiessen (1925) . the test results in a color change of the colloidal gold solution from orange-red to blue when there is incomplete stabilization. the minimum amount of protein a required to prevent this effect is taken as an estimation for the stabiliza tion of the gold solution. it is common practice to add 10 to 100% excess protein a to the solution to ensure stabilization geuze, 1981, 1985) . the protein a complex is then stored in a buffer (e.g., phosphatebuffered saline, pbs) containing an excess of unrelated protein such as bovine serum albumin (bsa); this further stabilizes the gold complex. the protein α-gold method is a two-stage immunolabeling procedure. the antigenic site within or on the specimen is revealed (indirectly) by the addition of a primary antibody. the excess antibody is removed and protein α-gold added. protein a has two functional fc binding regions (langone, 1982) , which interact predominantly at the ch 2 and ch 3 do mains of the fc region of the antibody (forsgren and sjöquist, 1966) . the binding is strong in most igg classes of several mammalian species such as human, rabbit, guinea pig, and dog; weaker with igg classes from horse, cow, and mouse; and weakest in igg from goat, sheep, rat, and chicken (forsgren and sjöquist, 1966; kronvall et al., 1970 kronvall et al., , 1974 biberfeld et al., 1975; lindmark et al., 1983) . binding to different classes of igg, human iga, and ige and igm from various species has also been shown (goudswaard et al., 1978; langone, 1982; lindmark et al., 1983) . it is obvious from the above that antibodies used in protein α-gold proce dures should be generated from species which produce high-affinity anti body-protein a complexes. furthermore, the antibodies should be of the one class, that is, they should be affinity-purified or monoclonal antibodies. labeling of antigens can occur prior to embedding (preembedding immunocytochemistry) or postembedding of whole or sectioned specimens. labeling can therefore occur either on or within tissue sections or whole mounts (for example, whole cells and complete cytoskeletal matrices). irrespective of the mode of antigenic presentation, the labeling protocol generally follows a set format such as that outlined below. labeling of structures in fig. 2.1a ,b was produced by the same protocol, the details of which are shown in parentheses. the incubations were performed in plastic petri dishes and solutions changed with micropipettes ( fig. 2. 2). 1. wash (pbs, 3 min). 2. fixation (0.1% glutaraldehyde in pbs, 2 min; when cytoskeletons are prepared the fixative includes 1% of the non-ionic detergent np40). 3. wash (pbs, 3x3 min). 4. blocking step [pbs containing 1% bsa (pbs-bsa), 10 min]. 5. primary antibody (1 : 10 dilution in pbs-bsa, 37°c (310 κ), 1 hr). 6. wash (pbs-bsa, 6x3 min). 7. protein a-gold (1 : 20 in pbs-bsa, 37°c (310 κ), 1 hr). 8. wash (pbs, 6x3 min). 9. stain (2% phosphotungstic acid adjusted to ph 6.8 with 1 μ koh). provided the antibodies are affinity purified or are monoclonal, the anti gen-antibody interactions will be specific and indicative of the presence and localization of the antigen. this assumes that the antigen has not been leached or translocated from or within its biological matrix due to inap propriate fixation. the possibility of "unwanted" antibodies, from whole sera, reacting with cell constituents is discussed in the following section. gold particles of varying sizes can be produced by different methodolo gies (handley, 1989) . when the gold particles vary in size, i.e., their coef ficient of variation (cv) is greater than 15% (slot and geuze, 1985) , the solutions are considered polydispersive in size; when the cv is less than 15% they are referred to as monodispersive. the production of monodispersive colloidal gold is dependent on the method for synthesis (han dley, 1989) and subsequent treatment (bendayan, 1984) . the ability to produce solutions of monodispersive gold particles is of paramount im portance in multiple-labeling immunoelectron microscopy. the advantage of protein a is that it can be readily complexed to a gold probe of any size and possesses an affinity for most igg of most mammals. protein α-gold probes therefore provide a potential means whereby colocalization of antigens within a single sample can be achieved. in the literature there are numerous reports which claim success in double labeling of anti gens within/on the one biological section. the techniques include colocalization of antigens on one face of a section with probes of different sizes (geuze et al, 1981; roth, 1982; hisano et al, 1984) or the labeling of both faces of a section (bendayan, 1982) . in the first technique addition of each antibody is followed by protein α-gold probes of specific sizes in a defined sequence. the first protein α-gold probe to be applied is the smaller of the two (3-5 nm), followed by the larger probe (12-15 nm). when this sequence is followed, little cross-contamination occurs (roth, 1982) . if free protein a is added during the last minutes of step 7 (see above) little cross-contamination is re ported to occur irrespective of which probe is applied first (slot and geuze, 1984) . if, however, the order of probes is reversed without the addition of free protein a, cross-contamination can occur (roth, 1982) . the protocol for this form of double labeling is a simple extension of that outlined for single labeling. 1-7. same as on p. 22. 8. free protein a (= 0.05 mg/ml) in the last few minutes of step 7. 9. wash (pbs, 6x3 min). 10. repeat steps 5 to 7. while this procedure has enjoyed some success, there have been nu merous reports of problems associated with it (bendayan, 1982; ben dayan and stephens, 1984; hyatt et al., 1988 ). an alternative procedure is described by bendayan (1982) . in this procedure both sides of the sec tion are used; that is, there are t w o independent labeling steps. this sec ond technique avoids any cross-contamination b e t w e e n the different im munolabeling steps. n o t all double labeling is performed on biological sections. w h e n only one aspect of a sample is available for labeling (for example, viruses ad sorbed to a grid substrate), then successful double labeling, as illustrated in fig. 2 .3, may not be possible with either of the above m e t h o d s . h y a t t et al. (1988) , for example, found that protein α -g o l d could not be used for the multiple epitope mapping of a k a b a n e virus as it resulted in colabeling of the primary antibody. successful double labeling could be achieved only with specific igg-gold probes (fig. 2.3) . f o r additional in formation on multiple labeling, the reader is referred to doerr-schott (1989) . some potential problems associated with protein α -g o l d labeling are also associated with other immunocytochemical techniques, namely nonspecific staining (for example, nonspecific antibodies and electrostatic at tachment; taylor, 1978; behnke et al., 1986; gosselin et al., 1986; birrell et al., 1987) . double immunolabeling with the protein a method can in duce further sources of error, namely cross-contamination of antibodies with protein α-gold. the problems of cross-contamination and nonspe cific labeling are discussed below (also see birrell and griffith, 1989; park etal., 1989) . as stated previously, it is advisable to use monospecific antibodies raised in the appropriate mammalian species for the primary localization of antigens. from a practical viewpoint, affinity-purified and monoclonal antibodies generally yield specific labeling with a clean background ( fig. 2.1a) . the labeling in fig. 2 .1b, on the other hand, was produced with a whole porcine serum as the primary antibody. although 1% bsa was used to minimize nonspecific labeling (as per fig. 2.1a) , the background in fig. 2 .1b was considerably greater. an explanation for this is that the use of whole serum (containing numerous natural antibodies, targeted an tibodies, and antibodies to any carrier molecules or extraneous material carried over or used in the immunization) with protein α-gold produces many immunocomplexes. the overall effect can result in the adsorption of unwanted antibodies and subsequent labeling of various cellular con stituents in addition to the targeted antigen. if normal serum is used to block nonspecific binding of primary antibodies to the reactive sites of tissues, an effect similar to that described above may occur. in many instances the problem of such nonspecific binding may be reduced by decreasing the effective antibody concentration. if the studies involve in fectious agents, hormones, or the like (i.e., cause-and-effect experi ments), then nonspecific staining can be further reduced by preadsorption of the serum with the normal (nonexperimental) biological tissue. biological samples bathed in serum may also provide another source of error (for example, tissue culture cells and cells of the blood). it is not unreasonable to expect that during specimen preparation some antibodies may fortuitously bind to the sample surface. such antibodies may provide additional binding sites for protein a. these samples should be thor oughly washed (e.g., with pbs) before labeling is attempted. in double-labeling experiments cross-contamination is now recognized as a problem which arises from the affinity which protein a possesses for different regions of igg antibodies, namely the fc and fab regions (roth, 1982; endresen, 1979; zikan, 1980) . protein a can bind two igg mole cules; thus, after the primary antibody-protein a complex has been formed, free igg binding sites can still be available on the bound protein a. addition of the second antibody can result in its binding to the free sites on protein a molecules and target specified antigens. a second pro tein α-gold probe can potentially bind not only to any protein a-gold free igg antibodies from the primary incubation (the fc region of the secondary igg complexes) but also to the fab regions of igg antibodies. reports which claim success with the "one-face" protein a-gold method use either a defined sequence of protein a-gold probes or free protein a (described above). if free protein a is not incorporated, then the success of the method depends on the smaller of the protein a-gold probes being used first. the small probes (= 3 nm) have approximately one associated protein a molecule, whereas larger probes (= 15 nm) can adsorb -60 such molecules (roth, 1982) . if the larger of the gold probes is used for the visualization of the first antigen, many potential fc binding sites (unoccupied protein a) are available for the second immunoglobulin and significant cross-contamination may therefore result. if excess free protein a is added, the primary antibody-protein a interactions may reach saturation and so minimize interactions with secondary protein a-gold probes. the binding of secondary antibodies to the existing pro tein a fc binding sites should not be recognized by the secondary protein a-gold probe as it is assumed that igg can bind only one protein a mole cule. cross-contamination can still occur, however, via interactions with the fab regions of igg. if cross-contamination is a continued problem, alternative techniques to the protein a method should be investigated. such techniques generally involve direct labeling and species-specific antibody-gold indirect labeling techniques. the use of small (3-6-nm) gold probes has the advantage of improving immunocytochemical resolution. anyone who has worked with small gold probes has observed them (in some specimens) to be extremely "sticky" (fig. 2.4a,b) . behnke et al. (1986 ), birrell et al. (1987 ), and birrell and griffith (1989 have reported the problem to be largely associated with exposed areas (areas not covered with protein) on the gold particles themselves and secondarily with the method by which the colloidal gold is produced. since small protein a-gold complexes are used widely, the problems associated with small probes merit discussion. flocculation of colloidal gold can be prevented by partial saturation of the particles (goodman et al., 1980; horisberger and vauthey, 1984) . the stabilization of colloidal gold therefore does not ensure its saturation. it is because of this that excess unrelated protein (for example, bsa) is (1 : 10) . n o stabilizer was included within the added to these protein-gold complexes; the subsequent complex is then assumed to be saturated. however, it would appear that some immuno gold labeling patterns are independent of protein-protein interactions and result from interactions between specimen constituents and the gold par ticles (behnke et al., 1986) . the occurrence of such nonspecific binding is obvious in whole cytoskeletons (fig. 2.4a ) and thick polyethylene glycolextracted sections (a. d. hyatt, unpublished observations) . strategies in colloidal gold labeling of cytoskeletal elements are discussed at length by birrell and griffith (1989) . nonspecific binding is attributed to electro static interactions between exposed areas on the gold particles (areas of negative charge) and cationic structures within the specimen (behnke et al., 1986) . the method used to prepare colloidal gold has also been considered a source for nonspecific labeling (birrell et al., 1987) . birrell et al. found that 5-nm gold particles produced by the trisodium citrate-tannic acid method exhibited a higher degree of nonspecificity than particles of simi lar size produced by other methods. this nonspecificity was attributed to the chemical agents used in colloidal gold production (birrell et al., 1987) ; for example, colloidal gold produced by the white phosphorus method was found to contain phosphate groups. tannic acid may therefore also be a source of contamination for colloidal gold produced by the trisodium citrate-tannic acid procedure (birrell et al., 1987; birrell and griffith, 1989) . such contaminants on gold particles may, as with tannic acid, form a series of complex interactions with biological specimens, particularly those fixed with an aldehyde and/or os0 4 (simionescu and simionescu, 1976) . observations in this laboratory and by birrell et al. (1987) and bir rell and griffith (1989) that nonspecific labeling is more pronounced with smaller probes may be explained by the greater accessibility of these probes to tissue constituents within open and/or permeabilized specimens. nonspecific binding due to protein-protein and electrostatic interac tions can be minimized by including in the washing and incubation steps a noncompeting protein which has a high affinity for gold particles. these proteins (e.g., bovine skin gelatin, fish gelatin, and bsa) will bind to protein-reactive constituents within the specimen and will minimize electro static interactions by covering the exposed areas on the gold particles. the choice of an appropriate stabilizer will significantly reduce the level of nonspecific labeling, as is illustrated in fig. 2 .4c (compare with fig. 2.4b ). an alternative or concomitant procedure would involve the incuba tion of protein α-gold with the biological sample. immunolabeling with protein α-gold has several distinct advantages: (1) it is easy and quick to prepare complexes of various sizes; (2) the complexes interact with antibodies from many mammalian species; and (3) the method is sensitive, clean, and can yield valuable information pro vided the parameters of immunolabeling are understood. in other words, the investigator must be familiar with the characteristics of the primary antibody, protein α-gold probes, and the specimen itself. generally, the use of the protein α-gold method should involve (1) the use of highly specific antibodies; (2) the use of such antibodies at their lowest effective concentration; (3) the absence of normal serum as a blocker; and (4), if using open matrix specimens, inclusion of an effective stabilizer in the incubation steps. these observations, if followed, will reduce the level of nonspecific labeling and cross-contamination. it should also be noted that at no stage during the labeling protocol should the sample be allowed to dry. in addition, the sample should be thor oughly rinsed between antibody and protein α-gold steps; this will re move unbound antibodies and protein a. failure to comply with the above precautions will result in high backgrounds. there are no detailed protocols for protein α-gold labeling which have universal applicability. for each different experiment, detailed protocols should be developed to produce optimum labeling characteristics. these protocols must be based on results of control immunocytochemical ex periments. if protein α-gold is the label, and nonspecific binding and cross-contamination are persistent problems in spite of the addition of excess free protein a, high-affinity stabilizers, adsorption of contaminat ing antibodies, and colloidal gold, then alternative labeling techniques should be pursued and evaluated. the author gratefully acknowledges drs. β. t. eaton and a. r. gould for reading the manuscript and mr. t. wise for skillful technical assistance. non-specific binding of protein-stabilized 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α-gold complexes with 3nm and 15nm gok particles and their use in labelling multiple antigens on ultra-thin sections the colloidal gold marker system for light and electron microscopic cyto chemistry galloylglucoses of low molecular weight a: mordant in electron microscopy. i. procedure, and evidence for mordanting effect sizing of protein α-colloidal gold probes for immu noelectron microscopy gold markers for single and double immunolabellinj of ultrathin cryosections preparation of colloidal gold immunoperoxidase techniques: practical and theoretical aspects ultrastructure of the mem brane attack complex of complement: detection of the tetramolecular c9-polymeriz ing complex c5b-8 interactions of pig fab gamma fragments with protein a from staphylococ cus aureus das kolloidale gold key: cord-014368-4nasrbs6 authors: nan title: gene chip for viral discovery date: 2003-11-17 journal: plos biol doi: 10.1371/journal.pbio.0000003 sha: doc_id: 14368 cord_uid: 4nasrbs6 nan comparing the genomes of related organisms, researchers can see what parts of the genomes are conserved-highly conserved genes tend to be important-and then focus on these regions to track down genes and determine how they function. to construct a draft sequence of the c. briggsae genome, the researchers merged genomic data from three sources-one derived from whole-genome shotgun sequencing, another from physical genome mapping, and the third from regions of a previously "finished'' sequence. for the shotgun sequence, the researchers extracted dna from worms, randomly cut it into short pieces, sequenced them, and then assembled overlapping sequences to create thousands of stretches of contiguous dna sequence. to help fill in the gaps between these "contigs,'' stein and colleagues developed a "fingerprint'' map of the genome as a guide for aligning the shorter fragments. the map also helped them identify inconsistencies and misalignments in the genome assembly. finally, they integrated the previously finished sequence to improve the draft genome sequence. using these massive datasets, the authors produced a high-quality genome sequence; although it does not quite meet the gold standard of a "finished'' sequence, it covers 98% of the genome and has an accuracy of 99.98%. after confirming the accuracy of the draft, the researchers turned to the substance of the genome. examining two species side by side, scientists can quickly spot genes and flag interesting regions for further investigation. analyzing the organization of the two genomes, stein et al. not only found strong evidence for roughly 1,300 new c. elegans genes, but also indications that certain regions could be "footprints of unknown functional elements.'' while both worms have roughly the same number of genes (about 19,000), the c. briggsae genome has more repeated sequences, making its genome slightly larger. because the worms set out on separate evolutionary paths about the same time mice and humans parted ways-about 100 million years ago, compared to 75 million years ago-the authors could compare how the two worm genomes have diverged with the divergence between mice and humans. the worms' genomes, it seems, are evolving faster than their mammalian counterparts, based on the change in the size of the protein families (c. elegans has more chemosensory proteins than c. briggsae, for example), the rate of chromosomal rearrangements, and the rate at which silent mutations (dna changes with no functional effect) accumulate in the genome. this would be expected, the researchers point out, because generations per year are a better measure of evolutionary rate than years themselves. (generations in worms are about three days; in mice, about three months.) what is surprising, they say, is that despite these genomic differences, the worms look nearly identical and occupy similar ecological niches; this is obviously not the case with humans and mice, which nevertheless have remarkably similar genomes. both worm pairs-as well as mouse and human-also share similar developmental pathways, suggesting that these pathways may be controlled by a relatively small number of genes and that these genes and pathways have been conserved, not just between the worms, but also between the nematodes and mammals. this question, along with many others, can now be explored by searching the two species' genomes and comparing those elements that have been conserved with those that have changed. with the nearly complete c. briggsae genome in hand, worm biologists have a powerful new research tool. by comparing the genetic makeup of the two species, c. elegans researchers can refine their knowledge of this tiny human stand-in, fill in gaps about gene identity and function, as well as illuminate those functional elements that are harder to find, and study the nature and path of genome evolution. some 200,000 people live with partial or nearly total permanent paralysis in the united states, with spinal cord injuries adding 11,000 new cases each year. most research aimed at recovering motor function has focused on repairing damaged nerve fibers, which has succeeded in restoring limited movement in animal experiments. but regenerating nerves and restoring complex motor behavior in humans are far more difficult, prompting researchers to explore alternatives to spinal cord rehabilitation. one promising approach involves circumventing neuronal damage by establishing connections between healthy areas of the brain and virtual devices, called brain-machine interfaces (bmis), programmed to transform neural impulses into signals that can control a robotic device. while experiments have shown that animals using these artificial actuators can learn to adjust their brain activity to move robot arms, many issues remain unresolved, including what type of brain signal would provide the most appropriate inputs to program these machines. as they report in this paper, miguel nicolelis and colleagues have helped clarify some of the fundamental issues surrounding the programming and use of bmis. presenting results from a series of long-term studies in monkeys, they demonstrate that the same set of brain cells can control two distinct movements, the reaching and grasping of a robotic arm. this finding has important practical implications for spinal-cord patients-if different cells can perform the same functions, then surgeons have far more flexibility in how and where they can introduce electrodes or other functional enhancements into the brain. the researchers also show how monkeys learn to manipulate a robotic arm using a bmi. and they suggest how to compensate for delays and other limitations inherent in robotic devices to improve performance. while other studies have focused on discrete areas of the brain-the primary motor cortex in one case and the parietal cortex in another-nicolelis et al. targeted multiple areas in both regions to operate robotic devices, based on evidence indicating that neurons involved in motor control are found in many areas of the brain. the researchers gathered data on both brain signals and motor coordinates-such as hand position, velocity, and gripping force-to create multiple models for the bmi. they used retraining the brain to recover movement if you have ever spent an evening hoisting brews with your pals at the corner pub, chances are you never stopped to think-gee, how do i lift my glass now that it's only half full? it seems like a simple task-you raise that glass reflexively, whether it is empty or full-yet the neural calculations that determine the force needed to lift your arm smoothly to your lips in each case are anything but simple. the brain, it seems, operates like a computer to process variable cues-such as the weight of a glass and the position of your arm-to generate an appropriate response: lifting the glass. neuroscientists believe the brain builds a kind of internal software program based on past experience to transform such variable cues into motor commands. the brain's software, or internal model, depends on specialized sets of instructions, or "computational elements,'' in the brain. but exactly how the brain organizes these elements to process sensory variables that affect arm movements is far from clear. eun jung hwang and colleagues predict that these computational elements are based on a multiplicative mechanism, called a gain field, through time after time in biology, revelations about structure lead to insights about corresponding functional mechanisms. while evolution throws in the occasional spandrel, more often organizational structure serves a practical purpose. so naturally, neuroscientists wonder, does the architectural organization of the motor system reveal an underlying functional organization? progress on this question has been complicated by the fact that there appears to be no clear correspondence between the development of motor neurons centrally and their target muscles in the periphery. in the visual system, for example, retinal ganglion cells send axons in an ordered manner into the brain, where they form connections with neurons of the primary visual center in the brain responsible for detecting visual targets. the arrangement of these connections mirrors the neighboring relationships of the neurons in the retina, and so the neural map of connections in the brain is an "anatomical correlate'' of the arrangements in the retina. the origin of these anatomical relationships can be traced through the process of development, allowing scientists to link the assembly of this sensory system with the function of the neurons involved. matthias landgraf and colleagues now report that in the fruitfly drosophila the arrangement of motor neurons corresponds to the distribution of their target muscles. thus, anatomical correlates also exist in the motor system, in the form of a "myotopic map,'' where the arrangement of motor neuron dendritic branches in the central nervous system reflects the distribution of their target body wall muscles in the periphery. starting with the larger question of how the neural networks governing locomotion are specified and assembled during development, the researchers decided to see if they could identify an elementary principle of motor system organization. working in drosophila, they examined motor neurons and the body wall muscles they innervate. with an eye toward understanding the mechanisms directing the assembly of the motor system, the researchers concentrated on the early stages of development, when the motor neurons first establish their characteristic dendritic territories. they found that the dendrites of motor neurons innervating internal muscles and that those innervating external muscles do in fact project into distinct regions, corresponding to the distinct mapping of the muscles themselves. surprisingly, the arrangement of the dendrites in the myotopic map forms independently of the muscles they innervate. it may be, the researchers suggest, that the initial signals charting the location of the dendrites are set very early in development, when the coordinates for other structural elements are established. but that question requires further investigation. the researchers are among the first to reveal such an orderly underlying principles of motor system organization revealed which sensory signals to the brain are amplified by signals from the eye, head, or limbs. in this way, the brain can rely on past experience of one kind of sensory cues to predict how to respond to new but similar situations. while previous studies had established that some visual cues are combined through a gain field, this study shows that motor commands may also be processed via gain fields. this finding, the researchers demonstrate, accounts for a range of behaviors. based on previous studies showing that when people reach to various directions in a small space, they can extrapolate what they learn about the forces in one starting position to a significantly different position, it has been proposed that the way the brain computes movement is not terribly sensitive to limb position. citing other research with seemingly contrary conclusions-that the brain can be highly sensitive to limb position in calculating force and movement-hwang et al. set out to investigate whether-and how-the brain creates a template to translate sensory variables (limb position and velocity) into motor commands (force). they created a computer model to mimic the reaching behaviors observed by people in their experiments and found that the most accurate model used computational elements that are indeed sensitive to both limb position and velocity. if the brain processes these two independent variables through a gain field, it can use the relationship of the two variables-that is, the strength of the gain field-to adapt information about the force needed to move or lift something in one situation to accomplish a wide range of similar movements. when the researchers compared their model to previously published results, they found their model accounted for seemingly disparate findings. they explain that the brain's sensitivity to limb position can be either low or high after a task has been learned because the gain field itself is adjustable. the authors note that neurophysiological experiments suggest that the motor cortex may be one of the crucial components of the brain's internal models of limb dynamics. the next step will be to track the motor cortex neurons to see whether their activity supports this model. hwang et al. predict they will. novel "checkpoint" mechanism mediates dna damage responses connection between patterns of motor neuron dendrites and patterns of muscles. this organization, in the form of the myotopic map, may be mirrored by the patterning of processes of higher-order neurons, which form connections with the motor neuron dendrites themselves. in vertebrates, studies have shown that motor neurons are grouped into "pools'' and "columns'' that correlate with the muscles they innervate. but because these pools and columns represent the location of the cell bodies and not the areas of the spinal cord where the neurons receive most of their inputs, that is, their dendritic branches, scientists could not say whether the pools and columns are simply spandrels-an incidental result of the way motor neurons are generated-or mirror a functional organization of the motor system. this novel fi nding in drosophila will pave the way for future studies on the relationship between anatomy and physiology during development. it will be particularly interesting to discover whether such myotopic arrangements of motor neuron dendrites are unique to insects or whether this organizational principle occurs in other motor systems, including vertebrates. of all the tasks a cell must accomplish day in and day out, protecting its genome may be the most important. genomes confront all manner of potential assaults, from the strand-splitting action of gamma-radiation to the simple copying mistakes sometimes made when dna replicates before a cell divides. though some mutations are harmless, others can disrupt gene action, leading to cancer and other diseases. to guard against such events, healthy cells maintain quality-control "checkpoints'' that sense and respond to dna injuries, as well as to defects in dna replication, and that prevent cell division until the dna can be repaired. if the damage is beyond repair, apoptosis pathways set about the business of destroying the affl icted cell. many of the genes and protein complexes involved in these checkpoint responses have been identifi ed, but the biochemical mechanisms that in some cases trigger cell cycle arrest are not fully understood. experiments by philip hanawalt and his student david pettijohn at stanford university in 1963 suggested that the molecular machinery of dna replication and repair-which they discovered at sites of damage-are quite similar and closely linked. while many studies have since supported that link, viola ellison and bruce stillman, the director of the cold spring harbor laboratory, have found new evidence that the two processes may indeed coincide by showing that protein complexes regulating a cellular checkpoint in dna repair operate much like similar complexes involved in dna replication. the molecular pathways governing the replication of dna before cell division are well known. as the double-stranded dna molecule unwinds, different protein complexes step in to ensure that each strand is faithfully reproduced. two protein complexes required for this process are replication factor c (rfc) and proliferating cell nuclear antigen (pcna). in the 1980s, stillman's laboratory isolated pcna and rfc and showed that they function together to "load'' pcna onto a structure in dna that is created after dna synthesis begins. pcna forms a clamp around the dna strand and regulates the dna polymerases that duplicate the dna double helix. studies in yeast had identifi ed a series of proteins required for the dna synthesis phase of the cell cycle and the dna damage checkpoint pathways; mutations in these proteins' genes make cells very sensitive to radiation (hence the name rad genes). a subset of these proteins, which are conserved in human cells, form two protein complexes-rsr and rhr-that function like rfc and pcna, respectively, with rsr loading the rhr clamp onto dna. ellison and stillman demonstrate that both pairs of "clamp-loading'' complexes follow similar biochemical steps, but, signifi cantly, rfc and rsr favor different dna structures for clamp loading. while it was known that the rsr/rhr complexes exist in human cells, it had not been established that the two types of clamps prefer different dna targets. the researchers also show that the rsr/rhr biochemistry depends on rpa, a protein known to be involved in the dna damage-response pathway. the discovery that rsr loads its rhr clamp onto a different dna structure was unexpected; it suggests not only that the two clamp loaders have distinct replication and repair functions, but also how the checkpoint machinery might work to prevent dna damage from being passed on to future generations. by establishing the chemical requirements of rsr/rhr interactions as well as the preferred dna-binding substrate, the researchers have charted the way for determining the different functions of these cell cycle checkpoint complexes and how the complexes' different subunits affect these functions. the researchers propose that the role of this checkpoint machinery is not as an initial sensor of dna damage, but rather as a facilitator of dna repair, stepping in after preliminary repairs to dna lesions have been made. ellison and stillman's work helps establish a biochemical model for studying how both of these checkpoint complexes function to coordinate replication and repair-and promise to help scientists understand how cancer develops when the checkpoint repair mechanisms fail. during animal development, cells gradually grow, multiply, and specialize to create the tissues and organs that shape and sustain multicellular organisms. the progression from a single cell to a thousand-, million-, or trillion-celled animal follows an exacting schedule and plan involving an elaborate network of genes and proteins. one of the primary mechanisms coordinating this process is cell-to-cell communication. cellular signaling regulates two crucial development mechanisms, apoptosis (programmed cell death) and cell proliferation, which work like chisel and clay to sculpt multiplying masses of cells into, say, a fly wing or a human finger. controlled by multiple signals operating at fixed intervals, the entwined pathways can be steered off-course by a single defect in the communication network, resulting in the death of a healthy cell, for example, or the survival of a damaged cell. such disruptions can lead to physical abnormalities, such as webbed hands and feet, when cells that should die remain alive; degenerative nerve disease, when healthy cells are killed; and cancer, when damaged cells survive and evade normal growth limitations. researchers have uncovered some of the mechanisms underlying these processes by studying genes involved in fruitfly (drosophila) development. following that tradition, stephen cohen and david hipfner have identified a gene critical to drosophila development that juggles cell growth and survival signals to help promote cell growth and prevent inappropriate apoptosis. they searched for genes associated with changes in tissue growth in fruitfly wings and identified some that can cause tissue "overgrowth''-abnormally large masses resulting either from cells growing faster than they divide or from cells escaping proliferation controls when they are overexpressed. among these is a gene that encodes a newly divide, and differentiate, most respond to the defect by killing themselves, even under conditions that normally promote survival. thus, cells without slik appear to have an intrinsic survival defect, suggesting that slik prevents apoptosis. when slik is overexpressed, cell proliferation increases, but so does apoptosis. only when apoptosis was blocked did the cells form tumor-like growths. this coupling of cell growth and cell death is characteristic of oncogenes (cancercausing genes), and slik also seems to function in both pathways. the authors point out that the signal to proliferate may inherently sensitize cells to apoptosis, as has been shown previously for some cancer cells. this may keep an individual cell under the control of its neighbors, who collectively monitor the needs of the organism. for a cell to respond to a signal by dividing rather than dying, it must get the appropriate signs from its comrades. slik, the authors demonstrate, is a key factor in determining whether a cell lives or dies. whether its mammalian counterparts play a similar role is yet to be determined. identified kinase that contributes to the regulation of cell proliferation and survival (or death, depending on the circumstance) during drosophila development. cohen and hipfner called the gene slik based on its similarity to two human kinase-coding genes (slk and lok). little is known about these human proteins, though previous studies suggest they may affect cytoskeletal dynamics and cell adhesion. in this paper, the authors report preliminary evidence supporting the notion that slik may regulate the cytoskeleton, the "backbone'' of the cell that confers structure and motility. interestingly, disturbances to cell adhesion and cytoskeletal structure are known triggers of apoptosis and are being explored as potential anticancer agents. kinases make up one of the largest families of proteins and are important regulators of cell signaling. to investigate the function of slik in drosophila, the researchers removed the gene and then studied the physical and cellular effects. they found striking delays in growth and developmental timing and showed that these effects result largely from the demise of the slik-deficient cells. while cells deprived of slik can grow, one might not expect that yeast lead terribly eventful lives, yet the singlecelled fungus must struggle to survive just like everyone else. and for yeastlike everyone else-survival means being able to detect and coordinate a rapid response to changes in its environment. though survival for humans is a bit more complicated, our cells use the same regulatory networks, which maintain cell growth and health when they work and contribute to diseases, from asthma to cancer, when they break down. given the variety of conditions even the lowly yeast is likely to encounter during its life, one might expect to find a multitude of molecules mobilizing a response. but yeast cells, it turns out, are fairly resourceful. as erin o'shea and colleagues report, just one protein in yeast activates different groups of genes in response to different amounts of an environmental stimulus. the researchers focused on how yeast responds to various levels of phosphate, an essential nutrient for all cells. one way that cells regulate responses to environmental stimuli is through the transcription (activation) of genes. these transcriptional responses are often controlled by a multistep process that shuttles gene-activating proteins into the nucleus, where they can generate the appropriate response for a given stimulus, or confines them to the cytoplasm if their gene products are not needed. during this process, called phosphorylation, the addition of a phosphate group to a protein-such as a receptor or transcription factor-acts as a mechanism for controlling gene expression. o'shea's team demonstrated that phosphorylation of a transcription factor called pho4 controls gene expression by controlling where that protein resides in the cell-in the cytoplasm or in the nucleus. as is the case with many proteins, pho4 can accept phosphate groups at multiple sites. to see whether the location of phosphorylation affects the action of pho4, o'shea's team exposed yeast to different levels of phosphate and tracked the cellular response. they found that when yeast is deprived of phosphate, pho4 has no phosphate groups at any of its binding sites and enters the nucleus, where it binds to dna and activates a set of genes whose products can scavenge for phosphate or otherwise compensate for the scarcity. when yeast has ample supplies of phosphate, pho4 is phosphorylated and remains in the cytoplasm-unable to influence transcription-suggesting that the cells can absorb plenty of nutrients from their environs without having to engage a specialized foraging team. when the researchers exposed the yeast to intermediate amounts of phosphate, the results were surprising. middling concentrations of phosphate produced different forms of phosphorylated pho4, which varied in their ability to activate genes, and so added to the number of possible responses. pho4 partially phosphorylated at one site, for example, could still enter the nucleus, but activated only one type of phosphate-recovery gene and not others. while it is not unexpected that differential phosphorylation could have different functional outcomes, the authors say, it is surprising that one enzyme acting on one transcription factor can create different phosphorylation patterns-and therefore different gene-expression patterns-in response to different amounts of a single stimulus. their results show that cells rely on a highly regulated series of interactions that induce subtle changes in gene expression to fine-tune their response to small environmental changes. and they do this in a remarkably efficient manner, relying on a small cast of characters to orchestrate the responses essential for survival. extraction, amplification, and decoding of viral sequences rapidly identify known viruses and classify new ones based on their genetic makeup. this was validated in march when the viral chip contributed to the identification of the cause for severe acute respiratory syndrome (sars) as a novel coronavirus. in the article published in this issue, the researchers describe the chip (or microarray), how it was used in the classification of the sars virus, and how it provides direct access to viral genomic sequence. microarray technology works by taking advantage of the structural properties of dna. dna molecules normally exist as double helices, two complementary strands of nucleotides wrapped around each other. the microarray consists of a large number of single dna strands attached to a solid base. these probes (which in case of the viral chip represent sequences from all fully sequenced reference viruses) can be used to interrogate unknown sequences: if a solution containing such sequences is passed over the chip, similar sequences will "hybridize,'' or bond in a signature double helix. known viruses hybridize in a characteristic pattern and can be identified quickly. because bonding occurs even when the match between probe and sample sequence is not perfect, new relatives of known viruses can be identified as belonging to a particular family (such as coronaviruses, in the case of sars). to quickly obtain more information on a novel virus, it is then possible to "syphon off'' those viral sequences that stuck to their respective counterparts on the chip and to use the material to determine part of the genomic sequence. such sequence information provides more detail on how the new virus relates to known ones, which might provide clues about its origin and possible treatment strategies. faced with all manner of potential threats in the form of billions of different viral, bacterial, and chemical pathogens, the mammalian immune system relies on a "safety in diversity'' strategy for protection. with two distinct subsystems-one innate, the other adaptive-the immune system can recognize some 100 trillion antigens. the innate system deploys cells programmed to quickly recognize microbes with a particular set of conserved molecular structures. the adaptive system relies on billions of uniquely outfitted lymphocytes (white blood cells) to identify just as many pathogens through their protein fragments, or antigens. a human being grinds out billions of these cells every day. in the absence of threats, the immune system maintains a quiescent state and many of these cells are discarded. but for the immune system, doing nothing takes a concerted effort. lymphocytes originate in the bone marrow, though not all differentiate there. one class of lymphocytes, called t cells, develops in the thymus, where every t cell acquires a one-of-a-kind receptor, called a t cell receptor (tcr), designed to recognize a different antigen. when an antigen gets bound by a tcr (a bound molecule is called a ligand), the antigen triggers a signaling cascade that tells the t cell either to attack the infected cell or to alert other immune cells of the infiltrator. but as jeroen roose, arthur weiss, and colleagues report, signaling pathways activated by bound tcrs appear to influence gene expression even in the absence of antigen or other receptor ligands, a process called ligand-independent signaling. these findings lend support to the notion that cellular signaling pathways regulated by surface receptors, like tcrs, exhibit a continuous low-level signaling (known as basal signaling) in the absence of a stimulus and that this continuous signaling, by influencing gene expression, has significant influence on cellular differentiation. roose, weiss, et al. focused on the tcr signaling pathway that regulates the expression of a group of genes, including rag-1 and rag-2, that are activated in two distinct waves during t cell development. rag genes play a crucial role in t cell development, a highly complex, multistage process that involves a reshuffling, or recombination, of tcr genes and the activation of different proteins and genes at different stages. rag genes regulate the genetic recombination and ultimate cell surface expression of tcrs. using chemical inhibitors and mutant human t cell lines deficient in critical signaling components involved in antigen receptor-dependent pathways, the researchers found that the loss of specific functions or specific proteins affected an unexpected set of target genes. notably, when downstream components (the protein kinases erk and abl) were disabled in the basal signaling pathway, the researchers saw a resurgence of rag gene expression. while erk was already known to play a prominent role in signaling pathways downstream of the tcr, it now appears that abl may also be regulated in tcr pathways. most importantly, these findings suggest that signaling pathways thought to be triggered only by ligated receptors can influence gene expression on their own. and it may be through this type of signaling that tcr pathways help regulate t cell development by repressing rag gene activity. these basal signals, the researchers postulate, may in effect save the rag expression machinery until recombination is called for. if rag genes were expressed at the wrong time, they could cause inappropriate genetic recombination and create t cells that either lack function or attack healthy cells, as happens in immunodeficiency and autoimmune diseases. elucidating the mechanisms and components of this basal pathway will contribute important insights into the development and function of the immune system. but these studies also establish a model for investigating other signaling systems, to determine whether biologically functional basal signaling is a rare phenomenon or whether it is a fundamental cell process needed to control the profile of gene expression in the quiescent state. a multicellular organism can have more than 200 different types of cells and as many as 100 trillion altogether. during the process of development, an organism enlists the service of hundreds of signaling molecules and thousands of receptors to direct cell growth, differentiation, and morphological destiny. any given cell has no use for most of these signals and gets by with just a limited repertoire of receptors on its surface. once a signal reaches a receptor, it triggers a series of biochemical reactions as different molecules transform the external signal into a biological response, in a process called signal transduction. one cell type controls all of its cellular functions-both universal and specialized-with just a few dozen receptors; each receptor elicits a wide range of responses by triggering a small number of interacting pathways. exactly how a receptor produces the right response at the right time is a fundamental question in biology. of particular interest is a class of receptors-called receptor tyrosine kinases (rtks)that regulate cell proliferation, differentiation, and survival and play an important role in embryonic development and disease. growth factor receptors are an important subset t he protozoan parasite plasmodium falciparum causes falciparum malaria, a fatal parasitic disease in humans, and is transmitted by anopheles mosquito vectors (predominantly the anopheles gambiae complex and an. funestus in africa). there are about 300 million malaria cases and 1-2 million deaths annually, the brunt of which are borne mostly in africa by children under 5 years of age and by pregnant women. in many african countries, malaria poses a formidable challenge to an overburdened and underfunded public health system. the current malarial control strategies consist of chemotherapy directed against the malaria parasite and prevention of mosquito vector/human contact using insecticide-impregnated bednets and, to a lesser extent, indoor residual insecticide spraying and environmental control for reducing mosquito breeding sites. there are still no malaria vaccines in clinical practice. chemotherapy (the use of drugs to target disease) is used for both treatment and prevention. drug resistance is increasingly becoming a problem. some of the antimalarial drugs in current use include quinolines, artemisinins, antifolates, atovaquone/ proguanil, and antibiotics. chloroquine of rtks. the platelet-derived growth factor receptor (pdgfr) family activates downstream signaling enzymes that stimulate the growth and motility of connective tissue cells, such as vascular smooth muscle cells (vsmcs), oligodendrocytes (cells of the tissue encasing nerve fibers), and chondrocytes (cartilage cells). the pdgf beta receptor is essential for directing the differentiation of vsmcs. while studies of signal transduction of this growth factor have established a model of how receptor tyrosine kinases function, the role of individual downstream signaling components in a living organism is still unclear. using mouse molecular genetics, michelle tallquist and colleagues set out to determine the function of individual components in the pdgfr beta pathway. they discovered a quantitative correlation between the overall amount of signal produced by the receptor and the end product of the signal, formation of vsmcs. receptor responses, they report, are controlled in two ways: signaling was influenced both by the amount of receptors expressed and by the number of specific pathways engaged downstream of the receptor. surface receptors have "tails'' that project into a cell's interior. when a surface receptor is activated, a number of potential binding sites-modified amino acid residues-are exposed on its intracellular tail. ten of these sites can bind to proteins with a specific amino acid sequence, called an sh2 domain; proteins with these domains can then initiate a signal transduction pathway. by introducing mutations in the sh2 domain-binding sites in mice, the researchers could evaluate how the loss of a particular binding site-and therefore pathway-affected the function of the receptor. they had previously investigated the functions of two other downstream signaling proteins in similar experiments. surprisingly, tallquist et al. found that losing some of the individual components did not produce a significant negative physiological effect. only when multiple downstream signaling pathways were disrupted did the researchers see a significant effect on the population of the cells. reductions in the numbers of both activated receptors and activated signal transduction pathways produced reductions in the population of vsmcs. these results have not been seen in tissue culture before, suggesting that signal transduction is more complex in vivo and that future studies would benefit from incorporating a global approach, rather than targeting a single signaling component. the next step will be to investigate exactly how the individual pathways contribute to this result. it is also unclear whether these results apply only to these growth factor receptors or explain how rtks operate in general. such questions have significant clinical relevance. overexpression of the pdgfr beta pathway has been linked to a variety of serious diseases, including atherosclerosis and cancer. understanding how cells control the action of this growth factor is an important step in developing targeted therapies. since many of these conditions result from a growth factor stuck in the "on'' position, inhibiting overactive receptors promises to be an effective clinical intervention. (cq) is a cheap and widely used aminoquinoline, but cq-resistant parasites have become ubiquitous in endemic countries and other drugs are now used much more frequently (ridley 2002) . fansidar, a combination of sulphadoxine and pyrimethamine (sp), is a first-line treatment in several african countries, but resistance to sp is spreading rapidly. targeting the mosquito vector with pyrethroidimpregnated bednets, in addition to chemotherapy, is an effective method of controlling malaria transmission. however, pyrethroid resistance has been reported in an. gambiae s.s. in west africa, and there is concern about its emergence in east africa (chandre et al. 1999) . thus, the public health problem due to malaria is exacerbated by the emergence of drug-resistant parasites and insecticide-resistant mosquitoes. the clinical application of efficacious intervention tools is therefore an urgent imperative for malarial control. this brings into sharp focus the importance of genomics research for drugs, vaccines, diagnostics, and insecticides. the unraveling of the genomes of humans, p. falciparum, and an. gambiae has ushered in a new era of hope that genomics research will result in the development of new and better tools for malaria control. the p. falciparum genome of 22.8 megabases (mbp) distributed among 14 chromosomes consists of 5,300 protein-coding genes (gardner et al. 2002) . p. falciparum possesses a relict plastid, the apicoplast, homologous to the chloroplasts of plants and algae. the apicoplast is essential for parasite survival and functions in the anabolic synthesis of fatty acids, isoprenoids, and heme (seeber 2003). these essential metabolic pathways are not present in humans and are therefore ideal targets for the development of safe antimalarial drugs. inhibitors of type ii fatty acid biosynthesis (triclosan and thiolactomycin) and mevalonateindependent isoprenoid biosynthesis (fosmidomycin and fr900098) with potent antimalarial activities have been identified by computational mining of the genome data. the fact that fosmidomycin has rapidly entered into clinical trials underscores the great utility of genomics research in the control of malaria (lell et al. 2003) . about 3,200 proteins (60%) in p. falciparum have no known functions (gardner et al. 2002) . the greatest challenge of malarial functional genomics (the elucidation of the functions of genes encoded by an organism's genome) is to assign functions to these proteins, thus comprehensively identifying the proteins that function at various lifecycle stages and that function together to carry out particular cellular processes, e.g., red blood cell invasion, signal transduction, growth, vesicular trafficking, etc. the application of functional genomics approaches allows the properties of many genes and proteins to be assessed in parallel on a large scale. these approaches are being used to address specific questions about the biology of p. falciparum. gene profiling (determining which genes are expressed) by microarray technology allows a rapid, parallel analysis of genome-wide changes in gene expression over a variety of experimental conditions (e.g., chloroquine versus saline control), tissues, and cell types; these genes can be clustered (ordered by expression pattern) to identify those that function in the same process. one of the most promising applications of microarrays is the study of differential gene expression during the complex p. falciparum lifecycle, specifically the formidable and challenging task of determining which subset of the 5,300 genes is represented in the transcriptome of each stage (bozdech et al. 2003; le roch et al. 2003) . these approaches are beginning to yield invaluable insights about new vaccine candidates, novel drug targets, and the molecular basis of drug resistance. proteomics is the study of all the proteins expressed in an organism. global protein analysis offers a unique means of determining not only protein expression, but also interacting partners, subcellular localizations, and post-translational modifications of proteins of whole proteomes. analyses of the proteomes of parasites that have been exposed to distinct environmental stimuli (e.g., chloroquine versus saline control) or that manifest distinct phenotypes (drug resistant versus drug sensitive) might also facilitate the identification of biochemical drug targets and of the specific proteins involved in drug resistance. comparative genomics (the comparison of genomes of related species), on the other hand, will yield invaluable insights about the biology of and the pathogenesis of disease associated with different parasites, i.e., p. falciparum doi: 10.1371/journal.pbio.0000039.g001 on the one hand and p. vivax on the other. the biology and pathology of the two parasites are quite distinct, e.g., the preference for reticulocytes (p. vivax) versus mature red blood cells (p. falciparum), the ability to cause severe (p. falciparum) versus mild (p. vivax) disease, and the implication of amino acid substitutions in pfcrt in cq resistance in one (p. falciparum) but not in the other (p. vivax). the 278 mbp sequence of the nuclear genome of the pest strain of an. gambiae s.s. has been published in draft form and is considerably larger than the 122 mbp assembled sequence of the fruitfly drosophila melanogaster (holt 2002) . the an. gambiae genome includes a treasure trove of 79 odorant receptor genes and about 200 genes that encode glutathione-s-tranferases, cytochrome p450s, and carboxylesterases. these and possibly other genes probably play a critical role in human host finding and detoxification of insecticides, respectively, and could be exploited, using gene profiling, proteomics, and comparative genomics, for the development of novel mosquito repellants or traps and insecticides. the ability to introduce foreign genes into anopheles vectors is an exciting advance that might facilitate the development of transgenic mosquitoes that do not transmit malaria parasites (moreira et al. 2002) . however, the future implementation of this control strategy, if current technical hurdles can be overcome, must take into consideration concerns about the environmental impact of releasing genetically altered mosquitoes. scientists in endemic countries must be active participants in malaria genomics research and not just conduits for field materials for northern partners. however, the reality is that there is an increasing technological gap between endemic-and developedcountry researchers in the field. this needs to be urgently addressed. the world health organization special programme for research and training in tropical diseases have initiated a series of training workshops in bioinformatics in endemic countries; the howard hughes medical institute has supported one such workshop. the training must extend to other aspects of genomics and include infrastructure development. there is considerable optimism that genomics research will result in new drugs, vaccines, diagnostics, and tools for malarial vector control. strong linkages between genomics research and national malarial control programs will facilitate the translation of research findings into intervention tools. as it is for all new technologies, it might also be important for the communities in endemic countries to have a greater awareness and understanding of genomics research. this will enhance acceptance of the products and improve informed consent. there is therefore a unique opportunity for collaborations between social-economic scientists and genomics researchers. the challenges of searching the scientific literature t he standard "front end" for biomedical literature search is medline and its entrez query system. huge, well-managed, and nearly exhaustive, medline and its 11 million references provide incredible ease and facility for anyone who can type a boolean query. though not quite a parallel for google-which runs a kind of popularity contest for web links in real time-the entrez search has opened up the literature to anyone with a web browser. to those who grew up chasing citations and papers through the aisles of a scientific library, entrez is a dream come true. and yet. suspend disbelief and imagine for a moment a kind of literature search dream-tool. "find me all references citing my gene of interest," you could ask. but why stop there? "find me all references citing some or all of my four genes of interest with expression or in vitro data." and then, "bring up the text of the paragraph in which these citations occurred so i can view them in context. and do it in real time." tools that can perform such searches would go beyond google because they avoid the repetitiveness involved in multiple searches. and they would go beyond entrez because they would search the entire medical literature in full-text format and not, as medline does, just the abstracts. furthermore, they would go beyond both types of searches in that they would be at least somewhat intelligent. such text-mining efforts are the next frontier for both academic and commercial groups that have sprung up from pasadena to boston to tel aviv. but how realistic is this venture? text-mining and its more universal relative "information retrieval" are still in their infancy. the first paper on text-mining for biology was published only in 1997. furthermore, because biological text-mining comes so close to the challenge of comprehending human language-arguably the most complex invention in the history of the planet-it is what computer scientists call a "hard problem." so even here, at the embryonic and fun stage in this technology's history, the outcome and especially the timing of improvement are impossible to predict. language-processing software tools have been successfully applied in text-mining of nonscientific sources, especially to newswire content. computer programs can already perform all three levels of text-mining ( figure 1 ) effectively: retrieving documents relevant to a given subject; extracting lists of entities or relationships among entities; and answering questions about the material, delivering specific facts in response to natural-language queries. information retrieval and extraction can be performed on news data at success rates of 90%-95%, says lynette hirschman, a structural linguist. question-answering has been reported in the literature at 85% accuracy, she notes, which is "amazingly good." the question is, how soon can these levels be achieved for biology? good thing for biologists that hirschman has turned her energies in their direction. hirschman works in massachusetts at mitre corporation, a government-funded institution that pursues projects in the national interest, be they in defense and intelligence or, as in the case of textmining, "anywhere we can move an entire field forward," says hirschman. the good news from news-mining is that improvement seems to arrive in direct proportion to the time and energy expended by the research community. similar improvement has occurred in speech recognition by computers, she adds ( figure 2 ). when people took successively harder problems and worked on them for four or five years, she explains, it caused error rates to drop, as a rule, by a factor of two every two years. one might think tackling the biomedical literature would be relatively easy, remarks hirschman: biology jargon has a lot of prefixes and suffixes, which can be parsed more easily than verbs and adverbs; it is highly regular, with greek-letter addons to gene or protein names signifying relatives or subtypes of the original proteins; and there are many resources available, such as databases and ontologies linking different biological terms. but whereas extraction of person and place names from news text routinely reaches 93%, results in biology remain mired in the 75%-80% range. "it's a little depressing," warns hirschman. "even something as simple as a slash may imply two different entities or a single compound." a chorus of assent greets her observation. programmers eager to codify the rules of biology have been stymied by what one bioinformaticist calls "a sea of exceptions." moreover, there is a chronic lack of data that have been "marked up" by software or humans to indicate the roles played by some of the key words. this marking-up process, however it is done, is crucial for machine-learning tasks. getting these data is both hard and expensive, says hirschman. to move biology text-mining forward, she believes, requires organizing different academic and commercial groups so that they are at least working on the same problem. only then can standards emerge that will allow progress in the field even to be measured. this type of shared problem-known as a "challenge evaluation"-has become something of a "religion" in the speech and language community since the 1980s, says hirschman. by putting out a set of data to train on and then issuing a "challenge" for each group to extract the same information or answer the same questions, "you compare apples to apples. in the process you build a research community." last year, hirschman and others ran the very first challenge evaluation in biology, the kdd cup (officially called the knowledge discovery and data-mining challenge cup). six weeks in advance, the organizers gave participants a training set of 862 journal articles already included in the model organism database flybase, along with associated lists of genes and gene products, as well as relevant data fields from flybase. after building their software tools, the entrants were then asked to take a test set of 213 articles and pretend they were curators: the tools were supposed to determine whether the articles were appropriate for curation, based on whether they contained experimental evidence for gene expression products, including both rna transcripts and proteins. eighteen participants took a shot at the kdd cup and their results speak of the infant state of the field. on average, they could assign only 58% of the papers correctly and could determine whether relevant gene products were present only 35% of the time. the winning entrant, a joint group from the israeli company clearforest ("see the forest and the trees") and marylandbased celera genomics, did better. doi: 10.1371/journal.pbio.0000048.g001 the four levels of information retrieval: google and medline both use keywords to direct a searcher to documents. but the next level has been tough to crack. improved software would allow biologists to jump from the web or medline to specifics with a single query. (adapted with permission from the mitre corporation.) information retrieval and extraction can be performed on news data at success rates of 90%-95%. the question is, how soon can these levels be achieved for biology? their entry made the right decision to curate 78% of the time and the right call on the presence of gene products 67% of the time. the winning group did so well by using a clever "trick," says hirschman admiringly. their program searched for figure captions and then applied multiple techniques to find those gene products they were looking for. the techniques applied by clearforest and others fall into two broad categories, statistical and heuristic. statistical techniques are the next step up from keyword searches. they count words such as genes or gene products appearing close to one another, but apply no linguistic insights, such as whether an adjective modifies a noun. by contrast, heuristic approaches use hand-crafted rules designed for specific datasets: e.g., january, february, march, etc., are months; the word following "mr." is a name; and so forth. this approach is labor-intensive but especially useful when there is only a limited amount of data-as is the case with single scientific papers or small groups of papers. some statistical approaches have been labeled with the nickname "bag of words" because they fail to account for grammatical relationships; e.g., "man bites dog" and "dog bites man" would drop the same three words in the bag. a key observation at the kdd cup was that the most basic statistical approach, which counts word occurrences at the document level, is not sufficient unless it takes into account at least some higher-level context, such as the part of the paper from which the search terms are extracted. furthermore, the more hand-crafted rules there were, the better. many of the top teams included biologists who applied their expertise to help create empirical rules that became part of the program instructions. this points to a general theme in machine learning: the greater the degree of human intervention, the better. the best programs are covered with fingerprints. although the march toward better text-mining systems is building momentum, there are two issues that could stop it in its tracks. the first is access. experts in text-searching uniformly cite access as a key obstacle for developing better search tools. "access is a bigger problem than algorithms" is how one machine-learning expert puts it, and a half-dozen others agreed. the present "balkanized" situation for text-processing is filled with "dead ends" and "short circuits" in information flow among biologists, says david lipman, head of the united states' national center for biotechnology information, which runs pubmed, the medline database, as well as the national library of medicine and other critical resources in biology and bioinformatics. it is as if readers are marine biologists on a coastline whose beaches are 98% private. at best, asking permission to view every article slows down the work. at worst, there are some important tools one can never build owing to the missing context. medline itself would be much more powerful if it were based on full text, experts say. owing to lack of access, says hirschman, "we miss a great deal by not having large corpora of full-text articles" included in the design of both the kdd cup and the next challenge evaluation, called biocreative, being held later this year. many of the relevant biological data are found outside abstracts, but getting access to full text is complicated at best. for manual searching, researchers traditionally fall back on portalhopping: jumping from one full-text subscription (to nature, science, or cell, for example) to another, or from one portal (highwire, web of science) to another. that way, many scientists routinely obtain access to as many as 80% of the journals they need. the rest they can usually request via interlibrary loan or order as photocopies online. however, this approach fails for most automated search programs. just sorting out the permissions and keeping up with changes in the portals dramatically increase the headaches for anyone trying to build a search tool. the second threat to text-searching programs ever becoming widely useful has more of the ring of linguistics jargon. the so-called " ontology problem" threatens successful searching based on the very specific nature of biological terminology. the issue here is not only that scientists are truly terrible about sticking to established terminologies. "scientists would rather share each other's underwear than use each other's nomenclature," as biochemist keith yamamoto is fond of saying. consequently, the scientific literature is a hodgepodge of identical or overlapping terms. a naã¯ve text-parsing program does not know whether "cat" refers to the catalase gene, the chloramphenicol transferase gene, or a household animal. the challenge is to build an ontology describing all the important relationships so your computer program can navigate among them without asking you what to do. consequently, an ontology would prescribe rules for understanding the interactions among genes based on the appearance of certain verbs ("inhibit," "express"), nouns ("agonist"), or phrases. although within each narrow scientific subdiscipline it may be possible to build exquisitely useful textmining tools, as soon as programmers broach the borders of the narrowest subfields, they will run into a kind of heisenberg uncertainty principle of linguistics and science. every toolmaker doi: 10.1371/journal.pbio.0000048.g002 is faced with the ontology problem in one respect or another, especially when the tool is meant to be a general one. david gilmour, chief executive officer of tacit inc., a knowledge management company in palo alto, california, is an industry veteran of exactly this war "and i have scars all over my body to prove it," he says. the issue in a nutshell, he explains, is that "ontologies scale poorly, and by the time they are useful," that is, large enough to capture most of the possible relationships among words, "they are unmaintainable." hirschman acknowledges that keeping up with the literature and new terminologies is challenging. adapting tools to new domains has traditionally been one of the "critical stumbling blocks" for text-processing technology, she says. the dynamic growth of biological terminology does not help. there are 50-100 alterations every week to the nomenclature section of mouse genome database web page. staying within one's narrow domain, then, could be a recipe for success, as long as the vocabulary and user questions remain tightly constrained, especially if there is a way to tiptoe around the access problem. that is apparently the case at wormbase, though the newly available tool there, called textpresso, is still being built. the motivation for textpresso was simple, says hans-michael mueller, a postdoctoral fellow in the lab of paul sternberg at caltech in pasadena, california, where wormbase-the genetic database for the nematode worm caenorhabditis elegans-is curated. "we want the user to be able to avoid going to the library to read all those papers [on genes and proteins] that your favorite gene interacts with. that is very tedious." the other goal is equally recognizable in the biology community: no mere mortal can hope to keep up with the burgeoning literature, even in the relatively narrow field of worm biology. mueller, a nuclear physicist by background, called textpresso "a search engine for full-text searches of abstracts and articles" that can help find answers to more challenging queries than simple keyword searches. mueller and his team use human "taggers" to mark up the corpus of text to indicate categories like "biological processes" ("late larval activation"), "genes" (let-7), and "molecular functions." then, like the clearforest-celera program, textpresso searches for combinations of categories in the same or neighboring sentences. the ontology relating the expressions and categories to one another is based both on scientific and common sense as well as linguistic components. in less than two years of work, mueller and his team have already marked up 3.9 million terms in 16,000 abstracts and 2,000 full-text papers. a typical search asks a question such as "what can be found out about the negative regulatory aspects of a genetic network in the pharynx?" answers emerge in the form of citations, abstracts, and, if available, a paragraph or so from the text of the relevant paper. textpresso went up-unpublicized-on the web in february this year and already receives a couple of hundred hits a day, a big number in a field of about 2,000 researchers. mueller estimates that textpresso is 95% accurate and that about 35% of the relevant papers have been included. textpresso needs full-text access to be as good as it is, says mueller. "we noticed" that drawing on full text "greatly increased the chances of a true hit," not a false positive. he managed to avoid the access issue by claiming a kind of "curator's privilege." only the curators see the full text. once the data are on the web, users can only get at most a paragraph, which falls within fair use, said mueller. if a user happens to subscribe to the journal in question, it is possible for him to click through, the publisher's portal and see the paper. whereas textpresso works exclusively on worm genetic data and commercial players like clearforest are just beginning to hunt for biological applications, a handful of companies have begun to market text-searching products to academic biomedical scientists. one such product is called quosa, for query, organize, share, and analyze. the software had its commercial launch in late 2002. put simply, the program-available on an institution-wide basis and already installed for hundreds of researchers at massachusetts general hospital and the dana-farber cancer institute in boston-allows a search across one's own documents. a front end for the literature that cooperates with medline, quosa pulls in and prioritizes full-text papers. the program first allows the user to search for the relevant files and download them in full-text format to the extent permitted by her library's subscription agreements and licenses. once it becomes second nature to users, they rave about it. like the best of the firstgeneration software, quosa allows users to make connections they would not have otherwise made. like so many other early software products, its longterm success will hinge on demand as well as improvements made in the upgrades. because of the ontology problem, improvements in searching in the next couple of years are likely to result from the application of ever-better techniques within existing domains. collaborations among wormbase, flybase, and other model-organism database groups will help improve all their search tools. medline itself may benefit from more advanced search techniques, though these will be restricted to abstract searches. the big unknown for predicting further development of text-search tools is the path publishers will take. if each publisher or portal such as reed-elsevier or highwire were to license or develop its own tool for searching its own content, the result might be better than the status quo, but would still be unsatisfying. running the same search three times on three different subsets of content might be better than running it 15 times-but wouldn't it be easier to run it just once? î�­ the transcriptome of the intraerythrocytic developmental cycle of plasmodium falciparum we are grateful to drs. lisa ranford-cartwright (university of glasgow, glasgow, united kingdom), ayoade oduola and yeya toure (world health organization, geneva, switzerland), and wilfred mbacham (university of yaounde, yaounde, cameroon) for critical comments on the manuscript. key: cord-009959-erh8ggh3 authors: bentley, william e.; wang, min‐ying; vakharia, vikram title: development of an efficient bioprocess for poultry vaccines using high‐density insect cell culture date: 2006-12-17 journal: ann n y acad sci doi: 10.1111/j.1749-6632.1994.tb44387.x sha: doc_id: 9959 cord_uid: erh8ggh3 nan infectious bursa1 disease virus (ibdv) is a pathogen of major economic importance to the world's poultry industries. it causes severe immunodeficiency in young chickens by destroying the precursors of antibody-producing b cells in the bursa of fabricius.' ibdv is a member of the birnaviridae family whose genome consists of two segments of double-stranded rna. presently, the principle method of controlling ibdv infection in young chickens is by vaccination with an avirulent strain of ibdv or by the transfer of high levels of maternal antibody to breeder hens.* recently, virulent strains that are antigentically different from previously established ibdvs have been isolated from vaccinated flocks on the delmarva peninsula. consequently, present vaccines afford only partial protection against infection. in figure 1, the large segment indicated is comprised of a precursor polyprotein that is processed into mature vp2, vp3, and vp4.3 vp2 and vp3 are the major structural proteins of the virion. vp2 is the major host-protective immunogen of ibdv and contains the antigenic regions responsible for the induction of neutralizing antibodies. for this reason, we have focused on vp2 as a potential vaccine. when expressed in baculovirus as one of a cassette (vp2, vp3, and vp4), the coat proteins self-assemble into empty virions, which subsequently afford protection in challenged chickens (fig. 2) : this article presents the current status of our efforts to develop a cost-efficient vaccine for ibdv using the recombinant baculovirus and insect cell culture process. although the baculovirus expression system has proven quite successful for use in laboratories, its use as an industrial expression system has yet to occur, although products for human use are in clinical trials. briefly, the system entails replacing the gene for the acnpv coat protein, polyhedrin, with the gene for the protein of interest. this coat protein is not required for the replication and maintenance of the virus in cell culture due to its biphasic life cycle. about three days after infection with a recombinant virus, the protein of interest is expressed instead of polyhedrin, which would normally constitute the majority of the total cellular protein. the system is attractive because it is nonpathogenic towards humans, and it involves the in virro propagation of cells for which technology has advanced due to previous mammalian and hybridoma culturing techniques. it provides for an abundant supply of the protein of interest in part due to the strong polyhedrin promoter and also because insect cell lines reported to date provide for glycosylation and other posttranslational processing. in addition, the construction of recombinant baculoviruses has become simplified due to commercial expression kits. several general articles, manuals, and review articles have been published that describe both the expression system'" and novel process engineering aspects.%" in this work, we subdivide the entire bioprocess into three key areas: (1) metabolism of infected cells and specific heterologous protein yield; (2) bioreactor configuration and high cell density continuous culture; and (3) integration of expression and product separation. in each of the results subsections, we briefly review the recent literature as well as present our efforts to understand and advance these areas. the stock virus solutions were developed as described previ~usly.~ virus titer was determined by the end-point dilution method. the infections were performed by adding different volumes of virus solution after viable cell count. thus, the time postinfection commences from the addition of the virus solution. the multiplicity of infection (moi) was evaluated separately in spinner flask culture. samples (2 ml) were taken from fermentors during the infection process, divided in two tubes, and centrifuged for 20 min at 12,000 rpm. the supernatants were separated from cell pellets and stored at -20°c until measurement of glucose and lactate. the cell pellet was resuspended in 250 ml 10 mm kh,po, (ph 7.4) buffer solution and stored at -20°c until assayed (page) for quantification. another two 10 ml samples were centrifuged for 20 min at 3500 x g. the cell pellets were stored at -20°c without resuspending in any buffer and used for the measurement of proteolytic activity and total protein. total cell counts were performed with a vwr-brand hemacytometer, and viability was determined by trypan blue dye exclusion using a 0.04% solution (sigma). cell pellets resuspended in 250 ml ehbs were sonicated on ice for 10 s with a microtip and a 30% pulsed duty cycle. glucose and lactate were measured using a ysi model 27 analyzer (yellow springs instruments), and ammonia was determined by an enzyme-based assay kit (sigma, no 170-uv). total protein was measured using a biorad protein assay kit i. alkaline protease activity was assayed using the synthetic substrate azocasein (sigma) at a concentration of 2% (w/v) in 0.1 m tris/hci buffer (ph 9.0). the cell pellets were resuspended using 250 ml of 0.1m tris/hci buffer (ph 9.0) and sonicated. one milliliter of azocasein solution was added to this 250 ml sample, and this mixture was incubated at 28°c for 1.5 hours. the reaction was stopped by the addition of 10% trichloroacetic acid (1.2 ml). after standing at 40°c for 30 min, the mixture was filtered, and 0.5 ml 0.5 m naoh was added to 0.5 ml of the filtrate. one unit of alkaline protease activity is defined as the amount of enzyme that gives an increase in am of 0.1 in 30 min at 28°c. cells were harvested by centrifuging 70 ml of culture and then resuspending them in 10 ml native binding buffer (20 mm sodium phosphate, 500 mm sodium chloride, ph 7.8). egg white lysozyme (100 pg/ml) was added to this mixture, which was incubated for 15 minutes on ice. the cells were sonicated on ice three times for 10 s with a 30% pulsed duty cycle (branson sonic power, co.) and then flash frozen in a -80°c freezer. after thawing the sonicated cells at 37"c, a final concentration of 5 pg/ml, rnase was added to the crude cell lysate and incubated on ice for 15 minutes. insoluble cell debris was removed by centrifugation at 3,000 g for 15 minutes. the column resin (invitrogen, ca) was washed with distilled water and then equilibrated with native binding buffer. cell lysate (7 ml) was then loaded onto the column and washed by native binding buffer until od,,, was less than 0.01. a step down to ph 6.3 was performed using native wash buffer (20 mm sodium phosphate, 500 mm sodium chloride, ph 6.3), and the od,,, of supernatant was monitored until its value was smaller than 0.01. protein was then eluted by loading native-ph elution buffer (20 mm sodium phosphate, 500 mm sodium chloride, ph 4.0) onto the column and collecting 1 ml fractions. hundreds of laboratories have expressed heterologous proteins using recombinant acnpv and sf-9 or bm-5 cell lines, yet only several laboratories have focused attention on maximizing yield by examining cellular functions during the growth and expression phases. factors such as moi, timing of infection, and cell density have been well documented. for example, neutra et al. l 2 examined the effects of reactor volume, infection timing, moi, and cell density on p-galactosidase production in shake flasks. king et al. 13 found that low moi was favorable for a temperaturesensitive baculovirus. conversely, for a more typical baculovirus with polyhedrin promoter control and an rcd4 product, lazarte et alt4 found high moi was most favorable. perhaps these systems differed by changes in cell metabolism that resulted from medium condition and infection temperature. in the lazarte et a1.i4 study, fresh medium added at infection tripled the yield. indeed, several groups have demonstrated that infected cells retain significant metabolic activity, and this is essential for producing the recombinant protein. caron et al. noted that postinfection cell physiology was critical and suggested that different growth and production medias might be required. zhang et a1. 16 noted that for bm-5 cells, serum concentrations for optimal growth were different than for viral production. additionally, kamen et al. "quantified differences in principal nutrients and waste products during the growth and production phases. reports demonstrating the effects of nutrient limitation and by-product inhibition on protein yield have been limited, however. wang et a1.i8 demonstrated that apparent glucose limitations could be offset by subsequent sucrose decomposition. lindsay and betenbaughi9 noted that the oxygen demand was higher for infected cells and adjusted aeration properties, which facilitated dramatic increases in yield. likewise, scott et a1.2" reported a significant increase in protein yield by maintaining dissolved oxygen tension at 50% of saturation. however, at high cell densities, maintaining oxygen was not sufficient for increasing yield. caron el al." restored recombinant protein production at higher cell density by renewing the medium at the time of infection. feeding the specific rate-limiting nutrients is less expensive, however, and aides in elucidating insect cell physiology under viral infection. two batch cultures were conducted to examine the effects of do during viral replication and protein production.*' the first batch culture was in a fermentor without do control (fig. 3) , while the other was conducted with do regulated near 35% (fig. 4) . virus infection was performed for the fermentor without do control by adding 15 ml virus solution (moi 5) at 91 hours, after the cells had attained a concentration of 6.15 x lo5 cells/ml. this moi and cell density were chosen so that other factors, such as nutrient depletion, were eliminated and differences due to oxygen level were discernible. shortly after infection, the do dropped to zero and remained zero for another 90 hours. the viable cell concentration remained near 6.55 x l@ cells/ml and then started to decrease simultaneously with an increase in do after 190 hours (fig. 3a) . the onset of cell lysis, however, is marked by both the increment in lactate dehydrogenase (ldh) activity and decrement in cell viability near 150 hours (fig. 3b) . note that after the do dropped to zero, the lactate increased to a maximum of 2.5 mm, reflecting the inadequacy of oxidative phosphorylation to supply the required atp. in these energy-limited cells, the final eh activity was 0.015 units per milliliter. for the batch culture with regulated do, the growth kinetics are shown in figure 4 . virus infection was also performed at 90 hours and 6.55 x 105 cells/ml. unlike the oxygen-uncontrolled culture, the viable cell concentration continued to increase to a maximum of 1.05 x lo6 cells/ml. the increased demand in oxygen immediately following infection was met in this experiment, and the do was maintained above 10% and averaged 35% for the remainder of the experiment. the continuous addition of oxygen likely provided the requirement for continued cell growth. note that the lactate concentration remained below 1 mm for the entire experiment, whereas the glucose concentration dropped almost 45%, from 14 mm to 8 mm after infection. the epoxide hydrolase activity reached 0.044 u/ml, which was 200% higher than in the oxygen-uncontrolled experiment. the specific productivity increased by 100% as well. this increase in protein synthesis is accomplished only by continued glucose uptake after infection. in the uncontrolled experiment, infected cells consumed little glucose (-1 mm) after the do dropped to zero. on the other hand, both the uninfected and infected cells in the do-controlled experiment continued to uptake glucose and consumed almost 1 g/l after infection for cell growth and viral replication. note, therefore, that the effects of oxygen, like moi and cell density, are delineated and realized when sufficient nutrients are available in the extracellular environment. three experiments were performed to evaluate glucose and glutamine feeding in spinner flasks at high cell density (fig. 5) . the use of 50 ml spinner flasks with 30 ml working volume prevents oxygen limitation. the cells were grown in a 250 ml spinner flask with 80 ml working volume until a density of 3.85 x lo6 cells/ml was attained; they were then divided and infected in three smaller spinner flasks at an moi of 10 and a cell density of 3.3 x lo6 cells/ml. one of the spinner flasks was diluted 3:l with fresh medium as a control (total working volume, 30 ml). previously, this method was reported as resulting in the highest specific yield attainable in spinner flasks.l5.iy this spinner flask is denoted an oxygen and substrate-sufficient control. one spinner flask was then fed 0.5 ml of 200 mm glutamine and 80 pl of 2 m glucose. subsequent daily glucose feeding maintained glucose supply (fig. 5a) . the last spinner flask was a control high cell density culture, without glucose/glutamine feeding. the epoxide hydrolase activity (fig. 5b) continued to increase for the culture with fresh medium (oxygenhubstrate control) until 118 hours postinfection (hpi). by contrast, in the high cell density cultures, epoxide hydrolase activity increased more rapidly initially and then decreased at 88 hpi for the glucose/glutamine fed culture, and at 48 hpi for the unfed culture. a significant improvement in protein production from glucose and glutamine feeding was seen as early as 12 hpi. coincidentally, the glucose concentration in the unfed culture had dropped dramatically by this time (fig. 5a) . the increase in activity continued well into the production phase and because the viable cell concentration in the fed culture was significantly lower after 48 hpi, the increase in activity was due primarily to an increase in specific productivity. when comparing the overall performance of the effects of oxygen, glucose, and glutamine feeding, the total protein activity of the do-controlled culture was three times that of the uncontrolled culture. further, the glucose/glutamine fed culture was twofold higher in yield than the do-controlled culture. ultimately, the epoxide hydrolase yield was 40-60 milligrams per liter.22 as seen above, cell metabolism may play a very important role in recombinant protein production when cells are infected at high cell density. the metabolic stress responses of bacterial and mammalian cells to oxidation, heat shock, and amino acid starvation have been well documented, and many stress proteins have been ider~tified.~"?~ insect stress, however, has been quantified by previous researchers only by measuring the bulk proteolytic activity of whole insect homogenates.25,26 in those reports, increased proteolytic activity corresponded to increased cellular stress. in many recent reports, degradation of the recombinant product has been s i g n i f i~a n t .~~.~~.~~ thus, another important aspect of cell metabolism concerns the elicitation of protease activity. we have used azocasein to investigate the appearance of alkaline proteases that may breakdown the product, particularly if allowed to follow in the separation and purification processes. 29 in another fermentor experiment, we controlled the dissolved oxygen to 50% and infected cells (moi 5) by the addition of virus stock solution when the cell density was about 1 . 2~1 0~ cells/ml. this lower cell density was chosen in order to avoid other cellular stress effects (for example, nutrient starvation). the cells (fig. 6) kept growing after viral infection due to a low mo121-30 for a period of 40 hours and then the viable cell density decreased later due to a secondary infection. the total cell density decreased due to cell lysis, and the viability was 20% at cell harvest. two recombinant protein activities and the total specific protease activity are also shown. in this system, p-galactosidase expression is under promoter p10 control; epoxide hydrolase (eh) is our desired recombinant product and is under the polyhedrin promoter control. 0-galactosidase is a cytoplasmic protein and will be liberated to the medium during expression and lysis. however, eh is membrane bound and not released to the medium. expression of both proteins (fig. 6) started simultaneously. the p-galactosidase activity increased until 79 hpi, was constant for a short time, and then decreased by 12% until harvest. on the other hand, eh increased more slowly initially, remained constant for 20 hours and increased slowly by 20% until harvest. the specific protease activity was shown to increase dramatically starting at about 70 hpi. this time just corresponds to the reduction of p-galactosidase activity and the slowdown in eh production. we should note that the viability was near 70% at this moment. inasmuch as the viability was high and.there was no nutrient limitation (not shown), there was no apparent reason for cells to stop producing recombinant protein. the protease (measured as specific activity in the cell pellet) increased at least 15-fold even when considering partial loss from cell lysis. we also measured proteolytic activity in supernatant, and no increment was found, which suggests that the proteolytic activity was either associated with the cell membrane or was too dilute to detect in the supernatant. although there is no specific connection between the protease activity and the shift in protein activities (p-gal, eh), the coincident timing warrants further consideration. because it is unlikely that protease inhibitors will be used in a commercial process, the specific nature and timing of this proteolytic activity is significant. hence, characterization of the identity and consequences of this and other proteases is under investigation. in addition to the many productivity advantages that continuous suspension culture has over stationary (t-flask) or batch-suspension culture, continuous culture provides homogeneous and constant environmental conditions that facilitate the modeling of cell growth and metabolism. research with hybridoma cells has demonstrated this p~i n t .~' .~~ due to infected cell lysis, most continuous insect cell systems have employed two reactor stages: one for cell growth and the other for virus propagation and recombinant protein expre~sion."*~*~~ however, it has been re ported that there is eventually an almost complete loss of productivity from continuous insect cell culture with a continuously passaged v i r~s .~~.~~.~~ limited stability of the virus upon serial passage may limit the application of two-stage continuous reaction systems. this may be improved, however, by a three-stage continuous bioreactor system proposed by taticek and shuler,3x or by a cycling-batch reactor scheme." at present, batch and perfusion systems have achieved significantly higher viable cell density than continuous cultures. further, there is some question as to the long-term productivity of the cell lines in continuous cultures, independent of the virus.ls consequently, we have investigated methods for maximizing cell number in extended continuous cultures.3y dilution rates were examined from 0.014 to 0.0403 hr-l, which is close to the maximum specific growth rate obtained in batch cultures.'' in figure 7, our small-volume glass-jacketed spinner flask reactorjy is depicted. cultures were stirred at 130 rpm and contained 0.1% pluronic f-68.40 the airsparging system was cyclic*with 60 min on and 30 min off. two volummetric air flow rates were examined: 2.056 ml/min (0.0093 vvm) and 2.76 ml/min (0.0125 vvm) for low-and high-sparging rates, respectively. the viable and total cell densities for both sparging rates are plotted as a function of dilution rate in figure 8. at the low sparging rate, a monotonic decrease in viable cell density with increasing dilution rate was observed. this is consistent with the semicontinuous culture of mouse ls cells (without ph and do control) by s i n~l a i r .~~ the high sparging rate exhibited a maximum in total and viable cell densities at a dilution rate of 0.0287 hr-i. the decrease in viable cell density at lower dilution rate (0.0251 hr-i) may have been due to low nutrient and/or high toxin concentrations."note that at 0.0332 hr-l, the cell density for the high sparging rate (0.0125 vvm) was five times higher ( 3 . 6 4~1 0~ vs. 7x105 cells/ml) than the low sparging rate. thus, this sparging rate provided much better oxygenation for cell growth with little contribution to cell death. the trends shown in figure^ are also similar to a continuous sp2/0-derived mouse hybridoma cell culture with ph and do control31 as well as an oxygen-limited continuous culture (ph control only) of mouse nbi h y b r i d o m a~.~~ the data depicted was collected in one continuous culture of over 3100 hours in duration. at several times, the dilution rate was reset to a previous setting, and the cell density returned to the previous steady state value.3y thus, the spinner flask reactor performed well, was reproducible, and will be very useful for further development of the additional protein expression stages. in figure 8, we demonstrated that a cell density of 4.2 x loh cells/ml with 94% viability could be achieved at a dilution rate of 0.0287 hr-i. in order to check the protein expression level of infected cells grown continuously for an extended period, cells at dilution rate of 0.0251 hr-l with low (0.0093 vvm, 800 h) and high sparging rate (0.0125 vvm, 2600 hr) were taken from the bioreactor and directly infected with an moi of 5 in two 50 ml spinner flasks (30 ml working volume). the maximum recombinant protein yields were 0.15 u/ml at 71 hpi and 0.05 u/ml at 69 hpi for cells from the 2600 hr and 800 hr samples, respectively. the maximum cell densities reached were 3.7 x lo6 and 1.6 x loh cells/ml for the 2600 hr and 800 hr samples. flow diagram and setup of continuous bioreactor system. the total volume is 250 ml, with working volume up to 230 ml. air is introduced by way of peristaltic pump and hplc solvent porous sparger. air is introduced in on/off mode with a home timer. temperature is maintained by water bath and glass jacket. agitation is provided by magnetic stir plate (bellco).3" the average specific eh activity, calculated as the maximum enzyme activity divided by the maximum cell density, was slightly lower for cells from the lower sparging rate (3.125 vs. 4.05 x lo--' u/1q5 cells). perhaps this was due to a lower growth rate (0.022 hr-i vs. 0.025 hr-i) or lower oxygen supply during the continuous culture. however, the specific eh activity from these long-term cultured cells is comparable to that from freshly prepared cells (3.34-5 x lo-' u/1@ cells"). this demonstrates that high cell density (more than 3 x loh cells/ml) can be achieved in continuous culture, even at a dilution rate of 0.0403 hr-i. in addition, the specific protein yield from long-term cultured cells (over three months) is not compromised. the results of this work can be combined with the previous discussion on maximizing specific productivity in designing the best reactor strategy. this may involve incorporating feeding policies in the second or third stage of a multistage continuous system, or the inclusion of several alternating batch reactors that are filled by one continuous or semicontinuous reactor. an attractive feature of the baculovirus system has been its potential for disease diagnostics and vaccine development. again, this is due to the high expression level, posttranslational processing, and nonpathogenicity. in addition, recent reports have shown that expression of virus coat proteins often results in self-assembled virus-like particles (vlp) that are essentially empty whole virion^.^,^"' of these vlp-producing systems, vaccines have been proposed for p~liovirus,"~ parvovirus,5" ibdv,4 flock house virus,s' and bluetongue virus. 44 in addition, vaccines have been proposed or tested from non-vlp proteins, including hiv,s2 yellow fever," feline herpes trypanosoma cru~i,'~ mokola and rabies viruses:h newcastle disease vi-us,^^ japanese encephalitis:s hepatitis c,59 and bovine coronavirus.60 of these, only two research groups have considered the advantages of integrating initial separation with product expression by employing imac.59*h' we have developed the bioprocess so that imac can be employed as a potentially single-step separation procedure. imac was introduced for the selective adsorption of protein by porath et ~1 . ~~ who used divalent cations, zn2+ and cu2+, chelated to a chromatographic matrix to fractionate serum proteins. metal ion ligands most often used in imac are first-row transition metals (zn, ni, cu, and fe) chelated by iminodiacetate (ida). in resin, they distinguish affinities exhibited by functional groups on the surface of proteins. these interactions between particular surface amino acids and immobilized metal ions provide the basis for metal-affinity protein to a first approximation, proteins are retained on metal-affinity columns according to the number of accessible his ti dine^.^*^' histidine is not a common residue in protein sequences and it accounts for only 2.1% of amino acids in globular proteins.hr thus, the chance that a desired protein, engineered to contain additional histidines at its c-or n-terminus, will be preferentially separated from other proteins is quite good. a description of the construction of the vp2 baculovirus that expresses his,-vp2 (denoted vp2h) is given elsewhere.6' monoclonal antibodies (mabs) recognizing ibdv were produced and characterized using protocols previously d e s~r i b e d .~~,~~' identification of ibdv antigens by modified antigen-capture elisa (ac-elisa) was also carried out as described by snyder et al." recombinant vp2 proteins from insect cells infected with vedlh-22 (for vp2h) and vedl-8 (for vp2) were identical using the array of mabs and ac-elisa. however, these two proteins (vp2, vp2h) were distinguished and resolved on a 12.5% sds-polyacrylamide gel, and detected immunologically following western blotting with polyvalent chicken anti-ibdv ~e r u m .~~,~* this indicated that vp2 protein was fused with histidine residues. the vp2h was purified by affinity chromatography using a niz+ immobilized resin column provided in a protein purification kit as described in the methods section (invitrogen, ca). the final elution was monitored by odzs0 readings of the fractions, using the native-ph elution buffer as a blank. in f l g u r e 9 , the optical density (odzso) of the column eluate is shown for the wash and vp2 eluate steps. the eluted pool (fractions 18-26) from the ph 4 buffer was collected, concentrated, and desalted by a 10 kda membrane (amicon) and checked by western blotting!' in figure 10a, the purified vp2h protein (molecular weight near 43 kda, lane 2) comigrated with two vp2 proteins: one derived from gls ibdv (lane 1) and the other from sf-9 cells infected by ibdv-7 recombinant baculovirus4 encoding entire vp2, vp3, vp4, vp2x proteins of ibdv (lane 5). this demonstrates that the vp2h protein can bind the ni2+ ion on the resin and was effectively eluted by the ph 4 elution buffer. the purity of purified vp2h protein was also checked by sds-page (fig. lob) . the intense band with a molecular weight of about 43 kda showed that this vp2h protein was dominant in the mixture (roughly 40%). some small proteins or peptides were still in this purified mixture which may have been due to partial degradation of vp2h (data not shown) or additional elution by this strong low-ph buffer. in this work, a step change from ph 6.3 to ph 4, which is much lower than the pka value commonly observed for surface histidines (-6.0):3 may have coeluted some peptides with a stronger binding ~apacity.'~ because we demonstrated that the recombinant viruses express immunoreactive vp2 proteins, the next step was bench-scale production of vp2h protein. i proteins were about 120 and 70 mg/l, respectively. although we have not used imac for a large volume purification, we have obtained sufficient data to estimate protein production costs using the bioprocess presented. advantages for the recombinant poultry vaccine, as compared to the injection of an avirulent strain or the transfer of maternal antibody to breeder hens, include potential for complete and passive protection, higher reactivity per milligram of vaccine protein, smaller dose quantities, and more convenient processing. at this point, results on the optimum dose composition and quantity are incomplete, so a direct economic comparison between the processes is unavailable. however, given both the attractiveness of the baculovirus insect cell expression system and the optimization research activity, one might anticipate commercialization of ibdv vaccines and other baculovirus-based products in the near future. the work reviewed and presented in this paper highlights specific areas where further advancements will lead to increased productivity and therefore lower processing cost, namely maintaining the maximum cell number and specific protein productivity, developing convenient and stable bioreactor operating strategies, and integrating unique lowcost separations steps. structural and growth characteristics of infectious bursal disease virus maternally derived antibody-effect on susceptibility of chicks to infectious bursal disease genomic structure of the large rna segment of infectious bursal disease virus infectious bursal disease virus structural proteins expressed in a baculovirus recombinant confer protection in chickens insect cell culture technology in baculovirus expression systems expression of foreign genes in insects using baculovirus vectors baculovirus as vectors for foreign gene expression in insect cells baculovirus expression vectors: a laboratory manual large-scale insect cell culture for recombinant protein production production of (recombinant) baculoviruses in insect-cell bioreactors optimization of protein-production by the baculorivus expression vector system in shake flasks assessment of virus production and chloramphenicol-acety i-transferase expression by insect cells in serum-free and serum-supplemented media using a temperature-sensitive baculovirus optimization of the production of full-length rcd4 in baculovirus-infected sf9 cells high-level recombinant protein production in bioreactors using the baculovirus insect cell expression system a two-stage bioreactor system for the production of recombinant proteins using a genetically engineered baculovirus/insect cell system culture of insect cells in a helical ribbon impeller bioreactor expression of epoxide hydrolase in insect cells: a focus on the infected cell quantification of cell culture factors affecting recombinant protein yields in baculovirus-infected insect cells effects of oxygen on recombinant protein production by suspension cultures of spodoptera fiugiperda (sf-9) insect cells effects of oxygen/glucose/glutamine feeding on insect cell baculovirus protein expression: a study on epoxide hydroxylase production interaction of hepatic microsomal epoxide hydrolase derived from a recombinant baculovirus expression system with an azarene oxide and an aziridine substrate analogue escherichia coli and sulrnonella typhirnuriurn cellular and molecular biology coordinated regulation of a set of genes by glucose and calcium ionophores in mammalian cells alterations in proteases, protease inhibitors and ecdysone levels: a profile of stress in insects effect of developmental derangements on the proteolytic and protease-inhibitory activities in galleria rnellonella (insecta) ecdysteroids increase the yield of recombinant protein produced in baculovirus insect cell expression system production of recombinant sarcotoxin ia in bonbyx rnori cells kinetic analysis of protease activity, recombinant protein production, and metabolites of infected sf-9 cells under different do levels factors influencing recombinant protein yields in an insect cell-baculovirus expression system: multiplicity of infection and intracellular protein degradation a kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: effect of nutrient concentration, dilution rate, and ph a structured kinetic modeling framework for the dynamic hybridoma growth and monoclonial antibody production in continuous suspension culture a model for baculovirus production with continuous insect cell cultures continuoue production of baculovirus in a cascade of insect-cell reactors continuous p-galactosidase production with a recombinant baculovirus insect-cell system in bioreactors a structured dynamic model for the baculovirus infection process in insect-cell reactor configurations a continuous process for the production of baculovirus using insect-cell cultures a continuous flow bioreactor system for the production of recombinant proteins using the insect cell-baculovirus expression system continuous insect cell (sf-9) culture with aeration through sparging in a novel low-volume bioreactor scale-up of insect cell cultures: protective effects of pluronic f-68 response of mammalian cells to controlled growth rates in steady-state continuous culture the baculovirus-integrated retrotransposon ted encodes gag and pol proteins that assemble into virus-like particles with reverse transcriptase development of baculovirus triple and quadruple expression vectors: co-expression of three or four bluetongue virus proteins and the synthesis of bluetongue virus-like particles in insect cells three-dimensional reconstruction of baculovirus expressed bluetongue virus core-like particles by cryo-electron microscopy synthesis of bluetongue virus (btv) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of btv analyses of the requirements for the synthesis of virus-like particles by feline immunodeficiency virus gag using baculovirus vectors assembly and release of hiv-1 precursor pr55eg virus-like particles from recombinant baculovirus-infected insect cells synthesis of immunogenic, but non-infectious, poliovirus particles in insect cells by a baculovirus expression vector canine parvovirus empty capsids produced by expression in a baculovirus vector: use in analysis of viral properties and immunization crystallization of virus-like particles assembled from flock house virus coat protein expressed in a baculovirus system cell-surface expression and purification of human cd4 produced in baculovirus-infected insect cells 17d yellow fever vaccine virus envelope protein expressed by recombinant baculovirus is antigenically indistinguishable from authentic viral protein the use of feline herpesvirus and baculovirus as vaccine vectors for the gag and env genes of feline leukemia virus trypanosoma cruzi flagellar repetitive antigen expression by recombinant baculovirus: towards an improved diagnostic reagent for chagas' disease structure and expression in baculovirus of the mokola virus glycoprotein: an efficient recombinant vaccine vaccination against newcastle disease with a recombinant baculovirus hemagglutinin-neuraminidase subunit vaccine protection of mice against lethal japanese encephalitis with a recombinant baculovirus vaccine secretion and purification of hepatitic c virus nsl glycoprotein produced by recombinant baculovirus-infected insect cells primary structure of the s peplomer gene of bovine coronavirus and surface expression in -insect cells purification of a recombinant protein produced in a baculovirus expression system by immobilized metal affinity chromatography metal chelate affinity chromatography, a new approach to protein fractionation imac-immobilized metal ion affinity based chromatography purification of proteins by imac the sage of imac and mit cu(i1)-binding properties of a cytochrome c with a synthetic metal-binding site: his-x3-his in an a-helix evaluation of the interaction of peptides with cu(ii), ni(ii), and zn(i1) by high-performance immobilized metal ion affinity chromatography the independent distribution of amino acid near neighbor pairs into polypeptides group and strainspecific neutralization sites of ibdv defined with monoclonal antibodies differentiation of infectious bursal disease viruses directly from infected tissues with neutralizing monoclonal antibodies: evidence of a major antigenic shift in recent field isolates naturally occurring-neutralizing monoclonal antibody escape variants define the epidemiology of infectious bursal disease viruses in the united states comparative studies on structural and antigenic properties of two serotypes of infectious bursal disease virus a mathematical model for metal affinity protein partitioning metal affinity precipitation of protein carrying genetically attached polyhistidine affinity tails we thank gerard h. edwards and sarah milczanowski for technical assistance. key: cord-008556-oetrdm8g authors: kozak, marilyn title: regulation of protein synthesis in virus-infected animal cells date: 2008-03-01 journal: adv virus res doi: 10.1016/s0065-3527(08)60265-1 sha: doc_id: 8556 cord_uid: oetrdm8g this chapter summarizes the structural features that govern the translation of viral mrnas: where the synthesis of a protein starts and ends, how many proteins can be produced from one mrna, and how efficiently. it focuses on the interplay between viral and cellular mrnas and the translational machinery. that interplay, together with the intrinsic structure of viral mrnas, determines the patterns of translation in infected cells. it also points out some possibilities for translational regulation that can only be glimpsed at present, but are likely to come into focus in the future. the mechanism of selecting the initiation site for protein synthesis appears to follow a single formula. the translational machinery displays a certain flexibility that is exploited more frequently by viral than by cellular mrnas. although some of the parameters that determine efficiency have been identified, how efficiently a given mrna will be translated cannot be predicted by summing the known parameters. the translation of viral mrnas: where the synthesis of a protein starts and ends, how many proteins can be produced from one mrna, and how efficiently. the next section focuses on the interplay between viral and cellular mrnas and the translational machinery. that interplay, together with the intrinsic structure of viral mrnas, determines the patterns of translation in infected cells. the final section points out some possibilities for translational regulation that can only be glimpsed at present, but are likely to come into focus in the future. to keep the project manageable, i have concentrated on animal viruses. plant viruses are mentioned, however, when they provide the best (or sometimes the unique) example of a given mechanism. the structural requirements for mrna function have been determined by inspection'of natural eukaryotic mrnas, followed by manipulation of features that looked suspicious. the general structural characteristics of eukaryotic mrnas have been reviewed previously (kozak, 1983a) and will not be elaborated here. the discovery of the m7g cap on a wide variety of viral and cellular mrnas (shatkin, 1976 ) was a provocative clue that the mechanism of initiation in eukaryotes differs from prokaryotes. although the list of plant virus mrnas that are translated without a cap has grown in recent years, picornaviruses and caliciviruses are still the only animal viruses known to be translated without a cap (nomoto et al., 1976; ehresmann and schaffer, 1979) . indeed, the near-indispensibility of the m7g cap may be inferred from the fact that animal viruses that replicate in the cytoplasm routinely encode their own capping and methylating enzymes. this is true not only for poxviruses (moss et d., 1976) , where the vast coding capacity of the genome allows room for frills, but also for reovirus (furuichi et al., 19761, vesicular stomatitis virus (vsv) (abraham et al., 19751, and alphaviruses (cross, 1983) ) in which the small size of the genome limits the encoded proteins to the barest essentials. the m7g cap enhances both the stability and translatability of mrnas. transcripts that are capped but not methylated are stable, but nonetheless untranslatable (furuichi et al., 1977; horikami et al., 1984) . much of the discussion that follows assumes that a scanning mechanism underlies the initiation process. the scanning model postulates that a 40 s ribosomal subunit binds initially at the 5' end of the mrna viral translation 23 1 and migrates until it reaches the first aug triplet. if the first aug codon occurs in the optimal context (accaugg-see kozak, 1981a kozak, , 1984a kozak, , 1986a all 40 s subunits stop there, and that aug serves as the unique site of initiation. if the first aug triplet occurs in a suboptimal context, only some 40 s subunits will initiate there; some will migrate beyond that site and initiate at an aug codon farther downstream. the scanning hypothesis is not universally accepted, but it is supported by extensive evidence from many laboratories (reviewed by kozak, 1980 kozak, , 1981b kozak, , 1986a . two alternative models have been suggested from time to time. one is that ribosomes bind directly to the sequence around the aug codon, but experiments designed to distinguish between scanning and direct binding do not support the latter (kozak, 1979a (kozak, , 1983b . a hybrid mechanism in which 98% of the ribosomes scan from the 5' end, while 2% of the binding occurs directly at the aug start site, is difficult to rule out, however. another suggestion is that secondary structure might guide the choice of aug codons (this idea is evaluated a few paragraphs hence). one consequence of the scanning mechanism is that deleting the "ribosome binding site" (i.e., the normal initiator codon and flanking sequences) will not abolish translation; ribosomes will simply use the next aug codon downstream, which, in some cases, has been shown to direct the synthesis of a biologically active, truncated protein (downey et al., 1984; halpern and smiley, 1984; katinka and yaniv, 1982) . conversely, introducing spurious upstream aug codons will reduce initiation from the authentic start site-a prediction that has been verified many times with laboratory constructs (bandyopadhyay and temin, 1984; lomedico and mcandrew, 1982; smith et al., 1983; zitomer et al., 1984) as well as with naturally occurring variant forms of mrna from the early1 and late regions of simian virus 40 (sv40) (barkan and mertz, 1984) . when the context around an upstream aug codon conforms closely to the accaugg consensus sequence, initiation from the downstream site is suppressed almost completely (kozak, 198313, 1984b; liu et al., 1984; m. scott and h. varmus, personal communication) . when the context around the upstream aug codon is less ideal, initiation from the downstream site is reduced but not abolished (kozak, 1986a) . stated in a more positive way, when the 5'-proximal aug codon occurs in a suboptimal context, ribosomes are able to initiate at the first and the second aug codons. this "leaky" scanning process is further explained and documented in section i1,c. the scanning mechanism predicts that translation should be downregulated by any ploy that interferes with the linear movement of 40 s ribosomal subunits from the cap to the aug codon: binding of a protein to the 5'-noncoding sequence; introducing spurious out-of-frame aug codons, as mentioned above; annealing cdna fragments that are complementary to the 5'-untranslated sequence (haarr et al., 1985; perdue et al., 1982; privalsky and bishop, 1982; willis et al., 1984) ; or creating a stable hairpin anywhere upstream from the aug codon, as described in the next section. on the other hand, the simplicity of the scanning mechanism suggests few possibilities for enhancing translation. although we know what features should be absent from the leader for a message to be efficient, the only features known to contribute in a positive way are the m7g cap and the sequence directly flanking the initiator codon. a promising place to look for other positive effectors is the tripartite leader on late adenovirus mrnas. transposition of the 200-nucleotide tripartite leader sequence to heterologous mrnas stimulates their translation 20-fold (berkner and sharp, 1985; logan and shenk, 1984) , but the feature responsible for the stimulation has not been pinpointed, and could turn out disappointingly to be a long sequence that simply lacks all of the negative effectors cited above. the impression that the leader sequences on most viral mrnas do not contain unidentified translational "enhancers" is reinforced by the ease with which 5'-noncoding sequences can be deleted without deleterious effects (bendig et al., 1980; spindler and berk, 1984a; villarreal et al., 1979) .2 if our intuition is correct that "extra" 5'aoncoding sequences are more likely to inhibit than to help, the trend toward short 5'-noncoding sequences on many viral mrnas becomes significant (reviewed by kozak, 1981b ; see also rose1 and moss, 1985) . indeed, the 24-nucleotide leader sequence on the mrna that encodes adenovirus polypeptide ix seems to mediate translation more efficiently than the long tripartite leader that has received so much attention (lawrence and jackson, 1982) . the synthesis of polyoma virus t antigen was significantly reduced in only one of the mutants studied by bendig et al. (1980) -a mutant in which the deletion extended to within two nucleotides of the aug codon. this fits with evidence from other sources that (only) the nucleotides immediately preceding the aug codon are part of the ribosome recognition sequence. secondary structure in viral mrnas might have various effects on translation. 1. one might expect secondary structure to inhibit more when it occurs near the cap, which is the presumptive entry site for ribosomes, than when a hairpin occurs farther downstream, because 40 s ribosomal subunits once bound must be able to melt secondary structure to some extent. (one knows for sure that 80 s ribosomes melt secondary structure during the elongation phase of protein synthesis; the triplet code could not be read linearly otherwise.) the prediction that 40 s ribosomal subunits can melt their way through secondary structure within the interior of the leader sequence has been verified: introducing a 13-base-pair hairpin (ag -30 kcal/mol) 60 nucleotides downstream from the cap did not impair the translation of preproinsulin mrna in uzuo (kozak, 1986b) . the effects of secondary structure close to the cap have not yet been tested systematically, but it has been noted that the 5' end of alfalfa mosaic virus rna-4 is unfolded (gehrke et al., 1983) and rna-4 is a notoriously efficient message. godefroy-colburn et al., (1985b) claim more generally that the degree of cap accessibility of the four alfalfa mosaic virus mrnas correlates with their translational efficiency, but the correlation appears weak. the cap was indeed least accessible on rna-3, which ranks lowest in translational efficiency, but the cap was equally accessible on rnas 1,2, and 4, which differ 15fold in competitive efficiency (godefroy-colburn, 1985a) . 2. although we expect ribosomes to melt secondary structure to some extent, there must be a limit to that ability. whereas a hairpin of -30 kcalimol at the midpoint of the leader sequence (involving neither the cap nor the aug codon) did not reduce the synthesis of preproinsulin under normal culture conditions, a hairpin of -50 kcal/mol nearly abolished translation (kozak, 198613) . because the hairpin did not encroach on the aug codon, the observed inhibition seems incompatible with the direct-binding hypothesis, but is consistent with the scanning hypothesis. pelletier and sonenberg (1985) have also shown that translational efficiency decreases as secondary structure in the 5'noncoding region increases. 3. there is no experimental support for the idea that secondary structure orients the cap and the aug codon, thus determining which aug will initiate translation. were that true, denaturation should impair translation; in fact, denaturation often enhances (payvar and schimke, 1979) . nor is there support for the idea that downstream cistrons are silent due to conformational constraints: attempts to activate internal initiation sites by denaturing viral mrnas invariably fail (collins et al., 1982; monckton and westaway, 1982) . a popular idea is that when secondary structure sequesters the 5'-proximal aug triplet, it might be skipped by ribosomes in favor of the next exposed aug codon (darlix et al., 1982; ghosh et al., 1978; hay and aloni, 1985; nomoto et al., 1982) . the results of a direct test contradict that notion, however, when the primary sequence around the 5'-proximal aug codon in a chimeric preproinsulin mrna was favorable for initiation, no translation from a downstream site could be detected irrespective of whether the first aug codon was single stranded or base paired (kozak, 1986b) . thus, 40 s ribosomal subunits appear to scan linearly, melting the secondary structure (ag 5 -30 kcal/mol) t o reach each aug codon in turn. if a hairpin is too stable to be melted (ag 2 -50 kcal/mol), the 40 s subunit apparently stalls, but it does not "jump over" the barrier. 4. in some viral mrnas, sequences at the 3' end are complementary, to a limited extent, to those at the 5' end (antczak et al., 1982; dasgupta et az., 1980) . that arrangement might be expected to inhibit translation-an expectation that has been confirmed recently using mrnas with artificially constructed terminal complementary sequences (spena et al., 1985) . some viruses seem to take measures to preclude such inhibition. whereas the genomic rnas of influenza (robertson, 1979) and bunyaviruses (eshita and bishop, 1984) have complementary 5'and 3'4erminal sequences, that potentially deleterious structure is not copied into mrna, inasmuch as the 3' terminus of each mrna stops short of the 5' end of the template strand (bouloy et al., 1984; eshita et al., 1985; hay et al., 1977) . arenaviruses also produce mrnas that lack the complementary sequences present at the termini of genomic rna (auperin et al., 1984) . 5 . incubation in hypertonic culture medium has been used often to study protein synthesis in virus-infected cells (see yates and nuss, 1982, and references therein) . hypertonic shock results in the rapid and reversible inhibition of protein synthesis at the level of initiation (saborio et al., 1974) . an intermediate concentration of salt or sucrose permits a residual low level of translation, under which circumstance viral protein synthesis nearly always predominates over cellular protein synthesis (cherney and wilhelm, 1979; nuss et al., 1975; oppermann and koch, 1976) . it is difficult to deduce the mechanism of this differential response from inspection of natural forms of viral and cellular mrnas. however, a cloned preproinsulin gene has been experimentally converted from hypertonic resistant to hypertonic sensitive by inserting into the 5'-noncoding sequence the oligonucleotide agcttgggccgtggtgg, thereby creating a 13base-pair hairpin around the aug initiator codon (mutant b13hp in kozak, 1986b) . a reasonable interpretation is that the hairpin structure (ag -30 kcal/mol), which does not inhibit translation under nor-ma1 culture conditions, is stabilized under hypertonic conditions to the point where it becomes inhibitory. an alternative explanation, currently under investigation, is that the primary sequence of the oligonucleotide insert underlies the enhanced sensitivity of mutant b13hp to hypertonic stress. if the first explanation turns out to be correct, one might suggest by extrapolation that most viral mrnas are less structured near the 5' end than are most cellular mrnas, and for that reason viral mrnas are more resistant to hypertonic stress. herpes simplex virus mrnas are a notable exception: they are unusually sensitive to hypertonic inhibition (stevely and mcgrath, 19781 , perhaps because their high g + c content generates extensive secondary structure. 6. the mechanism of action of interferon is too complex to discuss here, except to mention that double-stranded regions of rna, either free or incorporated into the mrna structure (debenedetti and baglioni, 1984; knight et al., 19851 , are critical in activating and targeting the interferon-induced enzymes. the deleterious effects of interferon on the stability and translation of viral mrnas have been reviewed by lengyel (1982) . the monocistronic rule means more than simply producing one protein from one mrna. a number of viral mrnas encode two or more proteins in nonoverlapping reading frames; with few exceptions, however, (see section ii,c), it is exclusively the 5'-proximal cistron that gets translated (shih and kaesberg, 1973; reviewed by kozak, 1978; . to cope with the usual inability of eukaryotic ribosomes to initiate at internal sites in mrna, the genomes of animal viruses are punctuated at one of four levels, as described below. the structures of plant virus rna genomes and their patterns of expression have been reviewed by davies and hull (19821 , and they are not exceptional. the mode of expression of cauliflower mosaic virus, which has a circular dna genome, is exceptional indeed, and is discussed in section i1,c. the following descriptions are generalized; additional details and references have been published elsewhere (kozak, 1981b) . each virus is classified according to its major mode of punctuation, which is often not the exclusive mode. 1. the genome itself is segmented. each segment typically consists of one gene, which is transcribed end to end, or nearly so. there is usually a simple correspondence between the size of the mrna and the size of the mature protein derived therefrom. reoviruses, influenza viruses, and bunyaviruses fit this description. arenaviruses and nodaviruses (e.g., black beetle virus) have segmented rna genomes but rely also on other mechanisms. 2. the viral genes are linked, but internal start and stop sites for transcription generate a separate mrna for each protein. punctuation is accomplished for the most part at the level of transcription rather than by posttranscriptional processing. again, the size of the mrna usually corresponds to the size of the mature p r~t e i n .~ this group includes poxviruses, herpesviruses, rhabdoviruses (vsv), and paramyxoviruses. here the genome lacks internal transcriptional and translational stoplstart sites. the genome-sized mrna is translated end to end to produce a "polyprotein," more than 2000 amino acids in length, which is cleaved to generate the mature viral proteins. the extreme situation in which all viral proteins are derived from a single precursor is characteristic of picornaviruses and flaviviruses (castle et al., 1986 ; c . m. . [rice et al. (1986) present a lucid explanation of some older data that had suggested a different translational strategy for flaviviruses.] posttranslational cleavage supplements other modes of punctuation in many animal virus systems, and is especially important in the maturation of retrovirus and alphavirus proteins. 4. the fourth, rather heterogeneous group of viruses characteristically produce big transcripts that cannot be translated completely: ribosomes bind at the 5' end and translate only up to the first stop codon, and the downstream cistrons in these polycistronic mrnas are usually silent. the downstream cistrons become translatable when they are moved closer to the 5' end, which is accomplished by producing truncated or subgenomic mrnas. various mechanisms generate these shortened transcripts. conventional splicing of nuclear transcripts is used by retroviruses, papovaviruses, and parvoviruses. adenoviruses also use splicing, on a rather grand scale (nevins, 1982; ziff, 1985) . coronaviruses use a novel cytoplasmic fusion mechanism to transfer a common leader sequence to each of six, progressively shorter, subgenomic mrnas (budzilowicz et al., 1985; lai et al., 1984; spaan et al., 1983) . in the case of alphaviruses and parvoviruses, initiation at a n 3 whereas the molecular weight correlation between mrnas and proteins holds for most early vaccinia virus genes (cooper and moss, 1979; hruby and ball, 1982) , late vaccinia mrnas are notoriously heterogeneous in size, apparently because transcription does not terminate discretely (mahr and roberts, 1984; rose1 and moss, 1985) . the 3'-proximal portions of such transcripts are assumed to be translationally silent. in the case of herpes simplex virus, the size of many mrnas corresponds simply to the size of the encoded protein, but more complex mrnas also exist (wagner, 1985) ; the functional significance of the latter is not yet clear. internal transcriptional promoter produces the subgenomic mrnas that encode the major capsid proteins (brzeski and kennedy, 1978; janik et al., 1984) . hepadnaviruses (hepatitis b and others) cannot yet be classified, since mrnas have been identified for some but not all of the viral proteins (tiollais et al., 1985) . the major subgenomic mrna is initiated at an internal promoter, and there is no evidence for splicing. the heterogeneous initiation sites for transcription in hepatitis viruses might be a means to regulate translation, as suggested by laub et al. (1983) and enders et al. (1985) . arenaviruses are a special case. the genomic s-rna segment codes for two structural proteins, n and gpc, but only gpc can be translated conceptually directly from the 5' half of virion rna; the 3' half of the sequence is an antisense version of the n gene (auperin et al., 1984) . thus, a subgenomic complementary mrna is produced to translate the n protein. although gpc could in theory be translated from the full-length viral s-rna, a subgenomic rna corresponding to the 5' portion of s-rna is also present in infected cells. this might be necessary to avoid "hybrid arrest" which could occur if translation were attempted with full-length viral and antiviral transcripts. whereas most eukaryotic mrnas are functionally monocistronic, certain viral mrnas have been shown to synthesize two separately initiated polypeptides. with few exceptions4 we can rationalize the 4 the mechanisms outlined herein cannot explain the (inefficient) internal initiation that occurs in a mutant form of rous sarcoma virus src mrna (mardon and varmus, 1983) . poliovirus mrna also initiates translation at more than one site, at least in uitro (celma and ehrenfeld, 1975) , but one cannot attempt an explanation until the sites have been identified. [dorner et al. (1984) claim to have localized an internal initiation site, but they did not prove that the template rna was intact. the fact that they could demonstrate "internal initiation" in extracts from reticulocytes but not from poliovirusinfected cells hints of an artifact.] because the poliovirus 5'-noncoding sequence has eight aug triplets upstream from the major translational start site (kitamura et al., 1981; racaniello and baltimore, 1981) , spurious initiation events are expected in that region. on the other hand, the upstream aug triplets would not preclude initiation of the polyprotein from the ninth aug codon, because seven of the upstream aug triplets lie in a weak context; the only one that lies in a favorable context is followed by an inframe terminator codon, which would allow reinitiation. the same explanations are compatible with the genomic sequences of many other picornaviruses callahan et al., 1985; forss et al., 1984; linemeyer et al., 1985) . the two structural peculiarities of picornavirus mrnas-presence of upstream aug codons and absence of production of two proteins from a single mrna by invoking one of the following mechanisms, each of which is experimentally supported. these mechanisms (with the exception of reinitiation) might be considered errors, i.e., the results of imprecise execution of some step in translation. a system that functions with less-than-perfect fidelity apparently gains the advantage of versatility. the scanning model postulates that, when the 5'-proximal aug codon occurs in a suboptimal context, ribosomes will initiate at that site as well as at another aug codon farther downstream. several nucleotides near the aug codon are known to affect the efficiency of initiation, but the most important determinants are a purine (preferably a) in position -3, and g in position +4; we can predict the occurrence of leaky scanning by focusing on those two positions. in each of the bifunctional viral mrnas listed in fig. 1 , the more 5'-proximal initiation site lies in a suboptimal context, thus rationalizing the ability of some ribosomes to reach the start of the second cistron. (in sv40 16 s mrna, influenza b, and adenovirus-12, which are bracketed in the center of the figure, the sequence flanking the first aug codon is not really weak, but it is not perfect; thus, some 10-20% of the 40 s subunits are expected to bypass the first aug codon and reach the second. that may be adequate to produce the second protein in the case of adenovirus and influenza virus, but it does not seem adequate to explain the synthesis of sv40 vp1, which is an abundant protein. in sv40 16 s mrna, however, ribosomes can reinitiate at the vp1 start site, as explained below.) the scanning model does not necessitate that the second aug codon lie in a stronger context than the first, although that usually is the case; it is necessary only that the first aug codon lie in a context that is less than optimal. each mrna listed in the upper part of fig. 1 produces two unrelated proteins, translated from two different reading frames. the mrnas in the lower part of the figure initiate at two aug codons in the same reading frame, thereby producing long and short versions of the same protein. whereas the relaxed scanning mechanism accounts qualitatively for the dual function of the mrnas listed in fig. 1 , the model is not very a cap-might be related it is possible that, when cap binding proteink) are not part of the 40 s initiation complex, aug codons in suboptimal contexts are recognized even less efficiently than usual, and the barrier effect of the upstream aug codons in poliovirus mrna would thus be minimized. perhaps p220 is cleaved (see section iii,d) to directly facilitate viral translation, rather than to inhibit host translation. good a t predicting the frequency with which ribosomes initiate a t each site. one problem is that the ratio of initiation a t sites 1 and 2 in vivo is often different from that i n vitro (bos et al., 1981; clarke et al., 1985; dethlefsen and kolakofsky, 1983; , and the ratio changes when salt or other reaction conditions are varied. that is hardly surprising because the fidelity of initiation in vitro is sensitive to reaction conditions (jense et al., 1978; kozak, 1979b; petersen and hackett, 1985) . on the other hand, the in vivo ratio might be skewed if one protein is less stable or less efficiently extracted than the other. in addition to the obvious economy of using one mrna to make two proteins, in a fixed ratio, their simultaneous production might allow the polypeptides to interact as the nascent chains grow. it would be amusing to determine whether complementation is less efficient when two proteins that are normally translated from one mrna are instead synthesized from separate templates. the hundreds of eukaryotic cellular genes that have been sequenced to date invariably initiate translation a t aug. when alternate initiator codons were tested experimentally, however, they were not inert. eukaryotic ribosomes can initiate a t gug (kozak, unpublished data) and uug (zitomer et al., 1984) , but the efficiency is a t least 30-fold lower than at an aug codon in the same context; initiation at gug, uug, or other nonstandard codons is (barely) detectable only when the codon is preceded by the optimal a in position -3 (m. k., unpublished data). there is credible, albeit not definitive, evidence that alternate initiator codons are used in two virus systems to produce minor virion components. one is adeno-associated virus capsid protein b, which probably initiates a t an acg codon that lies upstream from the major aug start site (becerra et al., 1985) . [an acg codon in coliphage t7 mrna is also recognized as an initiator codon by wheat germ ribosomes i n vitro (anderson and buzash-pollert, 1985) . although the template is unnatural in that case, the evidence for initiation a t acg is irrefutable.] the second natural example is gpr80gag, a nonessential but nonetheless conserved form of gag produced by moloney murine leukemia virus (edwards and fan, 1980; the nucleotide sequence of the region is given by shinnick et al., 1981) . gpr80gag is analogous to the elongated form of gag produced by feline leukemia virus, except that the latter is presumably initiated at an upstream aug codon in a weak context (fig. l ) , whereas in murine leukemia virus the most likely initiation site(s) are upstream gug and/or cug codons that lie in a favorable context. charles van beveren has shown that gpr80gag is produced not only by the left-most column shows that in most cases the sequence around the first functional initiator codon is suboptimal with respect to the nucleotides in positions -3 and +4, thus explaining how some 40 s ribosomal subunits can reach the second initiation site. in the case of the coronaviruses and black beetle virus, the indicated proteins are predicted but have not yet been demonstrated. although the 6.7-kda protein predicted from adenovirus region e3 has not been seen, its ribosome binding site has been proven functional by demonstrating the synthesis of a fusion protein from an appropriately engineered mutant virus (wold et al., 1986) . all of the other proteins listed here have been detected in infected cells, and most have also been synthesized in cell-free translation systems. notes: %since the 5' ends of hepatitis virus and some bunyavirus mrnas are heterogeneous (laub et al., 1983; patterson et al., 19831 , the second protein could be translated, without invoking leaky scanning, from the portion of the mrna population that lacks upstream aug codons. binfluenza virus rna-6 is unusual in that the first and second aug codons are separated by only four nucleotides (shaw et al., 1982) , but that probably does not explain the ability of ribosomes to initiate at both sites. in a version of preproinsulin mrna in which the first and second aug codons (both in the perfect context for initiation) were moloney virus, but also by two other murine leukemia viruses that have no aug codons upstream from the major (pr65gag) start site (personal communication). thus, there is no alternative to believing that nonstandard codon(s) are used to initiate the elongated form ofgag. experiments to pinpoint the start sites are in progress in van beveren's laboratory. although reinitiation was documented years ago in prokaryotes, there was no reason to suspect a similar phenomenon in eukaryotes until laboratory manipulations with cloned genes yielded some results separated by five nucleotides, ribosomes were unable to initiate a t the second member of the pair (kozak, 1984b) . 'because the first reading frame terminates upstream from the second in sv40 mrnas, ribosomes could reach the start site for vp1 by a combination of leaky scanning and reinitiation. dthe arrangement of aug codons near the 5' end of sv40 late 19 s mrna is gccaugg (out-of-frame at position 253-255) . . . uccaugg (start of vp2) . . . ccuaugc (out-of-frame a t position 679-681) . . . ggaaugg (start of vp3) . we postulate that leaky scanning allows some 40 s ribosomal subunits to bypass the first aug triplet (position 253-255) in order to initiate vp2. that does not contradict the fact that, in 16 s mrna, the aug codon in position 253-255 initiates the agnogene product. by extrapolating from the systematic measurements carried out in another system (kozak, 1986a) , we would expect 80-9070 of the ribosomes to initiate a t the aug codon in position 253-255, while 10-209 should reach the next aug; that seems sufficient to produce vp2, which is a minor component of the virion. synthesis of vp3 might depend on leaky scanning (bypassing the first three aug codons) as well as reinitiation, inasmuch as ribosomes that initiate a t the first aug codon would terminate before reaching the vp3 start site. ethe nucleotide in position -3 varies among strains of foot-and-mouth disease virus, and the relative yields of p20a and p16 vary accordingly (clarke et al., 1985) . fit is likely that two annaug sequences farther upstream are also used to produce longer forms of surface antigen (heermann et al., 1984) . most transcripts lack the extreme upstream aug codons, however. nthe two proteins postulated for feline leukemia virus are indeed seen in infected cells, but the mechanism of synthesis postulated here has not been proven. infrequent initiation at weak, upstream aug codons is also suspected with mrnas from some other retroviruses (gruss et al., 1981; willumsen et al., 1984) . references: sendai virus: giorgi et al., 1983 . measles virus: bellini et al., 1985 . reovirus: cashdollar et al., 1985 ernst and shatkin, 1985; kozak, 1982; sarkar et al., 1985. bunyaviruses: eshita and fuller et al., 1983 pasek et al., 1979; persing et al., 1985 . feline leukemia virus: laprevotte et al., 1984 . herpes simplex: haarr et al., 1985 wagner et al., 1981. that are difficult to explain ~t h e r w i s e .~ the principal observation is that eukaryotic ribosomes can initiate at an internal aug codon, when another aug codon occurs upstream and in a highly favorable context (thus ruling out leaky scanning), provided that a terminator codon occurs in-frame with the first aug codon and upstream from the second (kozak, 1984b; liu et al., 1984; m. scott and h. varmus, personal communication) . we envision that when a complete "minicistron," i.e., an aug triplet followed by a terminator codon, occurs upstream, it is translated; but the 80 s ribosome does not detach at the terminator codon. rather, the 60 s subunit dissociates while the 40 s subunit remains bound to the message and resumes scanning. when the 40 s subunit reaches the next aug codon, it reinitiates translation. reinitiation is more eficient when the terminator codon precedes, rather than when it overlaps, the aug codon (m. kozak, unpublished) . with respect t o natural mrnas rather than laboratory constructs, elegant genetic manipulations implicate reinitiation in the translation of rous sarcoma virus src mrna (hughes et al., 1984) and cauliflower mosaic virus mrna , dixon et al., 1986 . the latter is the most striking example to date of a functionally polycistronic mrna in eukaryotes. the overlapping arrangement of cistrons rules out the possibility of reinitiation with many other viral mrnas (contreras et al., 1977; meshi et al., 1983; schwartz et al., 1983; . however, in some instances in which adjacent cistrons do not overlap, and reinitiation is therefore expected, it has not been observed (barker et al., 1983; goelet et al., 1982; knowland, 1974; ou et al., 1982) . reinitiation, together with leaky scanning, could theoretically account for translation of the sv40 agnogene pro-5 the alternative to reinitiation is to postulate that eukaryotic ribosomes can initiate directly at an internal aug codon, and that they usually fail to do so only because the downstream site is occluded by the stream of 80 s ribosomes advancing from upstream. occlusion indeed occurs during the translation of polycistronic prokaryotic transcripts, but the inhibitory effect of an overlapping upstream cistron is sometimes only twoor threefold (das and yanofsky, 1984; hoess et al., 1980) . berkhaut et al. (1985) claimed to see complete inhibition of translation of the ms2 lysis protein when the coat protein cistron overlapped, but the unknown sensitivity of their biological assay complicates the interpretation. moreover, their claim that a strong upstream initiation site (for coat protein) suppresses initiation from the much weaker site for lysis protein hardly compares with the situation in eukaryotes, where an upstream aug codon can completely suppress initiation from an equally favorable downstream site (kozak, 1983b, 198413) . the essential difference between the occlusion and reinitiation mechanisms is that the former postulates direct binding of ribosomes to internal aug codons, while the latter prohibits such binding. there is experimental evidence against direct binding (kozak, 1979a (kozak, , 1983b and against occlusion (kozak, 198413) . tein and vp1 from the same mrna, although neither mechanism has been experimentally demonstrated with sv40. (the simultaneous occurrence of two phenomena complicates the task of demonstrating either one.) reinitiation is expected within the leader region of rous sarcoma virus genomic rna, where three small open reading frames (orfs), one of them headed by an aug codon in a highly favorable context, precede the gag coding sequence (schwartz et al., 1983) . it has been difficult to demonstrate synthesis of the predicted leader peptides, perhaps because their small size makes them unstable. with admirable persistence, however, hackett et al. (1986) have devised a sensitive assay with which they have detected small amounts of the peptide encoded in the first minicistron of rous sarcoma virus. parenthetically, when one is designing experiments to probe the function of a particular viral or cellular product, one must remember that introducing a nonsense codon near the beginning of a gene might not abolish its function. if an in-frame aug codon occurs downstream from the nonsense codon, ribosomes will probably reinitiate and the truncated polypeptide might be functional. the mechanism by which reverse transcriptase is synthesized has long puzzled retrovirologists. the pol coding sequence is not preceded by an initiator codon; rather, reverse transcriptase is derived by cleavage from a joint gag-pol precursor (murphy et al., 1978; oppermann et al., 1977) . the problem is that the genomic arrangement of gag and pol sequences would seem to preclude their joint translation. in avian retroviruses, gag and pol are in different, partially overlapping, reading frames (schwartz et al., 1983) ; in murine retroviruses, gag and pol are in the same frame but are separated by a terminator codon (shinnick et al., 1981) . in both cases, the solution involves a translational "error." with avian retroviruses, about 5% of the ribosomes shift reading frames somewhere near the end of the gag sequence, thereby producing from one message both gag and a small amount of the gag-pol fusion protein. jacks and varmus (1985) have shown beyond reasonable doubt that frameshifting occurs near the gag-pol junction in a cell-free translation system from reticulocytes. by using mrna that was transcribed in uitro from cloned rous sarcoma virus dna, they excluded the possibility that a low-abundance, spliced transcript served as the template for the fusion protein. inspection of the gag-pol junction sequences in several other retroviruses leads one to expect that frameshifting is not limited to the avian system. neither is it limited to eukaryotes, of course. frameshifting occurs under intrigu-ing circumstances in a few bacterial and phage genes (craigen et al., 1985; dunn and studier, 1983; kastelein et al., 1982) . the excitement that accompanied the old discovery of a "readthrough" version of coliphage qp coat protein (weiner and weber, 1973) has been rekindled recently by finding a similar phenomenon in eukaryotic systems. in murine retroviruses, for example, the gag and pol sequences are separated by a single uag terminator codon, the occasional suppression of which generates a gag-pol fusion protein. the first hint of this came from supplementing a cell-free translation system with yeast suppressor trna, which indeed enhanced the synthesis of the gag-pol precursor (philipson et al., 1978) . the notion was confirmed for both murine and feline leukemia viruses when yoshinaka et al. (1985a,b) directly determined the amino acid sequence of the protease that constitutes the nh,-terminal portion of the pol gene product. suppression of a terminator codon is not peculiar to retroviruses, for it occurs also with alphaviruses (lopez et al., 1985; strauss et al., 1983) , tobacco mosaic virus (pelham, 1978) , and probably carnation mottle virus (guilley et al., 1985) . suppression of the uag codon in tobacco mosaic virus rna has been traced to the major tyrosine-specific trnas which, in tobacco cells, have the anticodon sequence gjia (beier et al., 1984a,b) . the most abundant trnaer from wheat germ has the highly modified queuine base (q) in place of g in the wobble position of the anticodon, and it is not able to suppress. thus, minor differences in trna structure can be an important determinant of host range for some viruses. in one sense, suppression solves the problem of how to produce a full-length protein from an interrupted coding sequence. but that probably misplaces the emphasis. the real problem might be how to produce only a small amount of an essential protein that might be toxic if overproduced. an inefficient mechanism, such as suppression or frameshifting, is an ideal solution. whereas the features described in the preceding section are intrinsic to viral mrnas, and can be demonstrated readily in a "universal" reticulocyte lysate, the translation of viral mrnas in uiuo is influenced by specific conditions that prevail in the cytoplasm of infected cells. the way in which the translational machinery is partitioned viral translation 245 between viral and host mrnas is one important consideration. because the literature concerning inhibition of host protein synthesis by animal viruses has already been reviewed at length (fraenkel-conrat and wagner, 1984; kaariainen and ranki, 1984; shatkin, 1983) , i shall be selective in my coverage. an overview of the phenomenology is presented in table i . the general mechanisms of host shutoff defined by these phenomena are described briefly in sections b and c, which are followed by a detailed discussion of two viruses-poliovirus and adenovirus-that seem to merit more attention. the phenomenon of host shutoff is not as widespread as might appear from table i . retroviruses, paramyxoviruses, parvoviruses, and flaviviruses do not suppress host translation, and papovaviruses actually stimulate host protein synthesis. because host shutoff is interesting, and because it is easier to detect viral protein synthesis against a clean background, virologists understandably have focused on systems that demonstrate the phenomenon. the inhibition of host protein synthesis may be of more interest to virologists than to viruses, however. in many cases, the yield of infectious progeny from a virus that fails to shut off host protein synthesis is the same as from another virus strain (or the same virus in a different cell line) in which host protein synthesis is obliterated (detjen et al., 1982; gillies and stollar, 1982; jen and thach, 1982; lodish and porter, 1981; minor et at., 1979; munemitsu and samuel, 1984; read and frenkel, 1983; sharpe and fields, 1982) . a virus strain that suppresses host macromolecular synthesis sometimes replicates faster in culture than one that does not, however. whether the inhibition of host protein synthesis is beneficial or harmful or irrelevant to the virus during the course of natural infections is not known. in short, with a few viruses inhibition of host protein synthesis might be a strategic move, necessary for efficient expression of viral genes, but no unequivocal example can be cited. in most instances, host shutoff is likely to be an unintentional side effect of viral gene expression-an effect of no real value, and possibly even harmful, to the virus. it is interesting that poliovirus replicates better during coinfection with cytomegalovirus than during single infection; cytomegalovirus stimulates the cell functions that are turned off by poliovirus (furukawa et al., 1978) ! there are examples of nonpermissive virus-cell systems in which macromolecular synthesis is inhibited so effectively that neither host nor viral proteins can be made (brown and moyer, 1983; drillien et al., 1978; jones et al., 1982) . in such cases the wild-type virus must have a way to throttle the shutoff mechanism. that notion will be pursued in the section on adenoviruses. throughout this section i have tried to point out wrinkles in the data, uncertainties in some popular interpretations, and alternative mechanisms. this critical slant is intended not to minimize the value of the work that has been done, but to stimulate reconsideration of some paradigms that may have been accepted or rejected too quickly. experiments probing the mechanism of host shutoff are difficult. some of the pitfalls and caveats might be stated at the outset. certain techniques that are used to block virus infection at a particular step, in order to define the extent of viral expression that is needed to effect host shutoff, might inadvertently create a new inhibitory mechanism. in the resulting confusion one learns little about the physiological mechanism of inhibition. for example, treatment of poliovirus-infected cells with guanidine not only blocks the synthesis of progeny rna (which is the intended purpose), but also causes double-stranded rna to accumulate to higher-than-normal levels (baltimore, 1969); and double-stranded rna is a potent inhibitor of translation. experiments showing that a temperature-sensitive mutant virus which makes no progeny rna nevertheless shuts off host protein synthesis as effectively as wild-type poliovirus suffer the same defect. the mutant-infected cells accumulate massive amounts of partially double-stranded "replicative intermediates" which are likely to inhibit translation, irrespective of the normal shutoff mechanism (hewlett et al., 1982) . in short, the problem with many experiments is that translation can be inhibited in a variety of ways, and in the process of blocking one pathway, another can be activated. for the same reason, the assumption that the mechanism of host shutoff is the same at high multiplicities of infection as at low multiplicities is untenable. in the case of encephalomyocarditis (emu virus, the effect on host protein synthesis has been shown to differ qualitatively as a function of multiplicity (alonso and carrasco, 1981) . with poliovirus, the familiar statement that guanidine does not prevent host shutoff is true only when the cells are infected at a high multiplicity (helentjaris and ehrenfeld, 1977) . at a normal multiplicity of infection, guanidine does block host shutoff, and therefore it is not clear that viral rna synthesis (which is the guanidine-sensitive step) is uninvolved in the normal mechanism of host shutoff by poliovirus. the specific deficiency or alteration in the translational machinery can sometimes be pinpointed by studying protein synthesis in extracts prepared from virus-infected cells, provided that one appreciates the limitations of that approach. the notion that one can study the mechanism of host shutoff by one virus by using a second virus as a stand-in for host mrna is questionable, because proteins encoded by two differhelentjaris and ehrenfeld (1978) ; nuss et al. (1975) . (2) bienz et al. (1978) . (3) bossart and bienz (1981) ; femandez-munoz and darnell (1976) . (4) celma and ehrenfeld (1974) . (5) h e a l and . (6) etchison etal. (1982) ; a. dasgupta, personal communication. (7) helentjaris and ehrenfeld (1977) . emc uirus in hela cells: (1) jen e t d . (1980) . (2) carrasco and lacal (1983). (3) alonso and carrasco (1981) . (4) jen et al. (1980) . (5) alonso and carrasco f1982b); lacal and c a r r a m (1982). (6) mosenkis et al. (1985) ; a. p. rice etal. (1985) . sindbis and sfv: (1) lachmi and kaiiriiiinen (1977) ; wengler and wengler (1976) . (2) and (3) simizu (1984) . (4) van steeg et al. (1981) ; wengler and wengler (1976) . (5) carrasco and lacal(1983) ; gamy et d. (1979) . (6) van steeg et al. (1981) . (7) simizu (1984) . vsv: (1) lodish and porter (1981) ; mcallister and wagner (1976) . (2) grinnell and wagner (1985) . (3) jaye et al. (1982) ; lodish and porter (1980) ; nisbioka and silverstein (1978a) . (4) lodish and porter (1980) ; otto and lucas-lenard (1980). (5) francoeur and stanners (1978) ; . (6) centrella and lucas-lenard (1982) ; dratewka-kos etal. (1984) . reouirus in l cells: (1) zweerink and joklik (1970) . (2) sharpe and fields (1982) . (3) (1984) . skup and millward (1980) . (7) sharpe and fields (1982) . influenza virus: (1-3) inglis (1982) ; . (4) lazarowitz etal. (1971) . (5) carrasco and lacal (1983) . (6) katze et al. ( , 1986 . adenouirus: (1) castiglia and flint (1983) . (2) babich et al. (1983) ; beltz and flint (1979) . (3) babich et al. (1983) . (4) castiglia and flint (1983) . (6) see text. (7) babiss and ginsberg (1984) . vaccinia uirus: (1) hruby and ball (1981) ; oppermann and koch (1976) . (2) salzman etal. (1964) . (3) cooper and moss (1979) ; rice and roberts (1983) . (4) oppermann and koch (1976) ; rice and roberts (1983) . (5) norrie etal. (1982) . (7) bablanian etal. (1981) . herpes simpler: (1) pereira et al. (1977) . (2) fenwick and walker (1978) ; stenberg and pizer (1982) . (3) stage 1-see text; stage 2inglis (1982) ; nishioka and silverstein (1978b) . (4) silverstein and engelhardt (1979) . (5) fenwick and walker (1978) ; hackstadt and mallavia (1982) . (7) fenwick and walker (1978) ; nishioka and silverstein (1978b) ; read and frenkel (1983) . frog virus 3: (6) cited in mosenkis et al. (1985) . all other entries are from willis et al. (1985) . bthe timing of host shutoff relative to the onset of viral translation is indicated. a capitalized entry in this or any other column identifies the probable major mechanism of host shutoff. "coincident" in capitals means that competition probably underlies host shutoff. cfunctional stability is usually evaluated by the ability of host mrnas, extracted from infected cells, to be translated in a cell-free reticulocyte lysate. dthis column indicates the presence or absence of a temporal correlation between the inhibition of host protein synthesis and the influx of sodium ions that often accompanies virus infection (carrasco and lacal, 1983) . ea change in cap binding protein was postulated because extracts from sfv-infected cells were unable to translate most capped mrnas, with the exception of emc and sfv late 26s mrnas (van steeg et al., 1981 1. although it is true that efficient mrnas like emc and sfv 26 s can be translated without benefit of the m7c cap, it does not follow that cap binding protein(s1 are deficient in every instance where translation of those mrnas persists in the face of an overall decline. efficient mrnas will be selectively translated when any component of the translational machinery is made limiting. the best evidence for this is the ability of both emc and sfv 26 s mrna to be translated in emc virus-infected cells, in which host translation is drastically inhibited by a mechanism that has not been difined, but that clearly does not involve cap binding protein (mosenkis et al., 1985) . wan steeg et al. (1984) have postulated that capsid protein is responsible for host shutoff by sfv, but the evidence is not compelling: the binding of host mrna to ribosomes was only slightly inhibited in fig. 4 of their paper, and the inhibition was a t the level of 80 s rather than 40 s ribosomes. the fact that translation of late viral 26 s mrna was unaffected is not adequate evidence of specificity, since 26 s mrna-by virtue of its high efficiency-would be relatively resistant to any inhibitor, physiological or otherwise. gwith type-2 reovirus in l cells, infection at a multiplicity of infection (moi) of 10 caused no significant decrease in translation; a t mol of 20, translation gradually declined by -40% (sharpe and fields, 1982) . with type-3 reovirus (moi of zo), overall protein synthesis was initially stimulated in both hela and l cells; translation declined later only in l cells (munoz et al., 1985a) . hrecent data do not corroborate an earlier hypothesis concerning inzctivation of a cap-specific translation factor (skup and millward, 1980) . although extracts from reovirus-infected cells translate capped reovirus mrnas poorly, other cap-dependent mrnas, such as globin and tobacco mosaic virus, are translated efficiently in such extracts (lemieux et al., 1984) ; and capped sv40 mrnas are translated in cells coinfected with reovirus (daher and samuel, 1982) . perhaps translation of capped reovirus mrnas is inhibited (artificially) in extracts from infected cells because viral structural proteins, which must be abundant in those extracts, adsorb to the homologous mrnas and sequester them from ribosomes. 'in contrast with most other host mrnas, the synthesis of histone mrnas is inhibited in adenovirus-infected cells (flint et al., 1984) . ]the 55-kda e1b protein probably functions only indirectly to shut off host translation. the protein is required for efficient cytoplasmic accumulation of late viral -as, which might in turn shut off host protein synthesis by competition (see text). proteins from regions e1b and e4 may function as a complex. khost transcripts were stable by hybridization when hela cells were infected in the presence of actinomycin d (rosemond-hornbeak and moss, 1975) but were degraded during productive infection of l cells by vaccinia virus (rice and roberts, 1983) . the second observation seems more pertinent. 'ben-hamida et al. (1983) have purified a component from vaccinia virions that blocks the binding of met-trna to 40 s ribosomes in uitro, but the physiological (in uiuo) mechanism of host shutoff seems to require the expression of viral genes. there is no evidence that eif-2 function is impaired in infected cells. it is possible, however, that some component in the eif-2 cycle is altered in a positive way, i.e., a way that prevents inactivation by eif-2 kinase (whitakerdowling and youngner. 1984) . "it is clear that host translation can be inhibited rapidly in the presence of drugs that preclude the synthesis of viral mrna (moss, 1968) . but it is not clear that the normal shutoff mechanism is at work in such cases (see text). "the virion-mediated rapid shutoff of host translation is not usually seen with herpes simplex type 1. except in vero cells; type 2 virus displays the early shutoff function in all cell types. an important, albeit undeciphered, clue is that type 1 virus interferes with the early shutoff by type 2 virions in doubly infected friend erythroleukemia cells (hill et al., 19851. ent viruses can often be produced simultaneously in cells in which host protein synthesis is suppressed (alonso and carrasco, 1982a,c; otto and lucas-lenard, 1980) . some features of the intracellular environment, such as ionic changes that favor the translation of viral over host mrnas, are inevitably lost when the cells are lysed, and other features are not easily preserved. for example, phosphorylated initiation factors have sometimes been inadvertently restored to normal during their purification (centrella and lucas-lenard, 1982; wong et al., 1982) . phosphorylation of eif-2 has also been missed on occasion because eif-2(ap), retains the ability to function stoichiometrically, and the defect is evident only if one assays for catalytic function (safer, 1983) .6 on other occasions, phosphorylation of eif-2 has been missed because a high concentration of gtp in the lysate masks the functional defect (schneider et al., 1985) . extreme care is needed also to preclude the artifactual modification of initiation factors-by proteolysis, for example-during the preparation of cell-free extracts. the fact that one can reproduce in vitro the preferential translation of viral over host mrnas does not necessarily mean that one is studying the physiological mechanism of host shutoff. if viral mrnas are even slightly more efficient than host mrnas, as is often the case, any manipulation that establishes competition will favor the viral mrnas. one cannot define how competition is established in uiuo by showing that competition occurs in uitro. for example, the fact that translation of vaccinia mrnas is more resistant than host mrnas to inhibition by poly(a) when translation is studied in cell-free extracts from reticulocytes (bablanian and banerjee, 1986; coppola and bablanian, 1983) does not mean that vaccinia virus inhibits host translation by flooding the cytoplasm with short, polyadenylated transcripts. such transcripts are indeed produced in infected cells, but only when drugs are used to block the synthesis of normal viral mrnas (rosemond-hornbeak and moss, 1975) . the aforementioned problem of an experimental manipulation creating a new inhibitory mechanism, rather than exposing the normal mechanism, almost certainly applies here. the tendency to attribute functional significance to foreign agents that cosediment with polysomes should be resisted. everything cosediments with polysomes to some extent. the presence of a trace of ade-6 eif-2, eukaryotic initiation factor 2, is responsible for binding initiator methionyl-trna to the 40 s ribosomal unit, and eif-2(ap) is eif-2 phosphorylated on its a-subunit. when eif-2 is phosphorylated, the reaction in which gdp is exchanged for gtp fails. that reaction is mediated by an accessory protein called gef, which becomes trapped in an inactive complex with eif-2(ap). because the pool size of gef is small, phosphorylation of only 30% of the eif-2 pool can completely inhibit translation (safer, 1983; siekierka et al., 1984) . 25 1 novirus va-rna in the polysome region of sucrose gradients (schneider et al., 1984) , for example, is almost certainly unrelated to the function of va-rna. it is common to find viral capsid proteins stuck to ribosomes, and it is wise to treat such contamination as contamination, until it is proven otherwise. the known and suspected mechanisms by which translation of viral mrnas is facilitated, usually to the disadvantage of host mrnas, fall into four categories. 1. competition may be suspected when the decline in host protein synthesis and the onset of viral protein synthesis coincide. on the other hand, competition is an insufficient explanation when host protein synthesis is severely inhibited before the onset of viral translation, as occurs with poliovirus, herpes simplex virus, and frog virus 3. often competition is exacerbated by a decline in the overall translational capacity, which may be brought about by changes in the ionic environment or in the translational machinery. when initiation is limiting, most mrnas accumulate in small polysomes, the size of which increases upon exposure to a low concentration of cycloheximide. (cycloheximide slows elongation, thus causing the number of ribosomes to increase on mrnas that were previously limited at the initiation step.) the characteristic shift in polysome size upon exposure to cycloheximide is seen, for example, in cells infected by vsv (jaye et al., 1982) or adenovirus (perlman et al., 1972) . the competition between host and viral mrnas that takes place in uiuo is sometimes not maintained when the translation of endogenous mrnas is studied in extracts from infected cells, probably because the concentrations of critical components change during the preparation of such extracts. 2. inactivation of a normal component of the translational machinery. the resulting deficiency enables only a subset of mrnas, mostly viral, to be translated. the hallmark of this mode of regulation is the ability to restore translation to cell-free extracts by adding back the missing factor, in practice, this is not as easy as it sounds. there is evidence for inactivation of initiation factor eif-2 in several virus systems, as noted in table i . alterations in the initiation factor that mediates the translation of capped mrnas have been postulated for several viruses (table i) , but the story seems credible only in the case of poliovirus, which is described below. identifying characteristic here is that extracts from infected cells cannot be reactivated by the addition of normal initiation factors, but can be reactivated by washing the ribosomes to remove the inhibitor. based on these criteria, pensiero and lucas-lenard (1985) have postulated the production of an inhibitor during mengovirus infection. because mengovirus and host mrnas are equally sensitive to the inhibitor in cell-free extracts, one must invoke competition (for the residual functional ribosomes) t o explain the selective persistence of viral translation in uiuo. this seems justified in view of the extraordinary efficiency of mengovirus mrna when translation is carried out in uitro under conditions of competition (abreu and lucas-lenard, 1976) . until the postulated inhibitor has been identified, however, we cannot be certain that mengovirus belongs in category 3. the hallmark here is that viral mrnas should be translated more efficiently in cell-free extracts from infected than from uninfected cells. frog virus 3 meets this criterion (raghow and granoff, 1983) . it is possible that some other animal viruses alter the translational machinery in a "positive" way.7 the best evidence t o date comes from plant viruses, however. a genetic analysis of temperature-sensitive mutants of alfalfa mosaic virus strongly suggests that rnas-1 and -2 encode or induce a factor that facilitates the translation of coat protein from rna-4 (huisman et al., 1985) . extrapolating that mechanism to brome mosaic virus would explain why rna-4 fails to synthesize coat protein when it is injected (without rnas-1, -2, and -3) into barley protoplasts (kiberstis et al., 1981) . unfortunately, cellfree systems from infected plant cells are not available to test the hypothesis. the aforementioned hints are only hints. no virus has yet been proved to produce a new or alter an old translational factor in a way that specifically promotes its own translation. since competition is the most common mechanism of translational regulation in virus-infected cells, that topic merits more attention. there are at least three variations on the theme. although the overall ability to translate poliovirus mrna is about the same when cell-free systems are reconstituted with factors from infected or uninfected cells (brown and ehrenfeld, 1980) , there is a qualitative difference in the selection of initiation sites when factors from infected cells are used (brown and ehrenfeld, 1979) . other experiments support the idea that poliovirus (bernstein et al., 1985) as well as vaccinia (moss and filler, 1970) and human t-lymphotropic virus type i11 (rosen et al., 1986) produce something that enhances the synthesis of viral proteins. the enhancing substance could viral translation 253 in l cells infected by reovirus type 2, host protein synthesis is dramatically shut off. the elegant genetic studies of sharpe and fields (1982) revealed that the s4 gene, which encodes the major capsid protein u3, is responsible for the inhibition. the effect of u3 might be indirect, inasmuch as the same gene product is responsible for inhibiting host rna synthesis. although host shutoff by type 3 reovirus in sc-1 cells is less dramatic than with type 2 virus in l cells, the mechanism of type 3 shutoff is better understood due to the careful quantitative studies of thach and colleagues (walden et al., 1981) . their conclusion was rather surprising: the intrinsic translational efficiency of reovirus mrnas is not higher than that of host mrnas, but rather, reovirus translation dominates because viral mrnas accumulate in massive amounts-up to 45% of the total mrna in the cell! the evidence that reovirus mrnas initiate translation less efficiently than most host mrnas is twofold: (1) the size of reovirus polysomes is smaller than host polysomes that code for proteins of comparable size; and (2) whereas a low concentration of cycloheximide reduces the synthesis of host proteins (which is the result expected for mrnas of "normal" efficiency), the translation of reovirus proteins is actually enhanced by a low concentration of cycloheximide. whereas reovirus mrnas appear to be less efficient than most host mrnas, vsv mrnas are probably translated as efficiently as host mrnas, but not more so. competition is simply proportional to the concentration of viral mrnas in the cytoplasm (lodish and porter, 1981) ,8 and vsv and host mrnas that encode the same-sized proteins are on polysomes of the same size (lodish and porter, 1980) . in some cell lines infected by some strains of vsv, a portion of the eif-2 pool seems to be inactivated (centrella and lucas-lenard, 1982; dratewka-kos et al., 1984) . although that would intensify the competition, it is obvious that lowering the eif-2 level per se cannot explain the selective inhibition of host translation. selective shutoff requires that viral mrnas be more abundant than host mrnas, or more efficient, or be a virus-specific translation factor, or a protease inhibitor that stabilizes viral proteins, or a nuclease inhibitor, or something else. recent evidence indeed suggests that vaccinia encodes a function that protects late viral mrnas against degradation (pacha and condit, 1985) . both. the aforementioned experiments argue against vsv mrnas being unusually efficient, but other experiments have been taken as evidence for the contrary view. because vsv mrnas are more resistant than host mrnas to hypertonic stress, nuss et al. (1975) have suggested that the viral mrnas are intrinsically more efficient. their interpretation seems reasonable, but it must be carefully circumscribed. if high salt exacerbates some deleterious feature in the mrna (such as secondary structure) to the point where it becomes inhibitory, then the hierarchy of mrna strengths that one observes under hypertonic conditions might be irrelevant to normal growth condition^.^ the progressive inhibition of host protein synthesis during infection by vaccinia virus is probably due to competition, in proportion to the concentration of each mrna. viral mrnas are not apparently more efficient than host mrnas (cooper and moss, 1979; . degradation of host mrnas (table i ) and the massive synthesis of viral transcripts probably tip the balance in favor of viral protein synthesis. have suggested that differential association of mrnas with the cytoskeleton might also play a role, but that is a difficult hypothesis to test. in contrast with reovirus and vsv, the concentration of emc virus mrna in infected cells may be too low for simple competition to effect the observed switch from host to viral translation, even though emc mrna is translated more efficiently than host mrnas both in uiuo (jen et al., 1978) and in vitro (golini et al., 1976; svitkin et al., 1978) . in view of the overall decline in translation that begins 3 hours postinfection, however, the idea that emc virus mrna outcompetes host mrnas for the low, residual translational capacity seems reasonable. the overall decline is most likely due to an influx of monovalent cations, since the two events are temporally correlated (lacal and carrasco, 1982) . host translation is restored when emc virus-infected cells are shifted to hypotonic medium carrasco, 1981, 1982b1 , and excess salt, sufficient to inhibit the translation of host mrnas in uitro, dramatically stimulates the translation of emc rna (carrasco and smith, 1976 ). 9recall that, although reovirus mrnas are not more efficient than host mrnas in unperturbed cells, reovirus translation, like that of vsv, dominates when cells are subjected to hypertonic stress (nuss et al., 1975) . in other studies, the creation of a hairpin (ag -30 kcalimol) within the 5'-noncoding region of preproinsulin mrna impaired translation only in hypertonic medium; the hairpin did not inhibit translation under normal culture conditions (kozak, 1986b ). a similar mechanism might mediate the switch from host to viral translation during infection by alphaviruses (see sindbis and sfv, i.e., semliki forest virus, in table i) , since the influx of sodium ions exactly coincides with the overall decline in protein synthesis. the magnitude of the ion influx remains controversial (gray et al., 1983; munoz et al., 1985b) . translation of sfv mrna is more resistant than host protein synthesis to hypertonic conditions (garry et al., 19791 , but the resistance is not as dramatic as with emc virus (alonso and carrasco, 198213 . the notion that alphaviruses inhibit host translation by competition seems viable even if something more than enhanced permeability to monovalent cations is needed to explain the overall decline. the fact that sfv mrna can be translated in emc-infected cells (alonso and carrasco, 1982~1 , in which the overall translational capacity is very low, identifies sfv late 26 s mrna as an efficient message. consistent with the competition hypothesis, the time of host shutoff coincides with the production of viral mrna (lachmi and kaariainen, 1977) and the severity of inhibition correlates with the yield of viral rna in mutant-infected cells (atkins, 1976) . polysomes containing sfv (wengler and wengler, 1976) or emc virus mrna do not increase in size upon exposure to cycloheximide, suggesting that those mrnas are efficient enough to be fully loaded with ribosomes even when the overall translational capacity is low. influenza virus mrnas are translated with extraordinarily high efficiency i n uitro (katze et al., 1986) . because the shutoff of host protein synthesis coincides with the onset of influenza virus protein synthesis and there is no overall decline in translation, simple competition would seem adequate to explain the switch from host to viral translation. in the case of adenovirus, competition is probably exacerbated by a reduction in functional eif-2 levels. these issues are discussed in more detail in section ii1,e. in some virus systems, competition might dictate the switch from synthesis of early to late viral proteins. picornaviruses, rhabdoviruses, and influenza virus are uninteresting in this regard, as they display little or no temporal control over protein synthesis. the existence of a temporal switch is questionable for reoviruses, but all of the other entries in table i , as well as the papovaviruses, show a striking earlyto-late transition. in every case, the switch is effected primarily a t the level of transcription: the mrnas that encode late proteins are not synthesized until late. in several cases, however, early mrnas persist in the cytoplasm at late times, and some form of translational regulation seems to limit their expression (hruby and ball, 1981; johnson and spear, 1984; lachmi and kaariainen, 1977; vassef et al., 1982) . with black beetle virus that phenomenon can be attributed to competition, because translation of the late mrna predominates over early mrna in cell-free extracts under conditions of competition (friesen and rueckert, 1984) . the same explanation probably holds for alphaviruses. on the other hand, late vaccinia virus mrnas do not appear to be more efficient than early viral mrnas (cooper and moss, 1979; oppermann and koch, 1976) . instead, degradation of some early vaccinia transcripts (hruby and ball, 1981) might be part of the switching mechanism. the translation of early mrnas could be further reduced by the accumulation of "anti-early mrna" (boone et al., 19791 , which could inhibit translation much as antisense rna does in other experimental systems (izant and weintraub, 1985) . lo with baculoviruses, temporal switching involves the sequential activation of upstream promoters, such that the small, early mrnas are replaced by progressively longer overlapping transcripts (friesen and miller, 1985) . the resulting relegation of early protein coding sequences to the 3' ends of late transcripts probably prohibits their translation. promoter switching late in sv40 infection also generates forms of mrna from which t antigen is translated inefficient1y.l thus, although transcription plays the dominant role, translational mechanisms-involving competition or other ploys-contribute to the temporal switch in expression of viral genes in some systems. the current thinking is that poliovirus selectively shuts off host protein synthesis by inactivating a 220-kda protein (~2 2 0 ) which is a subunit of the initiation factor that mediates the translation of capped mrnasll because the 5' end of poliovirus mrna is uncapped, inac-10 few systems other than vaccinia show much potential for regulating translation by "hybrid arrest." complementary transcripts accumulate in the nuclei of many virusinfected cells, but the complementary sequences are usually edited from cytoplasmic mrnas. in the case of adenoviruses, for example, where transcription switches periodically from one dna strand to the other, the 3' ends of the juxtaposed mature mrnas rarely overlap (lemoullec et al., 1983) . the 3' ends of papovavirus early and late mrnas do characteristically overlap, however. 11 although the proteins that can be cross-linked to the m7g cap have a disturbing tendency to change from year to year, two proteins in mammalian cells that reproducibly cross-link are p24-cbp and p46-cbp. p220 is not a "cap binding protein" inasmuch as it does not cross-link to the cap, but p220 does copurify with p24-cbp and p46-cbp. "he aggregate, called eif-4f, is considered by most people to be the functional "cap binding factor." the functions of cap binding proteins have been reviewed by shatkin (1985) . tivation of the cap binding factor should not impair the translation of viral mrna. the idea is appealing because it is straightforward, but some of the supporting data are less so. the experiment that gave birth to the hypothesis was provocative. using an antiserum against initiation factor eif-3, etchison et al. (1982) showed by immunoblotting that p220 is clipped during the first few hours after infection of hela cells by poliovirus. because affinitypurified antibodies against p220 recognized a protein of the same size in some preparations of cap binding factor, the working hypothesis was that cleavage of p220 inactivated the cap-binding initiation factor. indeed, an activity from uninfected cells that was subsequently purified, based on its ability to restore translation to poliovirus-infected cell-free extracts, contained p220 and the cap binding proteins. these experiments are described below in more detail. this is the most promising explanation to come forth, but there are some irregularities and many lacunae in the supporting data. 1. there is a discrepancy between the kinetics of degradation of p220 and the kinetics of host shutoff (etchison et al., 1982) . the same anomaly occurs during infection by rhinovirus, which degrades p220 in a manner similar to poliovirus (etchison and fout, 1985) : the rate of translation is still half-maximal a t the time when p220 disappears from the polyacrylamide gels. in many of the experiments carried out with cell-free extracts, the question of timing was disregarded, and cells were routinely harvested 3 hours postinfection (lee and sonenberg, 1982; lee et al., 1985a) , which is well beyond the point when host translation is precipitously shut off. 2. the extent of cleavage is difficult to evaluate quantitatively. it seems dangerous to accept the recommendation of bernstein et al. (1985) to focus on the accumulation of the 115-kda cleavage products without also monitoring the disappearance of p220, because cleavage need not always be arrested at the 115-kda level. in some experiments the concentration of cleavage products in immunoblots from infected cells greatly exceeded the concentration of intact p220 in uninfected cells (bernstein et al., 1985) . 3. a p220 cleavage pattern qualitatively similar to that which occurs in infected cells, although not nearly as extensive, is evident in some extracts from uninfected cells (bernstein et al., 1985; fig. 5 in lee et al., 1985a) . because the link between degradation of p220 and virus infection is not tight, it was not surprising to find that the virus-encoded protease 3c is not responsible for cleaving p220 (lee et al., 1985b; lloyd et al., 1985) . it would seem wise to include a spectrum of protease inhibitors during the preparation of extracts. phenylmethylsulfonyl fluoride is the only one routinely used, at concentrations ranging from 5 mm (which is adequate; etchison et al., 1982) to 1 mm (bernstein et al., 1985) or 0.2 mm (lee and sonenberg, 1982) . one is reminded of the old excitement concerning "processing" of sv40 t antigen (ahmad-zadeh et al., 1976) which turned out to be an artifact of extraction (smith et al., 1978) . 4. in uninfected hela cells, the concentration of p24-cbp is 10-fold lower than p220 (duncan and hershey, 1985a,b) . [the concentration of p24-cbp is also low in reticulocytes (hiremath et al., 1985) .1 if p24 and p220 (together with p46) function as the cap binding complex, large changes in the p220 pool-although easy to detect by immunoanalysis-are unlikely to alter the rate of translation; but small changes in the pool of p24, which might go undetected with immunological or biochemical probes, would significantly impair translation. 5. a mutant of poliovirus called hf121 has been described in which the synthesis of viral rna is normal in cv-1 cells, but viral protein synthesis is inefficient, host translation is inhibited more slowly than usual, and p220 is not rapidly cleaved (bernstein et al., 1985) .12 (the phenotype of hf121 in hela cells, which are a more natural host than cv-1 monkey cells, is more complex. the synthesis of viral rna is greatly reduced and all protein synthesis, host and viral, is inhibited very early, again without the concomitant cleavage of p220.) the authors argue cogently that wild-type poliovirus appears to encode a function, absent from hf121, that promotes (or "avoids the early inhibition of") viral translation, and they argue less cogently that hf121 is translated poorly as a consequence of the failure to selectively inhibit host translation. to me, the second postulate seems redundant. lack of the putative positive factor would be sufficient to account for the poor translation of viral mrna, and the failure to inhibit host translation with normal kinetics could as likely be the result of ineffkient viral translation as the cause.i3 if the slow inhibition of translation by 12 although cleavage of p220 is not detectable at all in hela cells infected by mutant hf121, cleavage products are clearly evident in cv-1 cells a t 5 hours postinfection (fig. 8a, lane 7, in bernstein et al., 1985) . that is later than normal, and the cleavage is less extensive than normal, but some cleavage does occur. 13 one could argue similarly that cleavage of p220 in wild-type infected cells is the consequence of the abundant accumulation of viral proteins, rather than a precondition. the issue might be resolved by treating wild-type infected cells with guanidine, which allows only limited synthesis of viral proteins while host translation is inhibited with the usual rapid kinetics. it would be informative to know whether p220 undergoes cleavage under those circumstances. hf121 in cv-1 cells is a delayed version of the normal shutoff mechanism, then cleavage of p220 must not be central to the normal shutoff mechanism. the authors argue, to the contrary, that shutoff by hf121 is mechanistically different, inasmuch as the inhibition affects both host and viral mrnas in hf121-infected cells, whereas host translation is preferentially inhibited in wild-type-infected cells. however, selective stimulation of viral translation (mediated by the product that is defective in hf121) superimposed on a general inhibition of translation, would mimic selective inhibition. the authors contend that the ability of guanidine to block host shutoff by hf121 distinguishes it from the normal shutoff mechanism, but the experiment (in cv-1 cells) was done without testing wild-type virus in parallel, as could have been done by adding guanidine at the start of the infection rather than after 3 hours. 6. the assumption that tobacco mosaic virus, sindbis virus, and vsv mrnas are appropriate stand-ins for host mrna in the restoring assay is questionable rose et al., 1978; tahara et al., 1981) . in cells singly infected by vsv, viral mrnas are translated in preference to host mrnas under some conditions (nuss et al., 1975) ; thus, vsv mrnas are not equivalent to most host mrnas. when cells are doubly infected with poliovirus and sfv (which is akin to sindbis), or with poliovirus and vsv, conditions can be found that allow the simultaneous translation of poliovirus mrna and the capped mrnas of vsv or sfv (alonso and carrasco, 1982a) . thus, the factor that restores to poliovirus-infected extracts the ability to translate vsv or alphavirus mrnas might not be sufficient to restore the translation of most host mrnas. globin is the only cellular mrna that has been shown to work in the restoring assay (edery et al., 19841 , and its translational efficiency resembles viral mrnas more than the average cellular mrna. it would be reassuring to omit the usual micrococcal nuclease pretreatment of lysates, and show that the addition of cap binding factor to poliovirus-infected cell-free extracts restores the translation of authentic endogenous host mrnas. the association of p220 with p24-and p46-cbps does not prove that p220 is a necessary component of the complex. in early studies, preparations of p24-cbp that lacked the p220 subunit did preferentially stimulate the translation of capped mrnas in hela cell-free extracts (tahara et al., 1981; sonenberg et al., 1980) . those results were considered wanting because p24-cbp failed to reproducibly restore translation to extracts from poliovirus-infected cells, whereas an aggregate of p220, p46, and p24 could restore (tahara et al., 1981) . but there is no reason to reject the aforementioned demonstration that p24-cbp by itself does stimulate in uninfected systems. using the restoring assay to define the structure of cap binding factor would be acceptable only if one knew that cap binding factor was deficient in infected-cell extracts. grifo et al. (1983) showed that translation of globin mrna was stimulated by the p22o-p46-p24 aggregate, even when the system was saturated with p24-and p46-cbps. those data prove only that the system which they reconstituted from partially purified subfractions of a reticulocyte lysate was deficient in p220; the data do not prove that p220 is an essential component of cap binding factor. (indeed, translation of the uncapped mrna from satellite tobacco necrosis virus was stimulated to the same extent as capped globin mrna.1 the function of p220 would be clearer if one could show that antibodies against p220 inhibit the function of cap binding factor. such experiments have not been reported. indeed, the immunological evidence is far from convincing even for the original p24-cbp. a monoclonal antibody "directed against cap binding proteins" was shown to inhibit the translation of capped mrnas, but the antibody reacted with higher molecular weight proteins and not with p24-cbp (sonenberg et al., 1981) . the claim that the higher molecular weight polypeptides were related to p24-cbp no longer seems valid, because polyclonal antibodies obtained recently against p24-cbp react only with that polypeptide (hiremath et al., 1985) . in extracts from uninfected hela cells, p220 copurifies to some extent with both cbps and eif-3 (etchison et al., 1982) . whereas it is known that p220 restores translation to poliovirus-infected extracts when it is introduced in association with cap binding proteins, it is not known whether p220 would also enhance were it introduced in association with eif-3. in several studies, eif-3 failed to restore translation to extracts from poliovirus-infected cells, but it was usually tested on an equal weight basis, vis-a-vis the other initiation factors (table iv in grifo et al., 1983; rose et al., 1978) . because eif-3 is so massive, it must be tested on an equal molar basis. an experiment which intended to show the eif-3 from poliovirusinfected cells is fully functional failed to prove the point, because the assay for eif-3 was carried out in the presence of cap binding factor from uninfected cells (etchison et al., 1984) . the exogenous cap binding factor may have contributed a component (such as p220) which was necessary for, and absent from, infected-cell eif-3. the assay would have been more meaningful had an uncapped mrna been used, thus allowing the function of eif-3 to be evaluated without the necessity of adding cap binding factor. whether p220 should be considered a component of eif-3 or of the cap recognition factor involves more than semantics. whereas inactivation of cap binding factor would be sufficient to explain the selective inhibition of host translation, eif-3 is apparently needed for translating all mrnas; were eif-3 activity low, poliovirus would have to outcompete host mrnas for the residual activity. casting a wider net might identify other components that are involved in host shutoff by poliovirus. a few candidates have been ruled out. initiation factors eif-4a and eif-4b, for example, appear to be unaltered (duncan et al., 1983) . the normal association of host mrnas with the cytoskeleton is disrupted shortly after infection by poliovirus (lenk and penman, 1979) . whether that is the cause, or the effect, or is unrelated to inhibition of host translation remains unclear. such a dramatic effect seems unlikely to be gratuitous, but in other systems disruption of the cytoskeleton does not preclude all translation (welch and feramisco, 1985) . follow-up studies in the poliovirus system have not significantly extended penman's original, provocative observation. when bonneau et al. (1985) infected cv-1 cells first with vsv (which does not dissociate host mrnas from the cytoskeleton) and then superinfected with poliovirus, translation of vsv g and m proteins was inhibited and those mrnas were released from the cytoskeleton; unfortunately, it was not shown that vsv n and ns mrnas, which continued to be translated, remained bound to the cytoskeleton. the conclusion that translation requires association with the cytoskeleton hardly seems warranted. carrasco has suggested that the increased permeability of virusinfected cells to monovalent cations might mediate the switch from host to viral translation (carrasco and lacal, 1983; carrasco and smith, 1976) . when infected cells are incubated in medium containing sufficient excess nacl to inhibit the translation of most other proteins, poliovirus translation is fairly resistant (alonso and carrasco, 1982a; nuss et al., 19751 , but the resistance is not as striking as with emc virus (alonso and carrasco, 1982b) . the stimulation of in uitro translation by high salt is also less obvious with poliovirus mrna than with some other picornaviruses (bossart and bienz, 1981) . in the natural course of infection by poliovirus, the precipitous decline in host translation occurs within the first 2 hours, prior to the observed increase in intracellular sodium ions (nair et al., 1979) . moreover, the synthesis of cellular proteins cannot be reactivated by incubating poliovirusinfected cells in hypotonic medium (alonso and carrasco, 1982b) , a manipulation that works beautifully with emc virus. thus, hypertonicity does not appear to underlie the shutoff of host protein synthesis by poliovirus. morrow et al. (1985) made the astonishing discovery recently that the host-encoded kinase that is responsible for phosphorylating eif-2 binds to and mediates the replication of poliovirus rna. although that seems about as auspicious as a sheep shaking hands with a wolf, one can think of ways to rationalize such a dangerous move. if the pool of viral rna that serves as template for replication has to be kept free of ribosomes, for example, the presence of eif-2 kinase in replication complexes could help by phosphorylating the local pool of eif-2. indeed, eif-2 might become globally phosphorylated in infected cells, and the resulting eif-2 deficiency could contribute to the inhibition of host protein synthesis. whereas older studies suggested that eif-2 was not deficient in polio-infected cells (brown and ehrenfeld, 1980; helentjaris and ehrenfeld, 1978) , the translation of heterologous mrnas in infected-cell extracts was restored to a limited extent by the addition of eif-2 (rose et al., 1978) -an effect that the authors chose to ignore. asim dasgupta has reopened the question, and his careful measurements reveal extensive phosphorylation of eif-2(a) in poliovirus-infected cells (personal communication). the adenovirus system has generated considerable excitement recently because genetic manipulations, pioneered in shenk's laboratory, have revealed a regulatory mechanism that is novel, and yet connects to the extensive older literature on inactivation of initiation factor eif-2. the focal point is a small virus-encoded rna called va-rna,. thimmappaya et al. (1982) found that, in cells infected by an adenovirus mutant that produced no va-rnai, late viral mrnas were synthesized, processed, and transported, but failed to be translated. in the absence of va-rna,, translation was blocked at the level of initiation (schneider et al., 1984) and the defect was ultimately localized to eif-2. overwhelming evidence now supports the hypothesis that, in the absence of va-rna,, a kinase becomes activated that phosphorylates, and thus inactivates, eif-2 schneider et al., 1985; siekierka et al., 1985) . eif-2 kinase (one of the enzymes involved in the antiviral action of interferon; see lengyel, 1982) exists in uninfected hela cells in an inactive state, and is apparently activated by double-stranded rna that accumulates in infected cells as a by-product of adenovirus transcription (o'malley et al., 1986) . l4 the exact mechanism by which va-rnai blocks the action of eif-2 kinase is not yet known. an intriguing scenario can be extrapolated from a model that was proposed by rosen et al. (1981) in another context. their model proposes that high molecular weight double-stranded rna activates and targets eif-2 kinase: because both the kinase and eif-2 have binding sites for dsrna, high molecular weight dsrna could link the two proteins.15 by virtue of its small size, va-rna, might be able to bind to eif-2 or to eif-2 kinase, but not simultaneously to both. va-rnai would thus block the phosphorylation of eif-2 much as a monovalent hapten blocks antigen-antibody interactions. the proposal rationalizes the known properties of va-rna,: its small size (about 160 nucleotides), doubled-stranded structure (monstein and philipson, 19811 , and the high concentration that is required to confer protection. whether the double-stranded regions of va-rnai are crucial for its function is not yet clear. have mutated va-rna, and found that extensive regions could be deleted without affecting biological activity, although certain other mutations were deleterious. further experiments will be needed to pinpoint the essential region(s) in va-rnai. a second adenovirusencoded species called va-rnai, rescues translation far less efficiently than va-rna, (thimmappaya et al., 19821 , and va-rnaii appears less extensively base paired (mathews and grodzicker, 1981) . in addition to its proven protective effect on eif-2, it has been suggested that va-rna, might interact directly with viral mrnas to promote their translation (kaufman, 1985; schneider et al., 1984; svennson and akusjarvi, 1985) . a sequence-specific interaction seems unlikely, however, because the small rnas encoded by epstein-barr virus (which are related to adenovirus va-rnas by size but not sequence) can substitute to some extent for va-rnai (bhat and thimmappaya, 1983, 19851 , and the facilitating effect of va-rna, extends 14 a virus that is protected (by va-rna or some other mechanism) from the deleterious effects of its own symmetrical transcription process would also have some resistance to interferon. an interesting story along these lines is emerging with vaccinia virus (rice and kerr, 1984; whitaker-dowling and youngner, 1984) . 15 sen et al. (1978) showed that, once kinase has been activated by binding dsrna, incubation with ribonuclease i11 does not abolish the ability of kinase to phosphorylate histone h1 (which was chosen as a convenient substrate), but neither the extent of trimming by the nuclease, nor the activity of the trimmed kinase on eif-2, were determined. thus, the experiment does not contradict the targeting hypothesis for dsrna. not only to late adenovirus mrnas that carry the standard tripartite leader, but also to early adenovirus mrnas and late mrnas with a truncated version of the tripartite leader (svensson and akusjarvi, 19841, adeno-associated virus mrnas (janik et al., 19821, and various heterologous mrnas (svensson and akusjarvi, 1985) . the protective effect of va-rna, and the shutoff of host translation in adenovirus-infected cells might be two aspects of a single mechanism. if one postulates that the production of va-rna, by wild-type adenovirus is sufficient to protect only a portion of the eif-2 pool, the switch from host to viral protein synthesis could occur because late adenovirus mrnas outcompete host mrnas for the small residual translational capacity. the hypothesis that competition occurs during the late stage of infection by adenovirus is not entirely ad hoc. the overall translational capacity is low late in the infection (castiglia and flint, 1983) ; a portion of the eif-2 pool is phosphorylated (m. mathews, personal communication) ; polysomes are small and their size increases in response to a low dose of cycloheximide (perlman et al., 1972) ; and the decline in host translation correlates with the temporal onset and magnitude of late viral translation (castiglia and flint, 1983) . every mutation that has been shown to prevent host shutoff also prevents the cytoplasmic accumulation of late viral mrnas (babiss et al., 1985; halbert et al., 1985) . an interesting set of experiments by logan and shenk (1984) can be rationalized in terms of competition during the late stage of infection. they observed that transposition of the late tripartite leader to the early e1a genes had no effect on the efficiency of translation of e1a products at early times, but significantly enhanced the translation of e1a mrnas at late times. this is understandable if there is no competition early in the infection, allowing efficient and inefficient mrnas to be translated equally well. the facilitating effect of the tripartite leader would become evident only late in the infection, when eif-2 has been partially inactivated and competition has set in. one might think along similar lines to explain the surprising ability of influenza virus mrnas to be translated in adenovirus-infected cells . in wild-type adenovirus-infected cells, in which host protein synthesis is drastically reduced, both adenovirus and influenza virus mrnas are translated efficiently. in cells infected by d1331, a deletion mutant that produces no va-rna,, adenovirus proteins are translated inefficiently, as noted above, but influenza virus proteins are still synthesized in abundance. despite that striking observation, there is little support or necessity for the notion that influenza virus establishes its own translational system. a simpler explanation is that the very low capacity for protein synthesis that persists in the absence of va-rna, is sufficient for the translation of influenza virus mrnas.16 for that explanation to be correct, influenza virus mrnas would have to be translated with extraordinary efficiency, and that prediction has recently been confirmed (katze et al., 1986) . what makes all this so intriguing is that the 5' ends of influenza virus mrnas, which presumably dictate their high translational eficiency, are derived from host mrnas (krug, 1981) . it appears as if the viral capspecific endonuclease (which selects the cellular mrnas that will serve as donors) is biased toward the same features that facilitate translation. indeed, that deduction has been verified directly, at least in vitro. bouloy et al. (1978 bouloy et al. ( , 1980 found that p-globin mrna, which is translated more efficiently than a-globin mrna, is also a more efficient primer for influenza transcription; and alfalfa mosaic virus rna-4, which translates in vitro with extraordinary eficiency, is the best known primer for influenza transcription. [from the fact that mrnas with 2'-o-methyl groups in the penultimate position of the cap function better than monomethylated caps as primers for influenza virus transcription (bouloy et al., 1980) , one is tempted to suggest that 2'-0methyl groups enhance translation, although there is little direct evidence for that view!] if the selection of primers in infected cells follows the pattern that is seen in vitro, the influenza virus takeover scheme is indeed remarkable: the most efficient cellular mrnas would be sacrificed to construct viral mrnas that ips0 facto translate most efficiently. a few other possibilities for regulating translation in virus-infected cells are discussed briefly below. none of these topics is well understood at present, and the musings should be considered little more than that. cauliflower mosaic virus seems to be the only case in which the ability of eukaryotic ribosomes to reinitiate is fully exploited to produce several full-length proteins from one mrna. the structure of a few 16 katze et al. (1986) have shown that influenza virus partially suppresses the activation of eif-2 kinase, and they suggest that this underlies the ability of influenza virus to replicate in cells infected by adenovirus mutant d1331. that interesting hypothesis would be stronger if it could be shown that influenza virus replicates even in cells infected by the adenovirus double mutant (vai -vaii -), and if superinfection with influenza virus could be shown not just to reduce the level of activated kinase, but to increase the level of functional eif-2. other viral and cellular mrnas leads one t o predict that reinitiation is necessary for ribosomes to reach the major protein coding sequence, but the upstream open reading frames (orfs) in such mrnas are characteristically short. in some cases, however, the small peptides encoded near the 5' end of the message might be biologically important. genetic studies indicate that this is certainly the case with the agnogene product of sv40 (margolskee and nathans, 1983) . in contrast, the three peptides encoded within the avian retrovirus leader sequence probably are not functional because there is little conservation of amino acid sequences among virus strains (hackett et al., 1986) . in retrovirus mutants that lack most of the leader sequence, the only known deficiency is the absence of a cis-acting packaging signal (mann et al., 1983; nishizawa et al., 1985) . comparison of different strains of poliovirus reveals that the number of upstream aug codons varies and the coding properties of the small orfs are not conserved (toyoda et al., 1984) . upstream minicistrons that do not encode anything interesting might nevertheless be important for regulation. several possibilities come to mind for retroviruses. the least interesting idea is that upstream aug codons accumulate, not from design, but from defaultbecause the deleterious effects on translation can easily be compensated by using efficient transcription signals to mass-produce retrovirus mrnas. the opposite view is that upstream aug codons are deliberately retained to throttle the synthesis of a protein that would be harmful if overproduced (tarpley and temin, 1984) . while that seems a reasonable ploy to use for oncogenes, it makes little sense when extended to viral structural genes. a third possibility derives from the observation that reinitiation usually is not 100% efficient. with preproinsulin constructs, for example, in which the efficiency of reinitiation is routinely 20% (kozak, 1984b) , one might ask what the remaining 80% of the ribosomes are up to. one scenario is that, after 80 s ribosomes have moved through the 5'-proximal orf, 80% of the 40 s subunits detach at the terminator codon while the rest remain on the message, resume scanning, and reinitiate at the second aug codon. a more interesting possibility is that all 40 s subunits remain bound and resume scanning, but only 20% reinitiate at the closest aug codon, perhaps because the codon-recognition step in inefficient in the absence of met-trna,, cap binding proteins, and/or other initiation factors-all of which were presumably released at an earlier step. [we do not know the precise sequence of events during initiation, but it seems likely that the factors that mediate the binding of met-trna, and mrna to the 40 s ribosomal subunit are released prior to or during the joining of the 60 s subunit at the first aug codon (moldave, 1985) .] if the factor-deficient 40 s subunits that are unable to reinitiate at the second aug codon eventually become competent, they might reinitiate father downstream. thus, the effect of an upstream minicistron could be to loosen the process of initiation in a way that permits ribosomes to reach otherwise inaccessible internal aug codons. there is no evidence for this, as yet. we know only that reinitiation at the closest aug codon (following a terminator codon) is less than 100% efficient. yet another way in which ribosomes might gain access to internal aug codons, even in a message in which the major open reading frame initiates with a "strong" aug codon, relies on the presence of weak, out-of-frame initiator codons in the retrovirus leader sequence and the ability of ribosomes to reinitiate. this hypothetical scheme is best illustrated by using as an example an avian retrovirus mrna that encodes the e m glycoprotein (fig. 2) . katz et al. (1986) have studied the effects of mutations in the leader region of this mrna, using as an a hypothesis whereby minor initiation sites in the leader sequence of retroviruses create a shunt that directs ribosomes to internal initiation sites. the diagram represents a subgenomic mrna that encodes the enu protein of avian leukosis virus (katz et al., 1986) . messenger rna is represented by a wavy line, the pathway followed by 40 s ribosomal subunits is shown above the mrna, and the pathway of 80 s ribosomes is shown below the mrna. a solid black line traces the pathway followed by most 40 s subunits: they scan from the m7g cap to the start of the enu coding sequence, marked "major start site," where a 60 s subunit joins and translation begins. (some 40 s subunits will stop and initiate at three upstream aug codons, but in each instance there is a nearby terminator codon, enabling ribosomes to reinitiate. thus, the upstream aug codons are irrelevant for the present discussion and are not shown.) of more significance are the many nonstandard codons (gug, uug, etc.) that lie in the standard context for initiation. such codons occur frequently in the -1 reading frame which is open (in the functional ev-2 viral genome) over a stretch of about 200 nucleotides preceding the major enu start site; the open -1 reading frame ends 125 nucleotides beyond the start of the enu coding sequence at uaa51ss-5190, which is labeled t ~ 1 in the figure. were a few 40 s subunits to recognize the nonstandard upstream codons as initiation sites, the resulting 80 s ribosomes-translating in theframe-would bypass the normal enu start site. a dashed line traces the pathway of this shunt. the main point is that ribosomes that terminate at t-1 could reinitiate a t an internal site which would be inaccessible were it not for the shunt. assay the ability to complement a replication-defective (env-) strain of rous sarcoma virus. their results are provocative. point mutations in positions -4 and -7 (i.e., 4 and 7 nucleotides upstream from the aug codon that initiates enu) caused a 10-fold reduction in complementation. on the other hand, the translational efficiency of deletion mutants varied from 5 to 106% of the wild type level, and the variation did not correlate with the presence or absence of any particular portion of the leader sequence. to explain this puzzling pattern (or rather the absence thereof), skalka suggests that the mutations perturb some aspect of secondary structure that is critical for translation. because that idea is difficult to formulate in a way that can be tested, it can do no harm to consider an alternative explanation. the biological assay that was used has the advantage of being exquisitely sensitive, but it has the disadvantage of measuring the yield of enu protein only indirectly: the authors did not show a 10-fold reduction in enu synthesis; they showed a 10-fold drop in complementation. what if complementation were to require, in addition to enu, a second minor protein-either a truncated form of enu that initiates a little farther downstream or a small protein encoded in an alternate reading frame? (there is an open reading frame beginning at aug,,,,-,,,,, for example, that could direct the synthesis of a 10-kda protein.) because the context at the major enu start site is highly favorable, all of 40 s ribosomal subunits that reach that site should initiate there; production of the putative internally initiated protein would therefore require a mechanism for shunting some ribosomes beyond the major enu start site. the hypothesis illustrated in fig. 2 is that a small fraction of the ribosomes initiate within the leader sequence at weak sites (nonstandard codons that lie in a favorable context for initiation) in the -1 reading frame, and translate in that frame past the major enu start site, terminating at the site labeled tin fig. 2 . the small fraction of ribosomes that follow this shunt could reinitiate to produce the second protein postulated above. the notion that the enu gene encodes two products is certainly ad hoc, but it rationalizes the behavior of skalka's mutants. the deleterious mutation in position -4 creates a terminator codon in the -1 reading frame, which would short-circuit the shunt and prevent synthesis of the internally initiated protein. in all of the deletion mutants that fail to complement effkiently, the weak upstream start sites are either in-frame with the major enu start site or terminate upstream from it-again abolishing the shunt. on the other hand, all of the deletion mutants that retain the ability to complement efficiently retain one or more weak upstream start sites (such as gug in position 132-134 in mutant 1371349, or uug in position 22-24 in mutant 65/349) which can feed ribosomes into the shunt. the hypoth-viral translation 269 esis could be tested in two ways. one is to directly measure the yield of the major enu protein-which we predict will not vary, because it is the internally initiated protein that is deficient in these mutants. the best test would make use of a null mutant called pd99/394 that lacks the major enu start site: that mutant should still make the second protein encoded within the enu gene, and therefore should complement all of the other mutants that have lost the shunt. viruses that replicate in the cytoplasm have the potential for coupling transcription with translation. for example, if ribosomes were to bind the 5' end of reovirus mrnas as the nascent chains emerge from the subviral particle, the mrnas would be recruited for translation before the chains grew long enough to fold. that might enhance translation considerably, because the pattern of cleavage by t, rnase suggests that the capped terminus might be sequestered in mature reovirus mrnas (kozak and shatkin, 1978b) . it would be fun to know whether reovirus mrnas are translated more efficiently in naturally infected cells than in cells transfected with cloned viral genes which are transcribed from a plasmid vector. the idea of coupling is ad hoc for reovirus, but there is a glimmer of evidence in the case of silkworm cytoplasmic polyhedrosis virus; whereas performed viral mrnas were inactive in reticulocyte or wheat germ translation systems, viral proteins were synthesized during coupled transcription-translation in frog oocytes (ikegami et al., 1985) . payne and mertens (1983) obtained somewhat different results, in that some viral proteins were made in vitro in the absence of transcription; but the polyhedron protein that predominates in vivo was still not produced in vitro. in the vaccinia virus system, cooper and moss (1978) observed more efficient synthesis of vaccinia proteins when transcription and translation were coupled. synergism could also occur in the opposite direction; i.e., viral transcription might be facilitated by translation. during the early hours of reovirus infection in l cells, transcription is mainly from genome segments m3, s3, s4, and one of the l segments (nonoyama et al., 1974) . because mrnas from segments m3, s3, and s4 bind ribosomes very efficiently (shatkin and kozak, 19831 , one wonders whether preferential transcription is the consequence of preferential tran~1ation.l~ the 17 the hypothesis is complicated, but not necessarily contradicted, by the finding that m3, 53, and s4 are preferentially transcribed in viuo even in the presence of cycloheximide (shatkin and kozak, 1983) . although cycloheximide blocks elongation by 80 s ribosomes, 40 s ribosomal subunits could still bind to the nascent transcripts. possibility that coupled translation enhances transcription was fleetingly entertained for some other cytoplasmic viruses (ball and white, 1978; cooper and moss, 1978) , but the reticulocyte lysate appeared to enhance transcription only because it conferred protection against nucleolytic degradation . it remains possible that transcription and translation are obligatorily coupled in some less well studied rna viruses, as has been hinted for bunyaviruses (patterson and kolakofsky, 1984; pattnaik and abraham, 1983) . in the case of viruses that replicate in the nucleus, the possibility that movement of mrnas out of the nucleus might be coupled with translation has been raised from time to time. coupling clearly is not obligatory, because viral mrnas accumulate in the cytoplasm under many circumstances in which translation is blocked. a good example is the cytoplasmic accumulation of late adenovirus mrnas in the absence of va-rna,. on the other hand, the transport and translation of mrnas are sometimes coordinated. a striking example occurs in adenovirus-infected hela cells that are superinfected with influenza virus : whereas adenovirus blocks both the transport and translation of host mrnas, influenza virus mrnas escape both blocks. the probable mechanism that enables influenza virus mrnas to be translated was discussed in section ii1,e. what mechanism enables influenza mrnas to bypass the block that retains host mrnas in the nucleus? suggested one possibility, namely, an influenza virus-specific transport system. but it seems simpler to look for a single explanation that would account for both the preferential transport and translation. there could be competition at the level of transport, and the same features that make a message highly translatable might make it highly transportable. an alternative view is that the two processes are coupled. one might envision 40 s ribosomal subunits monitoring the nuclear pores, such that only mrnas that can be translated under given circumstances will be transported. along those lines, babiss et az. (1985) have noted that, whereas host mrnas are neither transported nor translated in wildtype adenovirus-infected cells, transport and translation of host mrnas are coordinately restored by mutations in early viral genes that reduce the cytoplasmic accumulation of late viral mrnas. as an extension of the idea that a message will be transported only if it can be translated, one might suggest that mrnas are transported as soon as they become translatable. the consequence would be that translation could sometimes regulate the extent of splicing. some splicing events that could occur, were the transcript kept longer in the nucleus, would be prevented by "prematurely" pulling the mrna out. svensson et al. (1983) invoked this notion to explain some of their observations on the processing of adenovirus early mrnas. coupling of splicing with transport, and transport with translation, would explain why few if any incompletely processed transcripts enter the cytoplasm: no matter how many introns are present in a primary transcript, it remains in the nucleus until every intron has been removed-in effect, until it becomes translatable. it would seem as if the easiest way to judge whether a transcript is translatable is to attempt to translate it. the shutoff of host protein synthesis by herpes simplex virus might not involve a modification in the translational machinery per se. late (second-stage) shutoff is clearly caused by the massive degradation of host mrna. the puzzle of how the nuclease is targeted, such that it degrades host but not viral mrnas, has not yet been solved. a partial explanation might be that herpes virus mrnas are more highly structured, by virtue of their high g + c content. the unusual sensitivity of herpes virus mrnas to hypertonic stress is consistent with the hypothesis that they have extensive secondary structure. the irreversible (read and frenkel, 1983 ) early shutoff of host translation by a structural component of the herpes virion also seems to involve cleavage of host mrnas-enough to inactivate them for translation (fenwick and mcmenamin, 19841 , although they can still be detected by hybridization, more or less (nishioka and silverstein, 1978b; schek and bachenheimer, 1985) . since mutants that are defective in stageone shutoff can still induce secondary shutoff of host protein synthesis (read and frenkel, 19831 , two distinct viral gene products, either nucleases or activators thereof, are apparently involved. a herpes virus mutant that is defective in stage-one host shutoff is defective in switching off the translation of early viral mrnas as well (read and frenkel, 1983) . the differential accumulation of adenovirus early mrnas is also mediated, in part, by the regulated degradation of some transcripts (wilson and darnell, 1981) . degradation of host mrnas might be part of the mechanism by which vaccinia and influenza viruses reduce host translation (see table i ), although clear-cut genetic evidence, such as that described for herpes virus, is lacking in those systems. the extent to which gene expression is regulated by posttranslational proteolytic degradation is probably not fully appreciated. there are striking, isolated examples, for example, the selective degradation of measles virus m protein (sheppard et al., 19851 , the rapid turnover of some early adenovirus proteins (spindler and berk, 1984b) , and stabilization of the cellular protein p53 by its interaction with sv40 t antigen (oren et al., 1981) . given the intricacies of the ubiquitin pathway for proteolysis, it might be surprising were that pathway not perturbed by virus infection. some animal viruses might encode a function that protects foreign (i.e., viral) proteins from degradation, analogous to the pin function of bacteriophage t4 (simon et al., 1983) . although the pattern of codon usage in viral genes is sometimes different from that of the cellular genome, imbalances in the trna pool probably do not affect the yield of most viral proteins because the rate-limiting step is usually initiation rather than elongation. moreover, while there is convincing evidence for a preferred pattern of codon usage in highly expressed bacterial and yeast genes (bennetzen and hall, 1982; ikemura, 1981 ikemura, , 1982 , codon preference seems to be more relaxed in higher eukaryotes (tso et al., 19851 , and therefore the cellular trna pool might not be markedly skewed. consistent with the idea that codon usage is not a major regulatory factor in viral gene expression, the close conservation of amino acid sequences between some viruses is not always accompanied by conservation in the choice of codons (ou et al., 1982) . the degree to which expression might be limited by trna deficiencies has been tested in escherichia coli by using cloned genes that are rich in rare codons. the availability of trna was found to limit translation only when the mrna concentration was extraordinarily high (pedersen, 1984; robinson et al., 1984) . codon usage might regulate translation in more subtle ways, however. one possibility with some experimental justification is that ribosomes pause briefly at rare codons (lizardi et al., 1979; misra and reeves, 1985; varenne et al., 1984) . la discontinuous elongation is not incompatible with efficient translation, as pausing has been detected during the synthesis of some very abundant proteins (cepko and sharp, 1982; lizardi et al., 1979) . slowing translation in certain positions might facilitate folding of the polypeptide and/or its interaction with other components, however. the pattern of codon usage in the signal peptide portion of some genes encourages this notion (spieth et al., 1985) . the suppression of nonsense codons and the occurrence of frameshifting (see section ii,c) might also be facilitated by an imbalance between the cellular trna pool and the viral pattern of codon usage. 1* an alternative explanation for discontinuous elongation is that ribosomes pause when they encounter hairpin structures in the mrna, but that idea is without experimental support. what we have learned about the structure and function of animal virus mrnas can often be extrapolated to cellular mrnas. the mechanism of selecting the initiation site for protein synthesis certainly appears to follow a single formula. the translational machinery displays a certain flexibility (leaky scanning, frameshifting, etc.) that is exploited more frequently by viral than by cellular mrnas. that no (doubt reflects the limited coding capacity of most viral genomes. in contrast, it would seem easier and more efficient for the expansive cellular genome to separately encode two versions of a protein than to attempt to skirt the "monocistronic rule" in the ways described for viruses.lg it is important to remember that there are rules for breaking the monocistronic rule. using those principles, we can correctly predict the qualitative aspects of viral protein synthesis, with very few exceptions. we understand much less about the quantitative aspects of translation, however. although some of the parameters that determine efficiency have been identified in the preceding pages, or at least surmised, we usually cannot predict how efficiently a given mrna will be translated by summing the known parameters. future studies will almost certainly uncover other features that affect translational efficiency: "repressor" proteins, perhaps, or helix-unwinding proteins, or effects of 3'-noncoding sequences, or aspects of mrna primary and secondary structure that are not yet obvious. the suggestion that it is easier to block translation than to enhance it merits repetition. the most efficient mrnas might be those that cannot interact with regulatory rnas, proteins, etc. it is sometimes but not always true that viral mrnas are translated more efficiently than cellular mrnas. i persist in believing that many viruses inhibit cellular protein synthesis inadvertently, and gain little thereby. understanding the mechanism of host shutoff is nonetheless interesting. it might aid in designing virus vectors, and in our understanding of the conditions that promote persistent virus infections (ahmed and fields, 1982) . acknowledgments i thank many colleagues who kindly sent reprints and preprints, and especially those who gave me permission to cite their unpublished observations. in several instances the the 5'-terminal structure of the methylated mrna synthesized in vitro by vsv cellular protein synthesis shutoff by mengovirus: translation of nonviral and viral mrnas in extracts from uninfected and infected ascites tumor cells two forms of sv40 tantigen in abortive and lytic infection role of the s4 gene in the establishment of persistent reovirus infection in l cells reversion by hypotonic medium of the shutoff of protein synthesis induced by emc virus translation of capped viral mrnas in poliovirus-infected hela cells protein synthesis in hela cells double-infected with emc virus and poliovirus translation of capped virus mrna in emc virus-infected cells can acg serve as an initiation codon for protein synthesis in eukaryotic cells? sequences at both termini of the 10 genes of reovirus serotype 3 (dearing) the effect of infection with sindbis virus and its temperature sensitive mutants on cellular protein and dna synthesis sequencing studies of pichinde arenavirus s rna indicate a novel coding strategy, an ambisense viral s rna effect of adenovirus on metabolism of specific host mrnas: transport control and specific translational discrimination adenovirus type 5 early region l b gene product is required for efficient shutoff of host protein synthesis adenovirus e1b proteins are required for accumulation of late viral mrna and for effects on cellular mrna translation and transport polyriboadenylic acid preferentially inhibits in vitro translation of cellular compared to vaccinia virus mrnas inhibition of protein synthesis by vaccinia virus coupled transcription and translation in mammalian and avian cell-free systems the replication of picornaviruses expression from an internal aug codon of herpes simplex thymidine kinase gene inserted in a retrovirus vector the number of ribosomes on sv40 late 16s mrna is determined in part by the nucleotide sequence of its leader complete nucleotide sequence of alfalfa mosaic virus rna 3 sequence analysis of hepatitis a virus cdna coding for capsid proteins and rna polymerase direct mapping of adeno-associated virus capsid proteins b and c: a possible acg initiation codon structure of the fmdv translation initiation site and of the structural proteins uag readthrough during tmv rna translation: isolation and sequence of two trnastyr with suppressor activity from tobacco plants the molecular basis for differential translation of tmv rna in tobacco and wheat germ measles virus p gene codes for two proteins inhibition of hela cell protein synthesis during adenovirus infection regulatory mutants of polyoma virus defective in dna replication and the synthesis of early proteins solubilization of a protein synthesis inhibitor from vaccinia virions codon selection in yeast translational interference a t overlapping reading frames in prokaryotic mrna effect of the tripartite leader on synthesis of a nonviral protein in an adenovirus 5 recombinant poliovirus mutant that does not selectively inhibit host cell protein synthesis two small rnas encoded by epstein-barr virus can functionally substitute for the virus-associated rnas in the lytic growth of adenovirus 5 construction and analysis of additional adenovirus substitution mutants confirm the complementation of vai rna function by two small rnas encoded by epstein-barr virus structural requirements of adenovirus vai rna for its translation enhancement function differential inhibition of host cell rna synthesis in several picornavirus-infected cell lines effect of viral infection on host protein synthesis and mrna association with the cytoplasmic cytoskeletal structure intermolecular duplexes formed from polyadenylated vaccinia virus rna the 2.2 kb e1b mrna of human ad12 and ad5 codes for two tumor antigens starting at different aug triplets regulation of protein synthesis in hep2 cells and their cytoplasmic extracts after poliovirus infection globin mrnas are primers for the transcription of influenza viral rna in uitro both the 7-methyl and the 2'-o-methyl groups in the cap of mrna strongly influence its ability to act as primer for influenza virus rna transcription a transcript from the s segment of the germiston bunyavirus is uncapped and codes for the nucleoprotein and a nonstructural protein sequencing of coronavirus ibv genomic rna: three open reading frames in the 5' "unique" region of mrna d translation of poliovirus rna in uitro: changes in cleavage pattern and initiation sites by ribosomal salt wash initiation factor preparations from poliovirusinfected cells restrict translation in reticulocyte lysates the white pock mutants of rabbit poxvirus: in uitro translation of early host range mutant mrna synthesis of alphavirus-specified rna complex regulation of sv40 earlyregion transcription from different overlapping promoters three intergenic regions of coronavirus mouse hepatitis virus genome rna contain a sequence that is homologous to the 3'-end of the viral mrna leader sequence molecular cloning and complete sequence determination of rna genome of human rhinovirus type 14 permeabilization of cells during animal virus infection sodium ions and the shut-off of host cell protein synthesis by picornaviruses sequences of the s1 genes of the three serotypes of reovirus effects of adenovirus infection on rrna synthesis and maturation in hela cells sequence analysis of the viral core protein and membrane proteins of the flavivirus west nile virus primary structure of the west nile flavivirus genome region coding for all nonstructural proteins effect of poliovirus double-stranded rna on viral and host-cell protein synthesis translation of poliovirus rna in uitro: detection of two different initiation sites regulation of protein synthesis in vsvinfected cells by decreased initiation factor 2 activity assembly of adenovirus major capsid protein is mediated by a nonvirion protein differential translation in normal and adenovirus type 5-infected cells and cell-free systems two initiation sites for foot and mouth disease virus polyprotein in vivo synthesis and processing of sindbis virus nonstructural proteins in uitro overlapping of the vp2-vp3 gene and the vp1 gene in the sv4q genome transcription of vaccinia virus mrna coupled to translation in uitro in uitro translation of immediate early, early, and late classes of rna from vaccinia virus-infected cells discriminatory inhibition of protein synthesis in cell-free systems by vaccinia virus transcripts bacterial peptide chain release factors: conserved primary structure and possible frameshift regulation of release factor 2 identification of a unique guanine-7-methyltransferase in semliki forest virus infected cell extracts mechanism of interferon action: differential effect of interferon on the synthesis of sv40 and reovirus polypeptides in monkey kidney cells structure-function relationship of rous sarcoma virus leader rna a ribosome binding site sequence is necessary for efficient expression of the distal gene of a translationally-coupled gene pair nucleotide sequence of a viral rna fragment that binds to eukaryotic ribosomes sequence of the 3'-untranslated region of brome mosaic virus coat protein messenger rna genome expression of plant positive-strand rna viruses inhibition of mrna binding to ribosomes by localized activation of dsrna-dependent protein kinase in vitro synthesis of the nonstructural c protein of sendai virus translational specificity in reovirus-infected mouse fibroblasts gene organization of the transforming region of adenovirus type 7 dna initiation of translation of the cauliflower mosaic virus genome from a polycistronic mrna: evidence from deletion mutagenesis oligonucleotide directed mutagenesis of cauliflower mosaic virus dna using a repair-resistant nucleoside analogue identifies an agnogene initiation codon in uitro translation of poliovirus rna: utilization of internal initiation sites in reticulocyte lysate peptide maps and nterminal sequences of polypeptides from early region 1a of human adenovirus 5 catalytic utilization of eif-2 and mrna binding proteins are limiting in lysates from vsv infected l cells host range restriction of vaccinia virus in cho cells: relationship to shutoff of protein synthesis regulation of initiation factors during translational repression caused by serum depletion cellular levels and covalent modification of the subunits of the cap binding protein complex, eif-4f protein synthesis factors 4a and 4b are not altered by poliovirus infection of hela cells complete nucleotide sequence of bacteriophage t7 dna and the locations of t7 genetic elements functional characterization of eukaryotic mrna cap binding protein complex: effects on translation of capped and naturally uncapped rnas sequence relationship of glycosylated and unglycosylated gag polyproteins of moloney murine leukemia virus mapping the major transcripts of ground squirrel hepatitis virus: the presumptive template for reverse transcriptase is terminally redundant reovirus hemagglutinin mrna codes for two polypeptides in overlapping reading frames the complete sequence of the m rna of snowshoe hare bunyavirus reveals the presence of internal hydrophobic domains in the viral glycoprotein analyses of the mrna transcription processes of snowshoe hare bunyavirus s and m rna species human rhinovirus 14 infection of hela cells results in the proteolytic cleavage of the p220 cap binding subunit viral translation proteolysis of a 220,000 dalton polypeptide associated with eif3 and a cap binding protein complex early virion-associated suppression of cellular protein synthesis by herpes simplex virus is accompanied by inactivation of mrna suppression of the synthesis of cellular macromolecules by herpes simplex virus structural difference between the 5' termini of viral and cellular mrna in poliovirus-infected cells: possible basis for the inhibition of host protein synthesis effect of adenovirus infection on expression of human histone genes nucleotide sequence and genome organization of foot and mouth disease virus evidence against the role of k + in the shutoff of protein synthesis by vsv temporal regulation of baculovirus rna: overlapping early and late transcripts early and late functions in a bipartite rna virus: evidence for translational control by competition between viral mrnas bunyavirus nucleoprotein, n, and a nonstructural protein, nss, are coded by overlapping reading frames in the s rna reovirus messenger rna contains a methylated, blocked 5'-terminal structure: m7g(5')ppp(s')gmpcp mechanism of formation of reovirus mrna 5'-terminal blocked and methylated sequence, m7gpppgmpc 5'-terminal structure and mrna stability enhanced poliovirus replication in cytomegalovirus-infected human fibroblasts na+ and k + concentrations and the regulation of the interferon system in chick cells na+ and k + concentrations and the regulation of protein synthesis in sindbis virus-infected chick cells 5'-conformation of capped alfalfa mosaic virus rna 4 may reflect its independence of cap structure or of cap-binding protein for efficient translation sv40 early mrnas contain multiple 5'-termini upstream and downstream from a hogness-goldberg sequence; a shift in 5' termini during the lytic cycle is mediated by large t antigen heterogeneity and 5'4erminal structures of the late rnas of sv40 protein synthesis in lysates of aedes albopictus cells infected with vsv sendai virus contains overlapping genes expressed from a single mrna translational discrimination between the four rnas of alfalfa mosaic virus cap accessibility correlates with the initiation efficiency of amv rnas competition between cellular and viral mrnas in uitro is regulated by a messenger discriminatory initiation factor protein synthesis in cells infected with semliki forest virus is not controlled by intracellular cation changes new initiation factor activity required for globin mrna translation inhibition of dna-dependent transcription by the leader rna of vsv sv40 recombinant molecules express the gene encoding p21 transforming protein of harvey murine sarcoma virus sequence of the black beetle virus subgenomic rna and its location in the viral genome nucleotide sequence and genome organization of carnation mottle virus rna utilization of internal aug codons for initiation of protein synthesis directed by mrnas from normal and mutant genes encoding herpes simplex virusspecified thymidine kinase synthesis in vitro of a seven amino acid peptide encoded in the leader rna of rous sarcoma virus sodium and potassium transport in herpes simplex virus-infected cells adenovirus early region 4 encodes functions required for efficient dna replication, late gene expression, and host cell shutoff effects of deletions on expression of the hsv thymidine kinase gene from the intact viral genome: the amino terminus is dispensable for catalytic activity influenza virus mrnas are incomplete transcripts of the genome rnas attenuation of late sv40 mrna synthesis is enhanced by the agnoprotein and is temporally regulated in isolated nuclear systems large surface proteins of hepatitis v virus containing the pre-s sequence inhibition of host cell protein synthesis by uvinactivated poliovirus control of protein synthesis in extracts from poliovirus-infected cells isolation and preliminary characterization of temperature-sensitive mutants of poliovirus type 1 virion component of hsv type 1 kos interferes with early shutoff of host protein synthesis induced by hsv type 2 immunological detection of the mrna cap-binding protein site-specific recombination of bacteriophage a: dna sequence of regulatory regions and overlapping structural genes for int and xis characterization of the infections of permissive cells by host range mutants of vsv defective in rna methylation control of expression of the vaccinia virus thymidine kinase gene mapping and identification of the vaccinia virus thymidine kinase gene mutation of a termination codon affects src initiation alfalfa mosaic virus temperature sensitive mutants transcriptioncoupled translation of silkworm cytoplasmic polyhedrosis virus genomic rna in xenopus oocytes correlation between the abundance ofe. coli transfer rnas and the occurrence of the respective codons in its protein genes correlation between the abundance of yeast transfer rnas and the occurrence of the respective codons in protein genes inhibition of host protein synthesis and degradation of cellular mrnas during infection by influenza and hsv constitutive and conditional suppression of exogenous and endogenous genes by anti-sense rna expression of the b u s sarcoma virus pol gene by ribosomal frameshifting biosynthesis of reovirus-specified polypeptides: the reovirus sl mrna encodes two primary translation products biosynthesis of reovirus-specified polypeptides polypeptide cleavages in the formation of poliovirus proteins requirement for adenovirus dna-binding protein and va-i rna for production of adeno-associated virus polypeptides adeno-associated virus proteins: origin of the capsid components identification of the sv40 agnogene product: a dna binding protein further studies on the inhibition of cellular protein synthesis by vsv inhibition of host translation in emc virus-infected l cells: a novel mechanism comparison of initiation rates of emc virus and host protein synthesis in infected cells shutoff of hela cell protein synthesis by emc virus and poliovirus: a comparative study two initiation sites for translation of poliovirus rna in uitro: comparison of lsc and mahoney strains evidence for translational regulation of hsv type 1 gd expression restriction of vsv in a nonpermissive rabbit cell line is at the level of protein synthesis inhibition of cell functions by rna-virus infections lysis gene expression of rna phage ms2 depends on a frameshift during translation of the overlapping coat protein gene deletions of n-terminal sequences of polyoma virus t-antigens reduce but do not abolish transformation of rat fibroblasts role of the avian retrovirus mrna leader in expression: evidence for novel translation control metabolism and expression of rna polymerase i1 transcripts in influenza virus-infected cells nuclear-cytoplasmic transport and vai rna-independent translation of influenza viral mrnas in late adenovirusinfected cells translational control by influenza virus: suppression of the kinase that phosphorylates the alpha subunit of initiation factor eif-2 and selective translation of influenza viral mrnas identification of the components necessary for adenovirus translational control and their utilization in cdna expression vectors viral protein synthesis in barley protoplasts inoculated with native and fractionated brome mosaic virus rna primary structure, gene organization and polypeptide expression of poliovirus rna interferon regulates c-myc gene expression in daudi cells at the post-transcriptional level protein synthesis directed by the rna from a plant virus in a normal animal cell how do eucaryotic ribosomes select initiation regions in messenger rna? inability of circular mrna to attach to eukaryotic ribosomes migration of 40s ribosomal subunits on mrna when initiation is perturbed by lowering magnesium or adding drugs evaluation of the "scanning model" for initiation of protein synthesis in eucaryotes possible role of flanking nucleotides in recognition of the aug initiator codon by eukaryotic ribosomes mechanism of mrna recognition by eukaryotic ribosomes during intiation of protein synthesis analysis of ribosome binding sites from the sl message of reovirus: initiation a t the first and second aug codons comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles translation of insulin-related polypeptides from mrnas with tandemly reiterated copies of the ribosome binding site compilation and analysis of sequences upstream from the translational start site in eukaryotic mrnas selection of initiation sites by eucaryotic ribosomes: effect of inserting aug triplets upstream from the coding sequence for preproinsulin point mutations define a sequence flanking the aug initiator codon that modulates translation by eucaryotic ribosomes influences of mrna secondary structure on initiation by eucaryotic ribosomes migration of 40s ribosomal subunits on mrna in the presence of edeine identification of features in 5'-terminal fragments from reovirus mrna which are important for ribosome binding priming of influenza viral rna transcription by capped heterologous rnas relationship between membrane integrity and the inhibition of host translation in virus-infected mammalian cells characterization of leader 127, 359-366. virus-infected cells rna sequences on the virion and mrnas of mouse hepatitis virus, a cytoplasmic rna virus nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus synthesis of hepatitis b surface antigen in mammalian cells: expression of the entire gene and the coding region translation of adenovirus serotype 2 late mrnas influenza virus structural and nonstructural proteins in infected cells and their plasma membranes inactivation of cap-binding proteins accompanies the shut-off of host protein synthesis by poliovirus isolation and structural characterization of cap-binding proteins from poliovirus-infected hela cells poliovirus protease 3c (p3-7c) does not cleave p220 of the eucaryotic mrna cap-binding protein complex expression of vaccinia virus early mrnas in ehrlich ascites tumor cells. part of the polysomes at an early stage of virus infection are not bound to the cytoskeleton translation of cellular and viral early mrna in cell-free systems from uninfected and (vaccinia) virusinfected cells a t the early stage mrna discrimination in extracts from uninfected and reovirus-infected l-cells polyadenylic acid addition sites in the adenovirus type 2 major late transcription unit biochemistry of interferons and their actions the cytoskeletal framework and poliovirus metabolism molecular cloning and partial sequencing of hepatitis a viral cdna initiation of translation at internal aug codons in mammalian cells discontinuous translation of silk fibroin in a reticulocyte cell-free system and in intact silk gland cells poliovirus protease does not mediate cleavage of the 220,000-da component of the cap binding protein complex translational control of protein synthesis after infection by vsv vsv mrna and inhibition of translation of cellular mrna-is there a p function in vsv? adenovirus tripartite leader sequence enhances translation of mrnas late after infection eukaryotic ribosomes can recognize preproinsulin initiation codons irrespective of their position relative to the 5'-end of mrna the nonstructural proteins of sindbis virus as studied with an antibody specific for the c terminus of the nonstructural readthrough polyprotein differential inhibition of host protein synthesis in l cells infected with rna-temperature-sensitive mutants of vsv arrangment of late rnas transcribed from a 7.1-kb ecori vaccinia virus dna fragment construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus frameshift and intragenic suppressor mutations in a rous sarcoma provirus suggest src encodes two proteins suppression of a vp1 mutant of sv40 by missense mutations in serine codons of the viral agnogene virus-associated rnas of naturally occurring strains and variants of group c adenoviruses nucleotide sequence of the coat protein cistron and the 3'-noncoding region of cucumber green mottle mosaic virus rna influence of the host cell on proteins synthesized by different strains of influenza virus intermediates in the synthesis of tolc protein include an incomplete peptide stalled at a rare arg codon eukaryotic protein synthesis restricted translation of the genome of the flavivirus kunjin in vitro the conformation of adenovirus vai-rna in solution the host protein required for in vitro replication of poliovirus is a protein kinase that phosphorylates eif2 shutoff of host translation by emc virus infection does not involve cleavage of the eif4f polypeptide that accompanies poliovirus infection inhibition of hela cell protein synthesis by the vaccinia virion irreversible effects of cycloheximide during the early period of vaccinia virus replication formation of the guanylylated and methylated 5'-terminus of vaccinia virus mrna biosynthesis of reovirus-specified polypeptides. multiplication rate but not yield of reovirus serotypes 1 and 3 correlates with the level of virus-mediated inhibition of cellular protein synthesis the regulation of translation in reovirus-infected cells modification of membrane permeability during semliki forest virus infection cell-free synthesis of a precursor polyprotein containing both gag and pol gene products by rauscher murine leukemia virus 35s rna guanidine-sensitive na+ accumulation by poliovirus-infected hela cells adenovirus gene expression: control at multiple steps of mrna biogenesis the complete sequence of the chicken 61 crystallin gene and its 5' flanking region alterations in the protein synthetic apparatus of friend erythroleukemia cells infected with vsv or herpes simplex virus requirement of protein synthesis for the degradation of host mrna in friend erythroleukemia cells infected with hsv type 1 unusual features of the leader sequence of rous sarcoma virus packaging mutant tk15 the 5'-end of poliovirus mrna is not capped with m'g(5')ppp(s') complete nucleotide sequence of the attenuated poliovirus sabin 1 strain genome control of transcription of the reovirus genome vaccinia virusinduced changes in "a+] and [ k + ] in hela cells selective blockage of initiation of host protein synthesis in rna-virus-infected cells a mechanism for the control of protein synthesis by adenovirus va rnal on the regulation of protein synthesis in vaccinia virus infected cells a joint product of the genes gag and pol of avian sarcoma virus: a possible precursor of reverse transcriptase post-translational regulation of the 54k cellular tumor antigen in normal and transformed cells the influence of the host cell on the inhibition of virus protein synthesis in cells doubly infected with vsv and mengovirus sequence studies of several alphavirus genomic rnas in the region containing the start of the subgenomic rna characterization of a ts mutant of vaccinia virus 25,422-426. novel function that prevents virus-induced breakdown of rna hepatitis b virus genes and their expression in e. coli characterization of lacrosse virus smallgenome transcripts multiple leader rnas and mrnas are transcribed from the lacrosse virus small genome segment identification of four complementary rna species in akabane virus-infected cells the reoviridae methylmercury hydroxide enhancement of translation and transcription of ovalbumin and conalbumin mrnas e . coli ribosomes translate in viuo with variable rate leaky uag termination codon in tobacco mosaic virus rna characteristics of a coupled cellfree transcription and translation system directed by vaccinia cores insertion mutagenesis to increase secondary structure within the 5'moncoding region of a eukaryotic mrna reduces translational efiiciency evidence for the presence of an inhibitor on ribosomes in mouse l cells infected with mengovirus regulation of herpesvirus macromolecular synthesis. properties of a polypeptides made in hsv-1 and hsv-2 infected cells utilization of messenger in adenovirus-2-infected cells at normal and elevated temperatures a frameshift mutation in the pre-s region of the human hepatitis b virus genome allows production of surface antigen particles but eliminates binding to polymerized albumin characterization of ribosome binding on rous sarcoma virus rna in uitro translation of mulv and msv rnas in nuclease-treated reticulocyte extracts: enhancement of the gag-pol polypeptide with yeast suppressor trna proteins specified by avian erythroblastosis virus: coding region localization and identification of a previously undetected erb-b polypeptide molecular cloning of poliovirus cdna and determination of the complete nucleotide sequence of the viral genome cell-free translation of 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of adenovirus 2 early region 1a mrnas is non-sequential a cell-free model of the emc virus-induced inhibition of host cell protein synthesis two forms of purified m7g-cap binding protein with different effects on capped mrna translation in extracts of uninfected and poliovirus-infected hela cells the location of v-src in a retrovirus vector determines whether the virus is toxic or transforming adenovirus vai rna is required for efficient translation of viral mrnas a t late times after infection the hepatitis b virus complete nucleotide sequences of all three poliovirus serotype genomes isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cdnas: genomic complexity and molecular evolution of the gene shutoff of neuroblastoma cell protein synthesis by semliki forest virus: loss of ability of crude initiation factors to recognize early sfv and host mrnas infection of neuroblastoma cells by sfv translation is a nonuniform process. effects of trna availability on the rate of elongation of nascent polypeptide chains translational control of early protein synthesis at the late stage of vaccinia virus infection mutational alterations within the sv40 leader segment generate altered 16s and 19s mrnas individual hsv transcripts: characterization of specific genes. jn "the herpesviruses nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1 the role of mrna competition in regulating translation a single uga codon functions as a natural termination signal in the coliphage q p coat protein cistron disruption of the three cytoskeletal networks 199-203 marilyn kozak in mammalian cells does not affect transcription, translation, or protein translocation changes induced by heat shock protein synthesis in bhk-21 cells infected with semliki forest virus characterization of a specific kinase inhibitory factor produced by vaccinia virus which inhibits the interferon-induced protein kinase nucleotide sequence of an immediateearly frog virus 3 gene macromolecular synthesis in cells infected by frog virus 3 further genetic localization of the transforming sequences of the p21 v-ras gene of harvey murine sarcoma virus control of mrna concentration by differential cytoplasmic half-life evidence that a g u a u e a and ccaagmga initiate translation in the same mrna in region e3 of adenovirus differential phosphorylation of soluble versus ribosome-bound eif2 in the ehrlich ascites tumor cell resistance to inhibitors of mammalian cell protein synthesis induced by preincubation in hypertonic growth medium murine leukemia virus protease is encoded by the gag-pol gene and is synthesized through suppression of an amber termination codon translational readthrough of an amber termination codon during synthesis of feline leukemia virus protease splicing in adenovirus and other animal viruses s. cereuisiae ribosomes recognize non-aug initiation codons studies on the intracellular synthesis of reovirus-specified proteins key: cord-000264-o80duxhs authors: chandramouli, kondethimmanahalli; qian, pei-yuan title: proteomics: challenges, techniques and possibilities to overcome biological sample complexity date: 2009-12-08 journal: hum genomics proteomics doi: 10.4061/2009/239204 sha: doc_id: 264 cord_uid: o80duxhs proteomics is the large-scale study of the structure and function of proteins in complex biological sample. such an approach has the potential value to understand the complex nature of the organism. current proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. advances in protein fractionation and labeling techniques have improved protein identification to include the least abundant proteins. in addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. however, the major limitation of proteomic investigations remains the complexity of biological structures and physiological processes, rendering the path of exploration paved with various difficulties and pitfalls. the quantity of data that is acquired with new techniques places new challenges on data processing and analysis. this article provides a brief overview of currently available proteomic techniques and their applications, followed by detailed description of advantages and technical challenges. some solutions to circumvent technical difficulties are proposed. the term proteomics describes the study and characterization of complete set of proteins present in a cell, organ, or organism at a given time [1] . in general, proteomic approaches can be used (a) for proteome profiling, (b) for comparative expression analysis of two or more protein samples, (c) for the localization and identification of posttranslational modifications, and (d) for the study of protein-protein interactions. the human genome harbors 26000-31000 protein encoding genes [2] ; whereas the total number of human protein products, including splice variants and essential posttranslational modifications (ptms), has been estimated to be close to one million [3, 4] . it is evident that most of the functional information on the genes resides in the proteome, which is the sum of multiple dynamic processes that include protein phosphorylation, protein trafficking, localization, and protein-protein interactions [5] . moreover, the proteomes of mammalian cells, tissues, and body fluids are complex and display a wide dynamic range of proteins concentration one cell can contain between one and more than 100000 copies of a single protein [6] . in spite of new technologies, analysis of complex biological mixtures, ability to quantify separated protein species, sufficient sensitivity for proteins of low abundance, quantification over a wide dynamic range, ability to analyze protein complexes, and high throughput applications is not yet fulfilled [7] . biomarker discovery remains a very challenging task due to the complexity of the samples (e.g., serum, other bodily fluids, or tissues) and the wide dynamic range of protein concentrations [8] . most of the serum biomarker studies performed to date seem to have converged on a set of proteins that are repeatedly identified in many studies and that represent only a small fraction of the entire blood proteome [9] . processing and analysis of proteomics data is indeed a very complex multistep process [10, 11] . the consistent and transparent analysis of lc/ms and lc-ms/ms data requires multiple stages [12] , and this process remains the main bottleneck for many larger proteomics studies. to overcome these issues, effective sample preparation (to reduce complexity and to enrich for lower abundance components while depleting the most abundant ones), state-of-the-art mass spectrometry instrumentation, and extensive data processing and data analysis are required. a wide range of proteomic approaches are available such as gel-based applications include one-dimensional and twodimensional polyacrylamide gel electrophoresis [13, 14] , and gel-free high throughput screening technologies are equally available, including multidimensional protein identification technology [15] , isotope-coded affinity tag icat [16] ; silac [17] ; isobaric tagging for relative and absolute quantitation (itraq) [18] . shotgun proteomics [19] and 2de dige [20] as well as protein microarrays [21, 22] are applied to obtain overviews of protein expression in tissues, cells, and organelles. large-scale western blot assays [23] , multiple reaction monitoring assay (mrm) [24] , and label-free quantification of high mass resolution lc-ms data [25] are being explored for high throughput analysis. many different bioinformatics tools have been developed to aid research in this field such as optimizing the storage and accessibility of proteomic data or statistically ascertaining the significance of protein identifications made from a single peptide match [26] . in this review we attempt to provide a overview of the major developments in the field of proteomics, some success stories as well as challenges that are currently being faced. about 20-30% of all genes in an organism encode integral membrane proteins, which are involved in numerous cellular processes [27] . membrane proteins constitute 30% of the typical proteome, yet their propensity to aggregate and precipitate in solution confounds their analysis [28] . the target residues for tryptic cleavage (i.e., lysine and arginine) are mainly absent in transmembrane helices and preferentially found in the hydrophilic part of these lipid bilayer-incorporated proteins. because of the protein aggregation step of ief, 2de is unsuitable for the separation of integral membrane proteins and is limited to detection of membraneassociated proteins and membrane proteins with a low hydrophobicity [29] . membrane solubilization methods have been deployed to analyze enriched membrane fractions and address the solubility issue by using detergents [30] , organic solvents [31] , and organic acids [32] compatible with subsequent proteolytic digestion/chemical cleavage, separation and analysis by lc/ms. in this approach, (1) an enriched yeast membrane fraction is solubilized with 90% formic acid in the presence of cyanogens bromide. the concentrated organic acid provides the solubilization agent, and cyanogen bromide, functional under acidic conditions, allows many embedded membrane proteins to be cleaved, (2) a membrane-enriched microsomal fraction is solubilized by boiling in 0.5% sds and, following isotope-coded affinity tag (icat) labeling, is diluted to reduce the concentration of sds, and (3) by using an enriched membrane sample, the proteins are thermally denatured and sonicated in 60% organic solvent (methanol) in the presence of trypsin. the resultant peptide mixture is then analyzed by lc/ms. all three of these methods are effective and optimize the identifications of membrane proteins. another method using high ph and protenase k is optimized specifically for the global analysis of both membrane and soluble proteins [33] . high ph favors the formation of membrane sheets, while proteinase k cleaves exposed hydrophilic domains of membrane proteins. commercially available nonionic detergents, dodecyl maltoside, and decaethylene glycol mono hexadecyl are proved most efficient membrane protein solubilizers [34] . another more successful approach to isolate membrane proteins relies on cell surface labeling in combination with high resolution two-dimensional (2d) lc-ms/ms [35] . in addition, improved analytical tools should be developed, that is, multidimensional liquid chromatography of peptide mixtures generated from membrane proteins, nanoflow chromatographic techniques for hydrophobic transmembrane peptides, and native electrophoresis of membrane protein complexes, which, in combination with mass spectrometry, should lead to the identification of the majority of proteins in the membrane proteome of simple microorganisms. it is important to quantify not only the identified membrane proteins but also to determine the levels of interacting partners. subcellular fractionation techniques that employ a combination of centrifugation steps are a common choice for preparing plasma membrane-(pm-) enriched fractions including detergent-resistant membrane fractions, commonly known as lipid rafts. these methods can offer a significant improvement in specificity for pm proteins over approaches that do not perform any subcellular fractionation, but rather use whole-cell or tissue preparations [36] . chemical-tagging methods [37] have been a more applied technique used to enrich for pm proteins and are often used in conjunction with physical separation strategies. this method allows for a specific class of protein or modification of interest to be physically separated from other nontagged proteins. importantly, when chemical tags are attached to the extracellular domain of pm proteins on intact cells, they offer an unrivaled specificity for pm proteins, because they offer a manner to distinguish true pm proteins from intracellular contaminants. cell-surface biotinylation, the covalent attachment of a biotin tag to the extracellular domain of pm proteins, is also a popular choice [38] [39] [40] . serum is a complex body fluid, containing a large diversity of proteins. more than 10000 different proteins are present in the human serum and many of them are secreted or shed by cells during different physiology or pathology processes [41] . serum is expected to be an excellent source of protein biomarkers because it circulates through, or comes in contact all tissues. consequently, serum proteomics has raised great expectations for the discovery of biomarkers to improve diagnosis or classification of a wide range of diseases, including cancers [42] . however, serum has been termed as the most complex human proteome [43] with considerable differences in the concentrations of individual proteins, ranging from several milligrams to less than one pictogram per milliliter [44] . the analytical challenge for biomarker discovery arises from the high variability in the concentration and state of modification of some human plasma proteins between different individuals [45] . albumin is a protein of very high abundance in serum (35-50 mg/ml) that would be a prime candidate for complete selective removal prior to performing a proteomic analysis of lower abundance proteins. thus, removal of albumin from serum may also result in the specific removal of low abundance cytokines, peptide hormones, and lipoproteins of interest. immunoglobulins, and antibodies are also abundant proteins in serum that function by recognizing "foreign" antigens in blood and initiating their destruction [46] . the presence of higher abundance proteins interferes with the identification and quantification of lower abundance proteins (lower than ng/ml in serum). complexity and dynamic range of protein concentrations can be addressed with a combination of prefractionation techniques that deplete highly abundant proteins and fractionate. heparin chromatography coupled with protein g appears to be an efficient and economical strategy to pretreat serum for serum proteomics [47] . protein prefractionation by immunodepletion and reversed-phase separation of the depleted plasma on mrp-c18 column provide methods compatible with lc-ms-based analysis. a polyclonal antibody-based system to rapidly deplete multiple high abundant proteins in serum, plasma, csf, and other biological fluids. individual antibody materials are mixed in selected percentages and packed into a column format. albumin can be removed by immunoaffinity columns [48] , isoelectric trapping [49] , dye-ligand chromatography [50] , and peptide affinity chromatography [51] . another approach involves the removal of igg by affinity chromatography using immobilized protein a or protein g [52] . a recently developed depletion method that mixes 6 high-specificity polyclonal antibodies (mars) to remove the top 6 proteins in a single purification step is commercially available [53] . human-14 multiple affinity removal column depletes the top 14 abundant proteins from human serum, plasma, csf, and other biological fluids. to address 2d limitations several types of mass spectrometry, in conjunction with various separation and analysis methods, are increasingly being adopted for proteomic measurements [54] . in contrast, 2d-page analysis, seldi-tof ms is a rather new method which is especially valuable for the identification of serum-derived biomarkers [55] . this method is based on proteinchip arrays which carry various chromatographic properties, such as anion exchange, cation exchange, and hydrophilic or hydrophobic surfaces [56] . for the analysis of serum, only 5-10 μl of serum sample is applied to these surfaces; after washing off unbound material, the protein fingerprint can be determined and visualized by time-of-flight mass spectrometry. the advantages of this method are the low amount of sample necessary for analysis, its speed, and high throughput capability. many different groups have used this method and related methods based on prefractionation of serum proteins by beads and subsequent maldi analysis for the identification of biomarkers in serum, urine, pancreatic juice, and other biological fluids [57] . the necessity of this removal or separation is also illustrated that many proteins found useful as biomarkers [58] . different fractionation steps (such as electrophoresis, seldi, and liquid chromatography) have been developed to reduce the complexity of serum proteome and to allow the detection and the identification of single proteins [59] . 2de and maldi ms had applied to identify candidate biomarkers at early and late stages of lung cancer disease. this method identified 46 proteins in tumor bearing mice this included disease regulated expression of orosomucoid-8, a-2-macroglobulin, apolipoprotein-a1, apolipoprotein-c3, glutathione peroxidase-3, plasma retinol-binding protein, and transthyretin [60] . recently 1065 proteins were identified by stable isotope labeled proteome (silap) standard coupled with extensive multidimensional separation with tandem mass spectrometry of which 121 proteins were present at 1.5-fold or greater concentrations in the sera of patients with pancreatic cancer [61] . specimen collection (blood, serum, plasma samples) is an integral component of clinical research. access to high-quality specimens, collected and handled in standardized ways that minimize potential bias or confounding factors, is key to the "bench to bedside" aim of translational research [62] . variables that may impact analytic outcomes include (1) the type of additive in the blood collection tubes; (2) sample processing times or temperatures; (3) hemolysis of the sample; (4) sample storage parameters; (5) the number of freeze-thaw cycles [63, 64] . the key variable in any analysis is that the case and control samples are handled in the exact same manner throughout the entire analytical process from study design and collection of samples to data analysis [63, 65] . these types of differences between samples could have a significant impact on the stability of proteins or other molecules of interest in the specimens. small differences in the processing or handling of a specimen can have dramatic effects in analytical reliability and reproducibility, especially when multiplex methods are used. a representative working group, standard operating procedures internal working group, comprised of members from across early detection research network should be formed to develop standard operating procedures (sops) for various types of specimens collected and managed for biomarker discovery and validation work. limitations of two-dimensional electrophoresis. figure 1 gives the general work flow in proteomics and table 1 addresses their strengths and limitations. two-dimensional electrophoresis (2de) was developed two decades before the term proteomics was coined [66, 67] . the 2de entails the separation of complex protein mixtures by molecular charge in the first dimension and by mass in the second dimension. 2de analysis provides several types of information about the hundreds of proteins investigated simultaneously, including molecular weight, pi and quantity, as well as possible posttranslational modifications. 2de is extensively used but mostly for qualitative experiments and this method falls short in its reproducibility, inability to detect low abundant and hydrophobic proteins, low sensitivity in identifying proteins with ph values too low (ph < 3) or too high (ph > 10) and molecular masses too small (mr < 10 kd) or too large (mr > 150 kd) [2] [3] [4] [5] . poor separations of basic proteins due to "streaking" of spots and membrane proteins resolution [68] are limiting factors in 2de. however, 2de is the only technique that can be routinely applied for parallel quantitative expression profiling of complex protein mixtures such as whole cell and tissue lysates [69] and most widely used method for efficiently separating proteins, their variants and modifications (up to 15000 proteins). there are two ways to study posttranslational modifications by means of 2de. first, posttranslational modifications that alter the molecular weight and or pi of a protein are reflected in a shift in location of the corresponding protein spot on the proteomic pattern. second, in combination with western blotting, antibodies specific for posttranslational modifications can reveal spots on 2de patterns containing proteins with these modifications [70] . protein extraction and solubilization are key steps for proteomic analysis using 2de, highly hydrophobic proteins tend to precipitate during isoelectro focusing (ief), low copy number and the insolubility of transmembrane proteins renders quantitative analysis of these peptides and polypeptides are very challenging [71] . in order to enhance protein extraction and solubilization, different treatments and conditions are necessary to efficiently solubilise different types of protein extracts [72, 73] . the major challenge for protein visualization in 2de is the compatibility of sensitive protein staining methods with mass spectrometric analysis. therefore, several fluorescent staining methods have been developed for the visualization of 2de patterns, including sypro stainings and cy-dyes [74] . although sypro ruby [75] and silver staining [76, 77] have a comparable sensitivity, sypro ruby staining allows much higher reproducibility, a significantly wider dynamic range and less false-positive staining. in addition, sypro ruby allows for the detection of lipoproteins, glycoproteins, metalloproteins, calcium-binding proteins, fibrillar proteins, and low molecular weight proteins that are less "stainable" using other methods. finally, a large number of protein spots on 2de patterns contain several proteins with a similar pi. a ph gradient with a narrow range allows zooming into different proteins with the same molecular weight. increased separation distance 40 × 40 cm gels using ca-ief [78] could increase the proteome coverage up to 5000 proteins. use of overlapping narrow range ipgs "zoom" gels and increase in separation area could yield better membrane protein separation [79] . this technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. proteins can also be fluorescently labelled with cy2, cy3, or cy5 prior to 2de [80] . cydyes are cyanine dyes carrying an n-hydroxysuccinimidyl ester reactive group that covalently binds the e-amino group of lysine residues in proteins. during dige [81] , proteins in each of up to 3 samples can be labelled with one of these fluorescent dyes, and the differentially labelled samples can be mixed and loaded together on one single gel, allowing the quantitative comparative analysis of three samples using a single gel ( figure 2 ). the dige technique has exhibited higher sensitivity as well as linearity, eliminated postelectrophoretic processing (fixing and destaining) steps and enhanced reproducibility by directly comparing samples under similar electrophoretic conditions [81, 82] . the resulting images are then analyzed by software such as de-cyder which are specifically designed for 2d-dige analysis [83] . the major advantages of 2d-dige are the high sensitivity and linearity of its dyes, its straightforward protocol, as well as its significant reduction of intergel variability, increasing the possibility to unambiguously identify biological variability, and reducing bias from experimental variation. moreover, the use of a pooled internal standard, loaded together with the control and experimental samples, increases quantification accuracy and statistical confidence [84] . the dige technique has dramatically improved the reproducibility, sensitivity, and accuracy of quantitation; however, its labeling chemistry has some limitations; proteins without lysine cannot be labeled, and they require special equipment for visualization, and fluorophores are very expensive [83, 85] . tag (icat) . gel-free, or ms based, proteomics techniques are emerging as the methods of choice for quantitatively comparing proteins levels among biological proteomes, since they are more sensitive and reproducible than two-dimensional gel-based methods. icat is one of the most employed chemical isotope labeling methods and the first quantitative proteomic method to be based solely on using ms [86, 87] . each icat reagent consists of three essential groups: a thiol-reactive group, an isotope-coded light or heavy linker, and a biotin segment to facilitate peptide enrichment. in an icat experiment, protein samples are first labeled with either light or heavy icat reagents on cysteine thiols. the mixtures of labeled proteins are then digested by trypsin and separated through a multistep chromatographic separation procedure. peptides are identified with tandem ms, and the relative quantifications of peptides are inferred from the integrated lc peak areas of the heavy and light versions of the icat-labeled peptides [88] . the icat concept has been widely used after its introduction [89] [90] [91] . different software programs were developed to analyze icat labeled ms data (e.g., proicat from applied biosystems, spectrum mill from agilent technologies, and sashimi from the institute of system biology [92] ). icat is extremely helpful to detect peptides with low expression levels, which is one of the bottleneck issues in analytic protein techniques [93, 94] . however, major limitations of this technique include selective detection of proteins with high cysteine content and difficulties in the detection of acidic proteins [95, 96] . the methods for direct comparison of dige and icat for the identification and quantification of proteins in complex biological mixtures are also being considered [97] . while the icat reagent only interacts with the free sulfhydryl of homocysteine and 8% protein is noncysteine, the silac has emerged as a valuable proteomic technique [98] which becomes more common for cell types and have been applied in many fields [99] [100] [101] . the silac technique can be effectively expanded to compare the differential expression levels of tissue proteome at different pathological states, which allows to identify new candidate biomarkers [102] . compared with the icat, a popular in vitro labeling, silac as an example of in vivo coding requires no chemical manipulation, and there is very little chemical difference between the isotopically labeled amino acid and its naturally occurring counterpart [103] . in addition, the amount of labeled proteins requires for analysis using silac technique is far less than that with icat. therefore, the silac-based method has broadly applied in many areas of cell biology and proteomics. except that the silac-based quantitative method is powerful in comparative/differential proteomics, it has been widely used in analyzing protein posttranslational modification, such as protein phosphorylation, detection of protein-protein or peptide-protein interactions and investigating signal transduction pathways [104, 105] .though there are numerous advantages for using silac-based methods compared to chemical labeling, a major drawback of silac is that it cannot be applied to tissue protein analysis directly. to overcome this shortcoming, silac has been successfully applied to tissue proteome based on 15 n isotope labeling [106] . microorganisms such as malaria parasite can be labeled with isoleucine [107] . latterly the culturederived isotope tags (cdits) method was developed as an alternative quantitative approach for studying the proteome of mammalian tissues based on the application of silac [108] . 18o stable isotope labeling. differential 16o/18o coding relies on the 18o exchange that takes place at the cterminal carboxyl group of proteolytic fragments, where two 16o atoms are typically replaced by two 18o atoms by enzyme-catalyzed oxygen exchange in the presence of h218o [109] . the resulting mass shift between differentially labeled peptide ions permits identification, characterization, and quantitation of proteins from which the peptides are proteolytically generated. in contrast to icat, 18o labeling does not favor peptides containing certain amino acids (e.g., cysteine), nor does it require an additional affinity step to enrich for these peptides [110] . unlike itraq, 16o/18o labeling does not require a specific ms platform nor does it depend on fragmentation spectra (ms2) for quantitative peptide measurements. it is amenable to the labeling of human specimens (e.g., plasma, serum, tissues), which represents a limitation of metabolic labeling approaches (e.g., silac). taken together, recent advancements in the homogeneity of 18o incorporation, improvements made on algorithms employed for calculating 16o/18o ratios and the inherent simplicity of this technique should result in increased use of 18o labeling [111] . in general, 18o labeling suffers from two potential drawbacks, inhomogeneous 18o incorporation and inability to compare multiple samples within a single experiment. a dual 18o labeling using a non-gel-based platform has been developed to overcome the major problems of existing proteolytic 18o labeling methods [112] . (itraq). the itraq reagent is well known for relative and absolute quantitation of proteins. the itraq technology offers several advantages, which include the ability to multiplex several samples, quantification, simplified analysis and increased analytical precision and accuracy [113] [114] [115] . the interest of this multiplexing reagent is that 4 or 8 analysis samples [116] can be quantified simultaneously. in this technique, the introduction of stable isotopes using itraq reagents occurs on the level of proteolytic peptides ( figure 3 ). this technology uses an nhs ester derivative to modify primary amino groups by linking a mass balance group (carbonyl group) and a reporter group (based on n-methylpiperazine) to proteolytic peptides via the formation of an amide bond [117] . due to the isobaric mass design of the itraq reagents, differentially labelled peptides appear as a single peak in ms scans, reducing the probability of peak overlapping. when itraq-tagged peptides are subjected to ms/ms analysis, the mass balancing carbonyl moiety is released as a neutral fragment, liberating the isotope-encoded reporter ions which provides relative quantitative information on proteins. an inherent drawback of the reported itraq technology is due to the enzymatic digestion of proteins prior to labelling, which artificially increases sample complexity and this approach needs a powerful multidimensional fractionation method of peptides before ms identification. prefractionation of proteins based on electrokinetic methodologies in free solution essentially relaying on the isoeletric focusing (ief) has gained wide acceptance. many commercial devices are now constructed to take the advantage of this principle ( table 2 ). reproducible fractionation steps will break down the sample complexicity while concentrating low abundant species, resulting in more confident protein identifications and quantification by 2d gels, mass spectrometry, and protein arrays. a good example of a innovation is liquid-phase isoelectric focusing (ief) as a prefractionation tool before the first dimension of 2d gel electrophoresis [118, 119] . for more consistent pi separation, the zoom ief fractionator [120] and multicompartment electrolyser (mce) [121] are being used to prefractionate the proteins. the fractionated samples can be directly applied on standard narrow range ipg strips for 2d electrophoresis. this allows at least 10000 to 15000 separate proteins to be analyzed, including proteins of very low abundance. ief, a highresolution electrophoresis technique, has been widely used in shotgun proteomic experiments [122] . ief runs in a buffer-free solution containing carrier ampholytes or in immobilized ph gradient (ipg) gels. the use of ipg-ief for the separation of complex peptide mixtures has been applied to the analysis of plasma and amniotic fluid [123, 124] as well as to bacterial material [125] . the ipg gel strip is divided into small sections for extraction and cleaning up of the peptides. this technique recovers the sample from the liquid phase and was demonstrated to be of great interest in shotgun proteomics [126] . ief is not only a high resolution and high capacity separation method for peptides, it also provides additional physicochemical information like their isoelectric point [127, 128] . the pi value provided is used as an independent validating and filtering tool during database search for ms/ms peptide sequence identification [129] . the recent introduction of commercially available offgel fractionator system by agilent technologies provides an efficient and reproducible separation technique [130] . this separation is based on immobilized ph gradient (ipg) strips and permits to separate peptides and proteins according to their isoelectric point (pi) but is realized in solution [131] . its micropreparative scale provides fraction volumes large enough to perform subsequent analyses as reverse phase (rp)-liquid chromatography (lc)-maldi ms/ms. the combined use of itraq labeling and offgel fractionation methods for the proteomic study of complex sample is also being considered [132, 133] . in this procedure, a large well is used to separate the sample by page and lanes are created on the membrane containing immobilized protein with the use of a manifold [134] . compatible combinations of primary antibodies are predetermined, with the criterion of being able to identify proteins that do not comigrate. different combinations of primary antibodies are added to each well, with appropriate dilutions of each primary antibody so that expressed proteins are detected in a single condition. the scalability of the system depends on defining suitable combinations of primary antibodies, with up to 1000 antibodies in 200 lanes being used in the largest screens. detection software is used to identify proteins based on their expected and observed gel mobility. unlike 2d page and hplc-ms/ms, large-scale western blotting only identifies proteins for which antibodies are already available. while this is not an appropriate screen for identifying uncharacterized proteins, it greatly simplifies the verification and functional analyses of proteins that are detected. in addition, this approach is highly flexible, and can be focused to particular sets of proteins or protein function, such as cell signaling molecules. importantly, the foundation of this approach is the large amount of data on individual antibodies, which are already available and characterized in the literature [135] . another approach to analyse proteomes without gels is "shotgun" analysis using mudpit [136] . in the mudpit approach, protein samples are subject to sequencespecific enzymatic digestion, usually with trypsin and endoproteinase lysc, and the resultant peptide mixtures are separated by strong cation exchange (scx) and reversed phase (rp) high performance liquid chromatography (hplc) [137, 138] . peptides from the rp column enter the mass spectrometer and ms data is used to search the protein databases [138] . the mudpit technique generates an exhaustive list of proteins present in a particular protein sample, it is fast and sensitive with good reproducibility however, it lacks the ability to provide quantitative information [139] [140] [141] . a combination of hplc, liquid phase isoelectric focusing, and capillary electrophoresis provides other multimodular options for the separation of complex protein mixtures [142] . high throughput production of human proteins using different methods is being developed to make protein array approach more practical. recently simple and efficient production of human proteins using the versatile gateway vector system has been developed [143] . in this approach, protein expression system is applied to the in vitro expression of 13364 human proteins and assessed their biological activity in two functional categories and developed "human protein factory" infrastructure which includes the resources and expression technology for in vitro proteome research. in another approach, dna array to protein array (dapa) is utilized, which allows the "printing" of replicate protein arrays directly from a dna array template using cellfree protein synthesis [144] . based on the nucleic acid programmable protein array (nappa) concept, high-density self-assembling protein microarray is developed to display thousands of proteins that are produced and captured in situ from immobilized cdna templates [145] . this method will enable various experimental approaches to study protein function in high throughput. the adventage of protein-based microarrays allows the global observation of biochemical activities on an unprecedented scale, where hundreds or thousands of proteins can be simultaneously screened for protein-protein, proteinnucleic acid, and protein-small molecule interactions, as well as posttranslational modifications [146, 147] . the microarray format provides a robust and convenient platform for the simultaneous analysis of thousands of individual protein samples, facilitating the design of sophisticated and reproducible biochemical experiments under highly specific conditions [148] . the principal challenges in protein array development are 3-fold: (1) creation of a comprehensive expression clone library; (2) high-throughput protein production, including expression, isolation, and purification; (3) adaptation of dna microarray technology to accommodate protein substrates [149] . functional protein microarrays differ from analytical arrays in that functional protein arrays are composed of arrays containing fulllength functional proteins or protein domains (figure 4) . these protein chips are used to study the biochemical activities of an entire proteome in a single experiment. they are used to study numerous protein interactions, such as protein-protein, protein-dna, protein-rna, proteinphospholipid, and protein-small molecule interactions [150, 151] . companies have introduced protein arrays aimed not only at proteomic analysis but also functional analyses of proteins (e.g., biacore ab, ciphergen biosystems inc., phylos inc.). affinity proteomics aim to produce antibodies to every protein expressed by the human genome and these will be characterized against purified antigens and tested on tissue arrays to collect information about their specificity for tissue antigens [152] . companies are focused to produce various binding partners, for example, affibodies, monoclonal antibodies, and their fragments [153] . protein chips will likely be the next major manifestation of the revolution in proteomics and offer another solution to analyze low abundant proteins and have the potential for high throughput applications to identify biomarkers [154] . protein chips differ from previously described methods; whereas screening by 2de or lc ms/ms can potentially detect any protein, and protein chips can only provide data on set of proteins selected by the investigator [155] . the development and application of high throughput, multiplex immunoassays that measure hundreds of known proteins in complex biological matrices, is becoming a significant tool for quantitative proteomics studies, diagnostic discovery, and biomarker-assisted drug development. two broad categories of antibody microarray experimental formats have been developed [156] , direct labelling, single antibody experiments [157] , dual antibody, sandwich immunoassays are described [158, 159] . in the direct labelling method, all proteins in a complex mixture are tagged, providing a means for detecting bound proteins following incubation on an antibody microarray. in the sandwich immunoassay format, proteins captured on an antibody microarray are detected by a cocktail of detection antibodies, each antibody matched to one of the spotted antibodies. in addition, a variety of microarray substrates have been described, including nylon membranes, plastic microwells, planar glass slides, gel-based arrays and beads in suspension arrays. much effort has been expended in optimizing antibody attachment to the microarray substrate. finally, various signal generation and signal enhancement strategies have been employed in antibody arrays, including colorimetry, radioactivity, fluorescence, chemiluminescence, quantum dots and other nanoparticles, enzyme-linked assays, resonance light scattering, tyramide signal amplification, and rolling circle amplification. each of these formats and procedures has distinct advantages and disadvantages, relating broadly to sensitivity, specificity, dynamic range, multiplexing capability, precision, throughput, and ease of use. in general, multiplexed microarray immunoassays are ambient analyte assays [160] . given the heterogeneity of antibody array formats and procedures currently in use in proteomics studies, and the absence of a "gold standard," there exists an urgent need for development and adoption of standards that permit platform comparisons and benchmarking. regardless of the choice of a given proteomic separation technique, gel-based or gel-free, a mass spectrometer is always the primary tool for protein identification. during the last decade, significant improvements have been made in the application of ms for the determination of protein sequences [161] . mass spectrometers consist of an ion source, the mass analyzer, and an ion detection system. analysis of proteins by ms occurs in three major steps (a) protein ionization and generation of gas-phase ions, (b) separation of ions according to their mass to charge ratio, and (c) detection of ions [162] . in gel-free approaches such as icat and mudpit, samples are directly analyzed by ms whereas, in gel-based proteomics (2de and 2d-dige), the protein spots are first excised from the gel and then digested with trypsin. the resulting peptides are then separated by lc or directly analyzed by ms. the experimentally derived peptide masses are correlated with the peptide fingerprints of known proteins in the databases using search engines (e.g., mascot, sequest). there are two main ionization sources which include matrix assisted laser desorption/ionization (maldi) and electrospray ionization (esi) and four major mass analyzers, which are time-of-flight (tof), ion trap, quadrupole, and fourier transform ion cyclotron (ftic) which are currently in use for protein identification and characterization [163] . a combination of different mass analyzers in tandem such as quadrupole-tof and quadrupole-ion trap has combined the individual strengths of different types of mass analyzers and greatly improved their capabilities for proteome analysis [162] . simple mass spectrometers such as maldi-tof are used for only measurement of mass, whereas tandem mass spectrometers are used for amino acid sequence determination [164] . in maldi the sample of interest is crystallized with the matrix on a metal surface and a laser ion source causes excitation of matrix along with the analyte ions, which are then released into the gas phase. maldi measures the mass of peptides derived from a trypsinized parent protein and generates a list of experimental peptide masses, often referred to as "mass fingerprints" [165, 166] . in esi, the analyte is ionized from a solution and transferred into the gas phase by generating a fine spray from a high voltage needle which results in multiple charging of the analyte and generation of multiple consecutive ions. tandem mass spectrometry or ms/ms is performed by combining two different ms separation principles. in tandem ms, individual trypsin-digested peptides are fragmented after a liquid phase separation. tandem ms instruments such as triple quadrupole, quadrupole ion trap, fourier transform ion-cyclotron resonance, or quadrupole time-of-flight are used in lc-ms/ms or nanospray experiments with electrospray ionization (esi) to generate peptide fragment ion spectra [167] . ion mobility spectrometry (ims) has been utilized as a rapid gas-phase separations strategy for biomolecular ions [168, 169] . the strategy provides high sensitivity because the gas-phase dispersion of peptide ions separates features corresponding to low abundance species from interfering chemical noise [170] . reduced spectral congestion also allows for the use of shorter experimental run times (lc separations) without sacrificing throughput; short analysis time scales are key to measuring the large numbers of samples required to determine normal protein variability prior to realizing individual plasma profiling. additionally, mobility-dispersed ions can be fragmented and mobility linked to fragment ions without ion loss from precursor mass selection [171] . these advantages have been demonstrated in head-to-head comparisons with conventional lc-ms/ms technology using rapid (21 minutes) lc gradients [169] . accurate mass and time (amt) tag approach [172] addresses an analogous situation in lc-ms-based proteomics studies. in this approach, initial lc-ms/ms analyses are performed on prefractionated peptide samples in order to provide peptide sequence identifications. these experiments are relatively low throughput because the peptide prefractionation can be quite extensive and require separate lc-ms/ms analyses for each fraction. the high-throughput accurate mass and time (amt) tag proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-negative bacterium rhodobacter sphaeroides 2.4.1. there has been a recent trend in proteomics toward the development and application of technologies for the targeted analysis of proteins within complex mixtures [173] . selected reaction monitoring (srm) is a powerful tandem mass spectrometry method that can be used to monitor target peptides within a complex protein digest [174, 175] . the specificity and sensitivity of the approach, as well as its capability to multiplex the measurement of many analytes in parallel, has made it a technology of particular promise for hypothesis driven proteomics. the use of tandem mass spectrometry data acquired on an ltq ion trap mass spectrometer can accurately predict which fragment ions will produce the greatest signal in an srm assay using a triple quadrupole mass spectrometer [176] . one of the biggest benefits of a targeted assay on a triple quadrupole mass spectrometer is high throughput. using the selectivity of multiple stages of mass selection of a tandem mass spectrometer, these targeted srm assays are the mass spectrometry equivalent of a western blot [173] . an advantage of using targeted mass spectrometry-based assay over a traditional western blot is that it does not rely on the creation of any immunoaffinity reagent. while its application is novel in the proteomics community, srm has been utilized for several decades in the toxicology and pharmacokinetics disciplines [177] . peptidebased immunofractionation methods show potential for proteome wide screening approaches but are limited by the availability of antibodies [178, 179] . the stable isotope standards with capture by antipeptide antibodies (siscapa) approach is based on the addition of stable isotope labeled standard peptides to the digested clinical sample followed by immunoaffinity enrichment of standard and analyte peptide by highly specific antipeptide antibodies [180, 181] . this approach enables the absolute quantification of selected diagnostic peptides from digested clinical samples down to physiologically relevant analyte concentrations (ng/ml) at high precision (10% cv) and accuracy [178, 179] . further improvement of mrm-based biomarker quantification should be possible if whole sets of analyte peptides can be enriched by immunofractionation. since this method relies on one specific antibody per target protein/peptide the generation of more than 10000 antibodies is necessary for proteome wide screening approaches. novel peptide affinity enrichment strategies enabling proteome wide analyses of signature peptides may provide an important addition to future proteome workflows. undoubtedly, the accuracy, high throughput, and robustness of ms technologies have made the characterization of entire proteomes a realistic goal [180, 181] . the major bottlenecks in proteomics research today are related to data analysis to create an environment where computer scientists and biologists and the people who collect data can work closely together, so they can develop the necessary analytical tools that will help interpret the data [182] [183] [184] . processing and analysis of proteomics data is indeed a very complex multistep process ( figure 5 ). the meaningful comparison, sharing, and exchange of data or analysis results obtained on different platforms or by different laboratories remain cumbersome mainly due to the lack of standards for data formats, data processing parameters, and data quality assessment. accurate, consistent, and transparent data processing and analysis are integral and critical parts of proteomics workflows [185] . we can now generate huge amounts of data, and currently there is an enormous challenge to figure out how to actually analyze this data and generate real biological insights. the necessity of an integrated pipeline for processing and analysis of complex proteomics data sets has therefore become critical. validation. this step consists of the assignment of ms/ms spectra to a database search using one of several engines available (e.g., sequest, mascot, comet, x!tandem, etc.). one of the difficulties related to the use of sequest for peptide identifications is the lack of methods to globally evaluate the quality of data and the lack of methods to access global changes created by filtering schemes and/or database changes [186] . most approaches are matching and scoring large sets of experimental spectra with predicted masses of fragment ions of peptide sequences derived from a protein database. results are scored according to a scheme specific to each search engine that also depends on the database used for the search. usually tools are linked to one specific platform or were optimized for one instrument type. the various search engines do not yield identical results as they are based on different algorithms and scoring functions, making comparison and integration of results from different studies or experiments tedious [187, 188] . peptide identification via database searches is very computationally intensive and time-demanding. high quality data allow more effective searches due to tighter constrains, that is, tolerance on precursor ion mass and charge state assignment, which will drastically reduce the search time in case of an indexed database. in addition, accurate mass measurements of fragment ions further simplify the database searches and add confidence to the results. the association of identified peptides with their precursor proteins is a very critical and difficult step in shotgun proteomics strategies as many peptides are common to several proteins, thus leading to ambiguous protein assignments. therefore it becomes critical to have an appropriate tool that is able to assess the validity of the protein inference and associate a probability to it. protein prophet database tool combines probabilities assigned to peptides identified by ms/ms to compute accurate probabilities for the proteins present [189] . 5:1921-1926, 2006. impossible. the lack of common standards and protocols has led to this situation and often resulted in duplication of efforts. results were usually reported as a set of identified proteins (i.e., list of peptides identified and associated proteins) with minimal supporting data. obviously the large volume of such data sets has made publication of detailed results using classical mechanisms very challenging. sharing and exchange of data and results requires the definition of standard formats for the data at all levels (including raw mass spectrometric data, processed data, and search results) as well as a better definition (and/or standardization) of the parameters used for the data processing or the database searches. organellar proteomics aims to describe the full complement of proteins of subcellular structures and organelles. identification of the proteins contained in subcellular organelles has become a popular proteomics endeavor [190] . when compared with whole-cell or whole-tissue proteomes, the more focused results from subcellular proteomic studies have yielded relatively simpler datasets from which biologically relevant information can be more easily extracted [191] . subcellular fractionation consists of two major steps, disruption of the cellular organization (homogenization) and fractionation of the homogenate to separate the different populations of organelles. such a homogenate can then be resolved by differential centrifugation into several fractions containing mainly (1) nuclei, heavy mitochondria, cytoskeletal networks, and plasma membrane; (2) light mitochondria, lysosomes, and peroxisomes; (3) golgi apparatus, endosomes and microsomes, and endoplasmic reticulum; (4) cytosol. each population of organelles is characterized by size, density, charge, and other properties on which the separation relies [192] . analyzing subcellular fractions and organelles allows tracking proteins that shuttle between different compartments, for example, between the cytoplasm and nucleus. a high dynamic range of proteins can be partially achieved by fractionation of the proteome into subproteomes by applying affinity purification may allow proteomic analysis of low copy number proteins [193] . the nuclear, chloroplast, amyloplast, plasma membrane, peroxisome, endoplasmic reticulum, cell wall, and mitochondrial proteomes were successfully characterized in arabidopsis [194] . several groups have taken advantage of this approach to recover a higher percentage of membrane proteins from subcellular extracts using various nonionic and zwitterionic detergents or phase-partitioning methods. these efforts resulted in the successful determination of the protein complement of the thylakoid and envelope membrane systems of the chloroplast [195] . by enriching for the protein class of interest based on a particular chemical/physical characteristic(s), offer the advantage of reducing sample complexity and access to lower abundance proteins in a discoverydriven experimental approach [196] . free flow electrophoresis (ffe) utilizes differences in electrophoretic mobility rather than density to separate cells or subcellular organelles [197] . ffe has previously been used in separating endosomes from hamster ovary cells [198] , plasma membrane from human platelets [199] , and insulin transporting vesicles in liver cells. the separation is based on the electrophoretic motility of cells or cell organelles suspended in a vertical free flowing buffer film on which an electric field is applied at a right angle to the flow direction. ffe has been a most valuable tool in the investigation of the composition of secretory vesicles and in addition, it has clarified how the membrane of plasma membrane vesicles is oriented after nitrogen disruption of human neutrophils [200] . importantly, subcellular fractionation is a flexible and adjustable approach that may be efficiently combined not only with 2d gel electrophoresis but also with gelindependent techniques. however, they do have limitations of considerable cross-contamination with other subcellular organelles. ptms of proteins are considered to be one of the major determinants regarding organisms complexity [201] . to date, at least more than 200 different types of ptms have been identified of which only a few are reversible and important for the regulation of biological processes. specific functions are usually mediated through ptms, such as phosphorylations, acetylations, or glycosylations, which places additional demands on the sensitivity and precision of the method [202] . one of the most studied ptms is protein phosphorylation, because it is vital for a large number of protein functions that are important to cellular processes spanning from signal transduction, cell differentiation, and development to cell cycle control and metabolism. enzymes and receptors can be switched "on" and "off " by phosphorylation and dephosphorylation. it was estimated that 10-50% of proteins are phosphorylated. phosphorylation often occurs on serine, threonine, and tyrosine residues in eukaryotic proteins [203] . analysis of the entire cellular phosphoproteome has been an attractive study subject since the discovery of phosphorylation as a key regulatory mechanism of cell life. unfortunately, phosphoproteins analysis is not straightforward for five main reasons. first, the stoichiometry of phosphorylation is generally relatively low, because only a small fraction of the available intracellular pool of a protein is phosphorylated at any given time as a result of a stimulus. second, the phosphorylatation sites on proteins might vary, implying that any given phosphoprotein is heterogeneous (i.e., it exists in several different phosphorylated forms). third, many of the signaling molecules, which are major targets of phosphorylation events [204] , are present at low abundance within cells and, in these cases; enrichment is a prerequisite before analysis. fourth, most analytical techniques used for studying protein phosphorylation have a limited dynamic range, which means that although major phosphorylation sites might be located easily, and minor sites might be difficult to identify. finally, phosphatases could dephosphorylate residues unless precautions are taken to inhibit their activity during preparation and purification steps of cell lysates. in addition, various methods for protein phosphorylation site determination have been developed, yet this task remains a technical challenge [205] . western blot has been widely used to determine the presence of ptms. however, this technique relies on the prior knowledge of the type and position of specific modifications and the availability of antibodies. it has low throughput and not ideal for studying highly complicated samples. specific chemical or affinity enrichment steps are usually incorporated into the sample preparation or fractionation stages of the general scheme of proteomic studies [206, 207] . well established methods involving the analysis of 32p-labeled phosphoproteins by edman degradation and two-dimensional phosphopeptide mapping have proven to be powerful but not without limitations. consequently, mass spectrometry (ms) has emerged as a reliable and sensitive method for the characterization of protein phosphorylation sites [208] and may therefore represent a method of choice for the analysis of protein phosphorylation [209] . immobilized metal affinity chromatography (imac), metal oxide affinity chromatography (moac), and covalent methods are all capable of selectively enriching phosphopeptides [210] . moac based on adsorption to tio2 is especially attractive, but as with all techniques, loading, rinsing, and elution solutions must be carefully selected to minimize nonspecific adsorption and to maximize the detection of both monophosphorylated and multiphosphorylated species. imac might not provide the selectivity available with tio2 enrichment, but with appropriate reagents, imac can be selective and sensitive for monophosphorylated and tetraphosphorylated peptides. however, some buffers and reagents such as edta are not compatible with imac, so hplc purification may be needed prior to this technique [211] . when trying to isolate and identify as many phosphoproteins as possible in a cell lysate, chromatographic column-based methods are required. multiple elutions from imac or moac columns or even gradient elutions can help to simplify fractions of proteins and reveal more peptides [212, 213] . a combination of techniques can reveal large numbers of phosphopeptides in complex samples, but comprehensive phosphoproteomics is still not possible. for the highest protein coverage, future phosphoproteomic techniques will likely employ multiple enrichment techniques along with two-dimensional separations, but such studies are time consuming. combinations of affinity-based enrichment and extraction methods, multidimensional separation technologies, and mass spectrometry are particularly attractive for systematic investigation of posttranslationally modified proteins in proteomics [214] . organisms. the application of proteomics and related technologies for the analysis of proteome is severely hampered by the lack of publicly available sequence information for most of the unsequenced organisms [215] . despite the precision of the mass information yielded by the seldi technique, a significant number of proteins were found to have no similarity to known peptides, an aforementioned weakness of proteomics studies in nonmodel organisms [216] . in order to circumvent this limitation, different strategies and tools were developed to make unsequenced organisms amenable to high-throughput proteomics [217] (figure 6 ). however, an evaluation of their performance in an integrated proteomics strategy using high-throughput shotgun ms data is currently missing. in principle, two different approaches can lead to an increase in protein identifications from unsequenced organisms. in the first approach, ms/ms data are searched against a protein database of an evolutionarily closely related organism. however, as a matter of principle of database-dependent searches, only proteins can be identified that contain at least one peptide with exactly the same sequence as the peptide from a protein in the database. with increasing evolutionary distance this will be an increasingly severe restriction [218] . in the second approach, the amino acid sequence of a peptide is extracted from the ms/ms spectrum for de novo sequencing, that is, in a fully databaseindependent manner using exclusively the information contained in the ms/ms spectrum. several software tools for peptide de novo sequencing are now available and some of them provide sufficiently good results when applied to high-quality spectra [219] . a basic limitation of ms de novo sequencing methods is the necessity for backbone cleavage between each pair of adjacent amino acids; a mass value representing a terminal fragment containing only one of the two residues is a first requirement for ordering of a specific pair [220, 221] and this limitation urged the need for bioinformatics approaches that can help interpret the proteomics data [219] . in the past several years there have been very important extremely useful advances in proteomics methods based on bottom-up display and bottom-up identification using peptides [222] . these methods offer more sensitivity, greater rapidity and greater proteome coverage are often made with the explicit or implicit assertion that these methods are bound to replace more traditional methods based on topdown analysis, especially using 2d gels [223, 224] . the combination of bottom-up display and bottom-up identification has achieved very important successes in detecting the presence of large numbers of different proteins in cells or subcellular organelles [225, 226] . the use of specific fractionation schemes and prudent adoption of methods to increase the number of proteins able to be identified and quantified is enabling significant biological advances to be made. further technological developments that enable a larger proportion of the proteome to be visualized will further enhance our ability to characterize biological systems. as such, these advances in proteomics will impact not only academic pursuits but also pharmaceutical, biotechnology and diagnostic research and development [227] . in the future gel-free techniques mudpit, itraq and 18o stable isotope labeling could be expected to gain more importance as they become more established. sample prefractionation system provides a highly valuable tool to fractionate proteins and peptides from complex eukaryotic samples like plasma. this approach has a positive influence on the number of proteins identified compared to scx method [228] . itraq is a very powerful tool, recognised form its ability to relatively quantify proteins. itraq reagent improves maldi ionisation, especially for peptides containing lysine. although silac labelling is easy for any laboratory that uses cell culture, the ms technology that is required is still beyond the capabilities of most groups. one of the factors that contributed to the rapid acceptance of the silac technology was the availability of an open-source program, msquant, for interpreting results. protein microarrays offer the ability to simultaneously survey multiple protein markers in an effort to develop expression profile changes across multiple protein analytes for potential use in diagnosis, prognosis, and measurement of therapeutic efficacy [229] . this technology is an excellent high-throughput method used to probe an entire collection of proteins for a specific function or biochemistry. it is an exceptional new way to discover previously unknown multifunctional proteins, and to discover new functionalities for well-studied proteins [230] . a systematic and efficient analysis of vast genomic and proteomic data sets is a major challenge for researchers today. to overcome limitations of current proteomics strategies in regard to the dynamic range of peptides detected and alternative mass spectrometrybased approaches are being explored. targeted strategies exemplified by multiple reaction monitoring detect, quantify, and possibly collect a product ion spectrum to confirm the identity of a peptide with much greater sensitivity because the precursor ion is not detected in the full mass spectrum [231] . a systematic and efficient evaluation of large-scale experimental results requires (1) automatic retrieval of user defined information to construct a customized, queryable database; (2) an intuitive graphical and query platform to display and analyze experimental data in the context of the customized database; (3) efficient utilization of webbased bioinformatics software tools for data interpretation, prediction of function, and modeling; (4) scalability and reconstruction of the database in response to changing user needs and an ever-expanding base of knowledge and bioinformatics tools [232] . creating a software tool to encompass the four crucial features outlined above is a challenging and ongoing task, particularly with respect to the ever-expanding publicly available base of knowledge and bioinformatics tools. the data processing and analysis bottleneck 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affinity tag technology mass spectrometry and proteomics quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry quantitative analysis of redox-sensitive proteome with dige and icat stable isotope labeling by amino acids in cell culture, silac, as a simple and accurate approach to expression proteomics mass spectrometricbased approaches in quantitative proteomics a proteomics strategy to elucidate functional protein-protein interactions applied to egf signaling unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors stable isotope labeling of arabidopsis thaliana cells and quantitative proteomics by mass spectrometry stable isotope labeling with amino acids in cell culture (silac) for studying dynamics of protein abundance and posttranslational modifications functional and quantitative proteomics using silac identifying dynamic interactors of protein complexes by quantitative mass spectrometry metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis quantitative proteomics of the human malaria parasite plasmodium falciparum and its application to studies of development and inhibition quantitative mouse brain proteomics using culture-derived isotope tags as internal standards 18 o stable isotope labeling in ms-based proteomics proteolytic 18 o labeling for comparative proteomics: model studies with two serotypes of adenovirus differential protein expression in the cytosol fraction of an mcf-7 breast cancer cell line selected for resistance toward melphalan non-gel-based dual 18 o labeling quantitative proteomics strategy shotgun proteomics using the itraq isobaric tags itraq is a useful method to screen for membrane-bound proteins differentially expressed in human natural killer cell types a perspective on the use of itraq reagent technology for protein complex and profiling studies eight-channel itraq enables comparison of the activity of 6 leukaemogenic tyrosine kinases multiplexed protein quantitation in saccharomyces cerevisiae using amine-reactive isobaric tagging reagents protein fractionation in a multicompartment device using off-gel tm isoelectric focusing efficient fractionation and improved protein identification by peptide offgel electrophoresis combining microscale solution-phase isoelectric focusing with multiplexed proteomics dye staining to analyze protein posttranslational modifications solution phase isoelectric fractionation in the multi-compartment electrolyser: a divide and conquer strategy for the analysis of complex proteomes a comparison of immobilized ph gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics two-stage off-gel tm isoelectric focusing: protein followed by peptide fractionation and application to proteome analysis of human plasma proteome analysis of human plasma and amniotic fluid by off-gel tm isoelectric focusing followed by nano-lc-ms/ms exploring glycopeptide-resistance in staphylococcus aureus: a combined proteomics and transcriptomics approach for the identification of resistance-related markers identification of brain cell death associated proteins in human post-mortem cerebrospinal fluid the focusing positions of polypeptides in immobilized ph gradients can be predicted from their amino acid sequences ingel isoelectric focusing of peptides as a tool for improved protein identification gel based isoelectric focusing of peptides and the utility of isoelectric point in protein identification isobaric tags for relative and absolute quantitation (itraq) reproducibility: implication of multiple injections technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (itraq) neuronal differentiation and long-term culture of the human neuroblastoma line sh-sy5y the proteome of the human neuroblastoma cell line sh-sy5y: an enlarged proteome from transcriptome to proteome: differentially expressed proteins identified in synovial tissue of patients suffering from rheumatoid arthritis and osteoarthritis by an initial screen with a panel of 791 antibodies a large-scale proteomic analysis of human embryonic stem cells mudpit analysis: application to human heart tissue multidimensional separation of peptides for effective proteomic analysis large-scale analysis of the yeast proteome by multidimensional protein identification technology multidimensional protein identification technology: current status and future prospects multiplexed protein quantitation in saccharomyces cerevisiae using amine-reactive isobaric tagging reagents reproducibility of quantitative proteomic analyses of complex biological mixtures by multidimensional protein identification technology proteomics: from gel based to gel free human protein factory for converting the transcriptome into an in vitro-expressed proteome printing protein arrays from dna arrays nextgeneration 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methods and applications of antibody microarrays in cancer research multiplexed sandwich assays in microarray format evaluating sandwich immunoassays in microarray format in terms of the ambient analyte regime mass spectrometry and protein analysis analysis of proteins and proteomes by mass spectrometry use of mass spectrometry-derived data to annotate nucleotide and protein sequence databases current initiatives in proteomics research: the plant perspective laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons the characteristics of peptide collision-induced dissociation using a high-perfformance maldi-tof/tof tandem mass spectrometer mass spectrometry and proteomics gas-phase separations of protease digests development of high throughput dispersive lcion mobility-tofms techniques for analysing the human plasma proteome formation of peptide aggregates during esi: size, charge, composition, and contributions to noise mobility labeling for parallel cid of ion mixtures advances in proteomics data analysis and display using an accurate mass and time tag approach selective detection of membrane proteins without antibodies: a mass spectrometric version of the western blot analysis of protein phosphorylation by hypothesis-driven multiple-stage mass spectrometry expediting the development of targeted srm assays: using data from shotgun proteomics to automate method development quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution current affairs in quantitative targeted proteomics: multiple reaction monitoringmass spectrometry peptidomics: a new approach to affinity protein microarrays antibodybased enrichment of peptides on magnetic beads for massspectrometry-based quantification of serum biomarkers mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (sis-capa) mass spectrometry-based proteomics bioinformatics meets proteomics-bridging the gap between mass spectrometry data analysis and cell biology statistical and computational methods for comparative proteomic profiling using liquid chromatography-tandem mass spectrometry protein identification by mass spectrometry: issues to be considered challenges and opportunities in proteomics data analysis toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry the need for guidelines in publication of peptide and protein identification data: working group on publication guidelines for peptide and protein identification data reporting protein identification data: the next generation of guidelines a statistical model for identifying proteins by tandem mass spectrometry proteome analysis at the level of subcellular structures complementary methods to assist subcellular fractionation in organellar proteomics subcellular fractionation, electromigration analysis and mapping of organelles affinity purification-mass spectrometry: powerful tools for the characterization of protein complexes the swiss-prot protein knowledgebase and expasy: providing the plant community with high quality proteomic data and tools proteomic study of the arabidopsis thaliana chloroplastic envelope membrane utilizing alternatives to traditional twodimensional electrophoresis subcellular fractionation and proteomics of nuclear envelopes 54 improved subcellular fractionation of the heavy mitochondria pellet using free flow electrophoresis system rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis characterization of human platelet surface and intracellular membranes isolated by free flow electrophoresis chemoattractant-regulated mobilization of a novel intracellular compartment in human neutrophils large-scale analysis of the yeast proteome by multidimensional protein identification technology phosphoproteomics for the discovery of kinases as cancer biomarkers and drug targets mass spectrometry-based proteomics and peptidomics for biomarker discovery in neurodegenerative diseases the extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions abrf-prg03: phosphorylation site determination interpreting the protein language using proteomics proteomic analysis of posttranslational modifications phosphoamino acid analysis signaling-2000 and beyond techniques for phosphopeptide enrichment prior to analysis by mass spectrometry evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques improved enrichment strategies for phosphorylated peptides on titanium dioxide using methyl esterification and ph gradient elution simac (sequential elution from imac), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides modification-specific proteomics: characterization of post-translational modifications by mass spectrometry a workflow to increase the detection rate of proteins from unsequenced organisms in high-throughput proteomics experiments application of genomics and proteomics for study of the integrated response to zinc exposure in a non-model fish species, the rainbow trout sequence similarity-based proteomics in insects: characterization of the larvae venom of the brazilian moth cerodirphia speciosa a workflow to increase the detection rate of proteins from unsequenced organisms in high-throughput proteomics experiments improving gene annotation using peptide mass spectrometry proteomics studies confirm the presence of alternative protein isoforms on a large scale plips, an automatically collected database of protein lists reported by proteomics studies proteome analysis by mass spectroscopy detection technologies in proteome analysis two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains is proteomics heading in the wrong direction? proteomic mapping of brain plasma membrane proteins how much of the proteome do we see with discovery-based proteomics methods and how much do we need to see? peptides offgel electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative itraq labeling development and standardization of multiplexed antibody microarrays for use in quantitative proteomics protein microarray technology integrating biological databases new paradigms in cellular function and the need for topdown proteomics analysis key: cord-003144-nqkw5v3w authors: qu, zehui; gao, fei; li, liwei; zhang, yujiao; jiang, yifeng; yu, lingxue; zhou, yanjun; zheng, hao; tong, wu; li, guoxin; tong, guangzhi title: label‐free quantitative proteomic analysis of differentially expressed membrane proteins of pulmonary alveolar macrophages infected with highly pathogenic porcine reproductive and respiratory syndrome virus and its attenuated strain date: 2017-11-24 journal: proteomics doi: 10.1002/pmic.201700101 sha: doc_id: 3144 cord_uid: nqkw5v3w significant differences exist between the highly pathogenic (hp) porcine reproductive and respiratory syndrome virus (prrsv) and its attenuated pathogenic (ap) strain in the ability to infect host cells. the mechanisms by which different virulent strains invade host cells remain relatively unknown. in this study, pulmonary alveolar macrophages (pams) are infected with hp‐prrsv (hun4) and ap‐prrsv (hun4‐f112) for 24 h, then harvested and subjected to label‐free quantitative ms. a total of 2849 proteins are identified, including 95 that are differentially expressed. among them, 26 proteins are located on the membrane. the most differentially expressed proteins are involved in response to stimulus, metabolic process, and immune system process, which mainly have the function of binding and catalytic activity. cluster of differentiation cd163, vimentin (vim), and nmii as well as detected proteins are assessed together by string analysis, which elucidated a potentially different infection mechanism. according to the function annotations, prrsv with different virulence may mainly differ in immunology, inflammation, immune evasion as well as cell apoptosis. this is the first attempt to explore the differential characteristics between hp‐prrsv and its attenuated prrsv infected pams focusing on membrane proteins which will be of great help to further understand the different infective mechanisms of hp‐prrsv and ap‐prrsv. porcine reproductive and respiratory syndrome virus (prrsv) has been a leading economically significant viral pathogen of swine worldwide for almost 28 years. [1] [2] [3] prrsv, equine arteritis virus, simian hemorrhagic fever virus, and lactate dehydrogenaseelevating virus are members of the family arteriviridae. [4, 5] prrsv is a positive-sense, single-stranded, rna virus with a full-length genome of 15 kb that has a 5 cap and a 3 poly (a) tail. [4, [6] [7] [8] prrsv was first reported in the united states in the late 1980s. [9] in 2006, several large-scale, severe outbreaks of highly pathogenic (hp) atypical prrsv (hp-prrsv) were reported in china and neighboring asian countries. [2, 3, 10] reported hp-prrsv morbidity and mortality rates were much higher than previous pandemic prrsv strains and associated with more severe clinical presentations and higher rectal temperature (>41°c). [11] the emergence of hp-prrsv has caused great economic loss to the swine industry in china and made preventing and controlling prrsv outbreaks difficult. therefore, elucidating the causes of the greater virulence of prrsv and the differences between the hp and attenuated pathogenic (ap) strains has become even more important. to this end, several trials have been conducted to identify virulence factors; these studies have resulted in some successes. [12, 13] however, changes in virulence and pathogenic mechanisms are difficult to discern. other than virulence in vivo, many distinctions in the biological aspects of hp-prrsv and ap-prrsv have been noted, such as in viral binding and entry into pulmonary alveolar macrophages (pams). prrsv exhibits highly restricted cell tropism both in vivo and in vitro. [14] the virus can be detected only in well-differentiated macrophages of lungs, lymph nodes, peyer's patches, spleen, tonsils, and thymus. pams are the main target cells of prrsv. [15] prrsv can also replicate in vitro in the african www.advancedsciencenews.com www.proteomics-journal.com membrane proteins (mps) of pams infected by highly proteomic (hp)-and attenuated proteomic (ap)-prrsv have been elucidated by lc-ms/ms for label-free quantitative proteomics. ninety-five differentially expressed proteins were identified and characterized. the most significant difference in the biological process between pams infected with hp-and ap-prrsv is the metabolic process. most different molecular functions were classified as binding and catalytic activities. cellular component categories showed that 26 differentially expressed proteins were confirmed as mps based on the annotation of uniprot database, such as rap2a, vcl (vinculin), ifitm3 function in cell-cell junctions, erk signaling pathway, g protein signaling pathways, biotic stimulus, and so on. among them, vcl is a kind of f-actin-binding protein which is involved in cell-matrix adhesion and cell-cell adhesion in humans. it was demonstrated that over expression of vcl could inhibit the replication of both hp-prrsv and the attenuated prrsv in the mrna level. there were obvious differences in the inhibiting ability for hp-prrsv and its attenuated strain. this is the first attempt to explore the differential characteristics between hp-prrsv and its attenuated prrsv-infected pams focusing on mps which will be of great help to further understand the different infective mechanisms of hp-prrsv and ap-prrsv. green monkey kidney cell line ma-104 and its derivatives, marc-145 and cl-2621, which are considered permissive cell lines for prrsv. [16, 17] reportedly, prrsv targets cellular membrane proteins (mps) and enters target cells through receptormediated endocytosis during viral infection. [18] studies have investigated possible mechanisms employed by prrsv to infect pams. [19] elucidating the characteristics of viruses and interactions between viruses and host cells is increasingly important. however, exploring individual proteins within the many proteins in cells is difficult. nonetheless, gradual advancements have come through use of proteomic techniques. of these, ms-based quantitative proteomic techniques offer the advantage of better accuracy and sensitivity, and have been widely used to analyze host cell responses to viral infection. among them, liquid chromatographytandem ms (lc-ms/ms) for label-free quantitative proteomics (lfqp) is an important mass spectrometric tool to detect and quantify large amounts of proteins. [20] compared with quantitative proteomics using stable isotope labeling such as stable isotope labeling by amino acids in cell culture and isobaric tags for relative and absolute quantitation, lfqp detects greater amounts of proteins and important signaling pathways and networks. the aim of this study was to determine potentially different infection mechanisms used by the hp strain vhun4 [10] and its derivative attenuated strain vhun4-f112 [21, 22] using lfqp. the animal study protocols were approved by the animal care and use committee of shanghai veterinary research institute, chinese academy of agricultural sciences. hp-prrsv strain vhun4 [3, 10] at a titer of 10 5 50% tissue culture infective dose (tcid 50 ) ml −1 and cell-passaged attenuated virus strain vhun4-f112 (ap-prrsv) [21, 22] at a titer of 10 5 tcid 50 ml −1 were stored as viral stocks. porcine circovirus 2, classical swine fever virus, prrsv antibody, and antigen-free 15-day-old piglets were used. animals were sacrificed in accordance with the ethics statement. lungs were dissected and lavaged with pbs (pbs; life technologies, inc., gibco/brl division, grand island, ny, usa) supplemented with 1% penicillinstreptomycin (gibco/brl), then centrifuged at 1000 × g for 5 min, resuspended in pbs, centrifuged, and resuspended in pbs. pams were collected in roswell park memorial institute (rpmi) 1640 medium (gibco/brl) containing 10% fetal bovine serum (gibco/brl) [23] and incubated in 10 cm dishes (corning, inc., corning, ny, usa) for 12 h at 37°c in a 5% co 2 atmosphere. after pams were washed with pbs three times, dead and nonadherent cells were removed when confluency exceeded 95%. three dishes were inoculated with vhun4 and another three with vhun4-f112 at multiplicity of infection 1. an additional three dishes were inoculated with dmem (gibco-brl) as a blank control. all dishes were incubated at 37°c in an atmosphere of 5% co 2 , as described previously. [13] after incubation for 1 h, inocula were discarded and pams were washed with pbs three times. cell monolayers in all dishes were overlaid with rpmi-1640 medium containing 2% fetal bovine serum and incubated at 37°c in a 5% co 2 atmosphere for 24 h. pams were digested with 2 ml 0.25% trypsinethylenediaminetetraacetic acid solution (gibco/brl), collected by gently pipetting, centrifuged at 1000 × g for 5 min and lysed using the proteoextract transmembrane protein extraction kits (novagen, emd biosciences, inc., madison, wi, usa), [24, 25] according to the manufacturer's instructions. cells were resuspended in extraction buffer 1 and protease inhibitor cocktail, incubated for 10 min at 4°c with gentle agitation, and centrifuged at 1000 × g for 5 min at 4°c. after removing supernatants, pellets were resuspended in 0.2 ml extraction buffer 2a and protease inhibition cocktail, incubated for 45 min at room temperature with gentle agitation, and centrifuged at www.advancedsciencenews.com www.proteomics-journal.com 16 000 × g for 15 min at 4°c. supernatants were precipitated with 1 ml acetone and centrifuged at 12 000 × g for 10 min. after evaporating to dryness, 150 μl sdt buffer (4% sodium dodecyl sulfate, 100 mm tris/hcl at ph 7.6, 0.1 m dithiothreitol) was added and mixtures were heated in boiling water for 5 min. after centrifugation, supernatants were collected and quantified with a bca protein assay kit (bio-rad, usa). fig. 1 digestion of protein (250 μg for each sample) was performed according to the fasp (filter-aided sample preparation) procedure. briefly, the detergent, dtt and other low-molecular-weight components were removed using 200 μl ua buffer (8 m urea, 150 mm tris-hcl ph 8.0) by repeated ultrafiltration (microcon units, 10 kd) facilitated by centrifugation. then 100 μl 0.05 m iodoacetamide in ua buffer was added to block reduced cysteine residues and the samples were incubated for 20 min in darkness. the filter was washed with 100 μl ua buffer three times and then 100 μl 25 mm nh 4 hco 3 twice. finally, the protein suspension was digested with 3 μg trypsin (promega) in 40 μl 25 mm nh 4 hco 3 overnight at 37°c, and the resulting peptides were collected as a filtrate. the peptide content was estimated by uv light spectral density at 280 nm using an extinctions coefficient of 1.1 of 0.1% (g l −1 ) solution that was calculated on the basis of the frequency of tryptophan and tyrosine in vertebrate proteins. [26] the peptide of each sample was desalted on c18 cartridges (empore spe cartridges c18 (standard density), bed id 7 mm, volume 3 ml, sigma), then concentrated by vacuum centrifugation and reconstituted in 40 μl of 0.1% (v/v) trifluoroacetic acid. ms experiments were performed on a q exactive mass spectrometer that was coupled to easy nlc (proxeon biosystems, now thermo fisher scientific). five microgram peptide was loaded onto a c18-reversed phase column (thermo scientific easy column, 10 cm long, 75 μm inner diameter, 3 μm resin) in buffer a (2% acetonitrile and 0.1% formic acid) and separated with a linear gradient of buffer b (80% acetonitrile and 0.1% formic acid) at a flow rate of 250 nl min −1 controlled by intelliflow technology over 120 min. ms data was acquired using a datadependent top ten method dynamically choosing the most abundant precursor ions from the survey scan (300-1800 m/z) for hcd fragmentation. determination of the target value is based on predictive automatic gain control. dynamic exclusion duration was 25 s. survey scans were acquired at a resolution of 70 000 at m/z 200 and resolution for hcd spectra was set to 17 500 at m/z 200. normalized collision energy was 30 ev and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%. the instrument was run with peptide recognition mode enabled. ms experiments were performed triply for each sample. [27] the ms data were analyzed using maxquant software version 1.3.0.5. ms data were searched against the uniprot sus scrofa sequence database (including 34 253 sequences downloaded on 12/27/2014). an initial search was set at a precursor mass window of 6 ppm. the search followed an enzymatic cleavage rule of trypsin/p and allowed maximal two missed cleavage sites and a mass tolerance of 20 ppm for fragment ions. carbamidomethylation of cysteines was defined as fixed modification, while protein n-terminal acetylation and methionine oxidation were defined as variable modifications for database searching. the cutoff of global false discovery rate for peptide and protein identification was set to 0.01. label-free quantification was carried out in maxquant as previously described. protein abundance was calculated on the basis of the normalized spectral protein intensity (lfq intensity). all statistical analyses were performed using unpaired t-tests. a p-value <0.05 and ratio >2 or <0.5 were considered to indicate significant differences. gene ontology (go) annotation and functional classification of identified proteins was with blast2go ver. v2.6.2 with the current public database b2g_aug 12 (www.blast2go.com). identified proteins were classified www.advancedsciencenews.com www.proteomics-journal.com using blast2go steps under default parameters: blast, mapping, and annotation. protein-protein interaction networks were analyzed using (string software string-db.org/) . confidence view was assigned a score of 0.4, indicating medium confidence. samples of prrsv-infected and dmem-inoculated pams were lysed at 24 h post infection and protein concentrations were determined. samples (20 μg) were separated by 12% sds-page and transferred to 0.22 μm nitrocellulose membranes (bio-rad laboratories, hercules, ca, usa). membranes were blocked with 5% skim milk in tris-buffered saline containing 0.05% tween-20 and incubated overnight at 4°c with monoclonal antibodies against heat shock protein 70 (hsp70; ab5439; abcam plc, cambridge, uk) or kdel receptor (ab69659; abcam plc). after washing three times, membranes were incubated at 37°c for 60 min with horseradish peroxidase-conjugated anti-mouse igg or antirabbit igg (abcam plc). detection used chemiluminescence luminal reagents (pierce biotechnology, waltham, ma, usa). the porcine vinculin (vcl) were amplified from the cdna obtained from pam cells. restriction enzyme sites were incorporated into the primer sequences to facilitate molecular cloning. pcr products were cloned into the pcaggs vector to produce the porcine vcl expression vector. for transfection, cells were seeded in 6-well plates (corning) and transfected at 70-80% confluency with respective constructed plasmids dna by using lipofectamine 3000 (life technologies), according to the manufacturer's instructions. the hp-hun4 or hun4-f112 was infected in marc-145 cells at 36 h post transfection. empty vector transfection samples served as controls in the experiment. at 48 h post infection, total rnas of prrsv-infected cells were extracted using the rneasy mini kit (qiagen), the viral rnas in supernatants were isolated using qiaamp viral rna mini kit (qiagen), according to the instruction manual. all the isolated rnas were used as the template for synthesis of firststrand cdna by rt-pcr using rt primed by oligo (dt) 18 primer using the primescript rt master mix (perfect real time, takara), according to the manufacturer's instructions. then the cdna templates were quantified using prrsv-specific real-time rt-qpcr. [28] 3. results a total of 2849 proteins were detected by lfqp and are displayed in a heatmap (figure 2) . statistical significance was determined using unpaired t-tests. for all tests, a p-value of <0.05 and ratio of >2 or <0.5 was considered to indicate a significant difference. glyceraldehyde 3-phosphate dehydrogenase was the internal normalization control and the ratio of glyceraldehyde 3-phosphate dehydrogenase between hp and ap-prrsv was 0.95 ± 0.55. the "only one exists" group indicated that proteins were detected only in one group but not another group due to low expression level ( table 1) . correlation analysis indicated good repeatability of the technology (figure 3) . a total of 95 differentially expressed proteins were identified ( table 2) . a control group was used to exclude false-positive interference. data from the control group was also used to elucidate functions of target proteins. differentially expressed proteins between control and hp-prrsv-infected cells (con/hp) and the www.advancedsciencenews.com www.proteomics-journal.com p <0.01 and ratio >2 or <0.5 indicated quantitative difference between two groups. the "only one exits" group indicated that proteins were detected three times in one, but not in the other group. hp-prrsv group means proteins identified in the hp-prrsv infected pams, while ap-prrsv was proteins detected in the attenuated pathogenic prrsv infected pams. control was displayed as the mock. control and ap-prrsv-infected cells (con/ap) are in supporting information. to extend the molecular characterization of quantitative differences and only-one-exists groups, uniprot and go databases were used to characterize information about biological processes (bps), molecular functions (mf), and cellular components (cc). bps of h (vh/va >2, including 41 proteins) and a (vh/va < 0.5, including 54 proteins) groups are in figures 4a and b bp. in group h, go annotations were primarily distributed in response to stimulus (70.7%), metabolic process (68.3%), and immune system process (43.9%). ratios were 63.0% response to stimulus, 90.7% metabolic process, and 33.3% immune system process in group a. the most significant difference in bp between pams infected with hp-prrsv and ap-prrsv was seen for metabolic process, which may be the major reason for the large differences among animals challenged with different virulence of prrsv. molecular function categories of h group and a group were shown in figure 4a ,b mf. most different molecular functions were classified as binding (87.8 and 90.7%) and catalytic activity (31.7 and 44.4%). binding of h group included enzyme binding (31.7%), nucleic acid binding (31.7%) and protein complex binding (26.8%), while group a mainly involved nucleic acid binding (31.5%), nucleoside phosphate binding (27.8%) and nucleotide binding (27.8%) from the analysis of go distribution by level 4. enzyme code distribution suggested there were five transferases (12.2%), one hydrolase (2.4%), one lyase (2.4%), and two ligases (4.9%) detected in group h. while four oxidoreductases (1.9%), five transferases (9.3%), five hydrolases (9.3%), three lyases (5.6%), and three isomerases (5.6%) in group a. cc categories were illustrated in figure 4a b cc. ninety-five detected differentially expressed proteins were annotated and categorized to ccs of macromolecular complex (58.9%), membrane (57.9%), membrane-enclosed lumen (57.9%), and extracellular region (44.2%). because of technological problems, we were unable to conclude that all the detected proteins were indeed mps. however, 26 were confirmed based on the annotation of uniprot database and there also existed proteins partially anchored on the membrane or binding with the mps. to verify the differentially expressed proteins via lc-ms/ms for lfqp, western blots were conducted for two proteins partially located on membranes. expression of hsp70 and the kdel receptor from cell lysates of vhun4-infected and vhun4-f112-infected pams, and dmem-inoculated pams were tested with antibodies to the proteins. lfqp showed that the ratios between vhun4infected and vhun4-f112-infected pams reached 0.67 and 0.51, respectively. western blots confirmed lfqp results ( figure 5 ). to examine whether the differentially expressed proteins detected affects virus infection, the vcl transient overexpression vector was transfected into marc-145 cells, followed by the hp hun4 or its attenuated strain infection. the prrsv-specific rt-qpcr results showed that vcl protein could inhibit both viruses, especially for hp-hun4 strain. compared with the empty vector in this study, lfqp of mps of hp-prrsv-, ap-prrsv-infected pams and the control was performed. important information about target proteins related to virus infection was obtained. the hp-prrsv strain vhun4 and its derivative, the serially cellpassaged attenuated strain vhun4-f112, [29] revealed different infection mechanisms in pams. a total of 2849 proteins were identified among the control, ap-prrsv-infected, and hp-prrsv-infected pams. of these, 2400 were detected in all three groups (figure 7) . we focused on 95 differentially expressed proteins of ap-prrsv-and hp-prrsv-infected pams; among these, 43 were detected in only one group. prrsv has been a threat to the global pig economy for several years because of its persistent infection, immune escape, and high mortality from inflammation and high fever. [30] the attenuated prrsv vhun4-f112 vaccine strain attenuated from hp-prrsv vhun4 by serial passages, which is now used in china. [31] we analyzed mps to identify factors associated with immunological effects and determine differences between hp-prrsv and ap-prrsv. mps classified based on go analysis are in table 3 . a heatmap based on the uniprot database was constructed to comprehend the functions and bps of differently expressed proteins ( figure 8b) , which revealed clustering and abundance of the 26 detected proteins that existed confidentially on the membrane based on uniprot database annotation. we first focused on proteins associated with immunology and inflammation with higher abundance in ap-prrsv-infected pams. ptpn2 (vh/va = 0.419), a member of the protein tyrosine phosphatase family, functions as signaling molecule that regulates cellular processes related to the jak-stat, il-3, il-5, and granulocyte-macrophage colony-stimulating factor (gm-csf) signaling pathways; dephosphorylation of nonreceptor kinases including jak1, jak3, stat3, and stat6; and negative regulation of il-2-, il-4, il-6, and ifn-mediated signaling. ptpn2 also functions in the response to inflammation via nf-κb. [32] [33] [34] sipa1 (h-/a+) is a mitogen-induced gt-pase activating protein for ras-related regulatory proteins. it was related to g-protein signaling and blood-brain barrier and immune cell transmigration: vcam01/cd106 (cluster of differentiation) signaling pathways. [35, 36] c5ar1 (h-/a+) is the receptor for the chemotactic and inflammatory peptide anaphylatoxin c5a, which stimulates chemotaxis, granule enzyme release, intracellular calcium release, and superoxide anion production, participates in the innate and adaptive immune responses to www.advancedsciencenews.com www.proteomics-journal.com table 3 . statistics analysis of proteins that existed definitely on the membrane. the lectin-induced complement pathway. [37] [38] [39] [40] [41] [42] polyribonucleotide 5 -hydroxyl-kinase clp1 (clp1) (vh/va = 0.136) mainly acts as a kinase that binds atp hosting 5 -hydroxyl-kinase activity, and functions in mrna cleavage and in sirna loading onto the rnainduced silencing complex involved in rna interference and its destruction. the kinase hclp1 phosphorylates and licenses synthetic sirnas to assemble into an rna-induced silencing complex for cleavage of target rna. [43] expression of these molecules was higher in the ap-prrsv-infected group and had a similar level to the control group. therefore, we concluded that they might be related to the immune escape of hp-prrsv and that the attenuated vaccine strain would not have side effects on these immune factors expression in the host cell, and these proteins above would contribute to the resistance of prrsv. some proteins related to immunology and inflammation had higher expression in hp-prrsv-infected cells. raftlin (vh/va = 2.554) protein is pivotal for maintenance of lipid rafts and may be involved in regulation of b-cell antigen receptor-mediated signaling. raftlin promotes binding of double-stranded rna, activations of b cell receptors and toll-like receptor 3 signaling pathways, and is involved in il-17 production to release proinflammatory cytokines. hence, the higher abundance of raftlin in the hp-prrsv group compared to the ap-prrsv and control groups may explain the more severe inflammation triggered by hp-prrsv. [44, 45] iars (h+/a-), isoleucyl-trna synthetase is a target of autoantibodies in autoimmune diseases. [46, 47] vcl (h+/a-), vinculin is a cytoskeletal protein associated with cellcell and cell-matrix junctions, and is related to the il-3, il-5, and gm-csf signaling pathways. [48] rap2a (h+/a-) is a member of the ras oncogene family, small gtp-binding protein, of which active form interacts with several effectors [49] [50] [51] related to the erk and g protein signaling pathways. [52, 53] it was reported that prrsv could induces prostaglandin e2 production through cyclooxygenase 1 and is related to erk signaling. [54] the differential expression of rap2a may provide information for future research on the interaction between prrsv and the erk signaling pathway. the abundance of rap2a was lower in ap-prrsv-infected pams than in hp-prrsv-infected pams and control cells, which had similar levels. thus, we hypothesized that rap2a was associated with immunization with the attenuated vaccine. ifn-induced transmembrane protein 3, ifitm3 (vh/va = 2.861), responds to biotic stimulus and had the highest expression levels among these proteins in the hp-prrsv group, as compared to the proteins identified in the ap-prrsv group but it was not detected in the control group. in humans, this protein negatively regulates the entry of multiple viruses, including influenza a virus, sars coronavirus, marburg virus (marv), ebola virus, dengue virus, west nile virus (wnv), human immunodeficiency virus (hiv) type 1, and vesicular stomatitis virus (vsv), into host cells. [55, 56] it could inhibit hemagglutinin protein -mediated entry of influenza virus, gp1, two-mediated viral entry of marburg virus and ebola virus, s protein-mediated viral entry of sars coronavirus, and g protein-mediated viral entry of vsv, thereby playing a critical role in the structural stability and function of vacuolar atpase (v-atpase). establishing physical contact with the v-atpase of endosomes is critical for proper clathrin localization and is required for v-atpase to lower the ph in phagocytic endosomes, thus establishing an antiviral state. [57] high expression of both hp-prrsv and ap-prrsv suggests that ifitm3 may help inhibit prrsv entry and promote resistance to hp-prrsv infection. peptidyl-prolyl cis-trans isomerase b (ppib, vh/va = 0.406) has peptidyl-prolyl cis-trans isomerase activity and is involved in protein folding and protein peptidyl-prolyl isomerization, which can accelerate protein folding. [58] ppib is positively regulated by viral genome replication, is involved with the viral processes of hepatitis c virus (hcv), and interacts with and stimulates the rna-binding activity of hcv ns5b. ppib is critical for efficient replication of the hcv genome. [59] compared with the control group, ppib was downregulated in the hp-prrsv and ap-prrsv groups. the abundance of ppib was lower in the hp-prrsv group than the ap-prrsv group, suggesting an association with prrsv replication. string network analysis was used to elucidate interactions among differentially expressed proteins. the essential factors and receptors involved in the entry of prrsv are reportedly cd163 (q2vl90), nmhc ii-a (myh9, f1skj1), sialoadhesin (sn, a7lcj3), cd151 (f1ryz1), and vimentin (vim) (p02543). [60] [61] [62] [63] [64] [65] we assessed the involvement of these mps together with proteins detected by string analysis on viral entry. receptor proteins involved in prrsv entry are in figure 9 to better understand the characteristics of pathway-like regions, the genomrnai database was analyzed to annotate detected proteins. [66] regions containing proteins with higher abundance in hp-prrsv-infected pams are green and in ap-prrsv-infected pams are red ( figure 9a) . a search of the genomrnai database determined that linked proteins ik (ik cytokine), wd repeat domain 12, dyskerin pseudouridine synthase 1, and adenylosuccinate lyase (adsl) in the green region of figure 9a shared similar annotations and functioned to decrease expression of nf-κb or il-8, [67] which are related to inflammation. [44, 45] hence, further analysis of these linked proteins may help explain differential mechanisms between hp-prrsv and ap-prrsv infection. other proteins in the green-colored region of 9a are reported to influence virus infection. nucleolar and coiled-body phosphoprotein 1 increases sindbis virus infection [68] and adsl increases human papilloma virus 16-gfp infection. [69] the lower abundance of nucleolar and coiled-body phosphoprotein and adsl in the hp-prrsv-infected group may be related to the host immune response repression of prrsv and may be used by hp-prrsv during infection to self-upregulate. the red region of figure 9a contains some linked proteins with some figure 9 . protein-protein interaction network determined by string software showing interactions among differentially expressed proteins between two virulent groups with cd163, vim, and myh9 added. red, protein abundance higher in hp-prrsv than ap-prrsv; green, protein abundance in hp-prrsv lower than in ap-prrsv. line colors represent type of evidence for association: green, neighborhood; red, fusion; purple, experimental; light blue, database; black, expression; blue, co-occurrence; yellow, text mining. gene abbreviations are shown. vh, protein abundance in hp-prrsv; va protein abundance in ap-prrsv. vh/va = 0, detected only in ap-prrsv; vh/va = null, detected only in hp-prrsv. information. uridine monophosphate synthetase decreases hiv-1 infection, [70] while programmed cell death 5 decreases hcv replication, [71] both downregulate nf-κb expression. [67] the lower abundance of these proteins in the hp-prrsv group suggested that hp-prrsv escaped inhibition through an unknown mechanism. myosin-9 (myh9) appears to function in cytokinesis, cell shape, and specialized functions such as secretion and capping. [72] myh9 is an important factor for prrsv infection and interacts with gp5 of prrsv, [65] although the underlying mechanisms remain unclear because of limited research. as shown by our results, myh9 was related to vcl in ap-prrsv and actl6 in hp-prrsv. vcl is an actin filament (f-actin)binding protein involved in cell-matrix adhesion and cell-cell adhesion in humans, regulation of cell-surface e-cadherin expression, and potentiation of mechanosensing, and may be important in cell morphology and locomotion by promoting binding with actin, alpha-catenin, cadherin, dystroglycan, and ubiquitin protein ligase. [73] in hiv research, transient overexpression of vcl reduced the susceptibility of human cells to infection with hiv-1 and negatively affected paxillin phosphorylation and limited retroviral infection. [74] just like hiv-1, in our study, it was demonstrated that over expression of vcl could inhibit the replication of both hp-prrsv and the attenuated prrsv in the mrna level. there were obvious differences in the inhibiting ability for hp-prrsv and its attenuated strain. actl6a, which is an actin-like protein 6a, is involved in transcriptional www.advancedsciencenews.com www.proteomics-journal.com activation and repression of select genes by chromatin remodeling (alteration of dna-nucleosome topology) and mainly functions in chromatin binding and transcription coactivator activities. [75] we found that myh9 may regulate ap-prrsv and hp-prrsv infection via different pathways. through super pathways annotation (www.genecards.org), vcl and actl6a were identified as participants in the il-3, il-5, gm-csf, and tnf-α/nf-κb signaling pathways, where they may help with differential mechanisms of prrsv infection with different virulence. using the genomrnai database, some linked proteins were found to be related to viruses or inflammation ( figure 9b ). in the green region, gnas complex locus (gnas) increased human papilloma virus 16-gfp, [69] and serine/arginine repetitive matrix 2 decreased hcv infection, influenza a replication and viral numbers, and il-8 expression, [76, 77] small nuclear ribonucleoprotein u1 subunit 70 decreased influenza a replication and viral numbers. [77] irf-3 decreased infection by hcv, west nile virus, and dengue virus. [76, 78] in the red region, hsf1 decreased hcv replication. [76] protein kinase camp-activated catalytic subunit alpha decreased vsv infection. [79] signal transducer and activator of transcription 6 decreased il-8 expression. rap2a, mink1, and gnas are regulatory proteins in the ras pathway. rap2a and mink1 were detected only in the hp-prrsv group, whereas gnas was detected only in the ap-prrsv group, in accordance with a previous report that stimulation of ras increases the replication ability of hcv by reducing ifn-jak-stat pathway activity. [80] we proposed that prrsv was also related to the ras pathway, and differed between hp-prrsv and ap-prrsv. creb-binding protein (crebbp) is composed of doublestranded rna-activated transcription factor with irf-3, and double-stranded rna-activated transcription factor is activated in many virus-infected cells to promote apoptosis. [81, 82] crebbp may interact with human herpes virus 8 virf-1, which could inhibit the binding of crebbp to irf-3. [83] our results showed that irf-3 abundance was tenfold lower in the hp-prrsv than the ap-prrsv group, which we proposed was a protective mechanism of prrsv to escape from host immunity and ensure survival after viral infection. the ability of hp-prrsv hun4 to induce cell apoptosis is stronger than classical prrsv (ch-1a) in immune organs and lungs of piglets. [84] we concluded that hp-prrsv might interact with crebbp and decrease irf-3 expression to escape from host immunity and cause severe damage to the cell, highlighting a difference from ap-prrsv. notable differences exist during the infection of ap-prrsv and hp-prrsv. hp-prrsv could inhibit host immune function and evade the immune response via unknown mechanism. [85, 86] label-free ms was performed using ap-prrsv-infected and hp-prrsv-infected pams. this is the first attempt to explore the differential characteristics between hp-prrsv and its attenuated prrsv infected pams focusing on membrane proteins. by analyzing detected proteins in hp-infected, ap-prrsv-infected, and control group, proteins related to the immune response or virus replication were identified that may elucidate unique pathways used by different virulent prrsvs for cell entry, virus replication, and immune escape mechanisms. researches on these detected proteins will help with the elucidation of the identity, the expression abundance, and significance of them, future study will be focused on functions of these key membrane proteins to deepen our understanding of differential mechanisms between hp-prrsv and ap-prrsv infection. proc. natl. acad. sci proc. natl. acad. sci supporting information is available from the wiley online library or from the author. the authors declare that they have no conflicts of interest associated with this report. keywords attenuated, highly pathogenic, infection, label-free quantitative proteomics, membrane proteins key: cord-021393-9loesliv authors: meade, h.m.; echelard, y.; ziomek, c.a.; young, m.w.; harvey, m.; cole, e.s.; groet, s.; smith, t.e.; curling, j.m. title: expression of recombinant proteins in the milk of transgenic animals date: 2007-09-02 journal: gene expression systems doi: 10.1016/b978-012253840-7/50015-8 sha: doc_id: 21393 cord_uid: 9loesliv nan introduction of foreign dna into the murine genome by microinjection and the generation of transgenic offspring were first reported by gordon et al. (1980) and gordon and ruddle (1981) . this technique has been applied to the production of transgenic livestock. using mammary gland-specific promoters, a wide range of proteins of biopharmaceutical interest have been expressed in rodents, pigs, and dairy animals. an expression vector, comprising a gene encoding the target protein of interest fused to a milk promoter gene, is introduced by microinjection into the pronucleus of a one-cell embryo. upon germ line integration and expression, the transgene becomes a dominant mendelian genetic characteristic that is inherited by the progeny of the founder animal. the transgenic offspring may express the target protein in gram per liter quantities, most frequently as a soluble whey protein. mammalian mammary epithelial cells have the capacity to carry out complex protein synthesis with a variety of posttranslational modifications and proper folding. coexpression of modifying enzymes in the epithelial cell golgi apparatus may allow the heterologous protein to be engineered to confer specific or desired pharmacokinetic characteristics. the milk of transgenic livestock presents an excellent starting material from which human diagnostic or pharmaceutically active, therapeutic proteins may be purified using established technologies of the dairy and biopharmaceutical industries. to date (1997) , probably more than 50 proteins have been expressed in the milk of transgenic mice, rats, rabbits, goats, sheep, pigs, and dairy cows. phase i clinical trials with antithrombin iii produced in transgenic goats were completed successfully in 1996, and phase ii clinical trials are currently being performed in the united states. ~l-antitrypsin produced in transgenic sheep is currently in phase i clinical trials in the united kingdom. in addition, human lactoferrin and fibrinogen expressed in the milk of transgenic cows and sheep, respectively, are in the late stages of development or preclinical evaluation. the field of transgenic research has been reviewed periodically since 1990. the reader will find the following articles, which trace the development of transgenic technology applied to dairy animals, of interest: henninghausen (1990) , henninghausen et al. (1990) , bialy (1991) , wilmut et al. (1991) , wall et al. (1992) , j~inne et al. (1992, 1994) , logan (1993) , ebert and schindler (1993) , lee and de boer (1994) , houdebine (1994 houdebine ( , 1995 , and echelard (1996) . this chapter discusses the expression of therapeutically useful proteins in mammalian milk with a focus on dairy animals as the production systems of choice and reviews expression constructs, milk-specific transgenes, transgene insertion, transgenic animal production, protein biosynthesis and secretion, lactation, milk, protein purification, and quality and regulatory issues. laboratory mice have served as a model expression system for foreign proteins since the inception of animal transgenesis and are used frequently for feasibility studies concomitant with or prior to the generation of larger, founder transgenic animals. many recombinant proteins of interest in human therapy are, by their very nature, biologically active in most mammals. the murine model is useful in determining, at an early stage of research, the transgene expression characteristics and potential effects on animal health. transgenic mice are generally predictive of what will be observed in larger animals. many transgenic proteins have been expressed in the milk of transgenic livestock. table 1 summarizes the published results of expression in transgenic farm animals during the period from 1990 to 1996. several of these proteins have been expressed at high levels, demonstrating the usefulness of the production system. examination of a great number of transgenic lines has shown that mammary gland-specific expression of a target protein is associated with increased plasma levels of this protein, even in the absence of ectopic expression. the most likely explanation is leakage of the protein from the milk into the circulation through the junctional complexes of the mammary epithelial cells. therefore, certain proteins and peptides, such as highly active hormones and cytokines, cannot be expressed in the mammary system as their secretion into the blood may have severe detrimental effects on the host. lonnerdal and iyer (1995) agene insertion via teat canal using retroviral vectors galv and momlv. bdata from genzyme transgenics corp. (1996) . transgenes containing sequences of several milk protein genes, reviewed by maga and murray (1995) and echelard (1996) , have been used to direct the expression of exogenous proteins to the lactating mammary gland. these transgenes are usually chimeric, being derived from the fusion of a target protein gene and mammaryspecific regulatory sequences. although both genomic dnas and cdnas coding for target proteins have been used for expression, higher levels are normally obtained with genomic dnas. the incorporation of untranslated exons and introns may contribute to increased expression of the transgene . addition of a signal sequence is necessary if the exogenous protein is not normally secreted. this will cause the protein to be secreted out of the mammary tissue into the milk. regulatory sequences from several milk-specific genes have been isolated and tested in transgenic animals: ovine ~-lactoglobulin; murine, rat, and rabbit whey acidic protein (wap); bovine ~-sl casein; rat, rabbit, and goat f~-casein; and guinea pig, ovine, caprine, and bovine ~-lactalbumin. of these promoters, several have permitted grams per liter expression of target proteins in the milk of transgenic offspring, sometimes in large dairy animals; some of this work is summarized next. the ovine f~-lactoglobulin gene contains seven exons and six introns spanning a 4.2-kb region (harris et al., 1988) . the first reported ovine ~-lactoglobulin chimeric transgenes (archibald et al., 1990) were composed of 4 kb of 5' flanking fused to the c~l-antitrypsin genomic sequences. other configurations using variable amounts of 5'-and 3'-flanking sequences have also been used (whitelaw et al., 1992) and reviewed by maga and murray (1995) . with the ovine ~-lactoglobulin gene, high-level expression (g/liter) of ~l-antitrypsin, fibrinogen, and hsa have been reported (wright et al., 1991 , shani et al., 1992 prunkard et al., 1996) . however, similar results have not been observed with transgenes containing cdna sequences (clark et al., 1989 , shani et al., 1992 , hansson et al., 1994 , yull et al., 1995 . rodent wap genes consist of four exons and three introns: the middle two exons encode the two cysteine-rich regions, which probably form separate protein domains (campbell et al., 1984) . wap is present in the milk of mouse, rat, rabbit, and camel, but is absent from the milk of cow, sheep, pig, goat, and human. no recognizable wap gene homologues have been isolated from these species. rat (wei et al., 1995 , yarus et al., 1997 , mouse (gordon et al., 1987; ebert et al., 1991 , reddy et al., 1991 velander et al., 1992; drohan et al., 1994; hansson et al., 1994; limonta et al., 1995) , and rabbit (bischoff et al., 1992; devinoy et al., 1994; th4pot et al., 1995) wap regulatory sequences have been used to direct expression of exogenous proteins to the mammary gland. high-level expression of the target protein was observed with mouse and rabbit wap transgenes. surprisingly, relatively high expression levels (up to 1 g/liter)were observed in transgenic pigs with a construct containing 2.6 kb of 5' and 1.3 kb of 3' mouse wap sequences linked to a human protein c cdna (velander et al., 1992) . this result seems to indicate that mwap regulatory sequences can function efficiently in species that do not have an endogenous wap gene. the bovine asl-casein gene contains 9 exons and spans 17.5 kb (koczan et al., 1991) . originally (meade et al., 1990) , a transgene containing 21 kb of 5' and 2 kb of 3' flanking sequence fused to the genomic sequences of human urokinase was shown to direct high milk expression levels (1-2 mg/ml) in mice. promising results were also obtained in transgenic rabbits with a construct containing the human igf-1 cdna (brem et al., 1994) , in mice with the human lysozyme cdna (maga et al., 1994) , and in human lactoferrin and human granulocyte-macrophage colony-stimulating factor genomic constructs (nuijens et al., 1995; uusi-oukari et al., 1997) . conversely, a bovine ~sl-casein-human lactoferrin cdna construct only permitted low-level expression in milk of transgenic mice (platenburg et al., 1994) , as was the case with a human tpa cdna construct fused to 1.6 kb of bovine ~-sl casein 5'-flanking sequences (riego et al., 1993) . the bovine ~-lactalbumin gene contains four exons and three introns. early reports (vilotte et al., 1989; stinnakre et al., 1991) indicated that a construct containing 750 bp of 5' and 336 bp of 3' flanking region was sufficient to direct intermediate expression levels in transgenic mouse milk when fused to bovine ~-lactalbumin or ovine trophoblastin cdnas. by using a construct containing the same amount of 5'-flanking sequence linked to hgh genomic sequences, higher levels of the target protein (up to 4.3 mg/ml) were obtained in the milk of transgenic rats (ninomiya et al., 1994) . however, at this point, results obtained with ~-lactalbumin-driven transgenes in large animals have not been reported. the caprine ~-casein gene (csn2) has been cloned and sequenced (roberts et al., 1992; persuy et al., 1992) . the intron/exon organization of the 9-kb goat gene is similar to that of other csn2 genes and its expression is limited principally to the mammary gland during lactation. high-level expression was observed in goats transgenic for a construct containing 6.2 kb of 5' and 7.1 kb of 3' goat ~casein flanking noncoding sequence fused to a variant of the human tpa cdna . high-level expression with caprine f~-casein-containing transgenes has also been observed, in mouse milk, with bovine k-casein (persuy et al., 1995; gutierrez et al., 1996) , antithrombin iii (cdna and genomic, mice and goats), hsa (cdna and genomic), ~l-antitrypsin (genomic, mice and goats), and both heavy and light chains of several humanized antibodies (h. meade et al., unpublished data) . transgenic animals may be generated by direct microinjection of the foreign gene into the pronuclei of one-cell stage embryos. microinjection techniques have been reviewed exhaustively by hogan et al. (1986) and pinkert (1994) , among others. techniques initially developed for gene insertion into murine pronuclei (gordon et al., 1980; gordon and ruddle, 1981; brinster et al., 1985; palmiter and brinster, 1986) have been adapted to gene transfer into the pronuclei of ruminants and pigs (hammer et al., 1985; pinkert, 1994) . if the microinjected dna integrates into the genome of the recipient before the first cell division occurs, a heterozygous founder can be created. later integration leads to genetic mosaics consisting of normal cells with a normal genome and cells with transgenomes. generally, fertilized eggs are flushed from the oviduct of a superovulated female donor, microinjected with a few hundred copies of the transgene, transferred to the oviduct or uterus of a pseudopregnant recipient animal, and developed to term. the first transgenic offspring, or founder animals, are at best hemizygous as the transgene is not integrated into both copies of a pair of homologous chromosomes. in the case of mosaic founders, germ line transmission is not always observed. in addition, multiple transgene integration sites have been detected in 10-20% of transgenic founders. to optimize the collection and transfer of microinjectable goat embryos, selgrath et al. (1990) established regimens for superovulation/synchronization and timing of pronuclear embryo collection. does were synchronized with norgestomet ear implants and superovulation was induced with pregnant mare serum gonadotropin (pmsg) or follicle-stimulating hormone (fsh-p). does were hand mated with the average female being mated six to eight times by two different males over a 24-to 36-hr period. embryos were recovered surgically and pronuclei could be visualized without centrifugation. after microinjection the embryos were transferred to the reproductive tracts of recipient females using a surgical procedure similar to that used for the embryo collection. ewes may be similarly synchronized and superovulated during the breeding season using progestin (30 mg fluorogestone acetate) pessaries and fsh injection (rexroad and wall, 1987) . fertilized sheep oocytes are semiopaque and not readily visible. however, differential interference contrast microscopy allows visualization, and successful microinjection may be determined by the swelling of the pronucleus, which occurs on injection of dna (simons et al., 1988) . microinjected embryos are then transferred to recipient ewes. bovine oocytes are generally collected from slaughtered heifers or obtained from cows superovulated with pmsg or prostaglandin. to circumvent surgical procedures and in vivo fertilization, krimpenfort et al. ( 1991 ) have used an/n vitro fertilization and embryo production procedure. the technique for dna microinjection is similar to those described earlier: centrifugation, e.g., at 12,000 • g for 10 min, is necessary to visualize pronuclei. microinjected cow embryos are usually cultured/n vitro to the morula-blastocyst age at which time they are transferred nonsurgically to suitable recipients. the techniques and efficiency of gene transfer in cows have been described by roschlau et al. (1989) and mcevoy and sreenan (1990) . synchronization of sows is accomplished with hormonal treatment and superovulation induced with pmsg or human chorionic gonadotropin. synchronization, however, has been shown to affect the farrowing rate (pursel et al., 1990) . pig ova are opaque and no nuclear structures can be seen, even using interference contrast microscopy. centrifugation at 15,000 • g for 5 min leaves pronuclei visible in the equatorial segment of the cytoplasm (brem et al., 1985; hammer et al., 1985) . microinjected embryos are then transferred to recipient pigs. factors affecting the success of microinjection as a gene insertion technique have been reviewed by rexroad et al. (1990) . experience in microinjection is an important factor as well as dna concentration and the gene construct. the stage of egg development and the quality of eggs may affect the efficiency of producing transgenic animals. three factors contribute to problems of microinjection of livestock embryos compared to mice. cytoplasmic vesicles may obscure the view of pronuclei, fewer embryos are available for microinjection, and there is considerable variability in the stage of embryo development at the time of embryo collection. despite these challenges, there has been considerable success in developing transgenic goats, sheep, pigs, and cows. key reproduction parameters and the success rate of transgenesis are summarized in table 2 . in summary, oocytes may be fertilized in vivo or in vitro. in vivo fertilization may be controlled by artificial insemination of a superovulated animal at the stage where the oocyte has matured in the ovary. an alternative pathway is to isolate follicular oocytes from the ovary of the donor animal and proceed to in vitro maturation and fertilization prior to microinjection and implantation in the recipient female. in goats, sheep, and cows the rate of transgenic births is 5-10%. embryonic stem (es) cells may offer an alternative to pronuclear microinjection for achieving transgenesis. however, pluripotent es cells able to contribute to the germ line have only been described in mice. there have been descriptions of chimeric animals generated with rat, pig, and cow es cells (iannaconne et al., 1994; wheeler, 1994; stice et al., 1996) , but in the case of rats and pigs no evidence of germ line transmission from these cells has been reported. cow fetuses obtained following nuclear transfer of es cell-derived nuclei in recipient oocytes died in utero, exhibiting major defects in placental development (stice et al., 1996) . in sheep, the first large animals derived from cultured cells were described in 1996 by campbell et al. two healthy phenotypicaly female lambs were born from embryos generated by transferring nuclei isolated from embryo-derived cells into enucleated oocytes. dna analysis demonstrated that all the nuclear transfer lambs and fetuses were derived from the cell line. it is not yet clear whether foreign dna can be introduced in this type of cell line, and there are questions about the health and reproductive fitness of the recovered offspring. nevertheless, this experiment is certainly a step toward the possibility of replacing pronuclear microinjection as the method of choice for the generation of transgenic large animals. another potential alternative to the pronuclear microinjection of the transgene is the use of replication-defective retrovirus vectors. archer et al. (1994) have described a procedure in which the construct is infused directly into the mammary gland, via the teat canal, during a period of hormone-induced mammogenesis. a gibbon ape leukemia virus was used to deliver the structural gene encoding for human growth hormone resulting in expression of the hormone in goat mammary epithelial cells. the advantage of this method is that expression of the recombinant protein is obtained quickly, without the delay caused by the generation interval required to generate a producing transgenic animal (18 months for goats). however, reported production levels (archer et al., 1994) are very low (see table 1 ) and at this point in time can only be used for analytical purposes. transgenic animals for the production of therapeutic and diagnostic proteins are produced by transferring fertilized, transgene-carrying embryos to recipient animals. following natural gestation and birth, offspring are subject to tissue biopsy and blood sampling: usually ear tissue and blood are screened by polymerase chain reaction and/or southern analysis for the presence of the transgene. animals identified as being transgenic are mated with nontransgenic animals: transgenic founder females will produce the protein for which the dna codes in their blood or milk depending on the tissue-specific, regulatory promoter sequence of the transgene. subsequently, transgenic female progeny derived from breeding founder females and males will also express the transgenic protein. in goats, 16-18 months are required before the first milk is obtained from a natural lactation of a female transgenic animal. however, milk samples can be obtained from founder transgenic females, as well as approximately 30% of male transgenic animals by hormonally induced lactation at about 13 months after microinjection. induced lactation is useful in checking the expression level and integrity of the heterologous protein. the original precursors of most of the milk constituents are cellulose, starch, protein, fat, minerals, and vitamins of the plant materials of the ruminant diet. water is also a prerequisite, and dairy cows require 3-4 liters of water per liter of milk produced. rumen microorganisms synthesize amino acids, which are adsorbed into the bloodstream. the milk protein precursor amino acids are adsorbed from the bloodstream via the extracellular fluid between the capillaries and the epithelial cells, across the basement membrane of the mammary epithelial cells. protein biosynthesis is carried out in epithelial cells, and milk proteins are discharged into the lumen of the alveolus by exocytosis and then into the ducts. in ruminants, the ducts empty into a single, primary duct or cistern, which provides extra milk storage capacity. up to 30% of the milk in the udder is held in the cistern. as measured by the concentration difference in arteriovenous blood, the mammary gland is particularly efficient at extracting amino acids: 80% of the arterial methionine; 70% of the phenylalanine, leucine, and threonine; 60% of the lysine, arginine, and isoleucine; 55% of the histidine; and 50% of valine are adsorbed. more than 25 % of the arterial blood glucose is removed during passage through the mammary gland and is used to power protein synthesis, fat, and lactose production. intermediates required for protein synthesis are produced in the cytosol and mitochondria. protein is synthesized at polyribosomes in the rough endoplasmic reticulum where removal of signal peptides occurs. glycosylation is carried out in the endoplasmic reticulum and the golgi apparatus where phosphorylation, other posttranslational modifications, and the assembly of casein micelles are carried out. studies with radiolabeled amino acids indicate that proteins are synthesized in 3-15 min. radioactivity is seen in the golgi apparatus after 15-30 min, and the label concentration in the lumen increases after a further 30-60 min. immunoglobulins and albumin present in milk are not synthesized in the mammary gland but by plasma cells and hepatocytes, respectively, and enter the milk by active transport or filtration. during lactation, b lymphocytes migrate to the mammary gland where they become plasma cells. plasma cells lodged in the interstitial space may contribute to the high igg concentration present in the colostrum of early lactation. in the cow, approximately 500 liters of blood is required to provide the precursors for 1 liter of bovine milk: the blood flow in the udder is ca. 280 ml per second. the goat uses about 400 liters of blood to produce 1 liter of milk: the blood flow in the udder is ca. 1200 liters per day. the mammary gland is able to secrete about 2 g of milk, containing approximately 18 mg of whey protein, per gram of tissue per day. a gram of tissue contains about 2 x 108 cells and the milk output is therefore of the order of 10 -8 g per cell per day. lymph drains from the udder of the cow at the rate of 1300 ml per hour and in the goat at the rate of 6.5-35 ml per hour. leukocytes, which account for the major part of the somatic cells found in milk, are derived from lymph. morc61 et al. (1994) have calculated the synthesis rate of human recombinant protein c in transgenic swine expressing the protein at 0.1-1 mg/ml milk to be approximately 14 mg per gram of mammary cell per day or about 14 pg/cell/day. in contrast, the rate of normal synthesis in hepatocytes was calculated to be 0.02 mg protein c per gram cells per day. it has been suggested that mammalian species phylogenetically close to humans may be expected to have more elements of the glycosylation machinery in common (jenkins et al., 1996) . initial reports indicate that human glycoproteins expressed in the mammary gland of transgenic animals contain glycosylation patterns that differ from those found on human plasma-derived proteins. in general, the glycosylation found on human proteins secreted into transgenic animal milk has been generally similar to plasma protein glycosylation. sites are mainly biantennary complex oligosaccharides with some variations consistent with the tissue and species of origin. cole et al. (1994) have shown that human an-tithrombin iii (at iii) and a long-acting form of htpa expressed in goat milk had some galnac replacing galactose on complex nlinked oligosaccharides. both latpa and at iii were shown to be more fucosylated than their recombinant or plasma counterparts. goat plasma at iii contains n-glycolylneuraminic acid and nacetylneuraminic acid, as do transgenically expressed at iii and latpa. an additional difference observed between plasma and transgenic goat at iii is in the degree of sialylation , with the transgenic protein less sialylated than plasma at iii. denman et al., (1991) have also noted that significantly lower levels of galactose, n-acetylglucosamine, and sialic acid are present in goat transgenic latpa compared to the murine c 127 cell line and chinese hamster ovary (cho) cell-derived latpa. the glycosylation of interferon-~ at asparagine 25 and 97 is influenced dramatically by cho cell culture conditions (curling et al., 1990) ; considerable variations in site occupancy are seen. transgenic mouse-derived interferon-~ has predominantly complex sialated biantennary n-glycans at asparagine 25, and oligosaccharides are ~ 1-6 core fucosylated similar to the asn2s of cho cellderived interferon-~ (james et al., 1995) . there is an increased incidence of oligomannose at asn97 compared to the cho-derived counterpart, suggesting that murine mammary epithelial cells may be deficient in the ~ 1-2 mannosidase i and glcnac transferase i activities in the endoplasmic reticulum. although an ultimate goal may be to produce proteins with authentic human glycosylation patterns, a more realistic and possibly more desirable objective is the production of proteins with defined, engineered glycosylation characteristics and therefore predictable pharmacokinetics. glycoproteins can be remodeled in situ by the transgenic coexpression of human glycosyltransferase. prieto et al. (1995) have demonstrated that the heterologous, transgenic expression of human ~l,2-fucosyltransferase results in expression of both transgene and secondary gene products. their work also suggests that the mammary gland may be a unique bioreactor for the production of biologically active oligosaccharides and glycoconjugates. in studies of the expression of rh protein c in transgenic pigs, subramanian et al. (1996) have shown that there are rate limitations of ~-carboxylation in mice and pigs, partly dependent on the transgene. their study indicates that a rate limitation of ~-carboxylation in mammary epithelial cells occurs at expression levels of >20 ~g/ml in mice and at >500 ~g/ml milk in pigs. mammary glands are skin glands that have no counterpart in nonmammals and are located in the inguinal region of cows, sheep, and goats. in the sow they are located along the thoracic, abdominal, and inguinal walls. streak canals link the internal milk secretory system with the external environment. the number of teats, teat orientation, and length of the let-down reflex affect the ease and periodicity of milking. milk is released as a result of a neuroendocrine reflex of the nervous system to tactile, auditory, and visual stimuli. negative psychological and environmental disturbances have a detrimental effect on milk production. milk let-down is a response to the release of oxytocin, synthesized in the hypothalamus, by the posterior pituitary gland. oxytocin is transported to the mammary gland in the arterial blood and binds to myoepithelial cells that contract, causing a release or let-down of the milk. sheep and goats have let-down periods of 1-2 and 2-4 min, respectively. the let-down reflex in the cow lasts for 5-8 min. in the sow, however, the reflex is extremely short, of the order of 10-20 sec, with a frequency of 1 hr or less. from the point of view of primary milk production, goats and cows are preferred animals because of the relatively long let-down reflexes, vertical teat orientation, milk volume, and duration of lactation. however, other factors, such as protein concentration, may favor a choice of sheep. goats, sheep, and cows respond to familiar signals, such as the sound of a milking machine, whereas lactating, nonsuckling sows require injections of oxytocin to elicit the let-down response. at parturition, a series of programmed hormonal changes take place that transform the mammary cells to the fully secretory state. stage 2 lactogenesis, or the copious production of milk, is brought about by a synchronous drop in progesterone, an increase in estrogen, and the release of prolactin from the anterior pituitary and follows the immediate postpartum production of colostrum. in the cow, milk production increases in the first 3-6 weeks of lactation and then slowly declines. a similar pattern is seen in goats, sheep, and pigs. milk secretion continues as long as milk is regularly withdrawn, although production declines during lactation. dairy animals are capable of undergoing estrus and pregnancy while maintaining their lactation. this enables dairy management to breed the animal such that it will give birth and begin lactation on a yearly basis. in order to maximize production, the animals are allowed to lactate until 2 months before they are due to give birth. they are then "dried off" by cessation of milking. the 2-month rest before the restart of lactation allows the animal to rebuild her energy reserves for the coming birth and lactation. following birth of the progeny, the animal once again begins the yearly lactation cycle. in all species, yield and energy content are related to body size. the expression level of exogenous protein tends to follow the normal milk output as can be seen from the plots in fig. 1 which shows (a) the production of a recombinant monoclonal antibody over a 220-day lactation of a transgenic goat and (b) the levels of antithrombin iii in the milk of a transgenic goat during a 300-day lactation. it has been noted that "considering the yearly milk output of dairy cattle (6000-8000 liters)and the milk content of as 1-casein (10 g/liter), one cow carrying a transgene under the control of as 1casein promoter would theoretically produce 60-80 kg/year of the transgene-derived protein" (j~inne et al., 1992) . at an expression level of 5 g/liter a transgenic goat is capable of producing about 4 kg of target protein per year; at 20 g/liter the output is 16 kg. it is, therefore, quite conceivable to produce 100-kg quantities of target protein in small goat herds or sheep flocks. milk is a multiphasic fluid composed of a fat emulsion, a micellular casein dispersion, a colloidal suspension of lipoproteins, and a solution of proteins, mineral salts, vitamins, organic acids, and minor components. when collected, milk is not sterile and contains bacteria derived from the teat as well as somatic cells derived mostly from the lymphatic ducts of the udder. according to the pasteurized milk ordinance (u.s. department of health and human services, 1993) , the bacterial plate count should be less than 100,000 per milliliter. milk composition is species specific; major differences are seen between human and ruminant milk. within a species, the composition varies with breed, diet, and other factors. volume and composition also vary during lactation. table 3 gives the average percentage compositions of livestock milk. milk is approximately 85-90% water; the ph is 6.5-6.7 and as high as ph 6.8 in ewe's milk. it is important from a purification point of view that the fat is present in globular form in the size range of 0.1-10 ~m and with a density higher than the other constituents. the fat globule is enclosed in a membrane of polar lipids and proteins. triglycerides make up 97% of the fat. the three subgroups of casein, as-, 13-, and k-casein, display genetic polymorphism and consist of two to eight variants. casein is present as 10 to 300-nm micelles formed of submicelles held together by phosphate and hydrophobic bonding. each submicelle has a polar core and a heterogeneous distribution of ~s-and ~-casein with surface k-casein, ~s-and ~-caseins are almost insoluble, whereas the glycoprotein, ~-casein is highly soluble in water. casein may be precipitated somewhat below the isoelectric point range (ph 5.1-5. 3) at about ph 4.6-4.7 by acidification or by the addition of chymosin, which attacks the 105 (phenylalanine)and 106 (methionine) peptide bond of the k-casein. the hydrophilic amino acid terminal peptide (106-169)of k-casein solubilizes in the whey fraction and all other caseins precipitate. the soluble whey proteins consist primarily of ~-lactalbumin and f~-lactoglobulin with serum albumin and immunoglobulins being derived from the bloodstream. ~-lactoglobulin is the major protein, accounting for 50% or more of the total whey protein in ruminants and pigs. ~-lactalbumin accounts for approximately 25 % of the whey protein fraction and is essential for lactose synthesis and the control of milk secretion: ~-lactalbumin binds calcium and zinc. both ~-lactalbumin and ~-lactoglobulin have an amino acid composition close to the nutritional optimum and provide amino acids that are essential for the neonate. whey acid protein is present only in rodent milk. the temperatures of raw milk at collection is about 3 7~ to prevent bacterial growth, oxidation, and proteolysis, milk should be chilled immediately to 4~ and processed within 48 hr unless it is frozen and stored. in the dairy, milk is standardized with regard to the fat content by the centrifugal separation of cream and skim milk: 100 kg of 4% (fat)bovine milk yields 90.35 kg of 0.05% fat skim milk and 9.65 kg of 40% fat cream. the cream fraction is remixed with the skim milk to the required fat content. in processing for a target protein in the whey it is clear that the bulk of the fat can be removed using this standard procedure. however, other standard dairy procedures, particularly microfiltration, may be used to remove fat, casein, and cellular components in a single step. the high lactose and salt content may also be reduced by ultrafiltration. membrane techniques are generally the initial recovery methods of choice and, when correctly used, may yield a 60% pure protein in a single or a tandem microultrafiltration step, thus providing a clarified whey concentrate for further processing. the use of such techniques also provides a barrier to the entry of adventitious viruses, bacteria, and other microorganisms into the final product. an alternative pathway is to apply expanded bed technology. after initial processing to remove fat, skim milk may be passed through an adsorbent for the target, allowing the casein, lactose, and the bulk of the whey proteins to pass through the bed. partially purified target protein may be recovered by desorbtion from the matrix in a fixed bed mode. this type of separation or direct feed capture may be used in a totally fixed bed mode applied to the whey fraction after tangential flow filtration. subsequent processing by various chromatographic techniques, including affinity, hydrophobic interaction, ion exchange, and metal chelate chromatography, is applicable to achieve a protein of required purity. the number of steps should be kept to a minimum as even small step losses can lead to a low yield over an extensive process. a procedure for the purification of human recombinant protein c expressed in porcine milk has been described (degener et al., 1996) . milk fat was removed by centrifugation, and casein was precipitated in the presence of zinc. direct feed capture was performed using an expanded bed, and the protein was purified by hydrophobic interaction and anion-exchange chromatography. farm activities associated with transgenic production include founder development, progeny testing, and dairying. to minimize production risks and achieve validatable procedures, "good agricultural practices" (gaps), which are in the spirit of good manufacturing practices and good laboratory practice, should be used. gaps are based on high standards of animal husbandry, adherence to standard operating procedures, on-site veterinary care, rigorous animal health monitoring programs, and state-of-the-art milking practices to maximize product quality. standard operating procedures should cover areas such as generating founder animals, herd maintenance, herd health, breeding, milk production, and other special procedures. master and working transgenic banks should be kept under strictly controlled conditions. animal feeds should be specially blended free of animal fat and protein. hay for transgenic production animals should be screened for residual chemicals that may have been used in the growing process. both written and computerized records should be kept to track animal lineages, health, and performance records. careful attention should be paid to milk facilities and personnel sanitation. production animals should be observed on a daily basis, and animal side testing of milk can be used to detect early indications of mastitis and other animal illnesses. milk collection and processing areas should be physically separated, and state-of-the-art pharmaceutical grade milking equipment and practices should be used in dedicated milking parlors. "points to consider in the manufacture and testing of therapeutic products for human use derived from transgenic animals" [food and drug administration (fda), 1995] is the result of an iterative process between the fda and the biopharmaceutical industry, and the fda is supportive of transgenic production. the european union's committee on proprietary medicinal products (cpmp) is similarly positive to the use of transgenic animals, stating in its guidance document: "transgenic animals may produce higher quantities of material in more concentrated form than existing culture methods, and therefore have considerable advantages in both the cost of producing the starting material and in its downstream processing. in some instances where very large amounts of material are required for therapy the use of transgenic animals may be one of the few viable production strategies" (cpmp, 1995) . the fda has addressed topics such as the structure of the transgene, creation, characterization, and maintenance of the transgenic herd, fidelity of transgenic inheritance, consistency of transgene expression, analysis of product identity and purity, and the avoidance of contamination by drugs, chemicals, and adventitious agents. many issues of purity, consistency, safety, and potency of transgenically produced therapeutic proteins overlap with various cber points to consider for monoclonal antibodies and other biologicals produced by recombinant dna technology. to quote the fda: "the considerations that apply are therefore a blend of those relevant to recombinant dna derived materials and materials from less defined sources" (fda, 1995) . despite the fact that goats and other farm animals are susceptible to species-specific viral and prion infections, viruses may be more of a concern for animal health than they are a human risk. a cber official has pointed out that prions, such as those responsible for bovine spongiform encephalitis and scrapie, have "less than a theoretical" risk of transmission via milk because they do not occur in the mammary gland and, if introduced, they do not persist (rudolph, 1995) . no transmission of scrapie has ever been reported in humans. milk and semen are noninfectious for scrapie according to the world health organization (1996) . although scrapie has been detected in significant numbers of sheep in the united states, only five goat scrapie cases have occurred in goats, all comingled with scrapie-infected sheep. the use of ani-mals from closed, scrapie-free herds or flocks has been adopted by the leading transgenic production companies to obviate any such concerns. goat viruses relevant in north america are shown in table 4 . the risk of viral and bacterial contamination of human biopharmaceutical products expressed in the milk of transgenic goats can be minimized by a multistage or combinatorial approach at three levels: the goat, the milk, and the final product (ziomek, 1996) . minimization of the risk of contamination of the goat production herd can be accomplished by strict selection, animal husbandry, and adherence to gaps. bacterial contamination can be minimized by careful, state-of-the-art milking procedures and rapid gmp processing. purification processes from raw milk are designed in a manner similar to the manufacturing schemes for recombinant therapeutics from fermentation and cell culture with the inclusion of steps to remove and/or inactivate adventitious agents, microbial contaminants, and pyrogens. for advantages and disadvantages of transgenic expression in the milk of dairy animals, see table 5 . afrom ziomek (1996) . bz, zoonotic: ss, single stranded; ds, double stranded. high level expression at multigram/liter level expression directed to and located in mammary gland expression can be tested in rodents genetic and lactation-to-lactation stability animal-to-animal consistency mammary epithelial cells carry out post-translational modification product recovered at high concentration in milk aseptic milk processing technology well proven bulk impurities removed easily low cost of pre-purification product low investment costs capability of combining transgenics with nuclear transfer (cloning) possible adventitious virus and prion issues in dairy animals control of animal environment and feed microinjection required as a technology for species where cloning is not yet available time to clinical product slower than cell culture (without process development) expression of therapeutically beneficial proteins in the milk of transgenic dairy animals present an unparalleled opportunity for the large-scale production of monoclonal antibodies, plasma, and other proteins. mammary gland epithelia have a cell density that is 100-to 1000-fold greater than the cell densities used, for example, in mammalian cell culture using cho cells. the cells are some of the most productive protein synthesis sources designed by nature to produce large amounts of correctly processed protein and which are switched on and off by hormonal changes. thirty-five transgenic goats expressing a monoclonal antibody at 8 g/liter in their milk are equivalent to a 8500-liter batch cho cell culture running 200 days/year with a 1-g/liter final expression level (young et al., 1997) . in production terms, 170,000 liters of culture is equivalent to 21,000 liters of milk; with the expression levels noted earlier and process yields of 60%, both of these systems would produce 100 kg of purified monoclonal antibody. it has also been discussed that the mammary bioreactor is capable of most posttranslational modifications and protein folding and can, therefore, be used to produce complex proteins. a current limitation of the technology is the low rate (5-10%) of transgenesis. nuclear transfer techniques, in which embryonic or adult cells are engineered and cultured to produce a master cell bank from which nuclei are transferred to recipient oocytes, are under development; these methods hold great promise for improved rates of transgenesis. because of concerns of transmissible spongiform encephalopathies, animal sourced proteins and amino acids are currently under scrutiny. however, it should be kept in mind that the expression system described in this chapter is the mammary gland. given the safety of milk and milk products in combination with the quality and regulatory precautions described, there is every reason to consider that proteins derived from transgenic animals should be safe with respect to possible virus and prion transmission. many of the proteins that are expression targets today are currently available as human plasma derivatives, which have a measurable degree of risk for the transmission of hiv, hepatitis, parvo-, and other viruses, and creutzfeld-jakob disease. as indicated, several proteins are in phase i and ii clinical trials. this number can be expected to increase dramatically in the near future. transgenic dairy animals provide a bulk protein production system that is capable of making proteins available that are either not available today or are only recoverable from other sources at very low yields. transgenic production may thus enable a move from "on-demand" patient treatment to prophylaxis and far wider indications and use of many proteins. in addition, the production of proteins in the milk of transgenic dairy animals is highly cost effective (young et al., 1997) , opening up real possibilities for nutraceutical product development. human growth hormone (hgh) secretion in milk of goats after direct transfer of the hgh gene into the mammary gland by using replication-defective retrovirus vectors high level expression of biologically active human al-antitrypsin in the milk of transgenic mice transgenic pharming comes of age a 17.2 kbp region located upstream of the rabbit wap gene directs high level expression of a functional human protein variant in transgenic mouse milk production of transgenic mice, rabbits and pigs by microinjection into pronuclei expression of synthetic cdna sequences encoding human insulin-like growth factor-1 (igf-1) in the mammary gland of transgenic rabbits factors affecting the efficiency of introducing foreign dna into mice by microinjection eggs dairy processing handbook sheep cloned by nuclear transfer from a cultured cell line comparison of the whey acidic protein genes of rat and mouse transgenics on trial. scrip mag. november pharmaceuticals from transgenic livestock expression of anti-haemophilic factor ix in the milk of transgenic sheep use of transgenic animals in the manufacture of biological medicinal products for human use recombinant interferon-gamma. differences in the glycosylation and proteolytic processing lead to heterogeneity in batch culture high selectivity purification of recombinant proteins from milk using expanded bed chromatography transgenic expression of a variant of human tissue-type plasminogen activator in goat milk: purification and characterization of the recombinant enzyme high level production of human growth hormone in the milk of transgenic mice: the upstream region of the whey acidic protein (wap) gene targets transgene expression to the mammary gland inefficient processing of human protein c in the mouse mammary gland transgenic farm animals: progress report transgenic production a variant of human tissue-type plasminogen activator in goat milk: generation of transgenic goats and analysis of expression induction of human tissue plasminogen activator in the mammary gland of transgenic goats recombinant protein production in transgenic animals points to consider in the manufacture and testing of therapeutic products for human use derived from transgenic animals integration and stable germ-line transmission of genes injected into mouse pronuclei genetic transformation of mouse embryos by microinjection of purified dna production of human tissue plasminogen activator in transgenic mouse milk expression of a bovine k-cn cdna in the mammary gland of transgenic mice utilizing a genomic milk protein gene as an expression cassette strategies to express factor viii gene constructs in the ovine mammary gland production of transgenic rabbits, sheep and pigs by microinjection expression and characterization of biologically active human extracellular superoxide dismutase in milk of transgenic mice complete nucleotide sequence of the ovine b-lactoglobulin gene the mammary gland as a bioreactor: production of foreign proteins in milk transgenic animals--production of foreign proteins in milk manipulating the mouse embryo production of pharmaceutical proteins from transgenic animals the production of pharmaceutical proteins from the milk of transgenic animals pluripotent embryonic stem cells from the rat are capable of producing chimeras n-glycosylation of recombinant human interferon-g produced in different animal expression systems transgenic animals as bioproducers of therapeutic proteins transgenic bioreactors getting the glycosylation right: implications for the biotechnology industry genomic organization of the bovine asl-casein gene generation of transgenic dairy cattle using "in vitro" embryo production production of biomedical proteins in the milk of transgenic dairy cows: state of the art transgenic rabbits as bioreactors for the production of human growth hormone transgenic animals: beyond 'funny milk lactoferrin molecular structure and biological function mammary gland expression of transgenes and the potential for altering the properties of milk expression of human lysozyme mrna in the mammary gland of transgenic mice the efficiency of production, centrifugation, microinjection and transfer of one-and two-cell bovine ova in a gene transfer program bovine as 1-casein gene sequences direct high level expression of active urokinase in mouse milk the porcine mammary gland as a bioreactor for complex proteins functions of milk protein gene 5' flanking region on human growth hormone gene characterization of recombinant human lactoferrin expressed in the milk of transgenic mice germ-line transformation of mice high expression of the caprine b-casein gene in transgenic mice high-level, stage-and mammary-tissue specific expression of a caprine k-casein-encoding minigene driven by a b-casein promoter in transgenic mice transgenic animal technology: a laboratory handbook expression of human lactoferrin in milk of transgenic mice remodeling of mouse milk glycoconjugates by transgenic expression of a human glycosyl transferase high-level expression of recombinant human fibrinogen in the milk of transgenic animals integration, expression and germ-line transmission of growth-related genes in pigs expression of human growth hormone in the milk of transgenic mice development of one-cell fertilized sheep ova following microinjection into pronuclei insertion, expression and physiology of growth regulating genes in ruminants production of transgenic mice and rabbits that carry and express the human tissue plasminogen activator cdna under the control of a bovine alpha $1 casein promoter cloning of the goat beta-casein gene and expression in transgenic mice gene transfer experiments in cattle regulatory issues relating to protein production in transgenic animal milk collection and transfer of microinjectable embryos from dairy goats expression of human serum albumin in the milk of transgenic mice gene transfer into sheep pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer the bovine a-lactalbumin promoter directs expression of ovine trophoblast interferon in the mammary gland of transgenic mice rate limitations in posttranslational processing by the mammary gland of transgenic animals rabbit whey acidic protein gene upstream region controls high level expression of bovine growth hormone in the mammary gland of transgenic mice department of health and human services public health service bovine a-s1 casein gene sequences direct high level expression of human granulocyte-macrophage colony-stimulating factor in the milk of transgenic mice high-level expression of a heterologous protein in the milk of transgenic swine using the cdna encoding human protein c efficient tissue-specific expression of bovine a-lactalbumin in transgenic mice making transgenic livestock: genetic engineering on a large scale production of human surfactant protein c in milk of transgenic mice development and validation of swine embryonic stem cells: a review targeting expression to the mammary gland: intronic sequences can enhance the efficiency of gene expression in transgenic mice position-independent expression of the ovine b-lactoglobulin gene in transgenic mice production of pharmaceutical proteins in milk report of a who consultation on public health issues related to human and animal transmissible spongiform encephalopathies high level expression of active human alpha-l-antitrypsin in the milk of transgenic sheep secretion of unprocessed surfactant protein b in milk of transgenic mice production of biopharmaceutical proteins in the milk of transgenic dairy animals fixing human factor ix (fix): correction of a cryptic rna splice enables the production of biologically active fix in the mammary gland of transgenic mice minimization of viral contamination in human pharmaceuticals produced in the milk of transgenic goats key: cord-003817-k3m72uxw authors: braun, elisabeth; sauter, daniel title: furin‐mediated protein processing in infectious diseases and cancer date: 2019-08-05 journal: clin transl immunology doi: 10.1002/cti2.1073 sha: doc_id: 3817 cord_uid: k3m72uxw proteolytic cleavage regulates numerous processes in health and disease. one key player is the ubiquitously expressed serine protease furin, which cleaves a plethora of proteins at polybasic recognition motifs. mammalian substrates of furin include cytokines, hormones, growth factors and receptors. thus, it is not surprising that aberrant furin activity is associated with a variety of disorders including cancer. furthermore, the enzymatic activity of furin is exploited by numerous viral and bacterial pathogens, thereby enhancing their virulence and spread. in this review, we describe the physiological and pathophysiological substrates of furin and discuss how dysregulation of a simple proteolytic cleavage event may promote infectious diseases and cancer. one major focus is the role of furin in viral glycoprotein maturation and pathogenicity. we also outline cellular mechanisms regulating the expression and activation of furin and summarise current approaches that target this protease for therapeutic intervention. the human genome encodes more than 550 proteases. these molecular scissors play important roles in essentially all physiological processes. they digest the proteins in our food, degrade misfolded or unwanted proteins and regulate the trafficking and activity of numerous cellular factors. proteolytic cleavage is certainly one of the most important post-translational modifications, generating a plethora of bioactive proteins and peptides with key roles in cell proliferation, immunity and inflammation. not surprisingly, mutations in proteases and/or aberrant protease activity are associated with numerous pathological processes including cancer, cardiovascular disorders and autoimmune diseases. 1 intriguingly, also many viral pathogens exploit cellular proteases for the proteolytic processing and maturation of their own proteins. similarly, activation of bacterial toxins frequently requires cleavage by proteases of the infected or intoxicated host. in recent years, modulation of protease activity has therefore emerged as a potential therapeutic approach in a variety of infectious and noninfectious diseases. one particularly promising target for therapeutic intervention is the cellular protease furin. this protease most likely cleaves and activates more than 150 mammalian, viral and bacterial substrates. 2 among them are viral envelope glycoproteins and bacterial toxins, as well as cellular factors that promote tumor development and growth if they are hyperactivated ( figure 1 ). in this review, we summarise our current knowledge of furinmediated protein processing in health and disease with a focus on the role of furin in viral protein processing. furthermore, we describe cellular mechanisms regulating furin activity at the transcriptional and post-transcriptional level. finally, we present approaches that aim at modulating furin activity for therapeutic purposes and discuss their suitability for clinical application. furin is a member of the evolutionarily ancient family of proprotein convertases. their similarity with bacterial subtilisin and yeast kexin proteases has coined the abbreviation pcsk (proprotein convertase subtilisin/kexin type). humans encode nine members of this protease family (pcsk1-9), with pcsk3 representing furin (table 1) . pcsks are well known for their ability to activate other cellular proteins. the proteolytic conversion of inactive precursor proteins into bioactive molecules has already been described in the 1960s. 3 however, it took more than 20 years until furin was identified as the first mammalian proprotein convertase. 4, 5 to date, more than 200 cellular substrates of pcsks have been described, including hormones, receptors, growth factors and adhesion molecules. although pcsks are frequently coexpressed in the same cell and may cleave the same substrates, there is no complete redundancy and the inactivation of individual pcsks results in specific knock-out phenotypes in mice. 6 pcsk1-7 cleave their substrates after basic residues, with the typical recognition motif k/r-x n -k/r↓ 6 ( table 1 ). in contrast, pcsk8 cleaves after nonbasic residues and is best known for its regulation of cholesterol and lipid metabolism by activating sterol regulatory element-binding protein (srebp) transcription factors. 6, 7 like pcsk8, pcsk9 also plays a key role in cholesterol metabolism as it regulates low-density lipoprotein (ldl) particle levels in the blood. however, this effect does not involve proteolytic cleavage of a specific substrate, but is mediated by a direct binding of pcsk9 to ldl receptors. 8 with two fda-approved inhibitors for the treatment of hypercholesterolaemia, pcsk9 is also the prime example of a protease that is successfully targeted for therapy. 6 the prototypical and best-characterised member of the pcsk family is furin/pcsk3. since it cleaves basic amino acid motifs, it has also been termed pace (paired basic amino acid cleaving enzyme). furin is expressed by the fur (fes upstream region) gene on chromosome 15. although furin is ubiquitously expressed, its mrna and protein levels vary depending on the cell type and tissue. high levels can be found in salivary glands, liver and bone marrow, whereas muscle cells express relatively low amounts of furin. 9 three promoters (p1, p1a and p1b), each harbouring an alternative transcription start site, have been described ( figure 2 ). however, the respective transcripts differ only in the first untranslated exon and are therefore predicted to express the same protein. 10 while the p1a and p1b promoters resemble those of constitutively expressed housekeeping genes, the p1 promoter binds the transcription factor c/ebpb and can be trans-activated upon cytokine stimulation. 10 in line with this, ifnc, tgfb, il-12 and pma induce furin expression. [11] [12] [13] [14] upon translation of the mrna, furin enters the secretory pathway as an inactive proenzyme and is integrated into the er membrane via its cterminal transmembrane domain ( figure 2 ). like most type i transmembrane proteins, it harbours a short n-terminal signal peptide that is cleaved off cotranslationally. similar to other proprotein convertases, furin contains an inhibitory nterminal 83-amino acid propeptide, whose chaperone function is required for correct folding of the catalytic domain. 15 during the transition of furin from the er to the trans-golgi network (tgn), the inhibitory propeptide is removed in a two-step autoproteolytic process and furin gains its enzymatic activity. 16 at the same time, n-linked oligosaccharides are added and trimmed. although furin accumulates in the tgn, it can be further transported to the cell surface and back via the endosomal pathway. 17, 18 finally, furin can also be shed and released into the extracellular space upon proteolytic separation of the catalytic domain from the membrane-bound c terminus. 19 whether this cleavage step is mediated by furin itself or another protease remains to be determined. 20 the presence of furin in the tgn and endosomal compartments, at the cell surface and in the extracellular space, may explain its ability to process a large variety of intra-and extracellular substrates. the canonical furin cleavage site is frequently described as r-x-k/r-r↓. however, variations of this motif may also be recognised and a stretch of 20 amino acids surrounding the cleavage site as well as post-translational modifications determine interaction with the furin binding pocket. 21 bioinformatic analyses and functional studies uncovered more than 100 furin cleavage sites in mammalian proteins. these comprise growth factors and cytokines (e.g. igf1, igf2, tgfb, pdgfa, pdgfb, vegf-c, ngf, cxcl10), hormones (e.g. pth, trh, ghrh), adhesion molecules (e.g. integrins, vitronectin), collagens, metalloproteinases, coagulation factors, receptors, membrane channels and albumin. 2 while most of these target factors are activated upon furin-mediated cleavage, furin also exerts inactivating cleavage steps. for example, the furin paralogue pcsk9 and endothelial lipase can be inactivated by furin. 22 maturation of the cellular protease furin. furin expression is driven by three different promoters, sharing characteristics of either cytokine-activated (p1) or housekeeping gene (p1a and p1b) promoters. during translation, furin is integrated into er membranes and glycosylated. after the n-terminal signal peptide (red) is removed, an autocatalytic cleavage event occurs, generating a short propeptide (light blue). this propeptide remains associated with furin and acts as an intramolecular chaperone and inhibitor. after transit to the golgi complex, the propeptide is removed and glycans are trimmed before furin gains its proteolytic activity. furin accumulates in the trans-golgi network (tgn), but can also traffic to the plasma membrane and cycle between these two compartments via endosomes. proteolytic cleavage at the c terminus of furin separates the transmembrane domain (orange) from the catalytically active domain. as a result, furin can be shed into the extracellular space as an active enzyme. the physiological importance of furin is reflected by furin knock-out mice, which die at embryonic day 11 because of cardial ventral closure defects and hemodynamic insufficiency. 24 similarly, endothelial cell-specific knock-out of furin results in cardiac malformation and death shortly after birth. 25 even mutations in the cleavage site of a single furin target protein may have detrimental effects and result in genetic disorders such as haemophilia b or x-linked hypohidrotic ectodermal dysplasia. 26, 27 because of its pleiotropic effects, variations in furin expression levels and/or its enzymatic activity may have detrimental effects and promote the pathogenesis of a variety of disorders, including rheumatoid arthritis, amyloid dementia and cancer. 17 furin has been termed a 'master switch of tumor growth and progression' 28,29 as its aberrant expression or activation can promote the formation and progression of various malignancies including colon carcinoma, rhabdomyosarcoma, head and neck cancers, lung, skin and brain tumors. 30 in some cases, furin levels positively correlate with aggressiveness, and increased furin expression has been proposed as prognostic marker for advanced cancers. 30 the oncogenic and prometastatic activity of furin has been ascribed to its ability to activate proteins that promote cell proliferation, angiogenesis, migration and tissue invasion. for example, furin cleaves and activates growth factors such as igfs, pdgfs or ngf that enhance cell proliferation and consequently tumor growth. similarly, angiogenic and lymphangiogenic factors such as vegf-c and vegf-d may be hyperactivated and promote the vascularisation and growth of solid tumors. 30 notably, furin expression is induced by hypoxia, as all three fur promoters harbour binding sites for the hypoxia-inducible factor-1 (hif-1). 31 thus, furin-mediated vascularisation may preferentially occur in otherwise growth-restricted hypoxic tumors. interestingly, hypoxia also results in subcellular relocalisation of furin to the cell surface, which may further enhance processing of growth factors and other extracellular tumorigenic precursor proteins. 32 besides effects on tumor growth, furin can also promote migration and extravasation of malignant cells as it processes adhesion molecules mediating cell-cell and cell-matrix interactions. the cleavage of integrins may be particularly relevant as they not only mediate adhesion of cells to the extracellular matrix, but also act as signal transducers regulating cell growth, division and survival. 33 in addition, furin activates matrix metalloproteinases (e.g. mmp14) that facilitate metastasis by degrading components of the extracellular matrix. 30 finally, increased furin activity may also promote cancer development by suppressing protective antitumor mechanisms. for example, increased furin-mediated activation of tgfb reduces immune surveillance by promoting the development of suppressive t reg cells and inhibiting effector t-cell functions. 34 the key role of furin in immunity is highlighted by t-cellspecific furin knock-out mice, which harbour inherently over-reactive effector t cells that secrete reduced levels of active tgfb. 35 notably, positive feedback loops can further enhance the oncogenic potential of furin. for example, the furin substrate tgfb not only increases furin mrna expression, but also enhances its proteolytic activity by an unknown mechanism. 36 similarly, furin enhances the secretion of ifnc, which in turn activates the fur promoter. 11, 14 this mutual enhancement seems particularly important given the key role of ifnc in tumor development and progression. 37 on the one hand, furin-driven ifnc release may have beneficial effects as it boosts the tumorlytic activity of natural killer cells and cytotoxic t lymphocytes. furthermore, ifnc may act as an antiangiogenic factor and directly inhibit tumor cell proliferation by inducing the expression of tumor suppressors such as p21 or p27. on the other hand, however, recent evidence suggests that ifnc may also exert tumor-promoting effects, for example by selecting for immune evasive phenotypes and promoting an immunosuppressive tumor microenvironment. 37 intriguingly, a study on laryngeal cancer patients suggests that the ifnc-furin feedback loop may be further boosted iatrogenically since radiotherapy increased furin expression in some patients. 38 in summary, aberrant furin activation promotes several steps of cancer development, including cell proliferation, vascularisation, metastasis and antitumor immunity. however, the relative contribution of individual furin substrates to tumor progression and the role of other proprotein convertases remain largely unclear. furin may not only promote disease upon aberrant expression, but also by activating a variety of pathogen-derived proteins. one example for cleavage of unwanted proteins is the proteolytic activation of bacterial toxins. particularly, the group of ab toxins comprises several well-described furin substrates. these exotoxins are secreted by bacteria and exert their effect in the cytoplasm of the target cell. they usually consist of an enzymatically active a subunit and a b subunit that mediates membrane binding and translocation. to exert its toxic effect, the a subunit has to be separated from the membrane-associated b subunit by proteolytic cleavage. 39 in case of diphtheria toxin and pseudomonas exotoxin a, furin cleaves r-v-r-r↓ and r-q-p-r↓ target sequences, respectively. 40, 41 cleavage most likely occurs in endosomes before the a subunit translocates into the nucleus where it inhibits protein synthesis by inhibiting the elongation factor eef2. 42 in line with an important role of furin in bacterial virulence, toxicity is highest if an optimal furin cleavage site is present. 43 similarly, furin cleaves shiga and shiga-like toxins expressed by certain shigella spp. and escherichia coli strains and enhances their ability to halt protein synthesis. although a furin target sequence is conserved among all shiga-like toxins, mutational analyses suggested that furin-mediated cleavage augments toxin activity, but is not essential. 39 another wellcharacterised example is anthrax toxin, a threeprotein exotoxin consisting of the receptor binding protective antigen (pa) and the enzymatically active components oedema factor (ef) and lethal factor (lf). upon binding to its receptor, pa is cleaved by furin at the cell surface. this cleavage step triggers the oligomerisation of pa into a prepore that binds ef and lf. subsequently, this toxin complex is endocytosed and pa forms a channel that allows the translocation of ef and lf into the cytoplasm. 39 although pa can be activated by different proprotein convertase family members, furin seems to be the major protease activating anthrax toxin. 44 these examples illustrate that several bacterial pathogens exploit furin and related convertases for the activation of their exotoxins. strictly speaking, however, some toxins produced by bacteria (e.g. diphtheria toxin and shiga toxins) represent viral gene products as they are encoded by bacteriophages. 45 in these cases, the term 'viral exotoxin' may be more appropriate. this strongly suggests that furin-mediated toxin activation confers a selection advantage to both, the bacterium and its phage. for example, induction of cell death by furin-activated toxins may promote tissue invasion, increase transmission rates (e.g. by causing diarrhoea) or suppress cellular immune responses. without the proteolytic activation of exotoxins, diseases such as dysentery or diphtheria would not occur. like bacterial and viral exotoxins, most viral envelope glycoproteins need to be proteolytically cleaved before they can mediate viral entry into host cells. in many cases, viruses exploit cellular trypsin-or subtilisin-like endoproteases for this purpose. while subtilisin-like proteases such as furin require polybasic cleavage sites, trypsin-like proteases also recognise monobasic motifs and cleave after single arginine or lysine residues. 46 notably, the dependency on specific proteases can also be an important determinant of tissue tropism and viral spread in an infected organism. for example, avirulent newcastle disease virus (ndv) strains harbour a monobasic cleavage site in their fusion (f) protein and result only in local infections (mainly in the respiratory tract) since expression of the respective host proteases is limited to a few cell types. in contrast, the f proteins of virulent ndv strains can be cleaved by furin or related proprotein convertases that are ubiquitously expressed. consequently, these viruses are able to spread systemically and cause high rates of mortality in infected birds. 47 another well-described example is the cleavage of influenza a virus hemagglutinin (ha). in contrast to low pathogenic avian influenza a viruses, their highly pathogenic counterparts harbour a polybasic furin cleavage site in the ha protein. 48 thus, the ability of viruses to exploit furin may have drastic effects on their pathogenicity. to date, furin-mediated cleavage has been described for envelope glycoproteins encoded by numerous evolutionarily diverse virus families, including herpes-, corona-, flavi-, toga-, borna-, bunya-, filo-, orthomyxo-, paramyxo-, pneumoand retroviridae (table 2) . although viral furin substrates generally harbour the canonical polybasic cleavage site, timing and subcellular localisation of furin-mediated activation may differ substantially between virus families. since furin and viral glycoproteins both enter the secretory pathway, proteolytic activation can occur at different steps of the viral replication cycle. while the envelope proteins of some viruses are cleaved in the producer cell, others are processed in the extracellular space or during entry into their target cells (figure 3 ). the following sections highlight the characteristics of a few selected viral glycoproteins, their processing by furin and the importance of furin-mediated cleavage for infection and pathogenicity. retroviral glycoprotein trimers, such as those of human immunodeficiency, rous sarcoma or murine leukaemia viruses, are proteolytically processed and activated in the producer cells (figure 3a , left panel). in case of hiv-1, the gp160 precursor of the viral envelope protein (env) is cleaved into gp120 and membrane-anchored gp41 that remain associated through noncovalent interactions. cleavage occurs in intracellular compartments, before the assembly of virions at the plasma membrane. notably, proteolytic processing of env depends on correct n-linked glycosylation as aberrant carbohydrate side chains may result in subcellular mistrafficking or sequestration of env. 49 most likely, hiv-1 takes advantage of the redundancy of several proprotein convertases recognising the polybasic cleavage motif in env. furin, pcsk5, pcsk6 and pcsk7 have all been shown to cleave gp160 in cells, albeit with different efficiencies. 49 notably, in vitro cleavage experiments using recombinant proteases did not always reflect cleavage efficiency in transfected cells and the relative contribution of individual pcsks to hiv-1 maturation in vivo remains unclear. 49 interestingly, hiv-1 env harbours a second polybasic cleavage site, about eight amino acid residues upstream of the major one. although cleavage at this site does not result in fusiogenic env species, about 15% of all gp160 molecules are cleaved at this position, at least in case of the cellculture-adapted hiv-1 clone lai. 50, 51 in some cases, gp160 escapes intracellular cleavage and may be incorporated as an unprocessed precursor into budding virions. whether these env molecules may be processed extracellularly by shed furin is unknown. in this context, it is noteworthy that membrane-bound plasmin has been shown to convert extracellular gp160 into gp120 and gp41. 52 however, it remains to be determined whether plasmin-mediated cleavage results in fully infectious hiv-1 particles. influenza a virus hemagglutinin (ha) can be cleaved and primed by a variety of cellular proteases. even bacterial proteases may promote influenza virus spread by cleaving ha 0 into ha 1 and ha 2. 53 both subunits remain linked via disulphide bonds and form trimeric structures. while ha 1 binds to the sialic acid receptor on viral target cells, ha 2 harbours the fusion peptide that mediates fusion of viral and cellular endosomal membranes. ha cleavage can occur within producer cells, upon release of virions from infected cells or directly prior to entry into new target cells. 54 as a general rule, hemagglutinins of mammalian and low pathogenic avian influenza a viruses cannot be cleaved by furin as they usually only harbour a mono-or dibasic cleavage site. instead, they depend on trypsin-like proteases such as transmembrane protease serine s1 member 2 (tmprss2) or human airway trypsin-like protease (hat). 55 expression of such trypsin-like proteases is largely restricted to the respiratory and gastrointestinal tract. in contrast, h5 and h7 hemagglutinins of a large number of highly pathogenic avian influenza a viruses (hpaiv) can be cleaved by furin or pcsk5, which are present in many cell types. 56, 57 this is because they acquired a polybasic cleavage site upon insertion of additional lysine and/or arginine residues. duplication of lysine and arginine residues in ha is facilitated by polymerase slippage as these amino acids are encoded by purine-rich codons. 58 instead of the prototypical r-x-k/r-r↓ motif, some hpaivs harbour a suboptimal k-x-k/r-r↓ cleavage motif. proteolytic cleavage at this site is only efficient if additional positively charged amino acids upstream of this cleavage motif are present or if attachment sites for masking oligosaccharide chains are missing. 59, 60 notably, a subset of h9n2 lowly pathogenic avian influenza a virus strains also harbour r-s-k-r↓ or r-s-r-r↓ sites that are not only cleaved by trypsin-like proteases, such as tmprss2 or hat, but also by pcsks. 61 however, their cleavage is only efficient in the presence of very high amounts of furin or upon mutation of a glycosylation site in ha. 62 thus, the ability to exploit furin for efficient ha cleavage and the associated increase in pathogenicity are not only determined by the presence of a furin consensus target site, but also by adjacent residues and the absence of masking oligosaccharide chains. furin cleaves the glycoproteins (gps) of marburg virus (marv) and all five ebolavirus species into a large n-terminal subunit (gp1) that mediates receptor binding and a small membrane-anchored c-terminal part (gp2) that contains the fusion , the viral envelope (env) glycoprotein precursor (green) migrates through the er to the golgi complex where it is cleaved by furin (pink scissors) into the functional mature env glycoprotein (blue). processed env glycoproteins are transported to the cell surface and incorporated into assembling viral particles. in contrast, dengue viruses bud into the er lumen and incorporate the uncleaved premembrane protein (prm) (right panel). during virus particle transit through the secretory pathway, virion-associated prm proteins can be cleaved by furin (dark blue to light blue). (b) some prm molecules escape furin-mediated cleavage in the producer cells resulting in the release of immature or partially mature dengue virus particles. in this case, processing can also occur in endosomes of new target cells, upon receptor-mediated endocytosis (left panel). during human papillomavirus (hpv) infection (right panel), attachment to heparan sulphate proteoglycans induces a conformational change that allows proteolytic processing of the minor capsid protein l2 (red) by furin, which is present at the cell surface. furin processing induces a structural rearrangement that allows binding to a secondary receptor and subsequent receptor-mediated endocytosis. peptide. 63, 64 marv and human pathogenic ebolavirus species harbour canonical furin cleavage sites (r-x-k/r-r↓). in contrast, the gp of the closely related reston virus, which causes asymptomatic infections in humans, is processed less efficiently by furin as it carries the suboptimal cleavage site k-q-k-r↓. 63 surprisingly, however, uncleavable gp mutants of highly pathogenic ebola virus (ebov) are able to mediate infection and furin-mediated cleavage is not required for replication in cell culture. 65 furthermore, an ebov mutant lacking the furin cleavage site replicated efficiently in nonhuman primates and showed no differences in disease progression or lethality compared to wild-type viruses. 66 thus, the high conservation of the furin cleavage site among different ebolavirus species is surprising and it remains to be determined whether furin-mediated gp processing plays a role in the natural reservoir hosts of these viruses. 65 notably, the ebov gp gene harbours an rna editing site sequence and may not only express fulllength gp, but also a soluble form of the glycoprotein (pre-sgp) that lacks the c-terminal transmembrane domain. 67 intriguingly, pre-sgp harbours another furin recognition site and is cleaved into mature sgp and a short so-called d peptide. both of them are ultimately released from infected cells. 68 although pre-sgp is produced in higher amounts than gp, its role in viral replication is under debate. among others, sgp has been suggested to serve as a decoy antigen, to act as a structural substitute for gp1 and to induce apoptosis of uninfected lymphocytes. 69 flaviviruses flavivirus rna is translated into a single large polyprotein that is cleaved by cellular and viral proteases into all structural and nonstructural proteins of the virus. the structural proteins comprise the envelope proteins prm and e that are incorporated as prm/e heterodimers into budding virions. 70 prm acts as a chaperone and facilitates correct folding of the e glycoprotein. 71 many flaviviruses bud into the lumen of the er and enter the secretory pathway. 70 in the acidic milieu of the trans-golgi network (tgn), a furin cleavage site is exposed and prm can be cleaved into mature pr and m proteins. 72 thus, furinmediated cleavage of the viral glycoprotein occurs only after its incorporation into newly formed virions (figure 3a, right panel) . this is in contrast to viruses such as hiv, whose envelope proteins are cleaved before assembly. the pr peptide remains associated with the e protein until the virion is released from the cell, thereby preventing premature unintended fusion with membranes of the producer cell. 73 furin-mediated prm cleavage is essential for replication of flaviviruses such as tick-borne encephalitis or dengue virus. 74, 75 in some cases, prm molecules escape furin processing in the producer cell and result in the release of immature or partially mature viral particles. partially mature virions are still infectious since low amounts of mature m are sufficient to mediate fusion with target cell membranes. 70 furthermore, uncleaved prm may participate in virion attachment to target cells and can be cleaved by furin during the entry process, in the acidic milieu of endosomes 76, 77 (figure 3b , left panel). the relative contribution of prm processing during viral entry into new target cells, however, remains to be determined. the ratio of mature to immature prm depends on a variety of factors, including the producer cell type and the flavivirus species. for example, dengue viruses are known to release high amounts of immature or partially mature viruses, most likely because of a conserved acidic residue within the furin recognition site 78 (table 2) . importantly, the content of prm in viral particles also affects antibody recognition and consequently antibody-dependent enhancement of dengue virus infection. 77 thus, furin-mediated protein processing may once again markedly affect the outcome of infection. while furin plays a key role in activating envelope glycoproteins of a variety of viruses, its activity is also exploited for the cleavage of other viral proteins. one example is the cleavage of the l2 protein of papillomaviruses. 79 together with the major capsid protein l1, this minor capsid protein builds the viral capsid. the furin cleavage site is located close to the n terminus of l2 and highly conserved among different human papillomavirus (hpv) strains. 80 cleavage is not required for virus assembly or release, but essential for infection of new target cells. for example, lovo and cho cells lacking furin expression are completely resistant to infection with pseudoviruses of hpv16, 79, 80 which is one of the high-risk hpv types causing cervical cancer. in contrast to flavivirus prm, which can be cleaved during egress and entry, papillomavirus l2 seems to be exclusively cleaved on target cells. 80 a model has been proposed, in which attachment of papillomaviruses to heparan sulphate proteoglycans induces a conformational change in l2 that exposes the polybasic cleavage site. 81 upon proteolytic processing of l2, l1 may engage a secondary cellular receptor and mediate infection 80 (figure 3b, right panel) . furthermore, interaction of cleaved l2 with an unknown intracellular receptor may be required for escape of l2 from the endosomal compartment and its ability to escort viral dna into the nucleus. 80 this illustrates that also nonenveloped viruses have evolved the ability to exploit furin or related pcsks for their own purposes. another nonenvelope protein that is cleaved by furin is the external core antigen (hbeag) of hepatitis b virus (hbv). 82, 83 cleaved hbeag is secreted from infected cells and exerts immunosuppressive effects. 84 it has been suggested to act as a t-cell tolerogen that prevents killing of infected hepatocytes by cytotoxic t lymphocytes. 85 in contrast, uncleaved hbeag may have the opposite effects as it is transported to the plasma membrane where it can trigger antiviral immune responses. 86 thus, furin-mediated cleavage of hbeag may affect the outcome of infection. intriguingly, han chinese frequently harbour a single nucleotide polymorphism in the p1 promoter of the fur gene that is associated with increased risk of developing persistent hbv infection with detectable amounts of hbeag in the serum. 87 this polymorphism increases the binding efficiency of the hepatic transcription factor nf-e2, thereby most likely increasing furin expression. 87 whether the observed increase in hbv persistence is the result of increased hbeag processing and/or other effects of furin remains to be determined. viral pathogens and their hosts are in a continuous arms race. 88 although viruses have evolved sophisticated strategies to exploit the metabolism, protein synthesis and trafficking pathways of an infected cell, the host is not defenceless. besides innate and adaptive immune responses that directly target components of the virus, infected cells may also restrict viral spread by limiting the availability of cellular factors that are critical for viral replication, so-called 'virus dependency factors'. for example, the host protein samhd1 restricts replication of several viral pathogens by depleting cellular dntp levels. 89 furthermore, ifi16 targets the cellular transcription factor sp1 to suppress viral gene expression. 90 intriguingly, accumulating evidence suggests that inhibition of the virus dependency factor furin represents another efficient and broadly active mechanism of antiviral immunity. in 2013, aerts and colleagues found that protease-activated receptor 1 (par1), a g-proteincoupled receptor, interferes with the expression of furin and furin-mediated processing of the human metapneumovirus f protein. 91 follow-up experiments revealed that par1 harbours an r 41 xxxxr 46 motif that mediates interaction with several pcsks. 92 in line with this, soluble pc5a/ pcsk5 and pcsk6 cleave par1 at r 46 ↓ and abrogate its ability to induce calcium signalling upon thrombin-mediated cleavage at the plasma membrane. surprisingly, however, membranebound pcsks such as furin fail to cleave par1 at this position. instead, furin traps par1 in the trans-golgi network and prevents its anterograde transport to the cell surface (figure 4a ). at the same time, par1 also blocks the proteolytic activity of furin, inhibiting for example the maturation of hiv-1 env. this inhibitory activity is not shared by its paralogue par2, which is efficiently cleaved by furin. 93 notably, expression of par1 is induced in proinflammatory environments such as the brain of hiv-1 infected individuals suffering from hiv-associated neurocognitive disorders (hand). 92 thus, par1mediated furin inhibition may represent a mechanism of innate immunity limiting the spread of hiv-1 and potentially additional furindependent viral pathogens. a similar inhibitory activity was recently described for two ifnc-inducible gtpases, termed guanylate-binding proteins 2 and 5 (gbp2 and gbp5). initially, gbp5 was described in a screening for novel restriction factors of hiv and shown to interfere with the maturation of the retroviral env protein. 94, 95 follow-up experiments revealed that this antiviral activity is shared by its paralogue gbp2 and that both proteins reduce the proteolytically active amount of furin. as a result, cleavage of the env precursor gp160 into mature gp120 and gp41 is reduced and newly formed virions are only poorly infectious (figure 4b ). since many viral pathogens rely on furin or related pcsks for the maturation of their own (glyco)proteins, gbp2 and gbp5 exert broad antiviral activity, inhibiting replication of highly pathogenic avian influenza a, measles and zika viruses. in contrast, gbp2 and gbp5 do not decrease infectivity of virions carrying the glycoprotein of vesicular stomatitis virus, which does not require a proteolytic activation step. notably, inhibition of furin in infected cells comes at a cost, since furin-mediated processing of matrix metalloproteinases and other cellular substrates is also reduced in the presence of increased gbp2 or gbp5 levels. 96 future experiments will reveal whether par-1-or gbpmediated inhibition of furin activity may also prevent the development or proliferation of certain cancers. remarkably, increased gbp2/5 expression is associated with favorable outcome in patients suffering from melanoma or breast cancer. 97, 98 thus, a better understanding of cellular mechanisms regulating furin activity will help to understand the pathogenesis of infectious diseases and cancer and may uncover novel targets for therapeutic intervention. because of the key role of furin in the pathogenesis of cancer and infectious diseases, its suitability as a therapeutic target has raised significant interest for several years. many laboratories have explored the possibility to limit tumor growth, viral replication or bacterial intoxication by reducing the amount or proteolytic activity of furin. initially, most studies focused on peptides or proteins that bind to the active site of furin and inhibit substrate binding in a competitive manner. for example, a variant of the naturally occurring serine protease inhibitor a-1 antitrypsin was modified to harbour a consensus furin cleavage site. this variant, termed a-1 antitrypsin portland (a1-pdx), inhibits furin and pcsk5 and has been shown to prevent the processing of hiv-1 env and measles virus f in vitro. 99, 100 similarly, peptides derived from the cleavage site of influenza a virus hemagglutinin and polyarginines compete with natural furin substrates. 101, 102 even exogenous addition of the autoinhibitory propeptide of furin has been shown to reduce its enzymatic activity, limiting for example the activation of mmp9 in breast cancer cells. 103, 104 however, the therapeutic potential of the propeptide has never been evaluated in vivo and the inhibitory effects are most likely limited as it is known to dissociate from furin in the tgn. several approaches, including incorporation of d-instead of l-amino acids, have been applied to increase the stability and hence efficacy of furin inhibitors. for example, hexa-d-arginine (d6r), one of the first furin inhibitors, exhibits good stability and prevents the cytotoxic effects of pseudomonas exotoxin a in vitro and in vivo. 105 similarly, topical application of nona-d-arginine (d9r) has been shown to reduce corneal damage in mice infected with pseudomonas aeruginosa. 106 interestingly, d9r also showed direct bactericidal activity, probably because of its polycationic nature. 107 besides d-amino acids, incorporation of amino acid analogs such as decarboxylated arginine mimetics or 4-amidinobenzylamide (amba) has been used to increase the stability of peptide-derived furin inhibitors. 108 furthermore, the addition of a chloromethyl ketone (cmk) moiety to the c terminus of a polybasic cleavage motif has proven useful as it results in the alkylation of the active site of furin that irreversibly blocks its enzymatic activity. 109 nevertheless, the cytotoxicity of cmkbased inhibitors and the instability of the cmk moiety may limit their use to topical applications such as the treatment of hpv skin infections. 110 finally, the elucidation of the crystal structure of furin enabled the targeted modelling of nonpeptidic inhibitors such as streptamine-based compounds. upon addition of guanidine residues, streptamine derivatives mimic the cationic furin cleavage site and inhibit its enzymatic activity in the nanomolar range in vitro. 111 dahms and colleagues describe an interesting example of a 2,5dideoxystreptamine-derived inhibitor, where two molecules of the inhibitor form a complex with furin. 112 while the first inhibitor molecule directly interferes with the conformation of the catalytic triad, the second molecule binds to an adjacent planar peptide stretch. besides stability, the subcellular localisation of furin inhibitors is a key determinant of their efficacy in vivo. notably, optimal localisation of the inhibitor strongly depends on the processing event targeted for therapeutic intervention. to inhibit activation of anthrax toxin, for example, the inhibitor does not need to enter the cells, as . par1 as well as gbp2 and gbp5 reduces hiv particle infectivity by inhibiting furin-mediated env processing. human immunodeficiency virus (hiv) particles containing functional mature envelope (env) glycoproteins fuse with the plasma membrane of the target cell to release the capsid core into the host cell cytoplasm. upon reverse transcription and integration of the retroviral genome, viral gene expression is initiated. (a) in a cytokine (e.g. il-1b)-induced inflammatory state, furin and protease-activated receptor 1 (par1) expression are induced. furin and par1 interact with each other and are trapped as inactive proteins in the trans-golgi network (tgn). as a consequence, par1 cannot traffic to the cell surface, where it is usually cleaved by thrombin to induce inflammatory signalling pathways. moreover, production of infectious hiv-1 particles is impaired because of reduced furin-mediated cleavage of hiv env. (b) at the same time, cells of the infected host may induce the expression of interferon-stimulated genes such as guanylate-binding proteins 2 and 5 (gbp2 and gbp5). both proteins colocalise with furin and inhibit its proteolytic activity. as a result, hiv env maturation is impaired and newly forming viral particles are poorly infectious since they incorporate immature env glycoproteins. ifn-i/ii, type i and type ii interferons; stat, signal transducers and activators of transcription. furin cleaves the toxin precursor at the cell surface. in contrast, penetration of the inhibitor into the cell is essential to prevent the maturation of hiv-1 env and other viral glycoproteins that are cleaved intracellularly. while some inhibitors (e.g. ha-derived peptides) efficiently enter cells, others were modified to increase their intracellular availability. for example, addition of a decanoyl moiety to cmk inhibitors increases their ability to penetrate cells. 113 streptamine derivatives may be particularly promising for targeted therapy as the positioning of the guanidyl substituents determines the localisation of the inhibitor to distinct subcellular compartments such as endosomes or the golgi complex. 29 while many of the inhibitors described above potently reduce furin activity both in vitro and in vivo, most of them also inhibit other proprotein convertases recognising the same or similar polybasic cleavage sites. this limitation is inherent to competitive inhibitors that aim at mimicking the target sequence of furin and may be overcome by allosteric inhibitors that bind furin-specific motifs outside the active site. one example is the nanobody nb14, which binds to the c-terminal pdomain of furin, thereby blocking the access of larger substrates to the active site. notably, nb14 specifically binds to the p-domain of furin and does not recognise other pcsks. 114 instead of targeting furin at the protein level, therapeutic approaches may also aim at targeting its rna. for example, the endogenous degradation of furin mrna by regnase-1 (zc3h12a) and/or roquin (rc3h1) 115 could be modulated to interfere with the expression and thus proteolytically active amount of furin. however, modulation of regnase-1 and roquin will most likely have off-target effects as both endoribonucleases also degrade additional mrnas. a more selective rna-based approach is the silencing of furin via shrna. in fact, shrnamediated suppression of furin expression is currently the clinically most advanced therapy targeting this protease. in a phase iii clinical trial, patients suffering from metastatic ewing's sarcoma family of tumors (esft) are treated with an immunotherapy that involves the silencing of furin and simultaneous overexpression of gm-csf. 116 more specifically, tumor cells are extracorporeally transfected and reintroduced as so-called furin knock-down and gm-csf augmented (fang) cancer vaccine, also known as vigil. while gm-csf boosts the antitumor response by dendritic cells and t cells, knockdown of furin prevents the proteolytic activation of tgfb, which may otherwise revert the beneficial effects of gm-csf. [117] [118] [119] in a phase ii clinical trial, the autologous fang/vigil vaccine has already proven successful as it increased relapse-free survival of ovarian cancer patients from 481 to 826 days and showed only limited adverse effects. 120 the proprotein convertase furin has become an attractive target for the treatment of various infectious and noninfectious diseases as it regulates the activity of numerous mammalian, bacterial and viral proteins. in recent years, several peptidic and nonpeptidic inhibitors have been developed that block the activation of bacterial toxins, prevent the maturation of viral proteins and suppress tumor growth in vitro. although some of them also yielded promising results in mouse models, there have been only a limited number of clinical trials in humans. one major challenge is the redundancy of furin with related proprotein convertases that also recognise polybasic cleavage sites. on the one hand, selective inhibition of furin may be beneficial as it limits unwanted side effects due to inhibition of other pcsks. on the other hand, treatment of some diseases may require the simultaneous inhibition of several pcsks to efficiently block pathological substrate conversion. to advance current approaches, a better understanding of the relative contribution of individual pcsks to (nonphysiological) proteolytic protein processing is urgently needed. therapeutic intervention needs to specifically target the convertase(s) that drive disease progression. currently, the most promising approaches for selective inhibition are shrnamediated silencing and nanobodies as they show no or only little off-target effects. even if selective inhibition of individual pcsks can be achieved, systemic long-term inhibition will most likely have detrimental effects, as pcsks are required for the activation of hundreds of cellular substrates. thus, local applications such as targeted treatment of tumors or topical treatment of bacterial and viral infections may be more feasible than systemic therapy. finally, the ability of tumor cells or pathogens to evolve resistance or evasion mutations remains poorly investigated. for example, several substrates such as dengue virus prm harbour suboptimal furin target sequences and may optimise their cleavage sites upon therapy to enable sufficient cleavage in the presence of inhibitors. although the therapeutic application of furin inhibitors may be full of pitfalls, it is certainly a promising approach that should be further pursued. future studies will elucidate the role of individual pcsks and their substrates in disease progression and a better understanding of cellular pathways regulating furin activity may uncover additional targets for therapeutic intervention. pathophysiological aspects of proteases furindb: a database of 20-residue furin cleavage site motifs, substrates and their associated drugs insulin biosynthesis: evidence for a precursor evolutionary conserved close linkage of the c-fes/fps 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transforming growth factor b 1 precursor by human furin convertase contrasting effects of tgf-b1 and tnf-a on the development of dendritic cells from progenitors in mouse bone marrow phase ii study of human cytomegalovirus strain towne glycoprotein b is processed by proteolytic cleavage identification and structure of the gene encoding gpii, a major glycoprotein of varicella-zoster virus epstein-barr virus glycoprotein homologous to herpes simplex virus gb coronavirus ibv: partial amino terminal sequencing of spike polypeptide s2 identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m41 cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution proteolytic activation of tick-borne encephalitis virus by furin nucleotide sequence of the 26s mrna of sindbis virus and deduced sequence of the encoded virus structural proteins furin processing and proteolytic activation of semliki forest virus mechanism of borna disease virus entry into cells processing of the borna disease virus glycoprotein gp94 by the subtilisinlike endoprotease furin crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by furin-like and ski-1 proteases to generate a novel 38-kilodalton glycoprotein molecular analyses of the hemagglutinin genes of h5 influenza viruses: origin of a virulent turkey strain complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned dna structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of newcastle disease virus fusion glycoprotein of human parainfluenza virus type 3: nucleotide sequence of the gene, direct identification of the cleavage-activation site, and comparison with other paramyxoviruses cloning and sequencing of the mumps virus fusion protein gene the nucleotide sequence of the mrna encoding the fusion protein of measles virus (edmonston strain): a comparison of fusion proteins from several different paramyxoviruses fusion protein of the paramyxovirus simian virus 5: nucleotide sequence of mrna predicts a highly hydrophobic glycoprotein nucleotide sequence of the gene encoding the fusion (f) glycoprotein of human respiratory syncytial virus endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus structural characterization of the avian retrovirus reverse transcriptase and endonuclease domains the role of envelope glycoprotein processing in murine leukemia virus infection furin-mediated cleavage of the feline foamy virus env leader protein we thank frank kirchhoff and dominik hotter for their helpful comments. this work was funded by the dfg priority programme 'innate sensing and restriction of retroviruses' (spp 1923) to ds; eb was supported by the international graduate school in molecular medicine ulm (igradu). the authors declare no conflict of interest. eb and ds performed literature research. eb drafted the figures, and ds wrote the initial version of the manuscript.this is an open access article under the terms of the creative commons attribution-noncommercial license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. key: cord-017968-17d37a2z authors: lewinski, martin; köster, tino title: systems approaches to map in vivo rna–protein interactions in arabidopsis thaliana date: 2018-08-30 journal: systems biology doi: 10.1007/978-3-319-92967-5_5 sha: doc_id: 17968 cord_uid: 17d37a2z proteins that specifically interact with mrnas orchestrate mrna processing steps all the way from transcription to decay. thus, these rna-binding proteins represent an important control mechanism to double check which proportion of nascent pre-mrnas is ultimately available for translation into distinct proteins. here, we discuss recent progress to obtain a systems-level understanding of in vivo rna–protein interactions in the reference plant arabidopsis thaliana using protein-centric and rna-centric methods as well as combined protein binding site and structure probing. rna-binding proteins (rbps) are a diverse class of proteins that control every step of rna processing and rna function in the cell. they are characterized by dedicated domains involved in rna binding and can have accessory domains engaged in protein-protein interactions or enzymatic activities. in higher plants, rbp function so far has been best studied in the reference plant arabidopsis thaliana. among the rbps present in the arabidopsis genome are 197 proteins with an rna recognition motif (rrm), the most abundant type of rna-binding domain, and 28 k homology (kh) domain proteins first identified in mammalian heterogeneous nuclear protein hnrnp k (silverman et al. 2013 ). in addition, 26 pumilio (pum) domain proteins, nine dead-box helicases as well as five proteins with cold shock domains (csds) have been identified (silverman et al. 2013) . another 450 proteins harbor pentatricopeptide repeat (ppr) domains. ppr domains consist of multiple 35-amino acid repeats of which two are known to be engaged in specific rna recognition (barkan and small 2014) . these proteins are imported into mitochondria or chloroplasts and regulate all aspects of rna metabolism, e.g., rna editing, splicing, rna cleavage, and translation in organelles (schmitz-linneweber and small 2008; barkan and small 2014) . a suite of arabidopsis rbps have been experimentally characterized, mainly through loss-of-function mutants and transgenic plants ectopically overexpressing rbps. these approaches revealed a crucial role for rbps in development (kalyna et al. 2003; ripoll et al. 2006; kupsch et al. 2012; völz et al. 2012; ferrari et al. 2017; foley et al. 2017; teubner et al. 2017) , timing of plant reproduction (macknight et al. 1997; streitner et al. 2008; hornyik et al. 2010) , responses to abiotic stress (kim et al. 2007b (kim et al. , c, 2008 (kim et al. , 2010 park et al. 2009 ), pathogen defense (fu et al. 2007; qi et al. 2010; jeong et al. 2011; lyons et al. 2013; nicaise et al. 2013 ), responses to phytohormones (lu and fedoroff 2000; hugouvieux et al. 2001; riera et al. 2006; carvalho et al. 2010; hackmann et al. 2014; löhr et al. 2014) , and circadian timekeeping (heintzen et al. 1994; staiger 2001; jones et al. 2012; schmal et al. 2013; perez-santángelo et al. 2014) . at the biochemical level, an impact of defined rbps on rna processing including pre-mrna splicing, 3 end processing, processing of microrna precursors, and translation has been described (lopato et al. 1999; simpson et al. 2003; vazquez et al. 2004; dong et al. 2008; stauffer et al. 2010; ren et al. 2012; rühl et al. 2012; juntawong et al. 2013; sorenson and bailey-serres 2014; staiger 2015; carvalho et al. 2016) . recent attempts to comprehensively identify rbps, summarized in sect. 2, provided experimental evidence for rna binding for most of the previously identified arabidopsis rbps and identified a plethora of proteins with noncanonical rbds. systems approaches to describe rna-protein interactions globally come in two main flavors (fig. 1) . in rna-centric approaches, proteins associated with mrnas are recovered by rna pull-down and identified by mass spectrometry, a technique referred to as mrna interactome capture (baltz et al. 2012; castello et al. 2012) (fig. 1a) . in protein-centric approaches, the focus is laid on a particular rbp. the rna complement associated with the rbp of interest, the ribonome, is identified via immunoprecipitation of the rbp from cell lysates and identification of the bound target rnas, initially by microarrays (tenenbaum et al. 2000; galgano and gerber 2011; guerreiro et al. 2014) or more recently via high throughput sequencing (licatalosi et al. 2008; könig et al. 2010; rossbach et al. 2014; müller-mcnicoll et al. 2016) (fig. 1b) . of all predicted rbps in arabidopsis, rna binding has only been experimentally confirmed for a limited number of them. a first attempt to globally identify proteins based on their ability to interact with mrnas in vivo was made for cultured arabidopsis cells (schmidt et al. 2010) . in this study, mrnas and interactors were recovered under native conditions by affinity chromatography on an oligo(dt) cellulose column followed by two-dimensional gel electrophoresis. the protein components were identified via maldi-tof. in the rna-bound proteome were a suite of rrm proteins including members of the family of glycine-rich rnabinding proteins like atgrp2 (arabidopsis thaliana glycine rich rna-binding protein 2), atgrp7 and atgrp8 (lewinski et al. 2016) , the two oligouridylatespecific rbp45 and rbp47 proteins (lorkovic et al. 2000) , and csd proteins. in 2012, mrna interactome capture was reported to comprehensively identify proteins interacting with mrnas in mammalian cells (baltz et al. 2012; castello et al. 2012 ). this technique employs in vivo cross-linking of mrna and bound proteins by uv light irradiation. the rna-protein complexes are recovered by pull-down of polyadenylated rnas using magnetic beads coated with oligo(dt). proteins are released by rnase treatment, subjected to tryptic digest and identified via mass spectrometry (fig. 1a) . following these pioneering studies, this technique was applied to a wide range of organisms including yeast, drosophila melanogaster, caenorhabditis elegans, leishmania, trypanosomes, and plasmodium (mitchell et al. 2013; beckmann et al. 2015; matia-gonzalez et al. 2015; bunnik et al. 2016; lueong et al. 2016; sysoev et al. 2016; wessels et al. 2016; nandan et al. 2017) . a minimal core mrna bound proteome occurring in both human and yeast was defined by beckmann and coworkers (beckmann et al. 2015) . lately, mrna interactome capture has also been successfully applied to arabidopsis (marondedze et al. 2016; reichel et al. 2016; zhang et al. 2016 ). the first mrna interactome capture experiments in arabidopsis employed widely differing tissues to catalog rbps. gueten and coworkers chose protoplasts, cells without a cell wall, assuming that uv cross-linking should occur as efficiently as xing et al. 2015 , or relative to polyadenylated rna, e.g., meyer et al. 2017 . in iclip (könig et al. 2010) , rna-protein complexes are subjected to rnase treatment. bound proteins are digested with proteinase, leaving a polypeptide at the crosslink site. reverse transcriptase stops there, allowing the detection of the cross-link site at the −1 position of the processed sequencing reads in mammalian cell monolayers (zhang et al. 2016) . leaf mesophyll protoplasts are also widely used in transient assays to study the regulation of gene expression. a mesophyll protoplast mrna interactome was defined with a total of 325 proteins based on enrichment in cross-linked samples vs. non-cross-linked controls with a log 2 fold change above 2 (zhang et al. 2016) . of these, one class was represented by 123 ribosomal proteins of which 52 were also present in the core mrna-bound proteome of human and yeast cells (beckmann et al. 2015) . the second class comprised 70 proteins with a known rbd. for 41 of them, a role in mrna binding and rna biology had already been described while the remaining proteins had a potential role in mrna processing. moreover, 12 of the rbps in the second class overlapped with the rbps identified in the native oligo(dt) affinity chromatography approach (schmidt et al. 2010) . the third class comprised 132 candidate rbps. of these, 49 were metabolic enzymes, mainly oxidoreductases. moreover, numerous proteins related to photosynthesis were found. as these are generally strongly expressed, their rna binding activity and the domains involved beg for an independent validation. one of the enzymes was the arabidopsis ortholog of phosphoglycerate kinase whose rna binding capacity has previously been validated in yeast and human cells (beckmann et al. 2015 ). another mrna interactome capture experiment employed 4-days-old etiolated arabidopsis seedlings (reichel et al. 2016 ). this was based on the rationale that uv-absorbing pigments present in green plant tissue may interfere with uv crosslinking in planta and their absence in etiolated tissue may allow more efficient uv cross-linking. around 300 of the 746 proteins identified altogether were significantly enriched in uv cross-linked samples vs. non-cross-linked controls with a false discovery rate below 1% and designated the "at-rbp set." eighty percent of these have a known rbd, and 75% have been linked to rna biology. more than 400 additional proteins did not meet the significance criteria applied for the "at-rbp set" and were classified as "candidate rbps." notably, of the 197 computationally predicted rrm proteins in arabidopsis 160 were detected in the input fraction in etiolated seedlings (silverman et al. 2013 ). half of these were recovered in the "at-rbd set" and another 50 were present among the "candidate rbps." similarly, seven of the predicted kh proteins were present in the "at-rbd set" and 12 were among the "candidate rbps." of the predicted 450 members of the ppr protein family only 60 were detected in the input fraction, likely due to low abundance (schmitz-linneweber and small 2008; reichel et al. 2016) . only six ppr proteins were found in the "at-rbp set" and another twelve in the "candidate rbps," likely because most rnas in the organelles lack poly(a) tails. a comparison of the identified proteins to the mrna interactome in other model organisms revealed that 52 were present in the interactomes of humans (baltz et al. 2012; beckmann et al. 2015) , mice (kwon et al. 2013; liao et al. 2016) , and yeast (beckmann et al. 2015) and were assigned to basic functions in rna metabolism such as translation, splicing, and rna unwinding. in addition to rbps with known rbds many arabidopsis proteins emerged that have not been linked to rna binding so far. among novel rbps were proteins harboring a yt521-b homology (yth) domain (li et al. 2014) . yth domain proteins have been shown to bind n 6 methyladenosine and thus serve as readers of the m 6 a mark in mammals (wang et al. 2014 ). in addition, alba domain containing proteins have been identified. alba domain proteins are well characterized in archaebacteria where they act as transcriptional repressors and in other eukaryotes where they control translation (goyal et al. 2016) . in plants, they have not yet been functionally characterized. the only observation pointing to rna binding is the recovery of an arabidopsis alba domain protein by rna-affinity chromatography (gosai et al. 2015) . whirly domain containing proteins have been characterized as single-stranded dna binding proteins in organelles (krause et al. 2009 ) and in maize, association of a whirly protein with chloroplast transcripts has been observed (prikryl et al. 2008 ). the identification of three whirly proteins in the etiolated seedling interactome (reichel et al. 2016 ) and of whirly1 upon oligo(dt) affinity chromatography in arabidopsis cells (schmidt et al. 2010 ) now provides evidence for global in vivo rna binding. in addition, a plethora of proteins with potential rna binding activity have been detected. to substantiate their rna-binding properties, independent replication is desirable. among those are proteins with the domain of unknown function 1296, cytoskeletal proteins, and photoreceptors. the identification of plasma membrane intrinsic proteins has led to the speculation that aquaporins may be involved in transport of rnas between cells (reichel et al. 2016 ). another mrna interactome capture experiment was performed on cell suspension cultures generated from roots of the arabidopsis accessions col-0 and landsberg erecta. in parallel, leaves of four-weeks-old arabidopsis col-0 plants were investigated (marondedze et al. 2016) . of 1145 proteins identified altogether in these three samples, 914 appeared only in uv cross-linked samples, and 233 proteins were significantly enriched upon uv cross-linking relative to non-cross-linked samples. more than 350 proteins were known rbds whereas 736 were novel candidate rbps not previously assigned an rna-related function or known rbd, including many enzymes of intermediary metabolism, and thus await further experimental proof (marondedze et al. 2016 ). the discovery of many novel rbps begs for further investigation of the rnabinding properties of these proteins. accordingly, methods to define rna targets of candidate rbps genome wide using protein-centric methods have recently been adapted for the use in arabidopsis, as discussed below. approaches to globally identify in vivo targets of an rbp in arabidopsis mostly rely on transgenic plants expressing an epitope-tagged version of the rbp. immunopurification is performed via an antibody directed against the epitope tag. to mirror-image the endogenous expression pattern, authentic promoters are used and the constructs are introduced into a loss-of-function mutant (köster and staiger 2014) . alternatively, endogenous rbps can be recovered with dedicated antibodies. to freeze the in vivo rna-protein interactions before cell lysis, cross-linking is performed by exposing plants to formaldehyde in rna immunoprecipitation (rip) or by uv irradiation in uv cross-linking and immunoprecipitation (clip) (fig. 1b) . formaldehyde efficiently cross-links nucleic acids and proteins in vivo but also cross-links proteins. thus, not only direct targets are recovered. this is circumvented by using 254 nm uv light that cross-links proteins directly binding to nucleic acids in the neighborhood of the excited nucleobase but does not cross-link proteins. to date, a comprehensive determination of in vivo targets, the ribonome, has been performed for only a few arabidopsis rbps, both nucleocytoplasmic proteins and chloroplast-localized proteins with different tasks in posttranscriptional regulation. in the subsequent sections, selected examples are presented. hlp1 is an arabidopsis rbp resembling mammalian hnrnp a/b-like proteins (zhang et al. 2015) . high throughput sequencing (hits)-clip of hlp1 fused to gfp and expressed under control of the strong, constitutive cauliflower mosaic virus 35s rna promoter identified above 5500 transcripts bound in vivo (zhang et al. 2015) . when endogenous hlp1 protein was precipitated by a specific antibody, 6850 transcripts bound in vivo were detected with an overlap of above 3000 transcripts to the hlp1-gfp precipitation. the prevalence of cross-linked regions near polyadenylation sites provoked the hypothesis that hlp1 may control polyadenylation. indeed, in more than 2000 transcripts the distal polyadenylation site was preferred over the proximal polyadenylation site in hlp1 mutant plants. around 19% of these transcripts were also recovered by hlp1 hits-clip, pointing to a role for hlp1 in the control of alternative polyadenylation, at least partly by direct binding. in line with this, meme motifs overrepresented in the crosslink regions, namely a-rich (5 -agaaaa-3 ) and u-rich (5 -uuuucu-3 ) motifs, resembled motifs enriched in the vicinity of the poly(a) site, 5 -aaagaaaa-3 and 5 -uguuuc-3 . the presence of cross-link regions in other parts of the transcripts apart from the 3 untranslated region (utr) suggests that hlp1 may also affect other aspects of pre-mrna processing in addition to polyadenylation. atgrp7 (arabidopsis thaliana glycine rich rna-binding protein 7) is another hnrnp-like protein with an n-terminal rrm and a c-terminus enriched in contiguous glycine residues. atgrp7 is regulated by the circadian clock and negatively autoregulates its own oscillations by alternative splicing and nonsense-mediated decay (staiger et al. 2003; schmal et al. 2013) . additionally, it is involved in several steps of posttranscriptional regulation including alternative splicing, nucleic acid chaperone function, and pri-mirna processing (kim et al. 2007a; streitner et al. 2012; . to gain insights into the breadth of its in vivo targets, individual nucleotide resolution cross-linking and immunoprecipitation (iclip) and rip-seq were performed . atgrp7 fused to gfp was expressed from its own promoter including all regulatory elements (5 utr, intron, and 3 utr) in the atgrp7-1 loss-of-function mutant. in parallel, transgenic plants expressing gfp alone or an rna-binding dead variant of atgrp7 with a single conserved arginine in the rrm mutated to glutamine (atgrp7 r 49 q) were used as negative controls. iclip identified 858 transcripts with significant iclip hits in four out of five biological replicates for atgrp7-gfp that were not present in the controls. rip-seq identified 2453 transcripts enriched by atgrp7-gfp relative to total polyadenylated rna. the higher number may be due to the higher cross-linking efficiency of formaldehyde compared to uv light, and the recovery of many indirect targets. 452 transcripts were common in both data sets, suggesting that they represent a set of high confidence binders. the iclip cross-link sites were observed in all transcript regions, the utrs, coding sequence and introns. after correcting for the length of the feature in the genome, cross-link sites in the 3 utr prevailed. conserved motifs in the vicinity of the cross-link sites generally were u/c rich. to determine how atgrp7 may impact its downstream targets, the binding targets were cross-referenced against transcriptome data from atgrp7 overexpressing plants or loss-of-function mutants. in both, the atgrp7 overexpressors or the mutant, a similar number of transcripts was expressed at elevated or reduced levels compared to wild-type plants. notably, significantly more differentially expressed iclip targets were downregulated in atgrp7-overexpressors than upregulated. in turn, more of the differentially expressed atgrp7 iclip targets were expressed at elevated in the mutant than at reduced levels. this indicates a predominantly negative effect of atgrp7 on its targets. among the targets were more circadianly regulated transcripts than expected. in particular, elevated atgrp7 levels lead to damping of circadian oscillations of target transcripts including dor-mancy/auxin associated family protein2 and ccr-like. this conforms with the idea that the circadian clock regulated atgrp7 functions as a molecular slave oscillator, conveying temporal information from the core circadian clock within the cell (rudolf et al. 2004 ). in addition, changes in splicing patterns were observed for iclip and rip-seq targets upon misexpression of atgrp7, confirming a role for atgrp7 in the control of alternative splicing. arabidopsis thaliana serine/arginine rich (sr)-like protein sr45, the counterpart of metazoan rnps1, is an sr-like protein with two rs domains, flanking either side of the rrm (badolato et al. 1995; golovkin and reddy 1999) . notably, recombinant arabidopsis sr45 can activate splicing of a β-globin splicing reporter in hela cell s100 extracts (ali et al. 2007 ). sr45 occurs in two splice isoforms that arise through differential usage of a 3 splice site in intron 6. this leads to two protein isoforms that differ by seven amino acid residues and in their function: sr45.1 is involved in petal development in flowers, whereas sr45.2 is important for root growth (zhang and mount 2009) . genome-wide targets for sr45.1 were determined during early seedling development (xing et al. 2015) and in inflorescences (zhang et al. 2017) , respectively. in seedlings, rip-seq identified 4361 transcripts from 4262 genes that were enriched upon precipitation of sr45.1-gfp from nuclei of transgenic plants compared to mock precipitation from wild type plants (xing et al. 2015) . these were designated sars, for sr45 associated rnas. a gene ontology term analysis showed that 43 of 147 abscisic acid (aba) signaling genes (30%) were among the sars, in line with a function for sr45 in the aba signaling pathway (carvalho et al. 2010) . hundred and forty-eight of the sars had an altered expression in the sr45-1 mutant, suggesting that binding of sr45 has functional consequences. a meme search for sr45 binding motifs revealed four overrepresented motifs within sar genes. two g/a rich motifs are largely positioned within exons and show strong similarity to the binding motifs of two metazoan splicing regulators transformer 2 (tra2) and serine/arginine-rich splicing factor 10 (srsf10). furthermore, one g/a rich motif closely resembles the gaag motif, a known cisregulatory element in regulating alternative splicing in plants. in contrast, two u/c rich motifs peak within intronic regions near 5 and 3 splice sites, in line with the observation that the majority of sars were from intron-containing genes and the known role as a splicing regulator (xing et al. 2015) . to gain insights into a potential role of sr45 in flower development, rip-seq was performed for sr45.1-gfp in inflorescence tissue (zhang et al. 2017) . the resulting reads were analyzed by two different bioinformatics pipelines, one based on mapping reads to the genome and one directly quantifying annotated transcripts. sars in inflorescence were defined based on a twofold enrichment compared to gfp only controls and the identification by both pipelines. of 1812 sars in inflorescence, 677 overlapped with the sars in seedlings. notably, 19 transcripts encoding splicing factors were among the sars including sr45 itself, the three sr proteins sr30, sr34, and scl35, the pre-mrna processing factors prp39, prp40a, prp40b, and prp2, and the rna helicase rh42, pointing to a hierarchical regulation of posttranscriptional regulators (keene 2007) . genes upregulated in the sr45-1 mutant are enriched for defense response genes. indeed, the sr45-1 mutant was more resistant to bacterial and fungal pathogens. of 68 upregulated defense response genes in sr45-1, 10 were sars. thus, sr45 has an additional role as a negative regulator of plant immunity. furthermore, 81 of the inflorescence sars were aberrantly spliced in the sr45-1 mutant. determination of potential sr45 binding sites in inflorescence sars uncovered an overrepresentation of the purine-rich motifs ggngg, gngga, and gnggnng. importantly, ggngg and related motifs are enriched in introns and exons that are alternatively spliced in the sr45-1 mutant, irrespective of the splicing event is favored or suppressed by sr45. this led to the suggestion that sr45 identifies regions for alternative splicing and acts as a facilitator for other splicing factors. however, the identified binding motifs for sr45 in inflorescences differ from that in seedlings, which might be in part due to the different bioinformatic tools used for motif determination. both rip-seq data sets nevertheless strengthen sr45's key role as an important splicing factor in arabidopsis. however, in both rip-seq experiments intron-less transcripts were identified in addition to intron-containing transcripts, pointing to functions of sr45 beyond its known role in pre-mrna splicing. interestingly, a comparison between the u/c-rich motifs of atgrp7 and the u/crich motifs of sr45 identified by meme in seedlings revealed a high degree of similarity . the functional significance remains to be tested. in bacteria, csps are upregulated upon cold stress and destabilize rna secondary structure at low temperatures (sommerville 1999) . to elucidate a potential involvement of arabidopsis csps in the regulation of cold responsive genes, rip followed by gene chip analysis was performed for csp1 (juntawong et al. 2013) . more than 6000 mrnas were identified. comparison of these csp1-associated transcripts in total rna and rna loaded onto polysomes revealed an enrichment of mrnas associated with ribosome biogenesis in the pool of actively translating rnas. the high gc content in 5 utrs of these mrnas suggested that csp1 is involved in removing secondary structures in the 5 utr to facilitate their translation. accordingly, these mrnas were less efficiently loaded onto polysomes at low temperature in the atcsp1-1 mutant compared to wild type plants or csp1 overexpressing plants (juntawong et al. 2013 ). the highly abundant chloroplast ribonucleoproteins (cprnps) have been well characterized for their role in regulating chloroplast transcripts (ohta et al. 1995) . the cprnps comprise an acidic domain and two rrms. they are encoded in the nucleus and imported into chloroplasts. mutants in distinct cprnps are widely affected in processing of transcripts in the chloroplast, leading to defects in chloroplast development and, consequently, plant performance owing to the essential role of the chloroplast in photosynthetic energy (ruwe et al. 2011) . for example, mutants deficient in cp29a (29 kda chloroplast protein a) and cp31a (31 kda chloroplast protein a) showed gross defects at low ambient temperature. rip performed with antibodies against the endogenous proteins and subsequent hybridization of coprecipitated rnas on tiling arrays covering the arabidopsis chloroplast genome (rip-chip) showed that cp29a and cp31a associate with large overlapping sets of chloroplast transcripts including strong enrichment for psbb, psbd, psaa/b, atpb, ndhb and intermediate enrichment for almost all chloroplast mrnas (kupsch et al. 2012) . both cp29a and cp31a are required for accumulation of chloroplast mrnas under cold stress. furthermore, binding of cp31a to 3 ends of certain transcripts serves to protect these transcripts against 3 exonuclease activity (kupsch et al. 2012) . together with the known role of cp31a in rna (tillich et al. 2009 ) this points to multiple functions in posttranscriptional regulation in chloroplasts. for cp33a (33 kda chloroplast protein a), rip-chip revealed an association with a large body of chloroplast mrnas (teubner et al. 2017) . a global reduction in mrnas and proteins making up the photosynthetic apparatus was found in the cp33a mutant. in line with a crucial role for cp33a in the development of the photosynthetic apparatus, cp33a null mutants have an albino phenotype and are not able to survive without external sucrose supply (teubner et al. 2017 ). in contrast to the broad substrate specificity of the cprnps, a very narrow substrate specificity was found for a representative of the ppr class of nuclear-encoded rbps that are imported into organelles. atcpr1 (arabidopsis thaliana chloroplast rna processing 1) is important for the production of subunits of the thylakoid protein complexes (ferrari et al. 2017) . atcpr1 mutants are yellow-white because the subunits of the photosynthetic apparatus do not accumulate. rip-chip was performed for atcpr1 under native conditions. hybridization of bound targets to chloroplast tiling arrays revealed specific binding of atcpr1 to only few transcripts, the psac transcript encoding a photosystem i subunit, petb-petd encoding cytochrome b 6 and the subunit iv of the cytochrome b 6 /f complex. because during rip rnase was used to digest unprotected rna, it was possible to delineate the binding regions. binding to the petb-petd intergenic region correlated with a requirement for processing of the polycistronic transcript comprising petb and petd (ferrari et al. 2017) , thus providing proof for the functional relevance of the observed in vivo binding. in addition to rna sequence, rna secondary structure also strongly influences the interaction of rbps with their cognate rna binding motifs (cruz and westhof 2009; vandivier et al. 2016) . rna structure may facilitate binding of rbds with a preference for double-stranded rna or inhibit binding of rbps with a preference for single-stranded rna. protein interaction profile sequencing (pip-seq) allows simultaneous delineation of in vivo rna secondary structure and protein-protected sites (ppss) (fig. 2) (gosai et al. 2015) . to identify ppss, samples are treated with a single-strand specific or double-strand specific rnase. proteins are then denatured before library preparation. to determine the rna secondary structure, proteins are denatured by sds and removed by protease digestion to make sites protected by proteins in vivo accessible for rnases. collectively, motifs that are enriched in the samples used to determine protein protected sites compared to the samples used for structure determination are in vivo target sites of rbds. gregory and coworkers applied pip-seq to the nuclei of two specific cell types in the arabidopsis roots that derive from epidermal cells through distinct differentiation, those cells bearing root hairs and those that do not (foley et al. 2017) . distinct protein binding patterns were detected, and binding motifs either specific to hair cells, non-hair cells or common to both cell types were determined. to identify candidate proteins, rna affinity chromatography was performed on immobilized oligonucleotides derived from enriched motifs. a ggn repeat motif enriched in sites protected in both hair cells and non-hair cells recovered serrate (se) from root lysates, a zinc finger containing rbp involved in processing of mirna precursors. a tg rich motif enriched in hair cell-specific protected sites identified atgrp2, atgrp7 and atgrp8. subsequently, atgrp8 was shown to regulate root hair development at the posttranscriptional level. an advantage of pip-seq is that it does not rely on an antibody to identify target sites within bound transcripts. in contrast, subsequent identification of the cognate binding proteins requires in vitro binding techniques. thus, binding in vivo has to be confirmed by independent means. the recent mrna interactome capture studies are very valuable in having established uv cross-linking and oligo(dt) affinity capture to determine the mrna binding proteome also in arabidopsis. a large number of previously predicted rbps in arabidopsis were now identified experimentally and many novel proteins without a previous assignment to rna biology unearthed. reichel and colleagues noticed a bias toward proteins with higher abundance in the interactome compared to the input (reichel et al. 2016) , suggesting that additional proteins with lower expression level may still be identified in the future. only few of the mrna interacting proteins were present in all three interactomes ). this may partly be attributed to the widely differing developmental stages investigated. among the commonly identified proteins are numerous cytoplasmic ribosomal proteins from the small and large ribosomal subunits, likely due to their high abundance, as well as the ubiquitously expressed glycine-rich rbps atgrp7 and atgrp8 . future applications are the dynamics of posttranscriptional networks in response to endogenous and exogenous stimuli cues by describing changes in the mrna bound proteomes. furthermore, as proteins binding to nonpolyadenylated rnas obviously remain elusive in these approaches, transcript-specific approaches have to be developed. transcriptome-wide identification of target transcripts bound by selected rbps in vivo has overcome a major limitation in research on plant rna-based regulation. nevertheless, except for the ppr proteins, we are still far from understanding the exact binding specificity of most proteins and the consequences in vivo binding has for the targets. to correlate in vivo binding with function, the impact of mutated candidate binding motifs on rbp binding and target gene expression has to be determined. most bioinformatics pipelines today discussing motif discovery are limited to sequence data. current efforts focus on developing bioinformatics pipelines for identifying conserved motifs taking rna structure context into consideration (maticzka et al. 2014) . molecular dynamics of rna molecules are still compute intensive but can shed light on possible interaction sites and three dimensional structures (tuszynska et al. 2015; boniecki et al. 2016) . finally, heterogeneous datasets and analyses, fusing several kinds of sources, can improve meta-analysis with in silico and in vivo datasets. this is yet limited in arabidopsis but will improve the information quality in the near future. additionally, it will be important to have comprehensive databases on rbp target sites linked to the arabidopsis information portal (the international arabidopsis informatics consortium 2012). such resources will be of great value to improve a systems understanding of rnaprotein interaction. regulation of plant developmental processes by a novel splicing factor identification and characterisation of a novel human rna-binding protein the mrna-bound proteome and its global occupancy profile on protein-coding transcripts pentatricopeptide repeat proteins in plants the rna-binding proteomes from yeast to man harbour conserved enigmrbps simrna: a coarse-grained method for rna folding simulations and 3d structure prediction the mrna-bound proteome of the human malaria parasite plasmodium falciparum the plant-specific sr45 protein negatively regulates glucose and aba signaling during early seedling development in arabidopsis the arabidopsis sr45 splicing factor, a negative regulator of sugar signaling, modulates snf1-related protein kinase 1 stability insights into rna biology from an atlas of mammalian mrna-binding proteins the dynamic landscapes of rna architecture the rna-binding proteins hyl1 and se promote accurate in vitro processing of pri-mirna by dcl1 crp1 protein: (dis)similarities between arabidopsis thaliana and zea mays a global view of rna-protein interactions identifies post-transcriptional regulators of root hair cell fate a type iii effector adp-ribosylates rna-binding proteins and quells plant immunity rna-binding protein immunopurification-microarray (rip-chip) analysis to profile localized rnas an sc35-like protein and a novel serine/arginine-rich protein interact with arabidopsis u1-70k protein global analysis of the rna-protein interaction and rna secondary structure landscapes of the arabidopsis nucleus the alba protein family: structure and function genome-wide rip-chip analysis of translational repressor-bound mrnas in the plasmodium gametocyte salicylic acid-dependent and -independent impact of an rna-binding protein on plant immunity a light-and temperature-entrained circadian clock controls expression of transcripts encoding nuclear proteins with homology to rna-binding proteins in meristematic tissue the spen family protein fpa controls alternative cleavage and polyadenylation of rna an mrna cap binding protein, abh1, modulates early abscisic acid signal transduction in arabidopsis structure function analysis of an adp-ribosyltransferase type iii effector and its rna-binding target in plant immunity mutation of arabidopsis spliceosomal timekeeper locus1 causes circadian clock defects cold shock protein 1 chaperones mrnas during translation in arabidopsis thaliana ectopic expression of at rsz33 reveals its function in splicing and causes pleiotropic changes in development rna regulons: coordination of post-transcriptional events cold shock domain proteins and glycine-rich rna-binding proteins from arabidopsis thaliana can promote the cold adaptation process in escherichia coli functional characterization of a glycine-rich rna-binding protein 2 in arabidopsis thaliana under abiotic stress conditions a zinc finger-containing glycine-rich rna-binding protein, at rz-1a, has a negative impact on seed germination and seedling growth of arabidopsis thaliana under salt or drought stress conditions functional characterization of dead-box rna helicases in arabidopsis thaliana under abiotic stress conditions glycine-rich rna-binding proteins are functionally conserved in arabidopsis thaliana and oryza sativa during cold adaptation process iclip reveals the function of hnrnp particles in splicing at individual nucleotide resolution rna-binding protein immunoprecipitation from whole-cell extracts regulation of pri-mirna processing by the hnrnplike protein atgrp7 in arabidopsis rna-binding proteins revisited: the emerging arabidopsis mrna interactome whirly proteins as communicators between plant organelles and the nucleus? arabidopsis chloroplast rna binding proteins cp31a and cp29a associate with large transcript pools and confer cold stress tolerance by influencing multiple chloroplast rna processing steps the rna-binding protein repertoire of embryonic stem cells genome-wide identification and phylogenetic analysis of plant rna binding proteins comprising both rna recognition motifs and contiguous glycine residues genome-wide identification, biochemical characterization, and expression analyses of the yth domain-containing rna-binding protein family in arabidopsis and rice the cardiomyocyte rna-binding proteome: links to intermediary metabolism and heart disease hits-clip yields genome-wide insights into brain alternative rna processing a glycine-rich rna-binding protein affects gibberellin biosynthesis in arabidopsis ) atsrp30, one of two sf2/asf-like proteins from arabidopsis thaliana, regulates splicing of specific plant genes rbp45 and rbp47, two oligouridylatespecific hnrnp-like proteins interacting with poly(a)+ rna in nuclei of plant cells a mutation in the arabidopsis hyl1 gene encoding a dsrna binding protein affects responses to abscisic acid, auxin, and cytokinin gene expression regulatory networks in trypanosoma brucei: insights into the role of the mrna-binding proteome the rna-binding protein fpa regulates flg22-triggered defense responses and transcription factor activity by alternative polyadenylation fca, a gene controlling flowering time in arabidopsis, encodes a protein containing rna-binding domains the rna-binding protein repertoire of arabidopsis thaliana conserved mrna-binding proteomes in eukaryotic organisms graphprot: modeling binding preferences of rnabinding proteins adaptation of iclip to plants determines the binding landscape of the clock-regulated rna-binding protein atgrp7 global analysis of yeast mrnps sr proteins are nxf1 adaptors that link alternative rna processing to mrna export comprehensive identification of mrna-binding proteins of leishmania donovani by interactome capture pseudomonas hopu1 affects interaction of plant immune receptor mrnas to the rna-binding protein grp7 three types of nuclear genes encoding chloroplast rnabinding proteins (cp29, cp31 and cp33) are present in arabidopsis thaliana: presence of cp31 in chloroplasts and its homologue in nuclei/cytoplasms cold shock domain proteins affect seed germination and growth of arabidopsis thaliana under abiotic stress conditions role for lsm genes in the regulation of circadian rhythms a member of the whirly family is a multifunctional rna-and dna-binding protein that is essential for chloroplast biogenesis a putative rna-binding protein positively regulates salicylic acid-mediated immunity in arabidopsis in planta determination of the mrna-binding proteome of arabidopsis etiolated seedlings regulation of mirna abundance by rna binding protein tough in arabidopsis arabidopsis rna-binding protein uba2a relocalizes into nuclear speckles in response to abscisic acid pepper, a novel k-homology domain gene, regulates vegetative and gynoecium development in arabidopsis crosslinking-immunoprecipitation (iclip) analysis reveals global regulatory roles of hnrnp l slave to the rhythm polypyrimidine tract binding protein homologs from arabidopsis are key regulators of alternative splicing with implications in fundamental developmental processes the rna-recognition motif in chloroplasts a circadian clock-regulated toggle switch explains atgrp7 and atgrp8 oscillations in arabidopsis thaliana a proteomic analysis of oligo(dt)-bound mrnp containing oxidative stress-induced arabidopsis thaliana rna-binding proteins atgrp7 and atgrp8 pentatricopeptide repeat proteins: a socket set for organelle gene expression genomic era analyses of rna secondary structure and rna-binding proteins reveal their significance to post-transcriptional regulation in plants fy is an rna 3 end-processing factor that interacts with fca to control the arabidopsis floral transition activities of cold-shock domain proteins in translation control selective mrna sequestration by oligouridylate-binding protein 1 contributes to translational control during hypoxia in arabidopsis rna-binding proteins and circadian rhythms in arabidopsis thaliana shaping the arabidopsis transcriptome through alternative splicing the circadian clock regulated rna-binding protein atgrp7 autoregulates its expression by influencing alternative splicing of its own pre-mrna polypyrimidine tract-binding protein homologues from arabidopsis underlie regulatory circuits based on alternative splicing and downstream control the small glycine-rich rna-binding protein atgrp7 promotes floral transition in arabidopsis thaliana an hnrnp-like rna-binding protein affects alternative splicing by in vivo interaction with target transcripts in arabidopsis thaliana global changes of the rna-bound proteome during the maternal-to-zygotic transition in drosophila identifying mrna subsets in messenger ribonucleoprotein complexes by using cdna arrays the rrm protein cp33a is a global ligand of chloroplast mrnas and is essential for plastid biogenesis and plant development taking the next step: building an arabidopsis information portal chloroplast ribonucleoprotein cp31a is required for editing and stability of specific chloroplast mrnas npdock: a web server for protein-nucleic acid docking the conservation and function of rna secondary structure in plants the nuclear dsrna binding protein hyl1 is required for microrna accumulation and plant development, but not posttranscriptional transgene silencing lachesis-dependent egg-cell signaling regulates the development of female gametophytic cells n 6 -methyladenosine-dependent regulation of messenger rna stability the mrna-bound proteome of the early fly embryo transcriptome-wide identification of rna targets of arabidopsis serine/arginine-rich45 uncovers the unexpected roles of this rna binding protein in rna processing two alternatively spliced isoforms of the arabidopsis thaliana sr45 protein have distinct roles during normal plant development integrative genome-wide analysis reveals hlp1, a novel rnabinding protein, regulates plant flowering by targeting alternative polyadenylation uv crosslinked mrna-binding proteins captured from leaf mesophyll protoplasts transcriptome analyses reveal sr45 to be a neutral splicing regulator and a suppressor of innate immunity in arabidopsis thaliana the work in tino köster's lab is supported by the dfg through grant ko 5364/1-1. martin lewinski is supported by the dfg through grant sta653/6-1 to dorothee staiger. key: cord-022955-vy0qgtll authors: nan title: proteases date: 2005-06-20 journal: febs j doi: 10.1111/j.1742-4658.2005.4739_4.x sha: doc_id: 22955 cord_uid: vy0qgtll nan the incretin hormones glp-1 and gip are released from the gut during meals, and serve as enhancers of glucose stimulated insu-lin release from the beta cells. furthermore, glp-1 also stimulates beta cell growth and insulin biosynthesis, inhibits glucagon secretion, reduces free fatty acids and delays gastric emptying. glp-1 has therefore been suggested as a potentially new treatment for type 2 diabetes. however, glp-1 is very rapidly degraded in the bloodstream by the enzyme dipeptidyl peptidase iv (dpp-iv; ec 3.4.14.5). a very promising approach to harvest the beneficial effect of glp-1 in the treatment of diabetes is to inhibit the dpp-iv enzyme, thereby enhancing the levels of endogenously intact circulating glp-1. the three dimensional structure of human dpp-iv in complex with various inhibitors creates a better understanding of the specificity and selectivity of this enzyme and allows for further exploration and design of new therapeutic inhibitors. the majority of the currently known dpp-iv inhibitors consist of an alpha amino acid pyrrolidine core, to which substituents have been added to optimize affinity, potency, enzyme selectivity, oral bioavailability, and duration of action. various compound series and their sar relative to alpha amino acids will be presented. memapsin 2 (b-secretase, bace1) is the membrane-anchored aspartic protease that initiates the cleavage of b-amyloid precursor protein (app) leading to the production of amyloid-b (ab), a major factor in the pathogenesis of alzheimer's disease (ad). since memapsin 2 is a major target for the development of inhibitor drugs for the treatment of ad, its structure and physiological functions are topics of intense research interest currently. here we discuss the structural features of memapsin 2 and how do they contribute to the activity and inhibition of the protease. structural and kinetic evidence support the presence of 11 subsites for substrate or inhibitor binding in the activesite cleft of memapsin 2. subsites p3 to p2' are most useful in the design of transition-state analogue inhibitors. recent data indicated that subsites p7, p6 and p5 have strong influence of hydrolytic rate or inhibition potency. these subsites are, however, too far from the transition-state isostere for the design of drug-like transition-state inhibitors but can be utilized for the design of non-transition-state inhibitors that compete for substrate binding. besides carrying out proteolytic activity, the ectodomain of memapsin 2 also interacts with app leading to the endocytosis of both proteins into the endosomes where app is hydrolyzed by memapsin 2 to produce ab. a phosphorylated motif in the cytosolic domain of memapsin 2 is responsible for the recognition of gga proteins as part of the recycling mechanism that transports memapsin 2 from endosomes to trans-golgi then back to cell surface. these interactions may also be considered for the design of small-molecular compounds that interfere with memapsin 2 trafficking and thus reduce the production of ab. identification of human carnosinase -a brainspecific metalloprotease m. teufel biochemistry, exploratory research, sanofi aventis, strasbourg, france. e-mail: michael.teufel@sanofi-synthelabo.com metalloproteases form a large and diverse family of proteases and are molecular targets that represent an opportunity for therapeutic intervention. in particular, the development of potent inhibitors has made progress for the family of matrix metalloproteases (mmp). the sequencing of the human genome revealed that a significant percentage of the drugable genome is represented by proteases, many of them still with unknown function. in this presentation, data will be presented on the deorphanization of two previously unknown genes by means of bioinformatics and classical biochemistry. this work led to the identification of human carnosinase, a dipeptidase specifically expressed in the human brain and an ubiquitously expressed close homologue, characterized to be a non-specific dipeptidase. stimulating serpins with synthetic tailor-made oligosaccharides: a new generation of antithrombotics m. petitou thrombosis & angiogenesis, sanofi-aventis, toulouse, france. e-mail: maurice.petitou@sanofi-aventis.com we will discuss our research on synthetic oligosaccharides able to selectively activate the inhibitory activity of antithrombin towards various serine proteinases. we first synthesized pentasaccharides closely related to the antithrombin binding domain of heparin [1] (the active site), as well as analogues displaying different pharmacokinetic profiles. selective inhibitors of coagulation factor xa were thus obtained that represent a new class of antithrombotic [2] drugs currently being evaluated worldwide. we then designed larger oligosaccharides [3] that inhibit both factor xa and thrombin in the presence of antithrombin. they are devoid of undesired nonspecific interactions with blood proteins, particularly with platelet factor 4. clinical trials are ongoing to prove the therapeutic benefits of this new type of coagulation inhibitors. slow tight binding inhibitors in drug discovery: in the case of dppiv and elastase inhibitors z. kapui, e. boronkay, i. bata, m. varga, e. mikus, k. urban-szabo, s. ba´tori and p. ara´nyi discovery research, chinoin member of sanofi-aventis group, budapest, hungary. e-mail: zoltan.kapui@sanofi-aventis.com enzyme are extremely potent causing significant inhibition at very low concentrations that may be comparable to the concentration of the target enzyme. when this inhibition is studied in vitro, complexities arise because the concentration of the inhibitor is so low that it is altered significantly as a result of combination with the enzyme. this situation is referred to as tight-binding inhibition. partly as a result of their low concentrations, tight-binding inhibitors often show slow-binding characteristics. unlike conventional inhibitors that act almost instantaneously (or at least within the ms time scale), slow-binding inhibitors may take several seconds, minutes or even hours for their effect to be fully exhibited. this association between slow-binding and tight-binding is relatively common and slow tight-binding inhibitors are extremely potent and specific. proteolytic enzymes are involved in a multitude of important physiological processes. their intrinsic properties and activities are in the focus of wide-ranging research and they have a valuable role in experimental and therapeutic purposes. serine proteases are attractive targets for the design of enzyme inhibitors since they are involved in the etiology of several diseases. within the class of serine proteases, human leukocyte elastase (hle) is one of the most destructive enzymes in the body. the enzyme dipeptidyl peptidase iv (dppiv) is a serine exopeptidase that cleaves xaa-pro dipeptides from the n-terminus of oligo-and polypeptides. inhibitors of dpp iv are of increasing interest to pharmaceutical industry alike, as they may become established as the next member of the oral antidiabetic class of therapeutic agents. objective of our work was to develop reversible, slow, tight-binding inhibitors against these serine proteases. ssr69071 is a potent inhibitor of hle, the inhibition constant (k i ) and the constant for inactivation process (k on ) being 0.0168 ± 0.0014 nm. this inhibitor is reversible, slow, tight-binding inhibitor with k on = 0.183 ± 0.013 10 6 /ms, and k off = 3.11 ± 0.37 10 )6 /s. ssr69071 inhibits the solubilization of elastin by hle with 13 nm of ic50 value. this inhibitor is one of the most effective inhibitor of a serine proteinase yet described. ssr162369 is a potent, competitive and slow tight binding type inhibitor of the human dipeptidyl peptidase-iv enzyme (k i = 2 nm, t½ = 8 h). on the basis of kinetic properties, ssr162369 forms stable enzyme-inhibitor complex. these slow tight-binding inhibitors have unique inhibitory properties, they are extremely active, and selective, form stable enzyme-inhibitor complex, therefore they have long-lasting effect. their oral activity and long lasting in vivo biological potency agreed very well with stable enzyme-inhibitor complex. the advantages in drug discovery of slow tight-binding inhibitors are discussed in this presentation. enzyme inhibition trend analysis -a new method for drug design m. shokhen, n. khazanov and a. albeck the julius spokojny bioorganic chemistry laboratory, chemistry, bar lan, ramat gan, israel. e-mail: albecka@mail.biu.ac.il many of the drugs that are currently in use or at different stages of development are enzyme inhibitors. therefore, enzyme mechanism-based inhibitors could be developed into highly selective drugs. our novel enzyme inhibition trend analysis method com-bines experimental enzyme kinetics data and high level quantum mechanical modeling of enzyme-inhibitor chemical interactions. the method utilizes the principal catalytic reaction scheme of the target enzyme and does not require its 3d structure (a ligand based approach). the method is valid for the prediction of the trend in binding affinity of inhibitors not only for the specific enzyme for which the qsar model was optimized, but also for the whole enzyme family. the methodology would contribute significantly to overcoming the problem of fast mutational resistance developed by pathogens in response to pharmaceutical treatment. it can be used as a computational tool for expert analysis of various hypotheses about structure-activity relationships formulated for the design of new inhibitors. angiotensin-converting enzyme (ace, ec 3.4.15.1) is a key enzyme for blood pressure control and water-electrolyte homeostasis. a large number of highly potent and specific ace inhibitors are used as oral drugs in the treatment of hypertension and congestive heart failure. somatic ace consists of two homologous domains (n-and c-) within single polypeptide chain, each one containing a catalytic site. the two catalytic sites within somatic ace molecule were long considered to function independently. however, recent investigations indicate the existence of negative cooperativity between ace active sites. we studied the properties of bovine ace active centers by use of separate ace n-domain (n-ace) obtained by limited proteolysis of parent somatic enzyme and testicular ace, which represents c-domain. these results were compared with the data obtained for full-length somatic ace from bovine lungs. the results obtained demonstrate strongly dependent mechanism of action of ace active centers in the reaction of the hydrolysis of tripeptide substrates. however, the hydrolysis of decapeptide angiotensin i proceeds independently on n-and c-domains. the mechanism of inhibition of ace activity is also dependent on the length of the inhibitor: (i) random binding of the ''short'' inhibitor molecule (such as captopril, lisinopril) to one of the active sites dramatically decreases binding of another inhibitor molecule to the second site; (ii) ''long'' nonapeptide teprotid binds to both active sites without any difficulties. since the main physiological ace substrates in the organism are ''long'' peptides angiotensin i and bradykinin, the development of new class of inhibitors with prolonged structure would be beneficial for abolishing of ace activity. synthetic peptide studies on severe acute respiratory syndrome coronavirus (sars-cov) extensive proteolytic processing of the replicase polyproteins, pp1a (486 kda) and pp1ab (790 kda), by the sars-cov 3clike protease (3cl pro ). besides, the structural spike protein of sars-cov contains two heptad repeat regions (hr1 and hr2) that form coiled-coil structures, which play an important role in mediating the membrane fusion process. in this study, we focused on both 3cl pro and the hr regions of sars. previous studies demonstrated that the coronavirus 3cl pro cleaves the replicase polyproteins at no <11 conserved cleavage sites, preferentially at the lq sequence. the reported crystal structure of sars-cov 3cl pro provides insights into the rational design of anti-sars drugs. in order to understand the molecular basis of the enzyme-substrate binding mechanism, we employ the synthetic peptide and mass spectrometry-based approaches to investigate the significance of selected amino acid residues that are flanking both sides of the sars-cov 3cl pro cleavage site. in addition, previous studies indicated that the relatively deep hydrophobic coiled coil grooves on the surface of sars-cov spike protein heptad repeat regions (hr1 and hr2) may be a good target site for the design of viral fusion inhibitors. we have designed and synthesized five truncated peptide analogs derived from hr1 and hr2 peptides based on both bioinformatics and structural analysis. the biological activities of these truncated analogs will be studied using circular dichroism spectroscopy, multidimensional chromatography, protein cross-linking and mass spectrometry-based approach. the above investigation will definitely broaden our knowledge on the sars research and will reveal the feasibility of rational design of synthetic peptide-based drug in combating with sars disease. ras-transfection-associated invasion: involvement of matrix metalloproteinase(s) confirmed using a chicken embryo model and real time pcr during metastasis tumorogenic cells leave the primary tumour and intravasate into the blood/lymphatic system, exiting at a secondary site to establish a secondary tumour. ras-transfection of a parental, non-invasive mcf-10a cell line, established from a patient suffering with benign fibrocystic disease, gave rise to an invasive derivative cell line (mcf-10a-neot) exhibiting the phenotype of a pre-malignant, invasive tumour. invasion and metastasis are protease-assisted processes, proteases either being secreted by the tumour, or by the stromal cells under the influence of the tumour. here we demonstrate the involvement of matrix metalloproteinase(s) in the invasion of the ras-transfected mcf-10a cell line. tumour cells were inoculated onto the damaged surface of the upper chorioamniotic membrane (cam) of a vasculated 9-day old chick embryo. the tumour cells were allowed to invade, and the number of invading cells quantified using real time pcr. inhibitors specific for various proteases were applied to the upper cam, to block invasion, and hence identify the proteinases involved. the number of tumour cells invading into the vascular system was established by sampling the lower cam and quantifying the numbers of alu sequences (present only in human cells) in the dna, isolated from the embryonic tissue, using real-time pcr. using this method, the key role of an mmp was demonstrated. spectrum ptk inhibitor, genistein (100 lm) abolished the release of neutrophil mmp-9, in the presence and absence of extracellular calcium, and reduced the release of timp-1. both pp2 (10 lm), a src family ptk inhibitor, and piceatannol (30 lg/ml), a syk family ptk inhibitor, reduced mmp-9 release substantially, indicating that multiple ptk families might be involved in mmp-9 release. inhibition of either syk or src ptks by piceatannol or pp2 did not appear to influence timp-1 release. low levels of wortmannin (100 nm, inhibition of pi3k) abolished the release of mmp-9 in the absence of calcium, and reduced mmp-9 release in the presence of calcium. investigations into the signaling pathways involved in timp-1 release are continuing. we conclude that mmp-9 release induced by extracellular calcium may be mediated through pi3k and multiple tyrosine kinases, including src and syk family ptks. timp-1 granule release may also be mediated by tyrosine kinases, although src and syk family ptks do not appear to be involved. thermodynamical and structural analysis of cruzain/cruzipain2 complexed with e-64 by molecular modeling and dynamics simulations peptidases represent one of the most relevant enzyme classes targeted by therapeutic intervention. to contribute to the assignment of a physiological role to genomic-derived peptidases and to make them more accessible for the drug discovery process, we have undertaken a program consisting of mrna expression profiling, full-length recombinant expression in insect cells, purification and determination of the catalytic activity for the human proteolytic enzymes. a milestone in the process was the construction of a non-redundant comprehensive database for all human peptidases comprising 443 unique annotated entries, by assembling and filtering public domain information and in-house generated data. in order to get an informative picture on their expression profiling, a transcriptome database for 375 human peptidases was created using the microarray (affymetrix tm ) and taqman ò (applied biosystems) technologies. in parallel, we have set up the procedure for pcr amplification and cloning of the peptidase genes in 96 mtp format and we have already created a repository of 231 full-length human cdnas encoding for peptidases. besides, the conditions for miniaturized insect cell cultures have been established. experimental trials have defined a validated, reliable and fully-automated robotic procedure for the purification of recombinantly expressed peptidases in 96 mtp format. in a pilot study using the high-throughput approach, 85% of the chosen reference hydrolases (14) were secreted into the insect cell medium. of them, 66% have been proven to be catalytically active using fluorescent homogeneous assays in 384well format compatible with the high-throughput screening criteria. the application of this procedure to genomic-predicted peptidases is discussed. comparison of putative glutamate racemases from bacillus species glutamate racemase catalyzes the interconversion between l-and d-glutamic acid and is the cell's source of d-glutamate, a key component in the synthesis of both the bacterial cell wall and the glutamyl capsule. bacillus subtilis has two glutamate racemases in its genome, race and yrpc, while b. cerus and b. anthracis have two race genes, race1 and race1. interestingly, race in b. subtilis is the isoform that is essential and has the greater catalytic efficiency, but both race1 and race2 have higher sequence homology to race, 70 and 79% respectively and share less homology with the yrpc isoform, both at 53%. we have cloned, overexpressed, purified, and are characterizing the kinetic and biophysical properties of the two putative glutamate racemases, race1 and race2 from b. cereus and b. anthracis, and will utilize kinetic and biophysical information to design inhibitors that may result in a novel antibiotic. although these two isoforms share a high sequence similarity, their properties are unique. kinetic data indicates a fivefold difference in catalytic efficiency of race2 compared to that of race1 in the l-to d-glutamate reaction. also, the absence or presence of substrate has an effect on the oligomerization state, details of which will be reported. finally, our collaborators have demonstrated through genetic knock out experiments that only one of the race isoforms is essential for the growth of b. anthracis. we have crystallized the race2 isozyme and x-ray data have been collected to 2.3 å . we are currently solving the structure via heavy-atom derivatives. acknowledgment: this research was funded by nih grant u19 ai056575. anti-inflammatory effects of methionine aminopeptidase 2 inhibition on human b lymphocytes e. janas 1 , r. priest 1 , s. ratcliffe 2 and r. malhotra 1 1 rheumatoid arthritis biology, glaxosmithkline, stevenage, uk, 2 high throughput chemistry, glaxosmithkline, stevenage, uk. e-mail: eva.x.janas@gsk.com processing of n-terminal methionine is an essential post-translational modification in both prokaryotes and eukaryotes regulating the subcellular localization, stability and degradation of proteins. the cleavage of the initiator methionine is catalysed by a highly conserved family of metalloproteases, methionine-aminopeptidase 1 and 2 (met-ap2). human met-ap2 is the molecular target of fumagillin, a natural product with antiangiogenic properties, which covalently binds to his 231 in the catalytic site of met-ap2. although fumagillin has been observed to inhibit proliferation and to cause cell cycle arrest in endothelial cells, the mechanism of inhibition is still poorly understood. recent studies describe high expression of met-ap2 in germinal centre b lymphocytes. here, we investigate the effect of the met-ap2 inhibitor fumagillin on b lymphocyte proliferation and cell cycle progression and compare these results to those observed in hu-vec. in addition our work sheds light on the mechanistic aspects of met-ap2 inhibition by fumagillin and its derivatives. effect of distal mutations on the molecular dynamics of the hiv-1 protease l10i and l90m are the most common distal mutations found in the protease gene of the drug resistant hiv-1 strains. these mutations do not confer resistance by themselves, however induce a large synergy effect when added to active site mutations. understanding the impact of the l90m and l10i mutations on the hiv-1 protease resistance profile is still a challenge. assuming that their contribution to the resistance profile could be mediated by conformational dynamics we have modeled l10i, l90m and l10i/l90m mutants of hiv-1 protease. these unbound mutated and wild type proteases were subjected to 10ns molecular dynamics simulations and compared using an essential dynamics (ed) analysis protocol. the first eigenvector of the native protease describes the flap openning motion. following eigenvectors describe ''the catalytic assisting motions'' (cam) of the protease that becomes dominant upon complex formation with a substrate (piana s et al. j mol biol 2002; 319(2): 567-583). mutation of luecine to methionine residue at position 90 perturbs the protein packing at the dimerization domain. such perturbations affect the dimerization domain motions which correlate with flap opening and the cam. as result the first eigenvector corresponds to the rotational of the one subunit relative to another along axis connecting residues 60 and 60'. in other words l90m mutation mistunes essential motions of the enzyme while retaining its flexibility. this could be the cause of the reduced structural stability of the l90m mutant. in contrast, l10i mutation causes only redistribution of the correlated motions amplitude. the catalytic assisting motion becomes the most influential that results in stabilization of the closed conformation. in turn, the flap opening motions are reduced in l10i mutant. essential dynamics of the double mutant l10i/l90m could be described in the following terms. a strong propagation of the cam induced by l10i mutation is coupled with the altered conformational space caused by l90m mutation. as result the double mutant prefers cam motions that are close to the native protease but also account for the perturbed packing within the dimerization domain. results presented may help understanding hiv-1 protease resistance pathways and in developing more efficient inhibitors of known drug resistant mutants. glutamate carboxypeptidase ii as a cancer marker and therapeutical target: two faces of an enzyme glutamate carboxypeptidase ii is a membrane-bound metallopeptidase expressed in a number of tissues such as jejunum, kidney, prostate and brain. the brain form of gcpii (also known as naaladase) is expressed in astrocytes and cleaves n-acetylaspartyl glutamate, an abundant neurotransmitter, to yield free glutamate. gcpii thus represents an important target for the treatment of neuronal damage caused by excess glutamate. animal model experiments suggest that specific inhibitors of gcpii could be useful for the treatment of several neuropathic conditions, such as brain stroke, chronic neuropathic pain or amyotrophic lateral sclerosis. in the same time, the enzyme is known as prostate-specific membrane antigen since it is upregulated in prostate cancer. it is used for the diagnosis and experimental therapy of prostate cancer using monoclonal antibodies and specific inhibitors. in order to analyze this important pharmaceutical target, we established an expression system based on drosophila schneider's cells. we have also cloned, expressed and characterized its human homolog gcpiii and homologous carboxypeptidases from pig and rat. using specific monoclonal antibodies, we have been able to study the expression of gcpii in various healthy and malignant tissues. we analyzed the substrate specificity of the enzyme using peptide libraries and identified two novel peptide substrates. availability of a recombinant protein enabled to introduce a simple fluorescent activity assay and test specific inhibitors. furthermore, we have biochemically characterized the recombinant protein in terms of pharmacologic properties, oligomeric status, ph dependence and activity modulation by metal ions. we have shown that the glycosylation is indispensable for gcpii carboxypeptidase activity and analyzed the role of each specific n-glycosylation site for the gcpii activity and folding. using site-directed mutagenesis, we are able to identify the domains sufficient and necessary for gcpii activity and also suggest structural explanation for the substrate specificity of the enzyme. dozens of chemicals feature inhibition of proteolytically important tyrosine residue of 20s proteasome by forming covalent bond to hydroxyl group that abolished its catalytic function. in contrary, the approach we utilize here is based on hydrogen and hydrophobic interactions reversibly inactivating all three sites of 20s complexes. we performed flexible docking studies of analogues of a natural product tmc-95a using 1jd2 crystal structure to describe the active site of protein and the position of the ligand. the search yielded several amide-like derivatives that have been screened for superimposition with tmc-95a. few of them revealed similar orientation of propylene groups to the active site of 20s. second screen was performed to reveal the chemicals with the strongest hydrogen-bonding of the ligand to the protein backbone of the receptor. this screen resulted in two chemicals that had strong h-contacts with tyr21, ser129 and, importantly, with proteolytically active tyr1 residue. to access the validity of the predicted chemicals we undertook in vitro studies measuring the hydrolyses of fluorogenic substrate by the sds activated 20s proteasome isolated from hela cells. we obtained more than 85% inhibition of 20s proteasome activity upon incubation the above chemicals (0.5 lg/ml) with proteasomes. we then demonstrated the effectiveness of the obtained chemicals to stabilize the level of oncosupressors, including p53 in benign (mcf10a) and highly metastatic (mda231) cell lines. treatment with these compounds greatly restored the level of p53 in cancer cells. finally, we performed proliferation assay and proved that adding of this artificially synthesized chemicals to mda231 cell line significantly reduced the level of proliferation, whereas mcf10a cells treated at similar conditions have not revealed any abnormal reduction of proliferation below control level. thus, we report of a strategy to predict highly suitable proteasome inhibitors that act via inhibition of protease activity and may lead to creation of a new class of drugs for cancer therapy. localization and trafficking of prostate specific membrane antigen (psma) and its variant form psḿ glutamate carboxypeptidase ii, also known as prostate specific membrane antigen (psma), is a transmembrane glycoprotein highly expressed in maligant prostate tissues. it was shown to represent very useful diagnostic marker and also potential therapeutic target for prostate cancer. two forms of the enzyme were identified in the prostate: full-length transmembrane form consisting of 750 amino acids and a truncated form (called psḿ ), believed to represent spliced variant of psma. the cdnas of both forms are identical except for 266-nucleotide region near 5´end of psma that is absent in psḿ . this deleted region codes for signal peptide as well as for intracellular and transmembrane domains. we are able to detect two protein forms in prostate cancer model cells (lncap cells) and we also show that both forms are glycosylated suggesting that this truncated form might originate from the processing of full length transmembrane psma. number of methods including differential centrifugation, pulse-chase experiments, immunochemistry and gfp-fusion protein analysis were used to analyze the origin, cell localization and trafficking of psma and psḿ in the mammalian cells. we have investigated the substrate specificity of the ns2b(h)-ns3pro protease by using internally quenched synthetic peptides representing both natural cleavage sequences and their recombinant chimeras. synthetic peptides incorporating the o-aminobenzoic acid/3-nitro-l-tyrosine fluorescence donor-quencher pair were used to analyze the minimum substrate length requirement, residue preferences and the contribution of prime side residues for enzymatic cleavage by the ns3 protease. a series of peptides derived from the ns3/ns4a cleavage site was designed for the substrate length mapping study. amino acid truncations in the non-prime and prime side region differently affected rates of substrate hydrolysis and binding as shown by their km and kcat values. the optimal substrate identified was a heptapeptide spanning p4-p3'. chimeric substrates with all possible combinations of non-prime and prime side sequences derived from 5 polyprotein cleavage sites (c, 2a/2b, 2b/3, 3/4a and 4b/5) were assayed for reactivity with the ns3 protease. kinetic parameters revealed a strong impact of the non-prime side residues on km, whereas variations in the prime side region had greater effect on kcat. the fluorogenic derivative of tetrabasic peptide rrrr/gtgn (c/ns5) demonstrated the highest affinity, whereas the peptide kkqr/sagm (2b/c) had the highest turnover number. the one with the greatest catalytic efficiency was identified as rrrr/sltl (c/4a). in addition, we have shown that a ser at p1' is the most preferred residue. the discovery of ns3 substrates with maximized reactivity will be useful for inhibitor development in sensitive high-throughput assays. inhibiting the mtor pathway with cci-779 results in decreased production of vascular endothelial growth factor in a head and neck squamous cell cancer cell line c.-a. o. nathan 1,2 , n. amirghahari 1,2 , x. rong 1,2 and y. sun 1,2 1 nathan, otolaryngology, otolaryngology/head and neck surgery, louisiana state university health sciences center, shreveport, la, usa, 2 nathan, cancer center, medicine, feist-weiller cancer center, shreveport, la, usa. e-mail: cnatha@lsuhsc.edu introduction: overexpression of the proto-oncogene eif4e in surgical margins of head and neck squamous cell cancer (hnscc) patients is an independent predictor of recurrence and is associated with increase in vascular endothelial growth factor (vegf) expression. activation of eif4e in margins through the mtor pathway has led us to determine that cci-779 an mtor inhibitor has both in vitro and in vivo growth inhibitory effects in hnscc cell lines. we wanted to determine if these effects were associated with decrease in vegf production. material and methods: a hnscc cell line fadu was treated with 1 and 10 ng/ml of cci-779 (previously established ic50 = 1 ng/ml). elisa was used to determine vegf protein levels in conditioned medium at 30', 1, 2, 4, 6, 24 and 48 h after treatment with the drug and compared to control cells treated with the diluent for each of the time points. results: a significant decrease in vegf production of 70% was noted at 24 h and maintained at 48 h in treated cells when compared to control cells at the same time points. the decrease in vegf levels (39-47%) was noted within 6 h of treatment with the drug. the percent decrease in vegf protein levels was the same for both doses of cci-779. conclusions: overexpression of eif4e in hnscc increases translation of mrnas with long 5'utrs, one of which is an important angiogenic factor vegf. inhibiting the mtor path-way with cci-779 can potentially decrease vegf production. this has future clinical implications for arresting tumor progression in hnscc patients with molecular positive margins identified by cells overexpressing eif4e, also known as minimal residual disease. proteases from cell culture of jacaratia mexicana m. c. oliver-salvador 1 , g. barrera plant proteases are important in food industry and food technology. the latex of jacaratia mexicana, caricaceae, fruits contains a high level of cysteine proteases. in this work was established a cell suspension culture of j. mexicana. callus culture was initiated from stem explants of j. mexicana on medium consisted of ¼-strength and full-strength ms mineral salts (murashige and skoog, 1965), full-strength ms organics and 6 g/l agar supplemented with cytokinins: 6-benzylaminopurine (bap) at 0.5 mg/l and 6-furfurylaminopurine (kinetin) at 0.25 mg/l and various concentrations (0.25, 0.5 and 1.0 mg/l) of auxins: 2,4-dichlorophenoxyacetic acid (2,4-d) 4-amino-3,5,6-trichloropiridin-2-carboxilic acid (picloram) indoleacetic acid (iaa) a-naphthaleneacetic acid (naa). all of the treatments induced callus except for the iaa, ana and without added phytohormones. the best auxin concentration for callus development was determined to be 0.5 mg/l. and the best condition medium for callus development and proteolytic activity of callus was determined to be 0.5 mg/l 2, 4-d + 0.5 mg/l bap. cysteine proteases were produced on callus culture of j. mexicana and liberated in the medium. also in the cell suspension culture these enzymes were secreted. our results support that is possible the synthesis of proteases in vitro culture of j. mexicana. since protease is a primary metabolite, further improvement in enzyme production is possible by increasing the growth rate and yield of cell culture of j. mexicana. and arginine, from peptides and proteins at neutral ph. it is known to play an important role in the control of peptide hormones, growth factor activity at the cell surface, and in the membrane-localized degradation of extracellular proteins. therefore, the present work was carried out to clone and express carboxypeptidase m in pichia pastoris, aiming at developing specific inhibitors and to evaluate the importance of the enzyme in different physiological and pathological processes. for this purpose, the enzyme's cdna was amplified from total placental rna by rt-pcr and cloned in the vector ppic9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in pichia pastoris. the results show that the cpm gene, after cloning and transfection, integrated in the yeast genome, which started to produce the active glycosylated protein. the recombinant protein was secreted into the medium and the enzymatic activity was measured with the fluorescent substrate dansyl-ala-arg. the enzyme was purified by a two-step protocol including gel filtration and ionexchange chromatography, resulting in a 1761-fold purified active protein in a concentration of 400 mg/l of fermentation medium. sds-page showed that recombinant cpm migrated as a single band with molecular weight similar to native placental enzyme (62 kda). these results demonstrate for the first time the establishment of a method using pichia pastoris to express human carboxypeptidase m. mutational analysis of active site of glutamate carboxypeptidase ii human glutamate carboxypeptidase ii (gcp ii) is a membrane metallopeptidase expressed predominantly in the nervous system, prostate and small intestine. in the brain, gcp ii catalyzes cleavage of the abundant neuropeptide n-acetyl-l-aspartyl-l-glutamate (naag) to n-acetylaspartate and glutamate. gcp ii is a type ii transmembrane glycoprotein with a short cytoplasmic nterminal region (amino acids 1-18), a transmembrane domain (amino acids 19-43) and a large extracellular domain (amino acids 44-750) where the active site of the enzyme is situated. gcp ii, as a cocatalytic zinc metallopeptidase, has two zn 2+ ions in the active site which are necessary for its enzymatic activity. recently, the crystal structure of gcp ii was determined in our laboratory and amino acids arg210, asn257, lys699 and tyr700 were proposed to bind c-terminal glutamate of naag (mesters et al., manuscript in preparation). in the presented study, we carried out site-directed mutagenesis to assess the influence of these amino acid residues on the activity of gcp ii. in addition, glutamic acid in the position 424 which is proposed to be involved in proton shift during the catalytical hydrolysis of peptide bond, was mutated to alanine. all the mutant proteins were expressed in insect cells, purified to near homogeneity and enzymatically characterized. it was shown that a mutation in any of these positions lead to significantly reduced naag-hydrolyzing activity. the substitution of glu424 almost completely abolished the enzymatic activity, thus suggesting glu424 is crucial for enzymatic activity of gcp ii. kinetic characterizations of mutant proteins and their substrate specificities will be presented in comparison with wild type gcp ii. comparative study of mammalian homologues of human glutamate carboxypeptidase ii glutamate carboxypeptidase ii (gcpii) is a membrane-bound metallopeptidase. in homo sapiens, gcpii was shown to be expressed in various tissues, mostly in the central nervous system, small intestine and prostate. in brain it hydrolyses n-acetylaspartylglutamate (naag), which is the most prevalent peptide neurotransmitter in the mammalian nervous system, to form glutamate and n-acetylaspartate. in small intestine gcpii plays an important role in folate absorption. in prostate its function is still unknown. it was shown that inhibition of gcpii is neuroprotective in many neurodegenerative states. according to current knowledge of this enzyme, its role may also be important in prostate (and possibly other) cancers, where its expression is dramatically changed in comparison with healthy tissue. gcpii is thus becoming an important therapeutic target and diagnostic molecule. in order to analyze structure-activity relationships in related glutamate carboxypeptidases, we set to study the mammalian homologues of human gcpii: gcpii of rattus norvegicus, sus scrofa and mus musculus, which have approximately 90% dna sequence similarity to human gcpii. information on the biochemical properties, expression pattern and structural similarity is crucial e.g. for testing of gcpii inhibitors in animal models. we have cloned and expressed recombinant gcpii of r. norvegicus and s. scrofa in insect cells with the aim to obtain pure recombinant protein sufficient for structural analysis. data on biochemical comparison of rat, pig and human gcpii forms will be presented and interpreted in the light of the gcpii structure. structural analysis of pla protein from y. pestis: docking and molecular dynamics of interactions with mammalian plasminogen systemz e. ruback and p. g. pascutti laborato´rio de modelagem e dinaˆmica molecular, departamento de biofisica, universidade federal do rio de janeiro, rio de janeiro, r.j. brazil. e-mail: eruback@biof.ufrj.br the plasminogen (plg) system is an important mechanism for the cell migration through the tissues in the mammalian organisms. some bacterial agents can activate this system by proteases and lead an uncontrolled degradation of extracellular matrix components (mec), and make an invasive character of these infections. the y. pestis protein pla is a plasmid coded outer membrane protein, with aspartic-protease activity and is closely related with the proteolytic activation of plg in the serine-protease form called plasmin. exactly how the pla activate plg in plasmin remains unclear. we performed in this work the predicted interaction between the plg and pla protein by rigid-body docking with hex and evaluate the complex stability by molecular dynamics (md) using the gromacs. to evaluate the docking accuracy we use the crystal structure of complex plg-streptokinase. the md results show more stability in the docked plg-streptokinase complex than in crystal complex observed by the rmsd and rmsf calculations after 2 ns in simulation box. the pla model was constructed with spdb-viewer using the pdb structure of ompt as template and quality of model was evaluated with prochek. the docked complex of plg-pla show same interaction site predicted in mutagenesis studies. after 8 ns md (72 083 atoms in box), we observed the relax of beta barrel structure of pla and the progressive approximation and stabilization between the cleavage site of plg into the extracellular loops of pla, followed of the increase of hydrogen bonds number. in this study we report the possible aminoacids that can be participant in the active site and the sub sites of interaction. the total understanding of these interactions can be a important tool for drug design against bacterial proteases. glutamate carboxypeptidase ii (gcpii), also known as naa-ladase i, folylpolyglutamate hydrolase (folh) or prostate specific membrane antigen (psma) is localized in number of tissues. in brain astrocytes, it regulates neurotransmission by cleaving neurotransmitter n-acetylaspatylglutamate (naag) into n-acetylaspartate and most common excitatory neurotransmitter glutamate. inhibition of gcpii activity protects against cell death after brain stroke. in animal models it has been also shown that specific inhibitors of gcpii could be useful for the treatment of chronic neuropathic pain, amyotrophic lateral sclerosis and other pathologic situations when excess glutamate is neurotoxic. gcpii is identical to prostate-specific membrane antigen (psma), a tumor marker in prostate cancer. gcpii is also found in the membrane brush border of the small intestine where it acts as a folate hydrolase. this reaction expedites intestinal uptake of folate through hydrolysis of folylpoly-gamma-glutamates to monoglutamyl folates. gcpii inhibitors might thus be useful in the imaging and treatment of tumors where folate is required for their growth. therefore it was of interest to investigate whether gcpii might be upregulated in brain tumors as well. in order to analyze this possibility, we took 57 samples from 49 patients with brain tumors treated in faculty hospital motol during 1999-2004 and determined expression and activity of gcpii by western blots and immunohistochemistry using monoclonal and polyclonal antibodies developed against extracellular epitopes of gcpii. moreover, we characterized the enzymatic activity of the enzyme in human samples and correlated the expression of gcpii with the type and grade of the tumor. search for optimal isosteres in beta-secretase peptidic inhibitors alzheimer's disease is a widespread, neurodegenerative, dementia-inducing disorder. it is ascribed to the presence of a lesion in several brain regions, the neuritic plaques, which are extraneuronal accumulations of b-amyloid protein (ab), a 42-aa insoluble peptide that mixed with axons and dendrites of neurons, interrupt the synaptic process and cause neuronal death. the peptide ab is a product derived by proteolitic cleavage from a larger transmembrane cell protein termed amyloid precursor protein, app. two enzymes are involved in this cleavage: b-secretase and a-secretase. the first one cuts app between met671 and asp672 of app to generate the n-terminus of ab in the rate limiting step of the process, while the second one cleaves at various places within a sequence between amino acids 712 and 717 to generate the respective c-terminus. using a combination of molecular modeling techniques, we have designed a set of novel b-secretase peptidic inhibitors with a variety of isosteres starting from the available crystallographic structure of this enzyme bound to the inhibitor om99-2. some of the resulting ligands are predicted to have higher affinity for this enzyme than the starting compound. these inhibitors have been synthesized, their b-secretase affinity tested and cell essays have been performed to determine their ability to preclude the formation of ab peptides in cell cultures. schizophrenia and bipolar affective disorder (bd) are two neuropsychiatric diseases with high social and economic costs. in spite of the prevalence of these diseases, no effective long-term treatments are currently available. the enzyme prolyl oligopeptidase (pop) shows increased activity in both illnesses. this serine protease hydrolyzes peptide hormones and neuropeptides at the carboxyl end of proline residues. because of the relevance of pop as a therapeutic target, many specific inhibitors of this protein have been developed in recent years. the inhibitors ono-1603, jtp-4819 and s-17092-1 are currently in clinical trial phase. s-17092-1 has been administered safely to humans and has been proposed as a potential treatment for cognitive disorders associated with cerebral aging. our aim is to develop new peptide human pop inhibitors. to obtain the human brain pop required for our studies, the cdna corresponding to the enzyme was cloned and subsequently expressed in e. coli. pop activity was monitored by 19f-nmr using a new synthesized pop substrate labeled with 19f. this substrate allowed us to perform the inhibition assay avoiding the interference problems of colorimetric and fluorimetric assays and was suitable for high throughput screening of new pop inhibitors. different strategies were used to find putative human pop inhibitors: in silico screening and solid phase synthesis of candidates and screening with chinese medicinal plants extracts. furthermore, nmr studies were performed with the purified human enzyme by labeling the protein isotopically with 15n and d2o and by selective labeling of the residues methionine and tryptophan with 13c. nmr spectra of the labeled protein were obtained at 800 mhz by applying trosy techniques. nmr will provide structural information to perform structure-based drug design of new pop inhibitors in the future as well as to study the interaction of the candidates with the active site of the enzyme. the crucial regulatory function of the membrane type 1-matrix metalloproteinase (mt1-mmp or mmp-14) in connective tissue metabolism, pericellular proteolysis of extracellular matrix (ecm) components, zymogen activation and angiogenesis was demonstrated with the severe phenotype of the mt1-mmp-deficient mice. this membrane-anchored enzyme is not only essential for normal development of hard tissues, but highly expressed in different human cancers where its level frequently correlates with malignant parameters. in most cases the high level of mrna or elevated level of protein can be predictive for disease development but these parameters only partly reflect the expression and forms of mt1-mmp in pathological conditions. biosynthesis, trafficking, intracellular activation, internalization, protein-protein interactions, and the level of physiological inhibitors (timps) strictly influence the activity of mt1-mmp in cells and tissues. in our experimental system, we followed mt1-mmp processing and shedding and characterized the cell-associated and released forms of the enzyme (jbc 2000; 275: 12080-12089; jbc 2002; 277: 26340-26350 and biochem j 2005; 386: 1-10). we found active and inactive truncated forms of mt1-mmp as a result of treatments or experimentally generated imbalance with timps. we have also developed approaches to identify mt1-mmp forms in tumor tissues. here we present and discuss different strategies to identify mmp-14 in diverse biological samples. because mt1-mmp endows tumor cells with the ability to invade and metastasize, these strategies can provide valuable information on the role and function of this key protease. contribution of calpain to cellular damage in human retinal pigment epithelium cultured with zinc chelator y. tamada 1 , t. nakajima 1 , t. r. shearer 2 and m. azuma 1,2 1 research laboratories, senju pharmaceutical co., ltd., kobe, hyogo japan, 2 departments of integrative biosciences, oregon health & science university, portland, or, usa. e-mail: yoshiyuki-tamada@senju.co.jp purpose: we previously showed involvement of calcium-dependent cysteine proteases (calpains, ec 3.4.22.17) in neural retina degeneration induced by hypoxia and ischemia-reperfusion. aged macular degeneration (amd) is one of the leading causes for loss of vision. amd showed degeneration of neural retina due to dysfunction and degeneration of the retinal pigment epithelium (rpe). rpe performs critical functions in neural retina, such as phagocytosis of shed rod outer segments. the purpose of the present study was to determine the contribution of calpain-induced proteolysis to damage in human rpe. zinc chelator tpen was used to induce cellular damage since zinc deficiency is a suspected risk factor for amd. methods: third-to fifth-passage cells from human rpe were cultured with tpen. leakage of ldh into the medium was measured as a marker of rpe cell damage. activity of calpains was assessed by casein zymography, and proteolysis of calpain substrates was detected by immunoblotting. to confirm calpain-induced proteolysis, calpain in homogenized rpe was also activated by addition of calcium. results: tpen caused ldh to leak into the medium from rpe cells, and calpain inhibitor sja6017 inhibited the leakage. casein zymography and immunoblotting for calpain and a-spectrin showed activation of calpain in rpe cultured with tpen. proteolysis by activated calpain was confirmed by addition of calcium to homogenized rpe. conclusion: these results suggested that activation of calpain contributed to rpe damage induced by tpen in vitro. acknowledgments: dr shearer has substantial financial interest (research contract and consulting fee) in senju pharmaceutical co., ltd., and dr azuma is an employee of senju pharmaceutical co., ltd., a company that may have commercial interest in the results of this research and technology. this potential conflict of interest has been reviewed and managed by the ohsu conflict of interest in research committee. in vivo and molecular risk factors of chloroquine or pyrimethamine-sulfadoxine treatment failure in children with acute uncomplicated falciparum malaria the risk factors associated with chloroquine (cq) or pyrimethamine-sulfadoxine (ps) treatment failure were evaluated in 691 children enrolled prospectively in six antimalarial drug trials between july 1996 and july 2004 in a hyperendemic area of southwestern nigeria. following treatment, 149 (39%) of 389 children given cq and 64 (22%) of 302 children given ps failed treatment by day 7 or 14. in a multiple regression model, four factors were found to be independent risk factors for cq treatment failure at enrolment: age <7 years [adjusted odds ratio (aor) = 0.46, 95% confidence interval (ci) 0.26-0.84, p = 0.01], asexual parasitaemia >100 000/ll (aor = 0.46, 95% ci 0.23-0.93, p = 0.03), presence of gametocytaemia (aor = 0.48, 95% ci 0.26-0.88, p = 0.02) and enrolment after 4 years of commencement of the study, that is, after 2000 (aor = 0.47, 95% ci 0.25-0.77, p = 0.003). following treatment with cq, two factors were independent risk factors for failure of treatment: delay in parasite clearance >3 days (aor = 0.26, 95% ci 0.1-0.7, p = 0.014) and presence of gametocytaemia on day 7 or 14 (aor = 2.5, 95% ci 1.1-6.0, p = 0.03). in those treated with ps, two factors were found to be independent risk factors for ps treatment failure at enrolment: age <1.5 years (aor = 2.9, 95% ci 1.3-6.4, p = 0.009) and presence of fever (aor = 0.3, 95% ci 0.14-0.78, p = 0.01). following treatment with ps, delay in parasite clearance >3 days (aor = 0.39, 95% ci 0.18-0.84, p = 0.016) was an independent risk factor for failure of treatment. the quintuple mutants made up of triple dhfr (asn-108, arg-59 and ile-51) mutant alleles and double dhps (gly-437 and glu-540) mutant alleles were found in isolates obtained from 33% of patients, was significantly associated with ps treatment failure (p = 0.001), while pfcrt and pfmdr-1 mutant genes did not significantly predict cq treatment failure in these patients. these findings may have implications for malaria control efforts in sub-saharan africa where control of the disease depends almost entirely on antimalarial monotherapy. development of high-throughput assay of lethal factor using native substrate m.-y. yoon department of chemistry, hanyang university, seoul, south korea. e-mail: myyoon@hanyang.ac.kr designing of inhibitors for anthrax lethal factor (lf) is currently of interest as an approach for the treatment of anthrax because lf plays major roles in cytotoxicity of target cells. lf is a zincdependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (mapkk) family. current assay system for the screening of lf inhibitor use the optimized synthetic peptide coupled with various kinds of fluorophores, which enables fast, sensitive, and robust assays suited to high-throughput screening. however, lines of evidence suggest that the regions beside the cleavage site are also involved in specificity and proteolytic activity of lf. in the present study, we tried to develop high-throughput assay for lf activity based on native substrate, mek1. the assay system relies on the ecl signal resulting from a specific antibody against the c-terminal region of native substrate. a glutathione-coated multiwell plate was used as a solid support to immobilize the native substrate by its n-terminal gst-moiety. immobilized substrate increases the specificity and sensitivity lf-catalyzed substrate hydrolysis compared to the solution phase assay. this assay system would be expected to discover a wide spectrum of anthrax inhibitor. while significant progress has been made over the past decade in elucidating the structure and enzymatic mechanism of the 20s proteasome, our understanding of its assembly pathway and the role of the propeptides in the maturation process is still substantially incomplete. similarly, the mechanisms involved in the translocation of substrates into the central nanocompartment are only dimly understood at present. we have used the rhodococcus proteasome to dissect the assembly pathway, combining mutagenesis and crystallographic studies. for the thermoplasma proteasome we have established a ''host-guest'' interaction system which allows us to follow the translocation of specific substrates into the interior of the proteasome by electron microscopy, mass spectroscopy and x-ray crystallography. transferring substrates to the 26s proteasome in the fission yeast schizosaccharomyces pombe c. gordon mrc human genetics unit, western general hospital, edinburgh, uk. e-mail: colin.gordon@hgu.mrc.ac.uk the ubiquitin pathway is found in all eukaryotes. in this pathway, target proteins are covalently modified by the addition of ubiquitin, a 76 amino acid protein, to specific lysine residues. the ability of multi-ubiquitin chains to function as a signal to target proteins for degradation by the 26s proteasome is well documented. a key question is how is the multi-ubiquitin chain is recognized as a signal? fission yeast rhp23/rad23 and pus1/ rpn10 represent two families of multi-ubiquitin chain binding proteins that can associate with the proteasome as well as some e3 ubiquitin ligases. they seem to provide a link to shuttle ubiquitinated substrates from the e3 ubiquitin ligases to the 26s proteasome. a detailed characterization of their proteasome binding will be presented along with their potential role in ubiquitin conjugate dynamics. finally data will be presented indicating that an additional substrate presentation pathway exists in fission yeast which is also conserved in higher eukaryotes. non-proteasomal rpn10 raises the threshold for association of a ubiquitin-binding protein with the proteasome the ubiquitin proteasome pathway is responsible for the removal of the vast majority of short-lived proteins in the cell. in order to be degraded, a protein substrate is tagged with polyubiquitin and delivered to the proteasome where it is proteolysed. a slew of shuttle proteins is thought to mediate the delivery of polyubiquitinated substrates, although the mechanism remains elusive. one such family of proteins is comprised of rad23, dsk2 and ddi1, which all bind polyubiquitinated substrates through a ubiquitinassociated domain (uba) as well as the proteasome through their ubiquitin-like domain (ubl). another potential shuttle structurally unrelated to the ubl-uba family is rpn10. rpn10 is found as an integral subunit of the proteasome as well as an in an unincorporated pool. we characterized the interactions of these proteins with individual proteasomal subunits, as well as between themselves. we find unique relationships between the putative shuttle proteins and the proteasome, pointing to functional dissimilarity among them. strikingly, unincorporated rpn10 interferes with binding of dsk2 to the proteasome. thus, we propose that rpn10 might play a negative role in proteolysis through its action on dsk2. proteins modified by multi-ubiquitin chains are usually targeted for degradation by the proteasome. in other cases, ubiquitylation mediates protein sorting or regulates other functions. a striking example for a non-proteolytic role of ubiquitin is the rad6 dna damage bypass at stalled replication forks. key elements of this pathway are two ubiquitin-conjugating enzymes, rad6 and the mms2/ubc13 heterodimer, which are recruited to chromatin by the ring-finger ubiquitin ligases, rad18 and rad5, respectively. moreover, also the sumo-conjugating enzyme ubc9 is affiliated with the pathway and we discovered that proliferating cell nuclear antigen (pcna), a dna-polymerase sliding clamp involved in dna synthesis and repair, is a substrate. pcna is (i) mono-ubiquitylated by rad6/rad18, (ii) modified by lysine (k) 63-linked multi-ubiquitylation, which additionally requires mms2/ubc13/ rad5, and (iii) sumoylated by ubc9. all three modifications affect the same lysine residue of pcna, indicating that they label pcna for alternative functions. indeed, we discovered that monoubiquitylation of pcna promotes an error-prone replication bypass, whereas k63-linked multi ubiquitylation mediates errorfree replication across the lesions. in contrast, sumoylation, which occurs even in the absence of dna damage, prevents recombination between homologs at the replication fork. these findings indicate that mono-ubiquitin, k63-linked multi-ubiquitin chains, and sumo are crucial for decision making at the replication fork. ubiquitin-mediated proteolysis is the primary mechanism in eukaryotes for degrading unwanted and misfolded proteins. through the cascade of e1, e2 and e3 enzymes, ubiquitin monomers are attached sequentially to the target proteins, which are then recognized and degraded by the 26s proteasome. the selection and specific timing of polyubiquitination of the target proteins are conferred by different e3 ubiquitin ligases. the anaphase-promoting complex (apc) is one of the most extensively studied e3 ubiquitin ligases that plays essential role in the cell cycle and specific developmental processes. the core apc is composed of 11-13 subunits. except for apc2 and apc11, relatively little is known about the role of the other apc subunits or the assembly of the complex. two wd40-repeat activator proteins, cdc20 and cdh1 determine stage-specific activation of the core apc as well as selection and binding of the apc substrates. in plants, the apc activators are present in multiple copies. arabidopsis contains 5 cdc20 genes, 3 cdh1-type activators known as ccs52a1, ccs52a2 and ccs52b. our work has been focused on the function of apc activators in the cell cycle and plant development, identification of novel apc substrates and on the assembly of the apc complexes. apc activities, based on the expression profiles of the cdc20 and ccs52 genes, will be presented at organism level. by detailed protein interaction studies in yeast two hybrid system and arabidopsis protoplasts or transgenic plants, we shall demonstrate how the core apc interacts with the activators and substrates, and propose a model for apc assembly. characterization of substrate delivery to the saccharomyces cerevisiae proteasome by quantitative shotgun proteomics the proteasome is the central protein degradation machinery in the eucaryotic cell. in conjunction with the ubiquitin system, it is responsible for constitutive bulk protein turnover as well as the controlled degradation of regulatory proteins. the system is very well characterized, but the mechanism by which poly-ubiquitinated substrates are delivered to the proteasome remains unclear. recently our lab has proposed a number of proteins to be proteasome-based receptors for poly-ubiquitinated substrates in s. cerevisiae (rpn10p, rad23p, dsk2p; verma et al., 2004). others (e.g. richly et al. 2005) have put forward a complex model for the delivery of substrates from the ubiquitinating machinery to the proteasome involving the aaa atpase cdc48p. by analyzing the composition of affinity purified proteasome complexes from s. cerevisiae cells lacking these factors and/or exposed to specific proteasome inhibition, we hope to further elucidate the substrate delivery pathway. ubiquitinated proteins recruited to the proteasome are identified utilizing capillary chromatography in-line to electrospray ion trap mass spectrometry (mudpit; link et al. 1999). using a reference strain grown in minimal medium solely providing heavy nitrogen ( 15 n) as an internal standard, we are able to record even gradual fluctuations in sample composition. differences in the recruitment of substrates to the proteasome in varying mutant backgrounds will shed light on the specificity of proteasome substrate receptors and the topology of the substrate delivery mechanism. oxidative protein damage by reactive oxygen species (ros) produces cross-linking, fragmentation and biochemical modification of the amino acids resulting in biological dysfunctions. quercetin, a widely distributed bioactive plant flavonoid, possesses anti-cancer, antioxidants and free radical scavenging activities, as well as it binds with dna causing dna fragmentation. a little is known about protein oxidative damage and its modifications by antioxidants. therefore, the aim of the present work was to investigate the molecular mechanisms of antioxidant and prooxidant activities of quercetin toward proteins. the antioxidant activities of quercetin, such as superoxide dismutase (sod)-and catalase (cat)-mimetic as well as hydroxyl radical (aeoh) scavenging activities were possessed. bovine serum albumin (bsa) was incubated with different concentrations of quercetin. quercetin has highly sod-and cat-like and hydroxyl radical (aeoh) scavenging activities. its activities are concentration dependent. quercetin fragmentized bsa into specific fragments which they detected by sds/polyacrylamide gel electrophoresis. oxidative protein damage was assessed as tryptophan oxidation, carbonyl, quenone and advanced oxidation protein products (aopp) generation. the increase of protein oxidation products was in concentration dependent manner. the carbonyl and quenone contents and aopp were highly significantly elevated in querce-tin-treated proteins when compared with the control sample. the tryptophan fluorescence was highly decreased in treated protein than in the control sample. the mechanisms of antioxidant and pro-oxidant activities of quercetin have been discussed. these results demonstrate that antioxidant quercetin may potentiate protein damage via oxygen free radical generation, particularly .oh radicals by quercetin. protein stability mediated by a hyaluronanbinding deubiquitinating enzyme is involved in cell viability protein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway. we have identified three novel genes encoding a deubiquitinating enzyme, vdub1, vdub2, and vdub3 (villi deubiquitinating enzyme 1, 2, and 3) from human chorionic villi by rt-pcr. their cdnas are 1,593 bp in length and encode an open-reading frame of 530 amino acids with a molecular weight of approximately 58 kda. expression analysis showed that vdub transcripts are highly expressed in the heart, liver, and pancreas. in addition, they are expressed in various human cancerous cell lines. amino acid sequence analysis revealed that they contain the highly conserved cys, his, and asp domains, which are required for the formation of active site for the deubiquitinating enzymes. in vivo and in vitro deubiquitinating enzyme assays indicated that vdub1, vdub2, and vdub3 have deubiquitinating enzyme activity. here, we show that the overexpression of vdub proteins leads to irregular nuclear morphology and apoptosis, suggesting that these vdubs play an important role in regulating signal transduction involved in cell death. interestingly, the sequence analysis showed that vdub proteins contain the putative hyaluronan/mrna-binding motifs, and cetylpyridinium chloride-precipitation analysis confirmed the association between vdubs and intracellular hyaluronan and rna. chemical cleavage of peptide (amide) bonds usually requires harsh conditions. as a result of side reactions and the lack of specificity, chemical amide bond hydrolysis is not a preferred means of protein digestion. we have discovered selective cleavage of peptide bonds in proteins under milder circumstances than any previously reported chemical method. hydrolysis takes place in aqueous buffers in a ph range of 410, and occurs c-terminal to the proteogenic non-natural amino acid azido-homoalanine (azhal), effected by a staudinger reaction after addition of the mild and biocompatible reagent tris(carboxyethyl)phosphine (tcep). key feature in the suggested reaction mechanism is the unprecedented nucleophilic substitution of the resulting gammaiminophosphorane by the flanking c-terminal backbone amide oxygen atom. after hydrolysis, the new c-terminal peptide is present as a homoserine lactone residue and the n-terminal peptide as its free amine. this new reaction may find application as a very mild and selective bio-orthogonal degradation pathway in biochemistry and biomaterials science. overexpression of proteasome b5 subunit increases amount of assembled proteasome and confers ameliorated response to oxidative stress and higher survival rates the proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. proteasomes play a fundamental role in retaining cellular homeostasis. alterations of proteasome function have been recorded in various biological phenomena including aging. we have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of btype subunits. in this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the b subunit. stable overexpression of the b5 subunit in wi38/t and hl60 cells resulted in elevated levels of other b-type subunits and increased levels of all three proteasome activities. immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of ''free''/not integrated b-type subunits identified to accumulate in vector-transfected cells. in support we have also found low pomp levels in b5 transfectants thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. functional studies have shown that b5 overexpressing cell lines confer enhanced survival following treatment with various oxidants. moreover we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. finally, as oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the b5 subunit into primary imr90 human fibroblasts and we have observed a delay of senescence by 45 population doublings. in summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights towards a better understanding of proteasome regulation. expression levels of the components of the ubiquitin/proteasome pathway in pisum sativum seedlings under anoxia stress change in gene expression: proteins produced under aerobic conditions are no longer synthesized and are replaced by the socalled anaerobic peptides. among those proteins synthesized under o 2 deficiency some enzymes of the glycolytic and fermentative pathways were identified in plants. upon reintroduction of air, the anaerobic mrnas disappear rapidly and the increased levels of those enzymes must return to the basal levels. the ubiquitin/proteasome system is a major pathway of proteolysis in eukaryotic cells and may contribute to controlling the intracellular levels of a variety of short-lived regulatory proteins. in this proteolytic pathway, proteins are covalently conjugated to ubiquitin, which flags them for rapid hydrolysis by the 26s proteasome. long polyubiquitin chains must be formed to target a protein for destruction by the proteasome. in plants, the ubiquitin-mediated proteolytic pathway is implicated in a variety of cellular processes, including stress responses. in this study, 3-dayold pisum sativum seedlings were subjected to: (i) 15 h of anoxia stress; (ii) 2 h of aerobic conditions after 15 h of anoxia stress and (iii) 4 h of aerobic conditions after 15 h of anoxia stress. the levels of free and conjugated ubiquitin were detected by immunoblotting using anti-ubiquitin polyclonal antibodies. the changes in the mrna levels of some components of the ubiquitin/proteasome pathway in the seedlings were determined by relative semiquantitative rt-pcr. the results suggest an involvement of the ubiquitin-mediated proteolytic pathway in the anoxia stress response. b2-012p involvement of the anaphase promoting complex in plant development controlled degradation of short-live proteins via ubiquitindependent proteolysis by the 26s proteasome is a key mechanism in eukaryotes that regulates nearly all fundamental cellular processes including cell cycle. polyubiquitination of the protein substrate is sufficient to target it for degradation by a large atp-dependent multicatalytic protease, the 26s proteasome. the selection and specific timing of ubiquitination of the target proteins are conferred by different e3 ubiquitin ligase. the anaphase promoting complex (apc) is one of the e3 ubiquitin ligases, which by ordered destruction of various cell cycle proteins has fundamental roles in the regulation of mitotic and endoreproduplication cycles. the apc functions also outside the cell cycle. in post-mitotic cells, the cdh1 adaptor protein ensures stage specific activation and substrate selection of the apc. in plants, two classes of the cdh1-type activators have been identified, ccs52a and ccs52b that display differential regulation during the cell cycle and plant development as well as differences in their substrate-specificities. in arabidopsis, transient and complimentary expression profiles of the atccs52a1, atccs52a2 and atccs52b genes indicate apc functions during flower development. to identify apc targets, yeast two hybrid screens were performed in the laboratory. out of about 200 interacting proteins, several proteins were transcription factors including a key a regulator of flowers development. data on the interactions of the ccs52 proteins and transcription factors in arabidopsis protoplasts will be presented as well as a model for the apc regulated pathways. novel effects of ubiquitin system and chaperone proteins on the prion ''life cycle'' in yeast t. a. chernova 1 , k. d. allen 2 , e. p. tennant 2 , k. d. wilkinson 1 and y. o. chernoff 2 1 department of biochemistry, emory university, atlanta, ga, usa, 2 school of biology and institute for bioengineering and bioscience, georgia institute of technology, atlanta, ga, usa. e-mail: tcherno@emory.edu yeast prion [psi + ], the self-propagated aggregated isoform of the translation termination factor sup35, is used as a model system to study neural inclusion disorders. prion aggregates and other neural inclusions in mammals were previously reported to sequester ubiquitin (ub). proteasome inhibitors affected the turnover of mammalian prion proteins. however, a role of ub-dependent proteolysis in the prion ''life cycle'' has not been clearly defined. chaperone proteins, which are also implicated in ub-dependent proteolysis, have been shown to influence the formation and propagation of the prion aggregates. our results uncover the connection between alterations of ub system and chaperone proteins in their effects on the maintenance of yeast prion. we have demonstrated that deletions of genes encoding deubiquitinating enzymes, that are critical for ub regeneration at the proteasome (ubp6) or the vacuole (doa4), cause pleiotropic phenotypic effects that are primarily due to decreased levels of free ub in the yeast cells. these alterations, as well as deletion of the gene encoding ub-conjugating enzyme, ubc4, decreases [psi + ] curing by the overproduced disaggregase hsp104, suggesting that ub system influences hsp104-dependent clearance of prion aggregates. spontaneous [psi + ] formation was also increased in the ubc4 depleted cells. we previously demonstrated that excess of cytosolic chaperone ssa of hsp70 family increases de novo formation of [psi + ]. both in vivo and in vitro experiments uncover direct interactions between sup35 and hsp70 proteins. the amount of sup35-bound to hsp70-ssa was increased in ubc4 deletion strain. we propose a model to explain roles of hsp104, hsp70 and ub system in the prion life cycle. effects of parkinson''s disease mimetics on proteasome activity and protein turnover in human sh-sy5y neuroblastoma cells it has recently been suggested that impairment of the ubiquitin/ proteasomal system contributes to the degeneration of dopaminergic neurons (dn) and lewy body (lb) formation in parkinson's disease (pd). mitochondrial dysfunction is also a key factor in pd and agents such as mpp + and dopamine, which inhibit mitochondrial electron transport, produce selective degeneration of dn in animal models. in this study the effects of treating sh-sy5y cells with mpp + or dopamine over 72 h on proteasomal chymotrypsin-like activity (cla) was monitored. mpp + (0.1mm) caused a sustained depletion of glutathione levels followed by a reduction in proteasomal activity. a reduction in atp levels, caused by higher levels of mpp + (2mm), exacerbated this effect. exposure to low dopamine concentrations (0.1mm) led to large reductions in atp without affecting cla or glutathione levels; whilst higher concentrations (2mm) caused marked reductions in cla, glutathione and atp levels. these results suggest that, under oxidative stress, glutathione levels are important regulators of proteasomal activity in this cell line. our group has shown that mpp + can destabilize the neurofilament network in shsy-5y cells, partly due to changes in phosphorylation of neurofilament (nf) chains. as nfs are important components of lbs, and their mode of turnover is uncertain, we tested the effects of proteasome inhibitors on nf levels. treatment with these inhibitors led to nf accumulation, which was enhanced when glutathione levels were artificially depleted, suggesting that nfs can be degraded via the proteasomal pathway. the effects of proteasome impairment on protein accumulation will be discussed. mitochondria and the hypoxia-inducible factor 1 (hif-1): regulation of hif-1 is independent of a functional mitochondrial respiratory chain k. doege, w. jelkmann and e. metzen insitute of physiology, university of luebeck, luebeck, germany. e-mail: doege@physio.uni-luebeck.de the hypoxia-inducible factor hif-1 is the ''master-regulator'' in adaptation to low oxygen concentration and induces the hypoxic expression of several target genes, e.g. erythropoietin and vascular endothelial growth factor (vegf). in normoxia hif-1a is constantly produced but also degraded by oxygen-dependent prolyl-hydroxylation. mitochondria consume most of the oxygen delivered to cells and have been implicated in oxygen sensing. firstly, mitochondria have been proposed to stabilize hif-1a by production of reactive oxygen species (ros) in hypoxia. secondly, inhibition of the respiratory chain, e.g. by nitric oxide, has been proposed to cause redistribution of intracellular oxygen followed by reactivation of the prolyl hydroxylases and inhibition of hif signalling. we have used cells depleted of mitochondrial dna (q0) and gas permeable cell culture dishes to eliminate all oxygen diffusion gradients affecting the cells. we show that these dishes neutralize all effects of mitochondrial inhibition. additionally, cellular hypoxia as assessed by pimonidazole staining has been evaluated in human osteosarcoma cells treated with inhibitors of the respiratory chain under hypoxia. these results demonstrate an elevated po 2 under hypoxic conditions after treatment with mitochondrial inhibitors correlating with an intracellular oxygen concentration which reduces hif-1 activation. thus, neither the absence of ros nor the redistribution of intracellular oxygen supply leads to the destabilization of hif-1a in hypoxia. our experiments provide evidence that an increased intracellular po 2 evoked by the absence of mitochondrial oxygen consumption reactivates the prolylhydroxylases and is therefore responsible for the degradation of hif-1a under hypoxic conditions. enzyme activity is generally higher in rhizosphere than in bulk soil, as a result of a greater microbial activity sustained by toot exudates or due to the release of enzymes from roots. negative effects of heavy metals on soil microorganisms and enzyme activities have been long recognized. the aim of this study was to assess the stimulatory effects of different low molecular weight organic compounds commonly present in root exudates (mres) on microbial activity and protease activities and , and how high cd concentrations affect such stimulatory effects. soils (arenic udifluvent) were sampled from the agir long-term field trials, contaminated with cd nitrate at rates of 0 (control soil), 20 and 40 mg cd per kg of soil. the mre solutions contained glucose, citric acid, oxalic acid, glutamic acid or a mixture of the four compounds, added to give a rate of 300 mg of mre-c per kg of soil. the effects were measured at 4 mm (bulk soil) distance from the mrs. protease activity was determined by hydrolysis of n-benzoylargininamide (baa). the results showed that different mres had different stimulatory effects on microbial growth and on the protease activities, mostly localized in the rhizosphere soil layer. in the control soil, the dsdna content was significantly increased by the addition of all mre in both rhizosphere and bulk soil layers. the 20 and 40 mg cd per kg of soil negatively affected on protease activity. the glucose, citric acid, oxalic acid, glutamic acid, mres mix in both rhizosphere and bulk soil layers, did not stimulate protease. the, microbial growth and protease activities were drastically reduced by high cd concentrations. participation of different digestive proteinases of the yellow mealworm, tenebrio molitor, in initial stages of hydrolysis of the main dietary protein insects generally have a wide spectrum of digestive proteinases. the knowledge about the impact of different proteinases to initial stages of hydrolysis of dietary proteins is essential for insect control by means of proteinase inhibitors and bacillus thuringiensis toxins. the larvae of a stored grain pest yellow mealworm, tenebrio molitor, were reared on milled oat flakes. the main dietary protein for these larvae was 12s globulin, the main storage protein of oat seeds. to study the initial stages of 12s globulin hydrolysis in vitro the reaction was performed in the physiological conditions of anterior midgut (am) (ph 5.6) by purified enzyme preparations from am: two fractions of cysteine proteinases cys ii and cys iii, chymotrypsin-and trypsin-like proteinases. total hydrolysis of 12s globulin was observed with cys ii. slightly less effective was hydrolysis by chymotrypsin-like enzyme. cys iii cysteine and trypsin-like proteinases produced only partial hydrolysis of seed globulin. in all cases high molecular mass (mm) intermediate products were formed testifying that hydrolysis of 12s globulin was sequential. incubation with both cysteine proteinase fractions led to formation of 31 kda product, while serine proteinases proin contrast to ''classical'' bioregulator peptides, peptides could be generated in the course of catabolic degradation of functional proteins. for 15 years, we have been interested in such particular group of peptides derived from blood hemoglobin, hemorphins. hemorphins consist in a family of opioid receptor-binding peptides from 4 to 10 amino acids that are released by proteolytic processing from the (32-41) segment of human hemoglobin betachain. they are prevalent throughout the peripheral and central nervous system and have been isolated in vivo from tissues or fluids. many in vivo physiological effects have been related (coronaro-constrictory, anti-tumorous, immunoregulatory activities) and several of the hemorphins interact at various levels of the reninangiotensin system (ras) by inhibiting angiotensin-convertingenzyme (ace), aminopeptidase n (apn) and dipeptidyl peptidase iv (dppiv) activities. in addition, some hemorphins and in particular lvv-hemorphin-7 (lvvypwtqrf), binds with high affinity to the brain (ic 50 = 4.15nm) and renal at4 angiotensin receptor subtype and is possible the main endogenous ligand from this receptor. in an attempt to characterize in vivo precise mechanisms for their release, our attention is focused towards tumoral and central nervous system environments. the last one is particularly interesting as all cellular components implicated in the release of hemorphins are present simultaneously: the haemoglobin precursor and localized brain proteases which might come in contact with blood haemoglobin. in this purpose, the examination of potentiality for this tissue to generate ''neuro''-hemorphins would be of interest since sources of hemorphins in the brain have not yet been definitively established. later, we showed that sgt1 interacts with calcyclin (s100a6) and other calcium-binding proteins of the s100 family (nowotny et al. j biol chem 2003). moreover, in collaboration with dr chazin's group, we found that in vitro sgt1 binds to hsp90 (lee y-t et al. j biol chem 2004). in this work we studied the expression and subcellular localization of sgt1 in mammalian cells by means of western and northern blots. among different cell lines examined human embryonic kidney hek293 and human glioma t98g cells exhibit highest expression of sgt1 protein. moreover, we found that in mouse and rat cells there is one isoform of sgt1, while in human cells two isoforms of this protein were found. to study the subcellular localization of sgt1 we chose the cells containing moderate level of sgt1 such as human epidermal hep-2 cells. by applying immunocytochemistry we found that this protein is present not only in the cytoplasm but also in the nucleus. at present we check the effect of intracellular ca 2+ concentration on subcellular localization of sgt1 and on its co-localization with target proteins. acknowledgements: this work was supported by grants: kbn 3 p04a 043 22 and firca/nih r13 tw006005. combining reverse genetics, reverse chemogenomics and proteomics to assess the impact of protein n-terminal methionine excision in the cytosol of higher eukaryotes in living organisms whatever the cell compartment, proteins are always synthesized with methionine (met) as the first residue. however, this first met is specifically removed from most mature proteins. in the course of protein n terminal met excision (nme), the free n terminal met is removed by met aminopeptidase (map) cleavage. three enzymes (map1a, map2a and map2b) have been identified in the cytoplasm of arabidopsis thaliana. by combining reverse genetics and reverse chemogenomics in transgenic plant lines, we have devised specific and reversible switches for the investigation of the role of cytoplasmic nme in a. thaliana and of the respective contributions of the two types of cytoplasmic map throughout development. in the map1a ko context (map1a-1), modulating map2 activity by treatment with various concentrations of the specific drug fumagillin impaired plant development. hence, (i) cytoplasmic nme is essential in plants, (ii) plant map1a and map2s are functionally interchangeable as a complete block of either map type activity does not cause any visible molecular or phenotypic effect, (iii) a minimal level of cytoplasmic map is required for normal development and (iv) the plant a. thaliana appears an excellent system to study nme and the associated-role of anti-cancer agents like fumagillin. proteomics was used to assess the impact of nme blocking induced by fumagillin. we used a wild-type plant and the map1a-1 variant grown in the presence of 100 nm fumagillin. the map1a-1 variant showed a dwarf phenotype. we compared by 2d gel electrophoresis the patterns of each protein extracts. protein spots were identified by tandem mass spectrometry. the data show that fumagillin induces many dedicated pathways, with a prevalence of those related to oxidative stress. prolyl endopeptidases from the midgut of the yellow mealworm tenebrio molitor side of proline residues. these enzymes were found in mammals, several higher plants, fungi and bacteria. it is suggested that the enzymes participate in the in vivo regulation of the action of biologically active peptides. we for the first time report about two prolyl endopeptidases in the larval midgut of a stored product pest yellow mealworm tenebrio molitor where they can participate in the proteolysis of one of the main dietary proteins of t. molitor larvae -rich in proline prolamines. characteristics of two prolyl endopeptidases are significantly different. optimum for hydrolysis of the substrate z-ala-ala-pro-pna (n-carbobenzoxy-l-alanyl-l-alanyl-l-prolyl-p-nitroanilide) by prolyl endopeptidase 1 was at ph 8.5, and prolyl endopeptidase 2 -at ph 5.6. prolyl endopeptidase 1 displayed high phstability in the ph range 7.0-10.0 and the rate of hydrolysis increased in the presence of kcl and cacl 2 . prolyl endopeptidase 2 demonstrated low stability in the whole ph range, the rate of hydrolysis strongly decreased in the presence of above mentioned salts, but increased in the presence of high concentrations of edta. the influence of cell growth media on the stability and antitumour activity of methionine enkephalin studies with cultured tumour cell lines are widely used in vitro to evaluate peptide-induced cytotoxicity as well as molecular and biochemical interactions. the objectives of this study were to investigate the influence of the cell culture medium on peptide metabolic stability and in vitro antitumour activity. the degradation kinetics of the model peptide methionine enkephalin (met-e, tyr-gly-gly-phe-met), demonstrated recently to play an important role in the rate of proliferation of tumour cells in vitro and in vivo, were investigated in cell culture systems containing different amounts of foetal bovine serum (fbs). the influence of enzyme inhibitors (bestatin, captopril, thiorphan) on the met-e degradation was also investigated. the results obtained in the dulbecco's modified eagle medium containing 10% fbs indicated a rapid degradation of met-e (t 1/2 = 2.8 h). pre-incubation of the medium with a mixture of peptidase inhibitors reduced the hydrolysis of met-e, as shown by increased half-life to 10 h. the in vitro activity of met-e against poorly differentiated cells from lymph node metastasis of colon carcinoma (sw620) and human larynx carcinoma (hep-2) cells was determined. tumour cells were grown 3 weeks prior to the experiment in a medium supplemented with 10, 5 or 2% fbs. statistically significant to mild or no suppression of cell proliferation was observed in all cultures. in both cell lines, a significant suppression of cell growth by a combination of peptidase inhibitors and met-e, compared with cells exposed to the peptide alone and cells grown in the absence of met-e, was observed. this study indicated that caution must be exercised in interpreting the antiproliferative effects of peptide compounds in conventional drug-response assays. protein metabolism in whole body and skeletal muscle of laboratory rats treated by proteasome inhibitors proteasome inhibitors are new agents which may be used in treatment of cancer and other severe disorders. one of the possible side effects of their administration is disturbance in protein metabolism which may affect outcome of the illness. two separate studies were performed using wistar rats. in the first study, m. soleus (sol) or m. extensor digitorum longus (edl) were incubated in medium containing 30 mmol/l mg 132 or 30 mmol/l adaahx3l3vs or without inhibitor (control). protein synthesis was evaluated using l-[1-14c]leucine. proteolysis was determined according to the rate of the tyrosine release into the medium during incubation. in the second study, proteasome inhibitor mg 132 diluted in dimethyl sulfoxide (dms) was administered intraperitoneally in dose 10 mg/kg b.w. controls consisted of dms treated animals. changes in protein and amino acid metabolism were estimated in steady-state conditions using continuous infusion of l-[1-14c]leucine 2 h later. mann-whitney (in vivo study) and paired t-test (in vitro study) were used for statistical analysis. in in vitro study, both mg 132 and adaahx3l3vs significantly decreased protein synthesis and proteolysis. however, in in vivo study, a significant increase in whole-body protein synthesis and proteolysis were observed in mg 132 treated animals. acknowledgements: the study was supported by a grant of gacr no. 303/03/1512. bioinformatical evidence for a prokaryotic ubiquitin-like protein modification system h. scheel, s. tomiuk and k. hofmann bioinformatics group, memorec biotec gmbh, ko¨ln, germany. e-mail: kay.hofmann@memorec.com until recently, the ubiquitin system has been considered a purely eukaryotic invention. by now, the bacterial moad/moeb and this/thif systems are known to be prokaryotic versions of a rudimentary activation system for ubiquitin-like proteins. however, similarities to the ubiquitin system end after the activation step, as moad and this are not conjugated onto target proteins but rather have a role in the biosynthesis of molybdopterin and thiamin, respectively. the eukaryotic protein urm1 is the closest homolog of moad and this. unlike its bacterial cousins, urm1 is conjugated onto target proteins and thus can be considered the founding member of the diverse eukaryotic ubiquitin family. by using a bioinformatics approach that integrates methods of sequence analysis, phylogenetics, phylogenomics and gene-order analysis, we were able to show that many bacteria possess a third ubiquitin-like activation system that most likely is used for protein modifications. the novel system uses a moad/this relative, which is more closely related to urm1 than the typical moad and this proteins. these bacterial urm1 (burm1) proteins typically require the proteolytic removal of a c-terminal extension, which masks the gg motif important for activation. many burm1 operons contain a mpn+/jamm domain protein (belonging to a bona fide ubiquitin-specific protease family), which is most likely responsible for this cleavage. as a third component, an e1-like enzyme is also part of typical burm1 operons. the burm1-associated e1 enzymes look more like uba4 (the eukaryotic urm1-e1) than like the bacterial moeb/thif e1 enzymes. interestingly, the mpn+/jamm protease is also conserved in those bacteria whose burm1 end with gg, suggesting that burm1 removal is important not only for the activation step b2-025p non-hypoxic induction of hypoxia-inducible factors by insulin and 2-deoxy-d-glucose hypoxia-inducible factors (hifs) are key mediators of the cellular adaptation to hypoxia, but also respond to non-hypoxic stimuli like insulin. to clarify involvement of all known hif subtypes in conditions resembling diabetes, we determined distribution of mrnas and proteins in rats subjected to in vivo hypoglycemia and glucoprivation. wistar rats were infused with either saline, insulin, or 2-deoxy-d-glucose (2-dg) to provoke hypoglycemia or impaired glucose assimilation. using real-time qpcr, mrna levels of hif subunits 1a, 2a, 3a, 1b, and of the target gene glut-1 were determined in various organs. cellular distributions of hif-a proteins were examined by immunohistochemistry. treatments with insulin or 2-dg resulted in a widespread increase in hif-3a mrna after 6 h, whereas mrna expression of other hif subunits remained unaffected, except for hif-2a which increased in lung and heart after 2-dg. in cerebral cortex and kidney, enhanced staining of all hif-a proteins was observed after insulin or 2-dg treatments. lung, heart and kidney showed enhanced levels of glut-1 mrna. both hypoglycemia and glucoprivation provoke functional activation of the hif system, with transcriptional up-regulation of hif-3a representing a typical response. our data indicate an involvement of the hif system, and hif-3a in particular, in the pathophysiology of diabetes. fragments of human salivary statherin and pb peptide underlying a furin-like pro-protein convertase action in the pre-secretory salivary fragmentation pathway the recent analysis of some derivatives of human salivary peptides and proteins [13], such as acidic and basic proline-rich proteins (prp) and histatins, allowed recognizing in the presecretory salivary fragmentation pathway the action of a furinlike pro-protein convertase of the kexin-subtilisin family, often followed by a carboxy-peptidase action. on the same line, the present study was carried out to search in human saliva the fragments generated from statherin and pb peptide by the action of furin-like proteinases, utilizing a selected-ion monitoring strategy based on hplc-it ms. the fragments and post-translational derivatives detected with high frequency in multiple samples were the following: (i) statherin (5380 amu), des-phe-43 (5232 amu), des-thr-42-phe-43 (5131 amu), des-asp-1 (5265 amu), mono-phosphor. (5300 amu), statherin sv2 (missing 6-15 residues; 4149 amu), fragm. 10-43 (4128 amu), fragm. 11-43 (3971 amu), fragm. 14-43 (3645 amu). moreover, the fragm. 6-57 (5215 amu) of pb peptide (5793 amu) was identified. the quantity of these fragments in salivary samples was usually <10% of the parent peptide. the identified fragments confirmed the action of a proprotein convertase on furin-like consensus sequences, being the cleavage at arg-9 (ekflr), arg-10 (lrr) and arg-13 (rrigr) for statherin, and at arg-5 (rgpr) for pb peptide. detection of statherin missing n-and c-terminus residues indicated also a pre-secretory exopeptidase action, already observed in other salivary peptides. the function of these statherin and pb derivatives in the oral cavity must be elucidated. cloning and expression of a pepstatin insensitive acid protease from thermoplasma volcanium in e. coli acid proteases, commonly known as aspartic proteases, are recognized by their specific inhibition by pepstatin. acid proteases are found in microorganisms both as intracellular and extracellular enzymes. there is very limited number of thermostable, pepstatin insensitive acid proteases isolated from bacterial sources. the only example of purified and cloned acid protease from archaebacteria is thermopsin, produced by sulfolobus acidocaldarius. this thermophilic enzyme represents a new class of acid proteases. to extend our knowledge on the microbial acid proteases with thermostable properties, in this study we have undertaken the cloning and expression of a thermostable, pepstatin insensitive acid protease from themoacidophilic archaeon thermoplasma volcanium. a primer set was designed based on nucleotid sequence of the predicted thermopsin gene and pcr amplification produced a 3080 bp fragment, which covered complete thermopsin gene with some upstream and downstream sequences. the amplified thermopsin gene was cloned in e. coli, using pdrive vector. the alignment of the amino acid sequences of thermopsins from various archaea revealed the highest homology (44%) between the tp. volcanium thermopsin and putative tp. acidophilum enzyme, thermopsin 1. there was a low degree of similarity (28%) between the tp. volcanium thermopsin and thermopsin from sulfolobus acidocaldarius. expression of the recombinant thermopsin was attempted using qia expression kit, where the cloned gene was ligated to pqe expression vectors to be expressed under the control of t5 promoter. in this system the protein was tagged with 6xhis residue at n-terminal end so that it could be selectively isolated using ni-nta metal-affinity chromatography. include various vital proteins with discrete functions in the propagation of apoptosis. our aim is to generate a caspase cleavage site predictor specific for each member of the caspase family in order to make subtype-specific predictions of new caspase substrates. we have used a set of experimentally verified proteins to generate sequence logos and train a neural network in order to predict caspase cleavage sites. machine learning techniques, such as artificial neural networks, are often well suited to integrate the subtleties of sequence variations. this approach also enables integration of structural information in the pattern recognition procedure which could possibly increase the predictive performance of the neural network. the identification of new caspase substrates can lead to further elucidation of several cellular processes involving caspases, including apoptosis, cell cycle regulation, cellular differentiation, and pro-inflammatory responses. in addition, the generation of caspase inhibitors could be greatly aided by a caspase cleavage site predictor. regulation of protein synthesis and autophagic-lyososomal protein degradation in isolated pancreatic acini a. l. kovacs and e. papp cell physiology laboratory, department of general zoology, eotvos lorand university, budapest, hungary. e-mail: alkova@cerberus.elte.hu a series of biologically active compounds (wortmannin, ly294002, 3-methyladenine, rapamycin, okadaic acid, theophyllin, insulin, glucagon, cholecystokinin) influencing protein synthesis and autophagic-lysosomal protein degradation by interfering with important signalization pathways were investigated. our results show that in exocrine pancreas cells phosphatidyl inositolkinases (pi3k-s) are activators, while the target of rapamycin protein (tor) is an inhibitor of autophagy. camp is an inhibitor of lysosomal protein degradation that acts through members of the pi3k family. okadaic acid inhibits lyososomal protein degradation without inhibiting the formation of autophagic vacuoles. the inhibition of pi3k-s and tor diminishes protein synthesis, inhibitors of these kinases reduce the synthesis stimulatory effect of insulin. cholecystokinin showed a biphasic stimulatory effect while glucagon was ineffective on protein synthesis. on the base of these results a possible signalization pathway is suggested for autophagic segregation and lysosomal protein degradation in pancreatic acinar cells. purification and characterization of a bifunctional protease from vibrio vulnificus in this study, we purified and characterized an extracellular protease showing dual functions as prothrombin activator and fibrinolytic enzyme from vibrio vulnificus atcc 29307. the purified enzyme had broad substrate specificity towards various bloodclotting associated proteins such as prothrombin, plasminogen, fibrinogen and factor xa. the cleavage of these proteins could be stimulated by addition of 1 mm mn 2+ . the protease could acti-vate prothrombin to active thrombin. however, the thrombin activity generated from prothrombin activation by the protease seemed to be transient, with further cleavage resulting in a loss of activity. interestingly, the enzyme could enhance the activity of thrombin during the initial rate of fibrin formation when purified fibrinogen was used as substrate. it could also actively digest fibrin polymer as well as cross-linked fibrin. these results suggest that the secreted protease functions as a prothrombin activator and a fibrinolytic enzyme to interfere with blood clotting as part of the mechanism associated with its pathogenicity in human. tumor invasion and metastasis are the major causes of treatment failure and death in cancer patients. one requisite for neoplastic cell invasion during tumorigenic processes is the remodeling events that occur within the stroma or extracellular matrix (ecm). cysteine cathepsins, most likely along with matrix metalloproteases and serine proteases, degradate the ecm, thereby facilitating growth and invasion into surrounding tissue and vasculature. clinically, the activity levels and localization of cysteine cathepsins and their endogenous inhibitors have been shown to be of diagnostic and prognostic value. the aim of our study was therefore both the determination of prognostic and diagnostic impact of cathepsins b, l and h from human tissues extracts (normal and tumor tissue) and extracellular fluids (such as plasma and urine) and a1-proteinase inhibitor (pi) in pathogenesis of different types of human brain tumors, and extraction and purification of cysteine cathepsin endogenous inhibitors from normal and tumor brains and studying of their physicochemical properties. it was found that the increasing of cysteine cathepsins b, l, h activity levels in brain tumors tissues depend on histostructure, histogenesis and tumor malignancy grade. increasing of cathepsins l and h activity levels was found in plasma and urine in depending on histogenesis. at the same time decrease in pi activity level was registered. besides, kinetic characteristics of extracted normal brain endogenous inhibitors of cysteine cathepsins were determined. in extracted tumor brain endogenous inhibitors, there were differences in physicochemical properties in comparison with normal. the data obtained contribute to understanding the participation of cysteine cathepsins and their inhibitors in mechanisms of cancer genesis and both become useful for solving the problem of improving of tumor therapy and provide the possibility of using their activity as diagnostic and prognostic markers. protein hydrolysates of sea origin as components for microbiological culture media dry hydrolysate was prepared from protein-containing waste of icelandic scallop chlamys islandicus processing (spw) by means of a proteinase complex from king red crabs hepatopancreas. the enzyme consist of the proteolytic enzyme complex from crab hepatopancreas, in which serine proteases dominate (collagenase, elastase and trypsin-and chymotripsin-like proteinases). as proteinases from king red crab hepatopancreas have high enzymesubstrate affinity to icelandic scallop proteins, a high degree of proteolysis can be achieved. the composition and properties of the material were investigated on enzymatic protein hydrolysate from spw obtained under the most technologically suitable conditions: 50-55 o c, ph 7.5, 6 h, the ratio between the protein material and the enzyme preparation being 1000:6. for comparison we examined the composition of commercial pancreatic hydrolysate from poor-quality fish species, mainly boreogadus and micromestistus. it was found that hydrolysate from spw significantly overpowered the commercial analog in the mass percentage of the target product (free amino acid and oligopeptides). the resulting product contains not <80% free amino acids and oligopeptides. predominant are aspartic acid, leucine, isoleucine, arginine and lysine, which account for >5% of the free amino acids. the potential usage of the protein hydrolysate as a nutrient for microorganism cultivation is estimated. microbiological studies have demonstrated that the hydrolysate from spw can be used as a protein component in nutrient media. the tested microbial strains satisfactorily grew on the media. the z variant alpha-1 proteinase inhibitor (a1piz) misfolds in the endoplasmic reticulum (er) and is a substrate for er-associated protein degradation (erad). we report here that a1piz degradation is also dependent on vps30/atg6, a gene that encodes a component of two pi3-kinase complexes that regulate membrane traffic; complex i is required for autophagy, complex ii is required for the cpy-to-vacuole pathway. to elucidate why vps30p participates in a1piz degradation, we tested the hypothesis that erad was saturated at elevated levels of a1piz expression and that excess a1piz was targeted to one of these alternative quality control pathways. overexpression of a1piz led to vacuole-dependent degradation and both complexes were required for delivery of the excess a1piz to the vacuole. when the cpy-to-vacuole pathway was compromised a1piz was secreted and the distribution of soluble vs. aggregated forms of a1piz was comparable with that of wild type yeast. however, disruption of autophagy led to an increase in levels of aggregated a1piz; suggesting that when erad is saturated the excess a1piz is selectively targeted to the vacuole via the cpy-to-vacuole sorting pathway, while excess a1piz that forms aggregates in the er is targeted to the vacuole via autophagy. together, these results reveal multiple pathways for recognition and removal of aberrant proteins and provide direct evidence that aggregated a1piz is removed by autophagy. our findings may have application in the understanding of, and treatment for, individuals with liver disease caused by the accumulation of er aggregates of a1piz. acknowledgements: the study was supported by national science foundation grants mcb-011079 and mcb-0110331. yeast and lactobacillus association generates peptides from acid goat whey proteins fermentation s. didelot, s. bordenave-juchereau, e. rosenfeld, l. murillo, j. m. piot and f. sannier laboratory of biotechnology and bioorganic chemistry, university of la rochelle, la rochelle, france. e-mail: lmurillo@univ-lr.fr our goal was to produce peptides from fermentation of unsupplemented acid goat whey by dairy micro-organisms. we used a lactobacillus, lactobacillus paracasei, and a yeast, candida parapsilosis, both previously isolated from a cheese microflora. when co-cultivated aerobically, both micro-organisms grew on unsupplemented goat whey and led to a medium acidification from 6 to ph 3.5. reversed phase (rp)-hplc analysis revealed a total alpha-lactalbumin hydrolysis after 96 h of fermentation, a modification of the beta-lactoglobulin elution peak, and 2.5-fold increase in peptide level compared with the non-fermented whey. in the absence of c. parapsilosis, l. paracasei grew poorly on whey and only a weak medium acidification from 6 to 4.5 was observed after 192 h of fermentation. rp-hplc analysis revealed a weak modification of beta-lactoglobulin elution peak, a truncated form of alpha-lactalbumin and no peptide generation. c. parapsilosis was able to grow on unsupplemented goat whey without modifying ph of the medium, but only 25% of proteins were hydrolysed (alpha-lactalbumin) or denaturated (beta-lactoglobulin) and, again, no peptides were detected. these results suggest that (i) c. parapsilosis is required for l. paracasei growth and (ii) the co-culture of both micro-organisms is needed to generate peptides from alpha-lactabumin hydrolysis. during co-culture on whey, the use of penicillin g and cycloheximide as bacterial and yeast growth inhibitors respectively, revealed that l. paracasei growth was required for medium acidification to ph 3.5 and alpha-lactalbumin hydrolysis. however, we demonstrated that the protease(s) responsible of alpha-lactalbumin hydrolysis was (were) synthesized by c. parapsilosis during the first stage of fermentation and that medium acidification (obtained either by l. paracasei growth or chemically) was required for yeast protease(s) activity. dengue virus causes widespread human diseases such as dengue fever, dengue hemorrhagic fever and dengue shock syndrome. the viral genome is a positive rna strand that encodes for a single polypeptide precursor. processing of the polyprotein precursor into mature proteins is carried out by the host signal peptidase and by ns3 serine protease. the three dimensional structure of ns3 protease domain ns3pro has been elucidated [1] . recently a new construct of the recombinant form of the ns3pro, was engineered [2] . we have expressed in e. coli the his-tag-cf40.gly.ns3pro protein a new construct of the recombinant form of the ns3pro linked to a 40 -residue co-factor, corresponding to a part of ns2b, via a non-cleavable, flexible non-apeptide (gly 4 sergly 4 ), and have currently optimized the purification procedure. chemically optimized substrates, peptides and depsipeptides, were designed and tested to afford an efficient in vitro activity assay, using hplc and fret spectroscopy. the data suggest that the amino-terminal region of the 40-amino acid co-factor domain is involved in additional charged interactions with ns3 that are essential for activity as previously described. this form showed catalytic activity and spectroscopic studies were performed to identify the folding of the protein. moreover, experiments of limited proteolysis have been performed to identify the essential enzymatic domain of the protein and to stabilize the role of the cofactor in the activity and in folding stabilization of the enzyme. after 2 h of the limited proteolysis with endoproteinase asp-n the product was analyzed by sds-page and activity assay, showing a high reduction of the molecular mass and only a loss of the activity of the 20%. cd and 15 n-1 h-hsqc spectra of this protein fragment were performed and other functional and structural characterizations are in progress in our laboratory. it is intended to obtain the structure in solution of the essential active domain of the uniformly 13 c, 15 n-labeled cf40.gly.n-s3pro by high-field 3d nmr spectroscopy. the solution structure of the enzyme will be used to answer yet unresolved questions about the mechanism of action, the role of its cofactor ns2b, and the observed substrate specificity. introduction: fish consumption is associated to nutritional benefits due to the presence of proteins of high biological value, minerals, vitamins and polyunsaturated fatty acids. most studies concerning the benefits of fish consumption on cancer prevention have focused on fish fatty acids but little is known about the potential bioactivity of fish peptides. the present study was then designed to assess the antiproliferative activity of various fish protein hydrolysates, in order to further purify and characterize anticancer peptides. methods: twenty-one fish hydrolysates (from seven species) produced within the framework of the european valbiomar programm. fish hydrolysates composition (protein, fat and salt content) was determined by standard methods (kjehldhal, soxhlet extraction and volhard respectively). cytotoxic and antiproliferative activity were assayed in vitro on mcf-7/6 and mda-mb-231 human breast adenocarcinoma cell lines, following a cell viability colorimetric assay (promega, france). antiproliferative activity of fish hydrolysates was compared with that of reference anticancer molecules with various cellular targets, namely actino-mycine d, cytosine-beta-d-arabinofuranoside, cyclophosphamide, etoposide, kenpaullone and roscovitine. results: composition analysis revealed that most hydrolysates contained more than 70% protein. three blue whiting hydrolysates containing 96% protein, 0.5% lipid and 0.2% salt induced a strong breast cancer cells growth inhibition when tested at 1 g/l for 72 h in cell culture medium. blue whiting hydrolysates 3, 4 and 5, respectively, induced a growth inhibition of 24.5, 22.3 and 26.3% on mcf-7/6, and 13.5, 29.8 and 29.2 % on mda-mb-231. these in vitro antiproliferative activities are in the range of that observed when the two breast cancer cell lines are treated for 72h with kenpaullone, roscovitine or cytosine-beta-d-arabinofuranoside 10 )6 m. further studies are engaged to fractionate and characterize the antiproliferative peptides contained in blue whiting hydrolysates. during recent years, it has been established that intracellular proteolysis in eukaryotic cells is largely accomplished by a highly selective non-lysosomal pathway that requires atp and a large (2.5 mda) multisubunit complex known as the 26s proteasome. the proteasome-mediated pathway plays vital regulatory functions. it degrades many important proteins involved in cell cycle control, in signaling pathway, and in general metabolism, including transcription factors and key metabolic enzymes. another function of the proteasomal system is the removal of abnormal, misfolded and oxidized proteins generated under normal and, in particular, stress conditions. to date, proteasomes from other than animal or plant cells were studied only in yeast. recently, in our laboratory, the proteasome-mediated pathway was shown to be involved in the regulation of ligninolytic activities in the white rot fungi trametes versicolor and phlebia radiata upon nutrient starvation (staszczak, enzyme microb technol 2002; 30: 537-540). it was the first report on proteasomes in fungi representing basidiomycota. white rot fungi are able to degrade lignin by the action of secreted enzymes, the best characterized of which are laccases, lignin peroxidases, and manganese peroxidases. the subject of lignin biodegradation has commanded attention for a considerable period of time mainly because of its ecological significance and wide industrial applications of bioligninolytic systems. heavy metal ions are important environmental pollutants which affect biodegradation processes performed by white rot fungi. in the present study, we investigated whether the proteasomal degradation pathway might be involved in the regulation of laccase production by t. versicolor in response to cadmium exposure. studies of cacybp/sip function using small interfering rna cacybp/sip was discovered as a protein that bound calcyclin (s100a6) in a calcium-dependent manner (filipek and wojda 1996; filipek and kuznicki, 1998) and its distribution and some biochemical properties have been studied. for instance, it has been shown that cacybp/sip binds calcyclin via its c-terminal fragment (nowotny et al. 2000) and that, beside calcyclin, it interacts with other calcium binding proteins of the s100 family (filipek et al. 2002) . originally, we identified cacybp/sip in ehrlich ascites tumour (eat) cells but it is also present in other mammalian tissues and cells. in particular, high expression of cacybp/sip was found in neuronal cells of mouse and rat brain (jastrzebska et al. 2000) . at present the distribution and structural properties of cacybp/sip are quite well described but its function remains obscure. there is only one paper published concerning the possible involvement of cacybp/sip in b-catenin ubiquitination and degradation (matsuzawa and reed 2001). to elucidate the biological role of cacybp/sip we have designed and synthesized sirna (small interfering rna) against this protein. this sirna was then used to transfect neuroblastoma nb-2a and embryonic kidney hek293 cells, expressing high and low amount of endogenous cacybp/sip respectively. the level of cacybp/sip was monitored in cell extracts by western blot technique. we found that sirna against cacybp/sip, which we designed, inhibited the expression of this protein, as its level in transfected cells was lower in comparison with control cells. at present, we checked the effect of diminished expression of cacybp/sip on b-catenin degradation and other cellular processes. acknowledgements: this work was supported by grants: kbn heavy metals are powerful poisons for living cells. it has been shown that exposure to arsenicals, either in vitro or in vivo, in a variety of model systems, causes the induction of a number of the major stress protein families, such as the heat shock proteins (hsp) (toxicol appl pharmacol 2001; 177: 132). the reasons for heavy metal toxicity in vivo are not fully understood, but they are known to contribute to the accumulation of aberrant proteins (bba,1995,1268, 59). in animal cells, arsenite has been reported to cause sulfhydryl depletion, to generate reactive oxygen species and increase the level of high molecular mass ubiquitin-protein conjugates (toxicol appl pharmacol 2003; 186: 101). in cells submitted to stress conditions, several components of the ubiquitin/proteasome pathway are activated. in this major, eukaryotic proteolytic pathway, multiple ubiquitin molecules are enzymatically ligated to proteins destined for catabolism by an enzyme system composed of three types of enzymes, commonly referred to as e1, e2, and e3. the large ubiquitinprotein conjugates thus formed are subsequently degraded by a very large protease complex, the 26s proteasome, in an atpdependent process. the changes in free ubiquitin (ub) and ubiquitin-protein conjugates (ub-p) levels were followed by immunoblotting during the incubation of the higher plant lemna minor l.(duckweed) in the presence of arsenite (as), at concentrations known to confer thermotolerance to the plants. the observed increase in the amount of large molecular mass ubiquitin-protein conjugates is indicative of a role for the ubiquitin/ proteasome pathway in the response of lemna to as stress. this outcome is primarily attributed to an increased availability in protein substrates during as treatment for three main reasons: an increase in protein carbonyl (a major marker for protein oxidation) content detected by immunoblotting; moderate increments (as determined by semi-quantitative rt-pcr) in the mrna levels of the codifying sequences for the ubiquitin pathway components: ubiquitin, e1, e2 and the b subunit and the atpase subunit of the 26 s proteasome; an identical pattern of variation for the large ubiquitin-protein conjugates is observed in the simultaneous presence of as and cycloheximide, indicating that the observed increase in ubiquitin conjugates does not depend on de novo protein synthesis. ageing and autophagy y. stroikin and a. terman experimental pathology, linko¨ping university, linko¨ping, sweden. e-mail: yurst@inr.liu.se life of aerobic cells is associated with continuous oxidative damage resulting in the formation of altered, non-functional macromolecules and organelles. intracellular accumulation of oxidized proteins defective organelles and lipofuscin inclusions are typical manifestations of ageing that preferentially affects long-lived post-mitotic or growth-arrested cultured cells. autophagy, an important biological mechanism for renewal of damaged intracellular structures, has been found decreased in ageing. to learn more about the role of autophagy in ageing, we studied the effect of the inhibitor of autophagic sequestration 3-methyladenine (3-ma) on human diploid fibroblasts and astrocytes. inhibition of autophagy in growth-arrested (confluent) fibroblasts for 2 weeks resulted in the accumulation of altered lysosomes displaying lipofuscin-like autofluorescence, especially when 3-ma exposure was combined with hyperoxia. the findings suggest that autophagy is indispensable for normal turnover of lysosomes, and lysosomal components may be direct sources of lipofuscin. the accumulation of oxidatively damaged intracellular structures (so-called biological ''garbage'') was associated with decreased cell viability. two-week-inhibition of autophagy with 3-ma resulted in a significantly increased proportion of dying cells when compared with both untreated confluent cultures and dividing (subconfluent) cells exposed to 3-ma. similar results were obtained when autophagic degradation was suppressed by the protease inhibitor leupeptin. the results support the idea that biological ''garbage'' accumulation is essential for ageing and age-related death of post-mitotic cells, which can be prevented by cell division. recently two family members of the tumour suppressor gene p53 have been described, p63 and p73, which seem to be necessary for specific p53-induced stress-response pathways. furthermore, p63 and p73 appears to be crucial to determine the cellular sensitivity to anticancer drugs, particularly in tumours lacking functional p53. here, we show that p63 and p73 isoforms are also regulated by proteasomal degradation. we have identified several e3-ubiquitin ligases responsible for the regulation of the stability of p63 and p73. we found that the regulation of p63 and p73 is isoform-specific. furthermore, we demonstrate that ubiquitination of p73 influences the cellular localization of p73 and of the respective e3-ubiquitin ligases. finally, we show that the expression of the various e3-ubiquitin ligases can be differentially induced by p73-isoforms. in addition, the e3-ubiquitin ligases can influence the apoptotic function of p73. our findings demonstrate that p63 and p73 are sent to degradation or stabilized by e3-ubiquitin ligases in an isoform-specific manner and we suggest a negative feedbackloop between p63, p73 and their regulators, as they also influence the function of p63 and p73. increased level of metalloproteases was shown to accompany tumor angiogenesis and active invasion in adjacent tissue [1] . development of different types of tumors is often accompanied by increased protease activity in blood [2, 3] . in the present study we compared protease activity of plasma and eluate from surface of blood cells in healthy donors and patients with breast tumor. we have demonstrated recently that in blood of healthy donors almost all circulating nucleic acids (cirna) are bound at the surface of blood cells. in patients with fibroadenoma cirna were found at cell surface whereas in breast cancer, no cell-surfacebound cirna were detected in blood [4] . conjugates of hydrophobic and hydrophilic peptides of cd34 receptor with biotin were incubated with avidin-coated 96-well eia microplates. avidinpeptide complex was incubated with samples under investigation and serial dilutions of proteinase k solution, which was used for calibration of protease activity. undegraded peptides were visualized by incubation with goat anti-peptide antibodies followed by conjugate of anti-goat immunoglobulins with peroxidase. blood plasma and eluate from surface of blood cells of cancer patients demonstrated increased level of anti-hydrophilic protease activity compared with healthy donors. increase of protease activity against hydrophilic peptide in blood correlate with decrease of cell-surface-bound cirna, indicating that blood proteases can affect concentration and distribution of circulated na. identification of cleavage site and natural substrate specificity of prta, a serralysin-type metalloprotease from the entomopathogenic microorganism photorhabdus prta, a secreted basic metalloprotease of photorhabdus, belongs to the m12b (serralysin) family of proteases. the biological function of these enzymes is not known, but in some cases they are supposed to have a role in virulence. serralysins are generally assumed to have broad substrate side-chain specificity. attempts toward the generation of a sensitive and specific substrate of these enzymes had limited success, and no such substrate is available for prt-a. through mass spectrometric analysis of prta cleavage products of oxidized insulin a and b chain, we found that prta has a welldefined cleavage site preference. based on this, we developed a sensitive and highly specific oligopeptide substrate through optimization of the amino acid composition and length. the kinetic parameters of prta isolated from photorhabdus luminescens ssp. laumondii strain brecon were measured on the best substrate, dabcyl-glu-val-tyr-ala-val-glu-ser-edans, giving a km of 8.8 · 10 )5 , a kcat of 2.1 · 10 )2 /s and a kcat/km of 2.4 · 10 6 . its poor hydrolysis by various proteases proved its specificity, while it was very sensitivity in measuring prta activity in hemolymph samples from photorhabdus infected galleria mellonella larvae. the substrate preference of prt-a was determined by in vivo digestion of hemolymph proteins from manduca sexta. six minor protein components were selectively cleaved, which were provisionally disthe epithelial sodium channel (enac) is an integral component of the pathway for na + absorption in epithelial cells. enac activity is mainly regulated by mechanisms that control its expression at the cell surface, such as ubiquitination. the ubiquitin ligases nedd4 and nedd4-2 have both been shown to bind to enac and decrease its activity. conversely, the serum-and glucocorticoid regulated kinase (sgk), a downstream mediator of aldosterone, is able to increase enac activity. this effect is at least partly mediated by direct interaction between sgk and nedd4-2. sgk binds both nedd4 and nedd4-2 but it is only able to phosphorylate nedd4-2. phosphorylation of nedd4-2 reduces its ability to bind to enac, and hence increases enac activity. the impact of the interaction between nedd4 and sgk remains unclear. nedd4-like proteins interact with enac via their ww-domains. these domains bind py-motifs (ppxy) present in enac subunits. nedd4 and nedd4-2 both have four highly homologous ww-domains. previous studies have shown that interaction between nedd4 and enac is mainly mediated by ww-domain 3. sgk also has a py-motif, therefore we tested whether the ww domains of nedd4 and nedd4-2 mediate binding to sgk. we show that single or tandem ww domains of nedd4 and nedd4-2 mediate binding to sgk and that, despite their high homology, different ww domains of nedd4 and nedd4-2 are involved. our data also suggest that ww domains 2 and 3 of nedd4-2 mediate the interaction with sgk in a concerted manner, and that in vitro the phosphorylation of sgk at serine residue 422 increases its affinity for the ww domains of nedd4-2. the stimulatory effect of sgk on enac activity is partly mediated via nedd4-2 and will decrease if competition between nedd4 and nedd4-2 for binding to sgk occurs. we show that nedd4 and nedd4-2 are located in the same subcellular compartment and that they compete for binding to sgk in vitro. the concerted or successive action of proteolytic enzymes has been described in a number of important biological processes in which proteins are degraded or matured, such as digestion, turnover (lysosomal, proteosomal...), blood coagulation, developmental remodeling or apoptosis, among others. the complementary action of proteases belonging to different families to achieve a more efficient o a better modulated hydrolytic mechanism is well documented. specific molecular associations or shared scaffolds between the involved proteases and/or protein inhibitors and defined three-dimensional structures have also been reported. however, only in a few cases such structures involved metallo.carboxy-peptidases or their inhibitors [1] . we shall review this subject and describe, in such a context, a new model found in a marine invertebrate organism in which such a fact takes place. in particular, the characteristics of a novel bifunctional molecule displaying the functionalities and structures of serine-and metallo.carboxy-peptidases will be presented. its structure is fully different than the ones previously reported by us and collaborative groups for metallocarboxypeptidase inhibitors [2] [3] [4] . regulating the activity of herpes virus proteases c. s. craik departments of pharmaceutical chemistry, pharmacology, and biochemistry and biophysics, ucsf, san francisco, ca, usa. e-mail: craik@cgl.ucsf.edu herpesviral proteases exist in a monomer-dimer equilibrium in solution. dimerization is required for activity and a comformational change communicates the oligomerization state of the enzyme to the active site of each intact monomer. each monomer has an active site, which is spatially separate from the dimer interface. kaposi's sarcoma-associated herpesvirus (kshv), encodes a protease (kshv pr), which is necessary for the viral lytic cycle. like those of other herpesvirues proteases, the dimer interface of kshv pr is composed primarily of a helix near the c terminus, of the protein. the helix of one monomer interacts with residues in the symmetrically related helix of the other monomer across the dimer interface as well as with neighboring helices. small molecule inhibitors, site directed mutagenesis and 2d nmr spectroscopy were used to compare the monomeric and dimeric forms of kshv pr and to investigate the relationship of the active site and the dimer interface of the enzyme. active site inhibition was shown to strongly regulate the binding affinity of the monomer-dimer equilibrium of the protease, shifting the equilibrium completely to the dimeric form of the enzyme. a previously undetermined conformational change provided insight in to the regulation of protease activity by dimerization as well as an explanation for the weak dimerization of a family of enzymes with a disparately large dimer interface compared to their measured binding affinities. using this information as a guide, protein grafting of the interfacial helix onto a small stable protein, avian pancreatic polypeptide, generated a small macromolecular inhibitor that successfully disrupted the dimer interface and inhibited enzymatic activity. these results provide direct evidence that peptide bond hydrolysis is integrally linked to the quaternary structure of the enzyme, validate the protease as a therapeutic target and suggest the dimer interface may be an alternative site for antiviral design. abteilung strukturforschung, max-planck-institut fu¨r biochemie, martinsried, germany. e-mail: huber@biochem.mpg.de proteolytic enzymes catalyze a very simple chemical reaction, the hydrolytic cleavage of a peptide bond. nevertheless they constitute a most diverse and numerous lineages of proteins. the reason lies in their role as components of many regulatory physiological cascades in all organisms. to serve this purpose and to avoid unwanted destructive action proteolytic activity must be strictly controlled. control is based on different mechanisms which i will discuss and illustrate with examples of systems and structures determined in my laboratory. the family of serine protease inhibitors known as the serpins is represented in all branches of life and predominate in the higher organisms, including man. they have evolved an extraordinary mechanism to inhibit proteases which distinguishes them from the 20 other families of serine protease inhibitors, and renders them uniquely qualified to control of the proteolytic pathways essential to life. the mechanism is best described as a spring-loaded mousetrap, where nibbling of the peptide loop bait springs the trap and crushes the unsuspecting protease. as with a mousetrap, the active state of a serpin is metastable, and the energy released upon conversion to its more stable form is used to trap the protease. the complexity of the serpin mechanism provides many advantages over the simpler lock-and-key type mechanism, utilized by all other serine protease families. serpins provide stoichiometric, irreversible inhibition, and the dependence on serpin and protease conformational change is exploited for signaling and clearance. the potential for regulation is also an inherent part of such a complex mechanism, as illustrated by the heparin activation of serpins antithrombin and heparin cofactor ii. however, with complexity of mechanism also comes susceptibility to disease causing mutations: both through loss-of-function, as with thrombosis caused by antithrombin deficiency; and gain-of-function, as with dementia caused by neuroserpin polymerization. many crystallographic structures of serpins have been solved over the past 20 years, and we now have a frame-by-frame cinematic view of the intricate conformational rearrangements involved in protease inhibition, modulation of specificity, and molecular pathology of the remarkable shape-shifting serpins. structural lessons of serine proteases: function and mechanism of the serine protease-like hgf as a growth factor in met signaling hepatocyte growth factor (hgf), a plasminogen-related growth factor, is the ligand for met, a receptor tyrosine kinase implicated in development, tissue regeneration and invasive tumor growth. hgf acquires signaling activity only upon proteolytic cleavage of single-chain hgf into its a/b-heterodimer, similar to zymogen activation of structurally related serine proteases. although both chains are required for activation, only the achain binds met with high affinity. recently, we reported that the protease-like hgf b-chain binds to met with low affinity this suggests that additional allosterically linked regions may be involved in the signaling process. furthermore, antibodies directed toward the b-chain or the hgf a-chain result in inhibition of met phosphorylation in a549 cells. these antibodies also inhibit proliferation in bxpc3 cells and baf3 cells. implications for dimerization mechanisms of hgf-dependent met receptor activation and signaling are presented. in addition, mutagenesis of the hgf b active site region has been investigated with respect to imparting enzymatic activity. thus while hgf has the function of a growth factor, the structural and receptor binding aspects of hgf are more akin to those of serine proteases. trypsinogen 4 with a 28 amino acid leader peptide on its n-terminus is the predominant form of the enzyme in human brain gene prss3 on chromosome 9 of the human genome encodes, due to alternative splicing, both mesotrypsinogen and trypsinogen 4. mesotrypsinogen has long been known as a minor component of trypsinogens expressed in human pancreas, while the mrna for trypsinogen 4 has recently been identified in brain and other human tissues. analysis of the gene encoding trypsinogen 4 predicted two isoforms of the zymogen: isoform a may have a 72 amino acid, while isoform b a 28 amino acid n-terminal leader sequence. the translation initiation site for isoform a is an atg codon, while the initiation site predicted for isoform b is a ctg codon. we measured the amount of trypsinogen 4 mrna and the quantity of the protein as well in 17 selected areas of the human brain. trypsinogen 4 could be localized in glial and neuronal cells using immunohistochemical methods. we purified human trypsinogen 4 by affinity chromatography. our results show that splice isoform b is the predominant if not the exclusive form of the zymogen in human brain. the n-terminal residue of the isolated protein was identified by amino acid sequencing as a leucine. at the same time the longest mrna we were able to isolate was barely longer than the one corresponding to splice isoform b. although the most trivial explanation of our results is that isoform a is proteolytically processed to result in isoform b, it cannot be excluded that leucine rather then methionine is used as translation initiator amino acid. search for endogenous substrates for prolyl oligopeptidase in porcine brain prolyl oligopeptidase (po) is a serine protease present in most tissues, which preferentially cleaves the peptide bond at the carboxyl site of proline residues. the function of po is unknown, but it has been associated with several disorders of the central nervous system, such as depression and alzheimer disease. the purpose was to look for endogenous substrates for the recombinant porcine po in porcine brain. we adapted a method to extract the proteins from the brain with special attention to the smaller polypeptides since po is not known to cleave peptides larger than 30 amino acids. subsequently we looked for a method to separate the protein mixture in less complex fractions. 2d-gelelectrophoresis, commonly used in proteomics, is only suitable for proteins with a molecular weight between 10 and 200 kda and an iso-electric point between 4 and 10. two-dimensional chromatography offers a suitable alternative for small peptides. we chose ion exchange chromatography as a first and reversed phase high pressure liquid chromatography as a second step. the resulting fractions were divided into two parts. one part was incubated with the purified po, the other served as a control. by looking for shifts in the mass spectrum between the control sample and the incubated sample, we identified peptides cleaved by po. different methods, such as esi-qtof-ms and maldi-toftof-ms, were used to sequence cleaved peptides by msms. these experiments allowed us to deduce the sequence requirements for po cleavage. serine protease subtilisin immobilized on novel mesoporous materials serine proteinases are widely used in protein mapping and peptide or ester bond formation. fixation of enzyme on solid support has many advantages, such as high stability, possibility of recovering and low product contamination by enzyme. subtilisin carlsberg, a protease from bacillus licheniformis, was immobilized on mesoporous silica (sba-15) and several organosilica supports via physical adsorption. the bifunctional mesoporous organosilicas containing ch2-ch2 or ch=ch bridges in combination with organic tethers bearing amino or hydroxyl functionalities were synthesized using supramolecular templating in the presence of non-ionic triblock copolymers and exhibited high surface area and large pore diameters in the range of 50-70 å suitable for the incorporation of subtilisin. the kinetics of immobilization was examined for six different carriers. it was shown that enzyme retained hydrolytic activity after the immobilization. the dependence of subtilisin loading on the starting concentration of the enzyme during adsorption shows the maximum loading (455 mg protein/g support) at [e] = 20 mg/ml. the ph dependences of loading and activity of immobilized biocatalysts were bell-shaped. for the organosilica support containing amino and hydroxyl groups the ph-dependence was shifted to the alkaline ph by 2 in comparison with the support containing ch2-ch2 bridges. the adsorbed subtilisin desorbs easily in aqueous media, while no leaching of the enzyme was observed in acetonitrile and dmf/acetonitrile mixture (6/4). the immobilized biocatalyst shows high hydrolytic activity after incubation in non-aqueous acetonitrile for 1 week and after 48 h incubation in 60% dmf/acetonitrile mixture. these data indicate a possible application of the obtained biocatalysts in low water media. purification, structural and biological characterization of protease inhibitors from acacia plumose seeds protease inhibitors have been used in many current medicines. therefore, there is a considerable interest inside the pharmaceutical industry in discovering new composites and mechanisms of protease inhibition, since these investments have led, for example, to new anti-hiv therapeutical tests, coagulation diseases treatment and tests with anti-carcinogenic drugs. serine protease inhibitors are found in all plant tissues, mostly in the seeds of the leguminosae subfamilies: mimosoideae, caesalpinoideae and papilionoideae. acacia genus is one of most important member of mimosoideae, and the presence of protease inhibitors in this genus was described in only three species and none of them were structurally characterized. in this sense, we are studying three new protease inhibitors from a. plumose seeds. from saline extract of triturated mature seeds the inhibitors were purified and presented anti-coagulant activity, serine protease inhibitory activity and action on growth of fitopathogenic microorganisms, in vitro. the purification steps included size exclusion chromatography on the superdex-75 column, equilibrated and eluted with pbs, a ionic exchange chromatography on mono-s (hr 5/5) column, equilibrated with the buffer sodium acetate 50 mm (ph 5.0), and eluted with the same buffer in a gradient of 0-0.5 m of nacl. three fractions (eluted around 0.18, 0.22 and 0.33 m of nacl) that presented anticoagulant activity and serine protease inhibition were separated and denoted apia, apib and apic. their apparent mws were around 20 kda, by sds-page in the absence of reducing agents. in the presence of reducing agents they shown two bands: between 14-22, and 8-6 kda. the n-terminal analyze of higher mw chains were tyafl (apia); kellvdne (apib) and telhdd (apic). the circular dichroism spectra of these inhibitors were very similar, presenting a maximum around 230 nm and a minimum in 202 nm, compatible with presence of unordered and beta elements of secondary structure. their nterminal, cd spectra and two-polypeptide chains linked by covalent bound, are compatible with kunitz type inhibitors. probably these inhibitors are three different isoforms that present different inhibition specificity degree on the serine proteases family. the ki to different serinoproteases (trypsin, plasmatic kalikrein, elastase, quimotrypsin) and specificity to the phytopatogenic fungus are being investigated. although the proteases were initially described as enzymes involved in the non-specific degradation of dietary proteins, today it is known that they can also act as highly specific enzymes that perform selective cleavage of specific substrates. thus, alterations in the structure, regulation or function of this type of enzymes underlie serious human disorders including cancer. to date, more than 550 protease and protease homologs are annotated in man, mouse, and rat genomes (www.uniovi.es/degradome). the increasing complexity of the proteolytic systems has led to the introduction of global concepts as the term degradome to define the complete set of proteases that are produced in a specific moment by a cell, tissue or organism. as part of our studies focused on the characterization of the mammalian degradomes, we have identified and cloned unusual mosaic proteases containing in tandem serine protease domains. the first, called polyserase-1 is synthesized as a transmembrane protein that undergoes post-translational events to generate three independent serine protease domains. the second polyprotease is the polyserase-2, a secreted protein that remains as integral part of the initial protein product. to date, it is difficult to understand the putative functional advantages derived from the complex polyproteases and, albeit extremely unusual, it is not an unprecedented situation. thus, the amphibians ovochymase and oviductin are polyserine proteases that contain three in tandem serine proteases. in humans, angiotensin-coverting enzyme and carboxypeptidase d are polymetalloproteases that exhibit some similarities to the polyserases. all these polyproteases constitute examples that illustrate an additional strategy for increasing the complexity of the degradomes. evolution of a genetic locus, expressing several protease inhibitors with homology to whey acidic protein (wap) a. clauss and å . lundwall department of laboratory medicine, lund university, malmo¨, sweden. e-mail: adam.clauss@klkemi.mas.lu.se we have previously described a locus on human chromosome 20 that gives rise to 14 proteins containing wap four disulphide core (wfdc) domains. among them are the elastase inhibitors elafin and secretory leukocyte proteinase inhibitor (slpi). both slpi and elafin are also known to be important components of the innate immune defence by displaying anti-microbial properties. in order to gain a deeper understanding of the biological role of the locus, we have now extended our investigations of its organization and evolution into non-human mammals. homologous loci were identified on mouse chromosome 2, rat chromosome 3 and dog chromosome 24. transcript sequences were generated by race technology or retrieved from the est databases. as in humans, the murine and canine loci are divided into two sub-loci separated by approximately 200 kb. the majority of genes are conserved in all species, but the comparison also showed gain and loss of genes, e.g. two human pseudogenes were identified due to the discovery of functional rodent genes, and in the rat several duplications has yielded four slpi genes. a most interesting finding was that there is no murine elafin gene. the different wfdc domains showed a highly variable species conservation. this was particularly striking in proteins containing multiple domains, where the aminoterminal wfdc generally displayed low conservation, whereas the opposite was true for the carboxyterminal wfdc. the difference could be due to the potential targets of the inhibitors, which might be either highly variable exogenous microbial proteases or conserved endogenous proteases. signaling mechanism of thrombin-induced human gingival fibroblast contraction thrombin is activated during gingival tissue injury and inflammation. thrombin and other bacterial proteases also affect the functions of adjacent periodontal cells via stimulation of proteaseactivated receptors (pars). we noted that thrombin and par-1 agonist peptide (20 lm) induced the gingival fibroblasts (gf)-populated collagen gel contraction within 2-h of exposure. however, par-3 and par-4 agonist peptide (<20 lm) show little effect on collagen gel contraction. u73122 (phospholipase c inhibitor) and 2-apb (ip3 antagonist) were effective in inhibition of gf contraction. thrombin-induced gf contraction was inhibited by 5 mm egta (an extracellular calcium chelator) and verapamil (a l-type calcium channel blocker). in addition, w7 (10 and 25 lm, a calcium/calmodulin inhibitor), ml-7 (50 lm, myosin light chain kinase, mlck inhibitor), and ha1077 (100 lm, rho kinase inhibitor) completely inhibited the thrombin-induced collagen gel contraction. thrombin also induced the phosphorylation of erk1/erk2 in gf. however, u0126 only partially inhibited the thrombin-induced gf contraction. similarly, wortmannin (100 lm), ly294002 (20 lm) (two pi3k inhibitors) and genistein, also showed partial inhibition. moreover, nac was not able to suppress the gf-contraction, as supported by slightly decrease in reactive oxygen species production in gf by thrombin. these results indicate that thrombin is crucial in the periodontal inflammation and wound healing by promoting gf contraction. this event is mainly mediated via par-1 activation, plc activation, extracellular calcium influx via l-type calcium channel, and the calcium/calmodulin-mlck and rho kinase activation pathway. survival of the anticarcinogenic bowman-birk inhibitor from soybean at the terminal ileum of cannulated pigs plant protease inhibitors (pi) of the bowman-birk class, a major pi class in legume seeds, have emerged as highly promising cancer chemopreventive agents, being capable of preventing or suppressing carcinogenic processes in a wide variety of in vitro and in vivo animal model systems. in order to exert their chemopreventive properties in vivo, plant pi have to resist and survive, at least to some extent, degradation by acidic conditions and digestive enzymes during gut passage. in this study, we have evaluated the survival rate of the bowman-birk inhibitor (bbi) in the terminal ileum of cannulated pigs fed defatted soybean. two different quantitative approaches have been carried out. firstly, a competitive indirect elisa assay using an antisera capable to detect bbi free and/or in complex with digestive proteases; secondly, we have carried out spectrophotometric measurements of trypsin and chymotrypsin inhibitory activities in ileal samples, where the presence of bbi metabolites and/or single active loops can be detected. according to the elisa method, ileal apparent digestibility of bbi was 58 %, which resulted in a recovery of 0.61 mg out of 1.5 mg/kg feed ingested. significantly higher ileal digestibility values (95 %) were found when trypsin and chymotrypsin inhibitor activities were evaluated. the results suggest that the immunoassay may be overestimating the presence of functional pi by detection of inactive bbi, but also that the presence of complexed bbi with digestive proteases, even if protein extraction was carried out under acidic conditions, could make bbi undetectable in activity assays. studies are in progress to overcome these drawbacks. the resistance of bbi to the acidic conditions and digestive enzymes of the upper gastrointestinal tract make these proteins very interesting candidates for evaluation as chemopreventive agents, in modulating cell viability and tumor progression within the gastrointestinal tract. a single amino acid change in a chymotrypsin prevents plant proteinase inhibitor binding plants have evolved economical strategies to combat insects, which on one hand involves the production of multi-domain pis that can target multiple enzymes with different specificities and on the other, pis that belong to structurally distinct families. solanaceous plants, produce both type i and type ii families of pis, which specifically target serine peptidases. this study showed that type i pis are better inhibitors of a particular class of chymotrypsins within the gut of helicoverpa species that is otherwise unaffected by the type ii class of inhibitors. homology models were used to identify a single amino acid substitution in the helicoverpa chymotrypsin that was likely to confer resistance to the type ii inhibitor. our hypothesis was further supported by recombinant expression and mutagenesis of this single amino acid in the type ii inhibitor-resistant chymotrypsin. we therefore propose that both type i and ii inhibitors are required to protect plants against lepidopteran insects. mobility of the sulphate protamin/ low molecular weight heparin complexes in an electrical field glycosaminoglycans low molecular weight heparin (lmwh) activated plasma serine proteases inhibitors. serine proteases play an important role in thrombogenesis, the process that leads to blood clotting and such as heart attack, stroke and other cardiovascular disorders. lmwh has been used to temporarily render the blood incoagulable during prophylaxis or treatment of thrombosis and sometimes result in serious bleedings and for the heparin anticoagulant activity neutralization used sulphate protamin. it was investigated relationship between new lmwh-sk derivatives (were generated through the controlled cleavage of porcine intestinal mucosa heparin with a mixture of chitinolytic complex from streptomyces kurssanovii) anticoagulant activities and lmwh-sk complexes with sulphate protamin mobility in an electrical field. with this purpose used biospecific electrophoresis in 1% agarose with protamin sulphate. precipitation zones (zones of the equivalent) in the ''rocket'' form were generated. scanning image was saved as jpg format. the ''rocket'' squares estimated with the help of bandscan program. results: lmwh-sk with molecular mass (mm) 14.0; 5.8; 5.4; 4.7; 4.0; 3.4 kd demonstrated antithrombin activities (aiia) 85-264 iu/mg, activities against factor xa (axa) has made 100-278 iu/mg, axa/aiia ratio -(0.8-2.2). correlation coefficients between mm and precipitation zone heights or squares consist 0.56-0.73 (p < 0.05), between axa activities and precipitation zone heights or squares consist 0.37-0.54 (p < 0.05). conclusion: lmwh-sk was obtained with the chitinolytic comlex hydrolisis help has ratio axa/aiia-2,2, it is necessary for antithrombotic preparations. with the mm decrease axa activity increase and precipitation zone heights or squares of the lmwh-sk complexes with sulphate protamin decrease. the role of extracellular proteases in supplying filamentous fungi with nutrient compounds is well understood and experi-mentally documented. however there is no definite answer on the question on the need and role of these proteases in pathogenesis. the study of differences in the spectra of extracellular enzymes of saprotrophic and pathogenic fungi performed on fusarium species revealed that activity of secreted serine proteinases of pathogenic f. culmorum strain was much higher (up to 20-fold) than that of saprotrophic strain. the use of f. culmorum strains differing in pathogenicity (strongly and weakly pathogenic) demonstrated that activity of secreted serine proteases of strongly pathogenic strain was significantly higher (1.5-8-fold) than that of weakly pathogenic strain. this tendency was preserved in calculations of activity towards protein content and dry weight of mycelium indicating on purposeful synthesis and secretion of extracellular proteases by strains with high pathogenicity. at that these differences were much higher when the substrate for trypsin-like proteinases bz-arg-pna was used than in the case of substrate for subtilisin-like proteinases glp-ala-ala-leu-pna. according to the data obtained it is proposed that the value activity of trypsin-like proteinases secreted by the fungi correlated with the degree of their pathogenicity and plays, apparently, an important role in pathogenesis. acknowledgment: this work was supported by grants from the russian foundation for basic research. conformational adaptation of a canonical protease inhibitor upon its binding to the target protease increases specificity atomic resolution crystal structure of sgti in complex with crayfish trypsin provided further data on the molecular basis of the inhibition mechanism of pacifastin type inhibitors. in complex with crayfish trypsin, sgti exhibits more or less continuous contacts in an extended region (through sites p 12 -p 5' ) of the molecule. the comparison of this complex with a simulated bovine trypsin-sgti one shows that more than half of the interaction energy surplus is originated from the extended region of binding. some of these contacts result from a conformational change of sgti that was induced by its binding to the enzyme which is strongly supported by the critical comparison of the crystal structure of crayfish trypsin-sgti complex with the free form of sgti. alignment of the nmr structure ensemble with the x-ray structure of complexed sgti and a careful comparison of the backbone j, w angles were carried out. additionally, noe-derived restraints and corresponding distances in the complex are also compared. local conformation of both p 12 -p 4 and p 4 '-p 5 ' regions of the inhibitor shows significant changes upon binding suggesting that either or both of these regions may act as molecular recognition sites. this comprehensive analysis of the local backbone properties of sgti in the free and in the complex form made possible to identify conformational similarities and differences responsible for its efficient binding to the enzyme, and provides a good basis for further studying the structural aspects of protease inhibitor specificity. as most of serine proteases enteropeptidase light chain contains four disulfide bonds and one nonpaired cysteine at 122 (chymotrypsinogen-derived residue numbering) position which forms disulfide bond linking the pro-and catalytic domains. a mutant of human enteropeptidase light chain cys122ser was constructed by site-directed mutagenesis. the recombinant wild type and mutant proteins were produced in escherichia coli bl21(de3) with expression vector pet-32a. the active proteins were obtained after solubilization and renaturation of the fusion protein thioredoxin/human enteropeptidase light chain from inclusion bodies. after autocatalytic cleavage of thioredoxin the active enzyme was purified on agarose linked soybean trypsin inhibitor. the yield of refolded active enzyme increased from 1.87 to 7.84% in case of cys122ser mutant. the wild type and c122s mutant showed similar kinetic parameters for cleavage of small synthetic substrate gly-asp-asp-asp-asp-lys-naphthylamide, small ester thiobenzyl benzyloxi-carbonil-l-lysinate (z-lys-sbzl) and fusion protein cleavage. both enzymes were inhibited by trypsin-like serine proteases inhibitors but not inhibitors of chymotrypsin-like, cysteineor metallo-proteinases. recombinant human enteropeptidase light chain and its mutant c122s were active between ph 6 and 9 with a broad optimum at about ph 7.54 and demonstrated quite high stability to different denaturating agents. both enzymes demonstrated secondary specificity to chromogenic substrate z-ala-phe-arg-na with km = 0.067 mm, kcat = 23 s-1. proteinaceous low molecular serine protease inhibitors from wood rotting fungi k. j. grzywnowicz and j. zuchowski biochemistry department, maria curie-sklodowska university, lublin, poland. e-mail: grzyw@hermes.umcs.lublin.pl proteolytic enzymes have been firmly established as main regulatory components in a number of cellular and physiological processes. the most important factors influencing the proteolytic enzymes are natural, proteinaceous protease inhibitors, which form complexes with target proteases. they have been extensively investigated from the points of view on physiological functions, as tools for protease enzymology, models for protein-protein interactions and on potential medical applications. there is growing interest in new inhibitors of proteases from various sources. among known protease inhibitors from fungi are, yeasts inhibitors of proteinases a (asparagine protease) and b (serine protease), and low molecular inhibitors of serine proteinases from fruiting bodies of mushrooms -pleurotus ostreatus and lentinus edodes as well as some undefined proteinase inhibitory activities from water extracts of some species of basidiomycetes. searching for new, bioactive metabolites of basidiomycetous fungi we isolated and characterized recently some low molecular, proteinaceous, natural inhibitors of serine proteases, from mycelia of wood rotting fungi -trametes versicolor, abortiporus biennis and schizophyllum communae. isolation of inhibitors was achieved by ion exchange and size exclusion chromatography. preliminary characterization of their inhibitory activity (against some serine proteases), ph and temperature optima of action, and molecular mass, were classically analyzed. analysis of n-terminal amino acid sequences of these inhibitors suggests a new family of serine protease inhibitors from fungi. more detailed characterization of inhibitors (including molecular modeling) and preliminary experiments with laboratory animals and with lines of human cells are in progress. the role of serine proteases in the lectin pathway of complement activation p. ga´l 1 , g. ambrus 1 , v. harmat 2 , b. ve´gh 1 , g. na´ray-szabo´2, r. b. sim 3 and p. za´vodszky 1 1 institute of enzymology, hungarian academy of sciences, budapest, hungary, 2 protein modeling group, hungarian academy of sciences, budapest, hungary, 3 department of biochemistry, university of oxford, oxford, uk. e-mail: gal@enzim.hu the complement system is a cascade of serine proteases, and mediates essential functions during infection as a part of the innate immunity. activation of the complement system culminates in the destruction and clearance of invading microorganisms and damaged or altered host cells. our view about the complement system has changed considerably in the recent years, due to the discovery of a new activation pathway of complement: the lectin pathway. we have recombinantly expressed and characterized the mannose-binding lectin associated serine proteases: masp-1 and masp-2. these are related mosaic serine proteases with similar domain organization but with different enzymatic properties. we showed that masp-2 is capable of autoactivation and it can cleave c2 and c4 complement subcomponents. masp-2, therefore, can initiate the complement cascade without the contribution of any other protease. we demonstrated that the complement control protein (ccp) modules, which associate directly with the serine protease domain, stabilize the structure of the catalytic region masp-2 and contain exosites for the large protein substrates. these results are in agreement with the crystal structures of activated and zymogen forms of masp-2. masp-1 is the most abundant mbl-associated serine protease but it cannot activate the complement system. we demonstrated that masp-1 has a more relaxed substrate specificity compared to masp-2 and the activity of both proteases can be blocked by c1-inhibtor. we concluded that the two mbl-associated serine proteases participate in evolutionary and functionally different pathways. comparative kinetic study on s2' trypsin variants l. gombos, j. tó th, p. medveczky, a. ma´lna´si csizmadia and l. szila´gyi laboratory of enzymology, department of biochemistry, eo¨tvo¨s lora´nd university, budapest, . e-mail: gl@ludens.elte.hu by far the most serine proteases have a glycine in position 193, which is part of the s2' subsite (the second subsite on the enzyme surface c-terminal from the scissile bond of the substrate). in contrast, human trypsin 4, the trypsin isoform expressed in human brain, possesses an arginine in that position. the bulky side chain of this amino acid is responsible for the inhibitor resistance, the most striking feature of this isoform, as it interferes with the binding of polypeptide inhibitors to the enzyme surface. a chimpanzee typsin also has an arginine193, while rat trypsin v bears a tyrosine in that position. there is also a snake venom plasminogen activator, a trypsin type serine protease, that contains an s2' phenilalanine. we created glycine, arginine, tyrosine and phenilalanine s2' variants of human and rat trypsins by site directed mutagenesis in order to investigate the effect of these amino acids on the kinetic behaviour. on small chromogenic substrates and synthetic inhibitors, which do not interact with the s2' residue, there is no signifi-cant difference between the various mutants in catalytic efficiency and inhibitory constants, respectively. however, on oligopeptide substrates the catalytic efficiency decreases 20-50-fold in the nonglycine variants. this effect is even more dramatic with polypeptide partners: the catalytic efficiency drops 200-500 times while inhibitory constants increase by 3-5 orders of magnitude. we conclude that the catalytic mechanism is not fundamentally influenced by the substitution of residue 193, although this amino acid is part of the oxyanion hole. bulky residues in the s2' subsite hinder mainly the binding to interaction partners. structural studies on masp-2: towards the understanding of the mechanism of autoactivation mannose-binding lectin-associated serine protease 2 (masp-2), is the key enzyme of the lectin activation pathway of complement, a major element of innate immunity. a dimer of masp-2 complexed with mannose-binding lectin (mbl) is able to perform its biological functions: upon recognition of the pathogen by mbl masp-2 undergoes autoactivation, and then initializes the complement cascade by cleaving c2 and c4. masp-2 is a mosaic protein containing a chymotrypsin like serine protease domain (sp) and further domains with binding sites of mbl or substrates. our present study focuses on the structural background of the ability of the zymogen form of masp-2 to undergo autoactivation. we solved the structures of catalytic fragment of masp-2 both in its zymogen and activated forms. comparison of the two structures reveals characteristic conformational differences in the classical activation domain and in some other loops lining the substrate binding region. loop 1 shows a unique conformation with arg192 blocking the s1 pocket. we docked the activation loop of masp-2 in the active site of the active enzyme and built a model of the complex of the active and zymogen forms. the model reveals extended regions of molecular recognition. while this model represents the second step of autoactivation (active form cleaves zymogen), the first step (zymogen cleaves zymogen) requires the stabilization of the zymogen enzyme in active-like conformation. we built a model of a zymogen-zymogen complex. favorable and unfavorable contacts of the two zymogen molecules help us to identify possible molecular switches, as well as contact regions stabilizing an active-like conformation of the zymogen enzyme in the complex. the deg/htra proteases are atp-independent serine endopeptidases which are present in most organisms, including bacteria, humans and plants. previous work in our laboratory has shown that the deg2 protease of the model plant arabidopsis thaliana selectively degrades the photodamaged d1 protein in the reaction center of photosystem ii (psii) in vitro. therefore, deg2 is thought to catalyze the primary cleavage of photodamaged d1 protein, which is an important step of the repair mechanism that restores functional psii. our present studies aim to elucidate the regulation of the deg2 protease activity, especially with regard to its d1 degrading activity. we found deg2 associated to the stromal side of the thylakoid membranes and as a soluble protein in the chloroplast stroma. the amount and distribution of deg2 protein remained unchanged after exposure to different light intensities, which suggest either a substrate regulation or a posttranslational regulation of the d1 degrading activity of deg2. recent advances on deg2 regulation and complex formation will be presented. novel peptide inhibitors of human kallikrein 2 (hk2) human kallikrein 2 (hk2) is a serine protease produced by the secretory epithelial cells in the prostate. it activates several other proteases that may participate in the proteolytic cascade mediating metastasis of cancer. thus, modulation of hk2 activity is a potential way of preventing tumor growth and metastasis. furthermore, specific ligands for hk2 may be potentially useful for targeting and imaging of prostate cancer. we used enzymatically active recombinant hk2 captured by a monoclonal antibody exposing the active site of the enzyme to screen phage display peptide libraries. six different peptides binding to hk2 were identified using libraries expressing 10 or 11 amino acids long linear peptides. three of these peptides were specific and efficient inhibitors of the enzymatic activity of hk2. alanine substitution analysis revealed that motifs of 5-7 amino acid determined the inhibitory activity of the peptides. the peptides are also of potential utility for development of immunopeptidometric assays for hk2, which is promising marker for diagnosis of prostate cancer. furthermore, these peptides are potentially useful for treatment and targeting of prostate cancer. the mechanism of autoactivation of the zymogen masp-2 residues on the surface of pathogens. we managed to recombinantly express and purify two forms of zymogen masp-2. one form is the wild type zymogen enzyme, which can be activated, while the other one is a stable zymogen mutant form of masp-2. we could prepare the zymogen form of wild type masp-2 under certain conditions which enabled us to examine the kinetics of activation. we demonstrated that activation of masp-2 is a true autocatalytic activation without the involvement of any other protease. we characterized the enzymatic properties of zymogen masp-2 using the stable zymogen form. we demonstrated that zymogen masp-2 cannot cleave small synthetic substrates but it can cleave large protein substrate (c4). a molecular model for the interaction between zymogen and activated masp-2 during activation has also been built based on the available 3d structures of zymogen and activated masp-2. influence of streptokinase on the fibrinolytic system proteins the present study is dedicated to the investigation of the effect of protein by bacterial origin -streptokinase (sk) on the activity and interaction regulation mechanisms of fibrinolytic system proteins. the study was carried out with use of porcine haemostasis system which plasminogen isn't activated by sk. especially we were interested in study of the changing fibrinolytic system parameters such as tissue type plasminogen activator (t-pa), plasminogen activator inhibitor (pai-1), plasminogen, a2-antiplasmin activities. also the main parameters of coagulation system such as fibrinogen, soluble fibrin, fibrin degradation products levels and thrombin activity and quantity were studied. it was used affinity chromatography, electrophoresis, western-blotting, elisa, determination of proteins activity. it has been determined an increased consumption of plasminogen on 15% in 4 h after streptokinase injection. it was shown that activity and concentration of t-pa were significantly increased in 3.5 times in 1 h. on the next stages of investigation this parameters tend to norm. after sk injection pai-1 quantity was increased in two times (16.7 ng/ml compared to normal 8.9 ng/ml). the interesting fact was the activation of prothrombin by sk without activation of coagulation system in vivo. the injection of sk causes the significant increase of t-pa activity and quantity possibly due to direct or/and indirect effect on endothelial cells. we can conclude that sk causes pai-1 secretion due to effect on platelets as 90% of pai-1 storage is in a-granules of platelets. thus analysis of the data displayed besides of well-known sk function the influence of sk on the changing of fibrinolytic system potential possibly due to its effect on endothelial cells and platelets. paracrystalline inclusions in the mitochondrial matrix or intermembrane compartment occur in several biochemically unrelated disorders such as myopathies, paragangliomas and steatohepatitis, and in various cell types under normal conditions, as well. however, little is known about the composition of the inclusions, the mechanism of their formation and their relation to disease processes. in this study we have described the helix-shaped structures in the intracristal compartments of rat liver mitochondria that have undergone ca 2+ -induced permeability transition. the filaments are anchored in opposing parts of the mitochondrial membranes and appear to support the cristae mechanically. a protein, that apparently is a component of these helical filaments, has been identified as serine protease lactb. this protein shows close sequence similarity to the class c bacterial beta-lactamases and is the only member of this class in animals. since lactb has not been studied previously we cloned its cdna for expression in e. coli as c-terminal his-tagged fusion protein. lactb underwent proteolytic processing in both e. coli and in isolated mitochondria resulting in several protein fragments. this is likely to be due to autocleavage and may be an activation/maturation process. 2d blue native gel electrophoresis indicated that lactb was part of a >600 kda protein supercomplex. in summary, the presence of the serine protease motive in lactb and its supposed ability to form helical filaments suggest that lactb might function not only as a component of 'mitoskeleton' in maintaining and rearranging the mitochondrial ultrastructure under certain conditions, but also might take part in apoptotic processes. novel psychrophylic trypsin-type protease from serratia proteomaculans proteinase with trypsin specificity from psychrophylic microorganism serratia proteomaculans was partly purified. it was shown that the properties of this enzyme (temperature and ph-stability, efficiency of substrate hydrolysis) correspond with the psychrophylic character. inhibitor analysis and study of substrate specificity indicate that this enzyme is serine trypsin-type protease. at the same time this enzyme is zinc-dependent. proteases of such type were unknown till now. secondary specificity of the studied enzyme differs from the bovine trypsin specificity -this protease hydrolyses the short substrates more efficient. zinc, cadmium (ii) and copper (ii) ions in mmolar concentrations inhibit the enzyme activity. the unusual character of calcium ions influence on substrate hydrolysis and inhibition by the bovine pancreatic trypsin inhibitor (bpti) was registered for the studied enzyme. in vitro by a neutral to basic ph change [2, 3] . the kinetics of the activation process can be followed by stopped flow fluorescence (sff) experiments while the structural features of the transition can be explored by in silico molecular dynamics (md) and targeted molecular dynamics (tmd) [4] simulations. to challenge the activation process, mutants were constructed and studied by sff measurements. subsequently, on these mutants multiple md/tmd simulations were carried out. our results indicate the existence of parallel activation pathways. they demonstrate the absolute necessity of multiple simulations and of proper statistics. they reveal the pros and cons of the tmd method. a simple method for the purification of a novel serine endoprotease from wheat triticum aestivum (cv. giza 164) has been developed. it consists of ion-exchange and gel filtration. the molecular mass of the enzyme was 58 kda by sds/page under reducing conditions and 57 kda by gel filtration on a sepharose 6b column. the enzyme had isoelectric point and ph optimum at 4.2 and 4.5, respectively. the substrate specificity of the enzyme was studied by the use of synthesized and natural substrates, azocasein, azoalbumin, hemoglobin, casein, gelatin and egg albumin. the enzyme appears to prefer azocasein with km 2 mg azocasein/ml. the enzyme had a temperature optimum at 50°c with heat stability up to 40°c. while co 2+ and mg 2+ accelerated the enzyme activity by 54 and 56%, respectively, ca 2+ and ni 2+ had very little effect. the enzyme was strongly inhibited by phenylmethylsulphonyl fluoride (pmsf), but not by the other protease inhibitors, suggesting that the enzyme is a serine protease. from the results it can be concluded from the characterization that the t. aestivum serine protease may be suitable for food processing. in vitro effects of a potent, selective dipeptidyl peptidase ii (dppii) inhibitor in leukocytes and u937-cells. the compound was able to penetrate the cell membrane and proved efficacy without evidence for acute cellular toxicity. there was a dosedependent inhibition of intracellular dppii activity without affecting the dppiv activity (maximal efficacy at 100 nm). these properties enable to differentiate between dppii and dppiv in biological systems and allow further investigation of the physiological function of dppii. in a second step, we have been investigating the involvement of dppii in apoptosis in human leukocytes by using this compound. preliminar results based on annexin v-/pi-staining using up to 1 lm inhibitor in u937-cells and pbmc did not show signs of apoptosis while dppii activity was inhibited for 90%. effect of calcium ions on hydrolysis of peptide substrates of general formula a-(asp/glu) n -lys(arg)-b, catalyzed by enteropeptidase (ec 3.4.21.9), differs depending on substrate type. for specific enteropeptidase substrates (n = 4) calcium ion exhibits the promotion of hydrolysis by the natural two-chain enteropeptidase. hydrolysis of atypical enteropeptidase substrates (n = 1-2) is as a rule less efficient; in addition calcium ion shows in this case the inhibition influence. therefore the regulation of the nondesirable side-hydrolysis during full-length enteropeptidase-catalyzed chimeric proteins processing is possible by means of calcium ions. on the contrary the hydrolysis of substrates of all type (n = 1-4) by enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 or 784-800 fragments) is inhibited by calcium ions. hydrolysis of the natural enteropeptidase substrate, trypsinogen, is at least two orders of magnitude more efficient than any artificial substrate hydrolysis. we propose that this effect is caused by participation in trypsinogen coordination with enzyme of the addition secondary substrate binding site and/or calcium-binding site; both sites located on the n-terminal half (118-465) of the enteropeptidase heavy chain. one more mechanism of the regulation of the enteropeptidase activity by calcium ion is the unusual calciumdependent autolysis of the enteropeptidase heavy chain leading to the drastic loss of its activity towards trypsinogen. autolysis of enteropeptidase heavy chain and well-known autolysis of trypsin were compared; the second one serves as the natural defense mechanism against the undesirable premature proenzymes activation in pancreas leading to pancreatitis. the corresponding enteropeptidase inactivation in low ca 2+ ion environment might be the component of the same protective mechanism. b3-034p human trypsin 4 selectively cleaves myelin basic protein: is this brain protease involved in the pathomechanism of multiple sclerosis? demyelination, the breakdown of the major membrane protein of the central nervous system, myelin is involved in many neurodegenerative diseases. proteases participating in this process are potential targets of therapy in neurodegenerative diseases. in the present in vitro study the proteolytic actions of calpain, human trypsin 1 and human trypsin 4 (the product of gene prss3) were compared on lipid-bound and free human myelin basic protein as substrates. digestions only with calpain and human trypsin 4 actions may be of some physiological or pathological relevance, since these two are expressed in human brain. the fragments formed were identified by using n-terminal amino acid sequencing and mass spectrometry. the analysis of the degradation products showed that human trypsin 4 of these three proteases cleaved myelin basic protein most specifically. it selectively cleaves the arg80-thr81 and arg98-thr99 peptide bonds in the lipid bound form of human myelin basic protein. based on this information we synthesized region 94-104 of myelin basic protein, peptide ivtprtpppsq that contains the specific trypsin 4 cleavage site arg98-thr99. in vitro studies on the hydrolysis of this synthetic peptide by trypsin 4 confirmed our results with intact myelin basic protein. what lends some biological interest to the above finding is that the major autoantibodies found in patients with multiple sclerosis recognize sequence 80-96 of the protein. our results suggest that human trypsin 4 may be one of the candidate proteases involved in the pathomechanism of multiple sclerosis. enteropeptidase is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of its activation peptide following the sequence asp-asp-asp-asp-lys. its light chain alone is sufficient for an effective cleavage of fusion proteins with trypsinogen activation peptide analog. human enzyme possesses 10-fold specificity coefficient compare to bovine one, and an explanation of this fact can contribute a lot to the attempts of improving or modulating enzymatic properties. highly pure and active recombinant human enteropeptidase light chain (l-hep) was obtained by renaturation from inclusion bodies expressed in escherichia coli cells and the active l-hep was purified on agarose-linked soybean trypsin inhibitor. enzymatic activity of purified l-hep was studied through the cleavage of the synthetic peptide substrates and several fusion proteins. l-hep associated with soybean trypsin inhibitor slowly and z-lys-sbzl cleavage was inhibited with ki* = 2.3 nm. comparison of l-hep and bovine enteropeptidase inhibition by bovine trypsin inhibitor aprotinin has shown almost an order difference in ki*. ph dependence of the enzyme activity was measured and ph optimum point was found to be 7.54. enteropeptidase light chain amino acid sequence and crystal structure were analyzed for the presence of target regions for mono-and bivalent ions. unlike trypsin with predicted and experimentally proved calcium-binding sites and sodium-activated thrombin, l-hep was predicted to be deprived of any of such sites and an influence of these ions on the cleavage of different substrates was found to be confined primarily to a substrate binding. as a continuation of our efforts to fully elucidate the antisnake venom properties of mucuna pruriens and to further understand the molecular changes that occurred in mouse plasma proteome as a result of in vivo challenge test with venom and mucuna pruriens proteins (mpe), two dimensional polyacrylamide gel electrophoresis was done. plasma was pooled and gels were run in triplicate to eliminate both biological and experimental variations. analysis using imagemaster 2d platinum software and other statistical analysis tools showed significant differences in protein expression between all the treatments and the control group. some proteins were down regulated, some up-regulated, some completely disappeared while new protein spots were identified. the protein expression of plasma of mouse immunized with mpe for 3 weeks before challenge with lethal dose of venom and that injected with venom alone was more complex. some venom proteins like ecarin are serine proteases that activate clotting factors like prothrombin, causing haemorrhage and disseminated intravascular coagulation, on the other hand, the protease inhibitors from mucuna pruriens must have acted to antagonize these effects by direct proteolysis (cleavage products/spots appearing in the protein map) or other immunological mechanisms. the results obtained represents the first proteomics approach in studying all the plasma proteins involved in this phenomenon. we have only concentrated on protein spots showing interesting variations with respect to control. it is also an important step in the identification of the affected proteins, the kind of modifications/molecular mechanisms involved which is likely the basis of the in vivo protection the plant extract showed against the venom. the use of enzymes at low temperatures has great potential in terms of lower energy costs, therapeutic applications and to lower microbial contamination in industrial processes. low temperature proteases (cryophilic -or psycrophilic -proteases) are of particular interest for detergents and as wound debriding agents. at present, we are studying cryophilic proteases from antarctic krill (euphausia superba), which normally lives in the sea at temperatures near 0°c. we have isolated several low temperature proteases by chromatography. enzyme activities and stability were characterized at low temperatures and as a function of ph to find optimum conditions for different applications. a particular enzyme, named kt1, showed particularly high specific activity at 20°c, several times that of commercial preparations of proteases such as subtilisins. this protein showed a high degree of similarity with digestive trypsins isolated from various arthropoda species. using mrna molecules obtained from abdominal sections of e. superba and subsequently subjected to a reverse-transcription reaction, we identified, isolated and sequenced a dna molecule that codes for an inactive zymogen of the enzyme. cloning of this dna sequence in escherichia coli strains allowed the recombinant expression of the zymogen, followed by purification and activation of the zymogen, which lead to an active cryophilic trypsin. we performed a homology modeling procedure that conducted us to obtain a molecular model of the mature enzyme. the 3d model thus obtained was refined using energy minimization, hydrogen network optimization and residue-residue contact optimization techniques, leading to a reliable model of the enzyme. we used this model to identify many interesting and novel features of the enzyme molecule that could be related with its cryophilic character, and to propose site-directed mutagenesis strategies that could be used to improve the enzyme performance at low temperatures, its ph-activity profile, specificity, inactivation resistance and recombinant expression. in addition, the 3d model allowed us to design and experimentally obtain mutants that are resistant to auto-degradation and more readily activated. molecular cloning and expression of lactba mitochondrial serine protease mitochondria are thought to have originated from a symbiotic relationship between a bacterium able to perform aerobic metabolism ant the ancestor of eukaryotic cells. lactb is the only mammalian protein showing sequence similarity to bacterial serine proteases and belongs to c class b-lactamases. mouse lactb is 551 amino acids long and compromises a predicted mitochondrial import sequence, a short putative transmembrane segment, a b-lactamase homology domain containing the serine protease motif, -sxxk-, and a c-terminal d-transpeptidase domain. the physiological role of mammalian lactb is unclear. therefore, the purpose of this research work was to clone the gene of lactb for expression of lactb in e. coli for further biochemical and cell biological study. the full length lactb gene was cloned into the entry plasmid pentr/sd/d-topo. expression clones were created performing a recombination reaction between the entry clone and four destination vectors. expression constructs resulting in n-or c-terminal gst fusion protein and in n-or c-terminal his6-tag fusion protein were transformed into bl21 (de3) competent cells which are designed for use with bacteriophage t7 promoter based expression systems. when lactb was expressed as an n-terminal gst fusion protein, full-length lactb protein was recovered by glutathione-agarose affinity chromatography. expression of lactb as a c-terminal gst fusion protein or with either an n-or c-terminal his6-tag resulted in proteolytic degradation of the protein and we were not able to detect full-length lactb. these results show that the n-terminal gst fragment protects lactb from proteolytic processing and that lactb can undergo autoproteolysis, which may be a part of a physiological maturation or activation process. design and synthesis of retro-binding peptides active site inhibitors of thrombin thrombin is an important pharmaceutical target for the treatment and prevention arterial and venous thrombosis. biological active peptides are recognized to have significant therapevtic potential but serious limitations especially for oral dosing. the peptide stereomers could differ when forming productive complexes with an enzyme. moreover, the replacement of l-amino acid residues forming the hydrolyzed p1-p'1 bond by their enantiomers is known to result in either an uncleavable or a very slowly hydrolyzed analogue. this phenomenon is often used for the synthesis of the peptide's inhibitors stable to the degradation by the enzymes of organism. as the peptides containing d-amino acids, nor are subject to an enzymatic hydrolysis, the purpose of researches was synthesis of a retro -d-analogues of thrombin's substrates constructed from d-amino acids. the di-and tripeptides of the general formula x-d-arg-d-phe-ome [where x = z, tos, ac h, and z-d-arg-d-ala-(d), l-phe-ome (otbu)] were synthesized by conventional methods of peptide synthesis in solution. special features of their interaction with thrombin are investigated. their inhibitory action on reaction of splitting of fibrinogen by thrombin and on reaction of a hydrolysis by thrombin baee showed, that their inactivating action depends on the substituent on n-end of dipeptides and configuration of phenylalanine in a molecule of tripeptides. the relationship between structure and inhibitory action of the synthesized peptides is discussed. the successful application of d-amino acids for designing of biologically active peptide's analogues as a potential medicinal agent, steady to enzymatic degradation is shown. substrate specificity of mannose lectin binding associated serine proteinase 3 n. s. quinsey and r. n. pike department of biochemistry and molecular biology, monash university, melbourne, victoria australia. e-mail: noelene.quinsey@med.monash.edu.au the innate complement system is involved with the neutralization of pathogenic microorganisms. it plays a comparative role to that of the classic immune complement cascade. in the innate complement system, the oligomers of mannose lectins are able to bind to microorganisms. these oligomers have been shown to have mannose lectin binding serine proteinase (masps) attached, which once activated lead to the activation of the c3 convertase complex, which finally leads to the formation of the membrane attack complex. there have been three active masps identified in the human innate immune system-masp-1, masp-2 and masp-3. there is high homology between these three serine proteases especially in the n-terserpins are protease inhibitors that present their reactive site loop (rsl) to target proteases, followed by drastic conformational changes that inactivate the protease. the sequence of the rsl of serpins determines the target specificity. the drosophila melanogaster gene spn4 encodes multiple serpin isoforms each containing an individual rsl, thus enabling the attack of different proteases. variant spn4a contains a consensus recognition/cleavage sequence of furin within its rsl and is equipped with a signal peptide and an endoplasmic reticulum (er) retrieval signal (hdel). this suggested that the protein resides in the secretory pathway, like furin, a proprotein convertase that activates many cellular proteins and pathogens. our experiments demonstrate that spn4a forms sds-stable complexes with human furin that is inhibited with a second order rate constant of 5.5 · 10 6 /m/s. the rsl of spn4a is cleaved c-terminally to arg-arg-lys-arg, in accord with the enzyme's cleavage site. furthermore, the serpin is retained in the er of transfected cos7 cells as shown by immunofluorescence staining. a hdel deletion mutant was detected mainly in the medium of trans-fected cos7 cells, demonstrating the necessity of the hdel signal for the observed cellular localization. further experiments show that furin 1 and 2 of drosophila melanogaster are physiological targets for spn4a, since secreted forms of both enzymes form stable complexes with the serpin. together, the results demonstrate that spn4a is a potent inhibitor of furin that may meet the target at its natural location. experiments with the other rsl variants show that the spn4 gene represents a multipurpose weapon that is directed against different families of proteases. formation of the covalent tetrahedral complex (tc) with substrate is the first step of the catalytic process in the active site of serine proteases. his57 (chymotrypsin numbering) plays a role of a general base catalyst, activating the ser195 nucleophile by abstraction of its proton. it was experimentally observed that the pka of his57 ne in tc formed by serine proteases with transition state analog inhibitors is about 5 units higher than the corresponding pka in the free enzyme. this work demonstrates that the environmental change of the his57 in tc, induced by the substrate binding in the enzyme active site, is the dominant factor in the pka increase of his57 ne, and triggers the enzymatic processing of the substrate. these results are based on quantum mechanical modeling of the active site of free chymotrypsin and tc complex of chymotrypsin with trifluoromethyl ketone inhibitor in dft b3lyp/6-31+g** level of theory. the polar environment of the enzyme active site is accounted for explicitly in the microscopic model. the combined environmental effects of the bulk water solvation and the rest of the protein is implicitly accounted for by our scrf(vs) continuous solvation approach. the role of local polar effects, such as the oxyanion and the asp102-his57 hydrogen bond, on the pka of his57 ne in tc is analyzed. genome-wide analysis of subtilase (subtilisinlike serine protease) genes in microbial genomes limited to regions surrounding the asp, his and ser catalytic residues. pattern-searching methods using hidden markov models, based on conserved sequences surrounding the catalytic residues, were used to search for subtilases encoded in >200 bacterial and archaeal genomes, representing 177 species. more than 350 subtilases were found to be encoded in 109 genomes. subtilases are more commonly found in grampositive bacteria than in archaea or gram-negative bacteria, and it is more common to have multiple subtilase-encoding genes than a single gene. the majority of the subtilases have a predicted signal peptide for translocation across the cell membrane, and a sub-group of these secreted subtilases are predicted to have a carboxy-terminal cell-envelope anchor, mainly of the lpxtg type for covalent anchoring to peptidoglycan. the genomic context of the subtilase-encoding genes was analyzed to gain insight in putative functions for these proteolytic enzymes. by also taking into account the predicted intracellular or extracellular location of the encoded subtilases, it was possible to predict a function for many subtilases in either nutrition/growth, spore germination, surface protein processing/activation, bacteriocin/toxin processing, or sigma factor activation/regulation. the poisoning by botropics species makes a similar physiologic, one of systemic effects is the blood coagulation for several mechanisms, as direct action on fibrinogen; factor x activation or platelet activation, by toxins of venoms. in the last years were identifies in botropics venoms, serine proteases. this toxins are responsible by coagulant activity with direct action on fibrinogen. serine proteases are utility for hemostatic system studies and for therapeutics use. looking for new molecules models is very important to show the mechanism of action and search structural characteristics responsible for its activities. the present work has the objective of purification and characterization of a coagulant factor (cf) from b. pirajai venom. the purification was made using a gel filtration, hydrophobic chromatography and an affinity chromatography. the molecular filtration was made in sephadex g-75 with ammonium bicarbonate buffer (ambic) 0.05 m ph 8.1, resulting four fractions (p1-p4), the coagulant fraction was named p1. the p1 fraction was submitted in phenyl sepharose chromatography using triz buffer 10 mm ph 8.6 in a decreasing gradient of nacl (4; 3; 2; 1; 0.5; 0 m), and to finish the chromatography it was used distilled water, resulting six subfractions (fp1-fp6), the coagulant subfraction was named fp1. the fp1 sub fraction was submitted in benzamidine sepharose chromatography and eluted in the solutions: distilled water, obtained the subfraction bfp1, sodium phosphate buffer 20 mm ph 7.8, obtained the subfraction bfp2 and glycine buffer 20 mm ph 3.2, obtained the sub fraction bfp3 that is the cf. the cf displayed one band in sds-page (11%) showing a pure protein, it has 58 kda, the minim coagulant dose is 1.75 lg and has action on fibrinogen beta chain. the genome of arabidopis thaliana encodes 16 putative proteases from the deg/htra family. this group of atp-independent serine-proteases was well examined in other organisms, especially e. coli and humans, but only limited data is available for members from this protease family in plants. deg1 and deg2 have been shown to act as proteases in the chloroplast, but no deg/htra proteases from other compartments have been examined so far. the putative protease deg15 is predicted to be localized in the peroxisome. we cloned the gene encoding deg15 (at1g28320) in an overexpression vector for heterologous expression in e. coli. the tagged protein was purified by affinity chromatography and used to raise polyclonal antibodies. with these antibodies we investigated the intracellular localization of deg15 and the protein level under various stress conditions in order to evaluate the in planta function of this protein. the effect of site-directed mutagenesis on cold adaptation of vpr; a subtilisin-like serine proteinase from a psychrophilic vibrio-species psychrophilic enzymes have very similar 3d structures as their homologous enzymes from mesophilic and thermophilic organisms. main characteristics of enzymes from psychrophiles are their high catalytic efficiency (kcat/km values) and thermolability. a subtilisin-like serine proteinase from a psychrophilic vibrio-species (vpr) shows these characteristics when compared to homologous enzymes from mesophilic and thermophilic organisms. the vpr gene was cloned, sequenced and expressed in e. coli and recently the crystal structure was determined at 1.84 å resolution [1] . structural comparisons have been carried out which have led to hypotheses about some of the structural factors which may contribute to cold adaptation of vpr. some of these hypotheses have been examined using site-directed mutagenesis. the specific residue exchanges were selected with the objective to incorporate stabilizing interactions into the cold adapted enzyme which were deemed to be present in related thermostable homologues. these include incorporation of pro into loops, a new potential salt-bridge, as well as substitutions aimed at improving packing in the hydrophobic core and decreasing apolar exposed surface. we have also introduced ser to ala substitutions at three different locations in the cold-adapted enzyme, but these were the most frequent amino acid exchanges observed in sequence comparisons of the enzyme to those of more thermostable homologues. here we report on the catalytic and stability characteristics of the selected mutants. engineering of gfp for the screening of serine protease inhibitors site specific proteolysis has been an attractive target for the development of antiviral therapies based on selective viral inhibitors. it has been previously demonstrated that reporter proteins like beta-galactosidase could be very useful for the high-throughput screening of hiv-1 protease inhibitors through the display of an accessible protease target site on the enzyme surface. in this work, by using structural analysis, we have engineered the gfp protein from jellyfish aequorea victoria to accommodate in its surface the hcv virus ns5a-5b protease cleavage site edvvccsmsytwtg, in a manner that proper proteolysis results in a fluorescent activity decrease. the three resulting gfp constructions, carrying the protease cleavage site in positions 23-24, 102-103 and 172-173, were soluble expressed in escherchia coli. moreover, the hcv ns4 cofactor residues 21-34 fused in frame via a short linker to the amino terminus of the hcv ns3 protease domain (residues 2-181) were also expressed in e. coli and under 1mm iptg induction, at least 60% of soluble protein was recovered and further purificated by an histidin tag. the analysis of gfp proteolysis in front of hcv recombinant protease were performed either with bacteria crude extracts and purificated proteins. the results presented here indicated that proper solvent exposure of target sites on gfp carrier protein may be a critical factor for protease cleavage and for the observation of fluorescence activity variance, being an aspect of absolute relevance for further design and implementation of newer analytical tests. various kinds of stressors cause the group of metabolic changes defined as the general stress response, initiated by some intracellular signals, such as production of abnormal or denaturated proteins, enhanced generation of reactive oxygen species and others. proteolytic enzymes quickly modify proteins and as a consequence can regulate cellular metabolism. although the stress defense mechanisms have been very often described in the recent literature, in very few works were estimated stress response abilities of white-rot basidiomycetes, which produce two kinds of very important ligninolytic enzymes -laccase and peroxidases. our previous results showed that the addition of menadione to abortiporus biennis idiophasic cultures caused the significant increase of the extracellular laccase activity in comparison to the control. the aim of this study was to determine activities of serine proteinases and natural serine proteinase inhibitors in idiophasic cultures of basidiomycete a. biennis grown under menadione-mediated oxidative stress conditions. we investigated the changes of intracellular serine proteinases activities in the presence and absence of atp, using hemoglobin and fluorogenic substrates. the level of natural serine proteinase inhibitors in mycelia was also measured. a fungal inhibitor of trypsin was partially purified and used to in vitro experiments. an interesting correlations between serine proteinases, serine proteinase inhibitors and laccase activities in prooxidant treated cultures were also observed. it can suggest that the proteolytic modifications under oxidative stress conditions can act as a regulation way of laccase activity. serine proteinases, inhibitory and laccase activities were additionally analyzed by native page. calpain is a ca 2+ -regulated cytosolic cysteine protease, functioning as a ''modulator protease'', i.e. regulating/modifying functions/activities of substrates by limited proteolysis to modulate cellular functions. human has 14 calpain genes and potential substrates extend to various cytosolic proteins such as kinases, transcription factors, cytoskeletal and er proteins. in skeletal muscles, expression of p94 (also called calpain 3) predominates, playing an indispensable role for muscle functions in cooperation with ubiquitously expressed conventional calpains. for, a defect of p94 proteolytic activity originated from gene mutations causes muscular dystrophy. p94 localizes in myofibrils binding to connectin/titin, a gigantic elastic muscle protein connecting the z-and m-lines of sarcomere, the repetitive unit of myofibril, with a single molecule. in mdm (muscular dystrophy with myositis) mice, connectin/titin with a small deletion caused by natural mutation of the connectin/titin gene is expressed, resulting in severe muscular dystrophy phenotypes such as body weight less than a half of that of wild type, severely affected limb muscles with impaired walking ability and only 2-3 months of life time. the deletion in the mdm allele of the connectin/titin gene overlaps one of the binding sites of p94 in the n2-line, another electron-microscopically visible line between the z-and m-lines of sarcomere. the mdm phenotypes clearly indicate that connectin/ titin or p94 or both are essential for proper muscle functions. to elucidate physiological roles of connectin/titin and p94, we analyzed mdm mice in relation to calpain system. as a result, mar-ps (muscle ankyrin repeat proteins) were shown to be up-regulated in mdm muscle. marps bind to the n2-and z-line regions of connectin/titin and function as transcriptional regulators translocating into the nuclei. carp (cardiac ankyrin repeat protein), one of marps, binding site in the n2-line region is proximate to the p94 binding site, thus suggesting interactions of both molecules. possible signal transduction systems to modulate muscle functions revealed by the analyses will be discussed based on the results. inhibition and activation of calpain by its disordered endogenous inhibitor, calpastatin p. tompa 1 , z. mucsi 2 , o. gyo¨rgy 2 , c. sza´sz 1 and p. friedrich 1 1 institute of enzymology, biological research center, budapest, hungary, 2 research group of peptide chemistry, university of eo¨tvo¨s lora´nd, budapest, hungary. e-mail: tompa@enzim.hu calpains are a family of intracellular calcium-activated cysteine proteinases, implicated in the regulation of key cellular processes, such as cell division and programmed cell death. their activity is under tight control by an intracellular protein inhibitor, calpastatin, an intrinsically unstructured protein that contains four equivalent inhibitory domains. each of these comprise three conserved subdomains, of which subdmomains a and c anchor the inhibitor in a calcium-dependent manner, whereas subdomain b binds at the active site and inhibits the enzyme. in this work it is shown that the consequence of this mode of binding is that isolated a and c peptides promote calcium binding to calpain and thus activate the enzyme. this activation is manifest in the sensitization to calcium ion: the calcium required for half-maximal activity is lowered from 4.3 to 2.4 lm for l-calpain and 250 to 140 lm for mcalpain. in the physiologically significant sub-micromolar and low micromolar calcium concentration range this sensitization leads to a more than tenfold activation, which is of potential physiological importance as isolated calpain requires high calcium concentrations never realized in vivo. here we suggest calpastatin is degraded in vivo in a way that generates the activator peptides. due to the structural disorder of calpastatin, this unprecedented mode of action raises intriguing questions with respect to the generality of this ambivalent behavior. to address this issue, we have collected extreme cases, when the same protein elicits opposing, inhibitory and activatory, responses within the same molecular setting: structural predictions show that these proteins are largely disordered. as a conclusion, the possible general implications of this finding are discussed. meprins are oligomeric, brush border membrane or secreted zinc proteases that have unique and complex structures. they are composed of multidomain, highly glycosylated evolutionarily-related a and b subunits that form disulfide-linked homo-or heterooligomeric dimers. the homooligomeric form of meprin a forms very high molecular mass multimers of 1 000 000-6 000 000 da, among the largest extracellular proteolytic complexes known. meprins cleave cytokines, growth factors, bioactive peptides and extracellular matrix proteins, important compounds in inflammatory intestinal disease and in cancer metastases. to investigate the role of meprins in intestinal immune responses, inflammation was induced in mice by oral administration of dextran sulfate sodium (dss). the results showed that wild-type mice (c57bl/6 · 129) had a more severe reaction to dss than meprin b null mice on the same genetic background, as determined by body weight loss, intestinal bleeding and mortality. this implies that the presence of meprin b increases host damage caused by dss and that meprin b plays an active role in intestinal pathophysiology. meprins are also expressed in colon cancer cells (e.g. sw480, sw620, and caco-2). expression of meprin a appears to increase with increasing metastatic potential. in addition, meprin a is highly expressed in the human liver hepatoblastoma cell line hepg2 and abundantly secreted into culture media. examination of human tumor samples showed that meprin a is expressed in primary colon tumors and in tumors that have metastasized to the liver. this indicates that meprin a expression in gastrointestinal tumor cells contributes to the progression of the disease. biochemical pathways mediating necrotic cell death and neurodegeneration in caenorhabditis elegans n. tavernarakis, p. syntichaki, c. samara and k. troulinaki institute of molecular biology and biotechnology, foundation for research and technology, heraklion, crete greece. e-mail: tavernarakis@imbb.forth.gr necrotic cell death plays a central role in devastating human pathologies such as stroke and neurodegenerative diseases. elucidation of the molecular events that transpire during necrotic cell death in simple animal models should provide insights into the basic biology of inappropriate neuronal death, and facilitate the characterization of mechanisms underlying degeneration in numerous human disorders. various cellular insults, including hyperactivation of ion channels, expression of human beta-amyloid protein implicated in alzheimer's disease, constitutive activation of certain g proteins, hypoxia and possibly the ageing process, can trigger a degenerative, necrotic cell death in the nematode caenorhabditis elegans. we are genetically and molecularly deciphering the c. elegans necrotic death program. we have isolated mutations in several distinct genetic loci that bock degenerative cell death initiated by various genetic and environmental insults. by characterizing such suppressors, we have discovered that neuronal degeneration inflicted by various genetic lesions in c. elegans, requires the activity of specific calcium-regulated calpain proteases and acidic ph-dependent aspartyl proteases. although, it is believed that these proteases become activated under conditions that inflict necrotic cell death, the factors that govern the erroneous activation of such-otherwise benign-enzymes are largely unknown. we identified novel factors that modulate cellular ph homeostasis, which are required for necrosis and showed that targeting these factors effectively protects from necrotic cell death in c. elegans. our findings demonstrate that two distinct classes of proteases are involved in necrotic cell death and suggest that perturbation of intracellular calcium levels may initiate neuronal degeneration by compromising ph homeostasis and deregulating proteolysis. search for regulatory proteins which are controlled by proteolysis in escherichia coli based on microarray analysis j. m. heuveling ag hengge, institute of microbiology, fu berlin, berlin, germany. e-mail: joheuvel@zedat.fu-berlin.de the impact of controlled proteolysis on regulatory events in prokaryotes is increasingly recognized over the last decade. as in eukaryotic cells, proteolysis is more than just a garbage disposal but has been found to be implicated in the regulation of many vital functions of the bacterial cells, like cell cycle, stress responses and development (hengge r and bukau b. mol microbiol 2003) . conditional degradation of regulators shows a high potential of integrating a great variety of signals as is well studied for the degradation of sigma s. this sigma subunit of the rna polymerase, which triggers the general stress response in escherichia coli is digested rapidly by the clpxp protease in association with the phosphorylated response regulator rssb under non-stress conditions (stuedemann a. embo 2004). several other regulatory proteins have been found to be subjected to proteolysis, as lexa, a regulator of the sos response. also lon protease is involved for example in the degradation of rcsa, a regulator of the capsule biosynthesis and of sula, a cell division inhibitor (as a review: hengge-aronis r, jenal u. curr opin microbiol 2003). in order to find other regulatory processes in which proteolysis plays a role we pursued a global approach using the microarray technique. in mutants lacking functional clpp or lon proteases or either one of the clp recognition factors clpa and clpx, we searched for genes, which are differentially transcribed compared to the wildtype. we found some interesting groups of genes belonging to common regulons governed by known regulators -candidates for clp or lon mediated proteolysis. after confirmation of these results through lacz fusion studies of representative genes of these regulons, these regulators are presently examined in in vivo degradation studies using immunodetection methods. a distinct group of serine peptidases cannot hydrolyze proteins, but can readily cleave peptides that are up to about 30 amino acid residues long. the representative member of the family, prolyl oligopeptidase is implicated in a variety of disorders of the central nervous system. the enzyme consists of a peptidase domain with an a/b-hydrolase fold and its catalytic triad is covered by the central tunnel of a seven-bladed b-propeller. this domain makes the enzyme an oligopeptidase by excluding large structured peptides from the active site. in most propeller domains the circular structure is ''velcroed'' together in a mixed blade, where both amino and carboxy terminus are involved to form a four stranded antiparallel b-sheet. non-velcroed or ''open topology'' propellers are rare, and prolyl oligopeptidase was the first protein structure exhibiting a domain of this nature. the apparently rigid crystal structure does not explain how the substrate can approach the catalytic groups. two possibilities of substrate access were investigated: either blades 1 and 7 of the propeller domain move apart or the peptidase and/or propeller domains move to create an entry site at the domain interface. engineering disulfide bridges to the expected oscillating structures prevented such movements, which destroyed the catalytic activity and precluded substrate binding. this indicated that concerted movements of the propeller and the peptidase domains are essential for the enzyme action. biochemical characterization of thermoplasma volcanium recombinant 20s proteasome and its regulatory subunit g. baydar and s. kocabiyik molecular genetics, biological sciences, middle east technical university, ankara, turkey. e-mail: gozde_baydar@hotmail.com proteasome associated energy dependent proteolysis is not only involved in rapid turnover of specific proteins that could be important during periods of stress, but also engaged in the turnover of the short-lived proteins that regulate a variety of cellular processes in both procaryotic and eucaryotic cell. the universal distribution of proteasome homologs in archaeal genome provide insight into the vital role of archaeal proteasomes. 20s catalytic core of archaeal proteasomes in combination with various aaa atpases and membrane associated lon proteases may play role in stress response or turnover of the regulatory proteins. however, little is known about the potential physiological roles of archaeal proteasomes. this study presents the data on biochemical and biophysical features of recombinant 20s proteasome of a thermoacidophilic archaeon thermoplasma volcanium (tpv). pcr was performed to amplify dna fragments containing tpv genes encoding the a -and b-subunits of the proteasome from tpv genomic dna. the amplified a-gene (tpva) and b-gene (tpvb) together with their upstream sequences were separately cloned and then combined in puc18 vector. the resulting recombinant puc-skba plasmid was used for heterologous production of in vivo assembled 20s proteasome in e. coli. the recombinant proteasome was purified by combination of ammonium sulfate precipitation, gel filtration chromatography (sepharyl s-300) and ion-exchange chromatography (q sepharose). molecular masses of purified protein subunits were estimated as 23.71 kda (b-subunit) and 21.13 kda (a-subunit). substantial post-glutamyl peptide hydrolyzing activity and chymotrysin-like activity were detected as associated with recombinant proteasome. maximum chymotrypsin-like activity was measured at 85°c and ph 8.5. crystallographic studies of the gtp-dependent transcriptional regulator cody from bacillus subtilis. cody is a gtp dependent transcriptional regulator of early stationary phase and sporulation genes in bacillus subtilis. it is activated by gtp, during rapid cell growth it represses several genes whose products allow adaptation to nutrient depletion. when the cells pass from rapid growth to stationary phase, the intracellular concentration of gtp drops thus releasing the repressed genes. cod y is a 259-residue polypeptide containing a helix-turn-helix motif for binding to dna. it also has motifs common with small gtpases, but cody has a much lower affinity for gtp. crystals of the full-length cody have been grown in the presence and absence of gtp from sodium citrate buffered solutions using lithium sulphate as a precipitant and diffraction data have been collected to 3.5 å resolution. attempts to solve the structure using anomalous data from the semet derivative crystals of cody have been hampered by the large number (70) of methionines in the asymmetric unit and difficulties in reproducibility of angiotensin-converting enzyme (ace) is a zinc metallopeptidase critical for the generation of the vasoconstrictor peptide angiotensin ii. a homologue of ace, ace-2, has recently been identified, which appears to play a counter-regulatory role to ace by inactivating angiotensin ii. like ace, ace-2 is a type i membrane protein with its active site contained within the extracellular domain. the expression of ace2 protein is normally low and restricted primarily to endothelial cells of the heart and kidney, kidney epithelium and testis. recent evidence from ourselves and others indicates that ace2 is significantly upregulated in a number of pathologies, such as myocardial infarction, renal disease and hepatitis c-induced cirrhosis. given that ace can be proteolytically released from the cell surface in culture, ace2 may likewise be shed into plasma or urine. detection of elevated levels of ace2 in plasma and urine may be a useful biomarker for the diagnosis of hepatic, renal and vascular disease. using a specific quenched fluorescent substrate, we have detected ace2 activity in human urine. in contrast, ace2 activity could not be detected in human plasma; interestingly, however, we noted that plasma markedly inhibited the activity of recombinant ace2, thus compromising the possibility of measuring plasma enzyme activity. we are in the process of purifying this inhibitor, which preliminary results suggest is small and hydrophilic. we are also currently optimizing methods for its removal from plasma samples, thus allowing detection of low levels of soluble ace2 activity in normal human plasma. the identification of a potential endogenous inhibitor of ace2, the first for this family of metallopeptidases, could have significant consequences for ace2 function in vivo and the regulation of angiotensin peptides. future studies will examine whether plasma or urinary levels of ace2 are elevated in cardiovascular, renal or liver disease. molecular determinants of proteolytic processing of non-structural polyprotein of semliki forest virus. institute of molecular and cell biology, university of tartu, tartu, estonia. e-mail: lulla@ut.ee semliki forest virus (sfv) is a positive-stranded rna virus. the replication of sfv is performed by the rna-dependent rna replicase complex (rc) and regulated by proteolytic processing. during the course of the infection template preference of rc changes from rna plus-strand to minus-strand. it has been known for several years that this preference switch is due to the proteolytic processing of sfv non-structural polyprotein p1234, mediated by viral cysteine protease located in the carboxy-terminal domain of the nsp2 protein. tight temporal regulation of this template specificity switch is crucial for the viral replication, but, nevertheless, its mechanism remains unsolved. therefore, the mapping of the essential molecular determinants of the site-specific cleavage consensuses may provide necessary information, concerning the cleavage regulation as well as regulation of the rna replication. the results of our studies indicate that as little as 5 amino acid residues from the c terminus of nsp3 protein determine the specificity of the proteolytic cleavage of the nsp3/nsp4 junction. at the same time sequences laying downstream of the cleavage point (in nsp4 region) have only minor effect on the cleavage efficiency. the exact region required for the cleavage of nsp2/nsp3 junction is yet not known but the sequences, required from c-terminal part of nsp2 protein, are likely short as well. in contrast, sequence lying within 80-240 n-terminal amino acid residues of nsp3 is vital for cleavage of the nsp2/nsp3 junction. this region may represent the cofactor of the nsp2 protease that activates processing at the nsp2/nsp3 cleavage site. thus, as the result of current research, a principally new function -regulation of the proteolytic processing and rna replication -was mapped to the conserved n-terminal region of the nsp3. this finding significantly improves our understanding about the role of nsp3, which was enigmatic till now, in the virus life cycle. activation occurs within the specific dibasic motif hsiirrsl, suggesting the involvement of the proprotein convertases (pcs) in these process. this family of endoproteases are responsible for the activation of a large variety of regulatory proteins by cleavage at multi-basic recognition sites exhibiting the general motif (k/r)-(x)n-(k/r)(n = 0, 2, 4 or 6). cotransfection of the furindeficient colon carcinoma cell line lovo with provegf-c and different pc members revealed that furin, pc5 and pc7 are vegf-c convertases. the processing of provegf-c is blocked by the inhibitory prosegments of furin, pc5 and pace4, as well as by furin-motif variants of alpha2-macroglobulin and alpha1antitrypsin. accordingly, mutation of the vegf-c pc-site (hsiirrsl to hsiisssl) inhibited provegf-c processing. following zebrafish caudal fin amputation, the injection of control vector or vector containing wild vegf-c did not affect fin regeneration. in contrast, injection of muted vegf-c (pro-vegf-c) inhibited fin regeneration. these data highlight the importance of vegf-c processing in zebrafish fin regeneration and suggest that zebrafish can be used as a simple and useful model for studying the role of protein maturation by the pcs in physiological processes. thimet oligopeptidase (top) hydrolyzes a variety of bioactive peptides and is implicated in the regulation of neurological and other physiological processes. top is composed of two ''clamshell'' domains, with the substrate-binding pocket and catalytic site lying between these domains. it is speculated that conformational changes in loops and coil regions connecting the domains lead to changes in substrate specificity. the loop region (residues 599-611) is close enough to the active site to interact with even the smallest substrate. it contains three glycine residues and is expected to be quite flexible. in an effort to trap intermediate conformations of the loop, we have replaced gly 599, 603, or 604 with ala and have compared the activities of the three resulting protein constructs towards two quenched fluorescent substrates. all three enzymes had lower activity than wild type towards a bradykinin analog, with g599a, the most active of the mutants, possessing 1/3 wild-type activity. however, utilizing a smaller substrate, g603a was the most active, surpassing even wild type (fivefold increase in activity). g604a had little activity towards either substrate. these results are consistent with data that revealed increases in activity towards the larger substrate, when the enzyme is partially denatured and presumably, more flexible and with increased accessibility of the binding loop to proteolytic enzymes, when partially denatured. acknowledgment: this work was supported by hhmi, nih-ns39892 (mjg). the proteasome: paradigm of a self-compartmentalizing protease self-processing of subunits of the proteasome crystal structures of the rhodococcus proteasome with and without its pro-peptides: implications for the role of the pro-peptide in proteasome assembly handbook of metalloproteins bovine chymotrypsinogen-a x-ray crystal-structure analysis and refinement of a new crystal form at 1.8 a resolution equilibrium and rate constants for the interconversion of two conformations of a-chymotrypsin. the existence of a catalytically inactive conformation at neutral ph refolding transition of alpha-chymotrypsin -ph and salt dependence e-mail: saleh38@hotmail.com reference 1. arnorsdottir j, kristjansson mm, ficner r. crystal structure of a subtilisin-like serine proteinase from a psychrotrophic vibrio species reveals structural aspects of cold adaptation (which is an excellent substrate for adam-17), we observed 35% increase in enzymatic activity in the media of 1 mm 5-ht-treated cells compared to untreated cells. however, we did not see any increase in the fluorescence when we used ''cate1'', an adam substrate, which does not recognize adam-17. to further support a role for adam-17/tace, we designed silencing rnas against the enzyme, which were introduced into the mesangial cells using lentiviral infection. successful silencing was confirmed by western blotting 4 days after infection. control and tace silenced human mesangial cells were stimulated with 2-10 lm of serotonin for 5 min, and erk activation was assessed by western blotting baron-ruppert 2 and e. heymann 1 1 department of physiological chemistry (fb8/gw) e-mail: rebecca.lew@med.monash.edu.au b4-015p functional properties of p94/calpain3 and connectin/titin in mdm mouse skeletal muscle y bunkyo-ku the lectin pathway of complement system is an important component of the innate immunity. it provides the first line of defence against infection, since it is activated on the surface of invading pathogens. the activation of the complement system results in the destruction and clearance of foreign microorganisms. mannose-binding lectin-associated serine protease-2 (masp-2) is the enzyme which is responsible for the initiation of the lectin pathway of complement activation. masp-2 is a multidomain serine protease, which is synthesized as an inactive zymogen and become activated upon mbl binds to carbohydrate plant proteinase inhibitors are widely spread in the different plant species being a significant component of a defense system. somewhere a significant diversity of the proteins related to the same structural family of the inhibitors in the same species may be observed. the family of potato kunitz-type proteinase inhibitors (pkpis) exemplifies a group of proteins with the diverse properties and may be divided into three major homology groups: a, b and c. a lot of genes encoding different pkpiproteins of each group were found in various potato cultivars (solanum tuberosum l.). inhibition activity of plant invertase, cysteine and serine proteinase was found in proteins subgroup c. a set of gene copies were isolated by pcr from potato cv. istrinskii genome. dna sequencing analysis of these resulted in identification of 24 different dna sequences with a high similarity to potato kunitz-type inhibitors of group c (pkpi-c). cluster analysis demonstrated that this clones represented multiple copies of six new genes denoted as pkpi-c1, -c2, -c3, -c4, -c5 and c6. it can be supposed that at least two alleles containing pkpi-c genes are harbored in tetraploid genome of potato. one of new genes, namely pkpi-c5, exhibited 99% identity with known invertase inhibitor cdna (1423) from cv. provita. another pkpi-c6 gene was similar (98% identical residues) with cdna (p340) from potato cv. bintje encoding for a putative trypsine inhibitor. four other new genes demonstrated as much as 89-92% identity with known pkpi-c proteins from other potato cultivars. the n-terminal sequence of the protein encoded by the pkpi-c2 gene was identical to the n-terminal sequence of specific subtilisin inhibitor pksi isolated from cv. istrinskii.b3-049p regional distribution of human trypsinogen 4 in human brain determined at mrna and protein level j. to´th 1 , l. gombos 1 , e. siklo´di 1 , p. ne´meth 2 , m. palkovits 3 , l. szila´gyi 1 and l. gra´f 1 1 laboratory of enzymology, department of biochemistry, eo¨tvo¨s lora´nd university, budapest, hungary, 2 institute of immunology and biotechnology, university of pe´cs, pe´cs, hungary, 3 laboratory of neuromorphology, department of anatomy, semmelweis university, budapest, hungary. e-mail: july@ludens.elte.huproteases play an important role in many physiological and pathological processes in the central nervous system such as development, neurite outgrowth, neuronal plasticity and degeneration and cell signaling. a gene coding for such an enzyme might be prss3 on chromosome 9 of the human genome. it encodes due to alternative splicing both mesotrypsinogen, which is expressed in pancreas, and trypsinogen 4 whose mrna has been identified in different human tissues (initially in brain, recently in different epithelial cell lines from prostate, colon and airway). analysis of the gene prss3 predicted two isoforms of the zymogen: isoform a may have a 72 amino acid, while isoform b a 28 amino acid n-terminal leader sequence. in order to gain information on the possible role of human trypsinogen 4 we have determined its amount at the mrna and the protein level as well in 17 selected brain areas using real-time quantitative pcr and elisa.the highest transcript levels could be detected in cerebellar cortex, while low amounts were found, e.g. in cerebellar white matter samples. the distribution of the mrna in different brain areas measured by real-time pcr is consistent with the protein levels detected with elisa. the usage of different monoclonal antibodies specific for the 28 amino acid leader sequence and the protease domain allowed the separate detection of the zymogen and the active enzyme. in e.g. the hypothalamus the zymogen is the dominant form, while a significant degree of activation was found in the cerebellar cortex. our data indicate that the extent of activation varies with different areas. as human trypsinogen 4 is ubiquitous in the brain we conclude that it might play a role in general neurological processes. serine proteases are enzyme involved in the maintenance of the cell homeostasis. thus, this type of enzymes must be extremely regulated and it has been highly reported that serine proteases are involved in the growth and expansion of different cancers. in this regard, the type ii transmembrane serine proteases (ttsps) constitute a subfamily of membrane anchored serine proteases that are ideally positioned to carry out different interactions with other cell surface or extracellular proteins. among them, tmprss2 and tmprss3 proteins have been reported to be overexpressed in most prostate and ovarian cancers respectively, matriptase/mt-sp1 is expressed in a wide variety of benign and malignant tumors and hepsin is overexpressed in ovarian and renal cancers. desc-1 is a ttsp member found differentially expressed in squamous cell carcinoma (differentially expressed in squamous cell carcinoma gene 1) and differentially from other ttsps, its expression is found to be reduced in tumor tissues respecting to the normal tissue at rna level in head and neck squamous cell carcinoma (hnscc), what suggests a possible tumor protective function for desc-1. in order to shed light about the role of desc-1 in these processes, we have carried out the molecular cloning of the human full-length cdna and expression of the recombinant protein to delineate the implication of this protease in hnscc.diffracting crystals. therefore we used limited proteolysis and mass-spectrometry analysis to identify the domain boundaries of the protein and were able to determine the sequence of two principal proteolytic fragments corresponding to the n-and c-terminal domains of cody. these individual domains which were successfully cloned in escherichia coli, overexpressed as histagged proteins, isolated and purified. both domains have been crystallized. the crystals of the n-terminal domain grow from bis-tris buffered solutions at ph 6.5 containing polyethylene glycol and calcium acetate. the crystals of the c-terminal domain were obtained using ammonium sulphate as a precipitant. the crystals of n-terminus domain diffract to at least 2.3 å and crystals of c-terminus domain -to 3.2 å using an in-house diffractometer with a mar research image-plate as a detector. progress towards the determination of cody structure will be presented. the gram-positive bacterium listeria monocytogenes is a facultative intracellular parasite. interactions of l. monocytogenes with the host cell are provided by a number of secreted and cell surface proteins. one of the most important virulence factors, actinpolymerizing protein acta, is surface attached via the hydrophobic c-tailed membrane anchor. despite, the membrane anchor acta was found in comparable amounts both on the cell surface and in the culture supernatant. the aim of the work was to investigate the mechanism of acta release and the role of this process in l. monocytogenes virulence. maldi-tof ms analysis of trypsin released acta suggested releasing due to proteolytic cleavage between histidine and threonine residues in the close vicinity of the membrane anchor predicted by the htmm analysis. the substitution of histidine with proline prevented acta release into the culture supernatant, although did not disturb its surface presentation. in silico analysis of eight other l. monocytogenes membrane-anchored surface proteins suggested the role for asparagine and threonine residues in specific proteolysis. the prediction was experimentally tested by substitution of the residues with alanine. the l. monocytogenes spontaneous mutant strain, unable to release membrane-anchored proteins into the culture supernatant, was isolated. the mutation was mapped outside the acta gene and presumably affected the corresponding peptidase. the mutation impaired the invasion of l. monocytogenes into the human epithelial-like hela cells that suggested the effect of the released proteins on signaling events that result in induced phagocytosis of the pathogen by normally non-phagocytic cells. angiotensin ii (ang ii) has been proposed to act as a regulatory peptide in the epidermal layer of human skin. while the expression of receptors and peptide precursors have been demonstrated in epidermis, the formation of ang ii and its inactivation have not been studied in detail. thus we have established a model system with cultured keratinocytes to examine the metabolism of ang i, ii and related peptides by intact epidermal cells. cultures were incubated with peptides in a minimal medium, which sustained cell viability for at least 24 h and the metabolism of peptides was monitored by chromatography (rp-hplc). with ang i as peptide substrate five major products were detected in keratinocyte culture media after 12 h incubation. a half-life of about 9 h was estimated for ang i and the slow degradation supports results of earlier studies revealing low activities of exopeptidases in a microsomal fraction from keratinocytes as compared to fibroblasts. the degradation of ang i was not affected by inhibitors of alanyl aminopeptidase, peptidyl dipeptidase a and neprilysin. since a peptide product formed from ang i in keratinocyte cultures resembled ang ii in hplc analysis, the activity of peptidyl dipeptidase a in these cells was assayed with hip-his-leu and the presence of the peptidase was confirmed by its sensitivity to captopril. further experiments showed that ang ii, iii and related peptides were degraded in keratinocyte cultures with rates similar to ang i and these reactions interfered severely with the formation of ang ii. immunohistochemical studies showed a strong positive staining for neprilysin and alanyl aminopeptidase in the dermal layer of human skin and at the epidermal-dermal junction confirming the results obtained with the cell cultures. soluble angiotensin converting enzyme-2 present in human plasma and urine p94/calpain 3 is the skeletal-muscle-specific calpain and is considered to be a modulator protease in various cellular processes. a defect in the p94 gene causes limb-girdle muscular dystrophy type 2a (lgmd2a), suggesting that p94 functions are indispensable for proper muscle functions. in sarcomeres, p94 localizes at z-, n2-and m line regions. although the binding partner for p94 at z-line has not been identified yet, n2-and m-line localization of p94 are considered dependent on its interaction with the n2a and m-line regions of connectin/titin, respectively. connectin is a gigantic sarcomeric protein playing an important role as a molecular template for sarcomeric organization, an elastic element generating passive tension, a platform for various protein ligands, etc. in this study, we focused on the molecular components associated with the n2a region of connectin/titin to extend our understanding on p94. intriguingly, a recessive mutation in the mouse connectin gene, mdm (muscular dystrophy with myositis), causes muscular dystrophy. there are two remarkable phenotypes consequential to mdm mutation. first, the mdm mutation abolishes p94 binding activity of connectin n2a fragment. second, in skeletal muscle from mice homozygous for mdm mutation, upregulation of cardiac ankyrin repeat protein (carp) is observed. carp also binds to n2a connectin at the n-terminal proximity of the region mutated by mdm. the effect of mdm mutation on p94 activity and the properties of n2a connectin as well as carp were analyzed using both animal model and cell culture systems. semliki forest virus (sfv) is well known model virus, which has been studied for decades. the main topic of this research was characterization and analysis of sfv replication machinery using approach based on use conditional-lethal mutants of viruses. the direct aim of the present study was to sequence and functionally characterize a panel of independent sfv temperature sensitive mutants. from all putative ts-mutations, identified in this study, two were mapped to nsp1 protein, four were mapped to nsp2 protein and one was founded in nsp4 region. number of assays were used to verify phenotypic effects of revealed mutations: titration of virus stocks at different temperatures, leak yield experiments, analysis of viral rna synthesis and viral polyprotein processing at different temperatures. nsp2 mutants had clear viral protease defect and accumulated non-cleaved polyproteins on different stages. besides all, biotechnological branch of our research is already developing. it includes improving of existing sfv based expression vector system by use of ts-mutations for the temperature regulation of foreign gene expression in mammalian cells. dipeptidyl peptidase iv activity and/or structure homologues (dash) in brain tumors pathogenesis of many diseases, including cancer, often involves improper proteolytic post-translational modification of biologically active peptides. association of dysregulated expression pattern of novel group of ''dipeptidyl peptidase (dpp)-iv activity and/or structure homologues'' (dash) with cancer development and progression has been suggested by several authors, including us [1] . dpp-iv enzymatic action as a common attribute of most of dash members modifies signaling potential of their substrates, biologically active peptides, not only quantitatively, but due to the changes in their receptor preferences also qualitatively. in this study, we have investigated expression (by real time rt-pcr and immunohistochemistry) and enzymatic activity (by biochemical assays and enzyme histochemistry) of plasma membrane localized dash members, in particular dpp-iv, fibroblast activation protein-alpha (fap) and attractin in human gliomas. it was revealed that varying quantities of dpp-iv, fap and attractin mrnas and proteins were coexpressed in the studied tumors. the majority of dpp-iv-like activity in the glioma tissue could be attributed to the canonical dpp-iv. this activity, assayed biochemically and expressed per mg of protein, was increased in high grade gliomas. inhibition studies suggested lack of enzymatically active attractin in the examined glioma tissues. the results of our pilot study demonstrate for the first time that both enzymatically active and inactive dash molecules are coexpressed in gliomas and suggest prevailing association of increased dpp-iv activity with high grade tumors. acknowledgment: this work was supported by iga nr/8105-3 and msmt 0021620808 vegf-c is involved in the neovascularization processes, steps essential for wound healing, cancer progression and many other physiological functions. zebrafish vegf-c processing and key: cord-016126-i7z0tdrk authors: dangi, mehak; kumari, rinku; singh, bharat; chhillar, anil kumar title: advanced in silico tools for designing of antigenic epitope as potential vaccine candidates against coronavirus date: 2018-10-14 journal: bioinformatics: sequences, structures, phylogeny doi: 10.1007/978-981-13-1562-6_15 sha: doc_id: 16126 cord_uid: i7z0tdrk vaccines are the most economical and potent substitute of available medicines to cure various bacterial and viral diseases. earlier, killed or attenuated pathogens were employed for vaccine development. but in present era, the peptide vaccines are in much trend and are favoured over whole vaccines because of their superiority over conventional vaccines. these vaccines are either based on single proteins or on synthetic peptides including several b-cell and t-cell epitopes. however, the overall mechanism of action remains the same and works by prompting the immune system to activate the specific b-celland t-cell-mediated responses against the pathogen. rino rappuoli and others have contributed in this field by plotting the design of the most potent and fully computational approach for discovery of potential vaccine candidates which is popular as reverse vaccinology. this is quite an unambiguous advance for vaccine evolution where one begins with the genome information of the pathogen and ends up with the list of certain epitopes after application of multiple bioinformatics tools. this book chapter is an effort to bring this approach of reverse vaccinology into notice of readers using example of coronavirus. it compelled us to apply the well-known reverse vaccinology (rv) approach on available proteome of coronavirus. rv approach has been successfully applied on many prokaryotes, but there are very few known applications on eukaryotes and viruses. so, it is worthwhile to explore the potential of this approach to identify potential vaccine candidates for coronavirus. rv basically does the in silico examination of the viral proteome to hunt antigenic and surface-exposed proteins. this approach was initially applied successfully to neisseria meningitidis serogroup b (kelly and rappuoli 2005) against which none of the prevailing techniques could develop a vaccine. the present book chapter is intended to explore the potential of rv approach to select the probable vaccine candidates against coronavirus and validate the results using docking studies. undoubtedly, the traditional approaches for vaccine development are fortunate enough to efficiently resist the alarming pathogenic diseases of its time. however, the traditional approach suffers from certain limitations like it is very timeconsuming, the pathogens which can't be cultivated in the lab conditions are out of reach, and certain non-abundant proteins are not accessible using this approach (rappuoli 2000) . consequently, a number of pathogenic diseases are left without any vaccine against them. all these limitations are conquered by reverse vaccinology approach utilizing genome sequence information which ultimately is translated into proteins. hence all the proteins expressed by the genome are accessible irrespective of their abundance, conditions in which they expressed. the credit of fame of reverse vaccinology should go to the advancements in the sequencing strategies worldwide. accordingly, improvement in the sequencing technologies has flooded the genome databases with huge amount of data which can be computationally undertaken to reveal the various crucial aspects of the virulence factors of the concerned pathogen. reverse vaccinology is based on same approach of computationally analysing the genome of pathogen and proceeds step by step to ultimately identify the highly antigenic, secreted proteins with high epitope densities. the best epitopes are selected as potential vaccine candidates (pizza et al. 2000) . this approach has brought the unapproachable pathogens of interest in spotlight and is evolving as the most reassuring tool for precise selection of vaccine candidates and brought the use of peptide vaccines in trend (sette and rappuoli 2010; kanampalliwar et al. 2013 ). bexsero is the first universal serogroup b meningococcal vaccine developed using rv, and it has currently earned positive judgement from the european medicines agency (gabutti 2014) . whether it is discovery of pili in gram-positive pathogens which were thought to not have any pili or the sighting of factor g-binding protein in meningococcus (alessandro and rino 2010), the reverse vaccinology steals all the credits from other conventional approaches. most of the applications of rv are against prokaryotes and very few against eukaryotes and viruses because of complexity of their genome. corynebacterium urealyticum (guimarães et al. 2015) , mycobacterium tuberculosis (monterrubio-lópez et al. 2015) , h. pylori (naz et al. 2015) , acinetobacter baumannii (chiang et al. 2015) , rickettsia prowazekii (caro-gomez et al. 2014) , neospora caninum (goodswen et al. 2014) and brucella melitensis (vishnu et al. 2017) are the examples of some pathogens that are recently approached using this in silico technique in order to spot some epitopes having potential of being a vaccine candidate. herpesviridae (bruno et al. 2015 ) and hepatitis c virus (hcv) (kolesanova et al. 2015) are the examples of the viruses that are addressed using this approach. (altschul et al. 1990; okonechnikov et al. 2012; golosova et al. 2014) . multiple sequence alignment (msa) was done via clustalw, and the phylogenetic tree was constructed using nj method from unipro ugene 1.16.1 bioinformatics toolkit (okonechnikov et al. 2012 ). analysis of secondary structure of the proteins of seed genome was done by means of expasy portal. the aim is to forecast the solvent accessibility, instability index, theoretical pi, molecular weight, grand average of hydropathicity (gravy), aliphatic index, number of charged residues, extinction coefficient etc. (http://web. expasy.org/protparam/; gasteiger et al. 2005) . virus-mploc was used to identify the localization of proteins of virus in the infected cells of host (http://www.csbio.sjtu.edu.cn/bioinf/virus-multi/; hong-bin shen and kuo-chin chou 2010) . this information is important to understand the destructive role and mechanism of the viral proteins in causing the disease. in total six different subcellular locations, namely, host cytoplasm, viral capsid, host plasma membrane, host nucleus, host endoplasmic reticulum and secreted proteins, were covered. these predictions could help in formulation of better therapeutic options against the virus. as per the protocol of rv, secreted and membrane proteins are of special interest, therefore, filtered for further analysis. to predict the number of transmembrane helices tmhmm server v. 2.0 (http://www.cbs.dtu.dk/services/tmhmm/; krogh et al. 2001 ) was used. signal peptides are known to impact the immune responses and possess high epitope densities. moreover, most of the known vaccine candidates also possess signal peptides. hence, it is worthwhile to predict signal peptides in proteins prior to epitope predictions. signal-blast web server is used to predict the signal peptides without any false predictions (http://sigpep.services.came.sbg.ac.at/signalblast.html; frank and sippl 2008) . the prediction options include best sensitivity, balanced prediction, best specificity and detect cleavage site only. we choose to make the predictions using each option, and the proteins predicted as signal peptide by all the four options were preferred for further investigation. the most appropriate targets as vaccine candidates are those which possess the adhesion-like properties because they not only mediate the adhesion of pathogen's proteins with cells of host but also facilitate transmission of virus. adhesions are known to be crucial for virulence and are located on surface which makes them promptly approachable to antibodies. the stand-alone spaan with a sensitivity of 89% and specificity of 100% was used to carry out the adhesion probability predictions, and the proteins with having adhesion probabilities higher than or equal to 0.4 were selected (sachdeva et al. 2004 ). betawrap motifs are dominant in virulence factors of the pathogens. if the proteins are predicted to possess such motifs, then they are appropriate to be taken under reverse vaccinology studies. betawrap server is the only online web server to make such predictions. the proteins having p-value lower than 0.1 were anticipated to contain betawraps (http://groups.csail.mit.edu/cb/betawrap/betawrap.html; bradley et al. 2001 ). for added identification of the antigenic likely of the proteins, they were subjected to vaxijen server version 2.0. it is basically an empirical method to hunt antigenic proteins. so, if the proteins are not found antigenic using other sequence-based methods, then they can be identified using this method. this step confirms the antigenicity of proteins selected using above-mentioned steps (http://www.ddgpharmfac.net/vaxijen/vaxijen/vaxijen.html; doytchinova and flower 2007). for being a probable vaccine candidate, the protein should not exhibit the characteristics of an allergen as they trigger the type-1 hypersensitivity reactions causing allergy. therefore, to escape out such possibilities, the proteins were also subjected to allergenicity predictions using allertop (http://www.pharmfac.net/ allertop; dimitrov et al. 2014) and algpred tools (http://www.imtech.res.in/ raghava/algpred/submission.html; saha and raghava 2006a, b). to check whether the filtered proteins possess any similarity to host proteins or not, the standard blastp (http://blast.ncbi.nlm.nih.gov/blast) searches were performed. in case of sequence similarity, there is a feasibility of generation of immune responses against own cells. predicting the epitopes binding to mhc class i is the main decisive phase of the rv to carry out valid vaccine predictions. the predicted epitopes were docked with receptor that is hla-a*0201 using cluspro (http://cluspro.bu.edu/login.php; kozakov et al. 2017 ) that is an automated protein-protein docking web server. the literature searches provided the information of conserved residues of the receptor site. the default parameters were used for docking (comeau et al. 2004a, b; kozakov et al. 2006 ). a total of 40 different sequenced strains of coronavirus are available at ncbi. among them 7 strains are pathogenic to humans. various information regarding source, host and collection of these strains are presented in table 15 .1 and 15.2. this information can be obtained from ncbi's genome database, the virus pathogen database and analysis resource and genomes online database (liolios et al. 2006; pickett et al. 2012) . the mers strain is taken as seed genome as it is the most prevalent and disastrous strain among others. its proteome consists of total 11 proteins as shown in table 15 .3. the results of sequence similarity to reveal orthologs using blastp are shown in table 15 .4. the sequences with greater than 30% identity score are considered as homologs. the phylogenetic tree is depicted in fig. 15 .1 and the mers-cov, taken as seed genome, found clustered with different bat coronaviruses. the results of analysis of secondary structure of the proteome using expasy tools are shown in the table 15 .5. from the analysis of charge on the residues and ph values, it is concluded that six of the proteins are basic and positively charged unlike allergens which are acidic in nature. however, five proteins are acidic and show negative charge. the negative gravy score of five proteins justify them to be of hydrophilic nature with majority of the residues positioned towards the surface. for the rest of six proteins, the gravy score is positive; it means that these are the accession number and identity of orthologs obtained in different strains is shown in the table hydrophobic proteins. the proteins with less than 40 value of instability index are quite stable than those with higher values. all the proteins are having the molecular weight less than 110 kda except 3 (yp_009047202.1, yp_009047203.1 and yp_009047204.1). this exhibits the effectiveness of lightweight proteins as targets as they can be easily purified because of their low molecular weights. the protein yp_009047204.1 is reported as a spike glycoprotein. it is acidic with prominent negative charge, with negative gravy score which suggests its hydrophilicity and figure 15 .2 depicts the subcellular localization of proteins of the seed genome, i.e. mers-cov. only one protein was predicted to be localized in host cytoplasm, four in host membrane, two in both host cell membrane and endoplasmic reticulum (er) while two in only er, and two are left unrecognized. the known spike protein is predicted to be localized in host er. from these results we decided to pick the proteins which are located in host membrane or were predicted to be localized in both host membrane and er. the two are known envelop protein and membrane protein from bibliographic studies, and along with that, the known spike protein was also included in the filtered results. out of the filtered proteins, only two (yp_009047210.1 and yp_009047208.1) contain more than two transmembrane helices, therefore filtered out. the results of transmembrane helices prediction are tabulated in table 15 .6. figure 15 .3 depicts the subcellular localization of proteins of all the four selected genomes using virus-mploc prediction tool. the proteins that are predicted to possess the signal peptides by signal-blast web server are yp_009047204.1 and yp_009047205.1. the results of signal-blast web server are tabulated in the table 15 .7. this step takes into account the concept of adhesion-based virulence. adhesions cause pathogen recognition and initiation of inflammatory responses by the host. spaan predicted 2 (yp_009047204.1 and yp_009047205.1) out of 11 proteins of mers strain as adhesive (table 15 .8). only one protein (yp_009047204.1) was predicted to contain betawrap motifs within it (table 15 .8). hence, it is considered virulent and might be responsible for initializing the infection in the host. a total of 9 out of 11 proteins of mers strain were predicted antigenic (prediction values greater than 0.4). the protein with accession number yp_009047206.1 and yp_009047208.1 were among the filtered proteins, however, not predicted antigenic, therefore filtered out. as a result, only four proteins (yp_009047204.1, yp_009047205.1, yp_009047207.1 and yp_009047209.1) were kept for further analyses. none of the 11 proteins of mers-cov possessed any clue of allergenicity as per prediction results from algpred and allertop tools; it means that no vigorous immune responses will be mounted if the epitopes from these proteins will be adopted as vaccine candidates. none of the protein of mers strain shows similarity with the proteins of host that demonstrates that the epitopes from these proteins can safely elicit the required immune response without the hazard of autoimmunity. in total 12 different 9-mer epitopes with potential to bind to receptors of both b-cell and t-cell were predicted. the list of the predicted epitopes can be found in the table 15 .9 and are specific for mers-cov strain. all these epitopes displayed no conservancy with proteins of other human and non-human pathogenic strains. docking permits to reveal the binding energy or potency of connection among epitopes and the receptor in appropriate orientation. the cluspro docking server was used to dock the predicted 90 epitopes against hla-a*0201. the structure of the receptor was available from pdb and was optimized before docking to free it from the complexed self-peptide (4u6y, resolution 1.47 å, bouvier et al. 1998 ). pepstr (peptide tertiary structure prediction server; kaur et al. 2007 ) was used to derive the tertiary structure of the predicted peptides. figure 15 .4 depicts the quaternary structure of the receptor hla-a*0201 with its conserved active site known to form complex with the peptides (bouvier et al. 1998 ). the binding energy results obtained after performing docking analysis are listed in table 15 .9. the 9-mer epitope vvcaitllv at site 21 of protein yp_009047209.1 docked to the receptor with smallest amount of binding energy (à951.7) and 12 hydrogen bonds. the next epitope in the list was also from the same protein yp_009047209.1 at site 27, i.e. tllvcmafl. the predicted structure of the top 5 potent epitopes on the basis of docking energy and the snapshots of docking results are displayed in figs. 15.5, 15.6, 15.7, 15.8 and 15.9 . the most chief restriction for developing a safe and sound vaccine against any of the virus is to identify the protective antigens. the present study is an effort of application of reverse vaccinology approach to investigate a choice of coronavirus proteomes to identify possible vaccine targets. this technique has demonstrated to be a competent way to forecast 12 different epitopes from the selected seed genome. these epitopes are from spike glycoprotein, ns3 protein, ns4b protein and envelope protein. unfortunately none of the epitope is found conserved in other strains, and all are specific to mers-cov. the docking analysis studies revealed perfect binding between hla-a*0201 receptor and epitopes. the conserved residues of the receptor site are also involved in h-bonding with epitope residues. further, the selected antigenic epitopes must be validated using in vitro and in vivo studies to confirm their potential as vaccine candidates. review: reverse vaccinology: developing vaccines in the era of genomics basic local alignment search tool the middle east respiratory syndrome coronavirus -a continuing risk to global health security crystal structures of hla-a*0201 complexed with antigenic peptides with either the amino-or carboxyl-terminal group substituted by a methyl group betawrap: successful prediction of parallel beta -helices from primary sequence reveals an association with many microbial pathogens geminiviridae. in: virus taxonomy-ninth report of the international committee on taxonomy of viruses lessons from reverse vaccinology for viral vaccine design discovery of novel cross-protective rickettsia prowazekii 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classifier for viral protein subcellular location prediction by incorporating multiple sites propred1: prediction of promiscuous mhc class-i binding sites identification of potential antigens from non-classically secreted proteins and designing novel multitope peptide vaccine candidate against brucella melitensis through reverse vaccinology and immunoinformatics approach key: cord-011184-ohdukhqt authors: patil, shital p.; goswami, ashutosh; kalia, kiran; kate, abhijeet s. title: plant-derived bioactive peptides: a treatment to cure diabetes date: 2019-07-22 journal: int j pept res ther doi: 10.1007/s10989-019-09899-z sha: doc_id: 11184 cord_uid: ohdukhqt abstract: recent advances in analytical techniques have opened new opportunities for plant-based drug discovery in the field of peptide and proteins. enzymatic hydrolysis of plant parent proteins forms bioactive peptides which are explored in the treatment of various diseases. in this review, we will discuss the identified plant-based bioactive proteins and peptides and the in vitro, in vivo results for the treatment of diabetes. extraction, isolation, characterization and commercial utilization of plant proteins is a challenge for the pharmaceutical industry as plants contain several interfering secondary metabolites. the market of peptide drugs for the treatment of diabetes is growing at a fast rate. plant-based bioactive peptides might open up new opportunities to discover economic lead for the management of various diseases. graphic abstract: [image: see text] biomolecules play an important role in any ligand-target interaction and their recognition in any biological system starts the cascade of signalling steps, which is an important part of the normal body function. nucleic acids, carbohydrates, and lipids are the important ligands that bind and cause conformational changes in targets (receptors) but the messengers that release after binding and conformation changes are peptides. any defect in these signal transduction processes cause diseases (otvos and wade 2014; gautam 1999) . proteins, under the influence of proteolytic enzymes get fragmented at particular catalytic sites to form bioactive peptides. bioactive peptides are made up of 2-20 amino acids and lack protein-protein interaction because of their small size. they have properties like tissue affinity, specificity, and efficiency (moller et al. 2008) . the structure and the overall charge and hydrophobicity/hydrophilicity of the any bioactive peptide depends on the nature of amino acids, their sequences in the peptide backbone along with the n and c terminals. the relation between structure and biological activity of the bioactive peptides are yet not confirmed but the structure is an important arbitrator for their activity (li and yu 2015) . bioactive and nutraceutical peptides have a positive effect in the regulation of the health of an individual (kitts and weiler 2003) . they boost the quality of human health by preventing various ailments and by improving medical conditions related to lifestyle, metabolism, and immunity (vitetta et al. 2005) . peptide and protein drugs are available from the different sources for therapeutic uses, but as with many drugs, they have several advantages and disadvantages of their own. peptide drugs are effective and specific to their biological target but devoided of some of the essential qualities of a drug to withstand in the current market of therapeutics. proteins have a large chemical structure which makes their synthesis complex, tedious and expensive, the further low oral bioavailability of proteins limit their oral delivery hence they are administered through parenteral route (uhlig et al. 2014) . the worldwide peptide therapeutics market size valued at usd 22,071.5 million in 2017 and assumes to grow at cagr of 9.1% over the period 2018 to 2026. (2016) . insulin is the first therapeutic peptide which has been used widely and shares good peptide market. it was first isolated by frederick banting and charles best from pancreatic islet extracts, further, j.b. collip developed the method for extraction and purification of insulin from other animal sources (quianzon and cheikh 2012) . eli lilly began producing insulin from the animal pancreas, later on, biosynthetic insulin was introduced in 1983 with the name humulin. novo nordisk, sanofi, and eli lilly together share 88.7 percent of the global insulin market. lantus (basal insulin) is a product of sanofi accounts for approximately 22.29% of the total sales of top 10 antidiabetic drug brands, in 2016 sale of lantus was usd 6.057 billion (dezzani 2017) . small molecules and peptide-based drug candidate belongs to the category of new chemical entities (nces) according to the food and drug administration, on the other hand, proteins used for the treatment of various diseases are classified under new biological entities (nbes). in recent years, therapeutic peptides are approved for the mitigation of various ailments mainly tumors, immunological disorders, blood disorders and metabolic disorders such as diabetes, obesity ( fig. 1) (loganathan 2016) . the peptide drug, liraglutide (victoza) is a glucagon-like peptide-1 receptor (glp-1r) agonist and used for the treatment of t2dm has gained popularity as a top-selling drugs in recent past, which is marketed by novo nordisk (lund et al. 2014) . glatiramer acetate (copaxone), developed by teva is an immunomodulator and used to treat multiple sclerosis (duda et al. 2000) . leuprolide (lupron), developed by abbott is useful for the treatment of cancer and estrogendependent conditions that respond to hormone therapy (brito et al. 2004) . goserelin (zoladex) from astra zeneca is a gonadotropin-releasing hormone agonist (gnrh agonist) that suppresses the release of different sex hormones and is useful in the treatment of breast and prostate cancer (magon 2011) . other innovator companies include amgen, eli lilly, roche, and pfizer have peptide and protein-based drugs in different developmental stages will reach the market soon for the treatment of various complicated diseases (loganathan 2016) . till date, plant secondary metabolites, mainly small molecules are the established source of new drugs to treat various diseases (verpoorte 1998) , however, advancement in analytical technique, sophisticated purification methodology and in vitro assay system pointed out the researchers fig. 1 uses of therapeutic peptides and proteins in various disease conditions (loganathan 2016) 1 3 to look beyond the small molecules. identification of plant primary metabolites including proteins and peptides for the management of diseases have opened up a new horizon in the development of plant-based peptides as a drug candidate (otvos 2008) . this review summarizes various plant-based peptides reported for the treatment of diabetes, challenges with the development of peptide-based drugs and the future prospect of peptides as a drug. diabetes is a metabolic disorder and can hit anyone at different stages of life. though it is considered as one of the top health killers, its treatment is a huge challenge. diabetes can be characterized with symptoms of prolonged hyperglycemia, glucose intolerance, disturbance in the regulatory systems for storage and utilization of metabolic energy, including catabolism and anabolism of carbohydrate, lipids, and proteins in a diabetic individual due to lack of insulin production and impaired insulin receptor functioning or both (piero 2015; votey and peters 2005) . based on the causes and clinical survey, diabetes mellitus is categorized into four types as mentioned in table 1 (deepthi et al. 2017) . ordinarily, the people below 18 age suffer from the type 1 diabetes, whereas type 2 diabetes mainly occurs in adult and old age group. according to the world health organisation, approximately 10% of all diabetes cases are type 1, and the remaining 90% cases of diabetes worldwide are of type 2 (baynes 2015) . diabetes is ruining the life of peoples worldwide. population suffering from diabetes is increasing and the imminence is expected to rise even more because of the current lifestyle issues. till the year 2017, 422 million people were affected by type 2 diabetes and the number is expected to increase up to 642 million people worldwide by 2040 (fig. 2) . the data indicates that approximately 50% of diabetic patients will increase in the next two decades. the threat is every 6 s a person dies with diabetes (roglic 2016) . from the statistics, it is clear that the population suffering from type 2 diabetes is increasing and it requires more promising therapies. still, no promising drug is available which has the ability to completely cure type 2 diabetes. generally, to treat type 2 diabetes, oral hypoglycemic agents are used. the first line treatment is metformin, which is assisted with other hypoglycemic agents from different categories like thiazolidinediones, alpha-glucosidase inhibitors, sulphonylurea, dipeptidyl peptidase-iv (dpp-iv) inhibitors, sodium glucose cotransporter 2 (sglt 2) inhibitors. the risk associated with these therapies include hypoglycemia, weight gain, tiredness, diarrhea, and anemia risk, etc. over the period of time, these side effects lead to further complications for a visceral system like cardiovascular system and central nervous system (nathan et al. 2009 ). peptide drugs used for the treatment of diabetes are derived from different sources, like exendin-4 was initially isolated from the heloderma suspectum (gila monster) and its synthetic version had come to the market in the form of exenatide (aramadhaka et al. 2013) . liraglutide and semaglutide are structurally very close to glp-1 with improved half-life and resistant to dpp-iv mediated degradation (edmonds and price 2013) . nowadays, these peptides are being produced in large scale by using recombinant dna technology. the peptides which were obtained via synthesis or by using the recombinant technology are comparatively expensive than small molecules and are not affordable to most of the patients. the interest in health-promoting products from the natural origin and pharmaceutical formulations involving bioactive peptides are remarkably increasing (henninot et al. 2018) . numerous scientific reports have been published on bioactive peptides obtained from animal proteins. expedition by researchers in the field of bioactive peptides is of great interest as it opens the way to find economic lead from plants for the better care of diabetic patients (daliri et al. 2017 ). plentiful bioactive peptides have been reported from the animal and plant sources. most of the peptides that had bioactivity are being derived from animal products such as milk, eggs, meat, and fish. plant peptides are still under exploration and few bioactive peptides are reported from soy, wheat, etc (hartmann and meisel 2007) . in animals, proteins are associated with high-fat content and lead to diseases, including high blood pressure and heart diseases if consumed in the large amount. on the other hand, plant proteins neither have associated fat nor have any side effects (nehete et al. 2013) . previously, plant hormones were considered the only player through which cells communicate, but from the last decade as research was driven towards an understanding of plants signaling, secreted peptides, small rnas and transcription factors emerging as a new and well-defined player in the cell to the cell communication network (lindsey et al. 2002; van norman et al. 2011; murphy et al. 2012) . however, it is now clearly defined that signaling peptides of plant and animal origin are biosynthesized through the pathway that is evidently similar, although some aspects of the biosynthesis are still not identified. peptides secreted by plants have well recognized role in preliminary processes of development like the growth of meristem, organ shedding, cell elongation, cell multiplication, cell differentiation, geotropism and protection from the invader (ghorbani 2014) . the ubiquitous presences of proteins, peptides as mediator components convey to the proposals that these messenger peptides may have emerged evolutionary in microbes and were further integrated into the complicated mediator systems of complex organisms during evolution (roth et al. 1982) . existence of hormone-like peptides in plants including insulin-like peptide in spinacia oleracea l. (spinach) and lemna gibba g-3, prolactin-like inhibitor in alfalfa, a substance with luteinizing hormone-releasing activity from leaves of avena sativa l. (oat) and somatostatin-like material in spinach support this assumption, it is speculated that the signaling peptides in plants existed from about 1 billion years ago (collier et al. 1987; fukushima et al. 1976; leroith et al. 1985; morley et al. 1980) . plant proteins containing essential amino acids are important in the food chain to fulfill human physiological needs. the proteins from plants are derived from leaves, seeds, and fruits, out of which seeds are considered as an economical source of proteins. as compared to vegetables and fruits, seeds (legumes and cereals) contain a higher amount of protein (creighton 1993) . proteins in a mature seed represent 18-20% in pisum sativum l. (pea), vicia faba l. (faba) bean and till 35-45% in lupinus albus l. (lupin) and glycine max l. (soybean) (gueguen and cerletti 1994) . many peptides are reported from different plants for the treatment of diabetes as shown in tables 2 and 3 through various known targets such as (a) alpha-glucosidase inhibitors (b) alpha-amylase inhibitors (c) dipeptidyl peptidase-iv inhibitors (d) inhibitors of the glucose transporter system (e) insulin mimetics alpha-glucosidase inhibitors competitively inhibit small intestinal enzyme alpha-glucosidase, which converts nonabsorbable polysaccharides into absorbable monosaccharide, these effects together postpone and minimize the rise in postprandial plasma glucose level. inhibitors of this enzyme decrease the glucose toxicity (improves insulin sensitivity), decreases stress on beta cells (decreases post-meal hyperglycemia), increases glucagon like peptide-1 (glp-1) production hence increases insulin secretion (scheen 2003; mooradian and thurman 1999) . in general, normalization of postprandial hyperglycemia is difficult as compared to fasting hyperglycemia, inhibitors act specifically on the postprandial part of 24 h blood glucose curve and causes a reduction in diabetes-associated hyperglycemia which is responsible for macrovascular complication in patients (mooradian and thurman 1999; ceriello et al. 2004 ). glycaemic control is achieved with these agents reduces glucotoxicity which increases insulin secretion from the beta cell (scheen 2003; salvatore and giugliano 1996) . all marketed alpha-glucosidase inhibitors are from natural origin, however, none of them belongs to peptide class. few research groups have reported peptides from plants having alpha-glucosidase inhibitory activity. peptides obtained from cannabis sativa l. (hemp) seeds contains hydrophobic amino acids (pro and leu), essential amino acids and branched chain amino acids in their structure and have reported to have enzyme inhibitory activity for this target (ren et al. 2016) . oat seed proteins hydrolysate obtained by protease (alcalase) digestion gives bioactive peptides which inhibits this enzyme. in vivo study also revealed that oat peptides, at a higher dose, showed the hypoglycaemic effect on stz-induced diabetic mice, reduces the food intake, stimulates insulin secretion, improves insulin sensitivity and elevate glycogenesis (zhang et al. 2015) . walnut hydrolyzed peptides (whps) from the fruit proteins of juglans mandshurica maxim. (walnut) showed anti-diabetic activity by inhibiting the enzyme. in vitro study of whps showed that peptides of molecular weight (3-10 kda) inhibit alpha-glucosidase with the inhibitory rate of (61.73%) and they remarkably raise extracellular glucose consumption in insulin-resistant hepg2 cells. whereas, in vivo results showed that whps reduce 64.82% of fasting blood glucose level by increasing insulin secretion, liver glucokinase, and glycogen level by 23.71%, 69.54%, and 76.19% respectively (wang et al. 2018) . vigna angularis wild. (adzuki bean) proteins have reported enzyme inhibitory properties in mice model kk-ay of diabetes. the extract reduces the postprandial (2015) 10 hook.f. in vitro and in vivo marella et al. (2016) 11 cucurbita pepo l. in vitro (vaštag et al. 2014) 12 lam. in vitro (gonzalez garza et al. 2017) 13 l. in vitro arise (2016) 14 theobroma cacao l. in vivo sarmadi et al. (2012) 15 l. in vitro mojica et al. (2017b) 16 willd. in vitro nongonierma et al. (2015) 17 l. in vivo and in vivo yibchok-anun et al. (2006) 18 momordica charantia l. insulin mimetic in vivo (khanna et al. 1981) 19 momordica charantia l. seed protein expressed in e.coli stimulated the phosphorylation of pdk1 and akt, enhanced expression of glut-4, stimulated both the uptake of glucose in cells and the clearance of glucose in vitro and in vivo lo et al. (2016) 20 hook.f. in vivo rajasekhar et al. (2010) 21 hook.f. molina. duchesne. in vivo teugwa et al. (2013) 22 zea mays l. enhance the secretion of glp-1 in vivo hira et al. (2009) 23 glycine max l. increases the glucose uptake, enhance the expression of p-ir, p-irs1, p-akt and membrane glut4 protein improve the insulin resistance hypoglycemic in vitro and in vivo lu et al. (2012) 24 oryza sativa l. improve insulin resistance in vivo boonloh et al. (2015) 25 roxb. in vivo poovitha et al. (2017) blood glucose in two sucrose challenge model i.e. normal and streptozocin-treated rats by 15.6% and 30.9% respectively. in vitro study of extruded adzuki bean proteins at 10 mg/ml, concentration inhibits 60.44% of rat intestinal alpha-glucosidase (yao et al. 2014 amylase (alpha-1,4-glucan-4-glucanohydrolase) is an important enzyme that speeds up the hydrolysis of (alpha-1,4) glycosidic linkage of carbohydrate and starch present in the diet and helps in absorption of non-absorbable polysaccharides after their conversion into absorbable monosaccharide (alagesan et al. 2012 ). inhibitors of amylase minimize the hydrolysis of (alpha-1,4) glycosidic linkage and help in slow digestion of carbohydrate and thus delay the absorption of glucose and reduces the postprandial glucose level in blood (jayaraj et al. 2013 ). the pinto beans are a rich source of proteins and other nutrients but are not well explored for its therapeutic benefits. pinto bean protein fraction was digested enzymatically using protease (protamex) at ph 6.5, and for the incubation period of 1-h with (e/s) ratio of 1:10 at 50 °c, hydrolysed fraction was dialysed using molecular weight cutoffs and the fraction obtained with molecular weight < 3 kda showed 62.10 ± 3.49% of enzyme inhibition (ngoh and gan 2016) . similarly digestion with protease (bromelain) and dialysis with molecular weight cut-off < 1 kda showed 49.9 ± 1.4% of enzyme inhibition (oseguera-toledo et al. 2015) . the peptide obtained from cuminum cyminum l. (cumin) seeds, cumin seed peptide1 (csp1) had shown 24.54% of alphaamylase inhibition (siow and gan, 2016) . in vitro study of triticum aestivum l. (wheat) albumin extract showed alphaamylase inhibition, wheat albumin restrained the peak of postprandial blood glucose levels in a dose-dependent manner. it showed the reduction in postprandial blood glucose by 31%, 47%, and 50% after administering 0.25 g, 0.5 g, and 1.0 g of wheat albumin respectively. in long-term administration study, 0.5 g of wheat albumin did not affect fasting blood glucose levels, while it reduces hemoglobin a1c levels which reflects the metabolic control of individual suffering from diabetes (kodama et al. 2005) . a study revealed that different protein fractions isolated from the hordeum vulgare l. (barley) flour have been subjected to pancreatic hydrolysis and the obtained peptides have shown 57-77% alpha-amylase inhibitory activity (alu'datt et al. 2012). cucurbitin protein was obtained from the cucurbita pepo l. (pumpkin) seeds, alcalase and pepsin hydrolysed fraction of it showed the enzyme inhibition (vaštag et al. 2014) . oligopeptides from the mulberry showed highest enzyme inhibition with ic 50 16.25 µg/ml. protein hydrolysates prepared from the seeds of moringa oleifera lam. by enzymes like trypsin, chymotrypsin and pepsin-trypsin for 2.5 and 5 h. pepsin-trypsin digested fraction showed alpha-amylase inhibition and the ic 50 was found to be 0.195 and 0.123 µg/ml for 2.5 h and 5 h of hydrolysis respectively (gonzalez garza et al. 2017) . protein hydrolysate of citrullus lanatus l. (watermelon) seeds was prepared by using enzymes like pepsin, trypsin and alcalase where alcalase hydrolysate gives the highest enzyme inhibition and the ic 50 was found to be 0.149 mg/ml (arise 2016) . autolysates of theobroma cacao l. fruits were prepared at ph 3.5 and 5.2. autolysate at ph 3.5 showed highest inhibition of alpha-amylase as well as helps to release the insulin, in vivo study with autolysates showed decrease in the blood glucose level (sarmadi et al. 2012) . quinoa proteins were enzymatically hydrolyzed to obtain bioactive peptides, hydrolyzed fraction thus obtained showed 6.86% inhibition of alpha-amylase at 250 µm concentration (vilcacundo et al. 2017 ). two main gut-derived hormones glp-1 and glucosedependent insulinotropic polypeptide (gip), also known incretin hormones are responsible for the attainment of normoglycemia. they stimulate insulin secretion, decrease glucagon, inhibit gastric emptying, reduce food intake and appetite (drucker and nauck 2006) . in diabetic individuals, reduced secretion of incretin hormones have been observed. these gut hormones bind with their respective receptors and show their biological action (barnett 2006) . treatment of diabetes requires to restore either normal secretion or reduce the degradation of these hormones. dipeptidyl peptidase-iv (dpp-iv) is a protease enzyme that cleave the glp-1 hormone within minutes after its secretion and makes it inactive for biological function. thus, inhibition of dpp-iv has the potential to revert the hyperglycaemic condition. dpp-iv inhibitors block the rapid degradation of glp-1 and enhance the postprandial level of active glp-1 which further reduces the liver glucagon production and stimulate beta cells of the pancreas and increase insulin level (ahrén 2007) . dipeptides obtained from oryza sativa l. (rice) bran protein hydrolysates showed inhibition of dpp-iv enzyme and have ic 50 1.45 ± 0.13 mg/ml (hatanaka et al. 2015) . in another study, rice bran protein fractions have been defatted and hydrolyzed with umamizyme g and bioprase sp. the dipeptides obtained after digestion with umamizyme g showed inhibition of enzyme and the ic 50 was found to be 2.3 ± 0.1 mg/ml (hatanaka et al. 2012) . the proteins extracted from amaranthus hypochondriacus l., have been subjected to simulated gastrointestinal digestion resulted into bioactive peptides. these peptides fraction have shown dose-dependent enzyme inhibition and have ic 50 1.1 mg/ml (velarde-salcedo et al. 2013) . glycine max l. (soybean) and lupinus albus l. (lupin) protein hydrolysate have been screened for the presence of bioactive peptides, and soy 1 and lup 1 were found to be efficient inhibitors of dpp-iv with ic 50 values 106 and 228 μm respectively (lammi et al. 2016) . peptides obtained (cpgnk and ggglhk) from the protein digestion of common beans showed enzyme inhibitory activity and the ic 50 (mg dw ml −1 ) for the pure peptides were found to be 0.87 ± 0.02 and 0.61 ± 0.10 respectively (mojica et al. 2017b) . gastrointestinal digestion of proteins obtained from phalaris canariensis l. (canary) seed showed 43.5% inhibition of dpp-iv (estrada-salas et al. 2014) . enzymatic digestion of quinoa proteins with papain showed enzyme inhibition and the ic 50 for the hydrolysate-papain was found to be 0.88 ± 0.05 mg/ml (nongonierma et al. 2015) . study was performed on quinoa protein simulated duodenal digestion and the fraction (˃ 5 kda) obtained after 120 min of digestion showed highest dpp-iv inhibition and the ic 50 (mg protein/ml) was found to be 0.84 ± 0.07 (vilcacundo et al. 2017 ). glucose, the metabolic fuel of any living cell, unable to cross lipid bilayer of the membrane due to its polar nature and membrane proteins (glucose transporter) associate with lipid bilayer helps glucose to travel across (abdul-ghani et al. 2011) . glut transporters facilitate the transport of glucose passively across the membrane along with its concentration gradient and do not require energy to operate, on the other hand sglt transporters actively transport glucose across the membrane against the concentration gradient thus these types of transporters require energy to operate. in order to receive energy for operation, sodium is transported along with its concentration or electrochemical gradient thus provide the needed amount of energy for the transportation of glucose across the membrane (musso et al. 2012; brown 2000) . in the human body, at least 12 different glut transporters and 7 sglt transporters exist which belongs to membrane class of proteins. excreted glucose is reabsorbed up to 90% in a convoluted segment of proximal tubule via high capacity, low-affinity sglt 2 transporter while remaining 10% of glucose is reabsorbed in the distal segment of proximal tubule via high affinity, low capacity sglt 1 transporter. in the case of diabetes, hyperglycemia causes the high load of glucose in order to compensate for this high load of glucose, an increase in expression of a glucose transporter gene in proximal tubule occurs (abdul-ghani et al. 2011) . plant peptides targeting glucose transporters are less explored. however, peptides having amino acid sequences aksplf, atnplf, feeln, and lsvsvl, isolated from the black beans have shown the ability to block the glucose transporters glut-2 and sglt-1. the results of in vitro studies by using the caco-2 cell model have shown reduced glucose re-absorption by 21.5% after 24 h treatment of these bioactive peptides. the oral glucose tolerance test in rats has shown a 24.5% decrease in postprandial glucose (p < 0.05) (50 mg hydrolyzed protein isolate/kg bw) (mojica et al. 2017a ). insulin hormone trigger several signaling events that control the metabolic fate of nutrients. insulin binds with alphasubunit of insulin receptor which causes the conformational changes in β-subunit of the insulin receptor, and these conformational changes in receptor activate the cytoplasmic tyrosine kinase domain. this activation further leads to the auto-phosphorylation which in turn trans-phosphorylate various intracellular substrate of the insulin receptor. these effects collectively control metabolic and mitogenic processes (nankar and doble, 2013) . tyrosine kinase domain activation by insulin mimetic, cause the auto-phosphorylation of receptor and trigger downstream signaling requires for metabolic activity of insulin. the other mechanism by which insulin mimetic (vanadate) works is by inhibiting the dephosphorylation of the insulin receptor via inhibition of tyrosine phosphatase (stapleton 2000) . the reports suggest that plant extracts of spinach and lemna gibba g-3 mimics like mammalian-insulin, which was confirmed by the radioimmunoassay. in an in vivo study on young rats indicated that the plant insulin-like material binds to insulin receptors on im-9 lymphocytes and stimulates glucose oxidation and lipogenesis in isolated adipocytes (collier et al. 1987) . vigna unguiculate l. (cowpea) containing bio-molecules have contributed in reducing the blood glucose level and enhancing the antioxidant status of patients. study on l6 rat skeletal muscle cells was performed where cells were treated with cowpea peptides at different doses (0.1, 1, 10 and 100 ng) for 20 h or insulin (100 nm) for 30 min. further, from the treated cells proteins were isolated for western blot analysis to check the phosphorylation of akt (a form of protein kinase b; pkb). the results of the study showed that the cowpea peptides have the ability to phosphorylated akt in the cell culture. this observation suggests that the treatment of cowpea peptides to the skeletal muscle cells can initiate the insulin signaling cascade. there is a possibility that cowpea peptides have the ability to mimic like insulin by inducing the same signaling cascade (uruakp, 2015) . linum usitatissimum l. (flax) seed peptides obtained from the protein hydrolysate (50kd) increases glucose uptake in l6 cells ). mc2-1-5 peptide fraction was obtained from momordica charantia l. showed hypoglycaemic effect in in vivo study. mc2-1-5 fraction decreases the blood glucose level by 61.70% and 69.18% in alloxan-induced diabetic mice with a time interval of 2 and 4 h respectively (yuan et al. 2008) . study on protein from the pulp of m. charantia l. showed increase glucose uptake in c2c12 myocytes and 3t3l adipocyte, the in vivo studies showed that the protein obtained from pulp helps in secretion of the insulin as well as act like insulin to lower the glucose level (yibchok-anun et al. 2006 ). polypeptide-p was isolated from the m. charantia l. acts like insulin mimetic (khanna et al. 1981) . another 68-residue insulin receptor (ir)-binding protein (mcirbp) was reported from the plant upon in vitro digestion yields mcirbp-19, spanning residues 50-68 of mcirbp which increases the binding of insulin to its receptor, stimulate the phosphorylation of pdk1 and akt, induces the expression of glucose transporter 4, and stimulate both the uptake of glucose in cells and the clearance of glucose in diabetic mice (lo et al. 2016) . a novel protein called mcy having molecular weight 17kd was isolated from momordica cymbalaria l. fruit which showed hypoglycemic effect in in vivo study (rajasekhar et al. 2010) . the peptide fraction obtained from the soybean showed increase in glucose uptake in muscular cells, it is reported that obtained peptide fraction enhances the glucose uptake via amk activation (roblet et al. 2014) . protein extract of cucurbitaceae family containing seeds like telfairia occidentalis hook.f., citrullus lanatus l., lagenaria siceraria molina., and cucurbita moschata duchesne., showed a significant decrease in blood sugar level in in vivo study (teugwa et al. 2013) . peptide reported from the leaves of bauhinia variegata l, mimics like insulin which is evidently proved from the immunological reactivity and via in vivo study (azevedo et al. 2006) . zea mays l. (zein) seed protein hydrolysate increase the secretion of glp-1 directly in the ileum and indirectly in the duodenum in the rat and also confirmed with the help of glutag cell line study (hira et al. 2009 ). aglycine which is a bioactive peptide obtained from the soy bean increases the glucose uptake in c2c12 cell, whereas in in vivo study on diabetic model it was observed that treatment with aglycine increases the expression of p-ir, p-irs1, p-akt and membrane glut4 protein which results into hypoglycemia (lu et al. 2012) . soymorphin-5 also called µ-opioid obtained from soy bean decreases the blood glucose level via activation of adiponectin and pparα systems in diabetic kkay mice (yamada et al. 2011) . rice bran protein hydrolysate helps to improve insulin resistance and prevent metabolic diseases (boonloh et al. 2015) . protein extracted from the fruit pulp of momordica dioica roxb. decreases the blood glucose level in a diabetic model (poovitha et al. 2017) . plant scientist believe that there are more plants left unexplored which may contain insulin-like peptides or peptides which will mimic insulin (xavier-filho et al. 2003) . the success of any therapy and its acceptability depends on the ease of its delivery. the oral route of drug administration remains the most accepted route of drug delivery and most of the available drugs in the market are delivered through the oral route (morishita and peppas 2006) . an orally active drug should be stable enough to withstand the gastric microenvironment and also it should be permeable through the gastrointestinal membrane. small molecules are well tolerant to the gastric ph and permeated through the membrane whereas the large macromolecules are vulnerable to degradation in the gastrointestinal tract and are not permeable in their intact form to reach specific target successfully without being degraded in git (morishita and peppas 2006) . as the medical field advances, the peptide drugs are widely explored for their therapeutic potential in the treatment of various diseases. though these drugs are specific to their target, efficacious and potent, the oral bioavailability of peptide and peptide-based drugs always remains a challenge that limits their wide acceptance and market value. high cost of therapy is a major limitation associated with peptide drugs (ayoub and scheidegger 2006) . with the advancement of biotechnology, the recombinant synthesis of a peptide of interest opens a door for cheap and cost-effective peptides. also, the commercial interest for the production of bioactive peptides from the natural sources is elevated to reduce the cost but due to the lack of suitable technologies which helps to retain or increase the activity of bioactive peptides, the large-scale production gets hampered (craik et al. 2013) . another limitation associated with peptide-based drugs is their short half-life. when the peptides come in contact with intestinal mucosa, gastric acid in stomach and peptidases that are present in the blood convert peptides into amino acids and gets quickly eliminated from the body, thereby reducing its half-life. in order to achieve prolonged action and to reduce the frequent dosing, it is necessary to increase the half-life of the peptides. as peptide therapeutics advances, several non-degradable polymers and polymeric matrix are available to increase the half-life of peptides and proteins thus reducing the frequent dosing (brown 2005) . another strategy to increase the half-life of the peptide is to modify the amino acids which are susceptible to cleavage by enzymes (adessi and soto 2002) . oral delivery of the peptides is explored in past years but limits its use as peptides are degraded in the git tract (lau et al. 2015) . novo nordisk developed a semaglutide oral formulation which is currently in clinical trial phase 3. other non-invasive and suitable routes for the peptide and protein-based drugs are under investigation includes the nasal and pulmonary route which directly deliver the drugs to the blood component hence, reduces the overall drug degradation which is the main drawback associated with oral delivery of peptides (davies et al. 2017 ). different therapeutic peptides have made their way to the market in recent years, and it is assumed that the market demand will increase further as peptides are specific, selective and safe in comparison to small molecules (fosgerau and hoffmann 2015; loffet 2002) . traditional synthesis and drug design approaches are used by researchers and pharmaceutical companies for the development of the peptide-based drug candidate. the recombinant protein is another tool for the synthesis of the desired peptide in bacteria, yeast, and fungi. the market of peptides has now moved from the isolation of peptides from an animal source that can mimic the endogenous peptide to synthetic, semi-synthetic and recombinant peptides. further, development in this area is drug conjugated peptides and multifunctional peptides (fosgerau and hoffmann 2015) . peptides are susceptible to degradation when given orally, which is supposed to be the most convenient route of administration. the preferred route for the peptide delivery is intravenous, however, more research is directed for the delivery of peptides. recently, alternative routes are explored, which include oral, transdermal and intranasal route. as peptides are excellent biomarkers, they can also be utilized for diagnosing diseases. further, peptides are also found to be advantageous in vaccine development (brown 2005; sheridan 2012 ). plant peptides have been isolated and characterized in different ways as described in fig. 3 . x-ray crystallography and nmr have contributed enormously in the field of structure elucidation of pure peptides (gomathi and subramanian fig. 3 the general scheme of isolation, purification, and characterization of plant peptides (gomathi and subramanian 1996; krishnan and rupp 2012; sinz et al. 2015; wysocki et al. 2005) 1996; krishnan and rupp 2012) . other techniques like electron microscopy, fluorescence resonance energy transfer, chemical cross-linking emerged in recent year for the characterization of proteins and peptides (sinz et al. 2015) . mass spectrometry is frequently used technique in analyzing peptide mixtures and identifying their proposed structures. amino acid sequencing in mass spectrometry can be performed by two methods namely top-down sequencing and down-top sequencing. in top-down sequencing, the proteins analysis is performed without hydrolysis and the molecular weight can be detected by fragmentation pattern of the whole protein. a common approach is down-top sequencing in which proteolytic digestion is performed followed by ms analysis. software based on the algorithm of the amino acid sequence is available for protein identification (wysocki et al. 2005 ). plants remain a valuable source for developing a new drug candidate. they are still sharing their fair share of the new drug candidates and lead. as research interest is now shifted from small molecules to bio-molecules by virtue of several added benefits, plants are now under exploration for the presence of proteins and bioactive peptides for the disease management. though plant proteins and their functions have not been defined earlier, plant hormones were only considered through which cells communicate, as our understanding to these signalling pathways become clear, several secreted peptides and small rnas now known which regulates molecular recognition and thus, controls cell communication. from our diet we consume proteins which after gastrointestinal digestion get converted to bioactive peptides with more tissue affinity to specific receptor. several research publications in recent years claimed plant proteins and their bioactive peptides as a potential candidate in the treatment of various diseases specially in metabolic and life style borne diseases including cancer, diabetes and obesity. sophisticated instrument and advancement in technology leads easy isolation, purification and characterization of proteins and peptides in a short period of time, which further reduces the overall time of discovery. apparently, most of the bioactive peptides available for the treatment of diabetes have emerged from the synthetic route or derived from recombinant technology, which further adds in the overall cost of the treatment. to make peptide therapy economic there is a demand to explore plant-based biomolecules. this review discusses bioactive peptide isolated from various plant parts like leaves, fruits and seeds. the trend indicates that most of the bioactive peptides are obtained from seeds, although reports are available where bioactive peptides have been also isolated from leaves, whole fruits and pulps. it was observed in various in vitro and in vivo studies that protein hydrolysate obtained from aqueous extracts of plant are capable of inhibiting the enzymes and transporter systems responsible for the diabetes. expedition to find molecular targets and mechanism of action of plant based bioactive peptides is needed to use these bioactive peptides as a potential drug candidate. epidemiological data reveals that type 2 diabetes requires promising therapy as the available synthetic drugs for the treatment have moderate to severe adverse effects. plant-derived bioactive peptides inhibit the enzymes like alpha-glucosidase, alpha-amylase, dipeptidyl peptidase-iv and glucose transporter systems involved in type 2 diabetes. in vivo studies of a certain plant peptides fraction showed insulin-mimetic action in animal models. it is clear that plant-derived bioactive peptides are not explored to their full potential in comparison to other classes of natural products. the reason might be the lack of suitable instrumental techniques to identify, purify and characterize the peptides from plant source. however, the recent advancement in lcms, lc-nmr, and x-ray crystallography has bridged the gap and this would be the right time to embark on plant peptides. in future, systematic studies including dereplication for the early identification of known peptides, target identification followed by in vivo studies would help to speed up the plant peptides research. dipeptidyl peptidase-4 inhibitors: clinical data and clinical implications amylase inhibitors: potential source of anti-diabetic drug discovery from medicinal plants anti-oxidant, anti-diabetic, and anti-hypertensive effects of extracted phenolics and hydrolyzed peptides from barley protein fractions connectivity maps for biosimilar drug discovery in venoms: the case of gila monster venom and the anti-diabetes drug byetta® in vitro antioxidant and α-amylase inhibitory properties of watermelon seed protein hydrolysates peptide drugs, overcoming the challenges, a growing business isolation and intracellular localization of insulin-like proteins from leaves of bauhinia variegata dpp-4 inhibitors and their potential role in the management of type 2 diabetes classification, pathophysiology, diagnosis and management of diabetes mellitus rice bran protein hydrolysates improve insulin resistance and decrease pro-inflammatory cytokine gene expression in rats fed a high carbohydrate-high fat diet a single luteinizing hormone determination 2 hours after depot leuprolide is useful for therapy monitoring of gonadotropin-dependent precocious puberty in girls glucose transporters: structure, function and consequences of deficiency commercial challenges of protein drug delivery postprandial glucose regulation and diabetic complications coherent market insights partial purification and characterization of an insulin-like material from spinach and lemna gibba g3 the future of peptidebased drugs proteins: structures and molecular properties effect of oral semaglutide compared with placebo and subcutaneous semaglutide on glycemic control in patients with type 2 diabetes: a randomized clinical trial a modern review of diabetes mellitus: an annihilatory metabolic disorder bloomberg markets top 20 diabetes drugs 2017 anti-diabetic and antihypertensive activities of two flaxseed protein hydrolysate fractions revealed following their simultaneous separation by electrodialysis with ultrafiltration membranes the incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes glatiramer acetate (copaxone®) induces degenerate, th2-polarized immune responses in patients with multiple sclerosis characterization of antidiabetic and antihypertensive properties of canary seed (phalaris canariensis l.) peptides peptide therapeutics: current status and future directions extraction and purification of a substance with luteinizing hormone releasing activity from the leaves of avena sativa defects in signal transduction proteins leading to disease. in: sitaramayya a (ed) introduction to cellular signal 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proteins and peptides from food sources. applications of bioprocesses used in isolation and recovery effects of single and long-term administration of wheat albumin on blood glucose control: randomized controlled clinical trials macromolecular structure determination: comparison of x-ray crystallography and nmr spectroscopy peptides derived from soy and lupin protein as dipeptidyl-peptidase iv inhibitors: in vitro biochemical screening and in silico molecular modeling study discovery of the once-weekly glucagonlike peptide-1 (glp-1) analogue semaglutide somatostatin-like material is present in flowering plants research progress in structure-activity relationship of bioactive peptides peptides: new signalling molecules in plants identification of the bioactive and consensus peptide motif from momordica charantia insulin receptor-binding protein peptides as drugs: is there a market? growth potential in peptide and protein therapeutics to enhance supplier capacities the soybean peptide 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pinto) quinoa (chenopodium quinoa willd.) protein hydrolysates with in vitro dipeptidyl peptidase iv (dpp-iv) inhibitory and antioxidant properties hard-to-cook bean (phaseolus vulgaris l.) proteins hydrolyzed by alcalase and bromelain produced bioactive peptide fractions that inhibit targets of type-2 diabetes and oxidative stress peptide-based drug design: here and now current challenges in peptide-based drug discovery diabetes mellitus-a devastating metabolic disorder protein extract from the fruit pulp of momordica dioica shows anti-diabetic, anti-lipidemic and antioxidant activity in diabetic rats history of insulin isolation and characterization of a novel antihyperglycemic protein from the fruits of momordica cymbalaria identification and characterization of two novel α-glucosidase inhibitory oligopeptides from hemp (cannabis sativa l.) seed protein enhancement of glucose uptake in muscular cell by soybean charged peptides isolated by electrodialysis with ultrafiltration 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unguiculata) peptides on insulin resistance intercellular communication during plant development bioactivity evaluation of cucurbitin derived enzymatic hydrolysates in vitro inhibition of dipeptidyl peptidase iv by peptides derived from the hydrolysis of amaranth (amaranthus hypochondriacus l.) proteins exploration of nature's chemodiversity: the role of secondary metabolites as leads in drug development release of dipeptidyl peptidase iv, α-amylase and α-glucosidase inhibitory peptides from quinoa (chenopodium quinoa willd.) during in vitro simulated gastrointestinal digestion mind-body medicine: stress and its impact on overall health and longevity diabetes mellitus, type 2-a review evaluation of the antidiabetic activity of hydrolyzed peptides derived from juglans mandshurica maxim fruits in insulin-resistant hepg2 cells and type 2 diabetic mice mass spectrometry of peptides and proteins plant insulin or glucokinin: a conflicting issue soymorphin-5, a soy-derived μ-opioid peptide, decreases glucose and triglyceride levels through activating adiponectin and pparα systems in diabetic kkay mice alpha-glucosidase inhibitory activity of protein-rich extracts from extruded adzuki bean in diabetic kk-ay mice slow acting protein extract from fruit pulp of momordica charantia with insulin secretagogue and insulinomimetic activities purification and characterisation of a hypoglycemic peptide from momordica charantia l. var. abbreviata ser peptides derived from oats improve insulin sensitivity the authors want to thank niper ahmedabad, under the aegis of the ministry of chemicals and fertilizers, department of pharmaceuticals for providing facilities and research fellowships. key: cord-243806-26n22jbx authors: vandelli, andrea; monti, michele; milanetti, edoardo; ponti, riccardo delli; tartaglia, gian gaetano title: structural analysis of sars-cov-2 and prediction of the human interactome date: 2020-03-30 journal: nan doi: nan sha: doc_id: 243806 cord_uid: 26n22jbx specific elements of viral genomes regulate interactions within host cells. here, we calculated the secondary structure content of>2500 coronaviruses and computed>100000 human protein interactions with severe acute respiratory syndrome coronavirus 2 (sars-cov-2). we found that the 3 and 5 prime ends are the most structured elements in the viral genome and the 5 prime end has the strongest propensity to associate with human proteins. the domain encompassing nucleotides 23000-24000 is highly conserved both at the sequence and structural level, while the region upstream varies significantly. these two sequences code for a domain of the viral protein spike s that interacts with the human receptor angiotensin-converting enzyme 2 (ace2) and has the potential to bind sialic acids. our predictions indicate that the first 1000 nucleotides in the 5 prime end can interact with proteins involved in viral rna processing such as double-stranded rna specific editases and atp-dependent rna-helicases, in addition to other high-confidence candidate partners. these interactions, previously reported to be also implicated in hiv, reveal important information on host-virus interactions. the list of transcriptional and post-transcriptional elements recruited by sars-cov-2 genome provides clues on the biological pathways associated with gene expression changes in human cells. a disease named covid-19 by the world health organization and caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has been recognized as responsible for the pneumonia outbreak that started in december, 2019 in wuhan city, hubei, china 1 and spread in february to milan, lombardy, italy 2 becoming pandemic. as of april 2020, the virus infected >2'000'000 people in >200 countries. sars-cov-2 is a positive-sense single-stranded rna virus that shares similarities with other betacoronavirus such as severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) 3 . bats have been identified as the primary host for sars-cov and sars-cov-2 4,5 but the intermediate host linking sars-cov-2 to humans is still unknown, although a recent report indicates that pangolins could be involved 6 . coronaviruses use species-specific proteins to mediate the entry in the host cell and the spike s protein activates the infection in human respiratory epithelial cells in sars-cov, mers-cov and sars-cov-2 7 . spike s is assembled as a trimer and contains around 1,300 amino acids within each unit 8, 9 . the receptor binding domain (rbd) of spike s, which contains around 300 amino acids, mediates the binding with angiotensin-converting enzyme, (ace2) attacking respiratory cells. another region upstream of the rbd, present in mers-cov but not in sars-cov, is involved in the adhesion to sialic acid and could play a key role in regulating viral infection 7, 10 . at present, very few molecular details are available on sars-cov-2 and its interactions with the human host, which are mediated by specific rna elements 11 . to study the rna structural content, we used cross 12 that was previously developed to investigate large transcripts such as the human immunodeficiency virus hiv-1 13 . cross predicts the structural profile of rna molecules (singleand double-stranded state) at single-nucleotide resolution using sequence information only. here, we performed sequence and structural alignments among 62 sars-cov-2 strains and identified the conservation of specific elements in the spike s region, which provides clues on the evolution of domains involved in the binding to ace2 and sialic acid. as highly structured regions of rna molecules have strong propensity to form stable contacts with proteins 14 and promote assembly of specific complexes 15, 16 , sars-cov-2 domains enriched in double-stranded content are expected to establish interactions within host cells that are important to replicate the virus 17 . to investigate the interactions of sars-cov-2 rna with human proteins, we employed catrapid 18, 19 . catrapid 20 estimates the binding potential of a specific protein for an rna molecule through van der waals, hydrogen bonding and secondary structure propensities allowing identification of interaction partners with high confidence 21 . the unbiased analysis of more than 100000 protein interactions with sars-cov-2 rna reveals that the 5' of sars-cov-2 has strong propensity to bind to human proteins involved in viral infection and especially reported to be associated with hiv infection. a comparison between sars-cov and hiv reveals indeed similarities 22 , but the relationship between sars-cov-2 and hiv is still unexplored. interestingly, hiv and sars-cov-2, but not sars-cov nor mers-cov, have a furin-cleavage site occurring in the spike s protein, which could explain the spread velocity of sars-cov-2 compared to sars-cov and mers-cov 23, 24 . yet, many processes related to sars-cov-2 replication are unknown and our study aims to suggest relevant protein interactions for further investigation. we hope that our large-scale calculations of structural properties and binding partners of sars-cov-2 will be useful to identify the mechanisms of virus replication within the human host. structured elements within rna molecules attract proteins 14 and reveal regions important for interactions with the host 25 . indeed, each gene expressed from sars-cov-2 is preceded by conserved transcription-regulating sequences that act as signal for the transcription complex during the synthesis of the rna minus strand to promote a strand transfer to the leader region to resume the synthesis. this process is named discontinuous extension of the minus strand and is a variant of similarity-assisted template switching that operates during viral rna recombination 17 . to analyze sars-cov-2 structure (reference wuhan strain mn908947.3), we employed cross 12 to predict the double-and single-stranded content of rna genomes such as hiv-1 13 . we found the highest density of double-stranded regions in the 5' (nucleotides 1-253), membrane m protein (nucleotides 26523-27191), spike s protein (nucleotides 23000-24000), and nucleocapsid n protein (nucleotides 2874-29533; fig. 1 ) 26 . the lowest density of double-stranded regions were observed at nucleotides 6000-6250 and 20000-21500 and correspond to the regions between the nonstructural proteins nsp14 and nsp15 and the upstream region of the spike surface protein s (fig. 1 ) 26 . in addition to the maximum corresponding to nucleotides 23000-24000, the structural content of spike s protein shows minima at around nucleotides 20500 and 24500 (fig. 1) . we used the vienna method 27 to further investigate the rna secondary structure of specific regions identified with cross 13 . employing a 100 nucleotide window centered around cross maxima and minima, we found good match between cross scores and vienna free energies ( fig. 1 ). strong agreement is also observed between cross and vienna positional entropy, indicating that regions with the highest structural content have also the lowest structural diversity. our analysis suggests the presence of structural elements in sars-cov-2 that have evolved to interact with specific human proteins 11 . our observation is based on the assumption that structured regions have an intrinsic propensity to recruit proteins 14 , which is supported by the fact that structured transcripts act as scaffolds for protein assembly 15, 16 . we employed crossalign 13 to study the structural conservation of sars-cov-2 in different strains (materials and methods). in our analysis, we compared the wuhan strain mn908947.3 with around 2800 other coronaviruses (data from ncbi) with human ( fig. 2) or other hosts (supp. fig. 1) . when comparing sars-cov-2 with human coronaviruses (1387 strains, including sars-cov and mers-cov), we found that the most conserved region falls inside the spike s genomic locus (fig. 2) . more precisely, the conserved region is between nucleotides 23000 -24000 and exhibits an intricate and stable to better investigate the sequence conservation of sars-cov-2, we compared 62 strains isolated from different countries during the pandemic (including china, usa, japan, taiwan, india, brazil, sweden, and australia; data from ncbi and in vipr www.viprbrc.org; materials and methods). our analysis aims to determine the relationship between structural content and sequence conservation. using clustal w for multiple sequence alignments 28 , we observed general conservation of the coding regions (fig. 3a) . the 5' and 3' show high variability due to experimental procedures of the sequencing and are discarded in this analysis 29 . one highly conserved region is between nucleotides 23000 -24000 in the spike s genomic locus, while sequences up-and downstream are variable (red bars in fig. 3a) . we then used crossalign 13 to compare the structural content (materials and methods). high variability of structure is observed for both the 5' and 3' and for nucleotides between 21000 -22000 as well as 24000 -25000, associated with the s region (red bars in fig. 3a) . the rest of the regions are significantly conserved at a structural level (p-value < 0.0001; fisher's test). we then compared protein sequences coded by the spike s genomic locus (ncbi reference qhd43416) and found that both sequence (fig. 3a) and structure (fig. 2) of nucleotides 23000 -24000 are highly conserved. the region corresponds to amino acids 330-500 that contact the host receptor angiotensin-converting enzyme 2 (ace2) 30 promoting infection and provoking lung injury 24, 31 . by contrast, the region upstream of the binding site receptor ace2 and located in correspondence to the minimum of the structural profile at around nucleotides 22500-23000 ( fig. 1) is highly variable 32 , as indicated by t-coffee multiple sequence alignments 32 (fig. 3a) . this part of the spike s region corresponds to amino acids 243-302 that in mers-cov binds to sialic acids regulating infection through cell-cell membrane fusion ( fig. 3b ; see related manuscript by e. milanetti et al.) 10, 33, 34 . our analysis suggests that the structural region between nucleotides 23000 and 24000 of spike s region is conserved among coronaviruses (fig. 2) and that the binding site for ace2 has poor variation in human sars-cov-2 strains (fig. 3b) . by contrast, the region upstream, which has propensity to bind sialic acids 10, 33, 34 , showed poor structural content and high variability (fig. 3b) . in order to obtain insights on how the virus replicates in human cells, we predicted sars-cov-2 interactions with the whole rna-binding human proteome. following a protocol to study structural conservation in viruses 13 , we first divided the wuhan sequence in 30 fragments of 1000 nucleotides each moving from the 5' to 3' and then calculated the protein-rna interactions of each fragment with catrapid omics (3340 canonical and putative rna-binding proteins, or rbps, for a total 102000 interactions) 18 . proteins such as polypyrimidine tract-binding protein 1 ptbp1 (uniprot p26599) showed the highest interaction propensity (or z-score; materials and methods) at the 5' while others such as heterogeneous nuclear ribonucleoprotein q hnrnpq (o60506) showed the highest interaction propensity at the 3', in agreement with previous studies on coronaviruses ( fig. 4a ) 35 . for each fragment, we predicted the most significant interactions by filtering according to the z score. we used three different thresholds in ascending order of stringency: z ³ 1.50, 1.75 and 2 respectively and we removed from the list the proteins that were predicted to interact promiscuously with more than one fragment. fragment 1 corresponds to the 5' and is the most contacted by rbps (around 120 with z³2 high-confidence interactions; fig. 4b ), which is in agreement with the observation that highly structured regions attract a large number of proteins 14 . indeed, the 5' contains multiple stem loop structures that control rna replication and transcription 36, 37 . by contrast, the 3' and fragment 23 (spike s), which are still structured but to a lesser extent, attract fewer proteins (10 and 5, respectively), while fragment 20 (between orf1ab and spike s) that is predicted to be unstructured, does not have binding partners. the interactome of each fragment was then analysed using clevergo, a tool for gene ontology (go) enrichment analysis 38 . proteins interacting with fragments 1, 2 and 29 were associated with annotations related to viral processes ( fig. 4c ; supp. table 1 ). considering the three thresholds applied (materials and methods), we found 22 viral proteins for fragment 1, 2 proteins for fragment 2 and 11 proteins for fragment 29 (fig. 4d) . among the high-confidence interactors of fragment 1, we discovered rbps involved in positive regulation of viral processes and viral genome replication, such as double-stranded rna-specific editase 1 adarb1 (uniprot p78563 39 ), 2-5-oligoadenylate synthase 2 oas2 (p29728) and 2-5adependent ribonuclease rnasel (q05823). interestingly, 2-5-oligoadenylate synthase 2 oas2 has been reported to be upregulated in human alveolar adenocarcinoma (a549) cells infected with sars-cov-2 (log fold change of 4.2; p-value of 10 -9 and q-value of 10 -6 ) 40 . while double-stranded rna-specific adenosine deaminase adar (p55265) is absent in our library due to its length that does not meet catrapid omics requirements 18 , the omixcore extension of the algorithm specifically developed for large molecules 41 attributes the same binding propensity to both adarb1 and adar, thus indicating that the interactions might occur (materials and methods). moreover, experimental works indicate that the family of adar deaminases is active in bronchoalveolar lavage fluids derived from sars-cov-2 patients 42 and is upregulated in a549 cells infected with sars-cov-2 (log fold change of 0.58; p-value of 10 -8 and q-value of 10 -5 ) 40 . we also identified proteins related to the establishment of integrated proviral latency, including xray repair cross-complementing protein 5 xrcc5 (p13010) and x-ray repair cross-complementing protein 6 xrcc6 (p12956; fig. 4b and 4e). in accordance with our calculations, comparison of a549 cells responses to sars-cov-2 and respiratory syncytial virus, indicates upregulation of xrrc6 in sars-cov-2 (log fold-change of 0.92; p-value of 0.006 and q-value of 0.23) 40 . nucleolin ncl (p19338), a protein known to be involved in coronavirus processing, was also predicted to bind tightly to the 5' (supp. table 1) 43 . importantly, we found proteins related to defence response to viruses, such as atp-dependent rna helicase ddx1 (q92499), that are involved in negative regulation of viral genome replication. some dna-binding proteins such as cyclin-t1 ccnt1 (o60563), zinc finger protein 175 znf175 (q9y473) and prospero homeobox protein 1 prox1 (q92786) were included because they could have potential rna-binding ability (fig. 4e) 44 . as for fragment 2, we found two canonical rbps: more than simple scaffold elements, gag proteins are versatile elements that bind to viral and host proteins as they traffic to the cell membrane (supp. table 1 ) 45 . analysis of functional annotations carried out with genemania 46 revealed that proteins interacting with the 5' of sars-cov-2 rna are associated with regulatory pathways involving notch2, myc and max that have been previously connected to viral infection processes ( fig. 4e) 47, 48 . interestingly, some proteins, including ddx1, ccnt1 and znf175 for fragment 1 and trim32 for fragment 2, have been shown to be necessary for hiv functions and replication inside the cell. recently, gordon et al. reported a list of human proteins binding to open reading frames (orfs) translated from sars-cov-2 58 . identified through affinity purification followed by mass spectrometry quantification, 332 proteins from hek-293t cells interact with viral orf peptides. by selecting 274 proteins binding at the 5' with z score ³1.5 (supp . table 1) , of which 140 are exclusively interacting with fragment 1 (fig. 4b) , we found that 8 are also reported in the list by gordon et al. 58 , which indicates significant enrichment (representation factor of 2.5; p-value of 0.02; hypergeometric test with human proteome in background). the fact that our list of proteinrna binding partners contains elements identified also in the protein-protein network analysis is not surprising, as ribonucleoprotein complexes evolve together 14 and their components sustain each other through different types of interactions 16 . we note that out of 332 interactions, 60 are rbps (as reported in uniprot 39 ), which represents a considerable fraction (i.e., 20%), considering that there are around 1500 rbps in the human proteome (i.e., 6%) and fully justified by the fact that they involve association with viral rna. comparing the rbps present in gordon et al. 58 table 2) . interestingly, srp72, larp7 and larp4b proteins assemble in stress granules [61] [62] [63] that are the targeted by rna viruses 53 . we speculate that sequestration of these elements is orchestrated by a viral program aiming to recruit host genes 49 protein kinase a radixin rdx (p35241; in addition to those mentioned above; supp. table 2 ). in the list of 274 proteins binding to the 5' (fragment 1) with z score ≥1.5, we found 10 hits associated with hiv (supp. table 3 (q06787) and rna polymerase-associated protein rtf1 homologue (q92541; supp. table 3 ). by contrast, no significant enrichments were found for other viruses such as for instance ebola. 65 , among other targets. in addition, hvb-related targets are nuclear receptor subfamily 5 group a member 2 nr5a2 (chembl3544), interferon-induced, double-stranded rna-activated protein kinase eif2ak2 (chembl5785) and srsf protein kinase 1 srpk1 (chembl4375). we hope that this list can be the starting point for further pharmaceutical studies. a number of proteins identified in our catrapid calculations have been previously reported to coalesce in large ribonucleoprotein assemblies such as stress granules. among these proteins, we found double-stranded rna-activated protein kinase eif2ak2 (p19525), nucleolin ncl (p19338), atp-dependent rna helicase ddx1 (q92499), cyclin-t1 ccnt1 (o60563), signal recognition particle subunit srp72 (o76094), larp7 (q4g0j3) and la-related protein 4b heterogeneous nuclear ribonucleoprotein q hnrnpq (o60506) 63 . to further investigate the propensity of these proteins to phase separate in stress granules, we used the catgranule algorithm (materials and methods) 66 . we found that the 274 proteins binding to the 5' (fragment 1) with z score ≥1.5 are highly prone to accumulate in stress-granules (274 proteins with the lowest z score are used in the comparison; p-value<0.0001; kolmogorov-smirnoff; fig. 4g ; supp. table 4 ). this finding is particularly relevant because rna viruses are known to antagonize stress granules formation 53 . indeed, the role of stress granules and processing bodies in translation suppression and rna decay have impact on virus replication 67 . spreading. using advanced computational approaches, we investigated the structural content of sars-cov-2 rna and predicted human proteins that bind to it. we employed cross 13, 68 to compare the structural properties of 2800 coronaviruses and identified elements conserved in sars-cov-2 strains. the regions containing the highest amount of structure are the 5' as well as glycoproteins spike s and membrane m. we found that the spike s protein domain encompassing amino acids 330-500 is highly conserved across sars-cov-2 strains. this result suggests that spike s must have evolved to specifically interact with its host partner ace2 30 and mutations increasing the binding affinity are highly infrequent. as the nucleic acids encoding for this region are enriched in double-stranded content, we speculate that the structure might attract host regulatory elements, thus further constraining the variability. the fact that the spike s region is highly conserved among all the analysed sars-cov-2 strains suggests that a specific drug can be designed against it to prevent interactions within the host. by contrast, the highly variable region at amino acids 243-302 in spike s protein corresponds to the binding site of sialic acids in mers-cov (see manuscript by e. milanetti et al.) 7, 10, 34 and could play a role in infection 33 . the fact that the binding region changes in the different strains might indicate a variety of binding affinities for sialic acids, which could provide clues on the specific responses in the human population. interestingly, the sialic acid binding site is absent in sars-cov but present in mers-cov, which represents an important difference between the diseases. both our sequence and structural analyses of spike s protein indicate high conservation among coronaviruses and suggest that human engineering of sars-cov-2 is highly unlikely. using catrapid 18, 19 we computed >100000 protein interactions with sars-cov-2 and found previously reported interactions such as polypyrimidine tract-binding protein 1 ptbp1, heterogeneous nuclear ribonucleoprotein q hnrnpq and nucleolin ncl 43 . in addition, we discovered that the highly structured region at the 5' has the largest number of protein partners including atp-dependent rna helicase ddx1 that was previously reported to be essential for hiv-1 and coronavirus ibv replication 54,55 , and the double-stranded rna-specific editases adar and adarb1 that catalyse the hydrolytic deamination of adenosine to inosine 49 . other predicted interactions are xrcc5 and xrcc6 members of the hdp-rnp complex associating with atp-dependent rna helicase dhx9 69 as well as and 2-5a-dependent ribonuclease rnasel and 2-5-oligoadenylate synthase 2 oas2 that control viral rna degradation 70, 71 . interestingly, ddx1, xrcc6 and oas2 are upregulated in human alveolar adenocarcinoma cells infected with sars-cov-2 40 . in agreement with our predictions, recent experimental work indicates that the family of adar deaminases is active in bronchoalveolar lavage fluids derived from sars-cov-2 patients 42 . a significant overlap exists with the list of protein interactions reported by gordon et al. 58 , and among the candidate partners we identified akap8l, involved as a dead/h-box rna helicase binding protein involved in hiv infection 59 . in general, proteins associated with retroviral replication are expected to play different roles in sars-cov-2. as sars-cov-2 massively represses host gene expression 49 , we hypothesize that the virus hijacks host pathways by recruiting transcriptional and post-transcriptional elements interacting with polymerase ii genes and splicing factors such as for instance a-kinase anchor protein 8-like akap8l and la-related protein 7 larp7 that is upregulated in human alveolar adenocarcinoma cells infected with sars-cov-2 40 . the link to proteins previously studied in the context of hiv and other viruses, if further confirmed fro, is particularly relevant for the repurposing of existing drugs 65 . the idea that sars-cov-2 sequesters different elements of the transcriptional machinery is particularly intriguing and is supported by the fact that a large number of proteins identified in our screening are found in stress granules 63 . indeed, stress granules protect the host innate immunity and are hijacked by viruses to favour their own replication 67 . moreover, as coronaviruses transcription uses discontinuous rna synthesis that involves high-frequency recombination 43 , it is possible that pieces of the viruses resulting from a mechanism called defective interfering rnas 72 could act as scaffold to attract host proteins 14, 15 . we predicted the secondary structure of transcripts using cross (computational recognition of secondary structure 13, 68 . cross was developed to perform high-throughput rna profiling. the algorithm predicts the structural profile (single-and double-stranded state) at single-nucleotide resolution using sequence information only and without sequence length restrictions (scores > 0 indicate double stranded regions). we used the vienna method 27 to further investigate the rna secondary structure of minima and maxima identified with cross 13 . we used crossalign 13,68 an algorithm based on dynamic time warping (dtw), to check and evaluate the structural conservation between different viral genomes 13 . crossalign was previously employed to study the structural conservation of ~5000 hiv genomes. sars-cov-2 fragments (1000 nt, not overlapping) were searched inside other complete genomes using the obe (open begin and end) module, in order to search a small profile inside a larger one. the lower the structural distance, the higher the structural similarities (with a minimum of 0 for almost identical secondary structure profiles). the significance is assessed as in the original publication 13 . the fasta sequences of the complete genomes of sars-cov-2 were downloaded from virus pathogen resource (vipr; www.viprbrc.org), for a total of 62 strains. regarding the overall coronaviruses, the sequences were downloaded from ncbi selecting only complete genomes, for a total of 2862 genomes. the reference wuhan sequence with available annotation (epi_isl_402119) was downloaded from global initiative on sharing all influenza data. (gisaid https://www.gisaid.org/). interactions between each fragment of target sequence and the human proteome were predicted using catrapid omics 18, 19 , an algorithm that estimates the binding propensity of protein-rna pairs by combining secondary structure, hydrogen bonding and van der waals contributions. as we used clustal w 28 for 62 sars-cov-2 strains alignments and t-coffee 32 for spike s proteins alignments. the variability in the spike s region was measured by computing shannon entropy on translated rna sequences. the shannon entropy is computed as follows: s(a) = -sum_i p(a,i) log p(a,i) where a correspond to the amino acid at the position i and p(a,i) is the frequency of a certain amino-acid a at position i of the sequence. low entropy indicates poorly variability: if p(a,x) = 1 for one a and 0 for the rest, then s(x) =0. by contrast, if the frequencies of all amino acids are equally distributed, the entropy reaches its maximum possible value. catgranule 66 was employed to identify proteins assembling into biological condensates. scores > 0 indicate that a protein is prone to phase separate. structural disorder, nucleic acid binding propensity and amino acid patterns such as arginine-glycine and phenylalanine-glycine are key features combined in this computational approach 66 . fig. 1 a novel coronavirus from patients with pneumonia in china coronaviruses and immunosuppressed patients. the facts during the third epidemic evaluation and treatment coronavirus (covid-19). in statpearls isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry isolation and characterization of 2019-ncov-like coronavirus from malayan pangolins structures of mers-cov spike glycoprotein in complex with sialoside attachment receptors cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein the structure and functions of coronavirus genomic 3' and 5' ends a high-throughput approach to profile rna structure a method for rna structure prediction shows evidence for structure in lncrnas rna structure drives interaction with proteins an integrative study of protein-rna condensates identifies scaffolding rnas and reveals players in fragile x-associated tremor/ataxia syndrome phase separation drives x-chromosome inactivation: a hypothesis identification of a coronavirus transcription enhancer catrapid omics: a web server for large-scale prediction of protein-rna interactions quantitative predictions of protein interactions with long noncoding rnas predicting protein associations with long noncoding rnas rnact: protein-rna interaction predictions for model organisms with supporting experimental data cloaked similarity between hiv-1 and sars-cov suggests an anti-sars strategy inhibition of furin-mediated cleavage activation of hiv-1 glycoprotein gp160 differential downregulation of ace2 by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl63 conserved structural rna domains in regions coding for cleavage site motifs in hemagglutinin genes of influenza viruses genome composition and divergence of the novel coronavirus (2019-ncov) originating in china viennarna package 2.0 the embl-ebi search and sequence analysis tools apis in 2019 rna-seq methods for transcriptome analysis the proximal origin of sars-cov-2 a pneumonia outbreak associated with a new coronavirus of probable bat origin t-coffee: a web server for the multiple sequence alignment of protein and rna sequences using structural information and homology extension distinct roles for sialoside and protein receptors in coronavirus infection silico evidence for two receptors based strategy of sars-cov-2 host cell proteins interacting with the 3' end of tgev coronavirus genome influence virus replication structural determinants and mechanism of hiv-1 genome packaging an overview of their replication and pathogenesis protein aggregation, structural disorder and rna-binding ability: a new approach for physico-chemical and gene ontology classification of multiple datasets uniprot: a worldwide hub of protein knowledge sars-cov-2 launches a unique transcriptional signature from in vitro, ex vivo, and in vivo systems omixcore: a web server for prediction of protein interactions with large rna evidence for host-dependent rna editing in the transcriptome of sars-cov-2 rna-rna and rnaprotein interactions in coronavirus replication and transcription insights into rna biology from an atlas of mammalian mrna-binding proteins hiv gag polyprotein: processing and early viral particle assembly the genemania prediction server: biological network integration for gene prioritization and predicting gene function viral interactions with the notch pathway what retroviruses teach us about the involvement of c-myc in leukemias and lymphomas the architecture of sars-cov-2 transcriptome. biorxiv (now cell in press) drllps: a data resource of liquid-liquid phase separation in eukaryotes liquid nuclear condensates mechanically sense and restructure the genome phase separation of signaling molecules promotes t cell receptor signal transduction a dead box protein facilitates hiv-1 replication as a cellular co-factor of rev the cellular rna helicase ddx1 interacts with coronavirus nonstructural protein 14 and enhances viral replication cyclin t1 domains involved in complex formation with tat and tar rna are critical for tat-activation role of the human and murine cyclin t proteins in regulating hiv-1 tat-activation a sars-cov-2-human protein-protein interaction map reveals drug targets and potential drug-repurposing the role of a-kinase anchoring protein 95-like protein in annealing of trnalys3 to hiv-1 rna the la-related protein larp7 is a component of the 7sk ribonucleoprotein and affects transcription of cellular and viral polymerase ii genes a stimulatory role for the la-related protein 4b in translation larp4b is an au-rich sequence associated factor that promotes mrna accumulation and translation context-dependent and disease-specific diversity in protein interactions within stress granules systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7sk capping enzyme chembl: towards direct deposition of bioassay data a concentration-dependent liquid phase separation can cause toxicity upon increased protein expression viral regulation of rna granules in infected cells a high-throughput approach to profile rna structure dna-dependent protein kinase (dna-pk) phosphorylates nuclear dna helicase ii/rna helicase a and hnrnp proteins in an rnadependent manner the nature of the catalytic domain of 2'-5'-oligoadenylate synthetases enzymatic characteristics of recombinant medium isozyme of 2'-5' oligoadenylate synthetase defective interfering rnas: foes of viruses and friends of virologists structure and interactions of sars-cov-2 population; p_bonf = p-value of the enrichment. to correct for multiple testing bias, use bonferroni correction) 38 ; (d) viral processes are the third largest cluster identified in our analysis; (e) protein interactions with the 5' of sars-cov-2 rna (inner circle) and associations with other human genes retrieved from literature (green: genetic associations number of rbp interactions identified by gordon et al. 58 for different sars-cov-2 regions (see panel a for reference). (g) proteins binding to the 5' with z score ≥ 1.5 show high propensity to accumulate in stress-granules the authors would like to thank dr. mattia miotto, dr lorenzo di rienzo, dr. alexandros armaos, dr. alessandro dasti and dr. claudia giambartolomei for discussions. we are particularly grateful to prof. annalisa pastore for critical reading, dr. gilles mirambeau for the rt vs rdrp analysis, dr. andrea cerase for the discussing on stress granules and dr. roberto giambruno for pointing to ptbp1 and hnrnpq experiments.the research leading to these results has been supported by european research council (ribomylome_309545 and astra_855923), the h2020 projects iasis_727658 and key: cord-103255-4k13re9y authors: daniell, henry; streatfield, stephen j; wycoff, keith title: medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants date: 2001-05-01 journal: trends in plant science doi: 10.1016/s1360-1385(01)01922-7 sha: doc_id: 103255 cord_uid: 4k13re9y abstract the use of plants for medicinal purposes dates back thousands of years but genetic engineering of plants to produce desired biopharmaceuticals is much more recent. as the demand for biopharmaceuticals is expected to increase, it would be wise to ensure that they will be available in significantly larger amounts, on a cost-effective basis. currently, the cost of biopharmaceuticals limits their availability. plant-derived biopharmaceuticals are cheap to produce and store, easy to scale up for mass production, and safer than those derived from animals. here, we discuss recent developments in this field and possible environmental concerns. research in the past few decades has revolutionized the use of therapeutically valuable proteins in a variety of clinical treatments. because most genes can be expressed in many different systems, it is essential to determine which system offers the most advantages for the production of the recombinant protein. the ideal expression system would be the one that produces the most safe, biologically active material at the lowest cost. the use of modified mammalian cells with recombinant dna techniques has the advantage of resulting in products that are identical to those of natural origin; however, culturing these cells is expensive and can only be carried out on a limited scale. the use of microorganisms such as bacteria permits manufacture on a larger scale, but introduces the disadvantage of producing products that differ appreciably from the products of natural origin. for example, proteins that are usually glycosylated in humans are not glycosylated by bacteria. furthermore, human proteins that are expressed at high levels in e. coli frequently acquire an unnatural conformation accompanied by intracellular precipitation, owing to lack of proper folding and disulfide bridges. the production of recombinant proteins in plants has many potential advantages for generating biopharmaceuticals relevant to clinical medicine. first, plant systems are more economical than industrial facilities using fermentation or bioreactor systems. second, the technology is already available for harvesting and processing plants and plant products on a large scale. third, the purification requirement can be eliminated when the plant tissue containing the recombinant protein is used as a food (edible vaccines). fourth, plants can be directed to target proteins into intracellular compartments in which they are more stable, or even to express them directly in certain compartments (chloroplasts). fifth, the amount of recombinant product that can be produced approaches industrial-scale levels. last, health risks arising from contamination with potential human pathogens or toxins are minimized. in the decade since the expression and assembly of immunoglobulin (ig) heavy and light chains into functional antibodies was first shown in transgenic tobacco, plants have proven to be versatile production systems for many forms of antibodies. these include full-sized igg and iga, chimeric igg and iga, secretory igg and iga, single-chain fv fragments (scfv), fab fragments and heavy-chain variable domains. recently, this list has been extended to include bispecific antibodies, which are made by the genetic fusion of two different scfvs via a flexible peptide linker 1 . plants have great potential as a virtually unlimited source of inexpensive monoclonal antibodies (dubbed 'plantibodies') for human and animal therapeutics (table 1) . there is not yet a consensus as to the best plant species or tissue for commercial antibody production. most antibodies expressed to date have been in tobacco, although recently potatoes, soybean, alfalfa, rice and wheat have also been used successfully 2-6 . the major advantage of using green tissue (tobacco, alfalfa, soybean) is sheer productivity. both alfalfa and tobacco can support several crops (cuttings) per year, with potential annual biomass yields of 25 tonne ha −1 and >100 tonne ha −1 , respectively. by contrast, the maximum yields of wheat, rice and corn seed are ~3 tonne ha −1 , 6 tonne ha −1 and 12 tonne ha − 1 , respectively. other advantages of tobacco include its relative ease of genetic manipulation, production of large numbers of seeds (up to a million per plant) and an impending need to explore alternate uses for this hazardous crop. however, seeds are likely to have fewer phenolic compounds and a less complex mixture of proteins and lipids than green leaves, which might be an advantage in purification. another advantage of seeds or tubers is their ability to be stored for long periods. levels of scfv in rice seeds did not show a significant decline after storage at room temperature for six months 5 . potato tubers in cold storage for 18 months lost only 50% of functional antibody 2 . for short periods of time (five to seven days), dried tobacco and alfalfa leaves can also be stored with little loss of scfv (ref. 7) or igg antibody 4 . purification of antibody from stored plant material has the advantages that the processing facility need not be near the field and can be used continually all year, rather than for just a few large batches. to date, only four antibodies have been made in plants that are potentially useful as human therapeutics. only one of these has been tested in humans: a chimeric secretory igg-iga antibody against a surface antigen of streptococcus mutans, the primary causal agent of tooth decay. this tobaccoproduced antibody was applied topically to teeth and found to be as effective as an igg produced in a murine hybridoma at preventing recolonization by s. mutans 8 . the second antibody, a humanized anti-herpes-simplex virus (hsv) antibody made in soybean, was effective in the prevention of vaginal hsv-2 transmission in a mouse model 3 . its activity was indistinguishable both in vitro and in vivo from the monoclonal antibody produced in cell culture. a third antibody, against carcinoembryonic antigen (cea), has recently been expressed in rice and wheat 5 . cea, a cell-surface glycoprotein, is one of the best-characterized tumor-associated antigens. antibodies against cea are used for in vivo tumor imaging, as well as in antibody-based cancer therapy. levels of scfv in seeds did not show a significant decline after storage at room temperature for six months. this same antibody has been expressed in a rice cell culture 6 . the fourth antibody is an example of both a novel use of plant-produced antibodies and an alternative production system. a plant virus vector has been used to produce a tumor-specific vaccine transiently in tobacco for the treatment of lymphoma 9 . the antibody genes for expression of an scfv were derived from a mouse b-cell lymphoma. the plantproduced scfv was used to immunize mice, which generated anti-idiotypic antibodies (antibodies against the binding portion of the antibody). these mice were protected against infection by the lymphoma that produced the original antibody. other groups have used modified plant viral vectors to produce therapeutically useful antibodies in plants, including an antibody against the colorectalcancer-associated antigen ga733-2 (ref. 10). although these vectors might find limited usefulness if the rapid production of an antibody is necessary (perhaps in greenhouse production), their acceptability to regulatory agencies (e.g. the us food and drug administration, dept of agriculture and environmental protection agency) has not been tested. there are no plantibodies yet in commercial production, therefore estimates of cost are difficult to find and involve many assumptions. the costs of producing an igg from alfalfa grown in a 250 m 2 greenhouse are estimated to be us$500-600 g −1 , compared with us$5000 g −1 for the hybridomaproduced antibody 4 . planet biotechnology (mountain view, ca, usa) has compared the cost per gram of purified iga made by cell culture, transgenic goats, grain (7.5 tonne ha −1 ) and green biomass (120.0 tonne ha −1 ) (fig. 1) . expression levels will have a significant impact on the costs but, at the best expression level reported [500 µg g −1 leaf for a secretory iga (ref. 11)], the final cost should be well below us$50 g −1 . this significantly undercuts the costs of cell culture (us$1000 g −1 ) or transgenic animal production systems (us$100 g −1 ). the biggest component of cost with plantibodies will be purification. however, expression in seeds of rice and wheat 5 opens up the possibility of oral administration of some therapeutic antibodies without the need for expensive purification. some of the properties of igs depend on their glycosylation (e.g. binding to monocyte fc receptors). there is one conserved n-glycosylation site in the ch 2 domain of igg. the structures of n-linked glycans on plant-and murine-produced guy's 13 (an igg1) have been determined and compared 12 . the plantibody n-glycans were more structurally diverse, with 40% being of the high-mannose type. the other 60% of the plantibody oligosaccharides had β-(1,2)-xylose and α-(1,3)-fucose linked to the man 3 glcnac 2 core. these linkages are typical of plants but are not found in mammalian n-glycans. the plantibody also lacked sialic acid, which represented ~10% of the sugar content of the mouse monoclonal antibody. these differences in glycan structure appear to have no effect on antigen binding or affinity in vitro 3,4,11,13 and might not be significant in vivo either. an igg produced in alfalfa had a serum half-life in balb/c mice that was indistinguishable from that of the hybridoma-produced antibody 4 . however, there is some concern about the potential immunogenicity and allergenicity of plantibodies used as human therapeutics. for mucosal applications, this is not likely to present problems for most people because plant glycoproteins are ubiquitous in the human diet. there has been no evidence of allergic reaction or of a human antimouse antibody (hama) response in 60 patients receiving topical oral application of a secretory iga specific to s. mutans 8 . proteins of microbial and viral pathogens were some of the earliest examples chosen to show the feasibility of transgenic plant expression systems 14-17 . the rationale was that key immunogenic proteins of major pathogens could be synthesized in plant tissues and then fed as edible . costs for plants compare green biomass (120.0 tonne ha −1 ) and seed production (7.5 tonne ha −1 ). cost differences are based primarily on production costs, and it was assumed that purification costs and losses during purification will be the same for all systems. subunit vaccines to humans or commercially important animals. the proof of this concept has since been shown using several bacterial and viral proteins ( table 2 ). the practical aspects of choosing particular foodstuffs in which to deliver defined doses of a vaccine are being explored, and efforts are under way to establish clear regulatory paths for the development of edible vaccines. oral delivery of vaccines is an attractive alternative to injection, largely for reasons of low cost and easy administration. the chances of acquiring mucosal immunity against infectious agents that enter the body across a mucosal surface are also increased with oral vaccines. however, a major concern with oral vaccines is the degradation of protein components in the stomach and gut before they can elicit an immune response. to guard against degradation, several delivery vehicles have been developed to ferry intact proteins to the gut. these include recombinant strains of attenuated microorganisms, bioencapsulation vehicles such as liposomes and transgenic plant tissues. early work with plant-based subunit vaccines used the readily transformed species tobacco, potato and tomato 14-18 . however, the most attractive species for expressing subunit vaccine components should have high levels of soluble protein that is stable during storage; seed crops such as cereals are particularly suitable. the embryo fraction is rich in soluble protein and can easily be separated from other seed tissue to increase the concentration of antigen and thus decrease the dose size. the choice of crop defines the type of material to be fed. many plant tissues can be consumed raw but others must be processed. processing facilitates the creation of a homogeneous sample, enabling a defined dose size, but it is important that any heat or pressure treatments involved do not destroy the antigen. alternative processing steps have been applied to a candidate vaccine component against enterotoxigenic strains of e. coli that consists of the b subunit of the heat-labile toxin (lt-b) expressed in corn. a typical 1 mg dose of lt-b could be delivered in an embryo fraction, to decrease the volume of the dose, or in a 'cooked' whole corn snack, to increase palatability and enhance stable storage (fig. 2) . in this case, neither treatment degrades the antigen. for commercial animal vaccines, the relevant protein can be expressed in a plant tissue that constitutes a major proportion of the diet, and heat and pressure treatments are not necessary. some key examples that illustrate the range of candidate proteins under investigation and plant expression systems being used are given in table 2 . plant-expressed antigens have been shown to able to induce mucosal and serum immune responses when administered parenterally or orally to experimental animals and, in some test cases, they have offered protection against a subsequent pathogen challenge or challenge model 14,16, [19] [20] [21] [22] [23] [24] . a few of these vaccine candidates have been successfully tested in clinical trials or, where appropriate, in commercial or native animal trials [25] [26] [27] [28] [29] [30] . thus, edible vaccines delivered in plant tissues or processed plant products show great potential for efficacy in target organisms. the bioencapsulation of lt-b in transgenic corn material results in an increased mucosal immune response compared with that achieved with naked antigen when fed to mice 30 . presumably, this is because the antigen is protected from degradation in the gut, and it augurs well for the development of plant-based edible vaccines. the quantity of plant tissue constituting a vaccine dose must be of a practical size for consumption. thus, achieving a high level of expression is crucial. the expression of vaccine components in plants has been increased by using a range of leader and polyadenylation signals 31 and by optimizing codon usage for plants 22, 29, 30 . expression could also be raised through crosses of transformed lines to various genetic backgrounds, an approach that has been successfully applied to boost protein production in corn. it is also important that any vaccine component should be present in its native form in the transgenic plant tissue. this has been assessed in several cases by examining the size of the synthesized protein, its ability to form higher-order complexes that mirror microbial or viral structures and, where relevant, by showing an enzymatic or receptor-binding activity 14,16-18, 22, 24, 29 . the stability of heterologous proteins and the assembly of multisubunit structures depend on the cellular environment and therefore on the subcellular location. favored locations for the expression of selected subunit vaccine components are the cell surface and the endoplasmic reticulum and golgi body 14,18,29,31,32 . as with antibodies, transient expression systems (in which candidate vaccine sequences are incorporated into plant viral surface proteins) have also been investigated extensively and high levels of expression have been achieved. a related strategy to that of edible vaccines uses transgenic plants expressing autoantigens, whereby a large oral dose of an autoantigen can inhibit the development of an autoimmune disease through the mechanism of oral tolerance. this approach has been successful in a mouse model for diabetes 33 . generally, levels of pharmaceutical proteins produced in transgenic plants have been less than the 1% of total soluble protein that is needed for commercial feasibility if the protein must be purified 34 . plantderived recombinant hepatitis-b surface antigen induced only a low level serum antibody response in a small human study, probably reflecting the low level of expression (1-5 ng g −1 fresh weight) in transgenic lettuce 27 . in spite of recent improvements in expression levels in potato with a view to clinical trials 31 , expression levels should be increased further for practical purposes. also, even though norwalk virus capsid protein expressed in potatoes caused oral immunization when consumed as food, expression levels are too low for large-scale oral administration (0.37% of total soluble protein) 16, 26 . expression of genes encoding other human proteins in transgenic plants has been disappointingly low: human serum albumin, 0.020% total soluble protein; human protein c, 0.001% total soluble protein; erythropoietin,~0.003% total soluble protein; and human interferon-β, <0.001% fresh weight (table 3) . a synthetic gene coding for the human epidermal growth factor was expressed only up to 0.001% of total soluble protein in transgenic tobacco 35, 36 . in spite of several successful reports of high-level expression of non-human proteins (e.g. phytase, glucanase) via the nuclear genome, there is a great need to increase expression levels of human blood proteins to enable the commercial production of pharmacologically important proteins in plants. one alternative approach is to express foreign proteins in chloroplasts of higher plants. foreign genes have been integrated into the tobacco chloroplast genome, giving up to 10 000 copies per cell and resulting in the accumulation of recombinant proteins at up to 47% of the total soluble protein 37 . chloroplast transformation uses two flanking sequences that, through homologous recombination, insert foreign dna into the spacer region between the functional genes of the chloroplast genome, thus targeting the foreign genes to a precise location. this eliminates the 'position effect' upon expression that is frequently observed in transgenic plants with genes inserted into the nuclear genome. in addition, gene silencing has not been observed with chloroplast transformation, whereas it is a common phenomenon with nuclear transformation. chloroplast genetic engineering is an environmentally friendly approach, minimizing several environmental concerns 38, 39 . importantly, chloroplasts can process eukaryotic proteins, including enabling correct folding and the formation of disulfide bridges. chaperonin proteins are present in chloroplasts and might function in the folding and assembly of non-native proteins of both prokaryotic and eukaryotic origins. also, chloroplast proteins are activated by disulfide bond oxidation-reduction cycles using the plastid thioredoxin system 40 or protein disulfide isomerase 41 . accumulation of large quantities of a fully assembled form of human somatotropin with the correct disulfide bonds (7% total soluble protein) 42 provides strong evidence for hyperexpression and assembly of pharmaceutical proteins using this approach. such folding and assembly of foreign proteins should eliminate the need for expensive in vitro processing of pharmaceutical proteins produced in recombinant organisms. for example, 60% of the total operating cost for the commercial production of human insulin in e. coli is associated with in vitro processing (formation of disufide bridges and cleavage of methionine) 43 . purification is likely to represent most of the cost of biopharmaceutical production in plants. for the commercial production of insulin in e. coli, chromatography accounts for 30% of operating expenses and 70% of equipment costs 43 . therefore, new approaches are necessary to minimize or eliminate chromatography in the production of pharmaceutical proteins. one successful recent approach is targeting pharmaceutical proteins to seed oil bodies. this was shown with hirudin, an anticoagulant first isolated from the leech hirudo medicinalis. an oleosin-hirudin fusion protein has been targeted to oil bodies of brassica napus seeds and purified by flotation centrifugation for commercial production in canada 44 . another novel approach is the use of gvgvp as a fusion protein to facilitate single-step purification without the use of chromatography. gvgvp is a protein-based polymer encoded by synthetic genes. at low temperatures, it exists as an extended molecule but, upon raising the temperature above the transition range, the polymer hydrophobically folds into dynamic structures called β-spirals that further aggregate by hydrophobic association to form twisted filaments 45 . using this approach, single-step purification of an insulin-polymer fusion has recently been shown. inverse temperature transition offers several advantages, including facilitating the scale-up of purification from grams to kilograms (o. carmona-sanchez and h. daniell, unpublished) . yet another recent approach is the use of a chaperonin protein to fold foreign proteins into cuboidal crystals, allowing their purification in a single step by centrifugation 37 . one additional advantage of this method is the protection of foreign proteins from cellular proteases. plant-derived biopharmaceuticals should meet the same standards of safety and performance as other production systems. however, many herbal medicines are now exempt from such close scrutiny and are not required to meet the same standards because of their classification as nutritional supplements. because several environmental concerns have been raised by interest groups to confuse public perception, it is of paramount importance that regulating agencies distinguish between real and perceived public concerns (scientific versus non-scientific). if biopharmaceuticals that are potentially harmful are capable of persisting in the environment and might accumulate in non-target organisms, precautionary measures should be taken. induction of biopharmaceutical production after harvesting (as was done in the case of glucocerebrosidase 36 ) might be one approach to minimize environmental exposure, provided that the use of viral vectors does not introduce additional environmental or regulatory concerns. expression of potentially harmful proteins in a form that must be treated for activation might minimize the risk of exposure. for example, hirudin is produced as a fusion protein and is inactive in this form; it is activated only after it is purified from seeds 36 . another hotly debated environmental concern has been the outcrossing of transgenic pollen to weeds or related crops 38, 39 . expression of harmful pharmaceutical proteins in non-target plants resulting from such outcrosses might create public concern and negative perception. several gene containment methods are currently being investigated, including apomixis, incompatible genomes, transgenic mitigation, control of seed dormancy or shattering, suicide genes, infertility barriers, male sterility and maternal inheritance. engineering foreign genes via the chloroplast genome has been shown to contain transgenes effectively, although there are a few exceptions in which the chloroplast genome shows biparental inheritance (e.g. pines) 46 . as an example of an alternative strategy, rnase genes have been expressed under the control of a tissue-specific promoter to destroy the tapetum selectively during anther development, resulting in male sterile plants 47 . there is also concern over the expression of harmful proteins in transgenic pollen. for example, the controversial observation of the toxic effect of bacillus thuringiensis (bt) corn pollen on milkweeds (asclepias spp.) fed to monarch butterfly larvae had a significant impact on public perception, even though the validity of this study has been repeatedly questioned. engineering biopharmaceuticals via the chloroplast genome might be a solution. although the cry protein of bt was expressed at high levels in leaves (up to 47% of total soluble protein), no toxicity was observed when milkweeds dusted with transgenic pollen were fed to monarch butterfly larvae 37 . however, to date, chloroplast genetic engineering has been shown only in tobacco and potato. more recently, several academic and industrial laboratories have initiated projects to extend this technology to other useful crops. also, there are no reports of the production of glycoproteins in transgenic chloroplasts. another public concern is the presence of antibiotic resistance genes or their products (which are used as selective markers) in edible parts of genetically modified crops. however, several approaches are now available to generate plants with transgenes in their nuclear 48 or chloroplast 49 genomes without the use of antibiotic selection. practical considerations will dictate the choice of biopharmaceutical proteins and the crop in which they are to be produced. these include yield, storage conditions, containment properties, initial set up and running costs, purification strategies, size of the market, environmental concerns, public perception and competing technologies. access to several alternative approaches to optimize protein synthesis in plants in an environmentally sound manner augurs well for the safe production of biopharmaceuticals in transgenic plants and for greater availability of these proteins to populations requiring them. toxin b subunit oligomers in transgenic potato plants efficacy of a food plantbased oral cholera toxin b subunit vaccine protective immune response to foot-and-mouth disease virus with vp1 expressed in transgenic plants expression of immunogenic glycoprotein s polypeptides from transmissible gastroenteritis coronavirus in transgenic plants edible vaccine protects mice against escherichia coli heat-labile enterotoxin (lt): potatoes expressing a synthetic lt-b gene immunogenicity of transgenic plant-derived hepatitis b surface antigen induction of a protective antibody response to foot and mouth disease in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein vp1 immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes a plant-derived edible vaccine against hepatitis b virus immunization with potato plants expressing vp60 protein protects against rabbit hemorrhagic disease virus immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants plant-based vaccines: unique advantages production of hepatitis b surface antigen in transgenic plants for oral immunization development of biopharmaceuticals in plant expression systems: cloning, expression and immunological reactivity of human cytomegalovirus glycoprotein b (ul55) in seeds of transgenic tobacco transgenic plants expressing autoantigens fed to mice to induce oral immune tolerance production of recombinant proteins in transgenic plants: practical considerations application of transgenic plants as production systems for pharmaceuticals transgenic plants for therapeutic proteins: linking upstream and downstream technologies hyper-expression of the bt cry2aa2 operon in chloroplasts leads to formation of insecticidal crystals gm crops: public perception and scientific solutions environmentally friendly approaches to genetic engineering regulation of chloroplast enzyme activities by thioredoxins: activation or relief from inhibition protein disulfide isomerase as a regulator of chloroplast translational activation high-yield production of a human therapeutic protein in tobacco chloroplasts computer-aided process analysis and economic evaluation for biosynthetic human insulin production: a case study molecular farming in plants: oil seeds as vehicles for production of pharmaceutical proteins hyperexpression of an environmentally friendly synthetic polymer gene containment of herbicide resistance through genetic engineering of the chloroplast genome induction of male sterility in plants by a chimaeric ribonuclease gene removing selectable marker genes: taking the shortcut engineering chloroplast genome without the use of antibiotic resistance genes transgenic plants as factories for biopharmaceuticals triple helix assembly and processing of human collagen produced in transgenic tobacco plants expression of full length bioactive antimicrobial human lactoferrin in potato plants key: cord-258363-gmgbus9i authors: kolla, venkatadri; chakravorty, maharani; pandey, bindu; srinivasula, srinivasa m; mukherjee, annapurna; litwack, gerald title: synthesis of a bacteriophage mb78 late protein by novel ribosomal frameshifting() date: 2000-08-22 journal: gene doi: 10.1016/s0378-1119(00)00264-x sha: doc_id: 258363 cord_uid: gmgbus9i mb78 is a virulent phage of salmonella typhimurium that possesses a number of interesting features, making it a suitable organism to study the regulation of gene expression. a detailed physical map of this phage genome has been constructed and is being extensively studied at the molecular level. here, we demonstrate the expression of two late proteins of bacteriophage mb78 derived from the same gene as a result of possible ribosomal frameshifting. in vitro transcription-translation yields a major protein that migrates as 28 kda, whereas in vivo expression using pet expression vectors yields two equally expressed proteins of molecular sizes 28 and 26 kda. a putative slippery sequence tttaaag and a pseudoknot structure, two essential cis elements required for the classical ribosomal frameshifting, are identified in the reading frame. mutations created at the slippery sequence resulted in a single 28 kda protein and completely abolished the expression of 26 kda protein. thus, we have produced the first evidence that ribosomal frameshifting occurs in bacteriophage mb78 of salmonella typhimurium. allow phages like p22 and 9na to grow in its presence. mb78 contains a 42 kb linear, double-stranded dna bacteriophage mb78 is a virulent phage of salmonella (molecular weight 28×106 da), which replicates through typhimurium (joshi et al., 1982; srinivasula, 1992) . concatemer formation, subsequently converted to full-morphologically, physiologically and serologically, it is length phage dna through 'headful' packaging mechadifferent from the well-known temperate phage p22 and nism. like p22, mb78 dna is circularly permuted and related phages as well as a virulent phage 9na (murthy, terminally redundant ( khan et al., 1991a; pandey, 1992; 1987) . mb78 cannot multiply in minimal medium consrinivasula, 1992 ). taining citrate. the chelating agent edta is an effective it is now known that two proteins can be expressed inhibitor of its dna synthesis, whereas egta and from a single open reading frame through 'ribosomal orthophenanthroline have practically no effect on the frameshifting'. if the ribosome shifts during translation, development of the phage ( verma and chakravorty, one base in either direction, i.e. towards 3∞ or 5∞ ends, 1987) . mb78 is a dominant phage in that it does not the reading frame will be changed. during the process of 'ribosomal frameshifting', two or more proteins can result, starting from a single initiation codon abbreviations: bp, base pairs; iptg, isopropyl b-thiogalactosidase; ( farabaugh and vimaladithan, 1998 direction (+1 frame shift) has been described in the k the nucleotide sequence reported in this paper has been deposited yeast retrotransposon ty (belcourt and farabaugh, in the embl/genbank database under accession no. x87092. 1990 ), copia-like elements of drosophila (saigo et al., been demonstrated for retroviruses (jacks et al., 1988; from bacteriophage mb78 is expressed by ribosomal frameshifting. vickers and ecker, 1992) , luteoviruses (brault and miller, 1992; prufer et al., 1992) , the bacterial transposon is 1 (sekine et al., 1992) and in potato leafroll virus ( kujawa et al., 1993) . a −1 frameshifting event often controls the levels of expression of viral reverse tran-2. materials and methods scriptase relative to viral core proteins in retroviruses. frameshifting is also known to effect gene expression in 2.1. bacterial strains coronaviruses and even in a bacterial system (chamorro et al., 1992) . in all these cases, frameshifting occurs as lt2, a salmonella strain, was originally obtained the ribosome passes a seven nucleotide sequence 5∞ from dr. myron levine, department of human xxxyyyz 3∞ ( x is a, u or g, y is a or u, and z is genetics, university of michigan, ann arbor, mi. e. coli strain kk2186 was a generous gift from dr. p. any nucleotide), known as the 'slippery site'. two of the berget, then at the department of biochemistry and three base pairs between the anticodons of each of the molecular biology, university of texas, houston, tx. two trnas and mrnas can be maintained after the all other bacterial strains were purchased commercially slip into the −1 reading frame. the slippery sequence from gibco-brl life technologies. all the chemicals is not the only determinant of frameshifting; secondary were obtained from sigma chemical company, st. signals are also required (larsen et al., 1995; atkinson louis, usa. et al., 1997) . secondary signals programmed in the mrna augment shifting at the slippery sequence to give high levels of frameshifting. these signals, called 2.2. purification of bacteriophage mb78 and isolation of 'stimulators', are very diverse. for example, the +1 its dna shift for decoding rf2 (release factor) of e. coli requires two stimulators: one is a uga terminator at codon 26 phage stocks were prepared as described earlier flanking the shift site on its 3∞ site ( weiss et al., 1988) ; ( kolla and chakravorty, 2000) . phage dna was isothe other is a shine-dalgarno sequence located three lated as per the method described by maniatis et al. nucleotides upstream of the shift site ( weiss et al., (sambrook et al., 1989; kolla and chakravorty, 2000) . 1988). these two stimulators act independently with substantial activity, but their effects are synergistic. 2.3. isolation of plasmid dna pseudoknots, a tertiary interaction involving base pairing between two regions of unpaired bases, are also plasmid dnas were isolated by either alkali lysis involved in frameshifting. the model for the pseudoknot method using standard protocols (sambrook et al., structure was based on biochemical analysis of the 3∞ 1989; kolla and chakravorty, 2000) or by qiagen and end of turnip yellow mosaic virus ( tymv ) rna (pleij promega columns according to the manufacturer's et al., 1985; dumas et al., 1987) . in e. coli, ribosomal instructions. dna from the gels was extracted using protein s4 represses its own synthesis in addition to the qiagen columns. synthesis of other ribosomal proteins viz. s11, s13 and l17 by binding to a pseudoknot structure. the structure resembles a 'double pseudoknot' linking a hairpin 2.4. nested deletions by exoiii upstream of ribosome binding site with sequences 2-10 codons downstream of the initiation codon ( tang and the deletions were created primarily as described by draper, 1989) . pseudoknots also play a role in the henikoff (1987) . briefly, cloned dna fragment (5structural mimicry of trna at the 3∞ termini of plant 10 mg) was digested with two different restriction viral rnas (pleij et al., 1985) . one of the most intrienzymes e.g. psti and bamhi. the enzyme psti produces guing functions of the pseudoknot structure in framea four-base 3∞ overhang, resistant to exoiii activity, shifting occurs during the translation of certain retroviral while the enzyme bamhi generates 5∞ protrusion, which mrnas (jacks et al., 1988; kujawa et al., 1993; is accessible to exoiii ( weiss, 1976) . after complete atkinson et al., 1997) . mutational analyses in mouse digestion with both the enzymes, the dna sample was mammary tumor virus (mmtv ) (chamorro et al., deproteinized by extracting with phenol-chloroform and 1992) and in infectious bronchitis virus (ibv ) (brierley precipitated with ethanol. the dna pellet was resuset al., 1992) provide strong evidence for the stimulator pended in 25 ml of 1× exoiii buffer and the exonuclease structural element being a pseudoknot. the autoregulatreatment carried out as per the recommendations of tion of gp32 in phage t4 also involves a pseudoknot the manufacturer (promega). finally, the deleted frag(shamoo et al., 1993) . in this investigation, we provide ments were ligated and used for completing the nucleotide sequence as well as for in vivo expressions. evidence for the first time that one of the two late genes 2.5. in vitro transcription and translation rc5c centrifuge. the supernatant was discarded, and the minicell pellet was suspended in 6 ml of m9 medium. the cell suspension was again layered on to sucrose the coding region for 26 and 28 kda proteins was amplified by pcr and cloned in bacterial expression gradient as before. again, two-thirds of the minicell layer was collected into a 15 ml of corex cup, and an vectors, pet21a and pet28a. recombinant dnas were in vitro transcribed-translated in the presence of equal volume of m9 was added. the optical density of this minicell suspension was measured at 600 nm in a [35s]-methionine in rabbit reticulocyte lysate with a t7-rna polymerase-coupled tnt kit (promega) hitachi spectrophotometer to determine the volume in which the minicells would be finally suspended to have according to the manufacturer's recommendations. briefly, reactions were set up in 50 ml volume in an a 600 =2.0/ml (2×1010 cells/ml ). the cells were finally suspended in m9 minimal medium containing 30% eppendorf tube containing 25 ml of rabbit reticulocyte lysate, 2 ml of reaction buffer, 1 ml of amino acid mix ( v/v ) glycerol, aliquoted into a number of tubes (200 ml in each) and stored at −80°c. the minicells thus stored minus methionine, 1 ml of rnasin (5u ), 1 mg of template dna, 4 ml of 35s-methionine (1000 ci/mmol ) and 1 ml could be used for at least a year. of t7 rna polymerase (20 u ). the final reaction was made up to 50 ml with sterile distilled water, and the 2.8. expression of plasmid encoded proteins tubes were incubated at 42°c for 90 min to allow the synthesis of proteins. one to 3 ml samples were applied purified minicells were labeled with 35s-methionine as described previously (reeve, 1979; kolla and on to sds-polyacrylamide gels after denaturing in a sample buffer by boiling. chakravorty, 2000). the frozen minicell suspension (0.1 or 0.2 ml ) was thawed slowly and centrifuged for 3 min in a microfuge. the pellet was suspended in 200 ml of 2.6. sequencing m9 minimal medium to which 3 ml of 10.5% ( w/v ) difco methionine assay medium were added and incu-nucleotide sequencing of ecori 'f' fragment was carried out by manual sequencing using sequenase kit bated at 37°c for 90 min, to complete the translation of bacterial mrnas in the minicells, received from the ( usb) and also by automated sequencing. forward and reverse sequencing primers were obtained from mother cell. then, 25 uci of 35s-methionine were added and incubated for 60 min at 37°c, followed by incuba-pharmacia ( uppsala, sweden). tion of 5 min after the addition of 10 ml of unlabeled methionine (1%). the cells were then centrifuged at 2.7. preparation of minicells 12 000 rpm for 3 min, the cell pellet was washed with 500 ml of 10 mm tris-hcl, ph 7.6, suspended in 20 ml minicells were prepared as described by reeve (1979) . e. coli strain ds410, the minicell producing of the same buffer to which 20 ml of 2× sample buffer were added. the labeled proteins were separated by strain, transformed with desired plasmid was grown overnight to stationary phase in 400 ml of terrific broth sds-polyacrylamide gel electrophoresis (sds-page) (laemmli, 1970) , at a constant voltage of 50 v. (sambrook et al., 1989) in the presence of the relevant antibiotics (ampicillin and tetracycline). the culture was examined under a light microscope to observe the forma-2.9. fluorography tion of long filamentous cells and minicells. when a reasonable number of minicells were visible under the to detect radio labeled proteins, the gels were fluorographed using water-soluble sodium salicylate (bonner, microscope, the culture was chilled on ice for 20 min and then centrifuged at 4°c for 5 min at 2,000 rpm 1984). the gel was soaked in methanol, acetic acid, and water (5:1:5) for 60 min, followed by a thorough wash (675×g) in gs3 rotor of sorvall rc5c centrifuge. the supernatant was transferred to fresh gs3 cups and with water (30 volumes of gel ), then immersed in 1 m sodium salicylate, ph 7.0 for 1 h with mild shaking. centrifuged at 7000 rpm (5,278×g) for 20 min in the same centrifuge. the cell pellet was resuspended in 9 ml finally, the gel was transferred to whatman no. 1 sheet, dried under vacuum and subjected to fluorography. of m9 minimal medium, and then 4 ml of the suspension were carefully layered on to 10-30% (w/v) sucrose gradients. the gradients were centrifuged at 4°c for 2.10. cloning and pcr amplification 18 min at 5000 rpm (4122×g) in the hb4 rotor of the sorvall rc5c centrifuge. the presumed coding regions for 26 and 28 kda proteins were amplified by pcr using the following the top two-thirds of the minicell layer was collected into a 30 ml corex cup using pasteur pipette. to this, forward and reverse primers with overhanging restriction sites. an equal volume of m9 minimal medium was added, and the suspension was centrifuged again at 10 000 rpm forward primer: 5∞ cccggatccatgaatcgtt-ttttacgttac 3∞ (11 953×g) for 10 min in the ss34 rotor of the sorvall reverse primer: 5∞ cccgaattcggcagggtt-agattt 3∞ the primers were commercially synthesized by gibco-brl life technologies (including the primers designed to create mutations at the 3∞ end of the fragment to avoid the frameshift by overlapping pcr). the primers were also used to identify the presence of inserts in the cloned vectors by pcr screening and to determine the nucleotide sequence. the amplified fragments were digested with appropriate restriction enzymes and cloned in-frame into pet21a and pet28a expression vectors at bamhi and xhoi restriction sites. restriction enzyme analyses and dna sequencing confirmed the sequence of all the constructs. cruz biotechnology) and resolved by sds-page (10%) and transferred electrophoretically (100 v constant for 1 h) to hybond ecl nitrocellulose paper (amersham were named according to the number of bases deleted; for example, +1518 means that 1518 bases were deleted, life science). the paper was blocked overnight in 10% nonfat milk, and incubated in 5% non-fat milk with and +1601 means that 1601 bases were deleted and so on from the full-length construct. to ascertain the sizes horse-radish peroxidase-conjugated t7-antibody (1:10 000, novagen) for 1 h at room temperature. after of the inserts, the deleted plasmid dnas were digested with hindiii, located 138 bases away from the ecori stringent washing, the filter was developed by chemiluminiscent ecl, as described (amersham, arlington site ( fig. 2) . the sequentially deleted dnas were further confirmed by dot-blot hybridization (data not pre-heights, il). sented ). the deleted fragments were cloned into puc18, and the deletions were confirmed by dna sequencing. in order to determine the expression of various deletion mutants, we performed in vivo minicell expression using 35s-methionine [only a few of the sequentially in order to understand more about the physiology and genetics of the phage mb78, the ecori 'f' fragment deleted plasmids are presented here ( fig. 1) ]. lane 6 (proteins expressed by vector puc18) shows two pro-(2.3 kb) of the phage was cloned in puc18 vector (data not shown), and the expression in minicells was exam-teins, including 30 kda b-lactamase protein. in lane 1, four major proteins of molecular weights 28, 26, 21 and ined. the ecori 'f' fragment of mb78 codes for four proteins of mass 28, 26, 21 and 11 kda ( fig. 1, lane 1) . 11 kda expressed from the cloned ecori 'f' fragment could be seen. lanes 2-5 show the proteins expressed the expression of b-lactamase was not strong in cells carrying the ecori 'f' fragment, suggesting that the by different sequentially deleted dnas. the 21 and 11 kda proteins could not be seen when 1438 bases presence of a strong promoter (pandey et al., 1997) in the 'f' fragment is interfering with the expression of the were deleted ( lane 2), suggesting that their promoters and orfs (open reading frames) reside within 1438 bp b-lactamase gene. to characterize the ecori 'f' fragment, sequencing of the fragment was carried out with from the original construct. the expression of 28 and 26 kda proteins was unaffected even after the deletion a nested set of deletions in the target dna. the clones of 1518 bp from the original construct ( lane 3). the ( fig. 3a) . computation also revealed the presence of a slippery sequence with a possible downstream pseu-present study focuses on the characterization of the 28 and 26 kda proteins. the clone f-1518 expressed 28 doknot structure ( fig. 3b) . the slippery sequence present in bacteriophage mb78 resembles that of the turnip and 26 kda proteins, suggesting that their promoters and orfs are present in 793 bp (1518-2311) of the ecori yellow mosaic virus ( kujawa et al., 1993) . 'f' fragment. after deletion of 1595 bp, the expression of both proteins was reduced simultaneously ( lane 4), 3.4. effect of 3∞ truncation on the expression and no expression was detected after the deletion of 1723 bp ( lane 5). these results suggest that the expres-we next examined whether the 28 and 26 kda proteins are expressed from overlapping open reading sion of these two proteins is driven by a common promoter that resides within 1518-1601 bp and that frames by ribosomal frameshifting. if they are expressed from overlapping open reading frames with the same both the proteins are possibly expressed from overlapping open reading frames. initiation codon, truncation of the gene from the 3∞ end should yield only a single protein. this part of the gene has an internal hindiii restriction site, located 138 bp 3.3. nucleotide sequence analyses away from the ecori site, at the 3∞ end of the fragment. when this portion is deleted, the coding region will be the nucleotide sequence of the ecori 'f' fragment was determined by sanger's dideoxy chain termination reduced to 555 bp, encoding a protein of approximately 22 kda. to test this, plasmid +1518 was truncated method (sanger et al., 1977) using a set of deletion mutants produced by exoiii (accession no. x87092). (+1518/138 h ) and used to transform the minicell producing strain ds410 to observe the expression of computer analysis of the nucleotide sequence indicated the possibility of encoding four proteins that could be proteins. the expression pattern of the deleted plasmid is presented in fig. 4 . deletion of 138 bp from the 3∞ expressed from three orfs (data not presented ). the orf for the 28 and 26 kda proteins starts from 1641 end of the ecori 'f' fragment resulted in complete abolition of 26 and 28 kda proteins, but a major protein, and does not have a stop codon in the 'f' fragment; it appears that the vector stop codon, located adjacent to smaller in size (22 kda, marked with triangle), was expressed ( lane 4). the deletion of 3∞ end of the gene, ecori site, might be used. analysis of the nucleotide sequence of ecori 'f' fragment revealed that expression resulting in the synthesis of a single protein instead of two proteins, supports the frameshift notion. the pres-of 28 and 26 kda proteins may have occurred through ribosomal frameshifting. we analyzed the sequence for ence of a slippery sequence and a pseudoknot structure downstream to the putative shift site strengthens the the formation of possible secondary structures, necessary for the process of classical ribosomal frameshifting argument. fig. 3 . sequence analysis. (a) probable secondary structure of mrna derived from 3∞ 138 nucleotides. (b) probable slippery sequence and downstream pseudoknot structure. the slippery sequence essential for the frameshift is marked in a box, and the bold nucleotides represent the stop codon where mutations were created. nucleotides involved in the pseudoknot structure are connected. in order to examine the phenomenon of frameshift further, we next performed coupled in vitro transcription and translation using rabbit reticulocyte lysate. the nucleotide sequence starting from atg to the end of the fragment was amplified by pcr with forward and reverse primers, as described in section 2.10. the amplified dnas were cloned in-frame into bacterial expression vectors, pet21a and pet28a, at bamhi and xhoi sites and named kvm21 and kvm28, respectively. these dnas were used to synthesize proteins by in vitro transcription and translation (promega). the results are presented in fig. 5 ( lanes 1 and 2) . in vitro transcription-translation of the fragment yielded a major protein (90%) of 28 kda, with he synthesis of a minor protein of apparent molecular mass 26 kda (arrows). we observed the formation of dimers from 26 and 28 kda proteins in the absence of reducing agents dtt (data not shown). these results suggest the synthesis of -methionine in rabbit reticulocyte lysate, as described in section 2. one to three microliters of these samples, as indicated, were applied on to sds-polyacrylamide gels after denaturing in a sample buffer by boiling. a major translated protein product (28 kda) and a minor protein are marked with arrows. the position of possible dimers is also marked with an arrow. lanes 3 and 4 represent the translation of empty pet28a and pet21a vectors, respectively. able volume of 2× sample buffer and subjected to sds-page fol-recombinant proteins with c-terminal his.tag lowed by western blot. the presence of 26 and or 28 kda proteins (pet21a) and c and n-terminal his.tags (pet28a) was detected using the ecl kit (amersham). the molecular weight of the proteins is marked with arrows. were expressed in e. coli bl-21 de3. purified proteins were separated on a 12% sds-polyacrylamide gel and transferred on to a nitrocellulose membrane, and vector (fig. 6b ). this suggests that frameshift occurs at the c-terminal region, resulting in the appearance of a western blotting was performed with t7 specific antibody. a single 28 kda protein was present in cells truncated 26 kda protein without a c-terminal his.tag in addition to the expression of a full-length 28 kda expressing c-terminal his.tag in pet21a vector (fig. 6a) , whereas 26 and 28 kda proteins were present protein with a c-terminal his.tag. mutations were created (deletion of three nucleotides) near the putative in cells expressing n and c-terminal his.tags in pet28a slippery sequence by overlapping pcr and were cloned 4. conclusions into pet28a. recombinant proteins were expressed and separated as described above. we predicted that this 1. we demonstrated that two late proteins of bacteriophage mb78 could be derived from the same gene as mutation should result in the loss of frameshift. as expected the mutated recombinant constructs did not a result of ribosomal frameshifting. 2. ribosomal frameshifting has been well established in yield the 26 kda protein, but only the 28 kda protein (fig. 6c, lane 1) . these results further support ribo-e. coli phages t2 (du et al., 1997) , t4 (groisman and engelberg-kulka, 1995) and t7 (condron et al., somal frameshifting. 1991; sipley et al., 1991; lewis and matsui, 1996) but not well documented in the case of salmonella 3.7. kinetic expression of 28 and 26 kda proteins bacteriophages except the previously reported observation in the phage p22 ( uomini and roth, 1974) . to examine the functions of the 28 and 26 kda 3. two cis elements are essential for ribosomal frameproteins, kinetic expression of phage mb78 was pershifting (jacks et al., 1988; blinkowa and walker, formed. the lt2 cells (host) were infected with phage 1990; tsuchihashi and kornberg, 1990), but the exact mb78 at a multiplicity of infection (m.o.i.) 10 and pulsemechanism is not clearly understood. it has been labeled with 35s-methionine for 2 min at different times postulated in −1 frameshift that an anti-shineafter infection. the labeled proteins were subjected to dalgarno-like sequence, present at 5∞ to the shift site 12.5% sds-page followed by fluorography. the (larsen et al., 1994) , pairs with the 16s rrna of kinetic study demonstrated that the two proteins are the elongating ribosome to make the ribosome pause, late proteins of bacteriophage mb78 ( fig. 7) . the proresulting in a frameshift. this feature is observed tein pattern of uninfected cells served as a control. the mostly in prokaryotes. expression of 28 and 26 kda proteins was low at 2 and 4. an aspect of the current study of bacteriophage 5 min after infection, but from 10 min onwards, their mb78 is that more than 35% of the ribosomes appear synthesis increased significantly until cell lysis. it may to be involved in frameshifting. reports in the literatherefore be assumed that these two proteins are late ture indicate that the proteins synthesized as a result proteins of phage mb78. of frameshifting are much less numerous, to a maximum of 10-20%. 5. amino acid sequence analysis (e.g. lc/ms, maldi-tof ) of the 28 and 26 kda two proteins, encoded by the ecori 'f' fragment is required to confirm the ribosome frameshift hypothesis from the present study. exponentially growing lt2 cells in minimal medium (m9) at 37°c were infected with phage atkinson ribosomal frameshifting in the yeast retrotransposon ty: trnas induce slippage on a 7 nucleotide centrifugation, washed with 500 ml of medium and finally suspended in 25 ml of m9 medium. samples collected at different times as indicated minimal site programmed ribosomal framewere lysed in an equal volume of 2× sample buffer and applied on to a 12.5% sds-polyacrylamide gel. lane 1, uninfected ( ui ) lt2 cells; shifting generates the escherichia coli dna polymerase iii gamma subunit from within the tau subunit reading frame. nucleic acids lanes 2-7, cells infected with phage mb78 after 2, 5, 10, 15, 30 and 45 min, respectively. brl low-molecular-weight markers are repre-res fluorography for the detection of radioactivity sented in the left lane. the 28 and 26 kda proteins are marked with two arrows. in gels translational frameshifting mediated phage 9na, a virulent phage of salmonella typhimurium identi-'slippery-sequence' component of a coronavirus ribosomal framefication of a strong promoter of bacteriophage mb78 that lacks shifting signal rohde, sequences stimulating frameshifting in the decoding of gene 10 of w., 1992. ribosomal frameshifting in plants: a novel signal directs bacteriophage t7 use of minicells for bacteriophage-directed polypepthe simian retrovirus-1 gag-pro frameshift site 3-d graphics modelling of the scriptase-like enzyme in a transposable genetic element in drosoph-trna-like 3∞-end of turnip yellow mosaic virus rna: structural ila melanogaster effect of frameshift-inducing press, cold spring harbor, ny. mutants of elongation factor 1alpha on programmed +1 frame unidirectional digestion with exonuclease iii in encoded by is1: identification of the site of translational frameshift-dna sequence analysis mb78, a specific recognition of an rna pseudoknot structure repesis and gene 10 frameshifting in escherichia coli showing different lication, maturation and physical mapping of bacteriophage mb78 degrees of ribosomal fidelity molecular analysis of bacteriophage mb78 cloning, sequencing, expression and ph unusual mrna pseudoknot strucpromoter analysis of a structural protein of bacteriophage mb78. ture is recognized by a protein translational repressor translational frameshifting gentural requirements for efficient translational frameshifting in the erates the gamma subunit of dna polymerase iii holoenzyme. synthesis of the putative viral rna-dependent rna polymerase proc cleavage of structural proteins during the mutants of bacteriophage p22 thesis is specifically inhibited by the chelating agent edta. fems rrna-mrna base pairing stimulates a programmed -1 ribosomal microbiol enhancement of ribosomal frame maldoshifting by oligonucleotides targeted to the hiv gag-pol region endonuclease ii of escherichia coli is exonuclease iii. 1123-1129 reading frame switch caused by base-pair formation 70, 2869-2875. between the 3∞ end of 16s rrna and the mrna during elongation of protein synthesis in escherichia coli biochemical characterization of the bacterio the authors are thankful to the department of biotechnology, government of india, the university grants commission and the national institutes of health (nih ) (to v.k ) for financial assistance and dr. ravikumar rallapalli for discussion. key: cord-014597-66vd2mdu authors: nan title: abstracts from the 25th european society for animal cell technology meeting: cell technologies for innovative therapies: lausanne, switzerland. 14-17 may 2017 date: 2018-03-15 journal: bmc proc doi: 10.1186/s12919-018-0097-x sha: doc_id: 14597 cord_uid: 66vd2mdu nan . a schematic representation of crispr based synthetic transcription factor technology. b mrna expression levels of protein transport related genes (napg, rab5a and arpc1b). background an increasing number of biologics are entering the development pipelines of pharmaceutical companies [1] . today, the preferred production host for therapeutic proteins is the cho cell line. however one of the major hurdles, especially for the production of non-antibody glycoproteins, is host cell-related proteolytic degradation which can drastically impact developability and timelines of pipeline projects. material and methods spike-in: cho cells were cultivated in a chemically defined culture medium at 36.5°c/10% co 2 in shake-flasks. when the cells reached their maximum viable density, they were removed by centrifugation and the conditioned medium was collected. a model mab was spiked into the conditioned medium and incubated at 37°c ± protease inhibitors. the amount of proteolytic degradation was analysed by western blot and lc-ms. transcriptomics: total rna was extracted after 3 days of cell cultivation. rna sequencing libraries were constructed and processed on the hiseq 2000 platform from illumina. generation of matriptase knockout: cho-k1 cells were transfected with mrna encoding "transcription activator-like effector nucleases" or "zinc finger nucleases" targeting matriptase exon 2. the transfected cells were subsequently sorted into single cells and analysed for frameshift mutations in both alleles via sanger sequencing. cell cultivation: fed batch cultivation was performed in 15-ml miniaturized bioreactors (ambr15). approximately 700 proteases are known in rodents. to reduce the number of candidate proteases we showed first that a model mab (prone to proteolytic degradation) incubated in conditioned medium of cho-k1 cells resulted in clipping of the mab, demonstrating the involvement of secreted/shedded proteases (fig. 1a) . broad spectrum inhibitors of the different protease classes revealed that only serine protease inhibitors prevented clipping. serine protease inhibitors of higher specificity highlighted the group of "s1a trypsin-like proteases" (fig. 1a) . comparison of the proteolytic degradation profile of several therapeutic proteins between cho-k1 with another cho cell line (cho-a) revealed less degradation in cho-a. therefore expression of the involved protease(s) is likely lower in cho-a. gene expression profile analysis of both cell lines showed five secreted/shedded "s1a trypsin-like serine proteases" more than 1.5 fold lower expressed in cho-cho-a versus cho-k1 (fig. 1b) . surprisingly, sirna knockdown experiments of these five candidates identified "matriptase" as the major protease involved in degradation of recombinant proteins expressed in cho-k1 cells ( fig. 1c upper panel) . next, we generated a cho-k1 matriptase knockout (ko) cell line. no proteolytic degradation product was detected when the model mab was spiked into conditioned medium of the ko cell line (fig. 1c lower panel) . also, stable expression of the model mab in the ko cell line resulted in no/significantly less clipping (fig. 1e) . the protein titer and the cell growth behaviour of the matriptase ko cells were similar to the corresponding wildtype (wt) cells (fig. 1d) as shown by comparative cultivation in ambr system. conclusions one major challenge for the production of recombinant proteins is cho host cell mediated proteolytic degradation which can negatively impact or even result in termination of projects [2; 3] . using a variety of techniques such as applying protease inhibitors, transcriptomics and sirna mediated knock-down we were able to identify "matriptase" as the major protease involved in degradation of recombinant proteins expressed in cho-k1 cells. subsequently we generated a matriptase deficient cho cell line. protein candidates of diverse formats, severely degraded in wt cho-k1 cell line, were not or significantly less cleaved in the matriptase ko cell line. furthermore cell growth, viability and productivity levels were comparable between the wt and the matriptase ko cell line. in summary, we have generated a superior platform-compatible cho production host cell line with the same favourable productivity properties as the parental host cell line [4; 6] , allowing expression of complex glycoproteins prone to clipping. background cho cell lines are common hosts for the production of biopharmaceutical proteins. so far, considerable progress has been made increasing productivity of cell culture to meet the rapidly growing demand for antibody biopharmaceuticals through increased cell densities and longer culture times. the downside is the increase of the process related impurities, bringing new challenges for process and harvest development. among the process related impurities such as host cell proteins (hcps) or dna the potential impact of lipids production and release during cell culture is still poorly understood due to the complex nature and diversity of this class of molecules. thanks to recent advances in analytical tools especially mass spectrometry, the advent of lipidomics offers now the feasibility to study several thousands of lipid species thus unraveling the possibility to understand and potentially control the interactions between high performance bioreactor processes, harvest conditions and purification. in order to analyze and quantify lipids, we developed a three steps method. in a first step, lipids were extracted with methyl tert-butyl ether (mtbe) according to matyash method [1] . lipids were then separated by liquid chromatography using either hilic of reverse phase column prior to detection and quantification by mass spectrometry. all lipid classes were detected by esi-ms/ms excepted cholesterol (apci-ms/ms). finally we applied this method to analyze the lipid content of different cell lines each expressing a different recombinant protein, during a 14 days fed batch process. lipid from cho cells were successfully extracted with a yield between 80% and 95% depending on the different lipid classes. stable isotope labeled lipids were used as internal standard in order to have comparable results between batches. the obtained results (fig. 1) show that for a given cell line, lipid distribution is changing over the process. moreover, this distribution may vary significantly depending on the cell line: cl-1 and in a lower extend, cl-3, show an accumulation of triglycerides from day 6 to the end of the process, while cl-2 doesn't seems to follow this trend. conclusion interestingly, in some cell lines/experimental conditions, we highlighted an overproduction of triglycerides and cholesterol leading to the accumulation of lipid droplets known as energy storage sink. at the metabolic level, these findings suggest a relative overflow of the carbon metabolism. from a process development perspective these findings can be considered on the one hand as a resource waste since the stored energy is not used for protein/biomass biosynthesis and on the second hand as the root cause of additional process challenges especially during the harvest and the first capture steps given the hydrophobic nature of these molecules. implementation of lipidomics analysis enables us to highlight a new type of process variability and to anticipate potential problems for the downstream steps. the application of this methodology on our platform has helped us to design tailor made solutions (pretreatment selection, filter selection,…) at the clarification step which are now implemented in our harvest development platform approach. . matriptase knock-out in cho cells prevents clipping of recombinant proteins. a serine protease inhibitors protect model mab from proteolytic degradation in cho-k1 cell derived conditioned medium. the model mab was incubated in conditioned medium for 0h or 48h at 37°c, subsequently samples were analyzed by western blot. broad spectrum serine protease inhibitors (aprotinin, leupetin) were added during incubation. aprotinin and leupetin are inhibiting proteolytic degradation. the intact mab (upper band) and the clipped mab (lower band) are indicated by arrows. b gene expression profiling of cho-k1 versus cho-a by ngs. shown is the gene expression profile of "secreted/shedded members of the s1a trypsin-like serine protease family" for cho-k1 and cho-a cell lines using next generation sequencing. the gene expression analysis highlights that five proteases were more than 1.5 fold higher expressed in cho-k1 cells (labelled with a red asterix). the y-axis shows the transcript abundance as rpkm (reads per kilobase of exon model per million mapped reads). c sirna knock-down identifies matriptase as major clipping protease and cho matriptase ko clone shows no detectable clipping activity. upper figure: sirnas directed against the five protease genes and scrambled (scr.) sirna were transfected and conditioned medium was collected three days after transfection. the model mab was incubated in fresh medium as control (first lane) and conditioned medium from the sirna transfected cells. samples were analyzed by western blot. only sirna targeting matriptase (st14) showed reduced proteolytic degradation. the intact mab (upper band) and the clipped mab (lower band) are indicated by arrows. lower figure: the model mab was incubated for 48h in conditioned medium collected from wt cho-k1 as well as the matriptase knockout clone. samples were analyzed by western blot. the intact mab (upper band) and the clipped mab (lower band) are indicated by arrows. no proteolytic degradation could be detected in the samples originating from the matriptase ko clone. d cell growth, viability and productivity in ambr (fed batch with temperature shift). cell growth, viability and volumetric productivity profiles of wt cho-k1 (red circles, n=2) and matriptase ko clone (blue squares, n=1) cultivated in 15-ml ambr. no significant differences were seen between wt and matriptase ko clone regarding cell growth and viability. comparable or slightly higher productivity was detected for the matriptase ko clone compared to the wt. e significant reduced proteolytic clipping applying matriptase ko clone. the model mab was stable expressed in cho-k1 (wt) as well as the cho-k1 matriptase ko clone. samples were analyzed by western blot. the intact mab (upper band) and the clipped mab (lower band) are indicated by arrows. significant reduced proteolytic degradation could be detected in the samples originating from the matriptase ko clone (3 samples each is shown for wt and ko cells) the glycosylation of therapeutic proteins is a critical quality attribute (cqa) and needs to be analyzed during cell line and bioprocess development. the current methods for analyzing glycosylation are mainly based on the enzymatic release of glycans. they are tedious and offer only limited throughput, which makes them unsuitable for cell line development work. in this study we evaluated a novel paia assay for measuring intact glycoproteins with capture beads and fluorescence labeled plant lectins to analyze glycans in a high throughput 384-well plate format. material and methods analytes: erbitux © , mabthera © , arzerra © and avastin © . two glycoengineered variants of one igg were kindly provided by merck (vevey, switzerland). all analytes were spiked into cho-k1 cell culture supernatant or buffer, diluted 1:1 with a denaturation solution and incubated at 65 or 70°c for 20 minutes to expose the fc glycans. erbitux samples were analyzed under denaturing conditions to detect fab-and fc-glycosylation and in native conditions for fab glycosylation only. 10μl of pretreated sample was added to each well of the special 384-well paiaplate, containing labeled lectin and capture beads. the microplate was incubated for 45 minutes at 1800 rpm on an orbital shaker at room temperature and spun down at 500 xg. the read-out was done on a fluorescence microscope (synentec, elmshorn, germany) in less than five minutes. results figure 1a : lectin binding profiles of different iggs. the analysis of different igg results in lectin binding profiles which show the different degrees in glycosylation. high abundance of sugars leads to high binding rates of the lectin for the respective sugar. avastin has a very low degree of galactosylation and high mannose species compared to mabthera and arzerra (fig. 1a) . only arzerra is carrying glycans with 2-6 linked sialic acids. these findings are in line with results from literature [1] . figure 1b : distinction between fc and fab glycosylation in erbitux. without denaturation only the fab glycans are detectable in erbitux. denaturation leads to additional exposure of the fc glycans and thus higher lectin binding rates compared to native erbitux. gna and npl only bind to denatured erbitux indicating that the high mannose glycans are only present on the fc part. the equal sna binding rates for both conditions confirm that the 2-6 linked sialic acids are almost exclusively found on the fab part. this is in agreement with published data [2] . figure 1c : lectin binding rates correlate with the levels of galactosylation and fucosylation. increasing degrees of glycosylation in the mixtures of the glycan variants from merck lead to higher lectin binding rates for all galactose and fucose markers proving that quantitative analysis can be performed with these assays. the cona lectin which binds to the common core mannose glycan motive remains at the same level, suggesting that the fc glycans were similarly exposed in all samples. the results demonstrate that paia assays are capable of quickly detecting differences in glycan patterns of different antibodies. in addition it was shown that glycan variants of the same igg can be analyzed quantitatively. and finally we could confirm the differences in fab and fc glycosylation in erbitux. we believe that bead-based assays with lectins have a great potential for monitoring product quality early in the development process. background gene-and cell therapy-based medicines are experiencing resurgence due to the introduction of "next generation" transfer viral vectors, which have demonstrated improved safety and efficacy. adeno associated viruses (aav) and lentiviruses are very commonly used in therapeutics and often produced by transient gene expression, using pei-mediated transient transfection in hek-293 or hek-293t cells [1] . the critical raw materials needed for cgmp vector production must be sourced from approved suppliers and should have gone through a rigorous testing program to reduce the risk of introducing adventitious agents into the production process. correspondingly, the pei transfection reagent must also be sourced from a qualified supplier, and have gone through rigorous testing to ensure reliable transfection efficiencies, and hence reproducible virus production yields. here, we present peipro® and peipro®-hq, the unique pei-based transfection reagents suitable for use in process development and in cgmp biomanufacturing, respectively. unlike commercially available peis, peipro® benefits from extensive research and development in polymer chemistry and formulation for mammalian cell transfection. we further demonstrate that peipro® and peipro®-hq are the reagents of choice for virus production runs in most cell culture systems, hence facilitating the transition from initial optimization during process development up to large-scale therapeutic viral vector production in adherent or suspension cells. manufacturing process of peipro® and peipro®-hq reagents. peipro® and peipro®-hq are fully synthetic reagents, free of any animal-origin components. in comparison to peipro®, a more extensive number of quality controls are performed on peipro®-hq to enable its use as a qualified raw material in gmp processes for the manufacturing of clinical batches of therapeutic products. lentivirus and aav production. irrespective of the cell culture vessel type, transfection using peipro® was performed following our recommandations. as an example, hek-293t (lentivirus) and hek-293 (aav) cells were thawed directly into each medium and passaged every 3 to 4 days before going into a 2 liter benchtop bioreactor. cells were resuspended and cultured for 3 days before transfection with peipro®. hek-293t cells were transfected with a third-generation system (four plasmids) for lentivirus production. hek-293 cells were co-transfected with three plasmids for aav production. lentiviral and aav titers were measured 48 and 72 hours post-transfection (data kindly provided by généthon). peipro® is the reagent of choice for virus production runs in most adherent and suspension cell culture systems from process development up to large scale clinical-grade virus production. irrespective of the cell culture-based system and production scale, peipro® and peipro®-hq have led to efficient viral vector yields in standard laboratory cell systems, such as in flasks, cell factories, and roller bottles, as well as in multilayers flasks or fixed-bed culture systems that take into account time and space concerns for the scaling-up process (table 1) . for example, high viral vector yields superior to 10 7 ig/ml and 10 11 -10 12 vg/ml were obtained respectively for lentiviruses and aavs in suspension hek-293t and hek-293 cells cultured in one of the commercially available synthetic cell culture medium balancd® hek293 (irvine scientific®). conclusion peipro® and its higher quality grade peipro®-hq are the unique pei suitable for efficient and reproducible production of therapeutic viral vectors. efficient viral vector production yields can be achieved in most cell culture systems, irrespective of the production scale. with appropriate and advanced quality controls, the highest quality grade peipro®-hq is commercially available to accompany academics and biopharmaceutical companies in terms of qualified raw material for their gmp-grade viral vector production needs. b distinction between fc and fab glycosylation in erbitux. erbitux was diluted to a concentration of 200 μg/ml in tris buffer and measured in native and denatured conditions to distinguish fab glycosylation (native erbitux) from fab and fc glycosylation (denatured erbitux). it could be confirmed that sialic acids are almost exclusively present on the fab part of erbitux and that the high mannose glycans are only found in the fc part. c lectin binding rates correlate with the levels of galactosylation and fucosylation. two glycan variants samples of the same igg from merck were mixed in different ratios to yield glycosylation rates of 9 to 55% in terminal β-galactose and 8 to 100% in core-fucose, based on data from 2-ab uplc analysis. the mixtures all contained 0.5 μg igg per well. the measured lectin binding rates for all galactose and fucose markers correlate very well with the respective degree of glycosylation in the mixtures. all measurements were performed in triplicates (irvine scientific®) . hek-293t (lentivirus) and hek-293 (aav) cells were thawed directly into each medium and passaged every 3 to 4 days before going into a 2 liter benchtop bioreactor. cells were seeded and cultured for 3 days before being transfected by peipro® (polyplus). for transfection, four plasmids were used for lentivirus and three plasmids were used for aav. lentiviral and aav titer were measured 48 and 72 hours post transfection (data kindly provided by généthon) table 1 (abstract p-004). peipro®, the reagent of choice for virus production runs in most cell culture systems in both adherent and suspension cells. irrespective of the cell culture-based system and production scale, peipro® and peipro®-hq have led to efficient viral vector yields superior to 10 7 ig/ml and 10 9 vg/ml, respectively for lentiviruses and aavs background continuous perfusion process is making a comeback as a competing upstream manufacturing technology for the production of biopharmaceuticals compared to the standard fed batch processes. this is primarily because of cost advantages such as reduced capital cost and increased product yield. the change in status of perfusion process from older perfusion to the new-age perfusion is due to availability of better cell retention devices leading to more efficient processes, improved cell lines, cell culture medium capable of supporting high cell densities and better bioreactor control strategies. in this work, we present the advantages, limitations and challenges of fed batch and perfusion type of processes through case studies. table 1 results the perfusion run yielded 5-fold higher titer compared to fed batch run (fig. 1a) . considering the number of runs that could be executed in a manufacturing facility within the same calendar days, about 1fold increase in product output can be achieved with the perfusion process (fig. 1a) . this difference is attributed to higher ivcc, higher pcd and longevity of cells because of decreased level of toxic metabolite concentrations such as lactate and ammonia. case 2: understanding product retention in perfusion process the new-age perfusion processes utilize hollow fiber filters. this has been observed to cause retention of product within the bioreactor especially towards the end of the production run. two types of experiments were conducted to study the factors contributing to product retention: -spiking studies: -role of product titer: product was spiked into chemically defined media -role of different cell viability: different broths with varying viability spiked with same product titer -evaluation of different hollow fiber membrane (m1, m2 and m3) on product retention. from spiking studies, it was evident that cell debris and poor quality cell broth (lower viability) were the major factors contributing to product retention (fig. 1c) . from the different membranes experiments, it was identified that at pilot scale, m1 showed much higher retention from the first perfusion cycle itself and it increased to more than 75% towards the end of the batch. however, with m2 membrane, product retention started only late (after 50% of batch duration) and it remained low (~20-40%). on the contrary, this difference was not observed at 1l scale due to the usage of membranes with larger filter area (2-3 folds higher compared to pilot scale). when the filter area per unit volume of perfusate was decreased by half (m2_batch 4) for the pilot scale, even m2 showed retention profile similar to m1 (fig. 1d ). we presented data to show that perfusion process has 5-fold increase in product yield on a per-batch basis and a 3-fold increase when facility throughput is considered. product retention is a technical challenge that requires optimization (perfusion rates and filter membrane types). we believe it is imperative that labs that develop processes for biologics can now consider both perfusion and fedbatch based processes as both these technologies can now closely compete with each other. the choice of the process format going forward should now solely be dependent on the requirement for the biologic rather than the earlier perception that fed-batch is the preferred choice because of its simplicity. background chinese hamster ovary (cho) cell culture has been widely used for production of monoclonal antibodies in the pharmaceutical industry. previous studies have shown that the cell specific productivity in cho cells can be increased by glucose limitation [1] . introducing a productivity enhancing effect it is possible that this also affects the quality of the product such as glycosylation or other posttranslational modifications. in this work, we are focusing on the impact of glucose limitation and increased productivity on the product quality of a monoclonal antibody produced in a fed-batch cultivation of cho cells. materials and methods cho cells were cultivated both under limiting and non-limiting nutrient conditions in fed-batch. for fed-batch cultivation the reduced range for glucose concentration was chosen between 0.2 and 0.5 g/ l. reference cultivation was performed between 1.5 and 3.0 g/l. both cultures were fed with similar volumes of a complex nutrient supplement. all cultivations were performed in chemically-defined, animalcomponent free cho growth media (xell ag). viable cell density and viability were determined using the automated cell counting system cedex (roche diagnostics), glucose and lactate concentrations were detected via ysi (ysi life sciences). amino acid were quantified using hplc-fld, vitamins were quantified using reversed phase chromatography coupled to a triple quadrupol mass spectrometer (varian 320, selected reaction monitoring). amounts of igg1 were quantified via protein a hplc, mab purified from another cho cell clone was used as a standard. the analysis of product quality was performed by intact mass analysis using reversed phase chromatography coupled to a microotof-q ii mass spectrometer (bruker daltonik). the cho cell culture cultivated under low nutrient conditions reached a 54% higher viable cell density than the reference culture (fig. 1a) . the product titer was even increased by 109% (fig. 1b) . the spent media analysis shows that some amino acids and vitamins were present at presumably limiting concentrations after day 5/6, mostly in the low nutrient level culture (down to 40 to 190 μm for tyr, gln, arg, and asn, below 1 μm for pyridoxine, data not shown). the product quality showed significant changes for the changed feeding strategy ( fig. 1c and d) . as expected, the glycation level decreased from 3% to 1% compared to the reference culture. the truncation level of c-terminal lysine at the heavy chain of the mab increased from 79% to 88%. the glycosylation was also significantly influenced by the low nutrient level (fig. 1e) : the nonfucosylated variants increased from 3% to 6% (fig. 1f) , the degree of galactosylation increased from 31% to 39% (fig. 1g) . cultivation under low nutrient level led to 54% higher viable cell density and a product titer increased by 109% when compared to reference culture grown under non-limiting nutrient conditions. the analysis of product quality reveals 75% less glycation of light chain for cho cells grown under low nutrient conditions (0.7% vs 2.7% in reference culture). the truncation of c-terminal lysine decreased by 10% (from 88% to 79%), the degree of galactosylation increased by 23% (from 31% to 39%, also observed by takuma et al. [2] ) and non-fucosylated glycans increased by 105% (from 2.8% to 5.8%) under low nutrient conditions. the product quality analysis by intact mass proved to be highly robust (average cv for four replicates = 2%). in summary, cultivation with alternative feed led to higher igg product titer and better product quality (glycation unwanted, higher amount of non-fucosylated glycans leads to higher antibody-dependent cellmediated cytotoxicity (adcc), higher amount of galactosylation to higher complement-dependent cytotoxicity (cdc) and adcc [3, 4] ). background we developed an automated, multiwell plate (mwp) based screening system for suspension cell cultures (fig. 1a) which is now routinely used in cell culture process development. it is characterized by a fully automated workflow with integrated analytical instrumentation. it uses shaken 6-24 well plates as bioreactors which can be run in batch and fed-batch mode with a capacity of up to 768 reactors in parallel [1] [2] [3] . a wide ranging analytical portfolio to monitor cell culture processes and also a cooperation with internal high throughput (ht) analytic groups to characterize product quality are available. in addition the use and the benefits of spectroscopic methods for cell culture automation were shown in the past [4, 5] . automated cell culture systems enable broader screening within a shorter time frame for many applications in upstream process development. the higher degree of parallelization and automation helps to screen for most promising parameters in a shorter time. the use of broad doe screening design allows in addition the identification of parameters that support high titers while keeping high product quality (multiple factors at the same time). the illustration (fig. 1b) shows an example how this combination can speed up process development steps. main applications of the cell culture automation are for example the identification of product quality levers and media or feed optimization. the application of the cell culture automation is shown for two examples. the goal in the first application was to identify levers to reduce trisulfides. by a screening of 39 conditions in parallel (in 4-fold replication, 158 wells in sum) the reduction of trisulfides by 97.5 % (normalized to start level) was possible. in addition the levers for trisulfide reduction were identified. the best and start conditions were verified in bioreactor scale (fig. 1c) . the goal in the second application was to increase product concentration without an impact on product quality. by a screening of 54 conditions in parallel (in 4-fold replication, 216 wells in sum) the increase of titer from 1.5 g/l to 3.7 g/l (> factor 2) was possible by media platform change and media optimization. an impact on product quality could not been shown. the best conditions were also verified in bioreactor scale (fig. 1d) . the benefits of using cell culture automation in late stage process development were shown based on two examples of current applications. for this purpose the experimental results of the development work of two late state projects using the in-house developed automated cell culture system were shown. the first example shows the capability of the automated cell culture system by reducing trisulfides significantly in just one experiment. for the other project the final product concentration could be increased by factor 2.5 by a media screening and changing to the in-house media platform. these two examples show the potential of cell culture automation as a routine tool in process development. the cell line development process has become faster and is simultaneously generating more clone-and product-related analytical data. in order to select the best producer cell line, extremely heterogeneous data types need to be systematically compared. the timely availability of all data needed to decide which cell line to pursue has become a bottleneck in the cell line development workflow. to ensure sound decision making, new integrated workflow support and data analysis methods are needed. we have developed a new end-to-end platform for bioprocess development, which includes a cell line development workflow system supporting seeding, selection, passaging, analyzing, cryo-conservation, and processing in (micro-) bioreactors. this platform, genedata bioprocess™, enables partially or fully automated cell line selection and assessment processes, and it increases process efficiency and quality. the system tracks the full history of all clones -from initial transfection all the way to their evaluation in bioreactor runs -and combines this information with analytics data on molecules, clones, and product quality. it can directly integrate with all instruments, such as pipetting robots, bioreactors, and bioanalyzers. the system is designed for a wide range of . a schematic illustration of the automated cell culture system. only the core system is shown with a robotic plate handler as key device connecting cultivation, processing and analytical parts. b illustration of an example how cell culture automation can speed up process development steps. c application in the identification of product quality levers. d application in titer optimization biologic molecules, including antibodies (iggs, novel formats) and other therapeutic proteins (e.g., fusion proteins). highlighted use cases describe the identification of top producer cell lines, decision making support, bioreactor data management, and full clone history report documentation (fig. 1 ). genedata bioprocess, which was developed in collaboration with top pharmaceutical companies, can flexibly support various (non-linear) workflows and structure the collected information in a way that fosters collaboration across an organization. while increasing throughput is crucial to ensure the timely availability of optimal producer cell lines, high-throughput is only possible when automated processes in the laboratory and the resulting data collection and aggregation can be streamlined. genedata bioprocess helps to establish more productive processes by offering support and integration for automation stations and measurement devices. thanks to the comprehensive workflow support and the possibility to integrate results from cell line stability experiments, product quality assessment, and bioreactor suitability tests, genedata bioprocess provides a unique way to evaluate cell lines. comprehensive analysis of all data collected in the process helps to ensure the highest possible quality and minimize the time and resources needed for data analysis and management. integration of bioreactor data analysis and visualization with other parameters measured in cell line development, streamlines clone evaluation in micro-bioreactors and supports highthroughput operations. genedata bioprocess comprehensively tracks the full clone history from the origin of the host cell line to the generation of the validated monoclonal producer cell line. for promising clones, the clone history report can be generated with one click. besides supporting cell line development, genedata bioprocess is a comprehensive platform capable of tracking the complete bioprocess development process. in 2012, 14.1 million people suffered from cancer [1] making it to a major concern of our society. since common cancer treatment is limited and not effective for late stage carcinoma, alternative methods are needed to reduce the high mortality rate of cancer patients. one alternative approach is the application of the oncolytic measles virus (omv), because omv has a natural affinity against cancer cells. the major drawbacks of omv is to produce the extremely high amount of at least 10 11 tcid 50 (50 % tissue culture infective dose) per dose [2] which is needed. to solve this problem, a high titer process must be established including an efficient downstream processing (dsp). we developed an appropriate upstream processing and are able to produce 10 10 -10 11 tcid 50 ml -1 in a bioreactor with 0.5 l working volume [3] . now, we focus on the dsp part. the following study tested the application of charged depth filters for the omv clarification. in contrast to common dsp schemes, a depletion of virus particles or a loss of infectivity is not desired. the aim is a reduction of protein content and dna with minimal loss of infective omv. further, we investigated the influence of the cell culture medium on the depth filtration process. to explore the influence of the surrounding cell culture medium on the depth filtration performance, omv was either produced in serum-free medium (vp-sfm) or serum-containing medium (dmem + 10% fcs). the production was done in a str with a working volume of 0.5 l as described in [3] . cells and carriers were separated with an opticap xl 1-module (polygard-cr; 5 μm; merck). for the depth filtration millistak+ ce50 filters (merck) were used. the filter material was autoclaved and rinsed with 25 ml of 20 mm tris-hcl (ph=7.4). the virus suspension was filtered with a load of 50 l m -2 using a peristaltic pump (ism931c; ismatec) applying a flux of 150 l m -2 h -1 (fig. 1 ). samples were collected at the beginning and end of a filtration run. the omv titer (tcid 50 ml -1 ) of the samples was determined according to kärber and reed [4, 5] . protein content was measured with the pierce bca protein assay kit (thermofisher scientific) according to the manufacturer's instructions. dna was measured by a microtiter assay using quant-it picogreen dsdna reagent (thermofisher scientific) according to the manufacturer's instructions. we found that positively charged depth filters were suitable to clarify omv suspensions. the cell culture medium, in which the omv was produced, influenced the outcome of the depth filtration. a log reduction value (lrv) of 0.87 was determined for omv present in serum-containing medium (scm), whereas the titer of omv in serumfree medium (sfm) was reduced 1.63 log levels. this indicates that without serum in the surrounding liquid, omv will adsorb to the filter material. however, we must evaluate if the missing serum or other components present in sfm are responsible for this effect. total protein was not relevantly reduced by the clarification using charged depth filters. for omv present in scm, the residual protein content was slightly less compared to omv present in sfm (table 1 ). in contrast, host cell dna (hcdna) was bound to the filter material. we achieved a 33% reduction of hcdna for an omv suspension in sfm. after clarifying an omv suspension in sfm, the remaining hcdna content was even lower being only 42 %. conclusions charged depth filters are suitable for the first clarification step of omv downstream processing. residual protein could pass the depth scheme of the complete cell line development workflow support in genedata bioprocess. showcasing integration of data from diverse measurement instruments, data visualization for decision making support as well as, tracking of full clone history filter almost unhindered, whereas the hcdna content was already reduced to 42% at maximum. however, the omv titer was also reduced by the depth filtration. this undesired effect was stronger for the omv present in sfm. because the agencies require avoiding serum in clinical-grade production processes, this is disadvantageous. nonetheless, because sfm will be soon standard for omv production, further experiments have to be done preventing the omv reduction during clarification. one option can be to reduce the adsorption strength of the virus to the filter material by the addition of salt. moreover, it is important to establish a standardized protocol for the upstream processing. we determined batch-to-batch variations within the clarification indicating a strong impact of upstream processing (usp) on the outcome of the dsp. therefore, further studies must investigate the influence of usp parameter e.g. time of harvest and ph of the harvest solution on the omv. fed-batch culture is commonly employed to maximize cell and product concentrations in upstream mammalian cell culture processes. typical standard platform processes rely on fixed-volume bolus feeding of concentrated feed supplements at regular intervals. however, such static approaches might result in over-or underfeeding. to mimic more closely the dynamics of a fed-batch culture, we developed a dynamic feeding strategy responsive to the actual nutrient needs of a mab-producing recombinant cho cell line. results and discussion improvements made at different steps during fed-batch development are shown in fig. 1 . at step 1, all eight cell boost supplements were added to cdm4ns0 according to a doe approach, and batch cultures were performed. this evaluation allowed us to select only those cell boost supplements that were beneficial to the overall culture performance. non-performing cell boost supplements were removed and not considered further. at step 2, the selected cell boost supplements were added daily to the cultures at different ratios according to a doe approach, and fedbatch cultures were conducted. as expected, daily feed additions to replenish consumed nutrients substantially improved mab and peak cell concentrations as well as viable cumulative cell days (vccd) compared to batch cultivation. further, the results enabled us to fine-tune the feed ratio of selected cell boost supplements. at step 3, we further optimized the best performing feed ratio by investigation of static and dynamic feed protocols. most fed-batch protocols rely on constant feed additions on distinct days. however, these approaches often lead to substantial over-or underfeeding during bioprocessing. to improve such "static" protocols, we investigated three different "dynamic" approaches as shown in table 1 by applying the selected cell boost supplements with the optimized feed ratio. this investigation allowed us to further improve the bioprocess performance. the best performing approaches, constant and retrospective feed, were further investigated in fully automated bioreactors under controlled conditions. in general, constant cultivation parameters in the bioreactor slightly enhanced mab titers compared to shake flask cultivation. the retrospective feed strategy yielded 10% higher titers than the constant strategy. overall, the established methodology for fed-batch development allowed us to obtain 2.5× higher mab titers (batch mean: 1.9 g/l vs. fed-batch 4.9 g/l) in a short time and three simple steps. in addition, the product quality was investigated. compared to the legacy fedbatch process, fed-batches that were conducted with the newly selected basal and feed media altered the distribution of charge and glycan variants. the amount of aggregated product was not altered. the established methodology for fed-batch development is a rapid protocol to select well-performing feed supplements and optimize their ratio to the culture requirements. in three steps, mab titers were boosted 2.5x from 1.9 g/l to 4.9 g/l. product glycosylation and charge variants could be influenced by the newly selected basal and feed media compared to a legacy fed-batch process. the amount of aggregated product was not altered. the present study investigates the beneficial effect of spiking hyclone™ actipro™ basal medium with hyclone cell boost™ 7a and cell boost 7b feed supplements on growth and productivity of a recombinant cho cell line. to evaluate the impact of feed-spiking compared with cultivation in basal medium only, the cell line was grown in bioreactors under controlled conditions to determine cellspecific metabolic rates, nutrient consumption, and byproduct accumulation over the process time. transcriptome analysis of the cultivated cells, using microarrays on four consecutive days to investigate differential gene expression, revealed the beneficial effect of feed-spiking compared with cells grown in basal medium. model cell line was a mab-expressing cho dg44 (licensed from cellca gmbh) cultivated either in actipro basal medium only (ge healthcare) or in actipro basal medium feed-spiked with additional supplementation with 7% cell boost 7a and 0.7% cell boost 7b (ge healthcare). both cultures were grown in batch mode using dasgip™ cellferm-pro™ stirred-tank bioreactors (eppendorf). the beneficial effect of feed-spiking was analysed by transcriptome analysis using microarray technology (8×60 k design, agilent). both basal and feed-spiked processes lasted for seven days with viabilities above 95% until day 6. on day seven, a sharp decline in viability indicated the end of the batch process (fig. 1a) . in feed-spiked medium, cells initially grew slower but reached almost twice as high peak cell concentrations (17.6 × 10 6 c/ml) than in basal medium only (9.79 × 10 6 c/ml). remarkably, the integral of the viable cell concentration over the total process time (viable cumulative cell days [vccd] ) was similar between both process strategies (fig. 1c) . while mab production plateaued after day 4 in basal medium only (final titer 0.8 g/l), a continuous increase to three-fold higher final titers (2.4 g/l) was observed in feed-spiked medium (fig. 1b) . the higher titers could be attributed to generally higher cell-specific productivities (qp), which remained rather constant (~70 pg/cell/day) in feedspiked cultures. in basal medium, the qp continuously dropped by 20% (day 0 to 3), 50% (day 4), and > 90% (day 5 to 7) from 70 to 10 pg/cell/day in basal medium cultures. in average, the qp was 70% higher in feed-spiked cultures (fig. 1d ). transcriptome analysis of differentially expressed genes between cells grown in basal medium or feed-spiked medium were used to identify relevant go terms that indicated a more active proliferative state for feed-spiked cultures (data not shown). the top go terms significantly related to cell cycle and primary metabolism, cellular division, as well as nucleobase formation or regulation. furthermore, gsea revealed several significantly enriched set of genes related to gene transcription, dna replication and repair, cell growth and proliferation, as well as inhibition of apoptosis in feed-spiked cultures. thus, feed-spiking increased the proliferative activity of cultivated cells. several of the identified genes appear as promising targets for cell line engineering, but have not yet been described in relation to high-producing recombinant cell lines and will need to be evaluated in future studies. feed-spiking of basal medium is a convenient and easy way to considerably increase product concentrations in a simple batch culture. differential gene expression revealed genes that appear important for high cell-specific production rates, and this knowledge can be leveraged into cell line engineering approaches or the design of high producing cho cell media. in the latter case, a maximized supply of high biosimilarity must be demonstrated by physicochemical and functional characterization for approval requirements of phase i and phase iii studies in terms of efficacy, safety and immunogenicity. in this study, rounds of upstream and downstream processes were run to reach the cqa limits of the originator molecule. after conducting many different development strategies, the mirror plot images of the intact deconvoluted mass were found to be identical corresponding to similar levels of glycoforms. the uv chromatogram of reversed phase ultraperformance liquid chromatography (rp-uplc) of tryptic peptide mapping demonstrated that the primary structure of tur01 is identical to the originator as shown in fig. 1a . post-translational modifications (ptms) such as oxidation, deamidation, n-terminal pyroglutamic acid, c-terminal lysine truncation levels were also comparable for two products. the glycosylation site (hc-asn301) was confirmed by peptide mapping analysis and 100% glycan site occupancy was proven for tur01 and originator. the glycosylation pattern for two products were highly similar in terms of major glycans (g0f, g1f, g0f-gn and etc.). man5 level was lower in tur01 compared to the originator product which may not have any clinical effect on the molecule. the secondary structure was determined by atr-ftir spectroscopy. absorption bands (amide i and amide ii) were overlapped completely and amounts of α-helix and β-sheet structures were comparable. furthermore; size-exclusion chromatography (sec) analysis revealed that both products have the same level of purity (>99%) and aggregate (<1%) levels. the level of impurities were determined as below 4% by ce-sds. the capillary isoelectric focusing (cief) experiments showed that the charge variant profiles of two products are indistinguishable and the isoelectric point of main peak is observed at 8.3 for both products. the association/dissociation rate constants and binding affinity for both tur01 and originator were highly similar and similarity score was calculated greater than 99%, as shown in fig. 1b . in this study, state-of-art analytical techniques were used to assess the biosimilarity of tur01 to the originator adalimumab. head-tohead comparison data clearly demonstrated that tur01 is highly similar to the originator adalimumab in terms of physicochemical and functional characteristics. based on the analytical similarities, we . process performance of basal medium (black) and feed-spiked (red) bioreactor batch cultures: a cell concentrations and viability, b viable cumulative cell days and specific growth rate, and c antibody concentrations and cell-specific productivity. error bars indicate standard deviation from three independent experiments. the black arrows on day 4 indicate the beginning of decreasing cell-specific productivities and lower cell-specific growth rates in basal medium cultures believe that tur01 will have comparable pk/pd, potency, and efficacy results to the originator adalimumab. the expanded interest in intensified continuous bioprocessing has highlighted the need to develop a small scale model for perfusion cell culture. the direction in the industry has been to increase target cell densities to ≥50x10 6 vc/ml and decrease perfusion rates to ≤3vvd. in order to increase the throughput of our perfusion media development capabilities we sought to develop a small scale model of perfusion using the ambr®15 instrument (sartorius, germany). we used a modified cell settling model from the previously published by kreye et. al. to achieve the cell retention necessary to reach perfusion relevant viable cell concentrations [1] . in this work, we will show the application of this small scale model for: (1) identification of specific productivity performance over a steady-state for tested media, (2) identification of cspr min for a specific cell line and medium combination, and (3) confirmation of consistent product quality profiles between the small scale model and benchtop perfusion (data not shown). a chozn® cell line producing an igg1 was evaluated in several proprietary chemically defined media prototypes generated during the development of the catalog excell® advanced hd perfusion medium: "fed batch medium", "early prototype", "mid prototype", "intermediate prototype" and "late prototype" [2] . small scale simulation of perfusion experiments were run in ambr®15. media exchange was performed 3 times per day in equal amounts. agitation, gassing, and liquid handling were stopped for an optimized period of time to allow cells to settle to the bottom. spent media was removed in an amount proportional to 1/3 rd daily exchange volume. agitation, gassing, and liquid handling were resumed and fresh media was added back to the vessels. for benchtop perfusion, cells were inoculated in 3l applikon bioreactors (applikon, netherlands). at a concentration of~6.0x10 6 vc/ml, perfusion was initiated using the atf2 (repligen, massachusetts). perfusion rate was limited at 1.2vvd during steady-state. using the cell settling method described above we have been able to achieve ≥90% cell retention efficiency. all media tested in this work were able to reach and maintain the 30x10 6 vc/ml target cell density at 1vvd (fig. 1) . performance of each media formulation was ranked based on specific productivity (table 1) . using "intermediate prototype", minimum steady-state cspr was determined to be 33.3pl/c/d for this cell line. n-glycan analysis of ambr®15 and bioreactor samples via intact mass spectrometry displayed only slight differences in product quality profile (data not shown). our work has shown a clear distinction between various prototype perfusion media and demonstrated a 50% increase in specific productivity over "fed batch medium" used in perfusion. additionally, we have shown the application to further characterize the process using this model to determine cspr min for a given medium and cell line. the transient process has been successfully operated at 500l in a sartorius biostat single use bioreactor (sub), yielding 0.4kg of crude product from a two-week expression culture (table 1) . successful scale up of the process to 500l creates the potential to supply transiently expressed products to support toxicology studies or even early gmp clinical supply, enabling accelerated biopharmaceutical development project timelines. the scale up from rocking bioreactors (rbr) to sub scale identified some scalability issues. lower specific productivity due to increased cell growth and decreased titres were observed in the sub ( fig. 1 iii & iv). to improve the predictability of scale up, a new process was developed and evaluated in the sub vessels utilising a modified transfection method, which resulted in comparable expression levels and specific productivity between rbr and sub scales. two sets of expression vectors comprising heavy chain and light chain plasmids expressing a human igg1 kappa mab, as previously described [1, 2] were used in the process optimisation study. the cell line used for transient expression and the pei mediated transfection method has been described previously [1] . transfected cultures were run under fed batch conditions for 14 days in 22l ge healthcare wave bioreactors (rbr), hyclone sub using 50l and 250l hyclone bioreactor bags (thermo scientific). the transfection process was modified to address the reduced titres and higher viable cell density (vcd) seen in the sub cultures. shake flask cultures were used to assess the standard (a) and modified transfection processes (b and c) (fig. 1 , i & ii). process c was identified as the process to be studied at sub scale, offering the potential to mitigate the high viable cell densities (vcd) observed. scaling up process c to 50l and 250l sub resulted in cultures producing titres exceeding 1g/l with desired cell growth profiles. scale up of process a into sub vessels resulted in decreased productivity compared to the rbr scale. after optimisation, the sub process c yielded increased specific productivities and expression titres comparable to those seen at rbr scale (table 1) . medimmune has successfully completed the first known successful cho transient culture at 500l scale producing > 800mg/l of mab at harvest. process optimisation has subsequently demonstrated reproducible titres at 50l to 250l scale exceeding 1g/l with comparable glycosylation profiles between sub and rbr cultures across scales. comprehensive analysis of the impact of trace elements in media on clone dependent process performance and product quality background state-of-the-art biopharmaceutical processes are accounting concomitantly for process performance and product quality. even though high yielding, robust processes are the cornerstones of any process development, product quality parameters such as structural integrity, charge variances and post-translational modifications are progressively becoming the focus of the developmental work. in conjunction with host cell line selection and process performance parameters, media components are crucial for the continued progress in rational modulation of product quality attributes affecting biological activity, immunogenicity, half-life or stability. among media components, trace elements (te) are of particular interest as they play a pivotal role in various cell metabolism pathways. based on a comprehensive doe approach, extensive process performance-and product quality evaluation combined with metabolic flux analysis, the impact of several trace elements on the biopharmaceutical process is assessed. in a comprehensive i-optimal doe approach ( fig. 1) , the effect of six te in various concentration levels and combinations in serum-free media was studied for four different cho-k1 cells lines in an ambr® 15 setup. a scrutiny of the process performance parameters such as cellular growth, productivity, amino acids and vitamins consumptions rates for each of the conditions was performed. the process performance evaluation was accompanied by extensive product quality analysis including size and charge variants, glycosylation patterns, oxidation and methylation. furthermore, a metabolic flux analysis was performed based on the nitrogen balance. based on extensive analytical data, the obtained response surface model provides a clear insight into the impact of particular te and their combinations on process performance and product quality. the high model quality enables discriminations between clone dependent and clone independent effects. with an elevation in titer up to 25% in the best condition of the cell lines clearly show, that even state-of-the-art media can be outperformed by trace element rebalancing. analyzing specific rates in combination with metabolic flux analysis improves the understanding of metabolic restructuring of the cell lines under distinct te levels and combinations. modulation of trace elements levels had a tremendous effect on the charge heterogeneity and glycosylation structure of the different proteins. this provides a toolbox for the fine tuning and control of product quality parameters. taken together, the data further paves the way to the rational fine tuning of process performance and product quality attributes. background due to regulatory concerns and economic impact, ensuring product quality and consistency is now one of the main challenge faced by the biopharmaceutical industry. for monoclonal antibodies (mab), glycosylation is one of the most important quality attributes as it impacts on mab structure integrity, and ultimately on both clinical efficacy and safety. many factors affect mab glycosylation and its inherent heterogeneity, including the host cell, the culture medium, the mode of operation and the operating conditions. in this context, the capacity to monitor and control on-line the antibody glycosylation, from early-to late-stage process development, would be of salient interest to reduce the time and cost to market. in order to address this unmet need, we have designed an improved spr biosensor assay to measure the kinetics of interaction between a mab and the extracellular domain of the fcγriiia receptor bound at the biosensor surface [1, 2] . of salient interest, we also demonstrated that various binding kinetic signatures, especially different dissociation kinetics could be correlated with distinct mab glycosylation patterns and with therapeutic efficacies, as deduced from mass spectrometry and a surrogate adcc assay, respectively. in parallel, we have also harnessed a spr biosensor directly to a bioreactor, which permitted the at-line determination of the concentration of antibodies by hybridoma cells during a bioreactor culture. we now plan on combining both approaches to determine on-line the glycosylation profile of the produced mabs. our ultimate goal is to design a unique and highly innovative bioprocess control tool that can be readily applied in an industrial bio-manufacturing setting. reducing timelines and costs are key factors for bio-pharmaceutical industries to accelerate process development and drug delivery to patients. enhancing throughput of bioprocess development has become increasingly important for the screening and optimization of cell culture processes. this challenge requires high throughput tools. in a previous study [1] , we showed that ambr® 15, a robotically driven mini-bioreactor system developed by tap-sartorius, could be advantageous to accelerate process development. the use of ambr® 15 system allows us to test a large number of experimental conditions in a single experiment. therefore, the large amount of production samples to be characterized for product quality attributes (pqa) increases as well: the bottleneck has moved from the generation of samples at the production bioreactor step to in-process analysis. for product quality attribute analysis at lab scale, protein purification is generally carried out on >5ml columns which is incompatible with the size of ambr® 15 bioreactors. moreover, the applied methods are relatively low throughput. the development of a high binding capacity resin (up to 70 mg/ml) [2] , combined with high performing new cell lines which are able to produce up to 5 g/l of recombinant monoclonal antibodies, allow require the development of an efficient and high throughput (hts) purification method robot. the use of robotic equipment for small scale purification purposes is a great opportunity for us to tackle this bottleneck, by enabling highthroughput sample purification at smaller scale (200μl). recombinant monoclonal antibodies were produced by a genetically engineered dihydrofolate reductase (dhfr)-/-dg44 chinese hamster ovary (cho) cell line. clarified cell culture fluid (cccf) was obtained from 2 and 2k liter bioreactors after three filtration steps. minipurifications were performed on tecan freedom evo® robot with predictor robocol-umns® containing 200μl mabselect sure® resin. larger scale purification were executed using an aktaxpress using hitrap column prota. to assess monoclonal antibody purification at small scale, we first tested the repeatability of the minipurification, purifying the samples 8 times on the same columns and using different columns, focusing on the yield of the purification and the impact on product quality attributes, especially the hmws. then, we compared those results to those obtained with the aktaxpress at larger scale purification, comparing the yield of the purification and the pqa of the protein-a eluates obtained with both purification systems. finally, we assessed the capability of the robot to perform hts of buffer and purification conditions, evaluating three different buffers at different concentrations and ph values, and also testing different loading column capacities. in this study we established that the tecan can be used as a robust platform for purification at small scale. we observed similar purification yields, intra and inter run. the analysis of the pqa1a level showed there is also very high reproducibility. and the ph of the eluate showed as well strong comparability as well. table 1 shows the coefficient of variation (cv) of the yield, hmws and eluate ph are low, demonstrating the good reproducibility of the purification. the strong reproducibility obtained between the different purifications showed that the tecan and the aktaxpress are similar in terms of purification performance and pqa (fig. 1a, b) . the tecan is a versatile automated liquid handler allowing the screening of huge purification conditions (fig. 1c) , the possibility to purify large quantities of samples, while the samples amount is limited. the tecan has the potential to purify more than 150 samples/day, reducing timelines and allowing us to deliver faster to the patients. viable cell density monitoring in bioreactor with lensless imaging geoffrey monitoring cell density and viability of mammalian cell culture bioreactors is a necessary task that presents today a number of remaining challenges. the traditional measurement for bioreactor cell count and viability rely on using the trypan blue exclusion method once a day. while automatic cell counters have reduced the statistical manual error, sampling the bioreactor remains a contamination risk and is prohibiting process control as the sampled volume becomes siginficant. lensless imaging technology is a new method for accurately determining cell concentration and viability without staining. this technique directly acquires the light diffraction properties of each individual cells through their holograms images without any objective, lens or focus settings. living and dead cells have significant holographic patterns that can be distinguished and precisely counted. lensless imaging technique directly acquires the light diffraction properties of each individual cells through their holograms images without any objective, lens or focus settings. living and dead cells have significant holographic patterns that can be distinguished and precisely counted. we compare cell counts and viability between the reference method and our lensless imaging device, the cytonote counter. measures are performed once a day on samples from 12 bioreactors, from the inoculation to the end of the culture. we also assessed the repeatability of our method. another lensless imaging prototype is setup as a measurement chamber directly connected to a perfusion bioreactor, for continuously receiving the bioreactor broth, and therefore reproducing an in situ measure. with a concentration range up to 40x10 6 cells/ml ( fig. 1) and viability range at 75-100%, we obtained a correlation factor of 0.98 between the two compared methods. the large field of view allows the analyze of several thousand cells within a single image, keeping the statistical variability of the measure as low as 3%. our measurement chamber prototype has demonstarted its capability for continuous viable cell density and viability monitoring. we are now working at designing a steam strerilizable probe, and we envision lensless imaging to become the future method of choice for on-line monitoring of suspension cells cultures. lensless imaging technology is capable of accurately and precisely monitoring viable cell density and viability with a combination of significant advantages starting from low sample volume use, label free detection, quick measure, simple device, to high number of cell analyzed which let us think that it is a good candidate for very smallcomparison between both purification systems and the ability of the system to be used as a high throughput tools for buffers screening. a purification yield (%). b pqa1.a (normalized). c impact of the ph and buffer concentration on the pqa1.a scale bioreactor and high-throughput measures. its high repeatability is also a key parameters in the effort to narrow batch to batch deviations. in addition we demonstrate that this technique is potentially powerful for in-line and continuous monitoring of a lab bioreactor. we envision lensless imaging to become the future method of choice for on-line and in-situ monitoring of suspension cells and a perfect tool for process control in fed-batch or perfusion mode in single-use bioreactors or traditional steam sterilized vessels. it can certainly become the first vcd measurement technique to work from cell line engineering, to process development, pilot scale, and up to manufacturing scale. time-dependent product heterogeneity in mammalian cell fermentation processes background a consistent product quality is a major goal in the production of biotherapeutics, especially recombinant glycoproteins. whereas it is unlikely that the polypeptide chain changes during a production process, posttranslational modifications and protein folding are sensitive to fluctuations in parameters and conditions. here we focus on protein glycosylation as one important indication for product quality [1] . during a batch process conditions change continuously. at the beginning, the supply situation for the cell is excellent, but the secreted material stays a long time in the culture fluid. later during cultivation substrate provision decreases, whereas the exposition time of the protein to the culture fluid is much shorter. altogether this leads to product heterogeneity of the secreted protein during a batch culture. four different cell lines, two of human origin and two cho clones, producing four different recombinant glycoproteins were investigated in this study. together with their respective parental cell line the clones were cultured in three replicates in shakers. supernatant from the cultures were harvested at four time points. the removed culture volume was replaced by culture supernatant of the identically cultured corresponding parental cell line. the product was isolated from the supernatant and the glycans were released. one part of the released glycans was labeled with 2-ab and separated by hilic-fld. the other part of the glycans was permethylated and analyzed by maldi-tof mass spectrometry (fig. 1a) . the investigated proteins were antibody, antithrombin iii from cho clones and α1-antitrypsin, c1-inhibitor from human clones. the antennarity of the glycans is quite stable in all production phases. the degree of core fucosylation is very high in all products. a low fucosylation degree of antibodies may be favorable for a higher adcc performance [2] . some of the products showed an antennary fucosylation, which seemed to change not very much in different cultivation phases. nevertheless, this might be an issue due to an antigenic impact in the patient. the antennary galactosylation changes noticeable for the antibody and α1-antitrypsin. in both cases the degree is highest in the first phase. an incomplete galactosylation leads to truncated glycans. this leads inevitable to undersialylated antennas to be seen for α1-antitrypsin. the sialylation is the highest in the early phases and decreases during cultivation time. sialylation of a therapeutical protein is important for the half-life in the patient. therefore highly sialylated products are desired [3] . in further studies the consistency of the galactosylation and the sialylation will investigated for fed batch and long term continuous cultures in comparison to batch cultures. due to the feed solution or the fresh media being present during such processes, the supply situation should be excellent for the whole cultivation time. the differences between the maldi-tof and hilic-fld data originate from complex and unresolved chromatograms (fig. 1b , chromatograms not shown). for that reason coupling of hilic-fld and ms is very much recommended. background novel biologics are often selected from a large library of lead candidates in the initial stage of preclinical and clinical developments. for this selection, there is a demand for high-throughput production of recombinant proteins of high quality and in sufficient quantity. transient gene expression offers a rapid approach to the production of numerous recombinant proteins for the initial-stage developments of biologics. mammalian cells are major host cells for transient gene expression, but they have the disadvantages of complicated operations and high cost of culture. insect cells are easy to handle and can be grown to a high cell density in suspension with a serum-free medium. insect cells can also produce large amount of recombinant proteins through post-translational processing and modifications of higher eukaryotes. hence, insect cells have been recognized as an excellent platform for the production of functional recombinant proteins [1, 2] . in the present study, the production of an antibody fab fragment through transient gene expression in lepidopteran insect cells was examined. the dna fragments encoding the heavy chain (hc) and light chain (lc) genes of an fab fragment of mouse anti-bovine rnasea [3] were respectively cloned into the plasmid vector pihaneo, which contained the bombyx mori actin promoter downstream of the b. mori nucleopolyhedrovirus (bmnpv) ie-1 transactivator and the bmnpv hr3 enhancer for high-level expression [4] . trichoplusia ni bti-tn-5b1-4 (high five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine (pei; mw 40,000). before transfection, the plasmids and pei were prepared in 150 mm nacl, ph 7.0 and incubated at room temperature for 5 min. when the transfection efficiency was checked, a plasmid vector encoding the enhanced green fluorescent protein (egfp) gene was also co-transfected. transfected cells were incubated with a serum-free medium in a static or shake-flask culture. culture supernatants were analysed by western blotting and enzyme-linked immunosorbent assay (elisa). the numbers of green fluorescent cells and total cells in culture broth was determined using a flow cytometer. western blot analysis and elisa of culture supernatants showed that transfected high five cells secreted the fab fragment with antigenbinding activity. in static cultures, transfection and culture conditions, such as hc:lc gene ratio, a serum-free medium, dna:pei ratio, and dna amount per cell, were successfully optimized by flow cytometry of egfp expression in transfected cells and the yield of the secreted fab fragment measured by elisa. the effects of culture temperature and initial cell density were also examined by comparing the cell growth and the production of fab fragments in shake-flask cultures. under optimal conditions (medium, psfm-j1 (wako pure chemical industries, japan); hc:lc gene ratio, 3:7; dna, 5 μg/(10 6 cells); pei, 10 μg/(10 6 cells); initial cell density, 1 x 10 6 cells/cm 3 ; temperature, 24°c), the yield of more than 100 mg/l of fab fragment was achieved in 5 days in a shake-flask culture ( fig. 1) . transfection did not significantly affect the growth of high five cells as compared with untransfected cells. transient gene expression using insect cells may offer a promising approach to high-throughput production of candidate proteins for the development of biologics. the increasing demand for biopharmaceuticals produced in mammalian cells has led industries to increase volumetric productivity of bioprocesses through different strategies [1, 2, 3] . in this context, fedbatch and perfusion cultures have attracted more interest than conventional batch processes. the efficient application of such alternative processes requires the availability of reliable on-line measuring tools for cell density and cell metabolic activity estimation [4] . the comparison of different culture strategies for hek293 cell line producing ifn-γ are presented below: batch, fortified batch and fed-batch. in this context, a new robust feeding strategy based on the monitoring of alkali buffer addition was applied for the estimation of nutrient requirements. this method allows to increase cell density and product titer compared with the other strategies assessed. three different culture strategies were carried out in 2-litre biostat b-dcu ii bioreactor. first, a reference batch and a batch using fortified medium (nutrient enriched medium) were run and assessed in terms of viable cell density (vcd) and product titer, and set as initial references. then, a fed-batch was performed applying a feeding strategy based on the nutrient requirements estimation by monitoring the alkali buffer addition used for the control of ph. results vcd and product titer achieved for the different culture strategies assessed (batch, fortified-batch and fed-batch) are presented in table 1. in fortified batch an increase in vcd of 145% and also 350% in product titer were obtained compared with batch. in the fed-batch culture carried out (fig. 1) , we observed that alkali buffer addition profile matched the vcd evolution trend. thus, the monitoring of alkali buffer addition was used for estimating the nutrients requirements (i.e. the volume of feeding medium) at any time during the fed batch phase. the feeding strategy based on alkali buffer addition enabled to maintain glucose concentration set point therein a narrow range during fed-batch phase (around 20 mm). as a result, higher vcd (16.6·10 6 cells/ml) was obtained when compared with both batch references: vcd was enhanced to 241% and 39% and an increase up to 381% and 7% in product titer in respect to batch and fortified batch respectively. the results prove that fed-batch strategy based on the alkali buffer addition is a robust on-line monitoring method that enables to optimize the feeding strategy in a fed-batch cultures. three different culture strategies have been tested in bioreactor with a hek293 cell line producing ifn-γ. results show as the higher vcd is reached, the higher product concentration is achieved. therefore, from bioprocess development point of view, it is very interesting to implement strategies with higher vcd outcome, such as fed-batch operation mode. in this context, a new robust method for vcd estimation in fed-batch was applied. the alkali buffer addition necessary for maintaining the ph set-point is an on-line reliable and easy measuring variable that provides information about by-products formation (mainly lactic acid). the monitoring of this variable can provide information about the cell concentration, activity and metabolism, to detect changes in culture. besides that, a relationship between alkali buffer addition and vcd can be established since the first is strongly correlated with cell growth and metabolites consumption/formation. the application of alkali buffer addition measure to implement an optimal feeding strategy in fed-batch permits to enhance vcd and product titer when comparing with batch strategies. a novel approach to high throughput screening for perfusion background perfusion systems for suspended mammalian cells raise growing interest in the biomanufacturing industry. continuous manufacturing is growing in the field and is encouraged by health authorities [1, 2, 3] . this work addresses scale down limitations inherent to continuous media exchange and cell retention by using a semi-continuous system. data was generated with a set of different clones that were previously studied in fed-batch mode [4] . materials and methods 4 cho-k1 cell lines expressing the same monoclonal antibody (mab) and issued from the same transfection were used as models. 3.5l bioreactors (sartorius) were used for fed-batch and perfusion production runs. the perfusion bioreactors were run using an alternating tangential flow filtration device (repligen, xcell™ atf 2 system). the cell biomass was controlled by removing cells through a bleed line and was controlled using a biocapacitance probe (hamilton, incyte). the perfusion rate (d) was fixed to one vessel volume a day (vvd -1 ). the semi-continuous runs were made in 50 ml shake tube (tpp, tubespin® bioreactor 50). once a day, the tubes were centrifuged (5 min, 200 g), the supernatant removed (to mimic a perfusion rate of 1 vvd -1 ), replaced with fresh media and cells were re-suspended. the clone's growth potential were preserved across the systems (fig. 1 ). clone #3 always reached the highest viable cell density (vcd), followed by clone #1. clone #2 and #4 showed similar growth characteristics. it is interesting to note that in the perfusion bioreactor different patterns in terms of vcd were observed although the cell biomass signals were similar for all 4 runs. this reflects the fact that the capacitance measures the biomass and not the absolute cell count [5] . to estimate the minimum cell specific perfusion rate (cspr min ) in the semi-continuous experiment, the perfusion rate was divided by the maximum viable cell density (vcd max ). this value was compared to the cspr obtained during the 4 th set-point (sp4) of the perfusion runs. as expected, the bleed fraction decreased when the capacitance set-point was increased and went down to 5% or less of the total perfusion rate (data not shown). since the bleed removes the excess biomass, it is an indication of how close to a limitation the system is. therefore, the cspr calculated at sp4 was considered as the minimum cspr. the cspr min obtained in both systems were very close ( table 1 ). the semi-continuous system can therefore be used to identify the cspr min before running a continuous bioreactor, it therefore facilitates the decision making early in the development (to define the target cell density for a defined perfusion rate). the specific productivity (q p ) of the 4 clones was quantified at the maximum vcd (semi-continuous) or at sp4 (perfusion). absolute values are not representative since the cell environment is so different in both systems. nevertheless, a relative ranking proved to be indicative of the respective performances ( table 1 ). the maximum cell growth in fed-batch, semi-continuous and perfusion were also compared, their ranking was always preserved. both indications can be used to assess a performance ranking for different clones. the performance of 4 clones was studied in 3 different cultivation systems: fed-batch/perfusion bioreactors and semi-continuous shake tube. the semi-continuous system was able to precisely predict the cspr min , an important process parameter for perfusion. specific productivity and maximum cell density ranking was preserved across the systems, therefore the scale down experiment can be used to assess a performance ranking for perfusion clone screening. modulating antibody galactosylation through cell culture medium for improved function and product quality jenny y. bang, james-kevin y. tan the production of therapeutic antibodies (abs) requires high titers and excellent product quality to ensure efficient manufacturing and potent drug efficacy. glycosylation, or the attachment of sugars to organic molecules, is a critical quality aspect that can significantly alter ab binding, function, and therapeutic effect [1] . galactose is a key sugar of interest due to its significant impact on ab function and the ability to control galactosylation through cell culture medium. herein, irvine scientific assessed the ability of media components to modulate galactose levels on a model therapeutic ab. various media compositions were able to modulate galactosylation levels without compromising cell growth and ab titers. in addition, an in vitro assay was utilized to evaluate the functional ability of abs to bind and activate complement-dependent cytotoxicity (cdc). differences in galactosylation significantly altered the abs' ability to induce cell cytotoxicity. furthermore, design of experiment analysis determined the optimal ratio of supplements to maximize galactosylation. this "optimized supplement" was verified and evaluated against other suppliers' galactosylation supplements in terms of growth, titer, glycan analysis, and ab function. the optimized supplement outperformed all other suppliers' supplements and resulted in the best overall cell growth, titer, galactosylation, and ab function. ab against cd20 were grown in balancd® cho growth a and were fed with balancd® cho feed 4 on days 3-7 of the cultures. viable cell density and cell viability were assessed by a beckman coulter vi-cell xr, ab titer was assessed by a pall fortébio qk e , and glycan analysis was assessed by a perkinelmer labchip gxii. for the functional cdc assay, abs were incubated with daudi b lymphoblast cells and normal human complement serum. cell cytotoxicity was assessed with a promega cytotox-glo kit. various supplements were evaluated in fed-batch cultures and resulted in 15-45% ab galactosylation without compromising cell growth and ab titers. design of experiment analysis determined an optimal composition, deemed "optimized supplement," which was evaluated against a panel of galactose-modulating supplements from other suppliers. the optimized supplement resulted in a similar viable cell density (vcd) and cell viability compared to the fed-batch culture control which had no supplements (fig. 1a) . supplements from supplier 1 resulted in similar to half the vcd of the control while supplements from supplier 2 resulted in very low vcd and percent viability. all of the supplements except those from supplier 2 resulted in ab titers similar to the control (fig. 1b) . due to the poor growth and subsequently low titer from supplier 2's supplement, supplier 2 was not further evaluated. the glycan profiles were analyzed and are presented in (fig. 1c ). all the evaluated supplements were able to raise galactosylation; however, only the optimized supplement and the 2x supplier 1 supplement resulted in over 40% galactosylation. the function of the abs was further evaluated in a cdc assay (fig. 1d) . abs from the optimized supplement were more effective than the control abs and had a significantly lower half-maximal effective concentration (ec 50 , 1.19 μg/ml) than the control (1.71 μg/ml). abs from the 2x supplier 1 supplement had a similar ec 50 to the control which may be due to the higher man5% of the abs. an optimized supplement was produced through fed-batch evaluation and design of experiment analysis. the optimized supplement outperformed all other supplenments from other suppliers and resulted in the best overall cell growth, glycan profile, and functional ab activity (table 1) . industry practice for mammalian cell culture media and feed development typically employs a high-throughput screening (hts) platform along with large sets of experiments [1] . modern hts systems often include robotic liquid handlers to replace labor intensive steps. to align with advancements in the field, a semi-automated hts platform was developed to facilitate in-house media and feed development for early stage biologics projects. selecting appropriate instruments and integrating them into a seamless system are the keys to a hts platform. the developed hts platform uses 24 deep well plates (dwps) for culture vessels, the liquid handler of the advance microscale bioreactor (ambr15) for media/ feed formulation preparation in an aseptic environment, nyone cell imager for viability and cell growth analysis, tecan freedom evo's liquid handler for activity assay sample preparation, and cedex bioht for high-throughput metabolite analysis. 24 dwps offer comparable cell growth to shake flasks and compatible layout to ambr15, which makes the 24 dwp an ideal candidate for the platform. in addition, the user friendly design of experiments (does) interface and liquid handler function of the ambr15 expediates the formulation preparation of varying doe conditions [2] . a macro program was written and developed in excel to enable the easy import of does design from major statistics software packages, such as jmp and simca, into ambr15. performance qualification of each component were performed prior to implementing the hts platform. comparable cell growth profile and productivity were achived between shake flasks and 24 dwps (fig. 1a ), indicating compatable cell culture environment for the cells. cell counts using nyone gave identical cell growth ranking as the traditional count from vi-cell xr (fig. 1b) . freedom evo's liquid handler was optimized to produce comparable activity results to manual operation while expediting the sample preparation with improved consistency (fig. 1c) . finally, implementing the liquid handler function of ambr15 to support media and feed formulation significantly reduced the labor for each experiment. summary of the capability comparison between the hts platform and the traditional method are listed in table 1 . a case study of a complex feed screening with definitive screening design was completed using the semi-automatic hts platform. this experiment, containing more than 60 feed formulations in duplicates, was handled by one operator and delivered a 40% improvement in productivity within a 4 week period (data not shown). in addition, implementing the hts platform for this study also resulted into~80% reduction in labor while improving the traceability of formulation preparation. a semi-automated hts platform was developed to support media and feeds screening and development for early stage biologics projects. the platform utilizes 24 dwps, nyone cell imager, ambr15, freedom evo liquid handler system, and bioht metabolic analyzer to accelerate the screening process. this screening platform not only improves process throughput, operational precision, and traceability of formulation preparation, but also reduces the labor for the media and feed formulation preparation. background a perfusion medium requires high concentrations of specific nutrients while balancing other components to support intensified perfusion processes. using a combination of design of experiment (doe), multivariate analysis (mva), and spent media analysis, we developed a catalog "de novo" perfusion medium by working with multiple cho cell lines and proteins. the optimization of the medium in bioreactors using alternating tangential flow (atf) cell retention devices reduced the minimum cspr from over 80pl/cell/d to under 35pl/cell/d for most cell lines while increasing specific productivity during 30 day steady states with stable growth rates, viability, volumetric productivity and product quality. high throughput screening (hts) was performed with seven cell lines, while four were used in bioreactors: cho-s, dg44, and two chozn® gs lines, each producing different monoclonal antibodies and include a fusion protein. for hts experiments, cells were inoculated at 2.0x10 6 vc/ml with a 30 ml working volume in 50 ml tpp® tubes and cultured for 7 days in a multitron shaken at 200rpm, 37°c, 80% rh, and 5% co 2 . for benchtop perfusion, cells were inoculated at 0.4-2.0x10 6 vc/ml in 3l applikon bioreactors (applikon, netherlands) with a 2l working volume. bioreactors were operated at 350 rpm, 37°c, 40% do, and a ph of 6.9 or 7.1±0.05 depending on the cell line. oxygen was supplied through an l-sparger or microsparger as needed, and excell® antifoam (milliporesigma, germany) was added at a maximum rate of 0.25% v/v to control foam. at a cell concentration of~6.0x10 6 vc/ml, perfusion was initiated using the atf2 (repligen, massachusetts), with a bleed set to maintain cell concentrations at 50 or 80*10 6 vc/ml. two "de novo" prototype media were developed using doe and mva in hts with tpps and an ambr®15 [1] and one was chosen for further development after comparing to a basal medium enriched with feed in bioreactors. eleven components were identified as significant effectors of critical parameters for perfusion processes across evaluated cell lines. doe central composite experiments were run and component concentrations were optimized in the selected prototype. in parallel, amino acid specific consumption rates were calculated from bioreactor spent media samples and used to adjust the concentration of amino acids to target a reduced cspr. increasing specific amino acids concentrations resulted in a significant reduction of the minimum cspr across all tested cell lines -for example the cspr of cho-s was reduced from 60 to 39pl/cell/day (table 1 ). however, even at the lower cspr, spent media analysis revealed excess concentration of some amino acids, so specific accumulating amino acids were reduced and components were streamlined for the final medium: excell® advanced hd perfusion medium. using this medium, a cho-s and a chozn® gs cell line producing a fusion protein were cultured at a cspr of less than 40pl/cell/day with a vcd of 50*10 6 vc/ml. metabolic profile, productivity, and product quality were constant over the 30 day steady state. the chozn® gs cell line was also tested at 80*10 6 vc/ml with a cspr of 33pl/cell/day (fig. 1 ). we have developed a catalog perfusion medium from first principles, ensuring broadness of application by using seven cell lines in scaleddown systems and four in perfusion bioreactors. the final catalog medium showed significant improvements in productivity across all cell lines, with reduced csprs when compared to enriched fed-batch medium or initial prototypes (table 1) . there is a rising demand for accelerated process development, increased efficiency and economics for biopharmaceutical production processes. furthermore, increased process understanding have evolved from the process analytical tool initiative (pat) and the quality by design (qbd) methodology. in contrast to one-factor-at-atime methods, statistical design of experiment (doe) methods are widely used to develop biopharmaceutical processes. even if highthroughput systems can handle these numbers of experiments in parallel, the heuristic restriction of boundaries and the high number of factors results in stepwise iterations with multiple runs. therefore, the combination of model-based simulations with doe methods (mdoe) for the development of sophisticated cell culture processes is a novel tool for process development [1] . it is used to reduce the number of experiments during doe and the time needed for the development of more knowledge-based cell culture processes. this concept was applied to the optimization of the initial glutamine and glucose concentrations of a cho batch process. a mechanistic model was adapted and modified from [2] and used to describe the dynamics of cell metabolism and antibody production of an il-8 antibody producing cho cell line (see abbreviation of fig. 1 for cultivation details). experiments were simulated and compared to a fully experimental doe. as can be seen from table 1 , user defined constraints were chosen to get a stable and reproducible process with the aim of maximizing the cell density but decreased lactate and ammonia production. at first, the experimental space was estimated by simulating the responses for broad concentration ranges and calculating the multiple response desirability function (fig. 1a) . this results in a small area (turquoise) suggested as experimental space. experiments were planned within these boundaries and responses were either simulated ( fig. 1b, 4 cultivations for fitting the model) or compared with the purely experimental responses (fig. 1c, 16 cultivations). optimal concentrations for glutamine and glucose with respect to the constraints are in the lower right corner and similar for both methods (red frame, fig. 1 ). compared with the fully experimental design, mdoe results in a reduction of 75 % in the number of experiments (4 experiments for modelling vs. 16 experiments in experimental doe). the method is intended to optimize cultivation strategies for mammalian cell lines and evaluated these before experiments have to be performed in laboratory scale. this results in a significant time and cost reduction during process development and process establishment. the strategy is especially intended for the use in multi-single-use-devices to speed up process development. . at a target cell density of 50*10 6 vc/ml, volumetric productivity was stable for a 30 day steady state with excell® advanced hd perfusion medium. shorter steady states were tested at 80*10 6 vc/ml background for the large-scale production of therapeutic glycoproteins, fedbatch culture has been widely used for its operational simplicity and high titer. however, repeated feeding of medium concentrates and/ or addition of a base to maintain optimal ph during fed-batch culture lead to increase in osmolality. the hyperosmolality affects glycosylation in a protein-specific manner. however, the mechanism behind such osmolality-dependent variations in glycosylation in recombinant chinese hamster ovary (rcho) cells remains unclear. in this study, to better understand the effect of hyperosmolality on the glycosylation of a protein produced from rcho cells, we investigated 52 n-glycosylation-related gene expression and n-linked glycan structure in fc-fusion protein-producing rcho cells exposed to hyperosmotic conditions. furthermore, to validate the effect of hyperosmolality on protein glycosylation, we performed hyperosmotic culture supplemented with betaine, an osmoprotectant, and then analyzed the n-linked glycan structure and mrna levels of n-glycan branching/antennary genes. after three days of hyperosmotic culture, nine genes (ugp, slc35a3, slc35d2, gcs1, manea, mgat2, mgat5b, b4galt3, and b4galt4) were differentially expressed over 1.5-fold of the control, and all these genes were down-regulated. n-linked glycan analysis by anion exchange and hydrophilic interaction hplc showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary n-linked glycans was significantly decreased upon hyperosmotic culture. addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra-antennary n-linked glycans (p ≤ 0.05), while it increased the expression of the n-glycan branching/antennary genes (mgat2 and mgat4b). thus, decreased expression of the genes with roles in the n-glycan biosynthesis pathway correlated with reduced sialic acid content of fc-fusion protein caused by hyperosmolar conditions. conclusions taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on n-glycosylation, especially sialylation, in rcho cells. the identified genes, particularly mgat2 and mgat4b, are potential targets for engineering in cho cells to overcome the impact of hyperosmolality on glycoprotein sialylation. disruptive cost-effective antibody manufacturing platform based on cutting-edge purification process v. medvedev, m. duyck, t. albano, j. castillo univercells sa, gosselies, belgium correspondence: v. medvedev (v.medvedev@univercells.com) bmc proceedings 2018, 12(suppl 1):p-093 background demand for high-quality monoclonal antibodies is growing exponentially, calling for new production capacities. overcoming current limitations of conventional manufacturing strategies, namely the high capital investment and production cost, can only be achieved through innovative process designs based on the latest technologies. this study presents a process design combining batch-fed technology with continuous multi-column capture. an advanced cell culture clarification method was introduced to simplify downstream operations and increase overall cost-effectiveness of the process, for an optimized production of recombinant proteins. this study was performed with cho cells expressing a monoclonal antibody targeted against the coronoavirus responsible for the middle east respiratory syndrome (mers), developed by organic vaccines tm and the nih, kindly provided to univercells. upstream process: -fed-batch, 12 days culture at 10l scale with cd-cho chemically defined media and feeds. harvest treatment: -precipitation of impurities in the production bioreactor using organic compounds (<1% v/v) and flocculation by electropositive organics (<0.1% w/v). -acidic ph and physiological conductivity. upstream processing and harvest treatment: culture reached 0.5 g/ l (8x10 6 cells/ml; 90% viability), harvest treatment was found to be very effective in terms of impurities clearance. capture: capture strategies were evaluated from the point of view of simplification of downstream operations, with hcp impurities content monitored as a key performance indicator. -protein a affinity chromatography: advanced harvest clarification enabled major improvements in affinity capture, in terms of eluate purity and reduction of host cell impurities (<35 ppm in all conditions tested). (fig. 1 ). -cation exchange chromatography: cex allows higher capacities (>100 g/l) than protein a, whilst being more affordable (from 2-to 6-fold cheaper). low residual hcp (<500 ppm) was observed with all cex resins tested. without harvest treatment and clarification preceding the capture studies (either affinity or cex), results showed a lower binding capacity of the resin, a higher content of hcp in the eluate (up to 2000 ppm), a higher content of hmw species in the elution fraction (up to 3-fold higher) and a significant turbidity of the neutralized eluate. -continuous multicolumn chromatography: further options to increase cost efficiency include using a continuous multicolumn setup (table 1) . two models were assessed based on two different static binding capacities (sbc), demonstrating that 4 to 6 columns of 100ml were able to process a 200l production in less than 24h. this method provides a great opportunity for designing simplified and low footprint mabs dsp processes, while maintaining similar or achieving superior quality profile compared to standard approaches: -harvests treatments followed by depth filtration proved to be a cost-efficient way to obtain pretreated feed and minimize the burden on downstream operations. -protein a resins exhibited advantages of extracting key contaminants during harvest treatment, while caex confirmed to be a competitive capture strategy. -switching from batch to continuous multicolumn mode allowed to process a complete batch in less than 24 hours, requiring lower media and resins volumes. followed by a single polishing step, such process set-up strongly supports the reduction of operations required to deliver a high-quality product. analyses of product quality of complex polymeric igm produced by cho cells background immunoglobulin m (igm) antibodies are secreted by b cells as the first defense against invading pathogens during primary immune response. some igm antibodies already gained the orphan drug status, which shows their unique capability in therapy of rare diseases. potential fields for applications are discovered with increasing knowledge about these molecules. it seems that the most active forms are pentameric and hexameric igms. unfortunately, recombinant production of igms is rather difficult as secretion and correct polymer formation results in low expression yields and mixtures of polymers. we established stable producing chinese hamster ovary (cho) dg44 cell lines to analyze cellular and extracellular factors that influence quantity and quality of the produced recombinant polymeric igm in future studies [1] . one quality parameter is polymer distribution, which can be measured directly in cell culture supernatant using densitometric analyses [2] . additionally, we developed a very efficient single-step-affinity purification strategy using the poros captureselect igm affinity matrix to analyze pure igms. for more precise measurements of the igm isoform distribution we separated the purified polymers by high performance liquid size exclusion chromatography (sec hplc). our cho dg44 cell lines grow to peak cell concentration of 4.5x10 6 cells/ml in erlenmeyer flasks and 4.0x10 6 cells/ml in bioreactors. similar productivity of approximately 50 mg/l was observed for cells cultivated in both cultivation vessels in a nonoptimized batch culture using chemically defined media. analysing how cultivation conditions affect the fraction of polymers may offer clues about the assembly of polymers and the challenges of igm production. we quantified polymeric distribution of igm directly in the supernatant using a densitometric method [2] . cultivated under standard conditions (37°c, ph 7) igm012 is produced as 90% pentamers, whereas igm012_gl only consists of approximately 80% pentamers. the purified igm012_gl was analysed with sec-hplc and contained 81 % pentamer and 19 % dimer, which is comparable to the results achieved with densitometry. the purification of the igm antibodies was quite challenging as the manufacturer recommend acidic elution, which led to aggregation and inefficient elution of our model igms. therefore, we screened for different elution buffers that prevent denaturation and aggregation. by combining high salt concentrations with moderate ph reduction we optimized elution conditions to 88-99% igm recovery, which corresponded to a five to six fold improvement compared to the manufacturers' conditions. sds-page analysis and sec-hplc showed that our elution strategy resulted in a very pure product after a single chromatographic step. the purification strategy was verified with the igm103, igm104 and igm617. our model igms were produced in a ratio of approximately 4:1 pentameric to dimeric igm, measured concordantly with both analytical methods. process development on igm purification using the poros capture select human igm affinity matrix enabled the recovery of highly pure fractions. through optimization, by combining mild ph and high salt concentrations, the relatively low elution yields were increased by a factor of 5-6. applying densitometry and sec-hplc we will investigate how culture conditions influence polymer formation in future. currently, no small scale (<0.5l) cell culture system is commercially available for high cell density perfusion cultivations to use in high throughput screening studies. to increase throughput for process characterization activities at janssen vaccines and prevention, a shaker flask-based scale down model was developed. though, the control possibilities of shaker flask cultures are technically very limited and different compared to a bioreactor controlled process. in addition, the sensitivity of the shaker flask model should allow the detection of the effects of process parameters on critical quality attributes (cqas) of the vaccine produced at large scale. iterative experiments were performed in shake flasks to evaluate the influence of cultivation parameters such as shaking speed, working volume, co2% in the incubator and daily base additions on cultivation parameters (as cell growth, ph and do). in addition, a medium exchange was tested to mimic the perfusion mode used in the bioreactor process. the presens shake flask reader was implemented to allow for ph and do monitoring. the conditions for which the performance as reflected in specific virus titer showed the best fit were selected. at these conditions, a series of parallel shaker flask infections were conducted to demonstrate statistical equivalence of performance parameter and cqas (as cell specific iu titer and vp/iu ratio) between the production scale and reduced scale processes and thus to qualify the shake flask as a scale-down model. a daily medium exchange by centrifugation was implemented and cultivation parameters for shake flasks were identified. based on performance parameter (cell specific vp titer) and the cqas of the vaccine (cell specific iu titer and vp/iu ratio), equivalence between the production-scale and scale down systems was confirmed. the scale down model data fall into the 95% prediction intervals calculated on manufacturing data whereas scale down model data from batch mode experiments (using non optimized cultivation conditions) do not. the shaker flask as a scale down model for the 10l bioreactor perfusion process was qualified. this model is a tool to screen a subset of process parameters at a higher throughput, thereby reducing process characterization timelines. background until today, the market for therapeutic proteins, especially monoclonal antibodies, is gaining more and more importance in the pharmaceutical field. to meet the increasing demand for these products, the industry made tremendous efforts to generate highly efficient production systems. one of the pharmaceutical industry's research focuses is the improvement of the secretion process in eukaryotic cells. in mammalian cells, the efficiency of protein transportation strongly depends on the translocation of a nascent protein into the er, which is mostly conducted by the signal peptide (sp) coupled to the nterminus. through the interchangeability of signal peptides between products and even species, a large variety can be used to enhance protein expression in already existing production systems materials and methods at first the influence of four different natural sps (sp (7), (8), (9) and (10)) was compared on the secreted amount of an igg4 model antibody (product a) in fed batches using a cho dg44 host cell line. in the second part, one promising sp-candidate showing improved secretion (sp (9)) was identified and the influence of this sp on four additional antibody products, which varied in their expressability from good to mid/bad, was investigated. in both approaches, the standard sp was implemented for comparative reasons. in the first approach, four signal peptides sp (7), sp(8), sp(9) and sp(10) were screened for their potential to improve the product secretion of cho dg44 cells expressing a model antibody (product a). the results revealed a 2.4-fold increase in average final fed-batch antibody titer of sp(9) when compared to the standard sp approach (standard sp = 0.44 g/l; sp(9) = 1.50 g/l). in the second approach, the enhancing capacity of sp(9) on secretion of four other igg products (named product b to e, table 1 ) was further evaluated. an improved performance was observed for all products when comparing sp(9) and the standard sp in a fed batch process (fig. 1) . with an increase in average final fed-batch titers ranging from 28 to 354 % and up to 290 % in cell-specific productivities. taken together, with a positive influence on the final concentrations of all tested products, the results obtained with sp(9) contribute to -signal peptide sp(9) was identified as a promising candidate with an average 2.4-fold titer increase during screening of four signal peptides. -sp(9) was able to improve production titers up to 354 % compared to standard sp. -sp(9) was able to improve cell-specific productivities up to 290 % compared to standard sp. -future usage of sp (9) contributes to the further optimization of sartorius stedim cellca's standard cell line development process. new platform for the integrated analysis of bioreactor online and offline data lukasz gricman 1 , milan ganguly 2 , amanda fitzgerald 3 , hans peter more and more experiments are used to assess bioreactor suitability and stability of clones, to evaluate media composition and other process parameters, and to start upscaling campaigns. this has resulted in a major bottleneck due to the increase in data capturing, processing, aggregation, visualization, and statistical analysis. in addition, the association of the data with the experimental context (e.g., fermentation protocols, media recipes, bioreactor control parameters) is not easily accomplished in high throughput. the data generated in the process must not only be analyzed, but also managed and stored to enable easy tracking and relating to historical records. furthermore, the processes are often developed by global teams interacting in complex enterprise it ecosystems. therefore, new and high performing systems for data capture, processing, and analysis need to be integrated in order to enable storage and correlation of experimental context information and various types of time course analytics data. we have developed genedata bioprocess™, a new enterprise platform for bioprocess development. the platform enables automatic capture and visualization of all online and offline data (e.g., ph, o2, metabolic data), auto-calculations and aggregations (e.g., ivcd, qp, consumption rates) and multi-parametric assessment of any type of time-series bioreactor data in the context of experimental protocol data (e.g., process parameters, feeds). genedata bioprocess comes with dedicated interfaces for integrating with relevant laboratory instruments, control systems, statistical analysis software packages and custom enterprise solutions. it enables the modeling and tracking of complex nonlinear workflows and supports decision making in bioprocess development. the data can be analyzed in the context of upstream process development, and also be correlated to other unit operations. automation support assists the ever increasing throughput of bioprocess development operations, and the analysis of experimental data and process parameters across unit operations or even different projects. this overall integration enhances process development workflows. highlighted use cases describe the selection of the best producer clones (fig. 1a) , the identification of optimized media feeding strategies (fig. 1b) , and the comparison of clone performance across different fermentation scales (fig. 1c) . a special focus is on the analysis of data from micro-and bench-top bioreactors (such as the ambr15™ and dasgip™ systems) operated in parallel. these bioreactors allow for increased throughput of clone selection and process optimization studies, which in turn leads to an increase in data generation. genedata bioprocess supports integration with such systems and enables a comparison of data regardless of the instrument provider or scale. automated bioreactor data analysis allows development groups to take advantage of even richer datasets and, as data management is built-in to the system, the data can be easily tracked and associated to historical records. another focus is on cross-reactor scale comparisons. data coming from different bioreactor scales can be easily imported into the platform and analyzed to establish the best conditions for upscaling. genedata bioprocess enables the correlation of process parameters (e.g., fermentation protocols, media recipes, bioreactor control parameters), with key performance indicators of the processes (e.g., titer, qp) and the product quality attributes (e.g., aggregation, glycosylation profiles). finally, bioreactor time course data can be tracked together with clone analytics and product quality parameters, which makes the platform uniquely able to support end-to-end biopharma development. upstream bioprocesses are at particular risk of contamination from adventitious agents. the typical 0.1 μm filters used at this step protect bioreactors from bacteria and mycoplasma but offer no protection from viral contaminations. a new polyethersulfone (pes) upstream virus filter, viresolve® barrier, has demonstrated high levels of microorganism retention -full retention for bacteria and mycoplasma (>8.0 lrv -log reduction value) and~5 lrv for small viruses, such as parvoviruses. it also has improved flow and capacity as compared to virus removal filters designed for monoclonal antibody purification. given the small pore size of virus retentive filters, implementing a virus filter upstream of the bioreactor raises the question of whether critical cell culture media components are removed. therefore, it is important to evaluate the cell culture performance and protein quality attributes using virus-filtered media to ensure that filtration does not negatively impact the process. materials and methods ex-cell® cho media and corresponding feeds were processed through either viresolve® barrier filters or 0.22 μm filters (control). media composition post-filtration was evaluated by high performance liquid chromatography (hplc), inductively coupled plasma/ optical emission spectrometry (icp-oes), and nuclear magnetic resonance (nmr). recombinant cho cells were cultured in fed batch culture. cell density and viability were measured by vi-cell tm cell viability analyzer while metabolites were analyzed by bioprofile® flex analyzer. shake flasks and bioreactors were utilized to verify that surfactants, such as poloxamer, (which are essential for shear protection in stirred tank bioreactors and can be difficult to filter) have not been removed during filtration. monoclonal antibody titer was quantitated by protein a hplc. characterization of the antibody product quality was assessed via weak cation-exchange chromatography (charge heterogeneity), size exclusion chromatography (aggregate profile), and 2-ab fluorescent labeling with np-uplc (glycan species). media and feed compositions were unaffected by filtration through the virus barrier filter. no significant differences in concentrations were observed with icp-oes (trace metals) or hplc (amino acids and water soluble vitamins). nmr showed no change in the organic composition of the media including poloxamer. the aromatic region with vitamin and amino acid signals is shown (fig. 1a) . cell cultures showed no differences in cell growth or titer, in either shake flasks or bioreactors (fig. 1b) . cell viability was unaffected, metabolite levels were within limits, and titer was consistent. the protein quality of the secreted antibodies showed no differences in the glycosylation pattern (fig. 1c) , amount of aggregates or charge variants. the risk of virus contamination in the bioreactor remains a concern for biotherapeutic manufacturers as there is no universal technology that provides a reliable, cost effective solution for virus removal that can be applied to all components of cell culture media. this study evaluated the viresolve® barrier filter that provides an efficient and easy way to protect bioprocesses from adventitious virus contamination. study results demonstrated that media and feed compositions, cell culture performance, and product quality were unaffected by filtration through the viresolve® barrier filter. implementation of vire-solve® barrier filters provides efficient filtration performance, high virus retention, and minimal cell culture impact and offers a viable option to improve the overall virus risk mitigation strategy for the manufacture of biotherapeutics. b tracking of process conditions together with online and offline performance analytics. the system allows to flexibly define tracked parameters and select optimal process conditions. c comparison of process performance across different reactor scales. the open architecture makes genedata bioprocess a provider agnostic system which allows to aggregate and compare data regardless of provider background bi-and multi-specific antibodies, antibody-cytokine fusion proteins, nonimmunoglobulin scaffolds, chimeric antigen receptors (cars), engineered t-cell receptors (tcrs) and tcr-based bispecific constructs can provide significant advantages for use in cancer immunotherapy. however, as highly engineered molecules they pose new challenges in design, engineering, cloning, expression, purification, and analytics. we have thus implemented an infrastructure that addresses these challenges and enables the industrialization of these various novel therapeutic platforms. in close collaboration with leading biopharmaceutical companies, we implemented a workflow, data management and analysis support system, genedata biologics™, enabling the automated design, screening, and expression of large panels of therapeutic candidates using these novel technologies. we have also built tools for developability and manufacturability assessments of these complex molecules. we have ensured that there is a seamless integration of all data generated and that functionalities such as bulk protein and vector generation using our in silico cloning engine, configurable library of template vectors and cloning strategies, fully annotated in silico protein molecules and dna constructs, and dna synthesis verification support, can be used for the newest protein formats and molecule topologies. we implemented data structures and data handling systems, which mirror how these complex next-generation biologics molecules and cell lines are being designed, screened, and analyzed. the result successfully addresses workflows for tcr optimization and engineering. we exemplified this with the generation and evaluation of a panel of engineered tcrs with an alpha chain cdr3 randomization and successfully supported the analysis and selection of beneficial mutations. the system also successfully supported workflows for the design and generation of a panel of tcr-based bispecifics (tcr coupled with anti-cd3) using automated molecule registration and in silico cloning tools and subsequent capture of expression, purification, and functional and analytical characterization data. on the car-t cell front, the system is able to provide traceability of the work from antibody generation, optimization, car engineering (e.g., attachment to the scfv with cd3-zeta and co-stimulatory domains to mimic the natural tcr complex) to the engineering of the t-cell. the genedata biologics platform successfully enabled automation, increased data integrity and traceability during research and development work, and will contribute towards the industrialization of these very exciting novel approaches for cancer immunotherapy. optimal selection of therapeutic antibodies and production cell lines by assessment of critical quality attributes and developability background the increasing cost of bringing a new drug to the market has put significant pressure on biopharma organizations. to increase efficiency in r&d processes and reduce costs, organizations need to evaluate potential drug candidates earlier in the r&d process, eliminate those with undesirable characteristics, and focus on the most promising candidates. after designing and thorough testing of successful candidates, efficient production of new biological entities in mammalian cell lines is necessary. the main goal here is to find a suitable cell line and optimal upstream and downstream processing conditions that not only lead to a satisfactory product yield, but also to a product with the desired biochemical properties. the evaluation of production cell lines, processes, and product quality attributes is performed earlier and in higher throughput for an increasing number of drug candidates. in addition, new methods in molecular and cell biology (e.g., novel genome engineering approaches such as crispr/cas9), in analytics [e.g., process analytical techniques (pats)], in process miniaturization, and in automation promise to make process development more efficient. however, the management and analysis of the increasing amount of experimental data during candidate selection and cell line and process development has become a bottleneck. in addition, quality-compromising steps in biopharma organizations can negatively impact the cost of goods and substantially prolong the drug candidate's time to market. therefore, systems for integrated management and analysis of wellstructured and curated data that comprehensively integrate molecule and sample information, manufacturing process parameters, and process and product quality attributes are needed. critical quality attribute (cqa) assessment should be enabled along the whole bioprocess development workflow, including cell line development, upstream and downstream process development, as well as analytical and formulation development. we have developed a comprehensive platform, genedata bioprocess™, which supports drug candidate developability and manufacturability assessment and bioprocess development. the platform captures and structures the cell line and process parameters together with analytical data for cell lines, processes, and protein products. the protein analytical data being tracked include biological data (such as bioactivity, immunogenicity), and physicochemical properties. these properties include glycosylation, chemical liabilities (such as deamidation and oxidation), aggregation, stability under different conditions (low ph, low and high temperature), solubility, and impurities. genedata bioprocess™ simplifies and streamlines laborious, manual process and supports tools for molecule, clone and process selection. furthermore, the platform allows for seamless integration with laboratory instruments, statistical software packages, and custom solutions. here, we present use cases showing how to identify and annotate liability sites prone to chemical modifications (fig. 1a) and how to monitor cqas of molecules allowing to assess developability more efficiently. we show how the analytical data generated in the course of a developability assessment are compiled to select the best drug candidate (fig. 1b) . implemented traffic-light systems indicate where molecules harbor issues such as in case of the antibody tpp-86, which is compromised by low temperature and repeated freeze-thaw operations. the same assessment views can also be applied on batches and cell lines. the underlying data can be visualized graphically. as an example, we show glycan types of products obtained from different cell line clones generated in a cell line development campaign for the molecule tpp-86 (fig. 1c) . even though the selected clone cli-35 meets the glycosylation criteria (e. g., <13% afucosylation, <40% galactosylation, <2% sialylation), the produced next-generation biologics molecules are composed from a number of specific subdomains. each type of molecule is composed of a specific set of domains, which must be mirrored in the registration and further research and development workflow. molecule registration and hit-selection using data from a number of assays is shown here using the example of car-t cells. the image is a screenshot from the genedata biologics™ software molecule harbors some stability issues as mentioned above. therefore, more attempts would be needed either in formulation or in reengineering of the complimentarity determining regions (cdrs) in order to provide a developable ttp-86-like drug candidate. background environmental process variables are often used as tools to optimize the performance of mammalian cell cultures to achieve higher cell densities and high productivities of r-proteins (q p ). the manipulation of culture temperature in the range of mild hypothermia (mh) (35-30°c) [1, 2] , as well as different glucose availability scenarios [3, 4] , has been shown to improve productivity in different cell lines. however, the manipulation of these variables individually or together has a concomitant effect on the rate at which cells grow, masking the net response exhibited by the cells. in order to identify the effects of these variables, we have taken advantage of the use of the chemostat culture. chemostat cultures were performed at two dilution rate (d)(0.010 or 0.018(h-1)), two temperatures (33 or 37°c) and three feed glucose concentrations (20, 30 or 40 mm). the response was analysed considering r-protein production, cell growth and key metabolites. r-tpa protein concentration was determined by immunoassay (trinilize tpa kit); cells were counted using a hemocytometer and cell viability was determined by the method of exclusion using trypan blue (t8154, sigma, usa); glucose, lactate and glutamate were determined by enzymatic assay using a biochemical analyser ysi (yellow spring instruments). statistical analysis of the results was performed by anova (design-expert 7 for windows). a decrease in cell density was observed in response to an increase of glucose feeding concentration, regardless of temperature or specific growth rate (in this case μ=d) evaluated. the maximum cell densities were reached at 20mm, achieving 1.65 and 1.50 x10 6 cells/ml at 37/33°c and 0.018(h -1 ); and 1.10 and 1.33 x 10 6 cells/ml at 37/33°c and 0.010(h -1 ) respectively (fig. 1a) . the increase in glucose concentration from 20 to 40mm resulted in an q p increase of 3 and 3.3 fold at 33°c/0.018(h -1 ) and 37°c/0.018(h -1 ) respectively. a lower increase of 2.4 and 1.8 fold was reached at 33°c/0.010(h -1 ) and 37°c/0.010(h -1 ) respectively (fig. 1b) . the highest q p s were reached at 37°c and 0.010(h -1 ). however, a positive effect of mh was not observed, in contrast to that observed in batch culture [1, 2, 3] . this behaviour suggests that low μ is a main factor on increased r-protein production in batch cultures exposed at mh condition. the specific consumption rate of glucose was significantly increased by the glucose increase from 20 to 40mm and reduced by mh (fig. 1c) . at 0.010 (h -1 ) the specific production rate of lactate (q lac ) was increased by glucose increase, independent of the culture temperature used. while at 33°c/0.018(h -1 ) the q lac decreased with increasing glucose concentration and at 37°c/0.018(h -1 ) a maximum consumption was observed at 30 mm glucose (fig. 1d) . the lactate-glucose yield ( fig. 1e ) not showed relevant changes at 0.010(h -1 ), while at 0.018(h -1 ) this yield showed a more efficient utilization of glucose, as glucose concentration was increased. however, this last behaviour was not reflected in an increase of r-protein production. the concentration of glucose has the greatest impact on the behaviour of the culture, and its increase affects positively the protein productivity. the mh did not improve proteins productivity of cho cells producing tpa under the different conditions evaluated; low dilution rate and at high glucose concentration impact positively the protein productivity and the metabolism exhibited by the cells. background mammalian cell cultures are the most commonly used bioprocess for the production of therapeutic recombinant proteins such as monoclonal antibodies (mabs). facing to the increasing demand of these biopharmaceuticals, the fda has initiated the process analytical technology (pat) framework in order to encourage pharmaceutical industries to use innovative technologies to monitor in real time the critical process parameters (cpps), and to ensure the final product quality [1] . one of the most important cpps for cell culture bioprocesses is the specific growth rate (μ), which is a direct indicator of cellular physiological state. indeed, μ is sensible to culture conditions and its value decreases when cells are in the unfavourable environment for growth [2] , which may greatly influence mab production and quality. however, until this day, the online monitoring of μ remains a great challenge for mammalian cell culture bioprocesses. igg-producing cho cells were cultured in 2 l stirred bioreactors equipped with an in situ dielectric spectroscopy (hamilton). operating conditions were fixed at 90 rpm, 50% of air saturation, ph 7.2 and 37°c. permittivity of cell culture was measured every 12 min, which allowed to calculate in real time the vcd by using a previously established linear correlation. then, a model of online estimation of μ was developed based on vcd prediction and cell mass balance equations. several signal noise filters and various calculation methods were evaluated to reach better model stability. cell cultures were performed in both batch and feed-harvest modes. feed-harvest cultures consisted of sequential renewals of 2/3 volume of the culture medium by following different strategies. this study proposed an innovative methodology based on dielectric spectroscopy to monitor in real time the cellular physiological state, by online estimating the specific growth rate (μ) of cells. model of online estimation of μ was developed from cultures in batch mode, and was validated by comparing online estimated μ with the experimental ones calculated at the end of the culture. with this model, the moment when μ started to decrease significantly, which indicated that cells were no longer in the exponential growth phase, was identified as the critical moment. to demonstrate the interest of online estimation of μ, the developed model was applied to a feedharvest culture, where the medium renewals were performed at the critical moments indicated by the model. this culture was then compared with the traditional feed-harvest culture where medium renewals were performed by following offline measurements of glucose and glutamine. we found that the online strategy allowed to maintain the value of μ by renewing the medium at the right time, while the values of μ varied a lot when using offline strategy. moreover, by using the online estimation of μ, the glycosylation of igg was kept at a high level (about 95%) throughout the whole culture. however, for the culture using offline strategy, the glycosylation level decreased progressively and was only about 75% at the end of the culture (fig. 1) . model of online estimation of μ was developed by using dielectric spectroscopy, which allowed to monitor the physiological state of cells in cell culture bioprocesses. implementation of this model in feed-harvest cell culture led to better mab glycosylation, which demonstrates clearly the potential of this methodology in mab production bioprocesses. background monoclonal antibodies are normally synthesised from transfected mammalian cells as heterogeneous mixtures of glycoforms [1] . however, clinical efficacy may depend upon single glycoforms which have been difficult to isolate [2] . we have now developed an efficient method for generating single glycoforms by solid phase re-modelling which is superior to previous methods because it allows a sequential series of enzymatic changes without the need for intermediate purification of the antibody. solidphase binding exposes the antibody glycans to enable easier access of the transforming glycosylation enzyme. the antibodies subjected to modification were a chimeric human/ camelid monoclonal antibody (eg2), a humanized monoclonal antibody (il8), a full size chimeric antibody (cetuximab) and polyclonal antibodies obtained from pooled human serum. the antibodies were bound to a protein a column using conditions typical of mab purification (fig. 1 ). after washing out non-bound impurities by a neutral ph buffer, each antibody was subjected to enzymatic modification directed to a targeted glycan profile ( table 1 ). the antibodies were then eluted with a low ph buffer and neutralized. the glycan profiles were analysed following glycan removal with pngase f, labelling with 2-aminobenzamide and separation on a hilic-hplc column [3] . prior to enzymatic modification glycan analysis of all 4 antibodies showed variable galactosylation and sialylation typical of human abs. this included a distribution of fg0, fg1, fg2, fs1 and fs2 with galactosylation indices ranging from 0.22 for il8 to 0.64 for eg2. there was minimal sialylation in il8 but up to 11% in eg2. glycan modifications were made as each antibody was held on a protein a column in accordance with procedures shown in table 1 . agalactosylated glycans were enriched by treatment with the single addition of galactosidase and neuraminidase. this resulted in 83-95% of agalactosylated structures in the mabs and 65% in the polyclonal antibody. galactosylated antibodies (>95% yield) were produced by a single stage reaction involving sialidase and by galactosyltransferase with udp-gal. breakdown of the glycans to a trimannosyl core was accomplished by treatment of the agalactosylated structures with hexosaminidase. this produced a yield of 76-80% of the fm3 structure with a small remainder of fa1. sialylated antibodies (>95%) were produced by a 2 stage reaction involving sialidase, galactosyltransferase and finally treatment with 2,6 sialyltransferase in the presence of cmp-nana. the latter reaction produced equimolar quantities of monosialylated and disialylated cetuximab and polyclonal antibodies. the results suggest that for human antibodies (150 kda) there may be a limitation for sialylation given the steric constraints between the two ch2 domains of the dimeric structure. the ability to sialylate the smaller camelid antibody (80 kda) was greater resulting in a high (>90%) level of disialylated glycans. this suggests that the steric constraints for glycosylation may be lower. these sialylated antibodies have significant potential clinical importance for their ant-inflammatory activities. we have modified the glycans of antibodies following immobilization on an affinity ligand column. this allows enzymatic transformation in a solid state that has a distinct advantage over the equivalent transformation in solution because the enzymes and buffers can be washed out on completion of the modification leaving the antibody still attached to the affinity ligand. this enables repeated rounds of an enzymatic reaction or sequential reaction steps without the need for intermediate antibody purification. the antibody can be removed eventually from the column by application of an elution buffer once all desired glycan modification have been made. since affinity ligand purification of antibodies is performed routinely as an initial step of purification after cell culture, the glycan modification can easily be incorporated into this process. the enrichment of the resulting antibody for a targeted glycoform can enhance the potential therapeutic efficacy as it is known that specific glycoforms are required for certain biological effects. [1, 2] . this is mainly because microvesicles can be enriched/deprived for specific proteins, based on their functional purpose and their cellular origin. recently, microvesicles purified from the supernatant of t24 bladder cancer cells were reported to be enriched for bcl-2 and cyclin d1 (anti-apoptotic proteins), but deprived for bax and caspase-3 proteins (pro-apoptotic proteins) contributing towards immunity against programmed cell-death [2] . however, impact of microvesicles on cho-based bioprocess has not been evaluated yet. therefore, in this investigation, we aimed to evaluate their impact on cell growth and recombinant protein production from cho cells. materials and methods cho-k1 cells were grown in chemically-defined protein-free culture medium (life technologies-1835273) in shake flask (gx-00125p). the different fractions of spent-media (microvesicles and microvesicle-free spent media) were collected using ultracentrifugation method [1, 3] . quality of different fractions was ensured using western blotting for exosomal marker, cd63 (sc-15363) and coomassie stained gel for loading control (fig. 1a) . to evaluate impact on cell growth, cells were seeded with microvesicles and microvesicle-free fraction collected from log-phase of culture and cell counts were performed by vicell using trypan-blue dye exclusion method. for impact on productivity, cell-free supernatant, collected from microvesicle-treated human igg secreting cho culture from stationary-phase of culture with respective control, was evaluated using elisa (ab100547). microvesicles collected from 10% of media (by volume) from routine maintenance cultures compared to working volume for microvesiclesupplementation were used in each experiment. the growth of microvesicle-supplemented cultures had shorter lag-phase and achieved 1.2 fold higher maximum cell density (1.46x10 6 viable cells/ml) compared to untreated standard culture (1.21 x10 6 viable cells/ml) and maintained higher for the remaining period of batch culture (fig. 1b) . however, microvesicle-free fraction did not had significant impact on growth. the viability of microvesicle-supplemented cultures, similar to microvesicle-free media supplemented, was also higher compared to standard culture suggesting potential use of microvesicles for regulating cho growth in production cultures. this could be possibily because microvesicles have already been reported to be enriched with cell growth/death-regulating proteins and hence facilitating cell growth [2, 3] . we have also observed abundance of cell cycle regulators including cyclin d1 in microvesicle-fraction compared to microvesicle-free spent-media in our laboratory (data not shown); however, further investigation are required to prove the hypothesis. the overall productivity of human igg secreting cho cells was also observed to increase bỹ 4 fold following supplementation of microvesicles to the culture without significantly affecting per-cell productivity. since microvesicle-supplementation facilitates cell growth, increased number of viable producer cells in the culture could be expected to be the basis of observed increase in the overall productivity of the culture [2, 3] . the further work is ongoing to in-depth explore the potential of microvesicles for improving recombinant protein production from cho cells. the data indicate that microvesicles secreted from cho cells can improve cell growth and hence recombinat protein production in culture. therefore, strategies need to be developed for sterile isolation of cho microvesicles from routine maintenance cultures and their supplementation into the production culture for improving the performance of cho-based production process. the glycosylation profile of a recombinant protein is one of the most important attributes when defining product quality. producing a protein with desired characteristics requires the ability to modify and target specific glycosylation profiles. traditionally the approach to modify the glycosylation profile of a protein involves supplementing a culture with components that can improve galactosylation. experimentation using this supplemental approach resulted in a dramatic increase in terminal galactosylation, but lacked the ability to easily and repeatedly target specific glycosylation profiles. using novel and proprietary technology, we have developed a feed (glycantune™) and a unique feeding process that will maximize growth and titer while being able to modulate glycan profiles. this new feed can be added as a standalone process that can result in a significant shift from g0f to g1f and g2f (maximum galactosylation). using a unique fed-batch process, glycantune can also be used with a standard feed to dial in targeted glycosylation profiles. through process development, we created a method where a transition point is used to switch from a standard feed to a glycan modulating feed. the timing of the transition point will determine the specificity of the glycan profile. ) . n-linked glycans were digested with pngase f and quantified using 100pmole maltohexose/maltopentose internal standards labeled with 8-aminopyrene-1,3,6-trisulfonic acid (atps) as described by laroy et al [1] or the user guide for the glycan labeling and analysis kit (glycanassure™ user guide, thermo fisher scientific). all ce separations were performed using the applied biosystems™ 3500xl. the timing of transition from efc+ to gtc+ made it possible to target specific glycosylation profiles. modulating g0f from 75% down to 32%, while increasing g1f and increasing g2f (fig. 1) . transitioning to gtc+ early in culture resulted in a greater shift from g0f to g1f and g2f. transitioning midway or late in culture resulted in a greater proportion of g0f compared to g1f and g2f. supplementation based approaches using glycosylation modulating media components to modify and target specific glycosylation profiles proved to be difficult. these approaches were able to increase terminal galactosylation (g1f and g2f), but lacked the ability to fine tune glycan profiles. this could result in numerous rounds of titration experiments to target specific glycan profiles that would likely remain inconsistent between cell lines, culture media and feeds, and process scale. the development of a unique process made it possible to predictably target specific glycosylation profiles. transition from standard feeding to glycantune allowed for precise targeting of glycan profiles. transition to glycantune early in culture resulted in an increased shift from g0f to g1f and g2f. a transition late in culture resulted in increased g0f and decreased g1f and g2f. growth performance during precultures and batch curves in plain shaking flasks did not show any differences among tested surfactants or lots thereof, and cell densities reached 10-12·10 6 cells/ ml ( fig. 1a and b) . experiments with hek 293-f cells at elevated power input in baffled shaking flasks revealed distinct differences between pluronic® f-68, f-127 and kolliphor® p188, with f-127 showing the best performance. peak viable cell densities reached with lots a and b of pluronic® f-68 and f-127 were comparable to those in plain shaking flasks, while those for kolliphor® p188 and lots c and d of pluronic® f-68 were significantly lower. peak viale cell densities were of 2 -12·10 6 cells/ml (fig. 1c) . similar transient transfection efficiency and mean fluorescence of transfected cells independent of applied surfactant and lot thereof indicated no major impact of respective poloxamer (fig. 1d) . interestingly, experiments using fluorescein-labelled pluronic® showed a time-dependent uptake into hek cells. visual tracking revealed an endocytic uptake of poloxamers by the cells (>10fold increase in signal after 96 h) and its co-localisation with cell membrane and lysosomes. sec (fig. 1e) analyses showed differences between the tested poloxamers. especially tested lots of pluronic® f-68 revealed notable deviations in the low molecular weight fraction (peak 2, fig. 1e ), compared to the other poloxamers. cultures subjected to varying levels of shear stress showed distinct growth differences depending on used poloxamer. while experiments in plain shake flasks did not show any differences in growth, cultivations under elevated shear stress in baffled shake flasks resulted in lower peak viable cell densities with kolliphor® p188 and some pluronic® f-68 lots. it remaines unclear whether this can be explained by different membrane protective activities alone, or if other mechanisms, occuring during and after cellular uptake, contribute to this effect. especially for the tested lots of pluronic® f-68, sec of surfactants showed differences in the low molecular weight fraction. this fraction mainly represents polyethylen oxide (peo) (revealed by nmr), which is likely to be a remnant from synthesis. these observations indicate that the use of different poloxamers and lots thereof should be carefully evaluated, especially under elevated shear stress. further experiments will focus on investigating distinct sec fractions of poloxamers. overcoming (fig. 1) . aurintricarboxylic acid (endonuclease inhibitor; enhancer used in e.g. salivary gland transfection) and polyvinylpyrrolidone (polymer; beneficial in electroporation) were both found to negatively impact peimediated transfection of cho cells, while another tested polymer enhanced growth as well as transfection efficiency. the use of a strong chelator led to a high transfection efficiency, but impaired cell growth. based on the results of the independent substance testings, the medium formulation was modified by the addition of a weak chelator and further components including vitamins. different osmolalities between 280 mosmol/kg and 340 mosmol/kg were tested for the final formulation, but no major impact was seen neither on transfection efficiency nor on viability 2 days post-transfection. the final cho tf medium formulation supported high cell growth of finally tested cho cell lines 2 and 3 with peak viable cell densities above 10⋅10 6 cells/ml in batch cultivations with an overall cultivation time of 7-8 days (fig. 1) . further improvements of the process might be achieved by adapting the protocol, as the results shown are based on a simple precomplexing of dna-pei. moreover, product yields could potentially be increased by using feeds, temperature shifts or commonly used enhancers (e.g. valproic acids). scaling of a cell culture process is an essential part in its development. in a typical approach scaling [1] is performed by keeping a (critical) process parameter constant throughout the complete bioreactor range. this can lead to non-beneficial results either on the high or the low end of the range. for instance, the specific power input [p/v] of 30 w/m 3 might result in a good agitation in production scale whereas it leads to a nonturbulent mixing behavior in process development scale. to overcome this issue a new approach for an easy scaling procedure was developed. this "utility function" approach for agitation scaling is based on individual functions with a value-based mapping independent of bioreactor scale. process insight information (established either from doe process investigation or existing experience with a process platform) is directly formalized into a set of mappings which transform bioprocess values into perceived benefits (0 to 1). at each bioreactor scale, parameters (e.g. stirring and gassing) are then chosen to maximize the product of resultant utility functions. the model cho fed-batch process in this trial comprised a cho dg44 cell line that was transfected to produce a humanized antibody igg1. a chemically defined media system was used. the process, including cell line, medium and feeding strategy was designed and developed by sartorius stedim cellca. the aim of the gassing scale-up was to achieve similar cell densities when the addition of pure oxygen starts. for all flexsafe str® bags oxygen was sparged via the micro sparger part of the combi sparger. all other systems used a ring sparger with holes face up. the initial air flow rate was set to an oxygen transfer rate (k l a) of 8 1/h at the corresponding agitation rate and volume. all process engineering characterization parameters were determined according to dechema guidelines [2] . with the use of the utility functions the discrete agitation rate was determined (table 1) . the utility functions led to discrete agitation rates where not only homogeneous mixing but also a turbulent flow pattern and a suitable specific power input was guaranteed. the initial gassing rate of air supplied enough oxygen for 5 x 10 6 cells/ml in all bioreactors. due to the used scaling methods the growth patterns in all bioreactor scales were comparable. peak viable cell densities (vcd) of 20 -26 x 10 6 cells/ml were achieved and viability at the point of harvest was above 80 % in all scales. the final product concentration was in an acceptable range of 2.9 -3.6 g/l. product quality attributes show comparability over the complete bioreactor range (fig. 1) . the harvest criteria of 12 days gave a combination of viability and product concentration that made it easy to process the cell broth during cell removal and other downstream steps. the process implementation of the cho production system -expressing mab 2 was successfully performed with the use of utility functions. cell growth, productivity and product quality is comparable over the complete bioreactor range. background endoplasmic reticulum (er), the central part of the secretory pathways in eukaryotic cells, is responsible for controlling the quality of secreted and resident proteins through the regulation of protein translocation, protein folding, and early post-translational modifications [1] . a number of physiological conditions such as oxidative stress, hypoglycemia, acidosis, and thermal instability can disturb the er functions, which triggers er stress [2] . prolonged er stress induces apoptotic cell death [3] . oxidative stress that naturally accumulates in the er as a result of mitochondrial energy metabolism and protein synthesis can disturb the er function [4] . because er has a responsibility on the protein synthesis and quality control of the secreted proteins, er homeostasis has to be well maintained. when h 2 o 2 , an oxidative stress inducer, was added to recombinant chinese hamster ovary (rcho) cell cultures, it reduced cell growth, monoclonal antibody (mab) production, and galactosylated form of mab in a dose-dependent manner. antioxidants can reduce the oxidative stress level and suppress the apoptotic cell death by scavenging oxygen free radicals, inhibiting chain reaction of oxidation, and detoxifying peroxide [5] . however, despite the importance of mass production of mabs, studies on the effect of antioxidants on the production and quality of mabs in rcho cell cultures have not been fully substantiated. to find a more effective antioxidant in rcho cell cultures, six different antioxidants including baicalein, which have used widely in mammalian cell cultures, were evaluated as chemical supplements with two different rcho cell lines producing the same mab in 6-well plates. then, batch and fed-batch cultures were performed in shake flasks with the supplementation of baicalein, which showed the best effect on culture performance among the 6 antioxidants. the reactive oxygen species (ros) and er stress levels were measured to study the effect of baicalein on mab production and quality. among these antioxidants, baicalein showed the best mab production performance. addition of baicalein significantly reduced the expression level of bip and chop along with reduced ros level, suggesting oxidative stress accumulated in the cells can be relieved using baicalein. as a result, addition of baicalein in batch cultures resulted in 1.7 -1.8-fold increase in the maximum mab concentration (mmc), while maintaining the galactosylation of mab ( fig. 1 and table 1 ). likewise, addition of baicalein in fed-batch culture resulted in 1.6-fold increase in the mmc while maintaining the galactosylation of mab. oxidative stress negatively affected the production and galactosylation of mab in rcho cell cultures. among the various antioxidants tested in this study, baicalein showed the best mab production performance in both batch and fed-batch cultures of rcho cells. baicalein addition significantly enhanced mab production while maintaining galactosylated forms of mab. thus, baicalein is an effective antioxidant for use in rcho cell cultures for improved mab production. background the production of many biopharmaceuticals (e.g. antibodies & proteins for diagnostic and therapeutic purposes) requires the cultivation of mammalian cell lines, which is demanding with respect to various aspects such as complex cell metabolism, variabilities in cell behavior, scale dependencies, influences of changes in cultivation conditions, medium composition etc. although an increasing number of measurement parameters is available, only a part of them is routinely utilized in industrial cell culture processes and their corresponding seed trains. nevertheless, the data base grows, statistical investigation of data gains importance and process data are more easily accessible in the context of industry 4.0. cell cultivation has to consider these complex requirements, e.g. for fed-batch control and seed train design. furthermore, cultivation strategies have to be adapted to new products, cell lines and clones as well as to different production plants when transferring processes. one approach to encounter the variabilities and to include actual information from the process and from data analysis is adaptive model-assisted control [1] . two software tools enabling adaptive model-assisted control applying unstructured, unsegregated models have been developed and implemented using matlab © , winers and fortran, one tool for fedbatch control and another one for seed train simulation and optimization. one key element of adaptive model-assisted control is the underlying process model. in order to provide an adaptive character, model parameters should be easily identifiable from routine cultivation data, which is available during seed train and fed-batch without additional sophisticated measurements. therefore, the usage of unstructured, unsegregated models is recommended. a) example of an unstructured, unsegregated cell culture model (for adaptive model-assisted control) one example, describing cell growth, cell death, uptake of substrates and production of metabolites via a first order system of ordinary differential equations and monod-type kinetics, is shown in table 1 . this mathematical model includes 13 cell specific model parameters [2] . ii) b) open-loop control sequence for seed train simulation and optimization [3] : using model, a priori identified model parameters and starting concentration values, the temporal concentration courses can be predicted for the first scale. subsequently, points in time for passaging and starting values for the next scale can be computed by adding a passaging strategy, seed train conditions and medium concentrations. prediction for the following scales can be obtained iteratively. integrating feedback from the process in terms of cultivation data enables increasing prediction accuracy and responding to possible changes in cell behaviour. process design and optimization, e.g. regarding seed train and fed-batch, is realized by adaptive model-assisted software tools using unstructured, unsegregated models. they enable feedback from the process via routine cultivation data and allow adaptation to diverse circumstances such as different cell lines, products, cultivation conditions, plant configurations etc. ) in polyelectrolyte capsules. significant advantages, such as great mechanical stability, good biocompatibility and good mass transfer properties characterized these capsules based on sodium cellulose sulfate/poly(diallyldimethyl) ammonium chloride (scs/pdadmac) [1, 2] . here, we present the possibility to cultivate human t cells, freshly isolated from blood, to high densities in similar semipermeable polyelectrolyte microcapsules within less than 10 days. cells were encapsulated in semipermeable scs/pdadmac polyelectrolyte microcapsules or confined in 1.5% alginate/poly-l-lysine (pll) beads, a standard approach for cell immobilization. the permeability of the microcapsules was estimated using dextran-based molecular weight standards (10 and 20 kda) and vitamin b12 (1.6 kda). gentle digestion with endocellulase allows an easy release of the cells out of the capsules. cell growth, cytokines production and phenotype were measured in non-encapsulated and encapsulated cells grown under standard culture conditions. moreover, we analyzed the interplay between the secreted cytokines and the scs within the capsules and its putative influence on cell growth. cells mixed in the cellulose sulfate solution under physiological conditions can be safely trapped within a liquid core during capsule formation. encapsulated cells can reached cell densities ≤ 40 x 10 6 cells ml capsule -1 , whereas cells confined in alginate/pll beads and non-encapsulated ones reached 11.3 x 10 6 cells ml bead -1 and 2.4 x 10 6 cells ml, respectively. one major advantage of these polyelectrolyte microcapsules (<1 mm) is the low mwco (<10 kda) (fig. 1a-b) . this restricted permeability allows for a conditioning of the capsule core by autocrine factors, which in turn permits the use of basal cell culture medium instead of expensive t cell specialized media, hence does not necessitate high amounts of rhil-2 and reduces the cultivation costs. moreover, co-encapsulation of rhil-2 had a beneficial effect on the growth kinetics in most cases (fig. 1c) . some evidence is presented that the scs used to form the polyelectrolyte microcapsules, specifically adsorbs il-2 (table 1 ) -a cytokine which provides an essential signal for t-cell proliferation and differentiation [3] . therefore, we postulate that the scs used for encapsulation has biomimetic properties, creating an artificial extracellular matrix mimicking heparin sulfate which in turn positively affect t cell proliferation via trans-presentation of il-2 (fig. 1d) [4] . primary t lymphocytes can be expanded under appropriate conditions outside the body. in the latter, t cells grow/expand in specific environments where the cells are tightly packed, leading to multiple cell-cell contacts and manifold interactions with the extracellular matrix. ex vivo suspension cultures of diluted cells cannot provide such a microenvironment. in the microcapsulesbased cultivation system presented, the cells are suspended in a viscous scs-solution. the low molecular weight cut off of the surrounding polyelectrolyte membrane assures that typical signaling molecules produced by the cells are retained thus facilitates the "conditioning" of the cellular microenvironment, while nutrients and metabolites can pass. expensive additives, such as interleukin-2 (il-2), can be co-encapsulated. expansion then no longer requires specialized t-cell media. moreover, the scs seems to have biomimetic properties, representing an artificial extracellular matrix mimicking heparin sulfate. we consider that the described method may be an appropriate alternative to expand t cells while creating a local microenvironment mimicking in vivo conditions. -175) . equations of balances and kinetics of an employed process model including x v viable cell density, x t total cell density, μ cell-specific growth rate, μ d cell-specific death rate, t time, k s and k monod kinetic constant and monod constant for uptake, k lys cell lysis constant, q cell-specific uptake rate or production rate, respectively, y kinetic production constant, c concentration, glc glucose, gln glutamine, lac lactate, amm ammonia, f feed rate, v volume balances with fed-batch terms kinetics ;uptake if c glc ≥ 0.5 mm : q lac,uptake = 0 if c glc < 0.5 mm : q lac,uptake = q lac,uptake,max q amm = y amm/gln • q gln background digital manufacturing (dm) is heightening the productivity and robustness of existing processes and facilities. it also enables the efficient development of previously unmanageable products or processes and provided the basis for a wave of innovations. dm is a resident and on-line source of continuous optimization of process performance. it relies upon the comprehensive, real-time interfacing of both human and machine sourced information through one centralized system. more than legacy distributed control system (dcs) and supervisory control and data acquisition (scada), it is an integral interconnection of real-time access to divergent sources of information. as such, it can promise deep analysis and predictions leading to shortened product cycle and advanced process control. this comprehensive analysis is extending beyond operations performance data from the production floor to data driving such activities as raw materials security of supply (sos) and business continuity management systems (bcms). digital biomanufacturing (db) can be viewed as yet another, larger, embodiment of digital biotechnology. db is similar to digital manufacturing in that it promotes innovations in the manufacturing of biologicals by using such things as computer aided design, manufacture, verification and deep process analysis using software sensors (fig. 1) . however, the fact that there are living components (cells) involved in the processes puts a distinctly different flavor to the systems employed. it is desirable to use a distinct term here to distinguish it because, as in the terms bioproduction and biopharmacology, db addresses many unique aspects of biologically-based activities. the reasons why the biotech and biopharma industry lags behind other sectors such as the automotive regarding the transformation to digital manufacturing are (i) the complexity and dissipative nature of biological systems, (ii) distributed heterogeneous data and (iii) limited at-line or on-line data sources. however, the costs of genomic sequencing, omics data generation, and computing resources are decreasing rapidly, and at the same time process analytical technologies, computational power and predictive modeling as well as data management infrastructures are greatly improving (table 1) . by removing roadblocks that used to limit approaches, these changes have paved the way to transforming the bioeconomy into an industry that is based on digital knowledge. such new and optimized manufacturing technologies as continuous biomanufacturing and 3d bioprinting can actually demand the interfacing of many sources of information, deep data prior to elisa, the various proteins were incubated at 37°c in scs prepared as for encapsulation. as control, the scs was replaced by pbs. shown are mean values ± sd, n = 3 analysis including software sensors for metabolic fluxes, and model-based predictions of digital biomanufacturing. the application of predictive models for bioprocess optimization greatly improves established platforms and finally leads to a massively increased mechanistic process understanding. four essential benefits result from the increased bioprocess understanding, development, and control of db. first, personnel are relieved of many manual and repetitive tasks. second, strategic planning and operational efficiency are improved. third, we see real-time optimization of end-to-end manufacturing based on such high-value criteria as projected product quality and profitability. fourth, it enables previously unmanageable operations and creates innovative solutions. monitoring between-batch behavior of real-time adjusted cellculture parameters xavier lories, jean-françois michiels arlenda, mont-saint-guibert, 1435, belgium correspondence: xavier lories (xavier.lories@arlenda.com) bmc proceedings 2018, 12(suppl 1):p-192 background cell-culture parameters (ccp), such as ph, may be continuously measured online and subject to real-time automated adjustment (e.g. automated addition of a base to prevent the ph to drop too low). this is an efficient method to maintain the parameter within specified limits. this type of control constraint the variability within the predefined limits and does not provide any information on the between-batch variability of the process. online measurements of ccp provide time-dependent curves presenting one or more transitions. different types of transition can be observed: -the process can shift from a state in which adjustment is needed to keep the ccp in range to a state in which it is not. typically, the ccp drifts away from a limit. -the process shifts from a state in which adjustment is not needed to one in which it is. for instance, a drifting ccp reaches the lower or upper limit of the accepted range. the timepoints at which those transitions take place are here called changepoints. those are aspects of the process and, as such, should be controlled. in the multiple changepoints cases, the approach allows the early termination of runs showing very early or very late first changepoint. the identification of the changepoints position is based on simple rules rather than complex statistical modeling to keep the identification methodology simple. once the changepoint are identified, a multivariate bayesian model is adjusted on the appropriately transformed data. prediction regions are obtained and used as control limits [1] . results obtained for a 2-changepoint case are shown on fig. 1 . points on the right-hand graph represent new batches. the red triangle represents a failed batch. it appears that the control strategy fails to identify the failed batch. two reasons can be considered: -the limits of the prediction region have been established based on 9 points, such a small sample size is likely to be insufficient for the definition of such a control chart. -the tested batches were produced out of set point. a control chart should be used on a stable process, ran in the same conditions, in order to be really relevant. this work was based on available historical data, which is never an ideal situation. the suggested strategy offers a simple approach to the monitoring of between-batch behavior for cell-culture. once the limits have been defined, the approach is quite straightforward and usable by nonstatistician. however, such strategy, as any other of this type, must be based on a sufficient number of batches for the definition of the control limits in order to have a good estimation of the batch-to batch variability. fig. 1 (abstract p-190) . intelligent software applications support digital biomanufacturing process development and control. • databases using data collected online, at-line, and offline from bioprocesses operating worldwide. • process data are used to generate metabolic network models that represent a specific host cell line in a bioprocess. • modelbased computational simulations improve process understanding and reduce experimental efforts for media design, clone selection, and metabolic engineering. • automated data import and processing allow for a streamlined and standardized metabolic process analysis. • identification of critical metabolic parameters is used for proactive steering and control of production processes background rabies is a zoonotic viral disease with a mortality close to 100% [1] . as there is not an efficacious treatment available, post-exposure vaccination is recommended for individuals in contact with the virus. on the other hand, the most common source of virus transmission is saliva of infected animals, mostly dogs, whereby mass vaccination of pets is the most cost-effective way to reduce human infections. in this context, availability of both human and veterinary vaccines is critical [2, 3] . our group had previously developed an effective vlp-based rabies vaccine candidate produced in high density hek293 cell cultures with serum free medium (sfm) [4, 5] . one of the aims in vaccine production process is the achievement of a good productivity with a low cost per dose, mainly in the case of vaccines for animal use in which case the sfm is one of the principal expenses. in this work, we show the adaptation of the producer clone to a non-expensive in-house developed culture medium, in order to reduce the global cost of the process and therefore the price per dose. experimental approach first, we compared a direct and a sequential adaptation protocol of our hek293 rv-vlps producer clone, from 100% of the commercial sfm (ex-cell293, safc) to a new formulation with only 50% of the sfm and a minimum essential medium (p2g), developed in our laboratory specifically for rv-vlps production. this new formulation was called rvpm (rabies vaccine production medium). the specific productivity of rv-vlps in culture supernatants was measured by sandwich elisa, using the 6 th international standard for rabies vaccine that quantify the glycoprotein content (nibsc, expressed in elisa units per ml). further, we evaluated both media for the production of the rabies vaccine, using stirred tank bioreactors operated in continuous mode (biostat qplus, sartorius). the production of the rv-vlps was daily evaluated by elisa and the obtained harvests analysed by the nih potency test for rabies vaccine. after the adaptation process, suspension cultures without aggregates or clumps were obtained, with the same specific growth rate. a lower maximum cell density with the rvpm was reached, achieving 5x10 6 cells.ml -1 , compared with the sfm that reach cell densities between 8 and 9x10 6 cells.ml -1 in batch mode. the specific rv-vlps productivity per cell was maintained, obtaining values of 0.88 and 0.90 eu.10 6 cells -1 .day -1 for the clone being cultured in sfm and rvpm, respectively. taking into account that this producer clone can be changed directly from one medium to the other without lag phase or cell damage, and that in rvpm the maximum cell density reached was lower, this medium was proposed to be analysed in high cell density in perfusion mode for a continuous culture in bioreactor. therefore, we performed two cultures in parallel to compare the efficacy of each media formulation in perfusion. as shown in fig. 1 , we obtained very similar culture performances in both bioreactors; 14.4 eu.ml -1 and 16.1 eu.ml -1 of rv-vlps for the commercial sfm and rvpm, respectively. after that, the harvests were evaluated by the nih potency test obtaining a rabies vaccine potency of 1.2 iu.ml -1 for both cultures (being 1 iu.ml -1 the minimum potency required for animal vaccine). thus, the results obtained represent an interesting advance in the optimization of this vaccine production process since the use of this new medium formulation represents a reduction of 40% of the total cost which will be reflected in a considerable reduction of the price of the vaccine dose. background vaccines are one of the most powerful and effective health inventions ever developedproviding tremendous economic and societal value; yet several factors hinder comprehensive immunization coverage. traditional methods of biologics production, based on stainless steel bioreactors, allow pharmaceutical companies to achieve economy of scale, but are limited by high capital expenditures. such approaches stifle manufacturing innovation and lack long-term cost-effectiveness and sustainability. current innovations can cut biologics' production costs to revolutionize the mainstream use of biologic treatments, focusing on developing fast, potent and cost-effective vaccine production. univercells' mission to make biologics affordable to all initiated a paradigm shift, targeting an innovative single-use manufacturing platform incorporating bioprocess into continuous operations. univercells employs process intensification, using high volumetric productivity bioreactors; and unit steps integration, coupling usp and dsp into continuous operations. the objective is a down-scaled high-productivity process for a cost-effective manufacturing solution. the resulting micro-facilities are easily-deployable in developing countries, breaking entry barriers to biomanufacturing (fig. 1) . manufacturing and distribution advancements, from centralized to distributed, foresee affordable treatments' obtainability via supplying local populations with local production units. -bench-scale fixed-bed bioreactor; -carriers made of 100% pure non-woven hydrophilized pet fibers; -vero cells grown in serum-free and serum containing media; -attenuated polio strains; -cell nuclei on carriers counted by the crystal violet method; -polio virus production estimated by elisa assay (d-antigen content). cultivation of vero cells in medium with serum and in serum-free medium, was carried out in bench-scale compact fixed-bed bioreactors, to determine which culture conditions result in the highest growth rate, the highest cell biomass by carriers and virus production. cells were inoculated at 0.05x10 6 cells/cm 2 and infected during the mid-exponential phase, following a complete media exchange. viral infection took place in serum-free media. in-line clarification and purification is targeted to be performed in only a few steps (maximum one of two) without intermediary diafiltration. in such configuration, we measured that vero cells can reach a cell density of 300-350x10 3 cells/cm 2 with pdl/day of 1.0-1.2 in serum-containing media. this new facility is expected to manufacture any type of viral vaccine at a very low cost and could be deployed at the site of the manufacturer in emerging countries, killing the two birds of cost of manufacturing and distribution with one stone. the presentation will feature the description of the engineering development, but also the preliminary results of cell growth, infections, and product quality, as well as a description of the cogs calculation. univercells developed a disruptive polio vaccine manufacturing technology exceeding expectations when compared to traditional methodsachieving a superior result via its all-in-one solution of a simple, scalable, and fully-disposable vaccine production platform resulting in long-term cost-effectiveness, flexibility and sustainability: -all upstream, downstream and inactivation steps take place within a closed system with all the equipment contained in a low footprint isolatorcreating a confined area for polio virus handling that facilitates the deployment of micro-facilities. -this leads to a dramatic reduction in capital investment, time required for development and increases production capacity. -in conclusion, this is a simple and elegant solution for the industrial production of human vaccines at a low cost in micro-facilities, making polio vaccines available to all. comparison of media formulations for the vaccine production in 1 l stirred tank bioreactors operated in continuous mode. both cultures were performed in parallel using the corresponding medium for the perfusion. a feeding was performed with the commercial sfm. b the first two days of perfusion feeding was performed with sfm until the cell density reach 10 7 cells.ml -1 and, after that, the bioreactor was fed using the rvpm formulation. (↓) on day number 10, 20% of the reactor volume was punctually bled maintaining the working volume background vectored vaccines based on modified vaccinia virus ankara (mva) are reported to stably maintain large transgenes, and to be safe, immunogenic and tolerant to pre-existing immunity. mva is usually produced on primary chicken embryo fibroblasts but continuous cell lines are being investigated as more versatile substrates. we have previously reported development of a continuous suspension cell line (cr.pix) derived from the muscovy duck and efficient production process for mva in chemically defined media [1, 2] . this process allowed isolation of an hitherto undescribed genotype (mva-cr19) that induced fewer syncytia in adherent cultures and replicated to higher infectious titers in the extracellular volume of suspension cultures [3] . replication of mva-cr19 remained restricted predominantely to avian cells, an important property of mva vectors. homologous recombination in cr.pix cells was used to generate viruses with various expression cassettes in deletion site iii [4] and combinations of the differentiating point mutations of mva-cr19 in a backbone of wildtype virus. all recombinant viruses were plaquepurified. successful introduction of the mutations was confirmed by sequencing and specifically designed restriction fragment length polymorphisms (rflps). viruses were analyzed by serial passaging, diagnostic pcrs accross deletion sites [4] , replication kinetics, plaque phenotype and electron microscopy. the genome was further investigated by anchored pcr and long pcr. efficiency of spread of recombinant viruses (fig. 1a) could be mapped to a point mutation in one of the genes, a34r. however, although mva-cr19 carries mutations in three structural proteins we detected no obvious differences to wildtype by electron microscopy (fig. 1b) . the replacement of the left viral telomere by the right counterpart was the most surprising result of our new study (fig. 1c) . this extensive rearrangement affects 15 % of the viral genome and has also increased the area of complementarity between the two telomeres. the recombination site was precisely located and shown via analysis of earlier and subsequent passages to be a stable property of mva-cr19. various viruses, including those with larger dual (dsred1 and gfp) expression cassettes, were serially passaged at least 20-fold. although the genotype of mva-cr19 is advantageous for replication, all genomic and genetic markers of wildtype and mva-cr19 were stably maintained in all passages of the recombinant viruses, independent of wildtype or mva-cr19 backbone. we confirmed our previous results that suggested that mva-cr19 replicates efficiently in single-cell suspensions and were able to connect this property with the d86y mutation in a34, a structural protein on the surface of the virions. mva-cr19 was also found to differ from wildtype mva by a recombination between left and right viral telomere. due to this event, several genes encoded at the left terminus have been deleted whereas the gene dosis of those originally encoded only at the right terminus may have increased. we do not currently know how much the various point mutations and changes in genomic structure combine to explain the improved replication of mva-cr19. as several of the affected genes have been reported in the literature to impact interaction of mva with the host we would expect that in vivo studies may reveal additional novel properties of mva-cr19. an extremely important distinction between our earlier study [3] and this one concerns the source of the viruses. here, we investigated plaque-purified viruses and confirm the high genetic and genomic stability of mva. different expression cassettes inserted into deletion site iii, all diagnostic rflps and pcrs over various sites of the genome and within the viral telomeres remained unchanged throughout at least 20 serial passages -independent of whether recombinant viruses with wildtype or cr19-derived backbones were characterized. fig. 1 (abstract p-225) . a one hallmark of mva-cr19 is a significantly reduced tendency to induce syncytia and an increased dispersion of plaques in cr.pix cell monolayers. this property appears to be supported by the mutation in a34r. b electron microscopy reveals no obvious differences between novel genotype and wildtype. background transient gene expression systems using polyethylenimine (pei) are considered to be fast, flexible and cost-efficient for recombinant protein production [1] . transfection efficiency depends on different factors; one of them is the type of media. production media support cell growth and protein production but not high transfection efficiency (te) mediated by pei [2] . therefore, media were selected for transfection followed by feeding of production media [3] to improve te and protein production. two different transfection strategies are compared: conventional transfection by preparing polyplex of a plasmid (pdna) and pei interaction before transfection and insitu transfection by direct addition both of them to the cell suspension and the polyplex formed spontaneously [4] . cells were seeded 24 hr in chomacs cd media before transfection. at transfection time point an equal amount of cells were resuspended in each media type. transfection was applied either insitu or conventional (polyplex prepared in 100 μl of 150 mm nacl and incubated for 20 min.), media addition was performed 5 hours post-transfection (hpt). media type and transfection condition were illustrated in table 1 . media screen result exhibits the highest transfection efficiency of around 50% transfected cells by opti-mem medium coming along with low cell growth and viability. to improve the transfection efficiency, basic parameters including cell density, pdna, and pei concentrations were varied and higher transfection efficiency was reached by reducing media or accordingly increasing cell density, pei and pdna concentration for transfection. further optimization results show that the transfection of cho-k1 cells in opti-mem (transfection medium) for 5 hours followed by addition of cho-macs cd (production medium) for further enhancing the transfection, cell count, and cell viability. the transfection efficiency (te) increased up to 85 ± 2.6% coincide with increases in viable cell concentration (vcc) in comparsion to transfection and cultivation in opti-mem media alone fig. 1a . both conventional and insitu methods are successfully transfected cho-k1 to the same similar high te as shown in fluorescence microscope images of fig. 1b . insitu transfection shows super-priority for suspension cell transfection concerning the reduction of handling steps (one step) compared to the conventional way (two steps). the insitu transfection avoiding the optimization step required for the incubation period to prepare transfection polyplex but require a higher amount of pdna and pei than conventional way as shown in table 1 . in order to deal with the growing demand of large quantities of therapeutic proteins in a timely fashion, expression systems are being optimized to reduce the time of generation of stable clones as well as to increase the levels of protein secretion. this can be achieved by a combination of expression cassette optimization, cell engineering and selection process. we have previously developed the cumate gene-switch, which is a very efficient expression system for protein production [1] . we have shown that the cumate-inducible promoter (cr5) was the strongest promoter we had tested so far in chinese hamster ovary (cho) cells. with this promoter, we were able to generate stable cho pools capable of producing high levels of a fc fusion protein (900 mg/l), outperforming by 3 to 4 fold those generated with cmv5 and hybrid ef1α-htlv constitutive promoters. besides the strength of the cr5 promoter, we demonstrated that the ability to control both the time and the level of expression during pool generation and maintenance gave a real advantage to the inducible expression system. indeed, we observed that keeping the expression off during selection enabled the generation of pools with superior productivity compared with the pools whose expression was maintained on. moreover, preliminary results suggest that keeping recombinant protein expression down increases the frequency of high producer clones [2] . knowing that one of the main bottlenecks of the successful bioprocessing of recombinant proteins using cho cells is the rapid isolation of a high producer, our data suggest that the cumate gene-switch system could be a valuable platform for the generation of stable clones. the main regulatory authorities and organizations demand proof of monoclonality for biotechnological producer cells. with increasing pressure to shorten timelines and to improve drug safety, technologically advanced methods have to be established to ensure that production cell lines are derived from a single progenitor cell. sartorius stedim cellca's single cell cloning approach is based on one round of fluorescence-activated cell sorting (facs) using becton dickinson (bd) facsariatm fusion cell sorter combined with photodocumentation by synentec cellavista microscopic imaging system. for the approach, critical process parameters such as different cell lines, viability and cell aggregation levels were investigated separately to assess their contribution to the probability of monoclonality. immediately after single cell cloning into 384-well plates (1 cell/ well) the plates were centrifuged followed by imaging using the cellavista (day 0). further cellavista images are taken on day 1, day 2 and on one day between day 5 and 7. outgrowth was defined at day 14. 8 cell lines expressing different recombinant products were investigated to calculate probability of having ≥ 2 cells/well after facs sorting p(d), the apparent probability p(i) of having ghostcells (cells that are out-of-focus and, thus, are not visible during initial microscopic imaging), and the apparent probability p(k) of having ghostcells that outgrow the 384-well stage (fig. 1 ). using these results, the probability of obtaining a monoclonal cell by using sartorius stedim cellca's single cell cloning approach was determined (table 1) by conservative examination: p(monoclonal, conservative) = 1 -(p(d) x p(i)) realistic examination: p(monoclonal, realistic) = 1 -(p(d) x p(k)) cell pools with low viability can theoretically impact the probability of monoclonality by e.g. diminishing microscopic imaging quality (cell debris). therefore, pool cell line 1 with very low viability (≥36 %) was used to demonstrate, that the probability of monoclonality is still 99.9 % in case of low viability on day of sorting: p(monoclonal, conservative) = p(d) x p(i) = 99.9 % p(monoclonal, realistic) = p(d) x p(k) = 99.9 % furthermore, cell pools with high aggregation levels can theoretically impact the probability of monoclonality by sticking together during facs sorting and therefore increase the probability p(d) of having ≥ 2 cells/droplet. therefore, pool cell line 8 with high aggregation levels (≥11.1 %) was used to demonstrate, that the probability of monoclonality is still ≥ 99.9 % in case of highly aggregated cell pools on day of sorting: p(monoclonal, conservative) = p(d) x p(i) = > 99.9 % p(monoclonal, realistic) = p(d) x p(k) = > 99.9 % conclusions in summary, there is no obvious correlation between protein product type and the determined probabilities for monoclonality. furthermore, pools with a viability as low as 36 % and pools with an aggregation level as high as 11.1 % can be used for scc resulting in acceptable probabilities of monoclonality. background ich guidance [1] requires that any cell line used to produce biopharmaceuticals originates from a single progenitor cell. recently, there has been increased scrutiny of the method(s) used to achieve this requirement. here, we review the suitability of the legacy capillary aided cell cloning (cacc) method in light of this changing landscape of expectations. the cacc method is based on the 'spotting' technique [2] and relies on independent visual conformation by two scientists of the presence of a single cell in a 1 μl droplet. this method achieves a high probability of monoclonality in one cloning round. although the method has since been replaced by facs single cell deposition for routine use, it remains a viable cloning method. -performed by trained scientists -dilute culture to 1500 ± 500 cells/ml with ≤2% doublets -draw cell suspension into pipette tip by capillary action; tap tip against the centre of the base of each well of a 48 well plate. -size of resulting droplet =~1μl (fig. 1a ) -two scientists independently view all wells using a microscope (initially use 40x magnification with the entire rim of the droplet visible within the field-of-view. next, examine particles using 100x or 200x magnification to confirm they are cells) and individually record the number of cells present in each well's droplet (fig. 1b to d) . -exclude droplet from further analysis if full visualisation is hindered (fig. 1e to h) . -add growth medium, and incubate plates. record all wells containing colonies; only progress colonies from wells that both scientists agree contains only one cell. -data analysis: -each scientist's observations categorised as: 0 cells, 1 cell or >1 cell -observed outcome for each well: growth or no growth -probability of monoclonality estimated from data using a statistical model cloning (ldc) increased accuracy of p(monoclonality) with cacc -ldc weakness: no visualisation after seeding (to check both well seeding and subsequent growth of colonies is well described by the poisson distribution), potentially overestimating p(monoclonality) -addressed by cacc: visual examination with colonies arising from wells seeded with 1 cell distinguished from those seeded with >1 cell -visualisation step further strengthened by: using controls for exclusion of wells; measuring errors based on the presence or absence of colonies in wells where two scientists independently reported 0 cells; and formally analysing the data using a suitable statistical model decreased time and resource requirements with cacc -high p(monoclonality) possible in single round as each well examined individually with only those containing a single cell progressed, and because the error rate for incorrect scoring is considered to be low two scientists miss a cell one cell sitting on top of another and the two thus appearing as one an experiment was performed to estimate error frequency [3] . conclusion -scientists miss a cell infrequently (in the range 0.4% to 1.3%, [3] ) -error frequency does not invalidate use of direct observation methods for cell cloning -single cell seen by both scientists is highly likely to be monoclonal -during method development, strategies established to control potential sources of error ( table 1 ) use of a contemporaneous visualisation approach, a strict control strategy, and a suitable statistical model (which takes into account potential errors) results in: -the cacc method being at least as robust as the ldc method -the cacc method being a reliable, single-step method for cloning to achieve a high p(monoclonality) background vector design is a key step in cell line development for the expression of therapeutic biologics. it is essential that the vector design results in high, stable expression of the encoded protein. other considerations include ease of cloning, stability for propagation in e. coli as well as in the mammalian host cell line, and ease of sequence amplification for verification of vector construction and for detection of insertion site and copy number in stably expressing cells. for these reasons, use of the same promoters and polya tails in dual cassette vectors, as is common for expression of the heavy and light chains of monoclonal antibodies, can be problematic. in order to minimize sequence similarities between the two expression cassettes, we have modified the promoters, introns, and polya tails of the light chain and heavy chain expression cassettes in the dual expression vector commonly used for the expression of therapeutic antibodies in the chozn® gs -/cell line development platform. gene synthesis and vector construction of igg1 and fluorophore-expressing vectors was done by atum. vectors were transfected into chozn® gs -/cells via electroporation. analysis of gfp and rfp expression was achieved using a macsquant instrument. selection and generation of stable pools and single cell clones from transfections with igg1-encoding vectors was performed as described in the chozn® platform technical bulletin. titer analysis was performed in static (96 well plate), in a 7 day tpp assay and in a 14 day fed batch assay using a qk fortebio. initial screening experiments identified a lead vector, #39, and a vector, #37, which produced very low titers and relatively few minipools expressing detectable levels of igg1. analysis of gfp and rfp expression from the modified vectors indicated relatively high expression from the rfp/hc expression cassette of vector #37. a stronger promoter resulting in overabundance of hc, known to be toxic to cells, provides a possible explanation for the poor results with this vector. interestingly, swapping the positions of the lc and hc in #37 resulted in a vector, #77, that outperformed the initially identified lead vector (fig. 1 ). this same change was made to vector #39 without any resulting improvement in titers (vector #78, fig. 1 ). interestingly, vector #39 had a smaller difference in relative promoter strengths, based on mean channel fluorescence ratio of gfp to rfp, suggesting that overabundance of hc was not an impediment to igg1 expression from #39. poor titers were also seen with a modified version of vector #39 (vector #79, fig. 1 ) in which the glutamine synthethase selection cassette was in the reverse orientation. this second screen identified vector #77 as the lead vector design (fig. 1) . a full comparative study of vector #77 and the control vector was performed, cumulating in the generation and comparison of single-cell clones from each. these studies have demonstrated the equivalence of these vectors in terms of igg1 titer. this work has resulted in the identification and characterization of a dual expression vector with minimized similarity between the two expression cassettes, easing the cloning, propagation and analysis of vector integration in stable cell lines while maintaining the high, stable expression of the encoded protein of the original vector design. background traditional cell line engineering strategies mainly include an antibiotics resistance selection. in this process, cells are transfected with the goi (gene of interest) together with an antibiotics resistance gene and those cells are selected that survive treatment with the respective antibiotic [1] . although the gene responsible for the survival of the cell is transfected together with the goi, resistance is not necessarily linked to high goi expression. thus, a significant proportion of resistant cells may not express the goi at all, necessitating the search for alternative, more closely linked selection systems. sirnas (silencing inducing rnas) are short, noncoding rnas that can bind to complementary mrna and inhibit their translation. this function has been used in many approaches to silence the expression of certain genes [2] . with their short length, sirnas can be hidden in introns (non-translating regions) of genes, making it possible to couple the expression of a sirna to a gene. this way a cell produces a correlating amount of sirna when transcribing the gene, without adding any further translational burden on the cell. the co-expression of the sirna can be used as a selective marker by one of the following methods: (1) knock-down of a suicide gene to enable a cell's survival after suicide gene mrna transfection, (2) down-regulation of a surface marker which is used in macs (magnetic cell separation) to filter out wanted or unwanted cells, and (3) inhibition of a fluorophore marker for selection using facs without product specific antibodies. for sirna based cell selection systems, sirnas replace the commonly used antibiotics resistance as a marker. cells that produce goi will also produce the sirna that protects the cell from a suicide gene. the selection protein (suicide genes, fluorophores, surface markers, etc.) is transfected as mrna and is only expressed during selection. the general process is outlined in fig. 1. (a) the traditional antibiotics resistance marker is replaced by an sirna, which is cotranscribed with the goi. unlike in antibiotic resistance, the marker here is not a protein, reducing the translational burden and providing more resources for goi production [3] . transfection with the suicide gene proved to be 100% lethal within 2 days, with no outgrowth over two weeks. protection by expression of the sirna was shown to be efficient. currently a comparison of stable cell line development programs based on sirna selection and neomycin selection is ongoing. conclusions the novel selection system should speed up cell line development, as the system kills rapidly and directly selects for cells transcribing the product gene on a high level. we expect to see more high producers earlier in the process, which will allow for an easier and faster selection in the following steps. sirna based selection offers great opportunities. by directly selecting based on goi transcription and not a proxy marker, we expect more relevant cells on a pool level. in addition, the elimination of an antibiotics resistance allows more cellular resources for goi production. the system offers multiple ways of application, either by enriching wanted, or depleting unwanted cells. background single-cell cloning is an essential step used in the upstream development of transformed cell lines for therapeutic protein production. while single-cell clones are typically used to ensure product consistency, such low cell density cultures present a survival challenge; cells grow more slowly or may even not survive at low densities in protein-free media, costing the industry time and money and limiting the pool of candidate colonies for choice of production clones [1, 2] . to address this problem, we aimed to develop a highly efficient serum-free medium suitable for optimising single-cell cloning efficiency by studying a range of conditioned media (cm) samples isolated from different chinese hamster ovary (cho) cell lines. materials and methods cho-s, dg44 and cho-k1 were adapted to cho-s sfm-ii (gibco) medium for a minimum of three passages. conditioned media was then collected when the cultures reached a cell density of 1x10 6 cells/ml (typically day 2-day 3 depending on the growth profiles of each cell line and whether they grew in suspension or attached conditions). samples were then centrifuged twice to remove cell pellet/debris and stored at -20°c. the ability of conditioned media to support cho colony formation was then assayed using 96-well plates, seeding the cells at low cell density (1-10cells/well) by diluting down cho cultures in media/conditioned media. after incubation at 37°c for 10 days, cloning efficiency was assayed using a standard xtt assay. initial screening of the nine cm samples was performed using cho-k1 cells due to their widespread use in industrial antibody production. successful media candidates were subsequently screened using additional cho cell lines. table 1) . the k1-sfmii-cm product improved cell cloning efficiency for dg44 cells (avg. increase>1.5-fold) and cho-s cells (avg. increase>3-fold) ( fig. 1) and also the adherent cho-k1 cell line growing in atcc +5%fbs. the ability of conditioned media to support cho growth in limiting-dilution conditions (1, 6 and 10 cells/ml) was investigated. from a range of nine conditioned media samples; four compelling products have been identified which improve low-cell density growth of cho-k1 cells, compared to sfm-ii control media. we feel that these early-stage conditioned media products may increase cloning efficiencies during upstream cho cell line development, resulting in financial savings for industry and increasing the possibilities of identifying particularly highperforming transformed clones. 12 (7):3496-3510. the main rate-limiting step in the upstream stages of protein biomanufacture is the isolation of stable, high producing cell clones. ubiquitous chromatin opening elements (ucoe®s) consist of at least one promoter region with associated methylation-free cpg island from housekeeping genes; they possess a dominant chromatin opening capability and thus confer stable transgene expression. ucoe®-viral promoter (e.g. cmv) based plasmid vectors markedly reduce the time it takes to isolate high, stably producing cell clones. although some ucoe®-viral promoter combinations have been tested, they have not been thoroughly evaluated in chinese hamster ovary (cho) cells. plasmid vectors containing combinations of either the human hnrpa2b1-cbx3 ucoe® (a2ucoe®) or murine rps3 ucoe® linked to different viral promoters (hcmv, gpcmv, sffv) driving expression of an egfp reporter gene were functionally analysed by stable transfection into cho-k1 cells and expression analysed by flow cytometry and qpcr to determine vector copy number. the results at 21 days post-transfection and selection clearly indicate that the rps3 ucoe®-gpcmv and -hcmv combinations give the highest transgene expression as shown in fig. 1 . the a2ucoe®-hcmv/gpcmv constructs were the next efficacious but 2-fold lower than the rps3 ucoe® vectors. the sffv promoter linked with either of the two ucoe®s was the least effective with expression levels 17-fold lower than the rps3-cmv constructs. the rps3 ucoe®-gpcmv/hcmv constructs are now being further modified to include elements that will provide optimal post-transcriptional pre-mrna processing (splicing, polyadenylation, transcription termination, mrna stability) thereby maximising stable cytoplasmic transgene mrna levels and protein production. in the last 20 years, growing number of innovator biologics and biosimilars have formed a competitive environment, where speed and efficiency of generating robust and highly productive cell lines needs to be improved continuously. through various advances, especially in media development and process optimization, product titers as high as 10 g/l were achieved in the pharmaceutical industry (kim et al., 2012) for standard products such as monoclonal antibodies. nevertheless, other proteins e.g. bispecific antibodies, fc-fusion proteins or fab-related products are difficult-to-express (dte) in chinese hamster ovary (cho) and may result in delays or even in termination of the cell line development process. we developed a new robust pool generation approach (cld 2.0) addressing both, easy-and difficult-to-express molecules, while reducing timelines down to 5 months (cld standard = 6 months), improving reliability of cell line development as well as clearly increasing obtained titers. in order to create stable cell lines, we transfected our cho dg44 host cells by electroporation. cells processed using the standard approach were cultivated in selective medium or medium containing additional 2.5 nm methotrexate (mtx) for three weeks. after an amplification step with 30 nm mtx for three weeks, stable individual cell pools were expanded and clones were generated by facs-sorting. clones were analyzed for growth performance and product concentration in fed-batch studies. in our new cld 2.0 approach, we increased mtx concentrations (2.5 nm, 5 nm and 10 nm mtx) during the first selection phase of three weeks. afterwards we omitted the 30 nm mtx amplification step. thereby, pool generation finished four weeks earlier than in the standard approach. to evaluate the stability of cell clones derived from mini pools (mps) generated according to the cld 2.0 approach, stability studies were performed for eight weeks, including stability fed batches at t=2 weeks and t=8 weeks. altogether three different proteins of interest with six cell clones each were tested. we adapted our cell line development process by increasing the initial selection during the first selection phase, thereby allowing the omission of the 30 nm mtx amplification step. we observed that the capacity of amplifiability varied for different products. cell lines with a protein titer ranging from >1 g/l to 1.5 g/l (dte) in shake flask fed-batch showed to be more susceptible to increased initial mtx levels and were thus not amplifiable with 30 nm mtx. in contrast, cell lines with high protein titer >1.5 g/l were observed to adapt to 30 nm mtx easily and were amplifiable. finale shake flask fed-batch data with cld 2.0 clones of highexpressing products showed comparable titers to clones from the standard approach. cld 2.0 clone titers for dte proteins revealed in average a 2.0-fold increase compared to clones generated in the standard approach. titers of top producing clones were in a range of 1.8 g/l to to 2.7 g/l (fig. 1) . furthermore, stability data of cld2.0 cell clones from different dte products showed a stable specific productivity in a range of +/-15 % over eight weeks cultivation. fed-batch titer from t=2 weeks and t=8 weeks were in a normal range of +/-20% of the standard 30 nm projects. our results demonstrate that cld 2.0 is a robust and reliable process for standard products (mab) and dte proteins. with our new process, we were able to increase titer of difficult-to-express proteins up to 200%. by omitting the amplification step (30 nm mtx) 96 % of generated clones were stable over eight weeks cultivation time. additionally using the cld2.0 approach, the time line from dna to rcb was reduced to 5 months. background cho cells have become the most popular platform for production of therapeutic proteins [1] . however, the generation of high-producer cells is a time-consuming and labor-intensive process that requires the screening of large amount of cells to get a clone of high titer and stability. since the expression titer and stability of clone is highly dependent on the site of integration, we demonstrated a new cell line development strategy by using ngs to identify the integration site and using crispr/cas9 to generate the target integrated high producing cell lines [1, 2] . to identify the high expression sites in the cho cells, we employed ngs to analyze the integration sites of a high producing cell line (titer > 3g/l). the pair-end reads with one read mapped to the vector and the other read mapped to the cho reference genome are extracted to identify the integration sites. to test the expression activity of the integration sites, we employed crispr/cas9 to specifically integrate the antibody gene into cho genome for expression. our data showed 4 integration sites are in the high producing cell line. among the 4 integration site, is1 integration site was tested by crispr/ cas9 for target integration of antibody gene for expression. the is1target integrated cell pool present higher expression titer than cell pool generated by target integration into other integration sites (fig. 1a) . the single cell clones derived from is1-target integrated cell pool had low copy number of goi (fig. 1b) . after normalization with copy numbers, the single cell clones derived from is1-target integrated cell pool showed high titer per copy (123~583 mg/l/copy) (fig. 1c) . this study demonstrated the generation of high-producing cell lines by crispr/cas9 mediated target integration. this approach will cost less time and labor than traditional method. the active integration site will serve as a platform like a cassette player for therapeutic antibody production. background cho, hek and sp2/0 are the dominant host cells for biologics drug production. achieving high level of recombinant protein production by these cell lines still remains a challenge. in order to understand the potential roles of lipids in protein production, secretion, vesicular transport and energy metabolism, we coupled high-throughput transcriptomics and lipidomics technologies. quantitative lipidomics is an emerging 'omics technology which can help us understand the physiological limitations of each cell line. the two types of major lipid groups in cells are non-polar and polar lipids. polar lipids such as glycerophospholipids (pls) include phosphatidylethanolamine (pe), phosphatidylcholine (pc), phosphatidylinositol (pi), phosphatidylserine (ps), phosphatidylglycerol (pg), and phosphatidic acid (pa). in this study; we integrated two dimensional high performance thin layer chromatography (2d-hptlc) and mass spectrometry (ms) lipid analysis of sp2/0, cho, and hek cell lines to understand the major differences in the lipid content of these hosts. bligh-dyer method was used to extract the lipids and extracts were analyzed by hp-tlc and ms. the polar lipids were separated into different categories by 2-d hp-tlc using a chcl 3 -meoh-h 2 o (71:25:2.5, v/v/v) solvent system in the first dimension and a chcl 3 -meoh-acetic acid-h 2 o (76:9:12:2, v/v/v/v) solvent system in the second dimension. non-polar lipids were separated by 1-d hptlc using hexane-diethyl ether-acetic acid. 2,7-dichlorofluorescein dye was used to visualize both polar and non-polar lipids. further detailed analysis was performed on a qqq mass spectrometer (thermo tsq vantage, san jose, ca) using negative-ion and positive-ion esi modes as well as negative-ion esi mode in the presence of lithium hydroxide. in this study, quantitative lipidomics was coupled with transcriptomics to further understand the physiological pathways of hek, cho-m and sp2/0 cells. initial hp-tlc analysis indicated that major lipids in these industrial cell lines were pe and pc. other polar lipids such as pi, ps, pg, pa, and sm were lower compared to pc and pe in exponential and stationary phases of each cell line. figure 1 represents 2d hp-tlc results of hek with the relative quantitation of polar lipids. in order to investigate the lipid subgroups, shotgun ms analysis was conducted for both exponential and stationary growth phases of the three cell lines. ms analysis indicated that lysophosphatidylethanolamine (lpe) and lyso-phosphatidylcholine (lpc) amounts were 4 -10 fold and 2-4 fold higher in hek cells compared to sp2/0 and cho cell lines. sphingomyelin (sm) was another lipid subgroup that was shown to have a major difference between sp2/0 and other mammalian cell lines. sm was 30-65 fold lower in sp2/0 cell line compared to cho and hek. to understand these metabolic differences, transcriptomics analysis using illumina highseq and gene expression omnibus was conducted on these mammalian cells. the kyoto encyclopedia of genes and genomes (kegg) database was used to map the transcriptomics data to the lipid synthetic pathways. transcriptomics data mapping to kegg pathways demonstrated that differences in lpe and lpc pathways correlate with the expression profiles of secretory phospholipase a2 (spla2), lysophospholipid acyltransferase (lpeat), lysophosphatidylcholine acyltransferase (lpcat), and lysophospholipase (lypla) [1] . the hp-tlc and lc/ms findings demonstrated that high levels of lpe and lpc existed in the hek cell line and low levels of sm were observed in the sp2/0 cell line. coupling lipidomics with transcriptomics provides us with an improved understanding of the physiological differences across sp2/0, cho, and hek cell lines that could be used to guide cell engineering efforts with the goal of increasing the recombinant protein expression capabilities of these three cell lines. biopharmaceuticals are a class of biological macromolecules that include antibodies and antibody derivatives, generally produced from cultured mammalian cell lines via secretion directly into the media. manufacturing at medimmune requires the generation of chinese hamster ovary (cho) clonal cell lines capable of producing the biopharmaceutical product at commercially relevant quantities with optimal product quality. the isolation of cell clones based on random single cell deposition via fluorescence activated cell sorting (facs) provides a heterogeneous panel of expressers. we hypothesize that the application of facs to provide an additional sorting step based on desirable cell attributes that correlate with productivity, product quality or cell growth attributes could lead to the isolation of higher producing cell lines with enhanced product quality attributes. a panel of 20 cell lines expressing a model recombinant monoclonal antibody were characterised in terms of growth, productivity, intracellular recombinant protein and mrna amounts. assays were also developed to investigate cell attributes using the commercially available imagestream instrument, an imaging flow cytometer, which enables the investigation of cellular characteristics that correlate with cell productivity at the single cell level. characterisation revealed the cell lines exhibited a range of values for productivity, growth, and intracellular (ic) antibody mrna and protein expression, ideal for further imagestream characterisation. western blot and qrt-pcr analysis demonstrated that final titre correlated with both ic heavy chain (hc) protein and mrna amounts (pearson correlation coefficient (pcc) = 0.70 and pcc = 0.80, respectively). to assess productivity at the single cell level, assays multiplexing ic hc protein and mrna with cell attributes were therefore developed. initial assay development focusing on hc mrna and protein amounts has revealed interesting results; four cell lines displayed two distinct populations, one producing the antibody and another nonexpressing population. the ratio of these populations varied amongst the cell lines. images obtained from the imagestream have shown the cellular localization and expression of hc and lc message and protein (fig. 1) . for both message and protein, hc and lc colocalize in the cell. whether there is any relationship between ic hc protein and cell attributes at the single cell level was then also investigated, as well as correlations with cell culture parameters at the population level. at the population level, correlations were found between titre and ic hc protein and mrna (pcc = 0.84 and pcc = 0.79, respectively) confirming the data obtained by western blot and qrt-pcr analysis. a panel of 20 cell lines has been characterised at the population level and show a wide range of antibody expression profiles at both the mrna and protein level. in parallel, assays have been developed for the imagestream to measure hc and lc message and protein amounts at the single cell level. protein and message quantification with the imagestream are consistent with more traditional approaches, such as western blots and qrt-pcr, that operate at the population level. the developed assays are now being used to investigate single cell productivity attributes and for the isolation of more productive clones. background productivity and stability are key factors for the selection of cell line in protein drugs production. large amount of target gene integrated in cell genome could lead to the instability of production. therefore, cells with low copies of target gene integrated in high yield sites could be an ideal production cells for manufacturing. it has been known that the transposon system can control the integrated copy number of target gene and can generate high yield producing cells, it could be a great approach to generate stable high yield producing cell lines carrying low copies of target gene through transposon system. we intended to develop a platform to generate high yield producing cell lines carrying 1-2 copy of the integrated target gene using transposon system. two cho cell lines, cho-s cells and dxb11 cells, have been applied. cells were co-transfected with transposon and target gene expression plasmids. after drug selection, the cell pool with highest productivity per target gene copy was applied to single cell cloning. the productivity and copy number of cell clones were determined, and the stability of cell clones was analysed after culture of about 60 generations. in the stable pools of cho-s and dxb11 cells, the productivities per integrated target gene copy were about 11-13 mg/l/copy and 68-75 mg/l/copy in a batch culture, respectively. after single cell cloning, the integrated copy numbers in most cell clones were less than three copies per cell. in cho-s and dxb11 cell clones, the productivities per integrated target gene copy were 20-60 mg/l/copy and 60-150 mg/l/copy in a batch culture, respectively. the productivity per integrated target gene in cell clones developed by the transposon system was much higher than that in cell clones developed by random integration (fig. 1a and b) . to evaluate the productivity stability of cell clones developed by the transposon system, ten cell clones at generation 0, 30, 60, and 100 were applied in the analysis. of interest, about 80% of cell clones were stable at generation 60, but lost the productivity at generation 100 (fig. 1c) , implying the most cell clones could maintain the stability within 2 months. using the optimized conditions of the transposon system to develop the stable gene expression cells, the productivity per integrated target gene was higher than random integration. these results suggested that our platform is capable to develop high yield producing cells with 1-2 copy of integrated target antibody gene and can be applied to identify high yield integration sites. background mammalian cells show an inefficient metabolism characterized by high glucose uptake and the production of high amounts of lactate, a widely known growth inhibition by-product [1] . recently, we have observed a different glucose-lactate metabolism in some cell lines. while some cell lines are unable to metabolize lactate, others can co-metabolize simultaneously glucose and lactate under certain culture conditions, even during the exponential growth phase [2] . these metabolic differences between different mammalian cell lines (cho, hek293 and hybridoma) have been studied by means of flux balance analysis (fba). three different cell lines were cultured in a 2-liter bioreactor: cho-s, hek293sf and hybridoma kb-26.5. for the fba, two adapted genome-scale metabolic models were used: a reconstruction of mus musculus for cho and hybridoma [3] , and a reconstruction of human metabolic model (recon 2) for hek293 [4] . in cultures where ph was not controlled, two different metabolic phases were observed for cho and hek293 cells. during the first phase both cell lines produced large amounts of lactate as a consequence of the high glucose consumption rates. interestingly, when ph dropped below 6.8, due to acid lactic secretion and accumulation, a second metabolic phase was identified, in which concomitant consumption of glucose and lactate was observed even during the exponential growth phase. conversely, hybridoma cells were unable to co-consume lactate and glucose simultaneously even under noncontrolled ph conditions. therefore, the hybridoma physiological data used for the fba corresponded to only phase 1 of phcontrolled cultures. a summary of the main cell growth and metabolic parameters obtained from the different experiments performed is presented in table 1 . fba shows ( fig. 1 for hek293 cell culture) that lactate is produced in phase 1 because pyruvate has to be converted to lactate to fulfill the nadh regeneration in the cytoplasm and only a small amount of pyruvate can be transported into tca through acetyl-coa. cell metabolism in phase 1 is highly inefficient, as the majority of the carbon source is not used for the generation of energy nor biomass. in phase 2, in which mitochondrial ldh was considered, tca fluxes could be maintained as in phase 1 at the maximal rate encountered; hence, the energy available for cells to grow was similar in both phases, obtaining similar growth rate. two different glucose and lactate metabolism behaviors have been observed in cho and hek293 cultures depending on the culture conditions: phase 1) glucose consumption and lactate production, and phase 2) glucose and lactate simultaneous consumption. in contrast, only phase 1 was observed in hybridoma cultures even when ph was non-controlled. fba showed that tca fluxes in phase 1 and phase 2 were similar, obtaining similar cell growth rate, but glucose uptake rate was much lower in phase 2 due to the lactate co-consumption. some authors hypothesize that cells metabolize extracellular lactate as a strategy for ph detoxification [2] . glucose and lactate co-metabolization resulted in a better-balanced cell metabolism, as can be seen from the metabolic fluxes calculated, with minor effects on cell growth. the observation of glucose and lactate co-consumption metabolic behavior and its deeper study and characterization could open the door of novel culturing strategies with the aim of increasing bioprocesses productivity. background transient protein expression in mammalian cell lines has gained increasing relevance as it enables fast and flexible production of high-quality eukaryotic protein. considerable efforts have thus been made to overcome existing limiting aspects of transient gene expression systems, in terms of cell lines, cell culture-based systems, and protein production in a cost-effective manner. milligram amounts of protein per liter can be produced within several days, allowing a significant shortening of the bioproduction process in comparison to protein production from stable clones. to ensure the robustness of the process, it is essential to have a reliable and easy-to-use transfection method. to palliate for the need of a reliable transfection reagent, we developed peipro®, the only commercially available pei optimised for mid to large-scale transient protein production during process development. peipro® is a non-polydiperse and fully-characterised polymer that has become the gold pei standard due to its reliability, reproducibility in high dna delivery efficiency and in ensuing high protein production yields. here, we present experimental data showing the benefits of using peipro® for protein production in comparison to other peis. we further demonstrate compatibility of using peipro® for recombinant protein production in most commonly used chemicallydefined media. materiel and methods suspension hek-293 and cho cells were cultured in shaker flasks in various synthetic media, as listed in table 1 . hek-293 and cho cells were resuspended at 1×10 6 cells/ml of serum-free medium, on the day before transfection. cells were transfected with 0.5-1 mg of plasmid dna encoding for the luciferase gene reporter using peipro®, pei "max" and l-pei 25 kda (polysciences, warrington, pa) resuspended at 1 mg/ml according to the manufacturer's recommendation. protein expression of the luciferase reporter gene was assayed 48 hours post-transfection by affinity chromatography using protein g (hplc). comparison of peipro® to other commercially available peis was achieved by transfecting suspension hek-293 and cho cells with plasmid dna encoding for the luciferase gene reporter. luciferase production yields obtained in hek-293 and cho cells were at least respectively 5-fold and 10-fold higher when using a similar amount of peipro in comparison to the other peis (fig. 1) . furthermore, peipro® was the only pei that led to similar luciferase production yields when decreasing the amount of plasmid dna per liter of cell culture. conversely, at least 1 mg of plasmid dna and 4-fold more of pei "max" and l-pei 25 kda were needed to obtain a similar luciferase expression range in both hek-293 and cho cells. we further assessed the compatibility and versatility of peipro® by measuring protein production yields obtained in most commonly used animal-free synthetic media. as shown in fig. 2 , peipro® leads to high protein production yieds in several commercially avaialble media formulations for hek-293 anc cho cell lines. peipro® is the only fully characterised pei transfection reagent that is suitable for reliable and reproducible recombinant protein production, irrespective of the scale of production and of the type of adherent and suspension cell culture system. fig. 1 (abstract p-274) . peipro® requires less reagent and similar to lower dna amount compared to other peis. suspension hek-293 and cho cells were seeded at 1×10 6 cells/ml in serum free medium and transfected with peipro®, pei "max" and l-pei 25 kda (polysciences, warrington, pa) resuspended at 1 mg/ml. luciferase expression was assayed 48 h after transfection using a conventional luciferase assay fig. 2 (abstract p-274) . peipro® is optimized for transfection of hek-293 and cho cells in several specific synthetic culture media. suspension hek-293 and cho cells were seeded following the recommended protocol in serum-free media and transfected with peipro® using the standard conditions. igg3-fc production was assayed 48 h after transfection using protein g affinity quantification (hplc) monoclonal antibodies (mabs), which are widely used in anticancer therapies, are mainly produced by mammalian cell lines. mab conjugation to biological molecules for enhancing their antitumor activity offers a new powerful tool for anticancer therapies. we have assessed the production of commercially approved anti-her2 therapeutic antibody trastuzumab (tzmb) [1] and also its fusion with interferon-α2b (ifnα2b). two cloning strategies consisting in transfecting cho-s and hek293 cell lines with two bicistronic or with a single tricistronic plasmids have been assessed. the in vitro efficacy of both antibodies has been tested and compared side by side. tzmb heavy and light chains were cloned in two bicistronic plasmids (pirespuro3 and piresneo3, clontech) and in a tricistronic plasmid derived from pirespuro3. ifnα2b was spliced to tzmb heavy chain by overlap extension pcr and the resulting tzmb-ifnα2b fusion protein was also cloned in the expression vectors in the same way than non-modified tzmb. selected cell pools were cultured in 125 ml shake flasks containing sfmtransfx supplemented with 10% v/v of cell boost 5 (hyclone), 4 mm of glutamax (gibco) and 2 μg/ml of puromycin and also with 700 μg/ml neomycin in the case of the cells transfected with pires-neo3. cells were cultivated in the same conditions as described elsewhere [2] . purified products (using protein a chromatography (hitrap mabselect sure, äkta avant 150)) were quantified by both elisa and sds-page. antigen binding test was performed in sk-br-3 breast cancer cell line by means of flow cytometry analysis. the biological activity of the different candidates was tested with mtt assay. both tzmb and the fusion protein tzmb-ifnα2b have been successfully expressed in cho-s and hek293, which use for heterologous protein expression have previously been optimized in prior works [3] . the tricistronic strategy resulted in the most efficient, showing a 3.5fold increase in terms of productivity with respect to the bicistronic double-transfection for tzmb in cho-s cells and a 5-fold increase in hek293 cells (fig. 1a) . in the case of tzmb-ifnα2b, the tricistronic strategy also allowed to achieve higher productivities than the bicistronic one (fig. 1b) . regarding the differences of specific productivity between both cell lines tested, hek293 emerged as the best production host candidate, for the two tested strategies (tricistronic and bicistronic) and for the two produced proteins, showing a 1.5-fold increase in terms of productivity with respect to cho-s cells for tzmb using the tricistronic strategy. tzmb and tzmb-ifnα2b were analysed in terms of their antigen binding capacity, and both were find to efficiently bind to her2+ skbr-3 cells (fig. 1c) . thus, the antibody affinity to her2 antigen has not been affected when fused to inf-α2b. finally, antiproliferative activity of tzmb and tzmb-ifnα2b were assessed on the same sk-br-3 cells. at a concentration of 500 nm of tzmb, and after a 72-hour incubation, sk-br-3 cells presented a 83% growth with respect to the untreated control. however, no antiproliferative effect was observed for tzmb-ifnα2b (fig. 1d) . the tricistronic strategy provides higher productivity yields in hek293 and cho-s cell lines for both recombinant proteins (trastuzumab and tzmb-ifnα2b). regarding which cell line is the best production host candidate, hek293 achieved higher productivity than cho-s cells for the two proteins tested. all constructions performed preserved the binding affinity to its antigen, trastuzumab and tzmb-ifnα2b bind efficiently to the her2 antigen present in skbr-3 cells. finally, tzmb-ifnα2b does not present an improved antiproliferative effect with respect to trastuzumab when compared by means of an in vitro assay. the genetic engineering of patient-specific t cells with lentiviral vectors (lvv) expressing chimeric antigen receptors (car) for late phase clinical trials requires the large-scale manufacture of high-titer vector stocks. the state-of-the-art production of lvv is based on 10-to 40layer cell factories transiently transfected in the presence of serum. this manufacturing process is extremely limited by its labor intensity, open-system handling operations, its requirements for significant incubator space plus costs and patience risk due to presence of serum. to circumvent these limitations, this study aims to develop a stable and serum-free process to produce lvv with pei-mediated transfection. in addition, this study also focuses on the development of a a c b d fig. 1 (abstract p-276) . expression of trastuzumab (a) and trastuzumab-ifnα2b (b) from bicistronic strategy (bc) and tricistronic strategy (tri) with cho-s and hek293 cells. relative specific productivity units are used for comparing the different strategies. c antigen binding analysis of trastuzumab and trastuzumab-ifnα2b. d antiproliferative activity of trastuzumab and trastuzumab-ifnα2b on sk-br-3 cells production system not only using a gfp marker but also a therapeutically relevant transgene (cd20-car) [1] . therefore, three different cell lines (hek 293, 293t, 293ft) were investigated concerning their productivity of lvv and their growing behavior in the in-house serum-free medium transmacs. as part of this, design of experiment was used to investigate the optimal conditions for pei/ dna-transfection. furthermore, this statistical approach was used focusing an ideal ratio between the 3rd generation plasmids (transfer plasmid cd20-car or gfp, envelope plasmid, packaging plasmids). in addition, different enhancers (sodium butyrate, lithium acetate, caffeine, trichostatin a, cholesterol, hydroxyurea, valproic acid) were investigated concerning their effects on productivity comparing hek cultures producing lvv encoding for gfp-marker or cd20-car. concerning productivity and growing behavior, hek 293t was the favored cell line for our serum-free lv manufacturing process. in addition, an additive screen revealed that sodium butyrate alone had the most promising effect on both gfp-lvv and cd20-car-lvv production. after pei/dna titration, we finally could increase lvv productivity by lowering pei/dna amount at higher cell densities referred to our standard transfection protocol. furthermore, the titration for the optimal plasmid ration revealed, that for large transfer constructs higher amounts of transfer plasmid are required than for smaller constructs to achieve a high productivity (fig. 1) . the outcome of these experiments enabled the development of a robust hek293t based process to produce clinical relevant lvv under serumfree conditions. furthermore, it provides an insight how therapeutic genes and the expression of its transgene can influence cell productivity. led to a vast increase in productivity, cho cells yield less than other expression systems like yeast or bacteria [1] . to improve yields and find beneficial bioprocess phenotypes, genetic engineering plays an essential role in recent research. the mir-23 cluster with its genomic paralogues (mir-23a and mir-23b) was first identified as differentially expressed during temperature shift, suggesting its role in proliferation and productivity [2] . the common approach to deplete mirnas is the use of a sponge decoy which, requires the introduction of reporter genes. as an alternative this work aims to knockdown mirna expression using the recently developed crispr/cas9 system which does not require a reporter transcript. this system consists of two main components: the single guide rna (sgrna) and an endonuclease (cas9) which induces double strand breaks (dsbs). these dsbs can result in insertion or deletion (indels) of base pairs which can disrupt mirna function and processing [3] . a cho-k1 cell line stably expressing an igg was used for knockdown experiments. sgrnas were designed to target the seed region of each mirna member and stable mixed populations were generated (fig. 1a) . total rna form each mixed population was reverse transcribed into cdna using mirna specific stemloop primers. the expression was quantified by rt-qpcr. to further analyse the range of indels the mir-23a and mir-23b clusters were amplified by a standard high-fidelity pcr. amplicons were cloned into pcr tm -topo® vector and positive clones were analysed by sanger sequencing. cell growth was monitored using viacount tm viability stain on a guava tm benchtop flow cytometer. productivity was assessed by elisa. students t-test was used for statistical analysis. it was shown that mirna expression was significantly reduced in mixed populations. a knockdown up to 95% was achieved for mir-23a, mir-23b and mir-24. the knockdown in mir-27a and mir-27b expression was considerably less -between 70-90% (n=3, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) (fig. 1b) . furthermore, it was shown that various sizes of indels were generated by targeting the seed region. smaller indels (+1/ +2/-1/-2 bps) seemed to be more common but larger deletions were detected as well (fig. 1c) . mir-23a, mir-23b and mir-27b showed increased viability in late stages of the culture. depletion of mir-27a reduced growth significantly whereas knockdown of mir-24 showed increased proliferation as well as boosting igg titers (table 1) . in this work, we have shown that crispr/cas9 can be successfully applied as a tool to knockdown mirna expression in cho cells. the data was generated using mixed pools and it remains to be established if both alleles can be successfully targeted e.g. using nextgeneration sequencing of individual clones. background chinese hamster ovary (cho) cells are the most widely used host cell line for the production of therapeutic antibodies. pre-and posttranslational modifications and optimization of culture methods contributed to increase the productivity, resulting in a very high titre [1, 2] . however, it has been pointed out that the intracellular secretion process is a bottleneck in the production of therapeutic antibodies [3] . in addition, the details of the process of secretion of humanized recombinant antibodies from cho cells have not been well investigated. in this study, we thus analysed the detailed process of secretion of therapeutic antibodies using cho cell lines, which have already been established as high producers, with the aim of obtaining information for the more rational and efficient establishment of high-producer cells. we performed 1) chase assay, 2) immunofluorescent microscopy observation, and 3) size exclusion chromatography (sec) analysis to investigate the duration of secretion, bottleneck position, and formation of recombinant igg, respectively. high-producer cho cells expressing humanized igg1 [4] and igg3 were used. for the chase assay, cells were cultivated in shake flasks with serum-free medium containing 50 μg/ml cycloheximide (chx) to stop nascent peptide synthesis. the amounts of igg both remaining in the cell and secreted into the medium at each time point were measured by quantitative western blotting. for immunofluorescent microscopy observation, cells were cultivated on coverslips with chx for 4 h. immunofluorescent staining against the recombinant igg, endoplasmic reticulum (er), and golgi apparatus was performed after chemical fixation. for sec, cells cultured with chx were re-suspended in a buffer containing tritonx-100 and injected into a column. the amount of igg in each fraction was measured by quantitative western blotting. the amount of igg3 in the supernatant increased until 4-6 h after the inhibition of protein synthesis by chx; however, it hardly changed thereafter (fig. 1, upper panel) . at this point in time, however, around 40% of igg still remained in the cells (fig. 1, lower panel) , meaning that all of the synthesized igg could not be secreted into the medium and remained in the cells for several hours. this result was almost the same as that of studies using igg1-expressing cells [5, 6] . the localization of igg in the cells was checked before and after the addition of chx, with the results showing that igg1 remained in the er and was hardly seen in the golgi apparatus [5] [6] [7] ; igg did not seem to be efficiently transported to the golgi apparatus. the sec experiment showed that most of the igg1 remaining in the cell seemed to form full-sized antibodies [5, 6] , but it could not be secreted despite this. the high-producer cells could not secrete all of the synthesized igg, and around 40% of igg remained in the cells for several hours. this incomplete secretion is a common phenomenon among cho cells producing different types of recombinant igg. the igg could not be transported from the er to the golgi despite its formation of fullsized antibodies. solving this bottleneck in the transportation of igg from the er to the golgi and/or achieving more efficient glycosylation of igg after the formation of full-sized antibodies might be the next target to improve productivity. background humanized monoclonal antibodies (mabs) are among the most promising drugs, but defined strategies for their modification are still not available. our work deals with humanization of murine mab2/ 3h6. the superhumanization approach leads to a loss of binding affinity which was partially restored by a single human-to-mouse backmutation (t98hr). [1] this residue was selected by synergistic combination of sequence analyses of antibody framework regions and structural information using novel in silico simulations. for structural stabilization, a conglomeration of tyrosine residues surrounding t98hr was identified, the so called "tyrosine cage". [2] analysis of the "tyrosine cage" was done by alanine scanning mutations with a double mutation variant t98hr + y27ha (bm09) and a triple mutation variant t98hr + y27ha + y32ha (bm10). in a recent series of experiments we tried to enhance binding affinity by three new variants with backmutations in the variable light chain (vl). originating from t98hr, residues in the vl were selected based on their spatial proximity to the cdr3 loop of the variable heavy chain. affinity improvement of t98hr was evaluated by vl-double backmutation variants t98hr + f46ll (su01) and t98hr + q49ls (su02) and a triple backmutation variant t98hr + f46ll + q49ls (su03). all five variants were expressed transiently in hek293-6e cells and binding affinities were investigated in two individual settings with bio-layer interferometry. in the first approach concentrated cell culture supernatants were directly applied and mabs were captured on protein a tips, blocked with 3d6scfv-fc and the association and dissociation of 2f5 igg was measured. for the second approach, the culture supernatants were purified and the affinity was determined with streptavidin biosensors. first, biotinylated 2f5 igg was bound and then the association/dissociation of the purified 3h6 variants was measured. affinity evaluation of concentrated culture supernatants with protein a sensor tips showed a decrease of binding affinity of bm09 and a loss of binding of bm10. the protein a measurement showed an increased binding strength of su01, su02 and su03 compared to su3h6 and bm07. su01 and su03 result in a higher binding affinity compared to su02. these results can be confirmed with purified variants by the streptavidin bio-assay (fig. 1) . alanine scanning of the tyrosine cage demonstrated a reduction of binding affinity (bm09) and a severe loss of binding (bm10), concluding that the tyrosine cage plays an important role for supporting a correct cdr loop conformation. further affinity improvement of the single mutation variant t98hr could have been reached via mutations in the vl. it demonstrates the underestimated role of the vl for the interaction with its binding partner. although cho cells are a major expression system for production of recombinant biopharmaceuticals, the molecular and cellular background characterizing a high producer is largely unknown. it has been observed that in producer cell lines important signaling pathways like the akt-signaling are altered in characteristical ways. thus analyzing according signaling events should lead to identification of key elements characterizing high producer cells. to investigate this, our emphasis lies on the phosphorylation status of involved proteins as reversible switches in all signaling pathways. we aimed to establish a workflow for cho-specific phosphoproteomics and focused on igf signaling, as cell culture media often are supplemented with this growth factor. two producer cell lines and the according parental cells were cultivated in a stable isotope labeling with amino acids in cell culture (silac) experiment, followed by quantitative ms phosphoproteomic analysis including chospecific data evaluation. the chosen cho cell lines were cultivated in triplicates in silac media containing isotopically-labeled lysine/arginine (hlys/harg) and in parallel in identical standard media (llys/larg, tcx10d, xell). cell density, viability, metabolism and cell cycle distribution were monitored during 50 ml batch culture for 7-8 days. at day 3.25 igf was added into hlys/harg cultures. 5 min later a part of the cells was harvested. for ms analysis igf-treated (hlys/harg cultures) and control cultures (llys/larg cultures) were combined. the following ms sample preparation workflow included digestion of whole protein lysate and phosphopeptide enrichment via tio 2beads. nanolc-esi-orbitrap ms (q exactive plus, thermo fisher scientific) of phosphopeptides was excecuted with subsequent identification and quantification in maxquant [1] . in addition to silac quantification of h/l ratios for investigation of igf effects, aquired data was also used to perform label-free quantification (lfq) in maxquant [1] for comparison of cell lines. statistical significance was calculated via t-test (p<0.05) or anova (permutation-based fdr<0.05) in perseus [2] . results igf effects on growth and production the igf treatment resulted in a prolonged viability for all cell lines. however, an increased vcd was only observed for producer cell line 1, yielding in an enhanced integral of vcd (ivcd). for the parental cells growth was inhibited by igf, although s-phase cells were enriched at least temporary (fig. 1a) . regarding antibody production igf led to a decreased qp and product titer, concomitantly with an increase in s-phase cells (fig. 1a) . this inverse correlation of proliferation and cell specific productivity is known from different productivity enhancing molecules, like butyrate [3] . ms investigation of signaling events the phosphoproteomic experiment resulted in the identification of 10.485 class i-phosphorylation sites. statistical evaluation of phosphopeptide abundances in perseus showed up 144 significant differences between the cell lines and led to producer vs. parental classifications (fig. 1b) . the quantitative evaluation via silac yielded in about 2.408 quantifiable phosphosites in at least 6 biological replicates. rapid phosphorylation changes after growth factor treatment indicated signaling towards protein synthesis, cell cycle and regulation of actin cytoskeleton amongst others. for 201 phosphosites significantly different h/l ratios were calculated between the two groups parental vs. producer, four of them are listed (table 1) . the workflow to study phosphorylation states revealed differences in the related cell lines and gave insights into signal transduction as a response on igf. on the one hand, igf-treatment resulted in a fast and widespread upregulation of phosphorylation sites within aktand mapk-signaling. on the other hand, a different phosphorylation status for producer compared to parental cell lines uncovered distinctions in biological processes like rna-and dna-binding and regulation of cytoskeleton. in sum, our sucessfully established phosphoproteomic approach allows to detect important signaling key players in cho cells that subsequently can be targeted through cell engineering or small molecule treatment. to improve antibody production in the cho cell expression system, it seems to be useful to up-or downregulate gene expression including antibody folding, secretion, and cell metabolism. many cell engineering approaches, including gene introduction, knockout and knockdown, have been employed to enhance recombinant antibody production [1] . however, identifying production enhancer genes is the rate-limiting step for cho cell engineering, because the conventional method requires a series of experiments including genomic integration of the tested genes, selection of stable cell clones and cell culture experiments of several clones. in this study, we propose an approach for rapid evaluation of production enhancer genes based on an episomal expression system. plasmid vector carrying the epstein-barr virus (ebv) encoded nuclear antigen 1 (ebna1) was transfected into cho cell line producing igg1 antibody. after g418 selection and single colony isolation, ebna1 expression was checked with capillary electrophoresis system wes (proteinsimple). ebv ebna1-antibody (1eb12) was used for detection as the primary antibody. the expression vector for the gene of interest was prepared by inserting 1508 bp of an orip dna sequence into a plasmid vector carrying cag promoter, resulting in the potc vector. pei max (polysciences, inc.) and balancd transfectory cho (irvine scientific) were used for the transfection. the number of viable cells and gfp-positive cells were counted using countess ii fl automated cell counter (thermo fisher scientific). the transfected cells were cultured in cellstar cellreactor tubes. the tubes were incubated in a climo-shaker isf1-x (kuhner). antibody production was measured using biolayer interferometry with an octet qk system (fortebio). we constructed four cho cell lines stably expressing ebna1, termed igg1-eb01 to eb04. in capillary electrophoresis analysis, we observed a clear peak corresponding to the ebna1 expression in all four cell lines. we tested the transfection efficiency by potc-gfp plasmids. in the best transfection condition, pei/dna ratio of 1/1, igg1-eb01 cell showed the highest gfp-positive cell number (1.07×10 7 cell/ml) and transfection efficiency (95%) among the four cell lines. therefore, igg1-eb01 cell lines were selected for further study. after the transfection, the number of gfp-positive cells continued to increase even after the passage (fig. 1) , suggesting that the potc-gfp plasmid was stably retained and replicated by ebna1/orip system in igg1-eb01 cell lines. in preliminary experiments, we introduced three genes, mdh2, gss and gclm, into igg1-eb01 cell lines. cotransfection of these three genes led to an increase in igg1 production from 287±18 mg/l (control) to 334±21 mg/l at day 8 (p<0.05, t-test, n=3). this result suggests that these three genes work as production enhancer genes. conventional methods based on stable cells take up to 6 months to determine whether the gene of interest is beneficial for recombinant igg1 production. in contrast, identification of production enhancer genes is achievable within 10 days by our proposed method based on ebna1/orip system. the proposed method makes it possible to evaluate production enhancer genes in a rapid manner. the proposed method is a promising approach to identify genes enhancing recombinant antibody production. background 2g unic™ (2gun) technology comprises a set of protected genetic elements that improve protein production by acting on transcription as well as on translation. the elements can either be inserted into existing (platform) vectors or be provided as complete ready-to-use vectors. the technology can be used in stable and in transient transfection to boost protein production for product development and is being applied in cld for pharmaceutical proteins. in combination with antibiotic selection or dhfr selection, 2gun technology routinely results in 2-3 fold increase in expression of client antibodies or fusion proteins, both in pools and after clonal selection. previously, we have successfully combined 2gun technology with glutamine synthetase (gs) selection and the cho gs null cells of horizon discovery, resulting in clonal cell lines producing > 6 g/l of a biosimilar mab in fed-batch assay. here we present data on the successful application of the 2gun technology for the enhanced expression of a large (>300 kda) human heterotrimeric glycoprotein, a renowned difficult-to-express (dte) protein. all expression vectors comprised a hcmv promoter and bgh polyadenylation sequence in the expression cassettes for the gene of interest, and a selection marker gene with sv40 promoter and sv40 polyadenylation sequence. 2g unic™ vectors also contained genetic elements (2g unic™ technology, proteonic). cho gs null cells (horizon discovery) were transfected in duplicate with reference or 2gun expression vectors and selected in media lacking glutamine and containing the appropriate antibiotics. the bulk pools were seeded at equal viable cell density after obtaining maximum viability and cultured for 9 days without feeding (batch). expression of the target protein in cell culture supernatants of stable bulk pools was measured by elisa. the three protein subunit genes were expressed from vectors with different selection markers. in the reference constructs (without 2gun), the α, β, and γ chains were expressed from vectors with marker genes for zeocin, blasticidin, and gs, respectively. a similar 3 vector combination was also generated with 2gun elements integrated in each vector. in addition, 2 vectors with 2 subunits (γ-α and α-γ), each with a separate 2gun element, promoter and polyadenylation signal, were generated with a gs marker gene. cho gs -/cells were transfected with the 4 appropriate vector combinations in equimolar ratios and selected in bulk in medium lacking glutamine and 1 or 2 antibiotics. the 2-vector transfected cell pools recovered first, due to the presence of only 1 antibiotic in the medium (fig. 1a) . the pools transfected with three 2gun vectors recovered to maximum viability just a few days after the 2-vector 2gun pools. recovery of the reference pools took up to a week longer than the 2gun pools. production of each pool was assessed in a batch production run in shaker flasks. all 2-vector 2gun pools which recovered first produced titers around 0.1 g/l, which is almost 10-fold higher as compared to the production by reference pools (fig. 1b) . the highest titers of 0.5 g/l were obtained in the 3-vector 2gun pools. these data show that the 2g unic™ genetic elements can be successfully used to obtain a significant increase in the titer of difficult-to-express proteins. similar results have been obtained with other dte proteins, including fc-fusion proteins and bi-specific antibodies (not shown). the expression of a large, glycosylated multimeric difficult to express protein can be increased more than ten-fold in cho gs pools by application of 2g unic™ genetic elements. the highest expression of is obtained using a separate vector for each subunit. characterization of antibody-producing cho cells with chromosome aneuploidy noriko yamano 1,2 , sho tanaka background chinese hamster ovary (cho) cells are commonly used as host cells to produce biopharmaceuticals. however, the number of chromosomes in cho cells varies. previously, dg44-sc20 and dg44-sc39 cell lines with modal chromosome numbers of 20 and 39 were isolated from parental cho-dg44 cells, from which igg3-expressing cell lines named igg3-sc20 and igg3-sc39 were established, respectively. the igg3-sc39 cell pool showed a higher specific igg3 production rate than the igg3-sc20 cell pool [1] . even though all of the igg3-sc20 clones and half of the igg3-sc39 clones contained the same number of vector integration sites (single integration site), igg-sc39 cell clones produced more igg3 following the culture of single-cell clones than any of the igg3-sc20 clones [1] . in this study, we performed transcriptome analysis to investigate the characteristics of high-producer cells with chromosome aneuploidy. transcriptome analyses using amplified fragment length polymorphism (aflp)-based high-coverage expression profiling (hicep) and de novo mrna-seq were performed on dg44-sc20, dg44-sc39, igg3-sc20 and igg3-sc39. to compare cell lines with different numbers of chromosomes, transcriptome data from mrna-seq were adjusted for cell number using rna reference materials (nmij crm 6204-a; national institute of advanced industrial science and technology) mixed at equal amounts per cell. pathways related to differentially expressed genes were searched using keymolnet (km data). high-chromosome-number cho cells showed larger cell diameters, as determined by vi-cell (beckman coulter) measurement. the predicted volume ratios, based on these diameters, are 2.24 (dg44-sc39:dg44-sc20) and 1.59 (igg3-sc39:igg3-sc20). the levels of β-actin and the products of most other genes that were detected by mrna-seq differed by approximately 20% in the comparison between sc39 and sc20 (sc39 > sc20). based on the analysis of gene expression levels per cell volume, approximately 90% of detected genes showed lower expression in both dg44-sc39 and igg3-sc39 compared with the levels in dg44-sc20 and igg3-sc20, respectively. in addition, the number of genes whose expression level was decreased in igg3-sc39 compared with that in dg44-sc39 was larger than those showing the opposite pattern. the results of the comparisons between igg3-sc20 and igg3-sc39 indicate that differentially expressed genes were mainly related to cell growth (e.g. myc, smad), apoptosis (e.g. caspase), lipid metabolism (e.g. srebp, pparγ) and epigenetic histone modification (e.g. brca, hat) pathways. the mrna levels of myc, smad, caspase, brca and hat related genes were lower in igg3-sc39, while those of srebp and pparγ related genes were higher in igg3-sc39. the effects of these pathways on antibody production should be examined in future. in this study, we found that high-chromosome-number cho cells have lower amounts of mrna relative to their volume. a reduction per unit volume in the expression of genes that are required for survival might generate additional energy for recombinant protein production in high-chromosome-number cells. from an evolutionary perspective, an increased set of chromosomes underlies rapid evolutionary adaptation. although there are issues to be considered, such as stability, there may also be advantages to using high-chromosome-number aneuploid cho cells as a production host cells of recombinant proteins. background human growth factors have an enormous therapeutic potential. among them, the bone morphogenetic protein-2 (bmp-2) can induce de novo bone formation endowing the protein a high therapeutic potential. however, finding a suitable recombinant production system for such a protein still remains a challenge. recombinant expression of hbmp2 was investigated in transiently transfected hek-293 cells and in stable clones established in cho-k1 cells cultivated in excell and pro-cho5 medium, respectively. protein stability and interaction of the hbmp2 with the producer cells were investigated in vitro using commercially available rhbmp2. in addition, we investigated a cell-free protein synthesis system harboring translocationally active microsomal structures, hence having the potential to perform post-translational modifications, as an alternative production method. we showed that growth rates and viabilities of the rhbmp2producing cells were similar to those of the parent cell line, while entry into the death phase was delayed in case of the recombinant cells. the maximum rhbmp2 concentration detected in the culture supernatant was low for stable clones but can be greatly improved combining the hek-293 cells transient expression system and batch reactor cultivation which reflects a better compatibility of the codon usage in the human cells (table 1) . hbmp2 protein is sensitive to slightly acidic ph and to a lesser extend to proteases (fig. 1a ) and binds to both producers cell lines (fig. 1b) -all this could incidentally contribute to the low product titers. cell-free protein synthesis has been proposed as alternative for "difficult-to-express" proteins. since native hbmp2 is glycosylated, a cell-free system based on eukaryotic cell lysates is required for its production. cho cell lysates were chosen, since they had previously been established as the most productive eukaryotic system in our hands [1] , while concomitantly enabling a direct comparison to the production of hbmp2 in stable clones established in cho-k1. the ability to perform post-translational modifications is a major advantage of eukaryotic systems. the cho lysates prepared by the protocol used here have previously been shown to contain significant amounts of endogenous microsomes derived from the endoplasmatic reticulum during lysis [2] . to enforce translocation of the target protein into the microsomal structures, a melittin signal peptide was fused to the hbmp2 cdna. the glycosylation of the protein was assessed by enzymatic treatment (pngase, endoh) and confirmed using 14 c-mannose for the de novo protein synthesis. upon cell-free protein synthesis, the hbmp2 yield was 100-fold higher than the best one in the hek-293 cells. the difference becomes even more dramatic, when productivities are considered (table 1) , i.e. the fact that maximum product titers are reached within 3 h in the cellfree system compared to 120 h in the cell-based ones. this demonstrates that the cell-free expression system is most suitable compared to mammalian cell expression method for the production of glycosylated human bmp2 (table 1 ) [3] . human growth factors are complex molecules, which make their production in mammalian cells desirable. however, low product titers caused by a variety of both cell and process related effects may hinder the development of highly productive processes. in such cases, cell-free protein production using cho cell lysates containing endogenous microsomes for posttranslational processing, may eventually present an attractive alternative. in particular since these lysates can be used under tightly controlled conditions assuring a higher degree of reproducibility, than, e.g. transient transfection systems. cell-free systems are known to circumvent typical bottlenecks of cellbased ones, e.g. metabolic regulation and cell maintenance mechanisms. in consequence, the production of a recombinant protein is neither inhibited by its accumulation nor by any interaction with the cells, e.g. through the activation of inhibitory signaling pathways. core. preliminary studies showed that the corresponding polyplexes, but also some of the cells that came into contact with them, became magnetic and were manageable by magnetic fields [1] [2] [3] . here, we present a characterization of the influence of structure and composition on the function of these polymers using a library of highly homogeneous, paramagnetic nano-stars with varied arm lengths and densities [4] . the paramagnetic nano-stars library was synthesized by coating maghemite nanoparticles (γ-fe 2 o 3 ) with a thin silica-shell functionalized with an atomic transfer radical polymerization (atrp) initiator. pdmaema arms were grown from the core particles via atrp. in one case, the pdmaema arms was end-capped with pdegma blocks produced during a second atrp step. all nanostars were characterized by size exclusion chromatography and thermogravimetry to calculate number and length of the pdmaema arms. the core diameter was determined by transmission electron microscopy and dynamic light scattering (dls). the different variants (table 1) were analyzed for their ability to complex pdna (pegfp-n1) using various physicochemical methods (dls, zeta sizer). transfection efficiency/cytotoxicity in cho-k1 cells were determined by flow cytometry. transfected cells were placed in a magnetic field and the influence of the polymer architecture on the magnetic separation was investigated. nonparametric spearman analysis was used to correlate between arm length/arm densities, magnetic properties of the cells and transfection efficiency. based on the hydrodynamic radii of the polyplexes, the investigated nano-stars could be divided into three subgroups (table 1) . middle, but also high arm density nano-stars formed smaller polyplexes with hydrodynamic radii ≤ 300 nm, a size that is considered suitable for endocytosis and transfection. transfection efficiencies and cytotoxicities varied systematically with the nano-stars architecture, with viability showing a more pronounced dependency on the characteristics of the transfection agent than the transfection efficiency itself. the arm density was particularly important, with values of approximately 0.06 arms/ nm 2 yielding the best results (fig. 1a) . the end-capping the polycation arms with pdegma significantly improved the serum compatibility (fig. 1b) . the gene delivery potential of a given nano-star and its ability to render the cells magnetic did not correlate. although, compared to the non-separated cells, egfp-expressing cells were consistently more frequent in the magnetic cell fraction, while the non-magnetic fraction was slightly depleted. when the egfp-expressing cells were further divided into low, middle and high producers, a statistically significant shift towards the high producers was observed in the magnetic cell fraction (fig. 1c) . a nonparametric spearman correlation analysis was used to statistically evaluate possible links between the molecular characteristics of the nano-stars, the physicochemical properties of the corresponding polyplexes, the transfection conditions, and the cellular reactions. the resulting correlogram is shown in fig. 1d . transfection agents with magnetic properties enlarge the toolbox for studying non-viral gene delivery, since cellular magnetism is added as a new parameter. this allows, inter alia, a distinction between mere cellular interaction and actual uptake, which is otherwise difficult. viability showed a much more pronounced dependency on the characteristics of the transfection agent/polyplex than the transfection efficiency itself, which should be taken into account during method optimization. end-capping the polycationic pdmaema-arms with pdegma-blocks improved the compatibility of the polycationic nano-stars with serum components. in future optimized, blood-compatible, nano-stars, which can be retained/directed by magnetic fields, could become options for non-viral gene delivery in vivo. the increasing demand for monoclonal antibodies has necessitated the need to increase the productivity of current industrial cell lines. in our earlier study [1] , we had shown that treatment with er-stress inducer, tunicamycin significantly increased the titers and productivity in recombinant cho cell lines with a simultaneous upregulation of many genes from the unfolded protein response pathway (upr). however the loss in cell viability prevented a sustained increase in titers. in the current study we explore the effect of varying concentrations of tunicamycin and treatment times, such as to modulate the increase in protein folding capacity while preventing induction of apoptosis. anti-rhesus igg-secreting cho cells [2] were cultured in sf-cdm in 125 ml shake flasks. the cells were treated with varying concentrations (30-500 ng/ml) of tunicamycin in a batch culture. further, the effect of treatment with tunicamycin for short periods of time (24 hrs) was also evaluated. igg titers and mrna expression levels were quantified using elisa and qrt-pcr (illumina), respectively. results cho cells were treated with different concentrations of tunicamycin and cultured in a batch for 8 days (referred as continuous treatment/cte). figure 1a presents the maximum vcd and % drop in viability under treatment. a dose-dependent inhibitory effect is observed on growth and viability of cells in cte-cultures, with minimal inhibition as lower concentrations. contrastingly, igg titers (fig. 1b) were higher in treated cultures w.r.t. control in initial phase of the cultures at all the concentrations of tunicamycin. the per-cell productivity (fig. 1c) also showed a significant increase w.r.t control at all the concentrations of tunicamycin. however, the increased productivity due to tunicamycin was not sustained and levels become similar to control after day 3 (data not shown). to prevent loss of viability due to tunicamycin, the effect of short-term treatment (ste) with tunicamycin was explored. cells treated with tunicamycin for 24 hours were harvested (corresponding to day 2 of cte cultures) and inoculated in fresh media. the ste-cultures showed improved viability and higher maximum vcd as compared to cte-cultures (fig. 1a) . the fold increase in igg titers was not sustained beyond day 1-2 in stecultures ( fig. 1d ) but significant increase in productivity was seen in the initial phase (fig. 1e) . further, the cells were adapted over 25 continuous generations under 30ng/ml tunicamycin. the adapted cells had overall 1.3-fold higher productivity, as compared to control (fig. 1f) , in a batch culture. to understand the molecular basis of increase in productivity, mrna expression level of key genes was determined. xbp1s is a transcription factor involved in activation of chaperones (like grp78, calreticulin) and apoptotic genes (such as chop). significant increase in the levels of calreticulin was seen on treatment with tunicamycin (fig. 1g) . both xbp1s and grp78 were marginally induced when treated with 30ng/ml of tunicamycin in both cte-and ste-cultures (fig. 1h) , and significantly up-regulated when treated with 500ng/ml of tunicamycin. the chop mrna levels also increase with increasing tunicamycin concentrations, with levels in ste-cultures lower than cte-cultures (fig. 1h) . the results suggest that upr induction may be important to increase productivity in these cte/ste-cultures. note that, tunicamycin had no effect on the expression levels of igg heavy-chain, thus eliminating the involvement of igghc-mrna in increasing productivity (fig. 1i) . tunicamycin induced er-stress increased productivity in the initial phase of the culture and enhanced upr-mediated folding capacity can be attributed as one of the reasons for it. at lower concentrations of tunicamycin, a fine balance between optimum upr induction and apoptosis can be achieved, as seen in 30ng/ml tunicamycin ste-cultures. in summary, this study demonstrates an alternate approach to enhance productivity of current industrial cell lines. background chinese hamster ovary (cho) cells have been widely used for the large-scale production of biopharmaceuticals [1] . to construct antibody-producing cho cells, exogenous genes encoding antibodies are usually integrated into unspecified regions of chromosomes (random integration). however, the chromatin structure differs depending on the location of the chromosomal region, which affects the expression level of the gene of interest [2] . recently, gene-targeting methods that enable site-specific integration of expression vectors have been developed. however, the regions that are most efficient for exogenous gene expression have not been clarified. we previously constructed a cho genomic bacterial artificial chromosome (bac) library generated from the recombinant cho-dg44 cell line. it was expected to cover the entire cho genome five times. the 20 chromosomes in cho-dg44 cells were aligned in decreasing order of size and assigned letters from a to t [3] . three hundred and four bac clones were mapped to every chromosome of cho-dg44. among the karyotypes of cho-dg44, cho-k1 and primary chinese hamster cells, chromosomes a and b are considered as the sole paired chromosomes corresponding to chromosome 1 in primary chinese hamster cells. hence, chromosomes a and b are considered to be stable [4] . in this study, we constructed antibody-producing cells by using a gene-targeting method, which focused on the stable chromosomes. a gene map of chromosome 1 was constructed by combining the bac-fluorescence in situ hybridization (fish)-based chromosome physical map and sequence data of mapped bac clones. the sequences of bac clones were searched by blast with ncbi and chogenome.org databases. three different regions on chromosomes a and b were selected based on cho genomic bac library sequences as target sites. cho-k1 cells were stably transfected by lipofection. the target sequences were broken using the clustered regularly interspaced short palindromic repeats (crispr)/crispr-associated protein 9 (cas9) system and humanized igg1 genes were integrated by non-homologous end joining recombination. transfection without using the crispr/ cas9 system was also performed. these cell pools were cultivated for six days with serum-supplemented medium, and their levels of antibody productivity were evaluated by elisa. copy number analysis was also performed using real-time pcr. results and discussion construction of gene map of chromosome 1: eighty-three bac clones were mapped onto chromosomes a and b (each clone contained 100-150 kb of the cho genome sequence). as a result of annotations of 83 bac clone sequences, 91 genes were mapped on chromosome 1. investigation of the differences of productivity among antibodyproducing cells that were constructed by chromosome 1 targeting and/or random integration: cell growth was not affected by the gene targeting site. the specific production rates of antibodyproducing cell pools constructed by gene targeting of chromosome 1 were higher than those of the cell pool constructed by random integration. all cell pools constructed by gene targeting showed lower copy numbers of heavy chain and light chain in genomic dna than those in the cell pool constructed by random integration, despite showing high productivity. our results indicate that high productivity of the cells constructed by gene targeting of chromosome 1 does not depend on the increase of the antibody copy number, and that the environments around these target regions are suitable for exogenous gene expression. the approach of using gene targeting to chromosome 1 may be promising for constructing antibodyproducing cells. retroviral vectors have been widely used as gene delivery tools in various biotechnology fields. however, the random integration feature of retroviral vectors seems to cause problems such as insertional mutagenesis and gene silencing. we previously demonstrated cre-mediated retroviral transgene insertion into a pre-determined site of the founder cells using integrasedefective retroviral vectors (idrvs), where a cre expression plasmid was transfected into the cells prior to retroviral transduction [1] . recently, we reported novel hybrid idrvs (cre-idrvs) incorporating bioactive cre recombinase protein, and validated site-specific gene integration of an scfv-fc antibody expression unit into the chinese hamster ovary (cho) cell genome [2] . we also developed an accumulative site-specific gene integration system, which enables repeated integration of multiple transgenes into a pre-determined locus of the cell genome [3] . here, we attempted repeated integration of transgenes using cre-idrvs. a viral vector plasmid (pqmscv/hd[scfv-fc]) encoding reporter genes and an scfv-fc expression unit flanked with wild-type and mutant loxps was constructed for the production of idrvs. cre-idrvs were produced described previously [2] . results and discussion figure 1a shows a schematic drawing of each round of targeted transgene integration using cre-idrvs harboring an scfv-fc expression unit. (fig. 1b) . genomic dna extracted from the cells were subjected to pcr using specific primer pairs α and β, and γ and δ to confirm site-specific integration. dna fragments with expected sizes were amplified in each cell clone (fig. 1c) . these results indicate that site-specific repeated integration was achieved using cre-idrvs. in contrast, scfv-fc productivity in cho/hd[scfv-fc]×2 cells was slightly decreased compared with that of cho/ne[scfv-fc]×1 (data not shown). although the reason remains unclear, repeat-induced gene silencing might occur due to tandem repeat structure of expression units. we reported improved recombinant antibody production using a production enhancer element [4] . such a cis-regulatory element might be a feasible approach to enhance the productivity. we demonstrated site-specific repeated transgene integration into a pre-determined chromosomal locus using cre-idrvs for the production of an scfv-fc antibody. if lipids role in the cell have been reduced for a long time to cell membrane formation, it is now understood that lipids plays also a role into energy metabolism, vesicular transport, membrane structure, dynamics and signaling. however, the exact mechanism of how compositional complexity affects cell homeostasis remains unclear. thanks to recent advances in mass spectrometry, it is now possible to study a wide range of lipids, providing a better understanding of lipid homeostasis in high performance cell culture processes. the purpose of this work was to develop a robust lipidomics method applied to mammalian cell cultures in a three step method: extraction, separation and detection (fig. 1 ). both matyash [1] and folch [2] extraction method were performed on our cells to reach the highest yield. two separation techniques were also tested: hydrophilic interaction liquid chromatography (hilic) and reverse phase chromatography. finally lipid classes' identification was achieved by tandem mass spectrometry analysis thanks to structure-specific fragmentation ions. the yield obtained with matyash extraction method was higher than with folch method for each lipid class tested. besides, matyash method presents also the advantage to be less toxic and suitable for high throughput analysis since the organic layer is above the aqueous layer. lipids separation by hilic is based on their polar head. since lipid classes are defined by polar head, the lipids are eluted class by class, making their identification easier. the separation of lipids by reverse phase was correct but the method is longer and we observed a massive carryover of triglycerides on the column. finally each lipid class was screened in ms/ms parent ion mode. target daughter ion was set according to the lipid class structure and fragmentation pattern. this detection technique enabled the identification of 50 different lipids. to ensure the absolute quantification of the detected lipids and to guarantee comparable results between batches labeled internal standard were added prior to extraction. this method was optimized in a stepwise process to ensure a sensitive and selective measurement of the lipids. lipids were extracted by matyash method, separated by hilic and detected by tandem mass spectrometry. this method is suitable for both in process sample lipid analysis providing information on the cell lipid content, and for harvest samples, enabling to follow the lipid release during the different harvest steps. this non-targeted lipidomic quantitation method will enable us to better control lipid synthesis during biopharmaceutical fed batch production through clone selection, metabolomics studies and harvest development. background human mesenchymal stem/stromal cells (hmsc) can easily be isolated from e.g. bone marrow, fat tissue or umbilical cord blood and are therefore a central player in regenerative medicine, gene therapy and cell therapy [1] [2] [3] . the necessary gene shuttle is mainly provided by viruses associated with diseases, like retrovirus or adenovirus [4] [5] [6] [7] . these possible pathogen viruses demand for high safety standards. also, they are prone to genomic alterations and there is the possibility of virus inactivation, triggered due to pre-existing immunity in the patient [8] [9] [10] . in this context, the autographa californica multicapsid nucleopolyhedrovirus (acmnpv) is a safe alternative. the virus replication is hostspecific for insects [11] , but it is known since the mid-90s, that a temporary transduction of mammalian cells is possible [12] . some modifications of the virus increased the applicability in stem cells. pseudotyping the virus with the vesicular stomatitis glycoprotein (vsv-g) led to an expansion of the transducable cell [13, 14] and the integration of the woodchuck hepatitis virus post-transcriptional regulatory element (wpre) prolonged the recombinant protein expression [15, 16] . for achieving a baculovirus-induced differentiation of hmscs, the promotor and the expression strength of the recombinant protein are crucial factors. still, there are still few comparative promotor studies [17, 18] . however, a successful virus uptake is the prerequisite for a successful protein expression. we therefore investigated factors significantly influencing the transduction process by applying design of experiments (s. fig. 1a ). the experimental design comprises a two level factorial screening, set-up using design expert v9.for the transduction 60,000 c/cm 2 were seeded in 24-well plates with dmem + 10% fcs and incubated overnight at 37°c, 8% co 2 and humidified atmosphere. the recombinant baculovirus using an integrated ef1α promoter to control gfp expression, described elsewhere [18] , was diluted to the respective concentrations in the different surrounding fluids. after discarding the cultivation medium of the hmsc-tert, 1 ml of virus containing solution was added to the cells. the following incubation was varied in duration before replacing the virus solution with growth medium and an incubation overnight. 24 h post transduction (hpt) the cells were washed with pbs, trypsinized with 100 μl trypsin/edta and incubated for 5 min at 37°c. trypsination was then stopped applying 100 μl soybean trypsin inhibitor and the cells were analyzed using flow cytometry. as shown in fig. 1a , the virus concentration and incubation time exert the highest influence on the transduction efficiency. obviously, a higher concentration of viral particles and longer incubation of cells with virus increases the probability for hits between cells and virus particles. additionally, the surrounding fluid can have a negative impact on the transduction. this is due to the interaction of medium components with the baculovirus. therefore, pbs containing ca 2+ & mg 2+ is recommended as surrounding fluid for transduction experiments. in fig. 1b , the transduction conditions resulting in the highest percentage of gfp+ cells are displayed: 150 virus particles per cell (ppc) and an incubation time of 5 h with hmsc-tert. the experiments show, that especially the virus concentration and the incubation time of cells with virus influence the transduction efficiency. based on the results of the screening, further optimization of the transduction conditions will be done using a face centered central composite design with pbs containing ca 2+ & mg 2+ as surrounding fluid and at an incubation temperature of 37°c. background breast cancer is the second main cause of cancer related deaths for women worldwide and among them the triple negative subtype (tnbc) represents a clinical challenge by being associated with high mortality and having no effective therapies against it [1] , [2] . accordingly, there is an urgent need to design new and more effective drugs to treat breast cancer. notch signaling is an evolutionary conserved cell-to-cell communication pathway crucial during embryonic and breast development and tissue homeostasis. this pathway is often hyper-activated by overexpression of notch receptors and/or its ligands in several types of cancers, such as breast cancer (tnbc included), where it contributes to its development, progression and drug resistance [3] , [4] , [5] . our aim is to generate a function blocking antibody against the notch delta-like-1 (dll1) ligand with therapeutic efficacy against breast cancer. materials and methods dna of human dll1 full length extracellular domain (dll1-ecd) and a truncated version, containing the minimal binding region to the notch receptor (dll1-egf3), were cloned into pfuse-fc1-igg1, and expressed in hek293e6cells. recombinant proteins were purified from culture media by protein-a affinity and size exclusion chromatography. the human scfv phage display tomlinson i+j library was used to select specific scfv against peptides targeting dll1 binding regions to notch. the binding ability and specificity of the selected scfv clones was evaluated by scfv-on-phage elisa. our strategy allowed us to obtain 20 mg of pure (>95%) and stable dll1-ecd-fc as confirmed by sds page and thermofluor assay. dll1-egf3-fc yield was very low and buffer screenings are ongoing to optimize protein stability. functional studies performed in human breast cancer mcf7 cells showed that both ligands are biologically active as they increased the expression of the notch-dependent genes hes-1, hey-l and hey-1. recombinant dll1 and peptides were used to select for monoclonal antibodies by phage display. after three rounds of panning with dll1 peptides we identified 13 scfv positive clones, 2 of which presented high affinity to dll1-ecd-fc. currently we are performing more phage display selections to increase the number of positive clones. scfv with higher affinities will be reformatted into iggs and their ability to inhibit the notch pathway will be evaluated. the anti-oncogenic effects of anti-dll1 iggs will be assessed in breast cancer cells in viability/apoptosis, proliferation, migration, and invasion assays. an anti-dll1 igg with therapeutic efficacy against breast cancer will demonstrate that targeting dll1 could be one of the key factors for successfully targeting breast cancer. recombinant adeno-associated virus (raav) approaches have an outstanding reputation in gene therapy and are evaluated for cancer therapy [1] . advantages include long-term gene expression, targeting of dividing and non-dividing cells, and low immunogenicity. established raav production utilizes triple transfection of adherent hek 293 cells, which hardly meets product yield requirements for clinical applications. we transferred the aav production system to hek 293-f suspension cells. this process is scalable and uses serum-free media streamlining downstream procedures. after optimization of transfection efficiencies and shaker cultivations, we produced titers of 1×10 5 viral genomes per cell in a 2 l bioreactor. the suspension adapted hek-freestyle 293-f cell line was used for the experiments in chemically defined animal component free media (hek-tf, hek-gm (xell ag), freestyle f17 (thermo fisher scientific)). samples for viable cell density and viabilities were taken daily and analyzed using an automated cell counting system (cedex, roche diagnostics). transient transfection of 3×10 6 cells/ml was carried out with polyethylenimine max in a 1:4 dna-pei ratio (w/w) with 2 μg dna. three plasmids (pgoi, prepcap, phelper) were applied in a molar 1:1:1 ratio (fig. 1a) . pretests were performed in orbital shaking tube spin bioreactors. for scale-up, batch processes were carried out in 125 ml shake flasks as well as in 2 l stirred bioreactors at 30% air saturation and ph 7.1. transfection efficiencies and raav production were quantified by flow cytometry using a goi coding for a fluorescent protein and qpcr of genomic copies, respectively. by optimizing the dna amount for transfection of 293-f cells more than 90 % of the cells were reproducibly transfected. batch cultivations in shaker flasks revealed that raav were produced in the first 24-96 h after transfection. figure 1b shows viable cell densities and viabilities in relation to the genomic titer. genomic titers were determined from raw cell extracts and up to 10 9 copies/ml were repetitively achievable. a decrease in viability marked the decline in genomic copies per ml showing that a prolongation of the process e.g. by addition of a feed would probably not increase yield. in a first scale-up, the raav production was transferred to a 2 l bioreactor (fig. 1c) . transfection efficiencies in bioreactors of up to 55% were comparable to that obtained in a simultaneous shaker flask experiment. transfection efficiencies were lower compared to prior experiments due to controlled conditions in the bioreactor. nonetheless the titer with up to 1×10 5 genomic copies per cell was elevated compared to that of shaker flasks. first experiments with 293-f cells in hek tf medium showed promising results of transferring raav production from the adherent system to suspension. after improvement of transfections by the adjustment of dna amounts in small scale experiments, aav production was analyzed in shaker flasks. the batch process showed an expected increase in cell density with low variability between biological replicates (fig. 1b) . the genomic titer increased according to the viable cell density until day four where a sudden drop started. this observation was made for aav productions in hek-tf, hek-gm and freestyle f17 medium. for optimal yields, we assume that a slight decrease in viability marks the point in time for harvest. from optimized protocols, a batch process in a 2 l bioreactor was carried out. interestingly the bioreactor cultivation resulted in lower overall viable cell densities but in higher genomic copies per cell compared to shaker flasks (fig. 1c) . these results are comparable to already published data for suspension cells [2] . subsequent optimization of the bioreactor protocol will lead to further increase in raav yield. genethon and pall have collaborated to assess pall's single-use icellis fixed-bed bioreactor for viral vector production. clinical use of gene therapies to treat formerly incurable genetic diseases is advancing rapidly. viral vectors are an important tool for introducing genes into target cells. many gene therapies have been developed using adherent cells in 2-dimensional flatware or roller bottles but using these technologies to reach commercial-scale production represents a significant challenge. the icellis bioreactor enables large-scale viral vector production by providing a 3-dimensional matrix for cell growth in a compact configuration (fig. 1 ). up to 500 m 2 of surface area is available in a compact bioreactor measuring 88 mm in diameter in a total volume of 75 l with ph, do and temperature control. a key feature of the icellis bioreactor is that it scales by increasing the diameter of the fixed-bed while keeping the height constant with no change in aspect ratios. the height of the fixed-bed can be varied (2, 4 and 10 cm) as well as density of carrier packing (96 gm/l or 144 gm/l). the icellis system comes in two formats, the icellis nano bioreactor (0.53-4.0 m 2 ) and the icellis 500 bioreactor (66-500 m 2 ). processes developed in the bench top icellis nano bioreactor can be directly transferred to the corresponding icellis 500 system. the icellis nano bioreactor enables an efficient platform for process optimization. the genethon raav-8 process was transferred to an icellis nano bioreactor 0.8 m 2 (2 cm bed height, 144 gm/l density) bioreactor using freestyle media. the initial icellis nano process was established as (1) seed on day 1, (2) transfect at day 5, (3) harvest at day 8 and yielded <1x 10 9 vp/cm 2 (n=3). media exchange, cell density at transfection, pdna/cell ratio, and lysis method were then changed to determine the effect on productivity. the modified process was then scaled from 0.8 m 2 to 4.0 m 2 (10 cm bed height, 144 gm/l density) icellis nano bioreactor. -media: a media exchange at 5 hours post transfection with dmem substituted for freestyle medium resulted in an 8x increase in specific productivity. 1 (abstract p-349) . a schematic overview of raav production in hek293 cells with triple-transfection system. b viable cell densities (vcd), viabilities and genomic copies per ml (gc) of a raav production with 293-f batch cultivations in shaker flasks. genomic copies per ml refer to the titer determined in 1 ml culture volume. error bars represent biological and technical duplicate measurements of samples. c viable cell densities and genomic copies per cell of a raav production with 293-f batch cultivation in a 2 l bioreactor. for reasons of comparability between shaker and bioreactor data genomic copies are given per cell. error bars represent technical duplicate measurements of samples -cell density at transfection: cells were seeded at 6,000 cells/cm 2 and reached 200,000 cells/cm 2 at day 5 which was determined to be the optimal cell density for transfection. -pdna/cell ratio: reducing pdna by 50% had no significant effect on productivity. -lysis: use of trion x-100 at 0.5% with 100 mm nacl at ph 8 resulted in >100% virus recovery compared to sampled carriers. -scaling: specific productivity was maintained as the system was scaled from 0.8 m 2 to 4.0 m 2 . -overall, an average yield of 4x10 13 vg/m 2 was achieved. the icellis technology is being adopted widely for viral vector production. transferring a process to the icellis nano bioreactor can be easily achieved and once in place can be optimized to provide significant productivity increases and cost savings such as reduced pdna. the icellis nano bioreactor is an efficient bench-top system the results of which can be readily scaled to the icellis 500 system. background tissuse multi-organ-chip (moc) platform contributes to the ongoing advancement in systemic substance testing in vitro. current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, often do not accurately predict drug toxicity. especially, cardiotoxicity is one of the main reasons why new compounds are failing in clinical trials. therefore, we aimed to establish an autologous dynamic multiorgan-device integrating cardiomyocytes for substance testing. generic 2d monolayer and 3d suspension ipsc derived cardiomyocytes differentiation protocols were established. beating cardiomyocytes were first seen on day 8 in monolayer as well as in spheroid culture. cardiomyocytes show up to 64% cardiac troponin t positive cells and 44% myosin heavy chain positive cells by flow cytometry (fig. 1g, h) . myosin ii heavy chain, α-actinin, myosin 9/10, myosin 11 and caldesmon expression was shown by immunohistochemistry ( fig. 1a-d) . due to the exclusion of a lactate enrichment of cardiomyocytes, cardiac fibroblasts are also expressed in the spheroids shown by vimentin staining. those cardiac fibroblasts lead to a physiological heterologous cell population similar to the human heart. beating spheroids were cultivated for 7 days under dynamic culture conditions in the multi-organ-chip. the integrated on-chip micropump provides physiological-like pulsatile circulation at a microliter scale and leads to better nutrition and oxygen supply. the next significant step is to combine multiple autologous 3d organ equivalents in our multi-organ-chip using ipsc differentiation technology. differentiating all cell types from one ipsc donor is crucial to overcome source and rejection problems. combining our multi-organ-chip platform with ipsc differentiation technology will eventually lead to a personalized system for drug and substance testing. lab as a service -automated cell-based assays lena schober, moriz walter, andrea traube laboratory automation and biomanufacturing engineering, fraunhofer ipa, stuttgart, germany correspondence: lena schober (lena.schober@ipa.fraunhofer.de) bmc proceedings 2018, 12(suppl 1):p-365 background the use of cell-based assays in pharmaceutical industry and academic research is a growing trend that is a driving force to reduce costs for drug development. academic research is gaining information about intracellular targets or functional mechanisms through the variety of different assays. these benefits can be used in preclinical studies and furthermore costly late-stage drug failures may be reduced by the use of cell-based assays. the use of automated systems is also in great demand and will change the testing of substances and research activities. nevertheless, there are a lot of barriers at the moment limiting the successful application of automated systems in this field. by the lack of flexibility and the demand for skilled computer scientists & engineers just the two main aspects stated by experts shall be mentioned. our strong background on automated cell culture technologies and expertise, gained in several projects, let us rethink the overall process chain and overcome established principles. a new service orientated platform for the execution of cell-based assays that are commonly used will be introduced. the main idea is to give access to automated infrastructure for academic research or spin-offs which cannot afford the special infrastructure. nowadays it is known that the development of inhibitory antibodies by hemophiliac patients is closely related with immunogenic epitopes present in the coagulation factors. these proteins are produced in hamsters cells [1 -4] which insert a different posttranslational modification profile when compared with the human profile. patients with high-titer/high-responding inhibitors must be treated with bypassing agents that can achieve hemostasis. activated factor vii (fviia) is an attractive candidate for hemostasis, independent of fviii/fix, making this coagulation factor an alternative for hemophilia patients with inhibitory antibodies. however recombinant factor vii is produced in bhk-21 cells (baby hamster kidney cells) and as well as the others coagulation factors, it may contain immunogenic epitopes [5 -7] . in this context, becomes extremely important to produce recombinant proteins with complex posttranslational modifications in a cell line not yet used [8 -10] . we have been using the sk-hep-1 human cell line for the production of recombinant fvii. to generate the recombinant cell line we have used a bicistronic lentiviral vector, 1054-gfp, containing a fvii gene and the gfp selection marker gene. a master cell bank and a work cell bank were generated in gmp conditions. the rfvii analyses were made by elisa assay, western blot, gene expression quantification and biological activity using the prothrombin time (pt) assay. rfvii purification by affinity chromatography using viiselect (ge) column. after purification the rfvii was formulated and dry froze to be used in in vivo experiments. in static conditions sk-hep-1 cells showed, for a period of 6 months, a stable fvii production with an average of 8,03 iu/ml of fvii, 83% of cell viability and 77% of cells expressing the gfp gene. after purification with viiselect column it was possible observe a recover of 65% of the purified protein with 95% degree of purity (fig. 1) . this recombinant purified fvii is being used in in vivo experiments to determine the pharmacokinetics parameters and to evaluate the posttranslation modifications profile. in conclusion, this study reports the use of sk-hep-1 cell line for high-level production of recombinant factor vii. these cells have proven to be effective in the production of recombinant protein and can be used as a new platform for the production of recombinant proteins. fig. 1 (abstract p-366) . a determination of protein yield of egfr (epidermal growth factor receptor) synthesized in a cho cecf system. analysis of egfr protein yield obtained in a various batches of cecf formatted reaction. cecf synthesis was performed in the presence of 14 c leucine for radio labeling of target proteins. radio labeled proteins were precipitated using tca followed by scintillation measurement. b detection of radio labeled egfr by autoradiography. a no template control (ntc) was prepared containing no egfr dna template background emergence of stem cell-based regenerative medicine recently leaded to the necessity to reach a sustained production of such cells [1] . hence, new bioreactors and carriers were designed for cell expansion. however, to meet this increasing demand, improvement of both quality and quantity of stem cells remains necessary. soft biocompatible microcarriers mimicking extracellular matrix in term of structure and stiffness should be of valuable utility as substrate stiffness strongly influence in vitro stem cell fate and differentiation [2, 3] . our expertise in the field of microbeads design using jetcutting technology [4] enabled us to engineer +/-200 μm alginate beads of various g/m monomer ratio. we used jetcutter (genialab gmbh) with 100 μm nozzle at max speed 12000 rpm. alginate solutions with concentrations 2% to 4% were gelifyed in 2% cacl2 etoh 50% solution. alginates with estimated viscosity (@1%) from 30 to 720 mpa were tested. a further surface treatment with gelatine (0,1%, 1%) and poly-l-lysine (0,1%) was carried out to reach an optimal cell anchoring of human adiposederived mesenchymal stem cells (atcc-psc-500-011) in mesempro rs medium (gibco). jetcutter technology allowed us to obtain alginate microcarriers with a good homogeneity in size around 200 μm and sphericity comparable to commercial carriers (table 1) . best adhesion of human adipose-derived mesenchymal stem cells was obtained on 0,1% gelatine coated alginate carriers (fig. 1) . we observed limited apoptosis and human adipose-derived mesenchymal cells stemness was conserved after 14 days in culture (data not shown). cellular bioassays developed with functionally immortalized cell lines aileen bleisch 1 , aleksandra velkova 3 , tom wahlicht 2 , dagmar wirth 2 , tobias may 1 1 inscreenex gmbh, braunschweig, germany; 2 msys, helmholtz centre for infection research, braunschweig, germany; 3 greiner bio-one gmbh, frickenhausen, germany bmc proceedings 2018, 12(suppl 1):p-381 background a major challenge of current research is the limited availability of physiologically relevant cells [1] . thus the development of relevant cellular bioassays that are robust, reproducible and scalable is hindered. to overcome current limitations we developed an immortalization strategy allowing the efficient and reproducible establishment of novel cell lines showing an in vivo-like phenotype. the main feature of our ci-screen technology is the ability to combine the advantage of cell linesthe unlimited cell supplywith the advantage of primary cellsthe physiological relevance. using this technology we have immortalized, amongst others, a human osteoblast cell line (ci-huob) [2] . in the present study, the in vivo-like phenotype and functionality of the novel ci-huob was examined. therefore, ci-huob cells were used to develop a 3d cell culture model by using the magnetic 3d bioprinting technology (nano3d biosciences, houston, tx, usa) [3] . the ci-huob cell line was recently described and cultivated in huob maintenance medium (inscreenex, germany). for spheroid creation ci-huobs were grown in a monolayer, magnetized by adding a magnetic nanoparticle assembly (nanoshuttle, ns, nano3d biosciences, houston, tx, usa) at a concentration of 4μl ns/cm 2 growth area. after an overnight incubation magnetized ci-huob were detached and seeded into cellstar® cell-repellent 96-well plates (greiner bio-one, frickenhausen, germany). with the help of mild magnetic forces cells were printed into spheroids within 2h. these consist of 1.000-50.000 cells and were cultured for a period of up to 50 days. the cell viability was analyzed by a propidium iodide (pi) and calcein am staining. to improve spheroid functionality spheroids were cultivated with huob differentiation medium (inscreenex, germany). "mini bone" tissue functionality and thus mineralization was analyzed by an alkaline phosphatase (alkaline phosphatase activity) and an alizarin red s staining (ca 2+ deposits). the combination of ci-huob cells with the magnetic 3d bioprinting technology enabled the establishment of reproducible and consistent 3d spheroids. single spheroids per well were formed independent of the amount of cells (1.000-50.000 cells) (fig. 1a) . formed spheroids were stable for a culture period of up to 50 days (fig. 1b) . neither cell death nor cell proliferation were observed in the bioprinted spheroids which is indicated by the stable size of the spheroids throughout the cultivation (fig. 1c) . after treatment with a differentiation stimulus the 3d bioprinted spheroids became fully functional "mini bones". this was highlighted by the alkaline phosphatase activity and the ca 2+ deposits within the 3d bioprinted spheroids (fig. 1d,e) . taken together, these results demonstrated that the functional immortalization technology provides physiologically relevant cells in sufficient numbers and that the magnetic 3d bioprinting technology enabled a fast, consistent cell aggregation and the formation of stable uniform spheroids. importantly, these immortalized cells are capable to differentiate when a suitable stimulus is provided. for differentiation into mini bones, 3d spheroid cultivation and additional stimulation by small molecules are required. the combination of physiologically relevant cell systems with three dimensional culturing will help to generate in vitro test systems which closely resemble the in vivo physiology and thereby supporting future drug discovery approaches. fig. 1 (abstract p-381) . characterization of spheroid "mini bones". a different number (1.000-50.000 cells) of ci-huob cells were printed into spheroids. b 20.000 ci-huobs were printed into spheroids and cultivated for indicated time points. c for analyzing spheroid sizes, pictures were taken and quantified by imagej. (d/e) 20.000 ci-huob cells were printed into spheroids and cultivated with (huob differentiation medium) or without a differentiation stimulus for two weeks. afterwards, bioprinted spheroids were sectioned by a cryo microtome and d stained for ca 2+ deposits (alizarin red s) or e stained for alkaline phosphatase activity crispr/cas9, a novel genomic tool to knock down microrna in vitro and in vivo degrontagged dcas9/cpf1 effectors for multi-directional drug-inducible control of synthetic gene regulation assessing the variability of an innovator molecule n-glycan profile correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up esi and maldi mass spectrometry techniques 2d-dige screening of high productive cho cells under glucoselimitation -basic changes in the proteome equipment and hints for epigenetic effects dependence on glucose limitation of the pco2 influences on cho cell growth, metabolism and igg production fcgalactosylation modulates antibody-dependent cellular cytotoxicity of therapeutic antibodies fc glycans of therapeutic antibodies as critical quality attributes development of an automated, multiwell plate based screening system for suspension cell culture global cancer statistics remission of disseminated cancer after systemic oncolytic virotherapy screening different host cell lines for the dynamic production of measles virus beitrag zur kollektiven behandlung pharmakologischer reihenversuche a simple method of estimating fifty per cent endpoints webinar: ambr15 as a sedimentation-perfusion model for cultivation characteristics and product quality prediction de novo" high density perfusion medium: increased productivity and reduced perfusion rates a high-yielding cho transient system: co-expression of genes encoding ebna-1 and gs enhances transient protein expression an integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries towards the development of a surface plasmon resonance assay to evaluate the glycosylation pattern of monoclonal antibodies using the extracellular domains of cd16a and cd64 biotinylation of the fc gamma receptor ectodomains by mammalian cell co-transfection: application to the development of a surface plasmon resonance-based assay ambr™ mini-bioreactor as a high-throughput tool for culture process development to accelerate transfer to stainless steel manufacturing scale: comparability study from process performance to product quality attributes maximizing binding capacity for protein a chromatography protein glycosylation and its role in protein folding afucosylated antibodies increase activation of fcγriiia-dependent signaling components to intensify processes promoting adcc sialic acids and other nonulosonic acids production of antibody in insect cells suitability and perspectives on using recombinant insect cells for the production of virus-like particles cloning of cdna and characterization of anti-rnase a monoclonal antibody 3a21 production of functional antibody fab fragment by recombinant insect cells optimization of hek-293s cell cultures for the production of adenoviral vectors in bioreactors using on-line our measurements enhancing heterologous protein expression and secretion in hek293 cells by means of combination of cmv promoter and ifnα2 signal peptide hek293 cell culture media study towards bioprocess optimization: animal derived component free and animal derived component containing platforms comparison of control strategies for fed-batch culture of hybridoma cells based on on-line monitoring of oxygen uptake rate, optical cell density and glucose concentration continuous bioprocessing: the real thing this time? mabs white paper on continuous bioprocessing fda perspective on continuous manufacturing. ifpac annu. meet screening and assessment of performance and molecule quality attributes of industrial cell lines across different fed-batch systems amanullah: quantitative modeling of viable cell density, cell size, intracellular conductivity, and membrane capacitance in batch and fed-batch cho processes using dielectric spectroscopy optimal and consistent protein glycosylation in mammcalian cell culture journal of laboratory automation. designs and concept-reliance of a fully automated high content screening platform mini-bioreactor as a highthroughput tool for culture process development to accelerate transfer to stainless steel manufacturing scale: comparability study from process performance to product quality attributes perfusion media development using cell settling in automated cell culture system model-based design of process strategies for cell culture bioprocesses: state of the art and new perspectives model-based strategy for cell culture seed train layout verified at lab scale hyperosmotic stimulus study discloses benefits in atp supply and reveals mirna/mrna targets to improve recombinant protein production of cho cells effects of osmoprotectant compounds on ncam polysialylation under hyperosmotic stress and elevated pco 2 evaluating the bottlenecks of recombinant igm production in mammalian cells igm characterization directly performed in crude culture supernatants by a new simple electrophoretic method effect of culture ph on erythropoietin production by chinese hamster ovary cells grown in suspension at 32.5 and 37.0°c enhancing protein expression in hek-293 cells by lowering culture temperature relationship between tissue plasminogen activator production and specific growth rate in chinese hamster ovary cells cultured in mannose at low temperature a quantitative proteomic analysis of cellular responses to high glucose media in chinese hamster ovary cells process analytical technologies in the pharmaceutical industry: the fda's pat initiative effects of ammonium and lactate on growth and metabolism of a recombinant chinese hamster ovary cell culture glycosylation in cell culture structural mechanism of high affinity fcgammari recognition of immunoglobulin g inhibition of glycosylation on a camelid antibody uniquely affects its fcγri binding activity investigation of cho secretome: potential way to improve recombinant protein production from bioprocess bladder cancer cell-derived exosomes inhibit tumor cell apoptosis and induce cell proliferation in vitro exploring packaged microvesicle proteome composition of chinese hamster ovary secretome glycome mapping on dna sequencing equipment iron (iii) citrate inhibits polyethylenimine-mediated transient transfection of chinese hamster ovary cells in serum-free medium efficient high-throughput biological process characterization scale-up of a stirred single-use bioreactor family quality control: er-associated degradation: protein quality control and beyond pharmacological targeting of endoplasmic reticulum stress signaling in cancer when er stress reaches a dead end oxidative stress and antioxidant defense the antioxidant edaravone attenuates er-stress-mediated cardiac apoptosis and dysfunction in rats with autoimmune myocarditis advanced process and control strategies for bioreactors. book chapter model-based strategy for cell culture seed train layout verified at lab scale seed train optimization for cell culture. chapter high cell density cultivation of human leukemia t cells (jurkat cells) in semipermeable polyelectrolyte microcapsules. eng. life sci cell retention by encapsulation for the cultivation of jurkat cells in fixed and fluidized bed reactors the role of interleukin-2 during homeostasis and activation of the immune system creating a biomimetic microenvironment for the ex vivo expansion of primary human the era of digital biomanufacturing the multivariate normal distribution recovery from rabies: a call to arms the role of vaccination in rabies prevention eliminating canine rabies, the principal source of human infection: what will it take? rabies virus-like particles expressed in hek293 cells immunogenic virus-like particles continuously expressed in mammalian cells as a veterinary rabies vaccine candidate an avian cell line designed for production of highly attenuated viruses a chemically defined production process for highly attenuated poxviruses a genotype of modified vaccinia ankara (mva) that facilitates replication in suspension cultures in chemically defined medium easy and efficient protocols for working with recombinant vaccinia virus mva large-scale transfection of mammalian cells for the fast production of recombinant protein recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives high density transfection with hek 293 cells allows doubling of transient titers and removes need for a priori dna complex formation with pei regulation of recombinant protein expression during chobri/rcta pool generation increases productivity and stability qc, canada; 2 department of microbiology, infectiology and immunology the cumate gene-switch: a system for regulated expression in mammalian cells rapid protein production from stable cho cell pools using plasmid vector and the cumate gene-switch christoph zehe sartorius stedim cellca gmbh, 88471 laupheim animal cell technology: from target to market recent advances in mammalian protein production molecular mechanisms of rna interference. annual review of biophysics ribosome profiling-guided depletion of an mrna increases cell growth rate and protein secretion ubiquitous chromatin-opening elements (ucoes): applications in biomanufacturing and gene therapy the art of cho cell engineering: a comprehensive retrospect and future perspectives a novel bxb1 integrase rmce system for high fidelity site-specific integration of mab expression cassette in cho cells high-troughput lipidomic and transcriptomic analysis to compare sp2/0, cho, and hek-293 mammalian cell lines single cell characterisation of chinese hamster ovary (cho) cells eva pekle 1,2 , guglielmo rosignoli 1 generation of stable chinese hamster ovary pools yielding antibody titers of up to 7.6 g/l using the piggybac transposon system comparison of three transposons for the generation of highly productive recombinant cho cell pools and cell lines effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production lactate and glucose concomitant consumption as a self-regulated ph detoxification mechanism in hek293 cell cultures flux balance analysis of cho cells before and after a metabolic switch from lactate production to consumption reducing recon 2 for steady-state flux analysis of hek cell culture trastuzumab -mechanism of action and use in clinical practice hek293 cell culture media study towards bioprocess optimization: animal derived component free and animal derived component containing platforms enhancing heterologous protein expression and secretion in hek293 cells by means of combination of cmv promoter and ifnα2 signal peptide production of lentiviral vectors references 1. wurm f m: production of recombinant protein therapeutics in cultivated mamammalian cells initial identification of low temperature and culture stage induction of mirna expression in suspension cho-k1 cells small indels induced by crispr/cas9 in the 5' region of microrna lead to its depletion and drosha processing retardance production of recombinant protein therapeutics in cultivated mammalian cells improved antibody production in chinese hamster ovary cells by atf4 overexpression the vesicle-trafficking protein munc18b increases the secretory capacity of mammalian cells rapid evaluation of n-glycosylation status of antibodies with chemiluminescent lectin-binding assay intracellular secretion pathway analysis for constructing highly producible engineered cho cells. 16th annual peptalk intracellular secretion analysis of therapeutic antibodies in engineered high-producible cho cells analysis of intracellular recombinant igg secretion in engineered cho cells. the 29th annual and international meeting of the japanese association for animal cell technology humanization strategies for an anti-idiotypic antibody mimicking hiv-i gp41 antibody humanization by molecular dynamics simulations-in-silico guided selection of critical backmutations maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification the perseus computational platform for comprehensive analysis of (prote)omics data hoffrogge: label-free protein quantification of sodium butyrate treated cho cells by esi-uhr-tof-ms the art of cho cell engineering: a comprehensive retrospect and future perspectives gs system for increased expression of difficult-to-express proteins the netherlands correspondence: maurice van der heijden (heijden@proteonic.nl) bmc proceedings increased recombinant protein production owing to expanded opportunities for vector integration in high chromosome number chinese hamster ovary cells ires-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems cell-free protein expression based on extracts from cho cells comparison of cell-based vs. cell-free mammalian systems for the production of a recombinant human bone morphogenic growth factor. eng dual-responsive magnetic core-shell nanoparticles for nonviral gene delivery and cell separation pdmaema-grafted core-shell-corona particles for nonviral gene delivery and magnetic cell separation influence of polyplex formation on the performance of starshaped polycationic transfection agents for mammalian cells systematic study of a library of pdmaema-based, superparamagnetic nano-stars for the transfection of cho-k1 cells systems biology of unfolded protein response in recombinant cho cells dynamics of unfolded protein response in recombinant cho cells cells were transfected with np@(pdmaema 1037 ) 46 (n/p 15), separated 24 h post transfection (t = 0) by magnetically-assisted cell sorting and placed into separated cultures. the bars represent the overall transfection efficiency. distribution of low (light green), middle (green), and high (dark green) producers within the egfp-expressing cell fraction. data represent one experiment carried out in duplicate, with random experimental error shown. d correlogram between the molecular characteristics of the nano-stars (core diameter, arm density, arm length, number of monomeric units per nano-star), the physicochemical properties of the corresponding polyplexes (hydrodynamic radius, zeta potential), the transfection conditions (n/p ratio, amount of polymer), and the cellular reactions (transfection efficiency, magnetism, viability) production of recombinant protein therapeutics in cultivated mammalian cells position effects on eukaryotic gene expression bacterial artificial chromosome library for genome-wide analysis of chinese hamster ovary cells construction of bac-based physical map and analysis of chromosome rearrangement in chinese hamster ovary cell lines suguru imanishi 1 , akira ito 1 , masamichi kamihira 1,2 1 department of chemical engineering cre recombinase-mediated sitespecific modification of a cellular genome using an integrasedefective retroviral vector targeted transgene insertion into the cho cell genome using cre recombinase-incorporating integrase-defective retroviral vectors an accumulative site-specific gene integration system using cre recombinase-mediated cassette exchange improved recombinant antibody production by cho cells using a production enhancer dna element with repeated transgene integration at a predetermined chromosomal site lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics a simple method for the isolation and purification of total lipids from animal tissues using baculovirus as a gene shuttle in hmsc: optimization of transduction efficacy gundula sprick 1 clinical applications of mesenchymal stem cells concise review: mesenchymal stem cell treatment of the complications of diabetes mellitus wharton's jelly-derived mesenchymal stromal cells as a promising cellular therapeutic strategy for the management of graft-versus-host disease gene therapy: twenty-first century medicine state-of-the-art gene-based therapies: the road ahead viral vectors: a look back and ahead on gene transfer technology basic biology of adeno-associated virus (aav) vectors used in gene therapy biosafety challenges for use of lentiviral vectors in gene therapy adenoviral vector-mediated gene therapy for gliomas: coming of age manufacturing of viral vectors for gene therapy: part i. upstream processing the complete dna sequence of autographa californica nuclear polyhedrosis virus efficient gene transfer into human hepatocytes by baculovirus vectors efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus g glycoprotein recombinant baculoviruses as mammalian cell gene-delivery vectors post-transcriptional regulatory element boosts baculovirusmediated gene expression in vertebrate cells baculoviral vector-mediated transient and stable transgene expression in human embryonic stem cells systematic comparison of constitutive promoters and the doxycycline-inducible promoter baculovirus-induced recombinant protein expression in human mesenchymal stromal stem cells: a promoter study triple-negative breast cancer: an unmet medical need the therapeutic monoclonal antibody market. mabs a monoclonal antibody against human notch1 ligand-binding domain depletes subpopulation of putative breast cancer stem-like cells notch activation stimulates migration of breast cancer cells and promotes tumor growth notch-out for breast cancer therapies aav production in suspension: evaluation of different cell culture media and scale-up potential modular adeno-associated virus (raav) vectors used for cellular virusdirected enzyme prodrug therapy production of recombinant adenoassociated virus vectors using suspension hek293 cells and continuous harvest of vector from the culture media for gmp fix and flt1 clinical vector development of a cost-efficient scalable production process for raav-8 based gene therapy by transfection of hek-293 cells simon arias 2 , mustapha hohoud 1 , roel lievrouw 1 , fabien moncaubeig 1 b-1120 brussels, belgium; 2 généthon, rue de l'internationale 1 cell-free systems based on cho cell lysates: optimization strategies, synthesis of "difficult-to-express" proteins and future perspectives cell-free protein expression based on extracts from cho cells comparison of cell-based vs. cell-free mammalian systems for the production of a recombinant human bone morphogenic growth factor ires-mediated translation of membrane proteins and glycoproteins in production of recombinant factor vii in sk-hep-1 human cell line zip 14049-900, brazil; 3 department of clinical, toxicological and food science analysis, faculty of pharmaceutical sciences of ribeirão preto human cell lines for the production of recombinant proteins: on the horizon production of recombinant protein therapeutics in cultivated mammalian cells recombinant protein therapeutics from cho cells -20 years and counting establishment of a cell line expressing recombinant factor vii and its subsequent conversion to active form fviia through hepsin by genetic engineering method expression and fast preparation of biologically active recombinant human coagulation factor vii in cho-k1 cells implications of the presence of n-glycolylneuraminic acid in recombinant therapeutic glycoproteins uniquely human evolution of sialic acid genetics and biology production platforms for biotherapeutic glycoproteins. occurrence, impact, and challenges of non-human sialylation human cells: new platform for recombinant therapeutic protein production therapeutic glycoprotein production in mammalian cells masthering industrialization of cell therapy products tissue cells feel and respond to the stiffness of their substrate matrix elasticity directs stem cell lineage specification continuous cider fermentation with co-immobilized yeast and leuconostoc oenos cells eternity and functionality -rational access to physiologically relevant cell lines generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies biocompatibility of nanoshuttletm and the magnetic field in magnetic 3d bioprinting publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal • we provide round the clock customer support • convenient online submission • thorough peer review • inclusion in pubmed and all major indexing services • maximum visibility for your research submit your manuscript at www submit your next manuscript to biomed central and we will help you at every step authors thankfully acknowledge the biotechnology and biological sciences research council for funding this research work. sns thanks esact 2017 for providing her with the opportunity to present her work at the meeting. we would like to thank moritz frei for his support for the generation of the ngs transcriptomics data. many thanks to valentine chevallier for her precious advices, to stefanos grammatikos for his support and to the whole upstream process sciences team. we thank david bruehlmann and thomas vuillemin from merck (vevey, switzerland) for providing the igg glycan variants and the 2-ab-uplc glycan data. polyplus-transfection would like to thank généthon for their kindly provided data.acknowledgements asmita mukerji, reetesh pm, sasi kumar k, pilot plant team acknowledgment to cedric allier from cea leti, grenoble, france. we would like to thank a. schemel and a. ehrlich for technical assistance. the authors would like to mention that this research was supported by the fi-dgr (2017) from spanish government and the project was led by prof. jordi joan cairó badillo. the authors want to thank the biotech process sciences team at merck in corsier-sur-vevey for their support and also the members of the morbidelli group at eth zürich for their input and collaboration. authors would like to thank dr. benjamin youn in manufacturing science and technology (msat) at biomarin for his help on coding the excel macro program for ambr15, and dr. donald l. traul from tap biosystems (now part of the sartorius stedim biotech group) for his assistance on ambr15 operations. thanks to the bioreactor team of dustin davis, amer al-lozi, and jana mahadevan. we organic vaccines tm and the nih, who kindly provided to univercells. we thank polymun scientific immunbiologische forschung gmbh for providing the antibodies igm103, igm104 and igm617 as a kind gift. this allison kurz and gian andrea signorell, genedata ag, basel, switzerland allison kurz, gian andrea signorell, genedata, basel, switzerland. allison kurz, gian andrea signorell, genedata ag high glucose concentration and low specific cell growth rate improve specific r-tpa productivity in chemostat culture of cho acknowledgements r. boraston for photographs in fig. 1 . we acknowledge atum for their contributions to vector design and construction. austrian bmwfw, bmvit, sfg, standortagentur tirol, government of lower austria and business agency vienna through the austrian ffg-comet-k2. this research is supported by sfi grant 13/ia/1841. financial support of the austrian science fund (fwf; grant number p 25056) is gratefully acknowledged. we would like to thank the australian institute for bioengineering and nanotechnology, university of queensland-brisbane, australia (aibn) for providing the cho clones. this research is partially supported by the developing key technologies for discovering and manufacturing pharmaceuticals used for next-generation treatments and diagnoses both from meti and amed, japan. this work was supported in part by grants for developing key technologies for discovering and manufacturing pharmaceuticals used for next-generation treatment and diagnoses, both from the ministry of economy, trade and industry (meti), japan and from the japan agency for medical research and developments (amed). many thanks stefanos grammatikos for his support and to the whole upstream process sciences team. the authors thank the hessen state ministry of higher education, research and the arts within the hessen initiative for scientific and economic excellence (loewe program) for the financial support. the authors thank xell ag, bielefeld, for providing hek serum-free media (hek gm and hek tf) and for fruitful discussions. the authors would like to thank dana wenzel for cho lysate preparation (fraunhofer izi, potsdam-golm, germany). this work is supported by the european regional development fund (efre) and the german ministry of education and research (bmbf, no. 031b0078a). the authors acknowledge são paulo research foundation -fapesp (2015/ 19017-6), centro de pesquisa, inovação e difusão (cepid), and national institute of science and technology in stem cell and cell therapy -inctc for financial support. this work was supported by grants from the niedersächsisches ministerium für wissenschaft und kultur (80029155) and the german ministry for economic affairs and energy (igf 16153 n). the infrastructure which was modularly built up, consists of automated liquid handling robots, plate and tube handling robots as well as incubators, refrigerator and analysis systems as for example an imaging system. the aim is to address the need on reproducibility and reliability of results and to offer access to a maximal controlled and automated environment. with the help of a web-based configurator assay selection as well as parameterization of the assays can be done in an easy way. after the order process, test items can be shipped to the lab. assays will be executed on the fully automated platform. by capturing in process data as well as environmental conditions, a real complete data set is leading to comprehensively results. as soon as results are available during the process, the view and analysing can be done in a secure cloud. the service can be used for single experiments in low throughput applications and is therefore a benefit for labs which cannot afford automated infrastructure or the staff for the maintenance for such platforms. extensive monitoring and data capturing during the run leads to a gapless data trail and the possibility of detailed result analysis. due to automated processing the reproducibility is increased associated with direct reduction of costs and time. the centralized service paired with specific know-how allows up-scaling of processes at any time. the web-based interface provides a flexible guidance for the user and the online order gives 24/7 access on the infrastructure, leading to a fast reliable result generation. furthermore the secure interaction with additional services e.g. other specific data analysis tool is possible. this dynamic access to automation offers high flexibility for low throughput experiments and will push high quality research and drug development in early stage. development of alternative animal cell technology platforms: cho based cell-free protein synthesis systems for the production of "difficult-to-express" proteins lena thoring 1, 2 background nowadays, animal cell technologies are commonly used for a broad range of medical and pharmaceutical applications. one main topic of these technologies is the production of proteins used for therapeutical purposes. these in vivo production processes are often time consuming and limited in production of so called "difficult-to-express" proteins including the pharmaceutical relevant class of membrane proteins. to overcome these issues, novel cell-free protein synthesis platforms were developed based on the industrial working horse cho cells [1] . cell lysates provide a basis for this technology by including all components of the translational machinery and enabling protein production within a few hours. microsomal structures present in cho cell lysates enable posttranslational modification of target proteins and insertion of membrane proteins into lipid bilayer. in this study a cell-free protein synthesis platform was developed based on a combination of cho cell lysates and a continuous exchange reaction format. the continuous exchange reactor consists of a twochamber system, a reaction and a feeding chamber, separated by a semipermeable membrane. due to concentration gradients, energy components can diffuse to the reaction chamber, while inhibitory byproducts are continuously removed. different classes of proteins were selected to evaluate the quality of the cho cecf system including a transmembrane receptor, a single chain variable fragment and an ion channel. cell-free protein synthesis was performed in the presence of 14 c leucine for radio labeling of synthesized proteins. protein yield was quantified by tca precipitation of radio labeled proteins followed by scintillation measurement and molecular mass was detected by autoradiography. posttranslational modifications and activities of proteins were estimated by kinase assays, elisa, endoglycosidase treatment and electrophysiological measurements. the demonstrated results showed a protein production of up to around 1 g/l while detecting correct molecular weights by autoradiography. analysis of the productivity using different lysate batches by the production of the membrane protein egfr revealed only minimal batch-to-batch variations (fig. 1a) . posttranslational modifications of proteins, including phosphorylation and glycosylation, were detected using western blot and autoradiography (fig. 1b) . evaluation of localization of membrane embedded eyfp fusion proteins by confocal laser scanning microscopy resulted in the detection of proteins in the microsomal fraction of cho cell lysate. produced single chain variable fragments showed binding specificity in elisa experiments. the activity of synthesized ion channels was underlined by electrophysiological measurements and detected single channel activities. a cell-free system based on cho cell lysates for high yield production of proteins was developed that provides a platform for efficient production of "difficult-to-express" proteins. the combination of a cho lysate based cell-free system and a continuous exchange cell-free system leads to be a highly efficient production system for various classes of "difficult-to-express" proteins. this approach opens up a fast and cost-effective process pipeline for the production of "difficult-to-express" proteins and shows a high potential for industrial applications including screening technologies, protein structure determination and just-in-time protein production processes. key: cord-011602-hzqayt3n authors: chen, jianlin; liu, xiaorong; chen, jianhan title: targeting intrinsically disordered proteins through dynamic interactions date: 2020-05-11 journal: biomolecules doi: 10.3390/biom10050743 sha: doc_id: 11602 cord_uid: hzqayt3n intrinsically disordered proteins (idps) are over-represented in major disease pathways and have attracted significant interest in understanding if and how they may be targeted using small molecules for therapeutic purposes. while most existing studies have focused on extending the traditional structure-centric drug design strategies and emphasized exploring pre-existing structure features of idps for specific binding, several examples have also emerged to suggest that small molecules could achieve specificity in binding idps and affect their function through dynamic and transient interactions. these dynamic interactions can modulate the disordered conformational ensemble and often lead to modest compaction to shield functionally important interaction sites. much work remains to be done on further elucidation of the molecular basis of the dynamic small molecule–idp interaction and determining how it can be exploited for targeting idps in practice. these efforts will rely critically on an integrated experimental and computational framework for disordered protein ensemble characterization. in particular, exciting advances have been made in recent years in enhanced sampling techniques, graphic processing unit (gpu)-computing, and protein force field optimization, which have now allowed rigorous physics-based atomistic simulations to generate reliable structure ensembles for nontrivial idps of modest sizes. such de novo atomistic simulations will play crucial roles in exploring the exciting opportunity of targeting idps through dynamic interactions. proteins are central components of regulatory networks that dictate virtually all aspects of cellular decision-making [1] . demand for more sophisticated signaling in complex multicellular organisms has been met with increasing utilization of proteins that are highly flexible [2] [3] [4] . in particular, so-called intrinsically disordered proteins (idps) account for~50% of signaling-associated proteins in eukaryotes [5] . these proteins have lower sequence complexity compared to folded proteins, lacking large hydrophobic residues and enriched with charged and polar ones [6] . they do not have stable tertiary structures in the unbound state under physiological conditions, even though they frequently undergo folding transitions upon binding to specific targets [7] . the inherent thermodynamic instability of the structural features of this class of proteins allows their conformational properties to respond sensitively to numerous stimuli, including the binding of various small and large molecules, changes in cellular environments (e.g., ph), and post-translational modifications [8] [9] [10] [11] [12] [13] . multiple signals could also be naturally integrated through cooperative responses of the dynamic structure ensemble biomolecules 2020, 10, 743 2 of 16 (such as coupled binding and folding) [14] . these properties make idps uniquely suitable for fulfilling the complex signaling need of higher organisms. at the same time, deregulation of idps has been associated with many human diseases, including cancers, neurodegenerative diseases, heart disease, and diabetes [5, [15] [16] [17] [18] [19] [20] . for example, over two-thirds of cancer-associated proteins have been predicted to contain extensive regions of intrinsic disorder [5] , and predicted disordered regions have been estimated to house almost one quarter of disease-associated missense mutations [21] . there is thus tremendous interest in determining if and how idps may be targeted for therapeutic purposes. the dynamic and heterogeneous nature of unbound idps presents substantial challenges for characterization and this has proven to be a major bottleneck for establishing a reliable sequence-structure-function-disease relationship of idps [14, [22] [23] [24] [25] [26] . the lack of a clear understanding of the molecular basis of idp function and deregulation in diseases has created significant ambiguity on the druggability of most idps, including transcription factors [16] . most existing case studies of targeting idps have focused on extending the traditional structure-based screening and drug design strategies and emphasize exploiting residual structures and pre-existing potential binding pockets of the unbound state [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] . nonetheless, it is clear that the disordered nature of idps would require novel strategies for targeting as well as new conceptual frameworks for thinking about how small molecule binding could modulate idp structure and function. in particular, it has been recognized that it may be more useful to consider the problem of targeting idps in the context of structural ensemble modulation [44] , even though it is generally believed that one still needs to achieve specific interactions, such as by exploiting pre-existing structural features [45] . many outstanding reviews have already been dedicated towards existing examples along these lines and they also provide extensive discussion of the successes, opportunities, and challenges of targeting idps via specific interactions of small molecules in neurodegenerative diseases, cancers, and other diseases [18, [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] . in this review, we will first summarize important recent advances in physics-based de novo simulations of disordered protein ensembles, including graphic processing unit (gpu) computing, enhanced sampling, and re-balanced protein force fields, and then focus on emerging examples that suggest the exciting possibility of targeting idps by directly modulating the disordered ensembles through dynamic and transient interactions. we will discuss the promise of such a broader view of how idps may be targeted as well as key challenges and required methodological developments to support targeting idps via dynamic interactions. a principal challenge in understanding the druggability and best targeting strategy of idps resides in the difficulty of detailed characterization of disordered protein states [14, 23, 24, 56] . these states need to be represented using heterogeneous structure ensembles and are not amenable to traditional high-resolution structure determination methods. here, we briefly discuss the current status and challenges of disordered protein ensemble determination, which has a direct impact on the ability to devise effective strategies of designing idp binders and optimizing leads identified from traditional screening efforts. experimentally, a wide range of biophysical methods can be applied to characterize disordered protein states, including nmr, circular dichroism (cd), small-angle x-ray scattering (saxs), förster resonance energy transfer (fret), hydrogen/deuterium (h/d) exchange, mass spectrometry, and others [57, 58] . these methods can provide complementary information on the local, intermediate, and long-range structural organizations of idps. nmr in particular is arguably the most powerful technique for structural studies of idp. many nmr observables can be measured at residue and atomic levels to infer secondary and tertiary structural properties. saxs and fret are highly complementary to nmr and provide information on the long-range global organization of the disordered ensemble. yet, a fundamental limitation is that these experimental measurements generally reflects the average properties, which alone are not sufficient to uniquely define the underlying heterogeneous ensemble due to the severely underdetermined nature of the structural calculation problem [22, 23, 25, [59] [60] [61] . at present, the most robust methods generally involve first generating a large number of candidate random structures and then using experimental structural restraints to select and construct optimal sub-ensembles according to various statistical criteria [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] . nonetheless, these methods rely critically on the ability to generate initial candidate structures that are not only diverse enough to cover the range of accessible states of the protein but also specific enough to contain any nontrivial local (and long-range) structure features associated with a particular protein state. these two requirements are difficult to satisfy simultaneously for most proteins of moderate sizes (e.g., 50-100 residues or longer) and complexity. as a consequence, disordered ensembles constructed using these approaches depend critically on the underlying protein model and/or coil library. furthermore, these ensembles are generally not proper thermodynamic ensembles. they should be considered just as ensemble models and cannot be used to reliably quantify statistical properties and extract thermodynamic parameters. as such, these experimental restraint-based ensemble construction approaches are likely inadequate for capturing potentially subtle effects of ligand binding on the disordered ensemble. given the fundamental challenges of disordered ensemble modeling based on experimental restraints alone, physics-based atomistic simulations have a crucial role to play in helping elucidate the conformational properties of idps and establishing a reliable molecular basis of their function and regulation [72] [73] [74] [75] [76] . a particularly attractive approach is to first generate the atomistic ensemble using a transferable physics-based force field in absence of any experimental restraints and then use the experimental data for independent evaluation of the quality of the simulated ensemble. such a de novo simulation approach can effectively overcome the under-determined nature of disordered ensemble calculation, by leveraging the laws of physics that govern the nature of conformational fluctuation of the protein. successful simulations of disordered protein ensembles have proven to be very challenging, requiring both accurate description of the conformational dependence of energy and sufficient sampling of relevant conformational space of the protein. early idp simulations suffered from both systematic biases in the general-purpose protein force fields and severe limitations in conformational sampling [14] . nonetheless, these physics-based simulation approaches promise to provide rigorous thermodynamic ensembles required for reliable description of idp-ligand interactions. the accuracy and capability of de novo idp simulations can be expected to improve continuously over time, benefiting from robust advances in molecular simulation methodologies and high-performance computing hardware. indeed, important breakthroughs have been made in the last few years in force field accuracy and sampling capability, which arguably have now allowed reliable de novo simulations of at least moderate-sized idps in general. one of the most important recent advances in molecular dynamics (md) simulations is the widespread availability of efficient gpu-enabled algorithms in virtually all major molecular simulation packages [77] [78] [79] [80] [81] [82] . modern gpus can process thousands of threads in parallel to accelerate explicit solvent md simulations by up to 100× compared to traditional cpu computing, significantly boosting the sampling capability. for example, the most efficient gpu-enabled md codes can yield 100-200 ns per day for systems of~1,000,000 atoms on a single nvidia rtx 2080ti gpu card that costs onlỹ $1000. the ability to efficiently sample the protein conformational space has further benefitted from the emergence of enhanced sampling techniques [83] [84] [85] [86] [87] [88] [89] [90] , particularly various replica exchange (rex)-based methods. among the various rex methods, replica exchange with solute tempering methods (rest) [86, 91] is particularly suitable for atomistic simulations of idps in explicit solvent. in rest, a selected region of the system (e.g., the protein solute or a flexible segment of the protein) is subjected to tempering (i.e., random walk in the temperature space) while the rest of the system is maintained at a constant temperature (e.g., room temperature). this is achieved by scaling the interactions within the selected region and between it and the rest of the system. because the number of required replicas of replica exchange simulations scales as the square root of the number of atoms, rest can significantly reduce the number of replicas required for covering the needed temperature space (by~3-fold), thus overcoming a critical drawback in traditional temperature rex simulation in explicit solvent. the role of enhanced sampling in the recent successes of simulating dynamic idp-ligand interactions will become evident in the later sections of this review. the importance of enhanced sampling for the generation of converged ensembles cannot be over emphasized. for example, figure 1a shows the evolution of the per-residue β-sheet structure during a 30-µs conventional md simulation of an intrinsically disordered aβ40 peptide in explicit solvent at 300 k, performed with anton specialized hardware using the well optimized a99sb-disp force field [92] . note that this trajectory is one to two orders of magnitude longer than typical md simulations performed using general purpose cpu-or gpu-based high-performance computing platforms. yet, very few reversible transitions are observed. for example, a transient β-hairpin spanning residues 15 to 36 persists for about 5 µs from~10 to 15 µs and never appears again for the rest of the simulation. as a result, the final average residue helix and β-sheet probability profiles calculated from the first and second halves of the trajectory differ greatly, reflecting a very limited level of convergence. there is thus danger in relying on standard md simulations in deriving quantitative characterizations of disordered protein ensembles. it should also be emphasized that achieving a sufficient level of convergence required for resolving potentially subtle changes in the disordered ensemble, such as upon ligand binding, can be extremely challenging even with enhanced sampling. it is critical to carefully analyze and establish the level of convergence for proper interpretation of simulated ensembles. ideally, one should perform two or more independent simulations using distinct initial conformations and compare the resulting ensembles. the simulated ensemble from a single continuous run may appear to stop changing with respect to simulation time due to trapping in numerous local energy minima, giving rise to a misleading impression of convergence. biomolecules 2020, 10, x 4 of 15 temperature space (by ~3-fold), thus overcoming a critical drawback in traditional temperature rex simulation in explicit solvent. the role of enhanced sampling in the recent successes of simulating dynamic idp-ligand interactions will become evident in the later sections of this review. the importance of enhanced sampling for the generation of converged ensembles cannot be over emphasized. for example, figure 1a shows the evolution of the per-residue β-sheet structure during a 30-μs conventional md simulation of an intrinsically disordered aβ40 peptide in explicit solvent at 300 k, performed with anton specialized hardware using the well optimized a99sb-disp force field [92] . note that this trajectory is one to two orders of magnitude longer than typical md simulations performed using general purpose cpu-or gpu-based high-performance computing platforms. yet, very few reversible transitions are observed. for example, a transient β-hairpin spanning residues 15 to 36 persists for about 5 μs from ~10 to 15 μs and never appears again for the rest of the simulation. as a result, the final average residue helix and β-sheet probability profiles calculated from the first and second halves of the trajectory differ greatly, reflecting a very limited level of convergence. there is thus danger in relying on standard md simulations in deriving quantitative characterizations of disordered protein ensembles. it should also be emphasized that achieving a sufficient level of convergence required for resolving potentially subtle changes in the disordered ensemble, such as upon ligand binding, can be extremely challenging even with enhanced sampling. it is critical to carefully analyze and establish the level of convergence for proper interpretation of simulated ensembles. ideally, one should perform two or more independent simulations using distinct initial conformations and compare the resulting ensembles. the simulated ensemble from a single continuous run may appear to stop changing with respect to simulation time due to trapping in numerous local energy minima, giving rise to a misleading impression of convergence. the dramatically improved sampling capability has facilitated extensive efforts to reparametrize general-purpose protein force fields to achieve greater balance of describing protein conformational equilibria. studies of disordered protein states have been a key driver of these developments and several well-characterized idps have been widely used as training systems and/or benchmarks for force field optimization [92] [93] [94] [95] [96] [97] [98] [99] . many force field variants have been developed in recent years, including amber ff98sb [100] , ff99sb*-ildn [101] and variants [95, 102] , ff03ws [103] , ff14sb [104] , ff99sbnmr [105] , charmm22* [106] , charmm36m (and c36mw) [93] , a99sb-disp [92] , and others. a key focus of these optimization efforts has been to rebalance the protein-protein, protein-water, and water-water interactions. earlier versions of general-purpose protein force fields consistently over-stabilize nonspecific protein-protein interactions and lead to overly compact conformational ensembles for disordered protein states [107, 108] . it was demonstrated that such bias could be effectively compensated by directly increasing the strengths of protein-water dispersion interactions [95, 103, 109] , even though other components of the force field should also be reparametrized for self-consistency. the latest charmm36m and a99sb-disp force fields, in particular, have been systematically reparametrized based on extensive simulations of tens of globular and disordered proteins and achieve impressive levels of accuracy for describing both structured and unstructured proteins. in a recent benchmark study, six of the latest protein force fields were evaluated using the 61-residue n-terminal transactivation domain (tad) of tumor suppressor p53, which is a very challenging system due to its size and complex conformational features [99] . it has been extensively characterized by nmr, saxs, and single-molecule fret and shown to contain a range of nontrivial local and long-range residual structures [110] . the disordered ensemble of p53-tad was calculated using rest2-enhanced sampling using gpu-accelerated gromacs 5.1.4 [80, 111] patched with plumed 2.3.0 [112] [113] [114] . each rest2 simulation lasted 1.0 µs per replica, representing one of the most extensive atomistic simulations of idps of similar sizes. the results show that the ensembles generated using the force field a99sb-disp yield the best agreement with the experimental data at both secondary and tertiary structure levels. for example, the back-calculated nmr paramagnetic relaxation enhancement (pre) effects are highly consistent with the experimental results for all four available labelling sites (figure 2 ). this suggests that the simulated ensembles not only have the proper overall chain dimension but also recapitulate much of the transient long-range ordering within the unbound state of p53-tad. the latter is an extremely challenging task. the fact that this could be achieved by a99sb-disp represents an exciting breakthrough, suggesting that de novo atomistic simulations are now ready to provide a reliable approach for detailed characterization of the disordered ensembles of at least moderately sized idps. the dramatically improved sampling capability has facilitated extensive efforts to reparametrize general-purpose protein force fields to achieve greater balance of describing protein conformational equilibria. studies of disordered protein states have been a key driver of these developments and several well-characterized idps have been widely used as training systems and/or benchmarks for force field optimization [92] [93] [94] [95] [96] [97] [98] [99] . many force field variants have been developed in recent years, including amber ff98sb [100] , ff99sb*-ildn [101] and variants [95, 102] , ff03ws [103] , ff14sb [104] , ff99sbnmr [105] , charmm22* [106] , charmm36m (and c36mw) [93] , a99sb-disp [92] , and others. a key focus of these optimization efforts has been to rebalance the protein-protein, protein-water, and water-water interactions. earlier versions of general-purpose protein force fields consistently over-stabilize nonspecific protein-protein interactions and lead to overly compact conformational ensembles for disordered protein states [107, 108] . it was demonstrated that such bias could be effectively compensated by directly increasing the strengths of protein-water dispersion interactions [95, 103, 109] , even though other components of the force field should also be reparametrized for selfconsistency. the latest charmm36m and a99sb-disp force fields, in particular, have been systematically reparametrized based on extensive simulations of tens of globular and disordered proteins and achieve impressive levels of accuracy for describing both structured and unstructured proteins. in a recent benchmark study, six of the latest protein force fields were evaluated using the 61residue n-terminal transactivation domain (tad) of tumor suppressor p53, which is a very challenging system due to its size and complex conformational features [99] . it has been extensively characterized by nmr, saxs, and single-molecule fret and shown to contain a range of nontrivial local and long-range residual structures [110] . the disordered ensemble of p53-tad was calculated using rest2-enhanced sampling using gpu-accelerated gromacs 5.1.4 [80, 111] patched with plumed 2.3.0 [112] [113] [114] . each rest2 simulation lasted 1.0 μs per replica, representing one of the most extensive atomistic simulations of idps of similar sizes. the results show that the ensembles generated using the force field a99sb-disp yield the best agreement with the experimental data at both secondary and tertiary structure levels. for example, the back-calculated nmr paramagnetic relaxation enhancement (pre) effects are highly consistent with the experimental results for all four available labelling sites (figure 2 ). this suggests that the simulated ensembles not only have the proper overall chain dimension but also recapitulate much of the transient long-range ordering within the unbound state of p53-tad. the latter is an extremely challenging task. the fact that this could be achieved by a99sb-disp represents an exciting breakthrough, suggesting that de novo atomistic simulations are now ready to provide a reliable approach for detailed characterization of the disordered ensembles of at least moderately sized idps. . calculated (lines) and experimental (grey bars) nmr paramagnetic relaxation enhancement (pre) effects induced by paramagnetic spin labelling at residues d7, e28, a39, and d61 of p53-tad. red and green traces were calculated from an independent control and folding rest2 simulations of p53-tad in a99sb-disp, respectively, to evaluate the level of convergence. control and folding simulations were initiated from helical and fully unstructured structures, respectively, and the length of rest2 simulations were 1 μs per replica. this figure was adapted from [99] . see [99] for details on the simulation and analysis. red and green traces were calculated from an independent control and folding rest2 simulations of p53-tad in a99sb-disp, respectively, to evaluate the level of convergence. control and folding simulations were initiated from helical and fully unstructured structures, respectively, and the length of rest2 simulations were 1 µs per replica. this figure was adapted from [99] . see [99] for details on the simulation and analysis. we note that implicit solvent protein force fields have also been developed and deployed for atomistic simulations of idps with various levels of success [75, 96, 98, 115] . implicit treatment of solvent reduces the simulation system size~10-fold by direct estimation of the solvation free energy. it could provide important advantages for satisfying the simultaneous requirements of adequate sampling and sufficient force field accuracy for simulating disordered protein states. the absinth model, in particular, has demonstrated significant successes in mapping the sequence-conformational space relationship of idps [25] . an improved version named absinth-c was recently developed by including the backbone torsion cross-terms optimized based on experimentally derived statistics [98] . independently, the generalized born with the molecular volume 2 (gmmv2) model, which is considered one of the most accurate implicit solvent models, was recently implemented on gpu within the charmm/openmm interface [116] . gpu-accelerated gbmv2 is about 60-fold faster and provides a competitive alternative to explicit solvent simulations for studying the idp structure and interaction. this model was previously optimized based on enhanced sampling of model peptides and shown to be capable of accurately describing the conformational properties of both folded and unfolded peptides [96] . the development of the gpu-accelerated version thus removed a key bottleneck to broader application of gbmv2 for atomistic simulations of the idp structure and interactions. nonetheless, whether these implicit solvent models could provide a viable alternative to explicit solvent simulations in studies of idp-ligand interactions is yet to be demonstrated. to date, small molecular targeting of idps has mostly focused on proteins involved in neurodegenerative diseases, such as amyloid β (aβ) peptides, α-synuclein, and tau protein, and disordered regions of cancer-associated transcription factors, such as p53, c-myc, ews-fli1, klf5, and others [16, 117] . advances in experimental and computational methods for studying disordered protein ensembles have allowed examination in greater details the interactions between idps and small molecules. there has been an emergence of examples showing that small molecules could modulate the disordered ensemble itself through nonspecific and dynamic interactions and achieve specific functional effects, in complete contrast to the traditional paradigm of drug binding that emphasizes strong specific interactions. such a dynamic mode of idp-small molecule interactions is reminiscent of "fuzzy complexes" in protein-protein interactions involving idps [118] [119] [120] . the observation that small molecules could induce substantial effects through dynamic interactions is fascinating and suggests a broader and more effective strategy for targeting idps in general. [27, 28] . nmr, cd, and fluorescence studies initially suggested that these inhibitors bound specifically to multiple independent sites in the monomeric and disordered c-myc, which induced max-binding-incompatible conformations to disrupt the c-myc-max interaction [27] . subsequent explicit solvent simulations by michel and cuchillo in 2012 [121] revealed that the interaction between c-myc and one of the inhibitors, 10058-f4, was actually dynamic and involved many short-lived contacts. such a dynamic and nonspecific nature of the interaction could explain the observation that small modifications to the ligand had limited effects on the binding affinity [122] . a similar mode of interaction was proposed by jin et al. in 2013 [123] for another c-myc inhibitor, 10074-a4, where atomistic simulations in explicit and implicit solvent force fields revealed the ligand to form a "ligand cloud" and interact dynamically with the disordered "protein cloud". importantly, the dynamic nature of the interaction between 10058-f4 and c-myc 402-412 can confer sequence specificity, even though the interaction involves many transient contacts instead of well-defined specific ones [124] . it has been further suggested that such dynamic interactions may be driven by entropic expansion of the idp conformational space [51] . indeed, thermodynamics analysis showed that binding of 10058-f4 to c-myc 402-412 was dominated by the entropic contribution (−20.7± −4.2 kj/mol out of the total binding free energy of −27.6 ± −8.5 kj/mol) [124] . the caveat, however, is that binding of hydrophobic ligands is generally associated with large entropic contributions due to the release of restricted water molecules near the hydrophobic surface. it is thus not clear if c-myc indeed undergoes entropic expansion upon 10058-f4 binding. an in-depth nmr study of the interaction of a small molecule with intrinsically disordered p27 kip1 found little evidence of ligand-induced conformational space expansion [31] . instead, ligand binding was shown to mainly shift the populations of pre-existing states. dynamic interactions were also found to underlie the mechanism of α-synuclein aggregation inhibition by analogs of cyclized nordihydroguaiaretic acid (cndga) [125] . the structural basis of cndga inhibition was characterized using an array of biochemical and biophysical methods, including nmr and fluorescence correlation spectroscopy. the results revealed that cndga induced modest compaction of the conformational ensemble of monomeric α-synuclein, apparently mediated by dynamic and transient interactions with the protein and without hindering membrane association. cndga-treated α-synuclein is resistant to aggregation even when seeded with α-synuclein aggregates. importantly, cndga was further shown to be effective in reducing α-synuclein-driven neurodegeneration in c. elegans. the observation that dynamic interactions between a small molecule and idp could be functionally effective both in vitro and in vivo is very encouraging and supports the promise of targeting idps using dynamic interactions for therapeutics. induced compaction of the disordered conformational ensemble has also been predicted to underlie the inhibition mechanism of two drugs under clinical trials for treating alzheimer's diseases [126] . the disordered ensembles of aβ 42 with and without tramiprosate (homotaurine; ht) and scyllo-inositol (si) were calculated using rest2 simulations in the charmm36m force field that lasted 10 µs per replica, making them the most extensive atomistic simulations of aβ 42 to date. the resulting ensembles are well converged and appear consistent with the nmr chemical shifts. comparing the ensembles with and without the ligand showed that both ht and si mainly reduced the β propensity in the c-terminal region with minimal secondary structure perturbation in the rest of the peptide. intriguingly, both ht and si were found to induce modest compaction of the conformational ensemble, particularly in the c-terminal segment that is known to be important for amyloid fibril formation (figure 3a-c) . detailed analysis further revealed that the effects of both ht and si binding were achieved via dynamic and nonspecific interactions with various backbone and sidechain moieties of the peptide. it is noteworthy that the conformational modulation effects of both ht and si can be very difficult to detect at the ensemble level using bulk measurements, highlighting a critical role for reliable atomistic simulations that leverage recent advances in both protein force field quality and sampling capability. nonetheless, additional validation is needed to support the predicted conformational shifts induced by drugs and to establish the roles of such conformational changes in the mechanisms of drug action. fda-approved drugs and identified 15 compounds that could bind nupr1, a multi-functional idp involved in pancreatic ductal adenocarcinoma [133, 134] . nmr chemical shift analysis suggested that nupr1 remained disordered in complex with all compounds, which is consistent with an inhibition mechanism involving transient and dynamic idp-ligand interactions. importantly, these compounds showed efficacy in cell-based assays, the most effective of which was found to completely arrest tumor growth in a mouse model. . the conformational space of aβ42 is projected onto the number of backbone hydrogen bonds and end-to-end distance and that of p53-tad is projected onto the first two principal components. the conformational ensembles were calculated using long timescale rest2 simulations in explicit solvent (10 and 1 μs per replica for aβ42 and p53-tad, respectively). representative conformations are shown in backbone traces. this figure was adapted from [126, 127] . see [126, 127] for details on the simulation and analysis. idps have remained an extremely challenging class of proteins to target using small molecules. albeit limited, successful inhibitors have been discovered and designed for several idps involved in cancers and neurodegenerative diseases, suggesting that idps are not undruggable. nonetheless, the unstructured and dynamic nature of idps is distinct from typical protein targets with well-defined binding pockets. it requires new conceptional frameworks to guide the development of novel strategies for discovering and designing small molecules that can modulate idp functions. traditional structure-based screening and lead optimization strategies are clearly inadequate, even though some success has been demonstrated in deploying existing tools to identify possible binding pockets and target pre-existing structural elements. such structural elements are lightly populated and generally too small to harbor significant pockets for small molecular binding that relies on specific interactions to achieve high affinity. in fact, there is a great uncertainty on whether highaffinity binding to idps is feasible with small molecules (e.g., to meet the typical industrial standard of dissociation constants of nm or lower). it is encouraging that examples are emerging that small molecules could modulate the idp ensembles entirely through dynamic nonspecific interactions. importantly, there is evidence that high-affinity binding may not be necessary to induce functional responses in vitro and in vivo. this may reflect a fundamental nature of how idps mediate function in biology, in that the disordered ensemble of an idp is poised to respond sensitively to a wide array figure 3 . conformational ensembles of aβ42 with and without the ligands (a-c) and p53-tad with and without ligands (d,e). the conformational space of aβ42 is projected onto the number of backbone hydrogen bonds and end-to-end distance and that of p53-tad is projected onto the first two principal components. the conformational ensembles were calculated using long timescale rest2 simulations in explicit solvent (10 and 1 µs per replica for aβ42 and p53-tad, respectively). representative conformations are shown in backbone traces. this figure was adapted from [126, 127] . see [126, 127] for details on the simulation and analysis. de novo atomistic simulations have also been integrated with nmr and biophysical experiments to examine how an anticancer drug, epigallocatechin gallate (egcg), modulates the disordered unbound state of p53-tad [127] . egcg is a major active ingredient of green tea and has been reported to have anticancer effects in both animal studies and clinical trials [128] [129] [130] . the results suggested that egcg also interacted dynamically with p53-tad through numerous transient and nonspecific interactions, which appeared consistent with nmr chemical shift titration results. multiple hydrophobic and particularly aromatic sidechains contribute significantly to egcg binding. the dynamic interaction with egcg was predicted to induce significant conformational compaction of p53-tad in the n-terminal region (figure 3d ,e), which appears consistent with the saxs measurements. the compaction could shield the p53-tad site required for interacting with mdm2 and thus inhibit p53 degradation to promote its anticancer activities. it is noteworthy that egcg has also been shown to be active in inhibiting the aggregation of multiple proteins, including aβ peptides and α-synuclein [131, 132] . the implication is that dynamic interactions of egcg could provide a molecular basis for promiscuous selectivity, which is a fascinating property to investigate further using a combination of experiments and simulation. recently, neira et al. screened a set of fda-approved drugs and identified 15 compounds that could bind nupr1, a multi-functional idp involved in pancreatic ductal adenocarcinoma [133, 134] . nmr chemical shift analysis suggested that nupr1 remained disordered in complex with all compounds, which is consistent with an inhibition mechanism involving transient and dynamic idp-ligand interactions. importantly, these compounds showed efficacy in cell-based assays, the most effective of which was found to completely arrest tumor growth in a mouse model. idps have remained an extremely challenging class of proteins to target using small molecules. albeit limited, successful inhibitors have been discovered and designed for several idps involved in cancers and neurodegenerative diseases, suggesting that idps are not undruggable. nonetheless, the unstructured and dynamic nature of idps is distinct from typical protein targets with well-defined binding pockets. it requires new conceptional frameworks to guide the development of novel strategies for discovering and designing small molecules that can modulate idp functions. traditional structure-based screening and lead optimization strategies are clearly inadequate, even though some success has been demonstrated in deploying existing tools to identify possible binding pockets and target pre-existing structural elements. such structural elements are lightly populated and generally too small to harbor significant pockets for small molecular binding that relies on specific interactions to achieve high affinity. in fact, there is a great uncertainty on whether high-affinity binding to idps is feasible with small molecules (e.g., to meet the typical industrial standard of dissociation constants of nm or lower). it is encouraging that examples are emerging that small molecules could modulate the idp ensembles entirely through dynamic nonspecific interactions. importantly, there is evidence that high-affinity binding may not be necessary to induce functional responses in vitro and in vivo. this may reflect a fundamental nature of how idps mediate function in biology, in that the disordered ensemble of an idp is poised to respond sensitively to a wide array of cellular signals to support signal transduction and cellular regulation [10] . therefore, there is a great potential and promise for targeting idps through dynamic interactions with small molecules. it is noteworthy that idps have been found to play central roles in mediating liquid-liquid phase separation (llps) that underlies a range of cellular processes [135] [136] [137] . how small molecules may modulate the equilibrium and properties of these biological condensates is essentially unknown at this point. however, it is conceivable that llps may also be ideally targeted using dynamic interactions with small molecules that modulate the conformational flexibility and preference of disordered regions, which in turn modify the multivalent interaction profiles and entropic contribution that affect the condensation process as well as the properties of the condensate itself. elucidating the molecular details of dynamic interactions between idps and small molecules will require further development and integration of new experimental and computational methodologies. the capability for reliable disordered ensemble characterization with and without small molecules will almost certainly be required for any future design and optimization strategy to discover drugs that target idps through dynamic interactions. this unfortunately remains a formidable task. bulk experimental measurements on average properties alone are not sufficient to uniquely define the disordered ensemble. the dynamic and transient nature of molecular contacts can be very difficult to detect and resolve experimentally [138, 139] . leveraging significant recent advances in the protein force field quality, sampling techniques, and gpu computing, de novo atomistic simulations are now poised to help meet these challenges and play a pivotal role in establishing the molecular basis of dynamic idp-small molecule interactions. author contributions: data analysis, x.l.; writing-draft and edit, j.c. (jianhan chen); writing-review and edit, j.c. 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colorectal adenomas: a randomized clinical trial egcg remodels mature alpha-synuclein and amyloid-beta fibrils and reduces cellular toxicity green tea epigallocatechin-3-gallate (egcg) reduces beta-amyloid mediated cognitive impairment and modulates tau pathology in alzheimer transgenic mice designing and repurposing drugs to target intrinsically disordered proteins for cancer treatment: using nupr1 as a paradigm identification of a drug targeting an intrinsically disordered protein involved in pancreatic adenocarcinoma considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates polymer physics of intracellular phase transitions methods of probing the interactions between small molecules and disordered proteins characterization of the binding of small molecules to intrinsically disordered proteins this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank d. e. shaw research for giving us access to the md trajectories of aβ40. the authors thank chungwen liang for helpful discussions. we would also link to thank anonymous reviewers for their careful reading and insightful suggestions that have greatly improved both the content and writing of this review. the authors declare no conflict of interest. key: cord-255371-o9oxchq6 authors: nguyen, thanh thi; pathirana, pubudu n.; nguyen, thin; nguyen, henry; bhatti, asim; nguyen, dinh c.; nguyen, dung tien; nguyen, ngoc duy; creighton, douglas; abdelrazek, mohamed title: genomic mutations and changes in protein secondary structure and solvent accessibility of sars-cov-2 (covid-19 virus) date: 2020-07-10 journal: biorxiv doi: 10.1101/2020.07.10.171769 sha: doc_id: 255371 cord_uid: o9oxchq6 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a highly pathogenic virus that has caused the global covid-19 pandemic. tracing the evolution and transmission of the virus is crucial to respond to and control the pandemic through appropriate intervention strategies. this paper reports and analyses genomic mutations in the coding regions of sars-cov-2 and their probable protein secondary structure and solvent accessibility changes, which are predicted using deep learning models. prediction results suggest that mutation d614g in the virus spike protein, which has attracted much attention from researchers, is unlikely to make changes in protein secondary structure and relative solvent accessibility. based on 6,324 viral genome sequences, we create a spreadsheet dataset of point mutations that can facilitate the investigation of sars-cov-2 in many perspectives, especially in tracing the evolution and worldwide spread of the virus. our analysis results also show that coding genes e, m, orf6, orf7a, orf7b and orf10 are most stable, potentially suitable to be targeted for vaccine and drug development. biological investigations of the novel coronavirus sars-cov-2 are important to understand the virus and help to propose appropriate responses to the pandemic. scientists have been able to obtain genomic sequences of sars-cov-2 and have started analysis of these data. reference genome of sars-cov-2 deposited to the national center for biotechnology information (ncbi) genbank sequence database (isolate wuhan-hu-1, accession number nc_045512) shows that sars-cov-2 is an rna virus having a length of 29,903 nucleotides. comparative genomic analysis results obtained in [1] [2] [3] suggest that the covid-19 virus may be originated in bats. other studies show that pangolins may have served as the hosts for the virus [4; 5] . andersen et al. [6] furthermore believe that sars-cov-2 is not a purposefully manipulated virus or constructed in a laboratory but has a natural origin. a study in [7] using machine learning unsupervised clustering methods corroborates previous findings that sars-cov-2 belongs to the sarbecovirus subgenus of the betacoronavirus genus within the coronaviridae family [8; 9] . the whole genome analysis results also indicate that bats are more likely the reservoir hosts for the virus than pangolins. another study in [10] demonstrates that sars-cov-2 may have resulted from a recombination of a pangolin coronavirus and a bat coronavirus, and pangolins may have acted as an intermediate host for the virus. since the first cases were detected, the covid-19 virus has spread to almost every country in the world and has been linked to the deaths of more than 404,000 people of over 7 million confirmed cases [11] . tracing the evolution and spread of the virus is important for developing vaccines and drugs as well as proposing appropriate intervention strategies. monitoring and analysing the viral genome mutations can be helpful for this task. due to a strong immunologic pressure in humans, the virus may have mutated over time to circumvent responses of the human immune system. this leads to the creation of virus variants with possible different virulence, infectivity, and transmissibility [25] . this paper reports all point mutations occurring so far in sars-cov-2 and presents exemplified implications obtained from the analysis of these mutation pattern data. four types of mutations, which include synonymous, nonsynonymous, insertion and deletion, are detected. we use 6,324 sars-cov-2 genome sequences collected in 45 countries and deposited to the ncbi genbank so far and create a spreadsheet dataset of all mutations occurred across different genes. eleven protein coding genes of sars-cov-2 have been identified, namely orf1ab, spike (s), orf3a, envelope (e), membrane (m), orf6, orf7a, orf7b, orf8, nucleocapsid (n) and orf10. the order of these genes and their corresponding length are illustrated in fig. 1 . the genes s, e, m, and n produce structural proteins that play important roles in the virus functions. for example, the receptor-binding domain (rbd) region of the s protein can bind to a receptor of a host cell, e.g. the human and bat angiotensin-converting enzyme 2 (ace2) receptor, enabling the entrance of the virus into the cell [12] . predictions of protein structures may help understand the virus's functions and thus contribute to developing vaccines and therapeutics against the virus. in this paper, to evaluate the possible impacts of genomic mutations on the virus functions, we propose the use of the sspro/accpro 5 methods to predict protein secondary structure and relative solvent accessibility [13] . these predictors were built using deep learning one-dimensional bidirectional recurrent neural networks incorporated in the scratch-1d software suite (version 1.2, 2018) [14] . by comparing the prediction results obtained on the reference genome and mutated genomes, we are able to assess whether the detected mutations have the potential to change the protein structure and solvent accessibility, and thus lead to possible changes of the virus characteristics. because of the functional importance of structural proteins, we only report the prediction results of these proteins in this study. the next section reviews related works in the literature. we then present materials and methods for sars-cov-2 mutation detection, and protein secondary structure and solvent accessibility prediction. next we summarize statistics of sars-cov-2 mutations so far and implications of these mutations. details of mutations in nonstructural orf genes and structural s, e, m and n genes are presented after that. a full sars-cov-2 mutation spreadsheet report is provided in the supplemental information. since the first genomes were collected in december 2019, there have been many findings on the mutations of sars-cov-2. for example, phan [15] analysed 86 genomes of sars-cov-2 downloaded from the the global initiative on sharing all influenza data (gisaid) database (https://www.gisaid.org/) and found 93 mutations over the entire viral genome sequences. among them, there are three mutations occurring in the rbd region of the spike surface glycoprotein s, including n354d, d364y and v367f, with the numbers showing amino acid (aa) positions in the protein. that study also reveals three deletions in the genomes of sars-cov-2 obtained from japan, usa and australia. two of these deletion mutations are in the orf1ab polyprotein and one deletion occurs in the 3' end of the genome. likewise, a study in [16] shows that the sars-cov-2 genomes may have undergone recurrent, independent mutations at 198 sites with 80% of these are of the nonsynonymous type. tang et al. [17] investigated 103 genomes of covid-19 patients and discover mutations in 149 sites of these genomes. the study also shows that the spike gene s consistently has larger ds values (synonymous substitutions per synonymous site) than other genes. in addition, two major lineages of the virus, denoted as l and s, have been specified based on two tightly linked snps. the l lineage is found more prevalent than the s lineage among the examined sequences. korber et al. [18] tracked the mutations of spike protein s of sars-cov-2 because it plays an important role in mediating infection of targeted cells and is the focus of vaccine and antibody therapy development efforts [19] . they detected 14 mutations in the spike protein that are growing, especially the mutation d614g that rapidly becomes the dominant form when spread to a new geographical region. likewise, hashimi [20] analysed the mutation frequency in the spike protein s of 796 sars-cov-2 genomes downloaded from the gisaid and genbank databases. the study found 64 mutations occurring in the s protein sequences obtained from multiple countries. it suggests that the virus is spreading in two forms, the d614 form (residue d at position 614 in the s protein) takes 68.5% while the g614 form takes 31.5% proportion of the examined isolates. koyama et al. [21] on the other hand found several variants of sars-cov-2 that may cause drifts and escape from immune recognition by using the prediction results of b-cell and t-cell epitopes in [22] . typically, the mutation d614g occurring in the spike protein is found prevalent in the european population. this mutation may have caused antigenic drift, resulting in vaccine mismatches that lead to a high mortality rate of this population. a recent situation report [23] by nextstrain [24] on genomic epidemiology of novel coronavirus using 5,193 publicly shared covid-19 genomes shows that sars-cov-2 on average accumulates changes at a rate of 24 substitutions per year. this is approximately equivalent to 1 mutation per 1,000 bases in a year. this evolutionary rate of sars-cov-2 is typical for a coronavirus, and it is smaller than that of influenza (average 2 mutations per 1,000 bases per year) and hiv (average 4 mutations per 1,000 bases per year). shen et al. [25] conducted metatranscriptome sequencing for bronchoalveolar lavage fluid samples obtained from 8 patients with covid-19 and found no evidence for the transmission of intrahost variants as well as a high evolution rate of the virus with the number of intrahost variants ranged from 0 to 51 around a median number of 4. pachetti et al. [26] examined 220 genomic sequences of covid-19 patients from the gisaid database and discovered 8 novel recurrent mutations at nucleotide locations 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881. mutations at locations 2891, 3036, 14408, 23403 and 28881 are mostly found in europe while those at locations 17746, 17857 and 18060 occur in sequences obtained from patients in north america. likewise, a study in [27] on 95 sars-cov-2 complete genome sequences discovered 116 mutations. among them, the mutations at position c8782t in the orf1ab gene, t28144c in the orf8 gene and c29095t in the n gene are common. we use 6,324 sequence records downloaded from the ncbi genbank database on 2020-06-17. the latest collection date for the samples from which the sequences were derived was on 2020-06-05. the data, which were collected in 45 countries, include both nucleotide sequences and protein translations of coding genes. a proportion of the 6,324 records have sequences of only few proteins, i.e. these records do not annotate all 11 proteins (orf1ab, orf3a, orf6, orf7a, orf7b, orf8, orf10, s, e, m and n). the number of available sequences is thus different from one protein to another (see column "avai num" in table 1 ). genome sequences that do not specify country or aa sequences that contain letter "x" representing an unknown aa are excluded in our calculations. we use the genome obtained from the isolate wuhan-hu-1, accession number nc_045512 as the reference genome. for the mutation detection purpose, we apply a dynamic programming algorithm to protein aa sequences to get global pairwise alignments between a reference sequence and a query sequence. specifically, we use the python bio.pairwise2.align.globalms function (https://biopython.org/docs/dev/api/bio.pairwise2.html) where a match is given 2 points, a mismatch is deducted 0.5 points, 2 points are deducted when opening a gap, and 1 point is deducted when extending it. gaps are then inserted into nucleotide sequences corresponding to the resulted protein sequence alignments. using the resulted pairwise alignments, we are able to compare query sequences and the reference sequences at each position and identify locations of insertion, deletion, synonymous and nonsynonymous mutations. virus protein structure plays a key role in its functions and a change in structure shape may affect its functions, virulence, infectivity and transmissibility, possibly resulting in non-functional proteins. protein secondary structure is defined by hydrogen bonding patterns, which make an intermediate form before the protein folds into a three-dimensional shape composing its tertiary structure. eight types of protein secondary structure defined by the dictionary of protein secondary structure (dssp) include 3 10 helix (g), α helix (h), π helix (i), hydrogen bonded turn (t), extended strand in parallel and/or anti-parallel β-sheet conformation (e), residue in isolated β-bridge (b), bend (s) and coil (c). the dssp tool assigns every residue to one of the eight possible states. in a reduced form, these 8 conformational states can be diminished to 3 states: h = {h, g, i}, e = {e, b} and c = {s, t, c} [28] . the protein secondary structure represents interactions between neighboring or near-by aas as its functional three-dimensional shape is created through the polypeptide folding. we thus determine a change in protein secondary structure if any change happens in the structures of the mutated aa and its 10 neighboring aas compared to those of the reference sequence. in detail, we consider 5 aas ahead and 5 aas behind the mutated aa. the same approach is applied when considering a change of the protein relative solvent accessibility. solvent-exposed area represents the area of a biomolecule on a surface that is accessible to a solvent. accordingly, a residue is considered as exposed if at least 25% of that residue must be exposed, denoted as the "e" state. alternatively, the residue is determined as buried, i.e. the "b" state. there have been various protein secondary structure prediction programs in the literature and many of those were developed based on artificial intelligence models using protein aa sequences such as jpred4 [29] , spider2 [30] , porter 5 [31] , raptorx [32] , psspred [33] , yasspp [34] and sspro [13] . in this paper, we use the protein secondary structure and relative solvent accessibility prediction methods sspro/accpro 5 [13] within the scratch-1d software suite (release 1.2, 2018) [14] . these predictors were built using the bidirectional recursive neural networks and a combination of the sequence similarity and sequence-based structural similarity to sequences in the protein data bank [35] . prediction results of 8-class structure (sspro8 predictor) and 25%-threshold relative solvent accessibility (accpro predictor) are used for statistics on protein secondary structure and accessibility changes. we however also report in the spreadsheet supplemental information prediction results of 3-class structure (sspro predictor) and relative solvent accessibility on 20 thresholds, ranging from 0% to 95% with a 5% step (the accpro20 predictor within the scratch-1d software). table 1 summarizes statistics of sars-cov-2 mutations so far. "aa length" indicates the length of the protein aa sequence derived from the sars-cov-2 reference genome. "avai num" denotes the number of records among 6,324 ncbi genbank records that have the complete sequence of the corresponding protein. "no mu" refers to the number of sequences that do not have any mutations compared to the reference sequence. "delete" means the number of deletion mutations occurring in the aa sequences of the protein. this number may be larger than the number of sequences having deletion mutations because an aa sequence may have more than one deletion. likewise, "insert", "nonsyn" and "syn" show the number of insertion, nonsynonymous and synonymous mutations occurring in the protein aa sequences. "nonsyn/syn" demonstrates a ratio between the number of nonsynonymous mutations versus the number of synonymous mutations. "struct change" means the number of nonsynonymous mutations that have protein secondary structure change potential based on the sspro8 predictor of the scratch-1d software. similarly, "acc change" refers to the number of nonsynonymous mutations that have potential to change the protein relative solvent accessibility based on the accpro predictor of the scratch-1d software. insertion and deletion mutations alter protein secondary structure and solvent accessibility by default so that they are not included in the structure and solvent accessibility change statistics. table 1 shows that the orf3a and orf8 proteins have the number of nonsynonymous mutations significantly larger than that of the synonymous mutations. in contrast, this ratio in proteins e, m, orf7b and orf10 are very small (less than 1). these proteins could be targeted for vaccine and drug development as they have less variations than other proteins. these findings are supported by results presented in figs (fig. 3) , entire regions before and after the spike at position 614 are almost unchanged. fig. 4 presents variations of multiple proteins. in addition to proteins e, m, orf7b and orf10, we find that proteins orf6 and orf7a are also relatively stable without a large number of variations at any particular locations. protein n has 1,927 nonsynonymous mutations but 1,678 of them are likely to make changes in protein secondary structure, making a ratio of 87.08%. this is considerably larger than those of protein s (4.42%), protein m (24.79%) and protein e (64.29%). the number of solvent accessibility changes of protein s is larger than its structure changes: 184 vs 164. this however is opposite in other structural proteins: e (7 vs 18), m (8 vs 30) and n (37 vs 1,678). the orf1ab polyprotein has 7,096 aas. among 6,324 records deposited to the ncbi genbank database, only 3,726 genomes have the complete coding sequence (cds) of protein orf1ab, with 1,024 unique aa sequences. this is quite a large number compared to other proteins but understandable because orf1ab is the longest protein of sars-cov-2 and thus has a large number table 2 . table 2 ). the genbank accession numbers are presented on the left while isolate names and collected dates are on the right. the numbers on top show the positions of aas in the protein and isolates are ordered by collected dates. the first isolate having these deletions is usa-ca6/2020 (record mt044258 in second row), collected on 2020-01-27 in usa: ca. this is also the isolate having the largest number of deletions: five sequentially at g82-, h83-, v84-, m85-, v86-and three at k141-, s142-, f143-. the other patients followed were possibly infected by this first case but more data such as travel history are needed to confirm this hypothesis. (18) germany (16) taiwan (9) the orf3a protein has 275 aas with its complete cds appearing in 5,527 isolates (146 unique aa sequences). among these, 2,321 sequences have no mutation or only synonymous mutations, and 3,206 sequences have insertion, deletion or nonsynonymous mutations. table 4 . notably, the mutation q57h occurs in 2,795 sequences collected in many countries. this is an emerging and active mutation, which requires further investigation as the latest case of this mutation was on 2020-06-05, same as the latest collection date of the entire downloaded dataset. the mutation g251v occurring in 206 sequences is also a prevalent mutation in the orf3a protein. the orf6 protein has 61 aas, appearing in 5,792 isolates with 25 unique aa sequences. among these, 5,719 sequences have no mutation or only synonymous mutations and 73 sequences have insertion, deletion or nonsynonymous mutations. two insertion mutations occur in record mt520188 at positions -62r and -63t (end of the sequence). nine continual deletions occur similarly in 2 sequences: mt547814 (collected in hong kong on 2020-01-22 from an adult male patient [37] ) and mt609561 (usa: virginia in 2020-04). these deletions are f22-, k23-, v24-, s25-, i26-, w27-, n28-, l29-and d30-. alignment of these sequences with the reference genome is displayed in fig. 6 . the isolate mt547814 thus may have transmitted the virus to mt609561 but this implication needs to be corroborated by patients' travel history. there are 23 distinct nonsynonymous mutations and those occurring in 2 or more sequences are presented in table 5 . the orf7a protein has 121 aas in length, found in 5,321 isolates with 34 unique aa sequences. among these, 5,215 sequences have no mutations or only synonymous mutations, while the rest orf7a. there are 15 deletion mutations occurring in 2 records: mt520425 (collected in usa: massachusetts on 2020-03-27) and mt507795 (usa on 2020-04-06). the mt520425 sequence has 1 deletion at position l77-while the mt507795 sequence has 14 sequential deletions f63-, a64-, f65-, a66-, c67-, p68-, d69-, g70-, v71-, k72-, h73-, v74-, y75-and q76-. alignment of these sequences with that of the reference genome is shown in fig. 7 . there are 32 distinct nonsynonymous mutations with those occurring in 2 or more sequences are reported in table 6 . the orf7b protein has 43 aas with its complete cds appearing in 5,175 isolates, forming a set of 11 unique aa sequences. there are 5,151 sequences having no mutations or only synonymous mutations and 24 sequences having nonsynonymous mutations. no insertion or deletion mutations are found in gene orf7b. this along with a small number of nonsynonymous mutations indicate that orf7b is a stable gene. distinct nonsynonymous mutations (10 of them) include f19l, f28y, f30l, s31l, l32f, t40i, c41f, c41s, h42y and a43t. summary of nonsynonymous mutations in gene orf7b occurring in 2 or more sequences is shown in table 7 . the orf10 protein has 38 aas in length, appearing in 5,891 isolates with only 9 unique aa sequences. among them, 5,872 sequences have no mutation or only synonymous mutations and the rest 19 sequences have nonsynonymous mutations. no insertion and deletion mutations are found in gene orf10. similar to orf7b, this is a stable gene. there are 8 distinct nonsynonymous mutations, including i4l, a8v, s23f, r24l, r24c, a28v, d31y and v33i. those occurring in 2 sequences or more are presented in table 9 . the virus transmission may have happened between these two isolates but this needs further investigation. alignment of these sequences is shown in fig. 9 . the number of nonsynonymous mutations in gene s is 3,711, with 240 distinct mutations. mutations that occur in 10 or more cases are reported in table 10 . the number of synonymous mutations is 670, making a ratio between nonsynonymous versus synonymous mutations at 5.54. among the nonsynonymous mutations, mutation d614g is extremely common as it happens in 3,089 sequences, majorly collected in usa (2340), india (210) and australia (132). the first collected date of the d614g mutation cannot be identified precisely because some sequences deposited to the ncbi genbank did not record the full date details. the current data show that either of the following sequences, which have the d614g mutation, was first collected: mt326173 in usa in 2020, or mt270104, mt270105, mt270108 and mt270109 all in germany: bavaria in 2020-01, or mt503006 in thailand on 2020-01-04. it is however important to note that the first patient having the d614g mutation and his/her location may never be known because genome of that patient might not be sequenced and reported. therefore, information reported here can support for further investigation. our statistics show that among 4,434 sequences of the s protein, 3,089 sequences have the mutation d614g, taking 69.67%. this number has considerably increased compared to 31.5% in the previous analysis in [20] on a dataset downloaded on 2020-03-22. on the other hand, there are 37 a829t mutations that all occur in thailand. the first case of this mutation was collected on 2020-01-23 and its latest case was on 2020-04-07. this may indicate that the first case had probably transmitted to other cases having the same mutation a829t in thailand. alternatively, mutations h146y (24 cases), v483a (11 cases), e554d (14 cases), p681l (16 cases) and s939f (11 cases) all occur only in usa or mutation l8v (4 cases) occurs only in hong kong (refer to the attached spreadsheet). the "latest date" in tables 2-15 may be used to infer which mutations are inactive or still active. for example, in gene s (table 10) , the latest date of d614g was on 2020-06-05 (same as the latest collection date of the entire dataset) that indicates that this mutation is still active. the latest date of p681l was on 2020-04-03, indicating that this mutation may no longer occur. this kind of information may be useful for further research on vaccine and drug development as ongoing changes of the viral proteins need to be focused and addressed. we identify the rbd region within the residue range arg319-phe541 of protein s based on a study in [36] . in the rbd region only, the number of nonsynonymous mutations is 53 and that of synonymous is 46, making a ratio of 1.15. this is much smaller than the ratio of 5.54 for the entire gene s, suggesting that the rbd region may have been optimized for binding to a receptor of a host cell. this is complemented by fig. 9 showing all deletion mutations in gene s being outside the rbd region. note that the difference of these ratios is partly due to the large number of d614g mutations (3, 089) , which is outside the rbd region. table 11 summarizes nonsynonymous mutations in the rbd region occurring in 2 or more sequences. notable mutation in this region is v483a occurring in 11 isolates all collected in usa. the first and latest collected dates of these isolates were respectively 2020-03-05 and 2020-04-05, suggesting that the first isolate may have spread to others having the same mutation v483a. likewise, the mutation g476s occurs in 6 isolates all collected in usa: wa from 2020-03-10 to 2020-03-25. alternatively, the mutation y453f occurs in 5 sequences all in netherlands but the first collected date was on 2020-04-25 and the latest collected date was on 2020-04-29. these dates are too close, indicating that all the reported y453f cases may have been infected from another case, whose genome had not been sequenced and reported to the ncbi genbank. it is important to note that all the transmission implications need further investigation with more data from other aspects such as travel history, physical contacts and so on. in for the entire protein s, 134 nonsynonymous mutations (48 unique) have both structure and solvent accessibility change potentials. these mutations occurring in 2 or more sequences are reported in table 12 . mutation h146y occurs in 24 cases and mutation p681l occurs in 16 cases, which are all collected in usa. the most common mutation d614g does not have the potential to change either protein secondary structure or relative solvent accessibility. the envelope protein e has 75 aas, found in 5,852 genbank records with 15 unique aa sequences. among them, 5,824 sequences have no mutation or only synonymous mutations while 28 sequences have nonsynonymous mutations. gene e is thus relatively stable and could be targeted for vaccine and drug development. this is supported by the fact that no insertion or deletion mutations are found within gene e. there are 14 distinct nonsynonymous mutations in gene e and those occur in 2 or more sequences are presented in table 13 . five distinct nonsynonymous mutations in gene e have protein structure change potential: s68c, s68f, p71l, d72y and l73f. alternatively, 4 distinct mutations have potential to change relative solvent accessibility: l37h, l37r, d72y and l73f. therefore, d72y and l73f are two mutations in gene e that have a potential to change both protein structure and solvent accessibility. table 12 . gene s -nonsynonymous mutations that have both structure and solvent accessibility change potentials occuring in 2 or more sequences. the "query structure" (and "query accessibility") shows the unique structure (and accessibility) changes based on on prediction results. structure letter in parentheses is the predicted structure of the residue at the corresponding mutation position. five letters before and after parentheses are structures of neighbouring residues. likewise, letter "b" or "e" in parentheses shows the accessibility status of the residue at the mutation position. 2 ccccc(c)cbeee ccccc(c)ceeee bebbb(b)bbbbb beebb(b)bbbbb d253g 7 eecct(t)ccctc ccccc(c)cceec cccct(c)cceec bbbeb(e)bbbeb bbbeb(b)bbbbb s254f 2 ecctt(c)cctcc ecttc(e)eeecc bbebe(b)bbebb bbbbb(b)bbbbb w258l 4 tccct(c)cccse cccee(e)eeese ebbbe(b)bbbeb bbbbb(b)bbbeb g261d 4 ctccc(c)seeee eeccc(c)seeee bebbb(b)ebbbb bbbbb(b) the m protein has 222 aas and its complete cds appears in 5,677 genbank records, with 37 unique aa sequences. there are 5,557 sequences having no mutation or only synonymous mutations while other 120 sequences have nonsynonymous mutations. no insertion or deletion mutations are found in gene m. the number of distinct nonsynonymous mutations in gene m is 37, with those occurring in 5 or more sequences shown in table 14 . among these, 10 mutations are likely to make changes in protein secondary structure: c64f, a69s, a69v, v70f, n113b, r158l, v170i, d190n, d209y and s214i. alternatively, 6 mutations have the solvent accessibility change potential: n113b, p123l, p132s, h155y, d190n and t208i. n113b and d190n are thus two mutations having potential to change both protein structure and solvent accessibility in gene m. the n protein has 419 aas and its complete cds appears in 5,281 isolates, with 178 unique aa sequences. among them, 4,315 sequences have no mutation or only synonymous mutations while the rest 966 sequences have deletions or nonsynonymous mutations. there are no insertion in gene n. the sequence in mt434815 (collected in usa: ny on 2020-03-09) has three sequential deletions at q390-, t391-and v392-while the sequence in mt370992 (usa: ny on 2020-03-20) has six sequential deletions at t366-, e367-, p368-, k369-, k370-and d371-. two other sequences mt605818 and mt560525 (both collected in turkey on 2020-04-16) have three sequential deletions at r195-, n196-and s197-. there are 1,927 nonsynonymous mutations with 156 distinct ones and those occurring in 10 or more sequences are presented in table 15 . notable mutations are r203k occurring in 871 sequences and g204r occurring in 433 sequences. there are 15 mutations in this protein having the potential to change both protein structure and solvent accessibility, including g18v, d22y, g34w, r40c, r40l, r185c, a211s, p365h, t391i, t393i, a398s, d399e, d399h, d401y and d402y. analysing the virus genome sequences and their proteins is crucial for understanding the virus and proposing appropriate approaches to respond to and control the pandemic. this paper has reported all point mutations of sars-cov-2 since the virus's first genomes were obtained in december 2019. a sars-cov-2 mutation database is built using a large number of genome sequences (6,324) obtained across 45 countries. this database can enable scientists to monitor the evolution and spread of the virus although the use of these data needs to be corroborated with patients' clinical data and travel history for substantiated confirmations. we also predict the secondary structure and relative solvent accessibility of the virus proteins to evaluate whether the detected mutations have a potential to change the virus characteristics. these protein secondary structure and solvent accessibility change potentials are predicted results based on deep learning recurrent neural networks, which need to be experimentally verified. they however provide important insights about the virus and prompt further experimental biochemistry and molecular biology research into the genomic regions of these mutations. among 3,089 d614g mutations, our prediction results show that none of these mutations is likely to make changes in the protein secondary structure and relative solvent accessibility. in addition, we have shown regions of the sars-cov-2 genomes that have small variations such as those coding for proteins e, m, orf6, orf7a, orf7b and orf10. these regions could be targeted for vaccine and drug development. usa (10) australia (6) bangladesh (3) hong kong (2) taiwan (1) germany (1) kazakhstan (1) s202n 25 2020-01-30 china australia (203) greece (122) bangladesh (88) japan (38) czech republic (34) poland (26) germany (18) india (12) taiwan (10) turkey (8) france (8) thailand (6) serbia australia (101) greece (61) bangladesh (44) japan (19) czech republic ) serbia (3) italy (3) spain (2) russia (2) sri lanka (1) puerto rico (1) peru (1) nigeria (1) a new coronavirus associated with human respiratory disease in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin identifying sars-cov-2 related coronaviruses in malayan pangolins probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak the proximal origin of sars-cov-2 origin of novel coronavirus (covid-19): a computational biology study using artificial intelligence. biorxiv a novel coronavirus from patients with pneumonia in china the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 isolation of sars-cov-2-related coronavirus from malayan pangolins who coronavirus disease (covid-19) dashboard characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine sspro/accpro 5: almost perfect prediction of protein secondary structure and relative solvent accessibility using profiles, machine learning and structural similarity scratch: a protein structure and structural feature prediction server genetic diversity and evolution of sars-cov-2. infection emergence of genomic diversity and recurrent mutations in sars-cov-2. infection on the origin and continuing evolution of sars-cov-2 spike mutation pipeline reveals the emergence of a more transmissible form of sars-cov-2. biorxiv a short review on antibody therapy for covid-19 emergence of mutations and possible antigenic drift in the surface glycoprotein of sars-cov-2 (covid-19) emergence of drift variants that may affect covid-19 vaccine development and antibody treatment a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov-2 genomic analysis of covid-19 nextstrain: real-time tracking of pathogen evolution genomic diversity of sars-cov-2 in coronavirus disease 2019 patients emerging sars-cov-2 mutation hot spots include a novel rna-dependent-rna polymerase variant genomic characterization of a novel sars-cov-2 multi-output interval type-2 fuzzy logic system for protein secondary structure prediction jpred4: a protein secondary structure prediction server spider2: a package to predict secondary structure, accessible surface area, and main-chain torsional angles by deep neural networks porter 5: fast, state-of-the-art ab initio prediction of protein secondary structure in 3 and 8 classes. biorxiv raptorx: exploiting structure information for protein alignment by statistical inference a comparative assessment and analysis of 20 representative sequence alignment methods for protein structure prediction yasspp: better kernels and coding schemes lead to improvements in protein secondary structure prediction the protein data bank structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor a rare deletion in sars-cov-2 orf6 dramatically alters the predicted three-dimensional structure of the resultant protein key: cord-253844-y6xdcf20 authors: yesudhas, dhanusha; srivastava, ambuj; gromiha, m. michael title: covid-19 outbreak: history, mechanism, transmission, structural studies and therapeutics date: 2020-09-04 journal: infection doi: 10.1007/s15010-020-01516-2 sha: doc_id: 253844 cord_uid: y6xdcf20 purpose: the coronavirus outbreak emerged as a severe pandemic, claiming more than 0.8 million lives across the world and raised a major global health concern. we survey the history and mechanism of coronaviruses, and the structural characteristics of the spike protein and its key residues responsible for human transmissions. methods: we have carried out a systematic review to summarize the origin, transmission and etiology of covid-19. the structural analysis of the spike protein and its disordered residues explains the mechanism of the viral transmission. a meta-data analysis of the therapeutic compounds targeting the sars-cov-2 is also included. results: coronaviruses can cross the species barrier and infect humans with unexpected consequences for public health. the transmission rate of sars-cov-2 infection is higher compared to that of the closely related sars-cov infections. in sars-cov-2 infection, intrinsically disordered regions are observed at the interface of the spike protein and ace2 receptor, providing a shape complementarity to the complex. the key residues of the spike protein have stronger binding affinity with ace2. these can be probable reasons for the higher transmission rate of sars-cov-2. in addition, we have also discussed the therapeutic compounds and the vaccines to target sars-cov-2, which can help researchers to develop effective drugs/vaccines for covid-19. summary: the overall history and mechanism of entry of sars-cov-2 along with structural study of spike-ace2 complex provide insights to understand disease pathogenesis and development of vaccines and drugs. angiotensin-converting enzyme 2 ccl2 chemokine (c-c motif) ligand the sars-cov-2 is a single, positive-strand rna virus that causes severe respiratory syndrome in humans [1] . the coronavirus disease 2019 (covid-19) has emerged as a severe pandemic, claiming more than 0.8 million lives worldwide between december 2019 and august 2020 [2, 3] . compared to sars-cov, sars-cov-2 human-to-human infection is more readily transmitted and spread to almost all continents leading to the who's declaration of a public health emergency of international concern (pheic) on january 30, 2020 [3] [4] [5] . generally, coronaviruses can cause respiratory, gastrointestinal, and central nervous system diseases in humans and animals, threatening humans life and causing economic loss [6, 7] . these viruses also have the capacity to adapt to a new environment through mutations and are programmed to modify host tropism; thus, the threats are constant and long-term [6, 8, 9] . including sars-cov-2, other coronaviruses cross the species barrier into humans, which lead to outbreaks of severe and fatal respiratory diseases. the sars-cov was first identified in bats, and spread to other animals in different geographic regions. the sars-cov outbreak was first emerged in humans in 2003, through transmissions from animals in open-air markets in china [10, 11] . thereafter, a higher number of genetically related viruses were also identified in chinese horseshoes bats (rhinolophus sinicus) [11] [12] [13] . coronaviruses belong to the family coronaviridae and are divided into alpha (α-cov), beta (β-cov), gamma (γ-cov), and delta (δ-cov) coronaviruses. the alpha and betacoronaviruses can infect mammals, and the viruses found in humans are genetically similar to β-cov genus. the β-covs are further divided into different lineages (a, b, c, and d lineages): sars-cov and sars-cov-2 are grouped in lineage b, which has approximately 200 published virus sequences, whereas mers-cov belongs to lineage c, which has ~ 500 viral sequences [11] . the hcov-229e and hcov-nl63 belong to the alphacoronavirus family, whereas hcov-oc43, hcov-hku1 and sars-cov are betacoronaviruses [14] [15] [16] . the phylogenetic analysis shows that sars-cov-2 protein is firmly rooted in the β-genus lineage of bat coronaviruses [14] . the whole genome of sars-cov-2 shares 80% identity with that of sars-cov and is 96% identical to the bat coronavirus batcov-ratg13 [17] . the spike protein sequence similarity between sars-cov-2 and sars-cov is around 76-78%. the rbd alone shares a similarity of 73-76%, and rbm shares 50-53%. in contrast, the human mers-cov is related to tylonycteris bat coronavirus hku4, shares less sequence similarity (~ 54%) and recognizes dpp4 as their receptor. the sequence similarity between sars-cov-2 and sars-cov spike proteins explains the possibility of binding to the same receptor angiotensin converting enzyme 2 (ace2) in the host cell [14] . coronavirus is one of the largest genomes among all rna viruses ranging from 27 to 32 kb. receptor-mediated endocytosis is the main process of virus entry to the host cells. sars-cov-2 uses ace2, a cell-surface receptor that is present in the kidney, blood vessels, heart, and importantly, in the lung at2 alveolar respiratory tract epithelial cells for viral infection [18] . the spike protein, which is responsible for the viral entry, has n-terminal and c-terminal domains, and two major subunits s1 and s2 are present in almost all coronaviruses [6] . one of these s1 or s2 subunits binds with the host receptors and acts as a receptor-binding domain (rbd) (fig. 1a) . next-generation sequencing technology (ngs) revolutionized the biological sciences, including virus discovery. the ngs made it easy to recognize thousands of novel virus sequences from wild animal and human populations around the world. despite these vast coronavirus sequences are published, very little work has been performed for further studies. further, the lack of tools to test these novel viruses and their ability to infect humans hindered the efforts to predict the subsequent zoonotic viral outbreaks [11] . understanding the virology of coronaviruses and the methods to control their spread is currently a necessary task to maintain the global health and economic stability. in this review, we discuss the history of coronaviruses in both humans and animals, their transmissions, mechanism of host cell entry and the structural studies, explaining active and inactive receptor binding of spike protein and the key residues playing an important role in the receptor binding. the drug repurposing and the therapeutic targets for sars-cov-2 are also discussed. until the first identification of human coronaviruses 229e and oc43, in the late 1960s, coronavirus infections were witnessed as harmless for humans [14, 15] . the outbreak of sars-cov in southern china in the winter of 2002, took a fatality rate of 10% of the infected patients [18] [19] [20] . the virus had been rapidly spreading throughout the world, especially in asia, and controlled after july 2003 [21] . viral analysis of the outbreak of sars showed that bats are natural reservoirs for sars-covs, and civet cats and raccoon dogs are the intermediate hosts. in the year 2012, a novel highly pathogenic middle east respiratory syndrome coronavirus (mers-cov) was identified in humans, demonstrating that the coronaviruses are transmitted from animals to humans at any time and with unexpected consequences for the public health [22] . mers-cov, the slow-spreading virus, has affected ~ 1700 people with a fatality rate of ~ 36% [6, 22] . the animal sources of sars-cov-2 infections are bats and sars-cov-2 can be transmitted to cats, pangolins, and dogs [23] . other than humans, the coronaviruses have a big impact on the animal kingdom. animal coronaviruses can cause severe threat to their hosts; since 1984, an unrecognized infection massively spread among the swine population in europe, and later it was identified as porcine epidemic diarrhea coronavirus (pedv), which is derived from the porcine enteric coronavirus tgev [24] . in 2013, the same pedv in less than a year had caused a 100% fatality rate in piglets and rubbing out more than 10% of america's pig population [5, 25, 26] therefore, the total number of human coronaviruses identified has been increasing throughout the years. hcov-229e and hcov-oc43 are the first two discovered human coronaviruses in the 1960s, hcov-nl63 and hcov-hku1 human coronaviruses identified after the sars-cov outbreak in [22] . however, the story continues with the new identification of sars-cov-2 in december 2019 at the seafood wholesale market in wuhan, china. sars-cov-2 is the seventh member of the family of coronaviruses that infects humans and it is different from both mers-cov and sars-cov. although some infections caused by human coronaviruses are mild and associated with common colds, certain animal and human coronaviruses can make a severe impact on the human population. especially in young children, elderly people, and immune-deficient patients, the infections can be lethal [9, [27] [28] [29] . therefore, it is important to understand the mechanism of invading viruses to theirs hosts, transmission and prevention of these processes. the respiratory droplets are the main routes of transmissions; sars-cov-2 can be transmitted to a healthy person if he happens to have contact with the infected person or any of his belongings, including clothes, doorknobs, etc. studies have been reported that aerosol transmission (airborne transmission) is also possible for sars-cov-2, but there is no clear study on neonatal infections (mother to child) [30] [31] [32] [33] [34] [35] [36] . however, the transmission can be avoided by keeping a distance of 2 m between two people, wearing masks while going out, and the isolation of infected people. during the initial phase of the covid-19 outbreak, a dataset was obtained from 1099 patients with laboratoryconfirmed covid-19 from 552 hospitals in 30 provinces of china on january 29, 2020. only 2% of the patients had a history of contact with animals; more than three quarters have either visited the wuhan city or are residents. hence, the outbreak patterns or the source of infection could not be predicted from their study. the incubation period of the sars-cov-2 was from 1 to 12 days; however, the median incubation period was 4 days [32] . the most common symptoms are fever (43% on admission, and 88.7% during hospitalization), cough (67.8%), diarrhea (3.8%), and fatigue [32, 37] . the sars-cov-2 was detected in saliva, blood, sputum, and urine before the development of viral pneumonia, and some patients do not develop pneumonia at all. asymptomatic persons are potential sources of sars-cov-2 infection, which control the transmission dynamics of the current outbreak [32, 38] . the sars-cov-2 first identified in wuhan, china has spread all over the globe. as of august 20, 2020, more than 22 million confirmed infection cases and 0.8 million deaths had been reported across the world, including almost all the countries. the rate of infection or the average number of people getting infected by an individual (r0) was 2.75 in the case of sars pandemic in 2003. the r0 value of ebola 2014 was in the range of 1.51-2.53, and h1n1 influenza 2009 was from 1.46 to 1.48, and for mers, it was around 1. the sars-cov-2 r0 value was estimated to be in the range of 1.5-3.5. the comparison of r0 values of various coronaviruses shows that the difference is minimal. however, the difficulties arising for sars-cov-2 infection are due to the following: (1) basic properties of the viral infection and the infection periods are uncertain, (2) most of the infected individuals do not show symptoms, but are capable of spreading the infection, (3) changing susceptibility of the population in affecting the spread of infection remains unanswered. in addition, there are no control measures for this spread [39] . coronaviruses consist of four structural proteins: the nucleocapsid protein (n) forms the helical capsid to accommodate its genome. the whole structure is further surrounded by a lipid envelope, which is made of s (spike), e (envelope) and m (membrane) proteins. the membrane and the envelope proteins are needed for the virus assembly and the spike protein is for virus entry and host cell recognition [6] . the spike protein forms large protrusions (peplomers) on the virus surface (looks like the virus has crowns), and hence it is named as "corona" (corona is a latin word which means crown). it comprises three segments: (1) large ectodomain, (2) transmembrane domain and (3) intracellular tail. the receptor-binding subunits s1 and s2 are placed in the ectodomain region. during the infection, the s1 binds with the host receptor, and s2 fuses the host and viral membranes, thereby releasing the viral genome into the cell (fig. 1 ). the spike protein is a clove-shaped trimer with three s1 heads and a trimeric s2 stalk [6] . during viral infection, spike protein (~ 1300 amino acid residues) is cleaved by host proteases into receptor binding subunit s1 and membrane fusion subunit s2. during cell entry, the s1 subunit binds directly to the sugar receptors [40] and ace2 of the host cell surface, and the s2 subunit undergoes conformational changes and obtains post-fusion state [41] . during this state, the three pairs of heptad repeat region hr-n and hr-c in trimeric s2 form a six-helix bundle structure [42] . the buried hydrophobic fusion peptides become exposed and insert into the target host membrane. these fusion peptides and the transmembrane anchors are positioned at the end of a six-helix bundle structure, bringing the viral and host membranes to fuse [42, 43] . during this process, a large amount of energy is released, which accelerates the membrane fusion forward. along with this, receptor binding and low ph can also trigger this membrane fusion [6] . since the spike protein has a good binding affinity for sugar receptors of human cells, it uses them as a mechanism of cell entry [6] . notably, the sars-cov-2 has a higher affinity to human ace2 than the sars-cov virus strain. the ectodomain of the sars-cov-2 spike protein binds to the peptidase domain (pd) of ace2 with a k d (equilibrium dissociation constant) of ~ 15 nm [4] . spike protein priming is done by transmembrane protease serine 2 (tmprss2), which is also essential for the entry of sars-cov-2 [44] . generally, sars-cov enters host cells through endocytosis, where its spike protein is processed by cathepsin l and cathepsin b lysosomal proteases. the extracellular proteases, including elastase in the respiratory tract and tmprss2 on the surface of flung cells are also known to activate spike membrane fusion [6] . the viral entry to the host cells can be via: (1) the endocytic pathway and (2) non-endosomal pathway [45] (fig. 1b) . the endocytic pathway, especially clathrindependent endocytosis is extensively studied for sars-cov and mers-cov viral entry. since the sars-cov-2 also uses the same receptor as sars-cov, it is reported that sars-cov-2 also uses the same viral entry mechanism. wang et al. [46] reported clathrin-and caveolaeindependent endocytic pathways for sars-cov entry. despite the common use of endocytic pathway as a viral entry mechanism, the discrepancies about the same are unavoidable. thus, the exact nature of the viral entry is context-dependent, including the type of the virus and the host cells [47] . in addition, the macrophages can also act as a viral reservoir and support minimally for the sars-cov-2 entry and its replication. although the dendritic cells and other immune cells are not infected by sars-cov-2, they may serve as a transporter for the viruses, which is also responsible for the pathogenesis [48, 49] . the s1 subunit of sars-cov spike glycoprotein is predominantly composed of β-strand structures, composed of an n-terminal domain (ntd, residues 14-294) and three c-terminal domains (ctd1, residues 320-516; ctd2, residues 517-578 and ctd3, residues 579-663). the ntd is linked with ctd1 through the linker residues 295-319, where ctd1 acts as a potential rbd for sars-cov and binds explicitly with the ace2 receptor. gui et al. [43] reported that the sars-cov spike glycoprotein trimer could obtain different conformations, which are necessary for effective binding with ace2. all these conformations are termed based on the position of ctd1 of the spike glycoprotein (fig. 2) . the three spike glycoprotein monomers intertwine with each other and form densely packed homotrimer. the head portion of this trimer is taken place by ntds and ctd1s of s1 subunits, where the ctd1s are located at the center and the ntds are located outside of this triangular head. the s2 subunits represented as a stem for this trimer, which is further surrounded by ctd2s and ctd3s of the s1 subunits (fig. 2) . in the inactive state (fig. 2a, b) , s2 subunits (stem portion) are completely covered by the "down" position of ctd1s (head portion), which causes steric clashes for the binding between spike protein and ace2. in the active state (fig. 2c) , two ctd1s adapt the "down" conformation and one ctd1 rotates outward and obtains "up" conformation, which does not cover the s2 subunit and allows the interactions between spike protein and ace2. the "up" position also paves the way for the s2 subunit to expose and insert its fusion peptides into the host cell membrane [43] (fig. 2) . the sequence similarity between sars-cov-2 and sars-cov spike protein explains the possibility of having the same receptor ace2 in the host cell [14] . the sequence and the structural studies revealed the key residues, which are involved in spike-ace2 interactions. the key residue at position 493 in rbd of sars-cov-2 is gln, wherein sars-cov (479 is the corresponding residue in sar-cov) of civets and humans, it is lys and asn, respectively. since the residue 479 of rbd is near to the virus-binding hotspot residue lys31 of human ace2, the lys residue present in civet causes steric clashes and not favoring human ace2 receptor binding. however, the lys479asn mutation revealed that the asn present in 479 of human sars-cov enhances the viral binding with human ace2 receptors. the gln493 in sars-cov-2 rbd is compatible with the human ace2 receptor hotspot residue lys31, which explains its target cell identification [14] . similarly, the residue 501 in sars-cov-2 rbd is asn, wherein civet rbd, it is ser (487 is the corresponding residue in sars-cov), and for humans, it is thr. this residue also plays an important role in making interaction with the hot spot residue lys353 of receptor ace2. ser487thr mutation analysis shows encouraging results upon human ace2 receptor binding and plays a role in human-to-human transmission. thus, the interactions with lys353 will be favorable for threonine (human sars-cov) and asn (sars-cov-2) than serine (civet) [14] . the residues lue455, phe486, and ser494 in sars-cov-2 rbd (tyr442, leu472, and asp480 in human and civet sars-cov) are considered as important for the human ace2 receptor binding. tyr442 of human and civet sars-cov rbds shows unfavorable interactions with hotspot residue lys31 of human ace2; however, lue455 of sars-cov-2 provides favorable interactions. compared with leu472 of human civet sars-cov rbd, phe486 of sars-cov-2 provides better interaction with hotspot residue lys31 of human ace2. although ser494 provides positive support for the human ace2 receptor hotspot residue lys353, the sars-cov asp480 also makes favorable interaction with hotspot residue lys353. throughout this viral entry process, the lys31 and lys 353 of human ace2 receptors are termed as "hot spot" residues, which consist of a salt bridge buried in a hydrophobic environment and contribute critically for the virus and host cell receptor binding. thus, this key residue comparison of sars-cov-2 with the civet and human sars-cov explains how actively sars-cov-2 is choosing and binding with the human ace2 receptors, which is likely to cause the human-to-human transmission [14] . the heterogeneity of amino acids in ace2 receptors is also responsible for the wavering binding affinities between the host and the virus, which is associated with the viral transmission. however, the variants in the host cell receptors (ace2) can confer resistance against the invading pathogens. hussain et al. reported that the mutations s19p and e329g in ace2 disrupt the intermolecular interactions and have low binding affinity with viral spike protein. in addition to the variations in the viral spike protein, ace2 allelic variants can also drive the potential resistance against sars-cov-2 infection [50] . intrinsically disordered proteins (idps) and intrinsically disordered regions (idrs) also play major roles in a number of biological functions, including dna/rna binding, protein binding, and facilitating access to the binding sites between the binding partners [51, 52] . the rna-protein recognition often needs conformational changes in both rna and protein, which is facilitated by the structural flexibility of disordered residues [51] . also, the functional importance of intrinsically disordered regions in proteins includes transcription, translation, post-translational modifications, and cell signaling [51, 53] . the categorization of coronaviruses, based on the intrinsic disorder propensities, can represent useful identification for the viral life cycle and its pathogenicity. the idrs in sars-cov nucleocapsid protein comprise three segments, such as 1-44, 182-247 and 366-422 [54, 55] . the highly flexible intrinsic disordered linker region, which connects the ntd and ctd is rich in serine and arginine residues. an intrinsically disordered domain that flanks the ctd (c-terminal tail peptide) plays a significant role in dimer-dimer association in human coronavirus 229e (hcov-229e). likewise, the coronavirus hku1, has a partially disordered conserved linker loop (amino acids 428-587) structure [56] . hku1 s2 subunit also shows the presence of disordered residues at its protease cleavage site [57] . in sars-cov-2, the attachment of spike protein with the host cell is activated by the host cell enzymes trypsin, cathepsin l, furin and tmprss2 (fig. 1b) . the sequence comparison of sars-cov-2 against other lineage b betacoronaviruses shows that the unique amino acid pattern "rrar" is present at the s1/s2 junction of the spike protein, which is cleaved by the furin enzyme. however, the structure reported for sars-cov-2 spike protein (pdb code: 6vsb), shows that s1/s2 junction is in a disordered, solvent-exposed loop [4] . hence, it has been hypothesized that the unique amino acid sequence "rrar" present in sars-cov-2 is responsible for their effective transmission [58] [59] [60] . the binding with ace2 is governed by the intrinsically flexible receptor binding motif, and the binding interfaces along with the key residues are reported in the literature [61, 62] . in addition, based on our analysis (fig. 3) , the monomeric structure of the spike protein in the free form (pdb id: 6vsb; green color) [4] shows a number of missing residues in the structure. these missing residues of the spike protein attain a stable conformation upon binding to the ace2 receptor and hence, they are termed as disorderto-order transition (dot) residues (pdb id: 6lzg; light golden color). specifically, the regions, leu455 to pro491 and asn501 to val503 show disorder-to-order transitions [61] . these disordered-to-ordered residues are facilitating a better shape complementary and affinity between ace2 and spike protein. interestingly, the key residues responsible for transmission, and interaction with ace2 receptors (already discussed in section "key residues of sars-cov-2 and sars-cov") are overlapping with these mentioned disordered-to-ordered residues. based on these observations, the disorder-to-order conformational change is necessary to facilitate the spike protein binding with its receptor. thus, an in-depth analysis of these disordered residues will shed additional insights on the viral recognition and transmission mechanism. the studies by goh et al. [63] on 1918 h1n1 and h5n1 explain that it is very likely that disordered regions are important for host specificity and recognition, e.g., across species of birds. they also explained the changes created by disorder in crucial regions, increasing the virulence of both the h5n1 and the 1918 h1n1 viruses. therefore, the increasing reports for the intrinsically disordered regions in coronaviruses need to be pointed out. the importance and functional role of intrinsically disordered regions need to be thoroughly studied. this could identify additional targets for drugs to combat coronavirus through the disruption of their packing and assembly process. the receptor binding, along with its membrane fusion, is the initial and important step in the coronavirus infection and serves as primary targets for inhibiting the viral entry. the genome sequencing of sars-cov-2 and the comparison of the genomes of related virus proteins suggested the anti-hiv lopinavir plus ritonavir combination can be likely effective [15, 64, 65] . sars-cov-2 uses the ace2 receptor of at2 cells in the lung as its primary targets. since the viral entry is governed by receptor-mediated endocytosis, ap2-associated protein kinase 1 (aak1), a known regulator of endocytosis, can be a good target to interrupt the virus entry. the janus kinase inhibitor baricitinib, and its binding with the cyclin g-associated kinase (endocytosis regulator) is sufficient to inhibit aak1 [66, 67] . similarly, sunitinib and erlotinib, the oncology drugs, have been shown to inhibit viral infection of cells through the inhibition of aak1 [68] . however, these compounds bring serious side effects and cannot be considered for a safe therapy. fig. 3 structure of the monomeric spike protein (green)-ace2 receptor (blue) complex. the interface residues are shown in light golden. the disordered-to-ordered transition residues (leu455 to pro491 and asn501 to val503) have been marked in the figure based on the pathogenicity studies of sars-cov and mers-cov, an increased amount of inflammatory cytokines in serum is associated with the inflammation and extensive lung damage [69] . similarly, sars-cov-2 also has a high amount of il1b, ifnγ, ip10, and mcp1/ccl2, which lead to t-helper-1 (th1) activation. however, sars-cov-2 is shown to increase the t-helper-2 (th2) cytokines (il4 and il10) that are known to suppress inflammation, which differs from sars and mers-cov infection. in view of cytokines induced by sars-cov-2 and other sars-cov, mers-cov, corticosteroids were frequently used to reduce the inflammation-induced lung injury [2] . arabi et al. [70] examined the combination of interferon beta-1b, lopinavir, and ritonavir combination for the mers infection in saudi arabia. the antiviral nucleotide prodrug remdesivir showed a potent efficacy on mers-cov and sars-cov infections. since sars-cov-2 is an emerging virus, no effective treatment has been developed so far, yet the already available combination of lopinavir and ritonavir is being used [2] . since the sars-cov enters the cell through endocytosis and the lysosomal protease is priming the spike protein, targeting/inhibiting the endosomal acidification or lysosomal cysteine protease can block the sars-cov entry [6] . the view of the activation of sars-cov and sars-cov-2 by tmprss2 might also have therapeutic suggestions. a protease inhibitor, camostat mesylate, which is studied as a tmprss2 inhibitor and has been used to treat humans, is available and could be employed as a defense against the respiratory viruses [71, 72] . autophagy has been implicated in viral replications, which is also responsible for the formation of doublemembrane vesicles (dmv) in the host cells [73] . since the autophagosomes are degraded by lysosomes, inhibitors like lysosomotropic agents have been proposed for sars-cov-2 [74] . the antimalarial drugs hydroxychloroquine (hcq) and chloroquine (cq) have been demonstrated for sars-cov-2 antiviral activity. however, data to support the use of hcq and cq for covid-19 are incomplete [75] . these lysosomotropic agents are helpful for neutralizing the endosome-lysosomal acidic ph, thereby blocking protease activity and subsequently blocking the viral entry. however, how autophagy is implicated in the infection of covs is still under debate [47, 74, 76] . table 1 describes the different inhibitors used for blocking the viral entry. the most studied and the common pathway proposed for all coronaviruses is the endocytic pathway, so blocking that pathway is a big hallmark for treating the disease. identification of potential drugs for sars-cov-2 is a necessary task, so drug repurposing can also work for this scenario. the small molecules, which are already in clinical trials or used for some other diseases and the molecules from the expert's opinion are also listed in table 2 . the table is categorized into three groups: (1) compounds involving drug repurposing, (2) compounds in clinical and pre-clinical study, (3) compounds targeting the mechanism pathway. researchers in government and private sectors are making huge efforts to develop effective vaccines for sars-cov-2. the vaccine development approaches are mainly based on inactivated and attenuated viral protein particles, viral vectors and viral dna/rnas. a novel rna-based vaccine uses a part of the genetic code of spike protein mrna-1273 (clinicaltrials.gov: nct04283461) [139] . several other mrna-based vaccines by curevac (tübingen, germany), bnt162 by biontech (mainz, germany), pfizer (new york, ny, usa) and biontech mrna vaccine (mainz, germany) are in different stages of development [139, 140] . cansino biologics (tianjin, china), the company that developed the vaccine for ebola is also developing a vaccine named ad5-ncov for sars-cov-2. it is a spike protein-based vaccine that is undergoing phase i clinical trials in healthy individuals in wuhan china (clinicaltrials. gov: nct04313127) [141, 142] . the current status of the vaccines under development is available at the milken institute treatment and vaccine tracker: https ://milke ninst itute .org/sites /defau lt/files /2020-03/ covid 19%20tra cker%20032 020v3 -posti ng.pdf [141] . sars-cov-2 is a continuously growing life-threatening disease. this coronavirus has rapidly evolved and spread all over the world, having a mortality of more than 0.8 million lives so far. the exact origin and mechanism of attack and spatial distribution are still not completely explored. the viral and the host cell proteins assisting in invasion process can be a target for treating the infections. the available crystal structures and the binding mechanism proposed by other coronaviruses can help to study the sars-cov-2. the critical structural studies of the viral particles are also helpful to identify the drug targets. the positional changes (up and down conformation) of spike protein trimer determine the active to inactive state. thus, targeting these conformations and developing small molecules or peptides can also stop the viral entry. in addition, phylogenetic analysis and structural studies revealed that the hot spot residues, the role of gln493 in human ace2 receptor binding and the critical residue asn501 of rbd explain the human-tohuman transmission. the intrinsic disorder region and a precise furin-like cleavage site can be responsible for viral cycle and pathogenicity. however, in-depth studies are needed to address this issue, which may represent a potential antiviral strategy. the small molecules, which are in clinical trials, the drug compounds, which 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for entry into target cells identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus s protein-mediated membrane fusion using the split-proteinbased cell-cell fusion assay oxazolidinones inhibit cellular proliferation via inhibition of mitochondrial protein synthesis sars-cov-2 vaccines: status report the pandemic pipeline sars-cov-2/covid-19: viral genomics, epidemiology, vaccines, and therapeutic interventions the covid-19 vaccine development landscape acknowledgements we thank indian institute of technology madras, key: cord-256340-w4z5avld authors: bailer, sm; haas, j title: connecting viral with cellular interactomes date: 2009-07-24 journal: curr opin microbiol doi: 10.1016/j.mib.2009.06.004 sha: doc_id: 256340 cord_uid: w4z5avld genome-scale screens for intraviral and virus–host protein interactions and the analysis of literature-curated datasets are able to provide a novel, comprehensive perspective of viruses, and virus-infected cells. until now, large-scale interaction screens were predominantly performed with the yeast-two-hybrid (y2h) system; however, alternative high-throughput technologies detecting binary protein interactions or protein complexes have been developed. although many of the previous studies suffer from a rather poor validation of the results and few biological implications, these technologies potentially lead to a plethora of novel hypotheses. here, we will give an overview of current approaches and their technical limitations, present recent examples and novel developments. the y2h system as the standard assay for the evaluation of interactomes essentially all high-throughput approaches to identify binary protein interactions on a genome-scale currently rely on the gal4-based yeast-two-hybrid (y2h) system ( figure 1 ) developed in 1989 [1] . in principle, two proteins are fused to separately expressed and nonfunctional domains of the gal4 transcription factor, either the gal4 dna binding domain (bait) or the gal4 activation domain (prey). upon interaction of the two proteins of interest, the transcription factor activity is reconstituted in the yeast nucleus leading to the activation of one or several reporter genes. a major improvement was the introduction of a mating protocol in which pretransformed haploid yeast cells form diploids that carry both the bait and prey vector [2] . compared to the previously used transformation protocol, this novel strategy is easier to perform and allows automated screening and the crosscombination of a large number of pretransformed bait and prey pairs. another major improvement that further boosted genome-scale y2h approaches was the combination with the highly efficient gateway recombinational cloning system for the generation of larger clone collections [3] . over the last 10 years, the interactomes of several previously sequenced organisms like h. pylori [4] , c. jejuni [5] , m. tuberculosis [6] , p. falciparum [7] , t. pallidum [8] , s. cerevisiae [9, 10] , c. elegans [11] , d. melanogaster [12] , and humans [13, 14] could be generated using the y2h system. in most cases arrays were generated to test either individual or defined pools of open reading frames (orfs) for interaction with each other (matrix screens). alternatively, a number of individual baits were used to screen cdna libraries. more recently, several viral interactomes with the herpesvirus family being by far the largest group analyzed have been generated (refs. [15 ,16 ,17 ,18 ,19 ] and (e fossum et al., in revision). the reliability and the biological relevance of the y2h system in general have been challenged repeatedly. despite certain limitations the y2h system is used by the majority of groups because of its enormous efficacy and the data discussed in this review are all based on y2h screens as all currently published large-scale studies on intraviral or virus-host protein interactions are based on them. clearly, since based on the nuclear localization of a transcriptional reporter system, it is limited in the analysis of transcriptional activators and proteins localized to membrane compartments. however, although the proteins to be tested are forced into the yeast nucleus for interaction, no bias between nuclear or non-nuclear proteins was observed [20 ] . interestingly, while the yeast system is not expected to provide translational modifications comparable to the mammalian cell, it is nonetheless able to introduce and report modificationdependent interactions up to a certain extent. thus, the yeast cell offers an environment sufficiently natural for the analysis of protein interactions of other species [20 ] . a major concern of the y2h system particularly in its high-throughput application is the small overlap of identified interactions in comparative studies. this is exemplified by two s. cerevisiae proteome-wide y2h approaches in which the screening of 6000 gene products identified 682 (uetz-screen) and 843 (ito-core) binary interactions [9, 10] . however, only 19% of the uetz-screen and 15% of the ito-core interactions were found in the respective other screen [9, 10] . a similarly small overlap was observed in several independently performed genome-wide herpesviral protein interaction screens [15 ,16 ,19 ,21,22 ] (e fossum et al., in revision). recent large-scale efforts demonstrated by reinvestigating a set of random protein interactions of the s. cerevisiae and human interactomes that the low coverage is the result of low sensitivity rather than low specificity [20 ,23 ,24 ]. several validation protocols performed in parallel (see below) confirmed 25-30% of the y2h interactions suggesting that the y2h approach does not yield more false positives than other assays that detect binary interactions and is comparable in quality to literature-curated data [25 ] . several reasons are likely to account for the low sensitivity of y2h approaches [23 ] . first, only a fraction of all possible pairwise combinations are actually tested in a given screening situation. second, depending on the assay or screening protocol applied different spaces are screened. this is demonstrated by studies on hepatitis c virus (hcv) where the same set of proteins was screened by a yeast mating (imapi) as well as a transformation protocol (imapii) using two different human cdna libraries [18 ] . although performed in the same laboratory, imapi and imapii shared only 22 interactions indicating that different screening protocols, vectors, and yeast strains as well as the quality and composition of cdna libraries have a greater impact on the screening success than generally assumed. third, high-throughput approaches are often hampered by technical limitations that can be improved for example by multiple screenings of the same set of proteins. multiple sampling of the same search space allowed the identification of 80-90% while a single round revealed only 60% of all possible y2h interactions [23 ] . the orfeomes of five evolutionarily related herpesviruses were used to systematically address the low coverage of y2h screens by comparing the interactions identified in individual species followed by secondary methods [15 ] (e fossum et al., in revision). in the initial y2h screens 283 interactions of the 41 core orthologous proteins were observed and 113 interactions high-throughput technologies to detect protein interactions. graphic depiction of several approaches for the detection of binary protein interaction including the yeast-two-hybrid (y2h) system, the nucleic acid programmable protein array (nappa), the lumier (luminescence-based mammalian interactome mapping), the protein fragment complementation assay (pca), and the mappit (mammalian protein-protein interaction trap). multicomponent protein interactions can be analyzed by the tap (tandem affinity purification) of prota-tagged bait proteins followed by mass spectrometry of associated proteins. bait (x) and prey (y) proteins are indicated, fusion tags are shown in orange (ad: activation domain; dbd: dna binding protein; atg: start codon). were found in more than one species (e fossum et al., in revision). on the basis of 55 y2h interactions detected in kshv, 59 of 92 interactions predicted for the corresponding orthologs in hsv-1, mcmv, and ebv could be identified by coprecipitation. in conclusion, the low coverage of the y2h system -currently its major drawback -can be addressed either by technical improvements or the combination with other assays. to address the biological significance of an interaction identified by y2h, validation by one or more biochemical methods is required ( figure 1 ). technologies similar to the y2h, for example an assay based on closeness of two proteins if not their direct interaction likely leads to high confirmation rates, however be little complementary [20 ] . coexpression in mammalian cells of two proteins fused to distinct tags followed by immuno-coprecipitation or affinity-coprecipitation (cop) was successfully applied to validate several y2h studies [15 ,17 ] (e fossum et al., in revision). this method, however, is time consuming and thus not applicable to analyze large sets of interactions. recently, a tool-box for high-throughput validation of y2h interactions was developed (figure 1; [20 ]). expression of proteins on array-printed template dna using a coupled in vitro transcription-translation reaction (nucleic acid programmable protein array, nappa, [26] ) follows a principle similar to the cop. however, since performed in vitro under a defined but artificial environment it is technically challenging. a second method used on a rather large scale is the lumier (luminescence-based mammalian interactome mapping) pull-down assay where two proteins are expressed in mammalian cells. while the bait protein is expressed as a fusion to the protein a-tag or flag-tag to immobilize the complex, the prey protein is expressed as a fusion with luciferase allowing the detection of the coisolated prey [27] . protein fragment complementation assays (pca), for example the split-yfp system in which interacting bait and prey proteins are fused to yfp domains, reconstitute an enzyme or a fluorescent protein and generally do not require an enrichment of the interactors. thus, they might be more easily performed in an automated large-scale fashion [28] . finally, the mappit (mammalian protein-protein interaction trap) uses a bait protein fused to a hybrid erythropoietin-leptin receptor located to the plasmamembrane and a prey protein fused to gp130, which drives a signaling cascade resulting in the read-out of an endogenous transcriptional reporter [29] . proteomic approaches that are able to detect indirect interactions are complementary to the y2h system rather than confirmatory, and are therefore well suited to increase the coverage of a y2h interactome. in the tandem affinity purification (tap) approach individual proteins are fused to a cleavable tandem tag composed for example of protein a or g and a calmodulin binding peptide (cbp), and the isolated proteins present in the pulled-down complex are subsequently identified by mass spectrometry as done for yeast ( figure 1 ; [30] [31] [32] ). similarly, smaller subsets of human proteins have been analyzed in higher eukaryotic cells [33] [34] [35] . since the systematic tagging of chromosomal genes is currently not feasible in these cells, tagged proteins have been introduced in addition to the endogenous proteins and thus compete with them in the pull-down analysis. genetic systems are now available for many viral genomes (including large dna viruses), and systematic functional tap-tagging of viral proteins might be addressed in the near future. proteomic analyses of purified virus particles have recently been performed for a few virus species and may provide further evidence for the interaction between viral proteins, particularly in conjunction with y2h data (figure 2) [36, 37] . initial 'genome-wide' studies concentrated on intraviral protein interactions of small rna and dna viruses. however, owing to the rather small number of proteins and protein interactions identified (figure 3) , a detailed network analysis could not be applied (reviewed by [38] ). more recent approaches on intraviral interactomes include several members of the herpesvirus family [15 ,16 ,19 scheme of hsv-1 virus particle with protein interactions detected in a genome-wide y2h screen. capsid, tegument, and glycoproteins are indicated depending on their localization in the virus particle. proteins are colored according to their conservation, purple: conserved in all herpesviruses, red: in alpha herpesviruses, grey: in herpes simplex virus, pink: in alpha and gamma herpesviruses. first combined virus-host interactomes were investigated in hcv [18 ], ebv [16 ] , vzv and kshv (haas and collaborators). virus-host protein interactions have been included into several public databases, or novel databases specific for virus-host interactions like virhostnet have been set up [39] . in a systematic screen of 27 full-length proteins or domains of hcv against two human cdna libraries, 314 virus-host interactions were identified [18 ]. taking published interactions into account, this is the first analysis on rna viruses producing a large enough dataset to constitute a virus-human interaction network composed of 481 interactions involving 11 hcv and 421 human proteins. the most highly connected proteins were the ns3, ns5a, and core proteins with 214, 96, 76 interactors, respectively. intriguingly, the insulin, jak/ stat, and tgfb signaling pathways were particularly enriched, which might be consistent with the metabolic disorders observed during chronic hcv infection. focal adhesion complexes could represent a novel target of hcv suggesting a role of several hcv proteins in viral spreading, cell-cell interaction, and tissue reorganization. this analysis may thus help to identify potential new targets for hcv therapy. the largest high-throughput approaches involving viruses have been performed with herpesviruses. equipped with moderately large-sized genomes encoding a manageable but complex set of genes these viruses represent the ideal candidates for genome-wide y2h approaches followed by bioinformatical analysis. intraviral interactomes have been generated for several herpesviruses including the a-herpesviruses global view of the interaction of two herpesviruses, vzv and kshv, with the human proteome. two experimental y2h datasets were used to connect the two viral interactomes of vzv (red) and kshv (pink) into a predicted high-confidence human interaction network consisting of 10 636 edges between 3169 nodes. interactions between viral proteins are depicted in red or orange, interactions to cellular proteins in blue. of five herpesviral species including 1007 intraviral interactions a core set of highly conserved protein interactions has been identified. intriguingly, the interactions between the orthologous proteins were found to be conserved independent of their sequence homology. the topology of all herpesviral networks differed from cellular networks; however, it is difficult to judge whether this really reflects biological differences or artefacts caused by different setups used to evaluate them. in ebv [16 ] , hcv [18 ], vzv and kshv (haas and collaborators), and kshv (haas and collaborators) viral proteins tend to interact with highly connected cellular proteins, which could be a general hallmark of many pathogen-host interactions [40 ] . the datasets available to date are hampered by the low coverage of the y2h screens performed, which makes it difficult to draw general biological conclusions. to reveal significant differences how different pathogens interact with the host proteome, protein interaction data with a considerably higher coverage of the screening space have to be generated. to provide an example of the power of this approach, a comparison of the five herpesviral core networks identified the highly connected hsv-1 ul33 ortholog (vzv orf25, mcmv m51, ebv bfrf4, and kshv orf67.5), which interacted with 14 tegument proteins (figure 2 , e fossum et al., in revision). interestingly, 11 out of 14 interactions were found in more than one species, a majority of which was confirmed by cop. in addition, the interaction between ul33 orthologs and the hsv-1 ul31 orthologs (vzv orf27, mcmv m53, ebv bflf2, and kshv orf69) which in turn were found and previously published to interact with the hsv-1 ul34 orthologs (vzv orf24, mcmv m50, ebv bfrf1, and kshv orf67) was highly conserved [41] [42] [43] [44] [45] . likely these proteins form a large protein complex that mediates budding of capsids at the inner nuclear membrane of the host (figure 4 ). the role of ul33 in dna packaging and cleavage could point to a role in connecting capsid maturation with nuclear egress [46] . this is in line with these proteins being crucial for capsid formation and nuclear egress [41, [46] [47] [48] [49] . the identification of this protein complex thus demonstrates the potential of systems virology to reveal targets for alternative herpesviral therapies. this could be achieved by peptides or other small molecules introduced to interfere with targeting or assembly of the complex components. nuclear egress of hsv-1 capsids. herpesviral capsids are formed in the host nucleus and released to the cytoplasm by budding through the nuclear envelope. primary envelopment at the inner nuclear membrane (inm) requires the membrane anchored ul34/ul31 family of proteins. the ul33 protein family interacts with this nuclear egress complex and may connect capsid packaging and nuclear egress (er: endoplasmic reticulum; inm: inner nuclear membrane; onm: outer nuclear membrane; npc: nuclear pore complex). these could be confirmed by cop. the intraviral interaction network revealed network parameters similar to herpesviruses [15 ] and the combined published and experimental virus-host interactions suggest that sar-s-cov targets host functions like apoptosis, cell communication, and signaling. large-scale protein interaction screens are able to provide large amounts of novel and unbiased data, but the extraction of biological implications from these screens is difficult, particularly if they are based on a technology with a rather low coverage as the y2h system. in the near future, however, improved 'deep' screening technologies will lead to more comprehensive interaction maps of both intraviral and virus-host protein interactions, which, in combination with other genome-scale technologies like sirna knock-down screens, transcriptional profiling, and spatial/temporal distribution studies may allow to setup improved models of virus-infected cells. the comparison of different viruses and, possibly, other pathogens may allow the identification of common strategies for infection and replication used by divergent pathogen groups, and the identification of targets for novel broad-spectrum antibiotics. on the other hand, it might reveal strategies that are specific for individual pathogens and help explain the characteristics of the infection with this particular pathogen. in combination with a genetic profiling of the infected host this approach will be even more powerful and might potentially lead to a step change in our understanding of viral infections. a tool-box of high-throughput methods was tested for the validation of y2h interactions including the mammalian protein-protein interaction trap (mappit), the luminescence-based mammalian interactome (lumier), the yellow fluorescent protein (yfp) protein complementation assay (pca), and the nucleic acid programmable protein array (nappa). this analysis aims at defining a confidence score for binary proteinprotein interactions. these two sets of data define high-quality binary interaction maps of yeast and human protein networks. in particular, these datasets suggest that the y2h data are more reliable than generally believed and that the y2h screens currently performed suffer from low sensitivity rather than low specificity. moreover, the distinct nature of binary versus cocomplex derived interactions is pointed out. these two sets of data define high-quality binary interaction maps of yeast and human protein networks. in particular, these datasets suggest that the y2h data are more reliable than generally believed and that the y2h screens currently performed suffer from low sensitivity rather than low specificity. moreover, the distinct nature of binary versus cocomplex derived interactions is pointed out. curation of protein interactions from literature is a method applied to obtain comprehensive protein networks. however this analysis critically evaluates the current literature curation and shows that it is more errorprone and possibly of lower quality than generally assumed. a novel genetic system to detect proteinprotein interactions toward a functional analysis of the yeast genome through exhaustive two-hybrid screens gateway recombinational cloning: application to the cloning of large numbers of open reading frames or orfeomes the proteinprotein interaction map of helicobacter pylori a proteome-wide protein interaction map for campylobacter jejuni mycobacterium tuberculosis interactome analysis unravels potential pathways to drug resistance a protein interaction network of the malaria parasite plasmodium falciparum the binary protein interactome of treponema pallidum -the syphilis spirochete a comprehensive analysis of protein-protein interactions in saccharomyces cerevisiae a comprehensive two-hybrid analysis to explore the yeast protein interactome a map of the interactome network of the metazoan c. elegans a protein interaction map of drosophila melanogaster towards a proteome-scale map of the human protein-protein interaction network a human protein-protein interaction network: a resource for annotating the proteome herpesviral protein networks and their interaction with the human proteome see annotation to epstein-barr virus and virus human protein interaction maps in revision) this study provides important insights into intraviral interactions of herpesviruses. moreover, it presented the first virus-host interactome of a member of the herpesvirus family self-assembling protein microarrays highthroughput mapping of a dynamic signaling network in mammalian cells capturing protein interactions in the secretory pathway of living cells design and application of a cytokine-receptor-based interaction trap functional organization of the yeast proteome by systematic analysis of protein complexes systematic identification of protein complexes in saccharomyces cerevisiae by mass spectrometry global landscape of protein complexes in the yeast saccharomyces cerevisiae a physical and functional map of the human tnf-alpha/nf-kappa b signal transduction pathway an efficient tandem affinity purification procedure for interaction proteomics in mammalian cells large-scale mapping of human protein-protein interactions by mass spectrometry viral proteomics: global evaluation of viruses and their interaction with the host comprehensive characterization of extracellular herpes simplex virus type 1 virions from orfeomes to protein interaction maps in viruses virhostnet: a knowledge base for the management and the analysis of proteome-wide virus-host interaction networks the landscape of human proteins interacting with viruses and other pathogens this paper integrates protein interaction networks of several pathogens and identifies certain cellular processes that participate in interactions with different pathogen groups. this global view on infectious diseases may be a first step toward the identification of multiviral or multibacterial treatments budding events in herpesvirus morphogenesis the epstein-barr virus bfrf1 and bflf2 proteins interact and coexpression alters their cellular localization characterization and intracellular localization of the epstein-barr virus protein bflf2: interactions with bfrf1 and with the nuclear lamina deletion of epstein-barr virus bflf2 leads to impaired viral dna packaging and primary egress as well as to the production of defective viral particles identification and characterization of the product encoded by orf69 of kaposi's sarcoma-associated herpesvirus temperature-sensitive mutations in the putative herpes simplex virus type 1 terminase subunits pul15 and pul33 preclude viral dna cleavage/packaging and interaction with pul28 at the nonpermissive temperature comprehensive mutational analysis of a herpesvirus gene in the viral genome context reveals a region essential for virus replication bfrf1 of epstein-barr virus is essential for efficient primary viral envelopment and egress functional domains of murine cytomegalovirus nuclear egress protein m53/p38 the work of the authors has been supported by grants provided by baygene (bayerisches staatsministerium fü r wissenschaft, forschung und kunst jh), dfg (sfb 576 jh, ba 1165/5-1 sb/jh), bmbf (ngfn+ jh), mrc (g050145, jh), and scottish funding council (ichair, jh). key: cord-018265-twp33bb6 authors: becker, pablo d.; guzmán, carlos a. title: community-acquired pneumonia: paving the way towards new vaccination concepts date: 2007 journal: community-acquired pneumonia doi: 10.1007/978-3-7643-7563-8_10 sha: doc_id: 18265 cord_uid: twp33bb6 despite the availability of antimicrobial agents and vaccines, community-acquired pneumonia remains a serious problem. severe forms tend to occur in very young children and among the elderly, since their immune competence is eroded by immaturity and immune senescence, respectively. the main etiologic agents differ according to patient age and geographic area. streptococcus pneumoniae, haemophilus influenzae, respiratory syncytial virus (rsv) and parainfluenza virus type 3 (piv-3) are the most important pathogens in children, whereas influenza viruses are the leading cause of fatal pneumonia in the elderly. effective vaccines are available against some of these organisms. however, there are still many agents against which vaccines are not available or the existent ones are suboptimal. to tackle this problem, empiric approaches are now being systematically replaced by rational vaccine design. this is facilitated by the growing knowledge in the fields of immunology, microbial pathogenesis and host response to infection, as well as by the availability of sophisticated strategies for antigen selection, potent immune modulators and efficient antigen delivery systems. thus, a new generation of vaccines with improved safety and efficacy profiles compared to old and new agents is emerging. in this chapter, an overview is provided about currently available and new vaccination concepts. the mucosa of the human respiratory tract represents a primary target for a large number of microbial pathogens. typically, colonization is an asymptomatic process, resulting from the interplay between bacterial factors and host clearance mechanisms. clinical illness may result from either the local release of bacterial toxins or the systemic dissemination of the pathogen after breaching the mucosal barrier. in the course of respiratory infections adaptive immune responses could be significantly impaired. this might lead to more severe forms of disease or to super-infections, which in turn complicate the clinical management of the patient. the most severe forms of respiratory infection tend to occur in very young children and among the elderly, in whom immune competence is eroded by immaturity or immunesenescence, respectively. in addition, patients who are immunocompromised, as a result of disease or therapeutic interventions, have the greatest risk of developing a fatal infection. despite the availability of new antimicrobials and effective vaccines, community-acquired pneumonia remains a common and serious illness. in fact, it is a leading contributor to the nearly 4 million deaths occurring each year due to respiratory infections, especially in children from developing countries [1, 2] . the main causative agents of pneumonia differ according to the patient age and the geographic area. in addition, there are relatively few comprehensive studies on the specific aetiology of pneumonia [2] due to (i) overlaps in the clinical manifestations of the different syndromes, (ii) difficulties in establishing the precise aetiology, and (iii) frequent occurrence of co-infections. however, streptococcus pneumoniae, haemophilus influenzae, respiratory syncytial virus (rsv), and parainfluenza virus type 3 (piv-3) have been identified as the main agents responsible for acute respiratory infections in children, whereas influenza virus related pneumonia is the leading cause of disease-related deaths in the elderly. in addition, the availability of new and more sensitive diagnostic tests have contributed to the identification of hitherto unknown lower respiratory pathogens, such as the human metapneumonovirus (hmpv) and novel coronaviruses causing the severe acute respiratory syndrome (sars). significant efforts have been invested in the last two decades to develop new diagnostic tools, to elucidate the molecular mechanisms of microbial pathogenesis and to understand host clearance mechanisms. this resulted in an improved knowledge on host responses to infection and immuno-pathogenesis, which in turn have facilitated the establishment of new prophylactic and therapeutic interventions. however, despite our accomplishments in vaccine development, there are many pathogens for which vaccines or adequate therapies are not available or the existent ones are suboptimal. the main approach applied for vaccine development has radically changed in recent years. whole cell vaccines are systematically being replaced by subunit vaccines, in which purified antigens or their coding genes are exploited in combination with new adjuvants and/or delivery systems. as a result, many of the vaccines under development will exhibit consistently improved stability, safety and efficacy profiles. they will also be amenable for mucosal administration, thereby mimicking natural infections. influenza a viruses are the most commonly responsible for severe respiratory illness in humans, followed by influenza b. the population's susceptibly to infection is renewed annually, because of the rapid antigenic variation of this virus. the antigenic variation is due to the accumulation of point mutations in the two major surface glycoproteins of the virus, haemagglutinin (ha) and neuraminidase (na). this can lead to an antigenic drift of the virus, which often leaves current influenza vaccines outdated and ineffective. antigenic shift can also occur due to the segmented nature of the viral genome that favours the emergence of re-assorted strains, in which an entire glycoprotein can be acquired from a different animal influenza virus. both types of variation represent a critical bottleneck for the establishment of a robust vaccination strategy against influenza. in fact, when an influenza virus with the capacity to spread from person-to-person and a complete new glycoprotein subtype suddenly emerges, a worldwide pandemic outbreak can result [3] . the earliest vaccines against influenza were whole cell vaccines obtained in the 1940s by inactivating viruses grown in the allantoic cavity of embryonated chicken eggs with formalin. while contemporary inactivated influenza vaccines are still produced in embryonated eggs, improvements in manufacturing have resulted in a highly purified and less-reactogenic detergentsplit product. three viral strains are selected on the basis of the previous year's surveillance data on the most prevalent subtypes, therefore, vaccine composition may vary from year-to-year. vaccination has a high benefit:cost ratio, since influenza-related illness (e.g., hospitalizations and deaths) are effectively prevented [1] . the world's total vaccine production is approximately 300 million doses, with a maximum capacity of 900 million doses. however, the world health organization (who) estimates that there are about 1.2 billion people at high risk for severe influenza outcomes (e.g., elderly over 65 years of age, infants, health care workers, children and adults with underlying cardiopulmonary disease). furthermore, the global infrastructure would not be able to handle the timely manufacturing and distribution of a vaccine for a pandemic outbreak [4] . one alternative would be to lower the quantity of antigen per dose and add adjuvants to the vaccine formulation, but this needs to be tested in clinical trials [5] . another solution would be to improve current vaccine production technologies (i.e., egg-derived vaccines). however, there is the limited number of egg producers and viral strains can emerge, which could not be easily adapted to embryonated eggs. to overcome these problems, several pharmaceutical companies have embarked themselves on projects for the development of vaccines produced by growing the virus on cell lines. the influenza virus can be adapted to grow on a variety of mammalian cell lines, including vero, per.c-6, and madin-darby canine kidney (mdck) cells [6] [7] [8] . this strategy would also improve the possibility of up-scaling vaccine production in face of a pandemic spread. alternatively, it would be possible to develop a vaccine against any influenza virus, such as the avian h5n1 strain, by using reverse genetics techniques [9] (see below in advances in vaccinology). cold adaptation was found to be a reliable and efficient procedure for the derivation of live attenuated viral vaccine strains for humans. cold-adapted (ca) virus strains can grow in primary chick kidney cells or embryonated eggs at 25-33°c, however, they exhibit a reduced replication at 37°c. the process of genetic re-assortment with the transfer of the six internal genes from a stable attenuated ca master donor strain of influenza a or b to the new prevailing wild-type epidemic strain has been used to generate attenuated cold-reassorted vaccines with the proper level of attenuation, genetic stability and immunogenicity, which show low or absent transmissibility [10] . medimmune and wyeth have developed along these lines a trivalent live ca vaccine (flumist) for intranasal spray delivery, which was licensed in 2003. in contrast, to parenterally-administered vaccines, this formulation triggers immune responses resembling those observed after natural infections [11] . despite the moderate hemagglutination-inhibiting antibody titres observed in vacinees, flumist showed 92% efficacy over a 2-year period in children, including protection against antigenic variants that circulated during the second year [12] [13] [14] . this ca vaccine also stimulated the production of nasal iga, as well as t-cell and interferon responses [15] . the cell-mediated immunity against virus matrix and nucleoprotein antigens may favour viral clearance and early recovery from illness [3] . the advisory committee on immunization practices has recommended its use only in persons from 5 to 49 years of age, since side-effects were observed in young children (wheezing, nasal congestion) and there are no data available for elderly [16, 17] . despite its remarkable genetic stability, this vaccine has to be kept at -18°c. thus, a new heat stable derivative has recently been developed, which showed good efficacy in clinical trials [1] . a live vaccine based on a master virus strain developed at the institute of applied microbiology (austria) by growing wild influenza virus in vero cells at 25°c was also demonstrated to be safe, well-tolerated and immunogenic after intranasal immunization in young adults [18]. a number of subunit-or dna-based vaccines are also in various stages of development. an influenza vaccine formulated in virosomes has been commercialized by berna biotech (inflexal ® v); it contains the surface spikes of the three currently circulating influenza virus strains inserted in vesicle membranes of the three corresponding virus types (for more details see section "pseudoviruses as antigen delivery systems") [19] . this company has also developed a virosome-based nasal formulation. however, it was withdrawn from the market due to the presence of sideeffects (i.e., bell's palsy), which was assumed to be linked to the presence of the escherichia coli heat labile toxin (lt) as adjuvant. two companies, yeda and bionvax, are also developing a peptide-based influenza vaccine for nasal administration, which showed protective efficacy in humanized mice [1] . a subunit vaccine containing recombinant ha protein produced using a baculovirus system was successfully tested in a phase ii trial in 64 to 89-year-old volunteers. an epidermal dna-based influenza vaccine, which contained the ha gene from a/panama/2007/99 delivered by particle-mediated epidermal delivery was also tested in humans by powderject [20] . serum haemagglutination-inhibition antibody responses were observed in volunteers receiving a single dose of 1, 2 or 4 g of dna, with the strongest and most consistent responses in subjects vaccinated with the highest dosage. some immunization approaches aim at the development of a universal vaccine with a broad spectrum of protective activity against different influenza strains [21]. among them, the use of the highly conserved transmembrane m2 protein of the virion can be mentioned. a recombinant particulate vaccine has been engineered by genetically fusing copies of the m2 to the hepatitis b core antigen (hbc). the m2-hbc fusion protein spontaneously assembled into virus-like particles (vlp), which provided complete protection against a lethal challenge with influenza virus a in mice [22, 23] . promising results were also obtained after vaccination with a m2 peptide conjugated with a neisseria meningitidis outer membrane protein complex (ompc) in monkeys [24] . the human piv (hpiv) consist of four serotypes, with hpiv-3 being the second leading cause of bronchitis and pneumonia in infants. no vaccine has been licensed to date against piv, however, several approaches are currently under investigation. the initial attempts to provide protection by using vaccines based on formalin-inactivated viruses failed. subsequent work demonstrated that the glycoproteins haemagglutininneuraminidase (hn) and f, which are responsible for virus attachment and fusion, are able to stimulate the elicitation of neutralizing antibodies in animals. however, their poor immunogenicity in naïve subjects led to the currently favoured approach, which is based on the use of live attenuated piv. live attenuated piv vaccines have been developed from both human and bovine strains, which are amenable for delivery by the intranasal route. candidate vaccines should be able to replicate and induce a protective immune response in young infants, even in the presence of maternally acquired antibodies. two main attenuated strains have been studied in detail. one is the hpiv-3 strain cp45, which was selected after 45 passages of the virus in african green monkey cells at low temperature. the other is a bovine piv (bpiv)-3 strain, which is antigenically related to the hpiv-3, and replicates poorly in humans. both cp45 and bpiv-3 have been evaluated in phase i/ii trials in sero-positive and sero-negative children and in young infants. they were found to be over-attenuated in sero-positive children, but immunogenic in sero-negative children and infants [25] . however, the magnitude of the anti-hn response was lower in children who received the bpiv-3 vaccine [25]. this prompted the engineering of chimeric bovine/human piv-3 candidates (e.g., hpiv-nb strain in which the human nucleocapsid is replaced by the bovine counterpart, or a bpiv-3 strain that expresses the f and hn proteins of hpiv-3). attenuated, chimeric viruses that contain piv-3 cp45 internal genes with the f and hn genes from either piv-1 or piv-2 have also been tested in hamsters [26] . berna biotech is also developing a virosomal formulation of the piv-3 [1] . using the successful approach of the influenza vaccine, a formalin-inactivated candidate against the respiratory syncytial virus (rsv) was tested in children in the 1960s. the consequence was the hospitalization of 80% of vaccinees and two deaths [1] . moreover, vaccinated children also suffered more severe disease on subsequent exposure to the virus, as compared to unvaccinated controls [27] . this demonstrated that the elicitation of a strong immune response is not sufficient to confer protection against disease, and can even lead to immuno-pathological reactions. thus, it is essential to stimulate the "right" type of immune response. in the particular case of rsv, host responses play an important role in the pathogenesis of the disease, thereby making the development of a preventive vaccine extremely difficult. in addition, naturally acquired immunity to rsv is neither complete nor long-lasting, and recurrent infections often occur [28] . however, older children and adults are usually protected, suggesting that protection against severe disease develops after several consecutive infections. passive immunization with rsv-neutralizing immune globulins was also shown to prevent rsv infection in newborns with underlying cardiopulmonary disease [29] . this demonstrates that antibodies play a major role in protection against this disease, whereas t-cell immunity targeted to internal viral proteins appears to contribute to clearance. although live attenuated vaccines seem to be preferable for immunization of naïve infants, subunit vaccines may be of choice for elderly, high-risk children and pregnant women. candidate subunit vaccines based on purified f proteins (pfp-1, -2 and -3) were demonstrated to be safe and immunogenic, even during pregnancy [30] . maternal immunization using a pfp-based vaccine could be an interesting strategy to protect infants younger than 6 months of age [25] . however, no significant protection was reported in a phase iii trial performed on children 1-12 years of age with cystic fibrosis after vaccination with a subunit vaccine based on pfp-3 [31] . a formulation based on surface glycoproteins f and g together with the virion matrix protein m from rsv-a was tested in healthy adult volunteers in the presence of either alum or polyphosphazene as an adjuvant. short-live neutralizing antibody responses to rsv-a and rsv-b were detected in 76-93% of the vaccinees, suggesting that annual boosting will be needed [32] [33] [34] . the central domain of the g protein of rsv-a is relatively conserved among viruses from the groups a and b. thus, a recombinant vaccine candidate, bbg2na, was developed by fusing the g2na domain to the albumin binding region of streptococcal protein g. this candidate was shown to be moderate immunogenic in adult human volunteers, but its clinical development was interrupted due to the appearance of purpura in vaccinees [1] . the main two difficulties associated with the generation of live attenuated vaccines against rsv are over-or under-attenuation of the virus and limited genetic stability. temperature-sensitive (ts), ca and cold-passaged (cp) mutant viral strains have been generated. despite the attenuation shown in adults and sero-positive children, cpts mutants still caused moderate congestion in the upper respiratory tract of sero-negative infants (1-2 months old) [35] . recombinant rsv vaccines with deletions in non essential genes (e.g., sh, ns1 or ns2), which also carry cp and ts mutations in essential genes are currently being evaluated [1] . through recombinant dna technology chimeric viruses were engineered, which contain the genes of hpiv-3 surface glycoproteins f and nh together with those of rsv glycoproteins f and g in a bpiv-3 genetic background. one of these candidates was found to be attenuated and able to induce the elicitation of immune responses against both hpiv-3 and rsv in rhesus monkeys [36] . similarly, a bpiv-3 genome was engineered to express hpiv-3 f and hn proteins and either native or soluble rsv f protein [37] . the resulting strain, which induced rsv neutralizing antibodies and protective immunity against rsv challenge in african green monkeys, needs to be tested for safety and efficacy against rsv and piv-3 in infants. this emerging disease was originally described in the guangdong province of china in 2002. even when the global outbreak of sars was under control in 2003, new infections were reported in persons who had contacts with animals in 2003 and 2004 [38] . the typical sars-cov-like virus is not transmitted from animals to humans. however, under certain conditions the virus can evolve into the early human sars-cov, which has the ability to be transmitted from animals to humans or even humans to humans, thereby leading to localized outbreaks of mild disease. the early human sars-cov, under selective pressure in humans, may further evolve into the late human sars-cov, which can cause local or global outbreaks of typical sars [39] . sars can be easily grown in cell cultures [38] . thus, there is an urgent need for vaccines, not only to prevent naturally occurring epidemic outbreaks, but also as a tool against the threat of biological weapons. several structural proteins are expressed by sars-cov, including nucleocapsid, envelope and spike (s) proteins [38] . the latter is a type i trans-membrane glycoprotein, which is responsible for virus binding, fusion and entry, and being the major target of neutralizing antibodies [38, 40] . the extracelullar domain of the s protein consists of two subunits, s1 and s2 [40] . the s1 subunit possess a receptor-binding domain (rbd), which is responsible for viral binding to one of its receptors [41, 42] . vector-based vaccines expressing the s protein, as well as dna vaccines encoding full-length s protein have been assessed in preclinical studies [43, 44] . when modified vaccinia virus ankara (mva) coding for full-length s protein was administered by either intranasal or intramuscular route, neutralizing antibodies were elicited [45] . however, vaccination of ferrets resulted in liver damage after challenge, raising some concerns about the safety of this approach [46] . vaccines formulated using different synthetic peptides encompassing linear b cell epitopes from the s protein, which were identified using sera from convalescent patients, stimulated high antibody titres. nevertheless, none of them triggered the elicitation of neutralizing activity. on the other hand, some studies demonstrated that although antibodies against s protein of the late sars-cov (urbani strain) exhibit neutralizing activity, they can also enhance infection by an early human sars-cov isolate (gd03t0013) and the civet sars-cov-like viruses. a derivative of the s protein with a truncation at amino acid (aa) 1153 fails to cause antibody dependent enhancement of infection, but retains the ability to induce neutralizing antibodies. these findings suggest that the elimination of the putative heptad repeated 2 (hr2, aa 1153-1194), which is implicated in viral fusion, might abrogate the stimulation of virus infection-enhancing antibodies [47, 48] . the use of the nucleoprotein of the coronavirus in a dna vaccination protocol also led to the stimulation of a protective response [49] . in contrast, protection was not achieved when a recombinant piv-3 expressing the nucleoprotein alone or together with the matrix protein was used [50] . this demonstrates that the selection of the delivery system and immunization strategy play a critical role in vaccine efficacy. the human adenoviruses are divided into six subgroups (a-f). the adenovirus can cause large-scale epidemics of acute respiratory disease, and dissemination is especially favoured under conditions in which persons are housed communally. the subgroup a viruses, such as ad31, have been associated with pneumonia in immunocompromised patients. neutralizing antibodies directed against the capsid (hexon and fiber proteins) seems to be the main effector mechanism to prevent re-infections by adenovirus [3] . until 1998, military recruits in usa were administered enteric-coated capsules containing live viruses from the serotypes 4 and 7. the virus, which was not attenuated if delivered by respiratory route, was able to replicate in the gastrointestinal tract without causing disease, thereby stimulating protective responses in the respiratory tract [51] . when the vaccine went out of production, outbreaks of respiratory diseases caused by adenovirus reemerged among the military recruits [3] . since serotypes 1, 2, 3 and 5 cause the 80% of adenovirus associated respiratory disease in young children, the development of a tetravalent vaccine similar to the above mentioned might solve the problem in children [52] . however, the implementation of a vaccine (live or attenuated) against adenovirus should be carefully evaluated, since recombinant adenoviruses are proposed both as vaccine vectors and as tools for the transfer of foreign genes in gene therapy protocols. polysaccharide-based vaccines against s. pneumoniae in 1945, macleod et al. [53] reported the protective efficacy of a capsular polysaccharide (ps) vaccine in military personnel during an outbreak of pneumococcal pneumonia. the immunization with purified ps showed a drastically reduced reactogenicity, in comparison with the previously used inactivated whole cell vaccines. this was a major breakthrough, not only in terms of safety, but also because it demonstrated that a specific virulence factor can be purified and effectively implemented for the prevention of an infectious disease, thereby paving the road for modern non toxoid-based subunit vaccines. although the serological correlates of immunity are poorly defined, type-specific anti-capsular antibodies are responsible for protective immu-nity. however, immunity is serotype specific, rendering extremely difficult the development of a universal vaccine. this is in part due to the elevate number of serotypes, the regional variations in dominant serotypes and the lack of updated sero-prevalence data for certain regions. these problems have been partially solved by the use of ps-based polyvalent vaccines. the currently licensed formulations contain 23 serotypes of s. pneumoniae, which cover approximately 90% of serious pneumococcal disease, but only in western industrialized countries. relatively good antibody responses (60-70%) are elicited in healthy adults 2-3 weeks after a single intramuscular or subcutaneous immunization [54] . unfortunately, they are poorly immunogenic in children aged less than 2 years, in immune compromised individuals (e.g., aids patients) and in elderly people with concomitant disease, and they do not induce good immunological memory. randomized controlled trials in healthy elderly and young men also failed to show a beneficial effect against pneumonia [55] . however, vaccination is recommended for healthy people over 65 years of age to confer protection against invasive disease [54] . ps-based vaccines can be also used in pregnant women to stimulate the production of antibodies, which are transferred to the foetus via the placenta or to the newborns by breast-feeding. however, it is still a matter of controversy whether maternal vaccination can indeed protect newborns against pneumococcal infections [56] . the second generation of ps-based conjugate vaccines stimulates stronger antibody responses, even in infants, young children and immune deficient individuals, as well as immunological memory. these vaccines also suppress nasopharyngeal carriage of the pathogen and reduce bacterial transmission in the community leading to herd immunity, which adds considerable value to their implementation. the introduction of these vaccines in usa in 2000 resulted in a dramatic decline in the rates of invasive pneumococcal disease [1, 57, 58] . a significant reduction in the incidence rates among non vaccinated individuals was also observed as a result of herd immunity [59, 60] . however, the licensed seven-valent vaccine does not contain some of serotypes that cause severe disease in developing countries (i.e., serotypes 1 and 5). new conjugate vaccines including more serotypes, such as the ninevalent vaccine (wyeth) and two 11-valent vaccines (glaxosmithkline and sanofi-pasteur), should provide better serotype coverage. new approaches to develop protein-based subunit vaccines against s. pneumoniae are currently being pursued by different research groups. this is expected to enable the generation of a universal vaccine conferring protective immunity against a large number of serotypes, as well as to avoid the complexity of manufacturing a conjugate vaccine [61] . there are different pneumococcal candidate antigens, such as the pneumolysin, neuraminidase, autolysin, pneumococcal surface protein a (pspa) and adhesin a (psaa), which are in an early phase of clinical development [1] . in addition, several promising candidates have been identified, which are currently being tested in pre-clinical experimental models [1] . among them, the two iron uptake abc transporters of s. pneumoniae (piaa and piua), which trigger protective immunity against invasive pneumococcal disease in mice. through the screening of s. pneumoniae genomic expression libraries with sera from convalescent patients, bacterial surface proteins were identified (e.g., bvh-3 and bvh-11) that promote the elicitation of protective anti-pneumococcal antibodies in mice [1] . a recombinant hybrid protein, bvh3/11v, has successfully been tested in toddlers and elderly volunteers. this candidate vaccine should be able to trigger serotype-independent responses, since the bvh3 and bvh11 antigens are common to all serotypes of s. pneumoniae. the major obstacle for developing an effective vaccine against h. influenzae capsular ps was related to the inherently poor immunogenicity of this t-cell-independent antigen. antibody responses against ps are age-related, with extremely poor immunogenicity in infants during the first 18 months of life. unfortunately, this age group exhibits the highest risk for invasive infections caused by h. influenzae. a ps-based vaccine against the h. influenzae type b (hib) was licensed in the united states in 1985, for children more than 18 months old [62, 63] . the protective efficacy after licensure studies showed the inefficacy of this vaccine not only in infants, but also in older children [64] . this problem was solved by the generation of a conjugate hib vaccine. to this end, the hib ps (i.e., polyribosylribitol phosphate; prp) was covalently linked to an immunogenic carrier protein, thereby leading to t-cell-dependent responses against the ps. different conjugate hib vaccines currently exist. these vaccines are hboc, prp-t and prp-omp, which make use of the mutant diphtheria toxin crm197, the tetanus toxoid and the outer membrane protein from group b n. meningitidis as carriers, respectively. all of them trigger similar immune responses at the recommended doses. however, the dynamic of the elicited response may vary for each of them [65, 66] . efficacy studies of these vaccines showed that they confer protection not only against meningitis, but also against pneumonia [67] [68] [69] . although hib vaccines are highly effective, their cost is still prohibitive for the world's poorest nations. however, with the establishment of the global alliance for vaccines and immunization (gavi), we have moved consistently ahead in making them also available for developing countries. gavi has approved the establishment of a hib initiative to support countries wishing to sustain hib vaccination, as well as those exploring whether their introduction could be considered a priority in the near future. although the introduction of conjugated ps vaccines has significantly decreased the prevalence of invasive hib disease, paediatric infections due to non typeable h. influenzae (nthi) are still highly prevalent. nthi is most often associated with otitis media, sinusitis and bronchitis. in addition, nthi is an important cause of lower respiratory infection in adults with chronic obstructive pulmonary disease (copd). thus, the development of a vaccine against nthi is considered an important goal in public health. in contrast to hib, vaccines against the non-encapsulated nthi strains must be directed against alternative virulence factors. the lipoproteins d and p6 are widely distributed and antigenically conserved among h. influenzae strains, and also trigger the elicitation of protective immunity in animals vaccinated by mucosal route [70] [71] [72] [73] . thus, their incorporation in vaccine candidates might facilitate the generation of a universal vaccine against all typeable and non typeable h. influenzae. even in the age of vaccine availability, b. pertussis continues to be a major cause of childhood morbidity and mortality (i.e., approximately 50 million cases and 300,000 deaths occur annually worldwide). since the late 1940s, the incidence of whooping cough has dramatically decreased in most developed countries, as a result of widespread immunization. the first vaccine formulations, which are still in use, consist of preparations based on killed b. pertussis. the frequent incidence of minor adverse effects (e.g., fever, protracted crying and local erythematous reactions), as well as concerns raised by reports of serious neurological side-effects, resulted in a decline in vaccine acceptance and use [74] . this in turn led to a re-emergence of whooping cough and its complications. this serious problem prompted the development of a new generation of acellular vaccines. in 1981 japan was the first country to successfully introduce acellular vaccines against whooping cough in its immunisation programme [75] , leading to a consistent reduction in the reported side-effects. in the mid 1980's a major phase iii trial of acellular vaccines was undertaken in sweden, at a time when the banning of the whole cell vaccine had resulted in a pertussis epidemic in that country [76] . the first vaccine trials contained chemically detoxified pertussis toxin (pt) and filamentous haemagglutinin (fha), or detoxified pt alone. the results of these trials showed that whilst producing good antibody responses, the vaccines failed to give an adequate level of protection in infants. the mono-component vaccine conferred no protection against infection, whereas the use of the two component candidate only gave incomplete protection against infection [77] . the results obtained in japan and sweden stimulated vaccine companies in the usa and europe to establish vigorous research programmes aimed at the development of a new generation of acellular vaccines with higher efficacy. currently available vaccines have incorporated chemically or genetically inactivated pt and additional virulence factors, such as fha, the outer membrane protein pertactin (prn) and fimbrial proteins (fims). the efficacy studies of this second generation of acellular vaccines have demonstrated that they confer levels of protection equivalent to the whole cell vaccines. the advent of improved techniques for antigenic characterisation and the introduction of acellular vaccines containing genetically defined components also resulted in a reduction of lot-to-lot variation in comparison with conventional whole cell vaccines and the acellular formulation originally introduced in japan. however, despite the wide implementation of vaccination campaigns in infants and children, the disease continues to be endemic. in addition, in countries with high vaccine coverage we are now observing a consistent increment in the cases of pertussis in adolescents and adults [78] [79] [80] . these patients can then transmit the disease to infants, thereby now representing a primary reservoir for bacterial transmission and cycling in the community. the above-mentioned observations can be explained by one or more of the following factors: (i) improved detection techniques, (ii) major awareness on the possibility that bacteria may affect these age groups, (iii) vaccinedriven antigenic changes in circulating isolates, and (iv) reduction in vaccine efficacy over time. in this context, concerns have been raised about genetic variation between the strains used for vaccine preparation and circulating isolates. this seems to be true, since the currently used whole cell and acellular vaccines are prepared with strains that were isolated before mass vaccine introduction and show clear mismatches with respect to circulating strains. there is a steady tendency to decrease diversity in recent isolates, together with clonal expansion during epidemic outbreaks [81, 82] . over time, at least two surface proteins (pt and prn) may have changed sufficiently to allow for an increase in the incidence of disease. unfortunately, our global information on antigenic variation and disease in adults and adolescent is extremely limited. thus, despite widespread introduction of pertussis vaccines, it is essential to continue surveillance studies and collection of circulating strains. the present view is that successful control of pertussis in the community may require routine immunization of adolescents and adults with the new acellular vaccines, perhaps in combination with the diphtheria and tetanus toxoids (dtap). this intervention might help in turn to reduce the burden of disease and transmission to infants. chlamydia pneumonia is an intracellular bacterium transmitted person-toperson via respiratory droplets. this pathogen is a common cause of pneumonia, with infections usually being oligosymptomatic or asymptomatic in young age groups. however, the rate of asymptomatic carriage in the normal population is unknown. there is also a tremendous gap in our understanding of host response to infections caused by c. pneumoniae. most of the studies have been focused on the development of efficient diagnostic methods. however, less work has been done on vaccine development, and there is a paucity of knowledge on the microbial components which may serve as target antigens. in fact, at present there are no licensed vaccines against c. pneumoniae. however, the potential of different antigens, such as the major omp2 [83] have been assessed in experimental animal models. nevertheless, mice vaccinated with omp2 using a protocol based on priming with dna and boosting with recombinant vlp showed only partial protection [84] . recent studies also suggested that ctl responses play a role in protection and clearance [85] . animals immunized with a mini-gene encoding seven h-2(b)-restricted ctl epitopes fused to a endothelial reticulum-translocation signal showed protection following intranasal challenge with a virulent c. pneumoniae [85] . the current view is that multi-component vaccine will be required in order to induce a protective response [86] . using the promising approach of reverse vaccinology combined with proteomics (see section "reverse vaccinology"), the whole-genome of c. pneumoniae was screened searching for vaccine candidate antigens among exposed and immune accessible surface proteins [87] . the selected candidates were then expressed in a heterologous system and used in immunization studies. approximately 53 proteins were able to trigger the elicitation of c. pneumoniae-binding antibodies. when tested in secondary screenings, six of them were also able to neutralize bacteria in vitro, and four inhibited systemic dissemination of c. pneumoniae in a hamster model [86] . moraxella catarrhalis is the third most common bacterial etiologic agent of otitis media in children. furthermore, m. catarrhalis is an important cause of respiratory infections in patients with copd. thus, different studies have been carried out to characterize potential protective antigens. in this context, two major omp (cd and e) have been identified, which are considered prime candidate antigens for vaccine development. these proteins are expressed on the surface and show a high degree of conservation among circulating strains. both omp triggered the elicitation of bactericidal antibodies and protective immunity in preclinical models [88] . additional candidates are the uspa1 protein [89] , which seems to be required for bacterial colonization of the human upper respiratory tract, the iron-induced omp b1 and lbp, and the iron-repressed omp b2 [90] . a conjugate vaccine based on detoxified lipo-oligosaccharide was also tested in mice by intranasal route with encouraging results [91, 92] . some of these candidates are planned to be tested in clinical studies soon [90] . mycoplasmas are commensal microorganisms, as well as opportunistic pathogens. mycoplasma pneumoniae is one of the causative agents of acute and chronic human respiratory diseases and the main responsible for primary atypical pneumonia, accounting for approximately 20-30% of all community-acquired pneumonia [93] . there is a considerable underreporting for m. pneumoniae-associated diseases. this is in part due to the wide diversity of clinical manifestations, the difficulties associated with its cultivation from clinical specimens and the lack of adequate diagnostic tools. no vaccines are currently available against this pathogen. however, studies conducted in human volunteers in the late 1960s demonstrated that a formalin-inactivated whole cell vaccine and an acellular extract were able to confer moderately protective immunity against m. pneumoniae [94] . unfortunately, immune pathological reactions were observed following challenge with live organisms. therefore, studies are still needed to understand the underlying mechanisms to the observed autoimmune responses [95] . more specifically, we need to elucidate the specific role played by humoral and cellular response in protection against m. pneumoniae. m. pneumoniae is one of the smallest self-replicating prokaryotic pathogens (approximately 800 kb). the complete genome sequence is now available. this is expected to expand our knowledge on the physiological and virulence properties of this agent, as well as new hints for vaccine development. a previously unrecognized bacterium was isolated after the outbreak of legionnaires disease in 1976, which was designated legionella pneumophila [96, 97] . the spreading of l. pneumophila is increasing due to the use of air-conditioners and humidifiers, since infections can occur by inhalation of aerosolized contaminated water sources. several approaches have been developed in the fight against this facultative intracellular pathogen. infection and immunization induce a rapid increase of antibody titres. however, antibodies do not seem to play a significant role in host resistance, particularly after aerosol challenge [98] [99] [100] . some authors also suggested that these antibodies can promote bacterial phagocytosis, thereby favouring invasion and subsequent intracellular replication [101] . in contrast, cellular responses appear to be important for protection. different vaccine candidates were tested in the past. heat-, acetone-and formalin-killed l. pneumophila vaccines were not able to confer protective immunity in guinea pigs, whereas animals immunized with l. pneumophila membranes survive an aerosol challenge with virulent bacteria [98, 99] . additional work demonstrated that also purified antigens, such as the major secretory protein [98] , the major cytoplasmatic membrane protein [102] , the peptidoglycan-associated lipoprotein [103] , omps [104] and flagella [100] can confer protection against challenge with virulent l. pneumophila. finally, different live attenuated mutants of l. pneumophila were used in animal infection models with promising results [105] . cystic fibrosis (cf) patients are particularly susceptible to severe bacterial infections of the lung, being pseudomonas aeruginosa one of the most prominent etiologic agents. thus, significant efforts have been invested to develop a vaccine against this pathogen. surface ps are among the antigens that were most intensively assessed. berna biotech have developed an octavalent vaccine against the eight most prevalent serotypes based on o-ps conjugated with the exotoxin a [106] [107] [108] [109] [110] [111] [112] [113] . a consistent reduction in the number of cf patients with chronic p. aeruginosa lung infection was observed in a cohort receiving the basic immunization protocol, followed by yearly boosters over a period of 10 years [112, 113] . the conjugate vaccine induced the production of specific igg antibodies and increased the number of igg memory b cells. it is still unclear if cellular responses might contribute to the overall protection conferred by this vaccine. however, strong proliferative responses of lymphocytes with a th1 phenotype were observed in vaccinated individuals in response to the carrier exotoxin a protein [113] . alternative vaccination strategies are currently being tested in clinical trials. among them, formulations based on a fusion protein between the outer membrane proteins f and i, which have been administered by parenteral and mucosal routes [114, 115] . these formulations were demonstrated to be safe in volunteers and conferred increased protection against p. aeruginosa in cf patients. cell-surface alginate, flagella, components of the type iii secretion system, inactivated toxins and proteases are other proposed target antigens [116] . some of them are already in clinical trials alone or in combination [116] . when pasteur returned from his summer holidays in 1881 to continue with his studies on chicken cholera, he inoculated chickens with an old culture of pasteurella multocida, which was left during the whole summer on his bench. the animals that received the preparation were protected against a challenge performed with a fresh isolate. thus, pasteur developed the hypothesis that pathogens could be attenuated by exposure to environmental insults (e.g., high temperature, oxygen and chemicals) [117] . the strategy was then successfully extrapolated for developing anthrax vaccines in livestock in the 1880s, with significant economic benefits. this was followed by the generation of attenuated vaccines against rabies and other important pathogens towards the end of the nineteenth century. pasteur's approach for "attenuating" or "inactivating" a pathogenic organisms still constitutes a cornerstone in vaccine technology [117] . this exemplifies that until recently the major achievements in vaccinology have been facilitated by technological (e.g., adjuvants, delivery systems, reverse vaccinology, genetic engineering) rather than immunological advances [117] [118] [119] . however, it is expected that the impressive knowledge accumulated in recent years in the fields of immunology, immune pathology and microbial pathogenesis will pave the road to a new golden era in vaccinology, in which knowledge and technology will enable rational vaccine design. in the 20th century, pertussis vaccines progressed from crude bacterial preparations to the highly purified antigens used for acellular vaccines. a similar quantum jump in technology allowed the development of subunit vaccines against influenza, hib and s. pneumoniae, as well as the production of antigens by recombinant dna techniques (e.g., genetically inactivated pt). despite the fact that these techniques enable the production of almost any foreseeable antigen, the identification of suitable targets still remained as a main bottleneck for vaccine development [120] . the advent of genomics and its exploitation in the vaccinology field have rendered possible the implementation of a systematic and holistic approach for the screening, identification and prioritisation of candidate antigens. this new approach, called "reverse vaccinology" [121] , does not require cultivation of the original pathogen, thereby being amenable for highlypathogenic or non culturable micro-organisms. it is possible to predict and select the most promising candidates by the analysis of genomic sequences in silico, which will then be cloned and expressed in heterologous systems. the resulting proteins are then used to perform immunological and/or functional studies to select the most promising candidates (e.g., able to induce the production of microbicidal or neutralizing antibodies, capacity to confer protective immunity). flanking studies are usually carried out, such as molecular epidemiological analysis to assess their degree of conservation among circulating strains, or transcriptional profiling to evaluate their expression during natural infections [122] . the time-consuming process in which highly expressed components of an in vitro cultivable organism are identified (one at a time) and separated (different components between them) is one of the disadvantages that reverse vaccinology has solved. the conventional method usually requires 15-20 years to arrive to a clinical trial, whereas reverse vaccinology reduces the process to approximately 5 years. reverse vaccinology also allows the identification of hundreds of potential candidates in a few days, in comparison with the small number of antigens that conventional approaches have provided after decades of research. moreover, reverse vaccinology offers the possibility to select potential candidates independent of their expression levels or purification easiness. the reverse vaccinology approach has proved its usefulness in the field for both viral and bacterial pathogens (e.g. hepatitis c virus, group b meningococci, group b streptococci) [123, 124] . reverse vaccinology has also become an essential tool for several vaccine development projects against agents causing community-acquired pneumonia (e.g., c. pneumoniae, streptococci). the potential and speed of genomic-based approaches was also shown when the nucleotide sequence of the coronavirus causing sars was made available in less than one month. in addition, the increasing number of available genomes from bacteria and viruses would allow comparative genomic studies, thereby providing hints on conserved protein families and/or functional domains. this would facilitate the generation of vaccines using immunogens covering multiple micro-organisms [125] . despite the incredible potential of reverse vaccinology, this approach also has some important limitations (tab. 1). among them is the fact that it is not be possible to identify non-protein antigens (e.g., ps, glycolipids), which are the cornerstone for many successful vaccines (e.g., pneumococcal and hib vaccines). currently available influenza vaccines (see above) are based on inactivated viruses, and, more recently, attenuated ca viruses and virosomes. all these vaccines exploit the same starting material (wild-type virus), which is inactivated or attenuated. the last approach consists in the co-infection of chicken eggs with the new isolate and a master attenuated strain, and subsequent selection for re-assorted viruses with the desired genotype/phenotype. however, the virulence of certain virus strains, such as the h5n1, renders difficult the implementation of this traditional strategy. the use of reverse genetics represents a valid alternative for the generation of vaccines against rna respiratory viruses, such as the influenza virus, piv and rsv. it consists in the production of the virus from cloned dna [126] , thereby allowing the development of vaccines against any pandemic viral strain. in some cases (e.g., avian h5n1) an additional mutagenesis step would be required to attenuate its virulence [127] . then, the new ha and na segments would be transferred into an appropriate influenza a virus master strain adapted to grow in a cell line. the final re-assorted virus will have the antigenic specificity of the pandemic strain and the growth characteristics of the master strain [128, 129] . this technology would also allow production of the influenza vaccine in cells that are co-transfected with plasmids encoding for different frag[130] . therefore, the complete genome is inside the cell and virus can be produced and assembled. one of the main advantages is that a plasmid encoding for ha and na can be easily replaced. therefore, re-assortment and selection become unnecessary. this method would considerably reduce the time for vaccine production, from many months to only a few weeks. another advantage would be the simple manipulation of the genome (contained in plasmids), which would enable detoxification of specific virulence factors. similar approaches can be implemented for other viruses, such as rsv, piv and sars-cov. however, intellectual property and liability issues are still obstacles for the industrial development of reverse-genetics-based vaccines [131] . furthermore, since the resulting viruses are considered genetically modified organisms, additional problems may arise from the regulatory stand point [131] . most of the infective agents are either limited to the mucosal membranes, or need to transit across them in order to cause disease. therefore, it is highly desirable to elicit an efficient immune response at the local site in which the first line of defence is laid. the stimulation of a pathogen-specific response at the portal of entry is expected to impair infection (i.e. colonization), thereby reducing the risk of transmission to susceptible hosts. parenterally administered vaccines mainly stimulate systemic responses, whereas vaccines given by the mucosal route mimic natural infections, thereby leading to efficient mucosal and systemic responses. thus, there is a considerable interest in the development of mucosal vaccines. however, antigens administered by this route are usually poorly immunogenic. different strategies are being pursued to overcome this bottleneck, among them can be cited the use of (i) advanced synthetic delivery systems, (ii) live attenuated bacterial or viral vectors, (iii) bacterial ghosts, (iv) pseudoviruses and (v) mucosal adjuvants [132] [133] [134] [135] . advanced synthetic mucosal delivery systems particulate antigens are more immunogenic than those in solution, due to their vulnerability to degradation by enzymes and extreme ph. thus, it would be helpful to incorporate them into a protective vehicle. often, these vehicles do not serve only to protect them, but can also enhance their uptake, promote targeting to antigen presenting cells and serve as adjuvants [136] . the most commonly exploited delivery systems are: (i) gelatine capsules, which are dissolved at alkaline ph in the intestine but not in the stomach, (ii) muco-adhesive polymers that are highly viscous inert ps, (iii) eldexomer and carboxymethyl cellulose, which have been used for oral, nasal and vaginal delivery, (iv) lipid-based structures with entrapped antigens, such as immune stimulating complexes (iscoms) and liposomes, and (v) biodegradable micro/nano-spheres based on biocompatible materials such as starch, copolymers of lactic or glycolic acid [137, 138] . some of these approaches are currently being explored to develop vaccines against agents causing community-acquired pneumonia. encouraging results have been obtained, among others, using surface antigens from s. pneumoniae encapsulated in micro-spheres [139] and a iscom-adjuvanted vaccine obtained by reverse genetics against the influenza virus, in preclinical models [140] . attenuated viruses and bacteria can be used not only as vaccine candidates per se, but also as delivery systems for heterologous antigens. thus, many attenuated microorganisms have been exploited as a scaffold for the development of subunit vaccines against other agents, under the premise that the expression of the recombinant antigen(s) does not increase their pathogenic potential for humans or animals. the most frequently exploited bacterial vectors are attenuated derivatives of salmonella enterica and shigella spp., and the bacille calmette-guérin (bcg). for example, vaccination with an attenuated salmonella expressing the oprf-opri was also shown to be able to confer protection against p. aeruginosa in a murine experimental infection model [141] . in addition, it was also demonstrated that a recombinant bcgbased vaccine expressing the pspa confers protection against s. pneumoniae in an infection animal model [142] . the use of commensals represents an alternative to attenuated organisms (e.g., lactobacilli). in this context it was demonstrated that oral administration of lactobacillus expressing proteins from coronavirus can protect against a gastric infection [143] . thus, this approach has been also proposed to combat sars. promising results were also obtained using x chlamydia psittaci [144] . on the other hand, different attenuated viruses, such as mva, bovine or attenuated hpiv-3 and adenovirus can be used as delivery systems for heterologous antigens [25, 145] . in fact, mva has recently been exploited for antigens of the sars associated coronavirus [146] . an alternative approach to the use of live attenuated carriers is given by the use of bacterial ghosts. ghosts are generated by the conditional expression of the lethal lysis gene e from bacteriophage phix174 in gram-negative bacteria [147] [148] [149] [150] [151] . this leads to the formation of a trans-membrane tunnel through the bacterial cellular envelope [147] . due to the high internal osmotic pressure, the cytoplasm content is expelled through the tunnel, thereby leading to an empty bacterial cell envelope [152] . the presence of envelope components in the ghosts provides a strong danger signal through the activation of pattern recognition receptors [153] . in addition, bacterial ghosts are efficiently taken up by antigen-presenting cells, stimulating their maturation and activation [154] . bacterial ghosts retain all morphological, structural, and antigenic features of the cell wall and can be used as vaccine candidates per se. ghosts can also be externally loaded with purified antigens. alternatively, ghosts can be generated from recombinant bacteria expressing heterologous antigens, hence avoiding the difficulties associated with the purification steps. this technology also offers the possibility to manipulate the topology of the recombinant antigen (e.g., the antigen can be bound to the inner membrane, secreted into the periplasmic space or associated to the surface). encouraging results has been obtained in preclinical models using ghosts expressing chlamydial antigens [135, 155] . promising results have been reported using different types of pseudoviruses, such as virosomes and virus-like particles (vlp), which are non-replicating viral-like structures. virosomes are based on the principle of reconstituting empty viral envelopes through integration of viral envelope proteins in liposomes. they offer the versatility of liposomes in terms of lipid composition, with the advantage of including viral membrane proteins. virosomes are produced by disassembling the viral membrane envelope with detergents. then, the viral nucleocapsid is removed by ultracentrifugation before reconstitution (fig. 1) . in contrast, vlp exploit the capacity of recombinant viral coat proteins to spontaneously self-assemble, thereby mimicking at structural level the viral capsid. vlp can be isolated after protein expression in eukaryotic cells or by in vitro assemblage from subunits produced in an heterologous system [156] . their main advantages are the lack the viral genetic material with an "intact" envelope, and the fact that they are significantly more immunogenic than soluble proteins. they can be used as vaccines per se, as well as a delivery system for protein-or nucleic acid based vaccines, or as carriers for small molecules. foreign antigens can be expressed on their surface, or can be simply encapsulated. in addition, amphiphilic adjuvants can be incorporated into their membranes, thereby offering the advantage of combining an adjuvant and the antigen in one entity without a covalent attachment. pseudoviruses are especially attractive for mucosal vaccination protocols, since they offer the opportunity to use the natural route of transmission of the agents. induction of serum antibodies, secretory iga, t helper and ctl responses, and protection against mucosal pathogen challenge has been reported from studies in animals and humans [157] [158] [159] . the virosomes generated using the influenza virus retain membrane fusion properties very similar to the naïve virus. therefore, they are able to deliver material to the cytosol of target cells, offering the possibility to access the mhc class i-restricted pathway of antigen presentation to prime ctl activity [160] [161] [162] . bacterial toxins and their derivatives are among the first molecules that have been used as mucosal adjuvants. they are characterized by the presence of an a moiety with enzymatic activity, and a b moiety that mediates toxin binding to the target cells. cholera toxin and the closely related escherichia coli heat-labile toxin showed potent adjuvant activity when co-administrated with different antigens by the mucosal route [163] [164] [165] . however, their use in humans is hampered by their intrinsic toxicity. thus, mutated derivatives were developed, in which the a subunit was modified to remove the adp-ribosylating activity. the resulting polypeptides retain their adjuvanticity, in the absence of detectable toxicity [166] [167] [168] . however, additional studies have demonstrated that even these derivatives can lead to potential severe side-effects, such as retrograde homing of adjuvant and antigen to neural tissues [169] . this might explain, at least in part, the side-effects observed after intranasal vaccination against influenza with a virosomes-based formulation containing heat-labile toxin (i.e., bell's palsy), which in turn led to its retraction from the market. however, chimeric derivatives lacking the targeting moiety for neural tissues (i.e., b subunit) are now available [170] . they might allow the exploitation of the high potential of these molecules for the development of vaccines against respiratory pathogens. in fact, preclinical studies provided the proof-of-concept for the usefulness of derivatives of bacterial toxins in the generation of acellular vaccines against microorganisms, such as s. pneumonia and h. influenzae [171, 172] . other bacterial components were also explored for their activity as adjuvants. the monophosphoryl lipid a retains much of the immune stimulatory properties of lps, without the inherent toxicity [165] . on the other hand, extracellular matrix binding proteins, such as the fibronectin binding protein i of streptococcus pyogenes, also exhibit adjuvant activity [173] . this offers the possibility of using them as dual antigen/adjuvant moieties in the same formulation. recent reports also demonstrate that vaccine formulations containing adamantylamide dipeptide, a non-toxic compound obtained by linking the l-alanine-d-isoglutamine residue of the muramyl dipeptide to the antiviral drug amantadine, confer protection against non typeable h. influenzae in preclinical models [73] . the innate immune system plays a critical early role in host defence against pathogenic microorganisms through the recognition of pathogenassociated molecular patterns [174] . this is achieved through the stimulation of pattern-recognition receptors (prr) that sense a broad range of exogenous and endogenous danger signals [153, 174] . toll-like receptors (tlr) represent the best-characterized family of prr. natural and synthetic tlr agonists are being used as immune modulators to optimize responses after vaccination. since the identification of the tlr4, many mammalian tlr homologues have been identified (i.e., 10 in humans and 13 in mice) [175] . each tlr member binds specifically to different ligands (tab. 2), alone or in combinations (e.g., heterodimers formed by tlr2 with either tlr1 or tlr6). an example of tlr agonist is bacterial dna, but not vertebrate dna, and synthetic oligodeoxynucleotides containing unmethylated cpg motifs. they act on tlr9, thereby inducing a strong th1 responses by activation of dendritic cells [176, 177] . cpg motifs have been successfully used as adjuvants in preclinical studies of different candidate vaccines against agents causing community-acquired pneumonia [178] [179] [180] . another important adjuvant with tlr-binding capacities is the mycoplasma-derived macrophage-activating lipopeptide malp-2, which act a the level of the tlr heterodimer 2/6 [181, 182] . malp-2 promotes a global activation of cells from the innate and adaptive immune system [183, 184] , such as macrophages, dc, t-and b-lymphocytes [183, 185] . when coadministered with an antigen by either the parenteral or the mucosal route, malp-2 promotes the elicitation of humoral and cellular responses at systemic and mucosal level [186] . preclinical studies suggested that malp-2 could be exploited in vaccine formulations against the sars-associated coronavirus, m. catarrhalis and influenza virus, among others (unpublished data). dna vaccination offers some advantage over the normal antigen vaccination, such as the fact that it is not necessary to express any antigen. in contrast, it is the biosynthetic machinery present in the cells of the vaccinees that takes care of this work. furthermore, since eukaryotic cells are in charge of protein synthesis, their glycosylation and folding are optimal. however, the large-scale purification of dna might be associated with high costs. this can be solved by the use of attenuated or inactivated bacteria or viruses as delivery systems [187] . this approach can also lead to an enhanced induction of antibodies, which is otherwise poor using conventional naked dna vaccines. we have recently demonstrated that bacterial ghosts can be also exploited as a delivery system for dna vaccines for both in vivo and ex vivo applications [188] . the potential of this approach is demonstrated by the fact that it is possible to optimize performance by a broad range of manipulations, such as (i) choice of optimal promoters, (ii) use of codon optimized genes for expression in mammalian cells, (iii) addition of nuclear localization signals or ubiquitination signals to improve expression and processing, and (iv) co-delivery of dna constructs coding for immune modulatory molecules [189] . in addition, by the presence of immune stimulatory cpg motifs, the dna vaccine constructs has built-in adjuvant properties. this vaccination approach is particularly suited for the stimulation of cellular immune responses [190] . interestingly, several reports suggest that dna vaccines may represent a valid alternative to prime the neonatal immune system, even in the presence of passive transferred maternal antibodies [191, 192] . in fact, promising results were also obtained in preclinical models of community-acquired pneumonia, such as influenza [193] and s. pneumoniae [194] . furthermore, dna coding for vaccine antigens appears to induce excellent immunological memory, which can be reawakened by later immunization or exposure to the pathogen. the knowledge generated in several basic disciplines, such as immunology and microbial pathogenesis, has allowed the identification of critical bottlenecks for establishing a successful vaccination strategy. it is expected that in the coming years we will develop customized approaches to address each of them, in order to stimulate efficient protection against infective agents under specific clinical settings (i.e., newborns, aging individuals, immunocompromised patients). the importance of immunological memory b lymphocytes that have differentiated into plasma cells are the producers of antigen-specific igg antibodies. bone-marrow (bm) plasma cells have a short life, therefore, the bm reservoir needs to be replenished by the stimulation of memory b cells [195, 196] . the maximal life span of bm plasma cells is still debated. only few factors have been identified that control the differentiation of antigen-specific b cells toward short-or long-life plasma cells or to memory b cells [119] . beside the requirement of cd4 + t cells, the nature of the antigen [197] and the dose are also important. higher antigen doses, as well as rapid vaccination schedules (closely spaced vaccine doses) tend to favour the rapid induction of short-term effectors, whereas lower doses of antigens preferentially support the induction of immune memory [198] [199] [200] [201] . it was demonstrated that neonatal vaccination (priming) and infant boosting might be effective even when pathogen exposure occurs very early in life. in children in whom vaccine-induced hib antibody titres have fallen to undetectable levels, memory is readily demonstrated [202] . however, immune memory per se is not enough to protect against pathogens that required high levels of neutralizing antibodies. the delay between memory b-cell reactivation and differentiation may limit the ability to interrupt pathogen invasion. therefore, it is important to establish vaccination protocols in which the population is boosted at different ages in order to maintain the required levels of antibodies. this is particularly important in diseases in which antibodies play a central role in microbial clearance or toxin neutralization. in the particular case of community-acquired pneumonia, we should consider that aging individuals are neglected in many vaccination programs. however, the strategies proposed for elderly would be different from those used for small children, since the main factors affecting vaccine efficacy are immune senescence and immaturity, respectively. the attempts to give a rational solution to this issue are discussed in the next sections. the immune system in children immune responses to bacterial and viral antigens usually increase with age in a stepwise manner [203] . prompt immunization after birth is required to induce active immunity against diseases that may occur early in life. unfortunately, this strategy is limited by the relative immaturity of the neonatal and infant immune system. some factors implicated in this poor response are the limited switch from igm to igg2 antibodies, impaired complement-mediated reactions and deficient organization of the splenic marginal zone. vaccination studies performed in newborn mice suggested that limited germinal centre reactions may results from the delayed devel-opment of follicular dc and limit plasma cell differentiation [204] . it was also showed that the neonatal bm has a limited capacity to support the establishment of long-life antibody-secreting plasma cells [205] . thus, the responses to glyco-conjugates and to most t-cell-dependent antigens are usually affected [119] . therefore, only few and highly immunogenic vaccines show significant protective efficacy after a single dose in infants. the limited igg responses are extended all over the first year of life. in addition, the immune responses, particularly antibodies, elicited in the first year of life after vaccination rapidly decline [203] . however, the problem observed in infants in terms of magnitude and duration of immune response does not seem to affect efficient priming. in fact, the immune memory generated in neonates may be recalled later in life [119] . nevertheless, strategies to generate strong and long-lasting protective responses in infants are still needed. this is in part due to the presence of maternal antibodies, which inactivate and clear the vaccine antigens, thereby rendering difficult the stimulation of an immature immune system [203] . in addition, the effects of adjuvants reported in adults cannot be extrapolated to neonates [206] . a potential strategy to overcome these problems would be to implement vaccination during pregnancy, to provide the required antibodies by placenta and later by maternal feeding [30, [207] [208] [209] . this could be complemented with an early priming of the "immature" immune system of the newborn by dna vaccination, followed by a boost during the second half of the first year or later in life [203] . poly-pathology and multiple organ failure is the rule rather than the exception in aging individuals. thus, many systems are affected (e.g., endocrine, cardiovascular), and the immune system is not an exception. the mechanisms involved in the immune senescence process, which in turn may lead to poor response to vaccination, are not fully understood. however, it is clear that responses against certain vaccines are more affected by immune senescence than others (e.g., ps-based vaccines against s. pneumoniae) [210] . in contrast, the responses to a boost dose of the anti-tetanus vaccine are hardly affected by age [211] . a rapid decline of antibody responses, together with a relative restriction of the t-cell repertoire is characteristic of the immune senescence process. this restriction and the reduction in the pool of naïve cells can explain the poor cd4 + t cell responses against antigens that are cross-reacting with proteins which were seen earlier in life. in contrast, t-cells responses of healthy elderly individuals to new antigens are often unaffected. nevertheless, the overall response to vaccination in the elderly is less efficient than in young adults, making more vigorous approaches necessary (fig. 2) . in the case of influenza, the actual strategy is annual re-vaccination. however, there are concerns regarding the capacity to increase antibodies with proper specificity against re-assorted viruses in aging adults who have been repeatedly infected or immunized. after exposure to a new, but cross-reacting antigenic variant, such individuals may respond by producing antibodies. however, these antibodies could be primarily directed against influenza strains, which were encountered earlier in life. figure 2 . factors affecting the responses in young adults and aging individuals after vaccination. the process of immune senescence impairs host response to both infection and vaccination. this critical issue needs to be considered during vaccine design and will require the development of special approaches. for example, individuals previously exposed to the "old" h1n1 influenza strain (i.e., 50 years ago), may respond differently from naïve adults who are vaccinated with a "new" h1n1 strain which have accumulated different mutations. the former might produce antibodies against the ha of the "old" h1n1 strain rather than to the cross-reacting epitopes of the new strain [212] . this is phenomenon is the so-called "original antigenic sin" [119] . on the basis of this observations, it was proposed that variations in vaccine efficacy might be due to differences in the antigenic distance between the vaccine strains and the epidemic strains responsible for influenza outbreaks [213] . however, this hypothesis was not confirmed by epidemiologic studies [214] . even more, individuals aged 65 years or older who were annually vaccinated showed a significantly reduced mortality risk. therefore, until now, it seems that the antigenic sin does not represent a major practical obstacle in influenza vaccination and additional strategies may not be required. despite the broad availability of vaccines against agents causing community-acquired pneumonia, they still represent an important cause of death, human suffering and economic losses. however, we have dramatically expanded our knowledge on the pathophysiology of diseases caused by respiratory pathogens, their virulence factors and the effector mechanisms responsible for their clearance. it is becoming clearer which microbial components are attractive as vaccine targets, as well as the type of immune response needed to confer protection against disease. thus, it is now 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safety, and immunologic memory dose dependency of antibody response in infants and children to pneumococcal polysaccharides conjugated to tetanus toxoid accelerated hepatitis b vaccination schedule in childhood immunological response to conjugate vaccines in infants: follow up study neonatal and early life vaccinology unresponsiveness to lymphoid-mediated signals at the neonatal follicular dendritic cell precursor level contributes to delayed germinal center induction and limitations of neonatal antibody responses to t-dependent antigens delayed and deficient establishment of the long-term bone marrow plasma cell pool during early life influence of mycobacterium bovis bacillus calmette-guerin on antibody and cytokine responses to human neonatal vaccination transfer of antibody via mother's milk maternal immunization with pneumococcal polysaccharide vaccine in the philippines pneumococcal conjugate vaccines as maternal and infant immunogens: challenges of maternal recruitment the protective efficacy of polyvalent pneumococcal polysaccharide vaccine booster effect of low doses of tetanus toxoid in elderly vaccinees vaccine-induced antibodies to heterologous influenza a h1n1 viruses: effects of aging and "original antigenic sin variable efficacy of repeated annual influenza vaccination annual revaccination against influenza and mortality risk in community-dwelling elderly persons immunoinformatics: bioinformatic strategies for better understanding of immune function. introduction syfpeithi: database for mhc ligands and peptide motifs paproc: a prediction algorithm for proteasomal cleavages available on the www this work was supported in part by grants from the dfg (gu482/2-3) and the bmbf ("pathogenomik" -competence center for genome research of pathogenic bacteria "pathogenomik", 031u213b) to cag. we are particularly grateful to d. felnerova from etna biotech, who provided us with a micrograph from a transmission electron microscopy of a virosome, and to m. höfle for critical reading of the manuscript. key: cord-013178-li1x1m25 authors: hung, ling-chu title: the monoclonal antibody recognized the open reading frame protein in porcine circovirus type 2-infected peripheral blood mononuclear cells date: 2020-08-29 journal: viruses doi: 10.3390/v12090961 sha: doc_id: 13178 cord_uid: li1x1m25 the purpose of this study in the context of the open reading frame 3 (orf3) protein of porcine circovirus type 2 (pcv2) was especially its location and its relation to the capsid protein and the apoptosis protein in pcv2-infected porcine peripheral blood mononuclear cells (pbmcs). to detect the orf3 protein, monoclonal antibodies (mabs) were generated in this study. the mab 7d3 binds to the orf3 peptide (residues 35–66) and the native orf3 protein in pcv2-infected pbmcs, as shown by immunofluorescence assay (ifa). the data show that 3–5% of pbmcs were positive for orf3 protein or p53 protein. further, 78–82% of pbmcs were positive for the capsid. this study confirmed the orf3 protein not only colocalized with the capsid protein but also colocalized with the p53 protein in pbmcs. immunoassays were conducted in this study to detect the capsid protein, the orf3 protein, anti-capsid igg, and anti-orf3 igg. the data show the correlation (r = 0.758) of the orf3 protein and the capsid protein in the blood samples from the pcv2-infected herd. however, each anti-viral protein igg had a different curve of the profile in the same herd after vaccination. overall, this study provides a blueprint to explore the orf3 protein in pcv2-infected pbmcs. the porcine circovirus (pcv) is a small virus and contains closed circular single-stranded dna [1] . previous studies indicated that porcine circovirus type 1 (pcv1) is non-pathogenic to porcine herd [2, 3] . however, porcine circovirus type 2 (pcv2)-infected pigs showed dullness, thinness, lymphadenopathy, jaundice, hepatomegaly, and other manifestations [4] [5] [6] [7] [8] . lymphocyte depletion and apoptosis of lymphocytes were the significant histopathological lesions in lymphoid tissues of pcv2-infected pigs [9, 10] . pcv2 infection causes a reduction of t-and b-lymphocytes in the pbmc [11] . likewise, pcv2-associated disease (pcvad) manifested as severe swine herd problems [4, [6] [7] [8] . recently, pcvad has become one of the significant swine diseases worldwide, and it impacts pork production [7] . at least four commercial pcv2a vaccines have been widely used to reduce viremia and pcv2 infectious pressure in swine herds [12] [13] [14] [15] . that might have caused the global trend shift from pcv2a to pcv2b first [16] [17] [18] [19] , and then pcv2d (pcv2b mutant strain) has become a predominant genotype globally [20] [21] [22] [23] [24] [25] [26] [27] . the open reading frame 1 (orf1), open reading frame 2 (orf2), and open reading frame 3 (orf3) genes are the three principal orfs among 11 orfs in pcvs [28] . the orf1 encodes for the replicase peptide c3 and peptide n1 were appended with an n-terminal cysteine during synthesis, which was required for conjugation with maleimide-activated carriers. animal experiments in this study were performed following the current legislation on ethical and welfare recommendations. the experiments followed the standards of the guide of the care and use of laboratory animals. all animal work was approved by the institutional animal care and use committee (iacuc) of the livestock research institute, and the iacuc of the animal health research institute. the iacuc approval numbers lriiacuc100-33 (for swine and murine experiments), a00027 (for murine and rabbit experiments), and a02023 (for murine and rabbit experiments) were given in this study. antisera against pcv2 were generated by immunizing new zealand white rabbits (from livestock research institute, council of agriculture, taiwan). rabbits were maintained in isolation rooms, and the room temperature was at 20-26 • c. they were fed with a commercially pelleted diet (fwusow in., taiwan), and pure water was available ad libitum. then, animals were immunized with peptide immunogen (the peptide n1 conjugated with klh) or commercial vaccine five times at two-week intervals. for peptide immunization, an initial dose of 150 µg of the conjugated peptide was mixed with complete freund's adjuvant (sigma-aldrich, st. louis, mo, usa) at the first injection. each rabbit was re-immunized with the same amount of immunogenic mixture with incomplete freund s adjuvant (sigma-aldrich, st. louis, mo, usa). for virus-like particles (vlp) of pcv2 immunization, the rabbit was injected intramuscularly in the legs with 0.5 ml of the pcv2 vaccine (circoflex ® , boehringer ingelheim). the blood was harvested two weeks after the last injection. the antibody titers were measured by indirect enzyme-linked immunosorbent assay (ielisa) [48] (performed as described previously for mouse anti-pcv2 elisa, with the exception that peroxidase-conjugated donkey anti-rabbit igg (h + l, jackson immunoresearch, west grove, pa, usa) secondary antibody was used). hybridomas were generated following established methods, as previously described [49] . briefly, four five-week-old, female, balb/cbyjnarl mice were purchased from a specific pathogen-free (spf) colony (the national applied research laboratories, taiwan). the mice were maintained in isolation rooms in filtertop cages, and the room temperature was at 20-26 • c. mice were fed with a commercially pelleted diet (labdiet, 5002, st. louis, mo, usa), and pure water was available ad libitum. mice were inoculated with 65 µg of immunogen (the peptide n1 conjugated with klh) emulsified in freund's adjuvant. subsequently, mice were boosted fortnightly with the same amount of immunogen. three days after the final booster, the spleens of the mice were harvested for hybridoma generation. hybridomas were screened for the secretion of anti-orf3-specific mabs by ielisa [49] using the peptide n1 as a coating antigen. that secondary antibody solution (hrp-conjugated goat anti-mouse iga/igg/igm, h/l chain (novus, saint charles, mo, usa) was used at 1:2500 dilution for hybridoma screening. the hybridomas that secreted anti-n1 mabs were subcloned by limited dilution of the cells. then, ascites containing mabs were collected as previously described [49] . subsequently, the mabs were purified from the ascites by using the nab protein g spin column (thermo scientific, rockford, il, usa), then collecting solutions were exchanged for the buffer and concentrated using the amicon ultra 50k (millipore, burlington, ma, usa). the concentration of mabs was determined using the nano photometer ® (implen, munich, germany). the heavy chain and the light chain of the two mabs secreted by each hybridoma were determined by using an sba clonotyping system/hrp (southern biotechnology, birmingham, al, usa). it was performed according to the manufacturer's instructions and a previously published method [49] . briefly, the nunc maxisorp flat-bottom 96-well plate (thermo fisher scientific, inc., waltham, ma, usa) was coated with the capture antibody (goat anti-mouse ig, 5 µg/ml) in 0.05-m bicarbonate buffer (sigma-aldrich, st. louis, mo, usa) at 4 • c overnight. after three washes with pbs containing 0.05% tween 20 (pbst), the plate was blocked with the blocking buffer (pbst containing 5% casein hydrolysate) at 37 • c for 30 min. after washing, 100 µl of each culture supernatant of hybridoma were added sequentially, and the plate was incubated at 37 • c for 2 h. after washing, 100 µl of hrp labeled goat anti-mouse igg1, -igg2a, -igg2b, -igg3, -iga, -igm, -κ light chain, and -λ light chain were added to appropriate wells of the plate. pbst served as background. the plate was then incubated at 37 • c for 1 h and after that washed with pbst. finally, 100 µl of 2, 2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts, sigma-aldrich, st. louis, mo, usa) buffer were added to each well of the plate. after 30 min, the optical density (od) of each well was measured at 405 nm using a spectramax m5 microplate reader (molecular devices, san jose, ca, usa). the epitope mapping of these mabs was performed by ielisa [49] using truncated peptides (as shown in table 1 ) as coating antigens. briefly, peptides contained the sequence of orf3 protein of pcv2b between residues 35 and 66, associated 10-mer peptides, truncated derivatives, and the control (peptide c3). ninety-six-well maxisorp plates were coated with each peptide (5 µg/ml) in bicarbonate buffer and incubated at 4 • c overnight. after three washes in pbst, the plates were blocked with the blocking buffer at 37 • c for 30 min. after washing, the culture supernatant of hybridoma was added, and plates were again incubated at 37 • c for 2 h. the anti-peptide n1 mouse serum (at a 1:1000 dilution) was used as the positive control, and the anti-orf2 (peptide c3) mab (1h3) was served as the negative control. after rinsing three times with pbst, the secondary antibody solution was applied to the appropriate wells. peroxidase-conjugated goat anti-mouse igg (subclasses 1 + 2a + 2b + 3, fcγ, jackson immunoresearch, west grove, pa, usa) was used at a 1:2500 dilution for binding to the mab 7d3. hrp-conjugated goat anti-mouse lambda light chain (novus, saint charles, mo, usa) was used at a 1:5000 dilution for binding to the mab 6d10. after 1 h, the plates were washed three times. the colorimetric reaction was developed by using the abts solution. following a 30-min incubation, the od was measured at 405 nm. both of the isotype and molecular weight of the mab 6d10 were determined by western blotting. the culture supernatant of the hybridoma (clone 6d10) was separated by bolttm bis-tris plus gel (invitrogen, carlsbad, ca, usa). one gel was stained with fastain protein staining solution (yeastern biotech, taiwan). the other gel was transferred to the polyvinylidene difluoride (pvdf) membranes (millipore, burlington, ma, usa) with fast semi-dry transfer buffer (thermo, waltham, ma, usa) by using a yrdimes semi-dry transfer system (wealtec, new taipei city, taiwan). the membrane was blocked with the blocking buffer for 30 min at 37 • c. after three washes in pbst, each strip of the membrane was incubated with one appropriate horseradish peroxidase (hrp)-conjugated antibody to detect mab at 4 • c overnight and then at room temperature for 2 h. peroxidase-conjugated goat anti-mouse iga/igg/igm, h/l chain (novus, saint charles, mo, usa) at 1:2500, hrp-conjugated goat anti-mouse iga (southern biotechnology, birmingham, al, usa) at 1:2500, hrp-conjugated goat anti-mouse lambda light chain (novus, saint charles, mo, usa) at 1:5000, hrp-conjugated goat anti-mouse igm, µ chain (jackson immunoresearch, west grove, pa, usa) at 1:5000, and hrp-conjugated goat anti-mouse igg (subclasses 1 + 2a + 2b + 3, fcγ, jackson immunoresearch, west grove, pa, usa) at 1:2500 were used in this assay. then, they were washed three times with pbst, 5 min each wash. each strip was incubated with 3, 3 -diaminobenzidine (dab, horseradish peroxidase substrate, millipore, burlington, ma, usa) buffer for 5 min. protein molecular weight markers were included in each western blot analysis. after revelation with the dab substrate, the target protein was visualized and calculated the molecular weight by using molecular weight markers. homemade immunofluorescence assay (ifa) slides were performed as previously described [49] . pcv2-infected peripheral blood mononuclear cells (pbmcs) were collected from the pcv2-infected conventional piglet. the pig serum was detected as seropositive for pcv2 by anti-capsid peptide (c3) specific immunoassay [48] . the whole blood sample was confirmed as positive for pcv2 antigen by using the ingezim pcv das kit (ingenasa, madrid, spain). briefly, pbmcs were isolated from ethylenediaminetetraacetic acid (edta)-treated whole blood samples by ficoll-paque plus (ge healthcare bio-sciences, uppsala, sweden) and according to the manufacturer's instructions. then, mononuclear cells were drawn from the interface of the ficoll-paque plus-containing tube and resuspended in 45 ml pbs for washing and centrifugation (100× g for 10 min at 4 • c). after washing three times with pbs, the cell pellet was resuspended in 0.5 ml of fetal bovine serum. an aliquot (25 µl) of cell suspension was loaded onto a glass slide for the cell smear. after the cell smear, the slides were air-dried at room temperature. thereby, the pbmcs were stuck on slides. these slides were fixed with pbs containing 2% paraformaldehyde at 4 • c for 10 min and then washed three times with pbs. following that, the slides were soaked in pbs containing 0.1% triton x-100 at 4 • c for 3 min. subsequently, slides were washed with pbs and prepared for ifa. two anti-capsid (the peptide c3) mabs (1h3 and 6b8) [49] , one anti-pcv2 mab 36a9 (ingenasa, madrid, spain), one anti-p53 protein (wt-p53) rabbit polyclonal antibody (bioss, woburn, ma, usa), the homemade anti-orf3 peptide (n1) mab (7d3), and rabbit antisera (as the method mentioned above) were used in this study. the anti-capsid peptide mabs (1h3 and 6b8) recognized native capsid proteins and reacted with core peptides (p59, 227 kdpplnp 233 ); however, the mab 1h3 bound the three minimal linear epitopes (dpplnp, dpplnpk, and lkdpplkp) and the mab 6b8 bound two minimal linear epitopes (kdpplnp and kdpplnpk). these mabs produced a variable definite staining pattern in pbmcs by ifa [49] . the pcv2-infected pbmcs slides were incubated with a 1:100 dilution of rabbit antiserum and a 1:100 dilution of mab (ascites). after incubation at 37 • c for 1 h, the slides were gently rinsed briefly in pbs and then soaked for 15 min in pbs at 4 • c. the slides were then incubated at 37 • c with tritc-conjugated goat anti-mouse igg (subclasses 1 + 2a + 2b + 3, fcγ) at a 1:100 dilution and fitc-conjugated goat anti-rabbit igg (h + l) (all from jackson immunoresearch, west grove, pa, usa, and each minimal antibody cross-reaction to other animal's serum protein) at a 1:100 dilution. after 30 min of incubation, the slides were washed with pbs and then incubated with 4, 6-diamidino-2-phenylindole, dihydrochloride (dapi, aat bioquest, sunnyvale, ca, usa) at a 1:2300 dilution in pbs at room temperature for 15 min. the slides were mounted under 50% glycerol and observed with an olympus bx51 fluorescence microscope and spot flex camera (diagnostic instrument, model 15.2 64mp, usa). terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay was performed according to the manufacturer's instructions (the cell meter™ tunel apoptosis assay kit, aat bioquest ® , inc., sunnyvale, ca, usa) to detect excessive dna breakage in the pbmc. briefly, the pcv2-infected pbmcs slides were incubated with the tf3-dutp/reaction buffer. after 60 min of incubation, the slides were washed with pbs. they were then incubated with hoechst solution at room temperature for 13 min. the slides were mounted under 50% glycerol and observed with an olympus bx51 fluorescence microscope and spot flex camera. blood samples were collected from the pcv2-infected herd with the following vaccination at two weeks of age with a pcv2 vaccine (2 ml, porcilis ® pcv, msd animal health) in the conventional pig farm with the farrow-to-finish operation. pigs were selected from animals of different ages (1, 3, 6, 9, 12, 15, 18, 21, 24 , and ≥34 weeks of age) at one time. then, four samples were randomly chosen per age group. blood was collected in the vacutainer ® edta tubes (becton and dickinson bv) and sored at −20 • c. all whole-blood samples had been treated by the freeze-thaw process twice before the test. the capsid protein was detected from 50 µl of whole-blood sample by using the ingezim pcv das kit (ingenasa, madrid, spain) according to the manufacturer's instructions, and the absorbance of each well was measured at 450 nm. meanwhile, the homemade antigen-elisa was conducted in this study for detecting the orf3 protein in whole-blood specimens. it was based on the double-antibody-sandwich principle. ninety-six-well maxisorp plates were coated with 1 µl/ml (100 µl per well) of the trapping sera (rabbit anti-orf3 peptide) in bicarbonate buffer. the plates were incubated at 4 • c overnight and washed three times with pbst. then, the plates were blocked with viruses 2020, 12, 961 7 of 22 the blocking buffer at 37 • c for 30 min. after the plates were washed, 50 µl of pbst and 50 µl of whole-blood samples were mixed and added to the well. the plates were incubated at 37 • c for 2 h and then washed in pbst. then, 100 µl/well of mab 7d3 at 1 µg/ml were added to the plates, and the plates were incubated at 37 • c for 1 h. after the plates were washed, 100 µl/well of peroxidase-conjugated goat anti-mouse igg fcγ at a 1:2500 dilution were added, and the plates were incubated at 37 • c for 1 h. after washing, abts buffer was added to each well. following a 30-min incubation, the od was measured at 405 nm. 2.9. detection of the specific antibodies against the capsid protein or the orf3 protein in plasm samples from the pcv2-infected herd with a pcv2 vaccine plasm samples were separated from the aforementioned fresh whole-blood samples and sored at−20 • c. the antibody titers were measured by ielisa [48] (performed as described previously for pig anti-peptide elisa, with the exception that the capsid protein (circoflex ® , boehringer ingelheim) or peptide p126 was used as the coating antigen, and pig plasm at a 1:200 dilution was used). the subsequent procedures of blocking, pig antibody, washing, secondary antibody, substrate, and the od reading, were the same as described previously. the data were analyzed by using one-way analysis of variance (anova) and tukey's studentized range (hsd, honestly significant difference) multiple comparisons test using the sas enterprise guide 7.1 ® software (sas institute inc., cary, nc, usa). a p-value < 0.05 was considered significant. the orf3 peptide n1 or vlp of pcv2 was capable of inducing each specific antibody. the antibody titer of each post-immune serum (two weeks after the fourth booster) was detected at the dilution 1:10000 ( figure s1 ). the binding of the rabbit antiserum to the virus was confirmed by using an immunofluorescence assay (ifa) ( figure s2 ). although pre-immune serum had a low background (od 405 value less than 0.4) at a dilution of 1:100, the pre-immune serum did not bind the virus as detected by ifa (data not shown). the author checked for the cross-reactivity of post-immune antiserum to other antigens. the result shows they have a minor cross-reaction at a dilution of 1:100 ( figure s1 ), but the od 405 value of cross-reaction has not significantly higher than that of the negative control serum (p ≥ 0.05) ( figure s1 ). following the fusions, hybridoma supernatants were screened for the presence of anti-orf3 (peptide n1) specific antibodies by ielisa. among them, 34 hybridomas supernatants reacted with the peptide n1 at the first screening (data not shown). after repeatedly subcloning by the limiting dilution and selection, two stable hybridomas secreting anti-orf3 mabs (7d3 and 6d10) were obtained. the hybridoma produced igg1 mab (7d3) with kappa-light chains. the other produced mab (6d10) with a lambda-light chain ( figure 1a) ; however, no heavy chain was detected in the supernatant of clone 6d10 by sba clonotyping system/hrp assay. the supernatant of a hybridoma (clone 6d10) was separated directly by bolttm bis-tris plus gel to confirm the heavy chain of this mab. the gel showed a broad staining region within 40-98 kda. the author speculated that the supernatant of a hybridoma contains abundant serum proteins from the fetal bovine serum-containing medium. that is to say, the supernatant of a hybridoma (clone 6d10) contains a little mab 6d10 (light chain). further, the western blotting was used to confirm the heavy chain of mab 6d10 and its molecular weight. the hrp-conjugated antibodies (goat anti-mouse iga/igg/igm, h/l chain, and goat anti-mouse lambda light chain) gave specific reactions with the supernatant. two bands were observed at about 33 (between 28 and 38) and 6 kda, respectively ( figure 1 ). the lower band (6 kda) implied that some degradation of this mab. however, other anti-ig antibodies (goat anti-mouse ig a (α chain), goat anti-mouse igm (µ chain), and goat anti-mouse igg (fcγ)) gave negative reactions with the supernatant. this study confirmed mab 6d10 contains a lambda light chain, but it does not include any intact heavy chain. since the molecular weight of the light chain was 25 kda, it may be assumed that the mab 6d10 might contain a short fragment of a protein. a peptide scan analysis was performed to determine the binding site for each mab ( figure 2 ). the mab 7d3 bound to two linear peptides (peptide n1 and p126). the peptide n1 was appended with a cysteine residue at the n-terminal of the orf3 peptide (residues 35-65, p126). both of them contained the sequence of the orf3 protein of pcv2b between residues 35 and 66. however, the non-igg mab (6d10) not only reacted with peptide n1 but also showed minor reactions against other peptides, which compared with the mab 7d3 in the elisa test ( figure 2) . interestingly, the anti-peptide n1 mouse serum reacted all test peptides and bound firmly to six linear peptides (peptide n1, p32, p33, p69, and p126). notably, anti-n1 poly antibodies reacted with these peptides (expect p33) which contained the common core peptide (p32 and 45 tllhfpahfq 54 ). nevertheless, this study did not get any mab which bound firmly to the peptide p32. viruses 2020, 12, x for peer review 9 of 21 a peptide scan analysis was performed to determine the binding site for each mab ( figure 2 ). the mab 7d3 bound to two linear peptides (peptide n1 and p126). the peptide n1 was appended with a cysteine residue at the n-terminal of the orf3 peptide (residues 35-65, p126). both of them contained the sequence of the orf3 protein of pcv2b between residues 35 and 66. however, the non-igg mab (6d10) not only reacted with peptide n1 but also showed minor reactions against other peptides, which compared with the mab 7d3 in the elisa test ( figure 2) . interestingly, the antipeptide n1 mouse serum reacted all test peptides and bound firmly to six linear peptides (peptide n1, p32, p33, p69, and p126). notably, anti-n1 poly antibodies reacted with these peptides (expect p33) which contained the common core peptide (p32 and 45tllhfpahfq54). nevertheless, this study did not get any mab which bound firmly to the peptide p32. anti-orf3 mabs (7d3 and 6d10) bound the linear peptide spanning from residues 35 to 66. the antipeptide n1 mouse serum was used as the positive control, and the anti-orf2 (peptide c3) mab (1h3) served as the negative control. the mab binding was tested by using an ielisa. peptides contained the sequence of the orf3 protein between residues 35 and 66, associated 10-mer peptides, truncated derivatives, and the control (peptide c3) (as shown in table 1 ). the experiments were performed three times. data represent the mean ± standard error (se). for these homemade pbmcs slides, all slides were made from the same batch and contained the same proportion of pcv2-infected cells. all wells contained both negative and positive cells (pcv2infected cells) confirmed by ifa, as previously noted in a similar study [49] . in this study, pcv2 viral protein (orf3 protein and capsid protein) locations were initially assessed by ifa. the previous study indicated that the mab 1h3 bound the three minimal linear epitopes (dpplnp, dpplnpk, and lkdpplkp), which were located at carboxyl-terminus (cterminus) of the capsid proteins of pcv2b, pcv2d, and pcv2a, respectively [49] . likewise, the mab 6b8 bound two minimal linear epitopes (kdpplnp and kdpplnpk), which were located at cterminus of the capsid proteins of pcv2b, and pcv2d, respectively [49] . first, the percentage was determined by counting the number of positive cells per 100 nuclei in an ifa slide. the data show that 78-82% of pbmcs were positive for anti-capsid peptide mab (1h3) staining (pcv2a-, pcv2b-, the anti-peptide n1 mouse serum was used as the positive control, and the anti-orf2 (peptide c3) mab (1h3) served as the negative control. the mab binding was tested by using an ielisa. peptides contained the sequence of the orf3 protein between residues 35 and 66, associated 10-mer peptides, truncated derivatives, and the control (peptide c3) (as shown in table 1 ). the experiments were performed three times. data represent the mean ± standard error (se). for these homemade pbmcs slides, all slides were made from the same batch and contained the same proportion of pcv2-infected cells. all wells contained both negative and positive cells (pcv2-infected cells) confirmed by ifa, as previously noted in a similar study [49] . in this study, pcv2 viral protein (orf3 protein and capsid protein) locations were initially assessed by ifa. the previous study indicated that the mab 1h3 bound the three minimal linear epitopes (dpplnp, dpplnpk, and lkdpplkp), which were located at carboxyl-terminus (cterminus) of the capsid proteins of pcv2b, pcv2d, and pcv2a, respectively [49] . likewise, the mab 6b8 bound two minimal linear epitopes (kdpplnp and kdpplnpk), which were located at cterminus of the capsid proteins of pcv2b, and pcv2d, respectively [49] . of pbmcs were positive for anti-orf3 mab (7d3) staining ( figure s3 ). it might be due to different abundance of the protein, the degradation of the protein, different pcv2 strains, conformational epitopes, or linear epitopes, which interacted with the antibody. interestingly, the anti-vlp of pcv2 rabbit serum produced cytoplasmic staining in pcv2-infected pbmcs ( figure 3b,f) . when pcv2-infected pbmcs were co-stained with anti-vlp of pcv2 rabbit serum and anti-capsid peptide mabs (6b8), some vlp/capsid peptide dual-positive cells were shown ( figure 3d ). the overlap between the anti-vlp rabbit serum staining and the anti-orf3 mab (7d3) staining is presented in figure 3h , and the anti-orf3 polyclonal antibody and anti-capsid mab (36a9) overlapped the same in figure 3p ; however, the polyclonal antibody staining was brighter than the mab staining. this result shows orf3 protein overlaps the capsid protein (or vlp) of pcv2 ( figure 3h,p) . both the capsid proteins and the orf3 protein were detected mainly in the cytoplasm. according to the previous study, the n-terminal half of orf3 protein (residues 1-53) was discovered in the cytoplasm, and the c-terminal half of orf3 protein (residues 53-104) was majorly located in the nucleus [50] . the medial region of orf3 protein (residues 35-66) might be primarily located in the cytoplasm in this study. of pcv2 ( figure 3h,p) . both the capsid proteins and the orf3 protein were detected mainly in the cytoplasm. according to the previous study, the n-terminal half of orf3 protein (residues 1-53) was discovered in the cytoplasm, and the c-terminal half of orf3 protein (residues 53-104) was majorly located in the nucleus [50] . the medial region of orf3 protein (residues 35-66) might be primarily located in the cytoplasm in this study. a previous study suggested that orf3 protein led to increased p53 protein levels and apoptosis of the infected cells [42] . to identify the subcellular location of the orf3 protein in pcv2-infected pbmcs, anti-capsid, anti-orf3, and anti-p53 antibodies were used. the p53 protein also colocalized with capsid peptide (figure 3l ), and the orf3 peptide ( figure 3x ). the p53 protein was mainly distributed in the cytoplasm and around the nucleus ( figure 3j,v) . the orf3 protein, as detected with mab 7d3, seemed to colocalize quite nicely with the p53 protein ( figure 3x ). the p53/orf3 dual-positive cell presented a segmented nucleus ( figure 3w,y) . the nuclei of negative cells were round or oval, and nuclear segmentation was very rare in negative cells ( figure 3y ). the apoptosis in pcv2-infected pbmcs was observed by using a terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay (figures 4 and s4) . the data show that 3-4% pbmcs were positive for tunel staining. it is very similar to the percentage of anti-orf3 mab (7d3) staining. however, these similar percentages are not sufficient proof to show that the orf3 protein of pcv2 causes dna damage. a more careful analysis would be needed to reveal whether pcv2 infection causes dna damage. unfortunately, it was not easy to label both the orf3 protein and the signals of dna breakage simultaneously by ifa and tunel assay (data not shown). further research would be needed to determine whether the orf3 protein colocalizes with the signals of tunel. a previous study suggested that orf3 protein led to increased p53 protein levels and apoptosis of the infected cells [42] . to identify the subcellular location of the orf3 protein in pcv2-infected pbmcs, anti-capsid, anti-orf3, and anti-p53 antibodies were used. the p53 protein also colocalized with capsid peptide (figure 3l ), and the orf3 peptide ( figure 3x ). the p53 protein was mainly distributed in the cytoplasm and around the nucleus ( figure 3j ,v). the orf3 protein, as detected with mab 7d3, seemed to colocalize quite nicely with the p53 protein ( figure 3x ). the p53/orf3 dual-positive cell presented a segmented nucleus ( figure 3w,y) . the nuclei of negative cells were round or oval, and nuclear segmentation was very rare in negative cells ( figure 3y ). the apoptosis in pcv2-infected pbmcs was observed by using a terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay ( figure 4 and figure s4 ). the data show that 3-4% pbmcs were positive for tunel staining. it is very similar to the percentage of anti-orf3 mab (7d3) staining. however, these similar percentages are not sufficient proof to show that the orf3 protein of pcv2 causes dna damage. a more careful analysis would be needed to reveal whether pcv2 infection causes dna damage. unfortunately, it was not easy to label both the orf3 protein and the signals of dna breakage simultaneously by ifa and tunel assay (data not shown). further research would be needed to determine whether the orf3 protein colocalizes with the signals of tunel. 6b8 (a); mab 1h3 (i); and mab 36a9 (m)), and the orf3 peptide of pcv2 (mab7d3 (e,u)). staining with spf mouse serum was used as the negative control (q). (b,f,j,n,v) staining with rabbit antiserum (green) was used to identify the vlp of pcv2 (b,f), the p53 (j,v), and the orf3 peptide of pcv2 (n). (c,g,k,o,s,w,y) nuclei were stained with dapi (blue). the arrow points to the positive staining cell (z) and its irregular shaped nucleus (y), and the enlarged image in the sixth row (x,w). (d,h,l,p,t,x,z) the merged images are also shown. the scale bar is 10 μm. the viral proteins were detected in the whole-blood samples using antigen-elisa techniques. the cut-off value in the commercial pcv2 capsid elisa and the orf3 protein elisa were 0.15 and 0.22, respectively. the capsid protein of pcv2 was mainly detected in pigs at 1, 3, 6, and ≥34 weeks of age in this study farm ( figure 5a ). noticeably, these groups (at 1, 3, 6, and ≥34 weeks of age) showed more elevated standard error of od 405 value than other groups in this statistical analysis. this result implies that pigs at that age would be susceptible to pcv2 infection. interestingly, non-vaccinated piglets showed the highest od 450 value of capsid protein at one week of age. after piglets were inoculated with the pcv2 vaccine at two weeks of age, the od 450 value of capsid protein gradually decreased until 24 weeks of age. however, the od 450 value of capsid protein was raised at ≥34 weeks of age (two gilts and two sows). there is evidence to suggest that the vaccine could not protect pigs against pcv2 infection at 32 weeks postimmunization. the orf3 protein of pcv2 showed the same result except for od values of the orf3 protein were lower than the capsid protein. the correlation coefficient (r) was 0.7583. the mean od 405 values of the orf3 protein were significantly higher for suckers at one week of age than postweaning ones at 9-24 weeks of age (p < 0.05; figure 5b ). there is an apparent probability that the amount of orf3 protein was fewer than the amount of capsid protein in pcv2-infected blood from suckers at one week of age. protect pigs against pcv2 infection at 32 weeks postimmunization. the orf3 protein of pcv2 showed the same result except for od values of the orf3 protein were lower than the capsid protein. the correlation coefficient (r) was 0.7583. the mean od405 values of the orf3 protein were significantly higher for suckers at one week of age than postweaning ones at 9-24 weeks of age (p < 0.05; figure 5b ). there is an apparent probability that the amount of orf3 protein was fewer than the amount of capsid protein in pcv2-infected blood from suckers at one week of age. figure 5 . assessment of the capsid protein and the orf3 protein in the whole-blood samples from pigs of different age groups. this study used the commercial capsid kit (a) and the orf3 protein elisa (b) to measure each specimen, and the absorbance was measured at 450 or 405 nm, respectively. the peptide n1 and psbt were used as a positive (pos) control and a negative (neg) control, respectively, in the orf3 protein elisa. the data represent the mean ± se. treatments with different letters have statistically significant differences (p < 0.05). figure 5 . assessment of the capsid protein and the orf3 protein in the whole-blood samples from pigs of different age groups. this study used the commercial capsid kit (a) and the orf3 protein elisa (b) to measure each specimen, and the absorbance was measured at 450 or 405 nm, respectively. the peptide n1 and psbt were used as a positive (pos) control and a negative (neg) control, respectively, in the orf3 protein elisa. the data represent the mean ± se. treatments with different letters have statistically significant differences (p < 0.05). the anti-viral protein-specific antibodies were detected in the plasm samples using ielisa techniques. the cut-off value of in the anti-capsid igg elisa and the anti-orf3 peptide (p126) igg elisa were 0.20 and 0.18, respectively. non-vaccinated suckers showed the highest mean od 405 value of anti-capsid igg and anti-orf3 igg at one week of age. it may mean that non-vaccinated suckers absorbed large amounts of maternally derived antibodies after birth. notably, the non-vaccinated group showed the most elevated standard error of od 405 value (anti-capsid igg) in this statistical analysis. a gradual decrease of the mean od 405 value of anti-capsid igg for piglets from the suckling period (one week of age) to the weaning period (six weeks of age) was observed. from 9 to 12 weeks old, the mean od 405 value of anti-capsid igg increased significantly (p < 0.05; figure 6 ). the mean od 405 value of anti-capsid igg also increased significantly for pigs from 15 to 18 weeks old (p < 0.05) and reached a plateau at 18 weeks old. the same trend was observed in the result of the anti-orf3 igg elisa test, except for od 405 values of anti-orf3 igg were lower than anti-capsid igg. notwithstanding, it is worth mentioning that there was a sharp decrease of the mean od 405 value of anti-orf3 igg for suckers from one to three weeks old. the mean od 405 value of anti-orf3 igg increased significantly for pigs from six to nine weeks old (p < 0.05; figure 6 ) and reached a plateau at nine weeks old. there was a weak correlation (r = 0.3771) between the od 405 value of anti-capsid igg and the od 405 value of anti-orf3 igg in the plasm samples. it points to the probability that the amount of anti-capsid igg reflects humoral immunity in pcv2-infected pigs before and after the injection of a pcv2 vaccine. besides, the amount of anti-orf3 igg reflects humoral immunity in pigs, which were caused by pcv2 infecting only. the mean od405 value of anti-capsid igg also increased significantly for pigs from 15 to 18 weeks old (p < 0.05) and reached a plateau at 18 weeks old. the same trend was observed in the result of the anti-orf3 igg elisa test, except for od405 values of anti-orf3 igg were lower than anti-capsid igg. notwithstanding, it is worth mentioning that there was a sharp decrease of the mean od405 value of anti-orf3 igg for suckers from one to three weeks old. the mean od405 value of anti-orf3 igg increased significantly for pigs from six to nine weeks old (p < 0.05; figure 6 ) and reached a plateau at nine weeks old. there was a weak correlation (r = 0.3771) between the od405 value of anti-capsid igg and the od405 value of anti-orf3 igg in the plasm samples. it points to the probability that the amount of anti-capsid igg reflects humoral immunity in pcv2-infected pigs before and after the injection of a pcv2 vaccine. besides, the amount of anti-orf3 igg reflects humoral immunity in pigs, which were caused by pcv2 infecting only. figure 6 . assessment of the anti-capsid igg and the anti-orf3 peptide (p126) in the plasm samples from pigs of different age groups. the experiments were repeated three times, and data represent the mean ± se. treatments with different letters at the same kind of antibody assay have statistically significant differences (p < 0.05). according to a previous study, the orf3 peptide (residues 35-65) of pcv2 interacts with the p53-binding domain of ppirh2 [42] . the peptide n1 (residues 35-66) of the orf3 protein was synthesized and used in this study. subsequently, anti-orf3 mabs were generated and characterized in this study. this work created one hybridoma producing the mab 7d3 with igg1, and the other producing the defective-ig mab (6d10) with a lambda-light chain. the mab 7d3 (igg1) bound to the linear peptide n1 (c35hndvyislpi tllhfpahfq kfsqpaeisdkr66) and p126 (35hndvyislpi tllhfpahfq kfsqpaeisdkr66). however, the defective-ig mab 6d10 minorly reacted with all truncated peptides. this finding is similar to the previous study [49] . the defective-ig mabs likely have broad binding, moderate specificity, and low affinity with the associated peptide. there were concerns about this defective-ig mab since this seems to be very rare. further, the molecular weight of the mab 6d10 was determined by western blotting. two bands were observed figure 6 . assessment of the anti-capsid igg and the anti-orf3 peptide (p126) in the plasm samples from pigs of different age groups. the experiments were repeated three times, and data represent the mean ± se. treatments with different letters at the same kind of antibody assay have statistically significant differences (p < 0.05). according to a previous study, the orf3 peptide (residues 35-65) of pcv2 interacts with the p53-binding domain of ppirh2 [42] . the peptide n1 (residues 35-66) of the orf3 protein was synthesized and used in this study. subsequently, anti-orf3 mabs were generated and characterized in this study. this work created one hybridoma producing the mab 7d3 with igg1, and the other producing the defective-ig mab (6d10) with a lambda-light chain. the mab 7d3 (igg1) bound to the linear peptide n1 (c 35 hndvyislpi tllhfpahfq kfsqpaeisdkr 66 ) and p126 ( 35 hndvyislpi tllhfpahfq kfsqpaeisdkr 66 ). however, the defective-ig mab 6d10 minorly reacted with all truncated peptides. this finding is similar to the previous study [49] . the defective-ig mabs likely have broad binding, moderate specificity, and low affinity with the associated peptide. there were concerns about this defective-ig mab since this seems to be very rare. further, the molecular weight of the mab 6d10 was determined by western blotting. two bands were observed at about 33 and 6 kda, respectively. the small one (6 kda) implied some degradation of this mab. since the molecular weight of the light chain was 25 kda, the author suggested that the mab 6d10 might contain a short fragment of a protein. interestingly, the author also tested the molecular weight of another defective-ig mab 6c3 [49] (which against peptide c3), and two molecules at about 33 and 6 kda were again shown (data not shown). according to previous documents, human patients with abnormal serum immunoglobulin-free light chain production should only be due to monoclonal plasmaproliferative disorders (included multiple myeloma, light chain myelomas, and light chain amyloidosis) [51] [52] [53] . arguably, the defective-ig mab (6d10) might be related to the phenotype of splenocytes, which fused with the myeloma cell. further research should be carried out using advanced techniques (such as liquid chromatography with mass spectrometry and spectroscopic techniques) to investigate the constitution of the mab 6d10. based on previous reports, lymphocyte depletion and apoptosis in lymphoid tissues are histological hallmarks in pcv2-infected pigs [54] [55] [56] . interestingly, these histopathological lesions are similar to marek's disease and african swine fever [57, 58] . more recent evidence shows that these viruses caused an early cytolytic infection in lymphocytes by apoptosis [57] [58] [59] . therefore, this study hypothesized that lymphocyte and pbmcs lineage cells should be the primary target cells for pcv2 infection. to the best of the author's knowledge, the study of the orf3 protein by indirect immunofluorescence assay in pcv2-infected pbmcs has never been performed, and only a single article mentions the transient expression of the orf3 in porcine pbmcs and detecting apoptosis with a tunel assay [50] . therefore, the author explored the relation of the capsid, p53 protein, and the orf3 protein by ifa and shed light on p53 protein (the marker of apoptosis) accompanied the peptide (residues 35-66, p126) of the orf3 protein in pcv2-infected pbmcs. it is worth highlighting that 3-5% of pbmcs were positive for some antibodies staining, including anti-orf3 mab (7d3) staining, anti-p53 protein rabbit polyclonal antibody staining, and tunel assay ( figure s3 ). there is a strong probability that 3-5% of pbmcs were undergoing the process of apoptosis. however, the data revealed that the variable percentage (4-82%) of pbmcs was positive for anti-capsid mabs (1h3, 6b8, and 36a9) staining and anti-vlp rabbit serum staining. it was because these mabs or antiserum recognized different epitopes of proteins presented in pcv2-infected cells. that means that the variety of interaction between antigen-binding sites of antibodies and epitopes, which includes linear form epitopes, conformational epitopes, degradation, and different pcv2 strains. curiously, this study ( figure s3 ) showed that the percentage of pcv2-infected pbmcs (by mab 1h3 staining) was about 20-fold the rate of orf3-positive pbmcs (by mab 7d3 staining) or the percentage of p53-positive pbmcs. it seems likely that not all pcv2-infected cells contained orf3 proteins or p53 protein. this finding concurs with the previous study, which indicated that the apoptosis statistically decreases in the initial pcv2-infected pigs [60] . on the other hand, this study showed the orf3 protein colocalized with the capsid protein marker of pcv2. moreover, the p53 protein was also colocalized with the orf3 protein. remarkably, the p53/orf3 dual-positive cell presented a segmented nucleus ( figure 3w ). however, these p53/orf3 dual-positive cells were very few in these samples. this finding will be confirmed by flow cytometry in the future. although a previous study indicated that pcv2-infected pigs had a reduction of lymphocytes in the peripheral blood [11] , the mechanism of lymphocyte lysis was still unclear. this study confirmed that the orf3 protein was related to the p53 protein (apoptosis marker) in pcv2-infected pbmcs, while other factors (proliferative activity [60] , caspases 8 and 3 [34, 61] , granzymes [62] , or corticosteroids [63, 64] ) causing lymphocyte lysis or depletion need to be considered. although the experiment on apoptosis in pcv2-infected pbmcs was carried out by the tunel assay (figure 4) , it did not clarify the relationship between the tunel result and the orf3 protein in pcv2-infected cells. only the ifa data confirm previous reports that exogenous orf3 protein was related to the accumulation of p53 protein [37, 42] , but more proof are needed to make sure the orf3 protein is a major factor leading to apoptosis in pcv2-infected cells [34, 37, 42] and depletion of lymphocytes. pcv2 is one of the most critical pathogens in modern swine production and causes endemic disease in pig farms [7] . the commercial vaccines were confirmed protective in the field against pcv2 and mainly administered to suckers in herds with pcv2 infection [13, 14, [65] [66] [67] . most researchers utilized the quantitative real-time pcr to detect pcv2 nucleic acid, and then they evaluated the efficacy of vaccination in regard to pcv2 viremia in pigs [13, 14, 66] . however, little is known about the capsid protein or the orf3 protein in blood from the pcv2-infected herd with vaccination. although the capsid antigen-elisa could detect capsid protein, this assay could not differentiate from native viral proteins or vaccine ones in blood. the orf3 protein-elisa, by contrast, only identified native orf3 proteins in pig blood since no commercial vaccine contains the orf3 protein of pcv2. for these purposes, this study used the commercial capsid antigen-elisa and homemade orf3 protein-elisa (anti-n1 polyclonal antibodies and mab 7d3 based) to detect viral proteins in pig blood. this study found non-vaccinated suckers had the highest of pcv2 proteins in blood at one week of age. the author suggests that in these suckers it was caused by pcv2 infection, and these viruses majorly stemmed from the sow-to-newborn transmission [68] . after inoculated with the pcv2 subunit vaccine, the capsid protein, and the orf3 protein were gradually decreased. however, these proteins were detected again in gilts and sows (≥34 weeks of age). that means pcv2 viral proteins (the capsid protein or the orf3 protein) in gilts and sows were higher than that of pigs at 9-24 weeks of age. to put it another way, vaccinated-pigs reduced viremia compared to non-vaccinated suckers, and the immunity could not continue to protect vaccinated pigs after 32 weeks post-vaccination. since pcv2 is highly resistant to environmental conditions, being able to remain in the farm environment and thus represent a risk for infection maintenance [69] , even mass pcv2 vaccination (without implementing further farm management practices or biosafety measures) was not able to clear out pcv2 infection, and the virus became detectable again when vaccination was stopped [12, 67] . another key point to mention is that pcv2-specific antibodies response play roles in the pcv2-infected herd. it is worth noting that there were two different antibody profiles in this pcv2-infected herd with the pcv2 vaccine. the data show that pcv2-infected herd had a higher od 405 value of anti-capsid igg at age 1, 3, and ≥12 weeks, compared with 6-9 weeks. in addition, pcv2-infected herd had a higher od 405 value of anti-orf3 igg at age 1 and ≥9 weeks, compared with 3-6 weeks. according to previous studies, newborn suckers received colostral antibodies from seropositive sows and showed various levels of maternally derived antibodies [48, 68] . these maternally derived antibodies might interfere with the viral protein-specific antibodies response while suckers immunized with the pcv2 subunit vaccine. it may be assumed that the artificial capsid protein (pcv2 vaccine) elicited anti-capsid igg producing while maternally derived antibodies progressed to degrade. the total level of anti-capsid igg decreased slowly but still maintained a high level of anti-capsid igg during the age of 1-6 weeks. this phenomenon could neutralize the pcv2 virus and prevent piglets from suffering pcv2 infection. in contrast with dual-source capsid proteins (pcv2 virus and subunit vaccine), the piglet's immunity response to the orf3 protein was only caused by pcv2 virus infection. the total level of anti-orf3 igg decreased quickly at 1-3 weeks of age. then, it increased significantly at 6-9 weeks of age. the weak correlation (r = 0.3771) between the anti-capsid igg and the anti-orf3 igg is worth mentioning. it might reflect two kinds of antibody responses in virus infection and post-immunization. these data interpret the interaction of the viral protein with the host immune system. according to previous reports, orf2 encodes a 30-kda protein that is involved in viral capsid formation and contributed to self-assembled virus-like particles (vlp) [32] . the monomer structures were assembled into a vlp model consisting of 60 capsid subunits to form an icosahedron [70] . vaccination with this vlp (subunit vaccine) induced both humoral and cell-mediated responses against pcv2 [71, 72] . however, orf3 encodes an 11.9-kda protein [28] that is the non-structural protein and involved in pcv2-induced apoptosis [34, 37, 42] . until now, no report mentions that the orf3 protein was solely used as the vaccine against pcv2, reducing viremia or pathological lesions in pcv2-infected herds. these differences contributed to their application in different ways [36, 73] . in this study, the percentage of orf3-positive pbmcs was significantly lower than capsid-positive pbmcs. similarly, the antigen-elisa result shows that the amount of orf3 protein was less than the amount of capsid protein in pcv2-infected blood from suckers at one week of age. that might be because mab 7d3 only recognizes the orf3 protein (peptide p126) for pcv2b strain (genbank: aac35332.1), and it causes low detection. however, pcv2d (pcv2b mutant strain) and pcv2b are still predominant genotypes in the field farms. besides the similar percentages of pbmcs were positive for these antibodies (mab 7d3 and anti-p53 protein rabbit polyclonal antibody) staining or tunel assay. in contrast to the orf3 protein, the detection of the capsid protein could be the variable result in pcv2-infected pbmcs via different antibodies, due to different pcv2 strains [49, 74] , conformational epitopes [75] , or linear epitopes [49, 76] . previous studies indicated that antibody binding residues were often on the exterior of the capsid of pcv2 [48, 49, 70, 74, 77, 78] . although an epitope might be on the surface of the single capsid unit, it could bury in the vlp and be inaccessible to the antibody [79, 80] . in general, these results confirm that mab 7d3 recognized the native orf3 protein in pcv2-infected pbmcs. this study used mab 7d3 or its minimal linear epitope to design various immunoassays. these assays evaluate the viral load and the immune response of viral proteins stimuli. these may serve as surveillance tools for monitoring natural pcv2 infection in the herd. overall, the author generated anti-orf3 mabs against the orf3 peptide (residues 36-66) of pcv2. this study confirmed the defective-ig mab (6d10) with a lambda-light chain, and its molecular weight was about 33 kda. the mab 7d3 contained heavy chains (γ 1 ) and kappa-light chains, and it bound to one minimal linear epitope (hndvyislpitllhfpahfq kfsqpaeisdkr). the data show that 3-5% of pbmcs were positive for orf3 protein or p53 protein. otherwise, 78-82% of pbmcs were positive for anti-capsid peptide mab (1h3) staining. this study confirmed the orf3 protein colocalized with the p53 protein in pcv2-infected pbmcs. the author devised the orf3 protein-elisa (anti-orf3 antisera and mab 7d3-based) to detect the orf3 protein in blood samples. the results show that the amount of orf3 protein was less than the amount of capsid protein in pcv2-infected blood from suckers at one week of age. the correlation between the orf3 protein and the capsid protein is worth noting (r = 0.7583). furthermore, the antibody level of anti-capsid igg and anti-orf3 igg could imply the immunity response of pig herd, but the sample size needs to be considered. the author declares no conflict of interest. a very small porcine virus with circular single-stranded dna characterization of novel circovirus dnas associated with wasting syndromes in pigs studies on epidemiology and pathogenicity of porcine circovirus experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus enteritis associated with porcine circovirus 2 in pigs porcine circovirus type 2 associated disease: update on current terminology, clinical 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proteins can act as architectural transcription factors by promoting the assembly of higher-order protein-dna complexes which can either activate or repress gene expression. the structural organisation of both classes of protein is similar with either a single or repeated dna binding domain preceding a short negatively charged c-terminal tail. in the hmgb class of proteins the hmg dna-binding domain binds non-specifically and introduces a sharp bend into dna whereas the at-hook in the hmga protein binds preferentially to a/t rich regions of dna and stabilises a b-dna structure. the acidic tails are hypothesised to facilitate the interaction of the proteins with nudeosomes by binding to the positively charged histone tails. both classes of protein also interact with a large number of transcription factors that bind to specific dna sequences. the eukaryotic nucleus contains three classes of abundant chromatin associated proteinsthe high mobility group proteins of the hmga, hmgb and hmgn classes (originally termed hmgia^, hmg 1/2 and the hmg 14/17; for recent nomenclature changes see re£ 1), so called because they were initially identified on the basis of their rapid migration through starch gels. the hmga and hmgb classes of chromosomal proteins in general share some common characteristics, notably a conserved acidic region and the ability to interact with several different transcription factors. a major role is to organise the struaure of dna-protein complexes in the context of chromatin. in both the eukaryotic nucleus and the bacterial nucleoid the trajectory of the dna double helix is normally tightly constrained so that not only can the dna be compacted without entanglement but also to provide an appropriate environment for the enzymatic machinery involved in dna transcription, replication and recombination. this organisation is normally effected by abundant dna binding proteins, termed architectural dna-binding proteins, that either induce dna bending or facilitate the formation of multicomponent dna-protein complexes. the term ^architectural' in this context implies that the protein is required for organising dna but the proteins that fall within this definition are often otherwise functionally distinct and would include, for example, the histone octamer, abundant eukaryotic dna conformation and transcriptiony edited by takashi ohyama. ©2005 eurekah.com and springer science+business media. chromosomal proteins, such as the hmga and hmgb proteins, abundant proteins associated with the prokajyotic nucleoid, such as fis, h-ns, ihf and hu, as well as bona fide transcription factors exemplified by the tata-binding protein (tbp). some of these proteins have more than one architectural function. for example fis can stabilise particular configurations of supercoiled dna plasmids and also act to promote the assembly and activity of transcription, replication and recombination complexes. many of the more generalised architectural proteins may be regarded as facilitators and are ofiien not essential for viability while some more 'specialised' proteins, such as the histone octamer and tbp, are clearly essential. the bending of dna by transcription factors and by other protein complexes is a major component in the establishment of the overall morphology of protein-dna complexes. this bending is usually a consequence of indirect readout, a mechanism by which the selectivity of binding is dependent not on making direct contacts between the aminoacids and bases, i.e., direct readout, but instead on the physicochemical properties of the dna molecule itself recognition of dna by transcription factors often involves both direct and indirect readout. however, the principles of indirect readout are well illustrated by the histone octamer which, although not a transcription factor itself, completely lacks direct contacts between the aminoacid side chains and the bases of the boimd dna. the octamer binds 147 bp of dna which are wrapped in a left-handed superhelix with a total curvature of approximate 10 radians. this curvature contrasts with the stiffness of dna in solution where the average persistence length (p), defined as the length over which the average deflection of the polymer axis caused by thermal agitation is one radian, is 140-150 bp,^ i.e., the same length as that bound by the histone octamer. for dna molecules that are not anisotropically curved the affinity of the dna for the octamer is direcdy proportional to the flexibility (the inverse of the stiffiiess). however the dependence of the binding energy on p is some 10-fold lower than the dependence of the bending energy in solution on p. this implies that the histone octamer increases the apparent flexibility substantially to compensate for the average increase in dna curvature on binding. how might this change in flexibility be effected? the histone core provides a dna binding siu-face in the form of a positively charged ramp. on binding to this ramp the negative charges on one side of the dna are neutralised. this asymmetric neutralisation, which can be mimicked in free dna,^ creates an imbalance in charge distribution on opposite sides of the double helix so that repulsion between the opposing sugar-phosphate backbones on the unneutralised side facilitates bending by increasing the width of the grooves. concomitandy, the reduction in this repulsion on the inside of the bend permits greater freedom in the motions of the basepairs, with a corresponding reduction in the width of the grooves. the greater flexibility of the motions between base-pairs is reflected in the periodic variation of twist and roll with groove width such that the ranges of values assumed for both are substantially larger than the corresponding ranges observed for dna molecules free in solution.^ the correlation between flexibility and affinity for the histone octamer only applies strictly when a dna molecide does not possess intrinsic anisotropic curvature. when it does the affinity may be relatively higher or lower. for example, the intrinsically curved tata dna sequence whose curvature is compatible with the surface of the histone octamer binds with an affinity that would normally be characteristic of a substantially more flexible isotropic binding site. ' in this case binding is favoured by the lower entropic penalty on binding relative to an isotropically flexible molecide.^ however if the intrinsic bend is too great and therefore less compatible with the protein binding surface the affinity is reduced relative to an isotropically flexible molecule.^^ an extension of this principle of asymmetric alteration of the ionic environment of dna is provided by the transcription factor tbp and the hmg-domain, found in hmgb proteins, a class of abundant chromosomal proteins and certain transcription factors such as sry and lef-1.^^ the hmgb proteins consist essentially of a small l-shaped protein domain with a cluster of hydrophobic residues on its inner surface and an extended unstructured basic region. when these proteins bind to dna they produce a bend of 95-120° over about six base-pairs and decrease both the axial and torsional stiffness. ^ on the outer surface of the bend the hydrophobic 'wedge' towards the apex of the l binds in and widens the minor groove, concomitandy untwisting the dna. this effect is believed to be facilitated by a local reduction in the dielectric constant which increases the repulsion between opposing sugar-phosphate backbones on the approach of the protein to same time the basic region neutralises the phosphates bounding the major groove on the inside of the bend thus decreasing the repulsive forces and permitting the narrowing of the groove. additionally the protein inserts, or intercalates, hydrophobic aminoacids into either a single base-step or into two base-steps that are themselves separated by a single base-step. the extent to which this intercalation increases or simply stabilises the induced bend is unclear. the bend induced by the intercalation contrasts with the smooth dna bending induced by the histone octamer since the intercalation effectively introduces a kink in the dna such that the stacking interactions between adjacent base-pairs are very substantially reduced. in the tata-binding protein this same principle of hydrophobic interactions predominates. here two pairs of phenylalanine residues are intercalated at steps separated by 6 bases, kinking the dna by --45° at each intercalation site.^^' between these pairs of phenylalanine residues a hydrophobic surface rests snugly within the minor groove. again the minor groove is widened and untwisted. however, unlike the hmgb proteins there is no charge neutralisation on the opposing major groove face of the bent dna and indeed the sharpness of the induced curvature is less than that for the hmgb proteins. in other transcription factors there is substantial variation in the degree of induced bending. the escherichia coli cap (aka crp) factor is a good example of mixed direct and indirect readout. this dimeric protein induces a bend of --45° per monomer.^^ in this case the major bend occurs where the recognition helix of the helix-turn-helix motif binds in the major groove on the inside of the bend, concomitandy making direct contacts with the dna bases and neutralising the sugar-phosphate backbone in the immediate vicinity. ^^ flanking the central recognition palindrome is a basic ramp which binds dna and increases the overall dna bend by indirect readout in a manner analogous to the histone octamer. although one of the principal roles of dna bending in the living cell is to maintain the compaaion of dna, it also has important functions in transcriptional control and, in particular, in the assembly of regulatory complexes. a major consequence of introducing a tight bend into dna is to bring dna sequences which are far apart on a linear representation of a dna molecide into close spatial proximity. this effect, which is also characteristic of plectonemically supercoiled dna, is mediated in chromatin by the hmg-domain transcription factors, such as tcf-1, lef-1 and sry. in the case of tcf-1 acting at the enhancer of the tcr promoter, the bend induced by the factor brings together a normally unstable complex of the ets-1 and pebp2a dna-binding proteins and atf/creb activator proteins to form a stable complex. ^^ this example is probably a particular case of the more general phenomenon in which the dna between a transcription factor and its target protein partner must be bent for protein-protein contacts to occur. the ease of bending will depend critically on the distance and the helical phase difference between the binding sites of the factor and its target. normally unless one or both of the partner proteins are flexible contact will be facilitated when the binding sites are in helical phase, primarily because of the constraints on the torsional flexibility of dna. however, at least in vitro, the constraints imposed by both torsional and axial rigidity can be overridden by the abundant dna-bending proteins of the hmgb class. in the presence one of these proteins a requirement for an integral number of helical turns between binding sites is no longer crucial.^^ furthermore the involvement of the hmgb protein in the formation of the complex need only be transient. to what extent are variations in dna flexibility reflected in genomic organisation? an excellent example of the dependence of biological function on dna structure is provided by genome of the enteric bacterium e. colt. in this organism the strongest promoters for dna transcription, often those directing the synthesis of rrna and trna, are almost invariably associated with a/t rich, and hence flexible, dna sequences extending upstream for 100-300 base-pairs from the transcription startpoint."^^ the activity of many of these promoters is strongly dependent on a high negative superhelical density stored in the dna. this would in principle favour both dna untwisting at sequences such as tataat close to startpoint"^^ and also lefthanded dna wrapping aroimd the protein complex responsible for initiating transcription. in many of these highly active promoters the dna sequence also imparts curvature to the region, a featiue that correlates both with the presence of multiple activating binding sites for the abundant dna bending protein fis (factor for inversion stimulation).^^'^^ these sites are often organised in helical phase such that the binding of fis could constrain a negative superhelical loop. indeed in the rma pi regulatory region the presence of a far upstream fis site centred at position -222 from the transcription startpoint results in the constraint of an additional supercoil in the initiation complex.^"^ a primary function of this fis-induced dna looping is to promote the wrapping of dna around the rna polymerase prior to the initiation of transcription, a phenomenon that has also been proposed for the activation of the lac promoter by the cap dna-bending transcription factor,^ and consequendy to facilitate the extended wrapping characteristic of the open complex.^'^ in turn the fis-induced constraint of negative superhelicity buff^ers this type of promoter against changes in the unconstrained superhelical density.^ ' '^^ in other such promoters an alternative model proposes that the upstream activating sequence contains regions that are highly susceptible to dna untwisting. for both dna wrapping and untwisting in the upstream region both models predict that the topological unwinding is transmitted to the 'tata sequence and promotes its untwisting. dna bending may also be required for the establishment of repressive regulatory complexes. here, a dna loop is often formed by the binding of an oligomeric repressor to two sites that are distant from each other along the dna sequence. this loop, which can be as tightly bent as nucleosomal dna, prevents the binding of rna polymerase to the regulated promoter. examples of this mode of regulation include repression by the arac, lad and galr proteins.^^'^^ the vertebrate hmga proteins are small proteins of -100-110 aminoacids and contain tandem copies, usually three, of a characteristic dna-binding domain, the at-hook, together with a c-terminal acidic region (fig. 1) .^^ the at-hook is not restricted to the hmga proteins as such as it is also found in a related drosophila chromosomal protein, dl, which contains multiple copies of the motif,^^'^ in the motor subunits of various atp-dependent chromatin remodelling complexes and in certain transcription factors that also contain a primary sequence-specific dna-binding domain where it is assumed to act as an auxiliary dna-binding element.^^ the at-hook is an unstructiu'ed short motif with the consensus sequence arg/lys-pro-arg-gly-arg-gly-pro-arg/lys^^'^^ and selectively binds in the minor groove of a/t-rich regions of dna.^^'^^ the central arg-gly-arg core adopts an extended conformation deep within the groove with the arginine side chains making extensive hydrophobic contacts along the base of the groove. ^ the proline residues change the trajectory of the backbone allowing the basic residues flanking the core mediate electrostatic and hydrophobic contacts with the dna backbone. when bound to dna the surface of the core motif contacting the dna is concave and resembles that of the dna binding drug netropsin, which has a similar selectivity for a/t-rich sequences. ^ for both netropsin and an at-hook one consequence of dna binding is a modest widening of the minor groove with a concomitant stabilisation of b-dna structure. in some cases this widening results in a change in the direction of bending of dna, particularly when that bending is dependent on a narrow minor groove width in a/t-rich sequences. ' thus a small intrinsic bend of -20° towards the minor groove in the ifn-p enhancer is reversed on binding hmgal. this could then facilitate recognition of the opposing major groove by transcription factors binding to specific sequences. nevertheless although the hmga proteins induce only small changes in dna structure they bind tightly to dna ligands v^ith distorted or unusual features. these include supercoiled dna, four-way junctions and baseunpaired regions of at-rich dna (reviewed in ref. 34) . strikingly in vitro these proteins can also introduce supercoils into relaxed dna, possibly by stabilising cross-overs and thereby stabilising dna loops. this ability has been suggested as an explanation of the observation that in vitro hmgal represses the chick globin p gene promoter in the absence of the 3' enhancer but strongly activates transcription in its presence, regardless of whether or not the substrate is free dna or is assembled into nucleosomes. this stabilisation of loops could be mediated by the presence of multiple at-hooks on each protein. in mammals the hmga proteins are encoded by two functional genes, hmgal and hmga2. alternative splicing of the transcripts of these genes increases the variety of protein products, of which the most abundant are hmgal a (hmg-i) and hmgal b (hmg-y). these proteins appear to perform a variety of functions, of which the most studied are related to chromatin structure and to the facilitation or inhibition of transcription factor binding. in vitro hmga proteins bind to nucleosomes, notably at the exit and entry points to the nucleosome core particle where they are in close proximity to histones h2a, h2b and h3. '^ the proteins can also bind to internal sites where they can induce local changes in the rotational setting of the wrapped dna. the binding to the nucleosomal dna is mediated by the athooks but (by analogy to the hmgb class of proteins) it is conceivable that the acidic tail may also be involved in contacting the histone octamer and could perform a similar role to that of the hmgb proteins. in mitotic chromosomes the hmga proteins are associated with particular bands and these proteins are localised to the base of large chromatin loops in close proximity to scaffold-attachment regions (sars). as a consequence it has been suggested that the hmga proteins are involved in the maintenance of the condensed mitotic chromosome structure in these regions. evidence supporting this view was adduced from the observation that synthetic 'math' proteins containing at-hooks interfere with chromosome condensation during mitosis. in contrast another model suggests that co-operative binding of hmga molecules to a looped chromatin domain in interphase nuclei will facilitate the formation of an 'open' chromatin structure that is competent for transcription by competing with histone hi. although some experiments demonstrate that the math proteins can counteract the spreading of heterochromatin, as shown in particular by suppressing position-effect variegation in flies. the mechanism by which this is accomplished remains to be established. however the expression of the hmga proteins is strongly correlated with cell growth and is characteristically high in neoplastically transformed cells. hmgal has been shown to interact directly with a large variety of transcription factors including at-1, atf-3, nf-y, irf-1, srf, nf-kb, p50, tst-l/oct-6 and c-jun.^^ in some cases the protein regulates the formation of an enhanceosome. thus at the virus-inducible (3interferon enhancer a complex containing both hmga proteins and transcription factors forms and then acts to recruit rna polymerase ii and its associated general transcription factors.^^'^ in other cases hmga proteins can block enhanceosome formation.^^ this modulation of transcription factor binding may be integrated with the regulation of chromatin organisation. thus hmgal a enhances the binding of the atf-3 to a site at the edge of a nucleosome positioned on the hiv-1 promoter.^ this combination of bound proteins can then recruit the remodelling complex hswi/snf. the interactions of hmga with these factors can be modulated by covalent modifications including phosphorylation and methylation. hmgb proteins are characterised by the hmg-box, a dna-binding domain specific to eukaryotes. a major characteristic of this domain is to introduce a sharp bend into dna (fig. 2) . accordingly the domain also binds preferentially to a variety of distorted dna structures, especially those in which the distortion itself induces a bend. these include negatively supercoiled dna, small dna circles, cruciforms, dna bulges and cisplatin modified dna.^^ the hmg-box domain is also found in several related types of protein, for example transcription factors such as sry and lef-1, and subunits of many chromatin remodelling complexes. all these proteins are predominantly nuclear and appear to act primarily as architectural facilitators in the manipulation of nucleoprotein complexes; for example, in the assembly of complexes involved in recombination and the initiation of transcription, as well as in the assembly and organisation of chromatin. the archetypal hmgb proteins are highly abundant (--10-20 copies per nucleosome in the mammalian nucleus^^) and often occur in two major forms, hmgbl and hmgb2, originally termed hmgl and hmg2, in vertebrates.^ the two distinguishing features of these highly homologous proteins are two similar, but distinct, tandem hmg-box domains (a and b) , and a long acidic c-terminal 'tail', consisting of-30 (hmgl) or 20 (hmg2) acidic (aspartic and glutamic acid) residues, linked to the boxes by a short, predominantly basic linker (fig. 1) . however the most abundant hmg-box domain proteins in saccharomyces cerevisiae, nhp6ap and nhp6bp (non-histone proteins 6a and 6b respectively), contain only a single hmg box, and lack an acidic tail. likewise the two major hmg-box domain proteins in drosophila melanogaster, hmg-d and hmg-z, have only a single hmg box but, unlike the yeast proteins, contain a short c-terminal acidic tail in addition to a basic region (fig. 1) . these abundant proteins in yeast and drosophila may be the general functional counterparts of hmgbl and 2 in vertebrates. the precise functions of the chromosomal hmgb proteins in vivo for a long time remained obscure. however there is now substantial evidence that they interact directly with both transcription factors and with the histone octamer. these interactions can affect transcription factor access to chromatin either directly or by promoting chromatin remodelling. in the latter case the proteins may facilitate repression or activation. there are two established cases in which the assembly of nucleoprotein complexes containing sequence-specific dna-binding proteins is promoted by the dna-bending properties of hmgbl and 2, i.e., the proteins have a classical architectural role. first, in v(d)j recombination the lymphocyte-specific proteins ragl and rag2 (human recombination activating genes 1 and 2) appear to recruit hmgbl and 2 to the appropriate sites in chromatin ' ^ presumably by protein-protein contacts with the ragl homeodomain. here they ensure the "12/23 rule". this requires that v(d)j recombination occurs only between specific recombination signal sequences (rss). each rss is made up of a conserved hep tamer and nonamer sequence separated by a non-conserved spacer of either 12 or 23 base pairs. hmgb 1 (in concert with rag 1,2) facilitates recombination probably by bending the dna between the two conserved sequences spaced by 23 bp and stabilising a nucleoprotein complex. the hmgb protein plays the dual role of bringing critical elements of the 23-rss hep tamer into the same phase as the 12-rss to promote rag binding and of assisting in the catalysis of 23-rss cleavage. recent footprinting experiments indicate that the hmgbl (or hmgb2) protein is positioned 5' of the nonamer in 23-rss complexes, interacting largely with the side of the duplex opposite the one contacting the rag proteins. a second instance in which an abundant hmgb protein may facilitate nucleoprotein complex assembly is in the formation of an enhanceosome containing the epstein-barr virus replication activator protein zebra and hmgb 1,^^ the two proteins bind cooperatively, hmgbl binding to, and presumably bending, a specific dna sequence between two zebra recognition sites. bending of dna by hmgbl and 2 has also been invoked to explain the essential role of these proteins in initiating dna replication by loop formation at the mvm (minute virus of mice) parvovirus origin of replication. in vitro hmgb proteins can enhance the binding of various transcription factors (e.g. adenovirus mltf, oct-1 and 2, hoxd9, p53, steroid hormone receptors, rel proteins, p73, dof2 and the epstein-barr activator rta) to their cognate dna binding sites (reviewed in ref gg). similarly rat ssrpl has been shown to facilitate the dna binding of serum response factor and human ssrpl is associated with the y isoform of p63 in vivo at the endogenous mdm22inap2nfl'"^^ promoters.^^ in most of these cases, the interaction of the hmg protein with the transcription factor has been detected in vitro and could, in principle, serve as the mechanism for recruitment of hmgb 1 or 2 to particular dna sites. in some cases transfection experiments indicate functional interactions in vivo. direct interactions between nhp6p and the gal4p and tup ip transcription factors have also been inferred in vivo by a split-ubiquitin screen and confirmed by a pull-down assay.^^ although the demonstrated interactions in vitro so far involve an hmgb protein and a single transcription factor, it is entirely possible that in vivo, in a natural regulatory context, the bending of dna by hmgbl and 2 could potentially allow the recruitment of a second transcription factor to the complex, in an analogous manner to the action of sequence-specific hmg-box transcription factors^ such as lef-1 in the enhanceosome at the t cell receptor alpha (tcra).^^ hmgbl may play a catalytic, chaperone role, since it does not appear to be stably incorporated into the final complex. although a role for hmgbl and hmgb2 induced dna bending in the facilitated binding of transcription factors, while being entirely plausible, has not been directly established, it is strongly suggested by the ability of hu to substitute for the hmgbl-stimulated binding of the epstein barr virus transactivator rta to its cognate binding sites.'^^ a possible role for an hmgblinduced change in dna conformation in facilitation of transcription factor binding is also suggested by the observation that hmgbl promotes binding of p53 to linear dna but not to gg bp dna circles. however, in this case the data do not distinguish between possible effects of dna bending or untwisting. the biological roles of the hmgb proteins have been studied using gene knock-outs. in mice the loss of hmgbl but not of hmgb2 is lethal although in the former knock-out there are pleiotropic effects on glucose metabolism while in the latter spermatogenesis is impaired. ' this suggests a functional redundancy between members of the hmgbl and 2 family. a similar situation occurs with nhp6ap and nhp6bp in yeast.'^^ however, the different phenotypes of the hmgbl and hmgb2 null mice probably reflect specific roles for the two proteins in different tissues."^^'"^ in s. cerevisiae the transcriptional effects o(nhp62irc not general but gene-specific. at the chal locus, loss of nhp6results both in an increase in the basal level of transcription and in a substantial decrease in the induced level.^^ this suggests an effect at the level of chromatin. the chal regulatory region contains a positioned nucleosome which occludes the tata box under non-inducing conditions. on induction the tata region becomes accessible.''^ however in the mutant strain, consistent with the increased basal level transcription, the chromatin structure of the tata region in the uninduced state is similar to that in the induced wild-type strain. nhp6thus appears to be required for establishment of the organised chromatin structure characteristic of the uninduced state. the rsc remodelling complex is also required for this process''^ suggesting that rsc and nhp6p may cooperate to remodel chromatin. further insights into how nhp6ap and nhp6bp function were provided by studies on the ho gene. loss of a^//p6^ function can be suppressed by mutations that increase nucleosome accessibility and mobility, and enhanced by those with the opposite effect. mutations both in the sin3 and rpd3 genes, encoding components of a histone deacetylase complex, and in sin4, partially restore wild-type function in cells lacking both nhp6ap and nhp6bp, while loss of the histone acetylase gcn5p (also a component of the saga histone acetylase complex) in the same cells results in a more severe phenotype. rpd3p and gcn5p contribute to the dynamic balance between histone acetylation and deacetylation.^^ both histone acetylation and the sin (swi/snf independence) phenotype are correlated with chromatin unfolding ' and/or enhanced nucleosome accessibility^^ while histone deacetylation would be expected to favour folding. on this argument one role of the nhp6p proteins would be to antagonise folding and possibly promote nucleosome accessibility. the ability of the hmgb proteins to promote both transcription factor binding to their cognate sites and also chromatin remodelling implies that these activities could be coordinated to alter chromatin structure in the vicinity of a factor binding site. like the hmga proteins the abundant hmgb proteins bind to nucleosomes at sites close to the dna exit and entry points. an insight into how hmgb proteins might alter the accessibility of nucleosomal dna was provided by the observation that hmgbl could facilitate the binding and subsequent remodelling function of the acf remodelling complex in vitro.^ further observations showed that hmg-d, a drosophila hmgb protein, when bound to nucleosome core particles increased the accessibility of nucleosomal dna to restriction endonucleases at particular sites. these sites were asymmetrically distributed, one site being located at one end of the bound dna and the other in the vicinity of the nucleosome dyad. this effect required the acidic tail of the hmgb protein: without it the hmg-d reduced accessibility at all sites tested on the nucleosome. this result argues that certain hmgb proteins can alter the structure of nucleosomes and to do so presumably by interacting with an available basic region of the histone octamer. from the distribution of the sites with increased accessibility a prime candidate would be one (but not both) of the n-terminal tails of histone h3 or one of the c-terminal tails of histone h2a. it is important to note that the yeast nhp6 proteins lack an acidic region and so could not interact with histones direcdy in this way. however they can associate with two other proteins, pob3p and sptl6p, to form a complex, spn, involved in chromatin remodelling. ' both these proteins contain extensive acidic regions and so, in principle, could substitute for the lack of an acidic region in nhp6p. the abimdant hmga and hmgb chromosomal proteins share several common features. both interact with nucleosomes, both can also bind a set of transcription factors, both are involved in enhanceosome formation and both can facilitate the recruitment of chromatin remodelling complexes. interestingly the hmgn class of hmg proteins shares with the hmga and hmgb classes the ability to interact with nucleosomes and also possesses a c-terminal region with a net negative charge. revised nomenclature for high mobility group (hmg) chromosomal proteins studies on nuclear proteins. the binding of extra acidic proteins to deoxyribonucleoprotein during the preparation of nuclear proteins hmg domain proteins: architectural elements in the assembly of nucleoprotein structures crystal structure of the nucleosome core particle at 2.8 a resolution flexibility of dna the structural basis of dna flexibility dna bending by asymmetric phosphate neutralization the structure of dna in the nucleosome core identification and characterization of genomic nucleosome-positioning sequences dual role of dna intrinsic curvature and flexibihty in determining nucleosome stabiuty sequence-dependent dna curvature and flexibility from scanning force microscopy images hmgl and 2, and related architectural dna-binding proteins reading the minor groove the low dielectric interior of proteins is sufficient to cause major structural changes in dna on association co-crystal structure of tbp recognizing the minor groove of a tata element crystal structure of a yeast tbp/tata-box complex comparative gel electrophoresis measurement of the dna bend angle induced by the catabolite activator protein crystal structure of a cap-dna complex: the dna is bent by 90° the hmg domain of the lymphoid enhancer factor 1 bends dna and facilitates assembly of functional nucleoprotein structures hmg proteins and dna flexibiuty in transcription activation a dna structural atlas for escherichia coli negative supercoiling induces spontaneous unwinding of a bacterial promoter dna microloops and microdomains: a general mechanism for transcription activation by torsional transmission promoter protection by a transcription factor acting as a local topological homeostat fis modulates the kinetics of successive interactions of rna polymerase with the core and upstream regions of the e. coli tyrt promoter mechanism of activation of transcription by the complex formed between cycuc amp and its receptor in escherichia coli wrapping of dna around the e. coli rna polymerase open promoter complex mechanism of transcriptional activation by fis: role of core promoter structure and dna topology dna topology-mediated control of global gene expression in escherichia coli an operator at -280 base pairs that is required for repression of the arabad operon promoter: addition of dna helical turns between the operator and promoter cyclically hinders repression atomic force microscopic demonstration of dna looping by galr and hu lac repressor forms loops with linear dna carrying two suitably placed lac operators in vivo thermodynamic analysis of repression with and without looping in lac constructs. estimates of fi-ee and local lac repressor concentrations and of physical properties of a region of supercoiled plasmid dna in vivo molecular biology of hmga proteins: hubs of nuclear function protein dl preferentially binds a + t-rich dna in vitro and is a component of drosophila melanogaster nucleosomes containing a + t-rich satellite dna isolation and sequencing of cdna clones encoding drosophila chromosomal protein dl. a repeating motif in proteins which recognize at dna at-hook motifs identified in a wide variety of dna-binding proteins the at-dna-binding domain of mammalian high mobiuty group i chromosomal proteins: a novel peptide motif for recognizing dna structure protein motifs that recognize structural features of dna a mammalian high mobility group protein recognizes any stretch of six a-t base pairs in duplex dna the solution structure of an hmg-i(y) dna complex defines a new architectural minor groove binding motif refinement of netropsin bound to dna: bias and feedback in electron density map interpretation reversal of intrinsic dna bends in the ifn p gene enhancer by transcription factors and the architectural protein hmg i(y) changes in superhelicity are introduced into closed circular dna by binding of high mobility group protein la^ hmg i/y regulates long-range enhancer-dependent transcription on dna and chromatin by changes in dna topology interaction of high mobility group-i(y) nonhistone proteins with nucleosome core particles substrate structure influences binding of the non-histone protein hmg-i(y) to free and nucleosomal dna metaphase chromosome structure: bands arise from a differential folding path of the highly at-rich scaffold sars are cis dna elements of chromosome dynamics: synthesis of a sar repressor protein sar-dependent mobilization of histone hi by hmg-i/y in vitro: hmg-i/y is enriched in hi-depleted chromatin in vivo analysis of scaffold-associated regions in drosophila: a synthetic high-affinity sar binding protein suppresses position effect variegation hmgi(y) and hmgi-c dysregulation: a common occurrence in human tumors ordered recruitment of chromatin modifying and general transcription factors to the ifn-p promoter the mechanism of transcriptional synergy of an in vitro assembled interferon-p enhanceosome hmg i(y) interferes with the dna binding of nf-at factors and the induction of the interleukin 4 promoter in t cells recruitment of swi/snf to the human immunodeficiency virus type i promoter a deoxyribonucleic acid unwinding protein isolated from regenerating rat liver. physical and functional properties v(d)j recombination: modulation of ragl and rag2 cleavage activity on 12/23 substrates by whole cell extract and dna-bending proteins stimulation of v(d)j cleav^e by high mobility group proteins accessibility of nucleosomal dna to v(d)j cleavage is modulated by rss positioning and hmgl the rag-hmgl complex enforces the 12/23 rule of v(d)j recombination specifically at the double-hairpin formation step the ragl homeodomain recruits hmgbl and hmgb2 to facilitate recombination signal sequence binding and to enhance the intrinsic dna-bending activity of rag1-rag2 fine structure and activity of discrete rag-hmg complexes on v(d)j recombination signals mechanism for specificity by hmg-1 in enhanceosome assembly two widely spaced initiator binding sites create an hmgldependent parvovirus rolling-hairpin replication origin chromosomal hmg-box proteins cooperative transcriptional activation by serum response factor and the high mobility group protein ssrpl ssrpl functions as a co-activator of the transcriptional activator p63 a new screen for protein interactions reveals that the saccharomyces cerevisiae high mobility group proteins nhp6a/b are involved in the regulation of the gall promoter assembly and function of a tcra enhancer complex is dependent on lef-1-induced dna bending and multiple protein-protein interactions the dna architectural protein hmgbl displays two distinct modes of action that promote enhanceosome assembly efficient specfic dna binding by p53 requires both its central and cterminal domains as revealed by studies with high-mobility group 1 protein the lack of chromosomal protein hmgbl does not disrupt cell growth but causes hypoglycaemia in newborn mice reduced fertility and spermatogenesis defects in mice lacking chromosomal protein hmgb2 nhp6a and nhp6b, which encode hmgl-uke proteins, are candidates for downstream components of the yeast slt2 mitogen-activated protein kinase pathway chromatin-mediated transcriptional regulation by the yeast architectural factors nhp6a and nhp6b nucleosome structure of the yeast chal promoter: analysis of activation-dependent chromatin remodeling of an rna-polymerase-ii-transcribed gene in tbp and rna pol ii mutants defective in vivo in response to acidic activators transcriptional repression of the yeast chal gene requires the chromatin-remodeung complex rsc architectural factors and the saga complex function in parallel pathways to activate transcription hyperacetylation of chromatin at the adh2 promoter allows adrl to bind in repressed conditions disruption of higher-order folding by core histone acetylation dramatically enhances transcription of nucleosomal arrays by rna polymerase iii the sin domain of the histone octamer is essential for intramolecular folding of nucleosomal arrays effects of histone acetylation on the equilibrium accessibility of nucleosomal dna target sites the dna chaperone hmgbl facilitates acf/chracdependent nucleosome sliding hmg-d and histone hi alter the local accessibility of nucleosomal dna sptl6-pob3 and the hmg protein nhp6 combine to form the nucleosome-binding factor spn a bipartite yeast ssrpl analog comprised of pob3 and nhp6 proteins modulates transcription hmg-d complexed to a bulge dna: an nmr model key: cord-257802-vgizgq2y authors: uttamchandani, mahesh; neo, jia ling; ong, brandon ngiap zhung; moochhala, shabbir title: applications of microarrays in pathogen detection and biodefence date: 2008-11-12 journal: trends biotechnol doi: 10.1016/j.tibtech.2008.09.004 sha: doc_id: 257802 cord_uid: vgizgq2y the microarray is a platform with wide-ranging potential in biodefence. owing to the high level of throughput attainable through miniaturization, microarrays have accelerated the ability to respond in an epidemic or crisis. extending beyond diagnostics, recent studies have applied microarrays as a research tool towards understanding the etiology and pathogenicity of dangerous pathogens, as well as in vaccine development. the original emphasis was on dna microarrays, but the range now includes protein, antibody and carbohydrate microarrays, and research groups have exploited this diversity to further extend microarray applications in the area of biodefence. here, we discuss the impact and contributions of the growing range of microarrays and emphasize the concepts that might shape the future of biodefence research. natural outbreaks and the wanton use of pathogenic organisms for acts of terror have had tremendous impact on human populations, especially when considering the events over the past decade. modern travel and trade have further dissipated traditional geographical boundaries that once curbed the spread of disease. infectious agents capable of spreading from human to human, such as the severe acute respiratory syndrome (sars) coronavirus, have shown us just how quickly (in a matter of weeks) a threat anywhere could become a threat everywhere [1] . the lessons learnt from sars, avian flu and the anthrax letter attacks have emphasized the need for improved preparedness for, response to and treatment of both known and emergent biological threats [2, 3] . this has also prompted heightened funding initiatives worldwide to build up national and international biodefence capabilities [3] . among the systems and protocols to be put in place, platforms that facilitate the rapid and accurate identification of agents are particularly vital, both in confirming whether an attack has occurred and in instituting prompt measures to secure public health. the intrinsic ability of microarrays to perform multiplexed, low-volume and sensitive biological assays in a highly scalable manner is a significant advantage in biological threat analysis [4] . developed in the early to mid 1990s, dna microarrays stirred a technological revolution that continues to propel genomics research today [5] . applications included the identification and comparison of mrna or dna from across tissues or organisms by hybridization against thousands of oligomeric dna probes immobilized on planar surfaces, such as glass slides (figure 1 ). this provided researchers with an unprecedented ability to quantify genome-wide differences in gene expression or sequence changes using minimal amounts of sample. in the context of biodefence, these studies have dramatically extended our capability beyond merely detecting known pathogens; now we are able to rapidly profile and characterize pathogens, as well as identify novel strains or 'genetic islands' that manifest changes in virulence (or the evolution of resistance). such comparative phylogenomic profiling using microarrays facilitates the understanding of pathogen diversification by providing a bird's-eye view of the gene content present or absent within a given microbial genome [6] . microarray technology has also been brought outside the laboratory to the point-of-care, a development that has widening implications for threat detection. dna-based detection systems generally rely on the ability of pcr to amplify and fluorescently tag the tiny amounts of target dna present in the specimen. advances in miniaturizing this initial pcr step, for instance the development of review glossary biodefence: defensive measures against biological threats, including natural/ emerging pathogens and bioterror agents, that have significant potential to endanger public health detection: identifying the presence of target pathogen(s) from clinical or environmental samples. diagnostics: tests used to detect a medical condition, for example to test for the causative pathogen responsible for an infection. sandwich immunoassay: a biochemical assay for detecting the presence and/ or abundance of a target substance using the antibody-antigen reaction. two antibodies are used; the first is immobilized and the other, free antibody carries a reporter group, thereby providing a positive readout when the target is recognized and 'sandwiched' between the two antibodies. sensitivity: probability of a positive result when the pathogen is indeed present; high sensitivity is related to a low type 2 error. serovars: a group of microorganisms distinguished by the presence of specific surface antigens. specificity: probability of a negative result when the pathogen is not present; high specificity is related to a low type 1 error. tiling arrays: tiling arrays are a type of dna microarray in which short probe segments that have been designed to cover the entire genome are used. the extent of probe overlap will translate to the mapping resolution and might range from highly overlapped probes across each individual nucleotide base (for resequencing applications) to non-overlapping probes (for genome-wide expression analysis). vaccinia virus: a poxvirus that is closely related to the virus that causes cowpox. it was the first human vaccine and has been extensively used for vaccination against smallpox. corresponding author: uttamchandani, m. (mahesh@dso.org.sg). micro-pcr (mpcr), followed by hybridization and identification against probes on dna microarrays have spun-off viable chip-based platforms that are able to successfully perform biological threat detection and analysis. such portable systems, as will be described, are already appearing on the market and competing to attract lucrative biodefence funding. alternative detection platforms have exploited quantitative pcr in 'fieldable' real-time detectors, where positive pcr amplification would indicate the presence of the corresponding target dna [4] . these have included devices such as genexpert (cepheid), rapid (idaho technologies) or bioseeq (smiths detection), but these systems provide relatively limited throughput [7] . microarray systems are, by contrast, more definitive and highly scalable because hundreds to tens of thousands of possible dna elements can be interrogated in a single experiment. their performance nevertheless hinges on both the adoption of robust panels of probes that can accurately identify dna from organisms of interest and the successful extraction and amplification of pathogen dna from the relevant clinical sample or isolates [8, 9] . more recent developments in microarray technologies have significantly broadened the horizon for their use in the biodefence arena well beyond the confines of dnabased profiling or detection. newer microarray formats developed at the turn of the 21st century provide a host of other biomolecules that can be presented on chip, in-cluding proteins (whole proteomes, enzymes and antibodies) [10, 11] , small molecules (drug-like molecules, peptides and carbohydrates) [12, 13] and even whole cells and tissues for simultaneous, multiplexed experimentation [14] . each format offers unique ways in which microarrays can be harnessed towards improving our understanding of pathogen biology ( figure 1 ). the microarray paradigm has over the years contributed significantly towards these goals by not only providing a robust tool for molecular diagnostics but also by advancing biodefence capabilities in protection and therapy. this review will describe microarray-based applications for biodefence, as well as exciting new concepts and approaches that will drive future advancement and growth. probe selection and design is usually an important first step in microarray-based pathogen detection, and many issues associated with probe design for dna microarrays can impact the overall fidelity of the assay, in particular with regard to levels of specificity and sensitivity attained [15] . these issues and considerations include cross-hybridizations, orthogonal probe binding to target dna from the specific organism(s) of interest and vice versa, uniformity of annealing temperatures (or gc content) and probe length. the occurrence of false positives and false negatives is highly problematic because they might cause an (a) as discussed in this article, dna microarrays can be applied to test for dna from pathogenic organisms or for the resequencing of pathogen genomes. (b) antibody microarrays can be used to detect pathogen proteins or antigens that might be present in environmental samples as an indication of contamination or for diagnostic purposes to determine pathogen infection in human tissues. (c) small-molecule microarrays offer novel approaches for differentiating between pathogens, for example by clustering the binding signatures obtained for each pathogen. they can also be used to identify therapeutics that could potentially disrupt the infection cycle. trends in biotechnology vol. 27 no.1 unwarranted response (and potentially panic) or a missed response (and a lost opportunity for intervention), which are unacceptable when dealing with deadly biological threats. for this reason, biodefence detection platforms strive to reach near perfect accuracies, which are comparable to, if not better than, the usual gold-standard assaystypically culture-based methods. with microarrays, redundancy can, however, be easily engineered into the system to improve accuracy and analytical power simply by over-representation. the us centers for disease control (cdc) have identified a list of 36 agents that, if spread uncontrollably, have the potential to cause a major public health crisis with heavy morbidity and mortality rates (box 1). these pathogens are further subclassified into categories a, b and c according to the degree of risk imposed (table 1 ) [16] . in 2002, wilson et al. fabricated a customized affymetrix microarray containing 53 660 probes to detect dna amplified from 18 different pathogenic microorganisms simultaneously, including pathogens from the us cdc's list of bioterrorism agents, such as bacillus anthracis (which causes anthrax), clostridium botulinum (which generates the botulinum toxin), yersinia pestis (which causes bubonic plague) and the ebola virus [17] . specific multiplexed primer sets were designed to amplify unique diagnostic regions specific to each organism for hybridization against tiling arrays comprising 20-mer probes. the highly redundant system facilitated the accurate identification of each organism tested, with over 91% of the probes working as predicted. impressively, as little as 10 fg, equivalent to the 'mass' of just two genomes (or dna from two cells), of b. anthracis could be detected after multiplexed pcr on these microarrays. even though culture methods should in theory be able to amplify and detect a single organism, the procedures are usually time-consuming and challengingdetection could take from 18 h to several days depending on the microbe type and even longer for fastidious microbes (e.g. mycobacterium tuberculosisa very slow growing microbe) that might require specialized, or as yet undiscovered, laboratory growth conditions. molecular identification using random pcr followed by multiplexed resolution over a microarray offers a particularly attractive alternative for detection and diagnostics, generating results in 2-6 h once assays are optimized ( figure 2a ). wang et al. [18] developed such a microarray approach for the screening of viral pathogens from across broad viral families. a randomized primer was used to amplify any viral rna that was present in the sample using reverse transcriptase-pcr followed by hybridization on a microarray comprising 1600 70-mer probes, representing nearly 140 virus genomes. degeneracy of probes on the microarray and cross-hybridization of certain viruses across expected patterns indicated the emergence of novel, uncharacterized strains, which could hence be identified with the aid of microarrays. similarly, sengupta and colleagues [19] developed microarrays with 476 probes to distinguish among various influenza viruses. primers were designed against characteristic sequences specific to influenza that targeted the viral fusion protein haemagglutinin and the glycosidase neuraminidase segments. using the affymetrix respiratory pathogen microarray, lin et al. [20] detected the respiratory viruses influenza a and adenovirus, which were then further differentiated according to strain and species. this setup made use of a so-called 'resequencing' microarray that contained one perfectly matched and three mismatched probes per base and thus was able to identify genetic mutations at the sequence level [20] . the array's large screening capacity meant that both the forward and reverse strands of the dna targets could be sequenced to provide added sequencing accuracy. pooled primer pairs have also been optimized for use in detecting both dna and rna targets for pathogens that cause upper respiratory tract infections, including viruses (influenza, corona viruses and others) and bacteria (bordetella pertusis, streptococcus pyogenes and others) [21] . the small subunit ribosomal rna (ssu rrna) gene is used widely as a microbial marker for taxonomy and species classification [22] . desantis and colleagues [23] generated a high-density tiling microarray with 62 358 oligonucleotide probes of ssu rrna with sufficient coverage to detect 18 different orders of microbes from environmental samples, as well as novel variants that exhibited mutations in their ssu rrna. the array fluorescence intensities correlated well with the spiked pathogen concentrations, providing a means of quantifying the pathogen box 1. bioterror agent classification biohazardous agents that pose a significant threat require special attention, especially in the implementation of regulatory measures and controls to limit their access and distribution and to prevent them from falling into the wrong hands. in addition, such measures also aim to raise awareness within national healthcare systems and amongst healthcare providers, especially in cases where these bioterror threats are only rarely seen. the us centers of disease control (cdc) have compiled a list of 36 biohazardous agents, which are divided into the categories a, b and c according to the level of threat they impose, and their characteristics are described here. examples of pathogens from these three categories are provided in table 1 . the cdc defines category a agents as those that are of the highest priority for biodefence research. these organisms can be easily disseminated from person to person. they result in high mortality rates and have the potential to cause a major impact on public health. the accidental or deliberate release of these agents could cause public panic and social disruption. these agents thus require particular attention in ensuring public health preparedness because they pose a significant risk to national security. the cdc defines category b agents as those that are moderately easy to disseminate. these pathogens result in moderate morbidity and low mortality. these agents would hence require specific improvements of diagnostic capabilities as well as enhanced measures for disease surveillance. the cdc defines category c agents as those that have the potential for mass dissemination in the future because of their availability, ease of production and dissemination. this category also includes emergent threats that have the potential of causing high morbidity and mortality rates, as well as a major impact on public health. further details on the classification and prioritization of biological threat agents are available on the cdc website: http:// www.bt.cdc.gov/agent/agentlist-category.asp. levels in the original sample. a similar method was used to detect staphylococci. out of 201 isolates taken from 33 staphylococci species, 185 were correctly assigned by using only 16s rrna sequences on microarrays, conferring a sensitivity of 92% [24] . the ability to refine and expand on the existing probe set, for example by the incorporation of additional informative genetic loci, is a key advantage provided by the dna microarray platform to increase redundancy and hence improve assay performance and fidelity. miller and colleagues [25] applied microarrays in clinical diagnostics and were able to identify pathogens in a panel of 36 patient specimens with a 94% accuracy score, calculated from 76% sensitivity and 100% specificity. the microarray was designed to detect up to 35 rna viruses, including the sars coronavirus and dengue viruses, using 40-mer probes. a total of 53 555 such probes in replicates of seven were printed on nimblegen tiling microarrays. the authors studied ways to minimize pcr bias to ensure uniform amplification and developed a unique statistical approach to infer pathogen identity from the resulting microarray fingerprints. apart from the highdensity microarrays described here, more focused biodefence platforms have been developed for selected panels of environmental [26] or blood-borne pathogens [27] , using inhouse microarrays that comprise only several hundred relevant probes. such panels are designed to selectively identify targets of interest with enough redundancy to reduce the occurrence of false positives, which is infrequently controlled in other diagnostic platforms that often use either one or a handful of primer sets specific to each pathogen of interest (as is the case in the genexpert, rapid and cepheid real time pcr platforms). there are several instructive examples where dna microarrays have advanced our understanding of disease etiology and epidemiology through their use in studies into molecular evolution, host-pathogen interactions and modes of virulence. in the following section, we will review salient studies that have provided such fundamental insight, focusing on agents from the cdc categories a to c. one important application of microarrays is in the resequencing of pathogen genomes, which has been successfully applied to interrogate inter-species and/or interstrain differences (figure 2b ). this provides a vital alternative to traditional shotgun sequencing approaches because it is quicker and more cost-effective; however, the disadvantage is that the sequence information for the organism of interest must available beforehand. this is not a severe limitation given that many pathogenic organisms have or are being sequenced, and with the advances in de novo sequencing capabilities, new pathogens such as sars can be readily sequenced in a matter of weeks from the time of discovery [28] . subsequent resequencing using microarrays would only take several days. early experiments on resequencing arrays showed high rates of false discovery of up to 12-45% [29] , but experimental improvements and the development of robust algorithms, such as the abacus (adaptive background genotype calling scheme) software package [30] , have now greatly improved sequencing quality. adopting this approach for variation analysis, wong and colleagues [31] tracked strains of the sars coronavirus that were rapidly resequenced using high-density microarrays. a total of 383 102 probes (27-mers) were designed to cover the complete 29.7 kb genome on nimblegen microarrays [31] . virus samples from cell cultures and patient tissues could be amplified by reverse-transcriptase pcr using optimized primers and resequenced with accuracy greater than 99.99%. the platform was used to confirm that a lone case of a sars virus infection of a graduate student had most likely come from a laboratory source because a unique 47-bp deletion was detected that was not present in other sars isolates. wang et al. [32] also developed a sars microarray that helped to characterize a novel coronavirus variant. the microarray platform is thus ideal in virus tracking should outbreaks occur and, furthermore, is readily applicable to other pathogens evolutionary perspectives lend unique insight into the genetic changes that result in differences in pathogenicity. plague-causing y. pestis is estimated to have diverged from the non-lethal yersinia pseudotuberculosis an estimated 1500-20 000 years ago [34] . bearing identical 16s rrna sequences, more comprehensive genetic analysis was required to understand the differences in disease morbidity manifested by these two bacterial species, as well as in the different modes of transmission to humans: y. pestis by flea bites and y. pseudotuberculosis by food or water. a microarray specific for y. pestis revealed three unique genomic islands and the inactivation of several genes, including those encoding the cell-adhesion proteins adhesin (yada) and invasion (inv), as well as the o-antigen biosynthetic operon. these unique characteristics potentially led to y. pestis' enhanced virulence and its adaptation from an enteric organism (y. pseudotuberculosis) to a mammalian blood-borne pathogen [35] . equivalent findings were reported by hinchliffe et al. [36] using a similar figure 2 . the use of microarrays for studying emergent pathogens, exemplified by severe acute respiratory syndrome (sars)-associated coronavirus and avian influenza (h5n1). various approaches have been developed for detection and diagnostics with dna and protein microarrays. (a) specific dna probes, for instance co-v (orange) for sars and h5 (green) for h5n1, can be used to detect the presence of pathogen dna or rna using pcr or reverse transcriptase (rt)-pcr, thus enabling multiple pathogens to be detected simultaneously [26] . (b) tiling arrays can be used to resequence pathogens so that any mutations in evolving pathogens can be rapidly detected and tracked. as illustrated, four probes, one for each nucleotide base, are used to determine the genotype at a given loci. a set of probes designed for an arbitrary position xyz is shown to reveal the unknown base to be adenine (a). multiple probe sets are 'tiled' across the whole pathogen genome and, upon hybridization and analysis, can confer the complete sequence information of the pathogen with great accuracy [17, 23, 25] . (c) antibody arrays make use of the sandwich immunoassay to screen for the presence of a pathogen. as depicted, the pathogen sample is first applied to the microarray, followed by the reporter antibody. as a result, the pathogen is sandwiched in between two antibodiesan immobilized antibody (blue) and a tagged antibody (purple) capable of reporting the presence of a pathogen on the microarray [43, 45] . (d) proteome microarrays, which contain immobilized proteins from the target pathogen, can be screened against sera obtained from infected individuals. if the individual has been exposed to the pathogen, the sera will contain antibodies (blue) against specific antigens of the pathogens that will react with the immobilized protein and that can be detected using tagged secondary antibodies (purple) [49] . this procedure also facilitates the identification of specific immunodominant antigens present in the pathogen proteome. these proteins are considered to be largely responsible for triggering the host immune response and thus have a high potential for the development of vaccines against this particular pathogen. trends in biotechnology vol.27 no.1 y. pestis gene-specific microarray. these also showed that, of the three major y. pestis subspecies, the antiqua and mediaevalis biovars showed greater divergence from the orientalis strains [36] . similarly, variation analysis has been performed on burkholderia pseudomallei microarrays to differentiate amongst virulent and avirulent burkholderia species, specifically bioterror agents such as b. pseudomallei (which causes meliodosis), b. mallei (which causes equine glanders) and the avirulent (but closely related) b. thailandensis [37] . in an interesting approach to reveal molecular mechanisms for pathogenesis, weiss and colleagues [38] developed a microarray-based negative-screening approach for francisella and obtained surviving bacterial samples from infected mice to reveal genes responsible for bacterial survival and growth in vivo. in a related study, genome-wide microarrays also revealed loci that are only present in the highly virulent franscicella tularensis subspecies tularensis strain [39] . these loci could be used to identify potential pathogenicity islands as well as to provide unique genetic markers that could be used for strain typing and identification. microarrays have also been applied to distinguish among variants of influenza a that are resistant to the antiviral drug amantidine by detecting mutations in the viral ion channel m2 protein [40] . such molecular forensics experiments using microarrays have extended our capabilities to profile new or emergent threats. the differences identified might provide an understanding of the causes of increased virulence, as well as reveal vulnerabilities that can be exploited for building therapeutic defences. beyond laboratory-based applications, dna microarrays have been tested successfully in epidemic outbreak surveillance (eos). project silent guardian was a 10-week trial launched by the naval research laboratory during the 2005 us presidential inauguration [41] . the challenge was to take laboratory-based microarray technology to a production-scale system capable of operationally screening up to 300 samples per day. a custom dna microarray was designed with the capability of detecting 20 natural (including avian flu) and biothreat agents with strain-level resolution. in total, 10 000 samples were collected and screened by civilian and military laboratory personnel within the stipulated period. the trial included blinded samples that were spiked with pathogens, which were all successfully detected. this exercise showcased the feasibility of implementing microarrays as a screening tool for eos and demonstrated their use as a rapid and robust means of sample processing and pathogen identification. recommendations from the study included the development of a more user-friendly system with greater automation of the steps involved. these features are being incorporated by several recently launched commercial platforms. microarray-based platforms for biothreat screening from akonni biosystems (http://www.akonni. com) and veredus laboratories (http://www.vereduslabs. com) are now on the market for multiplexed threat detection. new advances include (i) greater integration of the sample preparation (which is considered to be one of the major bottlenecks), (ii) the use of mpcr and (iii) the development of small handheld microfluidic chips and portable readers to take microarray technology to the point-ofcare. veredus has also recently launched an influenza test chip that enables the amplification and discrimination of influenza a and b subtypes, including avian flu (h5n1), within just two hours. this great improvement in detection time has been attributed to the mpcr step, which, through rapid thermocycling, significantly reduces the duration of pcr to under 30 min. similar devices might be developed in the future for protein and other types of non-dna microarrays for applications in serodiagnostics. the national institute of allergy and infectious diseases (niaid) and the j. craig venter institute fund a programme that enables researchers to obtain, through an approval process and material transfer agreement (mta), pathogen dna microarrays for select category a-c threats at no cost to promote and accelerate research on these pathogens. the pathogen functional genomics resource centre (pfgrc) currently fabricates and distributes a range of these 70-mer microarrays covering 39 agents, including b. anthracis, coronaviruses, f. tularensis, y. pestis and b. pseudomallei (http://pfgrc.tigr.org) ( table 1) . such collaborative and valuable initiatives augur favourably for the future of the biodefence community worldwide and will hopefully lead to greater sharing of resources in the pursuit of knowledge on global and regional pathogens. emerging roles of protein, peptide and carbohydrate microarrays newer microarray formats have already significantly extended the scope of microarray applications. the advantages of parallelization, miniaturization and automation hold true for whichever biomolecule is presented on highdensity microarrays. it was initially difficult to believe that proteins would retain their functionality once anchored on a solid support. however, schreiber and colleagues [42] erased this concern by developing the first small-molecule microarray in 1999 and the first protein microarray in 2000, simultaneously demonstrating that proteins could retain their activity despite being covalently attached to glass slides. soon afterwards, other array types were developed, such as cell arrays, carbohydrate arrays and proteome arrays. some of these newer applications have brought vital capabilities to pathogen detection and biodefence, as will be described below. as before, examples are provided for key biothreat agents and are categorized according to their consequent applicationseither in detection or profiling. pathogen detection using non-dna-based microarrays antibodies have become an established denominator for disease detection, through the time-honoured use of enzyme-linked immunosorbent assays (elisas), agglutination and lateral-flow assays. as a next-generation tool, antibody microarrays are increasing in popularity, and not without reason, for they offer unparalleled throughput, minimal reagent consumption and sensitive detection of multiple targets simultaneously [10] . the accuracy conferred is nevertheless closely linked with the quality of review trends in biotechnology vol. 27 no.1 antibodies employed, and there are issues relating to crossreactivity and antibody availability, both of which are particularly crucial for finer and more accurate strain typing. notwithstanding these matters, a plethora of antibody microarrays have been developed that have growing potential for clinical, biothreat and point-of-care applications (figure 2c ). rucker et al. [43] applied antibody microarrays for the detection of toxins with low-nanomolar sensitivities, such as anthrax lethal factor (lf, a metalloendopeptidase), protective antigen (pa) and tetanus toxins (endopeptidases), through the co-application of a competitive fluorescently-labelled toxin reporter. in this setting, the samples can be tested without the need for labelling by monitoring the depletion and competitive displacement of reporter signals generated by the labelled reporter. thirty-five antibodies were also arrayed to subtype the 20 most common salmonella serovars [44] , and a similar study was undertaken for escherichia coli [45] . in an impressive demonstration of the detection throughput attainable, huelseweh and colleagues [46] built an antibody microarray to detect several category a and b agents simultaneously, including f. tularensis, y. pestis, brucella melitensis (which causes brucellosis) and b. mallei, using sandwich immunoassays (figure 2c) . conversely, antigen microarrays have also been used to identify seropositive individuals by using the presence of defensive antibodies in the serum as a means of detecting exposure to a pathogen. pathogen proteins are typically orthologously expressed using high-throughput cloning and expression and then affinity purified to provide protein sets. in special cases, it might be necessary to consider the post-translational modifications or the way the pathogen presents itself to the host to identify and use the correct immunogenic protein variants in serodiagnostics. it is also desirable to purify the protein and check for efficient and generally uniform immobilization on the microarrays as a quality control, especially when comparisons are made across slides. in one such example, sundaresh et al. [47] narrowed down an initial panel of 1741 f. tularensis antigens to just the top 244 in terms of their ability to predictively discriminate seropositive sera and then used these antigens to establish a diagnostic microarray comprising whole proteins. mezzasoma et al. [48] also developed protein microarrays for simultaneous diagnostics using parasitic and viral antigens. zhu et al. [49] monitored the antibody profiles of sars patients using protein microarrays containing 82 purified coronavirus proteins. antibodies present in human serum samples were detected on the protein microarrays with fluorescently labelled goat anti-human immunoglobulins. it was found that immunoreactivity against the coronavirus nucleocapsid proteins remained high for 120-320 days post infection. this provided a means to check for exposure long after infection might have occurred. over 400 human sera samples were screened with an accuracy of 91% in differentiating infected and normal specimens [49] . nevertheless, caution should be applied in protein-based diagnostics because incubation periods vary greatly amongst diseases. during the incubation period, the patient might not express antibodies at detectable levels but might still be capable of spreading the infection. in the case of sars, the incubation period (the time between exposure and onset of symptoms) has a median of 4-5 days (up to a maximum of 10 days). antibodies might be detectable only 6-7 days after first symptoms are presented, but pcr testing might be able to pick up the virus more quickly, just 1-2 days after symptoms become apparent. it might become possible in the future to integrate dnaand protein-based microarray methods to extend the range of rapid clinical diagnostics from detection of current pathogen infections to testing for exposure long after the actual infection has occurred. as an alternative, short peptides have also been applied for serodiagnostics, and this was facilitated by the robust and routine procedures established to chemically synthesize peptide sequences up to 50 amino acids in length. peptide microarrays have been applied for detecting immunoreactive sera against sars, amongst other pathogens, enabling the detection of low-picomolar concentrations of antibodies [50] . lipopolysaccharide, carbohydrate-based and whole-cell microarrays have also been used for antibody-based detection of pathogens such as f. tularensis [51, 52] , b. anthracis [53] and b. pseudomallei [54] . the surface antigens on these microbes are frequently responsible for immunoreactivity, so these methods rely on detecting the presence of such immunogenic antigens. further improvements in fabrication techniques and greater knowledge of surface chemistries might lead to the development of hybrid microarray platforms in which multiple types of antigens, such as peptides, proteins and carbohydrates, are presented simultaneously to detect host antibodies with improved efficiency and sensitivity. pathogen profiling using non-dna-based microarrays protein microarrays with a high pathogen proteome content offer a valuable platform for high-throughput serology. proteins that are immunoreactive to patient sera represent antigens that can be applied as markers in serodiagnostics or as therapeutic proteins for vaccine development. in one such example, the immunogenic epitopes of the sars coronavirus were traced to the cterminal fragments of the nucleocapsid protein by screening against 52 human sera samples from infected individuals ( figure 2d) [55, 56] . to address the bottleneck of protein expression, davies and colleagues [57, 58] applied pcr recombinant cloning to accelerate the process of cloning and expression. this enabled more than 190 proteins from vaccinia virus and over 1700 proteins from f. tularensis to be expressed and profiled using microarrays [57, 58] . these arrays have also helped to identify antibodies against the vaccinia virus h3l envelope protein, which confers protection in vivo (in a mouse model) [59] . the vaccinia protein microarrays have also been used to derive antibody profiles in humans inoculated with the licenced dryvax 1 smallpox vaccine [60] , and these profiles were found to be very similar to those induced by the attenuated modified vaccinia virus ankara strain, an alternative vaccine candidate [61] . various protein microarrays have similarly been generated for identification of the immunodominant antigens of y. pestis [62, 63] . trends in biotechnology vol. 27 no.1 other microarray platforms have demonstrated valuable potential in biodefence. for instance, small-molecule microarrays have been applied to functionally screen anthrax lf through activity-based binding signatures against a library of 1400 immobilized peptide-hydroxamate inhibitors. putative drug candidates were discovered that bound selectively to the anthrax lf (with a low dissociation constant, k d = 0.81 mm) and inhibited its activity in vitro [64] . carbohydrate microarrays have also been developed to elucidate the receptor preferences of influenza viruses. stevens and colleagues [65, 66] applied glycan arrays to establish the a2-3 and a2-6 binding selectivity of the h5 and h1 haemagglutinins, which determine influenza virulence. the selectivity was altered by specific mutations in the protein, providing a useful way of functionally monitoring viral adaptation. non-dna microarray formats have thus contributed unique ways in which we can explore pathogen biology and develop countermeasures in the event of an outbreak. it is clear that microarrays have significantly strengthened our biodefence capabilities. although the upstream cost of library creation and microarray fabrication, as well as the initial infrastructure investment of several hundreds of thousands of dollars (for microarray spotters and scanners), might represent significant hurdles for small laboratories with tight budgets, the provision of printed microarray slides through commercial or research avenues heralds a favourable trend towards the reduction of exclusivity. greater access to the technology, such as the increased availability of microarrays together with established array designs, is helping researchers worldwide to move more quickly towards the downstream application phase, for example to perform regional studies of endemic variants. the applications of microarrays described here are broad-ranging and span from dna-or protein-based detection of pathogens to epidemic outbreak surveillance and vaccine development, thus showcasing the full spectrum of microarray potential ( table 1 ). the progress made in the past several years has brought microarrays to the forefront of rapid diagnostics and medical research. further integration of upstream sample preparation with downstream data processing is expected to transform microarrays into compact, field expedient solutions for analysis and monitoring in the near future. the large assortments of biomolecules now utilized in microarrays provide novel opportunities in molecular forensics and comparative profiling, especially for the newer and less well-understood pathogens. as we equip ourselves with better capabilities and countermeasures against potential biological threats, we hope to become more agile and responsive whenever such threats emerge in the future. microarrays have improved our confidence in this respect and will continue to play a decisive part in biodefence research and technology. molecular evolution of the sars coronavirus during the course of the sars epidemic in china chemical and biological weapons: current concepts for future defenses turning biodefense dollars into products current and developing technologies for monitoring agents of bioterrorism and biowarfare microarrays -status and prospects comparative phylogenomics of pathogenic bacteria by microarray analysis nucleic acid approaches for detection and identification of biological warfare and infectious disease agents potential applications of dna microarrays in biodefense-related diagnostics oligonucleotide microarrays in microbial diagnostics progress in protein and antibody microarray technology advances in functional protein microarray technology small molecule microarrays: recent advances and applications peptide microarrays: next generation biochips for detection, diagnostics and high-throughput screening microarrays in infection and immunity highly parallel microbial diagnostics using oligonucleotide microarrays microbiological threats to homeland security sequence-specific identification of 18 pathogenic microorganisms using microarray technology microarray-based detection and genotyping of viral pathogens molecular detection and identification of influenza viruses by oligonucleotide microarray hybridization broad-spectrum respiratory tract pathogen identification using resequencing dna microarrays identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray potential of dna microarrays for developing parallel detection tools (pdts) for microorganisms relevant to biodefense and related research needs rapid quantification and taxonomic classification of environmental dna from both prokaryotic and eukaryotic origins using a microarray high-density dna probe arrays for identification of staphylococci to the species level optimization and clinical validation of a pathogen detection microarray multipathogen oligonucleotide microarray for environmental and biodefense applications a multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood the genome sequence of the sars-associated coronavirus microarray-based resequencing of multiple bacillus anthracus isolates high-throughput variation detection and genotyping using microarrays tracking the evolution of the sars coronavirus using high-throughput, high-density resequencing arrays viral discovery and sequence recovery using dna microarrays genechip resequencing of the smallpox virus genome can identify novel strains: a biodefense application microevolution and history of the plague bacillus, yersinia pestis dna microarray analysis of genome dynamics in yersinia pestis: insights into bacterial genome microevolution and niche adaptation application of dna microarrays to study the evolutionary genomics of yersinia pestis and yersinia pseudotuberculosis patterns of large-scale genomic variation in virulent and avirulent burkholderia species in vivo negative selection screen identifies genes required for francisella virulence genome-wide dna microarray analysis of francisella tularensis strains demonstrates extensive genetic conservation within the species but identifies regions that are unique to the highly virulent f. tularensis subsp. tularensis detection of adamantane-resistant influenza on a microarray nrl uses microarray technology to detect natural and bio-threat pathogens during silent guardian project printing proteins as microarrays for high-throughput function determination antibody microarrays for native toxin detection development of a novel protein microarray method for serotyping salmonella enterica strains use of miniaturized protein arrays for escherichia coli o serotyping a simple and rapid protein array based method for the simultaneous detection of biowarfare agents from protein microarrays to diagnostic antigen discovery: a study of the pathogen francisella tularensis antigen microarrays for serodiagnosis of infectious diseases severe acute respiratory syndrome diagnostics using a coronavirus protein microarray functional peptide microarrays for specific and sensitive antibody diagnostics lipopolysaccharide microarrays for the detection of antibodies bacterial cell microarrays for the detection and characterization of antibodies against surface antigens photogenerated glycan arrays identify immunogenic sugar moieties of bacillus anthracis exosporium polysaccharide microarray technology for the detection of burkholderia pseudomallei and burkholderia mallei antibodies antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein screening of specific antigens for sars clinical diagnosis using a protein microarray immunodominant francisella tularensis antigens identified using proteome microarray profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery vaccinia virus h3l envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice proteome-wide analysis of the serological response to vaccinia and smallpox antibody profiling by proteome microarray reveals the immunogenicity of the attenuated smallpox vaccine modified vaccinia virus ankara is comparable to that of dryvax protein microarray for profiling antibody responses to yersinia pestis live vaccine quorum sensing affects virulence-associated proteins f1, lcrv, katy and ph6 etc. of yersinia pestis as revealed by protein microarray-based antibody profiling quantitative inhibitor fingerprinting of metalloproteases using small molecule microarrays structure and receptor specificity of the hemagglutinin from an h5n1 influenza virus glycan microarray technologies: tools to survey host specificity of influenza viruses the authors acknowledge funding support from dso national laboratories. key: cord-000642-mkwpuav6 authors: moreira, rebeca; balseiro, pablo; planas, josep v.; fuste, berta; beltran, sergi; novoa, beatriz; figueras, antonio title: transcriptomics of in vitro immune-stimulated hemocytes from the manila clam ruditapes philippinarum using high-throughput sequencing date: 2012-04-19 journal: plos one doi: 10.1371/journal.pone.0035009 sha: doc_id: 642 cord_uid: mkwpuav6 background: the manila clam (ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. diseases affecting this species can result in large economic losses. because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced rna from immune-stimulated r. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. methodology and principal findings: high-throughput deep sequencing of r. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. the reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. the 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. we have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. conclusions: this study represents the first transcriptome analysis using 454-pyrosequencing conducted on r. philippinarum focused on its immune system. our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. the discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of ruditapes philippinarum. the manila clam (ruditapes philippinarum) is a cultured bivalve species with important commercial value in europe and asia, and its culture has expanded in recent years. nevertheless, diseases produced by a wide range of microorganisms, from viruses to metazoan parasites, can result in large economical losses. among clam diseases, the majority of pathologies are associated with the vibrio and perkinsus genera [1] [2] [3] . although molluscs lack a specific immune system, the innate response involving circulating hemocytes and a large variety of molecular effectors seems to be an efficient defense method to respond to external aggressions by detecting the molecular signatures of infection [4] [5] [6] [7] [8] ; however, not many immune pathways have been identified in these animals. although knowledge of bivalve immune-related genes has increased in the last few years, the available information is still scarce and fragmentary. most of the data concern mussels and eastern and pacific oysters [9] [10] [11] [12] [13] [14] , and very limited information is available on the expressed immune genes of r. philippinarum. recently, the expression of 13 immune-related genes of ruditapes philippinarum and ruditapes decussatus were characterized in response to a vibrio alginolyticus challenge [15] . also, a recent 454 pyrosequencing study was carried out by milan et al. [16] , who sequenced two normalized cdna libraries representing a mixture of adult tissues and larvae from r. philippinarum. even more recently ghiselli et al. [17] , have de novo assembled the r. philippinarum gonad transcriptome with the illumina technology. moreover, a few transcripts encoded by genes putatively involved in the clam immune response against perkinsus olseni have been reported by cdna library sequencing [18] . currently (19/12/ 2011) , there are 5,662 ests belonging to r. philippinarum in the genbank database. the european marine genomics network has increased the number of ests for marine mollusc species particularly for ecologically and commercially important groups that are less studied, such as mussels and clams [19] . unfortunately, most of the available resources are not annotated or well described, limiting the identification of important genes and genetic markers for future aquaculture applications. the use of 454-pyrosequencing is a fast and efficient approach for gene discovery and enrichment of transcriptomes in non-model organisms [20] . this relatively low-cost technology facilitates the rapid production of a large volume of data, which is its main advantage over conventional sequencing methods [21] . in the present work, we undertook an important effort to significantly increase the number of r. philippinarum ests in the public databases. specially, the aim of this work was to discover new immune-related genes using pyrosequencing on the 454 gs flx (roche-454 life sciences) platform with the titanium reagents. to achieve this goal, we sequenced the transcriptome of r. philippinarum hemocytes previously stimulated with different pathogen-associated molecular patterns (pamps) to obtain the greatest number of immune-related transcripts as possible. the raw data are accessible in the ncbi short read archive (accession number: sra046855.1). the r. philippinarum normalized cdna library was sequenced with 454 gs flx technology as shown in figure 1 . sequencing and assembly statistics are summarized in table 1 . briefly, a total of 975,190 raw nucleotide reads averaging 284.1 bp in length were obtained. of these, 974,976 exceeded our minimum quality standards and were used in the mira assembly. a total of 842,917 quality reads were assembled into 51,265 contigs, corresponding to 29.9 megabases (mb). the length of the contigs varied from 40 to 5565 bp, with an average length of 582.4 bp and an average coverage of 5.7 reads. singletons were discarded, resulting in 37,093 contigs formed by at least 2 ests, and 26,675 of these contigs were longer than 500 bp. clustering the contigs resulted in 1,689 clusters with more than one contig. the distribution of contig length and the number of ests per contig, as well as the contig distribution by cluster are all shown in figure 2 . even though the knowledge of expressed genes in bivalves has increased in the last few years, it is still limited. indeed, only 41,598 nucleotide sequences, 362,149 ests, 24,139 proteins and 704 genes from the class bivalvia have been deposited in the genbank public database (19/12/11) , and the top entries are for the mytilus and crassostrea genera. for ruditapes philippinarum, these numbers are reduced to 5,662 ests, 612 proteins and 12 genes. this evidences the lack of information which prompted the recent efforts to increase the number of annotated sequences of bivalves in the databases. for non-model species, functional and comparative genomics is possible after obtaining good est databases. these studies seem to be the best resource for deciphering the putative function of novel genes, which would otherwise remain ''unknown''. ncbi swissprot, ncbi metazoan refseq, the ncbi nonredundant and the uniprotkb/trembl protein databases were chosen to annotate the contigs that were at least 100 bp long (49, 847) . the percentage of contigs annotated with a cut off evalue of 10e-3 was 44.7%. contig sequences and annotations are included in table s1 . of these contigs, 3.26% matched sequences from bivalve species and the remaining matched to non-bivalvia mollusc classes (4.13%), other animals (81.38%), plants (2.58%), fungi (1.78%), protozoa (1.50%), bacteria (4.95%), archaea (0.20%), viruses (0.21%) and undefined sequences (0.01%). as shown in figure 3a , the species with the most sequence matches was homo sapiens with 3,106 occurrences. the first mollusc in the top 35 list was lymnaea stagnalis at position 11. the first bivalve, meretrix lusoria, appeared at position 17. r. philippinarum was at position 25 with 124 occurrences. notably, a high percentage of the sequences had homology with chordates, arthropods and gastropods ( figure 3b and c), and only 343 contigs matched with sequences from the veneroida order ( figure 3d ). these values can be explained by the higher representation of those groups in the databases as compared to bivalves and the quality of the annotation in the databases, which has been reported in another bivalve transcriptomic study [22] . the data shown highlight, once again, the necessity of enriching the databases with bivalve sequences. a detailed classification of predicted protein function is shown for the top 35 blastx hits ( figure 4a ). the list is headed by actin with 903 occurrences, followed by ferritin, an angiopoietin-like protein and lysozyme. an abundance of proteins directly involved in the immune response was predicted for this 454 run; ferritin, lysozyme, c1q domain containing protein, galectin-3 and hemagglutinin/amebocyte aggregation factor precursor are immune-related proteins present on the top 35 list. ferritin has an important role in the immune response. it captures circulating iron to overcome an infection and also functions as a proinflammatory cytokine via the iron-independent nuclear factor kappa b (nf-kb) pathway [23] . lysozyme is a key protein in the innate immune responses of invertebrates against gram-negative bacterial infections and could also have antifungal properties. in addition, it provides nutrition through its digestive properties as it is a hydrolytic protein that can break the glycosidic union of the peptidoglycans of the bacteria cell wall [24] . the c1q domain containing proteins are a family of proteins that form part of the complement system. the c1q superfamily members have been found to be involved in pathogen recognition, inflammation, apoptosis, autoimmunity and cell differentiation. in fact, c1q can be produced in response to infection and it can promote cell survival through the nf-kb pathway [25] . galectin-3 is a central regulator of acute and chronic inflammatory responses through its effects on cell activation, cell migration, and the regulation of apoptosis in immune cells [26] . the hemagglutinin/amebocyte aggregation factor is a single chain polypeptide involved in blood coagulation and adhesion processes such as self-nonself recognition, agglutination and aggregation processes. the hemagglutinin/ amebocyte aggregation factor and lectins play important roles in defense, specifically in the recognition and destruction of invading microorganisms [27] . other proteins that are not specifically related to the immune response but could play a role in defense mechanisms include the following: angiopoietin-like proteins, apolipoprotein d and the integral membrane protein 2b. in other animals, angiopoietin-like proteins (angptl) potently regulate angiogenesis, but a subset also function in energy metabolism. specifically, angptl2, the most represented angptl, promotes vascular inflammation rather than angiogenesis in skin and adipose tissues. inflammation occurs via the a5b1 integrin/rac1/nf-kb pathway, which is evidenced by an increase in leukocyte infiltration, blood vessel permeability and the expression of inflammatory cytokines (tumor necrosis factor-a, interleukin-6 and interleukin-1b) [28] . apolipoprotein d (apod) has been associated with inflammation. pathological and stressful situations involving inflammation or growth arrest have the capacity to increase its expression. this effect seems to be triggered by lps, interleukin-1, interleukin-6 and glucocorticoids and is likely mediated by the nf-kb pathway, as there are several conserved nf-kb binding sites in the apod promoter (apre-3 and ap-1 binding sites are also present). the highest affinity ligand for apod is arachidonic acid, which apod traps when it is released from the cellular membrane after inflammatory stimuli and, thus, prevents its subsequent conversion in pro-inflammatory eicosanoids. within the cell, apod could modulate signal transduction pathways and nuclear processes such as transcription activation, cell cycling and apoptosis. in summary, apod induction is specific to ongoing cellular stress and could be part of the protective components of mild inflammation [29] [30] [31] . finally, the short form of the integral membrane protein 2b (itm2bs) can induce apoptosis via a caspase-dependent mitochondrial pathway [32] . to avoid redundancy, the longest contig of each cluster was used for gene ontology terms assignment. a total of 23.05% of the representative clusters matched with at least one go term. concerning cellular components ( figure 4b ), the highest percentage of go terms were in the groups of cell and cell part with 25.9% in each; organelle and organelle part represented 19.67% and 11.38%, respectively. within the molecular function classification ( figure 4c ), the most represented group was binding with 49.25% of the terms, which was followed by catalytic activity (29.12%) and structural molecular activity (4.60%). with regard to biological process ( figure 4d ), cellular and metabolic processes were the highest represented groups with 16.78% and 12.43% of the terms, respectively, which was followed by biological regulation (10.18%). similarities between the r. philippinarum transcriptome and another four bivalve species sequences were analyzed by comparative genomics (crassostrea gigas of the family ostreidae, bathymodiolus azoricus and mytilus galloprovincialis of the family mytilidae and laternula elliptica of the family laternulidae). this analysis could identify specific transcripts that are conserved in these five species. a venn diagram was constructed using unique sequences from these databases according to the gene identifier (gi id number) of each sequence in its respective database: 207,764 from c. gigas, 76,055 from b. azoricus, 121,318 from m. galloprovincialis and 1,034,379 from l. elliptica. c. gigas was chosen because is the most represented bivalve species in the public databases. the other three species are bivalves that have been studied in transcriptomic assays. figure 5 shows that of the total 29,679 clusters, 72% were found exclusively in the r. philippinarum group, while only 7.59% shared significant similarity with all five species. the number of coincidences among other groups was very low (4.14% to 0.31% of sequences), suggesting that 21,454 new sequences were discovered within the bivalve group. the percentage of new sequences is very high compared to previous transcriptomic studies [33] [34] , in which the fraction of new transcripts was approximately 45%. one possible explanation for this discrepancy is the low number of nucleotide and est sequences currently available in public databases for r. philippinarum, but these transcripts could also be regions in which homology is not reached, such as 59 and 39 untranslated regions or genes with a high mutation rate. on the other hand, a comparison between our 454 results and the milan et al. [16] transcriptome using a blastn approach is summarized in table 2 immune-related sequences r. philippinarum hemocytes were subjected to immune stimulation using several different pamps to enrich the est collection with immune-related sequences. the objective was to obtain a more complete view of clam responses to pathogens. a keyword list and go immune-related terms were used to find proteins putatively involved in the immune system. after this selection step, we found that more than 10% of the proteins predicted from the contig sequences had a possible immune function. some sequences were found to be clustered in common, well-recognized immune pathways, such as the complement, apoptosis and toll-like receptors pathways, indicating conserved ancient mechanisms in bivalves ( figures 6, 7, 8 ). the complement system is composed of over 30 plasma proteins that collaborate to distinguish and eliminate pathogens. c3 is the central component in this system. in vertebrates, it is proteolytically activated by a c3 convertase through both the classic, lectininduced and alternative routes [35] . although the complement pathway has not been extensively described in bivalves, there is evidence that supports the presence of this defense mechanism. ests with homology to the c1q domain have been detected in the american oyster, c. virginica [36] , the tropical clam codakia orbicularis [37] , the zhikong scallop chlamys farreri [38] and the mussel m. galloprovincialis [39] [40] . more recently, a novel c1q adiponectin-like, a c3 and a factor b-like proteins have been identified in the carpet shell clam r. decussatus [41] [42] . these data support the putative presence of the complement system in bivalves. our pyrosequencing results, using the blastx similarity approach, showed that the complement pathway in r. philippinarum was almost complete as compared to the kegg reference pathway ( figure 6 ). only the complement components c1r, c1s, c6, c7 and c8 were not detected. i. lectins. lectins are a family of carbohydrate-recognition proteins that play crucial self-and non-self-recognition roles in innate immunity and can be found in soluble or membraneassociated forms. they may initiate effector mechanisms against pathogens, such as agglutination, immobilization and complement -mediated opsonization and lysis [43] . several types of lectins have been cloned or purified from the manila clam, r. philippinarum [44] [45] [46] , and their function and expression were also studied [18, 47] . also, a manila clam tandemrepeat galectin, which is induced upon infection with perkinsus olseni, has been characterized [46] . lectin sequences have been found in the stimulated hemocytes studied in our work: 23 of the contigs are homologous to c-type lectins (calcium-dependent carbohydrate-binding lectins that have characteristic carbohydrate-recognition domains), 115 are homologous to galectins (characterized by a conserved sequence motif in their carbohydrate recognition domain and a specific affinity for bgalactosides), 4 contigs have homology with ficolin a and b (a group of oligomeric lectins with subunits consisting of both collagen-like and fibrinogen-like domains) and 34 contigs have homology with other groups of lectins such as lactose-, mannoseor sialic acid-binding lectins. ii. b-glucan recognition proteins. b-glucan recognition proteins are involved in the recognition of invading fungal organisms. they bind specifically to b-1,3-glucan stimulating short-term immune responses. although these receptors have been partially sequenced in several bivalves, there is only one complete description of them in the scallop chlamys farreri [48] . two contigs with homology to the beta-1,3-glucan-binding protein were found in our study. iii. peptidoglycan recognition proteins. peptidoglycan recognition proteins (pgrps) specifically bind peptidoglycans, which is a major component of the bacterial cell wall. this family of proteins influences host-pathogen interactions through their pro-and anti-inflammatory properties that are independent of their hydrolytic and antibacterial activities. in bivalves, they were first identified in the scallops c. farreri and a. irradians [49, 50] and the pacific oyster c. gigas, and from the latter four different types of pgrps were identified [51] . peptidoglycan-recognition proteins and a peptidoglycan-binding domain containing protein have been found for the first time in r. philippinarum in our results and were present 4 and 1 times, respectively. iv. toll-like receptors. toll-like receptors (tlrs) are an ancient family of pattern recognition receptors that play key roles in detecting non-self substances and activating the immune system. the unique bivalve tlr was identified and characterized in the zhikong scallop, c. farreri [52] . tlr 2, 6 and 13 were present among the pyrosequencing results. tlr2 and tlr6 form a heterodimer, which senses and recognizes various components from bacteria, mycoplasma, fungi and viruses [53] . tlr13 is a novel and poorly characterized member of the toll-like receptor family. although the exact role of tlr13 is currently unknown, phylogenic analysis indicates that tlr13 is a member of the tlr11 subfamily [54] suggesting that it could recognize urinary pathogenic e. coli [55] . it has been demonstrated that tlr13 colocalizes and interacts with unc93b1, a molecule located in the endoplasmic reticulum, which strongly suggests that tlr13 might be found inside cells and might play a role in recognizing viral infections [56] . figure 7 summarizes the tlr signaling pathway with the corresponding molecules found in the r. philippinarum transcriptome. pathogen proteases are important virulence factors that facilitate infection, diminish the activity of lysozymes and quench the agglutination capacity of hemocytes. because protease inhibitors play important roles in invertebrate immunity by protecting hosts through the direct inactivation of pathogen proteases, many bivalves have developed protease inhibitors to regulate the activities of pathogen proteases [1] . some genes encoding protease inhibitors were identified in c. gigas [57] , a. irradians [58] , c. farreri [59] and c. virginica; in the latter a novel family of serine protease inhibitors was also characterized [60] [61] [62] . a total of 23 contigs with homology to serine, cystein, kunitzand kazal-type protease inhibitors and metalloprotease inhibitors were found among our results. lysozyme was one of the most represented groups of immune genes in this transcriptome study with 208 contigs present. it is an antibacterial molecule present in numerous animals including bivalves. although lysozyme activity was first reported in molluscs over 30 years ago, complete sequences were published only recently including those of r. philippinarum [24] . antimicrobial peptides (amps) are small, gene-encoded, cationic peptides that constitute important innate immune effectors from organisms spanning most of the phylogenetic spectrum. amps alter the permeability of the pathogen membrane and cause cellular lysis [63] . in bivalves, they were first purified from mussel hemocyte granules [64, 65] . in mussels, the amp myticin c was found to have a high polymorphic variability as well as chemotactic and immunoregulatory roles [66, 67] . in clams, two amps with similarity to mussel myticin and mytilin [68] and a big defensin [69] are known. we were able to detect 36 contigs with homology to different defensins: defensin-1 (american oyster defensin), defensin mgd-1 (mediterranean mussel defensin) and the big defensin previously mentioned. four contigs were similar to an unpublished defensin sequence from venerupis ( = ruditapes) philippinarum. the primary role of heat shock proteins (hsps) is to function as molecular chaperones. their up-regulation also represents an important mechanism in the stress response [70] , and their activity is closely linked to the innate immune system. hsps mediate the mitochondrial apoptosis pathway and affect the regulation of nf-kb [71] . hsps are well studied in bivalves. for r. philippinarum, several assays have been developed to better understand the hsps profile in response to heavy metals and pathogen stresses [72] [73] [74] . the most important and well-studied groups of hsps were present in our r. philippinarum transcriptome (hsp27, hsp40/ dnaj, hsp70 and hsp90), but other, less common hsps were also represented (hsp10, hsp22, hsp83 and some members from the hsp90 family). recently, several genes related to the inflammatory response against lps stimulation have been detected in bivalves. such is the case of the lps-induced tnf-a factor (litaf), which is a novel transcription factor that critically regulates the expression of tnfa and various inflammatory cytokines in response to lps stimulation. it has been described in three bivalve species: pinctada fucata [75] , c. gigas [76] and c. farreri [77] . other tnf-related genes have been identified in the zhikong scallop, such as a tnfr homologue [78] and a tumor necrosis factor receptor-associated factor 6 (traf6), which is a key signaling adaptor molecule common to the tnfr superfamily and to the il-1r/tlr family [79] . figure 7 shows that several components of the tlr signaling pathway that are present in our transcriptomic sequences (myd88, irak4, traf-3 and -6, tram, btk, rac-1, pi3k, akt, btk and tank). a total of 1,918 contigs, 8.43% of those annotated, had homology with the main groups of putatively pathogenic organisms such as viruses (47 hits), bacteria (1,126 hits), protozoa (341 hits) and fungi (404 hits). figure 9 displays the taxonomic classification of these sequences and table 3 summarizes a list of the known bivalve pathogens found in our results. bacteria constitute the main group found among the sequences not belonging to the clam. as filter-feeding animals, bivalves can concentrate a large amount of bacteria and it could be one of their sources of food [24] . because vibrio spp. are ubiquitous in aquatic ecosystems, it was expected that the vibrionales order, with 141 hits, would be the most predominant. several species of the vibrio genus are among the main causes of disease in bivalves specifically causing bacillary necrosis in larval stages [80] . is noticeable that sequences belonging to the causative agent of brown ring disease in adults of manila clam, vibrio tapetis, have not been found. perkinsus marinus, with 2 matches, is the only bivalve pathogen found within the protozoa (alveolata) group. perkinsosis is produced by species from the genus perkinsus. both p. marinus and p. olseni have been associated with mortalities in populations of various groups of molluscs around the world and are catalogued as notifiable pathogens by the oie. viruses were the least represented among pathogens. the baculoviridae family was the most predominant with 21 matches, but the corresponding sequences were inhibitors of apoptosis (iaps) [81] that could also be part of the clam's transcriptome. five viral families were found in our transcriptome study: iridoviridae, herpesviridae, malacoherpesviridae, picornaviridae and retroviridae. a well-known bivalve pathogen was also identified, the ostreid herpesvirus 1, which has been previously been found to infect clams [82] . fungi had 404 matches in our results. it is known that bivalves are sensitive to fungal diseases, which can degrade the shell or affect the larval bivalve stages [83, 84] . this study represents the first r. philippinarum transcriptome analysis focused on its immune system using a 454-pyrosequencing approach and complements the recent pyrosequencing assay carried out by milan et al. [16] . the discovery of new immune sequences was effective, resulting in an enormous variety of contigs corresponding to molecules that could play a role in the defense mechanisms. more than 10% of our results had relationship with immunity. this new resource is now gathered in the ncbi short read archive with the accession number: sra046855.1. our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and gene expression studies as well as for the identification of genetic markers for various applications including the selection of families in the aquaculture sector. we have found sequences from molecules never described in bivalves before like c2, c4, c5, c9, aif, bax, akt, tlr6 and tlr13, among others. as a part of this work, three immune pathways in r. philippinarum have been characterized, the apoptosis, the toll like signaling pathway and the complement cascade, which could help us to better understand the resistance mechanisms of this economically important aquaculture clam species. animal sampling and in vitro stimulation of hemocytes r. philippinarum clams were obtained from a commercial shellfish farm (vigo, galicia, spain). clams were maintained in open circuit filtered sea water tanks at 15uc with aeration and were fed a total of 100 clams were notched in the shell in the area adjacent to the anterior adductor muscle. a sample of 500 ul of hemolymph was withdrawn from the adductor muscle of each clam with an insulin syringe, pooled and then distributed in 6-well plates, 7 ml per well, in a total of 7 wells, one for each treatment. hemocytes were allowed to settle to the base of the wells for 30 min at 15uc in the darkness. then, the hemocytes were stimulated with 50 mg/ml of polyinosinic:polycytidylic acid (poly i:c), peptidoglycans, ã�-glucan, vibrio anguillarum dna (cpg), lipopolysaccharide (lps), lipoteichoic acid (lta) or 1610 6 ufc/ml of heat-inactivated vibrio anguillarum (one stimulus per well) for 3 h at 15uc. all stimuli were purchased from sigma. pyrosequencing. after stimulation, hemolymph was centrifuged at 1700 g at 4uc for 5 minutes, the pellet was resuspended in 1 ml of trizol (invitrogen) and rna was extracted following the manufacturer's protocol. after rna extraction, samples were treated with turbo dnase free (ambion) to eliminate dna. next, the concentration and purity of the rna samples were measured using a nanodrop nd1000 spectrophotometer. the rna quality was assessed in a bioanalyzer 2010 (agilent technologies). from each sample, 1 mg of rna was pooled and used for the production of normalized cdna for 454 sequencing in the unitat de genã²mica (sct-ub, barcelona, spain). full-length-enriched double stranded cdna was synthesized from 1,5 mg of pooled total rna using mint cdna synthesis kit (evrogen, moscow, russia) according to manufacturer's protocol, and was subsequently purified using the qiaquick pcr purification kit (qiagen usa, valencia, ca). the amplified cdna was normalized using trimmer kit (evrogen, moscow, russia) to minimize differences in representation of transcripts. the method involves denaturation-reassociation of cdna, followed by a digestion with a duplex-specific nuclease (dsn) enzyme [85, 86] . the enzymatic degradation occurs primarily on the highly abundant cdna fraction. the single-stranded cdna fraction was then amplified twice by sequential pcr reactions according to the manufacturer's protocol. normalized cdna was purified using the qiaquick pcr purification kit (qiagen usa, valencia, ca). to generate the 454 library, 500 ng of normalized cdna were used. cdna was fractionated into small, 300-to 800-basepair fragments and the specific a and b adaptors were ligated to both the 39 and 59 ends of the fragments. the a and b adaptors were domain; pkc: protein kinase c; pten: phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase pten; raidd: caspase and rip adapter with death domain; tnf r1: tumor necrosis factor receptor 1; tnf-a: tumor necrosis factor alpha; tradd: tnf receptor type 1-associated death domain protein; traf2: tnf receptor-associated factor 2; trail: tnf-related apoptosis-inducing ligand; trail decoy: decoy trail receptor without death domain; trail-r: trail receptor. doi:10.1371/journal.pone.0035009.g008 used for purification, amplification, and sequencing steps. one sequencing run was performed on the gs-flx using titanium chemistry. 454 sequencing is based on sequencing-by-synthesis, addition of one nucleotide, or more, complementary to the template strand results in a chemiluminescent signal recorded by the ccd camera within the instrument. the signal strength is proportional to the number of nucleotides incorporated in a single nucleotide flow. all reagents and protocols used were from roche 454 life sciences, usa. pyrosequencing raw data, comprised of 975,190 reads, were processed with the roche quality control pipeline using the default settings. seqclean (http://compbio.dfci.harvard.edu/tgi/software/) software was used to screen for and remove normalization adaptor sequences, homopolymers and reads shorter than 40 bp prior to assembly. a total of 974,973 quality reads were subjected to mira, version 3.2.0 [87] , to assemble the transcriptome. by default, mira takes into account only contigs with at least 2 reads. the other reads go into debris, which might include singletons, repeats, low complexity sequences and sequences shorter than 40 bp. ncbi blastclust was used to group similar contigs into clusters (groups of transcripts from the same gene). two sequences were grouped if at least 60% of the positions had at least 95% identity. the 51,265 contigs were grouped into a total of 29,679 clusters. an iterative blast workflow was used to annotate the r. philippinarum contigs with at least 100 bp (49,847 contigs out of 51,265). then, blastx [88] with a cut off value of 10e-3, was used to compare the r. philippinarum contigs with the ncbi swissprot, the ncbi metazoan refseq, the ncbi nr and the uniprotkb/trembl protein databases. after annotation, blast2go software [89] was used to assign gene ontology terms [90] to the largest contig of a representative cluster (minimum of 100 bp). this strategy was used to avoid redundant results. default values in blast2go were used to perform the analysis and ontology level 2 was selected to construct the level pie charts. to make a comparison between r. philippinarum and other bivalve species, the nucleotide sequences and ests from c. gigas, m. galloprovincialis, l. elliptica and b. azoricus were obtained from genbank and from dedicated databases, when available. [93] . unique sequences from these databases (based on gi number) were used from each of the databases. these sequences were compared by blastn against the longest contig from each of 29,679 r. philippinarum clusters with a cut off e-value of 10e-05. hits to r. philippinarum sequences were represented in a venn diagram. the comparison between our 454 results, the longest contig from each of 29,679 clusters, and the milan et al. [16] transcriptome, contigs downloaded from ruphibase (http:// compgen.bio.unipd.it/ruphibase/query/), was made by blastn with a cut off e-value of 10e-05. another analysis was carried out to compare just the longest contig from each of 2,005 clusters identified as immune-related and the milan et al. contigs as well. the results were summarized in a table ( table 2 ). the percentage of coverage is the average % of query coverage by the best blast hit and the percentage of hits is the % of query with at least one hit in database, in parenthesis were added the total number of hits. identification of immune-related genes all the contig annotations were revised based on an immunity and inflammation-related keyword list (i.e. apoptosis, bactericidal, c3, lectin, socsâ�¦) developed in our laboratory to select the candidate sequences putatively involved in immune response. the presence or absence of these words in the blastx hit descriptions was checked to identify putative immune-related contigs. the remaining non-selected contigs were revised using the go terms at level 2, 3 and 4 assigned to each sequence after the annotation step that had a direct relationship with immunity. selected contigs were checked again to eliminate non-immune ones and distributed into functional categories. immune-related genes were grouped in three reference immune pathways (complement cascade, tlr signaling pathway and apoptosis) to describe each route indicated by our pyrosequencing results. to identify and classify the groups of organisms that had high similarity with our clam sequences, the uniprot taxonomy [94] was used except for the protozoa group. because protozoa are a highly complex group, a specific taxonomy [95] was followed. briefly, after the blastx annotation step all the hit descriptions included the species name (i.e. homo sapiens) or a code (i.e. human) meaning that protein has been previously identified as belonging to that species. with such information sequences were classified in taxonomical groups and represented in pie charts. table s1 list of contigs (e-value,10-3) of ruditapes philippinarum including sequence, length, description (hit description), accession number of description (hit acc), e-value obtained and database used for annotation (blast). study of diseases and the immune system of bivalves using molecular biology and genomics bacterial disease in marine bivalves, review of recent studies. trends and evolution perkinsosis in molluscs: a review bacteria-hemocyte interactions and phagocytosis in bivalves role of lectins (c-reactive protein) in defense of marine bivalves against bacteria modulation of the chemiluminescence response of mediterranean mussel (mytilus galloprovincialis) haemocytes immune parameters in carpet shell clams naturally infected with perkinsus atlanticus nitric oxide production by carpet shell clam (ruditapes decussatus) hemocytes generation and analysis of a 29,745 unique expressed sequence tags from the pacific oyster (crassostrea gigas) assembled into a publicly accessible database, the gigasdatabase immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster, crassostrea gigas sequence variability of myticins identified in haemocytes from mussels suggests ancient host-pathogen interactions mytibase, a knowledgebase of mussel (m. galloprovincialis) transcribed sequences insights into the innate immunity of the mediterranean mussel mytilus galloprovincialis development of expressed sequence tags from the pearl oyster, pinctada martensii dunker gene expression analysis of clams ruditapes philippinarum and ruditapes decussatus following bacterial infection yields molecular insights into pathogen resistance and immunity transcriptome sequencing and microarray development for the manila clam, ruditapes philippinarum: genomic tools for environmental monitoring de novo assembly of the manila clam ruditapes philippinarum transcriptome provides new insights into expression bias, mitochondrial doubly uniparental inheritance and sex determination analysis of est and lectin expression in hemocytes of manila clams (ruditapes phylippinarum) (bivalvia, mollusca) infected with perkinsus olseni increasing genomic information in bivalves through new est collections in four species, development of new genetic markers for environmental studies and genome evolution rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing sequencing technologies -the next generation transcriptomic analysis of the clam meretrix meretrix on different larval stages ferritin functions as a proinflammatory cytokine via iron-independent protein kinase c zeta/nuclear factor kappab-regulated signaling in rat hepatic stellate cells cloning and characterization of an invertebrate type lysozyme from venerupis philippinarum c1q and tumor necrosis factor superfamily: modularity and versatility the regulation of inflammation by galectin-3 isolation, cdna cloning, and characterization of an 18-kda hemagglutinin and amebocyte aggregation factor from limulus polyphemus angiopoietin-like proteins: emerging targets for treatment of obesity and related metabolic diseases modulation of apolipoprotein d expression and translocation under specific stress conditions neuroprotective effect of apolipoprotein d against human coronavirus oc43-induced encephalitis in mice apolipoprotein d itm2bs regulates apoptosis by inducing loss of mitochondrial membrane potential transcriptomic signatures of ash (fraxinus spp.) phloem transcriptomics of the bed bug (cimex lectularius) complement and its role in innate and adaptive immune responses potential indicators of stress response identified by expressed sequence tag analysis of hemocytes and embryos from the american oyster, crassostrea virginica analysis of a cdna-derived sequence of a novel mannose-binding lectin, codakine, from the tropical clam codakia orbicularis a novel c1q-domaincontaining protein from zhikong scallop chlamys farreri with lipopolysaccharide binding activity the c1q domain containing proteins of the mediterranean mussel mytilus galloprovincialis: a widespread and diverse family of immune-related molecules mgc1q, a novel c1q-domain-containing protein involved in the immune response of mytilus galloprovincialis differentially expressed genes of the carpet shell clam ruditapes decussatus against perkinsus olseni characterization of a c3 and a factor b-like in the carpet-shell clam, ruditapes decussatus structural and functional diversity of lectin repertoires in invertebrates, protochordates and ectothermic vertebrates purification and characterisation of a lectin isolated from the manila clam ruditapes philippinarum in korea characterization, tissue expression, and immunohistochemical localization of mcl3, a c-type lectin produced by perkinsus olseni-infected manila clams (ruditapes philippinarum) noble tandem-repeat galectin of manila clam ruditapes philippinarum is induced upon infection with the protozoan parasite perkinsus olseni lectin from the manila clam ruditapes philippinarum is induced upon infection with the protozoan parasite perkinsus olseni cdna cloning and mrna expression of the lipopolysaccharide-and beta-1,3-glucan-binding protein gene from scallop chlamys farreri molecular cloning and characterization of a short type peptidoglycan recognition protein (cfpgrp-s1) cdna from zhikong scallop chlamys farreri molecular cloning and mrna expression of peptidoglycan recognition protein (pgrp) gene in bay scallop (argopecten irradians, lamarck 1819) distribution of multiple peptidoglycan recognition proteins in the tissues of pacific oyster, crassostrea gigas molecular cloning and expression of a toll receptor gene homologue from zhikong scallop, chlamys farreri pattern recognition receptors and inflammation the evolution of vertebrate toll-like receptors a tolllike receptor that prevents infection by uropathogenic bacteria unc93b1 delivers nucleotide-sensing toll-like receptors to endolysosomes cg-timp, an inducible tissue inhibitor of metalloproteinase from the pacific oyster crassostrea gigas with a potential role in wound healing and defense mechanisms molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (argopecten irradians, lamarck 1819) molecular cloning and expression of a novel kazal-type serine proteinase inhibitor gene from zhikong scallop chlamys farreri, and the inhibitory activity of its recombinant domain a novel slowtight binding serine protease inhibitor from eastern oyster (crassostrea virginica) plasma inhibits perkinsin, the major extracellular protease of the oyster protozoan parasite perkinsus marinus evidence indicating the existence of a novel family of serine protease inhibitors that may be involved in marine invertebrate immunity serine protease inhibitor cvsi-1 potential role in the eastern oyster host defense against the protozoan parasite perkinsus marinus antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? innate immunity. isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, mytilus edulis a member of the arthropod defensin family from edible mediterranean mussels (mytilus galloprovincialis) evidence of high individual diversity on myticin c in mussel (mytilus galloprovincialis) mytilus galloprovincialis myticin c: a chemotactic molecule with antiviral activity and immunoregulatory properties analysis of differentially expressed genes in response to bacterial stimulation in hemocytes of the carpetshell clam ruditapes decussatus: identification of new antimicrobial peptides molecular characterization of a novel big defensin from clam venerupis philippinarum heat shock proteins: facts, thoughts, and dreams heat shock proteins, cellular chaperones that modulate mitochondrial cell death pathways djla, a membrane-anchored dnaj-like protein, is required for cytotoxicity of clam pathogen vibrio tapetis to hemocytes alternation of venerupis philippinarum hsp40 gene expression in response to pathogen challenge and heavy metal exposure identification of two small heat shock proteins with different response profile to cadmium and pathogen stresses in venerupis philippinarum molecular characterization and expression analysis of a putative lps-induced tnf-alpha factor (litaf) from pearl oyster pinctada fucata cloning, characterization and expression analysis of the gene for a putative lipopolysaccharide-induced tnf-alpha factor of the pacific oyster molecular cloning and characterization of a putative lipopolysaccharide-induced tnf-alpha factor (litaf) gene homologue from zhikong scallop chlamys farreri first molluscan tnfr homologue in zhikong scallop: molecular characterization and expression analysis identification and expression of traf6 (tnf receptor-associated factor 6) gene in zhikong scallop chlamys farreri diversity and pathogenecity of vibrio species in cultured bivalve molluscs an apoptosis-inhibiting baculovirus gene with a zinc finger-like motif detection of ostreid herpesvirus 1 dna by pcr in bivalve molluscs: a critical review synopsis of infectious diseases and parasites of commercially exploited shellfish a fungus disease in clam and oyster larvae a novel method for snp detection using a new duplex-specific nuclease from crab hepatopancreas simple cdna normalization using kamchatka crab duplex-specific nuclease using the miraest assembler for reliable and automated mrna transcript assembly and snp detection in sequenced ests basic local alignment search tool blast2go, a universal tool for annotation, visualization and analysis in functional genomics research gene ontology, tool for the unification of biology. the gene ontology consortium pyrosequencing of mytilus galloprovincialis cdnas: tissue-specific expression patterns insights into shell deposition in the antarctic bivalve laternula elliptica: gene discovery in the mantle transcriptome using 454 pyrosequencing highthroughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel bathymodiolus azoricus newt, a new taxonomy portal the new higher level classification of eukaryotes with emphasis on the taxonomy of protists key: cord-022262-ck2lhojz authors: gromeier, matthias; wimmer, eckard; gorbalenya, alexander e. title: genetics, pathogenesis and evolution of picornaviruses date: 2007-09-02 journal: origin and evolution of viruses doi: 10.1016/b978-012220360-2/50013-1 sha: doc_id: 22262 cord_uid: ck2lhojz the discovery of viruses heralded an exciting new era for research in the medical and biological sciences. it has been realized that the cellular receptor guiding a virus to a target cell cannot be the sole determinant of a virus's pathogenic potential. comparative analyses of the structures of genomes and their products have placed the picornaviruses into a large “picorna-like” virus family, in which they occupy a prominent place. most human picornavirus infections are self-limiting, yet the enormously high rate of picornavirus infections in the human population can lead to a significant incidence of disease complications that may be permanently debilitating or even fatal. picornaviruses employ one of the simplest imaginable genetic systems: they consist of single-stranded rna that encodes only a single multidomain polypeptide, the polyprotein. the rna is packaged into a small, rigid, naked, and icosahedral virion whose proteins are unmodified except for a myristate at the n-termini of vp4. the rna itself does not contain modified bases. the key to ultimately understanding picornaviruses may be to rationalize the huge amount of information about these viruses from the perspective of evolution. it is possible that the replicative apparatus of picornaviruses originated in the precellular world and was subsequently refined in the course of thousands of generations in a slowly evolving environment. picornaviruses cultivated the art of adaptation, which has allowed them to “jump” into new niches offered in the biological world. the discovery of viruses heralded an exciting new era for research in the medical and biological sciences. many contemporary virologists do not know, however, that the first animal virus described was a picornavirus, the etiological agent of the dreaded foot-and-mouth disease in cloven-footed animals. the discovery of footand-mouth disease virus (fmdv) by e loeffier and p. frosch in 1898 (loeffier and frosch, 1898) occurred at the same time as m.w. beijerinck described the amazing "contagium vivum fluidum" in 1898. this liquid was a filtered leaf extract derived from tobacco plants suffering from tobacco mosaic disease. free of bacteria, it was yet able to transmit the disease to uninfected plants. already in 1892, i. ivanovski had made a similar observation with tobacco mosaic virus but apparently he was unable to fully convince his peers of the significance of his discovery (waterson and wilkinsen, 1978) . research on viruses, now formally in its hundred-and-first year, has yielded an immense harvest of biochemical and biological information. the studies were driven not only by an urgent need to understand, and possibly prevent, viral disease; they were also fueled by a strong curiosity about the minute biologicals called viruses, which we can view as chemicals, on the one hand and as "living" entities on the other. poliovirus is an exquisite example of a chemical with a known empirical formula (molla et al., 1991) that can be crystallized (schaffer and schwerdt, 1955) yet causes a devastating disease in humans. poliovirus was discovered 90 years ago by landsteiner and popper (1909) to be the causative agent of poliomyelitis. the current knowledge of its chemical and three-dimensional structure and of its life cycle and pathogenesis is second to none. indeed, the intense research efforts on poliovirus over a period of nine decades will lead to its demise in the near future: global eradication of poliovirus is considered possible by the year 2000. following the identification of fmdv and poliovirus, a deluge of other viruses with similar properties were uncovered. these viruses have now been classified, as picornaviridae, a large family of small (lat. pico) rna (rna) viruses. currently, picornaviridae consists of six genera: enterovirus, rhinovirus, hepatovirus, parechovirus, cardiovirus and aphthovirus (table 12 .1). the first four genera include predominantly human pathogens, which cause a bewildering array of disease syndromes. although a disease syndrome may be considered characteristic for a specific picornavirus group, the same syndrome can possibly be also produced by b coxsackieviruses a9 (see cav above), poliomyelitis, c~v~ 3 (1) coxsackieviruses b1-6 myocarditis, pleurodynia, meningitis, hcar*, daf (2) hand-fix)t-and-mouth" disease, respiratory disease, neonatal, infections meningitis, encephalitis, pleurodynia, exanthema echoviruses 1-9, 11-21, 24-27, 29-33 vla-2(=0c2~i) daf (=cd55) enterovirus 69 nd c poliovirus types 1-3 poliomyelitis, meningitis cd155 (5) coxsackieviruses 1, 11, 13, 15, 17, [18] [19] [20] [21] [22] 24 common cold, infantile diarrhea icam-lt the following viruses have been recognized as picornaviruses on the basis of their genome sequences and physico-chemical properties as well as the result of comparative sequence analyses (see the section on evolution): equine rhinovirus types i and 2, aichi virus, porcine enterovirus, avian encephalomyelitis virus, infectious flacherie virus of silkworm clusters of enteroviruses refer to groups of enteroviruses arranged predominantly according to genotypic kinship (hyypia et al., 1997) . more clusters, including mainly animal enteroviruses, have been proposed. list of human syndromes adapted from melnick, 1996 . common syndromes in humans caused predominantly by one and/or other member(s) of the cluster but member viruses of other clusters may cause the same syndrome. receptors may be specific for specific serotypes. for details, see text. references describing the identification of receptors: (1) roivainen et al., 1994, (2) tomko and philipson, 1997; shafren et al., 1997, (3) bergelson et al., 1992; (4) bergelson et al., 1994; ward et al., 1994; (5) mendelsohn et al., 1989; (6) shafren et al., 1997; (7) staunton et al., 1989; greve et al., 1989; (8) hofer et al., 1994; (9) feigelstock et al., 1998; (10) neff et al., 1998; berinstein et al., 1995; (11) jackson et ai., 1996; (12) huber, 1994 . * shared with adenovirus type 2. t daf (decay accelerating factor) may function as non-essential (infection-augmenting) coreceptor. coxsackie virus a24v is a genetic variant of coxsackie virus a24. ** pringle, 1996. other picornaviruses. it has been realized that the cellular receptor guiding a virus to a target cell cannot be the sole determinant of a virus's pathogenic potential. indeed, it is a major challenge of the day to decipher the molecular mechanism(s) that determine viral tissue tropism and disease. what is the identity of picornaviruses? it relates to ancestral viruses whose identity we will never know. comparative analyses of the structures of genomes and their products, however, have placed the picornaviruses into a large "picorna-like" virus family, in which they occupy a prominent place (discussed in the section on evolution). these same analyses have led to an evolutionary tree of picornaviruses that reveals the extent of kinship (figure 12.1a) . one result of these phylogenetic investigations was a radical reorganization of the taxonomy of enterovirus, a genus of picornaviridae comprising numerous members infecting the gastrointestinal tract. the enteroviruses have now been divided into clusters (table 12. 1; figure 12 .1b) grouping the viruses mainly corresponding to genotypes (hyypia et al., 1997) . earlier classifications were based (1) on specific properties of the virions, (2) on disease patterns, (3) on the apparent absence of pathogenesis (echo is an acronym for "enteric cytopathic human orphan" because no disease was originally correlated with these viruses), or (4) in reference to the site of discovery (e.g. the town of coxsackie in new york state) and pathogenesis in suckling mice. as the number of known enteroviruses increased and the properties of these new isolates were elucidated, the need for a modified classification became apparent. however, even the latest dendrograms are likely to be modified again. principally, viruses that have been classified as belonging to a specific genus may be further divided into serotypes. a serotype is defined by the virus's ability to elicit a set of neutralizing antibodies ("antiserum") in a host animal; this set of neutralizing antibodies will generally not neutralize any other virus, regardless of the origin of the antiserum. neutralizing antibodies, in turn, are elicited by structures specific for a virus's capsid, and they have been referred to as neutralization antigenic determinants (or sites). the poliovirion carries at its surface four distinct neutralization antigenic determinants minor, 1990) . however, poliovirus expresses only three unique sets of these four determinants; hence poliovirus occurs in three serotypes. hepatitis a virus, on the other hand, expresses only one set of neutralization antigenic determinants; hence, it occurs in only one serotype. in contrast, human rhinoviruses (hrv) can express more than 100 unique sets of four antigenic determinants. rhinoviruses, therefore, occur in more than 100 serotypes. it should be noted that a poliovirus has been constructed that expresses neutralization antigenic determinants of all three serotypes. this virus, which is severely handicapped in proliferation, is trivalent as it can be neutralized by all three serotype-specific antibodies (murdin et al., 1992) . a genus consisting of viruses that cause the same disease syndrome can be subdivided further on the basis of receptor use. for example, all member viruses of the genus rhinovirus cause the common cold, yet they use two different receptors (icam-1 and ldl receptor; table 12 .1). on the basis of genotypes, however, this division no longer holds up (figure 12.1~) . as mentioned, the enteroviruses have now been subdivided into clusters based on genotypes (table 12 .1, figure 12 .1b). for example, the ccluster consists of the three serotypes of poliovirus and of serotypes 1, 11, 13, 15, 17, [18] [19] [20] [21] [22] . originally the c-cavs were not considered related to polioviruses because of the profound difference in pathogenesis (common cold and poliomyelitis, respectively) and the different use of receptor (icam-1 and cd155, respectively). however, their very close kinship was revealed by genome sequence. this proximity has led to the interesting question of whether the c-cavs are genetic variants of poliovirus (harber et al., 1995) or vice versa (discussed in detail in evolution an interesting recent variant of cav24 is cav24v, an agent that emerged in the early 1970s and that causes acute hemorrhagic conjunctivitis. this syndrome is also associated with a new variant of enterovirus 70, a d-cluster enterovirus (table 12.1; yin-murphy, 1973) . the phenomenon of the sudden appearance of enterovirus strains causing human diseases not previously associated with picornaviruses is of greatest interest with respect to the dynamics of picornavirus diversification, particularly in view of the eradication of poliovirus. what are the mechanisms by which the picornaviruses and other rna virus families have diversified? clearly, the genetic program inscribed into the viral genome is being changed as the viruses acquire new genetic traits. the predominant driving force of the changes in the genotype is largely an adaptation to new opportunities to proliferate. in the following, we will discuss some mechanisms and rules of genetic diversification and evolution of picornaviruses. sequences involved in rna replication, and the internal ribosomal entry site (ires), controlling translation. the virus-encoded 5'-terminal protein, vpg (viral protein genome-linked) is covalently linked to the 5'-terminal uridylic acid via a 04-(5'-uridylyl)tyrosine bond (lee et al., , 1977 nomoto et al., 1977b; rothberg et al., 1978) . picornavirus vpgs are 22-24 amino acids long; their third amino acid (from the n-terminus) is always a tyrosine, the residue linking vpg to the genome. genomelinked proteins are quite common amongst viruses belonging to the picorna-like super family (see figure 12.10) picornavirus vpgs are attached to 5'-terminal nucleotide sequences that form complex structures typical for entero-and rhinoviruses on the one hand, or cardio-, aphtho-and hepatoviruses on the other. these sequences are important signals in genome replication. entero-and rhinoviruses share a cloverleaf structure (rivera et al., 1988; andino et al., 1990 ) that has been subject to intense studies (see below). relatively little is known about the role of corresponding structural elements (which do not form cloverleaves) of cardio, aphtho-and hepatovirus genomes. the cloverleaf is followed by the internal ribosomal entry site (ires), arguably the most complex cis-acting element in any rna virus genome known (figures 12.2, 12.3; wimmer et al., 1993) . picornavirus ires elements, which are approximately 400 nt long, regulate the initia-tion of polyprotein synthesis. in deviation to c a p -d e p e n d e n t "scanning", ireses promote internal ribosomal entry, i.e. they allow initiation of translation independently of a capping group and even a free 5' end (jang et al., 1988 (jang et al., , 1989 pelletier and sonenberg, 1988; molla et al., 1992; chen and sarnow, 1995) . remarkably, ires elements are defined by their function, not by their sequences or apparent higher-order structure(s). this is illustrated in figure 12 .3, which depicts the sequence and folding pattern of the ires elements of poliovirus and encephalomyocarditis virus (emcv; pilipenko et al., 1989a,b) . in spite of these differences, the poliovirus ires has been exchanged with that of emcv, leading to a novel chimeric virus with excellent growth properties . similarly, the ires of hepatitis c virus (hcv), a flavivirus, was found to functionally substitute for the poliovirus ires, yielding a p o l i o / h c v chimeric virus (lu and wimmer, 1996; zhao et al., 1999) . finally, a construct in which the ires of h u m a n rhinovirus type 2 (hrv2) replaced that of poliovirus yielded a p v / h r v chimeric virus (pvi(ripo)) that is figure 12.3 sequences and secondary structures of ires elements of poliovirus and encephalomyocarditis virus. a. poliovirus ires; individual domains have been labeled with roman numerals. b. encephalomyocarditis virus (emcv) ires; domains have been labeled with capital letters. both ireses contain a conserved ynxmaug motif, of which the oligopyrimidine stretch (yn) and the aug triplet are indicated by solid bars. note that in the emcv ires, the aug triplet of the ynxmaug motif is the initiating codon of the polyprotein. in the poliovirus ires, this aug triplet is silent and is separated from an aug codon initiating the synthesis of the polyprotein by a "spacer sequence" of 154 nt (jang et al., 1990) . single attenuating mutations in the poliovirus vaccine strains map to domain v (wimmer et al., 1993) . indistinguishable from wt poliovirus with respect to replication in hela cells yet is highly attenuated in poliovirus-receptor-transgenic mice and in monkeys (gromeier et al., 1996 (gromeier et al., , 1999a ; discussed in the section on pathogenesis). the properties of this interesting novel virus will be discussed in a later section. the mechanism by which ires elements function is still obscure. translation of picornavirus mrna is initiated downstream of the ires to yield an unstable "polyprotein" that is rapidly cleaved by virusencoded proteinases to proteins involved in viral proliferation (figure 12.4 ; see also evoiution and figure 12 .10). it is important to note that the mrna found in viral polyribosomes that encodes the polyprotein differs from virion rna in one important aspect: it is terminated with pup ... (hewlett et al., 1976; nomoto et al., 1976) . apparently, the terminal protein vpg has been cleaved from incoming or from newly synthesized rna. it has been suggested that the enzyme cleaving the vpg-pup phosphodiester bond is of cellular origin but the reason for the removal of the protein and the nature of the enzyme catalyzing it remain unknown. moreover, it is not clear whether the incoming vpg-linked virion rna will be processed immediately after entry or whether the removal of vpg will occur only after the first round(s) of viral protein synthesis. entero-and rhinoviruses encode the two proteinases 2a pr~ and 3c/3cd p~~ aphthoviruses the two proteinases l pr~ and 3c pr~ and cardioviruses only the proteinase 3c p~~ interestingly, both cardioviruses and aphthoviruses have evolved a peculiar cleavage mechanism between 2a and 2b that occurs only in cis and is an enzyme-independent reaction (reviewed by ryan and flint, 1997) . a similar as yet unknown mechanism of proteolytic cleavage is that between vp4 and vp2 ( figure 12 .4d), which occurs only during maturation of the virion (maturation cleavage) and appears also to be proteinase-independent (harber et al., 1991; see below) . the origin of these fascinating enzymes and of specific cleavage events are discussed in the section on evolution. since most details of proteolytic processing have been accumulated for poliovirus, much of the following discussion will center on this viral system. the two poliovirus proteinases 2a pr~ and 3c/3cd pr~ cleave at different sites, as determined by the sequences of the scissile bond ( figure 12 .4b, c). theoretically, the poliovirus polyprotein could give rise to 77 different cleavage products if proteolytic processing by these enzymes and the maturation cleavage were entirely random (wimmer et al., 1993) . in fact, only roughly 29-30 cleavage products have been identified in poliovirus-infected cells (nicklin et al., 1986) . it has thus been concluded that processing of the picornavirus polyproteins is not random but follows a pathway that is determined by protein folding (masking of cleavage sites) and by the amino-acid sequences surrounding the scissile bond ( figure 12 .4b, c; harris et al., 1990) . for example, the precursor 3cd pr~ can be cleaved into 3c pr~ and 3cd p~ by a (cis?) cleavage in which the 3c/3cd pr~ proteinase is involved. both 3c pr~ and 3d p~ are quite stable end-products of processing. however, in the case of poliovirus type 1 (mahoney) (pv1 (m)), 3cd pr~ can also be efficiently processed in trans by 2a pr~ to 3c' and 3d' (figure 12 .4c), two polypeptides with no apparent function in viral proliferation (lee and wimmer, 1988) . just like 3c pr~ and 3d p~ 3c' and 3d' are quite stable endproducts of processing, even though 3d p~ harbors a perfect cleavage site for 2a pr~ and 3c' harbors a cleavage site for 3c/3cd pr~ (figure 12.4) . indeed, in pvl(m)-infected cells, nearly equal amounts of the four cleavage products of precursor 3cd pr~ are observed. it is assumed that structural constraints mask one or the other cleavage site from recognition and processing once the cleavage product has been formed (lee and wimmer, 1988) . the preferred cleavage sequence for 3c/3cd pr~ in the poliovirus polyprotein is axxq*g; hence, cleavage sites with this sequence are usually rapidly processed. numerous mutational studies have supported the identity of this 3c/3cd pr~ cleavage motif (reviewed in dougherty and semler, 1993, and wimmer et al., 1993) . an intriguing genetic analysis has made use of a viral construct that 12.4 processing scheme and cleavage sites of the poliovirus polyprotein. a. proteolytic cleavages of the polyprotein. triangles indicate cleavage by 3c pr~ and/or 3cd pr~ note that both enzymatic entities can efficiently cleave the non-structural proteins. in contrast, the p1 capsid precursor can be processed by 3cd pr~ only. solid triangles represent efficient cleavage sites, whereas open triangles represent slowly cleaved sites resulting in stable precursor proteins. the 2apr~ cleavages are depicted with circles. only the cleavage between p1 and p2-p3 (solid circle) is essential, whereas the cleavage of 3cd p'~ to 3c' and 3d' is dispensable (open circle). the maturation cleavage is indicated by the open diamond. the mechanism by which this cleavage occurs is unknown. numbers in brackets indicate the molecular weight in kda. b-d. amino-acid residues at sites cleaved by (b) 3c p~~ and/or 3cd pr~ (c) by 2a pr~ and (d) during the maturation cleavage are shown in a single-letter code. the positions of the amino-acid residues are designated p1, p2, p3 .... at the newly generated c-termini, or pi', p2', p3', ... at the newly generated n-termini. the fastest cleavages catalyzed by 3cp~~ pr~ occur at sites in which the p4 position is a small aliphatic amino acid (e.g. axxq*g). cleavage at tqsq*g between 3c and 3d is slow, giving rise to the 3cd p~~ cleavage intermediate with a long half-life (cao and wimmer, 1996) . mutated this axxq*g cleavage motif at a specific site in order to avoid proteolytic processing. the amino acids placed by the mutants into the motif confirmed the proposed interaction between substrate and enzyme during cleavage (cao and wimmer, 1996 , and references therein). as will be discussed later, poliovirus is a purist with respect to cleavage signals, since the scissile bond in all cleavages, catalysed by 3c/3cd pr~ is q*g (kitamura et al., 1981; semler et al., 1981a semler et al., , 1981b . in other picornaviruses, or viruses of the large picoma-like superfamily, the cleavage site may differ from the canonical q*g signal. a most important observation in studies of picomavirus proliferation is that cleavage intermediates may have important functions that in some cases may even be distinct from that of their end-products (e.g. 3cw pr~ yielding 3c pr~ and 3dp~ the structure of picornavirus 3c p~~ enzymes has been accurately predicted by gorbalenya et al. (1986) , leading to the genetic analyses alluded to above. the structures were proved to be correct by x-ray crystallographic studies of 3c p~~ of human hepatitis a virus (allaire et al., 1994) and human rhinovirus 14 (matthews et al., 1994) . following the orf, there is a heteropolymeric region that may be different with respect to length (72-126 nt) and structure in different picornavirus genomes (xiang et al., 1997) . however, all picornavirus genomes terminate with poly(a), as was shown first for poliovirus (yogo and wimmer, 1972) . the role these sequences play in replication will be discussed below. the genomic rna of picornaviruses can serve as mrna and, consequently, it is of the same polarity as cellular mrna. by convention, this polarity has been designated plus-strand polarity (baltimore, 1971) . fortunately, the genomic rna of picornaviruses is infectious; that is, upon transfection into suitable host cells, virion rna will initiate a complete infectious cycle (wimmer et al., 1993) . interestingly, poliovirus and its purified genome will replicate even in enucleated cells (morgan-detjen et al., 1978) , an observation suggesting that the nucleus does not contribute factors essential for viral proliferation. using reverse transcriptase, racaniello and baltimore (1981) generated full-length "complementary" dna (cdna) that contained the entire genetic information of the viral genome (currently, cdna refers to double-stranded dna generated from the original complementary dna strands). transfections into hela cells of the cdna that contained heterologous dna sequences at either end of the virus-specific sequences generated, surprisingly, poliovirus. with this experiment, "reverse genetics" of rna viruses was born as the rna genome was now amenable to manipulations developed for dna. the efficiency with which the original cdna clones induced an infectious cycle in hela cells was very low (about 10 pfu/~tg dna; racaniello and baltimore, 1981) . construction of plasmids that could replicate in transfected cells dramatically increased the specific infectivity to 103 pfu/~tg dna; semler et al., 1984) . however, reverse genetics was made more practical when the cdna was cloned downstream of the phage t7 rna transcriptase promoter and, using purified t7 transcriptase, virtually unlimited amounts of highly infectious transcript rna could be produced in a simple test-tube experiment (>10 s pfu/~tg of transcript rna; van der werf et al., 1986) . this was important because mutant genomes with highly debilitating replication phenotypes could not be recovered by the inefficient cdna transfection method. it was known before that vpg is not required to be at the 5' end for poliovirus rna to be infectious (nomoto et al., 1977a) . the 5' end of the t7 transcripts is pppgguuaaaa.., whereas that of virion rna is vpg-puuaaaa... the extra g residues do not prevent transfection but they reduce the specific infectivity of the transcript. in any event, picornavirus rna is quite tolerant of modifications of the 5' end of its genome and, in all cases, the virion rnas isolated after transfections have the authentic terminus restored (wimmer et al., 1993) . infectious cdnas have now been generated from members of all picornaviruses. the method of choice to generate virus remains transfection of t7 transcripts. recently developed methods of rt/pcr allow researchers to generate infectious cdna clones in less than 1 month (tellier et al., 1996) . in general terms, genome replication proceeds in two steps: synthesis of a complementary rna strand (-strand) that then serves as template for plus rna strands (+strands; figure t q a b c d e f g h figure 12.5 steps in the replication of the poliovirus genome. parental, positive-stranded virion rna (solid line) is transcribed, yielding -rna (broken line) after protein (vpg)-priming by the viral rna-dependent rna polymerase 3d pr~ (enzyme or any other proteins involved are not shown). a replicative intermediate (ri) form consisting of a single +strand template and multiple nascent -rna strands (a) has not been detected, so that, more probably, intermediates in -rna synthesis are either mainly single-stranded (b) or double-stranded (c). elongation of the nascent-rna (c) yields the replicative form (rf) double-stranded rna (d). available evidence suggests that the rf is an intermediate in genome replication (discussed in xiang et al., 1997) . accordingly, a cloverleaf/rnp is formed at the end of the rf that promotes vpg-primed synthesis of +rna (e). the structures formed after multiple initiation could either be "closed" (entirely base-paired; f) or "open" (g). available evidence suggests that structure f is the correct intermediate (note that 3d p~ is an unwindase). for details, see wimmer et al., 1993, and xiang et al., 1997 . modified from wimmer et al., 1993. 12.5). the validity of this scheme has been known for almost three decades yet only very few details of the individual steps have been elucidated . because the vast majority of studies have been carried out with poliovirus, this review will concentrate predominantly on this viral system. with the exception of the capsid proteins, all viral non-structural proteins and even processing intermediates have been implicated in genome replication (xiang et al., 1997) . the evidence for the involvement of these proteins (2a pr~ 2b, 2bc, 2c, 3a, 3ab, vpg, 3c/3cd pr~ 3d p~ is based largely on genetic data or on biochemical experiments assumed to be indicative of genome replication (wimmer et al., 1993; xiang et al., 1997) . for example, genetic and bio-chemical analyses of 3ab strongly suggest that this protein, a non-specific rna binding protein, and the proteinase 3cd pr~ participate in the formation of an initiation complex for +strands (xiang et al., 1997) . another example is the involvement of 2c in rna replication. briefly, poliovirus rna synthesis is highly sensitive to the presence of 2 mm guanidine hydrochloride (gua hc1); poliovirus mutants resistant to 2 mm gua hc1 harbor a single amino-acid exchange (n179a/g) in polypeptide 2c. it has recently been established that 2c is an atpase (and not a gtpase) and we now refer to it as 2c awpase (pfister and wimmer, 1999) . the atpase activity of purified 2c awpase is inhibited by 2 mm gua hc1, whereas that of purified 2c atpase with a n179/g mutation is resistant to this concentration of the drug (pfister and wimmer, 1999) . on the basis of these considerations, it can be assumed that the atpase activity of 2c awpase is essential for genome replication. just as with 3ab or 3cd pr~ however, the step(s) by which 2c awpase is exerting its essential function are still unknown. the only proteins whose role in genome replication has been firmly established are vpg and 3d p~ the crystal structure of 3d p~ has recently been solved (hansen and schultz, 1997) , a result that will greatly advance our (limited) understanding of this important enzyme. importantly, 3d p~ was established already in 1977 as being a primer-dependent and rnadependent rna polymerase . although a deluge of circumstantial evidence suggested that a uridylylated form of vpg might serve as primer for 3d p~ (nomoto et al., 1977b; wimmer, 1982; takeda et al., 1986; toyoda et al., 1987) , direct evidence for this mechanism has been obtained only very recently (paul et al., 1998b) . briefly, vpg is being uridylylated to vpg-pu(pu) by the viral rna polymerase 3d p~ in the presence of template (poly(a)). vpg-pu(pu) then primes the transcription of poly(a), leading to the synthesis of poly(u), which is the 5' terminus of-strands (paul et al., 1998b) . in spite of these seemingly simple experiments (paul et al., 1998b) , the mechanism of initiation of rna synthesis was a matter of controversy for almost two decades. baltimore's and flanegan's groups presented evidence favoring "hairpin priming", whereas wimmer's group accumulated data suggesting "protein priming" (reviewed by richards and ehrenfeld, 1990) . the controversy has finally been settled in favor of protein priming. at low concentration of enzyme, poliovirus polypeptide 3ab stimulates the transcriptional activity of 3d p~ up to 100-fold plotch et al., 1989; paul et al., 1994) . indeed, biochemical and genetic evidence suggests that 3d p~ and 3ab form a complex in solution (molla et al., 1994) . the significance of these observations is not yet known. an important additional property of 3d p~ is its ability to unwind double-stranded rna. that is, the enzyme, while transcribing a template, can replace a dormant rna strand that is hybridized to the template with the new strand that is just being synthesized (cho et al., 1993) . it should be noted, however, that 3d p~ is not a helicase as it will not separate two strands without transcribing one of them (cho et al., 1993) . the participation in picornavirus replication of cellular proteins, referred to by investigators as "host factors", has also had a history of controversies. several polypeptides were proposed to be involved in replication (e.g. a kinase or a uridylic acid transferase) but these proteins have disappeared after further analysis (richards and ehrenfeld, 1990 ). ehrenfeld's and semler's groups have recently identified a cellular 38 kda rna binding protein, poly(rc) binding protein 2 (pcbp2), that is not only required for the function of the poliovirus ires but it has also the propensity to bind, together with 3cd pr~ to the poliovirus 5'-terminal cloverleaf (blyn et al., 1996) . pcbp2 (or pcbp1, a protein related to pcbp2; gamarnik and andino, 1997) is undoubtedly the "host factor p36" that was originally proposed by baltimore's group to effect the binding of 3cd pr~ to the poliovirus cloverleaf (andino et al., 1990 (andino et al., , 1993 . andino et al. (1993) provided first evidence suggesting that the formation of a specific protein/cloverleaf rnp complex consisting of viral protein 3cd, a cellular protein ("p36") and the viral rna is required for the initiation of +strand synthesis (andino et al., 1993) . this hypothesis has been further supported by the discovery of pcbp2 (gamarnik and andino, 1997; parsley et al., 1997) . pcbp2 is therefore a sensible candidate for a "host factor" involved in poliovirus rna replication. however, poliovirus protein 3ab can replace pcbp2 in all biochemical reactions characteristic of the formation of a 5' terminal rnp. moreover, 3ab and 3cd pr~ both cleavage products of the p3 precursor ( figure 12 .4a), are associated in solution (molla et al., 1994) . finally, the phenotypes of mutants of 3ab in vivo and in vitro support the conjecture that 3ab is involved in the formation of a cloverleaf/3cdpr~ complex xiang et al., 1995a,b) . currently, there is no compelling evidence in favor of the cloverleaf/3cdpr~ complex over that of cloverleaf/3cdpf~ with respect to poliovirus genome replication (see a discussion in xiang et al., 1997) . recognition of rna signals located somewhere in the rna genome is a prerequisite for specificity in genome replication. this review will concentrate only on cis-acting elements of entero-and rhinoviruses because, as mentioned earlier, the overwhelming number of experiments deal with these viruses. currently, only the 5'-terminal cloverleaf has been firmly established as a cis-acting signal in enterovirus genome replication (see previous section), although the mechanism by which it functions is still obscure. clearly, the formation of a specific rnp plays a role the significance of which will be discussed below. more complicated is the recognition of the +strand template for the initiation of-strands. since replication of picornavirus rnas commences at the 3'-terminal poly(a), a homopolymeric sequence found also in most cellular mrnas, poly(a) alone cannot be a determinant for virus-specific -strand synthesis. vpg-pu(pu) can be synthesized in the presence of poly(a), and vpg-poly(u), the 5' end of -strands, will follow the synthesis of the primer (paul et al., 1998) . this reaction, however, does not reveal the mechanism of specificity. mutational analysis of the heteropolymeric sequence of the 3'ntr of enteroviruses indicated that this region was critically important for replication (pilipenko et al., 1996; melchers et al., 1997) . however, the poliovirus 3'-terminal heteropolymeric sequence can be replaced with that of hrv14, a hairpin with no apparent homology with the poliovirus structure, and the resultant poliovirus/hrv14 hybrid genome replicated with wt kinetics (rohll et al., 1995) . even more startling was a report from semler's group that presented evidence that the heteropolymeric region could be deleted altogether without loss of viability (todd et al., 1997) . currently, the paradox intrinsic to these findings remains unsolved . it is possible that the 3' heteropolymeric region plays an important role in the efficient formation of an initiation complex for replication but to a much lesser extent in +strand template recognition. the authentic recognition signal may reside in rna-internal sequences, as proposed by mcknight and lemon (1996) . these authors reported that, surprisingly, a stem loop structure mapping to the coding region of the hrv14 capsid proteins was absolutely necessary for genome replication. fittingly, a stemloop rna structure that has been uncovered in poliovirus rna also appears to play a role in genome replication; it maps to the coding region of 2c awpase (goodfellow et al., 1998) . the mechanism by which these new elements influence replication has yet to be resolved. finally, evidence has been presented suggesting that sequences within the ires play a role in genome replication (borman et al., 1994; shiroki et al., 1995) . this is difficult to comprehend if one considers chimeric ires viruses. as mentioned above, the cognate ires of poliovirus can be replaced with ires elements from different viruses whose ires are merely related (hrv2, hrv14, cbv4, cav9, cav24, ev71; gromeier et al., 1996, 1999a and unpublished results) or entirely different (emcv; alexander et al., 1994; hcv, lu and wimmer, 1996; zhao et al., 1999) without loss of genome replication. defective interfering particles (di particles; see below) of poliovirus are naturally occurring variants with deletions (of varying sizes) in the p1 region, encoding the capsid proteins. di particles can replicate their rna without helper function but they need wt virus for encapsidation. sequence analyses of genomic rnas of di particles led nomoto and his colleagues to the surprising observation that in all cases the deletions were in-frame of the polyprotein coding sequence. on the other hand, artificial genomes engineered with out-of-frame deletions were unable to replicate their rna, even in the presence of wt helper virus (kuge et al., 1986; hagino-yamagushi and nomoto, 1989) . it was concluded that translation was necessary for the cognate genome to replicate. that is, translation had a cis effect on replication that could not be complemented in trans by a helper genome. these observations were later confirmed and extended (wimmer et al., 1993; novak and kirkegaard, 1994; agol et al., 1999) . there are several hypotheses that are used to explain the phenomenon. the least likely is that certain replication proteins can only function in cis. if so, only viral mrna could serve as template in rna synthesis. since viral mrnas lack vpg (hewlett et al., 1976; nomoto et al., 1976 ; see above), every +strand rna that functions as template in rna synthesis should also lack vpg. available evidence suggests that all rna templates involved in replication are terminated with vpg (nomoto et al., 1977b; petterson et al., 1977; wu et al., 1978; larsen et al., 1980) . furthermore, rna replication occurs in a tight membranous environment (bienz et al., 1992) . thus, it is unlikely that these genome replicating membranous complexes also harbor viral polysomes (wimmer et al., 1993) . indeed, crude, membranous replication complexes can be isolated from infected cells that can replicate poliovirus rna yet they are free of ribosomes (takegami et al., 1983; takeda et al., 1986; toyoda et al., 1987) . an alternative explanation is that the observed cis effect is operating only during the very first round of translation at the onset of infection. clearly, translation of an infecting genome will have to be somehow arrested to allow the template to switch from translation to transcription. it is possible that, once the switch has been made, replication can proceed independently of translation. this does not exclude the possibility that viral proteins, perhaps intermediates with a short half-life or short-lived protein complexes, must be continuously supplied to the rna synthesizing machinery. the question of the switch from translation to rna synthesis of infecting + stranded genomic rna has been subject of much speculation. the classical study of kolakofsky and weissmann (1971) on phage q~ replication solved the dilemma by showing that the phage replicase (a complex of four proteins) can repress translation of viral mrna. a similar model has been proposed for poliovirus by gamarnik and andino (1998) : the formation of an rnp consisting of cloverleaf/3cdpr~ at the 5' end of the viral mrna inhibits further translation, thereby switching the template to replication. one problem with this model is that at the peak of poliovirus replication, translation and rna syn-thesis occur concomitantly in the presence of an excess of 3cd pr~ molecules (note that for each virus particle, 60 molecules of 3cd pr~ are synthesized; the ratio of viral +strand rna to unprocessed 3cd pr~ may be 1:100 through most of the replicative cycle). moreover, if the genome has to be translated for replication to occur, how can inhibition of translation promote rna synthesis? a very schematic representation of steps in genome replication is shown in figure 12 .5 (wimmer et al., 1993) . the possible rna structures involved in replication have been divided into three categories: (1) we have argued before that the cumulative evidence favors the "closed forms" for rf and ri but this view may not be shared by others (wimmer et al., 1993; xiang et al., 1997) . since 3d p~ is an "unwindase" (cho et al., 1993 ; see above), the scheme does not necessarily require a helicase. indeed, so far no picornaviral helicase has been identified, and purified 2c atpase has stubbornly refused to exhibit such activity (pfister and wimmer, 1999) . briefly, vpg will be uridylylated at the 3'-terminal poly(a). vpg-pu(pu), in turn, will then prime synthesis of-strands ( figure 12 .5c). it is unlikely that multiple initiation of-strands on the same template (prior to completion of the first-strand) occurs, since an ri with multiple -strands (such as in figure 12 .5a) has not been found in infected cells (bishop and koch, 1969) . it is even possible that initiation at the poly(a) tail of poliovirus rna occurs only once. completion of the-strand will thus yield rf ( figure 12 .5d), which we consider an intermediate in replication and not a byproduct (wimmer et al., 1993) . one compelling argument in favor of this assumption is that in the rf the 5' end of +strands is in the close vicinity of the 3' end of -strands, a prerequisite first proposed by baltimore and his colleagues (andino et al., 1993; harris et al., 1994) . destabilization of this end of the rna will lead to the formation of an rnp consisting either of cloverleaf/3cdpr~ or cloverleaf/3cdpr~ which, in turn, will free the 3' end of the-strand for vpgprimed +strand synthesis to occur ( figure 12 .5e). multiple initiation at this end will lead to the multistranded ri ( figure 12 .5f), the nascent or full-length +strands being replaced during transcription by the 3d p~ unwindase. initiation of +strands may be more efficient than initiation of-strands; hence the large excess of +strands over-strands in infected cells. note that a reconstituted replication system of purified viral and cellular components capable of synthesizing +strands from input +strands has not been achieved; thus many of the hypotheses put forward in this scheme have not yet been tested. gamamik and andino (1996) have described a novel system to study poliovirus replication in xenopus oocytes by injecting poliovirus rna into these cells. however, virus will replicate only if a hela cell $10 extract was co-injected with the rna. interestingly, the authors have been able to separate the hela supporting activities ($10) into two factors, one necessary for poliovirus ires-driven translation, the other for poliovirus rna synthesis. this system offers an excellent opportunity to separate and characterize viral and cellular factors involved in virus replication. viruses, lacking the genetic information as well as the tools to provide most of the essential components to replicate, are obligatory intracellular parasites. the complexity of viral proliferationmacromolecular synthesis of polypeptides and genomic nucleic acid, and encapsidation-has led to the text book wisdom that viruses are obligatory intracellular parasites unable to proliferate outside living cells. however, poliovirus rna (obtained either from virions or by transcription with phage t7 rna polymerase from plasmid dna), when incubated in an extract of uninfected hela cells void of nuclei, mitochondria and cellular mrna, will direct translation, genome replication and genome encapsidation such that infectious particles are formed. these newly synthesized virions are indistinguishable from poliovirus isolated from tissue cultures. thus, a picornavirus (poliovirus) is the first virus that has been synthesized de novo in a cell-free extract of mammalian cells (molla et al., 1991) . this experiment has nullified the notion that viruses can proliferate exclusively in living cells. moreover, the novel approach can be used to study individual steps of viral replication in the absence of cell-membrane barriers. several interesting observations regarding protein-protein interactions, the role of membranes, of cellular membranous components or soluble cellular factors, or of inhibitors of viral rna synthesis, have been published (barton and flanegan, 1993; molla et al., 1993c molla et al., , 1994 barton et al., 1995; parsley et al., 1997; cuconati et al., 1998; towner et al., 1998) . the use of the cell-free cellular extract for studies of poliovirus rna replication, however, is still in the early stages of exploitation. nevertheless, it has been possible to even achieve genetic recombination of poliovirus in cell-free hela extracts (duggal et al., 1997; tang et al., 1997; duggal and wimmer, 1999 ; see below). in the course of transcription, all templatedependent nucleic acid polymerases make errors in incorporating nucleotides with roughly the same frequency (10-3-10-4). as is discussed in chapter 7, this phenomenon has profound biological consequences for rna viruses. because rna viruses have chosen not to develop mechanisms by which misincorporations of nucleotides can be recognized and corrected, the average number of "spontaneous" mutations per replication of the genome, referred to as error rate, is around 10 -4 . the high error rate in the absence of mechanisms of proofreading and editing has several consequences. first, the average genome length of animal rna viruses is small (12000nt). notwithstanding the genome of the exceptional coronavirus (30 000nt), rna viruses with genomes exceeding 150 kb (e.g. the dna viruses, herpes viruses, poxviruses, iridoviruses) are inconceivable because of the high probability that each genome would carry multiple mutations after each round of synthesis. it should be noted that these considerations by no means imply that dna viruses with very small genomes do not exist. in fact, the animal virus with the smallest known genome is hepatitis b virus (3.2 kb). as to picornaviruses, their average genome length is 8000 nt (see also wimmer et al., 1993) . second, rna viruses replicate near the threshold of error catastrophe (holland et al., 1990) . that is, the artificial increase of misincorporation of nucleotides (e.g. by chemical mutagens) may lead to a rapid decline of the viability of the entire virus population. third, plaque-purified clones of rna viruses are not homogeneous but populations of many different, albeit very closely related genotypes; hence the term "quasispecies" (eigen, 1993) . fourth, the genetic heterogeneity allows an rna to rapidly adapt to a changing environment. a simple example should demonstrate the ease with which a drug-resistant mutant of poliovirus can be isolated. as mentioned, poliovirus rna replication is highly sensitive to the presence of 2 mm guanidine hydrochloride (gua hc1). after plating a stock of plaque-purified poliovirus on a monolayer of hela cells in the presence of 2 mm gua hc1, a few plaques will arise corresponding to resistant variants (gr) with mutations mapping to 2c awpase (pincus et al., 1986; tolskaya et al., 1994) . in the case of the selection of gr poliovirus mutants, it should be noted that the resistant variants already existed in the population of the inoculating virus. if the virus inoculum had been entirely free of gr variants, no selection of gr mutants could have occurred since the drug inhibits rna synthesis; hence, there would have been no misincorporation of nucleotides to generate the gr mutations in 2c atpase. although it may be a trivial thing to repeat, it is important to remember that genetic variation by misincorporation of nucleotides (just as recombination) requires replication. no replication, no mutants. the genetic plasticity of genotypes and the dynamics of genetic variation can be studied conveniently when transcript rna, produced by transcription of cdna with t7 rna polymerase, is transfected on to hela cell monolayers and the corresponding plaque phenotype of progeny virus is analysed. in the case of wt virus, the plaques are, by convention, "large". if mutant rnas are analysed in plaque assays, one may observe only "small plaques" with a rare "large" plaque emerging on the plate. this rare large plaque may signal a reversion (either directly or through suppresser mutations) to a fast-replicating genotype. passage of the population of small and large plaque phenotype viruses (at multiplicities of infection of more than 5) will rapidly yield populations of only the faster-growing virus because the impaired genomes are eliminated by competition. an example of this phenomenon has been described by lu et al., (1995b) , who analysed a hybrid poliovirus in which the cognate 2a pr~ coding sequence was exchanged to that of coxsackie b4 virus. a special case of a genetic phenomenon is that of a "quasi-infectious" genome. this term was originally introduced by agol and his colleagues (gmyl et al., 1993) to describe the following phenomenon. genetically engineered poliovirus variant rna was transfected into hela cells. progeny virus was harvested, sometimes only after prolonged incubations of the transfected tissue cell cultures. analysis of the genotypes of progeny virus genomes (by rt/pcr) revealed only revertant or pseudorevertant rnas. none of the original mutant genotypes were detectable. this phenomenon can be explained if the original mutant genotype was able to replicate its rna, albeit only at levels too low for virus production or even for the development of cpe. nevertheless, the slowly replicating mutant genome allowed for mutation (either misincorporation or deletions, insertions), eventually leading to fast-growing genotypes. by definition, the progeny of quasiinfectious genomes will not yield virus with the parental genotype. if a mutation (point mutation, linker insertion, etc.) engineered into the genome rna is lethal, the lesion may effect complete abrogation of genome replication. hence, reversion to viability cannot be expected. an interesting example of quasi-infectious versus lethal mutations in the poliovirus genome was described when mutations in vpg were studied (kuhn et al., 1988; reuer et al., 1990; cao and wimmer, 1995) . as mentioned, vpg is linked to the genome via a o4-(5'-uridylyl) tyrosine (the tyrosine in position three of vpg). a mutation of tyrosine to phenylalanine (y3f) was originally described as being lethal (reuer et al., 1990) . this conclusion made sense, since phenylalanine lacks a 04 hydroxyl group for phosphodiester formation. however, cao and wimmer (1995) later observed that cells transfected with vpg(y3f) variant rna produced viable virus, albeit only at very low frequency and only after prolonged incubation of the cultures. all of the progeny genomes carried a f3y reversion. the possibility of contamination of the cultures with wt virus was excluded. the only explanation for this surprising result was that the vpg(y3f) variant was quasi-infectious, presumably in that the threonine residue in position four of vpg may have served as a (poor) surrogate acceptor for uridylylation and protein priming of rna synthesis (it should be noted that genome-linked terminal proteins are often attached to serine residues; salas, 1991) . further analyses supported this hypothesis. a vpg(t4a) variant was found to be viable, expressing good growth kinetics. in contrast, vpg(f3y, t4a) variant rna never yielded progeny virus and was, therefore, considered unable to replicate its rna. this mutation then can be considered to be lethal. genetic analysis of mutant genomes and their revertants has been an invaluable tool to study the structure and function of picornavirus genetic elements and picornavirus proteins (wimmer et al., 1993) . genetic recombination of picomaviruses is the exchange of genetic elements between two viruses that may occur during replication in the same cell. discovered by hirst (1962) , and used first by cooper and his colleagues (cooper, 1977) to map poliovirus genetic units, lingering skepticism about the phenomenon was dispelled through biochemical analyses of poliovirus recombinant proteins (romanova et al., 1980; tolskaya et al., 1983) or fmdv recombinant genomes (king et al., 1982) . (for a detailed review of recombination, the reader is referred to wimmer et al., 1993.) picornavirus recombinants have been detected because (1) they acquired genetic traits from the parental strains allowing them to proliferate under conditions restricting the growth of either parent and (2) they arose in excess over (replication competent) revertants of the parental strains (cooper, 1977) . restricting conditions for the selection of recombinants can include specific drugs, such as 2 mm gua hc1, monoclonal neutralizing antibodies (emini et al., 1984) or host-cell specificity (duggal et al., 1997 ). an elegant method to study poliovirus recombination under normal growth conditions (without selection) has been developed by jarvis and kirkegaard (1992) . a wealth of experimental data has shed light on the most important steps in recombination. details of individual steps in recombination, however, remain to be elucidated. the current knowledge can be summarized as follows. 1. recombination is homologous and it occurs by copy choice; i.e. an incomplete (nascent) rna strand may switch template strands during genome replication. the probability of crossover depends strongly upon the degree of homology between the two recombining viral genomes. 2. genetic analyses have indicated that template switching occurs (predominantly?) during-strand synthesis (kirkegaard and baltimore, 1986) . 3. recombination is precise: no deletions or insertions at sites of homologous recombination have been observed. this is true even if crossover occurred in the 154 nt long noncoding region (spacer region) between ires and initiating aug of poliovirus (jarvis and kirkegaard, 1992) even though the sequence of this "spacer" is not conserved amongst the three poliovirus serotypes (toyoda et al., 1984) . indeed, deletions in the "spacer" could conceivably be tolerated in view of the observations by kuge and nomoto (1987) and others that the spacer can be partially or completely deleted without loss of viability. 4. template switching requires that the replication complex pauses, allowing a heterologous (invading) +strand to offer its service as template. an unsolved question is whether pausing and crossover is random (jarvis and kirkegaard, 1991) or non-random (romanova et al., 1986) . the latter is in all probability true. agol and his colleagues have proposed that higher order structures formed on template rnas may favor pausing of rna synthesis and crossover (romanova et al., 1986) in addition, king (1988) suggested that there are preferred sites of recombination in poliovirus rna, and that crossover may be favored immediately after synthesis of two uridylate residues (uu) in the nascent strand. duggal and wimmer (1999) observed that crossover patterns changed significantly when recombination occurred at different temperatures. specifically, crossover between two genetically marked rna strands at 34~ occurred over a wide range of the genome with preference for sequences coding for structural proteins in the 5'-terminal half of the genome. in contrast, recombination in vivo at 37~ and 40~ yielded crossover patterns that had shifted dramatically to a region encoding nonstructural proteins (duggal and wimmer, 1999) . preferential selection of recombinants at 37~ and 40~ was ruled out by analyses of the growth kinetics of the recombinants. the reason for the temperature effect is unknown. temperature-dependent stability of higher order rna structures seems possible. recombination frequencies are calculated by dividing the yield of the recombinant virus by the sum of the yield of the parental virus. for picornaviruses with linear genomes, the distance between genetic markers used to determine recombination is proportional to the recombination frequency. as mentioned above, the degree of homology between the parental genomes strongly influences the probability for crossover. the frequency of recombination between homologous genomes is remarkably high (2 x 10 -3 between markers only 600 nt apart; jarvis and kirkegaard, 1992) . it has been estimated that 10-20% of the homologous viral genomes may undergo genetic recombination within a single growth cycle (king, 1988) . this would mean an unprecedented genetic shuffling between genotypes of which the fittest retain the "wt" phenotype. experimental results that support a high frequency of recombination between sibling strands were obtained with engineered, quasiinfectious poliovirus genomes carrying two adjacent vpg sequences (cao and wimmer, 1996) . after transfection, all progeny viruses had lost the downstream vpg, most probably by homologous recombination during-strand synthesis. on studying recombination using genetically marked genomes, 90% of the recombination events occurred between sibling strands. finally, jarvis and kirkegaard (1992) have demonstrated that the frequency of recombination increases with the progression of the infectious cycle; i.e. the larger the concentration of intracellular viral rna the higher the probability of recombination. recombination in a cell-free extract of uninfected hela cells (molla et al., 1991) has recently been reported by two groups. recombination of parental viruses in the cell-free medium was detected either by rt/pcr in the absence of selection (tang et al., 1997) , or by plating the progeny virus under conditions that were restricted for either parent (duggal et al., 1997) . the recombination frequencies were found to be roughly the same as that in vivo (in tissue culture cells). the crossover pattern of recombination in vitro and in vivo at 34 ~ was the same, lending credibility to the cell-free system as reflecting an in-vivo environment (duggal and wimmer, 1999) . the in-vitro approach has the potential to decipher some basic steps in recombination, as, for example, the invasion of heterologous template strands into the replication complex. as mentioned before, the pattern of recombination changed significantly when recombination was carried out in vivo at 37 ~ or 40 ~ unfortunately, this effect at higher temperature cannot be analysed in vitro because cell-free synthesis of poliovirus is highly inefficient or completely absent at temperatures above 36~ (molla et al., 1993c) . picornavirus genomes are extremely "plastic" in that any change in their genotype can lead to unexpected nucleotide rearrangements. this has been observed, for example, in analyses of ires elements, where deletions or insertions lead to unexpected new genotypes with excellent growth properties (see, for example, dildine and semler, 1989; gmyl et al., 1993; pilipenko et al., 1992; alexander et al., 1994; charini et al., 1994; cao and wimmer, 1995) . genetic plasticity is particularly apparent when poliovirus genomes are constructed harboring foreign sequences. the analysis of genetic rearrangements and deletions is especially important when picornavirus genomes are to be used as vectors for the delivery of foreign genes. generally, polioviruses respond to the insertion of foreign sequences by rapidly deleting these sequences either partially or completely. the driving force behind the selection of deletion variants may be: (1) excessive length of the genome, restricting encapsidation; (2) interference with efficient processing of the polyprorein; (3) interference with initiation of translation; (4) alteration of rna structures necessary for replication; or others. there are examples, however, where poliovirus, or deletion mutants thereof, appear to tolerate a foreign sequence inserted into the genome. an interesting approach to studying ires elements was the insertion of the emcv ires into the orf of poliovirus, thereby making unnecessary the primary cleavage between pi*p2 catalyzed normally by 2a pr~ (figure 12 .6b; molla et al., 1992) . the resulting rna transcripts proved highly infectious (molla et al., 1992) . although this dicistronic virus expressed a small plaque phenotype, neither the plaque size nor the genotype surrounding the insertion changed over six passages. these observations suggested that the insertion was stable, at least under the conditions studied. the emcv ires has no apparent sequence homology with any sequence of the poliovirus genome, the poliovirus ires included. this eliminated the possibility that the emcv ires was rapidly removed by homologous recombination. perhaps, illegitimate recombination (see below) to delete the ires without debilitating the virus was a very rare event and not apparent in progeny virus. it should be noted in parenthesis that the viability of the dicistronic virus shown in figure 12 .6b proved for the first time the function of the emcv ires as a true internal ribosomal entry site (molla et al., 1992) . on the basis of these experiments, a specific version of novel expression vectors was constructed ( figure 12 .6c,d) that included, in addition to the foreign ires, a foreign orf lu et al., 1995a) . none of these vectors was genetically stable over extended numbers of passages, and in some cases the deletion occurred during first passage (lu et al., 1995a) . nevertheless, the insertion of a foreign orf between two ireses in the 5'ntr, the foreign gene (dark stippled box) was inserted upstream of 2a pr~ which now delivers the foreign protein by a cis cleavage. d. dicistronic poliovirus generated by inserting a foreign gene and the emcv ires into the 5'ntr. in this case the foreign gene is synthesized independently from the polyprotein. e. generation of an expression vector by fusing the coding sequence of a foreign gene to the n-terminus of the poliovirus polyprotein. the foreign gene product is liberated through trans cleavage, by either 3dpr~ pr~ or 2a pr~ f. expression vector based on mengovirus, a cardiovirus that carries a small leader sequence preceding the p1 region of the polyprotein (see figure 12 .9). in this case, the foreign gene is inserted into the l coding sequence. note that the organization of the genomes in e and f is identical. g. encapsidation-incompetent poliovirus expression vector in which a portion of the p1 coding sequence has been replaced by a foreign gene. this genome can be encapsidated in trans but, by itself, it can only go through one cellular cycle of replication. as shown in figure 12 .6d, yielded a replicating poliovirus vector that efficiently expressed the cat gene over several passages . no deletion was apparent after the first passage. remarkably, the genome of this construct is 17% larger than that of the wt genome, an observation indicating that the capsid of naturally occurring polioviruses is not "full". however, attempts failed to encapsidate and express the larger luciferase gene instead of the cat gene in the context of the dicistronic virus. the luciferase activity was clearly detectable in cells transfected with the appropriate dicistronic transcript, but the genome harboring luciferase was not encapsidated . apparently, an increase of genome length to 31% (luciferase gene plus emcv ires) was not tolerable for encapsidation . a different strategy to convert poliovirus to an expression vector was the fusion of a foreign orf directly to the poliovirus polyprotein (figure 12.6e; andino et al., 1994) . in these experiments, the strategy of altmeyer et al. (1994) was mirrored, which made use of the genetic make-up of cardioviruses (mengovirus or emcv). specifically, the cardiovirus polyprotein is preceded by a small leader protein (67 aa) that is cleaved from the capsid region p1 by the viral 3c pr~ proteinase, thereby allowing maturation and encapsidation of the virion. altmeyer et al. (1994) inserted into the leader sequence of the mengovirus genome a foreign gene ( figure 12 .6f) and expressed the product of this fusion protein over several passages in tissue culture (altmeyer et al., 1994 (altmeyer et al., , 1995 . andino et al., (1994) generated a similar "leader" protein in front of the poliovirus polyprotein. in this case, however, it was necessary to engineer a novel 3c/3cd pr~ cleavage site between the foreign orf (the new "leader") and the viral polyprotein such that the foreign polypeptide can be cleaved from the poliovirus capsid precursor (figure 12 .6e). although these poliovirus constructs were originally claimed to express excellent growth properties and, more importantly, were reported to be genetically highly stable (andino et al., 1994) , the poliovirus-based vectors proved, in fact, impaired in replication and prone to rapid deletions, at least if the insert was more than 500 nt in size (mueller and wimmer, 1998 , and references therein; see below). it should be noted that the cardiovirus-based vectors also suffered from loss of the inserts of a foreign gene upon repeated passage, an observation suggesting that even cardioviruses do not tolerate an extended leader protein for the purpose of gene therapy (altmeyer et al., 1994 (altmeyer et al., , 1995 . in a third strategy of the construction of picornavirus vectors, the p1 capsid region of the picornavirus genome is partially replaced with a foreign orf (figure 12 .6g), yielding proliferation-incompetent replicons that appear to be genetically quite stable (see, for example , porter et al., 1993) . for a possible application as vectors in gene therapy, the replicons are trans-encapsidated via a vaccinia virus-based p1 expression vector, with relatively low yields of proliferation-incompetent virions. the apparent genetic stability of these replicons may be due to the fact that the rnas are similar in size when compared to the wt genome, and that the naturally occurring cis cleavage (between pi*p2) catalyzed by 2a pr~ is highly efficient, placing no restriction on this step of polyprotein processing. however, the rapid selection of faster growing variants that lost the foreign gene is unlikely, since the trans-encapsidated replicons can only proceed to a one-step infectious cycle. this is very different from the selection pressure in proliferation-competent vectors, which engage in second-round infections. what is the mechanism by which poliovirus may eliminate foreign sequences? homologous recombination cannot function because there is not enough sequence homology to engage in crossover. pilipenko et al. (1995) have nevertheless proposed that short sequences may serve as parting and anchoring sites for template switching in illegitimate (non-homologous) recombination. an alternative mechanism is "loop-out" deletion, in which the nascent strand skips endogenous sequences, jumping to an upstream sequence that serves as anchoring sequence. given the high frequency by which recombination occurs among sibling strands, a crossover mechanism may be favored, but decisive experiments to decide between these two mechanisms are lacking. in any case, a detailed study of genetic variations of polyprotein fusion vectors (figure 12 .6d) strongly supports the model of parting and anchoring sites for template switching or loop-out deletion (mueller and wimmer, 1997; see below) . briefly, when expression vectors ( figure 12 .6e) consisting of a gag gene (encoding p17-p24; 1161 nt) of human immunodeficiency virus that was fused to the n-terminus of the poliovirus polyprotein (andino et al., 1994; mueller and wimmer, 1998) were analysed after transfection into hela cells, the genomes were not only found to be severely impaired in viral replication but they were also genetically unstable (mueller and wimmer, 1997) . upon replication, the inserted sequences were rapidly deleted as early as the first growth cycle in hela cells. interestingly, the vector viruses did not readily revert to wt sequences but rather retained some of the insert plus the artificial 3c/3cd pr~ cleavage site (to allow processing at the n-terminus of the polyprotein). thus, variants of different genotypes that replicated nearly as well as wt poliovirus had followed an evolutionary pathway towards the genetic organization of cardioviruses (mueller and wimmer, 1998) . that is, the poliovirus polyprotein of these variants was preceded by gag-derived "leader" proteins of different but distinct sizes (predominantly between 20 and 50 aa long), the most prominent leader size reflecting the length of that in cardioviruses (67 aa). in the immediate vicinity of the deletion borders of several isolates, short direct sequence repeats were observed that are likely to allow alignment of rna strands for non-homologous (illegitimate) recombination during-strand synthesis (figure 12.7; mueller and wimmer, 1998) . interestingly, the selection of the leader size occurred during the very first rounds of replication of the transfected rna; in most cases, as sequential shortening of the leader sequence was not observed. defective interfering particles an interesting phenomenon of naturally occurring deletion mutants of picornaviruses are defective interfering particles (di particles) that can be (rarely) discovered in laboratory stocks of virus or generated (with difficulty) by passage of virus at high multiplicities (reviewed in wimmer et al., 1993) . all naturally occurring di particles carry deletions in the p1 capsid precursor region (cole et al., 1971; cole and baltimore, 1973; lundquist et al., 1979; nomoto et al., 1979; kajigaya et al., 1985; kuge et al., 1986) . as mentioned before, nomoto and his colleagues have found that the deletions in all di particles are in-frame (kuge et al., 1986) . since genetically engineered di genomes (replicons) with an out-of-frame deletion in the p1 region are unable to replicate, nomoto and his colleagues (kuge et al., 1986; hagino-yamagushi and nomoto, 1989) correctly concluded that poliovirus rna replication requires, at some stage of the replicative cycle, translation of the replicating rna (cis requirement of translation), and that the di particles with out-of-frame deletion cannot be complemented in trans. novak and kirkegaard (1994) confirmed this hypothesis in that replicons with translation termination codons downstream ofthe p1 coding region could not be rescued in trans. for hypotheses to explain this phenomenon, see above. chetverin et al. (1997) have recently made the startling observation that certain rna fragments can join to one another via a molecular pathway determined by the intrinsic chemical properties of the rna molecules (chetverin et al., 1997) . the fragments that formed chemically stable duplexes were selected by the replicase of phage q[3: only those dimers that had acquired signals from two different rna molecules were able to replicate. gmyl et al. (1999) have now reported that viable recombinants could also be generated from non-replicating and non-translatable segments of the poliovirus genome. these fragments by themselves were unable to generate the viral rna-dependent rna polymerase necessary for a replication-dependent recombination event. the "crossovers" were targeted to the highly variable segment of 154 nucleotides located within the 5'ntr upstream of the initiating aug codon for the polyprotein. a great number of recombinants have been obtained by transfection of mixtures of rna fragments. analyses of viruses that evolved after the mix (+) figure 12.7 two models of illegitimate recombination during -rna synthesis as was observed with an expression vector shown in figure 12 .6e (mueller and wimmer, 1998) . both models require a partial dissociation of the nascent-rna from the template +rna, caused presumably by pausing of the rna polymerase. the free 3' end of the nascent-rna can re-anneal to a short complementary sequence further upstream on the same template strand, thereby looping out the intervening sequence (a), or it can re-anneal to the same complementary sequence but on a sibling +strand, and complete synthesis on this second template (b; strand switching). in both cases, the resulting strands would have excised the same sequence and could now, in turn, give rise to truncated +rna genomes. note that this deletion event can occur even during the first round of replication of the expression vector leading to partial or complete deletion of the foreign coding sequences. reproduced, with permission, from mueller and wimmer, 1998. ture of the rna species strongly suggested that the connection between two fragments was the result of chemical reactions between the fragments rather than of template-switching (gmyl et al., 1999) . the mechanism by which the chemical linkage between two fragments is formed is obscure but it could involve structures reminiscent of ribozyme-like activities in the viral rnas. nevertheless, this observation could have pro-found implications for the generation of novel genotypes in nature (gmyl et al., 1999) . genetic complementation is the compensatory action of gene products of two homologous genetic systems to alleviate defects of mutant genes. genetic complementation has been firmly established in picornavirus replication. however, because of the complexity of diverse function(s) of precursor proteins and their cleavage products, it has not been possible to define complementation groups (wimmer et al., 1993) . complementation groups are indicative of genetic elements that can function independently, and they have been the basis of the definition of cistrons (benzer, 1957) . a cistron, therefore, may be equated with a gene, i.e. a functional unit of genetic material specifying a single protein. on the basis of these definitions, the picornavirus genome, encoding only the polyprotein whose products function in many cases in overlapping or even opposing fashions, cannot be called multicistronic. in general genetics, mutations affecting the same polypeptide can occasionally complement each other, a phenomenon referred to as intracistronic complementation (schlesinger and levinthal, 1963) . based on these considerations, we have suggested that the picornavirus genome be considered "monocistronic" (wimmer et al., 1993) . it follows that the genome encodes only one gene product, the polyprotein. the polyprotein, in turn, contains multiple genetic units whose products may or may not be capable of intracistronic complementation. if this definition is accepted, one should avoid referring to individual coding regions of the picornavirus genome as "genes". thus, there would be no "3d p~ gene". this convention makes good sense if one considers that a "gene for 3d p~ is for the most part also the gene for 3cd pr~ a proteinase with properties unrelated to the polymerase 3d p~ it should be noted that theiler's virus is an exception to the monocistronic nature of picornaviruses in that it encodes a small protein in a separate reading frame, mapping towards the n-terminus of the polyprotein . it is interesting to consider that there is no absolute requirement that picornaviruses must exist as monocistronic (single-polyprotein-producing) entities. for example, the insertion of a second ires into the genome would represent a viable dicistronic virus (figure 12.6b) , an entity artificially generated by molla et al. (1992, 1993b) . apparently, during the evolution of picornaviruses, the elimination of genetic elements regulating the expression of different picornavirus proteins was favored over retaining them. similarly, there was no pressure to generate such regulatory sequences and insert them into the genome. in other words, proteolytic processing of a single polyprotein evolved not only to be highly efficient but also as a means to regulate the temporal appearance of viral proteins (e.g. precursor proteins versus end-products of proteolytic cleavage). in contrast, in prokaryotic rna phages or in-strand rna viruses, the expression of proteins is regulated by sequence elements located between different cistrons. interestingly, the dicistronic poliovirus depicted in figure 12 .6b resembles the genetic composition of cow pea mosaic virus (cpmv), a plant virus (hellen et al., 1989; molla et al., 1992) . indeed, cpmv and the dicistronic poliovirus shown in figure 12 .6b have similar gene order and amino acid sequences, the main difference being that the genome of cpmv is bipartite. that is, rather than inserting a sequence such as an ires into the genome, cpmv preferred to divide the genome into two portions, one coding for the capsid proteins the other for the replication proteins. such a genetic arrangement, which requires two particles to initiate a complete infectious cycle, may be suitable for a plant virus (where the yield of virus per host can be extremely high and, equally importantly, the local concentration of host organisms can be high) but it would be highly disadvantageous for an animal virus. the fascinating topic of the structure and evolution of polyproteins within the rna-like virus superfamily is discussed later. evidence for genetic complementation in vivo has existed for decades, the best-known involving guanidine-generated mutants. complementation of guanidine mutants seemed unidirecional (wimmer et al., 1993) . bernstein et al. (1986) provided the first conclusive evidence for symmetric complementation, using mutants that were generated in 2a pr~ and 3a. these authors, however, also made the unexpected observation that mutants mapping to 2b or 3d p~ (which they had also generated by genetic engineering) could not be complemented (bernstein et al., 1986) . on the other hand, charini et al. (1991) clearly showed that mutations in 3d p~ mapping to a different region of the coding sequence could be rescued in trans. this example and many others (wimmer et al., 1993) support the notion that the polyprotein is a single genetic unit that does not consist of non-overlapping genes whose functions can be separated by complementation grouping. a special case of complementation was tested using dicistronic polioviruses. briefly, cao and wimmer (1995) constructed a virus with a genotype shown in figure 12 .6b, the extra cistron being the coding region for poliovirus 3ab. the dicistronic construct yielded a virus expressing a small plaque phenotype but it was genetically unstable, losing its inserts after several passages. nevertheless, if the lethal mutation vpg(y3f, t4a) was engineered into the second cistron of the dicistronic virus (see above), the first cistron (3ab) could rescue the genome, albeit inefficiently. a much more efficient rescue of lesions in the 3ab coding sequence was reported by towner et al. (1998) , who used the cell-free system of poliovirus replication developed by molla et al. (1991) . apparently, the supply in trans of p3 polypeptides in vitro is more efficient that in vivo, a phenomenon that remains as yet unexplained. interestingly, it appeared as if a mutation in 3ab could be rescued only if the complementing polypeptide was a precursor of 3ab, preferably p3 (see figure 12 .4). perhaps, efficient complex formation between 3ab/3cd pr~ (see above) in this system depends on the cleavage of the p3 precursor in situ. picornaviridae combines species that infect animals with exceedingly varied pathogenic features affecting almost every organ system (table 12 .1). in the following, we will concentrate only on human picornavirus infections, which range in severity from protean symptoms associated with the common cold (e.g. rhinoviruses), mild gastroenteritis (e.g. echoviruses), hepatitis (e.g. hepatitis a virus), to fatal cns manifestations (e.g. pv, enterovirus 71) or lethal myocarditis (coxsackievirus b group). despite the enormous variety in organ tropis m observed with different species of the picornavirus group, pathogenic features of every single species are recognized in the form of highly distinct disease syndromes (exceptionally, coxsackieviruses can cause fatal disseminated infections in neonates with widespread viral propagation in multiple organs). .we will define pathogenic properties of picornaviruses as a combination of different viral traits: (1) those that affect tropism (determining the target cell type of, and influencing spread in, the host); (2) those that affect virulence (determining kinetics of particle propagation); and (3) those determining the progression of a disease syndrome ("pathogenicity proper", the propensity to cause clinical symptoms). there is a fourth parameter, which is strictly related to a condition of the host. an example is injury-provoked ("provocation") poliomyelitis, which will also be discussed below. surprisingly, the disparity in pathogenic properties may be contrasted with a high degree of sequence conservation among certain groups of picornaviruses. this is most evident with the cluster c enteroviruses (figure 12 .1b). for example, on the basis of sequences of the 3d p~ poliovirus serotype 2 (lansing) (pv2(l)) shares more than 90% sequence homology with its close relative coxsackievirus a24 (cav24). indeed, their sequence similarity exceeds that between pv2(l) and pv1 (mahoney) or pv3 (leon) (figure 12.1b) . yet, whereas all pv serotypes are associated with poliomyelitis, a severe and frequently fatal infection of the cns, cav24 causes mild upper respiratory tract infections only. since minor sequence variations of picornaviruses can account for drastically different disease syndromes it may be assumed that the pathogenic phenotype of picornaviruses is encrypted within a few crucial genetic determinants. these basic determinants of pathogenic features appear to be dynamic, leading occasionally to the emergence of novel virus variants causing clinical syndromes not previously observed with their ancestors. this was the case when widespread epidemics of acute hemorrhagic conjunctivitis ravaged africa and the pacific rim (yin-murphy, 1973) and quickly expanded worldwide. two picornaviruses were associated with this previously unknown clinical syndrome, coxsackievirus a24 variant (cav24v) and enterovirus 70 (ev70; table 12 .1). the former evolved from its ancestral cav24, causing mild upper respiratory tract infections, whereas ev70 was primarily recognized for its association with a poliomyelitis-like neurological disorder (melnick et al., 1974) . the deviation of tropism toward ocular tissues resulting in acute hemorrhagic conjunctivitis suggests a switch or an expansion in receptor specificity. this hypothesis, however, awaits confirmation, since the cellular receptor(s) of ev70 is unknown ( the circumstances and conditions that may favor a switch in host cell tropism with resulting changes in the pathogenic phenotype are unknown. a detailed discussion of our current view of the evolution of enteroviruses, however, is presented in the following section on evolution. this is particularly relevant in the context of the imminent global eradication of poliovirus. it is known that coxsackieviruses a7 and a9 (cav7, cav9) as well as enteroviruses 70 and 71 (ev71) occasionally cause a clinical syndrome with striking resemblance to poliomyelitis (melnick et a!., 1974) . occurrence of poliomyelitis caused by these virus species has only rarely been reported in epidemic proportions (voroshilova and chumakov, 1959; melnick et al., 1980) ; generally they occur as isolated incidents. fortunately, preliminary evidence (da silva et al., 1996) suggests that to date no surge in non-pv-caused poliomyelitis has occurred in response to the eradication of poliovirus in latin america. however, the time elapsed since the eradication of wt polioviruses in the western hemisphere is too short in terms of evolution to conclude that the incidence of non-pv-caused poliomyelitis and poliovirus eradication are unrelated (see section on evolution). the observation of diverse specific clinical syndromes caused by closely related picornaviruses (particularly enteroviruses) has sparked interest amongst virologists in identifying those factors that may determine the clinical outcome of picornaviral infections. generally, signals for pathogenic phenotypes can be found in all parts of the viral genome. however, factors that determine cell and tissue tropism are not necessarily the same as factors determining virulence or attenuation. for example, the capsid mutations of the live attenuated strains of pv (the sabin strains) have an attenuating effect without altering the tropism of the sabin strains for the prime target of pv: spinal anterior horn motor neurons. a different effect of capsid proteins on an extended host tropism has been reported for several poliovirus type 2 strains, e.g. pv2(l). a small segment in capsid protein vp1 of pv2(l) (the b-c loop) has been identified as carrying determinants of host range extension from primates to rodents (murray et al., 1988) . however, whereas pv2(l) infection causes poliomyelitis in primates and in cd155 tg mice, normal mice developed histopathology indicative of panencephalomyelitis, which was radically distinct from poliomyelitis observed in primates and cd155 tg mice (gromeier et al., 1995) . this observation suggests pv2(l) tropism toward a cell type in mice that is not targeted in primates. it is likely that the mouse-adapted pv2(l) acquired additional receptor specificity but the nature of the receptor for pv2(l) in normal mice is unknown. it should be noted that although pv2(l) causes disease in mice after intracerebral injection, cultivated mouse l cells cannot be infected with this strain (gromeier et al., 1995) . the fact that single determinants of pathogenicity (e.g. the pv capsid) can carry signals that influence either tropism (e.g. pv2(l)) and/or virulence (sabin strains of poliovirus) indicates different dimensions of picornaviral pathogenesis. taking multiple determinants of tissue tropism and virulence (shared between the capsid, non-structural viral proteins and non-coding sequences) into account, the enormous complexity of the molecular basis of picornaviral pathogenesis comes into perspective. capsid protein structure determines the interaction of a virus with its cellular receptor. as pointed out for the poliovirus sabin strains and pv2(l), small differences in the structure of viral capsids are critical for cell and organ tropism as they ultimately determine the pathognomic features of the resulting infection. similarly, the capsid was determined to harbor sequences critical for cardiotropism (tracy et al., 1995; cameron-wilson et al., 1998) as well as diabetogenicity (kang et al., 1994) of coxsackie b viruses (cbv). moreover, diabetogenicity of emcv in mice mapped to the capsid (jun et al., 1998) . as pointed out, the mechanism by which the changes in the capsid may affect pathogenesis may be related to differences in the interaction between virus and receptor. apart from direct receptor switching (or extending receptor specificity to more than one cell surface molecule), virus capsid alterations may affect the kinetics of virus/receptor binding or particle stability. this would influence the virulence of that virus without a concurrent change in host cell tropism. reduced particle integrity has been proposed to participate in the attenuation phenotype of the sabin strains of poliovirus (filman et al., 1989) . however, theories linking capsid mutations within the sabin strains with structural elements important for protomer cohesion and capsid integrity remain inconclusive. in contrast to a likely relationship between capsid structure and pathogenic phenotype, the role of non-structural viral gene products in the determination of disease has been less obvious. non-structural viral proteins are cell-internal and, hence, do not influence tropism in a strict sense. cell-type-specific restrictions in viral replication regulated by non-coding regions of the viral proteins or by non-structural proteins will not change the spectrum of target cells infected but may critically influence virulence. viruses normally evolve to adapt to host cells offering adequate portal of entries (receptors), thereby exposing the viral particles to an intracellular milieu supportive of particle propagation. thus, favorable cell-internal conditions for viral replication would ideally be matched by a suitable viral receptor to avoid virus entry into cells that do not permit replication. for most viruses invading a host organism, the match is not perfect and, hence, the viruses are restricted to replication in fewer cells or organs than the distribution of receptor molecules would suggest. cell-internal determinants mapping to the viral genome of various picornaviruses have been suggested to influence virulence. these can be divided into loci mapping to viral proteins or to non-coding regions (e.g. the 5'ntr). for example, mutations within the coding region for the rna-dependent rna polymerase 3d p~ of the pv1 sabin vaccine have been implicated in the attenuation phenotype (toyoda et al., 1987; tardy-panit et al., 1993) . it was found that these mutations contributed to the ts phenotype of this sabin vaccine strain (toyoda et al., 1987) . mutations in the p2 region of hepatitis a virus have been correlated with an attenuated phenotype of hav (raychaudhuri et al., 1998) . in many instances the genetic loci of pathogenesis mapping to viral non-structural proteins have been identified through sequence comparison. this approach, of course, did not reveal mechanisms to account for reduced virulence. the non-coding genetic elements of picornaviruses have also been shown to carry signals determining virulence (evans et al., 1985; duke et al., 1990; gromeier et al., 1996; tracy et al., 1996) . as has been discussed in the section on genetics, the 5'ntr harbors the internal ribosomal entry site (ires) that, on the basis of the early observation by evans et al. (1985) with the sabin type 3 vaccine strain, has been identified as a major determinant of virulence for a number of picornaviruses. sequence comparison of the sabin strains of poliovirus with their wt progenitors revealed point mutations within a confined region of the 5'ntr in all three serotypes, known as domain v (figure 12 .3; reviewed in wimmer et al., 1993) . in analyses of viral strains recovered from patients who acquired paralytic polio after vaccination, a point mutation at position 472 (direct reversion in domain v) of the ires of pv3(sabin) was proposed to contribute to the neurovirulence of the isolate (evans et al., 1985) . recent analyses led to a different hypothesis stressing a co-operative attenuating effect of capsid mutations with mutations in the ires and other locations in the sabin vaccine genomes (mcgoldrick et al., 1995) . the mechanism responsible for ires-mediated attenuation of neurovirulence remains obscure. analyses of cell-type-specific growth restrictions in cell lines of neuronal origin and biochemical studies of cell-type-specific ires function suggested impairment of initiation of translation in a cell-type-specific manner (haller et al., 1996; agol et al., 1989; la monica and racaniello, 1989) . following the example of poliovirus, ires elements or surrounding sequences of a large number of picornaviruses were found to contain genetic markers with a role in the determination of a pathogenic phenotype. this was most impressively demonstrated by the drastic attenuating effect of a deletion within the poly(c) tract of the 5'ntr of emcv (duke et al., 1990) . how do the multiple mechanisms alluded to interlace to produce a specific picornavirus disease syndrome? the intricacies of dual cell external and internal determinants of viral pathogenic features are best illustrated using the example of poliovirus. this most thoroughly studied prototype picornavirus is characterized by pathogenic properties of specificity untypical of viral pathogens of the cns. poliovirus host range is limited to primates only. within the primate organism poliovirus replicates within an unknown site in the gastrointestinal tract and within associated lymphatic structures, leading to viremia (bodian, 1972) . at this stage, the virus causes hardly any disease symptoms. however, viremia may, in only about 1% of infections, lead to cns invasion, presumably through passive passage of the blood-brain barrier (yang et al., 1997) . on muscle injury, the virus may also reach the cns via retrograde axonal transport wimmer, 1998, 1999) . within the cns, poliovirus uniquely targets spinal cord anterior horn motor neurons. lytic destruction of anterior horn motor neurons results in flaccid paralysis, the hallmark clinical sign of paralytic poliomyelitis (bodian, 1972) . whereas efficient poliovirus proliferation occurring in the human gastrointestinal tract produces few or no symptoms, this virus's pathological potential is expressed in a small and relatively inaccessible subpopulation of neurons in the cns. this peculiar restriction has been the subject of research interest for many years. available evidence clearly suggests that the restriction of the host range of poliovirus to primates is determined predominantly by the receptor. in humans, this receptor is the immunoglobulin superfamily molecule cd155 (mendelsohn et al., 1989) and two proteins closely related to cd155 function as poliovirus receptor in simians (koike et al., 1990) . clearly, the observed organ and cell tropism are co-determined by the virus's dependence on the cellular receptor. we believe that, at least in part, the expression of cd155 must also determine the highly restrictive cell tropism of poliovirus within the cns, because mice transgenic for the cd155 gene develop a neurological condition with pathologic and clinical features identical to those observed in primates (ren et al., 1990; koike et al., 1991; gromeier et al., 1996) . furthermore, support for this hypothesis comes from studies of transcriptional control (solecki et al., 1999) and developmental expression of the cd155 gene (gromeier et al., 1999b) . it has been reported that cd155 expression is restricted to structures in close anatomical and functional relationship with spinal cord anterior horn neurons during embryonic development of the cns (gromeier et al., 1999b) . it is thus likely that the restrictive expression pattern of cd155 may indeed direct poliovirus tropism toward a specific cellular compartment of the cns. analyses of pathogenesis related to genetic determinants mapping to the capsid, proteins, ires, or 3d p~ have recently been extended using poliovirus hybrid viruses. specifically, polioviruses have been constructed in which the cognate ires was replaced by that of other picornaviruses (gromeier et al., 1996) . by exchanging the cognate ires element of pv by that of other picornavirus species, it could be shown that neuropathogenicity of pv can be eliminated without affecting growth properties in non-neuronal cell types normally susceptible to poliovirus (gromeier et al., 1996) . thus, it was determined that the ires of rhinovirus type 2, a virus species never associated with neurological disease, confers the attenuation phenotype to poliovirus. significantly, this chimeric virus, called pvi(ripo), did not carry any attenuating mutations in the poliovirus-specific sequence of its genome. the neuropathogenic potential of a picornavirus ires cannot be predicted but it is innate, of course, in its sequence. certainly, ires elements of enteroviruses known to cause poliomyelitis (cav7, cav9, ev70, and ev71) are candidates for "neurovirulent ireses" and, indeed, a corresponding pv/cav chimera has proved this hypothesis (gromeier et al., unpublished results) . c-cluster enteroviruses, on the other hand, never cause poliomyelitis. however, because of their close genetic kinship, the ireses of c-cluster coxsackieviruses confer a highly neurovirulent phenotype to the pv/cav chimeras (gromeier et al., unpublished) .. finally, ires elements of the genus rhinovirus ablate neuropathogenesis in poliovirus chimeric viruses (gromeier et al., 1996) . these observations indicate that, indeed, cellexternal restriction in cell tropism as well as cellinternal factors exert powerful limitations toward enterovirus pathogenesis. many relatives of poliovirus of the enterovirus genus (particularly the c cluster; table 12 .1) presumably would equal the neuropathogenic properties of pv if their capsid structure allowed interaction with neuronal cells. thus, non-neurovirulent ccluster enteroviruses with high sequence homology to pv and "neurovirulent" ires elements may gain tropism for neurons in the future (see under evolution). in addition to virus-encoded factors of pv neuropathogenicity, the circumstances within the host organism at the time of infection or shortly thereafter may influence the outcome of poliovirus infection. trivial muscle injury has been shown to increase the probability of neurological complications of concurrent poliovirus infection (mccloskey, 1950) . a proposed pathogenic mechanism for provocation polio identified a deviation of the route of cns invasion toward retrograde axonal transport to account for the increased risk of polio among individuals who received intramuscular injections wimmer, 1998, 1999) . picornaviruses have adapted to a wide variety of cellular components of their hosts. the diverse spectrum of disease syndromes associated with human picornaviruses provides an excellent field of study to examine the factors that determine the clinical outcome of a viral infection. the enormous amount of sequence information, combined with a broad knowledge of the molecular biology of many of these agents, have sparked hopes of a rapid elucidation of the molecular basis for their pathogenic properties. initial optimism that sequence comparison of virulent strains with their attenuated variants alone would rapidly identify those elements responsible for a pathogenic phenotype and unravel mechanisms of pathogenesis is, however, unjustified. this is particularly true for poliovirus, the most thoroughly studied picornavirus. progress in the analysis of poliovirus neuropathogenicity has revealed that the interactions of poliovirus with the host are characterized by a degree of complexity not previously appreciated. mechanistic concepts of viral pathogenesis, combined with one-dimensional views of virus replication and its relation to the host organism, have helped little in increasing our understanding of the selective susceptibility to poliovirus of motor neurons. viral infections result in complex clinical syndromes that are difficult to explain in terms of single viral genetic elements. using poliovirus as an example, picornavirus-induced disease is the result of an intricate interplay of numerous factors, of both viral and host origin, that coordinately affect the ability of the virus to propagate in any particular cell type or organ. numerous investigations, particularly those of j.j. holland, e. domingo and their colleagues (see chapter 7), have led to the realization that the rapid evolution of rna viruses results from high mutation rates combined with exceedingly large heterogenic populations. this is true for picornaviridae that encode variants of conserved protein folds as well as catalytic systems not found in the cellular world and, hence, have explored an enormous evolutionary space. it is less appreciated, although equally true, that it is the host environment that has provided new opportunities for viruses to proliferate and select new variants out of a huge number of mutants. without this "co-operation" between host and parasites, picornavirus evolution would not have resulted in the tremendous diversity of genotypes whose number, now counting in the hundreds, is currently biased towards those infecting mammals. the key role of virus-host interaction is evident in the evolution of rna viruses. indeed, the relatively slow pace by which the cellular environment is changing imposes severe restrictions on the mode of rna virus evolution. a model has been proposed suggesting that, in each moment of protein evolution, mutations could only be accepted in a very limited number of positions of a polypeptide chain; otherwise, protein structure and function would have been severely compromised. these limited places of acceptable variation have been called covarions (fitch, 1971) . it has been argued that the fast evolution of rna viruses in a constrained environment has to proceed through the exploitation of constantly emerging but vastly overlapping covarions . this model of virus evolution provides a sensible hypothesis as to how picornaviral proteins have managed to accept a heavy load of mutations that are quite frequently unique and rarely seen at sites in cellular proteins, while still not entirely losing some discernible similarity with other viral and cellular homologs. this has important practical consequences, since structural similarities can be used to reconstruct the evolution of rna viruses, an undertaking impossible just 20 years ago. apart from the biological properties generally associated with all rna viruses, the current end-product of evolution makes each virus species unique. this is engraved in the virus' genetic plan: the organization of the genome and the mode of gene expression. during the past years we have learned that +strand rna viruses employ variations of surprisingly few basic genetic plans. the genetic organization and genetic expression of picornaviruses has been outlined in detail above. in the following, the genetic organization of different picornaviruses will be put into an evolutionary perspective. in addition, we will discuss in a rather speculative manner some hypothetical implications of evolutionary consequences of the eradication of poliovirus. numerous rna viruses have been combined into a picorna-like supergroup of which the picornaviridae comprise a rather compact domain (figure 12 .8). the viruses of the picorna-like supergroup, a taxon not yet recognized as higher-than-family rank, share a conserved array of replicative proteins (see below). they infect plants, insects, birds and mammals. most of the established members of picornaviridae are mammalian viruses, and they fall principally into six genera (table 12 .1). the newly characterized avian encephalomyelitis virus (aev), which is a member of picornaviridae, is most closely related to hepatitis a virus (marvil et al., 1999) . the latest revision of the classification of picornaviruses, although closely related to the original version, has a clear evolutionary flavor since it tends to combine viruses in accord with phylogenetic kinship rather than relying on phenotypic properties (see the introduction). the largest number of picornaviruses fall into the most closely related genera, enterovirus and rhinovirus (table 12 .1). cardiovirus and aphthovirus comprise two other genera that have most probably emerged from a common ancestor. it appears that the two pairs of picornavirus genera diverged after hepato-and parechoviruses split from the main trunk of the picornavirus tree (figures 12.1, 12.8) . the exact phylogenetic interrelationship between hepatoand parechoviruses remains somewhat uncertain and may be different from that shown in figure 12 .1 (see also below). in addition, three other picornaviruses, equine rhinovirus types 1 and 2 (erv1 and 2 respectively) and aichi virus (aiv), remain to be classified. erv1 was shown to be a distant relative of fmdv (wutz et al., 1996) , while erv2 (li et al., 1996; wutz et al., 1996) and aiv (yamashita et al., 1998) appear to be related to the cardio-and cardio/aphthovirus branches respectively. they are not recognized, however, as cardio-or aphthoviruses. in addition to mammalian viruses, a number of insect viruses have been previously included into the picornaviridae on the basis of phenotypic criteria. during the last 2 years, however, the complete genome structure of six picornalike insect viruses has been reported (van der wilk et al., 1997; isawa et al., 1998; johnson and christian, 1998; moon et al., 1998; sasaki et al., 1998) . some of these viruses feature different genome organizations, and only infectious flacherie virus of silkworm (infv; isawa et al., 1998) was shown to possess a genotype and gene organization that may justify placing it in the picornaviridae (a.e. gorbalenya, unpublished results) . it is likely that an ancestor of infv separated from the main branch of picornaviridae before the radiation of mammalian viruses (figure 12.8) . the rapid accumulation of new virus genotypes has not been matched by an understanding of its evolutionary meaning. therefore, the basis of picornavirus classification may need to be revisited. moreover, the relationship between picornaviridae and other genetic systems may have to be defined within a new classification. although confusing for virologists on first sight, any reclassification into hierarchically organized taxa will ultimately aid our understanding of evolution, host range of viruses and pathogenesis. it should be kept in mind, however, that any classification is, at best, an approximation of true phylogenetic relationships, and the current classification of picornaviridae should be treated as such. picornaviridae have evolved by speciation from a common ancestor. this plausible statement has been supported by computer analyses of nucleotide and protein sequences as well as by studies of the tertiary structure of capsid proteins and 3c pr~ proteinases. there is every reason to believe that the putative ancestral viral entity had a genetic organization that has been conserved largely in contemporary picornaviruses. its signature is a long 5'ntr-long open reading frame-3'ntr-poly(a) (figure 12 .2). with the sole exception of a strain of theiler's virus (tmev; see below), all picornavirus proteins are generated by autocatalytic processing of the gigantic polyprotein (figure 12 .3). the backbone of the polyprotein is formed by a set of polypeptides conserved in all known picornaviruses. in addition, the backbone may be decorated with a few optional proteins unique to a particular virus or virus group. among the proteins, the conservation increases in the order (figures 12.4, 12 .9): this order can be deduced from analyses of virus groups belonging to different phylogenetic ranks-clusters of closely related viruses, of distinct genera, of an entire family, or of the entire picorna-like supergroup (figure 12 .10; a.e. gorbalenya, unpublished results) . slight deviations can only be seen upon analysis of some small taxonomic groups. as already noted, tmev encodes a small, unique l* polypeptide outside the main reading frame at a locus overlapping the l reading frame. additionally, the insect picornavirus infv was predicted to encode unique domains as part of the polyprotein (isawa et al., 1998; a.e. gorbalenya, unpublished results) . a closer look at these proteins reveals the following. the l polypeptide preceding the capsid coding region (figure 12 .9) is encoded by all picornaviruses except the entero-and rhinoviruses. the l protein is the most variable of all picornavirus proteins, and it exists in five different versions. l proteins with proteinase activity are encoded only by fmdv, erv1 and erv2 (skern, 1998) . cardioviruses and aiv encode three different versions of l polypeptides containing a putative zn finger a.e. f i g u r e 12.9 primary cleavage in picornavirus polyproteins. open boxes at the left end depict l proteins, of which only that of aphthoviruses is a proteinase. of the 2a coding sequences, only 2a pr~ of entero-and rhinoviruses is a proteinase. in cardio-and aphthoviruses, processing at the c-terminus of 2a is strictly a cis cleavage event. in hepatoviruses, even this cleavage is catalyzed by 3c pr~ modified from ryan and flint, 1997. gorbalenya, unpublished observations), while hepato-, parechoviruses and infv appear to encode unique l proteins (najarian et al., 1985; hyypia et al., 1992; isawa et al., 1998) . some evolutionary characteristics of the 2a polypeptides parallel those of the l proteins. entero-and rhinoviruses encode 2a of the same protein family, known as 2a cysteine chymotrypsin-like proteinases (bazan and fletterick, 1988) , whereas each of the three groups of hepato-and parechoviruses and infv encodes a unique 2a with unknown function(s). the other picornaviruses, cardio-and aphthoviruses and aiv, encode a 2a protein having a characteristic c-terminal motif (or a derivative thereof) that has been implicated in the spontaneous separation of 2a and 2b proteins during polyprotein synthesis (reviewed in ryan and flint, 1997) . besides l and 2a, the only other protein of the family not conserved at the primary structure is vp4 (palmenberg, 1989; a.e. gorbalenya, unpublished observation) . it may therefore not come as a surprise that vp4 has been poorly resolved in x-ray analyses of picornavirions whose structure has been solved (lentz et al., 1997) . it is interesting to note that this small pro-tein, which occupies a position upstream of vp2, has moved its position to between vp2 and vp3 in insect infv . other picornavirus proteins are conserved, albeit to varying degrees. these polypeptides therefore may play similar role(s) in the life cycle of different picornaviruses. for example, 2b and 3a are of variable sizes but they contain hydrophobic regions thought to be involved in the anchoring of these proteins to membranes in rna replication complexes. only the hydrophobic patches of 2b and 3a polypeptides, however, have been conserved (a.e. gorbalenya, unpublished results) . in capsid proteins, the most pronounced conservation is evident in residues critically important for fold maintenance. finally, in the key replicative enzymes 2c awpase, 3c pr~ and 3d p~ as well as in 3b vpg, the active site residues are amongst the most highly conserved (gorbalenya and koonin, 1993a) . the majority of proteins of picornaviruses, regardless of how well they have been conserved within their own family, have homologs among cellular and other viral proteins. first, the three capsid proteins, vp1, vp2 and vp3, have adopted different versions of an eight-f i g u r e 12.10 comparison of the genome organizations of the main groups of the picorna-like supergroup. for each picorna-like family group, excluding apv, the conserved organization of an "averaged" genome typical for this group is shown and compared with that of picornaviruses. the genomes are aligned with respect to the position of the 3d (-like) locus (the rna polymerase). "averaging" was carried out with respect to genome size so that most conserved genome features could be shown. note that bymoviruses, comprising a genus of potyviridae, have a bipartite genome. it is believed that all picoma-like viruses contain vpg at the 5'-end, although a vpg has not yet been demonstrated for every group of viruses. in picoma-like viruses, proteins were designated so as to reflect their similarity to the prototype picornavirus enzymes, although other nomenclatures may be in use by other investigators. apart from studies with picomaviruses, enzymatic activities have been ascribed to some proteins of como-, poty-, calici-and sequiviruses, but the complete processing map of polyproteins has been established only for como-and potyviruses. the conserved 0~/~ rossmann-fold and palm-like fold comprise only one of the domains of 2c and 3d, respectively, or their homologs. for further details, see legends to figures 12.2 and 12.4, and the text. stranded antiparallel beta-barrel fold, dubbed "jelly-roll" (rossmann and johnson, 1989) . amongst rna and dna viruses of different families, this fold is the most common to build icosahedral capsids (rux and burnett, 1998) . it is also conserved in a number of cellular proteins (rossmann, 1987; orengo et al., 1997) . second, the core domain of 3d p~ containing several highly conserved sequence motifs, is related to a number of polynucleotide polymerases includ-ing rna-dependent rna polymerases of rna viruses, reverse transcriptases of viral and cellular origins, and dna-dependent dna polymerases (hansen et al., 1997 ). an analysis of the crystal structure of pv 3d p~ has also identified a (palm) subdomain adopting a rrm-like fold conserved among a number of functionally different proteins, including ribosomal proteins l7/l12 and $6 as well as the uia splicing factor (hansen et al., 1997) . third, two picomavirus proteinases, the ubiquitous 3c pr~ and entero/rhinovirus-specific 2a pr~ have adopted 12-stranded antiparallel two beta-stranded barrel folds, conserved in cellular serine proteases with chymotrypsin as the prototype (reviewed in skern, 1998) . these picornavirus proteinases have also relatives that are encoded by (+)rna viruses belonging to dozens of different species (gorbalenya and snijder, 1996; ryan and flint, 1997) . unlike cellular proteases, the picornaviruses 3c pr~ and 2a pr~ employ cysteine as the principal catalytic nucleophile and, in some lineages, have another unique replacementinstead of the catalytic asp they use a glu . the other small family of picornavirus proteinases, l pr~ of aphthoviruses, erv1 and erv2, is related to cellularpapain-like proteases (gorbalenya et al., 1991; skern, 1998) whose homologs have been identified in many animal and plant rna viruses as well (gorbalenya and snijder, 1996) . finally, 2c atpase, whose structure is yet to be solved, belongs to the so-called helicase superfamily iii. this protein group includes polynucleotidestimulated atpases, some with helicase activity, which are encoded by (+)rna and small dna viruses as well as proteins of cellular origin (gorbalenya and koonin, 1990, 1993b) . the 2c awpase has been predicted to be a three-domain protein. two (x/(x domains flank an atp-binding domain adopting a variation of the (x/j3 "rossmann" fold, which is widespread in the protein world (teterina et al., 1997) . with respect to details, our current understanding of the function of picornavirus proteins is rather fragmentary. nevertheless, a preliminary functional profile of picornavirus proteins fits patterns of conservation evident at the structural level. the most conserved non-structural proteins provide the basic enzymatic activities needed for the synthesis and expression of viral rnas inside the cell. the three conserved capsid proteins form the scaffold of virions shielding virus rna from the detrimental environment outside the cell. all these activities appear to be virus-specific, although they may be modulated by cell-encoded components. in contrast, non-conserved viral proteins seem to sense and modify the host environment in addition to serving basic biosynthetic processes pro-grammed by the viral genomes (for instance, see piccone et al., 1995; zoll et al., 1996; svitkin et al., 1998; ventoso et al., 1998) . virions also have host-dependent functions, such as the recognition of the cellular receptor, entry and, possibly, virion maturation. different lines of evidence have shown that the least conserved regions of three capsid proteins, as well as vp4, may mediate these early activities of host cell entry (for recent work, see hadfield et al., 1997, and lentz et al., 1997) . much of what has been said about proteins applies also to the terminal ntrs of the picornavirus genomes. these regions are conserved within related genera but they may diverge when groups are compared (e.g. enterovirus/ rhinovirus versus cardiovirus /aphthovirus) even although they play identical roles in viral proliferation (discussed in the section on genetics). variants of two very different conserved secondary structure organizations of ires elements are shown in figure 12 .3, prototyped by those of pv and emcv. it is unclear what type of 5'ntr was encoded by an ancestor of picornaviruses -that resembling one of the contemporary prototypes or rather a "consensus" one (le and maizel, 1998) . in contrast, the 3'ntr region has diverged profoundly amongst picornaviruses and it is not conserved even within the otherwise closely related entero-and rhinoviruses (poyry et al., 1996) . the polyprotein of numerous +strand rna viruses has evolved such that its organization reveals an additional level of conservation-the order of mature proteins in this large precursor (figure 12 .10; see also the order of protein domains in the prototype pv in figure 12 .4). this order is inflexible and none of the picornaviruses violates it, although entero-and rhinoviruses do not encode l proteins (see above). despite a near absolute conservation of the order of protein domains, there is some plasticity (figure 12 .10). upon computer sequence analyses of the picorna-like viruses, it has become evident that the polyprotein can be divided into two parts, one comprising capsid proteins and the other the non-structural proteins. these parts are expressed rather independently. in a group of picorna-like insect viruses, rhopalosiphum padi virus (rhpv), drosophila c virus (dcv), plautia stali intestine virus (psiv) and cricket paralysis virus (crpv), non-structural and capsid proteins are encoded by two orfs, separated by a ntr (koonin and gorbalenya, 1992; johnson and christian, 1998; moon et al., 1998; nakashima et al., 1998; sasaki et al., 1998) . in comoviridae, a family of plant viruses, the capsid and non-structural proteins are encoded by two distinct rnas, rna2 and rna1 (goldbach, 1986) . remarkably, in the dendrogram shown in figure 12 .8, comoviridae, sequiviridae, a plant virus family having the same polyprotein organization as picornaviridae (turnbull-ross et al., 1993) and picorna-like insect viruses form a division immediately adjacent to the picornaviridae. it is important to stress that comparison of the sequences of many viruses of the picorna-like supergroup has revealed a profile of sequence conservation that parallels that observed for picornaviridae. thus, two groups of highly conserved clusters can be distinguished in polyproteins. the first group comprises the capsid proteins vp2-vp3-vp1, the second the non-structural proteins 2c atp .... (vpg)-3cpr~ p~ (or equivalents). the functions assigned to the individual members of the group of non-structural proteins remain provisional for the majority of known viruses. they have been inferred largely on the basis of sequence similarities with proteins of well-characterized viruses like poliovirus. among the positionally highly conserved non-structural proteins, the genomelinked protein vpg has a special standing (highlighted by bracketing) since it is conserved functionally rather than structurally in the picornalike viruses (figure 12 .10; gorbalenya and koonin, 1993a) . the combination of conserved non-structural proteins of picorna-like viruses has been termed "replicative module" (goldbach, 1986) . such module of related proteins has been recognized also as "capsid modules" built of three "jelly-roll" proteins. animal caliciviridae, plant potyviridae and insect acyrthosiphon pisum virus, all of which are distantly related to picornaviridae, encode a distinct variety of the replicative module that is associated with one of three unique sets of capsid protein(s) encoded in the 3'-region of viral genomes (domier et al., 1986; meyers et al., 1991; van der wilk et al., 1997; figure 12.10) . the conservation of protein order in the picornavirus polyprotein and the patterns of expression (proteolytic processing) have been conserved. pairs of neighboring proteins are separated at scissile bonds cleaved by a virus proteinase or, in case of the vp4/vp2 junction, by an unknown mechanism. it could be hypothesized that the position of protein domains could be changed as long as the corresponding proteins were released more or less independently from the precursor. this, however, is not the case, as the pathway of proteolytic processing in picornavirus polyproteins is not random. furthermore, at least some intermediate precursors, e.g. 2bc, 3ab and 3cd pr~ have essential functions that differ from those of the end-product of processing (see the section on genetics). these considerations provide a biological reasoning for the observed conservation of the protein order in polyproteins. we have already pointed out that the order of two conserved units within the polyprotein, the capsid precursor and replicative modules, is flexible. the two least conserved proteins, l and 2a, flank the capsid precursor at the n-and ctermini, respectively, and bring additional plasticity to the organization of the polyprotein. this is reflected also in terms of expression, i.e. the mechanism of proteolytic processing. processing of the capsid precursors as well as of the replicative module at junctions separating conserved proteins involves exclusively the conserved 3c/3cd pr~ proteinases, a mechanism functioning not only in picornaviruses but also in other picorna-like viruses (figure 12.10; ryan and flint, 1997) . in contrast, the three cleavages separating the poorly conserved l and 2a pro-teins from the neighboring polypeptide chains (l/vp4, vp1/2a and 2a/2b) are processed by a range of mechanisms in a genus-specific manner. furthermore, whereas picornaviruses use two general pathways of cleavages -3c pr~ pr~ versus distinct mechanisms involving l and 2a-this genetic repertoire may be further diversified in some picorna-like viruses. for instance, in comoviridae and insect viruses, capsid precursor and non-structural proteins are encoded by distinct orfs (figure 12 .10), which eliminates the need for cleavages separating these polypeptide chains. 3cpr~ pr~ have emerged as the major enzymatic factors in the regulation of protein expression in all picorna-and related viruses. interestingly, the primary structure of sites recognized by these proteases is virus-specific rather than position-specific. among picornaviruses, entero-and rhinoviruses employ sets of structurally uniform sites while viruses of the other genera use more diversified sets. poliovirus and hav exemplify the most extreme diversity. in poliovirus, all eight cleavage sites have the same ("canonical") q/g structure ( figure 12.4) , whereas in hav, six variations of this structure were described in different sites (palmenberg, 1990) . poliovirus proteins produced from its replicative module seem to have been exceptionally strongly constrained not only with respect to the type of the terminal amino acids but also with respect to size. mature poliovirus proteins (except 3cpr~ as well as processing intermediates, have sizes that can be divided by 11 without remainder or with only a small remainder (gorbalenya et al., 1986) . this feature separates poliovirus proteins from the overwhelming majority of cellular and viral proteins. the latter are heterogeneous both in size and sequence, particularly at their termini, because of a relative abundance of mutations, including insertions and deletions. structural regularities documented for poliovirus can be visualized in a form of weak primary structure periodicities with the common denominator of 11 comprising the major portion of the replicative module. on the basis of these observations, it has been proposed that the replicative module of picornaviruses has originated from a primitive self-replicating rna molecule through consecutive multistep duplications (gorbalenya, 1995; gorbalenya et al., 1986) . how it is likely to evolve in the future? we have briefly described different levels of evolutionary conservation in picornaviruses by using results of comparative sequence analyses. the conservation of different properties is the result of a long evolutionary process, accompanied by numerous radiations. does the history of the polyprotein determine how picornaviruses may evolve in the future? we are unaware that this question has ever been directly addressed in experimental studies, although many results obtained by using genetic engineering seem to be quite relevant. these data can technically be separated into two setsthose obtained in studies using site-directed mutagenesis and those aimed at constructing chimeras. in numerous studies of the first type, it has been observed that different regions of the picornavirus genome express a differential tolerance to replacements (wimmer et al., 1993) . it can be predicted that a profile of the "accepted" mutability, drawn over the entire genome, would fit the conservation profiles described above. such a result would support the hypothesis that the past of picornaviruses influences their future in terms of evolution. however, mutagenesis saturating the genome has never been systematically carried out. therefore, the available "mutagenesis profile" can only be used as a rough approximation of the yet-to-bedefined "accepted" mutability profile in relation to the conservation of modules. the "resolution" of the mutagenesis studies that remained unresolved is potentially relevant to an understanding of the evolution of contemporary picornaviruses, regardless of whether this relates to recent evolutionary events or to the complete historical past. the second group of data involving genome engineering complements the mutagenesis studies and helps to address the question posed above. in wt genomes of entero-and rhinoviruses, the orf of the capsid precursor is preceded by the cognate 5'ntr ( figure 12 .2). as was observed in studies of poliovirus expression vectors ( figure 12 .5), genetically stable variants of poliovirus have been selected (mueller and wimmer, 1997) in which an additional leader peptide is encoded that is fused to the n-terminus of the polyprotein, just downstream from the 5'ntr. this organization may look unique on first sight, but in fact it resembles that of all other picornaviruses distantly related to enteroand rhinoviruses. these terminal appendices in the poliovirus variants resemble the l proteins and, hence, these poliovirus chimeras have a "cardio-like" organization ( figure 12 .6e). we can speculate that pv has "accepted" an artificial l peptide because a similar event has already happened in the past history of its ancestors. in a different set of experiments, several poliovirus chimeras have been generated in which the heterologous emcv ires was placed into the sequences specifying scissile bonds of the polyprotein, thereby dividing the polyprotein into two parts (figure 12 .6b). this insertion radically modified the conserved protein expression mechanism of picornaviruses, since it functionally replaced a proteolytic cleavage event by an event of internal initiation of translation directed by the alien ires. in all, poliovirus genomes were constructed in which the emcv ires was placed between the y*g cleavage site of 2a pr~ (figure 12 .6b) or all possible q*g cleavage sites involving the 3cpr~ pr~ proteinase (molla et al., 1992 (molla et al., , 1993b paul et al., 1998a) . only two poliovirus-emcv dicistronic chimeras, specifically those carrying emcv ires between vp1 and 2a and between 2a and 2b, have given rise to viable and stable virus progeny (molla et al., 1992 (molla et al., , 1993b paul et al., 1998a) . although the genome organizations of these chimeras do not match anything found in nature, immediate parallels come to mind with genomes of picorna-like viruses in which capsid and replicative modules are encoded by different orfs (for example comoviridae, see above and the section on genetics). these considerations imply that the conserved and non-conserved features in organization and structure of genomes of picornaviruses and even picorna-like viruses are indicative of an evolutionary plasticity and of possible future changes of a picornavirus. perhaps an "evolutionary space" of a picornavirus can be approximated from the past. mechanistically, this can be seen as if the past evolution of the entire family has been "imprinted" in the organization of the genome of each of the contemporary picornaviruses. phylogenetic trees that have been built for different picornaviral proteins (most often vp3, 2c awpase and 3d p~ by employing parsimonious and maximum-likelihood methods proved roughly topologically equivalent even though different regions of the polyproteins have definitely evolved at different rates (stanway, 1990; rodrigo and dopazo, 1995; hyypia et al., 1997) . these observations strongly favor a concerted evolution of (the majority of) the picornavirus proteins. this conclusion is not compromised by some incongruity in the tree topology of closely related viruses, e.g. the c cluster of the enteroviruses (poyry et al., 1996) , or very distantly related groups, e.g. hepato-and parechoviruses. it is likely that some trees generated for different regions look different, as a result of technical limitations related to phylogenetic and biopolymer sequence analyses as well as a biased representation of some groups. also, possible recombination events between closely related viruses may have complicated phylogenetic analyses. we shall analyse sequence alignments of picornavirus proteins and polynucleotides aimed at deducing the mechanisms functioning in picornavirus evolution. uniform sizes of each of the vp3, 2c awpase, 3c pr~ or 3d p~ polypeptides have been maintained in all picornaviruses. the diversity of the proteins is therefore most probably the result of numerous in-frame mutations. for the other proteins, some additional mechanism of diversification may have been functioning in the course of evolution. among the viruses encoding 2a proteins sharing the npgp motif, the two viruses fmdv and erv1 encode a 2a consisting of only 16 amino acids, whereas the cardioviruses erv2 and aiv encode a 2a ranging between 67 and 150 residues. it can be speculated that deletion events in the 2a coding region of fmdv and erv1 are the result of "jumping" of 3d p~ perhaps by loop-out deletion or by illegitimate recombination (figure 12.7) . on the other hand, the three adjacent coding regions for vpg uniquely found in all strains of fmdv suggest duplication events. in other viruses, e.g. erv2 or tmev, genetic events such as local duplication and deletions may have occurred, leading to considerable size heterogeneity of the corresponding vpgs and adjacent sequences (wutz et al., 1996; a.e. gorbalenya, unpublished results) . duplications have also been discovered in the 5'ntr of enteroviruses (pilipenko et al., 1989a) . picornavirus genomic redundancy, known as duplications, may have been generated by intragenomic recombination. after duplications, however, the sequences must have undergone some variation so as to avoid elimination by homologous recombination. indeed, the nucleotide sequences (and to a small extent also the amino acid sequences) of the three vpgs of fmdv differ such that homologous recombination at this locus is unlikely (cao and wimmer, 1996) . in spite of lack of evidence, duplications by intragenomic recombination might have been involved in the production of large differences in size found in capsid proteins vp1 and vp2, or in non-structural 3a and 2b proteins of some picornaviruses. the capsid proteins contain long extra loops while the 2b protein of erv1 has an enormous size relative to the 2b proteins of all other picornaviruses (283 versus 100-150 amino acids; wutz et al., 1996) . on the other hand, charini et al. (1994) have reported that, surprisingly, a viable poliovirus isolate they selected from a swarm of revertants had captured a short segment of cellular ribosomal rna. thus, capture of entirely foreign rna sequences, although very rare, cannot be excluded from the mechanisms of diversification. at least two different mechanisms could have given rise to the contemporary diversity of 2a and l protein families. the diversity includes, amongst others, chymotrypsin-like proteinase and npgp motif-containing polypeptides for 2a and papain-like proteinase and zn-finger proteins for l. phylogenetic analyses suggest that "new" unrelated 2a and l proteins have emerged in the course of evolution of picornaviruses on several occasions, following the split of the major groups of the picornavirus tree. it is logical to assume that, following each split, one of the two descendants has arisen from an ancestral viral source, the other from an "independent" source. as to the latter, the coding sequence of either 2a or l could have recombined with a gene of either another virus or of the cell, leading to the replacement of the ancestral coding sequence. for example, this replacement mechanism could have resulted in the capture of cellular chymotrypsin-like (2a pr~ or papain-like (l pr~ activities. this hypothesis is, of course, purely speculative since no potential partners in recombination have been identified as yet. alternatively, the diversity of the 2a and l families may be the result of frame-shifting events. for example, enteroviruses have a "spacer sequence" between the ires and the orf of the polyprotein ~. this spacer commences with an unused ("silent") aug at the 3' border of the ires. in poliovirus, it is 154 nt long and represents a small out-of-frame orf terminating inside the polyprotein orf. if the silent aug at the 5' end of the spacer were to trigger initiation of translation and, in addition, a frame-shift mutation connected the small orf with the main orf, a small "leader" peptide would be created fused to the polyprotein. all that is then necessary is a 3c pr~ cleavage site to sever the "leader" from vp4-and a genetic arrangement would have been created resembling that of cardio-and aphthoviruses (jang et al., 1990) . indeed, the silent aug of poliovirus can be turned on by changing its kozak context (pestova et al., 1994) , and stable poliovirus variants can be isolated that carry short foreign leaders (see above; mueller and wimmer, 1998) . thus, the conversion of an enterovirus to a cardiovirus genotype with respect to an l protein can be envisioned by relatively simple genetic changes. similarly, it should be possible to convert a cardiovirus genotype in this region into an enterovirus genotype by silencing its l orf. it is relevant that, as already mentioned, a strain of theiler's virus has been identified that, just like the normal cardioviruses, synthesizes a polyprotein-fused l protein and, in addition, a polypeptide l* in a separate orf. l* synthesis is initiated at its own aug initiation codon (takata et al., 1998) . apparently, the synthesis of l* may present the virus with an advantage in the natural host, a fact that may have contributed to its selection. by comparison with the l protein region in tmev, two 2a proteins may have existed in the ancestral picornavirus genome, one active in the polyprotein, the other "silent". in the course of subsequent speciation, each of these 2a variants may have been used in separate picornavirus lineages. the activation of the "silent" 2a may have led to a concomitant inactivation of the other 2a. it should be mentioned that the presence of multiple alternative orfs in ancestral picornavirus genomes may have been the rule rather than the exception, particularly if the polyprotein evolved by amplification of 11-mers (gorbalenya, 1995 ; see also above). ohno (1984) has demonstrated that periodicity-organized polynucleotides with a period that cannot be divided by 3 (11-long periodicity included) have an identical coding capacity in each frame. in other words, if one orf is open the two other frames are open also. in the course of evolution, two out of three reading frames may have deteriorated or may have given rise to genetic variation as speculated for the generation of the diversity in 2a and l proteins. numerous studies attest to a remarkable stability of the picornavirus genotype if grown under identical conditions (wimmer et al., 1993) . on the other hand, if exposed to altered conditions in the environment, a shift to new variants can be readily observed. just like other biological systems, it can be assumed that picornavirus speciation has been driven by a changing environment. circumstances upon which a picornavirus may encounter a "new" environment include: (1) horizontal or vertical transfer to a new (different) host; (2) entering a natural host through a non-natural gate; (3) infecting immunized (natural) hosts previously exposed to the same virus. although there is no proof, it is intuitively highly likely that all three scenarios have played a role in picornavirus speciation. in the following, a speculative reconstruction of forces will be presented that may have contributed to the evolution of picornaviruses. picornaviruses belonging to a genus or a cluster may have almost identical phenotypes with respect to growth properties and even in regard to pathogenic potential. a most important characteristic, however, does further divide a group of very closely related picornaviruses (e.g. polioviruses): the susceptibility to activation by different neutralizing antibodies and, hence, the separation into serotypes (see the introduction). it is logical to assume that the (negative) pressure of the immune system may be largely accountable for serotype diversification of picornaviruses. that is, the immune response can lead to the selection of viral variants resistant to the neutralizing immune response produced by the surviving host. such variants would form a pool from which a new serotype could be further selected. in fact, such mechanism of virus evolution seems to dominate in the case of influenza a virus or immunodeficiency virus (hiv). however, the sheer unlimited degree of serotype diversification observed in influenza viruses or hiv is an exception rather than the rule amongst viruses. indeed, not all picornaviruses seem to be able to easily produce new serotypes. for example, the genus hepatovirus encompasses only one serotype while others are restricted to a few serotypes (e.g. poliovirus). new viral variants that have escaped the immune surveillance must, of course, interact with multiple host components at virtually every stage of their reproduction in order to survive. this includes virus entry into the host cell, translation and processing, genome replication, encapsidation and maturation, spread in the host. each of these steps are checkpoints and every new viral variant must be fit to pass these barriers. the earliest events in the infectious cycle-receptor interaction, uptake, uncoating-and the mechanisms of neutralization are amongst the least understood in the molecular biology of picornaviruses. the crystal structures of some member viruses of four picornavirus genera have been solved; examples are: enterovirus, poliovirus 1 and 3 (hogle et al., 1985) ; rhinovirus, human rhinoviruses 2, 3, 14, 16 (rossman et al., 1985) ; cardiovirus, mengovirus (luo et al., 1987) , theiler's virus (luo et al., 1996) ; aphthovirus, fmdv (acharya et al., 1989) . (for a complete list, see lentz et al., 1999) . however, the precise localization and structures of different neutralization antigenic sites (the structures interacting with neutralizing antibodies) is known only for polioviruses, rhinoviruses and aphthoviruses. for aphthoviruses and for polioviruses, the available evidence suggests that the same structures that determine in part the serotype identity are also involved in receptor recognition (domingo et al., 1993; mason et al., 1994; harber et al., 1995) . thus, immuneescaping viral mutants are likely to be enriched in those variants that have maintained the ability to efficiently interact with the cognate receptor and follow the pathway of uptake and uncoating. this is, of course, only speculative but, if correct, it would explain in part serotype restriction (harber et al., 1995) . in this respect it may be informative to compare receptor specificities with serotype diversities of human enteroviruses, on the one hand and rhinoviruses on the other. these two genera encompass viruses that have diverged from an immediate common ancestor and radiated during the same time period (figure 12.1) . in the course of evolution, different serotypes in roughly the same numbers have been generated in these two picornavirus branches: there are about 66 enterovirus and over 100 rhinovirus serotypes. this implies that viruses of the two genera are similarly prone to accumulation of changes in those capsid structures giving rise to new serotypes. but what about receptor specificity of these viruses? at the time of writing, two receptors have been assigned for human rhinoviruses (which is probably all that will be found) and six receptors for human enteroviruses (at least four more are awaiting identification; table 12 .1). thus, in contrast to the quite similar extent of serotype diversification in both genera, adaptation to new receptors is significantly more restricted in rhinoviruses than in the closely related enteroviruses. importantly, there is an overlap between the two receptor patterns and, taken together, the icam-1 receptor specificity appears to be dominant among entero-and rhinoviruses. this can be interpreted to mean that the immediate common ancestor of both enteroand rhinoviruses may have used a receptor related to icam-1. regardless of whether this is true or not, the subsequent evolution of icam-1-recognizing picornaviruses has proceeded differently, as seen in the disparity of the current use of this cellular receptor (>90 for rhinoviruses versus 11 for c-cluster coxsackieviruses). given that the serotype diversification has proceeded at a similar pace in entero-and rhinoviruses, enteroviruses may have had greater opportunities -or a greater need -to adapt to new receptors in order to initiate an infection. this may be related to the function(s) of receptors in viral docking and uncoating: whereas rhinoviruses may need the receptor only for docking and uptake (because of their inherent sensitivity to the acidic ph inside late endosomes), the exceedingly stable enteroviruses do need the receptor (and possibly a co-receptor) for docking, uptake and uncoating. with poliovirus, a particle stable to detergents, proteases and low ph (ph 2), this is exemplified in the formation of a-particles, a labile product of receptor/virion interaction and an intermediate in uncoating . a-particle formation appears to involve also sequences of neutralization antigenic sites (harber et al., 1995) . thus, the intercourse between receptor and enterovirion may be much more complex than that between receptor and rhinovirion. consequently, a change in the serotype may have forced enteroviruses to search for new receptors to retain the uncoating capacity of the cellular receptor. the unusually large serotype diversity of the major receptor group human rhinoviruses may then be explained as follows. it seems possible that the initiation of an infectious cycle of hrv does not critically require an interaction between structures of the neutralization antigenic sites of the virion and icam-1. that is, the n-terminal domain of icam-1, by inserting itself into the virion's canyon, can effect docking, uptake and uncoating of the particle. progression through any of these events is not critically dependent on sequences of the neutralization antigenic sites. if correct, it follows that variation of the antigenic sites does not restrict viral proliferation and serotype evolution. consistently, in other picornaviruses the neutralization antigenic sites and the determinants recognizing the receptor would be much more overlapping and mutually dependent. it is likely that an initial immune-driven selection might also finally result in a virus variant with changed or extended tissue tropism. this might have happened with cav24v, a c-cluster human enterovirus. immune pressure might have initiated the selection of the cav24v mutant derived from a cav24 swarm. as mentioned before, cav24v is a very recent variant of cav24 and, unlike its parent and the other members of the c-cluster, it can cause acute hemorrhagic conjunctivitis. apart from the possibility that cav24v emerged through immune selection, it could also have been selected from a swarm when the parental cav24 was accidentally inoculated into the eye. another type of selection might have been responsible for the emergence of swine vesicular disease virus (svdv). phylogenetic analysis of genomes of human enteroviruses identified svdv as being interleaved with human viruses comprising the cbv-like cluster (poyry et al., 1996) . this observation is strongly indicative of selection of svdv from a mutant of a human coxsackie-b virus entering the new host through frequent contacts of these domestic animals with (infected) humans. we have discussed different aspects relevant to picornavirus evolution, but we did not address one crucial question: are picornaviruses a successful family? we believe that the answer is: yes. in discussing this issue, we will also formu-late considerations regarding the worldwide eradication of poliovirus. one of the strongest criteria of biological prosperity is the diversity of a taxonomic group. despite some bias inherent in current analyses, phylogenetic studies of picornaviral genomes suggest that picornaviridae have radiated densely over the course of evolution, at both early and late stages (figure 12.1) . furthermore, picornaviruses are members of a superfamily with numerous distant relatives (figure 12 .8) that infect a wide range of organisms, including both plants and animals. some of these viruses, like sequiviridae, employ a genetic plan that is basically a variation of the genetic plan used by picornaviruses (figure 12 .10). prosperity of the host is another prerequisite for a virus to be successful. by this criterion also, picornaviruses are successful, since the majority of them, representing different branches of the picornavirus tree, infect humans. humans are arguably one of the most successful species in the biological world. in truth, picornaviruses are relatively harmless even though few humans, if any, can escape picornavirus infections. this too can be viewed as evidence that these viruses have adapted well to their host, as they have not significantly undermined human affairs. this is true even for poliovirus, an agent that is commonly regarded as a deadly virus following epidemics of poliomyelitis. however, prior to this century, poliovirus did not cause epidemics, even though it infected humans at rates approaching 100%. epidemics emerged because human behavior changed through the invention of modern hygiene. hygiene broke the chain of natural immunization through infant infection combined with infant protection by maternal antibodies. even in this century's devastating epidemics, however only 1-2% of infected individuals developed poliomyelitis. the poliovirus-human relationship alluded to above deserves to be discussed in more detail. humans, who occupy a unique niche in the bio-logical world (because they care about each human life), did not accept their potential defeat as poliomyelitis became an epidemic. unprecedented efforts combining medical research with modern technologies led to the development of two highly effective poliovirus vaccines, the inactivated poliovirus vaccine by jonas salk and the live attenuated vaccine by albert sabin (wimmer et al., 1993) . through education of the populace and advanced healthcare measures, mass vaccinations have gradually eliminated wild-type poliovirus, first in the developed countries and later in most of the world. incredibly, the few cases of poliomyelitis in the western hemisphere now result from vaccination with the live sabin strains. overall, polio vaccination is a success story of greatest consequence. indeed, through worldwide efforts led by the world health organization, it is likely that wild-type polioviruses will be eradicated globally by the turn of the century (who, 1985) . do these considerations allow us to safely conclude that, after its global eradication, poliovirus will have no chance to re-emerge through enterovirus evolution? for discussion of this issue, we will first summarize hypotheses about the possible origin of polioviruses and their closest relatives, the c-cluster coxsackieviruses. the three serotypes of poliovirus belong to the c-cluster of enteroviruses (table 12 .1; figure 12 .1b). the most comprehensive analysis of the c-cluster has been performed with sequences of the vp4-vp2 capsids and with sequences of the 3d p~ rna polymerase (pulli et al., 1995) . results of these analyses are consistent with data obtained in a study of the other regions of the viral genome using a less representative set of sequences (poyry et al., 1996) . therefore, these relationships shown in figure 12 .1b can be assumed to be quite reliable. a phylogenetic analysis of the capsid vp4-vp2 region of c-cluster viruses indicated that the tree has split at least twice, perhaps before the emergence of an immediate ancestor of polioviruses. the first split led to the separation of a branch encompassing cav1, cav21 and cav24 from the main c-cluster trunk, and the second, more recent one resulted in the separation of the ancestor for pv and the ancestor for cav11, cav13, cav17, cav18, cav20 and cav20b. the results obtained with sequences of 3d p~ favor an even more complex evolutionary history of poliovirus, including more than five intermediate steps (pulli et al., 1995) . consistent with the results of the analysis of the capsid region, cav21 and cav24 were among those viruses that diverged from the main trunk relatively early in evolution while the three poliovirus serotypes clustered together with cav11, cav13, cav17 and cav18. remarkably, in the tree based on 3d p~ sequences the latter four coxsackieviruses (as well as several other coxsackieviruses) are interleaved with, rather than separated from, the three poliovirus serotypes (figure 12 .1). this stands in contrast to the tree of the capsid region. assuming the most parsimonious scenario of evolution, the combination of these results strongly implies that coxsackieviruses that recognize the icam-1 receptor formed a pool from which polioviruses, interacting with the cd155 receptor, have evolved. this conclusion is compatible with a hypothesis of the immune-driven evolution of entero-and rhinoviruses presented above. furthermore, the analyses do not indicate that three polioviruses comprise a monophyletic subgroup within the c-cluster enteroviruses and, hence, have emerged from an ancestral virus by speciation, as one could expect from a distinct phenotypic profile of these viruses. we have previously hypothesized that the coxsackiviruses may have derived from polioviruses by switching receptors from cd155 to icam-1 (harber et al., 1995) . this possibility may be supported from the fact that the ireses of c-cluster coxsackieviruses are highly 'neuropathogenic". on the other hand, the assessment presented above favors an evolutionary relationship in the opposite direction. regardless of the direction in which these viruses emerged, the receptor switch has profound consequences for their pathogenic properties: whereas the c-cluster coxsackieviruses cause respiratory disease, poliovirus can cause deadly neurological disease. these considerations may also have important practical implications. for the sake of the argument, we will assume that the poliovirus eradication campaign has been successfully completed and no more poliovirus particles, including those of the vaccine strains, are circulating worldwide. furthermore, we will assume that all vaccination against poliovirus (including vaccination by inactivated vaccines) has been terminated, a scenario that has been envisioned to be a reality by the end of the next decade. these measures would mark the beginning of a new era in the history of mankind: there will be no human exposure to polioviruses and their antigens. generations of humans will be born that have not been infected with wild-type or vaccine polioviruses and, gradually, they will replace the older generations who carry anti-poliovirus antibodies. at that point, the world will not only be free of poliovirus, but its human population will also no longer carry anti-poliovirus antibodies. thus, a new environment will emerge for human viruses, in particular for c-cluster coxsackieviruses, which are the closest genetic relatives of poliovirus. these c-cavs are expected to circulate widely in the human population, exploring a new evolutionary space. within the human space populated by the c-cavs, there will then exist also a free space that was previously occupied by the three (extinct) poliovirus serotypes. it is possible that mutations in antigenic sites of the c-cavs may (re)generate affinity to cd155. prior to eradication, c-cavs carrying such mutations could conceivably be eliminated by anti-poliovirus antibodies (harber et al., 1995) but in the poliovirus-free world they may remain unchecked. this means that, once emerged, these new viruses carrying poliovirus-like neutralization antigenic sites with cd155 receptor affinity are less likely to be eliminated from the human population after eradication than before. since all enteroviruses, the variants included, lead to enteric infections, these variants may find a passage to the cns and, mediated by their affinity to cd155, may cause neurological disease. it is relevant to point out (gromeier and wimmer, unpublished results ) that poliovirus chimeric viruses in which the poliovirus ires has been replaced with that of c-cluster ires elements have been found to be highly neurovirulent in cd155 tg mice (see the section on pathogenesis). thus, there is reason to fear that in a poliovirus-free world new coxsackievirusrelated, poliovirus-like pathogens that can cause poliomyelitis may emerge in the course of natural viral evolution. the time frame, however, cannot be predicted. it could be one generation or 1000 years. the human condition favors an increasing rate of diversity of human viruses simply because of the increasing size of the human population (estimated to stabilize at 8-12 billion during the next century). this population explosion will lead to a dramatic increase of human contacts, either in cities, particularly megacities (harboring more than 50% of the world's population), through travel or otherwise. clearly, this presents a fertile ground for proliferation and diversification of the highly infectious human picornaviruses. thus, the possibilities of genetic variation of picornaviruses leading to new or renewed human pathogens, such as cav24v, must always be kept in mind. at this point, however, our considerations of the possible re-emergence of poliovirus-like pathogens in the post-eradication era pale in the face of mankind's heroic attempt to eradicate an rna virus for the first time. after all, poliovirus has caused, and is still causing, terrible human suffering. picornaviruses have been discovered because they cause diseases in animals and humans. fortunately, most human picornavirus infections are self-limiting. yet the enormously high rate of picornavirus infections in the human population can lead to a significant incidence of disease complications that may be permanently debilitating or even fatal. the case of poliovirus has taught us that a change of human behavior, which, paradoxically, was the invention of modern hygiene, has greatly aggravated the impact of infection by this specific agent. clearly, this scenario could repeat itself with other human picornavirus species. the terror of this century's poliomyelitis epidemics has driven picornavirus research forward more than any other factor. this work has led to a wealth of discov-eries in biology in general, and to an abundance of data describing the unique biology of picornaviruses and their evolution in particular. picornaviruses employ one of the simplest imaginable genetic systems: they consist of single-stranded rna that encodes only a single multidomain polypeptide, the polyprotein. the rna is packaged into a small, rigid, naked, icosahedral virion whose proteins are unmodified except for a myristate at the n-termini of vp4. the rna itself does not contain modified bases. thus, picornaviruses travel with light baggage. on first sight, the replication of picornaviruses is exceedingly simple. after having chosen a receptor from a large menu of cell-surface proteins, the virion enters the cytoplasm and immediately translates its genome, controlled by its ires element. thereafter, the polyprotein is processed by its own proteinases. rna replication occurs by a unique, proteinprimed mechanism catalyzed by the rnadependent rna polymerase. assembly appears to be linked to rna synthesis, and release of the progeny virions follows a passive mechanism. there is no need for a cellular nucleus. indeed, the entire replication cycle can occur in a cellfree system free of nuclei, mitochondria and perhaps of all other cellular organelles. yet as of now we understand only a small fraction of these viruses' life cycle, and we are awed by the sophistication with which the viruses express their genetic information. the ires, arguably one of the most complex cis-acting signals known in rna systems, has freed picornaviruses from the cellular constraint of cap-dependent translation. this, in turn, allows the primer-dependent rna polymerase, an enzyme with properties generally ascribed only to dna polymerases or reverse transcriptase, to prime with vpg and leave the rna uncapped. polyprotein processing proceeds in a controlled manner yielding cleavage intermediates and end-products that can be used for different functions. thus, the menu of gene products is expanded through the temporal regulation of proteolytic processing. details of all of these steps in replication are still obscure . the key to ultimately understanding picornaviruses may be to rationalize the huge amount of information about these viruses from the perspective of evolution. it is possible that the replicative apparatus of picornaviruses originated in the precellular world and was subsequently refined in the course of thousands of generations in a slowly evolving environment. picornaviruses cultivated the art of adaptation, which has allowed them to "jump" into new niches offered in the biological world. also, by having chosen humans as an additional host, they were offered an abundance of opportunities to proliferate in different tissues, which has contributed to their diversification. these opportunities have further increased through the human population explosion and through changes in human behavior. we suggest that, in addition to drastic and expansive measures such as global eradication, strategies should be developed that aim at predicting the possible evolution of new picornavirus pathogens and preparing for their control. the results reviewed in this article may contribute to achieving this tantalizing and desirable goal. the threedimensional structure of foot-and-mouth disease virus at 2.9 a resolution restricted growth of attenuated poliovirus strains in cultured cells of a human neuroblastoma paradoxes of the replication of picornaviral genomes polioviruses containing picornavirus type 1 and/or type 2 internal ribosomal entry site elements: genetic hybrids and the expression of a foreign gene picornaviral 3c cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases attenuated mengo virus as a vector for immunogenic human immunodeficiency virus type i glycoprotein 120 attenuated mengo virus: a new vector for live recombinant vaccines a functional ribonucleoprotein complex forms around the 5' end of poliovirus rna poliovirus rna synthesis utilizes an rnp complex formed around the 5'-end of viral rna engineering poliovirus as a vaccine vector for the expression of diverse antigens expression of animal viral genomes coupled translation and replication of poliovirus rna in vitro: synthesis of functional 3d polymerase and infectious virus complete replication of poliovirus in vitro: preinitiation rna replication complexes require soluble cellular factors for the synthesis of vpg-linked rna viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications genetic fine structure decay-accelerating factor (cd55), a glycosylphosphatidyylinsitol-anchored complement regulatory protein, is a receptor for several echoviruses identification of the integrin vla-2 as a receptor for echovirus 1 antibodies to the vitronectin receptor (integrin alpha v beta 3) inhibit binding and infection of foot-andmouth disease virus to cultured cells genetic complementation among poliovirus mutants derived from an infectious cdna clone structural and functional characterization of the poliovirus replication complex infectious replicative intermediate of poliovirus: purification and characterization poly(rc) binding protein 2 binds to stem-loop iv of the poliovirus rna 5' noncoding region: identification by automated liquid chromatography-tandem mass spectrometry poliomyelitis sequences within the poliovirus internal ribosome entry segment control viral rna synthesis nucleotide sequence of an attenuated mutant of coxsackievirus b3 compared with the cardiovirulent wildtype: assessment of candidate mutations by analysis of a revertant to cardiovirulence intragenomic complementation of a 3ab mutant in dicistronic polioviruses genetic variation of the poliovirus genome with two vpg coding units trans rescue of a mutant poliovirus rna polymerase function transduction of a human rna sequence by poliovirus initiation of protein synthesis by the eukaryotic translational apparatus on circular rnas a picornaviral protein synthesized out of frame with the polyprotein plays a key role in a virus-induced immune-mediated demyelinating disease nonhomologous rna recombination in a cell-free system: evidence for a transesterification mechanism guided by secondary structure rna duplex unwinding activity of poliovirus rna-dependent rna polymerase 3d p~ defective interfering particles of poliovirus defective interfering particles of poliovirus. 1. isolation and physical properties genetics of picomaviruses brefeldin a inhibits cell-free, de novo synthesis of poliovirus role of enterovirus 71 in acute flaccid paralysis after the eradication of poliovirus in brazil the deletion of 41 proximal nucleotides reverts a poliovirus mutant containing a temperaturesensitive lesion in the 5' noncoding region of genomic rna the nucleotide sequence of tobacco vein mottling virus rna new observations on antigenic diversification of rna viruses: antigenic variation is not dependent on immune selection expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes temperaturedependent alteration of cross-over sites in poliovirus recombination. virology, submitted attenuation of mengo virus through genetic engineering of the 5' noncoding poly(c) tract the origin of genetic information: viruses as models recombinants of mahoney and sabin strain poliovirus type 1: analysis of in vitro phenotypic markers and evidence that resistance to guanidine maps in the nonstructural proteins increased neurovirulence associated with a single nucleotide change in a noncoding region of the sabin type 3 poliovaccine genome the human homolog of ha vcr-1 codes for a hepatitis a virus cellular receptor structural factors that control conformational transitions and serotype specificity in type 3 poliovirus rate of change of concomitantly variable codons poliovirus-specific primer-dependent rna polymerase able to copy poly(a) covalent linkage of a protein to a defined nucleotide sequence at the 5'-terminus of virion and replicative intermediate rnas of poliovirus replication of poliovirus in xenopus oocytes requires two human factors two functional complexes formed by kh domain containing proteins with the 5' noncoding region of poliovirus rna switch from translation to replication in a positivestranded rna virus functional and genetic plasticities of the poliovirus genome: quasi-infectious rnas modified in the 5'-untranslated region yield a variety of pseudorevertants molecular evolution of plant rna viruses abstract. europic'98 origin of rna viral genomes: approaching the problem by comparative sequence analysis superfamily of uvra-related ntp-binding proteins. implications for rational classification of recombination/repair systems comparative analysis of the amino acid sequences of the key enzymes of the replication and expression of positive-strand rna viruses. validity of the approach and functional and evolutionary implications helicases: amino acid sequence comparisons and structure-function relationships viral cysteine proteinases poliovirus induced proteinase 3c: a possible evolutionary link between cellular serine and cysteine proteinase families cysteine proteases of positive strand rna viruses and chymotrypsin-like serine proteases: a distinct protein super-family with a common structural fold putative papain-related thiol proteases of positive-strand rna viruses. identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha-and coronaviruses interaction of rhinovirus with its receptor, icam-1 mechanism of injury-provoked poliomyelitis prophylactic injections and the onset of paralytic poliomyelitis mouse neuropathogenic poliovirus strains cause damage in the central nervous system different from poliomyelitis internal ribosomal entry site substitution eliminates neurovirulence in intergeneric poliovirus recombinants dual stem loops within the poliovirus internal ribosomal entry site control neurovirulence the human poliovirus receptor/cd155 promoter directs reportergene expression in floor plate and optic nerve of transgenic mice in vitro construction of poliovirus defective interfering particles attenuation stem-loop lesions in the 5' noncoding region of poliovirus rna: neuronal cell-specific translation defects structure of the rna-dependent rna polymerase of poliovirus the catalysis of the poliovirus vpo maturation cleavage is not mediated by serine 10 of vp2 serotype polymorphism of poliovirus-cellular receptor interaction: separation of events of viral attachment and uptake proteolytic processing in the replication of picornaviruses interaction of the polioviral polypeptide 3cd pr~ with the 5' and 3' termini of the poliovirus genome: identification of viral and cellular cofactors necessary for efficient binding proteolytic processing of viral polyproteins in the replication of rna viruses 1976) 5'-terminal structure of poliovirus polyribosomal rna is pup genetic recombination with newcastle disease virus, polioviruses and influenza. cold spring harbor symp members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus the three dimensional structure of poliovirus at 2.9 a resolution mutation frequencies at defined single codon sites in vesicular stomatitis virus and poliovirus can be increased only slightly by chemical mutagenesis vcam-1 is a receptor for encephalomyocarditis vitrus on murine vascular endothelial cells a distinct picornavirus group identified by sequence analysis classification of enteroviruses based on molecular and biological properties analysis of genetic information of an insect picorna-like virus, infectious flacherie virus of silkworm: evidence for evolutionary relationships among insect, mammalian and plant picorna(-like) viruses efficient infection of cells in culture by type o foot-and-mouth disease virus requires binding to cell surface heparan sulfate a segment of the 5' nontranslated region of encephalomyocarditis virus rna directs internal entry of ribosomes during in vitro translation initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus rna in vitro cap-independent translation of picornavirus rnas: structure and function of the internal ribosomal entry site the polymerase in its labyrinth: mechanisms and implications of rna recombination poliovirus rna recombination: mechanistic studies in the absence of selection the novel genome organization of the insect picoma-like virus drosophila c virus suggests this virus belongs to a previously undescribed virus family determination of encephalomyocarditis viral diabetogenicity by a putative binding site of the viral capsid protein isolation and characterization of defective-interfering particles of poliovirus sabin i strain complete nucleotide sequence of a strain of coxsackie b4 virus of human origin that induces diabetes in mice and its comparison with nondiabetogenic coxsackie b4 jbv strain preferred sites of recombination in poliovirus rna: an analysis of 40 intertypic cross-over sequences recombination in rna the mechanism of rna recombination in poliovirus primary structure, gene organization and polypeptide expression of poliovirus rna the poliovirus receptor protein is produced both as membrane-bound and secreted forms transgenic mice susceptible to poliovirus q[3 replicase as repressor of q[3 rna-directed protein synthesis evolution of rna genomes: does the high mutation rate necessitate high rate of evolution of viral proteins? an insect picornavirus may have genome organization similar to that of caliciviruses construction of viable deletion and insertion mutants of the sabin strain type i poliovirus: function of the 5' noncoding sequence in viral replication primary structure of poliovirus defective interfering particle genomes and possible generation mechanism of the particles mutational analysis of the genome-linked protein vpg of poliovirus properties of purified recombinant poliovirus protein 3ab as substrate for viral proteinases and as co-factor for viral polymerase 3d p~ differences in replication of attenuated and neurovirulent poliovirus in human neuroblastoma cell line sh-sy5y ubertragung der poliomyelitis acuta auf affen the structure of poliovirus replicative form evolution of a common structural core in the internal ribosome entry sites of picornavirus proteolytic processing of poliovirus polyproteins: elimination of 2a pro-mediated, alternative cleavage of polypeptide 3cd by in vitro mutagenesis the genome of poliovirus is an exceptional eukaryotic mrna the genome-linked protein of picornaviruses. i. a protein covalently linked to poliovirus genome rna structure of poliovirus type 2 lansing complexed with antiviral agent sch48973: comparison of the structural and biological properties of three poliovirus serotypes equine rhinovirus i is more closely related to foot-and-mouth disease virus than to other picornaviruses berichte der kommission zur erforschung der maul-und klauenseuche bei dem institut fuer infektionskrankheiten in berlin poliovirus chimeras replicating under the translational control of genetic elements of hepatitis c virus reveal unusual properties of the internal ribosomal entry site of hepatitis c virus construction and genetic analysis of dicistronic polioviruses containing open reading frames for epitopes of human immunodeficiency virus type 1 gp 120 analysis of picornavirus 2a(pro) proteins: separation of proteinase from translation and replication functions characterization of a new isolate of poliovirus defective interfering particles the atomic structure of mengo virus at 3.0 a resolution the structure of a highly virulent theiler's murine encephalomyelitis virus (gdvii) and implications for determinants of viral persistence the relation of prophylactic inoculations to the onset of poliomyelitis role of mutations g-480 and c-6203 in the attenuation phenotype of sabin type 1 poliovirus capsid coding sequence is required for efficient replication of human rhinovirus 14 rna avian encephalomyelitis virus is a picornavirus and is most closely related to hepatitis. a virus rgd sequence of foot-and-mouth disease virus is essential for infecting cells via the natural receptor but can be bypassed by an antibody dependent enhancement pathway structure of human rhinovirus 3c protease reveals a trypsin-like polypeptide fold, rna-binding site, and means for cleaving precursor polyprotein kissing of the two predominant hairpin loops in the coxsackie b virus 3' untranslated region is the essential structural feature of the origin of replication required for negative-strand rna synthesis enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses identification of bulgarian strain 258 of enterovirus 71 enteroviruses 69, 70, and 71 cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily rabbit hemorrhagic disease virus -molecular cloning and nucleotide sequencing of a calicivirus genome antigenic structure of picornaviruses cell-free, de novo synthesis of poliovirus cardioviral internal ribosomal entry site is functional in a genetically engineered dicistronic poliovirus inhibition of proteolytic activity of poliovirus and rhinovirus 2a proteinases by elastase specific inhibitors studies on dicistronic polioviruses implicate viral proteinase 2apro in rna replication effects of temperature and lipophilic agents on poliovirus formation and rna synthesis in a cell free system stimulation of poliovirus proteinase 3cpro-related proteolysis by the genome-linked protein vpg and its precursor 3ab nucleotide sequence analysis shows that rhopalosiphum padi virus is .a~member of a novel group of insect-infecti~ig rna viruses poliovirus single-stranded and dou pathogenesis and evolution of picornaviruses ble-stranded rna: differential infectivity in enucleated cells expression of foreign proteins by poliovirus polyprotein fusion: analysis of genetic stability reveals rapid deletions and formation of cardioviruslike open reading frames expression of foreign proteins by poliovirus polyprotein fusion: analysis of genetic stability reveals rapid deletions and formation, of cardiovirus-like open reading frames poliovirus antigenic hybrids simultaneously expressing antigenic determinants from all three serotypes poliovirus host range is determined by a short amino acid sequence in neutralization antigenic site 1 primary structure and gene organization of human hepatitis a virus properties of a new picorna-like virus of the brown-winged green bug, plautia stali foot-and-mouth disease virus virulent for cattle utilizes the integrin alpha(v)beta3 as its receptor proteolytic processing in the replication of polio and related viruses genetic studies of the antigenicity and the attenuation phenotype of poliovirus the 5' end of poliovirus mrna is not capped with m7g(5')pppnp the 5' terminal structures of poliovirion rna and poliovirus mrna differ only in the genome-linked protein vpg the location of the polio genome protein in viral rnas and its implication for rna synthesis defective interfering particles of poliovirus: mapping of the deletion and evidence that the deletions in the genome of di coupling between genome translation and replication in an rna virus repeats of base oligomers as the primordial coding sequences of the primeval earth and their vestiges in modern genes cath -a hierarchic classification of protein domain structures sequence alignments of picornaviral capsid proteins proteolytic processing of picornaviral polyprotein poly (rc) binding protein 2 forms a ternary complex with the 5' terminal sequences of poliovirus rna and the viral 3cd proteinase studies with poliovirus polymerase 3dp~ stimulation of poly (u) synthesis in vitro by purified poliovirus c protein 3ab internal ribosomal entry site scanning of the poliovirus polyprotein: implications for proteolytic processing protein-primed rna synthesis by purified poliovirus rna polymerase internal initiation of translation of eukaryotic mrna directed by a sequence derived from poliovirus rna a conserved aug triplet in the 5' nontranslated region of poliovirus can function as an initiation codon in vitro and in vivo 1977) 5'-terminal nucleotide sequences of polio-virus polyribosomal rna and virion rna are identical characterization of the nucleotide triphosphatase activity of poliovirus protein 2c reveals a mechanism by which guanidine inhibits replication of poliovirus the foot-and-mouth disease virus leader proteinase gene is not required for viral replication conserved structural domains in the 5'-untranslated region of picornaviral genomes: an analysis of the segment controlling translation and neurovirulence conservation of the secondary structure elements of the 5'-untranslated region of cardioand aphothovirus rnas prokaryotic-like cis elements in the cap-independent internal initiation of translation on picornavirus rna a model for rearrangements in rna genomes cis-element, orir, involved in the initiation of (-) strand poliovirus rna: a quasiglobular multi-domain rna structure maintained by tertiary guanidine-selected mutants of poliovirus: mapping of point mutations to polypeptide 2c purification and properties of poliovirus rna polymerase expressed in escherichia coli encapsidation of genetically engineered poliovirus minireplicons which express human immunodeficiency virus type i gag and pol proteins upon infection genetics and phylogenetic clustering of enteroviruses virus taxonomy 1997 molecular comparison of coxsackie a virus serotypes cloned poliovirus complementary dna is infectious in mammalian cells utilization of chimeras between human (hm-175) and simian (agm-27) strains of hepatitis a virus to study the molecular basis of virulence transgenic mice expressing a human poliovirus receptor: a new model for poliomyelitis characterization of poliovirus clones containing lethal and nonlethal mutations in the genome-linked protein vpg poliovirus rna replication comparative sequence analysis of the 5' noncoding region of the enteroviruses and rhinoviruses evolutionary analysis of the picornavirus family the 3"untranslated region of picornavirus rna: features required for efficient genome replication intestinal trypsin can significantly modify antigenic properties of polioviruses: implications for the use of inactivated poliovirus vaccine biochemical evidence for intertypic genetic recombination of polioviruses the primary structure of intertypic poliovirus recombinants: a model of recombination between rna genomes the evolution of rna viruses icosahedral rna virus structure structure of a human common cold virus and functional relationship to other picornaviruses the genome-linked protein of picornaviruses v. 04-(5' uridylyl)-tyrosine is the bond between the genome-linked protein and the rna of poliovirus spherical viruses virus-encoded proteinases of the picornavirus super-group protein-priming of dna replication an insect picorna-like virus, plautia stali intestine virus, has genes of capsid proteins in the 3' part of the genome crystallization of purified mef-i poliomyelitis virus particles hybrid protein formation of e. coli alkaline phosphatase leading to in vitro complementation cleavage sites in the polypeptide precursors of poliovirus protein p2-x poliovirus replication proteins: rna sequence encoding p3-1b and the site of pioteolytic processing production of infectious poliovirus from cloned cdna is dramatically increased by sv40 transcription and replication signals a decay-accelerating factor-binding strain of coxsackievirus b3 requires the coxsackievirus-adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells a new cis-acting element for rna replication within the 5' noncoding region of poliovirus type 1 rna picornain 3c identification and characterization of the cis-acting elements of the human cd155 gene core promoter structure, function and evolution of picornaviruses a cell adhesion molecule, icam-1, is the major surface receptor for rhinoviruses rapamycin and wortmannin enhance replication of a defective encephalomyocarditis virus l* protein of the da strain of theiler's murine encephalomyelitis virus is important for virus growth in a murine macrophagelike cell line initiation of poliovirus plus-strand rna synthesis in a membrane complex of infected hela cells membrane fractions active in poliovirus rna replication contain vpg precursor polypeptides poliovirus rna recombination in cell-free extracts a mutation in the rna polymerase of poliovirus type 1 contributes to attenuation in mice amplification of the full-length hepatitis a virus genome by long reverse transcription-pcr and transcription of infectious rna directly from the amplicon poliovirus 2c protein determinants of membrane binding and rearrangements in mammalian cells translation and replication properties of the human rhinovirus genome in vivo and in vitro intertypic recombination in poliovirus: genetic and biochemical studies genetic studies on the poliovirus 2c protein, an ntpase. a plausible mechanism of guanidine effect on the 2c function and evidence for the importance of 2c oligomerization hcar and mcar: the human and mouse cellular receptors for subgroup c adenoviruses and group b coxsackieviruses rescue of defective poliovirus rna replication by 3ab-containing precursor polyproteins complete nucleotide sequences of all three poliovirus serotype genomes: implication for genetic relationship, gene function and antigenic determinants analysis of rna synthesis of type 1 poliovirus by using an in vitro molecular genetic approach genetics of coxsackievirus b3 cardiovirulence genetics of coxsackievirus b cardiovirulence and inflammatory heart muscle disease sequence analysis of the parsnip yellow fleck virus polyprotein: evidence of affinities with picornaviruses synthesis of infectious poliovirus rna by purified t7 rna polymerase nucleotide sequence and genomic organization of acyrthosiphon pisum virus mutational analysis of poliovirus 2apro. distinct inhibitory functions of 2apro on translation and transcription poliomyelitis-like properties of ab-iv coxsackie a7 group of viruses role for beta2-microglobulin in echovirus infection of rhabdomyosarcoma cells who (1985) expanded programme on immunization, global poliomyelitis eradication by the year 2000: manual for managers of immunization programmes on activities related to polio eradication genome-linked proteins of viruses genetics of poliovirus poliovirus receptors an electron microscope study of proteins attached to poliovirus rna and its replicative form (rf) equine rhinovirus serotypes 1 and 2: relationship to each other and to aphthoviruses and cardioviruses molecular dissection of the multifunctional poliovirus rna-binding protein 3ab interaction between the 5'-terminal cloverleaf and 3ab/3cdpro of poliovirus is essential for rna replication rna signals in entero-and rhinovirus genome replication complete nucleotide sequence and genetic organization of aichi virus, a distinct member of the picornaviridae associated with acute gastroenteritis in humans efficient delivery of circulating poliovirus to the central nervous system independently of poliovirus receptor viruses of acute haemorrhagic conjunctivitis polyadenylic acid at the 3-terminus of poliovirus rna polivirus/hepatitis c virus (internal ribosomal entry site-core) chimeric viruses: improved growth properties through modification of a proteolytic cleavage site and requirement for core rna sequences but not core-related polypeptides mengovirus leader is involved in the inhibition of host cell protein synthesis we are indebted to leena kinnunen for providing figure 12 .1, and to steffen mueller for figure 12 .6. we thank astrid wimmer for editing parts of the manuscript. work by m.g. and e.w. described here has been supported in part by grants from the national institutes of health, the national cancer institute, and the centers for disease control. key: cord-016448-7imgztwe authors: frishman, d.; albrecht, m.; blankenburg, h.; bork, p.; harrington, e. d.; hermjakob, h.; juhl jensen, l.; juan, d. a.; lengauer, t.; pagel, p.; schachter, v.; valencia, a. title: protein-protein interactions: analysis and prediction date: 2009-10-01 journal: modern genome annotation doi: 10.1007/978-3-211-75123-7_17 sha: doc_id: 16448 cord_uid: 7imgztwe proteins represent the tools and appliances of the cell — they assemble into larger structural elements, catalyze the biochemical reactions of metabolism, transmit signals, move cargo across membrane boundaries and carry out many other tasks. for most of these functions proteins cannot act in isolation but require close cooperation with other proteins to accomplish their task. often, this collaborative action implies physical interaction of the proteins involved. accordingly, experimental detection, in silico prediction and computational analysis of protein-protein interactions (ppi) have attracted great attention in the quest for discovering functional links among proteins and deciphering the complex networks of the cell. proteins represent the tools and appliances of the cellthey assemble into larger structural elements, catalyze the biochemical reactions of metabolism, transmit signals, move cargo across membrane boundaries and carry out many other tasks. for most of these functions proteins cannot act in isolation but require close cooperation with other proteins to accomplish their task. often, this collaborative action implies physical interaction of the proteins involved. accordingly, experimental detection, in silico prediction and computational analysis of protein-protein interactions (ppi) have attracted great attention in the quest for discovering functional links among proteins and deciphering the complex networks of the cell. proteins do not simply clump togetherbinding between proteins is a highly specific event involving well defined binding sites. several criteria can be used to further classify interactions (nooren and thornton 2003) . protein interactions are not mediated by covalent bonds and, from a chemical perspective, they are always reversible. nevertheless, some ppi are so persistent to be considered irreversible (obligatory) for all practical purposes. other interactions are subject to tight regulation and only occur under characteristic conditions. depending on their functional role, some protein interactions remain stable for a long time (e.g. between proteins of the cytoskeleton) while others last only fractions of a second (e.g. binding of kinases to their targets). protein complexes formed by physical binding are not restricted to so called binary interactions which involve exactly two proteins (dimer) but are often found to contain three (trimer), four (tetramer), or more peptide chains. another distinction can be made based on the number of distinct proteins in a complex: homo-oligomers contain multiple copies of the same protein while hetero-oligomers consist of different protein species. sophisticated "molecular machines" like the bacterial flagellum consist of a large number of different proteins linked by protein interactions. the focus of this chapter is on the computational methods for analyzing and predicting protein-protein interactions. nevertheless, some basic knowledge about experimental techniques for detecting these interactions is highly useful for interpreting results, estimating potential biases, and judging the quality of the data we use in our work. many different types of methods have been developed but the vast majority of interactions in the literature and public databases come from only two classes of approaches: co-purification and two-hybrid methods. co-purification methods (rigaut et al. 1999 ) are carried out in vitro and involve three basic steps. first, the protein of interest is "captured" from a cell lysatee.g. by attaching it to an immobile matrix. this may be done with specific antibodies, affinity tags, epitope tags along with a matching antibody, or by other means. second, all other proteins in the solution are removed in a washing step in order to purify the captured protein. under suitable conditions, protein-protein interactions are preserved. in the third step, any proteins still attached to the purified protein are detected by suitable methods (e.g. western-blot or mass spectrometry). hence, the interaction partners are co-purified, as the name of the method implies. the two-hybrid technique (fields and song 1989 ) uses a very different approachit exploits the fact that transcription factors such as gal4 consist of two distinct functional domains. the dna-binding domain (bd) recognizes the transcription factor (tf) binding site in the dna and attaches the protein to it while the activation domain (ad) triggers transcription of the gene under the control of the factor. when expressed as separate protein chains, both domains remain fully functional: the bd still binds the dna but lacks a way of triggering transcription. the ad could trigger transcription but has no means of binding to the dna. for a two-hybrid test, two proteins x and y are fused to these domains resulting in two hybrids: x-bd and y-ad. if x binds to y, the resulting protein complex turns out to be a fully functional transcription factor. accordingly, an interaction is revealed by detecting transcription of the reporter gene under the control of the tf. in contrast to co-purifications, the interaction is tested in vivo in the two-hybrid system (usually in yeast, but other systems exist). the above description refers to small-scale experiments testing one pair of proteins at a time, but both approaches have successfully been extended to large-scale experiments testing thousands of pairs in very short time. while such high-throughput data is very valuable, especially for computational biology which often requires comprehensive input data, a word of caution is necessary. even with the greatest care and a maximum of thoughtful controls, high-throughput data usually suffer from a certain degree of false-positive results as well as false-negatives compared to carefully performed and highly optimized individual experiments. the ultimate source of information about protein interactions is provided by high-resolution three-dimensional structures of interaction complexes, such as the one shown in fig. 1 . spatial architectures obtained by x-ray crystallography or nmr spectroscopy provide atomic-level detail of interaction interfaces and allow for mechanistic understanding of interaction processes and their functional implications. additional kinetic, dynamic and structural aspects of protein interactions can be elucidated by electron and atomic force microscopy as well as by fluorescence resonance energy transfer. fig. 1 structural complex between rhoa, a small gtp protein belonging to the ras superfamily, and the catalytic gtpase activating domain of rhogap (graham et al. 2002) 3 protein interaction databases a huge number of protein-protein interactions has been experimentally determined and described in numerous scientific publications. public protein interaction databases that provide interaction data in form of structured, machine-readable datasets organized according to well documented standards have become invaluable resources for bioinformatics, systems biology and researchers in experimental laboratories. the data in these databases generally originate from two major sources: large-scale datasets and manually curated information extracted from the scientific literature. as pointed out above, the latter is considered substantially more reliable and large bodies of manually curated ppi data are often used as the gold standard against which predictions and large-scale experiments are benchmarked. of course, these reference data are far from complete and strongly biased. many factors, including experimental bias, preferences of the scientific community, and perceived biomedical relevance influence the chance of an interaction to be studied, discovered and published. in the manual annotation process it is not enough to simply record the interaction as such. additional information such as the type of experimental evidence, citations of the source, experimental conditions, and more need to be stored in order to convey a faithful picture of the data. annotation is a highly labor intensive task carried out by specially trained database curators. ppi databases can be roughly divided in two classes: specialized databases focusing on a single organism or a small set of species and general repositories which aim for a comprehensive representation of current knowledge. while the former are often well integrated with other information resources for the same organism, the latter strive for collecting all available interaction data including datasets from specialized resources. the size of these databases is growing constantly as more and more protein interactions are identified. as of writing (november 2007) , global repositories are approaching 200,000 pieces of evidence for protein interactions in various species. all of these databases offer convenient web interfaces that allow for interactively searching the database. in addition, the full datasets are usually provided for download in order to enable researchers to use the data in their own computational analyses. table 1 gives an overview of some important ppi databases. until relatively recently, molecular interaction databases like the ones listed in table 1 acted largely independently from each other. while they provided an extremely valuable service to the community in collecting and curating available molecular interaction data from the literature, they did so largely in an uncoordinated manner. each database had its own curation policy, feature set, and data formats. in 2002, the proteomics standards initiative (psi), a work group of the human proteome organization (hupo), set out to improve this situation, with contributions from a broad range of academic and commercial organizations, among them bind, cellzome, dip, glaxosmithkline, hybrigenics sa, intact, mint, mips, serono, and the universities of bielefeld, bordeaux, and cambridge. in a first step, a community standard for the representation of protein-protein interactions was developed, the psi mi format 1.0 (hermjakob et al. 2004) . recently, version 2.5 of the psi mi format has been published , extending the scope of the format from protein-protein interactions to molecular interactions in general, allowing to model for example protein-rna complexes. the psi mi format is a flexible xml format representing the interaction data to a high level of detail. n-ary interactions (complexes) can be represented as well as experimental conditions and technologies, quantitative parameters and interacting domains. the xml format is accompanied by detailed controlled vocabularies in obo format (harris et al. 2004 ). these vocabularies are essential for standardizing not only the syntax, but also the semantics of the molecular interaction representation. as an example, the "yeast two-hybrid technology" described above is referred to in the literature using many different synonyms, for example y2h, 2h, "yeast-two-hybrid", etc. while all of these terms refer to the same technology, filtering interaction data from multiple different databases based on this set of terms is not trivial. thus, the psi mi standard provides a set of now more than 1000 well-defined terms relevant to molecular interactions. figure 2 shows the intact advanced search tool with a branch of the hierarchical psi mi controlled vocabulary. figure 3 provides a partial graphical representation of the annotated xml schema, combined with an example dataset in psi mi xml format, reprinted from kerrien et al. (2007b) . for user-friendly distribution of simplified psi data to end users, the psi mi 2.5 standard also defines a simple tabular representation (mitab), derived from the biogrid format (breitkreutz et al. 2003) . while this format necessarily excludes details of interaction data like interacting domains, it provides a means to efficiently access large numbers of basic binary interaction records. the psi mi format is now widely implemented, with data available from biogrid, dip, hprd, intact, mint, and mips, among others. visualization tools like cytoscape (shannon et al. 2003) can directly read and visualize psi mi formatted data. comparative and integrative analysis of interaction data from multiple sources has become easier, as has the development of analysis tools which do not need to provide a plethora of input parsers any more. the annotated psi mi xml schema, a list of tools and 359 databases implementing it, as well as further information, are available from http:// www.psidev.info/. however, the development and implementation of a common data format is only one step towards the provision of consistent molecular interaction data to the scientific community. another key step is the coordination of the data curation process itself between different molecular interaction databases. without such synchronization, independent databases will often work on the same publications and insert the data into their systems, according to different curation rules, thus doing redundant work on some publications, while neglecting others. recognizing this issue, the dip, intact, and mint molecular interaction databases are currently synchronizing their curation efforts in the context of the imex consortium (http://imex.sf.net). these databases are now applying the same curation rules to provide a consistent high level of curation quality, and are synchronizing their fields of activity, each focusing on literature curation from a non-overlapping set of scientific journals. for these journals, the databases aim to insert all published interactions into the database shortly after publication. regular data exchange of all newly curated data between imes databases is currently in the implementation phase. to support the systematic representation and capture of relevant molecular interaction data supporting scientific publications, the hupo proteomics standards initiative has recently published "the minimum information required for reporting a molecular interaction experiment (mimix)" , detailing data items considered essential for the authors to provide, as well as a practical guide to efficient deposition of molecular interaction data in imex databases . the imex databases are also collaborating with scientific journals and funding agencies, to increasingly recommend data producers to deposit their data in an imex partner database prior to publication. database deposition prior to publication not only ensures public availability of the data at the time of publication, but also provides important quality control, as database curators often assess the data in much more detail than reviewers. the psi journal collaboration efforts are starting to show first results. nature biotechnology, nature genetics, and proteomics are now recommending that authors deposit molecular interaction data in a relevant public domain database prior to publication, a key step to a better capture of published molecular interaction data in public databases, and to overcome the current fragmentation of molecular interaction data. as an example of a molecular interaction database implementing the psi mi 2.5 standard, we will provide a more detailed description of the intact molecular interaction database ), accessible at http://www.ebi.ac.uk/intact. intact is a curated molecular interaction database active since 2002. intact follows a full text curation policy, publications are read in full by the curation team, and all molecular interactions contained in the publication are inserted into the database, containing basic facts like the database accession numbers of the proteins participating in an interaction, but also details like experimental protein modifications, which can have an impact on assessments of confidence in the presence or absence of interactions. each database record is cross-checked by a senior curator for quality control. on release of the record, the corresponding author of the publication is automatically notified (where an email address is available), and requested to check the data provided. any corrections are usually inserted into the next weekly release. while such a detailed, high quality approach is slow and limits coverage, the provision of high quality reference datasets is an essential service both for biological analysis, and for the training and validation of automatic methods for computational prediction of molecular interactions. as it is impossible for any single database, or even the collaborating imex databases, to fully cover all published interactions, curation priorities have to be set. any direct data depositions supporting manuscripts approaching peer review have highest priority. next, for some journals (currently cell, cancer cell, and proteomics) intact curates all molecular interactions published in the journal. finally, several special curation topics are determined in collaboration with external communities or collaborators, where intact provides specialized literature curation and collaborates in the analysis of experimental datasets, for example around a specific protein of interest (camargo et al. 2006) . as of november 2007, intact contains 158.000 binary interactions supported by ca. 3,000 publications. the intact interface implements a standard "simple search" box, ideal for search by uniprot protein accession numbers, gene names, species, or pubmed identifiers. the advanced search tool (fig. 2) provides field-specific searches as well a specialized search taking into account the hierarchical structure of controlled vocabularies. a default search for the interaction detection method "2 hybrid" returns 30,251 interactions, while a search for "2 hybrid" with the tickbox "include children" activated returns more than twice that number, 64,589 interactions. the hierarchical search automatically includes similarly named methods like "two hybrid pooling approach", but also "gal4 vp16 complement". search results are initially shown in a tabular form based on the mitab format, which can also be directly downloaded. each pairwise interaction is only listed once, with all experimental evidence listed in the appropriate columns. the final column provides access to a detailed description of each interaction as well as a graphical representation of the interaction in is interaction neighborhood graph. for interactive, detailed analysis, interaction data can be loaded into tools like cytoscape (see below) via the psi 2.5 xml format. all intact data is freely available via the web interface, for download in psi mi tabular or xml format, and computationally accessible via web services. intact software is open source, implemented in java, with hibernate (www.hibernate.org/) for the object-relational mapping to oracle tm or postgres, and freely available under the apache license, version 2 from http://www.ebi.ac.uk/intact. on a global scale, protein-protein interactions participate in the formation of complex biological networks which, to a large extent, represent the paths of communication and metabolism of an organism. these networks can be modeled as graphs making them amenable to a large number of well established techniques of graph theory and social network analysis. even though interaction networks do not directly encode cellular processes nor provide information on dynamics, they do represent a first step towards a description of cellular processes, which is ultimately dynamic in nature. for instance, protein-interaction networks may provide useful information on the dynamics of complex assembly or signaling. in general, investigating the topology of protein interaction, metabolic, signaling, and transcriptional networks allows researchers to reveal the fundamental principles of molecular organization of the cell and to interpret genome data in the context of large-scale experiments. such analyses have become an integral part of the genome annotation process: annotating genomes today increasingly means annotating networks. a protein-protein interaction network summarizes the existence of both stable and transient associations between proteins as an (undirected) graph: each protein is represented as a node (or vertex), an edge between two proteins denotes the existence of an interaction. interactions known to occur in the actual cell ( fig. 4a ) can thus be represented as an abstract graph of interaction capabilities (fig. 4b ). as such a graph is limited by definition to binary interactions, its construction from a database of molecular interactions may involve arbitrary choices. for instance, an n-ary interaction measured by co-purification can be represented using either the clique (all binary interactions between the n proteins are retained) or the spoke model (only edges connecting the "captured" protein to co-purified proteins are retained). once a network has been reconstructed from protein interaction data, a variety of statistics on network topology can be computed, such as the distribution of vertex degrees, the distribution of the clustering coefficient and other notions of density, the distribution of shortest path length between vertex pairs, or the distribution of network motifs occurrences (see for a review). these measures can be used to describe networks in a concise manner, to compare, group or contrast different networks, and to identify properties characteristic of a network or a class of network under study. some topological properties may be interpreted as traces of underlying biological mechanisms, shedding light on their dynamics, their evolution, or both and helping connect structure to function (see the "network modules" section below). for instance, most interaction networks seem to exhibit scale-free topology (jeong et al. 2001; yook et al. 2004) , i.e. their degree distribution (the probability that a node has exactly k links) approximates a power law p(k) $ k -g , meaning that most proteins have few interaction partners but some, the so-called "hubs", have many. as an example of derived evolutionary insight, it is easy to show that networks evolving by growth (addition of new nodes) and preferential attachment (new nodes are more likely to be connected to nodes with more connections) will exhibit scale-free topology (degree distribution approximates a power-law) and hubs (highly connected nodes). a simple model of interaction network evolution by gene duplication, where a duplicate initially keeps the same interaction partners as the original, generates preferential attachment, thus providing a candidate explanation for the scale-free nature and the existence of hubs in these networks . interacting proteins are denoted as p1, p2, etc. (b) a graph representation of the protein interactions shown in a. each node represents a protein, and each edge connects proteins that interact. (c) information on protein interactions obtained by different methods. (d) protein interaction network derived from experimental evidence shown in c. as in a, each node is a protein, and edges connect interactors. edges a colored according to the source of evidence: red -3d, green -apms, brown -y2h, magenta -prof, yellow -lit, blue -loc a corresponding functional interpretation of hubs and scale-free topology has been proposed in terms of robustness. scale-free networks are robust to component failure, as random failures are likely to affect low degree nodes and only failures affecting hub nodes will significantly change the number of connected components and the length of shortest paths between node pairs. deletion analyses have, perhaps unsurprisingly, confirmed that highly connected proteins are more likely to be essential (winzeler et al. 1999; giaever et al. 2002; gerdes et al. 2003) . most biological interpretations that have been proposed for purely topological properties of interaction networks have been the subject of heated controversies, some of which remain unsolved to this day (e.g. (he and zhang 2006; yu et al. 2007 ) on hubs). one often cited objection to any strong interpretation is the fact that networks reconstructed from high-throughput interaction data constitute very rough approximations of the "real" network of interactions taking place within the cell. as illustrated in fig. 4c , interaction data used in a reconstruction typically result from several experimental methods, often complemented with prediction schemes. each specific method can miss real interactions (false negatives) and incorrectly identify other interactions (false positives), resulting in biases that are clearly technology-dependent (gavin et al. 2006; legrain and selig 2000) . assessing false-negative and false-positive rates is difficult since there is no gold standard for positive interactions (protein pairs that are known to interact) or, more importantly, for negative interactions (protein pairs that are known not to interact). using less-than-ideal benchmark interaction sets, estimates of 30-60% false positives and 40-80% false negatives have been proposed for yeast-two-hybrid and copurification based techniques (aloy and russell 2004) . in particular, a comparison of several high-throughput interaction datasets on yeast, showing low overlap, has confirmed that each study covers only a small percentage of the underlying interaction network (von mering et al. 2002 ) (see also "estimates of the number of protein interactions" below). integration of interaction data from heterogeneous sources towards interaction network reconstruction can help compensate for these limitations. the basic principle is fairly simple and rests implicitly on a multigraph representation: several interaction networks to be integrated, each resulting from a specific experimental or predictive method, are defined over the same set of proteins. integration is achieved by merging them into a single network with several types of linksor edge colors-each drawn from one of the component networks. some edges in the multigraph may be incorrect, while some existing interactions may be missing from the multigraph, but interactions confirmed independently by several methods can be considered reliable. figure 4d shows the multigraph that corresponds to the evidence from fig. 4c and can be used to reconstruct the actual graph in fig. 4b . in practice, integration is not always straightforward: networks are usually defined over subsets of the entire gene or protein complement of a species, and meaningful integration requires that the overlap of these subsets be sufficiently large. in addition, if differences of reliability between network types are to be taken into account, an integrated reliability scoring scheme needs to be designed (jansen et al. 2003; von mering et al. 2007 ) with the corresponding pitfalls and level of arbitrariness involved in comparing apples and oranges. existing methods can significantly reduce false positive rates on a subset of the network, yielding a subnetwork of highreliability interactions. the tremendous amounts of available molecular interaction data raise the important issue of how to visualize them in a biologically meaningful way. a variety of tools have been developed to address this problem; two prominent examples are visant (hu et al. 2005) and cytoscape (shannon et al. 2003) . a recent review of further network visualization tools is provided by suderman and hallett (2007) . in this section, we focus on cytoscape (http://www.cytoscape.org) and demonstrate its use for the investigation of protein-protein interaction networks. for a more extensive protocol on the usage of cytoscape, see (cline et al. 2007) . cytoscape is a stand-alone java application that is available for all major computer platforms. this software provides functionalities for (i) generating biological networks, either manually or by importing interaction data from various sources, (ii) filtering interactions, (iii) displaying networks using graph layout algorithms, (iv) integrating and displaying additional information like gene expression data, and (v) performing analyses on networks, for instance, by calculating topological network properties or by identifying functional modules. one advantage of cytoscape over alternative visualization software applications is that cytoscape is released under the open-source lesser general public license (lgpl). this license basically permits all forms of software usage and thus helps to build a large user and developer community. third-party java developers can easily enhance the functionality of cytoscape by implementing own plug-ins, which are additional software modules that can be readily integrated into the cytoscape platform. currently, there are more than forty plug-ins publicly available, with functionalities ranging from interaction retrieval and integration across topological network analysis, detection of network motifs, protein complexes, and domain interactions, to visualization of subcellular protein localization and bipartite networks. a selection of popular cytoscape plug-ins is listed in table 2 . in the following, we will describe the functionalities of cytoscape in greater detail. the initial step of generating a network can be accomplished in different ways. first, the user can import interaction data that are stored in various flat file or xml formats such as biopax, sbml, or psi-mi, as described above. second, the user can directly retrieve interactions from several public repositories from within cytoscape. a number table 2 ). third, the user can utilize a text-mining plug-in that builds networks based on associations found in publication abstracts (agilent literature search; table 2 ). while these associations are not as reliable as experimentally derived interactions, they can be helpful when the user is investigating species that are not well covered yet in the current data repositories. fourth, the user can directly create or manipulate a network by manually adding or removing nodes (genes, proteins, domains, etc.) and edges (interactions or relationships). in this way, expert knowledge that is not captured in the available data sets can be incorporated into the loaded network. generated networks can be further refined by applying selections and filters in cytoscape. the user can select nodes or edges by simply clicking on them or framing a selection area. in addition, starting with at least one selected node, the user can incrementally enlarge the selection to include all direct neighbor nodes. cytoscape also provides even sophisticated search and filter functionality for selecting particular nodes and edges in a network based on different properties; in particular, the enhanced search plug-in (table 2) improves the built-in search functionality of cytoscape. filters select all network parts that match certain criteria, for instance, all human proteins or all interactions that have been detected using the yeast two-hybrid system. once a selection has been made, all selected parts can be removed from the network or added to another network. the main purpose of visualization tools like cytoscape is the presentation of biological networks in an appropriate manner. this can usually be accomplished by applying graph layout algorithms. sophisticated layouts can assist the user in revealing specific network characteristics such as hub proteins or functionally related protein clusters. cytoscape offers various layout algorithms, which can be categorized as circular, hierarchical, spring-embedded (or force-directed), and attribute-based layouts (fig. 5 ). further layouts can be included using the cytoscape plug-in architecture, for example, to arrange protein nodes according to their subcellular localization or to their pathways assignments (bubblerouter, cerebral; table 2 ). some layouts may be more effective than others for representing molecular networks of a certain type. the spring-embedded layout, for instance, has the effect of exposing the inherent network structure, thus identifying hub proteins and clusters of tightly connected nodes. it is noteworthy that current network visualization techniques have limitations, for example, when displaying extremely large or dense networks. in such cases, a simple graphical network representation with one node for each interaction partner, as it is initially created by cytoscape, can obfuscate the actual network organization due to the sheer number of nodes and edges. one potential solution to this problem is the introduction of meta-nodes (metanode plug-in; table 2 ). a meta-node combines and replaces a group of other nodes. meta-nodes can be collapsed to increase clarity of the visualization and expanded to increase the level of detail (fig. 6 ). an overview of established and novel visualization techniques for biological networks on different scales is presented in (hu et al. 2007 ). all layouts generated by cytoscape are zoomable, enabling the user to increase or decrease the magnification, and they can be further customized by aligning, scaling, or rotating selected network parts. additionally, the user can define the graphical network representation through visual styles. these styles define the colors, sizes, and shapes of all network parts. a powerful feature of cytoscape is its ability of visually mapping additional attribute values onto network representations. both nodes and edges can have arbitrary attributes, for example, protein function names, the number of interactions (node degree), expression values, the strength and type of an interaction, or confidence values for interaction reliability. these attributes can be used to adapt the network illustration by dynamically changing the visual styles of individual network parts (fig. 7) . for example, this feature enables highlighting trustworthy interactions by assigning (table 2 ). all protein nodes with subcellular localizations different from plasma membrane are combined into meta-nodes. these meta-nodes can be collapsed or expanded to increase clarity or detailedness, respectively different line styles or sizes to different experiment types (discrete mapping of an edge attribute), to spot network hubs by changing the size of a node according to its degree (discrete or continuous mapping of a node attribute), or to identify functional network patterns by coloring protein nodes with a color gradient according to their expression level (continuous mapping of a node attribute). hence, it is possible to simultaneously visualize different data types by overlaying them with a network model. in order to generate new biological hypotheses and to gain insights into molecular mechanisms, it is important to identify relevant network characteristics and patterns. for this purpose, the straightforward approach is the visual exploration of the network. table 2 lists a selection of cytoscape plug-ins that assist the user in this analysis task, for instance, by identifying putative complexes (mcode), by grouping proteins that show a similar expression profile (jactivemodules), or by identifying overrepresented go terms (bingo, golorize). however, the inclusion of complex data such as time-series results or diverse gene ontology (go) terms into the network visualization might not be feasible without further software support. particularly in case of huge, highly connected, or dynamic networks, more advanced visualization techniques will be required in the future. fig. 7 visual representation of a subset of the gal4 network in yeast. the protein nodes are colored with a red-to-green gradient according to their expression value; green represents the lowest, red the highest value, and blue a missing value. the node size indicates the number of interactions (node degree); the larger a node, the higher is its degree. the colors and styles of the edges represent different interaction types; solid black lines represent protein-protein, dashed red lines protein-dna interactions in addition to the visual presentation of interaction networks, cytoscape can also be used to perform statistical analyses. for instance, the networkanalyzer plug-in (assenov et al. 2008 ) computes a large variety of topology parameters for all types of networks. the computed simple and complex topology parameters are represented as single values and distributions, respectively. examples of simple parameters are the number of nodes and edges, the average number of neighbors, the network diameter and radius, the clustering coefficient, and the characteristic path length. complex parameters are distributions of node degrees, neighborhood connectivities, average clustering coefficients, and shortest path lengths. these computed statistical results can be exported in textual or graphical form and are additionally stored as node attributes. the user can then apply the calculated attributes to select certain network parts or to map them onto the visual representation of the analyzed network as described above (fig. 7) . it is also possible to fit a power law to the node degree distribution, which can frequently indicate a so-called scale-free network with few highly connected nodes (hubs) and many other nodes with a small number of interactions. scale-free networks are especially robust against failures of randomly selected nodes, but quite vulnerable to defects of hubs (albert 2005) . how many ppis exist in a living cell? the yeast genome encodes approximately 6300 gene products which means that the maximal possible number of interacting protein pairs in this organism is close to 40 million, but what part of these potential interactions are actually realized in nature? for a given experimental method, such as the two-hybrid essay, the estimate of the total number of interactions in the cell is given by where n measured is the number of interactions identified in the experiment, and r fp and r fn are false positive and false negative rates of the method. r fn can be roughly estimated based on the number of interactions known with confidence (e.g., those confirmed by three-dimensional structures) that are being recovered by the method. assessing r fp is much more difficult because no experimental information on proteins that do not interact is currently available. since it is known that proteins belonging to the same functional class often interact, one very indirect way of calculating r fn is as the fraction of functionally related proteins not found to be interacting. an even more monumental problem is the estimation of the total number of unique structurally equivalent interaction types existing in nature. an interaction type is defined as a particular mutual orientation of two specific interacting domains. in some cases homologous proteins interact in a significantly different fashion while in other cases proteins lacking sequence similarity engage in interactions of the same type. in general, however, interacting protein pairs sharing a high degree of sequence similarity (30-40% or higher) between their respective components almost always form structurally similar complexes (aloy et al. 2003) . this observation allows utilization of available atomic resolution structures of complexes for building useful models of closely related binary complexes. the total number of interaction types can then be estimated as follows: where the interaction similarity multiplier c reflects the clustering of all interactions of the same type, and e all-species extrapolates from one biological species to all organisms. aloy and russel (2004) derived an estimate for c by grouping interactions between proteins that share high sequence similarity, as discussed above. c depends on the number of paralogous sequences encoded in a given genome. for small prokaryotic organisms it is close to 1 while for larger and more redundant genomes it adopts smaller values, typically in the range of 0.75-0.85. the multiplier for all species e allspecies can be derived by assessing what fraction of known protein families is encoded in a given genome. based on the currently available data this factor is close to 10 for bacteria, which means that a medium size prokaryotic organism contains around one tenth of all protein families. for eukaryotic organisms e all-species lies between 2 and 4. for the comprehensive two-hybrid screen of yeast by (uetz 2000) in which 936 interactions between 987 proteins were identified, aloy and russell (2004) estimated c, r fp, and r fn , and e all-species to be 0.85, 3.92, 0.55, and 3.35 respectively, leading to an estimated 1715 different interaction types in yeast alone, and 5741 over all species. based on the two-hybrid interaction map of the fly (giot 2003 ) the number of all interaction types in nature is estimated to be 9962. it is thus reasonable to expect the total number of interaction types to be around 10,000, and only 2000 are currently known. beyond binary interactions, proteins often form large molecular complexes involving multiple subunits (fig. 8) . these complexes are much more than a random snapshot of a group of interacting proteinsthey represent large functional entities which remain stable for long periods of time. many such protein complexes have been elucidated step by step over time and recent advances in high-throughput technology have led to largescale studies revealing numerous new protein complexes. the preferred technology for this kind of experiment is initial co-purification of the complexes followed by the identification of the member proteins by mass spectrometry. as the bakers yeast s. cerevisiae is one of the most versatile model organisms used in molecular biology, it is not surprising that the first large-scale complex datasets were obtained in this species (gavin et al. 2002; ho et al. 2002; gavin et al. 2006; krogan et al. 2006 ). the yeast protein interaction database mpact (guldener et al. 2006 ) provides access to 268 protein complexes based on careful literature annotation composed of 1237 different proteins plus over 1000 complexes from large-scale experiments which contain more than 2000 distinct proteins. these numbers contain some redundancy with respect to complexes, due to slightly different complex composition found by different groups or experiments. nevertheless, the dataset covers about 40% of the s.cerevisiae proteome. while many complexes comprise only a small number of different proteins, the largest of them features an impressive 88 different protein species. a novel manually annotated database, corum (ruepp et al. 2008 ) contains literature-derived information about 1750 mammalian multi-protein complexes. over 75% of all complexes contain between three and six subunits, while the largest molecular structure, the spliceosome, consists of 145 components (fig. 9 ). modularity has emerged as one of the major organizational principles of cellular processes. functional modules are defined as molecular ensembles with an autonomous function (hartwell et al. 1999) . proteins or genes can be partitioned into modules based on shared patterns of regulation or expression, involvement in a common metabolic or regulatory pathway, or membership in the same protein complex or subcellular structure. modular representation and analysis of cellular processes allows for interpretation of genome data beyond single gene behavior. in particular, analysis of modules provides a convenient framework for studying the evolution of living systems (snel and huynen 2004) . multiprotein complexes represent one particular type of functional modules in which individual components engage in physical interactions to execute a specific cellular function. algorithmically, modular architectures can be defined as densely interconnected groups of nodes on biological networks (for an excellent review of available methods see (sharan et al. 2007 ). statistically significant functional subnetworks are characterized by a high degree of local clustering. the density of a cluster can be represented as a function q(m,n) = 2m/(n(n à 1)), where m is the number of interactions between the n nodes of the cluster (spirin and mirny 2003) . q thus takes values between 0 for a set of unconnected nodes and 1 for a fully connected cluster (clique). the statistical significance of q strongly depends on the size of the graph. it is obvious that random clusters with q ¼ 1 involving just three proteins are very likely while large clusters with q ¼ 1 or even with values below 0.5 are extremely unlikely. in order to compute the statistical significance of a cluster with n nodes and m connections spirin and mirny calculate the expected number of such clusters in a comparable random graph and then estimate the likelihood of having m or more interactions within a given set of n proteins given the number of interactions that each of these proteins has. significant dense clusters identified by this procedure on a graph of protein interactions were found to correspond to functional modules most of which are involved in transcription regulation, cell-cycle/ cell-fate control, rna processing, and protein transport. however, not all of them constitute physical protein complexes and, in general, it is not possible to predict whether a given module corresponds to a multiprotein complex or just to a group of functionally coupled proteins involved in the same cellular process. the search for significant subgraphs can be further enhanced by considering evolutionary conservation of protein interactions. with this approach protein complexes are predicted from binary interaction data by network alignment which involves comparing interaction graphs between several species (sharan et al. 2005) . first, proteins are grouped by sequence similarity such that each group contains one protein from each species, and each protein is similar to at least one other protein in the group. then a composite interaction network is created by joining with edges those pairs of groups that are linked by at least one conserved interaction. again, dense clusters on such network alignment graph are often indicative of multiprotein complexes. an alternative computational method for deriving complexes from noisy large-scale interaction data relies on a "socio-affinity" index which essentially reflects the frequency with which proteins form partnerships detected by co-purification (gavin et al. 2006) . this index was shown to correlate well with available three-dimensional structure data, dissociation constants of protein-protein interactions, and binary interactions identified by the two-hybrid techniques. by applying a clustering procedure to a matrix containing the values of the socio-affinity index for all yeast protein pairs found to associate by affinity purification, 491 complexes were predicted, with over a half of them being novel and previously unknown. however, dependent on the analysis parameters distinct complex variants (isoforms) are found that differ from in terms of their subunit composition. those proteins present in most of the isoforms of a given complex constitute its core while variable components present only in a small number of isoforms can be considered "attachments" (fig. 10) . furthermore, some stable, typically smaller protein groups can be found in multiple attachments in which case they are fig. 10 definitions of complex cores, attachments, and modules. redrawn and modified with permission from (gavin et al. 2006) called "modules". stable functional modules can thus be flexibly used in the cell in a variety of functional contexts. proteins frequently associated with each other in complex cores and modules are likely to be co-expressed and co-localized. in this section, we offer a computational perspective on utilizing protein network data for molecular medical research. the identification of novel therapeutic targets for diseases and the development of drugs has always been a difficult, time-consuming and expensive venture (ruffner et al. 2007) . recent work has charted the current pharmacological space using different networks of drugs and their protein targets (paolini et al. 2006; keiser et al. 2007; kuhn et al. 2008; yildirim et al. 2007 ) based on biochemical relationships like ligand binding energy and molecular similarity or on shared disease association. above all, since many diseases are due to the malfunctioning of proteins, the systematic determination and exploration of the human interactome and homologous protein networks of model organisms can provide considerable new insight into pathophysiological processes (giallourakis et al. 2005) . knowledge of protein interactions can frequently improve the understanding of relevant molecular pathways and the interplay of various proteins in complex diseases (fishman and porter 2005) . this approach may result in the discovery of a considerable number of novel drug targets for the biopharmaceutical industry, possibly affording the development of multi-target combination therapeutics. observed perturbations of protein networks may also offer a refined molecular description of the etiology and progression of disease in contrast to phenotypic categorization of patients (loscalzo et al. 2007 ). molecular network data may help to improve the ability of cataloging disease unequivocally and to further individualize diagnosis, prognosis, prevention, and therapy. this will require a network-based approach that does not only include protein interactions to differentiate pathophenotypes, but also other types of molecular interactions as found in signaling cascades and metabolic pathways. furthermore, environmental factors like pathogens interacting with the human host or the effects of nutrition need to be taken into account. after large-scale screens identified enormous amounts of protein interactions in organisms like yeast, fly, and worm (goll and uetz 2007) , which also serve as model systems for studying many human disease mechanisms (giallourakis et al. 2005) , experimental techniques and computational prediction methods have recently been applied to generate sizable networks of human proteins (cusick et al. 2005; stelzl and wanker 2006; assenov et al. 2008; ram ırez et al. 2007 ). in addition, comprehensive maps of protein interactions inside pathogens and between pathogens and the human host have been compiled for bacteria like e. coli, h. pylori, c. jejuni, and other species (noirot and noirot-gros 2004) , for many viruses such as herpes viruses, the epstein-chapter 6.2: protein-protein interactions: analysis and prediction barr virus, the sars coronavirus, hiv-1, the hepatitis c virus, and others (uetz et al. 2004) , and for the malaria parasite p. falciparum (table 3) . those extensive network maps can now be explored to identify potential drug targets and to block or manipulate important protein-protein interactions. furthermore, different experimental methods are also used to expand the known interaction networks around pathway-centric proteins like epidermal growth factor receptors (egfrs) (tewari et al. 2004; oda et al. 2005; jones et al. 2006) , smad and transforming growth factor-b (tgfb) (colland and daviet 2004; tewari et al. 2004; barrios-rodiles et al. 2005) , and tumor necrosis factor-a (tnfa) and the transcription factor nf-kb (bouwmeester et al. 2004 ). all of these proteins are involved in sophisticated signal transduction cascades implicated in various important disease indications ranging from cancer to inflammation. the immune system and toll-like receptor (tlr) pathways were the subject of other detailed studies (oda and kitano 2006) . apart from that, protein networks for longevity were assembled to research ageing-related effects (xue et al. 2007 ). high-throughput screens are also conducted for specific disease proteins causative of closely related clinical and pathological phenotypes to unveil molecular interconnections between the diseases. for example, similar neurodegenerative disease phenotypes are caused by polyglutamine proteins like huntingtin and over twenty ataxins. although they that are not evolutionarily related and their expression is not restricted to the brain, they are responsible for inherited neurotoxicity and age-dependent dementia only in specific neuron populations (ralser et al. 2005) . yeast two-hybrid screens revealed an unexpectedly dense interaction network of those disease proteins forming interconnected subnetworks (fig. 11) , which suggests common pathways affected in disease (goehler et al. 2004; lim et al. 2006) . some of the protein-protein interactions may be involved in mediating neurodegeneration and thus may be tractable for drug inhibition, and several interaction partners of ataxins could additionally be shown to be potential disease modifiers in a fly model (kaltenbach et al. 2007) . a number of methodological approaches concentrate on deriving correlations between common topological properties and biological function from subnetworks around proteins that are associated with a particular disease phenotype like cancer. recent studies report that human disease-associated proteins with similar clinical and pathological features tend to be more highly connected among each other than with other proteins and to have more similar transcription profiles xu and li 2006; goh et al. 2007 ). this observation points to the existence of disease-associated functional modules. interestingly, in contrast to disease genes, essential genes whose defect may be lethal early on in life are frequently found to be hubs central to the network. further work focused on specific disease-relevant networks. for instance, to analyze experimental asthma, differentially expressed genes were mapped onto a protein interaction network ). here, highly connected nodes tended to have smaller expression changes than peripheral nodes. this agrees with the general notion that disease-causing genes are typically not central in the network. similarly, a comprehensive protein network analysis of systemic inflammation in human subjects investigated blood leukocyte gene expression patterns when receiving an inflammatory stimulus, a bacterial endotoxin, to identify functional modules perturbed in response to this stimulus (calvano et al. 2005) . topological criteria and gene expression data were also used to search protein networks for functional modules that are relevant to type 2 diabetes mellitus or to different types of cancer (jonsson and bates 2006; cui et al. 2007; lin et al. 2007; pujana et al. 2007 ). moreover, it was recently demonstrated that the integration of gene expression profiles with subnetworks of interacting proteins can lead to improved prognostic markers for breast cancer outcome that are more reproducible between patient cohorts than sets of individual genes selected without network information (chuang et al. 2007 ). in drug discovery, protein networks can help to design selective inhibitors of protein-protein interactions which target specific interactions of a protein, but do not affect others (wells and mcclendon 2007) . for example, a highly connected protein (hub) may be a suitable target for an antibiotic whereas a more peripheral protein with few interaction partners may be more appropriate for a highly specific drug that needs to avoid side effects. thus, topological network criteria are not only useful for characterizing disease proteins, but also for finding drug targets. the diversity of interactions of a targeted protein could also help in predicting potential side effects of a drug. apart from that, it is remarkable that some potential drugs have been found to be less effective than expected due to the intrinsic robustness of living systems against perturbations of molecular interactions (kitano 2007) . furthermore, mutations in proteins cause genetic diseases, but it is not always easy to distinguish protein interactions impaired by mutated binding sites from other disease causes like structural instability induced by amino acid mutations. nowadays many genome-wide association and linkage studies for human diseases suggest genomic loci and linkage intervals that contain candidate genes encoding snps and mutations of potential disease proteins (kann 2007) . since the resultant list of candidates frequently contain dozens or even hundreds of genes, computational approaches have been developed to prioritize them for further analyses and experiments. in the following, we will demonstrate the variety of available prioritization approaches by explicating three recent methods that utilize protein interaction data in addition to the inclusion of other sequence and function information. all methods capitalize on the above described observation that closely interacting gene products often underlie polygenic diseases and similar pathophenotypes (oti and brunner 2007) . using protein-protein interaction data annotated with reliability values, lage et al. (2007) first predict human protein complexes for each candidate protein. they then score the pairwise phenotypic similarity of the candidate disease with all proteins within each complex that are associated with any disease. the scoring function basically measures the overlap of the respective disease phenotypes as recorded in text entries of omim (online mendelian inheritance in man) (hamosh et al. 2005 ) based on the vocabulary of umls (unified medical language system) (bodenreider 2004) . lastly, all candidates are prioritized by the probability returned by a bayesian predictor trained on the interaction data and phenotypic similarity. therefore, this method depends on the premise that the phenotypic effects caused by any disease-affected member in a predicted protein complex are very similar to each other. another prioritization approach by franke et al. (2006) does not make use of overlapping disease phenotypes and primarily aims at connecting physically disjoint genomic loci associated with the same disease using molecular networks. at the beginning, their method prioritizer performs a bayesian integration of three different network types of gene/protein relationships. the latter are derived from functional similarity using gene ontology annotation, microarray coexpression, and proteinprotein interaction. this results in a probabilistic human network of general functional links between genes. prioritizer then assesses which candidate genes contained in different disease loci are closely connected in this gene-gene network. to this end, the score of each candidate is initially set to zero, but it is increased iteratively during network exploration by a scoring function that depends on the network distance of the respective candidate gene to candidates inside another genomic loci. this procedure finally yields separate prioritization lists of ranked candidate genes for each genomic loci. in contrast to the integrated gene-gene network used by prioritizer, the endeavour system (aerts et al. 2006 ) directly compares candidate genes with known disease genes and creates different ranking lists of all candidates using various sources of evidence for annotated relationships between genes or proteins. the evidence can be derived from literature mining, functional associations based on gene ontology annotations, co-occurrence of transcriptional motifs, correlation of expression data, sequence similarity, common protein domains, shared metabolic pathway membership, and protein-protein interactions. at the end, endeavour merges the resultant ranking lists using order statistics and computes an overall prioritization list of all candidate genes. finally, it is important to keep in mind that current datasets of human protein interactions may still contain a significant number of false interactions and thus biological and medical conclusions derived from them should always be taken with a note of caution, in particular, if no good confidence measures are available. a comprehensive atlas of protein interactions is fundamental for a better understanding of the overall dynamic functioning of the living organisms. these insights arise from the integration of functional information, dynamic data and protein interaction networks. in order to fulfill the goal of enlarging our view of the protein interaction network, several approaches must be combined and a crosstalk must be established among experimental and computational methods. this has become clear from comparative evaluations which show similar performances for both types of methodologies. in fact, over the recent years this field has grown into one of the most appealing fields in bioinformatics. evolutionary signals result from restrictions imposed by the need to optimize the features that affect a given interaction and the nature of these features can differ from interaction to interaction. consequently, a number of different methods have been developed based a range of different evolutionary signals. this section is devoted to a brief review of some of these methods. these techniques are based on the similarity of absence/presence profiles of interacting proteins. in its original formulation (gaasterland and ragan 1998; huynen and bork 1998; pellegrini et al. 1999; marcotte et al. 1999a ) the phylogenetic profiles were codified as 0/1 vectors for each reference protein according to the absence/presence of proteins of the studied family in a set of fully sequenced organisms (see fig. 12a ). the vectors for different reference sequences are compared by using the hamming distance (pellegrini et al. 1999) between vectors. this measure counts the number of differences between two binary vectors. the rationale for this method is that both interacting proteins must be present in an organism and that reductive evolution will remove unpaired proteins in the rest of the organisms. proposed improvements include the inclusion of quantitative measures of sequence divergence (marcotte et al. 1999b; date and marcotte 2003) and the ability to deal with biases in the taxonomic distribution of the organisms used (date and marcotte 2003; barker and pagel 2005) . these biases are due to the intuitive fact that evolutionarily similar organisms will share a higher number of protein and genomic features (in this case presence/absence of an orthologue). to reduce this problem, date et al. used mutual information from sequence divergent profiles for measuring the amount of information shared by both vectors. mutual information is calculated as: miðp1; p2þ ¼ hðp1þ þ hðp2þ à hðp1; p2þ; where hðp1þ ¼ p pðp1þ ln pðp1þ is the marginal entropy of the probability distribution of protein p1 sequence distances and hðp1; p2þ ¼ à p p pðp1; p2þ ln pðp1; p2þ is the joint entropy of the probability distributions of both protein p1 and p2 sequence distances. the corresponding probabilities are calculated from the whole distribution of orthologue distances for the organisms. in this way, the most likely evolutionary distances between orthologues from a pair of organisms will produce smaller entropies and consequently smaller values of mutual information. this formulation should implicitly reduce the effect of taxonomic biases. in an interesting work, published recently by barker et al. (2007) , the authors showed that detection of correlated gene-gain/gene-loss events improves the predictions by reducing the number of false positives due to taxonomic biases. the phylogenetic profiling approach has been shown to be quite powerful, because its simple formulation has allowed the exploration of a number of alternative interdependencies between proteins. this is the case for enzyme "displacement" in metabolic pathways detected as anti-correlated profiles (morett et al. 2003) , and for complex dependence relations among triplets of proteins (bowers et al. 2004) . phylogenetic profiles have also been correlated with bacterial traits to predict the genes related to particular phenotypes (korbel et al. 2005) . the main drawbacks of these methods are the difficulty of dealing with essential proteins (where there is no absence information) and the requirement for the genomes under study to be complete (to establish the absence of a family member). fig. 12 prediction of protein interactions based on genomic and sequence features. information coming from the set of close homologs of the proteins p1 and p2 from the organism 1 in other organisms can be used to predict an interaction between these proteins. (a) phylogenetic profiling. presence/absence of a homolog of both proteins in different organisms is coded as the corresponding two 1/0 profiles (most simple approach) and an interaction is predicted for very similar profiles. (b) similarity of phylogenetic trees. multiple sequence alignments are built for both sets of proteins and phylogenetic trees are derived from the proteins with a possible partner present in its organism. proteins with highly similar trees are predicted to interact. (c) gene neighbourhood conservation. genome closeness is checked for those genes coding for both sets of homologous proteins. interaction is predicted if gene pairs are recurrently close to each other in a number of organisms. (d) gene fusion. finding the proteins containing different sequence regions homologous to each of the two proteins is used to predict an interaction between them similarity in the topology of phylogenetic trees of interacting proteins has been qualitatively observed in a number of cases (fryxell 1996; pages et al. 1997; goh et al. 2000) . the extension of this observation to a quantitative method for the prediction of protein interactions requires measuring the correlation between the similarity matrices of the explored pairs of protein families (goh et al. 2000) . this formulation allows systematic evaluation of the validity of using the original observation as a signal of protein interaction (pazos and valencia 2001) . the general protocol for these methods is illustrated in fig. 12b . it includes the building of the multiple sequence alignment for the set of orthologues (one per organism) related to every query sequence, the calculation of all protein pair evolutionary distances (derived from the corresponding phylogenetic trees) and finally the comparison of evolutionary distance matrices of pairs of query proteins using pearsons correlation coefficient. protein pairs with highly correlated distance matrices are predicted to be more likely to interact. although this signal has been shown to be significant, the underlying process responsible for this similarity is still controversial (chen and dokholyan 2006) . there are two main hypotheses for explaining this phenomenon. the first hypothesis suggests that this evolutionary similarity comes from the mutual adaptation (co-evolution) of interacting proteins and the need to retain interaction features while sequences diverge. the second hypothesis implicates external factors. in this scenario, the restrictions imposed by evolution on the functional process implicating both proteins would be responsible for the parallelism of their phylogenetic trees. although the relative importance of both factors is still not clear, the predictive power of similarities in phylogenetic trees is not affected. indeed, a number of developments have improved the original formulation (pazos et al. 2005; sato et al. 2005 ). the first advance involved managing the intrinsic similarity of the trees because of the common underlying taxonomic distribution (due to the speciation processes). this effect is analogous to the taxonomic biases discussed above. in these cases, the approach followed was to correct both trees by removing this common trend. for example, pazos et al. subtracted the distances of the 16s rrna phylogenetic tree to the corresponding distances for each protein tree. the correlations for the resulting distance matrices were used to predict protein interactions. additionally some analyses have focused on the selection of the sequence regions used for the tree building (jothi et al. 2006; kann et al. 2007) . for example, it has been shown that interacting regions, both defined as interacting residues (using structural data) and as the sequence domain involved in the interaction, show more clear tree similarities than the whole proteins (mintseris and weng 2005; jothi et al. 2006) . other interesting work showed that prediction performance can be improved by removing poorly conserved sequence regions ). finally, in a very recent work (juan et al. 2008 ) the authors have suggested a new method for removing noise in the detection of tree similarity signals and detecting different levels of evolutionary parallelism specificity. this method introduces the new strategy of using the global network of protein evolutionary similarity for a better calibration of the evolutionary parallelism between two proteins. for this purpose, they define a protein co-evolutionary profile as the vector containing the evolutionary correlations between a given protein tree and all the rest of the protein trees derived from sequences in the same organism. this co-evolutionary profile is a more robust and comparable representation of the evolution of a given protein (it involves hundreds of distances) and can be used to deploy a new level of evolutionary comparison. the authors compare these co-evolutionary profiles by calculating pearsons correlation coefficient for each pair. in this way, the method detects pairs of proteins for which high evolutionary similarities are supported by their similarities with the rest of proteins of the organism. this approach significantly improves the predictive performance of the tree similaritybased methods so that different degrees of co-evolutionary specificity are obtained according to the number of proteins that might be influencing the co-evolution of the studied pair. this is done by extending the approach of sato et al. (2006) , that uses partial correlations and a reduced set of proteins for determining specific evolutionary similarities. juan et al. calculated the partial correlation for each significant evolutionary similarity with respect to the remaining proteins in the organism and defined levels of co-evolutionary specificity according to the number of proteins that are considered to be co-evolving with each studied protein pair. with this strategy, its possible to detect a range of evolutionary parallelisms from the protein pairs (for very specific similarities) up to subsets of proteins (for more relaxed specificities) that are highly evolution dependent. interestingly, if specificity requirements are relaxed, protein relationships among components of macro-molecular complexes and proteins involved in the same metabolic process can be recovered. this can be considered as a first step in the application of higher orders of evolutionary parallelisms to decode the evolutionary impositions over the protein interaction network. this method exploits the well-known tendency of bacterial organisms to organize proteins involved in the same biochemical process by clustering them in the genome. this observation is obviously related to the operon concept and the mechanisms for the coordination of transcription regulation of the genes present in these modules. these mechanisms are widespread among bacterial genomes. therefore the significance of a given gene proximity can be established by its conservation in evolutionary distant species (dandekar et al. 1998; overbeek et al. 1999) . the availability of fully sequenced organisms makes computing the intergenic distances between each pair of genes easy. genes with the same direction of transcrip-tion and closer than 300 bases are typically considered to be in the same genomic context (see fig. 12c ). the conservation of this closeness must be found in more than two highly divergent organisms to be considered significant because of the taxonomic biases. while this signal is strong in bacterial genomes, its relevance is unclear in eukaryotic genomes. this is the main drawback of these methodologies. in fact, this signal only can be exploited for eukaryotic organisms by extrapolating genomic closeness of bacterial genes to their homologues in eukaryotes. obviously, this extrapolation leads to a considerable reduction in the confidence and number of obtained predictions for this evolutionary lineage. however, conserved gene pairs that are transcribed from a shared bidirectional promoter can be detected by similar methods and can found in eukaryotes as well as prokaryotes (korbel et al. 2004) a further use of evolutionary signals in protein function and physical interaction prediction has been the tendency of interacting proteins to be involved in gene fusion events. sequences that appear as independently expressed orfs in one organism become fused as part of the same polypeptide sequence in another organism. these fusions are strong indicators of functional and structural interaction that have been suggested to increase the effective concentration of interacting functional domains (enright et al. 1999; marcotte et al. 1999b ). this hypothesis proposes that gene fusion could remove the effect of diffusion and relative correct orientation of the proteins forming the original complex. these fusion events are typically detected when sequence searches for two nonhomologous proteins obtain a significant hit in the same sequence. cases matching to the same region of the hit sequence are removed (these cases are schematically represented in fig. 12d ). in spite of the strength of this signal, gene fusion seems to not be a habitual event in bacterial organisms. the difficulty of distinguishing protein interactions belonging to large evolutionary families is the main drawback of the automatic application of these methodologies. 13 integration of experimentally determined and predicted interactions as described above, there are many both experimental techniques and computational methods for determining and predicting interactions. to obtain the most comprehensive interaction networks possible, as many as possible of these sources of interactions should be integrated. the integration of these resources is complicated by the fact that the different sources are not all equally reliable, and it is thus important to quantify the accuracy of the different evidence supporting an interaction. in addition to the quality issues, comparison of different interaction sets is further complicated by the different nature of the datasets: yeast two-hybrid experiments are inherently binary, whereas pull-down experiments tend to report larger complexes. to allow for comparisons, complexes are typically represented by binary interaction networks; however, it is important to realize that there is not a single, clear definition of a "binary interaction". for complex pull-down experiments, two different representations have been proposed: the matrix representation, in which each complex is represented by the set of binary interactions corresponding to all pairs of proteins from the complex, and the spoke representation, in which only bait-prey interactions are included (von mering et al. 2002) . the binary interactions obtained using either of these representations are somewhat artificial as some interacting proteins might in reality never touch each other and others might have too low an affinity to interact except in the context of the entire complex bringing them together. even in the case of yeast two-hybrid assays, which inherently report binary interactions, not all interactions correspond to direct physical interactions. the database string ("search tool for the retrieval of interacting genes/ proteins") (von mering et al. 2007) represents an effort to provide many of the different types of evidence for functional interactions under one common framework with an integrated scoring scheme. such an integrated approach offers several unique advantages: 1) various types of evidence are mapped onto a single, stable set of proteins, thereby facilitating comparative analysis; 2) known and predicted interactions often partially complement each other, leading to increased coverage; and 3) an integrated scoring scheme can provide higher confidence when independent evidence types agree. in addition to the many associations imported from the protein interaction databases mentioned above (bader et al. 2003; salwinski et al. 2004; guldener et al. 2006; mishra et al. 2006; stark et al. 2006; chatr-aryamontri et al. 2007 ), string also includes interactions from curated pathway databases (vastrik et al. 2007; kanehisa et al. 2008 ) and a large body of predicted associations that are produced de novo using many of the methods described in this chapter (dandekar et al. 1998; gaasterland and ragan 1998; pellegrini et al. 1999; marcotte et al. 1999c) . these different types of evidence are obviously not directly comparable, and even for the individual types of evidence the reliability may vary. to address these two issues, string uses a two-stage approach. first, a separate scoring scheme is used for each evidence type to rank the interactions according to their reliability; these raw quality scores cannot be compared between different evidence types. second, the ranked interaction lists are benchmarked against a common reference to obtain probabilistic scores, which can subsequently be combined across evidence types. to exemplify how raw quality scores work, we will here explain the scoring scheme used for physical protein interactions from high-throughput screens. the two funda-mentally different types of experimental interaction data sets, complex pull-downs and binary interactions are evaluated using separate scoring schemes. for the binary interaction experiments, e.g. yeast two-hybrid, the reliability of an interaction correlates well with the number of non-shared interaction partners for each interactor. string summarizes this in the following raw quality score: logððn 1 þ1þ á ðn 2 þ1þþ; where n 1 and n2 are the numbers of non-shared interaction partners. this score is similar to the ig1 measure suggested by saito et al. (2002) . in the case of complex pulldown experiments, the reliability of the inferred binary interactions correlates better with the number of times the interactors were co-purified compared to what would be expected at random: where n 12 is the number of purifications containing both proteins, n 1 and n 2 are the numbers of purifications containing either protein 1 or 2, and n is the total number of purifications. for this purpose, the bait protein was counted twice to account for bait-prey interactions being more reliable than prey-prey interactions. these raw quality scores are calculated for each individual high-throughput screen. scores vary within one dataset, because they include additional, intrinsic information from the data itself, such as the frequency with which an interaction is detected. for medium sized data sets that are not large enough to apply the topology based scoring schemes, the same raw score is assigned to all interactions within a dataset. finally, very small data sets are pooled and considered jointly as a single interaction set. we similarly have different scoring schemes for predicted interactions based on coexpression in microarray expression studies, conserved gene neighborhood, gene fusion events and phylogenetic profiles. based on these raw quality scores, a confidence score is assigned to each predicted association by benchmarking the performance of the predictions against a common reference set of trusted, true associations. string uses as reference the functional grouping of proteins maintained at kegg (kyoto encyclopedia of genes and genomes (kanehisa et al. 2008) . any predicted association for which both proteins are assigned to the same "kegg pathway" is counted as a true positive. kegg pathways are particularly suitable as a reference because they are based on manual curation, are available for a number of organisms, and cover several functional areas. other benchmark sets could also be used, for example "biological process" terms from gene ontology (ashburner et al. 2000) or reactome pathways (vastrik et al. 2007 ). the benchmarked confidence scores in string generally correspond to the probability of finding the linked proteins within the same pathway or biological process. the assignment of probabilistic scores for all evidence types solves many of the issues of data integration. first, incomparable evidence types are made comparable by assigning a score that represents how well the evidence type can predict a certain type of interactions (the type being specified by the reference set used). second, the separate benchmarking of interactions from, for example, different high-throughput protein interaction screens accounts for any differences in reliability between different studies. third, use of raw quality scores allows us to separate more reliable interactions from less reliable interactions even within a single dataset. the probabilistic nature of the scores also makes it easy to calculate the combined reliability of an interaction given multiple lines of evidence. it is computed under the assumption of independence for the various sources, in a na€ ıve bayesian fashion. in addition to having a good scoring scheme, it is crucial to make the evidence for an interaction transparent to the end users. to achieve this, the string interaction network is made available via a user-friendly web interface (http://string.embl.de). when performing a query, the user will first be presented with a network view, which provides a first, simplified overview (fig. 13) . from here the user has full control over parameters such as the number of proteins shown in the network (nodes) and the minimal reliability required for an interaction (edge) to be displayed. from the network, the user also has the ability to drill down on the evidence that underlies any given interaction using the dedicated viewer for each evidence type. for example, it is possible to inspect the publications that support a given interaction, the set of protein that were fig. 13 protein interaction network of the core cell-cycle regulation in human. the network was constructed by querying the string database (von mering et al. 2007 ) for very high confidence interactions (conf. score > 0.99) between four cyclin-dependent kinases, their associated cyclins, the wee1 kinase and the cdc25 phosphatases. the network correctly recapitulates cdc2 interacts with cyclin-a/b, cdk2 with cyclin-a/e, and cdk4/6 with cyclin-d. it also shows that the wee1 and cdc25 phosphatases regulate cdc2 and cdk2 but not cdk4 and cdk6. moreover, the network suggests that cdc25a phosphatase regulates cdc2 and cdk2, whereas cdc25b and cdc25c specifically regulate cdc2 co-purified in a particular experiment and the phylogenetic profiles or genomic context based on which an interaction was predicted. protein binding is commonly characterized by specific interactions of evolutionarily conserved domains (pawson and nash 2003) . domains are fundamental units of protein structure and function , which are incorporated into different proteins by genetic duplications and rearrangements (vogel et al. 2004) . globular domains are defined as structural units of fifty and more amino acids that usually fold independently of the remaining polypeptide chain to form stable, compact structures (orengo and thornton 2005) . they often carry important functional sites and determine the specificity of protein interactions (fig. 14) . essential information on fig. 14 exemplary interaction between the two human proteins hhr23b and ataxin-3. each protein domain commonly adopts a particular 3d structure and may fulfill a specific molecular function. generally, the domains responsible for an observed protein-protein interaction need to be determined before further functional characterizations are possible. in the depicted protein-protein interaction, it is known from experiments that the ubiquitin-like domain ubl of hhr23b (yellow) forms a complex with de-ubiquitinating josephin domain of ataxin-3 (blue) (nicastro et al. 2005) the cellular function of specific protein interactions and complexes can often be gained from the known functions of the interacting protein domains. domains may contain binding sites for proteins and ligands such as metabolites, dna/rna, and drug-like molecules (xia et al. 2004) . widely spread domains that mediate molecular interactions can be found alone or combined in conjunction with other domains and intrinsically disordered, mainly unstructured, protein regions connecting globular domains (dunker et al. 2005) . according to apic et al. (2001) multi-domain proteins constitute two thirds of unicellular and 80% of metazoan proteomes. one and the same domain can occur in different proteins, and many domains of different types are frequently found in the same amino acid chain. much effort is being invested in discovering, annotating, and classifying protein domains both from the functional (pfam (finn et al. 2006) , smart (letunic et al. 2006 ), cdd (marchler-bauer et al. 2007 , interpro (mulder et al. 2007 ) and structural (scop (andreeva et al. 2004) , cath (greene et al. 2007 )) perspective. notably, it may be confusing that the term domain is commonly used in two slightly different meanings. in the context of domain databases such as pfam and smart, a domain is basically defined by a set of homologous sequence regions, which constitute a domain family. in contrast, a specific protein may contain one or more domains, which are concrete sequence regions within its amino acid sequence corresponding to autonomously folding units. domain families are commonly represented by hidden markov models (hmms), and highly sensitive search tools like hmmer (eddy 1998 ) are used to identify domains in protein sequences. different sources of information about interacting domains with experimental evidence are available. experimentally determined interactions of single-domain proteins indicate domain-domain interactions. similarly, experiments using protein fragments help identifying interaction domains, but this knowledge is frequently hidden in the text of publications and not contained in any database. however, domain databases like pfam, smart, and interpro may contain some annotation obtained by manual literature curation. in the near future, high-throughput screening techniques will result in even larger amounts of protein fragment interaction data to delineate domain borders and interacting protein regions (colland and daviet 2004) . above all, three-dimensional structures of protein domain complexes are experimentally solved by x-ray crystallography or nmr and are deposited in the pdb database (berman et al. 2007) . structural contacts between two interacting proteins can be derived by mapping sequence positions of domains onto pdb structures. extensive investigations of domain combinations in proteins of known structures (apic et al. 2001 ) as well as of structurally resolved homo-or heterotypic domain interactions (park et al. 2001) revealed that the overlap between intra-and intermolecular domain interactions is rather limited. two databases, ipfam (finn et al. 2005 ) and 3did (stein et al. 2005) , provide pre-computed structural information about protein interactions at the level of pfam domains. analysis of structural complexes suggests that interactions between a given pair of proteins may be mediated by different domain pairs in different situations and in different organisms. nevertheless, many domain interactions, especially those involved in basic cellular processes such as dna metabolism and nucleotide binding, tend to be evolutionarily conserved within a wide range of species from prokaryotes to eukaryotes (itzhaki et al. 2006) . in yeast, pfam domain pairs are associated with over 60% of experimentally known protein interactions, but only 4.5% of them are covered by ipfam (schuster-bockler and bateman 2007) . domain interactions can be inferred from experimental data on protein interactions by identifying those domain pairs that are significantly overrepresented in interacting proteins compared to random protein pairs (deng et al. 2002; ng et al. 2003a; riley et al. 2005; sprinzak and margalit 2001) (fig. 15) . however, the predictive power of such an approach is strongly dependent on the quality of the data used as the source of information for protein interactions, and the coverage of protein sequences in terms of domain assignments. basically, the likelihood of two domains, d i and d j , to interact can be estimated as the fraction of protein pairs known to interact among all proteins in the dataset containing this domain pair. this basic idea has been improved upon by using a maximum-likelihood (ml) approach based on the expectation-maximization (em) algorithm. this method finds the maximum likelihood estimator of the observed protein-protein interactions by an iterative cycle of computing the expected likelihood (e-step) and maximizing the unobserved parameters (domain interaction propensities) in the m-step. when the algorithm converges (i.e. the total likelihood cannot be further improved by the algorithm), the ml estimate for the likelihood of the unobserved domain interactions is found (deng et al. 2002; riley et al. 2005 ). riley and colleagues further improved this method by excluding each potentially interacting domain pair from the dataset and recomputing the ml-estimate to obtain an additional confidence value for the respective domain-domain interaction. this domain pair exclusion (dpea) method measures the contribution of each domain pair to the overall likelihood of the protein interaction network based on domain-domain interactions. in particular, this approach enables the prediction of specific domain-domain interactions between selected proteins which would have been missed by the basic ml method. another ml-based algorithm is insite which takes differences in the reliability of the protein-protein interaction data into account (wang et al. 2007a) . it also integrates external evidence such as functional annotation or domain fusion events. an alternative method for deriving domain interactions is through co-evolutionary analysis that exploits the notion that mutations of residue pairs at the interaction interfaces are correlated to preserve favorable physico-chemical properties of the binding surface (jothi et al. 2006) . the pair of domains mediating interactions between two proteins p1 and p2 may therefore be expected to display a higher similarity of their phylogenetic trees than other, non-interacting domains (fig. 16) . the degree of agreement between the evolutionary history of two domains, d i and d j , can be computed by the pearsons correlation coefficient r ij between the similarity matrices of the domain sequences in different organisms: where n is the number of species, m i pq and m j pq are the evolutionary distances between species, and m i and m j are the mean values of the matrices, respectively. in figure 16 the evolutionary tree of the domain d2 is most similar to those of d5 and d6, corroborating the actual binding region. a well-known limitation of the correlated mutation analysis is that it is very difficult to decide whether residue co-variation happens as a result of functional co-evolution directed at preserving interaction sites, or because of sequence divergence due to speciation. to address this problem, suggested to distinguish the relative contribution of conserved and more variable regions in aligned sequences to the co-evolution signal based on the hypothesis that functional co-evolution is more prominent in conserved regions. finally, interacting domains can be identified by phylogenetic profiling, as described above for full-chain proteins. as in the case of complete protein chains, the similarity of evolutionary patterns shared by two domains may indicate that they interact with each other directly or at least share a common functional role (pagel et al. 2004) . as illustrated in fig. 17 , clustering protein domains with similar phylogenetic profiles allows researchers to build domain interaction networks which provide clues for describing molecular complexes. similarly, the domainteam method (pasek et al. 2005) considers chromosomal neighborhoods at the level of conserved domain groups. a number of resources provide and combine experimentally derived and predicted domain interaction data. interdom (http://interdom.i2r.a-star.edu.sg/) integrates domain-interaction predictions based on known protein interactions and complexes with domain fusion events (ng et al. 2003b) . dima (http://mips.gsf.de/genre/proj/dima2) is another database of domain interactions, which integrates experimentally demonfig. 16 co-evolutionary analysis of domain interactions. two orthologous proteins from different organisms known to interact with each other are shown. the first protein consists of two domains, d1 and d2, while the second protein includes the domains d3, d4, d5, and d6. evolutionary trees for each domain are shown, their similarity serves as an indication of interaction likelihood that is encoded in the interaction matrix strated domain interactions from ipfam and 3did with predictions based on the dpea algorithm and phylogenetic domain profiling ). recently, two new comprehensive resources, domine (http://domine.utdallas.edu) (raghavachari et al. 2008 ) and dasmi (http://www.dasmi.de) (blankenburg et al. 2008, submitted) , were introduced and are available online. these resources contain ipfam and 3did data and predicted domain interactions taken from several other publications. predictions are based on several methods for deriving domain interactions from protein interaction data, phylogenetic domain profiling data and domain coevolution. with the availability of an increasing number of predictions the task of method weighting and quality assessment becomes crucial. a thorough analysis of the quality of domain interaction data can be found in schlicker et al. (2007) . beyond domain-domain contacts, an alternative mechanism of mediating molecular recognition is through binding of protein domains to short sequence regions (santonico et al. 2005) , typically from three to eight residues in length (zarrinpar et al. 2003; neduva et al. 2005) . such linear recognition motifs can be discovered from protein interaction data by identifying amino acid sequence patterns overrepresented in proteins that do not possess significant sequence similarity, but share the same interacting partner (yaffe 2006) . web services like eml (http://elm.eu.org (puntervoll et al. 2003) ), support the identification of linear motifs in protein sequences. as described above, specific adapter domains can mediate protein-protein interactions. while some of these interaction domains recognize small target peptides, others are involved in domain-domain interactions. as short binding motifs have a rather high probability of being found by chance and the exact mechanisms of binding specificity for this mode of interaction are not understood completely, predictions of proteinprotein interactions based on binding domains is currently limited to domain-domain interactions for which reliable data is available. predicting ppis from domain interactions may simply be achieved by reversing the ideas discussed above, that is, by using the domain composition of proteins to evaluate the interaction likelihood of proteins (bock and gough 2001; sprinzak and margalit 2001; wojcik and schachter 2001) . in a naive approach, domain interactions are treated as independent, and all protein pairs with a matching pair of interacting domains are predicted to engage in an interaction. given that protein interactions may also be mediated by several domain interactions simultaneously, more advanced statistical methods take into account dependencies between domains and exploit domain combinations (han et al. 2004 ) and multiple interacting domain pairs (chen and liu 2005) . exercising and validating these prediction approaches revealed that the most influential factor for ppi prediction is the quality of the underlying data. this suggests that, as for most biological predictions in other fields, the future of prediction methods for protein and domain interactions may lie in the integration of different sources of evidence and weighting the individual contributions based on calibration to goldstandard data. further methodological improvements may include the explicit consideration of cooperative domains, that is, domain pairs that jointly interact with other domains (wang et al. 2007b ). basic interactions between two or up to a few biomolecules are the basic elements of the complex molecular interaction networks that enable the processes of life and, when thrown out of their intended equilibrium, manifest the molecular basis of diseases. such interactions are at the basis of the formation of metabolic, regulatory or signal transduction pathways. furthermore the search for drugs boils down to analyzing the interactions between the drug molecule and the molecular target to which it binds, which is often a protein. for the analysis of a single molecular interaction, we do not need complex biological screening data. thus it is not surprising that the analysis of the interactions between two molecules, one of them being a protein, has the longest tradition in computational biology of all problems involving molecular interactions, dating back over three decades. the basis for such analysis is the knowledge of the three-dimensional structure of the involved molecules. to date, such knowledge is based almost exclusively on experimental measurements, such as x-ray diffraction data or nmr spectra. there are also a few reported cases in which the analysis of molecular interactions based on structural models of protein has led to successes. the analysis of the interaction of two molecules based on their three-dimensional structure is called molecular docking. the input is composed of the three-dimensional structures of the participating molecules. (if the involved molecule is very flexible one admissible structure is provided.) the output consists of the three-dimensional structure of the molecular complex formed by the two molecules binding to each other. furthermore, usually an estimate of the differential free energy of binding is given, that is, the energy difference dg between the bound and the unbound conformation. for the binding event to be favorable that difference has to be negative. this slight misnomer describes the binding between a protein molecule and a small molecule. the small molecule can be a natural substrate such as a metabolite or a molecule to be designed to bind tightly to the protein such as a drug molecule. proteinligand docking is the most relevant version of the docking problem because it is a useful help in searching for new drugs. also, the problem lends itself especially well to computational analysis, because in pharmaceutical applications one is looking for small molecules that are binding very tightly to the target protein, and that do so in a conformation that is also a low-energy conformation in the unbound state. thus, subtle energy differences between competing ligands or binding modes are not of prime interest. for these reasons there is a developed commercial market for protein-ligand docking software. usually the small molecule has a molecular weight of up to several hundred daltons and can be quite flexible. typically, the small molecule is given by its 2d structure formula, e.g., in the form of a smiles string (weininger 1988) . if a starting 3d conformation is needed there is special software for generating such a conformation (see, e.g. (pearlman 1987; sadowski et al. 1994) ). challenges of the protein ligand problem are (i) finding the correct conformation of the usually highly flexible ligand in the binding site of the protein, (ii) determining the subtle conformational changes in the binding site of the protein upon binding of the ligand, which are termed induced fit, (iii) producing an accurate estimate of the differential energy of binding or at least ranking different conformations of the same ligand and conformations of different ligands correctly by their differential energy of binding. methods tackling problem (ii) can also be used to rectify smaller errors in structural models of proteins whose structure has not been resolved experimentally. the solution of problem (iii) provides the essential selection criterion for preferred ligands and binding modes, namely those with lowest differential energy of binding. challenge (i) has basically been conquered in the last decade as a number of docking programs have been developed that can efficiently sample the conformational space of the ligand and produce correct binding modes of the ligand within the protein, assuming that the protein is given in the correct structure for binding the ligand. several methods are applied here. the most brute-force method is to just try different (rigid) conformations of the ligand one after the other. if the program is fast enough one can run through a sizeable number of conformations per ligand (mcgann et al. 2003) . a more algorithmic and quite successful method is to build up the ligand from its molecular fragments inside the binding pocket of the protein (rarey et al. 1996 ). yet another class of methods sample ligand conformations inside the protein binding pocket by methods such as local search heuristics, monte carlo sampling or genetic algorithms (abagyan et al. 1994; jones et al. 1997; morris et al. 1998 ). there are also programs exercising combinations of different methods (friesner et al. 2004 ). the reported methods usually can compute the binding mode of a ligand inside a protein within fractions of a minute to several minutes. the resulting programs can be applied to screening through large databases of ligands involving hundreds of thousands to millions of compounds and are routinely used in pharmaceutical industry in the early stages of drug design and selection. they are also repeatedly compared on benchmark datasets (kellenberger et al. 2004; englebienne et al. 2007 ). more complex methods from computational biophysics, such as molecular dynamics (md) simulations that compute a trajectory of the molecular movement based on the forces exerted on the molecules take hours on a single problem instance and can only be used for final refinement of the complex. challenges (ii) and (iii) have not been solved yet. concerning problem (ii), structural changes in the protein can involve redirections of side chains in or close to the binding pocket and more substantial changes involving backbone movement. while recently methods have been developed to optimize side-chain placement upon ligand binding (claußen et al. 2001; sherman et al. 2006) , the problem of finding the correct structural change upon binding involving backbone and side-chain movement is open (carlson 2002) . concerning problem (iii), there are no scoring functions to date that are able to sufficiently accurately estimate the differential energy of binding on a diverse set of protein-ligand complexes huang and zou 2006) . this is especially unfortunate as an inaccurate estimate of the binding energy causes the docking program to disregard correct complex structures even though they have been sampled by the docking program because they are labeled with incorrect energies. this is the major problem in docking which limits the accuracy of the predictions. recent reviews on protein-ligand docking have been published in sousa et al. (2006) and rarey et al. (2007) . one restriction with protein-ligand docking as it applies to drug design and selection is that the three-dimensional structure of the target protein needs to be known. many pharmaceutical targets are membrane-standing proteins for which we do not have the three-dimensional structure. for such proteins there is a version of drug screening that can be viewed as the negative imprint of docking: instead of docking the drug candidate into the binding site of the proteinwhich is not availablewe superpose the drug candidate (which is here called the test molecule) onto another small molecule which is known to bind to the binding site of the protein. such a molecule can be the natural substrate for the target protein or another drug targeting that protein. let us call this small molecule the reference molecule. the suitability of the new drug candidate is then assessed on the basis of its structural and chemical similarity with the reference molecule. one problem is that now both the test molecule and the reference molecule can be highly flexible. but in many cases largely rigid reference molecules can be found, and in other cases it suffices to superpose the test moelcule onto any low-energy conformation of the reference molecule. there are several classes of drug screening programs based on this molecular comparison, ranging from (i) programs that perform a detailed analysis of the three-dimensional structures of the molecules to be compared (e.g. (lemmen et al. 1998; kr€ amer et al. 2003) ) across (ii) programs that perform a topological analysis of the two molecules (rarey and dixon 1998; gillet et al. 2003) to (iii) programs that represent both molecules by binary or numerical property vectors which are compared with string methods (mcgregor and muskal 1999; xue et al. 2000) . the first class of programs require fractions of seconds to fractions of a minute for a single comparison, the second can perform hundreds comparisons per second, the third up to several ten thousand comparisons per second. reviews of methods for drug screening based on ligand comparison are given in (lengauer et al. 2004; k€ amper et al. 2007 ). here both binding partners are proteins. since drugs tend to be small molecules this version of the docking problem is not of prime interest in drug design. also, the energy balance of protein-protein binding is much more involved that for protein-ligand binding. optimal binding modes tend not to form troughs in the energy landscape that are as pronounced as for protein-ligand docking. the binding mode is determined by subtle side-chain rearrangements of both binding partners that implement the induced fit along typically quite large binding interfaces. the energy balance is dominated by difficult to analyze entropic terms involving the desolvation of water within the binding interface. for these reasons, the software landscape for protein-protein docking is not as well developed as for protein-ligand docking and there is no commercial market for protein-protein docking software. protein-protein docking approaches are based either on conformational sampling and mdwhich can naturally incorporated molecular flexibility but suffers from very high computing demandsor on combinatorial sampling with both proteins considered rigid in which case handling of protein flexibility has to be incorporated with methodical extensions. for space reasons we do not detail methods for protein-protein docking. a recent review on the subject can be found in hildebrandt et al. 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proline recognition domains protein-protein interactions: analysis and prediction key: cord-104279-choywmwd authors: nan title: membrane protein sorting in the yeast secretory pathway: evidence that the vacuole may be the default compartment date: 1992-10-01 journal: j cell biol doi: nan sha: doc_id: 104279 cord_uid: choywmwd the targeting signals of two yeast integral membrane dipeptidyl aminopeptidases (dpaps), dpap b and dpap a, which reside in the vacuole and the golgi apparatus, respectively, were analyzed. no single domain of dpap b is required for delivery to the vacuolar membrane, because removal or replacement of either the cytoplasmic, transmembrane, or lumenal domain did not affect the protein's transport to the vacuole. dpap a was localized by indirect immunofluorescence to non-vacuolar, punctate structures characteristic of the yeast golgi apparatus. the 118-amino acid cytoplasmic domain of dpap a is sufficient for retention of the protein in these structures, since replacement of the cytoplasmic domain of dpap b with that of dpap a resulted in an immunolocalization pattern indistinguishable from that of wild type dpap a. overproduction of dpap a resulted in its mislocalization to the vacuole, because cells expressing high levels of dpap a exhibited vacuolar as well as golgi staining. deletion of 22 residues of the dpap a cytoplasmic domain resulted in mislocalization of the mutant protein to the vacuole. thus, the cytoplasmic domain of dpap a is both necessary and sufficient for golgi retention, and removal of the retention signal, or saturation of the retention apparatus by overproducing dpap a, resulted in transport to the vacuole. like wild type dpap b, the delivery of mutant membrane proteins to the vacuole was unaffected in the secretory vesicle-blocked sec1 mutant; thus, transport to the vacuole was not via the plasma membrane followed by endocytosis. these data are consistent with a model in which membrane proteins are delivered to the vacuole along a default pathway. ar~y proteins that reside in the organelles of the secretory pathway of eukaryotic cells have targeting information that directs retention in or sorting to the appropriate compartment (pfeffer and rothman, 1987) . in the absence of retention or sorting signals, soluble proteins of the secretory pathway are secreted; thus, the default pathway for these proteins is secretion (burgess and kelly, 1987; pelham, 1989) . in saccharomyces cerevisiae, mutations in the targeting signal of the soluble vacuolar protein, carboxypeptidase y (vails et al., 1990) , or the retention signal of the soluble er protein, bip (hardwick et al., 1990) , result in the secretion of these proteins. likewise, the flow of membrane proteins to the cell surface of nonpolarized mammalian cells is apparently by default, because mutations that disrupt the retention of er or golgi-retained membrane proteins (machamer et al., 1987; machamer, 1991; jackson et al., 1990) or the sorting of a lysosomal membrane protein (williams and fukuda, 1990) result in localization to the plasma membrane. little is known regarding membrane protein sorting in s. cerevisiae, although a previous study suggested that the cell surface is the default compartment for membrane proteins (fuller et al., 1989b) . in this paper, we characterize the targeting of two membrane proteins of the yeast secretory pathway, dap dipeptidyl aminopeptidases (dpaps) ~ dpap a and dpap b of the golgi apparatus and vacuole, respectively. the biogenesis of two membrane proteins of the yeast vacuole, dpap b (see fig. 1 a), and alkaline phosphatase (alp), has been characterized (klionsky and emr, 1989; roberts et al., 1989) . both dpap b and alp are type ii membrane glycoproteins (nomenclature of singer, 1990) , consisting of nh2-terminal cytoplasmic domains of approximately 30 amino acids, single hydrophobic membrane anchors, and cooh-terminal lumenal catalytic domains. these proteins transit the early compartments of the secretory pathway (i.e., er and golgi), but not the later compartments (i.e., secretory vesicles), indicating that the proteins do not transiently reside at the plasma membrane before delivery to the vacuole. the localization signals of these two proteins have not been identified, although the lumenal domain of alp has been shown to be unnecessary for vacuolar targeting (klionsky and emr, 1990) . the biosynthesis of several membrane proteins that reside this paper is dedicated to the memory of david merrill stevens. in the yeast golgi apparatus has also been examined. in yeast, three membrane-bound proteases, kex2p, kexlp, and dpap a (see fig. 1 a) process the mating pheromone a-factor precursor polypeptide as it traverses the secretory pathway (bussey, 1988; fuller et al., 1988) . the biosynthetic pathways of kex2p and kexlp have been characterized (fuller et al., 1989a,b; cooper and bussey, 1989) , and kex2p has been shown to function in a late golgi compartment (julius et al., 1984; graham and emr, 1991) . kex2p has been localized by indirect immunofluorescence to three to six punctate structures per cell that exhibit a somewhat random distribution within the cytoplasmic compartment (franzusoff et al., 1991; redding et al., 1991) . thus, the golgi apparatus of yeast is not localized to a perinuclear location, as in mammalian cells, but rather is dispersed throughout the cell. both kexlp (a. cooper and h. bussey, manuscript submitted) and dpap a (see below) have been immunolocalized to punctate bodies that are similar to those containing kex2p in size, abundance, and distribution. in this paper, a combined gene fusion and mutational analysis was used to show that no single domain of dpap b is required for vacuolar localization. furthermore, both overproduction of dpap a and a mutation in the cytoplasmic domain of dpap a resulted in mislocalization of this protein to the vacuole. finally, the fusion proteins analyzed in this work are shown to be transported directly to the vacuole from the golgi, and not to the plasma membrane. the bacterial strain mc1061 (casadaban and cohen, 1980) was used for all subcloning steps. oligonucleotide-directed mutagenesis was carded out in strain cj236 (kunkel et al., 1987) . the yeast strains used were jhry20-1aaia3 (mata, dap2a: : h1s3, stel3a : :leu2, ura3-52, leu2-3, 1eu2412, his3-a200, pep4-3; roberts et al., 1989) , sey2012zs (match, dap2:: leu2, emr et al., 1983) , cjry25-6b (mata dap2a : :leu2 mnn9 ura3-52 leu2-3 leu2412, and sey-5016 ( mata, s e cl-l , dap 2 : : le u2 , ura3-5 2 , le u2-3, leu2412) . yeast cultures were grown in yepd or minimal (sd) medium supplemented with the appropriate nutrients as previously described (sherman et al., 1982) . for the simultaneous induction of the gal/ promoter and the secl4 secretion defect, ceils were grown to log phase in minimal media plus raltinose and then harvested and resuspended in yep-raitinose. after a 1-h incubation at 25~ galactose was added directly to the cultures and at the same time the cultures were shifted to 34~ after 2 h, the cells were fixed immediately and prepared for indirect immunofluorescence as described below. oligonucleotides for mutagenesis were prepared by the university of oregon biotechnology laboratory on an applied biosystems 380b dna synthesizer (foster city, ca) as described (ito et al., 1982) . tran35s-label and zymolyase 100t were from icn biomedicals (irvine, ca), endo h was from boehringer marmheim (indianapolis, in), ultra-pure sds was from bdh biochemicals (san francisco, ca), glusulase was from dupont pharmaceuticals (wilmington, de), and all antibodies (except anti-dpap b, anti-dpap a, anti-alp, and anti-vat2p antibodies) used for indirect immunofluorescence were from jackson immunoresearch (west grove, pa), cappel products (malvern, pa), or promega biotech (madison, wi). all other reagents were from sigma chemical co. (st. louis, mo). dpap and invertase assays were performed as previously described (gildstein and lampen, 1975; roberts et al., 1991) . restriction endonuclease digests and ligations were performed as recommended by the suppliers. plasmid purification, agarose gel electrophoresis, fill-in reactions of sticky-ended dna fragments using t4 dna polymerase, and dna-mediated transformation of escherichia coli were done according to standard procedures (maniatis et al., 1982) . lithium acetate transformations of yeast were performed as described (jto et al., 1983) . a disruption of the chromosomal ste/3 locus was constructed by onestep gene disruption (l~thstein, 1983) , using the plasmid pslk349 (kindly provided by dr. george sprague). psl349 consists of pbr322 containing a 7.2 kbp bamhi ste/3 fragment, from which a 1.6-kbp bcli fragment within the coding region of the dpap a lumenal domain (c. a. flanagan, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication) was replaced by the 2.9-kbp bglii leu2 fragment. to create a strain lacking both dpap a and dpap b, a disruption of the chromosomal dap2 locus with the his3 gene was made using the plasmid pgp6, which contains the 1.2-kbp ecori-bamhi his3 fragment (sikorski and hieter, 1989) in place of the l3-kbp bsteii-kpni portion of the coding region of dap2 (roberts et al., 1989) . the c~-factor signal sequence was fused to the lumenal domain of dpap b as follows: a sail linker was inserted at the hincll site of plasmid p771, which contains a portion of the 5' region of the mfcd gene (kurjan and herskowitz, 1982) , including 70 bp of non-coding region (not including the uas) and 66 bp of coding region, including the signal sequence, signal peptidase cleavage site (waters et al., 1988) , and 3 nh2-terminal residues of pro-a-factor, fused to the kre/gene (boone et ai., 1990) . the acci site at position +140 of the dap2 gene was changed to a sali site using oligonucleotide-directed mutagenesis (kunkel et al., 1987) . mutagenesis of dap2 was performed using the vector pcjr27, which contains the 3.3-kbp bamhi-hindiii da/'2 fragment in the plasmid ks + (stratagene, san diego, ca). the 2.7-kbp sali-hindui fragment, encoding the lumenal domain of dpap b, was inserted into the sali-hindiii sites of p771/sali, fusing the coding regions of mfal and dap2 in frame, c~fss-b was placed under the control of the ga/_j promoter (johnston and davis, 1984) by inserting the 2.9-kbp bamhi-hindiii fragment from this plasmid into the bamhi-hindiii sites of pc jr52, which contains the 822-bp ecori-bamhi ga/_,/ promoter fragment inserted into ecori-bamhi sites of the cen plasmid pseyc68 ca modified version of pseyc58; emr et al., 1983) . the resulting fusion protein consists of the nh2-terminal 22 residues of prepro-afactor and two residues arising from linker sequences fused to residue 49 of the lumenal domain of dpap b (fig. l b ; amino acid sequence nh2-mrfpsiftavlfaassala-apvgrphh . . . ). the bb-inv fusion protein expression vectors, pcjr13 and pc jr15 (2p and cen plasmids, respectively), were constructed by inserting the 0.6-kbp bamhi-acci fragment (acci blunt ended) into the invertase fusion vectors psey304 (a derivative of the 2/z plasmid psey303; emr et al., 1986) and the cen plasmid pseyc306 (johnson et al., 1987) at the bamhi and hin-diii sites (hindlij blunt ended). the resulting fusion protein contains the 48 nh2-terminal residues of dpap b fused to residue three of cytoplasmic invertase ( fig. 1 b) . bb-inv was placed under the control of the gal/promoter by cutting pcjr15 with ecori and hiediii and ligating in the 822-bp gal/fragment (johnston and davis, 1984) . this fused the 3' end of the gal/fragment at nucleotide -92 of the dap2 sequence. vectors encoding a20-bb and a27-bb were constructed as follows. oligonucleotide-directed mutagenesis (kunkel et al., 1987) was used to create 60-and 81-bp deletions in dap2 (a20-bb and a27-bb, respectively) in pcjr27. the ~3.2kbp bamhi-hindiii mutant dap2 fragments were inserted into the bamhi-hindiii sites of the cen plasmid pseyc58, creating pmnh1 (620-bb) and pc jr45 (a27-bb). &20-bb and a27-bb were over-produced by replacing the dap2 promoter with the gal/promoter as follows. the hindiii site at -92 of dap2 was blunt ended and religated, creating an nhei site. the ~3-kbp nhei-hindiii fragments of pc jr43 and pc jr44 were inserted at the xbai-hindiii sites of pc jr52, creating pc jr56 and pc jr54, respectively. the a20 and a27 deletions removed residues 2-21 and 2-27 of dpap b, changing the nh2-terr~nal acid sequence of dpap b from nh2-meg-geeeveripdelfdtkkkhlldklirv30 to nh2-mhlldklirv and nh2-mirv, respectively ( fig. 1 b) . the 2pt plasmid encoding dpap a, pcjr46, was constructed as follows: the 5.9-kbp xbai-bamhi fragment, containing the stf_j3 gene (c. a. flanagan, d. a. barnes, m. c. flessel, andj. thorner, manuscript submitted for publication) was isolated from the plasmid p13-3 (julius et al., 1983) and inserted into puc13. the 5.9-kbp sali-bami-ii fragment from this vector was inserted into the xhoi-bglii sites of the 2# plasmid pckr201 (c. raymond and t. stevens, unpublished results) . the cen plasmid pcjr78 was made by inserting the 4.5-kbp eagi-pvuii ste/3 fragment into the eaglecorv sites of prs316 (sikorski and hieter, 1989) . the plasmid pc jr64, encoding the fusion protein aa-b, was constructed by inserting the 2.5-kbp bamhi-mlui (mlui blunt ended) fragment from p13-3, encoding the stf.j3 promoter and the cytoplasmic and transmembrane domains of dpap a, and the 2.7-kbp sall-hindlii fragment of dap2 (sail blunt ended), encoding the lumenal domain of dpap b, into the bamhi-hindlii sites of pseyc58. the 5.2-kbp bamhi-hindlii fragment of the resulting vector (pcjr41) was cloned into the bamhi-hindlii sites of the 2# plasmid, psey18 (emr et al., 1986) . the resulting plasmid encodes aa-b, which consists of the nh2-terminal 150 residues of dpap a to residue 47 of dpap b (fig. 1 c) . for the construction of the fusion protein a-bb, xbai sites were created in both the dap2 and ste/3 genes just upstream of the coding regions of the transmembrane domains of dpap b and dpap a, respectively, using oligonucleotide-directed mutagenesis (kunkel et al., 1987) . nucleotides 69 and 70 of the dap2 gene were changed from gt to tc (roberts et al., 1989) , and nucleotides 342-344 of ste/3 were changed from gcc to aga (with the a of the initiation codon as 1) (c. a. flanagan, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication). a 1.1kbp saci-xbai fragment (encoding the ste13 promoter and the cytoplasmic domain of dpap a) and a 2.8-kbp xbal-hindlii dap2 fragment (encoding the transmembrane and lumenal domains of dpap b) were ligated into the saci-hindlii sites of psey18. the resulting plasmid (pcjr67) encodes a protein consisting of 113 residues of the nh2-terminal cytoplasmic domain of dpap a fused to amino acid 24 of dpap b (fig. 1 c) . the fusion protein b-a-b was constructed as follows: the 3.3-kbp bamhi-hindui dap2 fragment, including the xbal site at +70, was ligated into the bamhi-hindlii sites of pseyc58. a 4.3-kbp xbal-hindlii fragment of ste/3, encoding the transmembrane and lumenal domains of dpap a, was inserted into the xbai-hindlii sites, resulting in the plasmid pc jr58. the 2.7-kbp sali-hindiii dap2 fragment (sali blunt ended), encoding the dpap b lumenal domain, was inserted into the mlui-hindlii sites (mlui blunt ended) of pcjr58, resulting in pcjr69, a cen plasmid encoding b-a-b. the 4.8-kbp bamhi-hindlii fragment from pcjr69 was inserted into the same sites of psey18, resulting in pcjrt0. b-a-b was placed under the control of the gal/promoter by cloning the nhei-hindlii b-a-b fragment into the xbai-hindlii sites of pcjr52, creating pc jr79. the b-a-b fusion protein consists of residues 116-150 of dpap a in place of residues 26-46 of dpap b ( fig. 1 c) . oligonucleotide-directed mutagenesis of the portion of ste/3 encoding the 118 amino acid cytoplasmic domain of dpap a ( fig. 1 a) was performed in pcjr71, which consists of the 0.65-kbp eagi-psti fragment of ste/3 inserted into the eagi-psti sites of ks +. a 66-bp in-frame deletion, removing the amino acids 85-106 (z~22), was created, and the sacl-mlui fragment from this plasmid was inserted into the saci-mlui sites of pcjr64, creating the 2# plasmid psn58, which encodes a92-aab ( fig. 1 c) . the same saci-mlui fragment was ligated with the 4-kbp mlui-hindlll (encoding the lumenal domain of dpap a) into the saci-hindiii sites of psey18, creating psn59, which encodes a22-aaa ( fig. 1 c) . a22-aa-b was placed under the control of the gal/promoter by fusing a saci-eagi fragment from pcjr125 (contains the 360 bp hindlii-ecorl gal/fragment in pgem-5zf) to an eagl-hindlii fragment from psn58, both of which were ligated into the saci-hindlii sites of psey18, creating psn89. the fusl-laczp expression plasmid pcjrll4 as follows: a 6-kb nhei-hindlli fragment, encoding the 254 nh2-terminal amino acids of the fus1 protein fused to ~-galactosidase, under the gall promoter (isolated from pcjrll3, a derivative of psb231 (trueheart and fink, 1989) ) was cloned into the xbal-hindlll sites of pvt105u, a 2# plasmid (vernet et al., 1987) , creating pcjr114. for the production of dpap a antigen to be used to generate dpap a antiserum, the plasmid pcjr24 was constructed by inserting the 0.8-kbp mlui-hpal (mlui blunt ended) ste/3 fragment into the sinai site of pexp1, an e. coli expression vector containing the tac promoter just upstream of the translational start codon of t4 lysozyme and a multiple cloning site (raymond et al., 1990) . a 27 amino acid peptide, corresponding to the 26 nh2-terminal residues of dpap b followed by a cooh-terminal cysteine residue, was synthesized on an applied biosystems peptide synthesizer. the peptide was coupled to carboxymethylated bsa through the cooh-terminal cysteine with the hifunctional crosslinking agent, mbs, following the manufacturer's recommendations (pierce chemical co., rockford, il), and through lysine residues with gluteraldehyde as previously described (kagen and glick, 1979) . a 1:1 mixture of bsa-peptide conjugates prepared by the two methods was used to immunize rabbits as described previously (vaitukaitis, 1981) . for affinity purification of the antibody, the poptide was cross-linked to tresyl-activated sepharose 4b (pharmacia fine chemicals), and affinity purification was carded out as described (raymond et al., 1990) . dpap a antigen was produced in e. coli cells containing the plasmid pc jr24. induction with iptg resulted in the production ofa 31-kd protein that corresponds to the nhe-terminal portion of the lumenal domain of dpap a (see fig. 6 ) fused to seven residues of t4 lysosyme and seven residues encoded by the pexpi polylinker. antigen purification was performed as described previously (raymond et al., 1990) . immunoprecipitations were performed by growing cultures to log phase in supplemented minimal media lacking methionine and cysteine, then pulse labeling in the same media with tran3ss-label and chasing by adding 50 /~g/ml methionine and 50/~g/ml cysteine, followed by the addition of sodium azide to 10 ram. the cells were converted to spheroplasts (stevens et al., 1986) which were lysed in 1% sds, 8 m urea plus a protease inhibitor cocktail (0.5 mm pmsf, 1/~g/ml leupeptin, and 1 /~g/ml pepstatin) for 5 rain at 100~ and adjusted to 1 ml in ip buffer (pbs, 0.1% sds, 0.1% triton x-100, 1 mivl edta). for precipitating ~fss-b from the extracellular fractions, the medium was supplemented with 2 m4,,/ml bsa and 50 mm potassium phosphate, ph 5.7. after spberoplasting, the periplasmic and media fractions were pooled and adjusted to 1 ml in ip buffer plus protease inhibitors. after pre-adsorbtion to 0.5% iggsorb, anti-dpap b coohterminal antibody (roberts et al., 1989) , or anti-dpap a antibody was added, and samples were incubated one hour on ice. iggsorb was added to 0.5%, followed by 1 h on ice, and the immune complexes were precipitated and washed twice with ip buffer. the immune complexes were solubilized, and half of the samples were treated with endoglycosidase h (endo h) overnight at 37~ as described (orlean et al., 1991) . samples were analyzed by sds-page and fluorography as described previously (stevens et ai., 1986) . the secretion of t~fss-b was quantified using an ambis radioanalytic imaging system (ambis systems, inc., san diego, ca). the fractionation of membranes in the presence of high ph sodium carbonate was performed as described (roberts et al., 1989) . preparation of fixed, spheroplasted cells for indirect immunofluorescence was carried out essentially as previously described (roberts et al., 1991) , except that the fixed spheroplasts were treated with 1% sds for 1-5 minutes. antibody adsorption against fixed spheroplasts harboring null mutations in either dap2 or stej3 was performed as described elsewhere (raymond et al., 1990; roberts et ai., 1991) . the fixed spheroplasts were stained with a 1:10 dilution of adsorbed anti-dpap a or anti-dpap b affinity-purified antibody in pbs-bsa (roberts et al., 1991) . for co-localization with the 60-kd subunit of the vacuolar h+-atpase (vat2p, the product of the vat2 gene; yamashiro et al., 1990) , the antibody solution also contained a 1:10 dilution of the mab, 13dll (kane et al., 1992) . the dpap b or dpap a staining pattern was amplified by subsequent incubations with goat antirabbit antibody conjugated to biotin, followed by a streptavidin-fitc conjugate. the last antibody incubation also contained goat anti-rabbit antibody conjugated to rhodamine. co-detection of alp and fusl-laczp was performed by staining cells with a 1:10 dilution of a rabbit polyclonal anti-alp antibody (raymond et al., 1990 ) and a 1:1,000 dilution of a mouse monoclonai anti-/~-gaiactusidase antibody, followed by antibody amplification identical to that used for dpap a and dpap b. the cells were mounted in media containing dapi for staining nuclei, and photomicrographs were made as described previously (roberts et al., 1989) . the role of the lumenal domain in the sorting of dpap b to the vacuole was addressed in two ways. first, to determine if the lumenal domain was sorted to the vacuole when expressed in the secretory pathway as a soluble protein, a gene fusion was used to create the protein ctfss-b, consisting of the nh2-terminal er-targeting signal sequence of prepro-t~ factor fused to the lumenal domain of dpap b at residue 49 ( fig. 1 b) . the signal sequence should direct the translocation of the protein into the er lumen and be cleaved, rendering the lumenal domain a soluble protein in the secretory figure 2 . immunoprecipitations of c~fssb from mnn9 and mnn9 strains. jhry20-1a ( dap2a mnn9 ) or cjry25-6b ( dap2a mnn9 ) cells were labeled with tran3ss-label for 15 rain and chased for 60 min in the presence of 50 izg/ml methionine and 50/~g/ml cysteine. cultures were separated into two fractions, intracellular (i.e., spheroplasts) and extraceuular (i.e., periplasmic and media), and immunoprecipitated with affinity purified dpap b antibody. half of each immunoprecipitation sample was treated with endo h, and equal amounts were analyzed by sds-page and fluorography. positions of the molecular weight standards are indicated (in kd). pathway. the construct was analyzed in a strain which contains a null allele of dap2, the structural gene for dpap b (suarez rendueles and wolf, 1987) . fig. 2 shows the results of immunoprecipitating o~fss-b from intraceuular and ex-traceuular (i.e., combined periplasmic and medium) fractions of 35s-labeled cells using an antibody that recognizes the cooh-terminal half of dpap b (roberts et al., 1989) . analysis of the samples by sds-page and fluorography showed that 64 % of txfss-b was secreted as a heterogeneous population of highly glycosylated species (fig. 2, lane 2) , similar to the secreted protein invertase (esmon et al., 1981) , whereas the intracellular fraction contained a tightly migrating species (fig. 2 , lane /). treatment of the immunoprecipitates with endo h to remove n-linked carbohydrate demonstrated that the difference in the apparent mobilities was due to glycosylation (fig. 2, lanes 3 and 4) . the glycosylation pattern of the secreted material differs from that of wild type dpap b, which receives only modest glycosyl modifications of the core oligosaccharides in the golgi apparatus (roberts et al., 1989) . to test whether the alteration in glycosylation caused the secretion of the lumenal domain, otfss-b was expressed in an mnn9 mutant, which is deficient in the addition of the extensive t~1,6 outer chain glycosyl groups (kukuruzinska et al., 1987) . fig. 2 , lanes 5 and 6, show that t~fss-b was secreted to the same extent (59%) from mnn9 cells, even though the protein was not aberrantly glycosylated. indirect immunofluorescence microscopy showed that the portion of the lumenal domain that remained intracellular was retained in the er, and no staining of the vacuole was observed (data not shown). unlike the secreted material, the er-retalned material was enzymati(suc2-a9) cells containing a plasmid encoding bb-inv (pcjr13) were labeled with tran35s-label for 30 rain and chased for 60 rain in the presence of 50/~g/ml rrw, thionine and 50 /~g/ml cysteine. the cells were converted to sphemplasts, and extracts immunoprecipitated with either affinity-purified dpap b antibody (lanes 1-8) or affinity-purified invertase antibody (lanes 9 and 10) . half of each immunoprecipitation sample was treated with endo h, and equal amounts were analyzed by sds-page and fluorography. positions of the molecular mass standards for lanes 1-8 are indicated (in kd) on the left, whereas standards for lanes 9 and 10 are indicated on the right. cally inactive (data not shown), and thus may be unfolded and incompetent to exit the er (rose et al., 1989; rothman, 1989) . the role of the lumenal domain of dpap b in vacuolar targeting was also tested by constructing the fusion protein bb-inv, in which the 48 nh2-terminal residues of dpap b were fused to the non-vacuolar protein, invertase (fig. 1 b) . immunoprecipitations of bb-inv using anti-invertase antibody, followed by sds-page and fluorography, showed that the fusion protein was glycosylated, and treatment with endo h showed that the protein was of the expected size (fig. 3, lanes 9 and 10) . bb-inv fractionated with membranes under high ph carbonate conditions, consistent with bb-inv being an integral membrane protein (data not shown). invertase enzyme assays of either permeabilized or non-permeabilized whole cells deleted for the invertase structural gene and expressing bb-inv from a cen plasmid showed that <2 % of the total invertase activity was extracellular. the subcellular localization of bb-inv was determined by indirect immunofluorescence microscopy using an antibody that recognizes the cytoplasmic domain of dpap b (see materials and methods). fig. 4 shows the staining pattern of wild type dpap b (fig. 4, a-c) and bb-inv (fig. 4 , g-i) when expressed from the high copy number (2#) plasmids, pgp3 (roberts et al., 1989 ) and pcjr13, respectively. for these and the other 2# plasmid constructs used in this study, the proteins were overproduced 10-20-fold as determined by enzyme activity. both dpap b and bb-inv were localized to the vacuolar membrane as judged by differential interference contrast (nomarski) optics and co-localization with a marker for the vacuolar membrane, the 60-kd subunit of the vacuolar h+-atpase (vat2p; yamashiro et al., 1990; table i ). dpap b and bb-inv were also localized to the vacuolar membrane when expressed from a single copy (cen) plasmid (data not shown). aside from the differences in signal intensity, no difference in subcellular localization was observed when bb-inv was expressed from cen or 2# plasmids. the results from the afss-b and bb-inv fusions indi-cate that, similar to alp (klionsky and emr, 1990) , the lumenal domain of dpap b is neither necessary nor sufficient for vacuolar targeting. the role of the transmembrane domain in the vacuolar targeting of dpap b was tested by constructing the fusion protein b-a-b (fig. 1 b) , in which the membrane anchor of dpap b was replaced by that of the non-vacuolar membrane protein, dpap a. the role of the cytoplasmic domain of dpap b was tested by constructing in-frame deletions in this domain using oligonucleotide-directed mutagenesis of the dap2 gene. two deletion variants of dpap b were constructed, a20-bb and a27-bb, in which 20 and 27 amino acids, respectively, were removed from the 29 residue cytoplasmic domain (fig. 1 b; see materials and methods). immunoprecipitations of b-a-b, a20-bb, and a27-bb from 35s-labeled cells, followed by sds-page and fluorography, showed that the mutant proteins were glycosylated, and that the deglycosylated proteins were of the predicted size ( fig. 3, lanes 3-8) . as with bb-inv, these proteins behaved as integral membrane proteins in high ph carbonate fractionation experiments (data not shown). dpap activity assays of cells expressing b-a-b, a20-bb, and a27-bb showed that the proteins were fully enzymatically active, and that 88-98 % of the total activity was intracellular. indirect immunofluorescence microscopy of ceils expressing b-a-b, a20-bb, and a27-bb from either 2# plasmids (fig. 5, c-h) or cen plasmids, using an antibody that recognizes the cooh-terminal half of dpap b, showed that each of the mutant proteins was predominantly localized to the vacuolar membrane as determined by nomarski optics (fig. 5) and co-localization with vat2p (data not shown). no staining of the plasma membrane was observed for any of these proteins. thus, the cytoplasmic and transmembrane domains of dpap b are not necessary for targeting to the vacuolar membrane. a significant fraction of cells expressing a20-bb and a27-bb also showed some er localization, as judged by staining of the perinuclear space and long cisternal compartments (table i; rose et al., 1989) , whereas b-a-b showed only vacuolar labeling. the z~20-bb and ~27-bb cells in fig. 5 show predominantly vacuolar staining. in many cases, the plane of focus had to be adjusted to observe staining of both vacuolar and er structures within a given cell. the increased residence time of a20-bb and a27-bb in the er was corroborated by immunoblotting analysis, which showed that a small amount of the er forms of a20-bb and a27-bb were seen in the steady state, along with the golgi-modified forms, whereas only the golgi-modified form was seen for wild type dpap b (data not shown). the increased er retention of a20-bb and a27-bb could be due to an impaired ability to achieve a native conformation competent for exiting the er, as has been observed with mutant membrane proteins in mammalian cells (gething et al., 1986; doms et al., 1988) . upon exiting the er, however, a20-bb and a27-bb were transported through the golgi complex to the vacuole, indicating that the cytoplasmic domain of dpap b is not necessary for vacuolar localization. these results, combined with those from the analysis of eefss-b, bb-inv, and b-a-b, demonstrate that, aside from a membrane anchor, no single domain of dpap b is required for transport to the vacuole. * for each construct, the percentages refer to the fraction of stained cells that exhibited staining of a particular organelle. depending on the protein being monitored, some cells showed staining of more than one class of organdie; thus, the percentages for a given protein may add up to more than 100. w vacuolar localization was determined both by coincidence of staining with the vacuole membrane as determined by nomarski optics, and by co-localization with vat2p. i) golgi localization was defined as punctate, non-vacuolar, and non-er staining, characteristic of dpap a, kex2p , and kexlp (cooper and bussey, manuscript submitted for publication). ** er localization was determined by staining of perinuclear (as determined by dapi staining of nuclei) and extended cisternal structures. several different models can explain the data presented above (see discussion), including a simple model in which the vacuolar membrane, not the plasma membrane, is the default compartment for membrane proteins of the yeast secretory pathway. to distinguish among these models, we analyzed the retention signal of the golgi membrane protein, dpap a. mutations in the retention signal of this protein should result in dpap a becoming localized to the default compartment for membrane proteins. dpap a is a protein that resides in the golgi apparatus, where it processes the c~-factor precursor polypeptide (julius et al., 1983) . the structural gene for dpap a (ste23) has been cloned and sequenced (julius et al., 1983 ; c. a. flanagun, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication), and the predicted structure of dpap a is similar to that of dpap b in several regards. both are type ii integral membrane proteins, and both have lumenal enzymatic domains of ,x,800 residues that share a high degree of sequence identity, including 48 % identical residues over the cooh-terminal 250 residues (fig. 1 a) ; however, there is no significant sequence similarity between the cytoplasmic and transmembrane domains of dpap a and dpap b. to assess the subcelhlar distribution of dpap a, an antibody specific for dpap a was generated (see materials and methods). the specificity of the antibody is demonst-rated in fig. 6 . immunoprecipitation of dpap a was carried out with 35s-labeled cells that varied with regard to the dosage of the ste/3 gene. sds-page and fluorography showed that the antibody immunoprecipitated a protein of 112 kd from wild type cells but not from ste13a ceils, and that this polypeptide was overproduced ~20-fold in a strain containing the ste/3 gene on a 2t~ plasmid (fig. 6 a) . a protein of *40 kd was also immunoprecipitated; however, the level of the 40-kd protein did not vary with the dosage of ste/3. the ste/3 dna sequence predicts three possible sites for addition of n-linked carbohydrate (c. a. flanagan, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication). treatment of dpap a with endo h resulted in an ~,5 kd decrease in apparent molecular weight (fig. 6 b) , suggesting that at least two of the three asn-x-ser/thr sites of dpap a were modified, assuming that the core oligosaccharides added were of the typical structure (kukuruzinska et al., 1987) , and that the carbohydrate moieties were only slightly modified in the golgi apparatus, as is the case for the glycosyl groups of kex2p (fuller et al., 1989a,b) and kexlp (cooper and bussey, 1989) . the apparent molecular weight of deglycosylated dpap a, 107 kd, is consistent with the molecular weight of dpap a predicted from the ste13 dna sequence, 107,200 d (c. a. flanagan, d. a. barnes, m. c. flessel, andj. thorner, manuscript submired for publication). indirect immunofluorescence microscopy of cells containing the $7f,/3 gene on a 2# plasmid showed that dpap a was localized to several non-vacuolar puncture patches dispersed throughout the cell, as judged by nomarski optics and in double staining experiments with vat2p antibody (fig. 7 , d-u). this signal was absent in a stei3a strain (fig. 7 , a-c). this staining pattern is typical of the golgi apparatus, as determined by the localization of kex2p and kexlp (a, cooper and h. bussey, manuscript submitted for publication). attempts to localize dpap a in cells containing only the chromosomal copy of ste/3 were unsuccessful due to the low abundance of the protein. because the copy number of yeast 2/~ plasmids can vary from 10-40 copies/cell within a population (rose and broach, 1991) , the fluorescence signal corresponding to dpap a when expressed from a 2# plasmid also varied from cell to cell, ranging from a weak signal to a very strong signal, whereas the intensity of the vat2p signal was consistent from cell to cell. all of the cells that showed a signal with the dpap a antibody displayed a golgi-staining pattern (fig. 7 , d-u; table i ); however, cells exhibiting a very strong signal, presumably due to a high dosage of the ste/3 gene, also showed vacuolar staining (5 % of the cells ofapep4-3 strain; table i ). an example of this is shown in fig. 7, (~ s, and u) . a single cell exhibiting a strong dpap a signal is shown, and the signal stains both vacuolar and punctate non-vacuolar structures, as judged by co-localization with vat2p. the percentage of cells showing vacuolar localization of dpap a decreased significantly in an otherwise isogenic pep4 strain, which contains the full complement of vacuolar proteases (i %; table i) . thus, cells producing high levels of dpap a showed mislocalization of the protein to the vacuolar membrane, with the mislocalized protein degraded in a vacuolar proteasedependent fashion. dpap enzyme assays on permeabilized and non-permeabilized cells expressing dpap a from either cen or 2g plasmids showed that ,088% of the total activity was intracellular; thus, it is possible that a small percentage of dpap a is present at the cell surface, even though no plasma membrane staining was detected by indirect immunofluorescence, however, the apparent extraceuular dpap a enzyme activity could in part be due to a small degree of cell lysis during the assay. to determine whether a specific domain of dpap a con-the journal of cell biology, volume 119, 1992 tained the signal for golgi retention, the fusion proteins aa-b and a-bb were constructed ( fig. 1 c) , in which the lumenal domain of dpap a was exchanged for that of dpap b (aa-b), and the cytoplasmic domain of dpap b was exchanged for that of dpap a (a-bb). immunoprecipitation of aa-b and a-bb from 35s-labeled cells, followed by sds-page and fluorography, showed that both fusion proteins were glycosylated and treatment of the immunoprecipitates with endo h showed that the proteins were of the expected size (fig. 6 b) . the broadly migrating species observed for aa-b and a-bb indicate that the proteins are more extensively modified in the golgi apparatus than dpap a. dpap activity assays on permeabilized or non-permeabilized whole cells demonstrated that the aa-b and a-bb proteins were enzymatically active, and that ~80% of the activity was intracellular. a-bb fractionated with membranes in the presence of high ph sodium carbonate, and thus behaves biochemically as an integral membrane protein (data not shown). indirect immunofluorescence microscopy using anti-dpap b lumenal domain antibody showed that aa-b and a-bb exhibited non-vacuolar punctate staining patterns indistinguishable from wild type dpap a (fig. 8, a-f) . again, no plasma membrane staining was detected. as with dpap a, all cells that showed a signal displayed a punctate staining pattern, and a subset of those cells that showed a strong signal also exhibited vacuolar localization (table i ). an example is shown in fig. 8 (a-c) for cells expressing aa-b. the cell in the bottom right corner shows a very intense fluorescein signal relative to the other cells in the panel, and clearly exhibits co-localization with vat2p. thus, as was seen for wild type dpap a, overproduction of these proteins resulted in some mislocalization to the vacuole. these experiments demonstrate that the cytoplasmic domain of dpap a is sufficient for the retention of an otherwise vacuolar membrane protein in punctate structures typical of the golgi apparatus in s. cerevisiae. the cytoplasmic domain of dpap a also has been shown to be sufficient for golgi retention when fused to the transmembrane and lumenal domains of the vacuolar membrane protein alp (s. nothwehr and 1". stevens, unpublished data). to determine if the cytoplasmic domain of dpap a was also necessary for the retention of dpap a in the golgi, a series of in-frame deletion mutations in the cytoplasmic domain were generated using oligonucleotide-directed mutagenesis (see materials and methods). deletion variants that lacked (fig. 9) , although both proteins also showed a small amount of er staining (table i). immunoblotting analysis of crude extracts prepared from cells expressing a22-aa-b showed a broadly migrating species on sds-page similar to aa-b and a-bb (data not shown; fig. 6 b) , suggesting that the a22-aa-b protein transits the golgi apparatus before its transport to the vacuole. similar to the deletions in the cytoplasmic domain of figure 8 . immunolocalization of aa-b and a-bb. jhry'20-1a (ste13a dap2a) cells containing plasmids expressing either aa-b (pcjr64, a-c) or a-bb (pcjr67, d-f) were fixed, spheroplasted, and stained with dpap a antibody and vat2p mab. the cells were viewed by nomarski optics (a and c) and by epifluorescence using filter sets specific for fluorescein (b and e) and rhodamine (c and f) fluorescence. dpap b, this deletion delays exit of the fusion proteins from the er; however, upon exit from the er, the enzymatically active a22-aaa and a22-aa-b fusion proteins were transported to the vacuole. thus, the cytoplasmic domain is both necessary and sufficient for the retention of dpap a in the golgi apparatus, and the signal for the retention of dpap a maps to a 22-amino acid segment within the ll8-residue cytoplasmic domain. we have previously shown that the transport of dpap b to the vacuole does not involve delivery to the plasma membrane followed by endocytic targeting to the vacuole (roberrs et al., 1989) . this was demonstrated by expressing dpap b in a secl mutant, which at 34~ is blocked at a late stage of the secretory pathway and accumulates secretory vesicles salminen et al., 1987) . to ad-dress the possibility that the mutant constructs analyzed in this study were mislocalized to the plasma membrane followed by rapid endocytic uptake to the vacuole, indirect immunofluorescence experiments were performed on bb-inv, b-a-b, a20-bb, and/x22-aa-b expressed in a secl strain at 34~ as a positive control for accumulation in secretory vesicles, the localization of the fusion protein fusl-laczp was analyzed (trueheart et al., 1987) . the fus1 gene product is required for the breakdown of the cell walls after the fusion of a and ct cells during conjugation (mccaffrey et al., 1987; trueheart et al., 1987) , and has been shown by protease treatment of whole yeast cells to be a plasma membrane protein (trueheart and fink, 1989) . the fusion protein fusl-laczp, consisting of the 254 nh2-terminal amino acids of fuslp fused to the/acz gene product (/3-galactosidase), has been shown by immunofluorescence microscopy to be localized to the plasma membrane after induction during mating (trueheart et al., 1987) . for these experiments, the expression of these proteins was under the control of the inducible gal/promoter (see materials and methods; johnston and davis, 1984) . neither fusl-laczp, bb-inv, b-a-b, or a20-bb showed any signal before addition of galactose (data not shown). fig. 10 (a-f) shows that fusl-laczp was localized to the plasma membrane in a sec ⧠strain after 2 h of induction at 34~ but accumulated intracellularly, presumably in secretory vesicles, in a secl strain under the same conditions. no colocalization with the vacuolar marker, alp, was detected. figure 10 . immunolocalization in sec* and secl strains. jhry20-1a (sec*) and sey-5016 (secl) cells containing plasmids encoding fusl-laczp (pcjrll4) or b-a-b (pcjr79) under the control of the gal/ promoter were grown to log phase in media containing raffinose, then shifted to 34~ at the same time that galactose was added to the cultures. after 2 h, the cells were fixed, spheroplasted, and prepared for immunofluorescence. cells expressing fusl-laczp were stained with anti-/~-galactosidase mab and anti-alp polyclonal antibody, and were viewed using filter sets specific for fluorescein and rhodamine fluorescence, respectively. cells expressing b-a-b were stained as in fig. 5 . typically, fusl-laczp accumulated in a large patch in the bud and bud neck region of budded cells, and near the cell periphery of unbudded cells. bb-inv, b-a-b, and a20-bb were each localized to the vacuole under these conditions (e.g., fig. 10 , g-l; table ii) , as defined by co-localization with vat2p. the intraceuular, non-vacuolar signal occasionally observed for b-a-b (23 %; table ii ) was different from that of fusl-laczp, in that the signal looked reminiscent of the golgi signal observed for dpap a (data not shown). this was also observed for b-a-b in sec ⧠cells at 34~ suggesting that the kinetics of delivery of this protein is slowed at elevated temperatures. the analysis of a22-aa-b in the secl strain was complicated by the fact that expression of the protein was leaky; ,~50% of the cells in the population showed a weak vacuolar staining pattern before galactose addition. however, addition of galactose to the culture after shifting the cells to 34~ resulted in '~90% of the cells exhibiting an intense vacuolar signal (data not shown). these data show that the bb-inv, b-a-b, a20-bb, and a22-aa-b proteins are targeted directly from the golgi apparatus to the vacuole without transient appearance at the cell surface; thus, these proteins follow the same route to the vacuole as wild type vacuolar membrane proteins (roberts et al., 1989) . the search for the vacuolar localization determinant of dpap b led to the surprising conclusion that neither the cytoplasmic, transmembrane, nor lumenal domain of the protein was necessary for vacuolar delivery. analysis of the golgi retention signal of dpap a led to the equally surprising observations that both overproduction of the protein and mutations in the cytoplasmic domain resulted in mislocalization of dpap a to the vacuole, not the plasma membrane. these results were unexpected, given that the targeting of soluble vacuolar proteins in yeast and soluble and membrane proteins of the lysosomes of animal cells require targeting information to prevent delivery to the cell surface (vails et al., 1990; kornfeld and mellman, 1989; williams and fukuda, 1990; machamer, 1991) . the fusion proteins analyzed in this study are appropriate tools for these experiments for the following reasons. all of the proteins were stable, enzymatically active, membranebound, and glycosylated when expressed in yeast, indicating that they have the correct membrane topology. in addition, all of the fusion proteins containing the dpap b lumenal domain, i.e., a20-bb, a27-bb, b-a-b, and a22-aa-b, were transported to the vacuole after receiving glycosyl modifications in the golgi apparatus. finally, each of these fusion proteins was transported to the vacuole directly from the golgi apparatus, without prior delivery to the plasma membrane followed by endocytic uptake to the vacuole. other golgi membrane proteins have also been shown to be mislocalized to the vacuole rather than the plasma membrane. both mutations in the cytoplasmic domain of kexlp as well as its overproduction result in mislocalization of the protein to the vacuolar membrane (a. cooper and h. bussey, manuscript submitted for publication). similarly, a single amino acid change in the cytoplasmic domain of kex2p results in its transport to the vacuole (c. wilcox, k. redding, r. wright, and r. fuller, manuscript submitted for publication). whereas this result might appear to conflict with earlier published data on kex2p, more recent analysis indicates that membrane-bound forms of kex2p that fail to be retained in the golgi are found in the vacuole rather than the plasma membrane, when these proteins are expressed at wild type or modestly elevated levels (c. wilcox, k. redding, r. wright, and r. fuller, manuscript submitted for publication). given that several golgi membrane proteins all show missorting to the vacuole when either mutant or overproduced, the model we favor to explain these data is the vacuolar default model, which states that vacuolar membrane proteins do not require sorting information, because the default pathway for membrane proteins of the secretory pathway leads to the vacuole. alternatively, one could argue that, given the similarity between dpap b and dpap a, both proteins could contain vacuolar targeting information in their transmembrane domains, even though no significant similarity is apparent from the amino acid sequence data (c. a. flanagan, 13. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication). mutations in the golgi retention signal of dpap a, or saturation of the retention apparatus, would then result in the delivery of dpap a to the vacuole. this model requires that both kex2p and kexlp must also have cryptic vacuolar targeting signals. with regard to the cryptic vacuolar targeting signal model, we have found that replacement of the membrane spanning domains of dpap a and a22-aaa with a 21 residue hydrophobic sequence, l(lalv)~, creating the proteins axa and a22-axa, results in the retention of axa in the golgi and the transport of a22-axa to the vacuole (s. nothwehr and t. stevens, unpublished data). these data suggest that the transmembrane domain of dpap a does not contain vacuolar targeting information, and thus support the vacuolar default model. the vacuolar default model is directly testable by analyzing potential localization signals of yeast plasma membrane proteins. in apparent conflict with the vacuolar default model is the observation that the majority of kex2p (70%) is missorted to the plasma membrane in cells deficient for clathrin heavy chain (payne and schekman, 1989) . however, dpap a is missorted to the cell surface to a lesser extent (30 %; seeger and payne, 1992a) . it is possible that the majority of dpap a and a significant percentage of kex2p are missorted to the vacuole in cells lacking clathrin heavy chain. mutations in clathrin may affect both the retention of golgi membrane proteins and the functional integrity of the sorting pathway (seeger and payne, 1992b) . this could obscure the default pathway for membrane proteins, resulting in the transport of golgi membrane proteins to the plasma membrane as well as the vacuole. an alternative explanation of the data is that one or more of the dpap a and dpap b fusion or mutant proteins analyzed in this study were in a partially unfolded state, and thus recognized as abnormal and transported directly from the er to the vacuole via a "garbage" pathway. there is no clear evidence for the existence of such a pathway in yeast or animal cells. however, several cases have been described in animal cells in which non-lysosomal membrane proteins were transported to lysosomes, either due to mutations (armstrong et al., 1990) , or in the case of the heptameric t-cell antigen receptor, complexes lacking the ~" subunit are degraded in lysosomes (minami et al., 1987) . in these cases, the proteins were transported through the golgi apparatus, and it is unclear whether the proteins were transported to lysosomes by a "garbage" pathway, or by the uncovering of a cryptic lysosomal targeting signal. in the great majority of cases, mislocalized membrane proteins of the secretory pathway in animal cells accumulate at the plasma membrane, which is presumed to be the default destination for these proteins (e.g., williams and fukuda, 1990) . it is well established that proteins that are slow to reach the folded state are retained in the er due to the action of proteins such as bip (pelham, 1989; rothman, 1989) , and proteins that are unable to fold or oligomerize properly are degraded in the er (klausner and sitia, 1990) . several of the proteins analyzed in this study (i.e., a20-bb, a27-bb, a22-aaa, and a22-aa-b) showed increased retention in the er. however, all of these proteins eventually exited the er, were enzymatically active, and were transported through the golgi complex to the vacuole. the 22-amino acid segment of the cytoplasmic domain of dpap a presumably contains the recognition domain for a "retention protein" of the golgi apparatus. this protein could be a permanent resident of the golgi apparatus; alternatively, the "retention protein" could reside in a post-golgi compartment and function as a salvage receptor, such as the proposed receptor for soluble er proteins (pelham, 1989) . it is interesting to note that the 22-amino acid stretch identified as the dpap a golgi retention signal contains five phenylalanine residues (fig. 1) . in animal cells, one or more aromatic amino acids in the cytoplasmic domains of cell surface receptors have been shown to comprise part of the signal for the clustering into coated pit regions of the plasma membrane (chen et al., 1990; johnson et al., 1990; lobel et al., 1989; mcgraw et al., 1991) . that the phenylalanine residues in the a22 region may play a direct role in golgi retention is supported by the observation that mutations in just two of these residues result in a substantial level of missorting of dpap a to the vacuole (s. nothwehr and t. stevens, unpublished data) . the results of this study suggest that the default pathway for membrane proteins of the yeast secretory pathway may be different from that of certain mammalian cell lines that have been examined, where mutant forms of er, golgi, and lysosomal membrane proteins are mislocalized to the plasma membrane with the bulk flow of membrane (machamer and rose, 1987; jackson et al., 1990; williams and fukuda, 1990; wieland et al., 1987; karrenbauer et al., 1990; machamer, 1991) . however, in polarized epithelial ceils, it remains unclear which membrane serves as the default destination for membrane proteins (simons and wandinger-ness, 1990; mostov et al., 1992) . even if the default compartments for membrane proteins of the 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the cytoplasmic tail we acknowledge cathy flanagan, jeremy thorner, antony cooper, howard bussey, celeste wilcox, robert fuller, and greg payne for communication of results prior to publication; scott ernr, charlie boone, and george sprague for plasmids; margaret ho and joe horecka for help in the construction of the a20-bb and fusl-laczp constructs, respectively; and jerry gleason and scan poston for the photographic work. we especially thank christopher raymond for numerous insightful contributions to this work, and nick davis, charlie boone, george sprague, antony cooper, carol vater, cynthia bauerle, and margaret ho for comments on the manuscript. received for publication 10 february 1992 and in revised form 30 june 1992. key: cord-256325-q70rky3r authors: stewart, cameron r.; deffrasnes, celine; foo, chwan hong; bean, andrew g. d.; wang, lin-fa title: a functional genomics approach to henipavirus research: the role of nuclear proteins, micrornas and immune regulators in infection and disease date: 2017-07-04 journal: roles of host gene and non-coding rna expression in virus infection doi: 10.1007/82_2017_28 sha: doc_id: 256325 cord_uid: q70rky3r hendra and nipah viruses (family paramyxoviridae, genus henipavirus) are zoonotic rna viruses that cause lethal disease in humans and are designated as biosafety level 4 (bsl4) agents. moreover, henipaviruses belong to the same group of viruses that cause disease more commonly in humans such as measles, mumps and respiratory syncytial virus. due to the relatively recent emergence of the henipaviruses and the practical constraints of performing functional genomics studies at high levels of containment, our understanding of the henipavirus infection cycle is incomplete. in this chapter we describe recent loss-of-function (i.e. rnai) functional genomics screens that shed light on the henipavirus–host interface at a genome-wide level. further to this, we cross-reference rnai results with studies probing host proteins targeted by henipavirus proteins, such as nuclear proteins and immune modulators. these functional genomics studies join a growing body of evidence demonstrating that nuclear and nucleolar host proteins play a crucial role in henipavirus infection. furthermore these studies will underpin future efforts to define the role of nucleolar host–virus interactions in infection and disease. paramyxoviruses (order mononegavirales) are single-stranded rna viruses of negative polarity that can cause diseases in humans (rabies, measles virus, mumps virus, respiratory syncytial virus, human parainfluenza virus, ebola virus) and animals (newcastle disease virus, canine distemper virus, borna disease virus). the family paramyxoviridae is divided into two subfamilies (paramyxovirinae and pneumovirinae), with hendra virus (hev) being the foundation member of the genus henipavirus in the subfamily paramyxovirinae. the discovery of the hev and nipah virus (niv) had a striking impact on our understanding of paramyxovirus biology. henipaviruses have a much wider host range and a significantly larger genome than other paramyxoviruses, and to date are the only biosafety level (bsl)-4 agents within the family. with mortality rates of human infection between 50 and 100%, hev and niv are among the most deadly viruses known to infect humans. hev emerged in 1994 in the brisbane suburb of hendra, queensland, australia, where it caused an outbreak of severe respiratory disease in horses that led to the natural death or euthanasia of 14 out of 21 affected animals. two people who had close contact with the infected horses were infected and one of these patients died (murray et al. 1995) . extensive sampling demonstrated that australian mainland flying foxes (family pteropodidae, genus pteropus) were seropositive for neutralising antibodies against hev (young et al. 1996) , while the virus was subsequently isolated from flying fox uterine fluid and urine (halpin et al. 2000) , providing strong evidence for australian mainland flying foxes as the hev reservoir. sporadic hev incidents occurred in horses between 1994 and 2010, with 14 events identified. an alarming number of hev incidents (34 in total) occurred between 2011 and 2013, with 18 of those occurring in 2011 alone, highlighting the unpredictable nature of hev outbreaks. seven human cases of hev disease have been observed, four of which resulted in fatal disease. all recorded cases of hev transmission to humans have occurred directly from affected horses. the horses are believed to have acquired hev infection following direct exposure to secretions from flying foxes. more recently, the decline of reported human cases of hev infection is potentially due to the development of a vaccine to inhibit hev disease in horses (middleton et al. 2014) . niv was first identified during a disease outbreak on the west coast of peninsular malaysia in late 1998. commercial pig farmers suffered disease characterised by febrile encephalitis that was linked to mild respiratory and neurological disease in pigs (mohd nor et al. 2000 ; from the centers for disease control and prevention 1999) . nucleotide sequencing demonstrated the virus was closely related to hev, whilst fruit bats of the pteropodidae family, pteropus genus, were confirmed as the natural reservoir (yob et al. 2001) . epidemiological evidence suggested that human infections were caused by transmission from pigs which likely had prior contact with fruit bats (update: outbreak of nipah virus-malaysia and singapore 1999). by mid-1999, cases of human infection were reported in singapore, where abattoir workers developed niv infection associated with contact with pigs imported from malaysia. this initial outbreak of niv in malaysia resulted in 265 human cases reported with 105 deaths. since 2001, niv outbreaks have been reported almost every year in selected districts of bangladesh (hossain et al. 2008; luby et al. 2009a) . unlike hev, human-to-human transmission of niv has been documented (luby et al. 2009b) , including in a hospital setting. an increasing focus on flying foxes as viral reservoirs has led to the discovery of new henipaviruses. the genus was expanded in 2012 upon the isolation and characterisation of cedar virus (cedpv), isolated from bat urine samples from a flying fox colony in cedar grove, south east queensland. cedpv shows a remarkably similar genome organisation to hev and niv, antigenic cross-reactivity of the nucleocapsid protein between henipaviruses, and shares the same predominant entry receptor molecule, ephrin-b2 (marsh et al. 2012 ). however, a critical difference between cedpv and hev and niv is that the cedpv p gene lacks coding capacity for the immune antagonising v protein, whilst the cedpv p protein shows an impaired capacity to bind and inhibit ifn signalling via signal transducer and activator of transcription (stat)1 and stat2 (lieu et al. 2015) . accordingly, cedpv infection induces a robust type i interferon (ifn) response in human cells in vitro and does not cause clinical disease in ferret and guinea pig models of disease. such findings highlight the importance of immune evasion in the context of henipavirus pathogenicity and demonstrate the diverse range of pathogenicity within the same genus. in addition to these three viruses, the henipavirus genus is likely to be expanded in the future to accommodate the discovery and characterisation of emerging viruses from bats and other reservoirs. west african fruit bats harbour neutralising antibodies against hev and niv in particular, demonstrating a wider geographical range for henipaviruses not limited to pteropid bats (hayman et al. 2008 ). furthermore, a novel henipa-like virus, mojiang paramyxovirus, was isolated from rats in the yunnan province of china in 2012 and may have caused fatal disease in three individuals (wu et al. 2014) . alarmingly, a recent study looking at bat and human serum samples from cameroon found that 3-4% of human samples were seropositive for henipaviruses, and that this was almost exclusively among individuals who reported butchering bat meat, providing the first evidence of human henipavirus spillover infections in africa (pernet et al. 2014 ). there are currently no licensed therapies to treat human cases of henipavirus infection. therefore, gaining a deeper understanding of host pathways exploited by henipaviruses for infection may identify targets for new antiviral therapies. viruses rely on the cell host machinery for completion of their infection cycle and therefore have adapted to interact with or exploit host molecules. retroviruses, most dna viruses, and many orthomyxoviruses replicate their genomes in the host nucleus. conversely, most positive-sense single-stranded viruses such as picornaviruses and flaviviruses and negative-sense, single-stranded viruses such as filoviruses, rhabdoviruses, and paramyxoviruses are perceived as cytoplasmic viruses and therefore are believed to not have a nuclear stage in their life cycle, replicating their genome entirely in the cytoplasm (lamb and parks 2007) . however, proteins of some of these viruses can traffic into nuclear compartments during infection (peeples 1988; yoshida et al. 1976; ghildyal et al. 2003; monaghan et al. 2014; wang et al. 2010) and this movement is sometimes critical for efficient infection (wang et al. 2010 ). this evidence indicates that the host nucleus may play a significant role in the infection cycle of henipaviruses and that the dynamics of virus-host interactions that occur in the nuclear compartments is an understudied area of molecular biology and virology. furthermore, since important discoveries in cell biology often follow studies of how viruses exploit normal host machinery, investigations into these nuclear interactions may reveal interesting novel insights into the cell biology of the mammalian nucleus. with this in mind, functional genomics provides a powerful and unbiased approach to study these biological questions. functional genomics refers to the development and application of global (genome-wide or system-wide) experimental approaches to assess gene function by making use of the information and reagents provided by sequenced genomes (hieter and boguski 1997) . a wide range of laboratory techniques can be considered as functional genomics, including genome interaction mapping (at the dna level), microarrays, transcriptomics and serial analysis of gene expression (sage) (at the rna level), yeast 2 hybrid systems and affinity chromatography and mass spectrometry (at the protein level) and loss-of-function studies such as mutational studies, rna interference (rnai) and clustered regularly interspaced short palindromic repeats (crispr) studies. functional genomics has demonstrated much power in its ability to dissect the dynamic interplay between host and viral factors during a virus infection, paving the way for novel drug targets. for instance, a haploid genetic screen resulted in the discovery of the once elusive entry receptor for ebola virus (carette et al. 2011 ). there have been many full-or partial-genome rnai screens of host-virus interactions, including orthomyxoviruses (brass et al. 2009; hao et al. 2008; karlas et al. 2010; konig et al. 2010; shapira et al. 2009 ), retroviruses (zhou et al. 2008; konig et al. 2008; brass et al. 2008 ) and flaviviruses (ang et al. 2010; sessions et al. 2009 ). until recently, such information was lacking for henipaviruses, and perhaps surprisingly, for paramyxoviruses generally. functional genomics screens can be technically challenging, laborious and involve the use of robotics and advanced imaging equipment. consequently there are technical and practical challenges to performing high-throughput screens at higher levels of containment. hev and niv are classified at bsl-4 agents due to their association with lethal human disease and the absence of preventive measures and effective treatments to combat infections. bsl-4 facilities feature additional precautions to protect workers from infections and prevent exposure, such as infectious work being conducted within class ii biosafety cabinets, limited access by secure, locked doors, hepa filtration of laboratory air, and additional primary containment (positive pressure air suits or class iii biosafety cabinets). due to these limitations, previous genome-wide screens for bsl-4 viruses used surrogate viruses, such as pseudotyped particles, and have been performed under bsl-2 conditions (kouznetsova et al. 2015; kleinfelter et al. 2015) . functional genomics have been employed to study henipavirus infection. for instance, the entry receptor of hev and niv, ephrin-b2, was identified by microarray analysis of infection-permissive and infection-resistant cell lines (bonaparte et al. 2005) . transcriptomics and proteomics have been utilised to uncover key differences in cellular responses to hev infection in hev disease-susceptible (human) and disease-resistant (bat) cells, and suggest that activation of apoptosis pathways via the innate immune pathway may contribute to the tolerance of henipaviruses by flying foxes (wynne et al. 2014 ). here we largely focus on findings from two recent rnai screens to identify protein-coding genes and host-encoded micrornas impacting the henipavirus infection cycle in human cells. not only can these findings be compared to published rnai screens of hostvirus interactions, but the identification of host genes required for infection (as opposed to those that are merely differentially expressed during infection) may deliver new targets for the development of antiviral therapies. the large number of hev incidents in australia from 2011 to 2013 prompted researchers at our laboratory to establish the capability to perform genome-wide rnai screens at bsl-4. central to this work was the development of a recombinant hev expressing the renilla luciferase construct, which allowed for high throughput and rapid measurement of virus infection (marsh et al. 2013 ). this recombinant virus was shown to be lethal in the ferret model of henipavirus disease and exhibited a pathogenesis profile comparable to the wild-type virus. functional genomics at high containment also required the establishment of protocols and/or safe work procedures for the operation and decontamination of liquid handling robots. a genome-wide analysis of host protein-coding genes required for henipavirus infection involved a primary screen assaying 18,120 protein-coding genes, followed by a secondary deconvolution screen and a tertiary screen determining whether screen results obtained using recombinant hev could be recapitulated using wild-type hev and niv (deffrasnes et al. 2016) . applying a robust z score normalisation method often used to interpret sirna screen results (birmingham et al. 2009; zhang et al. 2006) , 585 and 630 genes were identified that promoted or suppressed hev infection, respectively, without adversely impacting cell numbers. at the completion of the primary screen, 200 proviral genes were selected based on rank for the secondary deconvolution screen. by this measure, 20 high-and 46 medium-confidence genes (>2 standard deviations from mean mock values for 4/4 or 3/4, or 2/4 sirnas, respectively) were identified as being required for hev infection. the apparent reliance of henipavirus infection on the nuclear or nucleolar host proteins was particularly striking, as over 40% of high confidence hits localise in the nucleus or nucleolus, with many involved in ribosome biogenesis (table 1) . the nucleus is the site of gene expression and dna transcription into mrna, and houses the early steps of the rnai pathway. the nucleus is separated from the cell cytoplasm by the nuclear envelop which contains nuclear pores and import/export proteins allowing the passage of small molecules such as mrna. nuclear import/export proteins such as xpo1 and kpna3, which are required for trafficking of larger molecules like proteins, were identified by rnai screen as required for henipavirus infection (deffrasnes et al. 2016) . the nucleolus is a highly dynamic structure and has increasingly been shown to play a critical role in virus-host interactions (rawlinson and moseley 2015; xu et al. 2016 ). the nucleolus contains three regions composed of the fibrillar centre (fc) in the middle, surrounded by the dense fibrillary component (dfc) and the granular component (gc). this membrane-less structure contains a high concentration of proteins and rnas and is the site of ribosomal rna (rrna) synthesis and ribosome production but is also a multifunctional structure in eukaryotic cells. cell cycle progression, stress response, genetic silencing, regulation of apoptosis, cell migration and invasion are all functions associated with the nucleolus or partly regulated in this compartment (rawlinson and moseley 2015; xu et al. 2016; pederson 2010 ). fibrillarin is the main nucleolar protein responsible for the chemical modification of ribosomal rna (rrna). this 34-38 kda 2′-o-methyltransferase transfers methyl groups from its substrate, the s-adenosylmethionine (sam), to the 2-hydroxyl groups of ribose target in rrna. fibrillarin has also been shown to methylate glutamine residue 104 of the human histone h2a, weakening its binding to the fact (facilitator of chromatin transcription) complex and impacting chromatin remodelling and rdna transcription by rna pol i (tessarz et al. 2013) , which points at an additional role for fibrillarin in ribosome biogenesis and translation. pre-ribosome processing, modification of pre-rrna rpl7a component of the 60s ribosomal subunit sp7 transcriptional regulation xpo1 nuclear export fibrillarin itself is methylated on several arginine residues by protein arginine n-methyltransferase 1 (prmt1), which is thought to influence its activity (rodriguez-corona et al. 2015) . expression levels of fibrillarin have been shown to be regulated by p53 through direct binding to fibrillarin intron 1. abnormal levels of fibrillarin have been detected in p53-inactivated cancer cells and a decrease in p53 levels has been associated with an increase in fibrillarin expression, and conversely an increase in p53 expression results in decreased fibrillarin expression (marcel et al. 2013) . high levels of fibrillarin lead to changes in the rrna methylation pattern, diminished translation fidelity and increase in ires-mediated translation of some cancer genes. moreover, ribosome biogenesis is often dysregulated and over-activated in cancer cells that have a decreased or absent p53 expression (marcel et al. 2013) . in its n-terminal region, fibrillarin contains a glycine-and arginine-rich region (the gar domain) enabling interaction with cellular and viral proteins, and acting as a nucleolar retention signal. its c-terminal region (mtase) contains multiple rna-binding domains, a catalytic site allowing for fibrillarin methyltransferase function, and is the site for nop56/58 interaction. fibrillarin is a part of at least one nucleolar ribonucleoprotein (snornp) complex comprising the nop56, nop58 and 15.5 k nucleolar proteins. x-ray data have suggested that the methylation of rrna requires the formation of this complex with involvement of four fibrillarin molecules interacting with different regions of the target rrnas. the yeast equivalent of fibrillarin, nop1, has been more extensively studied than the human counterpart but fibrillarin is a well-conserved protein in most organisms, reinforcing the notion that all post-transcriptional processes involving fibrillarin such as chemical modification (methylation) of rrna, pre-rrna cleavage and ribosome assembly are essential for proper cellular functioning (rodriguez-corona et al. 2015) . in eukaryotes, ribosome biogenesis involves numerous nucleolar proteins and accessory factors, around 80 ribosomal proteins, many small nucleolar rnas (snornas), three rna polymerases (rna polymerase i, ii and iii) and four different species of rrnas. the process of assembly of elongation-competent 80s ribosomes is divided into three major steps: (1) ribosomal dna (rdna) transcription into precursor rrnas (pre-rrnas), (2) processing of pre-rnas into mature rrnas, and then (3) assembly of rrnas with ribosomal proteins into functional ribosomes. in the nucleolus, the rna polymerase i (rna pol i) is responsible for transcribing the 18s, 5.8s and 28s rrna from a single polycistronic pre-rrna, while rna pol iii transcribes the 5s rrna in the nucleus (xue and barna 2012) . the pre-rnas are then cleaved and modified during the pre-rrna processing phase. all ribosomal proteins (rp) are transcribed in the cytoplasm by rna pol ii and then translated before migrating to the nucleolus. these rp, along with nucleolar proteins such as fibrillarin and rpl13a, are responsible for modifying the rrnas (ribose 2′-o-methylation, pseudouridylation, etc.) with the activity of more than 100 snornas guiding the process in a site-specific manner. the main nucleolar protein involved in rrna modification is fibrillarin, which methylates more than 100 sites essential for ribosome biogenesis and stability. although these post-transcriptional modifications are crucial for ribosome functions, their roles are not yet fully understood. in eukaryotes, the large 60s subunit of ribosomes is made of the 5s, 5.8s, and 28s rrna along with multiple large subunit ribosomal proteins (rpl), while the small 40s subunit is made of the 18s rrna along with multiple small subunit ribosomal proteins (rps). the two subunits are assembled in the nucleolus into the 80s ribosomes before being transferred into the cytoplasm. deffrasnes and colleagues showed that sirna-mediated knockdown of fibrillarin expression dramatically reduced hev protein production and viral genome replication but did not impact viral fusion, and that fibrillarin catalytic activity was essential to henipavirus infection. on the other hand, overexpression experiment did not lead to an increase in viral titers, suggesting that a simple reduction or increase in overall ribosome production is unlikely to explain the reliance of henipaviruses on fibrillarin activity (deffrasnes et al. 2016 ). the requirement of fibrillarin and several other proteins from the ribosomal biogenesis pathway for henipavirus infection points a reliance on translation for efficient infection. however, while we tend to view ribosomes as homogenous, new studies reveal a more heterogeneous nature of ribosomes due to differences in the ribosomal proteins recruited, post-translational modifications of rrna and rrna composition. moreover, ribosomal proteins have been found to have additional functions outside of their primary roles in ribosomes and to be involved in other nucleolar functions such as regulation of cell proliferation, tumorigenesis and dna damage response (xu et al. 2016; xue and barna 2012; au and jan 2014) . in eukaryotes, most messenger rna (mrna) harbour a 5′ 7-methylguanosine cap structure and a 3′ poly(a) tail, which are both required for canonical, cap-dependent translation. a cap-independent translation mechanism also utilised by a subset of host proteins is called internal ribosome entry site (ires)-mediated translation. it is believed that most genes translated via an ires are related to stress response, cell proliferation, cell death/survival, and that ires-mediated translation happens when the canonical cap-dependent translation is inhibited either by the host reaction to environmental factors, damage, stress or infections. however, a group recently suggested that thousands of human genes are translated via this cap-independent mechanism, representing a 50-fold increase in the number of sequences previously associated with this translation pathway (weingarten-gabbay et al. 2016) . recently a new type of translation has been described in vesicular stomatitis virus (vsv)-infected cells. this non-canonical cap-dependent protein translation involves the ribosomal protein rpl40 acting as a constituent of the large subunit of ribosomal complexes and suggests a novel ribosome-specialised translation initiation pathway benefiting viral mrna translation (lee et al. 2012 ). translations of viral proteins from several other mononegaviruses, including the paramyxoviruses measles virus (mev) and newcastle disease virus (ndv), and a subset of cellular transcripts, are also rpl40-dependent. how henipavirus mrnas are translated is not fully understood. whilst the rpl40-dependent form of cap-dependent translation remains to be characterised in detail, one could speculate that fibrillarin, like rpl40, acts a novel initiation factor for henipavirus mrnas. the fact that depleting cells of fibrillarin did not impact synthesis of influenza a viral proteins (which occurs via the canonical cap-dependent pathway) suggests that henipavirus mrna translation occurs via a non-canonical pathway, perhaps used by a subset of cellular transcripts. such a concept would allow henipavirus protein synthesis to proceed in an environment where viruses may induce cellular translation shutdown in order to suppress host antiviral immune responses. there are several reports of paramyxoviruses blocking canonical translation pathways, including the mev n protein binding to the eukaryotic initiation factor 3 (eif3-p40) (sato et al. 2007 ), whilst the p and v proteins of simian virus 5 (sv5) limit activation of the double-stranded rna (dsrna)-dependent protein kinase (pkr) to limit both host and viral protein translation (gainey et al. 2008 ). similar to sv5, sirna-mediated depletion of pkr results in increased hev growth (robust z score 1.46), consistent with the notion that shutdown of host protein translation inhibits henipavirus infection. if future studies do indeed demonstrate a role of fibrillarin in influencing the synthesis of ribosome subtypes required for viral protein translation, this may explain the targeting of fibrillarin by several viral proteins. fibrillarin binds the hev matrix (m) protein during the early stages of infection, whilst the hiv-tat protein has been reported to bind fibrillarin and u3 snorna, both required for pre-rrna processing, and this interaction reduces the pool of cytoplasmic ribosomes (ponti et al. 2008) . intriguingly, the nucleoprotein of porcine reproductive and respiratory syndrome virus, the non-structural protein 1 (ns1) of a h3n2 influenza virus (melen et al. 2012 ) and the non-structural protein 3b of the severe acute respiratory syndrome coronavirus (yuan et al. 2005 ) all bind and co-localise with fibrillarin in the nucleolus; however, the reasons for this binding are yet to be determined. many negative strand viruses encode viral proteins that localise in the nucleus and/or nucleolus at some point in their infection cycle [reviewed in (rawlinson and moseley 2015; hiscox 2003; oksayan et al. 2012; flather and semler 2015; watkinson and lee 2016) ]. within the paramyxoviridae, nuclear localisation of matrix (m) protein has previously been described for ndv (peeples 1988) , sendai virus (sev) (yoshida et al. 1976 ), human respiratory syncytial virus (ghildyal et al. 2003) , hev (monaghan et al. 2014 ) and niv (wang et al. 2010) . during the early stages of henipavirus infection or when expressed ectopically (monaghan et al. 2014; wang et al. 2010) , the hev and niv m proteins traffic through the nucleolus to the cytoplasm. it has been recently shown that nuclear traffic is required for the henipavirus m protein to coordinate viral budding. the henipavirus m protein is a structural protein that mediates viral assembly and budding (liljeroos and butcher 2012; takimoto and portner 2004; eaton et al. 2007 ). indeed, for both hev-m and niv-m, overexpression of these proteins alone is sufficient to trigger viral-like particles (vlps) that bud into the supernatant. wang and colleagues (2010) demonstrated that mutation of niv-m nuclear localisation signals (nls) or nuclear export signals (nes) blocks nuclear/cytoplasmic traffic and impairs viral budding. furthermore, a highly conserved lysine residue in the nls (k258) serves two functions: its positive charge mediates niv-m nuclear import, while is also a potential site for monoubiquitination which regulates niv-m nuclear export. niv infection (deffrasnes et al. 2016 ). this raises the question: do henipavirus m proteins traffic through the nucleolus for other reasons? the multi-faceted roles of paramyxovirus proteins in replication-specific roles and various cellular processes, particularly immune evasion, would suggest so. to explore whether m binds to host proteins associated with infection efficiency, results from the genome-wide rnai hev screen were cross-referenced against a proteomics study by pentecost and colleagues cataloguing host proteins that bind hev-m and niv-m, among other paramyxovirus m proteins (pentecost et al. 2015) . that study revealed that the henipavirus m interactome spans hundreds of host proteins, with interactions with nuclear pore complex proteins, nuclear transport receptors and nucleolar proteins particularly prevalent. interestingly, niv-m and hev-m interactomes show notable overlap to other paramyxovirus m proteins, including sev and ndv, with over 60% of the proteins found in any single interactome also found in the interactomes of one or more of the other three viruses (pentecost et al. 2015) . whilst the binding of fibrillarin to hev-m was demonstrated by co-immunoprecipitation assays (deffrasnes et al. 2016 ) and this was not observed by proteomics, interactions were observed between hev-m and numerous nucleolar proteins such as nop58 (pentecost et al. 2015) which forms a complex with fibrillarin, supporting a functional interaction between fibrillarin and hev-m. the relative hev growth (presented as robust z scores) in cells depleted of the 389 hev-m-binding host proteins is shown in fig. 1a . of the 327 candidates assayed, hev-m binds to 22 protein-coding genes that have a large impact (robust z score −2 or ! 2) on hev infection, roughly evenly distributed between proviral (12) and antiviral (10) candidates. designating all candidates genes with z scores <0 as proviral and genes with z scores >0 as antiviral, host proteins that bind hev-m appears to be pro-and antiviral at approximately equal ratios with a slight enrichment of proviral genes (174 proviral candidates vs. 146 antiviral candidates). an assessment of whether the relative abundance of hev-m-host protein interactions indicated a likelihood of that host protein adopting a proviral or antiviral function was also carried out (fig. 1b) . the relative abundance of host proteins within the proteomics dataset is represented as the normalised spectral abundance factor (nsaf), with higher nsaf values presenting more abundant interactions. plotting nsaf values against robust z scores demonstrates that host proteins that bind hev-m with high abundance (nsafe5 scores between 250 and 938) were more proviral (11 candidates) than antiviral (4 candidates, z score sums: proviral 13.9, antiviral 2.6). these candidates are listed in table 2 and include several ribosomal proteins, further implicating m in host translation. akin to fibrillarin, the critical role of host molecules in henipavirus infection and pathogenesis can be inferred by their specific targeting by viral proteins. this is particularly true in the context of immune evasion, as the innate antiviral immune response is a known target for several henipavirus proteins. the henipavirus genome contains six transcriptional units, n, p, m, f, g and l, coding for nine proteins (eaton et al. 2007 ). the p gene alone codes for at least four of the proteins: p, w, v and c . all four of these proteins are involved in modification of the immune response in the host cell, through inhibition of the type i interferon (ifn) responses [reviewed in (audsley and moseley 2013) ]. intracellular detection of pathogen-associated molecular patterns (pamps) is mediated by membrane-bound toll-like receptors (tlrs) or cytoplasmic retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) and nucleotide-binding oligomerisation domain containing (nod)-like receptors (nlrs). engagement of these receptors with their agonists results in the activation of complex signalling pathways culminating in the production of cytokines and anti-microbial compounds. a critical component of this response is the type i ifn system, which induces a local antiviral state upon detection of viruses or intracellular bacteria or molecules associated with their replication (schoggins and rice 2011). genes with z scores <0 were designated proviral, while genes with z scores >0 were designated antiviral. values represent the sum of all the z scores. it should be noted that 43 genes were excluded from analysis due to ambiguous gene identification listings in the proteomics study, whilst the silencing of 19 additional gene targets resulted in cell death that prevented the measurement of virus growth. b plot of the z score of hev-m-binding proteins (x-axis) and relative abundance of hev-m interactions, represented by normalised spectral abundance factor (y-axis) viral replication is typically detected by tlrs 3 and 7/8 in endosomal compartments (alexopoulou et al. 2001; lund et al. 2004) , whilst rig-i and/or melanoma differentiation-associated gene 5 (mda5) recognise short or long viral dsrna intermediates in the cytosol (yoneyama et al. 2004; triantafilou et al. 2012) . tlr3 activates the tir-domain-containing adapter-inducing ifn-b (trif) (matsumoto et al. 2011) , whilst rig-i/mda5 interact via their caspase recruitment domains (cards) with mavs (mitochondrial activated signalling protein) (seth et al. 2005) to induce signalling. activation of trif or mavs promotes recruitment of multiple cytosolic effectors, resulting in the phosphorylation and dimerisation of interferon regulatory factor (irf) 3 or liberation of nf-jb from its inhibitory complex. these transcription factors then shuttle into the nucleus to form part of a large multiprotein complex that binds to the promoter region of ifn-b and initiates transcription (honda and taniguchi 2006) . the c-terminus of the hev v protein binds and sequesters mda5, thereby impairing ifn-b transcription in response to double-stranded rna (andrejeva et al. 2004) . this binding appears to be conserved amongst most paramyxoviruses including niv, sv5 and mumps virus (childs et al. 2007 ). intriguingly, rig-i is not targeted by paramyxovirus v proteins, and perhaps consistent with this, the genome-wide rnai screen suggested that depleting cells of mda5 increased hev infection (robust z score 2.02), whilst targeting rig-i had very little impact (z score −0.37). similar to the nlr cytoplasmic antiviral immune responses, tlr3-dependent antiviral signalling is also inhibited by henipaviruses, with the w protein localising to the nucleus via the importin molecules kpna3 and kpna4 to block irf3-responsive promoter activation by virus and intracellular dsrna (shaw et al. 2005). transfecting niv-w into cells in a dose-dependent manner sequesters inactive irf3 in the nucleus, thus depleting the pool of available irf3 for phosphorylation and activation. from the genome-wide screen, the impact of down-regulating tlr3 (z score 1.16) and irf3 (0.97) was a moderately antiviral phenotype. the best-characterised target of henipavirus immune evasion is the stat proteins, critical signalling molecules in the context of type i ifn cytokine production conferring the antiviral state [reviewed in (platanias 2005) ]. the binding of type i ifn (ifn-a and ifn-b) and type ii ifn (ifn-c) to their respective receptor complexes leads to the phosphorylation and association of stat1 and stat2 heterodimers (for type i ifn signalling), or stat1 homodimers (type ii ifn). this prompts the formation of stat1-stat2-irf9 (ifn-regulatory factor 9) complexes that translocate to the nucleus and bind ifn-stimulated response elements (isres) in dna to initiate transcription of ifn-stimulated genes (isgs). whilst there are hundreds, potentially thousands of isgs that collectively confer antiviral immunity, very few isgs have been functionally characterised in the context of henipavirus infection. one isg, cholesterol 25 hydroxylase (ch25h), inhibits infection by niv and a range of other rna viruses by blocking membrane fusion between host and viral membranes (liu et al. 2013a) . consistent with this observation, ch25h blocked hev infection in the genome-wide rnai screen (robust z score 1.05). henipaviruses, like other paramyxoviruses, generate multiple alternative mrnas from the p gene locus-p, v and w (thomas et al. 1988) . a fourth protein, c, is generated by alternate translation initiation site selection from all these mrnas and does not share sequence homology to the other proteins. the p, v, and w proteins share 407 amino acids in their n termini and all three proteins bind to stat1 and stat2 via this n-terminal region (ciancanelli et al. 2009; rodriguez et al. 2004) . virus-host interactions in this context prevent stat1/2 phosphorylation and activation, and lead to their sequestration in high molecular weight complexes (rodriguez et al. 2003; rodriguez et al. 2002; shaw et al. 2004) . interestingly, the sirna-mediated inhibition of stat1 increased hev infection in the genome-wide screen, but inhibition of stat2 did not (robust z scores of 1.01 and −0.67). this preliminary observation suggests that stat1 activity may have a greater impact on henipavirus infection than stat2, and may implicate type ii in antiviral immunity against henipaviruses. although the role of henipavirus p gene products in immune evasion is well-established, a recent study demonstrates the surprising ability of niv-m to antagonise the antiviral type i ifn response (bharaj et al. 2016) . the study by bharaj and colleagues shows that niv-m binds to and targets the e3-ubiquitin ligase trim6 for degradation. trim6 catalyses the synthesis of unanchored polyubiquitin chains that are used as a substrate for the activation of ikb kinase-e (ikke), which phosphorylates irf3 and activates irf3-dependent transcription of type i ifn, and tnf-a. trim6 targeting by niv-m occurs in the cytoplasm via an unknown mechanism not involving the proteasome or the lysosome, and requires nuclear/cytoplasmic trafficking of niv-m. similar to viral budding, this function of m is dependant on nuclear traffic, as k258 mutants of niv-m do not target trim6 for degradation. the study expands our understanding of immune antagonism and highlights the potential purpose of henipavirus m protein nuclear trafficking. . although far less frequent, mirna binding may also cause an increase in target mrna translation and thus up-regulation of protein expression (vasudevan et al. 2007 ). in terms of target complementarity, mirnas do not require perfect base pairing (tenoever 2013). as a result, one mirna has the potential to regulate a surprisingly broad network of genes (skalsky and cullen 2010; zhang et al. 2013) , with certain mirnas found to have binding sites located on several hundred different mrna sequences (guo and steitz 2014) . despite the potential for widespread impacts, studies have described the effects of mirna gene regulation on protein expression levels as generally 'subtle' (tenoever 2013) or 'typically relatively mild' (selbach et al. 2008 ). this is due to the fact that, in general, mirnas do not entirely silence but rather moderately repress translation and, hence, effectively fine tune rather than knock out gene expression (baek et al. 2008 ). the role of mirnas in the infection cycle of rna viruses is becoming increasingly apparent. certain mirnas may promote virus replication by directly interacting with the viral genome or, alternatively, by down-regulating the expression of host genes that suppress virus infection (skalsky and cullen 2010; roberts et al. 2011) . inhibiting specific 'proviral' mirnas, therefore, may have a direct negative impact on the viral life cycle (janssen et al. 2013) or alternatively render the intracellular environment unfavourable for virus replication (stewart et al. 2013) . in an example of the latter, mir-146a has been found to promote hev infection by repressing ring finger protein 11, a negative regulator of nf-ĸb activity (stewart et al. 2013) . furthermore, inhibiting mir-146a has been found to significantly reduce hev replication in vitro (stewart et al. 2013 ). on the other hand, mir-122 is an example of a mirna that promotes hepatitis c virus (hcv) replication by directly interacting with the viral genome-this activity is the basis of the first mirna inhibitor drug to enter phase ii clinical trials (janssen et al. 2013; wilson and sagan 2014) . the functional genomics platform established as part of the screen of protein-coding genes associated with hev infection was recently adapted to study the impact of host-encoded mirnas on hev growth (foo et al. 2016) . the screen involved the use of synthetic mirna mimics and inhibitors targeting 834 micrornas. mimic and inhibitor screens identified 35 and 61 micrornas, respectively, that promoted hev infection, and 19 and 83 micrornas, respectively, that inhibited virus infection. a major finding from this study was that all four members of the mir-181 family (-a to -d) promote infection by hev and niv. infection promotion was primarily mediated via the ability of mir-181 to significantly enhance henipavirus-induced membrane fusion. cell signalling receptors of ephrins, namely epha5 and epha7, were identified as novel negative regulators of henipavirus fusion. the expression of these receptors, as well as ephb4, was suppressed by mir-181 overexpression, suggesting that simultaneous inhibition of several ephs by the mirna contributes to enhanced infection and fusion. to our knowledge, this study represented the first evidence of a host-encoded mirna promoting virus cell entry. previous studies have reported that members of the mir-181 family are involved in different aspects of immune regulation (hutchison et al. 2013; galicia et al. 2014; zietara et al. 2013) . specifically, mir-181 has been found to play a central role in the regulation of b cell differentiation and t cell selection, maturation and sensitivity (sun et al. 2014) . for instance, induction of mir-181a has been found to occur at the cd4(+)-cd8(+) double-positive stage of t cell development, inhibiting the expression of cd69, bcl-2 and t cell receptor-all involved in positive selection and t cell maturation (neilson et al. 2007 ). in addition, mir-181c has been found to suppress cd4+ t cell activation by targeting interleukin 2 (il-2) (sun et al. 2014; xue et al. 2011 ). in addition, mir-181a expression levels have been shown to correlate with pro-inflammatory signals (e.g. il-1b, il-6 and tnf-a) in blood and various tissues of humans with chronic inflammation, as well as in the blood of lps-treated mice (xie et al. 2013) . consistent with the notion that mir-181 expression is immune-responsive, levels of mir-181 were up-regulated in the biofluids of ferrets and horses infected with hev, suggesting that the host innate immune response may promote henipavirus spread and exacerbate disease severity. the study of both mirnas and protein-coding genes associated with hev infection allows an assessment whether genes required for virus infection (i.e. proviral genes) are regulated by mirnas that inhibit virus infection (i.e. antiviral micrornas). multiple members of the let-7 mirna family inhibited hev infection. there are 10 mature let-7 sequences in humans, with multiple roles described, including negative regulation of tumorigenesis (shi et al. 2008; esquela-kerscher and slack 2006) . in a transcriptome-wide study in hela cells, genes significantly down-regulated by let-7b at either the mrna level, protein level or both, included fourteen validated genes required for wild-type hev infection, including akt1 (selbach et al. 2008 ). furthermore, six proviral genes contain putative let-7b binding sites in their 3′ utr (akt1, c6orf106, eif2s3, hmga1, ifitm3 and serpinh1), as identified by diana-mirextra (alexiou et al. 2010) . collectively, these data suggest that let-7 mirnas inhibit hev by suppressing host proteins required for virus infection. cross-referencing results from the protein-coding screen study showed that the majority of verified target genes for mir-181 and mir-17-92 mirnas (proviral in the mirna screen) were predominately antiviral, demonstrating a level of congruency between mirna and protein-coding gene screens. in contrast to let-7, all six members of the mirna precursor mir-17 family (mir-17, -20a, -20b, -106a, -106b and -93), part of the oncogenic mir-17-92 polycistron, strongly promoted hev infection. interestingly, other mirnas of the mir-17-92 cluster with distinct "seed" families (based on sequence identity at positions 2-7)-mir-18, mir-19 and mir-92) did not impact virus replication to a similar extent. the mir-17-92 cluster is a known oncogene locus-it is amplified in b cell lymphomas (ota et al. 2004 ) and accelerates tumour development in a mouse b cell lymphoma model (he et al. 2005) . members of the mirna precursor mir-17 family are expressed in almost all human tissues (liang et al. 2007 ). in addition, mir-106a and -106b are expressed in peripheral blood mononuclear cells (pbmcs), platelets and exosomes derived from peripheral blood (hunter et al. 2008) . henipaviruses are dangerous pathogens and control of disease caused by these viruses will critically rely on the development of new antiviral therapeutics and vaccination strategies. currently, there is requirement for renewed research into the host immune responses to henipavirus infection and how competent immune responses may fight disease. a major challenge is to ascertain the molecular mechanisms of virus replication and immunity associated with protection to infection. the improved knowledge of functional genomics approaches and immune response to viral infection means that we now have the tools to further progress our understanding and knowledge. nevertheless, this must be implemented to develop advanced infection control approaches. the diana-mirextra web server: from gene expression data to microrna function recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the ifn-beta promoter small interference rna profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus novel viral translation strategies 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identification of 53 compounds that block ebola virus-like particle entry via a repurposing screen of approved drugs a ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mrnas characterization of microrna expression profiles in normal human tissues the non-pathogenic henipavirus cedar paramyxovirus phosphoprotein has a compromised ability to target stat1 and stat2 matrix proteins as centralized organizers of negative-sense rna virions mechanism of t cell regulation by micrornas interferon-inducible cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-hydroxycholesterol recurrent zoonotic transmission of nipah virus into humans transmission of human infection with nipah virus recognition of single-stranded rna viruses by toll-like receptor 7 acts as a safeguard of translational control by regulating fibrillarin and rrna methylation in cancer cedar virus: a novel henipavirus isolated from 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for hit selection in rna interference high-throughput screening experiments progress in microrna delivery genome-scale rnai screen for host factors required for hiv replication critical role for mir-181a/b-1 in agonist selection of invariant natural killer t cells key: cord-009636-5kddituy authors: shirbaghaee, zeinab; bolhassani, azam title: different applications of virus‐like particles in biology and medicine: vaccination and delivery systems date: 2015-12-22 journal: biopolymers doi: 10.1002/bip.22759 sha: doc_id: 9636 cord_uid: 5kddituy virus‐like particles (vlps) mimic the whole construct of virus particles devoid of viral genome as used in subunit vaccine design. vlps can elicit efficient protective immunity as direct immunogens compared to soluble antigens co‐administered with adjuvants in several booster injections. up to now, several prokaryotic and eukaryotic systems such as insect, yeast, plant, and e. coli were used to express recombinant proteins, especially for vlp production. recent studies are also generating vlps in plants using different transient expression vectors for edible vaccines. vlps and viral particles have been applied for different functions such as gene therapy, vaccination, nanotechnology, and diagnostics. herein, we describe vlp production in different systems as well as its applications in biology and medicine. © 2015 wiley periodicals, inc. biopolymers 105: 113–132, 2016. v irus-like particles (vlps) known as viral "empty shells" maintain the same structural properties of virions, without genome. these constructs are considered very efficient as vaccine platforms and therapeutic delivery systems. 1 many antigens can readily be displayed on the surface of vlps. these antigens can be genetically or chemically fused to the vlp. 2 regarding to the reports, the immune stimulation by vlps contains: (a) stimulation of innate immunity through tlrs and pattern recognition receptors (prrs) due to the expression of multivalent structures; (b) induction of strong humoral response and also igm in t-cell independent way; and (c) enhancement of the uptake, processing and presentation by apcs through mhc i and mhc ii cross-presentation pathway due to the particulate nature of vlps. 3 vlps can be subcutaneously or intramuscularly injected. their small diameter facilitates entry into lymphatic vessels and direct drainage into local lymph nodes. once in the lymph node, vlps are taken up by lymph node resident dendritic cells (dcs). this uptake is enhanced by the size and form of vlps. vlps stimulate cd4 t cells via the mhc ii pathway, as well as highly efficient cross-presentation on the mhc class i pathway. 3 generally, viral-like particles, are considered as vaccine candidates because their natural properties such as multimeric antigens and their specific structures are suitable for the stimulation of efficient humoral and cellular immunity. currently, the development of recombinant subunit vaccines (suvs) has been significantly increased using heterologous expression systems. antigens derived from many bacterial, viral, fungal and parasitic pathogens were used for safe and effective vaccination. five vlp-based vaccines have been already approved including three for hbv and two for hpv, while in the veterinary field; a vlp-based vaccine against porcine circovirus type 2 (pcv2) has been approved. some vlp-based vaccines targeting human and animal diseases are recently in late stages of clinical trials. vlps have a positive value as academic, industrial, and commercial systems especially in gene therapy and design of nanomaterials. however, the study of the vlp-based applications (vaccination, gene and drug delivery, and imaging) must be followed to show the reliability, and cost efficiency of this technology. furthermore, the expression systems would be improved to achieve the best strategy for vlp production from different viral genes. this review will focus on vlp characteristics and its applications especially as vaccines or delivery systems for dna, sirna and drugs. it should be noted that in the vaccination field whenever a viral-like particle carries genetic material is called "vectored vaccines 4 " and in gene therapy, they are called viral vectors. however, for simplicity in this review, we called all particles entitled as viral-like particles (vlps). viral-like particles (vlps) have been generated for over thirty various infectious viruses in animals and humans. 5 vlps are composed of one or more structural (/capsid) proteins possessing natural properties for self-assembly, and are morphologically similar to authentic viruses. 5, 6 comparing to live viruses, vlps are non-replicating and non-infective due to the lack of infectious genetic material. 5 virus-like particles have the potential to be used as safe vaccine candidates without the need for any adjuvant. 7, 8 different viruses present different structures for generation of viral-like particles such as: a. simple viral capsids with one or two major proteins (e.g., parvoviruses, papillomaviruses, circovirses, calciviruses, hepatitis e virus (hev) and polyomaviruses). b. complex viral capsids with various protein layers, encoded by many distinct mrnas, or generated from a single polyprotein (e.g., picornaviruses). c. viral capsids with lipid envelopes including a lipid bilayer obtained from the host cell, as well as viral glycoprotein spikes (e.g., influenza, hiv and hepatitis c). 5, 9 figure 1 shows the general model of vlp along with its applications. the selection of expression vector is one of the major factors in vlp generation. the reports showed the successful production of 174 vlps indicating that bacterial systems, yeast and insect systems are used in 28%, 20%, and 28% of the cases. in addition, mammalian cells (15%) and plants (9%) were usually applied to produce vlps with special properties. 8 bacterial systems are often included the commercial e.coli strains and expression vectors, to produce non-enveloped vlps in high levels compared to other systems (table i) . 5 in addition, bacterial cells have been applied for generation of vlps which need several types of structural proteins, such as the avibirnavirus ibdv vp2, vp4, and vp3-polyproteins. 54 the reports indicated that the expression of the hepatitis b virus (hbv) capsid protein in e. coli leads to the formation of structures similar to the hbv core (hbc) particle. 55 bacterial figure 1 general model of vlp along with its applications: the picture shows the recombinant hpv16 l1 pentamers assembled in vitro into capsid-like structures. self-assembly of recombinant viral coat proteins into empty capsids is a promising strategy for production of virus-like particles (vlps) in vaccine design. the resulting vlps can induce a protective immune response by mimicking the authentic epitopes of virions. systems are not always a desired plan for vlp production due to several factors, such as (a) lack of ability to generate recombinant proteins with mammalian-like post-translational modifications (ptm), (b) failure to produce the correct disulfide bonds, (c) drawbacks of protein solubility, and d) the existence of lipopolysaccharides (lps)/or endotoxins in production of recombinant proteins (rp). 5 viral coat proteins (cps) can be efficiently produced as insoluble inclusion bodies, purified under denaturing conditions, refolded, and selfassembled, as indicated in the parvovirus b19 and the ccmv and cmv plant viruses. 56 a simple change in the cultivation conditions such as low-temperature can solve the problem of inclusion bodies and induce the formation of soluble vlps, as performed for two viral systems, the densovirus ihhnv, 57 and the potyvirus pvy. 58 some factors including the resistance markers of the expression plasmids and the composition of the cultivation medium can also change the vlp assembly (e.g., bacteriophage qb). 59 another strategy applied to increase expression levels and solubility involves the use of different fusion protein systems, e.g., glutathione-s-transferase (gst) fusion proteins such as the papillomavirus l1, the polyomavirus mupyv, and the picornavirus fmdv. [60] [61] [62] [63] other prokaryotic hosts have been recently used to generate vlp, e.g., lactobacillus. 8 the intracellular assembly of hpv16 l1 vlp was reported in lactobacillus casei, a lactose-inducible expression strain. 5 furthermore, the production of l1 vlps using lactobacillus developed new live mucosal prophylactic vaccines (table i) . 29, 30 a pseudomonas fluorescens (p. fluorescens) expression system is an efficient choice against e. coli, because of simple manipulation, high yields of active and soluble proteins, and largescale cultivation. some differences between p. fluorescens and e. coli including the various sizes of genome, and diverse metabolic approaches can influence the generation of recombinant proteins. 64 the capsid protein of a plant bromovirus, the cowpea chlorotic mottle virus (ccmv), has been recently expressed as a soluble form in p. fluorescens, and assembled into vlps in vivo. this construct was structurally similar to the natural viral particles provided from plants. 64 eukaryotic expression systems are a striking alternative to bacteria, especially for solving the problem of bacterial endotoxins in vaccine development. some structural genes of mammalian viruses expressed in yeast are able to form the vlp. this expression host has been efficiently applied to generate the first licensed hbv vaccine. 65 hbsag is one of the antigens commonly utilized for production of vlp-based hbv vaccine. hbsag has been expressed in pichia pastoris (p. pastoris), sac-charomyces cerevisiae (s. cerevisiae) and hansenula polymorpha (h. polymorpha) (table i) . 5, 16 it is critical to consider that the viral-like particles are not always formed during the cultivation procedure of the yeast cells. these studies showed that the selfassembly of the vlps in pichia system should be completed during the protein purification. 16, [66] [67] [68] the expression and selfassembly of recombinant bacteriophage q coat protein (q-cp) was indicated in saccharomyces cerevisiae and pichia pastoris. the yeast-derived q-vlps were greatly immunogenic in mouse similar to that in e.coli-derived q-vlps. 69 ms2 vlps produced in saccharomyces cerevisiae could package functional heterologous mrnas. for example, the linkage of the ms2 packaging sequence to the human growth hormone mrna allowed the packaging of the mrna in ms2 vlps. indeed, the high stability of ms2 vlps suggests them as an efficient delivery system for rna-based vaccines. 70 the p. pastoris system was also utilized as a potent alternative for expression of ccmv coat protein vlps due to easy manipulation and high expression levels. 71 in addition, this system has been utilized to express efficiently the premembrane and envelope glycoproteins of dengue virus type 2 (denv-2), 72 hbsag, 73, 74 hccag 75 resulting in the generation of vlps. 72 the major advantage of yeast systems is the ptm including phosphorylation or glycosylation, as indicated in hbv vlps. 8 the studies indicated that hbc phosphorylation plays a major role in viral replication and capsid formation. such yeast-derived hbc vlps are valuable for vaccination and diagnostics. 76 furthermore, the potent multigene expression systems have been constructed in yeasts. for example, the expression of three rotavirus structural genes from a single plasmid vector led to the generation of triple layered vlps in saccharomyces cerevisiae. 8, 77 however, the multimerization of protein into vlps is not supported for the enveloped viruses (e.g., gag vlps of hiv-2), suggesting that yeast does not have the essential factors of host. 8 thus, the generation of enveloped hiv-1 pr55gag vlps has been performed using s. cerevisiae spheroplasts, morphologically similar to immature viral particles. 5, 78 in general, the construction of yeast expression systems, especially hansenula and pichia strains, are more difficult than bacterial vectors. in addition, the yield of vlp production is less than that in e.coli. 8 other limitation of yeast system is its dissimilarity with mammalian cells in the ptm of proteins, especially glycosylation. 79,80 therefore, this system is more suitable for the generation of non-enveloped viral-like particles. another attractive system utilized broadly for production of vlp is the baculovirus-insect cell expression system, due to 118 shirbaghaee and bolhassani some advantages, such as the rapid growth ratios, the culture preparation in large-scale, and the ptm of the target proteins similar to mammalian cells. [81] [82] [83] the results showed that both yeast and insect cells were previously used for the vp1 expression of several polyomaviruses, and its assembly into viral-like particle. 84 in addition, insect cells were used to provide vlpbased vaccines, e.g., the approved hpv vaccine, cervarix. indeed, insect cells are able to generate both vlp types (i.e., enveloped and non-enveloped). there are enveloped vlps in clinical trials. 9 the main limitation of insect cell system is protein contamination with the enveloped baculovirus particles, suggesting the development of efficient plans for purification of vlps. 85 recently, co-expression of four genes of human influenza h3n2 virus (i.e., ha, na, m1, and m2) in insect cells led to generate influenza vlps which protected mice against h3n2 virus challenge. 86 these data suggested that viral-like particles are a hopeful vaccine candidate for h9n2 influenza and probably other subtypes of virulent avian influenza viruses. 87 the non-infectious viral-like particles of the alphavirus sav was also generated using the recombinant baculoviruses expressing sav capsid protein and two major immunodominant viral glycoproteins (e1 and e2) in insect cells. 88 moreover, baculovirus expression system was utilized to generate vlps from cowpea mosaic virus (cpmv), tomato bushy stunt virus, and entorovirus271 (ev71). 8, 89, 90 recently, non-replicative baculovirus have been developed to cope with the problem of baculovirus contamination. 91 stable systems using insect cells have been also tested. 92 moreover, silkworm expression systems were efficiently applied to generate vlps and the surface of vlps could be changed by some strategies, irrespective if their constructs are enveloped or not. silkworms show a high capability for production of recombinant proteins, in comparison with insect cells, and also easy and inexpensive protein preparation similar to e.coli expression system. 81 for over two decades, different mammalian cell lines have been developed as a source of commercial therapeutic proteins for clinical applications, 93 because of their ability for proper protein folding, assembly and ptm (e.g., the correct glycosylation pattern). 8, 93 however, high costs of production and potential safety concerns remained a challenge for these systems. the mammalian cells were progressively utilized to produce vlpbased vaccines 5,94 , e.g., for influenza viruses. for instance, the generation of a stable mammalian cell line (e.g., vero cells) expressing four influenza structural proteins (ha, na, m1, and matrix 2 (m2)) led to form hybrid vlps containing matrix proteins, and surface glycoproteins of h3n2 and h5n1 influenza types, respectively. 8, 95 another examples are the produc97 and hiv-1 vlp in cos-7/vero cells, 98 and hbv vlp in cho cells. [99] [100] [101] plant systems plants were successfully used to express specific gene products. the feasibility of recombinant plants for generation of vaccine antigens were shown in tobacco plants, potato tubers, and others. 102 this approach develops vaccine strategies which can stimulate mucosal as well as systemic immune responses. in addition, it can be delivered orally as part of a normal biologic function in human. 102 the antigen expressed in plant systems shows extensive disulphide crosslinking and oligomerization for formation of virus-like particles. for example, the hepatitis b major surface antigen has been expressed in several plant systems. 103 plants are able to express and assemble both types of vlps (i.e., enveloped and non-enveloped) as multimeric and chimeric proteins. the high expression of vlps in plant is easy and rapid (e.g., 1-2 weeks) using a tobacco mosaic virus (tmv) rna replicon system and/or a bean yellow dwarf virus (beydv) dna replicon system. 104 another advantage of plants is the use of plant virus particles as a delivery system to present foreign epitopes. furthermore, the problem of plant-specific glycans has been partially solved using the development of transgenic plants with "humanized" glycosylation pathways. 104 plantderived vlps can be used for oral delivery of vaccines. virallike particles are more resistant to digestive enzymes than soluble proteins in body, because of their highly ordered and packed structures. for example, the gastrointestinal virusesderived vlps including noroviruses and rotaviruses were utilized orally as potent candidates for mucosal immunization. 105 plant-derived vlps showed the same structures with vlps generated in other expression systems accompanied by a comparable or higher immunogenicity. some plant-derived vlps could induce protective humoral and cellular immunity and also safety in clinics. 105 the studies showed that the level of protein expressed in the recombinant plants is variable and often low. therefore, further increase in expression will be necessary for practical and efficient products. 102 recent progress in the glyco-engineering of plants allows human-like glycol-modification and optimization of desired glycan structures for increasing safety and functionality of recombinant pharmaceutical glycoproteins. 1 some plantbased systems can stabilize antigen and thus reduce storage and distribution costs. 103 different applications of virus-like particles 119 toxoplasma gondii (t. gondii) is an obligate intracellular parasite infecting the nucleated cells of warm-blood vertebrates. this parasite is able to stimulate strong humoral, cellular and mucosal immunity, and thus it can be used as an efficient delivery system for heterologous antigens. t. gondii was applied as a vector for live vaccination against infectious pathogens. [104] [105] [106] [107] [108] [109] recently, a non-pathogenic kinetoplastida, leishmania tarentolae, was utilized to express heterologous proteins. the studies showed that expression of mammalian glycoproteins in this parasite leads to their modification with mammalian-like oligosaccharides. [110] [111] [112] [113] recently, our group has focused on its use as a live vector or killed vaccine, [114] [115] [116] and also generation of viral coat proteins and their assembly as vlp in this system. 117 virus-like particles show an efficient strategy to deliver antigens to the immune system, inducing both arms of the adaptive immunity. 118 indeed, vlps present antigenic epitopes in the proper conformation, leading to induce humoral responses. 5 for example, preclinical trials with influenza vlps indicated their capacity to induce both humoral and cellular immune responses at low antigen doses. several authors have reported antibody response to parenterally or orally administered plant-derived antigens. 119, 120 as exogenous antigens, vlps are taken up by professional antigen presenting cells (apcs), especially dcs, followed by antigen processing and presentation via mhc class ii molecules, dc activation and maturation through up-regulation of co-stimulatory molecules and cytokine production, and stimulation of cd41 t helper cells. all these events can efficiently induce both humoral and cellular immunity. 5 in addition, the exogenous vlps can enter the cytosol of dcs, be processed and presented by mhc class i molecules to cytotoxic t lymphocytes (ctls) using cross-presentation. 5, 121, 122 furthermore, the b-cell activation using vlps is robust enough to induce t cell-independent igm antibodies. 7, 8 dcs loaded with yeast-derived hiv vlps can alter gag-specific memory cd81 t cells into effector cells through cross-presentation in chronically hiv-infected individuals, although some gag-specific t cells in these patients did not show any response. 123 the reports showed that the expression system used for generation of vlp might significantly affect direction, type and outcome of immune responses. 121 for example, potent and specific immunomodulatory effects were assigned to yeast-derived hiv vlps in comparison with other expression systems. 123 on the other hand, plant-or insectderived vlps, consisting of the l1 capsid protein of hpv, were both immuno genic to an equal degree. half of mice fed trans-genic potatoes expressing hpv vlps developed l1-specific antibodies. 124 the studies indicated that the vlps are taken up by clathrin-dependent macropinocytosis and phagocytosis before being degraded in acidic lysosomal compartments. vlp-derived peptides are loaded onto mhc i that have been recycled from the cell surface. 125 a study showed that uptake and activation of dc by vlp involves proteoglycan receptors, tlr4 and nf-kb, and can be inhibited by heparin. 126 several data suggest different routes of vlp uptake by dc and langerhans cells (lc). 127 for example, lcs and dcs internalize similar amounts of hpv-vlps in vaccine design, albeit through different uptake mechanisms. 128, 129 vlp uptake by dcs results in activation and cross-presentation of mhc i-restricted peptides with co-stimulation to t-cells. on the other hand, vlp uptake by lc leads to cross-presentation in the absence of costimulation. efficient vlp cross-presentation by lcs with costimulation can be achieved by addition of cd40 ligand. 128 the lack of a protective immune response after viral contact with lcs may explain why some women fail to induce an immune response against the virus. lcs endocytose hpv vlps via a non-clathrin, non-caveolae, actin-independent pathway, whereas dcs take up hpv vlps both by a clathrin-mediated mechanism and via macropinocytosis in an actin-dependent manner. this difference in endocytosis resulted in processing and presenting hpv vlp peptides by lcs similar to that by dcs on their surface, but in the absence of co-stimulation. with the addition of cd40l, lcs incubated with hpv vlps generated the efficient amounts of the pro-inflammatory cytokine (il-12) and could stimulate a hpv-specific immune response after incubation with t cells. 128 despite these differences, vlps taken up by dc and lc were able to prime naive cd81 t cells and induce cytolytic effector t cells in vitro. 127 furthermore, hiv-1 pr55 gag virus-like particles could stimulate strong humoral and cellular immune responses. vlp expressed by recombinant baculoviruses activated human pbmc to release pro-inflammatory (il-6, tnf-a), antiinflammatory (il-10) and th1-polarizing (ifn-c) cytokines as well as gm-csf and mip-1a in a dose-and time-dependent manner. furthermore, vlp-induced monocyte activation was shown by up-regulation of molecules involved in antigen presentation (mhc ii, cd80, and cd86) and cell adhesion (cd54). exposure of vlp to serum inactivated its capacity to stimulate cytokine production. 130 the linking of vlps to adjuvant molecules was also shown to improve the immunogenicity of the nano-bioparticles. 131 adjuvanted vlps [e.g., cpg odn1826 or poly (i: c) adjuvants] elicited a higher titer of total specific igg compared to vlps alone. furthermore, while vlps alone induced a balanced th2 pattern, vlps formulated with adjuvant elicited a th1-biased igg subclasses (igg2a and igg3), with poly (i: c) more potent than cpg odn1826 in 120 shirbaghaee and bolhassani animal model. 118 in addition, mice immunization with chimeric simian immunodeficiency virus (siv) vlps containing gm-csf significantly induced siv env-specific antibodies as well as neutralizing activity at higher levels than those elicited by standard siv vlps, siv vlps containing cd40l, or standard vlps mixed with soluble gm-csf. on the other hand, the incorporation of immunostimulatory molecules showed significantly increased cd41 and cd81 t-cell responses to siv env, compared to standard siv vlps. 132 formulation of vlps with rough lps (r-lps) adjuvant as well as dna primed-vlp boosted regimen were led to increase specific immune responses as compared to vlps alone, but among them the vlp/r-lps highly enhanced immune response. 133 recent studies demonstrated the potential of the hbc vlps as an oral immunogen. intraperitoneal immunization with the hbc vlp induced a strong, mixed th1/th2 response. in contrast, oral administration of the hbc vlp generated a high humoral response with mainly igg2a antibodies, directing toward a th1 response which is essential in the control of intracellular pathogens. 134 in addition, the intranasal monovalent adjuvanted norwalk vlp vaccine was well tolerated and highly immunogenic. 135 the studies showed that chimeric hpv-vlps are able to elicit potent ctl responses in mice against hpv16transformed tumors; however, the mechanism of t cell priming has remained obscure. hpv vlp could bind to human mhc class ii-positive apcs through interaction with fccriii, and immature dcs were activated after incubation with hpv vlp. 136 it was shown that binding and uptake of vlp by dc from fccrii, fccriii, and fccrii/iii deficient mice are reduced by up to 50% compared with wild-type mice. in addition, maturation of murine dc from fccrii/iii-deficient mice by vlp is also reduced, indicating that dc maturation, and thus ag presentation, is diminished in the absence of expression of fccr. 136 poor immunogenicity of mucosally administered proteins has been a major barrier for development of efficient oral vaccines. one way to overcome this obstacle is the use of appropriate adjuvants. also, delivery of antigen to mucosal surfaces as vlp provides an efficient way of inducing mucosal immunity. after oral or intranasal immunization with norwalk vlp, or rotavirus vlp without adjuvant, intestinal iga was detected in immunized mice, which were protected from virus challenge. 137 in addition, the plasma cell precursors that migrate to the genital tract are derived primarily from mucosal lymphoid tissues and often secrete iga. 138 the studies indicated that immune responses generated by mucosal administration of vlp were generally weaker than systemic administration. vlp specific iga was higher in intestine washes following intrarectally (i.r.) than intravaginally (i.va.) immunization, and higher in vaginal washes following intramuscularly (i.m.) than i.r. or i.va immunization. some studies suggested that the immunogenicity of virus particles at mucosal surfaces is probably a property of particulate antigens assembled as multimers of subunits. indeed, vlp might be actively taken up by mucosal apc through the integrin receptors. 137 lipoparticles are stable, highly purified, homogeneous, and specialized vlps containing high concentrations of an integral membrane protein. integral membrane proteins are involved in different biological functions and are targeted by 50% of existing therapeutic drugs. however, because of their hydrophobic domains, membrane proteins are difficult to manipulate outside of living cells. lipoparticles can incorporate a wide variety of the membrane proteins, including g proteincoupled receptors, ion channels, and viral envelopes. lipoparticles provide a platform for different applications such as antibody screening, production of immunogens, and ligand binding assays. [139] [140] [141] during the assembly of enveloped viruses, lipid ordered domains of the host cell plasma membrane, known as lipid rafts, frequently function as a natural target for viral proteins. the role of lipid rafts in the organization of complex combinations of immune receptors during antigen presentation and t cell signaling is extensively recognized. 142 on the other hand, in order to improve the immunogenicity of hiv-1 envelope glycoproteins, the fusion of gp120 was performed to a carrier protein, hepatitis b surface antigen (hbsag) which is capable of spontaneous assembly into viruslike particles. the hbsag-gp120 hybrid proteins assembled efficiently into 20-30 nm particles. the particles resembled native hbsag particles in size and density, consistent with a lipid composition of about 25% and a gp120 content of about 100 per particle. particulate gp120 folded in its native conformation and was biologically active, as shown by high affinity binding of cd4. because the particles are lipoprotein micelles, an array of gp120 on their surface closely mimics gp120 on the surface of hiv-1 virions. these gp120-rich particles can enhance the quality, and also quantity of antibodies elicited by a gp120 vaccine. 143 virus-like particles show an expanding spectrum of applications such as gene therapy, nanotechnology, vaccination, and diagnostics. 55, 77 recently, the studies showed a pattern of direct conjugation of some ligands, including nucleic acids and proteins attached to vlp surface. 144, 145 in addition, because of the superior accessibility of cysteine and lysine residues on vlps, bio-conjugation has been performed by commercial homo-or hetero-bifunctional linkers. [146] [147] [148] [149] for example, three foreign proteins were chemically conjugated to the vlp surface of cpmv by proper bifunctional cross-linkers. 147 on the other hand, the researchers could produce an alphavirus vlp surrounding a functional gold nanoparticle. 150 vlps have been also used to stimulate immune responses and generate antitumor responses, e.g., alphavirus-based virus-like replicon particles (vrp) expressing various melanoma antigens. [151] [152] [153] it is interesting that the first viral-associated cancer vaccines were founded on hbv vlp and hpv vlp to prevent hbvassociated hepatocellular carcinoma (hcc) and hpvassociated cervical carcinoma, respectively. 153, 154 it should be noted that these vlp formulations are viral vaccines that prevent a viral infection that may progress to carcinoma after a long time. we indicate some applications of vlps against viral diseases as following: in several studies, specific vaccine antigens were generated by various expression systems to induce protective immune responses and apply in licensed recombinant viral vaccines. 100, 155 some examples of preventive vlp-based vaccines are recently commercialized worldwide including glaxos-mithkline's engerixv r (hbv) and cervarixv r (hpv), and merck and co., inc.'s recombivax hbv r (hbv) and gardasilv r (hpv). other vlp-based vaccines undergo preclinical evaluation or clinical trials, including parvovirus-, influenza-, norwalk-derived vlps and also different chimeric vlps. 156 for generation of immunogenic vlps, eukary otic expression hosts including yeast (s. cerevisiae, p. pastoris and h. polymorpha) and mammalian cells (chinese hamster ovary cell line [cho]) were used. the studies indicated that the recombinant hbsag generated in cho and h. polymorpha have significant differences in size, molecular weight (mw), and monomer number. furthermore, the cho-derived viral-like particles include a combination of glycosylated and non-glycosylated hbsag proteins, similar to those in patients' sera, while yeastderived antigens were reported to be non-glycosylated. chobased vaccines were provided by pasteur-m erieux aventis in france (genhevac bv r ) and scigen in israel (sci-b-vac tm ). both vaccines contained not only the hbsag s pro tein but also the m protein (genhevac b) or the m and l protein (sci-b-vac). 156 on the other hand, gardasil approved by the fda in 2006 is a quadri valent hpv types 6/11/16/18 l1 vlp vaccine produced in s. cerevisiae. cervarix is the other licensed hpv vaccine approved by the fda in 2009. 156 cervarix is a bivalent hpv types 16/18 l1 vlp vaccine expressed via a recombinant baculovirus vector. 25, 157 different experiments have been concentrated on hpv vlp vaccination in mouse and human models including: (a) activation of immature human dcs by chimeric hpv16 vlps, (b) determination of systemic cytokine pattern elicited by hpv l1 vlps, (c) identification of gene expression signatures in hpv16 l1 vlp-induced human pbmcs, (d) generation of potent and prolonged neutralizing l1 antibodies using a single intramuscular (im) mice injection with recombinant adenoassociated virus encoding hpv16 l1 protein (raav-16l1), (e) augmentation of immunogenicity of hpv l1 dna vaccines using genetic linkage to a chemokine and secretory signal peptide sequences, (f) potent stimulation of systemic and mucosal immune responses to vlp vaccines using the encapsulation of a genetic cytokine adjuvant (e.g., il-2), (g) improvement of hpv16 vlp immunogenicity by linkage to the modified adjuvant, and m) nasal immunization of mice with hpv16 vlps. 158 hpv16 l1-e7 chimeric virus-like particles (cvlp) could induce e7-and l1-specific ctls. the therapeutic potential of the cvlp also indicated a considerable safety in high grade cervical intraepithelial neoplasia patients (cin 2/ 3). 159 several improvements in vaccine design by vlp are still in preclinical trials. some main examples are referred as following: a. co-injection of the hpv16 l1 vlp with e. coli heatlabile enterotoxin (lt) as an adjuvant significantly increased the levels of serum igg and vaginal iga after nasal or bronchial mice immunization. 160 antigens (hiv-1 p17/p24: ty vlp) was also immunogenic and well-tolerated in phase i clinical trials. 24,169 g. several groups have focused on improving bacteriophagebased vlp vaccines, e.g., rna bacteriophage qb. these chimeric vlp vaccines were targeted against noninfectious diorders including hypertension, allergy, neurodegenerative and autoimmune diseases (e.g., diabetes mellitus type ii and alzheimer), cancer (e.g., melanoma). the vaccine candidate against alzheimer (cad-106) was constructed to display chemically coupled amyloid beta (ab1-6) peptide derived from the n-terminal b cell epitope of ab protein, on the surface of qb-cp vlps. this vaccine could stimulate ab-specific igg and decrease amyloid accumulation in animal models expressing ab precursor protein, without eliciting t-cells or inflammatory reactions in brain tissue. 170, 171 in addition, the angiotensin ii vaccine was synthesized by covalently conjugation of a peptide derived from angiotensin ii to the rna bacteriophage qb vlp capsid. this modified vlp could decrease blood pressure in spontaneously hypertensive rats. 172 table i shows preclinical and clinical studies of vlps in vaccine development. generally, a major application of vlps is the stimulation of immunity against foreign protein epitopes by genetically fusing or chemically conjugating them to vlps entitled as chimeric vlp (cvlps). 173 antigens can be fused to vlps through either covalent or non-covalent bonds. the most common covalent bond is generated by the heterobifunctional chemical cross-linkers with amine and sulfhydryl-reactive arms. 104 for instance, cysteine-containing antigens can be conjugated to lysine residues of vlps surface at a high density (e.g., three peptides per coat protein molecules). the non-covalent conjugation strategy contains the use of streptavidin as linkers to attach biotinylated antigens and vlps through their efficient and specific interactions. 104 sv40 vlps can also encapsulate various materials such as dna (5 kb) and proteins as antigens. insertion of a special exogenous peptide into the surface loops of vp1 produced sv40 vlps with the ability of cell targeting. moreover, sv40 vlps stimulated innate immunity as a natural adjuvant. indeed, sv40 vlps may be a promising vaccine candidate to deliver heterologous antigens followed by the induction of ctls without synthetic adjuvants. 174 several chimeric vlp vaccines have entered clinical trials, such as the anti-influenza a m2-hbcag vlp vaccine (hbcag vlps displaying m2 epitope of influenza a), the anti-hiv p17/p24: ty vlp, two anti-malaria vaccines (hbcag vlps displaying malaria epitopes), the nicotine-qb vlp and the anti-ang ii qb vlp. 175 genetic linkage contains a stable bond between vlp and antigen. the studies showed that only peptides shorter than 30 amino acids (small peptides) can be presented without interfering with the correct assembly of vlps. other limitations contain the improper folding of displayed antigens and the formation of cvlps with heterogeneous size. to prevent these issues (e.g., assembly problems), structural studies have identified domains for different vlps such as hbcag, hbsag and hiv gag that were not necessary for vlp assembly as well as allowed insertion of foreign antigens. 104 the simplest way for generation of single component cvlps, is the insertion of peptides at the n-or c-terminal regions of chimeric vlps. multiple fusion positions should be identified to produce multicomponent cvlps inducing broad immune responses. 104 the direction and intensity of the immune responses are significantly influenced by the vlp type, the foreign antigen density, and its accessibility on or within the vlp. furthermore, preexisting immunity against the epitopes of the vlp as a delivery system may importantly change the response against the heterogenous antigen. for example, hbcag was also utilized to display a neutralizing epitope of hpv16 l2 protein. the nasal delivery of hbcag-hpv16 l2 epitope cvlps expressed in tobacco induced antigen-specific antibody responses in mouse model. on the other hand, an hpv16 l1-based chimeric vlp was generated in transgenic tomato to present several t-cell epitopes from hpv16 e7 and e6 proteins. the hpv l1-e7/e6 vlps elicited a neutralizing antibody response similar to that from an equal amount of the commercial vaccine (gardasil) in preclinical study. moreover, the chimeric vlps induced ctl responses against the e7 and e6 epitopes. chimeric hpv l1 vlps were also designed using genetic fusion to display epitopes of influenza m2 protein. 104 to overcome the problems associated with genetic fusion including the antigen size, conformation and vlp assembly, different applications of virus-like particles chemical conjugation approaches were applied to construct cvlps. in this strategy, target antigens and native vlps were generated individually and coupled together by attachment of the antigen to the surface of the pre-assembled vlps. two main advantages of this strategy include: (a) various sizes and types of antigens can be exposed, and (b) the antigen-vlp binding site can be manipulated for further presentation of the conjugated antigen. for example, vlps were used to display full-length and correctly folded proteins, such as interleukin-17 (il-17). 104 generally, vlps were used for delivery of protein/peptide, dna, sirna and drugs as a brief description in following: viral-like particles were used as a peptide/protein carrier, in vitro and in vivo. there are several examples for delivery of protein/peptide using vlps as following: a. chimeric vlp vaccines have been improved based on rna bacteriophage ap205, presenting peptides of selfantigens or pathogens fused to either the n-or cterminal regions of ap205 coat protein. ap205-derived vlps were highly immunogenic in mice. furthermore, influenza m2 vlps stimulated an efficient m2-specific antibody response and full protection against lethal influenza virus challenge. 176 b. vlps containing flt3 ligand (fl-vlps), a dc growth factor, could effectively increase immunogenicity in mice. dcs exposed to vlps also produced high levels of il-6. 177 c. a plant vlp-based approach was used to develop respiratory syncytial virus (rsv) vaccine. a target peptide displaying amino acids 170-190 of the rsv g protein was delivered on the surface of recombinant alfalfa mosaic virus (almv) particles. this construct induced high pathogen-specific immune responses in immunized animals. 178,179 d. in a recent study, a peptide from an external loop of mouse ccr5 protein was inserted into a neutralizing epitope of hpv l1. the particles generated by this chimeric l1 could elicit high levels of ccr5 antibodies that specifically recognized the surface of ccr5transfected cells and blocked in vitro infection of an mtropic hiv strain in mice. 161 in addition, chimeric vlps containing the full length hpv16 e7 oncoprotein linked to l2, or the n-terminal region of e7 fused to l1, could induce antigen-specific protection of mice from lethal challenge with e7-expressing tumor cells. 180-182 e. a pre-s1 epitope of hbv was also inserted into the ef loop of hpv vlp recognized by hbv-specific antibody. 6 chimeric vlps produced in e.coli carried a virus-neutralizing hbv pre-s1 epitope in the major immunodominant region (mir) and a highly conserved n-terminal hcv core epitope (aa 1 to 60) at the c-terminal region of the truncated hbv core vlps (hbc). the presence of two different foreign epitopes within the hbc molecule did not interfere with its vlp-forming potential, with the hbv pre-s1 epitope exposed on the surface and the hcv core epitope buried within the vlps. mice vaccination showed a specific t cell activation by both foreign epitopes and a highlevel antibody response against the pre-s1 epitope, whereas an antibody response against the hbc carrier was inhibited. 183 f. the researchers have shown that the nanosized hbc-vlps bearing mycobacterial antigen cfp-10 (hbc-vlp: cfp-10 fusion protein) induced an increased immune response in balb/c mice compared to mixtures of native antigen. 184 g. chimeric papillomavirus vlps based on the bovine papillomavirus type 1 (bpv-1) l1 protein were designed by replacing the 23-carboxyl-terminal amino acids of the bpv1 major protein l1 with a synthetic "polytope" minigene, containing known ctl epitopes of human pv16 e7 protein, hiv iiib gp120 p18, nef, and reverse transcriptase (rt) proteins, and an hpv16 e7 linear b epitope. the chimeric l1 protein assembled into vlps in insect cells. polytope vlps could deliver multiple b and t epitopes as immunogens to the mhc class i and class ii pathways. this study has demonstrated that hybrid vlps can be used as an efficient antigen delivery system to transfer more than one ctl epitope through mhc class i pathways. 185 h. the chimeric hpv vlps were generated in which hpv16 l2 neutralization epitopes (l2 residues 69-81 or 108-120) are inserted within an immunodominant surface loop (between residues 133 and 134) of the l1 major capsid protein of bpv1. immunization of rabbits with assembled particles elicited high l2-specific serum antibody responses. 186 193 l. the studies showed that the c-terminal region of gag fused by t cell epitopes from human cytomegalovirus pp65 led to the formation of hybrid vlps activating antigen-specific cd81 memory t cells ex vivo. 161 regarding to previous studies, the gag polyprotein is the only retroviral protein required for vlp formation. [194] [195] [196] vlps, derived from an avian retrovirus, were applied to deliver proteins to cells, either as part of gag fusion proteins (intracellular delivery) or on the surface of vlps. the construct is an effective system because the vlps are completely made of the gag fusion protein, and a single vlp will deliver 2000-5000 copies of gag fusion protein into a transduced cell. 197 delivery of foreign genes to the digestive tract mucosa by oral administration of non-replicating gene transfer vectors would be a very useful method for vaccination and gene therapy. 198 the studies indicated that plasmid dna could be packaged in vitro into a vlp composed of open reading frame 2 (orf2) of hev, which is an orally transmissible virus. these vlps could deliver this foreign dna to the intestinal mucosa in vivo, eliciting high mucosal and systemic immunity in mice, without the use of adjuvants. an orally administered hiv dna vaccine encapsulated in hev-vlps could induce mucosal and systemic cellular and humoral immune responses. 198 moreover, the ability of hpv vlps was examined to mediate delivery and expression of dna plasmids in vitro and in vivo. 199 hpv pseudoviruses were provided by disrupting hpv-vlp, mixing them with dna plasmids and reassembling them into the pseudoviruses (vlps with plasmids inside). the pseudovirus induced more potent immune responses than dna vaccines. the pseudovirus could be used in gene therapy by transferring the therapeutic genes into lymphoid tissues in human. 5 in addition, the recombinant hpv16 l1 vlps, produced in insect cells, could efficiently encapsulate a plasmid harboring either a gene for the gfp or b-galactosidase during in vitro disassembly-reassembly of vlps. 200 vlp-mediated delivery of a gfp reporter construct in vitro showed to be highly dependent on the presence of full-length l2 protein within the vlps. similarly, expression of gfp and luciferase reporter plasmids in vivo was efficiently enhanced by co-administration of l1/l2 vlps. in addition, co-administration of vlps with a hpv16 e6-expressing plasmid increased significantly e6-specific cellular immune responses. 201 the reports indicated that the recombinant major structural protein of the bk polyomavirus (bkv vp1) was shown to self-assemble into vlps with a diameter of 45-50 nm. the potential of bkv vp1 vlps was investigated to transfer gene into cos-7 cells using three methods for the formation of pseudovirions: disassembly/reassembly, osmotic shock and direct interaction between vlps and plasmid dna. the most efficient method is the direct interaction between vlps and linearized plasmid dna. the findings generally demonstrated that bkv vlps have exogenous dnabinding activity, as a promising vehicle for gene transfer studies. 200 sirna delivery there is a major challenge to identify novel approaches for specific and effective delivery of new types of drugs like sirnas and peptides. systemic delivery of small interfering rna (sirna) was restricted by its poor stability and low cellpenetrating properties. to overcome these limitations, an efficient sirna delivery system was designed using polyethyleneimine (pei)-coated vlps derived from adeno-associated virus type 2 (pei-aav2-vlps). generally, one of the strategies to integrate sirna into nanoparticles was to coat these particles with positively charged polymers, including pei, poly b-amino different applications of virus-like particles ester, or poly l-lysine. electrostatic coating could increase the efficiency of systemic sirna delivery due to its protective effects and improved cellular uptake. an insect/baculovirus expression system was used to generate aav2-vlps. pei-aav2-vlps could condense sirna, protect it from enzymatic degradation, transfer it with high efficiency and induce cell death in mcf-7 breast cancer cells, for breast cancer therapy. 201 furthermore, micrornas (mirnas) play an essential role in immunoregulation and may be involved in the pathogen esis of systemic lupus erythematosus (sle). among these sle-related mirnas, mir-146a, acts as a significant inhibitor of autoimmunity, myeloproliferation, and cancer. a novel mirna-delivery approach was described via bacteriophage ms2 vlps for evaluation of the therapeutic effects of mir-146a, in bxsb lupus-prone mice. treatment with ms2-mir-146a vlp increased the level of mature mir-146a, leading to a significant reduction in the expression of autoantibodies and total igg. furthermore, the levels of inflammatory cytokines, including ifn-a, il-1b and il-6 were decreased in mice. the stimulation of dysregulated mirnas by an ms2 vlp-based delivery system may be considered as a novel therapy. [202] [203] [204] the use of ms2 vlps was reported for selective delivery of nanoparticles, chemotherapeutic drugs, sirna cocktails, and protein toxins to human hcc. 205 in addition, the researchers used jc virus (jcv) vlps as a vector for delivering rnai in silencing the il-10 cytokine gene. jcv vlps were non-toxic, and showed the therapeutic use as a gene therapy approach for autoimmune diseases (aid) including sle. 206, 207 drug delivery a major challenge in pharmacology is to find methods that drugs (especially anti-cancer drugs) can be delivered specifically to target tissues. a potential strategy would be to package or encapsulate the drug molecules inside a particle which is bound to the cancerous tissue. such encapsulation would protect the drug from degradation in blood. for this purpose, it will be necessary to develop particles which can be modified on their outer surface to carry drug molecules into the target cells. novel nanocarriers such as dendrimers, liposomes, polymersomes, micelles, and vlps indicated high potency in improving drug delivery, and targeting strategies. all of these delivery systems make drugs more biocompatible, watersoluble, or colloidal, indicating low toxicity and high uptake in cells. 208 different virus-based materials were studied for drug delivery such as: the ccmv, the cpmv, the red clover necrotic mosaic virus (rcnmv), ms2 rna-containing bacteriophage, the bacteriophage qb, m13 bacteriophage, the tmv. 208 drug cargo can be loaded through covalent attachment of drugs or their analogs to particular reactive residues on the capsid pro-teins. 209 several cancer cell targeting ligands were attached to different types of vlps, including small molecules, antibodies, peptides and proteins, as well as dna aptamers. folic acid (fa) was broadly used in drug delivery targeted to cancer cells. uptake of fa into cells is mediated by the folate receptor (fr). 210 recently, lactobionic acid (la) was applied for the specific targeting of a rotavirus capsid vp6 to hepatocytes or hepatoma cells bearing asialoglycoprotein receptors (asgprs). 211 human holo-transferrin (tfn) is essential for iron homeostasis. tfn is especially recognized by the tfn receptor (tfnr), which is over-expressed on the surface of various tumor cells and efficiently taken up by cells in the clathrinmediated endocytosis. 212, 213 tfn has been conjugated to cpmv 214 and bacteriophage qb. 215 the cellular uptake of the qb-tfn particles was relative to the tfn density; while the internalization was prevented by comparable concentrations of free tfn. antibodies contain another group of targeting proteins that could be chemically linked to vlps. for instance, a single-chain (scfv) antibody that recognizes the carcinoembryonic antigen (cea) over-expressed in a variety of tumor cells, has been attached to cpmv. 216 an important strategy to improve cellular uptake of therapeutic molecules is the use of cell-penetrating peptides (cpps). 217 the hiv-1 tat peptide is one of the cpps that were extensively used in the delivery of vlps. 218, 219 in general, virus-like particles represents an attractive system for drug delivery in vitro. 220 the efficient delivery of hydrophobic drugs into target cells without the use of organic solvents or chemical linkage to delivery carriers is a critical issue in the biological field. recently, the intracellular delivery of hydrophobic dyes or drugs encapsulated in vlps through cyclodextrins (cds) showed high efficiency. as a model anticancer drug, paclitaxel (ptx)-cd complexes encapsulated inside vlps exhibited a dose-dependent cytotoxic effect with a 20-fold smaller ic50 than that of free ptx dissolved in dmso. 221 cell targeting is aimed to effective uptake of therapeutic and/ or diagnostic reagent in a special location such as a tumor. 222 targeting can also be achieved using proteins (mainly antibodies), peptides, nucleic acids (aptamers), small molecules, vitamins and carbohydrates. by attachment of targeting ligands, specificity for cell targeting was obtained by receptor-mediated endocytosis. for instance, bacteriophage ms2 vlps, were chemically conjugated to a targeting peptide (sp94) for the selective delivery of nanoparticles, chemotherapeutic drugs, sirna cocktails and protein toxins to human hcc. [223] [224] [225] recently, the chemical conjugation of human epidermal growth factor (egf) to simian virus 40 vlps allowed for cell 126 shirbaghaee and bolhassani selective targeting. 226 simian viruses 40 vlps have attracted a great attention in gene delivery due to their high stability and low toxicity in blood. 172 in design of polymeric nanoassemblies, chemical modification is necessary to conjugate the dye or probe for in vitro and in vivo imaging. however, in the case of nanobioassemblies, chemical or genetic modification can be applied for bioconjugation of fluorescent dyes or other probes. another advantage of nanobioassemblies such as vlps for bioimaging is their biological compatibility. quantum dots (qds) and gfp were used broadly for in vitro and in vivo imaging as alternatives to labelling. for example, fluorescent chimeric vlps of canine parvovirus were expressed in insect cells. 227 to create the fluorescent chimeric vlps of canine parvovirus, gfp was genetically engineered onto the n-terminal region of the viral protein vp2, as a visualization tool to understand mechanisms of viral infections. gfp was also used to design chimeric hiv vlps allowing protein to be followed during assembly and transmission using live-cell imaging. 228, 229 advantages of vlps include: (a) no need to propagate pathogenic organisms, (b) repetitive and ordered surface structures, (c) multivalent as well as particulate in nature, (d) safer than other vaccines because of non-infectious and non-replicating properties: the studies showed that there is no risk of disease progress in vaccinated groups with vlp-based vaccines as compared to attenuated viral vaccines, because they lack the genomic material needed for the replication and the spread of the viruses, (e) stable in extreme environmental conditions, depending on vlp structure (i.e., envelope or non-envelope), and (f) as carrier to express foreign antigen. 230 the potential of vlps to target dcs is a main advantage of vlp vaccines, for activating the innate and adaptive immune responses. they have a special benefit against other delivery systems in size, stability, and capacity to transfer biological molecules across cell barriers. particles in the 20-200 nm range can stimulate cd41, cd81 cells and especially generate th1 responses. in addition, despite a limited number of vlp vaccines approved for human use, they represent a promising platform for the development of novel mucosal vaccine strategies. indeed, vlps are sufficiently small, and the composition of their surface chemistry can be designed to minimize hydrophobic and electrostatic adhesive interactions with mucus. they can also be engineered for recombinant expression of multiple antigenic epitopes and for incorporation of co-stimulatory and immuno-regulatory proteins. however, vlp technology can be limited by difficulties of scale-up and the need for purification from the expression systems. 231 other limitation in chimeric vlp vaccine is to determine the compatibility of peptide with assembly of vlp and its immunogenicity property. under the host immune defence, pathogens undergo mutation which render the vlp vaccine ineffective and will be effective for only highly conserved b or t cell epitopes. 230 the major challenge is to develop novel production platforms that can deliver vlp vaccines while significantly reducing production times and costs. 104 viral-like particles (vlps) have shown high ability for the improvement of vaccines against infectious and non-infectious diseases. several recombinant expression systems were successfully applied for vlp production, with different efficiency. the use of vlps in vaccine development showed that they are considered safe. in addition, nano-sized vlps, can act as an adjuvant as well as antigen delivery system through increasing the antigen uptake by apcs. thus, it is not necessary for the use of adjuvants along with vlps to stimulate potent immune responses. vlps have shown a natural affinity to target host cells, and this property has been used for cell-targeting applications. regarding the advantages of vlps, it is necessary for further studies in various aspects especially easy and low-cost purification of vlps as well as their application as a delivery system in vivo. different applications of virus-like particles hum vaccine hepatitis b virus vaccine ip recombinant (genetically engineered): enivac hb hepavax-genev r . summary of product characteristics revac-b1tm. available at prescribing information. merck prescribing information. glaxosmithkline medicago to present additional positive clinical data at the 2011 eswi influenza conference medicago inc. news release exp rev vaccine hum vaccine immunother different applications of virus-like particles antimicrob agents chemother intranasal norwalk vaccine hum alves, p. m. exp rev vaccine exp rev vaccines different applications of virus-like particles curr top microbiol immunol the authors are grateful to elnaz agi and negar zohrei (dept. of hepatitis and aids, pasteur institute of iran) for technical assistance. key: cord-023865-6rafp3x3 authors: surjit, milan; lal, sunil k. title: the nucleocapsid protein of the sars coronavirus: structure, function and therapeutic potential date: 2009-07-22 journal: molecular biology of the sars-coronavirus doi: 10.1007/978-3-642-03683-5_9 sha: doc_id: 23865 cord_uid: 6rafp3x3 as in other coronaviruses, the nucleocapsid protein is one of the core components of the sars coronavirus (cov). it oligomerizes to form a closed capsule, inside which the genomic rna is securely stored thus providing the sars-cov genome with its first line of defense from the harsh conditions of the host environment and aiding in replication and propagation of the virus. in addition to this function, several reports have suggested that the sars-cov nucleocapsid protein modulates various host cellular processes, so as to make the internal milieu of the host more conducive for survival of the virus. this article will analyze and discuss the available literature regarding these different properties of the nucleocapsid protein. towards the end of the article, we will also discuss some recent reports regarding the possible clinically relevant use of the nucleocapsid protein, as a candidate diagnostic tool and vaccine against sars-cov infection. by definition, nucleocapsid is a viral protein coat that surrounds the genome (either dna or rna). nucleocapsid protein is the major constituent of a viral nucleocapsid. it is capable of associating with itself and with the genome, thus packaging the genome inside a closed cavity. in some viruses, nucleocapsid protein may also be assisted by other viral cofactors to form the capsid. however, in coronaviruses (including sars-cov), the nucleocapsid protein alone is capable of forming the capsid. the primary advantage of the virus for encoding the nucleocapsid protein is that the latter encloses and protects the viral genome from coming into direct contact with the harsh environment in the host. in fact, in some simple viruses like hepatitis e virus and polio virus, the nucleocapsid protein is the only coat that protects the genome from the outside world. however, in complex viruses, like hepatitis b virus and coronaviruses (including sars-cov), the nucleocapsid is covered by an additional coat composed of other viral proteins (spike protein is a major component of this coat). besides this property, nucleocapsid proteins of several viruses have been demonstrated to play multiple regulatory roles during viral pathogenesis. they are equipped with specific structural motifs and/or signature sequences, by which they associate with other viral/ host factors and skew the host cellular machinery in such a manner that it becomes more favorable for the survival of the virus. nucleocapsid protein is also one of the most abundantly expressed viral proteins and it is the major antigen recognized by convalescent antisera. hence, it is tempting to evaluate its potential as a candidate diagnostic tool or vaccine against the virus. therefore, understanding the properties of the nucleocapsid protein is of utmost importance to any virologist in order to understand the biology of the virus and develop effective tools to control the infection. since the identification and isolation of sars-cov in 2003, several laboratories around the world have focussed their research on characterization of various properties of the nucleocapsid protein. an indirect measure of the curiosity among sars-cov researchers to study the nucleocapsid protein is revealed from the fact that in pubmed the number of sars-cov research publications focussed on nucleocapsid protein is second only to those on spike protein. evidence accumulated from these articles has helped us gain substantial understanding of the properties of this protein. in this article, we will provide a comprehensive description of all the different properties of the nucleocapsid protein, as established by independent workers from several laboratories. we will conclude this article with the discussion of some of the remaining challenges in this field that need to be addressed in future. the nucleocapsid (n) protein is encoded by the ninth orf of sars-cov. the same orf also codes for another unique accessory protein called orf9b, though in a different reading frame, whose function is yet to be defined. the n-protein is a 46-kda protein composed of 422 amino acids (rota et al. 2003) . its n-terminal region consists mostly of positively charged amino acids, which are responsible for rna binding. a lysine-rich region is present between amino acids 373 and 390 at the c-terminus, which is predicted to be the nuclear localization signal. besides these, an sr-rich motif is present in the middle region encompassing amino acids 177-207. biophysical studies done by chang et al. (2006) have suggested that this protein is composed of two independent structural domains and a linker region. the first domain is present at the n-terminus, inside the putative rna binding domain, and the second domain consists of the c-terminal region that is capable of selfassociation. between these two structural domains, there lies a highly disordered region, which serves as a linker. this region has been reported to interact with the membrane (m) protein and human cellular hnrnpa1 protein (fang et al. 2006; luo et al. 2005) . besides, this region is also predicted to be a hot spot for phosphorylation. hence, in summary, the n-protein can be classified into three distinct regions ( fig. 9 .1), which may serve completely different functions during different stages of the viral life-cycle. a similar mode of organization has been reported for other coronavirus nucleocapsid proteins. in-vitro thermodynamic studies done by luo et al. (2004b) using purified recombinant n-protein have shown it to be stable between ph 7 and 10, with maximum conformational stability near ph 9. further, it was observed to undergo irreversible thermal-induced denaturation. it starts to unfold at 35 c and is completely denatured at 55 c . however, denaturation of the n-protein induced by chemicals such as urea or guanidium chloride is a reversible process. as in other coronavirus n-proteins, sars-cov n-protein has been predicted and later experimentally proven to undergo various posttranslational modifications such as acetylation, phosphorylation, and sumoylation. acetylation is the first modification of the n-protein to be experimentally proven. by mass spectrometric analysis of convalescent sera from several sars patients, it has been shown that the n-terminal methionine of n is removed and all other methionines are oxidized and the resulting n-terminal serine is acetylated. however, the functional relevance of this modification, if any, remains to be elucidated (krokhin et al. 2003) . another unique modification of the n-protein is its ability to become sumoylated. studies done by li et al. (2005a) have clearly established that heterologously expressed n in mammalian cells is sumoylated. using a site-directed mutagenesis approach, the sumoylation motif has been mapped to the 62nd lysine residue, which is present in a putative sumo-modification domain (gk 62 ee). their data further suggests that sumoylation may play a key role in modulating homo-oligomerization, nucleolar translocation and cell-cycle deregulatory property of the n-protein. further experimental support regarding sumoylation of n-protein came from another independent study carried out by fan et al. (2006) wherein they have demonstrated an association between the n-protein and hubc9, which is a ubiquitinconjugating enzyme of the sumoylation system. they have also mapped the interaction domain to the sr-rich motif, which is in agreement with the earlier report. however, they failed to detect the involvement of the gkee motif in mediating this interaction (fan et al. 2006) . initially, the sars-cov n-protein was predicted to be heavily phosphorylated. later on, from results obtained in our laboratory as well as by other researchers, it is now clear that the n-protein is a substrate of multiple cellular kinases. first experimental evidence for the phosphorylation status of the n-protein came from the study done by zakhartchouk et al. (2005) in which, using [ 32 p]orthophosphate labelling, they were able to observe phosphorylation of adenovirus-vectorexpressed n-protein in 293t cells. further studies done in our laboratory clearly confirmed this observation. the majority of the n-protein was found to be phosphorylated at its serine residues (although the involvement of threonine and tyrosine residues could not be detected; they may be occurring in vivo). in addition, using a variety of biochemical assays, it was proved that, at least in vitro, the n-protein could become phosphorylated by mitogen-activated protein kinase (map kinase), cyclin-dependent kinase (cdk), glycogen synthase kinase 3 (gsk3), and casein kinase 2 (ck2). also, this data provided preliminary indication regarding phosphorylation-dependent nucleo-cytoplasmic shuttling of the n-protein (surjit et al. 2005) . a recent report published by wu et al. (2008) has further confirmed that n-protein is a substrate of gsk3 enzyme, both in vitro and in vivo. using a variety of biochemical and genetic assays, it was clearly demonstrated that serine 177 residue of n-protein was phosphorylated by gsk3. an antibody specific to phospho 177 residue of the n-protein could efficiently detect the phospho n-protein both in vitro and in sars-cov infected cells. interestingly, biochemically mediated inhibition of gsk3 activity in sars-cov infected cells also leads to around 80% reduction in viral titer and subsequent induction of a virus-induced cytopathic effect. the authors proposed that gsk3 may be a major regulator of sars-cov replication, possibly by virtue of its ability to phosphorylate the n-protein. however, phosphorylation of other viral and/or host proteins by gsk3 may also be a determinant of the observed cytopathic effect. in contrast to the n-protein of many other coronaviruses, the sars-cov n-protein is predominantly distributed in the cytoplasm, when expressed heterologously or in infected cells (surjit et al. 2005; you et al. 2005; rowland et al. 2005) . in infected cells, a few cells exhibited nucleolar localization (you et al. 2005) . as reported by you et al. (2005) , the n-protein contains pat4, pat7 and bipartite-type nuclear localization signals. it has also been predicted to possess a potential crm-1dependent nuclear export signal. however, no clear experimental evidence could be obtained regarding the involvement of these signature sequences in regulating the localization of the n-protein. interestingly, studies done in our laboratory revealed that the majority of n-protein localized to the nucleus in serum-starved cells. this phenomenon could be reproducibly observed both in biochemical fractionation as well as immunofluorescence studies. in addition, treatment of cells with specific inhibitors of different cellular kinases such as ck2 inhibitor and cdk inhibitor resulted in retention of a fraction of the n-protein in the nucleus, whereas gsk3 and mapk inhibitor had very little effect. further, n-protein was found to be efficiently phosphorylated by the cyclin-cdk complex, which is known to be active only in the nucleus. the n-protein was also found to associate with 14-3-3 protein in a phospho-specific manner and inhibition of the 14-3-3y protein level by sirna resulted in nuclear accumulation of the n-protein. although these experiments are too preliminary to conclusively provide any answer regarding the intracellular localization of n-protein, nevertheless they do provide substantial clues regarding the physical presence of the n-protein in the nucleus, under certain circumstances, which may be a very dynamic phenomenon. another study done by timani et al. (2005) using different deletion mutants of the n-protein fused to egfp showed that the n-terminal of n-protein, which contains the nls 1 (aa 38-44), localizes to the nucleus, whereas the c-terminal region containing both nls 2 (aa 257-265) and nls 3 (aa 369-390) localizes to the cytoplasm and nucleolus. using a combination of different deletion mutants, they concluded that the n-protein may act as a shuttle protein between cytoplasm-nucleus and nucleolus. taken together, all these results further suggest that the n-protein per se has the physical ability to localize to the nucleus. whether this localization is regulated through phosphorylation-mediated activation of a potential nls or piggy-backing by association with another cellular nuclear protein or through any other mechanism remains to be established. being the capsid protein, the primary function of the n-protein is to package the genomic rna in a protective covering. in order to achieve this structure, the n-protein must be equipped with two different characteristic properties; such as (1) being able to recognize the genomic rna and associate with it, and (2) selfassociate into an oligomer to form the capsid. the n-protein of sars-cov has been experimentally proven to possess these properties in vitro, as discussed below. 9.6.1 recognition and binding with the genomic rna the first experimental evidence regarding the rna binding property of the n-protein came from the work of huang et al. (2004) , in which, by nmr studies, they proved the ability of the n-terminal domain to associate with several viral 3 0 untranslated rna sequences. additionally, chen et al. (2007) reported the presence of another rna binding domain at the c-terminal region (residues 248-365) of the n-protein, which was proposed to be a stronger interaction than that at the n terminus. based on structural analysis of the rna-protein interaction, they have further suggested that the genomic rna is packaged in a helical manner by the n-protein. in another report published by luo et al. (2006) , the rna binding motif of the n-protein was mapped to amino acid residues 363-382. in summary, the rna binding ability of the n-protein was attributed to its two distinct structural domains: the n-terminal domain (residues 45-181) and the c-terminal dimerization domain (residues 248-365). these two domains are spatially separated by long stretches of disordered region. a recent study done by chang et al. (2008) has demonstrated rna binding ability of these disordered regions. they have proposed that different rna binding domains of the n-protein may cooperate to enhance the overall rna binding efficiency of the n-protein and may also serve as interaction hubs for the association of n-protein with other viral and/or host nucleic acid and/or proteins. perhaps the most convincing proof to date regarding the ability of the n-protein to package the genomic rna came from the work of hsieh et al. (2005) . they have established a system to produce sars-cov vlps by cotransfection of spike, membrane, and envelope and nucleocapsid cdnas into vero e6 cells. while testing the packaging of an rna-bearing gfp fused to sars-cov packaging signal into this particle, they observed that presence of the n-protein is an absolute requirement. however, the n-protein was not essential for the assembly of the empty particle per se. further, by performing a filter binding assay using recombinant n-protein, they were able to identify two independent rna binding domains in the n-protein; one at the n terminus (aa 1-235) and the other at the c terminus (aa 236-384). these results are in agreement with previous findings and further suggest that these two regions may be functional in vivo. future experiments using a model infection system will confirm these observations. one of the most crucial properties required by the n-protein for genome encapsidation is its ability to self-associate. therefore, many laboratories have focused on characterizing this phenomenon, with an eye on developing possible interference strategies that may help in limiting virus propagation. initial studies done in our laboratory using a yeast two-hybrid assay revealed that n-protein is able to self-associate through its c-terminal amino acid 209 residues (surjit et al. 2004a) . a parallel study done by he et al. (2004) using the mammalian two-hybrid system and sucrose gradient fractionation also proved the ability of the n-protein to self-associate to form an oligomer. they further mapped the interaction region to amino acid 184-196 residues, encompassing the sr-rich motif. however, there were some discrepancies regarding the interaction domain mapped in these two studies. later on, extensive biophysical and biochemical analysis done by chen's laboratory and jiang's laboratory (luo et al. 2006 (luo et al. , 2005 have enriched our understanding of the oligomerization process of the nprotein. in summary, the sr-rich motif does possess binding affinity, but this is specific for the central region (aa 211-290) of another molecule of n-protein, instead of the sr-rich motif itself. the c-terminal region (aa 283-422) possesses binding affinity for itself and to associate into a dimer, trimer, tetramer or hexamer, in a concentration-dependent manner. the essential sequence for oligomerization of the n-protein was identified to be residues 343-402. interestingly, this region also encompasses the rna binding motif of the n-protein, which prompts us to speculate that there might be mutual interplay between rna binding and oligomerization activities of the n-protein. further, the oligomerization was observed to be independent of electrostatic interactions and addition of single strand dna to the reaction mixture containing tetramers of the n-protein promoted oligomerization. thus, it has been proposed that once the tetramer is formed by protein-protein interaction between nucleocapsid molecules, binding with genomic rna prompts further assembly of the complete nucleocapsid structure. besides being the capsid protein of the virus, the n-protein of many coronaviruses is known to double up as a regulatory protein. the n-protein of the sars-cov too has been shown to modulate the host cellular machinery in vitro, thereby indicating its possible regulatory role during its viral life-cycle. some of the major cellular processes perturbed by heterologous expression of the n-protein are discussed below. three different groups have reported the ability of the n-protein to interfere with the host cell cycle in vitro. work done by li et al. (2005a li et al. ( , 2005b proved that mutation of the sumoylation motif in the n-protein leads to cell cycle arrest. work done in our laboratory has shown the inhibition of s phase progression in cells expressing the n-protein (surjit et al. 2006 ). further, s-phase specific gene products like cyclin e and cdk2 were found to be downregulated in sars-cov infected cell lysate, which suggested that the observed phenomenon may be relevant in vivo. in an attempt to further characterize the mechanism of cell cycle blockage induced by the n-protein, several biochemical and mutational analysis were carried out. results thus obtained demonstrated that the n-protein directly inhibits the activity of the cyclin-cdk complex, resulting in hypophosphorylation of retinoblastoma protein with a concomitant downregulation of e2f1-mediated transactivation. analysis of rxl and cdk phosphorylation mutant n-protein identified the mechanisms of inhibition of cdk4 and cdk2 activity to be different. whereas the n-protein could directly bind to cyclin d and inhibit the activity of the cdk4-cyclind complex, inhibition of cdk2 activity appeared to be achieved in two different ways: indirectly by downregulation of protein levels of cdk2, cyclin e, and cyclin a, and by direct binding of n-protein to the cdk2-cyclin complex. a third piee of evidence supporting the ability of n-protein to deregulate the host cycle came from the work of zhou et al. (2008) . they observed slower transition from s to g2/m phase and slower growth rate in n-protein-expressing 293t cells. they also observed a similar phenomenon in human peripheral blood lymphocyte and k 562 cells infected with a retrovirus expressing sars-cov n-protein. while searching for interaction partners for the c terminus of n-protein (aa 251-422) by following a yeast two-hybrid library screening approach, zhou et al. (2008) discovered human elongation factor 1 alpha (ef1a) as a candidate partner. the specificity of the interaction was confirmed by various in-vitro and in-vivo assays. further, expression of n-protein induced aggregation of ef1a. it is known that the majority of cellular ef1a is bound to f-actin and promotes f-actin bundling, which is a key event during cytokinesis (kurasawa et al. 1996; yang et al. 1990) . hence, the authors tested whether n-protein-induced aggregation of ef1a affected f-actin bundling and cytokinesis. as expected, they observed significantly fewer f-actin bundles in n-protein-expressing cells. in fact, a similar f-actin distribution pattern was also observed by surjit et al. (2004b) in cos-1 cells. further, the authors observed multinucleated cells in n-protein-expressing cells at a later time point (72 h post-transfection), indicating inhibition of cytokinesis in those cells. specificity of the above data has been confirmed by the use of different deletion mutants of the n-protein, in which only the c-terminal domain of the n-protein (responsible for binding with ef1a) was able to reproduce the above results. thus, it has been suggested that ef1a binding by the n-protein leads to its aggregation, resulting in inhibition of f-actin bundling and subsequent blocking of cytokinesis. ef1a is known to play a key role during the peptide elongation stage of translation. therefore, it is an attractive candidate for pathogen proteins to manipulate its activity in order to skew the host translation machinery. for example, hiv-type 1 gag polyprotein has been shown to interact with ef1a and impair translation in vitro (cimarelli and luban 1999) . since zhou et al. (2008) observed an interaction between ef1a and sars-cov n-protein, they further tested whether it interfered with the host translation machinery. indeed, presence of the n-protein inhibited total cellular translation, both in vitro and in vivo, in a dose-dependent manner. moreover, exogenous addition of excess ef1a could reverse the n-proteininduced translation inhibition, thus suggesting that n-protein exerts its effect by interfering with ef1a function. however, it remains to be confirmed whether a similar effect is recapitulated in vivo. production of interferon (ifn) is one of the primary host defense mechanisms. however, sars-cov infection does not result in ifn production. nevertheless, pretreatment of cells with ifn blocks sars-cov infection (spiegel et al. 2005; zheng et al. 2004 ). based on this observation, palese's laboratory has studied the ifn inhibitory property of different sars-cov proteins, which revealed that orf3, orf6 as well as the n-protein have the ability to independently inhibit ifn production through different mechanisms. the n-protein was found to inhibit the activity of irf3 and nfkb in host cells, resulting in inhibition of ifn synthesis. irf3 activity was also blocked by orf3, orf6 proteins, but inhibition of nfkb activity was a property unique to the n-protein. in addition, orf3, orf6 proteins were able to block stat1 activity through different mechanisms (kopecky-bromberg et al. 2007 ). all these data suggest that sars-cov may employ multiple factors to check the activity of the host immune system and n-protein may be one of the major partners in this process. it may be possible that these different factors act independently during different stages of the viral life cycle. in that case, regulatory activity of the n-protein will be as indispensible as its structural activity. during the sars outbreak, a large number of patients developed severe inflammation of the lungs, which subsequently led to acute respiratory distress syndrome (ding et al. 2003; nicholls et al. 2003) . acute respiratory distress syndrome is characterized by pulmonary fibrosis, which results in lung failure and subsequent death of the patient. the tgfb signaling pathway plays a critical role in pulmonary fibrosis (roberts et al. 2006; border and noble 1994) . it enhances the expression of extracellular matrix (ecm) proteins, accelerates the secretion of protease inhibitors and reduces the secretion of proteases, thereby leading to deposition of ecm proteins. tgfb may also induce pulmonary fibrosis directly by stimulating chemotactic migration and proliferation of fibroblasts as well as by fibroblast-myofibroblast transition. hence, it is worth speculating that some of the sars-cov encoded factors may be modulating the tgfb signaling pathway. in fact, proteins of several other viruses, such as hepatitis c virus core, ns3 and ns5 protein, adenovirus e1a, human papilloma virus e7, human t-lymphotropic virus tax and epstein-barr virus lmp1, have been reported to modulate the tgfb pathway. in general, these proteins directly bind with smad proteins and alter the innate signaling pathway. interestingly, a recent report published by zhao et al. (2008) revealed that n-protein of sars-cov also interacts with smad3 and modulates the activity of the tgfb pathway. by performing a smad binding element (sbe)-driven reporter assay, rt-pcr and immunohistological analysis of tgfb target genes such as pai-1 (plasminogen activator inhibitor 1) and collagen in a variety of cell lines and sars patients, the authors have clearly proved that n-protein indeed enhanced the activity of the tgfb signaling pathway. further, they observed that the effect of n-protein on tgfb signaling was mediated through smad3 only (independent of the involvement of smad4). while trying to unravel the mechanism behind this phenomenon, they observed that n-protein specifically associated with the mh2 domain of smad3 (stronger binding affinity for phospho smad3) interrupted the interaction between smad3 and smad4, and enhanced the interaction between smad3 and transcriptional coactivator p300 in a dose-dependent manner. to further confirm the above data, they performed a chromatin immunoprecipitation assay at the sbe region of pai-1 promoter in hpl1 cells and detected the presence of n-protein in the complex of smad3 and p300. interestingly, however, n-protein inhibited tgfb-induced apoptosis of hpl1 cells (it is a well established fact that smad3 activation induces apoptosis of hpl1 cells). thus, n-protein appears to employ a clever mechanism whereby, on the one hand, it enhances the activity of the tgfb signaling pathway, thus leading to enhanced expression of a subset of genes (such as ecm protein coding genes), and on the other hand, it blocks the programmed cell death of the host cell. it would be interesting to unravel the mechanism behind this unique property of the n-protein. another major proinflammatory factor induced during viral infection is the cyclooxygenase-2 (cox2) protein. using 293t cells expressing the n-protein, yan et al. (2006) have shown that expression of the n-protein leads to upregulation of cox2 protein production in a transcriptional manner. they have further demonstrated that the n-protein directly binds to the nfkb response element present in the cox2 promoter through a 68 aa residue binding domain (aa 136-204) and activates its transcription. although the n-protein is known to associate with stretches of nucleic acids, to date there is no other documentation or prediction of its sequence-specific dna binding activity (as a transcription factor). in such a scenario, the above observation, if reproducible in vivo, may really be a unique property of the n-protein and may further add to the established regulatory functions of the n-protein. exogenously expressed n-protein has been reported to enhance the dna binding activity of c-fos, atf-2, creb-1, and fos b in an elisa-based assay, thus suggesting an increase in ap1 activity in these cells (he et al. 2003) . the mechanistic details and functional significance of this phenomenon remain to be elucidated. earlier work done in our laboratory has shown that n-protein, when expressed in cos-1 monkey kidney cells, induces apoptosis in the absence of growth factors. attempts to understand the mechanism of programmed cell death revealed that the n-protein downmodulated the activity of prosurvival factors such as extracellular regulated kinase, akt and bcl 2, and upregulated the activity of proapoptotic factors like caspase-3 and caspase-7 (surjit et al. 2004b ). however, this phenomenon was not observed in another cell line of epithelial lineage (huh7). the above observation was further confirmed by zhang et al. (2007) . they reported that serum starvationinduced apoptosis of n-protein-expressing cos-1 cells involved activation of mitochondrial pathway. another elegant study done by diemer et al. (2008) has further extended our understanding regarding the apoptotic property of the n-protein. through a series of experiments involving both a model infection system of sars-cov and transient transfection of n-protein, the authors have confirmed that n-protein induces an intrinsic apoptotic pathway resulting in activation of caspase-9, which further leads to activation of caspase-3 and -6. their data further revealed that these activated caspases cleave the n-protein at residues 400 and 403 and that nuclear localization of n-protein is an absolute requirement for cleavage. in addition, the authors have reported that the apoptosis-inducing ability of the n-protein is highly cell type specific. only in cells where n-protein localizes to both nucleus and cytoplasm (vero e6 and a549 cells), is it able to activate caspase and become cleaved; however, in cell lines where it localizes to the cytoplasm only (caco2 and n-2a cells), no activation of caspase is observed. it remains to be studied whether this phenomenon is actually recapitulated in vivo. a recent report by han et al. (2008) revealed that, of all the sars-cov structural proteins, only n-protein specifically induced the transcription of prothrombinase gene in thp-1 and vero cells. by performing luciferase reporter assay of hfgl2 promoter in n-protein-expressing cells and electrophoretic mobility shift assay using n-protein-transfected cell lysate, they demonstrated that n-protein expression induced the binding of transcription factor c/ebpa to its cognate response element present in hfgl2 promoter, leading to enhanced transcription of hfgl2 gene. since lungs of sars patients have been shown to contain high amount of fibrin, the authors proposed that n-protein-mediated enhanced production of prothrombinase gene may contribute to the development of thrombosis in sars patients. luo et al. (2005) have reported the interaction between hnrnpa1 and n-protein by using a variety of biochemical and genetic assays. the interaction was found to be mediated through the middle region (aa 161-210) of n-protein. if relevant in vivo, this interaction may play a significant role in regulation of the viral rna synthesis. another interesting study done by luo et al. (2004a) has reported association between the n-protein and human cyclophylin a. by spr (surface plasmn resonance) analysis they have shown it to be a high affinity interaction. although the significance of this interaction is not known in vivo, they have proposed that this interaction might be crucial for viral infection. notable is the fact that hiv-1 gag also binds with human cyclophylin a and this interaction is crucial for hiv infection (gamble et al. 1996) . recently, zeng et al. (2008) have reported that n-protein associates with b23, a phosphoprotein in the nucleus. by performing in vivo coimmunoprecipitation in hela cells and gst pull-down assay using purified recombinant n-protein, the authors have demonstrated direct interaction between b23 and n-protein. the interaction domain has been mapped to amino acid residues 175-210 of n-protein, which include the sr-rich motif. b23 plays a key role in centrosome duplication during cell division. phosphorylation of b23 at threonine-199 residue is known to regulate its function (okuda et al. 2000 , tokuyama et al. 2001 . in order to demonstrate the functional significance of n-protein interaction with b23 protein, the authors tested the phosphorylation status of threonine-199 residue of b23 in the presence of n-protein. interestingly, n-protein was able to block threonine-199 phosphorylation. based on this observation, the authors have proposed that n-protein exerts its effect on cell cycle deregulation by modulating the activity of b23 protein. in summary, although several regulatory roles have been proposed for the sars-cov n-protein using a variety of in-vitro experimental systems, no clear evidence exists for their occurrence in vivo. in the absence of a suitable in-vivo experimental system, all these functions remain speculative. one of the most essential steps to limit the outbreak of any infectious disease is the ability to diagnose the causative agent at the earliest possible time, which can be achieved by detecting some of the markers that are specifically expressed by the pathogen or by identifying some of the host factors that are specifically produced during infection. n-protein, being one of the predominantly expressed proteins at the early stage of sars-cov infection, against which a strong antibody response is initiated by the host, has been proposed to be an attractive diagnostic tool. in serum of sars-cov patients, the n-protein has been detected as early as day one of infection by elisa using monoclonal antibodies against it (che et al. 2004 ). further, a comparative study to detect sars-cov-specific igg, sars-cov rna, and the n-protein during early stages of infection has demonstrated that the detection efficiency of the n-protein is significantly higher than the other two markers (li et al. 2005b) . researchers have been mainly focussing on two different strategies by which nucleocapsid can be used as a diagnostic tool: (1) development of efficient monoclonal antibodies against the n-protein, and (2) production of recombinantly expressed, highly purified n-protein for detection of n-protein-specific antibody in the host. using a phage display approach, flego et al. (2005) have identified human antibody fragments that recognize distinct epitopes of the n-protein. these may help develop efficient reagents to detect n-protein in the infected host. further, several laboratories have been trying to develop efficient monoclonal antibodies against the major immunodominant epitopes of the n-protein, that can be used in elisa to detect sars-cov at an early stage of infection (shang et al. 2005; liu et al. 2003; he et al. 2005; woo et al. 2005) . in another interesting study, liu et al. (2005) have developed an immunofluorescence assay using antirabbit n-protein antibody that can specifically detect n-protein from throat wash samples of sars-cov patients at day two of illness. several other workers have focussed on economical production of highly purified recombinant n-protein using a variety of heterologous expression systems that can be used in elisa to detect n-protein-specific antibody in the patient sample. n-protein has been produced in abundant quantity using a codon-optimized gene in e. coli (das and suresh 2006) . saijo and coworkers have successfully expressed recombinant n-protein using a baculovirus expression system, which was found to be 92% efficient in neutralizing antibody assay (saijo et al. 2005) . in another study, liu et al. have expressed full length n-protein using a yeast expression system ). however diagnostic use of recombinant n-protein has been a problematic issue because of several reasons as discussed below. bacterially expressed n-protein has been reported to produce false seropositivity owing to interference of bacterially derived antigens (leung et al. 2006; yip et al 2007) . in addition, several studies have shown cross-reactivity between full-length n-protein of sars and polyclonal antisera of group 1 animal coronaviruses, which may lead to faulty detection (sun and meng 2004) . another study done by woo et al. also reported cross-reactivity of full-length recombinant n-protein with antisera of hcov-oc43 and hcov-229e infected patients, thus giving false positive results. they were able to minimize this false positivity by further verifying the elisa results with western blot assay using recombinant n and spike proteins of sars-cov (woo et al. 2004) . later, studies done by qiu et al. and bussmann et al. showed that the recombinantly expressed c-terminal of the n-protein acts more specifically in detecting sars-cov-specific antisera in comparison to full-length n-protein (qiu et al. 2005; bussmann et al. 2006) . it is noteworthy that this region is predicted to encompass major antigenic sites of the n-protein. in a recent report, shin et al. (2007) demonstrated significantly higher efficacy of phosphorylated n-protein as a diagnostic antigen. they expressed the n-protein in insect cells, where it was phosphorylated by posttranslational modification. when the antigenicity of this protein was compared to that of a bacterially expressed n-protein (unphosphorylated) or to that of a dephosphorylated n-protein (by treatment with protein phosphatase 1) using sars-positive or -negative patient serum, phosphorylated n-protein did not show any cross-reactivity with sars-negative serum, thereby reducing the number of false positives. also, the phosphorylated protein showed considerably stronger cross-reactivity with an n-protein-specific monoclonal antibody. based on these observations, the authors have proposed the use of a phosphorylated n-protein as a better diagnostic agent. also, several reports have been published dealing with the detection of n-protein-specific igm by elisa or indirect immunofluorescent assay (chang et al. 2004; hsueh et al. 2004; woo et al. 2004 ). however, in these studies, igm antibodies became detectable later than igg antibodies, which is in contrast to the phenomena observed in most other pathogens. a recent report published by yu et al. (2007) attempted to solve this problem by using a truncated n-protein (aa 122-422) as an antigen in igm elisa. they found the igm response appeared three days before detection of the igg response, which is in agreement with the results obtained from other known pathogens. further, their results showed 100% specificity and sensitivity of the truncated protein in detecting n-protein-specific igm from patients with laboratory confirmed sars cases in comparison to healthy volunteers. the authors have suggested that the igm capture elisa using this truncated n-protein may be more effective in serodiagnosis of sars-cov at an earlier time. in another interesting report, woo et al. (2005) carried out comparative studies to evaluate the relative diagnostic efficacy of recombinantly expressed n and spike proteins. they observed sensitivity of recombinant n-igg elisa to be significantly higher than that of recombinant s-igg elisa. the reverse was true in the case of igm elisa using recombinant n and s proteins. based on this data, they have suggested the practise of elisa for detection of igm against both s and n proteins instead of n alone (woo et al. 2005) . taken together, all this data does support the notion that the n-protein may be used as an efficient diagnostic tool for detection of sars-cov infection. nevertheless, production scale-up and further validation of specificity using patient samples will determine the possible clinical use of these reagents. one of the most clinically relevant uses of the n-protein can be its use as a protective vaccine against sars-cov infection. n-protein is one of the major antigens of the sars-cov. also, n-protein analyzed from different patient samples shows least variation in the gene sequence (tong et al. 2004) , therefore indicating it to be a stable protein, which is a primary requirement for an efficient vaccine candidate. earlier studies carried in collins', rao's, and li's laboratories have clearly shown that antiserum to the n-protein does not contain neutralizing antibodies against sars-cov (buchholz et al. 2004; pang et al. 2004; liang et al. 2005) . this may be attributed to the localization of n-protein inside the viral envelope, which will not be accessible to the antibody during infection. it is noteworthy that the most effective sars-cov structural protein that can induce neutralizing antibody production is the s-protein (buchholz et al. 2004 ). the s-protein antibody could block viral infection with 100% efficiency. on the other hand, although unable to induce humoral immunity, expression of n-protein induced significant cytotoxic t-lymphocyte (ctl) response (buchholz et al. 2004; gao et al. 2003; zhu et al. 2004) . induction of n-protein-specific ctls will help limit the infection by lysing virus infected cells. this will also limit the spread of virus. thus, n-protein-based vaccines may further augment the protection efficiency when coadministered with s-protein-based vaccine. several laboratories have been exploring various strategies to evaluate the potential of n-protein as a vaccine candidate. in an elegant work done by kim et al. (2004) , calreticulin-fused n-protein expressing vaccinia virus has been shown to generate potent n-protein-specific humoral and t-cell immune responses in mice. as reported by the authors, fusion with calreticulin specifically enhanced the efficiency and significantly reduced the titer of the challenging vector (vaccinia virus). the authors have proposed that n-protein may be the logical choice as a target antigen in the event of s-protein antibody-dependent enhancement (ade) of infection. however, the ade phenomenon has not been observed during spike-mediated vaccination (buchholz et al. 2004 ). another study done by wang et al. (2005) has attempted to use plasmid dna expressing s, m, and n proteins as an efficient vaccine candidate. although they report the production of some b-cell and t-cell responses against n-protein, strong immune response was obtained for the s and m proteins, thus scaling down the choice of n-protein as a suitable candidate vaccine . a similar plasmid-mediated vaccination approach has also been reported by zhao et al. (2004) , in which they immunized mice with the dna construct (pci vector) expressing the n-protein. they too reported the generation of a robust b-cell and t-cell immune response in animals. another group of workers has also reported successful use of the n-protein as a dna vaccine. they immunized mice by intramucosal injection of the n-protein-expressing plasmid vector and were able to obtain specific humoral and t-cell responses (zhu et al. 2004) . the n-protein has also been reported to be of potential interest as a peptidebased vaccine. a systematic study done by liu et al. (2006) has revealed the immunodominant epitopes of the n-protein which could efficiently stimulate immune response. they have also deduced some conserved immunodominant epitopes in mouse, monkey, and humans, which may help in design of the vaccine. a recent report published by gao's laboratory provides further evidence regarding the efficiency of an n-protein-based vaccine (zhao et al. 2007 ). by using overlapping synthetic peptides spanning the n-protein, they have identified dominant helper t-cell epitopes in the n-protein of sars-cov. immunization of mice with peptides emcompassing these dominant th cell epitopes resulted in strong cellular immunity in vivo. priming with the helper peptides significantly accelerated the immune response induced by the n-protein. further, by fusing with a conserved neutralizing epitope from the spike protein of sars-cov, two of the th cell epitope-bearing peptides assisted in the production of higher titer neutralizing antibodies in vivo, in comparision to spike epitope alone or its mixture with th epitope of n. thus, it is practically possible to generate a better immune response by using a fusion of n and s protein. however, the th epitopes identified in their report are specific to mouse, and will therefore not be useful for human. nevertheless, their data provides useful information for the design of peptide-based anti-sars-cov vaccines. another interesting study conducted by pei et al. (2005) reports the possible use of the n-protein as a mucosal vaccine candidate. they expressed the n-protein in lactobacillus lactis, which is a food-grade bacteria, and challenged the mice either orally or intramucosally. as preliminary evidence, they were able to observe significant n-protein-specific igg in the sera of orally challenged animals. it is a significant achievement for the research community that, within a short span of time, we have been able to obtain a more-or-less clear understanding regarding the structural and functional properties of the n-protein. however, it is a fact worth mentioning that all the studies done here were performed with in-vitro experiments, using recombinantly expressed n-protein, in isolation. so at present, all we can conclude is that the n-protein per se has the physical ability to perform the above described functions, in other words n-protein does bear the necessary signature sequence or motifs or conformation to perform these functions under suitable circumstances. whether a similar event is recapitulated in vivo during viral infection will be dependent on several criteria: (1) the net effect of other viral factors on the activity of n-protein, (2) the net translation and turnover rate of n-protein, (3) a conducive intracellular milieu, and (4) the net modulation of an already skewed cellular pathway by other viral factors. hence, it will be interesting to reevaluate the properties of n-protein in a sars-cov infection model. however, owing to the limited user-friendliness and accessibility of an infection system, we must probably still resort to in vitro systems for further analysis of the characteristics of n-protein. one of the better experimental systems has already been established by chang's laboratory (hsieh et al. 2005) , in which all the structural proteins were coexpressed to form vlp in 293t cells. if this system can be further improved to optimize the rate of synthesis of these different proteins to a level near that in vivo, it will at least enable us to study the net effect of the n-protein with respect to other viral proteins. further establishment of a replicon system may also be helpful. in addition, some of the interesting preliminary observations reported by several laboratories need to be analyzed in detail. to begin with, the reported interaction of the n-protein with the genomic rna packaging signal needs to be further characterized and mapped. since the oligomerization domain and the rna binding regions of the n-protein overlap with each other, the suggested possibility of regulated genome incorporation and capsid assembly should be further characterized with the aid of a replicon system or a particle assembly system. in addition, the reported ability of the n-protein to modulate different cellular pathways should be further characterized in the particle assembly system or at least in the presence of other viral accessory proteins. the most unique and significant property of the n-protein revealed by preliminary studies is its ability to act as a sequence-specific dna binding factor. it has been shown to bind the nfkb response element of cox2 promoter and to enhance cox2 gene expression. this activity may be further empowering the n-protein to manipulate the entire gene expression programme of the infected cell. therefore, studies should be initiated to analyze this phenomenon in detail. it seems to deserve so much attention because another study done by palese's laboratory has proved the ability of the n-protein to inhibit nfkb activity, which results in inhibition of ifn synthesis. further, liao et al. (2005) have reported the activation of nfkb by n-protein in vero e6 cells and he et al. (2005) failed to detect any change in nfkb activity in the same cells. therefore it needs to be clarified whether n-protein enhances nfkb activity and, if so, whether upregulation of cox2 transcription by direct dna binding is a property specific to that promoter or whether it is a global phenomenon. in such a scenario, there may be complicated cross-talk between the ability of n-protein to deregulate the expression of cox2 and ifn in infected cells. lastly, the n-protein is known to be the most abundantly expressed protein of the sars-cov. therefore, any information generated from the analysis of this protein, whether in vivo or ex vivo, will definitely help to increase our understanding of the biology of sars-cov and may someday help to design better protective tools against it. transforming growth factor beta in tissue fibrosis contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity antigenic and cellular localisation analysis 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nucleocapsid protein of sars-cov induces transcription of hfgl2 prothrombinase gene dependent on c/ebp alpha activation of ap-1 signal transduction pathway by sars coronavirus nucleocapsid protein analysis of multimerization of the sars coronavirus nucleocapsid protein characterization of monoclonal antibody against sars coronavirus nucleocapsid antigen and development of an antigen capture elisa assembly of severe acute respiratory syndrome coronavirus rna packaging signal into virus-like particles is nucleocapsid dependent chronological evolution of igm, iga, igg and neutralisation antibodies after infection with sars-associated coronavirus structure of the n-terminal rna-binding domain of the sars cov nucleocapsid protein generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon 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methylotrophic yeast pichia pastoris immunofluorescence assay for detection of the nucleocapsid antigen of the severe acute respiratory syndrome (sars)-associated coronavirus in cells derived from throat wash samples of patients with sars immunological characterizations of the nucleocapsid protein based sars vaccine candidates nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a in vitro biochemical and thermodynamic characterization of nucleocapsid protein of sars the nucleocapsid protein of sars coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein a1 carboxyl terminus of severe acute respiratory syndrome coronavirus nucleocapsid protein: self-association analysis and nucleic acid binding characterization lung pathology of fatal severe acute respiratory syndrome nucleophosmin/b23 is a target of cdk2/cyclin e in centrosome duplication protective humoral responses to severe acute respiratory syndrome-associated coronavirus: implications for the design of an effective protein-based vaccine expression of sars-coronavirus nucleocapsid protein in escherichia coli and lactococcus lactis for serodiagnosis and mucosal vaccination use of the cooh portion of the nucleocapsid protein in an antigen-capturing enzymelinked immunosorbent assay for specific and sensitive detection of severe acute respiratory syndrome coronavirus smad3 is key to tgf-beta-mediated epithelial-to-mesenchymal transition, fibrosis, tumor suppression and metastasis characterization of a novel coronavirus associated with severe acute respiratory syndrome intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells recombinant nucleocapsid protein-based igg enzyme-linked immunosorbent assay for the serological diagnosis of sars characterization and application of monoclonal antibodies against n protein of sarscoronavirus antigenic characterization of severe acute respiratory syndrome-coronavirus nucleocapsid protein expressed in insect cells: the effect of phosphorlation on immunoreactivity and specificity inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor 3 antigenic cross-reactivity between the nucleocapsid protein of severe acute respiratory syndrome (sars) coronavirus and polyclonal antisera of antigenic group i animal coronaviruses: implication for sars diagnosis the nucleocapsid protein of the sars coronavirus is capable of self-association through a c-terminal 209 amino acid interaction domain the sars coronavirus nucleocapsid protein induces actin reorganization and apoptosis in cos-1 cells in the absence of growth factors the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks s phase progression in mammalian cells nuclear/nucleolar localization properties of c-terminal nucleocapsid protein of sars coronavirus specific phosphorylation of nucleophosmin on thr(199) by cyclin-dependent kinase 2-cyclin e and its role in centrosome duplication direct sequencing of sarscoronavirus s and n genes from clinical specimens shows limited variation low stability of nucleocapsid protein in sars virus immune responses with dna vaccines encoded different gene fragments of severe acute respiratory syndrome coronavirus in balb/c mice longitudinal profile of immunoglobulin g (igg), igm, and iga antibodies against the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in patients with pneumonia due to the sars coronavirus differential sensitivities of severe acute respiratory syndrome (sars) coronavirus spike polypeptide enzyme-linked immunosorbent assay (elisa) and sars coronavirus nucleocapsid protein elisa for serodiagnosis of sars coronavirus pneumonia glycogen synthase kinase-3 regulates the phosphorylation of sars-coronavirus nucleocapsid protein and viral replication nucleocapsid protein of sars-cov activates the expression of cyclooxygenase-2 by binding directly to regulatory elements for nuclear factor-kappa b and ccaat/enhancer binding protein identification of an actinbinding protein from dictyostelium as elongation factor 1a naturally occurring anti-escherichia coli protein antibodies in the sera of healthy humans cause analytical interference in a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for serodiagnosis of severe acute respiratory syndrome subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein recombinant severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein forms a dimer through its c-terminal domain crystal structure of the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona-and arteriviridae recombinant truncated nucleocapsid protein as antigen in a novel immunoglobulin m capture enzymelinked immunosorbent assay for diagnosis of severe acute respiratory syndrome coronavirus infection severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice the nucleocapsid protein of sars-associated coronavirus inhibits b23 phosphorylation sars-cov nucleocapsid protein induced apoptosis of cos-1 mediated by the mitochondrial pathway immune responses against sars-coronavirus nucleocapsid protein induced by dna vaccine identification and characterization of dominant helper t-cell epitopes in the nucleocapsid protein of severe acute respiratory syndrome coronavirus severe acute respiratory syndrome-associated coronavirus nucleocapsid protein interacts with smad3 and modulates transforming growth factor-beta signaling potent inhibition of sars-associated coronavirus (scov) infection and replication by type i interferons (ifnalpha/ beta) but not by type ii interferon (ifn-gamma) the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha induction of sarsnucleoprotein-specific immune response by use of dna vaccine acknowledgments the authors wish to thank ms. alisha lal for helping out in typing and formatting this review. we apologize to all those colleagues whose work we might have omitted to cite in this article. key: cord-257465-9yrf7ofy authors: finlay, william j. j.; bloom, laird; grant, joanne; franklin, edward; shúilleabháin, deirdre ní; cunningham, orla title: phage display: a powerful technology for the generation of high-specificity affinity reagents from alternative immune sources date: 2016-05-23 journal: protein chromatography doi: 10.1007/978-1-4939-6412-3_6 sha: doc_id: 257465 cord_uid: 9yrf7ofy antibodies are critical reagents in many fundamental biochemical methods such as affinity chromatography, enzyme-linked immunosorbent assays (elisa), flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry techniques. as our understanding of the proteome becomes more complex, demand is rising for rapidly generated antibodies of higher specificity than ever before. it is therefore surprising that few investigators have moved beyond the classical methods of antibody production in their search for new reagents. despite their long-standing efficacy, recombinant antibody generation technologies such as phage display are still largely the tools of biotechnology companies or research groups with a direct interest in protein engineering. in this chapter, we discuss the inherent limitations of classical polyclonal and monoclonal antibody generation and highlight an attractive alternative: generating high-specificity, high-affinity recombinant antibodies from alternative immune sources such as chickens, via phage display. the rapid expansion of the genomics, proteomics , and biotechnology fi elds has led to a growing demand for affi nity reagents that can specifi cally recognize proteins, peptides, carbohydrates, and haptens . affi nity reagents of high specifi city are routinely required for diverse protein drug targets, members of newly discovered biochemical pathways, posttranslationally modifi ed proteins, protein cleavage products, and even small molecules such as drugs of abuse and toxins. individual biomedical researchers will often need to monitor, quantify, and purify proteins of interest via affi nity chromatography , but there may not be any commercially available antibody reagents to allow them to do so [ 1 ] . indeed, even in situations where there are commercially available antibodies, these reagents are often expensive, poorly characterized, and/or simply not appropriate for demanding applications. compounding this problem, the technical diffi culty of monoclonal antibody generation by the untrained researcher and the high cost (~$15,000) of a commercial monoclonal antibody generation program leads many researchers to the default solution of producing polyclonal hyperimmune sera in hosts such as rabbits. the net result of this is that researchers often settle for reagents that lack the necessary specifi city to perform the applications for which they were intended. in this review, we will outline the limitations of classical antibody generation technologies and illustrate an attractive alternative: the use of phage display libraries of recombinant antibodies built on immunoglobulin repertoires from nonmammalian animals. in particular, we will highlight the advantages of libraries derived from the domestic chicken gallus gallus , which offers a relatively inexpensive and technically accessible route to high-quality monoclonal reagents [ 2 ] . if, like many people, you have purchased (or paid to generate) a costly and "specifi c" antibody, but subsequently found that it is actually polyreactive and of dubious quality, phage display from immunized chickens may offer an attractive alternative. hyper-immune sera from rabbits, sheep, or other mammals may be produced in large quantities, but they do not offer the consistency of monoclonal antibodies and need to be regularly replenished and recharacterized. serum antibodies are also polyclonal and frequently polyspecifi c, even when purifi ed over an antigen column, rendering them suboptimal for the specifi c recognition of a single component in a complex matrix. one illuminating study has demonstrated that when used to probe a comprehensive yeast proteome chip, unpurifi ed polyclonal antibody preparations could recognize up to 1770 different proteins, with some monoclonal antibodies and antigen column-purifi ed polyclonal antibodies also recognizing multiple proteins (related and unrelated) [ 3 ] . the arrival of monoclonal antibody technology [ 4 ] was a major step forward in generating high-specifi city reagents, but the reliance on the murine immunoglobulin system frequently leads to a number of practical diffi culties: (1) monoclonal antibodies are raised on the basis of an ineffi cient fusion of splenic b-cells to an immortalized mouse myeloma line, followed by limiting dilution of the cell population. target-specifi c antibodies are randomly identifi ed, often by a simple direct elisa , where few preconditions can be set to determine which antibodies are identifi ed and one must "take what one can get" during the screening process. (2) it is often desirable to have multiple monoclonal antibodies with specifi city for different epitopes on the same target molecule, but the diffi culty in sequencing monoclonals does not allow the rapid identifi cation of unique clones early in the screening process. (3) humans and rodents are relatively closely related phylogenetically. many proteins of interest are highly conserved among mammals and this can frequently lead to thymic tolerance, restricting the antibody response after immunization. (4) when an immune response to a human protein is raised in mice, the large regions of sequence similarity between murine and human proteins may lead to a restricted number of immunogenic epitopes. (5) to generate antibodies that cross-react with orthologues from multiple species of mammal is particularly tricky, as the common epitopes among mammals are the very ones that are unlikely to provoke a strong immunoglobulin response in the mouse. (6) tolerance issues can become even harder to circumvent when the protein of interest is from a mouse or rat. creating "knockout" mice, in which the endogenous copy of the gene for the target protein has been disabled, can often break tolerance, but this is a highly laborious and time-consuming process that few laboratories have the resources to undertake. these factors all hinder the generation of high-quality antibody reagents and thereby limit one's experimental options when developing antibodies for purifying or tracking novel proteins. to bypass the limitations in polyclonal and monoclonal antibody generation, several groups have turned to in vitro display technologies such as phage display, ribosome display, or yeast display libraries to generate recombinant antibodies. the more recent technologies of yeast and ribosome display are becoming highly established, but phage display is currently the most robust, well characterized, and reliable of these methods. antibodies derived from these technologies are cloned in microbial hosts and are therefore monoclonal from the start, with their production being easily scaled up. critically, selection and screening efforts can be directed toward specifi c epitopes or species cross-reactivity and away from polyspecifi c binding. phage display allows a researcher to do in a single tube what would be unfathomable in traditional hybridoma work: interrogate libraries of millions to tens of billions of antibodies on the basis of their binding specifi cally to a target of interest. all of this is possible due to the ingenious concept of "genotypeto-phenotype linkage," which is exemplifi ed by phage display. phage display was originally described as a rapid method for cloning gene fragments that encode a specifi c protein [ 5 ] . by cloning gene fragments into the genome of a fi lamentous e. coli bacteriophage, smith et al. were able to generate libraries of gene fusions with the key phage coat protein p3. after transformation into e. coli , the viral replication system packaged the genome (and therefore the cloned gene) into a highly stable complex carrying the gene product on the tip of the phage particle, as a fusion protein with p3 . by subsequently selecting expressed virions on an immobilized antibody with specifi city for a known protein that had been cloned in the phage genome, they were able to show 1000-fold enrichment of the gene product. this set of experiments showed that "genotypeto-phenotype linkage" could be achieved and thereby defi ned the basis of all display technologies developed since. the subsequent development of a method for the effective cloning of antibody v-gene sequences via pcr allowed the capture of antibody sequences in a recombinant form [ 6 ] , removing the need to immortalize b-cells via hybridoma fusion as required in traditional monoclonal antibody generation. this discovery was combined with the phage display process to make an effi cient method of isolating antibodies and their corresponding gene sequences simultaneously [ 7 ] . in the antibody phage display process, libraries of diverse v-gene sequences are cloned into an appropriate expression vector in e. coli , creating an in-frame fusion with the p3 protein or, as favored in most recently described libraries, a truncated form of p3. the phage display of protein libraries was originally performed using "phage" vectors (i.e., built upon the phage genome itself), but due to practical diffi culties in handling these libraries, more recent phage methods have mostly used so-called phagemid expression vectors. in this case, the dna backbone is a stable, small plasmid such as puc, and the p3 gene plus f1 phage packaging origin are the only phage-derived dna sequences [ 8 ] . these phagemid libraries are more easily handled and more stable than phage libraries, as the plasmid is incapable of causing phage production by itself. the libraries can therefore be more simply cloned, expanded, and controlled than phage libraries. by infecting "helper" phage (based on m13) into a growing culture of e. coli harboring a phagemid library, the phage propagation machinery is provided, but due to a mutation introduced in the origin of replication on the helper phage genome, preferential packaging of the phagemid dna and p3 fusion occurs during phage replication [ 8 ] . phage production is thereby induced, genotype-to-phenotype linkage is created, and the expressed phage particles are interrogated for the presence of useful protein sequences via target binding. this selective step may be performed by simply immobilizing the target protein on, for example, a protein-binding plastic surface such as an elisa plate, adding phage and allowing binding to occur via the antibody-p3 fusion proteins. nonbinding phages are removed by washing the immobilized surface, and the remaining bound phages are eluted. the eluted phages are then reinfected back into a fresh culture of e. coli to retrieve the selected gene sequences. this process is not perfect, however, and two to four rounds of selection/re-expression/selection are normally performed iteratively to remove all unwanted clones and enrich the binding population from the background of the library. nonetheless, this process can be spectacularly powerful, massively enriching specifi c antibodies in a single selection round [ 9 ] . furthermore, the use of multiple forms of the target antigen in sequential selection rounds and the inclusion of competitor proteins can drive the selected pool toward a highly specifi c set of epitopes. much of the evolution and progression of phage display technology has been driven by the remarkable discovery that this method can be used to mine large libraries of combinatorial human antibody diversity, theoretically removing the need for animals in antibody production [ 10 ] . by random recombination of v h and v l sequences from human lymphocyte cdna [ 10 ] , or, by using degenerate oligonucleotides to create diversity of dna sequence in the loops associated with target binding [ 11 ] , several groups have created very large libraries of antibody gene sequences for phage display. these libraries are analogous to the naïve antibody repertoire in an animal, and selecting from them can result in the identifi cation of antibody fragments that exhibit high specifi city and occasionally high affi nity for the target protein. several major studies have proven that with appropriate application of this technology, specifi c antibodies can be raised to proteins, peptides, haptens and even carbohydrates. in the most exemplary studies, antibodies have been raised with equivalent affi nities to those associated with a strong humoral immune response [ 12 -15 ] . additionally, antibodies with suboptimal affi nities can provide useful starting molecules for the construction of mutagenized libraries which can be further screened for affi nity matured variants [ 16 -18 ] . in addition to the affi nity maturation process, phage display also provides a useful tool for the production of ultra-humanized antibodies from nonhuman sources. it is widely accepted that there are intrinsic diffi culties associated with the humanization of nonhuman antibodies using cdr grafting. frequently, such grafted antibodies suffer from immunogenicity associated with high non-germ-line amino acid content, the propensity for v-domain destabilization, and very often downstream expression and formulation issues. in a recent study, ultra-humanized antibodies from three nonhuman sources were generated via singlestep complementarity-determining region (cdr) germ-lining in a process termed " augmented binary substitution" (abs) [ 19 ] . this process utilizes phage display for selection from germline targeting combinatorial libraries which results in the identifi cation of cdr residues amenable to human germ-lining without compromising on specifi city and affi nity. a further degree of complexity is incorporated into the library through the random substitution of cdr-h3 residues in tandem with the germ-lining process. this simple single pass process generates signifi cantly lower non-germline sequence content, on cdr grafts from nonhuman hosts, rendering them virtually indistinguishable from fully human antibodies. this process opens up new possibilities for the affi nity maturation and humanization of antibodies from alternative sources such as chickens. recombinant antibodies are typically displayed and expressed in "fragment" forms. the simplest and most commonly used fragment is the single-chain fragment variable (scfv) where a fl exible peptide sequence links the v-regions of antibodies between the c-terminus of one domain and the n-terminus of the other, thereby combining both v-domains into a single polypeptide [ 20 ] . the scfv may be assembled in v l -v h or v h -v l orientations, with v h -v l being the most heavily used format historically. the fl exible linker helps to make the scfv simple to express, but must be sufficiently long and fl exible to allow effective association of the v-regions to form a functional antigen-combining site. as long as this is true, the classical hydrophobic pairing of the v-regions will stabilize the structure. by far, the most common linkers are based on glycine-serine repeat structures such as ggggs × 3. the second most commonly used recombinant antibody format is the fab (fragment antigen binding) molecule. this structure is a complete binding "arm" of an antibody and is comprised of the full immunoglobulin light chain, expressed in conjunction with the v h -c h 1 region from the heavy chain [ 21 ] . fabs obligately form predominantly monomeric, monovalent fragments. they are the most "natural" of the recombinant antibody fragments and it has been shown that the presence of the constant regions can often help to stabilize antibody variable regions [ 22 ] . the fab format is the less commonly used of the two main recombinant antibody formats, however, as its dual polypeptide structure is generally more diffi cult to express and display in e. coli than the scfv [ 22 ] . a recent engineered scfab platform however attempts to address some of the limitations associated with scfab expression [ 23 ] . much of the under-use of phage display may be due to experiences with the early libraries derived from naïve or synthetic human antibody diversity, which were donated to academic laboratories that were not specifi cally invested in antibody engineering. unfortunately, these forays into display technologies have often left investigators somewhat disappointed. many people have accepted the viewpoints of recombinant antibody technology experts, that these libraries can yield useful high-affi nity antibodies to any form of antigen . in general, (mostly to those highly skilled in the fi eld), this is indeed true, but the average antibody generated from these libraries is often of disappointingly low affi nity to those who are used to high-sensitivity antibodies from immunized sources. human recombinant antibodies from naïve library sources can require technically challenging in vitro molecular evolution if they are to perform the demanding "real world" functions required of many reagent antibodies [ 9 ] . molecular evolution is far from trivial to perform and is usually beyond both the scope and interest level of the average researcher. for such reasons, it is of little wonder then that most people either ignore, or at worst disparage, phage display technology itself. nevertheless, phage display can be a relatively simple technology to use and when employed to harness natural repertoires of antibodies from immunized animals, it can offer a rapid path to highly specifi c, high-affi nity antibodies against problematic antigens . while the most successful naïve antibody libraries contain over 10 10 members and are often the domain of biotechnology companies, typical immune libraries are in the 10 7 -10 8 range and are easily assembled by a single investigator [ 24 , 25 ] . when an immunized rabbit or sheep has raised a signifi cant serum immunoglobulin titer, the common end-point to the experiment is to exsanguinate the animal and harvest the serum. however, harvesting b-cell rich lymphoid tissues from the animal, such as the spleen and bone marrow, allows the isolation of total rna and the subsequent generation of cdna [ 24 ] . this is a simple method with which many biomedical researchers are familiar, and commercial kits are available to simplify most steps of the process. the immunoglobulin gene sequences of many animals are now known and the cdna from immune tissues can subsequently be used for the rt-pcr amplifi cation and cloning of the animal's variable region sequence repertoire [ 24 ] . these cloned variable region sequences can then be assembled into a display library format such as scfv or chimeric fab (using human c h 1 and c k/λ regions) [ 26 ] . these targeted immune libraries thereby offer a potentially huge advantage over monoclonal antibodies , as libraries of >10 8 variants may be built, allowing the effective sampling of a much broader range of antibodies than the hundreds (occasionally thousands) of clones usually examined in a monoclonal antibody screen. the resulting library can be interrogated for specifi c binding proteins via phage display and the retrieved antibody fragments expressed very simply in bacteria [ 26 ] . this process has been used to successfully harness the antibody repertoires of a large number of immune host species, including mice [ 24 ] , rabbits [ 25 ] , sheep [ 27 ] , camelids [ 28 ], and sharks [ 29 ] . of greater interest to us, however, is to exploit this approach to harvest the novel immunoglobulin repertoires of the domestic chicken ( gallus gallus ), which is as simple to use as mice and rabbits, but also highly phylogenetically distant from mammals. avians can circumvent many of the common problems encountered with mammalian immunizations described above. as a fully domesticated small animal, chickens are an attractive host for immunization as they are highly accessible, very affordable, and easily housed in a generic animal house. most importantly, however, the amino acid homology between the mammalian and avian orthologues of a given protein is typically lower than between the mammals commonly used for antibody generation, and indeed, some mammalian proteins may not even exist in avians. the immunoglobulin response of chickens to highly conserved mammalian proteins is reliably robust, generally exhibits high avidity, and potentially targets a broad spectrum of epitopes on protein immunogens [ 30 -32 ]. chickens therefore have a potentially major advantage over other common immune hosts: they can produce a high-affi nity cross-reactive antibody response targeting an epitope that is conserved across multiple orthologues of a mammalian protein. this can lead to signifi cant savings in time and resources as, if a single, broadly applicable cloned reagent can be identifi ed, it can then be used to generate a single affi nity column for the capture of the target protein from multiple species. chicken immunoglobulins have also shown benefi cial biophysical properties: they exhibit high stability to changes in ph and temperatures up to 70 °c [ 33 , 34 ] , provide functional coating on latex microspheres [ 35 ] , and demonstrate functional direct covalent coupling to a dextran layer for the detection of serum proteins by surface plasmon resonance [ 36 ] . furthermore, as chickens are small animals, very little protein immunogen is required to raise a strong immunoglobulin response. approximately 200 μg/bird of purifi ed protein is suffi cient to carry out a full immunization regime [ 37 , 38 ] . these observations have led to the regular use of chickens as an immune host for production of the polyclonal antibody termed igy (egg yolk antibody), in both research and commercial settings. laying hens will export signifi cant quantities of polyclonal igy into the egg (~100 mg of igy per yolk), in a process analogous to mammalian placental igg transfer, which allows direct screening of their antibody response without the need for serum sampling [ 38 ] . once a strong immune response has been raised, large quantities of polyclonal antibody are easily prepared from the yolk. these polyclonal antibodies have been successfully applied in research immunochemistry [ 39 ], diagnostics [ 40 ] , and affi nity column purifi cation [ 41 ] . indeed, immunodepletion resins based on chicken igy can be used to remove high abundance proteins from serum and are now commercially available (sigma-aldrich seppro ® igy14 spin columns). unfortunately, polyclonal igy does still suffer from the same issues of ill-defi ned specifi city that all polyclonal antibody preparations do. in addition, there have been several studies describing successful chicken hybridoma monoclonal antibody generation to antigens such as human peptides [ 42 ], sporozoite proteins [ 43 ], and prion protein [ 44 ], but the low antibody expression and instability associated with chicken myeloma cell lines [ 42 , 45 ] led to the under-use of this species as a source of monoclonal antibodies. today however, the progress in chicken antibody phage display has circumvented these problems and made recombinant chicken antibody reagents readily accessible, as we describe below. the chicken immunoglobulin repertoire is almost ideally suited to antibody phage display, as chickens generate their immunoglobulin repertoire from a single set of v h and v l germ line sequences [ 46 ] , with the majority of positions in the framework regions maintained as germline [ 2 ] . diversity in the v-regions is created by both v-d-j recombination and somatic hypermutation, with the additional infl uence of "gene conversion," where multiple upstream pseudogenes are recombined into the functional sequence. this germline v-gene system means that the entire chicken antibody repertoire can be captured using only four pcr primers [ 47 ], making chicken libraries highly representative of the induced immunoglobulin response. this is in direct contrast with immune hosts such as mice, which have diverse germline v-gene sequences and therefore require complex mixes of pcr primers [ 24 ] . additionally, the two v-gene germline sequences found in chickens are highly homologous to the human v λ and v h 3 germline families [ 48 ] , which are both associated with creating v-domains with high stability and solubility. indeed, chicken scfvs can be stable in crude bacterial culture supernatants for up to 1 month at room temperature [ 49 ] . the initial work of davies et al. [ 47 ] showed that a simple recombinant chicken antibody library could be displayed on phage. while this small library was nonimmune and derived from the bursa cells of a single young chicken, the group was able to select target-specifi c scfv sequences recognizing lysozyme , serum albumin and thyroglobulin. the potential of chicken recombinant antibodies was further highlighted by a study [ 50 ] which used an scfv library derived from the spleens of immunized chickens and successfully generated highly specifi c scfv antibodies that targeted both mouse and rat serum albumins, where tolerance issues limit the ability to generate murine monoclonal antibodies . in addition, an in-depth analysis of the avian immunoglobulin v h repertoire demonstrated that naïve repertoire features were fully replicated in the resulting target-selected, phage display repertoire [ 2 ] . a major study [ 26 ] subsequently demonstrated that chickens could be a useful source of scfv and chimeric fab antibodies with specifi city for hapten molecules. however, none of these early studies characterized the antibodies for their affi nity, or their function as practical reagents. more recent studies have shown that scfv antibodies derived from immunized chickens are highly effective reagents in diverse settings such as diagnostic elisa for infectious bursal disease virus [ 51 ], the diagnosis of prion disease [ 52 ], immunodetection of haptenic shellfi sh toxins [ 53 ], immunostaining of sars-infected cells [ 54 ], biosensing of cardiac biomarkers [ 55 ] , and the measurement of apob protein in mouse and human sera [ 56 ] . raats et al. [ 57 ] have also illustrated that antiidiotype scfvs from an immune chicken scfv library were of considerably higher sensitivity than those derived from a human antibody library in the same study. the generation of highly selective scfvs toward the prp protein, which is highly conserved in mammals demonstrates the advantage of the chicken as an immune model, as isolated scfvs were shown to react with murine, ovine, and bovine orthologues of the protein [ 52 ] . the cloning of chicken antibodies via phage display has also allowed the precise dissection of the specifi city and affi nity of the chicken immunoglobulin response. high-throughput affi nity measurements for panels of chicken scfvs to the infl ammatory biomarker c-reactive protein have identifi ed clones that preferentially recognize the multimeric and monomeric forms of the protein [ 50 ]. in the same study, clones with affi nities as high as 350 pm were generated from an immune phage display library of only 3 × 10 7 total clones. in addition, chicken anti-prp scfv have been reported to have affi nities up to 15 pm, making them among the highest affi nity scfvs reported to date [ 58 ] . what may be of particular practical interest to many researchers is that chickens can serve as a host for simultaneous immunization with multiple proteins of interest, with as many as eight proteins being used successfully in a single immunization scheme [ 37 , 49 , 59 ] . the target proteins of interest are mixed in a single adjuvant preparation and each immunized animal receives all multiplexed proteins simultaneously. spleen and bone marrow tissues from the immunized animals are then used to generate relatively small phage display libraries and specifi c antibodies are derived via selection of the library separately on the individual proteins originally used for immunization [ 37 ] . the immunized chickens appear to react to the proteins fully independently, as the phage display libraries generate individual scfv antibody clones that are fully specifi c by western blot and elisa , showing no reactivity to their co-immunogens [ 37 , 59 ] . this approach has major benefi ts practically and ethically, as it allows the use of a single library to derive high-affi nity antibodies to a group of proteins of interest. multi-immunization methods also simultaneously minimize animal use and raise the likelihood of success in generating an immediately useful reagent [ 37 , 59 ] . multi-target immunization regimes should be designed with one of two objectives in mind. firstly, the simplest scenario combines multiple unrelated proteins, which leads to unrelated b-cell responses after immunization. to derive antibodies of greatest specifi city during a multi-target immunization of this kind, it is important to ensure that each of the protein immunogens is highly purifi ed and that no closely related proteins are co-immunized into a single animal. secondly, to derive antibodies that are cross-reactive to orthologues of a conserved protein from multiple species, it is likely to be benefi cial, but not necessarily essential, to include each orthologue in the mix of immunogens given to each animal. iterative selection rounds that change orthologue each time can then be used to bias toward the isolation of cross-reactive antibodies. the generation and selection of chicken recombinant antibodies is extremely reliable using the methods described in detail in the accompanying chapter. the subsequent identifi cation and sequencing of antibodies displaying the characteristics desired can also be performed simply. in general, scfvs isolated from immunized chicken libraries exhibit high affi nity and can be assayed via a direct elisa , using crude periplasmic extracts from the protein expressing e. coli clones. the level of further downstream analysis carried out on positive hits identifi ed during the binding elisa depends on what the end user requires. specifi c binding function may suffi ce for scfvs that are to be used simply as reagent antibodies for in vitro analysis of samples via elisa or western blotting . for antibodies to be used in affi nity chromatography , the antibody fragments must be purifi ed and tested for their function after being coupled to a solid matrix and for their specifi city during purifi cation. few studies have been performed using antibody fragments in affi nity chromatography, but several potential approaches have been described. for the antibody binding site(s) to be fully solventexposed and active, the antibody fragment must be directionally captured onto the solid matrix. even for full-length igg , nonspecifi c adsorption or covalent coupling onto solid supports can lead to denaturation, reducing or negating antigen-binding function [ 60 ] . antibody fragments may be slightly more prone to chemical or physical denaturation than full-length immunoglobulins , but scfv and fab have been used successfully in affi nity chromatography, and in the creation of spr sensing surfaces which can go through serial rounds of binding and regeneration [ 61 ] . mcelhinney et al. [ 62 ] created a simple scfv-based affi nity column for the concentration and cleanup of microcystin toxins from environmental samples, by transiently coupling the his-tagged scfv to a disposable nickel chelate column. the scfv was thereby coupled directionally, maximizing the functional antibody content on the column, and the analyte for purifi cation was co-eluted with the scfv before quantifi cation by reverse-phase hplc. however, this method can only be used under a limited number of conditions, as the interaction of the his-tag with nickel is noncovalent and ph dependent. other possibly useful low affi nity expression tags include e. coli maltose binding protein and glutathione s -transferase , which have both been successful in protein purifi cation [ 63 , 64 ] ( see also chapter 8 for a discussion on protein tagging ). high affi nity, highly stable linkage via affi nity tagging may also be achieved by site-specifi c biotinylation of antibody fragments and their immobilization onto a matrix that has been passively or covalently coated with avidin. bacterial expression vectors are now available which introduce biotin into specifi c peptide tags ( avitag ), which can be produced on the termini of recombinant proteins. a similar method was proven to be effi cient in the production of fabs that are specifi cally biotinylated in vivo during bacterial expression, via c-terminal fusion of the fabs to the e. coli acetyl-coa carboxylase [ 65 ] . importantly, these biotinylated fabs were successfully used to purify recombinant tnf-alpha from bacterial lysates, via a streptavidinated column. the peptide tagging method has also been used successfully to label both fab and scfv antibodies for their oriented immobilization and use as capture antibodies in clinical diagnostic elisas [ 66 , 67 ] . these studies suggest that biotinstreptavidin coupling is a simple and rapid method for the stable, directional capture of recombinant antibody fragments. while the covalent coupling of recombinant antibody fragments via their reactive lysine side chains is likely to be disruptive to their function, some alternative covalent coupling methods have been identifi ed. in the simplest example, the disulfi de bonds linking the two constant regions of a fab can be reduced using a mild agent to expose cysteine thiols. these thiol groups can then be used to covalently couple the fragment to a thiol-activated surface [ 68 ] . more elegant versions of this approach have expressed antibody fragments with a c-terminal cysteine group, then gently applied the same chemistry, to preferentially reduce the exposed disulphide groups [ 61 ] . the exposed terminal thiols are again an effi cient reactive group for covalent attachment. it is also possible to express scfv fused to the constant regions of human igg light chains as another source of usable cysteine residues external to the v-regions [ 69 ] . whether any of the above attachment methods are appropriate for a given affi nity purifi cation application may be decided upon by the individual investigator. in cases where stable linkage has been achieved and the column is to be reused, it is prudent for the investigator to examine multiple clones for their stability under repeated cycles of elution and regeneration. while chicken scfvs are built upon naturally stable frameworks, the stability of different clones cannot be taken for granted. in cases where stability remains an issue, the appropriate chicken v-regions can be cloned into an fc-fusion [ 70 ] or igg [ 71 ] expression vector to produce full-length antibody in mammalian, yeast, and even plant culture systems [ 72 ] . in conclusion, this chapter demonstrates that phage display is a powerful technology that can be used to generate highly specifi c reagents from immune sources. it is an attractive alternative to classical antibody generation technologies that can be employed in many fi elds for reliable antibody and antibody fragment generation . antibody microarrays: promises and problems fundamental characteristics of the immunoglobulin vh repertoire of chickens in comparison with those of humans, mice, and camelids analyzing antibody specifi city with whole proteome microarrays continuous cultures of fused cells secreting antibody of predefi ned specifi city filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface cloning immunoglobulin variable domains for expression by the polymerase chain reaction phage antibodies: fi lamentous phage displaying antibody variable domains multi-subunit proteins on the surface of fi lamentous phage: methodologies for displaying antibody (fab) heavy and light chains selecting and screening recombinant antibody libraries human antibodies from v-gene libraries displayed on phage semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem human antibodies with sub-nanomolar affi nities isolated from a large non-immunized phage display library fully synthetic human combinatorial antibody libraries (hucal) based on modular consensus frameworks and cdrs randomized with trinucleotides generation of high-affi nity human antibodies by combining donor-derived and synthetic complementarity-determiningregion diversity high-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries picomolar affi nity antibodies from a fully synthetic naive library selected and evolved by ribosome display affi nity maturation of a humanized rat antibody for anti-rage therapy: comprehensive mutagenesis reveals a high level of mutational plasticity both inside and outside the complementarity-determining regions in vitro selection and affi nity maturation of antibodies from a naive combinatorial immunoglobulin library augmented binary substitution: single-pass cdr germ-lining and stabilization of therapeutic antibodies protein engineering of antibody binding sites: recovery of specifi c activity in an anti-digoxin single-chain fv analogue produced in escherichia coli human monoclonal fab fragments derived from a combinatorial library bind to respiratory syncytial virus f glycoprotein and neutralize infectivity domain interactions in the fab fragment: a comparative evaluation of the singlechain fv and fab format engineered with variable domains of different stability an improved single-chain fab platform for effi cient display and recombinant expression reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system high affi nity scfvs from a single rabbit immunized with multiple haptens methods for the generation of chicken monoclonal antibody fragments by phage display the isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep high throughput ranking of recombinant avian scfv antibody fragments from crude lysates using the biacore a100 determination of lox-1-ligand activity in mouse plasma with a chicken monoclonal antibody for apob generating recombinant anti-idiotypic antibodies for the detection of haptens in solution humanization of chicken monoclonal antibody using phagedisplay system multipleantigen immunization of chickens facilitates the generation of recombinant antibodies to autoantigens profi ling receptor tyrosine kinase activation by using ab microarrays oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-lr by phage display and its use in the immunoaffi nity concentration of microcystins from water advances in recombinant antibody microarrays comparison of affi nity tags for protein purifi cation in vivo biotinylated recombinant antibodies: construction, characterization, and application of a bifunctional fab-bccp fusion protein produced in escherichia coli in vitro enzymatic biotinylation of recombinant fab fragments through a peptide acceptor tail use of an in vivo biotinylated single-chain antibody as capture reagent in an immunometric assay to decrease the incidence of interference from heterophilic antibodies sitedirected immobilisation of antibody fragments for detection of c-reactive protein recombinant antibody expression vectors enabling double and triple immunostaining of tissue culture cells using monoclonal antibodies production of anti-prion scfv-fc fusion proteins by recombinant animal cells whole igg surface display on mammalian cells: application to isolation of neutralizing chicken monoclonal anti-il-12 antibodies plant expression of chicken secretory antibodies derived from combinatorial libraries key: cord-003020-q69f57el authors: farhadi, tayebeh; hashemian, seyed mohammadreza title: computer-aided design of amino acid-based therapeutics: a review date: 2018-05-14 journal: drug des devel ther doi: 10.2147/dddt.s159767 sha: doc_id: 3020 cord_uid: q69f57el during the last two decades, the pharmaceutical industry has progressed from detecting small molecules to designing biologic-based therapeutics. amino acid-based drugs are a group of biologic-based therapeutics that can effectively combat the diseases caused by drug resistance or molecular deficiency. computational techniques play a key role to design and develop the amino acid-based therapeutics such as proteins, peptides and peptidomimetics. in this study, it was attempted to discuss the various elements for computational design of amino acid-based therapeutics. protein design seeks to identify the properties of amino acid sequences that fold to predetermined structures with desirable structural and functional characteristics. peptide drugs occupy a middle space between proteins and small molecules and it is hoped that they can target “undruggable” intracellular protein–protein interactions. peptidomimetics, the compounds that mimic the biologic characteristics of peptides, present refined pharmacokinetic properties compared to the original peptides. here, the elaborated techniques that are developed to characterize the amino acid sequences consistent with a specific structure and allow protein design are discussed. moreover, the key principles and recent advances in currently introduced computational techniques for rational peptide design are spotlighted. the most advanced computational techniques developed to design novel peptidomimetics are also summarized. different diseases may be caused by pathogens or malfunctioning organs, and using therapeutic agents to heal them has an old recorded history. small molecules are conventional therapeutic candidates that can be easily synthesized and administered. however, many of these small molecules are not specific to their targets and may lead to side effects. 1 moreover, a number of diseases are caused due to deficiency in a specific protein or enzyme. thus, they can be treated using biologically based therapies that are able to recognize a specific target within crowded cells. 2 under the biologic conditions, some macromolecules such as proteins and peptides are optimized to recognize specific targets. 3 therefore, they can override the shortcomings of small molecules. 3 recently, pharmaceutical scientists have shown interest in engineering amino acid-based therapeutics such as proteins, peptides and peptidomimetics. [4] [5] [6] theoretical and experimental techniques can predict the structure and folding of amino acid sequences and provide an insight into how structure and function are encoded in the sequence. such predictions may be valuable to interpret genomic information and many life processes. moreover, engineering of novel proteins or redesigning the existing proteins has opened the ways to achieve novel biologic macromolecules with desirable therapeutic functions. 7 protein sequences comprise tens to thousands of amino acids. besides, the backbone and side chain degrees of freedom lead to a large number of configurations for a single amino acid sequence. protein design techniques give minimal frustration through precise identification of sequences and their characteristics. [8] [9] [10] [11] considering energy landscape theory, the adequately minimal frustration in natural proteins occurs when their native state is adequately low in energy. 7 the de novo design of a sequence is difficult because there are huge numbers of possible sequences: 20 n for n-residue proteins with only 20 natural amino acids. 12 peptide design should incorporate computational approaches. it can benefit from searching the more advanced fields used for small molecules and protein design. 13 however, the straightforward adoption of computational approaches employed to small-molecule and protein design has not be accepted as a reasonable solution to the peptide design problem. [14] [15] [16] in the peptide drug design, the conformational space accessible to peptides challenges the small-molecule computational approaches. besides, the necessity for nonstandard amino acids and various cyclization chemistries challenges the available tools for protein modeling. 13 furthermore, the aggregation of peptide drugs during production or storage can be an unavoidable problem in the peptide design procedure. rational design of a peptide ligand is also challenging because of the elusive affinity and intrinsic flexibility of peptides. 17 peptide-focused in silico methods have been increasingly developed to make testable predictions and refine design hypotheses. consequently, the peptide-focused approaches decrease the chemical spaces of theoretical peptides to more acceptable focused "drug-like" spaces and reduce the problems associated with aggregation and flexibility. 13, 18 for the discussions that follow, peptides can be defined as relatively small (2-30 residues) polymers of amino acids. 18 in physiological conditions, several problems such as degradation by specific or nonspecific peptidases may limit the clinical application of natural peptides. 19 moreover, the promiscuity of peptides for their receptors emerges from high degrees of conformational flexibility that can cause undesirable side effects. 20 besides, some properties of therapeutic peptides, such as high molecular mass and low chemical stability, can result in a weak pharmacokinetic profile. therefore, peptidomimetic design can be a valuable solution to circumvent some of undesirable properties of therapeutic peptides. 21, 22 in the biologic environment, peptidomimetics can mimic the biologic activity of parent peptides with the advantages of improving both pharmacokinetic and pharmacodynamic properties including bioavailability, selectivity, efficacy and stability. a wide range of peptidomimetics have been introduced, such as those isolated as natural products, 23 synthesized from novel scaffolds, 24 designed based on x-ray crystallographic data 25 and predicted to mimic the biologic manner of natural peptides. 26 using hierarchical strategies, it is possible to change a peptide into mimic derivatives with lower undesirable properties of the origin peptide. 27 over the past 10 years, computational methods have been developed to discover peptidomimetics. 28 in a part of this review, novel computational methods introduced for peptidomimetic design have been summarized. peptidomimetics can be categorized as follows: peptide backbone mimetics (type 1), functional mimetics (type 2) and topographical mimetics (type 3). 29 the first generation of peptidomimetics (type 1) mimics the local topography of amide bond. it includes amide bond isosteres, 30 pyrrolinones 31 or short fragments of secondary structure, such as beta-turns. 32 such mimetics generally match the peptide backbone atom-for-atom, and comprise chemical groups that also mimic the functionality of the natural side chains of amino acids. a number of prosperous instances of type 1 peptidomimetics have been reported. 33 the second type of peptidomimetics is described as functional mimetics or type 2 mimetics, which include small, non-peptide compounds that are able to identify the biologic targets of their parent peptide. 34 at first, they were assumed to be conservative structural analogs of parent peptides. however, using site-directed mutagenesis, their binding sites to biologic targets were investigated. the results indicated that type 2 peptidomimetics routinely bind to protein sites that are different from those selected by the original peptide. 35 therefore, type 2 mimetics maintain the ability to interfere with the peptide-protein interaction process without the necessity to mimic the structure of the natural peptide. 28 type 3 peptidomimetics reveal the best conception of peptidomimetics. they consist of the necessary chemical groups that act as topographical mimetics and contain novel chemical scaffolds that are unrelated to natural peptides. 36 here, theoretical and computational techniques to design proteins, peptides and peptidomimetics are reviewed. however, the current review does not deeply highlight the computational aspects of amino acid-based therapeutic design, but only discusses the methods used to design the mentioned therapeutics. figure 1 summarizes the key concepts presented in this study. as some examples, the structures of aldesleukin, leuprolide and spaglumic acid, important amino acid-based therapeutics approved by the us food and drug administration (fda), are shown in figure 2a computer-aided design of amino acid-based therapeutics figure 2a ) and leuprolide (pdb id: 1yy2; figure 2b ) were obtained from the protein data bank (pdb; http://www. rcsb.org/) and visualized by pymol tool. the structure of spaglumic acid was retrieved (in mol format) from pub-chem database (https://pubchem.ncbi.nlm.nih.gov/) with the pubchem id 188803 ( figure 2c ) and visualized using pymol. aldesleukin, a lymphokine, is a recombinant protein used to treat adults with metastatic renal cell carcinoma (https://www.drugbank.ca/drugs/db00041). leuprolide, a synthetic nine-residue peptide analog of gonadotropin releasing hormone, is used to treat advanced prostate cancer (https://www.drugbank.ca/drugs/db00007). spaglumic acid is used in allergic conditions such as allergic conjunctivitis. the drug belongs to a class of peptidomimetics known as hybrid peptides. hybrid peptides contain at least two dissimilar types of amino acids (alpha, beta, gamma or delta) linked to each other via a peptide bond (https://www.drugbank.ca/ drugs/db08835). in the current study, all fda-approved therapeutics (in 2018) were retrieved from drugbank (https://www.drugbank. ca/biotech_drugs) and an analysis was conducted to compare their percentages. protein-based therapies, gene or nucleic acid-based therapies, vaccines, allergenics and cell transplant therapies made up 8.05%, 0.17%, 2.64%, 16.20% and 0.14% of total approved therapeutics, respectively. small-molecule drugs made up 72.76% of the approved therapeutics ( figure 3 ). computational designing of proteins can be classified as follows: 1) template-based designing in which three-dimensional (3d) farhadi and hashemian structure of a predefined template is adapted to design a sequence and 2) de novo designing in which the amino acids' arrangement is changed to generate both sequence and 3d structure of a completely novel protein. 3 the problem of predicting the fold of an unknown sequence could be solved by utilizing templates. since the fold is unaltered, the backbone atoms are directly located on this framework. 3 moreover, to generate a functional protein, the side chains that can effectively stabilize the structure are added to the backbone. 37, 38 routine concerns and methods for template-based protein design are reviewed below. selecting the template (scaffold) protein the template (also named as scaffold protein) contains a group of backbone atom coordinates. the coordinates can be retrieved from an available x-ray crystal structure or cautiously from a nuclear magnetic resonance (nmr) structure. 39 computer-aided design of amino acid-based therapeutics fixing the backbone decreases the computational complication, but it may inhibit the main chain modifications to adjust sequence alternation. 7 backbone flexibility can generate designed functionalities over the protein's normal function. the backbone flexibility is introduced through incorporating other closely associated conformations to an existing structure. [40] [41] [42] recently, new functionalities were effectively introduced into the tim-barrel topology. 43 this fold has been detected as one of the most shared structures in 21 distinct protein superfamilies. 44 sequence search and characterization in a design procedure, a protein sequence is selected such that it meets the energetic and geometric constraints established by the chosen fold. sequence search techniques sample different sequences and estimate their energies to gain the one owing the minimum energy. 3 in order to identify the sequences subject to an objective function or a specific energy, a diverse strategies including optimization and probabilistic approaches have been developed. 45 optimization processes may recognize candidate sequences using stochastic or deterministic methods. 45 probabilistic approaches focus on characterizing the sequence space probabilistically. deterministic methods: to achieve a sequence folded into a global minimum energy conformation, deterministic methods search the whole sequence space and identify the global optima. 3, 7 these methods include dead-end elimination (dee), 46 self-consistent mean field, 47 graph decomposition and linear programming. 48 stochastic algorithms search the sequence space in an exploratory manner. 3 these algorithms include monte carlo algorithms (simulated annealing), 49 graph search methods 50 and genetic algorithms. 51 some of the most commonly used methods are discussed below. dee has been considered as a thorough search algorithm. to find and remove sequence-rotameric positions that are not portions of the global minimum energy conformation, dee compares two amino acid rotamers and removes the one with greater interaction energy. 52 interaction energies are computed for each rotamer of the test amino acid, along with all rotamers of every other amino acid. 3 the situation is repetitively examined for total amino acid states as well as their rotamers until it no longer holds true. 52, 53 expanding the sequence length increases the combinatorial complication of dee exponentially. therefore, to design sequences of 30 amino acids or larger, application of dee may be restricted. 54 details of the theorem are explained elsewhere. 3, 7 stochastic search algorithms: as mentioned before, deterministic approaches are perfect to design proteins with small sizes, but show the applied disadvantages with extension of sequence size. stochastic or heuristic methods are valuable to design large proteins. 3 the most widely used method for protein design includes monte carlo sampling. 3, 7 monte carlo method samples positions of complicated proteins in a way related to a selected probability distribution such as boltzmann distribution. boltzmann distribution specially weighs low-energy configurations. the monte carlo algorithm performs iterative series of calculations. at the primary step of each search, a partially accidental test sequence is generated, and its energy is calculated via a physical potential. during the primary step, both rotamer state and amino acid identity are adjusted and an efficient temperature controls the probable energy alterations. in the next step, named simulated annealing, the temperature gradually decreases and permits favorable sampling of lowerenergy configurations. 55 multiple independent calculations are carried out to converge the system to a global minimum. 3, 7 for more explanation about the theorems and details of the formulation of the probability distribution and weights, readers are referred to study previous reports. 3, 7 probabilistic approach: probabilistic approaches are frequently employed when thorough information is not accessible for protein design. in a probabilistic approach, sitespecific amino acid probabilities may be utilized, rather than particular sequences. the procedure is partially motivated by the uncertainties to find sequences consistent with a specific structure. briefly, the backbone atoms are fixed or greatly constrained, side chain conformations are discretely handled, energy functions are estimated and solvation is handled by simple models. 7 however, in order to offer valuable sequence information for design experiments and to find structurally significant amino acids, probabilistic techniques leverage structural characteristics of interatomic interactions. 7 generally, monte carlo methods give a probabilistic sampling of sequences. 49, 55 in addition, an entropy-based formalism has been defined to predict amino acid probabilities for a certain backbone structure. 56, 57 the method employs concepts from statistical thermodynamics to assess the sitespecific probabilities. to address the whole space of existing compositions, the theory is not restricted by the computational enumeration and sampling. large protein structures with .100 variable residues can be supplied simply. 7 sampling sequence space to generate conformations the chemical variability of a sequence and the number of various amino acids permitted at each position are defined as "degrees of freedom for each amino acid". moreover, each of the 20 natural residues search the whole sequence space. 58 drug design, development and therapy 2018:12 submit your manuscript | www.dovepress.com to decrease the degrees of freedom for each amino acid and searching the sequence space, diverse approaches such as hydrophobic patterning have been proposed. 58 monomers can be used to probe a protein structure 59 and improve its function, 60 other than the naturally occurring amino acids. 61 sampling of side chain conformational space to form conformations side chain conformations are typically consistent with the energy minima of molecular potentials and can be obtained from a structural database. 62 rotamer statuses are related to the repeatedly detected values of dihedral angles in the side chain of each amino acid. for example, the simplest amino acids including alanine and glycine have only one rotamer status, while the bigger amino acids have .80 diverse rotamer statuses. 62 a variety of rotamer libraries including backbonedependent, secondary structure-dependent and backboneindependent libraries have been developed for protein design. 62, 63 by using a rotamer library, one can discretize a meaningful state space to decrease the computational difficulty. rotamer libraries can be extended beyond the 20 natural amino acids. the effective rotamers can model cofactors, ligands, water and posttranslational modifications. for example, to improve the modeling of protein-protein interactions and model water within proteins interiors, the structurally definite water molecules can be inserted as a solvated rotamer library. 61 energy functions have been employed to quantify sequencestructure compatibilities. 64 they include linear associations of hydrogen bonds made by backbone atoms, repulsion among atoms, hydrophobic attraction among non-polar groups and electrostatic interactions among sequential neighbors. 65 the sequence of a protein is selected so that it can adjust the energetic and geometric constraints enforced by the favorite fold. constraints typically contain several intramolecular interactions such as van der waals, hydrophobic, polar and electrostatic interactions, as well as hydrogen bonds. generally, by using a scoring function, it is possible that energetic contributions of the mentioned parameters are taken into account. 3, 7, 65 de novo design: designing the sequence and 3d structure through assembly of proteins fragments 66, 67 or secondarystructure elements, 68,69 novel structures can be modeled de novo. in the design procedures, the backbone coordinates are generally constrained. summary and important findings of some proteins designed using computational approach including a retroaldol enzyme, 43 the kemp elimination enzyme, 70 a novel βαβ protein, 71 a redesigned procarboxypeptidase, 72 a novel α/β protein structure and the top7 73 are shown in table 1 . peptide design methods have been categorized as ligand-and target-based design methods. in the ligand-based designing procedure, information derived from peptides is used to design novel therapeutic peptides. in the target-based method, information derived from target proteins is specifically utilized. typically, a hybrid approach including both ligand-and target-based design is utilized. 13 ligand-based peptide design the ligand-based design has been classified as follows: 1) sequence-based, 2) property-based and 3) conformationbased design. sequence-based approach uses the information of conserved regions and analyzes the multiple sequence alignments. this method is directed by the hypothesis that conserved regions are functionally and structurally significant. 13 computational tools allow the ligand-based peptide design, although they lag behind bioinformatics strategies developed for protein designing. 13 recently, using a method based on a pam250 matrix, the relationship between a series of 35 collagen peptides and antiangiogenic activity including proliferation, migration and adhesion was analyzed. 74 the pam250 matrix captured information of mutation rates among all pairs of amino acids. based on the results, regions at the c and n termini of the peptides were detected to be significant for an ideal activity and suggested as two distinct binding sites. the approach showed the potential worth of the sequence-based peptide design. 74 in another report, a computational platform called sarvision was developed to support sequence-based design. sarvision signifies an important step for peptide sequence/activity relationship (sar) analysis. moreover, it pools the improved visualization abilities with advanced sequence/activity analysis. 75 compared to small molecules, property-based design methods for peptides are in the early stages of development. in a recent study, the δg decomposition per residue and the physicochemical characteristics of amino acids, such as hydrophilicity, hydrophobicity and volume, were used computer-aided design of amino acid-based therapeutics to model peptide binding to targets of interest. 76, 77 finally, a model was built to estimate peptide δg values for binding to the class i major histocompatibility complex (mhc) protein hla-a*0201. 78 furthermore, in a wide range of studies, antimicrobial peptides were successfully analyzed by using the property-based approach. 79 for example, a machinelearning method was employed to design novel antimicrobial peptides. 80 the victory of the property-based methods with antimicrobial peptides may be explained by the fact that the desired biologic activity of membrane disruption is relatively nonspecific. 13 in the case of conformation-based peptide design, computational techniques were developed to predict the conformational ensembles or structure of peptides and analyze the sars. 81,82 pep-fold is an online tool used to predict the 3d structures of peptides of length 9-36 residues. 81 a remarkable suggestion from the data is that pep-fold seems to solve the conformational sampling problem. 13, 81 in order to search conformational spaces of a peptide, long timescale molecular dynamic simulations have been employed. 83, 84 besides, quantum mechanical calculations are promising to address the scoring deficiency in the peptide conformational examination. 85 apparently, to affect the peptide design processes positively, improving the major theoretical and technical issues is necessary before such computationally sophisticated and costly procedures. conformation of a peptide may be modeled to generate a 3d pharmacophore hypothesis. a certain pharmacophore hypothesis is useful to determine the adme/tox activities or particular potencies of a peptide. 86 for example, screening of a peptide library was jointed to generate a pharmacophore hypothesis to identify potent agonists of melanocortin-4 receptor isoforms. a combinatorial tetrapeptide library was screened, and sar and ligand-derived pharmacophore templates were generated. the pharmacophore hypothesis was proposed to allow continuous attempts in the rational design of melanocortin receptor molecules. 86 target-based peptide design compared to ligand-based peptide design, target-based design appears to be in a more improved level. 13 targetbased design is initiated with the computer-aided survey of a ligand-bound or unbound protein target to recognize its potential binding sites, prospective specificity surfaces and other pharmacologic activity elements. the phase is generally followed by an in silico design phase where computational methods perform, refine and evaluate peptide design ideas. some recently developed computational methods for targetbased peptide design are reviewed below. recently, an increase in the number of protein-peptide 3d structures deposited in the pdb has assisted to search the molecular mechanism and structural basis of peptide recognition and binding. 87 information of crystal structures of protein-peptide complexes can improve our knowledge of the farhadi and hashemian chemical forces involved in the binding and special modes of binding. dynamic data of the complexes can be partially extracted from the solution nmr structures deposited in the pdb. to record the structures and functions of various protein-peptide complexes, the experimentally resolved structure data were gathered, annotated and analyzed, and several distinctive databases such as pepx, 88 pepbind 89 and peptiddb were generated. 90 the pepx database, derived from the pdb, comprises unique protein-peptide interface collections. 88 the pepbind database contains 4,986 proteinpeptide complex structures from the pdb. 89 peptiddb is a curated database of 103 protein-peptide complexes. 90 the abundance of the structural information specifically on monomeric proteins could be gathered to design proteinpeptide interactions with no requirement for their sequence homology. 91 protein-peptide docking precise docking of a highly flexible peptide is a major challenge. 18 traditional docking protocols, such as autodock, vina 92,93 and moe-dock, 94 developed for docking of small molecules, were also used to dock a peptide to a protein receptor. however, comparative studies revealed that these techniques would face failure if the docked peptides were .3 residues long. 95 therefore, development of peptide-focused docking protocols is very important. 96 other protein-protein docking tools such as z-dock and hex have been used for the computational peptide design in some studies. 96 below, details of recently developed peptide-focused docking approaches are discussed. first, heuristic evolution procedures were applied to search the large conformational space of linear peptides before the binding. 97 however, these procedures were not efficient and their use was limited. 18 then, a scheme based on conformational sampling became common in the peptide docking. besides, several illustrative approaches were proposed to balance between the accuracy and efficacy of the flexible peptide docking. in this aspect, docscheme, 98 dynadock 99 and pepspec 100 were integrated to online userfriendly interfaces and introduced. recently, pepcrawler 101 and flexpepdock 102 were developed as the peptide docking tools. 18 it is reported that flexpepdock 102 has sub-angstrom accuracy in reproducing the crystal structures of protein-peptide complexes. 103 all of the flexpepdock-based methods assume previous information about the peptide-binding site. 13 anchordock, a recently described algorithm, allows powerful blind docking calculations through relaxing the constraint. 104 the program predicts anchoring origins on a protein surface. following recognition of the anchoring origins, an assumed peptide conformation is refined using an anchor-constrained molecular dynamic process. 105 haddock, a well-known protein-protein docking tool, has been recently expanded to run the flexible peptide-protein docking. 105 to handle a docking procedure, haddock uses ambiguous interaction restraints based on the experimental information about intermolecular interactions. this rigid body peptide docking is followed through a flexiblesimulated annealing process. the novel haddock strategy initiates docking computations from an ensemble of three dissimilar peptide conformations (eg, α-helix, extended and polyproline-ii) that are high informative inputs. 105 cabs-dock is a recently introduced protein-peptide docking tool and runs a primary docking procedure whose outcomes can be refined by other tools such as flexpepdock. 106 in the primary phase of the procedure, random conformations of a peptide are predicted and located around the protein target of interest. the process is followed by replica exchange monte carlo dynamics. subsequently, 10 models are selected for the last optimization using the modeller tool to gain accurate scoring and ranking poses. 13, 106 galaxypepdock was developed to use experimentally resolved protein-peptide structures for running the template-based docking pooled by flexible energy-based optimization. 107 atomistic simulation atomistic monte carlo and molecular dynamics simulations are accurate, but they are meticulous techniques to investigate peptide-protein binding interactions. these techniques can also detect the thermodynamic profile and trajectory included in protein-peptide identification. these methods predict the association among conformations of a peptide in solution or protein. 108 in a study, in order to describe the binding of a decapeptide to the cognate sh3 receptor, a long-term molecular dynamic simulation was used and a two-state model was built. 109 in the first step, a relatively quick diffusion phase, nonspecific encounter complexes were generated and stabilized by using electrostatic energy. the secondary step was a slow modification phase, in which the water molecules were emptied out from the space between the peptide ligand and the receptor. 109 in another report, by using monte carlo method, the mentioned two-state model was verified to trace some oligopeptide routes for binding to various pdz (post synaptic density protein, drosophila disc large tumor suppressor, and zonula occludens-1 protein) domains. 110 drug design, development and therapy 2018:12 submit your manuscript | www.dovepress.com computer-aided design of amino acid-based therapeutics the affinity of bh3 peptides to bcl-2 protein was investigated, and results showed the higher affinity of bound peptides occurred when the corresponding peptides were in a lower degree of disorder in unbound states and vice versa. 111 these results showed that the highly structured peptides could increase their affinity through reducing the entropic loss associated with the binding. overall, in addition to the electrostatic and hydrophobic forces, protein-peptide interactions can be affected by the entropic effect and conformational flexibility that could be willingly examined with atomistic simulations. 111 very recently, using a fast molecular dynamics simulation, the energetic and dynamic features of protein-peptide interactions were studied. in most cases, the native binding sites and native-like postures of protein-peptide complexes were recapitulated. additional investigation showed that insertion of motility and flexibility in the simulation could meaningfully advance the correctness of protein-peptide binding prediction. 112 peptide affinity prediction most features of computational peptide design are based on the accuracy and efficacy of affinity prediction. hence, the fast and reliable prediction of peptide-protein affinity is significant for rational peptide design. 18 in this aspect, two categories of prediction algorithms including sequence-and structure-based approaches were developed. the sequencebased method uses the information derived from primary polypeptide sequences to approximate and evaluate the standards of the binding affinity. the structure-based process takes the information derived from 3d structures of proteinpeptide complexes to predict the binding affinity. 113 at the sequence level, the quantitative structure-activity relationships (qsars) have been widely utilized to forecast the binding affinity of peptides and conclude the biologic function. 114 to model the statistical correlation between sequence patterns and biologic activities of experimentally assessed peptides, machine-learning methods such as partial least squares (pls), artificial neural networks (ann) and support vector machine (svm) have been used. the obtained correlations have been used to infer experimentally undetermined peptides. 115 the relationship between the biologic activity and molecular structure is an important issue in biology and biochemistry. qsar is a well-established method employed in pharmaceutical chemistry and has become a standard tool for drug discovery. however, the predictive capacity of qsar techniques is generally weaker than statistics-based approaches. therefore, a combination of the qsar method with a statistic-based technique may bring out the best in each other and can be a trend in future developments of drug discovery. 114 at the structural level, numerous reports on affinity prediction have addressed the mhc-binding peptides. plentiful mhc-peptide complex structure records have been deposited in the pdb. 116 the significance of domain-peptide recognition has been recently illustrated in the metabolic pathway and cell signaling. 117 to predict the protein-peptide binding potency, a number of strict theories were suggested based on the potential free energy perturbation. the theories computed the alteration of free energies upon the interaction between phosphor-tyrosine-tetra-peptide (pyeei) and human lck sh2 domain. 118 furthermore, to obtain a deep insight into the structural and energetic aspects of peptide recognition by the sh3 domain, a number of molecular modeling experiments such as homology modeling, molecular docking and mechanism dynamics were used. 119 peptide array strategies confirmed that some peptide candidates may be potent binders of the abl sh3 domain. 120 very recently, an approach including quantum mechanics/molecular mechanics, semiempirical poisson-boltzmann/surface area and empirical conformational free energy analysis was developed to quantitatively illustrate the energetic contributions involved in the affinity losing of pdz domain and oppa protein to their peptide ligands. 121, 122 de novo peptide design recently, in order to de novo target-based peptide design, two remarkable methodologies including the vital method and an approach developed by bhattacherjee and wallin were introduced. the vital method pools verterbi algorithm with autodock to design peptides for the binding sites of a target. 123 the "bhattacherjee and wallin" approach explores both peptide sequence and conformational space around a protein target at the same time. 124 this approach was tested on three dissimilar peptide-protein domains to assess its ability. 13 a brief list of the existing computational resources employed in peptide design is presented in table 2 . in recent years, some computational methods have been proposed to design peptidomimetics. these methods can be classified based on their specificity to translate peptides to peptidomimetics. 28 to select the best method, drug design, development and therapy 2018:12 submit your manuscript | www.dovepress.com farhadi and hashemian awareness about the structure of peptide-protein complexes is important. 28, 96 herein, recently introduced methods for computer-aided design of peptidomimetics are presented. growmol is a combinatorial algorithm employed in the peptidomimetics design. growmol searches a variety of probable ligands for the binding sites of a target protein 125 and produces molecules with the chemical and steric complementarity for the 3d structure of binding sites. this method was used to generate peptidomimetic inhibitors of thermolysin, hiv protease and pepsin. by using the x-ray crystal structures of pepstatin-pepsin complexes, growmol predicted therapeutic peptidomimetics against the aspartic proteases. the algorithm created some cyclic inhibitors bridging the side chains of cysteine residues in the pl and p3 inhibitor subsites. the binding modes were checked using x-ray crystallography. 125, 126 ludi is another interesting software referring to the de novo methodology. 127 by using natural and non-natural amino acids as building blocks, the software designed peptidomimetics against renin, thermolysin and elastase. 127 conformational flexibility of each novel peptidomimetic was searched through sampling the multiple conformers of each amino acid. 127 peptide-driven pharmacophoric hypothesis is the most perceptive computational technique discovered in the peptidomimetics design. the method is especially useful when the x-ray structures of protein-protein complexes exist. 28 the main idea is to adapt the hot spot concept into the associated pharmacophoric feature concept. with a pharmacophorebased virtual screening process, this strategy can determine novel type 3 mimetics. 128 in fact, the side chains of each amino acid can be simply categorized based on the conventional pharmacophoric characteristics, such as hydrogen bond donors and acceptors, aromatic ring and charged and hydrophobic centers. for example, in a report, pharmacophore model directed synthesis of the non-peptide analogs of a cationic antimicrobial peptide identified an anti-staphylococcal activity. 129 to make a pharmacophore hypothesis, a model of rna iii-inhibiting peptide (rip), a well-known heptapeptide inhibitor of the staphylococcal pathogenesis, was utilized. through the virtual screening of 300,000 commercially available small molecules based on the rip-based pharmacophore, hamamelitannin was discovered as a non-peptide mimetic of rip. hamamelitannin is a tannin derivate extracted from hamamelis virginiana. 28, 129 in another study, two rounds of in silico screening were performed to discover potential peptidomimetics able to mimic a cyclic peptide (cyclo[cpfvktqlc] ) that is known to bind the anb3 integrin receptor. 130 at the end of the process, the most potent representatives were at least 2,000 times better than the original cyclopeptide (around 2 mm). 130 in a prosperous instance, virtual screening was done by using multi-conformational forms of a large commercial library. a target-based pharmacophoric model mapped the cd4-binding site on hiv-1 gp120. the pharmacophore hypothesis was made based on a homology model of the protein cavity. in a cell-based assay, two of the top scoring molecules were detected as micromolar inhibitors of hiv-1 replication. 131 computer-aided design of amino acid-based therapeutics the pharmacophore-based screening was used to find the novel alzheimer's therapeutics as mimetics of neurotrophins. 132 the therapeutic utilization of neurotrophins might be restricted because of several deficiencies such as its reduced central nervous system penetration, decreased stability and potency to enhance neuronal death through interaction with the p75ntr receptor. the mimetism of particular nerve growth factor domains could inhibit neuronal death. peptidomimetics of the loop 1 and loop 4 domains of nerve growth factor can prevent neuronal death induced by p75ntr-dependent and trk-related signaling. 132 in another study, a full-computational pharmacophorebased approach assessed the fda-approved drugs as valuable candidates to inhibit protein-protein interactions. 133 peptide structures were designated in terms of pharmacophores and searched against the fda-approved drugs to detect same molecules. the top ranking drug matches contained several nuclear receptor ligands and matched allosterically to the binding site on the target protein. the top ranking drug matches were docked to the peptide-binding site. the majority of the top-ranking matches presented a negative free energy change upon binding that was comparable to the standard peptide. 133 geometry similarity method geometry similarity methods create a geometric similarity between non-peptide templates and peptide patches. in a study, the supermimic tool was developed to recognize peptide mimetics. 134 in the program, a complex library of peptidomimetics composed of several protein structure libraries has been deposited. moreover, supermimic includes the d-peptides, synthetic components (reported as betaturn or gamma-turn mimetics) and peptidomimetic ligands obtained from the pdb. 134 in the program, the searching process allows scanning a library of small molecules that mimic the tertiary structure of a query peptide followed by scanning of a protein library where a query for small molecule can adopt into the backbone. 28, 134 sequence-based method recently, a method has been developed to rank peptide compound matches that are limited to short linear motifs in proteins and compounds with amino acid substituents. 135 the algorithm allows mapping the side chain-like substituents on every compound of a large chemical library. the complete molecule can be signified by a short sequence, and each fragment in the molecule can be represented as a distinct letter abbreviation. 28 a cross-search between the pubchem database (about 5.4 million molecules) and a non-redundant collection of 11,488 peptides obtained from pdb demonstrated that the algorithm can be useful for high-throughput measurements. 28 to recognize a true positive, the method explored identified protein motifs against the national cancer institute developmental therapeutic program compound database. 135 in another study, the similarity of amino acid motifs to compounds web server was developed to ease screening of identified motif structures against bioactive compound databases. 136 the methodology was reported to be efficient since the compound databases were preprocessed to maximize the accessible data, and the necessary input data was minimal. 136 in similarity of amino acid motifs to compounds, motif matching can be full or partial that may decrease or enhance the number of potential mimetics, respectively. using a novel search algorithm, the web service can perform a fast screening of known or putative motifs against ready compound libraries. the classified results can be examined by linking to appropriate databases. 28, 136 fragment-based method replacement with partial ligand alternatives through computational enrichment is a fragment-based approach. 137 by using structures of peptide-bound proteins as design anchors, the program can computationally find a non-peptide mimetic for specific determinants of known peptide ligands. 137 hybrid peptide-driven shape and pharmacophoric method development and application of strategies for pharmacophore modeling indicate that the medicinal chemistry community has broadly accepted the intuitive nature of the pharmacophore concept. besides, shape complementarity has been identified as a significant element in the molecular identification between ligands and their targets. 28 in virtual screening efforts, using the pharmacophore-and shape-based techniques distinctly may increase the rate of false-positive results. 128 therefore, incorporating both pharmacophore-and shape-matching techniques into one program can potentially diminish the rate of false positives. 128 recently, to discover novel peptidomimetics, a weboriented virtual screening tool named pepmmsmimic 138 was developed to pool the conventional pharmacophore matching with shape complementarity. a library of 17 million conformers were extracted from 3.9 million commercially available chemicals and gathered in the mmsinc database. the database was used as a skeleton to develop farhadi and hashemian pepmmsmimic. 139 in the pepmmsmimic interface, the 3d structure of a protein-bound peptide is used as an input. then, chemical structures able to mimic the pharmacophore and shape similarity of the original peptide are proposed to involve in the protein-protein recognition. 139 a list of in silico methods used to design potential peptidomimetics along with their strengths and weaknesses is presented in table 3 . overall, design and development of therapeutics are tedious, expensive and time-consuming procedures. therefore, using modern approaches including computer-aided design methods can lessen the examination phase, price and failure of therapeutics discovery. computational methods used to design amino acid-based therapeutics can increase the range of available biotherapeutics. benefiting from the dramatic advance in bioinformatics, computational tools can be used to find and develop therapeutic proteins, peptides and peptidomimetics. 140, 141 moreover, using the computational tools decrease the cost of therapeutics development, from concept to market, by up to 50%. 140 however, in the computational protein designing, there are some challenges such as our inadequate knowledge of folding and physical forces that stabilize protein structures. moreover, sequences and local structures have many degrees of freedom that can complicate the sequence search. therefore, there is a requirement for effective methods to find sequences related to a particular structure and measure essential protein folding criteria. overall, in silico design of amino acid-based therapeutics includes many challenges that should be removed to improve the overall performance of the design processes. for example, although structure determination of all disease-related proteins through crystallography and nmr is a laborious task, it is necessary to gather much structural information of peptide-protein interactions. besides, development of vigorous algorithms to calculate protein-protein binding energies is essential. the estimation of binding constant between two macromolecules with an appropriate speedaccuracy tradeoff needs millisecond scale molecular dynamics. moreover, understanding of both protein-protein and protein-peptidomimetics recognition processes in a molecular level can be improved using higher accurate force fields such as quantum mechanical polarizable force. in recent years, there are growing examples on the approval of monoclonal antibodies (therapeutic antibodies) by the fda for treatment of various diseases. this important area of amino acid-based therapeutics has been covered in more depth elsewhere. 142, 143 for more explanation about the theorems and details of antibody informatics for drug discovery as well as the computer-aided antibody design, readers are referred to study previous reports. 142, 143 the authors report no conflicts of interest in this work. computer-aided design of amino acid-based therapeutics what is the future of targeted therapy in rheumatology: biologics or small molecules protein therapeutics: a summary and pharmacological classification in silico methods for design of biological therapeutics constructing novel chimeric dna vaccine against salmonella enterica based on sopb and groel proteins: an in silico approach in silico phylogenetic analysis of vibrio cholera isolates based on three housekeeping genes designing of complex multi-epitope peptide vaccine based on omps of klebsiella pneumoniae: an in silico approach theoretical and computational protein design annu evaluation of in silico protein secondary structure prediction methods by employing statistical techniques inhibition of mycobacterial cyp125 enzyme by sesamin and β-sitosterol: an in silico and in vitro study theory of protein folding: the energy landscape perspective toward an outline of the topography of a realistic protein-folding funnel artificial diiron enzymes with a de novo designed four-helix bundle structure computer-enabled peptide drug design: principles, methods, applications and future directions docking small peptides remains a great challenge: an assessment using autodock vina empirical estimation of local dielectric constants: toward atomistic design of collagen mimetic peptides recent work in the development and application of protein-peptide docking rational design of peptide drugs: avoiding aggregation computational peptidology: a new and promising approach to therapeutic peptide design strategies employed in the design and optimization of synthetic antimicrobial peptide amphiphiles with enhanced therapeutic potentials multifaceted roles of disulfide bonds. peptides as therapeutics peptidomimetics, a synthetic tool of drug discovery an in silico pipeline for the design of peptidomimetic proteinprotein interaction inhibitors (order no. 10188557) natural products as sources of new drugs over the last 25 years diversity-oriented synthesis of macrocyclic peptidomimetics structure-based design, synthesis, and biological evaluation of peptidomimetic sars-cov 3clpro inhibitors advances in amino acid mimetics and peptidomimetics a hierarchical approach to peptidomimetic design mimicking peptides… in silico peptidomimetic design rational design for peptide drugs peptidomimetics as a cutting edge tool for advanced healthcare an unusual functional group interaction and its potential to reproduce steric and electrostatic features of the transition states of peptidolysis low molecular weight, non-peptide fibrinogen receptor antagonists neurotrophin small molecule mimetics: candidate therapeutic agents for neurological disorders design of peptides, proteins, and peptidomimetics in chi space molecular technology. designing proteins and peptides molecular engineering: an approach to the development of general capabilities for molecular manipulation x-ray versus nmr structures as templates for computational protein design high-resolution protein design with backbone freedom prediction of protein-protein interface sequence diversity using flexible backbone computational protein design backbone flexibility in computational protein design de novo computational design of retro-aldol enzymes one fold with many functions: the evolutionary relationships between tim barrel families based on their sequences, structures and functions search and sampling in structural bioinformatics the dead-end elimination theorem and its use in protein side-chain positioning application of a self-consistent mean field theory to predict protein sidechains conformation and estimate their conformational entropy design of protein-interaction specificity gives selective bzip-binding peptides computational methods for protein design and protein sequence variability: biased monte carlo and replica exchange exploring the conformational space of protein side chains using dead-end elimination and the a* algorithm side-chain and backbone flexibility in protein core design dead-end elimination with a polarizable force field repacks pcna structures improved prediction of protein side-chain conformations with scwrl4 trading accuracy for speed: a quantitative comparison of search algorithms in protein sequence design using self-consistent fields to bias monte carlo methods with applications to designing and sampling protein sequences computational design and characterization of a monomeric helical dinuclear metalloprotein statistical theory of combinatorial libraries of folding proteins: energetic discrimination of a target structure achieving stability and conformational specificity in designed proteins via binary patterning photophysics of a fluorescent non-natural amino acid: p-cyanophenylalanine an expanded eukaryotic genetic code a "solvated rotamer" approach to modeling watermediated hydrogen bonds at protein-protein interfaces rotamer libraries in the 21st century improved side-chain prediction accuracy using an ab initio potential energy function and a very large rotamer library potential energy functions for protein design de novo design of foldable proteins with smooth folding funnel: automated negative design and experimental verification assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and bayesian scoring functions structure by design: from single proteins and their building blocks to nanostructures computational de novo design and characterization of a four-helix bundle protein that selectively binds a nonbiological cofactor using α-helical coiled coils to design nanostructured metalloporphyrin arrays kemp elimination catalysts by computational enzyme design de novo design of a βαβ motif high-resolution structural and thermodynamic analysis of extreme stabilization of human procarboxypeptidase by computational protein design design of a novel globular protein fold with atomic-level accuracy novel peptide-specific quantitative structure activity relationship (qsar) analysis applied to collagen iv peptides with antiangiogenic activity development of an informatics platform for therapeutic protein and peptide analytics two-level qsar network (2l-qsar) for peptide inhibitor design based on amino acid properties and sequence positions recent development of peptide drugs and advance on theory and methodology of peptide inhibitor design predicting the affinity of epitope-peptides with class i mhc molecule hla-a*0201: an application of amino acid-based peptide prediction a brief overview of antimicrobial peptides containing unnatural amino acids and ligand-based approaches for peptide ligands machine learning assisted design of highly active peptides for drug discovery pep-fold: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides in silico predictions of 3d structures of linear and cyclic peptides with natural and nonproteinogenic residues long-timescale molecular dynamics simulations of protein structure and function how fastfolding proteins fold bond distances in polypeptide backbones depend on the local conformation identification of tetrapeptides from a mixture based positional scanning library that can restore nm full agonist function of the l106p, i69t, i102s, a219v, c271y, and c271r human melanocortin-4 polymorphic receptors (hmc4rs) the protein data bank protein design with fragment databases pepbind: a comprehensive database and computational tool for analysis of protein-peptide interactions the structural basis of peptide-protein binding strategies protein-peptide interactions adopt the same structural motifs as monomeric protein folds highly flexible protein-peptide docking using cabs-dock computer-aided design of amino acid-based therapeutics virtual screening for potential inhibitors of ctx-m-15 protein of klebsiella pneumoniae in silico panning for a non-competitive peptide inhibitor comparative evaluation of eight docking tools for docking and virtual screening accuracy in silico designing of peptide inhibitors against pregnane x receptor: the novel candidates to control drug metabolism computation of the binding of fully flexible peptides to proteins with flexible side chains a flexible docking procedure for the exploration of peptide binding selectivity to known structures and homology models of pdz domains dynadock: a new molecular dynamics-based algorithm for protein-peptide docking including receptor flexibility structure-based prediction of proteinpeptide specificity in rosetta pepcrawler: a fast rrt-based algorithm for high-resolution refinement and binding-affinity estimation of peptide inhibitors rosetta flexpepdock ab-initio: simultaneous folding, docking and refinement of peptides onto their receptors sub-angstrom modeling of complexes between flexible peptides and globular proteins anchordock: blind and flexible anchordriven peptide docking a unified conformational selection and induced fit approach to proteinpeptide docking cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site galaxypepdock: a proteinpeptide docking tool based on interaction similarity and energy optimization predicting peptide structures in native proteins from physical simulations of fragments mechanism of fast peptide recognition by sh3 domains binding free energy landscape of domain peptide interactions molecular dynamics simulations of pro-apoptotic bh3 peptide helices in aqueous medium: relationship between helix stability and their binding affinities to the anti-apoptotic protein bcl-xl structural and dynamic determinants of protein-peptide recognition quantitative sequenceactivity model (qsam): applying qsar strategy to model and predict bioactivity and function of peptides, proteins and nucleic acids recent advances in qsar and their applications in predicting the activities of chemical molecules, peptides and proteins for drug design comprehensive comparison of eight statistical modelling methods used in quantitative structure retention relationship studies for liquid chromatographic retention times of peptides generated by protease digestion of the escherichia coli proteome prediction of mhc-peptide binding: a systematic and comprehensive overview domain mediated protein interaction prediction: from genome to network calculation of absolute protein-ligand binding free energy from computer simulations prediction of binding affinities between the human amphiphysin-1 sh3 domain and its peptide ligands using homology modeling, molecular dynamics and molecular field analysis characterization of domain-peptide interaction interface: a generic structure-based model to decipher the binding specificity of sh3 domains why oppa protein can bind sequence-independent peptides? a combination of qm/mm, pb/sa, and structure-based qsar analyses characterization of pdz domain-peptide interactions using an integrated protocol of qm/mm, pb/sa, and cfea analyses computational design of peptide ligands exploring protein-peptide binding specificity through computational peptide screening multiple highly diverse structures complementary to enzyme binding sites: results of extensive application of a de novo design method incorporating combinatorial growth transformation of peptides into non-peptides. synthesis of computer-generated enzyme inhibitors towards the automatic design of synthetically accessible protein ligands: peptides, amides and peptidomimetics structure-based pharmacophores for virtual screening antimicrobial activity of small β-peptidomimetics based on the pharmacophore model of short cationic antimicrobial peptides small molecule inhibitors of hantavirus infection a dynamic target-based pharmacophoric model mapping the cd4 binding site on hiv-1 gp120 to identify new inhibitors of gp120-cd4 protein-protein interactions alzheimer's therapeutics approved drug mimics of short peptide ligands from protein interaction motifs supermimic-fitting peptide mimetics into protein structures identification of potential small molecule peptidomimetics similar to motifs in proteins drug design, development and therapy 1254 web server to identify similarity of amino acid motifs to compounds (saamco) replace: a strategy for iterative design of cyclin-binding groove inhibitors swimming into peptidomimetic chemical space using pepmmsmimic mmsinc: a large-scale chemoinformatics database computational drug discovery developability assessment as an early de-risking tool for biopharmaceutical development antibody informatics for drug discovery computer-aided antibody design tumorhope: a database of tumor homing peptides drug-permeability and transporter assays in caco-2 and mdck cell lines pepx: a structural database of non-redundant protein-peptide complexes rosetta flexpepdock web server -high resolution modeling of peptide-protein interactions pdock: a new technique for rapid and accurate docking of peptide ligands to major histocompatibility complexes predicting peptide binding sites on protein surfaces by clustering chemical interactions protein-peptide complex prediction through fragment interaction patterns pep-sitefinder: a tool for the blind identification of peptide binding sites on protein surfaces vital: viterbi algorithm for de novo peptide design drug design, development and therapy 2018:12 submit your manuscript | www.dovepress.com submit your manuscript here: http://www.dovepress.com/drug-design-development-and-therapy-journal drug design, development and therapy is an international, peerreviewed open-access journal that spans the spectrum of drug design and development through to clinical applications. clinical outcomes, patient safety, and programs for the development and effective, safe, and sustained use of medicines are the features of the journal, which has also been accepted for indexing on pubmed central. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord-104282-90t1m430 authors: nan title: microsomal aldehyde dehydrogenase is localized to the endoplasmic reticulum via its carboxyl-terminal 35 amino acids date: 1994-09-02 journal: j cell biol doi: nan sha: doc_id: 104282 cord_uid: 90t1m430 rat microsomal aldehyde dehydrogenase (msaldh) has no amino-terminal signal sequence, but instead it has a characteristic hydrophobic domain at the carboxyl terminus (miyauchi, k., r. masaki, s. taketani, a. yamamoto, a. akayama, and y. tashiro. 1991. j. biol. chem. 266:1953619542). this membrane-bound enzyme is a useful model protein for studying posttranslational localization to its final destination. when expressed from cdna in cos-1 cells, wild-type msaldh is localized exclusively in the well-developed er. the removal of the hydrophobic domain results in the cytosolic localization of truncated proteins, thus suggesting that the portion is responsible for membrane anchoring. the last 35 amino acids of msaldh, including the hydrophobic domain, are sufficient for targeting of e. coli beta-galactosidase to the er membrane. further studies using chloramphenicol acetyltransferase fusion proteins suggest that two hydrophilic sequences on either side of the hydrophobic domain play an important role in er targeting. domain results in the cytosolic localization of truncated proteins, thus suggesting that the portion is responsible for membrane anchoring. the last 35 amino acids of msaldh, including the hydrophobic domain, are sufficient for targeting of e. coli ~5-galactosidase to the er membrane. further studies using chloramphenicol acetyltransferase fusion proteins suggest that two hydrophilie sequences on either side of the hydrophobic domain play an important role in er targeting. great deal of attention has been paid to resolve the mechanism for sorting and targeting of newly synthesized proteins to their final destinations, and this is one of the fundamental problemsin cell biology. newly synthesized proteins are destined to follow two distinct routes, depending on the presence or absence of a signal sequence at their amino termini (37) . the signal recognition particle (srp) t in the cytosol recognizes and binds to a signal sequence of nascent peptides. the resulting srp-ribosome-nascent peptides complexes are targeted to the er membrane through their interactions with the docking protein complex (35, 36) . after translocation across the er membrane, secretory and plasma membrane proteins follow a common pathway from the er, through the golgi complex, to the cell surface by default with bulk flow of lipids (33) . resident proteins in the central vacuolar system are localized and retained in their final destinations with the aid of either specific targeting (18) or retention signals (25) (26) (27) 44) . on the other hand, proteins without a signal sequence at their amino termini are synthesized on free polysomes and 1. abbreviations used in this paper: cat, chloramphenicol acetyltransferase; fp2, nadph-cytochrome p-450 reductase; msaldh, microsomal aldehyde dehydrogenase; pbs(+), pbs containing 1 mm caci2 and 0.5 mm mgci2; pdi, protein disulfide isomerase; ptp, protein tyrosine phosphatase; ste, sucrose solution containing 10 mm tris-hcl, ph 7.4, 1 mm edta, 10 t~g/ml leupepdn a, 0.5 mm pmsf, and 10 u/ml trasyol; srp, signal recognition particle. are posttranslationally directed to intracellular organdies such as mitochondria, peroxisomes, the nucleus, and the er, or they remain in the cytosol. much is known regarding targeting signals (14, 16, 39) and cytosolic protein factors (1, 15) by which proteins are imported into these organelles. however, less is known regarding posttranslational targeting of integral membrane proteins to the outer mitochondrial membrane, the peroxisomal membrane, or the er membrane. cytochrome b5 has a hydrophobic domain instead of an amino-terminal signal sequence at its carboxyl terminus (42) . for a long time, cytochrome b5 has been noted to be a typical membrane protein inserted posttranslationally into microsomal membranes through the hydrophobic domain. besides, it has been reported that cytochrome b5 is localized in any membranes of intracellular organdies, including the er membrane, the golgi membrane, the plasma membrane, and the outer mitochondrial membrane (47) . experiments using srp-depleted or -supplemented in vitro systems have shown that this protein does not require srp for insertion into microsomal membranes (3) . therefore, the carboxylterminal hydrophobic sequence was termed an "insertion" sequence through which spontaneous integration of cytochrome b5 into any exposed membranes could occur not only in vitro, but also in vivo (5) . however, recent studies have shown that cytochrome b5 is restricted in the er in vivo (11) , and that the last 10 amino acids of this protein adjacent to the hydrophobic domain are important for its targeting to the er (23) . recently, we have isolated and sequenced a full-length cdna for rat microsomal aldehyde dehydrogenase (msaldh) (24) . the deduced amino acid sequence of this enzyme prediets a similar molecular structure to that of cytochrome b5, a bulky amino-terminal domain without an amino-terminal signal sequence and a short hydrophobic domain at the carboxyl terminus. we report here that msaldh is localized exclusively in the er in cos-1 cells when expressed from cdna, and that expression of this protein apparently alters the structure of the er from a reticular to large vesicular one. in addition, we report that the hydrophobic domain is responsible for membrane anchoring and that msaldh is likely to have two er targeting sequences on either side of the membrane anchoring domain. our results suggest a novel mechanism for the posttranslational er targeting of this tail-anchored protein. (toyama, japan), respectively. since the/3-subunit of prolyl 4-hydroxylase has been shown to be identical to protein disulfide isomerase (pdi) (34) , this mab is referred to as pdi mab in this paper. rabbit antibodies to rat msaldh, nadph-cytoehrome p-450 reductase (fp2), and pdi have been prepared and characterized as described (2, 21, 24) . rabbit antiserum to bovine mitochondrial complex hi was a generous gift from dr. takamasa ozawa (nagoya university, nagoya, japan). the eukaryotic assay vectors, pch110 and psv2cat, were from pharmaeia lkb biotechnohigy, inc. (uppsala, sweden) and stratagene (la joua, ca), respectively. the eukaryotic expression vector pmiw (43) was kindly provided by dr. akihiro inoue (national institute for physiological sciences, okazaki, japan). restriction enzymes and dna modifying enzymes were purchased from nippon gene (toyama, japan) and takara co., ltd. (kyoto, japan). all other chemicals were of the highest purity commercially available. all constructions were verified by the dideoxy chain termination method (38) . insertion of a full-length edna encoding rat msaldh into the unique ecori site of the sv-40-based vector pcd (30) has been described previously (24) . the gapped duplex method of oligonueleotide-directed mutagenesis (19) a chimeric edna for e. coli/~-gaiactosidase fusion protein was constructed as follows. first, a dna fragment encoding the earboxyl-terminal 53 amino acids (amino acids 994-1046) of/5-galaetosidase, followed by five amino acids (amino acids 450-454) of msaldh, was created by pcr using oligonuclcotides no. 6 (5' agccatcgccatctgctg 3') and no. 7 (5' ggaagaatttcgaccatrttw_tacaccagacc 3'). similarly, a second dna fragment corresponding to the last five amino acids (amino acids 1042-1046) of/5-galactosidase followed by the last 35 amino acids (amino acids 450-484) and 3' untranslated sequence of msaldh was amplified using oligonueleotides no. 8 (5' c_~'ictc.~tg'ic_aaaaa'iugtcgaaat-tcticc 3') and no. 9 with mutated nucleotides underlined for introduction of an ecori site (5' aacaacttagaattcac~gttc 3'). these two fragments were then used as the templates to create a chimeric edna with the oligonucleotides nos. 6 and 9. the resultant pcr fragment was digested with ecori and ligated in-frame to the ecori site (amino acids 1029-1030) of pchii0 to construct pchii0/aldh. since it was difficult to construct a series of various fusion genes using ~-gaiactosidase edna according to the method described above, we used cat edna for further analyses on the role of the carboxyl-terminai portion of msaldh in the intracellular localization. chimeric cdnas for cat fusion proteins (cat/aldh chimeras) were constructed essentially by combination of oligonucleotide-directed mutagenesis and pcr. a 1.8-kbp hindhlbamhi edna fragment encoding cat in the psv2cat was cloned into ml3tvl8. oligonuclnotides no. 10 (5' ag~ggc~ggtacct-aattttttta 3') and no. 11 (5' ataagtgatatcaagcggatga 3') with mutated nucleotides underlined were used for generation of a kpni site at the carboxyl terminus and an ecorv site in the 3' untranslated sequence of cat, respectively. the mutated edna was cloned into hindlh/ bamhi-digested pmiw expression vector to construct pmiwcat. pcr was then used to generate dna fragments corresponding to the carboxylterminal regions of msaldh, including the 3' untranslated sequence. pcr reactions used the following primers and templates to amplify dna fragments termed aldh1-7 and 9: aldh1; oligonueleotides no. 12 (5' gagtccaagggtacctggtcgaaattc 3') with mutated nueicotides underlined for introduction of a kpni site and no. 9 (pcdaldh as template), aldi-i2; oligonucleotides no. 12 and no. 9 (pcdaldha481-484 as template), aldh3; oligonucleotides no. 13 (5' aaacagticaac-ggtaccagc~tgcagctg 3') and no. 9 (pcdaldh as template), aldh4; oligonucleotides no. 13 and no. 9 (pcdaldha481-484 as template), aldh5; oligonucleotides no. 14 (5' gaaattcttcggtacc-aaacagttcaac 3') and no. 9 (pcdaldh as template), aldh6; oligonucleotides no. 14 and no. 9 (pcdaldha481-484 as template), aldh7; oligonucleotides no. 12 and no. 9 (pcdaldha457-459 as template), and aldh9; oligonucleotides no. 12 and no. 9 (pcdaldha460-463 as template). these amplified fragments were then digested with kpni and hpai and ligated into the kpni-ecorv site of pmiwcat. the resultant plasmids were designated pmiwcat/aldh1-7, and pmiwcat/aldh9, respectively. for construction of pmiwcat/aldhs, pmiwcat/aldh2, which has the unique psti site in the coding sequence for the hydrophobic domain of msaldh, was digested with psti and bamhi, and ligated into pstl/bamhi digested pmiwcatialdh7. similarly, the same fragment was ligated into psti/bamhi digested pm1wcat/aldh9 to construct pmiwcat/aldh10. cos-1 cells were maintained in dme with 10% fbs, 50 u/ml penicillin, and 50 ~g/ml streptomycin at 37"c in a 5% coz incubator, and were replated the day before transfection by trypsinization. transfection was performed 4 h after the medium was replaced by the fresh one. for subeellniar fractionation experiments, cells plated in a lo0-mm dish (50-70% confluem) were transfected with an expression plasmid (20 ~g) using the caisequences of the carboxyl termini of wild-type and truncated forms of msaldh. the single amino acid code is used, and the amino acid numbers of msaldh are shown at the top. positively and negatively charged amino acids are marked + and -at the bottom, respectively. the hydrophobic domains are underlined. the edna for msaldh was converted to code for three truncated proteins, msaldha450-484, msaldha471-484, or ms-aldha481-484 by oligonucleotide-directed mutagenesis. cium phosphate precipitation method (48) . for immunottuorescent experiments, cells were grown on a 22 × 22-ram coverslip in a 35-ram dish (10-20% confluent) and were transfected with 4 /~g plasmid dna per 35-mm dish. for immunogold localization of cat/aldh chimeras, cells plated in a 60-ram dish (50-70% confluent) were transfected with 10 ttg plasmid dna per dish. 4 h after application of dna-calcium phosphate precipitate at 37°c, cells were shocked with 15% glycerol for 2 rain at room temperature, then incubated again at 370c for an additional 44 h before harvesting for subcellular fractionation or fixation for indirect immunofluorescence microscopy or immunoelectmn microscopy. cell fractionation was performed essentially as described previously for cos-i cells by clark and waterman (10) with slight modifications. briefly, cells were washed once with pbs and harvested in 5 ml of ice-cold 0.5 m sucrose containing 10 mm tris-hcl, ph 7.4, 1 mm edta, 10 #g/ml leupeptin, 10/~g/ml pepstatin a, 0.5 m_m pmsf, and 10 u/ml trasyol (0.5 m ste). after centrifugation at 800 g for 5 rain, the pellet was suspended in 0.5 ml of 0.5 m ste, homogenized with a teflon-glass homogenizer, then diluted with an equal volume of 10 mm tris-hcl, ph 7.4, 1 mm edta, 10/tg/nd leupeptin, 10/=g/ml pepstatin a, 0.5 mm pmsf, and 10 u/rni trasyol to obtain isotonic conditions. the total homogenate (designated h) was layered over 0.5 ml of 0.5 m ste and centrifuged at 800 g for 10 rain at 4°c using a swing but bucket, yielding a pellet (p1) consisting mainly of nuclei and unbroken cells. the supernatant and the interface were again layered over 0.5 m ste and centrifuged as above at 9,000 g for 10 min to isolate mitochondrial fraction (p2). the resultant supernatant was centrifuged at 88,000 g for 80 rain at 40c to sediment microsomal fraction (p3, mostly of the er membrane). the final supernatant, consisting mostly of cytosol, was designated $3. membrane fractions, p1, p2, and p3 were resuspended by hand homogenization in 0.25 m ste. endogenous enzyme activities of fp2 (an er marker) and succinate-cytochrome c reductase (a mitochondrial marker) were assayed by the methods of omura and takesue (31) and king (17) , respectively. protein was measured by bradford's method (7) . membrane fractions (100/tg protein) were resuspended in 0.8 ml of 100 mm na2co3 (ph 11.5), and were incubated for 30 rain at 0*c (13). the suspension was then centrifuged at 88,000 g for 80 rain at 40c, and the pellet (p) was suspended in 100/~i of sds-page sample buffer (20) . the supernatant (s) was precipitated with 10% tca, washed twice with 90% ethanol and once with diethyl ether, and dried. the resultant pellet was suspended in 100/d of sds-page sample buffer. all procedures were done at room temperature. proteins were separated on 8.5 % polyacrylamide gels (20) and electrophoretically transferred to a dora-pore membrane according to the method of burnette (8) using a semidry transfer blotter for 2.5 h at 36 v. after blocking with 3% skim milk in tbs for 1.5 h, blots were incubated with primary antibody in 3 % skim milk/ tbs for 1.5 h, and washed four times (5 rain each) with 0.05% tween-20 in tbs. they were then incubated with secondary antibody (pemxidaseconjugated goat anti-rabbit igg or anti-mouse igg) in 0.05 % tween-20/ tbs for 1.5 h, followed by four washes (5 rain each) with 0.05% tween-20/tbs. blots were stained using the enhanced chemiluminescence western blotting detection system (amersham corp., arlington heights, il). protein bands were quantitated using an enhanced laser densitometer (lkb instruments inc., bromma, sweden) to evaluate the relative level of proteins in each subcellular fraction. the densitometric scan value was then estimated by multiplying the relative level by the protein yield (milligrams of protein) in corresponding subcellular fraction. in calculating the percent distribution of each immunodetectable protein, the densitometric scan value of the homogenate was defined as 100%. all procedures, except for the incubation with antibodies or fitcconjugated lectin at 37°c, were carried out at room temperature. cells grown on coverslips were washed gently three times (5 rain each) with pbs, fixed with 4% paraformaldehyde in pbs containing 1 mm cac12 and 0.5 mm mgci2 [pbs(+)] for 20 rain, and permeabilized with 0.1% triton x-100 in pbs(+) for 1 h. they were then rinsed twice with pbs(+), and incubated with 2 % fbs in pbs(+) to block nonspecific binding of primary antibodies for 1 h followed by 45 rain incubation with primary antibody in 2 % fbs/pbs(+). after washing four times (5 rain each) with pbs(+), cells were incubated with secondary antibody in 2% fbs/pbs(+) for 45 min. for localization of the golgi complex, cells were incubated with fitcconjugated wheat germ agglutinin in 2% fbs/pbs(+) for 45 rain. after washing with pbs(+), they were then mounted on glass slides with 90% glycerol in pbs containing 1 m~/ml paraphenylenediamine, examined, and photographed on a microscope (bh-2; olympus corp., tokyo, japan) with ektachrome 400 film (kodak, rochester, ny). frozen ultramicrotomy was performed as described by tokuyasu (46) . transfected cells were harvested by centrifugation at 1,000 g for 3 rain, and the pellet was fixed with 4% paraformaldehyde and 0.1% glutaraldehyde discell fractionation was performed as described in materials and methods. in calculating the percent distribution, each value of the homogenate was defined as 100%. the distribution of fp2 and succinate-cytochrome c reductase(scr) is determined from the specific activities measured. the specific activities of the homogenate fractions are 52.3 + 11.0 nmol cytechrome c reduced/rain per mg protein (the mean + sd n = 4) and 26.9 + 2.8 nmol cytoehrome c reduced/min per mg protein for fp2 and succinate-cytochrome c reductase, respectively. in the previous study (24), we cloned and sequenced the fulllength cdna for rat msaldh. the nucleotide sequence predicts a polypeptide of 484 amino acids, and the most characteristic feature of this membrane-bound aldh is carboxyl-terminal 35 amino acids, consisting of a stem region (amino acids 450-463) and a hydrophobic domain (amino acids 464-480) followed by a short hydrophilic tail region (amino acids 481-484) as shown in fig. 1 . since rat cytosolic tumor-associated aldh, which is 65.5 % identical to msaldh (24) , lacks the carboxyl-terminal portion, we asked whether this portion played an important role in the intracellular localization of msaldh. for this purpose, we constructed three mutant proteins truncated at either amino acid 450 (msaldha450-484, which corresponds to the tumor-associated aldh in size), 471 (msaldha471-484, deletion of more than half of the hydrophobic domain together with the tail region), or 481 (msaldha481-484, deletion of solely the tail region) by oligonucleotide-directed mutagenesis (fig. 1) . these mutant proteins and wild-type msaldh were expressed transiently in cos-1 cells under the control of sv-40 promoter in the pcd expression vector. this transient expression system in cos-1 cells was chosen as the most rapid method for evaluating the intracellular localization of expressed proteins. cells were allowed to express these proteins for 44 h, harvested, and the expressed proteins were analyzed by immunoblotting using anti-msaldh antibody. as shown in fig. 2 a, msaldh and three truncated proteins were expressed efficiently in the total homogenates. as expected, molecular masses of the truncated proteins were smaller than that of msaldh (54 kd). in addition, no crossreactive protein was detected in untransfected cos-1 cells (data not shown), indicating the absence of endogenous msaldh. these results allowed us to investigate the intracellular localization of msaldh by transfection experiments. we analyzed the intraceuular distribution of msaldh and three truncated proteins by subcellular fractionation accordfigure 3 . effect of sodium carbonate treatment on the membrane association of wild-type msaldh and two truncated proteins. (,4) each p3 fraction was treated with 100 mm na2co3 for 30 rain at 0°c, and centrifuged at 88,000 g for 80 min to separate the pellets (p) from the supernatants (s). the distribution of msaldh, msaldha481-484, msaldha471-484, fp2, or pdi in the p and s fractions was assayed by immunoblotting. polyclonal anti-pdi antibody was used for immunodetection of endogenous pdi. (b) the relative level of each protein in the p and s fractions was quantitated by a scanning densitometer. in calculating portions recovered in the p fraction, the total level of each protein recovered in the p and s fractions was defined as 100 %. the bars show the mean + sd (n = 3). ing to the method of clark and waterman (10) . the separation of er membranes from mitochondria was checked by assaying two typical marker enzymes, fp2 (nadph-cytochrome p-450 reductase), which has been shown to be an integral er membrane protein (22) , and a mitochondrial marker succinate-cytochrome c reductase. as shown in table i, fp2 and succinate-cytochrome c reductase were enriched in the p3 fraction and the p2 fraction, respectively. in addition, when equal amounts of protein were immunoblotted, the highest levels of fp2 and mitochondria rieske iron-sulfur protein were found inthe p3 fraction and the p2 fraction, respectively (fig. 2 b) . the percent distribution of the two proteins in each subcellular fraction also confirmed a good separation of er membranes from mitochondria (fig. 2 c) . the subceuular distribution of msaldh and msaldha481-484 was almost identical to that of fp2 (fig. 2, b and c) , suggesting the er localization of these proteins in transfected cos-1 cells. on the other hand, msaldh a450-484 was found exclusively in the $3 fraction. this result indicated that the mutant protein lacking the last 35 amino acid8 of msaldh was no longer associated with intracellular membranes. curiously, msaldha471-484 was recovered not only in the $3 fraction, but also in the p3 fraction, showing an intermediate distribution between msaldh and msaldha450-484. we explored the nature of the association of the expressed proteins with microsomal membranes. upon treatment of the p3 fraction with 100 mm na2co3 (ph 11.5) (13), fp2 (the integral er membrane protein) remained attached to microsomal membranes as judged by immunoblotting (fig. 3, a and b) . wild-type msaldh and msaldi-i~481-484 were also resistant to alkali extraction, although ,x,35 % of msaldha481-484 was released. under the same conditions, >80% of msaldha471-484 and pdi (a luminal er residen0 were released, indicating a loose association of these proteins with microsomal membranes. similar results were obtained upon triton x-114 cloud point extraction (6) (data not shown). these data, together with those from subcellular fractionation, suggested that the carboxyl-terminal portion of msaldh including the hydrophobic sequence (amino acid 464-480) was necessary for both its er localization and the tight association with the er membrane. we next determined the intracellular localization of msaldh and the truncated proteins using indirect immunofluorescence microscopy. cells transfected with cdnas in the pcd vector were fixed and permeabilized 44 h after transfection, and the expressed proteins were detected by incubation with anti-msaldh antibody followed by tritc-conjugated secondary antibody. as shown in fig. 4 a, anti-msaldh antibody stained a number of large vesicular structures that surrounded the nucleus, in addition to diffuse reticular ones in cos-1 cells transfected with msaldh edna. no staining was seen at the plasma membrane and in the nucleoplasm. to explore this strange structure in more detail, we used double indirect immunofluorescence microscopy using two mabs or fitc-conjugated lectin. expressed msaldh was found to colocalize with endogenous pdi (fig. 4 b, arrow) , although pdi displayed a characteristic reticular staining pattern in cells not expressing msaldh (fig. 4 b, arrowhead) . in contrast, msaldh colocalized neither with a mitochondrial 65-kd protein (fig. 4 , c and d) nor with the perinuclear golgi complex visualized by fitc-conjugated wheat germ agglutinin (fig. 4, e and f) . these results strongly suggest the er localization of msaldh and the drastic morphological change of the er by expression of msaldh. a similar er staining pattern was observed for msaldha481-484 (fig. 4, g and h) , whereas both msaldha471-484 and msaldha450-484 were found distributed diffusely throughout the cytoplasm, and they did not colocalize with pdi (fig. 4, i-l) . in addition, a significant amount of msaldha450-484 appeared to enter the nucleus (fig. 4 k) . these immunolocalization data were consistent with those from subcellular fractionation, suggesting strongly the important role of the carboxyl-terminal portion of msaldh in its er localization. we focused our attention on the role that the carboxylterminal portion of msaldh might play in er targeting, and we asked whether this portion could direct a heterologous protein to the er membrane. for this purpose, we constructed an expression plasmid shown in fig. 5 a. the control vector (pchll0) contains e. coli/3-galactosidase, which the chimeric edna fragment with ecori sites shown by the bar was amplified by pcr and cloned in-frame to the unique ecori site in pchll0 as described in materials and methods. to the right, the intracellular location of each protein is noted as e for er or c for cytosolie. (b) cos-1 cells were transfected with pchll0 (~gal) or pchll0/aldh ~3gai-aldh), and harvested 44 h after transfection. the membrane (p) and cytosol (s) fractions were prepared by centrifugation of the postnuelear fractions at 88,000 g for 80 i rain. the membrane fraction containing/3gai-aldh was treated • with 100 mm na2co3 at 0*c for 30 min, and was centrifuged at 88,000 g for 80 min to separate the pellet (p) from the supernatant (s). each fraction was assayed by immunoblotting using mab to /3-galactosidase. was supposed to remain in the cytoplasm when expressedin cos-1 cells under the control of the sv-40 promoter. the constructed expression plasmid (pchll0/aldi-i) contains the last 35 amino acids of msaldh cloned in-frame to the 3' end of/3-galactosidase. cos-1 cells were transfected with these dnas and subjected to a crude subcellular fractionation. in this case, the postnuclear supernatant was centrifuged at 88,000 g for 80 min to separate the membrane fraction (p, containing mostly mitochondria and microsomes) from the cytosol fraction (s). immunoblotting using mab to e. coli ~-galactosidase revealed that wild-type ~-galactosidase was recovered, as expected, in the s fraction, whereas ~-galactosidase/aldh chimera was found concentrated in the p fraction (fig. 5 b) . in addition, the chimera in the p fraction was resistant to alkali extraction, indicaring a fight anchoring of this protein to intracellular membranes (fig. 5 b) . indirect immunofluorescence microscopy showed that ~-gaiactosidase was distributed diffusely throughout the cytoplasm (fig. 6 a) . however, attachment of the carboxylterminal 35 amino acids of msaldh to ~-galactosidase resulted in the reticular pattern of staining that surrounded the nucleus and extended throughout the cytoplasm (fig. 6 b) . double indirect immunofluorescence microscopy using polyclonal anti-pdi antibody showed the colocalization of /3-galactosidase/aldh chimera and pdi in the er (fig. 6 c) . these results suggested that the carboxyl-terminai 35 amino acids were sufficient for targeting of e. coli #-galactosidase to the er membrane. we attempted to define the sequence requirement for er targeting within the last 35 amino acids of msaldh, which is composed of three regions as shown in fig. l , and we constructed a series of cat/aldh chimeras (cat/aldh1-4) (fig. 7) . cat/aldh1 contains the last 35 amino acids of msaldh at the carboxyl terminus of cat, while cat/ aldh2-4 chimeras lack either the tail, stem, or both regions of msaldh. cos-1 cells were transfected with these cdnas in the pmiw expression vector that possesses /3-actin promoter and rous sarcoma enhancer. the intracellular localization of the expressed proteins were determined by indirect immunofluorescence microscopy using anti-cat antibody. wild-type cat was distributed, as expected, throughout the cytoplasm. in addition, it was also detected in the nucleoplasm (fig. 8 a) . cat/aldh1 showed the characteristic er staining pattern (fig. 8 b) , which was confirmed by double indirect immunofluorescence microscopy using pdi mab (fig. 8 c) . this result was similar to that obtained with/5-gaiactosidase/aldh (fig. 6 , b and c). both cat/aldi-i2 and cat/aldh3 were also found to colocalize with endogenous pdi in the er (fig. 8, d-g) . on the other hand, cat/aldh4 containing only the hydrophobic domain of msaldh at its carboxyl terminus showed a similar pattern of staining to that of wild-type cat. this chimera remained not only in the cytoplasm, but in the nucleoplasm as well (fig. 8 h) , and it did not colocaiize with pdi (fig. 8 i) . the simplest interpretation of these results is that both the stem and tail regions appear to contain ertargeting information of msaldh. to elucidate more narrowly the sequence requirement within the stem region for er targeting, we constructed an additional series of cat/aldh chimeras (cat/aldh5-10) (fig. 9 ), in which three to seven amino acids of the stem region of either cat/aldh1 or cat/aldi-i2 are deleted. the intracellular localization of these chimeras expressed in cos-1 cells was analyzed by indirect immunofluorescence microscopy. three chimeras, cat/aldh5, cat/aldh7, and cat/aldh9, were supposed to be directed to the er by virtue of the tail region, even if they lack an er targeting sequence within the stem region, and indeed they showed the reticu~lar staining pattern including the nuclear membrane (fig. 10 , a, c, and e). among the mutant proteins lacking the tail region, cat/aldh6 and cat/aldh10 showed the reticular pattern (fig. 10, b and f) . these reticular compartments were confirmed to be the er by colocalization with pdi (data not shown). in contrast, cat/aldh8, which lacks the lys-gln-phe sequence (amino acids 457-459) within the stem region and the tail region, was found distributed diffusely throughout the cytoplasm and the nucleoplasm (fig. 10 d) . we investigated in more detail the intracellular localization of cat and cat/aldh chimeras in transfected cos-1 cells using an igg-gold immunoelectron microscopic technique on frozen ultrathin sections. gold particles were distributed in the cytoplasm and in the nucleoplasm in cos-1 cells transfected with pmiwcat ( fig. 11 a) . a similar distribution of gold particles was observed in cos-1 cells expressing either cat/aldh4 (data not shown) or cat/ aldh8 (fig. 11 d) . in marked contrast, gold particles were predominantly detected on the cytoplasmic surface of the er membrane in cos-1 cells expressing cat/aldh1 (fig. 11 b) , cat/aldh7 (fig. 11 c) , or the other cat/aldh chimeras (data not shown). these immunoelectron microscopic data are consistent with indirect immunofluorescent data, and these immunolocalization data together strongly suggest that the er-targeting sequences of msaldh exist within the lys-gln-phe sequence in the stem region and within the lys-asp-gln-leu sequence of the tail region. we have shown here by subcellular fracfionation and indirect immunofluorescence experiments that not only msaldh, but also msaldha481-484, colocalizes with two er marker proteins, fp2 and pdi, when expressed from cdnas in cos-1 cells. surprisingly, indirect immunofluorescence microscopy revealed the apparent alteration of the er structure in cells expressing either msaldh or msaldh-a481-484. the altered er structure was characterized by large vesicular structures mainly located near the nucleus. since the unusual staining pattern was observed by expression of msaldh or msaldha481.484, it appeared that the er anchoring of these proteins led to the morphological alteration of the er. we are now investigating in more detail the altered er structure by immunoelectron microscopy, and we have recently found that the er tubules in cos-1 cells expressing msaldh are packed in crystalloid hexagonal arrays (yamamoto, a., r. masaki, and y. tashiro, manuscript in preparation). the elucidation of the altered er structure would provide interesting information on the morphological change of the intracellular organelle induced by overexpression of a resident membrane protein. we showed previously that msaldh has no amino-terminai signal sequence, but instead, that it has a longer carhoxylterminal portion than cytosolic tumor-associated aldh (24) . our truncation and domain replacement experiments have shown here that the last 35 amino acids of msaldh are necessary for its er localization and also sufficient enough for localization of two beterologous proteins, e. coli /~-galactosidase and cat, to the er. we therefore conclude that the carboxyl-terminal portion contains an er-targeting sequence of msaldh. further analyses on the intraceilular distribution of various cat/aldh chimeras have defined the er targeting sequence ofmsaldh. both cat/aldh4 and cat/aldh8 were distributed in the cytoplasm and nucleoplasm similar to wild-type cat in spite of possessing the whole hydrophobic domain (amino acids 464-480) of msaldh at their carboxyl termini, whereas the other chimeras were predominantly localized to the er. the two chimeras are lacking the lys-gln-phe sequence within the stem region and the lys-asp-gln-leu sequence of the tail region, thus suggesting that both sequences represent the er-targeting sequences of msaldh. we are now in the process of determining through mutagenesis which amino acids within these two regions are required for er targeting. on the other hand, it seems likely that the hydrophobic sequence of msaldh functions mainly as the er membrane anchor because this portion is essential for membrane anchoring. to the best of our knowledge, our data characterize the first intracellular protein that appears to have two targeting sequences separated by a membrane anchor. the ambiguous behavior of msaldha471-484 could be explained as follows. a portion of this truncated protein could be targeted to the er by virtue of the lys-gln-phe sequence within the stem region, hut because of the shortened hydrophobic domain, the portion would he either released into the cytosol or maintain only a loose association with the membrane. the significance of the presence of two separated targeting sequences is not yet clear. a small portion of msaldha481-484 was, in fact, recovered in the $3 fraction, whereas only insignificant amounts of msaldh or fp2 (fig. 2, b and c) , which is efficiently targeted to the er through the aminoterminal signal sequence, were recovered in the $3 fraction. therefore, it is likely that the presence of two targeting sequences in the same protein might be a means to ensure the more efficient er localization of msaldh. in addition, the hydrophilic tail region might also serve to increase the strength of the membrane anchoring, as indicated by the partial membrane extraction of msaldha481-484 with na2co3 (fig. 3) . although it is not yet clear whether the (24) . the single amino acid code is used. positively and negatively charged amino acids are marked + and -at the top, respectively. each hydrophobic domain is underlined and the er targeting sequences of either cytochrome b5 (23) or msaldh are indicated by broken lines. (b) two possible models for er targeting of msaldh. in model i, the aggregation of the newly synthesized msaldh polypeptides in the cytoplasm is prevented by the hydrophilic er targeting sequences adjacent to the hydrophobic anchoring sequence. the monomeric msaldh would be inserted into the er membrane with the aid of a receptor protein on the er membrane. in model ii, the complex of msaldh and a cytosolic • receptor protein would be targeted to the er, and msaldh would be inserted correctly into the er membrane spontaneously or through the interaction with a receptor protein on the membrane. hydrophobic segment fully transverses the membrane, this transmembrane disposition seems more likely to explain the tight anchoring of the protein in the er membrane than a loop model where the hydrophilic tail remains on the cytoplasmic surface. the lys-asp-gln-leu sequence of the tail region strongly resembles the well-characterized lys-asp-glu-leu retention signal found in the er luminal proteins (25) , suggesting that the lys-asp-gln-leu sequence, if translocated into the lumen of the er, serves as an er retention signal of msaldh. however, andres et al. have shown that proneuropeptide y mutant bearing the lys-asp-gln-leu sequence at the carboxyl terminus is processed and secreted like wild-type proneuropeptide y when expressed in att-20 cells, despite the intraceuular retention of the unprocessed proneuropepnuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors distribution of protein disulfide isomerase in rat hepatocytes mechanisms of integration of de novo-synthesized polypeptides into membranes: signalrecognition particle is required for integration into microsomal membranes of calcium atpase and of lens mp26 but not of cytochrome b5 variants of the carboxylterminal kdel sequence direct intracellular retention intracellular protein topogenesis phase separation of integral membrane proteins in triton x-114 solution a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein a cloning of a cdna for a major human protein-tyrosinephosphatase the hydrophobic amino-terminal sequence of bovine 17~-hydroxylase is required for the expression of a functional hemoprotein in cos-i cells the specific subcellular localization of two isoforms of cytochrome b5 suggests novel targeting pathways the nontransmembrane tyrosine phosphatase ptp 1b localizes to the endoplasmic reticulum via its 35 amino acid c-terminal sequence isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum a conserved tripeptide sorts proteins to peroxisomes a mitochondrial import factor purified from rat liver cytosol is an atf-dependent conformational modulator for precursor proteins sequence requirements for nuclear location of simian virus 40 large t antigen preparations of succinate-cytochrome c reductase and the cytochrome b-c1 particle, and reconstitution of succinate-cytochrome c reductase the biogenesis of lysosomes oligonucleotide-directed construction of mutations via gapped duplex dna cleavage of structural proteins during the assembly of the head of bacteriophage t4 cylochrome p-450 and nadph-cytochrome p-450 reductase are degraded in the autolysosomes in rat liver immunoelectron microscope localization of cytochrome p-450 reductase on microsomes and other membrane structures of rat hepatocytes the carboxy-terminal 10 amino acid residues of cytochrome b5 are necessary for its targeting to the endoplasmic reticulure molecular cloning, sequencing, and expression of cdna for rat liver microsomul aldehyde dehydrogenase a c-terminal signal prevents secretion of luminal er proteins sequences within and adjacent to the transmembrane segment of c~-2,6-sialyltransferase specify golgi retention short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum ribosome-binding protein p34 is a member of the leucine-richrepeat-protein superfamily studies on the biosynthesis of microsomal proteins: site of synthesis and mode of insertion of cytochrome bs, cytochrome b5 reductas¢, cytochrome p-450 reductase and epoxide hydrolase. fur a cdna cloning vector that permits expression of cdna inserts in mammalian cells a new method for simultaneous purification of cytochrnme b5 and nadph-cytochrome c reductase from rat liver microsomes chemical structure of rat cytochrome bs: isolation of peptides by high-pressure liquid chrom~atography biosynthetic protein transport and sorting by the endoplasmic reticulum and golgi molecular cloning of the ~-subunit of human prolyi 4-hydroxylase. this subunit and protein disulfide isomerase are products of the same gene protein translocation across the er requires a functional gtp binding site in the a subunit of the signal recognition particle receptor transport of proteins across the endoplasmic reticulure membrane mechanisms for the incorporation of proteins in membranes and organelles dna sequencing with chain-terminating inhibitors signals guiding proteins to their correct locations in mitochoudria cloning and expression ofcdna for rat heine oxygenase mechanism of increase of heine oxygenase by hemin in cultured pig alveolar macrophages the binding of cytochrome b5 to liver microsomes a mouse embryonic stem cell line showing phiripotency of differentiation in early embryos and ubiquitous ~-galactosidase expression a goigl retention signal in a membrane-spanning domain of coronavirus el protein biogenesis of microsomal aldehyde dehydrogenase in rat liver application of cryoultramicrotomy to immunocytochemistry protein translocatiou across membranes biochemical transfer of single-copy eucaryotic genes using total cellular dna as donor we thank kimie masaki for valuable technical assistance, dr. shigeru taketani for helpful comments on the manuscript, dr. akihiro inoue, national institutes for physiological science, okazaki, japan, for the generous gift of the pmiw vector.this work was supported in part by a grant-in-aid for scientific research from the ministry of education, science, and culture of japan, and by a grant from naito foundation.received for publication 12 january 1994 and in revised form 1 june 1994. tide y with the lys-asp-glu-leu sequence (4), thus suggesting the important role of the acidic glu residue in er retention. we are now studying the membrane topology of msaldh and the intracellular localization of both msaldh and msaldha481-484 in more detail by immunoelectron microscopy to check the potential role of the tail region for er retention.novel er targeting sequences have recently been reported for two other tail-anchored proteins. frangioni et al. have found that the er targeting sequence of protein tyrosine phosphatase 1b (ptp 1b) exists within the carboxyl-terminal 35 amino acids (12). mitoma and ito have shown that the last 10 amino acids of cytochrome b5, which include the hydrophilic tail of this protein, are important for its targeting to the er (23). fig. 12 a shows an alignment of the last 35 amino acids of ptp 1b (9), cytochrome b5 (32) , and msaldh (24), where each hydrophobic domain is underlined and the er-targeting sequences of cytochrome b5 and msaldh are indicated by the broken line. although the sequence required for er targeting within the last 35 amino acids of ptp 1b has not been defined, ptp 1b and cytochrome b5 have the common leu-x-tyr-arg motif ( fig. 12 a) within their last 10 amino acids. in addition, ptp 1b and cytochrome b5 have tails of similar length (seven and eight amino acid residues, respectively), which are longer than that (four amino acid residues) of msaldh. it therefore seems likely that ptp 1b possesses a similar er-targeting sequence to that of cytochrome b5. we have not found in the carboxyl-terminal portion of msaldh a motif similar to that in those two proteins, despite the fact that the ertargeting sequences of cytochrome b5 and msaldh are both hydrophilic and adjacent to the membrane anchors. in addition, the presence in msaldh of two er-targeting sequences separated by the membrane-anchoring domain suggests that this protein, with respect to the er-targeting sequence, belongs to a different class than the other two. in addition to the three proteins described above, several other er membrane proteins are known that also have carboxyl-terminal anchors, such as heine oxygenase (40) and ribosome-binding protein p34 (28), but er-targeting sequences of these proteins have not yet been identified. cytochrome bs, heme oxygenase, and msaldh are synthesized on free polysomes (29, 41, 45) and posttranslationally targeted to the er. indeed, the srp-independent integration of cytochrome b5 into microsomal membranes has been demonstrated (3). our present study, together with these findings, suggests a novel pathway by which a class of tailanchored proteins with a novel targeting sequence is directed to the er.how might the er-targeting sequence function? fig. 12 b shows two possible models to explain the er targeting of msaldh. one possibility (/) is that the hydrophilic targeting sequences serve to prevent the aggregation of the newly synthesized msaldh polypeptides through their hydrophobic anchoring sequences, which makes it easier for them finding a. specific receptor on the er membrane. another possibility (i/) is that a cytosolic receptor binds to the targeting sequences of msaldh to form a complex that effects the correct insertion of the carboxyl-terminal anchor into the er membrane spontaneously or through their interactions with a receptor on the membrane. the elucidation of the mechanisms by which er localization of msaldh occurs should provide valuable clues for an understanding of the posttranslational targeting and insertion of a class of membrane proteins with a carboxyl-terminal anchor. key: cord-193489-u6ewlh16 authors: wang, rui; hozumi, yuta; yin, changchuan; wei, guo-wei title: decoding sars-cov-2 transmission, evolution and ramification on covid-19 diagnosis, vaccine, and medicine date: 2020-04-29 journal: nan doi: nan sha: doc_id: 193489 cord_uid: u6ewlh16 tremendous effort has been given to the development of diagnostic tests, preventive vaccines, and therapeutic medicines for coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). much of this development has been based on the reference genome collected on january 5, 2020. based on the genotyping of 6156 genome samples collected up to april 24, 2020, we report that sars-cov-2 has had 4459 alarmingly mutations which can be clustered into five subtypes. we introduce mutation ratio and mutation $h$-index to characterize the protein conservativeness and unveil that sars-cov-2 envelope protein, main protease, and endoribonuclease protein are relatively conservative, while sars-cov-2 nucleocapsid protein, spike protein, and papain-like protease are relatively non-conservative. in particular, the nucleocapsid protein has more than half its genes changed in the past few months, signaling devastating impacts on the ongoing development of covid-19 diagnosis, vaccines, and drugs. the ongoing pandemic of coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) poses crucial threats to the public health and the world economy since it was detected in wuhan, china in december 2019 [1] . as of april 24, 2020, more than 2.6 million cases of covid-19 have been reported in 185 countries and territories, resulting in more than 184,000 deaths [2] . tragically, there is no sign of slowing down nor relief at this monument partially due to the fact there is no specific anti-sars-cov-2 drugs and effective vaccines. sars-cov-2 is a positive-strand rna virus and belongs to the beta coronavirus genus. the genomic information underpins the development of antiviral medical interventions, preventative vaccines, and viral diagnostic tests. the first sars-cov-2 genome was reported on january 5, 2020 [3] . it has a genome size of 29.99 kb, which encodes for multiple non-structural and structural proteins. the leader sequence and orf1ab encode non-structural proteins for rna replication and transcription. among various nonstructural proteins, viral papain-like (pl) proteinase, main protease (or 3cl protease), rna polymerase, and endoribo-nuclease are common targets in antiviral drug discovery. however, it typically takes more than ten years to put an average drug to the market. the downstream regions of the genome encode structural proteins including spike (s) protein, envelope (e) protein, membrane (m) protein, and nucleocapsid (n) protein. notably, s-protein uses one of two subunits to bind directly to the host receptor angiotensinconverting enzyme 2 (ace2), enabling the virus entry into host cells [4] . the nucleocapsid (n) protein, one of the most abundant viral proteins, can bind to the rna genome and is involved in replication processes, assembly, the host cellular response during viral infection [5] . the e protein is a small integral membrane protein, a virulence factor, regulating cell stress response and apoptosis, and promoting inflammation [6] . the structural proteins, especially, the spike protein and the n protein, are the candidate antigens for vaccine development. developing safe and effective vaccines is urgently needed to prevent the spread of sars-cov-2. however, it typically takes over one year to design and test a new vaccine. furthermore, the sars-cov-2 genome undergoes rapid mutations partially stimulated as a response to the challenging immunological environments arising from the covod-19 patients of different races, ages, and medical conditions. sars-cov-2 exists as heterogeneous and dynamic populations because of their error-prone replication [7] . the vaccine developed at one time may not be effective for mitigating the infection by new mutated virus isolates. an alarming fact is that many of these mutations may devastate the on-going effort in the development of effective medicines, preventive vaccines, and diagnostic tests. accurate identification of the antigens and their mutations represents the most important roadblock in developing effective vaccines against covid-19. for example, different vaccines are needed for different geographic locations due to predominant mutations in the corresponding regions. in covid-19 diagnosis, the diagnosis kits are designed using two major methods, i.e., specific serological tests and molecular tests. serological tests are to detect specific covid-19 proteins. molecular diagnoses test specific covid-19 pathogenic genes, which usually rely on the polymerase chain reaction (pcr).because of the fast mutations of the sars-cov-2 genome, genotyping analysis of sars-cov-2 may optimize the pcr primer design to detect sars-cov safely and reduce the risk of false-negatives caused by genome sequence variations. in addition, the genotyping analysis may also reveal those regions that are highly conserved with very few mutations, which can be selected as a target sequence for reliable drug therapy and general diagnosis. the evolution pattern through the highly frequent mutations of sars-cov-2 can be observable on short time scales. in the early infection period (i.e., february 2020), the sars-cov-2 variants were clustered as s and l types [8] . recent genotyping analysis reveals a large number of mutations in various essential genes encoding the s protein, the n protein, and rna polymerase in the sars-cov-2 population [9] . monitoring the evolutionary patterns and spread dynamics of sars-cov-2 is of grace importance for covid-19 control and prevention. although mutations occur randomly, most preserved mutations can be regarded as virus responds to the host immune system surveillance. as a result, the faster and the wider the sars-cov-2 spread is, the more frequent and diverse the mutations will be. the tracking and analysis of covid-19 dynamics, transmission, and spread is of paramount importance for winning the on-going battle against covid-19. genetic identification and characterization of the geographic distribution, intercontinental evolution, and global trends of sars-cov-2 is the most efficient approach for studying covid-19 genomic epidemiology and offer the molecular foundation for region-specific sars-cov-2 vaccine design, drug discovery, and diagnostic development [10] . for example, different vaccines for the shell can be designed according to predominant mutations. this work provides the most comprehensive genotyping to reveal the transmission trajectory and spread dynamics of covid-19 to date. based on genotyping 6156 sars-cov-2 genomes from the world as of april 24, 2020, we trace the covid-19 transmission pathways and analyze the distribution of the subtypes of sars-cov-2 across the world. we use k-means methods to cluster sars-cov-2 mutations, which provides the updated molecular information for the region-specific design of vaccines, drugs, and diagnoses. our clustering results show that globally, there are at least five distinct subtypes of sars-cov-2 genomes. while, in the u.s., there are at least three significant sars-cov-2 genotypes. we introduce mutation hindex and mutation ratio to characterize conservative and non-conservative proteins and genes. we unveil the unexpected non-conservative genes and proteins, rendering an alarming warning for the current development of diagnostic tests, preventive vaccines, and therapeutic medicines. tracking the sars-cov-2 transmission pathways and analyzing the spread dynamics are critical to the study of genomic epidemiology. temporospatially clustering the genotypes of sars-cov-2 in transmission provides insights into diagnostic testing and vaccine development in disease control. in this work, we retrieve and genotype 6156 sars-cov-2 isolates from word as of april 24, 2020. there are 4459 single mutations in 6156 sars-cov-2 isolates. based on these mutations, we classify and track the geographical distributions of 6156 genoytype isolates by k-means clustering. the sars-cov-2 genotypes, represented as snp variants, are clustered as five groups in the world table 2 . the genotypes in the u.s. are clustered as three groups table 2 . the optimal clustering groups are established using the elbow method in the k-means clustering algorithm (see supporting material). the detailed distribution of the snp variants from the world for each cluster is provided in the supporting material. the snp variant clusters from 11 countries that have the highest number of cases recorded in are listed in table 2 figure 1 . the geographic distribution of the snp variant clusters reflects the approximate transmission pathways and spread dynamics across the world. several findings can be read from the table 2: 1. two early subtypes of sasr-cov-2 (cluster i and ii) are epidemic in the asian countries (cn, jp, kr). 2. the subtypes of sars-cov-2 in cluster iii is not spreading in the european countries (uk, de, fr, it). 3. all of the subtypes of sars-cov-2 in five different clusters can be found in the us, au, and canada. moreover, we analyze the statistic of snp variants located in different states of the united states. in table 3 , we list the number of cases in three different clusters with respects to the west coast states we note that cluster c in the u.s. is derived from cluster iii in the world, with an additional mutation at the leader sequence 241. the high spread in new york is consistent with the high transmission of sars-cov-2 in the european countries, where the subtype in cluster iii is predominated. table 5 presents the statistics of single mutations on various sars-cov-2 proteins that occurred in the recorded genomes between january 5, 2020, and april 24, 2020. the papain-like protease has the highest number of mutations of 599 while the envelope protein has the lowest number of mutations of 13. since the sizes of proteins vary dramatically from 1945 for the papain-like protease to 75 for the envelope protein, it is useful to consider the mutation ratio, i.e., the number of mutations per residue. in this category, the envelope protein still has the lowest score of 0.17, whereas the nucleocapsid protein has the highest score of 0.56, i.e, 235 mutations on its 419 residues. note that 3cl protease has the second-lowest mutation ratio of 0.22, indicating its conservative nature. another relatively conservative protein judged by the mutations ratio is the rna-dependent rna polymerase. it has 223 mutations over its 932 residues. counting the number of single mutations and mutation ratio does not reflect the fact some mutations occur numerous times over genome samples while other mutations may happen only on a few genome samples. to account for the frequency effect of mutations, we introduce a mutation h-index to measure both the number of mutations and the frequency of mutations of a given protein or genetic section. it is defined as the maximum value of h such that the given protein genetic section has h single mutations that have each occurred at least h times. it is very interesting to note from table 5 that the mutation h-index correlates very well with the number of mutations per residue. specifically, nucleocapsid protein has both the highest number of mutations per residues of 0.56 and the highest h-index of 27, suggesting that it is the most non-conservative protein in sars-cov-2 genomes. in contrast, the envelope protein has the lowest number of mutations per residues of 0.17 and the lowest h-index of 5, indicating its relatively conservative nature. by combining the number of mutations per residue and the mutation h-index, we report that the three most conservative sars-cov-2 proteins are 1) the envelope protein, 2) the main protease, and 3) the endoribonuclease. it is found that the most non-conservative sars-cov-2 proteins are 1) the nucleocapsid protein, 2) the spike protein and 3) the papain-like protease. real-time rt-pcr (rrt-pcr) is routinely used in the qualitative detection of nucleic acid from sars-cov-2 for diagnostic testing covid-19 [11, 12] . the primers used in the rrt-pcr are critical for the precise diagnosis of covid-19 and the discovery of new strains. the primer sequences are specially designed for amplifying the conserved regions across the different existing strains for high specificity and sensitivity, and also are subject to genotype changes as the sars-cov-2 coronavirus evolves. in diagnostic testing covid-19, many rrt-pcr primers are designed to detect for three perceived conservative sars-cov-2 regions: (1) rna-dependent rna polymerase (rdrp) gene in orf1ab region, (2) the e protein gene, and (3) the n protein gene [11] . our genotyping statistics given in table 5 indicates that the nucleocapsid protein is the worst choice. among four structural proteins of sars-cov-2, the spike surface glycoprotein (s) of 1273 amino acid residues, nucleocapsid protein (n) of 419 amino acid residues, membrane protein (m) of 222 amino acid residues, envelope protein (e) of 75 amino acid residues, the s protein is the most divergent with 385 unique mutations among the 6156 sars-cov-2 genomes. the n protein has 235 unique mutations, the e protein has 13 mutations. considering the lengths of the proteins, all the four structural proteins undergo high mutations. the rdrp gene, which is often used in diagnostic testing covid-19, also has 223 mutations. therefore, all the three regions in routine rrt-pcr target, namely rdrp, the n protein gene, and the e protein genes, have significant mutations. precise and robust diagnosis tools must be re-established according to the conserved regions and predominated mutations in the sars-cov-2 genomes detailed in the supporting material. notably, sars-cov-2 has a unique furin cleavage site, where four amino acid residues (prra) are inserted into the s1-s2 junction region 681-684 of the s protein [13] . the furin cleavage site is crucial for zoonotic transmission of sars-cov-2 [14] . this study reveals crucial mutations near the s1-s2 junction region in the s protein, including 23403a>gmoreover, these mutations of the s protein sars-cov-2 are located at the epitope region, corresponding to the regions 469-882 and 599-620 in sars-cov) [15] . additionally, many mutated amino acids are on the surface of the s protein as shown in fig. 5 . unfortunately, the s protein is the second most non-conservative protein in the genome based on the number of mutations per residue and mutation h-index. in fact, about half of the receptor-binding domain residues of the s proteins have had mutations in the past few months as shown in fig. 6 . because the surface accessibility of epitope is also important for the interaction of antibody and antigen, these mutations are critical for the antigenicity of the s protein. the convalescent covid-19 patients show a neutralizing antibody response after infection, which are directed against the s protein or the n protein [16] . the neutralizing antibody responses against sars-cov-2 could give some defense against sars-cov-2 infection and thus, having implications for preventing sars-cov-2 outbreaks. the divergence of spike proteins, the non-conserved regions of the spike proteins might contribute to the antigenicity. the high frequent mutations identified in the s protein and the n proteins must be considered when designing a vaccine. unfortunately, there is no specific effective drug for sars-cov-2 at this point. much of the drug discovery effort focuses on sars-cov-2 non-structural proteins. among the major non-structural proteins of sars-cov-2, the main protease of 306 amino acids has 68 mutations with 0.22 mutations per residue and the mutation h-index of 9, rna polymerase of 932 amino acids has 223 mutations with 0.24 mutations per residue and the mutation h-index of 13, and papain-like protease of 1945 amino acids has 599 mutations with 0.31 mutations per residue and the mutation h-index of 15. in fact, the main protease is the most popular drug target because there are no similar known genes in the human genome, which implies sars-cov-2 main protease inhibitors will be likely less toxic [17] . the present study suggests that the main protease is the second most conservative protein. therefore, it remains the most attractive target for drug discovery. the sars-cov-2 spike glycoprotein, or s protein, comprised of two subunits, s1 and s2, of very different properties [13] , see fig. 5 . among them, the s1 subunit, as shown in fig. 5 , contains the receptor-binding domain (rbd) responsible for binding to the host cell receptor angiotensinconverting enzyme 2 (ace2). the rbd is also the common binding domain for antibodies. the s2 subunit offers the structural support of the s protein and mediates fusion between the viral and host cell membranes. after the fusion, the virus releases the viral genome into the host cell. the s1 rbd protein plays key parts in the induction of neutralizing-antibody and t-cell responses, as well as protective immunity. however, s2 and extracellular domain (ecd) of spike protein and their combination are commonly used in recombinant proteins in sars-cov-2 antibody development. table 5 , the s protein is the most heterogeneous structural protein with a significant number of mutations as shown in figs. 5 and 6 and table 6 . the divergence of the spike protein, the non-conserved regions of the spike protein might contribute to the antigenicity difference in sars-cov-2 isolates. we found that most of the high frequent mutations of the s protein are located in the s1 subunit. figure 6 indicates that near half of the amino acid residues have had mutations since january 5, 2020. one of the important mutations at s1 is 23010 (v483a) within the rbd for ace2 binding. the structural study revealed that the amino acids 442-487 in the s1 subunit may impact viral binding to human ace2 [18, 19] . the mutations identified in this study imply the change in ace2 binding affinity and the transmissibility of sars-cov-2 as well as negative impacts in preventive vaccine and diagnostic test development. top1 10323 k90r 52 1 8 43 0 0 top2 10097 g15s 51 0 1 0 50 0 top3 10851 a266v 44 0 2 16 0 26 top4 10582 d176d 19 0 0 5 0 14 top5 10771 y239y 15 15 0 0 0 0 top6 10507 n151n 11 0 10 1 0 0 top7 10948 r298r 11 0 0 0 11 0 top8 10265 g71s 9 0 0 0 9 0 top9 10870 l272l 9 0 0 1 4 4 top10 10319 l89f 8 0 1 4 0 3 top11 10450 p132p 8 main protease sars-cov-2 main protease, or 3cl protease, is essential for cleaving the polyproteins that are translated from the viral rna [17] . it operates at multiple cleavage sites on the large polyprotein through the proteolytic processing of replicase polyproteins and plays a pivotal role in viral gene expression and replication. sars-cov-2 main protease is one of the most attractive targets for anti-cov drug design because its inhibition would block viral replication and it is unlikely to be toxic due to no known similar human proteases. another reason for the focused drug discovery efforts in developing sars-cov-2 main protease inhibitors is that this protein is relatively conservative as shown in table 5 . figure 7 illustrates the main protease mutation patterns. figure 8 further highlights the inhibitor binding domain (bd). indeed, the main protease is relatively conservative compared to the spike protein. table 7 lists top 11 mutations and their frequency in our dataset. it is interesting to see that many mutations, such as y239y, n151n, r298r, l272l, and p132p, are degenerate ones. one possible explanation is that nondegenerate may be non-silent and likely cause unsurvivable disruption to the virus. note that mutation g15s mostly occurs in cluster iv. mutation y239y is restricted to cluster i. some other mutations, such as r298r, g71s, and p132p, are specific to certain clusters. nonetheless, some mutations at the bd shown in fig. 8 are worth noting. they can undermine the ongoing drug discovery effort. top1 3037 f106f 3889 0 80 1800 964 1045 top2 2891 a58t 120 0 119 0 1 0 top3 3177 p153l 72 0 69 2 1 0 top4 4540 y607y 60 0 60 0 0 0 top5 7011 a1431v 45 0 43 2 0 0 top6 6312 t1198k 44 0 42 1 1 0 top7 7438 y1573y 34 0 9 21 4 0 top8 3373 d218d 29 0 3 0 26 0 top9 4002 t428i 26 0 1 0 25 0 top10 6040 f1107f 26 0 10 12 0 4 figure 9 : illustration of sars-cov-2 papain-like protease mutations using 6w9c as a template [20] . papain-like protease sars-cov-2 papain-like protease (plpro) is a cysteine cleavage protein located within the non-structural protein 3 (ns3) section of the viral genome [20] . like, the main protease, plpro activity is required to cleave the viral polyprotein into functional, mature subunits and, thereby, contributes to the biogenesis of the virus replication. additionally, plpro possesses a deubiquitinating activity. the sars plpro is also a major therapeutic and diagnostic target. as shown in table 5 , the sars plpro is prone to mutations. figure 9 shows that mutations are all over the places in plpro. table 8 lists top ten mutations in plpro. five of these mutations are degenerate ones, including one of the highest frequented mutations. note that none one of the top mutations occurred in cluster i. on the contrast, cluster ii has many different mutations. [21] . as one of the non-structure proteins, rdrps located in the early part of orf1b section. like most other rna viruses, sars-cov-2 rdrps are considered to be highly conserved to maintain viral functions and thus targeted in antiviral drug development as well as diagnostic tests. on the other hand, the sars-cov-2 rna polymerase lacks proofreading capability and thus its mutations are deemed to happen as shown in table 5 . figure 10 illustrates the sars-cov-2 rdrp mutations since january 5, 2020. surprisingly, there are many mutations in sars-cov-2 rdrp. table 9 describes the top ten mutations. as in other cases, five of these mutations are degenerate ones. cluster i has no nondegenerate mutations. endoribo-nuclease (nendou) protein is a nidoviral rnauridylate-specific enzyme that cleaves rna [22] . it contains a c-terminal catalytic domain belonging to the endou family rna processing. the nendou protein is presented among coronaviruses, arteriviruses, and toroviruses. the many aspects of the detailed function and activity of sars-cov-2 nendou protein are yet to be revealed. figure 11 depicts sars-cov-2 nendou protein mutations. like in most other sars-cov-2 proteins, mutations have occurred over different parts. table 5 shows that nendou is relatively conservative. table 10 lists the top twelve high-frequency mutations of the sars-cov-2 nendou protein that occurred in the past few months. three of these mutations are degenerate ones. the frequencies of these mutations range from 38 to 6. note that cluster i do not have any of these mutations. total frequency cluster i cluster ii cluster iii cluster iv cluster v top1 26319 v25v 8 0 7 1 0 0 top2 26340 a32a 7 0 5 2 0 0 top3 26326 l28l 5 0 5 0 0 0 top4 26256 f4f 4 0 0 2 2 0 top5 26301 l19l 3 0 2 1 0 0 top6 26433 k63k 2 0 0 1 0 1 top7 26370 y42y 1 0 1 0 0 0 top8 26392 s50g 1 0 1 0 0 0 top9 26313 f23f 1 the sars-cov-2 envelope (e) protein is one of sars-cov's four structural proteins. as a transmembrane protein, it involves in ion channel activity, and thus facilitates viral assembly, budding, envelope formation, pathogenesis, and release of the virus [23] . the e protein may not be essential for viral replication but it is for pathogenesis. figure 12 illustrates e protein as a very small pentamer with a few mutations. table 11 shows its top thirteen mutations. note that the first 7 mutations are degenerate ones. all other mutations have very low frequencies. as shown in table 5 , the sars-cov-2 e protein is very conservative. total frequency cluster i cluster ii cluster iii cluster iv cluster v top1 28881 r203k 989 1 20 4 964 0 top2 28882 r203r 983 0 18 1 964 0 top3 28883 g203r 983 0 18 1 964 0 top4 28657 d128d 125 1 124 0 0 0 top5 28311 p13l 102 0 101 1 0 0 top6 28688 l139l 91 0 90 1 0 0 top7 29045 p258t 67 0 65 2 0 0 top8 29046 p258r 67 0 65 2 0 0 top9 29047 p258p 67 0 65 2 0 0 top10 29049 r259l 67 0 65 2 0 0 top11 29050 r259r 67 0 65 2 0 0 top12 29051 q260e 67 0 65 2 0 0 top13 29052 q259r 67 0 65 2 0 0 top14 29053 q260h 67 0 its primary function is to encapsidate the viral genome. to do so, it is heavily phosphorylated (or charged) and thereby, can bind with rna. additionally, sars-cov-2 n protein confirms the viral genome to replicasetranscriptase complex (rtc) and plays a crucial role in viral genome encapsulation. therefore, it may function completely differently at different stages of the viral life cycle. sars-cov-2 n protein is considered to be one of the most conservative sars-cov-2 proteins in the literature and is a popular target for diagnosis of vaccine development [11] . the present works shown in table 5 indicates the sars-cov-2 n protein is the worst target of any drug, vaccine, and diagnostic development. table 12 presents the top fourteen mutations of the sars-cov-2 n protein since january 5, 2020. note that only three out of fourteen top mutations are degenerate ones, which is a significantly lower ratio than that of other proteins. the frequency of 14th mutation is 67, which suggests there are many mutations associated with these mediate-sized proteins. most top mutations occurred to clusters ii and iv. cluster v has none of the top fourteen mutations. membrane protein sars-cov-2 membrane (m) protein is another structural protein and plays a central role in viral assembly and viral particle formation. it exists as a dimer in the virion and has certain geometric shapes to enable certain membrane curvature and binding to nucleocapsid proteins. similar to other sars-cov proteins, m protein is also a popular target for viral diagnosis and vaccines. table 5 gives sars-cov-2 m protein the meddle ranking for its conservation. table 13 details the top eleven mutations in sars-cov-2 m protein occurred in the past few months. seven of these mutations are degenerate. clusters i and v have relatively a few of these mutations. on january 5, 2020, the complete genome sequence of sars-cov-2 was first released on genbank (access number: nc 045512.2) by zhang's group at fudan university [3] . since then, there has been a rapid accumulation of sars-cov-2 genome sequences. in this work, 6156 complete genome sequences with high coverage of sars-cov-2 strains from the infected individuals in the world are downloaded from the gi-said database [25] (https://www.gisaid.org/) as of april 24, 2020. all the records in gisaid without the exact submission date will not take into considerations. to rearrange the 6156 complete genome sequences according to the reference sars-cov-2 genome, multiple sequence alignment (msa) is carried out by using clustal omega [26] with default parameters. snp genotyping measures the genetic variations between different members of a species. establishing the snp genotyping method for the investigation of the genotype changes during the transmission and evolution of sars-cov-2 is of great importance. by analyzing the rearranged genome sequences, snp profiles which record all of thesnp positions in teams of the nucleotide changes and its corresponding positions can be constructed. the snp profiles of a given genome of a covid-19 patient capture all the differences from a complete reference genome sequence and can be considered as the genotype of the individual sars-cov-2. the jaccard distance measures dissimilarity between sample sets. the jaccard distance of snp variants is widely employed in the phylogenetic analysis of human or bacterial genomes [9] . in this work, we utilize the jaccard distance to compare the difference between the snp variant profiles of sars-cov-2 genomes. the jaccard similarity coefficient, also known as the jaccard index, is defined as the intersection size divided by the union of two sets a, b [27] : the jaccard distance of two sets a, b is scored as the difference between one and the jaccard similarity coefficient and is a metric on the collection of all finite sets: therefore, the genetic distance of two genomes corresponds to the jaccard distance of their snp variants. in principle, the jaccard distance measure of snp variants takes account of the ordering ofsnp positions, i.e., transmission trajectory, when an appropriate reference sample is selected. however, one may fail to identify the infection pathways from the mutual jaccard distances of multiple samples. in this case, the dates of the sample collections offer useful information. additionally, clustering techniques, such as kmeans described below, enable us to characterize the spread of covid-19 onto the communities. k-means clustering is one of the fundamental unsupervised algorithms in machine learning which aims at partitioning a given data set x = {x 1 , x 2 , â· â· â· , x n , â· â· â· , x n }, x n â�� r d into k clusters {c 1 , c 2 , â· â· â· , c k }, k â�¤ n such that the specific clustering criteria are optimized. more specifically, the standard k-means clustering algorithm starts to pick k points as cluster centers randomly and then allocates each data to its nearest cluster. the cluster centers will be updated iteratively by minimizing the within-cluster sum of squares (wcss) which is defined by: where âµ k = i â�� c k x i /n k is the mean of points located in the k-th cluster c k and n k is the number of points in c k . here, â· 2 denote the l 2 distance. the algorithm above only provides a way to obtain the optimal partition for a fixed number of clusters. however, we are interested in finding the best number of clusters for the snp variants. therefore, the elbow method is applied. by varying the number of clusters k, a set of wcss can be calculated in the k-means clustering process, and then the plot of wcss according to the number of clusters k can be carried out. the location of the elbow in this plot will be considered as the optimal number of clusters. to be noticed, the wcss measures the variability of the points within each cluster which is influenced by the number of points n . therefore, as the number of total points of n increases, the value of wcss becomes larger. additionally, the performance of k-means clustering depends on the selection of the specific distance. in this work, we propose to implement k-means clustering with the elbow method for analyzing the optimal number of the subtypes of sars-cov-2 snp variants. the jaccard distance-based and locationbased representations are considered as the input features for the k-means clustering method. suppose we have a total of n snp variants concerning a reference genome in a sars-cov-2 sample. the location of the mutation sites for each snp variant will be saved in the set s i , i = 1, 2, â· â· â· , n . the jaccard distance between two different sets (or samples) s i , s j is denoted as d j (s i , s j ). therefore, the n ã�n jaccard distance-based representation will be: suppose we have n snp variants with respect to a reference genome in a sars-cov-2 sample. among them, m different mutation sites can be counted. for i-th snp variant, v i = [v 1 i , v 2 i , â· â· â· , v m i ], i = 1, 2, â· â· â· , n is a 1 ã� m vector which satisfies: therefore, an n ã� m location-based representation will be: hundreds of complete genome sequences are deposit to the gisaid every day, which results in an evergrowing massive quantity of high dimensional data representations for the k-means clustering. for example, if the dataset of an organism involves 10,000 snps, the initial representation will be a 10,000dimensional vector for each sample, which can be computationally difficult for a simple k-means clustering algorithm. therefore, a dimensionality reduction method is used to pre-process the data. the essential idea of pca-based k-means clustering is to invoke the pca to obtain a reduced-dimensional representation of each sample before performing the k-means clustering. in practice, one can select a few lowest dimensional principal components as the k-means input for each sample. in ref. [28] , the authors proved that the principal components are the continuous solution of the cluster indicators in the k-means clustering method, which provides us a rigorous mathematical tool to embed our high-dimensional data into a low-dimensional pca subspace. the rapid global transmission of coronavirus disease 2019 (covid-19) has offered some of the most heterogeneous, diverse, and challenging mutagenic environments to stimulate dramatic genetic evolution and response from severe acute respiratory syndrome coronavirus 2 (sars-cov-2). this work provides the most comprehensive genotyping of sars-cov-2 transmission and evolution up to date based on 6156 genome samples and reveals five clusters of the covid-19 genomes and associated mutations on eight different sars-cov-2 proteins. we introduce mutation h-index and mutation ratio to qualify individual protein's degree of non-conservativeness. we unveil that sars-cov-2 envelope protein, main protease, and endoribonuclease protein are relatively the most conservative, whereas, sars-cov-2 nucleocapsid protein, spike protein, and papain-like protease are relatively the most non-conservative. we report an alarming fact that all of the sars-cov-2 proteins have undergone intensive mutations since january 5, 2020, and some of these mutations may seriously undermine ongoing efforts on covid-19 diagnostic testing, vaccine development, and drug discovery. the nucleotide sequences of the sars-cov-2 genomes used in this analysis are available, upon free registration, from the gisaid database (https://www.gisaid.org/). eighteen tables are provided in the supporting material for snp variants of 6156 sars-cov-2 samples across the world, snp variants of 1625 sars-cov-2 samples in the us, snp variants in five global clusters, snp variants in three us clusters, and mutation records for eight sars-cov-2 proteins. the acknowledgments of the sars-cov-2 genomes are also given in the supporting material. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 who. coronavirus disease 2019 (covid-19) situation report 93. coronavirus disease (covid-2019) situation reports a new coronavirus associated with human respiratory disease in china the sars-cov s glycoprotein: expression and functional characterization the coronavirus nucleocapsid is a multifunctional protein severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis the population genetics and evolutionary epidemiology of rna viruses origin and evolution of the 2019 novel coronavirus genotyping coronavirus sars-cov-2: methods and implications models of rna virus evolution and their roles in vaccine design detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr diagnosing covid-19: the disease and tools for ddtection structure, function, and antigenicity of the sars-cov-2 spike glycoprotein furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry a strategy for searching antigenic regions in the sars-cov spike protein coronavirus infections: epidemiological, clinical and immunological features and hypotheses structure of mpro from covid-19 virus and discovery of its inhibitors. biorxiv sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus the crystal structure of papain-like protease of sars cov-2 structure of the rna-dependent rna polymerase from covid-19 virus structural model of the sars coronavirus e channel in lmpg micelles crystal structure of rna binding domain of nucleocapsid phosphoprotein from sars coronavirus 2. center for structural genomics of infectious diseases (csgid) gisaid: global initiative on sharing all influenza data-from vision to reality clustal omega. current protocols in bioinformatics distance between sets k-means clustering via principal component analysis this work was supported in part by nih grant gm126189, nsf grants dms-1721024, dms-1761320, and iis1900473, michigan economic development corporation, bristol-myers squibb, and pfizer. the authors thank the ibm tj watson research center, the covid-19 high performance computing consortium, and nvidia for computational assistance. key: cord-014852-6friw2ek authors: chumakov, s. p.; prassolov, v. s. title: organization and regulation of nucleocytoplasmic transport date: 2010-04-24 journal: mol biol doi: 10.1134/s0026893310020020 sha: doc_id: 14852 cord_uid: 6friw2ek separation of dna replication and transcription, which occur in the nucleus, from protein synthesis, which occurs in the cytoplasm, allows a more precise regulation of these processes. selective exchange of macromolecules between the two compartments is mediated by proteins of the nuclear pore complex (npc). receptor proteins of the karyopherin family interact with npc components and transfer their cargos between the nucleus and cytoplasm. nucleocytoplasmic transport pathways are regulated at multiple levels by modulating the expression or function of individual cargoes, transport receptors, or the transport channel. the regulatory levels have increasingly broad effects on the transport pathways and affect a wide range of processes from gene expression to development and differentiation. since eukaryotic genetic material is localized in a separate compartment (the nucleus), dna replica tion and transcription are spatially separated from protein synthesis, which occurs in the cytoplasm. the spatial separation provides the cell with additional opportunities to finely regulate the processes and, on the other hand, requires the highly selective exchange of many macromolecules between the nucleus and cytoplasm. exchange is due to a variety of receptor transport proteins, which interact with components of the nuclear pore complex (npc) to transport bound proteins between the two compartments. each of the receptors is involved in its specific transport pathway, transferring a certain set of substrates (cargoes). the state of a substrate also plays an important role in the transfer, since a cargo must have necessary signal sequences to properly bind to its receptor. the major ity of cell signaling pathways are involved in signal transduction between the nucleus and cytoplasm. thus, detailed knowledge of the organization and reg ulation of nucleocytoplasmic transport is essential for the understanding of cell metabolism and functional activity. macromolecular exchange between the nucleus and cytoplasm is mediated by nuclear pores. selective transport of molecules is due to the npc. nuclear pores were first identified in 1950, when the nuclear membrane was examined by electron microscopy [1] . further studies showed that holes in the nuclear mem brane are occupied by the npc, whose structure is evolutionarily conserved [2] . the npc is a protein complex of 40-60 mda that has sevenfold symmetry and consists of more than 30 proteins known as nucle oporins (nup). the spatial structure of the npc was established by tomographic reconstruction. the npc consists of a cylindrical central framework, eight microfilaments attached to it at the cytoplasmic side, and a nuclear basket of eight filaments, which are attached to the framework at the nuclear side and are distally bound with each other. the central part of the npc has a channel, which resembles a sand glass in shape and has a diameter of approximately 45 nm in the narrowest region ( fig. 1 ) [3] . the cytoplasmic filaments of the npc are approx imately 35 nm in length; the nuclear filaments, which form the nuclear basket, are 60 nm. the length of the npc including the central framework of 50 nm is approximately 150 nm; its outer diameter is 125 nm [4] . npc components slightly vary in size among organisms but the general structure and proportion are the same [5] . the npc number on the nuclear membrane broadly varies depending on the cell size and activity. one yeast cell has approximately 200 npcs, while actively proliferating human cells have an npc den sity of 10-20 npcs/μm 2 , corresponding to 2000-5000 npcs per nucleus. mature oocytes of the spur toed frog xenopus have 60 npcs/μm 2 , corresponding to 5 × 10 7 npcs per cell [6] . the npc density increases as the cell progresses through the cell cycle [7] . one npc transmits 1000 molecules per second on average; i.e., at least ten molecules simultaneously pass through one nuclear pore. proteins of up to 39 nm can pass through the nuclear pore. proteins of less than 30-40 kda freely diffuse through the nuclear pore, while larger proteins (40-100 kda) must have a nuclear localization signal (nls) or a nuclear export signal (nes) to be transferred from the cytoplasm into the nucleus or backwards. the signals are bound by transport receptors, which are capable of passing through the npc both alone and in complex with another protein [8] . highly efficient (more efficient than simple diffu sion) transfer of a protein of any size into the nucleus requires the npc mediated active transport mecha nisms [9] . this transport depends on either affinity of the target protein for npc components or its com plexation with the receptor proteins that have such affinity. kariopherins, which are the largest class of transport receptors, include importins, exportins, and transportins, which are involved in both nuclear import and export. importin β (karyopherin 95 in yeasts) is one of the best studied karyopherins. this protein binds with cargo molecules through the adap tor protein importin α (karyopherin 60 in yeasts) and is capable of interacting with several nup proteins [10] . according to a typical mechanism of interactions of karyopherins with a target protein and nup, a short motif (nls or nes) is recognized in the target pro tein and is bound by karyopherins or intermediate adaptor proteins, which then interact with karyo pherins. the resulting protein complex may interact with the nup proteins contained in the npc to enter the nucleus or leave it. proteins of the same size that lack a nls/nes are incapable of passing across the npc barrier. energy for transportation of complexes through the npc is generated by a high gradient of the ran gtpase between the nucleus and cytoplasm. nuclear rangtp is capable of releasing the target protein from an imported complex upon binding with karyo pherins. exportins occurring in the nucleus interact with their targets in the presence of rangtp, which is involved in transferring the complex through the npc into the cytoplasm, where rangtp is hydrolyzed to disassemble the transport complex and to replenish the cytoplasmic rangdp pool [11] . proteins of the karyopherin family are involved in the majority of specific nucleocytoplasmic transport pathways. there are at least 20 karyopherins in human cells and 14 in yeast cells [12] . target proteins that are transferred by karyopherins into or out of the nucleus usually have an nls or nes. the main, or classical, nls structure was established in studies of the nls of the sv40 large t antigen. this nls consists of one or two positively charged amino acid clusters, which are connected via a neutral linker [6] . further studies revealed many nlss that lack this classical structure. functional activity of an nls may be regulated via posttranslational modification or signal masking as a result of conformational changes. the classical nls binds with the adaptor protein karyopherin α (importin α), which forms a hete rodimer with importin β. in turn, importin β ensures nuclear import [13] . nls binding is due to a karyo pherin α region that contains ten arm repeats, each consisting of 40 amino acid residues forming three α heli ces. the region assumes a superhelical structure, which has a shallow groove, which accommodates a nls. an nls is accommodated in the acceptor pocket of karyopherin α owing to its certain charge and hydrophobicity. binding with karyopherin α, one nls contacts several arm repeats [14, 15] . most proteins of the karyopherin β family bind with a target protein directly. the primary structures of nlss recognizable by karyopherin β are far more diverse than the structures of nlss interacting with importin α. an nls often has a consensus sequence of several positively charged amino acid residues. such sequences were found in the nlss of histones, riboso mal proteins, and certain rna binding proteins. in some cases, a minimal functional nls is rather large. for instance, the m9 nls consists of 38 amino acid residues, is enriched in glycine, and includes only a few positively charged residues. even longer nlss are known, indicating that a certain spatial structure of the karyopherin binding region plays an important role in complex formation [16] . karyopherins that mediate nuclear export recognize ness [9, 11] . minimal func tional ness have been identified for several proteins. a moderately conserved short motif with three or four hydrophobic amino acid residues (lpplerltl in the rev nes) has been studied in most detail to date. this nes is utilized in all eukaryotes and serves as a binding site for the karyopherin crm1. the nes was found in at least 75 proteins, including many tran scription factors, cell cycle regulators, and virus pro teins, such as rev and proteinase k inhibitor of the human immunodeficiency virus type 1 (hiv 1), where this signal was identified for the first time. like importin β1, crm1 is capable of transferring com plexes through the npc, binding its target via an adaptor protein [18] . certain proteins have nonhydrophobic ness. yeast msn5p is the best known karyopherin that binds to such sites [12] . this transportin is capable of medi ating both nuclear export and import of various pro teins [19] . export of all known targets of msn5p is induced by their phosphorylation, indicating that a phosphate group is contained in the nes or that phos phorylation indirectly affects the recognition of the nes by a receptor [20] . some exportins utilize other karyopherins as tar gets. for instance, cas exports karyopherin α from the nucleus, thus, bringing it back into the cytoplasm [21] . in addition to proteins, certain rnas are subject to karyopherin dependent export. at least two export ins, exportin t and exportin 5, are capable of directly binding rna. exportin t exports trna from the nucleus by binding to a trna structural element act ing as a signal. in higher eukaryotes, exportin 5 trans fers microrna precursors from the nucleus upon recognizing their hairpin structure with a protruding 3' end [22] . the main properties that allow karyopherins to ensure efficient nuclear transport are their capabilities of binding with a target protein (directly or through an adaptor) and interacting with nup or rangtp [12] . almost all karyopherins known in humans and yeasts are involved in either export or import. two proteins (human importin 15 and yeast msn5p) are exceptions, having both exporting and importing activities. known importins are more numerous than exportins. for instance, ten importing, two exporting, and one universal karyopherins were identified in yeast cells. karyopherin targets are far more numerous than karyopherins. a clear view of the situation is just emerging, since karyopherins recognize the localiza tion signals having low structural homology ( table 1) . all karyopherins are similar in molecular weight (96-146 kda), charge, and domain structure. a ran binding domain is usually at the n end, a nup binding domain is in the center, and a target protein binding domain is at the c end. structural studies of four dif ferent karyopherins showed that a karyopherin mole cule contains approximately 20 heat repeats, each consisting of 40 amino acid residues that form two antiparallel α helices linked by a turn. the repeats stack together to form superhelical arches at both ends of the protein [23] . one karyopherin may form differ ent complexes with different target proteins; i.e., the karyopherin molecule has many binding sites, each recognizing a certain motif. in addition, karyopherins may change their conformation depending on the tar get protein [24] . this circumstance explains why one karyopherin transfers several proteins lacking homol ogous regions [25] . transport both importins and exportins bind with rangtp. importins bind with rangtp with high affinity, and the target protein is consequently released from its complex with an importin. exportins, whose function requires the formation of a ternary complex with rangtp and a cargo, have low affinity for rangtp when not bound with a target protein [9, 11] . a study of the atomic structure for the complexes of importin β1 and karyopherin β2 with rangtp showed that rangtp binds similarly with both receptors. rangtp interacts with a concave on the n terminal arch of importins, and its binding site has two spatially separate domains, one being closer to the n end of the arch, and the other closer to its c end [26] . the c terminal domain that contacts rangtp is a negatively charged loop between two heat domains. the same loop was found to bind with proteins transported by karyo pherin β2. this circumstance may explain why the target protein is released from its complex with impor tin once transferred into the nucleus (and in contact with rangtp). mutations of the c terminal rangtp binding domain result in a loss of the asso ciation between karyopherin binding with rangtp and the release of a cargo. thus, a contact of rangtp with this region may regulate the formation of the importin-cargo complex [23] . structural studies of the exportin cse1p in complex with rangtp and its cargo karyopherin α clearly demonstrate the difference in rangtp binding between import and export complexes. cse1p forms a superhelix with n and c terminal arches, which clamp around rangtp on both sides. simulta neously, cse1p binds with karyopherin α and fixes it in a conformation that prevents cargo binding. karyo pherin α also forms bonds with rangtp in this com plex. a feature of cse1p-rangtp interaction is that the n terminal arch alone can form a weak bond with rangtp, but the complex becomes sufficiently stable only when rangtp binds with both n and c termi nal arches, which is conformationally possible only when cse1p is bound with cargo. the resulting ternary complex resembles a tight spring, which stretches in the cytoplasm upon rangtp hydrolysis to release the cargo karyopherin α [27] . the exportin crm1 is similar in several structural features to karyopherin β2. like the c terminal rangtp binding site of karypherin β2, crm1 has a flexible loop in the middle of the polypeptide chain. the domain is thought to shield the binding site for target proteins and, simultaneously, to prevent crm1 from forming a stable complex with rangtp. how ever, when crm1 binds with a target protein or rangtp, the loop changes its position and stimulates the formation of a stable ternary complex. it is possible that conformational changes of a flexible loop underlie a general mechanism of the formation and dissocia tion of transport complexes [28] . at the same time, the exportin cse1p lacks regions similar to the crm1 flexi ble loop, indicating that the mechanism is not univer sal [27] . structural studies of karyopherin β1 in complex with the phenylalanine-glycine (fg) repeat contain ing fragment of yeast nup showed that the interactions between the two proteins are mostly hydrophobic and involve the phenylalanine residues of nup. the karyo pherin molecule forms two hydrophobic pockets: between heat repeats 5 and 6 and between repeats 6 and 7. the pockets capture the amino groups of the phenylalanine residues contained in the fg repeats of nup [29] . a similar nup binding region occurs at the c end of karyopherin β1 [30] . there is evidence that importing karyopherins have higher affinity for the nup proteins located at the nuclear side of the npc, while exporting karyopherins have higher affinity for cytoplasmic nup proteins [31] . a deletion analysis of all fg containing nup proteins (with a yeast model) made it possible to construct npcs having minimal sets of the fg domains. it was found by this means that asymmetrical fg domains (which occur exclusively at the cyptoplasmic or nuclear side of the npc) have no functional role. the role of asymmetrical nup proteins in nuclear transport is still unclear. the main role in nuclear transport is ascribed to the ran gtpase, which controls the formation and disso ciation of transport complexes. ran activity is strongly regulated by evolutionarily conserved proteins, which regulate both the intracellular distribution of ran and the transition between the gtp and gdp associated forms [6] . although ran occurs predominantly in the nucleus, it is continuously delivered into the cyto plasm at a high rate (10 5 molecules per second), mostly as a component of export complexes [32] . re import of ran is due to ntf2. ntf2 interacts with rangdp, which is the main cytoplasmic form of ran. the ntf2-rangdp complex is transferred into the nucleus, which is due to the ability of ntf2 to interact with low affinity with fg containing nup proteins, as karyopherins do. the rangdp transfer is unidirec tional because rangdp is rapidly converted to rangtp on the nuclear surface of the npc. since ntf2 binds to the switchii region of rangdp and this region has another conformation in rangtp, the ntf2-rangtp transport complex dissociates, and ntf2 is recycled into the cytoplasm [33] (fig. 2) . rangdp is converted to rangtp in the nucleus, where ran interacts with the ran guanine exchange factor (rangef). rangef is associated with chro matin, occurs in the nucleus at approximately one copy per nucleosome, and directly interacts with nucleosomal histones h2a and h2b. rangef stimu lates the exchange of gdp for gtp because a rangef loop (β wedge) penetrates into ran and releases gdp. binding with rangef fixes ran in the free form for a period long enough for ran to bind gtp contained in the nucleoplasm [34] . although no preference for gtp is observed for nucleotide exchange in the presence of rangef in vitro, ran-gtp binding in vivo is more likely owing to a higher gtp : gdp ratio in the nucleoplasm. rangef does not always occur in a chromatin associated form. its free form is detectable in the cell both in the interphase and during mitosis [35] . how ever, only chromatin associated rangef is capable of stimulating gtp-gdp exchange in complex with ran. the t42n ran mutant, which is incapable of nucleotide exchange, leads to a fixation of free rangef on chromatin [35] . it is most likely that gtp binding to ran is necessary for a detachment of the two proteins from chromatin. in addition, the associ ation of such exchange complexes with chromatin plays an important role in mitosis, since a high local concentration of rangtp is necessary for the proper assembly of microtubules in the vicinity of the chro mosome surface [36] . high affinity interactions between rangtp and karyopherins leaving the nucleus take place both when cargoes are transported into the cytoplasm (with exportins) and when imported karyopherins are recyc led into the cytoplasm. in either case, a rangtpkaryopherin complex is moved onto the cytoplasmic surface of the npc and then interacts with rangap, which substantially (approximately by five orders of magnitude in vitro) increases the originally weak gtpase activity of ran. this interaction causes gtp hydrolysis to yield rangdp. rangdp in complex with a karyopherin is not fully accessible for rangap, and ranbp1 facilitates the rangtp-rangap bind ing via either generating a ranbp1-rangtp-karyo pherin intermediate complex or releasing rangtp from karyopherin [37] . the cytoplasmic localization of rangap and the nuclear localization of rangef underlie the mecha nism that maintains a high gradient of rangtp between the nucleus and cytoplasm [38] . a fluores cence resonance energy transfer (fret) analysis showed that the difference between rangtp concen trations in the nucleus and cytoplasm is more than two orders of magnitude [36] , which agrees well with mod eling results. rangtp plays an important role in nuclear trans port. a high rangtp concentration in the cytoplasm would distort nuclear import, causing premature dis sociation of import complexes. the cell has mecha nisms to ensure the proper location of the ran regula tors. rangef mediated exchange of gdp for gtp occurs exclusively in the nucleus owing to two import pathways [39] . rangtp hydrolysis occurs in the cyto plasm, since rangap is too large to enter the nucleus through the npc. rangap can be sumoylated (covalently bound with a protein of the sumo family), and the resulting form has affinity for cyto plasmic nup358 [40] . ranbp1 is small enough to enter the nucleus through the npc via diffusion. however, ranbp1 has a nes recognizable by the exportin crm1, which ensures continuous active export of ranbp1 appearing in the nucleus via diffu sion into the cytoplasm. rangtp and karyopherins are the main means of nuclear transport. it should be noted, however, that the eukaryotic cell has several accessory factors that affect the transport efficiency. for instance, the effi ciency of karyopherin α dependent nuclear import is improved upon npap60 binding, which increases karyopherin α affinity for importin β1. appearing in the nucleus, the npap60-importin β1-karyopherin α-nls cargo complex is affected by rangtp and cas, which acts as an exportin to recycle karyopherin α into the cytoplasm. the import complex is cleaved into two complexes, npap60-importin β1-rangtp and cas-karyopherin α-rangtp. a feature of the npap60 effect is that npap60 differently binds with importin β1 during import. it is thought that npap60 has a structure of a tri stable switch [41] . ranbp3, which is a npap60 analog involved in export, is struc turally similar to npap60 and binds with the exportin crm1 to increase its affinity for rangtp and nes containing proteins, thus improving the efficiency of crm1 dependent export [42] . nucleoporins: their structure and functions the npc consists of more than 30 different nup proteins, each occurring in at least eight copies [43] . some nup proteins are predominantly included in the npc, while some others shuttle between the nucleus and cytoplasm and are only associated with the npc for a short while [44] . the fg repeats, which are found in one third of all nup proteins, are particularly important in the func tioning of npc. there are approximately 128 fg domains containing 3500 fg repeats in one npc. fg domains are not distinctly structured and are thought to line the inner wall of the nuclear channel. all known transport receptors bind with fg repeats, which are essential for nuclear transport [11] . the interaction with a transport receptor involves mostly the phenyl alanine ring of the core fg region; the ring binds with hydrophobic amino acid residues on the surface of a transport receptor [45] . hydrophilic regions, which occur between individual fg motifs and account for a major part of an fg domain, allow several fg regions of one domain to bind with a receptor [46] and, pre sumably, are necessary for modulating the receptor binding (table 2) . structural studies of the fg domains in yeast nup proteins by biophysical methods showed that the domains are unfolded and lack a distinct secondary structure in natural conditions [47] . similar data were obtained for the fg domains of nup proteins of other organisms [48] . as was revealed by electron micros copy, structures containing fg domains are flexible and are capable of moving along the npc. atomic force microscopy (afm) showed that the human nup153 fg domain (700 amino acid residues) occurs as a long (180 nm) unstructured sequence [49] . the spatial structure of the npc changes dynami cally, adapting to nuclear transport requirements. two main types of npc conformations were identified by cryoelectron microscopy. conformations of one type are characterized by the cytoplasmic filaments extended towards the central channel of the npc. the filaments apparently interact with a protein complex transferred though the npc. conformations of the other type have stretched unfolded cytoplasmic fila ments [50] . the arrangement of certain functional domains of nup changes in the course of nuclear transport. for instance, the c terminal end of the fg domain of nup214 moves onto the nuclear side of the npc when polyadenylated rna is introduced into the nucleus, while the n end remains associated with cytoplasmic fibrils [50] . the fg domain of nup153 similarly moves along the npc as cargoes are transported [51] . in addition to their role in nucleocytoplasmic transport, the fg domains may perform other func tions. for instance, the fg repeats of the rrm domain of mouse nup35 have a distinct secondary structure and do not interact with transport receptors. these domains interact with the ndc1 transmembrane protein and, possibly, are involved in forming the cen tral framework of the npc [52] . nup proteins may play a role in transcriptional reg ulation by interacting with active genes. this assump tion is supported by the fact that npcs are not regu larly distributed through the nuclear membrane, but their distribution corresponds to the distribution of active chromatin within the nucleus. such a distribu tion is necessary for a correct rearrangement of the chromosomes and nuclear envelope during mitosis and for mrna export through specific npcs in the interphase [53] . further studies revealed that yeast genes whose transcription is increasing move to the periphery of the nucleus, which is due to their binding with certain nup proteins [54] . in addition, several nup proteins, such as nup2p, bind to loci at the boundary between euchromatin and heterochromatin, thus preventing heterochromatin from spreading to transcriptionally active regions. as was revealed more recently, nup2p binds to chromatin within a complex consisting of nup2p, nup60p (an element of the npc nuclear basket), prp20 (a yeast analog of rangef), and the htz1 histone protein [55] . htz1 is responsible for the prevention of hetero chromatin spreading [56] . nup2p interacts with the promoters of functionally active genes, and this inter action depends on transcriptional activators and the tata box located 5' of them [57] . thus, the npc may serve as a multifunctional regulator of gene expression by distributing transcription activation signals and checking the quality of spliced mrna. nup proteins can act as a platform for the attach ment of various transport factors. for instance, nup358, which is a component of the cytoplasmic fibrils of the npc, provides a platform for the cleavage of transport complex and a subsequent recycling of transport factors. the nup358-ranbp1 complex has four ran binding sites and a binding site for the sumoylated ran activator (rangap1). to bind to this complex, rangap1 must be sumoylated, while ranbp1 has sumoylating activity. acting in complex with the e2 ligase ubc9 similarly to the e3 ligase, ranbp1 stabilizes rangap1 on the cytoplasmic fibrils [40] . rangap1 is capable of stimulating hydrolysis of rangtp associated with export com plexes, which is essential for nuclear export. some nup proteins bind with proteins possessing desumoylating activity, such as senp2 (yeast ulp1) [58] . there is evidence that ulp1 binds with nup60p and mlps located on the nuclear side of the npc [59] . other data indicate that ulp1 is anchored on the npc by npc associated karyopherins [58] . this possibly facilitates desumoylation of hnrnps that were sumoylated on the cytoplasmic side of the npc and then transferred into the nucleus. yet this assumption disagrees with the data that ubc9 occurs on the nuclear side of the npc as well, possibly binding with nup153p [59] . apart from sumoylation, the dead box helicase dbp5 plays a substantial role in separating hnrnp from mrna. dbp5 also occurs on the cytoplasmic surface of the npc in complex with nup214 (yeast nup159p). atpase activity of dbp5 depends on the binding of gle1, an mrna export factor capable of binding to the npc, and is stimulated by inositol hexaphosphate (ip 6 ) [54] . in yeast cells, dbp5 is bound with mex67, which is an analog of tap/nxf1. the level of mex67 bound rna is elevated in cell lines with mutant dbp5, implicating dbp5 in the mex67 recycling to the nucleus [60] . thus, both karyopherin dependent export and mrna export depend on a protein stimulating ntpase activity (rangap1 in the case of ran gtpase and gle1/ip 6 in the case of atpase activity of dbp5). moreover, the interaction of these activators with a substrate is determined by specific binding sites of cytoplasmic nup proteins and is regulated by addi tional cofactors (sumoylation and ip 6 ). hierarchic regulation of nuclear transport nuclear transport is regulated by several mecha nisms, which are organized hierarchically. a flow of proteins transferred between the nucleus and cyto plasm changes in response to various signals, such as hormones, cytokines, and growth factors, as well as signals regulating the cell cycle, differentiation, and the immune response, and in stress. modification of signal molecules via phosphorylation or dephosphory lation, which is the most clearly understood mecha nism regulating nuclear transport, may involve many kinases and phosphatases [61] . kinases and phos phatases are regulated by many cell signals, directly linking external signaling factors and intracellular sig nals with changes in the nuclear import or export of signal molecules, such as cell cycle regulators, kinases, and transcription factors. many of these proteins have both nes and nls, which allow a fine regulation of their intracellular localization by changing the effi ciency of nuclear export or import [62] . one of the key steps of nuclear transport is the interaction of importins and exportins with the nls or nes of a target protein. changes in nls or nes accessibility via inter or intramolecular masking are one of the most common mechanisms modulating the efficiency of nuclear transport of a particular protein. intramolecular masking occurs when the accessibility of the nls or nes decreases as a result of conforma tional changes in the protein containing the given site. an example of such regulation is provided by the nf κb p50 transcription factor. the factor occurs in the cyto plasm in the form of a p105 precursor, which has an nls inaccessible for binding with importin α. during the immune response, phosphorylation and degrada tion of the c terminal fragment of p105 unmasks the nls, which then binds with importin α to allow active transfer of nf κb into the nucleus [63] . conformational changes that result from disulfide bonding between the amino groups of cysteine resi dues in one protein may also mask or unmask the nes or nls. for instance, a disulfide bond is formed in stress in the yeast transcription factor yap1p between cys598 and cys620, which belong to a cysteine rich region. an nes, which is in the same region, conse quently becomes inaccessible for xpo1p [64] . such masking takes place when binding with another protein makes the localization signals inac cessible for transportins. an example is provided by the nf at4 transcrip tion factor. at a higher ca 2+ concentration, nf at4 binds with the ca 2+ responsive phosphatase cal cineurin, which masks the crm1 binding nes of nf at4. when the ca 2+ concentration decreases, calcineurin dissociates from nf at4, the nes becomes accessible for crm1, and crm1 transfers nf at4 from the nucleus [65] . the nuclear localization of the p53 tumor suppres sor is regulated by several mechanisms. one of these is p53 homotetramerization in the nucleus, which occurs in response to dna damage [66] and masks the c terminal ness. it is essential for a nuclear export of p53 that the tetramer dissociate and the c terminal ness be unmasked [67] . ligand binding may also mask the nls or nes of a receptor. for instance, the nes of the androgen receptor is close to its ligand binding domain. upon binding with the ligand, the nes becomes inaccessi ble for crm1, and nuclear export is suppressed until the ligand dissociates from the receptor [68] . an inter molecular masking of an nls or nes is possible upon dna or rna binding. hiv 1 rev, which transfers nonspliced virus mrna from the nucleus into the cytoplasm, masks its own importin β2 dependent nls upon mrna binding [69] . a release of mrna restores the ability of rev to bind with importin β2, which then recycles rev into the nucleus. rev binds again with virus mrna, this binding facilitates rev dissociation from its complex with importin β2, and a new round of mrna nuclear export starts. the yeast gal4 transcription factor and human sry chromatin remodeling factor have dna bind ing domains that overlap their nlss [70] . binding to dna prevents their association with importin β2 and vice versa. it is possible that this mechanism is alterna tive to a rangtp mediated release of cargoes from transport complexes. the mechanism is effective when the local rangtp concentration is too low or ran activ ity is suppressed by high ca 2+ concentrations [71] . many various rnas are expressed in the nucleus. to be exported from the nucleus, rnas undergo post transcriptional modification, which is necessary for successful interactions with proteins of the transport complex. for instance, trnas acquire the capability of binding with exportin t at the last step of their maturation in the nucleus, and this capability ensures the export from the nucleus only for mature trnas. several cell mrnas are exported owing to cis regula tory elements. in mouse cells, certain retroviral tran scripts leave the nucleus owing to the constitutive transport element (cte), which is capable of binding directly to the mnxf1 export receptor [9] . the mrnas of several genes involved in the cell cycle con trol have a β1 untranslated region (utr) that is recogni zed by the translation initiation factor eif4e, which binds to the 3' utr and mediates the mnxf1 depen dent export of these transcripts [72] . in addition to an nes, mrna may contain an nls, which provides for an additional mechanism of gene expression regulation. the 3' utr of the msf cytokinin gene contains a sequence that holds this mrna in the nucleus. further posttranscriptional modification in response to tgf β1 activates the export of the msf mrna and msf synthesis [73] . signal elements may regulate the rna nuclear import as well. for instance, an 8 nt sequence is necessary and sufficient for the nuclear import of the mir 29b microrna [74] . the export of mature mrnas and ribosomal sub units results from a series of standard modifications essential for the binding with export receptors. during maturation, mrna interacts with various proteins to form mrnp. the composition of this complex changes in the course of mrna splicing, capping, and polyadenylation. mature mrna is capable of inter acting with the mnxf1-nxt1 (yeast mex67-mtr2) transport complex, which is necessary for cytoplasmic export [75] . the mrnp transfer through the npc depends on ip 6 , thus allowing phospholipase c to regulate the mrna nuclear export. similar modifica tions necessary for the export from the nucleus occur in rrna as well [75] . phosphorylation of proteins at sites close to an nls or nes is capable of not only masking these sig nals, but also improving protein affinity for importins and exportins. for instance, ck 2 kinase phosphoryla tion of the t antigen at ser111/112, which are adja cent to the nls, results in a 100 fold increase in nls affinity for importin α, and this leads to a 50 fold increase in the nuclear import of the t antigen [76] . phosphorylation may affect the nuclear export as well. for instance, phosphorylation of pho4 at ser114 and ser128 improves the recognition of its nes by the msn5p exportin [77] . in addition to phosphorylation, target proteins may experience other types of posttranslational modifica tion, such as methylation and ubiquitination, which are also capable of regulating the intracellular location of the protein. for instance, the proper nuclear import of rna helicase a requires methylation of its nls, and pten phosphatase does not efficiently enters the nucleus until monoubiquitinated at certain lysine res idues [78] . ubiquitinase ubcm2 appears in the nucleus only in an activated ubiquitinated form. this example sup ports the idea that the functional state of an enzyme may affect its intracellular localization [79] . another mechanism regulating nuclear transport is a binding of nls or nes containing proteins with cytoplasmic or nuclear factors that anchor the bound proteins to retain them in the cytoplasm or nucleus. for instance, p53 is retained in the cytoplasm in the absence of stress signals owing to its binding with parc ubiquitin ligase. excess synthesis or inhibition of parc modulates the cytoplasmic localization of p53 [80] . the hiv 1 transactivator tat, the vascular growth factor angiogenin, and the interferon dependent tran scription factor ifi16 are retained in the nucleus via nls dependent binding with nuclear or nucleolar components. angiogenin is small enough to enter the nucleus via passive diffusion, and its nls does not interact with importins, but binds with nuclear pro teins. as a result, angiogenin is retained in the nucleus, and its backward diffusion into the cytoplasm is pre vented [81] . such anchorage is due to nls phospho rylation in some cases. for instance, affinity of the ifi16 nls for nuclear proteins increases as a result of phosphorylation by the ck 2 kinase. in contrast to these two proteins, tat has an nls that has affinity for both nuclear and cytoplasmic anchoring proteins, and its affinity depends on protein modification [82] . different members of the importin family, espe cially importins α, have affinity for different groups of transport targets in higher eukaryotic cells. moreover, different transport complexes, for instance, β katenins or stat family proteins, pass through the npc via different pathways, binding exclusively with the fg repeats of a certain set of nup proteins [83] . thus, the presence or absence of a particular nup or karyopherin can determine whether a certain protein is transferred through the nuclear pore. a difference in the tissue distribution of transport proteins affects the efficiency of nuclear transport of the same proteins. an example is provided by the drosophila melanogaster heat shock protein dhsf, which is transferred into the nucleus by importin α3. importin α3 is absent in early embryo development, and, consequently, dhsf does not enter the nucleus [84] . differences in the tissue distribution of importins α are especially clear in higher eukaryotes. for instance, importin α4 accounts for more than 1% of total pro tein in human striped muscle cells (i.e., its content is 100 fold higher than that of importin α5) and is almost absent in heart, spleen, and kidney cells [85] . in contrast, importin α1 is abundant in the heart, testis, skeletal muscle, and ovary, while importins α3 and α7 occur at a high content in the ovary and brain. the levels of mrna expression in one tissue greatly vary among different importins α; however, a high content of impor tin α6 is only characteristic of ovarian cells [86] . competition between different karyopherins β for binding sites on the npc surface may also be subject to regulation. the contents and composition of karyo pherins β and their targets change during the cell life, affecting the efficiency of nucleocytoplasmic trans port. a higher content of a particular karyopherin β increases the transport efficiency of its targets [87] , the saturation point differing between individual karyo pherins β. since different transport receptors are capable of interacting with the same sites on the npc surface, an excess of one karyopherin β may inhibit the transport of proteins associated with another karyo pherins. for instance, the import in cultured cells is approximately tenfold lower than in systems in vitro, possibly, because artificially reconstructed systems lack the majority of competing karyopherins β [87] . the regulation of nucleocytoplasmic transport pathways changes in certain diseases. overproduction of karyopherin β and karyopherin α family proteins was observed in several colorectal, breast, and lung cancers. deregulation of karyopherin α2, which is frequent in melanoma and breast cancer cells, corre lates with a low survival. it is still unclear which karyo pherin α2 dependent proteins are redistributed to determine the high malignant potential of these tumors. a possible cause is that karyopherin β1 is sequestered by overproduced truncated karyopherin α2 to alter the karyopherin α1 dependent import of p53 into the nucleus [88] . the antiviral response is similarly altered in cells infected with the ebola virus or the avian influenza virus (sars cov) [89] . hodgkin's lymphomas are characterized by excess phosphorylation and degradation of i κb, which results in an extremely high nuclear content of nf κb p65 because the intermolecular masking of its nls is deregulated [90] . gametogenesis is well understood and is known to depend, to a substantial extent, on the nonuniform expression of various importin α genes. each of the three d. melanogaster importins α has its own expres sion pattern during spermatogenesis. importin α3 is syn thesized predominantly in the postmeiotic phase, reaching its maximum during spermatid elongation. the level of importin α1 remains low throughout the mitotic phase of spermatid development and is com pletely suppressed at spermatid elongation. importin α2 is actively synthesized in spermatogonia during the first four mitoses and during the two subsequent meioses. flies with a mutant importin α2 (imp α2 d14 ) have a low fertility. an elevated level of importins α1 and α3 in transgenic flies is capable of restoring the male fer tility, indicating that these importins may functionally substitute importin α2 to a certain extent during sper matogenesis. however, the fertility of imp α2 d14 mutant females is not restored at higher levels of other importins, suggesting a key role in oogenesis for importin α2 [91] . сaenorhabditis elegans importins are synthesized differentially in different cells. importins α1 and α2 occur mostly in germline cells, while importin α3 is found in somatic cells as well. suppression of importin α3 synthesis via rna interference blocks meiosis in pachytene. it seems that importins α1 and α2 are incapable of compensating for lack of importin α3, indicating that importin α3 is essential for successful meiosis. inhibition of importin α2 leads to aneup loidy, an improper chromatin organization during cell division, and an incomplete restoration of the nuclear membrane after meiosis [92] . in lower eukaryotes, different importins are critical at different stages of gametogenesis, indicating that the proper organization of nuclear transport is impor tant for meiosis. an essential developmental role of crm1 depen dent nuclear export was demonstrated in experiments with leptomycin b, which acts as a specific inhibitor of crm1. when nondifferentiated gonad explants from female mouse embryos were treated with leptomycin b, the sox9 chromatin remodeling factor was redis tributed into the nucleus, as characteristic of nondif ferentiated male gonads [93] . the sox9 content in the nucleus increased, which was apparently related to a role of crm1 in the export of sox9 into the cytoplasm. while crm1 and importins β2 and β3 occur in all cells in drosophila, exportin dcas, which is responsi ble for a recycling of importin α from the nucleus into the cytoplasm, is differentially expressed in different tissues. importin α synthesis is almost absent at the mid blastodermal stage. its level increases at subse quent stages, especially in embryonic nervous cells. mutations of dcas result in either a lethal phenotype or, in the case of hypomorphic dcas, developmental defects of the nervous system. such developmental defects are possibly caused by a lack of cytoplasmic importin α3 and a consequent decrease in the con centration of the notch regulated su9(h) protein in the nucleus [94] . nup proteins differ in affinity for importins and exportins, and, consequently, changes in the levels of certain nup proteins may affect the efficiency of nuclear transport. lack of nup98b alters the nuclear import of all proteins possessing the classical nls, except for the spliceosomal factor u1a [95] . nup bs 63, which is functionally associated with the ranbp2/nup358 complex, is synthesized exclu sively in spermatids [96] . nup bs 63 is capable of interacting with the af10 chromatin remodeling fac tor, which is contained in postmeiotic cells. factors similar to af10 may enter the nucleus via direct inter actions with certain nup proteins, as is the case with β catenins and smad family proteins. thus, nup bs 63 possibly provides a specific binding site for af10 in haploid sperms [97] . the npc, a dynamically changing macromolecu lar complex, can mediate both passive transport and active transport of specific complexes. some nup pro teins act as active components of a transport pathway. during mitosis, the npc presumably dissociates into several subcomplexes differing in nup composi tion. a functional npc is again reassembled from these subcomplexes in telophase [53] . in lower eukaryotes, whose mitosis proceeds with out a disassembly of the nuclear membrane, certain nup proteins (gle2/rae1 and nup98 in aspergillus) are separated from the npc to improve its permeabil ity [98] . in saccharomyces cerevisiae, the npc is not disas sembled in mitosis even partly, but nup53, which is bound with nup170 in the interphase, is phosphory lated and transferred onto nic96. as a result, a high affinity binding site for karyopherin 121 is opened in nup53, and karypherin 121 dependent nuclear trans port is suppressed [99] . during mitosis, several nup proteins (nup107-160, nup358, ranbp2, and gle2/rae1-nup98) bind with the kinetochores and play a role in regulating the interactions between the kinetochores and microtu bules, which are also necessary for the npc formation after mitosis [100] . as was revealed in experiments with xenopus, gle2/rae1 induces the assembly of microtubules into a mitotic spindle. according to other data, gle2/rae1-nup98 inhibits the anaphase promoting complex (apc) to delay anaphase [101] . dynamic changes in the npc composition affect the transmission of intracellular signals. the regula tion of nuclear transport is usually modulated by changes in the karyopherin interactions with target proteins. in particular, conformational changes in a protein or posttranslational modification of sites in the vicinity of or within the nls (or nes) may mask these signals. however, npcs are possibly capable of serving several independent transport pathways at the same time [102] . if so, structural and functional changes in the npc may provide for a fine regulation of the transmission of certain signals. this hypothesis is supported by the results of studying the independent changes in nuclear import of various proteins (fig. 3) . the npc contains approximately 128 fg domains with several thousands of fg repeats [103] . the fg repeats harbor various binding sites for transport pro teins. the multiplicity of fg associated transport posttranscriptional modification mrna splicing, rnp maturation, trna maturation nuclear retention due to the signal sequences of the 3' utr maturation of ribosomal subunits intra and intermolecular interactions nls or nes masking via homo and heterodimerization n p c t r a n s p o r t r e c e p t o r s c a r g o fig. 3 . nucleocytoplasmic transport may be regulated at the levels of npc components, transport receptors, or individual car goes. the regulations of higher hierarchic levels exert broader and less specific effects on transport processes. pathways is evident from the fact that only some of the nuclear import pathways are altered in yeast cells with functionally defective fg nup nsp1 s5 [104] . transport pathways that involve different karyo pherins need different sets of fg domains [105] . this finding well correlates with in vitro data. therefore, different pathways of transport through the npc have a common mechanism, but utilize different karyo pherin sites and may be regulated independently. experimental studies on amphibian oocyte nuclei: 1. investigation of the structure of the nuclear membrane by means of the electron microscope the ultrastructure of the nuclear envelope of amphibian oocytes: a reinves tigation: 2. the immature oocyte and dynamic aspects the nuclear pore com plex: nucleocytoplasmic transport and beyond snapshots of nuclear pore complexes in action captured by cryo electron tomography yeast nuclear pore complexes have a cytoplasmic ring and internal fila ments transport between the cell nucleus and the cytoplasm cell cycle dependent dynamics of nuclear pores: pore free islands and lamins the nuclear pore complex: oily spa ghetti or gummy bear? nucleocytoplasmic trans port: taking an inventory mechanisms of receptor mediated nuclear import and nuclear export regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle karyo pherins: from nuclear transport mediators to nuclear function regulators importin alpha: a multipurpose nuclear transport receptor crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin alpha autoinhibition by an internal nuclear localization signal revealed by the crystal structure of mammalian importin alpha nuclear import and the evolution of a multi functional rna binding protein the hiv 1 rev activation domain is a nuclear export signal that accesses an export path way used by specific cellular rnas nuclear export of ribosomal subunits the karyopherin kap142p/msn5p mediates nuclear import and nuclear export of different cargo proteins regulation of nuclear localization: a key to a door export of importin alpha from the nucleus is mediated by a specific nuclear transport factor microrna precursors in motion: exportin 5 mediates their nuclear export karyopherins and nuclear import molecular basis for the recognition of a nonclas sical nuclear localization signal by importin beta conformational variability of nucleo cytoplasmic transport factors structure of the nuclear transport complex karyopherin beta2 ran × gpp nhp structural basis for the assembly of a nuclear export complex architecture of crm1/exportin1 sug gests how cooperativity is achieved during formation of a nuclear export complex structural basis for the interaction between fxfg nucleoporin repeats and importin beta in nuclear trafficking importin beta contains a cooh terminal nucleoporin binding region important for nuclear transport gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import charac terization of ran driven cargo transport and the rangtpase system by kinetic measurements and computer simulation structural basis for the interaction between ntf2 and nucleoporin fxfg repeats structural basis for guanine nucleotide exchange on ran by the regulator of chromosome condensation (rcc1) a mechanism of coupling rcc1 mobility to rangtp production on the chromatin in vivo visualization of a ran gtp gradient in interphase and mitotic xenopus egg extracts co activation of rangtpase and inhibition of gtp dissociation by ran gtp binding protein ranbp1 the asymmetric distribution of the constituents of the ran system is essential for transport into and out of the nucleus nuclear import of the ran exchange factor, rcc1, is mediated by at least two distinct mechanisms the nucleoporin ranbp2 has sumo1 e3 ligase activity npap60/nup50 is a tri stable switch that stimulates importin alpha:beta mediated nuclear protein import ran binding protein 3 is a cofactor for crm1 mediated nuclear protein export the yeast nuclear pore com plex: composition, architecture, and transport mechanism nup98 is a mobile nucleoporin with tran scription dependent dynamics association of nuclear pore fg repeat domains to ntf2 import and export complexes structural basis for the high affinity binding of nucleoporin nup1p to the saccharomyces cerevisiae importin beta homologue disorder in the nuclear pore complex: the fg repeat regions of nucleoporins are natively unfolded rapid evolution exposes the boundaries of domain structure and func tion in natively unfolded fg nucleoporins from the trap to the basket: getting to the bottom of the nuclear pore complex nuclear pore complex structure and dynamics revealed by cryoelectron tomography nucle oporin domain topology is linked to the transport sta tus of the nuclear pore complex the crystal structure of mouse nup35 reveals atypical rnp motifs and novel homodimerization of the rrm domain pushing the envelope: structure, function, and dynamics of the nuclear periphery dynamic nuclear pore complexes: life on the edge the mobile nucleoporin nup2p and chromatin bound prp20p function in endogenous npc mediated transcrip tional control con served histone variant h2a.z protects euchromatin from the ectopic spread of silent heterochromatin nup pi: the nucle opore promoter interaction of genes in yeast unconventional tethering of ulp1 to the transport channel of the nuclear pore complex by karyopherins enzymes of the sumo modification pathway localize to filaments of the nuclear pore complex the dead box protein dbp5p is required to dissociate mex67p from exported mrnps at the nuclear rim nuclear target ing signal recognition: a key control point in nuclear transport? regulation of nuclear transport: central role in development and transfor mation? pro cessing of the precursor of nf kappa b by the hiv 1 protease during acute infection regulation of the yeast yap1p nuclear export signal is mediated by redox sig nal induced reversible disulfide bond formation nf at activation requires suppression of crm1 dependent export by cal cineurin con struction of chimeric tumor suppressor p53 resistant to the dominant negative interaction with p53 mutants a leucine rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by nes masking identification and characterization of a ligand regulated nuclear export signal in androgen receptor inhibition of nuclear import mediated by the rev arginine rich motif by rna molecules the c ter minal nuclear localization signal of the sex determin ing region y (sry) high mobility group domain medi ates nuclear import through importin beta 1 a sox9 defect of calm odulin dependent nuclear import in campomelic dys plasia/autosomal sex reversal eif4e is a central node of an rna regulon that governs cellular proliferation the expression of migration stimulating factor, a potent oncofetal cytok ine, is uniquely controlled by 3' untranslated region dependent nuclear sequestration of its precursor mes senger rna a hex anucleotide element directs microrna nuclear import exporting rna from the nucleus to the cytoplasm the protein kinase ck2 site (ser111/112) enhances recognition of the simian virus 40 large t antigen nuclear localiza tion sequence by importin roles of phosphoryla tion sites in regulating activity of the transcription fac tor pho4 ubiquitination regulates pten nuclear import and tumor suppression ubiquitin charging of human class iii ubiq uitin conjugating enzymes triggers their nuclear import parc: a cytoplasmic anchor for p53 novel properties of the nucleolar targeting signal of human angiogenin the hiv 1 tat nuclear localization sequence confers novel nuclear import properties nucleocytoplasmic shuttling by nucleoporins nup153 and nup214 and crm1 dependent nuclear export control the subcellular dis tribution of latent stat1 develop mental regulation of the heat shock response by nuclear transport factor karyopherin alpha3. develop ment cloning and characterization of hsrp1 gamma, a tissue specific nuclear transport factor evidence for distinct substrate specificities of importin alpha family members in nuclear protein import sim ple kinetic relationships and nonspecific competition govern nuclear import rates in vivo. in vivo truncated form of importin alpha identified in breast cancer cell inhibits nuclear import of p53 severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane nuclear transport and cancer: from mechanism to intervention drosophila melanogaster importin alpha1 and alpha3 can replace importin alpha2 during spermatogenesis but not oogenesis a role for caenorhabditis elegans importin ima 2 in germ line and embryonic mitosis a nuclear export signal within the high mobility group domain regulates the nucleocytoplasmic translocation of sox9 during sexual determination dcas is required for importin alpha3 nuclear export and mechano sensory organ cell fate specification in drosophila disruption of the fg nucleoporin nup98 causes selective changes in nuclear pore complex stoichiometry and function characterization and potential function of a novel testis specific nucle oporin bs 63 expression pattern and cellular distribution of the murine homo logue of af10 partial nuclear pore complex disassembly during closed mitosis in aspergillus nidulans cell cycle regulated transport controlled by alterations in the nuclear pore complex the nuclear pore comes to the fore the rae1 nup98 complex prevents aneuploidy by inhibiting securin degradation nuclear trans port of single molecules: dwell times at the nuclear pore complex pores for thought: nuclear pore complex proteins analysis of nucleo cytoplasmic trans port in a thermosensitive mutant of nuclear pore pro tein nsp1 minimal nuclear pore complexes define fg repeat domains essential for transport key: cord-016419-v1f6dx3e authors: gupta, varsha; sengupta, manjistha; prakash, jaya; tripathy, baishnab charan title: production of recombinant pharmaceutical proteins date: 2016-10-23 journal: basic and applied aspects of biotechnology doi: 10.1007/978-981-10-0875-7_4 sha: doc_id: 16419 cord_uid: v1f6dx3e the proteins produced in the body control and mediate the metabolic processes and help in its routine functioning. any kind of impairment in protein production, such as production of mutated protein, or misfolded protein, leads to disruption of the pathway controlled by that protein. this may manifest in the form of the disease. however, these diseases can be treated, by supplying the protein from outside or exogenously. the supply of active exogenous protein requires its production on large scale to fulfill the growing demand. the process is complex, requiring higher protein expression, purification, and processing. each product needs unique settings or standardizations for large-scale production and purification. as only large-scale production can fulfill the growing demand, thus it needs to be cost-effective. the tools of genetic engineering are utilized to produce the proteins of human origin in bacteria, fungi, insect, or mammalian host. usage of recombinant dna technology for large-scale production of proteins requires ample amount of time, labor, and resources, but it also offers many opportunities for economic growth. after reading this chapter, readers would be able to understand the basics about production of recombinant proteins in various hosts along with the advantages and limitations of each host system and properties and production of some of the important pharmaceutical compounds and growth factors. the proteins produced in the body control and mediate the metabolic processes and help in its routine functioning. any kind of impairment in protein production, such as production of mutated protein or misfolded protein, leads to disruption of the pathway controlled by that protein. this may manifest in the form of the disease. however, these diseases can be treated, by supplying the protein from outside or exogenously. the supply of active exogenous protein requires its production on large scale to fulfi ll the growing demand. the process is complex, requiring higher protein expression, purifi cation, and processing. each product needs unique settings or standardizations for large-scale production and purifi cation. as only large-scale production can fulfi ll the growing 7 demand, thus it needs to be cost-effective. the tools of genetic engineering are utilized to produce the proteins of human origin in bacteria, fungi, insect, or mammalian host ( fig. 4.1 ) . usage of recombinant dna technology for large-scale production of proteins requires ample amount of time, labor, and resources, but it also offers many opportunities for economic growth. after reading this chapter, readers would be able to understand the basics about production of recombinant proteins in various hosts along with the advantages and limitations of each host system and properties and production of some of the important pharmaceutical compounds and growth factors. in all living cells, the expression of gene occurs where genetic information contained in dna is passed on to rna in the process of transcription and from rna to protein in the process of translation . thus, the body synthesizes rna and then proteins according to the instructions from the dna. dna-dependent rna polymerase or rna polymerase carries the transcription . eukaryotes have three rna polymerases: polymerase i transcribes ribosomal rna genes (18s,5.8s and 28srrna), polymerase ii transcribes all proteincoding genes (mrna and small rna), and polymerase iii transcribes the genes for 5srrna and trna. in prokaryotic cells ( e. coli ), rna polymerase consists of fi ve subunits-two identical α subunits and one subunit each of β, β′, and σ subunit. the σ subunit dissociates after polymerization ensues. thus, the term "holoenzyme" is used for complete enzyme and "core enzyme" is used without σ subunit. the process of transcription is initiated by binding of rna polymerase to dna molecule at a very specifi c site called " promoters ." the promoters are critical for the start of the transcription. the promoter sites are nearly 40 bp long and are mostly located before the fi rst base, which is copied into rna ( fig. 4.2a ) . this fi rst base is called as start point of transcription and is denoted by +1. promoters for rnapol ii allow differential expression of genes and determine the rate at which the genes are transcribed. there are some for expression of the cloned gene, the gene is with all the essential regulatory elements required for transcription of the gene. the gene is attached to the selective gene, which helps in the selection of the clones with gene of interest. the cells are screened for the synthesis of desirable product and then processed for large-scale production in the bioreactor with optimum condition for high yields promoters that cause the inserted genes to be expressed all the time; in all parts of the system, they are known as "constitutive" promoters. others allow expression only at certain stages/ certain tissue/organ of individuals and at certain time points. gene expression is under temporal and spatial regulation. in prokaryotes the position before start site at −10 and −35 can interact with σ subunit of holoenzyme of rna polymerase. in eukaryotes the sequence (analogous to −10-consensus sequence of prokaryotes), tataaaa, is present at −30 position in the promoter region. eukaryotic promoter is shown (fig. 4.2a ) with tata box forming the core promoter at −30 position (from −30 to −100), upstream of transcription start site. caat box and gc box are at approximately −70-90 and −100, respectively. the location of promoter is always on the same dna molecule which they regulate. they are referred as cis-acting elements. the spacing of various elements is more important and much is dependent on locus-specifi c activators, either at core promoter or at distant sites. various other signals as enhancers are also involved which are far apart from the target gene. they exert stimulatory effects on promoter activity and can be upstream, downstream or in the middle of the gene. promoters of housekeeping genes or genes with complex patterns of expression have cpg islands rather than tata box. the gene to be transcribed has 5ʹ-untranslated region (5ʹ utr), exons (coding region) separated by introns (noncoding region). the end has 3ʹ-untranslated region (3ʹ utr). in the cloning for gene expression, usually intronless version of the gene is used. in eukaryotes, the transcription is further enhanced by enhancers, which may be 2,000-3,000 bases away from the promoter region but are able to affect the rate of transcription . the simplest host for the work of recombinant dna technology is prokaryotic bacterial system . in the early 1980s, the fi rst recombinant fdaapproved pharmaceutical, the human insulin (humulin-us/humuline-eu), was obtained from genetically engineered escherichia coli ( e. coli ) for treatment of diabetes . due to increasing demand, many strains of microbial species are being designed with increased throughput and better recovery of the therapeutic protein from large-scale culture. the recombinant proteins approved by fda are obtained either from escherichia coli or other prokaryotes; from saccharomyces cerevisiae or other fungal species; from insect cells , mammalian cells , or human cells ; or from transgenic plants or animals. cloning and production of protein in a particular host system are dependent upon a property of host to clone and express the desirable size of protein-encoding genes; production of correctly modifi ed, folded, and functional protein; high yield of the protein; and low-cost requirements. the choice of host systems requires best system, which can fulfi ll the requirements [ 17 ] . the advantages of producing proteins using recombinant dna technology are: • as human gene may be cloned and expressed, it minimizes the risk of immune reaction and the specifi c activity of the protein is high. • the therapeutic protein can be produced efficiently, maintaining its cost-effectiveness. • it minimizes the risk of transmission of unknown pathogens present in animal and human sources. • appropriate modifi cation with higher specifi city, increased half-life, and improved functionality. • allow to create critical changes for better specifi city and activity. expression and production of eukaryotic protein require cloning of its cdna in an expression vector and subsequent transfer of the vector in suitable host. dna is modifi ed, cloned, and expressed in other host for the production of the protein; thus, optimum production conditions are required in each host. however, there are yield variations in different expression systems but high-level expression of the protein may be achieved by considering the following points: • though various host systems are available for production of recombinant proteins, microbial hosts offer several advantages over other systems, as production is fast, cheap, and economic: 1. the molecular biology and physiology is well characterized and documented. 2. easy to maintain and manipulate. 3. utilizes inexpensive nutrition sources. 4. rapid growth and biomass accumulation to achieve high cell densities. 5. scale-up is easy and convenient. 6. their expression machinery can be with variety of strong inducible promoters . as with the cellular structure of bacteria, it can rapidly adapt to culturing conditions with very short replication time (20 min). the media requirement of bacterial cell is simple and consists of simple carbon and nitrogen source. thus, the overall inputs in bacteria are 90 % lower than for mammalian cells . some of the the inducible promoter may require an inducer, or the depletion or addition of a specifi c nutrient, or ph change or changes in physicochemical factors in order to initiate the process of gene expression. the inducible systems suffer from the disadvantage that chemical inducers may be expensive and toxic and would require elimination during downstream processing when the product is intended for human usage. thus, usage of thermoregulated systems has been used for production of recombinant pharmaceutical proteins as expression is dependent upon strong heatregulated promoter minimizing the risks of any addition of chemical agent. approved bacteria-derived (by either the european union or fda, usa) therapeutics include hormones (human insulin and insulin analogs, calcitonin, human growth hormone , glucagons, parathyroid hormone , somatropin, and insulin-like growth factor 1), interferons (alfa-1, alfa-2a, alfa-2b, and gamma-1b), interleukins 11 and 2, light and heavy chains raised against vascular endothelial growth factor-a, tumor necrosis factor alpha, cholera b subunit protein, granulocyte colony-stimulating factor, and plasminogen activator [ 3 ] . the microbe of fi rst choice for production of recombinant protein is enterobacterium e. coli . the system offers quick and easy modifi cations, ease of growing in manageable environmental conditions and short life cycle. the bacterial cell can tolerate and adapt to changes in the environment rapidly, thus scale-up is easier. however, the system suffers from some of the disadvantages [ 11 , 16 ] . 1. human or mammalian genes cloned in bacteria cannot undergo splicing due to lack of splicing machinery, thus intron-less version of the gene is cloned for optimum results (fig. 4.2b ). 2. the signals involved in transcription of genes may vary; thus, the gene of interest is usually fused with bacterial gene under the control of its promoter , and the protein is obtained as a fusion product, which can be later cleaved, purifi ed, and used. 3. lac promoter is one of the most popular bacterial promoters . however, for high-level expression, t7 promoter is also preferred (present in pet vectors . for addition of amino acids during the process of translation , as there are more than one codon for several amino acids, thus, codon biasing occurs which is the preference of a particular codon of amino acid in a particular species (fig. 4.2c ). 5. complexity: eukaryotic cells have the advantage of producing fully functional and properly folded proteins. the antibodies with four subunits may be secreted by eukaryotic cells in fully functional form. on the other hand, it is very diffi cult to obtain multidomain protein from e. coli . even if protein is obtained, the renaturation and folding in laboratory condition may either be very expensive or protein may lack its activity. 6. lack of posttranslational modifi cations (ptms) is the problem, which cannot be solved, and is mandatory for activity of many therapeutic proteins. the glycosylation is the most common modifi cations, and others are phosphorylation and formation of disulfi de bond, which are essentially required for the full functional capability of many human proteins. ptms play an important role in proper protein folding, processing, stability, tissue targeting, activity, immune reactivity, and half-life of the protein. lack of these results in insoluble, unstable, or inactive product. however, the n-linked glycosylation system of campylobacter jejuni has been successfully transferred to e. coli , thus opening a possibility for the production of glycosylated protein in it. certain mutant e. coli are being developed to promote disulfi de bond formation (ad494, origami, rosetta-gami) with reduced protease activity (blz1). 7. overproduction of recombinant protein in bacteria might result in the loss of solubility and deposition of many protein species as protein aggregates or inclusion bodies. alteration in growth conditions might render the product in insoluble form. many eukaryotic proteins are found trapped in inclusion bodies with resistance to further processing. success has been obtained in purifi cation of insulin and betaferon from inclusion bodies. the retrieval of proteins using denaturating condition with subsequent refolding and renaturation might not always be easy and prove to be extremely expensive. 8. with the e. coli host, it is very diffi cult to obtain protein larger than 60 kda in soluble form. due to certain limitations for production of proteins in e. coli , other host systems are being discussed for production of proteins. and two n-acetylglucosamine residues. the core may attach to different oligosaccharides to form different glycoproteins. man glcnac glcnac --asn-pentasaccharide core glycosylation is one of the important posttranslational modifi cations, which occurs inside the lumen of the endoplasmic reticulum (er) and in golgi complex. the er and golgi complex are important in protein targeting and transport. n-linked glycosylation starts in the endoplasmic reticulum and continues in the golgi complex after the polypeptide is synthesized on ribosomes. however, o-linked glycosylation exclusively occurs in the golgi complex. in the process, oligosaccharide to be attached to the protein associates with a specialized lipid present in er, dolichol phosphate, which consists of about 20 isoprene (c5) units. through phosphate of dolichol phosphate, oligosaccharide is transferred to specifi c asparagine residue of polypeptide chain on ribosomes. the enzyme responsible for glycosylating protein and activated oligosaccharide are located on the lumen side of er. then these are transported to the golgi complex, where the carbohydrate units are altered and fi nalized. golgi complex is responsible for o-linked sugar attachment and modifi cation of n-linked sugar. then the proteins are targeted and transported to their destination . covalent attachment of carbohydrate group to the protein to form glycoprotein is called glycosylation. in glycoproteins, proteins constitute a major fraction. these play important roles in various physiological processes and are components of cell membranes. the carbohydrates commonly attached to proteins may be fucose (fuc), galactose (gal), n-acetylgalactosamine (galnac), glucose (glc), n-acetylglucosamine (glcnac), mannose (man), and sialic acid (sia). these sugar moieties may associate through amide nitrogen atom of side chain of asparagine (asn) termed as n-linked glycosylation or to the oxygen atom in the side chain of serine (ser) or threonine (thr) termed as o-linked glycosylation. not all the asparagine (asn) present in polypeptide can accept the carbohydrate moiety. the residues with the sequence asn-x-ser or asn-x-thr, where x is any amino acid except proline, are targets for glycosylation. not only the residual sequence but other aspects of the structure of the protein and cell type determine the glycosylation site. all the n-linked sugar residues have a common core of pentasaccharides. these pentasaccharide consists of three mannose (continued) due to the problems encountered in e. coli for production of larger proteins or modifi ed proteins, the next cost-effective, fast, high-density, and easy to handle system is of fungi. saccharomyces cerevisiae ( yeast ) was the system of choice when it was diffi cult to obtain therapeutic protein in soluble form and with appropriate posttranslational modifications in bacterial host. in yeast , mutants are available which can give high yield. the approved products obtained from yeast are hormones , vaccines, recombinant granulocyte macrophage colony-stimulating factors (gm-csf), albumin, hirudin, and platelet-derived growth factor ( pdgf ). the advantages of s. cerevisiae are the following: (1) it secretes recombinant protein in the culture, (2) protein is properly folded, and (3) it performs most posttranslational modifi cations. with the yeast system, high amounts of recombinant protein are obtained, and yeast is also capable of performing posttranslational modifi cations as o-linked glycosylation, phosphorylation, acetylation, and acylation but differs drastically in patterns of n-linked glycosylation ( fig. 4.3 ). chaperones are family of highly conserved different proteins. the important functions of chaperones are: • prevention of aggregation and misfolding of newly synthesized polypeptide chain. • they prevent irreversible aggregation of nonnative conformation and maintain the protein on the productive folding pathway. • they prevent nonproductive interactions with other components of the cell. • they help and guide the direct assembly of multisubunit protein complexes and larger proteins. • the chaperones involved in folding recognize nonnative substrate proteins mainly via their exposed hydrophobic residues. the major classes of molecular chaperones are: heat shock proteins are present in a variety of systems and prevent damage to the proteins under high heat. (continued) differences in n-glycosylation in yeast are with high or hypermannose which is highly immunogenic. unmodifi ed proteins are suitable for production in yeast . other members of fungi are pichia pastoris , pichia methanolica , candida boidinii , and pichia angusta , which are facultative methylotrophic yeast having great potential. pichia pastoris is favored as high cell densities can be obtained; protein is secreted in high concentration (1 g/l), less hypermannosylation as compared to yeast and thus less immunogenic. however, the disadvantage is that it requires methanol to induce gene expression as transgene is under the control of the promoter of alcohol oxidase 1 (aox1) gene. methanol may be fl ammable and is toxic to cells and humans if not thoroughly removed. because of hypermannosetype glycosylation , the fungi are also unsuitable for production of many recombinant proteins . insect cells : insect cell can be infected with baculoviruses which are double-stranded circular dna viruses with arthropods as host. baculovirus-mediated gene expression in insects is a method of choice and is cost-effective, giving the much higher yield of recombinant protein compared to other systems. it is possible to produce large protein resulting in production of correctly processed and biologically active protein. a baculovirus autographa californica nuclear polyhedrosis virus (acmnpv) is used as a cloning vector for insect cell lines. in this viral polyhedron protein is used, which is required in its normal habitat and exhibits high rate of transcrip• it is a bacterial chaperone. • it binds with partially folded and misfolded proteins. • for its functionality groel requires its cofactor groes. • it is tetradecameric mitochondrial chaperonin. • it is implicated in protein import and macromolecular assembly. • required for folding of precursor polypeptides in atp-dependent manner. • prevents aggregation and mediates refolding of protein after heat shock. • they are central components of the cellular network of folding catalysts and molecular chaperones . • they assist in different types of processes of protein folding in the cell by transient association of their substrate binding domain with short hydrophobic peptide. • they bind and release their substrate by switching to low-affi nity atp-bound state and the high-affi nity adp-bound state. • they form complex network of folding machines [ 15 ] . • it is highly abundant chaperone. • it plays an important role in many cellular processes, for example, cell cycle control, cell survival, and hormone and other signaling pathways. • it is a key player in maintaining cellular homeostasis during stress . • has atpase activity, whose binding and hydrolysis affects conformational dynamics of the protein. • it has become a major therapeutic target for cancer, and its role is being explored in neurodegenerative disorders and infectious diseases [ 10 ] . • this is eukaryotic chaperonin tailless complex polypeptide 1 (tcp1) ring complex (tric). • it facilitates the proper folding of many cellular proteins. (continued) tion, but is not needed in cell culture. thus, the coding sequence of the gene is replaced with foreign dna. the gene is transcribed under the control of powerful polyhedron promoter with high yields (~30 % of total cell protein). the observed yield may be variable due to the course of virus infection and viral titer. the production of recombinant protein in insect cell is time consuming (as compared to bacterial system ) as cell growth is slow and the cost of medium is high. every time fresh cells are required, viral infection is lethal for cells. it also has limitations in performing posttranslational modifi cations as it performs non-syalated n-linked glycosylation . all the other optimizations need to be perfect as yield depends upon the virus titer and time taken from infection to expression. insect cells are preferred when active protein is diffi cult to obtain in e. coli system. genetic engineering has been used to select mimic™ (invitrogen) and sfswt-3, which are transgenic cell lines expressing all necessary enzymes to obtain humanized, complex n-linked glycosylation pattern. the system has been extensively used for structural studies as correctly folded eukaryotic proteins may be obtained in secreted form simplifying purifi cation protocols. some of the approved biopharmaceuticals from infected insect cell line hi five are cervarix (recombinant papillomavirus c-terminal truncated major capsid protein l1 types 16 and 18, used as cancer vaccine). glycosylation is a problem which is encountered when insect cells are used for production of recombinant human glycosylated proteins. lots of genetic engineering is required to produce humanlike glycosylation in insect cell . thus, the preferred system for therapeutic human protein production is mammalian system (chinese hamster ovary cell line ). due to time and diffi cultly in maintaining insect cells , the mammalian cells were explored for production of recombinant protein. mammalian cells , because of their properties of protein folding, assembly, and posttranslational modifi cations, have become the preferred system for protein production and are now accounting for major recombinant protein production . mammalian cells : production of complex proteins requires extensive processing and posttranslational modifi cations. mammalian cells have the advantage of performing ptms (fig. 4. 3 ) correctly; they secrete recombinant protein into the medium in their natural form, thus skipping the critical steps of renaturation and refolding which sometimes leads to inactive proteins. therefore, major therapeutic proteins (60-70 %) are produced in mammalian cells primarily chinese hamster ovary (cho) cells and baby hamster kidney cells (bhk). cho cells are relatively easy to manipulate and their properties favor large-scale production in them [ 24 ] . the proteins produced are safe to use in humans with no adverse reactions because of similar glycosylation pattern. chinese hamster ovary (cho) and baby hamster kidney (bhk) cells are the prominent producers of recombinant proteins (fig. 4.4 ) . the recombinant therapeutic product used for clinical applications was produced from mammalian cells. mammalian cells were maintained in serum-/blood-/plasma-based medium; therefore, the presence of any infectious agent in the product might be deleterious if not properly removed. infections may range from hiv , coronavirus (severe acute respiratory syndrome (sars), non-lipid-enveloped (nle) viruses as circoviruses (torque tenovirus (ttv) and torque tenominivirus (ttmv)), hbv, hcv, htlv (human t-cell lymphotropic virus), to west nile virus. prions that are self-replicating infectious proteins may also be present which may lead to variant creutzfeldt-jakob disease (vcjd). pathogen transmission was a major concern in the manufacture of blood-derived coagulation factor. in the early 1980s, the factor replacement products derived from plasma, which were used to treat hemophilia, were found to be contaminated with hiv , hbv, and hcv viruses. in the year 1984, up to 78 % of us-based hemophilic patients were infected with hiv and 74-90 % were infected with hcv. parvoviruses, b19 (b19v) and parv4, were present as con-in mammalian cells, genes can be expressed either transiently or stably. obtaining stably transformed cell lines requires the usage of some selectable marker. another major advantage of these cells is they can be grown in suspension, in serum-free (sf), protein-free, and chemically defi ned media . the product is safe without the risk of prions of bovine spongiform encephalopathy (bse) from bovine serum albumin and infections of variant creutzfeldt-jakob disease (vcjd) from the human serum. presently due to virus and prions in the donor plasma or blood samples, the manufacturers are opting for plasma-/serum-free growth medium for culturing the cell lines . cells require some of these components (albumin, transferring) for their growth. nowadays recombinant human albumin is available like human insulin (produced in e. coli and yeast ) and yeast-derived animal-free recombinant human transferrin is available. these support plasma-free mammalian cell culture. else, cho cells may be engineered to produce its own transferring or insulin-like growth factor. the therapeutic protein obtained by mammalian cell is treated and tested for the presence of any pathogenic agents or viruses as contaminant. the therapeutic products obtained during processing are treated with various agents to remove inactive virus, but the presence of asymptomatic virus poses a serious risk. common virus inactivation technique is using solvent/ detergent, which is effective against lipidenveloped viruses such as human immunodeficiency virus (hiv) , hepatitis b and c virus (hbv and hcv), human tcell lymphotropic virus (htlv), and west nile virus (wnv). non-lipidenveloped viruses such as parvoviruses, enteroviruses, and circoviruses are resistant to inactivation via solvent detergent treatment. human herpesvirus 8, responsible for kaposi's sarcoma, is shown to be transmitted through blood and blood product. following outbreaks, strict requirements were imposed on manufacturers of biologics and medical devices. such concerns prompted manufacturers to switch to sugar-based fi nal formulations and develop recombinant plasma-free albumin produced in yeast for usage in biopharmaceutical manufacturing. plasma-free manufacturing involves the elimination of plasma derivatives in every step (like cell line development, upstream processing, downstream processing, and fi nal formulations) of the process with appropriate postproduction checks. the manufacturers shifted from the use of serum to serum-free cell culture media with animal product-free media and ultimately to protein-free, completely synthetic chemically defi ned media . the media consists of protein hydrolysates derived from yeast , soy, and wheat with amino acids, peptides, carbohydrates, vitamins, and essential elements, which are ultrafi ltered to remove any unwanted contaminants [ 8 ] . (continued) ) , and replagal-alfa-galactosidase a (lysosomal hydrolase) have been approved by the european union (eu) or food and drug administration (fda, usa). as these products are fully glycosylated when expressed in human cell lines and used as therapeutics in human beings. transgenics for protein production the transgenic animals are successfully used for production of recombinant proteins (for details refer to chap. 5 ). protein production poses great risk in terms of safety as transmission of infectious agents, allergic responses, immune reactivity, and autoimmune responses might occur. atryn was the only approved (approved in 2006 by european medical agency and in 2009 by fda) recombinant biopharmaceutical using transgenic animals. it contains human antithrombin (432aa) with 15 % glycosylated moieties and is secreted into milk of transgenic goats. rhucin intended for acute attacks of angioedema in patients with congenital c1 inhibitor activity defi ciency, obtained from transgenic rabbit, was denied approval. transgenic plants are being explored as recombinant protein producers for research and diagnostic uses. obtaining therapeutic proteins from their natural source poses threat for spread of diseases. therefore, alternative systems for the production of therapeutic agents have their own benefi ts. molecular farming in plants has been widely explored for production of recombinant pharmaceutical proteins. their advantages are low cost, high mass production, scale-up, lack of human pathogens , and addition of eukaryotic ptms. the fi rst recombinant protein obtained in 1986 from tobacco plants was human growth hormone . however, sometimes plant-specifi c ptms might result in adverse immune reactivity. production of recombinant heterologous proteins in plants is simple and is used for production of non-naturally occurring proteins as single chain fv fragments (scfvs). high yield of recombinant protein is the main goal of production system in transgenic plants . therefore appropriate expression vectors and constructs are designed to achieve high yields of the engineered gene products [ 7 , 12 ] . taminant in plasma-derived factor viii. therefore, the production urgently required regulatory measures. then came fi rst recombinant factor viii , advate (baxter), in the usa in 2003. advate was produced in cho cells grown in serum-free and protein-free medium with ultrafi ltered soybean peptides with subsequent purifi cation by immunoaffi nity chromatography. the usage of advate helped in eliminating the risk of transmitting emerging blood pathogens . prions pose a serious risk, as they are highly resistant to physical/chemical inactivation. early stage of prion infection is almost impossible to detect in plasma donor. iatrogenic transmission of prions has occurred in patients who received humanderived pituitary hormones as human growth hormone (hgh) and gonadotropins. cjd was transmitted to over 160 recipients of cadaveric pituitary hgh before its withdrawal. cadaveric pituitaryderived gonadotropins for infertility were associated with iatrogenic transmission of cjd. later on cadaveric pituitary hgh and gonadotropins have been replaced with recombinant gh (produced in microbial system) and recombinant gonadotropins (produced in cho cell lines ). nowadays plant system is effi ciently engineered to produce human growth hormone , human serum albumin, erythropoietin, α-interferon, antibodies and scfvs, toxins, subunit vaccines , and insulin [ 20 ] . with increasing demand from the consumer, the companies are trying to increase the productivity [ 17 ] . few challenges with large-scale production of proteins are: the transgene in expression construct is chimeric structure as it is surrounded by various active regulatory elements. polyadenylation sites play an important role for the high level of expression of transgene. caulifl ower mosaic virus (camv) 35s promoter works well with dicots. it is a strong constitutive promoter that is made more active by duplicating the enhancer region. however, in monocots maize ubiquitin-1 promoter is the preferred promoter . the presence of an intron in the 5′-untranslated region (5′-utr) enhances transcription in monocots. for obtaining high yield of the protein, several factors may be appropriately considered: • the incorporation of polyadenylation sites may be from camv 35s transcripts or the agrobacterium tumefaciens nos gene or the pea ssu gene. • the yield can be controlled by placing the gene under the control of the promoter which is active in a particular tissue or developmental stage or particular environment (e.g., rice glutelin, pea legumin). • usage of inducible promoter (e.g., tomato hydroxyl-3-methylglutaryl coa reductase-2 (hmgr2)) which has mechanical gene activation system developed by cramer (crop tech corp., virginia, usa). transcription starts when harvested tobacco leaves are sheared during processing. • codon bias in the host plant may be overcome by engineering of transgene at positions, which might lead to truncation and/or misincorporation, or slowing the process. • subcellular targeting is also very important factor which affects the process of folding, assembly, and posttranslational modifi cation and can be effi ciently achieved by inclusion of an n-terminal signal peptide. • position of transgene integration. • structure of transgene locus. • gene copy number. • presence of truncated or rearranged transgene copies. • affi nity tags as his or the flag epitopes can be used to ease the process of purifi cation; however, these modifi cations not only affect the primary structure but also the properties of the protein. figure 4 .5 shows the general vector pcambia (it is small in size (7-12 kb) maintained in high copy number with pvs1 replicon that imparts chloramphenicol or kanamycin resistance and high stability in agrobacterium ) that is used for transgene expression in the plants. the modifi ed vector has shown success in insulin production. (continued) 4.9 challenges of production of therapeutic proteins important that gene of interest should give adequate protein production. however expression may be lost due to many factors, like, if there are structural changes in the recombinant gene or inactivation or disappearance of the gene from host cell. other factors infl uencing yield may be increase in the copy number of insert, maintenance of optimum temperature, and toxicity of the expressed protein to the host. chromosomal integration of the foreign gene might overcome the problem of expression stability, but in plasmid -based system, high copy number leads to increased yield. 2. posttranslational processing: protein folding requires foldases (accelerates protein folding) and chaperones (prevents protein formation of nonnative insoluble folding intermediates). glycosylation is complex ptm requiring consecutive steps and enzymes. glycosylation is important as it determines protein stability, solubility, antigenicity , folding, localization, biological activity, and circulation half-life. getting correctly glycosylated and folded protein is required for therapeutic usage. in prokaryotes, with the discovery of n-glycosylation system in campylobacter jejuni , several other systems of o-glycosylation were unraveled in both pathogenic and symbiotic bacteria. the production of recombinant proteins is commonly done by using e. coli , yeast , or cell lines derived from insects (sf9), mice (sp2/0), or cho, but obtaining fully human ptms is a challenging task. fig. 4 . 5 the fi gure shows the structure of pcambia vector (cambia.org) used for plant transformation. the vector has camv35s promoter , multiple cloning site, and reporter gene (gus or gfp may be used). the vector can be modifi ed to express genes for insulin (tomato) or hep-b surface antigen (hbsag) for recombinant therapeutics providing a human glycosylation pattern have increased attention, and the efforts are being made for the development of novel glycoengineered cell lines for production of fully glycosylated protein therapeutic. 3. overexpression of therapeutic protein might result in the formation of inclusion bodies in prokaryotic system. rapid intracellular protein accumulation and expression of large proteins increases the probability of aggregation. aggregation protects proteins from proteolysis and can facilitate protein recovery. when the expressed protein is toxic to the host, the presence of protein in the inclusion body tends to protect it. precautions recombinant protein production requires some precaution resulting in a loss of yield and/or product: 1. contamination: at any stage, contamination might occur from any source. it poses big challenge and may be adverse when contaminated material is put for human usage. 2. immune response: for the therapeutic agent, the body can mount the immune reactions which lead to deposition of immune complexes in various tissues, and condition of anaphylactic shock might occur (e.g., when some essential agent is lost since birth like factor viii , then the patient might raise antibody response against the treatment). this also occurs when antibodies are used as therapeutic agents in the treatment of variety of cancers. 3. protein aggregation: any nonfunctional condition might result in aggregation or loss of activity of recombinant protein. after purifi cation, the proper modifi cations and refolding are required for therapeutics. 5. disulfi de bond formation: it stabilizes protein structure; thus, strategies for specifi c extracellular excretion pathway or overexpression of chaperones is required for optimum production. 6. degradation: sometimes proteins, which are active in host cells like proteases or proteinmodifying chemicals, might degrade the recombinant protein (e.g., peg interferon is modifi ed to have polyethylene glycol with prolonged presence and reduction in enzymatic degradation and renal clearance, thus extending its presence with lesser immunogenicity ) [ 17 ] . ischemic stroke and myocardial infarction are one of the leading causes of cardiovascular morbidity and mortality in the world. important thrombolytic agents are urokinase (obtained from urine), tissue plasminogen activator (tpa), and streptokinase (obtained from bacteria). among these, t-pa is largely used commercially. plasminogen activators are serine proteases, which are responsible for conversion of inactive proenzyme plasminogen (plg-a single chain glycoprotein) to serine protease called plasmin (plm). the plasmin degrades the network of fi brin of the blood clots ( fig. 4.6a ). there are two immunologically unrelated groups of plasminogen activators, the 55 kda urokinase-type-pa (u-pa) and 72 kda tissue-type pa (t-pa) (ec 3.4.21.68). the t-pa is the physiological vascular activator consisting of single polypeptide chain of 72 kda consisting of 527 amino acids. it shows strong activity in the presence of fi brin [ 23 ] . the potential inhibitors of the thrombolytic cascade are type i plasminogen activator inhibitor (pai-1 or serpin e1) and pai-2 (secreted by placenta and present in signifi cant amount during pregnancy). they act by competing with t-pa for binding sites on fi brin thus preventing the fi brinolytic cascade. pai-1 complexes with t-pa for binding to fi brin. thus, truncated t-pa in which the residues responsible for interacting with pai-1 (296-299) are replaced with four alanine amino acids and three domains (fi nger, epidermal growth factor (egf) , and kringle 1 domains)) are deleted, and chimeric tetrapeptide gly-his-arg-pro (ghrp) with high affi nity to fi brin was added. reteplase is the deletion mutant with a prolonged half-life, in which the fi nger, egf , and kringle 1 domains of the full-length molecule are all deleted; thus, it is not inhibited. defi ciency of pai leads to over fi brinolysis and hemorrhagic diathesis (like defi ciency of clotting factors). tiplaxtinin is the inhibitor and is used for remodeling of blood vessels [ 2 ] . plasminogen activator has great clinical relevance for the management of stroke and myocardial infarctions. for production of t-pa, e. coli and yeast system did not work properly due to lack of posttranslational modifi cation and over glycosylation , respectively. a novel truncated form of t-pa with an improved fi brin affi nity and an increased resis-tance to pai-1 was expressed in a cho dg44 expression system. therapeutic protein was produced in stably transfected cho dg44 cell lines . these cell lines were maintained in serum-free medium, with glutamine, hypoxanthine, and thymine in stirred tank bioreactor . the cells were grown at 37 °c, 140 rpm with 5 % co 2 and 85 % humidity. the protein was then purifi ed, with higher yield (fig. 4.6b ). factor viii is one of the important factors of all blood clotting factors. defi ciency of factor viii causes bleeding disorder called hemophilia a. the hemophilia may be mild to severe depending upon factor viii concentration in the body. in moderate and severe factor viii defi ciency, there can be spontaneous bleeding episodes in the joints. hemophilia a affects 1 in 5,000-10,000 males. replacement therapy is the treatment option for hemophiliacs either with human plasma-derived factor viii (pdfviii) or recombinant fviii (rfviii) [ 21 ] . transfection of hek 293 cell cultures in serum-free suspension is being tried for optimal yield. recombinant factor viii (rfviii) is produced by culturing mammalian cells as baby hamster kidney (bhk) or chinese hamster ovary cells (cho), using large-scale bioreactors . standardizations are done to maximize yields . insulin is a peptide hormone consisting of 51 amino acids. it is secreted by β cells of islets of langerhans of pancreas. the hormone is responsible for maintaining normal blood glucose level in blood. insulin is stored in the form of proinsulin which contains two polypeptide chains, a and b, and is connected with a third peptide cchain), which before secretion is cleaved with production of insulin and c-chain. the cleavage results in the removal of c-chain, and the a (21 amino acids) and b chain (30 amino acids) are linked by disulfi de linkage to form mature insulin. in the beginning, efforts were made to isolate mrna for pre-and proinsulin from rat islets of langerhans of pancreas and to synthesize cdna. thereafter, it was inserted into a plasmid . the recombinant plasmids were transferred into the e. coli cells, which secreted proinsulin [ 4 , 5 , 6 , 19 ] . scientists have chemically synthesized dna sequences for two chains, a and b, of insulin and separately inserted into two pbr322 plasmids by the side of β-galactosidase gene. the recombinant plasmids were separately transferred into e. coli cells which secreted fused β-galactosidase-a chain and β-galactosidase-b chain separately. these chains were isolated in pure form by detaching from β-galactosidase with yields of about 10 mg/24 g of healthy and transformed cells. production of recombinant insulin is shown in (fig. 4.7a, b ) . growth hormone is produced by the anterior lobe of the pituitary gland and is released in multiple pulses. the hgh is encoded by ghn gene cluster (an array of fi ve closely related genes), which is localized on chromosome 17. it belongs to diverse gene family that has evolved by gene duplication events and has lots of structural similarity and some common functions. of the various forms, the predominant form of hgh is 22 kda protein of 191 amino acids with two disulfi de bonds. gh does not control the functions directly, but acts on certain hormones or somatomedins for its activity, for example, insulin-like growth factor 1 (igf-1). it has a wide spectrum of roles to play as promotion of long bone growth, promotion of normal sex organ development through puberty, regulation of metabolism, stimulation of tissue growth and repair, anabolic/anti-catabolic effect via improved nitrogen retention, modulation of bone mineral density and metabolism throughout life, proliferation of some cell types of the immune system, appetite stimulation, and breakdown of fat (lipolysis). in clinical conditions, the therapy of growth hormone is given in the treatment of dwarfi sm, bone fractures, skin burns, bleeding ulcers, and aids . recombinant human growth hormone (rhgh) is 22 kda consisting of long chain of amino acids. it is used in defi ciency disorders of growth hormone . in children, it is used for growth abnormality as short stature and is used in chronic renal insuffi ciency. the therapy of growth hormone is also approved for adult growth hormone defi ciency. gh is one of the most widely used hormones with the estimated market of more than 1.7 billion usd. long experience in its administration has proven the therapy as safe and effective in various conditions of growth abnormality. earlier pituitary-derived hgh was used but later on it was prohibited when found associated with creutzfeldt-jakob disease. because of recombinant dna technology , safe and abundant recombinant hgh was produced in various heterologous systems. as the non-glycosylated human growth hormone was biologically active, thus, the preferred system for its production is e. coli , which allows its rapid and economical production in large amounts [ 14 ] . recombinant hgh (rhgh) is now used to treat: • gh-defi cient (ghd) short-stature children. • acceleration of wound healing. • increase in insulin-like growth factor (igf)-1 levels. • rhgh increases igf-1, osteocalcin, type i procollagen pro-peptide (picp), and bone density, when administered to children with ghd. for optimal productivity, strong inducible promoters are preferred as ipl, ipr, trc, and t7 in e. coli . they are advantageous as they drive overproduction of recombinant proteins. apart from e. coli , human somatotropin (hst) expression was tried in a biologically active, disulfi de-bonded form in tobacco chloroplasts. the hormone is used for the treatment of hypopituitary dwarfi sm in children; additional indications are in treatment of turner syndrome, chronic renal failure, hiv wasting syndrome, and possibly treatment of the elderly. growth hormone defi ciency in human occurs both in children and adults [ 18 , 22 ] . hematopoietic growth factors consist of cytokines and protein hormones produced by the body which govern the production and maturation of the various cells produced during the process of hematopoiesis in the bone marrow from hematopoietic stem cell. the precursor cells in the presence of a particular growth factor differentiate and become a specialized kind of cell as monocyte, macrophage, lymphocytes, or red blood cell. one of the important growth factor is erythropoietin which is a protein hormone produced by a specifi c type of cells in the kidney. in the presence of erythropoietin, progenitor cells are stimulated in the bone marrow to form mature erythrocytes (red blood cells). thus the patients with chronic kidney disease are unable to maintain adequate amount of erythropoietin for normal development of erythrocytes in blood, resulting in low numbers of red blood cells and subsequent anemia. these patients either require blood transfusion or erythropoietin from outside. as supply is limited from the natural source that is kidney cells, thus a recombinant human erythropoietin epogen® which is amgen's trade name for epoetin alfa is marketed for anemic condition involving erythropoietin. the human gene encoding erythropoietin was cloned into the chinese hamster ovary cell line for production of the human protein. this cell line continues to be used today for the production of epogen®. the half-life of erythropoietin can be increased by incorporating the glycosylation of the protein growth factor. thus, darbepoetin-α is an analog which is engineered for two extra amino acids which are substrates for glycosylation . thus, production is done in cho cell lines ; the product has fi ve n-linked sugar chains and has almost three times longer life than erythropoietin . pdgf regulates cell growth and division and plays a signifi cant role in blood vessel formation (angiogenesis), the growth of blood vessels from already existing blood vessel in tissue, and may act in autocrine and paracrine stimulation of cell growth in vivo. pdgf plays the role in development, cell proliferation, cell migration, and angiogenesis and has been linked to atherosclerosis, fi brosis, and malignant diseases. pdgf has fi ve different isoforms: pdgfa, pdgfb, pdgfc, pdgfd, and ab heterodimer. pdgf-a and pdgf-b have 60 % similarity in amino acid sequence, but experiments suggest different biological functions for the two chains and different locations of these under different transcriptional controls. pgdf-aa is released in the medium, and pdgf-bb are insuffi ciently secreted and remain attached to the plasma membrane, and of these pgdf-bb and pdgf-ab are strong mitogens and are probably responsible for biological roles of pdgf. pdgf receptor (pdgfr) is receptor tyrosine kinase (rtk) (alpha and beta type). upon activation by pdgf, these receptors dimerize leading to autophosphorylation of several sites on their cytosolic domain. pdgf being a mitogen promotes the proliferation of fi broblasts and smooth muscle cells in vitro. pdgf shows considerable heterogeneity with sizes of 27-31 kda; however, purifi ed pdgf is cationic protein of 30 kda. recombinant human platelet-derived growth factor (rh-pdgf) was the fi rst recombinant protein to be approved by the us food and drug administration for treatment of chronic foot ulcers in diabetic patients (regranex, ethicon inc. somerville, nj). it has the potential for use in bone regeneration and increasing bone density in long bones and spine. pdgf is commercially produced by using e. coli and mammalian cell s . human egf protein has 53aa and three intracellular disulfi de bonds and plays an important role in the regulation of cell growth and proliferation. it shows strong sequential and functional homology with human type-alpha transforming growth factor (htgf alpha), which is a competitor for egf receptor site. egf acts by binding with high affi nity to egfr on cell surface and stimulates the intrinsic protein tyrosine kinase activity. egf has many biological activities. initial observations were centered around their proliferative effects on fi broblasts, keratinocytes, and epithelial cells. egf modulates luteinizing hormone and thyroid hormone . egf is produced commercially by engineered e. coli . the other systems are also being explored for optimum egf production [ 9 ] . fgfs are commonly mitogens with multifunctional proteins with a wide variety of regulatory, morphological, and endocrine effects. there are 18 mammalian fgfs (1-10 and 16-23) which affect growth and functions of a wide variety of mesenchymal, endocrine, and nerve cells. the functions of fgfs in developmental processes include mesoderm induction, anteroposterior patterning, limb formation, neural tube induction, and brain development and in mature tissue/systems angiogenesis, keratinocyte organization, and wound healing processes. fgf is very important during normal development of both vertebrates and invertebrates, and any irregularities in their function lead to a range of developmental defects [ 1 ] . fgf1 and fgf2 show strong angiogenic properties with the promotion of endothelial cell proliferation and the physical organization of endothelial cells into tubelike structures and the growth of new blood vessels from the preexisting vasculature. fgf7 and fgf10 (also known as keratinocyte growth factors (kgf) and kgf2, respectively) stimulate the repair of injured skin and mucosal tissues by stimulating the proliferation, migration, and differentiation of epithelial cells, and they have direct chemotactic effects on tissue remodeling. most fgfs are secreted proteins that bind heparan sulfates and can therefore be caught up in the extracellular matrix of tissues that contain heparan sulfate proteoglycans. this allows them to act locally in a paracrine fashion. however, the fgf19 subfamily (including fgf19, fgf21, and fgf23) which binds less tightly to heparan sulfates can act in an endocrine fashion on far away tissues, such as the intestine, liver, kidney, adipose, and bone. for example, fgf19 is produced by intestinal cells but acts on fgfr4-expressing liver cells to downregulate key genes in the bile acid synthase pathway; fgf23 is produced by the bone but acts on fgfr1expressing kidney cells to regulate the synthesis of vitamin d and in turn affect calcium homeostasis. fgf may be synthesized using e. coli as host system . fgf is involved in stimulating collateral vascularization and recovery from ischemia as well as enhancing wound healing, nerve regeneration, and repair of cartilage and has been alternately referred to as "pluripotent" (capable of developing into more than one cell type or tissue) growth factors and as "promiscuous" (biochemistry and pharmacology concept of how a variety of molecules can bind to and elicit a response from single receptor) growth factors due to its multiple actions on multiple cell types. the fgfs and small-molecule fgf receptor kinase inhibitor are used in the treatment of cancer and cardiovascular disease and have potential in the treatment of metabolic syndrome and hypophosphatemic diseases: • receptor tyrosine kinase inhibitor (sunitinib) is approved for indications in renal cell carcinoma and gastrointestinal stromal tumors. • small fgfr inhibitors, su5402, pd173074, and nordihydroguaiaretic acid, are effective in multiple myeloma cell lines . • pd173074 can induce cell cycle arrest in endometrial cancer cells with mutated fgfr2. • antibody against fgfr3 has been shown to effectively cause apoptosis in mouse models of multiple myeloma and bladder cancer. thus fgfr inhibition can be very effective in the treatment of cancer. the nerve growth factor (ngf) is an important member of the family of neurotrophins. five protein nerve growth factors of the neurotrophin family are important. they regulate the development of the nervous system and play an important role in maintaining the structure, plasticity , and repair of the adult nervous system. all the neurotrophins are basic proteins of about 120aa, share 50 % sequence homology, and are highly conserved in mammalian species. nerve growth factor (ngf) is a small secreted protein which induces the differentiation and survival of particular target neurons (nerve cells). little is known of the biological action of neurotrophin apart from ngf. the nucleotide sequence of cdna predicts that ngf is synthesized as pre-pro-ngf. upon removal of hydrophobic signal, either 34 kda or 27 kda pro-ngf is generated depending on the size of the transcript. however, processing of the precursors in different tissues is not well understood. they are essential for normal development, growth, and differentiation of the sympathetic and sensory neurons and are also essential to maintain the normal function of these cells in adults. thus it is important for maturation and survival of neurons and prevents degeneration of adult neurons. apart from its important role in the nervous system, it has been shown to possess protective action of human pressure ulcer, corneal ulcer, and glaucoma. reduced sensation may be observed in leprosy , wound healing, nerve injury, and diabetes . ngf may help to regulate the sensory fi ber sensitivity and function directly or indirectly by stimulating other effectors. administration of recombinant ngf may improve sensation and pain [ 13 ] . cholinergic neurons of the basal forebrain show receptors for ngf; specifi c mrnas for various ngfs have been identifi ed in different areas of the brain. cholinergic neuron loss is a cardinal feature of alzheimer's disease. nerve growth factor (ngf) stimulates cholinergic function, improves memory, and prevents cholinergic degeneration in animal models of injury, amyloid overexpression, and aging. ngf acts on intracellular calcium through tyrosine kinase receptor mechanism. nerve growth factor enhances early regeneration of severed exons and is also important in maintaining the biochemical and morphological phenotype of mature basal forebrain cholinergic neurons (bfcnls) after lesions or injury of the central nervous system (cns). thus, ngf may provide therapeutic option for preventing death of cholinergic neurons and other clinical conditions and is produced using e. coli . tgf-α exerts its function in an autocrine and endocrine fashion for various cell types of ectodermal origin, including most epithelial cells. it is normally a transmembrane protein and functions in cell communication through its ability to activate a receptor tyrosine kinase. ectodomain of tgf-α is cleaved in a highly regulated manner, releasing soluble tgf-α which activates paracrine signaling. the receptor is egf/tgf alpha receptor; therefore, the focus is on understanding the important roles of tgf-α and egf receptor signaling in carcinoma development. tgf-β is a large group of related proteins. some of its family members include bone morphogenetic protein (bmps), growth, and differentiation factors (gdp). it affects tissue remodeling, wound repair, hematopoiesis, morphogenesis, embryonic development, adult stem cell differentiation, immune regulation (is switch factor for iga), and infl ammation. it exists as multiple forms as tgf-β1, tgf-β2, and tgf-β3. it acts through transmembrane serine/ threonine receptor kinase leading to the activation of smads. it acts as tumor suppressor during cancer initiation but promoter during tumor progression. it has a role in the control of embryonic development, cellular differentiation, hormone secretion, and immune function. its role as mesenchymal differentiation factor, with focus on the muscle, fat, and bone cell, might provide insights into its deregulation in skeletal and developmental diseases and is the area of active research. it is produced using cho cell lines . there is huge potential for future therapies using proteins as therapeutic agents. the recombinant proteins are not only benefi cial, but the researchers can further engineer them to improve their activity and prolonged stay in the body, for example, engineering of monoclonal antibodies to have toxin or radioisotope or generation of bispecifi c antibody . still the technology is struggling hard to make the diseases completely a text of books and having a society free of diseases and the pathogens . • the production of proteins for therapeutic purpose is very important not only because of their specifi c action with minimum side effects but also due to their unique form and functions. • nowadays therapeutics (pancreatic enzymes from hog and pig pancreas or a-1-proteinase inhibitor from pooled human plasma) are not extracted from their native sources (from humans, animals) due to risk of transmission of pathogens . other problems faced for extraction from native sources were the scarce availability of animal tissue for production, high cost of purifi cation with less yield, and immunological reactions in the recipients. • majority of the biological therapeutic agents are produced by using various advance tools and technologies as cloning, selection , purification, and stability monitoring. it is also very important to monitor risks and side effects of therapeutics (safety analysis ) obtained from a wide range of cells. • the various biological systems are available for production of recombinant protein as bacteria, fungal system ( yeast ), insect cell , mammalian cell , human cell, and transgenic plants and animals . • the system of choice is dependent upon the total cost of production and obtaining fully functional and appropriately modifi ed (ptms) (glycosylation, phosphorylation, or properly folded) protein. all these are essential for the biological activity of the protein. • the bacterial system is the simplest with short cell division times, easy maintenance , easy to modify, and cost-effective production. the bacteria have limitation in production of (c) growth factors are required for progression of tumors (d) all of the above 11. darbepoetin is used for the treatment of: the fgf family: biology, pathophysiology and therapy nature reviews combined tge-sge expression of novel pai-1-resistant t-pa in cho dg44 cells using orbitally shaking disposable bioreactors microbial factories for recombinant pharmaceuticals the preparation and characterization of human insulin of recombinant dna origin chemistry and clinical use of insulin biosynthetic human proinsulin: review of chemistry, in vitro and in vivo receptor binding, animal and human pharmacology studies, and clinical trial experience biopharmaceuticals derived from genetically modifi ed plants emerging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells clinical applications of epidermal growth factor hsp90: structure and function engineering of therapeutic proteins production in escherichia coli the production of recombinant pharmaceutical proteins in plants nerve growth factor: basic studies and possible therapeutic applications genetic engineering and biotechnology of growth hormones, genetic engineering -basics, new applications and responsibilities hsp70 chaperones: cellular functions and molecular mechanism microbial factories for recombinant pharmaceuticals production of recombinant proteins optimization of production of recombinant human growth hormone in escherichia coli bacterial production of human insulin cloning, transformation and expression of proinsulin gene in tomato (lycopersicum esculentum mill) transient transfection of serum-free suspension hek 293 cell culture for effi cient production of human rfviii heat-induced production of human growth hormone by high cell density cultivation of recombinant escherichia coli the structure of the human tissue-type plasminogen activator gene: correlation of intron and exon structures to functional and structural domains production of recombinant protein therapeutics in cultivated mammalian cells large-sized protein and are unable to perform posttranslational modifi cation as glycosylation. the glycosylation is very important as it affects the activity, half-life, and immunogenicity of the recombinant protein.• due to limitations, the other host systems were used for production which could give better product with minimal immune reactions and are cost-effective. fungal and insect systems are utilized for protein production but sometimes result in inappropriate glycosylation. improper glycosylation may result in nonfunctional and highly immunogenic protein.mammalian cells are the most preferred system for production of these proteins. cho system is the system of choice for therapeutic protein production. it accounts for nearly 70 % production of all products available in the market. key: cord-024193-khdvj6t5 authors: zhang, hong; pelech, steven; ruijtenbeek, rob; felgenhauer, thomas; bischoff, ralf; breitling, frank; stadler, volker title: peptide arrays date: 2012-01-17 journal: microarrays in diagnostics and biomarker development doi: 10.1007/978-3-642-28203-4_7 sha: doc_id: 24193 cord_uid: khdvj6t5 despite the concern over the potential loss of structural information as a result of the use of peptides as opposed to proteins as molecular probes, peptide arrays have been implemented in a broad range of applications including antibody screening and epitope mapping, characterization of molecular interactions, and enzymatic activity profiling, and they have become a valuable tool for proteomics research. in this chapter, we first (sect. 7.1) recapitulate the development of these arrays and highlight a couple of key improvements in the array production and the application in proteomics research. for clinical and biomarker development applications, it is important to measure entities that are directly related to physiological function (and dysfunction). in this respect, the assessment of enzymatic activities is obviously preferable to genotyping, expression profiling, or even measurement of protein amounts. in sect. 7.2, an original technology based on peptides arrayed onto a porous support allows detailed profiling of kinase activities in a biological sample. the applications described range from kinase characterization to inhibition profiles, detection of off-target effects, and drug response prediction in a clinical setting, allowing rational choice of the drug to be used. such directly functional approaches will have an important role in the transition to more personalized medicine. finally, in sect. 7.3, a recently developed method for “laser printing” of peptide arrays that will make these approaches much more practical is presented. has proved to be unparalleled in its power in profiling gene expression and identifying single nucleotide polymorphisms (snps) in high-throughput manner, there is a poor correlation between mrna and protein expression and activity. this arises from regulation of mrna translation, for example, with micrornas, and from the involvement of posttranscriptional and posttranslational modifications (ptms), protein-protein interactions, and differences in subcellular localizations. these confounding factors have driven the development and the use of array technologies to directly study proteomes and have set the stage for the arrival of microarray-based proteomics. similarly to dna/oligonucleotide microarrays, arrays for proteomics studies feature a wide range of molecules including recombinant proteins, complex protein samples, antibodies, peptides, or small molecules that are assembled in an addressable fashion on planar surfaces to allow parallel interrogations for activity and interactions associated with biomolecules at the protein level. among various proteomics array types, protein microarrays and antibody microarrays have attracted most of attention in the field in the past decade, as illustrated by the rapid technological advancements and broad applications in numerous basic research and clinical studies (reviewed in chap. 6). even though synthetic peptides together with oligonucleotides were among the first to be explored as molecular probes in array format, the development and application of peptide arrays has been sluggish and has lagged far behind arrays of many other types (frank et al. 1983; geysen et al. 1984) . one of the concerns with the use of peptides as opposed to proteins as molecular probes is the potential loss of information as a result of the missing structural context (mahrenholz et al. 2010 ). there is a much higher degree of entropy in the structures of peptides than with macromolecules such as proteins which are much more constrained. arrays of proteins have the distinct advantage in being able to mimic their physiological counterparts through presenting full-length proteins that are likely in their native 3d conformations and with proper ptms and associated proteins, to facilitate investigation of protein-protein interactions and assay of physiological activities. however, the molecular cloning and expression of tens of thousands of proteincoding genes with optimal covalent modifications and in combination with activating subunits has been proven to be technologically challenging, which significantly hampers the development and application of protein microarrays. in contrast, the chemistry of solid-phase peptide synthesis (spps) was well defined about half a century ago (merrifield 1965a, b) . further technological advancement in peptide synthesis over the years coupled with the recent influx of genome sequencing information upon the completion of a number of genome-wide sequencing projects has made designing and synthesizing peptides with defined sequences corresponding to the segments of a protein easier than ever. furthermore, given the fact that the biological activity of a protein is often carried out through the coordinated actions of individual protein domains, it is reasonable to expect that the various functions of the protein can be recapitulated through the study of its constituent peptides representing individual functional domain sequences. since its advent about two decades ago, the peptide array has been implemented in a broad range of applications including antibody screening and epitope mapping, characterization of molecular interactions, and enzymatic activity profiling and has since become an increasingly important and versatile tool for proteomics research. more recently, with the improvements in array production techniques, peptide microarrays with higher peptide density and diversity can be produced en masse using the peptides generated from the parallel synthesis approaches such as the spot technology. the utility of these peptide microarrays has been demonstrated in some large-scale systems biology studies on the dynamics of protein interactions in cell signaling networks as well as kineome (also known as kinome) activity profiling. however, the clinical application of peptide arrays is still at its infancy despite potential and promises shown in early exploratory work. this chapter summarizes the development of peptide array technology and highlights the progress made toward its applications in proteomics research in recent years. a perspective of its future implementation in clinical practice is also presented. despite the fact that the concept of the array-format peptide synthesis was introduced about three decades ago, it was not until the introduction of the spot synthesis of peptides in the early 1990s that the peptide array finally took its shape (geysen et al. 1984; frank 1992) . currently, there are two main strategies (in addition to very new approaches, see sect. 7.2) being used to produce peptide arrays: in situ parallel on-chip synthesis or immobilization of presynthesized peptides on the array surface. each has its own advantages and shortcomings. hence, the choice of the approach for peptide array production is largely determined by the downstream applications of the resulting arrays. the in situ on-chip synthesis of peptides is affordable as a result of the requirement of small amounts of reagents and of the fact that no purification of individual peptides is required. however, by the same token, the resulting peptides from in situ synthesis may suffer from low purity due to the variability in coupling efficiency between amino acid residues, especially in the case of long peptides (>20 residues) or those with residues such as cys or met, or containing multiple hydrophobic residues or phosphorylated amino acid residues. the two most common techniques for in situ synthesis that have been described and routinely used are spot synthesis and photolithography. the spot synthesis was introduced by ronald frank back in 1992 and is essentially a stepwise synthesis of peptides through sequentially delivering small amounts of activated amino acids on functionalized cellulose or polypropylene membranes using standard fmoc-based peptide chemistry (frank 1992) . the resulting membrane-based arrays are usually at low to medium density and can be used directly for downstream assays such as antibody epitope mapping. recently, improvements have been introduced allowing peptides either to be cleaved from the membrane using strong bases or to be recovered from individual spots as soluble peptides. for this purpose, they are synthesized on acid-labile cellulose membranes (e.g., trifluoroacetic acid (tfa)-soluble), allowing postsynthesis printing of the recovered peptides on selected array surfaces at a higher density (zander et al. 2005; hilpert et al. 2007) . another technique used in preparing peptide arrays in situ is the photolithographic synthesis developed by fodor et al. (1991a, b) on the basis of the addressable surface activation concept. compared to spot synthesis, photolithographic synthesis of peptides on array surface is more suitable for generating high-density arrays. however, it is both laborious and expensive. it requires the use of special photolabile protected amino acid derivatives as building blocks and of photomasks through which a laser is then used to activate specific areas on the array to cleave photolabile protecting groups. even though the technique was initially developed for peptide synthesis, it was more easily adopted for the production of oligonucleotide arrays, due to the complexity of making masks for each of the 20 amino acids for every coupling cycle, as opposed to only four bases in oligonucleotide array production (pease et al. 1994; mcgall and fidanza 2001) . in recent years, a number of modifications have been introduced, including the uses of conventional amino acids and photogenerated reagents, resulting in the improvement of efficiency and the reduction in cost for array production (singh-gasson et al. 1999; gao et al. 2003; bhushan 2006; shin et al. 2010) . thanks to technological advances in microarray printing and array substrate production in the field of genomics, it has become a common practice to spot presynthesized peptides onto reactive planar array surfaces. the approach is particularly useful when multiple copies of the same array with high density are required. furthermore, peptides can be purified after synthesis and prior to array printing to avoid any complications that may stem from peptide impurity. various chemistries have been utilized for immobilizing peptides onto the array surfaces. one of the popular approaches is to attach n-terminal biotinylated peptides onto avidin/ streptavidin-coated microarray slides (lesaicherre et al. 2002) . covalently immobilizing peptides through a terminal cys onto a surface functionalized with maleimide groups or disulfide is also a method that is being used quite commonly (inamori et al. 2008) . the suitability of immobilization chemistry varies according to the nature of the peptide as well as the downstream applications of the array. in our hands, attaching peptides through the n-terminal free amino groups to a surface functionalized with epoxide groups worked very well for assaying protein kinase activity (see below). thus, it is very important and necessary to determine which strategy should be used for preparing peptide arrays already prior to peptide synthesis, according to the intended application of the resulting arrays. since the advent of the technology more than two decades ago, peptide arrays have been applied in a broad range of investigations including antibody epitope mapping, protein domain-mediated interaction screening, and enzymatic activity profiling. in recent years, the utility of peptide arrays has been further extended to system-wide proteomics studies, fuelled by the advances in genomics and proteomics. this can be exemplified by their roles in cell signaling studies. kineome activity profiling reversible phosphorylation and dephosphorylation of proteins mediated by protein kinases and protein phosphatases, respectively, are recognized as one of the most important and widespread molecular mechanisms in regulating cell signaling pathways involved in cell proliferation, division, differentiation, adherence, angiogenesis, and apoptosis (brognard and hunter 2011) . according to our most recent tallies, the total number of phosphorylation sites in the human proteome is estimated to exceed 650,000, which encompass over 100,000 phosphosites that have been experimentally characterized and those predicted based on evolutionary conservation (http://www. phosphonet.ca). the biological importance and potential clinical significance of protein phosphorylation, exemplified by the implication of deregulation of both kinase and phosphatase activity in a wide range of human diseases including cancer, autoimmune diseases, neurodegenerative diseases, and diabetes, has driven the development of strategies for the identification of physiological substrates for each of over 500 protein kinases within the human kineome, as well as for systematic profiling of kinase activity in biological samples. for the identification of physiological substrates of kinases, a protein microarray featuring all the proteins representing the entire proteome would seemingly be an ideal platform. however, it is technically challenging to create such a comprehensive array encompassing all of the proteins encoded by about 23,000 genes in the human genome with current cloning and expression technologies. the most comprehensive protein microarray currently available commercially, trademarked as protoarray® by life technologies (carlsbad, ca, http://www.lifetechnologies. com), consists of only 9,000 human proteins that have been expressed and purified from a baculovirus-based expression system. moreover, issues with protein conformation, autophosphorylation, and stability on the array surface, as well as complications in data interpretation as a result of the presence of multiple phosphorylation sites within a protein potentially targeted by different kinases, have limited the practicality of this approach. as a result, a number of peptide-based strategies have been devised including peptide libraries and peptide arrays, based on the notion that the substrate specificity of the kinase is largely defined by the flanking linear amino acid sequences around its target phosphorylation site(s) on the substrates. based on the consensus recognition sequences for protein kinases derived from the peptide-based studies, one can deduce potential physiological substrates for each of the kinases in a proteome, coupled with information about protein-protein interactions, subcellular colocalization, and correlations in expression or activation. back in 1995, luo and colleagues originally used the peptide array approach to identify and optimize substrate sequences for protein kinase a (pka) and transforming growth factor (tgf) b receptors (luo et al. 1995) . since then, the substrate specificities for a number of protein kinases have been elucidated and refined sequentially using peptide arrays (schutkowski et al. 2005) . there are two main strategies to be deployed for determining consensus phosphorylation sequences for protein kinases. on the one hand, peptide macroarrays featuring combinatorial peptide libraries or random peptide libraries such as those on the spot cellulose membranes were indispensable tools for elucidating the recognition sequences targeted by the kinases for which little information on their physiological substrates is available. the availability of expanding collections of recombinant active protein kinases in the past several years has facilitated the effort in this front. on the other hand, incorporation of increasing numbers of physiological phosphorylation sites uncovered through recent large-scale mass spectrometry-based phosphoproteomics studies into peptide microarrays has also significantly improved the efficiency of the substrate peptide screening process. currently, many large-scale peptide microarrays comprising a large number of experimentally verified phosphosites as well as those identified and optimized using the peptide library approach are readily available through various commercial sources such as pepstar™ from jpt peptide technologies (jpt peptide technologies gmbh, berlin, germany, http://www.jpt.com), pamchip® from pamgene (pamgene international b.v., hertogenbosch, the netherlands, http:// www.pamgene.com), and pepchip™ from pepscan presto (pepscan presto, lelystad, the netherlands, http://www.pepscanpresto.com), either as products or services, for profiling kinase activities in biological samples. various experimental protocols based on the same approach have been developed (schutkowski et al. 2005; thiele et al. 2010) . the approach was also adapted to kineome activity profiling in bovine samples by utilizing information gathered through bioinformatics analysis of the phosphorylation sites conserved in evolution (jalal et al. 2009 ). however, inferring endogenous kinase activities based on the data from such peptide microarrays is less straightforward than initially thought. one of the main issues associated with the current approach is the overlapping specificity among protein kinases dictated by the promiscuity in substrate recognition, especially for the kinases from the same or related families. phosphorylation of a peptide on each spot may represent the sum of activity of all the kinases targeting this particular peptide. indeed, a specific phosphosite sequence may be optimized through evolution to be recognized by a panel of kinases and phosphatases and not be optimized for an individual kinase or phosphatase. thus, under most circumstances, it is impossible to directly correlate the level of peptide phosphorylation on the array with the activity of a specific kinase. it is even more challenging when the activity of kinases in crude cell or tissue lysates is to be assessed, where the cell compartmentalization has been destroyed and the proper subcellular localization of proteins cannot be maintained. in light of these challenges, we set out to identify the optimal peptide substrate sequences unique to each kinase by combining the high-throughput capability of peptide microarrays with the power of a proprietary kinase-substrate prediction algorithm developed at kinexus (fig. 7 .1). the algorithm was built based on the information gathered through manual analysis of close to 10,000 confirmed kinase-substrate pairs for 229 typical kinases. coupling with the alignment of the primary amino acid sequences of the catalytic domains of protein kinases, the specificity-determining residues (sdrs) were identified, and the position-specific scoring matrix (pssm) was generated for each of the kinases for predicting their respective recognition sequences around the phosphorylation site. the pssms were then used to derive the optimal substrate peptide sequences. in total, 445 15-mer peptides corresponding to the predicted sequences with a single phosphorylatable residue (ser, thr, or tyr) in the middle were synthesized and immobilized onto an epoxysilane-coated glass microarray surface. the resulting peptide microarray was made up of four identical subfields to allowing four kinase assays to be run in parallel. phosphorylation of the peptides on the array was carried out by applying active protein kinases individually into each field under their respective assay conditions, and the extent of peptide phosphorylation was then detected with pro-q diamond (life technologies), a fluorescent dye that had been validated to bind specifically to phosphorylated residues including ser, thr, and tyr, regardless the context of sequences they are in. so far, over 200 protein kinases have been assayed. many highly reactive and selective peptides have been identified as substrates for the kinases tested. while most of the sequences conformed to those reported previously, some novel motifs were also uncovered. detailed analysis of the "hit" peptide sequences is expected to reveal the prototype optimal substrate peptides unique to each kinase, which will then be further optimized for their reactivity and selectivity using an oriented peptide library approach. it is expected in the near future a peptide microarray spotted with substrate peptides that are preoptimized to each of the kinases will become available from kinexus for kineome profiling in complex biological samples. compared to protein kinases, protein phosphatases have been less well characterized with respect to their regulation and physiological substrates. this can be attributed to the misconception that phosphatases are promiscuous in substrate recognition and a b c d fig. 7 .1 a bioinformatics algorithm-guided identification of the optimal peptide substrates unique to each kinase on peptide microarrays. panel a. schematic description of the workflow from peptide substrate sequence prediction using the kinase predictor 1.0 algorithm developed by kinexus to select test peptides for phosphorylation by kinases on the peptide microarray and, finally, to the deduction of the optimal substrate sequences for individual kinases. panel b. scanned image of the full kinex™ kinase substrate peptide microarray phosphorylated with three different kinases. the second field was incubated with atp in the absence of added kinase as a control. each peptide featured a phosphorylatable residue (ser, thr, or tyr) in the middle. the strong spots common among all four fields are the orientation markers designed for easy peptide localization. panel c. close-up scanned image of one field of the kinex™ kinase substrate peptide microarray. panel d. alignment of the top phosphorylated peptides detected following incubation with amp-dependent protein kinase alpha 1. peptides were ranked according to their respective phosphorylation signal intensity, and an optimal substrate peptide sequence is shown in the bottom row regulated in less stringent fashion in vivo, which might have arisen from that observation that a relatively small number of protein-serine/threonine-(ser/thr-) specific phosphatases are able to catalyze a myriad of dephosphorylation events, and that most protein phosphatases have not been found to recognize well-defined linear sequences or consensus motifs within their substrates so far. despite prevailing evidence that short synthetic phosphopeptides are poor phosphatase substrates compared to their parent proteins (zhao and lee 1997) , as supported by the role of regulatory subunits in forming the substrate-binding sites required for substrate recognition according to crystallography studies (virshup and shenolikar 2009) , several phosphopeptide-based studies have been reported that aimed at the delineation of substrate preferences using either activity-or interaction-based approaches (sun et al. 2009; wang et al. 2002) . among the two main classes of protein phosphatases, protein-tyrosine (tyr-) phosphatases (ptps), not protein-ser/thr phosphatases, had been the focus of early studies on substrate specificities, due to the availability of better characterized phospho-tyr antibodies than phospho-ser/thr antibodies. in those studies, phosphatase substrate specificities were commonly delineated using individually synthesized phosphopeptides (cho et al. 1993; zhang et al. 1994) . in recent years, peptide arrays, peptide microarrays in particular, have been demonstrated for their utility in protein phosphatase specificity mapping and activity profiling. in 2008, waldmann's and yao's groups independently used phosphopeptide microarrays for large-scale, high-throughput characterization of ptp and protein-ser/thr phosphatase substrate specificities, respectively (k€ ohn et al. 2007; sun, et al. 2008) , for the first time. while a fluorescently labeled phospho-tyr antibody was employed to monitor dephosphorylation of tyrosine in the peptides in waldman's study, yao and coworkers used pro-q diamond dye to detect dephosphorylation of ser/thr, circumventing the detection problem as a result of the lack of well-characterized generic antibodies for phospho-ser/thr. the dye has recently extended to the detection of dephosphorylation of tyrosine in place of anti-phospho-tyr antibodies on peptide microarray by the same group (gao et al. 2010) . a phosphopeptide microarray featuring the most evolutionarily conserved human phosphorylation sites is now being explored for its potential for the determination of phosphatase specificities and activity profiling in our laboratory. in addition to protein kinases and phosphatases, peptide microarrays have also been successfully used to characterize protease specificity, based on the notion that proteolytic cleavage can be monitored by the changes in fluorescent signals on fluorogenic peptides immobilized on the array upon the action of proteases. salisbury et al. (2002) used a fluorogenic peptide substrate array with 361 spatially addressable peptides to decipher the specificity of thrombin. gosalia and colleagues employed a solution-phase fluorogenic peptide microarray, in which peptides were spotted as spatially separate nanodroplets, to reveal the evolutionary conservation of substrate specificity of thrombin from human, bovine, and salmon (gosalia and diamond 2003) . the approach was also applied to determine substrate preferences of 13 serine and 11 cysteine proteases (gosalia et al. 2005) . winssinger et al. (2004) generated a library of 192 peptides tagged with peptide nucleic acid (pna) molecules and incubated it with protease in solution, followed by spatial deconvolution on a dna microarray to profile the substrate specificities of thrombin, plasmin, and caspase-3. the peptide array-based protease specificity profiling approach has now become an essential part of protease characterization platform, complementary and synergistic to other proteomic approaches used to detect alterations of substrate abundance and to identify and quantitate proteolytically generated neo amino-or carboxy-termini (auf dem keller and schilling 2010). the application of peptide arrays for protein-protein interaction characterization has been well documented since the advent of spot peptide synthesis. it is applicable to characterizing protein-protein interactions where the interface between the two interacting proteins can be recapitulated by linear peptide sequences derived from the parent proteins. it is even more advantageous compared to other proteomics techniques such as protein arrays when the protein-protein interactions mediated by ptms such as phosphorylation are concerned, as amino acids carrying corresponding ptms can be readily incorporated into specific sites during peptide synthesis. not only can the peptide array-based approach be used to map the consensus sequences recognized by these domains, it can also provide dynamic information on signal-dependent change in molecular networks for proteins defined by the peptides on the array and the proteins for which the binding is monitored (sinzinger and brock 2010) . intracellular signal networks are organized through the interactions of proteins, which are often mediated by a group of diverse modular protein interaction domains (pids) with defined specificity. among them, the src homology 2 (sh2) domains are the largest family recognizing tyrosine phosphorylated sequences, and thus play pivotal roles in relaying information flow emanating from receptor protein-tyr kinases (pawson 2007) . a phospho-tyr-oriented peptide library with only one amino acid introduced at the defined positions at a time, and a mixture of amino acids at the randomized position, was spotted on the array and was interrogated with 120 bacterially expressed human sh2 domains, and the phosphotyrosinecontaining peptide sequence motifs for 76 of them were defined (huang et al. 2008) . combining the power of phage-displayed libraries, spot technology, and bioinformatics, the peptide array-based approach was also successfully used to deduce the consensus sequences of yeast sh3 domains (tonikian et al. 2008 (tonikian et al. , 2009 . a peptide microarray featuring peptides with inverted configuration representing 6,223 c-terminal sequences of human proteins was probed with a pdz domain to screen for putative interaction partners (boisguerin et al. 2004 ). with the knowledge of consensus sequences for the pids, peptide microarrays that carry peptides corresponding to known sequences recognized by sh2, sh3, pdz, and other pids have been employed to profile the binding of proteins from complex biological samples to detect the differences in molecular interactions between different physiological states (stoevesandt et al. 2005; sinzinger and brock 2010) . a peptide microarray populated with peptides, in which kinase consensus sequences and caspase cleavage recognition motifs (identified through a search of the human proteome) are overlapping, was employed in a study to investigate the role of phosphorylation in the regulation of caspase signaling pathways. protein kinase ck2 emerged as the kinase with the most number of substrates that contained kinase consensus sequences that overlapped with caspase-3 cleavage motifs, indicating a role of phosphorylation in the inhibition of caspase-mediated apoptosis signaling pathways (duncan et al. 2011) . as a natural extension to their classical application in antigenic epitope mapping, peptide arrays displaying a collection of biologically active synthetic peptides have been demonstrated in recent years to be a very versatile tool for profiling the antibody repertoire in complex biological samples such as serum, urine, saliva, and other types of body fluids for the diagnosis of pathogen infections, allergy reactions, and autoimmunity, based on the notion that the immune response to pathogens, allergens, or autoantigens can be captured by the presence or absence of specific populations of antibodies. hence, serological mapping has become one of the most sought after applications of the peptide array technology, as it appears to have the greatest clinical potential. peptide libraries featuring fragments derived from autoantigens, allergens, or viral proteins presented on either the spot membrane-based peptide macroarrays or glass slide-based peptide microarrays have been used for antibody profiling in clinical samples. the clinical potential of such analyses have been shown by their use for antibody spectrum profiling in the sera from patients infected with hepatitis b and c, with a simian-human immunodeficiency virus (shiv), the severe acute respiratory syndrome (sars) corona virus, and with herpes virus. this provides crucial information not only for infection diagnostics but also for the development of vaccines (neuman de vegvar et al. 2003; duburcq et al. 2004; guo et al. 2004; andresen and gr€ otzinger 2009) . the use of peptide arrays in kineome profiling has also inspired the exploration of their application in the studies of human diseases. as increasing numbers of kinase substrate peptides have been identified in recent years, peptide microarrays with the capability of screening a broad range of protein kinases have been established and used to profile the aberrant kinase activity in clinical samples as well as for monitoring the response to kinase inhibitory compounds in a highthroughput manner. this underscores the potential of peptide arrays in disease diagnosis and drug discovery (piersma et al. 2010 ). among the handful of studies reported so far in this area, schrage et al. (2009) recently reported the activation of multiple pathways in relation to akt/gsk3b, pdgfrb, and src protein kinases in chondrosarcoma cells on a kinase substrate peptide array containing 1,024 peptides. supplemented with the cell viability data in vitro, the study indicated that the src inhibitor dasatinib is a potential treatment option for patients who are inoperable (schrage et al. 2009 ). tuynman and colleagues investigated the molecular mechanism underlying anticarcinogenic activity of celecoxib (celebrex), a selective cyclooxygenase-2 (cox-2) inhibitor, against colorectal cancer (crc) using a kinase substrate peptide array with 1,176 different kinase substrate consensus sequences and found that celecoxib represses c-met-dependent signaling, which in turn led to downregulation of oncogenic wnt signaling in crc, supporting the potential of targeting c-met and wnt signaling in crc therapy (tuynman et al. 2008) . recently, a cellulose membrane-based peptide array of 70 peptides derived from p160 peptide, a cancer cell targeting peptide identified by phage display, was employed to optimize the affinity of the peptides for human cancer cells using peptide-whole cell interaction assay (ahmed et al. 2010) . the binding of the three peptides with the highest affinity and selectivity for cancer cells was further confirmed using fluorescence imaging and flow cytometry. the study revealed the potential of the peptide array-based whole cell binding assay for screening and identifying cancer cell targeting peptides for cancer diagnosis and drug targeted delivery. peptide microarrays broaden a new field of research and applications often referred to as functional proteomics (thiele et al. 2011) . while dna and protein arrays mostly focus on determination of abundance of rna or protein molecules, peptide arrays allow the functional analysis of multiple proteins or protein families (fig. 7.2) . by functional analysis, we mean the detection of protein activity. clear examples are the detection of enzymatic activities, for example, of kinases, phosphatases, and proteases in lysates from cells or tissues. however, nonenzymatic functions, like the responses to hormone binding of nuclear receptors in terms of specific coregulator protein recruitment, are also currently being studied on peptide arrays (heneweer et al. 2007; koppen et al. 2009 ). peptide arrays enable the miniaturization and multiplexing of activity-based assays. in the context of pharmaceutical research, and in the field of translational medicine in particular, such array-based approaches are emerging. this makes sense since the majority of the drugs being developed target protein activity and function. these new and more targeted drugs act by effecting protein function rather than targeting dna or rna or interfering with the modulation of protein levels. because functional profiling of the interaction of drugs with cellular or tissue samples is of specific interest in pharmaceutical research, peptide microarrays are proving to be very useful with their ability to profile protein activity and its modulation by drugs. we focus here on the drug class of kinase inhibitors which have been reshaping the oncology field due to their high success rate. these molecules inhibit kinase function by reducing kinase activity, which can be monitored on a peptide array. kinases play a pivotal role in cellular biology by being the key regulators of signal transduction. signals being detected by a membrane-bound receptor are transduced to the inner parts of the cell to result in an appropriate response. this happens via highly complex cascades of events in which the signal is received and propagated using transphosphorylation reactions (fig. 7.3) . these reactions are catalyzed by protein kinases together with the crucial atp molecule. atp is important as not only does it provide the kinase's energy source, it also supplies the phosphate moiety, vital to the whole signal transduction cascade. a kinase becomes activated and places this phosphate group on a substrate protein; this being the subsequent link in the signal transduction pathway. often this substrate protein is a kinase as well. a signal transduction event can be compared to a relay in athletics, where each kinase gets activated by an upstream event and subsequently passes on the baton to the next member downstream in the pathway. most protein kinases have distinct preferences for the aromatic hydroxyl groups of tyrosine residues or for the aliphatic serines or threonines. it is this characteristic which divides this family of more than 500 members into two kinase subfamilies: proteintyrosine kinases (ptks) and protein-serine/threonine kinases (stks). while in classic kinase assays the activity is detected by the phosphorylation of a single substrate, multiple substrates can now be immobilized and monitored on a microarray. instead of placing multiple protein substrates on a chip, only the phosphosites (the sites within the protein which become phosphorylated) are immobilized in a peptide microarray. thus, the peptides represent the protein substrates. as has been discussed in the previous chapters, this can be done in a variety of ways, but all are based on a solid support. in most cases, the sequences are derived from known phosphorylation sites in the human proteome. as the human proteome is estimated to comprise more than a million proteins, of which more than two-thirds can be phosphorylated, this indicates the huge amount of different phosphosites that can be investigated by peptide arrays. the principle of the assay is that the kinase activities in the sample of interest phosphorylate the peptides. the phosphorylation event is detected by either radiography or fluorescence imaging of the array. in radio assays, the peptide is phosphorylated using radioactive atp as the phosphate source. this approach is increasingly being replaced by the use of fluorescence assays. in the latter case, the phosphorylation of the peptide is detected by a fluorescently labeled molecule which is either a chelator (e.g., phosphotag) or an antibody. ideally, the antibody needs to detect the phosphoamino acid in all the available peptide sequences on the chip equally well and independently of the adjacent amino acids. antibodies like py20 work very well in detecting tyrosine phosphorylated peptides, but for serine/ threonine phosphorylated peptides, cocktails are needed for full coverage of detection. the first peptide microarray applications for kinase profiling used glass as a solid support and radioactivity for readout at a single time point. later, protocols were developed based on the fluorescent readout of labeled antibodies (or cocktails of antibodies) binding to phosphorylated peptides. a second generation of this technology was developed by researchers in the netherlands and is referred to as the pamchip® technology (lemeer et al. 2007; hilhorst et al. 2009; versele et al. 2009) (fig. 7.4) . with this technology, antibody-based fluorescence detection has been combined with a change of solid support from glass slides to a porous ceramic. in this format, the sample is pumped up and down through the porous aluminum oxide ceramic sheets, in which the peptides are immobilized at designated spots. each spot comprises thousands of separated pores with diameters of 0.2 mm in which the peptides are site-specifically immobilized. each time the sample is below the solid support, the degree of phosphorylation is monitored by imaging the fluorescence intensities caused by the antibody binding to the phosphorylated peptides alone. these time curves, or kinetic readouts, appear to be instrumental in the enzymatic studies; a kinase is after all an enzyme which catalyzes the rate (the kinetics) of a phosphorylation reaction. in addition, the kinetic and multi(time) step readout for each of the 144 or 256 peptides on each single array allows much more comprehensive statistical analysis of the signals than data from a single time point per peptide spot on a glass array (thilakarathne et al. 2011) . the application of peptide arrays in biological, pharmaceutical, and medical studies often requires the analysis of many samples under variable conditions. for example, lysates from cells should be analyzed using a range of time points, varying concentrations, and with multiple different drugs. for this reason, a system has been developed which has the capability of analyzing 96 arrays at once. this latest technology for kinase activity profiling is based on a 96-well plate format, in which each well comprises a peptide microarray. bioinformatics for analysis of the vast datasets from such studies has been evolving in parallel. thilakarathne et al. (2011) developed a new method based on semiparametric mixed linear models to further enhance the amount of information that can be obtained from the multiparallel kinetic readouts from each microarray. a straightforward application is substrate identification using recombinant kinases. such studies have indicated that different kinases have their own preferences for the peptide sequences they phosphorylate. clear differentiation between the ptks and stks has been confirmed, although dual specificity kinases have also been found. in addition, it has become apparent that although each kinase has a preference for particular peptide sequences, they can also be promiscuous, resulting in multiple peptides being phosphorylated to different degrees in diverse peptide sets. in short, the degree of phosphorylation by purified kinases varies from peptide to peptide and can be profiled in hundreds per array, resulting in phosphorylation fingerprints. substrate profiling studies have revealed important biological information as described in the paper by schutkowski et al. where they showed that for optimal recognition by gsk3b, a peptide substrate should be prephosphorylated or primed . due to this porosity, the sample can be pumped up and down through this solid support. every time when the sample is positioned below the microarray, an image is taken of the microarray by a ccd camera (middle panel). via this realtime imaging of the microarray, the signal, developing in the peptide spots due to binding of fluorescent antibody detecting peptides phosphorylated by the kinases, can be monitored (schutkowski et al. 2004 ). another interesting application was explored by poot et al. who identified an optimal substrate for pkc isozymes and coupled this peptide to an atp-binding site inhibitor to generate a bisubstrate (poot et al. 2009; van ameijde et al. 2010) . the peptide microarrays were subsequently used to evaluate the resulting inhibitors, which were potent and selective toward the theta isozyme. with the development of protocols for profiling cell lysates of tissue homogenates, the application area has been broadened to signal transduction and pathway studies [well reviewed by the group of schutkowski (thiele et al. 2011)] . the effect of a stimulus or a kinase-inhibiting drug on cultured cells can now be investigated at the complexity level of a cell, where multiple kinases can be active in the context of the interacting networks that exist. at this point, peptide arrays provide a welcome extension to classical methods like (phospho) western blots and elisas, which monitor drug effects on a (single) kinase by detecting the variation in abundance of the downstream phosphorylated substrate. the peptide arrays monitor the enzyme activity of multiple kinases at once and not only the end result of this activity. an interesting feature of functional proteomics is found in the ability to study direct effects of the investigative drug. because activities of kinases can be monitored in cell lysates or tissues, drugs can be characterized in a complex, and probably more realistic, context than in classical single readout (singleplex) assays. this latter type of assay is limited as it can only investigate the activity of the isolated drug target. drug selectivity profiling is a clear example of an application which benefits from the combination of multiplexing and miniaturization (fig. 7.5) . another example of an application area is the unraveling of a drug's cellular mechanism of action. in this application, the peptides on the chip represent multiple different proteins involved in complex cellular pathways and signal transduction networks. in a small lysate sample, derived from just 10,000 to 100,000 cells or less than a tenth of a cubic millimeter of tissue, multiple diverse interactions can be studied at once. a recent development of peptide microarrays has been the application of new drugs in pharmaceutical research and clinical development. in the field of oncology in particular, fundamental progress has been made by so-called targeted medicine. previously, anticancer drugs were targeting cellular processes, like cell division, more globally. new insights into cell signaling and signal transduction cascades have changed the way novel oncological drugs are being developed, and it is the kinase enzyme class which is playing a crucial role in this progress. many of its members play a pivotal role in the mechanisms of tumor genesis, and some kinases are even the active protein products of oncogenes. examples of successful cancer drugs targeting protein kinases-or the signaling pathways they are involved with-are imatinib, erlotinib, gefitinib, and the previously mentioned sunitinib and sorafenib. these are all molecules that block the catalytic activity of protein kinases. a related class of therapeutics is antibodies, which intervene in a different way with cellular signaling: they act by blocking the initiation of receptor signaling. examples of the latter are trastuzumab and bevacizumab, which block egfr kinase and vegfr signaling, respectively. these drugs can be studied comprehensively with peptide arrays. in these studies, two formats are currently being used. using cell line models, the cells are either treated with the drug in culture or on the chip. in the first case, lysates are prepared from the cells before and after treatment and profiled for activities on the chip. in the second case, lysates can be treated directly by spiking the drug into the solution just before application onto the chip. in the latter instance, cell lines, tissue homogenates from animal models, or even clinical samples can all be used. the effects of the inhibitors on the kinase activities in these samples can all be directly assessed. although the highly important context of the cellular architecture is lost, which is surely a downside, the potential to profile all detectable, full-length kinases-with their relevant posttranslational modifications-in the same sample, opens up vast new fields of applications. in drug discovery, researchers screen for kinase-inhibiting compounds in chemical libraries. during such studies, they often use an abstracted model, the purified protein, but this protein is frequently truncated to its domain only. a major disadvantage of this approach is the absence of other domains, including those with a inhibition / activation sorafenib sunitinib fig. 7 .5 selectivity profiling of kinase inhibitor drugs using peptide microarrays. here sorafenib inhibition profiles are compared with sunitinib in extracts from both normal and tumor tissue from a renal cell carcinoma patient regulatory function. in the recently developed protocols for peptide array analysis of kinases in cell and tissue lysates, the drug target can now be studied more naturally as a full-length protein, in the way it is actually expressed in cells or tissues. at first, this was shown in a model system, but interestingly, this approach appears to be translatable to patient-derived tissues. this means that the kinase drug targets can now be studied in the same form as they are expressed in a patient's tumor, thus full length, fully decorated with all relevant posttranslational modifications and in the presence of stabilizing or activating cofactors (e.g., heatshock proteins). in addition, they can be studied in the presence of all other kinases expressed in the cell or tissue being investigated. such analysis of patient-derived tumor samples can result in the identification of tumor-specific kinase activities. when linked to pathological, diagnostic, and/or clinical data, this can lead to the identification of diagnostic or prognostic biomarkers. while the on-target effects are being monitored, the researchers can also obtain insights into the drug's effect(s) on other kinase targets, which are either intended-in the case of multitarget inhibitors-or unintended and can putatively cause side effects. the latter opens up new opportunities for the toxicologist in investigating and understanding adverse drug reactions leading to toxicological biomarkers. a very typical feature of activity-based assays is the capability of drug testing. with peptide microarray-based kinase assays, not only can multiple kinases in a patient sample be studied at once, their response to their inhibiting drugs can also be studied. this possibility links the presence and activity of the kinase drug targets to their responsiveness to the drug. drug response is a leading parameter in the clinical development of a drug. in the development of kinase inhibitors in cancer, the response rates are often very low, even in case of effective drugs. these drugs are developed against specific kinase targets, but these targets are not always equally present or active in the whole treated population. furthermore, in a subset of nonsensitive or resistant patients, the role of this target in tumorigenesis and growth or metastasis is not essential and can be overruled by other mechanisms. in order to identify the patient subpopulation that is likely to respond, tests need to be developed that match the right patient to the right drug and vice versa as more drugs are being developed. there are already examples of such companion diagnostic tests. for the prediction of response to trastuzumab (moelans et al. 2011) , targeting the receptor tyrosine kinase her2/neu, patients are tested for the presence of this target on their tumor cells, before they receive this breast cancer therapy. a recent example is the test for the elm4-alk translocation to select patients for pharmacotherapy with crizotinib (kwak et al. 2010) , an alk kinase inhibitor. if the whole lung cancer patient population would have been treated, only an extremely low percentage would have shown a clinical response because only 4% have this mutation. the availability of the companion diagnostic test was therefore essential for the success of the clinical trial. the identification of predictive biomarkers appears to become essential in many drug development programs. the classical technologies for biomarker discovery are based on testing for dna mutation or rna or protein expression levels. molecular data are obtained in biopsies taken before the patient is treated. if these data can be correlated or associated to the therapy response, this can be the start of generating a companion diagnostic test. peptide microarrays are also currently being used in this effort. while classical methods cannot involve the drug of interest in predose tissue samples, kinase microarrays can, as discussed above. in addition, the drug can actually be used in the test. this means that drugspecific data and information can be generated using predose biopsies. proof of concept of this approach was shown by versele et al. in a multiple cell line study. analogous to the way it is aimed to work in a clinical setting, they profiled the lysate of a cell line on a peptide microarray in the presence and absence of their drug candidate. the inhibition profiles were used to predict the response of the cell line to drug treatment in culture. from these profiles, they could identify a set of peptide phosphorylations of which the response (inhibition) on the chip was predictive for the tumor cell proliferation (versele et al. 2009 ). this concept (fig. 7.6 ) is now being explored in clinical studies by my research group in collaboration with the netherlands cancer institute (nki), the vu medical center, and other cancer centers in both the usa and japan. in a study presented at asco in 2011 on neoadjuvant treatment of non-small-cell lung cancer with the egfr kinase inhibitor erlotinib, we showed that candidate biomarkers could be identified. on-chip peptide phosphorylations and inhibitions were correlated to clinical responses. with no information on the pathological assessment of the resection tissues available to the testers, a model built on those profiles could still predict the pathological response (hilhorst et al. 2011) . it should be noted that resection tissue was used and not pretreatment biopsies which is needed to make this into a it could be possible to apply this principle of drug testing on patient-derived tissues to other targeted therapies as well. in addition, if other protein classes are targeted, for example, phosphatases, proteases, nuclear receptors, acetyltransferases, histone deacetylases, and methyltransferases, the target responses in patient-derived samples could be tested using a peptide microarray. the nonfocused, nonbiased, and global profiling nature of the arrays allows parallel monitoring of drug targets and class-related nontargets. these nontargets can be functional proteins involved in the mechanisms of resistance and are therefore possibly very useful markers for predicting resistance to targeted therapies. finally, nontargeted therapies such as chemoradiation could also be accompanied in the future by such testing methods, as was shown in a recent publication by a norwegian group. they generated kinase activity profiles of tens of biopsies taken before patients were treated and could identify peptide phosphorylation patterns that correlated to the tumor regression grade after therapy. they generated a response prediction model that could predict the responses of a newly tested set of patients with promising accuracy (folkvord et al. 2010) . thomas felgenhauer, ralf bischoff, frank breitling, and volker stadler several sophisticated methods are in use worldwide to produce peptide microarrays. each of these methods has its special advantages and drawbacks. high amounts of identical oligomers are achievable on cellulose supports via spot synthesis (frank 1992; dikmans et al. 2006) , but spot densities are very low due to droplet handling. with photochemical methods where chain growth is induced by a laser beam, very small spot sizes and high spot densities are possible (fodor et al. 1991a, b; lipshutz et al. 1999) . in this case, the drawback is in the sequential use of monomer solutions which might be acceptable in dna synthesis (four monomers), but the yield is dramatically reduced when the number of needed coupling cycles increases as in the case of standard peptide synthesis where minimum 20 individual cycles are needed to complete a fully combinatorial layer. the use of a laser printer as synthesis machine makes it possible to overcome the obstacles of the methods described above. solid particles (toners) carrying the reactive building blocks are printed in parallel in high resolution to a desired support. a full combinatorial layer is developed-like a color picture printoutat once, and the coupling cycles are reduced from 20 to a single one per layer (stadler et al. 2008) . a commercial laser printer uses small solid toner particles (~10 mm) that are triboelectrically charged by friction inside a toner cartridge drum system. because of the materials involved, this procedure leads to strong electrical charges on the particle surfaces, which enables the directional movement of the particles within electrical fields. a laser beam or an led row translates 2d light patterns into electrical patterns on top of an organic photoconductor drum. these images are developed with the charged toner particles that are finally transferred to the support. at office applications, a color laser printer system delivers four different color toners (black, cyan, yellow, and magenta) on a sheet of paper with a resolution of 1,200 up to 2,400 dpi. the polymer-based toner particles are fixed to the cellulose support by heat. the main challenge in combinatorial synthesis is to deliver different kinds of monomers with high accuracy to their designated reaction partner or reaction site. whereas a color laser printer delivers only four toners, a peptide synthesizer based on the xerographic technique should be able to handle at least 20 different building blocks for basic peptide synthesis or other feasible monomers for the production of peptide mimetics (amino acids in d-form, methylated, phosphorylated derivatives, nonnatural versions). in addition to the great flexibility of the synthesizer, an exact positioning of consecutively printed layers is the basic requirement for the parallel elongation of combinatorial assembled oligomer chains. with increasingly better printing accuracy, the spot density also increases, as well as the diversity of synthesized peptides. to benefit from the laser printer as delivery machine for monomers in combinatorial chemistry, the toner particles (delivery packages) have to be modified for this chemical purpose. in addition to their properties as solid, electrically charged particles, they also need the attributes of a solvent once melted. this change of properties happens after the particles have been addressed to their designated reaction site, where they are transformed into a liquid sphere simply by melting. thereby, activated monomers are mobilized, which allows them to diffuse to their reaction partner for chain elongation. these very special solid/liquid characteristics of the toner particle depend on the choice of the appropriate matrix material. on the one hand, this material should withstand the harsh mechanical treatment inside the printer (e.g., friction, charging, transport); on the other hand, the liquefaction at moderate temperatures (<100 c) is fundamental in order to perform as a solvent for a chemical reaction. in addition, the matrix material should protect the reactive monomers from ambient conditions during long-term storage in cartridges, and finally, the material itself must be inert toward the components inside. since all the different monomer particles are addressed in parallel by the printer, all the different activated amino acid derivatives within a completed layer of amino acid particles are activated at once in a single melting step. this feature is the main advantage of our technique. washing and deprotection steps that follow after the coupling step finish the cycle, and result, if repeated, in the combinatorial synthesis of a peptide array (fig. 7.7) . our method uses conventional fmoc chemistry (chan and white 2000) and differs from the spot synthesis only in the solvent we employ: it is solid at room temperature, which allows for the intermittent immobilization of chemically highly activated amino acid derivatives within particles. this activation is due to a c-terminal pentafluorophenyl ester in combination with n-terminal fmoc protection and standard side chain protecting groups. surprisingly, when embedded in particle matrix, these very reactive chemicals proved to be stable for months at room temperature, an exception being fmoc-arginine-opfp that shows a decay of 4% per month. however, if compared to the much faster decay of activated arginine esters in solution, this decay is negligible. the surface-coated solid support must provide free amino groups that react with preactivated amino acid derivatives. it must stand harsh conditions during peptide synthesis (solvents, bases, strong acids during final cleavage of side chain protecting groups) and postsynthesis; it must allow for the incubation of arrays with an analyte, for example, an antibody solution. we employ 30-100 nm thick 3d polymer coatings that have a high loading capacity (high density of amino groups). alternatively, essentially 2d layers are used as solid supports for peptide synthesis. these are generated from functionalized silanes that also stand the conditions during peptide synthesis and, dependent on the used assay system, sometimes perform better when compared to the polymer coating. such surfaces are described in detail elsewhere (beyer et al. 2006; stadler et al. 2007 ). the peptide laser printer at pepperprint gmbh (see fig. 7 .8a, b) has 24 printing units assembled in a row. twenty of these toner cartridges that are based on the oki system are equipped with fmoc-amino acid esters; the four remaining cartridges are used for nonstandard amino acid particles. the printer works with micron resolution which currently allows for the synthesis of 270,000 peptides on a (20 â 20) cm 2 glass substrate. this corresponds to a spot density of~800 spots per cm 2 . currently, the machine is used for the synthesis of up to 20meric peptides. our particle-based synthesis method makes available to the scientific community, for the first time, very high-density peptide arrays. this is, in technical terms, the main novelty of this method (table 7 .1). as such, this method certainly soon will approach the number of different molecules that nature's screening systems employ. these use, for example, millions of randomly generated antibodies to screen for binders against virtually any target molecule, among them nonnatural molecules that have never been encountered by evolution. peptide arrays with natural amino acids (l-form) have been used in the past mainly as protein fragment libraries in proteome research, or as diagnostic tools for serum screening and for antibody profiling. for the development of novel therapeutics, often nonnatural amino acids (e.g., d-amino acids) were integrated at critical positions within the peptide sequence in order to increase the metabolic stability of the peptides. however, due to the use of only low-density peptide arrays, up to now, such screens usually had to rely on extensive previously available knowledge. typically, a peptide sequence that was already known to bind to the target was then modified to improve the stability or the binding affinity, or an already known antigenic protein sequence was used to generate many overlapping peptides in order to narrow down an antibody's binding epitope. in the near future, we will certainly see that very high-density and affordable peptide arrays will be used to find binders without extensive previous knowledge about the sequence of a potential binder. similar to nature's screening systems, a vast number of different peptides should be sufficient to find binders against nearly any target molecule, and different from surface display techniques, the array format will allow for an easy and unequivocal discrimination of specific from nonspecific binders. very high-density peptide arrays will be used for such screens that have been cleared from all those peptides that were found to bind to more than a few different target proteins. it is practically impossible to avoid such nonspecific binders in all the surface display methods. thus, the high combinatorial diversity given by the laser printer method should increase the possibility to discover potent low-density peptide arrays have been used previously to narrow down the binding epitope(s) of binding antibodies by synthesizing many overlapping peptides derived from the sequence of a known antigen that has been used, for example, to immunize an animal; however, such experiments used to be prohibitively expensive. highdensity peptide arrays are cheaper-they simply allow more of these experiments. it should be feasible in the future, for example, to routinely monitor the kind of antibodies that evolved in a mouse that was immunized with a protein. the experimenter could then use only those mice that evolved an antibody specific for an especially interesting epitope within the protein that was used for immunization, thus saving a lot of time and money by using only selected mice for the generation of hybridomas. shown as an example for this statement are the results obtained when we used four different rabbit sera that were immunized with protein a and closely related protein b (fig. 7.9 ). only one of two rabbits immunized with protein a or with protein b revealed that specific antibodies have been generated, while the other two rabbits did not generate protein a-or protein b-specific antibodies. the staining pattern revealed that rabbit 1 developed several antibodies that were specific for the c-terminal region of protein a, while rabbit 3 developed antibodies that targeted the n-terminal region of protein b, and when scrutinizing the peptide sequences, both productive sera did not reveal cross-reacting antibodies against determinants from closely related proteins #a and #b. a biomarker is a substance that is used as an indicator of a biological state. the biomarker serves as an indicator to measure and evaluate normal or pathogenic biological processes, or pharmacologic responses to a therapeutic intervention. especially useful are biomarkers that are found in human sera. these are a rich and accessible source for the detection of diagnostic markers in human diseases. serum antibodies have been used extensively for diagnosis, for example, of flu antibodies in a patient. such antibodies often are found in a patient decades after infection and can easily be analyzed with peptide arrays. especially interesting are those (peptide-)antigens (and their corresponding antibodies) that are targeted by an immune response. these are useful as biomarkers for drug discovery and diagnostics. however, the most interesting scientific question in biomarker discovery is as follows: can we find novel antigens and their corresponding antibodies without previous knowledge about the antigen? figure 7 .10a shows that such a scientific question could be answered with very high-density peptide arrays. a randomized high-density array of 15meric stochastically chosen peptides (only a detail view of~5,000 structures is shown) was used to find peptide binders for the flag m2 antibody with its known binding epitope nnndyknnnd/ennn. indeed, we could find six weak binders in a first screen. sequences from all of these initial hits were then used in a follow-up screen that stained the completely permutated sequences from these initial binders. figure 7 .10b shows such a permutation screen that started with the sequence "ecwgdyksmecadwh" found as an initial hit. this sequence, and all the other five hits from the initial screen, then revealed either the sequence nnndyknnnennn or nnndyknnndnnn, i.e., the flag m2 epitope. all amino acid positions depicted by "n" could be exchanged by other amino acids. it remains to be seen if such a screen could be employed to also find novel biomarkers when staining stochastically chosen very high-density peptide arrays with serum antibodies derived from patients with enigmatic diseases. many protein-protein interactions are mediated by short linear motifs. some of these interactions are deregulated in diseases and thus are potential targets for modulating peptide-based drugs. these peptides should interfere with the protein-protein interactions by binding to one of the partners, either activating or inhibiting the signals that depend on the respective proteins. historically, and as stated above, peptide drugs have been based, for example, upon the optimization of natural peptide hormones but, more recently, novel peptides are being developed that have been isolated from combinatorial recombinant libraries. the idea is to offer such a large number of different potential peptide-based binders that simply by chance any protein would "find" at least one binder among a plurality of different peptides. however, the current size limits of peptide arrays and the associated costs had made it, until now, unrealistic to conduct such comprehensive profiling screens. the production of microarrays by laser printing uses a novel chemical concept where an activated monomer is encapsulated within solid particles that are sent to different "addresses" on a surface displaying reactive groups. thereby, an established technology is used for the rapid construction of a densely spaced pattern of different kinds of particles. these comprise different building blocks that are wild-type residues are highlighted by a red circle initially "frozen" at room temperature within solid particles. thawing these particles at once leads to a single coupling step per layer, which is the main advantage of this method when compared to sequential coupling, for example, in lithographic methods. our particle-based method is particularly well suited for automation and, thereby, results into drastically reduced cost per peptide spot. it brings affordable high-density peptide arrays within reach of normal laboratories and may have an impact similar to the one that high-density oligonucleotide arrays had in the field of genomics. peptide arrays for screening cancer specific peptides deciphering the antibodyome-peptide arrays for serum antibody biomarker diagnostics functional peptide microarrays for specific and sensitive antibody diagnostics proteomic techniques and activity-based probes for the system-wide study of proteolysis a novel glass slide-based peptide array support with high functionality resisting non-specific protein 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chemical synthesis on a membrane support bl€ ocker h (1983) a new general approach for the simultaneous chemical synthesis of large numbers of oligonucleotides: segmental solid supports light directed massively parallel on-chip synthesis of peptide arrays with t-boc chemistry activity-based high-throughput determination of ptps substrate specificity using a phosphopeptide microarray use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid printing chemical libraries on microarrays for fluid phase nanoliter reactions high throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays sars corona virus peptides recognized by antibodies in the sera of convalescent cases estrogenic effects in the immature rat uterus after dietary exposure to ethinylestradiol and zearalenone using a systems biology approach peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase a peptide arrays on cellulose support: spot synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion defining the specificity space of the human src homology 2 domain optimal surface chemistry for peptide immobilization in on-chip phosphorylation analysis genome to kinome: species-specific peptide arrays for kinome analysis a microarray strategy for mapping the substrate specificity of protein tyrosine phosphatase nuclear receptor-coregulator interaction profiling identifies trip3 as a novel peroxisome proliferatoractivated receptor gamma cofactor anaplastic lymphoma kinase inhibition in non-small-cell lung cancer proteintyrosine kinase activity profiling in knock down zebrafish embryos developing site-specific immobilization strategies of peptides in a microarray high density synthetic oligonucleotide arrays the specificity of the transforming growth factor beta receptor kinases determined by a spatially addressable peptide library a study to assess the cross-reactivity of cellulose membrane-bound peptides with detection systems: an analysis at the amino acid level photolithographic synthesis of high-density oligonucleotide arrays automated synthesis of peptides solid-phase peptide syntheses current technologies for her2 testing in breast cancer microarray profiling of antibody responses against simian-human immunodeficiency virus: postchallenge convergence of reactivities independent of host histocompatibility type and vaccine regimen dynamic control of signaling by modular adaptor proteins light-generated oligonucleotide arrays for rapid dna sequence analysis strategies for kinome profiling in cancer and potential clinical applications: chemical proteomics and array-based methods development of selective bisubstrate-based inhibitors against protein kinase c (pkc) isozymes by using dynamic peptide microarrays blind prediction of response to erlotinib in early-stage non-small cell lung cancer (nsclc) in a neoadjuvant setting based on kinase activity profiles peptide microarrays for the determination of protease substrate specificity kinome profiling of chondrosarcoma reveals src-pathway activity and dasatinib as option for treatment high-content peptide microarrays for deciphering kinase specificity and biology peptide arrays for kinase profiling automated maskless photolithography system for peptide microarray synthesis on a chip maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array peptide microarrays for a network analysis of changes in molecular interactions in cellular signalling multifunctional cmos microchip coatings for protein and peptide arrays combinatorial synthesis of peptide arrays with a laser printer peptide microarrays for the detection of molecular interactions in cellular signal transduction peptide microarray for highthroughput determination of phosphatase specificity and biology high-throughput screening of catalytically inactive mutants of protein tyrosine phosphatases (ptps) in a phosphopeptide microarray high density peptide microarrays for proteome-wide fingerprinting of kinase activities in cell lysates deciphering enzyme function using peptide arrays the use of semi-parametric mixed models to analyze pamchip peptide array data: an application to an oncology experiment identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries bayesian modeling of the yeast sh3 domain interactome predicts spatiotemporal dynamics of endocytosis proteins cyclooxygenase-2 inhibition inhibits c-met kinase activity and wnt activity in colon cancer preparation of novel alkylated arginine derivatives suitable for click-cycloaddition chemistry and their incorporation into pseudosubstrate-and bisubstrate-based kinase inhibitors response prediction to a multitargeted kinase inhibitor in cancer cell lines and xenograft tumors using high-content tyrosine peptide arrays with a kinetic readout from promiscuity to precision: protein phosphatases get a makeover screening combinatorial libraries by mass spectrometry. 2. identification of optimal substrates of protein tyrosine phosphatase shp-1 pnaencoded protease substrate microarrays a special cellulose membrane support for the combinatorial and parallel synthesis of peptide libraries suitable for the sc2-type manufacturing of high density multi-purpose chemical micro-arrays protein tyrosine phosphatase substrate specificity: size and phosphotyrosine positioning requirements in peptide substrates a protein phosphatase-1-binding motif identified by the panning of a random peptide display library key: cord-258468-52gej3co authors: marcekova, zuzana; psikal, ivan; kosinova, eva; benada, oldrich; sebo, peter; bumba, ladislav title: heterologous expression of full-length capsid protein of porcine circovirus 2 in escherichia coli and its potential use for detection of antibodies date: 2009-08-05 journal: j virol methods doi: 10.1016/j.jviromet.2009.07.028 sha: doc_id: 258468 cord_uid: 52gej3co a capsid protein of porcine circovirus 2 (pcv 2) serves as a diagnostic antigen for the detection of pcv 2-associated disease known as a postweaning multisystemic wasting syndrome (pmws). in this report, a bacterial expression system was developed for the expression and purification of the full-length pcv 2 capsid (cap) protein from a codon-optimized cap gene. replacement of rare arginine codons located at the 5′ end of the cap reading frame with codons optimal for e. coli was found to overcome the poor expression of the viral protein in the prokaryotic system. the cap protein was purified to greater than 95% homogeneity by using a single cation-exchange chromatography at a yield of 10 mg per litre of bacterial culture. despite the failure of the e. coli-expressed cap protein to self-assemble into virus-like particles (vlps), the immunization of mice with recombinant cap yielded antibodies with the same specificity as those raised against native pcv 2 virions. in addition, the antigenic properties of the purified cap protein were employed in a subunit-based indirect elisa to monitor the levels of pcv 2 specific antibodies in piglets originating from a herd which was experiencing pcv 2 infection. these results pave the way for a straightforward large-scale production of the recombinant pcv 2 capsid protein and its use as a diagnostic antigen or a pcv 2 subunit vaccine. porcine circoviruses (pcv) are small non-enveloped, singlestranded circular dna viruses of the circoviridae family . two distinct types of pcv have been described: the non-pathogenic pcv type 1 (pcv 1) (tischer et al., 1982) and the pathogenic pcv type 2 (pcv 2) nayar et al., 1997) , which is associated with a newly emerged disease called postweaning multisystemic wasting syndrome (pmws) (clark, 1997) . four-to twelve-weeks old piglets which are affected with pmws display clinical symptoms of wasting, respiratory distress, anaemia, diarrhoea, jaundice and enlarged lymph nodes (for review, see chae, 2004) . pmws is considered to be an important porcine disease worldwide which is reported to have a serious economic impact on the global pig farming industry. the 1.77 kb pcv 2 genome contains three functional open reading frames (orfs) . orf1 encodes several forms of non-structural replicase proteins (mankertz and hillenbrand, 2001; mankertz et al., 1998) , orf2 encodes the capsid protein (nawagitgul et al., 2000) , and orf3 encodes a 105-amino acid protein which appears to be involved in virus-induced apoptosis of infected cells (liu et al., 2005) . the capsid protein is a unique structural protein of the viral coat (nawagitgul et al., 2000) that is formed by 60 protein subunits in an icosahedral t = 1 capsid structure (crowther et al., 2003) . the n-terminus of the capsid protein is rich in basic amino acid residues and displays a nuclear localization signal (nls) which is important for capsid assembly (liu et al., 2001a) . the capsid protein is highly immunogenic and reacts strongly with serum from pcv 2-infected pigs (mahe et al., 2000; fenaux et al., 2003) , and thus, it is the preferred antigen in a variety of serological tests (nawagitgul et al., 2002; blanchard et al., 2003; liu et al., 2004; shang et al., 2008) . the expression of recombinant capsid protein was achieved successfully in the baculovirus system (nawagitgul et al., 2000; mahe et al., 2000; blanchard et al., 2003; fan et al., 2008) , where the expressed capsid protein assembled spontaneously into virus-like particles (vlps) (nawagitgul et al., 2000; liu et al., 2008) . vlps represent non-infectious structures which completely lack the dna or rna genome of the virus. therefore, vaccines based on vlps trigger an immune response to the host immune cells by mimicking native virus morphology (ludwig and wagner, 2007) . however, the production of vlps for vaccination in eukaryotic systems is costly for veterinary applications. on the other hand, heterologous protein expression in e. coli offers an alternative to the production of large amounts of protein. the expression of full-length capsid protein in a standard bacterial expression system, such as e. coli bl21 (de3), has not been reported. only certain regions of the cap protein (wu et al., 2008) or a fusion protein with maltose-binding protein (liu et al., 2001b) or truncated variant of cap lacking the nls (zhou et al., 2005; trundova and celer, 2007) have been expressed in e. coli. in this study, a genetic approach which takes advantage of a codon-optimized synthetic oligonucleotide and a synthetic codonoptimized gene is described to obtain the high-level production of the full-length pcv 2 capsid protein in e. coli bl21 (de3) cells. the purified capsid protein is used as antigen to develop an indirect elisa for monitoring the levels of pcv 2 specific antibodies in piglets originating from a herd experiencing pcv 2 infection. the czech field-strain isolate of porcine circovirus type 2 (l-14181, brno, czech republic) was used in this study. the virus stock was prepared from the supernatant of organ homogenate from a pig which fulfilled the diagnostic criteria for pmws (sorden, 2000) . samples of enlarged lymph nodes were pooled and homogenised in a fivefold volume of phosphate-buffered saline (pbs, ph 7.2). two volumes of chloroform were added to 10 volumes of the homogenate and the mixture was shaken at 20 • c for 10 min and centrifuged at 3000 × g for 15 min. the supernatant was subjected to a cushion of cscl density gradient (1.3 g/ml) and centrifuged at 60,000 × g for 4 h in a beckman sw 60ti rotor (beckman coul-ter, fullerton, usa). the purified pcv 2 virions were resuspended in pbs and subsequently used to infect the circovirus-free pk15 cells which were maintained in a d-mem medium (paa laboratories, pasching, austria) supplemented with 10% heat-inactivated fetal calf serum (gibco, invitrogen, carlsbad, usa) at 37 • c with 5% co 2 . the viral dna was purified from pk15-infected cells using a dnazol genomic dna isolation reagent (molecular research center, cincinnati, usa) according to manufacturer's instructions. the genomic dna of pcv 2 isolate was sequenced by abi prism 3130xl analyzer (applied biosystems, foster city, usa) using bigdye terminator 3.1 cycle sequencing kit (applied biosystems, foster city, usa). the nucleotide sequence encoding the cap protein was 99.9% identical with that of orf2 of pcv 2 strain fd4 (genbank accession no. ay321986) (de boisséson et al., 2004) . a 707 bp sequence encoding the cap protein was amplified using polymerase chain reaction (pcr) with the following primers: the upstream primer 5 -ccccatggcgatgacgtatccaaggaggc-3 containing the ncoi site and the downstream primer 5 -tgtctcgagagggttggggggtc-3 containing the xhoi site. the purified pcr product was cut with ncoi and xhoi (new england biolabs, ipswich, usa), cloned into the pet28b expression vector (novagene, merck kgaa, darmstadt, germany), and the vector was designated as pet28b-cap-his (fig. 1b) . to generate a truncated version of the cap protein ( cap-his) lacking the first n-terminal 16 amino acid residues, the pet28b-cap-his was used as a template for pcr with the following primers: the upstream primer 5 -agaccatgggcagcc-atcttggccagatc-3 containing ncoi site and downstream primer 5 -tgtctcgagagggttggggggtc-3 containing the xhoi site. the 669 bp pcr product was cut with ncoi and xhoi, cloned into the pet28b vector, and designated as pet28b-cap-his (fig. 1b) . for the construction of a vector carrying the codon-optimized nucleotide sequence of the first 19 amino acid residues of the cap protein, the pet28b-cap-his expression vector was cut with ncoi and msci. the resulting fragment was ligated with a dsdna adaptor (sense oligonucleotide 5 -catgacctatccgcgccgccgttacc-gccgccgccgccaccgcccgcgcagccatctggg-3 and anti-sense oligonucleotide 5 -cccagatggctgcgcgggcggtggcggcggcg-gcggtaacggcggcgcggataggt-3 ) creating the ncoi and msci overhangs at 5 and 3 end of the adaptor molecule, respectively. this construct was designated as pet28b-o-cap-his (fig. 1b) . codon-optimized gene encoding the pcv 2 capsid protein was obtained from genscript (piscataway, usa) and the nucleotide sequence was deposited in genbank (accession no. eu376523). the plasmid carrying the nucleotide sequence of the cap protein was cut with ncoi and xhoi and cloned into pet28b expression vector. to enhance the expression of the synthetic cap gene in e. coli, a pet42b expression vector was modified by replacement of the nucleotide sequence between the ndei and saci sites with a dna adaptor molecule annealed from a pair of synthetic oligonucleotides (sense oligonucleotide 5 -tatgcaccaccaccacc-accacgccatgggagct-3 and anti-sense oligonucleotide 5 -cccatggcgtggtggtggtggtggtgca-3 ) which encoded the n-terminal histidine tag of the synthetic cap gene. this construct was cut with ncoi and xhoi and ligated with the synthetic cap gene which had been cut with the same restriction enzymes to obtain the expression vector carrying s-cap with the flanking n-and cterminal histidine tags (pet42b-s-cap) (fig. 1b) . all the constructs were confirmed by dna sequence analysis with abi prism 3130xl analyzer (applied biosystems, foster city, usa) using a big dye terminator cycle sequencing kit. recombinant proteins were expressed in e. coli bl21 (de3) (novagene, merck kgaa, darmstadt, germany). cells containing the expression plasmid were grown at 37 • c in luria-bertani (lb) medium supplemented with 60 g/ml kanamycin to the optical density of 0.6 at 600 nm and then isopropyl ␤-dthiogalactopyranoside (iptg) (alexis corporation, lausen, switzerland) was added to a final concentration of 1 mm. after 4 h of growth, cells were harvested by centrifugation (4000 × g for 20 min) and resuspended in 100 mm tris-hcl (ph 8.0) containing 1 mm edta. the expression in e. coli bl21-codonplus(de3)-ripl strain (stratagene, agilent technologies, santa clara, usa) was done under the same conditions, except for antibiotics concentrations: kanamycin (30 g/ml), chloramphenicol (34 g/ml) and spectinomycin (75 g/ml). this strain contains extra copies of the argu, iley, prol, and leuw genes for rare trnas, which can rescue protein production restricted by either agg/aga or ccc codons. the expression of desired proteins was documented by sds-polyacrylamide gel electrophoresis (sds-page) and western blot analysis. bacterial pellet was washed and resuspended in sonication buffer (50 mm mes, ph 5.8, 50 mm nacl) containing 1 mm phenylmethylsulfonyl fluoride. the cells were disrupted by sonication (45 w, misonix sonicator 3000, misonix, farmingdale, usa) on ice and the homogenate was centrifuged at 15,000 × g at 4 • c for 30 min. solid urea was added to a final concentration of 8 m, and mixed gently for 30 min. after centrifugation at 15,000 × g for 10 min at 4 • c, the supernatant was loaded to a unosphere-s cation-exchange column (bio-rad, hercules, usa) which was connected to an akta prime chromatography system (ge healthcare, chalfont st. giles, united kingdom). after washing with 10 column volumes of sonication buffer, the cap protein was eluted with a continuous gradient of nacl (0-1 m) in a buffer containing 50 mm mes, ph 5.8 and 8 m urea. pooled fractions of purified cap protein were concentrated by using centricons (amicon mw 10000 cutoff, millipore, billerica, usa), and stored at −20 • c for further use. total protein concentration was determined by the bradford assay (bio-rad, hercules, usa) using bovine serum albumin as a standard. proteins were separated by sds-page using 12% polyacrylamide gels and either stained by coomassie brilliant blue r-250 or transferred to a nitrocellulose membrane (pall corporation, new york, usa) in a transfer buffer (20 mm tris-hcl, ph 8.3, 190 mm glycine, 0.1% sds, 20% methanol) using a te 70xp semi-dry transfer unit (hoefer, holliston, usa) at 1 v/cm 2 for 1 h. the membranes were blocked with 5% non-fat milk in pbs-t (phosphate-buffered saline containing 0.05% tween-20), incubated with anti-his monoclonal antibody (1:5000) (sigma, st. louis, usa) followed with a peroxidase-conjugated anti-mouse antibody. the signal was developed using an enhanced chemiluminiscence system (ge healthcare, chalfont st. giles, united kingdom). the experimental work with mice was conducted according to the certificate of animal welfare authority commission of the ministry of agriculture of the czech republic no. mze 926/08 in accordance with current czech legislation on animal welfare. a total of 15 balb/c mice (8 weeks of age) were used in the experiment. a group of 5 mice was bled before the immunization to obtain pre-immune serum. a group of 10 mice received intramuscular injection with recombinant s-cap (25 g protein per mouse) mixed with a complete freund's adjuvant (sigma, st. louis, usa). three weeks later, the mice were boosted intramuscularly with the same dose of recombinant cap protein mixed with incomplete freund's adjuvant (sigma, st. louis, usa). the mice were euthanised and bled 24 days after the last injection prior to the sera were screened for the presence of the cap-specific antibodies by using an immunoperoxidase monolayer assay (ipma). for ipma, confluent monolayers of pk15 cells were mixed with a reference pcv 2 isolate (stoon-1010, after 36 passages) and incubated at 37 • c for 48 h prior to fixation with 4% paraformaldehyde. the fixed cells were then incubated with mice sera (1:50 dilution in pbs-t buffer) at 37 • c for 1 h, followed by incubation with a peroxidase-conjugated goat anti-mouse secondary antibody (1:500 dilution in pbs-t buffer) and the peroxidase activity was detected with 4-chloro-1-naphthol (bio-rad, hercules, usa) as a substrate. e. coli cells were washed twice in 50 mm phosphate buffer (ph 7.4) and fixed in a solution containing 2% glutaraldehyde in 50 mm phosphate buffer (ph 7.4) for 2 h. after three washes, the cells were post-fixed in buffered 1% oso 4 (50 mm phosphate buffer, ph 7.4) for 1 h, washed three times in 50 mm phosphate buffer (ph 7.4), dehydrated in ethanol series, and then transferred into absolute acetone and embedded in vestopal w resin (sigma, hercules, usa). ultrathin sections were cut with glass knife using a lkb ultratome 1 (lkb, bromma, sweden) and mounted on formvar-coated copper grids. the grids were stained in saturated aqueous uranyl acetate followed by lead citrate. samples were examined under philips cm100 electron microscope (fei, eidhoven, the netherlands) at 80 kv. images were recorded by using a megaviewii slow scan camera controlled by analysis 3.2 software (olympus soft imaging solutions, münster, germany). pcv 2 antibodies were determined with an indirect enzymelinked immunosorbent assay (elisa) using the purified s-cap and cap-his proteins as coating antigens (cap-elisa). the 96-well elisa plates (nunc, thermo fisher scientific, waltham, usa) were coated with 50 l of 100 mm sodium carbonate buffer (ph 8.9) containing 10 g/ml of the proteins and incubated overnight at 4 • c. the plates were washed with phosphate-buffered saline (pbs) containing 0.05% tween-20 (pbs-t) and blocked with a blocking buffer (pbs containing 1% bovine serum albumin) at 37 • c for 1 h. after washing, 100 l of each serum sample (diluted 1:100) was added into each well and tested in quadruplicate: two wells for a negative control antigen glutathione s-transferase (sigma) and two parallel wells for the antigens. the plates were washed with pbs-t and 50 l of blocking buffer containing a peroxidase-conjugated goat anti-swine antibody (bethyl laboratories, montgomery, usa) was added into each well and incubated for 1 h. finally, the plates were washed with pbs-t three times and the colour reaction was developed with 3,3 ,5,5 -tetramethylbenzidine (tmb) as a substrate. the optical density at 450 nm (od 450nm ) was read using a microplate reader. the final od 450nm value was calculated by subtracting the mean value of od 450nm of wells containing a negative antigen from that of the parallel wells containing the capsid proteins. the cut-off value was determined by using specific pathogen free (spf) pig's serum samples diluted at 1:40 which were negative for anti-pcv 2 antibodies as determined by ipma. the mean od 450nm value was 0.098 with a standard deviation (s.d.) of 0.035 and the final cut-off value was calculated by adding the mean od 450nm value + 3 s.d. to the value of 0.200. pig serum with a high titre of pcv 2 antibodies 1:5120, as determined by ipma, was used as a positive control (kindly provided by annette mankertz from robert-koch institut, berlin, germany). the spf pig sera were negative for pcv 2 and used as negative controls in each plate. a limited serological and genomic load survey of specific pcv 2 antibodies and equivalent pcv 2 genome copy numbers, respectively, was carried out on pig sera collected at 4, 8, 12, 16 and 20 weeks of age. 22 selected piglets were selected from a pig herd experiencing pcv 2 infection, marked with tags and monitored during their nursery and fattening period. at the regular intervals, a minimum of 2 ml of blood was collected from each piglet and the sera were stored at −20 • c for further use. total genomic dna was extracted from a 200 l volume of each serum sample using a nucleospin blood isolation kit (macherey-nagel, düren, germany) according to manufacturer's instructions. quantification of pcv 2 dna levels was performed using a realtime pcr by roche light cycler 480 (roche, basel, switzerland) in a taqman format as previously described (brunborg et al., 2004) . in short, the taqman probe was labelled with 5 -fam and 3 -bhq1 fluorophores (generi biotech, hradec kralove, czech republic) and the primers were designed to amplify a 100 bp dna segment within the nucleotide sequence of the pcv 2 cap gene. the absolute quantification of pcv 2 dna was carried out using calibration curves generated by means of external standard dna obtained by cloning the pcv 2 cap gene into a pcr 2.1 vector (invitrogen, carlsbad, usa). standard curves for pcv 2 dna quantification were generated using tenfold dilution of the linearised plasmids in the range of 8 log 10 . statistical analyses were carried out using the one-way analysis of variance (anova) test running in spss software (base 14.0, spss, chicago, usa). values of p < 0.05 were considered significant. the open reading frame encoding cap protein was amplified using pcr from genomic dna of pcv 2 (l14181 isolate) and the 707 bp long dna fragment was cloned into pet28b expression vector (fig. 1b) . the expression was documented in crude cell lysate by sds-page and western blotting. as demonstrated in fig. 2 , no expression of cap-his was detected in coomasie-stained gels and by western blot using anti-his antibody. frequently, an insufficient production of heterologous proteins in e. coli is due to the presence of codons that are used rarely in bacterial proteosynthesis (zahn, 1996) . forced high-level expression of genes with rare codons may lead to the depletion of the endogenous pools of the corresponding trnas, resulting in the abortion of the translation process and the degradation of the mrna (mcnulty et al., 2003) . examination of the nucleotide sequence of the cap gene revealed a cluster of rare codons within the first 16 codons of the gene (fig. 1c) . the presence of such rare codon tandems near the 5 end of a coding sequence has been reported to cause ribosomal frameshifts and codon skipping with a strong inhibitory effect on protein translation (gurvich et al., 2005) . in order to eliminate the cluster of rare codons from the 5 end of the cap gene, the expression vector encoding a truncated variant of cap protein ( cap-his), which was lacking the first 16 amino acid residues, was constructed (fig. 1b) . expression of the truncated gene in iptg-induced e. coli bl21 (de3) cells resulted in the production of a protein with an apparent molecular weight of 26 kda ( fig. 2a) which corresponded to the cap-his protein. this suggested that the removal of the first 16 codons from the 5 end of the cap gene allowed the production of cap protein in e. coli. in previous report, the expression of the truncated variant of cap protein lacking the nuclear localization signal (nls) has been reported (zhou et al., 2005; trundova and celer, 2007) . the nls consists of 41 amino acid residues at the n-terminus of cap and the nls-defective constructs have been expressed also as fusion proteins with glutathione s-transferase (zhou et al., 2005) or maltose-binding protein (liu et al., 2001b) . based on these results, the authors concluded that the expression of the capsid protein in e. coli was inhibited by the nucleotide sequence encoding the nls domain. however, the expression of the truncated cap protein, which lacks the first 16 amino acids, was achieved which suggested that the production of the full-length capsid protein in e. coli was hindered only by the occurrence of rare arginine codons near the 5 end of the cap gene. the heterologous expression of the entire cap gene was examined also in e. coli bl21-codonplus(de3)-ripl cells. this strain contains extra copies of the argu, iley, prol, and leuw genes for rare trnas, which rescue protein production restricted by either agg/aga or ccc codons. the expression of the cap gene in iptginduced e. coli bl21-codonplus(de3)-ripl yielded the production of a protein with an apparent molecular weight of 27 kda which corresponded to the full-length cap protein ( fig. 2a) . this indicated that the expression of rare aminoacyl-trnas allowed the production of the full-length cap protein. however, the use of e. coli bl21-codonplus(de3)-ripl for a high-yield production of recombinant proteins is inconvenient as the expression system depends on using a combination of three different antibiotics: one for the maintenance of the expression vector, and the other for the maintenance of plasmids carrying trna genes. moreover, due to the presence of high amounts of antibiotics the growth rate of bacterial cells is reduced and expensive. a promising approach to maximise heterologous production of proteins in e. coli is codon optimization strategy which makes codon usage in the gene of interest to match the available trna pool within the cells (jana and deb, 2005; peti and page, 2007; burgess-brown et al., 2008) . to facilitate the cap gene expression in e. coli bl21 (de3), the sequence of the first 19 codons was optimized as described in fig. 1c . ten codons, rare in e. coli, were replaced by the most frequent ones, including six arginine, two proline, and single leucine and threonine codon substitutions. the codon-optimized sequence was introduced into pet28b-cap-his using ncoi and msci restriction sites to obtain pet28b-o-cap-his (fig. 1b) . the expression of the o-cap-his gene in e. coli bl21 (de3) cells yielded the production of a protein with a molecular weight of 27 kda (fig. 2a) . the corresponding band was recognised by the anti-his antibody which confirmed the presence of the o-cap-his protein (fig. 2b) . these data indicated that the replacement of the first 19 codons of the cap gene with a codon-optimized sequence enabled the production of cap protein in a conventional prokaryotic expression system, such as e. coli bl21 (de3). different bioinformatics tools are available for codon optimization which considers many factors, such as rna secondary structure, gc content, repetitive and rare codons (grote et al., 2005) . these approaches were used to design a synthetic cap gene with nucleotide sequence optimized for prokaryotic expression in e. coli (genbank accession no. eu376523). the synthetic gene with introduced ncoi and xhoi restriction sites was cloned into pet28b vector carrying the c-terminal polyhistidine tag and the expression of this construct was analyzed by sds-page. however, no protein product was detected in coomasie-stained gel (data not shown). to force the expression of the capsid protein from the synthetic gene, additional codons encoding the polyhistidine tag were added at the 5 end of the gene. the expression of this construct (pet42b-s-cap) in e. coli bl21 (de3) yielded the s-cap protein with a molecular mass of 28 kda, which was a slightly higher (+1 kda) than the o-cap-his protein due to the presence of the additional polyhistidine tag at the n-terminus of the protein (fig. 2a) . the levels of the s-cap protein expression were comparable to those of the o-cap-his protein which indicated that codon optimization of the entire sequence of the cap gene did not enhance significantly the heterologous expression of the capsid protein in e. coli. the analysis of protein distribution in e. coli cells revealed that all the cap proteins were recovered in the soluble (cytoplasmic) fraction of the iptg-induced cell lysate. however, initial attempts to purify native cap proteins gave unsatisfactory results. using standard chromatographic procedures, no significant binding of cap proteins was detected while loading the material on either cationexchange or nickel immobilised affinity columns in native buffer conditions (data not shown). to enable purification of cap proteins, denaturating conditions in the presence of 8 m urea were utilised. the cytosolic extract of the cell lysates was supplemented with solid urea to a final concentration of 8 m and the mixture was loaded onto a unosphere-s cation-exchange column at ph 5.8. a typical elution profile of protein fractions from the unosphere-s column is shown in fig. 3a . fractions 1-14 contained large amounts of unrelated bacterial proteins, while the later fractions, eluting after 38 min, were enriched with cap proteins (fig. 3b) . the identity of the cap proteins was confirmed also by western blotting with anti-his antibody (fig. 3c) . however, some apparent differences between the s-cap protein and the remaining capsid proteins were observed during purification procedures. while the s-cap protein was recovered in a high purity in the distinct peak eluting at 40 min (fig. 3b, fraction 17) , both the o-cap-his and cap-his proteins were eluted from the column in a shorter time (about 38-39 min). moreover, these fractions were enriched partially with a protein with a molecular weight of about 30 kda and some low molecular weight proteins below 15 kda (data not shown). the original aim of using the polyhistidine tag was to isolate the cap proteins on a metal affinity chromatography column. however, the presence of two polyhistidine tags within the s-cap protein resulted in the increase of the basicity of the protein, which enabled simple purification of the protein by a single step cation-exchange chromatography. the next purification steps did not increase the purity of proteins (data not shown) and these steps were omitted in the purification protocol. the purity of the s-cap protein was greater than 95% after the cation-exchange chromatography and approximately 10 mg of purified protein was obtained from 1 l of bacterial cell culture. these data suggested that the yield of the s-cap protein is much higher than the yield of the o-cap-his and cap-his proteins in terms of purity, time and costs for large-scale production of the recombinant pcv 2 capsid protein. to examine the immunogenicity of the s-cap protein, mice were immunized with the purified protein and the collected sera were analyzed for the presence of cap-specific antibodies. western blot analyses revealed that a single 28-kda band in the bacterial lysate enriched in the s-cap protein was recognised by the cap-positive sera, but not by the pre-immune sera (data not shown). moreover, a typical dense nuclear staining of pcv 2-infected pk15 cells was seen using the cap-positive sera compared to the pre-immune sera by in situ immunohistochemistry (data not shown). these results documented the immunogenicity of the s-cap protein isolated under denaturating conditions. the majority of vlps have been produced using insect or mammalian cell expression systems, but some vlps have been found to assemble also in e. coli cells (noad and roy, 2003) . in order to detect whether the pcv 2 capsid protein is able to self-assemble into vlps in e. coli, the ultrastructure of the bacterial cells expressing the o-cap protein, which lacks any of the additional tag (fig. 1) , was examined by using electron microscopy (em) of ultrathin sections. as shown in fig. 4 , no vlps were observed in either non-induced cells or e. coli cells expressing the o-cap protein. in contrast, in vivo vlps assembly was detected unambiguously following the expression of a fusion protein containing the capsid and nucleocapsid protein obtained from the gag polyprotein of mason-pfizer monkey virus (ulbrich et al., 2006) (fig. 4a and b ). in addition, the sucrose density gradient fractionation of cytosolic content of e. coli cells expressing o-cap protein did not reveal any presence of o-cap protein in the form of vlps. the absence of vlps was corroborated also by em of negatively stained preparations (data not shown). these results demonstrated that the pcv 2 capsid protein was unable to self-assemble into vlps in the cytosol of e. coli cells. in contrast, self-assembly of the pcv 2 capsid protein into vlps has been observed repeatedly in baculovirus expression systems (nawagitgul et al., 2000; kim et al., 2002; liu et al., 2008) . the vlps were of similar morphology as the intact pcv 2 virions and appeared to be empty capsids with a less ordered structure (nawagitgul et al., 2000) . however, the question why the pcv 2 capsid protein does form vlps in insect cells and not in e. coli remains to be addressed. sequence analysis of the pcv 2 capsid protein revealed amino acid residues with consensus patterns for potential posttranslational modifications (n-glycosylation, phosphorylation) (liu et al., 2001b) , which are known to occur in eukaryotic but not in prokaryotic expression systems. another explanation could be a specific need for chaperones, scaffolding proteins, or ssdna, which might play important roles in a proper pcv 2 assembly (ludwig and wagner, 2007) . a plausible hypothesis is that the n-terminal portion of the pcv 2 capsid protein is enriched with basic amino acids that could bind preferentially to the bacterial genomic dna, and therefore, prevent a regular arrangement of the capsid subunits into vlps. this hypothesis would be supported by the observation that the e. coli cells expressing cap protein produced large amounts of outer membrane vesicles (fig. 4d ) compared to the non-induced cells or the cells expressing the capsid protein of mason-pfizer monkey virus. indeed, the release of outer membrane vesicles has been attributed to a novel envelope stress response (mcbroom and kuehn, 2006) , which might result from the binding of the cap protein to genomic dna. specificity and sensitivity of both the full-length capsid protein (s-cap) and its truncated variant ( cap-his) to pig pcv 2-positive sera was determined using an indirect elisa format (cap-elisa). using a chequer-board titration of pcv 2-positive serum (ipma titre of 5120), optimal antigen concentrations and serum dilutions were selected to be 10 g/ml and 1:100, respectively. when the positive/negative cut-off value was set to 0.2 (see section 2), all the pcv 2-positive sera tested were 100% specific (data not shown). cap-elisa showed a low background, and significant differences between pcv 2-negative and pcv 2-positive pig sera were detected (fig. 5) . cap-elisa did not exhibit any cross-reactivity with pig sera obtained from spf pigs which were infected experimentally with coronavirus causing a transmissible gastroenteritis (tge) or a porcine epidemic diarrhoea (ped), respectively. moreover, no significant differences in specificity and sensitivity of cap-elisa were observed between the s-cap and cap-his proteins when used as coating antigens, indicating that the n-terminus of the capsid protein is not specifically recognised by antibodies in the pcv 2positive sera (wu et al., 2008) . this showed that the recombinant full-length cap protein could be used as coating antigen to develop the indirect cap-elisa for specific and sensitive detection of pcv 2 antibodies. pcv2 antibodies are detected currently by indirect immunofluorescence , ipma , competitive elisa (walker et al., 2000) and by indirect elisa based on either pcv2 viral particles (nawagitgul et al., 2002) or recombinant pcv 2 capsid protein expressed in baculovirus (nawagitgul et al., 2002; blanchard et al., 2003; liu et al., 2004) . however, while both the indirect immunofluorescence and ipma assays are highly demanding and not suitable for large-scale survey of pcv 2 infection, the cap-his proteins (10 g/ml) was analyzed by a colorimetric reaction at the optical density at 450 nm (od450nm). od450nm of 0.2 represents a cut-off value for seropositivity of the samples. tge-specific and ped-specific pig sera were obtained from spf pigs which were experimentally infected with the coronavirus causing transmissible gastroenteritis (tge) and porcine epidemic diarrhoea (ped), respectively. data represent the means ± s.d. for the three independent experiments. current elisa formats are less specific and display antigenic crossreactivity to non-pathogenic pcv 1 (magar et al., 2000) . recently, an indirect elisa based on the recombinant nls-truncated pcv 2 capsid protein expressed in e. coli has been established by shang et al. (2008) . although a direct comparison in the sensitivity and specificity of the full-length (this study) and the nls-truncated (shang et al., 2008) proteins as antigens would be interesting, it is very likely that both elisa formats are comparable in the detection of pcv 2 antibodies. considering these data, the cap-elisa based on the recombinant full-length cap protein as coating antigen would provide a simple and reliable tool for standard serodiagnosis of pcv 2 infection. to further analyze the capacity of cap-elisa to detect the pcv 2specific antibodies, a limited serological and genomic load survey of pcv 2 infection was monitored in piglets during the first 20 weeks of age. the piglet's antibody response along with total amounts of pcv 2 dna per 1 ml of serum was determined by cap-elisa and quantitative pcr, respectively. as shown in fig. 6a , the levels of pcv 2-specific antibodies showed an initial drop within the first 12 weeks of age, from then on the levels increased gradually until 20th week. in contrast, the dna copy numbers of pcv 2 increased gradually during the 1st weeks of age to reach the maximum level in the 12th week, after which they decreased slightly until the 20th week (fig. 6b) . these results are in good agreement with the time course dynamics of pcv 2 infection in newborn piglets (mckeown et al., 2005) . a dramatic decrease of the pcv 2-specific antibodies in the 12th week of age corresponds to the period in which the passive intake of maternal antibodies from breast milk during the weaning period (weeks 1-8) is discontinued. this results in the onset of pcv 2 infection after the 8th week of age prior to the active production of the intrinsic pcv 2-specific antibodies (weeks 12-20). taken the final values were expressed as a signal-to-positive (s/p) ratio. s/p value was determined according to the following formula: (od450nm of sample − od450nm of negative control)/(od450nm of positive control − od450nm of negative control). s/p value of 0.3 represents the cut-off signal for seropositivity of the samples. (b) time course analysis of the pcv2 dna copy numbers per 1 ml of serum as detected by the quantitative pcr. total genomic dna was extracted from 200 l of each serum sample and quantified by the pcr amplification of 100 bp dna segment within the nucleotide sequence of the pcv 2 cap gene using a taqman format. results of statistical analysis using one-way analysis of variance (anova) between serum samples obtained at given time intervals from 22 piglets are represented by the box plots. the median value for each dataset is indicated by the black center line. the vertical height of each box indicates the 25-75% data range. the upper and lower bars denote the largest and smallest data values. the marker ( ) denotes the extreme value of the individual serum sample that is >1.5 times the inter-quartile range from the upper and lower quartile. the marker (᭹) denotes the extreme value of the individual serum sample that is >3 times the inter-quartile range from the upper quartile. data represent the means ± s.d. for the three independent experiments. together, these data confirmed that cap-elisa was developed with a high specificity and sensitivity for detection of pcv 2 antibodies in pig sera and could be used for large-scale surveys of pcv 2 infection at low cost. in summary, a bacterial expression system has been developed for the production of the full-length recombinant capsid protein of porcine circovirus type 2. here, the codon optimization strategy was used to obtain high yields of the recombinant capsid protein in an inexpensive cultivation system. purification protocol based on the single step cation-exchange chromatography provided a cost effective procedure to obtain substantial quantities of the cap protein in a high purity. although the recombinant capsid protein expressed in e. coli did not self-assemble into vlps, the antigenic properties of the cap protein resembled that of intact pcv 2 virions. in addition, the recombinant full-length cap protein was used as antigen to develop the indirect cap-elisa for specific and sensitive detection of pcv 2 infection. porcine circoviruses: a review isolation of porcine circovirus-like viruses from pigs with a wasting disease in the united states of america and europe an orf2 protein-based elisa for porcine circovirus type 2 antibodies in post-weaning multisystemic wasting syndrome quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a taqman-based realtime pcr codon optimization can improve expression of human genes in escherichia coli: a multi-gene study postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology post-weaning wasting syndrome comparison of the structures of three circoviruses: chicken anemia virus, porcine circovirus type 2, and beak and feather disease virus molecular characterization of porcine circovirus type 2 isolates from post-weaning multisystemic wasting syndrome-affected and non-affected pigs isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome construction and immunogenicity of recombinant pseudotype baculovirus expressing the capsid protein of porcine circovirus type 2 in mice immunogenicity and pathogenicity of chimeric infectious dna clones of pathogenic porcine circovirus type 2 (pcv2) and nonpathogenic pcv1 in weanling pigs jcat: a novel tool to adapt codon usage of a target gene to its potential expression host expression levels influence ribosomal frameshifting at the tandem rare arginine codons agg-agg and aga-aga in escherichia coli strategies for efficient production of heterologous proteins in escherichia coli characterization of the recombinant proteins of porcine circovirus type 2 field isolate expressed in the baculovirus system nuclear localization of the orf2 protein encoded by porcine circovirus type 2 bacterial expression of an immunologically reactive pcv2 orf2 fusion protein development of an elisa based on the baculovirus-expressed capsid protein of porcine circovirus type 2 as antigen characterization of a previously unidentified viral protein in porcine circovirus type 2-infected cells and its role in virus-induced apoptosis efficient production of type 2 porcine circovirus-like particles by a recombinant baculovirus virus-like particles-universal molecular toolboxes retrospective serological survey of antibodies to porcine circovirus type 1 and type 2 differential recognition of orf2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes replication of porcine circovirus type 1 requires two proteins encoded by the viral rep gene identification of a protein essential for replication of porcine circovirus release of outer membrane vesicles by gramnegative bacteria is a novel envelope stress response effects of porcine circovirus type 2 (pcv2) maternal antibodies on experimental infection of piglets with pcv2 mistranslational errors associated with the rare arginine codon cgg in escherichia coli characterization of novel circovirus dnas associated with wasting syndromes in pigs detection and characterization of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs modified indirect porcine circovirus (pcv) type 2-based and recombinant capsid protein (orf2)-based enzyme-linked immunosorbent assays for detection of antibodies to pcv open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein virus-like particles as immunogens strategies to maximize heterologous protein expression in escherichia coli with minimal cost development and validation of a recombinant capsid protein-based elisa for detection of antibody to porcine circovirus type 2 update on porcine circovirus and post-weaning multisystemic wasting syndrome (pmws). j. swine health prod a very small porcine virus with circular single-stranded dna expression of porcine circovirus 2 orf2 gene requires codon optimized e. coli cells distinct roles for nucleic acid in in vitro assembly of purified mason-pfizer monkey virus canc proteins overexpression of an mrna dependent on rare codons inhibits protein synthesis and cell growth in vitro expression, monoclonal antibody and bioactivity for capsid protein of porcine circovirus type ii without nuclear localization signal expression of the porcine circovirus type 2 capsid protein subunits and application to an indirect elisa development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type 2 the excellent technical help of sona charvatova, hana kubinova, zuzana vecerkova and petra klodnerova (proteix, s.r.o.) is acknowledged. we also wish to thank pavel ulbrich (institute of chemical technology, prague, czech republic) for providing mason-pfizer monkey virus canc expression vectors. this work was supported by grants gacr 310/07/p115 (l.b.), npvii 2b06161 (p.s.) of the ministry of education, youth and sports of the czech republic, npv 1b53016 (i.p.) and 0002716201 (i.p.) of the ministry of agriculture of the czech republic, and institutional research concept av0z50200510 and av0z50520701. key: cord-262904-0b0ljjq1 authors: lon, jerome rumdon; bai, yunmeng; zhong, bingxu; cai, fuqiang; du, hongli title: prediction and evolution of b cell epitopes of surface protein in sars-cov-2 date: 2020-10-29 journal: virol j doi: 10.1186/s12985-020-01437-4 sha: doc_id: 262904 cord_uid: 0b0ljjq1 background: in order to obtain antibodies that recognize natural proteins, it is possible to predict the antigenic determinants of natural proteins, which are eventually embodied as polypeptides. the polypeptides can be coupled with corresponding vectors to stimulate the immune system to produce corresponding antibodies, which is also a simple and effective vaccine development method. the discovery of epitopes is helpful to the development of sars-cov-2 vaccine. methods: the analyses were related to epitopes on 3 proteins, including spike (s), envelope (e) and membrane (m) proteins, which are located on the lipid envelope of the sars-cov-2. based on the ncbi reference sequence: nc_045512.2, the conformational and linear b cell epitopes of the surface protein were predicted separately by various prediction methods. furthermore, the conservation of the epitopes, the adaptability and other evolutionary characteristics were also analyzed, the sequences of the whole genome of sars-cov-2 were obtained from the gisaid. results: 7 epitopes were predicted, including 6 linear epitopes and 1 conformational epitope. one of the linear and one of the conformational consist of identical sequence, but represent different forms of epitopes. it is worth mentioning that all 6 identified epitopes were conserved in nearly 3500 sars-cov-2 genomes, showing that it is helpful to obtain stable and long-acting epitopes under the condition of high frequency of amino acid mutation, which deserved further study at the experiment level. conclusion: the findings would facilitate the vaccine development, had the potential to be directly applied on the prevention in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus. in late december 2019, a novel coronavirus was officially named as sars-cov-2 by the international committee on taxonomy of viruses (ictv) and identified as the pathogen causing outbreaks of sars-like and mers-like illness in chinese city of wuhan, which was a zoonotic disease. as of august 13, 2020, the outbreak of sars-cov-2 has been reported in many areas of the world, with more than 20,423,000 people infected [1] . with an alarming epidemicity, the reproductive number of sars-cov-2 has been computed to around 3.28 [2] . according to the data in the national genomics data center (ngdc, https ://bigd.big.ac.cn/ncov/), 15,118 genomic variations of sars-cov-2 has been reported at 13:00(gmt + 8) on august 13, 2020, which has aroused widespread concern. the b cell epitope of viral surface protein can specifically bind to the host's b cell antigen receptor and induce the body to produce protective antibody and humoral immune response. the discovery of epitopes is helpful to the development of sars-cov-2 vaccine and the understanding of sars-cov-2′s pathogenesis [3] . 3 proteins embedded in the virus envelope of sars-cov-2 have been identified, including spike (s), envelope (e) and membrane (m) proteins. at present, due to the lack of study of the crystal structure of surface protein of sars-cov-2, the study of epitopes, is time-consuming, power-consuming, costly and difficult [4] , especially the conformational epitopes that depend on accurate protein structures. in this work, we analyzed the surface protein (s, e and m protein) of sars-cov-2 and predicted the structures with bioinformatics methods. on this basis, we predicted the linear and conformational b cell epitopes, analyzed the conservation of the epitopes, the adaptability and other evolutionary characteristics of the surface protein, which provided a theoretical basis for the vaccine development and prevention of sars-cov-2. however, the results still need some experimental confirmation to ensure the validity of the application. all of the analyses and prediction were based on the ncbi reference sequence: nc_045512.2. on the basis of previous research of our group, 3624 genome sequences from gisaid (up to april 6th, 2020) were downloaded to construct a dataset for conservation analysis (additional file 4: table s1 ) [5] . the structure of s protein(pdb id: 6 × 6p) was downloaded from rcsb pdb [6] , which has a resolution of 3.22 å [7] .the data sets for s, e, and m protein were obtained by extracting the corresponding locations of the reference genome. the physical and chemical properties of target protein were analyzed by the port-param tool in expasy(expert protein analysis system) [8] , an online practical analysis kit for proteomics, including the primary structure of the target protein, molecular formula, theoretical isoelectric point, the protein instability index(the index < 40 means the protein was stable) and the location information. online software, protscale, was used to deeply analyze the hydrophilicity and hydrophobicity of target protein and the distribution of hydrophilicity and hydrophobicity of polypeptide chains [8] . sars-cov-2 carried the s/e/m proteins through the virus envelope, the transmembrane region of the protein was predicted online by tmhmm 2.0 [9] . with the amino acid sequences of the surface protein of sars-cov-2 of nc_045512.2 as templates, we predicted the 3d structure of e and m protein through the online server swiss-model [10] based on homology modeling method, selected the optimal structure based on the template identity and gmqe value [10] , and the rationality of the structure was evaluated by ramachandran plot [11] with pdbsum server. the structures were displayed and analyzed by swiss-pdb viewer v4.10 [12] . based on the structures, the conformational b cell epitopes were predicted by seppa 3.0 [13] and ellipro [14] respectively, and the conformational b cell epitopes, which were predicted by all of the two methods were selected for the further analysis. the protean module of dnastar was used to predict the flexibility [15] , surface probability [16] and antigenic index [17] of the target protein of sars-cov-2. the linear b cell epitope was predicted by abcpred [18] and bepipred 2.0 [19] respectively and the common predicted linear b cell epitopes from two methods were selected for the further analysis. coupled with the secondary structure, the tertiary structure and the glycosylation sites [20] , the linear b cell epitopes were finally determined. based on the pdb model and the multiple alignment result, we used the consurf server to analyze the conservation of amino acid sites of the epitopes online [21] . the conservation of epitopes on the surface protein of sars-cov-2 was analyzed by multiple alignment with mafft and logo was drawn with weblogo [22, 23] . the primary structure and physicochemical properties of the s/e/m protein were analyzed. the results revealed that the s, e and m protein have average hydrophilic indexes of − 0.079, 1.128 and 0.446, respectively. on the basis of hydrophilicity, the s and m protein showed amphipathic properties, the e protein showed hydrophobic (additional file 1: figure s1 ). according to the prediction, there was an outside-in transmembrane helix in 23 residues from position 1214th to position 1236th at the n-terminal of the s protein, which was almost consistent with the study indicating that the transmembrane domain of s protein was at the position from 1213 to 1237th [24] , an inside-out transmembrane helix in 23 residues from position 12th to position 34th at the n-terminal of the e protein. two outside-in transmembrane helices of the m protein, one was in 20 residues from position 20th to position 39th, the another one was in 23 residues from position 78th to position 100th, and an inside-out transmembrane helix of the m protein in 20 residues from position 51st to position 73rd at the n-terminal, was predicted (additional file 2: figure s2 ). each transmembrane region corresponds to a hydrophobic peak in the hydrophilic index curve. the protein instability index of the s, e and m protein were 33.01 38.68 and 39.14, which revealed that all of the s, e and m protein was stable. the optimal template for homology modeling of the e protein of sars-cov-2 was the e protein of sars (pdb id: 5 × 29.1), with the sequence identity of 91.38% and the gmqe score of 0.73. according to the evaluation of the structure by ramachandran plot (fig. 1a) , 100% of the residues were located in the most allowed regions ( table 1 ), indicating that the structure was reliable. the e protein of sars-cov-2 is a pentamer (fig. 1b) , which can be divided into the concentrated transmembrane part and the head located outside the envelope. the head is mainly composed of α-helix, irregular curl and turn, which is exposed to the envelope and contributes to the formation of epitopes. the tail is mainly composed of long α-helix, most of which are embedded in the envelope, hindering the formation of epitopes. the optimal template for homology modeling of the m protein of sars-cov-2 was the effector protein zt-kp6-1(pdb id: 6qpk. 1. a), with the sequence identity of 20.00% and the gmqe score of 0.06. the sequence identity between the optimal template and the m protein of sars-cov-2 and the gmqe score are too low, so that the template is not suitable for homology modeling. all linear b cell epitopes of the surface protein were filtered according to the following criteria: (1) region with high surface probability (≥ 0.75), strong antigenicity(≥ 0) and high flexibility; (2) excluding the region with α-helix, β-sheet and glycosylation site (fig. 2) ; (3) in line with the prediction by bepipred 2.0(cut off to 0.35) and abcpred (cut off to 0.51). based on the results obtained with these methods and artificial optimization, we removed epitopes that are too long to be suitable for applicate, 4 potential linear b cell epitopes of the s protein were predicted ( table 2 , fig. 3a ), including 601-605 aa, 656-660 aa, 676-682 aa, 808-813 aa, and they were named as the epitope a, b, c, d, respectively; 1 epitope of the e protein was selected(60-65 aa) and named as the epitope e ( table 2 , fig. 3c ); 1 epitope of the m protein was selected (211-215 aa) and named as the epitope g ( table 2 ). the 3d structure prediction and ramachandran plot analysis of the e protein. a the ramachandran plot analysis of the 3d structure of the e protein (without gly and pro). all of the residues located on the allowed region. indicating that the structure was reliable from a thermodynamic point of view. b the 3d structure of the e protein predicted by homology modeling. it is a pentamer with ion channel activity [38] . its head is short, the middle of the tail is a transmembrane region which help the e protein embed in the envelope of sars-cov-2 residues in disallowed regions 0 0.00 with the structure of s protein (pdb id: 6 × 6p), the conformational b cell epitopes of surface protein were predicted with ellipro and seppa 3.0 with the default threshold of 0.063 and 0.5, respectively. one conformational b-cell epitope (60-65 aa) of e protein was predicted (table 2) , which is consistent with the linear epitope e. similarly, this region located on the outside (fig. 3c) , and we selected it as a dominant conformational epitope and named f. however, the conformational epitope of the m protein could not be predicted due to the failure of credible homology modeling. the consurf server was used to predict epitope conservative sites with the structure of surface proteins and the alignment results in our dataset. due to the lack of crystal structure of m protein, the epitope g only applies interestingly, the high antigenicity peaks of all three proteins were in the region where the α-helix is relatively sparse, which may be related to the fact that the α-helix structure of the helix prevents continuous residues from being located on the surface table 2 the composition and the antigenic index of the epitopes of sars-cov-2 the scores of the epitope e and the epitope g were calculated by ellipro, the others were calculated by bepipred 2.0. the epitopes a, b, c and d belong to s protein, the epitopes e and f belong to e protein and they are coincident, the epitope g belongs to m protein data sets to calculate conservation. in the dataset, the epitopes of s, e and m protein were basically conservative (table 3 , additional file 3: figure s3 ). further calculation of the conservatism of the epitopes in the dataset was carried out, and the average score of all the epitopes was less than 1, which could be considered as conservative epitopes. epitope e and epitope f from e protein had the lowest scores and showed the highest conservatism. however, it is worth noting that this value is an overall assessment of the epitopes. residues no. 808,809 of epitope d and 214 of epitope g, as a single residue, showed a conservative score greater than 1 respectively, which revealed a risk of mutation. sars-cov-2 caused huge impact to human production, living and even life, and has become a major challenge confronting the whole world. development of vaccine is one of the effective means of long-term prevention of the virus. epitope vaccine is the trend of development of vaccine due to the advantages of strong pertinence, less toxic and side effects and easy to transportation and storage [25] . a group founded in march 2020 by preston estep, calling themselves "the rapid vaccine partnership" (radvac), has developed a very simple vaccine. in early july, radvac published a white book detailing the vaccine they developed (https ://radva c.org/). the radvac vaccine is a "subunit" vaccine because it is composed of fragments of a pathogen, in this case it was peptide, which is essentially a short fragment of a protein that matches the sars-cov-2 section but does not cause disease. subunit vaccines are already used for diseases such as hepatitis b and human papillomavirus, and a number of companies are developing subunits for covid-19, including novavax biotechnology. reliable epitopes are particularly important for the development of subunit vaccines. (https :// www.techn ology revie w.com/2020/07/29/10057 20/georg e-churc h-diy-coron aviru s-vacci ne/). the determination of epitopes is the basis of the development and application of vaccine, and the clinical diagnosis.herrera et al. [7] reported antigenic analysis of s protein obtained by elisa, but did not study the epitopes. the conserved epitopes were predicted based on the calculation by us, which provided more reference for the immunological study of s protein. vashi et al. predicted some epitopes based on the structure of s protein [26] . although their studies predicted both b-cell and t-cell epitopes of s protein, they did not discuss the conservation of epitopes. we effectively supplement the study of epitopes with the conservative analysis based on a large amount of data, which can ensure the long-term effect and stability of epitopes in the application process. at the same time, their study is limited to s protein, while our study on e and m protein provides more options. moreover, walls et al. [27] reported the use of conservative glycosylation sequence in s protein of sars-cov can stimulate neutralizing antibody against sars-cov-2, the epitope g is the linear epitope and the f is the conformational epitope, which are coincide and the study of the yuan et al. [28] reported that they researched the recognition of epitopes and antibodies by parsing the structure of antibody cr3022 from rehabilitation in patients with sars. wang et al. [29] reported a kind of human monoclonal antibodies, which could neutralize the sars-cov-2, from the cell culture. what these studies have in common is that they are based on some immune responses that have already occurred. in contrast, our calculation in the computer environment is faster, but the accuracy still needs to be verified experimentally. the two methods form an effective complement. currently, the methods which were mainly used are x-ray scattering method, immune experiment method and bioinformatics method. the first two are time-consuming and laborious, while the bioinformatics method is gaining more and more credibility among researchers [3, 25, 30] . there are many factors to be considered in the prediction of epitopes by bioinformatics method, such as the surface probablity and flexibility of the epitopes. at the same time, it is necessary to exclude the structurally stable and non-deformable α-helix, β-sheet, glycosylation sites which may obscure the epitopes or alter the antigenicity, etc. [31] . even so, the predicted epitopes are still inaccurate [4] . our work takes the intersection of above methods to predict, which greatly improves the stability of the prediction. compared with the current study on sars-cov-2, this work adopted various prediction methods and 3d structure databases developed in recent years, which were based on artificial neural network, hidden markov model (hmm), support vector machine(svm), etc., such as abcpred, bepipred2.0, seppa 3.0, iedb, etc. compared with prediction by a single method [32] , on the basis of a single protein [33] or on the basis of epitopes of sars [28] , these methods and databases greatly improved the accuracy of prediction and had more bioinformatic meaning. we comprehensively analyzed the prediction results from the tools which were widely used, set up screening criteria on the basis of primary structure, secondary structure and tertiary structure, so that the prediction results would more accurate and reliable. the s protein, the e protein and the m protein are surface proteins of sars-cov-2 that form the outer table 3 the the calculation was independent and based on the sars-cov-2 data set layer of the coronavirus and protect the internal rna, which have the potential as antigenic molecules. however, considering the current study on the epitopes prediction of sars-cov-2 [34] and due to the fact that s protein has been reported to be the directly binding molecule of sars-cov-2 to ace2 [35] , the prediction of epitopes is mainly focusing on the s protein, with few studies on the e protein and the m protein. in this work, we analyzed the s protein, the e protein and the m protein and predicted their epitopes. on this basis, 7 b cell epitopes were predicted, including 1 conformational and 6 linear b cell epitopes, one of the conformational and one of the linear are coincide. all of the epitope a, b, c, d located on the surface of the tail of the s protein, which is relatively easy to bind. the epitope e and the epitope f located at the end of the head of the e protein coincide, and this may be explained by the fact that they are all consecutive and the secondary structure avoiding the α-helix and the β-sheet. the epitope g is derived from the m protein, and the structure and conservation could not be determined due to the inability to predict reliable structure. however, it could be inferred from the surface probability scores that the epitope g is more likely to be located on the surface of the m protein. the higher the conservation score calculated by the consurf server is, the more likely the site is to be mutated in the evolutionary process. when the score < 1, the site is likely to be a conservative site; when the score is between 1 and 2, the site is a site which is likely to be a relatively easy mutation; when the score > 2, the site is likely to be an easy mutation site [36] . in the 7 epitopes obtained, all the epitopes of the s, e, m protein were absolute conservative among all sars-cov-2 sequences. the conservation of the epitope g could not be calculated by the pdb file. our work provides identified and conserved sites for further study. mutations that occur during the spread of the virus can cause significant resistance to vaccine development. for example, the recently reported mutation of amino acid 614 of s protein [37] not only affects the ability of the virus to transmit, but also may affect the efficacy of vaccines involving this site. our work provides reliable candidates for the development of epitope vaccines, but the application value of the epitopes needed further experimental verification. for example, the antigenicity of the epitope could be tested. although the epitopes could be integrally considered to be conservative, the independent residues of these epitopes could still easy to mutate. epitopes d and e had two and one residues, respectively, with conservative scores greater than 1, meaning that they were at risk for a single point mutation. more attention should be paid to these two epitopes in application. the epitope detection in glycoproteins is significant to the study of the immunoreaction of sars-cov-2, but its challenge is less reliable than the epitope detection due to the presence of glycan [33] . in addition, sars-cov-2 would mutate frequently, and the epitopes predicted might mutate too, so conservative epitopes analyzed in the present study might be more reliable. according to the data from ngdc, the variation frequencies of s, e, and m proteins were 0.83, 1.02, and 0.73, respectively. under the condition of relatively high variation frequency, the conservation of the proteins was analyzed to identify the epitopes with low mutation risk, which were important for the development of long-term and stable vaccines. however, this work is limited. without the molecular dynamic analysis, the binding between epitopes and antibodies was not simulated to further determine the availability of epitopes, but researches from different perspectives can provide more epitopes choices for subsequent studies. in this work, we predicted 7 reliable epitopes: a, b, c, d, e/f and g. the reliability of the epitopes of the s protein was relatively better than that of the epitopes of the e protein and the m protein, indicating that the s protein is still the optimal choice for the prediction of epitopes and the development of vaccine. all of the 7 epitopes were able to achieve high conservation in sars-cov-2, therefore, the epitopes not only have the potential to be directly applied on the treatment in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus. in addition, although various factors of prediction were integrated in this work, more experimental data are needed to further verify whether all the 7 epitopes can induce the body to produce corresponding antibodies and generate specific humoral immunity, due to the limited data set and other factors. supplementary information accompanies this paper at https ://doi. org/10.1186/s1298 5-020-01437 -4. additional file 1. figure s1 : deep analysis of hydrophilicity and hydrophobicity of surface protein of sars-cov-2. the online software, protscale, was used to predict the hydrophilicity and hydrophobicity of the surface protein deeply. a. the s protein has a maximum score of hydrophobicity, 3.222 at the 7th site, which revealed a strong hydrophobicity; a minimum score of hydrophobicity, -2.589 at the 679th site, which revealed a strong hydrophilicity. the score of hydrophilicity and hydrophobicity on the polypeptide chain of s protein constantly fluctuates, with most of the scores being negative, which revealed the possibility that the protein had bisexual properties on the basis of hydrophilicity. b. the e protein has a maximum score of hydrophobicity, 3.489 at the 21st and the 25th site, which revealed a strong hydrophobicity; a minimum score of hydrophobicity, -1.550 at the 65th site, which revealed a strong hydrophilicity. most of the scores of the residues being positive, which revealed the possibility that the protein has obvious hydrophobicity. c. the m protein has a maximum score of hydrophobicity, 2.978 at the 84th site, which revealed a strong hydrophobicity; a minimum score of hydrophobicity, -1.956 at the 211th and the 212th site, which revealed a strong hydrophilicity. the scores of hydrophilicity and hydrophobicity on the polypeptide chain of m protein showed large fluctuations, and the number of positive scores and negative scores were similar, the positive scores accounted for the majority, which revealed the possibility that the protein had bisexual properties on the basis of hydrophobicity. additional file 2. figure s2 : the transmembrane region of the surface protein of sars-cov-2. the s, e and m protein are embedded in the envelope of sars-cov-2, the transmembrane helix was predicted by tmhmm 2.0 server. all of three amino acid indexes were higher than 18, indicating the reliability of the prediction. a. for the s protein, an outsidein transmembrane helix was predicted in the 23 residues of amino acids from position 1214th to position 1236th at the n-terminal. the amino acid index was 23.97303. b. for the e protein, an inside-out transmembrane helix was predicted in the 23 residues of amino acids from position 12th to position 34th at the n-terminal. the amino acid index was 25.72521. c. for the m protein, 2 outside-in transmembrane helices were predicted, which were a helix in the 20 residues of amino acids from position 20th to position 39th and a helix in the 23 residues of amino acids from position 78th to position 100th at the n-terminal. an inside-out helix was predicted in the 23 residues of amino acids from position 51st to position 73rd at the n-terminal. the amino acid index was 64.90522. the calculation of the transmembrane pattern and data has clarified the position and direction of the protein in the virus, which is of great significance for the understanding of the availability of the antigen when predicting the epitopes, the epitopes located outside the virus has significant application advantages. additional file 3. figure s3 : the antigenic conservation of the surface protein in sars-cov-2. the overall height of each stack is proportional to the sequence conservation, measured in bits, at that position, while the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position. all the epitopes in the data set are highly conservative, and the serial numbers (a-g) in the figure represent the epitopes a-g respectively. additional file 4. table s1 : the list of the id of genomes in dataset. coronavirus 2019-ncov: a brief perspective from the front line the reproductive number of covid-19 is higher compared to sars coronavirus recent advances in b-cell epitope prediction methods bioinformatics resources and tools for conformational b-cell epitope prediction comprehensive evolution and molecular characteristics of a large number of sars-cov-2 genomes reveal its epidemic trends rcsb protein data bank: biological macromolecular structures enabling research and education in fundamental biology, biomedicine, biotechnology and energy characterization of the sars-cov-2 s protein: biophysical, biochemical, structural, and antigenic analysis the proteomics protocols handbook sequence and structure-based prediction of eukaryotic protein phosphorylation sites swiss-model: homology modelling of protein structures and complexes procheck: a program to check the stereochemical quality of protein structures swiss-model and the swiss-pdbviewer: an environment for comparative protein modeling seppa 3.0-enhanced spatial epitope prediction enabling glycoprotein antigens ellipro: a new structure-based tool for the prediction of antibody epitopes prediction of chain flexibility in proteins induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide the antigenic index: a novel algorithm for predicting antigenic determinants convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year • at bmc, research is always in progress. learn more biomedcentral.com/submissions ready to submit your research ready to submit your research ? choose bmc prediction of continuous b-cell epitopes in an antigen using recurrent neural network improved method for predicting linear b-cell epitopes prediction of n-glycosylation sites in human proteins consurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules weblogo: a sequence logo generator sequence logos: a new way to display consensus sequences severe acute respiratory syndrome coronavirus-2 (sars-cov-2), a newly emerged pathogen: an overview immuno-informatics: mining genomes for vaccine components understanding the b and t cell epitopes of spike protein of severe acute respiratory syndrome coronavirus-2: a computational way to predict the immunogens structure, function, and antigenicity of the sars-cov-2 spike glycoprotein a highly conserved cryptic epitope in the receptor binding domains of sars-cov-2 and sars-cov a human monoclonal antibody blocking sars-cov-2 infection an introduction to epitope prediction methods and software the evolutionary pattern of glycosylation sites in influenza virus (h5n1) hemagglutinin and neuraminidase a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov-2 immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of 2019-ncov cross-reactive antibody response between sars-cov-2 and sars-cov infections potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody mutation feature analysis on epitope and receptor binding sites of influenza a h1n1 hemagglutinin spike mutation pipeline reveals the emergence of a more transmissible form of sars-cov-2 analysis of sars-cov e protein ion channel activity by tuning the protein and lipid charge publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the student entrepreneurship and innovation center of the school of biology and biological engineering, south china university of technology, also provided a lot of help during the preparation of the project. authors' contributions jl conceived the study and participated in its design and coordination. hd participated in the design of the study and helped draft the manuscript. yb participated in analysis of conservation, sequence alignment and manuscript drafting. bz participated in antigenic prediction. fc participated in drafting the manuscript. all authors read and approved the final manuscript. the viral genomes described in detail here were deposited in ncbi, genbank and gisaid. not applicable. not applicable. the authors declare that they have no competing interests.received: 1 may 2020 accepted: 20 october 2020 key: cord-023143-fcno330z authors: nan title: molecular aspects of viral immunity date: 2004-02-19 journal: j cell biochem doi: 10.1002/jcb.240591009 sha: doc_id: 23143 cord_uid: fcno330z nan mechanisms of t-cell mediated clearance of viruses from the central nervous system are poorly understood, but likely to differ from those employed in the periphery because the cns lacks lymphatic drainage and constitutive expression of mhc class i antigen, and the unique structure of the cns vasculature imposes constraints on access by leukocytes and soluble immune mediators. to study the mechanism by which viruses are cleared from neurons in the central nervous system, we have developed a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (oblv60). grew preferentially in the olfactory bulbs of balbk mice. using in situ hybridization, we found viral rna localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. virus was cleared rapidly from the olfactory bulb between 5 and 11 days. athymic nude mice failed to eliminate the virus demonstrating a requirement for t lymphocytes. immunosuppression of normal mice with cyclophosphamide also prevented clearance. both cd4+ and cd8+ t-cell subsets were important as depletion of either of these subsets delayed viral clearance. gliosis and infiltrates of cd4+ and cd8+ cells were detected by immunohistochemistry at 6 days. the role of cytokines in clearance was investigated using an rnase protection assay for il-la, il-lp, il-2, il-3, il-4, il-5, il-6, tnfa, tnfp and ifny. in immunocompetent mice there was upregulation of rna for il-la, il-lp, il-6, tnfa and ifny at the time of clearance. nude mice had comparable increases in these cytokine messages with the exception of ifny. induction of mhc-i molecules on cells in infected brains was demonstrated by immunohistochemistry in normal and nude mice, suggesting that ifny may not be necessary for induction of mhc-i on neural cells in vivo. luca g. guidotti, kazuki ando, tetsuya ishikawa, lisa tsui and francis v. chisari. the scripps research institute, la jolla, ca 92037 although cytotoxic t lymphocytes (ctl) are known to clear viral infections by killing infected cells, recent studies suggest that they can also suppress the replication of certain viruses by noncytolytic mechanisms. we have examined this area by monitoring the immunopathological and antiviral consequences of antigen recognition by hepatitis b virus (hbv) specific ctl in hbv transgenic mice that express the viral gene products in their hepatocytes. we have shown that intravenously injected ctl rapidly trigger their target hepatocytes to undergo apoptosis, but that the direct cytopathic effect of the ctl is minimal in comparison with the cytopathic effects of the antigen-nonspecific intrahepatic inflammatoly response that they activate. in addition to killing the hepatocyte, the same ctl also downregulate hbv gene expression and completely abolish hbv replication in the hepatocytes that they don't destroy. this noncytolytic antiviral ctl effect is mediated by at least two distinct processes in these animals. first, the ctl cause a quantitative reduction in the steady state content of all hbv mrna species in the hepatocyte, and this is followed by disappearance of all of the corresponding viral proteins in the liver and serum. the ctl initiate this process by secreting ifny and tnfa when they are activated by antigen recognition. since the regulatory effect of the ctl can he prevented completely by prior administration of the corresponding antibodies. nuclear run-on experiments reveal that viral mrna transcription is unaffected despite the profound reduction in hbv mrna content in the liver, suggesting that the ctl-derived cytokines accelerate viral mrna degradation in the hepatocyte. a second noncytolytic antiviral pathway is also activated by the ctl. we have recently shown that hbv nucleocapsid particles, and the replicative hbv dna intermediates that they contain, disappear from the transgenic mouse liver following either ctl administration or partial hepatectomy. the latter of which triggers hepatocellular regeneration without any change in hepatocellular hbv mrna content. these results suggest that preformed hbv nucleocapsid particles may be actively degraded during hepatocyte turnover, and they raise the possibility that similar events might also occur in nondividing hepatocytes that are activated by noncytolytic signals delivered by the ctl. we propose that, in addition to their pathogenetic effect, the comhined effects of the ctl response at die hbv mrna. nucleocapsid and rcplicative dna levels may represent a curative antiviral stimulus during hbv infection. since the virus must contain molecular elements that iespond to these ctl-induced antiviral signals. inactivating mutations at these loci could be very efficiently selected by immune pressure, because a single mutation could abrogate the antiviral effect of a wide spectrum of t cell responses, irrespectrve of epitope specificity. identification of these viral response elements and the intracellular pathways that interact with them may lead to the development of new strategies for antiviral drug design. human fibroblasts infected with hsv are resistant to lysis by cd8+ cytotoxic t lymphocytes (ctl), yet human b cell lines can be efficiently lysed by these ctl. the effect on human fibroblasts is rapid (within 2 hr of infection of cells), occurring before synthesis of mhc class i is altered by virus infection. a recombinant hsv, f-usbmhc, which expresses mouse mhc class i proteins does not render human fibroblasts sensitive to lysis by mouse ctl. mhc class i molecules are retained in the er of hsv-infected fibroblasts i n a misfolded, unstable form and stability of the mhc complex can be restored by addition of exogenous peptides. using a panel of hsv mutants and ad expression vectors we demonstrated that the hsv ie protein icp47 was both necessary and s f i c i e n t to cause retention of class i and icp47 expression in fibroblasts caused the cells to resist lysis by cd8+ t lymphocytes. icp47 is a soluble, cytosolic protein and we have found no evidence of membrane association. therefore, it appears that icp47 inhibits cytosolic stages of the antigen presentation pathway so that antigenic peptides do not reach the er. to date, polyclonal and monoclonal antibodies directed to icp47 have not specifically precipitated any of the previously described components of the antigen presentation pathway and we have not found icp47 associated with tap transporter proteins or proteosomes i n these experiments. the effects of icp47 are being assessed in proteosome and tap transporter assays. gst-icp47 fusion proteins tightly bind a 8.5 kda cytosolic cellular protein which is found in a number of adherent human cell lines but not lymphocytes. the protein has been purified and sequencing is in progress. in addition, radiolabelled icp47 binds to a single cellular protein of =55 kda on ligand blots. these proteins are good candidates as cellular targets of icp47 and as novel components of the antigen presentation pathway. preliminary experiments support the hypothesis that icp47 is very effective i n blocking cd8+ t lymphocyte responses in vivo, perhaps explaining the predominance of cd4+ vs. cd8+ anti-hsv ctl i n vivo. we expect that icp47 may be very useful, not only to elucidate antigen presentation pathways, but also to prevent immune recognition of gene transfer vectors and a s a immunosuppressive agent. susceftibility to polyoma virus-induced tumors is conferred by an endogenous mmtv superantigen. aron e. lukacherl, yupo ma2, john p. carroll2, sara r. abromson-leeman2, joseph c. laning2, martin e. dorf2, and thomas l. benjamin2. idepartment of pathology, emory university school of medicine, atlanta, ga 30322, and 2department of pathology, harvard medical school, boston, ma 02115. susceptibility to tumors induced by mouse polyoma virus varies among inbred mouse strains. we have previously shown that polyoma tumor susceptibility is controlled by products of mhc as well as non-mhc genes. in crosses between mhc-nonidentical strains differing in tumor susceptibility, resistance correlates with dominantkodominant inheritance of the resistant h-2 haplotype. we have observed the opposite pattern of inheritance of susceptibility in crosses between mhc-identical strains. in crosses between the highly susceptible c3wbida mouse and the highly resistant but mhc-identical (h-2k) c57bwcd.i mouse, polyoma tumor susceptibility is conferred by a single autosomal dominant gene, which we have designated pyvs. pyj does not encode cell receptors for the virus, affect viral dissemination or anti-viral antibody responses, or affect intracellular events essential for productive infection or cell transformation by the virus. whole-body irradiation renders cs7bwcd.i mice fully susceptible to polyoma-induced tumors, indicating an immunological basis for this strain's resistance. we hypothesized that p y j encodes an mtv superantigen (sag) that confers susceptibility to c3wbida mice by deleting precursors of polyoma-specific t cells. we found that tumor susceptibility in (c3wbida x c57bwcd.i) x c57bwcdj backcross mice cosegregated with mtv-7. inheritance of mtv-7 showed perfect concordance with absence of peripheral vp6+ t cells. genotyping of backcrossmice using markers of simple sequence repeat polymorphisms flanking mtv-7 showed no evidence of recombination between pyvs and mtv-7. strongly biased usage of vp6 by (a) polyoma-specific cd8+ ctl from virus-infected c57bwcdj mice and by @) cd8+ t cells infiltrating a polyoma tumor in a virus-immune c57bwcd.i host provide further evidence that t cells bearing this mtv-7 sag-reactive vp domain are critical anti-polyoma tumor effector cells. these results indicate identity between p y j and mtv-7 sag, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's t cell repertoire. infection of mice with lymphocytic choriomeningitis virus (lcmv) causes a transient to longlasting immunosuppression dependent upon virus-isolate dose of virus and age, h-2, non h-2, level of cd4+ t cells, of cd8+ t cells and kinetics of neutralizing antibodies of the host. the immunohistological analysis suggests that cd8+ t cell dependent disappearence of marginal zone macrophages of follicular dendritic cells and of virus infected cells in general correlates with immunosuppression. the details of mechanisms responsible for these findings are now being analysed. a role of this cd8+ t cell dependent immunosuppression in the establishment of a lcmv carrier state in immunocompetent mice is suggested by the following experiments: the otherwise slow and low neutralizing antibody response agamst lcmv is accelerated and enhanced by cd8+ t cell depletion at the time of infection, suggesting virus-specific immunopathology being responsible at least partially. the elisa antibody response is not significantly altered under the same conditions but is abrogated if lcmv-specific t cell receptor transgenic mice are infected with high doses of lcmv, indicating, that suppression of the specific antibody response depends upon the relative kinetics of ctl versus antibody responses. whether exhaustion of specific ctl responses is enhanced by similar mechanisms remains to be tested. the role of interleukins of the relative distribution of virus in the mouse and in the various aspects of immunosuppression are now being studied. immunosuppression, caused by cd8+ t cell-dependent immunopathology, may also be operational in hiv infection in humans. such a pathogenesis of hiv-triggered aids could explain several aspects of the disease process not readily fitting the (unproven) conventional idea that hiv is causing immunodeficiency via direct viral pathogenicity. the cellular immunity against two dna tumor viruses (i.e. human adenovirus type 5 (ad 5) and human papillomavirus type 16 (hpv16)) was studied with respect to possible immune escape mechanisms and to the development of ctl epitope based peptide vaccines. after identifying an immunorelevant ctl epitope in the ad 5e1a protein to which ctl clones were directed that could eradicate ad 5e1 induced tumors in nude mice, an amino acid replacement study of this epitope revealed a point mutation that totally eliminated the possibility to recognize the mutant peptide by the ctl clones directed against the wild-type peptide sequence. new viral constructs were made that contained this point mutation and used to transform mouse embryo cells. however, these mutant tumor cells were still immunogenic and ctl clones specific for these mutant tumor cells were shown to react with a peptide derived from the ad 5e1b protein. these ad 5eib specific ctl clones, however, were as effective as the ad 5e1a specific ctl clones in the eradication of ad 5e1 induced tumors in nude animals, indicating that a choice can be made of immunorelevant epitopes to which an immunization strategy could be developed. in addition, we discovered that by supertransfection of ad 5e1 induced tumor cells with the activated ras oncogen the possibility of ad 5e1b specific ctl to recognize the ad 5e1 induced tumors was eliminated whereas the ad 5e1a specific ctl could still kill these tumor cells. this might indicate a new mechanism of tumors to escape ctl. in an hpv16 induced mouse tumor model an immunosubdominant ctl epitope was identified in the e7 protein that could, upon immunization with that peptide,protect mice against a subsequent challenge with hpv16 induced tumor cells. by changing the anchor residues in that peptide an even more immunoprotective peptide could be generated. combined, these data indicated a successful use of a ctl epitope based peptide vaccine in the prevention of hpv16 induced tumors in mice. subsequently this led us to identify relevant ctl epitopes of hpv16, that is highly associated with cervical carcinoma in humans, for the major hla-a alleles (i.e. hla-a *0101, a *0201, a"0301, a*1101 and a *2401). together these alleles cover a majority of all humans. ctl epitopes were identified through peptide-mhc binding assays followed by in v i m peptide immunizations with high affinity binding peptides to induce primary ctl responses and immunogenicity studies in hla-a transgenic mice. thereafter, memory ctl responses were measured in cervical cancer patients against selected peptides. combined, these data led us to develop a ctl epitope based peptide vaccine that could be of use in hpv16 induced cervical cancer patients. a clinical trial for this disease is scheduled to start in the fall of 1994. class ii presentation of an endogenously synthesized glycoprotein. carol s. re is^'.'.^, shirley m. bartido', miriam stein', and stephanie diment3.4, biology department', and center in contrast to class i presentation which is well characterized to use peptide fragements of proteins sythesized in the cytoplasm, exogenously administered experimental antigens enter the class ii mhc pathway through endocytosis. we have been studying the recognition of the glycoprotein of vesicular stomatitis virus (vsv) which can enter either the exogenous or endogenous pathways for presentation to cd4 + t cells. investigations of the intracellular sites involved, the proteolytic processes involved in the epitope generation, will be discussed. the glycoprotein studied in detail is a truncated form of the wt type 1 glycoprotein, termed poison tail (gpt) . expressed with a vaccinia virus vector, the gpt remains endo h sensitive and never becomes endo d sensitive, indicating that it is restricted to the endoplasmic reticulum. gpt is degraded in the er, and w e believe the degradation products include the immunogenic epitopes recognized by a panel of lad and i-ed t cell clones and hybridomas. lmmunofluorescence studies have confirmed the er localization. flow cytometric evaluations s h o w that the gpt never appears on the cell surface, in contrast to the wt g. the peptides generated are not secreted; using an innocent bystander assay, gpt-infected cells are incapable of sensitizing 5'cr-labeled uninfected apc. this contrasts with the rapid ability of supernatants from wt g-vaccinia virus-infected cells to sensitize apc for t cell recognition. investigations of the characteristics of the enzymes contributing to the degradation of the gpt have shown that a reducing environment is essential, as diamide treatment of cells prevents degradation. lysosomotropic drugs (eg. nh,ci and leupeptin) d o not alter the half-life of the protein, but do prevent presentation of the peptides; this is inconsistent with an autophagic component to the proteolysis. ph optima are physiological, as ph8 environment inhibits the enzyme activity. inhibitors of enzyme classes are consistent with a trypsin-like, and not cystein-, cathepsin b-, or chymotrypsin-like class. supported by nih grant al 18083 to csr. (emcv) and mengovirus are related members of the cardiovirus genus of picomaviruses. their rna genomes encode a large polyprotein which is cleaved proteolytically in co-and post-translational reactions to yield all mature viral proteins necessary to establish an infection. although originally thought to be exclusively murine in host range, both viruses actually infect a wide range of mammals. emcv has caused devastating epizootics in captive primates (eg: macaques, chimps and baboons), domestic pigs and exotic zoo collections (elephants, lions and tigers). death, following ingestion of virus-contaminated material, is rapid, and caused by extensive meningoencephalitis and virus-induced damage to the cns. myocarditic lesions are common in older animals. when administered intracerebrally, the ld,, for emcv strain r is about 1 pfu. we are studying the pathogenesis of emcv and mengo with engineered cdna plasmids containing infectious viral sequences. many plasmids contain h'uncated versions of the unusual 5' noncoding homopolymeric poly(c) tract that is a hallmark of these cardioviruses. short poly(c) mengoviruses grow very well in tissue culture but are 106-10'z fold less pathogenic to mice than the wild-type strains. animals receiving sublethal doses of short-tract mengo strains develop high titers of neutralizing antibodies, exhibit potent ctl responses and acquire lifelong protective immunity against challenge with wild-type virus. the genetic stability of the short-tract strains, even upon serial brain passage, mark them as safe, efficacious live vaccines. currently, we believe the poly(c) phenomenon is due to interference by the wild-type virus sequences (long poly(c) tract) with normal cellular cytokine induction mechanisms (ie: ifa and ifp) during the initial stages of animal infection. the targeted cells are probably macrophages, and their singular ability to correctly respond or not respond to poly(c) tract length during the first few hours of infection determines whether an inoculated animal will live (protectively vaccinated) or die. the short-tract viruses probably induce if in the macrophages, and are consequently killed then rapidly cleared from the host in related experiments we've found that attenuated mengo strains can easily carry large heterologous insertions within their genomes, and express these sequences into protein them during replication in animals. the resulting immune response (b cell and ctl) to the chimeras is directed towards the foreign sequences (epitopes) as well as towards the mengo proteins. a chimeric hiv vaccine, a rabies vaccine and an lcmv vaccine have been developed and tested. the lcmv chimera seems especially effective, as a single pfu of this engineered mengo strain, administered orally to a mouse, is sufficient for complete immunogenic protection against intracerebral challenge with wild-type lcmv virus. rsv is the most common cause of serious viral lower respiratory tract disease in infants and children. we have recently renewed our efforts to generate a safe and effective live attenuated rsv vaccine for topical administration that will overcome the deficiencies of previously studied live and non-living rsv vaccines. this vaccine will be a bivalent vaccine consisting of subgroup a and b live attenuated virus components. since the peak incidence of severe disease caused by rsv is in the 2-month old infant, an rsv vaccine will need to be effective when given to 1-month old infants. based on the success of live poliovirus vaccines given early in infancy, it is anticipated that the intranasally administered live virus vaccine will infect and induce a protective local and systemic immune response even in infants with passively acquired maternal antibodies. the main approach that we have taken in this effort to develop the live rsv vaccine is to introduce one or more ts mutations by chemical mutagenesis into a cold-passaged virus (cprsv) that had been partially attenuated by the acquisition of host-range mutations selected by passage in cells of a heterologous host species. we have developed a large set of cprsv subgroup a rs mutants (termed cprs mutants) that contain the host-range mutations selected during cold passage and two or more ts mutations introduced by chemical mutagenesis. these mutants have been evaluated in virro for their level of temperature sensitivity and in vivo in rodents, chimpanzees, and humans. a large set of rsv subgroup b cpts mutants has been similarly produced and evaluated. the immunogenicity and protective efficacy of three candidate live attenuated rsv vaccine strains that represent a specaum of attenuation were evaluated for protective efficacy in chimpanzees. prior to infection some of these animals were given rsv immune globulin by the iv route to simulate the condition of the very young infant who possesses passively-acquired maternal rsv antibodies. the three candidate vaccine strains were immunogenic and induced significant resistance to rsv challenge in both groups of chimpanzees. interestingly, the chimpanzees infused with rsv antibodies prior to immunization were primed more effectively for an unusually high serum neutralizing antibody response to infection with challenge virus than chimpanzees which did not receive such antibodies. this high booster response occurred despite marked reshiction of replication of the challenge virus. the evaluation of two candidate vaccines in seronegative human infants will also be described. rs virus is immunologically interesting for at least t w o reasons: 1) upper respiratory reinfection occurs despite previous exposure and demonstrable immunological memory: 2) humans or rodents previously immunised against virus infection can show enhanced disease during reinfection. others have shown that passive transfer of antiviral antibody either protects against virus infection or has no effect, and there is no evidence of antibody enhancement of disease in vivo. by contrast, t cell immunity appears closely associated with disease augmentation. we have focused on examining the immunological mechanisms of disease enhancement in mice. initial studies showed that transfer of cd8+ cytotoxic t lymphocytes (ctl) causes rapid virus clearance from the lungs of rs virusinfected mice, but also increased disease severity with alveolar haemorrhage and polymorphonuclear (pmn) cell recruitment to the lung. this disease (reminiscent of shock lung) could sometimes be fatal, whereas normal mice recover well from similar doses of rs virus. next, we compared the effects of cd4' and cd8+ t cells, using polyclonal t cells separated immunomagnetically from mixed lines grown in vitro with viral antigen. cd4' t cells were more pathogenic than cd8+ t cells in a dose-for-dose comparison, but that the type of pathology varied depending on the type of cell injected. while testing recombinant vaccinia viruses expressing single rs viral proteins for their ability to protect mice against infection, we observed that animals sensitised t o the major surface glycoprotein g (attachment protein) developed lung eosinophilia after challenge with rs virus intranasally. t cell lines from the spleens of mice sensitised with various recombinant vaccinia viruses were established. those form mice primed with the m 2 (22k) protein were predominantly cd8' ctl, and that produced few cytokines. those from mice primed with fusion protein (f) generated mixed t cell lines with both t h l cd4+ t cells, and ctl. mice primed to g protein gave rise to predominantly cd4' t cells producing th2 cytokines. ln vivo transfer of these cell lines into na'ive rsv infected mice reproduces the patterns of disease seen in mice sensitised in vivo with the respective antigens. the mouse model of rs virus disease therefore has excellent potential for illustrating mechanisms of lung immunopathology. the eye is a complex organ whose function is to transmit light images through different cell and tissue layers and liquid media to a neurosensory retina. elements as could occur when invading pathogens arrive and an inflammatory response with its swelling, plasma protein extravasation, leukocyte infiltration and tissue damage results. inflammatory responses when possible and rely on immune defenses which do not involve tissue distortion and damage. restricting tissue damaging responses is not always effective and the process is best developed in response to agents delivered to locations such as the anterior chamber. where an inflammatory response is initiated which may result in ocular impairment. such herpetic stromal keratitis (hsk) is a common cause of blindness in man. animal model studies indicate that hsk is a multi-step process initiated by virus in an avascular structure. hsk fails to occur in the absence of t cells or replicating virus. disappears several days before a visible inflammatory response becomes evident. evidence will be presented that the secondary agonists which drive the inflammatory response may not be viral antigen(s) per s e . multiple cell types are involved in hsk, with the respective role of functional sets of lymphocytes changing according to the clinical phase of the disease. in addition, nonspecific inflammatory cells such as neutrophils and nk cells also influence the severity of lesions. basically the reaction begins with t cells that produce type one cytokines, particularly ifn-y, dominating the scene, but during remission type 2 cytokines, notably il-10, appear as mechanistically involved. from the use of knockout mice for various immunological parameters, evidence will be presented that numerous mechanisms of pathogenesis may be at play during hsk. damage to corneal tissues in all systems appear to involve tnfo. a second ocular damaging event in which immunopathology is at least partially involved is herpetic retinal necrosis. evidence that this disease may involve the immunopathological role of cd4 t cells and protective effects by cd8' t cells will be presented, as will be suggestions by which the pathological events are mediated at the molecular level. thus it is in the eye's functional interest to limit acute viral infections and live vaccines often confer long-term immunity the nature of t and b cell memory is different. b cell memory is manifested not only by the presence of memory b cells but also by continuous antibody production in contrast, the effector phase of the t cell response i s shortlived and long-term t cell memory is due to the presence of 'quiescent' antigen specific memory t cells that are present at higher frequencies and are able to respond faster upon re-exposure to virus due to increased levels of adhesion molecules in this talk i w i l l present our results on. (i) the bone marrow as a major site of long-term antibody production after acute viral infection, (ii) the role of c d 4 ' t cells and b cells (immune complexes) in maintaining cd8+ t cell memory, (iii) the role offas antigen in regulating t cell responses, and (iv) the efficacy ofvarious antigen delivery systems in inducing long-term t cell memory sendai virus is a natural respiratory viral pathogen of mice. intranasal infection of mice with the virus provokes a virus specific antibody-forming-cell reaction that exhibits a distinct kinetic pattern in the lymph nodes that drain the respiratory tract, in the spleen, and in the bone marrow. the bone marrow afc population is extremely long-sustained, and supports an active humoral response that essentially persists for the lifetime of the infected animal. thus the conventional categories of "primary" and "secondary" response may not apply to the humoral response of mice naturally exposed to respiratory viruses. paradoxically, the population of b cells that reacts most rapidly to sendai virus infection does not itself secrete antibody, but can he demonstrated by the recovery of hyhridomas that secrete "polyspecific" antibodies. the activation of this polyspecific b cell population is, like the humoral response, extremely persistant. viral infection thus sets in train multiple b cell "memory" processes. variation in the rules of development and turnover of different b cell populations constrains the mechanisms that may operate to generate these different forms of memory. establishment and maintenance of t cell memory to respiratory viruses, peter c. doherty, sam hou, christine ewing, david topham, anthony mcmickle, james houston, and ralph tripp, department of immunology, st. jude children's research hospital, memphis, tn 38105. the analysis of the development and memory phases of the cd4+ "helper" n h ) and cd8+ cytotoxic t lymphocyte (ctl) responses to the respiratory pathogens, influenza virus and sendai virus (parainfluenza type 1) have been characterized by a combination of limiting dilution analysis (lda) for determining th and ctl precursor @) frequency and facs separation of lymphocytes with different activation phenotypes. the interpretation at this stage, largely based on the analysis of the ctl response, is that the development phase of t cell memory and the primary response are synonymous. virus-specific ctlp are produced in considerable excess of the numbers required to provide the effector ctl that terminate the primary infection, with only a fairly small proportion localizing to the target organ (the lung) that supports virus growth. even when many of the proliferating ctlp are killed by administration of a small dose (20 mgkg) of the dna-targeted drug cyclophosphamide (cy), there is no indication of immune exhaustion. the cd4+ th response has, at this stage, not been analyzed through the course of the primary infection. use of the lda approach to determine thp frequencies is inherently more difficult, as the "read-out'' is lymphokine production and there is considerable "bystander" activation in these primary responses to respiratory viruses. memory thp and ctlp are characterized initially by the expression of an "activated" phenotype: cd44-high, l-selectin-low, cd49d (vla-4) high. after some months, an increasing proportion of the memory t cells revert to the l-selectin-high cd49d-low form typical of naive ctlp. the change, which is never absolute, seems to occur first with cd49d and the rate varies for different viruses. current experiments are addressing the possibility that intercurrent infection, particularly with the mouse y-herpesvirus 68 which causes persistent infection of lymphoid tissue, may be inducing a switch back to the activated pattern, as a consequence of "bystander" effects, or "low affmity" stimulation via the clonotypic tcr in responding lymphoid tissue. the question of such cross-reactivity and/or exposure to "high lymphokine" environments for the long-term maintenance of memory is also being addressed. to study the factors which regulate the generation and persistence of specific t cell memory we have used model systems utilizing t cell receptor transgenic mice as a source of enriched naive cells which can be either cultured in vitro to generate effector populations or restimulated in adoptive hosts. in either case one can visualize the development of an expanded effector population. we have documented that the proliferation and il-2 production of the naive t cells depends on their activation by apc expressing high levels of co-stimulatory molecules. we find that b7.1 and icam-i as costimulators strongly synergize and that increased t cell receptor triggering can both increase the magnitude of the response and decrease its dependence on costimulation. when cytokines il-4 vs il-i2/ifny are present at the initiation of the response of either cd8 or cd4 cells they dictate that the effectors generated will be polarized either towards il-4 and il-5 secretion or il-2 and ifny secretion, respectively. the fate of the effector population generated and followed in vitro, also is tightly regulated by ag, cytokines and probably by costimulation. cd4 effector cells not re-exposed to ag, produce no cytokines and they die within 3-4 days. effectors restimulated with ag make massive amounts of cytokines, regardless of the presence of cytokines, at low densities of ag and with little dependence on costimulation. when there is little il-2 produced and no cytokines added, effectors die rapidly by apoptosis. however the combination of il-2 and tgfp block apoptosis and support expansion of the effector population which is greatly enhanced by periodic ag stimulation. some conditions favor the reversion of effector-like cells to a more resting memory phenotype and these are being further explored. we have also examined the development and maintenance of memory after transfer of effector cells to adoptive hosts. long-lived polarized memory populations are generated from the polarized effectors and these persist for prolonged period in the absence of apparent ag stimulation. this supports the idea that factors other than antigenic stimulation, present in situ can support the expansion and maintenance of memory cells. the rabies glycoprotein (g) is the only external protein of the virion and is therefore responsible for any interaction that rabies makes with the host cell during the first steps of the virus cycle. the g protein is also the target of neutralizing antibodies. there are around 450 trimers of g at the virion surface which constitute the spikes visible by electron microscopy. upon exposure to slightly acidic ph, the glycoprotein undergoes a conformational change which results in ion er and less regular spikes. strikingly and quite differently from influenza hemagglutinin, this conformational change is reversible: if the p d is risen back to 7.0, the s ikes re ain their neutral configuration (1). probably as a consequence, the viral infectivity is totally preserved after an exposure of 2 hours at p 8 6.4 an cf 37t, which induces the conformational change, followed by an incubation at neutral ph. since the conformational change is reversible, there is a ph-dependant equilibrium between the native and the low-ph conformation: the higher the ph, the more spikes are in their native configuration. two main antigenic sites and several minor sites have been identified on the native rabies glycoprotein (2). specific amino acids belonging to each of the two major antigenic sites are important or essential for viral virulence. for instance a lysine in position 198, which is part of antigenic site 11, is important, although not essential, for the viral virulence. similarly, the arginine 333, which belongs to antigenic site 111, is essential for pathogenicity while dispensable for multiplication in cell culture (reviewed in 3). viral strains mutated at arginine 333 have lost the capability to penetrate certain categories of neurons, suggesting that this mutation affected the recognition of specific receptors or subsequent interactions necessaly for the penetration of the virus at nerve terminals. therefore the two main antigenic sites are regions of the glycoprotein which also interact specifically with neurons in the animals. we have found that neutralization requires the fixation of at least one or two igg for every three spikes, irrelevant of the anti enic site recognized by the antibody (4). most neutralizing antibodies recognize conformational epitopes which are accessible on the native configuration of the protein. some epitopes remain accessible also on the acidic configuration while others are not. in addition, a minority of antibodies recognize epitopes which are only accessible on the acidic conformation. this is not unlikely in view that each spike has a certain probability to undergo a conformational change, even at neutral ph. in consequence the surface of the virus probably fluctuates and g epitopes which are not accessible on the native glycoprotein could be transiently exposed. conformational flexibility at neutral ph and physiological temperatures has also been observed for poliovirus (5). structural flexibility of external proteins could have important implications in virus-host interactions. katpus, norlhwestern university medical school, chicago, il 6061 1 theiler's murine encephalomyelitis viruses (tmev) are endemic enteric pathogens of wild and colony-reared mice. lntracerebral inoculation of susceptible mouse strains leads to a chronic, progressive inflammatory demyelinating disease of the central nervous system (cns) characterized clinically by an abnormal gait, progressive spastic hind limb paralysis and urinary incontinence, and histologically by parenchymal and perivascular mononuclear cell infiltration and demyelination of cns white matter tracts. demyelination is related to persistent cns viral infection. due to the similarity in clinical and histological presentation, tmev-induced demyelination is considered to be a highly relevant model of multiple sclerosis (ms). our current interests are in determining the phenotype, fine specificity, lymphokine profile and tcr usage of cns-infiltrating cells involved in the effector stages of tmev-induced demyelination. based on a variety of experimental evidence, it is clear that demyelination induced in sjuj mice by infection with the bean strain of tmev is a thl-mediated event: (a) disease induction is suppressed in t cell-deprived mice and by in vivo treatment with anti-i-a and anti-cd4 antibodies; (b) disease susceptibility correlates temporally with the development of tmev-specific, mhc-class il-restricted dth responses and with a predominance of anti-viral lgg2a antibody; (c) activated (le., ll-2rc) t cells infiltrating the cns are exclusively of the cd4+ phenotype, and (d) proinflammatory cytokines (ifnq and tnf-p) are predominantly produced in the cns. we have mapped the predominant thl epitope on the virion to amino acids 74-86 of the vp2 capsid protein. a thl line specific for vp274-86 exacerbates the onset of demyelination in recipient mice infected with a suboptimal dose of tmev. tmev-infected sjuj mice fail to exhibit peripheral dth and t cell proliferative responses to the major myelin proteins, mbp and plp, and pre-tolerization with neuroantigens has no affect on the incidence or severity of tmev-induced demyelinating disease, whereas neuroantigen-specific tolerance prevents the induction of relapsing experimental autoimmune encephalomyelitis (eae). in contrast, tolerance induced with intact tmev virions specifically anergizes virus-specific thl responses and results in a dramatic reduction of the incidence and severity of clinical disease and cns dernyelination in sjuj mice subsequently infected with tmev. these results have important implications for a possible viral trigger in ms as they indicate that chronic demyelination in tmev-infected mice is initiated in the absence of demonstrable neuroantigen-specific autoimmune responses and are consistent with a model wherein early myelin damage is mediated via primarily by mononuclear phagocytes recruited to the cns and activated by pro-inflammatory cytokines produced by tmev-specific thl cells. the concept that prions m novel pathogens which are different fium both viroids and viruses has received increasing support from many avenues of investigation over the past decade. enriching fractions from syrian hamster (sha) brain for scrap= prion infectivity led to the discovery of the prion protein 0. prion diseases of animals include scrapie and mad cow disease; those of humans present as inherited, sporadic and w o r n neurodegenemive disorders. the inhecited human pion diseases m genetically linked to mutatim in the prp gene that result in non-conswative amino acid substitutions. transgenic v g ) mice expressing both sha and m o w @lo) prp genes were used to demonstrate that the "specie9 bank?' for -pie prions resides in the primary structure of pip. this concept was strengthened by the results of studies with mice expressing chimeric mdsha transgenes &om which "artificial" prions have been synthesized. similar chimeric mdhuman (hu) rp transgenes were constructed which differ from m o w by 9 amino acids between residues 96 and 167. au of the tg(mhu2m) mice developed neurologic drsease -200 days after inmulation with brain homogenates from three patients who died of creutzfeldt-jakob disease (cjd). inoculation of tg(mhu2m) mice with cjd prims produced mhu2mprpsc, inoculation with mo prions produced moprw. ihe patterns of meluzmprpc and mom% accumulation in the brains of tg(mhu2m) mice wen differenl about 10% of tg(huprp) mice expressing huf" and non-tg mice developed neurologic diseane >500 days after inoculation with cn, prions. the different susce@uies of tg(hw) and tg(mhu2m) mice to human prions indiate that additional species specific factors such as chaperone proteins are involved in prion replicaton. diagnosis, prevention and treament of human @on diseases should be faciliated by tg(mhu2m) mice. in other sindies, tg mice were compared expressing wt and mutant moprp. overexpression of the wtmoprp-a aansgene -8-fold was not deleterious to themiw but it did shorten scrapie incubation times from -145 d to -45 d after inoculation with murine m p i e pnons. in contrast, overexpression at the same level of l morp-a transgene mutated at codon 101 (corresponding to codon 102 in hurp) pmdnced spontaneous, fatal neurcdegeneration between 150 and 300 d of age in two lines of tg(mohp-pio1l) mice designated 2866 and 2247. genetic crosses of tg(moprp-p101l)2866 mice with gene targeted mice lacking both rp alleles ( p m -p ) produced anhats with a highly synchronous onset of illness between 150 and 160 days of age. the t g~o p r p -p l o l l ) 2 8 6~~ mice had numerous prp plaques and widespread spongiform degeneration in contrast 10 the tg2866 and 2247 mice that exhibited spongifonn degeneration but only a few prp amyloid plaques. another line of mice designated tg2862 overexpress the mutant transgene -32-fold and develop fatal neurodegeneration behveen 200 and 400 d of age. tg2862 mice exhibited the most severe spongiform degeneration and had numerous, large pip amyloid plaques. while mutant moprpccploll) clearly produces neurodegeneration, wtmoprpc profoundly modifies both the age of onset of illness md the mumpathology for a given level of transgene expression. our tidigs and those from other smdies suggest that mutant and wtprp interact, phaps through achaperone-like protein as noted above in sndies of tg(mhu2m) mice, to modify the pathogenesis of the dominantly inhe&ed prion diseases. anton, heidi t. link, and jonathan w. yewdell, laboratory of viral diseases, niaid, bethesda, md 20892-0440. cd8' lymphocytes (tcd8+) play an important role in host immunity to viruses and other intracellular parasites. virus-specific tcdi+ recognize mhc class i molecules in association with peptides of 8 to 10 residues derived from viral proteins. this presentation will focus on how and where antigenic peptides are generated by cells. to begin to characterize the nature of proteases involved in the generation of antigenic peptides from cytosolic proteins, we used a panel of recombinant vaccinia viruses expressing different forms of influenza virus nucleoprotein (np). we found that the efficiency of generation of two np peptides is related to the metabolic stability of the source gene product. there has been considerable speculation that such short lived proteins are degraded by proteasomes in a ubiquitin-targeted process. our observations, however, call into question the importance of ubiquitin targeted-proteolysis in generating antigenic peptides from exogenously provided or endogenously synthesized viral proteins. we also examined the extent to which antigenic peptides can be generated in the endoplasmic reticulum (er). we found that antigenic peptides could be produced from short precursors (17 residues) hut not from a number of full length proteins (influenza virus hemagglutinin, np, ovalbumin) that are targeted to the er by a nh2-terminal signal sequence. peptides were generated much more efficiently from the cooh-terminus of the 17 residue precursor than from the nh2-terminus. these findings indicate that the er has a much more limited capacity than the cytosol to generate antigenic peptides, but that er proteases (particularly aminopeptidases) could perform the final proteolytic steps in the generation of class i binding peptides from precursors imported from the cytosol by tap, the mhc encoded peptide transporter. potential advantages of synthetic peptide or engineered recombinant vaccines are that they can be limited to contain only the specific antigenic determinants for desired responses without other determinants that elicit unwanted responses, and that the sequences of the determinants themselves can be modified to enhance potency or breadth of crossreactivity. however, they can have the disadvantage that any single determinant may be presented by only a limited selection of major histocompatibility complex (mhc) molecules of the species. to overcome the problem of mhc polymorphism, we have identified determinants presented by multiple mhc molecules, and have also located multideterminant regions of the hiv-1 envelope protein that contain overlapping determinants each presented by different class i1 mhc molecules, so that the whole multideterminant region is presented by multiple mhc molecules of both mouse and human. we have made use of "cluster peptides" spanning these multideterminant regions of the hiv-1 envelope to provide help for neutralizing antibody (ab) and cd8+ cytotoxic t lymphocyte (ctl) responses to peptides attached to these helper regions. these synthetic peptide vaccine constructs containing the p18 peptide from the v3 loop of hiv-1 iiib or mn, elicited both neutralizing ab and ctl in multiple strains of mice. the cluster peptides inducing helper t cells were essential for elicitation of ab and ctl to the p18 segment of both iiib and mn strains of hiv-1 in mice of several mhc haplotypes. several adjuvants were compared for their ability to elicit both ctl and ab simultaneously, without one response inhibiting the other. a single formulation in incomplete freund's adjuvant (ifa) could elicit all 3 responses, neutralizing ab, ctl, and th1 helper cells. the ctl specific for the mn strain p18 peptide crossreacted with strains sc, sf2,2321, and cdc4. the peptides in f a also elicit high titers of antibodies in rabbits. boosting was found to enhance ctl responses as well as ab responses. these constructs are being prepared for a human immunotherapy trial. these vaccine constructs are potent and also avoid sites on gp160 that are known to elicit enhancing antibodies or autoimmune responses that might conmbute to disease pathogenesis. however, we can potentially improve on these by tinkering with the internal structure of the individual epitopes. we have found that replacing a negatively charged glutamic acid residue with an uncharged amino acid in one of the helper determinants makes it 10 to 100-fold more potent in binding to the class i1 mhc molecule and in eliciting murine helper t cells that still recognize the natural hiv-1 sequence. thus, such a modified peptide should be more potent as a vaccine, while retaining the ability to elicit t cells that will respond to hiv proteins that of course do not have the altered sequence. we are currently mapping the critical residues for presentation of one of these peptides by human hla-a2, with the intent of developing modified peptides that will be more potent as components of a human vaccine. thus, by leaming how these peptides bind to mhc molecules and tcell receptors, we can design internally modified determinants to construct more potent or more crossreactive second generation vaccines. we are testing these vaccine approaches in a mouse model in which mice can be protected against tumor cells expressing hiv proteins as would an hivinfected cell. dna vaccines, comprised of non-replicating plasmids encoding viral proteins, are capable of generating protective immunity in animal models of several viral diseases. in preclinical models of influenza infection, reduced viral shedding was observed in dna-vaccinated ferrets after challenge with the human clinical virus strain, a/georgia/93. cross-strain protection was conferred by dna encoding the major internal proteins (nucleoprotein, np, and matrix, m1) and the surface protein haemagglutinin (ha) from the antigenically-distinct previous virus strains, a/beijing/89 and a/hawaii/91. this protective efficacy was greater than that seen by immunization with the widely-used clinical vaccine composed of killed a/beijing/89 virus. thus, compared to a killed virus vaccine, protection seen with the dna vacane against a drifted virus strain was greater. we previously demonstrated that immunization of mice with np dna generated mhc class i-restricted cytotoxic t lymphocytes. mice likewise were protected from death and morbidity following cross-strain challenge'. ha dna vaccines generated neutralizing antibodies in mice, ferrets and primates, and provided protection in m i d and ferret models of influenza. in animal models of other viral diseases, immune responses and protection against viral challenge have been seen after immunization with dna encoding viral proteins. dna encoding hiv gp120 generated ctl and neutralizing antibodies in monkeys. antigen-specific proliferative responses and, in mice, secretion of high levels of yifn relative to levels of il-4, months after immunization were also observed . immunization of rabbits with dna encoding l1, the major viral capsid protein of cotton tail rabbit papilloma virus (crpv), resulted in neutralizing antibodies and protected against the development of warts after inoculation with crpv. mice immunized with dna encoding the glycoprotein gd from herpes simplex virus type 2 (hsv-2), developed neutralizing antibodies and were protected from death when subsequently challenged with hsv-2. dna vaccines were protective in animal models of various viral diseases. neutralizing antibodies, helper t cells (thl) and cytotoxic t cells were generated. cross-strain protection due to cellular immunity was demonstrated. ' science, 1993 2593745-1749 , 2dna cell biol, 1993 the profile of a neurovirulent virus is determined by its mechanism of entry into the cns (neuroinvasion), the type of cns cell in which it replicates (neurotropism) and its ability to cause pathologic effects in the brain (neurovimlence). whereas neuroectodermal cells, especially neurons, are the target cells of most neurovirulent viruses, the main target cell in the brain for siv and other lentiviruses is the macrophage. infection in, expression of viral antigens by and products of siv replication exported from these cells result in inflammation and degenerative changes in the brain and concomitant loss of neurons. siv strains that are mainly t-cell tropic cause transient activation of t-cells and during this period, infected t-cells cross the blood brain barrier and localize in the brain causing persistent hut minimally productive infection and minimal neuro pathologic effects. viral proteins but not virions are produced continuously. by virtue of the tropism of the virus for cd4 t cells, many infected animals eventually become immunosuppressed and develop aids, but not classical ueurological disease. viruses which are macrophage tropic invade the brain presumably also in t lymphocytes and the viruses infect macrophages in the brain. however, productive virus replication is minimized by antiviral cd8 t cells which suppress (kill?) all virus producing cells throughout the body, including the cns. productive virus replication in brain macrophages and accompanying inflammatory changes develop only when cd8 cells fail i.e. after profound immunosuppression sets in. the neurological disease that results from productive virus replication in macrophages in the brain therefore depends on presence of an appropriate macrophage-tropic viral phenotype invading the neuropil and development of immunosuppression in the host. the neurological disease could therefore be defined as one of the aids syndromes. the adenovirus (ad) early transcription region (e3) codes for more than 7 polypeptides, four of which have already been shown to alter the immune response to ad infection. the amount of the class i major histocompatibility complex (mhc) on the plasma membrane can be reduced by the binding of the ad e3 gpl9k protein to the mhc heavy chain, which prevents transport of the complex out of the endoplasmic reticulum. this process interferes with presentation of viral peptides to cytotoxic t lymphocytes. cytolysis by tumor necrosis factor-o (tnf) is inhibited by 4 distinct viral polypeptides, 3 of which (the ad e3 14.7k or the complex of the 10.4k and 14.5k proteins) are coded in the e3 region. the e3 polypeptides are translated from a family of viral mrnas, that are synthesized from a single viral promoter and processed by alternative splicing. we have studied the functions of the e3 polypeptides in several murine models. the goals of these experiments were to determine the effects of the ad e3 polypeptides in acute and persistent viral infections as well as in a transplantation model designed to measure whether these viral immunoregulatory proteins would abrogate allogeneic graft rejection. in a vaccinia virus (v.v.) pneumonia model, in which the isolated ad e3 14.7k or ad e3 gpl9k genes were inserted into the v.v. pathogen, the ad anti tnf polypeptide increased viral virulence but the ad anti mhc had no effects. in addition to manipulating the ad e3 genes in viral constructs, several transgenic mouse lines containing the ad e3 genes have been constructed for these experiments. the e3 genomic dna behind the rat insulin promoter (rip) has been used to generate transgenic animals. islets from rip-e3 transgenic animals (h-2b'd) have been transplanted allogeneically to h-2d recipients and remained viable, secreting insulin until the end of the experiment at 94 days; in contrast, control nontransgenic islets of the same genotype were rejected by 21-28 days. the e3 genes behind the native e3 promoter have been inserted into mouse embryos to generate transgenic animals, and the expression of the transgene monitored in multiple organs. the e3 promoter of the transgene is responsive to stimulation by the ad e1a following infection with an e3 minus ad 7001 and can also be upregulated by administration of bacterial lipopolysaccharide. the effects of this transgene on ad pathogenesis are currently being studied. thus, these viral immunoregulatory genes have been shown to alter viral pathogenicity during acute infection and to downregulate the host immune response sufficiently to permit islet cell transplantation. these results on manipulating the ad e3 genes for the control of the host immune response also have implications for designing adenovirus vectors for gene therapy. "emerging" infections can be defined as infectious diseases that either have newly appeared in the population, or that are rapidly increasing their incidence or expanding their geographic range. recent viral examples include aids, ebola, and hantavirus pulmonary syndrome (fnst identified in a 1993 outbreak in the southwestern u.s.). emerging viral infections show a number of common features. most "new" viruses derive from existing viruses that move into new areas or acquire new hosts ("viral traffic"). many are zoonotic (originating from animal sources) (even pandemic influenza appears usually to be a reassortant originating in wildfowl). ecological or environmental changes (either natural, or, often, man-made) may precipitate emergence of new diseases by placing people in contact with a previously unfamiliar zoonotic reservoir or by increasing the density of a mtud host or vector of a pathogen, increasing the chances of human exposure. upon introduction into a human population from a zoonotic reservoir, the newly introduced virus may cause localized outbreaks of disease. some may show rapid variation and evolution upon introduction, and some evidence suggests a role for immune selection in this process. a few viruses (such as hiv) may succeed in establishing themselves and disseminating in the human population, becoming truly "human" infections. human activities can also play an important role in establishment and dissemination. migrations from rural areas to cities, now an accelerating worldwide phenomenon, or other displacements, can introduce remote viruses to a larger population; the virus may then spread along highways and (globally) by air travel. the development of an effective system of surveillance and rapid response is essential, but resources for this are presently inadequate. vaccine development, production, and deployment problems also need to be addressed. immunopathology may be a key feature of many of these infections, a number of which manifest as hemorrhagic fevers. many of the life threatening complications are due to increased vascular permeability. the resemblances to septic shock suggest that cytokines (such as tnf) are likely to be important in the pathogenesis of these infections. the response of cells, such as the macrophage, that induce or synthesize key cytokines, may be an important element, and the ability to infect these cells may be one common denominator. why some viruses elicit this response, while other closely related viruses do not, cannot yet be predicted from molecular data. better understanding of these aspects of the immune response should lead to additional therapeutic strategies. (supported by nih grant roi rr03121.) genetic approaches have been used to detect and characterize numerous previously unidentified hantaviruses. puumalarrospect hilvsin nombre-like viruses or virus variants are present throughout north and south america, europe and russia. several of the american viruses identified are associated with the newly recognized hantavirus pulmonary syndrome (hps), a severe respiratory illness with high mortality. the genetic relationships of these and previously characterized hantaviruses have been studied by phylogenetic analysis of the nucleotide sequence differences located in pcr bgments amplified from the g2 encoding region of the virus m segments. the relationships observed are consistent with a long-term association of viruses with their primary rodent reservoirs and suggestive of coevolution of host and virus. a sin nombre virus isolate is now available and its genetic characterization has been completed. various virus antigens have been expressed and are being used to probe the interaction of the virus with the host immune system. hantaviruses cause significant morbidity and mortality throughout the world. more than 200,000 cases of hemorrhagic fever with renal syndrome (hfrs) are reported annually in asia, europe and scandinavia. the etiologic agents of hfrs are hantaan, seoul and puumala viruses, with hantaan virus causing the most severe form of the disease. in 1993, a new hantavirus was discovered in the united states (initially termed four comers virus), and was identified as the etiologic agent of hantavirus pulmonary syndrome (hi's). vaccines for hantaviruses are not readily available, although a number of inactivated viral preparations have been made and tested in asia. recurrent problems with inactivated hantaviral vaccines have been lot to lot variability, the need for repeated immunizations, and their inability to elicit long-lasting neutralizing antibody responses in immunized volunteers. because of such limitations on traditional vaccine development for these viruses, as well as the viruses' hazardous nature and slow, low-titer replication in cell culture, we used a recombinant dna approach to develop a vaccine for i-ifrs. our vaccine is a recombinant vaccinia virus expressing the m segment of hantaan virus under control of the vaccinia virus 7.5 k promoter and the s segment under control of the 11 k promoter. the m segment, which encodes the g1 and g2 envelope proteins, was included because of our findings that: (1) immunization with vaccinia or baculovirus-expressed g1 and g2 induced a neutralizing and protective immune response in hamsters; and, (2) neutralizing antibodies to g1 or g2 could passively protect hamsters from challenge with virulent virus. the s segment, which encodes the nucleocapsid protein (n), was included because of our finding that hamsters immunized with baculovirus-expressed n also were protected from subsequent infection. although the protective immune response to n is probably cell-mediated, the importance of such a response is presently not well defined. assessment of our vaccine in preclinical studies, indicated that immunized hamsters developed neutralizing antibodies and were protected from displaying viral antigen in their lungs after challenge. in a phase i, dose escalation, clinical study, the vaccine induced neutralizing antibodies in individuals immunized subcutaneously with approximately lo7 pfu of the recombinant virus. in addition to humoral responses, immunized volunteers developed a cell-mediated immune response as indicated in lymphocyte proliferation assays. larger clinical studies, including alternate routes or booster immunizations, are planned. based on these studies, we anticipate that the vaccine will be efficacious for preventing hfrs caused by hantaan and the antigenically closely related seoul virus. we are studying the cross-protective properties of this vaccine with more distantly related hantaviruses such as puumala virus. although we expect this vaccine to be safe as well as effective, we also are investigating the use of more attenuated pox-viruses as vaccine vectors. infection of mice with lymphocytic choriomenigitis virus (lcmv) results in a profound expansion in the number of spleen cd8 t cells and in the induction of virus-specific ctl activity. thereafter, the cd8 t cell number declines, and the ctl activity diminishes, though the frequency of lcmvspecific precursor ctl per cd8 cell, as assessed by limiting dilution assays (lda), is remarkably stable throughout long-term immunity. the decline in t cell and total spleen leukocyte number at the late stages of acute infection is associated with high levels of apoptosis, as detected by the in situ nucleotidyl transferase assay. apoptosis occurred in both the t cell and b cell populations, with the b cells dying in clusters. this apoptosis was also seen in tfansgenic mice ectopically expressing bcl-2 in the t and b cells and in c57bl/6 ipr/@r mice, which have a mutation in the fas gene. t cells from the infected animal underwent apoptosis in vitro when stimulated through the tcr with anti-cd3, thereby explaining some of the immunosuppression seen during acute viral infections. memory cells persisted for over a year and could be found in blast-size cell populations. challenge of lcmv-immune mice with either pichinde virus, vaccinia virus, or murine cytomegalovirus led to the reactivation of the lcmv-specific ctl response. lda analyses showed unexpectedly that these heterologous viruses crossreacted with subpopulations of lcmv-specific memory t cells. this memory t cell response to virus from an earlier infection was associated with enhanced immunopathology and enhanced clearance of virus during a heterologous virus challenge. over the course of the acute infection, ctl specific for the second virus were preferentially expanded over the crossreactive ctl, and after the acute infection, when the t cell response had subsided, ctl memory to the first infection had decreased. there is therefore a network of memory t cells which contribute to and are modulated by infections with putatively unrelated viruses, and apoptosis plays a homeostatic role in the course of these t cell responses. immune responses to live attenuated retroviral vaccines, r. paul johnson*?, cara wilsont, kelledy mansons, michael wyands, bruce walker?, ronald c. desrosiers* *new england regional primate research center, southborough, ma 01772 thfectious disease unit, massachusetts general hospital, boston, ma 021 14 â§tsi/mason, worcester, ma immunization of rhesus macaques with live attenuated retroviruses deleted in nef can induce protective immunity against challenge with pathogenic siv. development of protective immunity in these vaccinated animals occurs only after several months of infection, with maximal protection observed after one year. the specific immune responses responsible for mediating protection have not been defined, and little is known about the cellular immune responses in animals vaccinated with these live attenuated retroviruses. we have analyzed cellular and humoral immune responses in rhesus macaques and chimpanzees infected with live attenuated retroviruses. siv-specific neutralizing antibodies were present in vaccinated animals, but did not clearly correlate with protection against challenge. ctl specific for envelope and gag were identified in vaccinated macaques studied 12 or more months after vaccination. quantitation of siv-specific ctl activity in one of these animals using limiting dilution analysis revealed a relatively high precursor frequency of cytotoxic t lymphocytes, up to moo0 for gag and 1/8500 for envelope. cd8+ lymphocytes obtained from vaccinated macaques were also able to suppress siv replication in autologous cd4+ cells. suppression mediated by unstimulated cd8 autologous cells was maximal when cells were in direct contact with siv-infected lymphocytes, but cd8+ cells activated by an anti-cd3-specific monoclonal antibody were able to release. a potent soluble inhibitor of siv replication. in contrast to the relatively vigorous ctl response present in vaccinated macaques, we were not able to detect consistent ctl activity in chimpanzees infected with a hiv-1 molecular clone (nl43) or attenuated viruses at periods up to one year after infection, despite the use of a variety of stimulation techniques. proliferative responses to hiv p24 and gp160 were observed in chimpanzees infected with n u 3 and attenuated variants. although the relative contribution of these immune responses to protective immunity is not known, the relative vigor of the cellular immune responses observed in vaccinated macaques suggest they may play a role in mediating resistance to challenge. obiectives: to analyze the magnitude and specificity of the ctl response to hiv-1, and to determine the tcr usage by clonal ctl responses in infected persons, including persons with documented infection of up to 15 years with cd4 cells > 500/mm3. methods: hiv-l-specific ctl activity was evaluated in pbmc as well as in pbmc stimulated in vitro with hn-1 infected autologous cd4 cells, using target cells infected with recombinant vaccinia viruses expressing hn-1 proteins. ctl epitopes recognized by these individuals were determined using cloned effector cells. quantitative cultures were performed by endpoint dilution, and viral quantitation was determined by qc-pcr tcr analysis was performed by pcr, using both family-specific primers and anchored pcr, followed by sequencing. sequence analysis of ctl epitopes in autologous viruses was determined by pcr amplification and sequencing. clonal frequency was analyzed in pbmc by oligonucleotide probe to the cdr3 region of the tcr. studies performed in long-term non-progressing persons indicate the presence of a vigorous and broadly directed ctl response. detailed epitope mapping in a person infected for 15 years, who by qc-pcr had <i67 viral rna molecules per ml and who tested negative by bdna assay, revealed ctl clones directed at six different epitopes, with detectable recognition of three of these epitopes using freshly isolated pbmc as effector cells. the ctl clones were broadly reactive against many hown hiv-1 variants, and both the p17 and p24 epitopes were cross reactive with siv. sequence analysis of autologous virus within the three dominant ctl epitopes revealed the lack of immune escape variants in pbmc, plasma, and virus obtained from coculhue. supernatant. analysis of tcr usage by clones to defined epitopes in persons at different disease stages revealed heterogeneity in tcr usage and heterogeneity in recognition of virus variants withii these epitopes. analysis of clonal frequency using an oligonucleotide probe to the cdr3 region of the tcr in an individual with a vigorous response to gp41 suggested that approximately 6% of circulating t cells were a single hiv-l-specific clone. conclusions: our results indicate that cytotoxic t lymphocytes are part of the immune response in persistent non-progressive hiv-1 infection, and that prolonged infection can occur without the development of viral immune escape variation within defined ctl epitopes. in addition, vigorous circulating ctl responses can occur in the setting of an e x m e l y low viral load. heterogeneity in the ability of clones from different individuals to recognize sequence variation within ctl epitopes may be important in the pathogenesis of infection. ctl to conserved epitopes or ctl which are broadly reactive with multiple viral mains may be important in conmbuting to maintenance of the asymptomatic state. factor-i (irf-1) gene is essential for the transcriptional activation of inducible nitric oxide synthase (nos) in murine macrophages. it also plays an important role in the activation of interferon and il-6 and il-7 receptor genes. to investigate the role of irf-1 related systems of defense against viral myocarditis, the transgenic mice were innoculated with l o 5 pfu of coxsackie virus 83 (cvb3). homozygous irf-1 (+) and heterozygous irf-1 (+/-) mice were randomized into groups to determine heart and spleen viral titres, cardiac pathology and mortality rates at early and late stages of the disease. the mortality was dramatic in the irf-1 -imice. the hearts of the homozygous animals also had increased mononuclear cell infiltration, necrosis and viral replication. we condude that irf-1 regulated gene products such as inos and ifn are essential for the early defense against cvb3-induced myocarditis by attenuating viral replication and inflammatory responses. the flu season is a yeariy problem, especially in the elderly population. annual vaccination lacks broad protection and antibody titers induced in the elderly are frequently lower than those acheived by younger vaccinees. using the mouse model, we are investigating the differences in immune response between old and young mice to influenza vaccine and at the same time testing the ability of a potent adjuvant to boost the response in the the elderly. our studies show defects in the immune response of the elderly. antibody titers after immunization are lower in the elderly for both seronegative and seropositive mice. cmi studies show that helper t-cell response (proliferation) to in-vitro antigen stimulation is also decreased in the elderly. facs analysis of wbcs show the elderly mice have lower leukocyte and lymphocyte populations, lower cd4 and b-cell populations and a higher proportion of macrophages and cd8 cells than young mice. it was also noted that the young mice had very consistent wbc profiles while the elderly profiles had greater variability. serum cytokine studies show lower levels of il2, il4 and il5, but higher levels of il6 and ifng in the elderly as compared to the young mice. the addition of mf59 to the immunization increased the antibody titers of both naive and seropositive young and old mice. the helper t-cell response of the young mice was unaffected, while the response of the elderly group was increased. cytokine profiles show an increase in il2, il4 and il5 levels for both young and old, while il6 and ifng levels went up for young animals but remained constant for the elderly mice. coxsackievirus 8 3 (cvb3), a member of the picornavirus family, is a common cause of acute or chronic human myocarditis. however, whether cell damage results primarily from direct virusmediated effects or from immune-mediated destruction of the heart tissue during the infection remains unclear. we used a cardiovirulent variant of cvb3 which is able to infect several strains of mice. the infection results in a disease which strongly implicates immunopathogenetic mechanisms in myofiber necrosis. to characterize the role of the immune system in this disease we used various transgenic knockout (ko) mice with different immune defects. cvb3 is able to infect normal c57bl/6 mice and immunocompromised (cd4 ko and 62m ko) mice and causes a lethal disease after i.p. injection in a viral dose dependent manner. we found elevated ld5os in immunocompromised mice, which also die slightly later than normal mice. this virus replicates in the hearts of all mice used with maximal titers 3-5 days pi.. large pathological changes were detectable only in heart tissue of cd4 ko mice, and were maximal at 1-2 weeks after infection. these results suggest the possibility that cd4' t cells may limit tissue damage by an as yet undefined mechanism; the roles of cd4+ and cd8' t cells in this disease are under investigation. research supported by a grant of the daad, germany. mice lacking p56lck are resistant to coxsackievirus b3 induced heart disease. tamara martino, karen aitken, joseph penninger, jan0 nikhilanandhan, tak mak, fayez dawood, wen-hu wen, lily wee, martin petric, and peter liu, the toronto and princess margaret hospitals, university of toronto, canada. coxsackievirus 83 (cvb3) causes severe heart disease in adult mice, and is a useful model for the human cardiac conditions of myocarditis and dilated cardiomyopathy. in this study, we have demonstrated that transgenic mice lacking the t-cell signalling molecule p56kk are resistant to cvb3 induced heart disease. the virus replicates in the heart, and is cleared from the myocardium at a rate comparable to "normal" littermates, but there is only minimal mortality, or damage to the cardiac tissue. however, in ick-deficient mice depleted of natural killer (nk) cells prior to viral infection, there is increased cvb3 replication, and some infiltration by mononuclear cells in the myocardium. to further elucidate the role of the immune system in cvb3induced heart disease, cytokine expression in the hearts of cvb3 infected normal, ick-deficient, and nk-cell depleted mice is under investigation. we conclude that t-cell activation is critical to produce substantive myofiber necrosis after cvb3 infection, that nk-cells play an fundamental role in protecting the mice from severe virus infection, and that ick transgenic mice serve as an important model for understandina the dathogenic mechanisms involved. we now show that cd4+ t cells from 62m-/-mice exhibit antigen specific release of serine esterase and interferon-gamma. further, as quantitated by flow cytometry, the early decrease in cd4+ t cells seen in normal mice 3 days after lcmv infection, previously presumed to be due to the activity of cd8+ ctl, still occurs in 62m-/-mice. however, 621114mice show an earlier rise in percentage and absolute numbers of cd4+ t cells by 7 days after infection. cd4+ t cells from am-/mice rise to above baseline levels with kinetics paralleling the early rise in cd8+ t cells in normal mice after lcmv infection. cd4+ ciz activity is lower in 62m-/mice and have a later peak than cd8+ ctl from normal mice. these differences may explain the lower mortality and longer time course of immunopathologic meningitis in 621114-mice. mice can assume some of the cytotoxic functions of the cd8+ cell population from normal mice, including immune-mediated pathology. further, cd4+ t cells from 62m-/-mice have the capacity to release serine esterase, and show kinetics similar to cd8+ t cells from normal mice. mice genetically engineered to be deficient in 62-microglobulin in conclusion, these data suggest that cd4+ t cells from am-/australia. molecule is an essential component of the t cell help required for an antibody response. the interaction between cd40 and its ligand, in the presence of b cell acting cytokines, such as interleukin 4 (il-4), is sufficient to trigger b cells to proliferate and differentiate and to induce immunoglobulin class switching. a recombinant vaccinia virus encoding both factors, cd40l and il-4, did not, however, stimulate an enhanced antibody response in mice infected with the construct. instead, the antibody levels were lower than those of mice infected with a control virus. the reduced antibody response reflected the diminished growth of cd4ol-expressing viruses, to the extent that immunocompromised mice were able to resolve these infections. the kinetics of virus clearance indicate that the anti-viral mechanism of cwol is rapidly activated and has generalized tissue distribution. the cwolmediated clearance of virus is independent of the anti-viral cytokines, tnf and ifn-y. the anti-viral activity of c w l may represent a surprising and potent effector mechanism of t cells activated during a virus infection. mary's medical school, norfolk place, london w2 lpg, u. k. and *nationallnstirute formedica1 research, london, nw7 1aa. respiratory syncytial (rs) virus is the most important respiratory pathogen of infants, causing the majority of cases of bronchiolitis, it can be life threatening in very young infants and those with underlying cardiopulmonary disease or immunodeficiencies. t cell immunodeficiency can lead to prolonged infection in children and t cell transfers clear virus in the murine disease model. mice can be readily infected with rs virus and have proved a valuable model of its pulmonary pathology and the t cell response to it. il-3 has a broad spectrum of activities on proliferation and terminal differentiation of early blast cells and committed progenitors of many lineages. less is known about the effects of il-3 on t cell development and proliferation and its effect on the host response to infection by common pathogens. the il-3 gene was disrupted in 129/sv embryonic stem (es) cells by homologous recombination after transfection with omega-type constructs flanked with a herpes simplex thymidine kinase gene. these cells were used to produce a mouse strain deficient of il-3. mice were infected intranasally with a 2 strain rs virus; pathological changes in the lung were monitored by cytology of bronchial lavage, and virus persistence by nested rt-pcr of lavage fluid and lung homogenate. no differences were found in pathology or virus persistence between knockouts and normal mice. il-3 deficiency is therefore compatible with apparently normal responses to viral infection. recombinant adenoviruses are an atuactive vehicle for gene therapy to lung in the treatment of cystic fibrosis (cf). first generation viruses deleted of ela and elb transduce genes into airway epithelial cells in vivo, however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. in this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive nansfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. our studies indicate that mhc class i resmcted cd8+ cytotoxic t lymphocytes (ctls) are activated in response to viral antigens leading to destruction of virus infected cells and loss of transgene expression. mhc class ii associated presentation of viral antigens activates cd4+t helper (th) cells of the -1 subset and, to a lesser extent, of the tm subset. ldv establishes a life long viremic infection in mice which is not controlled by anti-ldv immune responses. anti-ldv antibodies are rapidly generated but they neutralize ldv infectivity only poorly. here we show that ldv-specific cytotoxic t cells (ctls)are generated within 7 days pi. c t l s were detected by h-2 specific lysis of ldv-specific target cells. these target cells were generated by transfection of 3t3-nm cells with a retrovirus vector carrying the gene (ow 7) for the ldv nucleocapsid (n) protein. spleen cells from ldv-infected mice were incubated in vitro with ldv-infected macrophages or transfected 3t3-nih cells and cultured for 5 days in the presence of il-2. the ctls had largely disappeared in mice by 30 days pi. the following experiments were conducted to further explore the mechanism of immune escape. in situ hybridization was used to localize ldvinfected cells in tissues of infected mice. these were found in persistently infected mice only in the liver. testis, and lymphoidal tissues. in addition large amounts of virion or virion debris accumulated in the germinal centers of the spleen and lymph nodes beginning about 3 days p.i. the accumulation of large amounts of ldv antigen in the germinal centers may be causally related to the suppression of the ctl response as well as to the polyclonal activation of b cells associated with ldv infection. to determine whether immune selection plays a role in ldv immune escape, ldv was reisolated from nude and immunocompetent balb/c mice at various times pi. the ows for the nand the two envelope glycoproteins were r t k r amplified and sequenced. the role of cytokines in initiating the immune response to venezuelan equine encephalitis (vee) was investigated. mice were infected in the left rear foot pad with either cloned virulent vee (v3000) or an isogenic single-site attenuated vee mutant (v3032) (single amino acid change at e2 glycoprotein position 209 from glu-lys) and at 1,6,24,48,72, and 96 hours post-infection, the popliteal lymph nodes and spleen were harvested and examined for tnf-a, il-12, ifn-y, il-2, il-4, il-6, and il-10 gene expression using a quantitative rt-pcr. in both strains elevations of tnf-a, il-12, ifn-y, il-10, and il-6 were detected in the lymph node, with no changes in either il-2 or il-4; however, whereas cytokine elevations were detected by 6 hours after injection of v3000, elevations were delayed until 24 hours following infection with v3032. cytokine mrna elevations in the spleen were less substantial, but elevated levels of ifn-y, il-10, and il-12 were also observed. these findings suggest that vee infection induces an early highly specific cytokine pattern associated with simultaneous elevations in both ifn-y and il-10 and that a single amino acid mutation can induce temporal changes in this pattern without alterring either the actual cytokines induced or the degree of their elevation. we are currently performing intervention experiments to investigate the effect of intravenous anti-il-12 andor anti-ifn-alp antibody administration on this early in vivo cytokine response to examine in particular whether these cytokines may be required for the observed elevations in either ifn-y or il-10. to confirm the importance of the immune system in clearing rotavirus infection we infected rag 2 knockout mice with murine rotaviruses. both knockout p u p s and adults developed chronic infections. to determine if a t cell independent antibody could be mediating the clearance of rotavirus infection in nude mice we measured igg, igm and iga in faeces and serum of rotavirus infected mice. a low and transient igm response was found in serum but not faeces of infected nude mice but not in age matched naive nude mice. the protein specificity of these antibodies is being determined. to determine if a primary infection in n u d e mice would induce protection (memory) against a secondary infection, we challenged six week old mice that had been infected as pups with the murine ew strain of rotavirus with the murine cambridge strain of rotavirus. the previously infected mice, as well as naive nude mice, both shed very low but equal levels of rotavirus in the stools. interestingly adult naive heterozygous littermates shed more virus than the nudes. the factors responsible for this relative resistance of adult nude mice to rotavirus infection as well as the mechanism for viral clearence in nude mice will be discussed. symp. 1990, 121:149-160) . the great majority of the poxvirus encoded proteins actively mediating the evasion of host defense are secretory proteins termed "virokines". viruses produce proteins which can either compete with or antagonizz molecules critically involved in generating immune responses. the vaccinia virus complement control protein (vcp) was the first virokine to be reported that has structural similarity to humadmouse complement control proteins, with the greatest similarity to the human complement 4b binding protein (bc4-bp) and the first protein to have a postulated role in the viral evasion of host defense (kotwal and moss, nature 1988, 355:176-179) . bioactive vcp, purified from serumfree medium has been shown to be more potent than the hc4bbp ( kotwal, am. biotech. lab., 1994) and has also been shown to bind to two important complement components, c3 and c4, and block the complement cascade at multiple sites in vitro (kotwal et al., science 1990, 2502327-830; mckenzie, kotwal et al., j. inf. dis. 1992 , 1661245-1250 . the importance of this protein was demonstrated in vivo using recombinant virus lacking an intact dna coding for vcp (isaacs, kotwal and moss, pnas 1992, 89:628-672) and further underscored by the smallpox virus genomic sequence analysis, indicating the presence of a highly conserved homolog of vcp ( massung et al., nature 1993,366:748-751) . in order to determine the precise effects of vcp at the site of infection, a mouse model has been developed. measurement of the specific swelling response and microscopic examination of the histological changes in balb/c and dbn2 mice has revealed that vcp is capable of regulating inflammatory response in vivo (jayaraman and kotwal, 1993 mol. immunol. 30:20) . in order to ensure that the possiblity of the unrelated mouse strains sharing identical h-2 haplotypes but differing at numerous other loci was not overlooked, we performed similar experiments in congenic strains. well characterized c5deficient mice injected with poxviruses in comparison to normal mice, produce a significant inflammatory response, accompanied with severe ulceration that persists for several days. the data suggests that the host complement response is a critical factor in egulating poxvirus pathogenesis. previous studies have suggested that immune response was one of the major factors that contributed to the host resistantlsusceptible mechanism to sendai virus infection (brownstein, 1981) . various immune regulatory molecules were investigated in inbred susceptible 129/j (129) mice and resistant c57bl/6j (b6) mice which share the same mhc haplotype. when they were given 200 eid, of sendai virus intranasally, 129 mice showed histologically diffuse interstitial pneumonia compared to the local moderate inflammation in 86 mice. virus was eliminated from the infected lung 3 days earlier in b6 mice than in 129 mice. the cytokine levels and cytokine-producing cells were investigated in the acute pneumonic lung. also the recall cytokine production in the draining mediastinal lymph node (mln) were studied. the maximal numbers of il-2, ifn-y, il-10, and il-4 producing cells were comparable for each strains of mice, while more il-6 producing cells were present in the lung of 129 mice. however, the numbers of these cytokine producing cells peaked in 129 mice 3 days later than in b6 mice. analysis of soluble cytokines in bronchoalveolar lavage fluid showed that at least two-fold higher levels of ifn-y, il-10, and il-6 were present in 129 mice than in 86 mice. recall cytokine production in the draining mln showed only the presence of il-2, ifn-y, il-10, and il-6, while no il-4 was detected. generally, higher levels of these cytokines were detected throughout the whole infection process in 129 mice. therefore, the susceptibility of 129 mice to sendai virus is not due to insufficient cytokine production in these two anatomical sites, yet heavier and prolonged virus burden in susceptible mice may drive the more rigorous and protracted induction of immune regulatory molecules. cytokines play a decisive role in determining the type of immune response elicited by an antigen, both in driving th cell differentiation along thl or th2 pathway and, ultimately, the effector and regulatory activity of these subsets. we have studied the role of cytokines in virus infections by constructing recombinant virus vectors encoding cytokine genes. during replication in mice, these vectors produce the encoded factor which is secreted from the infected cells with profound immunological consequences. cytokines studied todate fall into two categories; either altering viral pathogenesis or selectively stimulating specific immune responses. the expression of il-2, ifn-y, tnf-a or il-7 in recombinant vaccinia virus (rvv) dramatically alters pathogenicity, such that immunodeficient mice resolve the normally lethal infection. on the other hand, expression of il-4 by rvv suppresses the induction of cytotoxic t cells (ctl) inhibiting the clearance of virus which leads to an increase in viral pathogenicity. the expression of il-4 by rvv was shown to inhibit the host il-2 response required for the development of ctl's. no production was also significantly suppressed by il-4. rvvs expressing il-5 or il-6, although not affecting pathogenicity, preferentially stimulates iga reactivity to coexpressed antigens. encoding cytokines in recombinant viruses has clearly demonstrated the important role of cytokines as anti-viral effector molecules and in regulating appropriate immune responses. in mice infected with lymphocytic choriomeningitis virus (lcmv), interleukin-i2 (il-12) doses of >i00 ngld induce profound immunotoxicities characterized as almost complete inhibition of virus-induced cd8+ t cell expansion and ctl activation, and up to 2 log increases in viral replication [orange, wolf, and biron, j. immunol. 152:1253, 19941 . serum tumor necrosis factor (tnf) is also observed under these conditions. the studies reported here further characterize the expression and function of tnf in this context. northern blot and in sifu hybridization analyses demonstrated that il-12 induced tnf-cx expression and that lcmv infection synergized with il-12 for this induction. administration of antibodies neutralizing tnf reversed the il-12-induced immunotoxicities in lcmv-infected mice and restored anti-viral defenses. the tnf-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and cd8+ t cells isolated from lcmv-infected mice were more sensitive to tnfmediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. additional physiological changes were observed in il-12-treated uninfected mice and were dramatically elevated in il-12-treated virus-infected mice, including: 1) decreases in body weights; 2) elevation of circulating glucocorticoid levels: and 3) decreases in thymic mass. these changes were also reversed by anti-tnf. the results delineate a unique tnf-mediated immunotoxicity and have significant implications concerning detrimental consequences of in vivo tnf andlor il-12 for protective anti-viral responses. lactate dehydrogenase-elevating virus (ldv), a naturally occurring virus, causes a persistent infection in mice and presents an ideal model for the study of immune modulation during acute and persistent virus infections. within a few days following infection with ldv there is a pronounced polyclonal activation of b cells followed by the suppression of primary b cell responses to t-dependent ag. we investigated the effect of acute and persistent ldv infection on the development of a memory b cell response to the model protein antigen, horse cytochrome c (cyt), by employing a modification of the splenic fragment assay. about a 50% decrease in the frequency of responding agspecific memory b cells was observed in balb/c mice infected with ldv, whether the mice were immunized with cyt at the time of ldv infection or three weeks later. this may be due in part to a defect in t cell help, since in cultures of normal memory b cells and t cells derived from ldv acutelyinfected mice the frequency of responding b cells was also decreased two-fold. in situ hybridization using a cdna probe specific for ldv revealed two patterns of ldv rna within the spleen. twenty-four hr p.i. ldv rna was located within the marginal zone, surrounding each follicle. this pattern is consistent with permissive macrophages. during persistence viral rna could no longer be detected in the marginal zone, but was located within the follicles. the absence of ldv-permissive cells within the follicular region suggests that the source of ldv rna is not due to ongoing viral replication. one possibility is that circulating virus is trapped by a specific cell population within the follicle. the effect of virus trapping within the spleen provides a mechanism by which ldv and other viruses can modulate immune cell function during persistent infections. ifn-y can be produced by activated nk cells. this cytokine enhances immune responses by augmenting macrophage antigen presentation. viral infection induces ifn-dp and nk cell activation. changes in splenic architecture, cell trafficking, and cytokine expression were examined during viral infections of c57bu6 mice. at times coinciding with ifn-dp production and nk cell activation, there was a redistribution of nucleated cells from red pulp to white pulp regions in spleens isolated from mice infected with either lymphocytic choriomeningitis virus (lcmv) or murine cytomegalovirus (mcmv). cell transfer experiments with dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate-or pkh26-gl-labeled bone marrow cells isolated from normal mice demonstrated an infection-induced accumulation of non-t/non-b cell populations along recipient splenic marginal zones. flow cytometric analyses demonstrated that approximately 10% of the transferred bone marrow cells accumulated in spleens after 20 hrs and 30% of these expressed the nk cell marker, nkl.l+. in vivo antibody treatment procedures, to eliminate cell subsets in donor mice, demonstrated that the cells localizing at the marginal zone were derived from agmi+ and n k l . l + populations. a small subpopulation of marginal zone cells in infected mice were shown to be expressing high levels of ifn-7 mrna by in sifu hybridization. treatment with anti-agmi or anti-nk1 .i antibodies eliminated both endogenous nk cells and the ifn-y mrna positive cells. these data demonstrate that newly derived nk cells accumulate along marginal zones. the results also suggest that this trafficking pattern may act to enhance immune responses by facilitating delivery of cytokines to specialized antigen presenting cells. david segal, janet ruby, alistair ramsay and ian ramshaw. depamnent of cell biology, john curtin school of medical research, po box 334, canberra, act, 2601 australia cytokine expression has been shown to correlate with protective or ineffective immune responses in a number of disease models. recently there has been the suggestion that immunity to some retroviruses is associated with the production of ceaain patterns of cytokines. to explore this further we have have used rauscher murine leukemia virus (r-mulv) infection of c57bu6 (resistant) and balb/c (susceptible) mice to elucidate the role of cytokines immunity to retroviruses. initially the in viho proliferation of spleen and lymph node cells from infected mice was examined. in response to stimulation with immohilised anti-cd3 antibodies the proliferation of spleen hut not lymph node cells from infected mice. was found to he rapidly suppressed. suceptible balb/c mice exhibited a much greater suppression than resistant c57bu6 mice. the cause of this suppression is under investigation however, the immunosuppressive molecules nitric oxide and prostaglandins are not involved. in vitro cytokine production by spleen and lymph node cells from r-mulv infected mice was determined. in response to stimulation with immobilised anti-cd3 antibodies, spleen cells from infected balb/c mice produced diminishing amounts of ifn-7 and il-2. in contrast spleen cells from infected c57bu6 mice produced ifn-y and l-2 to levels that were only slightly less than uninfected controls. a-6 production by spleen cells from infected mice of both strains was at levels higher uninfected controls. anti-cd3 stimulated lymph node cells from infected mice produced elevated ifn-1 suggesting that suppressed cytokine production is spleen specific. expression of cytokine genes in vivo is currently being investigated using rt-pcr to detect cytokine mrna in the spleens of infected mice. we have previously shown that primary resting murine b lymphocytes are non-permissive for vesicular stomatitis virus (vsv), however, a productive infection can be induced when infected b cells are activated with anti-immunoglobulin (a-lg) plus il-4 or lipopolysaccharide (lps). we posit vsv in unactivated primary b cells provides a paradigm of persistently infected lymphocytes and activation dependent recall of an active infection. analysis of the behavior of virus in unstimulated b cells during long term culture and the requirements for subsequent induction of productive infection has been limited by the poor survival of primary cells in culture. we circumvented this limitation by using highly purified small b cells from mice transgenic for the bcl-2 proto-oncogene, expression markedly extends in vitro survival of unstimulated primary b cells. overexpression of bcl-2 does not alter b cell infection or induction of a productive infection by activators during acute infection. infection does not effect b cell survival in culture. unstimulated virus infected b cells produce primary viral mrnas but not viral proteins or infectious particles (pfu) during culture. persistently infected b cells stimulated with a-lg plus il-4 produced a fully productive vsv infection at all times analyzed, up to 3 weeks post infection. in contrast, vsv production in persistently infected b cells activated with lps markedly declined relative to acutely infected activated cells (50-1 00 fold by week 1 and 1,000 fold by week 2). cells were not completely refractory to lps activation as vsv protein was produced. the selective lps deficiency is unique to persistently infected cells as uninfected cultured b cells proliferate and differentiate to produce antibody upon lps activation. these data show that a persistent infection may selectively alter the host cell response to previously productive activators which may as a consequence interfere with immune regulation. rsv-g glycoprotein specific t cells preferentially secrete il-5 and predispose to pulmonary eosinophillia., anon srikiatkhachorn. and thomas j. braciale, the beirne b. carter center for immunology research and the departments of microbiology, pathology, and pediatrics, university of virginia health sciences center, charlottesville, va 22908 we studied the immune responses to two different glycoproteins of respiratory syncytial virus (rsv) in a murine model. balb/c mice were immunized with recombinant vaccinia virus expressing either rsv-fusion glycoprotein ( vac-f), attachment glycoprotein (vac-g) or 8-galactosidase (as a control). these mice were given rsv intranasally three weeks after priming and then sacrificed 5 or 14 days later. spleens and bronchial lymph nodes were harvested for in vitro culture and lungs were harvested for histologic studies . we found that bulk cultures obtained from both vac-f and vac-g immunized animals secreted both thl and th2 type cytokines when stimulated with rsv infected spleen cells . however, the levels of 11-5 and 1fn-y were higher in bulk cultures derived from vac-g primed animals while the levels of il-2 were higher in the bulk culture from vac-f primed animals. the il-4 and il-5 production was relatively short lived since spleen cells and bronchial lymph node cells obtaind form mice sacrificed 14 days after intranasal inoculation produced much lower levels of il-4 and 11-5 while the levels of il-2 and ifn-y production were comparable to bulk cultures obtained from mice at the peak of infection. there was little inflammatory response in the lungs obtained from mice immunized with the control vaccinia. in contrast , lungs from mice immunized with vac-f or vac-g showed significant infiltration of inflammatory cells. there was a striking infiltration of eosinophils in the lungs from mice primed with vac-g. these eosinophils could be detected aroud major bronchi and blood vessels, as well as, in some cases, in lung parenchyma. this study suggests that the immune responses to different viral glycoproteins may be distinct and may play important roles in viral pathogenesis. during infection of normal mice with lymphocytic choriomeningitis virus (lcmv), nk cell responses peak on day 3 and subside as cd8+ t cell responses are activated at day 7 post-infection. in contrast, 02m-/-mice, lacking cd8+ t cells, have dramatically elevated nk cell responses on day 7 postinfection. the 02m-/-response is evidenced by increased nk cell activity, as well as up to 5-fold increases in blast and total nki.i+cd3-cell numbers. nk cell responses in normal mice are cyclosporin a (csa)-resistant and interleukin (il)-2independent, whereas day 7 nk cell responses in 02m-/-mice are csa-sensitive and il-2-dependent. to investigate the role of additional cytokines in regulating cellular responses during acute viral infections, production and function of il-4 and transforming growth factor4 (tgf-0) were examined. induction of il-4 mrna, at late times post-infection of normal mice, was shown by in situ hybridization of t cell-enriched splenic leukocytes and polymerase chain reaction (pcr) amplification of cdna from rna. ellsas of media cor.aitioned with cells isolated on days 0, 3, 5, 7, 9, and 14 post-infection demonstrated delayed induction of il-4 protein as compared to ctl activation. tgf-0, evaluated in biological and elisa assays, was induced maximally at days 7 to 9 post-infection. the kinetics of tgf-0 production by cells from infected 82m-/mice was similar to that of normal mice. however, cells from 02m-/-mice produced il-4 at early but not at late times postinfection. together, these results suggest that either il-4 is a critical cytokine for shutting off nk cells during normal responses to viral infection, or that the 02m-l context modulates responsiveness of nk cell subsets to other late cytokines. studies are in progress to distinguish between these two possible mechanisms. the induction of fever in response to infection is an important host defense mechanism that enhances aspects of the immune response and restricts the replication of some microorganism. vaccinia virus, a member of the poxvirus family, is a complex cytoplasmic dna virus that encodes a variety of proteins that interfere with host immune functions, such as complement regulatory factors and soluble receptors for il-lp, tnf and ifny. here we show that expression of the vaccinia virus il-1p receptor (vil-lpr) in the w r strain prevents the febrile response and reduces the severity of infection in intranasally inoculated mice. fever was recorded on days 1-6 after infection of mice with a vil-lpr deletion mutant, but not in animals infected with wild type wr or a virus revertant. these studies were extended to other virus strains that were used as smallpox vaccines, and expression of the vil-lpr was consistently found to prevent the onset of fever. vaccinia virus induced a severe hypothermia after 6 days in infected mice that was independent on vil-lpr expression and correlated with virus replication in the brain, the organ that controls body temperature. these results represent the first example of a virus mechanism to inhibit the host febrile response and suggest a central role for soluble il-lp in the induction of fever in poxvirus infections. measles virus (mv) infection can depress cell-mediated immune responses for months following clinical disease. mv is known to infect the thymus during human illness and this may contribute to immune suppression. we have used the scid-hu m o w with co-implants of human fetal thymus and liver to determine the effect of virulent and avirulent strains of mv on the thymus. scid-hu mice were. infected by direct inoculation of the graft with 103 pfu of either a wild type strain of mv(chicago-1,chi-1) or an attenu-ated strain (moraten, mor) and sacrificed at intervals over 28 days. peak viral titers, as judged by plaque assay on vero cells, were reached by chi-1 on d4 (105.7 pfu/ third of implant), and moron d21 (103.2 pfu/ third of implant). hematoxylideosin stained sections of chi-1-infected thymuses showed marked distortion of the cortex and medulla by d4 with thymocyte poilolosis and decreased cellularity. by d14, these. implants were mostly devoid of normal thymocytes. mor-infected thymuses showed relatively preserved architecture and cellularity. suspensions of the cells from implants stained with mabs to cd3,cd4 and cd8 were analyzed by flow cytomehy. there were significant decreases in the cd4+cd8+ cell pop-ulation by d10 with complete loss of all such cells by d28 with chi-1, and only modest reductions with mor. immune fluorescence staining of sections with a mv mab to hemagluttinin(ha) and abs for either human cytokera-tins(ael/ae3) or cd15 co-localized mv predominantly to epithelial and monocytic cells. additionally, mv antigen was present diffusely by d4 in both cortex and medulla in chi-i infection whereas mor-infected implants had only patchy distribution by d21. only rare cells stained both with mv ha and cd2 or cd4. mv ha was not expressed over background on any cd4+ cells judged by facs. we conclude that mv replicates in the scid-hu thymic implant primarily in epithelial and monocytic cells, and that the attenuated virus reproduces more slowly and with less cellular disruption. little mv ha could be demonstrated in thymocytes, therefore the data suggest that significant infection of the thymic epithelial stroma disrupts the thymic microenvironment which normally supports and aids in selection of immature t cells. part of the long-term immune suppression seen in mv infection may be due to infection of the thymic epithelial stroma with subsequent loss of thymocytes. it is becoming increasingly evident that many poxviruses contain genes that enable the virus to evade the host's immune system. myxoma virus is a leporipoxvirus and is the causative agent of myxomatosis, a rapidly lethal disease in the european rabbit (oryctolagus cuniculus). one possible mechanism of immune evasion is virus-induced downregulation of cell-surface receptors important for an immune response. cell-surface levels of several receptors on a rabbit t cell lymphoma cell line (rl-5) were monitored by flow cytometry. following infection with myxoma virus, cellsurface levels of cd4 were found to drop dramatically. other cell surface antigens such as cd18, cd43, and cd45 were unaffected during infection with myxoma virus. further more, the downregulation of cd4 by myxoma virus could be inhibited by treating cells for an extended period of time with pma, suggesting that the downregulation was not simply a masking of the epitope via viral antigens. analysis of cd4 levels in the presence of cytosine arabinoside indicates that late gene expression is not necessary for the modulation. since the tyrosine specific protein kinase p56lck associates with the cytoplasmic domain of cd4 we have also examined the association of p56lck with cd4 as well as steady state levels of p56lck during viral infection. the modulation of surface cd4 has also been described in hiv infected t cells suggesting that the loss of cell-surface cd4 may be a common viral immune evasion tactic by lymphotrophic viruses. i n addition, stably-transfected cell l i n e s expressing e i t h e r u s 1 1 o r us2-6 gene products s i g n i f i c a n t l y reduced l e v e l s of mhc class i heavy chain. studies are i n progress t o f u r t h e r d e f i n e t h e mechanism by which t h e s e v i r a l gene products a l t e r immune recognition. cytotoxic t lymphocytes (ctl) may play a significant role in containing the spread of hiv in infected individuals. although hiv-infection is associated with immune suppression, a vigorous ctl response has been detected in infected adults. hiv can be transmitted from mother to child. one third of vertically infected children has a rapid evolution toward disease, with onset of aids before 18 months. the other two thirds remain asymptomatic for years. the bimodal course of disease evolution in hiv-infected children could be related to differences in the host immune control of viral replication. hiv-specific ctl response from fresh and in vitro activated pbmc of hiv-infected children was measured. the vast majority of infected chidren had detectable hiv-specific ctl, which where cds+cd8+. we previously showed that among children with a slow disease progression, fresh ctl were more frequent in the p2a(paucisymptomatic) group than in the pl(asymptomatic) and the p2b-f groups (symptomatic group). the cohort of children has now been followed during 4 years, and 46 children have been tested at least once. we found that ctl responses were less frequent in the children with a rapid disease progression than in the children with a slow disease progression at the same age. our data suggest that ctl response is an important factor in delaying disease evolution. we, as well as others. have proposed that sag function is critical to the ability of milk-borne m m n to infect mice. to determine whether this is the case, we created transgenic mice (hyb pro/cla) with a frameshift mutation int the sag gene. young hyb pro/cla mice (c 10 weeks of age) showed no deletion of their cognate vp14* t cells, unlike transgenic mice carrying a functional sag gene however, a slow, progressive loss was seen in the hyb prolcla mice as they aged, indicating that it was due to expression of wild type sag protein. thus, as the hyb pro/cla mice aged, there was production of virus that appeared to lose the cla mutation. the hyb pro/cla mice produced transgene rna in their lactating mammary gland and shed virus in their milk. their nontransgenic offspring of showed infection with transgene-encoded mmtv because they had the typical slow deletion of vp14+ t cells characteristic of c3h mmtv infection and because we detected transgene-derived m m n rna in their mammary glands. cloning and sequencing of the viral rna produced by the nontransgenic offspring of the hyb pro/cla mice showed that recombination between the mtv-1 endogenous viral rna and the transgene-encoded rna occurred, such that the frameshift introduced by the cia mutation was repaired. these results show that there is selection of infectious virus that contains a functional sag gene. thus, it appears that the only virus that is capable of being transmitted by the milk borne infection pathway is that which encodes a functional sag protein. hepatitis b virus (hbv) causes acute and chronic liver diseases and is closely associated with hepatocellular carcinoma. in order to understand the cellular immune response against hbv in chronic hbv infection, t cell proliferation, cytotoxicity and cytokine production were studied. we found that although the majority of asymptomatic hbsag carriers and patients of chronic hepatitis b (chb) had no proliferative response to hbsag, some individuals in both groups showed significant t cell proliferation against hbsag. in contrast, the proliferative t cell response to hbcag in asyrnpatomatic hbsag carriers was significantly stronger than that in patients of chb with acute exacerbation. in addition, the frequency of hbcag-reactive t cell precursors measured by limiting dilution assay was much higher in asymptomatic hbsag carriers than in patients of chb. therefore, t cell responses against hbsag and hbcag are regulated differently in chronic hbv infection. furthermore, we demonstrated hbsag-and hbcag-specific cytotoxic t lymphocyte (ctl) activity in asymptomatic hbsag carriers, using autologous hbsag-and hbcag-expressing lymphoblastoid cell lines (lcl) as target cells, respectively. the cloned ctl were able to produce ifn-y, tnf-a or gm-csf after stimulation. these findings demonstrate that t cell response to hbv is not completely suppressed in asymptomatic hbsag carriers. most of them have strong hbcag-specific response and some of them have hbsag-specific response. transcription and tax the human t-lymphotropic virus type i (htlv-i) promoter contains the structural features of a typical rna polymerase i1 (pol 11) template. the promoter contains a tata box 30 bp upstream of the transcription initiation site, binding sites for several pol i1 transcription factors, and long poly a+ rna is synthesized from the integrated htlv-i proviral dna in vivo. consistent with these characteristics, htlv-i transcription activity was reconstituted in v i m using tbp, tfiia, rtfiib, rtfiie, rtfiif, tfiih and pol 11. in hela whole cell extracts, however, the htlv-i ltr also contains an overlapping transcription unit (otu). htlv-i otu transcription is initiated at the same nucleotide site as the rna isolated from the htlv-i-infected cell line, mt-2, but was not inhibited by the presence of a-amanitin at concentrations which inhibited the adenovirus major late pol i1 promoter (6 pglml). htlv-i transcription was inhibited when higher concentrations of a-amanitin were used (60 pglml), in the range of a typical polymerase in (pol 111) promoter (va-i). purified tax, transactivates this promoter 5-to 10-fold in v i m . interestingly, basal and tax,-transactivated transcriptional activity of the htlv-i ltr could be reconstituted with the 0.5 m phosphocellulose fraction. these observations suggest that the htlv-i ltr contains overlapping tax,responsive promoters, a typical pol i1 promoter and a unique pol i11 promoter which requires a distinct set of transcription factors. tax, further in vifro transactivates a polymerase i1 template containing the 21 base pair repeats cloned upstream of the ovalbumin promoter and g-free cassette. tax,-transactivated transcription was concentration dependent and inhibited by low concentrations of a-amanitin. flaviviruses are arthropod-borne viruses whose route of infection is via the skin. they are mostly neurotropic and responsible for significant human morbidity and mortality. the classic cell-mediated immune response to a viral infection may be influenced by the ability of these viruses to modify expression of cell-surface molecules involved in the presentation of antigen to, and activation of, t cells. the skin langerhans cell is the prototypic nonlymphoid dendritic cell and as such is uniquely placed to participate in a response against epidermally-acquired viral infections. the migratory properties of these cells contribute to their role as initiators of t cell-mediated immune responses within the draining lymph node. we have previously shown infection of epidermal cells in vifro by the flavivirus west nile (wnv) results in an increase in mhc class i and i1 expression on the majority of epidermal cells and langerhans cells respectively. in this study a technique for infecting the epidermis with wnv in vivo was developed. tme-dependent increases in the surface expression of a number of antigens which are involved either directly or in a co-stimulatory capacity in initiating a cell-mediated immune response, were detected on both the majority of epidermal cells and the langerhans cell population using flow cytometry. these increases were detectable as early as 16 hours after infection. a significant decrease in the percentage of langerhans cells remaining in the epidermis was observed within 48 hours of infection. the phenotypic changes observed in vivo are analogous to those described following in vifro culture of langerhans cells. these results, together with the reduction in langerhans cell numbers, may represent the in situ maturation and concomitant migration of these. cells as a consequence of virus-induced cytokines within the skin microenvironment. which cause a wide variety of illnesses with high morbidity and mortality in humans throughout the world. their high genomic stability argues for a survival strategy related more to interaction with the vertebrate host immune response, than a dependence on viral genetic mutation. our previous work has shown that west nile virus (wnv) infection of many cell types directly induces functional increases in class i and 11 mhc expression. we report here that wnv infection of human embryonic fibroblasts (hef) results in the increased expression of cd54 by two distinct mechanisms. an early, direct cytokine-independent mechanism operates within 2 h of virus infection, while an indirect mechanism, regulated by type 1 interferon (ifn), operates within 24 h of virus infection. cd54 expression increased by 4-5 fold within 2h of wnv infection on hef, and by 6-7-fold within 24h. wnv-inactivated, conditioned supematants removed from infected hef cultures after 4 h incubation did not alter cd54 expression on unqimulated hef. whereas conditioned supernatants from 24 h-infected cultures increased cd54 expression by about 1.5-2-fold after incubation for 24 h, but not after 4 h, similar to cd54 induction by 200ulml of ifn-p. increased cd54 expression on hef by wnv was also cell-cycle dependent. cd54 increased only in quiescent, contact-inhibited infected hef in go phase. in contrast, induction of cd54 by types 1 and 2 ifn was not cell-cycle dependent. other viruses, including double-stranded dna viruses, vaccinia, and adenovirus 2 and 5, and the single, positive-stranded rna alphavirus, semiliki forest virus, did not induce cd54 expression on hef after 24 h. another alphavirus, ross river, was able to induce cd54 but only by the indirect mechanism of type 1 ifn-dependent release. poly i.c, also, increased cd54 expression to the same extent as ifn-p after 24 h, making it unlikely that the early increase was due to a nonspecific viral effect. the closely related flavivirus, kunjin, induced increased cd54 expression in a manner similar to wnv. the ability of flavivhses to induce increased cd54 expression directly within a few hours of infection may be an important virus-host survival strategy promoting cell-cell adhesion and hence possible further viral infectiodreplication. recognition of viral peptides presented on the cell surface in association with class i mhc molecules leads to lysis by cytotoxic t cells (ctl) and forms an important part of the immune response to hiv infection. hiv virus has a high mutation rate and variation in the region of the viral epitope may allow evasion of this immune response. variation could theoretically affect processing of the antigen, binding of the epitope to the hla molecule or recognition of the presented epitope on the cell surface. we have studied proviral sequence variation in gag and ctl responses in a number of hla b8 patients infected with hiv. amino acid substitutions, such as a lysine to arginine change at position 3 of the pi7 gag nonamer cckkkyklk, lead to loss of recognition of the peptide by ctl from the patient whose provirus contained this sequence. these variant peptides bind to hla 68 with comparable affinity to the index peptide suggesting that this loss of recognition is likely to be caused by changes in the interaction between the hla-peptide complex and the t cell receptor. other changes, such as lysine to arginine or glutamine at position 7, not only cause loss of recognition, but also lead to inhibition of lysis of targets bearing the index peptide. thus it appears that in addition to loss of recognition by cytotoxic t cells, naturally occurring epitope variants may act as "antagonists", as has been demonstrated in mhc class ii systems. antagonism may be an important mechanism allowing immune escape by the hiv virus. genes. subsequent complex formation between peptide, class i and p2microglobulin in the er results in stable cell surface expression of the trimeric mhc-1 molecule. in previous studies we showed that in hpv-16 positive cervical carcinomas there was a loss of mhc-1 protein expression, which correlated at the single cell level with loss of tap protein. in this study we investigated whether loss of tap and mhc-1 is mediated by an hpv-16 encoded protein. human keratinocytes were transfected withvarious hpv-16 constructs including pat16, the full length genome, pat16esx the full length genome with a premature stop codon in e5, puc.et16, the e6 and e7 oncogenes only, and pkve5, expressing e5 from mouse moloney ltr the different constructs were transfected into primary keratinocytes, cloned cells grown in medium supplemented with and without y-interferon ( y -a r ) for 48 hours. cells were harvested and total rna and protein harvested for northern and western blots respectively. western blots showed very low steady state levels of tap-1 and mhc-1 heavy chains in the cells with pat16 as well as those containing es alone, which was marginally increased by y-lfn. in contrast, primary keratinocytes, pat16esx and puc.et16 lines showed comparable tap-1 and mhc-1 protein levels, which increased a & y-ifn treatment. northem blots showed no differences in the amounts of tap-1 and mhc-1 mrna between the different cell lines. the data indicate that expression ofhf'v-16 e5 leads to post-transcriptional loss of mhc-1, presumably by interfering with tap. to map and characterize functional differences between e1a of ad5 and adl2, we previously constructed a series of hybrid ad5/12 e1a genes and used them with ad12 e1b to transform primary hooded lister rat kidney cells. at least two regions within the first exon of ad12 e1a were identified which influenced tumorigenicity. this study further examines the role of these regions in tumorigenicity by analyzing their affect on cell surface mhc class i expression and sensitivity to class i-restricted cd8+ as well as to non-class irestricted nks. the bcrfl open reading frame of epstein-barr virus exhibits remarkable sequence homology with the coding sequences of interleukin-10 from a variety of organisms. many of the numerous immunological properties ascribed to interleukin-10 are shared by the product of bcrfl and this has led to it being termed viral interleukin-10. in order to investigate the activity of viral interleukin-i0 (vil-10) and its interactions with the human interleukin-10 receptor we have expressed the protein in a bacterial and the eukaryotic cos-7 expression systems. the bacterially expressed vil-i0 was partially purified and used to set up two assays to measure i l l o activity: i)the increase in igm secretion from an ebv transformed b cell line -mt4.l and ii)the downregulation of class ii hla expression on the human monocytic cell line thp-1. a series of deletion mutants (both n-and c-terminal as well as an internal deletion to remove a putative heparin binding domain) were constructed to identify possible domains within the vil-10 protein that interact with the hil-10 receptor and confer its biological activity. a number of these mutants have been expressed in the cos-7 expression system and their structure and biological activity are currently being assessed. the identification of the domains within vil-10 that interact with the receptor or accessory proteins may aid in the understanding of the possible role of vil-i0 within the ebv life cycle and in the pathogenesis of the numerous diseases associated with the virus. generation. to further test the role of ctl in ad pathogenesis, viruses lacking the cll epitopes were tested when mutants that lack the immunodominate ctl epitope in eia where used, a second immun-ssive epitope in elb becorns the predominate target of clu. these findings arc important since human ad is currently being tested as a vector for gene therapy of cystic fibrosis. our data suggest that when consuucting ad vectors to be. used for gene therapy, one must retain either the 10.4k or 14.7k genes to decrease pathology and that meting the genes that encode the antigens that a n recognized by clu does not prevent the generation of ad specific clu. the interferons (ifns) a n ? a family of cytokines whose functions include the protection of cells against viral infection. type i ifns include the 15 ifna subtypes and ifnp that compete for binding to the same cell surface receptor, while type ii ifn (ifny) binds to a different receptor. the orthopoxviruses, of which vaccinia virus (vv) is the prototypic member, have developed a number of anti-ifn strategies. the vv e3l protein competitively binds dsrna and prevents the activation of ifninduced and dsrna-activated protein kinase (pkr), while the vv k3l protein shows sequence similarity to the eukaryotic initiation factor 2a (eif2a) that is phosphorylated and inactivated by pkr. the k3l protein competitively binds the kinase and blocks host eif2a phosphorylation and hence ifn-induced inhibition of host protein synthesis. onhopoxviruses also suppress cytokine action by expressing soluble cytokine receptors that bind and sequester the ligand; to date soluble receptors for interleukin-18, tumour necrosis factor and ifny have been described. supernatants from vv-infected cells were found to contain a soluble inhibitor of type i ifn that was conserved in most of the orthopoxviruses tested. the inhibitor was produced early in infection and did not inhibit ifny. the ifna/p inhibitor was mapped and the gene expressed from recombinant baculovirus. the inhibitor blocked the binding of 125i-ifna to u937 cells and binding of 125i-ifna to supernatants from baculovirus and vv-infected cells demonstrated that the inhibitor functioned as a soluble receptor for 1fnc1fp. direct binding of 1251-ifna to vv wr supernatants revealed that the soluble ifna/p receptor had a high affinity for type i ifn. deletion of the gene from the vv genome and ligand blotting of the soluble receptor demonstrated that ifn binding was encoded by a single protein. competitive binding curves using ifna from other species revealed that the poxvirus soluble ifndp receptor bound human and bovine ifn with high affinity but murine ifn with relatively low affmity. interestingly, the soluble ifncrip receptor is highly conserved in variola virus. given the importance of ifn in antiviral defense it is likely that the soluble ifndp receptor plays an important role in the virulence of the orthopoxviruses. endogenous processing of a viral glycoprotein for presentation t o cd4+ t cells has defined a previously under-investigated pathway in antigen processing and presentation. it may be important not only for pathogens, but also for self-proteins, and thus may be involved in self-tolerance. we have been characterizing the processing o f the er-restricted gpt glycoprotein of vesicular stomatitis virus (vsv) biochemically and enzymatically, by cellular localization using confocal immunofluorescence, cellular fractionation, and by t cell recognition assays. by flow cytometry, gpt is undetected on the plasma membrane; in contrast, the wild type protein (g) is readily found following infection of a20 cells with a vaccinia virus vector, leading t o endogenous synthesis. the gpt can be found exclusively in the er compartment using co-localization with markers for er (signal peptide binding protein, calnexin), and not in the golgi compartment (a-mannosidase 11, wheat germ agglutinin), endosome, lysosome, or surface plasma membrane. this is consistent with the characteristics o f the localization of the proteases which appear to be responsible for its degradation. work is in progress to localize the site of peptide binding to mhc heterodimers. supported by nih grant a118083 t o csr. presentation of an out-of-frame class i restricted epitope. t.n.j.bullock and l.c.eisenlohr, department of immunology, thomas jefferson university, philadelphia, pa 19107. antigen presentation by class i mhc molecules is thought to require the degradation of fully formed proteins in the cytosol. this degradative process supplies oligopeptide epitopes for transport into the endoplasmic reticulum (er) where they can interact with and stabilize class i molecules. stable class i molecules, associated with p2-microglobulin, can then proceed to the cell surface where they present the epitopes to t cell receptors. the generally accepted model for protein translation, the scanning hypothesis proposed by ko&, is thought to describe the traditional method of translation for the majority of proteins. we wished to test the hypothesis that any internal methionine that is in good translation initiation context can be a source of short peptides, which may then be processed into class i epitopes. nucleoprotein gene (np), the target of the ctl response of several inbred mouse strains. np contains three class i restricted epitopes at amino acids 50-57 (h2-kk), 147-155 (h2-kd) and 366-374 (h2-db). the frameshift was introduced 26 amino acids upstream of the h2-kd epitope. the mutated genes were then recombined with vaccinia virus and tested for presentation using ctl restricted to each of the epito s described above. we found that, whilst presentation of the h2-i@ epitope was unaffected by the frame shift, the epitope proximal to the frameshift (h2-kd) was no longer presented to appro riately restricted ctl. however, presentation of the distal h2-dg epitope was retained. therefore we have shown, using a viral protein and a viral expression system, that out-of-frame epitopes can be processed and presented to ctl. work is ongoing to c o n f m that internal methionines are capable of providing a platform for the initiation of translation for in-frame and out-of-frame epitopes. we have created a frameshift mutation in the influenza pr8 the fine specificity of t cell recognition of peptide analogues of the influenza nucleoprotein epitope np 383-391 srywairtr was studied using hla b27-restricted influenza-specific cytotoxic t cell (ctl) clones, of defined t cell receptor (tcr) usage, derived from unrelated individuals following natural infection. synthetic analogue peptides were synthesized containing single amino acid substitutions, and tested both for binding to hla b'2705 in vitro, and for presentation to ctl clones by hla 827positive targets. even conservative amino acid substitutions of the peptide residues p 4 , 7, and 8 profoundly influenced ctl recognition, without affecting binding to hla 8'2705. these amino acid side chains are thus probably directly contacted by the tcr. ctl clones which used the tcr v a l 4 gene segment (but not those using tcr va12) were also sensitive to p1 substitutions, suggesting that the tcr alpha chain of these clones lies over the n terminus of bound peptide, and that the "footprint" of certain tcrs can span all exposed residues of a peptide bound to mhc class 1. these results, taken together with previous structural and functional data, suggest that, for nonarner peptides bound to hla 827, p i , p4 and p8 are "flag" residues with tcr accessible side chains. the e3/19k protein of human adenovirus type 2 (ad2) is a resident transmembrane glycoprotein of the endoplasmic reticulum. its capacity to associate with class i histocompatibility (mhc) antigens abrogates cell surface expression and the antigen presentation function of mhc antigens. at present, it is unclear exactly which structure of the e3/19k protein mediates binding to mhc molecules. apart from a stretch of approximately 20 conserved amino acids in front of the transmembrane segment, e3/19k molecules from different adenovirus subgroups (b and c) share little homology. remarkably, the majority of cysteines is conserved. in this report, we examined the importance of cysteine residues (cys) for structure and function of the ad2 e3/19k protein. we show that e3/19k contains intramolecular disulfide bonds. by using sitedirected rnutagenesis, individual cysteines were substituted by serines and alanines, and mutant proteins were stably expressed in 293 cells. based on the differential binding of monoclonal antibody tw1.3 and cyanogen bromide cleavage experiments, a structural model of e3/19k is proposed, in which cys 11 and cys 28 as well as cys 22 and cys 83 are linked by disulfide bonds. both disulfide bonds (all four cysteines) are absolutely critical for the interaction with human mhc antigens. this was demonstrated by three criteria: loss of e3/19k coprecipitation, lack of transport inhibition and normal cell surface expression of mhc molecules in cells expressing mutant e3/19k molecules. mutation of the three other cysteines at position 101, 109 and 122 had no effect. this indicates that a conformational determinant based on two disulfide bonds is crucial for the function of the e3/19k molecule, namely, to bind and to inhibit transport of mhc antigens. previous studies have suggested that several abundant cmv proteins are major immunogenic targets in seropositive adults. we are interested in defining the major viral protein targets of a cd8' ctl response, in order to derive a vaccine strategy for individuals who are unable to mount immune responses which are lymphokinedependent because of immunosuppression. hla-typed and cmv-pgsitive normal volunteers who have hla-a alleles that represent -75% of the u.s. population are being tested to determine which of 5 abundant cmv proteins they recognize by a cd8' ctl response: p28, p65, p150, ie, and gb. t cell lines will be derived in order to unambiguously determine the hla restriction of the cd8' ctl response to each of these proteins. proteins which are recognized by the most hla diverse population will be further characterized in terms of mapping of class 1 epitopes through the use of t cell clones derived from the polyclonal cell lines by limiting dilution. the defined epitopes will form the basis of a vaccine strategy to augment the memory responses of seropositive volunteers against cmv. these epitopes will be used to boost the ctl precursor frequency of bone marrow transplant donors as a means to transfer cellular immunity to immunosuppressed hematologic transplant recipients. an alternative strategy is to immunize seropositive individuals with recombinant viral proteins as a means to boost immunologic memory. we are pursuing that strategy in a transgenic murine model of hla-a2.1 developed by dr. l. sherman (scripps institute, la jolla). we are vaccinating the transgenic mice with two well defined cmv proteins, p65 and gb together with either of two lipid-based adjuvants, commercially available d0tapm (bcehringer-mannheim) or mf5gth (chiron, emeryville, ca). our preliminary studies with hsv-2 gb demonstrate that both adjuvants are effective at eliciting murine class i restricted responses against the protein. current studies are evaluating the recognition properties of the adjuvant-cmv protein complexes by hwa2 as a restriction element in the transgenic model. the ctl response to sendai virus in c57by6 mice is directed almost exclusively to a single h-2kb-restricted epitope derived from the virus nucleoprotein, npj24-332 (sev-9). analysis of 18 independent t cell hybridomas generated from c57by6 mice following primary sendai virus infection has shown that a very diverse repertoire of tcr is selected in response to this epitope. crystallographic analysis of sev-9 bound to kb has shown that the side chaiis of peptide residues phpi, gl484, a d s , and alaps protrude towards the solvent and are potentially available for recognition by the tcr notably, residues gi484 and a d 5 protrude prominently from the peptide binding site due to their l o c a l i o n on a bulge in the center of sev-9. to determine the importance of each of these residues for t cell recognition, we analyzed hybridoma responses to sev-9 analogs substituted at each of these four positions. preliminary data showed there generally appeared to be dominant recognition of glyp4 and asnm. however, individual hybridomas exhibited distinct patterns of fine specificity for residues phep1 and alaps. thus, individual hybridomas were dependent on one, both, or neither of these residues for recognition of sev-9. these data are consistent with a critical role for the gi94 and a d 5 in governing tcr-sev-9eb recognition and suggest a structural basis for the diversity of the tcr repertoire selected by this @tope. previous results from this laboratoty demonstrated that the dominant influenza a epitope recognized by hla42.1 restricted ctl from hla-a2.1 uansgenic mice was the m1 peptide epitope that is immunodominant in human ctl responses. however, analysis of a large number of ctl lines revealed a subset of influenza a/pr/8/34-specific murine ctl that recognized an hla-a2.1 restricted epitope distinct from m1. using recombinant vaccinia viruses encoding werent influenza gene segments, the epitope recognized by these ctl was shown to be derived from the a/pr/8 nsl protein. because these ctl did not recognize targets infected with the a/alaska/6/77 saain of influenza, candidate peptide epitopes were synthesized based on sequences that included an hla-a2.1 specific binding motif and that differed between a/pw and nalaska all of these ctl recognized a nonamer and a decamer peptide which contained a common 8 amino acid sequence and two distinct sets of bmding mtif residues. however, the n0name.r peptide was able to sensitize ctl for half maximal lysis at 80-2500 fold lower doses than either the octamer or decamer. the homologous peptide derived from nalaska nsl contained conservative amino acid changes at positions 4 and 8 and was not recognized at any tested concentration, although it bound with higher &ity to hla-a2.1 than the peptide from a/pw8. the a/pr/8 nsl nonamer epitope was also recognized by human influenza a specific ctl derived from two individuals. these results substantiate the general utility of hla class i aansgenic mice for the identification of human cn epitopes for other pathogens. furthemore, the recombinant dhfr was functional in the induction of gb epitope-specific ctl response upon immunization of c57bv6 mice. these results indicate that an viral epitope expressed in a cellular protein can be. efficiently processed, presented and recognized by epitope-specific ctl, and suggest that the cellular proteins can be used to express ctl epitopes for induction of cd8+ immune responses. virus-specific cytotoxic t lymphocytes (ctl.) were generated a day later at this site. to determine which apc was capable of stimulating virusspecific ctl precursors in the mln, b, t and dendritic cells from the mln of influenza-infkcted mice were separated and examined for the presence of virus. the predominant cell type which contained infectious virus was the dendritic cell. b and t cells from the mln contained little, ifany, virus. the apc capacity ofthese populations was tested by their ability to stimulate vir~~-~pecific t cell hybridomas. only dendritic cells from the mln of influenza-infected mice were able to stimulate virusspecific t cell hybridomas, althwgh all apc populations from both naive and influenza-infected mice were effective stimulators after in y h pulsing with the appropriate intluenza peptide. potential apc populations were also separated from the lung. v i s was detected in bronchioalveolar macrophages and dendritic cells but not b or t cells. both macrophages and dendritic cells isolated from intlum-infected lungs could stimulate virus-specific t cell hybridomas. the ability of the mln and lung apc populations to stimulate naive cd8' t cells and generate virus-specific ctl is currently being examined. virus infected cells present only a very limited number of peptides intracellularly processed from a viral protein to ctl even when many peptides hearing the mhc class i-restricted binding motif are present in the protein. infection of h-2b mice w i t h lymphqtic choriomeningitis virus (lcmv) induces a cd8+ ctl response directed against three wellcharacterized epitopes presented by h-2db molecules: "396-404 (fqpq-ngqfi), gp33-43 (kavynfatcgi) and gp276-286 (sgven-pggycl). the h-2db motif is characterized by a sequence of 9 to 11 a.a. with two anchor residues: asn at position 5 and hydrophobic (met, ile, leu) at the c-terminus. the lcmv np and gp proteins contain thirly-one other peptides exhibiting the db motif. however, no ctl response against one (or more) of these peptides has been characterized. peptide binding to mhc is a critical step in antigen presentation. the aim of this study was therefore to analyze the binding properties of the potential db lcmv peptides. the 34 lcmv peptides and 11 known db-selective peptides were synthesized and their mhc binding affinities measured in two db-specific binding assays. most of the lcmv peptides (28/34) did not bind to db. the other 6 (including the 3 epitopes) and all the known db peptides showed good affinity. comparison of the sequences (good vs. non binders) allowed the identification of auxilliary anchors required for high binding affinity or of negative elements hampering mhc binding. in addition to the main anchors, the positive and negative factors at secondary residues play a crucial role in governing peptidemhc interactions. knowledge of such factors might he of importance for the prediction of mhcrestricted ctl epitopes. etienne joly, andrea gonzalez, carol clarkson, jonathan c. howard and geoffrey w. butcher. laboratory of immunogenetics, department of immunology, the babraham institute, cambs cb2 4at, uk. tap transporters from rats can be divided into two allelic groups, depending on their capacity to provide the rt1.aa molecule with an appropriate level of suitable peptidesl. recent results suggest that this might correlate with the rt1.aa molecule requiring arginine-ended peptides (powis et al., manuscript submitted), which the tapb allele of the transporter is unable to translocate across the er membrane efficiently2~3. rt1.a alleles are naturally linked with the tapa or the tapb allelic group4. we have set out to characterise various alleles for the rt1.a molecule, and find that, for the majority of tapaassociated rt1.a molecules, 3 acidic residues line the c/e pocket, dictating arginine as c-terminal anchor residue for the bound peptides. on the other hand, in tapb-associated rt1.a molecules, one acidic residue at the most is found in the c/e pocket, which certainly results in a different anchor residue for the bound peptides. the selective pressure of viral infections must have driven this coevolution which affects dramatically the array of peptides presented to cytotoxic t lymphocytes. cytotoxic t lymphocyte responses in hiv infection can be impaired due to variation in the epitope regions of viral proteins such as gag. we show here an analysis of variant epitope peptides in three gag epitopes presented by hla b8. seventeen variant peptides were examined for their binding to hla b8; all but one bind at concentrations comparable to known epitopes. all except two could be seen by ctl clones grown from hla b8 positive hiv-1 infected patients and were therefore immunogenic. however, in one haemophiliac patient studied in detail, there was a failure to respond to some of the peptides that represented virus present as provirus in his peripheral blood. in one case his ctl had previously responded to the peptide. thus there was a selective failure of the ctlresponse to variant epitopes. this impaired reaction to new variants and failure to maintain responses to some epitopes late in hiv infection could contribute to the loss of immune control of the infection. pira, anna ferraris, daniele saverino, peifang sun and annalisa kunkl; dept. immunology, san martino hosp. univ. of genoa, 16132 genoa, italy. th epitopes present on viral proteins can be recognized by specific th cells if appropriately expressed by antigen presenting cells (apc) as a result of uptake and processing. since viral epitopes are not simply present in the context of viral proteins, but also in the context of whole viral particles, it is important to determine the role of the molecular and/or structural context on antigen uptake-processing-presentation. therefore we have generated panels of cd4+ human t cell lines and clones specific for different hiv antigens (gp120, p66, p24), in order to test their ability to respond to the same epitopes present within synthetic peptides, recombinant proteins or inactivated virions (provided by g. lewis, dept. microbiology, univ. maryland, baltimore). we could identify t cell lines and clones that were able to discriminate the molecular and structural context of the epitops. certain t cells, in fact, responded to peptides and proteins, but not to viral particles, whereas other t cells were also able to proliferate when challanged in vitro with autologous apc and viral particles. the data suggest that in the human th cell repertoire specific for viral antigens t cells exist that can discriminate the molecularstructural context of th epitopes. it will be interesting to ascertain whether t cells specific for epitopes that can only be recognized when provided in the context of a soluble molecule, but not of a viral particle, have any relevance in viva protection, or are a simple by-product of the cellular immune response. eric g. pamer, merceditas s. villanueva, section of infectious diseases, yale university school of medicine, new haven, ct 06520 listeria monocytogenes is a gram positive bacterium that infects macrophages and secretes proteins into host cell cytosol. the murein hydrolase p60 is secreted by l. monocytogenes and is required for complete bacterial septation. in the infected macrophage secreted p60 is processed by the host cell into the nonamer peptide p60 217-225 and is presented to cytotoxic t lymphocytes by the h-2kd mhc class i molecule. we have used strains of l. monocytogenes that secrete different amounts of p60 to show that the rate of p60 217-225 production is proportional to the amount of antigen secreted into the host cell cytosol. p60 is degraded in the host cell cytosol with a half life of 90 minutes. the appearance of p60 217-225 is coupled to the degradation of newly synthesized p60. we have determined the rate of intracellular p60 secretion and by accounting for the rate of p60 degradation we estimate that approximately 35 p60 molecules are degraded to produce one p60 217-225 epitope. this ratio is maintained over a range of intracellular antigen concentrations. our findings provide an estimate of the efficiency of antigen processing and demonstrate the remarkable capacity of the mhc class i antigen processing pathway to accommodate new epitopes. we have isolated and characterized three cytotoxic t lymphocyte (ctl) clones from the peripheral blood of two acute seroconversion patients and one patient in the first trimester of pregnancy. these clones were cd8+ and class i hlarestricted by the b7 molecule. all three clones recognized lllb and rf but not mn strains of hiv-1. using vaccinia vectors expressing truncated versions of the hiv-1 envelope, the clones were found to recognize an epitope within amino acids 287-364, but not including 312-328 of gp120. further mapping of the epitope with synthetic 20-mer peptides overlapping by 10, or 25-mers overlapping by 8, was unsuccessful. the sequence of the region of gp120 recognized by these clones was compared to the predicted hla-87 peptide binding motif and a possible matching region was found. using shorter peptides corresponding to this potential epitope recognition site, the minimum epitope recognized by the clones was determined to be the 10 aa sequence rpnnntrksi spanning amino acids we have further pursued a strategy to define a minimal cytotoxic epitope for a vaccine against cmv infection using t cell clones derived from individuals who have the mhc 835 gene (kind gifts of drs. riddell and greenberg, fred hutchinson cancer research center and dr. robert siliciano, johns hopkins university medical center). we tested by chromium release assay (cra) the recognition of a series of 835 allelic variants of ebv-lcl. by 835 restricted and cmv or hiv-specific t cell clones. several conclusions quickly became apparent. the previously described 8'3501 peptide epitope from pp65 was not able to prime the autologous 835 ebv-lcl for killing by the pp65-specific ctl, whereas a recombinant vaccinia virus expressing whole pp65 could cause the same cell line to be recognized and killed in the same experiment. in addition, an hiv gp41-specific cd8' ctl which has a defined minimal cytotoxic epitope will only recognize and kill a subset of 835 ebv-lcl. the two t cell clones will not recognize each other's autologous ebv-lcl. the resolution of this interesting phenomena comes from sequence analysis of the hla class i b genes from both ebv-lcl. ebv-lcl which contain the b'3502 allele are recognized and killed by the pp65-specific t cell clone, and cell lines carrying 8'3501 alleles are recognized by the hiv gp41-t cell clone. we conclude that the reported cmv pp65 b"3501 restricted epitope is not correct, since the ctl in question will only recognize 6'3502 alleles in combination with the correct pp65 epitope. fragments with or without a signal sequence sensitize rma-s/kd to a similar limited extent. this data i s consistent with an inefficient movement of peptides from the cytoplasm into the er by a tap independent mechanism and does not reveal a processing competent compartment within the secretory pathway. peptide transport by the transporter associated with antigen processing (tap) was studied using a microsome system as previously reported by heemels et. al.. in this system, a radiolabeled synthetic peptide which can be n-link glycosylated is used as the indicator peptide for the transport studies. the transport efficiency of synthetic peptides corresponding to antigenic peptides restricted to the murine kd molecule was measured by inhibition of labeled peptide transported into the microsomes. the transport efficiency of three kd epitopes in the type a influenza virus "147-155, ha204-212 and ha210-219 was found to be similar. an 11 amino acid peptide corresponding to ha204-214 which contains the 204-212 epitope was transported at a similar efficiency as the 9 amino acid minimum epitope. however, when the peptide sequence is further extended by one amino acid to residue 215, this peptide is poorly transported. these results suggest that the flanking region of an epitope can dramatically influence the transport of the epitope. when the transport kinetics of tap was studied using the microsome system, the vmax for transporting the indicator peptide (a variant of np epitope that has the sequence tynrtrali) was found at 260.8 fmolelminute (+/-30.5). the km for this peptide was found to be 231.9nm(+/-31.8). bypassing a block in antigen processing for class i-restricted cytotoxic t cell recognition. amy j. yellen-shaw and laurence c. eisenlohr. thomas jeferson universitv. hiladelphia, pa., 19107. previous work from our laboratory showed that processing of an influenza nucleoprotein (np) epitope (amino acids 147-155) expressed endogenously from a recombinant vaccinia virus "minigene" is severely impaired when a flanking sequence (the dipeptide threonine-glycine) is appended to the cterminus of the construct (147-158/r-). the inhibition of processing is overcome by placing the unprocessed peptide in the context of the fulllength np molecule, demonstrating that regions of a protein outside the epitope itself critically affect the ability of the proteolytic machinery to fragment the protein appropriately. to determine the requirements for bypassing the block in antigen processing, we have constructed an array of "minigene"-expressing vaccinia recombinants in which the unprocessed epitope is extended by varying lengths toward either the c-terminus or the n-terminus of the np molecule. our results show that while an extension of the c-terminus by only one amino acid restores processability, a much longer extension of the n-terminus (75 < n < 100 amino acids) will also allow the substrate to be processed. it is therefore clear that a full-length, properly folded molecule is not required for liberation of the blocked epitope, and that probably more than one mechanism can contribute to enhancement of substrate proteolysis. we hypothesize that the c-terminal extension allows recruitment of an endopeptidase versus exopeptidase ("trimming") activity which is capable of cleaving the difficult bond. we considered the possibility that the n-terminal extension rescues processing by recruitment of the ubiquitin-dependent degradation system. to address this possibility we replaced all available ubiquitination sites (lysine residues) in one of the rescued constructs (50-158/r-) to see if the construct would still be processed and presented. the six available lysine residues were changed to arginine using pcr-based mutagenesis. the resulting construct (termed 6r) was recombined into vaccinia virus and tested for presentation to np-specific ctl. the 6r construct was presented at a level equivalent to that seen with the wild-type 50-158/rconstruct. this result provides clear evidence that entry into the ubiquitindependent degradation pathway is not responsible for rescue of presentation in this system and more importantly, that ubiquitination is not required for processing of all large substrates. chia-chi ku, li-jung chien ,and chwan-chuen king, institute of epidemiology, national taiwan university, taipei, taiwan, r.o.c. dengue virus (den) can cause dengue fever (df) and dengue hemorrhagic fever (dhf) i dengue shock syndrome (dss) and den-2 was the most common serotype found in dhf outbreaks globally. current hypotheses suggested that dhf may be associated either with antibodydependent enhancement (ade) or with viral virulence. den can replicate predominantly in monocytedmacrophages (mim), but whether peripheral blood lymhocytes (pbls) are the target cells of den still remain controversial. in order to compare whether various clinically derived den-2 will interact with mim and lymphocytes in different manners, we used two isolates --plo46 strain (obtained from a df patient during taiwan 1981 outbreaks) and 16681 strain (isolated from a dhf patient in thailand by cdc, usa) to infect primary mim and lymphocytes as well as several types of cell lines. primary lymphocyte culture was nonadherent cells obtained after 24 hr adherence of pbmcs, whereas the primary mim culture was collected by depletion of lymphocytes using anti-cd3icdi9 mab and complement prior to adherence procedure and the purity of mim culture was checked by cd14 surface marker staining. supernatants (sn) of virus were harvested at various time points post infection after with several or without treatments. our prelimanary data showed that dhf-associated den-2 strain had higher viral yield in certain age of mim and a promonocytic cell line (hl-cz) than taiwan df-associated den2 strain. in addition, this dhf-den2 strain was more likely to infect the promonocytic (hl-cz) than well differentiated monocytic (ctv-1) and lymphocytic (h9) cell lines and also had higher peak yields than den-i virus in hl-cz cells. interestingly, dhf-den2 strain replicated much more efficiently in primary lymphocytes no matter these cells were activated with pha or not, whereas taiwan df-den2 strain virus was hardly detectable in sn of both activated and non-activated lymphocyte cultures. therefore we conclude that (1) different strains of dengue virus could orchestrate quite differently with immune cells, (2) different stage of mim differentiation might be an important permissive determinants for dengue virus infection and replication, and (3) den virus strain virulence -a more important factor than lymphocyte activation status -seemed to determine whether this strain would infect human pbls. further studies should be focused on searching for detaied mechanisms of virus and immune cell interactions. (2) when viral yields were enhanced early than day5 post infection, it provided tremendous opportunity to attack the immune system and finally may lead to severe disease. hiv-1 using recombinant immunoglobulin molecules, marie-claire gauduin, graham p. allaway, paul j. maddon, carlos f. barbas, dennis r. burton, and richard a. university school of medicine, new york. ny 10016. primary isolates of hiv-1 have been shown to be less sensitive to neutralization by immune sera, monoclonal antibodies and cd4-based molecules than t cell line-adapted strains of hiv-i. we studied two immunoglobulin molecules for ability to neutralize primary isolates of hiv-i. lgg12 is an immunoglobulin molecule created from a combinatorial phage expression library and reacts with the cd4 binding site (cd4-bs) on gp120. cd4-lgg2 is a recombinant molecule in which the variable domains of both heavy and light chains of lgg2 were replaced with the first and second immunoglobulin-like domains of human cd4. both molecules have been previously shown to effectively neutralize hiv-i in vitro. ex vivo neutralizations were performed as follows: lgg12 and cd4-lgg2 were added at 25 pg/ml to wells containing serial dilutions of plasma from hiv-i-infected patients and phastimulated peripheral blood mononuclear cells from seronegative donors. p24 production was measured over 14 days of culture and an end-point titer of hiv-1 in the presence and absence of added antibody was determined. both igg12 and cd4-lgg2 were found to reduce the original hiv titer from seven plasma samples with high virus titer (>250 tcid50/ml) by up to 625-fold. this is in comparison to soluble cd4 which only reduced viral infectivity by 55-fold at the same concentration. in vitro binding and neutralization assays on isolates recovered from plasma confirm the potency and breadth of neutralization by these two molecules. these studies suggest that recombinant antibodies directed at the cd4-bs of hiv-1 gp120 are able to effectively neutralize primary isolates of hiv-1 and may be useful in dissecting the mechanisms of resistance to neutralization by other antibodies. dillner and p. heino, microbiology & tumor biology center, karolinska institute, stockholm, sweden hpv 16 the major cause of anogenital precancers in man. the search for neutralizing epitopes that could form the basis for a preventive vaccine has shown that the surface-exposed imunodominant epitopes of the capsid are strongly conformationdependent, which has precluded detailed epitope analysis. similarly, immunization with whole, denatured capsid proteins has only identified linear immunodominant epitopes positioned on the inside of the capsid. reasoning that linear surface-exposed epitopes should exist, but might be cryptic, a set of 66 overlapping synthetic peptides corresponding to the entire hpv16 capsid proteins was used to generate hyperimmune sera. several antisera against 3 different peptides were reactive with intact hpv16 capsids at titers up to 1:150.000. hiv-1 serum antibodies and mucosal iga. basil golding, john inman, paul beining, jody manischewitz, robert blackburn and hana golding. div. of hematology and viral products, cber, fda, and lab. of immunology, niaid, bethesda md 20892. previously, we showed that hiv-1 proteins conjugated to 8. abortus (ba) could generate anti-hiv-1 neutralizing antibodies in mice even after depletion of cd4* t cells. in this study a 14-mer peptide from the v3 loop of hiv-1 (mn) was synthesized 013) and coupled to ba and klh. balb/c mice were immunized twice i.p. with these conjugates at two week intervals. v3-klh induced mainly igg1, whereas v3-ba induced all igg isotypes but lgg2a predominated. fecal extracts from mice immunized with v3-ba were shown by elsa to contain iga antibodies. sera from these mice bound gp120, expressed on the surface of infected cells. sera from mice immunized with v3-ba inhibited syncytia formed between cd4' t cells and chronically infected [hiv-i (mn)] h9 cells. inhibition of syncytia, formed by other hiv-1 lab. strains correlated with the degree of their homology with the v3 region of hiv-i (mn). to mimic the efffect of hiv-1, mice were depleted of cd4' cells using anti-l3t4 at the time of primary or secondary immunization. following primary immunization, cd4+ t cell depletion abrogated v3-klh antibody responses, whereas responses to v3-ba were retained and sera from these mice were able to inhibit gp-120mediated syncytia. in secondary responses, cd4' t cell-depletion prevented boosting to v3-klh, but v3-ba increased anti43 and syncytia-inhibiting antibodies. these results suggest that: 1. 8. abortus, can provide carrier function for a peptide and induce both serum and mucosal antibody responses, and 2. that infection with hiv-1 with subsequent impairment of cd4' t cell function would not abrogate anti-hiv-1 antibody responses if 8. abortus is used as a carrier to stimulate memory responses. nucleotide sequence analysis of the vh genes revealed the usage of one particular vh germline element (vh61-1p) in all clones. this finding allowed the determination of somatically mutated positions in the vh regions. two vsv-ind neutralizing antibodies expressed vh and vl genes in complete germline configuration whereas the rest of the clones showed somatic mutations which obviously were antigen dependently selected for. however, binding affinities of mutated and unmutated antibodies were comparably high. in order to determine the influence of somatic point mutations on one single antibody we generated a monovalent single chain antibody (fv-ck) of a mutated clone and reversed it stepwise to germline configuration by means of site directed mutagenesis. surprisingly, already the germline configuration of fv-ck could neutralize vsv-ind, even though the binding affinty was lower than that of the mutated fv-ck. every single somatic point mutation tested improved the binding avidity although some mutations reduced affinity. thus, during the course of vsv-ind infection some antibodies are subjected to avidity maturation although this is not required for the generation of high affme, efficiently virus neutralizing antibodies, lisa hyland'", sam hou'.~, and peter c. doherty'. 'department of immunology, st. jude children's research hospital, memphis, tn 38 10 i, 2departments of immunology and microbiology, and 'pathology, university of otago,dunedin, new zealand. the b and t cell responses in c57bl/6j(b6) mice treated with the mab mel-14 to l-selectin have been analysed following i.n. infection with sendai virus. mel-14 treatment caused a 70-90% decrease in the lymphocyte recruitment to the mediastinal (h4ln) and cervical (cln) lymph nodes following infection with sendai virus. the cellularity of the spleen was unchanged. the clonal expansion of cd8+ ctl precursors in the mln was slightly delayed, but potent ctl effectors were present in the virusinfected lung by day 10 after infection and the overall magnitude of the response was not compromised. the prevalence of iga antibody forming cells (afcs) was greatly increased in both the mln and the cln of the mice given the mel-14 antibody. the igm response was prolonged and the igg response, particularly iggl, was delayed compared to controls. the altered pattern of the antibody response may reflect the limited availability in mel-14-treated mice of th cells secreting lymphokines which are involved in ig class switching, by blocking the entry of cd4+ th precursor cells into lymph nodes. facs sorting for l-selectin+, 8220+, and l-selectin-, b220+ cell populations from the mln and the cln of normal b6 mice 9 days post sendai virus infection, showed that the afcs were from the l-selectin-, b220+ cell population, a population which comprised 6-10% ofthe total cell population. we have distinguished targets of broadly neutralizing antibodies present in hiv-1 infected individuals by imunoselection in vitro and by the use of chimeric virus. one target of neutralizing antibodies, defined by an escape mutant with an ala to thr substitution at position 582 in gp41, is resistant to human monoclonal antibodies that map to a site closely congruent with that for cd4 binding. substitution of gly, ser, and val fail to confer resistance. a second, defined by an ala to val substitution at position 281, upstream from the v3 loop, does not involve the same site and does not involve v3. substitution of thr or ile also confers resistance. replacement of the v3 loop of hiv-l(mn) into a clone of hiv-l(iiib) allows the detection of two other broadly neutralizing targets. one recognizes the v3 peptide of mn but is affected by regions outside v3. the other appears to be conformational and outside v3, but its functional recognition is influenced by the v3 loop. all of these sites seem to depend on the overall conformation of the envelope protein rather than a single discrete linear epitope. antibodies against amino acids 579-613 of the hiv transmembrane (tm) glycoprotein have been shown to enhance hiv infection in vitro in the presence of complement. there has been no study demonstrating that enhancing antibodies to this region of hiv, despite increasing levels of infectious virus 10 to 100 fold in vitro, adversely affect disease pathogenesis. in two separate studies reported herein, it is shown that animals which have high levels of antibody against this region of siv, amino acids 603-622 of the envelope, fair poorly compared to animals with lower antibody levels against this region when subsequently challenged with siv. when actively immunized with a synthetic peptide from this region of siv, animals died earlier and failed to clear antigen at two weeks after infection compared to animals that received a control peptide (p<0.05). when animals were passively immunized with antibodies from a longterm survivor of siv infection, those animals that received higher levels of antibody against the tm peptide died within six months compared to longer intervals for those animals that had lower levels of antibody to this region. when taken together, these data suggest that antibody to the tm region of siv and hiv in general, and to this highly conserved peptide in particular, are detrimental to the host. therefore, immunization strategies that minimize the immune response against tm or treatment protocols that decrease antibody levels against tm may lead to prolonged survival following exposure to lentiviruses. we have developed a mouse model to examine the immune response to hpv 16 proteins when these proteins are presented to the immune system via the epithelial route. in this model animals are grafted with keratinocytes expressing hpv e6 a n d e7 genes using a transplantation procedure which permits epithelial reformation. animals so grafted when challenged intradermally with e7 either as protein or via a recombinant vaccinia virus exhibit a delayed type hypersensitivity response which is e7-specific and cd4+ t cell mediated. animals grafted with a sub optimal priming inoculum of cells develop immune non-responsiveness and have an abrogated dth response when challenged subsequently with a priming cell graft. in the present study w e have examined the antibody status in these animals. the e7 protein of hpv 16 was expressed in e. coli as a maltose binding fusion protein using the plasmid vector pmalc. after cleavage and affinity purification this protein was used in a n elisa assay to measure antibody levels in 4 groups of mice (1) those not challenged with e7 (2) mice not grafted but challenged with e7 protein in the ear (3) mice primed by grafting with 107 hpv e7 expressing cells and challenged with e7 protein (4) mice primed by grafting with 5 x 105 hpv 16 e7 cells on day 7, grafted again with lo7 hpv 16 e7 cells on day 14 and challenged with e7 protein in the ear. mice optimally grafted and challenged (group 3) exhibited high titres of igg antibodies, particularly elevated levels of iggza. mice sub-optimally grafted (group 4) exhibited igg antibody levels comparable to the control group (1). the possible mechanisms of this immune attenuation are discussed. the hepatitis c virus is a frequent cause of chronic liver disease. a proposed mechanism responsible for virus persistence is evasion of the host immune response through a high mutation rate of crucial regions of the viral genome. the portion of hcv genome coding for the amino-terminal part of the putative envelope protein (gp70) undergoes frequent mutation during the course of infection. we have cloned and sequenced the hypervariable region (hvri) of the virus isolated from an hcv asymptomatic patient at three time points during 18 months follow up. sequence analysis has allowed the identification of variants of this region and multiple antigenic peptides (map), corresponding to three hvrl variants, sequentially foundin the blood stream of the patient, have been synthesized. maps have been used as antigens for detection of specific antibodies in elisa. our results show that anti-hvri antibodies and their cognate viral sequence coexist in the blood stream but a viral sequence becomes undetectable when the specific antibodies reach maximum levels of reactivity. thus humoral immunity against the hvrl may play a role for virus clearence. the presence of anti-hvr1 antibodies was also investigated in 100 hepatitis c viremic individuals and 25 non-viremic patients. a high frequency of positive reaction (90%) against at least one of the three hvrl variants analysed in this study was detected in the viremic patients. finally, competition experiments show that antibodies crossreacting with more than one hvrl variant are produced by hcv infected individuals. this results suggest that complex cross-reactivity exist between hcv isolates for antibodies against the hvrl region as described for antibodies against the gp120 v3 loop of hiv. we propose as mechanism for viral escape in hcv chronic infections the one described as the "original antigenic sin", observed firstly in influenza, in togavirus, paramixovirus, enterovirus, and recently in hiv infection. using an adult mouse model to study active immunity against rotavirus infection, it was previously shown that oral immunization with some, but not all, animal rotavirus strains induced protection against subsequent infection following oral challenge witb the murine rotavirus strain edim (ward et al., 1992) . to determine i f a specific rotavirus protein could be associated with protection in this model, mice were immunized with a series of 18 reassortants between the fully protective edim strain and a partially protective heterologous rotavirus strain (rrv-g). reassortants that contained genes for edim proteins responsible for protection were anticipated to provide complete protection; however, no edim proteins were found to be both necessary and sd3cient for full protection. instead, protection was found to be highly correlated with viral shedding (p = ,005) and with serum rotavirus iga titers stimulated by the different reassortants (p < ,001). this indicated that protection was related to the intestinal replication properties of the different reassortants rather than to specific immunogenic properties of edim proteins. this conclusion was supported by the finding that the titers of serum rotavirus i& but not igg, stimulated in mice following oral immunization with a series of animal rotaviruses was directly related to protection against edim. if these findings can be extended to humans, they suggest that the efficiency of intestinal replication following oral inoculation with a live rotavirus vaccine candidate may be the primary determinant of successful immunization. h a l l medical center, 55 individuals with adequate serum samples were identified as either rapidly progressing (rp) or slowly progressing (sp) by clinical and surrogate marker criteria. anti-v3 profiles were determined using synthetic proteins derived from the amino acid sequences of the v3 region of 5 laboratory strains of hiv-1 in standard capture elisa format. serum obtained from each patient at multiple different time points was screened against these peptides. the majority of individuals in both groups demonstrated broad recognition, with reactivity to peptides corresponding to the v3 regions of mn, sf2, ny5 and han/sc. less than 50% of individual in each group recognized the v3 peptide derived from iiib, @=ns, between groups). as the rp progressed to aids there was significant nonspecific narrowing of response, while the sp remained broadly reactivity (p< .001). in v i m neutralizing activity of the homologous laboratory isolates was determined with cytotoxicity, cytopathic effect and p24 ag inhibition assays. although most patient serum was capable of inhibiting p24 ag production in homologous lab strains while aids-free, there was no relationship with the ability to inhibit homologous virus effects on target cells and anti-v3 profiles. model, we show that after resolution of the acute infection, when antiviral plasma cells in the spleen decline, a population of virus-specific plasma cells appear in the bone m w and constitute the major sou~ce of longterm antibody production. following infection of adult mice, wspecific antibody secreting cells (asc) peaked in the spleen at 8 days postinfection, but were at this time undetectable in the bone marmw. the infection was essentially cleared by 15 days and the asc numbers in the spleen rapidly declined while an increasing population of lcmv-specific asc appeared in the bone marrow. when compared to the peak response at 8 days post-infection, timepoints from 30 days to more than one year later demonstrated greater than a l@fold reduction in splenic asc. in contrast, jltvlv-specific plasma cells in the bone marrow remained at high numbers and correlated with the high levels of antiviral serum antibody. the prewnce of antiviral plasma cells in the bone marrow was not due to a persistent infection at this site, since virus was cleared from both the spleen and bone marrow with similar kinetics as determined by infectivity and pcr assays. the igg subclass profile of antibody m e t i n g cells derived from bone manuw and spleen correlated with the igg subclass distribution of lcmv-specific antibody in the serum. upon rechallenge with w , the spleen exhibited a substantial increase in virus-specific plasma cell numbers during the early phase of the secondary response, followed by an equally sharp decline. bone marrow asc populations and lcmv-specific antibody levels in the serum did not change during the early phase of the reinfection but both increased about 2-fold by 15 days post-challenge. after both primary and secondary viral infection, lcmv-specific plasma cells were maintained in the bone marrow showing that the bone marrow is a major site of long-term antibody production after acute viral infection. "memory" t cells, and associated with responsiveness to soluble and recall antigens. cd4+ lymphocytes staining bright, dim, or negative (equivalent to an isotype control) for cd29 were evaluated in 49 uninfected controls (group l), 84 hiv-1 positive patients with 220% cd4+ t cells (group 2), and 47 hiv-i-infected patients with ~2 0 % cd4+ t cells (group 3). most of these subjects also had 3-color staining for cd4\cd45ro\cd45ra. the appearance of positive cd29 and cd45ro on hivinfected and uninfected cells correlated well (r=.82 p<.ool). the percentage of cells staining cd4+\cd29+.(bright plus dim) was 43.3 (95%cl 37.3-49.4) in group 1, 28.9 (27.5-30.4 ) in group 2, and 10.2(8.6-11.9) in group 3. the respective values for these groups that were cd4+\cd2gbwm was 30.6 (26.9-34.3), 20.7 (1 9.3-22.2), and 7.4(6.3-8.6). values for cd4+\cd45ro+ were 33.7 (31.8-35.5), 21.8 (20.5-23.1), and 9.9 (8.5-11.3), respectively. in single factor discriminate function tests, the %cd4+\cd29+ cells best predicted subject group (87% correct), proving to be a better discriminator than %cd4+\cd29b'h' (77% ~orrect),cd4+\cd29~"" (5 l%), cd4+\cd45ro+ (75%) and cd4+\cd45ro+\cd45ra-(63%). overall, no advantage was seen to splitting the cd4+\cd29+ cells into bright and dim positive subsets in the subjects studied for the purpose of stratifying early vs. late hiv infection. likewise, splitting the cd4+\cd45ro+ compartment into cd45ra+ subsets did not improve the ability to distinguish between uninfected and early or late hiv-1 infected patients. the relationship between the virus-specific cytotoxic response in hiv infected patients and disease progression support the concept that a vaccine candidate should also induce a virus-specific ctl activity. immunization of uninfected adult volunteers by a hiv-gpl60 recombinant canarypox virus was carried out in a phase i trial.two injections of a recombinant canarypox expressing the hiv-l/mn gp160 were performed at month 0 and 1 and two boosts of recombinant gpl60mn/lai at month 3 and 6 in alum or incomplete freund adjuvant(1fa). hiv-envelope specific cytotoxic activities were detected from ctl lines derived from pbmc stimulated by specific stimulation with autologous hiv infected blasts. ctl lines were obtained from 18 out of 20 donors : seven out of eighteen (39%) were found to present envelope specific cytotoxic activity at months 2, 4, 7 or 12 post immunization ; this activity was characterized as a cd3+,cd8+, mhc class-i restricted cytotoxic activity, and for at least two volunteers, this activity was still present two years after the first canari-pox/env injection. because avian poxviruses are incapable of complete replication and undergo abortive replication in mammalian cells , this is a n example of the persistence of long term memory cd8+ cytotoxic t lymphocytes in the absence of the priming antigen, indicating that t-cell memory might be independent of continued antigenic exposure. the university of alabama at birmingham, al 35294. mhc class i restricted cd8' ctl activity plays an important role in the control of influenza virus infection as indicated in studies in mice and humans. cytokines such as il-2 and ifn-y regulate the generation of virus-specific ctl responses. we recently demonstrated a good correlation between the induction of influenza virus-specific ctl activity and the production of ifn-y by the cd8' t cells at the single cell level using an if?-specific elispot assay, secreted ifn-y by an elisa, and ifn-y specific mrna expression by rt-pcr. several recent studies have characterized cd4+ and cd8' t cells by their expression on the surface of distinct d45r isoforms. cd45ra is expressed on naive or virgin t cells, while cd45ro is expressed on memory t cells. in the present study, pbmc of healthy young adult subjects were stimulated with influenza a virus and then enriched for cd8+ t cells. the cd8' cells were stained for cd45ro' (pe) and cd45ra' (fitc) cells and sorted. ctl activity against virus-infected autologous target cells was determined in a 4 hour 'lcr release assay while ifn-y production and expression was assessed by elispot and quantitative rt-pcr, respectively. cds+/cd45ro+ (memory) cells exhibited significant mhc class i ctl while cds+/cd45ra+ cells exhibited no lytic activity. no activity was exhibited by freshly isolated or unstimulated cd8+/cd45ro+ t cells. similarly, cd8+/cd45rot t cells contained significantly higher numbers of ifn-y spot forming cells and higher quantity of ifn-y-specific mrna than cd8+/cd45rac cells. these data support our previous findings that ifn-y may serve as a useful surrogate marker for influenza virus-specific ctl activity in humans. in studying the kinetics of the cd8+ t cell response in lcmv infection we have observed a profound activation and proliferation of cd8+ t cells with a 10-40 fold increase in total number peaking at day 8-9 post infection. in c57bw6 mice, most of the viral antigen is cleared by day seven, and after day 9 the total cd8+ number per spleen drops about 10-fold. however, the relative specificity of the viral peptidespecific precursor ctl frequencies @ctwf) per cd8+ cell remains remarkably stable between day 7-8 of the acute infection and for many months thereafter. thus, the decline in the cd8' t cell number is not a function of the tcr specificities but is rather an across-the-board event. in contrast, we found that subsequent to the decline of the ctl response to a second heterologous virus infection such that the mouse was in a "resting, immune'' state, there often was a reduction in pctl/f to the first virus. for example, infections with w or mcmv substantially reduced the pctuf to lcmv or pv in all memory compartments, including spleen, lymph nodes, peritoneal exudate cells. reinfection with the original virus substantially elevated its pctuf and restored the pctuf that had been reduced by a heterologous viral infection. analyses of the progression of ctl responses during a heterologous virus challenge of a virus-immune mouse indicated a high frequency of crossreactive ctl appearing early during infection, but as the infection progressed there was a higher proportion of ctl specific only for the second virus. thus, we believe that when the across-the-board apoptosis of t cells occurs late in the infection, ctl specific for the first virus are diluted by those responding to the second virus. this may cause the reduction in memory to the first virus and may be one of the mechanisms contributing to the waning of secondary immune responses to certain viruses over time if there is no re-exposure to the original infectious agent. t-cells which arise after virus infection will aid our understanding of tcell memory and be useful in the design of vaccines which augment the memory response. to estimate the sendai virus specific precursor frequency in memory mice, cd4+ cells from c57bl6 female mice which had been infected with sendai virus intranasally (i.n.) more than two months earlier were subjected to limiting dilution analysis. responder cell populations were enriched for cd4+ cells either by magnetic bead depletion of non-cd4+ cells, or by facs after staining with anti-cd4 monoclonal antibody these enriched (>90% cd4+) responders were cultured with sendai virus-infected, irradiated, t-cell depleted splenic antigen presenting cells (apc). supernatants from these cultures were tested for activity on the cytokine-dependent ctll cell line. duplicate cultures of responders on uninfected apc were used to set the level of rejection (mean cpm + 3x std. dev.). using this type of analysis we were able to demonstrate a frequency of memory thp at 111600 cd4+ cells, compared to a frequency greater than 1/1ooooo in naive controls. the memory cd4+ cells were further characterized as cd45rb-low (1/472) , cd44-high (1/294), lselectin-low (1/364), and cd49d-high (vla-4-high) (v102). this is close agreement with other phenotyping studies on cd4+ memory cell specific for soluble antigens. t cells, ralph a. tripp, sam hou, anthony mcmickle, james houston and peter c. doherty, department of immunology, st. jude children's research hospital, memphis, tn 38105. the immune response of influenza a and sendai-virusspecific, memory cd8' cytotoxic t lymphocyte precursors (ctlp) have been analyzed in c57bu6 mice infected intranasally with unrelated or cross-reactive respiratory viruses. the numbers of influenza a-specific memory t cells increased in the regional lymph nodes (ln), spleen and bronchoalveolar lavage through the course of an irrelevant infection (influenza b). memory t cells showed evidence of enhanced steady-state activation. profiles of ctlp recruitment were analyzed in association with t cell proliferation and activation to determine whether signaling via the t cell receptor is necessary to induce "bystander" stimulation of the memory t cell pool. the extent of t cell proliferation was addressed by treating mice with low doses of cyclophosphamide (cy). "resting" sendai virus-specific memory t cells were unaffected by cy treatment, however upon challenge with influenza and treated 5 or 6 days later, the emergence of influenzaspecific ctlp was severely diminished. cell cycle analysis showed that cy eliminated the majority of cd8' t cells from the ln and spleen resulting in dna fragmentation of 12-18% ofthis lymphocyte subset. a decrease (though smaller) in the numbers of sendai virus-specific ctlp indicated that some of the cycling cells killed by cy were memory t cells, presumably activated in a "bystander" manner. the decrease in ctlp numbers for both influenza and sendai virus-specific ctlp was still apparent 9 days after cy treatment, long after the viral elimination. thus, immune responses to unrelated antigens may be a mechanism involved in maintaining the pool of memory t cells. experimentally vsv can result in an acute cns infection of mice. data from our in vitro experiments indicate that no has inhibitory effect on productive vsv infection. vsv infection at neuroblastoma nb41 a3 cells was significantly inhibited by loopm of a no donor s-nitro-n-acetylpencillamine (snap), while 1oopm of the control compound n-acetylpencillamine (nap) had no effect. when vsv infected nb41a3 cells were treated with 500pm of a constitutive no synthase (cnos) activator n-methyl-d-aspartate (nmda), a significant inhibition of vsv production was observed. inhibition by 500pm of nmda was reversed by 300pm of nos inhibitor n-methyl-l-arginine (l-nma). work is in progress to determine the effects of inducible nos (inos) in a glioma cell line c6 on vsv infection. levels of no and expressions of both cnos in neurons and inos in glial cells in the cns following vsv will be further investlgated. supported by nih grant a118083 to carol s. reiss. pediatrics, university of iowa, iowa city, ia. 52242 mouse hepatitis virus, strain jhm (mhv-jhm), is a neurotmpic coronavirus which causes acute encephalitis and acute and chronic demyelinating encephalomyelitis in susceptible rodents. 40.90% of suckling c57bu6 (kbdb) mice inoculated intranasally with mhv-jhm at 10 days and nursed by dams immunized against the virus develop a chronic demyelinating encephalomyelitis characterized clinically b hindlimb paralysis, at 3-8 weeks postinoculation. the chronic demyelinating encephalomyelitis nor the clinical symptoms. recently, it was shown that lymphocytes isolated from the central nervous system (cns) of c57bu6 mice both acutely and persistently infected with mhv-jhm display a cytotoxic t lymphocyte (ctl) response to the s protein of mhv-jhm. this response was further characterized by identifying the ctl epitopes that are recognized by a bulk population of ctls from the cns of mhv-jhm infected c57bv6 mice. three epitopes were identified using synthetic peptides and truncated forms of the s protein in primary ciz assays. the epitopes recognized were amino acids 510-518 (cslwngphl, db), 598-605 (rcqifani, kb), and 1143-1151 (nfcgngnhi, db). thus, the results indicate that cytotoxic t lymphocytes responsive to the s protein of mhv-jhm in c57bu6 mice recognize both kb and db-restricted cil epitopes. ctl lines and clones specific to these peptides and the entire s protein are being developed to test their biological significance in vivo with respect to the acute encephalitis and chronic demyelinating disease caused by mhv-jhm. a marked change in susceptibility to some neurotropic viruses during the first few postnatal weeks has long been recognised in rodents. infection of neonatal or suckling mice with the neurotropic alphavirus, semliii forest virus results in lethal encephalitis. infection of weaned animals is not lethal. earlier investigations focusing on changes in specific immunity have shown this not to be the explanation. infection of 3-4 week old mice with severe combined immunodeficiency does not result in acute rapidly fatal encephalitis. we have studied mortality, neuroanatomical distribution and spread of infection in mice of different ages and the effect of gold compounds on rendering infection of 3-4 week old mice lethal. neuroanatomical distribution of infection correlates with synaptogenesis. as this is completed in different systems within the first two weeks postnatal, systems no longer transmit virus and infection switches from disseminated to focal and restricted. complete productive replication and transmission of infection require smooth membrane synthesis which is present in neurones undergoing synaptogenesis, absent in mature neurones but inducible by administration of gold compounds. infection of neurones undergoing synaptogenesis is productive and virus is transmitted along neuralpathways, infection spreads rapidly around the brain, destroys cells and animlas die of a fulminant encephalitis. in mice infected after 14 days of age replication in mature neurones is restricted, nonproductive, cannot be transmitted, does not spread, is non-destructive and non-lethal. as a consequence, in the absence of immune responses virus can persist in isolated cns cells for life and can even be detected by reverse transcriptase pcr in immunocompetent mice months after infection. in the presence of an immune response, cd8+ t-cells recognise and destroy infected glial cells leading to dem yelination. a~k e r m a n n ,~ virology swine,' virology cattle,' and avian diseases3 research units, national animal disease center, usoa, agricultural research service, ames, ia 5001 0 a recombinant pseudorabies virus (prv) (lltbap) was constructed which contains a 3.0 kb deletion spanning the standard recombination junction of the unique long and internal repeat sequences replaced by e lacz expression cassette. this deletion interrupted the large latency transcript gene (llt) and truncated one copy of the diploid immediate early iel80 gene. replication and viral gene expression of lltbaz in madin-darby bovine kidney cells was similar to that of the parental virus and a virus rescued for the deleted sequences (lltbres). when inoculated intranasally in 4-week-old or 4-day-old pigs, lltba2 replicated efficiently at the site of inoculation yet caused markedly reduced fatality when compared to the parent or lltbres viruses. in particular, the lltba2-infected pigs did not exhibit neurological symptoms characteristic of prv infection. to further examine the pathogenesis of lltba2, 4-day-old pigs were infected intranasally with lltpa2 or lltbres and necropsied at various times postinfection. virus isolation from the nasal turbinate, tonsils, and trigeminal ganglia was comparable between the two viruses. although both viruses spread to the brain and induced an inflammatory response in cns tissues, virus isolation from brain tissues was reduced about 20-fold for lltpa2. abundant prv antigen was detected in the cerebrum and cerebellum of lltpresinfected pigs, but only a few antigen positive neurons were observed in the cerebrum of lltba2-infected pigs. while replication of lltbres in the brain progressed until death at 7 days post-infection, replication of lltpa2 in the brain ceased by 9 days post-infection and the pigs exhibited only mild clinical signs. since lltba2 is capable of spread to the cns, reduced neurovirulence of lltbaz is likely the result of its decreased ability to replicate in cns tissues. the cns is a target for hiv infection, and in individuals with aids this can lead to a devastatin dementia. only certain viral variants appear capable 07 invading the cns and infecting microglia and brain macrophages. in order to determine whether the virus entering the brain may be particularly pathogenic to the cns, we isolated microglia from the brains of siv-infected rhesus monkeys. transfer of these cells into naive animals indicated that productive siv infection could indeed be transferred. furthermore, cns infection occurred within a relatively short time span, and was associated with viral gene expression in the brain and pathology characteristic of hiv encephalitis. serial transfer of microglia into additional animals also resulted in successful transfer of infection, neuroinvasion, and neuropathology. behavioral analysis in a trained group of animals is ongoing. this result demonstrates that neuropathogenic virions partition into the cns during natural siv infection, likely driven by mutational events that occur during the course of infection. molecular characterization of the microglia-associated virus has revealed that a distinct pattern of sequence changes in the envelope gene occurs concomitantly with this in vivo selection. our approach will allow the dissection of functional neuropathogenic elements present in these viruses. in non-specific host defense mechanisms. ifn-y-induced nitric oxide (no) in murine macrophages was previously shown to inhibit the replication of poxviruses and herpes simplex virus type 1 (hsv-1) . we now demonstrate that murine macrophages activated as a consequence of vaccinia virus (vv) infection in viva express inducible nitric oxide synthase (ios). the vvelicited macrophages were resistant to infection with w and efficiently blocked the replication of w and hsv-1 in infected bystander cells of epithelial and fibroblast origin. this inhibition was arginine dependent, correlated with no production in cultures and was reversible by the nos inhibitor nqjmonomethyl-l-arginine. the mechanism of no mediated inhibition of virus replication was studied by treating vv-infected 293 cells with the noproducing compound, s-niuoso-n-afetyl-penicillamine. antibodies specific for temporally expressed viral proteins, a vv-specific dna probe and transmission electron microscopy were employed to show that no inhibited late gene protein synthesis, viral dna replication and virus particle formation, but not expression of the early proteins analyzed. further, we have also identified putative enzymatic targets of inactivation by no that results in inhibition vv replication. although antiviral ctl are important for virus elimination. they can only halt further virus spread, and cannot reduce the number of infectious particles already present. the beneficial effect of ctl-mediated lysis is apparent only if infected cells are lysed before assembly of progeny virus. if infectious virus was released from infected cells in solid tissues before the generation of neutralizing antibody or in sites where antibody did not readily penetrate, then recruitment of mononuclear phagocytes, which phagocytose and destroy infectious material and/or become non-productively infected, would definitely help control virus dissemination. in this context, inos induction in macrophages may be an important antiviral strategy. in addition, the inhibition of virus replication in infected contiguous cells by inos-expressing macrophages at infectious foci would prevent release of mature viral particles after lysis by nk cells and ctl. since viral early proteins are expressed in such infected cells, their recognition and subsequent lysis by ctls will not be hindered. cns persistence, tropism and genetic j. pedro s i s , anthony a. nash and john k. fazakerley, department of pathology, university of cambridge, cb2 iqp, uk theiler's murine enchephalomyelitis virus, a natural occuring enteric pathogen of mice, is a picomavirus belonging to the curdovirus genus. following intracerebral inoculation of 3-4 week old cba or balb/c mice, the bean strain causes a chronic persistent cns demyelinating infection in a proportion of the cba that survive acute infection. balb/c mice are resistant to chronic demyeliating disease. we have studied the tropism, persistence and genetic variability of bean, in cba and balb/c mice in the chronic phase of this disease. by in situ hybridisation and reverse transcription (rt) pcr and southern blot analysis, no viral rna could be detected in the cns of any balb/c mice later than day 60 post-infection. in contrast, in a large group of cba mice studied up until 393 days post-infwtion, viral rna could be detected by both techniques in 50% of mice until as late as 268 days post-infection. by employing a combination of, in situ hybridisation for viral genome followed by immunocytochemistry for cell phenotypic markers, bean rna was observed predominantly in oligodendrocytes and occasionally in astrocytes during persistent infection, in both brain and spinal cord. in the persistently infected mice, the striking total destruction of the pyramidal layer of the hippocampus, substantia nigra and anterior thalamic nuclei indicated that these were the mice that had had greatest dissemination of virus and highest virus titers during the preceeding acute phase of infection. direct pcr t h d cycle sequencing of uncloned rt-pcr products, revealed that during persistent infection, loops i and ii of the vpi capsid protein gene did not undergo any genetic variability. furthermore, no changes were detected in this region in sequenced pcr products amplified from the cns of mice with severe combined immunodeficiency in which no selective immunological pressure would have been operative. infection, thomas e. lane, michael j. buchmeier, dorota jakubowski, debbie d. watry, and howard s. fox, department of neuropharmacology, the scripps research institute, la jolla, ca 92037 our laboratory is interested in the effects of siv infection in the central nervous system of rhesus macaques. to enrich for neuroinvasive and neurovirulent viruses, microglia were isolated from infected monkeys and used t o infect new, uninfected monkeys. such microglia-mediated infection resulted in the production of neuropathological changes, including giant cells, macrophage infiltrates and microglial nodules in recipient animals within 4 months. microglial cells isolated from siv-infected monkeys produced virus in vitro as measured by reverse transcription (rt) and p27 production. treatment of microglia with recombinant human interferon alpha (rhulfn-a) resulted in a sharp decrease in viral activity (both rt and p27 production) suggesting that rhulfn-a is able t o modulate viral activity in infected microglia. we have analyzed slvenv sequences by pcr amplification directly from microglia dna preparations from monkeys. nucleotide sequence analysis results in an enrichment of unique sequences in the v1 region of the siv env gene. the majority (>95%) of nucleotide changes encoded amino acid changes, indicating that these envelope sequences evolved as a result of selection. moreover, sequential passage of sivassociated microglia resulted in an increase in potential n-linked glycosylation sites within the v1 region of the env gene when compared with the parental virus. these data suggest that sequential passage of microgliaassociated siv may select for neuroinvasive, neurovirulent variants. the adoptive transfer of ctl specific for an ld-restricted epitope within the nucleocapsid protein of the jhmv strain of mouse hepatitis virus both protect from acute infection and reduce virus replication in the mhc class 1 positive cells within the cns. the source of these ctl and the route of their delivery is critical in the outcome of this protection. for example, 10 fold less spleen cells activated in vitro with the pn peptide are required for protection via the direct i.c. route than the i.v. route. in addition, ctl clones are unable to protect via the i.v. route and are very efficient via the i.c. route. these data suggested the possibility that the cd4+ t cells within the polyclonal activated spleen cell population derived from in vitro culture on the pn peptide were facilitating access to the cns. to examine this question, polyclonal pn-specific t cells were either depleted of cd4+ t cells prior to transfer to infected recipients or untreated cells were transferred to recipients depleted of cd4+ t cells with monoclonal antibody gk1.5. both of these treatments eliminated the ability of the ctl to reduce virus replication within the cns, suggesting that cd4+ t cells in the peripheral compartment are required for the entry of ctl into the parenchyma of the cns during acute cns encephalomyelitis. division of retrovirology, walter reed army institute of research and henry m. jackson foundation, rockville, md 20850; department of retrovirology, armed forces research institute of medical sciences, bangkok, thailand background the hn-1 epidemic in thailand is largely due to two highly divergent subtypes of virus, b and e. dual infection with distinct hn-1 subtypes, which has not been reported previously, would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. merhoak: pcr typing and serologic typing were used to screen a panel of non-random convenience specimens from hiv-1 infected subjects in thailand. specimens that showed dual subtype reactivity in these assays were subjected to differential pmbe hybridization and nucleotide sequence analysis of multiple molecular clones to c o n f m the presence of dual infection. results. two individuals were shown to simultaneously harbor hiv-1 of env subtypes b and e (table) . additionally, both subtypes were identified in co-cultured pbmc from one individual. conclusions. these data provide the fmt evidence of dual hiv-i infection in humans and reinforce the need for polyvalent vaccines. infection by herpes simplex virus wsv) induces in man and in mice cytolytic t lymphocytes (ctl) which recognize the immediateearly protein icp27. because of its early expression during the hsv replication cycle, lcp27 represents a prime target for specific t cell responses susceptible of controlling virus replication. we have expressed in e. coli a remmbinant construct coding for a fusion protein consisting of a fragment of influenza virus non-structural protein4 (nsi) and the lcp27 sequence of hsv-2. the nsi-icp27 protein was purified by preparative eleclmplmresis and formulated in oil-in-water emulsions with monophosphoryl lipid a (mpl) and qszl adjuvants. balwc mice were immunized by two intrafootpad injections of formulations containing 5 pg of nsi-icp27. responder cells obtained from draining lymphnodes were re-stimulated in vitro with p815 cells lransfected with icp27 and then lesled for cytolytic activity on icp27-p815 and control p815. the induction of icp27 specific ctl by different formulations was observed and will be discussed. the induction of heterologous cytotoxic t lymphocytes (ctl) using cassettes of multiple conserved t cell epitopes derived from different proteins and/or virus strains is envisioned as a promising vaccine approach. to study the effects of antigen processing on peptide presentation from chimeric epitope precursors we are using a model system comprising two distinct viral epitopes which are immunodominant in the h-2d haplotype: a dd restricted epitope from the gp160 protein of hiv-1 and an ld restricted epitope from the murine hepatitis virus nucleocapsid protein (mhv n). the influence of proximity and flanking sequences of epitopes on antigen presentation was analyzed using vaccinia virus (vv) recombinants in which the epitopes were expressed as chimeras containing the individual epitopes in reverse order or separated by different spacer residues. whereas individually expressed epitopes were efficiently recognized by protein-specific ctl, recognition of peptides derived from tandem constructs varied significantly with closer epitope proximity and sequential order. following immunization with the recombinant viruses, the chimeras were all able to induce antiviral ctl specific for the native proteins. however, cn, frequency analysis indicated that the number of responder cells to the same epitope dramatically depends on its context within the chimera and correlates with antigen recognition in vitro. the profound effect of flanking regions on ctl induction suggests that the context of an epitope will require careful evaluation in the design of recombinant multivalent minigene vaccines to induce an optimal t cell mediated immune response. and robert e. johnston', departments of 'microbiology & immunology and %iochemistxy, univ. of north carolina, chapel hill, nc 27599 a hll-length cdna clone of venezuelan equine encephalitis virus w e ) has been altered to contain two strongly attenuating mutations and a second subgenomic rna promoter immediately downstream of the structural gene region. expression ofthe influenza ha protein from this second promoter in baby hamster kidney (bhk) cells was approximately 50?? of the level in influenza virus-infected cells, as measured by immunoprecipitation. fourweek-old cd-1 mice were inoculated subcutaneously with 2 x 10' p h of the ha vector, vector alone or diluent. expression of ha mrna was detected in the draining lymph node of ha vector-inoculated mice by in situ hybridization, consistent with the organ tropism of vee. mice were challenged three weeks after imnmization by intranasal administration of lo5 ed, of influenza h s . au 24 corn1 mice suffered severe disease and 50% died. only one of 12 ha vector-inoculated mice died, and another exhibited signs of disease for one day and recovered. the geometric mean elisa titer of anti-ha serum igg in the ha-vector inoculated mice was 246, while only three control mice had measurable serum reactivity, and that was at the lowest dilution tested, 150. in a parallel experiment, no influenza infectivity was detected in the lungs of 12 ha vector immunized mice at 4 days postchallenge. in contrast, 8/12 pbs-inoculated mice and 5/12 inoculated with vector alone were positive for influenza infectivity and had geometric mean titers of 3.04 and 1.93 x lo6 pwgm, respectively. this vector also has been used to express the h n w c a protein in a form recognized by patient sera and a specific antibody on western blots. these experiments demonstrate the feasibility of using vectors based on attenuated vee cdna clones for protective immunization against heterologous human and animal pathogens. dose/response curves have been used to compare different routes of immunization with plasmid dna encoding the h1 hemagglutinin glycoprotein of influenza virus. routes of inoculation included intramuscular, intradermal and gene gun delivery of dna. from 100 to 0.1 ug of dna was inoculated by intramuscular and intradermal routes. from 0.4 ug to 0.0004 ug of dna was inoculated by gene gun. each route was evaluated for single and boosted immunizations. antibody titers were followed over a 20 week period, following which animals were evaluated for protection against a lethal challenge. each of the routes raised both antibody and protective responses. gene gun-delivery of dna required 250 to 2,500 times less dna to raise responses than the intramuscular and intradermal inoculations. boosts did not have much of an effect on antibody titer or protection except at low dose inoculations (4 ng and lower for the gene gun). for each of the routes, antibody responses showed good persistence over the 20 weeks of the experiment. inoculation of mice with plasmid vectors carrying a microbial gene under the control of an appropriate promoter results in a full spectrum of immune responses to the vectorencoded antigen. using a murine rabies model a plasmid termed psgsrab.gp expressing the full-length rabies virus glycoprotein regulated by an sv40 promoter was shown to induce upon inmuscular inoculation a rabies virus specific t helper cell response. of the thl type, cytolytic t cells and virus neutralizing antibodies resulting in protection against a subsequent challenge with live rabies virus given either peripherally or directly into the cenaal nervous system. a response comparable in magnitude was also induce upon inoculation of a vector expressing a secreted form of the rabies virus glycoprotein. the immune response to the dna vaccine could be modulated by co-injection of the rabies virus glycoprotein-expressing vector with plasmids expressing mouse cytokines. inoculation of mice with the psg5rab.gp vector and a vector expressing granulocyte/macrophage colony stimulating factor (gm-csf) enhanced both the t helper and the b cell response to rabies virus thus improving vaccine efficacy. co-inoculation with vectors expressing interferon-g failed to improve the response. co-inoculation of the antigen-expressing vector with a plasmid encoding mouse i l 4 caused a reduction of both the t helper cell response and the b cell response to rabies vlns. hpvl6 e7 hpv associated cervical cancer cells express hpv16e7 protein and antibody to hpv16 e7 can be detected in the blood of cancer patients, yet the twnours are. not rejected. a mouse transgenic for the e7 protein of hpv16, and expressing e7 protein in the skin, has recently been described (1) and these mice develop spontaneous humoral immunity to e7 protein similar to patients with cervical cancer(2). to determine whether immunisation could induce immunity to e7 sufficient to allow tumour rejection, we firstly demonstrated that immunisation of h-zb mice with hpv16e7 protein with quil a as adjuvant could induce cytotoxic t cells able to kill hpv16 e7 expressing tumour cells in nrro. we then used similar immunisation with e7iquil a to induce e7 specific immunity in fvb (h-29) mice. h-2qskin @s expressing e7 were not rejected by e7 immunised h-zq mice, though immunisation induced antibody to e7, and similar grafts were rejected, as expected, across an doantigen mismatch in h-zb mice. we conclude either that hpvl6 e7 lack a tc epitope in the context of h-zq, or that expression ofe7 in the skin from the e7 transgenic mice is insufficient for recognition by primed effector cells, and further experiments will address this distinction. cervical carcinoma is strongly associated with infection by human papillomavirus (hpv) types 16 or 18, and continued expression of the e6 and e7 gene products. this provides an opportunity for an immunotherapeutic approach to the treatment of cervical carcinoma by activation of immune reponses directed against these virally encoded tumour specific antigens. we have constructed a recornbinant vaccinia virus expressing e6 and e7 from hpv16 and 18 with the aim of inducing e6 and e7 specific hla class i restricted cytotoxic t lymphocytes (ctl). the sequences have been inserted into the wyeth vaccine strain of vaccinia virus at a single locus in the form of two separate fused e6/e7 reading frames, each under the control of an early v a d n i a promoter, and each modified to inactivate the rb binding site. the virus has been characterised with respect to its ability to synthesise the expected hpv proteins, its genetic stability, and growth and virulence in a mouse model prior to use in human clinical trials. analysis of hpv16 â�¬7 specific ctl from c57bu6 mice immunised with this recombinant virus show the response to be equivalent to that generated by a control vaccinia recombinant expressing non-modified hpvi 6 e7 alone, with similar recognition of the defined immunodominant h-2db restricted epitope, e7 residues 49-57. ability of mice to resist influenza challenge, arthur friedman, douglas martinez, john j. donnelly and margaret a. liu, department of virus and cell biology research, merck research laboratories, west point, pa 19486 mice infected with the laboratory strains of a/pr/8/34 (hln1) or the mouse adapted a/hk/68 (h3n2) show complete protection against challenge with a different strain of influenza a. humans, however, undergo multiple influenza infections as previous infections appear to provide weak or short-lived protection against the continual antigenic change of strains. we have previously shown that immunization of naive mice with dna encoding the conserved internal antigen nucleoprotein (np) provides protection against both h1 and h3 strains of a/influenza. although such mice became infected they were resistant to weight loss and death this differed substantially from a/pr8 and a/hk recovered mice which were resistant to subsequent infection. to produce a more representative model of human infection, we infected the lungs of mice with currently circulating strains of human influenza. mice that had been given lung infections with a/beijing/92 were susceptible to subsequent infection with the a/hk/68 strain although they were resistant to weight loss and death. other strains such as a/beijing/89 or a/georgia/93 provided only marginal protection against weight loss and death against a/hk challenge. mice that were immunized with np dna had greater resistance to weight loss and death after a/hk/68 challenge than mice previously infected with a/bei/89 and a/ga/93, and were similar to mice that had been previously infected with a/bei/92. thus, infection with different virus strains provide various levels of cross strain protection and the level of protection provided by immunization with dna can exceed that induced by live influenza infection. the development of sendai virus-specific cytotoxic t lymphocyte (ctl) effectors and precursors (p) has been compared for mice that are homozygous (-/-) for a disruption of the h-21-ab class ii major histocompatibility complex (mhc) glycoprotein, and for normal (+i+) controls. the generation of cd8+ ctlp was not diminished in the (-/-) mice, although they failed to make virusspecific igg class antibodies. while the cellularity of the regional lymph nodes was decreased, the inflammatory process assayed by bronchoalveolar lavage (bal) of the infected lung was not modified and potent ctl effectors were present in bal populations recovered from both groups at day 10 after infection. there was little effect on virus clearance. as found previously with cd4-depleted h-2b mice, the absence of a concurrent class il-mhc-restricted response does not compromise the development of sendai virus -specific cd8+ t cell-mediated immunity. the importance of cytoxic t lymphocytes in defense against acute and chronic viral infections is gaining increasing recognition. our approach to investigating the structure-function relationship between immunogens and their in vivo ability to elicit cytotoxic t lymphocyte responses has been to formulate simple, well-defined structures that vary in their ability to introduce associated antigens directly into the cytoplasm of antigen presenting cells. we have introduced methods for the preparation of unique, lipid-matrix based immunogens, which are highly effective in mice and monkeys for stimulating strong cd8+ cytotoxic t cell responses, (ctl). antigens used have been proteins or peptides derived from influenza, parainfluenza, and hiv viruses, and whole formalin-fixed siv. ctl can be induced by parenteral as well as oral administration. comparing the physical and chemical nature of our formulations with those from other laboratories which have reported the use of subunit preparations to induce cd8+ ctl, leads us to propose that a minimal immunogenic formulation capable of eliciting cd8+, mhc class i restricted cytotoxic t lymphocytes includes: i) a peptide that represents a mhc class i epitope; ii) a component that enhances the aftinity of the immunogen for mhc class i positive antigen presenting cells ; iii) properties that can compromise the integrity of a lipid bilayer, facilitating delivery of the antigen directly into the cytoplasm for class i presentation. cd8+ responses to peptides, glycoproteins, and even whole fixed viruses, makes them attractive candidates for diseases where clearance of infected cells is important in protection and recovery. cani ne rabies is uncontrolled. rabies also is epizootifally active in several species in most areas of the wor!d. thm, vaccination of animals, both wild and domestic, as well as postexposure treatment of humans remains a global concern. unfortunately, in those countries in which,people most need postexposure prophylaxis, the best vaccines are expenswe and in limited supply, whereas available vaccines are of questionable immunogenic efficiency, are otten contaminated and may produce neurological complications. the goal of this study was to determine whether a rabies vaccine for global use is complete one round of replication have the potential to be used as vaccines. we have previously reported the abiliity of a ghdeleted herpes simplex virus type 1 (hsv-1) to protect mice and guinea-pigs from subsequent challenge with wild-type hsv. this virus, which we have called disc (disabled infectious single cycle) virus, can infect normal cells but the absence of gh in the progeny virus prevents further rounds of infection. as disc hsv clearly has potential as a vaccine, it is important to determine the durability of the immune response elicited by this virus. we have investigated the ability of disc hsv-1 to protect mice from a wild-type virus challenge six months post vaccination using the ear model of hsv infection. two immunisations on day 0 and day 21 resulted in a considerable reduction in virus titres in the challenged ears, and an almost complete absence of virus in the dorsal root ganglia. hsv-specific antibody titres as determined by neutralisation and ellsa were maintained for the six months period. it was possible to demonstrate an hsvspecific cytotoxic t-cell response in the disc hsv-1 vaccinated mice following challenge; this ctl activity was similar to that observed in mice vaccinated with wild-type virus and challenged after the same time period. animals vaccinated with inactivated virus or control mock-vaccinated mice showed a low level of ctl activity typical of a primary ctl response following challenge. these results indicate that an effective cell-mediated and humoral anti-hsv immune response can be maintained for at least six months following vaccination with disc hsv-1. viruses which lack an essential gene and thus can only the lmmunogenicity of two ctl @topes. influenza npl47-158 and plasmodium berghei cs protein 252-260 were studled in balblc mice. paptides were formulated as a) a iipopepudepeplkle conlugated to irlpalmltoyl-sgly~~l cysteine (pam3cyj) and dissolved in a 1% dmsolglycerol solution. b) micmparticles prepared with poly @.l ladide-coglywlide) using a solvent evaporation technique. the micropaltides were administered as a suspension in phosphate buffered saline or c) an emulsion prepared wilh egg lecithin and 10% soya oil in water. 1 wpg of peptide or controls (the welght equivalent of blanks) were administered to groups of 3 mice intra-peritoneally or subcutaneously at 1.10 and 20 days. 7 days following the last immunization splenocytes were cukured in vlro in the presence of appropriate pepwe or wntml m h rat con a supematant as a source of omwth fadors. ctl adivity was measured in a standard 4 hour chromium release assay and results expressed as % specific lysis. ctl could be elicited in vivo with all three formulations. at an ewedoctarget ratio of 1w:l the plasmodium berghei peptide encapsulated in micmpartides gave 47% iysls on peptide pulsed target calls. levels of lysis were similar for the peptide in emulslons. the iipopeplide p3ccs252-260 gave a level of lysis of 82% at an e:t ralioof1w1. these results demonstrate that peplides edminldered in a variety of formulations can induce a systemic ctl response in vivo. peplide vaccines using such formulations wuld be used to stimulate ctl responses as part of a prophyladic vaccines or as immunotherapeulics. attenuated the attenuated sabin strains of poliovirus have been used for many years to elicit protective immunity to poliovirus. oral vaccination with replicating polioviruses generates both mucosal and systemic immunity. therefore, use of recombinant polioviruses expressing heterologous antigens as vaccine delivery vectors should provide a system for generating protective immunity to those antigens. cdna copies of the poliovirus genome has been used to construct vectors containing a multiple cloning site for insertion of heterologous genes. a pilot enhanced-potency inactivated poliovirus vaccine (ejpv) with assumably improved immunogenicity containing win-treated type 3 poliovirus (shah sauketf) together with the regular type 1 and type 2 canponena was subjected to s*mdard safety and potency tesls in the labmatory and laken through wase i and i1 clinical aials. in balb/c mice, the lrypin-mcdit%d e-if' v cfryipv) was found to induce antibodies targeted ouaide the uypsin-sensitive bc-loop of capsid protein vp1. as previously shown for hypsin-mated type 3 poliovirus @vm alone. trypsin used to modify the type 3 component at the bulk phase was removed by the vaccine manufacturer (rivm) in the regular purification process. absence of uypsin in the final product was further confumed by immunizing mice and rabbits with 10-fold concentrated type 3 component of tryipv. assays for lrypin antibodies using eia and westem blot techniques were newve. in the clinical phase 1 aial six adult volunteers with existing immunity to poliovirus were given increasing doses of tryipv. already one tenth of the regular dose induced a booster effect in neuarlizing antibodies to both intact and mypsin-treated type 3 poliovirus. no unexpected sideeffects were recorded phase i1 trials comprised 50 adult volunteers with at least 5 years since the last dose of poliovirus vaccine and 50 children who were due to receive the third dose of the regular immunization schedule at about 2 years. in both groups. 25 individuals received tryipv and 25 were injected with the regular enhanced potency ipv (e-ipv). serum specimens drawn before injection and one month after were tested for neunalizing antibodies using standard microneuwlization assays (all mutypes) and the racina test (intact and uypsin-mated type 3 poliovirus). in all volunteers tryipv was at least as immunogenic t w the regular e-ipv according to all assays. no statistically significant differences in side effects were reported. a murine/influenza virus model has been used to evaluate the longevity of antibody and protective responses raised by gene gun delivery of a hemagglutinin-expressing dna. mice were immunized and boosted at one month with 0.4 ug of an h1 expressing plasmid dna (pcmvri1). antibody responses and protection against a lethal challenge were followed over the next year. antibody responses had good longevity exhibiting comparable titers at one year post boost as at 10 days post boost protection against the lethal challenge was complete at 10 days, 1 month and four months post boost, but only partial at one year. a transgenic mouse model for identifing htlv-1 t-cell epitopes: generation of hla-b*3501-restricted ctl directed against synthetic peptides and naturally processed viral antigens, christian schiinbach*, ai kariyone*, kiyoshi nokiharaa+, karl-heinz wiesmulle6 and masafumi takiguchil, departments of tumor biology* and immunology#, institute of medical science, university of tokyo, tokyo "tokyo university of agriculture and technology, tokyo +biotechnology instruments department, shimadzu corp., kyoto, japan $natural and medical science institute at the university of tubingen, reutlingen, germany the majority of human t-cell leukemia virus type-i (htlv-l), hla class i-resmcted t-cell epitopes have been identified by cloning htlv-1 patient-derived t cells. here we describe for the frst time a rapid method (reverse immunogenetics) for identifing t-cell epitopes, together with a transgenic mouse model as a guide for testing the cellular immune response to a mixture of the lipohexapeptide immunoadjuvant pamgcys-ser-(lys)4 and synthetic htlv-1 peptides which seem suitable for vaccine design. htlv-1 amino acid sequences were searched for eight to 14mer patterns carrying the anchor residues of the hla-b*3501 peptide motif at positions two and eight to fourteen. 65 candidate peptides were synthesized according to the matched sequence patterns. their hla-b*3501 affinity was quantitatively analyzed in an indirect immunofluorescence peptide binding assay using rma-s-b*3501 cells. the fourth group (controls) were inoculated with h3n2 (in) thereby providing heterotypic ctl immunity in the context of a natural infection without the confounding effects of humoral immunity against surface antigens. all four types of inoculations have been shown to protect normal (class i expressing) mice from a lethal challenge with influenza, presumably mediated by class i restricted cytotoxic t cells. the two groups inoculated via the intranasal route may gain additional protection by activating the mucosal immune system (iga). none of these types of inoculations has been evaluated in the context of class i1 restricted cytotoxic t cells, the only ctls found in class i deficient mice. for all four types of inoculations, mhc class i deficient mice lost significantly more weight than the class i expressing control groups (seven mice per group) indicating the importance of class i restricted t-cells in protection. within the class i expressing groups, there was no significant difference between the four types of inoculations; within the class i deficient groups the vac-np im immunized mice lost significantly more weight than the h3n2 group;the other two groups, vac-np in and genetically immunized groups had intermediary results. these data lend support for a protective role for mucosal immunity. results on both class i and class i1 ctl activity for the four types of inoculations will also be presented. we tested the pbmcs of patients participating in two vaccine therapy trials for their ability to recognize overlapping peptides of the gp120 la1 sequence. seventeen patients participating in a phase i gp160 protocol and 13 patients participating in a phase i gp120 protocol had their pbmcs isolated by ficoll separation of heparinized venous blood. the fresh pbmcs were plated, in triplicate, into 96 well plates containing peptides overlapping the la1 sequence of gp120, pulsed on day 7 with tritiated thymidine and harvested and counted on day 8. results: the percentage of patient's pbmcs from each trial with an lsi 2 5 to each peptide are depicted below. conclusions: the pbmcs of hiv-infected volunteers who have been multiply immunized with either gp160 or gp120 proliferate to multiple peptides within the gp120 molecule. reactivity from the end of c1 through early c2 (lai #i 12-21 1) is particularly prominent and contains previously undescribed th epitopes (asterisks). conspicuously missing is reactivity to the v3 loop peptide (la1 #300). although the percent reactivity to the entire gp120 molecule is similar between the immunization groups, there is differential recognition of some of the individual peptides, particularly peptides in early c3 (lai #319-348). the intracytoplasmic lifecycle of listeriu mmcytogenes (lm) enables it to be a convenient vaccine vehicle for the introduction of foreign proteins into the mhc class i pathway of antigen presentation. taking advantage of these properties, we have inserted the nucleoprotein (np) gene from lymphocytic choriorneningitis virus (lcmv) into the lm chromosome by site specific homologous recombination. infection of mice with recombinant lm expressing lcmv-np elicited a virus-specific ctl response. we were able to recover lcmv-np specific ctl precursers from recombinant lm vaccinated mice as shown by vigorous secondary ctl responses after in vitro stimulation. in contrast to mice immunized with wild type lm, mice vaccinated with np-recombinant lm were protected against challenge with immunosuppressive lcmv variants. protection was demonstrated by reduced viral titers or complete clearance of lcmv from serum and various organs including, spleen, liver, lung, kidney, and brain. the kinetics of the lcmv challenge indicate that mice vaccinated with recombinant lm were able to arrest viral growth early in the infection due to a strong ctl response and did not exhibit the immunopathology associated with infection of naive mice. since lm not only delivers antigens into the mhc class i pathway but also induces il-12 production, it has the potential to function simultaneously as a vehicle for expressing foreign antigens and as an adjuvant promoting cell mediated immunity. , latent membrane protein [lmp] 1 and 2a) in chromium release assays. we were fortunate in identifying one child from whom cryopresetved pbmc samples were available before. and during ebv seroconversion. ebv-specific ctl activity was demonstrated concurrent with initial detection of virus in the peripheral blood by ebv-dna pcr. in the absence of detectable serum antibody. ctl lines from all nine children recognized one or more ebv latent gene prcduct(s). all children demonstrated ctl responses against one or more ebna 3 proteins (3a, 3b, 3c). and ebna 3c was recognized most frequently. no ctl responses were detected against the ebv latent proteins ebna 1, 2, lp or lmp 1. the ebv-specific ctl lines expressed cd3/cd8 and mab blocking experiments demonstrated that the majority of target cell lysis was inhibited by antibody against mhc class-i but not antibody against mhc class-11. these results represent one of the first reports characterizing ebv-specific ctl responses in young children. the striking similarity between ebv-specific ctl responses described here in young children and those reported for adults suggests that the ebna 3 family of proteins and lmp 2a should be considered for inclusion in candidate ebv vaccines. evaluation of cellular immune responses to adenovirus vectors in the cotton rat. soonpin yei,' gary kikuchi,' ke tang' and bruce c. trapnell.' departments of virology' and immunology,2 genetic therapy, inc., gaithersburg, maryland 20878 replication deficient recombinant adenovirus (av) vectors are efficient gene delivery vehicles currently being developed for a variety of in vivo gene therapy strategies such a s for the fatal pulmonary component of cystic fibrosis. the cotton rat (sigmodon hispidus) is one of the most widely accepted animal models for studying these av vectors because wild type human adenovirus replicates in cotton rats and because of the histopathologic similarities of infected respiratory epithelial tissues from humans and cotton rats. despite this, methods for studying immunologic responses in the cotton rat have not been developed. importantly, recent studies in the cotton rat (gene tber. 1 :192-200; 1994) in our laboratory suggest that a dose-dependent specific immune response to av vectors can limit expression of the transgene. in this context, w e have established methods to evaluate cytotoxic lymphocyte (ctl) responses to av vectors in the cotton rat. to accomplish this, a ctl target cell line was established consisting of primary cotton rat lung fibroblasts (crlf). splenocytes from cotton rats exposed previously to an av vector were harvested, cultured in virro with irradiated, addb274nfected crlf. cultured (effector) splenocytes were then incubated with s'cr-labelled crlf (target) cells a t effectoctarget (e:t) ratios of 100, 50 and 10. in parallel, splenocytes from naive cotton rats served as negative controls. results demonstrated vector-specific ctl lysis of target cells significantly greater than controls: 80.3 f 1.3% vs 6.2* 0.5%. 49.6*1.6% v s 5.7*0.4%. and 22.8*3.5% vs4.8*0.5% (meanrts.e.m., n=3; p<o.ol, all comparisons) a t e:t ratios of 100, 50 and 10, respectively. thus, a specific ctl response does develop in cotton rats following in vivo av vector administration. this observation will be useful in guiding vector modifications in the rational development of improved vectors for clinical use. the identification of ctl epitopes is essential for subunit vaccine design against aids. a major portion of the anti-viral ctl responses after infection with htv in humans or siv in rhesus macaques is made up by anti-gag specificities. here, effector cell populations were established from hiv seropositive individuals by specific stimulation of pbl with pfa fixed autologous b-lcl infected with tw gag. expanded cultures were then tested for ctl activity against autologous b-lcl pulsed with overlapping 20-mer peptides derived from p24. two individuals (008 and 617) had ctl against p24.14 (a(mino) a(cids) 263-282, krwiilglnknrmysftsi), two others (038 and 157) recognized p24.17 (aa293-312, frdwdrfyktlraeqasqd). similarly we found two siv infected rhesus macaques reacting against the analogous aa sequences of siv; it. p26.14 (8653) and p26.17 (ivy). this led us to further fine map the human and rhesus ctl reactivities using sets of 9 aa overlapping 10-mers and to test for hiv-1isiv crossreactivity. both two p24.14 epitopes (possibly restricted by hla-a2) and the p26.14 epitope were non-overlapping, and all three epitopes were non-crossreactive. finemapping of the p24.17 epitopes is in progress. the sn p26.17 epitope was also non-crossreactive, despite only one aa difference (position 6, s+t) between the siv and hiv sequence. others have also mapped cl'l epitopes to the same regions of p24 in humans (johnson ji 147,1512 ,1991 , buseyne jv 67,694,1993 and to p26.17 in cynomolgus macaques (gotch jmprim 22.1 19,1993) . in conclusion, multiple non-cross reactive ctl epitopes are clustered within corresponding regions of hiv and siv gag. ctl hotspots may be important for inclusion into vaccines. forming virus) with cd-4 and cd-8 1-cells, b-cells and adherent cells, while an average of 5% of virus was associated with lps-stimulated 8-cells and adherent cells. in the second experiment the combined function of antigen presenting cells, 1-helper cells and 8-cells (humoral immune response) to a third party antigen (srbc) during the acute phase of enterovirus induced disease was increased at day 0, and decreased at days 2, 3 and 4 post-cvb3, infection relative to unlnlected animals. conclusions: these data indicate that cvb3, associates with and replicates in rnurine splenic immune cells, and that infection of susceptible mice with cvb3, modifies the immune response to a third party antigen during the acute stages of disease. an understanding of such cvb3,-i mmune cell interactions is criitical for gaining insight into the pathogenesis and persistence of enteroviral infection both within and outside of the immune system. understanding the pathogenic mechanisms of virus infections depends upon identification of viral gene products involved in host-virus interactions in vivo. many viral gene products which affect pathogenesis in vivo are nonessential for virus replication in vitro. therefore, we constructed insertion or deletion mutants in the hind i11 j and i regions of murine cytomegalovirus (mcmv) to assess the importance of these genes in in vivo replication of virus as well as interaction with immune regulatory and effector cells. three mutants replicated in nih 3t3 fibroblasts as efficiently as wild-type virus, thus confirming the nonessential nature of the genes. deletion of 2.8 kb in hind i11 j and of 7.7 kb in hind i11 j and i resulted in mutants with reduced replication in target organs in vivo. an insertion in hind i11 j had no significant effect on replication in mouse salivary gland, but curtailed growth of the virus in footpad fibroblasts and subsequent priming of mcmvspecific cpl precursors in the draining lymph node. in addition, the 7.7 kb deletion mutant was unique in its failure to replicate in a permissive, differentiated macrophage cell line. current studies aim to further define the role of this gene region in hcmv pathogenesis. that in vv5-4 progression o f v v life cycle is blocked at the the uncoating ii or replication stage. furthermore, host protein synthesis in v v 5 -4 cells is not shut-off after v v infection suggesting uncoatingll/replication is important for determining cell fate. since this mutant clone vv5-4 was isolated b y retroviral insertional mutagenesis. a cellular gene that was integrated by single retrovirus was isolate and codes for a 5kb transcript in northern blot analysis. a 2 kb cdna was isolated and codes f o r a novel polypeptide o f 800 amino acids that show no homology with known proteins. experiments are in progress to understand t h e role of this cellular gene in vv infection. in addition, a two-hybrid system will be employed to identify a n y viral gene product interacting with this cellular target u s i n g o u r laboratory's s t a n d a r d s t r a i n o f a/japan/305/57 in in uitro assays of viral infectivity of the mouse b-lymphoma a20-1.11. we have found that although infection sensitizes the a20 cells for recognition by both class i and class i1 restricted ctl.'s and leads t o high surface expression levels o f hemaglutinin (ha), the infected a20 cells grow through t h e infection, ha expression declines, and few of the infected cells die. in contrast, using a second s t r a b of a/japan/305/57 from a different passage history, we find that n o t only are the infected a20 cells sensitized for lysis by both class i and class ii restricted ctl's, exhibiting even higher levels o f ha surface expression, but in addition, most cells in the infected population die with characteristics of apoptosis. the two virus strabs appear to be highly related because in various assays we have found t h e m antigenically indistinguishable in both their t-and b-cell epitopes. using these two virus strains in a n in uiuo dose response assay, we have found that o u r laboratory's standard strain will kill all mice infected with a 10-2 the therapeutic and prophylactic antiviral efficacy of interleukin-12 (il-12) was studied using murine models of herpes simplex virus (hsv) and murine cytomegalovirus (mcmv) infection. therapeutic intraperitoneal administration of il-12 commenced 6 hours after mice were infected w i t h hsv, and was continued daily for a total of 5 days. il-12 therapy improved the survival rates of mice w i t h systemic hsv infection, compared to that of placebo treated infected mice. since neutrophilia and thrombocytopenia were common in dengue patients,we are investigating whether bm may be accompanied with abnormal hematopoiesis. using ficoll-hypaque separation, mononuclear cells prepared from the total bm cells. 1 .0x106we~ells/well of both adherent and nonadherent cell types were infected with two different den-2 virus strains : (1) thiland strain isolated from the dengue hemorrhagic fever(dhf1 patient and (2) taiwan strain isolated from the dengue fever(df) patient at moi=l and collected the suprnatants at various time points for assay. the preliminary data showed that : (a) adherent cells were infected with den2 and dhf virus strain replicated much more efficiently (2.3x104pfu/ml on day 3 p.i.1 than df strain; (b) nonadherent cells were 10 times less efficient to produce viral yields than adherent cells (dhf and2 df strains peaked at 6.3~10' pfu/ml and 1.5~10 pfu/ml on day 3 p.i., respectively); (c) addition of gm-csf to nonadherent cells increased virus infectivity and productivity. in conclusion, den viruses were able to infect bm cells and the more virulent virus strain isolated from dhf patients had better replication capability in human bone marrow cells. future studies on cytokines and immunologic evaluation are in progress. number of gene products whose function is to modify host responses. recent evidence suggests that a subclass of poxvirus-encoded host response modifiers interferes with host defense responses, and thus can be termed immune or inflammatory response modifiers (irms). several poxvirusencoded irms possess homology to mammalian proteins which are associated with host defenses, while other poxvirus irms have little sequence homology to immune-effector molecules or their receptors. the ultrecht strain of rabbitpox (rpv) virus induces a large reddish pock on the chorioallantoic membrane (cam) of the chicken embryo. rpv mutants lacking the spi-2/crmn38k gene trigger an inflammatory response by 36-48 hr after inoculation, similarly to that found for cowpox virus (brighton red strain) mutants. rpv mutant viruses with a deletion of the ps/hr gene, a gene which has recently been associated with virus host range and virion maturation, elicited an inflammatory response on the chicken embryo cam at 48-72 hr after virus inoculation. thus, another poxvirus-encoded gene product has been identified that has irm functional activity which would not have been predicted by sequence analyses. poxviruses have recently been found to possess a herbert w. virgin iv, center for immunology and division of infectious diseases, washington university school of medicine, st. louis, mo 63110 murine cytomegalovirus (mcmv) follows three stages of infection in the immunowmpetent host. following the initial acute infection, a persistent infection remains in which lytic virus resides in the acinar cells of the salivary glands. finally a third stage of infection exists which is characterized by a lack of detectable infectious virus. it is not known whether mcmv remains persistent at some low level during this thiid stage or establishes molecular latency. to test this, spleens, kidneys and salivary glands from mice 2-8 months post infection were evaluated by two sensitive assays. sensitivity was tested using virus co-cultured on murine embryo fibroblasts (mefs) for 14 days. by this method, 1 pfu mcmv was reproducibly detected in the presence of sonicated spleen, kidney and salivary glands diluted 1:lo. scid mice were used as a second detection assay for mcmv. using 46 scid mice and different doses of virus, the lds, of mcmv in scid mice was 2-3 pfu. mcmv infections were detected when 10-25 pfu were added to sonicated spleens, kidneys, and salivary glands prior to injection into scid mice. using both co-culture with mefs and injection of tissue into scid mice, we have tested a total of 34 spleens, 34 kidneys, and 37 salivary glands and have detected no persistent virus. while no preformed virus existed in these tissues, reactivation from explanted spleen, kidney and peritoneal exudate cells occurs in culture after 10-40 days. in addition, lytic mcmv was detected in scid mice 28 days after injection with 2(10)' kidney cells from latently infected mice. following identical transfers of latent spleen cells, mcmv was not detected in scid mice although explants from the same organs reactivated in vitro. using facs sorting, panning, magnetic bead separation, and reactivation assays, individual cell types have been analyzed for the presence of mcmv genome and the ability to reactivate virus. heat shock proteins (hsp) are expressed in all organisms in response to a variety of stresses, including virus infection. however, since viruses are not known to encode hsp genes, this represents expression of cellular hsps. we have found a dramatic induction of the 72kda hsp in the ovaries of w-infected mice, and using primary ovarian fibroblasts, demonstrated hsp72 mrna within virus-infected cells. the physiological role of hsps expressed during virus infection is unknown, although hsps act as molecular chaperones to facilitate protein folding and assembly in unstressed cells, and a number of bacterial hsps are essential for bacteriophage replication and morphogenesis. in order to determine whether hsp72 expression was beneficial to vv replication, we constructed a recombinant vv which encoded murine hsp72, but found that this virus grew to similar titres as control viruses, both in vitro and in vivo. in particular, kinetic studies of virus growth in an hsp72-negative cell line showed no difference from control viruses, and the presence of hsp72 did not influence the virulence of infection in immunocompromised nude mice. these results are interesting given that hsp70 proteins act as molecular chaperones and that hsp72 can be found in association with vv proteins. we suggest that this association may simply reflect the inherent affinity of hsp70 for non-native proteins, and the close physiological proximity of hsp and nascent viral proteins within the virus infected cell. our results indicate that the functional significance of these interactions are minimal with respect to the outcome of virus infection. furthermore, although the expression of hsw2 in vv-infected mice coincides with the peak anti-viral cellular immune response, hsp72 does not appear to be immunologically significant, as we cannot demonstrate any immunological responses to hsp72 during the course of vv infection. the effect of in vivo neutralisation of ifn-y on cytokine production during influenza infection was compared in cs7/bl6 and balb/c mice. similar cytokine profiles were obtained on in v i m restimulation of mln or spleen cells from virus-infected mice of each strain. at days 7 and 10 postinfection, mln or spleen cells from both strains of mice produced il-2, il-6, il-10 and ifn-y, but little or no il-4 or il-5. however, following in vivo treatment with anti-ifn-y antibody, production of 1l-4 and il-s was induced in mln and spleen cells of balb/c mice whereas those from cs7/bl6 mice showed little change in cytokine profile. an increase in il-6 and 1l-10 production was also observed on in virro restimulation of splenocytes from balb/c mice. however, significant amounts of 1l-2 and ifn-y were still secreted in these cultures. in v i m neutralisation of ifn-y in the restimulated cultures had little effect on the production of other cytokines, regardless of whether this cytokine had also been neutralised in vivo .despite the change in cytokine profile, anti-ifn-y treatment had no effect on the kinetics of viral clearance in balb/c mice. a similar situation was seen for c57/bl6 mice. congenic strains of mice are currently being used to determine whether the effect on cytokine profiles is associated with the mhc haplotype. wunner, and steven l. spitalnik, department of pathology and laboratory medicine, university of pennsylvania and the wistar institute, philadelphia, pa 19104 rabies virus glycoprotein (rgp) is the only glycoprotein on the viral surface, is the target of viral neutralizing antibodies, and is the viral protein that interacts with the host cell receptor. rgp of the era strain is a 505 amino acid type i membrane glycoprotein with 3 potential n-glycosylation sites at asn37, asn247, and asn319. due to rgps central role in the immunology and biology of rabies, it is important to determine its three-dimensional structure. towards this end we produced a recombinant form of rgp that may be more amenable to analysis by x-ray crystallography. to avoid the use of detergents, we constructed a soluble 434 amino acid form of rgp lacking a transmembrane domain (rgpt434). rgpt434 was secreted by transfected cells and was appropriately glycosylated, assembled, antigenic, and immunogenic. since n-glycosylation was required for rgpt434 secretion, we minimized the number of n-glycans by site-directed mutagenesis. interestingly, rgpt434 was secreted only when asn3l 9 was n-glycosylated. in contrast, full-length rgp was expressed at the cell surface a any of the three potential sites was n-glycosylated. soluble inhibitors of n-glycosylation were used to simplify the structures of the n-glycans on rgpt434. although inhibiting alpha-glucosidases with castanospermine blocked secretion, inhibiting any further processing with deoxymannojirimycin had no effect on secretion. finally, to simplify the purification process, a tail of 6 his residues was added to the cooh-terminus of rgpt434. this modification did not affect secretion, antigenicity, of assembly of the recombinant glycoprotein, but did allow purification using ni+2-agarose chromatography. in summary, we constructed a soluble form of rgp with a minimal number of homogeneous n-glycans and an "epitope" tag. this form of rgp is structurally similar to the extracellular domain of the full-length protein and may be amenable to analysis by x-ray crystallography. endogenous retroviral genes (ev) are well characterized in chickens. our laboratory has established chicken lines with single and multiple ev loci and, compared to ev negative birds, these lines exhibit a delayed and suppressed titer of neutralizing antibody in responses to avian leukosis virus (alv) challenge. to address the effects of ev genes on the cell mediated immune response we have developed an in-vitro assay to monitor the in-vivo induction of mhc restricted cytotoxic t cells (ctl) induced by alv. using this assay, we find the ctl response in birds expressing the ev-21 locus is reduced 85 to 95% compared to ev negative birds having nearly identical (eighth backcross) genetic backgrounds. other ev loci (1,6,10 and 11 ) also reduce the ctl response to alv in mhc matched chickens with different genetic backgrounds. ' national public health institute. helsinki. finland universities of tampere, qulu and 'helsinki, finland coxsackie b virus infections have been associated with clinical iddm in seveml previous studies but their initiating role in the slowly progressing beta-cell damage was for the fmt time directly implied in our recent prospective. nationwide dime study. siblings of the index iddm cases who progressed to clinical iddm experienced enterovim infections more frequently than nonprogressing siblings during prospective follow-up period. enterovim infections were initially demonstrated by detecting significant increases in serum class-specific cross-reactive enteroviral antibodies by using radioimmunoassay (ria) with the heavy-chain capture principle. causal association benveen these infections and the pathogenesis of iddm was suggested by temporal association of sercconvenions of viral antibodies with increases in islet cell antibody levels which are considered to be markers ok ficell damage. the possibility of the existmce of specific diabetogenic s & s of enternviruses was then considered. to identify the serotypzs responsible for the infections, successive sera from subjects exhibiting seroconvenion of autoantibodies coinciding with an increase in enteroviral antibodies were anlyzed by plaque n e u n a t i o n assay using a set of different enterovhes. the results indicate that several enternvirus serotypes may be associated with the progressive prediabetic p~ocess. not only different coxsackie b but also coxsackievirus a9 -infections were found to coincide with seroconvenions of islet-cell and insulin auwantibodies. it is known that an immunogenic region of gad 65. one of the major isletcell antigens, shows a high degree of homology with the 2c protein of cbv-4. studies on potential immunological cross-reactions between enteroviruses and two significant autoantigens, hsp60 and gad65, are in progress. this is carried out by using immunohistochemistry in infected cell lines and in frown sections of human pancreas with antibodies induced by synthetic peptides and natural and expressed viral proteins, the role of p53 alterations in human malignant pleural mesotheliomas (mpm) is unclear. immunostaining (ipox) of snap frozen mpm specimens revealed p53 expression in 31 of 52 (60%). p53 detection by ipox is usually associated with mutated p53. sscp for exons 5-9 revealed p53 alterations in 2 of 25 specimens, and loh at exon 4 was seen in only 2 of 12 evaluable specimens. thus, most of these specimens appeared to contain wild-type (wt) p53. we recently reported that sv40 induces mesotheliomas in rodents, and that sv40-like sequences are present and expressed in mpm. of note, sv40 large t antigen (tag) binds and inactivates wt p53 i n ! , abolishing its tumor suppressor function. we theorized that the immunohistochemical persistence of p53 may result from its physicdl binding with tag. tag was detected by ipox in 32 (62%) of these mpm specimens. expression of tag was significantly associated with coexpression of wt p53 (p2 = 0.008, fisher's exact text), and preliminary experiments suggest co-precipitation of p53 and tag. finally, we demonstrate that waf-1 is not detectable in mpm expressing wt p53 suggesting that p53 is inactive. we speculate that tag expression in mpm binds to and inactivates p53 contributing to the malignant phenotype. human papilloma virus (hpv) infection is involved in 90% of cervical carcinomas and possibly 95% of cervical intraepithelial neoplasia (cin). at present it is unclear whether patients infected with the transforming hpv types can mount efficient t cell responses. to address this issue we examined the ctl response of peripheral blood lymphocytes from patients attending a cervical neoplasia outpatient clinic. all were diagnosed hpv positive with various stages of c m and nine patients were selected for hla-a2 expression by facs analysis. pbmc were stimulated with caski, a cervical carcinoma cell line demonstrated to be hf' v type 16' and hla-a2'. culture media consisted of rpmi-10% human serum supplemented in three cases with 15 u/ml il-7, in another three cases with 3 u/ml il-2 and 15 u/ml il-7, and in the final three cases with 10 u/ml il-2 and 15 u/ml il-7. the ctl were screened for reactivity to caski and in addition the cervical carcinoma cell line c33a (hpv, hla-a2') transfected with either the hpv type 16 e6 or e7 genes. one patient's cells maintained with 10 uiml il-2 and 15 ulml il-7 showed hpv e7 protein specific cytotoxicity in a hla-a2 restricted fashion. these ctl lysed the caski stimulator cell line but not the nk sensitive target k-562. the ctl efficiently lysed a c33a-e7 clone but in contrast a vector only transfectant was not lysed. the lysis of c33a-e7 was blocked with a-cd3 and a-cd8 antibodies at 66% and 41% respectively. facs analysis determined that this ctl population was 92.8 % cd3'cds' and 7.3% cd3'cd4'. limited e6 recognition was also observed but has not been hlly characterized to our knowledge this report represents the first demonstration of hpv e7 responsive ctl isolated from the peripheral blood of hf' v infected patients. we have previously shown that proviral variants found in the peripheral blood mononuclear cells (pbmc) of hiv-1 infected individuals can interfere with recognition by autologous cytotoxic t-lymphocytes (ctl). abolition of recognition could be caused by interference with t h e processing of viral peptides, by a failure of the variant peptides to bind to mhc class-i molecules or by a failure of the mhc class-vpeptide complex to efficiently activate the tcr (t-cell receptor). these mechanisms could allow the virus to escape the immune surveillance by ctl. most studies so far have addressed the question of viral variation in t-cell epitopes by analysing proviral dna, extracted from the pbmc of hiv-1 infected individuals. however, the analysis of virion-associated rna offers several advantages. sequences found in viral rna are likely to be functional, a t least u p to the point of packaging into virus particles. furthermore, rna sequences represent virus which is currently actively produced. here, we present the sequence variation i n two hla-b8 restricted epitopes: p17-3 gag and amino acids 18-26 of the pol gene. both proviral dna from pbmc and viral rna sequences from plasma are discussed. the ability of viral variants to hind to mhc class-i molecules was assessed and the recognition of variant peptides by ctl was tested. variants were also tested for their ability to antagonize recognition of the index sequences. we show that viral variants with the ability to interfere with recognition by ctl are not only found in proviral dna but also in virion associated rna. objective: clinical course of hiv-1 infection may be influenced by an effective host immune response against hiv-1 and/or by viral properties. to investigate the cellular immune response to hn-1 we analyzed ctl activity against different hiv-1 proteins in long-term asymptomatic hiv-1 infection as well as during rapid progression to aids. methods: 10 longterm asymptomatic individuals with cd4 counts >500 celllpl after more than 8 years of infection were selected from the amsterdam cohort study on aids versus 10 subjects who progressed to aids < 5 years. ctl activity was measured on "cr labelled hla matched or autologous b-lcl, infected with rvv expressing hiv-1 ag. both bulk and limiting dilution ctl assays were performed longitudinally with pbmc after ag-specific stimulation. sequences of ctl epitopes were determined in homologous virus isolates resulrs: different kinetics of anti-gag ctl responses were observed in rapid progressors. in any case ctl responses disappeared during progression to aids. in long-term asymptomatic subjects persistent ctl responses were observed together with low viral load. conclusions: sustained, broad anti-hiv cellular immunity may correlate with maintenance of the asymptomatic state in long-term survival by controlling viral replication. enteroviruses are a large group of positive stranded rna viruses known to be responsible for a number of distinct disease entities. recombination is thought to be capable of generating new enterovirus strains that cause significant morbidity. for example, enterovirus 70 which was responsible for a pandemic of haemorrhagic conjunctivitis and poliomyelitis is thought to have originated by recombination between a coxsackie b like virus and another unidentified enterovirus. we are studying a group of echovirus 11 isolates from an outbreak of disease in southem india. sequence analysis within the 5' untranslated region reveals that these isolates fall into two groups that differ by -20% (equivalent diversity to that seen between between published sequences of poliovirus 1 and coxsackie a 9 virus). these two groups of viruses also differ in their cell tropism. isolates defined as group 1 by their 5'utr sequence grow equally well on ht29 cells (a human colon carcinoma cell line) and vero cells. isolates of group 2, with one exception, grow only on ht29 cells. analysis of the structural proteins of these isolates revealed differences in migration that correlated with their cellular tropism. thus, significant genotypic and biological diversity exists amongst these virus isolates. one virus isolate had the 5' untranslated region sequence of a group 1 virus but the protein profile and cellular tropism of a group 2 virus. the best explanation of these findings is that this anornolous isolate is a natural recombinant between the parented strains. both the ease with which viable recombinants are generated and the diversity present within this one enterovirus serotype increase the potential for the production of novel pathogenic enterovirus strains. dominant susceptibility to polyoma tumors in inbred wild mice, sharon r. nahill, yupo ma, john carroll and thomas l. benjamin, department of pathology, harvard medical school, boston, ma 021 15 polyoma virus (f' y) is a mouse dna tumor virus which, under appropriate conditions, causes tumors in a wide variety of cell types. generation of tumors is a function of both the viral and host genomes. lukacher et al. have recently described a dominant gene, pyv', carried by the c3-i mouse strain, which confers susceptibility to py-induced tumors mapping and immunological analyses indicate that py4 is the mouse mammary tumor virus 7 superantigen (mtv 7 sad gene, which deletes t cells required for py tumor immunosurveillance in h-2' mice. to determine the generality of endogenous superantigens as determinants of susceptibility and to reveal potentially novel mechanisms of susceptibility, we have looked for dominant susceptibility @ s ) gene(s) id newly established and genetically diverse inbred wild mouse strains, czech i1 and pedatteck (peru). both strains are susceptible to py as 100% of infected animals develop a full profile of tumors. crosses between cs7br, whose resistance is contributed by the major histocompatibility (mhc) locus, and susceptible peru or czech 11, yield f1 progeny which are fully susceptible, indicating a dominant inheritance pattern of susceptibility the incidence of tumor-bearing backcross animals [((peru x cs7br) x c57br) and ((czech i1 x cs7br) x c57br)i suggests that ds is due to at least one, but not more than two genes. amplification of genomic dna from the czech i1 and peru mice by pcr using primers specific for mtv 7 sag indicates that both strains are negative for proviral mtv 7 sag. furthermore, the mechanism ofds in these mice may be independent of all mtv sag as pcr using primers specific for the highly conserved region of mtv sag is unable to amplify mtv dna from peru or czech i1 genomic dna. these results indicate that, like the c3hibi, the pedatteck and czech i1 contain gene(s) which overide the resistance to py-induced tumors contributed by the mhc of the c57br parent and which may cause tumors via a novel, mtv sag-independent mechanism. we have initiated efforts to map the ds in peru and czech i1 mice using pcr and primer pairs flanking simple sequence length polymorphisms. fis-2 is a low leukemogenic, but relatively strong immunosuppressive variant of friend murine leukemia virus (f-mulv). this variant was originally isolated from t-helper cells of flc-infected adult nmrl mice. compared to f-mulv, fis-2 suppresses primary antibody response more efficiently in infected mice. some of the fts-2 infected adult nmri mice developed a disease resembling the acquired immunodeficiency syndrome induced by hiv. restriction mapping and nucleotide sequence analysis of fis-2 show a high degee of homology between this variant and the prototype f-mulv clone 57. in this study we have attempted to localize the genomic determinant of fis-2 which is responsible for induction of a strong suppression of primary antibody response. six chimeric viruses of fis-2 and f-mulv were constructed. the primary antibody response of the mice infected with these chimeric viruses were investigated. the results of these experiments will be presented. anti-fmdv antibodies, as measured in an elisa capture assay, were cross reactive. b) cellular: proliferative (cd4) t cell responses of peripheral blood mononuclear cells (pbmc) were low or undetectable during primary responses to vaccine or virus, and frequently low during secondary responses. for good t cell proliferation in vitro, multiple immunisation is required. this may reflect preferential stimulation of the th2 cd4 t cell subset. interestingly, when cd4 responses were observed, cd8 tcell responses were also detectable. 2 . recognition of individual viral proteins a) expression cloning: structural and non-structural protein pseudogenes were cloned from cdna by pcr. expressed in pgex-3xuc. and purified by sds-page. b) humoral: structural and non-structural proteins were recognised by infected animals. a good anamneetic antinon-structural response was only observed when the boosting serotype differed from the serotype stimulating the primary response. c) cellular: both structural and non-structural proteins were recognised and some were cross reactive. interestingly, vp1 was strain specific, and the polymerase (3d) was the most immunogenic and cross reactive. d) a construct comprising 3d and the immunodominant vp1 epitopes was prepared and tested. in common with other herpesviruses, the envelope glycoproteins of equine herpesvirus 1 (ehv-1; equine abortion virus) are major determinants of the infectious process and pathogenicity, and are inducers of humoral and cell-mediated immune responses. as such, they are candidates for components of subunit vaccines against ehv-1. to generate useful amounts of individual ehv-1 glycoproteins, we have constructed recombinant badoviruses capable of expressing glycoproteins c, d, h (gc, gd, gh ) in insect cells, and have evaluated the recombinant products as innnunogens in a murine model of ehv-1 infection. au three glycoproteins induced serum (elisa) antihodies to ehv-1, and ehv-1 gc and gd also induced neutralizing antibody responses. following intranad challenge with infectious ehv-1, protective immunity, as demonstrated by acelerated clearance of virus fiom respiratory tissues to below detectable levels, was evident in mice immunized with either recombinant gc or gd. in contrast, gh-immmkd mice did not develop detectable neutralizing antibody, and did not clear challenge virus more rapidly than controls. delayed type hypersensitivity and lymphoproliferation responses to ehv-1 antigen were observed for each of the ehv-1 glycoproteins, and in experiments with gdimmunized mice, a role for cell mediated immunity in protection was confirmed by adoptive transfer and t-cell depletion experiments. the data provide support for the potential of glycoproteins c and d as a subunit vaccine against ehv-1. molecular pathogenesis of ural infeetiom 52-331 enterovirus-immune cell interactions: implications in enterovirus-induced diseases we have also evaluated the effect of virus infection on the humoral immune response to cvb3, infection in adolescent c3h/hesnj mice. antigen presenting cell, 1-helper cell and 8-cell function were evaluated utllizlng a sheep red blood cell (srbc) plaque assay. mice were injected intrapentoneally (ip) with lo5 plaque forming units of cvb3, at day 0 and with lo7 srbc's at days 0, 2, 3 and 4 post-cvbb, infection. splenocytes were harvested 4 days post-srbc injection, mixed with target srbc's and guinea pig complement and incubated. plaques were then quantitated. results: cvb3, was associated with 12.9% to 17.4% of cd-8 positive t-cells and w a 11 % to 26% of adherent splenocytes. after mitogen (lps and con a) stimulation, b-cells and adherent cells were demonstrated to be permissive for viral replication. a 248% and 738% under non-stimulated conditions. an average of 1 % of virus is cell-associated (plaque north america. bruce anderson, teny yates, norah torrez-martinez, wanmin song, brian hjelle. university of new mexico, albuquerque, n.m.we recently identified a new species of hantavirus (hmv) associated with the harvest mouse reithrodontomys megalotis (hjelle b et al, j. viroj. 1994, in press ). an arizona woodrat (neotoma mexicana) was found to he infected with hmv, presumably through "spillover". hmv is most closely related to the four comers hantavirus (fcv) of deer mice (genus peromyscus). the nucleocapsid gene and protein of hmv differ from those of fcv by 24% and 15% of residues, and the 1896 nt s genome is shorter by 163 nt. we surveyed 174 reithrodontomys animals captured in the u.s. and mexico for hantavirus antibodies; 27 (15.6%) were positive. s segment cdnas were amplified and sequenced from seropositive animals captured in california (4), arizona (3), new mexico (l), and mexico (2). a monophyletic clade of hmv-like agents was identified at all sites, although an r. megalotis infected with an fcv-like virus was also identified in the state of zacatecas, mexico. nucleotide sequence distances among members of the hmv clade were up to 15.5%. but amino acid distances were less than 2%. hmv is enzootic in harvest mice throughout much of north america, and can also infect wood rats. htlv i-associated myelopathy/tropical spastic paraparesis (hamnsp) is a slowly pro ressive neurological disease characterized by perivascuaar mononuclear infiltrates in the cns. htlv i-specific cd8+ ctl are found in pbl and csf of infected patients with htlv i-associated neurological disease but not in htlv i seropositive individuals without neurological involvement. previous studies have shown that in hla-a2+ patients, htlv i-specific cd8+ ctl restricted by hla-a2 recognize a peptide derived from the htlv i tax protein (tax 11-19 llfgypvw). in the present study, we have analyzed the potential of these tax-specific ctl to recognize addtional peptides. our results demonstrate that a subpopulation of high affinity cd8' tax 11-19 specific ctl clones cross-react on a self peptide derived from the se uence of myelin-associated glycoprotein (mag 556-564 vl&sdfri) presented by hla-a2. these obsenatlons suggest that the demyelination process in hamltsp may be,due, in part, to virus-specific ctl recognition of a self myelin component that is independent of htlv i infection. development of pathology varies widely between different strains of mice after intracerebral inoculation with the so-called 'docile' isolate of lymphocytic choriomeningitis (lcm) virus. the c3hebfej and blo.br/sgsnj mouse strains have been of special interest because they display autoimmune hemolytic anaemia with varying degrees of apparent immunological involvement. in this study, we examined the role of cd4+ t helper cells in this autoimmune response by treating mice with the cw-specific gk1.5 monoclonal antibody. we also determined if polyclonal activation of b lymphocytes, induced either by lcm virus or by lactate dehydrogenase-elevating virus, another well known b cell activator, correlated with the development of anaemia in these mice. our results strengthened the central role of the immune system in the anaemia in c3h mice by showing that depletion of cd4+ cells largely, if not completely, abrogated this anti-erythrocyte autoimmune reaction. as reported by others, we found that the anaemia was more mild in b 1o.br mice than in c3h mice. however, we could not confirm the difference in the degree of b lymphocyte polyclonal activation between these mice. furthermore, lactate dehydrogenase-elevating virus had no apparent effect on erythrocytes, even though this virus also induced a sharp increase in plasma igg levels. one of the two class i mhc (h-pkd)-restricted immunogenic sites identified on the influenza strain aijapanl57 (h2n2) hemagglutinin (ha) encompasses two distinct partially overlapping epitopes, mapping to residues 204-212 and 210.219. when we investigated the magnitude of the ctl responses of balwc mice to the two overlapping epitopes, we found that while the nhrterminal nonamer epitope is immunodominant, eliciting vigorous ctl responses in njapanl57-immunized balb/c mice, the ctl responses to the cooh-terminal decamer epitope are weak and variable. the c-terminal epitope subdominance seems to be due to factors other than inefficient processing of the epitope in vivo because ctls generated by priming mice with recombinant sindbis viruses expressing only one of the ha 204-219 subsites displayed patterns of responsiveness similar to that of influenza virus primed ctls. limiting dilution ctl assays showed that the ctl precursor frequency (pctl) of the nterminal epitope is at least ten fold higher than the pctl of the cterminal epitope, implying that the low and variable pattern of cterminal specific responsiveness was due to the limited t cell precursors in the c-terminal specific ctl repertoire. this was further confirmed by the limited heterogeneity in the cross reactivity patterns displayed by the c-terminal specific ctl for an ig vh fragment and the ha 210-219 epitope of influenza strain a i m 5 7 in short term bulk cultures, and the facs analysis of tcr vg chain usage. taking these together with our previous observation that some jha 210-219 specific ctls can also crossrecognize an ig vh fragment. these studies had provided a strong evidence that ig gene products may influence t lymphocyte function and repertoire development. we have previously described the identification of homologous regions in the c-terminus of hiv-1 gp41 and in the n-terminus of hla class i1 beta chains. forty percent of patients infected with hiv-i virus were shown to have antibodies which bind to the homologous sequences, as well as to native hla class i1 molecules. affinity purified crossreactive antibodies (crab) were shown to have direct blocking effects on normal t cell responses to recall antigens, and could mediate adcc of hla class ii+ cell lines.in order to determine the contribution of such antibodies to disease progression, we obtained longitudinal plasma samples from patients in the macs study. in a first study, it was found that the presence of high titers crabs correlated with a more rapid disease progression (p = 0.027 by fisher two tail analysis)in a second, 7 year-longitudinal study of 12 progre.ssors and 12 stable patients we found: (1) the production of crab was seen in 70 -80% of rapid progresson, while the true stables produce only infrequent low-titers crab. (2) in rapid pmgressors, production of crab preceded by 2-3 years the marked drop in cd4 counts. (3) crab production did not correlate with the degree of hyperglobulinemia in these patients. (4) the presence of crab during the asymptomatic stage correlated with early loss of t-helper responses to recall antigens.we are currently establishing whether periodic measurements of crab in patients sera could be valuable in predicting a drop in cd4 counts and disease progression. the lymphokine ifn-y is i pleiotropic insnunomodulator and possesses intrinsic antiviral activity. we studied its significance in the development of antiviral immune responses using ifn-7 receptor deficient (ifn-yr-'.) mice. after inoculation with live attenuated pseudorabies virus (prv) the mutant mice showed no infectivity titers in various tissues and transient viral ag expression only in the spleen similar as in wild-type mice. however, the absence of the ifn-yr resulted in increased proliferative splenocyte responses. the prv-immune animals showed a normal ifn-1 and 11-2 production, without detectable 11-4, and with decreased 11-10 secretion in response to viral ag or con a. immunohistochemically, an increased ratio of ifny/i1-4 producing spleen cells was found. after immunization with either live attenuated or inactivated prv, ifn-yr"' mice produced significantly less antiviral antibody (ab), and more succumbed to challenge infection than the intact control animals. the reduction in ah titers in the mutant mice correlated with lower protection by their sera in transfer experiments. thes? findings are in line with the strong enhancing effect of exogenous ifn-y on rabies virusand prv-specific igg responses. our data demonstrate that a physiological ifn-y system is surprisingly not critical for the generation of antiviral th-i-type and the suppression of th-2-type cytokine responses. the lymphokine, however, is an important mediator in the generation of protective antiviral ab. key: cord-020101-5rib7pe8 authors: nan title: cumulative author index for 2008 date: 2008-11-17 journal: virus res doi: 10.1016/s0168-1702(08)00367-5 sha: doc_id: 20101 cord_uid: 5rib7pe8 nan dettori, g., see medici, m.c. (137) 163 devi, s., see osman, o. (135) murine leukemia virus reverse transcriptase: structural comparison with hiv-1 reverse transcriptase the gprlqpy motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus detection of ovine herpesvirus 2 major capsid gene transcripts as an indicator of virus replication in shedding sheep and clinically affected animals genetic characterization of equine influenza viruses isolated in italy between a new living cell-based assay system for monitoring genome-length hepatitis c virus rna replication unraveling the puzzle of human anellovirus infections by comparison with avian infections with the chicken anemia virus the contribution of feathers in the spread of chicken anemia virus cloning and subcellular localization of the phosphoprotein and nucleocapsid proteins of potato yellow dwarf virus, type species of the genus nucleorhabdovirus the p26 gene of the autographa californica nucleopolyhedrovirus: timing of transcription, and cellular localization and dimerization of product complete genomic sequence of turkey coronavirus recombinant l and p protein complex of rinderpest virus catalyses mrna synthesis in vitro molecular divergence of grapevine virus a (gva) variants associated with shiraz disease in south africa sequence analysis of a reovirus isolated from the winter moth operophtera brumata (lepidoptera: geometridae) and its parasitoid wasp phobocampe tempestiva (hymenoptera: ichneumonidae sars coronavirus replicase proteins in pathogenesis virus-induced gene silencing in medicago truncatula and lathyrus odorata evaluating the 3c-like protease activity of sars-coronavirus: recommendations for standardized assays for drug discovery hbx modulates iron regulatory protein 1-mediated iron metabolism via reactive oxygen species pathogenetic mechanisms of severe acute respiratory syndrome detection of a novel circovirus in mute swans (cygnus olor) by using nested broadspectrum pcr chimaeric hiv-1 subtype c gag molecules with large in-frame c-terminal polypeptide fusions form virus-like particles cross-species recombination in the haemagglutinin gene of canine distemper virus sapovirus-like particles derived from polyprotein cauliflower mosaic virus gene vi product n-terminus contains regions involved in resistance-breakage, self-association and interactions with movement protein adenovirus vector induced innate immune responses: impact upon efficacy and toxicity in gene therapy and vaccine applications interfering with cellular signaling pathways enhances sensitization to combined sodium butyrate and gcv treatment in ebv-positive tumor cells evidence for recombination between pcv2a and pcv2b in the field retroviral reverse transcriptases (other than those of hiv-1 and murine leukemia virus): a comparison of their molecular and biochemical properties mitochondrial plasmids of sugar beet amplified via rolling circle method detected during curtovirus screening appearance of intratypic recombination of enterovirus 71 in taiwan from the circulation of subgenogroups b5 and c5 of enterovirus 71 in taiwan from in vitro replication of bamboo mosaic virus satellite characterization of the interaction of domain iii of the envelope protein of dengue virus with putative receptors from cho cells intrahost evolution of envelope glycoprotein and orfa sequences after experimental infection of cats with a molecular clone and a biological isolate of feline immunodeficiency virus the sars-coronavirus plnc domain of nsp3 as a replication/ transcription scaffolding protein limited compatibility between the rna polymerase components of influenza virus type a and b serotype-specificity of recombinant fusion proteins containing domain iii of dengue virus very virulent infectious bursal disease virus isolated from wild birds in korea: epidemiological implications genetic analysis and evaluation of the reassortment of influenza b viruses isolated in taiwan during the enhanced immune responses of mice inoculated recombinant adenoviruses expressing gp5 by fusion with gp3 and/or gp4 of prrs virus effect of antiviral treatment and host susceptibility on positive selection in hepatitis c virus novel hiv-1 reverse transcriptase inhibitors synthesis of recombinant human parainfluenza virus 1 and 3 nucleocapsid proteins in yeast saccharomyces cerevisiae evolutionary analyses of european h1n2 swine influenza a virus by placing timestamps on the multiple reassortment events hepatitis b pregenomic rna splicing-the products, the regulatory mechanisms and its biological significance human papillomavirus 16 e6, l1, l2 and e2 gene variants in cervical lesion progression pathology and hematology of the caribbean spiny lobster experimentally infected with panulirus argus virus tomato leaf curl virus satellite dna as a gene silencing vector activated by helper virus infection down-regulation of sclerotinia sclerotiorum gene expression in response to infection with sclerotinia sclerotiorum debilitation-associated rna virus presence of p1b and absence of hc-pro in squash vein yellowing virus suggests a general feature of the genus ipomovirus in the family potyviridae the truncated virus-like particles of c6/36 cell densovirus: implications for the assembly mechanism of brevidensovirus tubulovesicular structures are a consistent (and unexplained) finding in the brains of humans with prion diseases interferon antagonist function of japanese encephalitis virus ns4a and its interaction with dead-box rna helicase identification of novel viral interleukin-10 isoforms of human cytomegalovirus ad169 cross-reactive and serospecific epitopes of nucleocapsid proteins of three hantaviruses: prospects for new diagnostic tools genetic diversity of the vp1 gene of duck hepatitis virus type i (dhv-i) isolates from southeast china is related to isolate attenuation the major tegument structural protein vp22 targets areas of dispersed nucleolin and marginalized chromatin during productive herpes simplex virus 1 infection mutational events during the primary propagation and consecutive passages of hepatitis e virus strain je03-1760f in cell culture the n protein of tomato spotted wilt virus (tswv) is associated with the induction of programmed cell death (pcd) in capsicum chinense plants evolutionary relationships of virus species belonging to a distinct lineage within the ampelovirus genus a novel genomic constellation (g10p[3]) of group a rotavirus detected from buffalo calves in northern india phylogeny of lagos bat virus: challenges for lyssavirus taxonomy dc-sign enhances infection of cells with glycosylated west nile virus in vitro and virus replication in human dendritic cells induces production of different modulation of cellular transcription by adenovirus 5, ⌬e1/e3 adenovirus and helper-dependent vectors involvement of cytoskeleton in junín virus entry hiv-1 reverse transcriptase inhibitor resistance mutations and fitness: a view from the clinic and ex vivo genomic characterization of novel marine vesiviruses from steller sea lions restricted quasispecies variation following infection with the gb virus b molecular characterization of vp4, vp6 and vp7 genes of a rare g8p[14] rotavirus strain detected in an infant with gastroenteritis in italy retroviral reverse transcription mechanisms of resistance to nucleoside analogue inhibitors of hiv-1 reverse transcriptase truncation of cytoplasmic tail of eiav env increases the pathogenic necrosis characterization of russian rabies virus vaccine strain rv-97 bacteriophage preparation inhibition of reactive oxygen species generation by endotoxin inhibition of autographa californica nucleopolyhedrovirus (acnpv) polyhedrin gene expression by dnazyme knockout of its serine/threonine kinase (pk1) gene serine/threonine kinase (pk-1) is a component of autographa californica multiple nucleopolyhedrovirus (acmnpv) very late gene transcription complex and it phosphorylates a 102 kda polypeptide of the complex amino acid at position 95 of the matrix protein is a cytopathic determinant of rabies virus phosphorylation of the tgbp1 movement protein of potato virus x by a nicotiana tabacum ck2-like activity comparative genomics of serotype asia 1 foot-and-mouth disease virus isolates from india sampled over the last two decades low dna htlv-2 proviral load among women in são paulo city antiviral potentials of medicinal plants a molecular epidemiological study of rabies in puerto rico the latent membrane protein 1 (lmp1) encoded by epstein-barr virus induces expression of the putative oncogene bcl-3 sars coronavirus accessory proteins hepatitis b viruses: reverse transcription a different way increase in proto-oncogene mrna transcript levels in bovine lymphoid cells infected with a cytopathic type 2 bovine viral diarrhea virus plantibody-mediated inhibition of the potato leafroll virus p1 protein reduces virus accumulation interferon ␤1-a and selective anti-5ht 2a receptor antagonists inhibit infection of human glial cells by jc virus the begomoviruses honeysuckle yellow vein mosaic virus and tobacco leaf curl japan virus with dna␤ satellites cause yellow dwarf disease of tomato genetic structure of a population of potato virus y inducing potato tuber necrotic ringspot disease in japan molecular characterisation and phylogenetic analysis of chronic bee paralysis virus inhibition of west nile virus replication in cells stably transfected with vector-based shrna expression system complete genome sequence analysis of dengue virus type 2 isolated in detecting molecular adaptation at individual codons in the glycoprotein gene of the geographically diversified infectious hematopoietic necrosis virus positive natural selection in the evolution of human metapneumovirus attachment glycoprotein sphingomyelin induces structural alteration in canine parvovirus capsid late steps of parvoviral infection induce changes in cell morphology molecular cloning and sequence analysis of the duck enteritis virus ul5 gene the interaction between kshv rta and cellular rbp-j and their subsequent dna binding are not sufficient for activation of modulation of hepatitis b virus replication by expression of polymerasesurface fusion protein through splicing: implications for viral persistence seroprevalence and genetic evolutions of swine influenza viruses under vaccination pressure in korean swine herds the cycle for a siphoviridae-like phage (vhs1) of vibrio harveyi is dependent on the physiological state of the host expression and biochemical characterization of nsp2 cysteine protease of chikungunya virus effect of sirna mediated suppression of signaling lymphocyte activation molecule on replication of peste des petits ruminants virus in vitro prevalence and molecular characterization of wu/ki polyomaviruses isolated from pediatric patients with respiratory disease in functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association genetic variation of hepatitis c virus in a cohort of injection heroin users in wuhan identification of a conserved linear b-cell epitope at the n-terminus of the e2 glycoprotein of classical swine fever virus by phage-displayed random peptide library lópez-galíndez, an hiv-1 215v mutant shows increased phenotypic resistance to d4t hiv-1 p17 binds heparan sulfate proteoglycans to activated cd4 ϩ t cells up-regulation of murid herpesvirus 4 orf50 by hypoxia: possible implication for virus reactivation from latency effective inhibition of japanese encephalitis virus replication by small interfering rnas targeting the ns5 gene size reversion of a truncated dna␤ associated with tobacco curly shoot virus f gene recombination between genotype ii and vii newcastle disease virus growth of tick-borne encephalitis virus (european subtype) in cell lines from vector and non-vector ticks dna recognition properties of the cell-to-cell movement protein (mp) of soybean isolate of mungbean yellow mosaic india virus emerging g9 rotavirus strains in the northwest of china implications of recombination for hiv diversity induction of apoptosis in vero cells by newcastle disease virus requires viral replication, de-novo protein synthesis and caspase activation subcellular localization of the triple gene block proteins encoded by a foveavirus infecting grapevines phylogenetic analysis of the ns5 gene of dengue viruses structural basis for drug resistance mechanisms for non-nucleoside inhibitors of hiv reverse transcriptase importance of cholesterol for infection of cells by transmissible gastroenteritis virus animal models and vaccines for sars-cov infection comparative full genome analysis revealed e1: a226v shift in oropouche virus entry into hela cells involves clathrin and requires endosomal acidification molecular evidence for polyphyletic origin of human immunodeficiency virus type 1 subtype c in transcriptomic analysis of responses to infectious salmon anemia virus infection in macrophage-like cells rnase h activity: structure, specificity, and function in reverse transcription a single nucleotide change in hop stunt viroid modulates citrus cachexia symptoms sequence analysis of mrna transcripts encoding jembrana disease virus tat-1 in vivo isolation of a type 3 vaccine-derived poliovirus (vdpv) from an iranian child with x-linked agammaglobulinemia a review of studies on animal reservoirs of the sars coronavirus genetic analysis of dengue 3 virus subtype iii 5ј and 3ј non-coding regions allopurinol, an inhibitor of purine catabolism, enhances susceptibility of tobacco to tobacco mosaic virus effects of acp26 on in vitro and in vivo productivity, pathogenesis and virulence of autographa californica multiple nucleopolyhedrovirus sars-cov replication and pathogenesis in an in vitro model of the human conducting airway epithelium rna transcription analysis and completion of the genome sequence of yellow head nidovirus mechanisms of inhibition of hiv replication by non-nucleoside reverse transcriptase inhibitors acute non-cytopathic bovine viral diarrhea virus infection induces pronounced type i interferon response in pregnant cows and fetuses extracellular vesicles containing virus-encoded membrane proteins are a byproduct of infection with modified vaccinia virus ankara analysis of jembrana disease virus mrna transcripts produced during acute infection demonstrates a complex transcription pattern complete sequence of the genome of avian paramyxovirus type 2 (strain yucaipa) and comparison with other paramyxoviruses cloning and sequencing of capsid protein of indian isolate of extra small virus from macrobrachium rosenbergii a member of a new genus in the potyviridae infects rubus molecular epidemiology of rabies in indonesia key: cord-260225-bc1hr0fr authors: sirpilla, olivia; bauss, jacob; gupta, ruchir; underwood, adam; qutob, dinah; freeland, tom; bupp, caleb; carcillo, joseph; hartog, nicholas; rajasekaran, surender; prokop, jeremy w. title: sars-cov-2-encoded proteome and human genetics: from interaction-based to ribosomal biology impact on disease and risk processes date: 2020-07-20 journal: j proteome res doi: 10.1021/acs.jproteome.0c00421 sha: doc_id: 260225 cord_uid: bc1hr0fr [image: see text] sars-cov-2 (covid-19) has infected millions of people worldwide, with lethality in hundreds of thousands. the rapid publication of information, both regarding the clinical course and the viral biology, has yielded incredible knowledge of the virus. in this review, we address the insights gained for the sars-cov-2 proteome, which we have integrated into the viral integrated structural evolution dynamic database, a publicly available resource. integrating evolutionary, structural, and interaction data with human proteins, we present how the sars-cov-2 proteome interacts with human disorders and risk factors ranging from cytokine storm, hyperferritinemic septic, coagulopathic, cardiac, immune, and rare disease-based genetics. the most noteworthy human genetic potential of sars-cov-2 is that of the nucleocapsid protein, where it is known to contribute to the inhibition of the biological process known as nonsense-mediated decay. this inhibition has the potential to not only regulate about 10% of all biological transcripts through altered ribosomal biology but also associate with viral-induced genetics, where suppressed human variants are activated to drive dominant, negative outcomes within cells. as we understand more of the dynamic and complex biological pathways that the proteome of sars-cov-2 utilizes for entry into cells, for replication, and for release from human cells, we can understand more risk factors for severe/lethal outcomes in patients and novel pharmaceutical interventions that may mitigate future pandemics. the sars-cov-2 (covid-19) pandemic has impacted every component of life, including research and medicine. in just a few months from the onset of infections to writing of this review, 10573 papers/objects have been published on sars-cov-2 ( figure 1 ). this body of literature primarily focuses on infectious diseases, the respiratory system, public environmental occupational health, biochemistry molecular biology, virology, immunology, pharmacology, microbiology, and healthcare science services, to name a few fields ( figure 1a ). title extraction of these papers reveals mainly clinically connected terms ( figure 1b) . the extensive infectious disease and clinical base of this literature has yielded knowledge of viral entry, replication, immune response, and transmission. however, in a short window of time, biochemical and molecular biology insights into sars-cov-2 have yielded a smaller body of literature that continues to grow (1267 out of the 10573 items), taking more time for data generation than clinical descriptions. of these 1267 biochemistry/molecular biology items, 934 are primary articles ( figure 1c ). title and abstract word extraction from these biochemistry/molecular biology items, followed by counting mentions of all human (20368) or sars-cov-2 proteins, shows a heavy focus on ace2 and spike (s) proteins ( figure 1d ). the virus primarily enters cells through the interaction of the sars-cov-2 surface glycoprotein, spike (s), interacting with the human encoded ace2, similar to that of the sars virus. 1,2 from the abstract/title terms, we identified 51/346 usages of ace2 and 76/295 of spike. other human proteins with repeated mentions include tmprss2 (13 titles/49 abstract), ace (1/32), furin (2/ 14), dpp4 (2/11), and c3 (2/2). additional sars-cov-2 proteins with mentions include nsp12 (rna-directed rna polymerase, 20/71), nucleocapsid (n, 17/71), membrane (m, 5/48), envelope (e, 4/31), nsp5 (3clpro/mpro, 7/26), nsp8 (3/19), nsp16 (2′-o-methyltransferase, 3/14), orf8 (1/10), nsp10 (3/9), nsp14 (guanine-n7 methyltransferase, 1/8), nsp3 (papain-like protease, 16/6), and nsp15 (uridylate-specific endoribonuclease, 16/4). only nsp6 and nsp11 for sars-cov-2 have no mentions within any of these titles or abstracts for biochemical linked papers on sars-cov-2. overall this suggests a few papers specifically related to sars-cov-2 proteins have been published; however, a large body of literature exists for the original sars and other coronaviruses that can give interpretation of the diverse functions performed by the viral-coded proteins and how they interact with human biology. the advancement of knowledge of the sars-cov-2 proteome has been slower than clinical insights due to the need for experimental work that is slow and that is being hampered by social isolation. the 29903 base-pair single-stranded rna genome of sars-cov-2 (ncbi nc_045512.2) has a 265 base-pair 5′ utr, multiple protein-coding segments, and a 228 base-pair 3′ utr. sars-cov-2 has a 79% genomic similarity with sars-cov, a known human pathogen, with both known to enter cells through the binding of human ace2. 3, 4 in addition to sars-cov and sars-cov-2, five other coronaviruses are capable of human-to-human transmission and infection (hku1, nl63, oc43, 229e, and mers-cov). 5 hundreds of coronaviridae family member genomes have been sequenced in human and other vertebrate hosts, 6,7 and many structures have been solved for coronaviridae species proteins, allowing for systematic assessments of the knowledge base. our group implemented a sequence-to-structure-to-function analysis 8, 9 to understand sars-cov-2 proteins, developing a robust understanding of protein conservation, structure, and molecular dynamics. 10 the data generated for each protein was then developed into the viral integrated structural evolution dynamic database (vistedd), a publicly released database of multiple tools for the virus. the database can be accessed at https://prokoplab.com/vistedd/. these tools consist of educational resources for the proteins coded by sars-cov-2 (molecular videos, 3d protein model prints, amino acid details of conservation, and dynamics), the mapping of critical sites to each protein, and the insights into how sars-cov-2 interacts with human proteins. generating this database has given our team a diverse understanding of sars-cov-2, particularly for host protein interactions of each of the viral proteins. multiple studies have begun building systemic insights for sars-cov-2 infections. multiple groups have performed systematic data assessment of ace2 expression and protein staining, suggesting the physiological cell types that can be targeted by the virus. they have shown expression in many tissues throughout humans, with expression within the lung found on the apical surface of polarized bronchial secretory epithelia cells. 11−14 once the virus enters the cells, it results in the alteration of broad biological pathways, including translation, splicing, protein homeostasis, and nucleic acid metabolism. 15 epithelial organoid cultures exposed to the virus produce a robust change in rna expression patterns for cytokine and interferon intracellular immune responses that give rise to tissue signals. 16 single cell profiling within the lungs of patients shows the intracellular cytokine/interferon response results in the recruitment of macrophages in severe cases and t-cells in moderate cases, with a high potential for therapeutic intervention. 17, 18 over activation of the cytokine/interferon response is connected to poor outcomes within patients, correlating with macrophage activation syndrome. 19 additional adverse outcomes for the activation of apoptosis within lymphocytes have been observed and may contribute to the noted lymphopenia. 20 proteomics and metabolomics of patient sera show the same macrophage dysfunction, while also elucidating platelet and complement dysregulation with the identification of severity classifiers. 21−23 in totality, the physiological response to the virus is likely mediated by a combination of immune system activation and the direct human interaction partners, altering cellular processes. an understanding of these detailed biological interactions can shed light on potential therapeutic opportunities while building a fundamental knowledge of viral biology. to date, few studies have been performed that systematically look at mapping how the sars or coronavirus proteins physically interact with human proteins. structural level insights for coronavirus proteins are surprisingly deficient of human interaction partners. 10 a few of these proteins have been targeted for interaction assessments, such as the nucleocapsid protein 24,25 (shown below). it has been speculated that the understanding of virus−host interactions represents a major untapped potential of viral inhibitors. 26 a 2018 review highlights the literature of viral−host interactions for coronaviruses, focused on synergizing the knowledge of independent experiments for virus receptors, translation, membrane dynamics, immune regulation, cell cycle control, and replication. 27 the more recent work by gordon et al. 28 covering the systematic affinity purification of 26 different sars-cov-2 proteins within human cells has elucidated many mechanisms and drug compounds for the regulation of viral processes. 28 bringing this data together with our vistedd tools, we provide a current snapshot of sars-cov-2 viral proteins ( figure 2 ). orf1ab is a large protein that is proteolytically cleaved to produce 16 different proteins, many involved in rna replication. the nmr structure of 2gdt has been solved, 29 and 250 sequences have been identified by basic local alignment search tool (blast). nsp1 interacts with proteins of the alpha dna polymerase ( figure 2 ) and is involved in regulating endonucleolytic rna cleavage of mrna, allowing the virus to enrich viral rna within a cell. 30, 31 nsp1 has been shown to interact with ribosomal subunits, resulting in the inhibition of translation, 5′ mrna capping changes, and mrna destabilization. 32−34 from a sars-cov yeast two hybrid screen, nsp1 was identified to interact with immunophilins, showing that it alters the intracellular immune response. 35 expression of nsp1 drives changes in interferon signaling. 36 these processes make nsp1 a potential virulence factor. 36−38 see prokoplab.com/ nsp1 for additional information. the protein has no solved protein structure, with itassergenerated predictions 39 that are mostly (67%) coiled, and 246 sequences have been identified by blast. all of the protein interaction partners are acetylated ( figure 2 ). the protein has been suggested to be dispensable to viral replication but does impact rates of replication. 40 see prokoplab.com/nsp2 for additional information. the protein has hundreds of solved x-ray crystal structures with a c4 zinc finger, and 3180 sequences have been identified by blast. the papain-like proteinase cleaves the first four nsp proteins, 41 where inhibition can block viral replication. 42 the proteinase can cleave proteins and has been shown to have deubiquitinase activity. 43−45 this deubiquitinase function has been linked to the regulation of immune system cytokine response, 46,47 specifically the type-i interferon signaling pathway, 48 and has connection to virulence. 49 see prokoplab. com/papain_like_proteinase for additional information. the protein has no solved protein structure, with itassergenerated predictions 39 that are mostly (58%) coiled, and 3325 sequences have been identified by blast. nsp4 interacts with several proteins involved in mitochondrial import for inner membrane insertion (figure 2 ). nsp4 and nsp3 interact and form within the membrane and are involved in transcription complex assembly anchoring. 50, 51 the complex is involved in the double membrane secretory vesicle formation 52, 53 in the endoplasmic reticulum, 54 conferring with human protein interaction partners. 28 see prokoplab.com/nsp4 for additional information. nsp5 has hundreds of solved x-ray crystal structures, with the protein found in a dimer form with a cysteine protease function, 55, 56 and 3397 sequences have been identified by blast. the enzyme cleaves most of the proteins of the larger rep protein with a highly conserved specificity, where inhibition is one of the most studied interventions. 57−59 see prokoplab.com/3c-like_proteinase for additional information. the protein has no solved protein structure, with itassergenerated predictions 39 that are mostly (63%) coiled, and 2558 sequences have been identified by blast. nsp6 interacts with multiple proteins involved in atp hydrolysis-coupled cation transmembrane transport ( figure 2 ). the protein is likely transmembrane-localized, along with nsp3/nsp4, 60 and is involved in autophagosome formations. 61−63 the few papers discussing nsp6 suggest a major future area of understanding and pharmaceutical intervention potential. see prokoplab. com/nsp6 for additional information. there are several solved structures for nsp7 that interact with nsp12/nsp8 (6nur, 2ahm, and 3ub0), 64−66 and 3256 sequences have been identified by blast. the nsp7 protein interacts with multiple small gtpases of the ras complex, many of which are prenylation-regulated ( figure 2 ). the nsp7/nsp8/nsp12 complex is a viral rna-directed rna polymerase unit, where nsp12 is enhanced through the binding of nsp7/nsp8. 67 see prokoplab.com/nsp7 for additional information. there are several solved structures for nsp8 that interact with nsp12/nsp7 (6nur and 3ub0), 64−66 and 3339 sequences have been identified by blast. the nsp8 protein interacts with proteins involved in translation, snrna 3′-end processing, 7s rna binding, and ribonucleoproteins ( figure 2 ). in addition to the information provided for nsp7, nsp8 has been suggested to also interact with the orf6 protein. 68 see prokoplab.com/nsp8 for additional information. nsp9 has many known protein structures, with the protein requiring dimerization to function, 69 and 3386 sequences have been identified by blast. nsp9 interacts with multiple proteins of structural constituents of the nuclear pore ( figure 2 ). nsp9 and nsp10 interact with the nuclear factor-κb repressing factor (nkrf) and may cause an interleukin (il)-8/il-6-mediated chemotaxis of neutrophils and an overexuberant host inflammatory response. 70 nsp9 is involved in viral rna synthesis and rna binding, which likely evolved from a protease. 71, 72 see prokoplab.com/nsp9 for additional information. nsp10 has many known protein structures, including those interacting with nsp14 and nsp16, and contains two zinc binding motifs; 73 3344 sequences have been identified by blast. nsp10 stimulates nsp14 3′−5′ exoribonuclease/ mismatch excision 74, 75 and nsp16 2′-o-methyltransferase activities. 76, 77 the interface of interaction with nsp14 and nsp16 overlaps, suggesting a dynamic regulation process 78 that may involve the linkage of functions through a spherical dodecameric structure. 79 a peptide-based inhibition of the nsp10 interaction has been proposed as a potential viral regulator. 80 see prokoplab.com/nsp10 for additional information. nsp11 is a little-known small 1.3 kda peptide with few interaction partners. 28 nsp12 has multiple known protein structures with a zinc active site and a structure that interacts with nsp7/nsp8, 64−66 and 5086 sequences have been identified by blast. nsp12 is involved in the replication of plus-strand rna through complement strand synthesis and then viral rna synthesis. 81 the enzyme is highly targeted for therapeutic inhibition of viruses. it is also known as rdrp and is the target of the drug remdesivir. see prokoplab.com/rna-directed_rna_polymerase for additional information. nsp13 has multiple known protein structures with a zinc active site, and 5598 sequences have been identified by blast. nsp13 interacts with multiple proteins involved in the centrosome−golgi apparatus and centrosome ( figure 2 ) and has a rna and a dna duplex-unwinding ability to separate strands with 5′ to 3′ polarity. 82−84 see prokoplab.com/helicase for additional information. nsp14 has multiple known protein structures with a zinc active site, and 2794 sequences have been identified by blast. nsp14 has an s-adenosyl-l-methionine (sam)-binding pocket and an exoribonuclease function that is involved in rna capping, 85−87 and it interacts with nsp10 and is known to interact with the human ddx1 rna helicase to enhance the virus replication. 88 see prokoplab.com/guanine-n7_ methyltransferase for additional information. nsp15 has multiple known protein structures, and 2489 sequences have been identified by blast. nsp15 is a mn 2+dependent toric monomer to the hexamer enzyme involved in uridylate-specific cleavage 89,90 that may be regulated by nsp7/ nsp8, 91 and it interacts with the retinoblastoma protein to impact the cell cycle 92 and is also known as nendou. see prokoplab.com/uridylate-specific_endoribonuclease for additional information. nsp16 has multiple known protein structures with na, mg, and s-adenosyl-l-methionine (sam), and 2495 sequences have been identified by blast. nsp16 is an sam-based enzyme for the methylation of ribose 2′-oh in viral rna capping 93, 94 and interacts with nsp10. 77 the protein is a critical component in the inhibition of the host type-i interferon response 95 and is also known as 2′-o-mtase. see prokoplab.com/2-omethyltransferase for additional information. the spike surface glycoprotein has multiple known protein structures that are heavily glycosylated and form a trimer complex 96, 97 and is a known structure of the interaction with the dimer of heterodimers ace2/slc6a19 (6m17); 3 6612 sequences have been identified by blast. s is a class-i viral fusion protein 98 and drives the specificity of cell targets through the interaction with ace2 to enter human cells. 99, 100 following binding to the receptor, s undergoes a conformational change to allow viral entry. 101 for the protein to function correctly, it must be proteolytically cleaved by trypsin and, upon cell-binding proteases such as tmprss2, elevate entry through the mediating tropism. 102 s is of interest to the development of immunizations and rapid detection of coronaviruses, as its surface is exposed. 103, 104 see prokoplab. com/spike for additional information. orf3a has no solved protein structure, with itassergenerated predictions, 39 and 65 sequences have been identified by blast. orf3a is a three transmembrane helix protein where the extracellular component localizes the protein to the golgi apparatus with a caveolin-1 binding potential 105 and is involved in the formation of viral particles. 106, 107 orf3a has been shown to impact the cell cycle. 108 see prokoplab.com/3a for additional information. the envelope protein (e) has no solved protein structure, with itasser-generated predictions, 39 and 94 sequences have been identified by blast. e is required for viral particle formation 109 with transmembrane helix-forming pentameric αhelical bundles with channel activity 110 that can contribute to the membrane permeability. 111 see prokoplab.com/e for additional information. the membrane protein (m) has no solved protein structure, with itasser-generated predictions, 39 and 1507 sequences have been identified by blast. m has human interaction partners that are involved in the mitochondrial matrix ( figure 2 ) and is a critical component of viral membranes that are involved in viral budding. 112 see prokoplab.com/m for additional information. orf6 has no solved protein structure, with itassergenerated predictions, 39 and 31 sequences have been identified by blast. two of the interaction partners are involved in the transcription-dependent tethering of rna polymerase ( figure 2 ). orf6 can function toward the inhibition of beta interferons 113 through the regulation of the signal transducer and activator of transcription 1 (stat1) 114 and endoplasmic reticulum (er) stress 115 and can interact with nsp8. 68 see prokoplab.com/orf6 for additional information. orf7a has multiple known protein structures, and 42 sequences have been identified by blast. orf7a has protein interaction partners involved in ribosomal large subunit biogenesis ( figure 2 ) and localizes to the er and golgi network. 116 it can regulate the cell cycle in g0/g1 progression. 117 see prokoplab.com/7a for additional information. orf8 has no solved protein structure, with itassergenerated predictions, 39 and 35 sequences have been identified by blast. orf8 has multiple interaction partners involved in the er lumen (figure 2) and is a protein shared with sarsr-batcov, with a high positive selection. 118 see prokoplab.com/ orf8 for additional information. the nucleocapsid protein (n) has multiple known protein structures, and 2261 sequences have been identified by blast. n has protein interaction partners involved in mrna binding, the ribonucleoprotein complex, and the mrna surveillance pathway (figure 2 ) and is critical for the viral replication 119 in multiple processes, including viral rna stability, replication, and packaging. 120 the protein is modified within the cell, including phosphorylation and adpribosylation. 121, 122 the protein consists of three domains, with the n-terminal domain involved in rna binding, the internal dynamic multimer structured unit, and the c-terminal domain, an acidic dimerization region. 123−125 the protein can interact with rna by serving as a rna chaperone 126 while also interacting with the m protein and human hnrnpa1 through the internal multimerization domain. 127, 128 see prokoplab.com/n for additional information. orf10 has no solved protein structure, with itassergenerated predictions, 39 and is unique to sars-cov-2. very little is known of its molecular function or cellular expression. see prokoplab.com/orf10 for additional information. ■ sars-cov-2 risk factors and genetics based on human protein interactions sars-cov-2 infection exhibits more adverse effects and outcomes in those with other comorbidities, including hypertension, diabetes mellitus, and coronary heart disease. the other risk factors for mortality include older age, elevated d-dimer levels, and a higher sequential organ failure assessment (sofa) score. 129 the mortality associated with sars-cov-2 infection is tied to the patient's progression to multiorgan dysfunction. the elderly are particularly susceptible to severe sars-cov-2 infection, which is most likely due to the immunosuppression and underlying comorbidities associated with advanced age. advanced age has been shown to have a depressive effect on both the innate and the adaptive immune system, known as immunosenescence. this is associated with decreased phagocytosis and the bactericidal effects of neutrophils 130 and is also associated with the downregulation of cytokine signaling 131,132 and innate immune receptors. 133 with sars-cov-2 infection, emphasis is placed on the adaptive immune system to aid in clearing virally infected cells. the elderly population has been shown to have a shift toward inhibitory pathways, particularly in cd8+ t cells and to a lesser degree in cd4+ t cells, 134 which may play a role in allowing disseminated viral spread. this reduction of t cell activity is also joined by the involution of the thymus with age, leading to less naive t cell output, 135 which further depresses immune functions. these accumulative effects on the immune system render the elderly population particularly susceptible to dispersed viral infection at baseline levels, which may ultimately result in viral sepsis. with the immunosenescence and increased prevalence of comorbidities associated with older age, it makes sense that this population is being hit the hardest by sars-cov-2; however, many younger adults who lack the above immunosenescence have also been killed from the infection, some of whom displayed no prior medical history. this aspect points to the idea that genetics may play a role in determining the severity of sars-cov-2 infection. the immune response to sars-cov-2 infection in severe cases characteristically induces lymphopenia, particularly of cd-8+ t cells, and increases il-2, il-6, il-10, and interferon (ifn)-γ levels. 136 this work is backed by multiple proteomic studies identifying biomarkers of severity that connect to the immune system. 21, 22 the cytokine storm induced by sars-cov-2 is not a new phenomenon and has been demonstrated in the pathogenesis of other novel human coronaviruses, including mers and sars-cov-1. 137 similar consequences in severe coronavirus infections appear to stem from the cytokine storm of proinflammatory chemokines and cytokines, eventually resulting in acute respiratory distress syndrome (ards) and multiorgan dysfunction. 138 a previous study on sepsis and cytokine storm indicates the presence of genetic variants in multiple pathways that have a polygenetic contribution. 139 in many patients with sars-cov-2 that have severe infection, the identification of hyperferritinemic sepsis often occurs. fever developed at day 1, sepsis developed at day 10, admission to the intensive care unit occurred at day 12 (for acute respiratory distress syndrome), and death occurred at day 19. critically ill patients, defined as those with septic shock, multiple organ dysfunction/failure, and/or respiratory failure, accounted for approximately 5% of the study population, yet the study population displayed a case fatality rate of 49.0% in early reports from wuhan, china. 129 hyperferritinemia on day 4 and day 7 predicts mortality long before the development of sepsis and intensive care unit admission. hyperferritinemia has been suggested to have genetic associations through pathogenic variants in genes targetable by il1rap and anti-c5 antibodies. 140 type-1 interferonopathies, like heterozygous null variants in irf7, have been shown to result in severe manifestations of seasonal influenza virus. 141 similar monogenetic variants likely exist that lead to the individual risk of severe disease onset from sars-cov-2 in previously healthy patients. much of the genetics around the immune activation leading to a cytokine storm and hyperferritinemic sepsis remains poorly defined and requires future initiatives and cohorts to define these genetic contributions adequately. initial sars-cov-2 infection is commonly associated with fever, cough, malaise, and fatigue. 142 in more severe cases, disseminated intravascular coagulation has been noted with elevated d-dimer levels in the serum of severe covid-19 patients, placing them into thromboembolic risk. 143 recent recommendations have been made to utilize thromboprophylaxis or full-anticoagulation therapy for patients in the thromboembolic risk category. 144 a specific protein−protein interaction was discovered between sars-cov-2's orf8 and the tissue plasminogen activator (tpa) protein of hosts. 28 the tpa, which is encoded by the plat gene, plays a crucial role in thrombolysis by catalyzing the conversion of plasminogen to plasmin, the major enzyme involved in lysis of blood clots. increased the activity of tpa can lead to excessive bleeding, whereas decreased activity is associated with thromboembolus formation, 145 increasing the chances of pulmonary embolism, stroke, and myocardial infarction. the extent to which orf8 interacts with tpa is not well understood, but its involvement may render a patient at risk for thromboembolism, as has been seen in the clinical setting. in a study by ladenvall et al., it was found that the discovered eight single nucleotide polymorphisms and the alu insertion polymorphism at the plat locus were not significant contributors to plasma tpa levels. 146 this finding indicates that inherited variants of the plat gene may not be directly involved with the coagulopathy in sars-cov-2 patients; however, the polymorphisms may render the host's tpa protein to a tighter binding by orf8, yielding greater repression during infection, placing the patient at higher risk for thromboembolism. it has also been shown that sepsis involves upregulation of platelet adhesion molecules and increased circulation of platelet−leukocyte aggregates. 147 this may point toward more of an immune-system-catalyzed coagulopathy, resulting in the presentation of strokes, 148 pulmonary embolisms, 149 myocardial infarctions, 150 and microvascular injury, 151 which impact severe sars-cov-2 patients. as coagulopathy has mainly been investigated in both viral and bacterial sepsis, there may be a dual effect of both the immune-mediated response and the protein−protein interaction of orf8 with the host tpa in cases of sars-cov-2 infection. further investigation is warranted to determine the extent of the interaction of orf8 with the host tpa to determine if it plays into the pathogenesis of sars-cov-2-related coagulopathy. sars-cov-2 has been associated with cardiac dysfunction, including myocardial infarction and heart failure. the underlying mechanisms for cardiac injury currently being hypothesized are indirect cardiac injury from the cytokine storm and inflammatory response, 152 severe hypoxia as a result of ards, 153 and direct viral invasion of cardiomyocytes. 154 interestingly, ace2, the host receptor for sars-cov-2, is expressed in the heart, 155 indicating direct viral invasion could be a potential cause of myocardial dysfunction. sars-cov-2's nonstructural protein 9 (nsp9) was found to interact with the e3-ubiquitin ligase mindbomb homologue 1 (mib1). 28 this ubiquitin ligase is a positive regulator of the delta-mediated notch signaling pathway, which is involved in multiple processes during cardiac development. 156 mutations in the mib1 have been associated with left ventricular noncompaction (lvnc) characterized by left ventricular trabeculations and reductions in cardiac systolic function. lvnc can range from being asymptomatic to presenting heart failure, depending on the extent the mutation has on the notch pathway. 157 the prevalence of lvnc in the general population is estimated to be around 1/5000 to 1/ 30000. patients with the asymptomatic form of lvnc may be at higher risk for exacerbation of cardiac dysfunction following sars-cov-2 due to involvement of this pathway, especially if they are unaware that they have this mutation. this may play a role in the cardiac dysfunction seen in younger sars-cov-2 patients who lack underlying comorbidities. aside from cardiac development, the notch pathway has also been implemented in cardiac repair, which was demonstrated in rat models where the notch 3 and notch 4 pathways were upregulated, thereby reducing postmyocardial infarctions in the setting of heart failure. 158 the mechanism behind the repair process is still under investigation; however, the disruption of the notch signaling pathway by the interaction of nsp9 with mib1 may prove to play a role in the cardiac involvement of sars-cov-2 infection. furthermore, although vertical transmission of sars-cov-2 has not been seen, 159 neonates who have tested positive for the infection may need to have their cardiac function assessed over time due to mib1's role in cardiogenesis and repair. while we present a detailed assessment of two interaction partners' connections to pathology and risk factors, many more likely exist. we postulate that if function of any protein diverges from normal biology to contribute to sars-cov-2 biology, it could result in a similar disease state within the cell as a loss of function or deleterious genetic mutation. thus, to journal of proteome research pubs.acs.org/jpr reviews understand the sars-cov-2-connected diseases through the human protein interactions, we assessed clinvar, a database of clinically identified variants. a query of the 332 sars-cov-2 human interaction partners through clinvar reveals 8311 protein-based variants within the list ( figure 3a ). in total, 188 of the queried 332 genes have a clinvar submission. of these clinvar-connected genes, there are a total of 111 that have a clinical annotation of pathogenic (pathogenic or likely pathogenic), with a total of 2386 different variants ( figure 3b ). the gene with the most pathogenic variants is fbn1, which is known to interact with sars-cov-2 nsp9 and is involved in autosomal dominant familial thoracic aortic aneurysms and aortic dissections and marfan syndrome. a further analysis of clinical disorders connected with those genes with 10 or more pathogenic-associated variants, excluding fbn1 (pkp2, acadm, ppt1, wfs1, col6a1, pcnt, fbn2, bcs1l, ngly1, cyb5r3, acad9, neu1, gnb1, nars2, tcf12, npc2, pigo, cdk5rap2, cenpf, ggcx, fkbp10, tbk1, fbln5, exosc3 , por, gpaa1, and rhoa), reveals a high connection to cardiac, neurological, diabetic, and syndromic biology. the sars-cov-2 orf8 has the most genes connected by interactions to pathogenic clinvar returns from queried genes, followed by protein m, nsp13, nsp7, and orf9c ( figure 3c ). orf8 is connected to 18 genes associated to human genetic diseases (col6a1, ngly1, neu1, npc2, fkbp10, dnmt1, plod2, smoc1, il17ra, adam9, sil1, lox, pofut1, tor1a, hyou1 , edem3, emc1, and hs6st2), with significant enrichment of these genes to protein folding (false discovery rate (fdr) = 0.00095) and endoplasmic reticulum lumen (fdr = 2.99 × 10 −5 ). while only associated with 3 pathogenic interaction partners, the nucleocapsid (n) protein has interesting disease genetics based on previous observations of a process known as viral-induced genetics. 160 the nucleocapsid (n) protein has the potential to impact and change cellular landscapes through the direct regulation of ribosomal biology. 161 the protein−protein interaction map by gordon et al. 28 supports the hypothesis that sars-cov-2 n proteins interact with multiple mrna-binding proteins and ribonucleoprotein complex proteins ( figure 2 ). in multiple viruses, proteins have been shown to interact with these complexes to regulate a process known as nonsense-mediated decay (nmd). 162 nmd is a cellular process involved in the removal of mrna that does not conform to the bulk of cellular mrna, where proteins accumulate on the transcript and direct the cellular degradation of the mrna. 163 the process is primarily used within cells to degrade mrna molecules with nonsense and frameshift genetic variants and those with improper splicing to prevent the cell from producing truncated proteins that can drive dominant-negative or deleterious gain of function outcomes. 164 viral rna is usually suppressed and degraded within cells through nmd, acting as a cellular immune system process. 165−167 thus, an evolutionary arms race has arisen where a virus can propagate more efficiently if it has a protein that can suppress nmd, keeping its rna levels elevated. 168−171 multiple lines of evidence for both sars-cov-2 and sars-cov suggested that the n protein is used to suppress nmd and evade cellular immune processes. nearly all of the coronaviruses and the larger nidovirales order genomes contain the n protein, which has been shown to interact with multiple ribosomal proteins, including crucial nmd factors. 28 directly inhibited by nmd, with the n protein expression blocking this inhibition. 162 positive-sense single-stranded rna viruses, including coronaviruses, are likely targets of nmd due to their many overlapping reading frames, retained introns, and long 3′ utrs present within the cytoplasm of human cells. the n protein interacts with three proteins annotated to the mrna surveillance pathway (upf1, pabpc1, and pabpc4) and several proteins involved in mrna binding and the ribonucleoprotein complex that are all known to have cellular interactions (figure 2 ). while the fine details of the n protein interaction on the factors are poorly understood, the three mrna surveillance genes are well-connected to nmd biology. pabpc1 is known to be critical for nmd, with its removal suppressing nmd. 175, 176 from plants to humans, upf1 is considered a key regulator of nmd through its recruitment of multiple proteins to rna. 177−180 the regulation switch of upf1 is known to be regulated/activated through phosphorylation at various sites to allow its protein interactions, 181−183 while the n protein has been shown in multiple viral species to be phosphorylated 184−188 and likely dynamic in modifications throughout the rna replication and viral lifecycle. 122, 189 these phosphorylation switches and the interaction of n to nmd proteins are potential sites of pharmaceutical or biological regulation that have been undervalued to this point. other notable interactions of the n protein are ras gtpaseactivating protein-binding protein homologues (g3bp1/2) and casein kinase 2 alpha (csnk2a2), 28 suggesting the regulation of stress granule formation. nmd is found at the intersection of a variety of cellular pathways beyond mrna surveillance and viral control. notably, it is closely associated with the integrated stress response requiring translation initiation factor eif2s1 for function. 190 cellular stresses such as hypoxia and er stress lead to the inhibition of nmd via phosphorylation of eif2s1. 190 this phosphorylation typically induces stress granule formation as well, which has been cited to aid viral replication in some cases and weaken them in others. 191 when g3bp1 is depleted within cells, there is a significant impairment of the replication for coronaviruses and respiratory syncytial viruses (rsvs). 191, 192 multiple viruses have been shown to regulate phosphorylation of eif2s1 at varying time points of infection with connection into nmd regulation. 193 stress granule formation can enhance nmd inhibition, such as hypoxic conditions modulating upf1 and eif2s1. 194, 195 the interaction of sars-cov-2 n protein human interactors promotes the inhibition of nmd and enhancement of both viral replication and truncated host polypeptides that can enhance viral pathogenicity (figure 4) . the regulation of nmd by viral proteins is crucial for allowing the viral rna to survive, but nmd processes within cells also regulate multiple endogenous transcripts, several in normal biology, and some based on genetic disease regulation. many genes, including isoforms with early truncation (frameshifts and nonsense codons) and genes involved in amino acid homeostasis, tumorigenesis, and cell cycle control, are activated when nmd is inhibited within a cell. 196−199 in total, this amounts to about 10% of transcripts within a cell that are regulated by nmd processes and could be altered within the cell by sars-cov-2. 196 on top of this, most individuals contain at least one gene where a nonsense or frameshift variant within the genome is being suppressed, being either inherited or somatic. assessments of human genomes reveal that every person has at least one variant regulated by nmd. 200 recently, our group has shown a complex involvement of this regulation with rare human variants, driving adverse outcomes through a process we have termed viral-induced genetics (vig). 160 in a patient with an epstein−barr virus (ebv) infection, they had an adverse immune response of classical hyperferritinemic sepsis like that of severe sars-cov-2 patients. this individual has both whole-exome sequencing in addition to multiple bloodbased rnaseq experiments performed throughout their clinical course. sequencing revealed a heterozygous splicing variant in the gene rnaseh2b, which is associated with recessive aicardi−goutieres syndrome 201 and has been connected to type-i ifn-mediated autoimmune disease. 202, 203 rnaseq of the patient, when healthy, showed that the splicing variant was present at very low levels, suggesting that the copy was being inhibited through nmd. while the patient was healthy for 16 years of life, the ebv was shown by rnaseq to inhibit nmd, resulting in the presence of the splice variant, which resulted in a dominant-negative rnaseh2b protein that drove cell dysfunction. this suggests that many human variants within genes connected to the immune system and viral response, which are usually suppressed by nmd and result in no cellular dysfunction, are activated by the virus through the inhibition of nmd and can give rise to severe viral outcomes. just like in a computer virus, the antivirus of the computer is often targeted. when additional computer code contains a risk to system failure that is inhibited by the antivirus, if the antivirus is shut down, the other system vulnerabilities become present and often contribute to the computer failure. the full extent of these variants and the disease process remain to be elucidated but is a promising avenue for exploration of viral-induced outcomes in sars-cov-2. the sars-cov-2 pandemic represents a unique challenge to scientists. unlike previous pandemics, our knowledge of the genome and its coded proteins was gleaned within weeks of the outbreak, now with thousands of sequences within a short window. this level of insight has allowed for a pivot to a more robust insight into the viral proteome and how it interacts with host proteins. the advancement of protein-based bioinformatics and previous coronavirus research studies have proved useful in defining the function of each protein coded by the virus. here, we show how many of these viral proteins interact with human proteins connected to biological pathways and disease connections, including numerous risk factors from immune to cardiovascular systems. most notably, we highlight literature on the role of the viral nucleocapsid (n) protein in nmd regulation, where the inhibition of nmd allows for viral rna stability while simultaneously activating genetics of cellular processes and viral-induced genetics. while we have seen thousands of publications on sars-cov-2 and other coronaviruses, the details of a proteome-wide knowledge base of sars-cov-2-coded proteins limit our ability to expand into the incredible potential of preventing and mitigating the current pandemic and future pandemics with a larger therapeutic toolset. characterization of coding/noncoding variants for shroom3 in patients with ckd molecular modeling in the age of clinical genomics, the enterprise of the next generation sars-cov-2 (covid-19) structural and evolutionary dynamicome: insights into functional evolution and human genomics expression of the sars-cov-2 cell receptor gene ace2 in a wide variety of human tissues ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia receptor ace2 and tmprss2 are primarily expressed in bronchial transient secretory cells hca lung biological network. sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes proteomics of sars-cov-2-infected host cells reveals therapy targets single-cell landscape of bronchoalveolar immune cells in patients with covid-19 eils, r. covid-19 severity correlates with airway epithelium-immune cell interactions identified by single-cell analysis imbalanced host response to sars-cov-2 drives development of covid-19 transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients proteomic and metabolomic characterization of covid-19 proteomic fingerprints for potential application to early diagnosis of severe acute respiratory syndrome the sars coronavirus nucleocapsid protein-forms and functions nucleocapsid phosphorylation and rna helicase ddx1 recruitment enables coronavirus transition from discontinuous to continuous transcription virus-host interactomes-antiviral drug discovery host factors in coronavirus replication novel beta-barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage suppression of host gene expression by nsp1 proteins of group 2 bat coronaviruses a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein severe acute respiratory syndrome coronavirus protein nsp1 is a novel eukaryotic translation inhibitor that represses multiple steps of translation initiation severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain unique sars-cov protein nsp1: bioinformatics, biochemistry and potential effects on virulence coronavirus nonstructural protein 1: common and distinct functions in the regulation of host and viral gene expression protein structure and sequence reanalysis of 2019-ncov genome refutes snakes as its intermediate host and the unique similarity between its spike protein insertions and hiv-1 the nsp2 proteins of mouse hepatitis virus and sars coronavirus are dispensable for viral replication identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication deubiquitinating activity of the sars-cov papainlike protease the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme sars coronavirus papain-like protease inhibits the tlr7 signaling pathway through removing lys63-linked polyubiquitination of traf3 and traf6 coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex the papain-like protease determines a virulence trait that varies among members of the sars-coronavirus species structure and cleavage specificity of the chymotrypsin-like serine protease (3clsp/nsp4) of porcine reproductive and respiratory syndrome virus (prrsv) mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication replication is supported by a reticulovesicular network of modified endoplasmic reticulum 3c-like proteinase from sars coronavirus catalyzes substrate hydrolysis by a general base mechanism dissection study on the severe acute respiratory syndrome 3c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase the substrate specificity of sars coronavirus 3c-like proteinase topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate evolutionary analysis of sars-cov-2: how mutation of non-structural protein 6 could affect viral autophagy coronavirus nsp6 restricts autophagosome expansion structure of the sars-cov nsp12 polymerase bound to nsp7 and nsp8 co-factors insights into sars-cov transcription and replication from the structure of the nsp7-nsp8 hexadecamer nonstructural proteins 7 and 8 of feline coronavirus form a 2:1 heterotrimer that exhibits primer-independent rna polymerase activity the sars-coronavirus nsp7+nsp8 complex is a unique multimeric rna polymerase capable of both de novo initiation and primer extension the nonstructural protein 8 (nsp8) of the sars coronavirus interacts with its orf6 accessory protein severe acute respiratory syndrome coronavirus nsp9 dimerization is essential for efficient viral growth virus-host interactome and proteomic survey of pmbcs from covid-19 patients reveal potential virulence factors influencing sars-cov-2 the nsp9 replicase protein of sars-coronavirus, structure and functional insights the severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded rna-binding subunit unique in the rna virus world crystal structure of nonstructural protein 10 from the severe acute respiratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs rna 3′-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/ nsp14 exoribonuclease complex structural basis and functional analysis of the sars coronavirus nsp14-nsp10 complex in vitro reconstitution of sars-coronavirus mrna cap methylation crystal structure and functional analysis of the sars-coronavirus rna cap 2′-o-methyltransferase nsp10/nsp16 complex coronavirus nsp10, a critical co-factor for activation of multiple replicative enzymes dodecamer structure of severe acute respiratory syndrome coronavirus nonstructural protein nsp10 coronavirus nsp10/nsp16 methyltransferase can be targeted by nsp10-derived peptide in vitro and in vivo to reduce replication and pathogenesis molecular anatomy of viral rna-directed rna polymerases multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase the severe acute respiratory syndrome (sars) coronavirus ntpase/helicase belongs to a distinct class of 5′ to 3′ viral helicases the human coronavirus 229e superfamily 1 helicase has rna and dna duplex-unwinding activities with 5′-to-3′ polarity structure-function analysis of severe acute respiratory syndrome coronavirus rna cap guanine-n7-methyltransferase functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase characterization of the guanine-n7 methyltransferase activity of coronavirus nsp14 on nucleotide gtp the cellular rna helicase ddx1 interacts with coronavirus nonstructural protein 14 and enhances viral replication crystal structure and mechanistic determinants of sars coronavirus nonstructural protein 15 define an endoribonuclease family crystal structure of a monomeric form of severe acute respiratory syndrome coronavirus endonuclease nsp15 suggests a role for hexamerization as an allosteric switch structural and biochemical characterization of endoribonuclease nsp15 encoded by middle east respiratory syndrome coronavirus binding of the methyl donor s-adenosyl-l-methionine to middle east respiratory syndrome coronavirus 2′-o-methyltransferase nsp16 promotes recruitment of the allosteric activator nsp10 coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2′o)-methyltransferase activity ribose 2′-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 assembly of coronavirus spike protein into trimers and its role in epitope expression cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex coronavirus spike proteins in viral entry and pathogenesis structure of sars coronavirus spike receptor-binding domain complexed with receptor characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry mechanisms of coronavirus cell entry mediated by the viral spike protein severe 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nucleocapsid phosphorylation nonsense-mediated mrna decay at the crossroads of many cellular pathways valiente-echeverría, f. strategies for success. viral infections and membraneless organelles innate immune evasion by human respiratory rna viruses mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies hypoxic inhibition of nonsense-mediated rna decay regulates gene expression and the integrated stress response possible roles in the control of translation and mrna degradation. cold spring harbor perspect nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise nonsense-mediated rna decay regulation by cellular stress: implications for tumorigenesis nonsense-mediated mrna decay factors act in concert to regulate common mrna targets nonsense-mediated mrna decay in health and disease the rules and impact of nonsense-mediated mrna decay in human cancers a novel rnaseh2b splice site mutation responsible for aicardi-goutieres syndrome in the faroe islands genetically defined autoinflammatory diseases rare adar and rnaseh2b variants and a type i interferon signature in glioma and prostate carcinoma risk and tumorigenesis key: cord-256156-mywhe6w9 authors: clausen, thomas mandel; sandoval, daniel r.; spliid, charlotte b.; pihl, jessica; perrett, hailee r.; painter, chelsea d.; narayanan, anoop; majowicz, sydney a.; kwong, elizabeth m.; mcvicar, rachael n.; thacker, bryan e.; glass, charles a.; yang, zhang; torres, jonathan l.; golden, gregory j.; bartels, phillip l.; porell, ryan; garretson, aaron f.; laubach, logan; feldman, jared; yin, xin; pu, yuan; hauser, blake; caradonna, timothy m.; kellman, benjamin p.; martino, cameron; gordts, philip l.s.m.; chanda, sumit k.; schmidt, aaron g.; godula, kamil; leibel, sandra l.; jose, joyce; corbett, kevin d.; ward, andrew b.; carlin, aaron f.; esko, jeffrey d. title: sars-cov-2 infection depends on cellular heparan sulfate and ace2 date: 2020-09-14 journal: cell doi: 10.1016/j.cell.2020.09.033 sha: doc_id: 256156 cord_uid: mywhe6w9 we show that sars-cov-2 spike protein interacts with both cellular heparan sulfate and angiotensin converting enzyme 2 (ace2) through its receptor binding domain (rbd). docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ace2 binding site. both ace2 and heparin can bind independently to spike protein in vitro and a ternary complex can be generated using heparin as a scaffold. electron micrographs of spike protein suggests that heparin enhances the open conformation of the rbd that binds ace2. on cells, spike protein binding depends on both heparan sulfate and ace2. unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic sars-cov-2 virus. we suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ace2. manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities. the covid-19 pandemic, caused by the novel respiratory coronavirus 2 (sars-cov-2), has swept across the world, resulting in serious clinical morbidities and mortality, as well as widespread disruption to all aspects of society. as of september 1, 2020, the virus has spread to 215 countries, causing more than 25.4 million confirmed infections and at least 851,000 deaths (world health organization). current isolation/social distancing strategies seek to flatten the infection curve to avoid overwhelming hospitals and to give the medical establishment and pharmaceutical companies time to develop and test antiviral drugs and vaccines. currently, only one antiviral agent, remdesivir, has been approved for adult covid-19 patients (beigel et al., 2020) and vaccines may be 12-18 months away. understanding the mechanism for sars-cov-2 infection and its mechanism of infection could reveal other targets to interfere with viral infection and spread. the glycocalyx is a complex mixture of glycans and glycoconjugates surrounding all cells. given its location, viruses and other infectious organisms, must pass through the glycocalyx to engage receptors thought to mediate viral entry into host cells. many viral pathogens have evolved to utilize glycans as attachment factors, which facilitates the initial interaction with host cells, including influenza virus, herpes simplex virus, human immunodeficiency virus, and different coronaviruses (sars-cov-1 and mers-cov) (cagno et al., 2019; koehler et al., 2020; stencel-baerenwald et al., 2014) . several viruses interact with sialic acids, which are located on the ends of glycans found in glycolipids and glycoproteins. other viruses interact with heparan sulfate (hs) (milewska et al., 2014) , a highly negatively charged linear polysaccharide that is attached to a small set of membrane or extracellular matrix proteoglycans (lindahl et al., 2015) . in general, glycan-binding domains on membrane proteins of the virion envelope mediate initial attachment of virions to glycan receptors. attachment in this way can lead to the engagement of protein receptors on the host plasma membrane that facilitate membrane fusion or engulfment and internalization of the virion. j o u r n a l p r e -p r o o f 5 like other macromolecules, hs can be divided into subunits, which are operationally defined as disaccharides based on the ability of bacterial enzymes or nitrous acid to cleave the chain into disaccharide units (esko and selleck, 2002) . the basic disaccharide subunit consists of α1-4 linked d-glucuronic acid (glca) and α1-4 linked n-acetyl-d-glucosamine (glcnac), which undergo various modifications by sulfation and epimerization as the copolymer assembles on a limited number of membrane and extracellular matrix proteins (only 17 heparan sulfate proteoglycans are known) (lindahl et al., 2015) . the variable length of the modified domains and their pattern of sulfation create unique motifs to which hs-binding proteins interact (xu and esko, 2014) . different tissues and cell types vary in the structure of hs, and hs structure can vary between individuals and with age (de agostini et al., 2008; feyzi et al., 1998; han et al., 2020; ledin et al., 2004; vongchan et al., 2005; warda et al., 2006; wei et al., 2011) . these differences in hs composition may contribute to the tissue tropism and/or host susceptibility to infection by viruses and other pathogens. in this report, we show that the ectodomain of the sars-cov-2 spike (s) protein interacts with cell surface hs through the receptor binding domain (rbd) in the s1 subunit. binding of heparin to sars-cov-2 s protein shifts the structure to favor the rbd open conformation that binds ace2. spike binding to cells requires engagement of both cellular hs and ace2, suggesting that hs acts as a coreceptor priming the spike for ace2 interaction. therapeutic unfractionated heparin (ufh), non-anticoagulant heparin and hs derived from human lung and other tissues blocks binding. ufh and heparin lyases also block infection of cells by s protein pseudotyped virus and authentic sars-cov-2. these findings identify cellular hs as a necessary co-factor for sars-cov-2 infection and emphasizes the potential for targeting s protein-hs interactions to attenuate virus infection. the trimeric s proteins from sars-cov-1 and sars-cov-2 viruses are thought to engage human ace2 with one or more rbd in an "open" active conformation (fig. 1a ) (kirchdoerfer et al., 2018; walls et al., 2020; wrapp et al., 2020) . adjacent to the ace2 binding site and exposed in the rbd lies a group of positively-charged amino acid residues that represents a potential site that could interact with heparin or heparan sulfate ( fig. 1a and suppl. fig. s1 ). we calculated an electrostatic potential map of the rbd (from pdb id 6m17 (yan et al., 2020) ), which revealed an extended electropositive surface with dimensions and turns/loops consistent with a heparin-binding site (fig. 1b) (xu and esko, 2014) . docking studies using a tetrasaccharide (dp4) fragment derived from heparin demonstrated preferred interactions with this electropositive surface, which based on its dimensions could accommodate a chain of up to 20 monosaccharides ( fig. 1b and 1c ). evaluation of heparin-protein contacts and energy contributions using the molecular operating environment (moe) software suggested strong interactions with the positively charged amino acids r346, r355, k444, r466 and possibly r509 (figs. 1a, 1d, and 1e) . other amino acids, notably f347, s349, n354, g447, y449, and y451, could coordinate the oligosaccharide through hydrogen bonds and hydrophobic interactions. notably, the putative binding surface for oligosaccharides is adjacent to, but separate from the ace2 binding site, suggesting that a single rbd could simultaneously bind both cell surface hs and the ace2 protein receptor. the putative hs binding site is partially obstructed in the "closed" inactive rbd conformation, while fully exposed in the open state (suppl. fig. s1 ). the amino acid sequence of s protein rbd of sars-cov-2 s is 73% identical to the rbd of sars-cov-1 s (fig. 1f) , and these domains are highly similar in structure with an overall cα r.m.s.d. of 0.929 å (fig. 1g) . however, an electrostatic potential map of the sars-cov-1 s j o u r n a l p r e -p r o o f 7 rbd does not show an electropositive surface like that observed in sars-cov-2 (fig. 1h ). most of the positively charged residues comprising this surface are conserved between the two proteins, with the exception of sars-cov-2 k444 which is a threonine in sars-cov-1 (fig. 1f ). additionally, the other amino acid residues predicted to coordinate with the oligosaccharide are conserved with the exception of asn354 in sars-cov-2, which is a negatively charged glutamate residue in sars-cov-1. sars-cov-1 has been shown to interact with cellular hs in addition to its entry receptors ace2 and transmembrane protease, serine 2 (tmprss2) (lang et al., 2011) . our analysis suggests that the putative heparin-binding site in sars-cov-2 s may mediate an enhanced interaction with heparin or hs compared to sars-cov-1, and that this change evolved through as few as two amino acid substitutions, thr lys444 and glu asn354. to test experimentally if the sars-cov-2 s protein interacts with heparin/hs, recombinant ectodomain and rbd proteins were prepared and characterized. initial studies encountered difficulty in stabilizing the s ectodomain protein, a problem that was resolved by raising the concentration of nacl to 0.3 m in hepes buffer. under these conditions, the protein could be stored at room temperature, 4 o c or at -80 o c for at least two weeks. sds-page showed that each protein was ~98% pure ( j o u r n a l p r e -p r o o f 8 recombinant s ectodomain and rbd proteins were applied to a column of heparin-sepharose. elution with a gradient of sodium chloride showed that the rbd eluted at ~0.3 m nacl, with a shoulder that eluted with higher salt (fig. 2a) . recombinant s ectodomain also bound to heparin-sepharose, but it eluted across a broader concentration of nacl. the elution profiles suggest that the preparations contained a population of molecules that bind to heparin, but that some heterogeneity in affinity for heparin occurs, which may reflect differences in glycosylation, oligomerization or the number of binding sites in the open conformation. the rbd protein from sars-cov-2 also bound in a saturable manner to heparin-bsa immobilized on a plate (fig. 2b ). the rbd domain from sars-cov-1 showed significantly reduced binding to heparin-bsa and a higher k d value (640 nm [95% c.i.; 282 -1852 nm] for sars-cov-1 rbd vs. 150 nm [95% c.i. 123 -173 nm]) for sars-cov-2 rbd), in accordance with the difference in electropositive potential in the proposed hs binding regions (fig. 1h) . a monomeric form of sars-cov-2 s ectodomain protein also bound in a saturable manner to heparin immobilized on a plate (suppl. fig. s3a ). the trimeric protein bound to heparin-bsa with an apparent k d value of 3.8 nm [95% c.i. 3.1 -4.6 nm] (fig. 2c ). binding of recombinant s ectodomain, mutated to lock the rbds into a closed (mut2) or that favors an open (mut7) conformation, showed that the heparin binding site in the rbd domain is accessible in both conformations (fig. 2d ). however, the k d value for mut7 is lower (4.6 nm [95% c.i. 3.8 -5.5 nm] vs. 9.9 nm [95% c.i. 8.7 -11.3 nm] for mut2), which is in line with the partial obstruction of the site in the closed conformation (suppl. fig. s1 ). as expected, only trimer with an open rbd conformation bound to ace2 (fig. 2e ). in contrast to spike protein, ace2 did not bind to heparin-bsa (fig. 2c) . ace2 also had no effect on binding of s protein to heparin-bsa at all concentrations that were tested (fig. 2c , inset). biotinylated ace2 bound to immobilized s protein (suppl. fig. s3b ) and a ternary complex of heparin, ace2 and s protein could be demonstrated by titration of s protein bound to immobilized heparin-bsa with ace2 (fig. 2f ). binding of ace2 under these conditions j o u r n a l p r e -p r o o f 9 increased in proportion to the amount of s protein bound to the heparin-bsa. collectively, these findings show that (i) spike protein can engage both heparin and ace2 simultaneously and (ii) that the heparin binding site is somewhat occluded in the closed conformation, but it can still bind heparin albeit with reduced affinity. the simultaneous binding of ace2 to spike protein and heparin suggested the possibility that heparin binding might affect the conformation of the rbd, possibly increasing the open conformation that can bind ace2. to explore this possibility, spike protein was mixed with ace2 (6-fold molar ratio) with or without dp20 oligosaccharides derived from heparin (9-fold molar ratio). the samples were then stained and analyzed by transmission electron microscopy, and the images were deconvoluted and sorted into 3d reconstructions to determine the number of trimers with 0, 1, 2, or 3 bound ace2 (fig. 2g -h and suppl. fig. s3c-d) . the different populations were counted and the percentage of particles belonging to each 3d class was calculated. two time points were evaluated after mixing ace2 and trimeric s: at 15 min 29,600 and 31,300 particles were analyzed in the absence or presence of dp20 oligosaccharides, respectively; at 60 min, 17,000 and 21,000 particles were analyzed in absence or presence of dp20 oligosaccharides, respectively. at both time points, the presence of dp20 increased the total amount of ace2 protein bound to spike . after 15 minutes in the absence of dp20 very few of the trimers had conformations with 1 or 2 bound ace2 (5% each), whereas the inclusion of dp20 oligosaccharides greatly increased the proportion of trimers bearing one (37%) or two (21%) ace2, with a proportional drop in the unbound conformers from 90% in the absence of heparin to 42% in its presence (fig. 2g ). extending the incubation to 60 minutes resulted in a mixture of trimers containing 1 (45%), 2 (11%) and 3 ace2 (13%) in the absence of heparin. inclusion of dp20 further increased the proportion of bound spike trimers bearing 2 (19%), and 3 (27%) ace2 (fig. 2h) . the imaging studies suggest that, under these j o u r n a l p r e -p r o o f 10 experimental conditions, heparin may stabilize the ace2 interaction, increasing the proportion of spike bound to ace2 as well as the occupancy of individual spikes. the sars-cov-2 spike protein depends on cellular heparan sulfate for cell binding. to extend these studies to hs on the surface of cells, s ectodomain protein was added to human h1299 cells, an adenocarcinoma cell line derived from type 2 alveolar cells (fig. 3a ). spike ectodomains bound to h1299 cells, with half-maximal binding achieved at ~75 nm. treatment of the cells with a mixture of heparin lyases (hsase), which degrades cell surface hs, dramatically reduced binding ( fig 3a) . the s ectodomain also bound to human a549 cells, another type 2 alveolar adenocarcinoma line, as well as human hepatoma hep3b cells (fig. 3b ). removal of hs by enzymatic treatment dramatically reduced binding in both of these cell lines as well (fig. 3b ). recombinant rbd protein also bound to all three cell lines dependent on hs (fig. 3c) . a melanoma cell line, a375, was tested independently and also showed hs dependent binding ( fig 3d) . the extent of binding across the four cell lines varied ~4-fold. this variation was not due to differences in hs expression as illustrated by staining of cell surface hs with mab 10e4, which recognizes a common epitope in hs ( we also measured binding of the s ectodomain and rbd proteins to a library of mutant hep3b cells, carrying crispr/cas9 induced mutations in biosynthetic enzymes essential for synthesizing hs (anower et al., 2019) . inactivation of ext1, a subunit of the copolymerase required for synthesis of the backbone of hs, abolished binding to a greater extent than enzymatic removal of the chains with hsases ( fig. 3f and suppl. fig. s4 ), suggesting that the hsase treatment may underestimate the dependence on hs. targeting ndst1, a glcnac n-j o u r n a l p r e -p r o o f 11 deacetylase-n-sulfotransferase that n-deacetylates and n-sulfates n-acetylglucosamine residues, and hs6st1 and hs6st2, which introduces sulfate groups in the c6 position of glucosamine residues, significantly reduced binding (figs. 3f and suppl. fig. s4 ). although experiments with other sulfotransferases have not yet been done, the data suggests that the pattern of sulfation of hs affects binding to s and rbd. to further examine how variation in hs structure affects binding, we isolated hs from human kidney, liver, lung and tonsil. the samples were depolymerized into disaccharides by treatment with hsases, and the disaccharides were then analyzed by lc-ms (experimental methods). the disaccharide analysis showed that lung hs has a larger proportion of ndeacetylated and n-sulfated glucosamine residues (grey bars) and more 2-o-sulfated uronic acids (green bars) than hs preparations from the other tissues (fig. 4a ). the different hs preparations also varied in their ability to block binding of rbd to h1299 cells (fig. 4b ). interestingly, hs isolated from lung was more potent compared to kidney and liver hs, consistent with the greater degree of sulfation of hs from this organ (suppl. table 1 ). hs from tonsil was as potent as hs from lung, but the overall extent of sulfation was not as great, supporting the notion that the patterning of the sulfated domains in the chains may affect binding. unfractionated heparin is derived from porcine mucosa and possesses potent anticoagulant activity due to the presence of a pentasaccharide sequence containing a crucial 3-o-sulfated nsulfoglucosamine unit, which confers high affinity binding to antithrombin. heparin is also very highly sulfated compared to hs with an average negative charge of -3.4 per disaccharide (the overall negative charge density of typical hs is -1.8 to -2.2 per disaccharide). mst cells, which were derived from a murine mastocytoma, make heparin-like hs that lacks the key 3-o-sulfate group and anticoagulant activity (gasimli et al., 2014; montgomery et al., 1992) . the 12 anticoagulant properties of heparin can also be removed by periodate oxidation, which oxidizes the vicinal hydroxyl groups in the uronic acids, resulting in what is called "split-glycol" heparin (casu et al., 2004) . all of these agents significantly inhibited binding of the s protein to h1299 and a549 cells ( fig. 4c and 4d ) yielding ic 50 values in the range of 0.01-0.12 µg/ml (suppl . table 1 ). interestingly, the lack of 3-o-sulfation, crucial for the anticoagulant activity of heparin, had little effect on its inhibition of s binding. in contrast, cho cell hs (containing 0.8 sulfates per disaccharide) only weakly inhibited binding (ic 50 values of 18 and 139 µg/ml for a549 and h1299, respectively) (suppl. table 1 ). these data suggest that inhibition by heparinoids is most likely charge dependent and independent of anticoagulant activity per se. the experiments shown in fig. 2g -h indicate that binding of heparin to spike protein can increase binding to ace2. to explore if hs, ace2 and spike interact at the cell surface, we investigated the impact of ace2 expression on s protein cell binding. initial attempts were made to measure ace2 levels by western blotting or flow cytometry with different mabs and polyclonal antibodies, but a reliable signal was not obtained in any of the cell lines tested (a375, a549, h1299, and hep3b). nevertheless, expression of ace2 mrna was observed by rt-qpcr (suppl. fig. 5a ). transfection of a375 cells with ace2 cdna resulted in robust expression of ace2 (fig. 5a) , resulting in an increase in s ectodomain protein binding by ~4fold (fig. 5b) . interestingly, the enhanced binding was hs-dependent, as illustrated by the loss of binding of s protein after hsase-treatment (fig. 5b ). crispr/cas9 mediated deletion of the b4galt7 gene, which is required for glycosaminoglycan assembly (suppl. fig. s5b ), also reduced binding of spike protein (fig. 5b ) despite the overexpression of ace2 (fig. 5a ). to explore the impact of diminished ace2 expression, we examined spike protein binding to a549 cells and in two crispr/cas9 gene targeted clones c3 and c6 bearing biallelic mutations in ace2 (suppl. fig. s5c ). binding of s ectodomain protein was greatly reduced in the ace2 -/-j o u r n a l p r e -p r o o f 13 clones and the residual binding was sensitive to hsases (fig. 5c ). these findings show that binding of spike protein on cells requires both hs and ace2, consistent with the formation of a ternary complex (figs. 2f-h). assays using purified components provide biochemical insights into binding, but they do not recapitulate the multivalent presentation of the s protein as it occurs on the virion membrane. thus, to extend these studies, pseudotyped vesicular stomatitis virus (vsv) was engineered to express the full-length sars-cov-2 s protein and gfp or luciferase to monitor infection. vero e6 cells are commonly used in the study of sars-cov-2 infection, due to their high susceptibility to infection. spike protein binding to vero cells also depends on cellular hs as binding was sensitive to hsases, heparin and split-glycol heparin (fig. 6a ). interestingly, hsase treatment reduced binding to a lesser extent than the level of reduction observed in a549, heparin very potently reduced infection more than ~4-fold at 0.5 µg/ml and higher concentrations (fig. 6g) . in contrast, studies of sars-cov-1 s protein pseudotype virus showed that hsase-treatment actually increased sars-cov-1 infection by more than 2-fold, suggesting that hs might interfere with binding of sars-cov-1 in this cell line (fig. 6h ). infection of h1299 and a549 cells by sars-cov-2 s pseudotype virus was too low to obtain j o u r n a l p r e -p r o o f 14 accurate measurements, but infection of hep3b cells could be readily measured (fig. 6i ). hsase and mutations in ext1 and ndst1 dramatically reduced infection 6-to 7-fold. inactivation of the 6-o-sulfotransferases had only a mild effect unlike its strong effect on s protein binding (fig. 3f) , possibly due to the high valency conferred by multiple copies of s protein on the pseudovirus envelope. hep3b cells were not susceptible to infection by sars-cov-1 s protein pseudotyped virus, but was infected by mers-cov s protein pseudotyped virus and infection was independent of hs (suppl. fig. s6 ). studies of pseudovirus were then extended to authentic sars-cov-2 virus infection using strain usa-wa1/2020. infection of vero e6 cells was monitored by double staining of the cells with antibodies against the sars-cov-2 nucleocapsid (n) and s proteins ( heparin inhibition (maroon and blue symbols). to rule out that the treatments caused a decrease in ace2 expression or a reduction in cell viability, vero cells were treated with heparin lyases and 100 µg/ml ufh, and ace2 expression was measured by western blotting and cell viability by celltiter-blue® (suppl. fig. s7a -b) . no effect on ace2 expression or cell viability was observed. these findings further emphasize the potential for using unfractionated heparin or other non-anticoagulant heparinoids to prevent viral attachment. j o u r n a l p r e -p r o o f 15 these findings were then extended to hep3b cells and mutants altered in hs biosynthesis using a viral plaque assay. virus was added to wildtype, ndst1 -/and hs6st1/2 -/cells for 2 hr, the virus was removed, and after 2 days incubation a serial dilution of the conditioned culture medium was added to monolayers of vero e6 cells. the number of plaques were then quantitated by staining and visualization. as a control, culture medium from infected vero e6 cells was tested, which showed robust viral titers. hep3b cells also supported viral replication, but to a lesser extent than vero cells. inactivation of ndst1 in hep3b cells abolished virus production, whereas inactivation of hs6st1/2 -/reduced infection more mildly, ~3-fold (fig. 7d) . hsase and ufh reduced infection more than 5-fold, but it had no effect on cell viability (suppl. in this report, we provide compelling evidence that hs is a necessary host attachment factor that promotes sars-cov-2 infection of various target cells. the receptor binding domain of the sars-cov-2 s protein binds to heparin/hs, most likely through a docking site composed of positively charged amino acid residues aligned in a subdomain of the rbd that is separate from the site involved in ace2 binding (fig. 1) . competition studies, enzymatic removal of hs, and genetic studies confirm that the s protein, whether presented as a recombinant protein (figs. 2-j o u r n a l p r e -p r o o f 16 5), in a pseudovirus (fig. 6) , or in authentic sars-cov-2 virions (fig. 7) , binds to cell surface hs in a cooperative manner with ace2 receptors. mechanistically, binding of heparin/hs to spike trimers enhances binding to ace2, likely increasing multivalent interactions with the target cell. this data provides crucial insights into the pathogenic mechanism of sars-cov-2 infection and suggests hs-spike protein complexes as a novel therapeutic target to prevent infection. the glycocalyx is the first point of contact for all pathogens that infect animal cells, and thus it is not surprising that many viruses exploit glycans, such as hs, as attachment factors. for example, the initial interaction of herpes simplex virus with cells involves binding to hs chains on one or more hs proteoglycans (shieh et al., 1992; wudunn and spear, 1989 ) through the interactions with the viral glycoproteins gb and gc. viral entry requires the interaction of a specific structure in hs with a third viral glycoprotein, gd (shukla et al., 1999) , working in concert with membrane proteins related to tnf/ngf receptors (montgomery et al., 1996) . similarly, the human immunodeficiency virus binds to hs by way of the v3 loop of the viral glycoprotein gp120 (roderiquez et al., 1995) , but infection requires the chemokine receptor ccr5 (deng et al., 1996; dragic et al., 1996) . other coronaviruses also utilize hs, for example nl63 (hcov-nl63) binds hs via the viral s protein in addition to ace2 (lang et al., 2011; milewska et al., 2018; milewska et al., 2014; naskalska et al., 2019) . in these examples, initial tethering of virions to the host cell plasma membrane appears to be mediated by hs, but infection requires transfer to a proteinaceous receptor. the data presented here shows that sars-cov-2 requires hs in addition to ace2. we imagine a model in which cell surface hs acts as a "collector" of the virus and a mediator of the rbd-ace2 interaction, making viral infection more efficient. hs varies in structure across cell types and tissues, as well as with gender and age (de agostini et al., 2008; feyzi et al., 1998; ledin et al., 2004; vongchan et al., 2005; warda et al., 2006; wei et al., 2011) . variation in competition by hs from different tissues supports this conclusion and raises the possibility that hs contributes to the tissue tropism and j o u r n a l p r e -p r o o f 17 the susceptibility of different patient populations, in addition to levels of expression of ace2 . coronaviruses can utilize a diverse set of glycoconjugates as attachment factors. human coronavirus oc43 (hcov-oc43) and bovine coronavirus (bcov) bind to 5-n-acetyl-9-oacetylneuraminic acid (hulswit et al., 2019; tortorici et al., 2019) , middle east respiratory syndrome virus (mers-cov) binds 5-n-acetyl-neuraminic acid (park et al., 2019) , and guinea fowl coronavirus binds biantennary di-n-acetyllactosamine or sialic acid capped glycans (bouwman et al., 2019) . whether sars-cov-2 s protein binds to sialic acid remains unclear. mapping the binding site for sialic acids in other coronavirus s proteins has proved elusive, but modeling studies suggest a location distinct from the hs binding site shown in fig. 1 (park et al., 2019; tortorici et al., 2019) . the s protein in murine coronavirus contains both a hemagglutinin domain for binding and an esterase domain that cleaves sialic acids that aids in the liberation of bound virions (rinninger et al., 2006; smits et al., 2005) . whether sars-cov-2 s protein, another viral envelope protein, or a host protein contributes to hs-degrading activity to aid in the release of newly made virions is unknown. the repertoire of proteins in organisms that bind to hs make up the so called "hs interactome" and consists of a variety of different hs-binding proteins (hsbps) (xu and esko, 2014) . unlike lectins that have a common fold that helps define the glycan binding site, hsbps do not exhibit a conserved motif that allows accurate predictions of binding sites based on primary sequence. instead, the capacity to bind heparin appears to have emerged through convergent evolution by juxtaposition of several positively charged amino acid residues arranged to accommodate the negatively charged sulfate and carboxyl groups present in the polysaccharide, and hydrophobic and h-bonding interactions stabilize the association. the rbd domains from the sars-cov-1 and sars-cov-2 s proteins are highly similar in structure (fig. 1g ), but the electropositive surface in sars-cov-1 s rbd is not as pronounced in sars-cov-2 s rbd (fig. 1h ). in accordance with this observation, recombinant rbd protein from sars-j o u r n a l p r e -p r o o f 18 cov-2 showed significantly higher binding to heparin-bsa, compared to rbd from sars-cov-1 (fig. 2b) . a priori we predicted that the evolution of the hs binding site in the sars-cov-2 s protein might have occurred by the addition of arginine and lysine residues to its ancestor, sars-cov-1. instead, we observed that four of the six predicted positively charged residues that make up the heparin-binding site are present in sars-cov-1 as well as most of the other amino acid residues predicted to interact with heparin ( fig. 1) . sars-cov-1 has been shown to interact with cellular hs in addition to its entry receptors ace2 and transmembrane protease, serine 2 (tmprss2) (lang et al., 2011) . our analysis suggests that the putative heparinbinding site in sars-cov-2 s may mediate an enhanced interaction with heparin compared to sars-cov-1, and that this change evolved through as few as two amino acid substitutions, thr444lys and glu354asn. further studies are underway to define the amino acid residues in the combining site for heparin/hs to test this hypothesis. the ability of heparin and hs to compete for binding of the sars-cov-2 s protein to cell surface hs and the inhibitory activity of heparin towards infection of pseudovirus and authentic sars-cov-2 illustrates the therapeutic potential of agents that target the virus-hs interaction to control infection and transmission of sars-cov-2. there is precedent for targeting proteinglycan interactions as therapeutic agents. for example, tamiflu targets influenza neuraminidase, thus reducing viral transmission, and sialylated human milk oligosaccharides can block sialic acid-dependent rotavirus attachment and subsequent infection in infants (hester et al., 2013; von itzstein, 2007) . covid-19 patients typically suffer from thrombotic complications ranging from vascular micro-thromboses, venous thromboembolic disease and stroke and often receive unfractionated heparin or low molecular weight heparin (thachil, 2020) . the findings presented here and elsewhere suggest that both of these agents can block viral infection (courtney mycroft-west, 2020; kim et al., 2020; liu et al., 2020; mycroft-west et al., 2020; tandon et al., 2020; wu et al., 2020) . effective anticoagulation is achieved with plasma levels of heparin of 0.3-0.7 units/ml. this concentration is equivalent to 1.6-4 µg/ml heparin (assuming that the activity of ufh is 180 units/mg). although this is sufficient to block spike protein binding to cells (fig. 4) , it would not be expected to prevent viral infection, but it should attenuate infection depending on the viral load (fig. 7) . the anticoagulant activity of heparin, which is typically absent in hs, is not critical for its antiviral activity based on the observation that mst derived heparin and split-glycol heparin is nearly as potent as therapeutic heparin ( figs. 4 and 6) . additional studies are needed to address the potential overlap in the dose response profiles for heparin as an anticoagulant and antiviral agent and the utility of nonanticoagulant heparins. antibodies directed to heparan sulfate or the binding site in the rbd might also prove useful for attenuating infection. in conclusion, this work revealed hs as a novel attachment factor for sars-cov-2 and suggests the possibility of using hs mimetics, hs degrading lyases, and metabolic inhibitors of hs biosynthesis for the development of therapy to combat covid-19. further information and request for resources should be directed to the lead contact, thomas mandel clausen (tmandelclausen@health.ucsd.edu) all developed sars-cov-2 expression plasmids produced in this study can be made available upon request to the lead contact. j o u r n a l p r e -p r o o f 28 this study did not generate any unique datasets or code. cell lines nci-h1299, a549, hep3b, a375 and vero e6 cells were from the american type culture collection (atcc). nci-h1299 and a549 cells were grown in rpmi medium, whereas the other lines were grown in dmem. hep3b cells carrying mutations in hs biosynthetic enzymes were previously derived from the parent hep3b line as described (anower et al., 2019) . all cell media were supplemented with 10% (v/v) fbs, 100 iu/ml of penicillin and 100 µg/ml of streptomycin sulfate, and the cells were grown under an atmosphere of 5% co 2 and 95% air. cells were passaged at ~80% confluence and seeded as explained for the individual assays. protein was produced in expicho or hek293-6e cells that were acquired from thermo fisher and grown according to the manufacturer's specifications. human bronchial epithelial cells were acquired from lonza. they were cultured in pneumacult-ex plus medium or to pneumacult-ali medium according to the manufacturer's instructions (stemcell technologies). specific details on the culture methods are described in the methods section. the collection of human tissue in this study abided by the helsinki principles and the an electrostatic potential map of the sars-cov-2 spike protein rbd domain was generated from a crystal structure (pdb:6m17) and visualized using pymol (version 2.0.6 by schrödinger). a dp4 fully sulfated heparin fragment was docked to the sars-cov-2 spike protein rbd using the cluspro protein docking server (https://cluspro.org/login.php) (kozakov et al., 2013; kozakov et al., 2017; vajda et al., 2017) . heparin-protein contacts and energy contributions were evaluated using the molecular operating environment (moe) software (chemical computing group). recombinant sars-cov-2 spike protein, encoding residues 1-1138 (wuhan-hu-1; genbank: mn908947.3) with proline substitutions at amino acids positions 986 and 987, a "gsas" substitution at the furin cleavage site (amino acids 682-682), twinstreptag and his 8x , was produced in expicho cells by transfection of 6 x10 6 cells/ml at 37 ºc with 0.8 µg/ml of plasmid dna using the expicho expression system transfection kit in expicho expression medium (thermofisher). one day later the cells were refed, then incubated at 32 ºc for 11 days. the conditioned medium was mixed with complete edta-free protease inhibitor (roche). samples of the recombinant trimeric spike protein ectodomain were diluted to 0.03 mg/ml in 1x tbs ph 7.4. carbon coated copper mesh grids were glow discharged and 3 µl of the diluted sample was placed on a grid for 30 sec then blotted off. uniform stain was achieved by depositing 3 µl of uranyl formate (2%) on the grid for 55 sec and then blotted off. grids were transferred to a thermo fisher morgagni operating at 80 kv. images at 56,000 magnification j o u r n a l p r e -p r o o f 31 were acquired using a megaview 2k camera via the radius software. a dataset of 138 micrographs at 52,000x magnification and -1.5 µm defocus was collected on a fei tecnai spirit (120kev) with a fei eagle 4k by 4k ccd camera. the pixel size was 2.06 å per pixel and the dose was 25 e − /å 2 . the leginon (suloway et al., 2005) software was used to automate the data collection and the raw micrographs were stored in the appion (lander et al., 2009) database. particles on the micrographs were picked using dogpicker , stack with a box size of 200 pixels, and 2d classified with relion 3.0 (scheres, 2012) . secreted human ace2 was transiently produced in suspension hek293-6e cells. a plasmid encoding residues 1−615 of ace2 with a c-terminal hrv-3c protease cleavage site, a twinstreptag and an his 8x tag was a gift from jason s. mclellan, university of texas at austin. briefly, 100 ml of hek293-6e cells were seeded at a cell density of 0.5 × 10 6 cells/ml 24 hr before transfection with polyethyleneimine (pei). for transfection, 100 µg of the ace2 plasmid and 300 µg of pei (1:3 ratio) were incubated for 15 min at room temperature. transfected cells were cultured for 48 hr and fed with 100 ml fresh media for additional 48 hr before harvest. secreted ace2 were purified from culture medium by ni-nta affinity chromatography (qiagen). filtered media was mixed 3:1 (v/v) in 4x binding buffer (100 mm tris-hcl, ph 8,0, 1,2 m nacl) and loaded on to a self-packed column, pre-equilibrated with washing buffer (25 mm tris-hcl, ph 8, 0.3 m nacl, 20 mm imidazole). bound protein was washed with buffer and eluted with 0.2 m imidazole in washing buffer. the protein containing fractions were identified by sds-page. j o u r n a l p r e -p r o o f sars-cov-2 spike protein in dpbs was applied to a 1-ml hitrap heparin-sepharose column (ge healthcare). the column was washed with 5 ml of dpbs and bound protein was eluted with a gradient of nacl from 150 mm to 1 m in dpbs. for binding studies, recombinant spike protein and ace2 was conjugated with ez-link tm sulfo-nhs-biotin (1:3 molar ratio; thermo fisher) in dulbecco's pbs at room temperature for 30 min. glycine (0.1 m) was added to quench the reaction and the buffer was exchanged for pbs using a zeba spin column (thermo fisher). heparin ( and incubated with s protein (100 nm). ace2 binding was measured to bound spike protein as described above. mixtures of stabilized (mut7) spike protein, 6x molar excess soluble ace2 ectodomain, with or without 9x molar excess an icosasaccharide (dp20) fragment derived from heparin were incubated at 4°c for 15 min or 1 hr. samples were diluted to 0.02 mg/ml with respect to spike protein in 1x pbs ph 7.4. carbon coated copper mesh grids were glow discharged at 20 ma for 30 s and 3 µl sample was applied for 20 s and blotted off. grids were washed five times in 10 µl 1x tbs ph 7.4 for 15 sec then stained and blotted twice with 3 µl 2% uranyl formate for 15 sec. grids were imaged with an fei tecnai spirit (120 kev) or fei tecnai f20 (200 kev) with an fei eagle ccd (4k) camera. data were collected on the fei tecnai f20 at 62,000x magnification, -1.5 µm defocus with a pixel size of 1.77 å per pixel. these datasets employed a box size of 256 and comprised 167 to 331 micrographs. data were collected on the fei tecnai spirit as described above. data collection on both microscopes was automated through leginon (suloway et al., 2005) . stored in the appion (lander et al., 2009 ) database, and particles were picked with dog picker . particles were 2d classified with relion 3.0 j o u r n a l p r e -p r o o f 34 (scheres, 2012) . trimeric 2d classes were selected for iterative 3d classification with relion 3.0. classifications were performed until 3d classes demonstrated ace2 occupancy throughout the relevant threshold-level of the spike protein as visualized using chimerax (goddard et al., 2018) . particle counts of final 3d classes were obtained with relion 3.0 (scheres, 2012) and the percentages of particles bound to 0, 1, 2, or 3 ace2 were calculated and visualized in graphpad prism 8. cells at 50-80% confluence were lifted with pbs containing 10 mm edta (gibco) and fresh human tissue was washed in pbs, frozen, and lyophilized. the dried tissue was crushed into a fine powder, weighed, resuspended in pbs containing 1 mg/ml pronase with 90% ethanol (esko, 1993) . for hs quantification and disaccharide analysis, purified hs was digested with a mixture of heparin lyases i-iii (2 mu each) for 2 hr at 37 °c in 40 mm ammonium acetate buffer containing the ace2 expression plasmid (addgene, plasmid #1786) (li et al., 2003) qpcr mrna was extracted from the cells using trizol (invitrogen) and chloroform and purified using the rneasy kit (qiagen). cdna was synthesized from the mrna using random primers and the superscript iii first-strand synthesis system (invitrogen). sybr green master mix (applied biosystems) was used for qpcr following the manufacturer's instructions, and the expression of tbp was used to normalize the expression of ace2 between the samples. the qpcr primers used were as follows: ace2 (human) forward: 5' -cgaagccgaagacctgttcta -3' and reverse: 5' -gggcaagtgtggactgttcc -3'; and tbp (human) forward: 5' -aacttcgcttccgctggccc -3' and reverse: 5' -gaggggaggccaagccctga -3'. to generate the cas9 lentiviral expression plasmid, 2.5 x 10 6 hek293t cells were seeded to a 10-cm diameter plate in dmem supplemented with 10% fbs. the following day, the cells j o u r n a l p r e -p r o o f 38 were co-transfected with the pspax2 packaging plasmid (addgene, plasmid #12260), pmd2.g envelope plasmid (addgene, plasmid #12259), and lenti-cas9 plasmid (addgene, plasmid #52962) (sanjana et al., 2014) in dmem supplemented with fugene6 (30µl in 600µl dmem). media containing the lentivirus was collected and used to infect a549 wt and a375 wt cells, which were subsequently cultured with 5 µg/ml and 2 µg/ml blasticidin, respectively, to select for stably transduced cells. a single guide rna (sgrna) targeting ace2 (5'-tggatacatttgggcaagtg -3') and one targeting b4galt7 (5'-tgacctgctccctctcaacg-3') was cloned into the lentiguide-puro plasmid (addgene plasmid #52963) following published procedure (sanjana et al., 2014) . the lentiviral sgrna construct was generated in hek293t cells, using the same protocol as for the cas9 expression plasmid, and used to infect a549-cas9 and a375-cas9 cells to generate crispr knockout mutant cell lines. after infection, the cells were cultured with 2 µg/ml puromycin to select for cells with stably integrated lentivirus. after 7 d, the cells were serially diluted into 96-well plates. single colonies where expanded and dna was extracted using the dneasy blood and tissue dna isolation kit (qiagen). proper editing was verified by sequencing (genewiz inc.) and gene analysis using the online ice tool from synthego (suppl. fig. 5 ). vesicular stomatitis virus (vsv) pseudotyped with spike proteins of sars-cov-2 were generated according to a published protocol (whitt, 2010) . briefly, hek293t, transfected to express full length sars-cov-2 spike proteins, were inoculated with vsv-g pseudotyped ∆gluciferase or gfp vsv (kerafast, ma). after 2 hr at 37°c, the inoculum was removed and cells were refed with dmem supplemented with 10% fbs, 50 u/ml penicillin, 50 µg/ml streptomycin, and vsv-g antibody (i1, mouse hybridoma supernatant from crl-2700; atcc). pseudotyped particles were collected 20 hr post-inoculation, centrifuged at 1,320 × g to remove cell debris and stored at −80°c until use. briefly, 100 µl of luciferin lysis solution was added to the cells and incubated for 5 min at room temperature. the solution was transferred to a black 96-well plate and luminescence was detected using an enspire multimodal plate reader (perkin elmer). data analysis and statistical analysis was performed in prism 8. fluor 594 labeling kits (invitrogen), respectively. zombie uv™ was used to gate for live cells in the analysis. cells were then analyzed using an ma900 cell sorter (sony). for 4 days. fresh medium, 100 µl in the apical chamber and 500 µl in the basal chamber, was added daily. at day 7, the medium in the apical chambers was removed, and the basal chambers were changed every 2-3 days with apical washes with pbs every week for 28 days. the apical side of the hbec ali culture was gently washed three times with 200 µl of phosphate buffered saline without divalent cations (pbs-/-). heparinase was added to the apical side for half an hour prior to infection. an moi of 0.5 of authentic sars-cov-2 live virus (usa-wa1/2020 (bei resources, #nr-52281)) in 100 µl total volume of pbs was added to the apical chamber with either dmso, heparinase (2.5mu/ml heparin lyase ii, and 5mu/ml heparin lyase iii (ibex)) or 100ug/ml of unfractionated heparin. cells were incubated at 37c and 5% co2 for 4 hours. unbound virus was removed, the apical surface was washed and the compounds were re-added to the apical chamber. cells were incubated for another 20 hours at 37c and 5% co2. after inoculation, cells were washed once with pbs-/-and 100 µl tryple (thermofisher) was added to the apical chamber then incubated for 10 min in the incubator. cells were gently pipetted up and down and transferred into a sterile 15 ml conical tube containing neutralizing medium of dmem + 3% fbs. tryple was added again for 3 rounds of 10 minutes for a total of 30 min to clear transwell membrane. cells were spun down and resuspended in pbs with zombie uv viability dye for 15 min in room temp. cells were washed once with facs buffer then fixed in 4% pfa for 30 min at room temp. pfa was washed off and cells were resuspended in pbs. zombie uv™ was used to gate for live cells in the analysis. infection was analyzed by flow cytometry as explained above. cell viability was assessed using the celltiter-blue® assay (promega). briefly, vero cells were seeded into a 96 well plate. the cells were treated with hsase mix (2.5 mu/ml hsase ii, and 5 mu/ml hsase iii; ibex) or 100 µg/ml ufh for 16 hrs. the viability of the cells using celltiter-blue® was measured according to the manufacturers protocol. briefly, the j o u r n a l p r e -p r o o f 42 celltiter-blue® reagent was added directly to the cell culture and the cells were incubated overnight. fluorescence was read at excitation 560nm and emission 590nm, using an enspire multimodal plate reader (perkin elmer). data analysis was performed in prism. the human bronchial epithelial cells were grown at an air-liquid interface as explained above. cell viability after treatment with hsase mix (2.5 mu/ml hsase ii, and 5 mu/ml hsase iii; ibex) or 100 µg/ml ufh for 16 hrs was measured by adding celltiter-blue® reagent directly to the transwell inserts and developed as explained above. all statistical analyses were performed in prism 8 (graphpad). all experiments were performed in triplicate and repeated as indicated in the figure legends. data was analyzed statistically using unpaired t-tests when two groups were being compared or by one-way anova without post-hoc correction for multiple comparisons. ic 50 values and confidence intervals were determined using non-linear regression using the inhibitor vs. response least squares fit algorithm. the error bars in the figures refer to mean plus standard deviation (sd) values. the specific statistical tests used are listed in the figure legends and in the methods section. experiments were evaluated by statistical significance according to the following scheme; ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001. after 48 hr, cell culture supernatants were collected and stored at -80°c. virus titers were determined by plaque assays on vero e6 monolayers greiner bio-one, #662160) and rocked for 1 hr at room temperature. the cells were subsequently overlaid with mem containing 1% cellulose the plaques were visualized by fixation of the cells with a mixture of 10% formaldehyde and 2% methanol (v/v in water) for 2 hr. the monolayer was washed once with pbs and stained with 0.1% crystal violet (millipore sigma # v5265) prepared in 20% ethanol the pennsylvania state university, following the guidelines approved by the institutional biosafety committees. human bronchial epithelial cell air-liquid interface generation and infection human bronchial epithelial cells (hbecs, lonza) were cultured in t75 flasks in plus medium according to manufacturer instructions (stemcell technologies) to generate air-liquid interface (ali) cultures, hbecs were plated on collagen i-coated 24 well transwell inserts with a 0.4-micron pore size (costar, corning) at 5x10 4 cells/ml. cells were maintained for 3-4 days in pneumacult-ex plus medium until confluence, then changed to pneumacult-ali medium triglyceride-rich lipoprotein binding and uptake by heparan sulfate proteoglycan receptors in a crispr/cas9 library of hep3b mutants remdesivir for the treatment of covid-19 -preliminary report guinea fowl coronavirus diversity has phenotypic consequences for glycan and tissue binding heparan sulfate proteoglycans and viral attachment: true receptors or adaptation bias? viruses 11 undersulfated and glycol-split heparins endowed with antiangiogenic activity the 2019 coronavirus (sars-cov-2) surface protein (spike) s1 receptor binding domain undergoes conformational change upon heparin binding identification of a major co-receptor for primary isolates of hiv-1 hiv-1 entry into cd4+ cells is mediated by the chemokine receptor cc-ckr-5 special considerations for proteoglycans and glycosaminoglycans and their purification order out of chaos: assembly of ligand binding sites in heparan sulfate age-dependent modulation of heparan sulfate structure and function bioengineering murine mastocytoma cells to produce anticoagulant heparin ucsf chimerax: meeting modern challenges in visualization and analysis structural analysis of urinary glycosaminoglycans from healthy human subjects human milk oligosaccharides inhibit rotavirus infectivity in vitro and in acutely infected piglets human coronaviruses oc43 and hku1 bind to 9-o-acetylated sialic acids via a conserved receptor-binding site in spike protein domain a loss of bcl-6-expressing t follicular helper cells and germinal centers in covid-19 stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis initial step of virus entry: virion binding to cell-surface glycans how good is automated protein docking? the cluspro web server for protein-protein docking appion: an integrated, database-driven pipeline to facilitate em image processing inhibition of sars pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans evolutionary differences in glycosaminoglycan fine structure detected by quantitative glycan reductive isotope labeling heparan sulfate structure in mice with genetically modified heparan sulfate production assessing ace2 expression patterns in lung tissues in the pathogenesis of covid-19 angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus proteoglycans and sulfated glycosaminoglycans sars-cov-2 spike protein binds heparan sulfate in a length-and sequence-dependent manner entry of human coronavirus nl63 into the cell human coronavirus nl63 utilizes heparan sulfate proteoglycans for attachment to target cells stable heparin-producing cell lines derived from the furth murine mastocytoma herpes simplex virus-1 entry into cells mediated by a novel member of the tnf/ngf receptor family heparin inhibits cellular invasion by sars-cov-2: structural dependence of the interaction of the surface protein (spike) s1 receptor binding domain with heparin membrane protein of human coronavirus nl63 is responsible for interaction with the adhesion receptor structures of mers-cov spike glycoprotein in complex with sialoside attachment receptors localisation and distribution of o-acetylated n-acetylneuraminic acids, the endogenous substrates of the hemagglutinin-esterases of murine coronaviruses, in mouse tissue mediation of human immunodeficiency virus type 1 binding by interaction of cell surface heparan sulfate proteoglycans with the v3 region of envelope gp120-gp41 improved vectors and genome-wide libraries for crispr screening relion: implementation of a bayesian approach to cryo-em structure determination cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans a novel role for 3-o-sulfated heparan sulfate in herpes simplex virus 1 entry nidovirus sialate-o-acetylesterases: evolution and substrate specificity of coronaviral and toroviral receptor-destroying enzymes the sweet spot: defining virus-sialic acid interactions automated molecular microscopy: the new leginon system effective inhibition of sars-cov-2 entry by heparin and enoxaparin derivatives. biorxiv the versatile heparin in covid-19 structural basis for human coronavirus attachment to sialic acid receptors the war against influenza: discovery and development of sialidase inhibitors structural characterization of human liver heparan sulfate dog picker and tiltpicker: software tools to facilitate particle selection in single particle electron microscopy function, and antigenicity of the sars-cov-2 spike glycoprotein isolation and characterization of heparan sulfate from various murine tissues site-specific glycan analysis of the sars-cov-2 spike a comprehensive compositional analysis of heparin/heparan sulfate-derived disaccharides from human serum generation of vsv pseudotypes using recombinant deltag-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines cryo-em structure of the 2019-ncov spike in the prefusion conformation vaccines and therapies in development for sars-cov-2 infections initial interaction of herpes simplex virus with cells is binding to heparan sulfate demystifying heparan sulfate-protein interactions structural basis for the recognition of sars-cov-2 by full-length human ace2 cov-2 spike protein interacts with heparan sulfate and ace2 through the rbd • heparan sulfate promotes spike-ace2 interaction • sars-cov-2 infection is co-dependent on heparan sulfate and ace2 • heparin and non-anticoagulant derivatives block sars-cov-2 binding and infection in brief provide evidence that heparin sulfate is a necessary co-factor for sars-cov-2 infection. they show that heparin sulfate interacts with the receptor binding domain of the sars-cov-2 spike glycoprotein we thank scott selleck (the pennsylvania state university), eugene yeo (uc san diego), john guatelli (uc san diego), mark fuster (uc san diego) and stephen schoenberger (la jolla institute for immunology) for many helpful discussions, and annamaria naggi and giangiacomo torri from the ronzoni institute for generously providing split-glycol heparin. this key: cord-272467-8heg5iql authors: armstrong, john; mccrae, malcolm; colman, alan title: expression of coronavirus e1 and rotavirus vp10 membrane proteins from synthetic rna date: 2004-02-19 journal: j cell biochem doi: 10.1002/jcb.240350206 sha: doc_id: 272467 cord_uid: 8heg5iql some viruses acquire their envelopes by budding through internal membranes of their host cell. we have expressed the cloned cdna for glycoproteins from two such viruses, the e1 protein of coronavirus, which buds in the golgi region, and vp10 protein of rotavirus, which assembles in the endoplasmic reticulum. messenger rna was prepared from both cdnas by using sp6 polymerase and either translated in vitro or injected into cultured cv1 cells or xenopus oocytes. in cv1 cells, the el protein was localised to the golgi region and vp10 protein to the endoplasmic reticulum. in xenopus oocytes, the e1 protein acquired post‐translational modifications indistinguishable from the sialylated, o‐linked sugars found on viral protein, while the vp10 protein acquired endoglycosidase‐h‐sensitive n‐linked sugars, consistent with their localisation to the golgi complex and endoplasmic reticulum, respectively. thus the two proteins provide models with which to study targeting to each of these intracellular compartments. when the rnas were expressed in matured, meiotic oocytes, the vp10 protein was modified as before, but the e1 protein was processed to a much lesser extent than in interphase oocytes, consistent with a cessation of vesicular transport during cell division. in the eukaryotic cell, the rough endoplasmic reticulum is the site of synthesis for both secretory proteins and integral proteins of the plasma membrane. to reach the cell surface, both types of protein must traverse the golgi complex. during this transport process, a series of post-translational modifications may occur. since the modifying enzymes are localised along the secretory pathway, the endoplasmic reticulum and golgi complex constitute a series of membrane-limited compartments, each apparently with a distinct complement of proteins [reviewed in . how are these proteins confined to their appropriate destinations, rather than migrating onward to the plasma membrane? we are investigating two viral model proteins, one for the endoplasmic reticulum and one for the golgi complex, with a view to determining the features of each molecule responsible for its correct localisation. rotaviruses are a class of animal viruses which characteristically assemble at the endoplasmic reticulum [5] . although assembly appears to be a budding process, the matured virion surprisingly lacks a visible envelope . the bovine uk strain encodes two glycoproteins: vp7, which forms part of the virion, and vpio, which does not [8, 9] . the latter is an integral membrane glycoprotein [9, 10] bearing "immature" n-linked sugars which imply a failure to leave the endoplasmic reticulum [ w . by contrast, with coronaviruses the envelope is acquired by budding initially at smooth membranes close to the golgi complex but continuous with the endoplasmic reticulum; as infection progresses budding may also occur in the golgi cisternae or the rough endoplasmic reticulum [ 11, 121 . the strain mhv-a59 bears two glycoproteins, the smaller of which, e l , is restricted within the cell and does not appear to reach the plasma membrane except as part of a budded virion 112,131. thus we have adopted the proteins vplo and e l as potential models for the class of membrane proteins which localise within compartments of the secretory pathway. previously we have reported the cloning and sequence analysis of both proteins [9, 14] . here we show that both cdnas can be expressed via artificial mrna prepared using sp6 polymerase [ 15, 161 . the mrna may be translated either in vitro or after injection into xenopus oocytes or cultured cvi cells. expressed in this way, vplo and e l prove to be valid model proteins for the endoplasmic reticulum and golgi complex, respectively. in addition, we have exploited the fact that xenopus oocytes can be matured into a meiotic state [ 17, 181 to investigate the transport of the two proteins to their destinations during cell division. all restrictions, ligations, and transformations were carried out according to standard methods 1191. full-length cdna for bovine rotavirus vplo (assembled and kindly provided by h. baybutt) was excised with the enzymes ahaiu and sali, to give a fragment corresponding to nucleotides 8-588 [in figure 3 of 91. this was inserted into the bglii site of the transcription vector psp64t [ 151. e l cdna [ 14, 203 was excised with aha111 and fom, representing nucleotides 59-780 [in 141, plus the following 13 bases of the adjacent nucleocapsid gene [21] and inserted into the bglii site of psp64t 1151. both plasmids were linearised with ecori and rna transcribed with sp6 polymerase in the presence of 0.5mm m7g (5')ppp(5')g exactly as described [16] . since the vector psp64t contains a poly-a tract between the bglii and ecori sites, this method results in the synthesis of capped, polyadenylated mrna in a single reaction. translation in vitro in reticulocyte lysates, injection of xenopus oocytes, immunoprecipitation of proteins, microinjection of cv 1 cells, and immunofluorescence analysis were all performed as before [16] . for analysis of vplo protein, rabbit antiserum to a fusion protein of bacterial 0-galactosidase and vpio, whose prepara-tion will be described in detail elsewhere, was used. to detect el, we used a rabbit serum to e l purified by detergent extraction of virus [22] , kindly provided by s. tooze, embl, heidelberg, who also provided stocks of coronavirus mhv-a59 and its host, sac-cells. radiolabelled virus was produced by infection of cells at a ratio of 10 pfukell, incubation for 16 hr in methionine-free medium to which was added 50 pci/ml 35s-methionine (amersham) , and collection of the culture supernatant. endoglycosidase h digestions were carried out as before [ 181. xenupus oocytes were matured into second meiotic metaphase by incubation in 10 p g / d progesterone as described [ 181. previously we showed that rna prepared with sp6 polymerase could be translated in cultured cells, provided it has both a 5' cap structure, and a 3' poly-a tract added using poly-a polymerase [16] . e l rna prepared in this way was found to be translated in both cv1 cells and xenopus oocytes (not shown). we have further simplified this method by using a vector which includes a sequence of 23 a's in the transcribed region [ 151, eliminating the requirement for a second reaction. rnas prepared by this method for vplo and e l were translated efficiencly in reticulocyte lysates, xenopus oocytes, and cultured cvl cells (figs. 1, 2). rough endoplasmic reticulum; these structures may be cleaved by endoglycosidase h [23] . vplo protein which had been immunoprecipitated from injected oxcytes was found to be completely sensitive to endoglycosidase h, yielding a species which approximately comigrated with the unprocessed form produced in the reticulocyte lysate (fig. 1, lanes a-d) . thus the vplo protein is likely to be localised within the endoplasmic reticulum of the oocyte. e l protein was synthesized in oocytes as a spectrum of forms, the most mobile of which comigrated with the unmodified protein from the reticulocyte lysate (fig. 1 , lanes e,f). the oocyte proteins were very similar in both mobilities and relative abundance to the species of e l found in virus particles (fig. 1, lane 8) . these have been shown to contain 0-linked sugars as their only posttranslational modification [24, 25] . since at least the later stages of 0-linked glycosylation are thought to occur in the golgi complex 1261, most or all of the el protein would appear to have reached this organelle in the oocyte. with both antibodies, no significant labeled proteins were precipitated from oocytes in the absence of the appropriate mrna. sp6 rnas for vplo and el were microinjected into cvl monkey kidney cells, and the resulting proteins were detected by immunofluorescence. vplo proteins was found in an elaborate pattern around the nucleus and throughout the cytoplasm, characteristic of the rough endoplasmic reticulum (fig. 2a) . by changing the plane of focus, it became clear that all of the nuclear envelope was labeled (not shown). this was expected as the outer nuclear membrane is continuous with, and usually considered as part of, the endoplasmic reticulum. in contrast, e l protein showed a much more localised fluorescence pattern (fig. 2b) . labeling was concentrated in a perinuclear area corresponding to the golgi region and coincident with the intracellular pattern shown by fluorescent wheat germ agluttinin, a golgi marker (not shown). the pattern also closely resembled the distribution of e l protein in virally infected cells 112,131. if injected cells were not permeabilised with detergent, no labeling of the cell surface was detected with either protein (not shown). incubation of xenopus oocytes in progesterone causes them to mature from their initial stage, first meiotic prophase, to second meiotic metaphase [17] . in this state, the golgi apparatus is broken down and protein secretion blocked [ 181. we investigated the synthesis of vplo and e l in such matured oocytes. vplo was electrophoretically indistinguishable from the form produced in nonmatured oocytes (fig. 3, lanes a,b) . e l , in contrast, showed a striking reduction in the extent to which it was processed in comparison to nonmatured oocytes (fig. 3, lanes c,d) , implying that most of the protein is denied access to the modifying enzymes of the golgi complex under these conditions. transcription of rna with sp6 polymerase has been used as a route for expression of cloned cdnas either in vitro or in the xenopus oocyte [ 151. if the rna incorporates a 5' cap structure and a 3' poiy-a tract, it may also be expressed in a b c d fig. 3. expression of vplo (a,b) and e l (c,d) in normal (a,c) or progesterone-matured (b,d) oocytes. proteins were analysed as for figure 1 , and the fluorograph was exposed for 1 wk. cultured cells [ 161. by using the vector psp64t [ 151, such rna can be prepared in a single reaction. we assume that the presence of an encoded poly-a sequence in the vector is the critical factor in allowing expression of the rna in cultured cells 1161; however, the transcribed region also contains 5' and 3' untranslated regions from a globin cdna, and a sequence of c bases at the extreme 3' end [ 151, and any of these elements may contribute to the stability of the mrna or the efficiency with which it is translated. whichever feature is important, the system provides a quick and versatile approach to the expression of cloned dnas. its principal limitation, at present, is the lack of a technique for the efficient introduction of rna into large populations of cultured cells. it appears that both of the proteins we have studied, vplo and el, are localised in a similar fashion whether they are expressed in isolation in cell types as diverse as oocytes and cvl cells, or in the course of viral infection. thus each is likely to be a legitimate model with which to study targeting of proteins to its respective intracellular destination. in the case of vpio, this is the rough endoplasmic reticulum, as judged by fluorescence microscopy and sensitivity of its n-linked oligosaccharides to endoglycosidase h (figs. 1, 2) . the corresponding protein of rotavirus sall , termed ncvps, bears oligosaccharides of the form man8glcnac2 [lo] . in contrast, the principal location of the e l protein appears to be the golgi complex. as the bulk of the protein made in oocytes has been modified to give forms of similar mobilities to the viral protein (fig. 1, lanes f,g; see also [27] ), it has presumably acquired 0-linked oligosaccharides, an indication of having reached the golgi complex [26] . most of the oligosaccharides on viral el contain terminal sialic acid [25] , whose presence is detectable by changes in electrophoretic mobility of the glycoprotein after neuraminidase digestion. however, we have repeatedly failed to observe any effect on mobility of oocyte el by treatment with neuroaminidases from various sources and under various conditions (not shown). the explanation for this is not clear; perhaps the oocyte adds exotic sialic acids which are not susceptible to cleavage. immunofluorescence of cultured cells expressing e l clearly showed a very localised perinuclear distribution (fig. 2b ) characteristic of the golgi region and similar to the pattern observed during viral infection [12] . however, not all of the viral protein is restricted to the flattened cisternae of the golgi complex; some at least is found in smooth membranes which are in the same region of the cell but are in fact continuous with the rough endoplasmic reticulum [ 12, 221 . these membranes are reminiscent of the transitional elements of specialised secretory cells [28] and are the earliest site of budding during viral infection [12] . at the resolution of light microscopy it is impossible to say whether the pattern in figure 2b includes this compartment. it will be of interest to resolve this point by immunoelectron microscopy, which should also allow us to determine how many of the golgi cisternae are labelled. nevertheless, it is probably safe to conclude that the behaviour of the e l protein is largely responsible for determining the intracellular budding site of coronavirus. the localisation of vplo might argue for a similar function in rotavirus morphogenesis. however, this protein's precise role in viral infection remains enigmatic. the other rotavirus glycoprotein, vp7, also appears to be restricted to the endoplasmic reticulum when it is expressed in the absence of other viral proteins [291. thus both proteins may be involved in determining the viral assembly site, but the subsequent fate of vplo is unclear, since it is not detectable in purified virions [9] . perhaps the simplest hypothesis is that the newly budded virus has a lipid envelope including vplo and vp7, which is somehow shed at a later stage to leave vp7 connected directly to components of the capsid [7] . an aspect of the biogenesis of the endoplasmic reticulum and golgi complex which has recently attracted attention is their fate during cell division. in mammalian cells, both organelles are thought to break up into vesicles which then partition randomly between the daughter cells and fuse together; concomitant with this disruption is a cessation of traffic of secretory and plasma mebmrane proteins to and through the golgi complex [reviewed in 301. the same phenomena appear to occur in the xenopus oocyte, which has the experimental advantage that its cell cycle state may be manipulated with hormones [mi. the results presented in figure 3 are entirely consistent with this model; vplo is synthesized and processed normally in meiotic oocytes, but e l is processed to a far lesser extent than in interphase oocytes, and thus has probably failed to traverse what is assumed to be the first vesicle-mediated step of membrane traffic, from the endoplasmic reticulum to the golgi complex. it might be of interest to study the vesicles in which e l becomes trapped under these conditions. what features of each protein might be involved in determining its intracellular destination, and what are the mechanisms involved in each case? the two proteins share a superficial topological similarity, in having a very short domain on the lumenal side of the membrane which is n-terminal but does not arise from cleavage of a signal sequence; e 1 has the additional unusual feature of hydrophobic regions large enough to span the membrane three times [9, 10, 14, 31, 32] . two mechanisms have been proposed for retention of proteins in the endoplasmic reticulum. one is that a species variously known as bip or grp78 attaches to improperly folded or assembled molecules and prevents their exit to the golgi complex [33-351. the second is that all of the membrane proteins of the endoplasmic reticulum form a continuous interacting network and are unable to diffuse in the lipid bilayer 134,371. in respect of either model, we have never observed any host species which coprecipitates from oocytes with vplo, although admittedly our labelling schedule is perhaps not optimal for detection. (note that the unidentified band in figure 1 , lanes b-d is made only in response to vplo mrna.) neither have we observed such species with our golgi model protein, e l , whose sorting mechanism is even more obscure. however, with the availability of cloned cdna and a reliable expression system, it is now possible to dissect each molecule and identify the characteristics involved in its correct targeting within the cell. molecular cloning: a laboratory manual molecular biology and pathogenesis of coronaviruses this work was supported by grants from the cancer research campaign (to a. colman) and the wellcome trust (to a. colman and m. mccrae). key: cord-254909-8zgvovu4 authors: srivastava, rajneesh; ray, sandipan; vaibhav, vineet; gollapalli, kishore; jhaveri, tulip; taur, santosh; dhali, snigdha; gogtay, nithya; thatte, urmila; srikanth, rapole; srivastava, sanjeeva title: serum profiling of leptospirosis patients to investigate proteomic alterations() date: 2012-12-05 journal: j proteomics doi: 10.1016/j.jprot.2012.04.007 sha: doc_id: 254909 cord_uid: 8zgvovu4 leptospirosis is a zoonotic infectious disease of tropical, subtropical and temperate zones, which is caused by the pathogenic spirochetes of genus leptospira. although this zoonosis is generally not considered as fatal, the pathogen can eventually cause severe infection with septic shock, multi-organ failure and lethal pulmonary hemorrhages leading to mortality. in this study, we have performed a proteomic analysis of serum samples from leptospirosis patients (n = 6), febrile controls (falciparum malaria) (n = 8) and healthy subjects (n = 18) to obtain an insight about disease pathogenesis and host immune responses in leptospiral infections. 2de and 2d-dige analysis in combination with maldi-tof/tof ms revealed differential expression of 22 serum proteins in leptospirosis patients compared to the healthy controls. among the identified differentially expressed proteins, 8 candidates exhibited different trends compared to the febrile controls. functional analysis suggested the involvement of differentially expressed proteins in vital physiological pathways, including acute phase response, complement and coagulation cascades and hemostasis. this is the first report of analysis of human serum proteome alterations in leptospirosis patients, which revealed several differentially expressed proteins, including α-1-antitrypsin, vitronectin, ceruloplasmin, g-protein signaling regulator, apolipoprotein a-iv, which have not been reported in context of leptospirosis previously. this study will enhance our understanding about leptospirosis pathogenesis and provide a glimpse of host immunological responses. additionally, a few differentially expressed proteins identified in this study may further be investigated as diagnostic or prognostic serum biomarkers for leptospirosis. this article is part of a special issue entitled: integrated omics. leptospirosis is caused by a gram-negative like, obligate, aerobic spirochete of genus leptospira, which is sustained in nature within a wide range of reservoir and carrier animals, including rodent and non-rodents. it is a contagious disease, transmitted through water or soil contaminated with urine of animal hosts containing a large number of this pathogenic spirochete. this zoonosis generally appears as a seasonal infection that initiates at the beginning of monsoon, occurs frequently during the rainy season and disappears when the rains recede; except few sporadic cases that may happen throughout the year [1, 2] . due to the increasing number of severe leptospirosis infections in developed and developing countries, it has been recognized as a major health problem [3] . additionally, this emerging infectious disease can adversely affect agricultural industry [4] . consequently, this fatal infectious zoonosis has a devastating impact on human health and corresponding impediment to economic improvement worldwide. leptospirosis is a systemic disease of humans and broad range of feral and domestic animals, which is characterized by fever, myalgia, conjunctival suffusion, renal and hepatic insufficiency, pulmonary manifestations and reproductive failure [5] . renal involvement is very common in leptospirosis patients and severe cases often lead to acute renal failure. pulmonary alveolar haemorrhage is the prime cause of leptospirosis related casualties [6] . successful completion of the genome sequence of leptospira interrogans [7] has paved the emergence of omics-based approaches in leptospirosis research [8] . proteome level analysis of different biological fluids has been found to be effective for investigation of disease pathobiology as well as identification of surrogate markers with diagnostic and prognostic significance [9, 10] . recent proteomic studies have generated valuable information regarding the global/differential proteome and immunome profile of pathogenic leptospira spp. [11] [12] [13] , protein expression and multiple modification system of the pathogen [14] , and analysis of leptospires excreted in urine of chronically infected hosts [15, 16] . to this end, notable achievements have been accomplished in deciphering the global and sub-cellular proteome profile of this pathogenic spirochete using quantitative mass spectrometric approaches [17, 18] . however, the molecular pathogenesis of leptospirosis is not clearly understood and requires further comprehensive studies at the molecular levels. the omics-based investigations can reveal the mechanism of disease pathogenesis and the pathogen induced alterations in host physiological system as well as identification of leptospiral immunogens useful for diagnostic applications and vaccine development. in the present study, we have performed a comprehensive proteomic analysis of serum samples from patients suffering from leptospirosis using two dimensional gel electrophoresis (2de) and 2d fluorescence difference gel electrophoresis (2d-dige), and compared with febrile (falciparum malaria) and healthy controls. identification of differentially expressed proteins was performed using high-sensitivity maldi-tof-tof mass spectrometry. in this differential proteomics analysis, we have identified 22 statistically significant (p < 0.05) proteins in leptospirosis patients compared to the healthy subjects. proteins showing differential expression in leptospirosis patients were subjected to further functional clustering by using database for annotation, visualization and integrated discovery (david) and protein analysis through evolutionary relationships (panther). the possible involvement of differentially expressed proteins in various physiological pathways and the host immune response against the pathogen has been studied. as far our knowledge goes, this is the first report of human serum proteome alterations in leptospiral infection and our results provide valuable insight into the host immune response against this acute bacterial infection and the underlying molecular mechanisms of the disease pathogenesis. materials and methods 2. (table s1 .1). blood specimens were also collected from age and sex matched healthy (n =18) (voluntary blood donors) and febrile controls (n= 8) with written informed consent to perform comparative analysis. patients with non-severe, uncomplicated, falciparum malaria diagnosed by microscopic examination and confirmed by rapid diagnostic tests (rdt) were selected as febrile controls (fc) for this proteomic study (table s1 .2). serum separation tubes (bd vacutainer®; bd biosciences) were used to collect blood samples from the antecubital vein of the subjects. subsequent to blood collection, samples were allowed to clot by keeping the tubes in ice for 30 min. after clotting, the samples were centrifuged at 2500 rpm at 20°c for 10 min to separate serum from the clotted blood. collected serum samples were labeled and stored in multiple aliquots at −80°c. to minimize any pre-analytical variations, exactly similar collection, processing and storage conditions were maintained for each control and diseased samples. previously described [19] . in brief, a combination of tcaacetone protein precipitation, sonication, albumin and igg depletion and desalting was used for processing serum samples from leptospirosis patients and controls for proteomic analysis. the 2d gel images were scanned by using labscan software version 6.0 (ge healthcare) and analysis was performed by using imagemaster 2d platinum 7.0 software (ge healthcare). 2d-dige gels were scanned using ettan dige imager scanner (ge healthcare) using proper wavelengths and filters for cy2, cy3 and cy5 dyes keeping the resolution at 40 μm. accurate cropping of the gel images were performed using imagequant software version 5.0 (ge healthcare). the cropped gel images were imported to imagemaster 2d platinum 7.0 dige software (ge healthcare) and subjected to comparative analysis for relative protein quantification across all the leptospirosis and control samples. the average ratio of expression was analyzed by student's t-test. protein spots showing differential expression with reproducibility and statistical significance (p< 0.05) were selected and excised for further ms analysis. in-gel trypsin digestion and mass spectrometry protein spots identified in 2de and 2d-dige gels exhibiting differential expression with statistical significance (p < 0.05) in leptospirosis patients compared to the healthy subjects were excised for in-gel digestion and further ms analysis. in-gel digestion of the proteins was performed as described previously [19] . the identity of differentially expressed proteins was established using ab sciex 4800 maldi-tof/tof mass spectrometer. the combined ms and ms/ms peak lists were searched using the gps™ explorer software version 3.6 (ab sciex). the mascot version 2.1 (http://www.martixscience.com) was used as the search engine for protein identification against the swiss-prot database. during the database search following parameters were specified: human taxonomy, trypsin digestion with one missed cleavage, carbamidomethyl (c) as fixed modification, oxidation (m) as variable modification, peptide mass tolerance set at 75 ppm and ms/ms tolerance of 0.4 da. western blot analysis was performed with serum samples from controls (healthy and febrile) and leptospirosis patients (n = 6) to validate the differential expression of some of the target proteins identified in 2de and 2d-dige experiments. prior to the western blotting experiment protein concentration in each sample (leptospirosis patients and controls) was estimated using the 2d-quant kit (ge healthcare) and bca protein assay kit (thermo fisher scientific) following the manufacturer's instructions. the serum proteins were separated on a 12% sds-page (50 μg of total proteins per lane) and then transferred onto pvdf membranes under semi-dry conditions by using an ecl semi-dry transfer unit (ge healthcare). protein migration was tracked by using pre-stained protein standard (fermentas). western blotting was performed by using monoclonal/polyclonal antibody against clusterin (santacruz biotechnology, sc-8354), ceruloplasmin (santacruz biotechnology, sc-365206) and appropriate secondary antibody conjugated with hrp (genei (merck)-621140380011730 or 621140680011730). imagequant software version 5.0 (ge healthcare) was used for quantitation of the signal intensity of the bands in western blots. the differentially expressed proteins in leptospirosis patients were subjected to functional pathway analysis using panther software, version 7 (http://www.pantherdb.org) [20] and david database version 6.7 (http://david.abcc.ncifcrf.gov/home.jsp) [21] for better understanding of the biological context of the identified proteins, their connection with disease pathobiology and participation in various physiological pathways. uniprot accession numbers of the 22 differentially expressed proteins identified in our study were uploaded and mapped against the homo sapiens reference dataset to extract and summarize functional annotation associated with individual or group of genes/proteins and to identify gene ontology terms, molecular function, biological process and important pathways for each dataset. this comparative proteomic profiling was performed to identify differentially expressed serum proteins in leptospiral infection. serum proteome profile of healthy subjects, febrile controls and leptospirosis patients were analyzed using two gel-based proteomic platforms classical 2de and advanced 2d-dige. in each analysis individual samples were studied (n = 32 for classical 2de and n = 18 for 2d-dige) and sample pooling was not executed since it cannot reflects the true pictures of biological variability effectively. differentially expressed protein spots identified in gel-based analysis, which full-filled the statistical parameters (t-test; p < 0.05) were subjected to further mass spectrometric and functional analysis. over 600 protein spots were detected in each 2d gel stained with gelcode blue safe protein stain using imp7 software. 13 statistically significant (p < 0.05) differentially expressed, 7 down-regulated and 6 up-regulated (with fold changes ranging from −4.39-fold to +2.7-fold) proteins were identified in 2de analysis ( fig. s1 and table s2 ). among the 6 up-regulated spots, 5 spots were between 1.1 and 2-fold, and 1 spot exhibited over 2-fold increase in expression level; while in the case of down-regulation, 2 spots were between 1.1 and 2-fold, 1 spot was between 2-and 3-fold, and 4 spots found to have over 3-fold reduced expression levels. representative 2de images of serum proteome profile of leptospirosis patients and healthy individuals, and bar-diagrammatic representation of the fold change and 3d views of some selected differentially expressed proteins are shown in fig. 1a and b. second level of gel-based proteomic analysis was performed using 2d-dige approach, where the test (leptospirosis) and control (healthy/febrile) samples, after pre-electrophoresis labeling with cydyes (ge healthcare), were separated on the same gel to reduce gel-to-gel variations. additionally, superior sensitivity of cydyes compared to routinely used cbb stains, allowed to visualize less abundance protein spots in 2d-gels, which were not detectable in classical 2de profiling, and thereby increased the overall coverage of serum proteome. approximately 1000 protein spots were detected on each 2d-dige gels in the image master 2d platinum 7.0 dige software analysis. in 2d-dige profiling, total of 98 (around 10% of the total detected spots) differentially expressed spots satisfied the statistical criteria (t-test and 1-way anova; p<0.05), among which, 48 were up-regulated (range from 1.15 to 2.71-fold) while, the remaining 50 protein spots showed reduced expression level (with changes from 1.19 to 7.5-fold) in the leptospirosis patients (table s3 ). 37 up-regulated spots were between 1.1 and 2-fold change range, and for the remaining 11 spots the range was between 2 and 3-fold; while among the 50 down-regulated spots, 33 were between 1.1 and 2-fold, 12 were between 2 and 3-fold, and 5 spots exhibited over 3-fold reduced expression level. owing to the superior sensitivity and reproducibility in dige technique, we obtained much higher numbers of differentially expressed protein spots from 2d-dige experiment ( fig. 2a) . fig. 2b depicts 3d views and graphical representation of few selected differentially expressed protein spots. identification of differentially expressed proteins using maldi-tof/tof ms alteration of human serum proteome due to the leptospiral infection was reflected by the differential expression of multiple serum proteins in leptospirosis patients detected by gel-based profiling. among the several differentially expressed protein spots detected in 2de, those 13 spots that satisfied the statistical criteria (p < 0.05) were subjected to in-gel trypsin digestion and subsequent maldi-tof/tof analysis to establish protein identity. ms and ms/ms analysis results revealed that 13 differentially expressed protein spots identified in regular 2de experiment correspond to 6 proteins, among which 2 were upregulated (α-1-antitrypsin and α-1b-glycoprotein precursor) and the remaining 4 were down-regulated (apolipoprotein a1 precursor, serum albumin precursor, apo-lipoprotein a-iv precursor, complement c4 precursor) ( table 1 and s4.1). in case of 2d-dige, selected spots were excised manually from preparative 2d gels stained with gelcode blue safe protein stain (thermo scientific, usa) containing higher amount of serum protein. among the 98 differentially expressed protein spots, 57 spots that could be excised from the gels were subsequently subjected to ms and ms/ms analysis, which successfully established the identity of 42 spots ( fig. 2a ; table s4 .2). for the remaining spots we were unable to establish ms identity most likely due to the very low intensity of those spots and insufficient amount of detectable peptides, probably beyond the sensitivity limit of the instrument. the 42 protein spots identified by ms correspond to 21 (10 down-regulated and 11 up-regulated) differentially expressed proteins in patients suffering from leptospiral infection ( fig. s2 ; table 2 ). in this study, we have performed two levels of gel-based proteomic analysis; initially classical 2de and latter more advanced 2d-dige technology using sub-sets of the patient and control (febrile and healthy) populations studied by 2de. almost all of the proteins (except complement c4 precursor) identified in 2de were again identified in 2d-dige profiling and exhibited similar trends of differential expressions which enhanced the confidence level of our study. the number of identified proteins in 2de and 2d-dige are 6 and 21 respectively, while 5 of the identified proteins (apolipoprotein a-i, serum albumin precursor, apolipoprotein a-iv, alpha-1-antitrypsin and alpha-1b-glycoprotein) found to be overlapping for 2de and 2d-dige, and overall 22 differentially expressed proteins (p < 0.05) have been identified in our study (tables 1 and 2) . pre-electrophoresis labeling of protein samples with highly sensitive cy dyes in 2d-dige attributed around 55% increase in spot number compared to the cbb stained 2de gels, which certainly enhanced the overall coverage of the whole serum proteome. in 2d-dige experiment we obtained 16 more differentially expressed proteins including complement c3 precursor, clusterin precursor, complement factor h, regulator of g-protein signaling 7 (down-regulated) and vitronectin precursor, ceruloplasmin precursor, leucine-rich α-2-glycoprotein (up-regulated) ( table 2) , which were not identified in classical 2de due to lower sensitivity and reproducibility issues. differentially expressed serum proteins identified in leptospiral infection (compared to the healthy subjects) were further investigated in falciparum malaria patients (febrile controls). around 60% of the identified proteins found to be commonly modulated in both of the infectious diseases. however, for most of the proteins levels of altered expression were found to be different in leptospirosis compared to malaria. interestingly, 3 of the identified proteins exhibited opposite trend of differential expression in leptospirosis compared to p. falciparum infection. moreover, 5 proteins found to be differentially expressed in leptospiral infection, but not in febrile controls (table s5) . bar-diagrammatic representation of altered expression levels of different serum proteins in leptospirosis and falciparum malaria has been depicted in fig.s3. two selected differentially expressed proteins; clusterin and ceruloplasmin identified in leptospirosis patients were measured using western blotting to confirm the results of proteomic analysis. equal loading of the samples was verified by cbb staining of the sds-page gels and ponceau staining of the transferred blots containing the resolved proteins ( fig. s4a 3a ) and 1.49-fold up-regulation of ceruloplasmin in the leptospirosis patients compared to the hc (fig. 3b) . additionally, comparative analysis performed between leptospirosis patients and febrile controls (n = 6 each), revealed 1.1-fold down-regulation of clusterin and 1.13-fold up-regulation of ceruloplasmin in leptospiral infection compared to falciparum malaria ( fig. s4c and d) . western blot analysis of each sample (l, fc and hc) were performed in duplicate to check the reproducibility and minimize technical artifacts. functional pathway analysis was performed with 22 differentially expressed serum proteins identified in the leptospirosis patients using gel-based and ms-based analysis to understand their biological context, involvement in diverse physiological pathways and association with leptospiral infections. panther analysis revealed involvement of identified proteins in blood coagulation system (33.3%), heterotrimeric g-protein signaling pathway-gq alpha and go alpha mediated pathway (33.3%) and heterotrimeric g-protein signaling pathway-gi alpha and gs alpha mediated pathway (33.3%) (table s6. 1; fig. 4a ). concerning biological process, the identified proteins were involved in 10 biological processes: the major five processes include metabolic process (17.3%), immune system process (16%), response to stimulus (13.3%), cell communication (13.3%) and cellular process (13.3%), (table s6. 1; fig. 4b ). five major go functions: binding (31.4%), receptor activity (17.1%), enzyme regulator activity (22.9%), catalytic activity (11.4%) and transporter activity (17.1%) were identified in panther analysis for the differentially expressed proteins in leptospirosis patients (table s6. 1; fig. s5 ). in david analysis, kegg category revealed complement and coagulation cascades (1.01 e − 09 ; 30.43%). reactome category revealed three biological pathways: signaling in immune system (p=0.010079; 21.74%), hemostasis (p= 0.035589; 17.39%), metabolism of lipids and lipoproteins (p=0.01712; 17.39%), while, different complement cascades (fig. 4c) leptospirosis has been categorized as a globally important infectious disease due to the frequent occurrence of leptospiral infections in both developing and developed countries, specifically the large outbreaks in tropical and subtropical regions leading to considerable adverse effects on human health and economy [3, 22] . incidence of this acute bacterial infection have been found to be high (annual incidence per 100,000 > 10) in quite a few countries of asia pacific region including india, indonesia, bangladesh, sri lanka, thailand etc. [23] . india being one of the most flood-stricken countries in the south asian region due to its geographical location and climate, leptospirosis remains as an important endemic environmental infectious disease in this country with multiple devastating outbreaks such as in orissa (1999), mumbai (2005) and several parts of the country [23] . serum and plasma are attractive biological fluids that contain diversity of proteins released by diseased tissue, and serum/plasma proteomics has gained considerable interest for the clinical studies, particularly in disease biomarker discovery [10] . both serum and plasma are components of blood; the liquid portion of blood is referred to as plasma, while removal of the fibrinogen and other clotting factors from plasma results in serum. applications of serum and plasma as biological fluid for clinical research have their own advantages and limitations. serum does not contain fibrinogen and other blood clotting factors and has a lesser total protein content compared to plasma, which reduces the unnecessary complexity to some extent making serum more ideal for disease diagnostic purposes. very recently, zimmerman et al. have performed a paired comparison of plasma and serum samples prepared from the same subjects and demonstrated negligible differences in the numbers of peptide and protein identifications or in the overall percentages of semi-tryptic peptides or methionine oxidized peptides between these two biological fluids indicating there is no significant difference between heparinized plasma and serum in terms of overall protein diversity except the depletion of fibrinogen in serum [24] . earlier reports suggest that proteome level analysis of different biological fluids specifically serum/plasma is very effective in studying disease pathogenesis, pathogen-induced alteration in host, and host responses in different vectorborne infectious diseases like malaria [19] , dengue [25] and leishmaniasis [26] . however, over the last decade, only few proteomic studies have been reported on this zoonotic infectious disease. again, most of the previous proteomic studies have focused on the proteome and immunome profile of the pathogen [11, 17, 18] , and very few studies have investigated the effect of leptospiral infection on host proteome. to this end, two recent studies have reported proteomic analysis of leptospires excreted in urine of chronically infected natural reservoir host rattus norvegicus [15, 16] , but, no proteome level analysis has been reported hitherto to describe alterations of human serum proteins and related biological pathways due to leptospiral infection. investigation of the pathogen induced alterations in host serum proteome has immense clinical relevance in light of diagnosis and prognosis. the present proteomic study aimed to perform human serum proteome analysis of leptospirosis patients from an endemic area in india, and comparative analysis with healthy subjects to investigate disease pathobiology and host responses against leptospiral infections. additionally, another clinically relevant infectious disease; falciparum malaria was analyzed to identify the generic febrile responses. leptospirosis and malaria have overlapping geographical distributions and coexistence of these two pathogenic infections have been reported from different part of the world, particularly in the tropics [27] [28] [29] [30] . although, these two infections have many similar clinical presentations, interestingly, quite a few serum proteins including complement c3 precursor, ceruloplasmin precursor, complement component c9, complement factor h and inter-alpha-trypsin inhibitor heavy chain h4 precursor exhibited altered expression levels in leptospirosis patients, but not in falciparum malaria (febrile controls) (table s5 ; fig. s3 ). again, few proteins like complement factor b precursor, ig kappa chain c region, ig mu chain c region found to exhibit opposite trend of differential expression between leptospirosis and falciparum malaria compared to the healthy controls. however, many of the identified targets found to be similarly modulated in leptospirosis and falciparum malaria ( table s5 ). all of the leptospirosis patients selected for this study were suffering from preliminary infection with 2-4 days of fever and treated with antibiotics and antipyretics prior to the sample collection. the mean age of the leptospirosis patients, selected for this proteomic analysis was 30.5 years (sd = 8.31; range of 23-42; median 26.5) ( table s1 ). selection of healthy and febrile control populations with comparable age distribution, with an average value of 28.7 years (sd = 5.98; range of 21-34; median 26.5) and 30.4 years (sd = 9.60; range of 20-45; median 28.5) respectively, allowed maintaining the uniform population profiles for differential protein expression analysis. since, rigid inclusion criteria were maintained (table s1 .1) during the selection of patients, and subjects with any significant past history of diseases were excluded, minimum variations were observed in serum proteome profile of the patients suffering from leptospiral infection. aiming at analysis of host serum proteome alteration due to leptospirosis, we have identified several differentially expressed proteins and modulation of multiple physiological processes and pathways, including inflammation mediated acute phase responses, complement pathways, heterotrimeric g-protein signaling pathway, coagulation cascade and hemostasis in patients suffering from leptospiral infection. some of our identified proteins such as ig mu chain c, clusterin precursor, different complement factors and associated pathways like acute phase response signaling, complement and coagulation cascades, even though not through proteomic studies, have been correlated with leptospiral infection earlier, supporting our findings and enhance the confidence in this study. further, proteomic analysis revealed differential expression of few serum proteins, such as α-1-antitrypsin precursor (2.29 to 1.81fold), vitronectin precursor (2.26-fold), α-1-antichymotrypsin precursor (1.40 to 1.68-fold), ceruloplasmin precursor (1.19 to 1.59-fold) up-regulated; and regulator of g-protein signaling 7 (1.62-fold), apolipoprotein a-iv (1.91-fold) down-regulated, which have not been reported previously in the context of leptospirosis (tables 1 and 2) . altered expression of multiple acute phase proteins (apps) including ceruloplasmin, α-1-antichymotrypsin, α-1-antitrypsin, apolipoprotein a-i precursor, inter-alpha-trypsin inhibitor heavy chain h4 precursor has been identified in leptospirosis patients ( table 2) . apps are a group of serum proteins that exhibit differential expression during various acute phase responses involved in host-adaptive and host-defense mechanisms including opsonizing activities, stimulate phagocytic killing of the invading pathogens and many other specific actions such as protein transport, antioxidant activity, and inhibition of serum serine proteinases [31] . inflammation mediated acute phase signaling has been reported previously in various parasitic and viral infectious diseases like malaria [19] , dengue [25] and severe acute respiratory syndrome [32] . cytokines play a pivotal role in stimulating the production of apps as a part of the host immune response against the infection. investigation of the precise biological significance of these apps and their association with leptospiral infection may provide some insight about the disease pathogenesis. our proteomic analysis reveals altered expression of few members of the blood coagulation cascade such as alpha-1-antitrypsin precursor, complement factors b and h etc. in the patients suffering from leptospiral infection (table s6 ). the thrombocytopenia has been found to be associated with majority of the leptospirosis patients, the precise reasons and pathophysiological mechanisms liable for bleeding in this zoonosis is not clear [33] . previous studies have demonstrated disseminated intravascular coagulation (dic) as an important feature of leptospirosis [34] . inflammation-induced coagulation activation leading to thrombocytopenia is a consistently detectable phenomenon in infectious diseases and reported in the context of falciparum malaria [35] and dengue virus infection [36] earlier. bleeding tendency in leptospirosis may arise due to an imbalance in the hemostatic equilibrium and toxic effect exerted by the pathogen on bone marrow leads to thrombocytopenia [37, 38] . however, the precise role of activation of coagulation system in the pathogenesis of leptospirosis needs to be established. another interesting finding is the modulation of complement pathways, alternative and lectin, in this zoonotic infectious disease (fig. s6 ). after being activated by innate immunity system of the host, the complement cascade plays an important role in recognition and destruction of invading pathogens. previous studies have also shown the activation of complement system's lectin pathway through elevated serum levels of mannose-binding lectin, which exhibit sound correlation with the severity of clinical signs of leptospirosis [39] . in order to survive within host cells and escape the defense system, pathogenic microorganisms adapt versatile mechanisms including inhibition of complement cascades [40] . in this proteomic analysis we have identified down-regulation of few complement factors, including complement c3 precursor (2.21-fold), complement c4 precursor (1.89-fold) and some complement regulatory proteins like clusterin (2.16-fold) in the leptospirosis patients ( table 2 ). in leptospiral infection, soluble complement regulator factor h prevents complement activation at the c3 stage by inhibiting the c3-convertase and inactivating c3b into ic3b [41] . to get rid of clearance and destruction by host complement system virulent pathogens sometimes acquire fluid-phase regulators of complement pathways such as factor h and c4bbinding protein (c4bp) on the cellular surface [42, 43] . previous studies have demonstrated immune evasion of leptospira species by acquisition of human complement regulator factor h, factor h-related protein 1 (fhr-1) and c4bp [41, 44, 45] . decreased expression level of complement factors and regulatory proteins might be a consequence of the leptospiral infection and probably through the deposition of complement inhibition molecules on the pathogen cell surface or by some other unknown mechanisms performed by this pathogenic spirochete after invasion. detailed investigation of functional aspects of complement factors and regulatory proteins might provide interesting insights about the disease pathogenesis and aid in identification of potential therapeutic targets. although, leptospiral infection in human generally remains at a subclinical level or results in mild self-limiting systemic illness, but, it may cause severe infection with lifethreatening complications like septic shock, multi-organ failure and lethal pulmonary hemorrhages leading to mortality if not diagnosed and treated timely. in the developing countries, lack of awareness of the disease and inaccessibility of appropriate laboratory diagnostic facilities are the major causes behind the rapid transmission and motility associated with this vector-borne infectious disease [46] . diagnosis of leptospirosis generally performed through the detection of igm antibodies against the pathogen by rapid screening tests [47] . microscopic agglutination test (mat) and elisa-based immunoassays are the most common approaches for diagnosis of this acute bacterial infection and carried out at different health facilities. since serological tests become positive only after one week, hematology and urine analysis is also executed to identify different physiological abnormalities such as increased erythrocyte sedimentation rate, breathlessness, bleeding tendencies, proteinuria, hematuria, thrombocytopenia, elevated levels of alkaline phosphatase, serum creatinine phosphokinase, sgot and sgpt etc. that can be utilized as the indicators of leptospiral infection [48, 49] . misdiagnosis of leptospirosis sometime occurs due to its broad spectrum of symptoms, which may mimic the clinical signs of many related infectious diseases, such as dengue fever, hantavirus infection and malaria [23] . moreover, mixed infections with malaria or dengue along with leptospirosis also create complications and difficulties for diagnosis process. establishment of a panel of serological markers that can confirm leptospiral infection with high accuracy and efficiently discriminate it from other related infectious diseases will be extremely precious from the diagnostic points of view. while, the major emphasis of the study was to provide better insight into the underlying molecular mechanisms of the disease pathogenesis and host immune response in leptospirosis, but, one of the possible outcomes of this study could be establishment of early detection surrogates for the disease to meet the need for better diagnostics and effective therapy. among the identified differentially expressed serum proteins, apolipoprotein a-i, clusterin, α-1b-glycoprotein precursor, vitronectin precursor, and α-1b-glycoprotein precursor could further be investigated as inflammation-related biomarkers of leptospiral infection. in summary, this is the first study to investigate leptospira induced alterations in human serum proteome. comprehensive proteomic analysis using classical 2de and 2d-dige in combination with maldi-tof/tof ms revealed differential expression of several serum proteins in leptospirosis patients compared to healthy individuals. our results suggest that identified proteins are associated with various essential physiological processes and biological functions. further investigation of functional properties of proteins identified in this study is likely to elucidate better understanding of disease pathobiology and may help to establish candidate surrogate markers proteins for detection of leptospiral infection and discrimination from other related infectious diseases. clinical presentation of leptospirosis: a retrospective study of 201 patients in a metropolitan city of brazil urban epidemic of severe leptospirosis in brazil salvador leptospirosis study group leptospirosis: a zoonotic disease of global importance epidemic leptospirosis associated with pulmonary hemorrhage-nicaragua unique physiological and pathogenic features of leptospira interrogans revealed by whole-genome sequencing proteomics in leptospirosis research: towards molecular diagnostics and vaccine development discovery of urinary biomarkers proteomic technologies for the identification of disease biomarkers in serum: advances and challenges ahead proteome and immunome of pathogenic leptospira spp. revealed by 2de and 2de-immunoblotting with immune serum global proteome analysis of leptospira interrogans analysis of differential proteomes in pathogenic and non-pathogenic leptospira: potential pathogenic and virulence factors high-coverage proteome analysis reveals the first insight of protein modification systems in the pathogenic spirochete leptospira interrogans comparative proteomic analysis of differentially expressed proteins in the urine of reservoir hosts of leptospirosis proteomic analysis of leptospira interrogans shed in urine of chronically infected hosts proteome-wide cellular protein concentrations of the human pathogen leptospira interrogans visual proteomics of the human pathogen leptospira interrogans serum proteome analysis of vivax malaria: an insight into the disease pathogenesis and host immune response applications for protein sequence-function evolution data: mrna/protein expression analysis and coding snp scoring tools systematic and integrative analysis of large gene lists using david bioinformatics resources leptospirosis: an emerging global public health problem leptospirosis in the asia pacific region global stability of plasma proteomes for mass spectrometry-based analyses two dimensional difference gel electrophoresis (dige) analysis of plasmas from dengue fever patients two-dimensional difference gel electrophoresis (dige) analysis of sera from visceral leishmaniasis patients severe sepsis due to severe falciparum malaria and leptospirosis co-infection treated with activated protein c co-infection of malaria and leptospirosis study of coinciding foci of malaria and leptospirosis in the peruvian amazon area coexistence of leptospirosis with falciparum malaria relation of serum retinol to acute phase proteins and malarial morbidity in papua new guinea children plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry thrombocytopenia in leptospirosis activation of the coagulation cascade in patients with leptospirosis does activation of the blood coagulation cascade have a role in malaria pathogenesis? activation of coagulation and fibrinolysis during dengue virus infection erythroid hypoplasia associated with leptospirosis what role do coagulation disorders play in the pathogenesis of leptospirosis high levels of serum mannose-binding lectin are associated with the severity of clinical signs of leptospirosis complement-resistance mechanisms of bacteria regulation of complement activation at the c3-level by serum resistant leptospires c4bp binding to porin mediates stable serum resistance of neisseria gonorrhoeae role of the hypervariable region in streptococcal m proteins: binding of a human complement inhibitor leptospiral immunoglobulin-like proteins interact with human complement regulators factor h, fhl-1, fhr-1, and c4bp immune evasion of leptospira species by acquisition of human complement regulator c4bp leptospirosis in madras-a clinical and serological study elisa for the detection of specific igm and igg in human leptospirosis world health organization. guidelines for prevention and control of leptospirosis strategies for diagnosis and treatment of suspected leptospirosis: a cost-benefit analysis we are grateful to all the leptospirosis patients and healthy volunteers who participated in our study. the active support from dr. srinivasarao chennareddy, wipro ge healthcare, mumbai 400093, india in data analysis process is gratefully acknowledged. we would also like to thank dr. priyanka parte and sumit bhutada, national institute for research in reproductive health (nirrh), parel, mumbai for support in performing the scanning part of 2d-dige experiment.funding: this research was supported by a start-up grant 09ircc007 from the iit bombay to ss. key: cord-000372-wzwpyvll authors: castelló, alfredo; álvarez, enrique; carrasco, luis title: the multifaceted poliovirus 2a protease: regulation of gene expression by picornavirus proteases date: 2011-04-14 journal: j biomed biotechnol doi: 10.1155/2011/369648 sha: doc_id: 372 cord_uid: wzwpyvll after entry into animal cells, most viruses hijack essential components involved in gene expression. this is the case of poliovirus, which abrogates cellular translation soon after virus internalization. abrogation is achieved by cleavage of both eif4gi and eif4gii by the viral protease 2a. apart from the interference of poliovirus with cellular protein synthesis, other gene expression steps such as rna and protein trafficking between nucleus and cytoplasm are also altered. poliovirus 2a(pro) is capable of hydrolyzing components of the nuclear pore, thus preventing an efficient antiviral response by the host cell. here, we compare in detail poliovirus 2a(pro) with other viral proteins (from picornaviruses and unrelated families) as regard to their activity on key host factors that control gene expression. it is possible that future analyses to determine the cellular proteins targeted by 2a(pro) will uncover other cellular functions ablated by poliovirus infection. further understanding of the cellular proteins hydrolyzed by 2a(pro) will add further insight into the molecular mechanism by which poliovirus and other viruses interact with the host cell. a great variety of animal viruses encode for proteases that accomplish crucial functions during the biological cycle of the virus [1] . usually, the main function of these proteases is to proteolyze viral polypeptide precursors to render mature viral proteins that form part of viral capsids or participate in virus vegetative processes [2] . although both dna and rna viruses can encode proteases, the proteolytic tailoring of polypeptide precursors is most common among viruses with positive single-stranded rna genomes, such as picornaviruses, flaviviruses, caliciviruses, and retroviruses [3] [4] [5] [6] [7] . this mechanism of gene expression by proteolytic processing serves to compress the genetic information of viruses in the limited space provided by the genome. in this manner, viruses reduce the genetic space occupied by 5 and 3 untranslated regions (utrs), the signals devoted for mrna transcription and to initiate translation are minimal, such that, for instance, in the case of picornaviruses or flaviviruses, only one 5 and 3 utr is necessary for viral replication, transcription, translation, and morphogenesis, despite the fact that several viral proteins are synthesized by the infected cells. in addition, a number of polypeptide precursors may exhibit functions that differ from those present in their mature products. in the case of poliovirus (pv), eleven mature proteins are produced from a single translation initiation event, and at least two precursors, 2bc and 3cd, accomplish functions which are not present in their mature proteins. taking together all these considerations, the "proteolytic strategy" provides the small rna viruses with an advantageous and efficient mechanism for distribution of the genome to accomplish all the viral biological functions with the smaller genetic space. apart from generation of active viral proteins that participate in capsid morphogenesis and genome replication, viral proteases may also target a number of cellular proteins. proteolysis of these cellular substrates can very much affect a variety of cellular processes and play an important role in virus-induced cytopathogenesis [8, 9] . in this regard, productive poliovirus infection induces rapid morphological alterations in host-cell. among them, the most prevalent is the accumulation of numerous membranous vesicles in the cytoplasm, derived from endoplasmic reticulum where the viral proteins 2c and 2bc play a central role [10] . in addition, cellular shape is modified upon viral replication giving rise to cell rounding, which is most probably induced by disorders in the cytoskeletal network [11] . finally, chromatin condensates at late times postinfection, associated with the nuclear envelope except for sites where nuclear pores are placed [11] . interestingly, individual expression of the viral proteases 2a pro and 3c pro leads to the induction of most of these cytopathic effects, supporting the idea that these proteases actively contribute to the viral-induced morphological changes [12] . indeed, long-term expression of either 2a pro or 3c pro triggers the activation of caspases and, thus, cell death by apoptosis [11, 12] , reflecting the strong cytotoxicity of both proteases. in addition to the cytopathic effects induced by 2a pro and 3c pro , hydrolysis of host proteins may impact on other cellular functions such as the antiviral responses to virus infection. activation of innate immunity pathways, as well as the establishment of an antiviral response, is absolutely dependent on signals traversing the nuclear membrane through the nuclear pore complex. therefore, many viruses block cellular gene expression at different levels, that is, translation, transcription or protein and rna trafficking between nucleus and cytoplasm. the blockade of active trafficking can inhibit the nuclear import of antiviral signals or prevent the export of cellular mrnas detrimental to virus processes. all these effects can be achieved by hydrolysis of specific cellular proteins. the precise number of cellular proteins degraded by a viral protease, which is known as the "degradome," still remains unknown for a given viral protease. perhaps, one of the beststudied proteases in this respect is pv 2a pro . the discovery that pv 2a pro bisects the initiation factor of translation eif4g leading to the regulation of translation in the infected cells has attracted much attention from many laboratories during the past three decades [13, 14] . more recently, 2a pro has been involved in the alteration of rna and protein trafficking between the nucleus and the cytoplasm upon proteolysis of several nucleoporins [15] [16] [17] . the present paper focuses on the multifaceted activities of 2a pro and its regulation of different viral and cellular processes. pv is a prototype member of the picornaviridae family that infects cells of human or simian origin cytolytically or persistently and is responsible for poliomyelitis in humans [18] . the rna genome is housed in a naked capsid formed by 60 copies of each of the four structural proteins: vp1, vp2, vp3, and vp4. the infectious cycle commences by the attachment of a viral particle to cellular receptors present at the cell surface [19, 20] . this interaction leads to virion internalization and destabilization of the capsid, which adopts a less compact structure. once the rna is released in the cytoplasm, it interacts with the translational machinery, directing the synthesis of viral proteins during the early phase of infection. the pv genome is composed of a single-stranded rna copy of positive polarity of about 7.4 kb [21, 22] . this rna molecule is uncapped and contains a poly(a) tail at its 3 end and a single open reading frame, which encodes for a polyprotein of about two thousands amino acid residues. this polyprotein is proteolytically processed giving rise to the mature viral proteins [23] (figure 1(a) ). three different cleavages can be distinguished on the viral polyprotein: (i) polysomal cleavages that are produced on the nascent polypeptide chain. the first of these cleavages is catalyzed by 2a pro at its amino terminus separating the p1 precursor that encodes for the structural proteins from the rest of nonstructural polypeptides (figure 1(b) ). the second cleavage still on polysomes is performed by 3c pro , releasing the p2 precursor (2abc) from p3 (3abcd); (ii) cytoplasmic cleavages that are mostly exerted by 3c pro and (iii) hydrolysis of vp0 (vp4-vp2), which is concomitant with the morphogenesis of virus particles [2, 23] . all these hydrolytic events lead to the formation of eleven mature proteins and several precursors such as p1, p2, p3, vp0, vp3, vp1, 2bc, 3ab, and 3cd. this last precursor, 3cd, can be used as substrate by 2a pro or 3c pro . the alternative cleavage carried out by 2a pro renders the mature products 3c and 3d , whereas 3c pro generates the canonical proteins 3c pro and 3d pol . however, the biological significance of this alternative cleavage is obscure because pv mutated at 2a pro -cleavage site on 3cd does not exhibit defects in virus replication [24] . the nonstructural proteins that are generated participate in the replication of viral genomes [25, 26] . to this end, the positive rna genome is recognized at its 3 end by proteins of the replication complex to synthesize the complementary rna strand of negative polarity. in this process, 3b protein, also known as vpg, acts as a primer to initiate viral rna transcription [27] . this leads to the formation of a doublestranded rna molecule, also known as the replicative form. the negative rna synthesized serves in turn as a template to direct the synthesis of several copies of positive rna, so this process leads to the production of several nascent rna molecules with a vpg molecule bound to their 5 ends on the negative rna molecule forming a replicative intermediate. the positive rna molecules synthesized may participate in three processes (i) to serve as templates for synthesizing more negative rna molecules; (ii) as mrnas that will be engaged in translation, and (iii) as genomes that will be encapsidated in new viral particles. in picornaviruses, the only type of mrna molecule known is exactly the same as the genome. once the synthesis of several thousands of positive rna molecules is performed, the late phase of translation takes place, giving rise also during this period to a great amount of viral proteins, some of which will participate in virus morphogenesis. this late phase of infection is preceded by the abrogation of cellular mrna translation, such that only viral proteins are being synthesized late in the pv life cycle [14] . in the case of picornaviruses, transcription is dependent on continuous viral protein synthesis [28] . thus, inhibition of viral mrna translation provokes the sudden blockade of viral rna synthesis. moreover, translation is coupled to transcription, such that viral rnas transfected journal of biomedicine and biotechnology figure 1 : (a) structure of poliovirus genome and proteolytic processing of its polyprotein. the pv genome consists of a single-stranded, positive-sense polarity rna molecule, which encodes a single polyprotein. the 5 nontranslated region (ntr) is covalently linked to the viral protein vpg. the pv genome is polyadenylated (a n ) in its 3 end. the polyprotein contains four structural (p1) and seven nonstructural (p2 and p3) proteins that are released from the polypeptide chain by proteolytic processing mediated by the viral-encoded proteinases 2a pro and 3c pro /3cd pro . the intermediate products of processing 2bc, 3cd, and 3ab exhibit functions distinct from those of their respective final cleavage products. the alternative cleavage carried out by 2a pro rendering the mature products 3c and 3d is also shown. (b) once the ribosome has synthesized the pv 2a pro sequence and continues translation on the p2 region, the autocatalytic activity of pv 2a pro is manifested by cleaving itself at its amino terminus still on the nascent polypeptide chain. this cleavage liberates the p1 precursor that will render the capsid proteins on subsequent proteolytic events catalyzed by 3c pro or 3cd pro . cleavage at the carboxy terminus of pv 2a pro on the p2 precursor is accomplished by pv 3c pro , leaving free 2a pro and generating the 2bc precursor. into picornavirus-infected cells are not able to direct protein synthesis [29] . therefore, these two processes of viral macromolecular biosynthesis are tightly coupled, making it difficult to determine exactly the function affected in some pv mutants. notably, continuous lipid and cellular membrane synthesis is also necessary for pv rna synthesis [30] . the morphogenesis of progeny virions in pv-infected cells is observed concomitantly with viral rna translation and replication. the release of new viral particles takes place by cell lysis, due to membrane permeabilization that occurs at the late phase of infection [31] . viroporin 2b and its precursor 2bc are responsible for this permeabilization upon the formation of pore channels in cellular membranes [32, 33] . pv has represented a useful model to gain insight into diverse aspects of molecular biology and gene expression. a number of discoveries concerning animal viruses with rna genomes were initially made in pv. for example, the presence of uncapped mrna, the sequencing and development of an infectious cdna clone, the three-dimensional structure of a virus particle, the discovery of the ires elements, the synthesis of an infectious virus in a cell-free system, the chemical synthesis of a complete viral genomes, among others, were initially reported in pv [34] [35] [36] [37] [38] . in addition, the first time that eif4g was found proteolytically cleaved was in pv-infected cells [39] . picornaviruses encode different proteases depending on the virus species although it is common to all of them to encode 3c pro and its precursor 3cd pro (figure 2 ). in pv, both these exhibit protease activity, and they execute most of the hydrolytic events on the viral polyprotein [2, 23, 40] . apart from these two proteases, 3c pro and 3cd pro , picornaviruses also contain a 2a gene, whose product in some species exhibits proteolytic activity, as is the case for pv ( figure 2) . 2a pro has a limited proteolytic effect on the polyprotein and its function is most probably one of altering cellular functions by the cleavage of a number of cellular proteins. in this regard, the best studied of these cleavages is the bisection of eif4g ( figure 3 ) [14] . the general organization of the picornavirus genomes is to encode for p1-p2-p3 precursors giving rise to 4-3-4 mature products. some picornavirus species, in addition, encode a leader protein (l) placed before p1 ( figure 2 ) [22] . in the case of aphthoviruses, such as foot-and-mouth disease virus (fmdv), the l protein has proteolytic activity and it is known as l pro [42, 43] . during polyprotein synthesis l pro is the first protein synthesized and its autoproteolytic activity releases itself from the rest of the polypeptide chain. thus, the only known hydrolysis executed by l pro on the polyprotein is to hydrolyze between its carboxy terminus and the amino terminus of vp4 ( figure 2 ). because l pro does not play a direct role in viral replication [44] and its protease activity has a limited impact in the viral polyprotein, this protease may be involved in the interaction with the hostcell [45] [46] [47] . indeed, l pro also exhibits proteolytic activity on eif4g acting at a position close to that of pv 2a pro (figure 3 ) [48] [49] [50] . in the case of fmdv, the 2a protein is reduced to a small peptide of 18 residues that does not hydrolyze eif4g; instead it induces the release of 2a from its carboxy terminus by a ribosomal skip mechanism [51, 52] . this model proposes that fmdv 2a modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2a)-trna(gly) ester linkage at the c-terminus of 2a, thereby releasing the polypeptide from the translational complex. however, not all l or 2a proteins from picornaviruses exhibit protease activity, since in the case of emcv, which encodes both proteins, neither has been demonstrated to possess proteolytic activity [53] . all known proteases have been classified in four classes and many subgroups according to three parameters: (i) their catalytic center, (ii) their substrate specificity, and (iii) their three-dimensional structure. the classification of the different picornavirus proteases initially relied upon the effect of protease inhibitors. compounds that blocked sulphydryl groups abrogated the proteolytic activity of 2a pro and 3c pro , suggesting that the nucleophilic aminoacid in the active site was cysteine [54, 55] . however, another cysteine inhibitor such as e64 had no effect on these proteases, while l pro activity was inhibited not only by sulphydrylactive compounds but also by e64 [56, 57] . these findings together with structural observations imply that l pro belongs to the class of papain-like cysteine proteinases [43, [58] [59] [60] . comparison of the structure of picornavirus proteases with prototypes of cellular ones revealed that both 2a pro and 3c pro are similar in structure to the chymotrypsin like group [61] [62] [63] . picornavirus 3c pro reflects similarities to the staphylococcus aureus proteinase, whereas 2a pro is more akin to streptomyces griseus proteinase a. pv 2a pro is a protein composed of 149 amino acids that belongs to the cysteine protease group [54] . pv 2a pro is autocatalytically processed at its amino terminus between the capsid protein vp1 and 2a [64] (see figure 1 (b)). the determinants of substrate specificity of picornaviral pv 2a pro have been investigated in detail by identification of cleavage sites by n-terminal edman degradation, mutational analysis and using synthetic peptides as substrates [34, [65] [66] [67] . pv 2a pro can recognize a wide variety of amino acid residues at the p1 position. the determinants of substrate specificity for pv 2a pro lie at positions p4, p2, p1 , and p2 , which are preferentially occupied with ile/leu, thr/ser, gly, and pro, respectively. moreover, the determinants of substrate specificity of hrv and coxsackievirus 2a pro are very similar to those found for pv 2a pro [65] [66] [67] . the yeast two-hybrid system has been used to identify the substrate sequence interacting with pv 2a pro . all the sequences identified contain the leu-x-thr-z motif (x for any amino acid; z for a hydrophobic residue) in positions from p4 to p1 suggesting the presence of a common interacting site on pv 2a pro substrates [68] . several 2a pro variants have been generated in the entire pv genome or in the isolated 2a gene. generation of pv 2a pro mutants was initially used to identify the cys 106 , his 18 , and asp 35 as the residues that form part of the catalytic triad of 2a pro [69, 70] . these data have been confirmed in the structure of hrv2 2a pro [71] . the role of the conserved cys and his residues in the structure-function relationship has also been studied by mutagenesis. the residues cys 55 , cys 57 , cys 115 , and his 117 play a critical role in the cis and trans proteolytic activity by maintaining 2a pro structure [72] . the structure of hrv2 2a pro shows that the zn 2+ ion is coordinated tetrahedrally by the side chains of these conserved cys and his residues. this zn 2+ ion is tightly bound near to the c-terminal domain and may be important for the stability of the 2a pro [71, 73, 74] . the yeast saccharomyces cerevisiae has been used as a system to obtain pv 2a pro variants [75] . the fact that this protease is very toxic for yeast has been exploited to generate 2a pro variants devoid of this cytotoxicity. using this approach, several pv 2a pro unable to cleave eif4g have been obtained. the characterization of these mutants revealed a region in 2a pro involved in the interaction with substrates but none of the mutations were found in the catalytic triad. a parallelism has been observed between the ability of these pv 2a pro variants to block protein synthesis and to cleave eif4g [76] . pv mutants in 2a gene that lack trans but not cis proteolytic activity have been also identified. normal journal of biomedicine and biotechnology processing of the viral polyprotein is observed upon infection with these pv variants, whereas eif4g remains intact in these cells [77] . interestingly, rna replication of those mutant viruses is hampered, suggesting that there is a correlation between pv rna replication and the trans activity of 2a pro . it is controversial whether pv 2a pro contributes directly to viral replication or not. although for many years it was thought that pv 2a pro plays a direct role in pv replication, more recent studies have shown that a full-length dicistronic pv construct lacking 2a pro is capable to give rise to progeny viruses. moreover, virus yields of pv variants lacking the p1 coding region is partially restored when p1 is expressed in trans, suggesting that cleavage of the viral polyprotein by pv 2a pro is not essential for viral replication. [78] . however, it is known that 2a pro is important for inducing the cytophatic effect and for avoiding the inhibition of pv replication in interferon (ifn) α treated cells [79] . in agreement with the idea that 2a pro participates in viral rna replication, a fraction of this protease localizes in pv replicative foci although the majority of 2a pro is associated with the matrix structure in the cytoplasm of infected cells [80] . however, the presence of 2a pro in the proximity of replication complexes does not demonstrate that it participates directly in the replication process. 5.1. structure and functioning of eif4g. the process of translation can be divided in different steps: initiation, elongation, termination and ribosome recycling. the synthesis of cellular proteins is highly regulated, and in this sense, the most precisely controlled step is the initiation of translation (for a recent review, see [81, 82] ). for most eukaryotic mrnas, the initiation of translation commences with the recognition of the cap structure ( 7 mgpppn) and the poly(a) tail by the heterotrimeric complex eif4f and the poly(a)-binding protein (pabp), respectively, followed by the recruitment of the 43s preinitiation complex containing the 40s ribosomal subunit, the ternary complex met-trna met i -eif2-gtp, and the eukaryotic initiation factors (eifs), 1, 1a, 3, and 5. then, the preinitiation complex scans along the 5 untranslated region until an aug initiation codon is encountered in a favourable context. the perfect complementarity between the aug start codon and the anticodon of met-trna i leads to the arrest of scanning and the hydrolysis of gtp in the ternary complex. the release of eif2-gdp and other factors triggers the interaction of the preinitiation complex with the 60s ribosomal subunit to form the 80s initiation complex, proceeding to translation of the mrna coding region. a number of eukaryotic initiation factors participate in both mrna binding and scanning of the 5 utr of the mrna by the small ribosomal subunit. the cap-binding protein eif4e, together with the dead-box helicase eif4a and the translation initiation factor eif4g, forms the protein complex eif4f. the heterotrimeric complex eif4f is required for recruiting the 43s preinitiation complex onto the cap structure located at the 5 end of the mrna [83] . in this sense, eif4g, the larger polypeptide of eif4f, functions as an adaptor molecule that bridges the mrnas to ribosomes via interactions with factors eif4e (which binds the 5 cap structure), pabp (which binds the poly(a) tail), and eif3, which interacts with the 40s ribosome subunit ( figure 3 ) [81, [84] [85] [86] . in addition, eif4g also contains binding sites for other polypeptides involved in translation, such as the rna helicase eif4a ( figure 3 ) [87] , which is required to unwind the secondary structure within the mrna 5 leader sequence that would otherwise inhibit ribosome scanning. the simultaneous interaction of eif4g with eif4e and pabp promotes circularization of the mrna in a closed loop that facilitates the initiation of new rounds of translation by the proximity of the 5 and 3 ends [88, 89] . furthermore, eif4g also interacts with the mitogen-activated protein kinase 1 (mnk1) (figure 3 ), which phosphorylates eif4e, although the role of this phosphorylation in the initiation of translation in still unclear [90] [91] [92] [93] [94] . two forms of eif4g, known as eif4gi and eif4gii, have been identified in mammalian cells. both forms show only 46% amino acid sequence identity but they are thought to be functionally interchangeable due to the high homology in key domains that interact with other factors [88] . evidence obtained by specific depletion of each eif4g form or differential cleavage of each of them by specific proteases (see below) points to the idea that both factors should be lacking for complete abolition of protein synthesis [95, 96] . eif4gi is the dominant form in hela cells, in which the ratio between eifgi and eif4gii is 9 : 1 [97] . however, the specific role of each form of eif4g in the initiation of translation remains unknown. it was proposed that both eif4g forms are differentially regulated by different kinases, supporting the hypothesis that eif4gi and eif4gii could drive differentially translation initiation [98] . eif4gi is phosphorylated in response to serum and in a rapamycin-dependent manner at ser 1148, 1188, and 1232, although the role of these posttranslational modifications is still under investigation [99] . in addition, eif4gi is phosphorylated by p21-activated protein kinase (pak-2) that is induced under stress conditions. this phosphorylation takes place in the eif4e-binding site of eif4g and avoids the interaction between these two factors, inhibiting cap-dependent initiation of translation [100] . on the other hand, eif4gii is phosphorylated during mitosis [101] by calmodulin-dependent kinase i at ser 1156 [102] . therefore, activity of eif4gi and eif4gii might be tightly and reversibly regulated by phosphorylation under different physiological conditions. nevertheless, this factor is also subjected to irreversible modifications such as caspasemediated proteolysis, which is triggered during apoptosis and leads to shutoff of protein synthesis. caspase-3 cleaves directly eif4gi in positions 532 and 1175 removing pabp, mnk1, and one eif4a-binding domain from the eif4gi core ( figure 3 ) [103, 104] . in contrast, eif4gii is degraded during apoptosis with a delayed kinetics in relation to eif4gi proteolysis, correlating with the shutoff of the protein synthesis [104] . furthermore, many eif4gi isoforms have been detected in hela cells and these are synthesized from several distinct mrnas via alternative promoter usage and alternative splicing [105] . the largest is the eif4gi-a isoform, which contains 1,600 residues, while the eif4gi-b, -c, -d, ande are shorter variants [106] . it has been described that the longer isoforms are more active in translation initiation, most probably because they contain the pabp-binding site [107] . pv results in a rapid shutoff of host-cell protein synthesis, whereas viral mrna translation takes place efficiently [108] . it was initially observed that the inhibition of host-cell translation in pv-infected cells correlated with the proteolysis of a component of the eif4f complex with a molecular mass of about 220 kda (later identified as eif4g) [39, 109] . this cleavage is exerted by 2a pro and can be prevented by both insertion of mutations that abolish the protease activity and addition of 2a inhibitors [76, 110, 111] . interestingly, this proteolysis is more effective when eif4e is interacting with eif4g, suggesting that pv 2a pro preferentially acts on the eif4g pool involved in translation [112] . cleavage of eif4gi also occurs in cells infected with other picornaviruses such as hrv, coxsackievirus, and fmdv [48, 113, 114] . interestingly, 2a pro from pv, hrv, and coxsackieviruses cleave eif4gi at positions 681/682 (figure 3 ), suggesting the conservation of the specificity of enterovirus 2a proteases for the substrate determinants present in eif4gi [114, 115] . cleavage at position 681/682 separates eif4e-and eif3binding sites of eif4gi, contained in n-terminal and cterminal fragments respectively, thus decoupling mrna and ribosome recruiting activities. the pv 2a pro cleaves eif4gi directly and does not require any additional proteins for this process to occur [116, 117] . however, the fact that pv 2a pro is not copurified with eif4gi fragments from pv-infected cell extracts suggest that 2a pro induced the activation of a host protease, which in turn cleaves eif4g during pv infection [112] . in addition, it has been proposed that eif3 and an unknown host-cell protein could act as cofactors for eif4gi cleavage by pv 2a pro [118] . in this sense, zamora and colleagues suggested that pv infection activates at least two host-cell proteases, which together with pv 2a pro , cleave eif4gi [119] . nevertheless, no additional evidence has been put forward to support this hypothesis and the identity of these host proteases has not yet been determined. many reports have demonstrated that the kinetics of protein synthesis shutoff and eif4gi cleavage are not correlated in pv-infected cells [120] [121] [122] . these data clearly indicate that additional translation factors may be cleaved to achieve an efficient inhibition of cellular mrna translation. in this regard, additional reports showed that eif4gii is also proteolyzed by 2a pro during pv and hrv infections and that this cleavage is exerted between amino acids 699/700 leading to a proteolytic pattern similar to eif4gi. interestingly, eif4gii is significantly more resistant to 2a pro -mediated cleavage than eif4gi and the kinetics of protein synthesis shutoff close correlates with eif4gii cleavage in pv-and rhv-infected cells [122, 123] . in those studies, gradi and colleagues proposed that hydrolysis of both eif4gi and eif4gii is required for achieving pv-and hrv-mediated inhibition of host-cell mrna translation and that the cleavage of eif4gii is the rate-limiting step in the shutoff of host-cell translation after infection with those viruses [122, 123] . a variety of approaches have been devised to express pv 2a pro in order to cleave eif4g in culture cells or in cellfree systems. of these approaches, the most straightforward system has been the addition of the purified pv 2a pro , usually as a hybrid protein such as mbp-2a pro , to cellfree systems such as rabbit reticulocyte lysates (rrls), hela and krebs-2 extracts [76, 118, [124] [125] [126] [127] . in this sense, the addition of about 1 to 5 μg mbp-2a pro suffices to hydrolyze eif4g in those cell-free systems [76, [125] [126] [127] . an alternative method to cleave eif4g in an in vitro system is the translation of an mrna encoding pv 2a pro in translation competent extracts [50] . this assay has the advantage of providing genuine and freshly made pv 2a pro , leading to total cleavage of eif4g in the test tube after several minutes of translation. many different approaches have been explored in culture cells, the most popular being transfection of plasmids encoding pv 2a pro in different eukaryotic cell types [75, 76, [128] [129] [130] [131] [132] [133] [134] . several plasmids have been utilized in this respect, and perhaps the most successful one is ptm1-2a, which is transfected in mammalian cells that transiently express t7 rna polymerase by infection with a recombinant vaccinia t7 virus [76, 130, 131, 133, 134] . the amount of protease synthesized in this system is similar to that found in pv-infected cells at late times of infection, but these amounts are reached 1-2 hours after transfection. similar results have been obtained in cells constitutively expressing t7 polymerase, which comprises a less pleiotropic system, because vaccinia virus proteins are not expressed (unpublished data). since pv 2a pro targets a number of different cellular proteins, which affect several cellular functions depending on the amount of protease synthesized (see below), in some instances, it is useful to express 2a pro at low levels. we have explored many alternative methods trying to get a system that allows us to control the levels of pv 2a pro into the cells. novoa and colleagues developed a protocol based on the addition of hybrid proteins bearing pv 2a pro . these recombinant proteins enter into the cytoplasm on cell membrane permeabilization by different methods such as addition of mbp-2a pro mixed with replicationally inactive chicken adenovirus particles [135] . cleavage of eif4g following these protocols takes place after incubation for 8-10 hours, suggesting that the amount of protease internalized is probably low but sufficient to hydrolyze eif4gi in virtually all culture cells [136] . probably one of the most attractive systems is a stable cell line that inducibly express pv 2a pro , obtained in two different laboratories including ours [137, 138] . in these cell lines, pv 2a pro is synthesized when tretracycline is removed from the culture medium, leading to low expression of pv 2a pro that induces efficient cleavage of eif4g after 13 h post induction correlating with a potent inhibition of cellular translation. finally, long term expression of pv 2a pro in tet off cell lines triggers apoptosis [12, 137, 138] . the main drawback of this cell line is the low pv 2a pro escape under repression conditions that gives rise to a basal cytotoxicity. probably, the most efficient method is electroporation of an mrna encoding 2a pro under the control of emcv leader sequence (ires-2a) [95] . the biggest advantage of this method is the capacity to regulate levels of 2a pro expression by controlling the amounts of ires-2a transfected. for example, electroporation of 9 μg of ires-2a into ∼ 1.5 · 10 6 hela cells leads to total cleavage of both eif4gi and eif4gii in only 2 h, resulting in an almost complete shutoff of cellular protein synthesis. in contrast, electroporation of low amounts of ires-2a (1 μg) into hela cells induces efficient cleavage of eif4gi, whereas eif4gii remains largely intact. therefore, 9-fold more ires-2a mrna is required to cleave eif4gii compared to eif4gi [95] . based on the ires-2a mrna electroporation method we were able to induce the differential proteolysis of eif4gi and eif4gii in a timeand dose-dependent manner. kinetics of protein synthesis shutoff and eif4gii cleavage is closely correlated in hela cells, resembling what was found in pv-infected cells [122] . in agreement with what was observed with the addition of exogenous recombinant proteins [136] , translation of de novo synthesized mrnas showed higher susceptibility to low doses of pv 2a pro than mrnas already engaged in translational machinery [95] . these results suggested a possible specific role of eif4gi in the pioneer round of translation in agreement with a previous report [139] . however, specific ablation of eif4gi using sirnas induced a moderate inhibition of luciferase synthesis from de novo synthesized and preexisting mrna (about 40% in both cases) [96] . these findings reported by welnowska and colleagues indicated that the higher susceptibility of de novo synthesized mrna translation to low doses of ires-2a might be produced by an additional effect of pv 2a pro on another gene expression step. in this regard, further studies demonstrate that the stronger impact of 2a pro on de novo synthesized mrnas is due to the concomitant inhibition of rna nuclear export by nucleoporin 98 cleavage, which is also achieved under these conditions (see below) [17] . interestingly, cellular mrnas are able to initiate translation after a polysome runoff with high salt treatment when eif4gi is totally cleaved by pv 2a pro , whereas it is completely abolished when both forms of eif4g are proteolyzed [95] . taken together these set of data from cell expressing 2a pro [95, 136] as well as from pv-infected cells [120] [121] [122] we can conclude that complete shutoff of the protein synthesis induced by pv 2a pro is achieved when both eif4gi and eif4gii are completely cleaved. therefore, when the levels of one of the two populations of eif4g remain unaffected either because it is not cleaved by pv 2a pro [95] or it is not depleted by sirnas [96] , extensive host protein synthesis takes place. the infection of pv and coxsackievirus also leads to hydrolysis of pabp [140, 141] . this cleavage is carried out by pv 3c pro and coxsackievirus 2a pro and 3c pro and it might actively contribute to the host translational shutoff induced by these viruses [142, 143] . in conclusion, the proteolysis of different components of the translation initiation machinery by picornavirus proteases can account for the shutoff of host translation induced after infection although the specific contribution of hydrolysis of eif4gi, eif4gii, and pabp remains still unclear. infection of animal cells with fmdv also leads to proteolysis of eif4g and to rapid inhibition of cellular translation [48] . the proteolysis of eif4g is carried out by the two virally encoded proteases l pro and 3c pro [144, 145] . l pro cleaves both eif4gi and eif4gii extremely rapidly at positions 674 ( figure 3) and 700, respectively, located seven and one amino acids upstream of the 2a pro cleavage sites on eif4gi and eif4gii [50, 146] . the cleavage of eif4g by fmdv l pro results in the rapid shutoff of host-cell protein synthesis [145] . although the initial cleavage of eif4gi can be carried out by fmdv l pro in the absence of virus replication [145] , a sequential cleavage of the c-terminal fragment of eif4gi by fmdv 3c pro also occurs in bhk cells at early stages of infection concomitant with the shutdown of viral translation. the 3c pro cleavage site on eif4gi has been located at position 712, 38 amino acids downstream of the l pro cleavage site [147] although the role of this sequential cleavage is still unclear. the amino acid segment of eif4g located between the l pro and 3c pro cleavage sites binds rna and was suggested to be critical for mrna scanning by the preinitiation complex [148] . interestingly, this secondary cleavage does not occur in human cell lines due to an amino acid substitution at the cleavage site on eif4gi [147] . infection of cells with other picornaviruses such as cardioviruses (emcv and mengovirus) leads to a shutoff of host-cell protein synthesis. however, eif4g remains unaffected in these cells. these findings indicate that apart from eif4g cleavage, there are other mechanisms that may block host translation by picornaviruses. in addition to picornavirus l pro and 2a pro , proteases from other viruses can also cleave eif4g. the protease of human immunodeficiency virus type-1 (hiv-1) hydrolyzes eif4gi during infection of human cd4+ cells [149] . the cleavage of eif4gi takes place at positions 718, 721, and journal of biomedicine and biotechnology 9 1125, separating it in three domains ( figure 3 ) [149, 150] . interestingly, hiv-1 protease efficiently cleaves eif4gi, but not eif4gii, both in cell-free systems and in mammalian cells [151] . the differential sensitivity of eif4gi and eif4gii to hiv-1 protease is more selective than that observed with picornaviral 2a proteases [122, 123] . hiv-1 protease also cleaves pabp at positions 237 and 477 separating the two first rna-recognition motifs from the c-terminal domain of pabp [152] . cleavage of eif4gi and pabp by hiv-1 protease is sufficient to inhibit the translation of capped and polyadenylated mrnas in cell-free systems, as well as in transfected cells [127, 151] . in contrast, ires-driven translation is unaffected or even enhanced by hiv-1 pr after cleavage of both eif4gi and pabp [127, 151] . moreover, the translation of capped and polyadenylated hiv-1 genomic mrna remains unaffected in hela extracts under these conditions suggesting that viral protein synthesis might persist at late phases of hiv-1 infection where those factors are cleaved [127] . in contrast, a previous report claimed that the hydrolysis of eif4gi impaired the translation of both capped and ires-driven mrnas in reticulocyte lysate assays [150] . however, the different effect observed by these authors on ires-driven translation can be due to differences already reported between hela extract and rrl [143, 153] . in addition, eif4g cleavage is executed by proteases from other retrovirus species, such as hiv-2, simian immunodeficiency virus (siv), human t-cell leukemia virus (htlv-1), moloney murine leukemia virus (momlv), and mouse mammary tumor virus (mmtv). these proteases hydrolyze eif4gi and eif4gii with different cleavage patterns and kinetics [134] . and indeed, several retroviruses, including hiv, siv, and momlv, promote the translation of their gag gene products by internal ribosome entry, indicating that eif4g cleavage could be compatible with viral protein synthesis in infected cells [154] [155] [156] . furthermore, cleavage of eif4gi and eif4gii also occurs in feline calicivirusinfected cells although the cleavages occur at different sites to those observed for picornavirus proteases [157] . in addition, the 3c-like protease of two caliciviruses, like pv 3c pro , cleaves pabp perhaps as a complementary strategy to inhibit cellular translation [158] . the fact that proteases from many picornaviruses, retroviruses and caliciviruses, target eif4g, and, in some cases, pabp, strongly suggest that those viruses may share a common mechanism to regulate cellular and viral translation. however, further investigation is required to determine the specific contribution of eif4gi, eif4gii and pabp to the shutoff of host-cell translation and virus protein synthesis. the biological cycle of picornaviruses is confined to the cytoplasm of infected cells. however, some of the pv proteins are able to target nuclear proteins such as transcription and splicing factors and proteins involved in nuclear-cytoplasmic trafficking. one of the best studied cases in this regard is the cleavage of nucleoporins (nups), components of the nuclear pore complex (npc), by pv and hrv 2a pro , which directly impacts on nuclear-cytoplasmic trafficking of proteins and rnas [15, 16, 159] . complementarily, emcv l protein affects on the phosphorylation status of nup62 and induces similar effects to those described for pv 2a pro in the transport of macromolecules through npc [160] . nevertheless, nups are not only targets for picornavirus proteins but also for matrix (m) protein from vesicular stomatitis virus (vsv) [161] and nonstructural protein 1 (ns1) from influenza virus [162] , which also impair components of the nuclear export-import host machinery following analogous mechanisms. taking into account that proteins from different positive and negative strand rna viruses target nups, we highlight in this paper npc as a key target for viral proteins, although the possible role of npc in virus biological cycle is still under intensive research. nucleus and cytoplasm are physically separated by a semipermeable barrier known as the nuclear envelope. due to this compartmentalization, a large number of macromolecules traverse the nuclear envelope to reach their biological destination. for example, proteins are synthesized by ribosomes in the cytoplasm, but some of them such as polymerases, transcription factors, nucleosome components and splicing factors, have to traverse the npc to reach the nucleoplasm. conversely, all rna species are transcribed and processed in the nucleus and later, most of mature rnas are exported through the npc to the cytoplasm, where their biological roles take place. therefore, the regulation of rna and protein trafficking between nucleus and cytoplasm directly impacts on gene expression [163, 164] . npc forms large structures (∼125 mda) embedded in the nuclear envelope with a polarized eightfold symmetrical core. it is sandwiched by a cytoplasmic and nuclear ring, which projects eight filaments of about 50 nm into the cytoplasm and a basket-like structure of about 100 nm into the nucleoplasm [165, 166] . the npc is composed of multiple copies (8, 16 or 32) of ∼30 different proteins, called nups, that are grouped in three major classes: (i) the phenylalanineglycine (fg)-containing nucleoporins that actively work in the nuclear-cytoplasmic trafficking of macromolecules; (ii) the structural components, which lack fg-rich domains and (iii) the membrane integral proteins, which anchor the npc to the nuclear envelope [163, 167] . whereas the two last groups of nucleoporins play a role in the architecture and localization of the npc, the fg-nucleoporins directly regulate the transport of rnas and proteins through the npc, and they are the main nucleoporin class targeted by viruses (figure 4) . movement of ions, metabolites and other small molecules between nucleus and cytoplasm takes place by passive diffusion; however, transport cargos larger than 40 kda require the participation of specific receptors and carriers [168, 169] . fg nucleoporins are placed on both cytoplasmic and nucleoplasmic sides of the npc and play a central role in the active transport of macromolecules. the fg domains of nucleoporins are unfolded regions that participate in energy-independent transient interactions with the cargo receptors during the docking and translocation processes [170] . nevertheless, the delivery of the cargo and some of the directional steps require hydrolysis of gtp. trafficking of proteins through npc is mediated by a family of conserved transport receptors named karyopherins, which recognize short peptides known as nuclear localization signal (nls) and nuclear export signal (nes) [169, 171] . in addition, karyopherins also recognize nucleotide sequences during the export of some classes of rnas [163] . due to their key role, karyopherins involved in cargo import are known as importins and those involved in export are known as exportins. the rangtp cycle also plays a central role in karyopherin activity and, therefore, in trafficking of macromolecules through the npc. importins bind the cargo in the cytoplasm and release it on binding to rangtp in the nucleus. in contrast, exportins bind the cargo in the nucleus together with rangtp; and then the ran-associated gtp is hydrolyzed in the cytoplasm by rangap and cargo is liberated [163, 164] . proteins containing constitutively active nls are predominantly nuclear; but in some cases, the accessibility of nls or the nls itself is modified to selectively regulate the localization of the protein [172] . a similar regulatory mechanism is also exerted for control of nes activity [173] . for example, the nls of the transcription factor nfκb is masked by the interaction with the inhibitor iκbα. however, iκbα is degraded on proinflammatory stimuli, exposing the nls of nfκb for importin recognition. therefore, under proinflammatory conditions, nfκb is imported to the nucleus, where it triggers a specific gene response [172] . in addition to "masking strategies," phosphorylation, ubiquitination or methylation of nls or nes also influences (negatively or positively) their recognition by importins or exportins. therefore, trafficking of proteins between nucleus and cytoplasm could be finely regulated by posttranslational modifications in the nls and nes [164, 174] . conceptually, export of most of nuclear rna follows a similar mechanism to that described above for protein trafficking. this process also involves cargo receptors, export factors, and nucleoporins to deliver mature rna to the cytoplasm, but in this case, structure and function of nuclear export signals are not well understood. aminoacylated trnas are necessary in the cytoplasm for protein synthesis, but trnas are transcribed in the nucleus. trna export to the cytoplasm is mediated by exportin-t, which belongs to the karyopherin family. exportin-t forms a complex together with rangtp in the nucleus, and once the exportin-t-rangtp-trna complex reaches the cytoplasm, rangap induces the hydrolysis of the ran-associated gtp and the release of the trna [175, 176] . it has been proposed that exportin-5 could act as an auxiliary protein in this process [177] . however, later reports in yeast have opened the possibility of alternative pathways for trna nuclear export [163] . traffic of snrna follows a complex mechanism involving adaptor proteins. snrnas are transcribed by the rna polymerase (pol) ii (with the exception of u6 snrna which is produced by poliii) and then they are capped but not polyadenylated. cap-binding proteins (cbp) 80 and 20 interact with the cap of snrnas in the nucleus and recruit an export adaptor known as phax [178] . this adaptor is phosphorylated and in this state, it is able to interact with crm1, another member of karyopherin family. this interaction together with the joining of rangtp is essential for snrna trafficking [163] . hydrolysis of rangtp and dephosphorylation of phax lead to the release of the snrna in the cytoplasm. ribosomal proteins are assembled together with the different rrnas in the nucleolus following a complex process of maturation to give rise to the ribosomal subunits 40s and 60s. although is known that preribosomal subunits are exported by separate routes that involve crm1 and rangtp, nowadays the exact mechanism followed by 40s preribosomal subunits to leave the nucleus remains unclear [163] . however, 60s preribosomal subunit relies on nmd3 adaptor, which mediates the interaction with crm1 [179, 180] . the release of the 60s preribosomal subunit requires two gtpase steps: (i) the hydrolysis of the ran-associated gtp induces the liberation of crm1; and (ii) the hydrolysis of gtp mediated by the cytoplasmic gtpase lsg1, which induces the release of nmd3 [181] . finally, mrnas compose the most heterogeneous group of rnas, varying in length and structure. thus, different export factors and adaptor proteins associate with each subpopulation of mrnas [163] . mrnas are transcribed by polii, and, concomitantly with this process, a number of rna-binding proteins assemble with them. these rnabinding proteins exert different modifications in immature mrnas such as polyadenylation, splicing and capping. in addition, export factors and adaptor proteins are also recruited to nascent pre-mrnas, playing a further function in nuclear export. trex (transcription-coupled export) complex is recruited to the 5 end of nascent pre-mrnas in a splicing-dependent manner by means of the interaction of one of its components, namely aly/ref, with the subunit of the nuclear cap-binding complex cbp80 [182] . the trex complex also recruits the conserved rna-helicase uap56 that is important for mrnp biogenesis [183] . tap-p15, (also known as nxf1-nxt1) directly binds the mrna immediately after splicing and actively participates in mrna export. aly/ref, tap-p15 and uap56 associate with exon junction complexes (ejc), which are deposited in a splicingdependent manner at 20-24 nt of every exon-exon junction [184, 185] . this interaction network makes spliced mrnas more susceptible to export and couple splicing and mrnatrafficking. in fact, unspliced mrnas are exported by an alternative and less efficient pathway that involves crm1 and rangtp. this alternative route is also followed by mrnas encoding some protoncogenes and cytokines [186, 187] . it is important to mention that although mrna can follow different nuclear export pathways, in all cases the interaction of export receptors with nucleoporins plays an essential role in the transport of the mrnps throughout the npc [170] . protein and rna trafficking by pv 2a pro . pv infection strongly impacts on hostcell protein localization, giving rise to an unusual cytoplasmic distribution of nuclear proteins [188] . this particular effect has been characterized by different laboratories for a number of nuclear factors involved in several cellular processes and containing different types of nlss [189] . nevertheless, not all nuclear proteins are re-localized after pv infection, evidencing the presence of a viral-specific mechanism affecting protein subcellular distribution [188] . gustin and colleagues proposed the inhibition of nuclear protein import machinery as the cause of the cytoplasmic accumulation of nuclear proteins in pv-infected cells (figure 4 ). they demonstrated that pv impairs protein trafficking across the npc by expressing gfp proteins encoding classical or transporting nlss in mock and infected cultured cells [15, 16, 159] . these recombinant proteins accumulate in the cytoplasm after pv infection, being almost completely depleted from the nucleus. nevertheless, pv does not affect gfp distribution when the nls is mutated or deleted, since the small size of gfp allows its inefficient efflux throughout the npc [15] . interestingly, cell-free nuclear import assays demonstrated that nls-containing gfp is unable to traverse the npc when cells are previously infected with pv [15] . in agreement with these findings, shuttling endogenous proteins, such as heterogeneous nuclear ribonucleoprotein (hnrnp) a1 and hnrnp k, are detected in the cytoplasm of infected cells from 3 hpi but are undetectable in the nucleus at 4.5 hpi. however, cytoplasmic accumulation of nuclear resident proteins such as hnrnp c requires longer times of infection [15] . these findings support the idea that the distribution of a protein that shuttles between nucleus and cytoplasm may be strongly altered by the disruption of protein trafficking pathways, as compared to nuclear resident proteins. however, not all the nuclear factors are redistributed to the cytoplasm of pv-infected cells. this is the case for sc35 (a serine/arginine-rich splicing factor), fibrillarin or tata-binding protein (tbp), which remain in the nucleus for the duration of infection. the different behaviour of these groups of proteins could arise as a consequence of different turnover; thus, the distribution of highly stable nuclear proteins might be less affected by the inhibition of protein nuclear import than those proteins with low stability. alternatively, pv might not impair all protein import pathways, and some might remain operative, thereby allowing the nuclear import of several families of nuclear proteins. interestingly, some nuclear factors such as la antigen [190] , ptb [191] , sam68 [192] , or nucleolin [188] have been shown to interact with pv rna or viral proteins. these proteins accumulate in the cytoplasm of pv-infected cells and, consequently, their availability for viral replication is increased [188, [193] [194] [195] . cytoplasmic accumulation of nuclear factors might be produced mostly by the blockade of nuclear-cytoplasmic trafficking. however, loss of the nls after the cleavage of ptb and la proteins by pv 3c pro may also contribute to the subcellular relocalization of those proteins in pv-infected cells [193, 195] . redistribution of nuclear proteins as well as impairment of cellular import machinery was also observed in cells infected with other picornaviruses such as hrv [159] or emcv [160] . these findings indicate that most picornaviruses might share similar strategies to impair nuclear-cytoplasmic trafficking machinery. an alternative hypothesis has been proposed as the cause of the redistribution of nuclear factors. belov and colleagues reported that the nuclear envelope is permeabilized on pv infection, allowing nuclear proteins to diffuse across the nuclear membrane [196] . indeed, electron microscopy revealed that pv 2a pro induces severe structural damage in npc [196] . however, this hypothesis did not clarify why other proteins resident in the nucleus do not diffuse to the cytoplasm after pv infection. both protein nuclear import and permeabilization of nuclear envelope could be integrated together as sequential steps in pv biological cycle. protein import blockade is detected early after infection [16] , correlating with the first modifications in the npc (see below). however, prolonged expression of viral proteins might induce nuclear membrane leakiness, reflected by stronger alterations in npc architecture [196] . nevertheless, what might the biological relevance of these events be for a cytoplasmic virus? the most evident answer, which was extensively commented above, is that inhibition of nuclear protein import and further nuclear envelope leakiness might increase the presence of nuclear proteins in the cytoplasm of infected cells, which has been proposed in some cases to play a relevant role in pv replication. in addition, many transcription factors are arrested in the cytoplasm of uninfected cells (e.g., nf-κb, irf7, and irf3), but they are immediately activated and imported to the nucleus after proinflammatory extracellular signals or on the activation of intracellular sensors as a consequence of the viral replication. once in the nucleus, these factors trigger the transcription of a set of genes involved in the antiviral response [197] . inhibiting the import of these transcription factors, pv might prevent or, at least attenuate, the establishment of a hostile intracellular environment. further effort will be made in the future to explore this attractive hypothesis. interestingly, nup98, nup153 and nup62, components of the npc belonging to fg nup family, were found to be degraded in pv-as well as hrv-infected cells (figure 4 ) [15, 16, 159] . these proteins are essential factors of the nuclear-cytoplasmic trafficking machinery since their nterminal fg-rich domains serve as docking sites for soluble transport factors [163, 198] . nup98, nup153 and nup62 are proteolyzed in pv-infected cells following different kinetics. thus, nup98 is the cleaved early after infection (from 1 hpi), whereas nup153 and nup62 are targeted at late times after infection (from 4 hpi) [15, 16] . in agreement with these findings, cleavage of nup98 is induced even in presence of inhibitors of pv replication, suggesting that small amounts of viral proteins are sufficient for this proteolysis to occur. however, cleavage of nup153, and nup62 are efficiently prevented on arrest of viral replication, probably because they are only efficiently achieved when large amounts of viral proteins are produced [16] . therefore, nup98, nup153, and nup62 exhibit different susceptibilities to pv replication, thus pv might have a gradual impact on the nuclear-cytoplasmic trafficking machinery. nup153 is also proteolyzed by caspase-3 and caspase-9 during apoptosis induction; however, the involvement of these cellular proteases in pv-induced nup cleavage has been ruled out by different laboratories. first, nup153 cleavage journal of biomedicine and biotechnology 13 products generated upon caspase activation differ to those found in pv-infected cells [159] . in addition, nup62 is cleaved in pv-infected cells, but it remains intact despite caspase activation [199] . most importantly, pv-induced npc structural damage takes place in cells lacking caspase 3 and 9 [196] , and nup153 and nup62 are efficiently cleaved in pv-infected cells even in presence of the caspase inhibitor z-vad [159] . all together, these data support the idea that one or more viral proteins play a direct role in the cleavage of those nups. in agreement with this hypothesis, pv-induced npc damage is prevented by pv 2a pro inhibitors such as elastatinal, elastase, and mpcmk, suggesting an involvement of this viral protease in the alteration of npc [196] . indeed, individual expression of pv 2a pro in hela cells as well as addition of this protease to cell-free systems gives rise to nup98, nup62, and nup153 cleavage [16, 17, 200] . in agreement with the data obtained from pv-infected cells, on pv 2a pro expression in hela cells, nup98 is cleaved faster than nup62 and nup153, which suggests the presence of optimal cleavage sites in this protein [17] . proteolysis of nup98 in pv-infected cells as well as in cell-free systems generates two different cleavage products of around 50-65 kda and 35 kda [16] . there are two optimal cleavage sites in nup98 for pv 2a pro located between aminoacids 373-374 and 551-552, containing gly at p1 , thr at p2, and leu at p4. hydrolysis at both sites results in n-and cterminal products with predicted molecular masses of 37 and 53 or 55 and 35 kda, in good agreement with the size of the peptides detected experimentally in pv-infected cells and 2a-treated hela extracts [16, 17] . an explanation for the delayed kinetics of nup62 and nup153 with respect to nup98 is that optimal pv 2a pro cleavage sites were not found in these nups (unpublished data). recently, park and colleagues have reported that pv 2a pro directly cleaves nup62 at six different positions rendering multiple proteolytic products. these cleavage sites are located between aminoacids 103 and 298, thus releasing the fg-rich region from the protein core [200] . functionally, loss of the fg-rich region might make nup62 inactive for interaction with cargo receptors. this hypothetical mechanism of nup62 functional decoupling could be extrapolated to nup98 and nup153 (figure 4) . however, it remains unknown whether pv 2a pro is able to directly cleave nup98 and nup153 or where cleavage might occur. nup98, nup153 and nup62 are also involved in rna export from the nucleus and therefore, cleavage by pv 2a pro might also impact on this process (figure 4) . however, oligo d(t) hybridization studies showed that pv infection does not affect distribution of the polyadenylated mrna bulk after 3 hpi [16] . a more detailed analysis revealed that expression of pv 2a pro in hela cells induces a number of disorders in rna location. nuclear export of cellular mrnas is inhibited in 2a pro -expressing cells in a dose dependent manner concomitantly with nup98, nup62 and nup153 cleavage [17] . interestingly, mrna export of constitutively expressed mrnas such as β-actin is less affected than that of newly synthesized mrnas. for example, tetracycline-induced luciferase mrna was almost totally retained in the nucleus when these nups are cleaved by pv 2a pro . this effect was also observed for endogenous mrnas such as il-6, c-myc or p53 mrnas which are induced on pv 2a pro expression. therefore, pv 2a pro could counteract the induction of proapoptotic (c-myc and p53) and proinflammatory (il-6) responses by accumulating the c-myc, p53 and il-6 mrnas in the nucleus [17] . this export blockage may prevent the establishment of a hostcell response against pv infection. these findings could explain why pv 2a pro is essential for replication of pv in cells pre-treated with ifn-α [79] . furthermore, impairment of mrna export strongly alters the localization of mrnas with high turnover as compared to constitutively expressed and highly stable mrnas such as β-actin [17] . as observed in pv-infected cells, oligo d(t) hybridization revealed that pv 2a pro expression hardly affects the distribution of the polyadenylated mrna pool after short times of expression (8 h). however, nuclear accumulation of polyadenylated mrna bulk is detected when cells are exposed to pv 2a pro for longer times (16 and 24 h) . in this regard, the progression of the alterations of polyadenylated mrna localization in 2a pro -expressing cells takes place as follows: (i) disruption of nuclear mrna-containing foci (ii) appearance of mrnacontaining granules in the cytoplasm (most probably stress granules) and (iii) depletion of cytoplasmic mrnas. these events were more clearly observed when high amounts of pv 2a pro are synthesized, reflecting that nuclear accumulation of mrnas is a time-and dose-dependent process [17] . nevertheless, pv 2a pro is not only able to block mrna export, but also rrna and snrna transport. both 18s rrna and u2 snrna accumulate in the nucleus of 2a proexpressing cells in a dose-dependent manner. most probably, cleavage of nup98, nup62 and nup153 is involved in these effects, since rrna, snrna, and mrna are exported using different cargo receptors and auxiliary proteins (see above) but all of them relay in nup activity to traverse npc [163] . importantly, trnas (val-trna) are exported normally despite pv 2a pro expression, indicating that some rna nuclear export pathways are not affected by this viral protease. in fact, nup98, nup62, and nup153 are not directly involved in trna export [163] , reinforcing the idea that nucleoporin cleavage plays a central role in the impairment of protein and rna trafficking by pv (figure 4) . notably, ifn-γ induced a specific increase of nup98 levels in hela cells that counteracts the inhibition of mrna export by pv 2a pro [17, 201] . collectively, these findings reflect the central role of nup98 in pv infection and in antiviral response, since its overexpression by itself prevents, at least in part, blockade of nuclear rna export. therefore, secretion of ifn-γ by immune cells might allow the induction of antiviral response by neighbouring cells by increasing the levels of nup98 in order to protect nuclearcytoplasmic protein and rna trafficking pathways. the physiological relevance of the crosstalk between nup98-pv 2a pro in pv (and other viruses) infection might be studied in the future with cellular and animal systems. that target the nuclear pore. as mentioned above, hrv also induces cleavage of nup62, nup153 and most probably nup98, leading to the impairment of nuclear protein import [159] . nevertheless, pv and hrv 2a pro exhibit high homology and both proteases are therefore expected to share common targets. in contrast, the 2a gene of cardioviruses encodes a short peptide with autoproteolytic activity but lacks trans-protease activity. however, emcv and mengovirus are able to damage npc [202] . as occurs in pv-infected cells, these cardioviruses induce both protein nuclear import inhibition and late membrane leakiness but they do not induce the cleavage of nups [160, 202, 203] . cardioviruses encode an additional protein known as l protein, which is highly cytopathic although it lacks protease activity (in contrast to aphthovirus l pro ). l protein contains a zinc finger domain, and an acidic region, which is proposed to be phosphorylated in infected cells [204] . individual expression of this protein in cultured cells or in cell-free systems induces several cellular disorders including the inhibition of protein nuclear import that resembles that observed in emcv-infected cells [160, 203] . several studies reported emcv l protein mutations that resulted in defective virus growth phenotypes in cell culture [202, 204] . in particular, mutations in the zinc finger domain (cys19ala and cys22ala) or in the acidic region (thr47ala) partially avoid blockade of protein trafficking between the nucleus and the cytoplasm [202] . taken together, these data support the involvement of cardiovirus l protein in npc damaging and in the inhibition of protein nuclear import, inducing nups phosphorylation rather than their cleavage. indeed, nup62 is quickly and strongly phosphorylated after emcv infection (2 hpi), and it was clearly detected by conventional western blotting. nevertheless, analysis with pro-q diamond phosphoprotein stain revealed that nup153 and nup214 are also phosphorylated to a certain extent upon emcv infection. notably, nups phosphorylation was avoided when cys19ala mutation was inserted in the l protein sequence, suggesting that the zinc finger domain is essential for this posttranslational modification to occur [160] . however, the exact role of nups phosphorylation in nuclear-cytoplasmic trafficking is still unknown. the idea that nups phosphorylation could regulate protein import and rna export as a switch that turns the different pathways on/off should be pursued in more detail. as mentioned above, ran is essential for the regulation of most of the nuclear export and import pathways, because it acts as a cofactor modulating the affinity of importins and exportins for the cargo. the rangtp cycle is described in detail in section 6.1. it has been described that emcv l directly interacts with ran, and this interaction is abrogated by the insertion of c19a mutation in l [203] . however, the potential role of l/ran interaction in the modulation of rangtp cycle and its impact in nuclear import and export pathways have not yet been studied. alteration of nuclear-cytoplasmic trafficking is not only restricted to picornaviruses but has also been observed with negative strand viruses such as vesicular stomatitis virus (vsv) and influenza virus. her and collaborators reported that rna export and protein import are strongly inhibited by vsv matrix (m) protein, by microinjection of oocytes with radiolabeled rnas and proteins. radiolabeled trnas, mrnas, u snrnas, and rrnas were injected directly into the nucleus of xenopus laevis oocytes, and the subcellular localization of those rnas was monitored by autoradiography. the conclusion of this work is that mrna, u snrna, and rrna but not trna trafficking is blocked by vsv m protein [205] , in agreement with our findings on 2a proexpressing hela cells [17] . in addition, protein nuclear import was monitored by microinjection of radiolabeled proteins containing nls in the oocytes cytoplasm. this assay revealed that vsv m protein abrogates protein nuclear import to the same extent as treatment with specific inhibitors of this pathway such as wga [205] . these interesting findings support the idea that pv 2a pro and vsv m protein could target similar host proteins to impair macromolecule trafficking between nucleus and cytoplasm. in agreement with this possibility, it was found that the vsv m protein interacts with nup98 [161] , which is one of the primary targets of pv 2a pro [16] . the n-terminal domain of vsv m is sufficient to block rna nuclear export and aa 52-54 may play an essential role in this blockade, because their mutations to ala completely abrogate this inhibitory effect. indeed, the n-terminal domain of m protein is involved in the interaction with nup98 and, in particular, aa 52-54, because their mutation to ala blocks the binding of vsv m to nup98 [161] . in addition, binding of m to nup98 requires active mrna export pathways since, treatment with inhibitors such as wga hampers this interaction. vsv m is also able to induce the accumulation of endogenous polyadenylated mrnas in the nucleus of hela cells, and this effect is prevented again by mutations in aa 52-54 of m [161] . furthermore, vsv m interacts with rae1, which plays an essential role in mrna nuclear export by its interaction with nup98 and mrnps. overexpression of either nup98 or rae-1 prevents the nuclear accumulation of polyadenylated mrnas, suggesting that both factors may play a role in the blockade of mrna nuclear export by vsv m protein [206] . interestingly, ifn-γ specifically increases the level of both nup98 and rae-1 and indicates a potential antiviral effect of these proteins. indeed, overexpression of both proteins by ifn-γ treatment counteracts the inhibitory effects of vsv m protein on mrna nuclear export, highlighting the possibility of a crosstalk between m and ifn-γ that might control the fate of the viral replication in infected animals [201, 206] . influenza virus replication also impacts on nuclearcytoplasmic trafficking and leads to the nuclear accumulation of host mrnas [162, 207] . influenza virus ns1 protein is a major virulence factor that is essential for pathogenesis, because it impairs innate and adaptive immunity by inhibiting host signal transduction and gene expression [208, 209] . ns1 forms a complex with nxf1/tap, p15/nxt [162] , rae1, and e1b-ap5, which are components of the mrna nuclear export machinery (see above). individual expression of ns1 in 293t cells induces the accumulation of polyadenylated mrnas in the nucleus, suggesting that the interaction of ns1 with these export factors yields an inactive complex for mrna export. influenza virus also induces a strong reduction of nup98 steady-state levels although the viral mechanisms involved in this process are still unknown [162] . expression of reporter luciferase mrna synthesized from a nuclear plasmid is inhibited by ns1. however, this inhibition is overcome by overexpression of nxf1, p15, rae-1 or nup98, evidencing the role of ns1 interaction with these factors in the impairment of mrna nuclear export [162] . furthermore, mouse cells expressing low levels of nup98 or/and rae-1 show greater susceptibility to influenza infection, resulting in a significant increase in cell death and virus production. in addition, mrnas encoding antiviral factors or immunomodulators such as irf-1, mhc i and icam1 accumulated more in the nucleus of those cells than in cells expressing normal levels of nup98 or rae-1 [162] . all these data support the physiological role of ns1 interaction with rna export factors as well as the reduction of nup98 levels in influenza pathogenicity. interestingly, vsv m, influenza ns1 and pv 2a pro expression gives rise to similar effects on mrna trafficking. all these viral proteins target nup98 and other components of the cellular machinery involved in nuclear-cytoplasmic trafficking. survival of motor neurons (smn) complex is composed by smn and a class of proteins called gemins, which localize in both cytoplasm and nucleoplasm [210, 211] . gemin7 and gemin8 constitute the core of the complex where the other gemins associate by means of numerous proteinprotein interactions from the periphery. the smn complex is involved in the biogenesis of uridine-rich small nuclear ribonucleoprotein (u snrnp) in the cytoplasm and then the u snrnp carries out the splicing of pre-mrnas in the nucleus [212] [213] [214] . the snrnps are composed of the major u snrnas u1, u2, u4, u5, and u6 as well as a group of seven proteins known as sm ribonucleoproteins that collectively make up the extremely stable sm core of the snrnp. gemins (except gemin-2) associate with sm proteins to form a heptameric ring structure in the presence of u snrnas [211] . after sm core assembly, the u snrnps are imported to the nucleus, localizing in foci known as cajal bodies, where further maturation processes take place [215] . gemin-3, one of the main components of smn complex, is cleaved in pv-infected hela cells leading to a 50 kda cleavage product. scission of gemin-3 negatively impacts on the kinetics of sm core assembly, which is prevented in presence of inhibitors of pv replication [216] . these results indicated that high levels of pv proteins are required for this process to occur, as is the case of nup153, nup62, and eif4gii, [16, 122] . pv 2a pro is able to hydrolyze purified gemin-3 in vitro, rendering a cleavage product similar to that found in pv-infected hela cells [216] . only one potential 2a procleavage site, between the amino acids tyr 462 and gly 463 (vhtyg), was found in this smn complex component. proteolysis of gemin-3 at this position would render two cleavage products of about 50-30 kda, in agreement with the polypeptide of about 50 kda found in pv-infected and 2a pro -expressing cells. in addition, g463e mutation avoids direct hydrolysis of gemin-3 exerted by pv 2a pro in vivo and in vitro [216] . taken together, these findings support the notion that vhtyg is the cleavage site for pv 2a pro in gemin-3. although hydrolysis of gemin-3 is exerted in cells transfected with plasmid encoding pv 2a pro , it does not take place when this protease is expressed from exogenous mrnas; contrary to that found with eif4gi, eif4gii, nup98, nup153 and nup62 [17] (and unpublished data). a probable explanation for this difference is that pv 2a pro is expressed at lower levels from transfected mrnas than from plasmids [95] (castello et al., 2006) . thus, gemin-3 cleavage may be a very late event in pv-infected cells because it requires expression of high amounts of pv 2a pro . gemin-3 hydrolysis may directly impact on pre-mrna splicing since this event reduces the availability of smn complexes, which is involved in u snrnps biogenesis. nevertheless, alstead and colleagues could not detect any apparent effect of gemin-3 proteolysis in splicing of cellular pre-mrnas [216] . therefore, the physiological relevance of gemin-3 cleavage in pv biological cycle remains unknown. in addition to eifs, nups and proteins from smn complex, pv 2a pro is able to cleave proteins involved in other cellular processes, such as transcription. tbp is cleaved by pv 2a pro between amino acids tyr 34 and gly 35 in vitro, although this cleavage only removes the first 34 aa located at the n-terminus and does not inhibit transcription carried out by rna polymerase ii [217, 218] . these findings are in agreement with the fact that host mrna transcription takes place in 2a pro -expressing cells when both translation and rna nuclear export are inhibited, upon cleavage of eif4g and nups [17] . one attractive hypothesis is that pv 2a pro could cleave specific initiation factors affecting specific rather than general mrna transcription in order to modulate host-cell response to viral infection. further studies in this direction can be carried out using microarray platforms to detect precise alterations in cellular transcriptome after pv-infection or 2a pro expression. these studies could be complemented by screening for new host factors cleaved by pv 2a pro using different in silico and experimental approaches. the study of viral proteases is crucial to understand the mechanism used by animal viruses to replicate their genomes and to translate viral mrnas at the molecular level. in addition, we wish to draw attention to the concept that viral proteases can be used as tools to reveal the exact functioning of their target cellular proteins. in this regard, pv 2a pro has been very useful for examining the requirements for eif4g to translate different cellular or viral mrnas. in addition to this, in this paper, we have highlighted the role of pv 2a pro not only in the processing of the poliovirus polyprotein but also in the interaction with the host-cell. interestingly, a single viral protein is able to modulate many steps of gene expression in order to generate an optimal intracellular environment for the viral biological cycle. in particular, pv 2a pro cleaves cell proteins involved in transcription, pre-mrna splicing, nucleus/cytoplasm transport and translation, in order to hijack those host functions and to concentrate the cellular resources on the production of the viral progeny. for example, pv 2a pro inactivates host translational machinery for capped cellular mrnas by cleaving eif4g, whereas viral protein synthesis takes place under those conditions by ires-driven translation [14] . because of the decrease of cellular mrna translatability in pv-infected cells, host ribosomes are available for viral protein synthesis. probably, the inhibition of host protein synthesis may prevent the production of antiviral proteins. similarly, the blockade of nucleus/cytoplasm transport of macromolecules might isolate nuclear processes from cytoplasmic cellular ones, hampering the arrival of specific proinflammatory transcription factors to the nucleus and of mrnas encoding proinflammatory, antiviral or proapoptotic proteins to the cytoplasm of infected cells. finally, transcription and pre-mrna splicing could be also modulated by pv 2a pro and might reduce the availability of mature mrnas. nevertheless, very little is known about the potential role of pv 2a pro in transcription and pre-mrna splicing, and further studies of these steps of gene expression in pv-infected and 2a-expressing cells should be carried out. making use of the "omics" technologies it would be possible to identify changes in the host transcriptome in those cells, allowing us to understand the readjustment of host gene expression to viral infection and to the cytotoxic effect of pv 2a pro . gene ontology tools can be used to cluster the pathways (kegg), molecular activities and biological functions of the genes which are transcriptionally up-or downregulated or not affected after these unfavourable stimuli. in silico analysis could be carried out in order to identify whether these gene clusters belong to particular networks controlled by specific transcription factors. this approach will give us additional information about potential host targets for pv 2a pro . complementarily, deep sequence (rnaseq), specific microarrays types and conventional rt-pcr could be used to screen for nonspliced or abnormal spliced mrna variants in pv-infected and 2a pro -expressing cells. finally, microarrays can be employed to identify the cytoplasmic and nuclear transcriptome in those cells to determine whether mrna nuclear export inhibition induced by pv 2a pro impacts on the distribution of the entire host mrna bulk or in specific mrna pools. taken together, all this information may provide us with a general and deep vision of the modification induced by pv 2a pro on the different steps of mrna metabolism. additionally, the physiological role of nups and gemin-3 cleavage might be studied using different models. one possible and interesting approach is to engineer stable cell lines expressing noncleavable versions of nup98, nup62, nup153 and/or gemin-3 and then to analyze the fitness of pv in the different cell types, especially in presence of extracellular antiviral stimuli such as ifns or interleukins, which will activate different epigenetic programs. these studies will provide essential information to help us understand the specific role that those host factors play in pv infection. the total number of cellular proteins targeted by pv 2a pro (degradome) remains unknown, but it can be anticipated that with the expanding use of proteomic methodologies, this analysis will be known soon not only for pv 2a pro , but also for other viral proteases of interest. furthermore, the analysis of the pv 2a pro -induced degradome in human cells will be of general interest for many researchers, including virologists and cellular biologists. this goal could be achieved combining in silico prediction of 2a pro cleavage sites and experimental tools such as proteomics. in the first case, blom and collaborators developed a bioinformatics tool using neural network algorithms to predict cellular targets for picornavirus proteases [219] . this approach has been successfully used to predict the cleavage of dystrophin by coxsackievirus 2a pro [220] although most of the predicted human targets for rhinovirus and enterovirus 2a pro have not been proved yet. in addition, the algorithm did not predict the cleavage of cellular targets that have been later demonstrated to be proteolyzed by picornaviral 2a pro such as nup98 and cytokeratin 8 [16, 17, 221] . thus, it would be necessary to develop an improved algorithm able to find optimal cleavage sequences in the host proteome by implementing the proteolytic sites known for newly described 2a pro targets. many parameters have to be taken into account, including the protein localization (cytoplasmic and nuclear protein will be considered, but not proteins resident in the lumen of other organelles such as re or peroxisomes), the exposure of the cleavage site to the solvent (the sequence must be accessible to the protease), and the secondary structure in which the proteolytic site is included (optimally, unstructured regions). potential targets could be ordered by their degree of homology with optimal cleavage sequences, as well as with the degree in which they fulfill the above prerequisites. on the other hand, novel pv 2a pro targets can be identified by proteomic tools such as two-dimensional differential gel electrophoresis (dige) or quantitative proteomics such as stable isotope labeling by amino acids in cell culture (silac) coupled to monodimensional electrophoresis. following these two methods, it will be feasible to identify proteins with reduced levels on 2a pro expression and, in addition, to detect the cleavage products that will appear as lower size peptides. by the uncovering of novel 2a pro targets we will be able to map the cellular networks impacted by pv 2a pro and to integrate them in the context of pv infection. in fact, the role of 2a pro hijacking host processes could be potentially expanded to other cellular pathways with direct impact on control of viral infection. such knowledge will provide more insight into our understanding of the cytopathogenicity of viral proteases at the molecular level. viral proteases structure and function of picornavirus proteinases hiv-1: fifteen proteins and an rna the structures of picornaviral proteinases organization and expression of calicivirus genes proteolytic processing 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required for viral replication the leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mrna and blocks the host innate immune response a conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function differential ifnα/β production suppressing capacities of the leader proteins of mengovirus and foot-and-mouth disease virus leader protein of foot-and-mouth disease virus is required for cleavage of the p220 component of the cap-binding protein complex relationship of p220 cleavage during picornavirus infection to 2a proteinase sequencing extremely efficient cleavage of eif4g by picornaviral proteinases l and 2a in vitro foot-and-mouth disease virus 2a oligopeptide mediated cleavage of an artificial polyprotein analysis of the aphthovirus 2a/2b polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip the cleavage activities of aphthovirus and cardiovirus 2a proteins purification and partial characterization of poliovirus protease 2a by means of a functional assay poliovirus proteinase 3c: large-scale expression, purification, and specific cleavage activity on natural and synthetic substrates in vitro multiple proteases in foot-and-mouth disease virus replication antiviral effects of a thiol protease inhibitor on foot-and-mouth disease virus putative papain-related thiol proteases of positive-strand rna viruses. identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi, α-and coronaviruses identification of the active-site residues of the l proteinase of foot-and-mouth disease virus identification of critical amino acids within the foot-and-mouth disease virus leader protein, a cysteine protease structural similarity of poliovirus cysteine proteinase p3-7c and cellular serine proteinase of trypsin cysteine proteases of 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poliovirus 2a protease the 2a proteinase of human rhinovirus is a zinc containing enzyme spectroscopic characterization of rhinoviral protease 2a: zn is essential for the structural integrity the yeast saccharomyces cerevisiae as a genetic system for obtaining variants of poliovirus protease 2a mutational analysis of poliovirus 2a(pro): distinct inhibitory functions of 2a(pro) on translation and transcription defective rna replication by poliovirus mutants deficient in 2a protease cleavage activity 2a protease is not a prerequisite for poliovirus replication proteinase 2a is essential for enterovirus replication in type i interferontreated cells viable polioviruses that encode 2a proteins with fluorescent protein tags regulation of translation initiation in eukaryotes: mechanisms and biological targets the mechanism of eukaryotic translation initiation and principles of its regulation eif4 initiation factors: effectors of mrna recruitment to ribosomes and regulators of translation molecular mechanisms of translational control eif4g: a multipurpose ribosome adapter elf4g: translation's mystery factor begins to yield its secrets human eukaryotic translation initiation factor 4g (eif4g) possesses two separate and independent binding sites for eif4a a newly identified n-terminal amino acid sequence of human eif4g binds poly(a)-binding protein and functions in poly(a)-dependent translation circularization of mrna by eukaryotic translation initiation factors mnk1, a new map kinaseactivated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates mitogen-activated protein kinases activate the serine/threonine kinases mnk1 and mnk2 human eukaryotic translation initiation factor 4g (eif4g) recruits mnk1 to phosphorylate eif4e phosphorylation and dephosphorylation events that regulate viral mrna translation regulation of capdependent translation by eif4e inhibitory proteins differential cleavage of eif4gi and eif4gii in mammalian cells: effects on translation translation of mrnas from vesicular stomatitis virus and vaccinia virus is differentially blocked in cells with depletion of eif4gi and/or eif4gii eukaryotic translation initiation factor 4g is targeted for proteolytic cleavage by caspase 3 during inhibition of translation in apoptotic cells selective modification of eukaryotic initiation factor 4f (eif4f) at the onset of cell differentiation: recruitment of eif4gii and long-lasting phosphorylation of eif4e serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic: translation initiation factor 4gi inhibition of capdependent translation via phosphorylation of eif4g by 20 journal of biomedicine and biotechnology protein kinase pak2 suppression of cap-dependent translation in mitosis phosphorylation screening identifies translational initiation factor 4gii as an intracellular target of ca 2+ /calmodulin-dependent protein kinase i cleavage of polypeptide chain initiation factor eif4gi during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage cleavage of eukaryotic translation initiation factor 4gii correlates with translation inhibition during apoptosis translation of eukaryotic translation initiation factor 4gi (eif4gi) proceeds from multiple mrnas containing a novel capdependent internal ribosome entry site (ires) that is active during poliovirus infection generation of multiple isoforms of eukaryotic translation initiation factor 4gi by use of alternate translation initiation codons specific isoforms of translation initiation factor 4gi show differences in translational activity regulation of protein synthesis in hela cells. 3. inhibition during poliovirus infection control of protein synthesis in extracts from poliovirus-infected cells. i. mrna discrimination by crude initiation factors poliovirus proteinase 2a induces cleavage of eucaryotic initiation factor 4f polypeptide p220 a poliovirus 2a(pro) mutant unable to cleave 3cd shows inefficient viral protein synthesis and transactivation defects direct cleavage of elf4g by poliovirus 2a protease is inefficient in vitro human rhinovirus 14 infection of hela cells results in the proteolytic cleavage of the p220 cap-binding complex subunit and inactivates globin mrna translation in vitro mapping the cleavage site in protein synthesis initiation factor eif-4γ of the 2a proteases from human coxsackievirus and rhinovirus 2a proteinases of coxsackie-and rhinovirus cleave peptides derived from eif-4 gamma via a common recognition motif the eif4g-eif4e complex is the target for direct cleavage by the rhinovirus 2a proteinase poliovirus 2a proteinase cleaves directly the eif-4g subunit of eif-4f complex relationship of eukaryotic initiation factor 3 to poliovirus-induced p220 cleavage activity multiple eif4gi-specific protease activities present in uninfected and poliovirus-infected cells lack of direct correlation between p220 cleavage and the shut-off of host translation after poliovirus infection monensin and nigericin prevent the inhibition of host translation by poliovirus, without affecting p220 cleavage proteolysis of human eukaryotic translation initiation factor eif4gii, but not eif4gi, coincides with the shutoff of host protein synthesis after poliovirus infection eukaryotic initiation factor 4gii (eif4gii), but not eif4gi, cleavage correlates with inhibition of host cell protein synthesis after human rhinovirus infection high level expression in escherichia coli cells and purification of poliovirus protein 2a(pro) cleavage of p220 by purified poliovirus 2a(pro) in cell-free systems: effects on translation of capped and uncapped mrnas poly(a)-binding protein interaction with elf4g stimulates picornavirus ires-dependent translation hiv-1 protease inhibits cap-and poly(a)-dependent translation upon eif4gi and pabp cleavage human immunodeficiency virus tat-activated expression of poliovirus protein 2a inhibits mrna translation the effect of poliovirus proteinase 2a(pro) expression on cellular metabolism. inhibition of dna replication, rna polymerase ii transcription, and translation expression of poliovirus 2a(pro) in mammalian cells: effects on translation efficient cleavage of p220 by poliovirus 2a(pro) expression in mammalian cells: effects on vaccinia virus hybrid proteins between pseudomonas exotoxin a and poliovirus protease 2a(pro) effects of poliovirus 2a(pro) on vaccinia virus gene expression the eukaryotic translation initiation factor 4gi is cleaved by different retroviral proteases hybrid proteins between pseudomonas aeruginosa exotoxin a and poliovirus 2apro cleave p220 in hela cells cleavage of eukaryotic translation initiation factor 4g by exogenously added hybrid proteins containing poliovirus 2a(pro) in hela cells: effects on gene expression a stable hela cell line that inducibly expresses poliovirus 2a(pro): effects on cellular and viral gene expression poliovirus 2a protease induces apoptotic cell death eif4g is required for the pioneer round of translation in mammalian cells cleavage of poly(a)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro cleavage of poly(a)-binding protein by coxsackievirus 2a protease in vitro and in vivo: another mechanism for host protein synthesis shutoff? efficient cleavage of ribosome-associated poly(a)-binding protein by enterovirus 3c protease cleavage of poly(a)-binding protein by poliovirus 3c protease inhibits host cell translation: a novel mechanism for host translation shutoff foot-andmouth disease virus leader proteinase: purification of the lb form and determination of its cleavage site on eif-4γ foot-and-mouth disease virus 3c protease induces cleavage of translation initiation factors eif4a and eif4g within infected cells cleavage of eukaryotic translation initiation factor 4gii within foot-and-mouth disease virus-infected cells: identification of the l-protease cleavage site in vitro sequential modification of translation initiation factor elf4gl by two different foot-andmouth disease virus proteases within infected baby hamster kidney cells: identification of the 3c cleavage site conducting the initiation of protein synthesis: the role of eif4g hiv-1 protease cleaves eukaryotic initiation factor 4g and inhibits cap-dependent translation in vitro cleavage of eif4gi but not eif4gii by hiv-1 protease and its effects on translation in the rabbit reticulocyte lysate system cleavage of eif4g by hiv-1 protease: effects on translation hiv protease cleaves poly(a)-binding protein cap-poly(a) synergy in mammalian cellfree extracts. investigation of the requirements for poly(a)-mediated stimulation of translation initiation characterization of an internal ribosomal entry segment within the 5' leader of avian reticuloendotheliosis virus type a rna and development of novel mlv-rev-based retroviral vectors an internal ribosome entry segment promotes translation of the simian immunodeficiency virus genomic rna the leader of human immunodeficiency virus type 1 genomic rna harbors an internal ribosome entry segment that is active during the g/m phase of the cell cycle cleavage of eukaryotic initiation factor elf4g and inhibition of host-cell protein synthesis during feline calicivirus infection calicivirus 3c-like proteinase inhibits cellular translation by cleavage of poly(a)-binding protein inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus leader-induced phosphorylation of nucleoporins correlates with nuclear trafficking inhibition by cardioviruses vesicular stomatitis virus matrix protein inhibits host cell gene expression by targeting the nucleoporin nup98 influenza virus targets the mrna export machinery and the nuclear pore complex exporting rna from the nucleus to the cytoplasm crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport the yeast nuclear pore complex: composition, architecture, transport mechanism proteomic analysis of the mammalian nuclear pore complex the part and the whole: functions of nucleoporins in nucleocytoplasmic transport nuclear pore complex is able to transport macromolecules with diameters of about 39 nm nucleocytoplasmic transport: taking an inventory the nuclear pore complex: bridging nuclear transport and gene regulation nuclear localization signal and protein context both mediate importin α specificity of nuclear import substrates regulation of nuclear transport: central role in development and transformation inhibition of crm1-p53 interaction and nuclear export of p53 by poly(adp-ribosyl)ation arginine methylation of rna helicase a determines its subcellular localization identification of a trna-specific nuclear export receptor identification of a nuclear export receptor for trna exportin-5-mediated nuclear export of eukaryotic elongation factor 1a and trna phax, a mediator of u snrna nuclear export whose activity is regulated by phosphorylation nmd3p is a crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit 60s pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm release of the export adapter, nmd3p, from the 60s ribosomal subunit requires rpl10p and the cytoplasmic gtpase lsg1p human mrna export machinery recruited to the 5' end of mrna splicing factor sub2p is required for nuclear mrna export through its interaction with yra1p the exon-exon junction complex provides a binding platform for factors involved in mrna export and nonsense-mediated mrna decay magoh, a human homolog of drosophila mago nashi protein, is a component of the splicing-dependent exon-exon junction complex a ranindependent pathway for export of spliced mrna delineation of mrna export pathways by the use of cell-permeable peptides viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells inhibition of nucleo-cytoplasmic trafficking by rna viruses: targeting the nuclear pore complex a cellular protein that binds to the 5'-noncoding region of poliovirus rna: implications for internal translation initiation a cytoplasmic 57-kda protein that is required for translation of picornavirus rna by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein human protein sam68 relocalization and interaction with poliovirus rna polymerase in infected cells intracellular redistribution of truncated la protein produced by poliovirus 3c(pro)-mediated cleavage kh domain integrity is required for wild-type localization of sam68 translation of polioviral mrna is inhibited by cleavage of polypyrimidine tractbinding proteins executed by polioviral 3c(pro) bidirectional increase in permeability of nuclear envelope upon poliovirus infection and accompanying alterations of nuclear pores cytoplasmic nucleic acid sensors in antiviral immunity versatility at the nuclear pore complex: lessons learned from the nucleoporin nup153 caspasedependent proteolysis of integral and peripheral proteins of nuclear membranes and nuclear pore complex proteins during apoptosis specific cleavage of the nuclear pore complex protein nup62 by a viral protease role of nucleoporin induction in releasing an mrna nuclear export block nucleocytoplasmic traffic disorder induced by cardioviruses a picornavirus protein interacts with ran-gtpase and disrupts nucleocytoplasmic transport leader protein of encephalomyocarditis virus binds zinc, is phosphorylated during viral infection, and affects the efficiency of genome translation inhibition of ran guanosine triphosphatase-dependent nuclear transport by the matrix protein of vesicular stomatitis virus vsv disrupts the rae1/mrnp41 mrna nuclear export pathway influenza virus ns1 protein inhibits pre-mrna splicing and blocks mrna nucleocytoplasmic transport rig-i-mediated antiviral responses to single-stranded rna bearing 5 -phosphates inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns1 protein of influenza a virus gemin8 is a novel component of the survival motor neuron complex and functions in small nuclear ribonucleoprotein assembly gemin8 is required for the architecture and function of the survival motor neuron complex a multiprotein complex mediates the atp-dependent assembly of spliceosomal u snrnps smnrp is an essential pre-mrna splicing factor required for the formation of the mature spliceosome specific sequence features, recognized by the smn complex, identify snrnas and determine their fate as snrnps biogenesis of small nuclear rnps inhibition of u snrnp assembly by a virus-encoded proteinase poliovirusencoded protease 2a(pro) cleaves the tata-binding protein but does not inhibit host cell rna polymerase ii transcription in vitro the interaction of cytoplasmic rna viruses with the nucleus cleavage site analysis in picornaviral polyproteins: discovering cellular targets by neural networks enteroviral protease 2a cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy 2a proteinase of human rhinovirus cleaves cytokeratin 8 in infected hela cells our group is supported by a dgicyt grant (no. bfu2009-07352). the institutional grant awarded to the centro de biología molecular "severo ochoa" by the fundación ramón areces is also acknowledged. a. castello is a beneficiary of marie curie ief fellowship (seventh framework programme). alfredo castelló and enriqueálvarez contributed equally to this work. key: cord-018018-2yyv8vuy authors: rybicki, ed title: history and promise of plant-made vaccines for animals date: 2018-07-04 journal: prospects of plant-based vaccines in veterinary medicine doi: 10.1007/978-3-319-90137-4_1 sha: doc_id: 18018 cord_uid: 2yyv8vuy plant-made vaccines are now a well-established and well-tested concept in veterinary medicine—yet the only product so far licenced was never produced commercially. this is puzzling, given the breadth of exploration of plant-made animal vaccines, and their immunogenicity and efficacy, over more than twenty years of research. the range of candidate vaccines that have been tested in laboratory animal models includes vaccines for e. coli, salmonella, yersinia pestis, foot and mouth disease virus, rabbit haemorrhagic disease virus, rabbit and canine and bovine papillomaviruses, mink enteritis and porcine circovirus, and lately also bluetongue virus, among many others. there are many proofs of efficacy of such vaccines, and regulatory pathways appear to have been explored for their licencing. this review will briefly explore the history of plant-made vaccines for use in animals, and will discuss the unique advantages of plant-made vaccines for use in a veterinary medicine setting in detail, with a proposal of their relevance within the “one health” paradigm. plant-produced vaccines for veterinary medicine are an exciting prospect, largely because of the possibilities of producing protein-based vaccines' including edible vaccines' at low cost, at almost any scale, and potentially locally and on demand. they have also been controversial because of the very real possibilities of contamination of the human food supply with vaccine-producing transgenic plants, and because of concerns around the possibility of immunological tolerance developing to oral or edible vaccines. however, one set of problems that many foresawregulatory and production problems-has not eventuated, and in fact the environment now seems primed very favourably for their introduction. the main justifications for plant-made vaccines are that vaccine antigen production in plants is safe; that it is both cheap and highly scalable; that plants produce and process eukaryote-derived proteins much better than can bacteria or even yeasts; that use of plants would allow for production of vaccines in the developing world where they are needed most; and of vaccines or therapeutics that will never be produced economically by other technologies. however, despite more than twenty years of development, there are still no plant-produced vaccines or biologics available for animals-although there are in fact products licenced for and in use in humans. this review will explore the early history of plant-produced vaccines with an emphasis on proofs of principle and of efficacy, what the recent development of robust, stable transient plant production systems for vaccine antigens could mean for veterinary medicine, and the potential of plant-produced vaccines to advance both animal and potentially human health' under the banner of the one health movement. while viral proteins have probably been the most common vaccine candidates made in plants (reviewed in rybicki 2014) , it was expression of a bacterial protein-escherichia coli heat labile enterotoxin (lt-b)-that first proved that veterinary-relevant antigens could be produced in plants, and provided the first proof of principle for edible vaccines. lt-b produced in transgenic tobacco or potatoes (haq et al. 1995) was functionally equivalent to e coli-produced protein in specific assays, and immunisation of mice by oral gavage with plant material elicited systemic and mucosal toxin neutralising antibodies. moreover, fresh potato containing lt-b was immunogenic in mice when eaten. an early virus vaccine candidate was one against mink enteritis virus (mev) disease: this was novel in that it comprised chimaeric cowpea mosaic virus (cpmv) virions incorporating a short linear epitope from mev vp2 capsid protein and displaying it on the surface of virions, produced by inoculation of bean plants with an infectious cdna clone of rcpmv (dalsgaard et al. 1997) . this conferred protection against clinical disease and virtually abolished virus shedding-and given that the epitope sequence used is found in mev, canine parvovirus, and feline panleukopenia virus, the same vaccine could potentially also protect against these viruses. another early virus vaccine candidate was against rabbit haemorrhagic disease virus (rhdv): this was made by expressing the whole rhdv vp60 capsid protein in transgenic potatoes; parenteral immunisation with plant extracts was protective in rabbits (castanon et al. 1999) . subsequently, another study demonstrated that an edible vaccine consisting of leaves of transgenic plants containing presumably partially-assembled vp60 subunits, was an effective priming vaccine for later baculovirus-derived parenterally-delivered vaccine (gil et al. 2006 ). the first report of a foot and mouth disease virus (fmdv) plant-made antigen was of expression in plant protoplasts of a vp1-derived peptide of fmdv as an insertion into the minor coat protein of a replicating cpmv as a demonstration of antigen presentation (usha et al. 1993) . however, the first proof of efficacy was done using transgenic arabidopsis thaliana expressing whole vp1: parenteral immunisation of mice with leaf extracts elicited antibodies that bound to vp1 and to intact fmdv particles, and all immunised mice were protected against virulent fmdv challenge (carrillo et al. 1998) . the wigdorovitz group went on to demonstrate that mice could be protected against fmdv challenge after oral or parenteral vaccination with extracts of transgenic alfalfa expressing vp1 (wigdorovitz et al. 1999) , or immunisation with leaf extracts of tobacco plants expressing vp1 via a recombinant tobacco mosaic virus vector (wigdorovitz et al. 1999) . a refinement of these achievements included transgenic expression in alfalfa of amino acid residues 135-160 of vp1 (vp135-160) fused to glucuronidase (gus), which both allowed selection of strongest expressers by assay of enzyme activity, and was protective in mice (dus santos et al. 2002) . another novel application of carrier technology was the insertion of vp1 amino acids 140-160 (g-h loop) in an interior region of the hepatitis b virus core antigen gene (hbcag), and expression of the chimaera in transgenic nicotiana tabacum. the chimaeric protein formed virus-like particles (vlps) in the tobacco leaves, and mice immunised intraperitoneally with a soluble extract were protected against viral challenge (huang et al. 2005 ). an early attempt at showing the feasibility of making an anthrax vaccine was the expression in transgenic n. tabacum of the protective antigen (pa) protein of bacillus anthracis, possibly the best target for a subunit vaccine because it alone is protective (aziz et al. 2002) , although it went no further than showing cytolytic activity of the protein. soon after, the same group went on to express pa in transplastomic n. tabacum, with significant yield increases but still no efficacy trial (aziz et al. 2005) . another investigation of transplastomic tobacco by henry daniell's group was more thorough: yields were high (2.5 g/kg in fresh leaf tissue), the protein was protected in chloroplasts from protease cleavage and was stable when stored in leaves or as crude extracts, and was biologically active (watson et al. 2004 ). while they did not show immunogenicity or protection, the authors speculated that "with an average yield of 172 mg of pa per plant using an experimental transgenic cultivar grown in a greenhouse, 400 million doses of vaccine (free of contaminants) could be produced per acre". the daniell group subsequently showed that chloroplast-derived pa was equal in potency to the natural product from b anthracis, and that mice immunised subcutaneously with partially purified chloroplast-derived pa with adjuvant produced high igg titres and survived challenge with lethal doses of toxin (koya et al. 2005) . a different sort of approach to anthrax, and one of the first attempts at making a therapeutic antibody in plants, was taken by vidadi yusibov's group, who used the technique of transient agrobacterium infiltration-mediated expression in n. benthamiana to produce a human-derived pa-specific monoclonal antibody (hull et al. 2005) . the antibody neutralised toxin activity both in vitro and in vivo at a comparable level to hybridoma-produced antibodies. the yusibov group at what became fraunhofer usa center for molecular biotechnology later used the same transient expression technology to separately express artificial antigens comprising domain 4 of pa or domain 1 of b anthracis lethal factor (lf), fused in-frame with lichenase (lickm), a thermostable enzyme from clostridium thermocellum (chichester et al. 2007) . mice immunised with a combination of the two antigens produced high titres of mainly igg1, and sera could neutralise the effects of anthrax lethal toxin (letx) in vitro. rabies vaccines made in plants included an early yet highly sophisticated candidate that was composed of the alfalfa mosaic virus (amv) cp fused to an artificial polypeptide containing rabies virus g protein amino acids 253-275, and n protein amino acids 404-418, and expressed either in n. tabacum plants transgenic for amv replicase, or via rtmv in either n. benthamiana or spinach (yusibov et al. 2002) . the plants made particles containing amv-derived rna, encapsidated with chimaeric cp: raw spinach leaves were orally immunogenic in mice and in human volunteers. a simpler candidate was the g protein alone, with plant signal peptide and er retention signal, made in transgenic n. tabacum (ashraf et al. 2005) . while yields were relatively low (0.38% of total soluble leaf protein), purified protein injected peritoneally in mice elicited protective immunity against lethal intracerebral challenge with live rabies virus-an excellent proof of both principle and efficacy. plant-made animal rotavirus vaccines were an early target, with a stand-out study by yu and langridge (2001) providing evidence that transgenic potato could produce fusion proteins consisting of cholera toxin (ct) b and a2 subunits fused with murine rotavirus enterotoxin and enterotoxigenic e coli fimbrial antigen, respectively. fusion antigens assembled in potato tubers into cholera holotoxin-like structures that bound enterocytes, and elicited serum and intestinal antibodies after oral immunisation in mice. moreover, passively immunised mouse neonates were partially protected against diarrhoea after rotavirus challenge, demonstrating that combination vaccines for viral and bacterial pathogens may be made in plants. a simpler approach to rotavirus prevention was expression of a his-tagged vp8 * fragment of bovine rotavirus (brv) vp4 in n. benthamiana via recombinant tmv, purification of the antigen by ni 2+ chromatography, and intraperitoneal immunisation of adult female mice ). eighty-five percent of suckling mice born from these mothers were protected from brv challenge, compared to 35% immunised with an irrelevant control antigen. the same group also showed that a fusion protein made in transgenic alfalfa consisting of a short peptide derived from brv vp4 fused to gus was immunogenic both when given intraperitoneally and orally to adult female mice, and their sucklings were protected against challenge ). another group used transgenic alfalfa to produce human rotavirus vp6, and showed that female mice gavaged with alfalfa extract containing oligocpg as an adjuvant developed high titres of antibodies both systemically and mucosally, and their pups were partially protected against simian rotavirus challenge. the same animal model first used to show the efficacy of insect cell-made papillomavirus virus-like particle (vlp)-based vaccines (breitburd et al. 1995) was also used to demonstrate the efficacy of two very different plant-made papillomavirus vaccines, a few years after the demonstration that human papillomavirus l1 major capsid protein virus-like particles could be produced in transgenic tobacco or potato (biemelt et al. 2003; varsani et al. 2003; warzecha et al. 2003) . cottontail rabbit papillomavirus (crpv), the cause of the famous "jackalope" sightings in the usa, provides an excellent model system in domestic rabbits for investigation of prophylactic and therapeutic papillomavirus vaccines (breitburd et al. 1997) . accordingly, in the first study crpv l1 major capsid protein-containing extracts were prepared either from transgenic n. tabacum or n. benthamiana infected with recombinant tmv, and used with freund's incomplete adjuvant to immunise rabbits that were subsequently challenged with live virus (kohl et al. 2006) . although the vaccines appeared to contain small aggregates of crpv l1 rather than intact vlps, and immune rabbit sera failed to neutralise crpv infectivity in an in vitro assay, the rabbits were protected from wart development (kohl et al. 2006 ). in the second study, infectious recombinant tmv was used to surface display, via fusion to the capsid protein, a peptide consisting of amino acids 94-122 of the l2 minor capsid protein from either crpv or the rabbit oral papillomavirus (ropv) (palmer et al. 2006) . groups of rabbits received either or both vaccines, and were challenged with live crpv or ropv. immune rabbit sera reacted with whole l2 protein, and crpv-specific sera neutralised crpv pseudovirion infectivity. rabbits receiving the crpv or crpv + ropv vaccines were completely protected against crpv infection, and those receiving ropv alone were weakly protected against crpv. these studies demonstrated for the first time that plant-made papillomavirus vaccines based on l1 protein or l2-derived peptide had real potential as prophylactic vaccines, for use in animals as well as in humans. strangely, given that bovine papillomaviruses (bpv) had been used for many years as model systems for anti-wart vaccination, it was not until 2012, with transient agroinfiltration-mediated expression of bpv-1 vlps in n. benthamiana, that a candidate plant-made bpv l1 vlp-based vaccine was successfully made, although no efficacy trials were done (love et al. 2012) . expression of animal vaccine components in seeds of transgenic plants was attempted quite early on, with lamphear et al. (2002) in 2002 reviewing their own earlier work on maize seed expression of the b subunit of e coli heat-labile enterotoxin and the tgev s protein, with data on the potency, efficacy, and stability of these vaccines. another report followed in 2002 on the expression in maize seed of the s envelope protein of transmissible gastroenteritis coronavirus (tgev) of swine, and its protective efficacy in piglets fed with the seed (jilka 2002) . this followed an earlier demonstration of oral immunogenicity of the s protein n-terminal domain in transgenic potato tubers (gomez et al. 2000) . rabies too was a target for maize seed expression, with a report of g protein expression in transgenic maize seed to 1% of total soluble protein, and complete protection in a heterologous rabies strain challenge of mice orally immunized with one dose of *50 lg of g protein in seed extract (loza-rubio et al. 2008) . the same group later showed that sheep orally given a single dose of the transgenic maize seed containing *2 mg of the g protein were protected to the same extent as those immunized with a commercial inactivated vaccine ). the authors claimed that "this is the first study in which an orally administered edible vaccine showed efficacy in a polygastric model", which was an important development. maize was a popular target for both production and storage of recombinant proteins in early molecular farming times (see streatfield et al. 2003) ; however, other hosts were used too. for instance, the haemagglutinin (h) protein of rinderpest virus was expressed in transgenic pigeon pea to 0.49% of total soluble protein (satyavathi et al. 2003) , and also in peanuts for a product that was both orally and parenterally immunogenic in mice (khandelwal et al. 2004 ); so too was glycoprotein b (gb) of human cytomegalovirus in seeds of transgenic tobacco (tackaberry et al. 2003) , the fusion (f) glycoprotein of newcastle disease virus in transgenic rice seed (yang et al. 2007) , and the serotype-specific vp2 protein of bluetongue virus in transgenic peanuts (athmaram et al. 2006) . most of these efforts were negated, however, by the one big scandal to have hit molecular farming as far as the use of food plants for vaccine production is concerned. in 2002, aphis inspectors found volunteer tgev cp-expressing maize growing in soybean fields in two locations that were used to grow prodigene inc's tgev transgenic maize in the previous season (aphis 2008)-and in one, the soybeans were harvested with the maize plants still standing and sent to a storage facility, where they were mixed with a large volume of other seeds. the company was fined and paid substantial cleanup costs, had to develop a new compliance implementation programme, and the us dept of agriculture issued new guidelines for trials of such products. this had an unfortunate knock-on effect for molecular farming, in that it resulted in an effectively voluntary moratorium on the use of food crops for recombinant protein production worldwide. the one major success story of early work on veterinary vaccines was the approval by the us department of agriculture's center for veterinary biologics of dow agrosciences' injectable newcastle disease virus (ndv) haemagglutin-based vaccine for poultry, that had been made in a suspension cultured n. tabacum cell line. sadly, the product was never sold: the company only wanted '… to demonstrate that our concert™ plant-cell-produced system is capable of producing a vaccine that is safe and effective and to demonstrate that it meets the requirements for approval under the rigorous usda regulatory system. ndv is well known and understood by the regulatory agency, so it served as an excellent model to prove this new technology' (rybicki 2009 ). the early historical account of molecular farming for veterinary vaccines given above gives an idea of the array of technologies available and used up to the mid-2000s: transgenic and transplastomic expression of subunit proteins; recombinant plant viruses either used to express whole vaccine candidate genes, or to display chosen peptides fused to their capsid proteins; fusion of vaccine protein genes to carrier proteins to improve immunogenicity, including by inherent adjuvant properties; candidate parenteral and oral vaccines to both viruses and bacteria; therapeutics for animals made in plants; use of plant cell cultures to make antigens. many proofs of principle were obtained, for candidate vaccines against a wide range of viral and bacterial disease agents; and proofs of efficacy for vaccines delivered orally or parenterally, in whole plant material or as extracts. while all of these aspects are still currently used in molecular farming, developments that have revolutionised the field were first, the widespread adoption of agrobacterium-mediated transient expression (agroinfiltration) of recombinant proteins; and second, the use of "deconstructed" plant virus-derived vectors delivered via agrobacterium to amplify expression (reviewed in rybicki 2010). these innovations enabled the advent of high-throughput testing of expression constructs, coupled with very rapid and generally higher yield production of vaccine antigens once optimal construct design had been determined. for example, our group investigated, via agroinfiltration techniques, three different codon usage schemes and three different intracellular localization strategies for optimization of human papillomavirus type 16 l1 protein expression in n. benthamiana, in one large experiment over only 7 days (maclean et al. 2007) . use of deconstructed tmv-based vectors delivered by agrobacterium routinely has allowed significant increases of antigen yield, up to grams per kilogram fresh tissue weight (gleba et al. 2014; klimyuk et al. 2014 ). the so-called tmv-based "launch vectors" of fraunhofer usa have also allowed significant yield increases and rapid production of antigens (chichester et al. 2013; shamloul et al. 2014) . improved non-replicating hyper-translational (ht) expression vectors derived from cowpea mosaic virus rna2 have also allowed significantly higher yields via agroinfiltration (sainsbury et al. 2008 (sainsbury et al. , 2009 and the possibility of multiple genes from the same vector (saxena et al. 2016) ; so too has the use of a ssdna geminivirus-derived set of vectors by different groups (huang et al. 2009; regnard et al. 2010) , and other ssdna plant (or other host) virus-derived vectors (rybicki and martin 2014) . the number of peptide display vectors/chimaeric protein fusion partners has multiplied: while self-replicating rtmv was once state of the art, now one may choose between tmv-and potato virus x (pvx)-based vectors (lico et al. 2015) , cucumber mosaic virus (cmv) cp (nemchinov and natilla 2007; zhao and hammond 2005) , bamboo mosaic virus (yang et al. 2007 ), pvx-vectored alternanthera mosaic virus (altmv) cp gene (tyulkina et al. 2011) , lichenase (lickm), cholera toxin b subunit (ctb), amv cp, and gus, as mentioned earlier. plant virus virions in particular are now seen as easily-made nanoparticles suitable for a number of vaccine-relevant purposes (steele et al. 2017) , including as selfadjuvanting peptide-based vaccine display vehicles (lebel et al. 2015; leclerc 2014) , and excellent inducers of cross-presentation by mhc receptors (hanafi et al. 2010) . the use of tags or small peptide fusion partners is now also considerably more sophisticated, with a variety of specialized tags to choose from. these include the now-ubiquitous 6xhis tag, used for ni 2+ or other immobilised metal affinity chromatography (imac) protein purification technique; a new "cysta-tag" for the same purpose ; the n-terminal proline-rich domain of maize seed gamma zein (zera) that induces the formation of er-located protein bodies (torrent et al. 2009 ); elastin-like polypeptides (elps) with repeating pentapeptide 'vpgxg' sequences, or hydrophobins-small fungal proteins which alter the hydrophobicity of the fusion partner-both of which also form protein bodies (conley et al. 2011) . as examples, our group has recently successfully used elp fusion to the cp of beak and feather disease virus (bfdv) of parrots to aid in both accumulation and purification of the protein as a candidate vaccine (duvenage et al. 2013) . we have also used the zera tag as a protein body display vehicle for an ectopic m2e moiety common to all influenzavirus a types, which could serve as a universal vaccine for these viruses (mbewana et al. 2015) . another potentially veterinary use of zera was in the enhancement of yersinia pestis f1-v antigen fusion protein accumulation: this was *3â higher than f1-v alone in three different host plant systems-namely, n. benthamiana, alfalfa and n. tabacum nt1 suspension-cultured cells (alvarez et al. 2010) . the expression vehicles themselves have also been subject to engineering: it is now possible to precisely control glycosylation of plant-made proteins. this can be done by knock-out modification via rna interference (rnai) technology of the plant glycosyltransferases beta1,2-xylosyltransferase (xylt) and core alpha1,3-fucosyltransferase (fuct). these enzymes are responsible for the transfer of beta1,2-linked xylose and core alpha1,3-linked fucose residues to glycoprotein n-glycans, which are plant-specific modifications not found in mammalian glycoproteins (strasser et al. 2008) . it is also possible to use transient co-expression technologies to modify glycosylation (castilho and steinkellner 2016) , as well as to achieve almost completely native sialylated recombinant proteins by expression of whole mammalian glycosylation pathways in plants (castilho et al. 2010; steinkellner and castilho 2015) . it is possible to abolish n-glycosylation entirely, by co-expression of bacterial pngase f (mamedov and yusibov 2013) . one can also control endogenous plant proteases that may limit recombinant protein accumulation: for example, transient co-expression of secreted a1/s1 protease inhibitor tomato cathepsin d inhibitor (slcdi) significantly lowered a1 and s1 protease activities in the n. benthamiana apoplast, while increasing recombinant protein content by *45% (goulet et al. 2012) . it was found that co-expression of tomato cystatin slcys8, which inhibits c1a proteases, increased the transient expression yield of a monoclonal antibody in n. benthamiana by nearly 40% (robert et al. 2013) . it is also possible to reduce protease activity in cell suspension cultures by expression of specific antisense rnas, resulting in significantly increased accumulation of recombinant antibodies (mandal et al. 2014) . while suspension-cultured plant cells have been used for many years for molecular farming-and in fact were used for the only usda-licenced plant-produced animal vaccine, against ndv-new developments have made them an even more attractive prospect for low-cost vaccine production. use of flow cytometry with cell sorting, formerly the province of mammalian cell culture work only, has allowed high-expressing mab-producing tobacco by-2 cell lines from a heterogeneous population of cells by selecting the co-expressed fluorescent marker protein dsred (kirchhoff et al. 2012 ). however, one of the most exciting recent developments with this technology is the advent of the "cell pack": this is a technique for getting highly efficient (up to 100%) agrobacterium-mediated transient transformation of suspension-cultured cells that have been captured by suction onto a filter (rademacher 2014) . cell packs can be tiny (eppendorf tube tips) or large (e.g.: centimetres deep in a 20 cm buchner funnel); protein expression occurs in immobilised cells in the presence of minimal liquid media, and can continue for days (https://tinyurl.com/k22da6q). the technology is ideal for rapid and high-throughput screening of expression-and the possibility exists for taking cells back into culture and selecting for permanent transfection. these are important developments, because of the acceptability of the products of plant cell cultures for production of biologics to regulatory bodies (see below). another production host highly suited to industrial-scale production is microalgae: they are easier to establish and use than plant cell cultures, and share all the same advantages of scalability, contained growth, and consistent transgene expression levels (specht and mayfield 2014) . a very important development for molecular farming has been the development of protocols for increasing yields and implementing industrial-scale production and downstream processing of vaccines and biologics, without which no large-scale trials could take place, or routine manufacturing occur. a useful development was use of a transgenic n. tabacum/n. glauca hybrid that does not synthesize alkaloids, is highly vigorous, can easily be propagated by vegetative cuttings and does not produce viable pollen, which greatly aids biocontainment (ling et al. 2012 ). the application of techniques more familiar to chemical engineers is also advantageous: for example, it proved possible, by sequential use of fractional factorial designs and response surface methodology, to optimize culture media for mab production in transgenic tobacco by-2 cells, and to increase mab yields up to 31-fold after 10 days of culture compared to use of standard media (vasilev et al. 2013 ). the fraunhofer ime group have described generic chromatography-based strategies focusing on the binding behaviours of host cell proteins to chromatography resins under varying conditions of ph and conductivity (buyel and fischer 2014) . another useful technique from that group is a comprehensive description of the use of heat treatment of either intact leaves or of plant extracts to facilitate the industrial-scale removal of host cell proteins, optimised by a design-of-experiments approach that will also be familiar to engineers (buyel et al. 2016 ). many of these and other strategies used to optimise yields in molecular farming are reviewed here (twyman et al. 2013) . the establishment by various companies and institutes of facilities suitable for manufacture of animal and clinical trial material is also a very welcome development. as examples, the long-established kentucky bioprocessing inc (kbp) is a contract manufacturer capable of production from transgenic plants or transiently transfected plants, using the u.s. food and drug administration's current good manufacturing practices (cgmp) for pharmaceuticals, at scales up to thousands of kilograms of plants per week (https://www.kentuckybioprocessing.com/). they have recently produced and stockpiled mabs against ebolaviruses. another contract manufacturing firm with large production capacity is ibio inc: like kbp, they have a wide range of patents on their proprietary gene expression technology (holtz et al. 2015) . they are also partnering with a range of agencies and companies, including with the brazilian oswaldo cruz foundation for plant-made yellow fever vaccine, and the us dept of defense and the bill & melinda gates foundation for influenza vaccines (http://www.ibioinc.com/). the fraunhofer usa center for molecular biotechnology (http://www.fhcmb. org/) is a not-for-profit research and development organisation, that offers "… plant-based protein production, purification, scale-up and gmp manufacturing to support the development of vaccines, therapeutics and diagnostics", also with proprietary expression platforms, and can take products right through to fill and finish. the fraunhofer ime in aachen also has a state-of-the-art mechanised plant production facility still under construction as of 2017. the regulatory environment has changed for the better, even though it was not in truth as inimical as first supposed: this was borne out by the fact that as early as 2006, the cuban regulatory agencies and the usda had approved plant-made mabs for the purification of an already-licenced yeast-made hepatitis b vaccine, and the tobacco cell-made ndv vaccines, respectively (rybicki 2009 ). as another early example, the fraunhofer ime molecular farming group published in 2004 that use of whole plants for biologics production lacks intrinsic benefits of cell culture techniques, such as precise control over growth conditions, batch-to-batch product consistency, sterile containment, and it being much harder to be in compliance with good manufacturing practice (gmp) (hellwig et al. 2004 ). they pointed out that plant cell suspension cultures have all the merits of microbial and animal cell cultures, have an established track record for secondary metabolite production, and are far cheaper to use. these justifications notwithstanding, the same group later noted, in a review on gmp issues for plant-made proteins in whole plants, that: "when [plant-derived] recombinant proteins are intended for medical use… they fall under the same regulatory guidelines for manufacturing that cover drugs from all other sources, and when such proteins enter clinical development this includes the requirement for production according to [gmp] . in principle, the well-characterized gmp regulations that apply to pharmaceutical proteins produced in bacteria and mammalian cells are directly transferrable to plants" . they subsequently were able to get gmp manufacturing authorisation from german authorities for making mabs from transgenic n. tabacum for a phase i clinical trial (ma et al. 2015) . other entities have also scaled and regularised production to allow production of materials for animal and clinical trial-and one of the most successful has been medicago inc., who presently has routine large-scale production of influenzavirus a haemagglutinin (ha)-based vlps for use in advanced human clinical trial (d'aoust et al. 2010) . in 2012, medicago inc. succeeded in manufacturing 10 million doses of an h1n1 vlp-based influenza vaccine candidate in one month, by phase 1-appropriate cgmp, as part of the us defense advanced research projects agency (darpa)-funded challenge (darpa 2012) . a group in japan has also recently developed a gmp-compliant production process for a transgenic rice seed-based cholera vaccine-mucorice-ctb-which is simply polished, powdered seed, now in clinical trial (kashima et al. 2016) . as evidence of the increasing maturity of veterinary molecular farming, one of the editors of this book has co-authored a recent article on regulatory and commercial hurdles hampering the advance to market of plant-produced veterinary vaccines, covering developing business plans, assessing market opportunities, manufacturing scale-up, financing, protecting and using intellectual property, and regulatory approval (macdonald et al. 2015) . at first sight, molecular farming appears the ideal way to make recombinant protein-based veterinary vaccines: production of active ingredients is markedly cheaper per unit mass than by use of any animal tissue-culture system, and generally cheaper than yeast or bacterial culture (rybicki 2010 ); partially-purified or unprocessed extracts are highly unlikely to contain any animal pathogens; edible and oral vaccines appear highly feasible; the financial barrier to entry for manufacture appears far lower than for conventionally-made vaccines. it is possible to efficiently make bacterial proteins using bacterial-derived translational machinery in chloroplasts in transplastomic plants, as well as to make other proteins at very high yield; conventional transgenics have been used to make many vaccine candidates, with many proofs of efficacy; transient expression technologies have revolutionised the field in terms of providing high yields and very rapid development times from concept to product. and yet, only one product-dow's ndv vaccine-is registered for use, and that is not sold. it is possible that heavy investment by big industry players in conventional manufacturing technologies has stalled their uptake of molecular farming technology for veterinary vaccines and biologics: this has certainly been true for human biologics. however, perhaps developments from the human field could be used as a spur for uptake of veterinary vaccines and biologics: an example here is the licencing of protalix biotherapeutics' elelyso ® or glucocerebrosidase, a therapeutic for a genetic mitochondrial enzyme deficit called gaucher disease, made using transgenic carrot cell lines in 800 litre plastic bag fermenters (http://protalix.com/ about/elelyso/). a contamination of genzyme's mammalian cell production facilities in 2009 with a mammalian calicivirus led to the fda allowing protalix to supply the drug to patients who needed it, and to accelerated licensure (bethencourt 2009 ). the company has also successfully tested oral administration of drugs in plant cells, which would be a highly welcome development: they claim that "oral delivery of protein therapies [is] possible due to the unique cellulose wall of plant cells that makes them resistant to degradation when passing through the digestive tract" (protalix 2017). another apposite example was the fortuitous availability of a plant-made anti-zaire ebolavirus mab cocktail known as zmapp™, at the height of the recent west african ebola disease outbreak (reviewed in rybicki 2014). this was made by transient expression in n. benthamiana, and only a few clinical trial doses were available: these were used under the humanitarian principle, and later the mabs were cleared for use by the fda in an efficacy trial just before the end of the epidemic (leafbio 2016). both these examples are of niche products that were not being made at large scale or for a large market by conventional techniques, and for which there was a sudden, pressing need that could not be supplied by other means. this could provide motivation for small companies to either develop inexpensive vaccines for emerging diseases, or to target niche vaccines or niche therapeutics, in the knowledge that large established entities are unwilling to take the risk. one example for the former possibility comes from the recent emergence of bluetongue virus (btv) disease in sheep and small ruminants in europe, due to northward spread of the insect vector with climate change (purse et al. 2008) : while attenuated live vaccines are available-south africa presently uses a cocktail of 24 such viruses-concerns in europe about reassortment of virus dsrna genome components between vaccinated and naturally diseased animals, as well as of the safety of the vaccines in terms of possible under-attenuation which may result in disease development in certain sheep breeds (niedbalski 2011) , mean these are not being used. the irregular occurrence of outbreaks, and the limited number of strains involved, mean that stockpiling vaccines is desirable. however, killed vaccines still require growing potentially dangerous viruses, and while it is possible to make vlps in cell cultures and these are effective (pearson and roy 1993; roy et al. 1994) , the technology is too expensive for farm animal use. it is fortunate, therefore, that it is also possible to make btv-8 vlps via transient expression in n. benthamiana, and these are as effective in a single injected dose as the commercial vaccine (thuenemann et al. 2013) . there are currently no plans to manufacture this or other plant-produced btv vaccines for the european or other markets; however, this may soon change. an example for a niche vaccine product comes from ours and others' work on beak and feather disease virus (bfdv) vaccines: psittacines are highly valued companion animals; however, there are very few vaccines for their diseases, and none yet available for bfdv. while some recent work in this area has shown that recombinant cp can be made in e coli and in insect cells (heath et al. 2006; patterson et al. 2013; stewart et al. 2007) , that it appears to be protective (bonne et al. 2009 ) and that this can apparently form vlps (sarker et al. 2015) , it still appears that the protein is too expensive to produce for use as a vaccine. while initial work with plant production of bfdv cp was disappointing due to low yields, recent work from our group (duvenage et al. 2013 ) showed a significant increase in bfdv yield due to fusion with elastin-like polypeptide (elp), and good immunogenicity in mice. this, coupled with a very simple purification protocol enabled by elpylation (conley et al. 2009 ), could allow scalable, cheap production of bfdv vaccines. while therapeutics such as mabs or other biologics for veterinary use are generally limited to high-value companion animals, plant production could open up a hitherto neglected market niche. one excellent example is the manufacture in japan of canine interferon-a (tabayashi and matsumura 2014) : this is done via transgenic strawberries in a completely enclosed gmp-compliant facility, and the product is powdered strawberry extract given orally, to combat canine periodontal disease. another very recent example in dogs, albeit with them being used as a model for human disease, was the proof that lyophilised transplastomic lettuce leaves expressing ctb fusions of coagulation factor ix (fix) could be used orally in feed for >300 days in haemophilia b dogs with no ill effects-and that this treatment resulted in robust suppression of igg/inhibitor and ige formation against intravenously-provided fix, and a marked shortening of blood coagulation times (herzog et al. 2017 ). an example for agricultural use is the oral dosing of pigs with transgenic arabidopsis thaliana seeds containing designer igas against enterotoxigenic e coli (etec) (virdi et al. 2013) : this product consisted of dimeric llama-derived heavy chain variable region fused to the fc portion of a porcine iga and the porcine iga j chain and secretory component, which allowed production of dimeric secretory iga-like antibodies (vhh-iga). in a piglet feed-challenge experiment with etec, dosing piglets with 20 mg/d per pig vhh-iga produced a progressive decline in bacterial shedding and a significantly higher weight gain than seen in control or other experimental pigs. a highly novel plant-made therapeutic product was the receptor binding domain of the tailspike protein gp9 from the p22 bacteriophage: this is known to reduce salmonella colonisation in the chicken gut (miletic et al. 2015) . purified elp-fused gp9 bound to salmonella enterica serovar typhimurium in vitro, and feeding lyophilized leaves containing gp9-elp to newly hatched chickens showed that it has the potential to control salmonella contamination in commercially-raised fowl. these and other experiments are reviewed here (juarez et al. 2016; topp et al. 2016) , in articles that make an excellent case for plant-made immunotherapeutics for veterinary use. the one health concept has as one of its central themes the integration of opportunities for vaccine-based approaches for the prevention of zoonotic and emerging diseases across veterinary and human medicine (monath 2013) a set of disease agents which exemplify the potential strength of the one health approach are influenza viruses, and they have in fact been the focus of a number of international meetings and planning sessions (chien 2013; dwyer and kirkland 2011; kahn et al. 2014; ludwig et al. 2014; powdrill et al. 2010; short et al. 2015) . the unique mix of hosts that occurs in intensive agricultural environments that could give rise to pandemics-swine, birds and humans-is a major cause of international concern; so too is the development of suitable vaccines for the prevention of infection in domesticated birds, farmed swine, and humans. plants have been shown to be highly useful for the production of influenza vaccines, and indeed possibly the fastest ever production at scale of an influenzavirus a strain vaccine-1 month for 10 million doses-was done by medicago inc. for h1n1pdm 2009 ha vlps in 2012 (rybicki 2014) . medicago also managed in 2013, as an exercise to demonstrate preparedness, to produce grams of cgmp-grade plant-made h7 ha-only vlps only 19 days after accessing the h7 ha gene cdna sequence, in response to an outbreak in china in the same year. the fact that plant-made influenza vaccines have worked very well in animal models means that they should be trialled extensively in domestic fowl and swine, to see if the maintenance of the viruses in these hosts can be curbed. as for companion animals, there is even a canine influenza vaccine candidate: following a 2004 h3n8 outbreak in the us, a group in canada used the plant-derived filamentous malva mosaic virus (mamv) nanoparticles as a vaccine platform to display the highly conserved ectopic m2e peptide and to increase its immunogenicity. together with the adjuvant ompc derived from salmonella typhi, the vaccine was protective against both the homologous virus and a heterosubtypic strain of influenza in mice, as well as eliciting antibodies reactive with m2e peptides derived from h9n2, h5n1 and h1n1 strains and being immunogenic in dogs (leclerc et al. 2013) . given that brucellosis is listed as a one health priority, it is worth noting that a transgenic plant-produced b abortus outer membrane protein (u-omp19) was an effective oral vaccine in mice against a systemic challenge, eliciting an adaptive il-17 immune response (pasquevich et al. 2011 )-and that the protein has significant adjuvant activity, and oral vaccination of mice with u-omp19 plus salmonella antigens was protective against virulent challenge with s typhimurium (risso et al. 2017) . it is important to realise that, while vaccines are the target of this review, one health products can also be reagents to be used in more effective or cheaper diagnostic kits, and in particular for point-of-care devices, or for research laboratory use-and especially proteins that could be both a reagent and used as a candidate vaccine in animals and possibly humans. a few of the best potential one health targets for plant-made dual-function proteins would be proteins from middle eastern respiratory syndrome (mers) coronavirus (wirblich et al. 2017) , nipah and hendra viruses (landford and nunn 2012; mackenzie et al. 2003) , diagnostic/ vaccine candidate proteins from rift valley fever and crimean-congo haemorrhagic fever viruses (kortekaas 2014; monath 2013) . inexpensive and abundant proteins made from these agents could first serve as reagents in the development of cheap point-of-care diagnostics, and then as vaccine candidates in animals, if appropriate, and then possibly in humans. a useful example here is of the expression both by agroinfiltration in n. benthamiana as a reagent, and in transgenic n. tabacum roots and leaves as a vaccine, of a fused gcgn envelope glycoprotein-encoding gene from crimean-congo haemorrhagic fever virus (ghiasi et al. 2011 ). the protein yield was 1-2 mg/kg fresh plant weight. transgenic material was orally immunogenic, and elicited humoral and mucosal antibody responses, and antibodies bound inactivated virus used as a vaccine booster in some experiments. agroinfiltration-produced gngc was used as a reagent in elisa to detect immune responses. another study from our group was of the production of cchfv n protein in n. benthamiana by agroinfiltration specifically as a reagent for use in diagnostic tests (atkinson et al. 2016 ): a plant codon-optimised and 6xhis tagged n protein gene was found to accumulate best as a soluble protein in the cytoplasm, from which it could be easily purified by ammonium sulphate fractionation and immobilised ni 2+ column chromatography. purified np was used in a validated indirect elisa to detect anti-cchfv igg in sera from convalescent human patients: this was successful for 13/13 samples, with no readings for samples from patients with no history of cchfv infection. the results were 100% concordant with those from a commercially available immunofluorescent assay. given that soluble n protein is hard to produce and difficult to purify from insect cell cultures, the plant-made product would seem to be a desirable replacement. while the same has been said in many venues over more than twenty years now, the field of molecular farming really does seem to be near to meeting its initial promise for veterinary use. all of the technology that is required for efficient, high-yield production of biologics is in place; downstream processing modalities have been well worked out by a number of near-and cgmp-compliant facilities; many candidate vaccines for a wide variety of pathogens have been tested; therapeutic biologics too for veterinary use are now feasible; regulatory agencies seem agreeable to considering plant-made products. the generally shorter regulatory path, the possibility of using less stringently purified products, and the very real possibility of using oral vaccines and therapeutics, should also be highly attractive for product developers. i sincerely hope, then, that realisation of the promise comes very soon. higher accumulation of f1-v fusion recombinant protein in plants after induction of protein body formation noncompliance history high level expression of surface glycoprotein of rabies virus in tobacco leaves and its immunoprotective activity in mice integration and expression of bluetongue vp2 gene in somatic embryos of peanut through particle bombardment method plant-produced crimean-congo haemorrhagic fever virus nucleoprotein for use in indirect elisa expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax transformation of an edible crop with the paga gene of bacillus anthracis virus stalls genzyme plant production of human papillomavirus type 16 virus-like particles in transgenic plants assessment of recombinant beak and feather disease virus capsid protein as a vaccine for psittacine beak and feather disease immunization with viruslike particles from cottontail rabbit papillomavirus (crpv) can protect against experimental crpv infection the rabbit viral skin papillomas and carcinomas: a model for the immunogenetics of hpv-associated carcinogenesis generic chromatography-based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts immunization with potato plants expressing vp60 protein protects against rabbit hemorrhagic disease virus in planta protein sialylation through overexpression of the respective mammalian pathway transient expression of mammalian genes in n. benthamiana to modulate n-glycosylation immunogenicity of a subunit vaccine against bacillus anthracis a plant-produced protective antigen vaccine confers protection in rabbits against a lethal aerosolized challenge with bacillus anthracis ames spores how did international agencies perceive the avian influenza problem? the adoption and manufacture of the 'one world, one health' framework optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants protein body-inducing fusions for high-level production and purification of recombinant proteins in plants the production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza plant-derived vaccine protects target animals against a viral disease a novel methodology to develop a foot and mouth disease virus (fmdv) peptide-based vaccine in transgenic plants expression in tobacco and purification of beak and feather disease virus capsid protein fused to elastin-like polypeptides influenza: one health in action gmp issues for recombinant plant-derived pharmaceutical proteins mice orally immunized with a transgenic plant expressing the glycoprotein of crimean-congo hemorrhagic fever virus successful oral prime-immunization with vp60 from rabbit haemorrhagic disease virus produced in transgenic plants using different fusion strategies plant viral vectors for delivery by agrobacterium oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus a protease activity-depleted environment for heterologous proteins migrating towards the leaf cell apoplast two distinct chimeric potexviruses share antigenic cross-presentation properties of mhc class i epitopes oral immunization with a recombinant bacterial antigen produced in transgenic plants the capsid protein of beak and feather disease virus binds to the viral dna and is responsible for transporting the replication-associated protein into the nucleus plant cell cultures for the production of recombinant proteins oral tolerance induction in hemophilia b dogs fed with transplastomic lettuce commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals immunogenicity of the epitope of the foot-and-mouth disease virus fused with a hepatitis b core protein as expressed in transgenic tobacco a dna replicon system for rapid high-level production of virus-like particles in plants human-derived, plant-produced monoclonal antibody for the treatment of anthrax an oral vaccine in maize protects against transmissible gastroenteritis virus in swine biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications swine and influenza: a challenge to one health research good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants systemic and oral immunogenicity of hemagglutinin protein of rinderpest virus expressed by transgenic peanut plants in a mouse model monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting production of recombinant antigens and antibodies in nicotiana benthamiana using 'magnifection' technology: gmp-compliant facilities for small-and large-scale manufacturing plant-produced cottontail rabbit papillomavirus l1 protein protects against tumor challenge: a proof-of-concept study one health approach to rift valley fever vaccine development plant-based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge delivery of subunit vaccines in maize seed good governance in 'one health' approaches leafbio announces conclusion of zmapp™ clinical trial plant viruses as nanoparticle-based vaccines and adjuvants a novel m2e based flu vaccine formulation for dogs plant viral epitope display systems for vaccine development the two-faced potato virus x: from plant pathogen to smart nanoparticle an interspecific nicotiana hybrid as a useful and cost-effective platform for production of animal vaccines in planta production of a candidate vaccine against bovine papillomavirus type 1 induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein influenza, a one health paradigm-novel therapeutic strategies to fight a zoonotic pathogen with pandemic potential regulatory approval and a first-in-human phase i clinical trial of a monoclonal antibody produced in transgenic tobacco plants bringing plant-based veterinary vaccines to market: managing regulatory and commercial hurdles managing emerging diseases borne by fruit bats (flying foxes), with particular reference to henipaviruses and australian bat lyssavirus optimization of human papillomavirus type 16 (hpv-16) l1 expression in plants: comparison of the suitability of different hpv-16 l1 gene variants and different cell-compartment localization in vivo deglycosylation of recombinant proteins in plants by co-expression with bacterial inhibition of protease activity by antisense rna improves recombinant protein production in nicotiana tabacum cv. bright yellow 2 (by-2) suspension cells production of h5n1 influenza virus matrix protein 2 ectodomain protein bodies in tobacco plants and in insect cells as a candidate universal influenza vaccine a plant-produced bacteriophage tailspike protein for the control of salmonella vaccines against diseases transmitted from animals to humans: a one health paradigm transient expression of the ectodomain of matrix protein 2 (m2e) of avian influenza a virus in plants bluetongue vaccines in europe protection of rabbits against cutaneous papillomavirus infection using recombinant tobacco mosaic virus containing l2 capsid epitopes an oral vaccine based on u-omp19 induces protection against b. abortus mucosal challenge by inducing an adaptive il-17 immune response in mice differential expression of two isolates of beak and feather disease virus capsid protein in escherichia coli genetically engineered multi-component virus-like particles as veterinary vaccines passive protection to bovine rotavirus (brv) infection induced by a brv vp8* produced in plants using a tmv-based vector one health approach to influenza: assessment of critical issues and options invasion of bluetongue and other orbivirus infections into europe: the role of biological and climatic processes high level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector u-omp19 from brucella abortus is a useful adjuvant for vaccine formulations against salmonella infection in mice protection of recombinant mammalian antibodies from development-dependent proteolysis in leaves of nicotiana benthamiana long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles: evidence for homologous and partial heterologous protection plant-produced vaccines: promise and reality plant-made vaccines for humans and animals plant-based vaccines against viruses virus-derived ssdna vectors for the expression of foreign proteins in plants expression of multiple proteins using full-length and deleted versions of cowpea mosaic virus rna-2 peaq: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants a chimeric affinity tag for efficient expression and chromatographic purification of heterologous proteins from plants an efficient approach for recombinant expression and purification of the viral capsid protein from beak and feather disease virus (bfdv) in escherichia coli expression of hemagglutinin protein of rinderpest virus in transgenic pigeon pea [cajanus cajan (l.) millsp.] plants virus-derived vectors for the expression of multiple proteins in plants optimization and utilization of agrobacteriummediated transient protein production in nicotiana algae-based oral recombinant vaccines synthetic plant virology for nanobiotechnology and nanomedicine n-glyco-engineering in plants: update on strategies and major achievements baculovirus expression of beak and feather disease virus (bfdv) capsid protein capable of self-assembly and haemagglutination generation of glyco-engineered nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like n-glycan structure corn as a production system for human and animal vaccines forefront study of plant biotechnology for practical use: development of oral drug for animal derived from transgenic strawberry increased yield of heterologous viral glycoprotein in the seeds of homozygous transgenic tobacco plants cultivated underground a method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles the case for plant-made veterinary immunotherapeutics protein body induction: a new tool to produce and recover recombinant proteins in plants optimizing the yield of recombinant pharmaceutical proteins in plants new viral vector for superproduction of epitopes of vaccine proteins in plants expression of an animal virus antigenic site on the surface of a plant virus particle expression of human papillomavirus type 16 major capsid protein in transgenic nicotiana tabacum cv optimization of by-2 cell suspension culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method orally fed seeds producing designer igas protect weaned piglets against enterotoxigenic escherichia coli infection oral immunogenicity of human papillomavirus-like particles expressed in potato expression of bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein vp1 protection of mice against challenge with foot and mouth disease virus (fmdv) by immunization with foliar extracts from plants infected with recombinant tobacco mosaic virus expressing the fmdv structural protein vp1 protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants one-health: a safe, efficient, dual-use vaccine for humans and animals against middle east respiratory syndrome coronavirus and rabies virus induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes expression of the fusion glycoprotein of newcastle disease virus in transgenic rice and its immunogenicity in mice a plant-based multicomponent vaccine protects mice from enteric diseases expression in plants and immunogenicity of plant virus-based experimental rabies vaccine development of a candidate vaccine for newcastle disease virus by epitope display in the cucumber mosaic virus capsid protein key: cord-266481-9afb0yvt authors: naskalska, antonina; dabrowska, agnieszka; szczepanski, artur; milewska, aleksandra; jasik, krzysztof piotr; pyrc, krzysztof title: membrane protein of human coronavirus nl63 is responsible for interaction with the adhesion receptor date: 2019-07-17 journal: journal of virology doi: 10.1128/jvi.00355-19 sha: doc_id: 266481 cord_uid: 9afb0yvt human coronavirus nl63 (hcov-nl63) is a common respiratory virus that causes moderately severe infections. we have previously shown that the virus uses heparan sulfate proteoglycans (hspgs) as the initial attachment factors, facilitating viral entry into the cell. in the present study, we show that the membrane protein (m) of hcov-nl63 mediates this attachment. using viruslike particles lacking the spike (s) protein, we demonstrate that binding to the cell is not s protein dependent. furthermore, we mapped the m protein site responsible for the interaction with hspg and confirmed its relevance using a viable virus. importantly, in silico analysis of the region responsible for hspg binding in different clinical isolates and the amsterdam i strain did not exhibit any signs of cell culture adaptation. importance it is generally accepted that the coronaviral s protein is responsible for viral interaction with a cellular receptor. here we show that the m protein is also an important player during early stages of hcov-nl63 infection and that the concerted action of the two proteins (m and s) is a prerequisite for effective infection. we believe that this study broadens the understanding of hcov-nl63 biology and may also alter the way in which we perceive the first steps of cell infection with the virus. the data presented here may also be important for future research into vaccine or drug development. navirus (bcov), and avian infectious bronchitis virus (ibv), reportedly use sialic acids for initial attachment to the cell (summarized in reference 10). the coronaviral particle consists of a dense core formed by a nucleocapsid (n) protein with viral genomic rna and an envelope decorated with the membrane (m), envelope (e), and spike (s) proteins. some coronaviruses contain other structural proteins, such as hemagglutinin esterase (he) or accessory open reading frame (orf) proteins (orf3, orf4a, and orf7) (59, 69) . the s protein is a class i viral membrane glycoprotein responsible for the interaction with the entry receptor and fusion (11) . hcov-nl63 employs ace2 as an entry receptor (3) . however, we recently reported that heparan sulfate (hs) proteoglycans (hspgs) are required for effective adhesion of the virus to the cell surface and that such an interaction enhances the infection process (12) . hspg binding was also demonstrated for mhv (13) , sars-cov (14) , and porcine epidemic diarrhea virus (pedv) (15) . hspgs are glycoproteins that are ubiquitous at the surface of the mammalian cell. binding to hspg is the initial event promoting subsequent recognition of a secondary receptor by increasing the local concentration of pathogens or triggering conformational changes of proteins involved in viral entry. as an example, binding to hspg induces structural rearrangements of proteins responsible for infection by adenoassociated virus 2 (16) , adenovirus types 2 and 5 (17) , human papillomavirus 16 (18) , and several herpesviruses (19) . furthermore, the majority of oncogenic viruses (hepatitis b and c viruses, kaposi's sarcoma-associated herpesvirus, human papillomaviruses, merkel cell polyomavirus, and human t cell lymphotropic virus type 1) initially attach to the hspgs (20) . hspgs can also enhance virulence by binding accessory viral factors necessary for viral replication. this is illustrated by hspg binding of the tat protein of human immunodeficiency virus, which after internalization activates transcription of the viral rna (21) . to better address the nature of the virus-hspg interaction, we constructed viruslike particles (vlps) composed of hcov-nl63 proteins. vlps structurally mimic the native virus and thus constitute a good model for studying virus-host interactions in the context of additional capsid components. importantly, vlps can be relatively easily tailored by molecular biotechnology techniques, facilitating the assessment of the role of individual viral proteins in receptor recognition. in the present study, we demonstrate that hcov-nl63 binds hspg via the m protein, which is to some extent responsible for virus attachment. we thus show that viral entry is an outcome of the concerted action of the m and s proteins. the presented findings improve the understanding of viral biology and may facilitate the development of improved antivirals and neutralizing vaccines. production and purification of vlps. hcov-nl63 vlps composed of the m, e, n, and (optionally) s proteins were produced in insect cells, as previously described (22) , with the modification that the n protein was also included. to evaluate protein expression, insect cells infected with bicistronic recombinant baculovirus (rbv) coding for m and e proteins (mϩe) and monocistronic n rbv (and optionally monocistronic s rbv) were immunostained and examined under a confocal microscope. colocalization of the m and n proteins was observed, suggesting the formation of a protein complex within the producer cells (fig. 1a) . to verify whether vlps were effectively assembled and released, the culture medium harvested from insect cells expressing m, e, n, and s proteins was analyzed by western blotting. the m (26-kda), n (42-kda), and s (150-kda) proteins were detected in the secreted fraction when insect cells were coinfected with (mϩe) rbv, n rbv, and, optionally, s rbv (fig. 1b) . in agreement with our previous studies, the m protein migrated as two bands, reflecting its two different glycosylation states. similarly, two bands of the n protein were observed, upon electrophoresis, resulting from protein degradation under denaturing conditions (23) . in contrast, the apparent molecular weight of the s protein band was higher than expected, most likely because of the glycosylation and/or incomplete denaturation of the protein trimer. both vlps, composed of m, e, and n (men) and m, e, n, and s (mens) proteins, were concentrated and purified using a heparin column according to a previously developed protocol (22) (fig. 1c ). the analysis of purified samples by transmission electron microscopy (tem) showed spherical particles of different diameters, which might reflect the amount of the n protein incorporated into vlps (fig. 1d ). spike protein is dispensable for hcov-nl63 vlp adhesion to llc-mk2 cells. previously, we have shown that hcov-nl63 vlps composed of the m, e, and s proteins penetrate llc-mk2 cells, which are naturally permissive to hcov-nl63 infection, and that this process is ace2 dependent. we have also observed that vlps composed of m and e proteins adhere to but do not enter the target cell (data not shown). in the present study, we evaluated the binding and endocytosis of men and mens hcov-nl63 vlps. llc-mk2 cells were incubated with the purified men and mens vlps, and we found that men vlps localize to the cell surface, whereas mens vlps were present on the cell surface and in the cytoplasm (fig. 2 ). this confirmed that the s protein is required for particle internalization but dispensable for vlp adhesion. to further validate this result, we blocked the virus-ace2 interaction with anti-ace2 antibody, which resulted in inhibition of mens vlp internalization but not adhesion to the cell surface (fig. 3) . interestingly, for mens vlps, we observed a higher number of viral particles on cells preincubated with anti-ace2 antibodies. we believe that this is an artifact, as during entry, vlps fuse with cellular membranes, which results in diffusion of the fluorescence signal and a decrease of the score. blocking of the interaction with the cellular receptor hampers vlp-cell fusion, and the scored number of particles remains high. adhesion of men vlps to cells was not affected by inhibition of the vlp-ace2 interaction. altogether, these observations indicate that s protein of hcov-nl63 is not involved in virus attachment to target cells. using flow cytometry, we next investigated whether the adhesion of men and mens vlps to the cell surface involves interaction with hspgs, as has been shown for hcov-nl63. for this, purified vlps were preincubated with soluble hs and used to inoculate llc-mk2 cells. identical samples were prepared using untreated vlps. the cells were then immunostained to label the vlps, and the signal was recorded. native, iodixanol-concentrated hcov-nl63 was used as a control. we observed that both men and mens vlps bound to the cell surface in the absence of hs and that hs pretreatment of vlps hampered adhesion (fig. 4) . peptides of the membrane protein are recognized by heparin. men vlps contain the n protein encapsulated in an envelope formed by the m and e proteins. we have a priori assumed that the m protein serves as a partner for cellular hspg during viral or vlp adhesion, and we verified this hypothesis experimentally. first, an array of synthetic, overlapping peptides covering the complete m protein was used to test binding with the labeled heparin. the m protein regions potentially interacting with heparin were hence identified as amino acids (aa) 25 to 51, aa 93 to 119, and aa 153 to 207 (fig. 5) . most of the peptides are arginine and lysine rich, which is in agreement with previously reported data (24, 25) . next, we predicted the m protein topology in silico, and based on these data and previous reports, we concluded that regions spanning aa 25 to 51 and aa 93 to 119 may not be involved in the interactions with hpsgs, as they are localized inside the virion or in transmembrane domains (tmds) (fig. 6 ). this led us to the conclusion that the hspg binding site is localized in the c-terminal region of aa 153 to 226, predicted to be exposed on the virion surface. data are presented as means ϯ 95% confidence intervals (ci). the total number of particles and the number of cells were quantified using the imagej fiji tool 3d objects counter, and internalized particles were counted manually (*, p ͻ 0.05; ****, p ͻ 0.0001; ns, not significant). ab, antibody. to further evaluate the interaction between the c-terminal domain of the m protein and hspgs, we expressed and purified a his-tagged fragment of the m protein consisting of amino acids 153 to 226 (6ϫhis-m 153-226 ) in bacteria. to investigate whether the protein adhered to cellular hspg, we performed an in situ enzyme-linked immunosorbent assay (elisa), with different amounts of the protein added to cells seeded in a microplate. the 6ϫhis-m 153-226 protein and an equal molar amount of 6ϫhis-n hku1, as a control, were serially diluted and incubated with llc-mk2 cells. a signal developed using an anti-his-horseradish peroxidase (hrp) antibody was detected only with the 6ϫhis-m 153-226 protein and was proportional to its concentration (fig. 7a) . importantly, when the 6ϫhis-m 153-226 protein was preincubated with soluble hs, the interaction with llc-mk2 cells was diminished (fig. 7b) , suggesting that the putative epitope is localized within the region from aa 153 to 226 of the m protein. inhibition of virus adhesion to the cell surface by anti-m antibody binding. to further validate the notion that hcov-nl63 employs the m protein to bind to the cell, we performed a "pseudoneutralization" assay using a polyclonal anti-m rabbit serum, obtained after immunizing rabbits with peptides corresponding to aa 181 to 195 cell infection is a complex process that involves viral attachment, which leads to viral enrichment on the cell surface and subsequent internalization. while initial binding is usually nonspecific and mediated by ubiquitous molecules, such as sugars or glycoconjugates (glycoproteins, glycolipids, and proteoglycans), the second step requires a highly specific interaction with the entry receptors, often involving their proteolytic processing. in some viruses, a single protein is responsible for both steps of this interaction, whereas in other viruses (i.e., paramyxoviruses), different structural elements of the virion are employed for the attachment to and fusion with the cellular membrane (26) . this sophisticated interplay of distinct receptor entities and their sequential engagement increase viral avidity and coordination in time for key events for efficient cellular uptake. for coronaviruses, it is generally believed that s protein is responsible for both virion attachment and internalization (reviewed in references 27 and 28). this large (ϳ150-kda) glycoprotein consists of three segments: an ectodomain, a transmembrane anchor, and a short endodomain. the ectodomain can be further divided into s1 and s2 subunits, although not all coronaviruses undergo enzymatic cleavage of the s ectodomain (29) . different experimental approaches demonstrating that the s1 domain encompasses one or more receptor binding sites (rbss), located at its c terminus, have been reported for almost all studied covs. while the c-terminal part of the s1 domain (s1-ctd) is highly divergent and responsible for the interaction with entry receptors, the more conserved n-terminal region of s1 (s1-ntd) is thought to function as a ligand for the initial attachment factors. indeed, it was demonstrated that the s1-ntds of several coronaviruses bind carbohydrates. examples include the alphacoronavirus (tgev), betacoronavirus (hcov-hku1 and bcov), and gammacoronavirus (ibv) genera (30) (31) (32) (33) . in contrast, the s2 domain presumably participates in the fusion of the cellular membrane with the viral envelope (34) . this was confirmed for a number of coronaviruses, including hcov-229e (35), sars-cov (36), mhv (37) , and hcov-nl63 (38) . the structure of the hcov-nl63 s protein and its role in viral infection were previously described (3, (39) (40) (41) (42) . the receptor binding domain (rbd) within the s1 subunit and the heptad repeat region within the s2 subunit were mapped (38) . some authors speculate that s1 also contains a domain responsible for binding to the attachment receptors on the cell surface, which triggers binding with ace2 (27, 41) . of note, it has been shown that purified full-length s protein of hcov-nl63 exhibits surprisingly low-affinity binding to its putative receptor (43) . at the same time, there is evidence that the rbd of the hcov-nl63 s protein binds ace2 with a higher affinity than its full-length counterpart and with efficiency comparable to that of s1 of sars-cov (40, 41, 44) . these observations raise questions about the existence of an additional, s-independent stimulus prerequisite for this interaction. in the present study, we provide evidence that the m protein may be responsible for the interaction of the virus with cellular hspg. for this, we have used previously developed vlps, produced in insect cells, which enable efficient and scalable expression of complex macromolecular structures. first, using confocal microscopy, we showed that adhesion to the cell surface was not abrogated in vlps missing the s protein. moreover, blocking the vlp-ace2 interaction with specific antibodies did not affect adhesion. next, using flow cytometry, we confirmed that adhesion is mediated by hspg, since soluble hs blocks the interaction. to identify the ligand involved in this interaction, we decided to first screen the m protein for the presence of putative heparin binding domains. using a simple peptide array overlay, we identified three regions that may potentially be responsible for such an interaction. the experimental data were consistent with the notion that these regions are rich in positively charged amino acids, lysine, and arginine (45, 46) . subsequently, we verified this result using the predicted topology of the m protein and literature data. only one site was predicted to localize on the virion surface, and consequently, we assumed that this particular site is responsible for the interaction. this assumption was then proven by an in situ elisa, which demonstrated that the region from aa 153 to 226 of the m protein binds to cellular hspg. of note, topology prediction algorithms detected four transmembrane domains (tmds) within the m protein, which is somewhat atypical for coronaviruses (fig. 6) . the c terminus of the m protein is essential for the m-m, m-n, and m-s interactions, and it is thought to be hidden within the virion envelope (6, (47) (48) (49) (50) (51) . however, the exact region of the m protein responsible for these interactions has not been defined for most coronaviruses. recently, it was suggested that this interaction might rely on structural motifs consisting of several residues dispersed throughout the protein (52) . furthermore, we have previously demonstrated that a c-terminally tagged m protein is assembly competent for vlp formation, which supports the four-tmd model of the m protein and the resulting n exo -c exo topology. this conclusion was validated by the observation that hcov-nl63 and nl63 vlp adhesion is hampered by an antibody raised against two peptides corresponding to the distal part of the m protein. interestingly, this observation was consistent with works of enjuanes and colleagues (53, 54) , who demonstrated a similar m protein topology of tgev cov belonging to the same genus (alphacoronavirus) as hcov-nl63. hence, we propose that such a unique organization of the nl63-hcov m protein might be one explanation for its engagement in hcov-nl63 infection. it has to be mentioned that some coronaviruses acquire the ability to bind hspg in the course of cell culture adaptation, as described for mhv and ibv (13, (55) (56) (57) . to exclude this possibility, we compared the m protein sequence used in the present study (amsterdam i strain) with those of clinical isolates, and no differences in this region were identified (not shown). we believe that the engagement of the m protein in cell attachment is hence a natural characteristic of hcov-nl63. the involvement of proteins other than the s protein in carbohydrate binding by coronaviruses has been previously described. for instance, he of an mhv strain binds cellular sialic acids (58) . he also appears to mediate the attachment of bcov and hcov-oc43 to the cell surface (33, 59, 60) . more generally, the involvement of multiple capsid proteins was described for a number of viruses, i.e., influenza virus (paramyxoviruses) or herpesviruses (reviewed in references 61 and 62). it is also worth mentioning that the presented results do not preclude the participation of s protein in the recognition of cellular hspg during hcov-nl63 infection. the observations presented here provide new insight into the understanding of cell receptors and their interplay with the viral ligands during hcov-nl63 infection. considering the role of the m protein, one could suggest novel strategies for inhibiting or preventing infection by this and potentially other coronaviruses. specifically, some experimental anticoronaviral vaccines that induce anti-s protein humoral responses cause serious adverse effects, probably related to an antibody-dependent enhancement mechanism (63) (64) (65) . in this context, vaccines, immunotherapeutics, or antivirals developed to inhibit the interaction between the m protein and cellular hspg may offer an interesting alternative. cell lines. sf9 (spodoptera frugiperda; atcc crl-1711) and hf (high five) (trichoplusia ni; atcc crl-7701) cells were cultured in esf medium (expression systems, ca, usa) supplemented with 2% fbs (fetal bovine serum; thermo fisher scientific, poland), 100 g/ml streptomycin, 100 iu/ml penicillin, 10 g/ml gentamicin, and 0.25 g/ml amphotericin b. the culture was maintained in a humidified incubator at 27°c. sf9 cells were used for baculovirus (bv) generation and amplification, while hf cells were used for recombinant protein expression. llc-mk2 cells (macaca mulatta kidney epithelial cells; atcc ccl-7) were maintained in minimal essential medium (mem) (2 parts hanks' mem and 1 part earle's mem; thermo fisher scientific, poland) supplemented with 3% fbs, 100 g/ml streptomycin, 100 iu/ml penicillin, and 5 g/ml ciprofloxacin. the culture was maintained at 37°c under 5% co 2 . vlp production. vlps composed of membrane (m), envelope (e), and spike (s) proteins of hcov-nl63 were produced as described previously (22) , but nucleocapsid (n) protein was additionally included for this work. for this, the n gene was subcloned from pet duet (23) to pfastbac dual, under the control of the polyhedrin promoter. recombinant baculoviruses (rbvs) were generated using the bac-to-bac system (thermo fisher scientific, poland) and titrated using a plaque assay. subsequently, hf cells were coinfected with (mϩe) bicistronic rbv, n monocistronic rbv, and s monocistronic rbv at a multiplicity of infection (moi) of 4 and cultured for 72 h. secreted vlps were then harvested by centrifugation (5,000 ϫ g for 30 min). for purification of vlps, the harvested culture medium was diluted (1:1) with binding buffer (20 mm k 2 hpo 4 -kh 2 po 4 [ph 6.2], 70 mm nacl) and loaded onto a 5-ml heparin ht column (ge healthcare, poland) using an äkta fast-performance liquid chromatography (fplc) system (äkta, sweden). before purification, the column was equilibrated with binding buffer. proteins were eluted with a linear nacl gradient (50 mm to 2 m nacl in binding buffer), and collected peak fractions were analyzed using sds-page and western blotting. sds-page and western blotting. insect cells or culture media were harvested, resuspended in denaturing buffer to final concentrations of 1.5% sds and 2.5% ␤-mercaptoethanol, and resolved by laemmli sds-page using 4-to-20% gradient precast gels (bio-rad, poland). a pageruler prestained plus protein ladder (thermo fisher scientific, poland) was used in this study as a protein size marker. gels were stained with coomassie brilliant blue or subjected to electrotransfer in buffer containing 25 mm tris, 192 mm glycine, and 20% methanol onto an activated polyvinylidene difluoride (pvdf) membrane. following transfer, the membrane was blocked with 5% skim milk in tris-buffered saline supplemented with 0.05% tween 20 (tbs-t), followed by 1.5 h of incubation with rabbit polyclonal anti-m serum (1:15,000; kindly provided by lia van der hoek and generated by rabbit immunization with peptides spanning aa 180 to 195 and aa 212 to 226 of the m protein), mouse monoclonal anti-n antibody (1:1,000; ingenansa, spain), and mouse polyclonal anti-s serum (1:250; eurogentec, belgium) and 1 h of incubation with anti-rabbit (1:20,000; dako, denmark) and anti-mouse (1:20,000; dako, denmark) secondary antibodies conjugated with horseradish peroxidase (hrp), respectively. the signal was developed using the immobilon western chemiluminescent hrp substrate (millipore, poland) and visualized by exposing the membrane to an x-ray film (thermo fisher scientific, poland). confocal microscopy. for assessment of protein colocalization in insect cells, hf cells were grown in 6-well culture plates on glass coverslips coated with 0.01% poly-l-ornithine (sigma-aldrich, poland). cells were infected with rbvs at an moi of 1, fixed at 48 h postinfection with 4% formaldehyde, permeabilized with 0.2% triton x-100 in phosphate-buffered saline (pbs), and blocked for 1 h, at room temperature with 5% bovine serum albumin (bsa) in pbs. expression of m and n proteins was detected with rabbit polyclonal anti-m serum (the same as described above; 1:1,000) and mouse monoclonal anti-n antibody (the same as described above; 1:2,000), respectively, followed by detection with alexa fluorophore secondary antibodies at a 1:400 dilution (santa cruz biotechnology, usa). cell nuclei were stained with dapi (4=,6=-diamidino-2-phenylindole) (0.1 g/ml in pbs; sigma-aldrich, poland). coverslips were mounted on glass slides with prolong diamond antifade mountant (sigma-aldrich, poland). for transduction and adhesion analyses of vlps, llc-mk2 cells were grown to 80% confluence for 48 h in 6-well culture plates on glass coverslips. cells were then washed with ice-cold pbs and inoculated with 1 ml of purified vlps. next, llc-mk2 cells were incubated for 2 h at 4°c and subsequently for 90 min at 32°c under 5% co 2 and further washed three times with pbs. subsequently, cells were fixed, permeabilized, and stained with anti-m polyclonal serum, as described above. additionally, actin filaments were visualized with phalloidin conjugated with alexa 647 (0.132 m; sigma-aldrich, poland). to verify the role of the ace2 protein during vlp entry, llc-mk2 cells were incubated with anti-ace2 polyclonal antibodies (catalog number af933; r&d systems) or an appropriate isotype control antibody (catalog number gtx35039; genetex) for 1 h at 37°c (5 g/ml). the anti-ace2 antibody concentration was determined based on hcov-nl63 neutralization experiments in llc-mk2 cells (not shown). furthermore, cells were overlaid with purified men or mens vlps and incubated for 2 h at 4°c and subsequently for 90 min at 32°c under 5% co 2 in the presence of antibodies. next, cells were washed three times with pbs, fixed, permeabilized, and stained with anti-m polyclonal serum, as described above. fluorescent images were acquired using a zeiss lsm 710 confocal microscope (carl zeiss microscopy gmbh), deconvolved using autoquant x3 software, and processed in imagej fiji (national institutes of health, bethesda, md, usa) (66) . the number of particles and number of cells were quantified using the built-in imagej fiji tool "3d objects counter." the numbers of internalized particles were counted manually from orthogonal views. for this study, the actin cortex was assumed to indicate the cell surface. electron microscopy. samples were prepared as described previously (22) . briefly, purified vlps were fixed in karnovsky solution and loaded onto copper grids coated with a support film (formvar 15/95e; sigma-aldrich, st. louis, mo, usa). after drying, the material was stained with uranyl acetate (polyscience, inc., warrington, pa, usa) and lead citrate (sigma-aldrich, st. louis, mo, usa). subsequently, the grids were washed with water and dried in air at room temperature. ultrastructural observations were performed by using a hitachi h500 transmission electron microscope at an accelerating voltage of 75 kv. flow cytometry. to evaluate adhesion of vlps to llc-mk2 cells, cells were grown for 48 h to reach 100% confluence in 6-well culture plates on glass coverslips. cells were washed twice with pbs and incubated with purified vlps, iodixanol-concentrated hcov-nl63 (12, 67) , mock supplemented with heparan sulfate (hs) (1 mg/ml; sigma-aldrich, poland), or pbs for 4 h at 4°c. the cells were then washed three times with pbs, fixed with 4% paraformaldehyde, permeabilized with 0.1% triton x-100 in pbs, and incubated overnight in 5% bovine serum albumin and 0.5% tween 20 in pbs. to examine hcov-nl63 or vlp adhesion, cells were incubated for 2 h at room temperature with a mouse monoclonal anti-n antibody (the same as described above; 1:1,000 in 2.5% bsa with 0.5% tween 20 in pbs), followed by 1 h of incubation with an alexa fluor 488-labeled goat anti-mouse antibody (1:400). cells were then washed, mechanically detached from the glass coverslips, resuspended in pbs, and analyzed by flow cytometry (facscalibur; becton, dickinson). data were processed using cellquest software (becton, dickinson) and flowjo v10. expression of the c-terminal domain of the m protein. the region coding for the c-terminal fragment (aa 153 to 226) of the m protein was pcr amplified (5= primer cca gga tcc gga tgg cca taa gat tgc tac tcg tg and 3= primer gca ctc gag tta gat taa atg aag caa ctt), digested with bamhi and xhoi enzymes (thermo fisher scientific, poland), gel purified, and cloned into the pet duet plasmid in a manner to include the 6ϫhis tag in frame at the n terminus. the sequence (6ϫhis-m 153-226 ) was verified by sequencing. the escherichia coli bl21 strain was transformed with the recombinant plasmid and precultured overnight at 37°c. lb medium (1 liter; bioshop-labempire, poland) was inoculated with the preculture, induced with isopropyl-␤-d-thiogalactopyranoside (iptg) (0.5 mm; bioshop-labempire, poland) at an optical density of 0.6, and harvested at 4 h postinduction. bacterial cell pellets were subsequently resuspended in 50 ml of lysis buffer (50 mm tris [ph 7.5], 5 mm urea, 250 mm nacl, 5 mm dithiothreitol [dtt], 1 mm edta, 1% triton x-100) and subjected to 2 cycles of cell disruptor (constant systems, uk) operation at 25 lb/in 2 . lysates were then centrifuged (40 min at 5,000 ϫ g), and the supernatant was diluted (1:1) with immobilized-metal affinity chromatography (imac) binding buffer (20 mm k 2 hpo 4 -kh 2 po 4 [ph 7.4], 500 mm nacl, 20 mm imidazole). diluted supernatants were loaded onto a 1-ml imac column (ge healthcare, poland) charged with ni 2ϩ and connected to an äkta fplc system (äkta, sweden) and preequilibrated with binding buffer. proteins were eluted with 500 mm imidazole in imac binding buffer. collected peak fractions were pooled and fractionated again to remove imidazole and excess nacl. for this purpose, a 26/10 desalting column (ge healthcare, poland) (preequilibrated with 20 mm na 2 hpo 4 -nah 2 po 4 [ph 7.7], 250 mm nacl, and 5% glycerol) was used. purified 6ϫhis-m 153-226 protein was analyzed by sds-page and western blotting. aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry angiotensin-converting enzyme 2 is a functional receptor for the sars 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external surface of the virion murine coronavirus with an extended host range uses heparan sulfate as an entry receptor cleavage of group 1 coronavirus spike proteins: how furin cleavage is traded off against heparan sulfate binding upon cell culture adaptation heparan sulfate is a selective attachment factor for the avian coronavirus infectious bronchitis virus beaudette attachment of mouse hepatitis virus to o-acetylated sialic acid is mediated by hemagglutinin-esterase and not by the spike protein structure, function and evolution of the hemagglutininesterase proteins of corona-and toroviruses the acetyl-esterase activity of the hemagglutinin-esterase protein of human coronavirus oc43 strongly enhances the production of infectious virus viral entry mechanisms: cellular and viral mediators of herpes simplex virus entry receptor binding and membrane fusion in virus entry: the influenza hemagglutinin evaluation of modified vaccinia virus ankara based recombinant sars vaccine in ferrets ezrin interacts with the sars coronavirus spike protein and restrains infection at the entry stage sars cov subunit vaccine: antibody-mediated neutralisation and enhancement fiji: an opensource platform for biological-image analysis entry of human coronavirus nl63 into the cell predicting transmembrane protein topology with a hidden markov model: application to complete genomes supramolecular architecture of the coronavirus particle key: cord-257584-v38tjof3 authors: fahmi, muhamad; kubota, yukihiko; ito, masahiro title: nonstructural proteins ns7b and ns8 are likely to be phylogenetically associated with evolution of 2019-ncov date: 2020-03-03 journal: infect genet evol doi: 10.1016/j.meegid.2020.104272 sha: doc_id: 257584 cord_uid: v38tjof3 the seventh novel human infecting betacoronavirus that causes pneumonia (2019 novel coronavirus, 2019-ncov) originated in wuhan, china. the evolutionary relationship between 2019-ncov and the other human respiratory illness-causing coronavirus is not closely related. we sought to characterize the relationship of the translated proteins of 2019-ncov with other species of orthocoronavirinae. a phylogenetic tree was constructed from the genome sequences. a cluster tree was developed from the profiles retrieved from the presence and absence of homologs of ten 2019-ncov proteins. the combined data were used to characterize the relationship of the translated proteins of 2019-ncov to other species of orthocoronavirinae. our analysis reliably suggests that 2019-ncov is most closely related to batcov ratg13 and belongs to subgenus sarbecovirus of betacoronavirus, together with sars coronavirus and bat-sars-like coronavirus. the phylogenetic profiling cluster of homolog proteins of one annotated 2019-ncov protein against other genome sequences revealed two clades of ten 2019-ncov proteins. clade 1 consisted of a group of conserved proteins in orthocoronavirinae comprising orf1ab polyprotein, nucleocapsid protein, spike glycoprotein, and membrane protein. clade 2 comprised six proteins exclusive to sarbecovirus and hibecovirus. two of six clade 2 nonstructural proteins, ns7b and ns8, were exclusively conserved among 2019-ncov, betacov_ratg, and batsars-like cov. ns7b and ns8 have previously been shown to affect immune response signaling in the sars-cov experimental model. thus, we speculated that knowledge of the functional changes in the ns7b and ns8 proteins during evolution may provide important information to explore the human infective property of 2019-ncov. in december 2019, the seventh human coronavirus, termed 2019 novel coronavirus (2019-ncov) or severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was found in wuhan, china. on february 8, 2020, the total number of infections and deaths due to 2019-ncov globally was 34,439 and 720, respectively, according to the johns hopkins university center for systems science and engineering. coronaviruses are enveloped rna viruses that infect many species, including humans, other mammals, and birds. after infection, the host may develop respiratory, bowel, liver, and neurological diseases (weiss and leibowitz, 2011; cui et al., 2019) . coronaviruses are members of the order nidovirales and subfamily orthocoronavirinae. this subfamily is divided into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. generally, alphacoronavirus and betacoronavirus tend to infect mammals, while gammacoronavirus and deltacoronavirus typically infect birds. however, some gammacoronavirus and deltacoronavirus can infect mammals under specific conditions (woo et al., 2012) . in immunocompromised individuals, infection with one of the four human coronaviruses-human coronavirus nl63 (hcov-nl63), human coronavirus 229e (hcov-229e), human coronavirus oc43 (hcov-oc43), and human coronavirus hku1 (ecov-hku1)-usually results in cold-like symptoms. these viruses can cause severe infections in some infants and the elderly. due to the frequent interaction between wild animals and humans, wild animals are a common source of human t zoonotic infections. sars-cov and middle east respiratory syndrome coronavirus (mers-cov) are zoonotic coronaviruses that can cause severe respiratory diseases in humans; both belong to betacoronavirus (su et al., 2016; forni et al., 2017; cui et al., 2019; luk et al., 2019; ramadan and shaib, 2019) . 2019-ncov is the seventh coronavirus discovered that infects humans. it causes acute respiratory disease in respiratory infections. immediately after its discovery, the complete genome sequence of 2019-ncov was determined. the sequence (mn908947) was released by genbank on 05 january 2020 (lu et al., 2020) . the sequence of 2019-ncov is 96% identical, at the wholegenome level, to a bat coronavirus (zhou et al., 2020) . the genomic characteristics and epidemiology of 2019-ncov have been analyzed (lu et al., 2020) . nine inpatient culture isolates were subjected to next-generation sequencing, and individual complete and partial 2019-ncov genomic sequences were obtained. phylogenetic analysis of these 2019-ncov genomes and other coronaviruses was performed to determine the evolutionary history of the virus and to explore the origin of 2019-ncov. at the first onset, homology modeling investigated the potential receptor-binding properties of the virus. however, sars-cov and mers-cov showed approximate similarities of 79% and 50% with 2019-ncov, respectively. these findings indicated that there is not a close evolutionary relationship of 2019-ncov with sars-cov and mers-cov. thus, 2019-ncov is considered the seventh novel human betacoronavirus (lu et al., 2020) . in this study, we comprehensively characterized the relationship of the translated proteins of 2019-ncov to other species of orthocoronavirinae. this was done using a combination of the phylogenetic tree constructed from the genome sequences and the cluster tree developed from the profiles retrieved from the presence and absence of homologs of ten 2019-ncov proteins. the genomes and the combination of genome and protein sequences were used to develop a phylogenetic tree and phylogenetic profiling, respectively. the dataset of the genomes of the orthocoronavirinae subfamily was collected from the refseq database using the orthocoronavirinae ncbi taxonomy id (txid2501931). this dataset contains representative complete genomes from each species of that subfamily. (pruitt et al., 2007; federhen, 2012) (supplementary table 1 ). additionally, we collected genome sequences from bat sarslike coronavirus (mg772934 and mg772933) from ncbi and betacov/ bat/yunnan/ratg13/2013 (epi_isl_402131) from gisaid (http:// www.gisaid.org). one species of the okanivirinae subfamily, the yellow head virus, was also collected as an outgroup (supplementary table 1 ). the genome sequences were aligned using mafft multiple sequence alignment program provided at the xsede portal in the cipres science gateway with an automatic selection strategy (miller et al., 2012; katoh and standley, 2013) . a phylogenetic tree was constructed using the maximum likelihood method with raxml-hpc blackbox in the cipres science gateway (stamatakis, 2006) . the analysis used an automatic bootstrapping option using a general timereversible substitution model with a gamma-shape parameter (gtr+ g). the model was selected as the best-fit model under the akaike information criterion using modeltest-ng (darriba et al., 2020) . phylogenetic trees were viewed using figtree v1.4 (http://tree.bio.ed.ac.uk/ software/figtree/). the annotated protein sequences of 2019-ncov were collected from the data of one representative genome from ncbi (mn996527). we built a blast database with the retrieved genome sequences data using blast+ version 2.2.30 (camacho et al., 2009) . we then determined the presence and absence of homolog proteins of one representative set of annotated 2019-ncov proteins against other genome sequences in a database using tblastn with a threshold of > 50 and > 25 bits score for protein sequences > 50 amino acids (aa) and < 50 aa in length, respectively. the results of the presence and absence of homolog proteins were converted into a binary matrix and used to build a clustering tree using ward hierarchical clustering method (ward jr, 1963) (supplementary table 2 ). nonstructural protein (ns) 7b and ns8 local alignments were only positive in the sarbecovirus subgenus sample, excluding the sars coronavirus. additionally, we predicted the structural properties of the 2019-ncov ns7b protein, including the secondary structure and order-disorder propensity, using jpred4 and dichot, respectively (fukuchi et al., 2014; drozdetskiy et al., 2015) . we also predicted the structure using the contact assisted protein structure prediction (c-i tasser) composite approach (zhang et al., 2018) . additionally, we specifically collected the sequences that produced significant alignments of ns7b using the mega x software (kumar et al., 2018) . the phylogenetic analysis using complete genome sequences showed that 2019-ncov was the most closely related to batcov ratg13 and belonged to the sarbecovirus subgenus of betacoronavirus, together with sars coronavirus and bat-sars-like coronavirus (bat-sl-covzxc21 and bat-sl-covzc45) with the full support of reliability (fig. 1) . additionally, hibecovirus with bat hp-betacoronavirus/zhe-jiang2013, as the representative species, was the most closely related subgenus of betacoronavirus to sarbecovirus as compared to other subgenera, including merbecovirus (under which mers-cov has been classified), nobecovirus, and embecovirus. these findings agree with previous phylogenetic tree and similarity plot data (paraskevis et al., 2020) . 2019-ncov was found to be more closely related to the batinfecting sarbecovirus species, bat sars-like coronavirus, and betacov ratg13 than to the sars coronavirus that infects humans. this indicated that 2019-ncov more likely originated from bats. however, the wuhan outbreak was first detected in december, which is a time of year when most bat species hibernate. moreover, the huanan seafood market, which is considered as ground zero of the outbreak, does not sell bats. instead, it has been suggested that there is an animal mediator for virus transmission from bats to humans, similar to the previous cases of sars-cov and mers-cov, wherein the masked palm civet (paguma larvata) and dromedary camel (camelus dromedarius) act as intermediate hosts, respectively (lu et al., 2020) . although coronaviruses can exchange genetic material during coinfection, a recent report described the lack of a mosaic relationship of 2019-ncov to the closely related sarbecovirus, indicating the lack of a recombination event in the emergence of 2019-ncov (paraskevis et al., 2020) . hence, 2019-ncov likely emerged from the accumulation of mutations responding to altered selective pressures or from the infidelity of rna polymerase perpetuated as replication-neutral mutations. these speculations need to be studied further. a previously reported comprehensive similarity plot revealed notable mutational hotspots and conserved regions of the genome nucleotide positions of 2019-ncov against closely related coronaviruses (lu et al., 2020; paraskevis et al., 2020) . the present findings provide a different perspective of the similarity among orthocoronavirinae species, using a cluster tree developed from the profiles retrieved from the presence and absence of homologs of ten 2019-ncov proteins. this cluster was combined with the cladogram of a previously constructed phylogenetic tree (fig. 2) . both the trees were consistent in their heatmap distributions. the tree of 2019-ncov proteins comprised two clades. the first, indicated with a blue bar in fig. 2 , contained a group of conserved proteins in most orthocoronavirinae species. these comprised orf1ab polyprotein, nucleocapsid protein, spike glycoprotein, and membrane protein. spike and orf1a regions of 2019-ncov were previously shown to have the lowest sequence identity as compared to the closely related coronavirus species (lu et al., 2020; paraskevis et al., 2020) . however, since the translated spike glycoprotein and orf1ab polyprotein from these regions are very long, the sequence similarity is still sufficient to classify them as homologs. in contrast, another clade, indicated by the green bar in fig. 2 , comprised proteins specific to sarbecovirus for all proteins in this clade and hibecovirus for envelope protein only. this clade included proteins that were not completely conserved by all orthocoronavirus. two (ns7b and ns8) of five nonstructural proteins were specific for 2019-ncov and its closely related species, batcov ratg13 and bat-sars-like coronavirus (bat-sl-covzxc21 and bat-sl-covzc45). the other three nonstructural proteins (ns3, ns6, and ns7a) were also detected in the sars coronavirus. based on these results, we propose that the comprehensive analysis of nonstructural proteins, especially ns7b and ns8, may provide new insight into the properties of 2019-ncov. as shown in fig. 2 , ns7b and ns8 of 2019-ncov, batcov ratg13, and bat-sars-like coronavirus were distinct from other species of orthocoronavirus. in sars-cov, ns7b is an integral protein localized in the golgi compartment. the protein is packaged into sars-cov particles (schaecher et al., 2007) . interestingly, open reading frame (orf) 7b, but not orf 7b deletion, induces interferon (ifn)-dependent reporter gene expression as well as apoptosis and the type i ifn response (pfefferle et al., 2009 ). moreover, the deletion of orf 7b enhance virus growth (pfefferle et al., 2009 ). thus, we speculate that the property of the non-conserved ns7b in 2019-ncov may affect the human infective property of the virus. similarly, the existence of 29 nucleotide deletions in orf 8b has been described in sars-cov (oostra et al., 2007) . a study involving mers-cov described that orf 8b strongly antagonizes the inf-beta (β) promoter and orf4b and 8b significantly suppress ifn induction (lee et al., 2019b) . accessory proteins 8b and 8ab of sars-cov can suppress the inf-β signaling pathway (and thus interferon production) by their participation in the ubiquitin-mediated rapid degradation of inf regulatory factor 3 (irf3) (wong et al., 2018) . in contrast, when we focused on mers-cov from bats and camels, orf 8b antagonized melanoma differentiation-associated protein 5-mediated nuclear factor kappa b (nf-κb) activation. orf 8b strongly inhibited tank-binding kinase 1-mediated induction of nf-κb signaling, but not iκb kinase epsilon and irf3-mediated activations (lee et al., 2019a) . thus, we speculate that the properties of the accessory proteins, ns7b and ns8, in 2019-ncov may affect its ability to infect humans. further studies are required to confirm this speculation. ns7b is a short peptide of 43 residues. a three-dimensional structure is often difficult to obtain from such a short peptide. we predicted the three-dimensional structure of the queried ns7b amino acid sequence using dichot and c-i-tasser ( supplementary fig. 1, fig. 3 ) (fukuchi et al., 2014; zhang et al., 2018) ; a protein family (pf11395) was found, but no known three-dimensional structure was found. the fahmi, et al. infection, genetics and evolution 81 (2020) 104272 secondary structure of this query was also predicted using jpred4 ( supplementary fig. 2 ) (drozdetskiy et al., 2015) . the secondary structure was predicted to be an α-helix, but that this very likely does not occur depending on the environment (fig. 3) . the alignment of this protein revealed three polymorphism sites between 2019-ncov, batcov ratg13, and bat-sars-like coronavirus sequences (bat-sl-covzxc21 and bat-sl-covzc45) ( supplementary fig. 3 ). in summary, some nonstructural proteins were conserved and others were not conserved between 2019-ncov and sars-cov. by focusing on the 2019-ncov-specific proteins, ns7b and ns8, we proposed a combination of phylogenetic profiling analysis and structural characterization of the genes that were specifically expressed in 2019-ncov and the closely related bat coronavirus. the data provide insight for further characterization of the infective properties of this virus. supplementary data to this article can be found online at https:// doi.org/10.1016/j.meegid.2020.104272. this work was supported by the mext-supported program for the strategic research foundation at private universities (grant number s1511028 to t.i) and the takeda science foundation. the authors declare no competing interest. okavirus as the outgroup. the heatmap indicates the binary matrix of the homolog proteins of 2019-ncov against other species in the dataset, with black and white colors as presence and absence, respectively. the bit pattern was arranged following the vertical and horizontal trees. the vertical tree is a phylogenetic profiling tree constructed from a binary matrix of the presence and absence of homolog proteins. it has two clades, indicated by blue and green bars. the horizontal tree is the cladogram of the maximum likelihood tree, as shown in fig. 1 , with a collapsed clade of 2019-ncov. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) fig. 3 . the model of nonstructural-structural transition of 2019-ncov nonstructural protein 7b. the predicted protein structure of 2019-ncov nonstructural protein 7b by c-i tasser is shown as the helix structure protein. blast+: architecture and applications origin and evolution of pathogenic coronaviruses modeltest-ng: a new and scalable tool for the selection of dna and protein evolutionary models jpred4: a protein secondary structure prediction server the ncbi taxonomy database molecular evolution of human coronavirus genomes ideal in 2014 illustrates interaction networks composed of intrinsically disordered proteins and their binding partners mafft multiple sequence alignment software version 7: improvements in performance and usability mega x: molecular evolutionary genetics analysis across computing platforms middle east respiratory syndrome coronavirus-encoded accessory proteins impair mda5-and tbk1-mediated activation of nf-κb middle east respiratory syndrome coronavirusencoded orf8b strongly antagonizes ifn-β promoter activation: its implication for vaccine design genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding molecular epidemiology, evolution, and phylogeny of sars coronavirus the cipres science gateway: enabling highimpact science for phylogenetics researchers with limited resources the 29-nucleotide deletion present in human but not in animal severe acute respiratory syndrome coronaviruses disrupts the functional expression of open reading frame 8 full-genome evolutionary analysis of the novel corona virus (2019-ncov) rejects the hypothesis of emergence as a result of a recent recombination event reverse genetic characterization of the natural genomic deletion in sars-coronavirus strain frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo ncbi reference sequences (refseq): a curated non-redundant sequence database of genomes, transcripts and proteins middle east respiratory syndrome coronavirus (mers-cov): a review the orf7b protein of severe acute respiratory syndrome coronavirus (sars-cov) is expressed in virus-infected cells and incorporated into sars-cov particles raxml-vi-hpc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models epidemiology, genetic recombination, and pathogenesis of coronaviruses hierarchical grouping to optimize an objective function coronavirus pathogenesis accessory proteins 8b and 8ab of severe acute respiratory syndrome coronavirus suppress the interferon signaling pathway by mediating ubiquitin-dependent rapid degradation of interferon regulatory factor 3 discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus template-based and free modeling of i-tasser and quark pipelines using predicted contact maps in casp12 a pneumonia outbreak associated with a new coronavirus of probable vat origin we want to thank dr. motonori ota, dr. satoshi fukuchi, dr. kota kasahara, and dr. takeshi kikuchi for their support and helpful comments. key: cord-196265-mvnkkcow authors: m'esz'aros, b'alint; s'amano-s'anchez, hugo; alvarado-valverde, jes'us; vcalyvseva, jelena; mart'inez-p'erez, elizabeth; alves, renato; kumar, manjeet; rippmann, friedrich; chemes, luc'ia b.; laboratory, toby j. gibson . european molecular biology; heidelberg,; germany,; embl, collaboration for joint phd degree between; university, heidelberg; biosciences, faculty of; estructural, laboratorio de bioinform'atica; leloir, fundaci'on instituto; aires, buenos; argentina,; chemistrybiology, computational; kgaa, merck; darmstadt,; biotecnol'ogicas, instituto de investigaciones; mart'in, universidad nacional de san title: short linear motif candidates in the cell entry system used by sars-cov-2 and their potential therapeutic implications date: 2020-04-21 journal: nan doi: nan sha: doc_id: 196265 cord_uid: mvnkkcow the primary cell surface receptor for sars-cov-2 is the angiotensin-converting enzyme 2 (ace2). recently it has been noticed that the viral spike protein has an rgd motif, suggesting that cell surface integrins may be co-receptors. we examined the sequences of ace2 and integrins with the eukaryotic linear motif resource, elm, and were presented with candidate short linear motifs (slims) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton and cell signalling. these slim candidates are highly conserved in vertebrates. they suggest potential interactions with the ap2 mu2 subunit as well as i-bar, lc3, pdz, ptb and sh2 domains found in signalling and regulatory proteins present in epithelial lung cells. several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, often involving tyrosine phosphorylation status. candidate lir motifs are present in the tails of ace2 and integrin beta3, suggesting that these proteins can directly recruit autophagy components. we also noticed that the extracellular part of ace2 has a conserved midas structural motif, which are commonly used by beta integrins for ligand binding, potentially supporting the proposal that integrins and ace2 share common ligands. the findings presented here identify several molecular links and testable hypotheses that might help uncover the mechanisms of sars-cov-2 attachment, entry and replication, and strengthen the possibility that it might be possible to develop host-directed therapies to dampen the efficiency of viral entry and hamper disease progression. the strong sequence conservation means that these putative slims are good candidates: nevertheless, slims must always be validated by experimentation before they can be stated to be functional. the covid-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), an enveloped, single-stranded rna virus. it had infected more than two and a half million people and caused circa 170,000 deaths globally by mid-april 2020. sars-cov-2 belongs to the coronaviridae family, whose members are common human pathogens responsible for the common cold, as well as for some emerging severe respiratory diseases. among them are the sars-cov and the middle east respiratory syndrome coronavirus (mers-cov), the former of which caused over 8,000 cases in 2003 with a fatality rate of ~10%, and the latter caused about 2,500 infections in 2012 with a fatality rate of 37% (de wit et al., 2016) . another coronavirus, infectious bronchitis virus (ibv), infects birds and has been used as a model in coronavirus research (sisk et al., 2018) . sars-cov-2, like sars-cov (li et al., 2003) , uses the angiotensin converting enzyme 2 (ace2) as a receptor to attach to the host cells (hoffmann et al., 2020; wrapp et al., 2020; p. zhou et al., 2020) . ace2 is a single pass type i membrane protein with a short cytosolic c-terminal region for which the functionality, however, is mostly unknown. earlier results clearly show that the sars-cov-2 receptor binding domain (rbd) of the spike protein interacts with ace2 for cellular entry. however, type ii alveolar cells (at2)the main targets of sars-cov-2 in the lung (zou et al., 2020) express a relatively low amount of ace2, which points to the existence of co-receptors being targeted by the virus in parallel. one such candidate are integrins that bind a large variety of ligands harbouring an rgd sequence motif, as recent analysis of the rbd identified a possibly functional rgd motif (sigrist et al., 2020) . integrins are major cell attachment receptors, which are known to be targeted by a range of viruses -including hiv, herpes simplex virus-2, epstein-barr virus (ebv) and the foot and mouth virus -for cell entry and activation of linked intracellular pathways (hussein et al., 2015; stewart and nemerow, 2007; triantafilou et al., 2001) . integrins are special types of receptors, as they propagate signals in both directions; extracellular ligands can induce cytoplasmic pathway activation, but intracellular binding on the cytosolic tails can influence the structure of the ectodomains and hence ligand-binding affinity. the complexity of integrin signalling stems in the dimeric structure of integrins, as they are composed of two subunits, α and β. for the rgd-binding integrins, the ligand binding surface lies at the interface of the two integrin subunits with both subunits making contacts with the ligand. these rgd motifs are recognized by at least 8 out of the 24 human integrins, and the flanking residues next to the core rgd motif are known to play a huge role in selectivity (kapp et al., 2017) . several viral proteins contain rgd (or rgd-like) short linear motifs (slims) for integrin modulation; and in addition, some viruses can not only use integrins on the host cell surface, but hiv and siv can also incorporate integrins into their own membranes for mediating interactions with the host (guzzo et al., 2017) . therefore, integrins can potentially be targeted at both the extracellular and the intracellular side to combat pathogenic hijacking. viruses, as obligate intracellular entities, need to interfere with major cellular processes like vesicular trafficking, cell cycle, cellular transport, protein degradation or signal transduction to satisfy their replication, enzymatic, metabolic and transport needs (davey et al., 2011) . to achieve this, a large number of host processes are hijacked using slims often located in intrinsically disordered regions to establish protein-protein interactions with host proteins or undergo post-translational modifications, like tyrosine phosphorylation. for example, cellular signalling relies heavily on the use of slims (van roey et al., 2013 . the low affinity and cooperativity of slim-based molecular processes allow reversible and transient interactions that can work as switches between distinct functional states and are regulated both in time and space (gibson, 2009; scott and pawson, 2009) . conditional switching of slims, for example through phosphorylation, can induce the exchange of binding partners for a protein, thus mediating molecular decision-making in response to signals reporting on the cell state (van roey et al., 2012) . the central resource for slims is the eukaryotic linear motif database (elm; http://elm.eu.org/), also serving as an exploratory server for over 280 manually curated slim classes with experimental evidence, each defined by a posix regular expression (kumar et al., 2020) . as explained above, a major strategy of viruses is to abuse the host system by using mimics of eukaryotic slims to compete with extracellular or intracellular binding partners or to sequester host proteins (davey et al., 2011) . this dependence of viruses and many other pathogens on slim-mediated functions suggests that there is an opportunity to drug the cell systems where these interactions are being hijacked (sámano-sánchez and . for example, tyrosine kinase inhibitors, often used in anticancer therapy, have shown promising coronavirus replication inhibition in infectious cell culture systems (coleman et al., 2016; dong et al., 2020; shin et al., 2018; sisk et al., 2018) . in the remainder of the introduction, we will describe some of the major pathways hijacked by viruses to accomplish cell attachment, entry and replication, which are suggested by our results to be relevant to sars-cov-2 infection. indicating that acidification is not a requirement per se, but acts by inducing the endosomal spike protein cleavage required for viral fusion (matsuyama et al., 2005; simmons et al., 2005) . spike protein cleavage can be done by the transmembrane protease serine 2 (tmprss2) at the cell surface, or by cathepsin l within endosomes (de wilde et al., 2018) . the same entry route and proteases are utilized by sars-cov-2, and the main entry route also seems to be endocytic (hoffmann et al., 2020; ou et al., 2020) . autophagy is an evolutionarily conserved process in eukaryotes with multiple cellular roles that include the regulation of cellular homeostasis through the catabolism of cell components, immune development and the host cell response to infection through pathogen phagocytosis (deretic and levine, 2009) . viruses have evolved mechanisms to block the host cell antiviral response, and can further hijack autophagy components to promote their survival and replication. this can be done through viral mimicry of host proteins coordinating autophagy or through the direct inhibition of the host autophagy machinery (kudchodkar and levine, 2009) . coronaviruses (covs) exploit the autophagy machinery through different mechanisms (cong et al., 2017; maier and britton, 2012) . for example, mers-cov targets the becn1 autophagy regulator for degradation, blocking the fusion of autophagosomes and lysosomes and protecting the virus from degradation (gassen et al., 2019) . coronaviruses repurpose cellular membranes to create double membrane vesicles (dmvs) onto which the replication-transcription complex (rtc) is assembled, a process that involves recruitment of multiple autophagy components (cong et al., 2017; prentice et al., 2004; v'kovski et al., 2019) . betacoronavirus mouse hepatitis virus (mhv) rtcs assemble by recruiting lc3-i, a nonlipidated form of the lc3 autophagy protein (cong et al., 2017; reggiori et al., 2010) , and sars-cov rtcs also colocalize with lc3 (prentice et al., 2004) . proximity-based mass spectrometry on the mhv replication complex further revealed that the rtc environment repurposes components from the host autophagy, vesicular trafficking and translation machineries (v'kovski et al., 2019) in the present work, we identify a set of conserved slim candidates in the ace2 and integrin proteins, which are likely to act in the cell entry system of sars-cov-2. these motifs can provide molecular links to understand how the virus recognizes target membranes, enters into cells, and how it repurposes intracellular membrane components to drive its replication. these molecular links might provide novel clues towards drugging sars-cov-2 infections. we first focus on the extracellular slims, before moving across the membrane to examine the cytosolic potential of the receptor tails. the ability of sars-cov-2 rbd to bind integrins via the rgd motif (see table 1 ) has not yet been assessed directly. however, there are several features that make the spike-integrin interaction plausible, including sequence-and structure-level information, expression profiles, the presence of accessory motifs and protein-protein interactions. aligning close homologues of the rbd from the coronaviridae family ( figure 1a ) shows that the motif candidate is located in a locally less conserved region, hinting at the rapid evolvability of the site. several coronavirus rbds contain kgd at this site, which is known to be a lower affinity integrin binding site, first identified in snake venom disintegrins, such as barbourin (minoux et al., 2000) . a kgd motif residing in the ebv gh/gl protein has also been shown to be essential for entry into epithelial and b cells . figure 1b shows the tree derived from the spike protein sequence alignment, highlighting that the sars-cov-2 rgd motif might have evolved from an earlier kgd motif, and might present a distinct step of adaptation if the motif is indeed an integrin attachment site. (kgd and rgd) shown in orange and red boxes, respectively. c) structure of the sars-cov-2 rbd as seen in the ace2-bound form (pdb id:6m17) . the rgd motif is shown in red sticks. regions in direct contact with ace2 are shown in blue. residues with missing atomic coordinates (indicating flexibility) in the unbound trimeric spike protein structures (pdb ids: 6vsb, 6vxx and 6vyb) are shown in transparency. alignment and tree prepared in jalview (waterhouse et al., 2009) with clustal colours. structure was visualized using ucsf chimera (pettersen et al., 2004) . at the time of reporting the rgd motif, no sars-cov-2 spike structures were available, so the authors used structural homology modelling to determine that the rgd motif is surface accessible (sigrist et al., 2020) . since then, several rbd structures have been determined, both in unbound (walls et al., 2020; wrapp et al., 2020) and ace2 complexed forms using electron microscopy ) and x-ray diffraction (lan et al., 2020) , allowing for the direct structural assessment of the possibility of binding to integrins. figure 1c shows the rgd motif together with the residues involved in direct binding of ace2. the rgd motif is located in the vicinity of the ace2 binding site, however, based on uncomplexed structures of the rbd, the residues that surround the rgd site are flexible. this indicates that while ace2 binding blocks the rgd motif, without ace2 the rgd is surface accessible and interaction with integrins are not sterically blocked. as spike exists as a trimer on the virion surface, different copies of the rbd can in theory interact with ace2 and integrins at the same time. there is no solved complex structure of the ace2-integrin complex. however, further structural consideration may indicate whether the spike-ace2 and the spike-integrin interactions can coexist. the ectodomains of both ace2 and integrins in the open conformation are roughly the same size measured from the membrane, being in the 150-200 å range (based on available structures pdb:6m17 and pdb:3ije (xiong et al., 2009) ). this means that the rgd-binding site of integrins and the rbd binding regions of ace2 are relatively close in space. in addition, in the more open 'up' conformation of the rbds, the distance between pairs of rbds is about 100 å (based on the structure pdb:6vsb reported in (wrapp et al., 2020) ), which is probably larger than the distance between the ace2 binding region and the integrin ligand binding site, estimated from the individual integrin and ace2 structures. the rgd motif is recognized by several integrins, and specificity is determined mostly by the flanking residues around the core motif. as evidenced by crystallized integrin dimer:ligand complexes, the residue preceding rgd is in contact with the α subunit, while the residue after the core motif interacts with the β subunit. the immediate context of the sars-cov-2 rbd motif is 402-irgde-406, which can give an indication about possible integrin targets. irgd can be found in several native integrin-binding partners, including frem1 , mfap4 (pilecki et al., 2015) and igfbp1/2 (cavaillé et al., 2006; wang et al., 2006) . these extracellular matrix proteins target integrins with αv, α5 and α8 subunits. rgde is present in the native human integrin ligands tgf-β1, osteolectin, collagen α-1(vi) chain, psbg-9 and polydom, and in vitro and in vivo binding studies on the specificity profiles of these proteins (cescon et al., 2015; rattila et al., 2019; sato-nishiuchi et al., 2012; shanley et al., 2013; shen et al., 2019; tumbarello et al., 2012) highlighted a post-rgd glu to be efficient in binding to β1, β2 and β3 integrin subunits. correlating these preferences with possible α and β integrin subunit pairings points to the most likely candidate target integrins for sars-cov-2 being αvβ1, αvβ3, α5β1 and α8β1. motif-domain interactions are typically under heavy spatio-temporal regulation. hence the sars-cov-2 rbd-integrin binding can only occur if the possible target integrins are expressed on at2 cells. both α5β1 (pilewski et al., 1997) and αvβ3 (caccavari et al., 2010; nakamura et al., 2002; singh et al., 2000) have been observed in lung epithelial cells and have been shown to be implicated in disease emergence and progression, including emphysema, non-small cell lung cancer and mechanical injury of the lungs (teoh et al., 2015) , marking these two integrins as prime suspects for targets of the rbd. it has been shown that in heart tissues, ace2 is able to bind the β1 subunit of integrins in an rgd-independent manner, enhancing cell adhesion and regulating integrin signalling via the focal adhesion kinase (fak) (clarke et al., 2012) . the rgd independence of the interaction means that while ace2 and integrins are in complex, the rgd binding site of the integrin is unoccupied, further supporting a potential integrin:ace2:spike ternary interaction. apart from the known interplay between ace2 and integrins, there are additional features that indicate an even tighter crosstalk between the two receptors. rgd-mediated binding to integrins is metal mediated (via divalent cations like mg 2+ or mn 2+ ), and all integrins have a so-called 'metal ion-dependent adhesion site' (midas) motif (dxsxs) (lee et al., 1995) . the integrin midas structural motif is located near the ligand binding site on the β subunit and is essential for binding, as sidechains belonging to the motif and an acidic residue from the ligand coordinate the metal ion together (zhang and chen, 2012) . ace2 also has a similar dxsxs motif (see table 1 ), that might facilitate interactions with ligands that are recognized by integrins, possibly creating an overlap between the ligand binding profiles and regulation of the two receptors. in the known structures where spike is bound to ace2 the rgd motif is not in contact with the ace2 midas . however, the midas motif is highly conserved across species (see figure 2) , and surface exposed. consequently it may still be involved in mediating an interaction with an rgd-like motif, potentially serving as a parallel mechanism for binding the spike protein. ace2 and several integrin subunits require proteolytic cleavage for biological activity (see table 1 ). integrin subunits α3, α5, α6 and αv are cleaved by furin or furin-like proprotein convertases (pcs) during maturation (lehmann et al., 1996; lissitzky et al., 2000) . nearly all pcs contain an rgd motif, and while its role in integrin binding is not clear, the motif has been shown to be required for proper functioning for several pcs (lou et al., 2007; lusson et al., 1997; rovère et al., 1999) . the sars-cov-2 spike protein contains a furin-like cleavage site that is absent from closely related spike proteins, immediately following the rbd (coutard et al., 2020) . this cleavage results in increased virulence, possibly by allowing greater movement of the rbd potentially aiding in exploring a larger space around the rbd-binding region of ace2. ace2 is cleaved by several proteases, including tmprss2 (heurich et al., 2014) . ace2 binds to tmprss2, forming a receptor-protease complex (shulla et al., 2011) . tmprss2 is also known to cleave the spike protein of both sars-cov and mers-cov (iwata-yoshikawa et al., 2019), augmenting their entry into the host cell (heurich et al., 2014) . furthermore, similar results have been found for sars-cov-2, where tmprss2 was found to be fundamental for cell entry (hoffmann et al., 2020) . this dependence is most probably two-fold: on one hand tmprss2 is needed for ace2 activation, on the other hand, sars-cov-2 spike protein also contains a tmprss2 cleavage site (meng et al., 2020) . the ace2 sequence (uniprot:ace2_human) was entered in the elm server (kumar et al., 2020) and returned several relevant candidate slims in the short cytosolic c-terminal tail. because slims are so short, it is difficult to obtain significant results in sequence searches. contextual information, including cell compartment localisation and functional relevance, is important in deciding whether a motif candidate is worth testing experimentally (gibson et al., 2015) . furthermore, in intrinsically unstructured protein sequence, amino acid conservation is indicative of functional interactions. therefore, an alignment was prepared of vertebrate ace2 proteins. all of the detected motif matches (shown in table 1 together with potential binding partner domains defined using pfam and interpro (mitchell et al., 2019) ) were conserved in mammals, most were conserved with birds and mammals and some were conserved with extant reptiles ( figure 3 ). these groups diverged from one another >300 million years ago (kemp, 2005) indicating a strong conservation of all candidate motifs. in addition, the functional contexts of these motifs are biologically coherent, involving signalling by tyrosine kinases, endocytosis, autophagy and actin filament induction (table 1 ). in the following subsections we briefly summarise each of the conserved motifs and their possible role in the viral entry mechanism. diagram of the ace2 protein. figure prepared with jalview using clustal colours. the yxxphi motif binds the μ2 subunit (uniprot:ap2m1_human) of the endocytosis ap-2 adaptors by β-augmentation (owen and evans, 1998) . it is found in numerous cell surface receptors which have intrinsically disordered c-terminal tails (bonifacino and traub, 2003) . a small selection is listed in the database entry elm:trg_endocytic_2, and while the motif has not been validated in ace2, it is highly conserved (figure 3 ). when the tyr is phosphorylated, this motif becomes an sh2 binding site, while in the apo form it binds the μ2 adapter. therefore, this motif can operate as a molecular switch. the residue following the tyr makes a β-strand interaction and therefore cannot be a proline (pdb:1bxx). the phi position requires a bulky hydrophobic residue. the motif pattern can be represented by the regular expression y[^p]. [lmvif] and this motif is conserved in ace2 from all mammals except monotremes. thus the mammalian ace2, which internalises the coronavirus, has a slim candidate for internalisation appropriately located within its cytosolic tail. the region encompassing the yxxphi motif overlaps with a src homology 2 (sh2) domain binding motif ( figure 3 ) that is created upon phosphorylation of tyr 781. sh2 domain binding motifs are characterized by an invariant phosphotyrosine (py) that is created following tyrosine kinase activation, and allows binding to more than 100 types of sh2 domains present in human proteins (tinti et al., 2013) . the py residue is accompanied by additional binding determinants that frequently involve hydrophobic residues at the py+3 position, but can also involve other combinations, such as asn at py+2 in grb2-specific sh2 motifs, or hydrophobic residues at py+4 in stap-1 sh2 motifs kaneko et al., 2010) . most motifs are also characterized by the exclusion of residues at certain positions following the py, and in general, sh2-binding motifs show a high degree of cross-specificity liu et al., 2010) . cell culture infection assays with different coronaviruses, including sars-cov, have shown susceptibility to tyrosine kinase inhibitors, indicating the involvement of host tyrosine phosphorylation (coleman et al., 2016; dong et al., 2020; shin et al., 2018; sisk et al., 2018) . the sequence found in ace2 (781-yasid-785) best matches the binding specificity for the sh2 domain present in nck1/2 proteins, which belong to the class ia sh2 domains (kaneko et al., 2010) . proteins known to contain this motif are listed in entry elm:lig_sh2_nck1_1. nck proteins are adaptor proteins that modulate actin cytoskeleton dynamics (buday et al., 2002) . for example, nck is recruited to the cell membrane by the nephrin protein, following tyrosine signalling that creates several nck sh2 binding motifs in nephrin (blasutig et al., 2008) . once recruited to the membrane, nck activates wiskott-aldrich syndrome (wasp) family proteins through the use of a helical binding motif (elm:lig_gbd_chelix_1) that relieves wasp autoinhibition and allows the recruitment of the actin regulatory protein complex arp2/3, which leads to the initiation of actin polymerization (okrut et al., 2015) . the nck binding motif is exploited by two known human pathogens, namely enteropathogenic escherichia coli and vaccinia virus (frese et al., 2006) . the residues present at py+1, py+2 and py+4 rule out that the ace2 yasid motif can be a strong grb2, crk or stap1 sh2 domain binder, and binding to stat1/2/3 sh2 domains is also unlikely due to the lack of adequate specificity determinants. other sh2 domains (e.g. src-related) could be recruited by ace2, and experimental validation will be required to test these hypotheses. tyrosine 781 in ace2 also overlaps a phosphorylation-independent npy motif (elm:lig_ibar_npy_1). this motif was initially described in the bacterial secreted protein translocated intimin receptor (tir) from pathogenic strains of e. coli like enterohaemorrhagic e. coli (ehec). the npy tripeptide recognizes and binds with a 60 μm affinity to inverse bin-amphiphysin-rvs (i-bar) domains in adaptor proteins like insulin receptor substrate protein of 53 kda (irsp53) and its homolog insulin receptor tyrosine kinase substrate (irtks) (campellone et al., 2006; de groot et al., 2011) . i-bar domains bind to the plasma membrane to favour weak membrane protrusions, and the preference of i-bar domains for negative membrane curvatures enables a positive feedback loop that can result in the formation of lamellipodia, filopodia and other types of membrane protrusions prévost et al., 2015; zhao et al., 2011) . irsp53 and irtks are modular proteins that contain sh3 domains which in turn recognize pxxp slims in actin filament regulators like mena, eps8 and mdia1 (ahmed et al., 2010) resulting in the formation of membrane protrusions through actin filament formation (campellone et al., 2006; chen et al., 2015; prévost et al., 2015; zhao et al., 2011) . moreover, irsp53 has an additional cdc42-binding motif that can result in a direct wasp activation (ahmed et al., 2010) . during ehec infection, the bacteria uses the npy motif in the transmembrane protein tir to recruit irsp53 (campellone et al., 2006) . irsp53 acts as a scaffold to localize the injected bacterial protein espfu to the bacterial attachment site, cytosolic side, through the binding of a pxxp motif in espfu to the irsp53 sh3 domain. through the use of the same helical slim present in nck (elm:lig_gbd_chelix_1), espfu acts as a potent wasp activator, inducing the actin polymerization that contributes to the pedestal formation characteristic of ehec infections (cheng et al., 2008; sallee et al., 2008) . the npy slim, although not yet experimentally validated in any human protein, is potentially functional in proteins like shank2 or the microtubule-binding clip-associating protein 1 (clasp1), based on protein conservation and functional association (de groot et al., 2011) . the putative npy motif in ace2 is conserved in all analysed mammalian and bird homologs (figure 3) , suggesting a direct interaction with host i-bar-containing proteins such as irsp53 or irtks, which are expressed in lung tissues (uhlén et al., 2015) . the i-bar domain-binding motif in the cytosolic region of ace2 could be relevant for sars-cov-2 infection in the following scenario. during viral cell entry, the npy motif could recruit i-bar-containing proteins such as irsp53 or irtks, resulting in membrane protrusion formation that could be exploited for viral entry or in cell to cell transmission. it is known that the hijack of the filopodia formation network is beneficial for the entry and spreading of many enveloped viruses (reviewed in chang et al., 2016) , but whether this process is active during coronavirus infection is still unclear. a second route might cooperate with the npy motif in the recruitment of actin cytoskeleton components. a direct interaction between the sars-cov spike protein cytosolic side c-terminal domain and the ezrin ferm domain can occur during the opening of the viral fusion pore and has been proposed to restrain viral infection (millet et al., 2012) . ezrin is a protein involved in cell morphology and apical membrane remodelling that acts as a membranecytoskeleton linker. ezrin recruits f-actin through its c-terminal domain, and can also bind to irsp53 located at negatively curved membranes (saleh et al., 2009; tsai et al., 2018) , suggesting that while the npy motif acts at earlier stages of viral attachment, the spike protein/ezrin interaction might work during or after viral fusion, to promote the recruitment of actin regulatory components to viral fusion sites. certain members of the ptb domain family were discovered to bind to phosphorylated npxy motifs, hence the designation phospho-tyrosine binding domain (zhou et al., 1995) . the npxy motifs in cytosolic tails of receptors, including integrins, are regarded as endocytosis sorting signals (bonifacino and traub, 2003) . it was later discovered that ptb domains in the internalisation adapter protein dab1 could also bind non-phosphorylated nxx[fy] motifs (apoptb motif) and that this might be the case for the majority of ptbs (uhlik et al., 2005) . representative receptors with apoptb motifs are in the database entry elm:lig_ptb_apo_2. in ace2, the core nxxf motif is conserved in all land vertebrates ( figure 3 ). for dab1 apoptb motifs, there is a hydrophobic requirement two residues before the asn. in ace2, this is predominantly a charged residue: therefore, if this strikingly conserved nxxf is an apoptb motif, it should bind a protein other than dab1 and proteins with related specificities. the apoptb motif binds as a short β-strand (β-augmentation) followed by a β-turn. proline is rejected at one strand-forming position and therefore the regular expression for this motif would be [^p] .n..f for land vertebrates or [eq] .n..f for mammals. as with the phosphorylated versions, the apo-motifs are tightly connected to endocytosis (uhlik et al., 2005) . autophagy, the recycling of cellular material, is vital for cellular homeostasis. many pathogens must control the autophagy response to establish productive infection (deretic and levine, 2009) . it has been shown that coronaviruses, including human covs, subvert autophagy components to promote viral replication at dmvs associated to the rtc (cottam et al., 2014; fung and liu, 2019; gassen et al., 2019; reggiori et al., 2010) . the lir motif is required for the interaction of a target protein with atg8 or its homologues lc3 and gabarap to facilitate autophagy of the target via the autophagosome (birgisdottir et al., 2013) . the lir motif has been catalogued in the elm resource entry elm:lig_lir_gen_1 which detected a candidate motif in the human ace2 cytosolic tail sequence (figure 3 ). after the lir motif was annotated in elm, a more recently solved lc3-lir structure (pdb:5cx3) showed that the interacting peptide is longer, with one or two more hydrophobic interactions (olsvik et al., 2015) . lir enters a hydrophobic groove bordered by positively charged residues. a core [wfy]xx[ilmv] enters the deepest part of the groove. on either side of the core, the interacting residues can be flexibly spaced. the core must be preceded by a negatively-charged residue (which might be enabled by phosphorylation). further, the motif core is followed by a flexibly spaced hydrophobic residue. there is often a negatively-charged residue preceding this hydrophobic position: it can make favourable interactions with counter charges but is not an absolute requirement, so is not included in the revised motif pattern. based on the structure (pdb:5cx3) and some spot arrays (alemu et al., 2012; olsvik et al., 2015; rasmussen et al., 2019) matches the motifs annotated in elm. the revised motif is conserved in the mammalian ace2 cytosolic tail, but not in birds or reptiles. the ace2 lir motif candidate can potentially enable the incoming coronavirus to attract autophagy elements such as lc3 to the structures where the virus replicates and assembles. in line with this, a nonlipidated form of the lc3 protein has been shown to be associated with the rtcs of mhv and sars-cov (cong et al., 2017; prentice et al., 2004; reggiori et al., 2010) . this brings up the interesting possibility that ace2 remains associated with the membranous structures where sars-cov-2 replicates at later infection stages, assisting in the repurposing of autophagy components required for viral replication. amongst other motif-binding modules, pdz domains come in great abundance in human and other multicellular animals (ernst et al., 2009) . pdz domains take part in a variety of biological processes including cellular signalling and activity at the synapse (manjunath et al., 2018) . these domains bind slims by β-strand augmentation which are called pdz-binding motifs or pbms, most commonly known to be found in the c-terminus of fully or partially disordered proteins. these interactions are widely studied and their link to various diseases and infections has been previously established (christensen et al., 2019) . a pbm candidate is also found in the very c-terminus of the cytosolic tail of vertebrate ace2 proteins (figure 3 ). motifs following a pattern [st] . [acvilf]$ are a common pdz-binding motif variant, described in the elm resource entry elm:lig_pdz_class_1. there are multiple functional examples of this motif. however, in the ace2 protein the matching sequence is not characterized. ace2 has a disordered tail facing the cytosol, where a number of different pdz domains could be its potential binders (manjunath et al., 2018) . two pdzs in two different adapter proteins -na(+)/h(+) exchange regulatory cofactor nherf3 and sh3 and multiple ankyrin repeat domains protein 1 shank1have been previously identified to be able to bind txf$ sequences ("$" stands for the cterminal end) (ernst et al., 2014) , which makes them both candidates for an interaction with the ace2 c-terminus. nherf3 is co-localised with ace2 in intestinal tissue, and its pdz domains were previously validated to interact with pbms in transmembrane proteins on the cytosolic side of the membrane (gisler et al., 2003) , so it is possible they come in proximity with the ace2 tail containing the txf$ motif, and possibly bind it as a part of ion exchange regulation of small molecule transport activities. nherf3 is known for its involvement in sodium ion-dependent transporter activity (srivastava et al., 2019) , and ace2 was also shown to interact with a sodium-dependent transporter , which could be one of the leads towards unravelling the possible interaction between nherf3 and ace2. nherf3 (gene name pdzk1) and ace2 share a network of experimentally validated protein-protein interactions localized at the cell membrane ( figure 4) . these proteins share localisation and function characteristics, and some of them could turn out to be the missing puzzle pieces to connect ace2 and nherf3 as possible interactors. all in all, whether nherf3 is the pdz-containing protein interacting with ace2 or not is an open question, but there should be very little doubt that ace2 exhibits pdz-binding activity in the cell, since the motif is highly conserved, very specific and already known to appear in membrane-associated proteins with c-terminal tails facing the cytosol. ace2. c-terminal pbms are displayed in white boxes. common functionalities of the proteins marked by different colours. thickness of nodes is proportional to the confidence behind the experimental evidence. the network was built using the string resource (https://string-db.org) (szklarczyk et al., 2019) . tyrosine 781 is a part of the motif patterns for four of the motifs listed above but must be phosphorylated to act as an sh2-binding motif. therefore, we searched the ace2 literature for reports of phosphorylation but were unable to find any with strong site identification. examination of the human ace2 entry in the database phosphositeplus (hornbeck et al., 2019) revealed that high-throughput (htp) phosphoproteomic studies, but no low-throughput (ltp) studies, identify ptyr781. as shown in figure 5 , thirteen htp measurements identified phosphorylation at tyr781 and this residue is the only ace2 phosphosite that is highly reproducible across multiple htp datasets. for example, ptyr781 was one of 318 unique phosphopeptides belonging to 215 proteins analysed from an erlotinib-treated breast cancer cell line model (tzouros et al., 2013) . therefore, this site indeed fulfils the phosphorylation requirement to be an sh2-binding motif. have only been reported once each and therefore are likely to be misidentified peptides. as described above, four sequence motifs overlap in the region surrounding tyr781: the yxxphi endocytic sorting signal (elm:trg_endocytic_2), an nck sh2 binding motif (elm:lig_sh2_nck_1), an npy i-bar binding motif (elm:lig_ibar_npy_1) and the lir autophagy motif (elm:lig_lir_gen_1). while the yxxphi, npy and lir motifs require a non-phosphorylated state of tyr781, the nck motif requires tyr781 phosphorylation, creating the opportunity for a four way molecular phospho-switch acting in this region of ace2 that directs different steps of the sars-cov-2 infection cycle. the state of this switch can be controlled by tyrosine kinase activity involving the src/abl and other tyrosine kinases known to be upregulated during endosomal processes (reinecke and caplan, 2014 ) and viral infection (davey et al., 2011) including in coronaviruses (coleman et al., 2016; dong et al., 2020; shin et al., 2018; sisk et al., 2018) . similar two-way switches have been described before, as with the ctla-4 receptors, where src-family tyrosine kinases dictate the binding preferences of overlapping yxxphi and sh2 binding motifs. in the non-phosphorylated state endocytosis is favoured, whereas t cell activation brings about tyr phosphorylation, shutting down endosomal recycling and initiating signalling through the recruitment of sh2-domain containing proteins (bradshaw et al., 1997; miyatake et al., 1998; ohno et al., 1996; owen and evans, 1998; shiratori et al., 1997) . during early stages of viral infection, and following viral attachment to the host cell membrane, the yxxphi motif could activate the early events of receptor-mediated endocytosis by binding the µ subunit of the ap2 complex, which in turn recruits clathrin and other endocytic components to the viral attachment sites. following the formation of the clathrin coat and initial invagination, actin polymerization is required to assist in the internalization of the endocytic vesicles. in addition, some viruses can 'surf' along filopodia by myosin-mediated actin cytoskeleton movements that transport the viral particles to the entry sites at the cell body, ultimately increasing their entry rate (reviewed in chang et al., 2016) . the actin hijack related to endocytic uptake and the formation of membrane protrusions could be coordinated in one or several stages by the nck sh2 and npy motifs respectively, by recruiting wasp and i-bar proteins to the viral attachment site. this might be enacted by a tyr781 phosphorylation switch that is regulated in time with early attachment characterized by non-phosphorylated tyr781 that allows the yxxphi and npy motifs to be active, and a later phase where tyr781 becomes phosphorylated, inactivating the yxxphi and npy motifs and bringing the nck sh2 motif into play. an alternative scenario might be enabled by the multimeric nature of the spike protein and by attachment of several viral particles to a membrane domain, leading to adjacent ace2 tails on the intracellular side that expose both phosphorylated and nonphosphorylated motifs, allowing these three signalling steps to take place simultaneously. the presence of several parallel routes for the recruitment of cytoskeleton components involving the npy and nck motifs could provide robustness needed to ensure the actinreorganization required for the uptake of virus-containing vesicles into the cytosol. following endocytosis and fusion, viral components are released into the cell and viral replication takes place. during this phase, the last component of the switch could come into play, when the ace2 protein that remains bound to spike protein-coated membranes could promote the hijack of autophagy components necessary to assemble the viral replication factories. at this time, non-phosphorylated ace2 tails would recruit lc3 to replication structures through their lir motifs. integrin β tails are short cytosolic c-terminal intrinsically disordered regions, similar to the analysed region of ace2. the two most probable integrin β subunit candidates at play in sars-cov-2 viral entry are β3 and β1. the c-terminal tail of both subunits share a high degree of sequence similarity, and similarly to ace2, contain several known and candidate slims (see table 1 and figure 6 ) that propagate signals in the cytoplasm and regulate integrin activity not just through intracellular pathways, but also changing the structural state of the ectodomains determining ligand binding capacity (anthis and campbell, 2011) . integrin β tails contain a highly charged patch in their membrane proximal region. this region is indispensable for the interaction between integrins and tyrosine kinases, including the src-family kinase fyn (reddy et al., 2008) and the focal adhesion kinase (fak), most probably via the direct interaction with paxillin (liu et al., 2002) . through these interactions, integrins regulate cytoskeletal remodelling (lv et al., 2016) and the promotion of cell survival (tang et al., 2007) , as well as regulation of focal adhesion assembly and cell protrusion formation (hu et al., 2014) . in turn, fak regulates integrin recycling and endosomal trafficking (mana et al., 2020; nader et al., 2016) . currently, there is no consensus sequence motif describing these interactions, although a definition of hdr[kr]e has been proposed (legate and fässler, 2009) , fitting integrins β1, β3, β5 and β6. this motif is under heavy regulation by several mechanisms. first, the interaction with tyrosine kinases seem to involve additional residues n-terminal of the charged motif coremost notably the conserved lysine preceding the hydrophobic patch (reddy et al., 1998) that are only accessible in the active state of the integrin dimer, as these regions are buried in the membrane otherwise (stefansson et al., 2004) . second, the d residue of the motif forms a salt bridge with the cytosolic tail of the α subunit of the integrin in the inactive conformation of the receptor. thus, this motif region is dependent on integrin activation regulated by ligand binding and intracellular interactions mediated by the c-terminally located npxy motifs. both the β1 and β3 tails contain two regions that match the apoptb motif defined in the elm resource (elm:lig_ptb_apo_2). furthermore, these regions are known to have tyr phosphorylation, matching the phosphorylated motif definition as well (elm:lig_ptb_phospho_1). these regions are known to be able to form β-turns, and are recognition sites for phosphotyrosine-binding domains. npxy motifs (or nxxy in the case of the second motif of the β3 tail) are the major sorting signals mediating interactions with ferm domains for regulating endosomal trafficking (ghai et al., 2013) . in integrin β tails these motifs recruit adaptor proteins and clathrin, serving as sorting signals (ohno et al., 1995) , and the npxy motifs in the β1 tail have a direct connection to viral entry for reovirus (maginnis et al., 2008) . the first npxy motif binds talin-1, serving as a connection between the plasma membrane and the major cytoskeletal structures (horwitz et al., 1986) . considering the expression profiles of talins, the most likely interaction partner of lung-expressed integrins is talin-1. talin-1 contains a ferm domain, similarly to ezrin, which establishes a direct interaction with the sars-cov spike protein upon viral fusion (millet et al., 2012) . however, the interaction between the rbd and integrins offers the virus an earlier point of interference with the cytoskeletal system, being able to modulate it cooperatively with the ace2 actin-regulatory elements (npy and nck sh2 motifs) before and during cellular entry. the talin/integrin interaction however presents a feedback loop: the binding of talin on the cytoplasmic side induces a structural rearrangement on the ectodomains of integrins, enabling a higher affinity interaction with rgd motif containing ligands (tadokoro et al., 2003) . the first npxy motif is also a binding site for dok1, a negative regulator of integrin activation. dok1 is in direct competition with talin for binding integrins (tadokoro et al., 2003) . the competition is fundamentally influenced by phosphorylation on tyr773 of the npxy motif. the non-phosphorylated motif has a higher affinity towards talin, whereas phosphorylation prefers dok1 (oxley et al., 2008) ; thus tyr773 acts as a phospho-switch that regulates integrin activation. the first npxy motif also presents a site for a largely phosphorylation-independent interaction with the integrin cytoplasmic domain-associated protein-1 (icap-1). icap-1 is a fundamental regulator of the assembly of focal adhesions (fa) and icap-1 knockdown reduced fa assembly (alvarez et al., 2008) , possibly working in conjunction with the membrane-proximal charged motif. icap-1 seems to be specific for β1, and hence the therapeutic considerations for targeting this pathway requires the verification of the type of integrins expressed on at2 cells (and other related cell types). the second npxy motif is a binding site for kindlin . this interaction requires the integrin tail to be non-phosphorylated and phosphorylation on tyr785 (for β3) can switch off the interaction with kindlin-2 (bledzka et al., 2010) . kindlin binding (together with talin binding) is a crucial step in integrin activation, and hence regulates the availability of integrins for extracellular ligands (herz et al., 2006) , and was also suggested to play a role in tgf-β1 signalling (kloeker et al., 2004) . the two npxy(-like) motifs in the integrin β tails not only constitute two separate phospho-switches, but also act in synergy to give rise to more complex regulation. filamin and the ptb domain region of shc1 each bind to both npxy motifs (deshmukh et al., 2010; liu et al., 2015) . shc is an adaptor protein playing a key role in mapk and ras signalling pathways, and its interaction with integrin β3 requires both phosphorylations on tyr773 and tyr785 (audero et al., 2004; deshmukh et al., 2010) . in contrast, binding of the ig domain of filamin-a requires both tyrosines to be in a non-phosphorylated state. the filamin-a interaction can be considered as a main shutdown switch in integrin signalling, as this interaction induces the closed conformation of the integrin ectodomains, decreasing the chance of ligand binding (liu et al., 2015) . in addition, binding partners utilizing both npxy motifs may also serve as stronger modulators of endosomal trafficking, switching on enhanced signals. the connection between autophagy and cell adhesion has already been described, showing that both reduced fak signalling (sandilands et al., 2011) and detachment from the extracellular matrix via integrins (vlahakis and debnath, 2017) enhances autophagy. atg deficient cells have enhanced migration properties, and at the molecular level there seems to be a direct connection between atg proteins and integrins as well: autophagy stimulation increases the co-localization of β1 integrin-containing vesicles with lc3stained autophagic vacuoles, while autophagy inhibition decreases the degradation of internalized β1 integrins (tuloup-minguez et al., 2013) . in drosophila cells it has been shown that the wiskott-aldrich syndrome protein and scar homolog (wash) plays a connecting role between integrin recycling and the efficiency of phagocytic and autophagic clearance (nagel et al., 2017) . however, molecular details about how this connection is brought about are unclear. sequence analysis of integrin β3 tails show a potential atg-interacting lir motif, similarly to the ace2 tail. low throughput phosphorylation assays have determined that both tyr773 and tyr785 (required for the functional ptb npxy motif) are in fact phosphorylated in vivo. however, such assays have also determined additional phosphorylation sites in the β3 tail, thr777, ser778, thr779 and thr784. these phosphorylations aren't connected to the npxy motif switches in any known way. however, the three phosphorylations between residues 777-779 and the following sequence region matches the lir autophagy motif also found in the ace2 tail. while the current motif definition does not exactly fit the β1 tail, there is also low throughput phosphorylation assay data (wennerberg et al., 1998) for the existence of these phosphorylations in the corresponding residues, hinting at the possibility of the presence of a slightly modified motif. for both β1 and β3 tails, the phosphorylation provides the negative charge required n-terminal of the fxxixy lir motif hydrophobic core. phosphopeptides spanning the candidate region should reveal whether the lir motif-like region is a functional atg-binding site in integrin β1, and also whether the multiple phosphorylations act as a rheostat, modulating the affinity of the interaction. the motif found in integrin β3 is also present in integrin β2, and the motif candidate identified in integrin β1 is also present in integrin β6. bringing together the candidate slims identified in the integrin β and ace2 tails potentially strengthens the functional links between them, and provides an emergent picture of slim-driven cooperative switches driving viral attachment, entry and replication (figure 7) . following attachment of spike to the receptors, the two npxy motifs in the integrin β subunit could act cooperatively with the apoptb and yxxphi motifs in ace2 as sorting signals that mediate the internalization of viral particles into endosomes. the presence of several endocytic motifs in close proximity would strengthen the interaction with the endocytosis apparatus creating a high-avidity environment for recruitment of rme components (bonifacino and traub, 2003) . during this time, the phosphorylated integrin npxy motifs would also reinforce viral attachment through inside out signalling, stabilizing the integrin ectodomain in the open, high affinity ligand-binding conformation. as discussed previously, rme also involves the recruitment of adaptor molecules that activate rearrangements of the actin cytoskeleton required for the internalization of the endocytic vesicle. at this stage, the npy and nck sh2 motifs in ace2 would recruit several molecules that mediate actin polymerization signalling, prominently i-bar containing proteins irsp53 and irtks, and the wasp-arp2/3 complex. while most of this actin signalling would serve to allow viral entry, additional actin recruitment processes could occur following viral fusion, such as that initiated by the interaction between the spike protein and ezrin. finally, at later stages of infection, both integrins and ace2 might remain attached to virus-associated dmvs and other replication-competent membranes where the rtc assembles. at this stage, ace2 and integrins might cooperatively mediate the recruitment of autophagy components such as lc3, through the lir motifs located in the cytosolic tails of both molecules. elements shown in one of the monomers of a homotrimer (spike) or homodimer (ace2) are also present in the other proteins forming that complex. lines below motif boxes represent each of the overlapping motifs in that specific region. arrows indicate the related cellular process and the protein known to interact with their respective motif is indicated in parenthesis. phosphorylation sites are shown as red circles with the respective sequence position indicated. for the integrin β tail, the ptb/apoptb phospho switch is depicted as two separate versions of the same motif region, and the subscripts represent the motif order in the sequence. the colour code is as follows: cleavage sites (brown), for motifs: apoptb/ptb (orange), endocytic sorting signal (purple), i-bar binding (red), lir (blue), midas (yellow), nck (green), pbm (magenta), rgd (light red). slims mediating interactions are marked with coloured aquares, protease cleavage sites are marked with hexagons, structural motifs are marked with ovals. motifs marked with † are experimentally validated. the analysis of candidate slims in ace2 and integrins suggests that sars-cov-2 hijacks both receptors, co-opting their slims to drive viral attachment, entry and replication. this creates an opportunity for drugging these interactions, or the processes they control through host directed therapies (hdts), to prevent viral entry. a list of potentially useful drugs is presented in table 2 , together with chembl accessions (mendez et al., 2019) . the sequence rgd is used by a large number of viruses for cell attachment, via integrins (hussein et al., 2015) . rgd mimics have been developed as inhibitors of integrin-extracellular matrix protein interaction for a variety of diseases. a cyclic rgd peptide (c-rgdfv, cilengitide) has been developed clinically for glioblastoma treatment and other cancers. it proved safe, but did not enhance the survival benefit (stupp et al., 2014) . sars-cov-2 has a unique rgd sequence in the ace2 binding region of its spike protein. it has been speculated that integrins may have a potential role for infectivity (sigrist et al., 2020) . therefore, integrin inhibitors like rgd mimetics might be able to block the rgd binding site(s) on target cells and block the attachment of the virus. another application that has been suggested is bacterial sepsis (sepsis is also a dreaded complication in covid-19 patients), and experimental evidence in animals is available (garciarena et al., 2017) . cilengitide is relatively specific for integrin αvβ3 and also active on αvβ5, αvβ6. the antibody abituzumab (aka di 17e6) is a pan-av antibody, i.e. also active against other αv integrins, and may consequently be better suited for blocking virus entry. it has been clinically tested in several cancer indications (élez et al., 2015; hussain et al., 2016) . cilengitide and abituzumab are made available for in vitro testing in the context of the sars-cov-2 pandemic by the company who developed it initially (contact: compound_donation@merckgroup.com). as discussed above, tyrosine kinase mediated phosphorylation plays an important role in virus entry and maturation, and several tyrosine kinase inhibitors have been developed in the clinic and show effects on viral infection. for example, saracatinib, a src and abl inhibitor which has completed several clinical trials, including some cancers, inhibited replication of different coronaviruses including mers-cov, sars-cov and hcov-229e in cell culture infection experiments (shin et al., 2018) . after internalization and endosomal trafficking, imatinib, an abl inhibitor, prevented fusion of sars-cov and mers-cov virions at the endosomal membrane in infected cell culture experiments (coleman et al., 2016) . using the avian model virus, ibv, imatinib and two other abl inhibitors, gnf2 and gnf5, prevented the fusion of the spike protein to the membrane of the target cell as well as cell-cell fusion and syncytia formation (sisk et al., 2018) . more recently, tyrphostin a9, a platelet-derived growth factor receptor (pdgfr) tyrosine kinase inhibitor came out from a high-throughput screening using cytopathic effect as readout, it also showed in vitro inhibitory capacity to transmissible gastroenteritis virus (tgev), an alpha coronavirus that infects pigs (dong et al., 2020) . the authors also showed that tyrphostin a9 has a broad spectrum, being active against three other tested coronaviruses: mhv in l929 cells, porcine epidemic diarrhea virus in vero cells and feline infectious peritonitis virus in ccl-94 cells. the mode of action was found to be through p38 mapk, at the post-adsorption stage. as fak has been implicated in viral entry for other viruses including influenza a (elbahesh et al., 2014) , experimental drugs targeting fak, including some in clinical trials (de jonge et al., 2019) , can be considered for studying the potential spike-induced integrin signalling. currently, 39 tyrosine kinase inhibitors are approved by the fda: eleven target non-receptor protein-tyrosine kinases and 28 inhibit receptor protein-tyrosine kinases (roskoski, 2020) . consequently, tyrosine kinase inhibitors may be good candidates to test for their effect on sars-cov-2. a number of protease inhibitors are currently discussed for sars-cov-2 treatment. serine protease inhibitor camostat mesylate is active against tmprss2 and blocks cell entry (hoffmann et al., 2020) . only the spike protein of sars-cov-2 contains a furin cleavage sequence (prrars|v). consequently, furin convertase inhibitors are considered as antiviral agents (shiryaev et al., 2007) . many viruses enter the cell via endocytosis, and a number of candidate slims relevant for sars-cov-2 infection are related to endocytosis (see above). chlorpromazine, an antipsychotic (via dopamine d2 antagonism) dating from the 1950s, is also a potent endocytosis inhibitor (which may explain some of its marked side effects, which can include low white blood cell levels). it promotes the assembly of adaptor proteins and clathrin on endosomal membranes thus depleting them from the plasma membrane, leading to a block in clathrin-mediated endocytosis (wang et al., 1993) . the potential use of endocytosis inhibitors such as amiodarone (stadler et al., 2008) and chlorpromazine in coronavirus infection is further discussed here . the situation with targeting autophagy seems unclear. autophagy activators might help the cell to consume incoming virus -or speed up the establishment of the viral replication complexes and accelerate disease. autophagy inhibitors might work in later stages of infection to dampen viral production, but this will depend on whether autophagy is active at the time or if the constituent components have been captured and effectively shut down. several inhibitors/activators have been reported which can target autophagy and multiple auxiliary signals feeding into the process of autophagy (table 2 ). one such axis is via the mtorc1 complex. active mtorc1 keeps the autophagy process inhibited by phosphorylating the ulk complex which is a key regulator in autophagy. inhibition of mtorc1 activates autophagy. multiple fda approved mtor inhibitors are known and include rapamycin and everolimus. rapamycin has been shown to be effective in cell culture for countering mers-cov infection and might assist in tackling sars-cov-2 infection (kindrachuk et al., 2015) although the stage of infection might be crucial for the desired outcome. simvastatin is another drug which is known to upregulate autophagy via the mtor pathway (wei et al., 2013) . simvastatin has also been reported to alleviate airway inflammation in a mouse asthma model (gu et al., 2017) . another autophagy modulator is niclosamide which regulates autophagy by targeting the autophagy regulator beclin1 via skp2 e3-ligase in mers-cov infection. in this case, reduced beclin1 levels lead to blocking fusion of autophagosomes and lysosomes and hence the virus protects itself in the host (gassen et al., 2019) . overall, inhibiting skp2 by niclosamide relieves beclin1, allowing autophagosome-lysosome fusion and resumption of autophagy to reduce the mers-cov production. in addition, niclosamide valinomycin has been shown to target sars-cov in cell cultures as well (wu et al., 2004) . a recent proteomic study expressed 26 tagged sars-cov-2 proteins individually to create a viral-human protein-protein interaction map (gordon et al., 2020) . as a result, 69 compounds, some being fda approved, are candidates for drug repurposing. the spike protein interacted with the golga7-zdhhc5 acyl-transferase complex, possibly for palmitoylation on spike's cytosolic tail. spike did not pull down ace2 or any cell surface receptor protein, but these experiments are not an assay for viral entry, and therefore many protein interactions related to the viral entry mechanism are likely missing from these data. therefore our observations of slim candidates in the viral attachment, entry and replication system reflect additional areas of the cell where drug repurposing for hostdirected therapy might be explored. although tyrosine kinase inhibitors have frequently been shown to dampen pathogen invasion and disease progression in cell culture, there has been little effort to move these findings into the clinic (sámano-sánchez and . because of their widespread use in cancer, the safety profiles of tyrosine kinase inhibitors are well known and we wonder whether this might be a neglected opportunity. drugging the cell to cure the pathogen using hdts is unlikely to fully remove a virus. this would also be undesirable, because the immune system must mount a defence in order to prevent viral reinfection. rather, dampening viral load during viral invasion or replication should be the target, to give the host defences time to respond. it is well known that drugs like tamiflu that slows influenza exit, and therefore entry into uninfected cells, can only have a strong effect when taken prophylactically or early in infection (bassetti et al., 2019) . depending on the importance of integrins in sars-cov-2 lung cell entry, reducing viral entry is a possible role for cilengitide or other molecules that hamper integrin or ace2 binding. an endocytosis inhibitor might play a similar role and is independent of receptor type. however, for any such inhibitor that passes the blood-brain barrier, effects on mood and other brain operations are an inevitable side effect: even so, the endocytosis inhibitor chlorpromazine is a widely used drug with a well-known safety profile (solmi et al., 2017) . due to the presence of the cell attachment motif rgd in sars-cov-2, integrin inhibitors seem worthwhile to explore further. cilengitide, a relatively selective integrin αvβ3 and αvβ6 inhibitor (a cyclic peptide that proved safe in patients, but failed to show a survival benefit in glioblastoma (stupp et al., 2014) ) might be useful in two phases: it could block virus attachment to target cells, and it has also been proposed as a potential treatment in sepsis (garciarena et al., 2017) . sepsis was the most frequently observed complication among covid-19 patients in wuhan (f. . another potential application of integrin inhibitors, especially integrin αvβ6, would be lung fibrosis -patients on respirators tend to develop lung fibrosis, and show increased αvβ6 levels (horan et al., 2008) . the antibody abituzumab (aka di 17e6) is a pan-αv antibody with high potency on αvβ6, i.e. also active against several αv integrins, and may consequently be better suited for blocking virus entry, or may be suitable for lung fibrosis or sepsis protection. it has been clinically tested in several cancer indications (élez et al., 2015; hussain et al., 2016) , and proved safe, but did not achieve a survival benefit in cancer. whether the enzymatic function of ace2 has a role in sars-cov-2 infection is unknown. however, it would be readily testable with available ace2 inhibitors, like captopril, on the market since the 1970s (enalapril would not be suited for in vitro testing, as it needs to be activated in the liver to become the active ingredient). it might be productive to test for synergy between molecules that block viral binding to ace2 and molecules that block binding to integrins. we have presented evidence at the sequence level for slims in ace2 and β integrins with the potential to function in viral attachment, entry and replication for sars-cov-2. we identified several candidate molecular links and testable hypotheses that might help uncover the (still poorly understood) mechanisms of sars-cov-2 entry and replication. because these motifs belong to host proteins acting as viral receptors, they are not revealed by virus-centred proteomic assays. most of these putative motifs lack direct experimental evidence. that they may well be functional, however, is indicated by sequence conservation, in some cases for hundreds of millions of years. in addition, these motifs are in appropriate cellular contexts to interact with their respective partner proteins. experimental validation will yield insights into receptor-mediated endocytosis for sars-cov-2 virus and, in addition, for the role of ace2 in the normal cell, where it surely has much more functionality than being an angiotensin converting enzyme. overall, the collection of candidate motifs in this system suggests that a range of host directed therapies might be explored including rgd inhibition, tyrosine kinase inhibition, endocytosis inhibition and autophagy inhibition and/or activation. or interpro (mitchell et al., 2019) , where applicable motif candidates 2 xany residue, p 5 pc: proprotein convertases 3 '*' marks cleavage points for protease-recognition motifs 6 not a slim but a structural motif i-bar domains, irsp53 and filopodium formation atg8 family proteins act as scaffolds for assembly of the ulk complex: sequence requirements for lc3 -interacting region (lir) motifs integrin cytoplasmic domain-associated protein-1 (icap-1) promotes migration of myoblasts and affects focal adhesions the tail of integrin activation adaptor shca protein binds tyrosine kinase tie2 receptor and regulates migration and sprouting but not survival of endothelial cells neuraminidase inhibitors as a strategy for influenza treatment: pros, cons and future perspectives the lir motif -crucial for selective autophagy phosphorylated ydxv motifs and nck sh2/sh3 adaptors act cooperatively to induce actin reorganization tyrosine phosphorylation of integrin beta3 regulates kindlin-2 binding and 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basis for endosomal trafficking of diverse transmembrane cargos by px-ferm proteins cell regulation: determined to signal discrete cooperation experimental detection of short regulatory motifs in eukar yotic proteins: tips for good practice as well as for bad pdzk1: i. a major scaffolder in brush borders of proximal tubular cells systems biology the cell biology of receptor-mediated virus entry simvastatin alleviates airway inflammation and remodelling through up-regulation of autophagy in mouse models of asthma virion incorporation of integrin α4β7 facilitates hiv-1 infection and intestinal homing membrane remodeling in clathrin-mediated endocytosis kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes tmprss2 and adam17 cleave ace2 differentially and only proteolysis by tmprss2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor partial inhibition of integrin alpha(v)beta6 prevents pulmonary fibrosis without exacerbating inflammation. a m 15 years of phosphositeplus®: integrating post-translationally modified sites, disease variants and isoforms interaction of plasma membrane fibronectin receptor with talin--a transmembrane linkage fak and paxillin dynamics at focal adhesions in the protrusions of migrating cells defining the specificity space of the human src homology 2 domain sars coronavirus, but not human coronavirus nl63, utilizes cathepsin l to infect ace2-expressing cells differential effect on bone lesions of targeting integrins: randomized phase ii trial of abituzumab in patients with metastatic castration -resistant prostate cancer beyond rgd: virus interactions with integrins tmprss2 contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection mechanisms of clathrin-mediated endocytosis loops govern sh2 domain specificity by controlling access to binding pockets a comprehensive evaluation of the activity and selectivity profile of ligands for rgd-binding the origin and evolution of mammals, oxford biology antiviral potential of erk/mapk and pi3k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis basement membrane assembly of the integrin α8β1 ligand nephronectin requires fraser syndrome-associated proteins the kindler syndrome protein is regulated by transforming growth factor-beta and involved in integrin-mediated adhesion viruses and autophagy elm-the eukaryotic linear motif resource in 2020 crystal structure of the 2019-ncov spike receptor-binding domain bound with the ace2 receptor crystal structure of the a domain from the alpha subunit of integrin cr3 (cd11b/cd18) mechanisms that regulate adaptor binding to beta-integrin cytoplasmic tails lack of integrin alpha-chain endoproteolytic cleavage in furin-deficient human colon adenocarcinoma cells lovo angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus endoproteolytic processing of integrin pro-alpha subunits involves the redundant function of furin and proprotein convertase (pc) 5a, but not paired basic amino acid converting enzyme (pace) 4, pc5b or pc7 sh2 domains recognize contextual peptide sequence information to determine selectivity structural insight into the mechanisms of targeting and signaling of focal adhesion kinase structural mechanism of integrin inactivation by filamin the transmembrane domain of the prohormone convertase pc3: a key motif for targeting to the regulated secretory pathway the integrity of the rrgdl sequence of the proprotein convertase pc1 is critical for its zymogen and c-terminal processing and for its cellular trafficking fyn mediates high glucose-induced actin cytoskeleton reorganization of podocytes via promoting rock activation in vitro kindlin-2 (mig-2): a 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system for exploratory research and analysis microfibrillar-associated protein 4 modulates airway smooth muscle cell phenotype in experimental asthma expression of integrin cell adhesion receptors during human airway epithelial repair in vivo coronavirus replication complex formation utilizes components of cellular autophagy irsp53 senses negative membrane curvature and phase separates along membrane tubules use of peptide arrays for identification and characterization of lir motifs interaction of pregnancy-specific glycoprotein 1 with integrin α5β1 is a modulator of extravillous trophoblast functions identification of an interaction between the m-band protein skelemin and beta-integrin subunits. colocalization of a skelemin-like protein with beta1-and beta3-integrins in nonmuscle cells analysis of fyn function in hemostasis and alphaiibbeta3-integrin signaling coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication 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required for the maintenance of adult skeletal bone mass saracatinib inhibits middle east respiratory syndrome-coronavirus replication in vitro tyrosine phosphorylation c ontrols internalization of ctla-4 by regulating its interaction with clathrin-associated adaptor complex ap-2 targeting host cell furin proprotein convertases as a therapeutic strategy against bacterial toxins and viral pathogens a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry a potential role for integrins in host cell entry by sars-cov-2 inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry vascular expression of the alpha(v)beta(3)-integrin in lung and other organs coronavirus s protein-induced fusion is blocked prior to hemifusion by abl kinase inhibitors safety, tolerability, and risks associated with first-and second-generation antipsychotics: a state-of-the-art clinical review identification of the multivalent pdz protein pdzk1 as a binding partner of sodium-coupled monocarboxylate transporter smct1 (slc5a8) and smct2 (slc5a12) amiodarone alters late endosomes and inhibits sars coronavirus infection at a post-endosomal level determination of n-and c-terminal borders of the transmembrane domain of integrin subunits cell integrins: commonly used receptors for diverse viral pathogens cilengitide combined with standard treatment for patients with newly diagnosed glioblastoma with methylated mgmt promoter (centric eortc 26071-22072 study): a multicentre, randomised, open-label string v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets talin binding to integrin beta tails: a final common step in integrin activation src-family tyrosine kinase fyn phosphorylates phosphatidylinositol 3-kinase enhancer-activating akt, preventing its apoptotic cleavage and promoting cell survival integrins as therapeutic targets for respiratory diseases the sh2 domain interaction landscape mechanisms of integrin-mediated virus attachment and internalization process ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partner autophagy modulates cell migration and β1 integrin membrane recycling ß3 integrin modulates transforming growth factor beta induced (tgfbi) func tion and paclitaxel response in ovarian cancer cells development of a 5-plex silac method tuned for the quantitation of tyrosine phosphorylation dynamics proteomics. tissue-based map of the human proteome structural and evolutionary division of phosphotyrosine binding (ptb) domains the switches.elm resource: a compendium of conditional regulatory interaction interfaces motif switches: decision-making in cell regulation determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling the interconnections between autophagy and integrin-mediated cell adhesion structure, function, and antigenicity of the sars-cov-2 spike glycoprotein an interaction between insulin-like growth factor-binding protein 2 (igfbp2) and integrin alpha5 is essential for igfbp2-induced cell mobility mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation jalview version 2--a multiple sequence alignment editor and analysis workbench enhancement of autophagy by simvastatin through inhibition of rac1-mtor signaling pathway in coronary arterial myocytes mutational analysis of the potential phosphorylation sites i n the cytoplasmic domain of integrin beta1a. requirement for threonines 788-789 in receptor activation cryo-em structure of the 2019-ncov spike in the prefusion conformation small molecules targeting severe acute respiratory syndrome human coronavirus crystal structure of the complete integrin alphavbeta3 ectodomain plus an alpha/beta transmembrane fragment structural basis for the recognition of sars-cov-2 by full-length human ace2 targeting the endocytic pathway and autophagy process as a novel therapeutic strategy in covid-19 the regulation of integrin function by divalent cations i-bar domain proteins: linking actin and plasma membrane dynamics clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study structure and ligand recognition of the phosphotyrosine binding domain of shc a pneumonia outbreak associated with a new coronavirus of probable bat origin single-cell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to 2019-ncov infection bm has received funding from the european union's horizon 2020 research and innovation programme under the marie skłodowska-curie grant agreement no. 842490 (mimic). jč is supported by the european union's horizon 2020 research and innovation programme under the marie skłodowska-curie grant agreement no. 675341 (pdznet). em-p is a phd student of conicet, argentina. ra is supported by bmbf-funded heidelberg center for human bioinformatics (hd-hub) within the german network for bioinformatics infrastructure (de.nbi #031a537b) and elixir germany. lbc is a national research council investigator (conicet, argentina). the work was supported by agencia nacional de promoción científica y tecnológica (pict 2017(pict -1924 grant to lbc. this paper is part of a project that has received funding from the european union's horizon 2020 research and innovation programme under the marie skłodowska-curie grant agreement no. 778247 (idpfun) to lbc and tjg. idpfun also funded em-p's placement at embl. key: cord-150183-zzzyewjb authors: phillips, j. c. title: synchronized attachment and the darwinian evolution of coronaviruses cov-1 and cov-2 date: 2020-08-27 journal: nan doi: nan sha: doc_id: 150183 cord_uid: zzzyewjb cov2019 has evolved to be much more dangerous than cov2003. experiments suggest that structural rearrangements dramatically enhance cov2019 activity. we identify a new first stage of infection which precedes structural rearrangements by using biomolecular evolutionary theory to identify sequence differences enhancing viral attachment rates. we find a small cluster of mutations which show that cov-2 has a new feature that promotes much stronger viral attachment and enhances contagiousness. the extremely dangerous dynamics of human coronavirus infection is a dramatic example of evolutionary approach of self-organized networks to criticality. it may favor a very successful vaccine. the identified mutations can be used to test the present theory experimentally. the standard tool for comparing two or more proteins is blast, which compares different protein sequences site-by-site. it is not enough to profile ψ(aa) site by site for two proteins, as this produces hundreds of oscillations, with no clear differences between the two proteins. instead we plot ψ(aa,w), where w is a rectangular box of length w = 35 over which ψ(aa) is averaged . this w value is chosen to maximize the hydropathic shape differences between cov-1 and cov-2, as measured by their variance ratio, which is dominated by extrema [11] . smoothing the profile over w enhances resolution by reducing the number of extrema to the level which corresponds to critical domain motion differences. choosing the best value of w to enhance resolution of evolutionary differences is similar to adjusting the focal plane of a microscope. our main result is shown in fig. 1 , which displays the 400-800 central region of the spikes. this region is part of the n-terminal domain (s1) responsible for cellular attachment [14] . fig. 1 shows the ψ(aa,w) hydropathic profiles of for cov-1 and cov-2, using the ψ(aa) values of mz [4] . while the two ψ(aa, 35) profiles are similar, there are important differences in their extrema. the maxima are most hydrophobic and are located in the globular interior, while the minima are most hydrophilic and are located on the globular surface. the two cleavage sites s1/s2 and s 2' [2] of cov-1 have moved lower (hydrophilically, further outside) in cov-2 ( fig. 1) , consistent with the very accurate mz scale. when a cleavage segment is further outside, there is more space for cleavage and reassembly, which will occur more rapidly. the insertion prra in cov-2 was identified [2] with blast (w = 1) as unique to cov-2, but with blast alone one cannot show that this change has made cov-2 more dangerous. a characteristic feature of second order phase transitions is that they break what physicists often call a "hidden" symmetry, here the key symmetry of extrema [15] . proteins are typically composed of domains of ~ 100 amino acids, and these domains can rotate from the resting state to the functional state, and then return reversibly to the resting state. this phase transition requires a balance between stability and flexibility [16, 17] . to maximize the reversibility and extend protein life one can synchronize this domain motion by leveling the pivotal hydrophobic (inside) extrema forming level sets [10, 11, [18] [19] [20] [21] . viruses must act rapidly before being destroyed by antibodies, and they could do this through synchronized motion differently, by leveling their hydrophilic (outside) extrema. as shown in fig. 1 , such a leveling of minima 1-3 occurs in cov-2, while it is absent from cov-1. the change in minimum 2 is especially striking: it is caused by a cluster of four critical mutations from cov-1 to cov-2. these are (cov-1 site numbering from uniprot p59594): 546gln to leu; 556 and 561ser to ala; and 568ser to leu. the differences associated with each of these mutations are hydropathically large (~50-100 in the mz scale [4] ; all 20 amino acids span a range from most hydrophilic to most hydrophobic of 170). how important is the fractal mz scale? in fig. 2 the mz panoramic profiles of cov-1 and cov-2 are compared across the entire 1255 amino acid spike. we see immediately a strong hydrophobic peak above 1200, which is responsible for anchoring the spike to the central cushion; it is almost unchanged from cov-1 to cov-2. the central hydrophilic level set, absent from cov-1 and present in cov-2, is our main result ( there is an excellent review of the principles of self-organized criticality in living matter [3] . these can be compared to relaxation of homogeneous glass alloys, whose composition has been adjusted to bring the glass network to a critical point. there the glass (commercial gorilla glass) is strong and flexiblenot brittle [21] . another broad and deep review includes fractals and their many implications for quantifying protein dynamic criticality [22] . in the genomic age abstract tools have many applications to analyzing the evolution of protein functions. some readers may be interested in the connections between hydropathic scaling theory of proteins and the more general synchronization of complex networks. the analogy is closest for networks that are scale-free because they are near a second-order phase transition. social networks contain many such examples, usually described by one or two fractals [23]. generally it is found that synchronizability is robust against random removal of nodes, but it is fragile to specific removal of the most highly connected nodes. in proteins the most highly connected nodes are hydrophobic extrema (deeply interior), where the number of van der waals contacts is largest. in coronavirus spikes, it is the hydrophilic extrema that are most exposed to water and most likely to attach to protein targets. thus the symmetry of extrema is broken between hydrophobic (proteins) and hydrophilic (cov-2). one can also regard darwinian selection as a kind of percolation [24] . further extensions to multiphase hydrodynamic level sets require a special theory [15] . our primary focus here has been on enhanced attachment caused by the synchronization of covthese grouped sites are all close to the hydrophilic minimum 1 in fig. 1 (mz scale) and fig. 3 (kd scale). fragmental binding is a feature common to both scales, and thus not sensitive to whether the phase transition is first-or second-order. instead, by synchronizing minima 2 and 3 with minimum 1, cov-2 is able to greatly increase its attachment to ace2 (angiotensinconverting enzyme 2) and probably other receptors as well; synchronization is specifically sensitive to allosteric interactions and is definitely a second-order effect. in conclusion, the answer to the question posed in [1] is that, compared to cov-1, cov-2 has moved closer to its functional critical point [3] . cov-2 has an added synchronized feature in its central attachment region that contributes to its extreme contagiousness in an asymptomatic phase [26] . the attachment of the s1 region precedes cleavage [2] , and it is likely that both mechanisms enhance the infectiveness of cov-2. an early estimate of the basic contagious reproductive number (r0) of coronavirus 2019 was 2.2-2.7, but this was later revised to 5.7 [27]. by comparison, the numbers for h1n1 flu (1918, 1.8; 2009, 1.5) were much smaller [28] the uncontrolled r0 for cov-1 was 3.6, and this dropped to 0.7 after the rapid implementation of control measures [29] . the new feature explains the unexpected yet often asymptomatic behavior of early stages of cov-2 infection, when the new feature is acting alone. it should be possible to desynchronize this added attachment feature and reduce r0 by attaching a small molecule in the region 530-575. in any case, the "hidden" symmetry of the cov-2 sequence should be of interest to both biologists and physicists. the effects of the four critical mutations on cov contagiousness could be tested on mice. if the predictions of their importance should prove to be correct, then the general principle of darwinian evolution of proteins would be confirmed. the level hydrophilic extrema of the spikes of cov-2 may be unique among viruses. these level extrema bring cov-2 contagion very close to a critical point. there very small differences in dna can cause large fluctuations in infection levels, not only between individuals, but also even in neighboring countries [30] . the quasi-linear spike morphology, with its large surface/volume ratio, is ideal for strong protein-water interactions, explaining [1] . the oxford vaccine is based on the cov-2 spike protein hydrophobically anchored to an adenovirus vaccine vector [31] [32] [33] . it seems likely that this vaccine will be more effective for cov-2 than for cov-1 because the synchronized attachment mechanism is stronger for cov-2 and thus more easily disrupted by induced antibodies. thus vaccines based on the entire spike are expected to be more effective than vaccines based on only part of the spike [34] . the most dangerous type of flu virus is h 3 n 2 , and it does not exhibit level sets [35] . flu viruses mutate significantly annually, while cov-2 has shown only a few mutations [36] . the spike stability against mutations is consistent with maximized attachment. there are many examples of scale-free networks. readers unfamiliar with the general properties of scale-free networks, such as (often only 1 or 2) fractals, preferential attachment, and synchronization, may find early reviews interesting [37, 38] . the sequences used here for cov-1 and cov-2 are the ones used in [2] , that is, aap13441.1 and yp_009724390.1. many other cov sequences are aligned (w = 1) in their supp. table [2] . similar w = 1 sequence alignments have been reported by others [39, 40] , but the genetic origin of cov-2 remains mysterious [1, 40] . historically it has long been the case that the amount of information that could be obtained from hydropathic scales was limited, because the fractals describing second-order phase transitions were not known. now that we are in the genomic age, with a very large sequence data base available for many proteins and many species, the discovery of these 20 fractals [5, 13] opens a new biophysics field of accurate thermodynamic analysis of small but medically important evolutionary differences. the methods used here on cov-1 and cov-2 do not require elaborate calculation to obtain new results. the hydropathic scaling methods are easily implemented on a spread sheet, but are still not routine, and vary between proteins. the modular nature of protein domains responsible for protein assembly is well known [41, 42] . here we maximized small and otherwise mysterious evolutionary differences by using hydropathic scaling to optimize w at 35. this enables us to focus on domain-scale motion responsible for stronger attachment in cov-2. evidence for large-scale motion has been found in the evolution of many proteins [4] [5] [6] [7] 9] , including molecular motors [11] , while it is not accessible to traditional w = 1 blast-based alignment methods [2, [43] [44] [45] . from detailed genomic studies it has been suggested that "a more comprehensive theory of molecular evolution must be sought" [46] . readers wishing to explore these novel scaling methods can obtain copies of sample excel spread sheets from the author. many physicists have long suspected that proteins must function near a critical point very near thermodynamic equilibrium [3] . however, to implement this idea in practice with protein sequences, one needs to compress the existing vast array of three-dimensional structural data into a one-dimensional form. if you are one in a million, namely among the small community of coauthors of the 500+ references of [3] , what comes next may not surprise you. as discussed above, this near-miracle compression has been achieved in the 21 st century by two brazilian physicists [13] , located far from the main stream. [13] described the discovery of 20 (not just one) universal amino-acid specific fractals in the solvent accessible surface areas (sasa) of proteins. this amazing 21 st century discovery was achievable only for proteins. it has made possible the quantitative analysis of darwinian evolution of many different proteins [4] [5] [6] [7] [8] [9] [10] [11] , which brings us to the present application. inserted at 681 in cov-1 (the s1/s2 cleavage interface) [2] , has an average mz hydropathicity 108.5. this lowers ψ(aa,35) to 140.9 at the 3 minimum, aligning it with ~140 minima 1 and 2. the three minima span ~ 250 amino acids sites, which makes their water-driven synchronization for cov-2, but not cov-1, outside the range of most simulation or modeling methods. why the coronavirus has been so successful structure, function, and antigenicity of the sars-cov-2 spike glycoprotein criticality and dynamical scaling in living systems scaling and self-organized criticality in proteins: lysozyme c 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impacts of control measures virus batters some areas, why does it spare others? oxford covid-19 vaccine begins human trial stage future prospects for the development of cost-effective adenovirus vaccines atomic structure of human adenovirus by cryo-em reveals interactions among protein networks subunit vaccines against emerging pathogenic human coronaviruses prediction (early recognition) of emerging flu strain clusters controlling the sars-cov-2 outbreak, insights from large scale whole genome sequences generated across the world collective dynamics of 'small-world' networks statistical mechanics of complex networks the proximal origin of sars-cov-2 a genomic perspective on the origin and emergence of sars-cov-2 assembly of cell regulatory systems through protein interaction domains arrangements in the modular evolution of proteins immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of 2019-ncov novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov-2 compared to sars-cov phylogenetic analysis and structural modeling of sars-cov-2 spike protein reveals an evolutionary distinct and proteolytically sensitive activation loop the neutral theory in light of natural selection with the mz scale panoramic spike profiles of cov-1 and cov-2 reveal a set of hydrophilic level extrema in cov-2, but not in cov-1. the new level set was identified with a even on the panoramic picture, it is clear that with the kd scale the three minima that were aligned in cov-2 are not level. this is shown in more detail in fig key: cord-259412-l8uta7du authors: mattossovich, rosanna; merlo, rosa; miggiano, riccardo; valenti, anna; perugino, giuseppe title: o(6)-alkylguanine-dna alkyltransferases in microbes living on the edge: from stability to applicability date: 2020-04-20 journal: int j mol sci doi: 10.3390/ijms21082878 sha: doc_id: 259412 cord_uid: l8uta7du the genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. alkylating agents cause dna damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. a class of enzymes known as agts (alkylguanine-dna-alkyltransferases) protects the dna from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in o(6)-position. the peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. in modern biotechnology, engineered variants of agts are developed to be used as protein tags for the attachment of chemical ligands. in the last decade, research on agts from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. this review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications. monofunctional alkylating agents, a class of mutagenic and carcinogenic agents present in the environment, induce dna alkylation in several positions including guanine at o 6 (o 6 -mg; 6% of adducts formed), the n 7 of guanine (n 7 -mg; 70%), and the n 3 of adenine (n 3 -ma; 9%) [1] . alkylation of guanine (o 6 -ag) is a cytotoxic lesion, although the specific mechanism of this cytotoxicity is not yet fully understood. it was proposed that the toxic effect occurs after dna replication, because the o 6 -ag incorrectly base-pairs with thymine generating a transition from g:c to a:t [2] . the mutations caused by o 6 -mg that occur at the time of replication are recognized by the post-replication mismatch repair system with potential harmful implications for cell viability. apart from conventional dna repair pathways as mismatch excision repair (mmr), nucleotide excision repair (ner), base excision repair (ber), alkylated-dna protein alkyl-transferases (called o 6 -alkyl-guanine-dna-alkyl-transferase (agt or ogt) or o 6 -methyl-guanine-dna-alkyl-transferase (mgmt); ec: 2.1.1.63) perform the direct repair of alkylation damage in dna [3, 4] . they represent the major factor in counteracting the effects of alkylating agents that form such adducts [4] . these are small enzymes (17) (18) (19) (20) (21) (22) that are widely present in organisms of the three kingdoms (bacteria, archaea, eukaryotes) but apparently absent from plants, schizosaccharomyces pombe, thermus thermophilus, and deinococcus radiodurans. the reaction mechanism of agts is based on the recognition of the damaged nucleobase on dna [5] , followed by a one-step sn 2 -like mechanism, in which the alkyl group of the damaged guanine is irreversibly transferred to a cysteine residue in its active site [5] [6] [7] [8] (figure 1 , blue path). in the c-terminal domain, a helix-turn-helix motif (in light green, or in orange in the snap-tag) is responsible of the dna-binding activity. the peculiar irreversible reaction mechanism of these enzymes plays a pivotal role in the physiological dna repair (blue path), and it has important repercussions in cancer cell treatment (red path) and biotechnological applications (green path). atoms are coloured by the cpk colour convention. for these reasons, they are also called suicide or kamikaze proteins, showing a 1:1 stoichiometry of their reaction with the natural substrate. the disadvantage of this elegant catalysis is that, upon alkylation, the protein is self-inactivated and destabilized, triggering its recognition by cellular systems to be degraded by the proteasome [8, 9] . alkylation damage to dna occurs in various living conditions, and for this reason the widespread presence of agt protects cells from killing by alkylating agents. however, human agt (hmgmt) is a double-edged sword-on the one hand, it protects healthy cells from these genotoxic and carcinogenic effects, but also counteracts alkylating agents-based chemotherapy by protecting cancer cells from the killing effect of these drugs [10, 11] . consequently, hmgmt has emerged as a crucial factor in anticancer therapies [12] -an inverse relationship has been discovered between the presence of hmgmt and the sensitivity of cells to the cytotoxic effects of alkylating agents, such as temozolomide (tmz), in different types of cancer cells, including prostate, breast, colon, and lung cancer cells [13] . the resistance to chemotherapy may be reduced by inhibition of these enzymes; as described before, after removing the lesion, the alkylated form of the protein is inactivated and enters the intracellular degradation pathways. hence, in order to counteract the action of hmgmt in chemotherapy regimens, a large number of studies aimed to the develop hmgmt inhibitors to be used in combination with alkylating agents. in view of this therapeutic relevance, much success has been obtained through the design of hmgmt pseudo-substrates, namely, the o 6 -benzylguanine (o 6 -bg) and the strong inactivator o 6 -[4-bromothenyl]-guanine (o 6 -btg, lomeguatrib) [13, 14] . these compounds mimic damaged guanine on dna and react with the protein by the covalent transfer of the alkyl adduct to the active site cysteine residue, thus irreversibly inactivating the enzyme (figure 1 , red path). therapeutically, o 6 -bg is not toxic on its own, but renders cancer cells 2 to 14 times more sensitive to alkylating agents' effects. the oligonucleotides containing several o 6 -bg are potent inhibitors and represent a valid alternative to the use of free modified guanines, thereby improving the activity of the alkylating chemotherapy drug in the treatment of some tumours [15] [16] [17] . the specific labelling of proteins with synthetic probes is an important advance for the study of protein function. to achieve this, the protein of interest is expressed in a fusion with additional genetically encoded polypeptides, called tags, which mediate the labelling. the first example of an autofluorescent tag was the aequorea victoria green fluorescent protein (gfp) allowing the in vivo localization of fusion proteins in cellular and molecular biology [18, 19] . among affinity tags, of particular importance are the poly(his)-tag, the chitin-binding protein, the maltose-binding protein [20] , the strep-tag [21] , and the glutathione-s-transferase (gst-tag) [22] , which allow fast and specific purification of proteins of interest from their crude biological source using affinity techniques. solubilization tags are especially useful to assist the proper folding of recombinant proteins expressed in chaperone-deficient species such as escherichia coli, avoiding protein precipitation and the use of alternative expression protocols [23, 24] -these include thioredoxin [25] and poly(nanp). however, all the tags listed above are limited by the fact that each of them can be used for one or only a few applications. the need therefore emerged to develop a universal tag that could widely cover several applications. in 2003, the group headed by kai johnsson pioneered the use of an engineered hmgmt variant as a fusion protein for in vitro and in vivo biotechnology applications, which led then to its commercialization, namely, the snap-tag (new england biolabs) [26] [27] [28] [29] . they started from the knowledge that hmgmt tolerates the presence of groups conjugated to the pseudo-substrate o 6 -bg (o 6 -bg derivatives)-the unusual covalent bond with the benzyl moiety can therefore be exploited for "biotech" purposes ( figure 1 , green path). thanks to its small size, the engineered hmgmt (snap-tag) can be fused with other proteins of interest. the expression of the fusion protein inside the cells followed by incubation with opportune fluorescent derivatives leads to in vivo labelling of fusion proteins with the probe, which can be used for localization studies [26] . the same principle has also been used for the immobilization of tagged fusion proteins in vitro [30] . this offers a delicate condition for fixing and disposing in a better orientation of a wide range of proteins/enzymes on a surface. the snap-tag technology was successfully applied to surface plasmon resonance (spr) for the covalent immobilization of proteins of interest [31] . another interesting application of this protein-tag is the possibility to produce new antibody fragments (scfv-snap) to be employed in the spr analysis [32] . despite the need to use a specific substrate, snap-tag offers endless applications-the possibility to covalently link a desired chemical group (conjugated to the o 6 -bg) to a protein of interest (genetically fused to it) makes it decidedly advantageous, if compared to traditional protein tags currently in use. table 1 shows a brief comparison between some examples of protein tags and the snap-tag in several application fields. as for organisms living under mesophilic conditions, environmental and endogenous alkylating agents also attack the genome of thermophilic and hyper-thermophilic organisms. additionally, high temperatures accelerate the process of alkylation, leading to dna breaks [33] , because alkylating agents are chemically unstable at the physiological conditions of these organisms, and their collateral decomposition may worsen the formation of dna alkylation products [34] . thus, the presence of agts and methylpurine glycosylases in hyperthermophilic organisms implies that they are naturally exposed to endogenous methylating agents [34] , thus supporting the crucial role of agts [35, 36] . apart from some studies on archaea using cell-free extracts, a few examples of biochemical studies of agts from thermophilic sources include the enzymes from pyrococcus sp. kod1 [35] conducted by imanaka and co-workers from aquifex aeolicus and archaeoglobus fulgidus performed by the group of prof. pegg in 2003 ( figure 2 ) [34] . intriguingly, a. aeolicus agt, whose organism was identified as the most primitive bacterium, is closer to the mammalian agts than other bacterial homologues in terms of o 6 -bg sensitivity [34] . despite the different primary structures (figure 2a) , thermophilic enzymes show a typical agt protein architecture, consisting of two domains [37] : a highly conserved c-terminal domain (ctd), surprisingly superimposable for all available agt structures (figure 2b) , and a n-terminal domain (ntd), which is very different among agts and whose function is not well understood (likely involved in regulation, cooperative binding, and stability [6, 38, 39] ). the ctd contains the dna binding helix-turn-helix motif (hth); the asn hinge, which precedes the -v/ipchrvv/i-amino acid sequence containing the conserved catalytic cysteine (except the caenorhabditis elegans agt-2 that has the -pchpsequence [40, 41] ); and the active site loop, responsible for the substrate specificity. a comparative structural analysis performed on agt proteins whose structures are in the protein data bank revealed significant differences of the intrinsic structural features that have been considered to be relevant for thermostability, such as helix capping, intramolecular contacts (hydrogen bonds, ion-pairs), and solvent-accessible surface areas. helix capping plays a central role in the stability of α-helices, due to lack of intra-helical hydrogen bonds in the first and last turn [42, 43] , and its effect results in an overall structural stabilization of protein folding [44] . by inspecting the crystal structure of ssogt (pdb id: 4zye), considered here as the thermophilic reference agt protein, we verified that the five α-helices of the composing the protein tertiary structure are characterized by the presence of helix capping, this possibly increasing the thermal stability. in particular, the helix h1 at the ntd is stabilised by a peculiar double serine sequence (s40-s41) and a glutamic acid (e54) at its ctd, the latter is strictly conserved in all agts from thermophilic organisms (see figure 2a) . the hth motif, built on helices h3 and h4, is stabilized at the level of h3 by a highly conserved threonine residue (t89) as n-cap and a serine (s96), distinctive of ssogt, as c-cap. furthermore, helix h4 contains two serine-based capping among which the one placed at ntd (s100) is strictly conserved in all thermophilic agts and is followed by a proline (p101) that fits well in the first turn of the helix thanks to its own backbone conformation. finally, the helix h5 is protected by glutamic acid capping that is present in all the agts from different species. another feature contributing to thermal stability is the solvent-accessible surface area (sasa). indeed, the decrease of sasa and the increase of hydrophobic residues that are buried from the solvent have stabilizing principles for thermostable protein [45] . as described in table 2 , ssogt shows the smaller total sasa value, in line with its exceptional stability. on the contrary, ogt from mycobacterium tuberculosis [38, 46] has a higher value due to the peculiar conformation of both the active site loop and the c-terminal tail that are exposed to the bulk solvent and are less heat stable (table 2) . finally, by comparing hyperthermophilic agts with the orthologs from mesophilic organisms, in terms of atomic contacts between charged residues as well as intramolecular hydrogen bonds (table 2 ), significant differences emerged in the number of charged residues contacts. as expected for thermostable proteins [47] , ssogt, as well as the proteins from sulfurisphaera tokodaii and pyrococcus kodakaraensis, shows a larger number of electrostatic contacts, characterized by higher bond-dissociation energy, with respect to hydrogen bonds for which we did not detect significant differences among the analysed structures, apart from mgmt of p. kodakaraensis (pk-mgmt) [48] . although the number of h-bonds is approximately similar across the agts from different organisms, there should be differences in the position-related role of such bonds, supporting overall stability of thermophilic variants. with reference to pk-mgmt, hashimoto and co-workers detected the same number of ion-pairs between the extremophilic protein and e. coli ada-c [49] ; however, more intra-and inter-helix ion pairs were found in pk-mgmt. although the absence of a correlation between ion pairs' position and stabilization in ada-c exists, the intra-helix ion pairs act in the secondary structure of pk-mgmt, stabilizing helices, and the inter-helix ion pairs consolidate the inter-domain interactions, enhancing the stability of the tertiary structure packing. in the last decade, ssogt has been characterized through detailed physiological, biochemical, and structural analysis. due to its intrinsic stability, the ssogt protein has proven to be an outstanding model for clarifying the relationships between function and structural characteristics. saccharolobus solfataricus (previously known as sulfolobus solfataricus) is a microorganism first isolated and discovered in 1980 in the solfatara volcano (pisciarelli-naples, italy) [51] , which thrives in volcanic hot springs at 80 • c and a ph 2.0-4.0 range. in order to protect its genome in these harsh conditions, s. solfataricus evolved several efficient protection and repair systems [33, 52] . s. solfataricus is highly sensitive to the alkylating agent methyl methane sulfonate (mms), showing a transient growth arrest when treated with mms concentrations in the range of > 0.25 mm to 0.7 mm [33, 52] . interestingly, although the ogt rna level increases after mms treatment, the relative enzyme concentration decreases, suggesting its degradation in cells in response to the alkylating agent and, in general, to a cellular stress [52] . under these treatment conditions, however, the protein level rises after few hours, and, in parallel, the growth of saccharolobus starts again [52] , indicating a role of ssogt in efficient dna repair by alkylation damage. various assays to measure agt activity are reported in the literature. the first methods were based on the use of oligonucleotides carrying radioactive ( 3 h or 14 c) o 6 -alkylguanine groups. proteinase k digestion was then carried out to measure the levels of marked s-methyl-cysteine in the lysate in an automatic amino acid analyser [53] . a very similar, but simpler and faster radioactive assay was used in another procedure with a 32 p-terminal labelled oligonucleotide containing a modified guanine in a methylation-sensitive restriction enzyme sequence (as mbo i). the agt dna repair activity thereby allowed the restriction enzyme to cut [54] . this procedure was also used by ciaramella's group to identify for the first time the activity of ssogt [52] . this test has the advantage of analysing the digested fragment directly by electrophoresis on a polyacrylamide gel [55] . it was therefore improved in terms of precision by the subsequent separation of the digested oligonucleotides by hplc. the chromatographic separation allowed the calculation of the concentration of active agt after measuring the radioactivity of the peak corresponding to the digested fragment [55] . similarly, moschel's group developed the analysis of hmgmt reaction products based on hplc separation in 2002. this test investigated the degree of inhibition of oligonucleotides with o 6 -mg or o 6 -bg in different positions that varied from the 3' to the 5' end and whether they could be used as chemotherapy agents. ic 50 values were obtained by quantifying the remaining active protein after the radioactive dna reaction [56] . although the assay measures the protein activity, the use of radioactive materials and chromatographic separations made these assays long, tedious, and unsafe. an alternative approach was proposed in 2010 by the group of carme fàbrega, who set up an assay based on the thrombin dna aptamer (tba), a single-stranded 15 mer dna oligonucleotide identified via systematic evolution of ligands by exponential enrichment (selex), which in its quadruplex form binds thrombin protease with high specificity and affinity [57] . in this assay, they put a fluorophore and a quencher to the tba-the quadruplex structure of this oligonucleotide is compromised if a central o 6 -mg is present, preventing the two probes to stay closer. an agt's repair activity on the oligonucleotide allows the folding of the quadruplex structure and the förster resonance energy transfer (fret) energy transfer takes place, resulting in a decrease of the fluorescence intensity [58] . recently, the introduction of fluorescent derivatives of the o 6 -bg (as snap vista green, new england biolabs) made possible the development of a novel dna alkyl-transferase assay. because agt covalently binds a benzyl-fluorescein moiety of its substrate after reaction, it is possible to immediately load the protein product on a sds-page-the gel-imaging analysis of the fluorescence intensity gives a direct measure of the protein activity because of the 1:1 stoichiometry of protein/substrate (figure 3 ). signals of fluorescent protein (corrected by the amount of loaded protein by coomassie staining analysis) obtained at different times are plotted, and a second order reaction rate is determined [38, 39, 46, 52, 59, 60] . this method can be applied to all agts that bind o 6 -bg, with the exception of the e. coli ada-c [61, 62] . innovative fluorescent agt assay. the substrate could be used alone for the determination of the agt catalytic activity, or in combination with a competitive non-fluorescent substrate (alkylated-dna). in the latter case, an indirect measure of the dna repair activity on natural substrates is determined (adapted from [63] ). furthermore, an alkylated double strand dna (dsdna) oligonucleotide can be included in a competition assay with the fluorescein substrate. this non-fluorescent substrate lowers the final fluorescent signal on gel imaging analysis, depending on its concentration. in this way, it is possible to measure the activity of agts for their natural substrate, giving an indirect measure of methylation repair efficiency (figure 3 ) [38, 39, 46, 52, 59, 60] . by using this methodology, it was even possible to discriminate the ssogt activity regarding the position of the o 6 -mg on dna (see below; [39] ), in line with previous data on hmgmt [64] . the recombinant ssogt protein, heterologously expressed in e. coli, has been fully characterized using the fluorescent assay described and summarized in section 3.1, and some results are compiled in table 3 . in agreement with its origin, the protein showed optimal catalytic activity at 80 • c, although retaining a residual activity at lower temperatures (table 3) , and in a ph range between 5.0 and 8.0. as for the most part of many thermophilic enzymes, ssogt is resistant over a wide range of reaction conditions, such as ionic strength, organic solvents, common denaturing agents, and proteases [52, 59] . interestingly, chelating agents do not affect the activity of this enzyme. crystallographic data clarified this observation, as the archaeal enzyme lacks a zinc ion in the structure [39] , whereas this ion is important for correct folding of hmgmt [6] . all catalytic steps of the agts' activity (alkylated dna recognition, dna repair, irreversible trans-alkylation of the catalytic cysteine, recognition, and degradation of the alkylated protein) have been structurally characterized. most information comes from the classic studies on hmgmt, as well as the ada-c and ogt from escherichia coli [5] [6] [7] [8] 49] . other agts' structures are also available in the protein data bank site (figure 2a) as shown in figure 1 , all agts are inactivated after the reaction and degraded via proteasome, whereas in higher organisms, the degradation is preceded by protein ubiquitination [9] . it is a common view that the recognition of alkylated-agts is due by a conformational change; however, data on structure and properties of alkylated agts are limited because alkylation greatly destabilizes their folding [39] . the methylated-hmgmt and benzylated-hmgmt 3d structures were only obtained by flash-frozen crystals, showing that alkylation of the catalytic cysteine (c145) induces subtle conformational changes [6, 7, 65] . consequently, these structures might not reflect the physiological conformation of the alkylated hmgmt [39] . concerning the interactions with the dna, ssogt binds methylated oligonucleotides. however, the repair activity depends on the position of the alkyl-group [39] . to efficiently repair the alkylated base on dna double helix, the protein requires at least three bases from either the 5 or the 3 end. this is due to the necessary interactions formed with the double helix. structural analysis confirmed these data [39] . to overcome the serious limitation to obtain structural data from mesophilic agts after reaction, studies have moved to thermostable homologues, based also on the knowledge that all agts share a common ctd domain structure (figure 2b) . in contrast to the human counterpart, alkylated ssogt was soluble and relatively stable, thus allowing in-deep analysis of the protein in its post-reaction form [39] . structural and biochemical analysis of the archaeal ogt, as well as after the reaction with a bulkier adduct in the active site (benzyl-fluorescein; [66] ), suggested a possible mechanism of alkylation-induced ssogt unfolding and degradation (figure 4) . on the basis of their data, perugino and co-workers suggested a general model for the mechanism of post-reaction agt destabilization-the so called active-site loop moves towards the bulk solvent as a result of the covalent binding of alkyl adduct on the catalytic cysteine and the extent of the loop movement and dynamic correlates with the steric hindrance of the adduct [39, 66] (figure 4) . the destabilization of this protein region triggers then the recognition of the alkylated protein by degradation pathway. as described in section 1.2, the introduction of the snap-tag technology enabled a wide in vivo and in vitro labelling variety for biological studies by fusing any protein of interest (poi) to this protein tag [67] . however, being originated from hmgmt, the extension to extremophilic organisms and/or harsh reaction conditions is seriously limited. [39] ), or with snap vista green substrate (in green; [66] ). by following the same approach used for the hmgmt as kai johnsson [26] [27] [28] [29] [30] , an engineered version of ssogt was produced [52, 59] . this protein, called ssogt-h 5 , contains five mutations in the helix-turn-helix domain, abolishing any dna-binding activity [52] . in addition, a sixth mutation was made-in the active site loop, where serine residue was replaced by a glutamic acid at position 132 (s132e). this modification increased the catalytic activity of ssogt [52, 59] , as it was observed in the engineered version of the hmgmt during the snap-tag development [26] . ssogt-h 5 shows slightly lower heat stability in respect to the wild-type protein (table 3) , whereas the resistance to other denaturing agents is maintained. moreover, ssogt-h 5 is characterised by a surprisingly high catalytic activity at lower temperatures, keeping the rate of reaction to the physiological ones (table 3) [52, 59] . these characteristics make this mutant a potential alternative to snap-tag for in vivo and in vitro biotechnological applications. the stability against thermal denaturation allowed miggiano and co-workers to obtain the structure of the protein after the reaction with the fluorescent substrate snap-vista green, revealing the peculiar destabilization of the active site loop after the alkylation of the active cysteine [66] . the saccharolobus ogt mutant has been firstly tested as protein tag fused to two thermostable s. solfataricus proteins heterologously expressed in e. coli. the chimeric proteins were correctly folded, and the tag did not interfere with the enzymatic activity of the tetrameric s. solfataricus β-glycosidase (ssβgly) [59] , nor with the hyperthermophile-specific dna topoisomerase reverse gyrase [68] [69] [70] [71] [72] . furthermore, the stability of h 5 made possible a heat treatment of the cell-free extract to remove most of the e. coli proteins and performing the β-glycosidase assay at high temperatures without the need of removing the tag [60] . as the applicability of the thermostable tag under in vivo conditions is very important, the ssogt-h 5 was also expressed in thermophilic organisms. the fluorescent agt assay allows for the detection of the presence of ssogt-h 5 both in living cells as well as in vitro in cell-free extracts [59, 72] . to assay the activity to ssogt-h 5 , it was necessary to choose models in which the endogenous agt activity is suppressed. thermus thermophilus is an ogtspecies, showing only one agt homologue (ttha1564), whose annotation corresponds to an alkyltransferase-like protein (atl) [73] . atls are a class of proteins present in prokaryotes and lower eukaryotes [74] , presenting aminoacidic motifs similar to those of agts' ctd, in which a tryptophan residue replaces the cysteine in the active site [75] . like agts, atls use a helix-turn-helix motif to bind the minor groove of the dna, but they do not repair it as they only recruit and interact with proteins involved in the nucleotide excision repair system [76, 77] . although t. thermophilus is a natural ogt knockout organism, sulfolobus islandicus possesses an ogt gene very similar to that of s. solfataricus, which was silenced by a clustered regularly interspaced short palindromic repeats (crispr)-based technique and then used as a host organism [72] . the fluorescent signal obtained by sds-page gel imaging revealed that ssogt-h 5 not only is efficiently expressed in these thermophilic microorganisms, but it also showed that this tag was correctly folded and active, demonstrating the fact that ssogt-h 5 might be used as an in vivo protein tag at high temperatures [59, 72] . as is the case with snap-tag in human cells, the utilization of ssogt-h 5 with different fluorescent substrates gives the opportunity to perform a multi-colour fluorescence study (see table 1 ), by following a poi inside living "thermo cells" at different stages and localization. as most biotechnological processes require harsh operational conditions, the immobilization of very robust enzymes on solid supports is often essential [78] . by definition, an immobilized enzyme is a "physically confined biocatalyst, which retains its catalytic activity and can be used repeatedly" [79] . protein immobilisation offers several advantages, such as the catalysts' recovery and reuse, as well as the physical separation of the enzymes from the reaction mixture. currently, different immobilisation strategies are available, from physical adsorption to covalent coupling [80] [81] [82] [83] . however, all these procedures require purified biocatalysts and suffer from problems related to steric hindrance between the catalyst, the substrate, and the solid support, with increasing of costs and time for the production processes. the introduction of "cell-based" immobilisation systems resulted in a significant improvement and reduces both time and costs of the process. one of the most widely used display strategies is the simultaneous heterologous expression of enzymes and their in vivo immobilisation on the external surface of gram-negative bacteria cells, by the utilisation of the ice nucleation protein (inp) from pseudomonas syringae [84, 85] . most recently, the n-terminal domain of inp (inpn) was used to produce a novel anchoring and self-labelling protein tag (hereinafter asl tag ). the asl tag consists of two moieties, the inpn and the engineered and ssogt-h 5 mutant ( figure 5 ) [86] . the novel anchoring and self-labelling protein tag (asl tag ) system. a protein of interest (poi) is genetically encoded with the tag, which in turn makes it anchored in the outer membrane and accessible for the covalent linkage to a desired chemical group (magenta sphere) by the activity of ssogt-h 5 (adapted from [87] ). the inpn allows an in vivo immobilisation on e. coli outer membrane of enzymes of interest and their exposition to the solvent. the significant reduction of the costs related to the purification and immobilization is added to the overcoming of problems related to the recovery of enzymes by simple filtration or centrifugation methods [88] . ssogt-h 5 , in turn, gives the unique opportunity to label immobilized enzymes with any desired chemical groups (opportunely conjugated to the benzyl-guanine; in magenta in figure 5 ) [27, 59] , dramatically expanding biotechnological applications of this new tool. depending of the chemical group of choice, modulating the activity of enzymes fused with the asl tag can be possible by introducing activator or inhibitor molecules ( figure 5 ). the asl tag system was successfully employed for the expression and immobilization of monomeric biocatalysts, such as the thermostable carbonic anhydrase from saccharolobus solfataricus (sspca), as well as the tetrameric ssβgly, without affecting their folding and catalytic activity [86] . moreover, sspca fused to the asl tag showed an increase in residual activity of up to 30 % for a period of 10 days at 70 • c [87] , representing a huge advantage in pushing beyond reactions in bioreactors and in the reutilization of biocatalysts. to extend the snap-tag technology to hyperthermophilic microorganisms for in vivo studies, an o 6 -alklylguanine-dna alkyltransferase has been recently characterized from the archaeon pyrococcus furiosus [89] . this extremophilic microorganism was originally isolated from hot marine sediments in vulcano island (italy) [90] , with an optimum growth temperature around 100 • c, thus thriving under extremely harsh conditions. like those of other thermophilic archaea, its enzymes are extremely thermostable and can be used in various biotechnological applications. for example, dna polymerase i, also known as pfudna polymerase, is one of the most famous and frequently used enzymes from p. furiosus because of its high activity, thermostability, and strong 3 -5 proof-reading activity [91] . the first demonstration of an ogt activity in p. furiosus was in 1998, when margison and co-workers identified a protein of 22 kda, whose catalytic activity was abolished by the o 6 -bg pseudo-substrate. the pf1878 orf is relative to a protein of 20.1 kda. from its primary structure, the relative polypeptide seems to be closely related to the mgmt from pyrococcus kodakarensis kod1 (pk-mgmt) [48, 89, 92] . the extreme thermostability was confirmed by in vitro biochemical studies on the heterologous expressed and characterized ogt protein from p. furiosus pfuogt. this enzyme was active on bg-fluorescent substrates, thus allowing the competitive assay with methylated dsdna. however, the experiments were performed at 65 • c instead of the standard procedure at 50 • c, as described for ssogt [39, 59, 60] , due to the strong thermophilicity of this enzyme. this behaviour was effectively confirmed by differential scan fluorimetry analysis where the temperature melting (t m ) of pfuogt was found to be 80 • c, much higher than that of ssogt (68 • c) [89] . it is worth noting that, in order to obtain a the sigmoidal melting curve for pfuogt, a slower heating rate (10 min/ • c × cycle) was set up, whereas the t m value measurement is usually performed at 1 min/ • c × cycle [93] . thermotoga neapolitana is a hyperthermophilic gram-negative bacterium of the order of thermotogales [94] [95] [96] , which are excellent models for genetic engineering and biotechnological applications [97] [98] [99] [100] . the ctn1690 orf shows a clear homology of the o 6 -alkylguanine-dnaalkyl-transferase. sds-page gel imaging analysis on lyophilized t. neapolitana cells incubated with the agt fluorescent substrate showed a strong fluorescent signal with a molecular weight close to that of ssogt. the observed molecular weight and, above all, the sensitivity to the o 6 -bg derivative, led to the cloning and heterologous expression of the thermotoga neapolitana ogt protein (tnogt) in e. coli [89] . this protein, like most agts, has a role in dna repair, as confirmed by competitive fluorescent assay in the presence of methylated dsdna. as shown in figure 3 , the ic 50 value was similar to that obtained for ssogt. surprisingly, the enzyme from t. neapolitana exhibited a very high activity at low temperatures [89] , similar to that possessed by the mutant ssogt-h 5 (table 3 ) [52, 59] . superimposition analysis between a tnogt 3d model and the free form of ssogt (id pdb: 4zye) revealed in both structures the presence of a serine residue in the active site loop (s132 in ssogt, see figure 2a ), which was replaced in ssogt-h 5 by a glutamic acid to improve its activity at lower temperatures. interestingly, some residues are missing in tnogt that play an important role in stabilizing ssogt. in particular, the ionic interactions that play a crucial role in the stability of the saccharolobus enzyme at high temperatures, such as the pair r133-d27 [39] and the k-48 network [60] , are largely replaced by hydrophobic residues in the thermotoga homolog. evidently, different residues and mechanisms of stabilization may contribute to its exceptional catalytic activity at moderate temperatures and the high thermal stability. the interest shown from the important insights of this class of small proteins led to novel biotechnological applications [101] . studies on thermophilic agts represent a unique opportunity for structural analysis and, in the case of the s. solfataricus protein, for the identification of conformational changes after the trans-alkylation reaction, which are detectable with mesophilic agts, as the alkylated form are rapidly destabilized [6] . these results could have a wide impact, especially in medical fields for the design of novel hmgmt inhibitors to be used in cancer therapy [102] . furthermore, given their small size, thermophilic enzymes are very useful for studying general stabilization mechanisms at high temperatures (as for pk-mgmt and ssogt), which can then be applied to mesophilic enzymes. searching for alternative ssogt homologues was clearly useful, leading to the identification of agts that are more resistant to thermal denaturation (pfuogt) or to enzymes with a higher reaction rate at all tested temperatures (tnogt). concerning biotechnology, the use of a modified hmgmt as protein tag opened the possibility to generalise this method-a targeted mutagenesis on a thermostable ogt by following a rational approach led to the characterization of ssogt-h 5 , applicable to in vitro harsh reaction conditions and to in vivo (hyper)thermophilic model organisms. on the other hand, by an irrational approach (random mutagenesis) it is also possible to enhance their catalytic activity [103] , or modify the substrate specificity of these enzymes, making them active on benzyl-cytosine (o 2 -bc) derivatives, such as that which happened for the production of the clip-tag [104] . this knowledge could be the starting point of developing a new engineered thermo-snap-tag to be employed in particular biotechnological fields, from in vivo studies in (hyper)thermophilic microorganisms (such as the in vivo crispr-cas immune system in p. furiosus [105, 106] ) to industrial processes that require high temperatures or, in general, harsh reaction conditions. targeted modulation of mgmt: clinical implications high-resolution structure of a mutagenic lesion in dna alkylation damage in dna and rna-repair mechanisms and medical significance multifaceted roles of alkyltransferase and related proteins in dna repair, dna damage, resistance to chemotherapy, and research tools the structure of the human agt protein bound to dna and its implications for damage detection active and alkylated human agt structures: a novel zinc site, inhibitor and extrahelical base binding dna binding and nucleotide flipping by the human dna repair protein agt dna binding, nucleotide flipping, and the helix-turn-helix motif in base repair by o 6 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for gene expression the green fluorescent protein vectors that facilitate the expression and purification of foreign peptides in escherichia coli by fusion to maltose-binding protein molecular interaction between the strep-tag affinity peptide and its cognate target walid qoronfleh, m. glutathione s-transferase pull-down assays using dehydrated immobilized glutathione resin structural investigations on orotate phosphoribosyltransferase from mycobacterium tuberculosis, a key enzyme of the de novo pyrimidine biosynthesis biochemical and structural investigations on phosphorribosylpyrophosphate synthetase from mycobacterium smegmatis a thioredoxin gene fusion expression system that circumvents inclusion body formation in the e. coli cytoplasm directed evolution of o 6 -alkylguanine-dna alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo a general method for the covalent labeling of fusion proteins with small molecules in vivo covalent and selective immobilization of fusion proteins directed evolution of o 6 -alkylguanine-dna alkyltransferase for applications in protein labeling how to obtain labeled proteins and what to do with them spr-based interaction studies with small molecular weight ligands using hagt fusion proteins snap-tag technology: a useful tool to determine affinity constants and other functional parameters of novel anti-body fragments selective degradation of reverse gyrase and dna fragmentation induced by alkylating agent in the archaeon sulfolobus solfataricus alkylation damage repair protein o 6 -alkyl-guanine-dna alkyltransferase from the hyperthermophiles aquifex aeolicus and archaeoglobus fulgidus the o 6 -methylguanine-dna methyltransferase from the hyperthermophilic archaeon pyrococcus sp. kod1: a thermostable repair enzyme thermostable archaeal o 6 -alkylguanine-dna alkyltransferases function of domains of human o 6 -alkyl-guanine-dna alkyltransferase biochemical and structural studies of the mycobacterium tuberculosis o 6 -methylguanine methyltransferase and mutated variants structure-function relationships governing activity and stability of a dna alkylation damage repair thermostable protein novel dna repair alkyltransferase from caenorhabditis elegans the dna alkylguanine dna alkyltransferase-2 (agt-2) of caenorhabditis elegans is involved in meiosis and early development under physiological conditions helix signals in proteins amino acid preferences for specific locations at the ends of α-helices importance of alpha-helix n-capping motif in stabilization of betabetaalpha fold structure of a hyperthermo-philic tungstopterin enzyme, aldehyde ferredoxin oxidoreductase crystal structure of mycobacterium tuberculosis o 6 -methylguanine-dna methyltransferase protein clusters assembled on to damaged dna insight into the molecular basis of thermal stability from the structure determination of pyrococcus furiosus glutamate dehydrogenase hyperthermostable protein structure maintained by intra and inter-helix ion-pairs in archaeal o 6 -methylguanine-dna methyltransferase crystal structure of a suicidal dna repair protein: the ada o 6 -methylguaninedna methyltransferase from e. coli structural studies of mj1529, an o 6 -methylguanine-dna methyltransferase the sulfolobus-"caldariella" group: taxonomy on the basis of the structure of dna-dependent rna polymerases activity and regulation of archaeal dna alkyltransferase: conserved protein involved in repair of dna alkylation damage repair of alkylated dna in escherichia coli. methyl group transfer from o 6 -methylguanine to a protein cysteine residue measurement of o 6 -alkylguanine-dna-alkyltransferase activity in human cells and tumor tissues by restriction endonuclease inhibition assay for o 6 -alkylguanine-dna-alkyltransferase using oligonucleotides containing o 6 -methylguanine in a bamhi recognition site as substrate repair of oligodeoxyribonucleotides by o(6)-alkylguanine-dna alkyltransferase selection of single-stranded dna molecules that bind and inhibit human thrombin development of a novel fluorescence assay based on the use of the thrombin binding aptamer for the detection of o 6 -alkyl-guanine-dna alkyltransferase activity a novel thermostable protein-tag: optimization of the sulfolobus solfataricus dna-alkyl-transferase by protein engineering interdomain interactions rearrangements control the reaction steps of a thermostable dna alkyltransferase differential inactivation of mammalian and escherichia coli o 6 -alkylguanine-dna alkyltransferases by o 6 -benzylguanine repair of o 6 -benzylguanine by the escherichia coli ada and ogt and the human o 6 -alkylguanine-dna alkyltransferase every ogt is illuminated... by fluorescent and synchrotron lights interactions of human o(6)-alkylguanine-dna alkyltransferase (agt) with short double-stranded dnas mechanism to trigger unfolding in o 6 -alkylguanine-dna alkyltransferase crystal structure of a thermophilic o 6 -alkylguanine-dna alkyltransferase-derived self-labeling protein-tag in covalent complex with a fluorescent probe labeling of fusion proteins with synthetic fluorophores in live cells dissection of reverse gyrase activities: insight into the evolution of a thermostable molecular machine inhibition of translesion dna polymerase by archaeal reverse gyrase reverse gyrase and genome stability in hyperthermophilic organisms positive supercoiling in thermophiles and mesophiles: of the good and evil in vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag an o 6 -methylguanine-dna methyltransferase-like protein from thermus thermophilus interacts with a nucleotide excision repair protein alkyltransferase-like proteins: brokers dealing with alkylated dna bases flipping of alkylated dna damage bridges base and nucleotide excision repair atl1 regulates choice between global genome and transcription-coupled repair of o(6)-alkylguanines mycobacterium tuberculosis uvrb forms dimers in solution and interacts with uvra in the absence of ligands progress in enzyme immobilization in ordered mesoporous materials and related applications chibata, i. studies on continuous enzyme reactions. i. screening of carriers for preparation of water-insoluble aminoacylase an overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes an overview of techniques in enzyme immobilization a general overview of support materials for enzyme immobilization: characteristics, properties chapter nine-enzyme immobilization: an overview on methods, support material, and applications of immobilized enzymes bacterial ice nucleation: significance and molecular basis ice crystallization by pseudomonas syringae an agt-based protein-tag system for the labelling and surface immobilization of enzymes on e. coli outer membrane thermostability enhancement of the α-carbonic anhydrase from sulfurihydrogenibium yellowstonense by using the anchoring-and-selflabelling-protein-tag system (asl tag ) a one-step procedure for immobilising the thermostable carbonic anhydrase (sspca) on the surface membrane of escherichia coli a journey down to hell: new thermostable protein-tags for biotechnology at high temperatures pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100 • c. arch. microb high-fidelity amplification using a thermostable dna polymerase isolated from pyrococcus furiosus mutational effects on o 6 -methylguanine-dna methyltransferase from hyperthermophile: contribution of ion-pair network to protein thermostability the use of differential scanning fluorimetry to detect ligand interactions that promote protein stability a new sulfur-reducing, extremely thermophilic eubacterium from a submarine thermal vent thermotoga neapolitana sp. nov. of the extremely thermophilic, eubacterial genus thermotoga microbial biochemistry, physiology, and biotechnology of hyperthermophilic thermotoga species site-directed mutagenesis of a hyperthermophilic endoglucanase cel12b from thermotoga maritima based on rational design engineering of tm1459 from thermotoga maritima for increased oxidative alkene cleavage activity engineering a switch-based biosensor for arginine using a thermotoga maritima periplasmic binding protein development of a pyre-based selective system for thermotoga sp. strain rq7 repair of o 6 -alkylguanine by alkyltransferases variability and regulation of o 6 -alkylguanine-dna alkyltransferase directed evolution of the suicide protein o 6 -alkylguanine-dna alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability an engineered protein-tag for multi-protein labeling in living cells rna-guided rna cleavage by a crispr rna-cas protein complex the rna-and dna-targeting crisp-cas immune systems of pyrococcus furiosus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: g.p. would like to thank all the authors, elena and elisa perugino for their efforts in writing this work and in their technical assistance, but mainly for their human support during the difficult and delicate period of staying at home following the covid-19 outbreak. all authors strongly thank the reviewers for their important corrections and changes, increasing the scientific level of this manuscript. the authors declare no conflict of interest. key: cord-260440-e63pgcir authors: dinjaski, nina; prieto, m. auxiliadora title: smart polyhydroxyalkanoate nanobeads by protein based functionalization date: 2015-02-24 journal: nanomedicine doi: 10.1016/j.nano.2015.01.018 sha: doc_id: 260440 cord_uid: e63pgcir the development of innovative medicines and personalized biomedical approaches calls for new generation easily tunable biomaterials that can be manufactured applying straightforward and low-priced technologies. production of functionalized bacterial polyhydroxyalkanoate (pha) nanobeads by harnessing their natural carbon-storage granule production system is a thrilling recent development. this branch of nanobiotechnology employs proteins intrinsically binding the pha granules as tags to immobilize recombinant proteins of interest and design functional nanocarriers for wide range of applications. additionally, the implementation of new methodological platforms regarding production of endotoxin free pha nanobeads using gram-positive bacteria opened new avenues for biomedical applications. this prompts serious considerations of possible exploitation of bacterial cell factories as alternatives to traditional chemical synthesis and sources of novel bioproducts that could dramatically expand possible applications of biopolymers. from the clinical editor: in the 21st century, we are coming into the age of personalized medicine. there is a growing use of biomaterials in the clinical setting. in this review article, the authors describe the use of natural polyhydroxyalkanoate (pha) nanoparticulates, which are formed within bacterial cells and can be easily functionalized. the potential uses would include high-affinity bioseparation, enzyme immobilization, protein delivery, diagnostics etc. the challenges of this approach remain the possible toxicity from endotoxin and the high cost of production. health-focused nanotechnologies have put under screening a growing spectrum of materials whose properties can be modified during fabrication. merging synthesis and smart functionalization of natural polymers allows straightforward cost-effective production of novel materials specifically designed for target application. 1, 2 the performance of polymers synthetic in origin has been investigated for nanotechnology applications, as well. 3 however, in this case production and functionalization are usually two separate processes. among natural polymers, polyhydroxyalkanoates (phas), the highly tunable bacterial polyesters, play an important role in the development of next generation biomaterials (figure 1 ). their properties are greatly influenced by the type (e.g., short chain length pha, scl-pha; medium chain length pha, mcl-pha) and homogeneity of hydroxyalkanoic monomer building blocks, and others ( figure 2 ). 4 the ability to edit and redirect bacterial cell system through metabolic or genetic engineering, enables the construction of platforms to produce versatile materials carrying wide range of functional groups which confer desired properties to the polymer. [4] [5] [6] alternatively, the direct use of highly structured natural pha nanoparticulate entities formed within bacterial cells opened new avenues for attractive biomaterial design where tailor-made beads are functionalized using intrinsic bacterial granule producing system. 1, 7, 8 these possibly phospholipidcoated inclusions carry granule-associated proteins (gaps) on their surfaces, such as: i) pha synthases, involved in the polymerization of the biopolyester; ii) pha depolymerases, responsible for pha mobilization; iii) phasins, the main structural components of gaps and iv) other proteins such as enzymes related to the synthesis of pha monomers, as well as transcriptional regulators not classified as gaps ( figure 3 ). 1, 9, 10 the implementation of these new assets, aside from broadening the potential, allows customizing and fine tuning to improve polymer performance for each specific application ( figure 4 ). nanostructured materials produced by bacteria are becoming increasingly recognized as functionalized beads with great biotechnological and biomedical potential. 11, 12 functionally complex architecture of pha inclusions, based on interacting proteins embedded/attached to pha core, 13 has been exploited as a toolbox to display molecules carrying out specific function (figure 4 ). under a wide scope of applications the performance of such engineered pha beads has been demonstrated in high-affinity bioseparation, 14 enzyme immobilization, 7, 15, 16 protein delivery to natural environments, 17, 18 diagnostics, 19 as an antigen delivery system 20 and many others (table 1) . 2, 20 herein, we revise the diversity of cell systems available to produce functionalized pha nanobeads and underline specific properties in context of their suitability for different applications. we highlight the advantages of different granule-associated proteins (gaps) and address the possible gaps that need to be fulfilled. importantly, powerful combination of synthetic biology and microengineering can create appropriate framework for future application of pha nanobeads. finally, we compare the properties of nanoparticles based on bacterial and selected synthetic polyesters. despite the fact naturally occurring nanoparticles have been present for millions of years, nanotechnology is first and . schematic representation of the currently used strategies for pha functionalization centered around added-value pha production. in vivo pha modification based on peptide functionalization of pha nano-beads using gaps for recombinant protein anchoring to the pha granule or nonspecific binding and in vivo chemical modification through incorporation of functional group in the side chain of the polymer applying metabolic engineering and systems biology approach. similarly to in vivo, in vitro approach for peptide functionalization can be based on the use of gaps or nonspecific binding, while the underlying principle of in vitro chemical modification might be based on polymer synthesis or modification. foremost focused on in vitro man-made particles. 11 nevertheless, dependently on the target application, in vivo biological or in vitro synthetic approach for fusion protein immobilization to the pha granule surface might better meet the requirements ( table 2 ). the in vivo pha granule functionalization consists of gap fusion immobilization onto the granule surface simultaneously with the granule formation inside the pha-producing host ( figure 4) . 7, 49 on the other hand, the production of these bioinspired constructs in vitro is based on pha extraction, followed by in vitro bead production and in vitro gap fusion protein immobilization via gap-bead interaction ( figure 4 ). 30 the main advantages of this in vitro cell-free system are: i) the possibility of tight control of nanoparticle disassembly and reassembly process; ii) absence of competition among the recombinant gap-fusion and wild type proteins; iii) tight control over particle size and immobilized protein/active agent concentration; iv) possibility of endotoxin removal, crucial for the design of every biomedical setup. nevertheless, pha isolation and in vitro nanobead production require more tedious methodology (e.g., to avoid pha particle aggregation) in comparison to isolation of in vivo produced pha granules. also, the use of non-environmentally friendly solvents is needed for in vitro technology. all mentioned significantly increase the costs of in vitro pha nanobead production and make the technology suitable mainly for added-value applications where tight control over particle size and active agent concentration is needed. 30 in the line of safety, in vitro approach is highly convenient for nanomedical purposes, including nanofabrication, imaging, drug delivery and tissue engineering, where the use of endotoxin-free pha is requisite. 4 importantly, the fabrication of endotoxin-free pha vehicles can also be achieved using in vivo settings (see below). some applications such as protein delivery to natural environments do not necessary acquire endotoxin free pha and can benefit from an in vivo approach where bacterial naturally produced nanoscale particulate entities can be used in a straightforward manner. 16 furthermore, as bacterial polymeric particles can be functionalized in vivo before isolation there is a clear environmental and economic advantage over those produced chemically. particle functionalization is achieved through the recombinant expression of fusion proteins, where natural gaps are used as anchoring tag for foreign protein immobilization. perfect example is biof tag from pseudomonas putida based on the use of intrinsic p. putida pha granules as scaffold to immobilize fusion proteins in vivo. once fermentation under optimal pha production conditions is accomplished, granules decorated with the biof-protein fusions are obtained as the end product ( figure 5 ). 8, 16 dependently on protein release treatment, up to 100% of fusion protein can be recovered with a good purity, since the phasins represent major gaps. 7 additionally, the possibility of minimizing the presence of gap proteins to increase the yield of fusion protein binding and purity has been investigated. 8 biof system was proven efficient for in vivo coating of mcl-pha granules with cry1ab derived insect-specific toxin protein. generation of bioplastic-biof-insect specific toxin complex indicated excellent performance of biof tag as a device for spreading active polypeptides to the environment without the need for active agent release and purification. 16 similarly, organophosphohydrolase from agrobacterium radiobacter immobilized on polyester inclusions of recombinant escherichia coli was shown suitable for bioremediation applications. 17 testing this new in vivo assets and analyzing their limits, indicated the possible room for improvement. current trends deal with implementation of new methodological platforms, as synthetic biology, to improve the production process and productivity. 57 this highlights the importance of re-programming approaches to optimize the system and design strategies focused on meeting the necessities of each specific application. in the line of fine tuning of biological interfaces and the use of pha as vehicles, addressing the key factors of pha machinery permitted overcoming biological barriers to reach maximal in vivo coating of pha nanobead and at the same time avoid side effects concerning disordered granule biodistribution after cell division (see below). 8 different gapsdifferent advantages: hydrophobic vs. covalent binding the diversity of gaps offers gentle alternatives through flexible and highly tunable design of specific tags suitable for personalized requirements of different application. thus, the window of possibilities that each specific gap offers implies different modes to connect recombinant protein and pha table 2 comparison of pha nanoparticles in vitro and in vivo production process, their applications and costs. in vitro ref. production by bacteria synthetic production 2, 20 use of renewable sources for production harsh chemical needed for polymer isolation and particle production 30, 53 simultaneous production and functionalization functionalization posterior to nanobead production 8, 20, 30 nanobead assembly and disassembly cannot be tightly controlled tight control over bead assembly and disassembly 10, 54 competition of recombinant and wild type gaps functionalization with target protein only, no other gaps 8, 30, 54 particle size can be controlled by biotechnological production process tight control over particle size 32, 54 immobilized protein concentration variation might represent challenge tight control over immobilized protein concentration 7, 30 in production cost total production cost includes in vivo particle production cost and particle purification, lower production cost compared to in vitro produced particles, since additional functionalization is not needed higher production costs compared to in vivo produced particles, total price accounts for polymer synthesis, isolation, endotoxin removal, in vitro particle synthesis and functionalization 30, 54, 56 nanobeads (covalent, hydrophobic or non-specific) ( figure 4 ). although so far very little is known about their structure and interaction with the pha granules, 58 phasins are highly attractive among gaps, largely due to the wide assortment of structurally different compositions compared to other gaps ( figure 4 ). phasins have been utilized as affinity tags and through protein engineering designed to build recombinant protein purification system. this provides low cost method for production and purification of high added value proteins in a continuous way. 49 significant improvements in bio-separation technology were made by upgrading the system interconnecting phasins and target proteins via self-cleaving intein. 47 this approach enabled in vivo recombinant protein immobilization onto the granule and the release of purified proteins once the native scl-pha particles were recovered, which in turn pushed bio-separation technology several steps ahead, toward convenience and economic production. in vivo immobilized correctly folded eukaryotic proteins on the surface of pha granules across phasin protein have been used for fluorescence activated cell sorting (facs) based diagnostics. 18 in completely different context to in vivo tag binding, in vitro synthesized pha nanoparticles and in vitro hydrophobic binding of phap fusion proteins with protein ligands (e.g., mannosylated human α1-acid glycoprotein (hagp) and human epidermal growth factor (hegf)) have been reported as another outstanding application of phasins for receptor-mediated drug delivery. 30 mostly utilized phasins are phap of ralstonia eutropha that bind scl-pha, 20 while the exclusive example of mcl-pha binding p. putida phaf phasin is for environmental application (biof system). 8, 16 other identified phasins as phap proteins of aeromonas hydrophila, phap of haloferax mediterranei, paracoccus denitrificans, bacillus megaterium, and others (revised in 10 ) have not been deeply studied for nanobiotechnology purposes. likewise, applying the in vitro approach the substrate binding domain of pha depolymerase has been used to hydrophobically anchor fusion proteins to pha nano and microbeads. 22, 59, 60 a different strategy to in vivo immobilize recombinant proteins onto pha nanobead surface relays on the advantage of covalent gap-pha binding using p. aeruginosa, p. putida, r. eutropha or b. megaterium pha synthase as a tag. 14,61-63 phasin-pha interaction usually results in a slow non-triggered protein release over time under physiologic conditions. moreover, specific environmental conditions can alter release rates. 64 in contrast, covalent attachment enables unique natural cross-linking of a protein and polymeric support and allows better control over protein release kinetics. pha synthase offers the possibility of covalent protein-pha conjugation. both n-and c-terminal of pha synthase were shown suitable for in vivo assembly of functionalized polyester beads. 14, 17, 26, 31, 44, 62, 65, 66 this approach based on pha nanobead functionalization through phac helps to circumvent the washing off of non-covalently bound fusion proteins during the process. 67 the particles with an intrinsic label can be tailored to covalently display proteins for applications in antibody capture-based diagnostic (e.g., immunochromatographic strips or bach-and-elute bioseparation applications). the modular arrangement of the protein domains provides a large figure 5 . in vivo immobilization of fusion proteins to bioplastics by biof tag. the procedure consists of: 1, the fermentation in p. putida under optimal pha production conditions; 2, 3, isolation of the granules carrying the biof-proteins fusions from the crude cell lysate by a simple centrifugation step; 4, release of fusion proteins via detergent treatment (modified from 16 ). design space for the production of custom-made materials. 20 by introducing enterokinase digestion site between the tag and target protein the latter can be efficiently released from polymer support providing efficient and cost-effective methodology to obtain added value product. 67 similarly, to facilitate target protein release from bio-bead, thrombin cleavage site was used as a linker, 68 as well as previously mentioned autolytic intein. this enables straightforward liberation of target protein. 52, 69 in addition, proteins can be unspecifically absorbed to pha. 59, 70 an alternative route to intracellularlly produce enzyme decorated pha beads consists of simultaneous synthesis of insoluble protein inclusion bodies and pha granules. charged particles are created by introducing acidic coil via n-terminal of phac. this structure has been used to capture an enzyme of interest that was co-expressed in the same host cell and contains a basic coil fused to its c-term. coils are held together by hydrophobic and electrostatic interactions. 65 therefore, it follows that understanding protein-pha interactions from a biophysical point of view will undoubtedly widen the biotechnological and clinical potential of these bioplastics. in fact, in some cases there are indications that phasin-pha interaction is influenced not only by the nature of these two components but also by the presence of other gaps that interfere and play the role of mediation elements facilitating the binding. 8, 10 for instance, the optimization of biof system by minimizing the dosage of natural phasins in p. putida kt2440 illustrates the importance of understanding the molecular basis underlying the pha-phasin interaction and its biological consequences. 8 also, the mechanistic study of the pha granule producing machinery functioning, the dynamics and factors that direct gap-pha binding together assist in overcoming technical hurdles and indicate bottlenecks important for the design of bioinspired nanoparticles (see "editing, streamlining and refactoring wild type strains for enhancement of protein immobilization" section for details). bug systems for scaling up: wild type over recombinant cells success in producing pha naturally or recombinantly in broad range of bacteria showed that many microorganisms with desirable properties could perform the function of cell factory for production of functionalized pha beads. e. coli is default host microorganism for recombinant protein production and often the first choice. the fact that this strain serves as a workhorse of basic and applied research worldwide is largely due to the possibility of high recombinant protein yield achievement. remarkably, e. coli, a previous non-pha producer, through pathway engineering has been set up to produce up to 150 g/l cell dry weight (cdw) with final pha content of more than 80%. 4 this was used to co-produce several tagged proteins (maltose binding protein (mbp), β-galactosidase (lacz), chloramphenicol acetyltransferase (cat)) with polyhydroxybuyrate (phb) granules in the e. coli cells. proteins were purified with yields of 3.17-7.96 mg/g cdw. 47 currently applying recombinant e. coli cells allows covering of the granule surface up to 20% of total proteins associated with the bead, 19 while using wild type such as p. putida strain as much as 2% can be achieved. 8 it should be noted that different bacterial strains have different pha producing capacities regarding polyester type (sclor mcl-pha) and relative amount to cdw. besides, the cause of altered final recombinant protein yield might be the consequence of the type of gaps used to immobilize recombinant protein, affecting the specific recombinant protein-pha interaction. importantly, r. eutropha naturally produces more than 200 g/l of phb, which gets to 80% of cdw similarly to recombinant production in e. coli, 40 while yields of mcl-pha obtained with p. putida reach 65%. 71 p. putida productivity can be upgraded to 84% of intracellular mcl-pha, incorporating knock-out mutations of beta-oxidation genes fada and fadb. 72 recombinant e. coli is able to produce 20% of mcl-pha when beta-oxidation is impaired due to the deletion of fadb, 4 whereas qi et al. used metabolic routing strategy to inhibit fatty acid beta-oxidation by acrylic acid in recombinant e. coli (fadr) and produce 60% mcl-pha. 73 additionally, phaj encoding (r)-specific enoyl-coa hydratase, was demonstrated to supply 3-hydroxyacyl-coa of c4-c6 for pha biosynthesis via beta-oxidation pathway. 74, 75 its co-expression with phac in e. coli led to production of pha with monomer composition containing c4, c6, c8, and c10 from unrelated carbon source. 76, 77 though, e. coli remains the most commercially valuable host for phb large-scale production as the polymer degradation is avoided, the down sides as endotoxin contamination and previously mentioned relatively low yields of mcl-pha, substantially limit its use for biomedical purposes. also, the overexpression of foreign genes over physiological rates usually triggers a spectrum of conformational stress responses and causes the accumulation of insoluble protein versions that do not reach their native conformation. 78 these pseudospherical protein aggregates, inclusion bodies, are considered undesired byproducts of protein production processes. other bottlenecks as the loss of the plasmid due to the instability of introduced genes, use of antibiotics and gene expression expensive inducers have been partially solved, however they still represent a challenge (reviewed in 53 ). taking all this together, the advantages of using wild type strains as host should not be overlooked. specific strategies applied on the components of pha machinery can drive productivities of high contents of pha immobilized recombinant proteins in wild type strains as reported for e. coli. 8 on the positive side, a great understanding of pha synthesis in model mcl-pha producer strains such as p. putida, has been gained through systems biology ("omics" data, genome-scale metabolic models, etc.). 57, [79] [80] [81] [82] [83] powerful genetic tools based on synthetic biology 84 support bottom-up approaches and might be used to design p. putida strains that generate added-value bioproducts, such as active mcl-pha based nanobeads. the great value of this bacterium as an autolytic specialized strain for mcl-pha production has also been demonstrated. 85 due to its broad metabolic versatility and genetic plasticity, which allow a variety of renewable carbon sources to be used for pha production, p. putida is one of the most prominent candidates for protein production. aside from pseudomonas, many other gram-positive and gram-negative eubacterial genera such as bacillus, ralstonia, aeromonas, rhodobacter, rhodospirillum, rhodococcus were shown suitable for production of pha nanobeads. 4, 86 editing, streamlining and refactoring wild type strains for enhancement of protein immobilization complex subcellular architecture and self-organizing nanoand micro-compartments of bacterial cell hold great promise, largely due to the possibility for their biofunctionalization. disturbing these highly coordinated systems might easily imbalance the physiology of the bacterial cell. pha granules take over the control of the carbon and energy storage and thus represent important element of bacterial metabolic network. 83 thereafter, from an energy flow and survival physiology standpoint, balanced distribution of pha between daughter cells after division has fundamental importance as competitive setting. understanding the pha machinery and interplay of its components was shown crucial for optimization of the in vivo system for production of protein functionalized pha nanobeads. 8, 9 different scenarios involving different molecular events and interactions as well as granule localization have been proposed by micelle, budding and scaffold model of granule formation. 7 in contrast to a micelle model where pha granules are assumed to be randomly distributed in the cytoplasm, budding and scaffold model suggests defined localization proposing granule-cell membrane interaction or phac-scaffold molecule interplay, respectively. recently proposed scaffold model suggest cooperative work of phac and phasins in granule formation. since, phasins-phac interaction has been spotted in some bacterial strains (e.g., pham, phasin-like protein that interacts with phac in r. eutropha), phasins were proposed as the main components forming network that interconnects granules, dna and enzymes involved in pha metabolism. 9, 87, 88 this network should serve as a mediation element responsible for granule localization within the cell and their balanced segregation between daughter cells during cell division. on some of gap interactions depends their activity, while the function of others is still to be discovered. for instance, homo-oligomerization of r. eutropha phac1 reu and phar reu 89, 90 and p. putida phac1 and hetero-oligomerization of phac bmeg with phar bmeg are known to be essential for accomplishing the function. meanwhile, the interaction of certain phasins with other pha players was identified, 90 but their exact function is to be unraveled. namely, p. putida phaf was proposed to form homoand hetero-tetramers interacting with phai through short leucine zipper. 58 another suggested role of phasins is the control of the access of pha depolymerases. indeed, weak phap2-phaz interaction was reported in r. eutropha. 90 all these interactions are taught to contribute to the formation of net-like structure found in the vicinity of pha granules 91 and provide a window into the system functioning. phaf has been shown to have a role as a central player in the machinery, controlling pha granule segregation and localization in the cell, since it shows a unique ability to bind at least two ligands (the pha granules and the nucleoid). 7, 9, 58, 92 the peculiar structural organization of phaf into two domains performing diverse functions (i.e., c-terminal histon-like domain, n-terminal phasin-like domain) supplies an explanation to its biological role. 8, 9 moreover, whether or not p. putida cytoskeletal or other gap proteins facilitate the organization of granules in needle array like structure (figure 4) , by direct or indirect interaction with phaf, is still an open question and currently the precise mechanisms by which intermediary phaf positions the pha granules are still unknown. 9 similarly, pham of r. eutropha can bind both dna and pha. 93 therefore, to refine the system it is needed to unravel the puzzle of how functionally diverse, or even a multifunctional set of gaps, should be combined to generate an optimal yield of in vivo immobilized protein onto the granule surface and engender a coherent cell phenotype. in a further step toward the use of pha granules as nanocarriers decorated with functionalized phasins, the information on phasin physiological function provided important insights into the critical factors needed to be targeted to improve existing models. 8, 9 for instance, phasin binding prevents unspecific attachment of not only proteins unrelated to the pha metabolism to the granules surface, but also limits the space for recombinant proteins to anchor. 94 therefore, the absence of wild-type phasins favors binding of recombinant tagged protein molecules anchoring to the granule surface. 8 this could be explained by limited surface for recombinant proteins to anchor wild-type pha granules and the need to compete with natural phasins. in this respect, the key phasin factors have been identified for optimal pha production in p. putida addressing the minimum amount of complete phasin proteins necessary to achieve adequate pha production and higher yield of immobilized recombinant protein. 8 applying this strategy maximum biof (n-terminal of phaf) fusion protein concentration was in vivo immobilized onto the pha beads (2.2% of recombinant protein/pha) without compromising phasins' intrinsic function. 8 also, this demonstrated the swappable nature of phai phasin and biof pha binding modules in terms of their physiological function and illustrated the utility of the phaf/phai structure redundancy, being autonomous modular cooperatively working units. 8, 58 altogether, these examples show that the escalating drive to identify the connections within the complex system of gaps network is fueled by the need to develop new strategies that will lead to improvement of protein immobilization onto the pha beads. metabolic and biotechnology capacities of p. putida, as well as global understanding of the capabilities of this strain are facilitated by metabolic models that enabled integration of experimental along with genomic and high-throughput data. 57 endotoxin free pha nanobead production bacterial lipopolysaccharides (lps) or endotoxins, also designated as pathogen associated molecular patterns (pamps) recognized by innate immune system are most potent identified microbial mediators implicated in the pathogenesis of sepsis and septic shock. lps is the most prominent 'alarm molecule' sensed by the host's early warning system of innate immunity presaging the threat of invasion by gram-negative bacterial pathogens. 95 thus, presence of lipopolysaccharide (lps) endotoxins in pha nanobeads produced in gram-negative bacteria makes these in vivo naturally produced particles unsuitable for biomedical applications. 96, 97 the problem occurs because co-purification of pyrogenic outer lps together with pha granules cannot be avoided. in vitro approach on the other hand offers the possibility of endotoxin removal from pha polymer. the concentration of endotoxins in pha is greatly influenced by purification strategy and might vary from more than 10 4 eu/g to less than 1 eu/g. 55, 98 the methodology for endotoxin elimination depends on type of pha (e.g., scl-pha, mcl-pha, presence of functional groups, etc.) and each results in different rates of polymer recovery. 55, 98 however, in vitro strategy remains hampered by the necessity of extensive and tedious purification methodology to achieve the levels in compliance with the endotoxin requirements for biomedical application according to the u.s. food and drug administration (fda). generally, for products that directly or indirectly contact the cardiovascular system and lymphatic system the limit is 0.5 eu/ml or 20 eu/ device, while for devices in contact with cerebrospinal fluid the limit is 0.06 eu/ml or 2.15 eu/device. 99 all mentioned factors together with the bacteria growth conditions significantly influence the total cost of the production of endotoxin-free polymer. to get around this limitation, alternative sources of functionalized pha granules free of lps contamination are gram-positive bacteria. they offer a platform for production of lps free tailored beads due to the difference in the structure of their cell envelopes compared to gram-negative bacteria. 100 even so, other pamps, such as lipoteichoic acid (lta) and peptidoglycan (pg), found in gram-positive bacterial pathogens are now appreciated to activate many of the same or similar host defense networks induced by lps. 95 subsequently their presence in phas isolated from gram-positive bacteria might have immunogenic activities similar to lps. 101 among pamps, lta predominates in the bacillus, whereas actinomycete bacteria typically synthesize lipoglycans. 102 importantly, certain gram-positive pha producing strains (e.g., bacillus circulans, bacillus polymyxa) lack both, lta and lipoglycans. 103 clostridium and staphylococcus citreus were reported to lack lta and may be considered for recombinant pha production. 104 hence, emerging area to be investigated are the mechanisms triggered by pamps of gram-positive pha producing bacteria regarding mammalian immune system. remarkably, gram-positive genera corynebacterium, nocardia and rhodococcus are the only wild-type bacteria, which naturally synthesize the commercially important copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), p(3hb-co-3hv), from simple carbon sources such as glucose. 105, 106 the genus bacillus, in common with many other pha-accumulating gram-positive bacteria, accumulates co-polymers of 3hb when grown on different substrates. 98 for instance, copolymers of p(3hb-co-3hv) are accumulated when the cultures are fed with odd-chain-length n-alkanoic acids such as propionic acid, valeric acid and heptanoic acid. 107 the generally-regarded-as-safe (gras) bacterium lactococcus lactis has been genetically engineered to produce pha beads. unfortunately this recombinant strain did not show feasibility for commercial-scale production, since the beads were both smaller in size and contributed less pha per cdw (6%) than other pha producing bacteria. 29 therefore, this platform was designated for added value medical product synthesis (e.g., vaccine development) instead the large scale production. 25 the improvement of the yield would likely require re-engineering metabolic flux to push carbon utilization away from lactate production and toward the pha biosynthesis pathway. 29 interestingly, the platform based on pha functionalized granules was used to develop a phap-based system for endotoxin removal from protein solution. an endotoxin receptor protein was fused with r. eutropha phasin, in vitro attached to phb beads and used to remove lps from the solution. 108 functionalized pha nanobead in vivo performance, cytotoxicity and biocompatibility numerous in vivo studies have clearly demonstrated that endotoxin and bacterial protein free phas provoke mild host reactions in different animal models, 96 which is not surprising when considering the fact that [r]-3-hydroxybutyric acid is a normal blood constituent 109 and is found in the cell envelope of eukaryotes. 110 in vitro based approaches have focused on enhancing growth of different eukaryotic cell lines using arginyl-glycyl-aspartic acid (rgd) tailored pha in form of a scaffold. as such, it showed excellent in vitro performance on supporting and promoting neural stem cell, human bone marrow mesenchymal stem cell and fibroblasts adhesion and growth. [111] [112] [113] phap-rgd fusion immobilization allowed evading tedious cross-linking processes and chemical immobilization that easily damage the biological activity of attached protein. new approaches based on nanoparticulate carriers with targeting capability for imaging and drug delivery to cancer cells are slowly replacing longstanding concepts. with this aim, posterior to synthesis of loaded pha particles, surface modification was performed via hydrophobic interaction between particle surface and growing pha chain from phac enzyme fusion with rgd that stabilized core-shell structure. 31 however, little attention was placed on endotoxin removal and scaffold performance in vivo. alternatively, the pha micelles synthesis was performed in vitro by mixing phac-rgd and 3hb-coa and therefore avoiding the incorporation of endotoxins. 66 bacterial polyester inclusions have been also engineered to display fusion protein of phac and the components involved in immune response to the infectious agent and used as a vaccine delivery system. 19 remarkably, particle-based carriers very closely mimic the physiochemical characteristics of natural pathogens, enhancing particle-displayed protein delivery to the immune system. [114] [115] [116] however, very few in vivo studies address essential issue of immunogenicity of soluble and pha granule bound gaps, considering that the main objective when using biomaterials and nanocarriers is to generate the most appropriate beneficial cellular or tissue response without eliciting any undesirable local or systemic effects in the recipient of the therapy. as the immune response and repair functions in the body are exceptionally complex, the biocompatibility of a material should not be described in relation to a single cell type or tissue. nevertheless, it is essential to consider in vitro and in vivo cellular behavior for further comprehensive biocompatibility evaluation of biopolymers. several studies report no toxic nor pyrogenic effect of wild type or functionalized non endotoxin free pha beads in mice, 19 which suggests that due to the profound differences between mice and human immune systems another animal model should be considered for these type of studies. 117 given the breadth of these functional differences, the discrepancies surely limit the usefulness of mouse models in mentioned studies and as such should be taken into account when choosing preclinical animal models. 118 the results of the study comparing immune response of pha-beads for vaccine application produced in l. lactis and e. coil support this hypothesis since no higher inflammation was spotted for e. coli produced particles. 26, 29 however, this might be due to the pamps, present in both gram-positive and gram-negative bacteria that induce similar immune reaction. in addition, overall impact of functionalized pha nanobeads on eukaryotic organism including levels of ketone bodies and other possible secondary effects are unknown. in vivo tracking of pha nanocarriers might give insight into environmentally-triggered structural changes of nanoparticles and provide additional information about their localization and pathway. in a very different context, complexed phas (cphas) were discovered representing different type of pha structures. unlike bacterial phas that play a major role in carbon and energy storing, these cphas found in mammalian cells are assumed to be involved in regulation of various cell functions through modification of target molecules. 119 complex of cpha with ca 2+ and inorganic polyphosphate is involved in formation of ion-conducting channels in mitochondrial membranes. 120 furthermore, cpha can interact with membrane proteins through hydrophobic and perhaps covalent interactions. 121, 122 it has been suggested that in case of protein channels these interactions might play an important role in regulation of channel function and selectivity. 123 previous studies indicate that cpha can be found in various subcellular compartments of the eukaryotic organisms 124 as well as associated with specific proteins. 125, 126 although, these structures are still not profoundly explored and are in very early stage of investigation, they definitely offer great possibility for functionalization and exploitation. additionally, they might give the critical piece of information on pha metabolism, their uptake and pathway inside the eukaryotic cell essential when dealing with functionalized pha nanobeads designed for biomedical application. besides natural polyesters such as pha, several synthetic polyesters have attracted considerable attention as materials for biomedical purposes due to their attractive properties (e.g., biocompatibility and biodegradability). currently majority of synthetic polyesters systems used in medicine are based on poly(lactic acid) pla, poly(glycolic acid) pga and their copolymer poly(lactic-co-glycolic acid) plga. this is mainly due to their well described formulations and methods for production, as well as their low toxicity and immunogenicity. even though such polyesters have been extensively used for resorbable sutures, bone implants, screws and others, 127 only small number of commercially available products are designed for nanoparticle based drug delivery. 128 nevertheless, synthetic polyesters such as plga have been profoundly tested for this application (reviewed in 128, 129 ) . synthetic polyesters are considered promising candidates for development of the nanoparticle delivery systems to release, target, uptake, retain, activate and localize the drugs at the right time, place and dose. 130 although natural and synthetic polyesters share many common properties (e.g., biocompatibility and biodegradability), due to their specific characteristic one or the other might be more suitable dependently on the application. the main characteristic of synthetic and natural polyesters, significant for nanoparticle production and drug delivery systems are outlined in table 3 . degradation of both, synthetic and natural polyesters, results in biologically compatible and metabolizable moieties. however, their degradation rates and patterns differ considerably. thereby, synthetic polyesters are suitable for sustained release due to their slow degradation rates. importantly, in the case of natural polyesters the drug release kinetics can be more easily controlled via conventionally engineering the pha matrix parameters to reach desired degradation rates. for instance, scl-phas are crystalline and hydrophobic, but many pores are formed on the surface and the drugs are released quickly without any polymer degradation. mcl-pha copolymers on the other hand, have low melting point and low crystallinity, therefore they are more suitable for drug delivery. plga found many applications in biomedical field, such as treatment of cancer, inflammation diseases, cerebral diseases, cardio-vascular disease as well as in regenerative medicine, infection treatment, vaccination and many others. 128, 133 they were also used for diagnostic purposes for magnetic resonance, cancer-targeted imaging 136, 137 and as ultrasound contrast agent. 138 similarly, the good performance of phas for variety of biomedical applications has been proven (table 1) . nevertheless, the main advantage of synthetic plga over natural phas is its fda approval as drug delivery platform and lower production costs. currently, the only fda approved pha is poly(4-hydroxybutyrate) p(4hb) for suture application, which might open the possibility for other phas to be tested and enter the investigations for fda approval. this would significantly influence the development of pha based drug delivery systems and enhance their application. at present, due to its large availability on the market and its relatively low price, pla shows one of the highest potential among polyesters, particularly for packaging and medical applications. for instance, cargill has developed processes that use corn and other feedstock to produce different pla grades (natureworks). 139 in this company, the actual production is estimated to be 140,000 tons/year. presently, it is the highest and worldwide production of biodegradable polyester. its price is lower than 2 €/kg. 140 although, the cost of production of phas is still quite high (3-5 €/kg), current advances in fermentation, extraction and purification technology as well as the development of superior bacterial strains are likely to lower the price of phas, close to that of other biodegradable polymers such as polylactide and aliphatic polyesters. 141 engineering biomaterial nanobeads has attracted much attention of the research community. ongoing efforts to push the boundaries are reflected in the design of wide range of nanostructured bacterial materials for innovative medicines. 1 apart from pha, biologically produced nanoparticles are highly diverse and omnipresent in prokaryotic (magnetosomes, storage paricles, etc.), but also in eukaryotic (e.g., exosomes, lipoproteins, etc.) systems giving the ground to the further development of bionanothechnology. 11 smart pha nanoparticles described in this review provide grounds on how these bacterial polymers, traditionally considered for industrial or conventional clinical applications, are progressively entering the most innovative biomedical fields as promising and highly flexible materials. the fact that pha can be produced from inexpensive waste carbon sources enhanced commercial interest in these polymers. on the other hand, interest in functionalized pha nanobead technology has been hampered by existing legislation in terms of endotoxin concentration allowed for biomedical application. 99 importantly, these technical hurdles were successfully surmounted following in vitro approach or using certain gram-positive strains for in vivo functionalized bead assembly. nevertheless, up-to-date phas are produced on the large-scale exclusively using gram-negative bacteria. 4 for simplicity and cost control the goal is to adapt the approach to a system in which maximal covering of pha granule surface with recombinant protein is achieved. different module swapping strategies and fine tuning were proven effective to reach this goal. 8 to meet the challenges new tendencies suggest multi-functionality. the concept behind multi-functional beads would allow the design of variety of biomedical systems with unique advantage of adaptability and subsequently responding to current trends of biomedicine. pha nanoparticles allow multifunctional tuning due to the possibility of the use of variety of gaps, as well as their both n-and c-terminal domains, to immobilize diverse proteins simultaneously. nevertheless, many nanotoxicological tests on their safety have to be performed before they can overtake the current stage of synthetic polyesters. aside from fda approval for biomedical applications, the production costs should be reduced. table 3 comparison of synthetic and natural polyesters production, processing, properties and application. bacterial polyesters (pha) ref. bio-production of la and chemical synthesis of pla, plga completely biosynthesized 4, 96, 131 no possibility of in vivo production and functionalization in vivo functionalization; one-step production of active agent and carrier, no need to produce, purify and conjugate active agent 26, 54, 131 use of harsh chemicals for production production from renewable sources 4, 132 difficulty to scale-up similar to bioprocesses for pha production; certain difficulties to scale-up 132, 133 production cost comparable with conventional plastics like pet high cost of production; at least twice that of pla 4, 131 high risk due to flammable and toxic solvents low risk level 132 production completed within days production duration 1-2 weeks 132 endotoxin contamination less probable due to synthetic origin endotoxins can be efficiently removed; use of gram+ strains allows endotoxin free production 20 properties lower number of copolymers that can be produced; only d-and l-lactic acids (la) more than 150 monomeric building blocks for polymer design 4, 131 approved by fda and european medicine agency as drug delivery system not approved by fda as drug delivery system 131, 133, 3 low drug loading no limitations regarding drug loading 32, 131, 133 protection of drug from degradation protection of drug from degradation 133, 3, 134 biodegradable, biocompatible, low cytotoxicity biodegradable, biocompatible, low cytotoxicity 30, 32, 96, 3 material properties poor, could be adjusted by regulating d-and l-la ratios good thermomechanical properties from brittle, flexible to elastic, fully controllable, easy processability 4, 30, 96, 135 degradation rate can be controlled degradation rate can be controlled 130, 3 drug delivery kinetics can be controlled drug delivery kinetics can be controlled 32, 130 easy particle size control size of in vitro produced particles might be controlled, in vivo production limits control over particle size 30, 32, 34, 134 application wind variety of biomedical applications applicable to a range of diseases 26, 133 lowering ph at the site of implantation that might lead to sterile sepsis no detected side effect of pha degradation 130, 131 best chance for clinical application due to fda approval. packaging, printing, coating, yet limited by t g of 65-75°c almost all areas of conventional plastic industry, limited by current higher cost and availability 4, 20, 131, 134 the big challenges that pha industry has to overcome 132 to lead to pha nanobeads successfully commercialization are: i) reduction of production costs; ii) construction of functional pha production strains to precisely control the structure of pha molecules increasing the consistency of structure and properties to reach the level of competitor synthetic polymers; iii) reach the simplicity of synthetic polymer processing; iv) use of alternative renewable sources for production to avoid use of expensive glucose; v) development of high value added applications. nanostructured bacterial materials for innovative medicines engineering bacteria to manufacture functionalized polyester beads biodegradable 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microspheres is capable of inducing a t helper type 1 immune response nanoparticles and microparticles as vaccine-delivery systems of mice and not men: differences between mouse and human immunology animal models have little to teach us about type 1 diabetes: 1. in support of this proposal identification of the polyhydroxybutyrate granules in mammalian cultured cells a large, voltage-dependent channel, isolated from mitochondria by waterfree chloroform extraction low molecular weight complexed poly(3-hydroxybutyrate): a dynamic and versatile molecule in vivo posttranslational modification of e. coli histone-like protein h-ns and bovine histones by short-chain poly-(r)-3-hydroxybutyrate (cphb) insight into the selectivity and gating functions of streptomyces lividans kcsa poly-beta-hydroxybutyrate/calcium polyphosphate complexes in eukaryotic membranes isolation and 1h-nmr spectroscopic identification of poly(3-hydroxybutanoate) from prokaryotic and eukaryotic organisms. determination of the absolute configuration (r) of the monomeric unit 3-hydroxybutanoic acid from escherichia coli and spinach gomes me, reis rl. biodegradable polymers and composites in biomedical applications: from catgut to tissue engineering. part 1. available systems and their properties plga nanoparticles in drug delivery: the state of the art nanoparticles-a review poly(3-hydroxyalkanoate)s: diversification and biomedical applications. a state of the art review biodegradable polyhydroxyalkanoate implants for osteomyelitis therapy: in vitro antibiotic release polyhydroxyalkanoates, challenges and opportunities plga-based nanoparticles: an overview of biomedical applications biodegradable polymeric nanoparticles as drug delivery devices polyhydroxyalkanoates: biodegradable polymers with a range of applications multifunctional nanoparticles for photothermally controlled drug delivery and magnetic resonance imaging enhancement designed fabrication of a multifunctional polymer nanomedical platform for simultaneous cancer-targeted imaging and magnetically guided drug delivery current advances in research and clinical applications of plga-based nanotechnology nano-biocomposites: biodegradable polyester/nanoclay systems environmental silicate nano-biocomposites, green energy and technology production of polyhydroxyalkanoates: the future green materials of choice key: cord-266543-ng9zr299 authors: klebe, gerhard title: virtual ligand screening: strategies, perspectives and limitations date: 2006-06-20 journal: drug discov today doi: 10.1016/j.drudis.2006.05.012 sha: doc_id: 266543 cord_uid: ng9zr299 in contrast to high-throughput screening, in virtual ligand screening (vs), compounds are selected using computer programs to predict their binding to a target receptor. a key prerequisite is knowledge about the spatial and energetic criteria responsible for protein–ligand binding. the concepts and prerequisites to perform vs are summarized here, and explanations are sought for the enduring limitations of the technology. target selection, analysis and preparation are discussed, as well as considerations about the compilation of candidate ligand libraries. the tools and strategies of a vs campaign, and the accuracy of scoring and ranking of the results, are also considered. in the late 1980s and early 1990s, experimental high-throughput screening (hts) and combinatorial chemistry were aggressively developed to overcome the lead discovery bottleneck in drug development. using sophisticated large-scale automation, it was anticipated that this would generate an unprecedented number of novel leads, resulting in a substantial increase in novel drug entities launched to the market per year. however, in reality, the opposite was the case [1, 2] . frequently, the discovered hits could not be validated and further optimized into actual leads and preclinical candidates. thus, the initial euphoria surrounding these approaches has subsided owing to the disappointingly low hit rates and significant costs involved [3, 4] . such situations fuel the consideration and development of alternative techniques. the expression 'virtual screening' (vs) was coined in the late 1990s; however, the techniques involved are much older. in an effort to show that searching for lead candidates using a computer is a serious alternative to hts, the term 'vs' was adopted by the community. in contrast to hts, which is largely phenomenological and technology driven, in vs, compounds are selected by predicting their binding to a macromolecular target using computer programs (in drug discovery, the term 'target' or 'receptor' is used frequently to describe the macromolecule to which a drug binds, which is usually a protein but can also be dna or rna). the compounds studied do not necessarily exist, and their 'testing' does not consume valuable substance material. experimental deficiencies, such as limited solubility, aggregate formation or any sort of influence that could possibly interfere with experimentally applied assay conditions do not need to be considered in the initial computational screen. in contrast to hts, vs requires as a key prerequisite knowledge about the spatial and energetic criteria responsible for the binding of a particular candidate ligand to the receptor under investigation. in consequence, either the three-dimensional (3d) structure of the macromolecular target -as given by crystal structure analyses, nmr or sophisticated homology modelling -or, at the very least, a rigid reference ligand with a known bioactive conformation mapping out the putative receptor binding site must be available [5] . this defines vs as a knowledgedriven approach. even though it is likely that multiple screening campaigns have been performed in parallel in industry [6] [7] [8] [9] , it was only recently that mcmaster university launched an open and unbiased competitive screening against escherichia coli dihydrofolate reductase to detect inhibitors [10] , to assess how well vs can enrich candidate ligands compared with hts random screening [10, 11] . the original sample set of compounds used for screening was split into two, and one fraction was tested by hts. obtained hits were reported and served as a training set to validate and tailor applied vs tools in different research groups, who entered into a competition to retrieve hits also detected by the experimental screen in the second portion of the screening sample [12] [13] [14] [15] [16] . subsequently, in a totally unbiased fashion, the second part of the data sample was evaluated by hts and vs in parallel. unexpectedly, the second portion did not show any hits as competitive inhibitors, as would be expected for ligands docked into the substrate binding site of a target protein. interestingly, brenk et al. [16] reported on some promising hits found by docking that were not detected as inhibitors in the initial hts. retesting at higher concentration indicated weak inhibition. this observation could be seen as supporting the view that vs and hts are complementary approaches and that they might find candidates missed by the other [7, 9] . as mentioned above, the methods involved in vs are much older than the approach itself. initial attempts to find ligands by docking or by mapping them onto ligand-based pharmacophore models were the generic prototypes of a vs approach. however, this term was not used at that time, probably because computers and algorithms were not fast enough to enable large scale applications. focusing on approaches that actually make use of an available protein structure, one of the first systems to be studied by docking was hiv protease. initial versions of the program dock, developed over many years by kuntz's group [17, 18] , tried to dock rigid entries from the cambridge crystallographic database into the protein receptor, focusing primarily on shape complementarity and later considering chemical complementarity. in 1990, the kuntz group retrieved the neuroleptic drug haloperidol as a potential 'lead' from a docking screen using a database of known drug molecules as input. however, this compound would have had to be administered at a very high dose -far beyond a toxicologically tolerable concentration -to be an effective inhibitor of the protease [19] . nevertheless, haloperidol as a 'lead' gave rise to some new ideas for developing a derivative possessing 15 mm inhibition of hiv protease [20] . later, at dupont-merck, a 3d database search retrieved a substituted terphenyl derivative as a putative lead for inhibiting this protease. further optimization via six-and sevenmembered rings resulted in the class of cyclic ureas that are able to replace the crucial structural water molecule in the protease, at the same time targeting the carboxy groups of the two catalytic aspartates via two appropriately placed hydroxy functionalities [21] . since these early vs attempts, a plethora of case studies has been performed and the list of success stories is steadily growing. despite the fact that vs is still a young discipline, it has been reviewed frequently [22] [23] [24] [25] [26] [27] [28] [29] [30] , most recently in a comprehensive overview by kubinyi [31] . if one excludes purely retrospective studies, in which the potential of a method is demonstrated by its ability to enrich putatively active molecules from a sample of anticipated nonactive ones, 50 targets have been studied to date, and reports on the discovery of mostly micromolar binding ligands in a truly predictive fashion are available (table 1) . still under development, and far from mature, the number of strategies followed in vs is nearly as large as the number of reported screening campaigns. owing to space constraints, this review is unable to give a comprehensive overview of success stories or investigated targets, or to review all details of currently applied vs protocols; accordingly, the reader is recommended to consult the abovementioned surveys [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] . instead, this review attempts to summarize and comment on the agreed concepts and prerequisites for performing successful vs runs. furthermore, it attempts to analyse the limitations of the approach in a frank and plain manner and seeks to explain why such limitations still exist. accordingly, in the following sections, first the target selection, analysis and preparation are discussed, followed by considerations about the compilation of a candidate ligand screening sample. this is followed by a discussion of the actual requirements, tools and strategies of a vs campaign, and, finally, remarks concerning the accuracy of the scoring and ranking of the screening results. being a knowledge-driven approach, the scope of vs strongly depends on the amount and quality of information available about the system under investigation. clearly, the availability of the target receptor is of great benefit compared with situations where only a rigid reference ligand is known. the target receptor could be any macromolecular biomolecule, either a protein, rna or dna. in terms of the rigid reference ligand, either a substrate, inhibitor molecule, agonist or antagonist (e.g. a steroid hormone) could serve. because the structures of target receptors are becoming increasingly available, this review concentrates solely on such situations. however, it does not mean that vs cannot be applied to situations in which no structural information about the target receptor is available [5] . with respect to dna and rna as target receptors, the development of appropriate docking and scoring tools has been initiated only recently, and, accordingly, vs applications on such targets are still rare [32, 33] . by contrast, the scope of applications addressing proteins ranges from enzymes to gprotein-coupled receptors and ion channels (table 1) . assessing the druggability of the target receptor the first issue to be considered is the druggability of the selected target. does the selected protein exhibit a binding pocket that can be successfully targeted by small molecule ligands? clearly, characteristics such as pocket size and geometry, surface complexity and roughness, exposure of recognition properties and their complementarity in shape and polarity with respect to a putative druglike ligand, are of importance. because such criteria are not immediately available, a pragmatic, but hardly generally applicable, approach would be to correlate gene families. if one member of such a family is able to bind a drug, other members might also be able to bind druglike ligands with related physicochemical properties [34] . this assumption is based simply on the fact that members of a gene family usually operate on related substrates or recognize similar endogenous ligands. however, the setup for a vs study requires more conclusive information about the actual bindingsite architecture. recently, hajduk et al. [35] suggested several very decisive indices that help to discriminate druggable from nondruggable binding pockets. for druggability, the total surface area and a portion of a polar contact area below 75 å 2 appear to be beneficial, along with an appropriate pocket compactness, surface roughness and complexity. this suggests that there is an optimal size and composition of a protein-binding pocket that is best suited to recognize and accommodate small organic ligands. however, the analysis by hajduk et al. also showed that no single index consistently dominates the correlation. this fact points to the complexity of the interrelationships among the various discriminatory indices and explains why, at present, our concepts for predicting druggability are still rudimentary. selection of the most relevant geometry of the target receptor the selection of an appropriate 3d geometry for the target is another important issue when setting up a vs run. the most powerful method for learning about the spatial structure of proteins is crystal structure analysis. the accuracy and reliability of this method strongly depends on the resolution of the diffraction data. an alternative experimental technique to determine protein structures is nmr; there the accuracy of the structural determination depends strongly on the local distribution of the nuclear overhauser effect distance information along the protein chain (this effect involves transfer of magnetization through space and gives information about distances between nuclei in a 3d structure). furthermore, in the past, significant methodological enhancements in homology modelling have improved the quality of protein models [36] [37] [38] ; accordingly, several successful vs screening campaigns have been reported based on model-built protein structures [39] [40] [41] . homology modelling strongly depends on the availability of related proteins for which a crystal structure has been determined. the model will be particularly precise in those areas where the homology with the experimentally determined references is high, which is usually the case in the conserved core regions. however, functional binding sites, to be addressed by vs, are generally located in loop regions where, even among homologues of a gene family, significant differences are experienced (apart from catalytic residues in enzymes that are spatially highly conserved). to enhance the accuracy of model-built structures in regions where the binding sites are found, new algorithms have been suggested that consider the putative binding orientation of known ligands during the homology modelling process. these algorithms feed the information about the binding properties of possibly bound ligands back into the homology building process. improved binding site geometries can be expected from such approaches [42, 43] . another obstacle that complicates vs attempts is molecular flexibility. ligands and proteins possess internal degrees of freedom and can adopt various conformational states. with respect to the target protein, several methods have been described to simulate flexibility [44] . for vs, it is important to obtain an estimate of the protein conformers competent at accommodating a ligand. once these conformers have been determined, a vs run can either be performed by considering the flexibility of the protein instantly during the calculation, or by addressing an ensemble of several rigid receptor conformations [45] [46] [47] [48] . ligand-binding competent conformers can either be sampled by exhaustive conformational searches (e.g. using molecular dynamics simulations [49, 50] ) or by examining multiple conformational states, observed in crystal structures with different ligands bound, to obtain insight into the relevant conformations [51, 52] . the latter approach appears tempting because it can be assumed that a sample of experimentally observed protein conformers corresponds to stable, low freeenergy states. even though the crystal structure is usually considered as the 'gold standard' for learning about the geometry of a protein, it is highly questionable how representative a single structure determination really is. mcgovern and shoichet [53] reported on increasing information decay, irrespective of whether the geometry of a ligand-bound or ligand-free crystal structure was used or a model-built structure was considered. principally, crystalline protein-ligand complexes can be prepared by exposing preformed crystals of a protein to the solution of a ligand, which can then diffuse into the crystals and find its way to the binding site. this process is called 'soaking'. alternatively, in a cocrystallization experiment, the protein-ligand complex is formed by equilibrating them in solution, and then the assembled complex is crystallized. we recently observed, depending on the soaking protocol applied, different conformational states of the protein aldose reductase complexed by the inhibitor zopolrestat [54] . in one structure, the protein forms a hydrogen bond with the ligand via one of its binding site-exposed amide bonds ( figure 1 ). in a second structure, the amide bond is rotated off from the binding site, and the same ligand can no longer form this hydrogen bond. such differences have a significant impact on docking and scoring results in vs. depending on the crystallization conditions, ligands have been observed to accommodate a binding pocket with reversed orientation [55] ; in other cases, ligand-induced conformational adaptations of the protein are observed [56] ( figure 2 ). increasing the concentration of a ligand in the soaking buffer will enhance the opportunity to accommodate it in the binding pocket. however, it is likely that soaking depicts some kind of kinetic trap for ligands; consequently, surprising differences in cocrystallization have been observed (several ligands bound at one time, multiple binding modes [57] ). before selecting a particular crystal structure as a reference for a vs run, detailed analysis of parameters such as population of the bound ligand, b-factors next to the binding site (b-factors indicate thermal motion in a crystal structure; however, they are highly correlated with the population of a ligand in the crystal) or consistency of the hydrogen bond network is advisable. in addition, most programs used for the actual computer screens require properly defined protonation states of the active-site residues. this is by no means easy to perform because local dielectric drug discovery today volume 11, numbers 13/14 july 2006 reviews induced-fit adaptations in two different crystal forms. two crystal structures of benzamidine (in both cases, accommodated deeply buried in the s1 pocket, which is indicated as a deep depression in the left-and right-hand images) bound to a trypsin mutant exhibiting a binding pocket related to factor xa. both crystal structures differ in terms of the conformational state of phe174 (in the left-and right-hand images at the left rim of the binding pocket with the red surface), which is exposed ('up') in one structure (orange and on the left) and buried ('down') in the other (green and on the right). this adaptation involves partial unwinding of a short three-turn helix (in the centre, helix at the left-hand side of the binding pocket) and rearrangement of a disulfide bond (in the centre, yellow bond to the back) [56, 60] . binding mode differs with soaking and co-crystallization conditions. crystal structure of aldose reductase with the inhibitor zopolrestat is shown. the crystal structure determined after one day of soaking (yellow) differs from the structure obtained after six days of soaking (magenta) but is identical to that obtained by cocrystallization (light blue). whereas in the one day-soaked structure (yellow) the amide bond between ala299 and leu300 orientates its nh group towards the inhibitor to form a hydrogen bond (dotted yellow line) with the bound ligand, in the latter two structures (magenta and light blue) this amide bond rotates away from the inhibitor and no such hydrogen bond is observed [54] . www.drugdiscoverytoday.com 583 conditions can modulate pk a values of functional groups by several orders of magnitude. even more complex and, at present, difficult to calculate are pk a shifts of residues during the course of ligand binding. isothermal titration calorimetry [58] measurements can make such changes apparent; these changes probably occur more frequently than we presently admit [59] , and can easily turn an acceptor functional group into a donor or a charge-assisted hydrogen bond into a neutral one. these changes are of significance during various validation steps of a vs run. we have recently observed that even the protonation states of the residues in the binding pocket of inhibitor-bound aldose reductase can change depending on whether the cofactor nadph/nadp + is present in the oxidized or reduced state ( figure 3 ) (o. krämer and g. klebe, unpublished results). with respect to protein flexibility, the decision has to be taken as to whether a vs run should consider one single protein conformer or multiple binding-competent states of the receptor. different strategies with respect to docking and scoring will be the consequence (see below). experience shows that conformational adaptations observed in a protein-binding pocket are usually related to the structural modulations potentially needed by the protein to accomplish its functional role [51] . such functional adaptations must correspond to low-energy transformations, otherwise dramatic shortcomings in the functional performance of the protein would be the consequence. accordingly, enzymes that operate on a large palette of structurally diverse substrates (cf. aldose reductase and short chain dehydrogenases) or perform substantial conformational adaptations during catalysis (cf. kinases) will experience multiple bindingcompetent states that have to be considered in vs. thus, a profound understanding of the functional properties of a protein might be the best option for predicting the conformational adaptability of a protein. such information is not usually available at the beginning of a drug development project, when vs is used. because proteins occur in gene families of closely related members, the analysis across different entries of the family might provide some insight into the flexibility properties inherently shared by the members of the family. however, it might also be that some family members display rigid solutions, as required by their function (e.g. trypsin and factor xa among the serine proteases), whereas others possess pronounced flexible behaviour, as appropriate to their function (e.g. the serine protease factor viia) [60] . protonation states can change upon ligand binding. depending on the oxidation state of the bound cofactor nadph (left) and nadp + (right), aldose reductase binds to a carboxylate-type inhibitor (e.g. idd594) by placing its acid functional group (red) into the catalytic centre; the complex thereby formed remains either in an unchanged protonation state (left) or picks up protons upon binding (right). in the centre, the crystal structure of the complex is shown. a short contact distance between the carboxylate function of the inhibitor (shown with a yellow surface) and the nicotinamide portion of the cofactor (shown with an atom-type coloured surface, where oxygen is red, nitrogen is blue, carbon is white, sulfur is yellow and phosphorous is orange) is formed. high resolution x-ray crystal structure analysis and neutron diffraction has provided evidence that the inhibitor binds with its carboxylate function in the deprotonated state, and the active site his is present in the neutral state. it remains unresolved where the proton goes in the case of the nadp + -bound complex, or whether the residues of the catalytic centre exhibit deviating protonation states in the uncomplexed and inhibited situation. with respect to vs, a unique and precise assignment of the protonation states of the ligand and protein functional groups in a pk a range between 3 and 11 is essential because, for example, for docking it is important whether such a group is considered as donor or acceptor of a hydrogen bond (krämer and klebe, unpublished). after the reliability and relevance of the protein conformer(s) of the selected target protein have been assessed, its binding-site properties should be mapped before blindly starting a vs run (see below). this strategy helps the user to evaluate the properties of the target protein better and subsequently to examine the relevance of docking solutions suggested by vs. several tools have been described to elucidate the 'hot spots' of binding in a particular binding pocket [61] . these methods are either based on thoroughly parameterized force fields [62] or on well-selected empirical information (superstar [63] , drugscore [64] ; figure 4 ). most importantly, this analysis has to be performed on all multiple conformational states of a binding pocket because this will provide a composite picture of how the molecular recognition properties of a binding site might change upon protein adaptation. water, the nasty third binding partner in proteinligand complexes finally, a very crucial decision with respect to the setup of the protein reference for a vs run concerns the consideration of water molecules in a binding site. the analysis of several thousand crystal structures of ligand-protein complexes using the waterbase module in relibase [65, 66] revealed that, in about two-thirds of all cases, a water molecule is involved in ligand binding, frequently mediating contacts between protein and ligand. thus, any approach based on the prediction of binding modes of putative candidate ligands, as required in vs, must take water into consideration. one possible approach for learning about water molecules tightly bound to the protein is the analysis of crystallographic data with respect to the repeated occurrence of water molecules in structurally related binding sites (e.g. in a gene family) or multiple structural determinations of the same protein with many distinct ligands [66] . if water is present in all structures analysed at more or less the same location, this is a strong indica-tion that this water molecule is tightly bound, and in a vs run could be considered as an integral part of the target structure. as with hts, vs needs a thoughtfully designed and thoroughly compiled sample of small molecule candidate ligands for screening. pharmaceutical companies will primarily screen their own proprietary compound collections, with the advantage that detected hits will be exclusive and will cover molecules for which the synthesis is well-established at their site. however, are such sample pools biased? how well is the available chemical space covered? these considerations have led large pharma companies to complement their inhouse collections with compounds offered through commercial suppliers. starting with the available chemical directory as the initial prototype (see mdl, http://www.mdli.com), there are currently more than 10 million unique purchasable compounds on offer [67] . how well do they cover chemical space, and which portion of this space represents druglike molecules? some dramatic figures have been proposed concerning the number of organic molecules that can be considered as druglike. usually, molecules meet these criteria if they are composed only of the elements h, c, n, o, p, s, cl and br, and possess a molecular weight <500 da [68] . how diverse should the entries of a compound database used for screening be? what physicochemical properties have to be met by the candidates to guarantee sufficient bioavailability? the concept of a well-balanced and homogeneously populated space of diverse druglike molecules appears very tempting; however, there is no proper definition of what descriptors to use as coordinates on the axes of such a compound space. what is 'diverse' in this context? as with the criterion 'similarity', the expression 'diversity' is a relative measure that relates to a reference point. in vs, the reference point is the target receptor and the difference in binding mapping the hot spots of binding. hotspot analysis using drugscore for the binding pocket of t-rna guanine transglycosylase (surface of the binding pocket indicated in white). regions energetically favourable for the binding of a hydrogen bond donor group (represented by an nh group) or an acceptor group (represented by a c=o group), or a hydrophobic molecular portion (represented by aromatic carbon atoms, c.ar) have been analysed and contoured on three subsequent levels (indicated using three different colours) above the deepest energy minimum found for each atom type in the maps [88] . the crystallographically determined binding mode of an inhibitor is shown superimposed. www.drugdiscoverytoday.com 585 affinity of two ligands with respect to this structure. this defines whether two molecules should be classified as 'similar' or 'diverse'. it can be imagined that, for one target, a correctly placed methyl group on a ligand might have a dramatic effect on binding affinity, whereas for another it will be tolerated with no change in binding affinity. in the first case, the ligands with and without a methyl group would have to be termed 'diverse', whereas in the second, they will be called 'similar'. however, consulting a 'diversity' scoring that concentrates solely on the topology of a ligand, the methylated and unsubstituted derivatives will probably be ranked as 'highly similar'. is there an optimal size for candidate ligands to be submitted to screening? in the past, combinatorial chemistry in particular has enabled pharmaceutical companies to develop screening libraries of several million candidate molecules; thus, in principle, the sheer number of test compounds is no longer an issue. although large in total count, the individual members of such libraries are usually large in terms of size and molecular weight. they are mostly in the range of typical drug-size molecules, having been synthesized in other drug development projects. however, if they turn out to be a micromolar screening hit, they still require optimization to improve their affinity towards the target protein by two or three orders of magnitude. this has to be accomplished while maintaining reasonable molecular size and appropriate absorption, distribution, metabolism and excretion properties. in consequence, it involves stripping down to a scarcely decorated core skeleton while subsequently building up this core again with novel well-tailored side chains. this is a challenging task, and is often difficult to achieve. experience from drug optimization programs has shown that small core fragments ('privileged templates' or 'fragments') known to bind with significant affinity are ideal starting points for further optimization [69, 70] . accordingly, the compounds selected for vs should leave some room for optimization, thus matching the range of socalled leadlike or fragment-like molecules [71] . verdonk et al. [72] have presented a very insightful discussion on approaches to be taken when constructing databases for vs, and have suggested that the methods involved should be validated (e.g. by assessing the enrichment rates of known binders). for this purpose, in a vs campaign, known binders are pooled with the sample set of screening candidates to assess how well the known binders 'enrich' during the course of the screening process. such considerations are important for the development of new methods; however, in an actual drug development project, at the end of the day, the medicinal chemist will ask for novel leads that are worthwhile pursuing with synthesis and optimization. impressive enrichment rates of known actives will not be convincing. in light of these considerations, what is a suitable compound collection? some general criteria have to be matched either by inhouse proprietary compounds or by substances offered by commercial suppliers. compounds can be validated with respect to drug likeness considering lipinski's rule of five, or lead likeness applying more stringent criteria (as defined by the rule of three) [73] [74] [75] . recently, martin tested lipinski's frequently applied rule with respect to experimentally determined bioavailability in the rat [76] . this study showed that the rule of five has predictive ability for neutral and positively charged molecules; however, anions obey different rules, and the size of their polar surface area confers some predictive power. similarly to the considerations concerning the druggability of binding sites, the criteria for bioavailability are multifactorial. irwin and shoichet took the initiative to set up the freely available database zinc with validated compounds for vs [67] . it is built from 2d compound information, generates 3d coordinates and curates, if possible, from stereo-and regioisomeric ambiguities. multiple states with respect to protonation, charges and tautomers are enumerated. however, as described for proteins, the properties of compounds might be altered upon protein binding. insoluble, reactive and aggregating compounds are not represented in the database. the rule of five is a straightforward filter to discard compounds with putatively undesired properties from the screening sample. depending on the strategy pursued in the subsequent vs campaign, multiple conformers can be precalculated and stored as separate entries in the database. limiting the search sample to purchasable compounds in vs is a pragmatic approach because screening hypotheses can be tested rapidly [67, 77] . however, vs can also scan over virtual compound libraries, and synthesis can be postponed to a later stage, considering only the most promising hits. reymond's group attempted to generate a database of all possible organic molecules up to 160 da under the constraints of defined chemical stability and synthetic feasibility [78] . this database contains 13.9 million entries. it is possible that such a sample could be used for fragment screening. for larger compounds, exhaustive sampling of possible molecular skeletons will end up in a combinatorial explosion with too many possible solutions. however, proper design criteria, defined by the architecture of the binding site used in vs as the target, might guide the generation of target-tailored virtual libraries for vs. in particular, considering the criteria of combinatorial chemistry and parallel synthesis, such vs strategies can actually help to synthesize only the most promising entries of a large virtual combinatorial library. principally, two strategies can be followed in a vs campaign: forward or backward filtering of hits obtained by docking. the most crucial step in vs is the docking of candidate molecules to the target protein [79] [80] [81] [82] [83] [84] [85] [86] . in forward filtering, various criteria are used to reduce the initial data sample, which might comprise several millions of test compounds, to the several hundred or thousand most promising candidates to be docked [87] . in backward filtering, all entries from the data sample are docked to the target protein, and filter criteria are subsequently applied to rank the generated docking solutions. nowadays, the speed of computers is no longer the limiting factor in selecting the strategy. the forward technique requires fewer computational resources. this is mainly because of the fact that at each hierarchical filtering drug discovery today volume 11, numbers 13/14 july 2006 step, a significant amount of the original data sample is discarded. however, with a decreasing number of compounds, the filtering becomes computationally increasingly demanding and sophisticated. flexible docking is the computationally most intensive step of all; thus, the fewer candidates to be considered here, the more effort can be spent in controlling, validating and assessing docking results. this is clearly an advantage of this strategy. forward filtering eliminates compounds initially according to simple descriptors such as molecular weight, number of rotatable bonds, lipophilicity (usually expressed by the logarithm of the partition coefficient, log p) or crude shape descriptors, such as the ellipticity of the overall structure. subsequently, information about the receptor's binding site is exploited. once a hotspot analysis of the most likely anchoring positions in the binding pocket has been performed, a protein-based pharmacophore can be derived [88] . this pharmacophore sets the constraints for the minimal requirement of functional groups to be matched by putative ligands (e.g. number of hydrogen bond donors, acceptors or hydrophobic groups). molecules satisfying such criteria can be retrieved by any database engine capable of a functional group substructure search. once the topographical arrangement of the protein-based pharmacophore has been incorporated into the search and the remaining candidates are requested to match this pattern, the study can be further focused. unity (unity chemical information software, version 4.1, tripos) and catalyst [89] are prototypes of such database engines supporting this screening step. an alternative tool is featuretrees, which can retrieve molecules of similar topology in feature space [90] . in this context, 'features' are considered as being similar types of functional groups or molecular building blocks. similarity can be considered as a further filter criterion in vs. for example, if candidate molecules are requested to match with a predefined protein-based pharmacophore, this condition already involves a selection in terms of similarity criteria; however, they are regarded very generally. furthermore, information about known binders can be used for filtering, although the danger then exists that, owing to biased filters and preconceived concepts, some unexpected and novel chemistry is discarded during the early filtering steps. finally, docking is pursued, usually considering only 1-10% of the initial sample collection. an advantage of the forward filtering approach is that it enables more elaborate docking protocols to be performed -for example, taking multiple protein conformational states into account or reflecting a protein-based pharmacophore as a restraint in docking. most importantly, this hierarchical filtering strategy enables the tracking of the performance at the various filter levels by human intervention, and, in particular, visual inspection of docking solutions remains feasible. backwards filtering starts with high-throughput docking and analyses the generated docking modes as subsequent steps. this approach is especially challenging because docking returns multiple solutions for most candidate molecules. strongly discriminative and reliable scoring functions must be available for analysing the computed results in an automated fashion because visual inspection for the large number of diverse compounds to be examined is hardly feasible. however, it is questionable whether the existing scoring functions are reliable enough to succeed with such heroic demands (see below). nevertheless, to proceed with the multiple docking solutions from a high-throughput run, the generated docking modes can be filtered with respect to achieved matching of the protein-based pharmacophore, contact complementarity of protein and ligand surfaces or the remaining residual unoccupied voids along the protein-ligand interface. because nature probably avoids gaps in molecular assemblies, the latter criterion could be a powerful indicator for irrelevant binding modes or the putative accommodation of interstitial water molecules. docking is the crucial step in vs. the seminal program dock, originally described in 1982 by kuntz et al. [17] , has evolved as the first vs tool. later, other programs were successfully applied in vs, such as gold [91] , flexx [92] , glide [93, 94] or autodock [95] , to name the most popular prototypes. these have been recently reviewed [96] [97] [98] [99] [100] [101] [102] . all docking tools follow slightly different concepts. this might give individual programs a particular advantage in one task with respect to another -for example, incorporating aspects such as flexibility of ligand and receptor or restraining the docking search engine to particular regions in configuration space (e.g. mapping a protein-based pharmacophore) [103] . however, all docking tools are still far from perfect. an even more challenging, but carelessly disregarded, aspect of docking is the appropriate consideration of water molecules. as indicated above, water molecules are involved in binding in about two-thirds of the known protein-ligand complexes. however, most docking tools ignore them simply because conclusive concepts of how to consider them correctly are missing. if structural evidence is given, preplacement of water molecules in a docking run is a feasible strategy [66] . the docking tool slide [104] treats preplaced water molecules in a way that still enables their replacement by ligand atoms in docking. the particle concept in flexx [105] enables the placement of water molecules 'on the fly' during the course of the generation of individual docking solutions. in the docking tool gold, water molecules can be switched on or off, and can spin around their principal axes to achieve good contacts with a docked ligand [106] . recently, the popular docking tools dock and flexx have been equipped with features that enable docking on a preconceived pharmacophore or property distributions [107, 108] . consideration of such criteria will drive docking solutions especially into regions either frequently trapped by other bound ligands or featured by complementary analytical tools as being particularly relevant for binding. the program autodock [95] performs docking on a precalculated grid, storing potential values from any sort of interaction field [109] . in the original implementation, lennard-jones and coulomb potential values are used. sotriffer et al. [110] replaced these by knowledge-based potentials, originally implemented into drugscore. the latter potentials have proven to be powerful for discriminating, and rank among multiple ligand poses. the potential grid approach in autodock also enables one to average across the fields produced by various protein frames, so that the conformational degrees of freedom of a protein can be considered. in addition, adapted fields optimized with respect to the binding properties of some known actives in the comparative molecular field-type approach 'adaptation of fields for molecular comparison' (afmoc) [111] can be used as target potential values in autodock [112] . the latter docking tool performs multiple stochastic searches on the potential hypersurface; accordingly, the frequency of occurrence of certain docking solutions can be used as an additional figure of merit for their relevance [113] . the issue of an optimal ligand size for screening has been addressed above. the complexity of the docking problem increases with the size of the ligand and its number of rotatable bonds. thus, smaller molecules in the typical range of ligand fragments should be simpler to dock. perplexingly, present experience indicates the opposite to be the case. fragments are easily scattered over a binding site by docking; reliably successful docking can only occur if the binding site itself is restricted in size and shows dimensions similar to the fragments [48] . interestingly, experimental approaches show the opposite: small molecular fragments (>200 da) usually populate, in a well-defined manner, in a very limited number of sites in binding pockets [69, 70] . enrichment rates to control the achievements of virtual screening vs runs are usually monitored and validated by comparing the performance of a set of known actives with a large number of 'randomly' picked compounds. the actives are pooled with the random entries. all compounds are submitted to the selected vs protocol, and the performance ranks of the known actives with respect to the remaining pool are converted into enrichment plots. such a process is essential for keeping control over the performance and achievements of vs. however, the choice of the random compound library is crucial and can strongly affect the enrichments obtained [72] . accordingly, it has to be examined whether the pooled, randomly picked decoy structures are actually nonbinders. in a real-life scenario, it should be noted that merging known actives with a set of candidate ligands should result in a gradually declining enrichment rate at the subsequent hierarchical filter steps because novel actives, retrieved by vs, will populate in prominent ranks and dilute the set of predefined known actives. independent of the actual vs strategy applied, docking and subsequent scoring of the suggested solutions is the key performancedetermining factor in vs. the discriminatory power of the applied scoring function is of utmost importance for ranking, and hopefully enriching, potentially active binders at the top of the list of docking solutions. in consequence, a myriad of scoring functions has been developed over the past few years [114] [115] [116] . approaches have been taken not simply to rely on one single function but to take on board the consensus picture of several scoring schemes [117, 118] . to characterize the binding affinity of putative lead candidates experimentally, the binding constant or its inverse, the dissociation constant, is determined (or approximated by values such as the ic 50 ). if we assume that the basic rules of equilibrium thermodynamics can be applied, we can define, according to the law of mass, an equilibrium constant that describes the formation of a protein-ligand complex. this equilibrium constant is logarithmically related to the gibbs free energy, comprising both an enthal-pic and entropic contribution. whereas the former relates to energetic features, the latter concerns configurational and ordering phenomena. the entropic contribution estimates how the energy content of the system is distributed over internal and external molecular degrees of freedom. up until now, three strategies have been followed to predict binding affinities on the basis of a given protein-ligand binding geometry. the most rigorous and theoretically most solid approaches are first-principle methods [59] . using quantum mechanics or computationally less demanding (however approximate) force fields, the partition function of a system is computed and the free energy differences between the bound and unbound state are determined. with the increasing speed of computers, such methods are becoming more accurate and obtaining a growing relevance for scoring [119] . however, screening large samples of docked solutions to estimate binding affinities is still far beyond tractability. nevertheless, first-principle methods do not need any calibration or training in experimentally determined affinity data; thus, they will not suffer from inherent experimental shortcomings or accuracy limits. this differs from the other two approaches described in the following section, the regression-and knowledgebased scoring functions, which are based on empirical concepts [59] . regression-based approaches assume additivity of individual terms considered in a master equation to describe the total gibbs free energy of binding. in this context, a 'term' can reflect any physicochemical property of relevance for the protein-ligand binding process -for example, the number of charged or uncharged hydrogen bonds, the size of the polar or nonpolar surface portion, the number of rotatable bonds or the enthalpy required to desolvate either the ligand or the protein, among others. they are assumed to be independent from each other, and their individual contribution in reproducing the known affinities of some training set ligands is extracted by regression analysis, partial least-squares analysis or neural networks [59] . however, independence of the 'terms' is unlikely, and fair to strong correlations between terms are probable. in the correlation analysis, this fact might point out that another, at first glance surprising, property pops up as the best explanation. however, it might be the case that the latter property is not on its own essential in a physical sense, but is highly correlated with another property that is actually crucial. we recently parameterized a new regression-based scoring function on the basis of a large sample of crystal structures with known affinities for the bound ligands. depending on the composition of the training set, different terms are found to be relevant in the analysis. this clearly shows that such empirical scoring functions reflect a best fit with respect to the training set used but they rarely achieve generality. an alternative to regression-based approaches are knowledgebased scoring functions [59] . these evaluate the occurrence frequencies of some properties of interest -for example, the mutual distance between particular atom types found across the proteinligand interface. the sample distributions describe occurrence probabilities and can be compared with a statistical mean reference situation. any deviations from this average can be translated using some type of mathematical relationship into statistical preferences that can be used to determine the geometry of protein-ligand complexes. conceptionally, the knowledge-based functions appear to be more general because no master equation with preconceived 'terms' is required. however, the data selected to derive the function, and the definition of atom types, together with many adjustable parameters needed to actually establish the method, also attenuates the generality of this approach. to estimate binding constants, both the regression-and knowledge-based scoring functions require experimental affinity data for internal calibration. thus, their prediction accuracy can never be better than the precision by which binding data can be measured. it is interesting to note that the estimated standard deviations of such empirical scoring functions are reduced if data for a selected number of targets, determined in one laboratory based on assay data recorded under strictly conserved conditions, are used or if broad-range data covering many targets are considered based on assay data determined in various laboratories. the relative differences in binding data within a series of compounds can be determined precisely -usually much better than across data from various systems for which the comparison has to be performed on an absolute scale. in consequence, the standard deviations of predicted binding affinities of presently available functions range between 0.7 and 1.5 logarithmic units in binding affinity. this range matches the experimental accuracy achieved for binding data across training sets of growing heterogeneity covering a broad spectrum of targets. in our experience, knowledge-based scoring functions are better able to extract binding geometries generated by a docking program that closely approximate the experimentally confirmed binding mode from a sample set of decoy placements. by contrast, regression-based scoring functions are better in the actual affinity prediction, provided that a fairly accurate binding geometry is given. unlike the professed opinion that in docking the geometry problem has been resolved to a sufficient extent, and that the scoring problem remains an open question [120, 121] , it appears that both are intimately related. we recently developed a knowledge-based scoring function based on accurate contact data from small molecule crystal data [122] . this function reliably recognizes the experimentally determined binding mode found in a crystal structure among a set of decoy poses. it appears selfevident that the scoring problem can only be alleviated if more relevant, near-native binding poses are produced by docking programs and reliably recognized by a scoring function. accordingly, it appears advisable to drive docking solutions as close as possible to the native geometries -that is, as they would show up in a corresponding crystal structure. this can also be achieved by optimizing them towards the near-native situation. at best, this involves minimization with respect to the function used for scoring. as a disadvantage, this process would be computationally demanding. binding affinity: a sufficiently well understood property? any discussion of scoring and ranking must ask the question as to how well we understand the target value gibbs free energy. is it advisable to focus scoring on free energy or would it be better to treat enthalpy and entropy as separate terms in the scoring [123] ? there is a mutual compensation between enthalpy and entropy owing to the fact that both entities scatter over much larger ranges than does the free energy itself. considering the binding of a ligand to a protein, enthalpy (dh) and entropy (-tds) can easily scatter over a 3-4-fold larger range than the spread of gibbs free energy (dg). the mutual compensation of enthalpy and entropy can even be found across closely related molecules [124] (figure 5 ). furthermore, many physicochemical phenomena of relevance for the binding process are not yet fully understood and, therefore, have not yet been correctly incorporated into scoring functions (e.g. the role of water, change in protonation states or an appropriate consideration of entropy). interestingly, microcalorimetry indicates that with increasing temperature, the protein-ligand binding process becomes more exothermic and entropically less favourable [58, 59] . because this observation applies to all targets, it suggests that general phenomena are involved which are not yet understood at a molecular level. despite our present deficiencies in understanding the physics of the binding process, scoring still works satisfactorily -most likely because we consider the binding of ligands to a protein on a relative scale to each other. accordingly, any unappreciated phenomena, similar across all complexes in the analysis, will simply be cancelled out. for example, as mentioned above, one approach for reflecting protein flexibility applies parallel docking into several rigid binding-competent conformers of the protein. the disadvantage of this strategy is the fact that additional degrees of scoring are created: what discriminates the scoring against different protein conformers? cancellation of unreflected internal protein energy contributions is no longer certain. studies have shown that a special scoring scheme or protocol is required [48, 52] . because dramatic energy differences between low-energy conformers are unlikely, a modulated pocket size for the different protein conformers might require individual scoring of the altered desolvation properties of the binding site. required accuracy of scoring: enrichment or hit identification? vs is used to enrich putative actives from a large sample set of test ligands. accordingly, the desired accuracy of scoring depends on whether, at first glance, prioritization of the sample set for testing is anticipated or whether putative actives are expected among the top ten or 100 of a hit list. the latter requires very powerful discrimination of actives over inactive decoy binders. present scoring functions have been optimized to discriminate for a particular ligand decoy binding mode from near-native ones. the discrimination of binders from decoy nonbinders still remains as major challenge for vs protocols [125] . at worst, in these cases the above-mentioned poorly understood contributions to binding become overwhelmingly important. perhaps the consideration of similarity criteria in the search [87, 107, 108, [110] [111] [112] enables the problem to be alleviated to some degree because this drives the search towards more closely related ligands for which some of the disregarded effects in scoring cancel themselves out. ultimate proof of concept: crystal structure analysis of vs hits only rarely, the crystal structure of detected vs hits has been determined and subsequently published. such case studies have recently been reviewed by shoichet [24] . cases have been described for which vs has correctly predicted the subsequently found binding mode. other examples point to deficiencies arising from the superficially understood phenomena described above. finally, it has also been reported that vs inappropriately identified an active compound because binding actually occurs in a totally different region of the protein surface. vs has been established as a powerful alternative and complement to hts. when performed optimally, impressive hit rates have been reported, which have been significantly higher (by a factor of 100-1000 [24] ) than those for hts. comparative studies of hts and vs indicate that the methods can capture alternative and complementary ligands. undoubtedly, vs is not yet a fully mature technology following a well-established process line. few of the foundations of proteinligand recognition are understood well enough to be deployed in a large scale, multi-compound effort such as that commonly undertaken in vs. this calls for further indepth research. in particular, protein flexibility and induced-fit adaptations, the role of water in solvation, desolvation and ligand binding, and the electrostatics involved, including changes in protonation states and an appropriate consideration of entropic changes, will need to be better understood to improve the hit retrieval rates in vs. frequently, experimental work performed in parallel or as a follow-up to a vs campaign provides a whole bunch of unexpected results pointing towards manifold deficiencies of the concepts applied. many more experimental studies are required. nevertheless, vs has proven successful and as a valuable alternative to hts, in particular if it is used as a tool to support and complement hit discovery. compared with hts, it is significantly cheaper and faster to use. it can be easily rerun under modified conditions -for example, if additional information about the target protein under consideration becomes available or novel filter criteria are taken into account. it is interesting to note that an experienced modeller or medicinal chemist can often figure out whether a particular binding pocket appears druggable or a certain molecule obeys the rules of drug likeness; however, putting such knowledge into computer algorithms makes the multifactorial nature of these rules apparent, complicating attempts to generalize them. the same multifactorial nature holds true for docking and scoring. it is therefore highly advisable to refrain from fully automated strategies in vs. experience and human intervention are of the utmost importance for keeping control over the various filter steps in a vs run. used in such a way, vs can be successful; in particular, if information about molecular similarity is considered in terms of generic physicochemical properties and not simply as chemical formulae. condrug discovery today volume 11, numbers 13/14 july 2006 similar ligands decompose differently into enthalpic and entropic binding contributions. crystal structures of two closely related thrombin inhibitors bearing a cyclopentyl or cyclohexyl moiety as terminal substituent to accommodate the s3/s4 pocket of the catalytic site (surface of the binding pocket is indicated in blue). whereas the five-membered ring (left) gives rise to a well-defined difference in electron density (white 'chicken-wire' contouring), the six-membered ring (right) cannot be assigned to any density (see inside white circles). it is likely that the latter fragment shows enhanced residual mobility and is scattered around several conformational states. interestingly, this deviating behaviour is well reflected in the thermodynamic properties (centre). both compounds exhibit the same free energy of binding (dg, blue columns). however, the cyclohexyl derivative (right) with the enhanced residual mobility is entropically (-tds, red columns) more favoured than the 'less-well clamped' five-ring derivative (left). the latter experiences better enthalpic contributions (dh, green columns) to binding [124] . sidering similarity concepts takes the risk that highly diverse molecules remain undetected; however, it probably makes the searches simpler because many parameters that actually matter in vs, and which are not properly considered, simply cancel each other out in a relative comparison. considering all of the mentioned limitations, and taking molecular similarity as some kind of work-around to evade existing shortcomings, a critical reviewer might raise the question of whether, under such restrained conditions, vs can really contribute something novel that an experienced medicinal chemist would not have thought about. only successful examples can convince. we applied vs to a trnamodifying enzyme for which the inhibitor replaces either guanine or preq1 (a precursor to the modified base queuine) as a substrate in the enzyme recognition pocket ( figure 6 ). initial vs searches retrieved structures that, admittedly, an experienced medicinal chemist might also have suggested as being 'substrate-like'. however, a follow-up vs screen in which several water molecules were allowed to be replaced in the binding site suggested a cyclic urea structure with a different orientation in the binding pocket [126] . synthetically, this lead appeared tempting and presently serves as a starting point for a new series of compounds ( figure 6 ). this unbiased approach, resulting, in chemical terms, in an unexpected lead, demonstrates that vs can make important contributions to drug discovery, providing some unexpected candidates. nevertheless, there is still a long way to go until it becomes an established tool for routine lead discovery. interestingly, nowadays, most aspects of contemporary drug discovery are optimized towards high throughput; by contrast, our current increase in the knowledge and understanding of protein-ligand recognition principles is still proceeding at a low-output rate. drug discovery today volume 11, numbers 13/14 july 2006 reviews an unexpected ligand skeleton from virtual screening. virtual screening (vs) has been used to search for putative inhibitors of t-rna guanine transglycosylase [88] . initial hits such as 1-4 (lower left box), which were followed up by chemical synthesis, showed structural similarity with the natural substrates of this enzyme guanine, preq 0 and preq 1 (upper box). these searches were based on the protein-based pharmacophore shown on the left (contours for hydrogen bond acceptor group) and in figure 4 . an experienced medicinal chemist could possibly have come up with similar suggestions for potential leads such as 1-4. however, in a second vs campaign we focused on the replacement of two water molecules at the lower right rim of the binding pocket of t-rna guanine transglycosylase (right, contours for hydrogen bond acceptor group [126] ). this screen suggested 5 as a potential hit. its cyclic urea-type skeleton is distinct in its structure and binding mode from any known natural substrate and it can serve as a synthetically easily accessible lead. several derivatives (e.g. 6-8) have been synthesized and show a reasonable structure-activity relationship (stengl et al., unpublished) . it is unlikely that this novel lead structure would have been suggested by comparative substrate considerations. this example underlines the power of vs as an alternative source for novel lead discovery. www.drugdiscoverytoday.com 591 reviews foundation review trends in development cycles a new grammar for drug discovery how many leads from hts? comment: how many leads from hts? chemical feature-based pharmacophores and virtual library screening for discovery of new leads virtual screening to enrich hit lists from high-throughput screening: a case study on small-molecule inhibitors of angiogenin molecular docking and high-throughput screening for novel inhibitors of protein tyrosine phosphatase-1b comparing performance of computational tools for combinatorial library design inhibitors of dihydrodipicolinate reductase, a key enzyme of the diaminopimelate pathway of mycobacterium tuberculosis high throughput screening identifies novel inhibitors of escherichia coli dihydrofolate reductase that are competitive with dihydrofolate experimental screening of dihydrofolate reductase yields a ''test set'' of 50,000 small molecules for a computational data-mining and docking competition mcmaster university data-mining and docking competition: computational models on the catwalk evaluating the high-throughput screening computations screening for dihydrofolate reductase inhibitors using molprint 2d, a fast fragment-based method employing the naïve bayesian classifier: limitations of the descriptor and the importance of balanced chemistry in training and test sets virtual ligand screening against escherichia coli dihydrofolate reductase: improving docking enrichment using physics-based methods here be dragons: docking and screening in an uncharted region of chemical space a geometric approach to macromolecule-ligand interactions docking flexible ligands to macromolecular receptors by molecular shape structure-based design of nonpeptide inhibitors specific for the human immunodeficiency virus 1 protease structure of a nonpeptide inhibitor complexed with hiv-1 protease. developing a cycle of structure-based drug design rational design of potent, bioavailable, nonpeptide cyclic ureas as hiv protease inhibitors structure-based virtual screening: an overview virtual screening in structure-based drug design. mini rev virtual screening of chemical libraries integration of virtual screening into the drug discovery process virtual screening in lead discovery and optimization virtual screening methods that complement hts recent development and application of virtual screening in drug discovery: an overview docking and scoring in virtual screening for drug discovery: methods and applications hit and lead generation: beyond high-throughput screening success stories of computer-aided design validation of an empirical rna-ligand scoring function for fast flexible docking using ribodock identification of ligands for rna targets via structurebased virtual screening: hiv-1 tar the druggable genome druggability indices for protein targets derived from nmr-based screening data comparative protein structure modelling all are not equal: a benchmark of different homology modelling programs utility of homology models in the drug discovery process protein-based virtual screening of chemical databases. ii. are homology models of g-protein coupled receptors suitable targets? successful virtual screening for a submicromolar antagonist of the neurokinin-1 receptor based on a ligand-supported homology model structure-based drug discovery using gpcr homology modeling: successful virtual screening for antagonists of the alpha1a adrenergic receptor docking ligands onto binding site representations derived from proteins built by homology modelling ligand-supported homology modelling of protein binding sites using knowledge-based potentials implications of protein flexibility for drug discovery molecular docking to ensembles of protein structures ligand docking to proteins with discrete side-chain flexibility flexe: efficient molecular docking considering protein structure variations testing a flexible-receptor docking algorithm in a model binding site accommodating protein flexibility in computational drug design incorporating protein flexibility in structure-based drug discovery: using hiv-1 protease as a test case probing flexibility and ''induced-fit'' phenomena in aldose reductase by comparative crystal structure analysis and molecular dynamics simulations unveiling the full potential of flexible receptor docking using multiple crystallographic structures information decay in molecular docking screens against holo, apo, and modeled conformations of enzymes expect the unexpected or caveat for drug designers: multiple structure determinations using aldose reductase crystals treated under varying conditions ph-dependent binding modes observed in trypsin crystals: lessons for structure-based drug design understanding protein-ligand interactions: the price of protein flexibility zz made ez: influence of inhibitor configuration on enzyme selectivity isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition approaches to the description and prediction of binding affinity of small-molecule ligands to macromolecular receptors reconstructing the binding site of factor xa in trypsin reveals ligand-induced structural plasticity predicting binding modes, binding affinities and ''hot spots'' for protein-ligand complexes using a knowledge-based scoring function a computational procedure for determining energetically favorable binding sites on biologically important macromolecules superstar: a knowledge based approach for identifying interaction sites in proteins knowledge-based scoring function to predict proteinligand interactions relibase: design and development of a database for comprehensive analysis of protein-ligand interactions utilising structural knowledge in drug design strategies: applications using relibase zinc-a free database of commercially available compounds for virtual screening the art and practice of structure-based drug design: a molecular modelling perspective fragment-based drug discovery fragment-based lead discovery current trends in lead discovery: are we looking for the appropriate properties? virtual screening using protein-ligand docking: avoiding artificial enrichment experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings a rule-of-three for fragment-based lead discovery admet in silico modelling: towards prediction paradise? a bioavailability score drug-like annotation and duplicate analysis of a 23-supplier chemical database totalling 2.7 million compounds virtual exploration of the small-molecule chemical universe below 160 daltons lead discovery using molecular docking small molecule docking and scoring high-throughput docking for lead generation docking: successes and challenges high-throughput docking as a source of novel drug leads comparing protein-ligand docking programs is difficult detailed comparison of current docking and scoring methods on systems of pharmaceutical relevance target-based scoring approaches and expert systems in structure-based virtual screening successful virtual screening for novel inhibitors of human carbonic anhydrase: strategy and experimental confirmation virtual screening for submicromolar leads of trna-guanine transglycosylase based on a new unexpected binding mode detected by crystal structure analysis pharmacophore modeling and three-dimensional database searching for drug design using catalyst feature trees: a new molecular similarity measure based on tree matching development and validation of a genetic algorithm for flexible docking a fast flexible docking method using an incremental construction algorithm glide: a new approach for rapid, accurate docking and scoring. 1. method and assessment of docking accuracy glide: a new approach for rapid, accurate docking and scoring. 2. enrichment factors in database screening automated docking using a lamarckian genetic algorithm and an empirical binding free energy function high-throughput docking for lead generation principles of docking: an overview of search algorithms and a guide to scoring functions molecular recognition and docking algorithms comparison of automated docking programs as virtual screening tools binding site characteristics in structurebased virtual screening: evaluation of current docking tools comparative evaluation of eight docking tools for docking and virtual screening accuracy a detailed comparison of current docking and scoring methods on systems of pharmaceutical relevance pharmacophore-based molecular docking to account for ligand flexibility virtual screening with solvation and ligandinduced complementarity the particle concept: placing discrete water molecules during protein-ligand docking predictions modeling water molecules in protein-ligand docking using gold flexible docking under pharmacophore type constraints similarity-driven flexible ligand docking automated docking to multiple target structures: incorporation of protein mobility and structural water heterogeneity in autodock docking into knowledge-based potential fields: a comparative evaluation of drugscore drugscore meets comfa: adaptation of fields for molecular comparison (afmoc) or how to tailor knowledge-based pair-potentials to a particular protein improving binding mode predictions by docking into protein-specifically adapted potential fields comparative docking studies on ligand binding to the multispecific antibodies ige-la2 and ige-lb4 detailed analysis of scoring functions for virtual screening comparative evaluation of 11 scoring functions for molecular docking assessing scoring functions for protein-ligand interactions consensus scoring: a method for obtaining improved hit rates from docking databases of three-dimensional structures into proteins how does consensus scoring work for virtual library screening? an idealized computer experiment large-scale validation of a quantum mechanics based scoring function: predicting the binding affinity and the binding mode of a diverse set of protein-ligand complexes evaluation of the casp2 docking section chris lipinski discusses life and chemistry after the rule of five drugscore(csd)-knowledge-based scoring function derived from small molecule crystal data with superior recognition rate of nearnative ligand poses and better affinity prediction structural parameterization of the binding enthalpy of small ligands library design based on privileged scaffolds through docking and direct design in the protein binding pocket. abstracts of papers decoys for docking crystallographic study of inhibitors of trna-guanine transglycosylase suggests a new structure-based pharmacophore for virtual screening you can access endeavour online on sciencedirect, where you'll find book reviews, editorial comment and a collection of beautifully illustrated articles on the history of science. featuring: waxworks and the performance of anatomy in mid-eighteenth-century italy by l. dacome representing revolution: icons of industrialization by p. fara myths about moths: a study in contrasts by d.w. rudge the origins of research into the origins of life by i. fry in search of the sea monster by the author is grateful to current and former members of his research group for their joint efforts in developing tools and strategies for virtual screening and in performing the experimental proof of concept. this article was written during a very stimulating sabbatical at the university of california, san francisco, usa, and many discussions with the author's host, brian shoichet, and his colleagues and research group are kindly acknowledged. endless bus rides on muni, san francisco, are appreciated for providing the opportunity to do the required reading for this review. the livingstone story and the industrial revolution by l. dritsas endeavour is available on sciencedirect, www.sciencedirect.com key: cord-262268-gm99cadh authors: wang, jingqiang; wen, jie; li, jingxiang; yin, jianning; zhu, qingyu; wang, hao; yang, yongkui; qin, e’de; you, bo; li, wei; li, xiaolei; huang, shengyong; yang, ruifu; zhang, xumin; yang, ling; zhang, ting; yin, ye; cui, xiaodai; tang, xiangjun; wang, luoping; he, bo; ma, lianhua; lei, tingting; zeng, changqing; fang, jianqiu; yu, jun; wang, jian; yang, huanming; west, matthew b; bhatnagar, aruni; lu, youyong; xu, ningzhi; liu, siqi title: assessment of immunoreactive synthetic peptides from the structural proteins of severe acute respiratory syndrome coronavirus date: 2003-12-01 journal: clin chem doi: 10.1373/clinchem.2003.023184 sha: doc_id: 262268 cord_uid: gm99cadh background: the widespread threat of severe acute respiratory syndrome (sars) to human life has spawned challenges to develop fast and accurate analytical methods for its early diagnosis and to create a safe antiviral vaccine for preventive use. consequently, we thoroughly investigated the immunoreactivities with patient sera of a series of synthesized peptides from sars-coronavirus structural proteins. methods: we synthesized 41 peptides ranging in size from 16 to 25 amino acid residues of relatively high hydrophilicity. the immunoreactivities of the peptides with sars patient sera were determined by elisa. results: four epitopic sites, s599, m137, n66, and n371-404, located in the sars-coronavirus s, m, and n proteins, respectively, were detected by screening synthesized peptides. notably, n371 and n385, located at the cooh terminus of the n protein, inhibited binding of antibodies to sars-coronavirus lysate and bound to antibodies in >94% of samples from sars study patients. n385 had the highest affinity for forming peptide-antibody complexes with sars serum. conclusions: five peptides from sars structural proteins, especially two from the cooh terminus of the n protein, appear to be highly immunogenic and may be useful for serologic assays. the identification of these antigenic peptides contributes to the understanding of the immunogenicity and persistence of sars coronavirus. the worldwide threat of severe acute respiratory syndrome (sars) 6 becoming an epidemic creates urgent challenges for the scientific community. several laboratories have unraveled the genetic information of the sars virus (1) (2) (3) (4) . the genome size of the sars coronavirus is ϳ29 kb and has 11 open reading frames, composed of a stable region encoding an rna-dependent rna polymerase with 2 open reading frames, a variable region representing 4 coding sequences for viral structural genes [spike (s protein), envelope (e protein), membrane (m protein), and nucleocapsid (n protein)], and 5 putative uncharacterized proteins. its gene order is similar to that of other known coronaviruses; however, phylogenetic analyses and sequence comparisons indicate that this virus does not closely resemble any of the previously characterized coronaviruses. the incubation period of sars is usually 2-7 days, but could be as long as 10 days. the disease progresses with unusual severity within a short time once a patient exhibits obvious clinical symptoms. therefore, an urgent task is to develop accurate and sensitive diagnostic tools for identifying sars, specifically for early diagnosis. a noninvasive diagnostic test for sars coronavirus has been reported recently that uses quantitative reverse transcription-pcr with detection of sybr green fluorescence (5 ) . the pcr primer design was based on a unique region within the rna-dependent rna polymerase-encoding sequence of the virus. the amplification was very specific and showed no cross-reaction with two serogroups of human coronavirus, 229e and oc43. in a total of 29 sars patients and 58 uninfected controls, the pcr assay showed positive identification for 79% of sars cases and negative identification for 98% of controls. however, this technique is limited in its clinical use. the samples for pcr were collected from nasopharyngeal aspirates of sars patients. because sars is a respiratory infection, any attempt to obtain a sample from a patient's pharynx and larynx increases the risk of infection to the healthcare worker. furthermore, 79% accuracy is below the level of acceptably when taking into account those in the early phase of infection, showing no symptoms, who were among the 20% excluded. this could certainly be cause for concern because these people could still pose a threat of transmitting the virus. serologic assays are used extensively for diagnosis of viral infection in a host (6 ) . in urgent situations, a common way to perform a serologic assay is to use complex viral lysates as antigens. the use of viral lysates, however, presents several disadvantages. viral lysates consist of many viral antigens that can not be clearly purified and classified. the lysates are prepared from cells infected with the virus; thus, cellular proteins can contaminate the preparation. moreover, sars-coronavirus lysates present a considerable risk of infection to laboratory workers. to overcome these problems, the discovery of antigenic fragments in sars-coronavirus proteins is expected to lead to the rapid development of new assays for the diagnosis of sars infection. it has been shown in many cases that several epitopes located in viral proteins can be successfully mimicked with synthetic peptides (7 ) . to expedite epitope mapping of the sars coronavirus, we have synthesized a group of peptides representing the most hydrophilic, as well as the most accessible, residue regions of the s, m, and n structural proteins of sars coronavirus. using sera from sars patients, we probed these peptides by elisa. sera from 31 sars patients from eight different hospitals in beijing and from 49 uninfected volunteers were collected for study. the clinical diagnostic criteria for sars followed the clinical description of sars released by who. confirmation of sars infection was evidenced by the presence of antibodies against sars coronavirus in the serum. the control sera were divided into two groups: 24 samples obtained from healthy volunteers and 25 samples obtained from patients suffering from respiratory symptoms but not infected with the sars coronavirus. on the basis of the published genome sequence of the sars coronavirus, we downloaded structural proteins s, m, and n into the protscale program at the swiss institute of bioinformatics to analyze the physical characteristics of the proteins, such as hydrophilicity, hydrophobicity, accessible residues, buried residues, molecular mass, and pi values. a total of 41 peptides ranging in size from 16 to 25 amino acid residues and in molecular mass from 2500 to 3000 da were selected for synthesis ( table 1 ). all of the peptides were synthesized commercially by chinese peptide co. the synthesized peptides were characterized by hplc and mass spectrometry. the sars coronavirus isolated from sars case bj01, whose genomic sequence was determined by the beijing genomics institute, was used as a viral source and propagated in vero-e6 cells as described previously (8 ) . after viral propagation, the cells were harvested and placed at 70°c for 2 h to inactivate the virus. the inactivated infected vero-e6 cells were completely lysed by freeze-thaw cycles followed by centrifugation. after the cell pellet was removed, the supernatant was loaded on a sephadex g-150 column for virus purification. the elution fractions were collected at 1 ml/min, and viral proteins in each fraction were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) with coomassie blue staining and confirmed by western blot using sars serum. before elisa the fractions containing virus were sonicated and concentrated with a centricon-10 biological filter. the reactivities of the various peptides with sars sera were determined by elisa. in brief, peptides (1 mg/l, 100 l/well) were adsorbed to duplicate wells of 96-well microplates in 0.5 mol/l carbonate buffer (ph 12.5) and incubated at 4°c for 12 h. after the wells were washed with phosphate-buffered saline (pbs), they were coated with 2 g/l bovine serum albumin at 37°c for 2 h and then incubated at 37°c for 30 min with 10 l of sera in sample buffer (total volume of buffer, 100 l). each well was then washed and incubated with peroxidase-conjugated goat anti-human igg at 37°c for 20 min. finally the wells were washed again with pbs containing 5 ml/l tween 20. the peroxidase reaction was visualized by use of o-phenylenediamine solution as substrate. after 10 min of incubation at 37°c, the reaction was stopped by the addition of 50 l of 4 mol/l sulfuric acid, and the absorbance at 450 nm (reference wavelength, 630 nm) was measured by an automatic elisa plate reader (multiskan microplate photometer). to determine the background resulting from nonspecific binding of viral lysates or peptides to normal sera, the cutoff value was calibrated for each viral preparation or peptide by incubation with negative control serum. a panel of 24 sera from healthy individuals was examined routinely for the calibrations. the mean absorbance was considered as the cutoff value for a viral preparation or a peptide. the cutoff values were ϳ0.2. western blot for each sample, 50 g of protein was subjected to 12% sds-page under reducing conditions and transferred to a bio-rad pvdf membrane. the membrane was blocked with pbs containing 50 g/l nonfat milk powder in tris-buffered saline at 37°c for 30 min before incubation with diluted sars serum. bound antibodies were detected by use of an appropriate alkaline phosphatasecoupled secondary antibody with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3ј-indolyphosphate p-toluidine salt added as the substrate for visualization. estimation of binding affinities of synthesized peptides to sera from sars patients to compare the affinities of the antigenic peptides to sars-coronavirus antibodies, we estimated the binding constants by the scatchard equation. each antibody has a limited number of binding sites that become saturated as the concentration of antigen increases. this can be quantified by the following equation: where ␣ is the proportion of bound antigens per antibody based on the elisa measurement at 450 nm; n is the number of binding sites on the antibody; k d is the dissociation constant of the antibody-antigen complex; and [p] is the antigen concentration. we used the student t-test to calculate whether the reactivities of the synthetic peptides and sars coronavirus preparations with the same serum in the elisa were significantly different. p values ͻ0.05 were considered statistically significant. results and discussion epitope mapping of the s, m, and n peptides to effectively develop synthetic peptide immunogens from sars-coronavirus structural proteins, we used several theoretical calculations for peptide design. for a potential epitopic peptide, we specifically looked for high local hydrophilicity, charged residues on the exposed protein surface, and accessible surface area. statistical analysis based on the linear amino acid sequences of the three proteins suggests that, in contrast to the other two viral structural proteins, the n protein contains a high percentage of accessible residues and low hydrophobicity (see figs. 1-3 in the data supplement that accompanies the online version of this article at http://www.clinchem. org/content/vol49/issue12/). a total of 41 peptides spanning multiple possible epitopic sites along the s, m, and n proteins were designed and synthesized in a solid-phase peptide synthesizer. these peptides were purified by hplc, incubated with panels of sera from both controls and sars patients, and then subjected to elisa for measurement of the immunoreactivities. the correlations between the length of amino acid sequence and immunoreactivity are shown in fig. 1 . the peptides representing the cooh terminus of the n protein, in particular n371 and n385, had high absorbance/cutoff value ratios with the highest positive detection rate and the lowest hydrophobicity score among all of the synthesized peptides (fig. 1c, and fig. 3 in the online data supplement). peptide n66 is located in a hydrophilic region and reacts with sars serum antibodies. however, compared with the peptide region n371-n404, which has a score of 9.4 for accessible residues and a hydrophobicity score of ϫ3.5, peptide n66 has relatively low antigenicity because of a correspondingly lower score for accessible residues (6.2) and higher score for hydrophobicity (ϫ1.8). these differences may partially explain the low number of true positives (58%) that we obtained when we used n66 as an antigen. according to the hydrophobicity analysis (figs. 1 and 2 in the online data supplement), most regions in both the s and m proteins are very hydrophobic. although many factors influence antigen-antibody interactions, two major factors, charge-charge interactions and hydrogen bonding caused by hydrophilicity, have been hypothesized to be crucial for an epitopic site (9 ) . of the 41 synthesized peptides, only 5 displayed significantly high immunoreactivity (table 1) . interestingly, all five immunoreactive peptides are located in regions with a relatively high hydrophobicity score (figs. 1-3 in the online data supplement). moreover, the number of polar amino acids in each peptide correlates well with the percentage of true positive, e.g., n371 and n385 reacted with ͼ94% of the samples from sars patients and contain nine and seven polar amino acids, respectively, whereas s599, m137, and n66 reacted with յ80% of the sera from sars patients and have four, three, and three polar amino acids, respectively. our findings thus support that both hydrophobicity and peptide charge are important in determining immunoreactive sites in sarscoronavirus structural proteins. the sars coronavirus propagated in vero-e6 was used as an antigen to test whether sars-coronavirus antibodies were raised in sera. we collected sera from 24 healthy controls and measured their immunoreactivities against sars coronavirus by elisa. none of the control sera reacted with the sars coronavirus. the same results were observed for 25 other control sera obtained from patients with respiratory diseases who did not have sars. however, sera from all 31 sars patients reacted with the sars coronavirus, with high absorbance/cutoff value ratios [mean (sd), 4.23 (1.85)]. the results of the peptide screening are summarized in table 1 . there are two key parameters listed in table 1 , p values, which indicate whether there was a significant difference between the synthesized peptide and sars coronavirus as antigen in the elisa, and x/31, which represents the detection rate when particular peptides were used as antigen. for the s protein, 18 peptides spanning the protein sequence were synthesized. only one peptide, s599, elicited a response in the elisa that was not significantly different from the response elicited by the sars coronavirus: ϳ80% (25 of 31) of the sera from sars patients reacted with this peptide. the other 17 peptides reacted only slightly with the sera from sars patients and gave low detection rates, suggesting that the regions of the s protein covered by these peptides have no epitopic site. for the m protein, a relatively small viral structural protein, nine peptides were synthesized. compared with the sars coronavirus, this peptide reacted with 80% (25 of 31) of the sera (p ϭ 0.1), indicating that it may contain a weak epitopic site. the highest immunoreactivities were for the peptides located within the n protein. three of 14 synthesized n-protein peptides, n66, n371, and n385, showed reactivities comparable to that of the sars coronavirus. interestingly, n66 is located at the nh 2 terminus of the n protein, whereas n371 and n385 are neighboring fragments, both at the cooh terminus. we thus reasoned that n protein has at least two epitopic regions. these three peptides showed low y/24 values-0/24, 1/24, and 0/24, respectively-and high x/31 values: n371 and n385 reacted with 97% (30 of 31) and 94% (29 of 31) of the sera from sars patients, respectively, whereas n66 reacted with ϳ58% (18 of 31). taken together, these data suggest that the most immunoreactive epitopic site in the sars coronavirus is located at the cooh terminus of the n protein. members of the coronavirus family all have three major structural proteins, s, m, and n, but the antigenicities and roles of these viral proteins in immunity remain unclear. different viruses often cause different immunoresponses. for example, in infectious bronchitis virus (10 ), the s glycoprotein is more antigenic than either the n or the m protein. because s protein is exposed to the outer viral surface, it is often used as the antigenic group for serologic testing. the m protein, embedded in the viral envelope, has highly variable sequences and is considered to exhibit weak antigenicity, but glycosylation of this protein may elicit antibody production. one common phenomenon is that the n proteins have been shown to be strong immunogens in several coronaviruses, such as murine coronavirus (11 ), turkey coronavirus (12 ) , and porcine reproductive and respiratory syndrome virus (13 ) . in these viruses, the n protein is a highly abundant and relatively conserved antigen. importantly, the n protein may be involved in stimulating cytotoxic t lymphocytes. it has been reported that the n protein accumulates even before it is packaged in the mature virus (14 ) . a cellular immune response elicited early in infection by internal viral proteins could therefore be an important defense mechanism. on the basis of the genomic sequences published to date, most of the sars-coronavirus n protein is highly conserved among strains. thus, the n protein, which has high antigenicity, is likely to have a great impact on the development of diagnostic tests for sars. the proteins extracted from vero-e6 cells infected by sars coronavirus were separated by sds-page (12% polyacrylamide), and the separated proteins were probed with antibodies in sera from sars patients. as shown in fig. 2 , some immunostained bands appeared only on the membranes incubated with patient sera, suggesting that these proteins are associated with sars. on the basis of genomic data, the structural proteins of the virus have molecular masses of 139 (s protein), 46 (n protein), 25 (m protein), and 8 (e protein) kda (4 ), respectively. in all of the western blots performed with sera from three sars patients as the primary antibody, no protein band ͻ40 kda was immunostained, suggesting that the m and e proteins either do not elicit antibody production or generate low antibody titers in sars patients. of the protein bands ͼ40 kda, most were located around two regions ( fig. 2) , 49 and 120 -240 kda. the protein band slightly smaller than 49 kda reacted consistently with all sars sera. because its location is close to the theoretical molecular mass of the n protein, 46 kda, we believe that this protein is the n protein. the immunostained bands around 120 -240 kda did not display a consistent pattern. although the theoretical value of the s protein is 139 kda, it has been reported to be glycosylated during infection of the host cells. it therefore is not surprising that sarscoronavirus s proteins have significantly higher molecular masses than the theoretical value. we thus deduced that these immunostained proteins with high molecular masses are the s protein. to further confirm the identities of these protein bands, we conducted competition experiments to determine whether the peptides from the s or n protein would inhibit the binding of the sera from sars patients in the elisa. the patient sera preincubated with 4 mg/l s599 or n385 gave a 25-30% lower response in the elisa (data not shown), suggesting that the two peptides could compete with sars coronavirus for binding to the antibodies in sars serum. these observations suggest that the s and n proteins account for the antigenicity of the sarscoronavirus structural proteins. affinity of the peptides located at the cooh terminus of n protein protscale analysis suggested that the cooh terminus of the n protein has fewer buried residues and higher hydrophilicity than the other regions of this protein. we designed four peptides around the region, n355, n371, n385, and n401. of these, n401, located at the end of the n protein, had low reactivity in the elisa; conversely, the other three peptides were significantly more immunoreactive in the elisa. to determine which peptides had higher affinities for antibodies from sars sera, we quantitatively measured the binding of the peptides to the antibodies in the elisa. in this experiment, the concentration of the antibodies was kept constant and the binding capacity was estimated as a function of peptide concentration (fig. 3) . using the scatchard equation, we calculated the constants n and k d ; the binding curves obtained for the three peptides gave similar n values but different k d values. on the basis of the definitions of the two parameters in materials and methods, the fact that the curves give similar n numbers indicates that the peptides share similar epitopic sites, whereas the different k d values indicate that the three peptides bind to the same epitope with different affinities. of the three peptides, the k d value for n385 was much lower than the k d values for n355 and n371, approximately one-fourth of the k d value for n371 and one-thirteenth of the k d value for n355, suggesting that n385 is able to bind more strongly with antibodies in sars sera. thus, the region around n385 is most likely a strong epitopic site within the n protein. among the 31 sars cases that were identified with use of the sars coronavirus as antigen, 1 and 2 cases were not detected by peptides n371 and n385, respectively, although both were the most immunoreactive antigens of the 41 synthesized peptides. a possible explanation for this failure may relate to the specific recognition of the antibodies raised from a sars case. in this case, the antibodies may not actively recognize the cooh terminus of the n protein. hence, combining the peptides that represent different structural proteins could improve the detection coverage. we selected n371 and n385 to use in combination with peptides s599 and m137, respectively, and used these combinations as antigens for the elisa. contrary to our expectations, the use of peptide combinations did not improve the detection coverage, whereas single peptides displayed higher immunoreactivities in the elisa ( table 1 in the data supplement). this unexpected behavior may be attributable to interactions between peptides n385 and s599 or between n371 and m137. if this hypothesis is correct, then generating a chimeric peptide by linking the two peptides could be a good solution for reducing interactions. we are currently pursuing this line of investigation. in summary, the data presented indicate that (a) in three sars-coronavirus structural proteins, a total of four epitopic sites, s599, m137, n66, and n371-404, were detected by screening with synthesized peptides; (b) of the five peptides with high reactivity against sars sera, n371 and n385 gave significant results compared with the sars coronavirus lysate in an elisa as well as a positive detection rate, suggesting that the cooh terminus of the n protein could be extremely antigenic and useful in clinical diagnosis based on the elisa; (c) of four peptides located at the cooh terminus of the n protein that were synthesized and tested for their binding with sars serum antibodies, n385 was confirmed to have the highest affinity for forming the peptide-antibody complex. taken together, these results show that synthesized peptides could be important in exploring epitopes of immunogens, specifically in urgent situations such as that presented by the emergence of the sars virus. the identification of these reactive peptides could aid in the development of diagnostic techniques for sars. the genome sequence of the sars associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection a complete sequence and comparative analysis of a sars-associated virus (isolate bj01) rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) new approaches and perspectives in cytomegagaovirus diagnosis use of peptide synthesis to probe vial antigens for epitopes to a resolution of a single amino acid isolation and identification of a novel coronavirus from patients with sars prediction of protein antigenic determinants from amino acid sequences evolution of avian coronavirus ibv: sequence of the matrix glycoprotein gene and intergenic region of several serotypes an immunodominant cd4ϩ t cell site on the nucleocapsid protein of murine coronavirus contributes to protection against encephalomyelitis nucleocapsid protein gene sequence analysis reveals close genomic relationship between turkey coronavirus and avian infectious bronchitis virus identification of a common antigenic site in the nucleocapsid protein of european and north american isolates of porcine reproductive and respiratory syndrome virus cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry key: cord-252576-1ec545o2 authors: wu, xiangli; sun, jian; zhang, guoqing; wang, hexiang; ng, tzi bun title: an antifungal defensin from phaseolus vulgaris cv. ‘cloud bean’ date: 2011-01-15 journal: phytomedicine doi: 10.1016/j.phymed.2010.06.010 sha: doc_id: 252576 cord_uid: 1ec545o2 an antifungal peptide with a defensin-like sequence and exhibiting a molecular mass of 7.3 kda was purified from dried seeds of phaseolus vulgaris ‘cloud bean’. the isolation procedure entailed anion exchange chromatography on deae-cellulose, affinity chromatography an affi-gel blue gel, cation exchange chromatography on sp-sepharose, and gel filtration by fast protein liquid chromatography on superdex 75. although the antifungal peptide was unadsorbed on deae-cellulose, it was adsorbed on both affi-gel blue gel and sp-sepharose. the antifungal peptide exerted antifungal activity against mycosphaerella arachidicola with an ic(50) value of 1.8 μm. it was also active against fusarium oxysporum with an ic(50) value of 2.2 μm. it had no inhibitory effect on hiv-1 reverse transcriptase when tested up to 100 μm. proliferation of l1210 mouse leukemia cells and mbl2 lymphoma cells was inhibited by the antifungal peptide with an ic(50) of 10 μm and 40 μm, respectively. fungi inflict tremendous damage to humans, other animals, and plants. some fungal diseases are devastating to plants leading to crop destruction and enormous economic losses. antifungal proteins have arrested the attention of investigators because of the tremendous economic implications. to date, many different types of plant antifungal proteins are known. among them are thaumatin-like proteins (chu and ng 2003; pressey 1997; wang and ng 2002; ye et al. 1999) , glucanases (vogelsang and barz 1993) , chitinases and chitinases-like proteins (lam et al. 2000; vogelsang and barz 1993) , ribosome inactivating proteins (roberts and selitrennikoff 1986) , defensins (thevissen et al. 2003; wong and ng 2003) , peroxidases , allergen-like proteins (ye et al. 2001a) , protease inhibitors (chilosi et al. 2000; ye et al. 2001a,b) , lectins (ye et al. 2001b) , lipid transfer proteins (cammue et al. 1995; wang et al. 2004 ), embryo abundant protein-like proteins , cyclophilin-like proteins (ye and ng 2000) , and others wang and ng 2005; . many of the aforementioned antifungal proteins are also referred to as pathogenesis related proteins (van loon 1990) . antifungal proteins have also been purified from animals including insects (iijima et al. 1993) . the aim of the present investigation was to isolate an antifungal protein from the seeds of the cloud bean cultivar of phaseolus vul-garis and to ascertain which type of antifungal protein it belongs to. its characteristics and activities were compared with those of other antifungal proteins. dried seeds of phaseolus vulgaris 'cloud bean' (250 g) from mainland china were purchased from a vendor in hong kong. they were authenticated by prof. shiu ying hu, honorary professor of chinese medicine, the chinese university of hong kong and then deposited in laboratory 302, school of biomedical sciences, under the voucher number pvcb 195. they were homogenized in distilled water (6 ml/g) and the homogenate was centrifuged at 12 000 × g for 20 min at 4 • c. the supernatant was collected and loaded on a 5 cm × 20 cm column of deae-cellulose (sigma) in 10 mm tris-hcl buffer (ph 7.2). following removal of unadsorbed proteins in fraction d1, adsorbed proteins were eluted from the column with 0.2 m nacl and then with 1 m nacl added to the 10 mm tris-hcl buffer, to yield fractions d2 and d3, respectively. fraction d1 was applied to a column (5 cm × 18 cm) of affi-gel blue gel (bio-rad) in 10 mm tris-hcl buffer (ph 7.2). following elution of unadsorbed proteins in fraction b1, adsorbed proteins were desorbed initially with 0.2 m nacl in the tris-hcl buffer to yield fraction b2, and subsequently with 1 m nacl in the tris-hcl buffer to yield fraction b3. fraction b2 was further purified by ion exchange chromatography on a column (2.5 cm × 20 cm) of sp-sepharose (ge healthcare) in 10 mm nh 4 oac buffer (ph 5.0). after elution of unadsorbed proteins (fraction s1), adsorbed proteins were desorbed with a linear concentration (0-1 m) gradient of nacl. the fraction with antifungal activity (s2) was then further fractionated by fast protein liquid chromatography on a superdex 75 hr 10/30 column (ge healthcare) in 0.2 m nh 4 hco 3 buffer (ph 8.5). the last absorbance peak (su3) represented purified antifungal peptide. it was conducted as described by laemmli and favre (1973) using 18% gel. following electrophoresis under non-reducing conditions, the gel was stained with coomassie brilliant blue. the molecular mass of the antifungal peptide was estimated by comparison of its electrophoretic mobility with those of molecular mass marker proteins from ge healthcare including horse myoglobin peptides of different molecular mass: 16 949, 14 404, 10 700, 8159 and 6214 da. the molecular mass was also determined using gel filtration on a superdex peptide column (ge healthcare). the amino acid sequence of the antifungal peptide was analyzed by means of automated edman degradation. microsequencing was carried out using a hewlett packard 1000a protein sequencer equipped with a high performance liquid chromatography system (lam et al. 1998 ). it was carried out by using the dye binding reagent (bio-rad). the assay for antifungal activity toward mycosphaerella arachidicola, physalospora piricola and fusarium oxysporum was conducted in 100 mm × 15 mm petri plates containing 10 ml of potato dextrose agar. after the mycelial colony had developed, sterile blank paper disks (0.625 cm in diameter) were laid at a distance of 0.5 cm away from the rim of the mycelial colony. an aliquot (15 l) of the antifungal peptide was added to a disk. the plates were then left at 23 • c for 72 h until mycelial growth had surrounded the disks containing the control and had produced crescents of inhibition around disks containing samples with antifungal activity . to determine the ic 50 value for the antifungal activity, three doses of the antifungal peptide were added separately to three aliquots each containing 4 ml potato dextrose agar at 45 • c, mixed rapidly and poured into three separate small petri dishes. after the agar had cooled down, the same amount of mycelia was added to each plate. buffer without the antifungal peptide was used as a control. after incubation at 23 • c for 72 h, the area of the mycelial colony was measured and the inhibition of fungal growth was calculated. from a graph plotting % reduction in area of mycelial colony versus concentration of antifungal peptide, the concentration bringing about 50% reduction in area of mycelial colony (ic 50 ) compared with the control was determined (wong and ng 2003) . the assay was conducted by the uptake of sytox green, a high-affinity nuclear stain that penetrates cells with compromised membranes as detailed by thevissen et al. (2003) . briefly, fungi were cultured in the presence or in the absence of cloud bean defensin. sytox green (invitrogen) was added to the fungal cultures (0.5 mm final concentration). after incubation for 10 min, the fungal cells were examined for the presence of the dye by using a fluorescence microscope (nikon te2000). an excitation wavelength of 500-540 nm was used. the assay was performed according to instructions provided with the assay kit from boehringer-mannheim (germany). the assay makes use of the ability of reverse transcriptase to synthesize dna, starting from the template/primer hybrid poly (a) oligo (dt) 15. the digoxigenin-and biotin-labeled nucleotides in an optimized ratio are incorporated into one of the same dna molecule, which is freshly synthesized by the reverse transcriptase (rt). the detection and quantification of synthesized dna as a parameter for rt activity follows a sandwich elisa protocol. biotin-labeled dna binds to the surface of microtiter plate modules that have been precoated with streptavidin. next, an antibody to digoxigenin conjugated to peroxidase binds to the digoxigenin-labeled dna. finally, the peroxidase substrate is added. the peroxidase enzyme catalyzes cleavage of the substrate, producing a colored reaction product. the absorbance of the samples at 405 nm, which is directly correlated to the level of rt activity, can be measured with a microtiter plate (elisa) reader. a fixed amount (4-6 ng) of recombinant hiv-1 reverse transcriptase was used. the inhibitory activity of the isolated peptide was calculated as percent inhibition as compared to a control without the peptide ng and wang 2001) . the defensin gymnin (wong and ng 2003) was used as a positive control. the plasmid that expressed his-tagged wild-type hiv-1 integrase, pt7-7-his (y|tx)-hiv-1-in, was a generous gift from professor s.a. chow (school of medicine, ucla). to express the protein, a 1-l culture of e. coli bl21 (de3) cells containing the expression plasmid was grown at 37 • c until od600 reached 0.7-0.8. cells were induced by addition of 0.8 mm iptg (isopropyl-␤-d-thiogalactopyranoside) and harvested, after 4 h of incubation, by centrifugation at 6000 × g for 10 min at 4 • c. cells were suspended at a concentration of 10 ml/g wet cell paste in 20 mm tris-hcl buffer (ph 8.0) containing 0.1 mm edtandash;hcl buffer (ph 8.0) containing 0.1 mm edta, 2 mm ␤-mercaptoethanol, 0.5 m nacl and 5 mm imidazole. lysozyme was added to a concentration of 0.2 mg/ml. after incubation at 4 • c for 1 h, the lysate was sonicated and centrifuged at 40 000 × g at 4 • c for 20 min. the pellet was homogenized in 50 ml buffer a (20 mm tris-hcl, ph 8.0, 2 m nacl, 2 mm ␤-mercaptoethanol) containing 5 mm imidazole. the suspension was rotated at 4 • c for 1 h, and cleared by centrifugation at 40 000 × g at 4 • c for 20 min. the supernatant was loaded onto a 1-ml chelating sepharose (ge healthcare) column charged with 50 mm imidazole. the column was washed with five column volumes of buffer a containing 5 mm imidazole, and the protein was eluted with three column volumes of buffer a containing 200 mm and 400 mm imidazole, respectively. proteincontaining fractions were pooled, and edta was added to a final concentration of 5 mm. the protein was dialyzed against buffer b (20 mm hepes, ph 7.5, 1 mm edta, 1 m nacl, 20% glycerol) containing 2 mm ␤-mercaptoethanol, and then against buffer b containing 1 mm dithiothreitol. aliquots of the protein were stored at −70 • c (loizidou et al. 2009 ). a non-radioactive elisa-based hiv-1 integrase assay was performed according to the dna-coated plate method. in this study, 1 g of smai-linearized pbluescript sk was coated onto each well in the presence of 2 m nacl as target dna. the donor dna was prepared by annealing vu5br (5 -biotin-gtgtggaaaatctcta-gcagt-3 ) and vu5 (5 -actgctagagattttccacac-3 ) in 10 mm tris-hcl, ph 8.0, 1 mm edta and 0.1 m nacl at 80 • c followed by 30 min at room temperature. integrase reaction was performed in 20 mm hepes (ph 7.5) containing 10 mm mncl 2 , 30 mm nacl, 10 mm dithiothreitol and 0.05% nonidet-p40 (sigma). after the integrase reaction, the biotinylated dna immobilized on the wells was detected by incubation with streptavidin-conjugated alkaline phosphatase (boehringer-mannheim, mannheim, germany), followed by colorimetric detection with 1 mg/ml p-nitrophenyl phosphate in 10% diethanolamine buffer (ph 9.8) containing 0.5 mm mgcl 2 . the absorbance due to the alkaline phosphatase reaction was measured at 415 nm. the ribosome inactivating protein trichosanthin was used as a positive control (loizidou et al. 2009 ). the antiproliferative activity of the purified peptide was determined as follows. the cell lines l1210 (mouse leukemia) and mbl2 (lymphoma) from american type culture collection were maintained in dulbecco modified eagles' medium (dmem) supplemented with 10% fetal bovine serum (fbs) and 100 mg/l streptomycin and 100 iu/ml penicillin, at 37 • c in a humidified atmosphere of 5% co 2 . cells (1 × 10 4 ) in their exponential growth phase were seeded into each well of a 96-well culture plate (nunc, denmark) and incubated for 3 h before addition of the peptide. incubation was carried out for another 48 h. radioactive precursor, 1 ci, [methyl-3 h] thymidine (ge healthcare) was added to each well and incubated for 6 h. the cultures were then harvested by a cell harvester. the incorporated radioactivity was determined by liquid scintillation counting (wong and ng 2003) . the activity of sars coronavirus (cov) protease was indicated by a cleavage of designed substrate which was composed of two proteins linked by a cleavage site for sars cov protease. the reaction was performed in a mixture containing 5 m sars cov protease, 5 m sample, 20 m substrate and buffer [20 mm tris-hcl (ph 7.5), 20 mm nacl and 10 mm beta-mercaptoethanol] for 40 min at 37 • c. after 40 min, the reaction was stopped by heating at 100 • c for 2 min. then the reaction mixture was analysed by sds-page. if sars cov protease is inhibited by the test sample, there is only one band, which is the intact substrate, shown in sds-page (leung et al. 2008) . the extract of cloud beans was fractionated into three fractions of approximately equal size: an unadsorbed fraction d1 with antifungal activity and two adsorbed fractions d2 and d3 without activity (table 1) . fraction d1 was resolved on affi-gel blue gel into a larger unadsorbed fraction b1 devoid of antifungal activity, an adsorbed fraction b2 with antifungal activity eluted with 0.2 m nacl in the tris-hcl buffer, and an adsorbed fraction b3 eluted with 1.0 m nacl in the tris-hcl buffer but without antifungal activity (table 1) . fraction b2 was separated on sp-sepharose into a broad unadsorbed fraction s1 and three sharp adsorbed fractions s2, s3 and s4 (fig. 1) . antifungal activity was detected only in the largest fraction s2 (table 1) . fraction s2 was separated by gel filtration on superdex 75 into three fractions su1, su2 and su3 (fig. 2) . su3, which displayed a molecular mass of 7.3 kda, was the only fraction with antifungal activity (table 1) . its amino acid sequence, which manifests pronounced homology to plant defensins, is recorded in table 2 . fraction su3 appeared in sds-page as a single band with a molecular mass of 7.3 kda (fig. 3) . the inhibitory action of the purified antifungal peptide represented by fraction su3 on the fungi mycosphaerella arachidicola and fusarium oxysporum is shown in figs. 4 and 5, respectively. there was a dose-dependent inhibition of mycelial growth. the antifungal peptide suppressed mycelial growth in m. arachidicola with an ic 50 value of 1.8 m (fig. 6) and fusarium oxysporum with an ic 50 value of 2.2 m. the antifungal peptide did not reduce the activity of hiv-1 reverse transcriptase. neither did it affect the activities of hiv-1 integrase and sars coronavirus proteinase (data not shown). but it inhibited the proliferation of l1210 and mbl2 tumor cells with an ic 50 of 1.0 m and 40 m, respectively (table 3 ). in the assay of sytox green uptake, cloud bean defensin (10 m) could induce membrane permeabilization in m. arachidicola and f. oxysporum as wong et al. 2008a,b; lin et al. 2010) . comparison with white cloud bean defensin table 4 presents a comparison of the characteristics and activities of cloud bean defensin with white cloud bean defensin. they are grossly similar. the antifungal peptide isolated from cloud beans in the present study is a defensin as judged by the remarkable homology of its n-terminal amino acid sequence to plant defensins (thevissen et al. 2003 ). it has a molecular mass and chromatographic behavior on cationic and anionic exchangers and affi-gel blue gel similar to those of plant defensins reported earlier. it is unadsorbed on deae-cellulose but adsorbed on sp-sepharose and affi-gel blue gel. this chromatographic behavior is also characteristic of antifungal proteins in general (lam et al. 2000; wang and ng 2000 , 2005 wang et al. , 2004 wong and ng 2003; ye et al. 2001a ye et al. ,b, 2002 ye et al. , 1999 ng 2002, 2000) . cloud bean defensin potently inhibits proliferation of tumor cells. antiproliferative activity toward tumor cells is also an attribute of defensins (wong and ng 2003) , defensin-like peptides and also other antifungal proteins including ribosome inactivating proteins (lam et al. 1998 ) and chitinase-like proteins (lam et al. 2000) . this activity of cloud bean defensin may be a consequence of the protein synthesis inhibitory activity which is a characteristic of antifungal proteins (ng and ye 2003) including ribosome inactivating proteins (ng and parkash 2002) . it is noteworthy that cloud bean defensin is inhibitory to both l1210 and mbl2 tumor cells. the antifungal protein passiflin (lam and ng 2009 ) inhibits proliferation in breast cancer mcf7 cells, but not in hepatoma hepg2 cells. some antifungal proteins including defensin-like peptides (wong and ng 2003) , protease inhibitors (ye et al. 2001a ), thaumatin-like proteins (wang and ng 2002) , chitinase-like proteins (lam et al. 2000) and ribosome inactivating proteins (wong et al. 2008a,b) demonstrate hiv-1 reverse transcriptase inhibitory activity. it is somewhat surprising that cloud bean defensin is devoid of inhibitory activity toward the retroviral enzyme. its lack of inhibition on hiv-1 integrase and sars cov protease is in concord with previous reports on other defensins (leung et al. 2008) . the present findings on cloud bean defensin is reminiscent of the observation that mungin, an antifungal protein from mung beans, is without hiv-1 reverse transcriptase inhibitory activity (ye and ng 2000) . the isolated defensin-like antifungal peptide potently inhibits fungal growth in both fungal species examined. some anti-fungal proteins are able to inhibit only one out of the several fungi tested . other antifungal proteins have a lower antifungal potency than cloud bean defensin (ye et al. 1999) . to recapitulate, a defensin with potent antifungal, and antiproliferative activities was isolated from cloud beans. like french bean defensin (leung et al. 2008) , it is devoid of inhibitory activity toward hiv-1 integrase and sars coronavirus proteinase. white cloud bean and cloud bean are two cultivars of phaseolus vulgaris. their defensins are similar in molecular mass, n-terminal sequence and antifungal potency. however, white cloud bean defensin, but not cloud bean defensin, has inhibitory activity toward hiv-1 reverse transcriptase. the former appears to be more potent in inhibitory activity toward tumor cells. hence similar, but not identical proteins are produced by different cultivars. a potent antimicrobial protein from onion seeds showing sequence homology to plant lipid transfer proteins antifungal activity of a bowman-birk type trypsin inhibitor from wheat kernel mollisin, an antifungal protein from the chestnut castanea mollissima purification, characterization, and cdna cloning of an antifungal protein from the hemolymph of sarcophaga peregrina (flesh fly) larvae maturation of the head of bacteriophage t4. i. dna packaging events passiflin, a novel dimeric antifungal protein from seeds of the passion fruit purification and characterization of novel ribosome inactivating proteins, alpha-and beta-pisavins, from seeds of the garden pea pisum sativum a robust cysteine-deficient chitinase-like antifungal protein from inner shoots of the edible chive allium tuberosum concurrent purification of two defense proteins from french bean seeds: a defensin-like antifungal peptide and a hemagglutinin a defensin with highly potent antipathogenic activities from the seeds of purple pole bean analysis of binding parameters of hiv-1 integrase inhibitors: correlates of drug inhibition and resistance inhibitory effects of antifungal proteins on human immunodeficiency virus type 1 reverse transcriptase, protease and integrase hispin, a novel ribosome inactivating protein with antifungal activity from hairy melon seeds panaxagin, a new protein from chinese ginseng possesses anti-fungal, anti-viral, translation-inhibiting and ribonuclease activities fabin, a novel calcyon-like and glucanase-like protein with mitogenic, antifungal and translation-inhibitory activities from broad beans two isoforms of np24: a thaumatin-like protein in tomato fruit isolation and partial characterization of two antifungal proteins from barley interactions of antifungal plant defensins with fungal membrane components the nomenclature of pathogenesis-related proteins purification, characterization and differential hormonal regulation of a beta-1,3-glucanase and two chitinases from chickpea (cicer arietinum l.) ginkbilobin, a novel antifungal protein from ginkgo biloba seeds with sequence similarity to embryo-abundant protein isolation of a novel deoxyribonuclease with antifungal activity from asparagus officinalis seeds isolation of an antifungal thaumatin-like protein from kiwi fruits purification of chrysancorin, a novel antifungal protein with mitogenic activity from garland chrysanthemum seeds an antifungal peptide from the coconut a non-specific lipid transfer protein with antifungal and antibacterial activities from the mung bean an antifungal protein from bacillus amyloliquefaciens gymnin, a potent defensin-like antifungal peptide from the yunnan bean (gymnocladus chinensis baill) marmorin, a new ribosome inactivating protein with antiproliferative and hiv-1 reverse transcriptase inhibitory activities from the mushroom hypsizigus marmoreus isolation of a novel peroxidase from french bean legumes and first demonstration of antifungal activity of a non-milk peroxidase mungin, a novel cyclophilin-like antifungal protein from the mung bean a bowman-birk-type trypsin-chymotrypsin inhibitor from broad beans cicerin and arietin, novel chickpea peptides with different antifungal potencies isolation of a homodimeric lectin with antifungal and antiviral activities from red kidney bean (phaseolus vulgaris) seeds first chromatographic isolation of an antifungal thaumatin-like protein from french bean legumes and demonstration of its antifungal activity this work was financially supported by national grants of china (2006bad07a01 and nyhyzx07-008) was gratefully acknowledged. key: cord-254107-02bik024 authors: hillisch, alexander; pineda, luis felipe; hilgenfeld, rolf title: utility of homology models in the drug discovery process date: 2004-08-31 journal: drug discovery today doi: 10.1016/s1359-6446(04)03196-4 sha: doc_id: 254107 cord_uid: 02bik024 abstract advances in bioinformatics and protein modeling algorithms, in addition to the enormous increase in experimental protein structure information, have aided in the generation of databases that comprise homology models of a significant portion of known genomic protein sequences. currently, 3d structure information can be generated for up to 56% of all known proteins. however, there is considerable controversy concerning the real value of homology models for drug design. this review provides an overview of the latest developments in this area and includes selected examples of successful applications of the homology modeling technique to pharmaceutically relevant questions. in addition, the strengths and limitations of the application of homology models during all phases of the drug discovery process are discussed. 1359-6446/04/$ -see front matter ©2004 elsevier ltd. all rights reserved. pii: s1359-6446(04)03196-4 the majority of drugs available today were discovered either from chance observations or from the screening of synthetic or natural product libraries. the chemical modification of lead compounds, on a trial-and-error basis, typically led to compounds with improved potency, selectivity and bioavailability and reduced toxicity. however, this approach is labor-and time-intensive and researchers in the pharmaceutical industry are constantly developing methods with a view to increasing the efficiency of the drug discovery process [1] . two directions have evolved from these efforts. the 'random' approach involves the development of hts assays and the testing of a large number of compounds. combinatorial chemistry is used to satisfy the need for extensive compound libraries. the 'rational', protein structure-based approach relies on an iterative procedure of the initial determination of the structure of the target protein, followed by the prediction of hypothetical ligands for the target protein from molecular modeling and the subsequent chemical synthesis and biological testing of specific compounds (the structure-based drug design cycle). the rational approach is severely limited to target proteins that are amenable to structure determination. although the protein data bank (pdb; http://www.rcsb.org/pdb) is growing rapidly (~13 new entries daily), the 3d structure of only 1-2% of all known proteins has as yet been experimentally characterized. however, advances in sequence comparison, fold recognition and protein-modeling algorithms have enabled the partial closure of the so-called 'sequence-structure gap' and the extension of experimental protein structure information to homologous proteins. the quality of these homology models, and thus their applicability to, for example, drug discovery, predominantly depends on the sequence similarity between the protein of known structure (template) and the protein to be modeled (target). despite the numerous uncertainties that are associated with homology modeling, recent research has shown that this approach can be used to significant advantage in the identification and validation of drug targets, as well as for the identification and optimization of lead compounds. in this review, we will focus on the application of homology models to the drug discovery process. homology, or comparative, modeling uses experimentally determined protein structures to predict the conformation of another protein that has a similar amino acid sequence. the method relies on the observation that in nature the structural conformation of a protein is more highly conserved than its amino acid sequence and that small or medium changes in sequence typically result in only small changes in the 3d structure [2] . generally, the process of homology modeling involves four steps -fold assignment, sequence alignment, model building and model refinement ( figure 1 ). the fold assignment process identifies proteins of known 3d structure (template structures) that are related to the polypeptide sequence of unknown structure (the target sequence; this is not to be mistaken with drug target). next, a sequence database of proteins with known structures (e.g. the pdb-sequence database) is searched with the target sequence using sequence similarity search algorithms or threading techniques [3] . following identification of a distinct correlation between the target protein and a protein of known 3d structure, the two protein sequences are aligned to identify the optimum correlation between the residues in the template and target sequences. the next stage in the homology modeling process is the model-building phase. here, a model of the target protein is constructed from the substitution of amino acids in the 3d structure of the template protein and the insertion and/or deletion of amino acids according to the sequence alignment. finally, the constructed model is checked with regard to conformational aspects and is corrected or energy minimized using force-field approaches. several improvements and modifications of this general homology modeling strategy have been developed and applied to the prediction of protein structures. to subject the available structure prediction methods to a blind test, community-wide experiments on the critical assessment of techniques for protein structure prediction (casp 1-5) have been performed and their results presented and published. as a result, the current state-of-the-art in protein structure prediction has been established, the progress made has been documented and the areas where future efforts might be most productively concentrated have been highlighted [4, 5] . homology modeling techniques are dependent on the availability of high-resolution experimental protein structure data. the development of effective protein expression systems and major technological advances in the instrumentation used for structure determination (x-ray crystallography and nmr spectroscopy) has contributed to an exponential growth in the number of experimental protein 3d structures. by may 2004, the pdb contained ~23,000 experimental protein structures for ~7400 different proteins (proteins with less than 90% sequence identity). a recent analysis of all protein chains in the pdb shows that these proteins can be grouped into 2500 protein families 660 ddt vol. 9, no. 15 august 2004 reviews research focus www.drugdiscoverytoday.com figure 1 . the steps involved in the prediction of protein structure by homology modeling. structure modeling of the bacterial transcriptional repressor copr is shown [28] . although the model is based on a low-sequence identity of only 13.8% between copr and the p22 c2 repressor, several experimental methods support this homology model. reproduced, with permission, from ref. [84] . abbreviation: copr, plasmid copy control protein. target sequence: homolog 1, no 3d structure: homolog 2, no 3d structure: template 1, 3d structure: template 2, 3d structure: comprising 900 unique protein folds [6] (updates can be found at http://scop.mrc-lmb.cam.ac.uk). the majority of the structures in the pdb (84%) were determined by x-ray crystallography, with 15% of the structures being characterized by nmr spectroscopy. the pdb database encompasses experimental information on an extensive array of ligands (small organic molecules and ions) bound to more than 50,000 different binding sites that can be analyzed using programs including relibase (http://relibase.ebi.ac.uk) [7] , ligbase (http://alto.compbio.ucsf.edu/ligbase) [8] and pdbsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum) [9] . although the experimental structure database is growing rapidly, there is still a substantial gap between the number of known annotated sequences [1, 182, 126 unique sequences in swiss-prot-trembl (http://www.expasy.org/ sprot) as of 29 august 2003] and known protein 3d structures (23,000). if only significantly different proteins are considered (~7400), which omits muteins, artificial proteins and multiple structure determinations of the same proteins (e.g. hiv-protease and carbonic anhydrase ii), then less than 1% of the 3d structures of known protein sequences have been elucidated. this sequence-structure gap can partly be filled with homology models. for example, the queryable database modbase (http://alto.compbio. ucsf.edu/modbase-cgi/index.cgi) provides access to an enormous number of annotated comparative protein structure models [10] . the program psi-blast was used to assign protein folds to all 1,182,126 unique sequence entries in swiss-prot-trembl. for 56% of these sequences, comparative models with an average model size of 235 amino acids could be built using the program modeller [11] . thus, by august 2003, 659,495 3d structure models of proteins were accessible via the internet. the models are predicted to have at least 30% of their c α atoms superimposed within 3.5 å of their correct positions. information on binding sites and ligands can be retrieved from this database using ligbase [8] . however, the majority of the models are built on a low sequence identity and it should be realized that this level of accuracy is, in most cases, not sufficient for a detailed structure-based ligand design. the swiss-model repository (http://swissmodel.expasy. org/repository) [12] is also a database of annotated comparative protein 3d structure models, which have been generated using the fully automated homology-modeling pipeline swiss-model. as of august 2003, this database contained models for 282,096 different protein sequence entries (26%) from the swiss-prot-trembl databases (1,073,566 sequences), with an average model size of ~200 amino acids. researchers from eidogen (http://www.eidogen.com) have created a database system called target informatics platform™ [13] that currently includes homology models for 55,000 proteins. homology modeling of 26,279 human protein sequences resulted in the construction of 17,442 models for 13,114 different sequences (50%). thus, putative and known ligand binding pockets can be detected, analyzed and compared and the resulting data used to support target prioritization and lead discovery and/or optimization procedures. accelrys (http://www.accelrys.com) produces discovery studio (ds) atlasstore™ as a complete oracle ® -based protein and ligand structural data management solution. currently, ds atlasstore™ contains 2,052,000 homology models that have been automatically generated from the sequences of 195,000 proteins from 33 different genomes. in conjunction with homology models, cengent therapeutics (http://www.cengent.com) offers dynamic structural information generated from molecular dynamics simulations for 5500 human drug target proteins. this structural information can be used for target prioritization and virtual screening. the quality of the homology models is dependent on the level of sequence identity between the protein of known structure and the protein to be modeled [14] . for a sequence identity that is greater than 30%, homology can be assumed; the two proteins probably have a common ancestor and are, therefore, evolutionarily related and are likely to share a common 3d structure. in this case, pairwise and multiple sequence alignment algorithms are reliable and can be used for the generation of homology models ( figure 2 ). if the sequence identity is below 15%, structure modeling becomes speculative, which could lead to misleading conclusions. when the sequence identity is between 15% and 30%, conventional alignment methods are not sufficiently reliable and only sophisticated, profile-based methods are capable of recognizing homology and predicting fold. for regions of low sequence identity, threading methods [15] are often applied. protein models that are built on such low sequence identities can be used for the assignment of protein function and for the direction of mutagenesis experiments ( figure 2 ). models that have a sequence identity between ~30% and 50% could facilitate the structure-based prediction of target drugability, the design of mutagenesis experiments and the design of in vitro test assays ( figure 2 ). if sequence identity is greater than ~50%, the resulting models are frequently of sufficient quality to be used in the prediction of detailed protein-ligand interactions, such as structure-based drug design and prediction of the preferred sites of metabolism of small molecules ( figure 2 ). there are numerous applications for protein structure information and, hence, homology models at various stages of the drug discovery process [16] . the most spectacular successes are clearly those where protein structural information has helped to identify or to optimize compounds that were subsequently progressed to clinical trials or to the drug market [17] . the applications of homology models that had an impact on target identification and/or validation, lead identification and lead optimization are reviewed here ( figure 3 ). it is clear that only a minute fraction of the entire proteome can be affected by drug-like (preferentially orally bioavailable) small molecules. based on the total numbers of known genes, disease-modifying genes and drugable proteins, the number of drug target proteins, for humans, has been estimated at 600-1500 [18] . for small molecules, sets of properties have been established that differentiate drugs from other compounds [19, 20] ; these properties can be used to identify compounds with, for example, poor oral absorption properties [21] . drug molecules and their corresponding target proteins are highly complementary, which suggests that some rules that distinguish good target proteins from others should be deducible [22] . deep lipophilic pockets that comprise distinct polar interaction sites are clearly superior to shallow highly charged protein surface regions. the inhibition of protein-protein interfaces as a valuable therapeutic principle has recently been shown with inhibitors of the p53-murine double minute clone 2 (mdm2) interaction [23, 24] . the binding site for these inhibitors is a distinct lipophilic pocket that normally interacts with the α-helical surface patches of the p53 tumor suppressor transactivation domain. advances in the rapid detection, description and analysis of ligand-binding pockets [25] [26] [27] , together with the availability of more than 0.5 million homology models, will open new possibilities for the prioritization of proteins with regards to drugability. in the pharmaceutical industry, structural aspects are being increasingly implemented as additional decision criteria on the drugability of potential drug targets. companies such as inpharmatica (http:// www.inpharmatica.com) have developed an integrated suite of informatics-based discovery technologies that contain software tools for the structure-based assessment of target drugability. the design of site-directed mutant proteins is one further important option for the application of homology models to target validation. introducing point mutations and subsequently studying the effects in vitro or in vivo is a common approach in molecular biology. this strategy enables the identification of amino acids that are functionally or relationship between target and template sequence identity and the information content of resulting homology models. arrows indicate the methods that can be used to detect sequence similarity between target and template sequences. applications of the homology models in drug discovery are listed to the right. the higher the sequence identity, the more accurate the resulting structure information. homology models that are built on sequence identities above ~50% can frequently be used for drug design purposes. superimpositions of x-ray crystal structures of the ligand-binding domains of members of the nuclear receptor family are shown to the left. these x-ray structures illustrate the increase in structure deviation with a decreased sequence identity. the pr is red, the gr is green, the erα is blue and the trβ is cyan. sequence identities: pr:gr, 54%; pr:erα, 24%; and pr:trβ, 15%. abbreviations: erα, estrogen receptor α; gr, glucocorticoid receptor; pr, progesterone receptor; trβ, thyroid receptor β. structurally important in the protein under investigation, which ultimately contributes to biological knowledge on, for example, potential target proteins. typically, the amino acids that are to be modified in these studies are selected on the basis of sequence alignments by focusing on conserved residues. however, if at least some structure information is available, the selection of the amino acids that are to be mutated can be much more precise and successful [28] . this approach is even more powerful when applied in conjunction with pharmacologically active compounds. site-directed mutants of the target protein can be made to render that target sensitive to an existing pharmacological agent. based on homology models, some members of the mitogen-activated protein (map) kinase family were mutated to make them sensitive to a kinase inhibitor from the pyridinyl imidazole class [29] . this enabled the use of the compound for broader target validation studies. one of the most attractive ways to validate a target protein is to administer a pharmacologically active compound that selectively acts on that protein and to study the effects in a relevant animal model. similar strategies have been described under the term 'chemogenomics' [30] . it has recently been shown that it is possible to design small molecules based on homology models and then to use these compounds as tools to study the physiological role of the respective target protein of that particular drug [31] . eight years after the discovery of estrogen receptor β (erβ), the distinct roles of the two er isotypes, erα and erβ, in mediating the physiological responses to estrogens are not completely understood. although knockout animal experiments have provided an insight into estrogen signaling, additional information on the function of erα and erβ was imparted by the application of isotype selective er agonists. based on the crystal structure of the erαligand-binding domain (lbd) and a homology model of the erβ-lbd (59% sequence identity to erα), hillisch et al. [31] designed steroidal ligands that exploit the differences in size and flexibility of the two ligand-binding cavities ( figure 4 ). compounds that were predicted to bind preferentially to either erα or erβ were synthesized and tested in vitro. this approach led directly to highly er isotype-selective (200-250-fold) ligands that were also highly potent. to unravel the physiological roles of each of the two receptors, in vivo experiments with rats were conducted using the erα-and erβ-selective agonists in parallel with the natural ligand of er, 17β-estradiol. the erα agonist was shown to be responsible for most of the known estrogenic effects (e.g. induction of uterine growth and bone-protection), in addition to pituitary (e.g. reduction of luteinizing hormone plasma levels) and liver (e.g. increase in angiotensin i plasma levels) effects [31] . however, the erβ agonist had distinct effects on the ovary, for example, the stimulation of early folliculogenesis [32] , which possibly presents clinicians with a new option for tailoring classical ovarian stimulation protocols. a comparison of the homology model with the x-ray crystal structure of the erβ-lbd complexed with genistein [33] revealed that the homology model had a root-mean-square deviation (rmsd) of the backbone atoms (not considering helix 12) of 1.4 å. the x-ray crystal structure confirmed the presence of essential interactions between the ligand and the erβ and did not reveal, at least in this case, any new aspects for the design of erβ agonists that were not covered by the homology model. these studies show that it is possible to design highly selective compounds, if structure information on all of the relevant homologs of the target is available, and 663 ddt vol. 9, no. 15 august 2004 reviews research focus www.drugdiscoverytoday.com figure 3 . applications of homology models in the drug discovery process. the enormous amount of protein structure information currently available could not only support lead compound identification and optimization, but could also contribute to target identification and validation. reproduced, with permission, from [84] . there are numerous examples where protein homology models have supported the discovery and the optimization of lead compounds with respect to potency and selectivity. currently, the structures of 40 of the 518 known different human protein kinases have been characterized by x-ray crystallography [34] . homology model-based drug design has been applied to epidermal growth factor-receptor tyrosine kinase protein [35, 36] , bruton's tyrosine kinase [37] , janus kinase 3 [38] and human aurora 1 and 2 kinases [39] . using the x-ray crystal structure of cyclin-dependent kinase 2 (cdk2), honma et al. [40] generated a homology model of cdk4. this model guided the design of highly potent and selective cdk4 inhibitors that were targeted towards the atp binding pocket. the diarylurea class of compounds were subsequently synthesized and tested. in an in vitro inhibition assay, the most potent compound had an ic 50 of 42 nm. the predicted binding mode of the lead compound was verified by co-crystallization with cdk2 [40] . vangrevelinghe et al. [41] identified a cdk2 inhibitor using a homology model of the protein and highthroughput docking. siedlecki et al. [42] have demonstrated the utility of homology modeling in the prediction of pharmacologically active compounds. alterations in dna methylation patterns play an important role in tumorigenesis; therefore, inhibitors of dna methyltransferase 1 (dnmt1), which is the protein that represents the major dna methyltransferase activity in human cells, are desired. known inhibitors from the 5-azacytidine class were docked into the active site of a dnmt1 homology model, which led to the design of n4-fluoroacetyl-5-azacytidine derivatives that acted as highly potent inhibitors of dna methylation in vitro. thrombin-activatable fibrinolysis inhibitor (tafi) is an important regulator of fibrinolysis, and inhibitors of this enzyme have potential use in antithrombotic and thrombolytic therapy. based on a homology model of tafi (~50% sequence identity to carboxypeptidases a and b), appropriately substituted imidazole acetic acids were designed and were subsequently found to be potent and selective inhibitors of activated tafi [43] . homology models of the voltage-gated k + -channel k v 1.3 and the ca 2+ -activated channel ik ca 1 were used to design selective ik ca 1 inhibitors that were based on the polypeptide toxin charybdotoxin. comparison of the two models revealed a unique cluster of negatively charged residues in the turret of k v 1.3 that were not present in ik ca 1. to exploit this difference, the homology model was used to design novel analogs, which were then synthesized and tested. research demonstrated that the novel compounds blocked ik ca 1 activity with ~20-fold higher affinity than k v 1.3 [44] . 4. (a,b) comparison of the two isotypes of the estrogen receptor. in the homology model, erα (blue) and erβ (green) ligand-binding pockets are shown in complex with the natural ligand of the er, 17β-estradiol. the binding of 8β-ve 2 , a highly potent and selective erβ agonist, modeled into the erβ ligand-binding niche is depicted to the right. reproduced, with permission, from ref. [31] . (c,d) a model of the antiprogestin ru 486 (mifepristone) bound to hpr. a single amino acid mutation renders this compound inactive at the cpr and hamster pr. steric clashes between ru 486 and cpr are shown on the right side. abbreviations: er, estrogen receptor; her, human estrogen receptor; cpr, chicken progesterone receptor; hpr, human progesterone receptor; pr, progesterone receptor; rba, relative binding affinity; 8β-ve 2 , 8β-vinylestra-1,3,5(10)-triene-3,17β-diol. the key proteinase (m pro , or 3cl pro ) of the new coronavirus (cov) that caused the severe acute respiratory syndrome (sars) outbreak of 2003 (sars-cov) is another example of successful homology model building; in this case, success is defined as the ability to use the model to propose an inhibitor that has significant affinity for the target enzyme. x-ray crystal structures for the m pro s of transmissible gastroenteritis virus (tgev, a porcine coronavirus) and of human coronavirus 229e [45, 46] have been characterized. these proteinases have 44 and 40% sequence identity, respectively, with the key proteinase of sars-cov. following publication of the genome sequence of the new virus, first on the internet and a few weeks later in print [46, 47] , the level of sequence identity between the proteinases enabled anand et al. [46] to construct a 3d homology model for the m pro of human cov. however, the 3d homology model generated was insufficient for the design of inhibitors with reasonable confidence. to establish the structural basis of the interaction with the polypeptide substrate of the m pro , anand and co-workers [46] synthesized a substrate-analogous hexapeptidyl chloromethylketone inhibitor that was complexed with tgev m pro . the x-ray crystal structure of the complex was then determined, which revealed that, as expected, the chloromethylketone moiety had covalently reacted with the active-site cysteine residue of the proteinase. the p1, p2, and p4 side chains of the inhibitor had bound to, and thereby defined, the specificity binding sites of the target enzyme. the experimentally determined structure of the inhibitor-tgev m pro complex was then compared with all inhibitor complexes of cysteine proteinases in the pdb, which revealed a surprisingly similar inhibitor binding mode in the complex of human rhinovirus type 2 (hrv2) 3c proteinase with ag7088 ( figure 5 ) [48] . at that time, ag7088 was in late phase ii clinical trials as a drug for the treatment of the strain of the common cold that is caused by human rhinovirus. the comparison of the crystal structures of hrv2 in complex with ag7088 and tgev m pro in complex with the hexapeptidyl chloromethylketone inhibitor revealed little similarity between the two target enzymes, except in the immediate neighborhood of the catalytic cysteine residue, but an almost perfect match of the inhibitors. to investigate these findings further, ag7088 was docked into the substrate-binding site of the sars-cov m pro model without much difficulty, although it was noted that there could potentially be steric problems with the p-fluorobenzyl group in the s2 pocket, and also with the ethylester moiety in s1′. therefore, it was proposed that, although ag7088 was not an ideal inhibitor, this compound should be a good starting point for the design of anti-sars drugs. indeed, only a few days after the on-line publication of these results in sciencexpress [46] , it was confirmed that ag7088 had anti-sars activity in vitro. derivatives of ag7088 with modified p2 residues have since been shown to have k i values in the lower µmolar range (rao et al., pers. commun.) . the crystal structure of the authentic sars-cov key proteinase was determined a few months later [49] . although the dimeric structure showed the expected similarity to the homologous enzymes of tgev and human cov 229e, there were interesting differences in detail. in particular, one of the monomers in the dimer was observed to be in an inactive conformation, which was thought to be the result of the low ph of crystallization. the overall rmsd for the entire dimer from the homology model of anand et al. [46] was >3.0 å (i.e. no residues excluded from the comparison), which dropped to 2.1 å when a few outliers at the carboxy terminus were excluded from the comparison, and to <1.8 å for each of the three individual domains of the enzyme. other homology models were generated (d. debe, unpublished and [50] ) and virtual screening has been performed using a sars-cov m pro model [51] . taken together, these findings confirm that homology modeling is often inadequate for the prediction of the mutual orientation of domains in multidomain proteins. however, the homology model generated by anand et al. [46] also shows that a reasonable model of a substrate-binding site can serve to develop useful ideas for inhibitor design that can inspire medicinal chemists to start a synthesis program long before the 3d structure of the target enzyme is experimentally determined. in the case of g-protein-coupled receptors (gpcr), homology-modeling approaches are limited by the lack of experimentally determined structures and the low sequence similarity of those structures that have been characterized with respect to pharmacologically important target proteins. the x-ray crystal structure of only one gpcr, bovine rhodopsin, has been determined [52] . this structure is complemented by bacteriorhodopsin, which is a transmembrane protein that comprises seven helices and is also of relevance for modeling approaches, even though this protein is not a gpcr. some examples of homology models for gpcrs and their utility have recently been reviewed [53] . high-throughput docking has been applied to verify the ability of homology models to identify agonists (glucocorticoid receptor agonists) [54] , antagonists of retinoic acid receptor α [55] , d 3 -dopamine-, m 1 -muscarinic acetylcholine-and v 1a -vasopressin-receptors [56] and inhibitors of thrombin [57] . in the identification of thrombin inhibitors, homology models of thrombin were built retrospectively and were based on homologous serine proteases (28%-40% sequence identity); the best docking solutions were yielded with those models that were derived from proteins of higher sequence identity. recently, the performance of docking studies into protein active sites that had been constructed from homology models was assessed using experimental screening datasets of cdk2 and factor viia [58] . when the sequence identity between the model and the template near the binding site was greater than ~50%, there was an approximate fivefold increase in the number of active compounds identified than would have been be detected randomly. this performance is comparable to docking to crystal structures. a further application of homology models is the design of test assays for the in vitro pharmacological characterization of compounds or hts. based on the structure of the coiledcoil domain of c-jun, models for α-helical proteins were designed such that they can be used as affinity-tagged proteins that incorporate protease cleavage sites [59] . the resulting 10.5 kda recombinant proteins were synthesized and used as molecularly defined and uniform substrates for in vitro detection of hiv-1 and iga endoprotease activity, which enabled the surface plasmon resonance-based screening of inhibitors. the enormous volume of structure information on entire target protein families that is available might also have an impact on screening cascades. many drug discovery projects endeavor to identify ligands that are highly selective for particular drug targets. selective compounds are supposed to be superior because such compounds typically lead to fewer adverse side effects (e.g. cox-2 inhibitors). however, the most important homologs that should not be targeted by the desired drug, with respect to the actual target, are not always clear, particularly within the larger target protein families. the sequence similarity of the full-length proteins or entire domains might not always be representative of the target protein when considering the conservation of the ligand-binding pockets. comparison of the shape and features of the binding pockets within a protein family could indicate which homologs should be included in the screening cascade for so-called 'counter screening'. the structure information that is currently available on entire protein families (e.g. proteases, kinases and nuclear receptors) could contribute to the design of selective compounds or better screening cascades, both of which could potentially advance the design of drugs that have fewer side effects. a detailed structural knowledge of the ligand-binding sites of target proteins was also shown to facilitate the selection of animal models for ex vivo or in vivo experiments. the proposal is that animals having target proteins with significantly different binding sites compared with human orthologs should be excluded as pharmacological models. many promising compounds showing high-potency in human in vitro assays have not reached clinical trials because efficacy could not be demonstrated in animal models. single amino acid differences between humans and animals might, in some cases, be sufficient to cause such effects. the er selectivity of ligands described by hillisch et al. [31] was shown by homology models and in vitro assays to be crucially dependent on the interaction of ligand substituents with one particular amino acid that differs between erα and erβ (figure 4a ) [31] . to ensure that this important interaction is present in estrogen receptors of all animal models that are used to characterize compounds [32] , homology models of murine, rat and bovine erβ were built and compared with the binding pocket of human erβ (herβ). a complete conservation of amino acids within the binding pockets of human, murine and rat erβ was observed. however, bovine erβ showed one amino acid difference at the exact position that was determined to be crucial for erβ ligand selectivity. the prediction that the herβ selective compounds should not bind to bovine erβ was later verified using transactivation experiments (unpublished results). thus, the implementation of uninterpretable experiments could be avoided at an early stage and the otherwise attractive bovine tissues (later available in larger amounts) could be excluded from ex vivo investigations. similarly, information on the structure of progesterone receptors (pr) can be used to explain the abolished binding of the progesterone antagonist mifepristone (ru 486) to chicken pr and hamster pr [60] . a single point mutation (human pr gly722 to chicken pr cys575) prevents antiprogestins containing 11β-aryl substituents (e.g. ru 486) from binding to chicken (and hamster) pr (figure 4c) , which therefore excludes hamsters, for example, from pharmacological studies with antiprogestins [61] . in the future, such effects could be predicted and particular species could then be excluded from pharmacological studies at an early stage, which would ultimately reduce attrition rates in the drug discovery process. one of the challenges in lead optimization is to identify compounds that not only show a high potency at the desired target protein but also have adequate physical properties to reach systemic circulation, to resist metabolic inactivation for a specific time period and to avoid undesired pharmacological effects. knowledge of the structure of the proteins that are involved in these processes, such as drugmetabolizing enzymes, transcription factors or transporters, could help to design molecules that do not interact with these 'non-target' proteins. the cytochrome p450s (cyp) are an extremely important class of enzymes that are involved in phase i oxidative metabolism of structurally diverse chemicals. only ~10 hepatic cyps are responsible for the metabolism of 90% of known drugs. recently, the x-ray crystal structures of three mammalian cyps, cyp2c5 [62] , cyp2c8 [63] and cyp2c9 [64] , have been solved and represent a solid basis for the homology modeling of this entire superfamily. models of cyp1a2, cyp2a6, cyp2b6, cyp2c8, cyp2c9, cyp2c19, cyp2d6, cyp2e1, cyp3a4 and cyp4a11 have been generated using different structure templates. these models have been used to explain and to predict the probable sites of metabolic attack in a variety of cyp substrates [65] [66] [67] [68] [69] [70] [71] [72] . however, the large lipophilic and highly flexible character of some cyp binding cavities renders pure in silico approaches towards the prediction of the occurrence and site of small molecule metabolism extremely difficult. if protein structure information is combined with pharmacophoric patterns and quantum mechanical calculations, some predictions concerning the preferred sites of metabolism within small molecules are possible [73] . regarding this aspect of homology modeling, cyp2d6 is a particularly interesting cyp because 5-9% of the caucasian population does not produce this polymorphic member of the cyp superfamily. the resulting deficiencies in drug oxidation can lead to severe side effects in these individuals. predictions on whether or not a lead compound could act as a cyp2d6 substrate could help to identify problematic cases early in drug discovery. combined homology modeling and quantitative sar approaches are able to predict such cyp inhibitors [74] . thus, in the future, protein structure information in conjunction with high-throughput docking and pharmacophore-based methods could be used to decide which compounds have the potential to inhibit particular cyps. this approach could facilitate the detection of potential drug-drug interactions early in the drug discovery process and measures could then be taken to avoid such interactions [75] . cyp substrates and inhibitors are not the only compounds to have been studied using homology models. these approaches have been used recently to investigate cyp inducers. the induction of cyps is primarily mediated via the activation of ligand-dependent transcription factors, such as the aryl hydrocarbon receptor (ahr) for the cyp1a family, the constitutive androstane receptor (car) for the cyp2d family and the pregnane x receptor (pxr), glucocorticoid receptor (gr) and vitamin d receptor (vdr) for the cyp3a family [76] . in principle, the in silico prediction of drug-metabolizing enzyme induction could be reduced to predicting the binding and activation of transcription factors (e.g. ahr and car). however, recent xray structure analyses of pxr have shown that the lbd of this nuclear receptor contains a large lipophilic and flexible binding pocket [77] . this renders pure in silico structure-based predictions concerning whether or not a small molecule will activate pxr difficult. the homology modeling of car [78, 79] and other members of the nuclear receptor family involved in cyp induction [80] have recently been described. these models predict reasonably shaped potential ligand binding pockets. however, further results on the utility of these models are needed. with respect to the structure-based prediction of adverse health effects, progress has been described with the human ether-a-go-go-related gene (herg). this tetrameric potassium channel contributes to phase three repolarization of heart muscle cells by opposing the depolarizing ca 2 + influx during the plateau phase. inhibition of this protein results in cardiovascular toxicity (qt-prolongation) and has caused several drugs to be withdrawn from the market. therefore, in silico predictions on the probability of the formation of an interaction between a drug and herg have gained enormous attention and have recently been reviewed [81] . homology models of herg, which are based on the x-ray crystal structures of the bacterial kcsa [82] and mthk channels [83] , have already shed light on some details of the molecular interactions that initiate herg inhibition. however, the complexity of this potassium channel signifies that detailed x-ray structure analyses of the protein in the open-and closed-state are required before these molecular interactions can be fully understood and predicted, which has implications for the prediction of cardiotoxicity. numerous examples for the successful application of homology modeling in drug discovery are described here. in the absence of experimental structures of drug target proteins, homology models have supported the design of several potent pharmacological agents. one of the advantages of homology models is that these models can be generated 667 ddt vol. 9, no. 15 august 2004 reviews research focus relatively easily and quickly. furthermore, such models could support the hypotheses of medicinal chemists on how to generate biologically active compounds in the important early conceptual phase of a drug discovery project. the design of compounds that are selectively directed at particular drug target proteins is one of the strengths of this technique. such selective compounds can even be applied to gain insights into the physiological role of novel drug targets. the in silico protein structure-based prediction of metabolism and toxicity of small molecules, particularly cyp inhibition and induction and herg inhibition, is currently in its infancy and predictive capabilities could be limited to classification only. however, while complete experimental structures of pharmacologically important proteins are missing, the homology modeling technique provides one approach to bridge the gap until this information becomes available. modern methods of drug discovery: an introduction the response of protein structures to amino-acid sequence changes fold recognition methods progress in protein structure prediction assessment of homology-based predictions in casp5 scop: a structural classification of proteins database for the investigation of sequences and structures databases for protein-ligand complexes ligbase: a database of families of aligned ligand binding sites in known protein sequences and structures pdbsum: summaries and analyses of pdb structures modbase, a database of annotated comparative protein structure models, and associated resources comparative protein modelling by satisfaction of spatial restraints the swiss-model repository of annotated threedimensional protein structure homology models supporting your pipeline with structural knowledge the relation between the divergence of sequence and structure in proteins casp5 assessment of fold recognition target predictions the role of protein 3d structures in the drug discovery process the impact of structure-guided drug design on clinical agents the druggable genome prediction of 'drug-likeness' a scoring scheme for discriminating between drugs and nondrugs drug-like properties and the causes of poor solubility and poor permeability structural bioinformatics in drug discovery inhibition of the p53-hdm2 interaction with low molecular weight compounds inhibition of the p53-mdm2 interaction: targeting a protein−protein interface a new method to detect related function among proteins independent of sequence and fold homology inferring functional relationships of proteins from local sequence and spatial surface patterns structural classification of protein kinases using 3d molecular interaction field analysis of their ligand binding sites: target family landscapes transcriptional repressor copr: structure model-based localization of the deoxyribonucleic acid binding motif use of a drug-resistant mutant of stress-activated protein kinase 2a/p38 to validate the in vivo specificity of sb 203580 chemogenomics: an emerging strategy for rapid target and drug discovery dissecting physiological roles of estrogen receptor alpha and beta with potent selective ligands from structurebased design impact of isotype-selective estrogen receptor agonists on ovarian function structure of the ligand-binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist the protein kinase complement of the human genome design and synthesis of novel tyrosine kinase inhibitors using a pharmacophore model of the atp-binding site of the egf-r rational design of potent and selective egfr tyrosine kinase inhibitors as anticancer agents rational design and synthesis of a novel antileukemic agent targeting bruton's tyrosine kinase (btk), lfm-a13 structure-based design of specific inhibitors of janus kinase 3 as apoptosis-inducing antileukemic agents targeting aurora2 kinase in oncogenesis: a structural bioinformatics approach to target validation and rational drug design structure-based generation of a new class of potent cdk4 inhibitors: new de novo design strategy and library design discovery of a potent and selective protein kinase ck2 inhibitor by high-throughput docking establishment and functional validation of a structural homology model for human dna methyltransferase 1 synthesis and evaluation of imidazole acetic acid inhibitors of activated thrombin-activatable fibrinolysis inhibitor as novel antithrombotics structure-guided transformation of charybdotoxin yields an analog that selectively targets ca 2+ -activated over voltage-gated k + channels structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs characterization of a novel coronavirus associated with severe acute respiratory syndrome structure-assisted design of mechanismbased irreversible inhibitors of human rhinovirus 3c protease with potent antiviral activity against multiple rhinovirus serotypes the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor evaluation of homology modeling of the severe acute respiratory syndrome (sars) coronavirus main protease for structure-based drug design a 3d model of sars cov 3cl proteinase and its inhibitors design by virtual screening crystal structure of rhodopsin: a g-proteincoupled receptor modeling the 3d structure of gpcrs: advances and application to drug discovery nuclear hormone receptor targeted virtual screening rational discovery of novel nuclear hormone receptor antagonists protein-based virtual screening of chemical databases. ii. are homology models of g-protein-coupled receptors suitable targets? docking ligands onto binding site representations derived from proteins built by homology modelling performance of 3d database molecular docking studies into homology models design of helical proteins for real-time endoprotease assays a single amino acid that determines the sensitivity of progesterone receptors to ru486 ru486 is not an antiprogestin in the hamster mammalian microsomal cytochrome p450 monooxygenase: structural adaptations for membrane binding and functional diversity structure of human microsomal cytochrome p450 2c8. evidence for a peripheral fatty acid binding site crystal structure of human cytochrome p450 2c9 with bound warfarin molecular modeling of human cytochrome p450-substrate interactions modelling human cytochromes p450 involved in drug metabolism from the cyp2c5 crystallographic template homology modelling of human cyp1a2 based on the cyp2c5 crystallographic template structure homology modelling of cyp2a6 based on the cyp2c5 crystallographic template: enzyme-substrate interactions and qsars for binding affinity and inhibition molecular modelling of cyp2b6 based on homology with the cyp2c5 crystal structure: analysis of enzyme-substrate interactions a molecular model of cyp2d6 constructed by homology with the cyp2c5 crystallographic template: investigation of enzyme-substrate interactions investigation of enzyme selectivity in the human cyp2c subfamily: homology modelling of cyp2c8, cyp2c9 and cyp2c19 from the cyp2c5 crystallographic template prediction of drug metabolism: the case of cytochrome p450 2d6 a novel approach to predicting p450 mediated drug metabolism. cyp2d6 catalyzed n-dealkylation reactions and qualitative metabolite predictions using a combined protein and pharmacophore model for cyp2d6 competitive cyp2c9 inhibitors: enzyme inhibition studies, protein homology modeling, and three-dimensional quantitative structure-activity relationship analysis molecular basis of p450 inhibition and activation: implications for drug development and drug therapy prediction of human drug metabolizing enzyme induction coactivator binding promotes the specific interaction between ligand and the pregnane x receptor a structural model of the constitutive androstane receptor defines novel interactions that mediate ligandindependent activity insights from a three-dimensional model into ligand binding to constitutive active receptor molecular modelling of the human glucocorticoid receptor (hgr) ligand-binding domain (lbd) by homology with the human estrogen receptor alpha (heralpha) lbd: quantitative structure-activity relationships within a series of cyp3a4 inducers where induction is mediated via hgr involvement predicting undesirable drug interactions with promiscuous proteins in silico a structural basis for drug-induced long qt syndrome characterization of herg potassium channel inhibition using comsia 3d qsar and homology modeling approaches modern methods of drug discovery we gratefully acknowledge fruitful discussions with mario lobell (bayer healthcare; http://www.bayerhealthcare.com), derek debe, sean mullen (eidogen) and sunil patel (accelrys). key: cord-256316-1odgm6hm authors: godet, murielle; l'haridon, rene; vautherot, jean-francois; laude, hubert title: tgev corona virus orf4 encodes a membrane protein that is incorporated into virions date: 1992-06-30 journal: virology doi: 10.1016/0042-6822(92)90521-p sha: doc_id: 256316 cord_uid: 1odgm6hm abstract the coding potential of the open reading frame orf4 (82 amino acids) of transmissible gastroenteritis virus (tgev) has been confirmed by expression using a baculovirus vector. five monoclonal antibodies (mabs) raised against the 10k recombinant product immunoprecipitated a polypeptide of a similar size in tgev-infected cells. immunofluorescence assays performed both on insect and mammalian cells revealed that orf4 was a membrane-associated protein, a finding consistent with the prediction of a membrane-spanning segment in orf4 sequence. two epitopes were localized within the last 21 c-terminal residues of the sequence through peptide scanning and analysis of the reactivity of a truncated orf4 recombinant protein. since the relevant mabs were found to induce a cell surface fluorescence, these data suggest that orf4 may be an integral membrane protein having a cexo-nendo orientation. anti-orf4 mabs were also used to show that orf4 polypeptide may be detected in tgev virion preparations, with an estimated number of 20 molecules incorporated per particle. comparison of amino acid sequence data provided strong evidence that other coronaviruses encode a polypeptide homologous to tgev orf4. our results led us to propose that orf4 represents a novel minor structural polypeptide, tentatively designated sm (small membrane protein). virology 188, 666-675 (1992) the coding potential of the open reading frame orf4 (82 amino acids) of transmissible gastroenteritis virus (tgev) has been confirmed by expression using a baculovirus vector. five monoclonal antibodies (mabs) raised against the 10k recombinant product immunoprecipitated a polypeptide of a similar size in tgev-infected cells. immunofluorescence assays performed both on insect and mammalian cells revealed that orf4 was a membrane-associated protein, a finding consistent with the prediction of a membrane-spanning segment in orf4 sequence. two epitopes were localized within the last 21 c-terminal residues of the sequence through peptide scanning and analysis of the reactivity of a truncated orf4 recombinant protein. since the relevant mabs were found to induce a cell surface fluorescence, these data suggest that orf4 may be an integral membrane protein having a cexo-nendo orientation. anti-orf4 mabs were also used to show that orf4 polypeptide may be detected in tgev virion preparations, with an estimated number of 20 molecules incorporated per particle. comparison of amino acid sequence data provided strong evidence that other coronaviruses encode a polypeptide homologous to tgev orf4. our results led us to propose that orf4 represents a novel minor structural polypeptide, tentatively designated sm (small membrane protein). transmissible gastroenteritis virus (tgev), an important pathogen of swine, is a member of the coronaviridae, a family of enveloped viruses with a large (-30 kb) continuous, positive rna genome. sequencing data have led to the identification of a number of large open reading frames (orfs). orfla and b, which account for the 5' two-thirds of the coronavirus genome, are assumed to encode nonstructural proteins including the viral replicase/transcriptase. the seven to eight remaining orfs are expressed through a set of 3' coterminal, subgenomic size mrnas of which only the unique region is translationally active. these include the orfs coding for the virion structural proteins, i.e., the nucleocapsid (n) and two or three envelope glycoproteins: the spike (s) and the membrane (m) proteins, and the hemagglutinin-esterase (he) present in a coronavirus subset. these orfs are distributed following the consensus gene order 5' pol-(he)-s-m-n 3'. the other orfs are interspersed within the genome and their number and position differ among coronavirus members (reviewed by spaan et a/., 1988; and lai, 1990) . they have been shown to be expressed by functionally mono-, di-, or tricistronic mrnas and were generally assumed to encode nonstructural proteins, the function of which is still unknown or conjectural. ' to whom correspondence and reprint requests should be addressed. in tgev genome, four such orfs have been deduced. two of them are expressed by the same mrna (mrna 3) in two out of the three virus strains sequenced (rasschaert et a/., 1987; kapke et a/., 1988; britton et al., 1989; wesley et al., 1989) . the predicted product of orf3a is 61 to 71 codon long, with a variable c-terminal end. it appears to be dispensable for virus replication since it was found to be absent in a tgev variant strain sp (wesley et a/., 1990) as well as in the closely related porcine respiratory coronavirus (prcv) (rasschaert eta/., 1990) . orf3b (expressed by a separate mrna species numbered 3-l in miller strain) has a constant length (244 residues); however, one clone of purdue-l 15 strain was reported to have an orf3b which is shortened by 79 codons at the 5' end and in several cdna clones by 67 codons at the 3' end (rasschaert et al., 1987) . orf4 was predicted to encode a 82 amino acid long hydrophobic polypeptide (rasschaer-t et a/., 1987; kapke et a/., 1988; britton et a/., 1989; wesley eta/., 1989) . so far, the putative products of the three above-mentioned orfs have not been identified in infected cells. the last orf, orf7, is located downstream of the n gene (kapke and brian, 1986; rasschaert et al., 1987; britton et al., 1988) an unusual feature among coronaviruses. a polypeptide of m, 14k, reacting with antibodies produced against an orf7 synthetic peptide, has been characterized in tgev-infected cells (garwes et al., 1989) . in this study we report the identification of a product of one of these orfs (orf4) in infected cells and its preliminary characterization. in particular, we show evidence that orf4 represents a novel virion-associated polypeptide with a possible counterpart in other coronaviruses. autographa cahfornica nuclear polyhedrosis virus (acnpv) and recombinant baculoviruses were grown and assayed in confluent monolayers of spocfoptera frugiperda (sf9) cells in medium containing 10% (v/v) fetal bovine serum, according to the procedures described by brown and faulkner (1977) . propagation of the high cell passage purdue-l 15 strain of tgev in swine cell lines pd5 or st was done as previously described (laude et al., 1986) . manipulations of plasmid dna were performed according to the procedures described by sambrook et a/. (1989) . restriction enzymes, t4 dna ligase, and calf intestine alkaline phosphatase (cip) were purchased from boehringer-mannheim. the baculovirus transfer plasmid containing the full-length cdna copy of tgev orf4 coding sequence was constructed following the general scheme outlined in fig. 1 . the ndel-sspl fragment (0.7 kbp) derived from plasmid ptg2-15 (rasschaert et al., 1987) was digested with the ddel restriction enzyme. ddel-sspl dna fragment was repaired with the klenow large fragment of dna polymerase i and cloned into the barnhi site of the pvl941 vector (luckow and summers, 1989) . the resulting plasmid, named pvlorf4, contained a 0.33 kbp insert. a second plasmid, pvlorf4a was constructed by inserting an orf4 gene in which the 3' last 63 nt were deleted through pcr mutagenesis @char-f et a/., 1986) on ptg2-15 using the oligonucleotides 5' g aagaagggatccatacctatgac and 5' clta-tagggatcctaagcatg as 5' and 3' amplimers, respectively. these amplimers were designed to introduce an additional stop codon tag at the 3'end of the gene and a barnhi cloning site at each end. the amplification product was digested by bamhl and ligated into the pvl941 cloning site. the orientation and sequence of the orf4 and orf4a inserts relative to the acnpv polyhedrin leader were determined by restriction analysis and partial dna sequencing. in these constructs, the initiation codon of the orf4 and orf4a sequences were positioned at 54 and 7 bp from the bamhl cloning site, respectively. transfer of the tgev orf4 gene into the acnpv genome was accomplished by transfection of sf9 cells using the calcium phosphate precipitation technique as described by summers and smith (1987) . recombinant baculoviruses were screened by dot blot hybridization using an orf4specific [32p]-labeled dna fragment as a probe. four polyhedrin-negative clones were tested for orf4 expression. tgev orf4a gene was introduced into a linear form of acnpv dna (kitts eta/., 1990) . circular acrpg-sc dna was linearized by digestion at the unique bsu361 site. two hundred nanograms of bsu361 or mock digest viral dna was mixed with 1 pg of pvlorf4a dna and transfected into sf9 cells using the lipofectin method (kitts et a/., personal communication) according to the procedure of the manufacturer (gibco-brl). after a 2-day incubation the culture supernatants were harvested and plated. a dozen well-isolated plaques were picked out and screened for orf4a expression using [35s]methionine-labeled cultures. three balb/c mice were injected threefold intraperitoneally at a 1 month interval with 1 x 10' acorf4-infected cells (disrupted in freund complete adjuvant for the first injection). three days before fusion, the mice were boosted both intraperitoneally and subcutaneously with orf4 protein purified from 4 x 10' infected cells by 15% sds-page. splenocytes from one mouse that tested positive by immunoprecipitation assay were fused with sp,o myeloma cells. supernatants of hybrid clones were tested in a comparative immunofluorescence assay (see below) using acorf4or acnpv-infected sf9 monolayers. subcloning of orf4-specific antibody-producing hybridomas and ig isotyping were done as described elsewhere (l' haridon et al., 1991) . iggs purified from ascites fluids by ammonium sulfate precipitation and gel permeation on a sephacryl-s200 column were used in all experiments. screening of hybridoma was performed on sf9 cell monolayers established in 96-well microplates, infected with baculovirus (m.o.i. 10 pfu) and fixed with acetone/ethanol (v/v) at 38 hr p.i. for surface fluorescence analysis, aliquots of cells in suspension were stained with mabs at 100 pglml in grace medium and then with fitc conjugate (each step 1 hr at 4") and spotted onto glass slides. alternatively, spotted cells were fixed with 4% paraformaldehyde and permeabil-ized or not with 0.1% triton x-l 00 before staining (1 hr at 37"). similar experiments were performed on st cell monolayers infected by tgev at a m.o.i. of 0.1 pfu and fixed 15 hr p.i. the procedures for metabolic labeling of insect and mammalian cells were as reported previously (godet et a/., 1991; . monolayers of 4 x 1 o5 sf9 cells or 6 x 1 o6 pd5 cells labeled with [35s]methionine were washed with pbs and lyzed in 4 ml of pbs-triton (tris, 50 mm, ph 8.5, 1 o/o triton x-l 00, 1 o3 kallicrein units of aprotinin per milliliter). resulting cytosols of sf9 cells and pd5 cells were centrifuged 30 min at 10,000 g or 1 hr at 30,000 rpm in a 50 ti rotor (beckman), respectively and stored in aliquots at -70". lmmunoprecipitation assay aliquots of radiolabeled cytosols or virions were adjusted to 0.5 ml with pbs-triton buffer containing the appropriate mab (100 pg/ml) or 3 ~1 of ascites fluid from a feline infectious peritonitis virus-infected cat (used as a source of anti-tgev polyclonal antibodies) and protein a-sepharose beads (50 ~1 of a 50% suspension); after a 2-hr incubation at room temperature with agitation, the immune complexes were extensively washed with pbs-triton, then with 0.5 m nacl + 50 mn/l tris (ph 8). beads were treated for 3 min at 100" in sample buffer. the immunoprecipitated material thus released was analyzed by 15% or 15-20% sds-page. virions in the supernatant of cultures labeled as above were purified following a described procedure (laude et a/., 1986) . the material pelleted by ultracentrifugation at 35,000 rpm in a 45 ti rotor was resuspended in distilled water and was applied to a linear sucrose gradient (16 ml, 20 to 45% sucrose w/v in distilled water). centrifugation was performed in an sw27 rotor for 3 hr at 25,000 rpm and 4". gradients were collected from top to bottom into 500-p.1 fractions. half of each fractions was run at 100,000 rpm for 30 min and the resulting pellets were analyzed by sds-page electrophoresis on a 8-20% gradient gel. the material remaining in the virus-containing fractions was pooled, pelleted as above, and split into three parts; one was analyzed directly by 15% sds-page; the two others were solubilized in pbs-triton, immunoprecipitated by a different mab each, and analyzed as above. in one experiment, virion-associated material was subjected to a second round of gradient purificalaude et al., 1986) prior to gel analysis. peptides were synthesized on polyethylene pins (geysen eta/., 1984) by using a commercially available kit, according to the procedure given by the manufacturer (cambridge research biochemicals). immunoreactivity of the immobilized peptides was assayed by elisa using anti-orf4 mabs (25 pglml) as primary antibody and an anti-mouse igg (h + l) peroxydase conjugate (biosys). insertion of the entire orf4 coding sequence into the genome of acnpv baculovirus was performed by using the transfer plasmid pvl941 (see methods and specific probe and a polyhedron-negative phenotype were selected, amplified, and tested for the expression of orf4. among four selected orflf-expressing clones, one, designated acorf4, was retained for subsequent studies. analysis of [35s]methioninelabeled acorf4-infected cells revealed the presence of a major polypeptide of i'@ 1 ok (fig. 2) in good agreement with the predicted m.w. of orf4 product (9.2k). the time course for its synthesis was found to be from 24 to 48 hr p.i. screening of positive hybridoma clones was achieved by comparative indirect immunofluorescence assay on acorf4-or acnpv-infected cells. among 268 hybridomas tested, 5 positive clones, all producing mabs of iggl isotype, were subcloned and used for the production of ascites fluids. when assayed by immunoprecipitation of acorf4-infected cells extracts, all 5 mabs recognized a single species of m, 1 ok, identical to the target protein (fig. 3a, lane 4 ). an immunoreactive species comigrating with recombinant orf4 protein was detected in tgev-infected mammalian cells as well (lane 2). analysis of the recombinant protein in nonreducing conditions revealed the existence of oligomeric forms, which were not observed with the authentic orf4 protein (fig. 3b) . orf4 product is a membrane-associated protein indirect immunofluorescence assays were performed to determine the subcellular location of orf4 protein. in acetone-fixed cultures of both acorf4-infected sf9 and tgev-infected cells, all anti-orf4 mabs induced a strong fluorescence, which were polarized in a juxtanuclear region consistent with golgi localization (fig. 4a) . in addition, a bright fluorescence was observed on unfixed (fig. 4b) positively stained cells were consistently observed (fig. 4d) , whereasvirtually all the cells showed the presence of orf4 antigen after permeabilization (not shown). the fluorescence observed in nonpermeabilized cells was unlikely due to cell damage since only a few of them were stained positively with a mab directed against an intracellular antigen (tgev n protein; data not shown). these data were interpreted as reflecting a late accumulation of orf4 at the outer membrane of tgev-infected cells. these observations showing that orf4 protein is found in association with cellular membranes are consistent with the prediction of a membrane-spanning domain in its amino acid sequence (fig. 5) . anti-orf4 monoclonal antibodies recognize the c-terminal domain of the protein assuming that orf4 was an integral membrane protein, it was of interest to determine whether exposed epitopes were located within the carboxy or amino domain of the polypeptide chain. the peptide scanning method (geysen et al., 1984) was used since two anti-orf4 mabs were shown to be reactive toward denatured and reduced protein. a set of peptides encompassing all overlapping linear nonapeptides homologous with orf4 sequence was synthesized and tested against each of the five mabs. as shown in fig. 6 , mab v27 strongly recognized a linear epitope centered on residues ayknf (positions 64 to 68; see fig. 5 ). mab s2 gave comparable results, although a lesser reactivity was observed when compared to mab v27. no reactivity was observed with the three remaining mabs, possibly because of a lower avidity. to test the possibility that these three anti-orf4 mabs may also recognize the carboxy-subterminal region of the molecule, a second recombinant baculovirus designated acorf4a, which expressed a truncated form of orf4 lacking the 21 c-terminal amino acids, was constructed via pcr mutagenesis. immunoprecipitation analysis revealed that a polypeptide of slightly reduced size was expressed by acorf4ainfected cells and recognized by polyclonal antibodies but not by any of the anti-orf4 mabs (partial data in fig. 7) . in order to determine whether the protein is incorporated within the virion as a possible envelope protein, labeled tgev particles were purified by centrifugation and analyzed by gel electrophoresis (fig. 8) . five welldefined major bands were visible, which corresponds to the previously recognized virion-associated polypeptides: (i) a 220k band (s protein), (ii) a 47k band (n peptides fig. 6. epitope mapping of anti-orf4 mab v27. a series of nonapeptides (overlapping 1) spanning the length of the entire orf4 sequence was tested for elisa reactivity toward mab v27. n-terminus is on the left. protein), and (iii) three bands corresponding to different species of m protein; 30-36k identified as complex type glycosylated forms, 29k (high mannose form) and 26k (unglycosylated form) (delmas and laude, 1991) . a single additional minor band of m, 1 ok, similar to that of orf4 polypeptide, was detected (fig. 8a) . the fact that very few, if any, other polypeptides copurified renders unlikely the possibility that the 1 ok polypeptide remained nonspecifically associated to the sedimenting virus particles. true association of the 10k band with virions was confirmed by showing that the observed polypeptide pattern remained unchanged after an additional round of purification by isopycnic centrifugation. lmmunoprecipitation with mab v27 of detergent-dissociated material from virus-containing fractions confirmed the identity between the 10k virion-associated and orf4 polypeptides (fig. 8b) . densitometric tracing of autoradiograms at different times of exposure was performed to evaluate the relative amounts of the virion-associated polypeptides. the resulting values were corrected according to the predicted number of met residues in the respective sequences. this led to an estimated molar ratio of 1:20:300 for orf4, s, and m polypeptides. a common feature of coronavirus genomes appears to be the existence, immediately upstream from the membrane protein m gene, of an orf 250 to 330 nucleotide long, which is predicted to encode a hydrophobic polypeptide. furthermore, recent studies on mouse hepatitis virus (mhv), infectious bronchitis virus (ibv), and bovine (bcv) coronaviruses have shown that the products translated from these orfs were associated with the membrane of infected cells (leibowitz et a/., 1988; abraham et a/., 1990; smith et al., 1990) , as evidenced now for tgev orf4. these observations prompted us to reexamine the relevant amino acid sequences for possible similarities that earlier comparative analysis by us and others failed to detect (except for the closely related mhv and bcv viruses). figure 9 shows a tentative alignment of the orf4-like sequences from 5 coronavirus members, which was outlined using the program multalin (corpet, 1988) and refined manually. on the basis of the deduced consensus sequence, in which 37 residues are conserved in at least 3 out of the 5 sequences aligned (87 positions), we conclude that these proteins share significant similarities. as a striking feature, a 19-20 residue-long hydrophobic stretch, followed by a cluster of 2 or 3 cysteines is present 15 to 20 residues from the n terminus the c-terminal part of the polypeptides seems to be more distantly related; in particular, ibv protein extended the consensus sequence by 14 to 28 residues, depending of the strain (see liu et a/., 1991 for ibv orf3c sequence data). in this study we have confirmed the coding potential of the orf encoded by tgev mrna 4 and characterized several properties of its translation product. baculovirus-vectored expression of orf4 resulted in the synthesis of a 1 ok polypeptide. the same species was identified in tgev-infected cells by immunoprecipitation using monoclonal antibodies (mabs) raised against the recombinant polypeptide. examination of its subcellular localization by immunostaining showed that orf4 translation product is a membrane-associated polypeptide. this finding is consistent with the prediction in the second n-terminal quarter of a stretch of uncharged residues with proper-ties of a membrane-spanning domain (fig. 5) . immunofluorescence data also suggested that orf4 may enter the exocytic pathway. in tgev-infected cells, however, orf4 could be detected in association with the cell surface only at a late stage of the infection. assuming that the observed surface fluorescence was related to externally exposed determinants, it was of interest to map the relevant epitopes on the amino acid sequence of the molecule. peptide scanning led to the identification of residues 64-ayknf-68 as the core sequence of the binding site of 2 of the mabs (cavanagh et a/., 1990) . pairwise homologies are marked by dots and bold letters; gaps are indicated by dashes. bottom line, consensus sequence showing residues identical in at least three of the five sequences. sequence data from rasschaert et al., 1987; raabe and siddel, 1989; skinner et al., 1985; woloszyn et al., 1990; and boursnell et al., 1985. the 22 last n-terminal residues of ibv orf3c (beaudette strain) are not shown. studied. furthermore, a recombinant orf4 protein truncated of its last 21 c-terminal amino acids was no longer recognized by any of the 5 mabs. these results support the view that an antigenic, possibly immunodominant site is expressed in the c-subterminal part of orf4 protein. in addition, they led us to speculate that the region of the molecule which is translocated across the membrane would correspond to its carboxy domain. recently, a striking correlation has been reported between the transmembrane orientation of eukaryotic proteins and the disposition of charged residues surrounding the most n-terminal membranespanning sequence. it has been proposed that the difference in the charge of the 15 residues flanking the presumed anchor segment determines its orientation with the more positive portion facing the cytosol (hartmann et a/., 1989) . applying such a rule to tgev orf4 sequence gives charges of -1 and +2 for the n and c flanking segments, respectively, which predicts a nexo-cendo orientation, in contrast to the available experimental evidence. thus further experiments, including the production of antibodies directed to the n-terminal part of the protein, are needed for a definite assignment of the transmembrane orientation of orf4. the possible role of the cysteine cluster immediately downstream the hydrophobic segment was also examined. gel analysis of recombinant orf4 protein under nonreducing conditions revealed the presence of multimeric, predominantly dimeric forms. in contrast, orf4 synthesized in tgev-infected cells could be detected in a monomeric form only, suggesting that the formation of disulfide-bridged species is an artifact, presumably linked to the high level of expression of orf4 in the insect cells (kiefhaber et al., 1991) . the possibility was tested that the cystein residues could serve as an acylation site, as demonstrated for coronavirus s protein (schmidt, 1982) . however, no incorporation of palmitic acid chains could be detected in recombinant orf4 protein (data not shown), as would be expected if the target residues belong to the orf4 ectodomain. an important implication of the above findings was that orf4 could represent a structural polypeptide present in the virus envelope. indeed, purified preparations of labeled tgev virions revealed the presence of a previously unrecognized 1 ok polypeptide which was specifically immunoprecipitated by anti-orf4 mabs. based on the estimated molar ratio and assuming that coronavirions bear 100 (roseto et a/., 1982) to 200 spikes, each composed of 3 s molecules (delmas and laude, 1990) it can be inferred that approximately 15-30 copies of orf4 protein are incorporated into tgev virions (purdue strain). such a small number of molecules in virus particles does not seem to reflect a selective exclusion since s and orf4 accumulated in infected cells at a ratio comparable to that found in virions (data not shown). these results lead us to conclude that orf4 may represent a minor structural polypeptide, which we propose to designate by the tentative acronym sm, standing for "small membrane" protein. several lines of evidence lend support to the view that a gene encoding an sm-like protein is a common feature of the coronavirus genomes: (i) an orf predicting a polypeptide with striking similarities to tgev orf4 was identified in the genome sequence of each of the 5 coronaviruses examined (fig. 9 ) and the fact that tgev sm was recognized by anti-fipv antibodies argues for the presence of a related gene also in feline infectious peritonitis virus genome; (ii) the product expressed from the relevant mhv, bcv, and ibv orfs was reported to have properties of a transmembrane polypeptide (leibowitz et a/., 1988; smith et al., 1990; abraham et a/., 1990) ; and (iii) although expressed through a mono-, di-, or tricistronic mrna (abraham et a/., 1990; budzilowicz and weiss, 1987; liu et al., 1991 ) the assumed sm-encoding genes are all located upstream and adjacent to the m protein gene. therefore, not only the sequences show significant similarities but the gene order 5'. . s sm/m . 3' is conserved, as would be expected for a structural protein. finally, preliminary experiments in this laboratory allowed us to detect orfs-encoded polypeptide in association with bcv particles (n. woloszyn, p. boireau, and j. f. vautherot, unpublished results). small integral membrane proteins have been described in several other enveloped rna viruses, including sindbis and semliki forest togaviruses, influenza a and b viruses, simian virus 5, and respiratory syncitial paramyxoviruses (garoff et a/., 1980; welch and sefton, 1980; lamb et al., 1985; hiebert, 1985; olmsted and collins, 1989) . the influenza virus m2 protein (15k) and the alphavirus 6k protein are both acylated, nexo-cendo transmembrane polypeptides. m2 and 6k have been shown to represent minor structural polypeptides, with an estimated number of 40 + 25 and 24 f 4 molecules per virion, respectively (zebedee gaedigk-nitschko and schlesinger, 1990 ). m2 has been reported to form tetrameric channels within the membrane and to be a target of the antiviral drug amantadine and of the ctl response to influenza virus infection (hay et al., 1985; lamb et al., 1985; surgrue and hay, 1991) . site-directed mutagenesis studies on sindbis and semliki forest viruses have demonstrated that 6k protein is dispensable for virus production but exerts a role late in the assembly, possibly during virus budding (gaedigk-nitschko and schlesinger, 1990; liljestrom et a/., 199 1). the sh protein of sv5 has been reported to be orientated in the mem-brane with its n-terminus domain exposed at the cytoplasmic face, as it might be the case for tgev sm. whether sh is incorporated into virions is still questioned, as well as its potential role (hiebert eta/., 1988) . the apparent conservation of sm gene in the coronavirus genome strongly implies that its product is essential for an efficient replication of the virus. based on its location and its low copy number in particles, we speculate that sm would more likely play a role in modulating assembly and/or release of the virion. thus, eluci-.dating the function of the coronavirus sm protein might contribute to a better understanding of an important aspect of the biology of enveloped viruses. finally, sm may be a potent surface antigen since murine antibodies recognized a domain of the protein possibly exposed on live infected cells. preliminary experiments indicated that anti-sm antibodies are readily detected in the serum from infected swines. therefore, the role of sm protein in humoral and cellular immune response to tgev infection should be worth investigating in the future. we thank j. gelfi for technical assistance, j. levin for revising the manuscript, and m. nezondb for the artwork, part of this work was carried out with the support of the e.e.c program eclair. note addedinproof. during the submission process of this article, a communication by (d. x. liu and s. c. lnglis (1991, virology 185, 91 1-917) reported the association of ibv orf3c protein with the virion envelope. this strengthens the view that sm-like proteins are a general feature of coronavirus. 5 kda encoded between the spike and membrane protein genes of the bovine 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virus. 1. viral nucleotide sequence of the bovine enteric coronavirus becv f15 mrna 5 and mrna 6 unique regions influenza a virus m, protein: monoclonal antibody restriction of virus growth and detection of m, in virions key: cord-260708-l9w5jhsw authors: lasecka, lidia; baron, michael d. title: the molecular biology of nairoviruses, an emerging group of tick-borne arboviruses date: 2013-12-11 journal: arch virol doi: 10.1007/s00705-013-1940-z sha: doc_id: 260708 cord_uid: l9w5jhsw the nairoviruses are a rapidly emerging group of tick-borne bunyaviruses that includes pathogens of humans (crimean-congo hemorrhagic fever virus [cchfv]) and livestock (nairobi sheep disease virus [nsdv], also known as ganjam virus), as well as a large number of viruses for which the normal vertebrate host has not been established. studies on this group of viruses have been fairly limited, not least because cchfv is a bsl4 human pathogen, restricting the number of labs able to study the live virus, while nsdv, although highly pathogenic in naive animals, is not seen as a threat in developed countries, making it a low priority. nevertheless, recent years have seen significant progress in our understanding of the biology of these viruses, particularly that of cchfv, and this article seeks to draw together our existing knowledge to generate an overall picture of their molecular biology, underlining areas of particular ignorance for future studies. new viral diseases appear with increasing frequency. just in the last two years we have seen the appearance of a new livestock virus (schmallenberg) and a new human pathogen (mers coronavirus). in some cases these viruses appear to be completely new, in others they have 'emerged' into our awareness as human use of different habitats changes, leading to increased contact with carriers of disease, whether those carriers are ''bush meat'' or the many insect and tick species that can act as vectors of disease. one such group of viruses that is rapidly becoming more important is the genus nairovirus in the family bunyaviridae. this genus includes a number of human and livestock pathogens, as well as a collection of other viruses about which little is known, not even the host in which they naturally circulate. the nairoviruses show a number of unique features, and the purpose of this article is to summarise our current knowledge of the molecular biology of these viruses and highlight areas where it is to be expected that research will soon bear fruit. a primary characteristic of the viruses of the genus nairovirus, distinguishing them from most of the other members of the family bunyaviridae, is that they are all transmitted by ticks [109] . based on antibody cross-reactivity, nairoviruses are classified into seven serogroups (table 1) [32, 39, 190] , of which the most important are the crimean-congo haemorrhagic fever (cchf) group, which includes the human pathogen crimean-congo haemorrhagic fever virus (cchfv), and the nairobi sheep disease (nsd) group, to which belong nairobi sheep disease virus (nsdv) dugbe virus (dugv) and kupe virus (kupv). kupv [43] and finch creek virus [102] are the most recently discovered viruses of this genus. cchfv is arguably the most important from a human perspective, causing haemorrhagic fever in humans, with mortality up to 30 % (reviewed in refs. [58] and [182] ). after dengue virus, this is the second most widespread of the arboviruses that are pathogenic to humans; cases of cchf have been reported in sub-saharan africa, the former soviet union, bulgaria, turkey, china, pakistan, india, the arabian peninsula, northern greece, iraq and iran [26, 53-55, 115, 121-123, 183] . although cchfv can infect several mammalian species, it appears to cause disease only in man [124, 158] . because of its importance as a human pathogen, most of the available data on nairoviruses come from studies on this virus, though studies on other members of the group have also contributed, notably on nsdv, a virus first identified nearly a hundred years ago as a tick-borne virus that causes severe haemorrhagic gastroenteritis in sheep and goats, with mortality rates of up to 90 % in susceptible populations [113, 167, 181] . interestingly, an asian virus causing a similar disease, ganjam virus (gv), was recently identified, based on genetic and serological studies, as being the same virus as that causing nsd in east haemorrhagic gastroenteritis in sheep and goats [106, 113] . antibodies to nsdv/gv have been reported in humans, but otherwise only laboratoryacquired infections have been seen [13, 45, 133] dugbe virus (dugv) frequently isolated from ticks infesting livestock in which dugv appears to be apathogenic [27, 46, 146] . while one case of human infection has been described [27] , the link to dugv was circumstantial kupe virus (kupv) crimean-congo haemorrhagic fever group crimean-congo haemorrhagic fever virus (cchfv) haemorrhagic fever in man [58, 179, 182] hazara virus (hazv) africa [47, 106, 187] . it is not possible to say yet whether gv is a variant of nsdv or vice versa, but it is clear that this virus also has a wide distribution. nairoviruses, like the members of the other genera of the family bunyaviridae, are enveloped viruses that appear spherical in the electron microscope, with a diameter of approximately 100 nm [21, 46, 142] . as with all bunyaviruses, the genome consists of three segments of negative-sense rna [39] (reviewed in refs. [109] and [175] ). these are termed the small (s) segment, encoding the nucleocapsid (n) protein, which forms a complex with each rna segment [24, 39] ; the medium (m) segment, which encodes a polyprotein that is processed into two mature glycoproteins (gn and gc) as well as one or more non-structural proteins [3, 17, 39, 107, 144] ; and the large (l) segment, which encodes the viral rna-dependent rna polymerase (rdrp) [85, 108] . viral replication takes place in the cytoplasm, and viral budding occurs in the golgi [57, 142] (fig. 1) . during viral replication, the genome segments are used as the template for synthesis of both mrna (transcription) and complementary rna (crna) (replication), where crnas are then used as the template for the synthesis of progeny genomic viral rna (vrna) [16] . the 5' and 3' untranslated regions (utrs) of the s, m and l segments contain the minimum cis-acting elements necessary for transcription, replication and packaging [16, 67] . the terminal 5' and 3' non-coding regions of each segment are complementary to each other and highly conserved among different nairoviruses [37] . the first nine nucleotides are usually the same in different segments, which suggests that this sequence is recognised by the viral polymerase to initiate viral transcription/ replication [107] . it is not clear whether the complementarity between 5' and 3' ends reflects an interaction between the ends of segments or the requirement for the same 3' sequence in genome and anti-genome rnas to act as the attachment site for the viral polymerase [14, 66] (reviewed in refs. [109] and [147] ). as with other bunyaviruses, the nairoviruses utilise capped primers snatched from host mrna for initiation of their mrna transcription [90] . the initiation of crna and vrna synthesis occurs by a different mechanism, which has not yet been fully worked out. the fact that polymerase slippage occurs during mrna synthesis [90] , coupled with the observation that the rna segments of cchfv, dugv and nsdv contain a 5'-terminal pyrimidine [90, 144, 187] , while viral rna polymerases can initiate rna synthesis only by attaching purines [12] , suggests that initiation of rna replication occurs in a prime-and-realign manner. the two membrane glycoproteins (gn and gc) are believed to determine cell tropism and the ability of the viruses to infect susceptible cells via recognition and binding of one or more cellular receptors. the specific cellular receptor(s) used by nairoviruses are currently unknown, but the gc protein is thought to be involved in virus attachment to the target cell receptor, as antibodies against gc, but not gn, appear to protect cells from cchfv infection in plaque reduction neutralisation assays [1, 17] . the ectodomain of cchfv gc contains an epitope that is highly conserved among strains [1] . this fragment is probably exposed in the virus, as antibodies against this epitope are neutralising for different strains of cchfv and protect mice from challenge with cchfv [1] . similarly, the gc of dugv was also demonstrated to be targeted by neutralising antibodies [107] . antibodies to the gn protein were found to be non-neutralising in vitro, though could still be protective in vivo [17] . based on the finding that an independently folding section of this gc protein bound to cchfv-susceptible cells, and using this part of gc as a probe [185] , nucleolin has been suggested as a putative cchfv receptor. nucleolin is a cellular protein that is abundant in the nucleus but can also be expressed on the cell surface of several cell types [36, 83, 148, 149, 151] , including mononuclear phagocytes, endothelial cells and hepatocytes which are known to be targets for cchfv [28, 185] . although nucleolin has also been suggested to act as a receptor for several other viruses, such as parainfluenza virus type 3 [22] , human immunodeficiency virus (hiv) [118] , coxsackie b virus [49] and respiratory syncytial virus (rsv) [162] , further studies are required to determine whether nucleolin acts as the primary receptor for cchfv; in particular, infection studies with wild-type (not cell-culture-adapted) viruses are required to confirm the biological relevance of any potential receptor candidates. after binding to their receptor, viruses fuse with the plasma membrane or internalise through one of several endocytosis pathways to gain entry into the intracellular environment (reviewed in refs. [40] and [153] ). cchfv entry is dependent on the low ph of the endosomal compartment, which appears to be required in the first steps of the virus entry post-internalisation [74, 156] , and the virus has been shown to use clathrin-dependent endocytosis, while caveolin-1 is dispensable for its entry [74, 156] . studies using dominant negative rab5 and rab7 indicate that cchfv fuses with early, but not late, endosomes to gain access to the cytoplasm [74] . cholesterol also appears to be important for cchfv replication, especially at early stages after binding and internalisation; it is possible that depletion of cholesterol from cells traps viral particles in endosomes [88] or interferes with clathrin-mediated endocytosis [140] . the internalisation of cchfv virions was shown to be dependent on intact microtubules [155] . further investigation of cchfv infection showed that disruption of the cell microtubules inhibited viral rna replication [155] , and both intact actin and microtubules are essential for correct distribution of the n protein to the perinuclear area [5, 155] . as nairoviruses enter via endocytosis and bud in the golgi compartment, the observation of a redistribution of viral proteins and suppression of cchfv assembly/egress by microtubule modification (depolarisation and stabilisation) [155] is not surprising, and this might be caused by interference with the endogenous secretory pathway, which also occurs along microtubules [101] (reviewed in ref. [87] ). disruption of actin filaments in cchfv-infected cells also drastically reduced replication of the virus [5] . while the n protein is often located in close proximity to the golgi apparatus, where generation of new viral particles occurs [16] , n does not interact directly with golgi membranes [5, 134] . the n protein appears to interact with the l protein, even in the absence of viral rna, as expressed n protein seems to redistribute most of the l protein into the perinuclear area containing n in transfected cells [16] . the n protein of nairoviruses, at approximately 53 kda, is almost twice of size of the n proteins of other bunyaviruses, with the exception of those of hantaviruses, which, at approx. 48 kda, are similar in size [39, 110] . the n protein is the most abundant protein in the virion and encapsidates newly synthesised vrna and crna; this process is necessary for completion of the replication cycle and packaging of the genome into virions [16, 65, 100] . viruses of different genera in the family bunyaviridae appear to adopt different mechanisms of rna encapsidation, e.g. some bunyavirus n proteins recognise generic ssrna, while others recognise specific structures in the vrna [110, 120, 135, 137, 150] . there is a need to understand the mechanism by which nairoviruses encapsidate genomic and antigenomic rna; inhibition of vrna encapsidation could be a target for potential antiviral drugs, e.g., using an rna decoy to bind the viral n protein, as has been suggested as an antiviral treatment for hepatitis b virus (hbv) [62] , or selecting rna-binding proteins that target specific packaging sequences in the viral genome [193] . the recently determined crystal structure of the n protein of cchfv shows that it is built from two major domains: a globular head and an extended stalk [31, 77, 174] . the globular domain is the larger and is formed from both n-terminal and c-terminal helices, while the stalk domain is formed by a group of internal helices (the exact numbering of the helices varies between the several papers publishing the structure of this protein). three potential rna-binding regions were identified in the n protein structure. two positively charged grooves are located in the globular domain: the smaller one is under the stalk domain, and the larger one is in the opposite site of the globular domain. the third positive groove is located in the stalk domain. the positively charged residues forming the rna-binding site appear to be well conserved across the genus nairovirus, and several residues were identified as crucial for rna binding and virus replication, e.g., k132 and q300 in the smaller groove and k411 and h456 in the larger groove of the head domain [31, 77] . the n protein of cchfv was also crystallised as a linear oligomer, where three monomeric n subunits were organised in head-to-tail manner, with the stalk domain of one monomer interacting with an oligomeric groove located at the base of the head domain of an adjacent molecule [174] . in addition, the linear oligomers of n were predicted to interact in a dimeric manner to form an antiparallel double superhelix [31, 174] . further predictions suggest that that there are nine n molecules per turn of the superhelix, which is 210 å in diameter [174] . additionally, a positively charged crevice located on the outside of the double superhelix is predicted to serve as an additional rna binding cleft [174] . studies performed by guo and collaborators [77] suggest that the cchfv n protein, when expressed without rna, predominantly exists as a monomer, and the nairovirus n protein is only a weak binder of nonspecific rna in this monomeric state [77, 80, 111, 135] . this suggests that nairovirus n binds rna only in the oligomeric state and/or that the n recognises specific structures of viral rna, as was shown for viruses of the genus hantavirus [110, 120] . however the fact that the nairovirus n protein does not form oligomers without rna suggests that its oligomerisation is rna-stabilised, where monomeric n protein requires binding to rna to form an oligomer, a mechanism that was previously suggested for influenza a virus [160, 161] . in a similar fashion, the n protein of rift valley fever virus (rvfv) can form only a short oligomeric form in the absence of rna [64] . superimposing the structures obtained for the cchfv n protein by two different groups, using two cchfv isolates, revealed the head and stalk with very similar folds, but with a transposition of the stalk domain when comparing these two molecules; results which suggest a flexibility of the stalk domain and the possibility of different conformations of n for different functions or different states of the n-rna complexes (e.g., transcription vs replication) [31] . additionally, comparison of the crystal structure of the n protein in the monomeric and oligomeric forms suggests that the stalk domain changes its conformation upon oligomerisation, probably by binding to the oligomerisation groove on the head domain of the adjacent molecule [174] . such flexibility of the stalk domain has also been shown for the n proteins of rvfv and lasv, which, during binding to an oligomerisation groove on an adjacent n molecule, undergo conformational changes exposing an rna-binding groove [64, 80] . these structural data have led to suggestions of possible models for the initiation of transcription and replication. transcription of viral mrnas by nairoviruses utilises short capped rna fragments (10-20 nt in length) derived from host mrnas as primers [90] . incubation of the cchfv n protein with primer-length ssrna resulted in a conformational change of the stalk domain, which resulted in disruption of the oligomeric interactions and release of the monomeric n protein from the antiparallel double superhelix [174] . as discussed by wang et al., presentation of the capped primers to the ribonucleoprotein (rnp) may initiate conformational changes in the stalk domain, leading to the release of monomeric n and exposing vrna to the primer and the viral polymerase. given that head-to-stalk interactions between two adjacent n molecules have also been observed for rvfv, lasv, and bunyamwera virus (bunv) [9, 64, 80] , it seems likely that the model proposed for cchfv may be biologically valid. the n protein of cchfv has also been shown to have nuclease activity specific for dsdna and ssdna (but not for rna); the importance of this dnase activity and its function in the virus life cycle are unknown [77] . the residues involved in the nuclease activity are located in the globular head domain of the n protein and are conserved in all nairoviruses [77] . in contrast, the n protein of lasv, the head domain of which exhibits high structural homology with that of cchfv, has rna-specific nuclease activity [79, 80, 130] . the n protein of nairoviruses also contains a conserved sequence signature specific for catalytic motif ii (cmii) of n-6 adenine-specific dna methylases (lasecka and baron, unpublished) (fig. 2) , where the conserved motif nppw could be involved in substrate binding or in catalytic activity [97, 164] . the motif is located on an exposed loop of the stalk domain, and the function and potential importance of this motif still need to be determined. the methylation of dna is used for regulation of gene expression, but so far there is no indication that the n protein of nairoviruses travels to the nucleus; the protein does not contain a classical nuclear localisation signal (nls), nor does it accumulate in the nucleus of infected or transfected cells. n6 methylation is also used as a posttranscriptional modification of mrna, and rna methyltransferases appear to contain motifs very similar to the cmi and cmii motifs identified in dna methyltransferases (reviewed in ref. [119] ); further studies are required to determine if the nairovirus n protein can modify its own or host cell rnas in this way. like those of members of the other genera of the family bunyaviridae, the m segment of nairoviruses contains a single open reading frame (orf) encoding a polyprotein that is co-and post-translationally cleaved into the mature viral glycoproteins [39] . the glycoproteins of most nairoviruses are still poorly characterised, and most of the available data come from studies on cchfv, largely through studies on proteins expressed from plasmids. the processing of the cchfv m polyprotein to generate the mature glycoproteins appears to be more complex than that of other bunyaviruses, as it involves first the generation of glycoprotein precursors through the action of the signal protease in the endoplasmic reticulum (er) followed by further cleavages to give rise to the full set of mature glycoproteins, a process that employs other cellular proteases [15, 144, 145, 172] (fig. 3) . the virions of most of the nairoviruses contain two mature glycoproteins, gn and gc [15, 33, 38, 107, 144, 145, 172] ; however, two nairoviruses, hazara virus and clo mor virus, have been shown to contain three structural glycoproteins [68, 175] . the full nairovirus m-encoded polyprotein appears to have six hydrophobic regions (tm 0 -tm 5 ), which could function as transmembrane helices [3, 144] and act either as classic secretory signal peptides (tm 0 ), membrane anchors (tm 1, 3 and 5 ), or a combination of both (tm 2,4 ) (fig. 3) . signal cleavage motifs are found after tm 0 (releasing the amino terminus of the gn precursor, pregn) and tm 4 (releasing the gc precursor pregc). sequence inspection revealed a signal cleavage signal immediately after tm 2 , suggesting that there might be a separate protein released, consisting of the sequence between the distal ends of tm 2 and tm 4 ; such a protein has been shown to be produced by cchfv and has been termed ns m [3] . further non-structural glycoproteins have been identified in cchfv-infected cells, referred to as the mucin-like domain (a highly o-glycosylated peptide), gp38, gp85 and gp160, all of which are released from infected cells; gp85 and gp160 contain both the gp38 protein and the mucin-like domain [145] . a model for the generation of the cchfv glycoproteins is summarised in figure 3 based on a number of studies [3, 144, 145, 172] . co-translational cleavage in the er by cellular signalase generates the 140-kda pregn (containing the mucin-like domain, gp38, and gn), the predominantly cytoplasmically oriented ns m , and the 85-kda pregc. the pregn is further cleaved, either in the er or the golgi compartment, to separate mucin-like domain/gp38 from [4, 161, 162] gn. this cleavage occurs at a conserved rrll; motif and is effected by the host's subtilisin kexin isozyme-1/site-1 protease (ski-1/s1p) [145, 172] . this cleavage has been shown to be critical for virus replication and subsequent infectivity, and lack of the cleavage prevents incorporation of the glycoproteins into viral particles [15] . a similar tetrapeptide (rkpl) is found 41 amino acids downstream of the signalase cleavage site in pregc, which, although it is not processed also by ski-1/s1p, is predicted to be utilised by a related subtilisin-like protease in the er/cis-golgi to generate the mature gc (75 kda) [144, 172] . the mature gn interacts (directly or indirectly) with the gc, and both are translocated to the virus-assembly sites in the golgi [17, 59, 145] . the interaction of mature gn with gc is essential for the gc to travel from er to golgi, as gn, but not gc, contains a golgi localisation signal [17] . the ectodomains of gn and gc appear to be sufficient for heterodimer formation and transport to the golgi, indicating that at least partial golgi-targeting information is located in the gn ectodomain [17] . the final processing of the glycoproteins takes place in the trans-golgi, where the mucin-like domain and gp38 are separated from each other by a furin-like protein convertase at the rskr; cleavage site [116, 145] . this cleavage is not required for cchfv gn maturation [145] , and it appears that not the entire pool of mucin-like domain/gp38 polyprotein is being cleaved, as the nonstructural proteins termed gp85 and gp160 contain both the mucin-like domain and gp38 and are resistant to resolution into smaller proteins by denaturation in sds and urea [145] . resistance to denaturation also suggests that gp160 is probably not a dimer of gp85 [145] . interestingly, the mucin-like domain/gp38 and gp38 can fold independently of the rest of gn and are secreted even when expressed on their own in transfected cells [145] . no specific biological function has been assigned to the mucinlike domain, gp38, gp85 or gp160. the mucin-like domain of the ebola virus glycoprotein has been shown to play a major role in pathogenesis, including involvement in the observed increase of endothelium permeability [154, 188] , and it will be important to see if this is also true of cchfv, especially given the changes visible in the endothelial cells of cchfv patients. the ns m protein, when expressed in transfected cells on its own, is transported to the golgi [3] . while the function(s) of the nairovirus ns m have still to be determined, the fact that gn requires ns m for maturation may mean that ns m is necessary for virus replication [3] . glycosylation is an important post-translational modification of secreted and membrane proteins that can influence protein folding, transport and function. n-linked glycosylation in particular is known to regulate protein folding, association with chaperones [112] , transport, cellular localisation [81, 82] , and even virus infectivity (reviewed in ref. [171] ). the n-terminal part of the nairovirus m polyprotein, which contains the mucin-like domain, is heavily o-glycosylated, while the adjacent gp38 domain appears to have few o-glycosylation sites [144, 145] . both domains are also n-glycosylated, the mucin-like domain containing five potential sites and gp38 two [145] . of two predicted n-glycosylation sites in each of the mature gn and gc proteins, only one is functional in gn (n557), while both sites in gc (n1054 and n1563) are glycosylated [59] . however, only the glycosylation of the gn is essential for gn maturation, correct localisation, and transport of itself and other cchfv glycoproteins [59] . given that the gn glycosylation sites are conserved among cchfv strains [51] , it is likely that correct glycosylation of the cchfv gn is critical for virus viability. recently, nmr has been used to determine a solution structure for the c-terminal (cytoplasmic) tail of cchfv gn, showing that this region contains a dual zinc-finger domain, the sequence of which is highly conserved among nairoviruses [60, 61] . classical bba-zinc finger domains have been shown to take part in protein-protein interactions (reviewed in ref. [71] ), and it is possible that the dual zincfinger domain in the c-terminus of the nairovirus gn protein is involved in interaction with rnps, which would help drive assembly/budding of the virus. analysis of the sequences of m segments of other nairoviruses suggests that the general model proposed for processing of the m polyprotein described above for cchfv holds true for the other nairoviruses; m polyproteins show similar membrane topology, with six transmembrane regions and conserved signalase cleavage sites after the transmembrane domains tm 0 , tm 2 and tm 4 (fig. 3) . glycoprotein maturation from precursor proteins has also been observed for dugv, where pregn is around 70 kda and pregc around 85 kda [107] . an exception to this general similarity is erve virus (ervev), which does not contain tm 3 and tm 4 , its m polyprotein lacking the entire amino acid sequence between tm 2 and the gc ectodomain. this suggests that ervev lacks an ns m protein. most of the nairoviruses for which we have sequence data appear to have a pregn domain that is about 120 amino acids shorter than that seen in cchfv, due to a much shorter mucin-like domain, as was previously described for dugv [144] . another difference is that other nairoviruses do not contain a furin-cleavage site (rskr) following the o-glycosylated mucin-like domain. the ski-1/s1p-like protease cleavage tetrapeptides in many nairoviruses appear to be different to those proposed for cchfv; however, all appear to fit the consensus subtilisin cleavage sequence (r/k)x(hydrophobic)z; (where x is any amino acid and z is preferably f, k, l or t but not v, p, e, d, c) [56] . this may reflect differences in adaptation to specific tick or mammalian hosts, and further studies on the importance of these various stages in the maturation of the glycoproteins for different hosts are clearly required. the l segment of nairoviruses, which contains a single open reading frame (orf) of approximately 12 kb, encoding a protein of approximately 450 kda, is almost twice as long as the l proteins of most other bunyaviruses, with the exception of the tospoviruses, in which the l segment is approximately 9 kb in length [48, 85, 94, 108] (fig. 4) . despite this difference in length and sequence, nairovirus l proteins still show the four conserved functional regions previously described for other bunyavirus l proteins [94, 108, 131, 139, 177] . the bunyavirus l proteins contain the rna-dependent rna polymerase (rdrp), and the most conserved region of the l segment among nairoviruses is the region corresponding to the coding sequence for the core catalytic domains of the rdrp [8, 85, 108] . within this polymerase module (also called region 3) can be distinguished six conserved motifs [94, 108] (fig. 4) . motifs a through d are conserved among all rna-dependent polymerases [50, 129] ; motif pre-a, upstream of motif a, is present in all rna-dependent rna polymerases and reverse transcriptases, and motif e, which is downstream of motif d, is conserved in segmented negative-strand rna viruses [114, 129, 186] . motifs a, c and d are predicted to bind nucleoside triphosphates (ntps) and are therefore likely to be involved in the catalytic functions of the polymerase [50, 114] , while motifs b and e are predicted to take part in template and/or primer positioning [114] . motif pre-a is also predicted to be involved in template positioning [94, 114] . the fact that the inter-motif distances are more or less constant suggests that the polymerase module functions in a structurally dependent manner [94] . upstream of the polymerase module are regions 1 and 2 (fig. 4) which are conserved in bunyaviruses and arenaviruses [94, 114] , while downstream of the polymerase module is region 4 (fig. 4) , which, although originally suggested to be specific for bunyaviruses [8] , appears to be conserved in other segmented negative-strand rna viruses [94] . protein sequence analysis shows that the distances between regions appear to be conserved among bunyaviruses, with the exception of the interval between regions 2 and 3, where nairoviruses appear to have much longer amino acid sequences than other bunyaviruses (fig. 4) . region 1, based on sequence similarity with other viruses, appears to be responsible for capsnatching endonuclease activity [52, 136, 189] ; however, this needs to be confirmed experimentally. in the case of nairoviruses, the rdrp accounts for only one-third of the entire l protein (fig. 4) , with regions of unidentified function in both the amino (n) and carboxy (c) termini [94] . all bunyaviruses have a significant c-terminal section after the rdrp, although the function of this region is so far unknown in any of the viruses of this family, and there appear to be no cross-genera-conserved motifs [136] . n-terminal to the rdrp motifs, the l proteins of nairoviruses contain several additional domains that are unique to this genus, of which the ovarian-tumour (otu)like protease domain is the most studied [2, 29, 85, 89, 94] . this domain belongs to a larger papain-like cysteine protease family also found in other viruses (e.g., blueberry scorch virus (blscv) of the genus carlavirus, and equine arteritis virus (eav) and porcine respiratory and reproductive syndrome virus (prrsv) of the genus arterivirus), in saccharomyces cerevisiae, in drosophila melanogaster and in mammalian cells [11, 103] . curiously, the region containing the otu domain also contains a sequence resembling a topoisomerase-like domain, located at amino acids , and therefore lying between amino acids that form part of the of the otu catalytic site -cysteine 40 and histidine 151 [85, 94] . the consensus topoisomerase motif the approximate size, expressed as the number of amino acids (aa), is indicated for each protein, and the diagram is drawn approximately to scale. see text for details of the various motifs. adapted from ref. [91] (skxxy) is not conserved across nairoviruses, being mostly slxxy in cchfv and nsdv/gv, but the activesite tyrosine is conserved across all nairoviruses so far sequenced. given the structural similarities between topoisomerases and strand-specific recombinases [35] , this motif may indicate a role for this region of l in rna strand manipulation as well as its function as a protease. alternatively, nairovirus l proteins may include their own topoisomerase activity, rather than having to recruit the host-cell topoisomerase i, as has been shown for at least one non-segmented rna virus [159] . downstream of the otu domain, the l protein of nairoviruses contains a c2h2-type zinc finger domain and a leucine zipper motif [85, 94] , both of which are highly conserved among nairoviruses, but the function of which in the viral replication cycle is still to be determined. interestingly, the zinc-finger domain and leucine zipper motif are located in region 1 and region 2, respectively, of the nairoviral l proteins but do not appear to be present in these regions in the l proteins of other bunyaviruses. the otu-domain protease activity is dispensable for virus genome replication [16] , and most recent studies have focussed on the effects of this enzymatic activity on host proteins. mammalian otu-domain proteins are primarily deubiquitinating enzymes (dubs), responsible for cleaving the modified peptide bond that links ubiquitin (ub) to hostcell proteins or to other ub molecules. most mammalian dubs are only able to deubiquitinate and usually have a limited set of targets that they de-conjugate in this way (reviewed in ref. [96] ). the otu domains of nairoviruses, in contrast, are capable of de-conjugating not only ub but also other ubiquitin-like peptides, notably the interferonstimulated gene 15 protein (isg15), removing these peptides from a variety of protein targets [10, 69, 84] . it has been shown that amino acids 1 to 169 of the l proteins of cchfv, nsdv or dugv are sufficient for enzymatic activity, and conserved active site residues that are critical for its catalytic activity have been identified [10, 69, 84] . in the last few years, the crystal structures of the otu domain with and without a ubiquitin molecule have been determined [2, 29, 89] . the cchfv otu showed an overall similar structure to yeast otu1, but with an additional domain formed by two antiparallel b-strands that allow the viral otus to bind both ub and isg15 [2, 89] . specific cleavage targets, other than host ubiquitinated or isgylated proteins, for the viral otu-like protease have not yet been described. as several potential cysteine-protease-like cleavage sites have been identified in the l protein sequence of nairoviruses [94] and some viral proteins containing an otu-like protease domain have also been shown to undergo autoproteolytic cleavage to generate multiple mature proteins, e.g., the replicase of blscv [98] , it has been suggested that the l proteins of nairoviruses may also be autoproteolytically cleaved into an active rna polymerase and protein(s) with additional function [85] . we have raised specific antibodies to the nand c-termini of the nsdv l protein and shown that such cleavage does not occur in infected cells (lasecka and baron, unpublished). early studies on nairoviruses showed that, unlike other bunyaviruses, they do not shut off host protein synthesis [33, 176] , so the viruses must have a different way of controlling the immediate host cell responses to infection, such as the innate immune response and apoptosis. cchfv infection in cultured cells induced apoptosis, albeit at late stages of infection [91, 141] . one of the ways in which nairoviruses might induce apoptosis has been suggested to be via the induction of er stress [141] . certainly cchfv and dugv have both been shown to induce er stress [141] , and we have observed the same in cells infected with nsdv (lasecka, unpublished data). during replication, nairoviruses synthesise large amounts of their glycoproteins, which mature in the er and golgi [17, 21, 142] and are likely to overload the normal er protein synthesis machinery. as long as the apoptosis occurs after the virus has completed the assembly and release of progeny virus, apoptosis is not a major problem, but it is unclear as yet whether nairoviruses take active steps to inhibit apoptotic pathways. there is the possibility that apoptosis may be delayed or inhibited in cchfv infections by the presence of a highly conserved caspase-3 cleavage site (devd) in the n protein [31, 91] , which may act as a decoy substrate for caspase-3. although this motif has been suggested to be involved in control of apoptosis [31, 91] , the cleavage of n protein by caspase-3 is not required for replication/transcription of a cchfv minigenome [31] . in addition, this devd motif appears to be inaccessible to caspase-3 in the oligomeric form of cchfv n, suggesting that only n monomers are caspase-3 sensitive [174] . cchfv also appears to be the only nairovirus that contains the devd motif [174] ; other nairoviruses, such as nsdv/gv, do not have the motif, yet are still highly pathogenic. the innate immune response to viral infection has been described in a number of recent reviews [72, 105, 132, 143] . cells detect viral pathogen-associated molecular patterns (pamps) (e.g., double-stranded rna (dsrna) or dna with unmethylated cpg motifs) using pattern recognition receptors (prrs), of which the most well-known are the toll-like receptors (tlrs), which scan extra-cytoplasmic spaces including the interior of endosomes and lysosomes, and the cytosolic proteins melanoma-associated differentiation gene-5 protein (mda-5) and retinoic acidinducible gene-i protein (rig-i), which detect virus-associated pamps in the cytoplasm. recognition of a pamp leads to activation of the prr, followed by an intracellular signalling cascade leading to the transcription of interferon b (ifnb) mrna. secreted ifnb works in an autocrine and paracrine manner by binding to the ifna/b receptor of both infected and uninfected cells. this in turn activates a signalling pathway, which up-regulates interferon-stimulated genes (isgs), including ifna, which increases the stimulus to other isgs. finally, the products of isgs (directly or indirectly) lead to the cell entering the so-called antiviral state, in which many proteins are present that can inhibit different viruses. interferon and its actions have strong inhibitory effects on the replication of nairoviruses. for instance, treatment of human endothelial cells with ifna significantly reduced the yield of cchfv [6] , while the replication of cchfv and dugv is impaired in vero cells stably expressing the human mx (mxa) protein; mxa is the product of an isg and appears to act through sequestration of the n protein of these viruses in a perinuclear region [4, 25] . however, as with other viruses, nairoviruses have developed mechanisms to evade this innate antiviral response. some of these mechanisms appear to be unique to one virus; others appear to be common to all the viruses in the genus that have been studied. for example, cchfv can delay ifna/b production in infected cells; andersson et al. showed that, in cchfv-infected cells, an increase in ifnb mrna could only be detected 48 h after infection, which leaves the virus a replication window with no antiviral state [7] . this delay is related to a delay in translocation of the transcription factor interferon regulatory factor-3 (irf-3) to the nucleus [7] and hence a delay in the induction of isg56, one of the genes whose transcription is regulated by irf-3. the isg56 protein is a cytoplasmic protein (p56) that is involved in the global inhibition of translation in response to infection (reviewed in ref. [63] ). the delay in isg56 expression correlates with earlier findings that nairoviruses do not appear to shut off cellular protein synthesis [33, 176] . in contrast to the findings with cchfv, ifnb induction was seen in nsdv/gv-infected cells after about 16 hours; however, during those 16 hours, the virus actively blocked induction of ifnb [84] . nsdv/ gv infection also blocked the signalling pathways activated by external ifna or ifnc, blocking the activation of transcription from promoters normally activated by one or the other of these cytokines by directly inhibiting the phosphorylation of the transcription factors stat1 and stat2 [84] . of the cytoplasmic prrs, rig-i is activated by rnas with a 5' triphosphate group, such as viral vrnas and crnas, while mda-5 is activated by binding dsrnas [44, 86, 92, 128] . like other negative-sense rna viruses, nairoviruses do not produce detectable amounts of dsrna during replication [178] and hence avoid activation of a number of dsrna-sensitive prrs (e.g., mda-5, tlr3) and dsrna-dependent enzymes (e.g., dsrna-activated protein kinase r [pkr] and 2'5'-oligoadenylate synthetase [2'5' oas] ). the newly synthesised vrna and crna of nairoviruses, as for other bunyaviruses, is cotranscriptionally encapsidated by the n protein, minimising the exposure of vrnas and preventing formation of dsrna intermediates [127, 135, 173] . it is thought that the major sensor for bunyavirus infection is therefore rig-i. 5'triphosphate groups are present on rna molecules generated during viral replication but are absent on cellular rnas, as cellular mrna contains a cap structure at the 5' end, and the transcription products of other cellular rna polymerases (rna polymerases i and iii) contain a monophosphate at the 5' end. by utilising capped, short nucleotide sequences snatched from host mrna to initiate viral mrna transcription [90] , nairoviruses prevent recognition of viral mrna by prrs. cchfv further avoids activation of rig-i as its vrna and crna have a monophosphate group rather than the triphosphate group found at the 5' end of most viral rnas [78] , though the mechanism of 5' monophosphate generation during replication of cchfv remains unknown. strategies to generate 5'monophosphates on viral rna have been shown for other viruses: htnv utilises a prime-and-realign mechanism to generate 5' and 3' complementary ends, where a viral endonuclease (which is probably also involved in cap-snatching) is proposed to remove 5'-terminal extensions, leading to a 5' monophosphate on the final rna [73] . interestingly, this mechanism is not common to all bunyaviruses, as rvfv was shown to contain triphosphate groups at the 5' ends of its vrnas, which can activate ifnb via the rig-i pathway [78] . it will be interesting to see if other nairoviruses adopt the rna processing seen for cchfv. it is possible that bunyaviruses that have developed methods of blocking the rig-i-activated pathway (e.g., by the activities of a nonstructural protein such as the phlebovirus nss protein) can have triphosphates at the 5' end of their vrnas while viruses that do not have an nss activity must generate monophosphates to avoid activating the rig-i pathway in the first place [18, 19, 23, 73] . ubiquitin and ubiquitin-like molecules play important roles in the initiation and maintenance of immune response. for example, they are essential for the action of cytokines such as ifna/b and tumour necrosis factor alpha (tnfa) (reviewed in ref. [95] ). ubiquitination allows for the activation of nuclear factor kappa b (nf-jb) by targeting the inhibitor of nf-jb (i-jb) for degradation [163] . k63-linked ubiquitination activates several molecules of the ifnb induction pathway, including rig-i, mitochondrial antiviral signalling protein (mavs), tank-binding kinase-1 (tbk1), ijb kinase-e (ikk-e), tumour necrosis factor receptor-associated factor 3 (traf3) and traf6 [70, 125, 126, 166] . in addition to modulation of the innate immune signalling, ubiquitination also plays an important role in antigen presentation by major histocompatibility complex (mhc) class i and ii proteins [152] . isg15 is an ifninduced 15-kda ubiquitin-like molecule that is composed of two-ubiquitin-like domains [20, 117] . although the precise role of isg15 in modulation of protein function is unknown, conjugation by isg15 is also known to modify hundreds of cellular proteins, including several of those involved in the antiviral response, e.g., pkr, mxa, stat1, rig-i, janus kinase 1 (jak1), and irf-3 [75, 104, 138, 192] . both ub and isg15 are synthesised as precursors, which are cleaved in order to expose the conjugation sequence (lrlrgg) by which they are attached to other proteins. the conjugation is mediated by a sequence involving activating enzymes (e1), conjugating enzymes (e2) and protein ligases (e3) [157, 180, 191] (reviewed in ref. [169] ). removal of these conjugated proteins is carried out by cellular deubiquitinating enzymes and deisgylating enzymes, which have roles, as expected, in negative feedback regulation of ifn induction and action [93, 99] . as described in the discussion of the nairovirus l protein, the n-terminus of these proteins contains an otu proteaselike domain [85] similar to that often found in mammalian dubs, and experimental evidence showed that this protease domain in the viral protein indeed deconjugated ub and isg15. global ubiquitination and isgylation levels were greatly reduced in nsdv/gv-infected cells [84] , and overexpression of the amino-terminal end of the l proteins of cchfv, nsdv or dugv in cell culture resulted in a similar reduction in ubiquitin-and isg15-conjugated proteins [10, 30, 69, 84] . the l protein otu domains of several nairoviruses have been shown to block ifnb induction and the actions of type i and type ii ifns [10, 69, 84] . interestingly, at high enough concentrations, even catalytically inactive otus are capable of blocking ifnainduced transcription [10, 69] . this suggests that catalytically inactive otu domain proteins are still capable of sequestering specific ubiquitinated or isg15ylated targets by binding to them. the nsp2 protein of eav and prrsv also contain otulike domains [69] . recently, it was shown that both the nsp2 of eav and the otu domain of cchfv l protein are able to deubiquitinate rig-i and hence block rig-i-mediated activation of ifnb [168] . interestingly, the rna of neither cchfv nor eav appears to activate rig-i [78, 168] , so the fact that these viruses have evolved specific mechanisms to inhibit the rig-i-mediated induction of ifnb suggests that rig-i still has a function in the antiviral response to these viruses. however, unlike cchfv, the vrna of nsdv/gv does activate transcription from the ifnb promoter (lasecka and baron, unpublished observations). the ability to directly block the rig-i pathway would therefore be particularly important for this virus. comparison of the otu domains of different nairoviruses revealed some differences between their affinity for different types of poly-ub and isg15 [30] . for instance, cchfv shows a higher affinity for k63-poly-ub than k48-poly-ub, and the cchfv otu was more active in the deubiquitination of host proteins than the otus of either nsdv or dugv [10, 30, 84] . on the other hand, while the ervev otu appears to bind any poly-ub weakly (when compared to those of cchfv and dugv), it had higher affinity for isg15 [30] . this may indicate that different nairoviruses have adopted slightly different ways of utilising their core deubiquitinating and deisgylating activities, which might reflect the wide range of pathogenicity caused by these viruses, or differences in the requirements imposed on these viruses by their arthropod hosts, about which we know very little. isg15 is not as strongly conserved as ubiquitin among different species, even mammals, so there can be real effects of species preference in isg15 binding/cleavage, e.g., the cchfv otu appears to show a preference for isg15 of human origin over that of mouse origin, while ervev appears to recognise both human and mouse isg15s equally [30] . the better binding of the murine isg15 by the ervev otu may be associated with the homology between the isg15 of mouse and the white-toothed shrew from which ervev is commonly isolated [34, 184] . nairoviruses share many of their features with other bunyaviruses, e.g., replication in the cytoplasm, budding in the golgi, and their coding and rna replication strategy. from phylogenetic studies, the members of the genus nairovirus appear to be most closely related to those of the genus phlebovirus of all bunyaviruses [108, 131, 139, 177] . however, nairoviruses possess many features not found in other bunyaviruses. nairoviruses appear to have complex processing of their glycoproteins, which involves the actions of cellular proteases such as ski-1/s1p-like proteases and furin. the proteins of nairoviruses also contain domains that have not been observed in other bunyaviruses, such as the l protein otu domain. nairoviruses express a secreted mucin-like domain, which may play an essential role in the pathogenicity of the virus [154, 188] . structural similarity between the nairovirus n protein globular domain or the rdrp region of its l protein and equivalent proteins of arenaviruses has been taken to suggest that the nairoviruses are more closely related to arenaviruses than to members other genera of the family bunyaviridae [31, 170] , with some authors even suggesting that the current classification of the nairoviridae might need re-evaluation in the future [31] . several areas for future study stand out. given that the nairoviruses are, in general, tick-borne, while most other bunyaviruses are insect-borne, it is to be expected that the interaction of these viruses with their arthropod hosts will be specific to the virus genus and need specific study. fortunately, expertise with handling ixodid ticks and tick cell lines is rapidly increasing, and it is to be hoped that our understanding of the replication of these viruses in their tick hosts will catch up with our knowledge of what is happening in mammals. the nairoviruses have been more resistant to the development of successful reverse genetics than e.g. the orthobunyaviruses or the phleboviruses, and development of such a system for nairoviruses will be invaluable in helping us understand the roles of various nairovirus-specific domains such as the topoisomerase-like domain, c2h2-zinc finger domain, leucine zipper motif, and otu domain of the l protein, or the ns m and mucinlike domain from the m segment, both in mammalian and arthropod hosts. the ability to create targeted mutations will also enable us to more rapidly develop stably attenuated viruses that could act as vaccines. presence of broadly reactive and group-specific neutralizing epitopes on newly described isolates of crimean-congo hemorrhagic fever virus molecular basis for ubiquitin and isg15 cross-reactivity in viral ovarian tumor domains identification of a novel c-terminal cleavage of crimean-congo hemorrhagic fever virus pregn that leads to generation of an nsm protein human mxa protein inhibits the replication of crimean-congo hemorrhagic fever virus role of actin filaments in targeting of crimean congo hemorrhagic fever virus nucleocapsid protein to perinuclear regions of mammalian cells type i interferon inhibits crimean-congo hemorrhagic fever virus in human target cells crimean-congo hemorrhagic fever virus delays activation of the innate immune response analysis of oropouche virus l protein amino acid sequence showed the presence of an additional conserved region that could harbour an important role for the polymerase activity nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and rna polymerization dugbe virus ovarian tumour domain interferes with ubiquitin/isg15-regulated innate immune cell signalling otubains: a new family of cysteine proteases in the ubiquitin pathway 5'-terminal cap structure in eucaryotic messenger ribonucleic acids viral infections in laboratory personnel role of the conserved nucleotide mismatch within 3'-and 5'-terminal regions of bunyamwera virus in signaling transcription crimean-congo hemorrhagic fever virus glycoprotein processing by the endoprotease ski-1/s1p is critical for virus infectivity crimean-congo hemorrhagic fever virus-encoded ovarian tumor protease activity is dispensable for virus rna polymerase function cellular localization and antigenic characterization of crimean-congo hemorrhagic fever virus glycoproteins nss protein of rift valley fever virus blocks interferon production by inhibiting host gene transcription la crosse bunyavirus nonstructural protein nss serves to suppress the type i interferon system of 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classical zinc fingers structural characterization of the crimean-congo hemorrhagic fever virus gn tail provides insight into virus assembly a selex-screened aptamer of human hepatitis b virus rna encapsidation signal suppresses viral replication the isg56/ifit1 gene family the hexamer structure of rift valley fever virus nucleoprotein suggests a mechanism for its assembly into ribonucleoprotein complexes reverse genetics system for uukuniemi virus (bunyaviridae): rna polymerase i-catalyzed expression of chimeric viral rnas mutational analysis of the uukuniemi virus (bunyaviridae family) promoter reveals two elements of functional importance reverse genetics for crimean-congo hemorrhagic fever virus structural polypeptides of hazara virus ovarian tumor domain-containing viral proteases evade ubiquitin-and isg15-dependent innate immune responses ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity sticky fingers: zinc-fingers as protein-recognition motifs 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traffic in the secretory pathway intracellular functions of n-linked glycans a multifunctional shuttling protein nucleolin is a macrophage receptor for apoptotic cells inhibition of interferon induction and action by the nairovirus nairobi sheep disease virus/ganjam virus crimean-congo hemorrhagic fever virus genome l rna segment and encoded protein ) 5'-triphosphate rna is the ligand for rig-i the role of motor proteins in endosomal sorting cholesterol is required for endocytosis and endosomal escape of adenovirus type 2 structural basis for the removal of ubiquitin and interferon-stimulated gene 15 by a viral ovarian tumor domain-containing protease non-viral sequences at the 5' ends of dugbe nairovirus s mrnas induction of caspase activation and cleavage of the viral nucleocapsid protein in different cell types during crimean-congo hemorrhagic fever virus infection differential roles of mda5 and rig-i helicases in the recognition of rna viruses duba: a deubiquitinase that regulates 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subtilisin/kex2p-like endoprotease involved in processing of a wide variety of precursor proteins crystal structure of the interferon-induced ubiquitin-like protein isg15 the anti-hiv pentameric pseudopeptide hb-19 binds the c-terminal end of nucleolin and prevents anchorage of virus particles in the plasma membrane of target cells n6-methyl-adenosine (m6a) in rna: an old modification with a novel epigenetic function rna binding properties of bunyamwera virus nucleocapsid protein and selective binding to an element in the 5' terminus of the negative-sense s segment genetic characterization of the m rna segment of crimean congo hemorrhagic fever virus strains crimean-congo hemorrhagic fever in bulgaria emergence of crimean-congo haemorrhagic fever in greece ecology of the crimean-congo hemorrhagic fever endemic area in albania tax1bp1 and a20 inhibit antiviral signaling by targeting tbk1-ikki kinases ubiquitin-regulated recruitment of ikappab kinase epsilon to the mavs interferon signaling adapter ribonucleoproteins of uukuniemi virus are circular rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates identification of four conserved motifs among the rna-dependent polymerase encoding elements cap binding and immune evasion revealed by lassa nucleoprotein structure sequence and phylogenetic analysis of the large (l) segment of the tahyna virus genome interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures laboratory infections with ganjam virus hantavirus nucleocapsid protein is expressed as a membrane-associated protein in the perinuclear region molecular biology of nairoviruses 1263 structure of the rift valley fever virus nucleocapsid protein reveals another architecture for rna encapsidation bunyaviridae rna polymerases (l-protein) have an n-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription characterization of the nucleic acid binding properties of tomato spotted wilt virus nucleocapsid protein isg15: the immunological kin of ubiquitin completion of the la crosse virus genome sequence and genetic comparisons of the l proteins of the bunyaviridae extraction of cholesterol with methyl-betacyclodextrin perturbs formation of clathrin-coated endocytic vesicles crimean-congo hemorrhagic fever virus-infected hepatocytes induce er-stress and apoptosis crosstalk ultrastructural studies on the replication and morphogenesis of nairobi sheep disease virus, a nairovirus antiviral actions of interferons characterization of the glycoproteins of crimean-congo hemorrhagic fever virus crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by furin-like and ski-1 proteases to generate a novel 38-kilodalton glycoprotein tickborne arbovirus surveillance in market livestock bunyaviridae: the viruses and their replication the v3 loop-mimicking pseudopeptide 5[kpsi(ch2n)pr]-tasp inhibits hiv infection in primary 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role in virulence and immune interactions crimean-congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase ski the inner structure of uukuniemi and two bunyamwera supergroup arboviruses structure of crimean-congo hemorrhagic fever virus nucleoprotein: superhelical homooligomers and the role of caspase-3 cleavage the proteins and rnas specified by clo mor virus, a scottish nairovirus synthesis of bunyavirus-specific proteins in a continuous cell line (xtc-2) derived from xenopus laevis tensaw virus genome sequence and its relation to other bunyaviridae double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses interferon and cytokine responses to crimean congo hemorrhagic fever virus; an emerging and neglected viral zonoosis ubiquitin and ubiquitin-like proteins as multifunctional signals nairobi sheep disease. in: 7th (ed) foreign animal disease crimean-congo hemorrhagic fever crimean-congo haemorrhagic fever: a seroepidemiological and tick survey in the sultanate of oman the erve virus: possible mode of transmission and reservoir identification of a putative crimean-congo hemorrhagic fever virus entry factor origin and evolution of retroelements based upon their reverse transcriptase sequences genomic analysis reveals nairobi sheep disease virus to be highly diverse and present in both africa, and in india in the form of the ganjam virus variant identification of the ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury crystal structure of an avian influenza polymerase pa(n) reveals an endonuclease active site electron microscopic and antigenic studies of uncharacterized viruses. ii. evidence suggesting the placement of viruses in the family bunyaviridae the ubch8 ubiquitin e2 enzyme is also the e2 enzyme for isg15, an ifnalpha/beta-induced ubiquitin-like protein human isg15 conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways rna-binding proteins that inhibit rna virus infection acknowledgments the laboratory of mdb receives funding from the uk biotechnology and biological science research council (bbsrc). ll was the recipient of a bbsrc phd studentship. key: cord-270594-62xotol3 authors: he, lei; hu, xiaolong; zhu, min; liang, zi; chen, fei; zhu, liyuan; kuang, sulan; cao, guangli; xue, renyu; gong, chengliang title: identification and characterization of vp7 gene in bombyx mori cytoplasmic polyhedrosis virus date: 2017-09-05 journal: gene doi: 10.1016/j.gene.2017.06.048 sha: doc_id: 270594 cord_uid: 62xotol3 the genome of bombyx mori cytoplasmic polyhedrosis virus (bmcpv) contains 10 double stranded rna segments (s1–s10). the segment 7 (s7) encodes 50 kda protein which is considered as a structural protein. the expression pattern and function of p50 in the virus life cycle are still unclear. in this study, the viral structural protein 7 (vp7) polyclonal antibody was prepared with immunized mouse to explore the presence of small vp7 gene-encoded proteins in bombyx mori cytoplasmic polyhedrosis virus. the expression pattern of vp7 gene was investigated by its overexpression in bmn cells. in addition to vp7, supplementary band was identified with western blotting technique. the virion, bmcpv infected cells and midguts were also examined using western blotting technique. 4, 2 and 5 bands were detected in the corresponding samples, respectively. the replication of bmcpv genome in the cultured cells and midgut of silkworm was decreased by reducing the expression level of vp7 gene using rna interference. in immunoprecipitation experiments, using a polyclonal antiserum directed against the vp7, one additional shorter band in bmcpv infected midguts was detected, and then the band was analyzed with mass spectrum (ms), the ms results showed thatone candidate interacted protein (vp7 voltage-dependent anion-selective channel-like isoform, vdac) was identified from silkworm. we concluded that the novel viral product was generated with a leaky scanning mechanism and the vdac may be an interacted protein with vp7. the genome of cytoplasmic polyhedrosis viruses (cpvs) normally consist of 10 dsrna segments (payne & mertens, 1983) , however, some cpvs have 11-12 dsrna segments (zhao, liang, hong, & peng, 2003a; zhao, liang, hong, xu, & peng, 2003b) . the genome of trichoplusiani cypovirus type 15 (tncpv-15) and antheraea mylitta cypovirus type 4 (amcpv-4) is composed of 11 dsrna segments (qanungo, kundu, mullins, & ghosh, 2002; rao, carner, scott, omura, & hagiwara, 2003) . each dsrna segment of bmcpv is composed of a single complete open reading frame (orf) . these open reading frames (orfs) code for the structural as well as non-structural proteins and polyhedrin. genome analysis of the bmcpv h and i strains revealed that segment s1, s2, s3, s4, s6 and s7 encode viral structural proteins such as vp1, vp2, vp3, vp4, vp6 and vp7, while segment s5, s8, s9 and s10 encode nonstructural proteins p101 (nsp5), p44 (nsp8), ns5 (nsp9) and polyhedrin, respectively (cao et al., 2012) . the eleventh dsrna segment was found in the bombyx mori cypovirus type 1 (bmcpv-1), which was termed as small polyhedron gene segment (sp) (arella, lavallee, belloncik, & furuichi, 1988) . the twelfth dsrna segment was also identified in the bmcpv-1 with novel rna extraction methods (nagae, miyake, kosaki, & azuma, 2005) . the functions of these newly found dsrna segments are still undefined. a unique characteristic of the dsrna viruses including bmcpvs, is their ability to accomplish transcription of the dsrna segments. 5′ end of bmcpv genome was processed by adding cap and methylation that form a cap like structure, mgpppampgp, protecting mrnas from degradation by exonuclease enzymes which cuts uncapped mrna in a 5′ to 3′ direction (furuichi, 1978) . all of the dsrna segments in the genome of bmcpv have the similar conservative sequence (guuaa……guuagcc) in the end (kuchino, nishimura, smith, & furuichi, 1982) , which were predicted as the recognize signal for rna transcriptase and replicase, or related with both ribosome and mrna, or assembly with viral structure (la, 2001) . these conservative sequences were also found in the genome of dendrolimus punctatus (zhao et al., 2003a; zhao et al., 2003b) . the size of complete segment 7 (s7) in bmcpv is 1501 bp and its orf is located at 25-1371 bp, which encode 448 amino acid residue. the prokaryotic expression product is 50 kda (p50). however, anti-p50 can detect one band with 56 kda from the bmcpv-1 infected bmn4 cells. by using the same antibody, the bands with 34, 36, 38 and 40 kda from the virion can be detected (jin, peng, wang, zhang, & zhang, 2014) . m2 gene of reovirus serotype 1 encoded protein μ1 could be cleaved into capsid protein μ1c, which can interact with s4 gene encoded protein σ3 to complete the assembly of the virion (wiener & joklik, 1988) . it is suggested that p50 of bmcpv might play a vital role in the process of assembly of virion. in the previous study, bmcpv-sz genome was cloned and found that vp7 enriched alanine and threonine, and a nuclear localization signal (nls) (cao et al., 2012) . the analysis of the cpv virion with frozen electron microscope and computer three-dimensional reconstruction technology showed that there are two different sizes of protuberance in the outer surface of the capsid. cpv caspid three-dimensional structure analysis showed that there are several protuberances (large protuberance and small protuberance) in the surface of caspid. it was suggested that p50 is processed by posttranslational modification to form the protuberance for the attachment of the virion to the targeted cells (cheng et al., 2011) . domain analysis of vp7 showed that it may also have role in nucleic acid binding. this study highlights the potential role of small vp7 gene-encoded proteins in the life cycle of bmcpv. three sirnas targeting the vp7 gene of bmcpv were designed and synthesized to reveal their interference effects on bmcpv replication in infected silkworm larvae and cells. the expression patterns and potential cleavage sites of vp7 were also investigated with the specific antibody, which were produced by the prokaryotic expression system. bmn cells of domestic silkworm were cultured in tc-100 medium at 27°c under standard conditions. the silkworms (strain p50) were cultivated on mulberry leaves at 25 ± 1°c temperature and 70-85% humidity, respectively, with a photoperiod of 12:12 ld. according to the vp7 gene (accession number: gq150538) sequence, three sirna were designed and termed as vp7-sirna1, vp7-sirna2 and vp7-sirna3 (shanghai integrated biotech solutions co., ltd). control sirna was designed with gfp gene (accession number: nc_011521.1) sequence and named as sirna-gfp (table 1) . 1 × 10 6 bmn cells were transfected with 2 μg sirna-gfp (control) and three bmcpv vp7 gene specific sirnas vp7-sirna1, vp7-sirna2 and vp7-sirna3 (test) using 2 μl lipidosome. after 24 h of transfection, bmcpv virions prepared by our lab (zhang et al., 2017) were added into the medium containing bmn transfected cells. after 48 h of infection, the infected cells were collected for the rna extraction from the control group and test group. two groups (control and test) were made randomly for third instar larva, followed by four replicates for each group. 1 μg sirna was injected into haemolymph of each silkworm. injected silkworms were fed with bmcpv (4.3 × 10 9 polyhedron/ml). 48 h later, midguts of the infected silkworms were taken for the rna isolation. total rna was extracted from the third instar larvae and bmn cells, infected with bmcpv, according to the standard protocol. the quantity and quality of rna was determined with both 260/280 absorbance ratio and gel electrophoresis, respectively. rna was stored at − 80°c for further experiments. 1 μg of total rna was used as template in the first-strand cdna synthesis. real-time quantitative pcr was performed using sybr green dye (bio-rad, hong kong, china) with c1000 thermal cycler system (bio-rad, hercules, ca, usa). the primers for the genes expression analysis are listed in table 2 . the 2 − δδct method was used for determination of relative expression level of the detected genes (livak & schmittgen, 2001) . experiments were performed in triplicates for each sample. to prepare polyclonal antibody against vp7 proteins of bmcpv, the cdna with primers listed in table 1 was amplified with pcr. the amplified pcr products were ligated into the prokaryotic expression vector pet28(a). the recombinant prokaryotic expression vector pet28(a)-vp7 ( supplementary fig. 1a ) was transformed into escherichia coli strain bl21 cells and induced with 1 mmol/l isopropyl-β-d-thiogalactopyranoside (iptg). after 4 h of induction, 1.5 ml of the transformed bl21 cells was collected for the recombinant protein detection. the proteins were detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and western blotting (supplementary fig. 1b) , using a primary antibody of rabbit anti-6 × his (sigma) and a secondary antibody of horseradish peroxidase (hrp)conjugated goat anti-rabbit (sigma). recombinant protein (vp7) was purified from the 1 l bl21 cells with ni-nta (ni 2 + -nitrilotriacetate) resin (invitrogen, carlsbad, ca) using standard procedure. mouse was immunized with recombinant protein (vp7) for preparing the vp7 polyclonal antibody. booster injections were administered according to the manufacturer's instructions. the collected serum was stored at − 70°c. 5′-ugcgcuccuggacguagccuu-3′ table 2 primers for real-time pcr. gene name primer name sequence puc-s7 plasmid, preserved in our lab, was used as a template for the amplification of s7 fragment with primers s7-ei/s7-xi. pcr amplicons were digested with digestion enzymes (ecoriand xba) and inserted into the vector pizt/v5-his to produce a recombinant plasmid pizt/v5-his-s7. bmn cells were transfected with recombinant plasmid pizt/v5-his-s7 by adding 2 μg of recombinant plasmid (pizt/v5-his-s7) and 5 μl of lipidosome into the 25 cm 2 cell culture flask containing bmn cells. after 3 days of transfection, transfected cells were examined with inverted fluorescence microscope for screening transfection in bmn cells. 400 μg/ml zeocin antibiotic was added for screening the positive transfected cells. transfected cells were screened continually for one month using zeocin antibiotic. 70-80% positive transfected cells were collected for the extraction of total protein. midguts were derived from bmcpv infected silkworm for the paraffin section. the sections of the midguts were fixed in 4% (v/v) paraformaldehyde at room temperature for 2 h and then rinsed with 0.01 m pbst (10 mm na 2 hpo 4 , 2.7 mm kcl, 140 mm nacl, 1.8 mm kh 2 po 4 , ph 7.4) containing 0.05% (v/v) tween-20. the fixed midgut sections were blocked with 3% (w/v) bsa at room temperature for 2 h followed by three washes (5 min each) in pbst, then incubated overnight at 4°c with vp7 polyclonal antibody (diluted 1:100 in blocking buffer). after three washes (5 min each) in pbst, cells were incubated with anti-rabbit fluorescein isothiocyanate (fitc)-labeled secondary antibody (huaan biotechnology) at a dilution of 1:200. fitc displays green fluorescence under blue light. the nuclei were labeled with 4′, 6diamidino-2-phenylindole (dapi), which exhibits blue fluorescence. cells were observed under a laser confocal scanning microscope (nikco, japan). silkworm midgut total proteins were extracted with the ripa buffer (sangon, shanghai). the total proteins were incubated overnight at 4°c with the specific murine polyclonal anti-vp7 antibody. the murine unimmune serum was used as the corresponding antibody. immune complexes were precipitated by adding protein a + g (sigma), washed three times in the lysis buffer and analyzed by sodium dodecyl sulfate 12% polyacrylamide gel electrophoresis (sds-page) followed by coomassie brilliant blue staining. co-ip was replicated three times and all the appeared bands were cut for the mass spectrum. proteins identification was carried out in shanghai applied protein technology co. ltd. peptidecutter online software (http://web.expasy.org/peptide_ cutter/) was used to predict the potential cleavage sites cleaved by proteases or chemicals in a given protein sequence (wilkins et al., 1999) . all data are presented as mean ± standard deviation (sd). statistical differences were evaluated using student's t-test for unpaired samples. the level of statistical significance was set at * p < 0.05, ** p < 0.01 and *** p < 0.001. after the successful construction of pizt/v5-his-s7 expression vector (fig. 1a) , bmn cells were transfected with recombinant vector (pizt/v5-his-s7) using liposome. 400 μg/ml zeocin antibiotic was added for screening the positive transfected cells. after 3 days of transfection, bmn cells were examined with inverted fluorescence microscope. the detection of green fluorescence in transfected cells indicated that expression vector was transferred into the bmn cells. transfected cells were screened continually for one month using zeocin antibiotic. the proportion of green fluorescence did not increase after 1 month of zeocin treatment (fig. 1b) . total protein was extracted from the transfected cells, and vp7 gene expression was confirmed with western blotting. the results showed that one band with 70 kda was detected from the transfected cells with 6*his antibody. while, with the vp7 antibody, three bands with about 70, 34 and 35 kda were detected from the transfected cells (fig. 1c) . vp7 recombinant protein was purified from the transfected cells. the purified product was also confirmed with sds-page and western blotting. the results showed that only one band with 70 kda was detected with 6*his antibody, while additional band with 35 kda was detected by vp7 antibody ( fig. 2a and b ). a novel protein was detected from the overexpression of vp7 gene in the bmcpv infected cultured cells, with vp7 antibody. to identify that whether other proteins could be present in the bmcpv, total proteins were extracted from the bmcpv infected cultured cells (bmn cells), midgut and bmcpv virion. a band with 55 kda was detected from the bmcpv infected bmn cells using western blotting technique. however, this band is similar with the hypothetical molecular weight of vp7, but smaller than overexpression product in bmn cells. additional band with 20 kda was also identified from the bmcpv infected bmn cells (fig. 3a) . proteins from bmcpv virion were also extracted and detected with vp7 antibody, and four clear bands (55, 38, 25 and 10 kda) were found in the bmcpv virion (fig. 3b ). total proteins from the bmcpv infected silkworm midguts (from the first day to the twelfth day) were also extracted, and detected with vp7 antibody. a single positive band was detected from the seventh day of infected silkworm. from the eighth day, three positive bands (55, 25 and 20 kda) were detected. however, from the ninth to the twelfth day, > 5 bands (55, 28, 25, 20 and 18 kda) were detected (fig. 4) . the expression level of bmcpv vp7 gene after transfection with specific sirna was evaluated by real-time pcr. virus replication in transfected cells and silkworm was analyzed. transfection with sirna-vp7 reduced the expression level of vp7 gene in infected cells and silkworm compared to the levels of the control (fig. 5a) . the transcript level of vp1 gene was decreased by 15.4 fold. silencing of the vp7 gene inhibited the multiplication of bmcpv (fig. 5b) . similar results were obtained from the bmn cells infected with bmcpv. when the expression of vp7 gene was down-regulated in bmn cells, vp1 gene transcript level was decreased by 13 fold (fig. 5c ). immunoprecipitation experiments using a polyclonal antiserum directed against the vp7 protein detected the vp7 protein and at least four additional shorter products in infected midguts. these shorter bands were cut for the mass spectrum (fig. 6) , and total 19 proteins were identified. except vp7, other 18 proteins were encoded by the genome of silkworm. all proteins are listed in the table 3 . only viral structural protein 7 and voltage-dependent anion-selective channel-like isoform (vdac) were identified with > 2 unique peptides among all of the identified proteins. these 4 bands with similar molecular weight of the products were identified from the bmcpv virion. it was suggested that these products may be translated from alternative in-frame aug codon in vp7 mrna. vdac was considered a potential interacted protein with the vp7, playing important role in the life cycle of bmcpv. the 3rd instar silkworm larvae were infected with bmcpv. 6 days later, the midguts were extracted to analyze with immunofluorescence technique. the results showed that the viral virion was localized in the cytoplasm of the infected cells (fig. 7) . bmcpv genome has ten dsrna segments (s1-s10), which are encoding structural and non-structural proteins and polyhedrin. vp7 gene is encoded by the s7 segment, and the function of it is still unclear. in this report, it was demonstrated that 4, 2 and 5 bands of vp7 gene products, recognized by a polyclonal anti-vp7 antibody, are present in bmcpv virion, bmcpv infected cells and bmcpv infected silkworm midguts and 3 bands in transfected cells overexpressed with vp7 gene. these polypeptides appeared may be encoded by a single open reading frame (orf) produced from the specific aug codons of vp7 gene. sequence analysis found that bmcpv vp7 gene contains nearly 17 aug codons in vp7 orf. all of these short mrna encoded > 100 codons, and only 3 short mrnas were inconsistent with the orf of vp7 gene. however, we cannot exclude the possibility that these short polypeptides were initiated from non-aug codons, or truncated vp7 was generated from the cleavage by the proteolytic enzyme. coding capacity of viruses is enhanced with the evolving overlapping reading frames. until now, the segmented dsrna genome of the viruses including reoviridae, coronaviruses and orbivirus families were thought to be monocistronic (i.e. ten genome segments encoding ten proteins), but the identification of novel orfs, translated in the same reading frame that begin at an alternative downstream aug, changed this assumption (gong, chen, chen, kuo, & shih, 2014) . rice black-streaked dwarf virus genome segments s5, s7 and s9 were bicistronic mrna, which were involved in the formation of tubular structures, the formation of viroplasms, virus replication and assembly occurrence. at least 9 novel orfs were found from the genome of coronaviruses. they were considered as the accessory proteins involved in viral pathogenicity (shukla & hilgenfeld, 2015) . a small orf in segment 10, overlapping the ns3 orf in the +1 position was identified from the genome of bluetongue virus (btv) (stewart et al., 2015) . in this study, we concluded that short products of vp7 were appeared in the gels, which might play a significant role in bmcpv life cycle. further experiments on these short vp7 products will be carried out in our future studies. voltage-dependent anion channels belongs to 6 . identification of the interaction proteins with the bmcpv vp7 with co-immunoprecipitation. co-ip was replicated with three time and all the disparity bands were cut for the mass spectrum. a class of porin ion channel and positioned on the outer mitochondrial membrane (hoogenboom, suda, engel, & fotiadis, 2007) . it act as a general diffusion pore for small hydrophilic (water attractant) molecules (benz, 1994) and helps in the exchanging of ions and molecules between mitochondria and cytosol and is regulated by the interactions with other proteins and small molecules (hiller, abramson, mannella, wagner, & zeth, 2010) . vdac has been shown to play a significant role in apoptosis. during apoptosis, increased permeability of vdac allows for the release of apoptogenic factors (tsujimoto & shimizu, 2002) . vdac-like protein in rhipicephalus microplus may play important role in the infection of babesia bigemina (rodriguez-hernandez et al., 2012) . previous studies suggested the apoptosis induction is carried out by the upregulation of vdac which results in enhanced permeability of outer mitochondrial membrane. the possible reason is thought to be a shift in the vdac equilibrium to an oligomeric form of the protein which forms large pores and thus allows the release of pro-apoptotic proteins (ghosh, pandey, maitra, brahmachari, & pillai, 2007) . furthermore, many viruses encode proteins that act on vdac to control apoptosis in infected cells (boya et al., 2003) , such as influenza a and hepatitis b viruses (boya et al., 2004) . whether vdac function is important during babesia bigemina invasion, propagation or replication must be determined in the near future. in the later period of bmcpv infected silkworm larvae, nearly 5 bands were found from the midguts. the alternative hypothesis was that vp7 was produced by the proteolytic enzyme. in our previous study, the analysis of vp5, encoded by the s5 fragment of bmcpv, showed that it has foot-and-mouth disease virus (fmdv) 2a pro -like protease domain which is a short polypeptide of 16 amino acids. the function of this domain is in breaking of the polyprotein of fmdv at the 2a/2b junction co-translationally. this cleavage occurs at its carboxyl terminus at a glycineproline amino acid pair (ryan, king, & thomas, 1991; mattion, harnish, crowley, & reilly, 1996) . whether it might be taking part in the cleavage of the vp7 still need further investigation. approximately 5% of gag gene (such as lentiviruses, human immunodeficiency virus, simian immunodeficiency virus) produced other proteins causing by the ribosomal frameshift with independent functions (rue, roos, tarwater, clements, & barber, 2005) . two mechanisms for generating more than one protein from a single mrna have been reported. one possibility is through leaky scanning, such as paramyxoviruses (kozak, 1989) and rabies virus. the other possibility is a cap-independent mechanism, such as picornavirus polyprotein and sendai virus proteins (pelletier & sonenberg, 1988; curran & kolakofsky, 1989) . why vp7 exited various protein expression patterns among virion, infected cells and larvae? peptidecutter online software was used to predict the potential cleavage sites cleaved by proteases in vp7. bnps-skatole, iodosobenzoic acid, ntcb (2-nitro-5thiocyanobenzoic acid) and caspase1 were predicted to be the potential enzymes (table 4 ). in the future, to better understand the potential cleavage mechanisms of vp7, these proteins with small molecular weight will be purified from the bmcpv virion and then will be identified with the mass spectrum analysis. in this study, we also conducted the experiments for revealing the function of vp7 gene in the viral life cycle. down-regulated vp7 gene transcript level decreased the transcript level of vp1 gene in bmcpv infected silkworm larvae and bmn cells. it was indicated that vp7 gene was associated with the bmcpv genome replication. the reason for the decline of genome replication due to the reduction in expression level of vp7 gene was that during the cell entry of the progeny virus, the vp7-vp4 outer layer un-coats releasing the intact double-layered particle (dlp) into the cytosol. vp4 mediates cell binding (fiore, greenberg, & mackow, 1991) and membrane disruption (denisova et al., 1999; kim, trask, babyonyshev, dormitzer, & harrison, 2010) , but the final steps of dlp delivery require un-coating of vp7 (cuadras, arias, & lopez, 1997; liprandi et al., 1997) . full un-coating of the outer layer is also required to activate the dlp (lawton, estes, & prasad, 2001) , which contains multiple copies of the viral polymerase complex that synthesize and extrude mrna transcribed from each of the 10 genome segments. the vp7 transcript was reduced because the vp7 layer anchors the vp4 spikes onto the underlying dlp was less, leading to the membrane disruption failure (chen & ramig, 1993; trask & dormitzer, 2006) . the new mrna transcribed from each segment was decreased, leading to the reduction of the genome replication. supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2017.06.048. our work was supported by the national natural science foundation of china (31602007 and 31072085), natural science foundation of jiangsu province (bk20140324) and a project funded by the priority academic program of development of jiangsu higher education institutions. molecular cloning and characterization of cytoplasmic polyhedrosis virus polyhedrin and a viable deletion mutant gene permeation of hydrophilic solutes through mitochondrial outer membranes: review on mitochondrial porins 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studies with human participants performed by any of the authors. key: cord-103709-86hv27vh authors: zhang, dong yan; wang, jian; dokholyan, nikolay v. title: prefusion spike protein stabilization through computational mutagenesis date: 2020-06-19 journal: biorxiv doi: 10.1101/2020.06.17.157081 sha: doc_id: 103709 cord_uid: 86hv27vh a novel severe acute respiratory syndrome (sars)-like coronavirus (sars-cov-2) has emerged as a human pathogen, causing global pandemic and resulting in over 400,000 deaths worldwide. the surface spike protein of sars-cov-2 mediates the process of coronavirus entry into human cells by binding angiotensin-converting enzyme 2 (ace2). due to the critical role in viral-host interaction and the exposure of spike protein, it has been a focus of most vaccines’ developments. however, the structural and biochemical studies of the spike protein are challenging because it is thermodynamically metastable1. here, we develop a new pipeline that automatically identifies mutants that thermodynamically stabilize the spike protein. our pipeline integrates bioinformatics analysis of conserved residues, motion dynamics from molecular dynamics simulations, and other structural analysis to identify residues that significantly contribute to the thermodynamic stability of the spike protein. we then utilize our previously developed protein design tool, eris, to predict thermodynamically stabilizing mutations in proteins. we validate the ability of our pipeline to identify protein stabilization mutants through known prefusion spike protein mutants. we finally utilize the pipeline to identify new prefusion spike protein stabilization mutants. the ongoing outbreak of the novel coronavirus [2] [3] [4] , which causes fever, severe respiratory illness, and pneumonia, poses a major public health and governance challenges. the emerging pathogen has been characterized as a new member of the betacoronavirus genus (sars-cov-2) 5,6 , closely related to several bat coronaviruses and to severe acute respiratory syndrome coronavirus (sars-cov) 7, 8 . compared with sars-cov, sars-cov-2 appears to be more readily transmitted from human to human, spreading to multiple continents and leading to the world health organization (who)'s declaration of a pandemic on march 11, 2020 9 . according to the who, as of june 9, 2020, there had been >7,000,000 confirmed cases globally, leading to >400,000 deaths. in the initial step of the infection, the coronavirus enters the host cell by binding to a cellular receptor and fusing the viral membrane with the target cell membrane 10, 11 . for sars-cov-2, this process is mediated by spike glycoprotein which is a homo-trimer 12 . in the process of viruses fusing to host cells, the spike protein undergoes structural rearrangement and transits from a metastable prefusion conformational state to a highly stable post-fusion conformational state 13, 14 . the spike protein comprises of two functional units, s1 and s2 subunits; when fused to the host cell, the two subunits are cleaved. the s1 subunit is responsible for binding to the angiotensin-converting enzyme 2 (ace2) [15] [16] [17] receptor on the host cell membrane and it contains the n-terminal domain (ntd), the receptor-binding domain (rbd) and the c-terminal domain (ctd). ntd in the s1 subunit assists recognize sugar receptors. rbd in the s1 subunit is critical for the binding of coronavirus to the ace-2 receptor [18] [19] [20] [21] . ctd in the s1 subunit could recognize other receptors 22 . the binding of rbd to ace2 facilitates the cleavage of the spike protein and promotes the dissociation of the s1 subunit from the s2 subunit 23 . s2 contains two heptad repeats (hr1 and hr2), a fusion peptide, and a protease cleavage site (s2'). the dissociation of s1 induces s2 to undergo a dramatic structural change to fuse the host and viral membranes. thus, the spike protein serves as a target for development of antibodies, entry inhibitors and vaccines 24 . coronavirus transits from a metastable prefusion state to a highly stable post-fusion state as part of the spike protein's role in membrane fusion. the instability of the prefusion state presents a significant challenge for the production of protein antigens for antigenic presentation of the prefusion antibody epitopes that are most likely to lead to neutralizing responses. thus, since the prefusion spike protein exists in a thermodynamically metastable state 1 , a stabilized mutant conformation is critical for the development of vaccines and drugs. computational mutagenesis is an effective approach to finding mutations that are able to stabilize proteins. we have previously developed a protein design platform, eris 25, 26 , which utilizes a physical force field 27 for modeling inter-atomic interactions, as well as fast side-chain packing and backbone relaxation algorithms to enable efficient and transferrable protein molecular design. originally, eris has been validated on 595 mutants from five proteins, corroborating the unbiased force field, side-chain packing and backbone relaxation algorithms. in many later studies, eris has been validated through prediction of thermodynamically stabilizing or destabilizing mutations [28] [29] [30] [31] [32] [33] , and direct protein design efforts [34] [35] [36] [37] . in this work, we propose a pipeline to automatically stabilize spike proteins through computational mutagenesis. within the pipeline, we first analyze the conservation score and solvent accessible surface area (sasa) of residues in the protein. we then perform discrete molecular dynamics (dmd) [38] [39] [40] [41] simulations to calculate the root mean square fluctuation (rmsf) of residues to analyze their flexibility. based on this information, we select appropriate residues (2 < conservation score < 5; sasa > 0.4; rmsf > 3.5 å; see discussion) as mutation sites. we subject the selected residues to computational redesign using eris to find the stabilizing mutations by calculating the change in free energy ∆∆ = ∆ − ∆ , where ∆ and ∆ are the free energies of the mutant protein and wild type proteins correspondingly. we utilize this pipeline to identify stabilization mutants of the spike protein. next, we describe our methods in detail and provide a list of stabilizing mutations for spike protein. we propose a pipeline to automatically find stabilized mutants of proteins ( figure 1 ). the pipeline can be divided into two stages. in the first stage, users designate the protein of interest, and then the pipeline will analyze the 3d structure of the protein by using different metrics. in the second stage, users designate the mutation sites according to the analysis of the 3d structure of the protein, and finally the pipeline will determine the stabilizing mutations of the protein. users can either upload the 3d structure of the protein or input the pdb id of the protein to designate the protein of interest. in the first stage, the first step is to remodel the 3d structure of the protein of interest to complete the missing atoms and residues. we integrate modeller into our pipeline to remodel proteins. next, the pipeline utilizes consurf 48 to calculate the conservation score of each residue in the protein of interest. the conservation score indicates the importance of the residue in maintaining protein structure and/or function. subsequently, the pipeline utilizes dmd to analyze the flexibility of each residue in the spike protein through rmsf. the technique has already been used to efficiently study the protein folding thermodynamics and protein oligomerization and allows for a good equilibration of the structures. then, the pipeline will calculate sasa of residues in the protein. in the second stage, users designate the mutation sites according to the conservation score, rmsf, and sasa. a high conservation score (≥ 7) indicates the residue may play important roles in the function or the stability of the structure of the protein; residues of high rmsf (> 3.5 å) are likely the culprit to undermine the stability of the structure of the protein, hence we select residues that have a low conservation score or high rmsf; residues with sasa < 0.5 are considered buried and residues with sasa ≥ 0.5 are considered exposed to solvent. after the designation of the mutation sites, the pipeline utilizes eris to determine the changes in free energies of the mutants. for each residue in the mutation sites, we utilize modeller 42 to re-model the 3d structure of the spike protein by using a structure deposited to protein databank (pdb), pdbid:6vsb 12 , the cryo-em structure of the prefusion state of the spike protein, as the template structure to complete the missing atoms and residues ( figure 2a&b ). next, we use chiron 44 all following computational mutagenesis study are performed using the structure with the 3 rbds in down conformation. to validate the ability of the pipeline to identify stabilization mutants, we use the pipeline to calculate the free energy changes of several known prefusion spike protein stabilization mutants. the 2p mutation strategy (k986p and v987p) has been proved effective for the stabilization of spike protein of sars-cov-2 and other betacoronavirus 12,20,50 . hsieh and coworkers 51 the spike protein is mainly composed of s1 and s2 subunits ( figure s1 ). we select residues for mutation from the ntd and rbd domains in s1, and we also select residues from the hr1 (heptad repeat 1) and ch (central helix) domains in s2. we don't select residues from hr2 domain in s2 because the structure of the hr2 domain has not been solved in the cryo-em structure 6vsb. at the outset, we calculate the conservation score ( figure 3a&d ) of all residues by using consurf. based on the conservation score, most residues in hr1/ch are conservative, while residues in ntd and rbd are prone to mutation in evolution. next, we use pymol 47 to calculate sasa of all residues in the spike protein ( figure 3b&e) . sasa indicates the level of residues exposed to the solvent in a protein and usually most of the functional residues are located on the protein structure's surface 52 . all four domains have both low sasa residues and high sasa residues. then, we perform 1,000,000 steps dmd simulation for the spike protein to calculate the rmsf of each residue ( figure 3c&f ). the residues in hr1/ch have extremely low rmsf, while the residues in ntd and rbd domains have moderate to high rmsf. we select residues that have different conservation scores in these four domains for mutagenesis. in the ntd, we select 5 residues (table s1 ) with the conservation score ranging from 1 (highly variable) to 9 (highly conservative). the sasa of these residues range from 0.01 (buried) to 0.75 (exposed). the rmsf range from 1.61 å (frozen) to 4.88 å (flexible). likewise, in the other three domains (table s23), we select 5 to 7 residues, respectively. these residues also have diverse conservation scores, sasa, and rmsf. of note, to avoid affecting the function of the spike protein, these residues are all not chosen from the functional sites of the spike protein, such as the ace2 binding site in rbd. we utilize eris to calculate the free energy changes of mutants relative to the wild type ( figure 4a , figure s2&3 , and table s4-6). in the ntd, 5 residues, e169, k113, i203, r246, and l270 are selected for mutagenesis. among them, the free energy changes of nearly all mutations of residues i203, r246, and l270 are positive, indicating that they are destabilizing the structure. in contrast, most mutations on residues e169 and k113 have negative free energy changes, suggesting that they are stabilizing the structure. the mutant that has the most negative free energy change is r246c. however, cysteine is prone to forming disulfide bond with other cysteine, which may affect the correct folding of the protein structure, so we recommend k113m to be a better choice as the stabilization mutant. in the rbd, 5 residues (a411, t415, y505, n439, and d428) (table s2) are selected for mutagenesis. the free energy changes calculated by eris ( figure s2 , in the hr1/ch domain, 7 residues (l948, i1018, a1026, y1007, s1003, t961, and v976) are selected for mutagenesis as shown in table s3 . in stark contrast to ntd and rbd, most residues have extremely high free energy changes (> 30 kcal/mol), suggesting that these residues are not very good choices for mutagenesis. the high free energy changes also implicate that they may play important roles in stabilizing the structure so that they are irreplaceable to some extent. this finding is also in concert with the high conservation scores of these residues. that said, we can still find stabilization mutants for these residues, such as t961a and s1003m ( figure s3 ). compared to experimental mutagenesis, such as random mutagenesis 53, 54 and site-directed mutagenesis 55, 56 , computational mutagenesis 25 is an efficient alternative that lays the foundation of large-scale mutation screening. however, performing computational mutation screening for all residues in the spike protein trimeric structure, which consists of 1288 residues in each monomer, is still timeconsuming and inefficient, so we seek to select critical residues to perform mutagenesis. in this work, to interrogate how to select residues as mutation sites, we select residues that have different conservation scores, sasa, and rmsf. we find that the probability of stabilizing mutations for specific residues is correlated with the conservation score, sasa, and rmsf of these residues ( figure 4b -d). we calculate the average and the minimum free energy change of all 19 mutations of each residue. we find that the average free energy change of mutations of residues with either high (>5) or low (<2) conservation score are typically larger than 0. only mutations in residues that have moderate (2~5) conservation score have negative average free energy change, thus indicating a possibility to find stabilizing mutations. we posit that conservative residues are typically playing critical roles in structural stability or protein functioning 57 , making them a likely target for finding stabilizing mutations. however, if the conservation score of the residue is too high, the residue may be irreplaceable and any mutation will destabilize the structure. on the other hand, residues with low conservation scores may be less critical to the structural stability, reducing the chances for finding stabilizing mutations at these positions. thus, we select residues that have moderate conservation score (2~5) for mutagenesis. similarly, we find that residues with large sasa or large rmsf have more stabilization mutants than residues with low sasa or low rmsf, respectively. overall, we select residues that have moderate conservation score (2~5), high sasa (>0.4), and high rmsf (>3.5 å) for mutagenesis. in addition, although we only select residues in ntd, rbd, and hr1/ch domains to perform mutagenesis, residues in other regions can also be used as mutation sites. for example, the known 2p mutation strategy (k986p and v987p) has been proved effective for the stabilization of spike protein of sars-cov-2 and other betacoronavirus 12, 20, 50 . in this work, we propose a pipeline to automatically stabilize proteins through computational mutagenesis. we analyze the conservation score, rmsf, and sasa of residues in the spike protein through the pipeline. we propose criteria based on the conservation score, rmsf, and sasa to identify residues for mutation. finally, we utilize eris to calculate the free energy change and find stabilizing mutants. all source codes are deposited in: https://bitbucket.org/dokhlab/proteinstabilization. the 3d structure of the spike protein colored sasa. the red/blue colors indicate exposed/buried residues. (f) the 3d structure of the spike protein colored by rmsf. red means flexible and blue means frozen. characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov a novel coronavirus from patients with pneumonia in china features, evaluation and treatment coronavirus (covid-19) coronavirus (covid-19) outbreak: what the department of endoscopy should know the proximal origin of sars-cov-2 a novel coronavirus genome identified in a cluster of pneumonia cases-wuhan severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and corona virus disease-2019 (covid-19): the epidemic and the challenges world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) fusion mechanism of 2019-ncov and fusion inhibitors targeting hr1 domain in spike protein sars-cov-2 infects t lymphocytes through its spike protein-mediated membrane fusion cryo-em structure of the 2019-ncov spike in the prefusion conformation. science (80-. ) structure, function, and evolution of coronavirus spike proteins the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structural basis for the recognition of sars-cov-2 by fulllength human ace2. science (80-. ) cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen unexpected receptor functional mimicry elucidates activation of coronavirus fusion structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion covid-19, an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics eris: an automated estimator of protein stability modeling backbone flexibility improves protein stability estimation emergence of protein fold families through rational design g protein mono-ubiquitination by the rsp5 ubiquitin ligase tyrosine phosphorylation switching of a g protein stabilization of μ-opioid receptor facilitates its cellular translocation and signaling large sod1 aggregates, unlike trimeric sod1, do not impact cell viability in a model of amyotrophic lateral sclerosis a phosphomimetic mutation stabilizes sod1 and rescues cell viability in the context of an als-associated mutation nonnative sod1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis computational design of chemogenetic and optogenetic split proteins engineering extrinsic disorder to control protein activity in living cells rational design of a ligand-controlled protein conformational switch rationally designed carbohydrate-occluded epitopes elicit hiv-1 env-specific antibodies discrete molecular dynamics studies of the folding of a protein-like model discrete molecular dynamics applications of discrete molecular dynamics in biology and medicine ab initio folding of proteins with all-atom discrete molecular dynamics comparative protein modelling by satisfaction of spatial restraints statistical potential for assessment and prediction of protein structures computational modeling of small molecule ligand binding interactions and affinities solving protein structures using short-distance cross-linking constraints as a guide for discrete molecular dynamics simulations ab initio rna folding by discrete molecular dynamics: from structure prediction to folding mechanisms convergent solutions to binding at a protein-protein interface consurf: identification of functional regions in proteins by surface-mapping of phylogenetic information mdanalysis: a toolkit for the analysis of molecular dynamics simulations stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis structure-based design of prefusion-stabilized sars-cov-2 spikes improving prediction of secondary structure, local backbone angles and solvent accessible surface area of proteins by iterative deep learning sequence saturation mutagenesis (sesam): a novel method for directed evolution evolving strategies for enzyme engineering directed mutagenesis site-directed mutagenesis: effect of an extracistronic mutation on the in vitro propagation of bacteriophage qbeta rna understanding hierarchical protein evolution from first principles we acknowledge support from the national institutes for health 1r35 gm134864,the huck institutes of the life sciences, and the passan foundation. the project described was also supported by the national center for advancing translational sciences, national institutes of health, through grant ul1 tr002014. the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih. the author declares no potential conflict of interest. key: cord-002967-yy3bennu authors: penna, fabio; ballarò, riccardo; beltrá, marc; de lucia, serena; costelli, paola title: modulating metabolism to improve cancer-induced muscle wasting date: 2018-01-29 journal: oxid med cell longev doi: 10.1155/2018/7153610 sha: doc_id: 2967 cord_uid: yy3bennu muscle wasting is one of the main features of cancer cachexia, a multifactorial syndrome frequently occurring in oncologic patients. the onset of cachexia is associated with reduced tolerance and response to antineoplastic treatments, eventually leading to clinical conditions that are not compatible with survival. among the mechanisms underlying cachexia, protein and energy dysmetabolism play a major role. in this regard, several potential treatments have been proposed, mainly on the basis of promising results obtained in preclinical models. however, at present, no treatment yet reached validation to be used in the clinical practice, although several drugs are currently tested in clinical trials for their ability to improve muscle metabolism in cancer patients. along this line, the results obtained in both experimental and clinical studies clearly show that cachexia can be effectively approached by a multidirectional strategy targeting nutrition, inflammation, catabolism, and inactivity at the same time. in the present study, approaches aimed to modulate muscle metabolism in cachexia will be reviewed. cancer-induced muscle wasting is one of the hallmarks of cachexia, a multifactorial syndrome that represents one of the most important comorbidities in oncologic patients. the occurrence of cachexia markedly complicates the management of cancer patients, negatively impinging on the tolerance and response to antineoplastic treatments, worsening the quality of life, and reducing survival. in particular, about 25% of deaths by cancer are due to cachexia, rather than to the tumor itself [1] . few years ago, a classification of cachexia was proposed, defining three different stages: precachexia, cachexia, and refractory cachexia [2] . prognosis progressively worsens going from patients with precachexia to those with refractory cachexia. in this regard, the earlier anticachexia treatments are set up, the better. for this reason, the research on cachexia is focused on two main goals: (i) to find out biomarkers useful to the early identification of a condition of still latent cachexia and (ii) to define treatment protocols useful to delay the progression from precachexia to refractory cachexia. skeletal muscle wasting in cancer patients has a good prognostic value, being predictor of reduced tolerance to chemotherapy and/or surgery, decreased ability to perform daily activities, and shortened survival. in addition, recent data report that loss of muscle mass negatively affects quality of life in cancer patients [3, 4] ; such correlation might occur irrespectively of survival rates [5] . being poor quality of life one of the most prominent and invalidating consequences of cancer cachexia, to investigate the mechanisms underlying cancer-induced muscle wasting is even more relevant to design therapeutic strategies that also take into account patient well-being. the possibility to underestimate the occurrence of muscle mass depletion exists, since the first approach to clinically evaluate a patient is to obtain information about body weight and body mass index (bmi). however, in face of no body weight loss and/or normal bmi, reduced muscle mass might well occur, being masked by fat or water content. another relevant point that frequently is poorly taken into consideration is that the loss of muscle mass is likely exacerbated by anticancer treatments. at present, different mechanisms have been proposed to lead to muscle wasting in cancer hosts, among which there are altered protein and energy metabolism and impaired myogenesis [1] . several factors may contribute to these alterations, such as reduced calorie intake, hormonal unbalance, and systemic inflammation. cancer-driven production of proinflammatory cytokines plays a relevant role in tumor progression and markedly contributes to cachexia. indeed, in cancer patients, systemic inflammation correlates with increased resting energy expenditure and with reduced survival rate [6] . along the same line, increased circulating levels of tumor necrosis factor α (tnfα), interleukin-6 (il-6), γ-interferon (inf), and, more recently, growth and differentiation factor 15 (gdf15) have been reported in cachectic cancer patients [1, 7] . the link existing between cytokines and cachexia has led to the inclusion of anti-inflammatory drugs in treatment protocols [8] . the present review will focus on strategies able to modulate metabolism that may reveal useful to prevent/delay cancer-induced muscle wasting. altered protein turnover is a general feature of muscle wasting in cancer cachexia. in particular, protein breakdown rates are persistently increased, while protein synthesis rates can be reduced, unchanged, or even increased, depending on the model system [1] . the different reaction kinetics that characterizes protein synthesis and degradation rates, zero and first order, respectively, implies that if degradation is higher than normal, the loss of total protein cannot be antagonized by simply increasing the rate of synthesis. taking this assumption for true, any anabolic approach should be associated with anticatabolic strategies in order to achieve a beneficial effect on muscle protein mass. 2.1. protein degradation. several pieces of evidence suggest that the intracellular proteolytic systems in the skeletal muscle of cancer hosts are poised towards activation above the physiological levels ( figure 1 ). particularly relevant in this regard are the pathways dependent on ubiquitinproteasome and autophagy. while the former mainly degrades short-lived and regulatory proteins, the latter gets rid of structural proteins and organelles [9] . both experimental and human cancer cachexia were associated with increased activity of the ubiquitinproteasome pathway [1] . of interest, alterations in molecular and biochemical markers of proteasome activation were observed in gastric cancer patients before any evidence of body weight loss, supporting the need to detect cachexia as early as possible [10] . modulations of the ubiquitinproteasome proteolytic system, however, are not a general finding in cancer cachexia, as shown by studies reporting that it is not differently activated with respect to controls in the muscle of patients affected by non-small-cell lung cancer (nsclc; [11] ) or esophageal cancer [12] . the involvement of the autophagic-lysosomal proteolysis in muscle wasting was recognized just in the last fifteen years. two main reasons account for such delay: (i) autophagy was not considered as typically operated by the skeletal muscle as a response to stress conditions. such belief was definitely abandoned when autophagy was clearly demonstrated in fasted mice overexpressing green fluorescent proteinlabeled lc3 [13] . (ii) the study and detection of autophagy was not easy since the atg genes were not discovered [14] . a number of studies reported that the autophagic system was overactivated, without reaching complete cargo degradation, in the muscle of both tumor-bearing animals and cancer patients [6, [15] [16] [17] . in particular, despite autophagic flux was enhanced in mice bearing the c26 tumor, autophagosomes accumulated, likely due to exhaustion of the lysosomal compartment [15] . a similar pattern could also be observed in cancer patients, as suggested by lc3b-ii and p62 accumulation [16, 17] . both proteasome and lysosomes, however, cannot directly degrade intact myofilaments. in this regard, a preliminary cleavage was proposed to be operated by other proteolytic systems, such as those dependent on caspases or calpains. these latter are ca 2+ -dependent cysteine proteases, normally inactive and localized in the cytosolic compartment. when intracellular ca 2+ concentrations increase, inactive calpains translocate to the cell membrane and become activated by autoproteolysis [18] . the system also includes calpastatin, a physiological inhibitor, which is a substrate of active calpain itself. increased calpain expression was reported in the muscle of tumor-bearing animals [19] , while rats transplanted with the yoshida ah-130 hepatoma showed a progressive reduction of calpastatin levels and increased in vitro cleavage of specific fluorogenic substrates [20] . more recently, calpain activation was also demonstrated in mice bearing the c26 colon carcinoma [21] . both increased or unchanged muscle calpain expression were reported in cancer patients [12, 22] . several lines of evidence show that proinflammatory cytokines act as triggers, or at least as contributors, of cancer-induced protein hypercatabolism [23] . briefly, data obtained in both experimental models and human pathology have demonstrated that cytokines such as tnfα and il-6 lead to reduced rates of protein synthesis paralleled by enhanced protein breakdown, both accounting for the loss of muscle mass [24] . such effects depend, at least in part, on activation of the transcription factor nf-κb, as shown in both experimental and human cancer cachexia patients [25, 26] . cancer-induced muscle wasting has also been associated with another proinflammatory cytokine, namely, tnf-like weak inducer of apoptosis (tweak) [27] . the therapeutic approaches mainly pursued by researchers to counteract enhanced muscle protein breakdown have long been those specifically targeting the different proteolytic systems. the results, however, did not really clarify the issue. since the discovery of muscle-specific ubiquitin ligases, these were considered a good target to interfere with protein breakdown, being involved in determining both substratespecificity and proteasome degradation rate. among the enzymes belonging to this family, the first described were mafbx/atrogin-1 and murf1/trim63, respectively, involved in the degradation of structural proteins and of proteins contributing to cell proliferation, differentiation, and survival [1] . subsequently, other members came out, such as trim32 and fbxo40. more recently, fbxo30/ musa1 was shown to contribute to denervation-and fasting-mediated muscle loss [28] , as well as to cancerinduced muscle wasting (unpublished data). genetic approaches specifically targeting these ubiquitin ligases proved effective in protecting the muscle against protein depletion [29] ; however, at present, the use of these enzymes as therapeutic targets for muscle wasting is not validated yet. on the other side, the inhibition of proteasome activity by means of pharmacological inhibitors was effective just in few models of muscle atrophy but totally unable to protect tumor-bearing animals against muscle wasting [30] . in contrast with these findings, few years ago, a study reported that inhibition of the ubiquitin-proteasome pathway by mg132 was able to improve experimental cancer cachexia [31] . however, mg132 is a rather unspecific inhibitor, being able to block also calpains and autophagy [31, 32] . finally, carfilzomib, an irreversible selective inhibitor of proteasome chymotrypsin-like activity, was shown to improve cachexia in tumor-bearing mice by inhibiting muscle protein breakdown [31] . such improvement, however, was associated with reduced tumor burden, which could be the real mechanism underlying the beneficial effect of the treatment. several lines of evidence proposed that modulations of autophagy could be useful to improve cancer-associated muscle wasting. in this regard, muscle-specific gene strategies aimed at silencing beclin-1, one of the proteins involved in autophagosome formation, showed that suppression of autophagy in the c26 hosts was unable to rescue myofiber diameter (unpublished data). in addition, pharmacological inhibition of autophagy in mice hosting the c26 tumor lead to death of the animals, suggesting that lysosomal degradation is mandatory to sustain the requirement of both energy and substrates in tumor hosts, at least when they are facing the terminal phase of cancer growth [15] . the other way round, excessive stimulation of muscle autophagy, experimentally obtained by the overexpression of tp53inp2/ dor, exacerbated muscle atrophy in tumor-bearing mice (unpublished data), while activation of autophagy by means of the mtor inhibitor rapamycin was shown to positively affect the skeletal muscle in the c26 hosts [16] . such discrepancy might depend on the fact that while mtor inhibition affects stress-induced autophagy, tp53inp2 hyperexpression targets basal autophagy. despite the literature report data supporting the involvement of ca 2+ -dependent proteolysis in the pathogenesis of cancer-induced muscle wasting, protein hypercatabolism was not downregulated in preparations of isolated muscles obtained from tumor-bearing animals and incubated in the presence of calpain inhibitors [19, 33, 34] . more recently, both pharmacological and genetic approaches aimed at inhibiting the ca 2+ -dependent proteolytic system were not able to prevent or delay cancer-induced muscle wasting [21] , although contrasting results were reported in this regard [35] . while interfering with specific proteolytic systems does not seem to be an appropriate approach to prevent/delay cancer-induced muscle wasting, the modulation of bulk protein turnover appears more promising. in this regard, anti-inflammatory approaches revealed able to improve muscle protein turnover in tumor-bearing mice [20] . more recently, administration of formoterol, a β 2 -adrenergic agonist, to tumor-bearing animals revealed able to reverse muscle wasting [36] . such an effect is mainly exerted by stimulating protein synthesis and inhibiting protein degradation rates. in particular, both the ubiquitin-proteasome and the autophagic-lysosomal proteolytic systems were downregulated in formoterol-receiving animals ( [24] ; unpublished data). at present, only one study evaluated the effectiveness of formoterol, combined with megestrol acetate, in cachectic cancer patients [37] . the results suggest that both muscle size and strength can be improved by the treatment, although more trials are needed to draw clear-cut evidence. 2.2. protein synthesis. as reported above, depending on the situation, reduced, normal, or even increased muscle protein synthesis rates were shown in cancer cachexia. due to the rapid development of cachexia, tumor-bearing animals frequently show reduced protein synthesis rates, although this is not a general finding. indeed, rats bearing the ah-130 hepatoma, that usually die about 10 days after tumor transplantation, showed muscle protein synthesis rates comparable to those of healthy animals [38] . the situation is more complex when studying human pathology. reduced muscle protein synthesis was reported many years ago in patients affected by different types of tumors [39] and more recently in prostate cancer patients [40] . on the contrary, van dijk et al. [41] reported that baseline protein synthesis rates were higher than control values in cachectic patients affected by pancreatic cancer. also, intermediate results are available in the literature: myofibrillar protein synthesis rates were analyzed in healthy people and weight-stable and weight-losing gastrointestinal cancer patients and found comparable among the different groups. similarly, no changes in whole body protein synthesis were reported in nsclc patients [42] . the possibility to modulate protein synthesis in order to correct muscle atrophy or simply to provide an environment permissive for the maintenance of muscle mass was long studied. many approaches were tested, most of them consisting in nutritional strategies or in molecular modulations aimed at pushing muscle metabolism towards anabolism. most of these approaches revealed unsuccessful, giving rise to the idea that anabolic resistance occurs in cancer cachexia. just to provide few examples, conventional nutritional supplementation or the infusion with an amino acid cocktail did not stimulate muscle protein synthesis in advanced cancer patients [42] . along this line, studies aimed at stimulating the anabolic pathway depending on igf-1, by both pharmacological and genetic means, did not succeed in improving muscle wasting in tumor-bearing animals [43, 44] . recently, however, patients not yet considered as refractory cachectic were proposed to display an anabolic window that could be exploited with nutritional interventions [42, 45, 46] or with other anabolism-inducing strategies. as an example, patients with stage iii and iv nsclc showed a normal anabolic response to hyperaminoacidemia but not to isoaminoacidemia, suggesting that high substrate availability is relevant to induce anabolism in cancer hosts [47] . consistently, muscle protein synthesis could be stimulated in advanced cancer patients by a high protein formula versus a conventional nutritional supplement [48, 49] . these observations point out the possibility to overcome the anabolic resistance that occurs in cancer patients by providing specifically enriched nutritional supplements. stimulation of anabolism can be exerted by several means. particularly interesting in this regard is ghrelin, a mediator released by the stomach during fasting or caloric restriction. modulations of ghrelin levels exert remarkable effects on both energy and protein metabolism, such as the inhibition of autophagy in conditions characterized by systemic inflammation [50] . the administration of ghrelin to tumor-bearing animals improved food intake, body weight, lean body mass, and chemotherapy-induced toxicity [51] . both ghrelin and ghrelin analogues are currently being tested in clinical trials. among these, anamorelin was recently shown to improve lean body mass, total body mass, and hand grip strength in patients affected by nsclc [52] . other studies, however, showed that anamorelin administration to cancer patients increased body weight and improved faact scores while did not enhance hand grip strength [53] . acids. in addition to be necessary to synthesize proteins, free amino acids (faa) also act as regulators of protein metabolism. in particular, plasma faa, that even represent a small fraction of the total amino acid pool, are the main source of metabolically active nitrogen compounds. among faa, the essential amino acids were reported to stimulate protein synthesis and to inhibit protein degradation. such a role is mainly played by the three branched chain amino acids (bcaa), leucine in particular. alterations of amino acid metabolism are frequent features in cancer-induced muscle wasting. reduced amino acid uptake was generally observed in cancer patients, mainly due to the occurrence of anorexia, which also leads to decreased insulin secretion. both decreased amino acid availability and insulin levels inhibit the anabolic pathway dependent on mtor, resulting in downregulation of protein synthesis rates and stimulating protein degradation. inhibition of mtor signaling in cancer cachexia is enforced by proinflammatory cytokines [1] . reduced amino acid uptake in the muscle was also reported to derive from altered amino acid transport. indeed, in the soleus muscle of tumor-bearing rats, the activity of system a was decreased, while no changes were observed for systems l and asc [54] . of interest, tnfα was shown to impair amino acid transport in tumor-bearing rats [55] . plasma glutamine levels were shown to be significantly reduced in tumor-bearing rats with respect to healthy animals [56] . of interest, reduced glutamine availability could activate the metabolic sensor adenosine monophosphateactivated protein kinase (ampk, see below; [57] ). finally, leucine oxidation was markedly increased in the muscle of rats bearing the yoshida ah-130 hepatoma [58] . consistently, enhanced activity of the bcaa dehydrogenase was reported in rats bearing the walker 256 carcinoma [59] . several studies have proposed amino acid supplementation as a mean to improve cancer-induced muscle wasting. in experimental models of cancer cachexia, bcaa were shown to attenuate the loss of muscle mass. the underlying mechanisms of such effect are not clear, although downregulation of protein degradation and stimulation of protein synthesis were hypothesized [60] . more recently, metabolomic alterations were proposed to be the basis of the positive effects exerted by a leucine-rich diet on cachexia in rats bearing the walker 256 carcinoma, in the absence of effects on tumor mass [61] . as for clinical studies, bcaa were proposed to improve anorexia [62] , thus removing, partially at least, one of the mechanisms accounting for reduced amino acid uptake. other studies supported a beneficial role of bcaas on muscle protein metabolism, although this should be confirmed by larger randomized, blind, placebocontrolled trials [63] . beta-hydroxy-beta-methylbutyrate (hmb), a metabolite of leucine, was shown to improve muscle wasting in experimental cancer cachexia, mainly by inhibiting protein degradation rather than stimulating protein synthesis [63] . recently, hmb was proposed to be more effective than leucine in preventing body weight loss in tumor-bearing animals [64] . such effect, however, could depend on the model system chosen, since hmb does not appear able to modulate muscle mass in mice bearing the c26 tumor (costelli et al., unpublished observations). the situation is even more confused in human cachexia. increase of both hemoglobin levels and fat-free mass were reported in advanced cancer patients administered a nutritional supplement containing hmb, arginine, and glutamine [65, 66] . another study, however, was not able to demonstrate a beneficial effect of the same supplement in cancer patients [67] , suggesting that the effectiveness of hmb in the clinical practice is still unclear and deserves further investigation. glutamine supplementation was reported to attenuate muscle protein wasting in cancer patients [68] , as well as to improve the energy balance in rats bearing the walker 256 tumor [69] . finally, promising data are available about the possibility to treat cancer hosts with l-carnitine, an amino acid derivative that plays a role in fatty acid metabolism and energy production [63] . a negative energy balance, generally resulting from both reduced production and increased expenditure, is a frequent occurrence in cancer patients. while resting energy expenditure (ree) frequently increases, likely due to enhanced thermogenesis, the occurrence of reduced physical activity, particularly in advanced cancer patients, paradoxically leads to a net decrease of total energy expenditure. the increased thermogenesis is consistent with the observation that in cachectic tumor-bearing animals, the expression of the brown adipose tissue-(bat-) specific uncoupling protein 1 (ucp1) is higher than in controls, while ucp2 (ubiquitous) and ucp3 (expressed in bat and muscle) levels increase in the skeletal muscle only [70] . similarly, muscle ucp3 mrna levels were higher in weight-losing than in non-weight-losing cancer patients or controls [71] . the increase of ree in cancer cachexia is not a new observation; however, just recently, the underlying mechanisms start to be clarified. a central point in this regard is played by muscle mitochondria compartment, which is markedly affected in tumor hosts. indeed, both morphological [72, 73] and functional alterations [74, 75] were reported in experimental tumor-bearing animals. in particular, mitochondrial uncoupling and reduced oxidative capacity were associated with myofiber shift from oxidative to glycolytic fibers [73] . impairment of the mitochondrial compartment results in decreased atp production, leading to an energy deficit that becomes even worse since it is coupled with steadily increased ree. consistently, reduced atp levels and increased activity of the energy sensor ampk were shown in the muscle of tumor-bearing animals [72, 73] . the lack of an appropriate energy supply results in compromised cell function and reduced contractile force generation, leading to loss of muscle mass and strength. several factors can lead to mitochondrial alterations in the skeletal muscle. among these, proinflammatory cytokines play a major role. indeed, the activation of nf-κb induced by tnfα was reported to reduce both muscle oxidative capacity and the expression of factors regulating mitochondrial biogenesis. similar observations were reported when other inflammation-driven pathways such as the il-6/stat3 or tgfβ/smad3 are activated above physiological levels [76] . in addition to proinflammatory mediators, also oxidative stress, due to reactive oxygen and nitrogen species levels exceeding the compensative capacity of the intracellular antioxidant systems, contributes to mitochondrial function impairment. in this regard, there are several studies sustaining the involvement of oxidative stress in cancerinduced muscle wasting, although a clear-cut causative evidence is still lacking [77] (ballarò et al., unpublished data; figure 2 ). being mitochondria crucial to the maintenance of muscle oxidative metabolism, emergency routes can be activated in order to avoid mitochondrial dysfunction. in particular, mitochondrial biogenesis and dynamics as well as the disposal of damaged organelles, mainly by mitophagy, are promoted ( figure 2 ). the other way round, impaired function of the emergency routes themselves could trigger the accumulation of altered mitochondria resulting in reduced muscle oxidative metabolism. in this regard, the expression of the peroxisome proliferator-activated receptor-γ (ppar-γ) coactivator-1α (pgc-1α), the master regulator of mitochondrial biogenesis and oxidative metabolism, was shown to be reduced in the skeletal muscle of tumorbearing mice [78] , although this is not a constant finding [73, 79] . mitochondria dynamics, representing the balance between fission and fusion processes, was shown to be altered in both experimental cancer cachexia and in cancer patients [78, 80] . in this regard, impaired mitochondrial dynamics could drive the hyperactivation of muscle protein breakdown, likely through pathways depending on ampk and foxo, eventually leading to muscle wasting [81, 82] . autophagy (mitophagy) is the main mechanism responsible for disposal of altered mitochondria. also, mitophagy was reported to be impaired in cancer cachexia, as shown by the observation that bnip3l and parkin1 mrna increased in the muscle of cancer patients [17] . similarly, bnip3l protein levels were increased in the muscle of mice bearing the lewis lung carcinoma [73] . on the whole, these observations suggest that in addition to mitochondria biogenesis and dynamics, also their disposal is perturbed in the skeletal muscle of tumor hosts, thus contributing to mitochondrial dysfunction and reduced muscle oxidative metabolism. several strategies were proposed to improve energy metabolism by acting on mitochondria. the first and perhaps simplest one, in theory at least, is exercise training, in particular a combination of both resistance and endurance exercise. these two types of training affect different but complementary targets, being able to improve force production and metabolic adaptations, respectively. of particular relevance is the observation that endurance training was reported to increase the number of mitochondria and to drive myofiber-type shift from glycolytic to oxidative, thus specifically targeting alterations that characteristically occur in the skeletal muscle of tumor hosts. however, these potentially favorable effects can be exploited just systematically practicing exercise, even if at a moderate level. this may not be an easy task in cancer patients that frequently present with chronic fatigue and comorbidities eventually leading to exercise intolerance. this point is supported by the observation that c26-bearing mice did not benefit from exercise training, suggesting that the effort to exercise in already compromised animals was damaging rather than protective [73] . consistently, excessive endurance exercise was associated with increased mitochondrial fission in the absence of mitophagy induction [83] . in the last years, the possibility to mimic the effects of exercise by drugs has been gaining a growing consensus. the positive side is that this strategy will allow to overcome both poor patient compliance to exercise training and possible occurrence of exercise intolerance. the negative part is that generally exercise mimicking drugs do not totally recapitulate the effects of exercise itself. in this sense, these drugs do not properly hit the target; however, they could be a good compromise when exercise training cannot be proposed to the patient. at present, several options were investigated as exercisemimicking strategies. while most of them are pharmacological, also a genetic approach was proposed. this latter consists in manipulations able to increase the levels of pgc-1α in the skeletal muscle. in this regard, improved exercise capacity and oxidative metabolism were reported in mice specifically overexpressing this factor in the muscle, resembling the phenotype induced by endurance training [84] . pgc-1α overexpression was shown to interfere with muscle atrophy induced by activation of the tweak-fn14 pathway [85] and to improve cancer-induced muscle wasting in tumor-bearing mice [73, 86] , although contrasting data were previously reported [87] . several classes of drugs were proposed to modulate energy metabolism, among which are activators of ampk, sirtuin 1 (sirt1), and trimetazidine (tmz). different compounds such as resveratrol, metformin, quercetin, and aicar can activate ampk [88] . in this regard, aicar administration was shown to impinge on exercise capacity, oxygen consumption, and fatty acid oxidation [89] . muscle atrophy induced by angiotensin ii was prevented by treatment with aicar. this drug also revealed able to activate autophagy and to improve muscle phenotype in both dystrophic mdx mice and animals bearing the c26 tumor [16, 90] . metformin administration was shown to improve sarcopenia of aging and muscle wasting in severely burned patients [91, 92] and was proposed to be useful to treat muscle wasting in cancer cachexia [93] . ampk activation can also be induced by resveratrol, as demonstrated by the observation that ampkα 1 -or ampkα 2 -deficient mice were refractory to resveratrol-induced increase of both mitochondrial biogenesis and endurance exercise capacity [94] . consistently, obese men receiving resveratrol showed improved inflammation, ampk activation, and increased expression of pgc-1α and sirt1 protein levels [95] . finally, an ampk-stabilizing peptide was reported to improve white adipose tissue wasting in tumor hosts [96] . sirt1 belongs to a class of deacetylases deregulated in aging and in different chronic diseases, including cancer. sirt1 is also involved in the regulation of energy homeostasis; its expression is induced in response to caloric restriction [97] and can be activated in the skeletal muscle by ampk [98] . specific overexpression of sirt1 in the muscle resulted in a fast-to-slow myofiber type transition, producing an oxidative phenotype. consistently, muscle-specific sirt1 transgenic mice exposed to fasting or denervation showed a reduced expression of atrogenes in comparison with wildtype littermates [99] . finally, improved muscle phenotype was reported in mdx/sirt1 double transgenic mice [100] . in addition to ampk, resveratrol also activates sirt1. in this regard, part of the above-described effects exerted by resveratrol derive from sirt1-dependent modulations of pgc-1α acetylation state [101] . synthetic selective sirt1 activators such as srt2104 are also available [102, 103] . plasma lipid profile and insulin sensitivity were improved in healthy volunteers by administration of srt2104 [102, 104] . very few studies are actually available about srt2104 action on both muscle mass and function. in this regard, srt2104 appeared to reduce the depletion of muscle mass due to fasting or inactivity [105] , at least in part by increasing pgc-1α expression [105] . tmz is a metabolic modulator that blocks fatty acid oxidation, shifting atp production to glucose oxidation and improving cell energy metabolism. indeed, atp synthesis through fatty acid β-oxidation requires more oxygen than glucose oxidation [106] . along this line, the shift to glucose oxidation improves the use of the available oxygen, possibly increasing metabolic efficiency and skeletal muscle function. tmz was shown to increase the size of cultured myotubes [107] and to improve both heart metabolism and exercise capacity in patients suffering from chronic stable angina [108] . when administered to aged animals, tmz resulted in increased muscle strength [109] . finally, treatment of c26-bearing mice with tmz resembled some of the benefits triggered by exercise, among which fast-to-slow myofiber phenotype shift, pgc-1α upregulation, oxidative metabolism enhancement, and grip strength increase (molinari et al., unpublished). the relevance of fatty acid oxidation to cachexia is also supported by a recent study showing that several tumor cell lines are able to release proinflammatory mediators, resulting in enhanced fatty acid oxidation and activation of p38-dependent signaling in the skeletal muscle, well before tissue wasting occurs. in addition, the same study also showed that treatment of tumor-bearing animals with etomoxir, an inhibitor of fatty acid oxidation, rescued both muscle mass and body weight [110] . a complex network of metabolic alterations sustained by hypercatabolism, energy deficit, and systemic inflammation is the milieu underlying cancer cachexia. while becoming overtly detectable in advanced cancer patients, such perturbations likely take place very early in the course of the disease, at least at the molecular level. protein and energy dysmetabolism in cachexia are quite well recognized; however, the available therapeutic strategies, although frequently promising from the preclinical point of view, have not yet reached validation to be used in the clinical practice. several drugs identified by experimental studies are currently tested in clinical trials for their ability to improve muscle metabolism in cancer patients. other emerging strategies are those aimed at interfering with the intestinal microbiota, previously reported to improve cachexia in a preclinical model [111] . the available results of both experimental and clinical studies, however, have clearly indicated that single-targeted therapies will hardly be successful in the treatment of cachexia. in this regard, the view of a multidirectional approach, selectively tailored and, whenever possible, personalized, is gaining a growing consensus. such an approach should not just rely on nutritional counseling and pharmacologic treatment with anti-inflammatory and anticatabolic drugs but also include exercise training and/or exercisemimicking agents. in this regard, exercise mimetics could not only merely replace exercise training in depleted patients but also improve exercise tolerance and effectiveness in precachectic individuals, thus amplifying the beneficial action of exercise itself. last but not least, treatments aimed at preventing/correcting the metabolic alterations underlying cancer-induced muscle wasting might also impinge on tumor-targeted therapies improving their effectiveness and/ or enhancing patient tolerance to chemotherapy. in addition, metabolic modulators could also directly affect tumor growth. this is the case, for example, of exercise, that was shown to prevent or at least delay tumor growth [112] . cancer cachexia: understanding the molecular basis definition and classification of 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treatment the authors declare that there is no conflict of interest regarding the publication of this paper. key: cord-273019-hbpfz8rt authors: glingston, r. sahaya; deb, rachayeeta; kumar, sachin; nagotu, shirisha title: organelle dynamics and viral infections: at cross roads date: 2018-06-25 journal: microbes infect doi: 10.1016/j.micinf.2018.06.002 sha: doc_id: 273019 cord_uid: hbpfz8rt viruses are obligate intracellular parasites of the host cells. a commonly accepted view is the requirement of internal membranous structures for various aspects of viral life cycle. organelles enable favourable intracellular environment for several viruses. however, studies reporting organelle dynamics upon viral infections are scant. in this review, we aim to summarize and highlight modulations caused to various organelles upon viral infection or expression of its proteins. a unique feature of eukaryotic cells is the presence of distinct membrane bound structures called organelles. this subcompartmentalization of eukaryotic cells is very essential for its optimum function. organelles achieve this due to the presence of a unique set of proteins and distinctive lipid composition that determines their function. they are dynamic and interact with the surrounding organelles to regulate their biogenesis and function [1, 2] . association of organelle dysfunction with human diseases/ disorders is reported and widely acknowledged [3] . it is interesting that not only organelles are required for proper functioning of cells, but are also required for the successful infection of a virus [4, 5] . it is well established that viruses develop alternate strategies to survive in the cells. the steps of the viral life cycle include entry, translation, replication, assembly and egress [6] . moreover, viruses have developed remarkable ways to complete their life cycle by targeting specific cell organelles and processes. organelles like mitochondria, er, peroxisomes play an important role in innate immunity and host defence [7] . recently lipid droplets have also been reported to be essential for innate response against viral infection [8] . the viral life cycle is also a dynamic process that leads to the extensive host cellular reorganization. characterization of this spatiotemporal reorganization is a key step in order to understand the molecular mechanism of viral infection. localization of the viral proteins to an appropriate sub-cellular compartment is part of the strategy to hijack the host machinery and necessary pathways leading to establishment of an infection [9, 10] . with the advent of several modern microscopy and proteomics methods, it is now clear that the localization and function of many host proteins are altered due to viral infections. not only the host proteins but the intracellular localization of the viral proteins and their interactions with the host proteins are also extensively studied using these methods [10] . however, currently our understanding of organelle dynamics during viral infections is still at its infancy. current review briefly summarizes our knowledge of the various cell organelles/compartments following virus infection. this topic at the interjection of virology and cell biology represents an emerging research area of molecular investigations in virology. presence of nucleus that houses the genome is what distinguishes eukaryotic cells from prokaryotes (fig. 1 ). it is a double membrane organelle and is the site for gene regulation and transcription. nuclear envelope comprising of nuclear pores is the barrier between the nuclear content and the rest of the cell. nucleolus is the region where ribosomal subunit assembly takes place in the nucleus. sub nuclear structures comprising of several proteins which regulate many cellular processes like apoptosis, dna damage, etc are called pml-nbs. many dna and rna viruses depend on the host nuclear proteins for their replication [11] . several strategies are used by viruses in order to deliver its genome to the host cells [12] . usually, when cells undergo mitosis, there is a temporary disassembly of the nuclear envelope (ne) which allows some viruses to enter into the nucleus [13] . entry and integration of murine leukemia virus (mlv) into host nucleus depends on the ne breakdown during mitosis [14] . various human immunodeficiency virus (hiv) preintegration complex proteins, such as the matrix [15] , vpr [16] , integrase [17, 18] were found to contain nuclear localization signal (nls) in their genome sequence. this nls interacts with the nuclear transport receptors, which transports the viral proteins through the nuclear pore complex (npc). similarly, the influenza virus nucleoprotein (np) present in the viral ribonucleoprotein complex (vrnp) contains nls1, nls2 and nls3, which helps in its binding to cellular importins resulting in transportation of the vrnp to the nucleus [19e21] . in another strategy, the capsid of certain viruses gets attached to the cytoplasmic side of the host npc either directly or with the help of importins. this interaction acts as a signal for capsid disassembly followed by entry of the viral genome along with their proteins through the npc into the nucleus [12] . studies on the herpes simplex virus-1 (hsv-1) infection on vero, bhk-21 and ptk 2 cells reported transportation of viral tegument-capsid by dynein to the cytoplasmic side of npc [22, 23] . the hsv-1 capsid binds to npc with the help of importin b and subsequently releases dna into the nucleus through the npc [24] . similarly, the capsid protein of adenovirus (ad) on binding with the nuclear transport protein nup214 attaches to the cytoplasmic side of npc and releases the genetic material into the nucleus [25] . some small viruses such as hepatitis b virus (hbv), baculovirus, etc were found to cross the npc and release their genome at the nuclear side of npc or within the nucleus [12] . the capsid protein of hbv was reported to interact with npc via the nuclear receptor nup153 in xenopus laevis oocytes [26] . furthermore, this binding depends on importin a, b and phosphorylated core protein present on its capsid [27] . upon infection, hbv capsid enters the nucleus through the npc and subsequently releases its genome into the nucleoplasm [28] . similarly, capsid proteins of simian virus 40 (sv40) were found to enter the nucleus through npc [29] . it has been reported that the disruption of the ne and nuclear lamina temporarily aid the nuclear entry of viruses such as parvoviruses [30] . disruption of ne in xenopus oocytes and both ne and nuclear lamina in mouse fibroblast cells upon parvovirus infection and minute virus of mice (mvm) was reported using electron microscopy analysis [30, 31] . presumably, viruses evolved different mechanisms to enter nucleus in order to facilitate their replication cycle and survivability inside the host cells. after the successful entry into the nucleus, both dna and rna viruses exploit various nuclear components such as nucleolus, promyelocytic leukaemia nuclear body (pln) and/or nuclear proteins in order to facilitate their replication (fig. 2) [11] . nucleolin, fibrillarin and b23 (nucleophosmin) are examples of nucleolar proteins reported essential for various functions like post transcriptional process, ribosome assembly, etc [32] . these multifunctional host nucleolar proteins were reported to be incorporated into the replication and translation complex of many viruses [11] . many dna viruses like hsv and ad were reported to be involved in relocalization or disruption of the host nucleolar proteins [11] . for instance, the transfection of the recombinant ul24 protein of hsv-1, tagged with an n-terminal hemagglutinin in vero cells resulted in the redistribution of nucleolin and b23 [33, 34] . another nucleolar protein fibrillarin was also found to be redistributed in spots throughout the nucleus but in an ul24 independent manner [33] . the core protein of ad was found to be associated with the nucleolus in hela cells [35] . further studies showed that this association results in the relocation of the nucleolin to the cytoplasm of the infected cells [36] . the ad-induced relocation of nucleolin was proposed to be a strategy used by virus to suppress its activity [36] . additionally, infection of hela cells with ad resulted in inhibition of synthesis and maturation of rrna [37] . nucleolar localization of the viral capsid and rna binding proteins of many rna viruses has been reported [11] . for example, the encephalomyocarditis viral (emcv) proteins 2a and 3bcd and human rhinovirus hrv 3c protease were found to localize to the nucleoli resulting in inhibition of cellular rna transcription [38, 39] . it has been reported that the expression of tat and rev proteins of hiv-1 in cos7 cells resulted in their accumulation in dense fibrillar component in the nucleolus [40] . moreover, rev protein was reported to be involved in deforming the nucleolar architecture [40] . similarly, the viral n protein was found to be localized to the nucleolus in addition to the cytoplasm upon infection of coronavirus resulting in delayed cell cycle to facilitate viral assembly [41] . poliovirus (pv) or rhinoviruses interact with nucleolin and blocks the nuclear import leading to accumulation of nucleolin in the cytoplasm of the infected cells [42] . the accumulated nucleolin interacts with the internal ribosome entry site (ires) element present in the upstream 5 0 end of the viral genome to stimulate its translation [43] . on the other hand, these changes result in the shutdown of cellular transcription leading to the downregulation of the host defence mechanism [42] . infection of human respiratory syncytial virus (hrsv) in a549 cells resulted in depletion of nucleolin [44] . although, both dna and rna viruses behave differently towards acquiring the nuclear niche however the goal being to establish control over cellular transcription and favour its genome over the host. pml nb is another subnucleolar component, which contains proteins such as pml and sp100. these proteins are induced by interferons and hence pml nb can act as a target for viruses to escape the antiviral signalling response [45] . redistribution of the pml nb has been reported upon infection of hsv-1 to bhk-21 cells [46] . similarly, ebna lp protein of epstein-barr virus (ebv) was found to displace sp100 from pml nbs in burkitt's lymphoma and hep2 cells [47] . the viral e4-orf3 protein induced reorganization of pml nbs into elongated track like structures upon infection of cv1 cells with human ad5 [48] . later, it was found that this reorganization was responsible for the downregulation of the host antiviral response [49] . another example is the infection of human foreskin fibroblasts (hffs) with human cytomegalovirus (hcmv) resulting in the accumulation of pp71 at pml nbs in the infected cells [50] . further studies showed that pp71 was responsible for the proteasomal degradation of pml-nb protein daxx, which is important for inducing intrinsic immune response against hcmv infection [50] . some rna viruses also result in the redistribution and degradation of host pml nbs in order to neutralize the antiviral response. the ring finger protein z of lymphocytic choriomeningitis virus interacts with pml protein and leads to the redistribution of pml-nbs from the nucleolus to the cytoplasm [51] . the expression of 3c protease of emcv in cho cells was reported to target pml-nbs and promote its degradation in proteasome and by sumo-dependent mechanism [52] . interestingly, cho cells infected with rabies virus were reported to contain enlarged pml nbs [53] . disruption of the nucleocytoplasmic trafficking in the host cells is another major alteration caused due to viral infections ( fig. 2 ) [54] . two conserved proteins pul50 and pul53 of hcmv were reported to remodel the nuclear lamina of helfs (primary human lung fibroblasts) for the budding of virions [55] . interaction between human papillomavirus (hpv) and host mitotic chromosomes has been documented [56] . infection of porcine bone marrow cells with african swine fever virus (afsv) resulted in fragmentation of the nucleus [57] . the organelle found in continuation with the nucleus in a cell is the er (fig. 1) . protein synthesis and folding (rough er), lipid synthesis, calcium regulation (smooth er), etc are some of the important functions of the er. multiple structural domains of the er are reported which enable it to serve as a site for various important functions in a cell. several morphological alterations of er such as vesicle formation, invagination of the membrane, zippered appearance, etc have been reported upon viral infection in different cell lines (fig. 2) . the large malleable surface of the er aids viruses to form protective compartments to set up their replication machinery. these compartments known as viroplasm not only concentrate viral and host proteins required for viral genome replication but also protect the viral genome from cellular nucleases [58] . in order to construct these compartments, viruses alter host's fatty acid metabolism, induce rearrangement of the membrane constituents and also recruit cellular machinery to produce proteins essential for its replication [59, 60] . infection of vaccinia virus (vacv) in bs-c-1, hela and rk-13 cells leads to the formation of vacv membranes derived from the er membrane [61] . in case of dengue virus (denv-2), the genomic rna was observed to localize over the rough er in c6/36 aedes albopictus cells [62] . denv infection in human hepatoma 7 (huh7) cells leads to invagination of er membrane resulting in the formation of spherules or vesicles containing double stranded rna [63] . hela cells and cos-1 cells on infection with pv 1 cause formation of single membrane tubules which later mature into double membrane vesicles (dmvs) [64, 65] . similarly, severe acute respiratory syndrome corona virus (sars-cov) induced formation of er derived dmvs upon infection of vero cells [66, 67] . zippered appearance of er was observed upon infection of infectious bronchitis virus (ibv) on mammalian, avian and tracheal epithelial cells [68] . studies with the plant viruses like the potato virus x (pvx) reported tgbp2 protein induced reorganization of the er and appearance of vesicular structures in tobacco plants [69] . effect of the viral protein expression on sterol biosynthesis apart from the alterations in er morphology have also been reported [59, 70] . several viral proteins need to be glycosylated at their n-terminal to ensure proper folding and for the incorporation into virions [71] . hence, a common phenomenon observed upon viral infection is interference with the host post translational machinery and competition with the cellular proteins to undergo these modifications [72] . for example, protein porf2 of hepatitis e virus (hev) gets glycosylated in the er upon its expression in cos-1 cells and huh7 mammalian cells [73, 74] . this increased biosynthetic load is proposed to increase in accumulation of malformed proteins resulting in er stress (fig. 2) . er gets relieved from this stress by inducing a pathway called unfolded protein response (upr) pathway [72] . upr serves to enhance the cell's degradation ability in order to establish er homeostasis [72] . transport of misfolded, misassembled proteins from the er to the cytosol and clearance by the ubiquitin proteasome system takes place via the er associated degradation (erad) pathway [72] . however, some viruses use erad pathway for their advantage by co-opting erad to disassemble and gain access to the cytosol of the host cell [75] . upon sv40 infection of cv-1, hela and 3t6 cells, induction of erad factors in turn induce er membrane reorganization into distinct subdomains called foci and the accumulation of sv40 in these foci was observed [76] . later, sv40 particles travel across the er membrane to reach the cytosol [76] . many enveloped and non-enveloped viruses that exploit er functions like upr, erad to facilitate their replication have been discussed elsewhere [71] . in addition to the above listed mechanisms, er mediated nlinked glycosylation plays a very important role in the survival of the viruses. it has been very elegantly demonstrated that the biogenesis of influenza could modulate the host immune response [77] . similarly, the role of n-glycans in the host immunity against hiv has been documented [77] . these studies corroborate to a common point where the level of glycosylation in the viral surface glycoproteins could alter their antigenicity. mitochondria are double membrane bound organelles that comprise their own genome ( fig. 1 ). they are involved in various functions like fatty acid metabolism, apoptosis, calcium homeostasis, etc. considered evolutionarily the oldest organelle of a eukaryotic cell, they are indispensable as power house of the cell. mitochondrial morphology is maintained by a series of interlinked events namely mitochondrial fusion, fission and mitophagy [78] . mitochondrial fusion helps in exchanging matrix metabolites and intact mitochondrial dna while mitochondrial fission helps in sorting impaired mitochondria from the healthy population which are further eliminated by a process called mitophagy [79] . all the above dynamic events are altered upon viral infection in order to facilitate its replication (fig. 2) [80] . for example, it was reported that hbv infection in the cell induces mitochondrial fission followed by mitophagy in order to attenuate apoptosis [81] . on the other hand, expression of orf-9b protein of sars-cov virus promoted mitochondrial fusion in hek293 cells [82] . upregulation of mitophagy and degradation of the mitochondrial antiviral signalling protein (mavs) in order to attenuate the antiviral immune response in non-small cell lung cancer (nsclc) cells was reported upon measles virus infection [83] . the expression of matrix protein (m) of human parainfluenza virus type 3 (hpiv3) in hek293t and hela cells was reported to induce mitophagy resulting in the suppression of type1 interferon response [84] . hbv induces mitophagosome formation which upon fusion with lysosomes leads to mitophagy and prevents apoptosis, thus facilitating persistent infection in the huh7 cells [81] . similarly, newcastle disease virus (ndv) was reported to induce mitophagy, which promotes ndv replication by preventing caspase dependent apoptosis in human non-small cell lung cancer a549 cells [85] . viruses can alter the intracellular distribution of mitochondria either to prevent the release of mediators of apoptosis or to meet their energy requirement during replication by concentrating them near their viral factories [86] . hbv x protein induces the perinuclear clustering of mitochondria in huh7 cells [87] and afsv leads to the transport of mitochondria to viral assembly sites in vero cells [88] . mitochondrial dna codes for proteins essential for respiratory functions of a cell. certain viruses evade the mitochondria associated antiviral response by damaging the host cell mitochondrial dna, which is essential for synthesizing enzymes for optimum mitochondrial function [86] . for example, mitochondrial dna degradation is induced in mammalian cells upon expression of ul12.5, an amino-terminally truncated ul12 isoform of hsv-1 [89] . raji cell lines infected with hepatitis c virus (hcv) also exhibit mitochondrial dna depletion [90] . maintenance of mitochondrial/cellular ca 2ã¾ homeostasis is vital for various cellular functions. many viruses are involved in altering the mitochondrial calcium homeostasis in order to meet their needs during replication [86] . hcmv upon infection causes calcium influx into mitochondria from er [91] . on the other hand, expression of 2b protein of coxsackievirus resulted in reduced signalling of ca 2ã¾ between the er-golgi and the mitochondria in hela cells resulting in suppression of apoptosis [92] . rotavirus was also reported to alter the calcium homeostasis in the host cell throughout its life cycle [93] . mitochondria are the major source of ros in a cell and a balance between ros production and scavenging is crucial for optimum functioning of the cells. upon viral infection mitochondria undergo oxidative stress and result in an increased production of ros which in turn reduces virus replication [86] . interestingly, ros is also involved in activating many host cellular pathways favourable for viral replication and pathogenesis. several viruses like hiv, hcv, adv, ebv, etc result in increased oxidative stress upon infection [86] . on the other hand, both increase and decrease of oxidative stress is employed as a survival strategy by hbv [94, 95] . many viral proteins reach mitochondria through the mitochondria-associated membrane [96] a sub-compartment of the er or are targeted directly from the cytosol and result in an altered mitochondrial permeability transition pore (mptp) [97] . mptp is also responsible for the maintenance of mmp and provides energy for atp synthesis. altering mptp leads to passive swelling, outer membrane rupture, osmotic water flux, and release of proapoptotic factors leading to cell death [98] . in general, increased mmp induces apoptosis, while decreased mmp prevents apoptosis [86] . viruses are proposed to decrease mmp to prevent cell death in order to promote their replication. however, in later stages, they may trigger an increase in mmp to release the progeny virions by apoptosis [86] . the m11l protein of myxoma pox virus prevents the loss of mmp in cos-7 monkey kidney cells, hela cells, thp-1 human monocytes and jurkat t lymphocytes [99] . on the other side, the r protein of hiv-1 induces the loss of mmp in cem-c7 and jurkat cells and results in apoptosis [100] . viruses alter the host mitochondrial metabolic pathways in order to maintain cellular energy homeostasis essential to ensure efficient replication and to avoid mitochondrial antiviral response particularly in case of slow replicating virus [101] . some viruses modulate the normal cells to increase aerobic glycolysis and use glucose biosynthetically, which helps especially enveloped viruses to increase their available pool of fatty acids, lipids and nucleotides during their replication [102, 103] the feline leukemia virus infection on lung fibroblasts (flf-3) resulted in a 30e40% increase in glucose uptake and lactic acid production [104] . an increase in the lactic acid production upon infection of the chick embryo cells with rous sarcoma virus was reported [105] . hcmv upon infection of hffs, human retinal pigment epithelial cells (arpe19), human embryonic lung fibroblasts (mrc5) and vero cells enhances glycolytic flux and directs the supply of carbon from glucose to tca cycle. this helps to facilitate fatty acid biosynthesis while hsv-1 upon infection of same cell lines directs the central carbon metabolism in order to induce the production of pyrimidine nucleotide components [106] . hcv core protein suppresses mitochondrial complex 1 activity and impaired function of electron transport cycle leading to ros accumulation in hepatoma cells of transgenic mice [107] . mitochondrial involvement in virus survival is quite relevant to the fact that viruses require a source of energy to favour the active processes involved in their life cycle. peroxisomes are single membrane bound dynamic organelles required for b-oxidation of fatty acids and ros metabolism (fig. 1) . they have developed diverse functions which are organism and environment dependent. they are unique with respect to proliferation, as they can increase in number by both growth and division of pre-existing organelles and formation of new organelles from the er. many viruses or viral proteins are reported to localize to peroxisomes and/or exploit their functions to facilitate their replication in the host cells [108] . presence of a peroxisome targeting signal (pts) in the protein sequence is reported to be essential for targeting proteins into the peroxisomes [109] . however, it was also proposed that viral proteins without pts may get associated with host peroxisomal proteins in the cytosol which then ferry the viral protein into the peroxisomes in a piggyback fashion [108] . for instance, hcmv encodes a protein called vmia reported to be localized to peroxisomes in hffs and hepg2 cells [110] . the vmia interaction with the host pex19 protein aids the viral protein localization to the peroxisomes. fragmentation of peroxisomes upon vmia expression in hepg2 was also reported [110] . in addition, peroxisomal localization of two cymbidium ring spot viral proteins p33 and p92 was reported in yeast [111] . a role for peroxisomes in the replication of tbsv has also been reported. the viral replication protein p33 interacts with the host peroxisomal protein pex19 for its targeting to the peroxisomal membrane and subsequent replication [112, 113] . the host hsp70 protein was reported to promote the localization of the viral replication proteins to the peroxisomes in yeast cells [114] . n-terminal protease n pro of pestivirus is another example of a viral protein that is associated with peroxisomes and facilitates its survival and replication [115] . some members of the family tombusviridae are involved in remodelling the peroxisomal membrane resulting in the formation of vesicular structures called "profoundly modified peroxisomes" or "peroxisome derived multivesicular bodies" [108] . for example, the infection of cymbidium ring spot virus resulted in the formation of small vesicles at the periphery of the peroxisomes in plant leaf tissues [116] . at the later stage of infection, the entire peroxisomal matrix is occupied by these vesicles [116] . similarly, the cucumber necrosis virus (cnv) induces the formation of peroxisomal vesicles in which the viral rna replication occurs in yeast cells [117] . studies revealed upregulation of peroxisomal biogenesis in endothelial cells upon latent infection of kaposi's sarcoma associated herpes virus [118] . it was also reported that proteins involved in peroxisomal lipid metabolism were essential for the survival of latently infected human dermal microvascular endothelial cells and lymphatic endothelial cells [118] . interestingly, hiv infection reduces the number of peroxisomes in infected cells due to upregulation in the levels of micrornas that inhibit production of peroxisome biogenesis factors [119] . it has been reported that the west nile virus (wnv) and denv infection on a549 and hek293t cells result in the degradation of peroxisomes [120] . the capsid proteins of both denv and wnv were reported to interact with the peroxisomal protein pex19 required for peroxisome biogenesis. degradation and redistribution of pex19 from perinuclear region to juxtanuclear region was reported in the infected cells. in addition, reduced level of the antioxidant enzyme catalase was observed in the infected cells. these alterations resulted in an impairment of early antiviral signalling of the peroxisomes. expression of the pts containing vp4 protein of the rotavirus, in ma104 cell lines resulted in peroxisomal localization [121] . the role for such a localization was speculated to utilize the peroxisomal lipid metabolism for the supply of cholesterol for lipid raft synthesis, required for the viral replication. peroxisomal b-oxidation metabolism leads to the production of myristoyl-coa by shortening the fatty acids chain which could be exported to the cytosol for nmyristoylation of the viral proteins vp2 and vp6 of rotavirus [108] . studies on hiv suggested an interaction between viral nef protein and the peroxisomal enzyme thio-esterase using yeast two hybrid system [122] . further studies reported an increase in the enzymatic activity of human acyl-coa thio-esterase 3 on binding with the nef protein [123] . the increased activity of the peroxisomal enzyme was speculated to contribute in alteration of the subcellular morphology and in downregulation of the host antiviral response [108] . extensive modifications of proteins for proper functioning and targeting in a cell takes place at the golgi apparatus. other functions of golgi inevitable for the cell are carbohydrate synthesis and lipid transport. the unique stacked structural organization of the golgi is essential for these functions (fig. 1) . the golgi apparatus is composed of three regions namely cis golgi network (cgn), medial and trans golgi network (tgn). various viruses and viral proteins have been identified to localize to these golgi regions during their life cycle. for example, hela cells upon infection with adeno-associated virus type 5 (aav-5) led to an accumulation of the viral particles in the tgn and in the golginetwork associated coated vesicles [124] . in sars-cov, the transmembrane domain of the orf7b protein contains the retention signal required for accumulation of the protein in the cgn and tgn [125] . reports suggest bunyaviruses assemble in the golgi as a result of its retention signal in the glycoprotein (gn) of the virus [82, 126] . fragmentation of golgi body is a common phenomenon that occurs as a result of various viral infections (fig. 2) . orf virus (a parapox virus) causes disruption and fragmentation of golgi in vero cells. this structural modification affects the late vesicular export machinery and results in downregulation of the host immune response [127] . similar phenomenon was also reported upon expression of 3c protease of foot-and-mouth disease virus in vero cells [128] . infection of hrv1a on wi-38, 293t, and h1-hela cells was reported to induce fragmentation of golgi body, rearrangement of the golgi membranes into vesicles which are utilized as sites of rna replication [129] . another incidence of the virus induced golgi body fragmentation was observed upon infection of sars-cov that resulted in death of vero cells [130] . many rna viruses were also found to alter the integrity of golgi complex of the host cells. for example, the expression of n-terminal non-structural protein of norwalk virus in crandell-rees feline kidney (crfk) and hela cells co-localizes to golgi complex and induces its disassembly into discrete aggregates [131] . another example is the pv infection on vero cells which results in complete disruption of the cgn into fragments scattered throughout the cytoplasm. the expression of pv protein 2b in vero, normal rat kidney (nrk) and cos-7 cells also resulted in golgi bodies disassembly [132] . similarly, expression of protein 3a of avian encephalomyelitis virus (aev) in chick embryo brain (ceb) cells and cos-7 cells resulted in depletion of golgi stacks and in severe disassembly of golgi bodies [133] . an interesting alteration in the morphology of golgi apparatus was observed upon infection of turnip mosaic virus (tumv) on n. benthamiana plant. tumv infection was observed to induce amalgamation of golgi apparatus, er and chloroplast [134] . mccoy mouse fibroblast cells led to accumulation of golgin-97 a tgn resident protein in the viral factories [135] . similarly, 3a protein of some picornaviruses were also found to interact with a golgi apparatus resident protein namely golgi adaptor protein acyl coenzyme a (acyl-coa) binding domain protein 3 (acbd3/gpc60), acts as an adaptor to recruit phosphatidylinositol 4-kinase class iii beta (pi4kiii) in infected cells [136] . evidence shows that the tomato spotted wilt virus (tswv) glycoprotein gn localizes to golgi membranes and induces deformation of the membranes into pseudo-circular and pleomorphic structures in tobacco plant cells [137] . upon infection of hela cells, rabbit kidney cells and mouse monocytes-macrophages cell lines with vacv, viral progeny becomes enwrapped in the membrane derived from tgn cisterna to form the enveloped virus [138] . several viral proteins localize to the golgi body and mature by undergoing post translational modifications such as glycosylation, phosphorylation, etc. rubella virus was reported to undergo a golgi dependent maturation upon infection of bhk-21 and vero cells [139] . the inner tegument protein pul37 of the dna virus hsv was identified to be responsible in directing the viral capsids to the tgn in order to undergo secondary envelopment in different cell lines [140] . the glycoproteins of bunyamwera virus undergo primary maturation by modifying their sugar composition in the tgn upon infection of the bhk-21 and vero cells [141] . lipid droplets are single membrane bound organelles with a lipid core that primarily consists of neutral lipids like triacylglycerols (fig. 1) . they are essential for lipid storage and metabolism in a cell. these stored lipids can be used to generate and maintain energy homeostasis and hence they are central to the cellular function. lipid droplets are dynamic intracellular organelles which are required for storing lipids in a cell. they play a major role in energy homeostasis and membrane trafficking [142] . many rna viruses exploit this energy storing capacity of lipid droplets to facilitate their replication [142] . upon expression, various viral proteins are reported to localize to the lipid droplets [143] . for example, upon hcv infection, the hcv ns5a protein localizes to the surface of the lipid droplets with the help of a host factor diacylglycerol acyltransferase 1 (dgat1) in huh7 and hek293t cells [143] . another study reported that hcv core 3a protein is involved in downregulating the expression of phosphoinositide 3-kinase (pi3k)/phosphatase and tensin (pten) which in turn induces the accumulation of enlarged lipid droplets in human huh7 and hepg2 cells [144] . in another example, denv c protein was reported to bind and interact with perilipin 3 a protein present on the surface of the lipid droplets in hepg2 cell lines [145] . a similar localization of denv c protein was also observed upon infection of denv2 in bhk-21, hepg2, and c6/36 ht mosquito cells of a. albopictus [146] . additionally, the denv c protein localization on lipid droplets was also found to be essential for the denv2 replication [146] . denv induces autophagy and results in a reduction in lipid droplet area in huh7.5 cells, a subline derived from huh7 cells [147, 148] . further analysis of the infected cells showed reduced levels of triglycerides in the lipid droplets and suggests that atp generation by b-oxidation of fatty acids is essential for robust replication of viral rna [147, 148] . another study reports that rotavirus recruits lipid droplets into their viroplasm compartments upon infection of ma104, caco-2, bsc-1, and cos-7 cell lines [149] . confocal microscopy studies reported that two ld-associated proteins namely perilipin a and adrp colocalize with rotaviral proteins present in the viroplasm [149] . overexpression of the hbv x (hbx) protein was found to induce lipid accumulation in hepatic cells [150, 151] . na and colleagues found that hbx induces a pathway that involves the expression of liver x receptor and its associated genes result in accumulation of lipid droplets in hepg2 cell lines [152] . lysosomes are single membrane-bound organelles which house enzymes involved in the degradation of various extracellular and intracellular macromolecules such as proteins, pathogens, etc through phagocytic or autophagic pathway. plant and fungal vacuoles have similar degradative and storage functions like the lysosomes (fig. 1) . the specialized acidic lumen is the unique feature of these organelles which is needed to keep the enzymes active. viruses utilize lysosomal enzymes in order to facilitate their replication and release within the host cell [153] . it has been suggested that lysosomal enzymes are involved at different stages of vacv and mouse hepatitis virus replication [153] . they proposed a possibility for viruses to recruit lysosomal enzymes inside phagosomes in order to uncoat and release their genome in the host cell. a possible role for the lysosomal enzymes in enhancement of glycolysis in viral infected cells was also proposed. recent studies reported the accumulation of hav progeny in the lysosome of the host hepg2 cells. the maturation of these viral particles was reported to be catalysed by the lysosomal protease [154] . sv40 infection in bsc-1 and 3t3 cell lines resulted in swelling up of lysosomes followed by the release of lysosomal enzyme into the cytoplasm [155] . since lysosomes play a major role in the host's antiviral response, they are likely to become a target for certain viruses. the x protein of the hbv leads to inhibition of lysosomal acidification leading to loss of functioning [156] . however, the lysosome's ability to fuse with autophagosomes was found to remain unaffected. this virus induced accumulation of immature lysosomes resulting in the suppression of autophagic degradation was followed by development of hbv-associated hepatocellular carcinoma [156] . deglycosylation of the lysosome-associated membrane proteins by neuraminidase (na) of h5n1 influenza virus resulted in destruction of lysosomes. this was followed by cell death due to the release of hydrolytic lysosomal enzymes to the cytoplasm [157] . shubin and colleagues studied the expression of 3c protease of hav in a549 and human lung epidermoid carcinoma (calu-1) cell lines and found that hav 3c protease induces the development of non-acidic cytoplasmic vacuoles which originate from several types of lysosomal/endosomal organelles [158] . similarly several viruses like hiv, adenovirus, pv have been reported to cause lysosomal rupture [159] . tobacco plant when infected with cucumber mosaic virus (cmv), the viral replicase complex that constitutes the replicaseassociated protein cmv1a and rna dependent rna polymerase protein cmv2a was reported to localize on the vacuolar membrane [160] . singapore grouper iridovirus (sgiv) infection on grouper embryonic cells (gecs) from the brown-spotted grouper epinephelus tauvina results in the formation of a large intracellular vacuole for viral accumulation. later, the virus recruits the host cytosolic membrane-bending proteins in order to induce tubulation of vacuolar membrane [161] . the individual vacuoles fuse together to form a large vacuole which in turn fuses with the cell membrane. these events aid in the release of virions [161] . semliki forest virus (sfv) targets the vacuolar atpase in order to cause acidification of vacuoles resulting in low intra luminal ph [162] . upon infection with the sfv the endocytic vacuoles with low ph trigger membrane fusion essential for the viral pathogenesis in bhk-21 cells [163] . vacuolar proton atpase activity leading to low intra endosomal ph was reported to be required for the entry of reovirus into the host cells [164] . acidification of vacuoles by cellular v-atpase in human dermal fibroblast cells upon infection of hcmv was reported to be required for the formation of the specialized compartment for virion assembly [165] . apart from the above discussed well defined organelles of a cell, viruses also modify and hijack various vesicular structures and protein complexes of the host cell to ensure efficient infection. we discuss these essential compartments/structures in this section. endosomes are required for internalization of extracellular material into the cells and lead them to lysosomes for degradation or recycle back to the plasma membrane. multi vesicular bodies are vesicular compartment of the endocytic pathway and contain intraluminal vesicles. autophagosomes are double membrane vesicular structures that sequester the cytoplasm containing proteins, organelles, etc and direct them to degradation (fig. 1) . efficient cellular entry of most viruses is via endocytic pathway that comprises of various endosomes. several viruses like influenza, sfv, vsv, sv40, ebola, etc use different endocytic pathways like clathrin, caveoli or micropinocytosis to gain entry into the cells [6,166e168] . viruses modify or induce the formation of vesicles like endosomes in order to facilitate their multiplication inside the host cells [169] . sfv modifies the endosomal and lysosomal membrane of the infected bhk-21 cells for the construction of its replication site [170] . further the endosomes and lysosomes were reported to fuse together resulting in the formation of cytoplasmic vacuoles [170] . sv40 was reported to trigger the formation of endocytic vacuoles that migrate and fuse with the nuclear membrane upon infection of cv-1 cells leading to the migration of virions into the infected cell nucleus [171, 172] . endosomal sorting complexes required for transport (escrt) catalyse the process of invagination of endosomal membrane and result in the formation of multiple vesicular bodies (mvb) in eukaryotic cells [173] . mvb act as an intermediate for transporting ubiquitinated or misfolded protein to lysosomes [173] . mvb are also reported to play an active role in endolysosomal transport and budding of the virus. several rna and dna viruses hijack the escrt machinery in order to facilitate their release from the infected cells [169] . the matrix protein vp40 recruits tsg101 protein and escrt-1 complex constituting of vps28, vps37b and vps4 in order to direct the ebola virus into multivesicular bodies that assists in their budding [174] . hoffmann and colleagues reported that upon infection of hepatoma derived cell lines by the dna virus hbv, cellular a-taxilin acts as an adaptor for the binding of large hbv surface antigen with escrt components. this aids in recruiting the escrt machinery for the release of hbv-dna containing particles [175] . various rna viruses alter autophagosomes and induce or suppress autophagy in order to complete their life cycle or to escape from the host antiviral response [176] . it has been reported that the coxsackie b3 infection triggers the formation of autophagosomes in hela and hek293 cells [177] . prevention of lysosomal fusion with autophagosomes also enhanced coxsackie virus replication in the host cells. wild type mouse embryonic fibroblast (mef) and huh7 cells also exhibit enhanced autophagosome formation upon denv 2 infection [178] . the viruses modulating the phenomenon of cellular autophagy for their advantage have been reviewed elsewhere [176] . proteasome is a complex of proteases that selectively degrades intracellular proteins. this complex is tightly regulated and identifies polyubiquitinated proteins for degradation process. recent studies identified that viruses hijack ups in several ways to enhance their infectivity [179] . this is achieved by degradation of host proteins of ups, enhancing the function of viral proteins by modifications, hampering the modifications of signalling molecules of innate immunity, etc [179] . virus induced ubiquitination and subsequent proteasomal degradation of p53 as a strategy is employed by dna viruses such as hpv, adv, etc [180] . viral protein x of hiv-2 was reported to be responsible for the ubiquitination and proteasomal degradation of the host protein samhd1 that inhibits hiv infection [181] . an interesting strategy by denv was reported where the viral protein ns5 stimulates proteasomal degradation of stat2 and blocks type 1 ifn signalling and thus evades the host immune mechanisms [182] . similarly selective degradation of ns3 (non-structural protein) of the zika virus via proteasome mediated pathway was recently reported [183] . this proteasome dependant degradation of the viral protein was proposed as a strategy for the host antiviral mechanism. a role for ups has also been reported in plant viral infections. components of rna silencing such as argonaute 1 are targeted to proteasomal degradation by viruses like potato virus x, enamovirus, etc for efficient infection [184, 185] . this review summarizes the modifications that organelles encounter upon viral infection in a cell. understanding organelle dynamics under various conditions is a fundamental question that has attracted researchers for a long time. the importance of organelle dynamics and function is highlighted by many examples of diseases/disorders where it is affected. alterations in organelles such as shape, content, dynamics and eventually the function as a result of viral infections is observed. not only viruses but pathogenic bacteria have also been reported to alter organelles for their survival and infection. as emphasized in this paper, recent studies have shown that many viruses encode proteins that are targeted to various cellular organelles and control their functions. certainly there exists a close relationship between organelle dynamics and viral infections but thorough characterization will highlight their relevance to pathogenesis. advanced methods in microscopy and proteomics have enabled such characterization and the molecular details of virus-host interactions and viral replication in host cells is now understood in detail for few viruses. it is important to determine how various organelle proteins are temporally and spatially regulated upon viral infections leading to altered functions. organelles not as independent entities but a role for inter-organelle communication/inter-organelle cross talk in a cell for optimum functioning is now unequivocally accepted. this is another very interesting aspect to be explored to enhance our understanding of the virus-host interaction mechanisms to enable design of new antiviral strategies. our understanding on the organelle-virus interaction has been rapidly increasing with the advent of new molecular biology tools and advance imaging techniques. although we know the basic modus operandi of the viruses, there might be a novel virus that behaves different than the existing dogma with respect 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characterization of hiv-1 vpr nuclear import: analysis of signals and pathways hiv-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway integrase interacts with nucleoporin nup153 to mediate the nuclear import of human immunodeficiency virus type 1 nuclear import and export of influenza virus nucleoprotein application of bioinformatics-coupled experimental analysis reveals a new transport-competent nuclear localization signal in the nucleoprotein of influenza a virus strain importin alpha nuclear localization signal binding sites for stat1, stat2, and influenza a virus nucleoprotein function of dynein and dynactin in herpes simplex virus capsid transport microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro import of adenovirus dna involves the nuclear pore complex receptor can/nup214 and histone h1 nucleoporin 153 arrests the nuclear import of hepatitis b virus capsids in the nuclear basket phosphorylationdependent binding of hepatitis b virus core particles to the nuclear pore complex nuclear import of hepatitis b virus capsids and release of the viral genome role of nuclear pore complex in simian virus 40 nuclear targeting pushing the envelope: microinjection of minute virus of mice into xenopus oocytes causes damage to the nuclear envelope parvoviral nuclear import: bypassing the host nuclear-transport machinery nucleoli: composition, function, and dynamics involvement of ul24 in herpes-simplex-virus-1-induced dispersal of nucleolin involvement of the ul24 protein in herpes simplex virus 1-induced dispersal of b23 and in nuclear egress adenovirus core protein v is delivered by the invading virus to the nucleus of the infected cell and later in infection is associated with nucleoli adenovirus protein v induces redistribution of nucleolin and b23 from nucleolus to cytoplasm effects of adenovirus infection on rrna synthesis and maturation in hela cells encephalomyocarditis virus (emcv) proteins 2a and 3bcd localize to nuclei and inhibit cellular mrna transcription but not rrna transcription rhinovirus 3c protease precursors 3cd and 3cd' localize to the nuclei of infected cells the post-transcriptional regulator rev of hiv: implications for its interaction with the nucleolar protein b23 localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus nucleolin stimulates viral internal ribosome entry site-mediated translation quantitative proteomic analysis of a549 cells infected with human respiratory syncytial virus ifn enhance expression of sp100, an autoantigen in primary biliary cirrhosis hsv-1 ie protein vmw110 causes redistribution of pml mediation of epstein-barr virus ebna-lp transcriptional coactivation by sp100 adenovirus replication is coupled with the dynamic properties of the pml nuclear structure adenovirus e4 orf3 protein inhibits the interferon-mediated antiviral response inactivating a cellular intrinsic immune defense mediated by daxx is the mechanism through which the human cytomegalovirus pp71 protein stimulates viral immediate-early gene expression two ring finger proteins, the oncoprotein pml and the arenavirus z protein, colocalize with the nuclear fraction of the ribosomal p proteins sumoylation promotes pml degradation during encephalomyocarditis virus infection rabies virus p and small p products interact directly with pml and reorganize pml nuclear bodies viral interactions with the nuclear transport machinery: discovering and disrupting pathways remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pul50 and pul53 papillomavirus interaction with cellular chromatin phenotypic and cytologic studies of lymphoid cells and monocytes in primary culture of porcine bone marrow during infection of african swine fever virus wrapping membranes around plant virus infection inhibition of sterol biosynthesis reduces tombusvirus replication in yeast and plants endoplasmic reticulum: the favorite intracellular niche for viral replication and assembly direct formation of vaccinia virus membranes from the endoplasmic reticulum in the absence of the newly characterized l2-interacting protein a30.5 intracellular localisation of dengue-2 rna in mosquito cell culture using electron microscopic in situ hybridisation composition and three-dimensional architecture of the dengue virus replication and assembly sites cellular origin and ultrastructure of membranes induced during poliovirus infection remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagylike origin for virus-induced vesicles ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes the potato virus x tgbp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection tombusviruses upregulate phospholipid biosynthesis via interaction between p33 replication protein and yeast lipid sensor proteins during virus replication in yeast opportunistic intruders: how viruses orchestrate er functions to infect cells arms race between enveloped viruses and the host erad machinery mutational analysis of glycosylation, membrane translocation, and cell surface expression of the hepatitis e virus orf2 protein cytoplasmic localization of the orf2 protein of hepatitis e virus is dependent on its ability to undergo retrotranslocation from the endoplasmic reticulum the expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis bap31 and bip are essential for dislocation of sv40 from the endoplasmic reticulum to the cytosol global sitespecific n-glycosylation analysis of hiv envelope glycoprotein the interplay between mitochondrial dynamics and mitophagy mitochondrial fusion, fission, and mitochondrial toxicity mitochondrial dynamics and viral infections: a close nexus hepatitis b virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis sarscoronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/traf3/traf6 signalosome mitophagy in viral infections the matrix protein of human parainfluenza virus type 3 induces mitophagy that suppresses interferon responses mitophagy promotes replication of oncolytic newcastle disease virus by blocking intrinsic apoptosis in lung cancer cells viruses as modulators of mitochondrial functions hepatitis b virus x protein induces perinuclear mitochondrial clustering in microtubule-and dyneindependent manners migration of mitochondria to viral assembly sites in african swine fever virus-infected cells herpes simplex virus eliminates host mitochondrial dna hepatitis c virus triggers mitochondrial permeability transition with production of reactive oxygen species, leading to dna damage and stat3 activation human cytomegalovirus pul37x1 induces the release of endoplasmic reticulum calcium stores the coxsackievirus 2b protein suppresses apoptotic host cell responses by manipulating intracellular ca2ã¾ homeostasis role of ca2ã¾in the replication and pathogenesis of rotavirus and other viral infections human hepatitis b virus-x protein alters mitochondrial function and physiology in human liver cells hbx sensitizes cells to oxidative stress-induced apoptosis by accelerating the loss of mcl-1 protein via caspase-3 cascade influence of cytosolic and mitochondrial ca2ã¾, atp, mitochondrial membrane potential, and calpain activity on the mechanism of neuron death induced by 3-nitropropionic acid viral product trafficking to mitochondria, mechanisms and roles in pathogenesis a pore way to die: the role of mitochondria in reperfusion injury and cardioprotection the myxoma poxvirus protein, m11l, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore the hiv-1 viral protein r induces apoptosis via a direct effect on the mitochondrial permeability transition pore a renewed focus on the interplay between viruses and mitochondrial metabolism systemslevel metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy dynamics of the cellular metabolome during human cytomegalovirus infection glycolysis during early infection of feline and human cells with feline leukemia virus glycolysis in chick embryo cell cultures transformed by rous sarcoma virus divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism hepatitis c virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ros) production viruses exploiting peroxisomes import of peroxisomal matrix and membrane proteins peroxisomes are platforms for cytomegalovirus' evasion from the cellular immune response expression of the cymbidium ringspot virus 33-kilodalton protein in saccharomyces cerevisiae and molecular dissection of the peroxisomal targeting signal localization of the tomato bushy stunt virus replication protein p33 reveals a peroxisome-to-endoplasmic reticulum sorting pathway the host pex19p plays a role in peroxisomal localization of tombusvirus replication proteins a key role for heat shock protein 70 in the localization and insertion of tombusvirus replication proteins to intracellular membranes the pestivirus n terminal protease n(pro) redistributes to mitochondria and peroxisomes suggesting new sites for regulation of irf3 by n(pro.) the fine structure of cymbidium ringspot virus infections in host tissues. iii. role of peroxisomes in the genesis of multivesicular bodies the role of the p33:p33/p92 interaction domain in rna replication and intracellular localization of p33 and p92 proteins of cucumber necrosis tombusvirus integrated systems biology analysis of kshv latent infection reveals viral induction and reliance on peroxisome mediated lipid metabolism micro-rnas upregulated during hiv infection target peroxisome biogenesis factors: implications for virus biology, disease mechanisms and neuropathology flavivirus infection impairs peroxisome biogenesis and early antiviral signaling identification of a type 1 peroxisomal targeting signal in a viral protein and demonstration of its targeting to the organelle binding of hiv-1 nef to a novel thioesterase enzyme correlates with nef-mediated cd4 down-regulation a novel acyl-coa thioesterase enhances its enzymatic activity by direct binding with hiv nef endocytosis of adeno-associated virus type 5 leads to accumulation of virus particles in the golgi compartment the transmembrane domain of the severe acute respiratory syndrome coronavirus orf7b protein is necessary and sufficient for its retention in the golgi complex a signal for golgi retention in the bunyavirus g1 glycoprotein orf virus interferes with mhc class i surface expression by targeting vesicular transport and golgi foot-and-mouth disease virus 3c protease induces fragmentation of the golgi compartment and blocks intra-golgi transport fragmentation of the golgi apparatus provides replication membranes for human rhinovirus 1a the open reading frame 3a protein of severe acute respiratory syndrome-associated coronavirus promotes membrane rearrangement and cell death norwalk virus n-terminal nonstructural protein is associated with disassembly of the golgi complex in transfected cells poliovirus infection and expression of the poliovirus protein 2b provoke the disassembly of the golgi complex, the organelle target for the antipoliovirus drug ro-090179 membrane-association properties of avian encephalomyelitis virus protein 3a impact on the endoplasmic reticulum and golgi apparatus of turnip mosaic virus infection a trans-golgi network resident protein, golgin-97, accumulates in viral factories and incorporates into virions during poxvirus infection the 3a protein from multiple picornaviruses utilizes the golgi adaptor protein acbd3 to recruit pi4kiiibeta tomato spotted wilt virus glycoproteins induce the formation of endoplasmic reticulum-and golgi-derived pleomorphic membrane structures in plant cells assembly of vaccinia virus: the second wrapping cisterna is derived from the trans golgi network structural maturation of rubella virus in the golgi complex inner tegument protein pul37 of herpes simplex virus type 1 is involved in directing capsids to the trans-golgi network for envelopment key golgi factors for structural and functional maturation of bunyamwera virus emerging role of lipid droplets in host/pathogen interactions lipid droplets and viral infections down-regulation of phosphatase and tensin homolog by hepatitis c virus core 3a in hepatocytes triggers the formation of large lipid droplets dengue virus capsid protein binding to hepatic lipid droplets (ld) is potassium ion dependent and is mediated by ld surface proteins dengue virus capsid protein usurps lipid droplets for viral particle formation dengue virus-induced autophagy regulates lipid metabolism dengue virus and autophagy rotaviruses associate with cellular lipid droplet components to replicate in viroplasms, and compounds disrupting or blocking lipid droplets inhibit viroplasm formation and viral replication hepatitis b virus x protein induces lipogenic transcription factor srebp1 and fatty acid synthase through the activation of nuclear receptor lxralpha hepatitis b virus x protein induces hepatic steatosis via transcriptional activation of srebp1 and ppargamma liver x receptor mediates hepatitis b virus x protein-induced lipogenesis in hepatitis b virusassociated hepatocellular carcinoma activation of lysosomal enzymes in virus-infected cells and its possible relationship to cytopathic effects lysosomes serve as a platform for hepatitis a virus particle maturation and nonlytic release lysosomal changes in lytic and nonlytic infections with the simian vacuolating virus (sv40) hepatitis b virus x protein inhibits autophagic degradation by impairing lysosomal maturation neuraminidase of influenza a virus binds lysosome-associated membrane proteins directly and induces lysosome rupture protease 3c of hepatitis a virus induces vacuolization of lysosomal/endosomal organelles and caspase-independent cell death lysosomal cell death at a glance a zinc finger protein tsip1 controls cucumber mosaic virus infection by interacting with the replication complex on vacuolar membranes of the tobacco plant visualization of assembly intermediates and budding vacuoles of singapore grouper iridovirus in grouper embryonic cells involvement of the vacuolar h(ã¾)-atpase in animal virus entry membrane and protein interactions of a soluble form of the semliki forest virus fusion protein the entry of reovirus into l cells is dependent on vacuolar proton-atpase activity cellular v-atpase is required for virion assembly compartment formation in human cytomegalovirus infection endocytosis via caveolae endocytosis of simian virus 40 into the endoplasmic reticulum cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes membrane dynamics associated with viral infection biogenesis of the semliki forest virus rna replication complex fusion of sv40-induced endocytotic vacuoles with the nuclear membrane interaction of endocytotic vacuoles with the inner nuclear membrane in simian virus 40 entry into cv-1 cell nucleus escrt complexes and the biogenesis of multivesicular bodies involvement of vacuolar protein sorting pathway in ebola virus release independent of tsg101 interaction identification of alpha-taxilin as an essential factor for the life cycle of hepatitis b virus divergent roles of autophagy in virus infection autophagosome supports coxsackievirus b3 replication in host cells autophagic machinery activated by dengue virus enhances virus replication irhom2 is essential for innate immunity to dna viruses by mediating trafficking and stability of the adaptor sting identification of three functions of the adenovirus e4orf6 protein that mediate p53 degradation by the e4orf6-e1b55k complex molecular determinants for recognition of divergent samhd1 proteins by the lentiviral accessory protein vpx ns5 of dengue virus mediates stat2 binding and degradation viperin restricts zika virus and tick-borne encephalitis virus replication by targeting ns3 for proteasomal degradation the silencing suppressor p25 of potato virus x interacts with argonaute1 and mediates its degradation through the proteasome pathway the enamovirus p0 protein is a silencing suppressor which inhibits local and systemic rna silencing through ago1 degradation we sincerely apologize to our colleagues for any research study or review publication not being cited in this review. this is only due to space limitation. research in the laboratory of sn is supported by grants from science and engineering research board ( key: cord-029957-q7v5gli8 authors: prabhu, d.; rajamanikandan, s.; anusha, s. baby; chowdary, m. sushma; veerapandiyan, m.; jeyakanthan, j. title: in silico functional annotation and characterization of hypothetical proteins from serratia marcescens fgi94 date: 2020-07-31 journal: biol doi: 10.1134/s1062359020300019 sha: doc_id: 29957 cord_uid: q7v5gli8 serratia marcescens, rod-shaped gram-negative bacteria is classified as an opportunistic pathogen in the family enterobacteriaceae. it causes a wide variety of infections in humans, including urinary, respiratory, ocular lens and ear infections, osteomyelitis, endocarditis, meningitis and septicemia. unfortunately, over the past decade, antibiotic resistance has become a serious health care issue; the effective means to control and dissemination of s. marcescens resistance is the need of hour. the whole genome sequencing of s. marcescens fgi94 strain contains 4434 functional proteins, among which 690 (15.56%) proteins were classified under hypothetical. in the present study, we applied the power of various bioinformatics tools on the basis of protein family comparison, motifs, functional properties of amino acids and genome context to assign the possible functions for the hps. the pseudo sequences (protein sequence that contain ≤100 amino acid residues) are eliminated from the study. although we have successfully predicted the function for 483 proteins, we were able to infer the high level of confidence only for 108 proteins. the predicted hps were classified into various classes such as enzymes, transporters, binding proteins, cell division, cell regulatory and other proteins. the outcome of the study could be helpful to understand the molecular mechanism in bacterial pathogenesis and also provide an insight into the identification of potential targets for drug and vaccine development. enterobacteriaceae family consists of more than 250 published bacterial species and serratia is one of the most clinically important genera of the family (alnajar and gupta, 2017) . the bacteria belonging to the enterobacteriaceae are most commonly encountered organisms which are isolated from water, soil and clinical specimens. serratia species is frequently found associated with animals and plants either detrimental or beneficial. among the genus, serratia marcescens (s. marcescens), gram-negative bacteria have been recognized as a crucial cause of healthcare associated infections (nosocomial infections) in humans (parente et al., 2016) . several strains of s. marcescens have been reported till date, and the complete genome sequence of serratia strain fgi94 has been sequenced and its 16s rrna has shown 99% highest nucleic acid identity with the pathogenic strain serratia rubidaea jcm1240 (aylward et al., 2013) . in recent times, the majority of the healthcare associated urinary, respiratory, eye infections, osteomyelitis, wound infections, endocarditis and pulmonary infections were reported by s. marcescens (padmavathi et al., 2014) . resistance to antimicrobial agents through intrinsic and acquired process is a notable feature in s. marcescens. a wide variety of gene cassettes containing resistance were identified in the chromosome and plasmids of s. marcescens. certain strains of s. marcescens have shown resistance to antibiotics (gentamicin, cephalosporins, fluoroquinolone, cefotaxime and ceftazidime) which already complicates the antibiotic therapy (iguchi et al., 2014) . according to the world health organization (who) report 2018, serratia spp. were classified under class-1 priority. the categorization was based on the antimicrobial resistance (amr) mechanism of the pathogens (who priority pathogens list for r&d of new antibiotics, 2018). amr has emerged as a serious threat to the public health authorities at global level, particularly in intensive care and surgical units. resistance leads to the long-term medication, higher medical cost, prolonged hospitalization, and on the severity, leading to death (vazirianzadeh et al., 2013) . the rapid emergence of antibiotic resistance is theoretical biology occurring worldwide as at least 2 million people acquire resistance each year and around 23,000 deaths were documented by cdc report (cdc-antibiotic resistant threats report, 2013) . according to the who report, amr is one of the three major challenging problems of the human race (who antimicrobial resistance, 2018) . global distribution of resistance plasmids among several bacterial species is alarming the entire world on amr (finley et al., 2013) . the recent technological innovations in the next generation sequencing (ngs) generates larger amount of genomic data from wide range of bacterial species. however, very limited bacterial species have their complete proteome information, but sound knowledge of the microbial proteome is essential to understand the disease pathogenesis, virulent determinants, and their survival and propagation (singh et al., 2015) . for instance, in most bacterial species, nearly 30-40% of genes within in the genomes are marked as unknown or hypothetical (hoskeri et al., 2010) . proteins with unknown function or conserved putative proteins, which are showing limited connection with functionally annotated proteins, are termed as hypothetical proteins (hps). hps are translated portions of nucleic acid sequences based on their sequence similarity, but for experimental existence functional and biochemical characterization has to be evaluated (shahbaaz et al., 2013) . moreover, hps also includes the low identity proteins, imprecisely described and vague functional proteins (hoskeri et al., 2010) . in general, hps are classified into two major classes: (i) uncharacterized protein families (upfs), and (ii) domains of unknown function (dufs). the ones whose protein structures are available but not been functionally characterized or linked to any known functional gene, are referred as upfs. whereas the ones whose experimental existence of protein is available although not related to any known functional or structural domain, are represented as dufs (varma et al., 2015) . annotating the hps have many advantages such as, deriving new structurefunctional relationship, novel protein structures, and also helps in decoding additional pathways for better understanding of the pathogens. functionally annotated hps may serve as potential biomarkers for the screening and diagnostics of diseases. they can also be used as a pharmacological target to design and discover novel drugs (singh et al., 2015) . function of the protein can be predicted using various strategies, such as phylogenetic profiling, mass spectrometry identification, sage analysis, lethal analysis, etc., (varma et al., 2015; gasperskaja and kučinskas, 2017) . in order to minimize the time and investment cost, various computational tools were developed to aid the functional annotation process (hawkins and kihara, 2007) . in the current study, we have used the power of computational tools to annotate the possible functions for the hps in s. marcescens. functional predictions of hps from various bacterial genomes (v. cholera, n. gonorrhoeae, c. difficile, s. aureus, m. tuberculosis, h. influenza) through computational approaches were widely used in successful proteome annotation (shahbaaz et al., 2013; singh et al., 2015; costa et al., 2018) . deciphering the function of entire gene coding regions in the genome is essential to merge the gaps in proteome to fully understand the pathogenicity and genome plasticity of s. marcescens. sequence retrieval the entire protein sequences of s. marcescens fgi94 were identified by searching in ncbi (http://www.ncbi.nlm.nih.gov/) database. s. marcescens genome possesses 4434 protein-coding genes out of which 690 proteins were marked as hps. all the hps sequences were retrieved for the biological functional assignment using various functional annotation servers. in order to minimize the misinterpretations in functional annotation pipeline, pseudo genes (proteins having less than 100 amino acid residues) were excluded in this study. the various tools used in the functional predictions of hps in s. marcescens are tabulated in table 1 . physicochemical characterization of the hps was performed using expasy proteomics tools (gasteiger et al., 2005) . parameters such as molecular weight (mw), theoretical isoelectric point (pi), grand average of hydropathicity (gravy), aliphatic and instability index were computed for the 483 hps in s. marcescens. localization of the hps was predicted using various tools including psortb (yu et al., 2010) , pslpred (bhasin et al., 2005) and cello (yu et al., 2004) . the signal peptide was predicted using signalp server (petersen et al., 2011) . secretomep (bendtsen et al., 2005) was used to identify the presence of hps in nonclassical secretory pathway. transmembrane information about the hps was predicted using tmhmm (krogh et al., 2001) and hmmtop (tusnády and simon, 1998) servers. functions of the hps were predicted using the web-based tools. ncbi-blast (altschul et al., 1990) , smart (letunic et al., 2012) , ebi-interproscan (hunter et al., 2011) and motif (kanehisa, 1997) were used to identify the functional domains/motifs present in the hps. family level classifications of the hps were identified using pfam (finn et al., 2014) , scop-superfamily (gough et al., 2001) and panther (thomas et al., 2006) families. structural level classification of protein super families was predicted using cath database (orengo et al., 1997) . domain architecture of the hps was predicted using svmprot (cai, 2003) , cdart (geer et al., 2002) and protonet tools (rappoport et al., 2012) . virulence activity of the hps was predicted using virulentpred (garg and gupta, 2008) and vcim-pred (saha and raghava, 2006) . characterization 483 hps were selected, which are having more than 100 amino acids residues for the physicochemical characterization and functional annotation. on the basis of amino acid sequences, predicted physicochemical properties of the 483 hps were tabulated in supplementary table 1. molecular weight is an important criterion in protein functional characterization. it can be seen that the proteins agb82318.1 and agb83651.1 have shown the lowest and highest mw of 106 48.04 and 137161.31 da respectively. isoelectric point (pi) is the ph at which the proteins have no net charge and the mobility based on charge will be zero. prediction of pi is essential in the development of buffer system for purification and in isoelectric focusing. the predicted pi value of the hps ranged from 3.53 to 11.83. out of 483 hps, 246 proteins were observed to be acidic and the predicted pi values ranged from 3.53 to 6.97. similarly, 236 proteins have shown the pi values between 7.01 and 11.83, are considered basic and agb83651.1 has shown neutral charge. extinction coefficient of the hps were measured in water at 280 nm based on the concentration of cystine, tryptophan and tyrosine amino acid residues in the protein sequences. higher occupancy of these amino acid residues in the protein results in higher extinction coefficient values. small proteins with 100 plus amino acid residues containing minimal number of cystine, tryptophan and tyrosine residues have shown very less extinction co-efficient values. especially, agb81485.1 and agb84113.1 have not shown the extinction co-efficient values due to absence of cystine, tryptophan and tyrosine amino acid residues. computing the extinction co-efficient values helps in the quantitative analysis of protein-protein and protein-ligand interactions for the drug development process. instability index of the hps were found to be minimum value of -0.24 to the maximum of 119.78. instability index value illustrates the stability of the proteins in test tube environment. the 228 hps having the instability index values greater than 40 are classified as unstable and 225 proteins having the instability index less than 40 are classified as stable. aliphatic amino acid residues present in the protein are directly proportional to the aliphatic index. proteins with higher aliphatic index have shown higher thermal stability, especially in globular proteins. the computed aliphatic index of the 483 hps falls between 38.03 and 157.52. the stability of the thermophilic proteins are mainly contributed by the aliphatic amino acids (a, v, i and l), which results in higher aliphatic index values to withstand the wide range of temperatures. gravy value represents the protein-water interactions and it illustrates the hydrophilic nature of the protein. the gravy values are computed based on the average sum of the hydrophilic and hydrophobic side chains in the amino acids. least gravy value among the hps is found to be -1.478 and the highest is 1.238. proteins were characterized as drug and vaccine targets are mainly based on the subcellular localization. proteins located in cytoplasmic matrix are capable to be potential drug targets, whereas both inner and outer membrane proteins can possibly act as potential vaccine targets. crucial step in determining the function of the proteins is underlying on the knowledge of localization. based on the knowledge of trained data sets, the hps were predicted for their presence in any of these locations (cytoplasm, periplasm, extracellular, inner membrane and outer membrane) in the cell. we predicted the localization of 356 hps out of 483 proteins subject for the study based on the concurrence hits. among the predicted 356 hps, 60% (214) of the proteins were shown to be present in cytoplasm. presence of hps in inner membrane and outer membrane is 17.41% (62) and 2.52% (9) respectively. it is predicted that 14% (50) hps are present in periplasm and 5.89% (21) in extra cellular matrix. however, locations of 127 proteins were not confirmed due to absence of concurrence results. signal peptides are the key players in determining the transport of proteins to the target location. hence, prediction of signal peptide is essential to know the transport system of the particular proteins and the cleavage sites. apart from the certain cytoplasmic proteins, all other proteins have the signal peptides to facilitate the transport of proteins in and out of the membrane to reach the target cellular location or organelles. we predicted the presence of signal peptide sequences in 116 hps out of 483. similarly, 142 proteins were predicted to be involved in non-classical secretory pathway. proteins which are secreted outside by the cells are secretomes, and these proteins are important to maintain the cell-cell communications, cell proliferation and pathogenesis. membrane proteins are involved in various processes such as transport, signaling and energy transduction; hence half of the targets used for drug development are membrane proteins. the predicted 136 proteins have shown transmembrane helices from tmhmm server and 235 proteins from hmmtop server. prediction of membrane proteins is vital for the better understanding of drug targets to develop potent drug molecules. the details of the 483 hps were shown in supplementary table 2 . annotating the function of hps is essential for the better understanding of the entire biological system towards the development of effective drugs. we have analyzed 483 hps of s. marscences to predict their possible functions. further, hps were analyzed for the presence of functional domains and signature motifs in context with the biological function. the results of the domain and motif analysis are shown in supplementary table 3 . results of the sequence and structural features of the hps were shown in the supplementary table 4 . we have successfully annotated the function of 108 hps with high confidence out of the 483 proteins. the list of 108 proteins with their functions assigned is illustrated in table 2 . based on the analysis, only 22.36% of hps functions were predicted and the remaining hps have not shown concurrence results, which indicates that suitable experimental strategy has to be coupled for better functional assignment. annotated proteins ( fig. 1) were majorly classified into the following six categories such as, enzymes, binding proteins, cell division proteins, cell regulatory proteins, transporters and the remaining proteins involved in different biological process. bacterial enzymes play a catalyst role in all the metabolic and catholic process, leading to the supply of essential nutrient for the growth and also responsible for the pathogenesis of the organism (gurung et al., 2013) . we have characterized 34 proteins which act as enzymes, out of which 9 were shown to be transferases. transferases catalyze the transfer of a functional group (methyl group or glycosyl group) from one molecule (act as donor) to another molecule (act as acceptor). six proteins were identified as endonucleases, which function in destroying the invaded foreign dna ( van den broek et al., 2005) . two proteins agb81206.1 and agb83112.1 were predicted as a member of the exonuclease-endonuclease-phosphate domain super family which plays a crucial role in the intracellular signaling activities in bacteria (dlakic, 2000) . disrupting the intracellular signaling activities either arrests the physiological role or even kills the organism, therefore it is widely considered as a potential target for drug development (kohanski et al., 2010) . the protein agb83394.1 is predicted to contain smr domain like. it is observed that, smr domain like protein is broadly classified into three sub-families. family-1 closely relates to the c-terminal domain of the muts, presumably found in deltaproteobacteria, firmicutes, bacteroidetes and epsilonproteobacteria phyla of bacteria and plants. the proteins under this family are responsible for the protection of cells from oxidative dna damages. family-2 closely relates to the c-terminal domain of muts (eukaryotes), whereas family-3 indicates the muts that are found in e. coli. overall, these three families of proteins are involved in endoculease activity and also responsible for the formation of the branched dna structures (fukui and kuramitsu, 2011) . the protein agb81307.1 is predicted to have acyl carrier protein phosphodiesterase and functions essentially in catalyzing the hydrolytic cleavage of the 4′ phosphopantetheine residue from acyl carrier protein phosphodiesterase with the generation of apo acyl carrier protein phosphodiesterase. it also plays a significant role in the regulation of fatty acid synthesis (fischi and kennedy, 1990) . the fatty acid metabolic regulation in bacteria is essential to maintain the lipid homeostasis in the growth and stationary phases as well as in various physical and nutrient states (fujita et al., 2007) . agb82203.1 is predicted as 2-methyl aconitate cis/trans isomerase prpf. these proteins catalyze the inter-conversion of 2-methy caa and 2methyl taa in the 2-methylcitric acid cycles (du et al., 2017) . we predicted agb83389.1 as an elongation factor p. the factor p is required for the synthesis of proteins containing polyproline motifs and functions in ribosomal stalling (hersch et al., 2013) . agb83655.1 is predicted as p-loop containing nucleoside triphosphate hydrolase superfamily. these families of proteins function as kinases in many pathways and also work as motor to drive reactions through conformational changes (leipe et al., 2004 agb84572.1 is found to be oxidoreductase molybdopterin-binding domain and these proteins are reported to be involved in the h2 metabolism and bioenergetic pathways. the protein involved in these pathways help in the production of atp molecules by using oxidation-reduction reactions and thereby providing energies to the bacteria for the normal cellular activities (li et al., 2009) . the observed function of the hps helps to understand the crucial role of new proteins in bacterial growth and can be targeted as a potential targets for drug discovery. we identified 11 hps under binding proteins. agb80434.1 is classified as pk beta barrel domain like protein which is reported to be involved in chromosome formation and prerequisite for the initiation of chromophore maturation. it is well understood that pk beta barrel domain like protein play a significant role in wide variety of cellular processes including dna replication, dna repair and horizontal gene transfer (stepanenko et al., 2013) . thus, such proteins are important for the survival of the pathogens in the environment. we have characterized agb82209.1 and agb84196.1 as leptospira immunoglobulin like pro-tein b and it is observed that these proteins plays a major role in binding with fibrinogen, collagen, laminin and elastin and inhibit fibrin clot formation (lin et al., 2011) . the molecular mechanism of protein binding plays a critical role in the coagulation cascade and platelet aggregation, tissue regeneration, and immune responses proves to be a potential target for many pathogens (choy et al., 2011) . cell division in bacteria is closely linked to bacterial multiplication. knowledge on the mechanism of cell division is essential to explore novel targets in drug development. the agb81074.1 and agb83861.1 are predicted to be cell division protein (zapd). it is understood that the members of the zapd family of proteins share a common role in cell division machinery and mechanism (durand-heredia et al., 2012) . the protein agb84389.1 is predicted to a capsule assembly wzi family protein. this protein is classified as an outer membrane protein which is involved in extracellular capsule formation in many pathogenic bacteria (bushell et al., 2013) , and can be used as novel drug target. cell regulatory process protein gene regulation in prokaryotes and eukaryotes includes wide range of mechanisms for the production of desired gene product. this regulatory process is a complex network that controls the expression of the various transcriptional units in the bacteria, presumably maintains the microbial pathogenesis, growth and survival. the agb80600.1 protein was predicted to be dna recombination protein rmuc. although the function of this protein is not clearly understood, it shows high level of sequence similarities with myosins and structural maintenance of chromosome proteins (gaudermann et al., 2006) . protein agb80981.1 is a crea (dna binding protein), which function in the repression and control of alc regulon expression, that are necessary for the ethanol utilization pathways (panozzo et al., 1998) . protein agb81139.1 is a recombination regulatory (reca) protein and functions in dna pairing, strand exchange and recombinational dna repair (cox, 1991) . reca are multifunctional proteins that are found in all forms of living organisms. in the form of recombinase, the protein exhibits dna dependent atpase activity and also plays a role in the regulatory system that controls the induction of the sos response. as in the case of nucleoprotein filament, the pair plays a role in dna strand exchange (gruenig et al., 2008) . we found agb82748.1 protein in association with stage v sporulation protein r related protein. beall and moran (1994) in their work described the involvement of spovr from bacillus subtilis in spore cortex formation (beall and moran, 1994) . proteins agb83410.1 and agb83544.1 are antitoxin systems (reii/pare) composed in all bacterial genome and play a significant role in the formation of persistence cells, involvement in biofilm formation and in the pathogenesis of the organisms (wen et al., 2014) . we identified agb84710.1 as der gtpase activating protein yihi; the families of these proteins are more conserved in eubacteria and involved in the cell survival and bacterial growth (verstraeten et al., 2011) . the findings are helpful to conclude that by inhibiting these proteins bang the normal cellular process and thereby reduce the bacterial pathogenecity. bacteria contains various transport proteins for the import and export of substances such as nutrients, ions, metabolites, amino acids, etc., through the cell membrane, to exclude the unwanted by-products, and modify their cytoplasmic content of protons and salts needed for growth and development of the microorganisms. we predicted agb81989.1 and agb83037.1 as type vi secretion system and these proteins play a major role in virulence, antibacterial activity and also participate in metal ion uptake conferring an advantage during bacteria-bacteria competition (gallique et al., 2017) . agb82622.1 belongs to multi-drug resistance efflux transporter. emre belongs to the smr family of small multidrug transporters (ninio et al., 2004) . recent data suggest that over expression of smr causes bacteria to become resistant to wide range of antiseptics and antibiotics such as ethidium bromide, methyl viologen and intercalating dyes (ma and chang, 2004) . agb82887.1 is predicted to be manganese efflux pump mntp. metals such as manganese, copper and zinc are essential for all micro-organisms and appropriate maintenance of metal homeostasis is important to prevent toxicity and mismetallation for both eukaryotes and bacteria (procheronet et al., 2013) . the identification of such pumps in various bacteria such as e. coli (mntp) (martin et al., 2015) , streptococcus pneumoniae (mnte) (turner et al., 2015) and neisseria spp. (mntx) (veyrier et al., 2011) play an important role in removing excess manganese before levels become toxic. the protein agb83371.1 is found to be tripartite tricarboxylate transporter permease. this kind of protein is most abundant in beta-proteobacteria and plays a significant role in the transport of carboxylate (rosa et al., 2018) . we identified agb84290.1 as lipopolysaccharides export abc transporter periplasmic protein lptc. it is found that the lipopolysaccharide acts as hydrophilic barrier to wide range of hydrophobic antibiotics and plays a counterpart in the pathogenicity of the organisms (hicks and jia, 2018) . we categorized 39 hypothetical proteins function in different biological process under one category named other proteins. the protein agb80728.1 was predicted as translocation and assembly module (tam), which plays a major role in outer membrane biogenesis and virulence mechanisms in bacterial kingdom (josts et al., 2017) . agb8163.1 is found to be sh3 domain and it has been identified in various bacteria and plays a critical role in the targeting domains involved in bacterial cell wall recognition and metal binding (kamitori and yoshida, 2015) . we identified agb82063.1 as brct domain, and it is recognized as a c-terminal region of brca1 gene which participates in the signal transduction and in protein targeting motif in the dna damage response system (woods et al., 2012) . further, structural analysis of brct domain reveals that it may appear either in singleton (single brct) or tandem pair (double brct) and it also has a phosphate binding site in which the phosphate molecules are bound either in the dna end or in the phosphopeptide (sheng et al., 2011) . we found the protein agb82090.1 belongs to ycel protein and to the subgroup of lipocalin superfamily, and plays a role in isoprenoid quinine metabolism, transport and storage (handa et al., 2005) . the proteins agb81427. involving sugar modification and also play a role in oxalate metabolism. we predicted agb82764.1 as outer membrane lipoprotein slp family which is encoded by the gene xac1113 (ferreira et al., 2016) . bacterial lipoproteins are a class of membraneanchored proteins and play important role in bacterial physiology and pathogenesis (zuckert, 2014) . de souza et al., 2004 reported slp involvement in the biofilm formation. the protein agb83581.1 is found to be tetratricopeptide repeat like domain. this kind of proteins are found in wide variety of prokaryotic and eukaryotic organisms and plays an vital role in cell processes and associated with virulence mechanisms of bacterial pathogens (cerveny et al., 2013) . agb83707.1 is predicted to be outer membrane lipoprotein rcsf, presumably involved in signal transduction pathway (shiba et al., 2012) . we identified agb84181.1 protein as antibiotic biosynthesis monooxygenases. increased production of reactive oxygen species (ros) causes oxidative damages to the cells. the primary ros generated within mitochondria are limited to flavoproteins and flavins that activate the molecular oxygens. the generated monooxygenases do variety of functions in all living organisms and catalyze a wide range of reactions such as participating in respiratory chain within the cytoplasmic membrane and protecting themselves against external reactive oxygen species (khan et al., 2017 jer, 2005) . these lipoproteins are universally distributed in the bacterial kingdom and accounts to about 1-3% of the total genome. these lipoproteins play pivotal roles in many physiological and cellular processes such as cell wall metabolism, antibiotic resistance, nutrient uptake, cell division, signal transduction and virulence. thus, the identified hps are predicted to play an important role in the survival and pathogenesis of the pathogens and the identified functions of hps may provide clue as a therapeutic target for the drug design and developments. virulence factors are essential to invade the bacteria to colonize, causes disease and to overcome the defenses of the host. therefore, understanding the molecular mechanisms of microbial virulence plays a central role in the pathogenesis of the bacteria. so, we used vicmpred and virulentpred, an svm based method to predict the bacterial virulence factor among the 483 hps. the predicted results are shown in supplementary table 5 . we found that 29 hps as virulent factors, 226 as a cellular process, 15 as information and storage and 120 as metabolism molecules. all these proteins can be used as potential targets for drug design and development. in general, about 30-50% of the sequenced genome are referred as hypothetical proteins, and the rapid accumulation of the genome data containing hps is one of the emerging challenges in modern biology. hps hamper the identification of novel drug targets which can specifically act on pathogens to combat the pathogenicity. in the case of bacterial pathogens, these hps play a crucial role in the identification of potential drug targets and also enhance our understanding of their virulence capacity and pathogenicity. in this study, we have characterized the functions for the 108 hps from s. marcescens with a high level of confidence using various bioinformatics approaches. various types of enzymes, transporters, cell division, binding proteins were characterized which play an essential role in the growth, survival virulence and pathogenesis of s. marcescens. characterization of proteins on the basis of physiochemical properties and subcellular localization helps in differentiating the vital drug targets from vaccine targets. in addition, we also identified 18 virulence proteins that are predicted to play a crucial role in the pathogenesis of the organisms. hence, this study may facilitate future studies on the predicted hps as novel therapeutic targets for the drug and vaccine development. the authors declare that they have no conflict of interest. this article does not contain any studies involving animals or human participants performed by any of the authors. 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https://doi.org/10.1134/s1062359020300019 and are accessible for authorized users. key: cord-275993-isff6lp2 authors: han, dong p; kim, hyung g; kim, young b; poon, leo l.m; cho, michael w title: development of a safe neutralization assay for sars-cov and characterization of s-glycoprotein date: 2004-08-15 journal: virology doi: 10.1016/j.virol.2004.05.017 sha: doc_id: 275993 cord_uid: isff6lp2 the etiological agent of severe acute respiratory syndrome (sars) has been identified as a novel coronavirus sars-cov. similar to other coronaviruses, spike (s)-glycoprotein of the virus interacts with a cellular receptor and mediates membrane fusion to allow viral entry into susceptible target cells. accordingly, s-protein plays an important role in virus infection cycle and is the primary target of neutralizing antibodies. to begin to understand its biochemical and immunological properties, we expressed both full-length and ectodomain of the protein in various primate cells. our results show that the protein has an electrophoretic mobility of about 160–170 kda. the protein is glycosylated with high mannose and/or hybrid oligosaccharides, which account for approximately 30 kda of the apparent protein mass. the detection of s-protein by immunoassays was difficult using human convalescent sera, suggesting that the protein may not elicit strong humoral immune response in virus-infected patients. we were able to pseudotype murine leukemia virus particles with s-protein and produce sars pseudoviruses. pseudoviruses infected vero e6 cells in a ph-independent manner and the infection could be specifically inhibited by convalescent sera. consistent with low levels of antibodies against s-protein, neutralizing activity was weak with 50% neutralization titers ranging between 1:15 to 1:25. to facilitate quantifying pseudovirus-infected cells, which are stained blue with x-gal, we devised an automated procedure using an elispot analyzer. the high-throughput capacity of this procedure and the safety of using sars pseudoviruses should make possible large-scale analyses of neutralizing antibody responses against sars-cov. during the first epidemic of severe acute respiratory syndrome (sars), which began in november of 2002 in guandong province of the people's republic of china, and lasted for about 7 months, close to 8100 people were infected worldwide, among which 774 people died (who, 2003) . the etiological agent of this atypical respiratory disease has been identified as a novel coronavirus (designated as sars-cov) fouchier et al., 2003; ksiazek et al., 2003; peiris et al., 2003; poutanen et al., 2003) . with a mortality rate of over 9%, sars-cov had a major health and socioeconomic impact. fortunately, there have been very few incidences of sars infections during the winter season of [2003] [2004] . however, with multiple modes of virus transmission and a wide range of potential nonhuman reservoirs including wild animals commonly found in markets (e.g., civet cats and raccoon dogs; guan et al., 2003) as well as domestic cats (martina et al., 2003) , it is highly likely that a virus of this nature will most certainly resurface in the future. currently, there are no antiviral drugs, immunotherapeutic agents, or vaccines available against the virus. to better control or prevent future epidemics, anti-sars-cov drugs and/or vaccines need to be developed. sars-cov belongs to coronaviridae family. the genomic organization of the virus is similar to that of other coronaviruses with a general order of replicase (rep; orfs-1a and 1b), spike (s)-glycoprotein, envelope (e), membrane protein (m), and nucleocapsid (n) from 5v to 3v direction (marra et al., 2003; rota et al., 2003) (fig. 1) . several openreading frames have also been identified, which may encode additional proteins (marra et al., 2003; rota et al., 2003; snijder et al., 2003) . their functions, however, are not known at the present time. the protein of a major interest as a target of antiviral drug development efforts as well as for developing vaccines is s-glycoprotein. s-protein of coronaviruses, which is thought to function as a trimer (delmas and laude, 1990) , is responsible for both binding to cellular receptors and inducing membrane fusion for virus entry into target cells (collins et al., 1982; godet et al., 1994; kubo et al., 1994) . mutations in the protein have been shown to alter virulence and cellular tropism (fazakerley et al., 1992; leparc-goffart et al., 1998; sanchez et al., 1999) . taken together, the s-protein plays a critical role in the biology and pathogenesis of coronaviruses. not surprisingly, it is an important target of virus-neutralizing antibodies (chang et al., 2002; collins et al., 1982; fleming et al., 1983; godet et al., 1994; kant et al., 1992; kubo et al., 1993 kubo et al., , 1994 takase-yoden et al., 1991) . moreover, mice immunized with a recombinant s-protein, or a peptide derived from it, are protected from lethal challenges with murine hepatitis virus (mhv) (daniel and talbot, 1990; koo et al., 1999) . s-protein is a type i membrane glycoprotein, which is translated on membrane-bound polysomes, inserted into rough endoplasmic reticulum (rer), cotranslationally glycosylated, and transported to the golgi complex. during the transport, s-proteins are incorporated onto maturing virus particles, which assemble and bud into a compartment that lies between the rer and golgi (lai and holmes, 2001) . virions are carried from golgi to plasma membrane in secretory vesicles. virions are released from cells when virion-containing vesicles fuse with plasma membrane. excess s-proteins not incorporated onto virus particles are transported to the surface of plasma membrane (lai and holmes, 2001; tsai et al., 1999; yamada et al., 1998) . s-protein of sars-cov is 1255 amino acids long (fig. 1) . it is predicted to have a 13 amino acid signal peptide at the amino-terminus, a single ectodomain (1182 amino acids) and a transmembrane region followed by a short cytoplasmic tail (28 residues) at the carboxy-terminus (marra et al., 2003; rota et al., 2003) . due to low sequence homology between the s-protein of sars-cov and that of the other coronaviruses (marra et al., 2003; rota et al., 2003) , the structural and immunogenic properties of sars-cov sprotein must be ascertained experimentally. the cellular receptor for sars-cov has recently been identified to be angiotensin-converting enzyme 2 (ace2; li et al., 2003) . the molecular interactions between the s-protein and ace2 are not yet known. better understanding of the interactions could lead to development of virus entry inhibitors. neutralizing antibodies (nabs) play a critical role in protection against a variety of viral diseases. an accurate assessment of nab responses in virus-infected patients is needed to determine immune correlates of protection. it is also an essential and integral part of a vaccine development process. conventional virus-neutralization assays require the use of replication-competent, infectious viruses. evaluating virus-neutralizing activity of a large number of antisera with these assays is undesirable due to safety concerns, especially for a biosafety level 3 (bsl3) pathogen like sars-cov. the same safety concerns have prompted our laboratory to utilize replication-defective pseudoviruses for hiv-1 neutralization assay (kim et al., 2001) . in this assay, nonreplicating moloney murine leukemia virus (mulv) particles pseudotyped with hiv-1 envelope glycoproteins are used (schnierle et al., 1997) . these pseudoviruses encode a h-galactosidase gene, which allows detection of individual infected cells when stained with x-gal (5-bromo-4-chloro-3-indolyl-h-d-galactopyranoside). in this study, we report development of a sars-cov pseudovirus neutralization assay, which should be particularly valuable for researchers who may not have easy access to bsl3 containment facility. additionally, we describe a high-throughput system for quantitative analyses of x-gal stained cells. this assay system should facilitate rapid evaluation of antibody responses to vaccine candidates and/or entry inhibitors against sars-cov. to express sars-cov s-glycoprotein, we initially cloned a dna fragment encoding the protein into pcdna-3 vector (pcdna-s; fig. 1b ). to detect s-protein, western blot was performed with convalescent sera from sars-cov-infected patients. however, no clear protein band was detected despite number of attempts. we reasoned that one of the possibilities for the inability to detect the protein is a low level of s-protein expressed from pcdna-s. to increase the amount of sprotein expressed, we subcloned the s gene into phcmv-g vector (burns et al., 1993) , which expresses high level of vesicular stomatitis virus (vsv) g glycoprotein. although we were able to express higher amount of s-protein (see below), this was not sufficient to detect a clear band on western blots. an alternative explanation is that antibodies against the protein in convalescent sera cannot recognize s-protein subjected to denaturing conditions of sds-page (viz. linear epitopes). however, because results from radioimmunoprecipitation and indirect immunofluorescence assays were also ambiguous, it is most likely that the antibody titer against s-protein is very low in convalescent sera. to further increase protein expression level, s gene was subcloned into a ptm vector (moss et al., 1990) . with this vector, a protein of interest is under the control of a strong t7 rna polymerase (t7rnap) promoter and the protein is expressed when cells transfected with the plasmid are infected with a recombinant vaccinia virus expressing t7rnap (vtf7-3; fuerst et al., 1986) . the presence of encephalomyocarditis virus internal ribosome entry site (ires) at the 5v end of rna transcripts allows efficient translation of mrna transcribed in cytoplasm. using ptm-s, we were able to detect a faint, but distinct band of approximately 160 -170 kda by western blot (fig. 2a , lane 3). we also reevaluated pcdna-s as this vector has a dual promoter system (cmv and t7 promoter). using t7 promoter, we were able to detect a protein band of a similar size, albeit less clear than using ptm-s (lane 2). the lower expression of the protein is likely due to the lack of ires in the pcdna vector. because the calculated molecular weight of s-protein without 13 amino acid signal peptide is about 138 kda, the result suggested posttranslational modification (e.g., glycosylation). to better demonstrate this, we generated another clone (ptm-eshis) that expresses the entire ectodomain of s-protein (amino acids 1 -1190) with a six-histidine tag at the carboxy terminus. the ectodomain of s-protein migrated with an approximate molecular weight of 163 kda while its calculated molecular weight is only 131.2 kda (fig. 2b , lane 2). to demonstrate that this difference is due to glycosylation, eshis protein was treated with endoglycosidase h (endo-h) or peptide: n-glycosidase f (pngase f). as shown in fig. 2b (lanes 3 and 4), treatment with either glycosidase increased the mobility of the protein to approximately 133 kda. because the mobility of the protein treated with either glycosidases was the same, s-protein is most likely modified with high mannose and/or hybrid, rather than complex, oligosaccharides. while s-glycoprotein of some coronaviruses is cleaved into two subdomains, s1 and s2, the fact that we observed only a single band suggests that sars-cov s-protein functions as a single unit. despite difficulties in detecting s-protein directly by immunoassays, proteins expressed from both pcdna-s and phcmv-s constructs were able to pseudotype mulv particles to produce sars pseudoviruses that could readily infect vero e6 cells (fig. 3a) . none of the other cell lines we tested, including hela, a549, 293t, and bs-c-1, were susceptible. this is in contrast to vsv-g pseudotyped viruses, which could infect all cell lines (data not shown). the fact that bs-c-1 cells, which, like vero e6 cells, are african green monkey kidney cells, were not susceptible was somewhat unexpected. however, when we performed infections with a high multiplicity of infection, we were able to detect infected bs-c-1, albeit at a significantly reduced titer (7 -8-fold compared to vero e6 cells; data not shown). this result was not too surprising because it has been shown that even 293t cells, which express small amounts of ace2, support some basal level of sars-cov replication . many pseudovirus-infected cells appeared as a doublet, which is the result of a cell division following integration of mulv pseudovirus genome encoding h-galactosidase. these doublets are counted as a single infectious unit. a typical yield of sars pseudoviruses was about 2 â 10 4 infectious units per milliliter of culture supernatant using phcmv-s, which was about fivefold greater than using pcdna-3. this yield is comparable to what we have been able to achieve for hiv-1 pseudoviruses (between 2 â 10 3 and 2 â 10 4 depending on envelopes; kim et al., 2001) , but lower than vsv-g pseudovirus yield (between 3 â 10 4 and 9 â 10 4 depending on target cell lines used). interestingly, sars pseudovirus production was about 20-fold less efficient when plasmid transfection was performed by calcium phosphate method compared with using cationic lipids (lipofection). this difference, however, was not observed for vsv-g pseudovirus production. an additional difference was that a longer incubation time was needed to achieve peak pseudovirus production for sars-s compared to vsv-g (3 vs. 2 days posttransfection, respectively). the reasons for these discordant results are unknown at the present time. cellular entry of coronaviruses can occur either by acidic ph-dependent or -independent pathway (gallagher et al., 1991; lai and holmes, 2001; cavanagh, 1990, 1992; nash and buchmeier, 1997; payne et al., 1990) . to investigate whether sars-cov infection requires low ph, we examined sensitivity of pseudovirus infections to lysosomotropic agents chloroquine and nh 4 cl. as expected, infectivity of viruses pseudotyped with vsv-g was reduced by chloroquine and nh 4 cl in a dose-dependent manner 3 and 4, respectively) . the protein was detected by western blot with anti-6âhis antibody. no band was detected from cells transfected with an empty vector (lane 1). acrylamide gradient gel (4 -12%) was used. ( fig. 3b and c, respectively) . in contrast, sars pseudovirus infection was virtually unaffected, suggesting that sars-cov infection proceeds in an acidic ph-independent manner. to assess whether sars pseudoviruses we generated could be used to quantify virus-neutralizing antibodies, we examined their susceptibility to convalescent sera from sars-cov-infected patients. as shown in fig. 4a , sera from two patients were able to specifically neutralize sars pseudoviruses; the same convalescent sera could not neu-tralize hiv-1 or vsv-g pseudoviruses and no neutralizing activity was observed with a normal serum. to determine neutralizing antibody titers in virus-infected patients, we performed the assay with serially diluted sera from seven patients. as shown in fig. 4b , antibody levels were quite similar in all patients with 50% neutralization titer between 1:15 and 1:25. although the pseudovirus neutralization assay is sensitive, quantitative, and safe, it has one disadvantage of having to count individual x-gal-stained cells through a microscope. to overcome this problem, we looked into a possibility of automating the data collection procedure using an elispot reader (immunospot analyzer, cellular technology ltd.). although this instrument is commonly used to quantify antigen-specific t cell cytokine responses by counting chromogenic immunospots (e.g., ifn-g), we rationalized that it might be able detect x-gal-stained blue cells. as shown in fig. 5 , there was no problem with using the instrument to count spots at a single-cell resolution and the analysis was highly efficient as the entire 96-well plate could be processed in less than 20 min. virus-infected cells appearing as doublets did not pose a problem because parameters on the analysis software could be adjusted to count two stained cells adjacent to each other as one. the number of infectious foci counted was quite linear as a function of virus inoculum (fig. 5c ), validating the methodology. this procedure could be used to quantify other assays based on x-gal staining of cells (e.g., recombinant vaccinia viruses that express h-galactosidase). in this study, we expressed sars-cov s-glycoprotein, which was able to pseudotype mulv particles. sars pseudoviruses were able to efficiently infect vero e6 cells, which have been shown to support sars-cov infection. the infection did not require low ph, suggesting viral entry is mediated by a direct fusion event between viral and plasma membranes. this result is consistent with a previous report that cell-to-cell fusion mediated by sprotein and its cellular receptor ace2 occurred at neutral ph (xiao et al., 2003) . however, our result is in direct disagreement with recently published article by simmons et al. (2004) . there are three major differences in exper-imental procedures between the two studies. first, we pseudotyped mulv particles whereas simmons et al. used hiv-1. second, we used an authentic s-glycoprotein whereas they used a c-terminal fusion protein that included a v5 epitope and polyhistidine tag, which totaled, by our estimation, 27 extra amino acids. whether the discrepant result is due to the use of different s-glycoproteins and/or different virus cores needs to be further investigated. the third difference between the studies is the concentrations of lysosomotropic agents used. while we used nh 4 cl at 0-50 am amounts, which are sufficient to inhibit vsv-g-mediated fusion ( fig. 3 ; picard-maureau et al., 2003) , they used millimolar (mm) amounts. at these concentrations, nh 4 cl could have a secondary effect on sglycoprotein. we were unable to find concentrations of chloroquine used in their study. it is interesting to note that while simmons et al. observed that pseudovirus infections required low ph, s-protein-mediated cell-to-cell fusion did not. the sars pseudoviruses we generated could be specifically inhibited by convalescent sera from sars-cov infected patients, indicating that s-glycoprotein of sars-cov is a target of neutralizing antibodies as it is for other coronaviruses. the major purpose of generating sars pseudoviruses was to devise an assay system to assess virus-neutralizing antibodies safely and rapidly without having to use infectious, replication-competent sars-cov. the results of our study indicate that sars pseudoviruses could be used to evaluate efficacy of various s-glycoprotein-based vaccine candidates to elicit virus-neutralizing antibodies. they could also be used to perform structurefunction analyses of s-glycoprotein. due to a large size of sars-cov genome, it would be difficult to perform such analyses directly in the context of the virus, not to mention potential safety hazards from working with it. in contrast, mutational analyses of the protein could be performed readily using pseudoviruses. our attempt to characterize biochemical and immunological properties of the s-protein was hampered by the fact that antibody titers against the protein in convalescent sera were extremely low; we were able to identify only a faint band on a western blot (with high background) and attempts to detect the protein by immunofluorescence and radioimmunoprecipitation assays were less than successful. in contrast, convalescent sera have been successfully used to detect sars-cov-infected cells by an immunofluorescence assay (hsueh et al., 2003; peiris et al., 2003) . together, the available data seem to suggest that s-protein might not be immunogenic, at least compared to other viral proteins. in fact, immunoreactivity analyses of a panel of synthetic peptides derived from s, membrane (m), and nucleocapsid (n) proteins suggested that n protein might be the most immunogenic protein . the nonimmunogenic nature of s-protein might present potential problems in developing a vaccine that can elicit potent neutralizing antibodies against sars-cov. in this regard, it is interesting to note that s-protein is highly glycosylated with 23 potential asparagine-linked glycosylation sites. based on our analyses of the ectodomain of the protein, carbohydrate residues account for approximately 30 kda (based on mobility in sds-page). the glycans were primarily high mannose and/or hybrid type. this, however, needs to be verified using proteins produced from nonvaccinia virus expression system, because the virus infection could possibly affect cellular glycosylation machinery. extensive glycosylation of hiv-1 envelope glycoprotein has been one of the major obstacles in eliciting good humoral responses against the protein and in developing an effective vaccine against the virus (cho, 2003) . it remains to be seen whether and to what extent glycans on s-protein affect immunogenic properties of the protein. interestingly, potential glycosylation sites are clustered into three regions of the protein (fig. 1a) : n-terminal, middle, and c-terminal. it has been shown that individual glycosylation sites on hiv-1 surface glycoprotein gp120 may have different functions; while some are important for evading immune responses, others are critical for maintaining proper protein structure necessary to interact with cellular receptors and mediate membrane fusion (ogert et al., 2001; reitter et al., 1998) . additional studies are needed to determine whether glycosylation sites in different clusters of s-protein serve different functions. in the absence of an effective vaccine and/or antiviral drugs against sars-cov, early detection of virus-infected patients would be critical for effective containment of future epidemics. quantitative rt-pcr-based diagnostic assays have been described for sars-cov (grant et al., 2003; lau et al., 2003; ng et al., 2003; poon et al., 2003a poon et al., , 2003b tang et al., 2004; yam et al., 2003) . despite high sensitivity, their utility has some limitations: (i) the detection rate varies widely between 20% and 80% depending on clinical sam-ples and protocols used for the assay; (ii) the window of detectability is limited to early stages of infection; and (iii) the assay is not suitable for routine surveillance. antibodies against sars proteins have been shown to appear as early as 9 days after the onset of illness (hsueh et al., 2003) . therefore, development of a high-throughput serologybased diagnostics could complement pcr-based assays. in this regard, a virus-neutralization assay could be used as a confirmatory test, which would enhance the accuracy of early diagnosis of sars-cov. because neutralizing antibodies are important for virus clearance, the assay could also be used to assess disease prognosis. in either case, the availability of sars pseudoviruses allows avoiding the use of infectious sars-cov. the overall cloning strategy is shown in fig. 1b . two parental plasmids encoding a sars-cov s gene (urbani strain), pentr-s and pcr-s, were obtained from the u.s. centers for disease control and prevention. two s-proteinexpressing plasmids (pcdna-s* and pcdna-s) were generated using pcdna-3 (invitrogen). the s gene in pcdna-s*, which was transferred from pentr-s (bamhi -ecori fragment), lacks the original translation stop codon taa because it was changed to aat of ecori restriction site (gaattc). pcdna-s with a stop codon was constructed by replacing a swai-ecori fragment of pcdna-s* with the same fragment from pcr-s. to generate phcmv-s, a bamhi -ecori fragment from pcdna-s was inserted into a bamhi site of phcmv-g following blunting ends with klenow. to construct ptm-s, a bamhi -xhoi fragment from pcdna-s was cloned into the corresponding sites of ptm-ndei (cho et al., 1994) . despite the fact that ptm-s has a small open-reading frame that encodes eight amino acids between the internal ribosome entry site of ptm-ndei vector and the s gene, s-protein was efficiently expressed and the plasmid was used as is without further modification. to generate ptm-eshis, 3v end of the ectodomain was pcr amplified using a sense primer 5v-gtc gtc aac att caa aaa gaa-3v (nts 3472 -3492 of s gene) and an antisense primer 5v-aat gaa gcg gat cccggg tta gtg atg gtg gtg atg atg ttg ctc ata ttt tcc caa-3v. base-pairing region (nts 3553-3570) is shown in bold and the six histidine residues are italicized. the amplified fragment was digested with swai (nt 3521) and smai (underlined) and subsequently cloned into ptm-s digested with swai and stui. cell culture, protein expression, and western blots all cell lines, except for vero e6, were maintained in dmem supplemented with 10% fetal bovine serum (fbs), 2 mm l-glutamine, and penicillin -streptomycin antibiotics. vero e6 cells were maintained in emem with the same supplements plus 0.1 mm nonessential amino acids. cells were cultured at 37 jc in 5% co 2 incubators. to express sprotein, cells were transfected with plasmids by a calcium phosphate precipitation method. briefly, 0.5 ml of 0.25 m cacl 2 solution containing 30 ag of plasmids was slowly mixed with 2â hbs (50 mm hepes, 1.5 mm na 2 hpo 4 , 280 mm nacl, ph 7.1) and the mixture was added to cells. after an overnight incubation, culture medium was replaced and cells were further incubated for two additional days. for expression from ptm-s and ptm-eshis, transfected cells were infected with vtf7-3 (fuerst et al., 1986 ) at a multiplicity of infection of 5. following 2 days of infection, cells were lysed with a hypotonic cell lysis buffer (10 mm tris, ph 8.0, 10 mm nacl, 1.5 mm mgcl 2 , 1% np-40). insoluble cell debris and nuclei were removed by a brief centrifugation in a microfuge. cell lysates were subjected to sds-page and western blot. s-proteins were detected with either a pool of convalescent sera (1:100 dilution) or anti-his (c-terminal) monoclonal antibody (invitrogen; 1:3000 dilution) followed by horseradish peroxidase-conjugated goat anti-human or anti-mouse igg antibody (pierce), respectively. protein bands were visualized using supersignal west pico chemiluminescence detection system (pierce). molecular weights of the protein bands were approximated by the mobility of standard molecular weight markers. pseudoviruses were generated as previously described (kim et al., 2001) . briefly, mulv packaging cell line telceb6 (schnierle et al., 1997) was transfected with pcdna-s, phcmv-s, phcmv-g (burns et al., 1993) , or pltr-gp140 (hiv-1 dh12 ; kim et al., 2001) using either calcium phosphate precipitation or lipofection (lipofectin; invitrogen) method. two days posttransfection (3 days for sars pseudovirus), cell culture medium was harvested and subjected to centrifugation (1700 â g, 10 min) to remove cell debris. supernatant was aliquoted, stored at à80 jc and used as a virus stock. virus titer was determined in vero e6 cells for sars-s and vsv-g or in hos-cd4-ccr5 (cheng-mayer et al., 1997; deng et al., 1996) for hiv-1 gp140 pseudotyped viruses. typically, cells were infected with 60-80 infectious units for 36 h. cells were washed with pbs and incubated with a fixative (1% formaldehyde, 0.05% glutaraldehyde in pbs) for 10 min at room temperature. the cells were washed twice with pbs and incubated with a freshly prepared staining solution (pbs containing 5 mm potassium ferricyanide, 5 mm potassium ferrocyanide, 2 mm magnesium chloride, and 1 mg/ml of x-gal) for >2 h at 37 jc. for routine analyses, x-gal-stained blue cells were manually counted using an inverted microscope. to determine ph-dependency of viral entry, vero e6 cells were incubated in culture medium containing 0 -100 am chloroquine for 1 h at 37 jc before adding viruses. vsv-g or sars-s pseudoviruses were allowed to adsorb to cells for 1 h at 37 jc in the absence of chloroquine. following adsorption, virus inoculum was removed, cells were washed, and infection was allowed to proceed for about 36 h in the absence of chloroquine. for nh 4 cl, cells were incubated with 0 -50 am. due to minimal cytotoxicity, nh 4 cl was present throughout the infection period including 1 h incubation before virus addition. all infections were done in duplicates. neutralization assay was performed as previously described (kim et al., 2001) using convalescent sera (13 -50 days post-onset of symptoms) obtained from cdc or from patients hospitalized in queen mary hospital, hong kong. approximately 60 -80 infectious units of pseudoviruses were incubated with serially diluted, heat-inactivated (56 jc, 30 min) convalescent or normal sera for 1 h at 37 jc. the mixture was subsequently added to vero e6 (for sars-s or vsv-g) or hos-cd4/ccr5 (for hiv-1 gp140) cells. virus infection was allowed to proceed for another 36 h. virus-neutralizing activity was determined relative to no serum control. the general pseudovirus infection procedure is the same as described above. the major difference was that 96-well 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1 the sars-cov s glycoprotein: expression and functional characterization evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus requirement of proteolytic cleavage of the murine coronavirus mhv-2 spike protein for fusion activity we are grateful to cdc for providing plasmids encoding sars-cov s gene and convalescent sera, to dr. bernard moss for vtf7-3, to dr. franc ßois-loïc cosset for telceb6 cell line, and to drs. jonathan silver and mario skiadopoulos for vero e6 cells. hos-cd4-ccr5 cell line was obtained from dr. nathaniel landau through the aids research and reference reagent program, division of aids, niaid, nih. we specially thank dr. magdalena tary-lehman for providing assistance with using immuno-spot analyzer. key: cord-103509-hynnba03 authors: wong, ten-tsao; liou, gunn-guang; kan, ming-chung title: a self-assembled protein nanoparticle serving as a one-shot vaccine carrier date: 2020-09-18 journal: biorxiv doi: 10.1101/2020.09.16.299149 sha: doc_id: 103509 cord_uid: hynnba03 in this paper, we are exploring the role of an amphipathic helical peptide in mediating the self-assembly of a fusion protein into a protein nanoparticle and the application of the nanoparticle as a one-shot vaccine carrier. out of several candidates, an amphipathic helical peptide derived from m2 protein of type a influenza virus is found to stimulate high antigenicity when fused to a fluorescent protein genetically. this fusion protein was found to form protein nanoparticle spontaneously when expressed and purified protein stimulates long-lasting antibody responses in single immunization. through modeling peptide structure and nanoparticle assembly, we have improved this vaccine carrier in complex stability. the revised vaccine carrier is able to stimulate constant antibody titer to a heterologous antigen for at least six months in single immunization. the immune response against a heterologous antigen can be boosted further by additional immunization in spite of high immune responses to carrier protein. subunit vaccine is a safe alternative to traditional inactivated or attenuated vaccines, but its efficacy is often hindered by the low antigenicity of recombinant protein. different approaches are utilized to resolve this issue, among them, virus like particle (vlp) and self-assembled protein nanoparticle (sapn) are considered the best platforms for subunit vaccine development(1). vlp is assembled from a recombinant capsid protein alone without the genomic nucleic acid and bound nucleocapsid protein so it is non-infectious (2) . the size of vlp is ranged between 20-200 nm that facilitates both draining efficiently to lymph node and also uptake by antigen presenting cells like dendritic cell and macrophage (3) . the other benefit of vlp based vaccine is the induction of b cell receptor clustering when presenting repetitive antigen to b cell, a function that can activate antibody class-switch and somatic hypermutation in a t cell dependent mechanism (4) . not only the virus like particle can be used for vaccine directly, heterologous antigen can be presented in the particle surface through genetic fusion (5) . the universal flu vaccine candidate, ectopic m2 domain (m2e), was genetically fused with hepatitis b core antigen(hbc) and assembled into a nanoparticle that provides full protection to homologous flu strain (6) . but the application of hbc based vlp in human vaccine development is restricted by the pre-existing anti-hbc antibody present in the 450 million chronic hbv carriers and the population exposed to hbv infection (7) . an artificially designed sapn may avoid the effect of the existing antibody. a sapn assembled from protein constituted by two coiled-coil domains that form trimer and pentamer respectively can be assembled into nanoparticles with specific sizes (8, 9) . this sapn stimulates strong immune responses to target protein fused to the terminal of constituting monomer after 3 immunizations even without adjuvant, but this immunity waned gradually (10) . the green fluorescent protein is a member of fluorescent protein family that are structurally conserved and emit fluorescent light from a chromophore when excited by photons of shorter wavelength (11) . the shared features of fluorescent proteins including a sturdy barrel shaped structure constituted by 11 β-sheets and an enclosed chromophore that emits fluorescent light when excited (12) . the function of the barrel shell is to provide a well organized chemical environment to ensure the maturation of chromophore and protects it from hostile elements (13) . so it is conceivable that the protein sequences among fluorescent protein family members in the barrel shell are highly variable and fluorescent proteins possess desirable biophysical properties can be selected using directed evolution (11, (14) (15) (16) (17) . the applications of fluorescent protein have been expanded into multiple areas beyond live imaging which includes serving as biological sensors (18, 19) , or detectors for protein-protein interaction or protein folding (20, 21) . amphipathic α-helical peptide (ahp) forms hydrophilic and hydrophobic faces when folded and is often identified in proteins related to phospholipid membrane interaction. the n-terminal amphipathic helical peptide is required for membrane anchorage of hepatitis c virus ns3 protein and the protease function of the ns3/ns4a complex (22, 23) . several anti-microbial peptides also possess amphipathic properties and function by forming membrane pores or causing membrane disruption (24) . the amphipathic α-helical of type a influenza virus m2 protein is required for m2 protein anchorage and induces membrane curvature required for virus budding (25, 26) . life protection from diseases through vaccination is considered a holy grail only achievable by some live attenuated viral vaccines with multiple doses up to date (27) . here in this report, we are describing the identification of a protein nanoparticle based on an amphipathic α-helical peptide (ahp) from m2 protein of type a influenza strain h5n1 and a green fluorescent protein. we have predicted the protein nanoparticle structure according to transmissive electronic microscope images using protein modeling and generated ahp mutants that provide higher stability and antigenicity to the protein nanoparticle. this modified protein nanoparticle is able to simulate long constant antibody response to an inserted heterologous antigen in single immunization without adjuvant. and this antibody response can be further boosted by additional immunization. the identification and application of this nanoparticle as a vaccine carrier was explored in this study. identification of an ahp-gfp protein complex with high antigenicity and stability. as described in our patent application filed in 2015, we have tested the immunogenicity of fusion proteins composed of an ahp and a gfp (28) . the results showed an increase of anti-gfp igg titer ranged between 2~3 log under a two immunizations regime ( figure s1 ). one of the peptides, ah3, that derived from m2 protein of type a influenza strain h5n1 gives extended stability to the gfp fusion protein when compared to another peptide, ah1 ( figure s2 ) as well as other peptides in our study (data not shown). since a stable protein is essential for vaccine carrier and may profits worldwide vaccination effort, we were interested in the mechanism of ah3-gfp stability and antigenicity. to study the potential mechanisms that contribute to the above mentioned properties of ah3-gfp fusion protein, we first checked the composition of ah3-gfp protein post expression and purification. one clue that led us to study the composition of ah3-gfp fusion protein is the difficulties encountered during protein purification. unlike other fusion proteins studied, both ah3-gfp and ah5-gfp fusion proteins are mostly expressed as insoluble inclusion body and the remaining soluble protein did not bind to ni-nta resin under normal condition of 300mm nacl. the ah3-gfp and ah5-gfp fusion proteins only started to bind to ni-nta resin after lowering the nacl concentration from 300mm to 50mm, an indication that hydrophobic interaction may induce a protein complex with n-terminal his tag hindered from binding to ni-nta ligand. also, the resistance of ah3-gfp fusion protein to hydrolysis suggested the linker between ah3 peptide and gfp is kept in a water tight complex. from these two clues, it was hypothesized that ah3-gfp fusion protein form stable protein complex through hydrophobic interaction mediated by n-terminal ah3 peptide. to test the hypothesis that ah3-gfp or ah5-gfp fusion protein forms a protein complex, we first used the protein concentration tube with different molecular weight cut off (mwco) to determine the protein complex sizes. as shown in figure 1a , when gfp protein with a molecular weight of 27kda is able to pass through membranes with mwco of 100kda, 300kda and 1000kda freely, ah3-gfp fusion protein purified from bacterial lysate was prevented from passing through the membrane with an mwco of 1000kda. with a molecular weight of 33kda, the purified ah3-gfp protein need to form a complex with more than 30 monomers to be excluded from passing a membrane with a 1000kda mwco. to explore further the geometric composition of the ah3-gfp protein complex, we examined the fusion protein under transmissive electronic microscope (tem). the tem results showed the ah3-gfp fusion protein forms a cylinder-like structure with length up to ~60 nm and a diameter around 10 nm (fig. 1b) . the difference in length suggests that the particle may be assembled along the long axis. when scanning along the long axis of the ah3-gfp particle, there is a repetitive pattern of two-one-two-one of white dots with two less visible dots on each side of the single dot. the predicted structure according to tem images is shown in fig.1d . we also examined protein geometric composition of ah5-gfp under tem, but there is no clear evidence of forming higher order protein complex, suggesting ah5-gfp protein complex is not as stable as ah3-gfp to withstand the conditions during negative staining. to find the correlation between protein complex formation and antigenicity, we immunized mice with purified ah3-gfp fusion protein and the recombinant gfp protein that pass through the membrane freely. proteins were prepared from lps synthesis defective e. coli strain, clearcoli bl21(de3), to avoid the interference of lps contamination, a known tlr4 ligand. the mice were immunized with purified proteins by single intramuscular injection and sera were collected at day 7, 14, 30 and 182 to evaluate anti-gfp igg titer by elisa. deoxycholate was added to test if deoxycholate in the concentration of 0.2% affects ah3-gfp antigenicity and related experiment was terminated at 30 days post immunization when it showed no effect on antigenicity of either gfp or ah3-gfp. these results suggest gfp alone is a poor antigen and only gained high antigenicity after fused with ah3 peptide (fig. 1c) . to understand the potential molecular mechanism leading to the assembly of ah3-gfp nanoparticle, we tried to build a protein model based on three observations: first, the particle was assembled through hydrophobic interaction and second, the particle assembled along the long axis and the third: ah3-gfp protein particle has a repetitive three-two pattern when observed under tem. we first assembled the two ah3 peptide as anti-parallel helices with hydrophobic sidechains of f4, f5, i8, l12 and l16 mediating intermolecular contacts. the assembled ah3 dimer created a hydrophobic core between two helices and the helix dimer is surrounded by hydrophilic sidechains from multiple lysine and arginine except one exposed hydrophobic patch, as predicted using deepview and marked by white mesh (fig. 2a) . this hydrophobic patch can be seen only to cover one face of the dimer (fig. 2b ). when the water accessible surface of ah3 dimer was calculated, two cavities could be seen located within the hydrophobic patch that provides contact points for two arg9 sidechains extruding from the opposite face of ah3 dimer. a second ah3 dimer can make close contact with the first dimer after turning counter clockwise looking down the hydrophobic patch for 36 o and forms a tetramer (fig. 2c) . the intermolecular energy between two dimers from this model was calculated to has a δg of -101 kcal/mol (fig. 2c) . after adding gfp protein structures onto the ah3 tetramer model, the ah3-gfp fusion protein tetramer will form a cross-shaped assembling unit and the stacking of every ah3-gfp tetramer on top of another tetramer will extend the particle length by 2.8 nm and turning the cross by 72 o . since the gfp protein barrel diameter is ranged between 2.7~3.5 nm, the out extending gfp from ah3-gfp tetrameric cross can spatially fit with the model (fig. 2d ). under this model, the protein nanoparticle will be extended continuously with a hydrophobic patch presenting on one end of the assembled particle constitutively and serving as a point for polymerization. after proving that the ah3-gfp protein complex possesses high antigenicity, we decided to explore the application of ah3-gfp protein nanoparticle as a vaccine carrier. for the hepatitis b core antigen, the amino acid 144 served as an insertion site for heterologous antigen fusion (29) . gfp protein has a thermal stable structure that constituted by 11 β-strands and 3 α-helices and some of the loops between strands have been explored as insertion sites for heterologous protein for various purposes (18, 19, 30) . among those candidates, loop173 linking strand 8 and strand 9 was chosen because it has a high capacity for foreign peptide insertion (fig 3a) (30) . the original ah3-gfp recombinant protein is constructed in pet28a vector with ah3 coding region inserted c-terminal to his-tag and thrombin cleavage site followed immediately by gfp cloned from pegfp-c2. this expression vector was low in soluble protein productivity and unable to express soluble recombinant protein when the peptide is inserted between d173 and g174. to resolve the expression and folding issues, we designed a new expression vector. first, we cloned ah3 peptide into the very n-terminal following methionine in the pet27 vector, and then to its c-terminal we inserted a synthesized sfgfp gene (31) with a created antigen insertion site following 175s of sfgfp. the antigen insertion site also included an 8xhis tag for recombinant protein purification. to verify vaccine carrier function, we inserted two copies of broad spectrum flu vaccine candidate, human m2 ectopic peptide (hm2e) separated by a 6 amino acid linker (fig. 3b) . the newly constructed vector was proven to be efficient for expressing soluble ah3-sfgfp-2hm2e fusion protein as a protein complex (data not shown). the ah3-sfgfp-2xhm2e protein complex under tem is not as stable as ah3-gfp unless first cross-linking the protein preparation with a heterobifunctional protein crosslinker, sulfo-smcc (fig. 3c ). following the previous established ah3-gfp protein model, we were seeking strategies to create a more stable ah3-sfgfp protein complex. first, we found the mutation of isoleucine 8 to leucine increase the intermolecular interaction(δg) from -16 kcal/mol to -37 kcal/mol (fig.3d) . second, we mutated lysine 13 to glutamic acid and generated additional electrostatic interactions between side chains of glu13 with arg10 and arg11 (fig. 3e ). to verify whether the protein modeling results are correct, including the presence of hydrophobic patch on ah3 peptide complex and a higher stability of ah3 based protein complex with i8l and/or k13e mutations. we first generated mutations in ah3 peptide in the context of ah3-sfgfp-2xhm2e construct (fig. 4a ). our hypothesis is that the hydrophobic patch of the ah3-gfp complex will bind bacterial membrane and co-sediment with it during ultracentrifugation. and then the methodology was verified by first centrifuge bacterial lysate prepared from clearcoli culture in a centrifuge tube preloaded with 15%(w/v), 45%(w/v) and 85%(w/v) sucrose solution in the volume of 7ml, 2ml, 1ml respectively ( fig. 4b left panel) . the distribution of bacterial membrane was marked by a lysochromic dye, sudan iii. the control sample contained sudan iii with lysis buffer alone (fig. 4b lane 1) . after ultracentrifugation, the bacterial membrane is sedimented to the junction of 15%/45% sucrose solution (fig.4b lane 2) . using the same protocol, ah3-gfp was found to co-sedimented with the bacterial membrane (fig. s3a) as well as the ah3-sfgfp-hm2e fusion protein but not a free gfp protein (fig s3b) these results are consistent with our protein structure modeling that arg11 serves as a main contact point for dimer stacking also it mediates the electrostatic interaction with mutated glu13 (fig. 3e ). as shown in the sucrose step gradient result, the presence of hydrophobic patch enables nonspecific interaction of the ah3-gfp protein complex with phospholipid membrane. which may restrict the free moving of protein nanoparticle and keep it from reaching draining lymph node for stimulating immunity (4) . to compare the antigenicity of protein complexes derived from either ah3-sfgfp-2xhm2e or lyrrle-sfgfp-2xhm2e, we immunized mice with a single injection of either recombinant proteins. post immunization, sera were collected at day 15, 50, 90 and 202 to evaluate anti-hm2e igg titer by elisa. the geometric mean titer of anti-hm2e igg reached the highest point for the ah3-sfgfp -2xhm2e group and then declined afterward. but of the lyrrle-sfgfp-2xhm2e group, the gmt reached highest point at day 50 and remained steady up to day 90 (fig. s4) . when the individual mouse serum result is observed separately, only one out of 5 mice from ah3-sfgfp-2xhm2e group has higher anti-hm2e igg titer at day 202 than day 15. but there are 4 out of 5 mice from the lyrrle-sfgfp-2xhm2e group shows a higher antibody titer in day 202 compared to day 15 (fig. 5a) . these results suggest that the two point mutations of ah3 in i8l and k13e enable the formation of a stable, high antigenic protein complex that stimulates long lasting immune responses in a single immunization. vaccine carrier like hbc based virus like particle (vlp) is often failed at boosting humoral immune responses after prime dose in a multiple doses protocol due to antigen competition. although gfp is a protein of low antigenicity, the fusion with ah3 strongly enhances its antigenicity as shown in figure 1c . to test if sfgfp backbone competes with inserted hm2e peptide for immune machinery, we immunized mice in a prime-boost protocol using the same protein preparations. the two consecutive injections were carried out 14 days apart and sera collected at day 14, 28 and 90 were subject to elisa assay using either hm2e peptide or sfgfp protein as coating antigen. the result shows the igg titer against hm2e elevated continuously after consecutive immunizations for both proteins as well as anti-sfgfp igg titer. the result suggests that although carrier protein ah3-sfgfp also has high antigenicity, it did not interfere with the immune response against the heterologous protein, hm2e (fig. 5b, 5c) vaccine as a tool to prevent infectious disease is the most cost effective strategy. especially for attenuated viral vaccines like vaccinia, mmr or oral polio vaccine, they produce long lasting even life time protective immune responses but these attenuated viral vaccines took decades for development. apparently, this strategy will not be able to timely develop a vaccine to ward off emerging global pandemic like covid-19. although the new vaccine technology like dna vaccine, mrna vaccine or adenovirus based vaccine that can quickly develop a subunit vaccine after the genomic information of pathogen become available, but the immune responses generated are often declined to base level within a year (32) (33) (34) (35) . this short lived immune response may expose vaccinated people to the risk of antibody dependent enhancement (ade) that is known to be devastating and leads to vaccine failure (11, 36) . in this study, we have created a self-assembled protein nanoparticle composed of ah3-fp and a more stable variant that stimulates long lasting antibody responses. the nature of this long lasting immune responses is not known but it may be mediated by long lasting plasma cell generated during ah3-fp immunization, the same mechanism that accounts for the lifelong protection of attenuated viral vaccines (27) . fluorescent protein family is a group of proteins with conserved barrel shaped structure with chromophore buried inside. since the function of this beta strand constituted barrel is to provide a suitable environment for chromophore maturation, the primary sequence of fluorescent protein barrel is prone to mutagenesis through either direct selection (11) or evolution (12) . also, fluorescent proteins of desired biophysical properties like thermal stability or folding efficiency can be obtained expert review of vaccines key: cord-265642-7mu530yp authors: syomin, b. v.; ilyin, y. v. title: virus-like particles as an instrument of vaccine production date: 2019-06-17 journal: mol biol doi: 10.1134/s0026893319030154 sha: doc_id: 265642 cord_uid: 7mu530yp the paper discusses the techniques which are currently implemented for vaccine production based on virus-like particles (vlps). the factors which determine the characteristics of vlp monomers assembly are provided in detail. analysis of the literature demonstrates that the development of the techniques of vlp production and immobilization of target antigens on their surface have led to the development of universal platforms which make it possible for virtually any known antigen to be exposed on the particle surface in a highly concentrated form. as a result, the focus of attention has shifted from the approaches to vlp production to the development of a precise interface between the organism’s immune system and the peptides inducing a strong immune response to pathogens or the organism’s own pathological cells. immunome-specified methods for vaccine design and the prospects of immunoprophylaxis are discussed. certain examples of vaccines against viral diseases and cancers are considered. immunoprophylaxis has long been used to control infectious diseases exerting an enormous impact not only on the global health but also on the safety of numerous populations of agricultural animals. in the past decades, the focus on vaccine production technologies has changed from handling intact pathogens to producing recombinant subunit vaccines based on isolated target antigens [1] . intensive development of recombinant vaccines began in the 1980s when it became possible to clone the desired dna sequences into expression plasmids and produce the target proteins. the construction of a recombinant vaccine takes advantage of the knowledge of the nucleotide sequences of genes encoded by a pathogen, and involves identification of the antigenic determinant, synthesis of the nucleotide sequence encoding the antigen, its cloning into an expression vector, and production of the target peptide in a certain expression system. the expression systems for individual heterologous proteins has been developed based on the prokaryotic and eukaryotic cells, which allow us to produce target proteins both on laboratory and industrial scales [2] . the interest in recombinant vaccines is a consequence of the emergence of new infectious diseases, most often zoonotic ones. among them are the outbreaks of human diseases caused by the ebola virus, zika virus, marburg virus, the middle east respiratory syndrome, and severe acute respiratory syndrome coronaviruses [3] . oncology also places great hopes in recombinant vaccines, which may help to overcome immune tolerance in the case of cancer treatment [4] . moreover, there exists a constant threat of the emergence of new highly virulent strains of well-known viruses due to unceasing mutational processes in virus genomes [5] . against this background, a new scientific concept, vaccinomics [6] , which consists of identifying the minimum subset of antigens, which are able to induce a competent immune response to a pathogen or tumor by their specific interaction with the b and t immune cells, is being actively developed. using this approach it appears possible to design vaccines based on the minimum subset of antigens which most specifically characterize the pathogen in its interaction with the immunome (the set of all immune receptor sequences, which are present in the individual organism) [7] . target detection of epitopes, the regions located on the surface of the protein, is possible with the aid of x-ray analysis, which is rather labor-consuming since it requires obtaining protein crystals. presently, the 3-d protein structure modeling has found broad use. this approach is based on identification of the physicochemical and electrostatic characteristics of the polypeptide chain regions and correlation of these characteristics with antigenic properties. two strategies are currently utilized, namely, modeling by homology and abbreviations: vlp, virus-like particles. udc 636.2+611:619: 616-006.446 de novo modeling. in the first case, the protein data bank (http://www.ncbi.nlm.nih.qov/genbank/) database of 3-d protein structures is used to model the protein. the commonly used modeller [8] , as well as other software such as i-tasser [9] , swiss-model [10] , esypred3d [11] , 3d-jigsaw [12] , phyre [13] , and cphmodels [14] , can be used as homology modeling tools. the limitation of this approach lies in the requirement that the structures of homologous proteins should demonstrate more than 30% identicality [15] . the de novo modeling strategy is based on the computer simulation of the protein folding process (rosetta [16] and tasser [17] software). in this case, all possible variants of polypeptide chain folding are considered, and the most energetically favorable conformation, i.e., the one with the lowest potential energy, is chosen. predicted antigenic determinants are synthesized using genetic engineering vector techniques and heterologous protein expression systems, and then are experimentally tested using, for example, the antibody neutralization assay. using protein expression systems it is possible to produce virus-like particles (vlps), which are made up of monomers, which are able to multimerize into vlps, and display the antigenic determinants of target pathogens on their surface. this point will be addressed further in the review, we will just observe here that even complex epitopes represented by trimers may be obtained using heterologous expression systems. this has been demonstrated, for instance, for the trimeres of the human immunodeficiency virus (hiv-1) envelope glycoprotein [18, 19] and influenza virus haemagglutinin [20] . in order to choose the most specific antigens of the pathological agent of a new infectious disease, it appears inevitable to work with the infected material if only to isolate and sequence the nucleic acids containing the genetic information of the pathogen. however, in order to produce a vaccine based on epitopes characteristic for the target pathogen, there is no need to work with the pathogen itself. therefore, apart from all other advantages of the vaccines of this kind, they appear to be improved in terms of biological safety. vaccines based on the presentation of a subset of antigenic determinants of the infectious agent are characterized by a high level of reproducibility when commercially manufactured and are highly effective [21] , since in this case, the immune response is directed exclusively against the most significant antigenic elements of the pathogenic microorganism or tumor. a number of vaccines specifically interacting with certain immune system receptors have been tested to date. for example, a vaccine containing only a single epstein-barr virus epitope specifically recognized by cd8 + t-cells is proposed for the prophylaxis of mononucleosis in human [22] . this vaccine prevents the development of the disease, although it does not protect the organism from the entry of the virus. another example is classical swine fever virus (csfv). it has been demonstrated that the e2 viral protein produced in the baculovirus expression system induces the synthesis of csfv-neutralizing antibodies in pigs [23] . vaccines are designed to produce immunity to a disease. although a single epitope is able to induce a strong immune response, it appears usually insufficient to induce protective immunity. the in vitro synthesized peptide properly representing the main antigenic determinant of the pathogen is highly likely not to be itself a strong immunogene primarily because of its small size (<10 nm) and high risk of proteolytic degradation. this problem may be overcome by the use of nanoparticles. it has been shown in a considerable number of works that in order to induce a strong immune response, antigenic determinants should be exposed on the surface of nanoparticles whose shape and size (20-150 nm) mimic those of the virus (fig. 1) . in this respect also, vlps, the nanoparticles composed by viral proteins capable of self-assembly (multimerization) into structures morphologically resembling viruses, are the choice selection for the role of a strong immunogene. antigenic determinants multiply repeatedly on the vlp surface and promote complement fixation and b-lymphocyte receptor clusterization leading to the activation of the immune response. additionally, the same antigen multiply displayed on the surface of the particle promotes the multiplication of the pool of autoreactive b-cells, which is the primary objective when designing vaccines against autoimmune diseases and tumors [29] . natural sources for vlp bioengineering and the specific features of their assembly structural proteins of various viruses are capable of autonomous self-assembly into vlps. they interact with the formation of globular, icosahedral, or rodlike structures. so far, the structural proteins of several dozen viruses have been obtained in heterologous expression systems, and almost all of them proved able to form vlps. the vlp size varies from 20 to 200 nm and is similar to the size of the corresponding viruses [30] . being similar to viruses allows vlps to penetrate into the lymph and to be efficiently entrapped by antigenpresenting cells [31] . currently, several heterologous expression systems and the corresponding expression vectors which can be used with them are available. these systems are based on both the bacterial cells (the pet system based on escherichia coli cells is often exploited) and different eukaryotic cells. in the case of eukaryotic cells, the most commonly used systems are yeast expression systems and the ones which rely on drosophila [32] and spodoptera frugiperda insect cell lines [33] . mammalian cells are also utilized, the most pop-ular among them being cho and hek293 [34] . the cost of heterologous protein production in bacterial systems is lower than in eukaryotic cells. however, when studying the functional activity of the protein or vlp associated, for example, with the potential glycosylation sites or interaction with ubiquitin, or other ubiquitin-like peptides [35] , no alternative to eukaryotic expression systems is available. it should be noted that the choice of the expression system is up to the researcher and is driven by the research task. for example, in different laboratories different eukaryotic systems for viral protein expression, including plant cells, are used to produce vlps which are used for vaccination against the hepatitis c virus (hcv) [36] . in most cases, vlps assembled from a virus protein are considered as a candidate vaccine against this very virus, since protein monomers in multimeric configuration (vlp) induce a more potent immune response than protein monomers [29, 30] . the strong immunogenic properties of vlps are determined by several factors. first of all, the dominant epitope of the structural protein is displayed as a part of the particle and is present in a multimeric form as is the case with a native virion [21] . second, vlps stimulate b-lymphocytes and induce t-lymphocytes in the same manner as does the virus infecting the host organism [30] . for example, vlps formed by the main capsid protein l1 of the human papilloma virus of several serotypes are successfully used as vaccines against cervical cancer [37] . a vaccine against hepatitis b virus has been produced which contains vlps in the lipid membrane envelope [38] . the vaccine against coxsackievirus a6 contains vlps assembled from the virus capsid proteins produced in the baculovirus expression system [39] . scientists from china [40] have demonstrated that the capsid protein (vp60) of the rabbit hemorrhagic disease virus (rhdv) efficiently multimerize into vlps, and a single intramuscular injection of the obtained vlp preparation completely protects the immunized rabbits against rhdv infection for at least 180 days. the porcine circovirus capsid protein is also able to efficiently assemble into vlps when synthesized either in the human embryonic kidney cell culture (hek293) [41] , or yeast [42] and baculovirus [43] expression systems, as well as in bacteria [44] . footand-mouth disease vlps are assembled from three structural polypeptides vp0, vp1, and vp3 (naturally produced as a result of processing the p1-2a precursor polypeptide), simultaneously produced in e. coli [45] . it has been recently demonstrated that the polyprotein 2) liposome-based particles [24] , (3) nondegradable spherical nanoparticles (for example, metal nanoparticles) [25] , (4) polymer nanoparticles [26] , (5) graphene nanosheets [27] and nanotubes [28] . antigen of the duck hepatitis a virus produced in the baculovirus expression system assembles into vlps immediately in the cultured spodoptera frugiperda (sf9) cells, while immunization of ducklings with the obtained vlps induces a high level humoral immune response and protects them from developing the disease [46] . the abilities of vlps resulting from multimerization of the cloned virus protein to play the role of an immunogen are not limited just to the presentation of their proper epitopes but may also be taken advantage of to display heterologous proteins. multimerization of capsid proteins into a particle requires the presence of nucleic acid, while for in vitro assembly, a short oligonucleotide (7-10 nt) is sufficient [47] . therefore, vpls formed by capsid proteins are free from the infectious virus rna or dna. moreover, the above-mentioned property, which triggers protein monomer multimerization by nucleic acid, may be taken advantage of to package rna or dna into a particle with two different objectives. hence, antisense rna can be used to suppress virus expression. for example, in the case of the vaccine against foot-and-mouth disease, researchers from china [48] not only displayed the vp1 epitope of the foot-and-mouth disease virus on the surface of vlps but also packaged the antisense rna complementary to the fragment of the viral genomic rna into a particle. another goal of nucleic acid packaging into a particle lies in the presentation of viral nucleic acids leading to the activation of specific immune receptors which induce the synthesis of type i interferons (inf) and other cytokines triggering the antivirus response [49] . it has been demonstrated that long dna and rna molecules may be incorporated into vlps. for example, mrna for the reporter protein, red fluorescent protein, was packaged into particles representing the hepatitis e capsids [50] . the plasmid containing the green fluorescent protein (gfp) gene was encapsidated into the vlps assembled in vitro from the main capsid protein of the hamster polyoma virus [51] . a strategy for the packaging of up to 17 kbp doublestranded circular dna into the particles was developed using the structural protein of the sv40 simian retrovirus expressed in baculovirus system [52] . there exist two fundamentally different approaches for nucleic acid incorporation into vlps: (1) in vitro vlp assembly from protein monomers in the presence of rna or dna, which is to be encapsidated into the vlp; (2) exposure of the assembled vlps to osmotic shock in the presence of nucleic acids. osmotic shock is produced by using a solution with a low ionic strength; nucleic acid enters into a vlp as a result of the shift in the surface structures [53] . however, the incubation of vlps in the presence of nucleic acids without exposure to osmotic stress results in a certain part of the nucleic acids becoming associated with the particles [54] . a far more predictable approach is the in vitro assembly of vlps from the mixture of protein monomers and nucleic acids which should be encapsidated in them. in this case, after synthesis in a heterologous system, the obtained protein monomers are purified to completely remove the contaminating nucleic acids from the protein preparation; then the proteins are denatured in 7-8 m urea, nucleic acids which should be packaged are added, and the proteins are assembled into the particles by eliminating urea from the solution. the use of this strategy was demonstrated, for instance, in the works [47, 55, 56] . protein association into a particle is a reversible process. vlp dissociation can be easily achieved by the addition of a denaturing agent. further, it may be removed and vlps may be reassembled in vitro [47] with the encapsidation of the target rna or dna into the particle. this approach was implemented for the p21 protein of the hepatitis b virus. virus proteins produced in the heterologous expression system were denatured in 7 m urea solution, and their assembly into vlps in the presence of the nucleic acid was further induced [57] . protective immunity is controlled by specific humoral and cellular mechanisms activated by the antigen. posttranslational protein modifications and covalent bonds between the modifying molecules and the functional groups in the polypeptide chains are of great importance for immune homeostasis during the antiviral response. the balance between phosphorylation, ubiquitination, methylation, acetylation, sumoylation, adp-ribosylation, and glutamilation of a certain antigen performs the fine adjustment of the host's antiviral response [49, 58] . the structural proteins for vlp production when synthesized in the eukaryotic expression systems undergo a number of posttranslational modifications. in particular, the gag gypsy monomer proved to be a natural substrate for type 2 casein kinase [59] . moreover, while produced in the eukaryotic cell, gag gypsy monomers become associated with ubiquitin and sumo, the cellular protein partners of the viral structural protein [33] . generally, ubiquitin and sumo can bind with any lysine residue within the protein molecule, although there are preferable binding sites; it should also be noted that phosphorylation determines which of these two partners binds with the protein [60, 61] . additionally, binding with a limited number of the above-mentioned signal peptides, such as monoubiquitination [62] , usually plays a regulatory role and controls the transport of the protein itself, or the particle formed by it. in the case of polyubiquitination, the protein is destined for proteasome degradation [60, 63] . by constructing recombinant polypeptides based on the viral capsid proteins it is possible to obtain vlps bearing several antigens. for example, dna encoding the vp2 capsid protein of the porcine parvovirus was fused with the dna fragment encoding 35 amino acid residues of the main antigen of porcine circovirus (pcv2) nucleoprotein. the resulting hybrid dna was used to produce protein in heterologous cells which further formed vlps. these vlps induced a significantly stronger immune response against pcv2 than the recombinant adenovirus encoding the open reading frame 2 (orf2) of pcv2 [41] . another interesting example is represented by the vlps formed by the influenza virus matrix protein and displaying a toxoplasma gondii antigen on their surface [64] . there exists another approach to designing therapeutic vaccines based on vlps. in this case, the vlp surface displays the variable fragment of an antibody specific to the antigen of the target virus [65] . moreover, knowing which receptor is bound by the virus proteins renders it possible to produce particles possessing tropism to the cells of certain tissues. for example, the pre-sprotein of the hepatitis b virus exposed as a ligand on the vlp surface provided for their specific binding with hepatocytes [66] . vlps assembled from the structural proteins of bacteriophages are also considered as carriers for human and animal vaccine production. for example, the dna fragment encoding foreign protein was inserted into the dna region encoding the n-terminal β-hairpin of the coat protein of the e. coli ms2 phage. it has been demonstrated that the expression of the obtained construct in bacterial cells resulted in the production of the recombinant protein in which a foreign peptide was present in the central part of the hairpin. the obtained chimeric protein monomers were able to self-assemble into particles morphologically similar to the phage capsid both in vivo and in vitro [67] . using the mentioned property of the ms2 coat protein, vlps displaying the ep141-160 epitope of the foot-and-mouth disease vp1 structural protein on their surface were obtained. these chimeric vlps induced string immune response in the animals, which allowed regarding them as a promising base for the development of a prophylactic vaccine [68] . it is worth noting that the exposure of ep141-160 on the vlp surface resolved a long-standing problem of how to make use of the beneficial properties of this peptide. researchers from several laboratories have demonstrated that this antigenic determinant of the footand-mouth disease not only induces the production of neutralizing antibodies but also stimulates t-lymphocytes [69] . however, when isolated, ep141-160 is not able to induce the immune response protecting animals against the foot-and-mouth virus infection [70] . many attempts were made to solve this problem, for example, by combining the antigenic determinant with large molecules, such as for instance, t cell-specific molecules [71] . however, only integration of ep141-160 into vlps resulted in the possibility of using this antigenic determinant as a strong immunogen for vaccine production [68] . practically all structural proteins of phages infecting various species of bacteria are able to autonomously form vlps. for example, salmonella typhimurium phages are able to autonomously multimerize into vlps on whose surface the epitopes of eukaryotic viruses, including the epitopes of the human influenza virus may be exposed [72] . at the same time, however, bacteriophages are apparently incapable of presenting large epitopes, whose length exceeds 24 amino acid residues [67] . structural proteins of retroviruses and retrotransposons have a higher capacity compared to bacteriophage proteins. this means that protein monomers forming the particle may be fused with a longer peptide and still retain the ability to multimerize. in particular, it has been shown for the gag gypsy capsid protein [73] , that at least 26% of its amino acid sequence (more than 100 amino acid residues) is of little importance for multimerization into vlps [55] . therefore, the truncated form of this protein may be fused with a heterologous peptide with the length similar to the length of the deleted fragments and a recombinant protein may be obtained which will retain its ability to self-assemble into vlps. in such a way, the truncated gag gypsy fused with the heterologous peptide formed particles when it was synthesized in bacterial cells. we were also able to assemble particles from the purified protein monomers in vitro [47, 55, 74] . the obtained particles resembled the native gypsy virus by their morphology [75] . the substitution of the deleted region in the dna encoding gag gypsy by the nucleotide sequence encoding the main antigenic determinant of the foot-and-mouth virus vp1 protein (ep141-160) and his 6 -tag resulted in the formation of particles which displayed ep141-160 as the target antigen. the advantage of the technique used is that vlps formed by the protein containing his 6 -tag can be readily isolated and purified by affinity chromatography [76] . chimeric vlps may be obtained through the construction of recombinant dna molecules encoding both the corresponding virus protein and a foreign peptide or protein. another way is vlp pseudotyping. this approach was elaborated using the hamster polyomavirus. the v1 capsid protein of this virus first forms pentamers which further assemble into vlps comprised of 72 pentamers. when v1 is expressed together with the minor capsid protein v2, the latter binds with the central part of each v1 pentamer. it has been demonstrated that the n-terminal part of v2 is not involved in this interaction and therefore may be removed and substituted by the target epitope [77] . another approach is also known. antigen is immobilized on the vlp via covalent binding between the reactive groups of the amino acid residues of the antigen and vlp. this approach proved to be ineffective due to the disruption of the native conformation of the antigen attached to the particle surface [77, 78] . this problem was countered in the following way. glygly-lysglygly sequence was inserted into the monomer subunits for vlp assembly. in this environment, the reactive ε-amino group of lys residue is exposed and ready to interact with the cys-group of any protein in the presence of the cross-linking agent, such as m-maleimidobenzoyl-n-hydroxysulfosuccinimide ester (sulfo-mbs) [79] , or succinimidyl-6-[(β-maleimidopropionamido)hexanoate] (smph) [80] . although the authors claimed that the described system of molecular assembly may be used to induce strong blymphocyte response against most antigens and prospective vaccine prototypes were suggested, this platform became the prototype only for a single prospective preparation based on vlps, the medication for lowering blood pressure in patients with hypertension [80] , due to the development of the new improved techniques for antigen immobilization of the vlp surface, which will be discussed below. one of them takes advantage of the ability of his 6tag to bind with multivalent tris-nitrilotriacetic acid (trisnta) which in turn binds with a broad range of molecules, in particular, with biotin. the possibilities of this elegant technique for the functionalization of noninfectious viral nanoparticles were demonstrated in the case of vlps formed by the norovirus (nov) structural proteins. using the baculovirus expression system, the authors obtained nov vlps, displaying his 6 -tag on their surface, which was first used to purify vlps, and further to bind trisnta molecules conjugated with a fluorescent dye, or biotin which in turn successfully bound streptavidin [81] . notwithstanding some authors suggesting the described technique to be promising for vlp vaccine production, this approach continues to be only a prototype and has not advanced further than model experiments. the search for techniques providing efficient and stable immobilization of the antigen on the vlp surface led to the development of a versatile platform which allows us to covalently attach large antigens to the vlp surface [82] . this molecular assembly system uses the tag-catcher conjugation system which was derived from the cnab2 domain of fibronectin-binding protein fbab from streptococcus pyogenes. as a result a highly reactive spytag peptide (13 amino acid residues) was obtained which efficiently interacted with the spycatcher protein with the formation of isopeptide bonds in a broad range of buffer solutions [83] . the spytag peptide was inserted into the vlp-forming polypeptide of the acinetobacter-infecting phage (these particles were called ap205), so that each monomer forming ap205 displayed two spytag peptides. a recombinant antigen consisting of the target protein and the spycatcher peptide was produced using bacterial or baculovirus expression systems [84] . the obtained antigen efficiently bound with the vlp surface and induced a very strong immune response. almost the same effect was observed in the case of the reverse combination of the conjugating peptides, that is when spycatcher was incorporated into vlps, and spytag was fused to the antigen. this system is already utilized to design different types of vaccines, including the vaccines against tuberculosis and malaria [84, 85] . a recent report has demonstrated that it was successfully used to develop a vaccine against breast cancer [86] . this work is remarkable due to the fact that the high density of the her2 antigen (human epidermal factor receptor 2) synthesized in the cultured drosophila melanogaster cells could be obtained on the ap204 vlp surface [32] . the obtained particles induced a strong immune response to the antigen in the patients, which rendered it possible to overcome the immune tolerance to her2 known for patients with the her2-dependent breast cancer [85, 86] . in summary, the analysis of the discussed published data allows us to conclude that the nature of the vlp-forming protein is not as important for vlp functionalization as the technique which makes it possible to obtain high antigen density on the surface of vlps (table 1) . the product range a number of vlp vaccines are available on pharmaceutical markets in many countries. the most wellknown are the cervarix® and gardasil® vaccines against cervical cancer, which have been successfully used for prophylaxis of this disease in girls for more than ten years. both vaccines are based on vlps formed by the main capsid protein l1 of the human papilloma virus belonging to several serotypes [37] . engerix and recombivax hb vaccines against the hepatitis b virus representing vlps enveloped in a lipid membrane [38] , as well as hecolin and xiamen innovax vaccines against hepatitis e [88] , were developed and commercialized. the anti-malaria vaccine malaria rts has been certified, which represents the c-terminal part of the cs protein of plasmodium falciparum attached to the surface antigen of the hepatitis b virus (hbsag), which is also used in the certified vaccines against hepatitis b. to stabilize recombinant vlps, the fused protein is expressed together with the hbsag protein in saccharomyces cerevisease [89] . a vaccine against the porcine circovirus representing vlps assembled from the vp2pcv2 capsid protein synthesized in a heterologous system has also been commercialized [41] . an increasing number of works have appeared that report on the development of vaccines based on nanoparticles, including vlps and/or replicationinactive viruses. among the apparent advantages of vaccines of this kind are their high specificity, efficiency, and good pharmacokinetic characteristics. it has been demonstrated that in an organism vlps reach lymphatic nodes in less than 10 min, while the particle mixture bearing different antigens may be pro-cessed by the same antigen-presenting cells simultaneously [90] . it should be noted that nanoprticle-based vaccines offer new possibilities in the development of immunoprophylaxis strategies, especially, the development of injection-free formulations, in particular, intranasal vaccines and inhalers. the administration of these vaccines may not involve humans, which is especially important in the case of industrial animal breeding in large agricultural enterprises characteristic of presentday russia [19] , since it proves to be extremely difficult to administer identical injections to a large number of animals at the same time. this problem may be resolved by nebulizing aerosol vaccines in the area where animals are kept. the potential of this approach was demonstrated for the first time in 1947 with mice [92] ; then, in the 1950s-1970s, attempts were made to introduce this approach into animal and poultry farms in many countries. starting from the 1960s, attempts were also made in the soviet union [93] . however, at that time, the method of inhaled administration of vaccines was not introduced in practice since protective immunity could only be obtained when the tolerable dose was exceeded many times. for example, the inhaled administration of the vaccine against the newcastle disease led to 10-20% lethality in birds [94] . however, due to the introduction of vaccines based on the exposure of the epitopes of a pathogen on the surface of nanoparticles, including recombinant vlps, together with the development of innovative approaches to aerosol production, inhaled vaccines again became a topical issue. for example, biodegradable polymer nanoparticles formed by poly(glyceroladipate-co-ω-pentadecalactone), pga-co-pdl, proved themselves as an effective antigen carrier for inhaled vaccination [95] , while the vaccine based on the adenovirus with the particles ranging from 4 to 10 µm induced stable protective immunity when administered via inhalation to monkeys [96] . lastly, a method for vlp-vaccination via inhalation was proposed as protection against the human papilloma virus [97] . one of the most important properties of vlps is mimicking virus particles and the consequent ability to induce a strong immune response to the antigen which they demonstrate irrespective of the source of the monomers which multimerize into vlps, these being either insect viruses, in particular the gypsy virus [56, 61] , or plant viruses [98] . vlps based on the structural proteins of plant viruses produced in plants [99] make it possible to obtain vaccines with another nonconventional way of administration, edible vaccines [100] . vaccines of this type may be synthesized directly in plant forage, with the oral vaccination of this kind inducing an immune response. expression vectors for foreign protein production in plants have been developed based on plant viruses, which allows obtaining plant-producing recombinant viruses or vlps displaying the target antigen on their surface [101, 102] . vlps assembled from the structural proteins of plant viruses are also used to design functionalization platforms for antigen binding. these platforms are based on the strategies described above. in particular, they take advantage of the reactivity of the lys ε-group to immobilize the antigen on the surface of the plant's vlps [102] [103] [104] [105] . using the tobacco mosaic virus [106] and potato x virus [104] , it has been shown that a considerably large antigen can be immobilized on the surface of the corresponding vlps via the formation of the streptavidin-biotin complex. finally, based on the structural protein of the plant virus, the platform for antigen immobilization using the tag-catcher conjugation system is being developed [107] . it should be noted that among the drawbacks of vlp vaccines manufactured using proteins which are not specific for mammals, for example, the plant virus proteins, include the development of the immune response to the structural component of the particles, which is the plant virus protein. undoubtedly, there is no therapeutical benefit in developing such immunity; however, whether there are adverse effects will be demonstrated by further studies. model molecules including biotin, alexa 488 (fluorescent dye), and gfp protein conjugated with trisnta [81, 87] isopeptide bond formation between spytag (integrated into vlp monomers) and spy-catcher (fused with antigen) peptides vaccine against breast cancer (her2 antigen presentation). it is also implemented to develop vaccines against malaria and tuberculosis [82] [83] [84] [85] [86] the use of vlps for vaccination in medicine is becoming increasingly widespread. this point is reinforced by the list of clinical trials of vlp vaccines prepared by the united states national institute of health. currently, more than hundred vlp vaccines, which are directed against human and avian influenza viruses, the norwalk virus, norovirus, hiv-1, and the chikungunya virus, the foot-and-mouth disease virus, as well as against melanoma, adenocarcinoma, papillomatoses, and cervical cancer, are undergoing clinical trials (http://clinicaltrials.gov/ct2/search/index). it may be expected that the variety of nanoparticle vaccines against different cancers and viral infections will grow steadily, and vlps which can be used for immunization against microorganisms belonging to different taxons as well helminthes will also be developed. we thank the designers oleg vasil'ev and galina podzolkova (institute for statistical studies and economics of knowledge (issek), national research university higher school of economics) for their assistance in preparing the illustrations. the article was prepared within the framework of the basic research program at the national research university higher school of economics (hse) and supported within the framework of a subsidy by the russian academic excellence project 5-100. the authors declare that they have no conflict of interest. this article does not contain any studies involving animals or human participants performed by any of the authors. assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile 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hans-joachim title: chicken grifin: structural characterization in crystals and in solution date: 2017-12-15 journal: biochimie doi: 10.1016/j.biochi.2017.12.003 sha: doc_id: 270273 cord_uid: a4iu9qg6 despite its natural abundance in lenses of vertebrates the physiological function(s) of the galectin-related inter-fiber protein (grifin) is (are) still unclear. the same holds true for the significance of the unique interspecies (fish/birds vs mammals) variability in the capacity to bind lactose. in solution, ultracentrifugation and small angle x-ray scattering (at concentrations up to 9 mg/ml) characterize the protein as compact and stable homodimer without evidence for aggregation. the crystal structure of chicken (c-)grifin at seven ph values from 4.2 to 8.5 is reported, revealing compelling stability. binding of lactose despite the arg71val deviation from the sequence signature of galectins matched the otherwise canonical contact pattern with thermodynamics of an enthalpically driven process. upon lactose accommodation, the side chain of arg50 is shifted for hydrogen bonding to the 3-hydroxyl of glucose. no evidence for a further ligand-dependent structural alteration was obtained in solution by measuring hydrogen/deuterium exchange mass spectrometrically in peptic fingerprints. the introduction of the asn48lys mutation, characteristic for mammalian grifins that have lost lectin activity, lets labeled c-grifin maintain capacity to stain tissue sections. binding is no longer inhibitable by lactose, as seen for the wild-type protein. these results establish the basis for detailed structure-activity considerations and are a step to complete the structural description of all seven members of the galectin network in chicken. the emerging versatility of physiological functions of animal and human lectins gives ample reason to characterize their structures in detail. in overview, more than 12 folds have been identified that convey ability to bind glycans [1e4] . sequence divergence after duplication events, starting from an ancestral gene, then led to forming families of homologous proteins. the case study on galectins (b-galactoside-binding proteins with b-sandwich fold and a sequence signature responsible for ligand contact [5] ) is describing such a network with overlapping and distinct expression profiles [6e8]. this emerging evidence poses the challenge of a complete characterization of the galectins of an organism. it would be a step forward towards delineating rules of network design and providing insights into the functional meaning of sequence variations. in this respect, the galectin fold presents remarkable adaptability for accommodating ligands of different biochemical nature. the crystallographic or nmr spectroscopical study of such domains of protozoan (toxoplasma gondii) micronemal protein 1 and protein 2-associated protein [9, 10] as well as the n-terminal modules of bovine and murine coronavirus spike proteins [11, 12] have taught the following instructive lesson: conversion of the concave topology of the carbohydrate-binding pocket established by surrounding loops to a predominantly hydrophobic surface shifts specificity from glycans to distinct proteins. looking at the assumedly crucial sequence signature for contact to a b-galactoside, a less dramatic deviation than just described may not necessarily impair glycan binding. for example, the change of a seemingly essential asn (in position 46 in human galectin-1 (gal-1)) to ala at the equivalent position 64 in a fungal (agrocybe cylindracea) galectin is not detrimental. it is neutralized by a five residue insertion at positions 42e46 (with the inserted asn46 taking the place of the asn lost at position 64) [13, 14] . the structural way how to maintain affinity for lactosides in the conger eel (conger myriaster) galectin from peritoneal cells (con-p), although even seven from eight conserved amino acids are replaced as reported in [15] , has not yet been characterized. alternatively, sequence deviation(s) can alter carbohydrate specificity. the trp81arg change implemented binding to bi-n-acetylated disaccharides (chitobiose, lac-dinac) in the third galectin protein (cgl3) from the inky cap mushroom coprinopsis cinerea [16] . a unique situation, i.e. a species-dependent loss of lectin activity, is encountered for the galectin-related inter-fiber protein, termed grifin. this protein has first been described in rat as lens-specific protein [17, 18] . it was found in the insoluble fraction of nuclear fiber cells and localized at the interface between adjacent fiber cells, representing about 0.5% of total protein in adult lens. its gene with an elaborate promoter region to facilitate lens-specific expression is a common constituent of vertebrate genomes [19] . given this sitespecific occurrence and conserved presence among vertebrates, it is exceptional and thus intriguing to see sequence deviations at canonical positions between mammalian and bird/fish grifins. especially, the equivalent of the already noted asn46 (in human gal-1) is turned to lys in mammalian grifins [17] . as consequence, a bead (lactosylated sepharose beads) assay revealed no lectin activity for rat grifin [17] . in contrast, grifins from zebrafish [20] and chicken [19] were bona fide lectins. two reasons prompted us to initiate structural analysis of grifins by studying chicken (c)-grifin: the mentioned plasticity within the galectin fold and our long-term interest to achieve complete crystallographic documentation of galectin structures in an organism. towards this end, chicken with its (only) seven family members is a favorable model. since c-grifin shares a deviation from the canonical sequence for lactose binding with mammalian grifins, i.e. the arg71val exchange, defining the resulting contact pattern to the ligand will enable a comparison to common features. we here combine crystallographical analysis, reaching atomic resolution, with studies of c-grifin in solution. they were started by determining its quaternary structure, a key feature of proto-type galectin functionality [21] . quaternary structure and tendency for aggregation were first examined in solution, up to a concentration of 9 mg/ml, by ultracentrifugation and small angle x-ray scattering (saxs). crystallographically, respective specimen from solutions at seven ph values ranging from 4.2 to 8.5 could be processed to monitor stability of structural features. the interaction with lactose was monitored in the crystals and also in solution. here, thermodynamic parameters (by isothermal titration calorimetry (itc)) and profiles of hydrogen/ deuterium exchange (hdx) in the absence and presence of the ligand were measured. faced with the conundrum that reestablishing the sequence signature in rat grifin with the lys-to-asn reconstitution did not repair the loss of lectin activity [17] , we finally probed into the effects of site-specific mutations on c-grifin's carbohydrate-binding activity by a histochemical assay. the wild-type protein was obtained after recombinant production directed by a pgemex-1 vector with the respective cdna insert and purified by affinity chromatography using lactosebearing resin as described [19] . cdnas of the mutants of c-gri-fin, i.e. the trp66lys and the asn48lys single-site mutants, the asn48lys/arg50val double mutant and the asn48lys/arg50val/ tyr66leu triple mutant, were prepared by using the quikchange™ site-directed mutagenesis protocol (agilent technologies, munich, germany). the following primer pairs were used: 5 0 c ctg gcc aac cac ctg ggg aag gag gag g 3 0 and 5 0 c ctc ctc ctt ccc cag gtg gtt ggc cag g 3 0 (trp66leu), 5 0 c gcc ttc cac tt t aag ccc cgc ttt gcc agc 3 0 and 5 0 gct ggc aaa gcg ggg ctt aaa gtg gaa ggc g 3 0 (asn48lys), 5 0 gct ggc aaa gac ggg ctt aaa gtg gaa ggc g 3 0 and 5 0 c gcc ttc cac ttt aag ccc gtc ttt gcc agc 3 0 (asn48lys/arg50val) (exchanged base pairs are underlined). the cdna of the triple mutant was generated by further altering the cdna of the double mutant (asn48lys/arg50val) by respective processing with the primers for the trp66leu mutant. successful implementation of the intended changes was checked by dna sequencing (sequiserve, vaterstetten, germany). mutant proteins of c-grifin were designed as fusion proteins with a glutathione s-transferase (gst) part in a pgex-6p-2 vector (ge healthcare, münchen, germany), they were purified after recombinant production by affinity chromatography using glutathione-presenting sepharose 4b (ge healthcare). thereafter, the linkage between both proteins was cleaved by gst-tagged human rhinovirus 3c protease (at a ratio of 1:100 (w/w)), then the c-grifin part was separated from released gst and the tagged protease by a second round of affinity chromatography as described [19] . protein was either precipitated by adding (nh 4 ) 2 so 4 or labeled by biotinylation for the histochemical analysis, as described for human gal-1 [22] . protein samples were diluted to final concentrations of 0.5 and 1.0 mg/ml in 5 mm phosphate buffer containing 150 mm nacl and 4 mm b-mercaptoethanol, and solutions were pre-cleared at 16 ,000 â g. sedimentation-velocity experiments were run at 293 k in an optima kl-i analytical ultracentrifuge (beckman coulter, indianapolis, usa) with an an50-ti rotor and standard doublesector epon-charcoal center pieces (1.2 cm optical path length). measurements were performed at 48,000 rpm, registering the course of protein migration every minute at 280 nm. rayleigh interferometric detection was used to monitor the course of development of the concentration gradient as a function of time and radial position, and the data were analyzed using the sedfit software (version 14.7). saxs data were collected at the bm29 beamline (esrf synchrotron, grenoble, france) using the biosaxs robot and a pilatus 1 m detector (dectris, baden-daettwill, switzerland) with synchrotron radiation at a wavelength of l ¼ 1.0 å and a sampledetector distance of 2.867 m [23] . each measurement consisted of 10 frames, each of 1 s exposure of a 100 ml sample flowing continuously through a 1 mm diameter capillary during exposure to x-rays. buffer scattering was measured immediately before each measurement of the corresponding protein sample at 277 k. the obtained scattering profiles were spherically averaged, and the buffer scattering intensities were subtracted using in-house software. protein samples were prepared at concentrations of 1, 2, and 9 mg/ml in 20 mm phosphate buffer (ph 7.0) containing 150 mm nacl and 5 mm lactose. particle envelopes were generated ab initio using the program dammif [24] . multiple runs were performed to generate 30 independent model shapes that were combined and filtered to produce an averaged model using the damaver software package [25] . suspensions of precipitated protein were extensively dialyzed against 5 mm phosphate buffer (ph 7.0) containing 150 mm nacl and 4 mm b-mercaptoethanol. protein was purified by affinity chromatography using a column packed with lactosylated sepharose 4b to remove inactive material. fractions after elution with 200 mm lactose were concentrated using amicon ultra 10,000 mcwo centrifugal filter units and then loaded on a hiprep 16/60 sephacryl s-200 column equilibrated with phosphate-buffered saline (ph 7.0) containing 4 mm b-mercaptoethanol and 5 mm lactose. the protein eluted as a single peak, the respective fractions were concentrated to a concentration of 15 mg/ml. separately, protein was purified in the absence of lactose by loading sample directly after dialysis on a gel filtration column as above equilibrated with phosphate-buffered saline (ph 7.0) containing 4 mm bmercaptoethanol. eluted protein fractions were finally concentrated as described above. crystallization was performed at 295 k using the sitting-drop vapour diffusion method. small crystals appeared in a wide range of crystallization conditions. in detail, crystal size and quality were optimal at the following conditions: 20% peg 8000, 0.1 m phosphate/citrate (ph 4.2); 30% peg 4000, 0. each crystal was flash-cooled by immersion in liquid nitrogen using the corresponding crystallization medium supplemented with 30% ethylene glycol as cryo-solution. all data collections were done at the xaloc beamline of the alba synchrotron (cerdanyola del vall es, spain) except for the crystal obtained at ph 4.6 that was taken to the proxima 2 beamline of the soleil synchrotron (gifsur-yvette, france). diffraction data were processed using xds [26] and aimless [27] . a summary of reflection data parameters is presented in table 1 . the molecular replacement method was used to solve c-gri-fin's structures, using the program phaser from the phenix suite [28] . a theoretical model generated by the swiss-model server (http://swissmodel.expasy.org) was applied as a search probe [29] . the phenix suite [28] was employed for structural refinements, and addition of water molecules and placement of lactose were carried out manually with the coot program [30] , if necessary. the statistical details of final models are given in table 1 . molecular illustrations of the structural models were generated using pymol. solutions of c-grifin (5 mm or 200 mm (at ph 8.5)) were prepared in phosphate-buffered saline (10 mm, ph 7.2), in tris-hcl (30 mm, ph 8.5) or in sodium acetate (10 mm, ph 5.2). the ligand-containing solutions (100 mm or 60 mm (at ph 8.5) for b-lactosides, 40 mm for the 3 0 -o-sulfated derivative of the 2-naphthyl b-lactoside) were also freshly prepared just before starting the experiment by injecting first 2 ml, then 10 ml (or 5 ml for the titration at ph 8.5) of ligand-containing solution into the cell of a microcal vp-itc calorimeter (ge healthcare), filled with the protein-containing solution, at 298 k. control experiments using buffer excluded lectin-independent heat generation. resulting data were fitted using the origin software package. a solution of lactose and c-grifin at a molar ratio of 2344:1 in equilibration buffer (20 mm potassium phosphate containing 97 mm sodium chloride in h 2 o, ph 7.5), with a lactose-free solution as control, was incubated for 1 h at room temperature. hdx was started at room temperature by adding 10-times deuteration buffer (20 mm potassium phosphate containing 97 mm sodium chloride in d 2 o, pd 7.9, ph 7.5) to each sample. after 0.5 min, 1 min, 10 min, 30 min, 60 min and 240 min, the deuterated samples were quenched by adding ice-cold quenching buffer (100 mm potassium phosphate buffer containing 5 m guanidinium chloride and 0.5 m tris [2-carboxyethyl] phosphine hydrochloride in h 2 o, ph 2.4) at a 1:1 ratio, followed by immediate freezing on dry ice, as previously performed for the n-terminal lectin domain of chicken galectin (cg)-8, referred to as cg-8n [31] . in parallel, protein solutions without lactose were kept in equilibration buffer without deuteration and quenched in the same way, this undeurated control sample used for identification of the peptic peptides. experiments were performed in triplicate. quenched undeuterated and deuterated sample solutions (320 pmol c-grifin) were injected into a nanoacquity uplc system with hdx technology (waters corporation, milford, usa), allowing an online peptic digest on a poroszyme-presenting pepsin cartridge (2.1 mm â 30 mm; applied biosystems, foster city, usa) at 15 c. peptic peptides were then trapped on an acquity uplc beh c18 vanguard pre-column (1.7 mm, 2.1 mm â 5 mm; waters) and separated with a linear acetonitrile gradient over 14 min at 0 c on an acquity uplc beh c18 column (1.7 mm, 1 mm â 100 mm; waters). the column outlet was directly connected to a synapt g2 hdms mass spectrometer equipped with a lockspray esi source (waters). mass spectra for the peptic peptides were acquired in the mse mode over the range of m/z 50e2000. identified peptic peptides obtained by the digestion of c-grifin were organized as list using the protein lynx global server software (waters) and the mse data for the undeuterated controls. by applying dynamx software (waters), the relative deuterium uptake for each identified peptide was calculated by subtracting the centroid masses of the corresponding peptides found in the undeuterated and deuterated samples, for both ligand-loaded and ligand-free c-grifin. the ligand-dependent difference in relative deuterium uptake was determined using dynamx, and its significance was evaluated by calculating a two-sided confidence limit with a significance level of 0.02 [32] . the result of this evaluation was visualized by color coding of b-strands in the threedimensional model of c-grifin. biotinylated c-grifin and its asn48lys, trp66leu, asn48lys/ arg50val and asn48lys/arg50val/trp66leu mutants were tested on paraffin-embedded sections (5 mm) of adult chicken kidney, obtained after fixation of tissue specimens for 24 h in bouin's solution and embedded, mounted on superfrost ® plus glass slides (menzel, braunschweig, germany). following optimized processing for deparaffinizing sections, blocking of sites for nonspecific binding, incubation of biotinylated probe in the absence and presence of lactose (200 mm) and signal development by bound avidin-alkaline phosphatase (ap) conjugate with vector ® red ap substrate (enzo life sciences, l€ orrach, germany) as described [33, 34] , documentation was recorded using an axioimager.m1 microscope (carl zeiss microimaging, g€ ottingen, germany) equipped with an axiocam mrc3 and mrc digital camera and axiovision (version 4.6) software. processing controls with an incubation step using plain buffer instead of buffer containing the labeled probe excluded probe-independent staining, titrations with probe concentrations of 4 mg/ml, 12 mg/ml, 24 mg/ml and 48 mg/ml were systematically performed for all proteins and for the pair of measurements in the absence and presence of lactose. at low concentrations in the course of gel filtration, grifins from chicken, mouse and rat eluted at the position of a dimer, with evidence for inter-subunit exchange when fractionating mixtures of tag-free and tagged proteins [17, 19, 35] . considering the high local concentration in the lens, we examined the protein status after further increasing the concentration. in sedimentationvelocity experiments at concentrations up to 1.0 mg/ml, c-grifin migrated as single sharp peak with a sedimentation coefficient of 2.6 ± 0.1 s. in comparison, homodimeric human gal-1 and cg-1a gave very similar values under these conditions, whereas cg-2 has an s-value of 2.03 ± 0.1 [36] . frictional ratios among homodimeric proto-type cgs can thus differ when measured by ultracentrifugation. it is about 1.3 for c-grifin and cg-1a but 1.63 for cg-2 [36] . of note for cg-2, heterogeneity in size distribution had been observed for this protein at 4 mg/ml, with the acquisition of the quaternary structure as trimer of dimers [37] . we proceeded to determine the quaternary structure of c-grifin at concentrations up to 9.0 mg/ml. when reaching the concentration of 9.0 mg/ml in saxs experiments, the particle distribution function of c-grifin is still in agreement with exclusive presence of a dimer. the ab initio model of the dimer calculated with these data is shown in fig. 1 . its elongated disc shape fits well with the frictional ratio of the dimer as calculated using data obtained by ultracentrifugation. overall, no evidence for aggregation up to this concentration was obtained. these results thus define the quaternary structure in solution as homodimer with no indication for higher-order aggregates at concentrations up to 9 mg/ml under the given conditions. structural analysis was next taken to the level of crystallography. in view of the remarkable long-term stability of c-grifin in lenses, it was attempted to prepare a panel of crystals from solutions over a broad ph range. a wide range of combinations of buffers and additives was tested and structural analysis became possible with crystals obtained at seven conditions. in addition to presence of different types of additives, they covered the ph range from 4.2 to 8.5. as summarized in table 1 , the resolution reached the level of 0.96 å for crystals grown at ph 7.5. space groups differed within this group, p21 seen in three cases, either p4 2 2 2 or p22 1 2 1 in two cases. the asymmetric unit cell content was variable in the seven crystals, being either one, two or four (table 1) the c-grifin monomer adopts the typical b-sandwich fold with two anti-parallel b-sheets of six (s1-s6) and five (f1-f5) b-strands (fig. 2) . the short 3 10 helix in the long loop connecting the s2/f5 strands is also a typical feature. among the proto-type cgs [37e39], the loop between the s3/s4 strands reaches the largest length, as highlighted by rectangles in fig. 3 . loop-length variation also holds true for the s4-s5 section. it is four amino acids shorter than in cg-1a/b, while superimposing with that of cg-2 (fig. 3) . as consequence, a flat surface is created in this region (fig. 3) . length, conformation and structure of this loop has been described as a discriminatory factor between human gal-1 and -3 when interacting with the tf antigen [40] , the core 1 disaccharide of mucintype o-glycosylation (cd176) [41] . overall, alignment is at a high level, as reflected by rmsd values at 2.9 å (to cg-1a, cg-2) and 2.1 å (cg-1b). for comparison, respective values are 2.4 å for cg-1a/cg-2 and 1.5 å for the paralogue pair cg-1a/b. the availability of crystals of c-grifin exposed to a wide range of ph values enabled a detailed comparison of the seven crystal structures. it revealed no marked structural changes, the common homodimer shown in fig. 4a . to substantiate this conclusion by a number, the rmsd value was 0.337 å for the pairwise analysis of the crystal structures at the tested ph minimum (4.2) and its maximum (8.5) . since the availability of the crystallographic homodimer structure made its placement into the sphere of the saxs-derived model possible, as done in fig. 1 , the fitting of crystal and solution structures could be tested and was found to be excellent. looking at the unit cell content of the seven crystals, it differs considerably, as indicated in table 1 . the example of cg-2, which is arranged as a non-crystallographic trimer of dimers (added to fig. s1 ) and occurs in part as hexamer in solution at 4 mg/ml in sedimentation-velocity analysis, underlines capacity of homodimer association of a galectin [13] . crystal packing of other galectins to a dimer of dimers [42, 43] , also seen for human gal-1 in solution in an aprotic solvent [44] , that can even be stable in solution [16,45e47] as well as the aggregation of a monomer (murine gal-9n) to a dimer [48] and na þ -mediated association of the n-terminal lectin domain of murine gal-4 to a tetramer around the crystallographic four-fold axis [49] indicate potential of galectin domains for building higher-order aggregates under special conditions. in this study, the availability of crystals of the same protein obtained at different conditions documents the very low degree of influence of the ph value on the galectin fold, with some variability in unit cell looking closely at the interface region between the two subunits of c-grifin's homodimer, it is established by the f1/s1 strands from the n-and c-termini of each subunit in the homodimer (residues 4e14/127-136) (fig. 4b) . around the s1 edge, the salt bridge between arg5/glu12 and hydrogen bonds between glu7/ leu9 stabilize the assembly. in the case of the anti-parallel f1 strands, hydrogen bonding between ser131/thr135 and also ser130, ile134 and lys136 serve this purpose. typically for cg homodimers, hydrophobic contacts via phe6, ile129, val132 and ile134 come into play, too (fig. 4b) . together, these contacts enable c-grifin to act as cross-linker, reported previously based on haemagglutination assays [19] , or as a kind of molecular glue (please see below). the susceptibility to abolish the cell bridging by presence of lactose at 1.0e1.5 mm, together with c-grifin's binding to lactose-bearing beads used in affinity chromatography, was indicative of lectin activity. here, we first confirm this conclusion by itc analysis and then characterize the contact site and its pattern of contact formation with lactose crystallographically. analysis of itc data for the association of c-grifin and the b-methyl derivative of lactose resulted in revealing this process to be enthalpically driven. homodimeric cg-1a and cg-2 had similar thermodynamics of binding, whereas ligand association to cg-1b produced a relatively small enthalpy gain [13] . per dimer, the number of binding sites of 1.76 ± 0.42 was reached at ph 7.2. it increased to 1.89 ± 0.45 by exchanging the methyl by a 2 0 -naphthyl group, together with the considerable enhancement of affinity from 140 mm to 8.6 mm. adjusting the ph to 8.5 (in tris buffer) lowered ligand loading to well below 1 and also the affinity. at the acidic side, no heat production was observed at ph 5.2 (in sodium acetate). this ph profile with rather sharp decreases in binding activity to both sides corresponded well with assays on human gal-1 using lactose-bearing beads or surface-immobilized asialofetuin [50, 51] . fittingly, binding sites for lactose in the homodimer were occupied by the ligand in the crystals obtained at ph values of 6.2, 6.5, 8.0 and 8.5, and they remained free when the ph was set to 4.2/4.6. interestingly, a special circumstance precluded complete loading of both sites of the homodimer at ph 7.5 in the crystal. at this ph value, crystallographic contacts with asn28 of a symmetry-related subunit impedes access to one lactose-binding site of the homodimer. this situation makes a comparison of lactose-free with lactose-loaded subunits possible at atomic resolution. the resulting rmsd value of this superimposition is 0.263 å, arguing in favor of a common shape irrespective ligand loading. the contact site in crystals at each ph value is composed of amino acids from b-strands s4-s6 (fig. 4a) . interestingly, the electron density map of the ligand shows a preference for presence of a-lactose instead of the natural b-anomer at the reducing end, as shown in fig. s2 , likely by crystallographic contacts. despite sequence variations among the homodimeric cgs, the canonical contact profile between the protein and lactose is maintained, except for the arg71val substitution (fig. 5) . as illustrated in this figure, the axial 4 0 -hydroxyl group of the galactose moiety is involved in hydrogen (h-)bonding with the conserved his46, asn48 and arg50 moieties, sequence conservation of asn59/glu69 enabling h-bonding with the 6 0 -hydroxyl group. in addition, the 3 0 -hydroxyl is in watermediated contact with glu32, and c-h/p interactions with trp66 complete the binding pattern for the galactose moiety. since trp oxidation to the oxindole impairs ligand binding, as first shown for electrolectin [52] , the substantial reactivity of this residue (up to 18.6% after 24 h when exposed to 0.05% hydrogen peroxide [19] ) is noteworthy. deamidation within peptide 58e70 (after seven days at 25 c/40 c reaching 9.2%/13.5% [19] ) may also have an impact on the protein's lectin activity, if not protected by bound ligand. in addition to galactose, the 3 0 -hydroxyl group of the glucose part of lactose contributes to ligand binding, as shown for cg-1a and cg-2 by measuring inhibitory capacity of methyl b-lactosides on lectin binding to asialofibrin [53] . b-methyl derivatives of lactose with 3-deoxy and 3-deoxy-3-methyl glucose have low or no inhibitory potency on cg-1a (and human gal-1). in c-grifin, this 3-hydroxyl connects by h-bonding with arg50 and glu69 (fig. 5) . val71, taking the place of the arg residue conserved in homodimeric cgs and zebrafish grifin, is not able to engage in h-bonding. when for example compared to cg-2 [13] , the arg71val substitution causes a loss in h bonding to this hydroxyl for c-grifin. this exchange of an amino acid, which is shared by mammalian grifins, yet does not appear to be detrimental for lactose binding. considering the ph dependence, contribution to affinity by glu69 could be reduced by an increasing degree of its protonation. its pk a was calculated to be 4.6 so that a loss of enthalpy generation may ensue at this site at low ph. going above neutral ph, his46 (at pk a 6.75) may be a factor in the observed affinity decrease at ph 8.5, whose origin appears to be more complex than a single-site alteration. when extending testing to the 3 0 -o-sulfated derivative of 2naphthyl b-lactoside in itc, the k d -value decreased from 8.6 to 0.13 mm. this marked enhancement of affinity by presence of the sulfate group is in contrast to 3 0 -sialylation, as observed in cell binding [19] . when comparing the binding mode of the negatively charged lactose derivative to cg-8n, reported previously [31] , with the situation in c-grifin, a lack of interaction with arg57 and also glu45 may have an influence on this grading of affinity to the lactoside and its 3 0 -modified derivatives. binding-site modeling with the 3 0 -o-sulfated lactose as ligand provides a set of putative contacts to the sulfate group that can adopt two orientations (fig. s3) . interestingly, these sets include contacts to asn48 and arg50 as well as trp66, albeit not with the same conformer, as is the case for the n-domains of gal-4 and -8 [54, 55] : gal-4c, in contrast, appears to employ a transient contact to trp256 for the slight affinity increase caused by the presence of the 3 0 -sulfate group [56] . when comparing in detail positions of amino acid of the ligandfree and -loaded subunits of the c-grifin homodimer, one liganddependent event of reorganization was revealed (fig. 6a) . the side chain of arg50 moves toward the glucose ring into the direction of trp66 from the position of pointing to asn48, in this place also in crystallographic interactions with ser54 and glu78 of a symmetryrelated protein. interestingly, the accommodation of ligand into the n-terminal lectin domain of human gal-9 triggers such a movement of the side chain of arg87 to let its nh1 become hydrogen bonded with o2 of the glucose moiety [57] . the movement in c-grifin was also seen in crystals obtained at ph 6.2 (fig. 6b) . when c-grifin in solution bound to lactose on beads and was tryptically fragmented, peptides covering the amino acid stretches of 26e52 and 58e70 had sufficient interactions to maintain their binding during repeated washes and could be eluted [19] . this result is in line with the contact pattern seen in crystals. taking analysis of c-grifin and lactose binding again to the level of a solution in this report, measuring extent and profile of hdx in the absence and presence of ligand can identify the contact site. beyond furnishing a sensitive means to do so, measuring the impact of ligand presence on amide deuteration can also detect alterations in this parameter at other sites. this has recently been accomplished for cg-8n when introducing application of this method to galectins [31] . measured for homodimeric human gal-1 and cg-1b/2 by small angle neutron scattering and fluorescence correlation spectroscopy, respectively [44, 58, 59] , ligand binding can lead to shape changes into both directions, depending on the type of protein. we thus performed hdx experiments with c-grifin. peptic fingerprinting of c-grifin and sequence coverage is illustrated in fig. 7 (for complete listing of peptides with sequence assignment, please see table s1 ; the n-terminal peptide without release of the methionine, i.e. malr, is detected in tryptic digests at an overall level of 2.1% together with the predominant alr tripeptide after release). the number of detected peptides was 94. the size of the panel ensured complete sequence coverage at a redundancy of 7.25. this value denotes the average frequency of a distinct amino acid position covered by the panel of identified peptides. presence of lactose reduced deuterium uptake into amides, predominantly in the sequence stretch covering canonical amino acids, with strong impact especially on the peptide 55e61 (fig. 8) . changes seen at position 1e17 and 21e38 should be viewed with caution, because no confirmation by overlapping peptides was possible. the quantitative data on the level of peptides given in fig. 8 can now be introduced by color coding to the crystal structure. their implementation into structural models of the binding site is shown in fig. 6c and d. accommodation of the binding site by lactose thus reduces extent of deuteration in the region of hydrogen/ch-p bonding, without a marked alteration at other sites. to pursue the delineation of relative importance of distinct amino acids in this region for ligand binding, site-directed mutagenesis was applied. the example of mammalian gal-1 had revealed that already the mutation at a single site was sufficient to abolish reactivity to glycans. in detail, the tested cases are asn46asp, arg48his, asn61asp, glu71gln and arg73his, the trp68leu substitution causing a drastic reduction [60e62]. interestingly, the natural occurrence of the arg71val substitution does obviously not preclude binding of lactose by c-grifin despite the documented reduction in h-bonding to the 6 0 -hydroxyl group of galactose (fig. 5) . as outlined in the introduction, a mutation can yet have different consequences. using a histochemical assay to probe lactose-inhibitable reactivity with the pattern of natural glycans in tissue sections, wild-type c-grifin and three mutants were tested. the application of tissue sections as assay platform enables to monitor the activity profile of the test proteins with cellular glycoconjugates, covering natural diversity in structural and spatial parameters. in view of the general importance of trp66's indole ring for c-h/p interactions, as shown in fig. 5 , a trp66leu mutant was designed, with the expectation of a substantial loss of signal. next, the structure of mammalian grifin at position 48 was mimicked by the asn48lys mutation. absence of asn at the equivalent site leads to knocking down lactose-dependent binding for human gal-1 noted above. this alteration was combined with an arg50val exchange, aimed at further harming hydrogen bonding to the 4 0 -hydroxyl group of the galactose moiety. of note, this pair of two sequence deviations is encountered in chicken galectinrelated protein that has no affinity for lactose (c-grp [63] ). to complete our series, a triple mutant was engineered. all four variant proteins failed to bind to lactose-bearing beads, precluding their purification by standard affinity chromatography, thus requiring the route over fusion-protein design. in the histochemical assay, the wild-type and the four mutant proteins were tested under identical conditions. titrations from 4 mg/ml to 48 mg/ml were performed in the absence and presence of 200 mm lactose, flanked by controls to exclude probeindependent staining. incubation of sections with the wild-type protein led to strong staining that was completely inhibited by the cognate sugar (fig. 9a) . the presence of trp66leu mutation decreased signal intensity and capacity of lactose for inhibition (fig. 9b ). in contrast, the substitution at position 48 even increased signal intensity that was not affected by lactose (fig. 9c) . by adding the arg50val to the asn48lys substitution, signal intensity fell back to the level of the trp66leu variant (fig. 9d) . as likewise seen in the case of the triple mutant (not shown), the effect of lactose presence on signal intensity by the double mutant was nearly comparable to that of the trp66leu mutant (fig. 9b,d) . the asn48lys substitution, the central difference between mammalian and bird/fish grifins with respect to direct ligand contact, therefore appeared to fundamentally affect relative signal intensity among mutants and susceptibility to presence of lactose. of course, other alterations present in mammalian grifin, too, could make their presence felt, because reconstitution at three sites of rat grifin (i.e. val46phe, lys47asn, val70arg) did not repair the defect in lectin activity [17] . furthermore, the examples of longrange effects of the r111h/c2s mutations in human gal-1, causing a shift in the positions of his52/trp68 [13] , and of the snp-based phe19tyr change in a natural variant of human gal-8 [64] attest remarkable plasticity and sensitivity for subtle changes. in this respect, it will be very informative to have the crystal structures of cg-3 and also of the c-terminal domain of cgfig. 8 . ligand-dependent reduction of deuterium uptake in c-grifin. summary of differences in deuterium uptake over a deuteration time course of 0.5 min, 1 min, 10 min, 60 min and 240 min between peptides of ligand-free and lactose-loaded c-grifin is presented in quantitative form. the peptide index given on the x-axis was calculated on the basis of midpoint values that reflect the position of the peptide within the amino acid sequence of the protein; the blue dotted lines represent the two-sided confidence limit (a 0.02) calculated as described [32] . fig. 9 . histochemical staining profiles of biotinylated wild-type c-grifin and three mutants in paraffin-embedded sections of fixed adult chicken kidney. wild-type c-grifin strongly stained the thick loops (tl), distal tubules (dt) and the apical part of the proximal tubules (pt) (a). binding was also detected in the collecting ducts (cd) and in the peripheral collecting tubules (ct). presence of 200 mm lactose led to complete abolishment of binding of c-grifin (inset to a). the introduction of the trp66leu mutation led to marked decrease of staining, with only weak signals in the thick loops (tl), distal tubules (dt) and the collecting tubules (ct) (b). very weak but significant staining was seen in the proximal tubules (pt). incubation with 200 mm lactose either slightly reduced signal intensity (thick loops (tl), distal tubules (dt), collecting tubules (ct)) or led to complete inhibition of binding (proximal tubules (pt)) (inset to b). in contrast, the asn48lys mutant led to very strong staining (c). no inhibitory effect was seen in the presence of lactose (inset to c). combining this mutation with an arg50val substitution (to obtain the asn48lys/arg50val double mutant) markedly decreased signal intensity in all areas of the section (d), with no effect by presence of 200 mm lactose (inset to d). the concentration of biotinylated proteins was 12 mg/ml. the corresponding category of signal intensity is given in the bottom left part of each microphotograph, according to the following grading system: -, no staining; (þ), very weak but significant staining; þ, weak staining; þþ, medium-level staining; þþþ, strong staining; þþþþ, very strong staining. scale bars: 20 mm. 8 available, which are closest to c-grifin in the phylogenetic family-tree diagram [19] . the same holds true for the identification of binding partners in the lens. "since grifin comprises approximately 0.5% of the watersoluble lens protein [17] , it is rather remarkable that so little is known about a possible physiological role of this protein in the lens" [35] . in transgenic mouse lenses, chemical cross-linking of a tagged version of human aa-crystallin led to detecting grifin in the complexes, supported by co-elution of the two proteins in gel filtration at a peak around 600 kda [35] . the k d -value measured in filtration binding (scatchard) assays with murine a l -crystallin was 13.6 ± 5.3 mm (9.4 mm) with a stoichiometry of 0.4 ± 0.08 (0.34) grifin monomer per crystallin monomer (0.25 ± 0.01 for bovine crystallin) [35] . this stoichiometry intimates the possibility that the grifin homodimer may help give order when packaging crystallin proteins. as consequence, grifin's presence contributes to establish the refractive index of the lens. that galectins of this design have been referred to as "bridging molecules" [65] and can indeed, especially at locally high concentrations, form vesicle aggregates [66e68] or lattices [21] makes them suited to become a molecular "glue" for binding partners [69] . intriguingly, di-or tetrameric (plant) lectins of the b-sandwich fold are known to assist in ordered packaging. this is an intracellular role of leguminous lectins. their association to storage proteins destined to fill protein bodies depends either on protein-glycan (for glycosylated vicilin) or protein-protein interactions (for nonglycosylated vicilin and legumin) [70, 71] . this dual reactivity is not uncommon, a lectin from slime molds even employing two contact sites for a glycan and for a protein to fulfill its role in ordered cell migration [72] . to take the comparison to grifin further, the leguminous lectins can bind to protein body membranes [73] . the finding of an association of grifin and a-crystallin with the plasma membrane of lens fiber cells [17, 74] has led to assuming an influence on "cell elongation and suture formation during lens development" [35] . the homodimeric design as well as high degree of stability and rigidity can thus be molecular means of grifins for helping to build and/or maintain the high-order organization of lens proteins. bridging of counterreceptors, an often encountered theme in translating glycan-encoded messages by lectins [8, 21, 75] , will in this context likely engender stability of aggregates in three dimensions. whether and how grifins and their yes/no variability of lectin activity in proteins from different vertebrates actually play a role in these processes remains to be clarified. equally important, mapping the course of grifin expression and localization in lens development is required to define physiological role(s) of respective proteins with/without lectin activity. the combined analysis of c-grifin in crystals (at seven ph values from 4.2 to 8.5) and in solution (up to 9.0 mg/ml in saxs) revealed c-grifin to be a stable and compact homodimer with no tendency for aggregation in solution. the contact profile to lactose compensated the arg71val deviation and maintained the enthalpically driven nature of binding. ligand association caused a shift in the position of the side chain of arg50, the only observed structural change in crystals. in solution, monitoring hdx in peptic fingerprints at 100% sequence coverage, too, confirmed location of the binding site and did not record any significant liganddependent change. localization of contact site(s) for ligands and monitoring for an impact of the association on solvent accessibility of protein regions are the strengths of this technique [31, 76] . implementing the asn48lys change, a hallmark of mammalian grifins, made binding of this mutant protein to tissue sections rather insensitive to lactose presence, intimating this position to be a key site for interactions. this result and the presented structural informations build a solid foundation to proceed in the quest to understand 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ligandinduced structural changes in crystal and solution hydrodynamic properties of human adhesion/growth-regulatory galectins studied by fluorescence correlation spectroscopy analysis of homodimeric avian and human galectins by two methods based on fluorescence spectroscopy: different structural alterations upon oxidation and ligand binding soluble 14-kda b-galactoside-specific bovine lectin. evidence from mutagenesis and proteolysis that almost the complete polypeptide chain is necessary for integrity of the carbohydrate recognition domain effect of amino acid substitution by sited-directed mutagenesis on the carbohydrate recognition and stability of human 14-kda b-galactoside-binding lectin further evidence by site-directed mutagenesis that 127e138 conserved hydrophilic residues form a carbohydrate-binding site of human galectin-1 galectin-related protein: an integral member of the network of chicken galectins. 1. from strong sequence conservation of the gene confined to vertebrates to biochemical characteristics of the chicken protein and its crystal structure natural single amino acid polymorphism (f19y) in human galectin-8: detection of structural alterations and increased growthregulatory activity on tumor cells factors mediating cell-cell recognition and adhesion. galaptins, a recently discovered class of bridging molecules mimicking biological membranes with programmable glycan ligands selfassembled from amphiphilic janus glycodendrimers dissecting molecular aspects of cell interactions using glycodendrimersomes with programmable glycan presentation and engineered human lectins reaction of a programmable glycan presentation of glycodendrimersomes and cells with engineered human lectins to show the sugar functionality of the cell surface galectin: intelligent glue, non-bureaucratic bureaucrat or almighty supporting actor interactions between lectins and other components of leguminous protein bodies in vivo binding partners of the lens culinaris lectin receptor for the cell binding site of discoidin i interaction of the soybean (glycine max) seed lectin with components of the soybean protein body membrane characterization of a-crystallin plasma membrane binding how to crack the sugar code exploration of the metal coordination region of concanavalin a for its interaction with human norovirus network analysis of adhesion/growth-regulatory galectins and their binding sites in adult chicken retina and choroid rr is grateful to the natural sciences and engineering research council of canada (nserc) for a canadian research chair and financial support. ar thanks the spanish ministry of economy and competitiveness for the financial support through the bfu2016-77835-r project. we are grateful for inspiring discussions with drs. b. friday and a. leddoz, for obtaining access to the itc equipment to prof. n. doucet, to m. l etourneau, j. gagnon and p.t. nguyen for helpful advice as well as for the outstanding technical assistance provided by the staff members of the soleil (france) and alba (spain) synchrotrons and for the thorough manuscript review leading to valuable recommendations. supplementary data related to this article can be found at https://doi.org/10.1016/j.biochi.2017.12.003. key: cord-256047-mabrmzd9 authors: jacomin, anne-claire; taillebourg, emmanuel; fauvarque, marie-odile title: deubiquitinating enzymes related to autophagy: new therapeutic opportunities? date: 2018-08-19 journal: cells doi: 10.3390/cells7080112 sha: doc_id: 256047 cord_uid: mabrmzd9 autophagy is an evolutionary conserved catabolic process that allows for the degradation of intracellular components by lysosomes. this process can be triggered by nutrient deprivation, microbial infections or other challenges to promote cell survival under these stressed conditions. however, basal levels of autophagy are also crucial for the maintenance of proper cellular homeostasis by ensuring the selective removal of protein aggregates and dysfunctional organelles. a tight regulation of this process is essential for cellular survival and organismal health. indeed, deregulation of autophagy is associated with a broad range of pathologies such as neuronal degeneration, inflammatory diseases, and cancer progression. ubiquitination and deubiquitination of autophagy substrates, as well as components of the autophagic machinery, are critical regulatory mechanisms of autophagy. here, we review the main evidence implicating deubiquitinating enzymes (dubs) in the regulation of autophagy. we also discuss how they may constitute new therapeutic opportunities in the treatment of pathologies such as cancers, neurodegenerative diseases or infections. autophagy is a lysosomal catabolic process that ensures the degradation and recycling of intra-cytoplasmic components and, therefore, highly contributes to the maintenance of cellular homeostasis. in order to adapt to various stresses and promote its survival under challenging conditions, the cell also uses autophagy to degrade a broad range of endogenous or exogenous substrates [1] . the best-studied endogenous substrates of autophagy are mitochondria and protein aggregates [2] [3] [4] . defects in the elimination of such substrates are often associated with human pathologies, including neurodegenerative diseases or cancers [5] [6] [7] . autophagy is a very dynamic process and has to be tightly regulated to provide a timed and efficient response to a multitude of signals. posttranslational modifications play a significant role in the induction and regulation of autophagy [8, 9] . ubiquitination of substrates and protein of the autophagy machinery has notably emerged as a central regulatory mechanism of autophagy acting at various levels to promote autophagy induction or shutdown [10, 11] . ubiquitination is a reversible protein modification that consists of the covalent attachment of one or several ubiquitin moieties to protein substrates [12] [13] [14] . while ubiquitination is achieved by the sequential action of e1 ubiquitin-activating enzyme, e2 ubiquitin-conjugating enzyme, and an e3 ubiquitin ligase, the removal of ubiquitin from a protein is catalyzed by deubiquitinating enzymes (dubs) [15] . ubiquitination can promote or interfere with protein-protein interactions, modifying the the main regulators of autophagy are ampk (amp-activated kinase) and mtorc1 (mtor complex (1) which act as autophagy activator and inhibitor, respectively. ampk and mtorc1 regulate the activation of the ulk1 complex which, together with the pi3k-ш complex, initiates the autophagosome formation. the formation of an autophagosome starts with the generation of a phagophore which elongates into an isolation membrane where the cargos to be degraded are gathered. the enclosure of the isolation membrane forms a double-membraned autophagosome that matures and eventually fuses with the lysosome, forming an autolysosome where lysosomal hydrolases degrade its content. the atg12 and lc3 conjugation systems are essential for the autophagy process and formation of the autophagosome. the atg12 conjugation system consists in the formation of the atg12-atg5-atg16l1 complex that then promotes the conjugation of lc3; pro-lc3 is cleaved by atg4 (lc3-i) which is then conjugated with phosphatidylethanolamine (pe) (lc3-ii) before being anchored to the nascent autophagosomal membrane. ubiquitin is a small globular protein with a -grasp superfold conformation [39, 40] . ubiquitin is covalently conjugated by its terminal glycine (g76) onto a lysine residue of a substrate protein. protein ubiquitination is a complex process requiring the successive activity of three types of enzymes: an e1 ubiquitin-activating enzyme, an e2 ubiquitin-conjugating enzyme, and an e3 ubiquitin-ligase enzyme. the ubiquitination process can be broken down in two main steps: (1) the atp-dependent activation of a ubiquitin molecule by conformational modification of its c-terminus extremity by an e1 enzyme followed by its transfer onto an e2 enzyme, and (2) the conjugation of the activated ubiquitin onto a substrate protein, mediated by an e3 enzyme that bridges the e2 to a the main regulators of autophagy are ampk (amp-activated kinase) and mtorc1 (mtor complex (1) which act as autophagy activator and inhibitor, respectively. ampk and mtorc1 regulate the activation of the ulk1 complex which, together with the pi3k-iii complex, initiates the autophagosome formation. the formation of an autophagosome starts with the generation of a phagophore which elongates into an isolation membrane where the cargos to be degraded are gathered. the enclosure of the isolation membrane forms a double-membraned autophagosome that matures and eventually fuses with the lysosome, forming an autolysosome where lysosomal hydrolases degrade its content. the atg12 and lc3 conjugation systems are essential for the autophagy process and formation of the autophagosome. the atg12 conjugation system consists in the formation of the atg12-atg5-atg16l1 complex that then promotes the conjugation of lc3; pro-lc3 is cleaved by atg4 (lc3-i) which is then conjugated with phosphatidylethanolamine (pe) (lc3-ii) before being anchored to the nascent autophagosomal membrane. ubiquitin is a small globular protein with a β-grasp superfold conformation [39, 40] . ubiquitin is covalently conjugated by its terminal glycine (g76) onto a lysine residue of a substrate protein. protein ubiquitination is a complex process requiring the successive activity of three types of enzymes: an e1 ubiquitin-activating enzyme, an e2 ubiquitin-conjugating enzyme, and an e3 ubiquitin-ligase enzyme. the ubiquitination process can be broken down in two main steps: (1) the atp-dependent activation of a ubiquitin molecule by conformational modification of its c-terminus extremity by an e1 enzyme followed by its transfer onto an e2 enzyme, and (2) the conjugation of the activated ubiquitin onto a substrate protein, mediated by an e3 enzyme that bridges the e2 to a specific substrate allowing for the subsequent transfer of ubiquitin from the e2 enzyme to the substrate through the formation of an isopeptide bond [41, 42] . alternatively, e3 ligases of the hect family possess e2 and e3 activities [43, 44] . there is also evidence for the addition of ubiquitin moieties onto non-lysine residues. the ubiquitination of cysteine or serine and threonine residues requires the formation of thiol-or oxy-ester bonds respectively [45] . ubiquitination was discovered in 1980 and first described for its essential role in targeting proteins to the proteasome for degradation [46] . it is now widely recognized that ubiquitination also acts in other cellular processes. indeed, a broad range of types of ubiquitination have been reported. substrates can be modified by the attachment of a single ubiquitin molecule (mono-ubiquitination) or several single ubiquitin molecules on different lysine residues of the substrate (multi-mono-ubiquitination). mono-ubiquitination is primarily described for its function in endocytosis of plasma membrane receptors [47] . alternatively, several ubiquitin molecules can be ligated to one another using ubiquitin internal lysine residues, forming chains of ubiquitin moieties that can elongate on the substrate or directly be attached to a target protein (poly-ubiquitination). because ubiquitin has seven lysine residues, there can be at least as many types of ubiquitin chains that can be generated (k6, k11, k29, k48, k63-linked ubiquitin chains) [48, 49] . poly-ubiquitin chains can also be assembled through n-to c-terminal interaction to form linear chains (m1) [50, 51] . the tridimensional structures of ubiquitin chains vary depending on the lysine in the ubiquitin used to generate the chain and affect the function or stability of the substrate. for instance, k48-linked ubiquitin chains have a compact conformation and target substrates for proteasomal degradation, while k63-linked ubiquitin chains display an open conformation and are mostly described to promote signal transduction through the assembly of large protein complexes [52, 53] . protein ubiquitination is a reversible posttranslational modification. the hydrolysis of ubiquitin linkages is conducted by a specific family of proteases: the dubs. these enzymes can act at different stages of the protein ubiquitination process: (1) at the "initial" stage, by cleaving the ubiquitin precursors to supply ubiquitin monomers to the ubiquitination enzymes; (2) at the "intermediate" stage, by the regulated removal of ubiquitin moieties from proteins to alter their fate (stabilization, conformational change); and (3) at the "final" stage by the removal of ubiquitin chains from substrates addressed to the proteasome to facilitate their degradation and processing into ubiquitin monomers, free to enter a new ubiquitination cycle ( figure 2 ) [54] [55] [56] . the hydrolysis of k48-linked ubiquitin chains, most well-known to induce the proteasomal degradation, can affect the fate of the protein they are added to either by protecting substrates from degradation or by supporting proteasomal degradation, as the removal of k48-linked ubiquitin chains, mostly by proteasomal dubs, is also required for protein entry into the proteasome [57, 58] . there are approximately 100 dubs encoded by the human genome [15, 59] , which are divided into two main families: the cysteine proteases and the metalloproteases. there are 12 dubs from the metalloprotease family characterized by a jamm (jab1/pab1/mpn domain-containing metalloenzymes) domain that catalyzes the hydrolysis of isopeptide bonds in the presence of zn 2+ . cysteine proteases are divided into five sub-families according to the sequence and structure of their catalytic domain: ubiquitin-specific proteases (usps), ubiquitin c-hydrolases (uchs), otubain proteases (otus), machado joseph disease proteases (mjds), and the most recently identified sub-family miu-containing novel dub family (mindys). the most abundant sub-family of dubs is the usps with over 50 members, come after the otus (18 members), uch, mjds and mindys (each with four members) [15] . protein posttranslational modifications are crucial in the dynamic regulation of the autophagy process. modifications of core components of the autophagic machinery are notably essential for the induction of autophagy. autophagy proteins that are ubiquitinated constitute substrates for dubs, therefore regulating their function and/or stability [60] . the mechanistic target of rapamycin complex 1 (mtorc1) plays a central role in the integration of various environmental signals to regulate cell metabolism, growth, proliferation, and survival. in nutrient-rich conditions, mtorc1 is active and promotes cell growth while down-regulating autophagy. conversely, down-regulation of mtorc1 activity during nutrient deprivation activates autophagy. the protease otub1 was recently reported to inhibit mtorc1 activity by deubiquitinating and stabilizing the inhibitor deptor in response to amino acid deprivation [61] ( figure 3a ). deptor stabilization by otub1 depends on its catalytic activity as the catalytically inactive mutant otub1-c91a fails to both remove ubiquitin moieties from deptor and protect it from proteasomal degradation. consistent with these observations, otub1 overexpression induces autophagy while its knockdown represses autophagy induction [61] . protein posttranslational modifications are crucial in the dynamic regulation of the autophagy process. modifications of core components of the autophagic machinery are notably essential for the induction of autophagy. autophagy proteins that are ubiquitinated constitute substrates for dubs, therefore regulating their function and/or stability [60] . the mechanistic target of rapamycin complex 1 (mtorc1) plays a central role in the integration of various environmental signals to regulate cell metabolism, growth, proliferation, and survival. in nutrient-rich conditions, mtorc1 is active and promotes cell growth while down-regulating autophagy. conversely, down-regulation of mtorc1 activity during nutrient deprivation activates autophagy. the protease otub1 was recently reported to inhibit mtorc1 activity by deubiquitinating and stabilizing the inhibitor deptor in response to amino acid deprivation [61] ( figure 3a ). deptor stabilization by otub1 depends on its catalytic activity as the catalytically inactive mutant otub1-c91a fails to both remove ubiquitin moieties from deptor and protect it from proteasomal degradation. consistent with these observations, otub1 overexpression induces autophagy while its knockdown represses autophagy induction [61] . the serine/threonine protein kinase ulk1 (unc51-like kinase 1) is a critical inducer of autophagy (see above and figure 1 ). dynamic phosphorylation and polyubiquitination regulate ulk1 activity. notably, the ubiquitination of ulk1 with k63-linked ubiquitin chains contributes to its stabilization and activity [38] . in contrast, the linkage of k48-linked ubiquitin chains leads to proteasomal degradation of ulk1, resulting in a blockade of starvation-induced autophagy [62, 63] . a loss-of-function screen of dubs in hela cells identified usp20 as the first dub to be involved in regulating ulk1 ubiquitination and stability. usp20 interacts with and deubiquitinates ulk1, thus protecting it from degradation. the maintenance of basal levels of ulk1 by usp20 contributes to rapid induction of autophagy under stress condition. indeed, silencing of usp20 encoding gene inhibits autophagosomes and autolysosomes formation in response to starvation ( figure 3b ). however, the interaction between ulk1 and usp20 is weakened upon prolonged induction of autophagy (4 to 8 h), leading to a reduction of the level of ulk1 protein while its ubiquitinated form accumulates [64] . the molecular mechanisms regulating the dissociation of usp20 and ulk1 are not known yet but could depend on posttranslational or allosteric modifications on usp20 as its stability is not affected by starvation; alternatively, the e3 ubiquitin ligase nedd4l, may compete with usp20 to interact with ulk1 and promotes its proteasomal degradation [63] . beclin1 is a multivalent adaptor protein and forms, with vps34 and vps15, the core components of the pi3k-iii signaling complex, which is essential for the maturation of the autophagosome (see above and figure 1 ). beclin1 also interacts transiently with accessory factors, such as atg14l, ambra1, uvrag or bcl-2 [65] . moreover, beclin1 versatile ubiquitination is tightly linked to its function and activation [37] . different types of ubiquitin chains, including k63-and k48-linked chains, were found on beclin1, and several dubs control beclin1 ubiquitination status and activity ( figure 3c ). usp14 is a ubiquitin-specific protease tightly associated with the proteasome which has been shown to cleave k48-linked ubiquitin chains [66, 67] ; however, other studies have shown that usp14 is also able to cleave k63-linked ubiquitin chains [68, 69] . knockdown of usp14 or its inhibition with the inhibitor iu1 (see below section 4.1) induces the activation of autophagy, indicating that usp14 is a negative regulator of autophagy in h4 (neuroglioma) cells. because silencing the usp14 encoding gene does not affect the stability of beclin1 or other components of the complex, this suggests that its ability to regulate autophagy is independent of its proteasomal function in h4 (neuroglioma) cells. according to this observation, it was shown that usp14 suppresses the activity of beclin1 complex and induction of autophagy by interacting with and controlling k63-rather than k48-linked ubiquitin chains of beclin1 [70] . autophagy is highly interconnected with immune processes and can be triggered by activated tlr4 (toll-like receptor 4). activation of tlr4 by microbial components contributes to the recruitment of adaptor proteins and enzymes required for the signal transduction and induction of the immune response by nf-κb (nuclear factor kappa b) transcription factors, such as the e3 ubiquitin ligase and scaffold protein traf6 (tnfr-associated factor 6). traf6 is then responsible for the induction of autophagy following the activation of the tlr4 in macrophages through the ubiquitination of beclin1 with k63-ubiquitin chains in murine macrophages raw 264.7 [71] . indeed, ubiquitination of the lysine residue 117 within the bh3 domain of beclin1 prevents its interaction with the inhibitor bcl-2 [71, 72] . in addition, min and colleagues showed that traf6 and usp14 compete for the interaction with beclin1 in hek293t (human embryonic kidney 293t) cells and that usp14 negatively regulates autophagy in thp-1 monocyte cells [73] . traf6 and beclin1 interact through their coiled-coil domains in the absence of usp14, whereas the interaction is gradually attenuated when the cells are co-transfected with increasing quantity of usp14. however, the study does not provide any evidence for the catalytic role of usp14 and instead suggests that the interaction of beclin1 with usp14 inhibit traf6-mediated ubiquitination of beclin1 [73] . additionally, the dub a20-a downstream target of nf-κb-is responsible for the catalytic removal of k63-linked ubiquitin chains on the lysine 117 of beclin1 to limit the induction of autophagy in the murine macrophage cell line raw 264.7 [71] . interestingly, beclin1 and the bcl-2 family member, mcl-1, compete for their interaction with usp9x, which contributes to their stabilization by protecting them from proteasomal degradation in hek293t cells [74] . in the same study, it was observed that mcl-1 levels were increased in malignant tissues from melanoma patients while beclin1 was destabilized, suggesting that usp9x promotes tumor progression. however, usp9x function in tumorigenesis appears to be more complex and context-dependent as independent studies have shown that usp9x can be either a tumor promoter or suppressor depending on the origin of the cells [75, 76] . the addition of k11-linked ubiquitin chains to beclin1 by the e3 ligase nedd4 triggers its degradation by the proteasome [77] . the enzymes usp13 and usp19 are known to process k11-linked ubiquitin chains resulting in the stabilization of beclin1 in hek293t cells (for both usp13 and usp19 roles), as well as in mef, hela and bcap-37 cells (role of usp13) [78, 79] . although several lysine residues in beclin1 may be ubiquitinated with k11-linked chains, usp19 seems to mainly target ubiquitin moieties bound to the lysine 437 of beclin1 in hek293t cells, suggesting that its function may not be redundant, but instead complementary to usp13 [78] . finally, the dub usp33 was found to be a regulator of early steps of starvation-induced autophagy activation by promoting the interaction between beclin1 and the ras-like gtpase ralb. indeed, the assembly of the complex ralb-exo84-beclin1 is made possible by the deubiquitination of ralb at its lysine residue 47 in hek293t and hela cells [80] . the identification of usp33 as indirectly regulating beclin1 activity through the deubiquitination of one of its partners reinforces the hypothesis that the modification and activation of autophagy machinery components are context-specific. the formation of protein aggregates is a continuous process in the cell, and their degradation by autophagy, referred to as aggrephagy, is one of the first types of selective autophagy that has been described. aggrephagy involves the autophagy receptors p62/sqstm1 and nbr1 which are recruited to ubiquitinated protein aggregates [81] [82] [83] [84] . the accumulation of protein aggregates that fail to be degraded in neuronal or glial cells is a hallmark of various neurodegenerative diseases. aggregation of α-synuclein is characteristic of the pathogenesis of parkinson's disease in which mono-and poly-ubiquitinated α-synuclein are major constituents of the lewy bodies [85, 86] . mono-ubiquitination of α-synuclein seems to be required for its targeting of proteasomal degradation and thus negatively regulates its autophagic degradation. usp9x interacts with α-synuclein in vitro and in vivo and contributes to the removal of mono-ubiquitin moieties and favors its targeting for degradation by autophagy rather than the proteasome [87] . conversely, uch-l1-a dub associated with parkinson's disease and whose gene is frequently mutated in familial forms of the pathology [88] -promotes the accumulation of α-synuclein aggregates in oligodendrocytes [89, 90] . another independent study also reported that membrane-associated uch-l1 contributes to α-synuclein neurotoxicity, possibly by negatively regulating its lysosomal degradation [91] ( figure 3d ). finally, a recent study demonstrates the prevalence of k63-linked ubiquitin chain conjugates in lewy bodies, suggesting that their elimination can be primarily ensured by the lysosomal route rather than the 26s proteasome. in addition, this study identifies usp8 as one of the best markers of lewy bodies in human pigmented neurons in sporadic cases of parkinson's disease and demonstrates the ability of usp8 to hydrolyze k63-linked ubiquitin chains from α-synuclein in vitro [92] . moreover, usp8 gene extinction significantly reduces the total level of α-synuclein both in a drosophila model of parkinson's disease and in human embryonic kidney (hek293t) cultured cells [92] . thus, the presence of usp8 on endosomal membranes (see below) and in lewy bodies, as well as its ability to deubiquitinate α-synuclein, makes it a preferred therapeutic target according to the assumption that inhibition of usp8 could directly promote the elimination of amyloid fibers by the endocytic and lysosomal pathways. it was recently shown that the selective autophagy receptor p62 binds to protein aggregates modified not only with k63-linked ubiquitin chains but also with k33-linked ubiquitin chains [93, 94] . zranb1/trabid is a k29-and k33-specific dub [95] . knockdown of zranb1 enhances the recruitment of p62 to k33-associated protein aggregates, suggesting that zranb1 is a negative regulator of aggrephagy; yet, the physiological function of k33-linked ubiquitin chains and the role of zranb1 are not entirely understood [94] . in drosophila, the p62 homolog ref (2)p is also implicated in the clearance of ubiquitinated protein aggregates [96, 97] . however, little is known about the regulation of autophagy-associated ubiquitination processes in flies. the only dub known to regulate ubiquitin-dependent autophagy negatively is dusp36 [98] . this protein negatively regulates the formation of ubiquitinated nuclear aggregates, while promoting cell growth. indeed, deletion of the dusp36 encoding gene results in the robust accumulation of ubiquitinated protein aggregates, which include the histone protein h2b, and in the activation of autophagy, independently of the tor pathway [98] (figure 3d ). protein aggregates clearance requires the action of selective receptors to be adequately targeted for autophagic degradation [3] . ndp52 is an autophagy receptor mostly described for its role in addressing ubiquitin-decorated bacteria for degradation by autophagy [99, 100] . however, a new role for ndp52 in the formation of traf6 aggregates was unveiled [101] . indeed, ndp52 mediates the aggregation and selective autophagic degradation of the tlr adaptor molecule trif and the signaling molecule traf6 in response to tlr4 stimulation. ubiquitination of ndp52, mediated by traf6, is necessary for its activity and is counteracted by a20 [101] ( figure 3d ). with the variety of proteins prone to aggregations, it is not surprising that different dubs are involved in the regulation of the formation and degradation of protein aggregates. protein aggregation is a hallmark of various pathologies, notably neurodegeneration and infection, and a better understanding of the regulatory mechanisms associated with each pathology will greatly benefit the development of appropriate and targeted treatments. mitophagy refers to the clearance of exhausted mitochondria by autophagy. the mitochondrial kinase pink1 and the e3-ubiquitin ligase parkin play a central role in the mitochondrial quality control. upon mitochondria damage and loss of membrane potential, parkin is translocated to the outer mitochondrial membrane (omm) and activated by stabilized pink1 [102] . active parkin ubiquitinates a myriad of substrates on the omm that can be recognized by ubiquitin-binding selective autophagy receptors [103, 104] . so far, three dubs-usp30, s-usp35, and usp15-have been reported to counteract parkin activity following acute mitochondrial depolarization, thus acting as a negative regulator of mitophagy. usp8 is the only dub identified so far as a positive regulator of mitophagy ( figure 3e ). usp30 is a mitochondrial enzyme, tethered to the outer membrane of the mitochondria with its catalytic domain facing the cytoplasm [105] . several independent studies point out that usp30 is one of the major dub regulating mitophagy. overexpression of usp30 reverses the ubiquitination of parkin substrates, such as tom20, and impairs mitophagy [106] [107] [108] [109] . usp30 function in autophagy is dependent on its catalytic activity as a catalytically inactive mutant usp30-c77a is ineffective at inhibiting mitophagy [106] . the depletion of usp30 was shown to enhance the degradation of mitochondria in neuronal and hela cell cultures [106, 109] . usp30 knockdown also increases the ubiquitination level on multiple parkin substrates, thus confirming that usp30 antagonizes parkin function. usp30 proteolytic activity is more efficient on k6-linked ubiquitin chains, even though it can also process k11, k48 and k63 chains [108, 110] . the ubiquitin chains targeted by usp30 are similar to the ones parkin adds to its substrates, suggesting that these two enzymes act as antagonists on shared substrates. the majority of the work carried out on the regulation of mitophagy have relied on cells overexpressing parkin along with the use of mitochondrial-depolarizing agents. such experiments simulate an extreme scenario of mitochondrial stress and interpretations may not be relevant to basal conditions of mitochondrial clearance [111] . however, recently published work by marcassa and colleagues describes the investigation into the role of usp30 in more physiological conditions [112] . the authors propose a new model in which usp30 acts upstream pink1 as the depletion of pink1 in cells lacking usp30 abrogated the increased mitophagy induced by usp30 knockdown in u2os cells [112] . in the same study, usp30 is revealed to regulate the degradation of peroxisome by autophagy (pexophagy) in a similar way to its role in mitophagy. like mitochondria, peroxisomes are the main source of reactive oxygen species (ros) that can be damaging to the cells if produced in high quantities [113] . moreover, contact sites between mitochondria and peroxisomes exist, and mitochondria were shown to play a role in the generation of peroxisomes [114] ; thus reinforcing the hypothesis of an intrinsic relationship between both organelles. the depletion of usp30 increases both pexophagy and mitophagy. however, the localization of usp30 on mitochondria and peroxisomes relies on distinct sequences, suggesting that the role of usp30 in pexophagy is independent to its mitochondrial function [112] . it is possible that usp30 acts at different levels during the mitophagy or pexophagy processes depending on the conditions. in basal condition, usp30 could serve as a safety check-point to avoid mitophagy or pexophagy to be triggered inappropriately. besides, other studies identified additional dubs which may also contribute to the regulation of mitophagy. usp35 short form (s-usp35) is another dub that is localized at the mitochondria [109] . in a similar manner to usp30, overexpression of s-usp35 impairs mitophagy while s-usp35 knockdown enhances mitochondrial degradation [109] . usp15 is the third dub identified to antagonize parkin-mediated mitophagy. overexpression of usp15 inhibits mitophagy dependently of its catalytic activity, while depletion of usp15 enhances mitophagy. unlike usp30 and s-usp35, usp15 is only rarely localized at the mitochondria [115] . only one dub, usp8, may act as a positive regulator of mitophagy through parkin regulation. usp8 knockdown impairs parkin-mediated mitophagy by preventing parkin recruitment of depolarized mitochondria. in this process, usp8 selectively removes k6-linked ubiquitin chains on parkin and counteracts parkin auto-ubiquitination and auto-catalytic activation [116] . whether and how these dubs act in concert or within different organs or situations remains to be determined to fully understand their specific requirements in physiological or stressed conditions. proteins can be degraded by autophagy independently of their aggregation in a way that can be either dependent or independent of their ubiquitination state. to date, only a few substrates, known to be directly targeted for degradation by autophagy in response to ubiquitination, have been shown to be regulated by a specific dub ( figure 3f ). the hypoxia-inducible factor 1, α subunit (hif-1α) is a transcription factor essential for cells to adapt rapidly to low oxygen levels (hypoxia). when oxygen is available, hif-1α is polyubiquitinated and rapidly degraded either by the proteasome or directly by the lysosome through chaperone-mediated autophagy [117, 118] . the dub cezanne/otud7b is itself induced by oxygen deprivation in cultured endothelial cells. moreover, loss of cezanne reduces the amount of hif-1α protein while cezanne overexpression stabilizes hif-1α and protects it from autophagic degradation in a catalytic-dependent manner by specifically processing k11-linked ubiquitin chains [119] . mutation of the cma-targeting motif (kferq motif) of hif-1α makes it insensitive to cezanne knockdown, thus suggesting that cezanne specifically regulates the degradation of hif-1α mediated by cma. cezanne is not the only dub to regulate the ubiquitination status of hif-1α. indeed, usp8 is essential for the removal of ubiquitin moieties on hif-1α in normoxia, contributing to the maintenance of a basal level of hif-1α. in this case, however, usp8 appears to protect hif-1α from proteasomal degradation [120] . these two studies show that different dubs can regulate the fate of a shared substrate depending on the physiology of the cell. connexin-43/cx43 is a member of the connexin family that forms the gap junction channels between adjacent cells, enabling direct intercellular exchanges between cells, which is another example of a substrate for at least two different degradative pathways. indeed, cx43 polyubiquitination triggers its degradation through either the proteasome or the lysosome via endocytosis and autophagy [121] [122] [123] [124] . usp8 interacts with and deubiquitinates cx43, removing monoubiquitin moieties as well as k63-and k48-linked ubiquitin chains. cx43 ubiquitination and degradation by autophagy are increased in usp8 knockdown cells [125] . even though usp8 regulates autophagic degradation of cx43 in basal condition, one cannot exclude that usp8 may also affect cx43 through the endolysosomal pathways in different conditions as usp8 is well described for its implication in the endocytosis of various plasma membrane receptors [126] [127] [128] . thus, there are several cases of proteins being degraded through different processes notably as a result of the linkage of different kinds of ubiquitin moieties and undergoing tight regulation by specific dubs. autophagy and endocytosis are two conserved and interconnected degradative pathways among eukaryotes. moreover, fusion events between autophagosomes and endocytic compartments have been observed and investigated [129, 130] . endocytosis and autophagy converge not only at the level of lysosomes but also at the level of early and late endosomes, forming another type of vesicle called amphisomes. several dubs, known for their role in endocytosis, also impact directly or indirectly the autophagic flux ( figure 3g ). amsh is a metalloprotease of the jamm type involved in the sorting of cell-surface receptors at endosomes [131] [132] [133] [134] . amsh localizes at the endosomes and promotes the recycling of internalized receptors [135, 136] . disruption of amsh in mice results in the loss of neurons in the hippocampus and severe atrophy of the cerebral cortex [137] . an independent study observed that the loss of amsh in neurons results in the accumulation of ubiquitinated protein aggregates associated with the autophagy receptor p62, indicating that the autophagy flux is impaired [138] ( figure 3d) . however, at this stage of the study, it is not possible to discriminate whether the blockade of the autophagy flux results from an impairment in the endocytic process or a lack of targeting of cargoes to autophagosome, independently of endocytosis. in the plant model arabidopsis thaliana, the ortholog amsh1 interacts with the escrt-iii protein vps2.1 and contributes to autophagic degradation [139] (figure 3g ). usp8 is a second dub playing a major role in endocytosis by regulating both the ubiquitination status of cargoes and members of the escrt machinery regulating membrane deformation and scission events [126] [127] [128] [140] [141] [142] . usp8 has been extensively studied for its role in the regulation of the trafficking and lysosomal degradation of receptors, such as egfr, through the endocytic process [135, 143] . in addition to its role in endocytosis, loss of dusp8 in drosophila blocks the progression of autophagy, resulting in the accumulation of ref(2)p/p62 and ubiquitinated proteins [144] . as for dusp8, dusp12 depletion affects both the autophagic flux and endocytosis process. indeed, silencing dusp12 encoding gene results in the accumulation of autophagosomes in drosophila [144] , and usp12 negatively regulates the endocytosis and translocation of the notch receptor to the lysosomes in both drosophila and mammalian cells [145] . although these studies cannot exclude a direct role of these dubs in the regulation of autophagy, they support a close imbrication of endocytosis and autophagy and suggest interdependence of the two processes. this hypothesis is reinforced by the fact that disruption of the endocytic process using dominant-negative rab or by their knockdown also results in impaired autophagy [146] . the particular case of the β2-adrenergic receptors (β2-ars) also illustrates a reliable interconnection between the endocytic and autophagic processes and their tight regulation by ubiquitination. β2-adrenergic receptors (β2-ars) availability on the plasma membrane is tightly regulated by balancing their internalization and recycling rates. misregulation of β2-ars trafficking has been associated in various pathologies, including heart failure and asthma [147] . β2-ars take an unconventional route to the lysosomes; indeed, after their endocytic internalization, ubiquitinated β2-ars are directed to the autophagosomes rather than the lysosomes [148] . the post-endocytic sorting of the receptor from the endosomes to the autophagosomes is modulated by the proteases usp20 and usp33 [149] . however, solely usp20 was shown to promote the deubiquitination of β2-ars and their post-endocytic trafficking to autophagosomes. in this process, phosphorylation of usp20 at serine residue 333 is required for its activity providing an additional level of regulation [148] . recently, the protein chmp2a of the escrt-iii complex was identified to be crucial for the closure of the autophagosome [150] . therefore, endosomes-associated dubs such as usp8 and amsh, or other dubs that remain to be identified, may also play a direct regulatory role in this process through the regulation of escrt-iii components activity, as recently shown for chmp1b during endocytosis [140] . the expression of a number of genes related to autophagy is activated upon starvation in mammalian cell culture. in this process, histone protein h2b monoubiquitination (h2bub1) is an essential modification for the regulation of gene transcription. the level of h2bub1 is controlled by the protease usp44 which is upregulated after starvation. knockdown of usp44 results in the maintenance of h2bub1 upon starvation and abolishes the change in expression of starvation-induced autophagy-related genes. moreover, downregulation of usp44 encoding gene blocks the induction of autophagy in mescs (mouse embryonic stem cells) ( figure 3h ). this study thus unveils a new role for dub in the transcriptional regulation of autophagy through the modulation of h2b monoubiquitination [151] . ubiquitination of microbial molecular patterns is used by eukaryotic cells to tag invasive pathogens and target them for autophagic degradation. this reaction leads to the accumulation of ubiquitinated protein aggregate known as ubiquitinated aggresome-like induced structures (alis). such aggregates contribute to the upregulation of autophagy and the removal of intracellular pathogens. in response to this host defense mechanism, intracellular pathogens, such as bacteria or viruses, have developed strategies to hijack the host ubiquitin pathway by expressing dub-like enzymes able to counteract ubiquitination and permit them to escape their elimination by autophagy. for instance, the intracellular pathogenic bacteria salmonella enterica serovar typhimurium (s. typhimurium) counteracts the alis-induced autophagy by translocating a dub-like enzyme, ssel (salmonella-secreted factor l), into the cytosol. lysates from mouse macrophages infected with ∆ssel mutant bacteria are enriched in ubiquitinated proteins, and immunofluorescence experiments revealed that these bacteria are more prone to ubiquitination and recognition by autophagy markers such as lc3 or p62 [152, 153] . secreted ssel deubiquitinates alis and the salmonella-containing vacuoles, reducing the induction of autophagy, further promoting the survival and replication of s. typhimurium [152] ( figure 3d ). like salmonella, legionella is an intracellular bacterium that can establish niches in cytoplasmic vacuoles which allows for the survival and replication of the bacteria. the legionella pneumophila effector protein ravz is a secreted cysteine protease that interferes with the autophagy machinery by irreversibly deconjugating lc3 from the autophagosome membrane [154] . lc3 is an autophagy-related ubiquitin-like protein anchored to the autophagosome membrane. the process leading to lc3 lipidation and association to the membrane is similar to the ubiquitination process and requires a ubiquitin-like conjugation system. ravz deconjugates lc3 from the autophagosome membrane by hydrolyzing the amide bond between the c-terminal glycine residue and an adjacent aromatic residue; the lack of terminal glycine residue prevents the conjugation of lc3 to the membrane [154, 155] . ravz can also process conventional ubiquitin chains and prevent the targeting of intracellular bacteria for autophagic degradation [156] . indeed, using a co-infection system with salmonella and legionella, kubori and colleagues showed that legionella ravz protease prevents the recruitment of the autophagy receptors p62 and ndp52 to the salmonella-containing vacuoles. the lack of autophagy receptors at the scvs is due to the removal of ubiquitin moieties from the scvs by ravz [156] . it was recently shown that ravz specificity toward lc3 anchored to the autophagosomal membrane depends on its interaction with pi3p [157] ; this observation suggests that ravz activity as dub on ubiquitin coats depends on the lipid structure of the nearby vacuole. autophagy also targets viruses; yet, many viruses exploit autophagy for their replication [158] . coronaviruses induce the formation of double-membrane vesicles that allow for their replication and are often decorated with lc3 and cell infection with coronaviruses is often accompanied with induction of autophagy [159] . the non-structural protein plp2-tm, which is a transmembrane papain-like protease with deubiquitinating activity [160] , is sufficient for the accumulation of autophagosomes in different cell lines. however, its role in the regulation of autophagy is independent of its protease activity [161] . plp2-tm interacts with lc3 and promotes the accumulation of autophagosomes by blocking their fusion with the lysosomes. in their study, chen and colleagues suggest that plp2-tm blocks autophagosome-lysosome fusion through its interaction with beclin1, a prime target for viruses that manipulate the autophagy pathway [162] . plp2-tm also promotes the interaction of sting (stimulator of interferon genes) with beclin1, possibly to impede the activation of downstream antiviral responses, accentuated by the deubiquitination of components of the signaling cascade such as rig-1 or traf3 [161, 163] . these studies provide fascinating examples of possible coevolution and adaptation of the pathogens with their host, where pathogens managed to bypass the host's defense to their own benefit. ubiquitination regulates major cellular functions by controlling protein stability and activity, and defects in this process contribute to the development of many diseases. in some cases, ubiquitin-dependent autophagic processes constitute entry points to design new treatments. depending on the context, however, autophagy can either be beneficial and contribute to survival and recovery or have adverse effects. as such, there is a need for in-depth understanding of autophagy function and regulation in pathological or physiological situations to define in which situation the inhibition of a particular dub will be beneficial or detrimental. interestingly, the design of chemical tools is also a powerful strategy to probe the effect of dubs inhibition to help both the understanding of their role in the regulation of autophagy and the design of future treatments to modulate autophagy in the corresponding pathologies. efficiency and usability of an inhibitor depends on its specificity. as such, the discovery of dub-focused drugs has been challenging [20] . indeed, although dubs have a catalytic pocket that is suitable for drug development, their sequence and structure are very similar. moreover, dubs are flexible enzymes, and the regulation of their activity can involve allosteric effects as described for several dubs that alternate between active and inactive conformations [164] [165] [166] [167] [168] [169] . for instance, the free catalytic domain of usp7 undergoes significant structural modifications when it is complexed to ubal (ubiquitin aldehyde, an irreversible dub inhibitor) [164, 170] . recent publications of the dynamic interaction of usp7 with specific small-molecule inhibitors demonstrated that the binding of the molecules into the active site of usp7 modifies the catalytic residue c223. this modification of the active site of usp7 results in its inability to change conformation and perform the cleavage of ubiquitin chains. these studies show that the development of specific inhibitors binding to the active site of dubs is a realistic approach, opening new avenues in the field [171, 172] . in addition to intrinsic modulation of their activity and substrate specificity, some dubs require cofactors. for instance, the full activation of usp19 requires its interaction with hsp90, which promotes the binding of ubiquitin to the catalytic domain of usp19 [173] . another example is the proteasome-associated enzyme usp14, whose activity is strongly enhanced when in association with the proteasome [66] . despite the complexity of the regulation of dubs activity, much effort has been placed in the identification and development of small-molecule regulating dubs catalytic activity. some of the most successful small-molecules affecting autophagy the process, through the inhibition of the activity of autophagy-associated dubs, are shortly introduced hereafter. screens for inhibitors of dubs sought to identify new small-molecules with potential in two main therapeutic fields, for cancer treatment and neurodegeneration (reviewed in [20] ). in both fields, some of the inhibitors' targets play a role in the regulation of autophagy that possibly contributes to pathogenesis. usp14 is an enzyme associated with the proteasome, which plays an essential role in the regulation of protein turnover. the role of usp14 is particularly important in neurons to maintain synaptic functions and constitutes an appealing target for drug development in order to modulate the activity of the proteasome [174] . a screen of 63,052 compounds using proteasome reconstituted with usp14, led to the identification of the first inhibitor of usp14. the small-molecule iu1 inhibits specifically usp14 with an ic 50 of 4-5 µm [66] . the inhibitor iu1 blocks the activity of usp14 only in the presence of the proteasome, suggesting that it binds only to the activated enzyme. moreover, the compound iu1 abrogates the catalytic activity of usp14 without affecting its noncatalytic regulatory function [66] . however, with the growing number of usp14 substrates identified, there was a need for the development of iu1 analogs with improved selectivity over the usp14-substrate complexes. a curated screen of 87 variants of iu1 led to the identification of iu1-47 as a new potent inhibitor of usp14. iu1-47 treatment of murine primary neuron cultures and in neurons derived from human-induced pluripotent stem cells (ipsc) accelerates the degradation of the microtubule-associated protein tau, which is implicated in many neurodegenerative diseases. besides, the inhibition of usp14 by iu1-47 induced an increase of the autophagy flux, consistent with the increased degradation rate of tau [175] . as mentioned above, uch-l1 is a negative regulator of autophagy widely studied for its implication in parkinson's disease and its contribution to the aggregation of α-synuclein as a result of autophagy blockade [86, 89] . uch-l1 is also expressed in various primary lung tumors while not detectable in normal, healthy lung tissue, suggesting a possible contribution to cancer [176] . because of the correlation between uch-l1 and tumor progression, as well as its implication in neurodegenerative disease, uch-l1 is a recognized target for the development of therapeutic inhibitors. as such, the compound ldn-57444 was identified in a high throughput drug screening as a specific uch-l1 inhibitor. treating h1299 lung cancer cell line with this compound significantly reduces the cell proliferation rate [177] . nsc632839 is another inhibitor that affects uch-l1 activity. however, nsc632839 activity is not specific to uch-l1, and it is already known to inhibit usp2 and usp7 [178, 179] . the amount of p62 in cells is reduced after treatment with both ldn-57444 and ncs632839, suggesting that these drugs could prevent the accumulation of protein aggregates [178] . therefore, these inhibitors may constitute new tools to investigate further the implication of uch-l1 in parkinson's disease and evaluate whether uch-l1 inhibition favors the clearance of α-synuclein aggregates in neurons. inhibition of early regulators is another strategy to inhibit autophagy in some situations. the inhibitor wp1130, which targets usp9x, was reported to lead to an increase in ulk1 ubiquitination, inducing its transfer to the aggresomes and its inhibition, further resulting in the blockade of autophagy in several cultured cell lines, including the bone osteosarcoma u2os cell line [180] . it was speculated that the inhibition of usp9x could be responsible for the accumulation and subsequent aggregation of ubiquitinated ulk1. however, silencing usp9x did not result in changes in ulk1 expression level when cells were treated with wp1130. this could be because wp1130 is only partially specific and could have other targets in vivo that remain to be discovered [180, 181] . in order to screen and select new small-molecules interfering with autophagy in mammalian cells, an imaging-based assay has been optimized by liu and colleagues that makes use of cells expressing the autophagy marker gfp-lc3 to quantify the accumulation of autophagosomes [79] . using this assay, they screened the iccb known bioactives library, a collection of 472 compounds, and they identified the inhibitor spautin-1 (specific and potent autophagy inhibitor 1). spautin-1 inhibits the catalytic activity of both usp10 and usp13 with an ic 50 of~0.6-0.7 µm. these two dubs are involved in the regulation of beclin1 ubiquitination in the vps34 complex and, therefore, constitute an entry point to modulate the initiation of autophagy. cancer cell lines treated with spautin-1 demonstrated an increased cell death rate under starvation conditions. as such, spautin-1 constitutes a potential lead for the development of autophagy inhibitors for anti-cancer therapies [79] . because of its essential function in mitophagy, which is crucial to clear damaged mitochondria notably in neuronal cells, several small-molecule inhibitors of usp30 have been developed in the past few years. for example, based on a phenotypic screening, it was shown that the inhibition of usp30 by the compound 15-oxospiramilactone enhances the activity of usp30's targets mfn1 and mfn2-two gtpases anchored at the omm and essential for tethering adjacent mitochondria-and promotes mitochondrial fusion, thus contributing to the restoration of the mitochondria network [182] . more recently, an in vitro study identified a new small-molecule mf-094, as a potent and selective inhibitor of usp30. this compound has the opposite effect of 15-oxospiramilactone, as mf-094-mediated inhibition of usp30 accelerates mitophagy [183] . these two studies highlight the fact that the same dubs can be involved in different processes, dependent on the signal they may receive and the interaction within different protein complexes. there is no doubt that new inhibitors of dubs will arise with problems related to the existence of several substrates or to poor selectivity, requiring in-depth analysis of the selected compounds in different cell types and stress situations before any preclinical assays. interestingly, these investigations may tell a lot about how dubs regulate autophagy and other cell processes, and may be used as molecular tools to unveil regulatory mechanisms. protein ubiquitination is an essential, reversible, posttranslational modification involved in virtually every cellular process. the past decades have seen remarkable progress in the understanding of the function of dubs, their mechanism of action and regulation. recently, there has been an increasing body of evidence that ubiquitination plays a crucial role in regulating autophagy, and dubs intervene at multiple steps in autophagy. deregulation in both autophagy and ubiquitination/deubiquitination processes have been linked to many pathologies such as neurodegenerative diseases, cancer onset and progression, and different kinds of viral or bacterial infections. also, considerable effort was placed on the development and optimization of small-molecules acting as dubs inhibitors. such molecules can serve not only as leads for the development of drug-like molecules but also as tremendous useful tools to investigate the molecular mechanism of autophagy and its regulation by the ubiquitin system. by the development of molecules targeting protein-protein interaction instead of the catalytic activity, it could be possible to manipulate and orientate precisely the function of a dub towards a given process and/or target to avoid pleiotropic effects. 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metabolism and signalling crosstalk two distinct vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase y sorting in saccharomyces cerevisiae a ubiquitin-like system mediates protein lipidation atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion gabarap and gate16 localize to autophagosomal membrane depending on form-ii formation regulation of the tumor-suppressor beclin 1 by distinct ubiquitination cascades mtor inhibits autophagy by controlling ulk1 ubiquitylation, self-association and function through ambra1 and traf6 structure of ubiquitin refined at 1.8 å resolution the ubiquitin domain superfold: structure-based sequence alignments and characterization of binding epitopes the ubiquitin system getting into position: the catalytic mechanisms of protein ubiquitylation ubiquitin ligases: structure, function, and regulation hect and ring finger families of e3 ubiquitin ligases at a glance 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interacting with the proteasome a genomic and functional inventory of deubiquitinating enzymes emerging roles of e3 ubiquitin ligases in autophagy otub1 protein suppresses mtor complex 1 (mtorc1) activity by deubiquitinating the mtorc1 inhibitor deptor chaperone-like protein p32 regulates ulk1 stability and autophagy fine-tuning of ulk1 mrna and protein levels is required for autophagy oscillation the deubiquitinating enzyme usp20 stabilizes ulk1 and promotes autophagy initiation conformational flexibility of becn1: essential to its key role in autophagy and beyond enhancement of proteasome activity by a small-molecule inhibitor of usp14 deubiquitinating enzyme ubp6 functions noncatalytically to delay proteasomal degradation deubiquitination of dishevelled by usp14 is required for wnt signaling phosphorylation and activation of ubiquitin-specific protease-14 by akt regulates the ubiquitin-proteasome system usp14 regulates autophagy by suppressing k63 ubiquitination of beclin 1 traf6 and a20 regulate lysine 63-linked ubiquitination of beclin-1 to control tlr4-induced autophagy disruption of the beclin 1-bcl2 autophagy regulatory complex promotes longevity in mice ubiquitin-specific protease 14 negatively regulates toll-like receptor 4-mediated signaling and autophagy induction by inhibiting ubiquitination of tak1-binding protein 2 and beclin 1 beclin 1 restrains tumorigenesis through mcl-1 destabilization in an autophagy-independent reciprocal manner deubiquitinase usp9x stabilizes mcl1 and promotes tumour cell survival the deubiquitinase usp9x suppresses pancreatic ductal adenocarcinoma nedd4-dependent lysine-11-linked polyubiquitination of the tumour suppressor beclin 1 usp19 modulates autophagy and antiviral immune responses by deubiquitinating beclin-1 beclin1 controls the levels of p53 by regulating the deubiquitination activity of usp10 and usp13 the deubiquitylase usp33 discriminates between ralb functions in autophagy and innate immune response essential 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neurotoxicity and is a therapeutic target for parkinson's disease deubiquitinase usp8 regulates α-synuclein clearance and modifies its toxicity in lewy body disease lysine 63-linked polyubiquitin potentially partners with p62 to promote the clearance of protein inclusions by autophagy novel polyubiquitin imaging system, polyub-fc, reveals that k33-linked polyubiquitin is recruited by sqstm1/p62 an ankyrin-repeat ubiquitin-binding domain determines trabid's specificity for atypical ubiquitin chains ref(2)p, the drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain ref(2)p and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects the deubiquitinating enzyme usp36 controls selective autophagy activation by ubiquitinated proteins lc3c, bound selectively by a noncanonical lir motif in ndp52, is required for antibacterial autophagy the tbk1 adaptor and autophagy receptor ndp52 restricts the proliferation of ubiquitin-coated bacteria regulation of toll-like receptor signaling by ndp52-mediated selective autophagy is normally inactivated by a20 parkin is activated by pink1-dependent phosphorylation of ubiquitin at ser65 emerging themes in mitochondrial homeostasis the three 'p's of mitophagy: parkin, pink1, and post-translational modifications regulation of mitochondrial morphology by usp30, a deubiquitinating enzyme present in the mitochondrial outer membrane the mitochondrial deubiquitinase usp30 opposes parkin-mediated mitophagy usp30 deubiquitylates mitochondrial parkin substrates and restricts apoptotic cell death usp30 and parkin homeostatically regulate atypical ubiquitin chains on mitochondria deubiquitinating enzymes regulate park2-mediated mitophagy ubiquitin linkage-specific affimers reveal insights into k6-linked ubiquitin signaling organelle turnover: a usp30 safety catch restrains the trigger for mitophagy and pexophagy dual role of usp30 in controlling basal pexophagy and mitophagy peroxisomal dysfunction in age-related diseases newly born peroxisomes are a hybrid of mitochondrial and er-derived pre-peroxisomes the deubiquitinase usp15 antagonizes parkin-mediated mitochondrial ubiquitination and mitophagy usp8 regulates mitophagy by removing k6-linked ubiquitin conjugates from parkin chaperone-mediated autophagy targets hypoxia-inducible factor-1α (hif-1α) for lysosomal degradation stub1/chip is required for hif1a degradation by chaperone-mediated autophagy cezanne (otud7b) regulates hif-1α homeostasis in a proteasome-independent manner hif1α deubiquitination by usp8 is essential for ciliogenesis in normoxia regulation of connexins by the ubiquitin system: implications for intercellular communication and cancer epidermal growth factor regulates ubiquitination, internalization and proteasome-dependent degradation of connexin43 gap junction turnover is achieved by the internalization of small endocytic double-membrane vesicles internalized gap junctions are degraded by autophagy the ubiquitin-specific protease usp8 deubiquitinates and stabilizes cx43 regulation of epidermal growth factor receptor ubiquitination and trafficking by the usp8.stam complex essential role of ubiquitin-specific protease 8 for receptor tyrosine kinase stability and endocytic trafficking in vivo the mit domain of ubpy constitutes a chmp binding and endosomal localization signal required for efficient epidermal growth factor receptor degradation induction of autophagy promotes fusion of multivesicular bodies with autophagic vacuoles in k562 cells new insights into autophagosome-lysosome fusion amsh, an escrt-iii associated enzyme, deubiquitinates cargo on mvb/late endosomes targeting of amsh to endosomes is required for epidermal growth factor receptor degradation activation of the endosome-associated ubiquitin isopeptidase amsh by stam, a component of the multivesicular body-sorting machinery amsh interacts with escrt-0 to regulate the stability and trafficking of cxcr4 endocytosis: the dub version amsh is an endosome-associated ubiquitin isopeptidase loss of neurons in the hippocampus and cerebral cortex of amsh-deficient mice amsh is required to degrade ubiquitinated proteins in the central nervous system the deubiquitinating enzyme amsh1 and the escrt-iii subunit vsp2.1 are required for autophagic degradation in arabidopsis chmp1b is a target of usp8/ubpy regulated by ubiquitin during endocytosis regulation of endocytic sorting by escrt-dub-mediated deubiquitination ubpy controls the stability of the escrt-0 subunit hrs in development the ubiquitin isopeptidase ubpy regulates endosomal ubiquitin dynamics and is essential for receptor down-regulation the deubiquitinating enzyme ubpy is required for lysosomal biogenesis and productive autophagy in drosophila the ubiquitin-specific protease 12 (usp12) is a negative regulator of notch signaling acting on notch receptor trafficking toward degradation a functional endosomal pathway is necessary for lysosome biogenesis in drosophila trafficking of β-adrenergic receptors: implications in intracellular receptor signaling phosphorylation of the deubiquitinase usp20 by protein kinase a regulates post-endocytic trafficking of β 2 adrenergic receptors to autophagosomes during physiological stress the deubiquitinases usp33 and usp20 coordinate β 2 adrenergic receptor recycling and resensitization an autophagy assay reveals the escrt-iii component chmp2a as a regulator of phagophore closure histone h2b monoubiquitination is a critical epigenetic switch for the regulation of autophagy the salmonella deubiquitinase ssel inhibits selective autophagy of cytosolic aggregates ssel, a salmonella deubiquitinase required for macrophage killing and virulence the legionella effector ravz inhibits host autophagy through irreversible atg8 deconjugation elucidation of the anti-autophagy mechanism of the legionella effector ravz using semisynthetic lc3 proteins legionella ravz plays a role in preventing ubiquitin recruitment to bacteria-containing vacuoles the legionella anti-autophagy effector ravz targets the autophagosome via pi3p-and curvature-sensing motifs viruses and the autophagy pathway involvement of autophagy in coronavirus replication deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases coronavirus membrane-associated papain-like proteases induce autophagy through interacting with beclin1 to negatively regulate antiviral innate immunity beclin-1 targeting for viral immune escape coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling crystal structure of a ubp-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde structure and mechanisms of the proteasome-associated deubiquitinating enzyme usp14 amino-terminal dimerization, nrdp1-rhodanese interaction, and inhibited catalytic domain conformation of the ubiquitin-specific protease 8 (usp8) structural basis for conformational plasticity of the parkinson's disease-associated ubiquitin hydrolase uch-l1 structural basis and specificity of human otubain 1-mediated deubiquitination structure validation of the josephin domain of ataxin-3: conclusive evidence for an open conformation mechanism of usp7/hausp activation by its c-terminal ubiquitin-like domain and allosteric regulation by gmp-synthetase usp7-specific inhibitors target and modify the enzyme's active site via distinct chemical mechanisms active site-targeted covalent irreversible inhibitors of usp7 impair the functions of foxp3+ t-regulatory cells by promoting ubiquitination of tip60 characterization of the deubiquitinating activity of usp19 and its role in endoplasmic reticulum-associated degradation synaptic defects in ataxia mice result from a mutation in usp14, encoding a ubiquitin-specific protease an inhibitor of the proteasomal deubiquitinating enzyme usp14 induces tau elimination in cultured neurons expression of the protein gene product 9.5, pgp9.5, is correlated with t-status in non-small cell lung cancer discovery of inhibitors that elucidate the role of uch-l1 activity in the h1299 lung cancer cell line ubiquitin c-terminal hydrolase l1 regulates autophagy by inhibiting autophagosome formation through its deubiquitinating enzyme activity characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities deubiquitinase inhibition by wp1130 leads to ulk1 aggregation and blockade of autophagy deubiquitinase inhibition by small-molecule wp1130 triggers aggresome formation and tumor cell apoptosis a small natural molecule promotes mitochondrial fusion through inhibition of the deubiquitinase usp30 novel highly selective inhibitors of ubiquitin specific protease 30 (usp30) accelerate mitophagy key: cord-000575-g1ob16b9 authors: xie, xiao-li; zheng, li-fei; yu, ying; liang, li-ping; guo, man-cai; song, john; yuan, zhi-fa title: protein sequence analysis based on hydropathy profile of amino acids date: 2012-01-27 journal: journal of zhejiang university science b doi: 10.1631/jzus.b1100052 sha: doc_id: 575 cord_uid: g1ob16b9 biology sequence comparison is a fundamental task in computational biology. according to the hydropathy profile of amino acids, a protein sequence is taken as a string with three letters. three curves of the new protein sequence were defined to describe the protein sequence. a new method to analyze the similarity/dissimilarity of protein sequence was proposed based on the conditional probability of the protein sequence. finally, the protein sequences of nd6 (nadh dehydrogenase subunit 6) protein of eight species were taken as an example to illustrate the new approach. the results demonstrated that the method is convenient and efficient. the comparative biological sequence is one of the issues in bioinformatics when analyzing similarities of function and properties of different sequences. similarly, evolutionary homology is analyzed by comparing dna and protein sequences. in general, there are two types of methodologies to conduct the comparison. one is an alignment-based method, and the other is an alignment-free method. sequence alignment is based on computeroriented and computer-intensive comparisons of sequences, and then a distance function or a score function is obtained. using the distance function, one can compare biological sequences. however, multiple sequence alignment of several hundred sequences always produces a bottleneck, firstly due to long computational time, and secondly due to possible bias of multiple sequence alignments for multiple occurrences of highly similar sequences (pham and zuegg, 2004) . therefore, the emergence of a study on alignment-free sequence analysis is obvious. until now, alignment-free sequence analysis is still in its early development. for most alignment-free methods, a biological sequence should be transformed into an object for which a linear algebra and statistical theory already has useful analytical tools. since 1983, dna sequence has been represented in different dimension spaces (hamori and ruskin, 1983; hamori, 1985; nandy, 1994; 1996; nandy and basak, 2000; randić et al., 2001; randić, 2003; randić and balaban, 2003; zhang et al., 2003; liao and wang, 2004; liao et al., 2005; nandy et al., 2006; bai et al., 2007; feng and wang, 2008) . each nucleotide of a given dna sequence is a point in different dimension spaces, and these graphical representations can allow us to qualitatively analyze dna sequences, and provide a way of viewing, sorting and comparing various genomic sequences. based on the graphical representation, it is possible to numerically characterize dna sequence and further quantitatively measure similarity of different dna sequences. although protein sequence and dna sequence belong to symbolic sequences, compared with dna sequence, there are fewer methods for the graphical representation of protein sequence. this is mainly because extension of dna graphical representation to protein sequences would enormously increase the number of possible alternative assignments for the 20 amino acids. the amino acid sequence is the key to understanding protein structure and function in the cell, so analysis of amino acid sequence is an important part of post-genomic studies. recently, several schemes have been proposed in protein graphical representation (randić and krilov, 1997; vinga and almeida, 2003; bai and wang, 2005; li j. et al., 2006; li c. et al., 2008; munteanu et al., 2008; yau et al., 2008; yao et al., 2008; wen and zhang, 2009) . in order to plot amino acid sequence, 20 amino acids in protein sequences are divided into different types, including protein sequence regarded as a word with three, four, or five different letters. since ordering amino acids based on their physicochemical properties may offer better insights into comparative study of protein than representation of protein based on the random ordering of amino acid, randić (2007) and yao et al. (2008; outlined different 2d graphical representations of protein sequence based on different physicochemical properties. the graphical representation of protein sequence cannot only describe amino acid sequence, but also measure similarity/ dissimilarity of different protein sequences. however, the methods only consider the string's information of protein, and do not consider adjacent string's information of amino acid sequence. here, we choose conditional probability to measure adjacent string's information. in this paper, we converted a protein sequence into three-letter sequence based on hydropathy profile of amino acid and defined the three curves to represent different hydropathy features. we then selected conditional probability as a new invariant for the protein sequences. to illustrate the proposed method, we made a comparison of the sequences belonging to eight nd6 (nadh dehydrogenase subunit 6) proteins from http://www.ncbi.nlm.nih.gov/: human , rat (ap_004903), and mouse (np_904339). according to the hydropathy profile of amino acids, the amino acids can be classified into three groups (nei and kumar, 2002; liu and wang, 2006) : internal group (f, i, l, m, v), external group (d, e, h, k, n, q, r), and ambivalent group (s, t, y, c, w, g, p, a). the amino acid of internal group tends to occur in the inner side of the protein's spatial structure, while the amino acid of external group tends to appear at the surface. in order to characterize the hydropathicity of a protein primary structure, we defined a primary protein sequence as a symbolic sequence including three letters according to the following rule: where s(i) is the letter in the ith position in the protein primary sequence, and f(s(i)) is the substitution for s(i). since the hydropathy profile can detect more evolutionary relationships, in the next section, we analyzed the new protein sequence containing three letters through different mathematical methods. given a protein primary sequence with length n, we transformed it into a new sequence according to the above definition. for example, for the protein sequence, s=mmyalfllsvglvmgfvgfs, then f(s)=iiaaiiiiaiaiiiaiiaia. to obtain more information, we defined three curves of the sequence. firstly, we let ie ea ia 1 if ( ( )) i, 0 otherwise, where i ranges from 1 to n. then, let y n u and n are y axis and x axis, respectively, and then we can draw three different curves, which are named as ie, ia, and ea curves of the protein sequence. the three different curves can give us some information about the protein sequence. according to the ie curve, we can compare the numbers of the amino acids belonging to the internal group and the external group at different positions. the ia curve can then be used to compare the numbers of the amino acids belonging to the internal group and the ambivalent group at different positions. finally, the ea curve can compare the numbers of the amino acids of the external group and the ambivalent group at different positions. according to the above definitions of three different curves, we drew three curves of nd6 proteins for the eight species (fig. 1 ). fig. 1 shows that the amino acids of the internal group in nd6 protein sequences are more than the amino acids of the external group, and the amino acids of the ambivalent group are more than the amino acids of the external group. furthermore, it is evident that g. seal and h. seal have similar curves, rat and mouse's curves are almost identical, and the three curves of human, gorilla, and chimpanzee are similar, but wallaroo's curve is different from curves of other species. protein sequence is composed of three parts, internal group, external group and ambivalent group, so we regard the random numerical sequence to be composed of three parts (+1, 0, −1). we calculated the conditional probability, which was invariant to quantity protein sequences. for example, let x i ie represents the state of the ith (i=1, 2, ..., n) moment, state space s={+1, 0, −1}. there are nine conditional probabilities as follows: ( 1 1 ), according to the above definition, we can obtain these conditional probabilities of a given protein sequence. the conditional probability of each of nd6 proteins is listed in table 1 . given two protein sequences, we can obtain two nine-component vectors whose elements are conditional probabilities for each protein sequence. based on the vectors, we can compare different protein sequences. in general, similarities of the two vectors can be obtained by calculating euclidean distance. the smaller the euclidean distance of two vectors is, the more similar are the protein sequences. the euclidean distance of two vectors u and v is as follows: where u i and v i denote the components of vectors u and v, respectively. k is the dimension of vectors u and v. yao et al. (2009) proposed a new similarity measure of sequences, and coefficient of determination (r 2 ), which is defined as: r 2 can vary from 0 to 1, and represents the percent of the data, which is the closest to the line of best fit. the larger the coefficient of determination of two vectors is, the more similar are the protein sequences. in tables 2 and 3, we give the similarity/dissimilarity matrices for the eight nd6 sequences based on euclidean distance and coefficient of determination amongst nine-component vectors. as shown in tables 2 and 3, it is obvious that nd6 proteins of human, gorilla, and chimpanzee are more similar to each other. in addition, nd6 proteins are more similar for (g. seal, h. seal) and (mouse, rat). however, nd6 protein of wallaroo is very dissimilar to others amongst the eight species. the results are consistent with the known fact of evolution (yao et al., 2009) . biology sequence analysis is a fundamental task in computational biology, whose aim is to detect similarity/dissimilarity relationships between molecular sequences. some alignment-free methods to analyze similarities/dissimilarities of dna sequences have been proposed. however, there are few alignmentfree methods to analyze protein sequences. the amino acid sequence of a protein is the key to understanding its structure and function in the cell, so we present a new method to analyze protein primary sequence in this paper. the method is based on the graphical representation and conditional probability taken as the numerical characterization of the protein sequence. the demonstrable significance of the new method is that it cannot only analyze similarity/dissimilarity of protein sequences, but also provide more biological information about the protein sequences. according to the ie curve, we can compare the numbers of amino acids of the internal and external groups at different positions. also the ia curve can be used to compare the numbers of amino acids of the internal and ambivalent groups at different positions. the ea curve can be used to compare the numbers of amino acids in the external and ambivalent groups at different positions. therefore the three curves show the distribution of the three types of amino acids. furthermore, the conditional probability reflected the distribution of the two adjacent amino acids. the new approach was applied to nd6 protein sequences of several species and results have shown that the introduction of hydropathy profile of amino acids into protein sequence is effectual and feasible. a 2-d graphical representation of protein sequences based on nucleotide triplet codons a representation of dna primary sequences by random walk a 3d graphical representation of rna secondary structures based on chaos game representation novel dna sequence representation h curves, a novel method of representation of nucleotide series especially suited for long dna sequences 2-d graphical representation of protein sequences and its application to coronavirus phylogeny simplification of protein sequence and alignment-free sequence analysis analysis of similarity of dna sequences based on 3d graphical representation application of 2d graphical representation of dna sequence protein-based phylogenetic analysis by using hydropathy profile of amino acids enzymes/non-enzymes classification model complexity based on composition, sequence, 3d and topological indices a new graphical representation and analysis of dna sequence structure: i. methodology and application to globin genes two-dimensional graphical representation of dna sequences and intron-exon discrimination in intronrich sequences simple numerical descriptor for quantifying effect of toxic substances on dna sequences mathematical descriptors of dna sequences: development and applications molecular evolution and phylogenetics a probabilistic measure for alignment-free sequence comparison condensed representation of dna primary sequences 2-d graphical representation of proteins based on physico-chemical properties of amino acids characterization of 3-d sequences of proteins on a four-dimensional representation of dna primary sequences on the characterization of dna primary sequences by triplet of nucleic acid bases alignment-free sequence comparison-a review a 2d graphical representation of protein sequence and its numerical characterization analysis of similarity/dissimilarity of protein sequences similarity/dissimilarity studies of protein sequences based on a new 2d graphical representation a protein map and its application the z curve database: a graphic representation of genome sequences we would like to thank dr. jian-gang wang (college of animal science and technology, northwest a&f university, china), jian-zhong luo (department of foreign languages, northwest a&f university, china), feng an and dr. jun-li du (college of sciences, northwest a&f university, china) for their helpful suggestions. key: cord-261961-u4d0vvmq authors: st-germain, jonathan r.; astori, audrey; samavarchi-tehrani, payman; abdouni, hala; macwan, vinitha; kim, dae-kyum; knapp, jennifer j.; roth, frederick p.; gingras, anne-claude; raught, brian title: a sars-cov-2 bioid-based virus-host membrane protein interactome and virus peptide compendium: new proteomics resources for covid-19 research date: 2020-08-28 journal: biorxiv doi: 10.1101/2020.08.28.269175 sha: doc_id: 261961 cord_uid: u4d0vvmq key steps of viral replication take place at host cell membranes, but the detection of membrane-associated protein-protein interactions using standard affinity-based approaches (e.g. immunoprecipitation coupled with mass spectrometry, ip-ms) is challenging. to learn more about sars-cov-2 host protein interactions that take place at membranes, we utilized a complementary technique, proximity-dependent biotin labeling (bioid). this approach uncovered a virus-host topology network comprising 3566 proximity interactions amongst 1010 host proteins, highlighting extensive virus protein crosstalk with: (i) host protein folding and modification machinery; (ii) membrane-bound vesicles and organelles, and; (iii) lipid trafficking pathways and er-organelle membrane contact sites. the design and implementation of sensitive mass spectrometric approaches for the analysis of complex biological samples is also important for both clinical and basic research proteomics focused on the study of covid-19. to this end, we conducted a mass spectrometry-based characterization of the sars-cov-2 virion and infected cell lysates, identifying 189 unique high-confidence virus tryptic peptides derived from 17 different virus proteins, to create a high quality resource for use in targeted proteomics approaches. together, these datasets comprise a valuable resource for ms-based sars-cov-2 research, and identify novel virus-host protein interactions that could be targeted in covid-19 therapeutics. the sars-cov-2 ~30 kb positive strand rna genome (genbank mn908947.3 1, 2 ) contains two large open reading frames (orf1a and orf1ab) encoding polyproteins that are cleaved by viral proteases into ~16 non-structural proteins (nsps). 13 smaller 3' orfs encode the primary structural proteins of the virus, spike (s), nucleocapsid (n), membrane (m) and envelope (e), along with nine additional polypeptides of poorly understood function (fig 1a) . to enable proteomics-based approaches for the analysis of complex biological samples, we analyzed both sars-cov-2-infected cell lysates and mature virions, generating a high confidence virus peptide spectrum compendium. this dataset can be used e.g. for the selection of virus peptides for use in targeted proteomics approaches (e.g. the identification of viral peptides in human clinical samples), or for the generation of peptide spectral libraries for increased sensitivity of detection. two sars-cov-2 -host protein-protein interaction (ppi) mapping efforts have utilized immunoprecipitation coupled with mass spectrometry (ip-ms) of epitope-tagged viral proteins to identify >1000 putative virus-host ppis in hek293t 3 and a549 4 cells. while extremely powerful for identifying stable, soluble protein complexes, ip-ms approaches are not optimal for the capture of weak or transient ppis, or for the detection of ppis that take place at poorly soluble intracellular locations such as membranes, where key steps in viral replication occur. to better understand how host cell functions are hijacked and subverted by sars-cov-2 proteins, we used proximity-dependent biotinylation (bioid 5 ) as a complementary approach to map virus-host protein proximity interactions in live human cells. this dataset provides a valuable resource for better understanding sars-cov-2 pathogenesis, and identifies numerous previously undescribed virus-host ppis that could represent attractive targets for therapeutic intervention. a sars-cov-2 peptide compendium. to identify high quality tryptic virus peptides for use in targeted proteomics analyses, data-dependent acquisition (dda) mass spectrometry was conducted on the mature sars-cov-2 virion. the toronto sb3 virus strain 6 was cultured in veroe6 cells (moi 0.1), and culture media was collected 48 hrs post-infection. virus was concentrated by centrifugation, inactivated by detergent and subjected to tryptic proteolysis. the resulting viral tryptic peptides were identified using nanoflow liquid chromatography -tandem mass spectrometry (lc-ms/ms; fig 1a, together, these data confirm and expand upon previous proteomic analyses of sars-cov-2 virions, infected cells 4, 7-11 and patient samples [12] [13] [14] , and provide a library of high quality virus peptide spectra covering 17 virus proteins that can be used for the creation of peptide spectral libraries and targeted proteomics approaches. a sars-cov-2 -host protein proximity interactome. based on standard transcript mapping algorithms and conservation with orfs in other coronaviruses 1, 2 , we created a sars-cov-2 open reading frame (orf) vector set 15 (fig 1a) . nine sars-cov-2 proteins are predicted to have one or more transmembrane domains (s, e, m, nsp3, nsp4, nsp6, orf3a, orf7a and orf7b). to better characterize sars-cov-2 -host membrane-associated ppis, these virus orfs (along with the remaining poorly understood open reading frames orf3b, orf6, orf8 and orf9b) were fused in-frame with an n-terminal bira* (r118g) coding sequence, and the resulting fusion proteins individually expressed in hek 293 flp-in t-rex cells. using these cells, a virus-host ppi landscape was characterized using bioid (as in 16 ; supp table 2 ). applying a bayesian false discovery rate of ≤1%, 3566 high confidence proximity interactions were identified with 1010 unique human proteins (all raw data available at massive.ucsd.edu, accession #msv000086006). 412 prey polypeptides were detected as high confidence interactors for a single sars-cov-2 bait protein in this analysis, underscoring the high degree of specificity in this virus-host proximity interaction map. bait-bait correlation analysis (fig 1b) , based on similarity between interactomes (jaccard index analysis conducted in prohits-viz 17 ) revealed high levels of correspondence between the s (spike), e, m, nsp4, nsp6, orf7a, orf7b, and orf8 bait proteins. the nsp2, nsp3, orf3a, orf3b, orf6, and orf9b interactomes shared a lower degree of similarity with the other bait proteins in this set. bait proteins with one or more predicted transmembrane domains thus largely clustered together, with the exception of orf3a (which clusters outside the main group of putative membrane baits, even though it is predicted to possess three transmembrane helices), and orf8 (which clusters with the putative membrane baits, but has no predicted transmembrane domain itself). a self-organized force-directed bait-prey topology map was next generated, in which map location is determined by the number and abundance (i.e. total peptide counts) of host cell interactors (fig 1c) . this approach similarly clustered all of the baits with one or more predicted transmembrane helices, along with orf8, in a dense "core" region of the map, indicating that these bait proteins share a large proportion of common interactors. nsp3 and orf3a occupy regions at the edge of this dense region of the map, indicating a lower number of shared interactors with the other membrane proteins. nsp2, orf3b and orf9b occupy peripheral regions of the map, indicating that they share far fewer interactors with the rest of the baits analyzed here. interestingly, orf6 occupies a region of the map near orf3a (these two baits were also clustered near each other in the bait-bait analysis, above). consistent with this location, more than half of the orf3a interactome (39 of 73 proteins) is also present in the larger orf6 interactome (217 proteins). this overlapping group of interactors is enriched in plasma membrane (pm) and er proteins. based on this observation, it will be interesting to explore similarities in orf3a and orf6 function. as a whole, the virus-host interactome is significantly enriched in proteins associated with the endoplasmic reticulum (er)/nuclear, golgi and plasma membranes, and er-golgi trafficking vesicles ( even amongst those viral proteins that appear to localize exclusively to the er-golgi-pm endomembrane membrane system, specificity in virus-host interactomes was observed, likely reflecting preferences for interactions with different subsets of membrane proteins and/or localization to unique membrane lipid nanodomains. for example, both the orf7a and orf7b interactomes are enriched in pm solute channels, but orf7a appears to interact uniquely with the anion exchanger slc4a2, the taurine transporter slc6a6, and the glycine transporter slc6a9, while orf7b interacts specifically with the amino acid transporter slc1a5, the sulfate transporter slc26a11, and the divalent metal transporter slc39a14. autophagy is an important part of the innate immune response, effecting the elimination of intracellular pathogens such as viruses (virophagy), and delivering them to the lysosome, which processes pathogen components for antigen presentation 26, 27 . many viruses have thus evolved strategies to inhibit the host autophagic machinery. notably, however, (+)rna viruses appear to be dependent on autophagic function for efficient replication, and hijack components of the autophagic machinery for use in membrane re-organization and the creation of ros 28 . consistent with these observations, our data highlight multiple interactions amongst sars-cov-2 proteins and the er-phagy receptors fam134b, tex2644 and sec24c. a number of virus protein interactions were also detected with components of the ufmylation system (ddrgk1, cdk5rap3, ufl1 and ufsp2), which was recently shown to play a key role in er-phagy 10 , highlighting interesting links between specific autophagy pathways and sars-cov-2. interactome is significantly enriched in mitochondrial proteins (supp table 2, supp table 3 ). amongst the high confidence orf9b interactors is the mitochondrial antiviral signaling protein (mavs), which acts as a hub for cell-based innate immune signaling. the cellular pattern recognition receptors (prrs) detect pathogen-associated molecular patterns (pamps, e.g. viral rnas). pamp-bound prrs interact with mavs, which activates the nf-kb and type i interferon signaling pathways 29 . many different viruses block the host antiviral response by interfering with mavs signaling. this may be accomplished by e.g. direct mavs cleavage by viral proteases (a strategy used by hav, hcv and coxsackievirus b3), or via 26s proteasomemediated degradation, a strategy used by sars-cov orf9b 30 , which recruits the hect e3 ligase itch/aip4 to effect mavs ubiquitylation. we did not detect any ubiquitin e3 ligases in the orf9b bioid interactome, but consistent with a recent report indicating that sars-cov-2 orf9b binds directly to tomm70 31 to block mavs-mediated ifn signaling, we detected tomm70 as a major component of the orf9b interactome. the sars-cov-2 orf6 interactome is uniquely enriched in nuclear pore complex (npc) components (supp table 2, supp table 3 ). sars-cov orf6 was shown to inhibit npcmediated transport by tethering the importin proteins kpna2 and kpnb2/tnpo1 to er/golgi membranes 32 , which effectively blocks the import of immune signaling proteins such as stat1 into the nucleus. sars-cov-2 orf6 shares 69% identity at the amino acid level with its sars-cov counterpart, and similarly displays potent immune repressor function 33 . it will be interesting to detemine if the sars-cov2 orf6 interaction with npc components leads to a similar disruption of immune signaling and nuclear transport. orf3b interacts specifically with lamtor1 and lamtor2, components of the ragulator complex, which is localized to the lysosomal membrane and regulates the mechanistic target of rapamycin complex 1 (mtorc1). mtor signaling is inactivated by amino acid starvation and other types of stress, to inhibit cap-dependent translation and upregulate autophagy 34 . many viruses have thus evolved mechanisms to maintain mtorc1 activity during infection 35 . recent work has shown that the lamtor1/2 proteins may also play important roles in xenophagy 36 . based on these observations, sars-cov-2 orf3b could play an important role in regulating mtorc1 activity and/or in the disruption of antiviral immune function. in addition to er/golgi proteins, sars-cov-2 nsp3 interacts with the cytoplasmic rna binding proteins fxr1 and fxr2 37 . the fxr proteins were identified as host cell components of (+)rna equine encephalitis virus (eev) rna replication complexes (rc) [38] [39] [40] . fxrs are recruited to the viral rc by eev nsp3 proteins, and the fxrs are required for rc assembly. it will be interesting to determine whether the fxrs play similar roles in sars-cov-2 rna replication. we and others have previously reported that orthogonal ppi discovery approaches such as proximity-dependent biotinylation (bioid) can provide information that is highly complementary to ip-ms datasets 16, [41] [42] [43] [44] . to this end, we applied bioid to identify proximity partners for sars-cov-2 proteins in the proteomics "workhorse" 293 cell system. this mapping project significantly expands upon the sars-cov-2 virus-host interactome, providing a rich resource that can be mined by the scientific community for better understanding sars-cov-2 pathobiology, and identifying virus-host membrane protein interactions that could be targeted in covid-19 therapeutics. the design and implementation of sensitive mass spectrometric approaches for the analysis of complex biological samples will be important for clinical and basic research proteomics focused on covid-19. to this end, we also undertook an analysis of sars-cov-2 virions and infected vero cell lsyates using data-dependent acquisition tandem mass spectrometry, and identified 189 unique tryptic peptides, assigned to 17 different virus proteins. this work provides a significantly expanded sars-cov-2 tryptic peptide compendium for use in targeted proteomics approaches such as parallel or selected reaction monitoring (prm/srm), or for use in spectral library building. bioid, mass spectrometry and data analysis were conducted exactly as in 16 supp table 1 . virus peptide identification dataset, complete raw data. data for viral preparation and infected cells is presented in individual tabs. table 2 genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan a new coronavirus associated with human respiratory disease in china multi-level proteomics reveals host-perturbation strategies of a promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells sequence, infectivity, and replication kinetics of severe acute respiratory syndrome coronavirus 2. emerg infect dis data, reagents, assays and merits of proteomics for sars-cov-2 research and testing shortlisting sars-cov-2 peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data shotgun proteomics analysis of sars-cov-2-infected cells and how it can optimize whole viral particle antigen production for vaccines a genome-wide er-phagy screen highlights key roles of mitochondrial metabolism and er-resident ufmylation proteomics of sars-cov-2-infected host cells reveals therapy targets mass spectrometric identification of sars-cov-2 proteins from gargle solution samples of covid-19 patients mass-spectrometric detection of sars-cov-2 virus in scrapings of the epithelium of the nasopharynx of infected patients via nucleocapsid n protein proteotyping sars-cov-2 virus from nasopharyngeal swabs: a proof-of-concept focused on a 3 min mass spectrometry window global interactomics uncovers extensive organellar targeting by zika virus prohits-viz: a suite of web tools for visualizing interaction proteomics data an intramembrane chaperone complex facilitates membrane protein biogenesis here, there, and everywhere: the importance of er membrane contact sites +)rna viruses rewire cellular pathways to build replication organelles rhinovirus uses a phosphatidylinositol 4-phosphate/cholesterol countercurrent for the formation of replication compartments at the er-golgi interface fat(al) attraction: picornaviruses usurp lipid transfer at membrane contact sites to create replication organelles host lipids in positive-strand rna virus genome replication the oxysterol-binding protein cycle: burning off pi(4)p to transport cholesterol hepatitis c virus replication depends on endosomal cholesterol homeostasis digesting the crisis: autophagy and coronaviruses autophagy enhances the presentation of endogenous viral antigens on mhc class i molecules during hsv-1 infection manipulation of autophagy by (+) rna viruses regulation of mavs expression and signaling function in the antiviral innate immune response sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/traf3/traf6 signalosome sars-cov-2 orf9b suppresses type i interferon responses by targeting tom70 viral subversion of nucleocytoplasmic trafficking sars-cov-2 nsp13, nsp14, nsp15 and orf6 function as potent interferon antagonists nutrient regulation of mtorc1 at a glance adapting the stress response: viral subversion of the mtor signaling pathway lamtor2/lamtor1 complex is required for tax1bp1-mediated xenophagy concise review: fragile x proteins in stem cell maintenance and differentiation mutations in hypervariable domain of venezuelan equine encephalitis virus nsp3 protein differentially affect viral replication hypervariable domain of eastern equine encephalitis virus nsp3 redundantly utilizes multiple cellular proteins for replication complex assembly new world and old world alphaviruses have evolved to exploit different components of stress granules, fxr and g3bp proteins, for assembly of viral replication complexes bioid-based identification of skp cullin f-box (scf)beta-trcp1/2 e3 ligase substrates getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles parallel exploration of interaction space by bioid and affinity purification coupled to mass spectrometry proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes saint: probabilistic scoring of affinity purification-mass spectrometry data key: cord-000674-36jzzy77 authors: reggiori, fulvio; komatsu, masaaki; finley, kim; simonsen, anne title: autophagy: more than a nonselective pathway date: 2012-05-15 journal: int j cell biol doi: 10.1155/2012/219625 sha: doc_id: 674 cord_uid: 36jzzy77 autophagy is a catabolic pathway conserved among eukaryotes that allows cells to rapidly eliminate large unwanted structures such as aberrant protein aggregates, superfluous or damaged organelles, and invading pathogens. the hallmark of this transport pathway is the sequestration of the cargoes that have to be degraded in the lysosomes by double-membrane vesicles called autophagosomes. the key actors mediating the biogenesis of these carriers are the autophagy-related genes (atgs). for a long time, it was assumed that autophagy is a bulk process. recent studies, however, have highlighted the capacity of this pathway to exclusively eliminate specific structures and thus better fulfil the catabolic necessities of the cell. we are just starting to unveil the regulation and mechanism of these selective types of autophagy, but what it is already clearly emerging is that structures targeted to destruction are accurately enwrapped by autophagosomes through the action of specific receptors and adaptors. in this paper, we will briefly discuss the impact that the selective types of autophagy have had on our understanding of autophagy. three different pathways can deliver cytoplasmic components into the lumen of the lysosome for degradation. they are commonly referred to as autophagy (cell "self-eating") and include chaperone-mediated autophagy (cma), microautophagy, and macroautophagy. cma involves the direct translocation of specific proteins containing the kferq pentapeptide sequence across the lysosome membrane [1, 2] . microautophagy, on the other hand, entails the invagination and pinching off of the lysosomal limiting membrane, which allows the sequestration and elimination of cytoplasmic components. the molecular mechanism underlying this pathway remains largely unknown. the only cellular function that so far has been indisputably assigned to microautophagy is the turnover of peroxisomes under specific conditions in fungi [3] . recently, it has been reported the existence of a microautophagy-like process at the late endosomes, where proteins are selectively incorporated into the vesicles that bud inward at the limiting membrane of these organelles during the multivesicular bodies biogenesis [4] . in contrast to cma and microautophagy, macroautophagy (hereafter referred to as autophagy) entails the formation of a new organelle, the autophagosome, which allows the delivery of a large number of different cargo molecules into the lysosome. autophagy is a primordial and highly conserved intracellular process that occurs in most eukaryotic cells and participates in stress management. this pathway involves the de novo formation of vesicles called autophagosomes, which can engulf entire regions of the cytoplasm, individual organelles, protein aggregates, and invading pathogens ( figure 1 ). the autophagosomes fuse with endosomal compartments to form amphisomes prior to fusion with the lysosome, where their contents are degraded and the resulting metabolites are recycled back to the cytoplasm (figure 1 ). unique features of the pathway include the double-membrane structure of the autophagosomes, which were originally characterized over 50 years ago from detailed electron microscopy studies [5] . starting in the 1990s yeast mutational studies began the genetic and molecular characterization of the key components required to initiate and build an autophagosome in response to inactivation of mtorc1 (but also other cellular and environmental cues), the ulk1 complex is activated and translocates in proximity of the endoplasmic reticulum (er). thereafter, the ulk1 complex regulates the class iii pi3k complex. atg9l, a multimembrane spanning protein, is also involved in an early stage of autophagosome formation by probably supplying part of the membranes necessary for the formation and/or expansion. local formation of pi3p at sites called omegasomes promotes the formation of the phagophore, from which autophagosomes appear to be generated. the pi3p-binding wipi proteins (yeast atg18 homolog), as well as the atg12-atg5-atg16l1 complex and the lc3-phosphatidylethanolamine (pe) conjugate play important roles in the elongation and closure of the isolation membrane. finally, the complete autophagosome fuses with endosomes or endosome-derived vesicles forming the amphisome, which subsequently fuses with lysosomes to form autolysosomes. in the lysosomes, the cytoplasmic materials engulfed by the autophagosomes are degraded by resident hydrolases. the resulting amino acids and other basic cellular constituents are reused by the cell; when in high levels they also reactivate mtorc1 and then suppress autophagy. [6] . subsequently, genetic and transgenic studies in plants, worms, fruit flies, mice, and humans have underscored the pathway's conservation and have begun to unveil the intricate vital role that autophagy plays in the physiology of cells and multicellular organisms. for a long time, autophagy was considered a nonselective pathway induced as a survival mechanism in response to cellular stresses. over the past several years, however, it has become increasingly evident that autophagy also is a highly selective process involved in clearance of excess or dysfunctional organelles, protein aggregates and intracellular pathogens. in this introductory piece, we will briefly discuss the molecular mechanisms of selective types of autophagy and their emerging importance as a quality control to maintain cellular and organismal health, aspects that will be presented in deep in the reviews of this special issue of the international journal of cell biology and highlighted by the research papers. 2.1. the function of the atg proteins. autophagosomes are formed by expansion and sealing of a small cistern known as the phagophore or isolation membrane ( figure 1 ). once complete, they deliver their cargo into the hydrolytic lumen of lysosomes for degradation. a diverse set of components are involved in the biogenesis of autophagosomes, which primarily includes the proteins encoded by the autophagyrelated genes (atg). most atg genes have initially been identified and characterized in yeast. subsequent studies in higher eukaryotes have revealed that these key factors are highly conserved. to date, 36 atg proteins have been identified and 16 are part of the core atg machinery essential for all autophagy-related pathways [7] . upon autophagy induction, these proteins associate following a hierarchical order [8, 9] to first mediate the formation of the phagophore and then to expand it into an autophagosome [10, 11] . while their molecular functions and their precise contribution during the biogenesis of double-membrane vesicles remain largely unknown, they have been classified in 4 functional groups of genes: (1) the atg1/ulk complex, (2) the phosphatidylinositol 3-kinase (pi3k) complex, (3) the atg9 trafficking system, and (4) the two parallel ubiquitinlike conjugation systems ( figure 1 ). the atg1/ulk complex consists of atg1, atg13, and atg17 in yeast, and ulk1/2, atg13, fip200 and atg101 in mammals [12] [13] [14] [15] . this complex is central in mediating the induction of autophagosome biogenesis and as a result it is the terminal target of various signaling cascades regulating autophagy, such as the tor, insulin, pka, and ampk pathways [16] (figure 1 ). increased activity of the atg1/ulk kinase is the primary event that determines the acute induction and upregulation of autophagy. it is important to note that ulk1 is part of a protein family and two other members, ulk2 and ulk3, have been shown play a role in autophagy induction as well [14, 17] . the expansion of this gene family may reflect the complex regulation and requirements of the pathway in multicellular long-lived organisms. stimulation of the ulk kinases is achieved through an intricate network of phosphorylation and dephosphorylation modifications of the various subunits of the atg1/ulk complex. for example, atg13 is directly phosphorylated by tor and the phosphorylation state of atg13 modulates its binding to atg1 and atg17. inactivation of tor leads to a rapid dephosphorylation of atg13, which increases atg1-atg13-atg17 complex formation, stimulates the atg1 kinase activity and induces autophagy [18, 19] . the matg13 is also essential for autophagy, but seems to directly interact with ulk1, ulk2 and fip200 independently of its phosphorylation state [13, 14] . in addition, there are several phosphorylation events within this complex as well, including phosphorylation of matg13 by ulk1, ulk2, and tor; phosphorylation of fip200 by ulk1 and ulk2; phosphorylation of ulk1 and ulk2 by tor [13, 14] . additional studies are required to fully characterize the functional significance of these posttranslational modifications. autophagy is also regulated by the activity of pi3k complexes. yeast contains a single pi3k, vps34, which is present in two different tetrameric complexes that share 3 common subunits, vps34, vps15, and atg6 [20] . complex i is required for the induction of autophagy and through its fourth component, atg14, associates to the autophagosomal membranes where the lipid kinase activity of vps34 is essential for generating the phosphatidylinositol-3-phosphate (pi3p) that permits the recruitment of other atg proteins [9, 21] (figure 1 ). complex ii contains vps38 as the fourth subunit and it is involved in endosomal trafficking and vacuole biogenesis [20] . there are three types of pi3k in mammals: class i, ii, and iii. the functions of class ii pi3k remains largely unknown, but both classes i and iii pi3ks are involved in autophagy. while class i pi3k is principally implicated in the modulation of signalling cascades, class iii pi3k complexes regulate organelle biogenesis and, like yeast, contain three common components: hvps34, p150 (vps15 ortholog), and beclin 1 (atg6 ortholog). the counterparts of atg14 and vps38 are called atg14l/barkor and uvrag, respectively [22] [23] [24] . the atg14l-containing complex plays a central role in autophagy and functions very similarly as the yeast complex i by directing the class iii pi3k complex i to the phagophore to produce pi3p and initiate the recruitment of the atg machinery ( figure 1 ). atg14l is thought to be present on the er irrespective of autophagy induction [25] . upon starvation, atg14l localizes to autophagosomal membranes [8] . importantly, depletion of atg14l reduces pi3p production, impairs the formation of autophagosomal precursor structures, and inhibits autophagy [8, 24, 26, 27] . the uvrag-containing class iii pi3k complex also regulates autophagy but it appears to act at the intersection between autophagy and the endosomal transport pathways. uvrag initially associates with the bar-domain protein bif-1, which may regulate matg9 trafficking from the trans-golgi network (tgn) [28, 29] . uvrag then interacts with the class c vps/hops protein complex, promoting the fusion of autophagosomes with late endosomes and/or lysosomes [30] . finally, the uvrag-containing class iii protein complex binds to rubicon, a late endosomal and lysosomal protein that suppresses autophagosome maturation by reducing hvps34 activity [26, 31] . importantly, both the atg14l-and uvrag-containing complexes interact through beclin 1 with ambra1, which in turn tethers these protein complexes to the cytoskeleton via an interaction with dynein [32, 33] . following the induction of autophagy, ulk1 phosphorylates ambra1 thus releasing the class iii pi3k complexes from dynein and their subsequent relocalization triggers autophagosome formation. therefore, ambra1 constitutes a direct regulatory link between the atg1/ulk1 and the pi3k complexes [32] . together with the atg1/ulk and the pi3k complexes, atg9 is one of the first factors localizing to the preautophagosomal structure or phagophore assembly site (pas), the structure believed to be the precursor of the phagophore [9, 34] (figure 1 ). atg9 is the only conserved transmembrane protein that is essential for autophagy. it is distributed to the pas and multiple additional cytoplasmic tubulovesicular compartments derived from the golgi [35] [36] [37] . atg9 cycles between these two locations and consequently it is thought to serve as a membrane carrier providing the lipid building blocks for the expanding phagophore [37] . one of the established functions of atg9 is that it leads to the formation of the yeast pas when at least one of the cytoplasmic tubulovesicular compartments translocates near the vacuole [34] . atg9 is also essential to recruit the pi3k complex i to the pas [9] . retrieval transport of yeast atg9 from the pas and/or complete autophagosome is mediated by the 4 international journal of cell biology atg2-atg18 complex [38] and appears to be regulated by the atg1/ulk and pi3k complexes [37] . mammalian atg9 (matg9) has similar characteristics to its yeast counterpart. matg9 localizes to the tgn and late endosomes and redistributes to autophagosomal structures upon the induction of autophagy (figure 1 ) [39] , further promoting pathway activity [29, [40] [41] [42] . as in yeast, cycling of matg9 between locations also requires the atg1/ulk complex and kinase activity hvps34 [39, 43] . the core atg machinery also entails two ubiquitin-like proteins, atg12 and atg8/microtubule-associated protein 1 (map1)-light chain 3 (lc3), and their respective, partially overlapping, conjugation systems [44] [45] [46] (figure 1 ). atg12 is conjugated to atg5 through the activity of the atg7 (e1like) and the atg10 (e2-like) enzymes. the atg12-atg5 conjugate then interacts with atg16, which oligomerizes to form a large multimeric complex. atg8/lc3 is cleaved at its c terminus by the atg4 protease to generate the cytosolic lc3-i with a c-terminal glycine residue, which is then conjugated to phosphatidylethanolamine (pe) in a reaction that requires atg7 and the e2-like enzyme atg3. this lipidated form of lc3 (lc3-ii) is attached to both faces of the phagophore membrane. once the autophagosome is completed, atg4 removes lc3-ii from the outer autophagosome surface. these two ubiquitination-like systems appear to be closely interconnected. on one hand, the multimeric atg12-atg5-atg16 complex localizes to the phagophore and acts as an e3-like enzyme, determining the site of atg8/lc3 lipidation [47, 48] . on the other hand, the atg8/lc3 conjugation machinery seems to be essential for the optimal functioning of the atg12 conjugation system. in atg3-deficient mice, atg12-atg5 conjugation is markedly reduced, and normal dissociation of the atg12-atg5-atg16 complex from the phagophore is delayed [49] . some evidences suggest that these two conjugation systems also function together during the expansion and closure of the phagophore. for example, overexpression of an inactive mutant of atg4 inhibits the lipidation of lc3 and leads to the accumulation of a number of nearly complete autophagosomes [47] . while controversial [50] , it has been postulated that atg8/lc3 also possesses fusogenic properties, thus mediating the assembly of the autophagic membrane [51, 52] . it has to be noted that mammals possess at least 7 genes coding for lc3/atg8 proteins that can be grouped into three subfamilies: (1) the lc3 subfamily containing lc3a, lc3b, lc3b2 and lc3c; (2) the gammaaminobutyrate receptor-associated protein (gabarap) subfamily comprising gabarap and gabarapl1 (also called gec-1); (3) the golgi-associated atpase enhancer of 16 kda (gate-16) protein (also called gabarap-l2/gef2) [53] . although in vivo studies show that they are all conjugated to pe, they appear to have evolved complex nonredundant functions [54] . membranes. the origin of the membranes composing autophagosomes is a long-standing mystery in the field of autophagy. a major difficulty in addressing this question has been that phagophores as well as autophagosomes do not contain marker proteins of other subcellular compartments [55, 56] . a series of new studies has implicated several cellular organelles as the possible source for the autophagosomal lipid bilayers. the plasma membrane and elements of the trafficking machinery to the cell surface have been linked to the formation of an early autophagosomal intermediate, perhaps the phagophore [57] [58] [59] [60] [61] . it is possible that early endosomal-and/or golgiderived membranes are also key factors in the initial steps of autophagy [34, 36, 39] . the golgi, moreover, appears also important for autophagy by supplying at least in part the extra lipids required for the phagophore expansion [29, [62] [63] [64] [65] . the endoplasmic reticulum (er) is also central in this latter event. while the relevance of the er in autophagosome biogenesis was already pointed out a long time ago [5, 55, 66, 67] , recently two electron tomography studies have demonstrated the existence of a physical connection between the er and the forming autophagosomes [68, 69] . these analyses have revealed that the er is connected to the outer as well as the inner membrane of the phagophore through points of contact, supporting the notion that lipids could be supplied via direct transfer at the sites of membrane contact. in line with this view, it has been found that atg14l is associated to the er and pi3p is generated on specific subdomains of this organelle from where autophagosomes emerge under autophagy-inducing conditions [25, 70] (figure 1 ). it has also been proposed that the outer membrane of the mitochondria is the main source of the autophagosomal lipid bilayers, but while the experimental evidences appear to show that mitochondria are essential for the phagophore expansion, it remains unclear whether these organelles play a key role in the phagophore biogenesis [71] . the discrepancy between the conclusions of the various studies has not allowed yet drawing a model about the membrane dynamics during autophagosome biogenesis. the different results could be due to the different experimental conditions and model systems used by the various laboratories. alternatively, the lipids forming the autophagosomes could have different sources depending on the cell and the conditions inducing autophagy [72, 73] . a third possibility is that the source of phagophore membrane could depend on the nature of the double-membrane vesicle cargo. additional investigations are required to shed light on these issues. despite the potential of curing, quite a substantial range of specific pathological conditions by inducting autophagy, there are currently no small molecules that allow to exclusively stimulate this pathway [74] . nevertheless, there is a variety of chemicals that by acting on signaling cascades that also regulate autophagy permit to trigger this degradative process. these agents fall into two distinct categories based on the mechanism of action; whether they work through an mtordependent (rapamycin or torin) or mtor-independent pathway (e.g., lithium or resveratrol) [74] . in addition to these compounds, there are biological molecules such as interferon î³ (ifnî³) and vitamin d that can be used to stimulate autophagy especially in experimental setups [75, 76] . international journal of cell biology 5 inhibition of autophagy can also be beneficial in specific diseases but as for the inducers there are no compounds that exclusively block this pathway without affecting other cellular processes. the small molecules inhibiting autophagy include wortmannin and 3-methyladenine, which hamper the activity of the pi3k; bafilomycin a and chloroquine, which impair the degradative activity of lysosomes [77] . they are currently solely used in the basic research on autophagy. it is becoming increasingly evident that autophagy is a highly selective quality control mechanism whose basal levels are important to maintain cellular homeostasis (see below). a number of organelles have been found to be selectively turned over by autophagy and cargo-specific names have been given to distinguish the various selective pathways, including the er (reticulophagy or erphagy), peroxisomes (pexophagy), mitochondria (mitophagy), lipid droplets (lipophagy), secretory granules (zymophagy), and even parts of the nucleus (nucleophagy). moreover, pathogens (xenophagy), ribosomes (ribophagy), and aggregate-prone proteins (aggrephagy) are specifically targeted for degradation by autophagy [78] . selective types of autophagy perform a cellular quality control function and therefore they must be able to distinguish their substrates, such as protein aggregates or dysfunctional mitochondria, from their functional counterparts. the molecular mechanisms underlying cargo selection and regulation of selective types of autophagy are still largely unknown. this has been an area of intense research during the last years and our understanding of the various selective types of autophagy is starting to unravel. a recent genomewide small interfering rna screen aimed at identifying mammalian genes required for selective autophagy found 141 candidate genes to be required for viral autophagy and 96 of those genes were also required for parkin-mediated mitophagy [79] . in general, these pathways appear to rely upon specific cargo-recognizing autophagy receptors, which connect the cargo to the autophagic membranes. the autophagy receptors might also interact with specificity adaptors, which function as scaffolding proteins that bring the cargo-receptor complex in contact with the core atg machinery to allow the specific sequestration of the substrate. the selective types of autophagy appear to rely on the same molecular core machinery as non-selective (starvation-induced) bulk autophagy. in contrast, the autophagy receptors and specificity adapters do not seem to be required for nonselective autophagy. autophagy receptors are defined as proteins being able to interact directly with both the autophagosome cargo and the atg8/lc3 family members through a specific (wxxl) sequence [80] , commonly referred to as the lc3-interacting region (lir) motif [81] or the lc3 recognition sequences (lrs) [82] . based on comparison of lir domains from more than 20 autophagy receptors it was found that the lir consensus motif is an eight amino acids long sequence that can be written d/e-d/e-d/e-w/f/y-x-x-l/i/v. although not an absolute requirement, usually there is at least one acidic residue upstream of the w-site. the terminal l-site is occupied by a hydrophobic residue, either l, i, or v [83] . the lir motifs of several autophagy receptors have been found to interact both with lc3 and gabarap family members in vitro, but whether this reflects a physiological interaction remains to be clarified in most cases. it should be pointed out that not all lir-containing proteins are autophagy cargo receptors. some lir-containing proteins, like atg3 and atg4b, are recruited to autophagic membranes to perform their function in autophagosome formation [84, 85] , whereas others like fyve and coiled-coil domain-containing protein 1 (fyco1) interact with lc3 to facilitate autophagosome transport and maturation [86] . others might use an lir motif to become degraded, like dishevelled, an adaptor protein in the wnt signalling pathway [87] . the adaptor proteins are less well-described, but seem to interact with autophagy receptors and work as scaffold proteins recruiting and assembling the atg machinery required to generate autophagosomes around the cargo targeted to degradation. examples of autophagy adaptors are atg11 and alfy [88, 89] . the list of specific autophagy receptors is rapidly growing and the role of several of them in different types of selective autophagy will be described in detail in the reviews of this special issue. here we will briefly discuss the best studied form of selective autophagy, the yeast cytosol to vacuole targeting (cvt) pathway, as well as the best studied mammalian autophagy receptor, p62/sequestosome 1 (sqstm1) (figure 2 ). the cvt pathway is a biosynthetic process mediating the transport of the three vacuolar hydrolases, aminopeptidase 1 (ape1), aminopeptidase 4 (ape4) and î±-mannosidase (ams1), and the ty1 transposome into the vacuole [90, 91] . ape1 is synthesized as a cytosolic precursor (prape1), which multimerizes into the higher order ape1 oligomer, to which ape4, ams1, and ty1 associate to form the socalled cvt complex, prior to being sequestered into a small autophagosome-like cvt vesicle. sequestration of the cvt complex into cvt vesicles is a multistep process, which requires the autophagy receptor atg19, which facilitates binding to atg8 at the pas, as well as the adaptor protein atg11 (figure 2(a) ) [92] . atg11 acts as a scaffold protein by directing the cvt complex and atg9 reservoirs translocation to the pas in an actin-dependent way and then recruiting the atg1/ulk complex [40, 93] . the pi3p-binding proteins atg20, atg21, and atg24 are also required for the cvt pathway [94, 95] , but their precise function remains to be elucidated. interestingly, atg11 overexpression was found to recruit more atg8 and atg9 to the pas resulting in more cvt vesicles. this observation indicates that atg11 levels could regulate the rate of selective autophagy, and maybe also the size of the cargo-containing autophagosomes in yeast [90, 96] . indeed, a series of studies has revealed that atg11 is also involved in other types of selective autophagy such as mitophagy and pexophagy. however, the autophagy receptors involved in the different atg11-dependent types figure 2 : representative selective autophagy. (a) the cytoplasm-to-vacuole targeting (cvt) pathway. ape1 is synthesized as a cytoplasmic precursor protein with a propeptide and rapidly oligomerizes into dodecamers that subsequently associate with each other to form a higher order complex. the autophagy receptor atg19 directly binds to the complex and mediates the recruitment of another cvt pathway cargo, ams1, leading to the formation of the so-called cvt complex. atg19 also interacts with the autophagy adaptor atg11 and this protein allows the transport of the cvt complex to the site where the double-membrane vesicle will be generated. at this location, atg11 tethers the atg proteins essential for the cvt vesicle formation and the direct binding of atg19 to atg8 permits the exclusive sequestration of the cvt complex into the vesicle. (b) a model for p62 and nbr1 as autophagy receptors for ubiquitinated cargos. p62 and nbr1 bind with ubiquitinated cargos via their ubiquitin-associated (uba) domain and this interaction triggers the aggregate formation through the oligomerization of p62 via its phox and bem1p (pb1) domain. furthermore, p62 interacts with both autophagy-linked fyve protein (alfy), which serves to recruit atg5 and to bind pi3p, and directly with lc3. this latter event appears to organize and activate the atg machinery in close proximity of the ubiquitinated cargos, which allows to selectively sequester them in the autophagosomes in analogous to the cvt pathway. of selective autophagy are different as atg32 is required for mitophagy [97, 98] , whereas atg30 is essential for pexophagy [99] . like atg19, these two proteins have an atg8-binding lir motif and directly interact with atg11. mammalian cells appear to not possess an atg11 homologue, and further studies are necessary to delineate the molecular machinery involved in sequestration and targeting of different cargoes for degradation by autophagy in higher eukaryotes. the mechanism of the cvt pathway is reminiscent of the selective form of mammalian autophagy called aggrephagy, which involves degradation of misfolded and unwanted proteins by packing them into ubiquitinated aggregates. in both cases aggregation of the substrate (prape1 or misfolded proteins) is required prior to sequestration into cvt vesicles or autophagosomes, respectively [100] [101] [102] . similar to cvt vesicles, aggregate-containing autophagosomes appear to be largely devoid of cytosolic components suggesting that the vesicle membrane expands tightly around its cargo [88] . aggrephagy also depends on proteins with exclusive functions in substrate selection and targeting [81, 88, 100, 103] . the autophagy receptors p62 and neighbour of brca1 gene (nbr1) bind both ubiquitinated protein aggregates through an ubiquitin-associated (uba) domain and to lc3 via their lir motifs and, thereby, promote the specific autophagic degradation of ubiquitinated proteins (figure 2(b) ) [81, 82, 100, 103, 104] . nbr1 and p62 also contain an nterminal phox and bem1p (pb1) domain through which they can oligomerize, or interact with other pb1-containing binding partners [83] . in addition to being a cargo receptor for protein aggregates, p62 has been implicated in autophagic degradation of other ubiquitinated substrates such as intracellular bacteria [105] , viral capsid proteins [106] , the midbody remnant formed after cytokinesis [107] , peroxisomes [108, 109] , damaged mitochondria [110, 111] , and bacteriocidal precursor proteins [112] . the pb1 domain was recently found to be required for p62 to localize to the autophagosome formation site adjacent to the er [113] , suggesting that it could target ubiquitinated cargo to the site of autophagosome formation or alternatively promote the assembly of the atg machinery at this location. international journal of cell biology 7 the large scaffolding protein autophagy-linked fyve (alfy) appears to have a similar function as the specificity adaptor atg11. alfy is recruited to aggregate-prone proteins through its interaction with p62 [101] and through a direct interaction with atg5 and pi3p it serves to recruit the core atg machinery and allow formation of autophagic membranes around the protein aggregate [88] (figure 2(b) ). interestingly, alfy is recruited from the nucleus to cytoplasmic ubiquitin-positive structures upon cell stress suggesting that it might regulate the level of aggrephagy [114] . in line with this, it was found that overexpression of alfy in mouse and fly models of huntington's disease reduced the number of protein inclusions [88] . it will be interesting to determine whether alfy, as p62, is involved in other selective types of autophagy such as the one eliminating midbody ring structures or mitochondria. it is well known that posttranslational modifications like phosphorylation and ubiquitination are involved in the regulation of the activity of proteins involved in autophagy and degradation of autophagic cargo proteins, respectively. however, little is known about how these modifications may regulate selective autophagy. the fact that the core atg machinery is required for both nonselective and selective types of autophagy gives raise to the question of whether these two types of autophagy may compete for the same molecular machinery. such a competition could be detrimental for the cells undergoing starvation and to avoid this, there might be a tight regulation of the expression level and/or activity of the proteins specifically involved the selective autophagy. it has recently been proposed that phosphorylation of autophagy receptors might be a general mechanism for the regulation of selective autophagy. dikic and coworkers noted that several autophagy receptors contain conserved serine residues adjacent to their lir motifs and indeed, the tank binding kinase 1 (tbk1) was found to phosphorylate a serine residue close to the lir motif of the autophagy receptor optineurin. this modification enhances the lc3 binding affinity of optineurin and promotes selective autophagy of ubiquitinated cytosolic salmonella enterica [115] . in yeast, phosphorylation of atg32, the autophagy receptor for mitophagy, by mitogen-activated protein kinases was found to be required for mitophagy [116, 117] . the atg8/lc3 proteins themselves have also been found to become phosphorylated and recent works have identified specific phosphorylation sites for protein kinase a (pka) [118] and protein kinase c (pkc) [119] in the nterminal region of lc3. interestingly, the n-terminal of lc3 is involved in the binding of lc3 to lir-containing proteins [120] . it is therefore tempting to speculate that phosphorylation of the pka and pkc sites might facilitate or prevent the interaction of lc3 with lir-containing proteins such as p62. it has been found that phosphorylation of the pka site, which is conserved in all mammalian lc3 isoforms, but not in gabarap, inhibits recruitment of lc3 into autophagosomes [118] . the role of ubiquitin in autophagy has so far been ascribed as a signal for cargo degradation. ubiquitination of aggregate prone proteins, as well as bacteria and mitochondria, has been found to serve as a signal for recognition by autophagy receptors like p62 and nbr1, which are themselves also degraded together with the cargo that they associate with [83] . the in vivo specificity of p62 and nbr1 toward ubiquitin signals remains to be established under the different physiological conditions. interestingly, it was recently found that casein kinase 2-(ck2-) mediated phosphorylation of the p62 uba domain increases the binding affinity of this motif for polyubiquitin chains leading to more efficient targeting of polyubiquitinated proteins to autophagy [121] . ck2 overexpression or phosphatase inhibition reduced the formation of aggregates containing the polyglutamine-expanded huntingtin exon1 fragments in a p62-dependent manner. the e3 ligases involved in ubiquitination of different autophagic cargo largely remains to be identified. however, it is known that the e3 ligases parkin and rnf185 both regulate mitophagy [122, 123] . smurf1 (smad-specific e3 ubiquitin protein ligase 1) was recently also implicated in mitophagy, as well as in autophagic targeting of viral particles [79] . interestingly, the role of smurf1 in selective autophagy seems to be independent of its e3 ligase activity, but it rather depends on its membrane-targeting c2 domain, although the exact mechanism involved remains to be elucidated. it is also not clear whether ubiquitination could serve as a signal to regulate the activity or binding selectivity of proteins directly involved in autophagy, and whether this in some way could regulate selective autophagy. the role of ubiquitinlike proteins as sumo and nedd in autophagy is also unexplored. acetylation is another posttranslational modification that only recently has been implicated in selective autophagy. the histone de-acetylase 6 (hdac6), initially found to mediate transport of misfolded proteins to the aggresome [124] , was lately implicated in maturation of ubiquitinpositive autophagosomes [125] . the fact that hdac6 overproduction in fly eyes expressing expanded polyq proteins is neuroprotective further indicates that hdac6 activity stimulates aggrephagy [126] . furthermore, the acetylation of an aggrephagy cargo protein, muntant huntingtin, the protein causing huntington's disease, is important for its degradation by autophagy [127] . hdac6 has been also implicated in parkin-mediated clearance of damaged mitochondria [128] . the acetyl transferase(s) involved in these forms of selective autophagy is currently unknown, but understanding the role of acetylation in relation to various aspects of autophagy is an emerging field and it will very likely provide more mechanistic insights into these pathways. basal autophagy acts as the quality control pathway for cytoplasmic components and it is crucial to maintain the homeostasis of various postmitotic cells [129] . while this quality control could be partially achieved by nonselective autophagy, growing lines of evidence have demonstrated 8 international journal of cell biology that specific proteins, organelles, and invading bacteria are specifically degraded by autophagy (figure 3 ). mice deficient in autophagy die either in utero (e.g., beclin 1 and fip200 knockout mice) [130] [131] [132] or within 24 hours after birth due, at least in part, to a deficiency in the mobilization of amino acids from various tissues (e.g., atg3, atg5, atg7, atg9, and atg16l knockout mice) [49, [133] [134] [135] [136] . as a result, to investigate the physiological roles of autophagy, conditional knockout mice for atg5, atg7, or fip200 and various tissue-specific atg knockout mice have been established and analyzed [133, 137, 138] . for example, the liver-specific atg7-deficient mouse displayed severe hepatomegaly accompanied by hepatocyte hypertrophy, resulting in severe liver injuries [133] . mice lacking atg5, atg7, or fip200 in the central nervous system exhibited behavioral deficits, such as abnormal limb-clasping reflexes and reduction of coordinated movement as well as massive neuronal loss in the cerebral and cerebellar cortices [137] [138] [139] . loss of atg5 in cardiac muscle caused cardiac hypertrophy, left ventricular dilatation, and systolic dysfunction [140] . skeletal muscle-specific atg5 or atg7 knockout mice showed age-dependent muscle atrophy [141, 142] . pancreatic î² cell-specific atg7 knockout animals exhibited degeneration of islets and impaired glucose tolerance with reduced insulin secretion [143, 144] . podocytespecific deletion of atg5 caused glomerulosclerosis in aging mice and these animals displayed increased susceptibility to proteinuric diseases caused by puromycin aminonucleoside and adriamycin [145] . proximal tubule-specific atg5 knockout mice were susceptible to ischemia-reperfusion injury [146] . finally, deletion of atg7 in bronchial epithelial cells resulted in hyperresponsiveness to cholinergic stimuli [147] . all together, these results undoubtedly indicate that basal autophagy prevents numerous life-threatening diseases. how does impairment of autophagy lead to diseases? ultrastructural analyses of the mutant mice revealed a marked accumulation of swollen and deformed mitochondria in the mutant hepatocytes [133] , pancreatic î² cells [143, 144] , cardiac and skeletal myocytes [140, 141] and neurons [138] , but also the appearance of concentric membranous structures consisting of er or sarcoplasmic reticulum in hepatocytes [133] , neuronal axons [137, 139] and skeletal myocytes [141] , as well as an increased number of peroxisomes and lipid droplets in hepatocytes [133, 148] . in addition to the accumulation of aberrant organelles, histological analyses of tissues with defective autophagy showed the amassment of polyubiquitylated proteins in almost all tissues (although the level varied from one region to another) forming inclusion bodies whose size and number increased with aging [149] . consequently, basal autophagy also acts as the quality control machinery for cytoplasmic organelles (figure 3(a) ). although this could be partially achieved by bulk autophagy, these observations point to the existence of selective types of autophagy, a notion that is now supported by experimental data. p62/sqstm1 is the best-characterized disease-related autophagy receptor and a ubiquitously expressed cellular protein conserved among metazoan but not in plants and fungi [83] . besides a role of p62 as the receptor, this protein itself is specific substrate for autophagy. suppression of autophagy is usually accompanied by an accumulation of p62 mostly in large aggregates also positive for ubiquitin (figure 3(a) ) [104, 150] . ubiquitin and p62-positive inclusion bodies have been detected in numerous neurodegenerative diseases (i.e., alzheimer's disease, parkinson's disease, and amyotrophic lateral sclerosis), liver disorders (i.e., alcoholic hepatitis and steatohepatitis), and cancers (i.e., malignant glioma and hepatocellular carcinoma) [151] . very interestingly, the p62positive aggregates observed in hepatocytes and neurons of liver-and brain-specific atg7 deficient mice, respectively, as well as in human hepatocellular carcinoma cells, are completely dispersed by the additional loss of p62 strongly implicating involvement of p62 in the formation of diseaserelated inclusion bodies [104, 152] . through its self-oligomerization, p62 is involved in several signal transduction pathways. for example, this protein functions as a signaling hub that may determine whether cells survive by activating the traf6-nf-îºb pathway, or die by facilitating the aggregation of caspase 8 and the downstream effector caspases [153, 154] . on the other hand, p62 interacts with the nrf2-binding site on keap1, a component of the cullin 3-type ubiquitin ligase for nrf2, resulting in stabilization of nrf2 and transcriptional activation of nrf2 target genes including a battery of antioxidant proteins [155] [156] [157] [158] [159] . it is thus plausible that excess accumulation or mutation of p62 leads to hyperactivation of these signaling pathways, resulting in a disease onset (figure 3(b) ). paget's disease of bone is a chronic and metabolic bone disorder that is characterized by an increased bone turnover within discrete lesions throughout the skeleton. mutations in the p62 gene, in particular in its uba domain, can cause this illness [160] . a proposed model explaining how p62 mutations lead to the paget's disease of bone is the following: mutations of the uba domain cause an impairment in the interaction between p62 and ubiquitinated traf6 and/or cyld, an enzyme deubiquitinating traf6, which in turn enhances the activation of the nf-îºb signaling pathway and the resulting increased osteoclastogenesis (figure 3(b) ) [160] . if proven, this molecular scenario could open the possibility of using autophagy enhancers as a therapy to cure paget's disease of bone. it is established that autophagy has a tumor-suppressor role and several autophagy gene products including beclin1 and uvrag are known to function as tumor suppressor proteins [161] . the tumor-suppressor role of autophagy appears to be important particularly in the liver. spontaneous tumorigenesis is observed in the livers of mice with either a systemic mosaic deletion of atg5 or a hepatocytespecific atg7 deletion [152, 162] . importantly, no tumors are formed in other organs in atg5 mosaically deleted mice. enlarged mitochondria, whose functions are at least partially impaired, accumulate in atg5-or atg7-deficient hepatocytes [152, 162] . this observation is in line with the previous data obtained in ibmk cell lines showing that both the oxidative stress and genomic damage responses are activated by loss of autophagy [163, 164] . again, it is clear that accumulation of p62, at least partially, contributes to tumor growth because the size of the atg7 â��/â�� liver tumors is reduced by the additional deletion of p62 [162] , which may cause a dysregulation of nf-îºb signaling [165] and/or a persistent activation of nrf2 [166] . almost all tissues with defective autophagy are usually displaying an accumulation of polyubiquitinated proteins [149] . loss of autophagy is considered to lead to a delay in the global turnover of cytoplasmic components [137] and/or to an impaired degradation of substrates destined for the proteasome [167] . both observations could partially explain the accumulation of misfolded and/or unfolded proteins that is followed by the formation of inclusion bodies. as discussed above, p62 and nbr1 act as autophagy receptors for ubiquitinated cargos such as protein aggregates, mitochondria, midbody rings, bacteria, ribosomal proteins and virus capsids [83, 168] (figure 3 ). although these studies suggest the role of p62 as an ubiquitin receptor, it remains to be established whether soluble ubiquitinated proteins are also degraded one-by-one by p62 and possibly nbr1. a mass spectrometric analysis has clearly demonstrated the accumulation of all detectable topologies of ubiquitin chain in atg deficient livers and brains, indicating that specific polyubiquitin chain linkage is not the decisive signal for autophagic degradation [169] . because the increase in ubiquitin conjugates in the atg7 deficient liver and brain is completely suppressed by additional knockout of either p62 or nrf2 [169] , accumulation of ubiquitinated proteins in tissues defective in autophagy might be attributed to p62mediated activation of nrf2, resulting in global transcriptional changes to ubiquitin-associated genes. further studies are needed to precisely elucidate the degradation mechanism of soluble ubiquitinated proteins by autophagy. concomitant with the energy production through oxidative phosphorylation, mitochondria also generate reactive oxygen species (ros), which cause damage through the oxidation of proteins, lipids and dna often inducing cell death. therefore, the quality control of mitochondria is essential to maintain cellular homeostasis and this process appears to be achieved via autophagy. it has been postulated that mitophagy contributes to differentiation and development by participating to the intracellular remodelling that occurs for example during haematopoiesis and adipogenesis. in mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. nix/bnip3l, an autophagy receptor whose structure resembles that of atg32, is also an outer mitochondrial membrane protein that interacts with gabarap [170, 171] and plays an important role in mitophagy during erythroid differentiation [172, 173] (figure 3(c) ). although autophagosome formation probably still occurs in nix/bnip3l deficient reticulocytes, mitochondrial elimination is severely impaired. consequently, mutant reticulocytes are exposed to increased levels of ros and die, and nix/bnip3l knockout mice suffer severe anemia. depolarization of the mitochondrial membrane potential of mutant reticulocytes by treatment with an uncoupling agent results in restoration of mitophagy [172] , emphasizing the importance of nix/binp3l for the mitochondrial depolarization and implying that mitophagy targets uncoupled mitochondria. haematopoietic-specific atg7 knockout mice also exhibited severe anaemia as well as lymphopenia, and the mutant erythrocytes markedly accumulated degenerated mitochondria but not other organelles [174] . the mitochondrial content is regulated during the development of the t cells as well; that is, the high mitochondrial content in thymocytes is shifted to a low mitochondrial content in mature t cells. atg5 or atg7 deleted t cells fail to reduce their mitochondrial content resulting in increased ros production as well as an imbalance in pro-and antiapoptotic protein expression [175] [176] [177] . all together, these evidences demonstrate the essential role of mitophagy in haematopoiesis. recent studies have described the molecular mechanism by which damaged mitochondria are selectively targeted for autophagy, and have suggested that the defect is implicated in the familial parkinson's disease (pd) [178] (figure 3(c) ). pink1, a mitochondrial kinase, and parkin, an e3 ubiquitin ligase, have been genetically linked to both pd and a pathway that prevents progressive mitochondrial damage and dysfunction. when mitochondria are damaged and depolarized, pink1 becomes stabilized and recruits parkin to the damaged mitochondria [122, [179] [180] [181] . various mitochondrial outer membrane proteins are ubiquitinated by parkin and mitophagy is then induced. of note, pd-related mutations in pink1 and parkin impair mitophagy [122, [179] [180] [181] , suggesting that there is a link between defective mitophagy and pd. how these ubiquitinated mitochondria are recognized by the autophagosome remains unknown. although p62 has been implicated in the recognition of ubiquitinated mitochondria, elimination of the mitochondria occurs normally in p62-deficient cells [182, 183] . when specific bacteria invade host cells through endocytosis/phagocytosis, a selective type of autophagy termed xenophagy, engulfs them to restrict their growth [184] (figure 3(d) ). although neither the target proteins nor the e3 ligases have yet been identified, invading bacteria such as salmonella enterica, listeria monocytogenes, or shigella flexneri become positive for ubiquitin when they access the cytosol by rupturing the endosome/phagosome limiting membrane [185, 186] . these findings raise the possibility that ubiquitin also serves as a tag during xenophagy. in fact, to date, three proteins, p62 [105, 185, 187] , ndp52 [188] , and optineurin [115] have been proposed to be autophagy receptors linking ubiquitinated bacteria and lc3. an ubiquitin-independent mechanism has recently been revealed; recognition of a shigella mutant that lacks the icsb gene requires the tectonin domaincontaining protein 1 (tecpr1), which appears to be a new type of autophagy adaptor targeting shigella to atg5-and wipi-2-positive membranes [189] . interestingly, the shigella icsb normally prevents autophagic sequestration of this bacterium by inhibiting the interaction of shigella virg with atg5 indicating that some bacteria have developed mechanism to inhibit or subvert autophagy to their advantage [190] . this latter category of pathogens also includes viruses such as herpes simplex virus-1 (hsv-1), which express an inhibitor (icp34.5) of atg6/beclin1 [106] . however, it was recently shown that a mutant hsv-1 strain lacking icp34.5 becomes degraded by selective autophagy in a smurf1dependent manner [79] , suggesting that selective autophagy plays an important role in our immune system. recently, a different antimicrobial function has been assigned to autophagy and this function appears to be selective. during infection, ribosomal protein precursors are transported by autophagy in a p62-dependent manner into lysosomes [112] . these ribosomal protein precursors are subsequently processed by lysosomal protease into small antimicrobial peptides. importantly, it has been shown that induction of autophagy during a mycobacterium tuberculosis infection leads to the fusion between phagosomes containing this bacterium and autophagosomes, and the production of the antimicrobial peptides in this compartment kills m. tuberculosis [112] . while the molecular mechanism is largely unknown, autophagy contributes at least partially to the supply of free fatty acids in response to fasting (figure 3(e) ). fasting provokes the increase of the levels of free fatty acids circulating in the blood, which are mobilized from adipose tissues. these free fatty acids are rapidly captured by various organs including hepatocytes and then transformed into triglycerides by esterification within lipid droplets. these lipid droplets appear to be turned over by a selective type of autophagy that has been named lipophagy in order to provide endogenous free fatty acids for energy production through î²-oxidation [148] . indeed, liver-specific atg7 deficient mice display massive accumulation of triglycerides and cholesterol in the form of lipid droplets [191] . agoutirelated peptide-(agrp-) expressing neurons also respond to increased circulating levels of free fatty acids after fasting and then induce autophagy to degrade the lipid droplets [192] . similar to the case in hepatocytes, autophagy in the neurons supplies endogenous free fatty acids for energy production and seems to be necessary for gene expression of agpr, which is a neuropeptide that increases appetite and decreases metabolism and energy expenditure [192] . originally, it was assumed that autophagy was exclusively a bulk process. recent experimental evidences have demonstrated that through the use of autophagy receptors and adaptors, this pathway can be selective by exclusively degrading specific cellular constituents. the list of physiological and pathological situations where autophagy is selective is constantly growing and this fact challenges the earliest concept whether autophagy can be nonselective. it is believe that under starvation, cytoplasmic structures are randomly engulfed by autophagosomes and delivered into the lysosome to be degraded and thus generate an internal pool of nutrients. in yeast saccharomyces cerevisiae, however, the degradation of ribosomes, for example, ribophagy, as well as mitophagy and pexophagy, and the transport of the prape1 oligomer into the vacuole under the same conditions requires the presence of autophagy receptors [97, [193] [194] [195] . as a result, these observations suggest that autophagy could potentially always operate selectively. this is a conceivable hypothesis because this process allows the cell to survive stress conditions and the casual elimination of cytoplasmic structure in the same scenario could lead to the lethal depletion of an organelle crucial for cell survival. future studies will certainly provide more molecular insights into the regulation and mechanism of the selective types of autophagy, and this information will also be important to determine if indeed bulk autophagy exists. agrp: agouti-related peptide ampk: amp-activated protein kinase alfy: autophagy-linked fyve protein ams1: î±-mannosidase 1 ape1: aminopeptidase 1 ape4: aminopeptidase 4 atg: autophagy-related gene bnip3l: b-cell leukemia/lymphoma 2 (bcl-2)/adenovirus e1b interacting protein 3 ck2: casein kinase 2 cma: chaperone-mediated autophagy cvt: cytoplasm to vacuole targeting er: endoplasmic reticulum fip200: focal adhesion kinase family interacting protein of 200 kd fyco1: fyve and coiled-coil domain-containing protein 1 gabarap: gamma-aminobutyrate receptor-associated protein gate-16: golgi-associated atpase enhancer of 16 kda hdac6: histone de-acetylase 6 hops: homotypic fusion and protein sorting hsv-1: herpes simplex virus-1 keap1: kelch-like ech-associated protein 1 lc3: microtubule-associated protein 1 (map1)-light chain 3 lir: lc3-interacting region lrs: lc3 recognition sequences nbr1: neighbour of brca1 gene ndp52: nuclear dot protein (ndp) 52 nf-îºb: nuclear factor îºb nix: nip-like protein x nrf2: nf-e2 related factor 2 pas: phagophore assembly site pb1: phox and bem1p international journal of cell biology pe: phosphatidylethanolamine pd: parkinson's disease pi3k: phosphatidylinositol 3-kinase pi3p: phosphatidylinositol 3-phosphate pka: protein kinase a pkc: protein kinase c ros: reactive oxygen species rubicon: run domain and cysteine-rich domain containing beclin 1-interacting protein smurf1: smad-specific e3 ubiquitin protein ligase 1 sumo: small ubiquitin-like modifier sqstm1: p62/sequestosome 1 tbk1: tank binding kinase 1 tecpr1: tectonin domain-containing protein 1 traf6: tumour necrosis factor receptor-associated factor 6 tor: target of rapamycin tgn: trans-golgi network uba: ubiquitin associated ulk1: unc-51-like kinase 1 uvrag: uv-resistance associated gen vps: vacuolar protein sorting. chaperone-mediated autophagy in protein quality control chaperone-mediated autophagy: molecular mechanisms and physiological relevance microautophagy in mammalian cells: revisiting a 40-year-old conundrum microautophagy of cytosolic proteins by late endosomes seeing is believing: the impact of electron microscopy on autophagy research eaten alive: a history of macroautophagy dynamics and diversity in autophagy mechanisms: lessons from yeast characterization of autophagosome formation site by a hierarchical analysis of mammalian atg proteins hierarchy of atg proteins in pre-autophagosomal structure organization autophagosome formation: core machinery and adaptations toward unraveling membrane biogenesis in mammalian autophagy ulk1â·atg13â·fip200 complex mediates mtor signaling and is essential for autophagy nutrient-dependent mtorcl association with the ulk1-atg13-fip200 complex required for autophagy ulk-atg13-fip200 complexes mediate mtor signaling to the autophagy machinery a novel, human atg13 binding protein, atg101, interacts with ulk1 and is essential for macroautophagy regulation mechanisms and signaling pathways of autophagy autophagy mediates the mitotic senescence transition tor-mediated induction of autophagy via an apg1 protein kinase complex an overview of the molecular mechanism of autophagy two distinct vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase y sorting in saccharomyces cerevisiae assortment of phosphatidylinositol 3-kinase complexes-atg14p directs association of complex i to the pre-autophagosomal structure in saccharomyces cerevisiae beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian atg14 and uvrag autophagic and tumour suppressor activity of a novel beclin1-binding protein uvrag identification of barkor as a mammalian autophagy-specific factor for beclin 1 and class iii phosphatidylinositol 3-kinase autophagy requires endoplasmic reticulum targeting of the pi3-kinase complex via atg14l two beclin 1-binding proteins, atg14l and rubicon, reciprocally regulate autophagy at different stages amino terminus of the sars coronavirus protein 3a elicits strong, potentially protective humoral responses in infected patients bif-1 interacts with beclin 1 through uvrag and regulates autophagy and tumorigenesis bif-1 regulates atg9 trafficking by mediating the fission of golgi membranes during autophagy beclin1-binding uvrag targets the class c vps complex to coordinate autophagosome maturation and endocytic trafficking distinct regulation of autophagic activity by atg14l and rubicon associated with beclin 1-phosphatidylinositol-3-kinase complex the dynamic interaction of ambra1 with the dynein motor complex regulates mammalian autophagy ambra1 regulates autophagy and development of the nervous system an atg9-containing compartment that functions in the early steps of autophagosome biogenesis apg9p/cvt7p is an integral membrane protein required for transport vesicle formation in the cvt and autophagy pathways membrane delivery to the yeast autophagosome from the golgi-endosomal system the atg1-atg13 complex regulates atg9 and atg23 retrieval transport from the pre-autophagosomal structure the atg18-atg2 complex is recruited to autophagic membranes via phosphatidylinositol 3-phosphate and exerts an essential function starvation and ulk1-dependent cycling of mammalian atg9 between the tgn and endosomes recruitment of atg9 to the preautophagosomal structure by atg11 is essential for selective autophagy in budding yeast atg17 recruits atg9 to organize the pre-autophagosomal structure coordinated regulation of autophagy by p38alpha mapk through matg9 and p38ip evolution of atg1 function and regulation a ubiquitin-like system mediates protein lipidation a protein conjugation system essential for autophagy mammalian autophagy: core molecular machinery and signaling regulation the atg16l complex specifies the site of lc3 lipidation for membrane biogenesis in autophagy the atg12-atg5 conjugate has a novel e3-like activity for protein lipidation in autophagy the atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice snare proteins are required for macroautophagy atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion lc3 and gate-16 n termini mediate membrane fusion processes required for autophagosome biogenesis atg8: an autophagy-related ubiquitin-like protein family lc3 and gate-16/gabarap subfamilies are both essential yet act differently in autophagosome biogenesis studies on cellular autophagocytosis. the formation of autophagic vacuoles in the liver after glucagon administration purification and characterization of autophagosomes from rat hepatocytes ralb and the exocyst mediate the cellular starvation response by direct 14 international journal of cell biology activation of autophagosome assembly quantitative analysis of autophagy-related protein stoichiometry by fluorescence microscopy autophagosome precursor maturation requires homotypic fusion molecular mechanisms and regulation of specific and nonspecific autophagy pathways in yeast plasma membrane contributes to the formation of pre-autophagosomal structures exit from the golgi is required for the expansion of the autophagosomal phagophore in yeast saccharomyces cerevisiae the golgi complex as a source for yeast autophagosomal membranes characterization of the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by lectin cytochemistry the conserved oligomeric golgi complex is involved in double-membrane vesicle formation during autophagy studies on the mechanisms of autophagy: maturation of the autophagic vacuole studies on induced cellular autophagy. i. electron microscopy of cells with in vivo labelled lysosomes a subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation 3d tomography reveals connections between the phagophore and endoplasmic reticulum autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum mitochondria supply membranes for autophagosome biogenesis during starvation the emergence of autophagosomes the origin of the autophagosomal membrane chemical modulators of autophagy as biological probes and potential therapeutics autophagy and cytokines vitamin d, vitamin d receptor, and macroautophagy in inflammation and infection guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes how shall i eat thee image-based genome-wide sirna screen identifies selective autophagy factors structural basis of target recognition by atg8/lc3 during selective autophagy p62/sqstm1 binds directly to atg8/lc3 to facilitate degradation of ubiquitinated protein aggregates by autophagy structural basis for sorting mechanism of p62 in selective autophagy selective autophagy mediated by autophagic adapter proteins the structure of atg4b-lc3 complex reveals the mechanism of lc3 processing and delipidation during autophagy autophagy-related protein 8 (atg8) family interacting motif in atg3 mediates the atg3-atg8 interaction and is crucial for the cytoplasm-to-vacuole targeting pathway fyco1 is a rab7 effector that binds to lc3 and pi3p to mediate microtubule plus end-directed vesicle transport autophagy negatively regulates wnt signalling by promoting dishevelled degradation the selective acroautophagic degradation of aggregated proteins requires the pi3p-binding protein alfy mechanism of cargo selection in the cytoplasm to vacuole targeting pathway the cvt pathway as a model for selective autophagy selective autophagy regulates insertional mutagenesis by the ty1 retrotransposon in saccharomyces cerevisiae autophagy in organelle homeostasis: peroxisome turnover autophagy: molecular machinery for self-eating cooperative binding of the cytoplasm to vacuole targeting pathway proteins, cvt13 and cvt20, to phosphatidylinositol 3-phosphate at the pre-autophagosomal structure is required for selective autophagy atg21 is a phosphoinositide binding protein required for efficient lipidation and localization of atg8 during uptake of aminopeptidase i by selective autophagy peroxisome size provides insights into the function of autophagy-related proteins atg32 is a mitochondrial protein that confers selectivity during mitophagy mitochondria-anchored receptor atg32 mediates degrada-tion of mitochondria via selective autophagy ppatg30 tags peroxisomes for turnover by selective autophagy p62/sqstm1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death p62/sqstm1 and alfy interact to facilitate the formation of p62 bodies/alis and their degradation by autophagy the propeptide of aminopeptidase 1 mediates aggregation and vesicle formation in the cytoplasm-to-vacuole targeting pathway a role for nbr1 in autophagosomal degradation of ubiquitinated substrates homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice the adaptor protein p62/sqstm1 targets invading bacteria to the autophagy pathway autophagy protects against sindbis virus infection of the central nervous system midbody ring disposal by autophagy is a post-abscission event of cytokinesis ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes peroxisomal dynamics nix is critical to two distinct phases of mitophagy, reactive oxygen species-mediated autophagy induction and parkin-ubiquitin-p62-mediated mitochondrial priming pink1/parkinmediated mitophagy is dependent on vdac1 and p62/sqstm1 delivery of cytosolic components by autophagic adaptor protein p62 endows autophagosomes with unique antimicrobial properties p62 targeting to the autophagosome formation site requires self-oligomerization but not lc3 binding alfy, a novel fyve-domain-containing protein associated with protein granules and autophagic membranes phosphorylation of the autophagy receptor optineurin restricts salmonella growth phosphorylation of serine 114 on atg32 mediates mitophagy two mapk-signaling pathways are required for mitophagy in saccharomyces cerevisiae regulation of the autophagy protein lc3 by phosphorylation protein kinase c inhibits autophagy and phosphorylates lc3 the nterminus and phe52 residue of lc3 recruit p62/sqstm1 into autophagosomes serine 403 phosphorylation of p62/sqstm1 regulates selective autophagic clearance of ubiquitinated proteins parkin is recruited selectively to impaired mitochondria and promotes their autophagy rnf185, a novel mitochondrial ubiquitin e3 ligase, regulates autophagy through interaction with bnip1 the deacetylase hdac6 regulates aggresome formation and cell viability in response to misfolded protein stress hdac6 controls autophagosome maturation essential for ubiquitin-selective quality-control autophagy hdac6 rescues neurodegeneration and provides an essential link between autophagy and the ups acetylation targets mutant huntingtin to autophagosomes for degradation disease-causing mutations in parkin impair mitochondrial ubiquitination, aggregation, and hdac6-dependent mitophagy autophagy: renovation of cells and tissues role of fip200 in cardiac and liver development and its regulation of tnfî± and tsc-mtor signaling pathways promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor impairment of starvation-induced and constitutive autophagy in atg7-deficient mice the role of autophagy during the early neonatal starvation period atg9a controls dsdnadriven dynamic translocation of sting and the innate immune response loss of the autophagy protein atg16l1 enhances endotoxin-induced il-1î² production suppression of basal autophagy in neural cells causes neurodegenerative disease in mice neural-specific deletion of fip200 leads to cerebellar degeneration caused by increased neuronal death and axon degeneration loss of autophagy in the central nervous system causes neurodegeneration in mice the role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress autophagy is required to maintain muscle mass suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in pompe disease autophagy is important in islet homeostasis and compensatory increase of beta cell mass in response to high-fat diet loss of autophagy diminishes pancreatic î² cell mass and function with resultant hyperglycemia autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice autophagy protects the proximal tubule from degeneration and acute ischemic injury inducible disruption of autophagy in the lung causes airway hyper-responsiveness autophagy regulates lipid metabolism autophagy in mammalian development and differentiation ref(2)p, the drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain p62 is a common component of cytoplasmic inclusions in protein aggregation diseases persistent activation of nrf2 through p62 in hepatocellular carcinoma cells cullin3-based polyubiquitination and p62-dependent aggregation of caspase-8 mediate extrinsic apoptosis signaling p62 at the crossroads of autophagy, apoptosis, and cancer physical and functional interaction of sequestosome 1 with keap1 regulates the keap1-nrf2 cell defense pathway keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy p62/sqstm1 is a target gene for transcription factor nrf2 and creates a positive feedback loop by inducing antioxidant response elementdriven gene transcription the selective autophagy substrate p62 activates the stress responsive transcription factor nrf2 through inactivation of keap1 a noncanonical mechanism of nrf2 activation by autophagy deficiency: direct interaction between keap1 and p62 recent advances in understanding the molecular basis of paget disease of bone autophagy regulation in cancer development and therapy autophagydeficient mice develop multiple liver tumors autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis autophagy suppresses tumor progression by limiting chromosomal instability autophagy suppresses tumorigenesis through elimination of p62 oncogene-induced nrf2 transcription promotes ros detoxification and tumorigenesis autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates biogenesis and cargo selectivity of autophagosomes ubiquitin accumulation in autophagy-deficient mice is dependent on the nrf2-mediated stress response pathway: a potential role for protein aggregation in autophagic substrate selection nix is a selective autophagy receptor for mitochondrial clearance nix directly binds to gabarap: a possible crosstalk between apoptosis and autophagy essential role for nix in autophagic maturation of erythroid cells nix is required for programmed mitochondrial clearance during reticulocyte maturation the autophagy protein atg7 is essential for hematopoietic stem cell maintenance a critical role for the autophagy gene atg5 in t cell survival and proliferation autophagy is essential for mitochondrial clearance in mature t lymphocytes identification of atg5-dependent transcriptional changes and increases in mitochondrial mass in atg5-deficient t lymphocytes mechanisms of mitophagy pink1 stabilized by mitochondrial depolarization recruits parkin to damaged mitochondria and activates latent parkin for mitophagy pink1 is selectively stabilized on impaired mitochondria to activate parkin pink1-dependent recruitment of parkin to mitochondria in mitophagy p62/sqstm1 is required for parkin-induced mitochondrial clustering but not mitophagy; vdac1 is dispensable for both p62/sqstm1 cooperates with parkin for perinuclear clustering of depolarized mitochondria ubiquitination-mediated autophagy against invading bacteria shigella phagocytic vacuolar membrane remnants participate in the cellular response to pathogen invasion and are regulated by autophagy recognition of bacteria in the cytosol of mammalian cells by the ubiquitin system listeria monocytogenes acta-mediated escape from autophagic recognition the tbk1 adaptor and autophagy receptor ndp52 restricts the proliferation of ubiquitin-coated bacteria a tecpr1-dependent selective autophagy pathway targets bacterial pathogens escape of intracellular shigella from autophagy chronic oxidative stress sensitizes hepatocytes to death from 4-hydroxynonenal by jnk/c-jun overactivation autophagy in hypothalamic agrp neurons regulates food intake and energy balance cvt9/gsa9 functions in sequestering selective cytosolic cargo destined for the vacuole mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the ubp3p/bre5p ubiquitin protease cvt19 is a receptor for the cytoplasm-to-vacuole targeting pathway the authors thank shun kageyama (tokyo metropolitan institute of medical science) for helping in the creation of the figures used in the paper. f. reggiori key: cord-258784-9bdd9krr authors: wei, chiming; lyubchenko, yuri l.; ghandehari, hamid; hanes, justin; stebe, kathleen j.; mao, hai-quan; haynie, donald t.; tomalia, donald a.; foldvari, marianna; monteiro-riviere, nancy; simeonova, petia; nie, shuming; mori, hidezo; gilbert, susan p.; needham, david title: new technology and clinical applications of nanomedicine: highlights of the second annual meeting of the american academy of nanomedicine (part i) date: 2006-12-31 journal: nanomedicine: nanotechnology, biology and medicine doi: 10.1016/j.nano.2006.11.001 sha: doc_id: 258784 cord_uid: 9bdd9krr abstract the second annual meeting of the american academy of nanomedicine (aanm) was held at the national acadmy of science building in washinton, dc, september 9–10, 2006. the program included two nobel prize laureate lectures, two keynote lectures, and 123 invited outstanding state-in-art lectures presenting in 23 special concurrent symposia. in addition, there were 22 poster presentations in the meeting addressing different areas in nanomedicine research. all of the presenters at the meeting are outstanding investigators and researchers in the field. the second annual meeting of the aanm was a great success. the meeting provides investigators from different world areas a forum and an opportunity for discussion. we believe that nanomedicine research will develop rapidly in the future. the aanm invites basic and clinical researchers from the world to join this exciting research. the second annual meeting of the american academy of nanomedicine (aanm) was held at the national academy of science building in washington, dc, september 9-10, the opening ceremony was held in the morning of september 9, 2006 . aanm president chiming wei, md, phd, welcomed all the attendees and extended his deep appreciation and thanks to all presenters and attendees for contributing to the meeting ( figure 1 ). mr. joe mok, chair of the governorts committee of maryland, gave a wonderful message welcoming everybody who came to maryland to attend this very important meeting. tachung yih, phd, the vice president of aanm, gave a thoughtful address encouraging attendees to continue their efforts in advancing nanomedicine research. donald a. tomelia, phd, the vice chair of the 2006 aanm organizing and program committee, summarized the aanm program and provided attendees information and presentations in aanm symposia. james castracane, phd, member of the board of directors of aanm, summarized the aanm membership and fellowship development and future strategy. marianna foldvari, phd, the co-chair of the aanm international committee, summarized the aanm international collaboration research projects and international research activities and developments. during the presidential lecture, chiming wei, md, phd, of johns hopkins university, summarized new published and unpublished findings and results in each nanomedicine research area, and also reviewed the new nanotechnologies and clinical applications in nanomedicine development. during the past year many important research results were published in nanomedicine research areas. the significance of these investigations lies in the development of platform technologies including nanoscale molecular imaging, drug delivery, gene delivery, and diagnostic approaches. dendrimer-based nanomedicine was developed for protein mimicry research, nanopharmaceuticals, diagnostic imaging with contrast agents, and targeted drug delivery in cancer cells. biosensors with nanoscale materials provide unique and powerful technology for detection of biological and chemical species to aid in disease diagnosis and in the discovery and screening of new drug molecules. the development of a cancer therapy and monitoring with diagnostic nanotechnology include cancer-related genotyping detection, gene expression, and immunochemical analysis, as well as nearly real-time monitoring of patient blood for cancer cells. nanotechnologies were developed for clinical applications in cardiovascular, neurological, pulmonary, skin, and renal diseases. in the first nobel prize laureate lecture, baquaporin water channels: from atomic structure to clinical medi-cineq, peter agre, md (2003 nobel prize laureate for chemistry), from duke university school of medicine, summarized the aquaporin (aqp) water channel structure and applications to clinical medicine (figure 2, a) . aqp water channel proteins permit high water permeability of certain biological membranes. in people with no aqp1, lack of water causes defective urine concentration and reduced fluid exchange between capillary and interstitium in lung. mutations in the gene encoding aqp0, expressed in lens fiber cells, result in familial cataracts. mutations in the gene encoding aqp2, expressed in renal collecting duct principal cells, result in nephrogenic diabetes insipidus. mistargeting of aqp5, normally expressed in the apical membranes of salivery and lacrimal gland acini, can occur in sjogrents syndrome. aquaporins also are implicated in brain edema and muscular dystrophy (aqp4), anhidrosis (aqp5), renal tubular acidosis (aqp6), conversion of glycerol to glucose during starvation (aqp7 and aqp9), and cystic fibrosis (several). dr. agre received the honor fellowship from the aanm (figure 2 , b). in the second nobel prize laureate lecture, bnanotechnology, biology and businessq, ivar giaever, phd (1973 nobel prize laureate for physics), from applied biophysics, inc., indicated that nanotechnology holds much future promise to make things both cheaper and better ( figure 3 , a). in particular, dr. giaever discussed a general immunology detector that utilizes small indium particles to detect antibodies. he also described a whole-cell biosensor using electrical fields to obtain information about the morphology of cells in tissue culture. finally, dr. giaever discussed means to bring nanotechnology to the market with many important examples and experiments. dr. giaever received the honor fellowship from the aanm (figure 3, b) . in the keynote lecture, belectron cryomicroscopy of biological nanomachinesq, wah chiu, phd, from baylor medical college, demonstrated the applications of electron cryomicroscopy in determination of multiple molecular components and performance of specific biological functions (figure 4, a) . image reconstruction methods can be used to reconstruct three-dimensional (3-d structures from single nanomachine images at sub-nanometer resolution. mining of salient structural features within the molecular components of a large nanomachine is a daunting task. computational and visualization tools have been developed to extract features such as a-helices and b-sheets of protein subunits with a high degree of reliability. 3-d structure at sub-nanometer resolution can be combined with sequence-based structure prediction methods to derive pseudo atomic models of molecular components of a nanomachine. dr. chiu received the presidential award from aanm (figure 4 , b). there were 23 concurrent symposia in the second aanm annual meeting. each symposium focused on a different nanomedicine research area, covering topics from basic nanomedicine to clinical nanomedicine. following are the summaries for each symposium. because of the journalts page limitations we can only publish summaries for several symposia in this issue of nanomedicine: nanotechnology, biology, and medicine. we will publish the summaries for other symposia that remain will appear in the next issue. this symposium focused on the nanoscale analysis of the protein misfolding and aggregation phenomena critically associated with such neurodegenerative disorders as alzheimer, parkinson, and huntington diseases, to mention a few [1, 2] . the field of medicine can be dramatically advanced by establishing a fundamental understanding of key events leading to the misfolding and self-aggregation of proteins involved in the various protein-folding pathologies. there were four speakers at the symposium. dr. yuri lyubchenko outlined approaches for detection and analysis of protein misfolded states. protein misfolding is a complex phenomenon that can be facilitated, impeded, or prevented by interactions with various intracellular metabolites and interprotein interaction with intracellular nanomachines controlling protein folding. a fundamental understanding of molecular processes leading to misfolding and self-aggregation of proteins will provide critical information to help identify appropriate therapeutic routes to control these processes. protein misfolding is the very fist link in this long chain of events eventually leading to neurodegeneration. therefore, availability of methods capable of detecting the pathological protein conformations facilitates the development of novel tools for diagnostic and treating the diseases at very early stages of development. single-molecule force spectroscopy atomic force microscope was proposed to measure the strength of the interprotein interactions before aggregation [3, 4] . the capability of the atomic force microscope to manipulate with the tip in the nanometer scale was used for development of the nanotweezers approach capable of single-molecule selecting of antibodies for a certain type of protein aggregates. the role of oxidative stress in protein misfolding and aggregation of a-synuclein was a topic of dr. jean-christophe rochet. to investigate the link between complex i impairment and sequence-specific, post-translational modifications of a-synuclein, the protein was isolated from rotenone-treated pc12 cells and analyzed by tandem mass spectrometry [5] . it was found that rotenone induced various modifications in the c-terminal region of a-synuclein, including oxidation of methionine, nitration and amination of tyrosine, and phosphorylation of tyrosine and serine. these modifications correlated with an increase in the levels of membrane-bound, oligomeric a-synuclein detectable by fluorescence lifetime imaging microscopy and lipid flotation. in parallel, it was found that a-synuclein aggregation and toxicity were suppressed by dj-1, an antioxidant protein that is dysfunctional in some cases of familial parkinson disease. a current goal in nanomedicine is to determine how the conformational behavior of a-synuclein is influenced by sequence-specific modifications using single-molecule approaches. the presentation of dr. boris akhremitchev focused on analysis of thermodynamics of a-synuclein fragments critically involved in protein misfolding and aggregation. interactions between individual molecules were studied using a scanning force microscopy-based technique [6, 7] . the reported energy landscape parameters of interactions, including the barrier width and dissociation rate, were obtained, illustrating the possibility for direct measurements of the molecular-level parameters for investigating the molecular origin of the misfolding-based protein aggregation and effects of chemical agents that prevent or hinder the aggregation. dr. robert tycko reviewed the advances in highresolution studies of fibrillated aggregates achieved with the use of solid-state nuclear magnetic resonance. this technique is capable of providing site-specific constraints on the secondary, tertiary, and quaternary structures of amyloid fibrils, as demonstrated by recent works on fibrils formed by the full-length b-amyloid peptide associated with alzheimer disease [8] and fragments thereof [9] . the recent results focused in the following areas [10] : (1) development of a structural models for b-amyloid fibrils grown under either agitated or quiescent conditions; (2) development of a structural model for fibrils formed by the 37-residue amylin peptide, which is associated with type 2 diabetes; (3) constraints on the molecular structures of fibrils formed by the ure2p and sup35 yeast prion proteins. dr. justin hanes from the johns hopkins university started the first session by describing a new biomaterial platform for targeted and intracellular drug delivery, the poly(ether-anhydrides). these polymers incorporate poly(ethylene glycol) (peg) into their backbone structure and are polymerized with one to several other monomers, such as sebacic acid (sa), that are also known to be safe in humans. the addition of peg to the polymer backbone allows classical poly(anhydrides) to be formulated into nanoparticles that are readily resuspended in liquids for injection or inhalation, or into dry powder microparticles that are easily aerosolized for inhalation. peg coats the surface of the particles naturally during particle preparation, allowing the linkage of targeting agents to the free end of peg (i.e., end not attached to the rest of the polymer). highly selective targeting of poly(peg-sa) particles to inflamed blood vessels was demonstrated following the attachment of antibodies against molecules that are upregulated on the luminal side of inflamed endothelial cells in a collaboration with prof. doug goetz from the ohio university. poly(peg-sa) nanoparticles (with transferrin attached to the free end of peg) were also able to penetrate targeted cancer cells and rapidly transit within the cell to the perinuclear region; these particles were more toxic to cancer cells at relevant doses than the free drug. dr. richard price from the university of virginia next discussed the use of ultrasound-assisted local permeation of blood vessels by microbubble destruction as a method to enhance the targeting of drugs, including those contained within nano-and microparticles. microbubbles (hollow microspheres with a shell composed of albumin) are currently used clinically as a contrast agent in diagnostic ultrasound. dr. price showed that ultrasound can be used at low frequencies to cause bubbles in targeted regions to explode (by cavitation), which causes a significant increase in the local permeability of the endothelium to drugs and even large particles. highly targeted delivery of polymer nanoparticles to muscle tissue was clearly demonstrated in an animal model. control over particle penetration depth was achieved by simply applying ultrasound without microbubbles, in which case particles penetrated only the endothelium and not into the interstitium, whereas the application of ultrasound with microbubbles led to particle delivery to both the endothelium and the interstitium. the results suggest that this technique may provide an alternative to molecular targeting for a variety of diseases. dr. lawrence tamarkin from cytimmune sciences, inc., next spoke about his companyts colloidal gold-based system for targeted drug delivery to tumors (aurimune). tumor necrosis factor a (tnf-a) is bound to the surface of colloidal gold nanoparticles with an average diameter of 30 nm. the nanoparticles are pegylated to reduce clearance by the reticuloendothelial system; evidence that liver and spleen uptake of the nanoparticles was minimal as compared with uptake by tumors in mice was provided. aurimune was administered to two dogs with naturally occurring tumors and to rabbits; in each case its administration caused fever but did not cause hypotension. hypotension has historically been dose limiting in the treatment of cancer with tnf-a. based on promising preclinical results, aurimune production was scaled up and certified by gmp it is currently being studied in a national cancer institute-sponsored phase i trial in patients with advanced-stage cancer. dr. esther chang from georgetown university next provided provocative results with targeted cationic liposomes for both the treatment and improved imaging of various tumor types. noting that tumors need iron to grow and that they therefore typically express high levels of transferrin receptor on their surfaces, dr. changts group uses an anti-transferrin receptor single-chain antibody fragment as the targeting ligand for their liposomal delivery platform. dr. chang gave an overview of her laboratoryts extensive experience with these liposomal drug carriers in a variety of tumor types, including head and neck, breast, prostate, kidney, and pancreatic, demonstrating antitumor effectiveness of several drugs and drug types, including dna, small interfering rna, and small molecules. dr. chang reported that the technology is now entering clinical trials in the united states. finally, dr. chang also showed that contrast agents for magnetic resonance imaging could be encapsulated into the liposomes, leading to improved sensitivity and resolution in detecting metastatic lesions. dr. hamid ghandehari from the university of maryland, baltimore, capped the first drug delivery session with an overview of ways that nanoscale medicines might be improved by controlling particle structure using specialized chemistries and fabrication methods. he noted that carriers used for the controlled temporal or spatial delivery of bioactive agents are typically made of polymers, liposomes, or inorganic particles with polydisperse size distributions that can make prediction of biodistribution or subcellular fate challenging. he also pointed out that most of the polymers being studied for drug delivery are made by random copolymerization techniques, which limits control over monomer sequence, polymer molecular weight, and molecular weight distribution that may affect the performance of a given nanomedicine platform technology. dr. ghandehari then described several means by which polymers and nanosystems could be made with better definition of structure and dimensions, including his laboratoryts experience with n-(2-hydroxypropyl)methacrylamide copolymers, poly(amido amine) dendrimers, recombinant polymeric gene carriers, and silica nanotubes, and discussed the relationship of carrier structure at the nanoscale and function in targeted drug delivery. the second session of the drug delivery nanomedicine symposium began with a talk by dr. kathleen j. stebe from the johns hopkins university on the importance of controlling interfacial phenomena in the production of nanoparticles to enhance particle stability and functionality. dr. stebe discussed how the interfacial properties of nanoparticles could be controlled during fabrication to influence the properties and performance of nanomedicine products. the final surface properties of nanoparticles dictate everything, from whether they will aggregate in vivo to how effectively they will circulate and find their target. understanding the complex kinetics and thermodynamics of nanoparticle systems during their synthesis and formulation is a critical and yet underappreciated science that can improve the performance of nanomedicine platforms. dr. jimmy yun from singapore nanomaterials technology next discussed his companyts efforts in developing an effective process for the large-scale production of pharmaceutical nanoparticles with excellent control over size, size distribution, and morphology by a high-gravity precipitation method. these formulation methods may be valuable in enhancing the performance of drugs used in aerosol and solid dosage forms. dr. tsuneya ikezu from the university of nebraska medical center discussed his groupts findings that amyloidb aggregation and s-tubulin kinase-1 induces s phosphorylation and reduces microtubule extension. this study has important implications in understanding a potential molecular mechanism underlying impaired neuronal plasticity in the brains of alzheimer patients and may provide a potential therapeutic target for treating alzheimer disease. dr. hai-quan mao from the johns hopkins university capped the second drug delivery session by discussing a new polyphosphoester block copolymer system that self-assembles into micelles with an average diameter of 80 to 150 nm. improved gene expression in the liver using this new polymer nanoparticle platform technology was demonstrated. polyphosphoester micelles have potential in treating liver-associated diseases by gene medicine approaches. the cell is the basic unit of animate matter, but the cell consists of an astonishing collection of different individual inanimate nanostructures and ordered aggregates of nanostructures ( table 1) . three of the five talks in the symposium had a basic biological bent; the others were more technological in character. one of the technological talks was from a start-up company that has spun out of a university; the others were from academia. dr. anja nohe (chemical and biological engineering, university of maine) spoke on mouse genetic models of bone stem cells and nanoscale receptor dynamics. methods in imaging and microscopy-in particular fourier imaging correlation spectroscopy, and fluorescence resonance energy transfer-allow assessment of receptor protein dynamics within microdomains of the plasma membrane. dr ari requicha (laboratory for molecular robotics, university of southern california) described how binstrumentedq cellular systems could be developed with sensor/actuator networks and distributed robotics to open new directions in biomedical research. the eventual realization of multiple nanosensors providing continuous streams of real-time, multimodal, and complementary data on the behavior of groups of cells or single cells will increase human understanding of life processes at the tissue, cellular, and molecular levels. dr you han bae (pharmaceutics and pharmaceutical chemistry, university of utah) spoke on the use of phsensitive polymeric mixed micelles constructed from the block copolymers poly(l-histidine)-b-peg and poly(llactide)-b-peg for targeting tumors and overcoming multiple-drug resistance. the approach could result in a paradigm shift for treatment of solid tumors from monoclonal antibody/receptor-based targeting to ph shift-based targeting. dr don haynie (bionanosystems engineering laboratory, artificial cell technologies, inc.) explained how the same polypeptide multilayer nanofilm platform technology is being adapted to the development of different biomedical applications: artificial extracellular matrixes for tissue regeneration, artificial red blood cells for oxygen therapeutics, and artificial viruses for synthetic vaccine development. in each case peptide design and nanofilm architecture is tailored to the specific purpose, mimicking key properties of cells, viruses, and biomacromolecules at the microscale and the nanoscale. dr denis wirtz (chemical and biomolecular engineering, johns hopkins university) showed how to probe cytoskeletal dynamics in ovarian cancer progression with multiple particle tracking bnanorheologyq. cells corresponding to the two known pathways of ovarian cancer progression vary distinctly in their cytoplasmic viscosity. further progress in medicine will require a deeper understanding of how cell, tissue, and system behavior depends on the properties of subcellular nanostructures and nanostructured components of molecular assemblies. in the pharmacological nanomedicine symposium, four talks were presented with the latest advancements in drug discovery and delivery. in the first talk, dr. jeanne hardy (university of massachusetts-amherst) presented a site-directed approach called btetheringq in which allosteric sites on key proteins are exploited for drug discovery. these sites are on the surface of proteins and can be redesigned to be controlled by a small molecule of choice. recent advances with the caspase family of cysteine proteases were presented, which revealed the pathway of inhibition for caspase regulation of apoptosis. dr. wafik s. el-deiry (university of pennsylvania school of medicine) presented novel optical imaging approaches to monitor the molecular events relevant to tumor progression and therapeutic response. the approach is to introduce genetic alteration into human cells along with bioluminescent reporter genes to determine the role of specific oncogenes and tumor suppressor genes in controlling tumor progression and therapeutic response. these molecular beacons are then used in high-throughput screens for small molecules that can modulate signaling leading to tumor cell death. other approaches included evaluation of bioluminescent tumors in animals for drug-target validation and therapeutic efficacy. the overall goal presented by dr. el-deiry is to develop new approaches in drug screening and target validation to accelerate the preclinical phase of anticancer drug development to bring new agents into clinical trials. dr. scott l. diamond (university of pennsylvania), director of the penn center for molecular discovery, reported on their breakthrough in microarray design and screening where nanoliter-scale biochemical reactions can be analyzed by matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry with 10-fmol sensitivity. dr. diamond described a multiplex screen across numerous proteases that identified a nanomolar inhibitor of human cathepsin l that was a potent inhibitor of severe acute respiratory syndrome coronavirus entry. dr. david needham (duke university) described the temperature-sensitive liposome approach for drug delivery that his group introduced in 1996 and is now in phase 1 the aanm is rapidly expanding into an international organization with membership from all over the world. symposium xvi was an exciting representation of the international scope of nanomedicine and bionanotechnology research. in addition to the united states, eight other countriest leading researchers gave an overview of the current state of research in nanomedicine in their respective countries. the following countries were represented: canada nanomedicine presents an explosive number of opportunities to improve peoplets lives. a great diversity of research areas is being developed worldwide, with the united states being the leader. in canada some of the important nanomedicine research areas include intelligent drug delivery systems; vaccine and gene delivery nanosystems; polymeric systems and devices; biochips; lab-on-achip; artificial cells; anti-cancer, immune-, neural-and musculoskeletal systems; nanoscale targeting; tissue and cellular engineering; novel biomaterials; and molecular imaging. in hong kong dna electronics, microfluidic devices, metallic nanoparticles, nanofibers, and biopolymeric materials are being investigated for diagnostic and tissue engineering applications. in india the nanomedicine research includes drug delivery with dendrimer-based nanoarchitecture for the controlled and targeted delivery of anticancer bioactives; mitochondrial drug targeting; gene therapy of genetic disorders like diabetes and cancer using dendrimer-based nano carriers; developing a dna vaccine against leichmania donovani; and stem cell research. korea is active in the design of intelligent microsystems and biomedical devices, whereas in japan imaging, drug delivery, and the application of nanoscience in medical treatments are already strong. the second international conference of america-japan nanomedicine society will be held in japan during april 2007. the nanotechnology program in russia has recently been launched and includes nanomedicine research across the country. in singapore the biomedical sciences are a leading area of nanotechnology and supported by a strong economy. the panel discussion after the presentations concluded with the future vision of a true international effort in the development of nanomedicine-related technologies and discoveries, with regular updates to be presented at the annual aanm meetings. this symposium dealt with highlights relating to the toxicology of skin, eye, respiratory, and cardiovascular effects as well as the interpretation of polymer toxicology. dr. nancy monteiro-riviere discussed the effects of multiwalled carbon nanotubes, fullerenes, derivatized fullerenes, and quantum-dot effects in human epidermal keratinocytes and penetration through skin. some of these nanomaterials depicted inflammatory effects, altered gene expression proteins, ultrastructural localization within skin cells, and irritation profiles. quantum dots of different sizes and surface chemistries depicted unique profiles in penetration through the skin. dr. gerard lutty talked about the use of different types of injected nanoparticles to deliver genes in rabbit eyes. chitosan, pcep, and magnetic nanoparticles did not show toxicity in vitro but demonstrated toxicity in vivo. all of these nanoparticles were capable of transfecting cells with chitosan, causing inflammation in the eyes. magnetic nanoparticles were the least toxic and can be transfected to specific cell types in the eye. dr. randall schneider discussed the consideration that must be taken into account when using polymer nanoparticles for biomedical applications. he emphasized the complexicity of nanoparticles and that the predictive models currently used may not be suitable for nanoparticles. therefore, it is important to study the behavior of different types of nanoparticles in the biological environment and to fully characterize these materials before full interpretations can be made. dr. petia simeonova discussed her research with singlewalled carbon nanotubes instilled into the lung of mice. she demonstrated a dose-dependent increase in oxidative vascular damage. her studies also demonstrated that singlewalled carbon nanotubes did not modify the lipid profiles but did generate an accelerated plaque formation in a mouse model of arteriosclerosis. these findings not only demonstrated lung toxicity but also depicted cardiovascular effects. all of these studies presented in the toxicological nanomedicine symposium showed how nanomaterials may be toxic under specific conditions and that extensive studies on interactions of these materials in the body are essential before full interpretations can be made. nanotechnology product development cycles should incorporate an evaluation of potential risk reduction from the earliest stages. hidezo mori, md, phd summarized his groupts national project in japan named nanolevel imaging of molecular structure and function. in this project mori and his group analyzed structures of fundamental protein in human diseases. they selected complexes of cardiac troponin and a calcium sensitizer, vascular apoptosis-inducing protein 1, and calcineurin homologous protein 2 as targets ( figure 5 ). proteins expressed in escherichia coli or isolated from crude snake venom are purified and finally crystallized by hangingdrop method. all the x-ray diffraction data were collected at spring-8 for the structural determination. such sub-nanolevel structural imaging will be useful for structure-based drug design to treat cardiovascular disease or cancer. shuming nie, phd talked about semiconductor quantum dots. when linked with biotargeting or biorecognition ligands such as monoclonal antibodies, peptides, or small molecules, these nanoparticles can be used to target tumor antigens (biomarkers) as well as tumor vasculatures with high affinity and specificity. in the bmesoscopicq size range of 10 to 100 nm (diameter), quantum dots and polymeric nanoparticles also have more surface areas and functional groups that can be linked to multiple diagnostic (e.g., optical, radiosotopic, or magnetic)and therapeutic (e.g., anticancer) agents. kenneth wong, phd reported that topical delivery of silver nanoparticles reduces systemic inflammation of burns and promotes wound healing. his team investigated the wound healing properties of silver nanoparticles in an animal model and found rapid healing and better cosmetic appearance in a dose-dependent manner. furthermore, through quantitative polymerase chain reaction, immunohistochemistry, and proteomic studies the team showed that silver nanoparticles exerted positive effects through their antimicrobial properties, reduction in wound inflammation, and modulation of fibrogenetic cytokines. these results have given an insight into the actions of silver and provided a novel therapeutic direction for wound treatment in clinical practice. yoshinobu bana, phd described nanofabrication, molecular nanotechnology, and nanomaterials for bioanalysis and biomedical application. arrays of nanopillars, which are 200 nm wide and 4,000 nm tall, are successfully applicable to fast separation of dna within 10 to 25 seconds. nanoball, with a diameter of 50 nm, has been developed and successfully applied for fast separation of dna fragments from 100 base pairs to 15 kilobase pairs. bacteria cellulose, a biofiber with a diameter of 50 nm, has been found to be a useful in highly sensitive detection of biomolecules based on the optical confined effect. the nanobiodevice is useful for fast separation of protein samples from several kilodaltons to 200 kda within 15 seconds. joe kao, phd, reported the possibility of controlling biology with light. a bphototriggerq or bcaged moleculeq is a biologically inactive but photosensitive precursor that is rapidly transformed into a fully bioactive molecule upon exposure to a flash of light. the bioactive molecule that is thus generated acts as a biochemical trigger; it can be a hormone, a neurotransmitter, a second messenger, or an enzyme modulator. therefore, in combination with focused light pulses, caged molecules represent a simple methodology for manipulating biology in living cells or tissues with excellent temporal and spatial resolution. specific applications of the technology to manipulate neurotransmission at single synapses and to stimulate single nerve terminals were discussed. this year there were nine finalists for the young investigator award (yia). the nine finalists each gave a 10-minute platform presentation on topics representing the most novel and exciting areas of nanomedicine. awards were presented in two categories: those scientists who were more senior and the junior scientists who were graduate students, postdoctoral fellows, or new investigators. for the junior yia scientists, jean-christophe rochet of purdue university received first place for his work to develop nanoimaging-based approaches for detection and analysis of protein misfolding states. gengfeng zheng of harvard university received second place for his development of nanowire transistor arrays for large-scale, label-free, real-time, parallel electrical detection of a variety of biomolecules ranging from small molecules to proteins and viruses. third place was awarded to winston timp of the massachusetts institute of technology for his development of living-cell microarrays in which arrays of optical traps are able to manipulate hundreds of bacteria or tens of mammalian cells into 3-d arrays. these living-cell microarrays can be observed over time to assay gene expression and cell viability. in the senior yia group, the first place award went to justin hanes of johns hopkins university. his group is developing a new family of polymers, poly(ether-anhydride nanoparticles, designed to be selectively adhesive to desired cell types, and nonadhesive to obstacles in the body that might prevent their targeted delivery. second place went to xiaohua huang of the university of california-san diego for his efforts in developing a technology that is capability of rapidly sequencing a human genome for as little as $1,000. the third place recipient was susan l. beamis rempe of sandia national laboratories for her contributions in understanding ion transport between aqueous and biological environments for the purpose of designing new transporter devices relevant to medical and environmental applications. there was the poster session in the second annual meeting of aanm. the presentations in these posters covered basic, biosensors, cellular, dendrimer-based, diagnostics, engineering, experimental, genetics, neurology, oncology, policy, and toxicology nanomedicine. the presentations in each poster were excellent, and the discussions were a positive step in building future pathways in various research areas. the first place of best poster award went to rutledge g. ellis-behnke of the massachusetts institute of technology. the second place of best poster award went to lajos p. balogh of nanobiotechnology center, roswell park cancer institute. the third place of best poster award went to winston timp of the massachusetts institute of technology. during the welcome reception, researchers and investigators had the opportunity to discuss the dayts lectures and presentations ( figure 6 ). wenchi wei (chiming weits daughter) performed the chinese music instrument gu-zheng during the reception (figure 7) . this year the aanm volunteer staff team worked very hard and provided the wonderful service for the conference. we greatly appreciate the aanm staff teamts excellent work. dr. ling xu, the senior coordinator of aanm staff team, received the aanm service award during the reception (figure 8) because the aanm will guide academic activities, provide leadership, and lead research in the field, the aanm fellowship committee approved and elected the 48 new fellows to join the academy. we would like to invite more qualified scientists and investigators to apply for the aanm fellowship and join us in the development of nanomedicine research. in conclusion, the second annual meeting of the aanm was a great success ( figure 9 ). the meeting provides investigators from different world areas a forum and an opportunity for discussion. we believe that nanomedicine research will develop rapidly in the future. the aanm invites basic and clinical researchers from the world to join this exciting research. we sincerely appreciate everyone who contributed to this conference, and we are looking forward to seeing everyone next year at the third annual meeting of the aanm, september 8-9, 2007, in san diego, california. nanoimaging for protein misfolding and related diseases nanotools for megaproblems: probing protein misfolding diseases using nanomedicine modus operandi nanomedicine and protein misfolding diseases protein interactions and misfolding analyzed by afm force spectroscopy identification of rotenone-induced modifications in a-synuclein using affinity pull-down and tandem mass spectrometry packing density and structural heterogeneity of insulin amyloid fibrils measured by afm nanoindentation single-molecule force spectroscopy measurements of bhydrophobic bondq between tethered hexadecane molecules experimental constraints on quaternary structure in alzheimer's b-amyloid fibrils polymorphic fibril formation by residues 10-40 of the alzheimer's b-amyloid peptide molecular structure of amyloid fibrils: insights from solidstate nmr key: cord-260057-2m6jdvtc authors: pandey, preeti; prasad, kartikay; prakash, amresh; kumar, vijay title: insights into the biased activity of dextromethorphan and haloperidol towards sars-cov-2 nsp6: in silico binding mechanistic analysis date: 2020-09-23 journal: j mol med (berl) doi: 10.1007/s00109-020-01980-1 sha: doc_id: 260057 cord_uid: 2m6jdvtc abstract: the outbreak of novel coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) virus continually led to infect a large population worldwide. sars-cov-2 utilizes its nsp6 and orf9c proteins to interact with sigma receptors that are implicated in lipid remodeling and er stress response, to infect cells. the drugs targeting the sigma receptors, sigma-1 and sigma-2, have emerged as effective candidates to reduce viral infectivity, and some of them are in clinical trials against covid-19. the antipsychotic drug, haloperidol, exerts remarkable antiviral activity, but, at the same time, the sigma-1 benzomorphan agonist, dextromethorphan, showed pro-viral activity. to explore the potential mechanisms of biased binding and activity of the two drugs, haloperidol and dextromethorphan towards nsp6, we herein utilized molecular docking–based molecular dynamics simulation studies. our extensive analysis of the protein-drug interactions, structural and conformational dynamics, residual frustrations, and molecular switches of nsp6-drug complexes indicates that dextromethorphan binding leads to structural destabilization and increase in conformational dynamics and energetic frustrations. on the other hand, the strong binding of haloperidol leads to minimal structural and dynamical perturbations to nsp6. thus, the structural insights of stronger binding affinity and favorable molecular interactions of haloperidol towards viral nsp6 suggests that haloperidol can be potentially explored as a candidate drug against covid-19. key messages: •inhibitors of sigma receptors are considered as potent drugs against covid-19. •antipsychotic drug, haloperidol, binds strongly to nsp6 and induces the minimal changes in structure and dynamics of nsp6. •dextromethorphan, agonist of sigma receptors, binding leads to overall destabilization of nsp6. •these two drugs bind with nsp6 differently and also induce differences in the structural and conformational changes that explain their different mechanisms of action. •haloperidol can be explored as a candidate drug against covid-19. electronic supplementary material: the online version of this article (10.1007/s00109-020-01980-1) contains supplementary material, which is available to authorized users. the current outbreak of corona virus disease 2019 (covid19) caused by a novel coronavirus sars-cov-2 was first reported from wuhan, china, in late december 2019 [1] , which has subsequently affected the entire world, reporting nearly 26 million of confirmed cases of covid-19 along with 9.0-lakh deaths as per data recorded in september 1st week, 2020, posing a global threat for human health and economy. with so many novel studies and findings surfaced, since its inception, we are still lagging behind in development of an effective treatment strategy to control the virus spread and prevent the disease [2] [3] [4] [5] [6] [7] . sars-cov-2 is an enveloped non-segmented large positive sense, single-stranded rna virus (~30 kb) with 5′-cap structure and 3′-poly-a tail belonging to β-cov category [8, 9] . its rna genome contains 29,891 nucleotides and encoding for~9860 amino acids [9] . the genome codes for both structural proteins like spike (s), envelope (e), membrane (m), and nucleocapsid (n), along with many nonstructural proteins (nsps 1-16) [10] . while these nsps linked to rna replication and processing of subgenomic rnas, the functions of some of the nsps are not known. a key component, nsp6, is a membrane protein of approximately 34 kda with eight transmembrane helices and a highly conserved cterminus. together with nsp3 and nsp4, nsp6 is involved in the formation of replication-transcription complexes (rtcs) or replication organelles (ro) by stimulating the rearrangement of host cell membranes [11] . these replication complexes serve many important functions during the virus life cycle and play an important role in infection [12, 13] . the expression of these three nsps has been associated with the formation of different membranous structure characteristic of cov-infected cells including the double-membrane vesicles (dmvs), large virion-containing vacuoles (lvcvs), cubic membrane structures (cmss), and zippered endoplasmic reticulum (er) spherules [14] [15] [16] . nsp6 protein is also involved in blocking er-induced autophagosome/ autolysosome vesicle formation that plays a protective role in checking viral production inside host cells. nsp6 through the activation of the omegasome pathway induces autophagy [17] . the autophagosomes produced by nsp6 are higher in number but smaller in size as compared with those induced by starvation [18] . this may favor coronavirus infection by limiting the ability of autophagosomes to deliver viral components to lysosomal degradation. recently, gordon et al. [19] presented a blueprint of sars-cov-2-human interactome to reveal potential drug targets against covid-19. the authors identified 332 interactions between viral and host proteins, largely targeting the innate immune signaling pathway. using this blueprint, they identified a series of drugs and compounds with high potential to fight covid-19-some of which are now being entered into clinical trials. the authors found that sars-cov-2 nsp6 protein interacts with the sigma receptor, which regulates er stress response [19] and blocks er-induced autophagosome/autolysosome vesicle that restricts viral production. they found that drugs or molecules targeting sigma-1 and sigma-2 receptors had effectively inhibited virus replication and growth. these identified drugs or molecules include antipsychotics, haloperidol, and melperone, which are used to treat schizophrenia; antihistamines like clemastine and cloperastine; compound pb28; and the female hormone progesterone. recently, serkan tulgar and co-authors [20] advocated the use of haloperidol, an anti-inflammatory agent, in preventing the progression and reduction of severity of covid-19. gordon et al. [19] in the same study also identified the sigma-1 benzomorphan agonist, dextromethorphan, that actually has pro-viral activity, which helped the growth of the virus in cells. the authors suggested that dextromethorphan might activate sigma-1, which could help the activation of the s t r e s s r e s p o n s e t h a t t h e s a r s -c o v -2 h i j a c k s . dextromethorphan is a common cough suppressant found in several over-the-counter cough medicines. it binds to nmda receptors and can modulate glutamatergic signaling [21] . it also binds to serotonin transporters [22] and several other protein targets, including sigma-1 receptors [21, 22] which have been considered therapeutic targets for antidepressant drugs [23] [24] [25] [26] . however, the consumer healthcare products association (chpa) in their statement says that "there is currently no clinical data indicating that the cough suppressant dextromethorphan has a pro-viral effect in people with covid-19 infection. the study results published by gordon et al. [19] are experimental, preliminary, and not conclusive." more importantly, theresa enkirch et al. [27] had evaluated the anti-influenza activity of dextromethorphan in vitro, and in mice as well as in animal models. the authors demonstrated that dextromethorphan was able to inhibit viral replication both in vitro and in vivo. all these findings suggest that haloperidol and dextromethorphan are potential candidate binding to sars-cov-2 nsp6 and exert different viral activity. haloperidol inhibits the sars-cov-2 with ki of 2-12 nm, while dextromethorphan activates the virus with an activity constant, ki of 118 nm [19] . however, it is still not clear about the theoretical basis of this contradictory and biased activity. to explore the molecular mechanism of antiviral activity of haloperidol and the pro-viral activity of dextromethorphan, a number of computational methods like molecular docking, all-atom molecular dynamics (md) simulation, and the molecular mechanics/poisson-boltzmann surface area (mm-pbsa) were employed in this study. these mechanism-relevant studies will explore the binding modes, examine the key residues in the binding process, and elucidate the detailed interaction mechanisms. the results are expected to provide insights into the binding mechanism of the nsp6drug complexes, which will be useful for future exploration of efficient drug targets in covid-19. the three-dimensional structure of nsp6 was determined by the deepmind algorithm aplhafold, based on deep neural network learning [28] . the nsp6 model structure was energy-minimized to remove steric clashes, and the lowest energy structure was selected based on the molprobity scores. in addition, the zhang group also predicted models for the sars-cov-2 proteins [29] using the novel c-i-tasser platform [30] . however, these models have poor local geometries, numerous atomic clashes, poor side-chain conformations, and bad backbone dihedral angles, as measured by the molprobity score. the stereochemical quality of the modeled protein was evaluated using the structural analysis and verification server version 5.0 (saves) (https://servicesn.mbi.ucla. edu/saves/). this meta server runs six programs for checking the stereochemical quality of a protein structure by analyzing residue-by-residue geometry and overall structural geometry. the chemical structures of the drugs were downloaded from the zinc database [31] as mol2 files and then converted into three-dimensional pdb files using open babel 2.4.1 suite. the swiss-pdb tool was used to energy-minimize the protein structure to get the stable and low-energy conformation state of protein. all the docking studies have been performed with autodock vina [32] . the autodock tools package [33] was used to prepare the protein structures for docking by adding hydrogen to the polar groups along with kollman charges. both the protein and ligand files were converted into pdbqt format using the autodock vina plugin with scripts from the autodock tools package. the autodock vina generates up to 9 poses for each drug containing the molecular coordinates as well the gibbs free energy variation (δg, kcal/mol) for each pose. finally, the obtained top-posed docking conformations were subjected to post-docking energy minimization in discovery studio (ds 3.531). after docking, the resultant receptor-ligand complexes were visualized and studied by the discovery studio visualizer (biovia) and pymol [34] , ucsf chimera 1.9 [35] , and ligplot+ [36] . the all-atom md simulations were performed using gromacs v5.1.4 on the atomic coordinates of sars-cov-2 nsp6, and nsp6 complexed with dextromethorphan and haloperidol. the force field was selected as charmm27, and the water model tip3p was used to solvate the systems [37] , and the ligand parameters were taken as described elsewhere [38] . the system was placed in the center of the octahedral simulation box with buffer distance (10 å) and the water molecule padding around the system. to neutralize the system, 0.15-m counter ions (na + and cl − ) were added [39] . all the md simulations were performed at physiological temperature, 300°k. the energy minimization of all three systems was performed using steepest descent as first, then conjugant gradients (50,000 steps for each). bonds involving hydrogens were treated with shake algorithm, pme (particle mesh ewald) was used to define long-range electrostatic forces, and pbc (periodic boundary condition) was applied to x, y, and z directions [40] . the ensemble processes, nvt and npt, were applied for the equilibration of the system for the period of 500 ps. during the simulation, berendsen thermostat [41] and parrinello-rahman pressure [42] were used to maintain the pressure and temperature, respectively. linc algorithm was used to constrain the bonds and angles [43] . the van der waals interactions were taken cared of by lj potential with a cutoff of 0.10 nm. using the npt ensemble, production runs were performed for the period of 100 ns, with time integration. the energy, velocity, and trajectory were updated at the time interval of 10 ps. all production runs were done on cuda-enabled tesla gpu machine (dell t640 with v100 gpu) and os centos 7 [44, 45] . gromacs utilities and python scripts with mdtraj [46] were used to analyze the global structural order parameters, rmsd (root mean square deviation), rg (radius of gyration), sasa (solvent accessible surface area), rmsf (root mean square fluctuation), pca (principal component analysis), and free energy landscape (fel), and dssp was used to examine the secondary structure conformational dynamics during the simulation [47] . we calculated the molecular mechanics/poisson-boltzmann surface area (mm-pbsa) from the obtained md trajectories of protein-ligand complexes to describe the structural stability of protein-ligand, spatial orientation ligands, and molecular interactions at binding site of nsp6. the mm-pbsa provides a robust estimation of free-binding energy, contacts, and the effect of solvent underlying the binding affinity of ligand molecules [48, 49] . the binding free energy is expressed as: where g complex represented the total free energy of proteinligand complex, g protein as the free energy of protein, and g ligand used as the free energy of ligand. neglecting entropy terms, the free energy for each entity can be represented as: where δe mm is the average molecular mechanics interaction energy change (gas-phase) upon ligand binding. δg solv is the solvation free energy change on ligand binding. e mm included the bonded, electrostatic, and van der waals energy, and g solv included polar and non-polar solvation terms. we have applied the fereiro and woylness [50] algorithm for the calculation of residual frustration in the nsp6 structures. the residual frustration index was calculated from the frustratometer server (http://www.frustratometer.tk) [51] . the results were explained based on the "single residual frustration index." a frustration value (denoted as z-score) greater than + 0.78 will be defined as minimally frustrated or stabilizing residue, while a frustration value less than − 0.78 will be defined as highly frustrated or destabilizing [52] . if the z-score lies in between these two limits, the residue will be defined as neutral. the secondary structure of nsp6 was predicted using alphafold, a deep learning algorithm, and shown in fig. 1 . the constructed model of sars-cov-2 nsp6 has been verified through the saves v5.0 server. the ramachandran plot showed that 91.7% and 8.3% of residues are located in the most favored, and additionally allowed, regions, respectively. the environmental profile for nsp6 was computed with verify3d, which showed that almost 80% of the amino acids have scored more than zero. the non-bonded interactions between the atoms were also predicted with the errat server, which showed the overall quality factor of 92.19 (supplementary figure s1 ). the overall three-dimensional structure of nsp6 ( fig. 1) consists of fourteen α-helices, a c-terminal, two antiparallel β-strands, and sixteen turns. the structure of nsp6 has eight transmembrane helices (tm1-tm8). to predict the binding site for performing the molecular docking, a ligand-independent binding site search was done on nsp6 using castp server [53] . this program provides comprehensive and detailed quantitative characterization of geometric and topological features of protein structures. the server identifies a putative binding site with a solvent accessible surface area of 1076 å 2 and volume of 1442 å 3 . this binding site of nsp6 is encompassed by helices h3 (residues 61-69), turn and coil region (residues 126-133) joining h6 and h7, h8-h9 (residues 170-179), and c-terminal h11-h12-β1 (residues 221-244) (supplementary figure s2) . we have used the other two web-servers, prankweb [54] and coach [55] , for predicting the binding sites. these two servers also predict similar residues in the binding pocket, with certain changes. for the molecular docking study, we utilized the autodock vina, which is an open-source program and the most commonly used docking software with an effective scoring function [32] . haloperidol is an antipsychotic drug and is an inhibitor of sigma-1, dopamine d2, and histamine h1 receptors; it inhibits sars-cov-2 with a ki of 2-12 nm [19] . dextromethorphan, an antidepressant and sigma-1 agonist, activates the virus with an activity constant, ki of 118 nm [19] . the molecular docking results showed that both haloperidol and dextromethorphan form a good complex with nsp6 with docking (affinity) scores of − 7.7 kcal/mol and − 6.5 kcal/mol, respectively, which indicates k d (k d = exp δg/ rt ) values in the nanomolar to low micromolar range. the binding mode for dextromethorphan based on the docking studies is presented in fig. 2a . dextromethorphan forms h-bonds with its methoxy group to lys61 of nsp6. both cys229 and tyr242 form a liophilic binding pocket with the formation of π-sulfur and π-alkyl interactions with aromatic rings. the morphian group binds with several hydrophobic residues, including his62, arg233, arg236, and leu245, and forms additional van der waals interactions from residues asn232, thr238, asp243, and pro282. this suggests that dextromethorphan-binding pocket lies mainly in cterminal α-helix, h12, and β1 strand. the docking results show the different binding pose of haloperidol compared with dextromethorphan (fig. 2b) . haloperidol is predicted to bind in the hydrophobic cavity formed by h7 (tm5), h9 (tm7), and c-terminal positively charged residues. the carbonyl oxygen of the phenone group forms an h-bond with ser176, while the hydroxy group of piperidino forms h-bonds with ser265. due to the close proximity of leu231, leu237 and leu239 show cation-π interactions with the chlorophenyl group of the drug and ala136 form π-stacked interactions with the aromatic ketone of the phenone group. additional van der waals interactions from the residues arg137, trp140, asn174, tyr175 val178, thr238, and gln290 are observed. thus, a higher number of hydrogen bonds and hydrophobic interactions in haloperidol are observed in comparison to dextromethorphan, indicating the former as a good inhibitor molecule. evaluating the stability and conformational dynamics of nsp6-systems through md simulation to further characterize the binding-induced structural and conformational changes, all-atom md simulations for 100 ns were performed to obtain the conformational sampling of the two systems. the selected docking conformations of nsp6 in complex with haloperidol and dextromethorphan were sampled by 100-ns md simulation, and the dynamic stability of the complex was elucidated by calculating the cα-rmsd values of the protein as the function of simulation time ( figure s3a ). the rmsd trajectory indicated that all the systems are stable and well equilibrated after~50 ns and well converged for further analysis. the rmsd plots showed that the nsp6 and nsp6haloperidol remained mostly stable with a rmsd of~0.5 nm, while nsp6-dextromethorphan showed a sharp increase in a rmsd at~45 ns reaches up to 1.25 nm and then remained stable throughout the simulation. the result thus indicated that the nsp6-haloperidol complex showed almost similar rmsd as unliganded nsp6 and formed much more stable complex as compared with nsp6-dextromethorphan complex. the analysis of the one-dimensional probability distribution function of the nsp6 without ligand showed a narrower distribution with a higher probability of 0.10 around the rmsd ≤ 5.0a and thus had a stable helical conformation (fig. 3a) . indeed, in the presence of dextromethorphan, the complex showed a broader rmsd distribution with the appearance of new populations with the maximum probability of 0.12 at a rmsd of 1.25 nm, indicating a change in conformation leading to destabilization of α-helix of h12 (fig. 3a) . the cα-rmsd beyond 0.12 nm indicates a transition from an α-helical conformation towards the extended configuration (fig. 3a) . however, the drug haloperidol showed a lower probability (p = 0.10) with a slightly broader distribution and a rightward shift of the rmsd population compared with the nsp6. at this rmsd, intrahelical h-bonds are still present to stabilize a helical conformation. thus, dextromethorphan has highly unstable binding to nsp6 which is evident with the rmsd plot, while haloperidol binds strongly. rg is an effective parameter to evaluate the structural integrity and compactness of the studied systems. the rg is defined as the mass-weighted root mean square distance of a collection of atoms from their common center of mass. the time evolution plot of rg showed that all systems were compact, with the nsp6-dextromethorphan having the lowest rg ( figure s3b ). the rg plot clearly suggests that the nsp6dextromethorphan complex having the least rg value forms a well compact complex than the nsp6-haloperidol complex. it also indicates that after binding of the drug, the nsp6 protein attains a compact conformation and the drug dextromethorphan could stay stably at the binding site, which provided a guarantee for stronger interaction between dextromethorphan and nsp6. the nsp6-haloperidol complex showed similar rg to nsp6. the rg differences between the nsp6 systems were minimal in haloperidol, indicating that haloperidol binding marginally changes the spatial packing of the residues. in case of the nsp6-dextromethorphan system, rg distribution shows the mixture of two normal distributions that correctly describes the sampled configurations (rg1 = 2.12 nm, p = 0.05 nm; rg2 = 2.25 nm, p = 0.04 nm) (fig. 3b) . interestingly, the first peak is slightly in lesser side than the nsp6 and nsp6-haloperidol systems, suggesting a more compact globular state. the second small peak falls near the nsp6-haloperidol system. a visual inspection revealed the fig. 3 probability distributions of structural parameters of nsp6 systems. a cα-rmsd. b radius of gyration (rg). c sasa. d rmsf for nsp6 (green), nsp6-haloperidol (blue), and nsp6-dextromethorphan (red) more compact globular shape (rg =~1.65 nm) in the haloperidol system as a consequence of the lesser fluctuations (see rmsf results). the sasa results showed stable trajectories without any fluctuations throughout the simulation, thus suggesting the structural stability of nsp6 in the presence of drugs ( figure s3c ). the marginal increment of sasa in the nsp6-dextromethorphan system indicates increased exposure of the residues to the surface of nsp6 (fig. 3c) . the rmsf displays the flexibility/mobility of each amino acid residue in the nsp6 and nsp6-drug complexes (fig. 3d) . it is worth noting that nsp6 alone showed lower rmsf (higher rigidity) in comparison with the complexes, maintaining lower flexibility in α-helices, except for the helix h5 (s89-d99). the nsp6-haloperidol complex does not increase the fluctuation and showed rmsf value similar to unliganded nsp6, suggesting a favorable protein-inhibitor association. from the figure, it can be observed that the rmsf values of residues 130-140 (correspond to h7) in nsp6-haloperidol were lower than unliganded nsp6, indicating the stabilization of these regions upon haloperidol binding. the residues 85-92 (correspond to h5), 96-110 (correspond to hinge between h5 and h6), and 245-260 (h13) showed a milder increase in fluctuations. however, dextromethorphan caused much higher fluctuations along the whole protein when bound to nsp6, indicating that dextromethorphan binding results in significant structural perturbation to nsp6 (fig. 3d) . the dextromethorphan binding, however, decreases the flexibility in residues 93, 94, 97, and 98 (correspond to h5), indicating the dominant role of these residues in drug binding. from the rmsf analysis, we also observed that the nsp6dextromethorphan complex showed maximum fluctuation in the c-terminal domain (residues 240-270 correspond to β1-h13-h14) while unliganded nsp6 and nsp6-haloperidol do not show this fluctuation. from the rmsf result, we thus conclude that the nsp6-haloperidol complex is more stable as compared with the nsp6-dextromethorphan complex. this has been further substantiated by the h-bonding analysis. the protein-drug interaction is transient, and h-bonds play a critical role in the stability of the protein-drug complex. we have analyzed the number of h-bonds for the last 60-ns trajectories ( figure s4) . the time evolution of h-bonds for the three systems has been monitored during the simulation and represented in figure s4 . the average number of h-bonds for the nsp6-haloperidol and nsp6-dextromethorphan complexes was 0-2 and 0-1, respectively. as shown in the figure, hydrogen bonding patterns in the nsp6-haloperidol system remained constant throughout the entire simulation. whereas, hydrogen bond formation in the nsp6-dextromethorphan was unstable and absent most of the time during the simulation. for the last 20 ns of simulation, the number of intermolecular h-bonds is greater in the case of the nsp6-haloperidol system, indicating the most stable binding. to further understand the drug binding-induced changes in secondary structure, the time evolution of the secondary structure profiles is shown in fig. 4 . as mentioned earlier, the nsp6 adopts a compact fold consisting of fourteen α-helices and two β-strands. notable changes in the secondary structure profile were observed in the nsp6-drug complex. most significant changes were an increase in helicity of the residues 89-99 (i.e., h5) and residues 200-205 (i.e., residues between h10 and h11). in the nsp6 and nsp6-drug complexes, the hinge region covering residues 100-110 can transform between helix or turn during simulation. in the case of the nsp6-haloperidol complex, the region covering residues 226-236 (i.e., h12) was lost to turn during the simulation. dssp information indicates that dextromethorphan binding to nsp6 decreases the helicity of residues 150-160 (h7 and h8) while the h12 helix remains stable as in unliganded nsp6. here, we use the principal component analysis (pca) to examine the conformational sampling of the nsp6 systems via examining their dominant modes of motion. the covariance matrix of atomic fluctuations was diagonalized for predicting the eigenvalues. the first few eigenvectors play a key role in the motions of protein. it is apparent from the result that the first 5 eigenvectors have a larger eigenvalue for the unliganded than for the drug-bound nsp6, reflecting larger collective atomic fluctuations of the unliganded nsp6 ( figure s5 ). in addition, the first five principal components (pcs) account for more than 70% of motions observed for the last 40-ns trajectories of the nsp6 and nsp6-haloperidol systems, whereas only the first three pcs account for~100% of the motions of the nsp6-dextromethorphan system ( figure s5 ). this indicates that the nsp6 and nsp6haloperidol complex showed lesser motions as compared with the nsp6-dextromethorphan complex, and the first few pcs are not the same in the studied three systems. the conformational sampling of the nsp6 systems in the essential subspace is shown in figure 5a which illustrates the global motions along with the pc1 and pc2 projected by the cα atom. the figure clearly indicates that modeled nsp6 showed a more stable cluster as compared with the nsp6drug complex. at the same time, we found that the nsp6dextromethorphan complex occupied a wide and different conformational subspace and showed fewer stable clusters as compared with nsp6-haloperidol. the pca thus supports the findings that show the nsp6-dextromethorphan complex less stable with increased conformational dynamics. to find the reasons how drug binding affects the motions described by pc1 and pc2, the displacements of pcs for the nsp6 systems were calculated and are shown in fig. 5b . average fluctuation for the nsp6-dextromethorphan in both the pcs were much higher as compared with nsp6haloperidol and unliganded nsp6. this indicated that nsp6-dextromethorphan might influence the protein motions during binding, whereas the nsp6-haloperidol complex showed fewer motions during binding, which is consistent with the rmsf analysis (fig. 3d) . to demonstrate the effects of drug binding on conformational redistributions, free energy landscapes (fel) for the three fig. 6 . each protein-drug complex has a different fel pattern. fel of nsp6 protein had multiple minima with small energy barriers in a single broad valley (basin). most of the minima with the lowest energy had a flat end showing the clustering of low-energy conformations (fig. 6a) . after haloperidol binding, the basins segregate to the different coordinates and show clustered lowest energy minima close to each other. some of the energy minima had a conical end suggesting the presence of stable conformation, while the minima with flat end indicating the absence of low-energy conformations (fig. 6b) . however, in the case of the dextromethorphan-bound nsp6, there exists only one deeper energy minima in the global free energy minimum region, indicating only one stable conformational state residing within this valley (basin) (fig. 6c) . the free energy valley is much deeper than the unliganded nsp6 and nsp6-haloperidol complex, indicating that the nsp6dextromethorphan complex has a lower free energy value. besides this, the nsp6-dextromethorphan complex also showed a reversed population shift relative to the unliganded nsp6. a comparison of the fels for these two drug complexes of nsp6 reveals that the fel of the haloperidol-bound nsp6 exhibits a more rugged free energy surface than that of the dextromethorphan-bound nsp6. furthermore, the fel of the nsp6-haloperidol complex contains a greater number of local free energy minima either in the global free energy minimum region (i.e., the funnel bottom) or in the region outside the global free minimum (i.e., the funnel wall), resulting in a more rugged and complex fel. evaluation of binding free energy is one of the important aspects of drug discovery as they describe the strength of non-bonded molecular associations during binding. the more negative free energy value signifies the stronger binding between the ligand and the protein. using the mm-pbsa method [56, 57] , binding free energy (δg binding ) was calculated as the difference between the free energy of the protein and figure s6) . the average binding free energies and detailed energetic contribution components of the last 20 ns of md trajectories were calculated and are shown in table 1 . it can be seen that haloperidol binds more strongly to nsp6 than dextromethorphan by − 12.74 kcal/mol, indicating enhanced binding affinity. while van der waals energy (evdw) largely contributed to the binding of haloperidol to nsp6, polar energy is unfavorable with the positive value. besides, hydrophobic interaction played a crucial role in hel binding to nsp6. for dextromethorphan binding to nsp6, both polar energy and van der waals interactions were favorable, while electrostatic energy is highly unfavorable with the positive value of 52.1 ± 3.39 kcal/mol (table 1 ). according to table 1 , it is apparent that the polar (charged interactions) component was the key contributor in the binding free energy of the nsp6dextromethorphan complex. the results indicated that the large unfavorable electrostatic term (δeele) was compensated by the large contribution of polar and van der waals component in the binding process of dextromethorphan to nsp6. therefore, the polar contribution was considered the main driving force in binding mechanism. to identify the key residues that contributed to the binding energy, we decomposed the binding energy at amino acid basis and the important ones with high contribution (≥ 0.1 kcal/mol) were identified and are shown in fig. 7 . as represented in fig. 7 , the values of amino acid energy with high contribution in each drug complex varied significantly. the decompositions of the relative binding energies of individual residues to the nsp6-haloperidol complex formation with the most favorable interactions were from trp140, trp165, ala166, and gly177, which comprised both charged and hydrophobic amino acids (fig. 7) . residues within dextromethorphan-binding sites of nsp6 includes mainly charged amino acids like w31, e39, d112, d133, d134, d159, e195, d243, e250, and d267 (fig. 7) . of note, the dextromethorphan interactions with nsp6, many charged residues like lysine (k61, 63, 109, 111, 151, 263, 270, 274 , 281, and 285) and arginine (r93, 187, 233, 236, and 252) , produced large positive unfavorable binding free energy values on average. thus, we have observed the large spatial shift of the binding residue clusters in both the drugs. overall, haloperidol binds to the residue clusters near trp165-gly177 (while there was no energy contribution in the nsp6-dextromethorphan system). also, the dextromethorphan binds to the c-terminal residue clusters arg236-asp267 (while there was no energy contribution in the nsp6-haloperidol system). calculation of correlation matrix is frequently utilized to illustrate dynamical information of proteins in two dimensions [52, 58, 59] . to observe the correlation in the dynamics, correlation matrices for each of the nsp6 systems were calculated through dynamut web server [60] and are plotted in fig. 8 . the red regions in the correlation maps represent the strongly correlated motion of the residues while the blue regions are associated with strong anticorrelated motion of the residues. from the comparison of the correlation map of the nsp6 systems, substantial loss of motions in the correlation maps of the nsp6-drug complexes had been identified and labeled as shown in fig. 7 the per-residue binding free energy decomposition for the simulated nsp6-haloperidol (blue) and nsp6dextromethorphan (red). the free energy values ≥ 0.1 kcal/mol contributes more to the binding interaction fig. 8 . in the presence of drugs, the helices of the hydrophobic core significantly destabilized and this is most apparent in the nsp6-dextromethorphan complex, whereby most of the helices showed the weakest correlation in motions compared with the nsp6-haloperidol complex. as can be seen, dextromethorphan binding leads to the complete loss of correlated motions of helices h9 and h11 in nsp6. the significant loss of correlated motions between h1-h6 and h1-h8 along with disruption of long-range motions between h5-h13, h6/h7-h11, and h5-β2, and shortrange motions between h8/h9-h11 was observed. interestingly, correlated motions between h9 and β2 have been observed in nsp6-dextromethorphan which is not present in the unliganded nsp6 protein. moreover, the long-range anti-correlated motions between n-and c-terminal regions were also lost in the nsp6-dextromethorphan complex. on the contrary, the nsp6-haloperidol complex displayed a somewhat better correlation in motion of residues of h8 and h11 compared with the nsp6-dextromethorphan system, hence indicating enhanced stability of the hydrophobic core (fig. 8) . however, similar to dextromethorphan, haloperidol binding also leads to complete loss of correlated motions between h1-h6, h1-h8, and h6/h7-h11, while retaining the motion between h8/h9-h11. there is also gain in long-range correlated motions between h2 and h13. thus, the lack of correlation in the motion of the helical domains for the nsp6-dextromethorphan complex suggested the loss of contacts among these helices, which might cause the greater accessibility of the hydrophobic core to the solvent molecules, and subsequently the destabilization of the protein. to examine the role of frustration in binding, we compute residual frustration in the protein by using the protein frustratometer server [51] . the frustration indices calculated through single residual frustration analysis indicate that sars-cov-2 nsp6 is more frustrated than a typical globular protein. using the cutoff values of the frustration indices, we found that nsp6 protein is highly frustrated molecules with almost 27% of residues are highly frustrated (compared with 10% observed in a typical protein), and 36% of the residues are minimally frustrated (40% in general). the frustration values range between − 2.9 and + 1.4. here, the minimally frustrated residues were found in the helices h3, h5, h6, h10, and h11, whereas the highly frustrated residues were largely scattered and seen mainly in h5 and c-terminal h13 and h14 ( figure s7a-c) . furthermore, we also monitored the frustration changes in nsp6 upon drug binding and the frustration profile showed some significant differences in protein-drug complex structure as shown in the supporting information table s1 . as can be seen, dextromethorphan binding increased the local destabilization of the protein by increasing the highly frustrated residues (29.3%) while the local frustration is similar to apo nsp6 (35.8%). haloperidol binding also increased the frustration to a little extent (28%) while keeping the minimal frustration unchanged (table s1 ). the drug binding thus increases the number of highly frustrated residues and thus increases the local frustration and flexibility of the protein, especially in dextromethorphan binding. moreover, the residues involved in drug binding were dominantly neutral to minimal frustration. however, some of the dextromethorphan-binding residues like ser32 and leu276 gain frustration upon binding, whereas the frustration of tyr132, tyr136, gly177, arg236, and lys270 decreased upon binding. also, haloperidol binding increases the frustration of leu231, leu237, and thr238 upon binding, and at the same time, it also decreased the frustration of residues like tyr136, phe184, and phe269 (table s1) . moreover, to find out how drug binding induced the local frustration changes, we performed a comparative analysis of the changes in highly frustration values in nsp6-drug complex. the secondary structural regions are represented as: h-α-helix and s-βstrands fig. 10 the structural snapshots of nsp6-drug complex observed during the md simulation (0 ns, 25 ns, 50 ns, 100 ns) for the most abundant structure of a-d nsp6-dextromethorphan and e-h nsp6-haloperidol the spatial distribution of local frustrations mapped onto the secondary structure of the protein (fig. 9 ). in particular, highly frustrated residues in the nsp6-dextromethorphan complex increased in the α-helix h1, h7, and h13, while the increase is seen in h11 for the nsp-haloperidol complex (fig. 9a) . the decrease in frustration was also seen for both the complexes mainly in c-terminal h11, h12, s1, and h14 for the nsp6dextromethorphan complex, and for nsp6-haloperidol, a decrease is observed in h5, h8, and s1 (fig. 9a) . additionally, the drug binding decreased the minimal frustration in h5, h7, h9, and h11 helices, and also increased the minimal frustration in h2 and h12 helices (fig. 9b) . thus, the coupling between structurally rigid c-terminal helices h10 and h11, and conformationally flexible helices, h5 and h13, is important for drug binding, and also the frustration index of the regions close to the binding site changes upon association. the all-atom md simulation shows significant differences in the tertiary structure of the nsp6-drug complexes. the structural snapshots of the protein-drug complex were used to analyze the differences in tertiary structure caused by each drug ( figure s8 ). the nsp6-drug complexes show the significant differences in h2, h5, h7, and c-terminal regions comprising h12, h13, β1, and β2 ( figure s8 ). the haloperidol and dextromethorphan complexes have a kink in h2, h5, and h7 at midway of simulation (50 ns) that causes a change in orientation when the drug binds ( figure s8 a, c). the dextromethorphan binding induces much larger kink at h2 and h5 with a much larger deviation also (rmsd = 3.59) ( figure s8c ). at the end of the simulation, the haloperidol and dextromethorphan complexes showed an increase in twist of h12 and h13, and both the strands of the c-termini along with the kinks present in h2, h5, and h7 ( figure s8b, d) . similar to what was observed at 50 ns, dextromethorphan binding induces much larger disruption (rmsd = 3.24). thus, although both drugs lead to bending of the helices, dextromethorphan binding induces larger disruption of the helices and thus more destabilization observed in the protein. the protein-drug binding analysis shows that dextromethorphan and haloperidol interact with many key residues and are shared between both the drugs. the residues interacting with each drug during the simulation were compared and visual 2d representations are shown in figures s9 and s10. figure 2 a shows the initial pose of the dextromethorphan molecule in the md simulation, which is also the best pose from the docking study. as can be seen, the dextromethorphan molecule formed hydrogen bonds with residue lys 61, and several van der waals interactions with his62, asn232, arg233, arg236, thr238, asp243, leu245, and pro282. figure 2 b shows the haloperidol binding sites in nsp6 which is comprised of helix h7, and h9 residues (i.e., h7: tyr132, asp134, ala136, arg137, trp140; h9: asn174, tyr175, ser176, and val178) and c-terminal positively charged residues leu231, leu237, leu239, and ser265. the structural snapshots of the drug-protein complexes have been shown in fig. 10 . it has been seen during simulation that dextromethorphan has drifted to the different locations inside the nsp6 during~20-100 ns of simulation. in the first ∼ 20 ns, dextromethorphan stayed in its initial location (fig. 10a) . after that, it drifted away from its initial location and moved into the water above the binding pocket and the interaction becomes minimal, only with tyr234, phe235, and leu276 (fig. 10b) . without drifting further into the water, the dextromethorphan molecule re-entered to the enlarged binding pocket, where it interacts mainly with h12 residues (phe225, leu230, arg233, tyr234, and arg236) through the hydrophobic interaction (fig. 10c) . during the second half of the md simulation (~50-100 ns), the dextromethorphan molecule stably remained at a new binding pose with much stronger interactions. at this new location, dextromethorphan coordinates with ser32 through h-bonds, and hydrophobic interactions by trp31, ile189, val190, met192, cys193, phe200, and phe201 (h9-h10) (fig. 10d) . in contrast, the drug haloperidol was stably bound inside the nsp6 pocket and explored two binding poses during the simulation. for the first 50 ns, it remained at its initial docking pose where it forms h-bonds to ser 176 and ser265, and several hydrophobic interactions mediated by arg137, trp140, asn174, tyr175, val178, and thr238 (fig. 10e, f) . after that, it drifted to another binding site and remained stable there, until the end of the simulation. at this new site, haloperidol interacts mainly with h7-h9 residues (trp140, asn144, trp165, ala166, ile169, ser170, ser176, val178, and phe184), leu237, leu239, and ile266, phe269 (h14) (fig. 10g, h) . to better understand the mechanism of biased activity of sars-cov-2 in the presence of two sigma-r1 binding drugs to nsp6, molecular dynamics simulation studies were employed. our data suggests dextromethorphan binds to cterminal helices while haloperidol binding sites are in the middle of the protein domain in helices h7 and h9. the disruption of the alpha helix h7 and h8 is significant for the dextromethorphan complex along with a large kink in h5 and h7. the nsp6-haloperidol complex showed a less significant change in the tertiary structure despite major disruption of the h12 helix. furthermore, the analyses of rmsd, rmsf, pca, fel, and dynamic cross-correlation matrix (dccm) indicated that dextromethorphan binding leads to destabilization of the protein with the loss of correlated motions and residual frustrations. in contrast, haloperidol binding brings milder alterations in these order parameters and thus showed minimal changes in stability compared with dextromethorphan system. besides, intermolecular hydrogen bonds were constantly formed in haloperidol with high occupation indicating the more stabilization of the nsp6-haloperidol system. in conclusion, the study elucidated the detailed interaction mechanism of dextromethorphan and haloperidol to nsp6 protein and the associated structural and dynamical changes upon drug binding. these results will significantly enhance our understanding of the working mode of these drugs at the molecular and structural level and will contribute to the future rational drug design for covid-19. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study novel therapeutic approaches for treatment of covid-19 covid-19: molecular diagnostics overview immuno-epidemiology and pathophysiology of coronavirus 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alignment calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models g_mmpbsa-a gromacs tool for high-throughput mm-pbsa calculations a computational analysis of binding modes and conformation changes of mdm2 induced by p53 and inhibitor bindings revealing origin of decrease in potency of darunavir and amprenavir against hiv-2 relative to hiv-1 protease by molecular dynamics simulations dynamut: predicting the impact of mutations on protein conformation, flexibility and stability publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations author contributions v.k. designed the study. p.p. and k.p. performed the experiments and calculations. v.k. and a.p. analyzed the data. p.p. and k.p. prepared figures of the results. v.k. wrote the manuscript with the contributions of k.p. and a.p. conflict of interest the authors declare that they have no conflict of interest. key: cord-269531-7gy4epzo authors: kumar, pankaj; van den hurk, jan; ayalew, lisanework e.; gaba, amit; tikoo, suresh k. title: proteomic analysis of purified turkey adenovirus 3 virions date: 2015-07-09 journal: vet res doi: 10.1186/s13567-015-0214-z sha: doc_id: 269531 cord_uid: 7gy4epzo turkey adenovirus 3 (tadv-3) causes high mortality and significant economic losses to the turkey industry. however, little is known about the molecular determinants required for viral replication and pathogenesis. moreover, tadv-3 does not grow well in cell culture, thus detailed structural studies of the infectious particle is particularly challenging. to develop a better understanding of virus-host interactions, we performed a comprehensive proteomic analysis of proteinase k treated purified tadv-3 virions isolated from spleens of infected turkeys, by utilizing one-dimensional liquid chromatography mass spectrometry. our analysis resulted in the identification of 13 viral proteins associated with tadv-3 virions including a novel uncharacterized tav3gp04 protein. further, we detected 18 host proteins in purified virions, many of which are involved in cell-to cell spread, cytoskeleton dynamics and virus replication. notably, seven of these host proteins have not yet been reported to be present in any other purified virus. in addition, five of these proteins are known antiviral host restriction factors. the availability of reagents allowed us to identify two cellular proteins (collagen alpha-1 (vi) chain and haemoglobin) in the purified tadv-3 preparations. these results represent the first comprehensive proteomic profile of tadv-3 and may provide information for illustrating tadv-3 replication and pathogenesis. electronic supplementary material: the online version of this article (doi:10.1186/s13567-015-0214-z) contains supplementary material, which is available to authorized users. hemorrhagic enteritis (he) is an economically important disease of turkeys characterized by depression, splenic enlargement, intestinal haemorrhages and sudden death [1] . the disease is caused by turkey adenovirus 3 (tadv-3), also known as hemorrhagic enteritis virus (hev), a member of genus siadenovirus a [2] . oral infection of susceptible turkeys with pathogenic tadv-3 strains results in well-characterized splenomegaly and intestinal bleeding in 4 to 6 days causing subclinical infections and mortality [3] . although tadv-3 remains one of the most important causes of economic loss to turkey industry, critical molecular determinants of virulence and factors affecting virus replication are not well understood. this may be in part because of unavailability of an efficient "in vitro" tissue culture system for propagation of tadv-3 [4] [5] [6] . the genome of tadv-3 is 26,263 bp [7] . although, tadv-3 genomic organization of central block of genus-common genes [8] appears similar to that of other adenovirus genomes [7] , the left (e1) and right (e4) terminal regions appear absent. interestingly, tadv-3 encodes a genus specific protein, which shows similarity to bacterial sialidase protein [8] . although western blot analysis of purified tadv-3 particles isolated from crude spleen extract revealed presence of eleven structural polypeptides with apparent molecular weight ranging from 9.5 to 96 kda [9] , no systematic study has been performed to identify the precise protein composition of purified tadv-3 particles. in recent years, mass spectrometry (ms) based proteomic characterization has revealed important insights into viral replication, tropism and virulence for a number of different enveloped viruses [10] [11] [12] [13] [14] . in contrast, a few proteomic studies have been reported for nonenveloped viruses [15] [16] [17] [18] . additionally, there is now compelling evidence suggesting that host cellular proteins incorporated in the virions play an important role in viral replication and pathogenesis [10, 13, 19, 20] . using ms based approaches, a number of host proteins have been reported to be incorporated into rna viruses ("human immunodeficiency virus-1 [10, 13] "; "simian immunodeficiency virus [21] "; "respiratory syncytial virus [22] ; hepatitis c virus [23] "; "swine hepatitis e virus [24] "; "coronavirus [25] " and "influenza [20, 26] ") or dna viruses ("herpes simplex virus 1 [27] "; "african swine fever virus [28] "; "kshv [29] "; "marek's disease virus (mdv) [30] ", and "mimivirus [31] "). however, to the best of our knowledge, characterization of the host cellular factors integrated into virions for any member of adenoviridae family including tadv-3 has not been reported so far. here, we report the protein composition of the purified tadv-3 particles by performing a comprehensive proteomic analysis utilizing liquid chromatography-mass spectrometry (lc-ms/ ms). our analysis resulted in successful identification of 13 viral structural proteins and 18 host-incorporated proteins. moreover, incorporation of two host proteins in purified virions was verified by western blot analysis using available immunological reagents. all turkey procedures were approved by university committee of animal care and supply (protocol # 19940211) at the university of saskatchewan, saskatoon, canada according to guidelines set by the canadian council of animal care. day-old hybrid poults obtained from chinook belt hatcheries, calgary, canada were housed in isolation rooms throughout the experiments. the avirulent tadv-3 isolate (pheasant origin) was passaged in sero negative turkeys by oral inoculation and purified from crude spleen extracts, as described earlier [32] . the tadv-3 virions were purified as previously described [9] . the proteinase k (pk) treatment of purified tadv-3 virions was performed as described previously [33] . briefly, double cscl-purified virions were incubated in 1 ml of mnt buffer (30 mm morpholineethanesulfonic acid [mes], 10 mm nacl, and 20 mm tris-hcl [ph 7.4]) containing proteinase k [0 to 20 μg] (roche, mannheim, germany) for 45 min at room temperature and subsequently treated with "2 mm phenylmethylsulfonyl fluoride" (roche) prior to purification by cscl density gradient centrifugation. purified virions were resuspended in 10% glycerol and stored at −80°c until further use. the experiments were performed in triplicate employing three independent virus preparations. electron microscopy was performed on cscl 2 gradient purified tadv-3 virions (proteinase k treated or untreated) at em facility at biology department, university of victoria, bc, canada, as described [34] . briefly, for negatively stained preparation, cscl 2 gradient purified virus was first applied onto carbon and formvar coated grids, washed with h 2 0 and stained with 2% aqueous phosphotungstic acid. the specimens were photographed using a charge-coupled device camera (advanced microscopy techniques, amt ccd camera equipped hitachi h7000 tem operating at 75 kv). production and characterization of anti-tadv-3 serum and monoclonal antibodies (mabs) recognizing tadv-3 hexon (15g4) and fiber (87-03) proteins has been described earlier [4, 9] . chicken polyclonal anti-human hemoglobin serum (ab28961) was purchased from abcam (cambridge, ma, usa). rabbit polyclonal antihuman collagen type vi alpha-1 serum (col6a1) was purchased from antibodies-online inc. (atlanta, ga, usa). alkaline phosphatase conjugated goat anti-rabbit (sigma aldrich) and peroxidase-conjugated goat "antiturkey" igg (kpl, maryland, usa) were used as described [4, 9] . proteins from purified tadv-3 were separated by sodium dodecyl sulphate (sds) polyacrylamide gel electrophoresis (page) on 10-15% or 4-15% precast gradient gels (bio-rad),transferred to nitrocellulose membrane and probed with protein specific antibodies as described previously [9] . proteins from cscl 2 gradient purified virion-enriched (proteinase k treated or untreated) samples were diluted with 200 mm ammonium bicarbonate prior to reduction with 200 mm dithiothreitol and incubated 30 min at 37°c. cysteine sulfhydryl groups were alkylated with 20 μl of 100 mm iodoacetamide (30 min at 37°c in darkness). each sample was digested with 5 μg of trypsin (promega) at 37°c for 16 h [33, 35] . finally, the samples were de-salted on a waters hlb oasis column, speed vac concentrated and stored at −80°c prior to lc-ms analysis. the peptide mixtures were separated by on-line reverse phase chromatography using a easy-nlc ii system (thermo scientific) with a reversed-phase magic c-18aq pre-column (100 μm i.d., 2 cm length, 5 μm, 100 å, michrom bio resources inc, auburn, ca, usa) and reversed phase nano-analytical column magic c-18aq (75 μm i.d., 15 cm length, 5 μm, 100 å, michrom bio resources inc, auburn, ca, usa) at a flow rate of 300 1/min. the resulting peptides were analyzed by the chromatography system, which was coupled on-line with a ltq orbitrapvelos mass spectrometer (thermo fisher scientific, bremen, germany) equipped with a nano-sprayflex source (thermo fisher scientific) as described previously [33, 35] . the data was acquired with keratin and trypsin peptide mass exclusion lists. raw files were analysed with proteome discoverer 1.4 software suite (thermo scientific). parameters for the spectrum selection to generate peak lists of the collisioninduced "dissociation (cid) spectra were activation type: cid"; (s/n cut-off: 1.5; total intensity threshold: 0; minimum peak count: 1; precursor mass: 350-5000 da). the peak lists were submitted to an in-house mascot 2.3 server against "the following databases": uniprot_trembl 20111103 (17 651 715 sequences; 5,747,683, 275 residues) and uniprot-swissprot 20110104 (523 151 sequences; 184 678 199 residues) all species taxonomy. database search parameters were as follows: precursor tolerance 8 ppm; ms/ms tolerance 0.6 da; trypsin enzyme 1 missed cleavages; fourier transform ion cyclotron resonance (ft-icr) instrument type; fixed modification: carbamidomethylation (c); variable modifications: deamidation (n,q); oxidation (m). the decoy database percolator settings: max delta cn 0.05; target fdr strict 0.01, target fdr relaxed 0.05 with validation based on q-value. additional virus only species searches were also performed with tolerances previously mentioned. all data were also searched against ncbi (gallus gallus (chicken)) database to detect viral and host proteins. only sequences identified with a mascot score value greater than 30 were considered as significant. protein identifications were accepted when the peptide probability was greater than 95.0% [33, 35] , the protein probability greater than 99.0%, and contained at least 2 identified peptides. peptide identifications were systemically evaluated manually. due to difficulty in propagating turkey adenovirus 3 in cell culture system, tadv-3 was propagated in six to 8 week old turkeys. tadv-3 virions were purified from spleens of turkeys inoculated orally with an avirulent vaccine strain of tadv-3 [4, 9] ( figure 1a ). following cscl 2 density gradient purification, two distinct bands were observed, the upper band (present at lower density) containing capsid and the lower band (at higher density, between 1.25 and 1.35) containing complete infectious viruses ( figure 1b , left panel). the lower band was subjected to second round of cscl 2 density gradient purification resulting in single band containing purified virions ( figure 1b, right panel) . virion-enriched preparations were checked for quality by negative stain transmission electron microscopy (tem) (figures 1c and d) . as seen, virions demonstrated uniform, intact tadv-3 virus particles of 100 nm diameter. these tem results were consistent with the quality and apparent purity reported earlier [33, 35] . the purity of the virion preparation was also determined by western blot analysis using turkey anti-tadv-3 sera. as seen in figure 1e , polypeptides of 96 k (hexon), 57 k (iiia), 52 k (penton base), 29 k (fiber) and 24 k (pvi) were detected in cscl 2 purified tadv-3 virions. these findings suggest that our enrichment procedure yielded a highly purified preparation of tadv-3 virions. the protein composition of tadv-3 virions was analyzed by the method of in-solution trypsin "digestion a gel-free approach" to ms that subject the entire sample to sequential one-dimensional reversed-phase chromatography coupled on-line to ms/ms analysis (1d-nanospray-lc-ms/ms). this method eliminates the problems reported with proteins that either enter gel poorly or extracted inefficiently from the gel slices. our lc-ms/ms analysis revealed a total of 15 virus-encoded proteins packaged in the purified tadv-3 virions. this included 13 proteins, which have been detected in human adenovirus 5 (hadv-5) virions [16] (table 1) , a novel uncharacterized hypothetical viral protein designated as tav3gp04 (table 1 , additional file 1) and a non-structural viral protein (22 k) to be associated with tadv-3 virions. in addition to tadv-3 encoded viral proteins, interestingly 26 cellular proteins appeared to be associated with purified tadv-3 virions (table 2) . to determine if the host proteins are actually incorporated into the virions, the purified tadv-3 virions were treated with proteinase k (20 μg/ml) and subjected to another round (third round) of cscl 2 purification. the proteinase k treated and untreated, purified virions were then analysed by western blotting. proteinase k treatment degrades fiber protein protruding from the capsid but does not degrade hexon protein not protruding from the capsid. as seen in figure 2a , hexon protein could be detected in proteinase k treated or untreated tadv-3 virions. in contrast, fiber protein could only be detected in untreated virions, but not in proteinase k treated virions. moreover, tem analysis suggested that the virions were intact and maintained virion integrity after proteinase k treatment and cscl 2 density gradient purification ( figure 2b ). the lc-ms/ms analysis of proteinase k treated cscl 2 density gradient purified tadv-3 virions identified eleven virus-encoded proteins (hexon, pvi, pvii, penton base, pviii, sialidase, iiia, adenain, px, iva2 and dbp) previously reported to be in other adenoviruses (table 3 and figure 3a ) [16] . in addition, a novel viral protein tav3gp04 remains an integrated part of proteinase k treated tadv-3 virions (table 3 , additional file 1). as expected, peptides representing fiber protein were not detected in proteinase k treated tadv-3 virions. in addition, ptp and 22 k virion proteins were not detected in proteinase k treated tadv-3 ( table 3 ). the high mascot scores and number of peptides observed for hexon, pvi and pvii presumably reflect the fact that they are perhaps the most abundant proteins in the tadv-3 particles. interestingly only 18 host proteins were exclusively detected in proteinase k treated tadv-3 virions (table 4 and figure 3b ). notably, thirteen of these host proteins were the same as detected in the untreated tadv-3 virions (table 4, figure 3b ) indicating that these proteins are part of the tadv-3 virions. among these proteins, promyelocytic leukemia protein (pml) isoform x6 (additional file 2), collagen alpha-1(vi) chain (additional file 3), haemoglobin subunit alpha (additional file 4) and haemoglobin subunit beta (additional file 5) appeared abundant. the pml protein appears as abundant as viral structural protein pviii or penton base peptide. in addition, five host proteins namely, vitronectin, collagen alpha-3 (vi) chain, collagen alpha-2 (vi) chain, tyrosine protein phosphatase and turkey heterophil peptide 2 (thp-2) were only detected in proteinase k treated tadv-3 virions. functional classification of the identified proteins revealed that many of these proteins participate in a common molecular pathway (table 4 and figure 3c ) and are involved in innate immunity, cell adhesion, cytoskeleton organization and virus replication. non availability of turkey host protein specific antisera made it difficult to verify the packaging of host proteins in tadv-3 virions. however, human collagen alpha-1(vi) peptides showed 70% identity to turkey collagen alpha-1(vi) and chicken collagen alpha-1(vi) (additional file 6). in addition, human haemoglobin peptides demonstrated 75% identity to turkey haemoglobin alpha and chicken haemoglobin alpha, 50% identity to turkey haemoglobin beta and 66% identity to chicken haemoglobin beta proteins (additional file 7). therefore, we attempted to determine the incorporation of collagen alpha-1(vi) and haemoglobin in purified tadv-3 using western blot assays. as shown in figure 4 , anti-collagen alpha-1 (vi) serum detected collagen alpha-1 (vi) chain specific band in proteinase k untreated tadv-3 (panel a, lane 1). similar protein could be detected in proteinase k treated purified tadv-3 (panel a, lane 2). antihaemoglobin serum detected haemoglobin specific band in proteinase k untreated tadv-3 (panel b, lane 1). similar protein band could be detected in proteinase k treated purified tadv-3 (panel b, lane 2). viruses exploit multiple host proteins for successful entry, establishment of infection, replication, and immune evasion. for a better understanding of the tadv-3-host interactions, we performed a comprehensive analysis of the protein content of tadv-3 virions, using a lc-ms/ms based proteomic approach. to the best of our knowledge, incorporation of host proteins in adenovirus has not been reported so far. the proteomic analysis of cscl 2 purified tadv-3 identified a total of 13 virion proteins and 18 host proteins. earlier, proteomic analysis has not reported the detection of host proteins in purified hadv-5 virions [15, 16] . it is possible that the observed host proteins identified by proteomic analysis of cscl 2 purified tadv-3 virions may not be actually incorporated in the purified virions but are loosely associated on the outside of the tadv-3 virion capsids. since proteinase k treatment has been traditionally used to remove any contaminating protein from the surface of enveloped viruses [33, 35] , we used protease treatment of non-enveloped tadv-3 to remove the potential contaminating proteins. several lines of evidence validate the approach and suggest that proteinase k treatment of tadv-3 appears successful in removing contamination proteins. 1) intact virions could be detected by tem after proteinase k treatment of tadv-3. 2) western blot analysis of protease k treated tadv-3 detected hexon protein but not fiber protein (protruding from the capsid). 3) the fiber and 22 k (non structural protein) could not be detected by ms analysis of proteinase k treated tadv-3. 4) only 18 of the 26 host proteins could be identified in proteinase k treated tadv-3. interestingly, all major viral proteins were identified in proteinase k treated virions (table 3 ) except viral ptp, possibly due to its low abundance and least mascot score values observed (table 1) . overall sequence coverage observed for different viral peptides ranged from 2 to 70%, with the majority between 10 and 35%. earlier, sequence analysis of turkey adenovirus-3 identified a hypothetical protein orf 4 (named tavgp04) [7] , which appears to be conserved in raptor adenovirus-1 [36] and south polar skua adenovirus [37] . in contrast, a hypothetical hydrophobic protein was identified in frog adenovirus 1 [8] , which shows no similarity to similar proteins identified in turkey adenovirus 3 [7] and raptor adenovirus 1 [36] . our results suggest that an orf4 of tadv-3 encodes a structural protein tavgp04, which is incorporated into virion capsid (additional file 1). in addition, this is the first report to suggest the existence of tavgp04 as a structural protein in siadenoviruses particularly of avian origin. the proteomic analysis of proteinase k treated purified virions identified eleven cellular proteins incorporated in tadv-3, which have been identified in other viruses (table 4 ). in addition, proteomic analysis identified seven host proteins incorporated in tadv-3 virions (table 4) , which have not been identified so far in any other virus. interestingly, of the 18 detected host proteins, five of the proteins were only detected in proteinase k treated tadv-3. it is possible that high abundance non-specific proteins might have masked the detection of these proteins in virions not treated with proteinase k that are truly virion associated, but present in low copy numbers. though earlier reports have demonstrated the packaging of viral [38] or non viral rnas [39] into purified adenovirus, recent reports have not described the detection of any cellular protein in purified lizard adenovirus-2 [40] , a member of atadenovirus genus and purified hadv-5, a prototype of mastadenovirus genus [15] . the absence of a cellular protein packaged in purified adenovirus virions could be due to variety of reasons. as stated, the difference could be due to the technique used for analysis [15] . alternatively, it is possible that packaging of the cellular proteins may be dependent on the type of adenovirus (tadv-3, a prototype of siadenovirus genus) and origin of cells used for virus cultivation [15] . the host proteins packaged intadv-3 are known to play important roles in enhancing the cell-to-cell spread of virus, transcription and virus replication (table 4 , figure 3 ). for example, extracellular matrix (collagen) has been shown to increase infectious sindbis virus titers from bhk cells by enhancing post-infection cell survival [41] . in another study, rotavirus-induced pi3k activation resulted in prolonged adherence of infected cells to collagen and increased virus production [42] . similarly, extracellular matrix vitronectin has been reported to enhance the growth of human adenovirus19 (hadv-19) [43] . however, the incorporation of antiviral host defense factors including, protein pml, haemoglobin and antimicrobial peptide (thp-2) into tadv-3 virions is particularly intriguing. all of these host defence factors have been implicated in establishing antiviral environments. recent studies have implicated pml in maintaining host antiviral defence and revealed different strategies developed by viruses to disrupt pml nuclear bodies [44] [45] [46] . in addition, protein pml has been shown to be important for the inhibition of adenovirus replication [47] . similarly, avian antimicrobial peptide thp-2, a member of beta-defensin family is effector of the innate defence system and play key functions during host defence by generating vigorous cytokine response [48, 49] . on the other hand, a novel role of haemoglobin in innate immunity has been recently reported for classical swine fever virus (csfv) [50] as silencing of haemoglobin expression using sirna promoted csfv growth and replication, whereas overexpression of haemoglobin antagonized csfv replication and growth by triggering ifn signalling [50] . although tadv-3 grows efficiently in spleen of infected turkey, virus grows poorly in primary or established cell lines. it is tempting to speculate that integration of certain established antiviral host restriction factors into viral particles may play a role in determining tadv-3 replication "in vitro". additional studies need to be performed in order to investigate whether these proteins are functionally required for virus entry, replication and pathogenesis. future availability of reagents and a reliable cell culture system to grow tadv-3 should make it possible to determine the role of individual host restriction factor in tadv-3 replication. comparison of 12 turkey hemotthagic enteritis virus isolates allows prediction of genetic factors affecting virulence ictv at paris icv: results of the plenary session and the binomial ballot avian adenoviruses characterization of group ii avian adenoviruses with a panel of monoclonal antibodies evaluation of cell culture propagated and in vivo propagated hemorrhagic enteritis vaccines in turkeys development and application of quantitative real-time pcr for the rapid detection of hemorrhagic enteritis virus in tissue samples the complete dna sequence and genome organization of the avian adenovirus, hemorrhagic enteritis virus genetic content and evolution of adenoviruses 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virions of porcine adenovirus type 3 presence of prepackaged mrna in virions of dna adenovirus molecular characterization of a lizard adenoivirus reveals the first atadenoivirus with two fiber genes and the first adenovirus with either one short or three long fibers per penton effects of collagen matrix on sindbis virus infection of bhk cells rotavirus replication in intestinal cells differentially regulates integrin expression by a phosphatidylinositol 3-kinase-dependent pathway, resulting in increased cell adhesion and virus yield vitronectin: a possible determinant of adenovirus type 19 tropism for human corneal epithelium effects of promyelocytic leukemia protein on virus-host balance rabies virus p and small p products interact directly with pml and reorganize pml nuclear bodies serum-dependent expression of promyelocytic leukemia protein suppresses propagation of influenza virus adenovirus e4 orf3 protein inhibits the interferon-mediated antiviral response human beta-defensin 2 induces a vigorous cytokine response in peripheral blood mononuclear cells antimicrobial activity of chicken and turkey heterophil peptides chp1, chp2, thp1, and thp3 hemoglobin subunit beta interacts with the capsid protein and antagonizes the growth of classical swine fever virus submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www authors thank other members of tikoo's laboratory for helpful suggestions. published as vido-intervac article # 725. the work was supported by grants from saskatchewan agriculture development fund, alberta livestock and meat agency, saskatchewan chicken industry development fund, canadian poultry research council and agriculture and agri-food canada. the authors declare that they have no competing interests. conceived and designed the experiments: pk, skt. performed the experiments; pk, jv, ag, lea. analyzed the data; pk, jv, skt; ag. wrote the manuscript: pk, lea, skt. all authors read and approved the final manuscript. key: cord-274080-884x48on authors: rumlová, michaela; ruml, tomáš title: in vitro methods for testing antiviral drugs date: 2018-06-30 journal: biotechnology advances doi: 10.1016/j.biotechadv.2017.12.016 sha: doc_id: 274080 cord_uid: 884x48on abstract despite successful vaccination programs and effective treatments for some viral infections, humans are still losing the battle with viruses. persisting human pandemics, emerging and re-emerging viruses, and evolution of drug-resistant strains impose continuous search for new antiviral drugs. a combination of detailed information about the molecular organization of viruses and progress in molecular biology and computer technologies has enabled rational antivirals design. initial step in establishing efficacy of new antivirals is based on simple methods assessing inhibition of the intended target. we provide here an overview of biochemical and cell-based assays evaluating the activity of inhibitors of clinically important viruses. viral pandemics remain serious threats to humankind. every year, known viruses such as hiv-1 and hepatitis b virus newly infect millions of people across the globe. in addition, recent outbreaks of ebola virus, influenza virus, severe acute respiratory syndrome (sars) coronavirus and middle east respiratory syndrome-coronavirus (mers-cov) serve as a reminder of the silent danger. the world health organization reported in 2015 that over 1.34 million causalities per year are connected with hepatitis b and almost 500,000 with hepatitis c. occasional epidemic outbreaks of other viruses are also striking. these include outbreaks of noroviruses, flaviviruses (zika and dengue viruses), new strains of influenza viruses, the re-emergence of west nile virus in italy and the united states. despite relatively long pauses, viruses such as influenza virus can re-emerge and cause global pandemic health problems. unlike cellular genomes, which consist of double-stranded (ds) dna, viral genomes can be formed by a broad variety of nucleic acids (nas), including ds or single-stranded (ss) circular or linear dna or positive-, negative-or sometimes ambi-sense rna. as viral life cycles are dependent on cellular factors and cellular metabolic and signaling pathways, the number of possible antiviral drug targets is limited. however, almost all viruses encode unique proteins and enzymes that may serve as specific targets for antiviral therapy. the overarching goal of modern drug development efforts is to design compounds that specifically inhibit viral targets or cellular targets essential for virus replication. the purpose of this review is to provide insight into the broad variety of cell-based and biochemical assays used for identification and evaluation of antivirotics, including high-throughput screening (hts) methods. an overview of available inhibitors and vaccines, which has been reviewed elsewhere [e.g. in (de clercq and li, 2016) ], is beyond the scope of this paper. to package their genomes into particles of very limited size, viruses encode few genes. virions consist of an rna or dna genome that is protected by an outer shell called a capsid (also nucleocapsid) formed by a lattice of capsid proteins. in enveloped viruses, the capsid is additionally surrounded by a lipid bilayer spiked with viral proteins. the size of animal viruses ranges from approximately 25 nm to over 300 nm (cohen et al., 2011) . the capsids and virions of rna viruses adopt various shapes. viral capsids may be icosahedral (e.g. picornaviridae, astroviridae, reoviridae, togaviridae), bullet-shaped (rhabdoviridae), helical (coronaviridae), helical filamentous (filoviridae, orthomyxoviridae, paramyxoviridae), or filamentous (arenaviridae, bunyaviridae). the retroviral capsid core may be conical, spherical, or rod-shaped. the morphology of dna viruses is similarly diverse, ranging from icosahedral (enveloped -hepadnaviridae, herpesvridae; nonenveloped -adenoviridae, parvovirdae, polyomaviridae and papillomaviridae) to rodshaped (baculoviridae) or pleomorphic (poxviridae). the viral life cycle is the process of viral replication in a host cell. first, viruses enter the host cell and replicate their genomes. following translation of viral proteins by the host cell machinery, viruses package their genomic material into protective proteinaceous capsids and exit the cell to infect another host cell. nonenveloped viruses consist only of a protein capsid shell enclosing the viral genome and enzymes, while the capsid shell of enveloped viruses is enclosed in a lipid envelope derived from the host cell membrane. regardless of whether the virus is enveloped, its surface must display (glyco)proteins suitable for specific interactions with host cell receptors. in contrast to the surface proteins of nonenveloped viruses, those of enveloped viruses usually serve another function in addition to host cell recognition and attachment. for example, they may enable fusion of the viral and cellular membranes, usually through an interaction with a secondary receptor(s) or co-receptor(s). the fusion domains of viral surface glycoproteins thus can lower the kinetic barrier for the energetically demanding membrane fusion. the viral particle must be sufficiently stable to protect the genome until its delivery into the host cell. simultaneously, it must be metastable, to allow its disassembly and release of the genome for replication in the infected cell at the appropriate time. the energetic barrier that prevents viral disassembly outside the cell is lowered upon infection by structural changes in the viral components. these changes may be induced by binding of a cellular ligand or by changes in the environment, such as ph change upon entering a specific cellular compartment. numerous viruses preassemble immature particles that undergo irreversible (usually proteolytic) transitions into mature structures of fully infectious virions. mutual interactions of viral capsid proteins are typically different from those of cellular proteins, which predominantly create binary interactions. viral structural proteins interact with multiple neighboring partners to form multicomponent macromolecular structures [reviewed in (cheng and brooks, 2015; jayaraman et al., 2016) ]. the economy of the packaged viral genomes due to the limited capsid size implies formation of only a few types of structural proteins, which are usually symmetrically organized [reviewed in (prasad and schmid, 2012; raguram et al., 2017) ]. in the initial stage of infection, viruses must overcome several obstacles. the first is the cellular plasma membrane with an actin cortex. then, they must traffic through dense cytoplasm to reach their final destination for replication and assembly. these pathways are specific for different viruses and often are dictated by the size of the particle and its structure. the viral life cycle can be divided into several common stages, including entry, uncoating, genome replication, genome packaging and assembly, release, and maturation ( fig. 1) . in general, the first phase of viral infection is specific recognition of the target host cell and binding to a surface receptor displayed on the cell membrane. this process is common to both enveloped and nonenveloped viruses. in enveloped viruses, the binding is mediated by viral surface components, typically oligomers of integral glycoproteins. nonenveloped viruses bind receptors through sites or projections on the capsid surface. viruses can use either a single receptor (e.g. tim-1 for hepatitis a virus, gm1 for sv40, cd155 for poliovirus, low-density lipoprotein receptor for human rhinovirus) or multiple receptors with equivalent roles (e.g., nectin-1/2 or hvem for herpes simplex virus 1/ 2, ace or l-sign for sars coronavirus). for other viruses (e.g. hiv, hcv, adenoviruses, rotaviruses, picornaviruses, and some herpesviruses), the presence of at least two cytoplasmic membrane components is required. for example, hiv-1 binds to the primary receptor cd4 and one of two co-receptors (ccr5 or cxcr4) [reviewed in (cossart and helenius, 2014; grove and marsh, 2011) ]. to infect a cell, viruses must overcome the plasma membrane to deliver their genetic material into the cytosol. viruses enter cells by two main mechanisms. a majority of animal viruses, both enveloped and nonenveloped, enter cells by one or two types of endocytosis, such as clathrin-mediated endocytosis (e.g. vsv, influenza a virus, rhinovirus), caveolin-mediated uptake (echovirus, polyoma virus), clathrin/caveolin-independent endocytosis such as caveolar or lipid raft-mediated (e.g. sv40, polyomavirus mouse), or macropinocytosis (e.g. vaccinia virus, respiratory syncytial virus, ebola virus, hiv-1) (blaas, 2016; cossart and helenius, 2014; fields and knipe, 2013; kirkham and parton, 2005; marechal et al., 2001; mayor and pagano, 2007; mercer and helenius, 2009; parton and simons, 2007; rasmussen and vilhardt, 2015; saeed et al., 2010) . these endocytic mechanisms enable the virus to be transported by the cell's machinery through the plasma membrane and to pass through the dense actin cortex. cellular entry of some viruses is coupled with receptor-mediated signaling, resulting in activation of molecules that facilitate virus entry by cytoskeleton reorganization, induction of long-distance transport of the virus-containing vesicles, or actin cortex disassembly. the second type of entry is used by some enveloped viruses (paramyxo-, herpes-, and retroviruses), which upon cell surface receptor binding, infect the cell by direct fusion of viral and plasma membranes at neutral ph. upon internalization, most viruses become trapped and enclosed in vesicles that pinch off the inner side of the plasma membrane and transport their cargo into the cytoplasm. on their way through the cell, these transport vesicles undergo maturation by fusing with other vesicles. to fulfill their task of genome replication, viruses have to escape from these endosomes. the "great escape" is triggered by activation of a fusion or penetration mechanism, such as changes in conditions in the endosomal interior [e.g. ph, ionic environment (e.g. calcium ion concentration), oxido-reducing conditions], changes in membrane composition, and other physico-chemical cues (blaas, 2016; cossart and helenius, 2014; inoue and tsai, 2013) . depending on the requirements for a particular virus, these events can occur in early endosomes (ph 6.5-6.0; e.g. hepatitis c virus, vesicular stomatitis virus), late endosomes (ph 6.0-5.0; e.g. influenza a virus, dengue virus, sars coronavirus), recycling endosomes, macropinosomes, the endoplasmic reticulum, the golgi apparatus, or lysosomes (ph 5.0-4.5) (blaas, 2016; cossart and helenius, 2014; grove and marsh, 2011; inoue and tsai, 2013) . for the majority of animal viruses, the activation of these fusion or penetration mechanisms occurs through conformational changes and structural rearrangements in viral surface proteins and/or the whole virion shell that may destabilize the capsid core. structural rearrangements in enveloped viruses usually mediate fusion of viral and endosomal membranes. in nonenveloped viruses, the structural changes uncover amphipathic or hydrophobic domains that may induce pore formation or disruption of endosomal membranes. to deliver genetic material to the replication site, these mechanisms ultimately release viral capsid structures from endosomal vesicles into the cytosol either by fusing with the endosomal membrane (enveloped viruses) or by penetrating the endosomal membrane (nonenveloped viruses). uncoating is the partial or complete disassembly of the protective capsid shell and/or lipid envelope to liberate the viral genome. for many viruses, this process is closely connected with conformational changes induced by the virus binding to the cell surface receptor; a low ph environment; or changes in oxido-reducing conditions, ion concentrations, or other factors. for enveloped viruses, uncoating involves a loss of the viral membrane by fusion either with the plasma membrane or with intracellular vesicles, followed by stepwise uncoating of the protective capsid shell. in nonenveloped viruses, the uncoating process typically involves conformational changes that result in the weakening of intermolecular interactions, loss of structural proteins, proteolytic cleavage, and so on (fields and knipe, 2013) . depending on the virus, uncoating can take place at the plasma membrane, in the cytoplasm, during endocytosis in early or late endosomes, in lysosomes, in the nucleus, or at the nuclear pore complex (npc). the ssrna genome of retroviruses, which is fully enclosed inside a protective capsid shell, must be reverse transcribed into dsdna and released from the capsid. although it is accepted that hiv-1 uncoating is linked to reverse transcription and nuclear import (ambrose and aiken, 2014) and is controlled by host factors (e.g. cyclophilin a, trim5α), the precise molecular mechanisms that trigger the uncoating remain unknown. several hypotheses have been proposed, including breakage of the capsid shell due to increased inner pressure caused by accumulation of the reverse transcription product (rankovic et al., 2017) , the requirement of intact microtubules and dynein and kinesin motors (lukic et al., 2014) , and phosphorylation of capsid shell protein by the host cell kinase melk (takeuchi et al., 2017) . in contrast to the majority of rna viruses, which replicate in the cytoplasm, most dna viruses (with the exception of large dna viruses including poxviridae, asfarviridae, and mimiviridae) and several negative stranded rna viruses enter the nucleus to replicate their genomes (kobiler et al., 2012; koonin and yutin, 2010) . passive diffusion into the nucleus is suitable for molecules smaller than 9 nm in diameter, but larger structures must enter through the nuclear pore complex (npc), which can accommodate molecules of up to 39 nm (pante and kann, 2002) . translocation through the npc is tightly regulated and requires the presence of a nuclear localization signal (nls) on the passing molecule and nuclear import receptors (importins or karyopherins) (cautain et al., 2015) . due to the diversity of viral particle sizes (from 25 nm to over 300 nm) and structures, viruses have evolved several strategies to export their dna or rna genome into the nucleoplasm. with regard to the npc, these pathways can be divided into npc-dependent and npcindependent mechanisms. due to size limitations, only a few, very small simplified scheme of common stages of viral life cycle targeted by antiviral drugs. these stages including: 1) attachment and entry, 2) uncoating, 3) genome replication, 4) genome packaging and assembly of viral particle and 5) virus release and maturation. m. rumlová, t. ruml biotechnology advances 36 (2018) 557-576 viral capsid particles, such as hepatitis b virus (hbv; 32-36 nm diameter), can pass through the npc (cohen et al., 2011; rabe et al., 2003) in an importin-dependent manner. hbv then disassembles at the nuclear side of the npc (the nuclear basket), releasing its genome into the nucleoplasm (fay and panté, 2015) . the capsid shells or ribonucleoprotein complexes (rnps) of some larger viruses, such as influenza a virus and hiv-1, disassemble within the cytoplasm. the viral genome, released from the shell and complexed with nls-containing components, is then translocated through the npc (ambrose and aiken, 2014; campbell and hope, 2015; cohen et al., 2011; fay and panté, 2015; hutchinson and fodor, 2013) . another mechanism, used by herpes simplex virus (hsv) and adenoviruses, involves cellular (importins, nup) or viral protein-mediated attachment (docking) of the capsid shell at the cytoplasmic side of the npc. this facilitates the passage of genomic dna into the npc either by ejection from an almost intact particle (through the capsid portal in hsv-1) or upon complete disassembly of the capsid shell (in adenoviruses) (kremer and nemerow, 2015; ojala et al., 2000; pasdeloup et al., 2009) . npc-independent mechanisms are used by some retroviruses (e.g. mlv) that enter the nucleus during the mitotic phase of the cell division cycle when the nuclear envelope is dissolved (matreyek and engelman, 2013; roe et al., 1993) . another mechanism, described for parvoviruses, involves partial disruption of the nuclear envelope (cohen and pante, 2005; fay and panté, 2015) . viral genomes can be encoded by various types of nas, as summarized in table 1 for dna viruses and table 2 for rna viruses. by convention, ssdna of equivalent polarity to mrna is designated as the positive (+) strand. the complementary ssnas are of minus polarity (−). the majority of dna viruses replicate in the nucleus, where cellular dna replication and transcription also occur. in contrast, rna viruses usually replicate in the cytoplasm. rna viruses are the only "organisms" that store their genetic information in the form of rna. replication of their genomes is accomplished either by rna-dependent rna synthesis ( fig. 2a , b) [reviewed in (ferron et al., 2017; lu and gong, 2017; menéndez-arias and andino, 2017; pietilä et al., 2017; tao and ye, 2010) ] or through rnadependent dna synthesis (reverse transcription) followed by dna integration, replication, and transcription ( fig. 2c ). as some of these enzymatic activities are not commonly found in uninfected host cells, rna viruses must encode enzymes to aid replication (table 2 ). rna viruses are divided according to the baltimore classification into dsrna viruses (birna-and reoviridae); positive-sense ssrna viruses (corona-, flavi-, and picornaviriade); negative-sense ssrna viruses (filo-, rhabdo-, paramyxo-, and orthomyxoviriade), and ambisense rna viruses (arena-and some members of bunyaviridae) with both positive-and negativesense rnas (nguyen and haenni, 2003 ) (see table 2 ). the nature of the genome not only dictates the mechanism of replication, but also has other important consequences. the genome of (+)rna viruses may serve directly as mrna for production of viral proteins. therefore, the mere introduction of genetic material (e.g. in exocytic vesicles) may result in productive infection. the rna polymerases that copy the genetic material of rna viruses are error-prone, which provides considerable genetic flexibility and the propensity to evolve drug-resistant mutants. these features are amplified in viruses with segmented genomes that undergo reassortment. in viruses with segmented genomes (orthomyxoviridae, arenaviridae and bunyaviridae), each segment is transcribed in an autonomous transcription-replication unit by viral rna polymerase that binds to the 5′ end cap structure. of note, the genomic rna (grna) is capped by a unique mechanism called cap-snatching (ferron et al., 2017) , in which the cap is cleaved from cellular mrna and transferred onto the viral grna by a subunit of viral rna polymerase (pflug et al., 2017) . some dsrna viruses (birnaviridae and reoviridae) also contain segmented genomes. upon replication, these viruses must ensure stoichiometric incorporation of single copies of each grna segment into new particles. this is guided by specific packaging signals on each segment of grna that interact with positively charged domains in the capsid proteins [reviewed in (isel et al., 2016; pohl et al., 2016) ]. although different models of packaging have been proposed for various segmented genome viruses, a common feature is co-assembly of the capsid proteins with the grna and rna polymerase. the genomes of dna viruses come in a considerable variety of sizes and shapes, from small ss to large ds molecules that may be linear or circular. the size range of these genomes (from 1.8 kb to 1200 kb) reflects the necessity for some viruses to encode specific proteins required for viral replication. small-genome dna viruses (polyoma-, papilloma-, and parvoviruses) use only host cell enzymes for replication and transcription. the only exceptions are some hepadnaviruses (e.g. hepatitis b virus) that despite having small genomes (approximately 3 kb), encode their own specific dna polymerase/reverse transcriptase that reverse transcribes pregenomic rna (pgrna) into genomic viral dna (fig. 2d , ii) (beck and nassal, 2007) . viruses with intermediate-size genomes (up to 35 kb) (e.g. adenoviruses) encode their own dna polymerase for genome replication, but they usually utilize cellular rna polymerases ii and iii for transcription (fields and knipe, 2013) . viruses with large genomes (larger than 100 kb) (e.g. herpesviruses) encode proteins for replication, including dna polymerase and dna helicase-primase, as well as some enzymes necessary for biosynthesis of deoxyribonucleotide triphosphates (dntps) and several transcription factors (boehmer and nimonkar, 2003) . poxviruses (e.g. vaccinia virus), are another type of virus with large dsdna genomes, and their replication, transcription, and translation take place entirely in the cytoplasm within discrete juxtanuclear sites called virus factories (moss, 2013) , (fig. 2d , iii). these viruses encode all enzymes and specific factors necessary for genomic replication and transcription. with their own replicative machinery, large-genome dna viruses can replicate in nondividing cells. in contrast, the replication of small-genome dna viruses, which depends on cellular dna polymerases, must occur simultaneously with the s-phase of the cell cycle (e.g. parvoviruses), or must express some viral protein/ oncogene to re-program the host cell-cycle regulatory proteins p53 or retinoblastoma protein (prb), triggering entry into the s-phase (e.g. polyomaviruses and papillomaviruses). by affecting the g1/s checkpoint (controlled by p53 and prb), the viruses ensure the production of host enzymes required for viral replication (fields and knipe, 2013) . production of viral proteins of dna viruses with intermediate and large size genomes is divided into early and late phases. in the early (prereplicative) phase, nonstructural proteins required for dna replication are translated. the late phase, during which the late structural proteins needed for assembly are translated, begins after viral dna replication ( fig. 2d , i). depending on the species, viruses assemble either in contact with the cellular membrane or independent of the membrane in either the nucleus or cytoplasm. the membrane-independent route is used by nonenveloped viruses and a few enveloped viruses (e.g. orthomyxoviruses, herpesviruses, and some retroviruses) that acquire the membrane envelope after intracellular assembly during budding from the cell. the limiting step for nuclear assembly is the size of npcs, which are large enough to transport rna and import proteins required for assembly into the nucleus; however, npc size limits the transport of larger assemblies. therefore, some viruses form assembly intermediates in the nucleus. these structures are then exported to the cytoplasm, where they come together to form viral particles. nuclear export is specific and depends on the presence of nuclear export signals (nes) in the transported proteins. one example of a nonenveloped icosahedral virus that assembles in the nucleus is adenovirus, the assembly of which has been studied intensely due to its potential use in gene therapies [reviewed in (ahi and mittal, 2016) ]. recent data suggest that upon accumulation of multiple copies of adenoviral dsdna genomes, coordinated assembly and packaging occur by two interlinked mechanisms that involve both capsid proteins and core components (condezo and san martín, 2017) . the assembly occurs in the so-called peripheral replicative zone with the assistance of scaffolding proteins that facilitate formation of adenoviral particles but are excluded from mature viruses. adenoviruses are finally released upon lysis of the infected host cell. orthomyxoviruses and herpesviruses are enveloped viruses that assemble their nucleoproteins in the nucleus. herpesviruses package their dsdna genome as head-to-tail concatemers and assemble icosahedral procapsids on scaffold proteins in the nucleus [reviewed in (heming et al., 2017) ]. however, subsequent steps of herpesvirus assembly proceed in the cytoplasm. the preassembled procapsid is too large to pass through the npc, but it exits the nucleus by viral proteindriven vesicular transport across the nuclear inner membrane leaflet. thus, the herpesvirus acquires an initial envelope from the inner nuclear membrane [for review see (fields and knipe, 2013; hellberg et al., 2016) ]. next, the herpesviral membrane fuses with the outer nuclear membrane, and the naked particle is released from the nucleus into the cytosol. here, the virus acquires tegument (a protein layer between the capsid and the envelope) and other proteins. final herpesvirus envelopment occurs at the golgi membrane containing the viral glycoproteins (fields and knipe, 2013) . preassembled orthomyxoviral ribonucleoproteins, upon their export from the nucleus, are driven to the plasma membrane to which they attach through electrostatic interactions of the matrix protein m1 with membrane phosphatidylserine. the virions then assemble simultaneously with budding during which they also acquire ha, na and m2 membrane proteins. poxviruses undergo an even more complicated pathway. they are enveloped with multiple membranes acquired from er/intermediate compartments and golgi or early endosomes (moss, 2015 (moss, , 2016 . these membranes also provide the poxviruses with their membrane proteins. most viruses assemble upon interaction of their structural proteins with cellular membranes. the target membrane for assembly differs according to the virus type. flaviviruses assemble at the surface of the er and then upon their budding into the er lumen, the immature particles are then transported into the tgn. some viruses, such as coronaviruses, assemble at the er-golgi intermediate compartment [reviewed in (ujike and taguchi, 2015) ]. the assembly of bunyaviruses occurs concurrently with replication of grna segments in virus factories located along the golgi (guu et al., 2012; strandin et al., 2013) . the presence of membrane glycoproteins at the golgi membrane determines the sites where assembling bunyavirus particles bud into the golgi lumen, similar to other enveloped viruses. numerous viruses, including paramyxoviruses (cox and plemper, 2017) , orthomyxoviruses (pohl et al., 2016) , alphaviruses (jose et al., 2009) , rhabdoviruses (okumura and harty, 2011) , and most retroviruses (freed, 2015) , assemble underneath the cytoplasmic membrane, which facilitates assembly by providing a scaffolding function. the virus then acquires a lipid envelope through budding across the plasma membrane. during this process, it also gains the envelope glycoproteins (env) that are anchored in the plasma membrane by hydrophobic transmembrane domains. env reaches the plasma membrane by a cellular secretory pathway upon synthesis in the er and subsequent glycosylation in the golgi. usually, a specific interaction between viral structural proteins and glycoproteins is required. this may be either direct or mediated through the interaction with matrix protein (e.g. in (-)rna viruses such as ortho-and paramyxoviruses or rhabdoviruses). most retroviruses, including hiv (so-called morphogenetic c-type), also assemble at the plasma membrane of the host cell. the interaction of the structural polyprotein precursor (gag) with the plasma membrane is usually facilitated by a bipartite signal in the n-terminal domain of gag (i.e. matrix protein) comprising both the basic patch and n-terminally linked myristoyl residue (added co-translationally to gag). in contrast to c-type assembly at the membrane, morphogenetic b/d-type retroviruses assemble at pericentriolar sites where gag polyproteins are transported along microtubules by dynein molecular motors (sfakianos et al., 2003; vlach et al., 2008) . for both morphogenetic pathways, the packaging of grna facilitates assembly. (+)rna viruses have adopted a mechanism of extensive rearrangement of intracellular membranes to provide a milieu for virus replication and assembly. this mechanism effectively protects dsrna intermediates from degradation by the host cell machinery (delgui and colombo, 2017; jackson, 2014) . this process has been well-documented for poliovirus, in which the newly formed membranous structures exclusively serve for virus production. various types of vesicles that are formed upon viral infection have different roles in viral replication (rossignol et al., 2015) . some nonenveloped mammalian viruses, such as reoviruses, assemble in the cytosol in so-called viroplasms or viral factories into icosahedral particles consisting of three concentric layers encircling segments of genomic dsdna (benavente and martinez-costas, 2006; jones, 2000; shah et al., 2017) . rotavirus seems to be the only exemption in the reovirus family, as it enters the endoplasmic reticulum to gain its outer protein shell (trask et al., 2012) . numerous viruses assemble from polyprotein precursors that must be specifically cleaved by a viral protease to generate infectious particles. this mechanism, which is an irreversible step in the virus life cycle, ensures equimolar packaging of structural proteins and proportional co-assembly of viral enzymes in the form of precursors. upon proteolytic release, the liberated proteins may undergo different trafficking pathways or fulfill various functions in the virus. maturation changes the energy of interaction forces among those interfaces required for intracellular assembly of immature particles to those suitable for viral stability in the environment and for disassembly and uncoating of genetic material for replication [reviewed in (veesler and johnson, 2012) ]. in poliovirus, the autocatalytic and subsequent viral proteasemediated cleavage of p1 precursor protein allows formation of pentamers that interact with grna. additional cleavage of vp0, yielding vp2 and vp4, is required to form infectious poliovirus particles. in retroviruses, proteolytic processing is initiated by the autocatalytic liberation of viral protease, which subsequently cleaves the polyproteins to trigger major structural rearrangements in the virus and release of other viral enzymes (reverse transcriptase and integrase) and structural proteins. in herpesviruses, maturation involves proteolytic processing of the scaffolding protein and recruitment of tegument proteins that stabilize the particle and mediate interactions with the membrane during envelopment (gibson, 2008; tandon and mocarski, 2012; tandon et al., 2015) . adenoviruses undergo a maturation process that involves processing of six structural components of the core and one non-structural precursor that initiates replication of gdna (gaba et al., 2017) . one interesting feature of adenoviral maturation is that dna is required as a co-factor for protease activity. this means that maturation occurs only in particles that have packaged gdna (mangel and san martín, 2014) . flaviviruses form icosahedral particles upon budding into the neutral milieu of the er. the particles then translocate to the more acidic environment of the trans-golgi network. this ph shift results in disassembly of the immature lattice and extensive rearrangement of the flaviviral particle. this is connected with the exposure of a viral structural glycoprotein (prm) that is specifically processed by the , retroviruses (c) and dna viruses (d) (the schemes a-c were modified from: (ahlquist, 2006) . a: following endocytosis, the genome of (+)rna viruses may directly serve as mrna for translation of virus encoded proteins. among them, there are proteins of rna-replication machinery that recruit (+)rna into a membrane-associated replication complex. the genomic rna is replicated by using (-)rna template, which is transcribed in a low copy number amplified (+)rna is then packaged into newly assembled virions that exit the host cell either through secretion or cell lysis. b: upon the virus attachment, a core containing both grna and virus encoded rna polymerase is delivered into the cytoplasm by endocytosis. cytoplasmic transcription of the (-)rna template provides (+)rna that serves as mrna for translation of viral proteins. in dsrna viruses, the (+)rna is packaged into new cores which undergo maturation by synthesizing (-) rna (dotted strand) and acquiring surface proteins. in the (-)rna viruses, the (+)rna strand is transcribed into genomic (-)rna in the cytoplasm and then packaged. the new virions egress the cell either through secretion or cell lysis. c: following fusion of viral and cell plasma membrane, the retroviral core is released into cytosol, rna genome is transcribed into dsdna by viral reverse transcriptase concomitantly with uncoating of the viral core, ds dna is transferred into nucleus where it is integrated into host cell genome by viral integrase, following translation of retroviral structural and enzymatic polyproteins, the unspliced grna is packaged into the immature particles that usually assemble at the plasma membrane and the viral particles bud from the cell. d: three mechanisms (i.-iii.) of dna viruses replication are shown: (i): following entry and uncoating, the dna genome is transported to the nucleus; products of early genes (regulatory proteins, transcription factors) regulate the synthesis of viral enzymes (e.g. dna polymerase) required for genome replication; expression of late genes encoding structural capsid proteins in the cytosol, they are then transported into nucleus where packaging and pre-assembly take place; preassembled procapsids exit the nucleus and leave the cell (e.g. herpesviruses). (ii) unique replication cycle of hepatitis b virus (hbv): following entry, the viral particle is internalized by endocytosis and the nucleocapsid is released into the cytoplasm; the genome (relaxed circular rcdna) is transported into the nucleus, where it is converted to a covalently closed circular form (cccdna); which serves as a template for transcription of pregenomic rna (pgrna) for translation of the core protein and the viral polymerase and three subgenomic mrnas used for translation of regulatory and envelope proteins; following viral transcription and translation, the hbv core proteins self-assemble in the cytoplasm into viral nucleocapsid with concurrent incorporation of pgrna and hbv polymerase, pgrna is reversely transcribed into a rcdna within the nucleocapsid; nucleocapsid containing rcdna can either re-enter the nucleus to amplify cccdna, or can be enveloped by hbv envelope proteins in the endoplasmic reticulum. the particles are then secreted from the infected hepatocyte by exocytosis. (iii) upon entering the cell, the replication, transcription and translation take place entirely in the cytoplasm, within discrete juxtanuclear sites called virus factories (e.g. poxviruses) adapted from: ahlquist, p., 2006. parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses. nat rev microbiol 4(5), 371-382. cellular protease furin in the trans-golgi network. the liberated globular heads of prm remain attached at the low ph, but are released when the virus enters neutral body fluids (rey et al., 2017) . one frequently used mechanism of release of nonenveloped and some enveloped viruses is lysis of the infected cell. this is the simplest release mechanism, although the transition to lysis from latent infection is delicately regulated (aneja and yuan, 2017; levings and roth, 2013; schmiedel et al., 2016) . however, viruses that are usually lysogenic may also use alternative release pathways (bird and kirkegaard, 2015a) . these include non-lytic spread of viruses mediated by vesicles, which has been observed for poliovirus (bird and kirkegaard, 2015b; jackson et al., 2005) , coxsackievirus (alirezaei et al., 2012) , and hepatitis a virus . another possibility is that vesicles released from a cell infected with (+)rna viruses contain naked viral grna. this (+)grna-containing vesicle functions itself as an infectious agent as it is transferred to another cell (bird and kirkegaard, 2015a) . the standard way for enveloped viruses to leave the host cell is budding, which includes membrane extrusion and separation of the viral and cellular membranes (so-called pinching off). the lipid envelope layer acquired during viral particle budding through the plasma membrane protects the virus particle. there are three basic mechanisms of budding: i) mediated by envelope glycoproteins, such as the e protein of coronaviruses; ii) independent of glycoproteins, in which the viral structural proteins trigger the extrusion of the plasma membrane, such as retroviral budding in which strong interactions between the gag n-terminal domain (matrix protein) and the plasma membrane induce bulging of the membrane; and iii) requiring interaction between the viral glycoproteins and the capsid for membrane extrusion, as in alphaviruses. the final step that results in the separation of virus from the cell surface is pinching off the particle. this is a controlled process that involves both viral protein domains and cellular factors. during this process, viruses apparently use cellular machinery similar to that used during the last step of cell division (the release of the daughter cell) called endosomal sorting complex required for transport (escrt) [reviewed in (campsteijn et al., 2016; hurley, 2015; olmos and carlton, 2016; scourfield and martin-serrano, 2017) ]. direct interactions of numerous viral domains with escrt complex subunits have been identified (bieniasz, 2006) . in retroviruses, short specific amino acid sequences (ptap or psap) interact with the escrt components, and the interaction of hiv with the escrt proteins nedd4 and alix is wellknown (freed, 2002; fujii et al., 2007; gomez and hope, 2005) . however, this mechanism has also been adopted by other viruses that interact with the same escrt components, such as filoviruses (han et al., 2015; jasenosky and kawaoka, 2004; liu and harty, 2010) . interactions with escrt proteins have also been reported for vesicular stomatitis virus luyet et al., 2008 ), rhabdovirus (taylor et al., 2007 , arenaviruses (ziegler et al., 2016) , picornaviruses , paramyxoviruses (park et al., 2016) , and hepatitis c virus, a representative of the flavivirus family (falcon et al., 2017) . the typical release pathways used by viruses may vary under some conditions. for example, during chronic infection, numerous viruses use alternative cell-to-cell transmission that may help the virus avoid host neutralization (hulo et al., 2017) . syncytium formation, an hivinduced cell fusion, was recognized in the early 1990s (callahan, 1994) . another type of cell-to-cell transmission through tight junctions was shown for hiv (hübner et al., 2009; wang et al., 2017) and murine leukemia virus (sherer et al., 2010) . receptors on tight junctions that specifically recognize hepatitis c virus (carloni et al., 2012; ploss et al., 2009 ) and reovirus (barton et al., 2001) have been identified. some viruses are able to modify cellular pathways to reprogram both the synthesis and metabolism of lipids and membrane compartmentalization for their transmission and to evade cellular defense mechanisms (mazzon and mercer, 2014) . despite a general understanding that poliovirus spreads through cellular lysis, it was recently found that it may also be transferred between cells by an autophagy-dependent mechanism, called autophagosome-mediated exit without lysis (bird and kirkegaard, 2015b; bird et al., 2014; lai et al., 2016; richards and jackson, 2012) . similar mechanisms have been described for varicellazoster virus and human cytomegalovirus (grose et al., 2016; meier and grose, 2017) . poxviruses encode several proteins that block the apoptotic cellular response to the presence of their dsdna in the cytoplasm. this allows cell-to-cell passage of poxviruses by a mechanism known as apoptotic mimicry (amara and mercer, 2015; nichols et al., 2017) . in this process, enveloped viruses expose surface phosphatidylserine to mimic apoptotic bodies. these cells are then macropinocytosed by either dendritic cells or macrophages (mercer and helenius, 2010) . among the plethora of compounds designed to inhibit infectious viruses, only a few (< 100) have been approved for clinical use (de clercq, 2004; de clercq and li, 2016) . nevertheless, some effective antiviral drugs have been on the market for several decades, such as the anti-influenza a virus drug amantadine, marketed under the trade name symmetrel (by dupont), which was approved in 1966. in 1982, burroughs-wellcome introduced acyclovir as an inhibitor of herpesviruses. its remarkable specificity is connected with its activation by viral thymidine kinase-catalyzed phosphorylation. however, due to development of drug resistance in a number of viruses, especially rna viruses, there is a continuous need to design and test new inhibitors, preferably targeting different steps of viral life cycles. here, we provide insights into the world of biochemical and cell-based assays that were developed to test antivirotics targeting various steps in the viral life cycle. different types of assays, including cell-cell fusion assays, cell-virus fusion assays with pseudotyped viral particles, and in vitro biochemical assays have been developed to screen inhibitors of viral entry. in enveloped viruses, entry is initiated by fusion of the viral envelope with the target cellular membrane. the entry mechanism has been well-described for hiv-1, in which it is mediated by env glycoprotein, consisting of transmembrane gp41 and surface gp120 subunits. binding of gp120 to its cellular receptor, cd4, and to one of two co-receptors, cxcr4 or ccr5, triggers a refolding of gp41 that promotes fusion of the viral and cellular membranes. the refolding involves oligomerization of the extracellular n-and c-terminal heptad repeat (hr) domains of gp41, which leads to the formation of a 6-helical bundle [reviewed in (melikyan, 2008) ]. jiang et al. established an in vitro system to quantify formation of the hiv-1 gp41 6-helical bundle (jiang et al., 1999 ). in their system, the bundle is formed by mixing equimolar concentrations of peptides derived from the n-and c-hr regions of gp41. elisa using the monoclonal antibody nc-1, which specifically recognizes and binds an epitope formed on the gp41 6-helix bundle but not the individual peptides, enables screening for compounds that prevent formation of fusion-active gp41. for hts of hiv-1 fusion inhibitors, this method was modified to use a fluorescence-linked immunosorbent assay (flisa), in which the c-hr peptide was replaced with fitc-conjugated c-hr peptide (boyer-chatenet et al., 2003) . cell-based assays are routinely used to screen viral entry inhibitors. high throughput formats have been developed for screening of hiv-1 fusion inhibitors. cell-cell fusion assays involve two types of cells: effector cells that stably (bradley et al., 2004) or inducibly (herschhorn et al., 2011; ji et al., 2006) express hiv-env glycoprotein and target cells that express cd4 and either cxcr4 or ccr5. co-cultivation of these cells leads to hiv-1 env-mediated cell membrane fusion, resulting in formation of multinucleated syncytia. membrane fusion induces expression of a reporter protein such as luciferase (herschhorn et al., 2011; ji et al., 2006) or β-galactosidase (bradley et al., 2004) . one such assay enables determination of both the efficiency and specificity of fusion inhibitors (herschhorn et al., 2011) . this approach uses effector cells that express both hiv-1 env and the renilla luciferase (r-luc) reporter protein using inducible tetracycline-controlled transactivator (tta) and target cells that express the hiv-1 receptor (cd4) and coreceptor (ccr5) and contain the firefly luciferase (f-luc) reporter gene under the control of a tta-responsive promoter. upon fusion of the effector and target cells, tta enters the target cells and activates the expression of the f-luc reporter. the inhibition of fusion of cellular membranes is determined as a decrease in f-luc luminescence, and the inhibitor specificity is measured as the r-luc activity (herschhorn et al., 2011) . based on an hiv-1 cell-cell fusion method that uses a computercontrolled digital image analysis system for automatic quantitation , a modified method was developed to screen inhibitors targeting mers-cov s protein (lu et al., 2014) . to test potential fusion inhibitors, effector cells stably expressing the mers-cov spike protein s2s and egfp were used to mediate fusion with target cells expressing the dpp4 receptor (lu et al., 2014) . virion-based fusion assays are another category of cell-based fusion assays. one such approach is based on production of chimeric hiv-1 virions carrying β-lactamase-vpr chimeric protein (blam-vpr). chimeric hiv released into the cell culture media is isolated by ultracentrifugation and used to infect target cells. entry of the virions into the cytoplasm is detected by cleavage of a fluorescent substrate by βlactamase. the fluorescence shift corresponds to the fusion efficacy and is measurable by fluorescence microscopy, flow cytometry, or fluorometric plate reader (cavrois et al., 2002; cavrois et al., 2004 cavrois et al., , 2014 . modification of this assay by constructing pseudotyped hiv-1 virions in which hiv-1 env was replaced with ebola virus glycoprotein (gp) has also been described (yonezawa et al., 2005) . tscherne et al. developed an approach to monitor viral entry using the blam reporter (tscherne et al., 2010) . their assay uses influenza virus-like particles (vlps) bearing the influenza membrane proteins hemagglutinin and neuraminidase. blam tagged with influenza matrix protein (m1) is incorporated into the vlps and delivered into target cells. upon release, blam can be detected by flow cytometry, microscopically, or fluorometrically. a rapid cell-based hts method was developed to assess sars-cov entry inhibitors (zhou et al., 2011) . this dual envelope pseudovirion (dep) assay employs two hiv pseudoviruses: one encodes an envelope protein from the target virus and firefly luciferase and the second encodes a control, unrelated viral envelope protein and renilla luciferase. reporter expression is determined with the dual-glo luciferase assay system (promega). inclusion of the unrelated envelope protein greatly reduced false positive hits (zhou et al., 2011) . the method was further used to screen compounds that inhibit entry of filoviruses, including ebola virus . a similar approach employing four pseudotyped hiv viruses, carrying marburg virus glycoprotein, hemagglutinin and neuraminidase isolated from a/goose/qinghai/59/05 (h5n1) influenza virus, ebolavirus zaire envelope glycoprotein, and lassa virus envelope glycoprotein, has been used for entry inhibitor screening . this screening identified multiple compounds with potent inhibitory activity against entry of both marburg and ebola viruses in human cancer cell lines, and confirmed their anti-ebola activity in human primary cells . other pseudotyped viral assays have been used to screen entry inhibitors of sars-cov, ebola, hendra, and nipah viruses. to establish infection, the glycoproteins of these viruses must be processed by the host intracellular lysosomal protease cathepsin l (catl). hts resulted in identification of several inhibitors that block the cleavage of viral glycoprotein but not catl itself (elshabrawy et al., 2014) . until recently, the development of anti-hbv therapeutics had been limited by the lack of an in vitro infection system. several aspects of the hbv life cycle have been elucidated using in vitro production of hbv particles after transfection of human hepatoma cell lines (hepg2) with recombinant hbv dna and by establishment of hepatoma cell lines with the entire hbv genome integrated, such as the hepg2.2.15 cell line (ladner et al., 1997; sells et al., 1987; sureau et al., 1986) . in addition to differentiated human (phhs) (gripon et al., 1988) and tupaia belangeri (glebe et al., 2003) hepatocytes, the heparg cell line, which exhibits hepatocyte-like characteristics, also supports hbv replication (gripon et al., 2002) . the identification of sodium taurocholate cotransporting polypeptide (ntcp) as an hbv receptor by yan et al. opened possibilities to use ntcp-complemented hepg2 cells not only for studies of hbv replication mechanisms but also for hts of inhibitors (yan et al., 2012) . in an infection competition assay, hbv particles were isolated and used to infect heparg and phhs cells that had been pre-incubated with hbv envelope protein-derived peptides to test their potential activity to block hbv infection. twelve days after infection, viral rnas were quantified by northern blot (gripon et al., 2002 (gripon et al., , 2005 . using this assay, researchers identified a peptide that specifically prevents hbv and hdv entry into heparg and phhs cell lines (gripon et al., 2005) , primary cultures of tupaia hepatocytes (glebe et al., 2005) , and cells in vivo (lütgehetmann et al., 2012; petersen et al., 2008; volz et al., 2013) . recently, a phase iia clinical trial of a first-in-class entry inhibitor (myrcludex-b) that functions as an ntcp inhibitor was promisingly completed (bogomolov et al., 2016) . development of cell culture systems producing hcv pseudoparticles (hcvpp) and infectious hcv particles (hcvcc) has shed light on hcv interactions and enabled discovery of antiviral drugs and vaccines (colpitts et al., 2016) . hcvpp consist of hcv e1 and e2 glycoproteins enveloping a retroviral particle that packages gfp mrna during assembly (bartosch et al., 2003) . the entry efficiency of hcvpp can be quantified by facs analysis as the number of gfp-positive cells. use of this screening system led to discovery of a triazine derivative that blocks the entry of hcv (baldick et al., 2010) . production of hcvcc is a robust system to generate infectious hcv in naïve cells (kato et al., 2006; zhong et al., 2005) . the anti-hcv activity of hundreds of compounds approved for a wide variety of indications was determined immunochemically with anti-hcv e2 antibody in 96-well plate format. over thirty compounds displayed anti-hcv activities, most of which were directed against hcv entry (gastaminza et al., 2010) . the majority of viruses enter cells by endocytosis. unfortunately, there are no suitable experimental techniques for endosome handling, making it difficult to study early steps in the viral life cycle such as uncoating and capsid disassembly. viruses that enter cells by direct membrane fusion, such as hiv-1, are an exception. there are several methods available to monitor and quantify hiv uncoating. as some of these were reviewed recently by campbell and hope (campbell and hope, 2015) , we will discuss them very briefly. two main techniques are used in these assays: ultracentrifugation or utilization of hiv-1 specific cellular restriction factors. the "in vitro core-stability assay" is based on ultracentrifugation of released hiv-1 virions through a detergent layer, where the viral membrane dissolves, into a sucrose gradient, where the viral cores are concentrated (shah and aiken, 2011) . the "fate of capsid" assay uses ultracentrifugation through a sucrose cushion to separate the hiv-1 core from a whole cell lysate prepared shortly after infection (stremlau et al., 2006) . the "csa-washout assay" involves specific cellular factors (trim-cyclophilin) that restrict hiv-1 infection by binding to the capsid core and cyclosporine a (csa), which can effectively turn off restriction (hulme et al., 2011) . recently, a novel entry/uncoating assay (eurt), an alternative to blam-vpr (described in section 3.1), was reported (da silva santos et al., 2016). it quantifies the protein product of a virion-packaged mrna reporter upon uncoating. a method to monitor the uncoating/disassembly of the capsid of influenza a virus, which enters the cell by endocytosis, also is based on ultracentrifugation (stauffer et al., 2016) . purified virions are separated using velocity gradient centrifugation through a two-layer glycerol gradient. similar to the "in vitro core stability" assay for hiv, the sedimenting viruses migrate through the detergent-containing layer of the gradient, which dissolves the membrane to release the viral core. moreover, modification of the detergent-containing glycerol layer-for example, by lowering ph, changing ionic strength, or adding putative viral uncoating factors-enables study of the factors or compounds that affect viral uncoating in vitro. depending on the virus type, either dna or rna polymerases replicate the viral genome. thus, these enzymes play a key role in viral life cycles. reverse transcriptase, an rna-dependent dna polymerase of retro-and hepadnaviruses, is unique among nucleic acid polymerases. despite the different mechanisms of viral replication, polymerases, which are essential for all viruses, are excellent targets for antiviral therapies. polymerase inhibitors represent the vast majority of clinically approved antiviral drugs, followed by protease inhibitors, immunostimulators, entry inhibitors, and integrase inhibitors [reviewed in (de clercq and li, 2016) ]. polymerases continue to be a preferred target for newly designed inhibitors (clercq, 2008; de clercq and li, 2016) . polymerase inhibitors may be nucleoside and nucleotide analogs, pyrophosphate analogs, or nonnucleosides, such as allosteric inhibitors (de clercq and li, 2016; öberg, 2006) . non-specific approaches such as plaque assays were initially used to monitor the effectiveness of polymerase inhibitors (tino et al., 1993; tisdale et al., 1993) . the activity of these inhibitors also can be evaluated based on their ability to prevent the cytopathic effects of the virus (zhang et al., 2017) . more straightforward assays involve measurement of incorporated radio-labeled nucleotides, which directly reflects the polymerase activity (coates et al., 1992; hirashima et al., 2006; joyce, 2010) ; these include hts methods using homopolymeric polycytidylic acid and [ 3 h]-gtp (amraiz et al., 2016) . alternatively, the pyrophosphate released during the polymerase reaction can be measured luminometrically by combining the primer extension with the commercial piper assay (malvezzi et al., 2015) . the pyrophosphate anion also can be detected with dna-attached magnetic nanoparticles (tong et al., 2015) or quantum dots (chai et al., 2015) . frequently used fluorescence methods avoid the use of radiochemicals. these methods exploit a fluorescent label, such as dsdna binding dyes like sybr green or pi-cogreen (driscoll et al., 2014; holden et al., 2009; zipper et al., 2004) , or fret between two nucleotides (schwartz and quake, 2009; weiss, 2000) . numerous kits for quantification of products of both dna and rna polymerases, including reverse transcriptase, are commercially available (bustin, 2000) . quantitative real-time pcr is a preferred method to monitor the activity of dna polymerases (cellular as well as viral) in the presence of inhibitors (beadle et al., 2016; zweitzig et al., 2012) , and quantitative real-time reverse-transcription pcr has become standard for screening inhibitors of viral rna polymerases okon et al., 2017; pelliccia et al., 2017; zhang et al., 2017) . white et al. described a hts method using a microfluidic apparatus combined with digital pcr for single-cell analysis (white et al., 2013) . in their method, a fluorescently labeled template also serves as a primer, due to stem formation. it emits a measurable polarization signal both upon binding of the polymerase and extension (mestas et al., 2007) . the assay has been used for hts of inhibitors of poliovirus rna polymerase (campagnola et al., 2011) . when available, viral genomes modified with reporter genes can be used for cell-based luminescent or fluorescent screening of viral inhibitors (beadle et al., 2016; edwards et al., 2015; fenaux et al., 2016; feng et al., 2014; lo et al., 2017; madhvi et al., 2017; wang et al., 2015c) . this approach can be extended to screen inhibitors of other enzymes. in the aptamer-based approach, a dna template encodes rna of interest joined to a fluorescence module and ribozyme sequence. when transcribed, the fluorescence module is released and detected (höfer et al., 2013) . hcv uses an rna-dependent rna polymerase (rdrp). a unique cell-based assay for monitoring hcv rna polymerase (ns5b) activity, based on the innate immune signaling molecule retionic acid-inducible gene i product (rig-i), has been developed (ranjith-kumar et al., 2011) . the rig-i-like receptors are cytosolic proteins that recognize viral rna and induce production of proinflammatory cytokines and interferon-activated genes (jensen and thomsen, 2012) . rig-i triggers cytokine production stimulated by various viral rnas from different families, including flaviviridae, paramyxoviridae, rhabdoviridae, orthomyxoviridae, and arenaviridae, as well as ebola virus rna (jensen and thomsen, 2012) . the assay is based on recognition of hcv rna produced by active ns5b by rig-i, followed by rig-i-mediated activation of firefly luciferase expression controlled by the interferon b promoter. renilla luciferase expression is used for normalization of transfection efficacy. the viral grnas of some rna viruses are modified at the 5′ ends with cap structures, which may be either acquired from the host cell mrna (e.g. in influenza virus) or newly synthesized (e.g. flaviviruses). the methylation of viral grna mimics that of cellular mrna cap structures to enhance the chances of the virus to escape from cellular defense mechanisms and also to increase the efficiency of translation of viral proteins. virus-specific methyltransferases are thus a promising therapeutic target. virus-encoded methyltransferases have been identified and characterized in flaviviruses such as zika virus coutard et al., 2017; duan et al., 2017; munjal et al., 2017; zhao et al., 2017) , west nile virus, and dengue virus (dong et al., 2012) ; rhabdoviruses such as vesicular stomatitis virus (vsv) (rahmeh et al., 2009 ); coronaviruses such as sars (wang et al., 2015b) ; and roniviruses (zeng et al., 2016) . in flaviviruses, the n-terminal part of ns5 methyltransferase catalyzes cap formation via both guanine n-7 and ribose 2′-oh methylations at the 5′ end of grna, and the c-terminus of the enzyme is responsible for the rna polymerase activity (ray et al., 2006; zhao et al., 2015) . the c-terminal part of the sars nsp14 protein exhibits guanine n7-methyltransferase activity, forming the grna cap, while the 3′-5′ exoribonuclease activity of the n-terminus enhances the fidelity of viral replication (case et al., 2016; minskaia et al., 2006) . in vsv, the methyltransferase activity resides in the conserved region vi of the multifunctional large polymerase protein (li et al., 2005; ma et al., 2014) . some cellular methyltransferases regulate viral infections, as shown for herpes simplex virus, for which epigenetic control is involved in viral latency (cliffe and wilson, 2017) . inhibition of human histone methyltransferases such as histone-lysine n-methyltransferase (ezh2/ 1) (arbuckle et al., 2017) or lysine-specific demethylase-1 (lsd1) induces antiviral signaling pathways (liang et al., 2009) . the inhibitory effect of histone demethylase activity has been demonstrated for human cytomegalovirus (gan et al., 2017) and herpes simplex virus (hill et al., 2014; liang et al., 2013) . the activity of purified recombinant methyltransferases can be determined by measuring the methylation by-product s-adenosyl homocysteine (sah) using commercially available kits. in one such assay, conversion of s-adenosyl methionine (sam) to sah is monitored luminometrically via luciferase reaction, in which measurable atp is generated through a sequence of reactions with mtase-glo™ reagent (promega). the epigeneous™ methyltransferase kit (cisbio bioassays) is based on competition of produced sah with fluorescently labeled sah (sah-d2 tracer) for binding to a terbium cryptate-labeled anti-sah antibody. the decrease in fret between the tracer and antibody is then evaluated. elisa using anti-5-methylcytosine antibody can be used to quantify methylation of immobilized cytosine-rich dna substrate (e.g. epiquik™ dna methyltransferase activity/inhibition assay kit; epigentek). this method was modified with homogenous time resolved fret (degorce et al., 2009) to screen a library of inhibitors against sars-cov nsp14 . flaviviral and human cap n7-mtases have been screened with radioactive assays using 3 h-labeled sam and gpppac4 or 7m gpppac4 synthetic rnas. the 3 h methyl transferred onto deae filter-bound rna can be measured by scintillation after multiple washings to remove unincorporated 3 h-sam . alternatively, in vitro transcription can be carried out using 3 h-sam. the radioactively labeled products are then resolved by thin-layer chromatography and developed using a phosphorimager (li et al., 2007) . a yeast cell-based method was established based on the finding that coronavirus methyltransferase can functionally replace a methyltransferase essential for yeast viability sun et al., 2014) . in this method, sun et al. constructed recombinant yeasts producing the viral methyltransferase instead of the yeast one (sun et al., 2014) . this strain was used for a hts of methyltransferase inhibitor activity that negatively correlated with the cell density after 20 h incubation. virus-encoded atpase-driven helicases have been identified in numerous human pathogens. helicases from several (+)rna viruses have been characterized, including ns3 helicases from flaviviruses such as dengue virus, hcv, west nile virus, yellow fever virus, and japanese encephalitis virus (cao et al., 2016; gu and rice, 2016; jain et al., 2016; lin et al., 2017; nedjadi et al., 2015; wu et al., 2005) . sars and mers coronaviruses encode nsp13 with helicase activity (adedeji and lazarus, 2016; hao et al., 2017; seybert et al., 2000) . in semliki forest virus, a representative member of the togavirus family, helicase activity is encoded by the n-terminal domain of nsp2 protein. helicases are also common in some dna viruses, including poxviruses. these include the vaccinia virus helicase-primase d5 (bayliss and smith, 1996; hutin et al., 2016) , e1 protein of bovine papilloma virus 1 (yang et al., 1993) , and the large tumor antigen of sv40 (stahl et al., 1986) . helicases appear to be attractive targets for antiviral drugs (briguglio et al., 2011; frick, 2003; reynolds et al., 2015) , but development of such compounds is challenged by cytotoxicity and bioavailability issues (kwong et al., 2005) . traditional methods for monitoring the activity of rna helicases use radioactively labeled dsrna substrates and follow the unwinding reaction by electrophoretic separation (nondenaturing page) of the ss reaction products, which are detected autoradiographically (adedeji et al., 2012; utama et al., 2000) . to determine whether the inhibitor affects binding of helicase to nucleic acid, a standard gel mobility shift assay is usually used (adedeji et al., 2012) . helicase activity is fueled by atp hydrolysis; thus, inhibition of atpase activity became another possible antiviral strategy. atpase activity can be determined either by the decrease in atp or formation of adp (using commercially available fluorescent anti-adp antibodies) or inorganic pyrophosphate. phosphates released by atp hydrolysis can form a molybdophosphate complex that can be measured colorimetrically using malachite green, quinaldine red, or rhodamine b (baykov et al., 1988; debruyne, 1983; miyata et al., 2010) or by lightscattering (oshima et al., 1996) . the colorimetric methods can be miniaturized for hts (zuck et al., 2005) . the absorbance-based assay was also converted into a hts method based on fluorescence quenching by a colored quinaldine red complex ). an alternative method employs a europium-tetracycline complex for luminescent determination of inorganic phosphate (schäferling and wolfbeis, 2007) . a more complex coupled enzyme colorimetric assay (with maltose phosphorylase, glucose oxidase, and horseradish peroxidase) was used for hts of atpase inhibitors (avila et al., 2006) . assays for luminescent and fluorescent screening of atpase activity, including an immunochemical method using fluorescently labeled anti-adp antibodies, have been reviewed by shadrick and colleagues (shadrick et al., 2013) . in a radioactive method using [γ-32 p]atp, the amp product was separated from unreacted atp by thin-layer chromatography on polyethyleneimine-cellulose and visualized autoradiographically (adedeji et al., 2012) . several research groups have described fluorescence assays to identify inhibitors targeting sars-cov helicase (nsp13) (adedeji et al., 2012; cho et al., 2015; özeş et al., 2014) . the substrate is usually a dsdna oligonucleotide consisting of a fluorescently labeled strand annealed to the complementary strand carrying a quencher. this approach was also adapted for real-time determination of the rna helicase activity of hcv (tani et al., 2010) . in this assay, an ssdna capture strand complementary to the strand carrying the quencher was used to prevent reannealing of the unwound duplex. recently, a colorimetric assay for monitoring helicase activity using dna-conjugated gold nanoparticles was developed (deka et al., 2017) . this method is based on shifts in the optical properties of nanoparticles due to dna unwinding and allows simple screening of inhibitor activity. the dsdna melting curves can be determined spectrophotometrically (at 524 nm and 260 nm) or even by the naked eye. another fluorescence hts of dengue virus ns3 helicase inhibitors measures the unwinding of a double-labeled molecular beacon (basavannacharya and vasudevan, 2014) . other approaches involve graphene oxide-based fluorescence monitoring of viral helicase activity [reviewed in (jang et al., 2013) ]. a gquadruplex-based method for label-free determination of hcv helicase ns3 activity measures changes in the luminescence of transition metal complexes with dna upon helicase-mediated quadruplex melting (leung et al., 2015) . both protein tyrosine kinases and protein serine/threonine (s/t) kinases have been found in viruses. tyrosine kinase function is wellunderstood in connection with oncogenic retroviruses, [for review on retroviral oncogenes, see (vogt, 2012) ]. in contrast to tyrosine kinases from oncogenic viruses, such as rous sarcoma virus src tyrosine kinase, viral s/t kinases share little homology with cellular enzymes. they are exclusively encoded by large dna viruses (e.g. herpesviruses), in which they play important roles in viral virulence, helping the virus to escape defense mechanisms such as those regulated by cytokine signaling pathways (jacob et al., 2011; sato et al., 2017) . their autophosphorylation and transphosphorylation activities mimic those of cellular cyclin-dependent kinases such as cdc2. for example, viral kinases can phosphorylate translation elongation factor 1 delta (ef-1δ) (jacob et al., 2011; kawaguchi and kato, 2003) . all herpesviruses encode the s/t protein kinase ul13, and us3 s/t kinases have been described in the alphaherpesvirus subfamily (kato, 2016; kawaguchi and kato, 2003) . in addition to protein kinases, hsv encodes a thymidine kinase. unlike cellular thymidine kinase, hsv thymidine kinase has a wide substrate specificity that includes pyrimidine and purine phosphonate analogs (e.g. acyclovir, ganciclovir, penciclovir) (de clercq and li, 2016) . in the body, ganciclovir is phosphorylated by cellular kinases and penciclovir and acyclovir by virus-encoded thymidine kinases to the active nucleoside triphosphate forms of the drugs that inhibit viral dna synthesis (de clercq and li, 2016; kokoris and black, 2002) . some cellular protein kinases appear to support viral replication. for example, polo-like kinases induce early stages in the influenza virus life cycle (pohl et al., 2017) , and human protein kinase c regulates the assembly of the ribonucleoprotein complexes in influenza virus (mondal et al., 2017) . inhibitors of abelson tyrosine-protein kinase 2 are active against sars and mers coronaviruses (coleman et al., 2016) . several in vitro approaches can be used to determine kinase activity as well as the activity of kinase inhibitors. analyses of the cellular phosphoproteome, sometimes accompanied by phosphopeptide enrichment, have become standard to determine kinase activity (lea and simeonov, 2011; meyer et al., 2017; vyse et al., 2017) . these assays can be used to assess the impact of inhibitors on the overall phosphoproteome in mammalian cells. although these methods provide complex information about the overall array of kinases and phosphatases in the cell (olsen et al., 2006) , they are not applicable to screening inhibitors of a single viral kinase. for these purposes, in vitro assays with recombinant kinases have been developed [for review see ], including some hts fluorescence methods (zegzouti et al., 2009 ) that can replace radioactive techniques using [γ 32 p]atp (sanghera et al., 2009) . mass spectrometric analysis also can be used to identify in vitro kinase inhibitors. for these analyses, synthetic peptides, proteins, or phosphatase-and heat-treated tissue samples (to dephosphorylate the proteins and inactivate all enzymes, respectively) are subjected to kinase treatment in the presence of inhibitors (huang et al., 2007; meyer et al., 2017; xue et al., 2012) . recombinant kinase activity in the presence of inhibitors can be quantified as atp consumption or adp production in the phosphorylation reaction by numerous commercial kits. other methods monitor binding of inhibitors to phage-displayed kinases in ligand competition assays (fabian et al., 2005) . fluorescence methods including fret, fluorescence polarization or intensity endpoint measurement, and lifetime imaging of fluorescence including fluorescence biosensors (zhang and allen, 2007) have been reviewed elsewhere. sulfonamido-oxine labeled peptides can be used as chromophores that bind mg 2 + upon phosphorylation and emit chelation-enhanced fluorescence (devkota et al., 2013; luković et al., 2008) . kinase-catalyzed phosphorylation of fluorescent peptides promotes their binding to metal-coated nanoparticles, which decreases their mobility and enhances measurable fluorescence polarization (lea and simeonov, 2011; sportsman et al., 2004) . tbiii complexes, in which phosphotyrosine induces fluorescence emission (wang et al., 2015a) , may be used to evaluate protein tyrosine kinase activity (akiba et al., 2015; sumaoka et al., 2016) . fluorescence polarization methods also can be useful for drug screening [reviewed in (hall et al., 2016) ]. other alternatives are immunochemical methods that use antibodies specific to the phosphorylated amino acids, such as phosphotyrosine (li et al., 2001; youngren et al., 1997) or phosphoserine/ phosphothreonine. these antibodies can be used to detect protein/ peptide phosphorylation by western blotting, elisa, or immunoprecipitation of phosphorylated proteins for further mass spectrometry-based analysis (grønborg et al., 2002; zhang et al., 2002) . an elegant approach that limits the false-positive hits in screening of specific kinase inhibitors is based on an in situ proximity ligation assay using both an antibody against the target protein and an anti-phosphotyrosine antibody (leuchowius et al., 2010) . both antibodies are coupled with oligonucleotides, which when brought together due to antibody binding, can be enzymatically ligated and replicated through rolling circle amplification to form a long linear tandem repeat of sequences detectable by a complementary fluorescent oligonucleotide. in addition to protein kinases, some lipid kinases have been targeted by antivirals. one example is sphingosine kinase 1, which affects replication of dengue virus (aloia et al., 2017) . its activity can be determined by measuring the production of 32 p-labeled sphingosine-1phosphate from sphingosine and [γ 32 p]atp (clarke et al., 2016; pitman et al., 2012) . retroviral integrase inhibitors are a new type approved new type of inhibitors imposed by the emergence of drug-resistant mutants. hiv integrase activities, integrase inhibitors, and drug resistance have been discussed in detail elsewhere (andrake and skalka, 2015; anstett et al., 2017; hajimahdi and zarghi, 2016; liao et al., 2010; podany et al., 2017; thierry et al., 2016) . methods to assess the two major activities of integrase-end processing of the reverse transcription product and its joining to target chromosomal dna-have been reviewed in detail by several groups (engelman and cherepanov, 2014; marchand et al., 2001; merkel et al., 2009) . initial methods used radioactively labeled dna oligonucleotides comprising the terminal cis-acting sequences of linear viral dna required for integration. the joining of the processed strand to the other strand (self-integration) or to supplemented target dnas can be analyzed by page (katz et al., 1990; katzman et al., 1989) . a less time-consuming, non-radioactive method involves timeresolved fluorescence anisotropy measurement using a 21-meric oligonucleotide fluorescently labeled on the terminal gt dinucleotide. this assay monitors the binding of integrase to the substrate as well as the subsequent 3′-processing reaction, which both change the anisotropy (guiot et al., 2006) . alternatively, the yields of both the processing and joining reactions can be measured upon separation of the radioactively labeled product from the rest of the dna molecule using adsorption to pei-cellulose (muller et al., 1993) . a real-time hts method measures fluorescence emission resulting from removal of the 3′-terminal dinucleotide, labeled with a quencher, by integrase (he et al., 2007) . han et al. described a fluorescence method to screen molecules that inhibit binding of integrase to viral dna . methods evaluating the integrase strand transfer reaction have been modified to a high-throughput format using magnetic beads (he et al., 2008) or streptavidin-coated microplates (john et al., 2005) . a method to assess strand transfer by time-resolved fret with a europium-streptavidin-labeled substrate has been optimized for 384-and 1536-well plate formats (wang et al., 2005) . the hbv capsid protein is the building block of the viral core, surrounding the viral nucleic acid (pre-genomic rna, pgrna) and reverse transcriptase. the hbv core is icosahedral, formed by 240 copies of capsid protein dimers. in vitro hts of hbv core assembly inhibitors using a modified hbv capsid protein has been described (stray et al., 2006) . the capsid protein was modified by deleting the nucleic acid binding domain, which is dispensable for capsid assembly, and the nterminal assembly domain alone was used in the assay. to fluorescently label the hbv capsid protein, all cysteine residues dispensable for assembly were replaced with alanines. a unique cysteine residue (c150) was c-terminally joined to the assembly domain and labeled with fluorescent bodipy-fl dye (c150bo). during assembly, capsid proteins dimerize, bringing c150bo residues close together and resulting in c150bo fluorescence self-quenching. following incubation of the labeled hbv protein with inhibitors, fluorescence was measured in black 96-well microtiter plates. development of in vitro assembly systems has contributed greatly to current understanding of the structure of retroviral particles and mechanisms of virion formation. these systems also became the base for several high throughput assays for screening assembly inhibitors. during the last 20 years, a number of in vitro assembly assays have been established, mainly for hiv-1 (campbell et al., 2001; campbell and rein, 1999; ehrlich et al., 1992; gross et al., 2000; lanman et al., 2002) , mason-pfizer monkey virus (bohmova et al., 2010; klikova et al., 1995; rumlova-klikova et al., 2000; rumlova-klikova et al., 1999; ulbrich et al., 2006) , rous sarcoma virus vogt, 1995, 1997; purdy et al., 2008 purdy et al., , 2009 , and murine leukemia virus (dolezal et al., 2016; hadravova et al., 2012; cheslock et al., 2003) . hts assays for inhibitors of hiv-1 assembly include several methods using purified hiv-1 ca or ca-nc proteins. one of them, the turbidimetric assay, is based on the observation that direct dilution of the hiv-1 capsid protein (ca) into a high-salt solution (1.6-2.2 m nacl) leads to the formation of tubular structures. as the tube formation is accompanied by an increase in light scattering, assembly can be detected as an increase in turbidity, and the rate of turbidity change is proportional to the rate of the assembly (lanman et al., 2002) . other method published by lemke et al., exploits the affinity of nucleocapsid (nc) to a short tg-rich deoxyribooligonucleotide, d(tg25), which is used as a scaffold (lemke et al., 2012) . this arrangement enables ca to assemble at much lower protein and salt concentrations than in the turbidimetric assay (lanman et al., 2002) . biotin-labeled d(tg25) bound on the surface of neutravidin-coated microtiter well plates nucleates assembly of complexes of ca-nc and soluble fluorescein-labeled d(tg25). fluorescence is measured after washing to remove the unbound and unassembled material from the captured assembly products (lemke et al., 2012) . similarly, the faith assay uses a dually labeled oligonucleotide (tqon). however, in this case, the ssdna oligonucleotide tqon is labeled with the reporter dye fluorescein (fam) as well as black hole quencher (bhq); thus, it does not emit any fluorescence. the assembly reaction is triggered by mixing hiv-1 ca-nc or a gagtruncated assembly-competent version with tqon. following incubation, during which tqon is incorporated into the particles, exonuclease is added to degrade unbound tqon, while co-assembled tqon is protected from cleavage. degradation of free unbound tqon with exoi results in separation of fam from its quencher, and the emitted fluorescence is measured (hadravova et al., 2015) . phage display has been employed to screen peptide inhibitors of hiv-1 assembly (sticht et al., 2005) . a commercial library of m13-derived phages presenting random 12-amino-acid peptides was analyzed for specific binding to purified ca or ca-nc proteins. the specifically bound phages were sequenced, and corresponding peptides were chemically synthesized and re-tested in an in vitro assembly assay (gross et al., 2000) . late in the retroviral life cycle, grna is incorporated into the nascent particle during assembly at the plasma membrane. nc contains two zinc-finger domains that are responsible for specific binding of grna. to screen for compounds that would prevent nc-rna/dna interactions, a hts system consisting of two sequential screens was developed (breuer et al., 2012) . the primary screen uses fluorescence polarization (fp), while the secondary one uses differential scanning fluorimetry (dsf). the combination and order of these two techniques were selected to first identify compounds that disrupt interactions between dna and nc, and then identify the compounds binding to nc during the secondary screen. a rapid and simple turbidimetric method was developed to screen inhibitors of assembly of hcv core protein (fromentin et al., 2007) . for the in vitro assembly reaction, two components, an n-terminal part of the hcv core protein corresponding to the minimal assembly competent domain and rna corresponding to the full-length 5′utr of hcv, were used. assembly of hcv nucleocapsid-like particles was initiated by mixing the purified protein and nucleic acid, and the assembly process was monitored by measuring turbidity at 350 nm. numerous viruses, including picornaviruses, retroviruses, alphaviruses, and flaviviruses, encode proteases that are essential for their virulence. the majority of viral proteases specifically cleave viral polyprotein precursors to liberate the functional proteins of the virion. some viral proteases, such as the papain-like proteases of coronaviruses, also reprogram cellular signaling pathways, including ubiquitination mechanisms and interferon controlled responses, to prevent degradation of viral components (clementz et al., 2010; frieman et al., 2009; randow and lehner, 2009; xing et al., 2013) . numerous viruses, including papillomaviruses (bronnimann et al., 2016; buck et al., 2005) and retroviruses (hallenberger et al., 1992) , use also host cell proteases, mainly furin, to trim their envelope and surface proteins. this triggers conformational changes required for interaction with cellular receptors and membrane fusion in enveloped viruses. some viruses use proteases other than furin to modulate their infectivity, as shown for viruses entering airway epithelial cells [reviewed in (laporte and naesens, 2017) ] such as influenza virus (böttcher-friebertshäuser et al., 2010; kühn et al., 2016) , newcastle disease virus (gotoh et al., 1992) , and respiratory syncytial virus (sugrue et al., 2001) . extensive research efforts have yielded detailed information about hiv-1 protease and its inhibitors [reviewed in (de clercq, 2004; konvalinka et al., 2015; midde et al., 2016) ]. large amounts of data also are available for hcv (foote et al., 2011; pawlotsky et al., 2007; razonable, 2011) and sars-cov 3cl protease inhibitors (pillaiyar et al., 2016) , although there is not yet an inhibitor of the latter target approved for clinical use. numerous approved drugs are synthetic peptides derived from the natural proteolytic substrates of target viruses modified to improve the in vivo effects related to bioavailability, stability, and so on. numerous in vitro assays to monitor the activity of proteases and their inhibitors, including commercial kits, have been developed. classical methods use synthetic peptides that mimic the target sites of the protease. although some of the methods described here were not originally designed to screen the activity of viral proteases in the presence of inhibitors, they can be adapted for this purpose by simply changing the peptide sequence to the target site of the protease of interest. the cleavage yield is usually monitored either colorimetrically (ding and yang, 2015; zhou et al., 2014) or as a change in fluorescence triggered by the release of fluorescent labels such as 7-amido-4-methylcoumarin (amc) or rhodamine (grant et al., 2002) . fluorogenic substrates have been used to determine the activity of coronavirus proteases and screen inhibitors (kuo et al., 2004; lee et al., 2014; park et al., 2017; song et al., 2014; tomar et al., 2015; wang et al., 2016; yang et al., 2005; zhao et al., 2008) . some recently described arrangements employ nanoparticles (feltrup and singh, 2012; khalilzadeh et al., 2016; udukala et al., 2016; wang et al., 2014; zeng et al., 2015) or quantum dot bioconjugates (lee and kim, 2015; li et al., 2014; medintz et al., 2006) with immobilized fluorescently or luminescently labeled peptide substrates. alternatively, cleavage products may be monitored by analysis of proteolytic products by mass spectrometric methods (hu et al., 2015; joshi et al., 2017; lathia et al., 2011; rumlová et al., 2003) , analytical hplc (teruya et al., 2016) , or electrochemical methods based on the difference in penetration of substrate and cleavage products through the membrane of a polyionselective sensor (gemene and meyerhoff, 2011; han et al., 1996) . to study the specificity of inhibitor binding and to extend the research to rational design of inhibitors, x-ray or nmr structures of proteases in complex with the inhibitor may be determined, as reported in numerous cases for the proteases of hiv-1 [reviewed in (ghosh et al., 2016) ], hcv (yilmaz et al., 2016) , and mers . cell-based assays can provide additional information, including the capability of the inhibitor to pass through the cell membrane and its stability in the cytoplasm. a general determination of infectivity, such as a plaque cytotoxicity assay in the presence of protease inhibitor, may be used for confirmation. one elegant example exploits the cytotoxicity of hiv protease. cells are transfected with a protease precursor fused to gfp. in the absence of inhibitor, hiv-1 protease is autocatalytically activated and cleaves a broad variety of cellular proteins, resulting in activation of apoptosis and cell death (cummins and badley, 2010; rumlova et al., 2014) . this toxic effect, when suppressed by active inhibitors, results in production of a gfp signal in surviving cells (lindsten et al., 2001) . another elegant approach for hiv and coxsackievirus b3 proteases, which both undergo autocatalytic cleavage, employs constructs in which the protease gene is inserted between sequences encoding the dna-binding domain and the domain that activates transcription of the gal1-lacz reporter gene (dasmahapatra et al., 1992; murray et al., 1993) . the protease-mediated cleavage separates the dna-binding domain from the trans-activating domain and results in failure of reporter gene transcription. this approach has also been modified with gfp as a reporter gene (hilton and wolkowicz, 2010) . co-expression of cleavage-activated luciferase substrate and mers-cov protease permits both live-cell imaging and quantification of the enzyme activity (kilianski et al., 2013) . the need to develop new antiviral compounds will likely persist over the long term, although there has been enormous progress in molecular biology methods, especially rna silencing, bioinformatics, imaging, and structural biology techniques. viruses present challenging targets for drug development due their flexibility and adaptability caused by the error-prone copying of their genomes, which can result in emergence of drug-resistant mutants. viral integration into the host genome and inhibitor toxicity are other obstacles. here, we provide an overview of in vitro methods, including cell-based assays, that may be suitable for screening of antivirotics that interfere with the key steps of viral life cycles and target either virus or 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resistance against strand transfer integrase inhibitors toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay inhibitors of the histone methyltransferases ezh2/1 induce a potent antiviral state and suppress infection by diverse viral pathogens highthroughput screening for hsp90 atpase inhibitors a novel small molecule inhibitor of hepatitis c virus entry junction adhesion molecule is a receptor for reovirus infectious hepatitis c virus pseudoparticles containing functional e1-e2 envelope protein complexes suramin inhibits helicase activity of ns3 protein of dengue virus in a fluorescence-based high throughput assay format a malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay vaccinia virion protein i8r has both dna and rna helicase activities: implications for vaccinia virus transcription -phosphonomethoxy)ethyl]guanine (ode-bn-pmeg), a potent inhibitor of transient hpv dna 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against phosphorylated motifs cell-based high-throughput screening assay identifies 2′,2′-difluoro-2′-deoxycytidine gemcitabine as a potential antipoliovirus agent structure of the main protease from a global infectious human coronavirus, hcov-hku1 molecular basis for specific viral rna recognition and 2′-oribose methylation by the dengue virus nonstructural protein 5 (ns5) structure and function of the zika virus full-length ns5 protein robust hepatitis c virus infection in vitro inhibitors of sars-cov entryidentification using an internally-controlled dual envelope pseudovirion assay a new colorimetric strategy for monitoring caspase 3 activity by hrp-mimicking dnazyme-peptide conjugates protease inhibitors targeting coronavirus and filovirus entry the lymphocytic choriomeningitis virus matrix protein ppxy late domain drives the production of defective interfering particles investigations on dna intercalation and surface binding by sybr green i, its structure determination and methodological implications miniaturization of absorbance assays using the fluorescent properties of white microplates characterization of a novel dna polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes this work was supported by ga čr (cz) ga17-25602s to mr, ga17-24281s to tr and the ministry of education, youth and sport of the czech republic(oppc cz.2.16/3.1.00/24503), through its "national program of sustainability i" npu lo 1601. key: cord-254957-jqp1gto6 authors: klann, kevin; bojkova, denisa; tascher, georg; ciesek, sandra; münch, christian; cinatl, jindrich title: growth factor receptor signaling inhibition prevents sars-cov-2 replication date: 2020-08-11 journal: mol cell doi: 10.1016/j.molcel.2020.08.006 sha: doc_id: 254957 cord_uid: jqp1gto6 summary sars-cov-2 infections are rapidly spreading around the globe. the rapid development of therapies is of major importance. however, our lack of understanding of the molecular processes and host cell signaling events underlying sars-cov-2 infection hinder therapy development. we employed a sars-cov-2 infection system in permissible human cells to study signaling changes by phospho-proteomics. we identified viral protein phosphorylation and defined phosphorylation-driven host cell signaling changes upon infection. growth factor receptor (gfr) signaling and downstream pathways were activated. drug-protein network analyses revealed gfr signaling as key pathway targetable by approved drugs. inhibition of gfr downstream signaling by five compounds prevented sars-cov-2 replication in cells, assessed by cytopathic effect, viral dsrna production, and viral rna release into the supernatant. this study describes host cell signaling events upon sars-cov-2 infection and reveals gfr signaling as central pathway essential for sars-cov-2 replication. it provides with novel strategies for covid-19 treatment. co-expression clustering severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a novel coronavirus, has been rapidly spreading around the globe since the beginning of 2020. in people, it causes coronavirus disease 2019 (covid-19) often accompanied by severe respiratory syndrome (chen et al., 35 2020) . to conquer the global health crisis triggered by covid-19, rapidly establishing drugs is required to dampen the disease course and relieve healthcare institutions. thus, repurposing of already available and (ideally) approved drugs might be essential to rapidly treat covid-19. many studies for proposing repurposing of specific drugs have been conducted in the last months, but mostly remain computational without tests in infection models (smith and smith, 40 2020; wang, 2020) . in addition, they are hindered by the lack of knowledge about the molecular mechanisms of sars-cov-2 infection and the resulting host-cell responses required to allow 2 viral replication. to rationally repurpose drugs, a molecular understanding of the infection and the changes within the host cell pathways is essential. experimentally identifying viral targets in the cell allows candidate drugs to be selected with high confidence for further testing in the 45 clinics to reduce the risks for patients resulting from tests with drugs lacking in vitro validation. growth factor receptor (gfr) signaling plays important roles in cancer pathogenesis and has also been reported to be crucial for infection with some viruses (beerli et al., 2019; kung et al., 2011; zhu et al., 2009) . gfr activation leads to the modulation of a wide range of cellular processes, including proliferation, adhesion, or differentiation (yarden, 2001) . various viruses, 50 such as epstein-barr virus, influenza, or hepatitis c, have been shown to use the epidermal growth factor receptor (egfr) as an entry receptor (eierhoff et al., 2010; kung et al., 2011; lupberger et al., 2011) . in addition, egfr activation can suppress interferon signaling and thus the antiviral response elicited in respiratory virus diseases, for instance influenza a and rhinovirus (ueki et al., 2013) . activation of gfr signaling might play an important role also in 55 other respiratory viruses, such as sars-cov-2. in the last years, it has been shown for many viruses that modulation of host cell signaling is crucial for viral replication and might exhibit strong therapeutic potential (beerli et al., 2019; pleschka et al., 2001) . however, how sars-cov-2 infection changes host cell signaling has remained unclear. we recently established an in vitro cell culture model of sars-cov-2 60 infection using the colon epithelial cell line caco-2, which is highly permissive for the virus and commonly used for the study of coronaviruses (herzog et al., 2008; ren et al., 2006) . here, we determine changes in the cellular phospho-protein networks upon infection with sars-cov-2 to gain insight into infection-induced signaling events. we found extensive rearrangements of cellular signaling pathways, particularly of gfr signaling. strikingly, inhibiting gfr signaling 65 using prominent (anti-cancer) drugs -namely pictilisib, omipalisib, ro5126766, lonafarnib, and sorafenib -prevented sars-cov-2 replication in vitro, assessed by cytopathic effect and viral rna replication and release. these compounds prevented replication at clinically achievable concentrations. due to their clinical availability, these drugs could be rapidly transitioned towards clinical trials to test their feasibility as covid-19 treatment option. 70 in a previous study, we analyzed the effect of sars-cov-2 infection on the host cell translatome and proteome (bojkova et al., 2020) . this study found the effects 24 hours after sars-cov-2 infection especially useful for identifying druggable host pathways. to evaluate changes in 75 intracellular signaling networks brought about by sars-cov-2 infection, we quantified phosphoproteome changes 24 hours after infection ( figure 1a ). caco-2 cells were mock-infected or infected with sars-cov-2 patient isolates (in five biological replicates at an moi of 1) for one hour, washed, and incubated for 24 hours before cell harvest. extracted proteins were digested and split to 1) carry out whole-cell proteomics of a tandem mass tag (tmt) 10-plex samples 80 using liquid chromatography synchronous precursor selection mass spectromety (lc-sps-ms 3 ), or 2) use fe-nta phosphopeptide enrichment (achieving 98% enrichment) for phospho proteome analyses of a tmt 10-plex analyzed by lc-ms 2 , due to the higher precision and identification rates of ms 2 based methods during phosphopeptide measurements (hogrebe et al., 2018) . we identified and quantified 7,150 proteins and 16,715 different phosphopeptides for 85 a total of 15,093 different modification sites ( figure 1b , c, s1, and table s1 , s2). the main fraction of phosphopeptides were modified serines (86.4%), followed by threonine (13.4%), and tyrosine (0.2%) ( figure 1d ). upon infection, 2,197 and 799 phosphopeptides significantly increased or decreased respectively (log 2 fc > 1, p value < 0.05). 3 viral proteins are produced in the host cell and underlie (and often require) post-translational 90 modification (ptm) by host cell enzymes (wu et al., 2009) . accordingly, we assessed viral proteins phosphorylated in the host cell. we identified 33 modification sites on 6 different viral proteins ( figure 1e -j). possible functions of the observed modifications largely remain unclear due to a lack of understanding of their molecular function and regulation. sars-cov-2 protein 3a was phosphorylated on the luminal side of this transmembrane protein ( figure 1e ) membrane 95 protein m was phosphorylated at three serines in close proximity, at the c-terminal, cytoplasmic region of the protein ( figure 1f ), suggesting a high-activity modification surface. sars-cov-1 protein 6 was described to accelerates infections in murine systems (tangudu et al., 2007) . we found a single phosphorylation of the sars-cov-2 protein homologue non-structural protein 6 in host cells ( figure 1g ) protein 9b was modified at two sites ( figure 1h ). however, its function in 100 sars-cov-1 or sars-cov-2 remains unknown. polyprotein 1b is a large 7,096 amino acid protein heavily processed to generate distinct proteins in sars-cov-1 (tangudu et al., 2007) . we found polyprotein 1b to be modified at three residues, two in a region of unknown function and one in the non-structural protein 11 (nsp11) part of the protein ( figure 1i ). our data cannot distinguish whether phosphorylation occurred before or after cleavage and whether 105 phosphorylation may affect processing. sars-cov-2 nucleoprotein was heavily phosphorylated ( figure 1j ). mapping phosphosites to the structure (residues 47 to 173, pdb: 6vyo) revealed a small surface region, suggesting specific regulation and interaction changes ( figure 1k ). to reveal host kinases potentially phosphorylating viral proteins, we bioinformatically assessed identified phosphorylation motifs using netphos 3.1 and gps5 (blom et al., 2004; wang et al., 110 2020) (table s3) . some motifs present in nucleoprotein were predicted to be modified by cmgc kinases. among several others, casein kinase ii (ck2) kinases are part of the cmgc family and have been independently identified as interaction partners of the nucleoprotein, when expressed in cells (gordon et al., 2020) . inhibition of ck2 kinases, could be employed to study possible functional interactions between kinase and viral protein. taken together, we identified extensive changes in phosphorylation of host and viral proteins after sars-cov-2 infection. the roles of viral protein modifications remain unclear. however, targeting the corresponding host kinases may offer new treatment strategies. to identify the key host signaling pathway networks modulated by infection, we carried out 120 protein-protein co-regulation analysis on all proteins quantified in phosphorylation and total protein level. we first standardized phosphorylation and total protein levels by individual zscoring to compare the different datasets. subsequently, to merge phosphorylation and proteome data, we collapsed all phospho-sites for each protein into one average profile and calculated combined z-scores. patterns of co-regulation were identified using protein-protein 125 correlation and hierarchical clustering ( figure 2a ). this generalized approach allows to study large scale patterns of dependencies of protein and phosphorylation levels, that then can be dissected into individual phosphorylation sites and protein levels for downstream analysis. the dynamic landscape of the proteome revealed three main clusters of co-regulated proteins, each one representing different sets of pathways (discussed in detail below): 130 the first cluster was mainly comprised of receptor signaling and endocytic pathways ( figure 2b ). prominent among these pathways were platelet derived growth factor receptor (pdgfr), erbb1 (egfr) signaling, metabolism, and various pathways associated with vesicle trafficking (table s4 ). as changes in phospho-peptide abundance can represent different ratios in phosphorylated versus non-phosphorylated peptide or a change in protein abundance (with the same ratio of 135 protein being phosphorylated), we integrated our phospho-proteome dataset with total proteome data ( figure 2c ). when comparing abundances of individual phosphopeptides and their protein levels, extensive changes were observed in the phospho-proteome; however, no general j o u r n a l p r e -p r o o f 4 changes were seen for the total proteome ( figure 2c , table s2 ). thus, phosphorylation changes were induced by signaling activity alteration resulting in increased phosphorylation and not due 140 to protein abundance differences. the second cluster was mainly comprised of proteins decreased in phosphorylation and was highly connected to cell cycle and translation initiation ( figure 2d and table s4 ). we reported recently that inhibition of cellular translation prevented sars-cov-2 replication in cells (bojkova et al., 2020) , consistent with regulation of translation by altering phosphorylation patterns. to 145 further distinguish the regulations within this cluster, we correlated protein levels with differential phosphorylation abundance ( figure 2e ) and found two groups of proteins: the first was contained translation related pathways (identified in figure 2e ) and was predominantly regulated by decreased modification. the second set of proteins was decreased in phosphorylation and on total protein level. the majority of proteins found in the second cluster belonged to diverse cell 150 cycle pathways. consistent with these findings, cell cycle pathways were also enriched in the set of proteins significantly decreased on protein level ( figure 2f , s2, and table s4 and s5). translation pathways were not regulated on protein level to this extent. analysis of the third cluster revealed signaling events of the splicing machinery (table s4) possibly explaining previously observed changes in splicing machinery abundance upon sars-155 cov-2 infection (bojkova et al., 2020) . consistent with previous literature (grimmler et al., 2005; ilan et al., 2017; mathew et al., 2008; mermoud et al., 1994) , we therefore hypothesized that the host splicing machinery is extensively reshaped during viral infection. this finding further supports splicing as a potential therapeutic target, in agreement with decreased sars-cov-2 pathogenic effects when inhibiting splicing by pladeinolide b. additionally, we found carbon 160 metabolism among the pathways showing significantly increased phosphorylation upon sars-cov-2 infection (table s4 ) in addition to previously described changes of total protein levels of enzymes part of glycolysis and carbon metabolism (bojkova et al., 2020 )( figure s3 ). taken together, we showed that, during sars-cov-2 infection, specific rearrangements of signaling pathways were elicited in the cellular proteome. regulation was mainly comprised of 165 cellular signaling and translational pathways as well as proteins regulated not only by phosphorylation, but also in total protein abundance. proteins exhibiting decreased protein levels were significantly enriched in cell cycle proteins. we observed over 2,000 phospho-peptides to be increased in abundance while their protein 170 levels stay constant upon infection ( figure 2c and s4). this reveals differential modification activity (e.g. signaling events) for these phospho-proteins. for many kinases in cellular signaling pathways there are already approved drugs available. hence, we investigated the potential to repurpose drugs to treat covid-19 by mapping already available drugs via reactomefi to the set of proteins increased in phosphorylation. we filtered the network for drugs and direct targets 175 and found egfr as one of the central hits, including a number of regulated proteins in the downstream signaling pathway of egfr ( figure 3a ). these downstream targets are also regulated by other gfrs and could thus also be explained by their observed activation upon sars-cov-2 infection ( figure 2 ). 28 clinically approved drugs (largely used in cancer therapy) are already available to target egfr or downstream targets. indeed, we found a subnetwork of 180 gfr signaling components remodeled ( figure 3b ). we mapped identified members of gfr signaling and their respective phosphorylation differences upon sars-cov-2 infection ( figure 3c ) revealing an extensive overall increase in phosphorylation of the whole pathway, including related components for cytoskeleton remodeling and receptor endocytosis. how gfr signaling might regulate sars-cov-2 infection is still matter for speculation. however, gfr signaling 185 j o u r n a l p r e -p r o o f 5 inhibition might provide a useful approach already implicated in sars-cov induced fibrosis therapy and might be a viable strategy to treat covid-19. since gfr signaling seemed to be central during sars-cov-2 infection, we examined the use of inhibitors as antiviral agents. since there are several gfrs integrating their signaling and 190 regulating a number of processes inside the cell, directly targeting downstream signaling components is likely to be more successful to prevent signaling of different gfrs and to avoid mixed effects of multiple pathways. gfr signaling, amongst others, results in activation of 1) the raf/mek/erk mapk signaling cascade and 2) integrates (via phosphoinositide 3 kinase [pi3k] and protein kinase b [akt]) into mtorc1 signaling to regulating proliferation ( figure 4a ). to 195 explore the antiviral efficiency of targeting proteins downstream of gfrs, we first tested the pi3k inhibitors pictilisib and omipalisib (ippolito et al., 2016; sarker et al., 2015; schmid et al., 2016) . both compounds inhibited viral replication, based on their propensity to prevent cytopathogenic e ect (cpe) and viral rna production in cells ( figure 4b -d, s5, and s6). our drug-target analyses identified mitogen activated protein kinase kinase (map2k2, better known as mek) 200 and the raf inhibitor sorafenib (wilhelm et al., 2006) as promising targets inhibiting downstream signaling of gfrs ( figure 4a ). thus, we tested sorafenib and the dual raf/mek inhibitor ro5126766 in our viral replication assays. both compounds inhibited cytopathic effects during infection and the viral replication ( figure 4b -d, s5, and s6). to validate our findings in another cell line, we repeated the treatments in ukf-rc-2 cells infected with sars-cov-2. quantifying 205 the viral rna copies in the supernatant, we observed that the compounds efficiently inhibited virus replication ( figure 4e and s7a) at non-toxic conditions ( figure s7b , except for sorafenib). overall, five compounds, inhibiting downstream signaling of gfrs, prevented sars-cov-2 replication at clinically achievable concentrations ( figure 4b and 5) (eskens et al., 2001; fucile et al., 2014; martinez-garcia et al., 2012; munster et al., 2016; sarker et al., 2015) , emphasizing 210 the importance of gfr signaling during sars-cov-2 infection and revealing clinically available treatment options as drug candidates for covid-19. with the rapid spreading of the covid-19 pandemic, investigating the molecular mechanisms underlying sars-cov-2 infection are of high importance. in particular, the processes underlying 215 infection and host-cell response remain unclear. these would offer potential avenues for pharmacological treatment of covid-19. here, we report global, differential phosphorylation analysis of host cells after infection with intact sars-cov-2 virus. we could identify phosphorylation sites on numerous viral proteins in cells, showing that they can undergo efficient modification in infected cells. until now, we can only speculate about the host kinases involved 220 and the functions driven by ptms, which will be an important topic for follow-up studies. for sars-cov-1, it was shown that modification of viral proteins can lead to regulation of rna binding of the nucleoprotein (wu et al., 2009 ) and is needed for viral replication. although similar effects in sars-cov-2 are likely, this remains to be studied in this novel virus. a recent paper analyzed the interaction profile of sars-cov-2 proteins expressed in hek293t cells (gordon et 225 al., 2020) . for the heavily phosphorylated nucleoprotein they could identify interactions the host casein kinases, which might indicate possible modification events by the latter. also for the orf9b/protein 9b that we found modified in cells, interaction mapping identified mark kinases as interaction partners. by exploring the signaling changes inside the host cell, we could gain important insights into host 230 cell signaling during infection. we found essential gfr signaling pathways activated such as egfr or pdgfr, together with a plethora of rhogtpase associated signaling molecules. we could furthermore show modulation of the splicing machinery, in line with previous results indicating dependency of viral in vitro pathology on the host spliceosome (bojkova et al., 2020) . the same is true for metabolic reprogramming, for which we found differential post-translational 235 modification of most members of the carbon metabolic pathways, namely glycolysis, pentose phosphate and tca cycle. these metabolic pathways were significantly up-regulated on total protein levels in the presented dataset, consistent with our previous study (bojkova et al., 2020) , suggesting that these key pathways are regulated on multiple levels. a number of drugs to treat covid-19 have been suggested, largely based on bioinformatics 240 analyses of genetics or cellular data (gordon et al., 2020; li et al., 2020; wang, 2020) . however, for many of these compounds, studies explaining their working mechanisms in the context of sars-cov-2 or viral assays to determine their efficacy of blocking viral replication in cell models of sars-cov-2 infection, are missing. while monitoring signaling changes in host cells, we observed activation of gfr signaling cascades after infection, consistent with other viruses 245 relying on the receptors themselves or elicited signal transduction (eierhoff et al., 2010; kung et al., 2011; lupberger et al., 2011; ueki et al., 2013; wu et al., 2017; zhu et al., 2009) . from our data we could not clearly conclude which gfr might be activated and thus tested whether gfr downstream signaling inhibition can prevent sars-cov-2 replication, as reported for some other viruses (baturcam et al., 2019; pleschka et al., 2001) . previously, temporal kinome analysis 250 identified antiviral potential of ras/raf/mek and pi3k/akt/ for mers-cov (kindrachuk et al., 2015) . by targeting the ras/raf/mek and pi3k/akt/mtor downstream axes of gfr signaling, we found efficient inhibition of viral replication in two different cell lines derived from different tissues (figure 4) . gfr signaling was shown to play a role in diverse virus infections as well as in fibrosis induction by sars-cov-1 (beerli et al., 2019; kung et al., 2011; lupberger et 255 al., 2011; pleschka et al., 2001; ueki et al., 2013; . thus, our results in cytopathic effects might indeed indicate cytoprotective roles for gfr signaling axes during sars-cov-2 infection and possible development of fibrosis (luo et al., 2020) . notably, some inhibitors used in our study such as omipalisib were shown to suppress fibrosis progression in patients with idiopathic pulmonary fibrosis, which may share deregulation of signaling pathways 260 involved in lung fibrosis of coronavirus patients . these findings suggest that inhibitors of gfr downstream signaling may bring benefit to covid-19 patients independently of their antiviral activity. taken together, this study provides new insights into molecular mechanisms elicited by sars-cov-2 infection. proteomic analyses revealed several pathways that are rearranged during 265 infection and showed that targeting of those pathways is a valid strategy to inhibit cytopathic effects triggered by infection. in this study, cancer cell lines were used to assess the effect of sars-cov-2 on host cells during infections. we chose the experimental time-point, based on previous analysis of the 270 infection course in these cells (bojkova et al., 2020) . notably, the kinetics of infection are likely to be different in other cell lines or primary material, since we also observed a different moi needed for ukf-rc-2 cells. in addition, we tested the efficiency of the presented drugs only in the context of in vitro cell line experiments. thus, the results do not represent direct evidence for the use of these therapeutics in patients, as the effects might differ in primary tissue. the results 275 presented indicate potential anti-viral effects that have to be further validated in other models and clinical trials to assess their usefulness for the treatment of covid-19. (a) experimental scheme. caco-2 cells were infected with sars-cov-2 for one hour (moi: 1), washed and incubated for additional 24 hours. proteins were extracted and prepared for bottomup proteomics. all ten conditions were multiplexed using tmt10 reagents. 250 µg of pooled samples were used for whole cell proteomics (24 fractions) and the remainder (~1 mg) enriched for phosphopeptides by fe-nta. phosphopeptides were fractionated into 8 fractions and 305 concatenated into 4 fractions. all samples were measured on an orbitrap fusion lumos. (b) volcano plot showing fold changes of infected versus mock cells for all quantified phosphopeptides. p values were calculated using an unpaired, two-sided student's t-test with equal variance assumed and adjusted using the benjamini hochberg fdr method (n = 5 biological replicates). orange or blue points indicate significantly increased or decreased 310 phosphopeptides, respectively. (c) volcano plot showing differences between sars-cov-2 and mock infected cells in total protein levels for all quantified proteins. p values were calculated using an unpaired, two-sided student's t-test with equal variance assumed and adjusted using the benjamini hochberg fdr method (n = 5). orange or blue points indicate significantly increased or decreased 315 phosphopeptides, respectively. (d) distribution of phosphorylation sites identified across modified amino acids. see also figure s1 and tables s1 and s2. see also table s1 , s2, s3 and figure s1 . (e) scatter plot showing correlation between fold changes of phosphopeptides compared to fold changes of total proteins levels. two subsets of phosphopeptides were detected: one was 340 j o u r n a l p r e -p r o o f 9 mainly regulated by differential modification (indicated in yellow), the second by changes in protein abundance. (f) string network analysis of proteins decreased in total protein levels ( figure 1c) . inserts indicate pathways found in the network. see also table s1 , s2, s4, s7, figure s2 and s3. 345 see also figure s4 . see also figure s5 . resource availability further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact: christian münch: ch.muench@em.uni-frankfurt.de (c.m.) 400 ukf-rc-2 human kidney cells derived from renal carcinoma are available upon request. the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride (perez-riverol et al., 2019) human caco-2 cells, derived from colon carcinoma, was obtained from the deutsche sammlung von mikroorganismen und zellkulturen (dsmz; braunschweig, germany). cells were grown at 37°c in minimal essential medium (mem) supplemented with 10% fetal bovine serum (fbs) and containing 100 iu/ml penicillin and 100 µg/ml streptomycin. all culture reagents were purchased from sigma. 415 cell line designated ukf-rc-2 was established from a tumor sample of a patient with a diagnosis of renal carcinoma hospitalized at department of urology, university hospital frankfurt. tumor tissue was cut in pieces and dissociated using 0.2% trypsin solution. primary tumor cells and passaging of cell line was performed using imdm medium supplemented with 10% fcs and antibiotics. ukf-rc-2 cells between passages 15 and 20 were used for antiviral 420 experiments. all culture reagents were purchased from sigma. sars-cov-2 was isolated from samples of travelers returning from wuhan (china) to frankfurt (germany) using human colon carcinoma cell line caco-2 as described previously 12 . sars-cov-2 stocks used in the experiments had undergone one passage on caco-2 cells and were 425 stored at -80° c. virus titers were determined as tcid50/ml in confluent cells in 96-well microtiter plates. confluent layers of caco-2 cells in 96-well plates were infected with sars-cov-2 at moi 0.01. virus was added together with drugs and incubated in mem supplemented with 2% fbs with different drug dilutions. cytopathogenic e ect (cpe) was assessed visually 48 h after infection. to assess effects of drugs on caco-2 cell viability, confluent cell layers were treated with different drug concentration in 96-well plates. the viability was measured using the rotitest vital (roth) according to manufacturer's instructions. data for each condition was collected for at 435 least three biological replicates. for dose response curves, data was fitted with all replicates using originpro 2020 with the following equation: ic50 values were generated by originpro 2020 together with metrics for curve fits. for ukf-rc-2 cells, the assay was performed as described above, except for usage of a moi of 440 0.1 as staining experiments for sars-cov-2 infection in ukf-rc-2 cells revealed the need of a higher moi to achieve comparable effects to caco-2 cells. sars-cov-2 rna from cell culture supernatant samples was isolated using avl buffer and the qiaamp viral rna kit (qiagen) according to the manufacturer's instructions. absorbance-based 445 quantification of the rna yield was performed using the genesys 10s uv-vis spectrophotometer (thermo scientific). rna was subjected to onestep qrt-pcr analysis using the luna universal one-step rt-qpcr kit (new england biolabs) and a cfx96 real-time system, c1000 touch thermal cycler. primers were adapted from the who protocol (corman et al., 2020) targeting the open reading frame for rna-dependent rna polymerase 450 (rdrp): rdrp_sarsr-f2 (gtg ara tgg tca tgt gtg gcg g) and rdrp_sarsr-r1 (car atg tta aas aca cta tta gca ta) using 0.4 µm per reaction. standard curves were created using plasmid dna (pex-a128-rdrp) harboring the corresponding amplicon regions for rdrp target sequence according to genbank accession number nc_045512. all quantification experiments have been carried out with biological replicates. 455 effect of selected compounds on viral replication was assessed by staining of double-stranded rna, which has been shown to be sufficient for measurement of sars-cov-1 replication (weber et al., 2006) . briefly, cells were fixed with acetone/methanol (40:60) solution 48 h post infection. immunostaining was performed using a monoclonal antibody directed against dsrna (1:150 460 dilution, scicons j2, mouse, igg2a, kappa chain, english & scientific consulting kft., szirák, hungary), which was detected with biotin-conjugated secondary antibody (1:1000 dilution, jackson immunoresearch) followed by application streptavidin, peroxidase conjugate (1:3000 dilution, sigma aldrich). lastly, the dsrna positive cells were visualized by addition of aec substrate. wells were imaged at different areas to visualize a larger area (presented in 465 supplementary figures). for all proteomics analysis, caco-2 cells were infected at an moi of 1 and the sample preparation was performed as described previously . briefly, lysates were precipitated by methanol/chloroform and proteins resuspended in 8 m urea/10 mm epps ph 470 8.2. concentration of proteins was determined by bradford assay and 300 µg of protein per samples was used for digestion. for digestion, the samples were diluted to 1 m urea with 10mm epps ph 8.2 and incubated overnight with 1:50 lysc (wako chemicals) and 1:100 sequencing grade trypsin (promega). digests were acidified using tfa and tryptic peptideswere purified by tc18 seppak (50 mg, waters). 125 µg peptides per sample were tmt labelled and the mixing 475 was normalized after a single injection measurement by lc-ms/ms to equimolar ratios for each channel. 250 µg of pooled peptides were dried for offline high ph reverse phase fractionation by hplc (whole cell proteome) and remaining 1 mg of multiplexed peptides were used for phospho-peptide enrichment by high-select fe-nta phosphopeptide enrichment kit (thermo fisher) after manufacturer`s instructions. after enrichment, peptides were dried and 480 resuspended in 70% acetonitrile/0.1% tfa and filtered through a c8 stage tip to remove contaminating fe-nta particles. dried phospho-peptides then were fractionated on c18 (empore) stage-tip. for fractionation c18 stagetips were washed with 100% acetonitrile twice, followed by equilibration with 0.1% tfa solution. peptides were loaded in 0.1% tfa solution and washed with water. elution was performed stepwise with different acetonitrile concentrations in 485 0.1% triethylamine solution (5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 50%). the eight fractions were concatenated into four fractions and dried for lc-ms. peptides were fractionated using a dionex ultimate 3000 analytical hplc. 250 µg of pooled and purified tmt-labeled samples were resuspended in 10 mm ammonium-bicarbonate (abc), 5% 490 acn, and separated on a 250 mm long c18 column (x-bridge, 4.6 mm id, 3.5 µm particle size; waters) using a multistep gradient from 100% solvent a (5% acn, 10 mm abc in water) to 60% solvent b (90% acn, 10 mm abc in water) over 70 min. eluting peptides were collected every 45 s into a total of 96 fractions, which were cross-concatenated into 24 fractions and dried for further processing. 495 all mass spectrometry data was acquired in centroid mode on an orbitrap fusion lumos mass spectrometer hyphenated to an easy-nlc 1200 nano hplc system using a nanoflex ion source (thermofisher scientific) applying a spray voltage of 2.6 kv with the transfer tube heated to 300°c and a funnel rf of 30%. internal mass calibration was enabled (lock mass 445.12003 500 m/z). peptides were separated on a self-made, 32 cm long, 75µm id fused-silica column, packed in house with 1.9 µm c18 particles (reprosil-pur, dr. maisch) and heated to 50°c using an integrated column oven (sonation). hplc solvents consisted of 0.1% formic acid in water (buffer a) and 0.1% formic acid, 80% acetonitrile in water (buffer b). for total proteome analysis, a synchronous precursor selection (sps) multi-notch ms3 method 505 was used in order to minimize ratio compression as previously described (mcalister et al., 2014) . individual peptide fractions were eluted by a non-linear gradient from 7 to 40% b over 90 minutes followed by a step-wise increase to 95% b in 6 minutes which was held for another 9 minutes. full scan ms spectra (350-1400 m/z) were acquired with a resolution of 120,000 at m/z 200, maximum injection time of 100 ms and agc target value of 4 x 10 5 . the 20 most intense 510 precursors with a charge state between 2 and 6 per full scan were selected for fragmentation ("top 20") and isolated with a quadrupole isolation window of 0.7 th. ms2 scans were performed in the ion trap (turbo) using a maximum injection time of 50ms, agc target value of 1.5 x 10 4 and fragmented using cid with a normalized collision energy (nce) of 35%. sps-ms3 scans for quantification were performed on the 10 most intense ms2 fragment ions with an 515 isolation window of 0.7 th (ms) and 2 m/z (ms2). ions were fragmented using hcd with an nce of 65% and analyzed in the orbitrap with a resolution of 50,000 at m/z 200, scan range of 110-500 m/z, agc target value of 1.5 x10 5 and a maximum injection time of 120ms. repeated sequencing of already acquired precursors was limited by setting a dynamic exclusion of 45 seconds and 7 ppm and advanced peak determination was deactivated. 520 for phosphopeptide analysis, each peptide fraction was eluted by a linear gradient from 5 to 32% b over 120 minutes followed by a step-wise increase to 95% b in 8 minutes which was held for another 7 minutes. full scan ms spectra (350-1400 m/z) were acquired with a resolution of 120,000 at m/z 200, maximum injection time of 100 ms and agc target value of 4 x 10 5 . the 20 most intense precursors per full scan with a charge state between 2 and 5 were selected for 525 fragmentation ("top 20"), isolated with a quadrupole isolation window of 0.7 th and fragmented via hcd applying an nce of 38%. ms2 scans were performed in the orbitrap using a resolution of 50,000 at m/z 200, maximum injection time of 86ms and agc target value of 1 x 10 5 . repeated sequencing of already acquired precursors was limited by setting a dynamic exclusion of 60 seconds and 7 ppm and advanced peak determination was deactivated. an ms 2 based 530 method was chosen, because of higher precision and identification rates (hogrebe et al., 2018) . phospho-peptide fractions intrinsically exhibit lower complexity, rendering them less prone to ratio compression by isolation interference. raw files were analyzed using proteome discoverer (pd) 2.4 software (thermofisher 535 scientific). spectra were selected using default settings and database searches performed using sequestht node in pd. database searches were performed against trypsin digested homo sapiens swissprot database, sars-cov-2 database (uniprot pre-release). static modifications were set as tmt6 at the n-terminus and lysines and carbamidomethyl at cysteine residues. search was performed using sequest ht taking the following dynamic modifications into 540 account: oxidation (m), phospho (s, t, y), met-loss (protein n-terminus), acetyl (protein nterminus) and met-loss acetyl (protein n-terminus). for whole cell proteomics, the same settings were used except phosphorylation was not allowed as dynamic modification. for phosphoproteomics all peptide groups were normalized by summed intensity normalization and then analyzed on peptide level. for whole cell proteomics normalized psms were summed for each 545 accession and data exported for further use. peptide and protein identifications were validated using a concatenated target-decoy strategy and fdr was estimated using q-values calculated by percolator applying 1% and 5% cut-offs for high and medium confidence hits, while only high confident proteins are reported. phosphosite localization probabilities were calculated using the ptmrs-node working in "phosphors mode" and using default settings. 550 unless otherwise stated significance was tested by unpaired, two-sided students t-tests with equal variance assumed. resulting p values were corrected using the benjamini-hochberg fdr 555 procedure. adjusted p values smaller/equal 0.05 were considered significant. for phosphoproteomics an additional fold change cutoff was applied (log2 > |1|), while for total protein levels, due to different dynamic range, a fold change cutoff of log 2 > |0.5| was applied. kinase motifs of phosphopeptides from sars-cov-2 proteins were predicted using netphos 3.1 560 (blom et al., 1999) and gps 5.0 (stand-alone version) using the fasta-file of the uniprot prerelease which was also used for the proteomics data analysis. (blom et al., 2004; wang et al., 2020) . for netphos, only kinases with a score above 0.5 were considered as positive hits. for gps 5.0, sequences were submitted separately for s/t-and y-kinases and the score threshold was set to "high". for the final list in supplementary table 3 , only the top hits with the highest 565 scores were considered. z-scores were calculated for each phospho-site and the total protein levels individually. phosphosites were collapsed by average. for merging phosphorylation and total protein levels z-scores for collapsed phosphorylation and protein level were added for each condition and 570 replicate. thus, both negative z-scores (downregulation) will produce a lower combined z-score and vice versa two positive z-scores will produce a larger combined z-score. next, euclidean distance correlation for all possible protein-protein pairs were calculated, taking all conditions and replicates individually into account. a heatmap was then build by euclidean distance hierarchical clustering of the correlation matrix. 575 pathway enrichment analysis was performed by reactomefi cytoscape plugin or by string functional enrichment analysis. both analysis used reactome database for pathway annotations. all proteins were loaded into reactomefi cytoscape plugin to visualize protein-protein functional 580 interaction network. next, drugs were overlaid by reactomefi and network was filtered for the drugs and the first interacting partners. layout was calculated by yfileslayout algorithm. all proteins showing significant regulation were loaded by omicsvisualizer cytoscape plugin and string interaction network was retrieved with a confidence cutoff of 0.9. for egfr 585 subnetwork, egfr was selected with first interacting neighbors. highlights: • phosphoproteomics of sars-cov-2 infected cells reveal the signaling landscape • sars-cov-2 proteins are extensively phosphorylated in host cells • infection leads to activation of growth factor receptor signaling • drugs inhibiting growth factor receptor signaling prevent viral replication in this study, klann et al. dissected the host cell signaling landscape upon infection with sars-cov-2. mapping differential signaling networks identified a number of pathways activated during infection. drug-target network analysis revealed potential therapeutic targets. growth factor receptor signaling was highly activated upon infection and its inhibition prevented sars-cov-2 replication in cells. j o u r n a l p r e -p r o o f growth factor receptor growth factor receptor growth factor receptor mek inhibition drives anti-viral defence in rv but not rsv challenged human airway epithelial cells through akt/p70s6k/4e-bp1 signalling vaccinia virus hijacks egfr signalling to enhance virus spread 620 through rapid and directed infected cell motility sequence and structure-based prediction of eukaryotic protein phosphorylation sites1 1edited by prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence proteomics of sars-cov-2-infected host cells reveals therapy targets epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus 630 pneumonia in wuhan, china: a descriptive study detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr the epidermal 635 growth factor receptor (egfr) promotes uptake of influenza a viruses (iav) into host cells phase i and pharmacokinetic study of the oral farnesyl transferase inhibitor sch 66336 given twice daily 640 to patients with advanced solid tumors measurement of sorafenib plasma concentration by highperformance liquid chromatography in patients with advanced hepatocellular carcinoma: is it useful the application in clinical practice? a pilot study a sars-cov-2-human protein-protein interaction map reveals drug targets and potential drug-repurposing phosphorylation regulates the activity of the smn complex during assembly of spliceosomal u snrnps plaque assay for human coronavirus nl63 using human colon carcinoma cells benchmarking common quantification strategies for large-scale phosphoproteomics pkr activation and eif2α phosphorylation mediate human globin mrna splicing at spliceosome assembly omipalisib (gsk458), a novel pan-pi3k/mtor inhibitor, exhibits in vitro anti-lymphoma activity in chemotherapy-sensitive and -resistant models of burkitt lymphoma antiviral potential of erk/mapk and pi3k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis functional translatome proteomics reveal 670 converging and dose-dependent regulation by mtorc1 and eif2α epstein-barr virus lmp1 activates egfr, stat3, and erk through effects on pkcδ network bioinformatics analysis provides insight into drug repurposing for clinical pathology of critical patient with novel coronavirus pneumonia egfr and epha2 are host factors for hepatitis c virus entry 680 and possible targets for antiviral therapy first-in-human, phase i dose-escalation study of the safety, pharmacokinetics, and pharmacodynamics of ro5126766, a first-in-class dual mek/raf inhibitor in patients with solid tumors phosphorylation of human prp28 by srpk2 is required for integration of the u4/u6-u5 tri-snrnp into the spliceosome multinotch ms3 enables accurate, sensitive, and 690 multiplexed detection of differential expression across cancer cell line proteomes regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism first-in-human phase i study of 695 gsk2126458, an oral pan-class i phosphatidylinositol-3-kinase inhibitor, in patients with advanced solid tumor malignancies the pride database and related tools and resources in 2019: improving support for quantification data influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade analysis of ace2 in polarized epithelial cells: surface expression and function as receptor for severe acute respiratory syndrome-associated coronavirus first-in-human phase i study of pictilisib (gdc-0941), a 710 potent pan-class i phosphatidylinositol-3-kinase (pi3k) inhibitor, in patients with advanced solid tumors phase ii randomized preoperative window-of-opportunity study of the pi3k inhibitor pictilisib plus anastrozole compared with anastrozole alone in 715 patients with estrogen receptor-positive breast cancer repurposing therapeutics for covid-19: supercomputer-based docking to the sars-cov-2 viral spike protein and viral spike protein-human ace2 interface severe acute 720 respiratory syndrome coronavirus protein 6 accelerates murine coronavirus infections respiratory virus-induced egfr activation suppresses irf1-dependent interferon λ and antiviral defense in airway epithelium the role of epidermal growth factor receptor (egfr) signaling in sars coronavirus-induced pulmonary fibrosis overactive egfr signaling leads to increased fibrosis after sars-cov infection fast identification of possible drug treatment of coronavirus disease -19 730 (covid-19) through computational drug repurposing study 0: an update on the prediction of kinase-specific phosphorylation sites in proteins double-735 stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses discovery and development of sorafenib: a multikinase inhibitor for treating cancer glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication human cytomegalovirus glycoprotein complex gh/gl/go uses pdgfr-α as a key for entry the egfr family and its ligands in human cancer: signalling mechanisms and therapeutic opportunities airway mucin production involves a novel tlr3-egfr-dependent pathway we thank the quantitative proteomics unit (ibc2, goethe university frankfurt) for support and expertise on lc-ms instrumentation and data analysis and christiane pallas and lena 280stegmann for support of with experimental work. key: cord-266977-5swwc6kr authors: secker, thomas.j.; leighton, timothy.g.; offin, douglas.g.; birkin, peter.r.; hervé, rodolphe.c.; keevil, charles.w. title: journal of hospital infection a cold water, ultrasonic activated stream efficiently removes proteins and prion-associated amyloid from surgical stainless steel date: 2020-09-19 journal: j hosp infect doi: 10.1016/j.jhin.2020.09.021 sha: doc_id: 266977 cord_uid: 5swwc6kr background: sterile service department decontamination procedures for surgical instruments struggle to demonstrate efficient removal of the hardiest infectious contaminants, such as prion proteins. a recently designed novel system, which utilises a low pressure ultrasonic activated, cold water stream, has previously demonstrated efficient hard surface cleaning of several biological contaminants. aim: to test the efficacy of an ultrasonically activated stream for the removal of tissue proteins, including prion-associated amyloid, from surgical stainless steel (ss) surfaces. methods: test surfaces were contaminated with 22l, me7 or 263k prion infected brain homogenates. the surfaces were treated with the ultrasonically activated water stream for contact times of 5 and 10 seconds. residual proteinaceous and amyloid contamination were quantified using sensitive microscopic analysis, and immunoblotting was used to characterize the eluted prion residues before and after treatment with the ultrasonically activated stream. findings: efficient removal of the different prion strains from the surgical ss surfaces was observed, and reduced levels of protease sensitive and resistant prion protein was detected in recovered supernatant. conclusions: this study demonstrated that an ultrasonically activated stream has the potential to be a cost-effective solution to improve current decontamination practices and has the potential to reduce hospital acquired infections. instruments struggle to demonstrate efficient removal of the hardiest infectious contaminants, 23 such as prion proteins. a recently designed novel system, which utilises a low pressure 24 ultrasonic activated, cold water stream, has previously demonstrated efficient hard surface 25 cleaning of several biological contaminants. 26 aim: to test the efficacy of an ultrasonically activated stream for the removal of tissue 27 proteins, including prion-associated amyloid, from surgical stainless steel (ss) surfaces. 28 methods: test surfaces were contaminated with 22l, me7 or 263k prion infected brain 29 homogenates. the surfaces were treated with the ultrasonically activated water stream for 30 contact times of 5 and 10 seconds. residual proteinaceous and amyloid contamination were 31 quantified using sensitive microscopic analysis, and immunoblotting was used to characterize 32 the eluted prion residues before and after treatment with the ultrasonically activated stream. at present, the reprocessing of surgical instruments utilises; a pre-wash, washer/disinfector 44 cycle (run at elevated temperature with detergents) and sterilisation in high heat/pressure 45 autoclaves 1 . decontamination protocols for reusable surgical instruments are very efficient 46 against microbiological contaminants. however, highly hydrophobic proteins such as prions, 47 responsible for the transmission of variant creutzfeldt-jakob disease (vcjd), are readily 48 adsorbed to surgical stainless-steel surfaces and poorly removed or inactivated by current 49 decontamination methods. this results in an impending risk of iatrogenic transmission of 50 vcjd 2-5 . a risk that has been experimentally demonstrated in both animal and cell-based 51 bioassays [6] [7] [8] [9] . 52 the latest estimated prevalence of asymptomatic carriers of the causative protein of vcjd 53 (prp sc ) in the uk is approximately 1/2000 10 . while the full impact of the genetic 54 susceptibility of the host remains unclear, the ostensibly long incubation periods and the 55 potential for disease transmission via infected blood [11] [12] [13] , imply that all surgical procedures 56 pose a risk of vcjd transmission. 57 improvements in the methodologies used for reprocessing surgical instruments, potentially 58 contaminated with prions, are required to diminish the risk of iatrogenic vcjd transmission. 59 novel, specialised prion decontamination protocols have been developed and in some cases 60 marketed for sterile service departments (ssds) 7, 14-22 . however, some of these protocols 61 are very aggressive and can be damaging to instrument surfaces and/or the 62 washer/disinfectors themselves 14 . simple methods to adopt into ssd's have been researched 63 and demonstrated improved efficiency over current practices, such as preventing instruments 64 from drying once contaminated, i.e. keeping them in a moist environment prior to cleaning 23-65 27 . 66 j o u r n a l p r e -p r o o f bjerknes forces 43 aid the scrubbing bubbles in efficiently removing contaminants from 92 microscopic crevices, such as those found on worn surgical instruments 44 , that are 93 traditionally difficult to clean by brushes, wiping, or by chemical means that rely on passive 94 diffusion for reagents to penetrate deep into the crevice 45 . the efficient removal of 95 contamination from crevices using a uas system has been demonstrated previously 40 . 96 furthermore, the microstreaming that radiates from the resonating bubbles can penetrate into 97 crevices present on the surfaces of the contaminant as shown in the insert in figure 1 46 . the 98 fact that such results can be obtained in cold water without chemical additives warrants 99 investigation of uas for the removal of infectious prion proteins from surgical surfaces. 100 high temperature decontamination using aggressive enzymatic or alkaline solutions, that are 101 currently adopted to clean expensive surgical items (such as intricate neurosurgical tools) are 102 ineffective at protein and prion removal, and can shorten the surgical item lifetime 47 . it is not 103 the purpose of this study to explore the replacement of such standard cleaning practices. 104 however, given the above properties of uas, it is important to explore the possible benefits 105 of including an innovative cold-water uas pre-wash (at the stage where ssds conduct hand 106 brushing of instruments under a stream of water) that can be introduced with minimal 107 operator training. this would be particularly beneficial if it could be conducted immediately 108 after instrument use (e.g. before contaminated tissue dries on the instrument and becomes 109 harder to remove), although in this trial the contaminant is tested in a dried-on state. the 110 question is whether such a uas pre-wash could remove a substantial proportion of the 111 contaminant, especially from microscopic crevices of the type associated with worn surgical 112 instrument surfaces, and break up aggregates in which the inner portion of biological 113 contaminant is partially protected from subsequent enzymatic cleaning chemistries. 114 a previous study demonstrated efficient tissue protein removal from surgical stainless steel 115 using the uas 39 . however, due to the globular nature of the predominantly β-sheet 116 structured infectious prion protein, it adheres to surgical stainless steel far more rigorously 117 than do normal brain tissue proteins, and therefore the ability of uas to remove brain tissue 118 protein cannot be taken as an indicator of any efficacy in reducing the iatrogenic transmission 119 risk of vcjd. therefore, this study involved the contamination of surgical stainless-steel 120 surfaces with several amyloid-rich brain homogenates from prion infected rodents. normal 121 tissue proteins and more hazardous prion-associated amyloid were differentially stained and 122 analysed using sensitive in situ microscopy, to compare the ability of uas to remove both 123 during the same cleaning operation. inoculating, tokens were decontaminated and analysed to be deemed free of any 131 contamination following a previously described protocol 49 . 132 murine scrapie me7-infected brain homogenate produced from c57bl mice (tse resource 134 centre, roslin institute, university of edinburgh, scotland, uk); murine scrapie 22l-135 infected brain homogenate produced from c57bl/6j mice (kindly donated from the 136 neuroscience department, school of biological sciences, university of southampton) and 137 syrian hamster scrapie 263k-infected brain homogenates (tse resource centre, roslin 138 institute, university of edinburgh, scotland, uk) were standardized to 1 mg/ml (bsa 139 equivalent) in phosphate buffered saline (pbs, gibco) with 0.1 % (v/v) tween 20 (sigma-140 aldrich) as previously described 50 . 141 pristine tokens were spiked with 1 µl (1 µg bsa equivalent) drops of 22l, me7 or 263k 143 infected brain homogenate, and dried at 37 o c for 2 hours or room temperature for 24 hours. 144 tokens were subjected to decontamination using a prototype recirculating uas device (the 145 mark i starstream ® system (f0030001)) using fresh dh 2 o for each sample, running at 2.32 ± 146 0.02 l/min at room temperature with the ultrasound on for 5 and 10 s contact times, with the 147 sample being 10 mm from the nozzle ( figure 1 ). once processed the tokens were dried at 148 37 o c for 1 hour prior to staining and analysis. 149 residual tissue protein and prion-associated amyloid on the control and processed surfaces 151 was quantified, in situ, using the total protein blot stain sypro ruby (sr; invitrogen, uk) 152 and the amyloid specific stain thioflavin t (tht [0.2% (w/v) in 0.01m hcl]; sigma-153 aldrich), as described elsewhere 50, 51 . fluorescent signal was visualised using episcopic 154 differential interference contrast (edic) microscopy coupled with epifluorescence (ef -155 best scientific, wroughton, uk) 50, 52 . full x/y scans of the contaminated areas were 156 acquired at x100 magnification showing the sypro ruby (excitation: 470nm; emission: 157 618nm) and tht (0.2% (w/v) in 0.01m hcl sigma-aldrich) signals. the captured images 158 were analysed using imagej software (national institutes of health). 159 to analyse the effects of the uas treatment on infectious prion proteins, immunoblot 161 analysis was used to determine the presence of prp c and proteinase k (pk) resistant prp sc in 162 both 22l-spiked distilled water, as an untreated control, and the effluent taken from the uas system post cleaning of 22l-spiked stainless-steel tokens. controls were prepared by spiking 164 1 l of sterile distilled water with 15 µg of 22l-infected brain homogenate. uas positive 165 samples were prepared from capturing the 1 l uas effluent post cleaning of 15 surgical 166 stainless-steel tokens contaminated with 1 µg 22l-infected brain homogenates each (dried for 167 24 hours at room temperature) as described above. the control and effluent solutions were 168 filtered through nitrocellulose membranes to capture the suspended protein aggregates. 169 after 24 h drying at room temperature the 22l brain homogenate, again demonstrated the 203 highest affinity for the stainless steel with the highest attachment of protein and prion-204 associated amyloid observed. when compared with 2 hours drying, the 263k-contaminated 205 homogenate resulted in higher protein attachment after 24 h drying and the me7 infected 206 brain homogenates demonstrated similar protein attachment but higher prion-associated 207 amyloid attachment (figure 3 ). the removal of 22l and me7 tissues was slightly more 208 difficult using a 5 s uas treatment with 91 and 90 % protein and 97 and 99 % amyloid 209 removal, respectively (figure 3 ). after 10 s uas treatment the removal was improved with 210 98 and 99 % protein and 99 -100 % amyloid removal, respectively. the 263k was harder to 211 remove after 24 hours drying with only 56 % protein and 90 % amyloid removal after the 5 s 212 uas treatment, however, after the 10 s uas treatment the cleaning was improved with 74 % 213 protein and 87 % amyloid removal (figure 3 ). the percentage of amyloid within the total 214 residual contamination was again very low with 4 -8 % amyloid remaining for all the 215 samples after 10 s uas contact time (figure 3) . 216 the effluent from the uas system after decontaminating the 22l spiked surfaces was filtered 218 and labelled for residual prion protein (both non-resistant and pk-resistant) and compared to 219 control samples of distilled water spiked with the equivalent amount of 22l brain 220 homogenate. a clear reduction of both the pk-sensitive and pk-resistant prion protein from 221 the tokens was observed (as demonstrated by the protein capture on nitrocellulose membranes 222 following the previously demonstrated 98 -99 % protein and 99 -100 % amyloid removal, 223 described above) after 10 s uas treatment (figure 4) . the reduction in immuno-labelled 224 prion proteins post uas treatment could be demonstrating that the uas treatment is 225 destructive to the antibody specific epitopes of the prion protein, therefore reducing the 226 immunochemical detection post uas treatment. furthermore, small protein aggregates could 227 be observed in the control samples but not in the samples post uas treatment, suggesting that 228 the uas may degrade and/or solubilize these aggregates. 229 230 current practices for the decontamination and sterilisation of surgical instruments within 232 ssds are not entirely efficient at removing all potentially infectious material, especially, 233 hardy prion proteins. therefore, surgical instruments which may have come in contact with 234 cjd-infected tissues cannot be deemed safe post cleaning 3, 7, 16, 53 and are subsequently 235 quarantined. simple, cost effective methods to prevent the initial attachment of bioburden to 236 surgical surfaces have been demonstrated [25] [26] [27] . ultrasonic baths provide efficient cleaning 237 using water alone, however; the limitations associated with water baths was described earlier 238 in this manuscript. this study has tested the efficacy of uas technology for the removal of 239 total protein and prion-amyloid from stainless steel, which is considered the most difficult 240 contaminant to decontaminate in the surgical field. 241 the uas technology demonstrated significant removal of the three prion strains tested after 242 differing drying and uas treatment times; however, increased uas treatment times are 243 required to further improve the efficacy of the uas treatment. the efficient removal of me7 244 and 22l, both murine adapted scrapie strains, was very similar following both drying and 245 uas treatment times. however, 263k, a hamster adapted scrapie strain, was harder to 246 remove and would require a longer uas treatment to reduce to the levels observed with the 247 two murine strains. this observation suggests that the hamster brain constituents and prp sc 248 conformation is different to the mouse brains and showed increased affinity to stainless steel. 249 this highlights the importance of studying different prion strains, from different hosts when 250 determining the efficacy of hospital decontamination tools. for comparison of the efficacy of 251 the uas system to that of cleaning chemistries used in ssds, the removal of me7-infected 252 brain homogenate from stainless steel tokens using the same methodology as this study have 253 been previously published 3, 25 . hervé et al (2010) , tested four different cleaning chemistries 254 marketed for proteinaceous decontamination which demonstrated total protein removals of 255 39%, 97.9%, 98.9% and 99.85%, respectively 3 . secker et al (2012) , tested two cleaning 256 chemistries, also marketed for proteinaceous decontamination, which demonstrated total 257 protein removal of 0% and 90.1%, respectively 25 . all the cleaning chemistries tested in these 258 studies required heating of the cleaning solution, whereas the uas system tested here 259 removed 97% total protein with cold water and only a 10s contact time. a recent nihr 260 health technology assessment (hta) has extensively compared studies quantifying the 261 efficacy of interventions to reduce the surgical transmission of vcjd 54 . the other important 262 observation was that the uas system favourably removed the prion-associated amyloid 263 (infectious prion proteins in the aggregated form) from the surfaces. demonstrated by the low 264 percentages of the total residual proteinaceous contamination being tht positive amyloid, 265 compared to the comparative treatment using commercially available cleaning chemistries 3, 266 25 . 267 immunoblot analysis of both pk-sensitive and -resistant residues of prp was carried out to 268 determine the presence and state of prion aggregates post uas decontamination. following 269 the predetermined 98 -99 % protein and 99 -100 % prion-amyloid removal, described 270 above, the supernatant from the uas treatment was filtered and the prion proteins were 271 labelled. the pk resistant and sensitive aggregates observed in the control immunoblots were 272 not present in the uas treated samples; suggesting that the uas mechanism of action is 273 causing the breakdown of the prp aggregates, reducing the available epitopes for antibody 274 binding, and therefore a reduction in antibody positive prp residues. furthermore, this would 275 explain why an increase in the removal of prion-amyloid using the uas system was 276 observed, as described earlier. further work is required to confirm and determine if the 277 breakdown of prp caused by the action of uas correlates with a reduction in prion 278 the results from this study demonstrated efficient removal of tissue proteins, and more 280 importantly prion-associated amyloid from surgical stainless-steel harnessing the power of 281 water at ambient temperature. while the cleaning efficacy demonstrated by this system is 282 improved compared to that of the best currently available cleaning chemistries tested on the 283 same contaminants, interestingly the uas appeared more effective at removing prion-284 amyloid as well as the total proteinaceous contamination. 285 this study has demonstrated the efficacious ability of the uas to clean with just cold water. 286 however, the uas system could work also with chemical cleaners, so that you can get a 287 synergistic effect of mechanical (acoustically activated bubbles) and chemical cleaning. 288 furthermore, previous studies have demonstrated that the uas efficiently removes microbial 289 contamination from rough, etched surfaces 40 . thus demonstrating that a uas has the ability 290 to clean items, such as surgical instruments, that contain dynamic differences in surface 291 topography 40 . in its current form, the uas system is designed as a hand-held device, and the 292 plan is to include this in a pilot to test as a pre-clean before the surgical instruments proceed 293 on to washer-disinfectors (i.e. at the stage where currently ssds conduct washing by hand, 294 brushing and pre-cleaning of surgical instruments). the mechanical removal by uas of 295 prion-associated amyloid embedded in dried-on brain homogenate, demonstrates an 296 interesting parallel with the problem of removing the sars-cov-2 virus responsible for the 297 current covid-19 from touch-surfaces. lacking an appropriate attachment mechanism, the 298 virus relies on the stickiness of respiratory secretions in which it resides (that are composed 299 mainly of mucin glycoproteins, surfactant and intercellular fluid) to attach to abiotic surfaces. 300 therefore, the efficient ability of the uas system for removing prion-associated amyloid by 301 cleaning away the biological material, in the case of this study brain homogenate, as well as 302 bacteria and lubricant contamination, previously published 40, 46 , highlights the importance of 303 testing this system against viruses. if viruses can also be removed by uas, then incorporation 304 of uas in society to clean these surfaces with just water could aid infection prevention, the datasets generated and analysed during this study will be openly available from the 322 university of southampton repository at http://dx.doi.org/10.5258/soton/[ note to editors: 323 the policy of the university of southampton is that they will grant a link for insertion once 324 the paper is accepted to avoid their repository referring to papers that were not published]. 325 ts carried out laboratory experimentation, data analysis interpretation, participated in the 327 design of the study and drafted the manuscript; tl conceived the study, coordinated across 328 disciplines, and helped draft the manuscript; do and pb helped conceive the study and 329 provided support for the set-up and running of the uas; rh helped with experimental design 330 and drafting the manuscript; ck oversaw the microbiological components and helped draft 331 the manuscript. all authors gave final approval for publication. 332 one of the authors (t.g.l.) is director and inventor-in-chief of the company (sloan water 334 technology, ltd.) that holds the patent to this technology but has drawn no salary from this. 335 j o u r n a l p r e -p r o o f tissue protein (dark grey bars) and prion-associated amyloid (light grey bars) attachment 545 from different prion-infected brain homogenates (22l, me7 and 263k) to surgical stainless 546 steel pre and post treatment with an ultrasonically activated stream (uas) (graph a). brain 547 homogenate was initially dried for 2h at 37 o c prior to cleaning (pos). the orange dashes 548 represent percentage protein removal and the blue dashes represent percentage prion-549 associated amyloid removal (graph a). graph b has an expanded y-axis scale to distinguish 550 the lower levels of contamination. data shows mean ± sem (n=9), however, in 551 decontamination and other research areas 55 , outliers are also important to assessing outcomes, 552 whether it be risk of infection, or the response of the most sensitive individuals to some 553 stimulus; ***: p=≤0.001 for total proteins; † †: p=≤0.01 for amyloid, when compared to the 554 corresponding positive controls, respectively. 555 556 557 tissue protein (dark grey bars) and prion-associated amyloid (light grey bars) attachment 560 from different prion-infected brain homogenates (22l, me7 and 263k) to surgical stainless 561 steel pre and post treatment (5 and 10s contact times) with an ultrasonically activated stream 562 (uas) (graph a). brain homogenate was initially dried for 24h at room temperature prior to 563 cleaning (pos). the orange dashes represent percentage protein removal and the blue dashes 564 represent percentage prion-associated amyloid removal (graph a). graph b has an expanded 565 y-axis scale to highlight the lower levels of contamination. data shows mean ± sem (n=9); 566 however, in decontamination and other research areas 55 , outliers are also important to 567 assessing outcomes, whether it be risk of infection, or the response of the most sensitive 568 individuals to some stimulus; *: p=≤0.05 and ***: p=≤0.001 for total proteins; † †: p=≤0.01 569 and † † †: p=≤0.001 for amyloid, when compared to the corresponding positive controls, 570 respectively. 571 572 immunoblot films showing captured proteins from 1 l of 22l-spiked solution containing 15 574 µg of 22l homogenate in distilled water (a and b) and from the uas system effluent after 575 treating surfaces contaminated with the equivalent amount of 22l homogenate (c and d). 576 proteins were detected using the primary antibody 6h4, without (a and c) or with pk 577 digestion (b and d). 578 minimise 337 transmission risk of cjd and vcjd in healthcare settings cjd): guidance, data and analysis reports by the uk 341 government department of health (22 october2015) crown copyright gajdusek dc 343 transmission of creutzfeldt-jakob disease to a chimpanzee by electrodes 344 contaminated during neurosurgery keevil cw current risk of iatrogenic creutzfeld-jakob disease in 346 the uk: efficacy of available cleaning chemistries and reusability of neurosurgical 347 instruments keevil cw diathermy forceps and pencils: reservoirs for 349 protein and prion contamination? surface 351 decontamination of surgical instruments: an ongoing dilemma highly sensitive, 354 quantitative cell-based assay for prions adsorbed to solid surfaces investigations 357 of a prion infectivity assay to evaluate methods of decontamination weissmann c transmission of 360 scrapie by steel-surface-bound prions infectivity of prion protein bound to 362 stainless steel wires: a model for testing decontamination procedures for transmissible 363 spongiform encephalopathies. infection control and hospital epidemiology : the 364 official journal of the society of detection 370 of prion infection in variant creutzfeldt-jakob disease: a blood-based assay. the 371 will rg creutzfeldt-jakob disease and blood 373 transfusion: results of the uk transfusion medicine epidemiological review study busick dn effects on instruments of the world 379 health organization-recommended protocols for decontamination after possible 380 exposure to transmissible spongiform encephalopathy-contaminated tissue decontamination of prion protein (bse301v) using a genetically engineered protease collinge j a 386 standardized comparison of commercially available prion decontamination reagents 387 using the standard steel-binding assay prion inactivation using a 390 new gaseous hydrogen peroxide sterilisation process decontamination 393 of surgical instruments from prions. ii. in vivo findings with a model system for 394 testing the removal of scrapie infectivity from steel surfaces the challenge of prion decontamination sklaviadis t photocatalytic degradation of 399 prions using the photo-fenton reagent keevil cw doped diamond-402 like carbon coatings for surgical instruments reduce protein and prion-amyloid 403 biofouling and improve subsequent cleaning halting the spread of human prion disease--exceptional measures for an 405 exceptional problem keevil cw application of a 407 fluorescent dual stain to assess decontamination of tissue protein and prion amyloid 408 from surgical stainless steel during simulated washer-disinfector cycles keevil cw effect of drying time, ambient 411 temperature and pre-soaks on prion-infected tissue contamination levels on surgical 412 stainless steel: concerns over prolonged transportation of instruments from theatre to 413 central sterile service departments adsorption of prion and tissue proteins to surgical 415 stainless steel surfaces and the efficacy of decontamination following dry and wet 416 storage conditions keevil cw efficacy of humidity retention bags for 418 the reduced adsorption and improved cleaning of tissue proteins including prion-419 associated amyloid to surgical stainless steel surfaces quantitative measurement of the efficacy of protein removal by cleaning 422 formulations; comparative evaluation of prion-directed cleaning chemistries offin d a new approach to ultrasonic cleaning the collapse of single bubbles 427 and approximation of the far-field acoustic emissions for cavitation induced by shock 428 wave lithotripsy white pr prediction of far-430 field acoustic emissions from cavitation clouds during shock wave lithotripsy for 431 development of a clinical device crawford ae the measurement of cavitation 435 characterisation of measures of reference acoustic cavitation (comorac): an 436 experimental feasibility trial acoustic fields: modern trends and applications ultrasonic cleaning: an historical perspective measurement of cavitation activity in ultrasonic cleaners acoustics: from whales to other worlds. proceedings of the 443 institute of acoustics transient processes near the threshold of acoustically 445 driven bubble shape oscillations the acoustic bubble: oceanic bubble acoustics and ultrasonic cleaning leighton tg electrochemical 'bubble swarm' 450 enhancement of ultrasonic surface cleaning cold water 453 cleaning of brain proteins, biofilm and bone -harnessing an ultrasonically activated 454 stream dental biofilms with an ultrasonically activated water stream bubbles vs 459 biofilms: a novel method for the removal of marine biofilms attached on antifouling 460 coatings using an ultrasonically activated water stream acoustic radiation force on a parametrically distorted 466 bubble evaluation 468 of stainless steel surgical instruments subjected to multiple use/processing leighton tg an electrochemical and high-speed imaging study 471 of micropore decontamination by acoustic bubble entrapment industrial 474 lubricant removal using an ultrasonically activated water stream, with potential 475 application for coronavirus decontamination and infection prevention for sars-476 current limitations about the cleaning of luminal endoscopes sidiropoulou t 480 a simple method for blocking the deep cervical nerve plexus using an ultrasound-481 guided technique keevil cw amyloid-specific 483 fluorophores for the rapid, sensitive in situ detection of prion contamination on 484 surgical instruments keevil cw a rapid dual staining procedure 486 for the quantitative discrimination of prion amyloid from tissues reveals how 487 interactions between amyloid and lipids in tissue homogenates may hinder the 488 detection of prions keevil cw rapid method for the 490 sensitive detection of protein contamination on surgical instruments keevil cw rapid detection of biofilms and adherent pathogens using scanning 493 confocal laser microscopy and episcopic differential interference contrast microscopy flan b in vitro infectivity assay for 496 prion titration for application to the evaluation of the prion removal capacity of 497 biological products manufacturing processes wong r interventions to 500 reduce the risk of surgically transmitted creutzfeldt-jakob disease: a cost-effective 501 modelling review analogies in contextualizing human response to airborne ultrasound and fish response 504 to acoustic noise and deterrents key: cord-257392-u6jy6w1m authors: zhao, yanfeng; ben, haijing; qu, su; zhou, xinwen; yan, liang; xu, bin; zhou, shuangcheng; lou, qiang; ye, rong; zhou, tianlun; yang, pengyuan; qu, di title: proteomic analysis of primary duck hepatocytes infected with duck hepatitis b virus date: 2010-06-07 journal: proteome sci doi: 10.1186/1477-5956-8-28 sha: doc_id: 257392 cord_uid: u6jy6w1m background: hepatitis b virus (hbv) is a major cause of liver infection in human. because of the lack of an appropriate cell culture system for supporting hbv infection efficiently, the cellular and molecular mechanisms of hepadnavirus infection remain incompletely understood. duck heptatitis b virus (dhbv) can naturally infect primary duck hepatocytes (pdhs) that provide valuable model systems for studying hepadnavirus infection in vitro. in this report, we explored global changes in cellular protein expression in dhbv infected pdhs by two-dimension gel electrophoresis (2-de) combined with maldi-tof/tof tandem mass spectrometry (ms/ms). results: the effects of hepadnavirus infection on hepatocytes were investigated in dhbv infected pdhs by the 2-de analysis. proteomic profile of pdhs infected with dhbv were analyzed at 24, 72 and 120 h post-infection by comparing with uninfected pdhs, and 75 differentially expressed protein spots were revealed by 2-de analysis. among the selected protein spots, 51 spots were identified corresponding to 42 proteins by ms/ms analysis; most of them were matched to orthologous proteins of gallus gallus, anas platyrhynchos or other avian species, including alpha-enolase, lamin a, aconitase 2, cofilin-2 and annexin a2, etc. the down-regulated expression of beta-actin and annexin a2 was confirmed by western blot analysis, and potential roles of some differentially expressed proteins in the virus-infected cells have been discussed. conclusions: differentially expressed proteins of dhbv infected pdhs revealed by 2-de, are involved in carbohydrate metabolism, amino acid metabolism, stress responses and cytoskeleton processes etc, providing the insight to understanding of interactions between hepadnavirus and hepatocytes and molecular mechanisms of hepadnavirus pathogenesis. the hbv, prototype of the hepadnaviridae family, is a noncytopathic hepatotropic dna virus replicating via reverse transcription [1] . more than 350 million individuals are hbv carriers worldwide and over one-third of them develop serious liver diseases such as chronic hepatitis, cirrhosis and primary hepatocellular carcinoma [2] . major obstacles in hbv research have been the inability of the virus to infect cells in vitro and lack of adequate animal models for hbv infection, though primary human hepatocytes and heparg cell line have been used to study hbv infection [3] . human primary hepatocytes and heparg cells can support hbv life cycle, but have limitations in accessibility, reproducibility and low level of hbv replication, and a large amount of input virus was needed to infect low proportion of cells [4] [5] [6] . dhbv and woodchuck hepatitis b virus (whbv) are classified into the family of hepadnaviridae. thus for hepadnavirus infection primary hepatocytes of ducks (dhbv) and woodchucks (whbv) are still considered as suitable models for investigating the viral replication and pathogenesis [7, 8] . the development of proteomic methods has enabled us to investigate the changes of cellular protein expression at a global scale to reveal virus-host interactions [9] [10] [11] [12] . the effect of hepadnavirus replication on the host cells, such as the carcinoma derived hepatocyte lines transfected with the hbv genome, heparg cell lines or hbv trans-genic mice, have been investigated by using 2-de analysis [13] [14] [15] . in the present study, we intend to utilize the dhbv-pdhs system to explore global protein expression changes during hepadnavirus infection by 2-de. a total of 75 differentially expressed protein spots were revealed by 2-de between dhbv infected and uninfected pdhs, and 51 protein spots have been identified by ms/ms analysis. differential expression of beta-actin and annexin a2 was confirmed by western blot analysis, and potential roles of some differentially expressed proteins in the viral infection have been discussed. pdhs isolated from the same liver of dhbv-negative cherry valley duckling were infected with dhbv at multiplicity of infection of 30 (moi, based on dhbv dna genome equivalents) and cultured for 12, 24, 72 and 120 h in l15 medium supplemented with 5% fetal bovine serum (fbs). the efficiency of dhbv infection in pdhs was determined by indirect immunofluorescence using anti-dhbv pres monoclonal antibody. pdhs inoculated with phosphate-buffered saline (pbs, ph 7.2) as a control. at 12 h and 24 h after infection, only a few cells showed fluorescence (data not shown), and at 72 h post-infection about 30% of cells expressed viral large surface antigen, indicating that cells were infected with dhbv, showed in figure 1 . dhbv dna in pdhs was analyzed by southern blot hybridization using an alpha-32 p-dctp labeled dhbv-specific probe, and dhbv in the supernatant was detected by real time polymerase chain reaction (pcr). single stranded forms of intracellular viral dna and dhbv copy number increasing in the supernatant indicated the replication of dhbv 72 h and 120 h post-infection (additional file 1 and 2). differentially expressed proteins between dhbv infected and uninfected pdhs at 24, 72 and 120 h post-infection were analyzed using the 2-de. the gels were stained by a modified silver staining method compatible with mass spectrometry (ms) analysis and processed for image analysis. on the 2-de gels (ph 3-10 nl, 24 cm), about 1150~1350 protein spots were detected. compared with the parallel uninfected pdhs, 91 differentially expressed protein spots were revealed by 2-de (p-values less than 0.05 with at least a 1.5-fold difference in percentage of the volume), shown in figure 2 and additional file 3 (see also additional file 4), and a total of 75 differentially expressed non-redundant protein spots were analyzed by ms/ms. differentially expressed protein spots between dhbv infected and uninfected pdhs, were excised, digested ingel with trypsin and determined by ms/ms. among 75 differentially expressed protein spots, 51 protein spots were identified corresponding to 42 proteins (table 1 and additional file 5). with a mascot cutoff score of 72 (pvalue less than 0.05), 51 spots were identified, and 37 spots were matched to orthologous proteins of avian species (26 protein spots to gallus gallus, 4 spots to anas platyrhynchos and 7 spots to other avian species), listed in table 1 . some of the differentially expressed protein spots such as annexin a2, beta-actin, lamin a, destrin, aconitase 2 and mn superoxide dismutase were illustrated in enlarged formats (figure 3 ), and representative mass spectrum of annexin a2 (spot 49) analyzed by maldi-tof/tof ms was shown in figure 4 . isoforms of annexin a2, alpha-enolase, lamin a, glyceraldehyde-3phosphate dehydrogenase (gapdh), heat shock protein 70 (hsp70) and elongation factor 2 have been identified. for example, protein spot 49 (mw 31 kda and pi of 5.45) and spot 50 (mw 10 kda and pi of 5.78) down-regulated in dhbv infected pdhs were both identified as annexin a2, and up-regulated protein spot 26 (mw 65 kda and pi of 6.61) and spot 27 (mw 66 kda and pi of 6.4) were matched to lamin a (theoretical mw 73.1 kda and pi of 6.5) showed in figure 2 . biological functions of the differentially expressed proteins in the dhbv-infected pdhs, were analyzed according to the gene ontology criteria and classified into carbohydrate metabolism (29%), amino acid metabolism (14%), cytoskeletal/structural protein (24%), stress response (18%) and other functions (16%), as shown in table 1 . the roles of selected differentially expressed proteins reported in viral infections were showed in table 2 . expression levels of annexin a2, beta-actin, hsp70, destrin, and lamin a were validated by western blot analysis to confirm the dynamic alterations of protein expression during dhbv infection. equal amounts (30 μg) of cell lysates of dhbv-infected and uninfected pdhs at 12, 24, 72 and 120 h post-infection were separated by sds-page. duck beta-actin and annexin a2 expression were detected down-regulated in the dhbv-infected pdhs at 12-120 h post-infection with mouse anti-beta-actin and anti-duck-annexin a2 as primary antibodies ( figure 5a ), that were consistent with the protein expression pattern revealed by the 2-de analysis. duck hsp70, destrin, and lamin a were not detected by western blot analysis with the rabbit anti-human or anti-mouse hsp70 polyclonal antibodies, rabbit anti-human destrin polyclonal antibody and rabbit anti-human lamin a polyclonal antibody. the same amount protein of each sample were applied to a parallel sds-page gel and stained with coomassie brilliant blue ( figure 5b ). hbv infection remains a public health problem worldwide. because the lack of appropriate cell lines that can support hbv infection efficiently, the cellular and molecular mechanisms of hepadnavirus infection remain incompletely understood. the hepadnavirus animal infection models such as ducks (dhbv) and woodchucks (whbv) have been used to investigate the viral replication, pathogenesis or hepadnavirus-associated hepatocellular carcinoma. dhbv-pdhs model is a valuable model of hepadnavirus infection with high reproducibility and efficiency [16] . in the present study, global changes in cellular protein expression in dhbv-infected pdhs were explored by 2-de combined with ms/ms. among the 75 differentially expressed protein spots, 51 spots have been identified by ms/ms corresponding to 42 proteins, in which 30 spots were matched to orthologous proteins of gallus gallus or anas platyrhynchos, 7 spots to other avian species, and 14 spots to non-avian species, while mass spectra of the other 24 protein spots did not match to any proteins in the current databases, possibly due to the incomplete genome sequence of anas platyrhynchos or low abundance of those protein spots. in previously studies, tong performed a proteomic analysis comparing hepg2 with hepg2.2.15 in which hbv genome integrated into cellular chromosome [13] , and narayan revealed 19 differentially regulated features in heparg cells by 2-de [14] . hepg2.2.15 is a hbv replication cell model but not an infection model, while the human hepatoma heparg cells are susceptible to hbv, but 10~20% of cells can be infected regardless of the amount of virus used (moi > 200) [4, 6] . in previous studies, it has been showed that at moi of 30, about 50%~60% pdhs can be reproducibly infected with dhbv [17] . some of differentially expressed proteins identified in the present study, such as alpha-enolase, lamin a, gapdh and cofilin2 have not yet been reported in hepadnavirus proteomic analysis. viruses depend on host cell metabolism for their replication. elucidation of the pathways/processes involving in the viral life cycle will help to understand the mechanisms of viral infection. in the 2-de analysis, the identified differentially expressed proteins were classified into carbohydrate metabolism, amino acid metabolism, cytoskeletal/structural protein, stress response and other functions according to the gene ontology criteria. some of differentially expressed proteins identified in the present study have been reported playing roles in viral infections, as shown in table 2 . in dhbv infected pdhs, the expression of some carbohydrate metabolic enzymes, such as phosphoglycerate kinase 1, triosephosphate isomerase, phosphoglycerate mutase 1 etc, was up-regulated. the differentially expressed proteins involving in carbohydrate metabolism, suggests perturbed energy metabolism in dhbv infections. hepatitis c virus (hcv) infection reprograms the cellular metabolisms to favor glucose fermentation and glycolytic intermediates toward the metabolite synthesis that supports the viral life cycle [18] . in lymphocytic choriomeningitis virus infection, there was a significant increasing in transcripts promoting gluconeogenesis for viral mediate synthesis, and a decreasing in transcripts promoting glycogenolysis in the early stage of infection [19] . however, gapdh and alpha-enolase, key enzymes involving in glycolysis and gluconeogenesis, are decreased in dhbv infected pdhs. gapdh and alphaenolase have been found associating with the cell membrane and in secreted viral particles of influenza virus, lentiviral vector etc [20, 21] . gapdh may phosphorylate the hbv core protein, and binds to the pres1 region of the hbv envelope antigen and posttranscriptional regulatory element in regulating expression of surface antigen, suggesting that gapdh plays an important role in the life-cycle of hbv infection [22] [23] [24] . the host cellular carbohydrate metabolism affected by dhbv infection may benefit viral replication. alterations of cytoskeleton networks were found in many viral infections [25] [26] [27] . hepadnavirus needs to manipulate and utilize the host cytoskeleton to promote viral infection like many viruses, although the mechanism is still unclear [28] . in dhbv infected pdhs, the microfilament-associated proteins, beta-actin and cofilin-2 were down-regulated, and three microfilament-associated proteins such as transgelin, destrin, and collapsin response mediator protein-2b were up-regulated. actin plays an active role in maturation of the viruses [29, 30] . many viruses require actin for viral entry and establishment of figure 2 . b) accession no. is the mascot result of maldi-tof/tof searched from the ncbinr database. c) protein score was from maldi-tof/tof identification. the proteins that had a statistically significant score great than 72 (p < 0.05) were considered identified. d) theoretical/experimental molecular mass. e) theoretical/experimental molecular pi. f) not applicable, because the spots on the gels were too weak or non-detectable. g) a represents the spot on one of the gels was detectable, n represents the spot on one of the gels was too weak to detect. [31] [32] [33] [34] . however, the actin cortex beneath the plasma membrane can also be an obstacle for virus entry or budding [35] . it has been reported that dhbv entry depends on both intact microtubules and their dynamic turnover but not actin cytoskeleton [28] . therefore the role of actin in dhbv replication is required to further investigation. lamin a is key structural components of the nuclear lamina and lamins, involving in dna replication and gene expression, as well as presenting a natural barrier against most dna viruses such as human cytomegalovirus (hcmv), kaposi's sarcoma-associated herpesvirus, herpes simplex virus (hsv) 1 and epstein-barr virus [36] . lamin a/c is phosphorylated in hsv-infected cells supporting a role in regulating virus capsid nuclear egress [37, 38] . infection of epstein-barr virus induced disassembly of the nuclear lamina and redistribution of nuclear lamin for the nuclear egress [39] . the expression of lamin a with different isoforms, were up-regulated in dhbv infected pdhs, suggesting that lamin a may play a role in dhbv replication. in dhbv infected pdhs, up-regulated expressions of amino acid metabolism enzymes, catalyzing interconversion of glutamate, histidine, and proline (glutamate dehydrogenase 1, urocanate hydratase, delta-1-pyrroline-5-carboxylate dehydrogenase, the orthologs in human referred to protein 16, 19 and 20, 22 in table 1 ), indicate that glutamine metabolism is enhanced. switching the anaplerotic substrate from glucose to glutamine to accommodate the biosynthetic and energetic needs of the viral infection and to allow glucose to be used biosynthetically was reported in hcmv infection [40] . hcvinfected cells exhibit increased levels of the enzymes catalyzing glutamine flux to replenish metabolic intermediates through the latter half of the citric acid cycle providing substrates for atp production [18] . thus simi-lar mechanism of glutamine metabolism may be at work in dhbv infection. stress response associated proteins including endoplasmic reticulum stress associated proteins such as hsp70, and chaperonin containing t-complex polypeptide 1 (tcp1) and oxidative stress associated proteins such as antioxidant enzymes mn superoxide dismutase and peroxiredoxin-3 (similar to antioxidant protein isoform 2) were found to be up-regulated post dhbv infection. hsp70 assists folding of many newly synthesized polypeptides, and refolding of the proteins misfolded [41, 42] . hsp70 can enhance flock house virus replication [43, 44] . hsp70 and hsp90 participate in dengue virus entry as a receptor complex [45] . moreover, hbv p protein activation in vitro is fundamentally dependent on heat shock protein 70 family hsc70/hsp40 [46] . in hbv-replicating hepad38 cell, expressions of heat shock proteins (hsp70 and hsp90) and mn superoxide dismutase increase, after hbv replication induced by tetracycline [47] . in humanized transgenic mice, inhibition of hbv replication results in suppression of mn superoxide dismutase expression in hepatocytes [47, 48] . it suggests that oxidative stress can be induced by hepadnavirus replication as epstein-barr virus [49] . annexin a2, belongs to a family of calcium-dependent, phospholipid binding proteins, is involved in many biological processes, such as the ca 2+ dependent exocytosis, calcium transport and cell proliferation. it participates in viral infection, including assisting in the assembly of hiv in monocyte-derived macrophages [50] , as a cellular cofactor supporting hiv-1 infection [51] , enhancing cytomegalovirus binding and membrane fusion [52] and supporting the replication of influenza viruses by mediating activation of plasminogen [53] . it has been reported that hbv polymerase activity was inhibited by interacted with s100a10, a protein binding to annexin a2 [54] . in hepg2.2.15 compared with hepg2, annexin a2 was revealed down-regulated [55] , which was consistent with our observation in dhbv-pdhs model and confirmed by western blot analysis. it indicated that annexin a2 may involve in hepadnavirus infection and warrants further investigation. beta-actin and gapdh are usually referred as the internal standards for detections of rna transcription and protein expression of genes. however, those proteins were found to be down-regulated post dhbv infection by 2-de analysis. recently, accumulated evidence showed that in hbv-related hepatocellular carcinoma or viral infections, beta-actin and gapdh are unsuitable controls in quantitative mrna expression or western blot analysis due to variations in expression [56] [57] [58] [59] [60] , though there are controversial observations [61] . these findings therefore highlight the importance of re-evaluating the housekeeping genes whose expressions may be affected by hepadnavirus infection. in summary, the present study explored global changes in cellular protein expression of hepadnavirus infection by 2-de analysis, using a natural dhbv-pdhs infection system. forty-two differentially expressed proteins in dhbv infected pdhs have been identified by ms/ms. most of them involve in carbohydrate metabolism, amino acid metabolism, stress responses and cytoskeleton processes including alpha-enolase, beta-actin, lamin a and annexin a2. it suggests that those proteins may play important roles in hepadnavirus infection. differential expressions of annexin a2 and beta-actin were confirmed by western blot analysis. further investigation of the roles of the differentially expressed cellular proteins will help to understand cellular and molecular mechanisms of hepadnavirus infection. cherry valley ducks (anas platyrhynchos) were purchased from breeding center of shanghai institute of veterinary medical sciences, china. animal protocols were approved by the ethics committee of fudan university. three-day-old ducklings with no congenital dhbv infection detected by pcr (sence: 5'-ctcactttgtg-gatctcattg-3', antisense: 5'-atcggatagtcg-ggttgg-3'), were used for pdhs cultures. duck hepatocytes were isolated with in situ liver perfusion method with modifications. after the duck was anesthetized with approximately 0.3 ml of 0.75% pentobarbital sodium, the liver was perfused via portal vein with prewarmed liver perfusion medium (gibco laboratories), then the inferior vena cava was cut to effuse the buffer liquid when the liver was engorged. at first, perfusion was maintained at 15 to 20 ml per minute with 100 ml of liver perfusion medium until the liver became blanch, then the liver became soft followed by 50 ml digestion buffer with 1 μg/ml of collagenase type iv (sigma) in l15 medium (gibco). after the perfusion, the gallbladder was removed and hepatocytes were dispersed in l-15 medium. hepatocytes were filtered through sterilized gauze, centrifuged at 40 g for 4 min, and washed three times with hepatocyte wash medium (gibco). then 4 × 10 6 hepatocytes were seeded onto 100-mm-diameter dish and incubated in l-15 medium containing 5% fbs (gibco laboratories), 15 mm hepes, 100 u penicillin per liter, 100 mg of streptomycin per liter, 1 mg of insulin (sigma) per liter and 10 -5 m hydrocortisone-hemisuccinate (sigma) at 37°c. the medium was changed every day. infectious dhbv were produced by lmh-d2 cell line which carries a stably integrated dhbv dimer and constitutively secretes dhbv virions (generous gift of william mason, fox chase cancer center, usa) [62] . dhbv particles were obtained from lmh-d2 cells by ultracentrifugation, and the virus pellet was suspended in pbs with 10% glycerol. dhbv was quantified using real time pcr as a titer of 2 × 10 9 copies per milliliter. pdhs cultured 16 h after plating were infected with purified dhbv viral particles at moi of 30, and incubated at 37°c overnight. then, they were washed with pbs three times and cultured for 12, 24, 72 and 120 h in l15 medium supplemented with 5% fbs. the efficiency of dhbv infection in pdhs was determined by indirect immunofluorescence and southern blot hybridization. monolayers of pdhs grown on glass coverslips were fixed directly by adding 4% polyoxymethylene at room temperature for 20 min, washed twice with pbs and preincubated with 3% bovine serum albumin for 30 min. after incubation with a 1:100 dilution of monoclonal mouse anti-dhbv pres (generous gift of john c. pugh and william mason, fox chase cancer center, usa) at 37°c for 60 min, the cells were washed three times with pbs, subsequently incubated with a 1:200 dilution of fitc-conjugated sheep anti-mouse igg (gghl-90f, immunology consultants laboratory) at 37°c for 30 min. cell nucleus were stained with 1 μg/ml 4',6'-diamidino-2-phenylindole (dapi, sigma) and mounted in 50% glycerol in pbs. efficiency of dhbv infection was observed by the confocal laser scanning microscope (leica). to detect dhbv replication, intrac-ellular dna was extracted from dhbv infected or uninfected pdhs. forty micrograms of dna from each sample was separated on a 1.5% agarose gel, and analyzed by southern blot hybridization with an alpha-32 p-dctp labeled dhbv-specific probe for detection as described previously [63] . the dhbv-infected and control (uninfected) pdhs at 24, 72, and 120 h post-infection were washed three times with ice-cold pbs before harvesting and stored at -80°c. pdhs for dhbv infection or the uninfected controls were from the same duck in order to avoid individual differences. approximately 2 × 10 7 cells were lysed in 1 ml lysis buffer (7 m urea, 2 m thiourea, 2% (w/v) chaps, 50 mm dithiothreitol (dtt), 2% (v/v) ph 3-10 nonlinear immobilized ph gradient (ipg) buffer (amersham biosciences) containing 1% protease inhibitor cocktail (roche) and 1 mm pmsf (sigma)), then sonicated on ice for 12 cycles, each consisting of 5 s pulse and 10 s pause. after centrifugation at 20,000 g at 4°c for 1 h, the supernatants of lysates were divided into aliquots and the protein concentrations were determined by the bradford assay. then, aliquots were stored at -80°c for further analysis. the 2-de gels were performed using 24-cm ipg strips (ph 3-10, nonlinear, ge healthcare) in ettan ipgphor isoelectric focusing system (amersham biosciences) plus ettan-dalt six system (amersham biosciences) according to the manufacturer's instructions. to compensate the variability of gel electrophoresis, at least three replicate gels were performed for each group. in the first dimensional isoelectric focusing (ief), 120 μg proteins of each sample were diluted to 450 μl with rehydration buffer containing 8 m urea, 2% (w/v) chaps, 50 mm dtt, 0.5% (v/v) ampholyte (ph 3-10, nonlinear, amersham biosciences), and ipg strips were allowed to rehydrate in the above solution under mineral oil. ief was performed as follow: 30 v for 6 h (active rehydration); 60 v for 6 h (active rehydration); 500 v for 2 h, rapid; 1,000 v for 2 h, rapid; 4,000 v for 2 h, linear; linear ramping to 8,000 v for 2 h, and finally 8,000 v for about 7 h with a total of 64 kvh at 20°c. then the ipg strips were incubated in equilibration buffer (75 mm tris-hcl (ph 8.8), 6 m urea, 29.3% (v/v) glycerol, 2% (w/v) sds and 0.002% (w/v) bromophenol blue) containing 2% (w/v) dtt for 15 min with gentle agitation, followed by incubation in the same equilibration buffer supplemented with 2.5% (w/v) iodoacetamide for 15 min at room temperature. the second dimension sds-page was performed on 1 mm thick 12.5% polyacrilamide vertical gels in ettan-dalt six system using 5 w/gel for 30 min, and followed by 12 w/gel at 10°c until the bromophenol blue dye front reached the end of the gels. the gels were stained by a modified silver staining method compatible with ms analysis [64] and scanned at 300 dpi (dots/inch) using imagescanner (umax, amersham biosciences). images were captured and analyzed by imagemaster 2d platinum 6.0 software (amersham biosciences). the percentage of the volume of the spots representing a certain protein was determined in comparison with the total proteins present in the 2-de gel. to select differentially expressed protein spots, quantitative analysis was performed using the student's t-test to compare the percentage volumes of spots between dhbv infected and uninfected groups at three time points. the differentially expressed protein spots with p-values less than 0.05 were considered as significant differences, and at least 1.5 fold difference in percentage of the volume for each spot was set as a threshold. these protein spots were selected and subjected to in-gel tryptic digestion and identification by ms. the differentially expressed protein spots were manually excised from the sliver-stained gels (each gel of 120 μg protein) and placed into a 96-well microplate. the gel pieces were destained with a solution of 15 mm potassium ferricyanide and 50 mm sodium thiosulfate (1:1) at room temperature for 10 min, then washed twice with deionized water, each for 30 min, and dehydrated in 80 μl of acetonitrile (acn) twice. then the samples were swollen in a digestion buffer containing 25 mm nh 4 hco 3 and 12.5 ng/μl trypsin (promega) at 4°c after 30 min incubation, and incubated at 37°c for more than 12 h. the peptide mixtures from the gel were extracted twice using 0.1% trifluoroacetic/50% acn at room temperature, re-suspended with 0.7 μl matrix solution (α-cyano-4-hydroxy-cinnamic acid (sigma) in 0.1% trifluoroacetic, 50% acn), allowed to dry in air under the protection of n2. the peptide mixtures from samples were analyzed by 4700 maldi-tof/tof proteomics analyzer (applied biosystems). the uv laser was operated at a 200 hz repetition rate with wavelength of 355 nm. the accelerated voltage was operated at 20 kv. myoglobin digested by trypsin was used to calibrate the mass instrument with internal calibration mode. all acquired spectra of samples were processed using 4700 series explore software (applied biosystems) in a default mode. the parent mass peaks with mass range 700-3200 da and minimum s/n 20 were picked out for tandem tof/tof analysis. combined ms and ms/ms spectra were submitted to mas-cot (v2.1, matrix science) by gps explorer software (v3.6, applied biosystems) and searched with the following parameters: ncbinr database (release date: 2009.11), taxonomy of bony vertebrates or viruses, trypsin digest with one missing cleavage, none fixed modifications, ms tolerance of 100 ppm, ms/ms tolerance of 0.6 da and possible oxidation of methionine. known contaminant ions (human keratin and tryptic autodigest peptides, etc.) were excluded. mascot protein scores (based on ms and ms/ms spectra) with greater than 72 were considered statistically significant (p < 0.05). the individual ms/ms spectrum with statistically significant (confidence interval >95%) and best ion score (based on ms/ ms spectra) was accepted. to eliminate the redundancy of proteins that appeared in the database under different names and accession numbers, the protein belonging to the species anas platyrhynchos or with the highest protein score (top rank) was singled out. duck cdna of annexin a2 was amplified by reverse transcription-pcr with the primers designed according to the sequence of the chicken annexin a2 (genbank accession no. gi|45382533, sense: 5'-ccgctcgaggtc-ctctccaccacacaggt-3' and antisense: 5'-ccg ctcgaggtcctctccaccacacaggt-3'). the fulllength of duck annexin a2 amplified from pdhs, was cloned into prokaryotic expression plasmid pet-28a (novagen). recombinant annexin a2 with c-terminal fusion his-tag was induced by iptg, and purified by ni-nta affinity chromatography (qiagen). balb/c mice were immunized by the purified recombinant duck annexin a2 with freund's complete adjuvant (sigma). the serum was collected 2 weeks following the final injection and the levels of anti-duck-annexin a2 antibody titers from immunized mice were determined by western blot. differential expression of duck beta-actin, annexin a2, hsp70, destrin and lamin a were confirmed by western blot analysis. the primary antibodies for detection were as follow: a monoclonal antibody against beta-actin (sigma), rabbit anti-human lamin a polyclonal antibody (santa cruz biotechnology and proteintech), rabbit antihuman hsp70 polyclonal antibody (santa cruz biotechnology), rabbit hsp70 polyclonal antibody (bios), rabbit anti-human destrin polyclonal antibody (protein-teck) and mouse anti-duck-annexin a2 polyclonal antibody prepared in our laboratory described as above. thirty microgram proteins from each sample were separated in 12% sds-page gels and transferred to pvdf membranes using the transfer system (biorad). the blots were blocked with 5% nonfat milk for 2 h at room temperature and incubated at 4°c overnight with 1:200-1:500 dilution of primary antibody. the blots were then washed four times with pbs containing 0.1% tween-20, and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (santa cruz biotechnology) 1 hour at room temperature. after washed four times with pbs containing 0.1% 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avian liver tumor cell line rapid resolution of duck hepatitis b virus infections occurs after massive hepatocellular involvement pharmacia a: a modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry proteomic analysis of primary duck hepatocytes infected with duck hepatitis b virus proteome science we would like to thank professor alastair i. h. murchie for careful reading and correcting the english of the revised manuscript. this work was supported by national natural science foundation of china (30670092, j0730860) and the program of ministry of science and technology of china (2010dfa32100, the authors declare that they have no competing interests.authors' contributions dq was responsible for the conception and design of the study. yz were responsible for pdhs culture, virus infection and sample preparation. hb and sq carried out the 2-de gels experiments, image analysis and excised the protein spots. xz performed the mass spectrometric analyses. yz and ly confirmed the differential expression by western blot. yz and hb carried out the analysis and interpretation of data. dq, yz and hb wrote the manuscript. bx, sz, ql, ry, tz, py have been involved in drafting the manuscript or revising it critically for important content. all authors read and approved the final manuscript. key: cord-259237-aty0vrat authors: frabutt, dylan a.; zheng, yong-hui title: arms race between enveloped viruses and the host erad machinery date: 2016-09-19 journal: viruses doi: 10.3390/v8090255 sha: doc_id: 259237 cord_uid: aty0vrat enveloped viruses represent a significant category of pathogens that cause serious diseases in animals. these viruses express envelope glycoproteins that are singularly important during the infection of host cells by mediating fusion between the viral envelope and host cell membranes. despite low homology at protein levels, three classes of viral fusion proteins have, as of yet, been identified based on structural similarities. their incorporation into viral particles is dependent upon their proper sub-cellular localization after being expressed and folded properly in the endoplasmic reticulum (er). however, viral protein expression can cause stress in the er, and host cells respond to alleviate the er stress in the form of the unfolded protein response (upr); the effects of which have been observed to potentiate or inhibit viral infection. one important arm of upr is to elevate the capacity of the er-associated protein degradation (erad) pathway, which is comprised of host quality control machinery that ensures proper protein folding. in this review, we provide relevant details regarding viral envelope glycoproteins, upr, erad, and their interactions in host cells. despite their vast diversity, animal viruses can be simply divided into two categories: non-enveloped viruses and enveloped viruses [1] . while non-enveloped viruses are wrapped with naked shells made of viral capsid proteins, enveloped viruses are covered with a lipid-bilayer, which is called a viral envelope. the viral envelope is obtained from progenitor host cells during the budding process, which can be a portion of plasma membrane or intracellular membrane. on the surface of the enveloped viruses, there are peplomers that project from the viral envelope, and play a critical role in viral infection. these peplomers are also described as spikes, which are made of viral envelope glycoproteins. envelope spikes serve to identify and bind to viral receptors on the host cell surface, allowing viral entry into cells and the initiation of infection by mediating virus-cell fusion. thus, the infectivity of enveloped viruses is absolutely dependent on the integrity of the viral envelope, and the functionality of the viral glycoproteins found therein. enveloped viruses are more stable than non-enveloped viruses under physiological conditions, at the expense of their sensitivity to high-temperature, low-ph, desiccation, or detergent-treatment, which limits their ability to withstand severe environments [2] . the entry of enveloped viruses requires the formation of a fusion pore between the viral envelope and the cell membrane, through which the viral genome is released into the cell. this fusion process is triggered by interactions between viral glycoproteins on the viral envelope and viral receptors on the cell surface, which can occur directly at the plasma membrane at neutral ph or in endocytic compartments at either low or neutral ph [3] . in addition, enveloped viruses can also enter cells through direct cell-to-cell contacts via virological glycosylation, which is one of the most common post-translational modifications in eukaryotic cells, is required for protein folding and maintaining protein structure. viruses have taken advantage of this benefit at nearly every step of the viral life cycle [12] . n-glycosylation significantly promotes their folding and solubility, enhances subsequent trafficking of these viral proteins to their destinations, and ensures that they are properly processed and incorporated into virions. nevertheless, glycosylation can have distinct effects that are both advantageous and detrimental to viral fitness. for example, if glycosylation occurs close to the glycoprotein processing sites, it may block the precursor cleavage by proteases and inhibit viral infection [13] ; if glycosylation occurs adjacent to the receptor-binding site, it may enhance the binding affinity and promote viral infection [14, 15] . in addition, the high density of glycans on virions may form a shield to impede antibody attack and promote immune evasion. however, these glycans can also become epitopes for stimulating neutralizing antibodies and the innate immune response, making viruses more vulnerable to immune clearance [16] . thus, there are multiple selective pressures on viral envelope glycosylation that can influence the pattern of glycosylation in order to achieve the optimal fitness in their hosts [17] . viruses are obligate intracellular parasites, and their glycoprotein biosynthesis and modification rely entirely on host cell machinery in the secretory pathway. therefore, viral and host proteins are glycosylated in a similar manner by the same mechanism. although glycans can be attached to polypeptide structures via several different mechanisms, asparagine n-linked glycosylation represents a fundamental and well characterized post-translational modification in eukaryotic organisms [18] . n-linked glycosylation starts from the membrane of the endoplasmic reticulum (er), where the tetradecasaccharide precursor is assembled. this precursor consists of two n-acetylglucosamine (glcnac), nine mannose (man, 4 are α1,2-linked), and three terminal glucose (glc) residues distributed on three extended man branches: a, b, and c (glc 3 man 9 glcnac 2 ) ( figure 1a ) [19, 20] . when nascent polypeptides enter the er lumen, the precursor is en bloc attached to asn residues of a nascent polypeptide in a consensus asn-x-(ser/thr) motif. after the attachment, these precursors are processed by a series of enzymes in both the er and the golgi apparatus to remold the core oligosaccharide into diverse n-linked glycan structures ( figure 1b ). the first step in this process is the sequential removal of the two outermost glc residues on branch a. the first glc residue is removed by glucosidase i (gi), resulting in the di-glycosylated oligosaccharide glc 2 man 9 glcnac 2 , which is recognized by an er transmembrane lectin malectin [21] . the second glc residue is then removed by glucosidase ii (gii), resulting in the mono-glucosylated oligosaccharide glc 1 man 9 glcnac 2 , which is recognized by two other er lectins, the membrane-bound calnexin (cnx) and/or soluble calreticulin (crt). interaction with these two chaperones segregates the newly formed glycoprotein and provides access to protein disulfide isomerases (pdis) such as erp57, which promotes disulfide bond formation, resulting in protein folding into a native conformation. once a protein is properly folded, gii cleaves the last glc residue on branch a, which releases the protein from the cnx/crt cycle. the er class i α-mannosidase (ermani) then cleaves the outermost man residue on branch b on native proteins, resulting in the oligosaccharide man 8 glcnac 2 . these high-man glycans are then recognized by lectins including er-golgi intermediate compartment-53 (ergic-53), vesicular integral membrane protein of 36kda (vip36), and vip36-like (vipl), which promote trafficking from the er to the golgi [22] . the remaining man residues are cleaved by the golgi mannosidases, and the glycan remolding process is continued through the remainder of the n-glycosylation pathway, which generates functional glycoproteins that are delivered to the cell surface ( figure 1b ). in addition to these chaperones and enzymes that promote protein folding, the er is also equipped with a unique quality control mechanism that extracts and degrades proteins that are not correctly folded or assembled into their native conformation, which is called er-associated protein degradation (erad) [23] . in fact, the folding efficiency of glycoproteins in the er is very low, which requires cycles of association and dissociation from cnx/crt to ensure proper glycoprotein maturation. if glycoproteins with the man 9 glcnac 2 oligosaccharide display non-native conformations, they are reglucosylated by the udp-glc:unfolded glycoprotein glucosyltransferase (ugt1 or uggt), and are subject to additional rounds of re-engagement with the cnx/crt machinery until folding is achieved. however, if a certain time frame for the folding is exceeded, proteins may never fold properly. misfolded proteins are sequestered into coat protein complex ii (cop-ii) -dependent, highly mobile er-derived quality control vesicles (qcvs), where ermani is enriched ( figure 1b ) [24] . because ermani is able to excise all α1,2-man residues when it is expressed at much higher levels in vitro [25] , the enzyme may catalyze extensive demannosylation, resulting in the production of low-man oligosaccharide man 5 glcnac 2 -containing glycoprotein precursors. the removal of the a branch terminal man residue, which is the acceptor for glc transferred by uggt, disables these proteins from reengagement with cnx/crt and re-entering into the folding cycle. importantly, the low-man n-glycans represent a tag for defective glycoproteins, targeting them to erad [26] . figure 1b ) [24] . because ermani is able to excise all α1,2-man residues when it is expressed at much higher levels in vitro [25] , the enzyme may catalyze extensive demannosylation, resulting in the production of low-man oligosaccharide man5glcnac2-containing glycoprotein precursors. the removal of the a branch terminal man residue, which is the acceptor for glc transferred by uggt, disables these proteins from reengagement with cnx/crt and re-entering into the folding cycle. importantly, the low-man n-glycans represent a tag for defective glycoproteins, targeting them to erad [26] . , and endoplasmic reticulum (er) stress pathways. nascent polypeptides are translocated through sec61 into the rough er, where the core oligosaccharide is transferred from a dolichol phosphate onto asparagine residues in asparagine-x-serine/threonine (nxs/t) motifs (i). the two terminal glucose residues on the core oligosaccharide are trimmed by glucosidase i, (gi) (ii), and gii (iii), respectively, allowing for the association with the chaperones, membrane-bound calnexin (cnx) and and/or soluble calreticulin (crt), which promote folding to a native conformation. eventually, the last terminal glucose residue will be trimmed by gii, and the glycoprotein will attain a native conformation (iv), or misfold (vii). glycoproteins that reach a native conformation will have the terminal α1,2-man residue on the b branch removed by er class i α-mannosidase (ermani) (v), as a signal to allow it to traverse the canonical secretory pathway for surface presentation or secretion (vi). polypeptides unable to reach a native conformation (vii) will engage in multiple rounds of the cnx/crt cycle, facilitated by reglucosylation of the terminal glucose by udp-glc:unfolded glycoprotein glucosyltransferase (uggt) (viii), and trafficking between quality control vesicles (qcv) (ix) and the the er-derived quality compartments (erqc) (x) under er stress. terminally misfolded glycoproteins will be demannosylated to remove all α1,2-man residues (xi), followed by association with lectins osteosarcoma amplified 9 (os9) and xtp3-transactivated gene b protein (xtp3-b) for erad (xii). figure 1 . (a) schematic presentation of the n-linked core oligosaccharide structure. the core is composed of two n-acetylglucosamine (glcnac, blue), nine mannose (man, red), and three glucose (glc, yellow) residues. a, b, and c are three oligosaccharide branches. (b) schematic description of n-glycosylation, endoplasmic reticulum-associated protein degradation (erad), and endoplasmic reticulum (er) stress pathways. nascent polypeptides are translocated through sec61 into the rough er, where the core oligosaccharide is transferred from a dolichol phosphate onto asparagine residues in asparagine-x-serine/threonine (nxs/t) motifs (i). the two terminal glucose residues on the core oligosaccharide are trimmed by glucosidase i, (gi) (ii), and gii (iii), respectively, allowing for the association with the chaperones, membrane-bound calnexin (cnx) and and/or soluble calreticulin (crt), which promote folding to a native conformation. eventually, the last terminal glucose residue will be trimmed by gii, and the glycoprotein will attain a native conformation (iv), or misfold (vii). glycoproteins that reach a native conformation will have the terminal α1,2-man residue on the b branch removed by er class i α-mannosidase (ermani) (v), as a signal to allow it to traverse the canonical secretory pathway for surface presentation or secretion (vi). polypeptides unable to reach a native conformation (vii) will engage in multiple rounds of the cnx/crt cycle, facilitated by reglucosylation of the terminal glucose by udp-glc:unfolded glycoprotein glucosyltransferase (uggt) (viii), and trafficking between quality control vesicles (qcv) (ix) and the the er-derived quality compartments (erqc) (x) under er stress. terminally misfolded glycoproteins will be demannosylated to remove all α1,2-man residues (xi), followed by association with lectins osteosarcoma amplified 9 (os9) and xtp3-transactivated gene b protein (xtp3-b) for erad (xii). ermani containing qcv are rapidly recycled through autophagy/lysosome pathways (xiii). without interactions with client glycoproteins, edemosome components are degraded through an autophagy-like mechanism (xiv). viruses can hijack edemosomes to form double membrane vesicles (dmvs) that serve as platforms for their replication (xv). with only one-tenth of the total cell volume, the er is responsible for the synthesis of the vast majority of the secreted or membrane proteins, which account for one-third of total cellular proteins. therefore, the er has extremely high protein concentrations (100 mg/ml), which renders this organelle very susceptible to protein aggregation [27] . in addition, the protein folding is error prone, and this process can be further compromised by physiological and pathological perturbations. moreover, genetic mutations may prohibit proteins from being folded properly. all these factors may cause the accumulation of unfolded or misfolded proteins. when the level of these aberrant proteins exceeds the folding and clearance capacity of the er, known as er homeostasis, it leads to a cellular stress response termed "er stress", which in turn activates the unfolded protein response (upr) to restore the er homeostasis [28] . er stress is sensed by three er transmembrane receptors: double-stranded rna (dsrna)-activated protein kinase (pkr)-like er kinase (perk), inositol-requiring enzyme 1 (ire1), and activating transcription factor 6 (atf6). perk and atf6 are in association with another er chaperone, the binding immunoglobulin protein (bip, or grp78), when the cell is not under stress. bip preferentially binds to misfolded proteins and dissociates from perk and atf6 under er stress, resulting in their activation and upr to mitigate this stress [29, 30] . ire1 is activated by the direct binding of unfolded proteins [31] . ire1 then activates the transcription factor x-box binding protein 1 (xbp-1), which in turn up-regulates er chaperones to assist in the folding capacity of the er as well as erad components to boost protein degradation. perk phosphorylates the eukaryotic initiation factor (eif)-2α and halts protein translation, and atf6 up-regulates protein expression to boost the er protein folding capacity and erad. however, if these objectives are not achieved within a certain time span or if the disruption is prolonged, upr also activates pathways leading to cell death. although perk activation causes global inhibition of protein translation by blocking eif-2α activity, it paradoxically enhances translation of the transcription factor atf4. atf4 then trans-activates the ccaat/enhancer-binding protein-homologous protein (chop), which is a pro-apoptotic transcription factor, resulting in cell death by apoptosis [32] . erad is a protein quality control mechanism conserved in all eukaryotic cells, which is an important arm of upr, necessary to alleviate er stress [33] . erad results in the selective dislocation of unfolded and misfolded proteins from the er to the cytosol via specific membrane machinery. erad targets are subsequently degraded by the cytosolic ubiquitin proteasome system (ups) [34] . quality control of functional proteins produced from the er is also critical for maintenance of the er homeostasis by eliminating unfolded and misfolded proteins. thus, erad is a central element of both the secretory pathway and upr, which targets a number of physiological and pathological substrates such as the t cell antigen receptor (tcr) [35] , 3-hydroxy-3-methylglutaryl coenzyme-a (hmg-coa) reductase (hmgcr) [35] , squalene monooxygenase (sqle) [36] , inositol 1,4,5-trisphosphate (ip 3 ) receptor [37] , diacylglycerol acyltransferase 2 (dgat2) [38] , heme oxygenase-1 (ho-1) [39] , alpha-1 antitrypsin [35] , and cystic fibrosis transmembrane regulator (cftr) [40] . so far, more than 60 human diseases have been attributed to this pathway [41] . although the vast majority of secreted proteins are glycosylated, the er is responsible for the folding and assembly of both glycosylated and non-glycosylated proteins into functional complexes, which are subjected to erad quality control if they are misfolded. the process of erad can be divided into three steps: substrate recognition, retrotranslocation, and ubiquitylation/proteasomal degradation. in fact, extensive excision of α1,2-man residues from n-glycans sends an important signal to trigger misfolded glycoprotein degradation, which is dependent on class i mannosidases [42] . class i mannosidases belong to the glycoside hydrolase family 47 (gh47), which are exo-acting α1,2-mannosidases that are divided into three subfamilies [43] . the first subfamily consists of ermani, which is supposed to cleave the outmost α1,2-man residue on the b branch from n-linked glycans in the er. the second subfamily consists of three golgi α-mannosidase i, including golgimania, golgimanib, and golgimanic, which cleave the remaining three α1,2-man residues in the golgi complex for n-glycan maturation. the third subfamily consists of the er degradation-enhancing α-mannosidase-like proteins (edem) 1, 2, and 3. although some edem orthologs in lower eukaryotes have detectable α1,2-mannosidase activity, such activity has not been reported for any mammalian edem proteins in vitro. nevertheless, there is evidence suggesting that these edem proteins should have enzymatic activity in vivo [44, 45] . indeed, the extent of man excision determines the fate of a glycoprotein, which could be either targeted to erad for degradation or sent to the golgi for normal trafficking. ermani exhibits a slow rate of enzymatic activity, which allows nascent proteins to perform multiple rounds of reglucosylation and achieve proper folding [46] . properly folded glycoproteins should have one man residue trimmed off from n-glycans by ermani. these glycoproteins then interact with the high-man binding lectin, er-golgi intermediate compartment 53 kda protein (ergic-53) [47] , for trafficking from the er to the golgi ( figure 1b) . however, if glycoproteins are misfolded terminally, the remaining three α1,2-man residues are excised from these molecules, which targets misfolded proteins for degradation [48] . it is still not completely understood how misfolded glycoproteins are subjected to such extensive demannosylation in the er and then targeted for erad. although ermani alone may be able to complete this task, there is evidence suggesting that additional gh47 enzymes are involved. elevation of the golgi mannosidases has been shown to accelerate erad, so these enzymes may possibly be responsible for such extensive excision, likely by trafficking back to the er via an unknown mechanism [49] . in fact, the localization of the golgimania has recently been observed in qcv with other canonical erad machinery such as ermani, and overexpression and knockdown can, respectively, increase or retard trimming of misfolded glycoproteins from man 9 glcnac 2 to man 5 glcnac 2 in vitro [50] . in addition, upon er stress, qcv converge to form the er-derived quality compartments (erqc), where edem proteins are also sequestered ( figure 1b ) [48] . edem1 and edem3 boost mannose trimming when overexpressed [45, 51, 52] . in addition, using a genomic knockout approach, it has been recently proposed that edem2 plays a central role in the trimming of the outmost man residue on the b branch, whereas edem1 and edem3 should be responsible for trimming of the remaining α1,2-man residues. accordingly, a "double check" model for misfolded glycoproteins has been proposed, which suggests that edem2 catalyzes the first step of man trimming, and edem1 and edem3 contribute the second step [44] . under these joint actions, all four α1,2-man residues are removed from the oligosaccharide, which is then recognized by the lectins osteosarcoma amplified 9 (os9) and xtp3-transactivated gene b protein (xtp3-b) via the mannose 6-phosphate receptor homology (mrh) domain ( figure 1b) [53, 54] . misfolded proteins are targeted to specific translocation channels (retrotranslocons) for retrotranslocation in an energy-dependent manner. this process is facilitated by p97, a member of the atpases associated with the diverse cellular activities (aaa) family, by catalyzing atp hydrolysis [55] . the p97 atpase is recruited by the ubiquitin-like (ubx)-domain-containing protein ubxd8, an er-membrane protein that plays a role in erad [56] . it is still mysterious how these retrotranslocons are formed and how integral membrane and lumenal erad substrates are exported across the er membrane through these retrotranslocons. the first candidate channel is composed of the sec61 complex, which is comprised of α, β, and γ subunits. the α subunit crosses the membrane 10 times, and forms a channel with the other subunits. the sec61 heterotrimeric channel is the main translocon involved in co-translational protein transport into the er [57] . however, there is evidence suggesting that the sec61 translocon is also involved in retrotranslocation of erad substrates, implying the non-specificity and bi-directional property of this channel [58] . the second candidate is a member of the derlin family (derlin-1, -2, and -3) [59, 60] . derlins are integral membrane proteins that likely span the er membrane four times and contain a rhomboid-like domain [61] . the third candidate includes the erad-specific e3 ligases. they have a large number of transmembrane domains, which are not only responsible for polyubiquitylation, but could act as potential exit channels for erad substrates [62] . in saccharomyces cerevisiae, there are two major types of really interesting new gene (ring)-finger e3 ligase complexes, hrd1 and doa10, which mediate erad by targeting discrete substrates [63] . hrd1 was the first e3 enzyme identified in the erad pathway during the study of hmg-coa reductase degradation (hrd) [64] . hrd1 has 6 transmembrane helices in its n-terminal transmembrane domain and a catalytic ring domain in the soluble c-terminal region extended to the cytosol. using an elegant erad assay reconstituted in vitro, the hrd1-mediated formation of ubiquitin-gated protein-conducting membrane channels has been demonstrated [65, 66] . hrd1 has two mammalian orthologs named hrd1 and gp78, and its functioning e2 enzyme is known as ubc7, which also has two mammalian orthologs, ube2g2 and ube2g1 [67] . hrd1 is unstable, and must be stabilized by its co-factor hrd3 in an equimolar ratio [68] . the mammalian ortholog of hrd3 is sel1l, which is required for erad substrate retrotranslocation [69] . hrd1 interacts with der1, and the der1-hrd1 interaction is bridged by another integral membrane protein usa1, which is also required for hrd1 oligomerization [70] . the usa1 mammalian ortholog is called herp, which also interacts with hrd1 and derlin-1 and plays an important role in erad [71] . doa10 was identified in degradation studies of mating type (mat)-α2-10 (doa), which is a yeast transcription factor. doa10 is a~150 kda protein that has 14 transmembrane domains, which requires both ubc6 and ubc7 as e2 enzymes. the doa10 mammalian ortholog is teb4 (march6), which functions in the erad pathway with similar subcellular distribution and topology [72] . the ubc6 e2 enzyme has two mammalian orthologs, ube2j1 and ube2j2; both are involved in erad [73, 74] . in saccharomyces cerevisiae, erad is executed synergistically by hrd1 and doa10 with minimal redundancy because they exhibit different substrate specificity. doa10 mainly triggers ubiquitylation of specific soluble proteins and membrane proteins with degrons exposed to the cytosol; a process referred to as erad-c [75, 76] . hrd1 interacts with two other types of substrates, whose degradation is termed erad-l and erad-m. erad-l includes soluble lumenal proteins in the er and transmembrane proteins with degrons exposed to the er lumen; erad-m includes transmembrane proteins with degrons embedded into the er membrane [63] . simultaneous inactivation of both genes has been shown to increase the sensitivity to heavy metal-induced cellular stress and exhibit an elevated upr. the regulation of protein folding and the functional relation between erad and the upr are much more complex in mammalian cells. in saccharomyces cerevisiae, the cnx cycle does not exist due to the lack of uggt. in addition, protein synthesis is tightly controlled at the translational level by determination of the stoichiometry to avoid surplus production, resulting in minimal dependence on the post-translational regulation of protein expression. moreover, although the yeast ermani ortholog mns1p and edem ortholog htm1p are indispensable for erad, only one edem ortholog is present in yeast [77, 78] . because overly active erad may interfere with the regular protein folding process in the er, mammalian cells have evolved mechanisms to tightly regulate this quality control device by a combination of compartmentalization and tuning. edem1 is segregated into er-derived, lc3-i-associated vesicles, which are called edemosomes, where edem1, os-9, and sel1l are concentrated when they lack client glycoproteins to dislocate ( figure 1b) [79] . notably, unlike chaperones and the other enzymes, many erad regulators including ermani, edem1, os-9, herp, and sel1l are short-lived proteins, and ermani, edem1, sel1l, and os-9 are targeted to the lysosomal pathway for degradation [79, 80] . thus, edemosomes are called erad tuning vesicles, which deliver their content to lysosomes for disposal via an autophagy-like pathway to reduce the erad capacity under natural conditions [81] . additionally, lysosomal inhibitors are able to cause the accumulation of an aggregating mutant of dysferlin in the er when compared to the wild-type, which was used as evidence to propose that large protein aggregates are disposed of via an autophagy/lysosomal pathway, dubbed erad ii [82] . however, under er stress, most of these factors are highly induced, including the edem proteins, but not ermani [45, 83, 84] . under stress, qcv are recruited to the erqc, resulting in the accumulation of ermani and its glycoprotein substrates [85] . moreover, many other erad components, including edem1, hrd1, derlin-1, sec61β, and herp, are also concentrated in erqc. importantly, it has been found that edem1 stabilizes ermani and increases its protein expression at steady-state levels [86] . such enrichment of these critical components accelerates efficient assembly of the erad machinery, potentiating the degradation of misfolded glycoproteins and alleviating er stress. during infection, viruses are able to hijack the host translational machinery and saturate the er with viral proteins. not only do viruses use the er to generate their glycoproteins, but some even utilize the er as their site to assemble progeny particles [87] . such accumulation of viral proteins in the er places a heavy demand on the protein folding machinery, which may cause er stress, and in turn, activate the upr, resulting in restoration of the er homeostasis or apoptosis. so far, at least 36 viruses have been found to be able to induce er stress, and activate the three upr stress signaling pathways [88] . enveloped viruses may bud through the plasma membrane or an intracellular compartment. in addition, their envelope glycoproteins are targeted to the er for post-translational modifications and folding. not surprisingly, many viral envelope glycoproteins are significant inducers of the upr, which includes hcv [89] , hepatitis b virus (hbv) [90] , coronaviruses [91] , chikungunya virus (chikv) [92] , and retroviruses [93] . as introduced earlier, the upr utilizes three different mechanisms to alleviate er stress: reducing global protein translation, increasing the er folding capacity, and enhancing erad by activating the perk, atf6, or ire1-xbp1 pathways, respectively. viral infections may activate these pathways, resulting in the inhibition or enhancement of viral replication (table 1) . for example, the perk-mediated global translation shutdown is a very effective antiviral mechanism, and a similar shutdown by pkr has been used in the interferon pathway to defend against viral infection [94] . conceivably, viruses have evolved a number of strategies to circumvent the detrimental effect of upr to establish productive infection. hcv is still able to produce viral proteins even when the cellular translational machinery is shut down, because these viruses have their own internal ribosome entry site (ires) to recruit and assemble the ribosomal initiation complex for protein expression [95] . epstein-barr virus (ebv), herpes simplex virus (hsv), and african swine fever virus (asfv) can counteract the perk-mediated eif2α phosphorylation by activating an eif2α phosphatase pp1 [96] [97] [98] . in another example, the hcv e2 protein directly interacts with perk to prevent er stress sensing by acting as a pseudo-substrate to block perk activity [99] . in addition to combating the upr, viruses also take advantage of the upr pathways to benefit their replication. for example, influenza a virus (iav) replication is promoted by activation of the ire1-xbp1 pathway [100] ; atf6 activation promotes asfv, lymphocytic choriomeningitis virus (lcmv), denv, human cytomegalovirus (hcmv), and japan encephalitis virus (jev) replication [101, 102] , and atf4 activation enhances hiv-1 replication [103] . thus, despite the detrimental effects, viruses have evolved to manipulate host upr signaling pathways to promote viral infections. below, we will focus on the roles of erad played in virus replication, which is the main target of this review. as introduced earlier, erad transports unfolded/misfolded proteins from the er into the cytosol for proteasomal degradation. conceivably, viruses can manipulate and exploit this cellular machinery to degrade several important host factors to promote their propagation. herpesviruses have evolved multiple mechanisms to suppress the host immune response via erad. major histocompatibility complex (mhc) molecules play an indispensable role in triggering an immediate immune response to inhibit virus infections. herpesviruses inhibit mhc class i (mhc-i) expression by targeting these molecules to erad for degradation. for example, hcmv produces two transmembrane proteins, us2 and us11, and each is sufficient to bind to mhc-i heavy chains, causing their dislocation from the er to the cytosol for degradation [108] . notably, us2 and us11 use different mechanisms to degrade mhc-i. us2-dependent mhc-i degradation is mediated through an interaction with the e3 ligase, trc8. this us2/trc8 complex has been implicated in the degradation of other membrane proteins including multiple alpha-integrins, the interleukin 12 receptor (il-12r), thrombomodulin (thbd), protein tyrosine phosphatase receptor type j (ptprj), and cd112 [109] . although the signal peptide peptidase (spp) has been shown to bind to trc8, the us2/trc8 complex maintains its mhc-1 degradation activity in spp−/− knockout cells, suggesting that spp binding is not related to mhc-1 degradation [110, 111] . recent reports now regard the us2/trc8 complex as a multifunctional hub that is able to degrade a multitude of targets in order to further hcmv immune evasion [109, 112] . a complex formed between us11, derlin-1, and the e3 ligase, tmem129, mediates mhc-i degradation via us11 [113] . initial reports concerning us11 found an association with sel1l and assumed that us11 mediated mhc-1 degradation could be sel1l/hrd1 dependent. recent literature has confirmed that while the us11/tmem129 complex degrades mhc-1, us11 itself is degraded through a sel1l/hrd1 axis in the absence of the client mhc-1 [113, 114] . recruitment of p97 by ubxd8 is also crucial for us11-mediated mhc-i degradation [115] . with regard to us11, hcmv utilizes erad to dispose of mhc-i and its own effector protein using discrete axes for ubiquitination. mouse gammaherpesvirus 68 (mhv68) uses another mechanism to inhibit mhc-i. mhv68 produces a protein termed mk3, which is a ring-finger e3 ligase anchored on the er membrane. mk3 interacts with mhc-i heavy chain molecules, and it also associates with the transporter associated with antigen processing (tap), p97, derlin-1, and the e2 ube2j2. association with ube2j2 results in an interesting pattern of ubiquitination of non-lysine residues (the mk3/ube2j2 complex can ubiquitinate serines as well as lysines) that leads to rapid degradation of the mhc-i by proteasomes [73, 116] . thus, herpesviruses have evolved numerous strategies to block the mhc antigen presentation and evade the host immune response to establish a persistent infection. primate lentiviruses also harness the erad pathway to promote their replication via downregulation of their receptor cd4. cd4 downregulation prevents superinfection and promotes viral release by interrupting viral receptor-envelope interactions on the plasma membrane, leading to a controlled and productive viral infection and immunodeficiency [117] . these viruses produce two accessory proteins, nef and vpu, to trigger cd4 degradation via two distinctive mechanisms [118] . nef uses the endocytic pathway to redirect cd4 from the cell surface, or to interfere with the transport of newly synthesized cd4 from the trans-golgi network (tgn) to the cell surface, resulting in cd4 dislocation to endosomes and degradation by lysosomes [119] . however, vpu interacts with cd4 in the er and induces cd4 proteasomal degradation via erad [120] . vpu is a small transmembrane protein encoded by hiv-1 and some simian immunodeficiency virus (siv) isolates. vpu forms ion conductive membrane pores; it also interacts with β-transducin repeat-containing proteins (βtrcp), which are f-box/wd repeat-containing proteins that are part of the skp1-cul1-f-box (scf) e3 ubiquitin ligase complex [121] . the vpu-induced cd4 degradation is strictly dependent on the scf-β-trcp complex [122] . notably, this e3 ligase complex is not associated with the er membrane, and therefore does not normally function in erad. however, the degradation also requires the cytosolic atpase p97 and its cofactors ufd1l and npl4, which are key components of the erad machinery, suggesting that cd4 is degraded via erad [122] . nevertheless, the degradation is not dependent on hrd1, sel1l, and ubc7. in addition to degradation, viruses may harness erad components to benefit their replication. first, erad can promote viral protein expression. mouse mammary tumor virus (mmtv) is a betaretrovirus, which expresses the rem protein in the er. rem has a n-terminal 98-amino acid signal peptide (sp), which is cleaved off by signal peptidase and retrotranslocated in a p97-dependent manner [123] . rem sp then promotes the nuclear export of viral unspliced rnas to the cytosol for protein expression. similarly, hepatitis e virus (hev) orf2 is an n-linked glycoprotein, but functions as the major capsid protein. although orf2 is expressed in the er, it depends on erad components to exit from the er to the cytoplasm without being polyubiquitylated [124] . second, erad can promote virus entry. polyomaviruses (pyv) enter cells through the er and then replicate in the nuclei [125] . to get from the er to the nucleus, these viruses can cross the er membrane into the cytosol via the erad retrotranslocons [126] . an example of this is mouse pyv, which uses derlin-2, whereas simian virus 40 (sv40) uses derlin-1 and the sel1l complex for dislocation [126, 127] . in addition, the proteasome machinery is also required for the human bk pyv exit from the er [127] . third, erad can promote virus replication. the replication of positive-strand rna viruses normally involves the formation of double-membrane vesicles (dmvs) and convoluted membranes (cms) by rearrangement of cellular membranes, which segregates and protects viral proteins and genomes from the host's innate immune response. as introduced earlier, the erad activity can be adjusted by erad tuning vesicles termed edemosomes ( figure 1b) , which display non-lipidated lc3 and segregate the erad factors edem1, os-9, and sel1l from the er lumen [81] . by comparing the similarity between dmvs and edemosomes, it has been discovered that mouse hepatitis virus (mhv), equine arteritis virus (eav), and jev indeed replicate in these erad tuning vesicles [128] . thus, these viruses can subvert edemosomes as their replication vesicles to promote infection [129] . although erad has been frequently manipulated by a number of viruses to promote infection or attenuate immune responses, it may also function directly as an antiviral device to protect host cells from infection. because viral envelope glycoprotein production and folding take place in the er, these viral proteins may become the primary targets for erad, resulting in the inhibition of viral infection. primate lentiviruses, including hiv and siv, have low levels of envelope glycoproteins on their surface, and the average copy number is~14 env trimers per virion [130, 131] . in contrast, ifa, sendai virus, hsv, and moloney murine leukemia virus (momulv) have much more envelope glycoproteins on their surfaces [132] [133] [134] [135] . the exceptionally low number of env spikes may protect hiv-1 from host immune responses [136] since almost 85% of env proteins are retained in the er and are degraded [137] [138] [139] . this degradation mechanism was not clear until we recently reported that hiv-1 env glycoproteins are targeted for erad. from completely unrelated studies, we isolated hiv-1 non-permissive (np) and permissive (p) t cell clones n2-np and n5-p from the original cem.nkr human t cell line [140] . our initial analysis uncovered that hiv-1 replication is restricted from the second round of the viral life cycle in n2-np cells, resulting in~1000-fold inhibition when compared to n5-p. further transcriptome analysis by microarrays revealed that n2-np cells overexpress the mitochondrial translocator protein (tspo), which strongly inhibits hiv-1 env expression [141] . tspo interacts with the mitochondrial permeability transition pore (mptp) complex, which includes the outer membrane protein voltage-dependent anion channel (vdac) protein, the inner membrane protein adenine nucleotide translocase (ant), and the mitochondrial matrix protein cyclophilin d (cypd) [142] . tspo binds to vdac and contributes to the regulation of the mitochondrial membrane permeability by the mptp complex [143] . our results suggested that tspo overexpression could reduce the oxidative redox status in the er, which interferes with the env oxidative folding process, resulting in env degradation. consistently, the rapid env degradation in n2-np cells was rescued by kifunesine, an effective inhibitor of glycoside hydrolase family 47 (gh47) enzymes [144] , suggesting that hiv-1 is degraded via erad in n2-np cells. to further explore the env degradation mechanism, we investigated which of those four er-associated gh47 enzymes was responsible for the env degradation. notably, when ermani, edem1, edem2, and edem3 were ectopically expressed in 293t cells, only ermani strongly inhibited env expression in a dose-dependent manner. in addition, when the endogenous ermani was knocked out by crispr/cas9, tspo was no longer able to suppress the env expression [145] . these results demonstrated that ermani should be responsible for the initiation of hiv-1 env degradation via erad. human ermani is a 699-amino-acid, 79.5-kda, type ii membrane protein, which is divided into an n-terminal cytoplasmic domain (cd), transmembrane (tm) helix, lumenal 'stem' region, and a catalytic domain [146, 147] . using an immunoprecipitation assay, we found that hiv-1 env interacts with the catalytic domain of ermani [145] . the structure of this catalytic domain shows an (αα) 7 -barrel composed of 14 consecutive helices [148] . in the catalytic domain, there are seven residues that are critical for ermani function. c527 and c556 form a highly conserved disulfide bond and were reportedly critical for protein folding [149] , whereas e330, d463, and e599 were proposed as catalytic residues [148] . r334c and e397k mutations are found in nonsyndromic autosomal-recessive intellectual disability (ns-arid) disease [150] , and the r334c mutation is also found in the congenital disorders of glycosylation [151] . all these residues are required for hiv-1 env degradation, suggesting that the mannosidase activity is important for the ermani activity. ermani also targets the terminally misfolded human alpha1-antitrypsin variant null (hong kong) (nhk) for degradation via erad, but neither its catalytic activity nor its catalytic domain is required for this degradation, suggesting that different mechanisms are involved in hiv-1 env and nhk degradation [152] . we have also found that the viral protein r (vpr) of hiv-1 enhances viral replication in monocyte-derived macrophages (mdms) and dendritic cells (mddcs) by rescuing env from erad degradation through the erad (ii) autophagy pathway. compounds known to facilitate glycoprotein folding (pk11195 and as 2 o 3 ) and inhibit er α-mannosidases crucial for erad (kifunensine), and those that block lysosomal proteases (bafilomycin) rescued envelope expression and infectivity in a ∆vpr background to that of wild-type virus [153] . as aforementioned, unlike ermani, whose expression is not responsive to upr, the expression of the edems is induced upon upr via the ire1/xbp activation pathway, which boosts erad and alleviates er stress. although ectopic expression of edems did not inhibit hiv-1 env expression [145] , these proteins inhibit the expression of some other envelope glycoproteins. hbv expresses three surface glycoproteins, the large (l), middle (m), and small (s), which are translated from different initiation codons within the same open reading frame (orf) and share the tetra-spanning transmembrane domains in the s protein. the n-terminus of the m and l protein contain additional pres2 and pres1-pres2 domains, respectively. the common s domain has an n-glycosylation site, and the m pres2 domain has another site. overexpression of the surface proteins is sufficient to activate the ire1/xbp1 pathway and elevate edem1, edem2, and edem3 expression. importantly, edem1 overexpression destabilizes s, m, and l, and edem1 silencing stabilizes their expression [154] . in addition, the autophagy/lysosomal pathway, but not the proteasomal pathway, is involved in the degradation of hbv surface glycoproteins, further complicating our understanding of the viral protein degradation process via erad [154] . hcv has two n-glycosylated envelope proteins e1 and e2 on the surface of virions, which are type i transmembrane proteins expressed from a common viral polyprotein precursor. hcv infection strongly induces the activation of the ire1 stress sensor, resulting in elevation of edem1, edem2, and edem3, but not the ermani expression. both edem1 and edem3, but not edem2, interact with e2, and overexpression of these two proteins induces e2 polyubiquitylation and degradation. conversely, knockdown of edem1 expression or treatment with kifunesine increases e2 expression, and also reduces the interaction of edem1 and edem3 with sel1l [155] . taken together, these results strongly suggest that edem proteins are able to extract viral polypeptides from the er quality control cycle, and degrade them via erad. however, since none of these proteins can target the jev e protein to erad for degradation, not every viral glycoprotein is recognizable by these proteins [155] . in vivo experiments on patients with chronic liver injury were unable to identify up-regulation of upr and erad elements in diseased versus control patients [156] . erad has also been implicated in the degradation of hcmv glycoproteins, gh and gl, via the 26s proteasome. hcmv produces at least 65 unique glycoproteins, with four homologues to the hsv glycoproteins, gh, gb, gl, and gm [157] . the glycoproteins, gh and gl, are constituents of the gcii type complexes found on the surface of hcmv virions. the gcii trimeric complex between gh, gl, and go can initiate ph independent fusion [158] . in addition, a pentameric complex between gh, gl, and the gene products u128, u130, and u131 is able to mediate entry into different cell types via ph-dependent receptor-mediated endocytosis; a process that requires the trimeric gh/gl/go complex [159] . although previous studies have shown that the glycoprotein gl stabilizes the expression of gh and potentiates its surface localization [160] , recent work revealed that gh is degraded via erad in the absence of gl [161] . replacement of the cytoplasmic tail of gh with that of the human cd4 protein subverted gh degradation via erad, potentiating surface expression. current studies describe two paradigms for erad to target viral glycoproteins for degradation: ermani-mediated, which targets hiv-1 env, and edem-mediated, which can target hcv and hbv surface glycoproteins. gh47 family members share a common catalytic mannosidase homology domain of~440-residues [52] , and the three catalytic residues e330, d463, and e599 found in ermani are all conserved in these proteins [43] . nevertheless, there is little protein sequence homology beyond this domain among these proteins. unlike ermani, all three edems are er-lumenal proteins, although the signal sequence of edem1 is resistant to cleavage [162] . edem3 has two novel features including an additional protease-associated domain of unknown function and a kdel signal for er retention [45] . whether or how the coordination between the edems and ermani facilitates erad is still a convoluted issue. due to lysosomal degradation mediated by the n-terminal cytoplasmic tail, ermani is expressed at very low basal levels in cells, and its expression is not induced by upr [86] . such proteolytically driven checkpoint control of ermani expression may contribute to establish glycoprotein quality control at a baseline level, which maintains er homeostasis without activation of ire1/xbp1. however, if this basic mechanism fails to restore er homeostasis, ire1/xbp1 is induced to elevate expression of the edems, which will increase erad. unlike hcv and hbv, hiv-1 induces upr, but barely activates the ire1/xbp1 pathway, which may explain why hiv-1 env is not directly targeted by edem proteins [93] . nevertheless, these two different arms of erad do not exclude the role of the edems in ermani-mediated degradation. edems may accelerate the release of terminally misfolded glycoproteins from the cnx/crt cycle, and thereby help ermani to conduct more extensive demannosylation [163] ; and the association of edem with sel1l may further accelerate the cytosolic delivery of misfolded proteins [164] . moreover, edem1 may form a complex with ermani, which stabilizes ermani by the suppression of its proteolytic degradation [86] . discrepancies concerning the localization of ermani with various labs determining colocalization with the er, golgi, or er-golgi intermediate compartments and quality control vesicles, lends credence to both current theories that ermani is either a golgi checkpoint in quality control that will return misfolded proteins back to the er for further processing, or that it resides in quality control vesicles with glycoprotein substrates as part of the cnx/crt cycle [24, 165] . it is well established that viruses have evolved to manipulate host upr and erad to optimize their replication, whether they are 'tuning' host quality control to ensure the proper folding of their envelope glycoproteins, circumventing erad in order to prevent degradation of their viral envelope glycoproteins, or hijacking erad to dispose of host proteins. there are still many questions left to be answered, including the identities of the dislocons that each envelope glycoprotein is targeted to, the motifs or patterns that allow α1,2-mannosidases to differentiate between native and misfolded glycoproteins, why some viral proteins are disproportionately targeted (hcmv gh), and the roles that the upr and erad play in vivo during viral infections. these exciting areas merit more extensive studies. virus entry: molecular mechanisms and biomedical applications the cell biology of receptor-mediated virus entry recruitment of hiv and its receptors to dendritic cell-t cell junctions penetration of nonenveloped viruses into the cytoplasm virus and cell fusion mechanisms both e protein glycans adversely affect dengue virus infectivity but are beneficial for virion release a systematic study of the n-glycosylation sites of hiv-1 envelope protein on infectivity and antibody-mediated neutralization playing hide and seek: how glycosylation of the influenza virus hemagglutinin can modulate the immune response to infection the hepatitis c virus glycan shield and evasion of the humoral immune response comprehensive functional analysis of n-linked glycans on ebola virus gp1 tracking global patterns of n-linked glycosylation site variation in highly variable viral glycoproteins: hiv, siv, and hcv envelopes and influenza hemagglutinin glycosylation affects cleavage of an h5n2 influenza virus hemagglutinin and regulates virulence importance of hemagglutinin glycosylation for the biological functions of influenza virus interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics the hiv glycan shield as a target for broadly neutralizing antibodies virus glycosylation: role in virulence and immune interactions vertebrate protein glycosylation: diversity, synthesis and function roles of n-linked glycans in the endoplasmic reticulum n-glycan-based er molecular chaperone and protein quality control system: the calnexin binding cycle malectin: a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein n-glycosylation the role of lectin-carbohydrate interactions in the regulation of er-associated protein degradation the long road to destruction mammalian er mannosidase i resides in quality control vesicles, where it encounters its glycoprotein substrates the specificity of the yeast and human class i er alpha 1,2-mannosidases involved in er quality control is not as strict previously reported endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to man6-5glcnac2 the unfolded protein response: integrating stress signals through the stress sensor ire1alpha endoplasmic reticulum stress sensing in the unfolded protein response dynamic interaction of bip and er stress transducers in the unfolded-protein response the unfolded protein response: from stress pathway to homeostatic regulation unfolded proteins are ire1-activating ligands that directly induce the unfolded protein response er-stress-induced transcriptional regulation increases protein synthesis leading to cell death endoplasmic reticulum-associated degradation regulation of endoplasmic reticulum-associated protein degradation (erad) by ubiquitin. cells how early studies on secreted and membrane protein quality control gave rise to the er associated degradation (erad) pathway: the early history of erad sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase doa10/teb4. elife 2013, 2, e00953 when worlds collide: ip(3) receptors and the erad pathway regulation of diacylglycerol acyltransferase 2 protein stability by gp78-associated endoplasmicreticulum-associated degradation ubiquitin-proteasome system mediates heme oxygenase-1 degradation through endoplasmic reticulum-associated degradation pathway selective inhibition of endoplasmic reticulum-associated degradation rescues deltaf508-cystic fibrosis transmembrane regulator and suppresses interleukin-8 levels: therapeutic implications the delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology flagging and docking: dual roles for n-glycans in protein quality control and cellular proteostasis family 47 alpha-mannosidases in n-glycan processing edem2 initiates mammalian glycoprotein erad by catalyzing the first mannose trimming step edem3, a soluble edem homolog, enhances glycoprotein endoplasmic reticulumassociated degradation and mannose trimming in vitro mannose trimming property of human er alpha-1,2 mannosidase i ergic-53 and traffic in the secretory pathway endoplasmic reticulum (er) mannosidase i is compartmentalized and required for n-glycan trimming to man5-6glcnac2 in glycoprotein er-associated degradation stimulation of erad of misfolded null hong kong alpha1-antitrypsin by golgi alpha1,2-mannosidases mannosidase ia is in quality control vesicles and participates in glycoprotein targeting to erad edem1 regulates er-associated degradation by accelerating de-mannosylation of folding-defective polypeptides and by inhibiting their covalent aggregation glycoprotein folding and the role of edem1, edem2 and edem3 in degradation of folding-defective glycoproteins os-9 and grp94 deliver mutant alpha1-antitrypsin to the hrd1-sel1l ubiquitin ligase complex for erad the mrh domain suggests a shared ancestry for the mannose 6-phosphate receptors and other n-glycan-recognising proteins in vitro analysis of hrd1p-mediated retrotranslocation of its multispanning membrane substrate 3-hydroxy-3-methylglutaryl (hmg)-coa reductase derlin-1 and ubxd8 are engaged in dislocation and degradation of lipidated apob-100 at lipid droplets structure of the native sec61 protein-conducting channel proteasome 19s rp binding to the sec61 channel plays a key role in erad a membrane protein required for dislocation of misfolded proteins from the er a membrane protein complex mediates retro-translocation from the er lumen into the cytosol derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant alpha-1 antitrypsin from the endoplasmic reticulum protein quality control in the er: balancing the ubiquitin checkbook the ubiquitylation machinery of the endoplasmic reticulum role of 26s proteasome and hrd genes in the degradation of 3-hydroxy-3-methylglutaryl-coa reductase, an integral endoplasmic reticulum membrane protein autoubiquitination of the hrd1 ligase triggers protein retrotranslocation in erad key steps in erad of luminal er proteins reconstituted with purified components selective ubiquitylation of p21 and cdt1 by ubch8 and ube2g ubiquitin-conjugating enzymes via the crl4cdt2 ubiquitin ligase complex endoplasmic reticulum degradation requires lumen to cytosol signaling. transmembrane control of hrd1p by hrd3p association of the sel1l protein transmembrane domain with hrd1 ubiquitin ligase regulates erad-l usa1 functions as a scaffold of the hrd-ubiquitin ligase the ubiquitin-domain protein herp forms a complex with components of the endoplasmic reticulum associated degradation pathway membrane topology of the yeast endoplasmic reticulum-localized ubiquitin ligase doa10 and comparison with its human ortholog teb4 (march-iv) ube2j2 ubiquitinates hydroxylated amino acids on er-associated degradation substrates hrd1 and ube2j1 target misfolded mhc class i heavy chains for endoplasmic reticulum-associated degradation distinct ubiquitin-ligase complexes define convergent pathways for the degradation of er proteins the yeast erad-c ubiquitin ligase doa10 recognizes an intramembrane degron htm1p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast the saccharomyces cerevisiae processing alpha 1,2-mannosidase is localized in the endoplasmic reticulum, independently of known retrieval motifs segregation and rapid turnover of edem1 by an autophagy-like mechanism modulates standard erad and folding activities human endoplasmic reticulum mannosidase i is subject to regulated proteolysis disposal of cargo and of erad regulators from the mammalian er two endoplasmic reticulum-associated degradation (erad) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome erad(i) and autophagy/lysosome erad(ii) a novel er alpha-mannosidase-like protein accelerates er-associated degradation a novel stress-induced edem variant regulating endoplasmic reticulum-associated glycoprotein degradation glycan regulation of er-associated degradation through compartmentalization the mammalian upr boosts glycoprotein erad by suppressing the proteolytic downregulation of er mannosidase i how viruses use the endoplasmic reticulum for entry, replication, and assembly the expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis unfolded protein response in hepatitis c virus infection modulation of the unfolded protein response by the human hepatitis b virus coronavirus infection, er stress, apoptosis and innate immunity differential unfolded protein response during chikungunya and sindbis virus infection: chikv nsp4 suppresses eif2alpha phosphorylation hiv infection and antiretroviral therapy lead to unfolded protein response activation signal integration via pkr the pathway of hcv ires-mediated translation initiation the lmp1 oncogene of ebv activates perk and the unfolded protein response to drive its own synthesis herpes simplex virus 1 infection activates the endoplasmic reticulum resident kinase perl and mediates eif-2alpha dephosphorylation by the gamma(1)34.5 protein the african swine fever virus dp71l protein recruits the protein phosphatase 1 catalytic subunit to dephosphorylate eif2alpha and inhibits chop induction but is dispensable for these activities during virus infection protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis c virus envelope protein e2 through the eukaryotic initiation factor 2alpha kinase perk influenza a viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (ier1) stress pathway the atf6 branch of unfolded protein response and apoptosis are activated to promote african swine fever virus infection er stress, autophagy, and rna viruses short communication: activating transcription factor 4 (atf4) promotes hiv type 1 activation pb1-f2 attenuates virulence of highly pathogenic avian h5n1 influenza virus in chickens dengue virus modulates the unfolded protein response in a time-dependent manner cytomegalovirus downregulates ire1 to repress the unfolded protein response the sars coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor the hcmn gene products us2 and us11 target mhc class i molecules for degradation in the cytosol plasma membrane profiling defines an expanded class of cell surface proteins selectively targeted for degradation by hcmv us2 in cooperation with ul141 cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins signal peptide peptidase is required for dislocation from the endoplasmic reticulum the trc8 e3 ligase ubiquitinates mhc class i molecules before dislocation from the er tmem129 is a derlin-1 associated erad e3 ligase essential for virus-induced degradation of mhc-i identifying the erad ubiquitin e3 ligases for viral and cellular targeting of mhc class i sel1l nucleates a protein complex required for dislocation of misfolded glycoproteins mhc class i ubiquitination by a viral phd/lap finger protein role of cd4 receptor down-regulation during hiv-1 infection mechanisms of cd4 downregulation by the nef and vpu proteins of primate immunodeficiency viruses a novel human wd protein, h-beta trcp, that interacts with hiv-1 vpu connects cd4 to the er degradation pathway through an f-box motif hiv-1 vpu -an ion channel in search of a job multilayered mechanism of cd4 downregulation by hiv-1 vpu involving distinct er retention and erad targeting steps retroviral rem protein requires processing by signal peptidase and retrotranslocation for nuclear function cytoplasmic localization of the orf2 protein of hepatitis e virus is dependent on its ability to undergo retrotranslocation from the endoplasmic reticulum the polyomaviridae: contributions of virus structure to our understanding of virus receptors and infectious entry murine polyomavirus requires the endoplasmic reticulum protein derlin-2 to initiate infection simian virus 40 depends on er protein folding and quality control factors for entry into host cells coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication how viruses hijack the erad tuning machinery electron tomography analysis of envelope glycoprotein trimers on hiv and simian immunodeficiency virus virions distribution and three-dimensional structure of aids virus envelope spikes retrovirus envelope protein complex structure in situ studied by cryo-electron tomography three-dimensional structure of herpes simplex virus from cryo-electron tomography paramyxovirus ultrastructure and genome packaging: cryo-electron tomography of sendai virus zernike phase contrast electron microscopy of ice-embedded influenza a virus why hiv virions have low numbers of envelope spikes: implications for vaccine development model for intracellular folding of the human immunodeficiency virus type 1 gp120 secretion of a truncated form of the human immunodeficiency virus type 1 envelope glycoprotein biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160 a novel hiv-1 restriction factor that is biologically distinct from apobec3 cytidine deaminases in a human t cell line cem the mitochondrial translocator protein, tspo, inhibits hiv-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway the mitochondrion in apoptosis: how pandora's box opens tspo interacts with vdac1 and triggers a ros-mediated inhibition of mitochondrial quality control elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation ermani (endoplasmic reticulum class i alphamannosidase) is required for hiv-1 envelope glycoprotein degradation via endoplasmic reticulum-associated protein degradation pathway identification, expression, and characterization of a cdna encoding human endoplasmic reticulum mannosidase i, the enzyme that catalyzes the first mannose trimming step in mammalian asn-linked oligosaccharide biosynthesis cloning and expression of a specific human alpha 1,2-mannosidase that trims man(9)glcnac(2) to man(8)glcnac(2) isomer b during n-glycan biosynthesis structural basis for catalysis and inhibition of n-glycan processing class i alpha 1,2-mannosidases role of the cysteine residues in the alpha1,2-mannosidase involved in n-glycan biosynthesis in saccharomyces cerevisiae. the conserved cys340 and cys385 residues form an essential disulfide bond mutations in the alpha 1,2-mannosidase gene, man1b1, cause autosomal-recessive intellectual disability somatic overgrowth associated with homozygous mutations in both man1b! and sec23a. cold spring harb. mol. case stud a golgi-localized mannosidase (man1b1) plays a non-enzymatic gatekeeper role in protein biosynthetic quality control hiv-1 vpr increases env expression by preventing env from endoplasmic reticulum-associated protein degradation (erad) activation of erad pathway by human hepatitis b virus modulates viral and subviral particle production role of the endoplasmic reticulum-associated degradation (erad) pathway in degradation of hepatitis c virus envelope proteins and production of virus particles no evidence of the unfolded protein response in patients with chronic hepatitis c virus infection human cytomegalovirus glycoproteins human cytomegalovirus penetrates host cells by ph-independent fusion at the cell surface human cytomegalovirus gh/gl/go promotes the fusion step of entry into all cell types, whereas gh/gl/ul128-131 broadens virus tropism through a distinct mechanism a novel herpes simplex virus glycoprotein, gl, forms a complex with glycoprotein h (gh) and affects normal folding and surface expression of gh human cytomegalovirus gh stability and trafficking are regulated by er-associated degradation and transmembrane architecture characterization of early edem1 protein maturation events and their functional implications role of edem in the release of misfolded glycoproteins from the calnexin cycle edem1 recognition and delivery of misfolded proteins to the sel1l-containing erad complex golgi localization of ermani defines spatial separation of the mammalian glycoprotein quality control system the authors declare no conflict of interest. key: cord-260869-rym2ik0o authors: lemmermeyer, tanja; lamp, benjamin; schneider, rainer; ziebuhr, john; tekes, gergely; thiel, heinz-jürgen title: characterization of monoclonal antibodies against feline coronavirus accessory protein 7b date: 2016-02-29 journal: vet microbiol doi: 10.1016/j.vetmic.2015.12.009 sha: doc_id: 260869 cord_uid: rym2ik0o feline coronaviruses (fcovs) encode five accessory proteins termed 3a, 3b, 3c, 7a and 7b of unknown function. these proteins are dispensable for viral replication in vitro but are supposed to play a role in virulence. in the current study, we produced and characterized 7b-specific monoclonal antibodies (mabs). a recombinant form of the 7b protein was expressed as a fusion protein in escherichia coli, purified by immobilized metal affinity chromatography and used as immunogen. two hybridoma lines, 5b6 and 14d8, were isolated that expressed mabs that recognized 7b proteins of both fcov serotypes. using an extensive set of nand c-terminally truncated 7b proteins expressed in e. coli and a synthetic peptide, the binding sites of mabs 5b6 and 14d8 were mapped to an 18-residue region that comprises the only potential n-glycosylation site of the fcov 7b protein. the two mabs were suitable to detect a 24-kda protein, which represents the nonglycosylated form of 7b in fcov-infected cells. we speculate that glycosylation of 7b is part of the viral evasion strategy to prevent an immune response against this antigenic site. feline coronaviruses (fcovs) are enveloped viruses with a large (approximately 30 kb) single-stranded rna genome of positive polarity. they belong to the family coronaviridae within the order nidovirales (de groot et al., 2012) . based on phylogenetic analyses, coronaviruses are divided into four genera termed alpha-,beta-, gamma-and deltacoronavirus. feline coronaviruses together with the closely related canine coronavirus (ccov) and porcine transmissible gastroenteritis virus (tgev) have been classified to the species alphacoronavirus 1 in the genus alphacoronavirus. the latter contains a large number of more distantly related alphacoronaviruses, including a number of important medical and veterinary pathogens, such as porcine epidemic diarrhea virus (pedv), human coronavirus 229e (hcov-229e), and human coronavirus nl63 (hcov-nl63) (de groot et al., 2012) . the 5 0proximal two-thirds of the coronavirus genome comprise orf 1a and orf 1b that together encode up to 16 nonstructural proteins (nsps) that form the viral replication/transcription complex but may also be involved in interactions with host cell factors and functions. the remaining part of the genome codes for the structural proteins s (spike), e (envelope), m (membrane) and n (nucleocapsid) (masters, 2006) and a varying number of so-called accessory proteins with poorly characterized functions. fcovs infect cats and other members of the felidae worldwide. based on serological properties, fcovs are separated into serotypes i and ii (heeney et al., 1990; kennedy et al., 2003) . serotype i viruses dominate in the field and account for up to 95% of fcov infections (hohdatsu et al., 1992; addie et al., 2003) , whereas the prevalence of type ii fcovs is much lower. serotype ii fcovs emerged through homologous recombination between serotype i fcovs and ccovs (herrewegh et al., 1998; terada et al., 2014) . in addition to a serology-based classification, fcovs may be divided into different biotypes based on their pathogenic potential. the avirulent biotype is generally referred to as feline enteric coronavirus (fecv) and causes inapparent persistent infections of the gut, while the virulent biotype, feline infectious peritonitis virus (fipv), causes a fatal disease called feline infectious peritonitis (fip) (pedersen, 2009) . according to the "internal mutation theory" fipv evolves from fecv through mutations in approximately 5-10% of the persistently infected cats (vennema et al., 1998) . so far, the genetic changes responsible for the biotype switch have not been identified, but there is increasing evidence that mutations in the accessory genes and the s gene are involved in the development of fip (vennema et al., 1998; kennedy et al., 2001; pedersen, 2009; chang et al., 2010 chang et al., , 2011 licitra et al., 2013; bank-wolf et al., 2014) . coronavirus genomes comprise a variable number of accessory genes at different positions in the 3 0 -proximal genome region. with only very few exceptions, homologs of specific accessory genes are only conserved in very closely related viruses (of the same species or sublineage) but not in the more distantly viruses (lai and cavanagh, 1997) . there is increasing evidence that accessory gene products are important for virulence in the natural host but the precise functions of the vast majority of accessory proteins remain to be investigated. alphacoronaviruses harbor accessory genes at two different positions in their genomes. between the s and e genes, fcovs and the very closely related ccovs possess three orfs (3a-3c), while tgev contains only two orfs (3a and 3b) in this genome region. recently, an additional orf, named orf3, was found between the s and e genes in ccov serotype i (lorusso et al., 2008) . other alphacoronaviruses, such as pedv, hcov-229e and hcov-nl63, have only one orf 3. sequence analyses suggest that fcov orf 3a is homologous to ccov orf 3a and tgev orf 3a while the fcov orf 3 c is a homolog of ccov orf 3c, tgev orf 3b and orf 3 of all other alphacoronaviruses. downstream of the n gene, all members of the species alphacoronavirus 1 contain a different number of additional accessory genes. thus, tgev contains only one orf (called orf 7), which is homologous to orf 7a of fcovs and ccovs, while the latter two coronaviruses contain yet another orf called 7b in the 3 0 -terminal genome region. altogether, the fcov genome encodes five accessory proteins termed 3a, 3b, 3c, 7a and 7b (dye and siddell, 2005; tekes et al., 2008) . using a reverse genetics approach, haijema et al. (2004) showed that the accessory genes of the fipv strain 79-1146 are dispensable for viral growth in vitro and recombinant viruses that lack orfs 3a-3c or 7a and 7b were unable to induce fip in vivo (2004) . so far, there is no evidence that 3a-3c accessory proteins are produced in infected cells. nevertheless, it has been proposed that 3c is essential for viral replication in the gut (as is the case for fecv) but dispensable for systemic infections (chang et al., 2010) . the functions of the fcov-accessory proteins 3a-3c remain to be determined. recently, it was suggested that the accessory protein 7a represents an interferon antagonist (dedeurwaerder et al., 2014) , although its expression in infected cells has not been confirmed. among the fcov-accessory proteins, the 7b protein has been studied most extensively. the 7b protein has a molecular mass of $26 kda, it is secreted from the cell and contains (i) a cterminal kdel-like endoplasmic reticulum (er) retention signal, (ii) an n-terminal signal sequence of 17 amino acids and (iii) a potential n-glycosylation site at aa position 68 (vennema et al., 1992a) . the presence of 7b-specific antibodies in both fecv-and fipv-infected cats indicates that this protein is produced during natural infections (vennema et al., 1992a,b; herrewegh et al., 1995; kennedy et al., 2008) . it was also reported that cell culture adaptation often leads to mutations in the 7b gene and loss of expression of this protein (herrewegh et al., 1995) . taken together, there is growing evidence that the accessory genes and their products are involved in fcov persistence and virulence but, up to now, their function is not known. one reason for our limited knowledge on fcov-accessory proteins is the lack of appropriate tools to study these proteins. to our knowledge, specific antibodies against the individual accessory proteins are not available. in this manuscript, we describe the production of fcov 7b protein-specific mabs. our data show that the mabs recognize the 7b protein of both serotype i and ii fcovs. moreover, using a panel of n-and c-terminally truncated 7b proteins and a synthetic peptide, we determined the binding sites of the mabs in 7b. the data further indicate that the mabs recognize in fcov-infected cells only the nonglycosylated form of 7b. all animal procedures were performed in strict accordance with the legal regulations of the german animal welfare jurisdiction (tierschutzgesetz). the animal experiment was approved by the giessen regional council (regierungspräsidium) and recorded after approval under reference number a15/2013. crandell rees feline kidney cells (crfk) were purchased from the american type culture collection (attc ccl-94). felis catus whole fetus 4 cells (fcwf-4) were kindly provided by r. j. de groot, university of utrecht, the netherlands. both cell lines were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal calf serum (fcs), penicillin (100 iu ml à1 ) and streptomycin (100 iu ml à1 ) in 5% co 2 at 37 c. the type-ii strain fcov 79-1146 was kindly provided by r. j. de groot, university of utrecht, the netherlands (genbank nc_002306). the recombinant type-i fcov strain recfcov-dstop-7b was generated by reverse genetics as described previously (tekes et al., 2012) . it comprises the type-i fcov strain black with a restored accessory gene 7b (genbank eu186072). the type-ii fcov 79-1146 and the type-i recfcov-dstop-7b were propagated on crfk cells and fcwf-4 cells, respectively. crfk or fcwf cells were incubated for 1 h with fcov 79-1146 (multiplicity of infection [moi] of 1) or recfcov-dstop-7b (moi of 0.001), respectively. to inhibit n-glycosylation of viral proteins, 2 mg ml à1 tunicamycin in dimethyl sulfoxide (dmso) were added 4 h post infection (p.i.). at 24 h p.i., the cells were harvested in ripa buffer (150 mm nacl, 50 mm tris, 1% np-40, 0.5% na-deoxycholate, 0.5 mm pefabloc sc [merck], ph 8.0) with 0.2% sds for sds-page and western blot analysis. following treatment with tunicamycin, cells were either fixed for immunofluorescence or harvested in ripa buffer for sds-page and western blot analysis at 15 h p.i. deglycosylation of cell lysates was carried out for 3 h with nglycosidase f (pngase f; new england biolabs, ma) according to the manufacturer's instructions. the full-length 7b genes of fcov 79-1146 (nucleotides (nts) 28462-29082) and recfcov-dstop-7b (nts 28337-28954) were amplified by reverse transcription polymerase chain reaction (rt-pcr). the amplified product of fcov 79-1146 was cloned with primers c32/c33 into the ncoi/xhoi sites of a modified pet-11a vector (novagen). the resulting plasmid was named pc18.2 and contained a c-terminal 12â histidine-tag (7b-his). the amplified product of recfcov-dstop-7b was cloned with primers c83/ c82 into a modified pgex-6p-1 vector (ge healthcare) by digestion with bamhi and xhoi. the resulting plasmid with an n-terminal gst and a c-terminal 13â his-tag was named pc80.1 (gst-7bdstop-his). for the generation of gst-7bdss-his (pc52.1; c50/ c47), a deletion mutant of 7b (amino acid 17-206, without the nterminal signal sequence [dss] ) was cloned into the same modified pgex-6p-1 vector. to determine the antigenic site of the monoclonal antibodies, n-and c-terminally truncated 7b proteins of fcov 79-1146 were generated as gst-fusion proteins. to this end, the sequences were amplified by pcr with specific primers and cloned into the modified pgex-6p-1 vector. the resulting plasmids were named pc71.2 (aa 112-206; c72/c47), pc72.1 (aa 1-111; c46/c73), pc82.1 (aa 65-206; c84/c47), pc88.1 (aa 26-206; c86/c47), pc89.2 (aa 34-206; c87/c47), pc90.1 (aa 42-206; c88/ c47), pc91.5 (aa 50-206; c89/c47), pc92.9 (aa 58-206; c90/c47), pc97.4 (aa 1-60; c46/c101), pc98.1 (aa 1-63; c46/c102), pc102.3 (aa 1-67; c46/c103), pc108.2 (aa1-70; c46/c107) and pc114.1 (aa 1-75; c46/c109). primer sequences are shown in table 1 . the pet-11a vector was modified by pcr with primers c17 and c18 to insert an ncoi and xhoi restriction site upstream of a bamhi restriction site. furthermore, a 7â his-tag followed by a stop codon behind the xhoi site was inserted and the existing bamhi site was deleted. in a second pcr with primers c12 and c18, the his-tag was extended with 5 histidines to generate a 12â his-tag. the pgex-6p-1 vector was modified by pcr with primers c36 and c37 to insert a 13x his-tag downstream of the existing xhoi site. plasmid constructs were verified by sequence analysis. the expression of the recombinant proteins was performed after transformation of the different plasmids into escherichia coli (e. coli) strain rosetta tm (de3) plyss (novagen). an overnight culture was diluted 1:10 in fresh lb medium supplemented with ampicillin (100 mg ml à1 ), chloramphenicol (34 mg ml à1 ) and glucose (1%) to a final volume of 500 ml and grown for 2-3 h at 37 c to od 600 of 1.0. protein expression was induced by the addition of isopropyl-b-d-thiogalactopyranoside (iptg) to a final concentration of 1 mm. after 3 h of growth at 28 c the cells were harvested by centrifugation and resuspended in 50 mm na 2 hpo 4 , 300 mm nacl and 1% triton x-100. cells were lysed by freeze/thaw treatment (3â) and sonication on ice. the insoluble protein fraction was separated from soluble proteins by ultracentrifugation (100,000 â g for 1 h). to solubilize inclusion bodies the pellet was resuspended in 8 m urea, 300 mm nacl and 50 mm na 2 hpo 4 , ph 8.0 or in 6 m guanidinium-hydrochloride (guhcl), 50 mm na 2 hpo 4 , 20% ethanol, 1% triton x-100 and 100 mm nacl, ph 8.0 and treated with ultrasonication. after an additional ultracentrifugation at 100,000 â g for 30 min, the solubilized proteins were applied on histrap hp columns (ge healthcare). the purification of the recombinant proteins was performed by metal ion affinity chromatography (imac) with ni 2+ sepharose as recommended in the supplier's protocol. the purified recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and coomassie staining. following electrophoresis the proteins were identified by western blot using anti-his mab. after dialysis overnight against pbs (gst-7b-dss-his) or acetone precipitation (7b-his), the proteins were used for the immunization of mice. protein concentrations were determined in bradford assay (bradford, 1976) . four 12 week old female balb/c mice (charles river breeding laboratories) were injected subcutaneously with 50 mg of purified protein gst-7bdss-his in freund's incomplete adjuvant (sigma) on days 0, 22 and 54. antibodies binding the recombinant protein were detected in serum samples by western blotting after the third injection. on days 70, 71 and 72 the mouse with the best immune response was immunized with 40 mg of purified protein 7b-his without adjuvant. one day after the final antigen application splenocytes were fused with sp2/0-ag14 myeloma cells (atcc crl-1581) at a ratio of 3:1 using polyethylene glycol 1.500 (roche) according to standard protocols (köhler et al., 1976) . the fused cells were cultured in 96-well plates and hybridoma cells were selected in dmem supplemented with 20% f10 (gibco), 20% f12 (gibco), 15% fcs, 2 mm l-glutamine, 0.35% 2-mercaptoethanol, 50 mm hepes, oxaloacetate-pyruvate-insulin supplement (opi; sigma), and hypoxanthine-aminopterin-thymidine supplement (hat; sigma). for the detection of specific antibodies against the 7b protein, hybridoma culture supernatants were screened by elisa (engvall and perlmann, 1971 ) 7-10 days after fusion. to avoid screening for antibodies that react with gst, the fusion protein 7b-his was used for coating. however coating with 7b-his resulted in od 450nm values <0.35. as an alternative flat-bottomed elisa 96-well plates (nunc maxisorp) were coated overnight at 4 c with 35 ng purified gst-7bdss-his protein in 0.1 m na 2 co 3 /nahco 3 (ph 9.5) per well. the supernatants were also tested against an irrelevant control protein (gst-yfp-his; 75 ng/well) in order to exclude gst-and hisreactive clones. this control protein was expressed in the same vector and purified as described for the gst-7bdss-his construct. after washing three times with pbst (pbs containing 0.1% tween-20), the plates were blocked with 10% fcs in pbst for 1 h at 37 c. then, the plates were washed again and incubated with 100 ml/ well of hybridoma culture supernatants. specific antibodies were detected using goat anti-mouse igg conjugated with horseradish peroxidase (dianova) at dilution 1:2500. the elisa was performed with 3,3 0 ,5,5 0 -tetramethylbenzidine (tmb; sigma) and enzyme reactions were terminated with 25% (v/v) h 2 so 4 solution. the optical density (od values) at 450 nm was measured in a microtiter plate reader (spectra ii, slt). hybridomas with elisa values >0.4 were cloned twice. reactivity was confirmed by elisa and specificity was determined by western blot analysis. to confirm the antigenic sites recognized by the mabs 5b6 and 14d8, elisa table 1 primer sequences used to amplify defined fragments of fcov orf 7b. sequence 5 plates were coated with 460 ng/well of bsa-7b-pep (c-rvece-giegfnctwpgfq [centic biotec, germany]). due to problems during the synthesis of the peptide, the internal cysteines were linked by a disulfide bond. therefore, the peptide was incubated with the reducing agent dtt (5 mm) for 1 h at room temperature before coating. as negative control, an irrelevant peptide (coupled to bsa), bsa-7b-clvg (1 mg/well; c-lvgavpkqkrlnvg, biogenes, germany) and, as positive control, the purified protein gst-7bdss-his (36 ng/well) were coated. the mabs 5b6 and 14d8 were used at a 1:3 dilution, anti-his mab was used at a 1:10 dilution. the elisa was performed as described above. the immunoglobulin subclass of both mabs was determined using the commercially available iso-gold rapid mouse-monoclonal isotyping kit (bioassay works) according to the manufacturer's instructions. sds-page was carried out using a 10% tricine-polyacrylamide gel system (schagger and von jagow, 1987) . for western blot analysis, the proteins were transferred to a nitrocellulose membrane (pall corporation). residual binding sites were blocked with 4% skimmed milk in pbst. the membrane was then incubated with monoclonal antibodies of the hybridoma cell culture supernatants (primary antibodies) at a 1:3 dilution or sera in pbst (1:1500 dilution). bound antibodies were visualized with hrpconjugated goat anti-mouse or goat anti-cat igg (dianova) and chemiluminescence reagent (western lightning plus-ecl; perki-nelmer). goat anti-cat igg was diluted in pbst with 1x roti 1 -block (roth) additionally. crfk cells were cultured on sterile cover slips in 24 well plates pretreated with rat tail collagen (sigma). cells were mock or virusinfected and treated with 2 mg ml à1 tunicamycin 4 h p.i. at 15 h p.i. the cells were fixed with pbs containing 4% paraformaldehyde for 20 min at 4 c, followed by three pbs washes. cells were permeabilized with triton x-100 (1% in pbs) for 5 min at room temperature. the cells were rinsed twice with pbs and residual binding sites were blocked with 1â roti 1 -immunoblock (roth) for 10 min at room temperature. the cells were then incubated with primary antibodies (1:3 dilution) or sera (1:1500 dilution) in pbs for 1 h at 37 c, followed by three pbs washes. subsequently the cells were incubated with secondary antibodies conjugated to cyanogen-3 (1:500 dilution, dianova) in pbs for an additional hour at 37 c. the dna was counterstained with 66 mg ml à1 4 0 ,6diamidino-2-phenylindole (dapi) for 5 min at room temperature. after three washes the cover slips were mounted with mowiol (sigma) containing the anti-fading reagent dabco (1,4-diazabicyclo(2.2.2)octane, roth). the immunofluorescent staining was visualized by fluorescence microscopy (axiovert 10, zeiss). the full-length 7b protein of fcov 79-1146 of 206 amino acid (aa) residues was expressed in e. coli with a c-terminal his-tag (7b-his). induction of expression of 7b-his resulted in insoluble protein aggregates that could be solubilized using 6 m guhcl-containing buffer and purified by ion metal affinity chromatography (imac). the identity of the purified protein was confirmed by sds-page and western blotting using anti-his mab. the protein had an apparent molecular mass of 24 kda as judged by sds-page analysis, which is predicted for this protein, and was recognized by the his-tag-specific antibody (fig. 1a) . western blot analysis further revealed a protein of approximately 48 kda, suggesting dimerization of the full-length protein. the overall yield for this protein was low. we therefore decided to express 7b as part of a gst fusion protein. to do this, the 5 0 region encoding a putative nterminal signal sequence (ss, residues 1 to 17) was removed from the 7b coding sequence and replaced by the gst coding sequence. the resulting construct was termed gst-7bdss-his. expression of gst-7bdss-his in e. coli resulted again in insoluble protein aggregates. the protein was purified under denaturing conditions using 8 m urea-containing buffer and analyzed by sds-page and western blotting using an anti-his mab. consistent with its calculated molecular mass, the major fraction of the purified protein migrated as a 51-kda protein in sds polyacrylamide gels. two additional minor bands in the sds-page were specifically recognized by western blotting using the his-tag-specific mab. these bands are consistent with a dimer and a 37-kda degradation product, respectively, of the gst-7bdss-his protein (fig. 1b) . the total yield of gst-7bdss-his was estimated to be 25 times higher compared to 7b-his. the elution fractions of 7b-his and gst-7bdss-his were pooled, concentrated, desalted and used for immunization ( fig. 1c and d, respectively) . the fusion protein 7b-his was used for the immunization of balb/c mice. however, even after four injections over a time period of 12 weeks, no immune reaction against 7b-his could be detected in sera obtained from immunized animals. we therefore decided to immunize another four balb/c mice using the gst-7bdss-his protein. two weeks after the third injection a specific seroconversion was detected. the mouse with the best immune response received an additional boost with 7b-his. splenocytes isolated from immunized mice were fused with myeloma cells and cell culture supernatants were screened by elisa using the gst-7bdss-his as capture antigen. gst-yfp-his was used as a control antigen to identify and exclude clones producing antibodies specific for one of the tags. two supernatants were confirmed to contain antibodies that bind to gst-7bdss-his but not the gst-yfp-his control protein. the respective hybridoma cells were subcloned twice and continued to produce 7b-specific antibodies as determined by elisa. antibodies produced by the two stable hybridoma cell lines (5b6 [igg2a], 14d8 [igg1]) were subsequently tested by western blotting. both mabs recognized the recombinant proteins 7b-his (fig. 2a) and gst-7bdss-his (data not shown). the data also suggest that the formation of the putative 7b dimer discussed above ( fig. 1c and text) involves one or more intermolecular disulfide bonds since the higher molecular mass band of >40 kda was not detectable if 2-mercaptoethanol was included in the protein sample buffer (fig. 2b) . to determine whether the two fcov serotype ii (strain 79-1146) 7b protein-specific mabs recognize epitopes that are conserved among serotype i and ii strains, the mab reactivities against the well-characterized serotype i strain black were analyzed. the 7b proteins of strains black and 79-1146 share 90% amino acid sequence identity. however, strain black contains an internal translation stop codon in orf 7b, resulting in a c-terminally truncated 7b protein. we therefore restored the orf 7b of fcov strain black and expressed the strain black 7b peptide sequence as part of a recombinant fusion protein (gst-7bdstop-his) in e. coli. western blot analysis of lysates obtained from iptg-induced e. coli cells transformed with the appropriate expression plasmid confirmed that the two mabs recognized the recombinant 7b protein of strain black (data not shown), confirming that the two mabs obtained in this study recognize epitopes that are conserved among the 7b proteins of representative fcov serotype i and ii strains. to further characterize the newly generated mabs, we sought to determine the binding sites within the 7b protein. as shown above, both mabs recognized 7b-his in western blot experiments after sds-page under non-reducing and reducing conditions, suggesting that they recognize a linear epitope on the 7b protein. first, we produced a set of bacterially expressed segments of 7b. appropriate coding sequences were cloned into the pc23.6 plasmid and expressed with n-terminal gst and c-terminal histidine tags. mab reactivities against these proteins were tested by western blotting (fig. 3) . all proteins containing aa residues 58-111 of 7b (pc72.1, pc88.1, pc89.2, pc90.1, pc91.5, pc92.9) were detected by the mabs. in contrast, proteins containing residues 112-206 (pc71.2) or 65 to 206 (pc82.1) were not recognized. furthermore, a protein containing residues 1-75 of 7b (pc114.1) was recognized while none of the slightly smaller proteins that lacked a few more residues at the carboxyl terminus (pc97.4, pc98.1, pc102.3, pc108.2) were recognized. the combined results summarized in fig. 3 led us to conclude that both mabs recognize a peptide segment that includes residues 58 to 75 of the 7b protein (fig. 3) . to further corroborate this hypothesis, a synthetic peptide coupled to bsa (bsa-7b-pep, c-58 rvecegiegfnctwpgfq 75 ) was specificities of 5b6 and 14d8 were characterized using total lysates of noninduced (à) and iptg-induced (+) e. coli rosetta cells transformed with the appropriate 7b-his expression plasmid (see materials and methods). proteins were separated by sds-page (10%) under nonreducing conditions and antibody reactivities were analyzed by western blotting using anti-7b mabs (5b6 and 14d8, respectively) and anti-his mab (control) as indicated. (b) the purified protein 7b-his/e100 was separated by sds-page (10%) in the absence (à2-me) or presence (+2-me) of the reducing agent 2-mercaptoethanol. western blot analysis was performed using anti-7b mab 5b6. arrowheads indicate the recombinant protein 7b-his. e100: elutate fraction obtained with 100 mm imidazole. used in an additional set of experiments. to resolve problems that occurred during peptide synthesis, an intramolecular disulfide bond was introduced. to further characterize the mab binding properties, bsa-7b-pep was used as antigen in an elisa that was performed under non-reducing and reducing conditions, respectively. bsa-7b-pep was treated with or without dtt and elisa plates were coated with the peptide. bsa-7b-clvg and purified gst-7bdss-his fusion protein were used as negative and positive control, respectively. as shown in fig. 4 , mabs 5b6 and 14d8 were confirmed to bind specifically to bsa-7b-pep in the absence and presence of dtt. the data provide direct evidence that both mabs recognize a region in the 7b protein that spans residues 58 to 75. 3.5. comparative sequence analysis of the peptide region recognized by the mabs as shown above, the epitope(s) recognized by both mabs are located within the amino acid sequence 58 to 75 of the fcov 7b protein. this part of the 7b sequence is highly conserved within fcov serotypes i and ii with sequence identities of 78-94%. interestingly, this part of the sequence comprises the only n-glycosylation motif ( 68 nct 70 ) within the 7b sequence (fig. 5) . this motif is conserved in almost all sequenced fcov strains. in order to detect the authentic 7b protein with our newly generated anti-7b mabs, crfk cells were mock-infected or infected with fcov 79-1146 and analyzed by indirect fig. 3 . determination of the 5b6 and 14d6 binding sites in the 7b protein. the scheme shows the full-length 7b construct and the truncated versions derived from this construct. all proteins were expressed as gst-and his-tag fusion proteins and mab reactivities were tested by western blotting under non-reducing conditions. +: specific binding detected; à: not detected. fig. 4 . reactivities of mabs 5b6 and 14d8 against bsa-7b-pep as determined by elisa. elisa plates were coated with bsa-7b-pep in the presence (+) or absence (à) of dtt. as controls, bsa-7b-cvlg and gst-7bdss-his were used. the data represent three independent experiments; standard deviations are indicated. immunofluorescence assay (ifa) 24 h post infection (p.i.). surprisingly, neither of the anti-7b mabs produced a positive signal, although there was a clear reactivity with an antiserum against fcov (data not shown). as the two mabs were shown previously to recognize fcov 7b fusion proteins in western blot experiments (fig. 2) , we used this method to analyze cell lysates obtained at 24 h p.i. both mabs detected a protein with an apparent molecular mass of approximately 24 kda in fcov-infected cells, whereas no signal was detected in mock-infected cells (fig. 6a) . it has been shown by others that orf 7b encodes a glycoprotein of 26 kda (vennema et al., 1992a) . in order to investigate whether our mabs detect a glycosylated form of the 7b protein, pngase f treatment of cell lysates was performed. surprisingly, incubation with pngase f did not affect the migration behavior of the 7b protein (data not shown), indicating that the protein detected by the mabs represents a nonglycosylated form of the 7b protein. since the only n-glycosylation motif in 7b is located at aa position 68-70 (nct), we speculated that the n-glycosylation site might be part of the epitope(s) recognized by the mabs. accordingly, the epitope(s) may be masked and thereby inaccessible for our antibodies. in order to test this hypothesis, the effect of an inhibitor of n-linked glycosylation was studied. crfk cells were infected with fcov 79-1146 or mock-infected and treated with tunicamycin at 4 h p.i. cell lysates were prepared at 15 h p.i. and probed by western blotting. the mabs recognized a single protein with an apparent molecular mass of 24 kda in the absence and presence of tunicamycin (fig. 6b) . interestingly, the signal intensity obtained with the mabs was much stronger after inhibition of nglycosylation. moreover, after treatment with tunicamycin, a dimer of 7b was recognized by both mabs which was not detectable in the absence of tunicamycin (fig. 6b) and disappeared after incubation with 2-mercaptoethanol (data not shown). these results support the idea that both mabs do not recognize the glycosylated form of fcov 7b. an anti-fcov serum, which served as positive control, detected the virus-specific proteins n and m (fig. 6b) . in addition, a protein band with an apparent molecular mass of 24 kda was detected by the anti-fcov serum. this protein may represent the nonglycosylated form of the m protein and/or the 7b protein. the results outlined above show that the anti-7b mabs recognize exclusively the nonglycosylated form of the viral protein in fcov-infected cells. this was particularly striking after treatment of the cells with tunicamycin. in light of this finding, another round of immunofluorescence experiments was performed with the mabs after infection with fcov 79-1146 and incubation with tunicamycin. as already mentioned, there was no staining after incubation with the mabs in the absence of tunicamycin. however, after tunicamycin treatment, clear signals were obtained in infected cells (fig. 7) . at this point, the observed cytoplasmic localization of 7b cannot be further narrowed down. an anti-fcov serum detected viral proteins in the cytoplasm of infected cells regardless of whether or not they were treated with tunicamycin ( fig. 7) . the genome of feline coronavirus (fcov) encodes five accessory proteins termed 3a-3c, 7a and 7b. among these, only 7b has been identified in virus-infected cells (vennema et al., 1992a) . the 7b protein was also expressed from a plasmid under the control of the t7 promoter in t7 polymerase-expressing vaccinia virus-infected cells. the produced 7b protein was identified with ascites from a fipv-infected cat. fcov 7b was shown to be a glycoprotein that to some extent may be secreted from infected cells. interestingly, a kdel-like sequence at its c-terminus has been shown to slow down the export of the protein (vennema et al., 1992a) . we are interested in accessory proteins encoded by fcov in particular with respect to potential roles in (i) establishing persistent infections by enteric feline coronavirus in the gut and (ii) the pathogenesis of feline infectious peritonitis (pedersen, 2009) . since there was already some information on fcov 7b, we first concentrated on this protein. for further characterization including its unambiguous identification in fcov-infected cells monoclonal antibodies against 7b protein were generated. using nand c-terminally truncated parts of the 7b protein expressed by bacteria as well as a synthetic peptide conjugated to bsa, the binding sites of two mabs were determined. according to this analysis, the mabs recognize a stretch of 18 amino acids (aa) between aa position 58 and 75. interestingly, the only putative nlinked glycosylation site as well as two of the seven cysteines of the fcov 7b protein are contained within this sequence. this finding led to the question whether the mabs recognize only the nonglycosylated form of 7b. in fact, both mabs detected in fcov-infected cells only recognized a protein with an apparent molecular weight of 24 kda and not the previously described glycosylated form of 26 kda (vennema et al., 1992a) . the exclusive recognition of the nonglycosylated form was supported by results obtained after tunicamycin treatment of fcov-infected cells. notably, immunofluorescence studies using the mabs led to negative results unless the fcov-infected cells were treated with tunicamycin. the difference to western blot experiments where the nonglycosylated form could be readily shown without inhibition of glycosylation most likely results from different sensitivities of the two methods. masking of epitopes by glycosylation has been shown for many other viral proteins including the hemagglutinin of influenza virus (skehel et al., 1984) . the authors also used tunicamycin to demonstrate recognition by mabs in the absence of n-linked glycosylation. accordingly, the antigenicity of influenza virus hemagglutinin and of other proteins can be modified by addition of carbohydrate chains. furthermore, since both mabs recognize the same or a nearby epitope in 7b, it can be assumed that the identified 18 aa stretch represents a highly antigenic site. masking of this region through glycosylation of asparagine 68 may prevent an immune response and could be relevant for establishment of persistent infection by fcov. this could be directly tested by the use of recombinant fcovs, where the n-linked glycosylation site of 7b is missing. it remains to be seen whether this finding has implications for vaccine development against fcov-induced diseases. another interesting aspect of our studies with fcov 7b concerns putative intra-and intermolecular disulfide bonds. in this context, it is noteworthy that expression of 7b in bacteria allowed the detection of disulfide linked dimers, which disappeared after treatment of extracts with a reducing agent. such dimeric forms were also detected in fcov-infected cells, but apparently only after treatment with tunicamycin. thus, it is tempting to speculate that intermolecular disulfide bridges represent an artifact only observed in the absence of glycosylation at asparagine 68 of fcov 7b. it will be interesting to determine whether one or both cysteines present in our synthetic peptide is/ are involved in intramolecular disulfide bonds. recognition of 7b by our mabs in the absence and presence of reducing agent may give some indirect evidence in this regard. pilot experiments actually indicate that our mabs recognize the nonreduced form of 7b from fcov-infected cells better than the reduced one; this result suggests that the native protein contains one or more intramolecular disulfide bridges, which are important for the antigenicity of 7b. another way to study the relevance of disulfide bonds for the structure of 7b is the exchange of cysteine codons in different expression systems together with western blots under nonreducing and reducing conditions. however, final proof for the presence of intramolecular disulfide bridges within fcov 7b will require the determination of its three-dimensional crystal structure. unfortunately, our mabs are not well suited to detect the secreted glycosylated form of 7b. to further study the synthesis, intracellular localization and secretion of 7b, we will use our reverse genetics system (tekes et al., 2008 (tekes et al., , 2012 to obtain recombinant viruses containing the desired changes in the 7b coding sequence. ultimately, these studies together with the three dimensional structure of 7b will give more insight into the biological role of the 7b protein. persistence and transmission of natural type i feline coronavirus infection mutations of 3c and spike protein genes correlate with the occurrence of feline infectious peritonitis a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral 3c gene sequence analysis of feline coronaviruses and the circulating virulent/avirulent theory family coronaviridae orf7-encoded accessory protein 7a of feline infectious peritonitis virus as a counteragent against ifn-alpha-induced antiviral response genomic rna sequence of feline coronavirus strain fipv wsu-79/1146 enzyme-linked immunosorbent assay (elisa): quantitative assay of immunoglobulin g live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis prevalence and implications of feline coronavirus infections of captive and free-ranging cheetahs (acinonyx jubatus) the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf7a/7b transcription unit of different biotypes feline coronavirus type ii strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type i and canine coronavirus the prevalence of types i and ii feline coronavirus infections in cats deletions in the 7a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis detection of feline coronavirus infection in southern african nondomestic felids evaluation of antibodies against feline coronavirus 7b protein for diagnosis of feline infectious peritonitis in cats fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines the molecular biology of coronaviruses mutation in spike protein cleavage site and pathogenesis of feline coronavirus gain, preservation, and loss of a group 1a coronavirus accessory glycoprotein the molecular biology of coronaviruses a review of feline infectious peritonitis virus infection: 1963-2008 tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kda a carbohydrate side chain on hemagglutinins of hong kong influenza viruses inhibits recognition by a monoclonal antibody genome organization and reverse genetic analysis of a type i feline coronavirus a reverse genetics approach to study feline infectious peritonitis emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses a novel glycoprotein of feline infectious peritonitis coronavirus contains a kdel-like endoplasmic reticulum retention signal genomic organization and expression of the 3 0 end of the canine and feline enteric coronaviruses feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses this study was supported by the "bundesministerium für bildung und forschung" of the german government (zoonosis network, consortium on ecology and pathogenesis of sars, project code 01ki1005a-f) as well as by the collaborative research centre 1021: "rna viruses: rna metabolism, host response and pathogenesis". key: cord-261472-qcu73sdu authors: yao, yong xiu; ren, junyuan; heinen, paul; zambon, maria; jones, ian m. title: cleavage and serum reactivity of the severe acute respiratory syndrome coronavirus spike protein date: 2004-07-01 journal: j infect dis doi: 10.1086/421280 sha: doc_id: 261472 cord_uid: qcu73sdu severe acute respiratory syndrome (sars) coronavirus (scov) spike (s) protein is the major surface antigen of the virus and is responsible for receptor binding and the generation of neutralizing antibody. to investigate scov s protein, full-length and individual domains of s protein were expressed on the surface of insect cells and were characterized for cleavability and reactivity with serum samples obtained from patients during the convalescent phase of sars. s protein could be cleaved by exogenous trypsin but not by coexpressed furin, suggesting that the protein is not normally processed during infection. reactivity was evident by both flow cytometry and western blot assays, but the pattern of reactivity varied according to assay and sequence of the antigen. the antibody response to scov s protein involves antibodies to both linear and conformational epitopes, with linear epitopes associated with the carboxyl domain and conformational epitopes associated with the amino terminal domain. recombinant scov s protein appears to be a suitable antigen for the development of an efficient and sensitive diagnostic test for sars, but our data suggest that assay format and choice of s antigen are important considerations. and is responsible for virus attachment to receptors, entry by fusion, and the development of neutralizing antibody [9] . of interest, most of the residue changes identified within s protein lie in a region that, in other covs, causes significant alteration in tropism [10] , suggesting that drift toward a virus more capable of using a human cell-surface receptor could occur. the structure and function of scov s protein is, therefore, an integral part of studies of viral tropism and of the development of a receptor-blocking antibody response. during the convalescent phase of sars, antibody against the whole virus can be detected at day 10 and increases to a peak titer by day 28 [11] . however, the spectrum of the antibody response, its role in viral clearance, and its use as a correlate of protection has not been described. because scov s protein is such a pivotal viral protein, we have applied a high-throughput expression strategy to the production of recombinant s protein for biochemical and antigenic characterization. here, we describe high-level expression of 6 overlapping fragments of s protein on the surface of insect cells, a context that allows high-level production and maintains sensitive conformation [12, 13] . recombinant s protein showed strong serum reactivity in convalescent-phase serum samples by use of both flow cytometry and western blotting (wb), suggesting that it could be used in the development of sars-specific diagnostic techniques. reactivity was not equivalent between fluorescence-activated cell sorting (facs) and wb, however, and was not equal on all fragments, providing evidence of a variety of antibodies in the host response. cell culture. spodoptera frugipurda (sf9) cells were routinely cultured in sf900-ii medium (life sciences) at 28њc in suspension culture. for plaque assays, cells were allowed to settle onto polystyrene dishes for 1 h before virus inoculation. after virus adsorption, cells were sequentially overlaid with 1.6% lowmelting-temperature agarose and then with sf900-ii supplemented with 5% fetal calf serum and were cultured for 4 more days before staining with neutral red. vector construction. a cdna encoding the full-length s protein minus the signal peptide and transmembrane (tm) domain (aa 18-1193) of the hong kong isolate of scov [7] was supplied by andrew davidson and stuart siddell (university of bristol, bristol, uk) as a reverse-transcription polymerase chain reaction-amplified fragment and was first cloned into ptopo (invitrogen). several overlapping fragments of s protein, with end points chosen after bioinformatics analysis, were amplified from the original clone. all fragments generated were flanked by dissimilar restriction sites for the enzyme sfii. each fragment was cloned into a set of 3 vectors (18 variants in all), to provide his-tagged, maltose binding protein-tagged, and cell surface-displayed forms of s protein. only the cell surface-displayed cassette is described in detail here. the baculovirus display transfer vector pacvsv gtm [12] , modified to include sfii sites at the junction of the signal peptide and the vesicular stomatitis virus (vsv) g protein tm domain, was used to provide expression at the cell surface. expression in insect cells. for expression in insect cells, recombinant baculoviruses were constructed by use of a new, rapid recombination technique after cotransfection of each transfer vector with a linearized, modified form of bacmid dna capable of growth only after recombination [14] . coexpression of mouse furin or human calnexin was done by coinfection of viruses at mois 15 [15] . wb. protein samples to be analyzed were separated on precast 10% tris-hcl sds-polyacrylamide gels (bio-rad) and transferred onto immobilon-p transfer membranes (millipore). wb was performed with human convalescent-phase serum or rabbit serum to the vsv g protein tm domain (research diagnostics), followed by peroxidase-coupled secondary antibodies (sigma). the membrane was finally developed by use of bm chemiluminescence (roche). facs analysis (flow cytometry). cells were plated in a 6well plate ( cells/well) and overlaid with 2 ml of me6 1 ϫ 10 dium. after virus inoculation, cells were cultured for a further 2 days at 28њc, harvested, washed with pbs, fixed with cellfix (becton dickinson), and stained with serum and conjugate. data acquisition and analysis were performed by use of a fac-scan flow cytometer and cellquest software (both from becton dickinson). human serum. serum samples from patients with sars and human cov 229e were obtained from the respiratory disease reference laboratory of the health protection agency (colindale, uk). nine probable sars cases occurred in the united kingdom, but only 4 conformed to the clinical definition of sars in use at the time. serum samples from patients known to be positive for sars were derived from the patients with clinically confirmed cases, whereas serum samples used as probable but unconfirmed sars cases were derived from the remaining 5 patients. all patients recovered from sars. no human cov oc43 serum samples were available for the present study, so the reactivity of cov oc43 with sars s protein could not be assessed. similarly, only 2 serum samples from cov 229e-infected patients were available. serum samples were used in facs and wb analyses at 1:50 and 1:400 dilutions, respectively, unless otherwise stated. the time-course serum samples, available from 1 of the patients with confirmed sars, were assayed by use of elisa using lentil lectin-captured, fulllength s protein, and the 50% end-point titer was compared. the class of antibody bound was ascertained by use of classspecific, secondary anti-human antibodies (sigma). the scov s protein is ∼1200 residues and has 23 glycosylation triplets clustered toward the amino and carboxyl termini. because glycosylation and protein folding are often linked, we focused our expression studies on the use of a highyielding but eukaryotic-based expression system: baculovirusmediated expression in insect cells. we examined a number of biochemical features, to characterize the protein expression before use for antibody binding. the s protein signal peptide and tm domain were substituted for regions known to function well in this expression system-a signal peptide from the major baculovirus glycoprotein gp64 and a tm domain from the vsv g protein already present in pacvsv g tm [12] . this vector efficiently displays proteins on the surface of insect cells and is a method of expression that maintains the conformational integrity of even problematic proteins [13] . in addition to the full-length s protein, fragments representing the predominantly b-sheet and a-helical domains (residues 18-713 and 714-1193, respectively), a fragment from the n terminus (residues 18-410) predicted to form a distinct folded domain when analyzed by folding predication software [16] , and fragments representing residues 411-1193 and 411-713 were expressed in the same way ( figure 1a) . abundant expression of all s protein fragments was achieved by use of this strategy, as shown by wb with a vsv g tm domain antibody that showed 2 predominant bands for each construct representing nonglycosylated and glycosylated proteins, with apparent molecular weights consistent with those predicted (figure 1b). interaction with calnexin and s protein cleavage. the s proteins of all covs are heavily glycosylated, and improper glycan trimming can result in poor surface-expression levels through retention of the viral glycoprotein by the glycoprotein chaperone calnexin [17, 18] . to assess whether the calnexin pathway was relevant to expression of s protein, we coinfected recombinant full-length s protein with a virus-expressing calnexin and re-examined the level of glycosylated and nonglycosylated protein at 3 days after infection. compared with expression of s protein only, coexpression of s protein with calnexin led to an increase in the ratio of glycosylated to nonglycosylated product, calculated by use of gel scan to be ∼20% ( figure 2a) , showing that s protein interacts with calnexin during glycosylation. however, no overall increase in s protein yield was observed, despite improved glycoprotein processing. some, but not all, cov s proteins are cleaved around the center of the molecule to form the receptor-binding outer domain s1 and the inner-membrane fusion domain s2 [19, 20] . the full-length s protein was not cleaved in insect cells, with the fully glycosylated form having an apparent molecular weight of ∼180 kda ( figure 1b) . we assessed the potential for s protein to be cleaved by 2 proteases, the subtilisin-like furin (provided by coinfection, as described elsewhere [15] ) and by trypsin (added exogenously). furin is a classic viral glycoprotein maturation enzyme [21] . the consensus furin cleavage site does not occur in the s protein sequence, but several dibasic sites, which also can be recognized by the enzyme [15] , are present, allowing the possibility of furin cleavage. recombinant s protein produced in the presence of coexpressed furin showed no evidence of cleavage (figure 2b), suggesting that furin is not associated with scov envelope maturation. s protein on the surface of infected insect cells was also treated with trypsin under conditions found to cleave murine cov s [19] and was reanalyzed after the cleavage reaction, by wb using the vsv g tm domain and patients' antibody. treatment with trypsin caused the loss of the ∼180-kda glycosylated s protein band and concomitant appearance of 2 new bands, the most intense of which was at ∼70 kda and whose mobility was indistin-guishable from that shown by s-f3r1 (figures 1b and 2c). the f3/r3 junction, which was chosen wholly on the basis of predicted secondary structure, is at residue 713, and a single lys residue occurs at position 714. on the basis of these data, we suggest that trypsin can access the s protein under nondenaturing conditions, to cleave at lys 714 and produce a b-sheetrich s1 domain and a a-helix-rich s2 domain. interestingly, molecular modeling of the scov s protein has also suggested that the s1-s2 junction lies within the amino acid sequence 680-727 [22] . no s protein was found in the nonpellet fraction after the addition of trypsin, suggesting that the 2 s protein domains remain associated even after cleavage (data not shown). when probed with patients' serum samples, the presumed s2 domain at ∼70 kda was also highlighted, but there were also a number of smaller breakdown products. no distinct band at the predicted size of the s1 domain was visible, suggesting that it is degraded by trypsin or is not detected efficiently by the available human serum samples (see below). whether s protein is cleaved before or during scov cell entry remains undetermined. reactivity with human serum samples. the abundant, stable, and characterized surface expression of a suite of s protein-related fragments prompted us to use these reagents to investigate the antibody response to s protein in the serum samples from infected individuals. serum samples used were obtained from several uk patients (for patients' details, see materials and methods); in addition, serum samples representing a time course from disease onset were obtained from 1 reference patient. we assessed the reactivity of each serum sample with each fragment by use of flow cytometry and wb, to represent native and denatured sources of antigen, respectively. cytometry profiles showed the greatest reactivity of patients' serum samples with the protein s-f1r2 and s-f1r1, but all other fragments also reacted, with the exception of s-f2r3 (aa 411-713), which represents the middle section of the s coding region and failed to react significantly with most patients' serum samples, despite high levels of expression ( figure 1b) . the same general pattern of binding was apparent for all the patients' serum samples, although the exact degree of reactivity with each fragment varied somewhat (of the 2 serum samples shown in figure 3 , the data for serum sample 1 were the more typical pattern). when serum reactivity was assessed by use of wb, however, the pattern of binding was substantially different. reactivity was seen with the full-length protein s-f1r1, but strong binding was also seen with s-f2r1 and s-f3r1 ( figure 3, bottom) . one serum sample (2908, the most broadly reactive of all the samples) showed some reactivity with s-f1r2, s-f1r3, and s-f2r3 but was very weak, compared with fragments spanning the c-terminal half of the s protein, and was wholly absent in other serum samples (typified by the wb using serum sample 1 in figure 3, bottom) . quantitation of the serum response to s protein by densitometry (wb) and relative fluorescence (facs) gave the reactivity orders and f3r1 1 f2r1 1 f1r1 111 others , respectively (figure 4). f1r1 p f1r2 1 f1r3 p f2r1 p f3r1 thus, there was a clear shift in reactivity with the same serum samples, depending on the assay format. specificity of serum reactivity with s protein fragments. the possible use of insect cell-displayed s protein for diagnostic application was assessed by examining fragment reactivity with serum samples from patients infected with human cov 229e and also with serum from a patient with suspected but clinically unconfirmed sars (serum sample 3118). preliminary data have indicated that serum samples from some cov 229e-infected individuals can cross-react with purified scov nucleocapsid protein (p.h. and m.z., unpublished data), so the use of a more specific test for seroconversion, based on s protein, may be valuable. serum samples from 2 patients with confirmed cov 229e infection did not react with s-f1r2 by use of facs (figure 5), whereas the serum sample from a patient with suspected sars showed a weak but clear shift in fluorescence. wb could also distinguish the serum samples, although the discrimination between the samples was not as good as that achieved by use of facs (data not shown), suggesting that the most discriminatory test in uncertain cases should use optimized s protein fragments presented in a nondenaturing assay format. time course of antibody response. a set of serum samples representing a time course from 6 to 40 days after the onset of sars for 1 patient was obtained and used in an s protein-specific elisa, to determine the increase in s protein titer over time. the titer of s protein antibodies was significant from day 10, similar to the serum responses reported for whole infected cells [11] and isolated n protein [23] , and increased to the latest time point (40 days after onset) ( figure 6 ). serum samples were also used in wb assays to examine the changes of reactivity with each individual s protein fragment over time. the earliest wb-positive serum sample (10 days after onset) gave a pattern of binding similar to that of the other serum samples tested (figures 3 and 6). the 40-day time point provided a much stronger signal, but the pattern of binding was not altered ( figure 6 ). blots were also probed with isotype-specific conjugates, and the results were compared with blots probed with an igg-specific conjugate. we found evidence for the development of some igm response late in the recovery period, but it was weak, compared with igg (data not shown). only serum antibodies were available for the present study, and it remains possible that secreted antibodies are also present in respiratory secretions early during the infection. our data provide the first profiles of antibody binding to the scov s protein. efficient expression of the s protein as a specific source of antigen was achieved by use of a highly productive expression system, recombinant baculoviruses, combined with the use of signal and tm sequences proven to efficiently direct proteins to the surface of the expressing cell [12] . this format offered assays based on antibody binding to the cell surface, mimicking infected cells but with singularly high levels of expression of s protein, as well as denatured antigen prepared by cell lysis. characterization of the expressed products provided some evidence for potential interaction with a known glycoprotein chaperone (calnexin), although the stimulation in glycan processing was marginal (20% of the total), and for cleavage of s protein by trypsin-like, but not furin, proteases. cleavage of surface glycoproteins is a key factor in pathogenesis for some viruses [24, 25] , and it will be interesting to evaluate the role of s protein cleavage, if any, in scov infectivity in both animal and human hosts. in the present study, full-length s protein on the surface of insect cells did not bind a variety of species of red blood cells, suggesting that it does not hemagglutinate. s protein released by detergent lysis also failed to bind to any discrete proteins in far-western blot assay of vero cell membranes (data not shown). accordingly, our data do not address the nature of the receptor for scov, in particular the early suggestion that it may be cd13 [14] . recent data show angiotensin-converting enzyme 2 to be at least 1 functional scov receptor [26] . efficient sources of several recombinant s protein fragments allowed us to evaluate the predominant antibodies present in convalescent serum samples. interestingly, the patterns of s reactivity with available serum samples was similar within each assay format, suggesting that the immune response to s protein varies only quantitatively between individuals. this result would be in keeping with the apparently low immunological pressure suggested from the sequence variation observed in several isolates [7] . of the 2 assay formats we used, nondenatured s protein present on the cell surface provided the most sensitive detection of antibodies, with clear shifts in fluorescence for serum samples from patients with suspected but clinically unconfirmed sars. no shift was apparent with human cov 229e serum samples, suggesting that this format is highly specific. when reactivity of positive serum samples to individual s protein fragments was compared, we observed a strong differential binding, depending on the assay used. thus, although facs showed the highest reactivity with fragments, including the amino terminus of s protein, wb with the same serum samples showed preferential reactivity to the carboxyl terminal half of the molecule. differential antibody binding was not influenced by the high-mannose glycans present on insect-derived glycoproteins, because s protein prepared in mimic cells (invitrogen), which add complex glycans to the polypeptide [27] , showed the same pattern of reactivity with the serum samples used (data not shown). our data, which were obtained with only the few serum samples from patients with sars available in the united kingdom, clearly require confirmation with larger sets of serum samples but would be consistent with a level of conformational antibody present in the serum samples preferentially directed toward the globular n terminus of the protein and detected by use of facs but not by use of wb. this class of antibody includes those most useful for diagnosis (see above), suggesting that the most suitable assay format for a final diagnostic will be best configured with authentically folded protein, rather than, for example, peptides. our preliminary work with purified soluble full-length s protein suggests that sensitivity is maintained with the soluble protein, indicating that assay formats simpler than flow cytometry will be feasible. confirmation that the class of conformational antibody directed at the globular s1 domain includes neutralizing antibody, and whether it has any bearing on the outcome of infection, will be important if recombinant s protein is also to be considered as part of a possible vaccine for scov infection. identification of a novel coronavirus in patients with severe acute respiratory syndrome koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars coronavirus: a new challenge for prevention and therapy pathogenesis of chimeric mhv4/ mhv-a59 recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study non-polar distribution of green fluorescent protein on the surface of autographa californica nucleopolyhedrovirus using a heterologous membrane anchor baculovirus surface display of theileria parva p67 antigen preserves the conformation of sporozoite-neutralizing epitopes putative hapn receptor binding sites in sars-cov spike protein legitimate and illegitimate cleavage of human immunodeficiency virus glycoproteins by furin why are "natively unfolded" proteins unstructured under physiologic conditions? role of n-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control folding and oligomerization of influenza hemagglutinin in the er and the intermediate compartment conformational changes in the spike glycoprotein of murine coronavirus are induced at 37њc either by soluble murine ceacam1 receptors or by ph 8 the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex furin at the cutting edge: from protein traffic to embryogenesis and disease molecular modelling of s1 and s2 subunits of sars coronavirus spike glycoprotein antibody response of patients with severe acute respiratory syndrome (sars) to nucleocapsid antigen of sars-associated coronavirus furin: a mammalian subtilisin/kex2p-like endoprotease involved in processing of a wide variety of precursor proteins the pathogenesis of influenza in humans angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus engineering the protein n-glycosylation pathway in insect cells for production of biantennary, complex n-glycans we thank robin gopal (health protection agency, colindale, uk), for providing the original viral rna; andrew davison and stuart siddell (university of bristol, bristol, uk), for providing spike protein cdna; malik peiris (university of hong kong, hong kong), for distribution of source materials; christian drosten (bernard noch institute, hamburg, germany); and members of the virology group, for constructive criticism. key: cord-264031-0y7xbgun authors: wierbowski, shayne d.; liang, siqi; chen, you; andre, nicole m.; lipkin, steven m.; whittaker, gary r.; yu, haiyuan title: a 3d structural interactome to explore the impact of evolutionary divergence, population variation, and small-molecule drugs on sars-cov-2-human protein-protein interactions date: 2020-10-13 journal: biorxiv doi: 10.1101/2020.10.13.308676 sha: doc_id: 264031 cord_uid: 0y7xbgun the recent covid-19 pandemic has sparked a global public health crisis. vital to the development of informed treatments for this disease is a comprehensive understanding of the molecular interactions involved in disease pathology. one lens through which we can better understand this pathology is through the network of protein-protein interactions between its viral agent, sars-cov-2, and its human host. for instance, increased infectivity of sars-cov-2 compared to sars-cov can be explained by rapid evolution along the interface between the spike protein and its human receptor (ace2) leading to increased binding affinity. sequence divergences that modulate other protein-protein interactions may further explain differences in transmission and virulence in this novel coronavirus. to facilitate these comparisons, we combined homology-based structural modeling with the eclair pipeline for interface prediction at residue resolution, and molecular docking with pyrosetta. this enabled us to compile a novel 3d structural interactome meta-analysis for the published interactome network between sars-cov-2 and human. this resource includes docked structures for all interactions with protein structures, enrichment analysis of variation along interfaces, predicted δδg between sars-cov and sars-cov-2 variants for each interaction, predicted impact of natural human population variation on binding affinity, and a further prioritized set of drug repurposing candidates predicted to overlap with protein interfaces†. all predictions are available online† for easy access and are continually updated when new interactions are published. † some sections of this pre-print have been redacted to comply with current biorxiv policy restricting the dissemination of purely in silico results predicting potential therapies for sars-cov-2 that have not undergone thorough peer-review. the results section titled “prioritization of candidate inhibitors of sars-cov-2-human interactions through binding site comparison,” figure 4, supplemental table 9, and all links to our web resource have been removed. blank headers left in place to preserve structure and item numbering. our full manuscript will be published in an appropriate journal following peer-review. the ongoing global covid-19 pandemic caused by the infection of sars-cov-2 has to date infected impact of mutations on interaction binding affinity and performed a comparison of protein-protein and 96 protein-drug binding sites. we compile all results from our structural interactome into a user-friendly 97 web server allowing for quick exploration of individual interactions or bulk download and analysis of the 98 whole dataset. further, we explore the utility of our interactome modeling approach in identifying key 99 interactions undergoing evolution along viral protein interfaces, highlighting population variants on 100 human interfaces that could modulate the strength of viral-host interactions to confer protection from or 101 susceptibility to covid-19, and prioritizing drug candidates predicted to bind competitively at viral-102 human interaction interfaces. enrichment of divergence between sars-cov and sars-cov-2 at spike-ace2 binding interface to highlight the utility of computational and structural approaches to model the sars-cov-2-human 106 interactome, we first examined the interaction between the sars-cov-2 spike protein (s) and human 107 angiotensin-converting enzyme 2 (ace2) (fig 1.a) . this interaction is key for viral entry into human 108 cells 3 and is the only viral-human interaction with solved crystal structures available in both sars-cov 47 109 and sars-cov-2 [48] [49] [50] . comparison between sars-cov and sars-cov-2 revealed that sequence 110 divergence of the s protein was highly enriched at the s-ace2 interaction interface (fig 1.a; log2oddsratio=2.82, p=1.97e-5), indicating functional evolution around this interaction. to explore the 112 functional impact of these mutations on this interaction, we leveraged the rosetta energy function 51 to 113 estimate the change in binding affinity (δδg) between the sars-cov and sars-cov-2 versions of the 114 s-ace2 interaction (fig 1.b and 1.c) . the predicted negative δδg value of -14.66 rosetta energy units 115 (reu) indicates an increased binding affinity using the sars-cov-2 s protein driven by better optimized 116 solvation and hydrogen bonding potential fulfillment along the ace2 interface. our result is consistent 117 with the hypothesis that increased stability of the s-ace2 interaction is one of the key reasons for 118 elevated transmission of sars-cov-2 52 . moreover, recent experimental energy kinetics assays have 119 shown that sars-cov-2 s protein binds ace2 with 10-20-fold higher affinity than that of sars-cov s 120 protein 53 supporting the conclusions from our computational modeling. among individuals 6, 7, 54 . several hypotheses for genetic predisposition models have been proposed 123 including that expression quantitative trait loci (eqtls) may up-or down-regulate host response genes 124 and that functional coding variants may alter viral-human interactions 55, 56 . for instance, a recent rna-in order to add a structural component to our interactome map, and thereby enable modeling of 149 the binding affinity for these interactions, we additionally performed docking in pyrosetta using our 150 eclair interface likelihood predictions to refine the search space (supplemental after constructing the 3d interactome between sars-cov-2 and human, we first looked for evidence 162 of interface-specific variation by mapping both gnomad 58 reported human population variants 163 (supplemental table 3 ) and sequence divergences between sars-cov and sars-cov-2 164 (supplemental table 4 ) onto the predicted interfaces. in general, conserved residues have been shown 165 to cluster at protein-protein interfaces 63 , and a recent analysis of sars-cov-2 structure and evolution 166 likewise concluded that highly conserved surface residues were likely to drive protein-protein 167 interactions 64 . consistent with these prior findings at an interactome-wide level, we observed significant 168 depletion for both viral and human variation along the predicted interfaces comparable to that observed 169 on solved human-human interfaces (fig 2.a) . nonetheless, considering each interaction individually, our analysis uncovered a 13 interaction 171 interfaces enriched for human population variants (fig 2.b) , and 7 enriched for recent viral sequence 172 divergences (fig 2.c) . a breakdown of variant enrichment on each interface is provide in supplemental 173 table 5 . the individual viral interfaces showing an unexpected degree of variation may-like the 174 previously discussed s-ace2 interface-be indicative of recent functional evolution around the viral-human interaction. considering the slower rate of evolution in humans, enrichment of population variants along the human interfaces is unlikely to be a selective response to the virus. rather, these 177 interfaces with high population variation along the interfaces may represent edges in the interactome whose strength may fluctuate among individuals or between populations. alternatively, enrichment and depletion of variation along the human-viral interfaces could help distinguish viral proteins that bind 180 along existing-and therefore conserved-human-human interfaces from those that bind using novel 181 interfaces-that would be less likely to be under selective pressure. to further explore the functional impact of naturally occurring variants on the human interactors 183 of sars-cov-2, we considered variants with phenotypic associations as reported in hgmd 65 , clinvar 66 184 or the nhgri-ebi gwas catalog 67 . interactors of sars-cov-2 were significantly more likely than the 185 rest of the human proteome to harbor phenotypic variants in each of these databases (fig 2.d) . notably, 186 among the individual disease categories enriched in this gene set, several were consistent with reported 187 comorbidities including heart disease, respiratory tract disease, and metabolic disease 68, 69 (fig 2. e; 188 supplemental table 6 ). disruption of native protein-protein interactions is one mechanism of disease 189 pathology, and disease mutations are known to be enriched along protein interfaces 70, 71 . human 190 population variants on the predicted human-viral interface were more likely to be annotated as 191 deleterious by sift 72 and polyphen 73 but showed identical allele frequency distributions compared to 192 those off the interfaces (supplemental figure 4) . however, mapping annotated disease mutations onto 193 the protein interfaces only revealed significant enrichment along known human-human interfaces; no 194 such enrichment was found on human-viral interfaces (fig 2.f) . this is likely because unlike with human-195 human interactions, mutations disrupting human-viral interactions would not disrupt natural cell function, 196 and therefore would be unlikely to be pathogenic. our finding that disease mutations and viral proteins 197 affect human proteins at distinct sites is consistent with a two-hit hypothesis of comorbidities whereby 198 proteins whose function is already affected by genetic background may be further compromised by viral 199 infection. we next sought to explore the impact of sequence divergence in sars-cov-2 relative to sars-cov on 202 viral-human interactions. mutations between the two viruses were identified by pairwise alignment and the 203 impacts of these mutations on the binding energy (δδg) for 250 interactions amenable to docking were 204 predicted using a pyrosetta pipeline 46, 59, 60 . although the binding energy for most interactions was 205 unchanged-either because no mutations occurred near the interface or because the mutations that did 206 had marginal effect-we observed an increased likelihood of the divergence from sars-cov to sarscov-2 resulting in decreased binding energy (i.e. more stable interaction) (fig 3. a; supplemental table 208 7). the significant outliers in these δδg predictions can help pinpoint key differences between the viral-209 human interactomes of sars-cov and sars-cov-2. we further note a wide range of affinity impacts 210 among various human interactors of a single viral protein (fig 3.d) and hypothesize that these differences 211 may help identify the most important interactions. to further explore the significance of these changes in interaction affinity, we considered those 213 interactions with the largest decrease in binding energy; corresponding to the largest predicted increase in 214 affinity. specifically, we highlight the interaction between coronavirus orf9c and human mitochondrial nadh scanning mutagenesis along all docked interfaces in pyrosetta. we identified as binding energy hotspot 254 mutations all mutations with a predicted δδg at least one standard deviation away from the mean for identical amino acid substitutions across the rest of the interface. in total, out of 2,241 population variants 256 on eligible interfaces, 161 (7.2%) were identified as hotspots predicted to disrupt interaction stability, and 257 116 (5.2%) were identified as hotspots predicted to contribute to interaction stability (fig 3.b) . most of the 258 hotspot mutations were predicted to be driven by solvation or repulsive forces, with disruptive hotspots 259 primarily being driven by repulsive forces and stabilizing hotspots primarily being driven by solvation forces 260 (fig 3.c) . results summarizing the predicted impact of all 2,241 population variants on the docked 261 interfaces are provided in supplemental table 8 . the current version of the sars-cov-2 human structural interactome web server describes 332 291 viral-human interactions reported by gordon et al. 20 . we will continue support for the web server with 292 periodic updates as additional interactome screens between sars-cov-2 and human are published. as we update, a navigation option to select between the current or previous stable releases of the web 294 server will be provided. overall, we present a comprehensive resource to explore the sars-cov-2-human protein-protein 297 interactome map in a structural context. analysis through this framework allows us to consider the recent 298 evolution of sars-cov-2 in the context of its interactome map and to prioritize for further functional 299 characterization key interactions. likewise, our consideration of underlying variation in the human 300 proteins that interact with sars-cov-2 may be valuable in explaining differences in response to 301 infection. we particularly note that our observation that perturbation from underlying disease mutations 302 and viral protein binding occur at distinct sites on the protein is of clinical interest. further investigation 303 into the combined role of these two sources of perturbation to better understand the mechanisms linked 304 to comorbidities is warranted. however, our work is not without limitation. firstly, we note that although structural coverage 306 from our homology modelling of sars-cov-2 proteins was robust (supplemental figure 1) , the same done to orient the most likely interface residues on each structure towards each other, protein-protein 309 docking using incomplete protein models introduces some bias and low coverage may exclude some 310 true interface residues. for this reason, the initial eclair interface annotations-which are less subject 311 to structural coverage limitations-may provide orthogonal value. we additionally note that direct perhaps most importantly, we emphasize the importance of further experimental 323 characterization to confirm the predictions made here. nonetheless we believe our 3d structural sarscov-2-human interactome web server will prove to be a key resource in informing hypothesis driven 325 exploration of the mechanisms of sars-cov-2 pathology and host response. the scope, and potential 326 impacts of our webserver will continue to grow as we incorporate the results of ongoing and future 327 interactome screens between sars-cov-2 and human. additionally, we note that our 3d structural 328 interactome framework can be rapidly deployed to analyze future viruses. homology-based modeling of all 29 sars-cov-2 proteins was performed in modeller 90 using a multiple 332 template modeling procedure. in brief, a list of candidate template structures for each protein to be 333 modelled was obtained by running blast 91 against a reference containing all sequences in the protein data bank (pdb) 92 . templates were filtered to only retain those with at least 30% identify to the protein 335 to be modelled, and the remaining templates were ranked using a weighted combination of percent 336 identity and coverage as described previously 93 . to compile the final set of overlapping templates for 337 modeling, first the top ranked template was selected as a seed. overlapping templates were iteratively 338 added to the set so long as 1) the new template increased the overall coverage by at least 10%, and 2) 339 the new template retained a total percent identity no more than 25% worse than the initial seed template. pairwise alignments between the protein to be modelled and the template set were generated using a regions with large gaps (at least 5 gaps in the alignment in a 10 residue window). finally, alignment was to accommodate predictions between sars-cov-2 and human, slight alterations were made. using the predicted interface probabilities reported by eclair, we set up the initial docking 379 conformation to explore a restricted search space for each docking simulation. in cases where multiple 380 structures were available for the human protein, all structures were weighted based on the eclair 381 scores for the covered residues in each structure to maximize both coverage age inclusion of likely 382 interface residues. for each protein in the interaction, we performed a linear regression classification in scikit-learn 102 to optimally separate the likely interface residues from likely non-interface residues. the plane defined by this linear regression served as a reference to orient the structures along the y-axis the x-z plane and separated a distance of 5 å along the y-axis. for each docking attempt, a series of 387 random perturbations from these initial conformations were made to search the nearby space. first, the 388 human protein was rotated up to 360° along the y-axis to allow full exploration of different rotations of 389 the two interfaces relative to each other. second, to apply some flexibility to the plane predicting the 390 interface vs. non-interface sides of each protein, up to 30° of rotation along the x-and z-axis were 391 allowed for both the viral and human proteins. finally, a random translation up to 5 å in magnitude was 392 applied to the human protein along the x-z plane so that the docking could explore contact points other 393 than the center of masses along these axes. after initializing these guided starting conformations, docking was simulated in pyrosetta 46 395 using a modified version of the protein-protein docking methodology provided by gray 2006 103 . the 396 initial demo (https://graylab.jhu.edu/pyrosetta/downloads/scripts/demo/d100 docking.py) takes two 397 chains from a co-crystal structure, applies a random perturbation, and re-docks them. because 398 randomized initial orientation was already handled as described previously, these steps were removed 399 from our docking runs. in brief, the protein models were converted to centroid representation, slid into 400 contact using the "interchain_cen" scoring function, and converted back to full atom representation, 401 before having their side-chains optimized using the predefined "docking" and "docking_min" scoring table 2 . to annotate interface residues from atomic resolution docked models, we used a previously described 407 and established definition for interface residues 45 . in brief, the solvent accessible surface area (sasa) 408 for both bound and unbound docked structures was calculated using naccess. 100 we define as accessibility) and 2) in contact with the interacting chain (defined as any residue whose absolute 411 accessibility decreased by ≥ 1.0 å 2 ). the full list of sars-cov-2 mutations is reported in supplemental table 4 . human population variants in all 332 human proteins shown to interact with sars-cov-2 430 proteins were obtained from gnomad 58 reported as the most general term with no more significant ancestor term (supplemental table 6 , sheet 465 1). raw enrichment values for all terms are also predicted (supplemental table 6 , sheet 2). for curation of disease and trait associations from nhgri-ebi gwas catalog (http://www.ebi.ac.uk/gwas/) 67 the scoring function used for these calculations is as described previously 59 using the following weights; protein-ligand docking using smina the previous viral-human interactome screen by gordon et al. 20 supplemental table 9 . list of all predicted drug-target binding sites . comparison of the percentage of human genes that interact with (green) or do not interact with (orange) coronaviruses: an overview of their replication and pathogenesis a pneumonia outbreak associated with a new coronavirus of probable bat origin including severe acute respiratory syndrome (sars) and 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interactions in virus-host systems. front microbiol interactome insider: a structural interactome browser for genomic studies pyrosetta: a script-based interface for implementing molecular modeling algorithms using rosetta stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis structural and functional basis of sars-cov-2 entry by using human ace2 sars-cov-2 and bat ratg13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects structure, function, and antigenicity of the sars-cov-2 spike glycoprotein the rosetta all-atom energy function for macromolecular modeling and design structural basis of receptor recognition by sars-cov-2 cryo-em structure of the 2019-ncov spike in the prefusion conformation who is most likely to be infected with sars-cov-2? the lancet infectious diseases comparative genetic analysis of the novel coronavirus (2019-ncov/sars-cov-2) receptor ace2 in different populations genetic predisposition models to covid-19 infection the mutational constraint spectrum quantified from variation in 141,456 humans a simple physical model for binding energy hot spots in protein-protein complexes spatial chemical conservation of hot spot interactions in protein-protein complexes tests of concrete strength across the thickness of industrial floor using the ultrasonic method with exponential spot heads the sequence of human ace2 is suboptimal for binding the s spike protein of sars coronavirus 2. biorxiv conserved residue clusters at protein-protein interfaces and their use in binding site identification sars-cov2 (covid-19) structural/evolution dynamicome: insights into functional evolution and human genomics. biorxiv human gene mutation database (hgmd): 2003 update clinvar: improving access to variant interpretations and supporting evidence the nhgri-ebi gwas catalog of published genome-wide association studies, targeted arrays and summary statistics 2019 characteristics associated with hospitalization among patients with covid-19 prevalence of comorbidities and its effects in patients infected with sars-cov-2: a systematic review and meta-analysis widespread macromolecular interaction perturbations in human genetic disorders three-dimensional reconstruction of protein networks provides insight into human genetic disease sift web server: predicting effects of amino acid substitutions on proteins a method and server for predicting damaging missense mutations analysis of intraviral protein-protein interactions of the sars coronavirus orfeome human mitochondrial complex i assembly is mediated by ndufaf1 cia30 complex i assembly factor: a candidate for human complex i deficiency? hum genet human genome-wide rnai screen reveals a role for nuclear pore proteins in poxvirus morphogenesis mitochondrial reactive oxygen species control t cell activation by regulating il-2 and il-4 expression: mechanism of ciprofloxacin-mediated immunosuppression the landscape of human cancer proteins targeted by sars-cov-2 genome-wide sirna screen identifies the retromer as a cellular entry factor for human papillomavirus the master regulator of the cellular stress response (hsf1) is critical for orthopoxvirus infection a genome-wide small interfering rna (sirna) screen reveals nuclear factor-kappab (nf-kappab)-independent regulators of nod2-induced interleukin-8 (il-8) secretion architecture of the human interactome defines protein communities and disease networks tmed2 potentiates cellular ifn responses to dna viruses by reinforcing mita dimerization and facilitating its trafficking role of the early secretory pathway in sars-cov-2 infection tom70 mediates activation of interferon regulatory factor 3 on mitochondria a whole-genome association study of major determinants for host control of hiv-1. science extensive disruption of protein interactions by genetic variants across the allele frequency spectrum in human populations comparative protein structure modeling using modeller basic local alignment search tool the protein data bank interactome3d: adding structural details to protein networks protein identification and analysis tools on the expasy server the proteomics protocols handbook divergence measures based on the shannon entropy predicting functionally important residues from sequence conservation direct coupling analysis for protein contact prediction evolutionarily conserved pathways of energetic connectivity in protein families modbase, a database of annotated comparative protein structure models and associated resources the interpretation of protein structures: estimation of static accessibility accelerating protein docking in zdock using an advanced 3d convolution library scikit-learn: machine learning in python high-resolution protein-protein docking uniprot: a worldwide hub of protein knowledge analysis of multimerization of the sars coronavirus nucleocapsid protein a new coronavirus associated with human respiratory disease in china genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan a general method applicable to the search for similarities in the amino acid sequence of two proteins amino acid substitution matrices from protein blocks the ensembl variant effect predictor explaining odds ratios. j can acad child adolesc psychiatry ldlink: a web-based application for exploring population-specific haplotype structure and linking correlated alleles of possible functional variants open babel: an open chemical toolbox lessons learned in empirical scoring with smina from the csar 2011 benchmarking exercise sars-cov-2 that contain disease annotations in hgdm (log2or=0.57, p=1.70e-4) sars-cov-2 proteins were significantly more likely to harbor disease mutations than non-interactors error bars indicate ± se. e, a sample of individual disease terms enriched in human genes targeted by 603 comparison of the enrichment of hgdm, clinvar, and gwas annotated mutations on human-vial 605 interfaces or human-human interfaces for the same gene set. although disease mutations were enriched 606 on human-human interfaces (hgmd, log2or=0 24), no 607 enrichment was observed on human-viral interfaces (hgmd, log2or=0.21, p=0.13; clinvar the gwas category was removed from this analysis because most lead gwas 609 snps occurred in non-coding regions. error bars indicate ± se predicted changes in binding affinity from sequence divergences between sars-cov and sars-613 an overall representation of 614 these δδg predictions is reported (mean=-1.40 reu, std=6.16 reu) with interactions sorted from those 615 with the largest decrease in binding energy (most stabilized relative to sars-cov) to those with the 616 largest increase in binding energy (most destabilized relative to sars-cov) < z-score ≤ -1, n=85), or strongly stabilizing (z-score ≤ -2, n=31) score < 1, n=1,964) showed minimal impact of binding affinity. c, breakdown of the contribution of each 623 term in the pyrosetta energy function used for in-silico scanning mutagenesis for all population variants a breakdown of which term contributed most heavily 625 to the classification of all 277 interface hotspot population variants is shown on the right. d, individual 626 sars-cov-2-human interactions involving the same viral protein can have distinct interfaces with 627 distinct predicted changes in binding affinity between sars-cov and sars-cov-2 versions of the 628 protein. an example involving orf9b is highlighted where some interactions (e.g. tomm70 and ptbp2) 629 are predicted to be more stabilized in sars-cov-2 whereas others (e.g. bag5, slc9a3r1, and 630 mark2) are predicted to me unaffected. e, docked structure for the interaction between sars-cov-2 631 orf9c and human ndufaf1 sars-cov-2 (bottom) orf9c. interface residues are colored by their predicted energy contribution 633 from blue (stabilizing) to white (no impact) to red (destabilizing) -2 are labeled in red, while other residues with a major contribution to the binding 635 affinity are labeled in green (ndufaf1) or blue (orf9c). the overall predicted change in binding energy 636 (δδg=-21.7 reu) suggests the interaction is more stable (lower energy) in the sars-cov-2 version of 637 the interaction supplemental figure 4. summary of human population variant frequency and deleteriousness summary of allele frequency for human population variants either on or off the predicted human-700 viral interface presented as either a raw distribution or a cumulative density respectively. variants in 701 either category had roughly identical allele frequency distributions. c, d, summary of the sift 702 deleteriousness score for human population variants either on or off the predicted human-viral interface 703 presented as either a raw distribution or a cumulative density respectively population variants on the 706 interface were significantly more likely to be classified deleterious. f, g, summary of the polyphen 707 deleteriousness score for human population variants either on or off the predicted human-viral interface 708 presented as either a raw distribution or a cumulative density respectively. plots are colored based on 709 the split between polyphen benign, possibly damaging, and probably damaging categories. e, pie chart 710 breakdown of these categories. pie char outlines distinguish interface (green) from non-interface 711 (orange) for each sars-cov-2-human interaction with 3d structure available for both proteins, 50 independent 680 guiding docking trials were used to select a final docked configuration. the structure for the viral protein 681 is colored from white to blue with darker blue corresponding to higher eclair prediction. the structure 682 for the human protein is colored similarly using a green to white gradient. initial semi-random docked 683 configurations were generated using five steps. first a plane separating eclair predicted likely 684 interface from likely non-interface residues was drawn to divide each protein. second, the two protein 685 chains were separated 5 å apart on the y-axis using the previously defined plane to orient the likely 686 interface sides of each protein towards each other. third, the human protein was randomly rotated up 687 to 360° along the y-axis to sample different orientations of the two interfaces relative to each other. key: cord-281005-6gi18vka authors: singh, praveen kumar; kulsum, umay; rufai, syed beenish; mudliar, s. rashmi; singh, sarman title: mutations in sars-cov-2 leading to antigenic variations in spike protein: a challenge in vaccine development date: 2020-09-01 journal: j lab physicians doi: 10.1055/s-0040-1715790 sha: doc_id: 281005 cord_uid: 6gi18vka objectives the spread of severe acute respiratory syndrome coronavirus-2 (sars-cov-2) virus has been unprecedentedly fast, spreading to more than 180 countries within 3 months with variable severity. one of the major reasons attributed to this variation is genetic mutation. therefore, we aimed to predict the mutations in the spike protein (s) of the sars-cov-2 genomes available worldwide and analyze its impact on the antigenicity. materials and methods several research groups have generated whole genome sequencing data which are available in the public repositories. a total of 1,604 spike proteins were extracted from 1,325 complete genome and 279 partial spike coding sequences of sars-cov-2 available in ncbi till may 1, 2020 and subjected to multiple sequence alignment to find the mutations corresponding to the reported single nucleotide polymorphisms (snps) in the genomic study. further, the antigenicity of the predicted mutations inferred, and the epitopes were superimposed on the structure of the spike protein. results the sequence analysis resulted in high snps frequency. the significant variations in the predicted epitopes showing high antigenicity were a348v, v367f and a419s in receptor binding domain (rbd). other mutations observed within rbd exhibiting low antigenicity were t323i, a344s, r408i, g476s, v483a, h519q, a520s, a522s and k529e. the rbd t323i, a344s, v367f, a419s, a522s and k529e are novel mutations reported first time in this study. moreover, a930v and d936y mutations were observed in the heptad repeat domain and one mutation d1168h was noted in heptad repeat domain 2. conclusion s protein is the major target for vaccine development, but several mutations were predicted in the antigenic epitopes of s protein across all genomes available globally. the emergence of various mutations within a short period might result in the conformational changes of the protein structure, which suggests that developing a universal vaccine may be a challenging task. since the rapid outbreak of 2019 novel coronavirus (2019-ncov, later named sars-cov-2 or severe acute respiratory syndrome coronavirus 2) in wuhan, china, the world health organization on january 30, 2020, declared the sars-cov-2 epidemic as a public health emergency of international concern. the enduring pandemic has caused nearly 5 million detected cases of coronavirus disease 2019 illness and claimed over 3,25,563 lives worldwide as of may 20, 2020 according to covid-19 resource center johns hopkins. 1 however, so far, no proven therapeutic or effective vaccine candidate has been found. 2 for developing a drug or vaccine, the protein profiling and/or genomic information of the pathogen is extremely crucial. to understand genetic landscape of sars-cov-2 virus, scientists have worked tirelessly and the complete genome sequences of virus isolates are published. now, many isolates have been sequenced completely or partially and are available in the database for scientific community. 3 it is found that the genome size of sars-cov-2 varies from 29.8 kb to 29.9 kb. specific genetic characteristic in its genome have also been found. 4 the genome consists of four structural proteins including spike protein (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. 3 of the four glycoproteins, the m protein is reported to have role in determining the shape of the virus envelope and stabilizing the nucleocapsids, the n protein is involved in processes related to the viral genome, the viral replication cycle, and the cellular response of host cells to viral infections. the e protein which is the smallest protein in the sars-cov-2 structure plays the role in the production and maturation of this virus. 4 however, the glycoprotein s is the core transmembrane monomer of approximately 180 kda size with two subunits s1 and s2. this glycoprotein mediates membrane fusion and finally facilitates virus entry (receptor-binding and entry of virion into the target cells). 5 the receptor binding domain (rbd) (residues 319-541) of the subunit s1 is known to interact with angiotensin-converting enzyme 2 (ace-2), which provides tight binding to the peptidase domain of ace-2. 6 this gives an impression of rbd being an important element of virus-receptor interaction and has an essential role in virus-host range, tropism, and infectivity. the rbd sequences of different sars-cov-2 strains that are circulating globally were initially thought to be conserved; however, with the availability of sequencing data several mutations have been reported. 7, 8 these findings emphasize the argument, that there may be correlation of the mutated strains circulating in a particular geographic setting with higher mortality rates and transmission patterns beside other combating factors. 9 the mutation rate in ribonucleic acid (rna) viruses is intensely high which can be million times higher than that of their hosts and this results in virulence modulation and evolutionary capability for better viral adaptation. 7 genetic depiction of virus mutations can thus offer valuable insights for assessing the fitness of drug resistance, immune escapism, and pathogenesis. due to its receptor binding property, the s protein is also supposed to be immunogenic and a putative target for developing the neutralizing antibodies and vaccines. it is reported that single-point mutations in the conserved amino acid residues in the rbd region completely abolishes the capacity of fulllength s protein to induce neutralizing antibodies. 10 thus, virus mutation studies can be crucial for designing new vaccines and antiviral drugs. in this study, we aimed to predict the mutations in the spike protein (s) of sars-cov-2 genomes available in the database (whole genome sequences as well as partial coding sequences of spike protein) and analyze the effect of each mutation on the antigenicity of the predicted epitopes. this information may be helpful in predicting the transmission and infectivity of various sars-cov-2 strains circulating worldwide. entrez direct (edirect) utilities were used to access the ncbi's nucleotide database by using in-house developed bash scripts to batch download the data. we used query keyword or phrase as "severe acute respiratory syndrome coronavirus 2" and "spike coding sequences (cds)" by applying esearch and efetch utilities implemented in bash scripts to download the dataset for genome and spike proteins that were available till may 1, 2020. a total of 1,325 complete draft genomic sequences of sars-cov-2 and additional 279 cds having partial genomes coding spike protein (total 1,604 cds of spike protein) were downloaded in fasta format available globally from ncbi database (►fig. 1). multiple sequence alignment (msa) of all the 1,325 complete genome sequences as well as 1,604 cds was performed using clustalw-mpi with default parameters. 11 generated snps were identified by in-house developed bash scripts to batch process the data using blade server (dell poweredge fc640 server) with 256 gb ram and 40 core processor with 2.30 ghz. after msa, each genome and spike protein were marked based on the location and clustering was done based on 100% similarity for ease of visualization and analysis. visualization was performed by using jalview 2.11.1.0. 12 the output snp alignment generated from msa was used to assemble a maximum likelihood phylogenetic tree using raxml (randomized axelerated maximum likelihood raxml 8.2.12). 13 phylogenetic trees were visualized using the interactive tree of life (itol) v5 with their respective metadata. 14 emboss antigenic software was used to predict the antigenic regions and the epitopes in the 88 unique spike proteins based on antigenic scores using the formula: where f(ag) = antigenic frequency; f(s) = surface frequency and antigenic score ≥ 1.0 is considered potentially antigenic. [15] [16] [17] the data for different epitopes were analyzed and the epitopes with high antigenicity were superimposed on the structure of spike protein. overall, 1,197 snps were found in 1,325 complete genome datasets. on the basis of similarity these were classified in 782 clusters. however, among cds of spike proteins (1,325 complete genomes and 279 partial cds) a total 140 snps in 88 clusters were found. further snp analysis resulted in identification of snps in a gene stretch of 21,563-25,384 bp in the s gene, encoding the spike (s) protein. the most predominant snp predicted in the gene encoding s protein was 23402a>g in 48.2% of overall genomes under study. in addition to several single mutations in the s gene of all available genomes, we also predicted double mutations such as 22436g>t, 22439c>g, 22444c>a, 22445c>t (corresponding to four amino acid antigenic drift aldp -> sves at position 292-295) and 21723t>g (l54w); 21726t>a (f55i) in two different genomes from the united states. list of all the snps in the rbd, antigenic sites, and double mutations among strains is shown in ►table 1. one deletion (21994-21996deltta) was also found in an indian strain (mt012098.1). no copy number variants were observed in this virus. the alignment of 1,604 spike proteins extracted from 1,325 complete genomes and 279 partial cds was performed and after clustering based on 100% identity, we identified 88 unique sequences with 88 hypervariable sites within these protein sequences. based on the variable sites the phylogeny was inferred showing two major clades a and b with many subclades in the s protein of sars-cov-2 circulating worldwide (►fig. 2). furthermore, the evaluation of the antigenicity of spike protein predicted 14 highest scoring antigenic epitopes (antigenic scores ≥ 1.0) due to variations in each (at positions l54f, l54w, f55i, s71f, d111n, f157l, l293m, l293v, d294e, d294i, a419s, v367f, a348v, and a653v). out of these, amino acid changes were noted at positions a348v, v367f and a419s in the rbd with v367f and a419s being novel. other speculated mutations in the putative epitopes lying within rbd showing less antigenicity were t323i, a344s, r408i, g476s, v483a, h519q, a520s, a522s, and k529e out of which t323i, a344s, a522s, and k529e are also novel. in addition, regions outside rbd (4-19, 1,215-1,256, 1,039-1,071, 123-146, 607-629, 1,123-1,136 , 857-867, 535-541) also infer high antigenicity (based on predicted antigenic score in the range 1.167-1.261) with variations in s protein of different genomes from similar locations as shown in ►fig. 3. the antigenic epitopes are depicted in the protein structure of spike protein (►fig. 4). two mutations a930v and d936y were observed in the heptad repeat domain 1 (hr1) and one mutation d1168h in heptad repeat domain 2 (hr2). several studies have shown that mutations within the spike protein influence virus-host interaction. 18 among the four proteins, viz., m, s, n and e, the m protein is known to play a significant role in virus assembly, role of e protein involves the production and maturation of this virus, the n protein is involved in processes related to viral replication cycle, and cellular response of host cells to viral infections, and s protein is the major target for neutralizing antibodies as it mediates the fusion and facilitates viral entry into host. 4, 5 in the present study, we found that although multiple genetic variants were identified in the same country, yet there were some unique mutations found in a particular country, which suggests that diversity of s protein mutations might have significant role in the pathogenicity of this virus in countries with high or low mortality rates, as proposed by others also. 19 we predicted 140 snps in the s protein with a>g and vice versa in sars-cov-2 genomes submitted from india, t>a and c>t from china and the interchange of all four nucleotides (c>t, t>a, a>g, g>t, c>g, c>a, g>c, t>c, a>t, g>a) in genomes submitted from the united states. the data from the united states is significant. however, it might be because maximum genomes were submitted from the united states only. the snp profile revealed that the s protein mutations were predominant at specific positions only. these mutations are expected to make the virus more capable to escape from the host immune and might help in natural selection and evolution of the sars-cov-2, as reported by andersen et al. 20 it is important to mention that double mutations in the s protein were found only in the strains from the united states but not in genomes from other regions. these double mutations probably could have helped in the increased virulence of the virus. 21 it has also been noticed that the death toll is comparatively higher in the united states than in other regions included in the study. 1 this might probably indicate that the prevalence of several mutated strains within the provinces would have either reduced or increased its severity. it may also help in understanding the antigenic and immunogenic changes but the correlation of mutation with regional virulence could not be established due to statistical imbalance of the available genomes in the database at the time the study was done. moreover, extensive research is required to correlate the mutations with the severity of the disease and mortality. out of 88 snp clusters, d614g was found in 34 (38.6%) sars-cov-2 genomes. the amino acid change in 23403a>g variant (p.d614g) involves a change of large acidic residue d (aspartic acid) into small hydrophobic residue g (glycine). this observation is important, as this large difference in both size and charge may help compromise the binding affinity of antibodies against s protein, due to electrostatic interactions in the tertiary structure of protein group. this may hinder the developments of vaccines and might potentiate the virus for antigenic drifts. 22 the effect of deletion variant (figured in one sars-cov-2 genome from india) on the viral phenotypes needs further investigation. the high frequency of genetic mutations in rna viruses is well known but in the genomes of sars-cov-2, we found a series of single amino acid variations. this can affect the virus evolution and emergence of the new strains. 21 the mutations in the rbd found in our study predicted conformational changes in the s1 domain of spike protein. the mutations in rbd play an important role while designing new drugs, as suggested in a recent study. 23 these mutations might affect the interaction of viral rbd with the host receptor. our study revealed 12 mutations of which six were novel mutations (►table 1). out of the six novel mutations two were exhibiting high antigenicity while others were in the less antigenic region. the amino acid change observed in the antigenic epitopes were from positively charged to uncharged amino acids (r->i, h->q), and negatively charged to uncharged (d->n, d->g, d->y) amino acids. we also found mutations in negative to positively charged (d->h) amino acids. these replacements might influence the tertiary structure of the proteins and facilitate the increased virulence by escaping host immune response. 24 the sequences in the hr1 (residues 902-952) and hr2 (residues 1,145-1,184) regions tend to form dimeric or trimeric helix bundles. 25 as the s protein of coronavirus are homodimers or homotrimers, these hr regions may undergo oligomerization and result in the conformational change of s protein during virus-host cell fusion. 26 these regions show different conformations in different fusion states and are known to be the most conserved among other regions in s protein of sars-cov. 25, 27 however, a previous study shows variations in hr1 domain which forms helical bundles with hr2 to facilitate fusion and entry of virus into the host and hypothesizes that the mutation a1168v in hr2 domain along with a930v mutation in hr1 domain confers peptide entry inhibitor resistance in mouse hepatitis coronaviruses. 28 hence the mutation a930v in hr1 domain and d1168h in hr2 domain found in our study might be relevant in explaining the pathogenesis of sars-cov-2. with the rapid spread of this virus and limitation of specific therapy, studies are being focused on exploring the potential of neutralizing antibodies (as in plasma therapy) against vulnerable epitopes of s protein. 29 our study predicts an interactive web-based dashboard to track covid-19 in real time a review of sars-cov-2 and the ongoing clinical trials genomic characterization of a novel sars-cov-2 coronavirus envelope protein: current knowledge cryo-em structure of the 2019-ncov spike in the prefusion conformation structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry emerging sars-cov-2 mutation hot spots include a novel rna-dependent-rna polymerase variant preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies real estimates of mortality following covid-19 infection single amino acid substitutions in the severe acute respiratory syndrome coronavirus spike glycoprotein determine viral entry and immunogenicity of a major neutralizing domain clustalw-mpi: clustalw analysis using distributed and parallel computing jalview version 2-a multiple sequence alignment editor and analysis workbench raxml-vi-hpc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models interactive tree of life (itol) v4: recent updates and new developments emboss: the european molecular biology open software suite a semi-empirical method for prediction of antigenic determinants on protein antigens new hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-ray-derived accessible sites potential therapeutic targeting of coronavirus spike glycoprotein priming sars-cov-2 viral spike g614 mutation exhibits higher case fatality rate the proximal origin of sars-cov-2 why are rna virus mutation rates so damn high? electrostatic interactions in protein structure, folding, binding, and condensation exploring the genomic and proteomic variations of sars-cov-2 spike glycoprotein: a computational biology approach lineage-specific differences in the amino acid substitution process interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion mechanisms of viral membrane fusion and its inhibition coronavirus escape from heptad repeat 2 (hr2)-derived peptide entry inhibition as a result of mutations in the hr1 domain of the spike fusion protein a highly conserved cryptic epitope in the receptor binding domains of sars-cov-2 and sars-cov most of the vaccines strategies against covid-2 are focusing on the predicted epitopes of sars-cov-2 spike protein. this protein is also proposed to be the most potent and specific drug target and for designing neutralizing antibodies. our findings indicate that vaccine designing against sars-cov-2 could be a challenging task. even though both rna based, and peptide-based vaccines are being developed in more than seven laboratories, our observations may be useful in the efficacy analysis of these vaccine candidates. none.conflict of interest p.k.s. and u.k. are research officers in a department of biotechnology funded unrelated project (bt/pr23016/ ner/95/581/2017). key: cord-259112-tkj5de7b authors: mandal, santi m; panda, souvik title: inhaler with electrostatic sterilizer and use of cationic amphiphilic peptides may accelerate recovery from covid-19 date: 2020-06-17 journal: biotechniques doi: 10.2144/btn-2020-0042 sha: doc_id: 259112 cord_uid: tkj5de7b we explore the design of a smart inhaler with electrostatic sterilizer and propose the utilization of cationic amphiphilic peptides, independently or in conjunction with a bronchodilator, for covid-19 patients to quickly improve wellbeing while maintaining a strategic distance to protect healthcare personnel from virus-containing aerosol or droplets during the process of inhalation. caps are known effector molecules synthesized in the host cell in response to immunological challenge by pathogens, and can eliminate microorganisms (bacteria, fungi and protozoan parasites) and acellular entities (viruses and viroids) [10] . the epithelial surfaces of the respiratory tract and lungs are shielded from pathogens by emitting safeguarding molecules, referred to as intrinsic mucosal resistance. this intrinsic mucosal resistant framework eliminates pathogens by forestalling their colonization in the epithelial layer. synthesis and discharge of a few caps -for example, collectin, ␤-defensins, cathelicidins, hydrophilic surfactant proteins -and other molecules plays a significant role in counteracting pathogen attacks [11] . caps have an expansive range of action against microorganisms and infections, neutralize endotoxins and exhibit other in vivo activity [12] . the utility of recombinant lung surfactant proteins has been demonstrated for the treatment of respiratory pain disorder in neonates, chronic obstructive pulmonary disease, emphysema, cystic fibrosis and asthma [13] . both in vitro and in vivo data reveal the significant impact of caps in epithelial host defense [14] . under inflammatory disease conditions, illness and morbidity are enhanced because of dysfunction in mucosal resistance [15] ; hence inhalation of caps or hydrophilic surfactant proteins should be an effective way to deal with receptor-binding proteins of viruses, interrupting or disrupting the membrane lipid bilayer or annihilating colonized virus particles from the epithelial surface. cap-induced disturbance/disruption of the membrane lipid bilayer could happen in various ways, as envisaged in various studies (e.g., barrel-fight model, toroidal model, cover model and cleanser model) [16, 17] . a few caps are notable for causing fusion of cell membranes and can control viral infections by intervening in the fusion process between the host cell membrane and the enveloped virus [18] . the fusogenic tat protein transduction area has been utilized to convey a wide scope of the naturally dynamic segments and medications by the immediate entrance over the lipid membrane [19] . membrane permeabilization is viewed as a significant trait of antiviral action. in poxviruses, rifampin is a compelling inhibitor of viral envelope arrangement; lipid layer-bound viral proteins might be targeted immunologically to expand its counterviral efficacy. for a successful and effective interaction, both electrostatic charge and hydrophobicity are significant. a positive charge is required initially to attract negatively charged membranes, and hydrophobic mass aides is required to disturb the membrane just as it makes contact with the hydrophobic site of hr1, hr2 area of viral combination protein and the receptor restricting space of s protein; this may lessen viral passage into the cell. the significant point here is that higher hydrophobicity increases hemolytic activity, which may hinder the utilization of caps. in any case, hemolytic action might be diminished by the alteration of residues [20] . a few lipopeptides, including maginin and gomesin, are known to be successful caps for antimicrobial action. nonetheless, the use of caps is managed here through an inhaler, allowing the caps to reach to the upper respiratory tract and lungs, which are the hotspot for sars-cov-2 because of their overwhelming hyperexpression of its main receptor molecule, ace2. their cationic property is apt to disrupt the viral the inhaler is an extremely common and valuable device for conveying medicine into the body via the lungs, and is commonly utilized for the treatment of asthma, chronic obstructive pulmonary disease and viral diseases. covid-19 patients often display symptoms of extreme respiratory complication and inhalers may be used to provide immediate relief. healthcare workers may risk infection from patients using inhalers and none can rule out the possibility of spreading contamination in the room during exhalation. here we have designed a unit to stop the spread of virus particles from patients. the inhaler is commonly utilized for quick alleviation of blocked airways. although it delivers short-acting medications, during its use aerosolized viral particles may be released into the environment and may affect health workers. for this reason, another device is proposed to kill viral particles inside the aerosol of covid-19 patients while inhaling air ( figure 2 ). to assemble the gadget, the two halves of the spacer need to be firmly pushed together to rotate the mouthpiece top. the spacer includes two locks which guarantee proper assembly of the two parts. a canister is put into the face of the inhalation chamber with one push. there is a one-way gate valve (owgv) which helps entry of the medicine during inhalation but does not let it out in this entryway. the full inhalator air is flown in this gadget and followed to the mouthpiece. there is another owgv in the guard of the face of inhalation chamber. the pressurized canned products from exhalation are not returned to this valve, which will be shut due to the owgv working head. the patient exhales completely, closing the lips immovably around the mouthpiece to make a good seal without biting on it, breathes in profoundly through the mouth, and in this manner takes in the medicine through the spacer. at that point, the patient removes the inhaler spacer from the mouth, holds the breath for about 10 s (or as long as possible) and breathes out gradually. a different parabolic chamber is attached with this gadget, where one cathode plate and one anode plate are placed independently. a high efficiency particulate air channel, battery, switch and other devices creating a high voltage circuit are additionally appended. a push switch is arranged outside of the parabolic chamber. an ordinary 4 v direct current (dc) battery is utilized in our device to create a high intensity power of 1000 v to enable the positive anode and negative cathode free to liberate charged particles separately. a negative voltage of 1000 v is applied between the cathode wire or plate. an electric discharge from the anode plate ionizes the air; aerosol concentrates around the electrode inside the parabolic chamber during the utilization of high voltage. this is then ionized, causing sparking-induced burning of virus particles due to huge electrostatic power. viruses in the inhaled air or aerosols within the gadget are killed instantaneously, and is burned right away inside the device. when patients breathe out slowly, the owgv will open and air goes into the parabolic chamber. this inletting air is charged by cathode and anode electrodes, leaving virus particles to be burned. at that point the charged air flowing through the channel can trap 99% of remaining virus particles. virus-free air will be discharged from the chamber, preventing dissemination of viruses. two strategies are proposed here for covid-19 patients. first, caps might be brought into the inhaler or medicine independently, alongside bronchodilators and may assist with diminishing the interaction propensity between ace2 receptors and the s1 protein of sars-cov-2. the virus is mainly abundant in the lung and respiratory framework. after inhalation of caps, viral membranes rupture and the resulting new molecular membrane arrangement should be disordered; this should loosen binding with ace2 receptors and diminish the viral burden. second, another plan of smart inhaler has been proposed for covid-19 patients. we have appended an extra parabolic chamber with battery-operated terminals to kill viral pathogens within a second by electrical contact. in this way, the risk of contaminating healthcare personnel with virus-loaded aerosols released from the exhaled air is diminished. structure, function, and evolution of coronavirus spike proteins the coronavirus nucleocapsid is a multifunctional protein structural insights into coronavirus entry host cell proteases: critical determinants of coronavirus tropism and pathogenesis coronavirus envelope (e) protein remains at the site of assembly analysis of sars-cov e protein ion channel activity by tuning the protein and lipid charge the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex sars-cov fusion peptides induce membrane surface ordering and curvature coronavirus envelope protein: current knowledge antimicrobial and host-defense peptides as new anti-infective therapeutic strategies collectins and cationic antimicrobial peptides of the respiratory epithelia cationic antimicrobial peptides and their multifunctional role in the immune system the potential of recombinant surfactant protein d therapy to reduce inflammation in neonatal chronic lung disease, cystic fibrosis, and emphysema antimicrobial peptides as mediators of epithelial host defense humoral immunodeficiency in recurrent upper respiratory tract infections. some basic, clinical and therapeutic features biomedical exploitation of self assembled peptide based nanostructures functional and structural insights on self-assembled nanofiber-based novel antibacterial ointment from antimicrobial peptides, bacitracin and gramicidin s cationic host defence peptides: potential as antiviral therapeutics transducible tat-ha fusogenic peptide enhances escape of tat-fusion proteins after lipid raft macropinocytosis cationic amphiphiles, a new generation of antimicrobials inspired by the natural antimicrobial peptide scaffold the authors are grateful to prof. ranadhir chakraborty (department of biotechnology, north bengal university, india) for his kind help in linguistic corrections and reframe the manuscript. the authors have no relevant financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.no writing assistance was utilized in the production of this manuscript. this work is licensed under the attribution-noncommercial-noderivatives 4.0 unported license. to view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ key: cord-276456-oa6hh7ky authors: collins, r.n.; holz, r.w.; zimmerberg, j. title: 5.14 the biophysics of membrane fusion date: 2012-05-03 journal: comprehensive biophysics doi: 10.1016/b978-0-12-374920-8.00523-3 sha: doc_id: 276456 cord_uid: oa6hh7ky a crucial interplay between protein conformations and lipid membrane energetics emerges as the guiding principle for the regulation and mechanism of membrane fusion in biological systems. as some of the basics of fusion become clear, a myriad of compelling questions come to the fore. is the interior of the fusion pore protein or lipid? why is synaptic release so fast? why is pip(2) needed for exocytosis? how does fusion peptide insertion lead to fusion of viruses to cell membranes? what role does the tmd play? how can studies on membrane fission contribute to our understanding of membrane fusion? what exactly are snare proteins doing? the biological membrane is ancient, and crucial to the emergence of life itself. while a single topology of membrane was sufficient for bacteria and archebacteria, eukaryotic cells contain specialized subcellular systems of more topologies, enclosing many discontinuous volumes. in order to mix these intracellular volumes, and to secrete such volumes to the extracellular space, or to add a volume to the cytoplasmic space, the membranes enclosing one of these volumes must either rupture or merge with another membrane, a process termed membrane fusion. topological membrane rearrangements, then, such as fusion, its inverse -fission, or formation of a membrane pore, are the most essential ingredients of the complex membrane dynamics of living cells. these membrane transformations are key elements of dynamic intracellular trafficking networks; they are also intimately linked to important pathological processes including cellular entry and egression of enveloped viruses and various parasites, membrane rupture and apoptosis. the topological membrane remodeling generally converges to highly bent intermediates. the characteristic length scales associated with key structural intermediates, such as fusion or fission pores, are typically of the order of tens of nanometers. it has become increasingly clear that critical properties of biological material at this nanoscale cannot be readily extrapolated from bulk measurements. neither can they be obtained from experiments with individual molecules, as in many cases, especially in most membrane processes, the functional unit is not a single protein (e.g., channel), but a selectively self-assembled cluster of proteins and lipids acting in a highly co-operative manner. therefore, for intuition and information about the nature of these transformations, we must create and study them where possible. since the biological membrane is thin (5 nm in thickness, that of two lipid molecules) we cannot see the process of membrane fusion under the microscope; we can only see the sequellae of fusion as organellar or cellular contents mix or are secreted. the simplest natural occurring instance of fusion is the coalescence of two miscible liquid droplets in air, whose two outer molecular layers merge into one. but these layers can dive into the interior, any given molecule is an ephemeral rather than integral part of the droplet's surface. films made of soap or protein (e.g., bubbles made by children from detergent or blowing bubbles in their milk) provide a better model, and we have all seen them fuse and lyse before our eyes. however, they also have surfaces that can exchange with a bulky interior, and their width can vary. these films are unlike the biological membrane in that their apolar moities face the low dielectric air rather than the high dielectric watery interior of the film. the biological membrane is composed of lipids, proteins, and carbohydrates of varying chemical structure. it exists within the context of an aqueous cellular environment that prefers to avoid the interior of the membrane fusion. this entropically-driven hydrophobic effect leads to two important constraints on topological transformations, (1) a tension at the interface of the polar head groups of the lipids to resist any stretching, and (2) a uniform thickness which is primarily determined by the lipid constituents. bilayers made from phospholipids have been used extensively to study the fusion process, and more recently the fission process. since the number of phospholipids molecules far outnumber the other constituents, membrane entropy is dominated by the thermodynamics of the lipids themselves, and thus it is likely that whatever is learned from investigations of lipid bilayer fusion will inform us about biological membrane fusion. indeed, it seems that many of the key intermediates of membrane fusion are the same in the fusion of synthetic and natural membranes. [1] [2] [3] we will not cover this ground, as many excellent reviews serve this purpose. [4] [5] [6] [7] [8] [9] [10] rather, in this chapter we will introduce a number of controversies that the reader may be stimulated to solve. in general there are a number of energy barriers that prevent fusion from proceeding spontaneously. first and foremost, there is the fact that in distilled water and dilute solutions of monovalent salt, all lipid bilayers resist close approach with a force that rises exponentially from an equilibrium distance of about 2 nm. with divalent cations in the solution bathing the membranes, close approach is possible, depending upon the lipid composition. this is readily seen as exchange of components in the contacting leaflets. 11, 12 presumably, this occurs through minute and transient contact sites in which the contacting leaflets are joined (hemifusion). however, this is not generally sufficient either for membrane fusion or the formation of an extended diaphragm composed of the outer, or non-contacting leaflets of the joining bilayers (termed hemifusion diaphragm), 13 for there are energetic restrictions to the widening of the hemifusion intermediate that joins the two leaflets (termed a stalk, [14] [15] [16] . the main way to facilitate the formation of a stable hemifusion diaphragm is to add hydrocarbon solvent to the membrane, so as to lower the energy of the three-way junction between the two bilayers and the joined diaphragm. 17, 18 once there is a sufficient junction, another way to complete fusion is via the formation of a lipidic pore within the hemifusion diaphragm. 19 this can be facilitated by either increasing membrane tension, or by adding lipids whose composition favors spontaneous pore formation. 20,21 (formally, such lipids would be defined as having positive monolayer spontaneous curvature. 22 ) thus it is possible to set up ionic and membrane compositional situations which allow the demonstration of membrane fusion and its intermediates without adding energy, thus spontaneous fusion is possible under a set of restricted conditions. what is not at all clear is the favorable effect of tension on the formation kinetics of hemifusion contacts, and how the huge hydration repulsion between contacting monolayers is breached during the tension-driven fusion of liposomes to bilayers. 20 the fusion pore links the interior of a vesicle to the external space, or the interior of a virus to the cytoplasm. a relatively long-lived fusion pore is universal in biological fusion, regardless of whether exocytosis, viral fusion, or cell-cell fusion is studied. since its discovery as a sub-ultrastructural entity, its architecture and composition has been in debate. in part, this is because of two very different views of the mechanism of membrane fusion: lipid centric and protein centric. in the lipid centric view, proteins surround a fusion site, and the conformational energy of the protein (as it transitions from a pre-fusion to a post-fusion form) is harnessed into stressing the encircled lipids to the point of a spontaneous transition in topology along a well-studied set of molecular intermediates, first a hemifusion intermediate -the stalk -and then the fusion pore ( figure 1) . 21, 24, 25 the prediction of this hypothesis is clear: the proteins should be outside of an hourglass-shaped pore scaffolded by proteins. in the protein-centric view, proteins link up the two membranes that are to fuse and make a solid proteinaceous connection, a potential channel that is first closed, then opens to form the initial fusion pore. 3 the prediction of this hypothesis is also clear: the proteins should be at the center of the pore, surrounded and later infiltrated by lipids as the proteins dissociate to guide the topological change of the lipids. a direct method is needed to determine which of these predictions is met during different instances of biological fusion. one of the abiding mysteries in biology is the great speed that synaptic transmission is capable of, with fusion of some vesicles beginning some tens of microseconds after ca 2 þ floods the presynaptic intracellular release site. 26, 27 this finding leads one to wonder if there is a physical state to a small population of vesicles whose fusion machinery is beyond the stages of priming and conformational change. 3, 28 there are two main proposals: 1. that synaptotagmin provides positive curvature stress that translates into hemifusion at the center of a ring of protein at the base of a dimple, 29 and 2. that ring assemblies of snare (soluble n-ethylmaleimidesensitive factor attachment protein (snap) receptor) and synaptotagmin complexes form to appropriately concentrate and orient c2b domains of synaptotagmin. 2 this ring of ordered domains effectively creates a tube-like scaffold of positively charged protein residues that span the two membranes that are to fuse, a favorable location for dimples of membrane to approach each other. in other words, it would be an electrostatic tunnel for membrane fusion that is extended by the polybasic linker regions of syntaxin and synaptobrevin. 3 variations of this model can account for many physiological pathways, including a small fraction of the vesicles that may already interacting at the level of the hemifusion prior to the entry of calcium. what is the role of calcium once it enters? first, ca 2 þ turns on an 'electrostatic switch' initially proposed for synaptotagmin-syntaxin interaction, but better suited to instantaneously stressing the phospholipid bilayers of the presynaptic membrane and the synaptic vesicle for the ultra-rapid exocytosis seen in the nervous system. second, even without synaptotagmin, ca 2 þ speeds up fusion of snare-reconstituted membranes considerably. perhaps divalent ions play a direct role, electrostatically complexing ps headgroups to promote fusion between negatively charged phospholipid bilayers. is this effect specific for calcium over magnesium? there are indications that the spontaneous curvature of ps in the presence of calcium, but not magnesium, is significantly more negative with calcium than with magnesium. 2 ultimately, synaptotagmin, snares, and the other proteins that comprise the exocytotic fusion machine must cajole lipids to move through a pathway that culminates in fusion pore opening. the snare proteins and synaptotagmin are the guides that walk and pull the membrane through a bumpy stalk-pore path, with electrostatic interactions playing a larger role than hitherto realized. 20 5.14.5 why is ptdins-4,5-p 2 needed for exocytotic fusion? experiments on the permeabilized chromaffin cell established a requirement for atp in membrane fusion in exocytotic secretion. 30, 31 further work revealed the product of atp in exocytosis to be ptdins-4,5-p 2 , which stands out as a key player amongst bilayer lipids. it has been almost 20 years since this minor plasma membrane constituent was directly implicated in exocytosis. the early studies used biochemical approaches in permeabilized cells that directly implicated the polyphosphoinositides 30 and subsequently pip kinase and ptdins-4,5-p 2 5 as key components late in the fusion pathway. imaging of ptdins-4,5-p 2 in secreting cells demonstrates that the lipid is located on the plasma membrane and not on the secretory granule. 32, 33 ptdins-4,5-p 2 associates with syntaxin puncta in plasma membrane lawns from pc12 cells. 7 the concentration of ptdins-4,5-p 2 in the cytoplasmic leaflet of puncta is surprisingly high,b6 mole%. 8 an initial fusion pore of 3 nm diameter and 10 nm 9,10 would have room for approximately 140 phospholipid molecules (area ¼ 70 å 2 ) facing the cytosol including nine ptdins-4,5-p 2 molecules at 6 mole%. clearly, ptdins-4,5-p 2 is absolutely required for exocytosis. ptdins-4,5-p 2 can have two, potentially interrelated functions: 1. as a scaffold for proteins of the exocytotic and endocytotic pathways, such as the exocyst and indirect effects through the actin cytoskeleton. 34-36 2. as a lipid involved in the topological rearrangement of the plasma membrane during fusion and fission. a distinguishing feature of ptdins-4,5-p 2 is its high negative charge of b à 4 at ph 7. 11 the highly charged lipid creates a highly dynamic electrostatic scaffold that interacts with unstructured (e.g., in marcks, gap43) and structured (e.g., in ph and c2 domains) basic moieties on a variety of proteins (for a review see ref. 12) . unstructured basic peptides cause lateral sequestration of ptdins-4,5-p 2 on membranes in vitro containing 1 mole% ptdins-4,5-p 2 with as much as 30 mole % ps 13, an effect that is explained by electrostatic considerations. 14 several proteins that play important roles in exocytosis have structured basic moieties in c2 domains that interact with ptdins-4,5-p 2 including synaptotagmin 15 and rabphilin. 16 stop-flow techniques demonstrate that ptdins-4,5-p 2 greatly speeds the ca 2 þ -dependent interaction of synaptotagmin with membranes in vitro 17 , suggesting that this interaction may have important physiological consequences. in addition, fret has been used to show that ptdins-4,5-p 2 directly interacts with syntaxin in vitro. 37 there are numerous proteins involved in exocytosis and endocytosis that contain a structured basic sequences in pleckstrin homology (ph) domains that interact with ptdins-4,5-p 2 . these include caps in the exocytosis pathway 19 and dynamin in the endocytosis pathway. ptdins-4,5-p 2 also plays an important regulatory role in conjunction with small gtpases and proteins that regulate the actin cytoskeleton. 20 these pathways also influence fusion and fission. while there is strong evidence for ptdins-4,5-p 2 interacting with proteins that are important for fusion and fission, there is little direct evidence at this time for a direct role of the lipid in the fusion or fission reactions. for example, both long term and acute modulation of ptdins-4,5-p 2 in chromaffin cells alter the size of the releasable granule pools but not the fusion kinetics, 21 consistent with a role prior to but not during fusion. nevertheless, it seems likely that ptdins-4,5-p 2 molecules with high charge and relatively high concentrations at fusion sites (as many as nine molecules in the cytoplasmic leaflet of the fusion pore) would directly influence lipid rearrangements. this is an important area for future investigation. it will be challenging to distinguish between the lipid simply being a scaffold for a multitude of proteins involved with trafficking at the plasma membrane, and having a direct function in the bilayer rearrangements of fusion and fission. in fact, these two roles may sometimes be indistinguishable, since one or more of the interacting proteins may have as its primary task the regulation of ptdins-4,5-p 2 function in fusion or fission. the superficially shared structural features of ha with the snare proteins essential for internal cellular fusion have stimulated the hope that there is a universal mechanism for protein-mediated fusion. as the prototype of class i fusion proteins, and the epitope for flu serotyping the influenza virus ha has been studied extensively. while the speed of lipid mixing of ha-mediated fusion is rapid in vitro relative to infection speeds ( figure 2 ), fusion pore formation has not been measured and thus we do not know the kinetics of complete fusion for ha in an intact virus. however, the accessibility of ha-mediated cell-cell fusion, and the large body of investigation into ha make this the best studied fusion protein that is sufficient for fusion. we learn the importance of guided conformational transitions together with protein-protein interactions in conjunction with clear phenotypic discrimination between intermediates of hemifusion and the fusion pore opening and subsequent widening. clearly there is much more work to be done, because we do not know why changing a single amino acid residue at the tip of the fusion peptide gives hemifusion instead of fusion. 38 ha is the first viral glycoprotein for which the structures of both pre-and post-fusion forms were solved at atomic resolution. 39, 40 ha is synthesized as a single-chain precursor protein ha0, which then oligomerizes into a trimer during protein transport through the secretory pathway 41 . the precursor ha0 (549 amino acids in a/hong kong/68/h3n2) then needs to be cleaved into the ha1 and the ha2 polypeptides after the conserved arginine at residue 329 to be primed for the subsequent low ph-triggered conformational changes ( figure 3) . 42 although the crystal structures of both pre-and post-fusion forms of ha have been available for more than a decade ( figure 4) , we still do not know exactly how conformational rearrangements occur step by step, how low ph environment triggers the rearrangements of the ha ectodomain, and which structural elements are crucial for ha fusion activity. it has been suggested that the low ph might lead to an enhanced protonation of the ha1 domain, and generate enough electrostatic repulsion force to partially dissociate the ha1 domain from the ha2 to allow the loop region in the ha2 to contact with water; this would cause the loop to transition into a helix and extend the pre-existing central coiled coil ( figure 5 ). [44] [45] [46] in this model, it seems that the only step requiring a low ph trigger is the exposure of the loop region connecting the two alpha helices in ha2 to water by partial disorientation of ha1 from ha2. consistent with this idea, spontaneous formation of extended coiled coils are observed in bacterially expressed ha2 polypeptide at neutral ph, indicating that the low ph trigger is not necessary for the extension of coiled coils. 47 the prevailing hypothesis for the mechanism of how conformational changes lead to the fusion of two membranes is that extension of coiled coils triggered by the low ph directs the fusion peptide to insert into the target membrane, and then a helix-to-loop conformational change reorients the protein to pull the fusion peptide toward the transmembrane domain. these molecular transitions result in a tight packing of the cooh-terminus of ha2 against grooves of the nh 2 -terminal coiled coil, proceeding to the fusion of the two bilayers. [48] [49] [50] this hypothesis emphasizes that both the extension of the coiled coil and the bending of the protein resulting from the helix-to-loop conformational change are important for fusion. the experimental results favoring this hypothesis demonstrate that a double proline substitution mutant (f63p/f70p) at the region supposedly undergoing loop-to-helix conformational change upon low ph failed to induce fusion, although the mutant still presented and inserted the fusion peptide to the target membrane. 50 the block to fusion was demonstrated to occur in the tight packing of cooh-terminal extended regions into the grooves between the helices of the nh 2 -terminal half of the coiled coil due to the splayed nh 2 -terminal half of ha2, suggesting that the insertion of fusion peptide into target membrane alone is not sufficient for fusion, and the tension caused by the packing of cooh-terminus against the nh 2 -terminus of ha2 may be the driving force for membrane merging. another piece of evidence for the role of the packing of the cooh-terminus to the nh 2 -terminus in viral fusion is that the alanine substitution mutants at five apolar residues after the short cooh-terminus of ha2 fails to cause both lipid and content mixing. 51 as a result of conformational changes of ha upon low ph exposure, the fusion peptide located at the tip of the ha2 molecule is exposed to the close proximity of the target membrane. when the target membrane is available, the fusion peptide inserts into the lipid bilayer to induce lipid mixing between the target cell membrane and the viral membrane. it has been experimentally determined that the free energy associated with the insertion of full-length fusion peptide into the lipid membrane is 4.5 kbt, and as many as 18 fusion peptides binding to the target membrane might generate enough energy to stabilize a stalk-like fusion intermediate, which has high membrane curvature. 52 while necessary for fusion, the packing of fusion peptides does not appear to be sufficient for fusion. lipids present in the membrane act together with ha to cause fusion. changing membrane lipid physical properties or composition in ways that well defined also blocks fusion, despite conformational-specific antibody binding indicating that ha is in the post-fusion state. 53 in other words the protein conformational change, while essential, is not sufficient, as there are post-protein conformational changes in lipid conformation that are needed for fusion to continue along its path. thus the pathway of ha-mediated membrane fusion involves conformational changes to induce lipids to undergo the more general pathway outlined above for lipid membrane fusion. 53 the role of the fusion peptide domain for ha-mediated membrane fusion has been the subject of many biophysical studies. 54 the fusion peptide of ha is rich in glycine; for example, influenza a/x31/h3n2 contains 6 glycine residues in 20 residues of fusion peptide (glfgaiagfiengwegmidg). extensive mutagenesis studies of fusion peptides have revealed that both the primary sequence and the length of fusion peptide are crucial for ha fusogenic activity. 55 for example, ha2 g1e substitution abolishes cell-cell and rbc-cell fusion activity of expressed ha, while g4e substitution decreases fusion efficiency and elevated the ph threshold for activation. 56 alanine can substitute for glycine at positions 1 and 4 without impacting ha-induced cell-cell fusion; however, the polar amino acid serine substitution for glycine at position 1 causes a hemifusion phenotype. 38 the requirements for specific amino acids at certain positions and for a defined length in the fusion peptide have been further supported by the nmr-solved structure of fusion peptide in detergent micelles and in model lipid membranes 43,52 stabilized by a charge-dipole interaction between the n-terminal gly and the dipole moment of helix 2 54 (figure 6 ). at acidic ph, the 20 residues of fusion peptide adopt a v-shaped 'boomerang' structure with an oblique nh 2 -terminal amphipathic helix spanning residues 2-10 and a turn formed by residues 11, 12 and 13 followed by a short cooh-terminal helix 43, 57 stabilized by a charge-dipole interaction between the nterminal gly and the dipole moment of helix 2 58 ( figure 6 ). the bent fusion peptide then may insert b16-17 å into the outer leaflet of the target membrane, almost to the mid-plane of the lipid bilayer with the residue leu 2 and phe 3 penetrating deepest into the membrane. 52, 57 in the solution structure, the hemifusion phenotype mutant g1s has a similar structure to that of wild type, but the glycine ridge on the outer surface of the nh 2 -terminal helical arm is disrupted. in contrast, the mutant g1v, where fusion is completely abolished, has a very irregular linear amphipathic helix instead of the fixed angled boomerang structure ( figure 6 ). 59,60 another fusion-defective mutant, w14a, has a more flexible kink than that of the wild type, in contrast, the alanine substitution at phenylalanine residue 9 (f9a) has a similar structure to that of the wild-type and has no defect in fusion ( figure 6 ). 61 these studies suggest that both the angled and deeply inserted structure as well as the glycine ridge make a contribution to the fusion activity. despite the fact that we have gained a large amount of information on the structure of the influenza fusion peptide over the past several years, there are still open questions surrounding how the insertion of fusion peptide leads to fusion of viral membrane and target membrane and also with regard to the thermodynamic profile during the mixing of two bilayers. it has been generally accepted that the transmembrane domain (tmd) of ha2 and cooperation of multiple ha molecules are required in the fusion pore initiation and fusion pore enlargement. 62 replacing the tmd of ha with a glycerylphosphatidylinositol (gpi) anchor or the deletion mutants with less than 17 residues in length cause a hemifusion phenotype, but a tmd with polar amino acids at the coohterminus still allows full fusion. 12,63 a synthetic peptide representing the transmembrane segment of x31/ha spans an artificial dmpc/dmpg bilayer as an a-helix that aligns roughly perpendicular to the bilayer membrane. the consequences of ha fusion peptide insertion are not well understood, although it is clear that the proposed boomerang structure avoids placing the polar amino acids within the hydrophobic phase, which would be disruptive or structure-forming. insertion of a charged amphipathic helix per se would tend to promote positive curvature, and might aid in ''nippling'' the membranes towards each other to contact prior to fusion, as in the proposed mechanism for synaptotagmin. 29 wt-ph 7. one suggestion is that the membrane helices of the fusion peptide and the tmd could interact with each other, so providing a driving force for the mixing of two membranes. 64 other open questions include the study of ha fusion (and other enveloped viruses) in the context of negative membrane curvatures found in the multi-vesicular bodies of endosomal compartments, 65 and lipids known to be enriched in endosomes. another open question is whether the ha conformational changes are strictly irreversible in the absence of a target membrane. the prevailing model that spring-loaded conformational changes are uni-directional is based in part on the fact that pre-treatment of virus particles from typical laboratory strains (e.g., the h3 strain x-31) with low ph effectively neutralizes infection. analysis of other viral subtypes (e.g., h2) show features consistent with reversible conformational changes 66 and many natural isolates of influenza do not show the irreversible conformational changes associated with x-31 in the absence of a target membrane (g. whittaker personal communication). tatulian and tamm 67 have demonstrated that the conformational change of the entire ha is reversible in the absence of bound target membranes. the formation of a six-helix bundle (6hb) is the structural feature that characterizes class i viral fusion proteins. because fusion peptides insert into target membranes and tmds span the viral envelope, the folding of fusion proteins into hairpins brings the viral envelope and host cell membrane into proximity. even for the two other classes of viral fusion proteins, which do not exhibit 6hbs and contain much b-sheet, in the final, post-fusion state, it is likely that fusion peptides and tmds are proximal. it is not experimentally certain that this proximity occurs, because the hydrophobic tmds and fusion peptides themselves are not present in the crystallized proteins. this apposition of the membrane imbedded tmd and fusion peptides of viral fusion proteins is similar to the proximity between tmds of v-and t-snares in their tetrameric coiledcoil. when the ubiquity of 6hbs was first demonstrated, it was assumed that bundle formation merely brought membranes into close contact, and fusion then occurred. we now know this is incorrect. the correlations between bundle formation and steps in the fusion process have been investigated for hiv-1 env, influenza ha, and aslv env. the precise steps of bundle formation depend on the precise protein. it appears that the longer the amino acid sequence that intervenes between tmds and the coiled-coil, the earlier the bundle can form. for hiv-1 env, which has a relatively short intervening sequence, not only does hemifusion occur prior to completion of bundle formation, but so does the creation of the initial fusion pore. bundle formation releases considerable energy and the late occurrence of formation indicates that considerable energy is required for pore enlargement -the last step in fusion. in fact, several lines of evidence lead to the picture that hemifusion is energetically easy to achieve, pore formation more difficult, and pore enlargement even more difficult. from the theoretical point of view of membrane mechanics, considerable work must be expended to enlarge a fusion pore because additional membrane must be bent from its relaxed state as the pore enlarges. the requirements for hemifusion are reasonably well understood: the standard helfrich concepts of spontaneous membrane curvature and bending energy are sufficient to account for experimental observation. the mechanisms by which fusion proteins create the pore formation is less understood and how fusion proteins contribute to pore enlargement remains a mystery. for topological reasons, tmds cannot span hemifusion diaphragms and can only enter the diaphragm from the junction between the diaphragm and the two original membranes. common sense suggests that this entry of tmds should destabilize the junction and thereby generate pores. this is consistent with demonstrations that tmds are directly involved in pore formation 68 and with theory that predicts that pores form at the junction. but why tmds are forced through the junction has not been addressed and the determinants of pore properties are virtually uncharacterized. for example, some viral fusion proteins, such as hiv-1 env, generate large initial pores than readily open whereas others, such as influenza ha, create small pores that generally flicker open and closed before they enlarge. it seems reasonable that differences in pores are conferred by the proteins (rather than by the lipids), but the field has not formulated guiding principles as to which structural features of a protein control pore properties. because viral nucleocapsids can only enter cytosol if a pore enlarges, these questions are potentially of practical, in addition to biophysical, importance. the snare complex formed between two fusing membranes are the principal fusogens of the eukaryotic molecular machinery that mediates membrane fusion in intracellular trafficking pathways. 69 the snare complex is a coiled bundle of four parallel helices provided by three or four individual snare protein molecules (figure 7) . perhaps the best studied is the snare complex mediating neuronal exocytosis containing four helices provided by three snare proteins: snap-25 (synaptosome-associated protein of 25 kd), synaptobrevin-2, and syntaxin-1. 70 the free energy generated during the assembly of a single ternary snare is estimated to be 20-35 k b t. [71] [72] [73] a key question is what proportion of the energy of snare complex formation is directed at membrane fusion and what the contribution of the energetics of this reaction, if any, contribute to membrane docking and tethering. several of the proteins engaged in and/or responsible for fusion have been studied at atomic resolution with biophysical structural approaches. these studies have greatly illuminated our understanding of the protein machines driving membrane fusion reviewed in refs. 75 and 76. the minimal domains of snare proteins that can spontaneously engage in a stable 4-helix snare complex revealed a characteristic packing at the core of the snare complex. the majority of the packing interactions are hydrophobic, however there is an ionic layer typically consisting of a single arginine and three glutamine residues. 70, 74 this ionic layer is found at the midpoint of the coiled-coiled bundle and hence is referred to as the zero ionic layer. the zero ionic layer is an evolutionarily conserved feature of all snare complexes examined to date, however the functional role of this feature is not known. biophysical characterization of snare complexes that perturb the zero ionic layer suggest that it may be important for the stability of snare complexes. 77 one idea was that this layer could provide an intervention point for snare complex disassembly, however perturbation of the layer in an in vitro disassembly assay 78 had no discernable impact on nsf/snap catalyzed snare disassembly. current hypotheses for how polar zero layer residues might impact snare function favor potential role(s) in snare complex assembly, such as the suggestion that a polar zero layer helps align assembly of the snare helices in register, or in other downstream functions of the snare complex. mutagenesis studies of the zero ionic layer in different systems suggest different effects, but they are all relatively subtle. 79, 80 clearly, the influence of particular residues may vary according to the individual snare complex in question and the local parameters governing the assembly of a particular cognate snare complex. structures of the individual snare proteins have been tremendously stimulating in posing novel questions. the coiled-coil a-helix of synaptobrevin extends to the most membrane proximal residue, lysine 87 and this residue is also part of the extended transmembrane helix. the energetics and topology of snare complex formation may influence local bending of the a-helix at the interfacial region, which in turn could generate local membrane destabilization to aid fusion ( figure 8) . it is not known how the membrane itself may locally influence the structure of cytoplasmic portions of synpatobrevin or other proteins involved in fusion. a recent structure of lipid-bound synpatobrevin suggests that the amphipathic helix 1 of synpatobrevin may lie on the surface of the membrane, 81 providing a molecular explanation of the observation that the membrane may influence the cytoplasmic portion of synaptobrevin to adopt conformations not observed in the absence of lipid. 82 some snares contain independently folded nh 2 -terminal domains together with additional unstructured linker regions of significant length. the presence of such domains and their ability to interact inter-and intra-molecularly significantly increases the complexity of snare complex formation and the ability of the snare proteins to drive membrane fusion. syntaxin contains a linker region connecting the snare domain with the h abc nh 2 -terminal domain. fully extended, this region may be up to approximately 120 å in length ( figure 9 ). it is currently unknown whether there are proteins that selectively bind to or regulate this region. in contrast to synaptobrevin, syntaxin has an slightly extended membrane proximal region that may not be part of the initial core snare complex structure (residues 260-265). the post-fusion snare complex shows this region adopts an a-helical structure that directly links the snare complex a-helix to the transmembrane a-helix. the local secondary structure adopted by these residues prior to fusion is unknown; secondary structural predictions suggest the region is unstructured and could conceivably act as a hinge facilitating the molecule to sample up to a 290 å radius of the membrane proximal area ( figure 10 ). there is evidence for an initial nh 2 -terminal interaction between the snare proteins, but the final low energy 4-helical bundle may not be an intermediate in fusion, but rather either a dead-end conformation or a post-fusion conformation. an experiment missing from the field is a demonstration of helical bundle formation before, or simultaneous with fusion pore formation, as described above for viral fusion. there is strong evidence that prior interaction between the plasma membrane snares syntaxin and snap25 increases rate of interaction with vamp. 83 the relationship of the post-fusion snare complex ( figure 11 ) to lipid rearrangements that occur during fusion and content mixing are currently not known. one study has placed syntaxin as a pore-forming molecule with 5-8 syntaxin molecules making up a fusion pore. 84 assembly of 5-8 snare complexes as a minimum number required to drive fusion events is in good agreement with theoretical considerations of the energetics of snare complex assembly and membrane fusion. b8 snare complexes were required to promote fast fusion in supported bilayer experiments, 85 and fewer in other lipid mixtures including pe, presumably because pe promotes fusion by curvature. 86 what is enormously fascinating is how the protein fusogens form such a pore, how the pore forms initially and how the pore dilates as fusion proceeds to completion. these pathways may have different kinetic and thermodynamic parameters for different types of biological fusion, depending on the physiological requirements of the particular fusion event. the snare machinery certainly appears to have the potential for adaptability, mediating types of fusion as diverse as ''kiss and run'', complete fusion during constitutive events of exocytosis and homotypic fusion events such as those between endosomes. 87 the snare complexes for which the structures have been solved all contain the four helix bundle in a parallel orientation. perhaps not surprisingly, given the versatility of coiledcoil structures, the snare domains are also capable of associating in anti-parallel bundles, which are also stable, although not as stable as the parallel bundles. 88 this is superficially reminiscent of the anti-parallel and parallel coiled-coil transitions experienced by different conformations of ha (figures 4 and 5) although caution should be exercised in extending these analogies given the topological constraints of the fusion machinery. it is not known what such associations may represent physiologically; they could possibly represent a means of tethering membranes independently of fusion, or may be unproductive molecules requiring reconditioning by accessory factors in order to participate in multiple rounds of membrane fusion. the profile of the energy landscape 89 traversed by the fusion reaction will be influenced by a multitude of exogenous factors. known factors include the membrane lipid composition, the availability of snare proteins in suitable pre-fusion states and the specific activity of snare accessory factors ( table 1) . these factors can have multiple influences, for example, membrane composition can play a role in providing molecular determinants for protein assembly and will also determine membrane elasticity. how conformational changes amongst snare proteins and their accessory factors control the thermodynamics and kinetics of docking, lipid mixing and content mixing after membrane fusion remain open questions. our understanding of the conformational plasticity of protein machineries and the hysteresis properties of biological fusion machines lead to an appreciation for the energetic complexities of the fusion reaction. the use of single molecule studies to unravel the complex energy landscape of membrane fusion will be an important biophysical approach with tremendous potential to relate the topography of the energy landscape to the mechanism and regulation of fusion. although the application of this approach to studying biological fusion machines is relatively new, 72, [125] [126] [127] [128] [129] [130] such experiments have some additional advantages -such as being able to distinguish fusion events from vesicle aggregation or vesicle rupture. single molecule approaches of fluorescently-labeled virus in living cells has allowed visualization of influenza fusion with intracellular compartments/endosomes, 131 and the use of solid supported lipid bilayers, 132 facilitates a more detailed analysis of the single-particle kinetics of ha-mediated fusion as well as snare-mediated fusion. 85 in combination with assays that reflect lipid and content mixing, together with pore formation and expansion, such approaches are expected to contribute substantially towards providing missing information regarding the intermediates and pathways involved in fusion. membrane fusion? recent work has shown that two rungs of a dynamin spiral is the minimal structural unit responsible for the formation of the fission neck and hemifission intermediate in model membrane nanotubes (figure 12) . 133 requires the same hemifusion event, but in a cylindrically symmetrical way, as the inner leaflet of the tube must touch itself in the center to allow for the hemifission intermediate. once again, it is relatively straightforward to calculate the ring conditions that will lead to constriction and narrowing of the tube towards the center, as long as the distance between the two rings is not too short. but once again, we are faced with the hydration force resisting any further constriction of the neck, since the surfaces of the inner leaflet lipids would have to come closer than the 2 nm equilibrium distance between attractive dispersion forces and repulsive hydration forces. but lo, there is a new modality for minimizing energy in this system; it is the tilt-like movement of lipid head groups away from each other at the very center of the hourglass constriction of the neck (figure 12 in formation of a narrow separation of heads exposing the hydrophobic interior of the bilayers, termed a hydrophobic ''belt'' to indicate its presence as a ring (you can visualize this ring by rotating the figures of figure 12 (a) around the axis of the horizontal dashed line under each bilayers). now the repulsive hydration force stabilizing inner aqueous diameters of 2 nm gives way to a newly developed hydrophobic attractive force, which is effectively the desire of water to desolvate the space between the tilting headgroups in the center. this water ejection leads to the close approximation and finally merger of the neck with itself at the midpoint; that is, its closure. figure 12 (d) shows the calculated energy of a 9-mm-long segment of neck, depending upon the width (h) and radius (rmid) of the hydrophobic belt discussed above. like the stalk, the belt width becomes that of a single monolayer at the point of merger. the energy barrier of 35 k b t is also similar to those calculated for membrane fusion, 24 so it is energetically feasible. the lipid bending and tilting needed to catalyze membrane merger are mainly motivated by high curvature stress in the neck inner leaflet. accordingly, the energy barrier depends sharply on the minimal radius of the thinned neck, rmin, which should approach 1-2 nm for the fission to occur, as seen in other estimations of hemifission. 135 thus the rings of protein acting on the outside of a 12 nm tube lead to boundary conditions whose effects propagate through the bilayer to influence and stress lipids facing each other across water, leading to their head groups parting and their oils merging in energetically feasible hemifission. the proteins act at length scales consistent with what we know about protein structural lengths, and the lipids respond fluidly to the protein's influence. mechanistic studies concerning dynamin offer not only a static view of the energetics of non-leaky fission/fusion reactions but also insights into the dynamics of the transition structures. dynamin assembly shapes bilayers into highly curved structures ( figure 12 ). assembly also greatly enhances the rate of gtp hydrolysis (100-fold), 136 which in turn leads to dynamin disassembly. thus, assembly is self-limited in the presence of gtp. recent experiments suggest that assembly-induced gtpase activity reduces the interaction of dynamin with the highly curved lipid membrane even before disassembly. 137 the result is the unstable, highly curved lipid neck described above that resolves either in membrane fission (endocytosis, described above), or reversal of high membrane curvature. from these studies, performed in model membranes, membrane fission may therefore be considered to be a stochastic result of gtp hydrolysis. 133, 139 studies in adrenal chromaffin cells suggest that ectopic expression can reverse the otherwise lethal neurodegeneration of cysteine string protein-a knockout mice. (123, 124) dynamin functions in a similar way to control the fate of a recently fused secretory granule. increasing the dynamin gtpase activity increases the rate of fusion pore expansion and the likelihood of rapid endocytosis. 138 the results are consistent with a function for dynamin in restricting fusion pore expansion. increased gtp activity catalyzes a more rapid stochastic decision that results in either fusion pore expansion or (less frequently) membrane fission and endocytosis. recent theoretical and experimental studies of membrane fission by dynamin and viral matrix protein reveal how protein complexes are arranged to effectively apply localized curvature stress to membranes without perturbing lateral membrane integrity; that is, without leakage. one conceptual approach is that sites of membrane remodeling are organized as membrane domains, both through membrane composition and membrane curvature; thus membrane remodeling is a collaborative effort accomplished by the entire domain, involving protein complexes and multiple lipids. we emphasize that despite decades of studies we still know only a little about fundamental physical principles underlying the spatial and temporal organization of membrane domains specialized in membrane remodeling. for example, the structure and composition of the fusion pore are unknown. new synergistic experimental and theoretical approaches are needed to resolve how proteins merge and separate membranes. for example, the ability to detect submicron deformations of the plasma membrane with the combination of polarization and tirfm techniques 138, 139 permits detection of the expanding fusion pore and may enable investigations of the molecular basis for membrane curvature changes in living cells. the key is to study membrane remodeling in the context of concrete biological processes, taking into account corresponding length scales for the key membrane intermediates, dynamic cooperation between protein machineries and lipids, 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effector eea1 interacts directly with syntaxin-6 involvement of lma1 and gate-16 family members in intracellular membrane dynamics structure of gate-16, membrane transport modulator and mammalian ortholog of autophagocytosis factor aut7p intracellular bacteria encode inhibitory snare-like proteins a phorbol ester/diacylglycerol-binding protein encoded by the unc-13 gene of caenorhabditis elegans an open form of syntaxin bypasses the requirement for unc-13 in vesicle priming modular architecture of munc 13/calmodulin complexes: dual regulation by ca 2 þ and possibility function in short-term synaptic plasticity structural basis for a munc13-1 homodimer to munc13-1/rim heterodimer switch cellular functions of nsf: not just snaps and snares isolation and characterization of a dual prenylated rab and vamp2 receptor structural basis of rab effector specificity: crystal structure of the small g protein rab3a complexed with the effector domain of rabphilin-3a a novel syntaxin 6-interacting protein, ship164, regulates syntaxin 6-dependent sorting from early endosomes synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin specificity and regulation of a synaptic vesicle docking complex sec1p binds to snare complexes and concentrates at sites of secretion tomosyn: a syntaxin-1-binding protein that forms a novel complex in the neurotransmitter release process structure of the yeast polarity protein sro7 reveals a snare regulatory mechanism amisyn, a novel syntaxin-binding protein that may regulate snare complex assembly snap family of nsf attachment proteins includes a brain-specific isoform the yeast sec17 gene product is functionally equivalent to mammalian alpha-snap protein doc2b is a high affinity ca 2 þ sensor for spontaneous neurotransmitter release synaptotagmin-ca 2+ triggers two sequential steps in regulated exocytosis in rat pc12 cells: fusion pore opening and fusion pore dilation synaptotagmin modulation of fusion pore kinetics in regulated exocytosis of dense-core vesicles synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine vesicle-associated membrane protein and synaptophysin are associated on the synaptic vesicle alpha-synuclein promotes snare-complex assembly in vivo and in vitro alpha-synuclein cooperates with cspalpha in preventing neurodegeneration a single-vesicle content mixing assay for snare-mediated membrane fusion discrimination between docking and fusion of liposomes reconstituted with neuronal snare-proteins using fcs single-molecule studies of the neuronal snare fusion machinery single molecule measurements of mechanical interactions within ternary snare complexes and dynamics of their disassembly: snap25 vs. snap23 single molecule mechanical probing of the snare protein interactions single molecule observation of liposome-bilayer fusion thermally induced by soluble n-ethyl maleimide sensitive-factor attachment protein receptors (snares) visualizing infection of individual influenza viruses single-particle kinetics of influenza virus membrane fusion gtpase cycle of dynamin is coupled to membrane squeeze and release, leading to spontaneous fission real-time visualization of dynamin-catalyzed membrane fission and vesicle release membrane fission: model for intermediate structures dynamin self-assembly stimulates its gtpase activity real-time detection reveals that effectors couple dynamin's gtp-dependent conformational changes to the membrane a new role for the dynamin gtpase in the regulation of fusion pore expansion localized topological changes of the plasma membrane upon exocytosis visualized by polarized tirfm this work was supported by nih grant no. 5r01gm069596 to r. collins, r56-ns38129 to rwh, and the intramural program of the nichd, nih. many thanks to fabio rinaldi for help with figure preparation, fred cohen for his discussion of the viral helical bundle timing experiments, and gary whittaker for helpful discussions. key: cord-265087-g4k6pc82 authors: munteanu, cristian robert; gonzález-díaz, humberto; borges, fernanda; de magalhães, alexandre lopes title: natural/random protein classification models based on star network topological indices date: 2008-10-21 journal: journal of theoretical biology doi: 10.1016/j.jtbi.2008.07.018 sha: doc_id: 265087 cord_uid: g4k6pc82 abstract the development of the complex network graphs permits us to describe any real system such as social, neural, computer or genetic networks by transforming real properties in topological indices (tis). this work uses randic's star networks in order to convert the protein primary structure data in specific topological indices that are used to construct a natural/random protein classification model. the set of natural proteins contains 1046 protein chains selected from the pre-compiled culledpdb list from pisces dunbrack's web lab. this set is characterized by a protein homology of 20%, a structure resolution of 1.6å and r-factor lower than 25%. the set of random amino acid chains contains 1046 sequences which were generated by python script according to the same type of residues and average chain length found in the natural set. a new sequence to star networks (s2snet) wxpython gui application (with a graphviz graphics back-end) was designed by our group in order to transform any character sequence in the following star network topological indices: shannon entropy of markov matrices, trace of connectivity matrices, harary number, wiener index, gutman index, schultz index, moreau–broto indices, balaban distance connectivity index, kier–hall connectivity indices and randic connectivity index. the model was constructed with the general discriminant analysis methods from statistica package and gave training/predicting set accuracies of 90.77% for the forward stepwise model type. in conclusion, this study extends for the first time the classical tis to protein star network tis by proposing a model that can predict if a protein/fragment of protein is natural or random using only the amino acid sequence data. this classification can be used in the studies of the protein functions by changing some fragments with random amino acid sequences or to detect the fake amino acid sequences or the errors in proteins. these results promote the use of the s2snet application not only for protein structure analysis but also for mass spectroscopy, clinical proteomics and imaging, or dna/rna structure analysis. one of the widely used methods for the predicting of the protein properties is quantitative structure activity relationship (qsar) (devillers and balaban, 1999) . graph theory can be used to obtain macromolecular descriptors named topological indices (tis). the branch of mathematical chemistry dedicated to encode the dna/protein information in graph representations by the use of the tis has become an intense research area with interesting works of liao (liao and wang, 2004a, b; liao and ding, 2005; liao et al., 2006) , randic, nandy, balaban, basak, and vracko (randic, 2000; randic et al., 2000; randic and basak, 2001; randic and balaban, 2003) , bielinska-waz team (bielinska-waz et al., 2007) or our group (perez et al., 2004; aguero-chapin et al., 2006) . using graphic approaches to study biological systems can provide useful insights, as indicated by many previous studies on a series of important biological topics, such as enzyme-catalyzed reactions (andraos, 2008; chou, 1989; forsen, 1980, 1981; chou and liu, 1981; chou et al., 1979; king and altman, 1956; kuzmic et al., 1992; myers and palmer, 1985; zhou and deng, 1984) , protein folding kinetics (chou, 1990) , inhibition kinetics of processive nucleic acid polymerases and nucleases (althaus et al., 1993a (althaus et al., , b, c, 1994a (althaus et al., , b, 1996 chou et al., 1994) , analysis of codon usage (chou and zhang, 1992; chou, 1993, 1994) , base frequencies in the anti-sense strands , and analysis of dna sequence (qi et al., 2007) . moreover, graphical methods have been introduced for qsar study prado-prado et al., 2008) as well as utilized to deal with complicated network systems (diao et al., 2007; gonzalez-diaz et al., 2007a . recently, the ''cellular automaton image'' (wolfram, 1984 (wolfram, , 2002 has also been applied to study hepatitis b viral infections (xiao et al., 2006a) , hbv virus gene missense mutation (xiao et al., 2005b) , and visual analysis of sars-cov (gao et al., 2006; wang et al., 2005) , as well as representing complicated biological sequences (xiao et al., 2005a) and helping to identify protein attributes (xiao and chou, 2007; xiao et al., 2006b) . the actual work presents for the first time a natural/random protein classification using only the chain sequence and amino acid connectivity protein structural data. the data are transformed into sequence and connectivity star graph's tis, which are then used as input for a statistical linear method in the construction of a simple classification model. two sets of proteins are compared in the new classification model: a set (nat) of 1046 natural protein chains as defined in the pre-compiled culledpdb list from pisces dunbrack's web lab (wang and dunbrack, 2003) and a second (rnd) with the same size formed by random amino acid sequences generated with python scripts (rossum, 2006) . the natural set is characterized by a homology of 20%, a structure resolution of 1.6 å and r-factor lower than 25%. the random set is composed by the same standard amino acid types and the average length of the chains is the same as that of the natural set. python scripts are used to download pdb files from the pdb data bank (berman et al., 2000) and to create the correspondent dssp file with the dssp application (kabsch and sander, 1983) . the chain sequences were extracted with a python script from these dssp files and were filtered with our prot-2s web tool (http://www.requimte. pt:8080/prot-2s/) by removing the chains that contain nonstandard amino acid (usually labelled x). each protein can be considered as a real network where the amino acids are the vertices (nodes), connected in a specific sequence by the peptide bonds. the graph is the abstract representation of the network and is a collection of n vertices and the connections between them. the star graph is a special case of trees with n vertices where one has got nà1 degrees of freedom and the remaining nà1 vertices have got one single degree of freedom (harary, 1969) . in addition, as a general property, there is a unique path between any pair of vertices. for proteins, each of the 20 possible branches (''rays'') of the star contains the same amino acid type and the star centre is a nonamino acid vertex. the same protein can be represented by different forms which are associated to distinct distance matrices (randic et al., 2007) . if the vertices do not carry a label, the sequence information will be lost; for that reason, the best method is to construct a standard star graph where each amino acid/vertex holds the position in the original sequence and the branches are labelled by alphabetical order of the three-letter amino acid code (randic et al., 2007) . in the present study we are using the alphabetical order of oneletter amino acid code. the standard star graph for a random virtual decapeptide (acadcefdgh) is illustrated in fig. 1 . if the initial connectivity in the protein chain is included, the graph is embedded (fig. 2) . in order to compare the graphs, it is necessary to transform the graphical representation in connectivity matrix, distance matrix and degree matrix. in the case of the embedded graph, the matrices of the connectivity in the sequence and in the star graph are combined. these matrices and the normalized ones are the base for the tis calculation. the protein chain sequences are transformed into star graph representations and then characterized by several tis using our new sequence to star networks (s2snet) application. s2snet is a wxpython (noel rappin, 2006) gui application with graphviz (koutsofios, 1993 ) as a graphics back-end. the user of this interactive tool is able to choose the level of calculations, such as: embedded graph, additional weights for each amino acid, markov normalization, power of the matrix connectivity, the input files (files with sequences, groups and weights), the output files, the level of details (files for summary and detailed results) and the type of graph visualization (dot, neato, fdp, twopi, circo). in particular, the calculations presented in this work are characterized by embedded and non-embedded tis, no weights, markov normalization and power of matrices/indices (n) up to 5. the summary file contains the following tis (todeschini and consonni, 2002) : shannon entropy of the n powered markov matrices (sh n ): where p i are the n i elements of the p vector, resulted from the matrix multiplication of the powered markov normalized matrix (n i â n i ) and a vector (n i â 1) with each element equal to 1/n i ; the trace of the n connectivity matrices (tr n ): where harary number (h): where d ij are the elements of the distance matrix, m ij are the elements of the m connectivity matrix, w j are the weight elements and nw is a switch to select (1) or not select (0) weights calculations; wiener index (w): -500 case number gutman topological index (s 6 ): where deg i are the elements of the degree matrix; schultz topological index (non-trivial part) (s): moreau-broto, autocorrelation of topological structure (ats n , n ¼ 1àpower limit), only with weights included: where dp ij n are the elements of the pair distance matrix when the distance is n; balaban distance connectivity index (j): where nodes+1 ¼ aa numbers/node number in the star graph+origin, p k d ik is the node distance degree; kier-hall connectivity indices ( n x): randic connectivity index ( 1 x): all these tis will be used to construct a natural/random classification model by statistical methods. general discriminant analysis (gda) (kowalski and wold, 1982; van waterbeemd, 1995) (statsoft.inc., 2002) has been chosen as the simplest and fastest method. in order to decide if a protein chain is classified as natural (if exists in the pdb database) or random, we added an extra dummy variable named nat/rnd (binary values of 0/1) and a cross-validation variable (cv). there are three often used crossvalidation methods to examine a predictor for its effectiveness in practical application: independent dataset test, subsampling test, and jackknife test (chou and zhang, 1995) . through a crystal-clear analysis, shen (2007, 2008) have shown that only the jackknife test has the least arbitrariness. therefore, the jackknife test has been increasingly used by investigators to examine the accuracy of various predictors (chen and li, 2007a, b; diao et al., 2007; ding et al., 2007; jiang et al., 2008; jin et al., 2008; li and li, 2008; lin, 2008; lin et al., 2008; niu et al., 2006 niu et al., , 2008 wang et al., 2008; xiao and chou, 2007; zhou et al., 2007; zhang et al., 2008) . in the actual work, the independent data test is used by splitting the data at random in a training series (train, 75%) used for model construction and a prediction one (val, 25%) for model validation (the cv column is filled by repeating 3 train and 1 val). all independent variables are standardized prior to model construction. using s2snet methodology, as defined previously we can attempt to develop a simple linear qsar, with the general formula where nat/rnd-score is the continue score value for the nat/rnd classification, t i ¼ tis described above, c 1 àc n ¼ tis coefficients, n is the number for the indices and c 0 is the independent term. gda models quality was determined by examining wilk's u statistics, fisher ratio (f), p-level (p), and canonical regression coefficient (r c ). we also inspected the percentage of good classification, cases/variables ratios, and number of variables to be explored in order to avoid over-fitting or chance correlation. the forward, backward and best subset model types are tested for the embedded, non-embedded and both data. eight variable selection methods were applied in order to find the best gda equation which is able to discriminate between natural and random chain proteins. eight models were constructed using embedded/non-embedded star graph tis obtained with s2snet application and forward, backward and best subset model types. the values obtained for the training/predicting accuracies are presented in table 1 . the forward stepwise selection variable method conjugated with the ne and e tis provides the best results for our data set with values of correctly classified compounds of 91.01%, 90.06% and 90.77% for the training, cross-validation and full sets, respectively, and using a minimum number of 12 parameters (eq. (15)). the embedded tis have the name of the non-embedded ones plus ''e'' as suffix: nat=rnd à score ¼ 0:1 þ 4:8sh0 þ 254:9h þ 1860:2w à 1931:0s þ 39:4j à 139:2x0 à 73:0x3 þ 146:7x4 à 159:3x5 à 6:6tr4e þ 7:1x2e, where n is the number of studied protein sequences (nat+rnd), r c is the canonical regression coefficient, u is the wilk's statistics, f is the fisher's statistics and p is the p-level (probability of error). the present r c value shows a high level of correlation between the input variables and the classification of proteins. wilk's u is used to measure the statistical significance of the discriminatory power of the model and has values from 1.0 (no discriminatory power) to 0.0 (perfect discriminatory power). the f value shows the statistical significance in the discrimination between groups, a measure of the extent to which a variable makes a unique contribution to a prediction of group membership. the values of the p-level of fisher's test for the gda is less than 0.05 and show that the hypothesis of group overlapping with a 5% error can be rejected (hua and sun, 2001 considered as excellent in the literature for lda-qsar models (garcia-garcia et al., 2004; marrero-ponce et al., 2004 . the parametrical assumptions such as normality, homoscedasticity (homogeneity of variances) and non-colinearity have the same importance in the application of multivariate statistic techniques to qspr (bisquerra alzina, 1989; stewart, 1998) as the correct specification of the mathematical form has. the validity and statistical significance of any model is conditioned by the above-mentioned factors. in our study, a simple linear mathematical form of the model has been chosen in the absence of prior information. figs. 3 and 4 show that the training cases against the residuals did not present any characteristic pattern (dillon and goldstein, 1984) . the protein nos. 632 and 864 are the only two cases not shown in fig. 4 because the corresponding raw residuals are clear distinct from the whole set, ca -7. they correspond to 1qwn, chain a (1014 aas) and 1jz8, chain a (1011 aas). one possible reason for the apparent different statistical behaviour could be the limitation of the model when the length of the chains is greater than 1000 amino acids. it is possible that the star net tis for large proteins become similar to the tis of the random proteins. a different and better threshold for the a priori classification probability can be estimated by means of the receiver operating characteristics (roc) curve (james and hanley, 1982) . as the fig. 5 clearly shows, one can see that the model is not a random, but a truly statistically significant classifier, since the area under the roc curve (for both training ¼ 0.98 and validation ¼ 0.96) is significantly higher than the area under the random classifier curve random ¼ 0.5 ¼ diagonal line (morales helguera et al., 2007) . the validity of the gda models depends on the normal distribution of the sample used as well as the homogeneity of their variances. thus, we carried out two significant tests for normality, chi-square and kolmogorov-smirnov tests, and we have found significant statistical differences (po0.01) on the respective values (chi-square, d). these results allow us to reject the hypothesis of normal distribution of the sample under study (fig. 6) (stewart, 1998) . the heteroscedasticity of a large set can be detected with the simple graphical method based on the examination of the residuals of the variable included in the model. fig. 7(a and b) shows that the nat/rnd gda model variables against the residuals plots do not present any pattern, which indicates that homoscedasticity assumption is fulfilled (stewart, 1998) . due to the robustness of the gda multivariate statistical techniques, the predictive ability and interference reached by using the proposed model should not be affected (see fig. 8 ). this study extends for the first time the classical tis to protein star network tis by proposing a model that can predict if a chain protein is natural or random. the results prove for the first time the excellent predictive ability (90.77%) of the simple and fast star network tis and gda statistics linear models in the case of natural/random protein model. this classification can help the study of the protein function by changing some fragments with random amino acid sequences or can detect the fake amino acid sequences or the errors in proteins. the s2snet application can be very useful to calculate the protein star network tis, which can be the base of a model for any other protein property. s2snet can also be used for mass spectroscopy, clinical proteomics and imaging or dna/rna structure analysis. novel 2d maps and coupling numbers for protein sequences. the first qsar study of polygalacturonases; isolation and prediction of a novel sequence from psidium guajava l steady-state kinetic studies with the non-nucleoside hiv-1 reverse transcriptase inhibitor 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nucleotides condensed representation of dna primary sequences on a four-dimensional representation of dna primary sequences characterization of dna primary sequences based on the average distances between bases on 3-d graphical representation of dna primary sequences and their numerical characterization on representation of proteins by starlike graphs python reference manual statistica (data analysis software system), version 6.0, /www handbook of molecular descriptors discriminant analysis for activity prediction pisces: a protein sequence culling server a new nucleotide-composition based fingerprint of sars-cov with visualization analysis predicting membrane protein types by the llda algorithm cellular automation as models of complexity digital coding of amino acids based on hydrophobic index using cellular automata to generate image representation for biological sequences an application of gene comparative image for predicting the effect on replication ratio by hbv virus gene missense mutation a probability cellular automaton model for hepatitis b viral infections using cellular automata images and pseudo amino acid composition to predict protein subcellular location graphic analysis of codon usage strategy in 1490 human proteins analysis of codon usage in 1562 e. coli protein coding sequences prediction protein structural classes with pseudo amino acid composition: approximate entropy and hydrophobicity pattern an extension of chou's graphical rules for deriving enzyme kinetic equations to system involving parallel reaction pathways using chou's amphiphilic pseudoamino acid composition and support vector machine for prediction of enzyme subfamily classes cristian r. munteanu thanks the fct (portugal) for support from grant sfrh/bpd/24997/2005. gonzá lez-díaz humberto acknowledges an isidro parga pondal research contract supported by xunta de galicia, university of santiago de compostela (spain). key: cord-262043-66qle52a authors: basit, abdul; ali, tanveer; rehman, shafiq ur title: truncated human angiotensin converting enzyme 2; a potential inhibitor of sars-cov-2 spike glycoprotein and potent covid-19 therapeutic agent date: 2020-05-20 journal: j biomol struct dyn doi: 10.1080/07391102.2020.1768150 sha: doc_id: 262043 cord_uid: 66qle52a the current pandemic of covid-19 caused by sars-cov-2 is continued to spread globally and no potential drug or vaccine against it is available. spike (s) glycoprotein is the structural protein of sars-cov-2 located on the envelope surface, involve in interaction with angiotensin converting enzyme 2 (ace2), a cell surface receptor, followed by entry into the host cell. thereby, blocking the s glycoprotein through potential inhibitor may interfere its interaction with ace2 and impede its entry into the host cell. here, we present a truncated version of human ace2 (tace2), comprising the n terminus region of the intact ace2 from amino acid position 21-119, involved in binding with receptor binding domain (rbd) of sars-cov-2. we analyzed the in-silico potential of tace2 to compete with intact ace2 for binding with rbd. the protein-protein docking and molecular dynamic simulation showed that tace2 has higher binding affinity for rbd and form more stabilized complex with rbd than the intact ace2. furthermore, prediction of tace2 soluble expression in e. coli makes it a suitable candidate to be targeted for covid-19 therapeutics. this is the first md simulation based findings to provide a high affinity protein inhibitor for sars-cov-2 s glycoprotein, an important target for drug designing against this unprecedented challenge. communicated by ramaswamy h. sarma the rapid spread of sars coronavirus 2 (sars-cov-2) demands an immediate public health emergency, and no fda approved treatment/vaccines are currently available. sars-cov-2 spike (s) protein (1267 amino acids) is essential for virus entry through binding with the host receptor angiotensin converting enzyme ii (ace2) and mediating virus-host membrane fusion (boopathi et al., 2020; sarma et al., 2020) . the s protein contains two functional domains (s1 and s2). the s1 (residues 14-685) domain performs the function of virion attachment with human ace2 receptor on epithelial membrane cell surface, followed by its internalization, hence initiating the infection (hasan et al., 2020) . this binding induces certain conformational changes in the s protein, which results the s2 (residue 686-1273) to mediate fusion with cellular membrane. the receptor-binding domain (rbd) of the sars-cov-2 s protein are highly conserved and directly involve in binding to human ace2 (yuan et al., 2020) . since, ace2 is not mutated/evolved to recognize s protein of sars-cov-2; therefore, using alternative of ace2 with more binding affinity for s protein than the wild type receptor, may inhibit entry of sars-cov-1& à2 into human cells. this strategy can play important role in devising therapeutics of sars-cov-2. several studies have proposed small compounds based inhibitors as therapeutic agents for covid-19 (aanouz et al., 2020; elmezayen et al., 2020; gupta et al., 2020; khan et al., 2020; wahedi et al., 2020) . the small compounds based drugs may not efficiently block the entire binding patch of s protein. on the other hand, the peptides based therapeutics can block the entire binding interface (rbd) of s protein (wan et al., 2020b) , as reported for hiv peptide based drug fuzeona (jenny-avital, 2003; w ojcik & berlicki, 2016) . there is growing interest in peptide based therapeutics for covid-19 treatment (pant et al., 2020) and approximately 140 peptide based drugs have been evaluated in clinical trials (fosgerau & hoffmann, 2015) . peptide based drugs have little side effects and little drug tolerance compared with chemical drugs (bruno et al., 2013) . in order to block the fusion of sars-cov-2 s protein with human cells, a recent study has reported a neck and transmembrane deficient ace2, called as soluble ace2 (sace2), that can block the entry of sars-cov-2 into the host cell (procko, 2020) , which is also found safe in healthy human subjects (haschke et al., 2013) and patients with lung disease (khan et al., 2017) . recombinant sace2 is under clinical trials for covid-19 treatment in guangdong province of china (clinicaltrials.gov #nct04287686). the study proposed that mutations in ace2 receptors interface may increase s/ace2 interaction. another study has proposed a 23 amino acid peptide, derived from ace2 (amino acid position 21-43), which can bind with sars-cov-2 s protein with a low nanomolar affinity, and can block the attachment of sars-cov-2 to human ace2 . since, the binding residues of ace2 involve in interaction with rbd are located at amino acid position 21-119 (wan et al., 2020a; yan et al., 2020) , therefore, we hypothesized that this fragment carrying all the binding residues will have better binding affinity for rbd and can hinder the interaction of sars-cov-2 with human ace2, hence blocking its entry into the epithelial cells. we designed a truncated version (tace2) of ace2 receptor covering the binding residues and performed protein-protein docking and molecular dynamic simulations to analyze its binding affinity for rbd and complex stability. the tace2 will compete with wild type human ace2 receptors for binding to sars-cov-2, as they will have more binding affinity for s protein. this will allow all sars-cov-2 viral particles to bind strongly with the tace2, blocking all its available binding sites for the host ace2 receptors, thus inhibiting its entry into the cell which will be eliminated through body defense mechanisms. we further determined the soluble expression for tace2 in e. coli, a suitable host for bulk production of tace2. the pdb structure of ace2 and rbd of sars-cov-2 s glycoprotein (pdb id: 6m17) was obtained from pdb database. in order to determine the variation in the sars-cov-2 s glycoprotein sequence reported from different regions of the globe, 61 s glycoprotein sequences of sars-cov-2 including reference sequence (nc_045512, reported from wuhan, china) were retrieved from ncbi. multiple sequence alignment of the sequences was performed through mega-x. the aligned sequences were then analyzed for amino acid variations. the pdb structures of ace2 and rbd were repaired for their missing loops and optimized for energy minimization and amino acid side chain clashes through foldx (schymkowitz et al., 2005) . side chains were optimized through foldx to remove vander waals' clashes by mutating residues with bad energy values into new rotamers with energy minimization (van durme et al., 2011) . the optimized three dimensional (3 d) structures of ace2 and rbd were used to design truncated ace2 and studying protein-protein interactions. based on protein-protein interactions between ace2 and rbd shown in ace2-rbd complex (pdb id 6m17), a truncated version of ace2 was produced by removing the c-terminus residues from amino acid position 116-768, leaving a truncated n-terminus fragment tace2, from 21-119 amino acid position. the first 20 residues of ace2 is the signal peptide (huang et al., 2003; turner & hooper, 2004) , therefore it was also removed. the structure of tace2 was produced through i-tasser, which build the model by assembling continues fragments of multiple threading templates, identified through replica exchange monte carlo (remc) simulations (yang et al., 2015) . in order to determine binding affinity of both intact and truncated ace2 with sars-cov-2 s glycoprotein, rigid body protein-protein docking tools; zdock (pierce et al., 2014) , cluspro (kozakov et al., 2017) , patchdock (schneidman-duhovny et al., 2005) and a flexible protein-protein docking tool, haddock (van zundert et al., 2016) were used. the energy function used by zdock is z score, which is a cumulative of pairwise shape complementarity function with desolvation and electrostatics. the zdock rank the top 10 predicted docking poses on the basis of z score (chen et al., 2003) . cluspro uses piper's scoring function, which contains terms of shape complementarity, electrostatics, and pairwise potentials applied on the top 1000 conformations produced and ranked on the basis of cluster size. patchdock uses patchdock score as the energy function which ranked the docked model based on desolvation energy, interface area size and geometric score (zhang et al., 1997) . haddock is a flexible docking method used for docking of protein-protein complexes. haddock drive the docking process by retrieving information from experimentally identified protein complex interfaces. the haddock scoring function consists on combination of various energies and buried surface area. the scoring of the models was performed according to the haddock score. all the generated docking poses of ace2 and spike protein were visualized through pymol (schrodinger, 2010) . based on the haddock score and the docking rmsd value, the docked complexes of ace2 and tace2 with rbd were analyzed for binding affinity dg (kcal mol à1 ) and stability using protein binding energy prediction (prodigy) server (xue et al., 2016) . the server predicts the binding affinity and stability on the basis of structural properties of the proteinprotein complexes. stability of the protein-protein complex is measured through dissociation constant k d (m). the run was performed at different temperatures ranging from 25 to 36˚c. the protein-protein docked complex with the minimum rmsd and higher binding affinity was considered for md simulation to further confirm stability of the complex. md simulation of the rbd domain in complex with intact ace2 and tace2 was performed through gromacs 5.0.4 (abraham et al., 2015) . simulation was performed by using charm 36.0 force field and tip3p cube box as water model. the protein complex in the cubic box was solvated with water molecules to provide an aqueous environment. the system was then neutralized with addition of 3 na ions followed by energy minimization for removal of conflict between the atoms. the system was then equilibrated through nvt and npt at constant temperature (300 k) and pressure (1 bar), respectively. langevin thermostat was applied to regulate temperature of the system. md simulation was then run for 20 ns. in order to determine post translation modifications (ptms) in ace2, the protein sequence was submitted to ptm-ssmp server, which combines the submitted sequence and site specific modification profile to predict ptm sites in mammalian protein (liu et al., 2018) . since, glycosylation is the most abundant and diverse posttranslational modification of proteins, therefore, we further determined the o-glycosylation sites in ace2 using netoglyc 4.0 server which specifically predict the galnac-type o-glycosylation site, unique to ser and thr (steentoft et al., 2013) . we further determined the n-glycosylation sites by using netnglyc-1.0 server using a threshold value of 0.5 (gupta et al., 2004) . in order to express the tace2 in e. coli, its soluble expressionat 37 c was determined through camsol intrinsic and camsol structurally corrected online solubility prediction tools (sormanni & vendruscolo, 2019) . camsol determines the solubility on the basis of amino acid sequence, while camsol structurally corrected tool determines the solubility profile on the basis of the structure, which accounts for amino acid distribution in the structure and their solvent exposure. both run was performed at ph 7.0. in both methods, the solubility profile scores higher than 1.0 denotes highly soluble regions, while scores lower than à1 indicates poor solubility in e. coli. in the current study, we have proposed a truncated version of ace2 that comprises the binding interface for receptor binding domain (rbd) of sars-cov-2 spike protein. recently, in-vitro binding assay have confirmed that rbd is mainly responsible for initial binding to ace2, which further mediate virus entry into the host cell (lan et al., 2020) . variation in the rbd sequence was analyzed in the sars-cov-2 genome reported from various region of the globe so far (shu et al., 2020) . the sequence alignment showed more than 99.99% homology for rbd domain, with only single variation r408i in the sars-cov-2 genome reported from india ( figure s1 ). the rest of the sars-cov-2 genome sequences submitted throughout the globe have identical rbd sequence, which indicate that the sars-cov-2 rbd is highly conserved globally. structural elucidation has also found the rbd domain as highly conserved (lan et al., 2020). in order to block the spike protein attachment to the cell, the ace2/rbd binding interface comprising residues from position 21-119 of ace2 was selected as truncated version of ace2.the structure of tace2 was built through i-taseer with c-score 1.22. the c-score value in range à5 to 2 shows correctness of the fold. the high c-score for tace2 suggest the highly likelihood of the structure. the tace2 fragment contains almost all binding residues involve in binding with rbd domain of sars-cov-2 (yan et al., 2020) , covering two complete helices (lan et al., 2020) . this suggests that rational design of a binder based on this interface with enhanced affinities to rbd may play vital role by blocking the sars-cov-2 spike protein interaction with ace2, thus inhibiting viral entry into the host cell. previously, peptides based strategies have been employed successfully to inhibit fusion of the sars-cov-1 s protein and membrane receptor (du et al., 2009) . another recent study has reported a 23 amino acid based peptide, a homologue of ace2 binding interface, which successfully bind with s protein with low nanomolar affinity . since, the binding residues for ace2 are located at distant location on rbd, thus providing a larger protein binding site, which is difficult for a small size therapeutic peptide to cover the entire binding sites on rbd. however, our proposed tace2 fragment carrying almost all the binding residues that can block the attachment of rbd with the intact ace2. rbd was docked with intact and truncated ace2 through haddock, a flexible protein-protein docking tool. the method allows the side-chains and backbone atoms of both the protein and receptor flexible during docking run . haddock scoring function (haddock score) is a linear combination of non-bonded intermolecular van der waals (vws), coulomb electrostatics energies and empirically derived desolvation energy term (vangone et al., 2017) .the haddock-score of ace2 and tace2 was à111 and à126.6, respectively, (the more negative the better). similarly the vws and electrostatic energy of tace2-rbd complex was also greater than the ace2-rbd complex, which shows higher binding affinity of tace2 for rbd than the intact ace2 (table 1 ). the rmsd value of ace2 and tace2 in complex with rbd were 0.7 and 0.8, respectively, showing the high likelihood of the docked complexes with native-one (vangone et al., 2017) . in order to further confirm these docking scores, rigid docking was also performed through patchdock, z-dock and cluspro protein-protein docking tools. the docking results obtained for ace2 was compared with tace2 in term of energy functions of each docking tool (table 2 ). all the three docking scores are higher for tace2 than that of the intact ace2, indicating high affinity of tace2 for rbd. our docking results showed that seven residues of ace2 glu23, thr27, asp30, glu35, tyr 83, asn 330 and lys 353 of ace2 interact with rbd residues lys417, lys458, asn487, tyr 489, gln493, tyr495, gly496, thr500 and gly502, respectively, which is almost similar to the binding residues profile of ace2 interface reported previously (yan et al., 2020) , with additional thr27 and glu35 reported by our docking results (figure 1(a, b) ). however, the tace2 form a different binding residues network than the intact ace2. our docking results showed that ser23, asn31, tyr30, glu36, gln40, gln76 and arg95 of tace2 are involved in binding with rbd ( figure 1(c, d) ). this seems that the truncation has produced the conformational changes in the tace2-rbd complex which results in exposure of buried binding residues , thus facilitate higher binding of tace2 to the rbd as compared to the native ace2, which are in agreement with previously reported peptides inhibiting viral attachment with the host cell (koehler et al., 2013) . since, docking methods are not reliable for predicting binding affinity between protein-protein complexes, due to their simple scoring functions (ram ırez & caballero, 2016). as binding affinity of protein-protein complex also depends on dissociation constants (k d ), ph and temperature (kastritis & bonvin, 2010) , while these parameters are not included in the benchmark of docking scoring functions. therefore, we determined the binding affinity of ace2 variants for rbd through prodigy server, which determine the binding affinity based on structural properties of the protein-protein complexes (vangone & bonvin, 2015) . the ace2 and tace2 complexes showed à10.7 and à12.7dg (kcal mol à1 ) binding affinity for rbd, respectively, at temperatures ranges from 20 to 37 c, showing higher binding affinity of tace2 for rbd than the intact ace2 (table 3) . similarly, the dissociation constant k d value of tace2-rbd complex was more than three-fold lesser than the intact ace2-rbd complex, showing that tace2 is more tightly bound to rbd. the smaller k d value indicates high stability and strong binding affinity between protein-protein complex (johnson et al., 2007) . the ace2 variants showed a significant decline in k d value when temperature was increased from 20 c to 36 c, leading to a lower k d (9.8 â 10 à10 m) for tace2 (higher affinity) than that of intact ace2 (2.6 â 10 à8 m) at 36 c.this k d value of tace2 is lesser than the previously reported k d value (47 nm) of sbp1 (an ace2 derived peptide of 23 amino acid) to rbd . the optimum stability of the complexes was found at 36˚c (table 3 ). the dramatic changes of binding kinetics might be caused by reduced stability of ace2 complex below optimum temperature 36˚c (zhao et al., 2018) . in order to determine the structural stability and dynamic behavior of intact ace2-rbd and tace2-rbd complexes, we performed md simulation for 20 ns using gromacs 5.0.4. the docking pose of each complex obtained from haddock with lowest energy was selected for md simulation run. to investigate structural stability of the complex, rmsd plot of the complex backbone was produced. a uniform rmsd plot signifying structural stability of tace2-rbd complex. the rmsd value for tace2 complex was 0.2-0.25 nm, while intact ace2 showed 0.25-3.0 nm rmsd (figure 2 ). the rmsd value of tace2-rbd complex is lesser than sbp1-rbd complex, reported previously, which is almost 0.8 nm , showing higher stability of tace2-rbd complex. root mean square fluctuation (rmsf) was determined to evaluate the residues flexibility of both ace2 and rbd in the docked complexes. the high rmsf values indicate the mobility of residue side chains in relation to their average position (kumar et al., 2014) .the rmsf plot shows the residues of rbd in tace2 complex are stable with a few peaks with rmsf more than 0.2 nm (figure 3(b) ), while rbd of ace2 complex shows many residues with rmsf above 0.35 nm (figure 3(d) ).the residues of tace2 at position 24, 30, 40, 76 and 95 showed reduction in rmsf value due to creating binding interactions with rbd (figure 3(a) ). the residues involved in binding with other protein, present lower rmsf values, reveal the most stable regions of the complex (ardalan et al., 2018) .similarly, the residues window of 470-480 of rbd showed higher fluctuation to 0.25 nm, while decrease in fluctuation at the binding residues positions (figure 3(b) ). the most violent fluctuation in the intact ace2 was observed at c-terminus, which was above 0.7 nm (figure 3(c) ). the overall rmsf values of both tace2 and rbd are below 0.2 nm, which indicate that tace2 complex with rbd is stable, which are in agreement with a previously reported rmsf value 0.4 nm, showing complex stability (maqsood et al., 2020) . the overall trajectories obtained after every 100 ps during a 20 ns md simulation run, very small backbone deviation for both the intact ace2 and tace2 complex was observed (figure 4) . however, the amino acid region 470-489 of rbd has shown backbone fluctuation highlighted as yellow (figure 4(c) ), which we suggest the region of binding site for ace2. previously, the amino acid region of the sars-cov-2 spike protein (480-488) was also reported as binding region for ace2 (ibrahim et al., 2020) . à185.2 ã the haddock score is defined as: 1.0 evdw þ 0.2 eelec þ 1.0 edesol þ 0.1 eair. ãã the z-score produced by haddock indicates standard deviations from the average cluster (the more negative the better). radius of gyration (r g) of both ace2 complexes describes overall spread of molecule during a 20 ns md run. a low rg value indicates better structural integrity and folding behavior (erva et al., 2016) . a slight increase in rg value of the intact ace2-rbd complex was observed during first 5 ns of the run, then after no further drifts till end ( figure 5 , red line), however, the tace2-rbd complex was found stable throughout the md run ( figure 5 , violet line), which indicates its structural integrity. overall, the md simulation results confirm that tace2 form a more stabilized complex with rbd and suggest its inhibitory features for sars-cov-2 spike glycoprotein. post-translational modifications (ptms) play important role in protein-protein interactions (su et al., 2017) . since, experimental methods are high-cost and time-consuming, therefore, it is necessary to theoretically predict ptms site on protein to be expressed heterologously. ptm-ssmp, which predict ptms sites on human protein based on local sequence and site specific modification profile (liu et al., 2018) . ace2 analysis through ptm-ssmp server predicted ubiquitination at position 74 and 304, phosphorylation at 606 and o-glycosylation at 720 residue position. the ptm site at 74 is important for protein degradation and have no role in ppis (lecker et al., 2006) . in transmembrane proteins, the extracellular domains may only be n-glycosylated (gupta et al., 2004) . however, there was no n-glycosylation and oglycosylation site predicted for tace2. these results conclude that there is no ptms site predicted on tace2, which is important for protein-protein interactions. interestingly, an experimental study reported that the lack of glycosylation do not affect the binding of sars-cov-1 rbd to human ace2 (chakraborti et al., 2005) , which strongly support our designed tace2 fragment, if expressed in e. coli may bind efficiently with rbd of sars-cov-2 s glycoprotein. figure 2 . rmsd plot of the ace2-rbd (red) and tace2-rbd complex (violet) backbone atoms. the tace2 complex showing less rmsd value than the intact ace2, indicating its higher complex stability than the intact ace2. since, there was no ptm site predicted in tace2, therefore, e. coli would be an ideal host for its large scale expression. e. coli is the easiest, quickest, and cheapest expression host with a fully known genome, most widely used for hetrologous expression of recombinant protein (basit et al., 2019) . since, ace2 is eukaryotic protein; therefore, its expression in its native form in e. coli will be uncertain, as most of the eukaryotic protein showed insoluble expression in e. coli, which need to be refolded invitro , which is costly and time consuming. that's why, the protein that express in soluble form in e. coli are referred as "low hanging fruit", as their bulk production is cost effective and easy to recover (maqsood et al., 2020) . both sequence and structure based solubility prediction tool using camsol software predicted expression of intact ace2 in a completely insoluble form in e. coli with intrinsic solubility score à1.027 and complete soluble expression of tace2 with a solubility score of 1.23. the software generate solubility profile with one score per residue, where regions with scores higher than 1 denote highly soluble regions, while scores lower than à1 showing poorly soluble ones (figure 6(a, b) ). these results propose e. coli as a suitable host for soluble expression of tace2 using pet28a (þ) as an expression vector, which favors single step purification. structure-based rational design of inhibitory protein with enhanced affinities to the sars-cov-2 spike glycoprotein may facilitate development of potential therapeutics. in this study, we have designed a truncated version of human angiotensin converting enzyme 2 as a potential inhibitor of spike glycoprotein. the truncated protein tace2 was extensively studied through protein-protein docking and md simulation for binding to rbd of sars-cov-2 spike glycoprotein. we found that tace2 can bind to rbd with a higher binding affinity and form more stabilized complex than the intact ace2. in addition, the tace2 sequence predicted soluble expression in e. coli, which makes it an easy target for rapid production at large scale for sars-cov-2 prevention. we believe that this study narrow down the region of interaction between sars-cov-2 s glycoprotein and human ace2 and paves the way to further enhance the binding affinity between tace2 and sars-cov-2 s glycoprotein through rational design. this will open a new path to covid-19 treatment. the authors declare no conflict of interest. moroccan medicinal plants as inhibitors of covid-19: computational investigations gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers novel mutant of escherichia coli asparaginase ii to reduction of the glutaminase activity in treatment of acute lymphocytic leukemia by molecular dynamics simulations and 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web server: user-friendly integrative modeling of biomolecular complexes contacts-based prediction of binding affinity in protein-protein complexes. elife, 4, e07454 sense and simplicity in haddock scoring: lessons from casp-capri round 1 stilbene-based natural compounds as promising drug candidates against covid-19 receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus peptide-based inhibitors of protein-protein interactions prodigy: a web server for predicting the binding affinity of protein-protein complexes structural basis for the recognition of sars-cov-2 by full-length human ace2 the i-tasser suite: protein structure and function prediction a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov determination of atomic desolvation energies from the structures of crystallized proteins the first-in-class peptide binder to the sars-cov-2 spike protein. biorxiv, 1-15 impact of temperature on heparin and protein interactions key: cord-147565-mtdhdkc1 authors: harmalkar, ameya; gray, jeffrey j. title: advances to tackle backbone flexibility in protein docking date: 2020-10-15 journal: nan doi: nan sha: doc_id: 147565 cord_uid: mtdhdkc1 computational docking methods can provide structural models of protein-protein complexes, but protein backbone flexibility upon association often thwarts accurate predictions. in recent blind challenges, medium or high accuracy models were submitted in less than 20% of the"difficult"targets (with significant backbone change or uncertainty). here, we describe recent developments in protein-protein docking and highlight advances that tackle backbone flexibility. in molecular dynamics and monte carlo approaches, enhanced sampling techniques have reduced time-scale limitations. internal coordinate formulations can now capture realistic motions of monomers and complexes using harmonic dynamics. and machine learning approaches adaptively guide docking trajectories or generate novel binding site predictions from deep neural networks trained on protein interfaces. these tools poise the field to break through the longstanding challenge of correctly predicting complex structures with significant conformational change. protein-protein interactions are involved in nearly all of the biological processes in human health and disease. understanding the dynamics of binding and the structure of protein complexes at the molecular level can be instrumental in delineating biological mechanisms and developing intervention strategies. computational protein-protein docking provides a route to predict the three-dimensional structures of protein assemblies or complexes from known structures of individual monomeric proteins [1] . docking methods are tested in the blind prediction challenge known as the critical assessment of prediction of interactions (capri) [2] , which in recent rounds pushed the field by including a wide array of target types such as transport proteins, higher order assemblies and host-virus interactions [3, 4] . out of the 28 protein-protein targets evaluated in capri over the past four years [4, 5] , predictors achieved high quality structures for 11 "easy" targets, defined as those with little backbone motion (unbound to figure 1 : performance of protein docking approaches on blind targets in capri rounds 38-46. [4, 5] distribution of dockq scores for the best model submitted by each predictor group (points) for each individual target (x-axis). dockq measures a combination of intermolecular residue-residue contacts, interface rmsd, and ligand rmsd on a scale of 0 (incorrect) to 1 (matching the experimental structure) [5] . targets are labelled by their capri target number and, when needed, interface number (after the decimal). the targets are classified into rigid (easy) targets (high-homology monomer templates and under 1.2 a unbound-bound backbone motion, and flexible targets (poor template availability and/or over 1.2å rmsd bu ). dockq scores are color-coded by capri model quality ranking: blue, high; green, medium; yellow, acceptable; gray, incorrect. data graciously provided by marc lensink [4, 5] . bound c α root mean square deviation (rmsd bu ) of less than 1.2å [6, 7] ; figure 1 ). the remaining 17 targets were categorized as "difficult" (rmsd bu over 2.2å and/or poor monomer template availability). for these targets, predictors only achieved acceptable quality in 8 of 17 targets (47%) and high quality in only 2 (12%) [4, 5] . thus, the intrinsic flexibility of biomolecules still confounds the protein docking community at large. in this review, we focus on the central docking challenge of capturing larger binding-induced conformational changes. we summarize progress by recent algorithms and frameworks, additionally augmented by growth in databases and computational power (cpu-and gpu-based). these new methods have achieved greater accuracy on more challenging targets and additionally yielded insight into binding mechanisms. we first present progress in binding site identification and then docking methods including molecular dynamics (md) and monte carlo (mc) approaches, normal modes, and machine learning. together, these techniques have helped better explore broader regions of conformational space and more thoroughly evaluate the energy landscape to improve protein-protein docking. to reduce the complexity of the immense conformation space of flexible proteins, coarse-grained models are frequently used to reduce the degrees of freedom ( figure 2 ). in the extreme, global docking approaches typically first treat protein partners as rigid bodies by restricting to six degrees of freedom (three rotational and three translational). a prime method to exhaustively sample the global 6d space is enumerating and scoring different rigid-body orientations on a dense grid. approaches such as clus-pro [15, 16] , zdock [17, 18] , piper [19] and hexserver [20] [21] . relative to traditional fft-based docking, fmft accelerates calculations ten-fold [13]*. another shape-based approach is geometric hashing, which indexes point sets or curves to match geometric features under arbitrary transformations like translations, rotations or even scaling [22] . local 3d zernike descriptor-based docking (lzerd), one of the top methods in capri, projects 3d surfaces onto spheres to efficiently capture complementarity of protein surfaces [23] . some rigid-body approaches exploit data from chemical cross-linking experiments [24] or small-angle x-ray scattering (saxs) [25] to further improve discrimination of generated structures. these approaches provide fast, global exploration of the energy landscape, and in recent capri rounds [4, 5] , many predictors incorporated these approaches as the first step to identify putative binding patches, and they supplement with other refinement tools to capture backbone flexibility. molecular dynamics (md) is one strategy that is often used after grid-search or template-based approaches for refinement ( figure 3 ) [26, 27] . unbiased, all-atom md simulations can provide a highresolution, time-resolved microscopic model of protein-protein interactions. md calculates newtonian trajectories using physics-based energy functions to simulate protein association and dissociation events. md use for protein docking has been limited because non-native local minima trap proteins, and dissociation is too slow [28] . over the past decade, two new modifications to capture conformational changes are steered molecular dynamics (smd) [29] , which utilizes external force constraints, and markov sampling, which breaks a long md simulation into multiple short trajectories [30] . to accelerate dissociation of protein partners at sub-optimal binding regions, ostermeir et al. developed a hamiltonian replica exchange md protocol (h-remd) for protein docking [31] *. in h-remd, biasing potentials are based on the shortest distance between protein partner atoms (defined as "ambiguity restraints"). as the biasing potential and associated ambiguity restraints vary across replicas, associated protein partners in one replica are forced to dissociate in another. pan et al. simulated long timescales in a global search space for a benchmark set of five targets on the special purpose machine anton [32, 33] . their "tempered binding" protocol updates energy function parameters throughout the simulation: a soft-core van der waals intermolecular potential is scaled so that long-lived states are dissociated more frequently, improving the in contrast to md approaches that target flexibility with newtonian dynamics; monte carlo (mc) methods sample by random moves often followed by minimization (mcm) [40, 41] . mc allows a wide variety of conformational move types to sample diverse conformations. mc algorithms have emulated the kinetic binding models, namely key-lock, conformer selection (cs) and induced-fit (if) mechanisms [42, 43, 44] . the cs model chooses protein backbones from a pre-generated ensemble, thus this approach has the advantage of docking one partner's conformations at a time. however, cs docking can fail if the ensemble is devoid of native-like backbone conformations [45] . docked from an ensemble of 10 structures). to diversify backbone conformations, the protocol generates monomer structures by three methods: (1) normal modes [46] (2) backrub motions [47] and (3) all-atom backbone refinement [48] . further, to discriminate between near-native and non-native structures, they developed a more accurate coarse-grained energy function with 6-dimensional residue-pair data obtained since intrinsic fluctuations in proteins contribute to conformational change, some docking approaches utilize harmonic dynamics to capture protein backbone motions [49, 50, 51] . normal modes of vibration represent internal motions of a protein based on a hookean potential between close residues. normal mode analysis (nma) is incorporated in docking approaches such as attract [52] , fiberdock [53] , swarmdock [54] and eigenhex [55] . to mimic induced-fit, schindler et al. developed iattract [56] by moving interface residues in cartesian coordinate space subject to nma-generated harmonic potentials. iattract served as a refinement stage and improved the fraction of native contacts predicted by 70%. for targets with unbound to bound interface rmsd over 4å, iattract can achieve acceptable quality models [56] . population-based methods such as particle swarm optimization (pso) have also [57, 4] . extending the swarm intelligence methods, the lightdock algorithm uses a "glowworm" swarm optimization to sample different backbone conformations in local regions of the protein surface with an anisotropic network model [58] . lightdock additionally uses multiscale modeling to combine all-atom and coarse grained scoring functions. while normal modes have typically been used on individual protein partners prior to docking, oliwa and shen introduced the complex nma in docking to also sample molecular complex fluctuations [60] . by calculating modes of an encounter complex, this approach focuses on the binding region as it reduces the dimensionality of the search space [61] . one of the problems of nma is that higher frequency modes often distort protein bonds. to overcome this limitation, frezza and lavery developed the internal coordinate nma (inma) approach to move in the torsion angle space, that is, with fixed bond lengths and angles ( figure 4 ) [62] . with a reduced protein model in an internal coordinate space, they captured larger conformational changes from eigenvectors of low-frequency modes [59] **. inma can generate structures within 3å of the bound state when starting from the unbound for 39% of single-domain and 45% of multi-domain proteins in their benchmark. although protein folding has been one prime focus of deep learning methods in biology (e.g., alphafold [63, 64] and raptorx [65] ), in recent years, a few studies have explicitly addressed challenges relevant to protein docking [66] . protein binding sites can be thought of as an information-rich molecular space that can be mined for elucidating protein interactions [67, 68, 69] . one approach is to use this information to create score functions for use with traditional docking approaches. for example, geng et al. used graph representations to train a support vector machine (svm) on native and non-native protein complex structures to develop a scoring potential (graphrank) to rank docked poses [70] . and iscore, composed of the graphrank and haddock [71] scores, achieved top performance in capri scoring rounds (medium or high quality structures for nine out of 13 targets). other teams have used deep learning techniques to identify protein interfaces by extrapolating image recognition tools to protein structures. raptorx-complexcontact [69] uses a deep residual neural network trained on single-chain proteins to predict contacts between binding partners, achieving the top contact prediction scores in casp [72] . another approach is to characterize interaction environments. townshend et al. created "voxels," i.e., volumetric pixels with local atomic information for every protein surface residue, and with this 3d representation, they trained a deep 3d convolutional neural network (sasnet) on a curated database of bound protein complex structures [73] . pittala et al. employed graph convolutions with the nodes representing the amino acid residues and edges connecting residues with a c β − c β distance under 10å [74] . they placed geometric and chemical features on both nodes and edges and used a graph neural network to predict epitopes and paratopes in antigen-antibody interfaces. in a unique approach by gainza et al., a geometric deep learning model (masif) used molecular interaction "fingerprints" calculated using geometric and chemical features of protein surfaces [14]** (2). their deep network was composed from geodesic convolutional layers, and they used it to predict binding sites, evaluate alternate docked interfaces, and assess likelihood of a given protein-protein interaction. relative to conventional rigid docking methods on protein targets, masif-search can perform ultra-fast scanning to identify true 'binder' with similar accuracy but significantly faster (4 cpu-minutes vs. 45 hours for patchdock and 93 days for zdock to evaluate a benchmark of 100 bound protein complexes). in a study to explore how neural networks might be used to generate structures with considerable backbone motion, degiacomi trained an autoencoder with conformations from md simulations, compressing the protein motion into a low-dimensional latent space [75] *. by training with simulations of both closed (bound) and apo conformations of a target protein, the autoencoder generated an intermediate closed-apo conformation at 0.8å rmsd [75] from the native state. however, when the autoencoder was trained only with open conformations, the generator could only create structures far from the closed state (over 4.2å), limiting the utility of this approach for blind docking. in an approach suitable for blind cases, cao and shen developed a bayesian active learning (bal) model to quantify uncertainty in protein 8 structure quality, and then they extended their model to flexible protein docking [76] *. the bayesian framework determines the posterior probability as it samples backbone conformations [60] . flexibility is captured with low-frequency complex-nma modes, and in principle it can be extended to higher frequencies that capture loop and hinge motions. compared to zdock [17] and pso, bal improves the interface rmsd of the near-native predictions by 0.5å. in conjunction with experimental data, docking has advanced a range of biological and health applications (e.g., alzheimer's disease [77] , celiac disease [78] , sars-cov-2 [79] , influenza [80] , cancer [81] , and heart disease [82] , to name just a few). over the past few years, docking success rates have improved on "difficult" blind prediction targets, but rates need to be higher for docking to be a reliable stand-alone tool in all cases. clearly, a diverse and impressive array of tools has steadily advanced toward reliably capturing large conformational changes in protein docking. docking will be even more impactful when the field finally overcomes this challenge. recent progress and future directions in protein-protein docking capri: a critical assessment of predicted interactions the challenge of modeling protein assemblies: the casp12-capri experiment blind prediction of homo-and hetero-protein complexes: the casp13-capri experiment modeling protein-protein, protein-peptide, and protein-oligosaccharide complexes: capri updates to the integrated protein-protein interaction benchmarks: docking benchmark version 5 and affinity benchmark version 2 dockground: a comprehensive data resource for modeling of protein complexes cluspro: a fully automated algorithm for protein-protein docking the cluspro web server for protein-protein docking zdock: an initial-stage protein-docking algorithm interactive docking prediction of protein-protein complexes and symmetric multimers piper: an fft-based protein docking program with pairwise potentials hexserver: an fftbased protein docking server powered by graphics processors efficient search for the possible mutual arrangements of two rigid bodies with the use of the generalized five-dimensional fourier transform prediction of protein-protein interactions by docking methods protein-protein docking using region-based 3d zernike descriptors integrating cross-linking experiments with ab initio protein-protein docking ultra-fast filtering using small-angle x-ray scattering data in protein docking modeling of protein complexes in capri round 37 using template-based approach combined with model selection performance and enhancement of the lzerd protein assembly pipeline in capri 38-46 atomic-level characterization of the structural dynamics of proteins implicit flexibility in protein docking: crossdocking and local refinement complete protein-protein association kinetics in atomic detail revealed by molecular dynamics simulations and markov modelling accelerated flexible protein-ligand docking using hamiltonian replica exchange with a repulsive biasing potential hamiltonian replica exchange (h-remd) modifies parts of the force field across different replicas. in this paper, a repulsive potential between receptor and ligand surface residues promotes transient dissociation on switching replicas, accelerating exploration of the protein surface to identify possible binding sites raising the bar for performance and programmability in a special-purpose molecular dynamics supercomputer atomic-level characterization of protein-protein association with long timescale md simulations using a "tempered binding" protocol that scales a soft-core energy across replicas to promote dissociation of longlived states, this work found that protein binding occurs through repeated association-dissociation events rather than prolonged in-contact exploration prediction of protein-protein complexes using replica exchange with repulsive scaling using a novel replica exchange scheme with variable van der waals radii for interface residue atoms, the rs-remd approach promotes dissociation in some replicas, which improves sampling for both global searches and refinement replica exchange with solute tempering: a method for sampling biological systems in explicit water replica exchange improves sampling in low-resolution docking stage of rosettadock umbrella sampling funnel metadynamics as accurate binding free-energy method holo-like and druggable protein conformations from enhanced sampling of binding pocket volume and shape high-resolution protein-protein docking sampling and scoring: a marriage made in heaven protein-protein docking with backbone flexibility conformer selection and induced fit in flexible backbone protein-protein docking using computational and nmr ensembles monte carlo replica-exchange based ensemble docking of protein conformations pushing the backbone in protein-protein docking anisotropy of fluctuation dynamics of proteins with an elastic network model backrub-like backbone simulation recapitulates natural protein conformational variability and improves mutant side-chain prediction alternate states of proteins revealed by detailed energy landscape mapping harmonic modes as variables to approximately account for receptor flexibility in ligand-receptor docking simulations: application to dna minor groove ligand complex flexibility and conformational entropy in protein-protein binding accounting for conformational changes during protein-protein docking flexible docking and refinement with a coarse-grained protein model using attract fiberdock: flexible induced-fit backbone refinement in molecular docking swarmdock and the use of normal modes in protein-protein docking flexible protein docking refinement using pose-dependent normal mode analysis iattract: simultaneous global and local interface optimization for protein-protein docking refinement enhanced sampling of protein conformational states for dynamic cross-docking within the protein-protein docking server swarmdock * a hybrid conformational-selection/induced-fit approach for dynamic crossdocking in swarmdock, a particle swarm optimization algorithm. ensembles are pre-generated with nma and undergo cross-docking while sampling alternate protein conformations using low frequency normal modes lightdock: a new multi-scale approach to protein-protein docking internal coordinate normal mode analysis: a strategy to predict protein conformational transitions this work employs nma in the internal coordinate space with a reduced protein model to capture large conformational changes of proteins with a faster compute time and no distortion of protein bonds cnma: a framework of encounter complex-based normal mode analysis to model conformational changes in protein interactions predicting protein conformational changes for unbound and homology docking: learning from intrinsic and induced flexibility internal normal mode analysis (inma) applied to protein conformational flexibility protein structure prediction using multiple deep neural networks in the 13th critical assessment of protein structure prediction (casp13) improved protein structure prediction using potentials from deep learning accurate de novo prediction of protein contact map by ultra-deep learning model deep learning in protein structural modeling and design (2020) recognition of functional sites in protein structures protein interface prediction using graph convolutional networks complexcontact: a web server for interprotein contact prediction using deep learning iscore: a novel graph kernel-based function for scoring protein-protein docking models haddock: a protein-protein docking approach based on biochemical or biophysical information critical assessment of methods of protein structure prediction (casp)-round xiii end-to-end learning on 3d protein structure for interface prediction learning context-aware structural representations to predict antigen and antibody binding interfaces coupling molecular dynamics and deep learning to mine protein conformational space this paper describes a unique method of generating plausible motions of a protein using a generative neural network (autoencoder) bayesian active learning for optimization and uncertainty quantification in protein docking * with a framework to quantify uncertainty in docked models, the bayesian approach uses a posterior distribution to guide sampling to likely low-energy conformations from monomer to fibril: abeta-amyloid binding to aducanumab antibody studied by molecular dynamics simulation plasma cells are the most abundant gluten peptide mhc-expressing cells in inflamed intestinal tissues from patients with celiac disease dna aptamers block the receptor binding domain at the spike protein of sars-cov-2, chemrxiv characterizing receptor flexibility to predict mutations that lead to human adaptation of influenza hemagglutinin targeting the corest complex with dual histone deacetylase and demethylase inhibitors protein docking and steered molecular dynamics suggest alternative phospholamban-binding sites on the serca calcium transporter this work was supported by the national institutes of health through grant r01-gm078221. we thank marc lensink for generously providing us with data from capri and sai pooja mahajan and sudhanshu shanker for helpful comments on the manuscript. dr. jeffrey j. gray is an unpaid board member of the rosetta commons. under institutional participation agreements between the university of washington, acting on behalf of the rosetta commons, johns hopkins university may be entitled to a portion of revenue received on licensing rosetta software including applications mentioned in this review. as a member of the scientific advisory board, dr. gray has a financial interest in cyrus biotechnology. cyrus biotechnology distributes the rosetta software, which may include methods mentioned in this review. key: cord-275023-0z219rcy authors: cerofolini, linda; fragai, marco; luchinat, claudio; ravera, enrico title: orientation of immobilized antigens on common surfaces by a simple computational model: exposition of sars-cov-2 spike protein rbd epitopes date: 2020-07-29 journal: biophys chem doi: 10.1016/j.bpc.2020.106441 sha: doc_id: 275023 cord_uid: 0z219rcy the possibility of immobilizing a protein with antigenic properties on a solid support offers significant possibilities in the development of immunosensors and vaccine formulations. for both applications, the orientation of the antigen should ensure ready accessibility of the antibodies to the epitope. however, an experimental assessment of the orientational preferences necessarily proceeds through the preparation/isolation of the antigen, the immobilization on different surfaces and one or more biophysical characterization steps. to predict a priori whether favorable orientations can be achieved or not would allow one to select the most promising experimental routes, partly mitigating the time cost towards the final product. in this manuscript, we apply a simple computational model, based on united-residue modelling, to the prediction of the orientation of the receptor binding domain of the sars-cov-2 spike protein on surfaces commonly used in lateral-flow devices. these calculations can account for the experimental observation that direct immobilization on gold gives sufficient exposure of the epitope to obtain a response in immunochemical assays. the activity and reactivity of an immobilized protein strongly depend on its orientation with respect to the surface of the support in -or on -which it is immobilized. this holds true for enzymes, as well as for antibodies and antigens. therefore, the possibility to control and manipulate the exposition of the relevant residues and protein surfaces plays an important role in the rational design of devices based on immobilized proteins. among these devices, immunosensors represent an expanding space for research and market opportunities. while the path to reach a technologically relevant product must rely upon a strong experimental characterization, [1, 2] possibly relying upon atomic-level methodologies, [3] [4] [5] [6] [7] [8] [9] the preparation/isolation of the protein of interest, its immobilization, and the characterization of the resulting composite are complex and time-consuming, therefore it is also true that guidelines for achieving optimal orientations could improve the efficiency of the r&d connected to protein immobilization. [10, 11] however, simplified simulation models that would allow for a rapid prediction of the most plausible orientations are not particularly common. in this manuscript we apply a very simple method based on a unitedresidue modelling of protein-surface interactions, to specifically address the problem of determining the orientation of the sars-cov-2 spike protein receptor binding domain (rbd) on a few prototypical surfaces for biomedical use. united residue modelling of protein-surface interactions is a rather effective model to screen the poses of protein molecules with respect to surfaces. [12, 13] the method we apply is based on the works by jiang, zhou and del monte-martinez, [10, [12] [13] [14] [15] [16] [17] [18] and encompasses van der waals and electrostatic interactions, as well as covalent immobilization. the choice of the target protein is motivated by the recent emergence of a new infectious disease (covid-19) caused by a coronavirus (sars-cov-2). [19] this infectious disease has spread significantly throughout the world, counting 13.841.890 infected people and a death toll of 590.845 as of july 2020. [20] models suggests that it will remain circulating and active for several months, [21, 22] and there is a marked possibility that reinfection is possible, [23, 24] thus increasing the time of the circulation of the virus. this pandemic outbreak has had a major impact on world economics, with a very long outlook. [25] a capillary control of the diffusion of the infection has proven crucial, [26] and serological tests are expected to have a key role in mass screening. [27, 28] methods j o u r n a l p r e -p r o o f the structures of the proteins were downloaded from the protein databank (pdb), [29] the pka values of reactive groups were calculated using propka, [30, 31] and the interfaces were calculated using the pdbe pisa server. [32] the non-bonded interaction of a residue of type i is represented with a lennard-jones (lj) potential: [12, 13] where r is the nearest distance between the residue and the surface, is the energy at the minimum position, is the equivalent van der waals radius of each residue and is a size parameter taken from the literature (see tables s2-s7, parameters are taken from [14, 18, [33] [34] [35] [36] 16, 37] , as indicated in the table captions). the electrostatic interaction is represented through the gouy-chapman potential [12, 13, 38] ( ) where r is the nearest distance between the i-th residue with charge and the surface, is the surface charge density, is the inverse debye length calculated from the ionic strength i as √ , and the relative permittivity of the medium is assumed to be distance-dependent ( ). [13, 38] a 1:1 buffer salt concentration of 0.15 mol dm -3 is assumed. for silica, the surface charge density is estimated to be -0.3 c m -2 . [37] for self-assembled charged monolayers (sam), the charge density is set to +0.02 c m -2 for the amino-capped monolayer (sam-nh 2 ) and to -0.02 c m -2 for the carboxyl-capped monolayer (sam-co 2 h). [14, 18] the formation of a covalent bond is treated with the following potential: where is the bond energy and is set to 600 kj mol -1 for imino bonds [39] and 100 kj mol -1 for gold-thiol bonds, [40] [41] [42] regardless of the starting oxidation state of the thiol. [43] the desolvation energy is already accounted for in the vdw term. lj parameters for epoxide-glyoxyl functionalization is assumed to be equal to sam-co 2 h, whereas for gold the parameters have been adapted from reference [36] . the sampling of the relative protein-surface orientations is performed by rotating a plane around the center of mass of the protein. the plane is initially parallel to the z=0 plane. only two rotations are necessary, as all the rotations around the normal to the plane will j o u r n a l p r e -p r o o f yield the same energy. the first rotation by an angle [ ] is applied around the y-axis, followed by another rotation of an angle [ ] around the z-axis, and then a translation is applied to optimize the position, similarly to what is done in the popular pales software. [44] [45] [46] the sampling of the pairs is made uniform by using repulsion angular sampling. [47] [48] [49] the distance of the plane to the protein is then set by minimizing the energy terms described above. the most important feature of a composite thought for immunochemical applications is that the orientation of the antigen with respect to the surface must ensure the accessibility of the epitope to the antibodies, to guarantee the recognition. therefore, we have selected the crystallographic structure of rbd in complex with a fragment (fab) of the human antibody cr3022 (pdb id: 6w41), [50] and identified the interface residues relevant for the interaction ( figure 1 and table s1 ). in the analysis of the orientations, we assume that the full length antibody will have the same accessibility as the fab because of the high flexibility of the linkers of the heavy chains (see figure s1 ). [51] [52] [53] [54] it is also important to note that, while the spike protein is highly glycosylated at n-and o-positions, [55, 56] the structured part of the rbd which is recognized by the antibody only carries one glycation at position 343 [55] (pink in figure 1 ), and the glycation site faces away from the antibody binding site. on these grounds, we have not considered glycation (experimentally, this would be done expressing recombinant rbd in prokaryotic cells, whereas glycation would be obtained in human cells [55] ). the fragment of the human antibody cr3022 is shown in blue. hydrophobic adsorption occurs selectively on hydrophobic carriers at low ionic strength. [57] it is a rather common immobilization protocol, because of its simplicity. the also this immobilization strategy is rather common because of its simplicity. it is slightly less general, because the outcome strongly depends on the nature of the protein and of the surface. the electrostatic interaction of the i-th residue with the uniformly charged surface with a given charge density is estimated by the gouy-chapman potential, [12, 13] which is added to a lj term. the parameters defining each system are listed in tables s3-s6. we have considered the following surfaces: 1) silica -a common chromatographic support with high negative surface charge; 2) positively charged self-assembled monolayer (sam), with amino capping of the chains; [14, 18] 3) negatively charged sam, with carboxylic capping of the chains. [14, 18] supports #2 and #3 imply the possibility of colorimetric detection through gold, [58] vide infra. the most probable orientations are shown in figure 3 . degrees from the one with highest relative population, except one that is more tilted, yielding a larger accessibility, with a relative population around 4% ( figure s2 ). it is apparent that only negatively charged surfaces allow for the exposition of the epitope, and this is anyway relatively marginal. these results suggest that it would be nontrivial to achieve a good orientation relying upon adsorption, either based on hydrophobic or on charge interactions. therefore, we have considered directed approaches based on stronger interactions. in particular, we have considered epoxide-glyoxyl (directed at primary amine moieties) [59] and gold (thiols and disulfide bridges). [58] the glyoxyl-based approach is quite popular for multipoint orientation-selective immobilization of proteins on surfaces. it involves a two-step mechanism, in the first step, the primary amine groups of the protein are allowed to react with the aldehyde groups to form schiff base bonds, in the second step the bonds are reduced with sodium borohydride. this kind of immobilization has been simulated in a similar way as described by del monte-martìnez et al., [11] assuming a working ph = 7.5, to maximize the reactivity of the n-terminus and at the same time limiting the reactivity of lysine residues (see table s7 ). the choice of gold is also extremely popular, because of two reasons: the strong plasmonic response of gold, which causes a purple coloring of the bioconjugate, and because of the relatively easy manipulation required. current sars-cov2 serological tests are indeed based on gold conjugates. [60] the conjugation to the surface is simulated in the same way as the amine-glyoxyl reaction, assuming that all cysteines are equally reactive towards gold (disulfide bridges can interact with gold to a comparable extent as thiols). [43] the resulting orientation has 100% relative population. colloidal gold has a net negative surface charge, [61] but including the electrostatic term has no impact on the recovered orientation. in the epoxide-glyoxyl strategy, the conjugation appears to be mostly directed at the nterminus, 1 which is facing away from the recognition interface but is not topologically very remote. therefore, the epitope will only be partially exposed, whereas for the gold conjugation, ample access to the epitope is possible in the most probable orientation. finally, a completely different strategy could be applied for conjugation to (e.g.) gold nanoparticles: the use of a avidin-biotin affinity system. [62] biotinylation can be achieved through amine-specific reagents, [63] and improvement in the selectivity can be achieved with minimal engineering of the sequence. [64] given that there is a rather substantial difference in the calculated pkas for the different amine sites (see table s7 ), it can be expected that, for ph values lower than 7, all lysine residues will be protonated and thus less reactive with probability higher than 99%. the n-terminus is not facing the interaction site (see figure 1 ). therefore, selective biotinylation at the n-terminus is expected to be possible. in this case, the accessibility of the epitope is warranted if the interaction between the antigen and streptavidin, if at all possible, is sufficiently weak. to explore this possibility we have performed an initial-stage docking using zdock, [65] and inspected the first two elements that had a significantly higher zdock score ( figure s3 ). the possible interaction between the rbd and streptavidin was investigated also using haddock2.4. [66] the protein-protein interface residues were predicted with cport, [67] and then used as "active" and "passive" residues in the haddock calculation. about 10 lowly populated clusters with weak energy were obtained; the most significant three with the lowest haddock-scores are reported in figure s3 and their energies in table s8 . both dockings indicate that, should the interaction occur, it would occur in a position that does not interfere with the antigen-antibody recognition. in this work, we describe the use of united-residue modelling for the prediction of the orientation of the receptor binding domain of the spike protein of the novel coronavirus sars-cov-2, a protein of high immunological relevance at the most commonly used surfaces for the preparation of lateral-flow immunochemical devices. with this simple, yet very flexible approach, we find that immobilization on silica, or through glyoxyl reaction of amine residues, or on gold yield orientations compatible with antibody recognition, with gold granting the highest exposition. in this way, we can explain why random conjugation of the rbd to a gold surface yields responsive immunosensors, which are now routinely used. a more detailed experimental verification of the predictions of protein orientation at surfaces represents a significant challenge for the current biophysical methodologies. [1] one can expect that cryo-electron transmission microscopy will be limited by the fact that, in most cases, the surface has higher electron density than that of the protein. confocal laser scanning microscopy can be used to assess the positioning of the protein with respect to the support, and super-resolution microscopic techniques, such as total internal reflection fluorescence microscopy also allow for the detection of discrete molecular events (e.g., desorption, unfolding, lateral diffusion, …), [2] but the orientation is still a high-hanging fruit by these methodologies. conversely, the interaction between the protein and the interface can be probed at the atomic level through the application of solid-state nmr, [4, [7] [8] [9] [68] [69] [70] effort which is being started in our lab. our results suggest that very simple modelling approaches can provide significant hints towards rationally orienting j o u r n a l p r e -p r o o f antigens in a way to maximize the exposition of epitopes, and therefore help in the initial moments of the design of conjugates for immunologic applications, when a rapid response to emergency is vital. this is also testified by the emergence of theoretical modelling of several molecular aspects of viral infection and inhibition mechanisms. 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biomolecular complexes cport: a consensus interface predictor and its performance in prediction-driven docking with haddock atomic structural details of a protein grafted onto gold nanoparticles 1h-detected solid-state nmr of proteins entrapped in bioinspired silica: a new tool for biomaterials characterization probing the transmembrane structure and dynamics of microsomal nadphcytochrome p450 oxidoreductase by solid-state nmr reckoning a fungal metabolite, pyranonigrin a as a potential main protease (mpro) inhibitor of novel sars-cov-2 virus identified using docking and molecular dynamics simulation identification of a novel dual-target scaffold for 3clpro and rdrp proteins of sars-cov-2 using 3d-similarity search, molecular docking, molecular dynamics and admet evaluation inhibition of the main protease 3cl-pro of the coronavirus disease 19 via structure-based ligand design and molecular modeling identification of potential binders of the main protease 3clpro of the covid-19 via structure-based ligand design and molecular modeling interaction of hydroxychloroquine with sars-cov2 functional proteins using all-atoms non-equilibrium alchemical simulations delving deep into the structural aspects of a furin cleavage site inserted into the spike protein of sars-cov-2: a structural biophysical perspective key: cord-254100-u6x5zd4i authors: taliansky, m.e.; brown, j.w.s.; rajamäki, m.l.; valkonen, j.p.t.; kalinina, n.o. title: involvement of the plant nucleolus in virus and viroid infections: parallels with animal pathosystems date: 2010-10-15 journal: adv virus res doi: 10.1016/b978-0-12-385034-8.00005-3 sha: doc_id: 254100 cord_uid: u6x5zd4i the nucleolus is a dynamic subnuclear body with roles in ribosome subunit biogenesis, mediation of cell-stress responses, and regulation of cell growth. an increasing number of reports reveal that similar to the proteins of animal viruses, many plant virus proteins localize in the nucleolus to divert host nucleolar proteins from their natural functions in order to exert novel role(s) in the virus infection cycle. this chapter will highlight studies showing how plant viruses recruit nucleolar functions to facilitate virus translation and replication, virus movement and assembly of virus-specific ribonucleoprotein (rnp) particles, and to counteract plant host defense responses. plant viruses also provide a valuable tool to gain new insights into novel nucleolar functions and processes. investigating the interactions between plant viruses and the nucleolus will facilitate the design of novel strategies to control plant virus infections. the nucleolus is a dynamic subnuclear body with roles in ribosome subunit biogenesis, mediation of cell-stress responses, and regulation of cell growth. an increasing number of reports reveal that similar to the proteins of animal viruses, many plant virus proteins localize in the nucleolus to divert host nucleolar proteins from their natural functions in order to exert novel role(s) in the virus infection cycle. this chapter will highlight studies showing how plant viruses recruit nucleolar functions to facilitate virus translation and replication, virus movement and assembly of virus-specific ribonucleoprotein (rnp) particles, and to counteract plant host defense responses. plant viruses also provide a valuable tool to gain new insights into novel nucleolar functions and processes. investigating the interactions between plant viruses and the nucleolus will facilitate the design of novel strategies to control plant virus infections. the interactions between a virus and its host cell play a central role in the viral infection cycle. the analysis of virus-host interactions is critical for understanding the mechanisms of viral infections and for the development of novel antiviral strategies. viruses are obligate intracellular pathogens with small genomes and, therefore, are reliant on subverting of the cellular the nucleolus and plant viruses functions and machineries to facilitate their own replication. the cell nucleus is one of the key features of eukaryotic cells. it is a highly dynamic, membrane-bound organelle that hosts major cellular events, including dna replication, messenger rna synthesis and processing, and ribosome subunit biogenesis (trinkle-mulcahy and . given the pivotal role of the nucleus in cell host function, it is not surprising that viruses interact with this organelle and its compartments, and that such interactions play a crucial role in the virus infection cycle. indeed, certain viruses including plant dna containing begomoviruses (rojas et al., 2001; sharma and ikegami, 2009) , rna reverse-transcribing caulimoviruses (camvs) (haas et al., 2005) , and negative-strand rna rhabdoviruses belonging to the genus nucleorhabdovirus (tsai et al., 2005) replicate in the nucleus, and therefore it makes sense that these viruses modify nuclear structures to usurp some of the nuclear functions and provide an appropriate environment for their own replication. in contrast, single-stranded positive-sense rna [(þ)ssrna] viruses replicate in the cytoplasm. therefore, the rational for rna viruses targeting the nucleus and its compartments is not so evident. however, an increasing number of reports clearly show that cytoplasmic rna viruses including plant viruses can also target nuclear compartments and, nucleoli, in particular (reviewed by hiscox 2002 hiscox , 2007 greco, 2009 ). thus, investigating why and how plant rna viruses interact with the nucleolus to meet their own needs will contribute to the better understanding of molecular biology of plant viruses and facilitate the design of novel strategies for virus control. these studies may also teach us about the fundamental principles of plant nucleolar structure and functions. the nucleolus is a prominent subnuclear compartment formed around the clusters of genes (rdna) coding for ribosomal rna (rrna), and is the site of rdna transcription, rrna processing, and ribosome assembly (olson, 2004; rubbi and milner, 2003; sirri et al., 2008) . indeed, for many years the exclusive function of the nucleolus was thought to be rrna synthesis and ribosome biogenesis. however, several insights from the past decade have dramatically changed this traditional view and implicated the nucleolus in many other aspects of cell function such as cellcycle regulation, gene silencing, telomerase activity, senescence, stress responses, and biogenesis of multiple ribonucleoprotein (rnp) particles (boisvert et al., 2007; olson and dundr, 2005; sirri et al., 2008) . this chapter briefly reviews the structure and composition of the nucleolus and, subsequently, data implicating the nucleolus in the infection cycles of animal and plant viruses and also viroids. most of the current knowledge on nucleolar localization and functions of viral proteins has been gained in studies on animal viruses and has also been reviewed comprehensively (greco, 2009; hiscox, 2002 hiscox, , 2007 . therefore, the main emphasis of this review will be on what is known about the different aspects of interactions between plant virus proteins and the nucleolus, of which the functional significance in control of virus movement and interference with host antiviral defense has started to appear only recently. we also discuss recent findings on the potential role of cajal bodies (cbs), another type of small subnuclear bodies structurally and functionally associated with the nucleolus. the nucleus is a highly structured organelle responsible for chromosome organization, replication and division, for gene expression and regulation, and the integration of a vast array of activities required for cellular function. the nucleus contains chromatin-rich regions, made up of condensed heterochromatin, more dispersed euchromatin, and interchromatin regions, and chromatin organization is responsible for regulating gene expression, dna replication, and chromosome segregation (schneider and grosscheld, 2007; trinkle-mulcahy and lamond, 2008) . in addition, it contains many other structures or bodies of different numbers and sizes which vary between cell types, at different stages in the cell cycle and under different conditions. the most prominent of these structures is the nucleolus but many other bodies [e.g., cbs, splicing speckles, paraspeckles, pml (pro-myeloid leukemia) bodies, etc.] have now been identified and are being characterized on the basis of their protein and rna components and functions (boisvert et al., 2007; cioce and lamond, 2005; lamond and spector, 2003; matera et al., 2009; rippe, 2007) . the nucleolus is where rrna genes are transcribed and processed, and rrnas are assembled, along with ribosomal proteins, into the small and large subunits of the ribosome (boisvert et al., 2007; fatica and tollervey, 2002; granneman and baserga, 2004) . the nucleolus contains three different regions on the basis of their appearance in the transmission electron microscope: fibrillar centers (fc), the dense fibrillar component (dfc), and the granular component (gc). in the mammalian nucleolus, the fcs are small, lightly staining structures surrounded by the more densely stained dfc. the dfc in turn is surrounded a particulate region-the gc. the plant nucleolus tends to be more regular in its structure (usually near to spherical) than animal nucleoli. the organization of the nucleolar regions is different in plant nucleoli in that the dfc is not as densely stained as in animal nucleoli and occupies much more of the nucleolar volume (up to 70%). transcription of precursor rrnas (pre-rrnas) occurs at multiple sites (200-400) within the dfc regions and pre-rrnas undergo processing in the dfc and gc. indeed, the localization of pre-rrnas and different small nucleolar rnas (snornas) and proteins has allowed early and late pre-rrna cleavage events to be correlated to the dfc and gc, respectively, suggesting a vectorial model for the production and maturation of rrnas. plant nucleoli also often contain a prominent central region called the nucleolar cavity (brown and shaw, 1998; shaw and brown, 2004) . the function of the nucleolar cavity is currently unknown, and it remains to be seen whether the differences in organization of plant and animal nucleoli influence the type and range of other functions which nucleoli carry out and interactions with different viruses. the major function of the nucleolus is the transcription and processing of precursor rrnas and ribosomal subunit assembly. the dynamic assembly pathway involves a series of intermediate pre-ribosomal complexes of the 40s and 60s ribosomal subunits and in addition to ribosomal proteins, requires up to 200 accessory proteins (grannemann and baserga, 2004) . processing of the pre-rrnas involves both cleavage of the precursor transcript and nucleotide modifications, the majority of which are 2 0 -o-ribose methylation and pseudouridylation. some cleavage reactions and the modifications require snornps. snornps consist of a snorna and associated proteins (kiss, 2002) . by the nature of the major function in rrna production and ribosomal subunit assembly, there is a huge flux of proteins and rna complexes into and out of the nucleolus. the highly dynamic movement of proteins and complexes has been illustrated in animal cells by quantitative nucleolar proteomic analyses lam et al., 2007) . in particular, ribosomal proteins are highly expressed and rapidly accumulate in the nucleolus where they are incorporated into ribosomal subunits or rapidly degraded (lam et al., 2007) . three of the most abundant and well-studied nucleolar proteins are fibrillarin, nucleolin, and b23. fibrillarin, one of the major proteins of the nucleolus, is a core component of box c/d snornps and is required for rrna processing (venema and tollervey, 1999) . fibrillarin has methyltransferase activity directing 2 0 -o-ribose methylation of rrna (barneche et al., 2000; cioce and lamond, 2005; matera and shpargel, 2006) . fibrillarin is highly conserved in sequence, structure, and function in eukaryotes. the n-terminal region of fibrillarin comprises a glycine-and arginine-rich (gar) domain (barneche et al., 2000) . the gar domain is methylated at arginine residues (liu and dreyfuss, 1995) and is responsible for interactions with various proteins including the survival motor neuron (smn) gene product ( jones et al., 2001) and the nuclear dead (asp-glu-ala-asp) box protein p68 (nicol et al., 2000) . fibrillarin contains a centrally located rna-binding domain which together with the c-terminal helix domain and the intervening spacer constitutes a methyltransferase-like domain that contains an s-adenosyl methionine binding motif and is responsible for fibrillarin methyltransferase activity (wang et al., 2000a) . the c-terminal helix domain appears to target fibrillarin to cbs (snaar et al., 2000) . although it is well established that fibrillarin plays a role in ribosome biogenesis within the nucleolus, its role in cbs is not well understood but it is presumably responsible for the 2 0 -o-ribose methylation of small nuclear rnas (snrnas). nucleolin is an abundant, ubiquitously expressed protein, which is highly phosphorylated, methylated, and also can be adp-ribosylated (ginisty et al., 1999) . nucleolin is found in various cell compartments, and it is especially abundant in the nucleolus. nucleolin has three well-defined domains. the n-terminal domain with alternating acidic and basic stretches is involved in rdna transcription by interacting with rdna repeats and histone h1 and in nuclear localization. the central portion is the rnabinding domain, whereas the c-terminal part contains a gar domain involved in interaction with the ribosomal proteins (tuteja and tuteja, 1998) . nucleolin is involved in ribosome biogenesis (mongelard and bouvet, 2007) , affects transcription, processing and modification of rrna and nuclear-cytosolic transport of ribosomal proteins and ribosomal subunits by shuttling between the nucleus and the cytoplasm (tuteja and tuteja, 1998) . b23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly, binding to other nucleolar proteins, nucleocytoplasmic shuttling (li et al., 1996) , and possibly regulating transcription of rdna by mediating structural changes in chromatin (okuwaki et al., 2001) . two isoforms of b23 have been identified: the major form (b23.1) is predominately located in the nucleolus and the minor form (b23.2) resides in the cytoplasm (reviewed by hiscox, 2002) . small proteins of less than 40-60 kda can enter the nucleus through nuclear pore complexes by passive diffusion (hiscox, 2007; nigg, 1997) . rna-binding proteins that diffuse into the nucleus may therefore nonspecifically target the nucleolus as it contains a large amount of rrna. the nuclear import of larger proteins is mediated by nuclear localization signals (nlss), composed of one (monopartite) or two (bipartite) stretches of basic amino acid (arginine and/or lysine) residues of a given size (hiscox, 2007; macara, 2001) . nucleocytoplasmic shuttling proteins also contain specific nuclear export signals (ness), usually leucine-rich amino acid motifs (hiscox, 2007; macara, 2001) . how proteins may be further delivered to the nucleolus is poorly understood. the nucleolus does not have apparent membrane or other barriers, and entry into it does not require energy, unlike entry to the nucleus. it seems conceivable that viral proteins localize to the nucleolus, firstly, as a result of targeting the nucleus via classical nls followed by direct or indirect interactions between the viral molecules (via various nucleolar localization signals, nolss) and components that make up the nucleolus (hiscox, 2002 (hiscox, , 2007 . the structure of nolss is not well defined and depends on different factors including whether the protein associates with another nucleolar-bound protein or alternatively traffics to the nucleolus on its own or associates with rna transcripts that are being transcribed in the nucleolus. at least in some cases, nols are rich in arginine and lysine residues and can overlap with nlss (hiscox, 2002 (hiscox, , 2007 . the second most studied nuclear body is cbs which are frequently associated with the nucleolus and found in animal and plant nuclei. cbs are involved in snrnp and snornp maturation and transport, with snrnps and snornps accumulating in cbs before appearing in speckles or the nucleolus, respectively (cioce and lamond, 2005) . as mentioned above, spliceosomal snrnas are also modified and contain 2 0 -o-ribose methylations and pseudouridines. modification of nucleotides in snrnas is guided by small cb-specific rnas (scarnas) (darzacq et al., 2002; jady et al., 2003) . a major component of cbs is the protein, coilin, which is required for their formation. in plants, mutants are available which knock out cbs or alter their size and number, two of which are due to mutations in coilin (collier et al., 2006) . the number of cbs per nucleus can vary in different cell types and is under developmental control (cioce and lamond, 2005) . besides rrna transcription and processing and the production of ribosomal subunits, the nucleolus is also involved in many other aspects of rna processing and rnp assembly as well as cellular functions (boisvert et al., 2007; olson, 2004; pedersen, 1998; raška et al., 2006; rubbi and milner, 2003) . for example, the nucleolus has a role in the maturation, assembly, and export of rnp particles. the signal recognition particle (srp) has a nucleolar phase in its assembly with particular protein constituents of the srp being localized to the nucleolus. similarly, telomerase rnp, required for chromosome replication, may be assembled in the nucleolus, and the nucleolus may also have a role in sequestering telomerase rnp to avoid inappropriate nucleation of telomere structures. spliceosomal snrnps may also undergo part of their assembly in the nucleolus, and processing of pre-trnas and u6snrna occurs in the nucleolus. in addition, the nucleolus is a site of sequestration of particular proteins to regulate, for example, the cell cycle or cell death, and acts as a sensor of cellular stress (boisvert et al., 2007; olson, 2004; pedersen, 1998; raška et al., 2006; rubbi and milner, 2003) . characterization of the protein composition of the nucleolus under different conditions has provided support for or suggested new functions for the nucleolus. initial proteomic analyses of human cells identified a few hundred proteins which included not only well-known nucleolar proteins such as ribosomal proteins, fibrillarin, nucleolin, b23, etc. but also, for example, splicing and translation factors (andersen et al., 2002) . advances in proteomics has now allowed the identification of around 4500 proteins in the human nucleolar proteome (ahmad et al., 2008) and the dynamic behavior of nucleolar components and complexes can now be determined lam et al., 2007) . in plants, a partial proteomic analysis of the nucleolus identified many expected ribosomal and nucleolar proteins but also found splicing and translation factors (pendle et al., 2005) . in particular, exon junction complex proteins (associated with mrnas following splicing) were identified in the nucleolar proteome and in the nucleolus by fluorescence microscopy (pendle et al., 2005) . one of these proteins, eif4a-iii, a core protein of the exon junction complex, was shown to redistribute from the nucleoplasm to the nucleolus and finally to splicing speckles under stress conditions of hypoxia . similar dynamic distribution, involving the nucleolus, of proteins that interact with mrnas has been demonstrated and the nucleolus and cbs have been shown to be involved in u1snrnp production in plants (lorković and barta, 2008; tillemans et al., 2006) . in plants, novel functions for the plant nucleolus, cbs, and another largely nucleolar-associated body, the d-body, have been described. firstly, the production of heterochromatic sirnas, which are involved in transcriptional silencing, is thought to occur in a region of the nucleolus or in d bodies due to the localization of protein components of the machinery and of sirnas (pontes and pikaard, 2008) . secondly, maturation of micrornas (mirnas) may occur in d-bodies as precursor mir-nas as well as dicer-like ribonuclease 1 (dcl1) have been located to these structures (pontes and pikaard, 2008) . similarly, in mammalian cells, some precursor and mature mirnas have recently been found in the nucleus with some being enriched in the nucleolus (politz et al., 2009; scott et al., 2009) . of particular interest was the enrichment of some precursors in the gc suggesting that mirna processing could occur in the nucleolus, or these mirnas may be involved in ribosome synthesis or other nucleolar functions (politz et al., 2009) . the link between the nucleolus and mirna production is illustrated by the evolutionary relationship between some snornas and mirna precursors. for example, a human snorna was shown to be processed by dicer to generate small rnas which were associated with argonaute proteins (agos) and caused reduced expression of gene targets (ender et al., 2008) . in addition, numerous snorna-derived small rnas from different organisms (including arabidopsis) were associated with components of rna silencing pathways (taft et al., 2009) and many mirna precursors have retained snorna features (scott et al., 2009 ). finally, a third novel function for the plant nucleolus is in mrna biogenesis, surveillance, or nonsensemediated decay (nmd). biochemical fractionation of nucleoplasm and nucleoli and subsequent sequencing of isolated cdnas have shown that the plant nucleolus not only contains mrnas but that aberrant mrnas are enriched in the nucleolus ). the aberrant mrnas show splicing defects, the majority of which would introduce premature termination codons and therefore be expected to be substrates for nmd. using upf mutants (affected upf proteins, key components of the nmd mechanism), the correlation between enrichment of aberrant mrnas in the nucleolus and turnover by nmd was corroborated and was further supported by the localization of the nmd proteins, upf2 and upf3, to the nucleolus . before the observation that the plant nucleolus contained mrnas and aberrant mrnas, some spliced mrnas (e.g., c-myc) were localized to the nucleolus while their unspliced versions were found in the nucleoplasm in mammalian cells (pedersen, 1998; olson, 2004) . the nucleolus has also been associated with mrna export on the basis of accumulation of polyaþ rna upon disruption of export factors, nucleolar structure, or stress conditions in yeast and animal cells, although this could reflect increased polyadenylation prior to degradation (ideue et al., 2004; pederson, 1998; schneiter et al., 1995) . more recently, the nucleolus has been proposed to be involved in the formation of mrnps which are localized to specific regions of the cytoplasm for translation. in yeast, ash1 mrna enters the nucleolus bound to specific rna-binding proteins at which time translation repressor proteins are loaded onto the mrna. the mrnp is then exported to the cytoplasm, transported to its final destination, and then translation is activated (du et al., 2008; jellbauer and jansen, 2008) . a similar trafficking pathway may also operate in mammals for mrnps associated with the nucleolar protein, staufen, which is involved in transport of mrnas in neurons . thus, the nucleolus has numerous functions related to rna biogenesis and different rna processing and rnp maturation pathways. it contains many rna-interacting proteins involved in these processes, including highly abundant rna-binding proteins (such as fibrillarin and nucleolin) involved in rrna and ribosomal subunit production. it is therefore a dynamic hub of rna processing activity, rna:protein interaction and complex formation. these characteristics, in addition to the potential involvement of the nucleolus in mrna biogenesis and, particularly, the transport of mrnas and mrnps to and from the nucleolus to other parts of the cell, make the nucleolus a prime target for exploitation by viruses. it is therefore not surprising that viruses have taken advantage of the nucleolus and nucleolar components for production and distribution of viral rnas and rnps. several excellent reviews have documented various aspects of the involvement of the nucleolus in the infection cycle of animal and human viruses (greco, 2009; hiscox, 2002 hiscox, , 2007 matthews and olson, 2006; stark and taliansky, 2009 ). this section therefore briefly summarizes the main findings in this research area to illuminate the nucleolar functions involved in virus infections that are conserved between plant and animal kingdoms. certain animal viruses such as dna containing adenoviruses that replicate in the nucleus interact with and disrupt the nucleolus. as a result, synthesis of rrna is disrupted in adenovirus-infected cells. adenovirus infection also causes the redistribution of nucleolin and b23 (matthews, 2001) . interestingly, b23 has been shown to stimulate adenovirus replication. rna virus replication may also be facilitated by nucleolar proteins. indeed, the hdag protein encoded by hepatitis delta virus (a (à)ssrna virus which is a subviral satellite of the dna virus, hepatitis b virus) also binds to nucleolin and b23. it has been proposed that hdag interacts with both these proteins in a complex that promotes viral rna replication, presumably as a result of the helicase activity of nucleolin (huang et al., 2001) . nucleolar proteins may also be involved in virus assembly and egress. for example, accumulation and assembly of structural (cap) proteins of adeno-associated virus take place in nucleoli (bevington et al., 2007) . further export of the assembled cap proteins from the nucleolus is mediated by another type of virus-encoded packaging (rep) proteins, which should form a complex with cap proteins. remarkably, formation of this complex is mediated by b23. after export of complexes containing rep, b23, and cap proteins from the nucleolus to the nucleoplasm, viral dna encapsidation occurs (bevington et al., 2007) . another animal virus, the (à)ssrna containing borna disease virus, uses the nucleolus as a site of genome replication (pyper et al., 1998 ). an rna-binding protein encoded by this virus has the appropriate trafficking signals for import and export to and from the nucleus (cros and palese, 2003) many proteins encoded by animal viruses that replicate mainly or exclusively in the cytoplasm have also been shown to localize to the nucleolus, cause the relocalization of nucleolar proteins and disruption of nucleolar architecture and function. these include the (þ)ssrna virus proteins such as nucleocapsid proteins encoded by coronavirus and arterivirus, the dengue virus core protein, the alphavirus capsid protein and nonstructural nsp2 protein, the (à)ssrna virus proteins, including the influenza virus nucleoprotein (np) and nonstructural protein 1 (ns1), newcastle disease virus matrix protein, and retrovirus proteins such as human immunodeficiency virus (hiv) rev and the transactivator tat proteins (reviewed by greco, 2009; hiscox, 2002 hiscox, , 2007 . although functional relevance of why these proteins localize to the nucleus or nucleolus and how this relates to their functions in virus replication in many cases is largely unknown, several reports reveal significant progress in this area as exemplified below. infection of cells with poliovirus (picornavirus) results in the inactivation of the nucleolar protein, rna polymerase i upstream binding (transcription) factor, which inhibits transcription of rrnas in the host cell (banerjee et al., 2005) . poliovirus is also able to interact with nucleolin causing its selective redistribution from the nucleolus to the cytoplasm (waggoner and sarnow, 1998) . after relocalization, nucleolin binds to the internal ribosome entry sites (iress) at the 5 0 untranslated region of poliovirus genomic rna, and this interaction stimulates ires-dependent translation (hellen and sarnow, 2001) . this also occurs in hepatitis c virus (izumi et al., 2001) and represents an alternative translation initiation strategy as compared to the classical eukaryotic cap-dependent translation (hellen and sarnow, 2001) . nucleolin is also able to interact with the 3 0 -untranslated region of poliovirus rna, which controls synthesis of negative strand rna (waggoner and sarnow, 1998) . interaction of picornaviruses with the nucleolus could also down-regulate host gene expression. for example, the human rhinovirus 3c protease precursors that are localized in the nucleolus at early stages of the infection inhibit cellular rna by cleavage of vital transcription factors (amineva et al., 2004) . the nucleocapsid proteins encoded by porcine arterivirus and avian coronavirus (infectious bronchitis virus, ibv) interact with nucleolin and fibrillarin (chen et al., 2002; yoo et al., 2003) , and as a result may disrupt the normal functions of these proteins. for instance, by altering the distribution of fibrillarin, viruses might be reducing poli transcription, that is, the synthesis of rrna, as blocking fibrillarin with antibody prevented its translocation to nucleoli and resulted in the reduction or inhibition of poli transcription (fomproix et al., 1998) . the ibv infection leads to disruption of the nucleolus (dove et al., 2006a) and arrest of the cell cycle and cytokinesis (chen et al., 2002; dove et al., 2006b; wurm et al., 2001) . therefore, disruption of nucleolar architecture and function might be common in cells infected with viruses interacting with the nucleolus. the loss of essential nucleolar function in its turn may play an important role during virus infection toward an active production of the virus. one of the most studied viruses in terms of viral interactions with the nucleolus is hiv, a retrovirus. hiv rnas are reverse transcribed in the cytoplasm of infected cells and trafficked to the nucleus. after transcription in the nucleus, progeny rna molecules are transported back to the cytoplasm. one of the functions of the rev protein is to export unspliced or partially spliced viral mrna from the nucleus (reviewed by greco, 2009; hiscox, 2007) . before nuclear export, hiv rna passes through the nucleolus. rev binds to a cis-acting rna element (rev-response element), which is found in all unspliced and incompletely spliced viral mrnas, and this promotes the translocation of these rnas from the nucleus (cantó-nogués et al., 2001; michienzi et al., 2000) . the nucleolus plays a central role in this process, and the nucleolar trafficking of rev and viral rna is critical for the outcome of infection. thus, many animal viruses, whether they replicate or not in the nucleus, have evolved a nucleolar phase for part of their infection cycle to prevent unwanted interference from the cell. alternatively, they use nucleolar functions for their own benefit. recruitment of nucleolar proteins is especially beneficial for viruses, and in particular for rna containing viruses, as these proteins possess many crucial functions in cellular rna biosynthesis, processing, translation, and trafficking. indeed, during virus infections of mammalian cells, various viral components traffic to and from the nucleolus where they interact with different host factors. certain nucleolar proteins are redistributed into other cell compartments or are modified, and some cellular proteins are relocalized in the nucleolus of infected cells (reviewed by greco, 2009; hiscox, 2002 hiscox, , 2007 . well-documented studies have established that several of these nucleolar modifications play a role in some steps of the viral infection cycle such as viral attachment and entry, intracellular trafficking, transcription, translation, replication, virus assembly, and regress (reviewed by greco, 2009 ). the virally induced nucleolar modifications could also affect fundamental cellular pathways including the initiation of transcription from the dna promoter of the rrna genes, cell-cycle regulation, and apoptosis (reviewed by greco, 2009) . although some steps (replication, translation) of the infection cycles of plant and animal/human viruses are essentially similar, there is a fundamental difference in some other mechanisms employed to enter host cells and spread from cell to cell between viruses infecting animal cells and viruses infecting plants. this is because animal cells are separated by barriers far less formidable than the thick, rigid, and impermeable cell walls consisting of cellulose and pectin that separate plant cells from one another. other differences relate to defense strategies employed by humans/animals and plants against viral infections. for example, in mammals the interferon (ifn) pathway plays a key role in the innate antiviral immune response, whereas plants do not display such an activity. instead, plants primarily rely on other natural defense strategies such as rna silencing. interestingly, functional links between plant virus infection cycle and the nucleolus have been described for both common and plant-specific virus infection steps. certain plant virus proteins localize to the nucleolus with examples from single-stranded dna viruses, para-retroviruses and negative-strand and positive-strand rna viruses (table i) . the most common technique for studying the nucleolar targeting of plant virus proteins is based on the confocal microscopy localization of the proteins which have been tagged with a fluorescent fusion protein (such as green fluorescent protein, gfp). such proteins are usually larger than the size-exclusion limit ($ 40-60 kda) and hence prevented from nonspecific protein diffusion into the nucleus. moreover, in many cases the specific nucleolar localization of plant virus proteins has been supported by identification of nolss (table ii) . although no overall conserved nucleolar trafficking motif has been identified in these nolss, they presumably resemble host nolss. thus plant viral nucleolar trafficking might use a form of molecular mimicry as has earlier been proposed for animal viruses (table ii) . however, in the case of grv orf3, in addition to the arginine-rich domain (nls), a leucine residue at position 149 (l149) residing inside the leucine-rich domain (nes) is also essential for nucleolar targeting of orf3 protein (table ii; kim et al., 2007a) . the fact that viral proteins contain nolss is a strong indication that viruses have evolved specific nucleolar functions. viral proteins might also traffic to the nucleolus through association with host proteins. one example of such an association may be the interaction of various plant virus proteins with the major nucleolar protein, fibrillarin. the first description of this interaction was the demonstration that the umbravirus grv orf3 long-distance movement protein (mp) binds to fibrillarin in vivo and in vitro (kim et al., 2007a,b) . for example, the leucine-rich domain (and l149, in particular) of orf3 is involved in direct interaction with fibrillarin (kim et al., 2007b) . mutations in the leucine-rich domain prevent fibrillarin from binding to orf3 and nucleolar trafficking (kim et al., 2007b) . by implication, this may relate fibrillarin binding to nucleolar trafficking. other plant virus mps which interact with fibrillarin are the pomovirus pmtv triple gene block protein 1 (tgb1) and hordeivirus pslv tgb1 (n. o. kalinina and d. rakitina, unpublished results). as their name suggests, mps are involved in virus spread in infected plants, and the potential role of fibrillarin in this process will be discussed in section iv.b. the multifunctional pva (potato virus a)-encoded viral genomelinked protein (vpg) is also able to interact with fibrillarin (rajamäki and bonfiglioli et al. (1996) , qi and ding (2003) valkonen, 2009). however, the role of this interaction is likely to be different from that in virus movement as this process is not compromised by fibrillarin depletion (rajamäki and valkonen, 2009 ; section iv.c). another example of a viral protein that interacts with fibrillarin in the nucleolus is a silencing suppressor of cmv (cucumber mosaic virus), the 2b protein (gonzález et al., 2010 ; section iv.c). collectively, these data indicate that interaction with fibrillarin is a general property of various plant virus proteins that is not restricted to one or two virus taxonomic groups. however, such interactions may have quite diverse molecular implications for different viruses being required at various phases of virus infection cycle. this may also suggest novel, unexpected natural functions for fibrillarin that are hijacked or affected by plant viruses at different stages of infection for needs of the viruses. viroids are small, circular, self-replicating, and non-coding rna molecules that cause plant diseases (reviewed by tabler and tsagris, 2004) . viroids do not replicate in the cytoplasm like conventional plant rna viruses but replicate in either the nucleus or the chloroplast. nuclear viroids such as pstvd and cccvd have a nucleolar phase in their replication cycle (table i ; qi and ding, 2003; schumacher et al., 1983) . virus acronyms and other abbreviations are as in table i . a nlss are also required for the nucleolar localization. b nuclear and nucleolar localization is controlled independently by the same nls regions. c l149 (italic) residing inside the orf3 nes is also essential for nucleolar localization of the orf3 protein. in early experiments using in situ hybridization, pstvd rna [including both (þ) and (à) rna strands] was localized in nucleoli of nuclei isolated from infected plants (harders et al., 1989) , suggesting that the nucleolus is the replication site of the viroid. however, later using improved sample preparation and in situ hybridization protocols, qi and ding (2003) have found that the (à) strand of pstvd localizes in the nucleoplasm but not in the nucleolus. by contrast, the (þ) strand of pstvd localizes in the nucleolus as well as in the nucleoplasm, with various distinct spatial patterns. these experiments are suggestive of successive stages of nuclear involvement in viroid replication: (1) synthesis of the (à) and (þ) strands of pstvd occurs in the nucleoplasm; (2) the (à) strand rna is anchored in the nucleoplasm; (3) the (þ) strand rna is transported selectively into the nucleolus; and (4) some (þ) strand rna traffics from the nucleolus back into the nucleoplasm and further into the cytoplasm for spreading into neighboring cells. the significance of (þ) strand pstvd rna trafficking into the nucleolus remains to be determined. however, nucleolar machineries for processing of rrnas, snornas, and trnas make the nucleolus an attractive candidate site for pstvd processing. the specific intranuclear localization of (à) and (þ) strands of pstvd may have some implication for pathogenicity. assuming that the (à) and (þ) strands each can interact with specific cellular factors to disrupt normal cell functions to cause symptoms, the differential subnuclear localization of the (þ) and (à) strands of pstvd may suggest different cellular targets for these rnas. (þ) strand of pstvd rna has been shown to interact with a bromodomain-containing protein (a member of a family of transcriptional regulators associated with chromatin remodeling), termed viroid rna binding protein 1 (virp1) (martínez de alba et al., 2003; tabler and tsagris, 2004) . virp1 contains an nls and therefore might transfer the viroid rna to the nucleus and bring it into contact with transcription units associated with chromatin (martínez de alba et al., 2003; tabler and tsagris, 2004) . however, mechanisms for selective nucleolar trafficking of (þ) strand pstvd are still unknown. the nucleolus and its factors may be used not only by viruses and viroids for their own needs in replication, but also by plant host defense systems. for example, one of the major nucleolar proteins, nucleolin, binds to the 3 0 non-coding region of the tomato bushy stunt virus, tombusvirus, rna ( jiang et al., 2010) . this leads to significant inhibition of tombusviral rna replication and may thus represent one of the innate immunity systems of plant hosts. plant viruses enter cells either through sites of mechanical injury to plant tissues or during the feeding by a specific vector organism (insect, nematode, or soil microbes belonging to protocists). to induce disease, after replication in the initially infected cells, plant viruses must spread to the rest of the plant. the systemic spread of plant viruses proceeds in two stages: (i) cell-to-cell movement through plasmodesmata (pd) and (ii) long-distance movement through vascular tissues. first, the virus (in the form of virions or nucleic acid protein complexes) moves intracellularly from the sites of replication to pd, which are unique intercellular membranous channels that span cell walls linking the cytoplasm of contiguous cells. the virus then transverses the pd to spread intercellularly. it is generally accepted that viral cell-to-cell movement involves virus-encoded mps as well as host-encoded components (reviewed by lucas, 2006) . virus systemic movement between organs (long-distance movement) occurs through the phloem, the specialized vascular system used by plants for the transport of assimilates and macromolecules (reviewed by lucas, 2006; oparka, 2004) . viruses enter, move through, and exit from the vascular system, which is usually surrounded by bundle sheath cells and contains various cell types including vascular parenchyma cells, companion cells, and enucleate sieve elements. thus, transport of a virus into and within vascular tissue implies movement from mesophyll cells to bundle sheath cells, from bundle sheath cells to vascular parenchyma and/or companion cells, and into sieve elements. virus exit from vascular tissue presumably involves the same steps in reverse order. coat protein (cp) is essential for efficient long-distance transport of plant viruses, with only few exceptions (reviewed by lucas, 2006 ). one of the most well-studied plant viruses in terms of viral interactions with the nucleolus is grv, an umbravirus [(þ)ssrna virus]. umbraviruses differ from most other viruses in that they do not encode a cp such that conventional virus particles are not formed in infected plants. umbraviral genomes encode at least three proteins. in grv, two orfs at the 5 0 -end of the rna are expressed by a frameshift mechanism as a single protein that appears to be an rna replicase . the other orfs (orf3 and orf4) overlap each other. orf4 encodes the mp that mediates the cell-to-cell movement of viral rna via pd (ryabov et al., 1998) . orf3 protein is the long-distance movement factor that facilitates trafficking of viral rna through the phloem (ryabov et al., 1999) . umbraviral orf3 proteins (26) (27) (28) (29) show no significant similarity with any other recorded or predicted proteins . the grv orf3 protein interacts with viral rna in vivo to form filamentous rnp particles, which have elements of regular helical structure, but not the uniformity typical of virus particles . the rnps accumulate in cytoplasmic inclusions which have been detected in all cell types and were abundant in phloem cells . they serve to protect viral rna and move it through the phloem to cause systemic infection. remarkably, in addition to its presence in the cytoplasm, the orf3 protein was also found in nuclei and predominantly in nucleoli (ryabov et al., 1998 (ryabov et al., , 2004 . studies of the biology of grv infection have provided molecular insights into how and why viruses may target the nucleolus (canetta et al., 2008; kim et al., 2007a,b) . it has been demonstrated that the grv orf3 protein traffics to the nucleolus via a mechanism involving the reorganization of cbs into multiple cajal body-like structures (cbl) and their fusion with the nucleolus. nucleolar localization and further trafficking of orf3 protein from the nucleolus back to the cytoplasm is essential for the umbravirus infection. the integral connection between nucleolar targeting of the orf3 protein and its biological function in virus long-distance spread is demonstrated by mutagenesis of the arginine-and leucine-rich domains that block nucleolar localization or nuclear export of the orf3 protein, and which prevent the formation of cytoplasmic viral rnps and their long-distance movement (kim et al., 2007a) . although the mechanisms by which the orf3 protein targets cbs and produces cbls are unknown, it could be suggested that targeting of cbs by the orf3 protein may utilize elements of existing cb-trafficking pathways. for example, part of the maturation pathway of snrnps in mammalian cells occurs in the cytoplasm and involves a complex containing the smn protein (smn complex) which together with the snrnp are reimported into the nucleus and targeted to cbs (matera and shpargel, 2006; navascues et al., 2004; sleeman and lamond, 1999) . particular snrnp proteins contain modified symmetric di-methyl arginines (sdmas) which enhance the formation of snrnps and interaction with smn (paushkin et al., 2002) . preliminary experiments have identified sdmas in the orf3 protein (m. taliansky, unpublished results) , suggesting that targeting of the orf3 protein to cbs could involve interactions with the smn protein. although smn protein has yet to be identified in plants, the existence of an orthologue has been suggested (collier et al., 2006) . the formation of cbls may involve either fragmentation of cbs into multiple bodies by the orf3 protein or the redistribution of cb components into new structures containing the orf3 protein. interestingly, the multiple cbl phenotype, described here, is similar to that of the poly cajal bodies (pcb) mutant of arabidopsis (collier et al., 2006) . as the protein normally encoded by a gene controlling the pcb phenotype, appears to regulate cb formation, orf3 protein may interfere with the function of this or other proteins to affect the integrity and number of cbs in nuclei. the second possibility is that the orf3 protein causes the redistribution of cb components, such as coilin, u2b 00 and fibrillarin, to form cbls with the orf3 protein. orf3 protein trafficking to the nucleolus uses a novel pathway of nucleolar import by causing the fusion of cbls with the nucleolus. the physical and functional association of the nucleolus and cbs is welldocumented and is controlled by complex molecular interactions among cb and nucleolar proteins such as coilin, smn, fibrillarin, and nopp140 (cioce and lamond, 2005; ogg and lamond, 2002) . expression of mutant versions of some of these proteins has profound effects on cb structure and function causing disruption or dispersal and compositional changes ( jones et al., 2001; pellizzoni et al., 2001; tucker et al., 2001) . moreover, phosphorylation of coilin is an important factor determining physical interactions and trafficking of cbs (cioce and lamond, 2005; ogg and lamond, 2002) . for example, cbs form within the nucleolus of hela cells upon treatment with okadaic acid (an inhibitor of protein phosphatase) and with transient expression of coilin mutated at a single serine residue (lyon et al., 1997; sleeman et al., 1998) . cbs have also been observed within nucleoli in human breast carcinomas (ochs et al., 1994) and in liver cells of hibernating dormice (malatesta et al., 1994) . therefore, the orf3 protein may interfere with normal protein-protein interactions or posttranslational modifications causing the reorganization and fusion of cbs with the nucleolus. the last stage of the nuclear voyage of the orf3 protein is its nuclear export leading to the redistribution of some fibrillarin from the nucleolus to cytoplasm (fibrillarin normally does not accumulate in cytoplasm) (kim et al., 2007a) . this redistribution is mediated by the direct interaction between the orf3 protein and fibrillarin (kim et al., 2007b) . taking into account the long-distance movement function of grv orf3, it could be expected that fibrillarin is directly involved in this particular viral function. further support of a role for fibrillarin in umbravirus systemic infection has been provided by the fibrillarin knock-down experiments (kim et al., 2007b) . silencing of the fibrillarin gene has indeed prevented formation of rnp particles and long-distance movement of grv but does not affect viral replication or cell-to-cell movement. thus, it has been concluded that fibrillarin is needed for formation of cytoplasmic rnp particles that are capable of long-distance movement and causing systemic viral infection such that the redistribution of fibrillarin with orf3 protein is a prerequisite to form rnps (kim et al., 2007a,b) . further experiments have shown that fibrillarin, in combination with orf3 protein and viral rna in vitro, produced filamentous rnp structures with structural properties similar to in vivo rnps (as discussed in detail in section iv.e). fibrillarin, an rna-binding protein, may bind the viral rna or act as a chaperone to permit or catalyze the regular assembly of proteins around viral rna. although fibrillarin and orf3 protein are sufficient to form functional viral rnp particles in vitro, additional proteins may also be associated with the in vivo particles. when formed in phloem companion cells, the viral rnps are able to enter sieve elements and move through the plant to cause systemic infection. hence formation of the fibrillarin-orf3 protein complexes appears to be the key prerequisite for formation of grv rnp particles and their long-distance movement through the phloem. poleroviruses are (þ) ssrna viruses that are mainly restricted to cells in the vascular system. besides a major cp plrv encodes a minor cp, an extended version of the major cp produced by occasional translational ''readthrough'' of the cp gene (cp-rt) (bahner et al., 1990) . both cp and cp-rt contain the same nols, and are targeted to the nucleolus when they are individually expressed in plant tissues (haupt et al., 2005) . however, cp-rt but not plrv cp loses its nucleolar localization in the presence of replicating plrv. it has been suggested that the cp-rt protein does not accumulate in the nucleolus in the presence of plrv infection because plrv rna or plrv-encoded or -induced factors retain this protein outside the nucleolus (haupt et al., 2005) . like grv, plrv has been unable to cause systemic infection in the fibrillarin-silenced plants, although accumulation of plrv in the inoculated leaves was not affected (kim et al., 2007b) suggesting that fibrillarin is also involved in plrv long-distance movement. a role of nucleolar functions in systemic infection has also been suggested for plant viruses that require tgb for movement. the genomes of some viruses, such as potexviruses, hordeiviruses, and pomoviruses, contain so-called triple gene block, tgb that encodes three proteins required for cell-to-cell and long-distance movement (reviewed by morozov and solovyev, 2003) . the tgb1 protein encoded by the tgb-containing virus, pmtv (pomovirus) localizes to the nucleus and nucleolus. deletion of 84 nterminal amino acids abrogates its nucleolar localization. northern blots of rna from inoculated and upper non-inoculated leaves of plants infected with clones carrying the tgb1 n-terminal deletion mutant reveal that long distance movement of viral rnas has also been abolished, but this mutant is still competent for cell-to-cell movement (l. torrance, personal communication). moreover, the pmtv tgb1 protein has been shown to interact with fibrillarin in vitro. interestingly, tgb1 protein encoded by the hordeivirus, pslv can also interact with fibrillarin both in vitro and in vivo (n. o. kalinina, unpublished results) . thus it appears that some tgb-encoding viruses such as pmtv and pslv may represent another example of plant viruses that require association with the nucleolus to control long distance movement of their genomes. potyviruses form the largest group of plant-infecting rna viruses (rajamäki et al., 2004) . they have a polyadenylated (þ) ssrna genome of ca. 9500-10,000 nucleotides that is encapsidated into a 680-900 nm long, filamentous particle. the genome is translated into a large polyprotein of about 3000-3350 amino acids, which is subsequently processed into up to ten mature proteins by three virus-encoded proteinases (rajamäki et al., 2004) . additionally, a 25-kda protein produced from the p3 cistron by frameshifting was recently identified (chung et al., 2008) . potyviruses replicate in membranous structures in the cytoplasm (cotton et al., 2009; schaad et al., 1997a) . however, two potyviral replication-associated proteins, the nuclear inclusion protein a (nia) and nuclear inclusion protein b (nib), accumulate in the nucleus of virus-infected cells (baunoch et al., 1991; dougherty and hiebert, 1980; hajimorad et al., 1996; knuhtsen et al., 1974; restrepo et al., 1990) . in addition, nia localizes in the nucleolus and cbs (baunoch et al., 1991; rajamäki and valkonen, 2009; restrepo et al., 1990) . the nib of tev (tobacco etch virus) has also been detected in the nucleolus (baunoch et al., 1991; restrepo et al., 1990) . potyviruses may also induce nuclear inclusions, which consist of nia and nib (baunoch et al., 1991; dougherty and hiebert, 1980; edwardson and christie, 1983; knuhtsen et al., 1974; martin et al., 1992) . the significance of these nuclear inclusions is unknown but they may simply represent inactivated protein complexes, because they seem to form only at late stages of virus infection (baunoch et al., 1991) . nia is multifunctional and consists of two domains. the n-proximal half of nia is the vpg that is covalently linked to the 5 0 -end of viral genomic rna (oruetxebarria et al., 2001; siaw et al., 1985) . the c-proximal part (nia-pro) is the main viral proteinase (dougherty et al., 1989) . the two domains are separated by a suboptimal proteolytic cleavage site, the slow processing of which is essential for efficient viral replication schaad et al., 1996) . the majority of full-length nia is found in the nucleus of virus-infected cells . however, nia can also exist as a polyprotein with the 6k2 protein located upstream of nia in the viral polyprotein. the 6k2 protein impedes nuclear localization of the 6k2-nia polyprotein (restrepo-hartwig and carrington, 1992) and directs nia to cytoplasmic membrane vesicles, the sites of viral replication cotton et al., 2009; restrepo-hartwig and carrington, 1994; schaad et al., 1997a) . many lines of evidence suggest that nia is part of the viral replication complex involving several viral and host proteins fellers et al., 1998; li et al., 1997; murphy et al., 1996; puustinen and mäkinen, 2004; schaad et al., 1996) . the vpg domain has ntp binding activity, is uridylylated by nib, and may act as a primer for synthesis of viral rna (anindya et al., 2005; murphy et al., 1996; puustinen and mäkinen, 2004; schaad et al., 1996) . in addition, vpg is involved in viral cell-to-cell and long-distance movement (nicolas et al., 1997; valkonen, 1999, 2002; schaad et al., 1997b) . the nlss and nols of potyviral nia map to the n-proximal part of vpg (carrington et al., 1991; rajamäki and valkonen, 2009; schaad et al., 1996) . the signal controlling nuclear localization of nia is bipartite (carrington et al., 1991; rajamäki and valkonen, 2009; schaad et al., 1996) . the regions of nia controlling nucleolar targeting of nia have been studied in pva and found to map to the same regions as the nlss. however, mutations in both nls regions are required to abolish nuclear targeting of pva nia, whereas mutations in either nls prevent nucleolar localization of nia (rajamäki and valkonen, 2009) . the most n-terminal nls controls also localization of pva nia to cbs (rajamäki and valkonen, 2009) . mutations that affect nuclear localization of nia are detrimental for genome amplification of tev and pva (rajamäki and valkonen, 2009; schaad et al., 1996) , suggesting that nuclear/nucleolar localization of nia has an important role in virus infection. potyviral nia (or vpg) has been shown to interact with several host proteins (dunoyer et al., 2004; huang et al., 2010; léonard et al., 2004; rajamäki and valkonen, 2009; schaad et al., 2000; thivierge et al., 2008; wittmann et al., 1997) . the structure of vpg is intrinsically disordered (grzela et al., 2008; rantalainen et al., 2008) , which may provide flexibility for many types of interactions. one of the interaction partners of vpg and/or nia is the eukaryotic translation initiation factor eif4e or its isoform (robaglia and caranta, 2006; schaad et al., 2000; wittmann et al., 1997) and the interaction appears important for virus infectivity (léonard et al., 2000; robaglia and caranta, 2006) . remarkably, interaction of tumv nia with eif(iso)4e and the poly(a) binding protein 2 (pabp2) has been detected in the nucleus and nucleolus using bimolecular fluorescence complementation (bifc) and colocalization experiments . by contrast, interaction with the 6k2-nia polyprotein targets eif(iso)4e and pabp2 to cytoplasmic membrane vesicles . the data suggest that these host proteins are needed in different viral processes. in healthy plants, most of pabp is cytoplasmic but some of the protein is redistributed to the nucleolus probably by the nia . nia appears to mediate also eif (iso)4e localization to the nucleolus and could interact simultaneously with eif(iso)4e and pabp2 as shown by in vitro binding assays and bifc and colocalization experiments . hence, all three proteins may be part of the same complex. although the role of interaction between nia and eif(iso)4e and/or pabp2 in the nucleus is currently unclear, some possibilities can be suggested. the nuclear pool of eif4e takes part in mrna export from the nucleus but may also be involved in nuclear translation and nmd of rna (strudwick and borden, 2002) . pabp regulates the initiation of protein synthesis, nuclear export of mature mrnas, and mrna stability and decay (mangus et al., 2003) . interaction with nia might disrupt some of these functions. as mentioned above, some animal viruses are known to modulate and inhibit activities of translation initiation factors in order to favor viral replication and translation (reviewed by thompson and sarnow, 2000) . recent data connect nuclear/nucleolar targeting of plant viral proteins to interference with host antiviral defense. for example, the potyviral vpg protein has been observed to accumulate in the companion cells of upper leaves of a wild potato species ahead of virus infection, which suggested that vpg might interfere with host defense and hence facilitate infection of the cells that receive the virus via systemic transport (rajamäki and valkonen, 2003) . indeed, this hypothesis gained support in the experiments in which overexpression of vpg in leaf tissues temporarily interfered with cosuppression of gene silencing (i.e., rna silencing), whereas nls mutants of vpg, which exhibited reduced nuclear and nucleolar localization, were not able to suppress rna silencing (rajamäki and valkonen, 2009) . rna silencing is a sophisticated, sequence-specific rna degradation mechanism operating in the cytoplasm (ruiz-ferrer and voinnet, 2009) . a key feature of the rna silencing pathway is the generation of dsrna that corresponds in sequence to the target (virus) rna. this dsrna is cleaved into sirnas by dcls and these mediate the target specificity for rna degradation (for reviews, see carrington, 2000; ruiz-ferrer and voinnet, 2009; vance and vaucheret, 2001; voinnet, 2001) . rna silencing is a natural anti-viral defense system of plants but is also involved in gene regulation in a wide range of developmental and pathogen defense processes (ruiz-ferrer and voinnet, 2009) . to combat host defense rna silencing, most plant viruses encode silencing suppressor proteins. the potyviral helper-component proteinase (hc-pro) was the first detected viral suppressor of rna silencing (anandalakshmi et al., 1998; brigneti et al., 1998; kasschau and carrington 1998) and the potyviral p1 protein may enhance its activity (rajamäki et al., 2005) . hc-pro acts in the cytoplasm and, therefore, the association of vpg with rna silencing suppression when localized in the nucleolus is intriguing because many host proteins involved in rna silencing as well as the processing centers for small rnas are located in the nucleus, nucleolus, and cbs as discussed in section ii (pontes and pikaard, 2008) . in contrast, nuclear/nucleolar localization of the p19 protein of tbsv, tombusvirus, mediated by plant aly proteins (mrna-processing and -export factors) may be a defense mechanism of the plant to down-regulate the silencing suppressor activity of p19 (canto et al., 2006) . previously, suppression of rna silencing has been found to be connected to nuclear localization of another viral protein: the 2b protein, silencing suppressor of cmv. cmv 2b localizes in the nucleus and the nucleolus where it interacts with argonaute 1 (ago1), the core component of the rna-induced silencing complex (gonzález et al., 2010; lucy et al., 2000; zhang et al., 2006) . more recently, it has also been shown that cmv 2b interacts with ago4 (gonzález et al., 2010) . however, these interactions are not sufficient for suppression of rna silencing, and hence their biological relevance remains so far unclear (gonzález et al., 2010) . the vpg of pva interacts with fibrillarin in the nucleolus and cbs as detected by bifc experiments (rajamäki and valkonen, 2009 ). depletion of fibrillarin reduces pva accumulation in nicotiana benthamiana, suggesting a role for fibrillarin in virus infection (rajamäki and valkonen, 2009 ). as mentioned above, in grv, fibrillarin is recruited for viral long-distance transport (canetta et al., 2008; kim et al., 2007a,b) , but in potyviruses the role is likely to be different as long-distance transport of pva is not compromised by depletion of fibrillarin (rajamäki and valkonen, 2009) . taking into account the silencing suppression activity of vpg, it could be suggested that vpg-fibrillarin might contribute to such an activity. alternatively, the fibrillarin-vpg interaction may disrupt certain nucleolar functions (e.g., host transcription or pre-mrna processing), which might explain the observed shutdown of host gene expression during potyvirus infection (wang and maule, 1995) . in mammals, the ifn pathway plays a key role in the innate antiviral immune response whereas involvement of rna silencing is still controversial (reviewed by hale et al., 2008) . however, emerging evidence suggests some parallels in how plant and animal viruses could use the nucleolus to counteract host defense. for example, the ns1 protein of influenza a virus is an important factor in counteracting ifn-based cellular antiviral mechanisms (hale et al., 2008) . in addition, ns1 is also able to suppress rna silencing in plant, insect, and mammalian cells (bucher et al., 2004; de vries et al., 2009; delgadillo et al., 2004; haasnoot et al., 2007; li et al., 2004) . remarkably, ns1 localizes to the nucleolus and interacts with nucleolin (murayama et al., 2007) . nib is a viral rna-dependent rna polymerase of potyviruses and hence involved in the replication complex (hong and hunt, 1996; schaad et al., 1997a) . while viral replication takes place in the cytoplasm on cellular the nucleolus and plant viruses membranes, nib is also targeted to the nucleus and the nucleolus (baunoch et al., 1991; restrepo et al., 1990) . nuclear targeting of nib appears to be highly sensitive to alterations in protein conformation and is eliminated by deletions, dipeptide insertions and amino acid substitutions introduced also into parts of nib other than the nlss (li and carrington, 1993) . nuclear and nucleolar localization of nib may be important for the viral infection cycle, because mutations in the nlss abolish infectivity of tev (li and carrington, 1995; li et al., 1997) . three host proteins, the pabp2, the heat shock cognate 70 protein (hsc70-3) , and the eukaryotic translation elongation factor 1a, eif1a, interact with nib thivierge et al., 2008; wang et al., 2000b) . however, none of these protein interactions was found in the nucleolus, shedding no light on the role of the nucleolar localization of nib. besides nuclear inclusion proteins, the p3 protein of tev is targeted to the nucleus and nucleolus of virus-infected tobacco cells, as detected by immunogold labeling (langenberg and zhang, 1997) . p3 is a nonstructural protein with no well-characterized function. however, it is involved in virus multiplication (kekarainen et al., 2002) and virus-host interactions (chu et al., 1997; eggenberger et al., 2008; jenner et al., 2003; johansen et al., 2001) . a role for nuclear/nucleolar localization of p3 is currently unknown. the multifunctional nucleocytoplasmic shuttling p6 protein encoded by plant para-retrovirus camv has also been found to localize to the nucleolus (haas et al., 2005) . although a nols has not been identified for this protein, its nucleolar import might be facilitated by its interaction with ribosomal proteins of the 60s ribosomal subunit (haas et al., 2008) . the presence of p6 in the nucleolus, where assembly of ribosomal subunits occurs, raises the possibility that p6 might interact directly with ribosomes before their export to render them competent for translation of the camv polycistronic mrna. indeed, p6 interacts with the ribosomal proteins l18 (leh et al., 2000) , l24 (park et al., 2001) , and l13 (bureau et al., 2004) , which hence may be the potential targets because they participate in the formation of the 60s subunit in the nucleolus (andersen et al., 2002) . another functional activity of camv p6 is suppression of rna silencing. this protein interferes with rna-directed rna polymerase 6 (rdr6)dependent rna silencing via inhibition of the dsrna-binding protein drb4, a protein normally enhancing dcl4-mediated dicing. however, unlike nuclear targeting, the nucleolar localization of p6 is completely dispensable for its silencing suppression function (haas et al., 2008) . transport of the viral genome into the nucleus is an obligatory step in the replication cycle of plant dna viruses such as the begomoviruses. cps of monopartite begomoviruses (such as tylcv and tolcjav) are nucleocytoplasmic shuttling proteins thought to be involved in this process (rojas et al., 2001; sharma and ikegami, 2009 ). interestingly, these proteins also contain nolss targeting them to the nucleolus (sharma and ikegami, 2009 ). however, the biological significance of nucleolar localization of begomoviral cp is unclear. nlss have been identified in three of the seven proteins encoded by plant nucleorhabdovirus, mfsv. remarkably, two of them, the nucleocapsid (n) protein and phosphoprotein (p), localize to the nucleolus but only when they are coexpressed in plant tissues (as fusions with gfp) using the agrobacterium system; when expressed individually these proteins do not target nucleoli (tsai et al., 2005) . this clearly indicates the interdependent character of nucleolar targeting for the n and p proteins. however, the molecular mechanisms responsible for this effect and its biological relevance remain to be explored. another plant virus protein with nucleolar localization is the cp of spmv (satellite panicum mosaic virus) (qi et al., 2008) . some of the functional and biochemical properties of the spmv cp including the nucleolar association of spmv cp, its rna binding activity (desvoyes and scholthof, 2000) , and the involvement of the cp in systemic movement (omarov et al., 2005) are similar to those of grv orf3. by analogy therefore, the authors have suggested that nucleolar localization of spmv cp may be essential for its role in the systemic movement of spmv as in the case of grv orf3. collectively, all of these findings support an active role of the nucleolus and fibrillarin in various aspects of the virus infection cycle and interactions with host cells promoting systemic infections with plant viruses belonging to various taxonomic groups. the nucleolus contains a complex machinery for rrna modification and rrnp assembly and may provide an environment which allows other forms of functional rnps, such as srp, telomerase, splicing snrnps, and viral rnps to exploit the nucleolus or nucleolar components in their biogenesis pathways (reviewed by boisvert et al., 2007) . for example, fibrillarin is one of the four core protein components of a box c/d snornp complex (reviewed by tran et al., 2004) . being a methyltransferase, fibrillarin (as a component of box c/d snornps) functions in the 2 0 -in vitro-methylation and processing of pre-rrna. in addition, human fibrillarin forms a subcomplex with splicing factor 2-associated p32, protein arginine methyltransferases, and tubulins a3 and b1 that is independent of its association with snornps, suggesting that fibrillarin may also coordinate protein methylation during the processes of ribosome biogenesis . furthermore fibrillarin interacts with some other cellular proteins such as smn protein ( jones et al., 2001) and the nuclear dead box protein p68, an rna-dependent atpase and rna helicase (nicol et al., 2000) . however, the physiological role of these interactions is unclear and may be based on novel natural functions of fibrillarin that remain to be established. the most studied viral rnp complex containing fibrillarin is the grv orf3 complex with elements of regular helical structure that is capable of long-distance movement via the phloem. assembly of grv rnp particles occurs in the cytoplasm and requires fibrillarin relocalized from the nucleolus (kim et al., 2007a,b; ; also see above). rnp particles similar in architecture and infectivity to the viral rnps formed in vivo, have been reconstituted in vitro from fibrillarin, the orf3 protein and viral rna (kim et al., 2007a,b) . taking the study further, the in vitro experiments have shown that the orf3-fibrillarin interaction occurs between the leucine-rich region (l149 in particular) of the orf3 protein and the gar domain of fibrillarin (kim et al., 2007b) known to be responsible for interaction with other proteins such as smn ( jones et al., 2001) . this interaction leads to formation of single-layered ring-like complexes of orf3 with fibrillarin as was shown by atomic force microscopy (canetta et al., 2008) . the diameter of these orf3 protein-fibrillarin rings is 18-22 nm which correlates with that of the filamentous rnp particles (canetta et al., 2008) . it thus appears that the orf3 protein fibrillarin rings interact with viral rna, encapsidating it and reorganizing it into helical structures, and thereby play a key role in the assembly of umbraviral rnp complexes. these results demonstrate that, in addition to traditional functions in rrna processing and modification, fibrillarin possesses completely novel functions in mediating assembly of umbraviral rnps. these functions are presumably based on the ability of the orf3 protein to interact and form ring-like complexes with fibrillarin such that the virus alters and exploits the properties of fibrillarin for successful virus propagation. other viral proteins, such as the nucleocapsid protein encoded by porcine arterivirus also interact with fibrillarin in an rna-dependent manner . however, the structure and architecture of these complexes and how they impact the viral infection cycle remain unknown. the nucleolus is a highly conserved feature of eukaryotic cells that has a key role as the site of ribosome subunit production. however, recent multiple lines of investigation have confirmed and characterized additional roles for nucleoli in other important cellular processes including cell-cycle control, stress responses, and coordination of the biogenesis of a number of other functional rnps. the nucleus has also been shown to play a crucial role in the infection cycle of various viruses, and the nucleolar localization of viral proteins has recently been described as a pan-virus phenomenon (hiscox, 2002 (hiscox, , 2007 . in this regard, plant viruses are not different from other eukaryotic viruses. the past few years have brought remarkable progress in our understanding of why and how some plant viruses (in particular, umbraviruses and potyviruses) target the nucleolus and the functional role of the interaction between viral and nucleolar proteins in the plant virus infection cycle. for many other plant virus proteins, nucleolar localization and interaction with nucleolar components have also been demonstrated, and functional implications of these findings is a challenge for future research. we anticipate that more information will emerge about the mechanisms involved in regulating nucleolar function and structure in response to plant virus infections and hijacking nucleolar functions for the virus infection cycle. there are now several examples in which the plant viruses also target other subnuclear bodies, such as cbs. in particular, for umbraviruses the role of cbs in nucleolar trafficking of the orf3 protein has been established. the potential role of sub-nuclear structures in other plant virus infections will be addressed in the future. the study of viral interactions with the nucleolus also provides unique and valuable tools to gain new insights into novel nucleolar functions and processes. for example, as previously discussed, the major nucleolar protein fibrillarin, is involved in formation and long-distance movement of umbraviral rnp particles. these functions as well as other potential yet unrecognized natural functions of nucleolar proteins will be the focus of much future research. on a practical level, both the plant cell and viral biology of the nucleolus can, and hopefully will be exploited for the design of novel strategies to control plant virus infections. nopdb: nucleolar proteome database -2008 update rhinovirus 3c protease precursors 3cd and 3cd' localize to the nuclei of infected cells the nucleolus and plant viruses a viral suppressor of gene silencing in plants directed proteomic analysis of the human nucleolus nucleolar proteome dynamics tyrosine 66 of pepper vein banding virus genome-linked protein is uridylylated by rna-dependent rna polymerase expression of the genome of potato leafroll virus: readthrough of the coat protein termination codon modifications of both selectivity factor and upstream binding factor contribute to poliovirus-mediated inhibition of rna polymerase i transcription fibrillarin genes encode both a conserved nucleolar protein and a novel small nucleolar rna involved in ribosomal rna methylation in arabidopsis thaliana a temporal study of the expression of the capsid, cytoplasmic inclusion and nuclear inclusion proteins of tobacco etch potyvirus in infected cells the poly(a) binding protein is internalized in virus-induced vesicles or redistributed to the nucleolus during turnip mosaic virus infection visualization of the interaction between the precursors of vpg, the viral protein linked to the genome of turnip mosaic virus, and the translation eukaryotic initiation factor iso 4e in planta adeno-associated virus interactions with b23/nucleophosmin: identification of sub-nucleolar regions the multifunctional nucleolus tissue and intra-cellular distribution of coconut cadang cadang viroid and citrus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy viral pathogenicity determinants are suppressors of transgene silencing in nicotiana benthamiana small nucleolar rnas and pre-rrna processing in plants the influenza a virus ns1 protein binds small interfering rnas and suppresses rna silencing in plants p6 protein of cauliflower mosaic virus, a translation reinitiator, interacts with ribosomal protein l13 from arabidopsis thaliana a plant virus movement protein forms ringlike complexes with the major nucleolar protein, fibrillarin, in vitro translocation of tomato bushy stunt virus p19 protein into the nucleus by aly proteins compromises its silencing suppressor activity ultrastructural localization of the rna of immunodeficiency viruses using electron microscopy in situ hybridization and in vitroinfected lymphocytes rna silencing. moving targets bipartite signal sequence mediates nuclear translocation of the plant potyviral nia protein internal cleavage and trans-proteolytic activities of the vpg-proteinase (nia) of tobacco etch potyvirus in vivo interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell two separate regions in the genome of the tobacco etch virus contain determinants of the wilting response of tabasco pepper an overlapping essential gene in the potyviridae cajal bodies: a long history of discovery a distant coilin homologue is required for the formation of cajal bodies in arabidopsis turnip mosaic virus rna replication complex vesicles are mobile, align with microfilaments and are each derived from a single viral genome trafficking of viral genomic rna into and out of the nucleus: influenza, thogoto and borna disease viruses cajal body-specific small nuclear rnas: a novel class of 2 0 -o-methylation and pseudouridylation guide rnas differential rna silencing suppression activity of ns1 proteins from different influenza a virus strains human influenza virus ns1 protein enhances viral pathogenicity and acts as an rna silencing suppressor in plants rna: protein interactions associated with satellites of panicum mosaic virus translation of potyvirus rna in a rabbit reticulocyte lysate: identification of nuclear inclusion proteins as products of tobacco etch virus rna translation and cylindrical inclusion protein as a product of the potyvirus genome characterization of catalytic residues of the tobacco etch virus 49-kda proteinase changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication nuclear transit of the rna-binding protein she2 is required for translational control of localized ash1 mrna heat shock 70 protein interaction with turnip mosaic virus rna-dependent rna polymerase within virus-induced membrane vesicles a cysteine-rich plant protein potentiates potyvirus movement through an interaction with the virus genomelinked protein vpg cytoplasmic cylindrical and nucleolar inclusions induced by potato virus-a gain of virulence on rsv1-genotype soybean by an avirulent soybean mosaic virus requires concurrent mutations in both p3 and hc-pro a human snorna with microrna-like functions making ribosomes in vitro interactions between a potyvirus-encoded, genome-linked protein and rna-dependent rna polymerase effect of anti-fibrillarin antibodies on building of functional nucleoli at the end of mitosis structure and functions of nucleolin cucumber mosaic virus 2b protein subcellular targets and interactions: their significance to its rna silencing suppressor activity ribosome biogenesis: of knobs and rna processing involvement of the nucleolus in replication of human viruses virulence factor of potato virus y, genome-attached terminal protein vpg, is a highly disordered protein the open reading frame vi product of cauliflower mosaic virus is a nucleocytoplasmic protein: its n terminus mediates its nuclear export and formation of electron-dense viroplasms nuclear import of camv p6 is required for infection and suppression of the rna silencing factor drb4 rna interference against viruses: strike and counterstrike nia and nib of peanut stripe potyvirus are present in the nucleus of infected cells, but do not form inclusions the multifunctional ns1 protein of influenza a viruses imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy nucleolar localization of potato leafroll virus capsid proteins internal ribosome entry sites in eukaryotic mrna molecules brief review: the nucleolus -a gateway to viral infection? rna viruses: hijacking the dynamic nucleolus rna polymerase activity catalyzed by a potyvirusencoded rnadependent rna polymerase the nucleolar phosphoprotein b23 interacts with hepatitis delta antigens and modulates the hepatitis delta virus rna replication a host rna helicase-like protein, atrh8, interacts with the potyviral genome-linked protein, vpg, associates with the virus accumulation complex, and is essential for infection the nucleolus is involved in mrna export from the nucleus in fission yeast nucleolin stimulates viral internal ribosome entry site-mediated translation modification of sm small nuclear rnas occurs in the nucleoplasmic cajal body following import from the cytoplasm a putative function of the nucleolus in the assembly or maturation of specialised messenger ribonucleoprotein complexes the dual role of the potyvirus p3 protein of turnip mosaic virus as a symptoms and avirulence determinant in brassicas nucleolin/nsr1p binds to the 3 0 noncoding region of the tombusvirus rna and inhibits replication recessive resistance in pisum sativum and potyvirus pathotype resolved in a gene-for-cistron correspondence between host and virus direct interaction of the spinal muscular atrophy disease protein smn with the small nucleolar rnaassociated protein fibrillarin a counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing functional genomics on potato virus a: virus genome-wide map of sites essential for virus propagation cajal bodies and the nucleolus are required for a plant virus systemic infection interaction of a plant virus-encoded protein with the major nucleolar protein fibrillarin is required for systemic virus infection aberrant mrna transcripts and the nonsensemediated decay proteins upf2 and upf3 are enriched in the arabidopsis nucleolus small nucleolar rnas: an abundant group of non-coding rnas with diverse cellular functions partial purification and some properties of tobacco etch virus induced intranuclear inclusions dynamic behaviour of the eif4a-iii putative core protein of the exon junction complex: fast relocation to nucleolus and speckles under hypoxia analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins nuclear speckles: a model for nuclear organelles immunocytology shows the presence of tobacco etch virus p3 protein in nuclear inclusions the cauliflower mosaic virus translational transactivator interacts with the 60s ribosomal subunit protein l18 of arabidopsis thaliana complex formation between potyvirus vpg and translation eukaryotic initiation factor 4e correlates with virus infectivity interaction of vpg-pro of turnip mosaic virus with the translation initiation factor 4e and the poly(a)-binding protein in planta nuclear transport of tobacco etch potyviral rnadependent rna polymerase is highly sensitive to sequence alterations complementation of tobacco etch potyvirus mutants by active rna polymerase expressed in transgenic cells c23 interacts with b23, a putative nucleolar-localization-signal-binding protein functions of the tobacco etch virus rna polymerase (nib): subcellular transport and protein-protein interaction with vpg/proteinase (nia) interferon antagonist proteins of influenza and vaccinia viruses are suppressors of rna silencing in vivo and in vitro arginine methylation of rna-binding proteins role of cajal bodies and nucleolus in the maturation of the u1 snrnp in arabidopsis plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus inhibition of protein dephosphorylation results in the accumulation of splicing snrnps and coiled bodies within the nucleolus transport into and out of the nucleus. microbiol ultrastructural location of non-structural protein 3a of cucumber mosaic virus in infected tissue using monoclonal antibodies to a cloned chimeric fusion protein cytochemical and immunocytochemical characterization of nuclear bodies during hibernation poly(a)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression intracellular localization of three non-structural plum pox potyvirus proteins by immunogold labelling a bromodomaincontaining protein from tomato specifically binds potato spindle tuber viroid rna in vitro and in vivo pumpimg rna: nuclear bodybuilding along the rnp pipeline nuclear bodies: random aggregates of sticky proteins or crucibles of macromolecular assembly? adenovirus protein v induces redistribution of nucleolin and b23 from nucleolus to cytoplasm what's new in the nucleolus? rybozyme-mediated inhibition of hiv 1 suggests nucleolar trafficking of hiv-1 rna nucleolin: a multifaceted protein triple gene block: modular design of a multifunctional machine for plant virus movement influenza a virus non-structural protein 1 (ns1) interacts with cellular multifunctional protein nucleolin during infection replacement of the tyrosine residue that links a potyviral vpg to the viral rna is lethal targeting smn to cajal bodies and nuclear gems during neuritogenesis the nuclear dead box rna helicase p68 interacts with the nucleolar protein fibrillarin and colocalizes specifically in nascent nucleoli during telophase variations in the vpg protein allow a potyvirus to overcome va gene resistance in tobacco nucleocytoplasmic transport: signals, mechanisms and regulation coiled bodies in the nucleolus of breast cancer cells cajal bodies and coilin-moving towards function identification of nucleophosmin/b23, an acidic nucleolar protein, as a stimulatory factor for in vitro replication of adenovirus dna complexed with viral basic core proteins nontraditional roles of the nucleolus the moving parts of the nucleolus the capsid protein of satellite panicum mosaic virus contributes to systemic invasion and interacts with its helper virus getting the message across: how do plant cells exchange macromolecular complexes? identification of the genome-linked protein in virions of potato virus a, with comparison to other members in genus potyvirus a plant viral 'reinitiation' factor interacts with the host translational machinery the smn complex, an assemblysome of ribonucleoproteins the plurifunctional nucleolus the survival of motor neurons (smn) protein interacts with the snornp proteins fibrillarin and gar1 proteomic analysis of the arabidopsis nucleolus suggests novel nucleolar functions micrornas with a nucleolar location sirna and mirna processing: new functions for cajal bodies uridylylation of the potyvirus vpg by viral replicase nib correlates with the nucleotide binding capacity of vpg the nucleolus is the site of borna disease virus rna transcription and replication differential subnuclear localization of rna strands of opposite polarity derived from an autonomously replicating viroid the complex subcellular distribution of satellite panicum mosaic virus capsid protein reflects its multifunctional role during infection the 6k2 protein and the vpg of potato virus a are determinants of systemic infection in nicandra physaloides viral genome-linked protein (vpg) controls accumulation and phloem-loading of a potyvirus in inoculated potato leaves localization of a potyvirus and the viral genome-linked protein in wild potato leaves at an early stage of systemic infection control of nuclear and nucleolar localization of nuclear inclusion protein a of picorna-like potato virus a in nicotiana species infection with potyviruses a novel insertion site inside the potyvirus p1 cistron allows expression of heterologous proteins and suggests some p1 functions potato virus a genome-linked protein vpg is an intrinsically disordered molten globule-like protein with a hydrophobic core structure and function of the nucleolus in the spotlight nuclear transport of plant potyviral proteins regulation of nuclear transport of a plant potyvirus protein by autoproteolysis the tobacco etch potyvirus 6-kilodalton protein is membrane associated and involved in viral replication dynamic organisation of the cell nucleus translation initiation factors: a weak link in plant rna virus infection functional analysis of proteins involved in movement of the monopartite begomovirus, tomato yellow leaf curl virus nucleolar-cytoplasmic shuttling of prrsv nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences disruption of the nucleolus mediates stabilization of p53 in response to dna damage and other stresses roles of plant small rnas in biotic stress responses intracellular location of two groundnut rosette umbravirus proteins delivered by pvx and tmv vectors a plant virus-encoded protein facilitates long-distance movement of heterologous viral rna identification of a nuclear localization signal and nuclear export signal of the umbraviral long-distance rna movement protein analysis of the vpg-proteinase (nia) encoded by tobacco etch potyvirus: effects of mutations on subcellular transport, proteolytic processing and genome amplification formation of plant rna virus replication complexes on membranes: role of an endoplasmid reticulum-targeted viral protein vpg of tobacco etch potyvirus is a host genotype-specific determinant for long-distance movement strain-specific interaction of the tobacco etch virus nia protein with the translation initiation factor eif4e in the yeast twohybrid system dynamics and interplay of nuclear architecture, genome organisation, and gene expression mrna transport in yeast: time to reinvestigate the functions of the nucleolus subcellular localization of viroids in highly purified nuclei from tomato leaf tissue human mirna precursors with box h/aca snorna features characterization of signals that dictate nuclear/nucleolar and cytoplasmic shuttling of the capsid protein of tomato leaf curl java virus associated with dnab satellite plant nuclear bodies identification of a protein covalently linked to the 5 0 -terminus of tobacco vein mottling virus rna nucleolus: the fascinating nuclear body newly assembled snrnps associate with coiled bodies before speckles, suggesting a nuclear snrnp maturation pathway dynamic interactions between slicing snrnps, coiled bodies and nucleoli revealed using snrnp protein fusions to the green fluorescent protein mutational analysis of fibrillarin and its mobility in living human cells old and new faces of the nucleolus. workshop on the nucleolus and disease the emerging roles of translation factor eif4e in the nucleus viroids: petite rna pathogens with distinguished talents small rnas derived from snornas molecular biology of umbraviruses: phantom warriors an umbraviral protein, involved in long-distance rna movement, binds viral rna and forms unique, protective ribonucleoprotein complexes eukaryotic elongation factor 1a interacts with turnip mosaic virus rna-dependent rna polymerase and vpg-pro in virus-induced vesicles regulation of host cell translation by viruses and effects on cell function insights into nuclear organisation in plants as revealed by the dynamic distribution of arabidopsis sr splicing factors evolutionary origins of the rna-guided nucleotide-modification complexes: from the primitive translation apparatus? toward a high-resolution dynamics view of nuclear dynamics nuclear functions in space and time: gene expression in a dynamic complete genome sequence and in planta subcellular localization of maize fine streak virus proteins residual cajal bodies in coilin knockout mice fail to recruit sm snrnps and smn, the spinal muscular atrophy gene product nucleolin: a multifunctional major nucleolar phosphoprotein rna silencing in plants -defense and counterdefense ribosome synthesis in saccharomyces cerevisiae rna silencing as a plant immune system against viruses viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells inhibition of host gene expression associated with plant virus replication crystal structure of a fibrillarin homologue from methanococcus jannaschii, a hyperthermophile, at 1.6 å resolution interaction between zucchini yellow mosaic potyvirus rna-dependent rna polymerase and host poly-(a) binding protein interaction of the viral protein genome linked of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4e of arabidopsis thaliana using the yeast two-hybrid system localisation to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division human fibrillarin forms a sub-complex with splicing factor 2-associated p32, protein arginine methyltransferases, and tubulins alpha 3 and beta 1 that is independent of its association with preribosomal ribonucleoprotein complexes colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rnaassociated protein fibrillarin cucumber mosaic virus-encoded 2b suppressor inhibits arabidopsis argonaute1 cleavage activity to counter plant defense key: cord-271642-i71g2tmd authors: mullen, lisa m.; nair, sean p.; ward, john m.; rycroft, andrew n.; henderson, brian title: phage display in the study of infectious diseases date: 2006-02-07 journal: trends microbiol doi: 10.1016/j.tim.2006.01.006 sha: doc_id: 271642 cord_uid: i71g2tmd microbial infections are dependent on the panoply of interactions between pathogen and host and identifying the molecular basis of such interactions is necessary to understand and control infection. phage display is a simple functional genomic methodology for screening and identifying protein–ligand interactions and is widely used in epitope mapping, antibody engineering and screening for receptor agonists or antagonists. phage display is also used widely in various forms, including the use of fragment libraries of whole microbial genomes, to identify peptide–ligand and protein–ligand interactions that are of importance in infection. in particular, this technique has proved successful in identifying microbial adhesins that are vital for colonization. microbial infections are dependent on the panoply of interactions between pathogen and host and identifying the molecular basis of such interactions is necessary to understand and control infection. phage display is a simple functional genomic methodology for screening and identifying protein-ligand interactions and is widely used in epitope mapping, antibody engineering and screening for receptor agonists or antagonists. phage display is also used widely in various forms, including the use of fragment libraries of whole microbial genomes, to identify peptide-ligand and protein-ligand interactions that are of importance in infection. in particular, this technique has proved successful in identifying microbial adhesins that are vital for colonization. phage display technology display on filamentous phage one of the first genetic techniques developed to study protein-ligand interactions was phage display, described by smith in 1985 [1] . this method displays recombinant peptides or proteins on the surface of phage particles, which can then be selected for ('panned' for figure 1 ) by enabling the phage to interact with selected immobilized ligands. the power of phage display lies in its ability to: (i) maintain a physical link between the displayed protein and the dna sequence encoding it; and (ii) screen libraries containing billions of unique peptides and proteins. escherichia coli filamentous phage of the ff class including strains m13, fd and f1 have been extensively used to develop and exploit this technology. these phage are composed of a circular single-stranded dna genome that is encased in a long tube composed of thousands of copies of a single major coat protein, with four additional minor capsid proteins at the tips ( figure 2 ). phage display involves the fusion of foreign dna sequences to the phage genome such that the resulting foreign proteins are expressed in fusion with one of the coat proteins. although all five coat proteins have been used to display proteins or peptides, gene viii protein (pviii) and gene-iii-encoded adsorption protein (piii) are by far the most commonly used [2] . a viable wild-type phage expresses w2700 copies of pviii and 3-5 copies of piii ( figure 2 ) [3] , although this does depend on the size of the phage genome. phage display libraries can be constructed using vectors based on the natural ff phage sequence (i.e. phage vectors) or by using 'phagemids', which are hybrids of phage and plasmid vectors [2, 3] . such phagemids are designed with the origin of replication (ori) from the ff phage, a plasmid origin of replication from e. coli, gene iii and/or gene viii for fusion formation, a multiple cloning site and an antibiotic resistance gene [2] . however, they lack all other phage genes that encode the structural and non-structural proteins that are required to produce a complete phage. phagemids can be grown as plasmids in e. coli and packaged as recombinant ff phage dna with the aid of helper phage, which provide all of the necessary components for phage assembly. the key feature of filamentous phage (as applied to phage display) is that, in contrast to the lytic bacteriophages, filamentous phage are assembled in the cytoplasmic membrane and secreted from infected bacteria without cell lysis [2] (figure 3 ). however, the characteristics of the filamentous phage life cycle has limitations for the display of proteins, the properties of which prevent the correct transfer of the hybrid capsid protein across the lipid bilayer of the inner membrane of e. coli [4] . alternative bacteriophage display systems have been developed using bacteriophage such as t4, t7, l [4] and p4 [5] , which have lytic life cycles so that the proteins of the phage capsids are assembled and folded in the cytoplasm rather than being secreted through the membrane [4] . because the most common approaches to phage display (described earlier) involve n-terminal fusion to the gene iii or gene viii products of filamentous phage, they are unsuitable for surface expression of proteins coded by intact cdna inserts that have stop codons [6, 7] . hence, most phage libraries of cdna fragments are constructed in alternative display systems. however, a modified filamentous phage display system based on the highaffinity interactions between the jun and fos leucine zipper proteins was developed by crameri and suter [6] . in this system, the jun leucine zipper protein is fused to the n terminus of the piii coat protein so that the jun protein is displayed on the surface of the phage, and the cdna libraries are cloned as a c-terminal fusion to the fos leucine zipper protein [8] . the piii-anchored jun protein is bound by the soluble fos fusion in the periplasm, thus, the cdna-encoded protein is bound indirectly to piii. there are two types of phage display library: random peptide libraries (rpls) and natural peptide libraries (npls). the repertoire of peptides displayed in rpls is encoded by synthetic random degenerate oligonucleotide inserts [9] [10] [11] and these libraries have been extensively used to identify linear antigenic epitopes (see later). the advantage of this type of library is their universal nature, which enables many applications of each library. the main disadvantage of rpls is that, because of the way in which they are constructed, peptide sequences that are not found within the antigen or intact pathogen can be displayed [12] . by contrast, npls are constructed from randomly fragmented dna from the genomes of selected organisms such as pathogenic microbes. thus, the phage particles in these libraries display fragments of natural proteins. although the majority of clones in npls are nonfunctional (only one in 18 clones will be correctly in frame with the vector sequences: one clone in three will start correctly, one clone in three will end correctly and one clone in two will be in the correct orientation), peptides or proteins selected from npls are more successful in eliciting an antibody response that crossreacts with the native intact pathogen than those selected from rpls [13] . therefore, npls provide important alternatives to rpls for applications such as the identification of vaccine components and, recently, have been used effectively in the identification of bacterial adhesins (see later). phage display technology has been used in a wide variety of applications including: the identification of peptide agonists and antagonists for receptors [14] , the gvii gii gix gviii giii gvi gx gv gi giv identification of targets for the inhibition of tumourspecific angiogenesis [15] , the identification of peptide drug candidates [16] , vaccine development [17] and the isolation and engineering of recombinant antibodies [18] . however, in recent years, numerous studies have used phage display to address specific aspects of infectious disease. the purpose of this review is to focus on the growing number of ways in which phage display technology can be applied to the study of infectious diseases and to evaluate the impact of the use of this technology. infectious diseases are absolutely dependent upon hostpathogen interactions, the extent of which determines the infectious process both for the pathogen and the host. as discussed later, epitope mapping of infectious agents has been used by numerous research groups, particularly to focus on the bacterial adhesins that enable host colonization. phage display technology has been used to good effect in malaria research to investigate host-pathogen interactions in both hosts of the malaria parasite: homo sapiens and the mosquito anopheles. panning of a plasmodium falciparum (the causative agent of malaria) cdna phage display library against immobilized human erythrocyte membrane proteins identified seven parasite proteins that are potentially involved in the entry into or exit from human erythrocytes by p. falciparum [19] . such proteins could become vaccine targets. the search for the mechanisms used by plasmodium species to cause infection has also prompted novel uses for phage display libraries. the plasmodium parasite completes its life cycle in the mosquito and it is this interaction, rather than that of the human host, that has been investigated by ghosh et al. [20] . because the development of the plasmodium parasite in the mosquito involves the crossing of both midgut and salivary gland epithelia, this study investigated the hypothesis that such trafficking requires specific host-pathogen interaction. a phage display library of random dodecapeptides fused to the n terminus of phage coat protein viii was injected into anopheles, followed by dissection of the organs of interest and elution of bound phage. a 12-residue peptide from the eluted phage was identified and designated 'salivary gland and midgut peptide 1 (sm1)' [20] . this peptide strongly inhibited plasmodium invasion of salivary-gland and midgut epithelia, thereby hindering the development of the parasite in anopheles. this study illustrates another variation of phage display technology -the use of phage display libraries to investigate specific phage-binding targets within the whole organism. in vivo panning was first described by pasqualini and ruoslahti [21] , who isolated phage-displayed peptides that bound to vascular beds in vivo. to date, there are limited reports of such in vivo panning but this could have an important future role for the identification of specific tissue receptors for microbes or their products. a variation on this theme is the panning of phage display libraries against ex vivo biomaterials that have been used in a range of clinical applications including prosthetic heart valves and intravenous catheters. bacteria such as staphylococcus aureus and staphylococcus epidermidis are frequently isolated from biomaterial infections [22] . a phage display library constructed from the genomic dna of s. aureus was panned against a central intravenous catheter that had been removed from a patient [23] . numerous clones from the s. aureus library were recovered through panning and, after the second and third rounds, the enriched phage encoded fragments of bacterial proteins known to bind to fibrinogen and b 2 -glycoprotein i, which, unsurprisingly, were the most abundant host proteins deposited on the catheter. laminin-binding motifs in the surface virulence factor [plasminogen activator (pla)] of yersinia pestis were mapped by benedek et al. [24] using an rpl. this approach involved identification of phage-displayed amino acid sequences that bound to laminin, then comparison of these sequences to the sequence of pla to identify the specific binding site of pla to laminin. this study demonstrates how phage display can be used to map specific binding sites within a protein of interest. epitope mapping and identification of potential vaccine candidate antigens the term epitope can be used to describe the contacting points of any molecular interaction but it is more often used to describe the region on an antigen that elicits an immune response. the identification of epitopes from microbial pathogens has obvious importance in the study of infectious disease, particularly for the development of novel vaccines. phage display technology is especially suited to epitope mapping and has been widely used in infectious-disease research (for review, see ref. [17] ). much of the work on epitope mapping and identification of mimotopes of infectious agents has used rpls. mimotopes are peptide sequences that are capable of inducing immune responses by mimicking the structural features of a non-linear protein epitope or of a non-protein (e.g. carbohydrate) epitope structure. these libraries have been panned against immune sera to identify diseasespecific epitopes. selected examples of these applications are listed in table 1 . in addition, phage display has successfully identified peptide mimics of polysaccharides. this approach has important implications for the development of novel vaccines against encapsulated bacteria because the polysaccharide capsules of these bacteria are often antigenic. peptide mimics of the capsular polysaccharide of three of the neisseria meningitidis serotypes a [25] , b [26] and c [27] have been identified using rpls. the results of these studies are encouraging in that the mimotopes identified in this way did elicit immune responses in murine models and could be useful in the development of novel vaccines against this bacterium. the pathogenesis of s. aureus has been investigated by panning an rpl against the rnaiii-activating protein (rap), which autoinduces toxin production in this bacterium [28, 29] . initial work demonstrated that selected peptides could attenuate infection by s. aureus in murine models [28] . more recent studies have identified a peptide mimic for the rap protein that, when displayed on e. coli and used to vaccinate mice, prevented mortality caused by s. aureus infection [29] . infection with pseudomonas aeruginosa is a major problem in patients with cystic fibrosis (cf). the development of vaccines against this organism must take into account that antibiotic treatment of chronic p. aeruginosa infections rarely results in clearance of the bacterium. this is in contrast to treatment of early p. aeruginosa infections in which the bacteria can be eradicated [30] . antigens expressed early in p. aeruginosa infections of cf patients were investigated by panning an rpl against sera from non-infected and infected patients [31] . in conjunction with gene-array data and bioinformatic analysis, this study identified several genes that encode secreted proteins and outer-membrane proteins that are potential targets for vaccine development against p. aeruginosa infection [31] . alterations in the panning technique used, such as subjecting the library to initial negative selection, further increases the potential uses of these libraries. gnanasekar et al. [32] constructed a t7 phage display library of the cdna of the parasite brugia malayi (a causative agent of lymphatic filariasis). to ensure that the clones of interest (i.e. those binding to infection-specific antibodies) were enriched, the phage library was first subjected to three negative-selection steps using non-infected human sera followed by a positive-selection step using sera from patients infected with b. malayi. further experiments revealed that one of the five antigens identified using this strategy, b. malayi [50] 12mer rpl displayed on piii monoclonal iga1 specific for the capsule of streptococcus pneumoniae identification of mimotopes of the capsular polysaccharide of type 8 s. pneumoniae. when conjugated to tetanus toxoid, mimotopes induced a type 8 capsular-polysaccharide-specific antibody response in mice [51] 7mer rpl displayed on piii sera from swine infected with nipah virus identification of several putative epitopes within the nucleocapsid protein of nipah virus [52] 12mer rpl displayed on piii polyclonal igg specific for neutral polysaccharides of mycoplasma tuberculosis isolation of phage clones that were antigenic mimotopes of b-cell epitopes of m. tuberculosis sugars. one clone invoked immune responses in rabbits [53] review trends in microbiology vol.14 no. 3 march 2006 nip3-like protein, conferred protection against challenge infections in animal models [32] . single chain variable fragment (scfv) libraries have been used in several ways to study infectious diseases. identification of specific scfvs that bind to microbial antigens forms the basis for the development of novel vaccines and diagnostic reagents [3] . panning of a scfv library against purified severe acute respiratory syndrome-associated coronavirus (sars-cov) identified two antibody fragments that bind to sars-cov with high specificity [33] . these could potentially be used in the development of detection assays for the virus or to elucidate potential vaccine candidate antigens. phagedisplay scfv libraries have also been used successfully to develop immunodiagnostic and detection methods for microbial products or spores. one of the most impressive aspects of these applications is the high specificity of selected clones for the microbe. for example, panning of a scfv library against spores of bacillus subtilis resulted in the selection of clones that bound to only one of 11 spore strains [34] . similarly, clones selected from a phage display scfv library screened against clostridium difficile toxin b were highly specific and showed no crossreactivity with strains of c. difficile that are toxin-b negative. the single-chain antibody could also be used to detect as little as 10 ng of toxin b when used in elisas [35] . microbial infections are initiated by molecular interactions between the pathogen (through cell surface adhesins) and receptor molecules on host cells and the extracellular matrix (ecm), resulting in microbial adhesion, which can be followed by internalization [36, 37] . it is well established that the adhesion of enteric, oral and respiratory bacteria is required for colonization [37] . furthermore, when bacteria adhere to surfaces, they are substantially more resistant to host antimicrobial defences [38] . adherence to structures such as the ecm is, therefore, a key step in the development of disease. the ecm consists of a complex mixture of macromolecules including collagens, fibronectin, fibrinogen, vitronectin, laminin and heparin sulfate [38] , all of which function as ligands for bacterial adhesion. with the continued rise in antibiotic resistance in bacteria, there is an urgent need to find alternative ways to combat microbial and, specifically, bacterial infection. inhibition of bacterial adhesion is one potential therapeutic approach but usually requires an intimate knowledge of the adhesins involved in infection. phage display lends itself perfectly to the investigation of possible binding partners for a particular ligand and this application of phage display was first exploited by jacobsson and frykberg [39, 40] to identify genes encoding bacterial proteins that interact with host proteins. by constructing libraries from random fragments of bacterial genomic dna (i.e. shotgun phage display) and subsequent panning against components of the host, such as proteins of the ecm, host serum or plasma, it is possible to identify bacterial genes that encode cell surface adhesins. microbial proteins (including cell-bound and soluble proteins) that bind to mammalian target proteins were termed 'receptins' by kronvall and jonsson [41] . following the initial description of gene-iii-based phage display to identify genes coding for ligand-binding domains of bacterial receptins by jacobsson and frykberg [39] , many genes involved in host-pathogen interactions (from a variety of bacterial species) have been discovered using phage-display systems ( table 2 ). the majority of these studies have been carried out using gene-viii-based vectors such as the pg8h6 or pg8saet phagemid vectors developed by jacobsson and frykberg [40, 42] . panning of piii-based libraries tends to select for fusion proteins with the strongest interaction with the ligand of choice but only a small fraction of piii libraries contain functional inserts [43] . use of pviii generates phage with multiple copies of recombinant proteins but the selection process tends to select for lower affinity interactions. shotgun phage display has also been used in the mapping of binding domains within proteins coded by genes of interest. instead of random fragmentation of genomic dna from a particular species, a previously identified gene of interest is fragmented by sonication and used to generate a single-gene phage display library. this approach has been used to map fibronectin-binding activity to two c-terminal domains of the fibronectinbinding protein fnz of streptococcus equi [44] , the igg and b 2 -glycoprotein-i-binding domains of the sbi protein of s. aureus [42, 45] and the igg and albumin-binding domains in two cell-surface receptors, mag and zag, from group c streptococci [46] . the application of shotgun phage display to the identification of bacterial virulence factors has been further extended by frykberg and colleagues, with the development of phagemid vectors that only enable the display of bacterial exported proteins [47, 48] . many exported microbial proteins are adhesins, enzymes or toxins, all of which might have a role in pathogenicity. the libraries constructed by the shotgun method might not achieve full coverage of bacterial genomes for two reasons: (i) the low transformation efficiency of e. coli limits the number of primary clones (i.e. those containing unique inserts); and (ii) only one in 18 clones displays fusion proteins (see earlier). however, despite these limitations, the success of this method is obvious considering the numbers of shotgun phage display libraries that have been used to identify novel genes that encode putative bacterial receptins and protein binding domains ( table 2) . use of this technique identifies multiple microbial genes encoding proteins that potentially interact with the host in a single panning experiment and such results provide a starting point to determine the importance of various bacterial genes in pathogenesis. it is likely that phage display npls will become even more widely used in the study of infectious diseases, particularly because the growing number of available bacterial genomes means that the dna fragments that encode phage-displayed peptides and proteins can be easily mapped to a particular bacterial gene. the study of infectious diseases has benefited greatly from phage display technology. this technique has many advantages: the link between phenotype and genotype, the enormous diversity of variant proteins displayed within a single library and the flexibility of selection that can be performed in vitro or in vivo. earlier limitations on this technique (such as the unsuitability of piii-or pviii-based filamentous phage display systems for the display of cdna coding for peptides or problems with protein conformation) have been circumvented by continual development of the technology. the versatility of phage display means that it can be adapted easily to many different areas of research, as highlighted by the variety of biological questions to which phage display has been applied. it has yielded interesting and important results in the study of infectious diseases, not least in epitope mapping, the identification of potential vaccine candidates and bacterial adhesins. however, the vast potential of phage display is such that there are many more ways of applying the technology to these areas of research. there is certainly scope for developing new strategies to identify host receptors for microbial products by panning in vivo in animal models or against ex vivo tissues or organs. there are numerous examples of synergistic relationships between opportunistic bacteria that could be examined by panning phage display libraries against each other to investigate interactions between microbes. perhaps the most exciting potential use of phage display in the study of infectious diseases is in comparative genomics. the number of sequenced microbial genomes has increased greatly in the past ten years and continues to do so as more genomes are sequenced. this will enable detailed bioinformatic analysis of genes identified by panning of npls and a comparison of the genes implicated in pathogenicity between groups of closely related bacteria. filamentous fusion phage -novel expression vectors that display cloned antigens on the virion surface introduction to phage display and phage biology phage display technology: clinical applications and recent innovations alternative bacteriophage display systems peptide presentation by bacteriophage p4 display of biologically-active proteins on the surface of filamentous phages -a cdna cloning system for selection of functional gene-products linked to the genetic information responsible for their production pjufo: a phage surface display system for cloning genes based on protein-ligand interaction display of expression products of cdna libraries on phage surfaces -a versatile screening system for selective isolation of genes by specific gene-product ligand interaction peptides on phage -a vast library of peptides for identifying ligands searching for peptide ligands with an epitope library random peptide libraries -a source of specific protein-binding molecules peptides isolated from random peptide libraries on phage elicit a neutralizing anti-hiv-1 response: analysis of immunological mimicry immunogenically fit subunit vaccine components via epitope discovery from natural peptide ligands peptides identify the critical hotspots involved in the biological activation of the insulin receptor in vivo phage display and vascular heterogeneity: implications for targeted medicine phage display-derived peptides as therapeutic alternatives to antibodies epitope identification and discovery using phage display libraries: applications in vaccine development and diagnostics biotechnological applications of phage and cell display construction and use of plasmodium falciparum phage display libraries to identify host parasite interactions targeting plasmodium ligands on mosquito salivary glands and midgut with a phage display peptide library organ targeting in vivo using phage display libraries pathogenesis of infections due to coagulasenegative staphylococci sorting a staphylococcus aureus phage display library against ex vivo biomaterial identification of laminin-binding motifs of yersinia pestis plasminogen activator by phage display selection of an immunogenic peptide mimic of the capsular polysaccharide of neisseria meningitidis serogroup a using a peptide display library peptide mimotopes of neisseria meningitidis group b capsular polysaccharide two different methods result in the selection of peptides that induce a protective antibody response to neisseria meningitidis serogroup c inhibition of staphylococcus aureus pathogenesis in vitro and in vivo by rap-binding peptides a novel peptide screened by phage display can mimic trap antigen epitope against staphylococcus aureus infections significant microbiological effect of inhaled tobramycin in young children with cystic fibrosis use of phage display to identify potential pseudomonas aeruginosa gene products relevant to early cystic fibrosis airway infections novel phage display-based subtractive screening to identify vaccine candidates of brugia malayi identification of single-chain antibody fragments specific against sars-associated coronavirus from phage-displayed antibody library peptide ligands that bind selectively to spores of bacillus subtilis and closely related species recombinant single-chain variable fragment antibodies directed against clostridium difficile toxin b produced by use of an optimized phage display system bacterial adhesins: common themes and variations in architecture and assembly bacterial adhesion to animal cells and tissues mscramm-mediated adherence of microorganisms to host tissues cloning of ligand-binding domains of bacterial receptors by phage display phage display shot-gun cloning of ligand-binding domains of prokaryotic receptors approaches 100% correct clones receptins: a novel term for an expanding spectrum of natural and engineered microbial proteins with binding properties for mammalian proteins a second igg-binding protein in staphylococcus aureus shotgun phage display -selection for bacterial receptins or other exported proteins fibronectin-binding protein of streptococcus equi subsp zooepidemicus staphylococcus aureus expresses a cell surface protein that binds both igg and b2-glycoprotein i shot-gun phage display mapping of two streptococcal cell-surface proteins phage display as a novel screening method to identify extracellular proteins identification of a novel collagen-like protein, sclc, in streptococcus equi using signal sequence phage display epitope mapping of burkholderia pseudomallei serine metalloprotease: identification of serine protease epitope mimics epitope mapping of mycoplasma hyopneumoniae using phage displayed peptide libraries and the immune responses of the selected phagotopes a peptide mimotope of type 8 pneumococcal capsular polysaccharide induces a protective immune response in mice identification of epitopes in the nucleocapsid protein of nipah virus using a linear phage-displayed random peptide library peptide mimotopes of mycobacterium tuberculosis carbohydrate immunodeterminants molecular cloning and characterization of two helicobacter pylori genes coding for plasminogen-binding proteins a novel von willebrand factor binding protein expressed by staphylococcus aureus staphylococcus aureus fibronectin-binding protein (fnbp)-mediated adherence to platelets, and aggregation of platelets induced by fnbpa but not by fnbpb interaction of staphylococcus aureus fibronectin-binding protein with fibronectin -affinity, stoichiometry, and modular requirements identification of a fibronectin-binding protein from staphylococcus epidermidis staphylococcus aureus fibronectin binding proteins a and b possess a second fibronectin binding region that may have biological relevance to bone tissues a fibrinogen-binding protein of staphylococcus epidermidis identification of novel adhesins from group b streptococci by use of phage display reveals that c5a peptidase mediates fibronectin binding a novel family of fibrinogen-binding proteins in streptococcus agalactiae m-like proteins of streptococcus dysgalactiae sfs, a novel fibronectin-binding protein from streptococcus equi, inhibits the binding between fibronectin and collagen a von willebrand factor-binding protein from staphylococcus lugdunensis a fibrinogen-binding protein of staphylococcus lugdunensis key: cord-262119-s6hc7fxs authors: ostaszewski, marek; niarakis, anna; mazein, alexander; kuperstein, inna; phair, robert; orta-resendiz, aurelio; singh, vidisha; aghamiri, sara sadat; acencio, marcio luis; glaab, enrico; ruepp, andreas; fobo, gisela; montrone, corinna; brauner, barbara; frischman, goar; monraz gómez, luis cristóbal; somers, julia; hoch, matti; gupta, shailendra kumar; scheel, julia; borlinghaus, hanna; czauderna, tobias; schreiber, falk; montagud, arnau; de leon, miguel ponce; funahashi, akira; hiki, yusuke; hiroi, noriko; yamada, takahiro g.; dräger, andreas; renz, alina; naveez, muhammad; bocskei, zsolt; messina, francesco; börnigen, daniela; fergusson, liam; conti, marta; rameil, marius; nakonecnij, vanessa; vanhoefer, jakob; schmiester, leonard; wang, muying; ackerman, emily e.; shoemaker, jason; zucker, jeremy; oxford, kristie; teuton, jeremy; kocakaya, ebru; summak, gökçe yağmur; hanspers, kristina; kutmon, martina; coort, susan; eijssen, lars; ehrhart, friederike; rex, d. a. b.; slenter, denise; martens, marvin; haw, robin; jassal, bijay; matthews, lisa; orlic-milacic, marija; senff ribeiro, andrea; rothfels, karen; shamovsky, veronica; stephan, ralf; sevilla, cristoffer; varusai, thawfeek; ravel, jean-marie; fraser, rupsha; ortseifen, vera; marchesi, silvia; gawron, piotr; smula, ewa; heirendt, laurent; satagopam, venkata; wu, guanming; riutta, anders; golebiewski, martin; owen, stuart; goble, carole; hu, xiaoming; overall, rupert w.; maier, dieter; bauch, angela; gyori, benjamin m.; bachman, john a.; vega, carlos; grouès, valentin; vazquez, miguel; porras, pablo; licata, luana; iannuccelli, marta; sacco, francesca; nesterova, anastasia; yuryev, anton; de waard, anita; turei, denes; luna, augustin; babur, ozgun; soliman, sylvain; valdeolivas, alberto; esteban-medina, marina; peña-chilet, maria; helikar, tomáš; puniya, bhanwar lal; modos, dezso; treveil, agatha; olbei, marton; de meulder, bertrand; dugourd, aurélien; naldi, aurelien; noel, vincent; calzone, laurence; sander, chris; demir, emek; korcsmaros, tamas; freeman, tom c.; augé, franck; beckmann, jacques s.; hasenauer, jan; wolkenhauer, olaf; wilighagen, egon l.; pico, alexander r.; evelo, chris t.; gillespie, marc e.; stein, lincoln d.; hermjakob, henning; d’eustachio, peter; saez-rodriguez, julio; dopazo, joaquin; valencia, alfonso; kitano, hiroaki; barillot, emmanuel; auffray, charles; balling, rudi; schneider, reinhard title: covid-19 disease map, a computational knowledge repository of sars-cov-2 virus-host interaction mechanisms date: 2020-10-27 journal: biorxiv doi: 10.1101/2020.10.26.356014 sha: doc_id: 262119 cord_uid: s6hc7fxs we hereby describe a large-scale community effort to build an open-access, interoperable, and computable repository of covid-19 molecular mechanisms the covid-19 disease map. we discuss the tools, platforms, and guidelines necessary for the distributed development of its contents by a multi-faceted community of biocurators, domain experts, bioinformaticians, and computational biologists. we highlight the role of relevant databases and text mining approaches in enrichment and validation of the curated mechanisms. we describe the contents of the map and their relevance to the molecular pathophysiology of covid-19 and the analytical and computational modelling approaches that can be applied to the contents of the covid-19 disease map for mechanistic data interpretation and predictions. we conclude by demonstrating concrete applications of our work through several use cases. the coronavirus disease 2019 (covid-19) pandemic due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1] has already resulted in the infection of over 40 million people worldwide, of whom one million have died 1 . the molecular pathophysiology that links sars-cov-2 infection to the clinical manifestations and course of covid-19 is complex and spans multiple biological pathways, cell types and organs [2, 3] . to gain the insights into this complex network, the biomedical research community needs to approach it from a systems perspective, collecting the mechanistic knowledge scattered across the scientific literature and bioinformatic databases, and integrating it using formal systems biology standards. with this goal in mind, we initiated a collaborative effort involving over 230 biocurators, domain experts, modelers and data analysts from 120 institutions in 30 countries to develop the covid-19 disease map, an open-access collection of curated computational diagrams and models of molecular mechanisms implicated in the disease [4] . to this end, we aligned the biocuration efforts of the disease maps community [5, 6] , reactome [7] , and wikipathways [8] and developed common guidelines utilising standardised encoding and annotation schemes, based on community-developed systems biology standards [9] [10] [11] , and persistent identifier repositories [12] . moreover, we integrated relevant knowledge from public repositories [13] [14] [15] [16] and text mining resources, providing a means to update and refine contents of the map. the fruit of these efforts was a series of pathway diagrams describing key events in the covid-19 infectious cycle and host response. we ensured that this comprehensive diagrammatic description of disease mechanisms is machine-readable and computable. this allows us to develop novel bioinformatics workflows, creating executable networks for analysis and prediction. in this way, the map is both human and machine-readable, lowering the communication barrier between biocurators, domain experts, and computational biologists significantly. computational modelling, data analysis, and their informed interpretation using the contents of the map have the potential to identify molecular signatures of disease predisposition and development, and to suggest drug repositioning for improving current treatments. covid-19 disease map is a collection of 41 diagrams containing 1836 interactions between 5499 elements, supported by 617 publications and preprints. the summary of diagrams available in the covid-19 disease map can be found online 2 in supplementary material 1. the map is a constantly evolving resource, refined and updated by ongoing efforts of biocuration, sharing and analysis. here, we report its current status. in section 2 we explain the set up of our community effort to construct the interoperable content of the resource, involving biocurators, domain experts and data analysts. in section 3 we demonstrate that the scope of the biological maps in the resource reflects the state-ofthe-art about the molecular biology of covid-19. next, we outline analytical workflows that can be used on the contents of the map, including initial, preliminary outcomes of two such workflows, discussed in detail as use cases in section 4. we conclude in section 5 with an outlook to further development of the covid-19 map and the utility of the entire resource in future efforts towards building and applying disease-relevant computational repositories. the covid-19 disease map project involves three main groups: (i) biocurators, (ii) domain experts, and (iii) analysts and modellers: i. biocurators develop a collection of systems biology diagrams focused on the molecular mechanisms of sars-cov-2. ii. domain experts refine the contents of the diagrams, supported by interactive visualisation and annotations. iii. analysts and modellers develop computational workflows to generate hypotheses and predictions about the mechanisms encoded in the diagrams. all three groups have an important role in the process of building the map, by providing content, refining it, and defining the downstream computational use of the map. figure 1 illustrates the ecosystem of the covid-19 disease map community, highlighting the roles of different participants, available format conversions, interoperable tools, and downstream uses. the information about the community members and their contributions are disseminated via the fairdomhub [17] , so that content distributed across different collections can be uniformly referenced. the biocurators of the covid-19 disease map diagrams follow the guidelines developed by the community, and specific workflows of wikipathways [8] and reactome [7] . the biocurators build literature-based systems biology diagrams, representing the molecular processes implicated in the covid-19 pathophysiology, their complex regulation and the phenotypic outcomes. these diagrams are main building blocks of the map, and are composed of biochemical reactions and interactions (further called altogether interactions) taking place between different types of molecular entities in various cellular compartments. as there are multiple teams working on related topics, biocurators can provide an expert review across pathways and across platforms. this is possible, as all platforms offer intuitive visualisation, interpretation, and analysis of pathway knowledge to support basic and clinical research, genome analysis, modelling, systems biology, and education. table 1 lists information about the created content. for more details see supplementary material 1. communicating to refine, interpret and apply covid-19 disease map diagrams. these diagrams are created and maintained by biocurators, following pathway database workflows or standalone diagram editors, and reviewed by domain experts. the content is shared via pathway databases or a gitlab repository; all can be enriched by integrated resources of text mining and interaction databases. the covid-19 disease map diagrams, available in layout-aware systems biology formats and integrated with external repositories, are available in several formats allowing a range of computational analyses, including network analysis and boolean, kinetic or multiscale simulations. both interactions and interacting entities are annotated following a uniform, persistent identification scheme, using either miriam or identifiers.org [18] , and the guidelines for annotations of computational models [19] . viral protein interactions are explicitly annotated with their taxonomy identifiers to highlight findings from strains other than sars-cov-2. moreover, tools like modelpolisher [20] , sbmlsqueezer [21] or memote 3 help to automatically complement the annotations in the sbml format and validate the model (see also supplementary material 2). the knowledge on covid-19 mechanisms is rapidly evolving, as demonstrated by the rapid growth of the covid-19 open research dataset (cord-19) dataset, a source scientific manuscript text and metadata on covid-19 and related coronavirus research [28] . cord-19 currently contains over 130,000 articles and preprints, over four times more than when it was introduced 10 . in such a quickly evolving environment, biocuration efforts need to be supported by other repositories of structured knowledge about molecular mechanisms relevant for covid-19, like molecular interaction databases, or text mining resources. contents of such repositories may suggest improvements in the existing covid-19 disease map diagrams, or establish a starting point for developing new pathways (see section "biocuration of database and text mining content"). interaction and pathway databases contain structured and annotated information on protein interactions or causal relationships. while interaction databases focus on pairs of molecules, offering broad coverage of literature-reported findings. pathway databases provide detailed description of biochemical processes and their regulations of related interactions, supported by diagrams. both types of resources can be a valuable input for covid-19 disease map biocurators, given the comparability of identifiers used for molecular annotations, and the reference to publications used for defining an interaction or building a pathway. table 2 text-mining approaches can help to sieve through such rapidly expanding literature with natural language processing (nlp) algorithms based on semantic modelling, ontologies, and linguistic analysis to automatically extract and annotate relevant sentences, biomolecules, and their interactions. this scope was recently extended to pathway figure mining: decoding pathway figures into their computable representations [30] . altogether, these automated workflows lead to the construction of knowledge graphs: semantic networks incorporating ontology concepts, unique biomolecule references, and their interactions extracted from abstracts or full-text documents [31] . the covid-19 disease map project integrates open-access text mining resources, indra [32] , biokb 15 , ailani covid-19 16 , and pathwaystudio 10 . all platforms offer keyword-based search allowing interactive exploration. additionally, the map benefits from an extensive protein-protein interaction network (ppi) 17 generated with a custom text-mining pipeline using opennlp 18 and gnormplus [33] . this pipeline was applied to the cord-19 dataset and the collection of medline abstracts associated with the genes in the sars-cov-2 ppi network [34] using the entrez gene reference-into-function (generif). for detailed descriptions of the resources, see supplementary material 3. molecular interactions from databases and knowledge graphs from text mining resources discussed above (from now on called altogether 'knowledge graphs') have a broad coverage at the cost of depth of mechanistic representation. this content can be used by the biocurators in the process of building and updating the systems biology focused diagrams. biocurators can use this content in three main ways: by visual exploration, by programmatic comparison, and by direct incorporation of the content. first, the biocurators can visually explore the contents of the knowledge graphs using available search interfaces to locate new knowledge and encode it in the diagrams. moreover, solutions like covidminer project 19 , pathwaystudio and ailani offer a visual representation of a group of interactions for a better understanding of their biological context, allowing search by interactions, rather than just isolated keywords. finally, indra and ailani offer assistant bots that respond to natural language queries and return meaningful answers extracted from knowledge graphs. second, programmatic access and reproducible exploration of the knowledge graphs is possible via data endpoints: sparql for biokb and application programming interfaces for indra, ailani, and pathway studio. users can programmatically submit keyword queries and retrieve functions, interactions, pathways, or drugs associated with submitted gene lists. this way, otherwise time-consuming tasks like an assessment of completeness of a given diagram, or search for new literature evidence, can be automated to a large extent. finally, biocurators can directly incorporate the content of knowledge graphs into sbml format using biokc [35] . additionally, the contents of the elsevier covid-19 pathway collection can be translated to sbgnml 20 preserving the layout of the diagrams. the sbgnml content can then be converted into other diagram formats used by biocurators (see section 2.3 below). the biocuration of the covid-19 disease map is distributed across multiple teams, using varying tools and associated systems biology representations. this requires a common approach to annotations of evidence, biochemical reactions, molecular entities and their interactions. moreover, the interoperability of layout-aware formats is needed for comparison and integration of the diagrams in the map. the covid-19 disease map diagrams are encoded in one of three layout-aware formats for standardised representation of molecular interactions: sbml 21 [36] [37] [38] , sbgnml [27] , and gpml [24] . these xml-based formats focus to a varying degree on user-friendly graphical representation, standardised visualisation, and support of computational workflows. for the detailed description of the formats, see supplementary material 1. each of these three languages has a different focus: sbml emphasizes standardised representation of the data model underlying molecular interactions, sbgnml provides standardised graphical representation of molecular processes, while gpml allows for a partially standardised representation of uncertain biological knowledge. nevertheless, all three formats are centered around molecular interactions, provide a constrained vocabulary to encode element and interaction types, encode layout of their diagrams and support stable identifiers for diagram components. these shared properties, supported by a common ontology 22 [39] , allow cross-format mapping and enable translation of key properties between the formats. therefore, when developing the contents of the map, biocurators use the tools they are familiar with, facilitating this distributed task. the covid-19 disease map community ecosystem of tools and resources (see figure 1 ) ensures interoperability between the three layout-aware formats for molecular mechanisms: sbml, sbgnml, and gpml. essential elements of this setup are tools capable of providing cross-format translation functionality [40, 41] and supporting harmonised visualisation processing. another essential translation interface is a representation of reactome pathways in wikipathways gpml [42] and sbml. the sbml export of reactome content has been optimised in the context of this project and facilitates integration with the other covid-19 disease map software components. the contents of the covid-19 disease map diagrams can be directly transformed into inputs of computational pipelines and data repositories. besides the direct use of sbml format in kinetic simulations, celldesigner sbml files can be transformed into sbml qual [43] using casq [44] , enabling boolean modelling-based simulations (see also supplementary material 3). in parallel, casq converts the diagrams to the sif format 23 , supporting pathway modelling workflows using simplified interaction networks. notably, the gitlab repository features an automated translation of stable versions of diagrams into sbml qual. finally, translation of the diagrams into xgmml format (the extensible graph markup and modelling language) using cytoscape [45] or ginsim [46] allows for network analysis and interoperability with molecular interaction repositories [47] . thanks to the community effort discussed above supported by a rich bioinformatics framework, we constructed the covid-19 disease map, focussing on the mechanisms known from other coronaviruses [48] and suggested by early experimental investigations [pmid:32511329]. then, we applied the analytical and modelling workflows to the contributed diagrams and associated interaction databases to propose initial map-based insights into covid-19 molecular mechanisms. the covid-19 disease map is an evolving repository of pathways affected by sars-cov-2. figure 2 . it is currently centred on molecular processes involved in sars-cov-2 entry, 22 http://www.ebi.ac.uk/sbo/main/ 23 http://www.cbmc.it/fastcent/doc/sifformat.htm replication, and host-pathogen interactions. as mechanisms of host susceptibility, immune response, cell and organ specificity emerge, these will be incorporated into the next versions of the map. the covid-19 map represents the mechanisms in a "host cell". this follows literature reports on cell specificity of sars-cov-2 [3, [49] [50] [51] [52] [53] . some pathways included in the covid-19 map may be shared among different cell types, as for example the ifn-1 pathway found in cells such as dendritic, epithelial, and alveolar macrophages [54] [55] [56] [57] [58] . while at this stage, we do not address cell specificity explicitly in our diagrams, extensive annotations may allow identification of pathways relevant to the cell type of interest. the sars-cov-2 infection process and covid-19 progression follow a sequence of steps ( figure 3 ), starting from viral attachment and entry, which involve various dynamic processes on different time scales that are not captured in static representations of pathways. correlation of symptoms and potential drugs suggested to date helps downstream data exploration and drug target interpretation in the context of therapeutic interventions. human host ilc1, ilc-2, ilc3 natural killer renin-angiotensinaldosterone system (raas) granulocytes nasal mucosa disease map golgi er cd8+ cd4+ ace2 tmprss2 integrative stress response dendritic cells transmission of sars-cov-2 primarily occurs through contact with respiratory drops, airborne transmission, and through contact with contaminated surfaces [66] [67] [68] . upon contact with the respiratory epithelium, the virus infects cells mostly by binding the spike surface glycoprotein (s) to angiotensin-converting enzyme 2 (ace2) with the help of serine protease tmprss2 [69] [70] [71] [72] . importantly, recent results suggest viral entry using other receptors of lungs and the immune system [73, 74] . once attached, sars-cov-2 can enter cells either by direct fusion of the virion and cell membranes in the presence of proteases • dendritic cells. • nk cells. • monocytes and macrophages. • t cells, th1 and th2 response. • b cells, antibody production. asymptomatic/pre -symptomatic. vaccine? pre-exposure prophylaxis? antivirals? sirs, shock. shortness of breath. anosmia, ageusia, cough, fever, diarrhea. multiple organ dysfunction ards, complications. host response • cellular stress. • apoptosis. systemic and ventilation support oxygen therapy host raas ards; acute respiratory distress syndrome. raas; renin-angiotensin-aldosterone system. sirs; systemic inflammatory response syndrome. pathophysiology virus-host cell interactions and host response disease map critical asymptomatic (lung, heart, kidney) (nasal and respiratory epithelium, alveoli, vascular endothelial) tmprss2 and furin or by endocytosis in their absence. regardless of the entry mechanism, the s protein has to be activated to initiate the plasma or endosome membrane fusion process. while in the cell membrane, s protein is activated by tmprss2 and furin, in the endosome s protein is activated by cathepsin b (ctsb) and cathepsin l (ctsl) [71, 75] . activated s promotes the cell-or endosome-membrane fusion [76] with the virion membrane, and then the nucleocapsid is injected into the cytoplasm. these mechanisms are represented in the corresponding diagrams of the map 25 . within the host cell, sars-cov-2 hijacks the rough endoplasmic reticulum (rer)-linked host translational machinery. it then synthesises viral proteins replicase polyprotein 1a (pp1a) and replicase polyprotein 1ab (pp1ab) directly from the virus (+)genomic rna (grna) [48, 77] . through a complex cascade of proteolytic cleavages, pp1a and pp1ab give rise to 16 non-structural proteins (nsps) [78] [79] [80] . most of these nsps collectively form the replication transcription complex (rtc) that is anchored to the membrane of the double-membrane vesicle [78, 81] endoplasmic reticulum stress and unfolded protein response as discussed above, the virus hijacks the er to replicate. production of large amounts of viral proteins exceeds the protein folding capacity of the er, creating an overload of unfolded proteins. as a result, the unfolded protein response (upr) pathways are triggered to assure the er homeostasis, using three main signalling routes of upr via perk, ire1, and atf6 [87]. their role is to mitigate the misfolded protein load and reduce oxidative stress. the resulting protein degradation is coordinated with a decrease in protein synthesis via eif2alpha phosphorylation and induction of protein folding genes via the transcription factor xbp1 [88] . when the er is unable to restore its function, it can trigger cell apoptosis [89, 90] . the results are er stress and activation of the upr. the expression of some human coronavirus (hcov) proteins during infection, in particular the s glycoprotein, may induce activation of the er stress in the host cells [91] . based on sars-cov results, this may lead to activation of the perk [92], ire1 and in an indirect manner, of the atf6 pathways [93] . processes of degrading malfunctioning proteins and damaged organelles, including the ubiquitin-proteasome system (ups) and autophagy [94] are essential to maintain energy homeostasis and prevent cellular stress [95, 96] . autophagy is also involved in cell defence, including direct destruction of the viruses via virophagy, presentation of viral antigens, and inhibition of excessive inflammatory reactions [97, 98] . sars-cov-2 directly affects the process of ups-based protein degradation, as indicated by the host-virus interactome dataset published recently [34] . this mechanism may be a defence against viral protein degradation [99] . the map describes in detail the nature of this interaction, namely the impact of orf10 virus protein on the cul2 ubiquitin ligase complex and its potential substrates. interactions between sars-cov-2 and host autophagy pathways are inferred based on results from other covs. a finding that covs use double-membrane vesicles and lc3-i for replication [100] may suggest that the virus induces autophagy, possibly in atg5-dependent manner [101] , although some evidence points to the contrary [102] . also, the cov nsp6 restricts autophagosome expansion, compromising the degradation of viral components [103] . recently revealed mutations in nsp6 [104] indicate its importance, although the exact effect of the mutations remains unknown. based on the connection between autophagy and the endocytic pathway of the virus replication cycle [105] , autophagy modulation was suggested as a potential therapy strategy, either pharmacologically [96, [105] [106] [107] , or via fasting [108] . apoptosis, a synonym for programmed cell death, is triggered by virus-host interaction upon infection, as the early death of the virus-infected cells may prevent viral replication. many viruses block or delay cell death by expressing anti-apoptotic proteins to maximize the production of viral progeny [109] . in turn, apoptosis induction at the end of the viral replication cycle might assist in viral dissemination while reducing an inflammatory response. for instance, sars-cov-2 [110] and mers [111] are able to invoke apoptosis in lymphocytes, compromising the immune system. apoptosis follows two major pathways [112] , called extrinsic and intrinsic. extrinsic signals are transmitted by death ligands and their receptors (e.g., fasl and tnf-alpha). activated death receptors recruit adaptors like fadd and tradd, and initiator procaspases like caspase-8, leading to cell death with the help of effector caspases-3 and 7 [113, 114] . in turn, the intrinsic pathway involves mitochondria-related members of the bcl-2 protein family. cellular stress causes bcl-2 proteins-mediated release of cytochrome c from the mitochondria into the cytoplasm. cytochrome c then forms a complex with apaf1 and recruits initiator procaspase-9 to form the apoptosome, leading to the proteolytic activation of caspase-9. activated caspase-9 can now initiate the caspase cascade by activating effector caspases 3 and 7 [114] . the intrinsic pathway is modulated by sars-cov molecules [115, 116] . as intrinsic apoptosis involves mitochondria, its activity may also be exacerbated by sars-cov-2 disruptions of the electron transport chain, mitochondrial translation, and transmembrane transport [34] . the resulting mitochondrial dysfunction may lead to increased release of reactive oxygen species and pro-apoptotic factors. another vital crosstalk is that of the intrinsic pathway with the pi3k-akt pro-survival pathway. activated akt can phosphorylate and inactivate various pro-apoptotic proteins, including bad and caspase-9 [117] . sars-cov uses pi3k-akt signalling cascade to enhance infection [118] . moreover, sars-cov could affect apoptosis in a cell-type-specific manner [119, 120] . sars-cov structural proteins s, e, m, n, and accessory proteins 3a, 3b, 6, 7a, 8a, and 9b have been shown to act as crucial effectors of apoptosis in vitro. structural proteins seem to affect mainly the intrinsic apoptotic pathway, with p38 mapk and pi3k/akt pathways regulating cell death. accessory proteins can induce apoptosis via different cascades and in a cellspecific manner [114] . sars-cov e and 7a protein were shown to activate the intrinsic pathway by blocking anti-apoptotic bcl-xl localized to the er [121] . sars-cov m protein and the ion channel activity of e and 3a were shown to interfere with pro-survival signalling cascades [114, 122] . the viral replication and the consequent immune and inflammatory responses cause damage to the epithelium and pulmonary capillary vascular endothelium and activate the main intracellular defence mechanisms, as well as the humoral and cellular immune responses. resulting cellular stress and tissue damage [123, 124] impair respiratory capacity and lead to acute respiratory distress syndrome (ards) [61, 125, 126] . hyperinflammation is a known complication, causing widespread damage, organ failure, and death, followed by a not yet completely understood rapid increase of cytokine levels (cytokine storm) [127] [128] [129] , and acute ards [130] . other reported complications, such as coagulation disturbances and thrombosis are associated with severe cases, but the specific mechanisms are still unknown [64, [131] [132] [133] , although some reports suggest that covid-19 coagulopathy has a distinct profile [134] . the sars-cov-2 infection disrupts the coagulation cascade and is frequently associated with hyperinflammation, renin-angiotensin system (ras) imbalance and intravascular coagulopathy [132, [135] [136] [137] . hyperinflammation leads in turn to detrimental hypercoagulability and immunothrombosis, leading to microvascular thrombosis with further organ damage [138] . importantly, ras is influenced by risk factors of developing severe forms of covid-19 [139] [140] [141] . ace2, used by sars-cov-2 for host cell entry, is a regulator of ras and is widely expressed in the affected organs [142] . the main function of ace2 is the conversion of angii to angiotensin 1-7 (ang1-7), and these two angiotensins trigger the counter-regulatory arms of ras [143] . the signalling via angii and its receptor agtr1, elevated in the infected [142, 144] , induces the coagulation cascade leading to microvascular thrombosis [145] , while ang1-7 and its receptor mas1 attenuate these effects [143] . the innate immune system detects specific pathogen-associated molecular patterns (pamps), through pattern recognition receptors (prrs). detection of sars-cov-2 is mediated through receptors that recognise double-stranded and single-stranded rna in the endosome during endocytosis of the virus particle, or in the cytoplasm during the viral replication. these receptors mediate the activation of transcription factors such as ap1, nfkappab, irf3, and irf7, responsible for the transcription of antiviral proteins, in particular, interferon-alpha and beta [146, 147] . sars-cov-2 reduces the production of type i interferons to evade the immune response [49] . the detailed mechanism is not clear yet; however, sars-cov m protein inhibits the irf3 activation [148] and suppresses nfkappab and cox2 transcription. at the same time, sars-cov n protein activates nfkappab [149] , so the overall impact is unclear. these pathways are also negatively regulated by sars-cov nsp3 papain-like protease domain (plpro) [150] . the map contains the initial recognition process of the viral particle by the innate immune system and the viral mechanisms to evade the immune response. it provides the connection between virus entry (detecting the viral endosomal patterns), its replication cycle (detection cytoplasmic viral patterns), and the effector pathways of pro-inflammatory cytokines, especially of the interferon type i class. the latter seems to play a crucial but complex role in covid-19 pathology: both negative [151, 152] and positive effects [3, 153] of interferons on virus replication have been reported. interferon type i signalling interferons (ifns) are central players in the antiviral immune response of the host cell [55] , specifically affected by sars-cov-2 [154] [155] [156] [157] . type i ifns are induced upon viral recognition of pamps by various host prrs [48] as discussed earlier. the ifn-i pathway diagram represents the activation of tlr7 and ifnar and the subsequent recruiting of adaptor proteins and the downstream signalling cascades regulating key transcription factors including irf3/7, nf-kappab, ap-1, and isre [48, 158] . further, the map shows irf3mediated induction of ifn-i, affected by the sars-cov-2 proteins. sars-cov nsp3 and orf6 interfere with irf3 signalling [159, 160] and sars-cov m, n, nsp1 and nsp3 act as interferon antagonists [48, 150, 158, 161, 162] . moreover, coronaviruses orf3a, orf6 and nsp1 proteins can repress interferon expression and stimulate the degradation of ifnar1 and stat1 during the unfolded protein response (upr) [163, 164] . another mechanism of viral rna recognition is rig-like receptor signalling [58] , leading to sting activation [165] , and via the recruitment of traf3, tbk1 and ikkepsilon to phosphorylation of irf3 [56] . this in turn induces the transcription of ifns alpha, beta and lambda [166] . sars-cov viral papain-like-proteases, contained within the nsp3 and nsp 16 proteins, inhibit sting and the downstream ifn secretion [167] . in line with this hypothesis, sars-cov-2 infection results in a unique inflammatory response defined by low levels of ifn-i and high expression of cytokines [58, 168] . the ifnlambda diagram describes the ifnl receptor signaling cascade [169] , including jak-stat signaling and the induction of interferon stimulated genes, which encode antiviral proteins [170] . the interactions of sars-cov-2 proteins with the ifnl pathway are based on the literature [171] or sars-cov homology [58] . metabolic pathways govern the immune microenvironment by modulating the availability of nutrients and critical metabolites [172] . infectious entities reprogram host metabolism to create favourable conditions for their reproduction [173] . sars-cov-2 proteins interact with a variety of immunometabolic pathways, several of which are described below. heme catabolism is a well-known anti-inflammatory system in the context of infectious and autoimmune diseases [174, 175] . the main effector of this pathway, heme oxygenase-1 (hmox1) was found to interact with sars-cov-2 orf3a, although the nature of this interaction remains ambiguous [34, 176] . hmox1 cleaves heme into carbon monoxide, biliverdin (then reduced to bilirubin), and ferrous iron [pmid:31396090]. biliverdin, bilirubin, and carbon monoxide possess cytoprotective properties, and have shown promise as immunomodulatory therapeutics [177, 178] . importantly, activation of hmox1 also inhibits the nlrp3 inflammasome [178] [179] [180] , which is a pro-inflammatory and prothrombotic multiprotein system [181] highly active in covid-19 [182] [183] [184] . it mediates production of the pro-inflammatory cytokines il-1b and il-18 via caspase-1 [181] . the sars-cov orf3a, e, and orf8a incite the nlrp3 inflammasome [185] [186] [187] [188] . still, the potential of the hmox1 pathway to fight covid-19 inflammation remains to be tested [176, 189, 190] despite promising results in other models of inflammation [176, 178, [191] [192] [193] . the tryptophan-kynurenine pathway is closely related to heme metabolism. the ratelimiting step of this pathway is catalysed by the indoleamine 2,3 dioxygenase enzymes (ido1 and ido2) in dendritic cells, macrophages, and epithelial cells in response to inflammatory cytokines like ifn-gamma, ifn-1, tgf-beta, tnf-alpha, and il-6 [194] [195] [196] . crosstalk with the hmox1 pathway also increases the expression of ido1 and hmox1 in a feed-forward manner. metabolomics analyses from severe covid-19 patients revealed enrichment of kynurenines and depletion of tryptophan, indicating robust activation of ido enzymes [197, 198] . depletion of tryptophan [173, 199, 200] and kynurenines and their derivatives affect the proliferation and immune response of a range of t cells [176, [201] [202] [203] [204] [205] . however, despite high levels of kynurenines in covid-19, cd8+ t-cells and th17 cells are enriched in lung tissue, and t-regulatory cells are diminished [206] . this raises the question of whether and how the immune response elicited in covid-19 evades suppression by the kynurenine pathway. the sars-cov-2 protein nsp14 interacts with three human proteins: gla, sirt5, and impdh2 [34] . the galactose metabolism pathway, including the gla enzyme [207] , is interconnected with amino sugar and nucleotide sugar metabolism. sirt5 is a naddependent desuccinylase and demalonylase regulating serine catabolism, oxidative metabolism and apoptosis initiation [208] [209] [210] . moreover, nicotinamide metabolism regulated by sirt5 occurs downstream of the tryptophan metabolism, linking it to the pathways discussed above. finally, impdh2 is the rate-limiting enzyme in the de novo synthesis of gtp, allowing regulation of purine metabolism and downstream potential antiviral targets [211, 212] . the pyrimidine synthesis pathway, tightly linked to purine metabolism, affects viral dna and rna synthesis. pyrimidine deprivation is a host targeted antiviral defence mechanism, which blocks viral replication in infected cells and can be regulated pharmacologically [213] [214] [215] . it appears that components of the dna damage response connect the inhibition of pyrimidine biosynthesis to the interferon signalling pathway, probably via sting-induced tbk1 activation that amplifies interferon response to viral infection, discussed above. inhibition of de novo pyrimidine synthesis may have beneficial effects on the recovery from covid-19 [215] ; however, this may happen only in a small group of patients. covid-19 pathways featured in the previous section cover mechanisms reported so far. still, certain aspects of the disease were not represented in detail because of their complexity, namely cell-type-specific immune response, and susceptibility features. their mechanistic description is of great importance, as suggested by clinical reports on the involvement of these pathways in the molecular pathophysiology of the disease. the mechanisms outlined below will be the next targets in our curation roadmap. cell type-specific immune response covid-19 causes serious disbalance in multiple populations of immune cells. some studies report that covid-19 patients have a significant decrease of peripheral cd4+ and cd8+ cytotoxic t lymphocytes (ctls), b cells, nk cells, as well as higher levels of a broad range of cytokines and chemokines [128, [216] [217] [218] [219] . the disease causes functional exhaustion of cd8+ ctls and nk cells, induced by sars-cov-2 s protein and by excessive pro-inflammatory cytokine response [217, 220] . moreover, the ratio of naïve-to-memory helper t-cells, as well as the decrease of t regulatory cells, correlate with covid-19 severity [206] . conversely, high levels of th17 and cytotoxic cd8+ t-cells have been found in the lung tissue [221] . pulmonary recruitment of lymphocytes into the airways may explain the lymphopenia and the increased neutrophil-lymphocyte ratio in peripheral blood found in covid-19 patients [216, 222, 223] . in this regard, an abnormal increase of the th17:treg cell ratio may promote the release of pro-inflammatory cytokines and chemokines, increasing disease severity [224] . sars-cov-2 infection is associated with increased morbidity and mortality in individuals with underlying chronic diseases or a compromised immune system [225] [226] [227] [228] . groups of increased risk are men, pregnant and postpartum women, and individuals with high occupational viral exposure [229] [230] [231] . other susceptibility factors include the abo blood groups [232] [233] [234] [235] [236] [237] [238] [239] [240] and respiratory conditions [241] [242] [243] [244] [245] [246] . importantly, age is one of the key aspects contributing to the severity of the disease. the elderly are at high risk of developing severe or critical disease [227, 247] . age-related elevated levels of pro-inflammatory cytokines (inflammation) [247] [248] [249] [250] , immunosenescence and cellular stress of ageing cells [125, 227, 247, 251, 252] may contribute to the risk. in contrast, children are generally less likely to develop severe disease [253, 254] , with the exception of infants [125, [255] [256] [257] . however, some previously healthy children and adolescents can develop a multisystem inflammatory syndrome following sars-cov-2 infection [258] [259] [260] [261] [262] . several genetic factors have been proposed and identified to influence susceptibility and severity, including the ace2 gene, hla locus, errors influencing type i ifn production, tlr pathways, myeloid compartments, as well as cytokine polymorphisms [156, 235, [263] [264] [265] [266] [267] [268] [269] . we aim to connect the susceptibility features to specific molecular mechanisms and better understand the contributing factors. this can lead to a series of testable hypotheses, including the role of vitamin d counteracting pro-inflammatory cytokine secretion [270] [271] [272] in an age-dependent manner [247, 273] , and modifying the severity of the disease. another example of a testable hypothesis may be that the immune phenotype associated with asthma inhibits pro-inflammatory cytokine production and modifies gene expression in the airway epithelium, protecting against severe covid-19 [245, 246, 274] . in order to understand complex and often indirect dependencies between different pathways and molecules, we need to combine computational and data-driven analyses. standardised representation and programmatic access to the contents of the covid-19 disease map enable the development of reproducible analytical and modelling workflows. here, we discuss the range of possible approaches and demonstrate preliminary results, focusing on interoperability, reproducibility, and applicability of the methods and tools. our goal is to work on the computational challenges as a community, involving the biocurators and domain experts in the analysis of the covid-19 disease map and rely on their feedback to evaluate the outcomes. in this way, we aim to identify approaches to tackle the complexity and the size of the map, proposing a state-of-the-art framework for robust analysis, reliable models, and useful predictions. visualisation of omics data can help contextualise the map with experimental data creating data-specific blueprints. these blueprints could be used to highlight parts of the map that are active in one condition versus another (treatment versus control, patient versus healthy, normal versus infected cell, etc.). combining information contained in multiple omics platforms can make patient stratification more powerful, by reducing the number of samples needed or by augmenting the precision of the patient groups [275, 276] . approaches that integrate multiple data types without the accompanying mechanistic diagrams [277] [278] [279] produce patient groupings that are difficult to interpret. in turn, classical pathway analyses often produce long lists mixing generic and cell-specific pathways, making it challenging to pinpoint relevant information. using disease maps to interpret omics-based clusters addresses the issues related to contextualised visual data analytics. footprints are signatures of a molecular regulator determined by the expression levels of its targets [280] . for example, a footprint can contain targets of a transcription factor (tf) or peptides phosphorylated by a kinase. combining multiple omics readouts and multiple measurements can increase the robustness of such signatures. nevertheless, an essential component is the mechanistic description of the targets of a given regulator, allowing computation of its footprint. with available sars-cov-2 related omics and interaction datasets [281] , it is possible to infer which tfs and signalling pathways are affected upon infection [282] . combining the covid-19 disease map regulatory interactions with curated collections of tf-target interactions like dorothea [283] will provide a contextualised evaluation of the effect of sars-cov-2 infection at the tf level. the virus-host interactome is a network of virus-human protein-protein interactions (ppis) that can help understanding the mechanisms of disease [34, [284] [285] [286] . it can be expanded by merging virus-host ppi data with human ppi and protein data [287] to discover clusters of interactions indicating human mechanisms and pathways affected by the virus [288] . these clusters first of all can be interpreted at the mechanistic level by visual exploration of covid-19 disease map diagrams. in addition, these clusters can potentially reveal additional pathways to add to the covid-19 disease map (e.g., e protein interactions or tgfbeta diagrams) or suggest new interactions to introduce into the existing diagrams. computational modelling is a powerful approach that enables in silico experiments, produces testable hypotheses, helps elucidate regulation and, finally, can suggest via predictions novel therapeutic targets and candidates for drug repurposing. mechanistic models of pathways allow bridging variations at the scale of molecular activity to variations at the level of cell behaviour. this can be achieved by coupling the molecular interactions of a given pathway with its endpoint and by contextualising the molecular activity using omics datasets. hipathia is such a method, processing transcriptomic or genomic data to estimate the functional profiles of a pathway conditioned by the data studied and linkable to phenotypes such as disease symptoms or other endpoints of interest [289, 290] . moreover, such mechanistic modelling can be used to predict the effect of interventions as, for example, the effect of targeted drugs [291] . hipathia integrates directly with the diagrams of the covid-19 map using the sif format provided by casq (see section 2.3), as well as with the associated interaction databases (see section 2.2). the drawback of approaches like hipathia is their computational complexity, limiting the size of the diagrams they can process. an approach to large-scale mechanistic pathway modelling is to transform them into causal networks. carnival [292] combines the causal representation of networks [13] with transcriptomics, phosphoproteomics, or metabolomics data [280] to contextualise cellular networks and extract mechanistic hypotheses. the algorithm identifies a set of coherent causal links connecting upstream drivers such as stimulations or mutations to downstream changes in transcription factor activities. analysis of the dynamics of molecular networks is necessary to understand their dynamics and deepen our understanding of crucial regulators behind disease-related pathophysiology. discrete modelling framework provides this possibility. covid-19 disease map diagrams, translated to sbml qual (see section 2.3), can be directly imported by tools like cell collective [293] or ginsim [46] for analysis. preserving annotations and layout information ensures transparency and reusability of the models. importantly, cell collective is an online user-friendly modelling platform 26 that provides features for real-time in silico simulations and analysis of complex signalling networks. the platform allows users without computational background to simulate or analyse models to generate and prioritise new hypotheses. references and layout are used for model visualisation, supporting the interpretation of the results. the mathematics and code behind each model, however, remain accessible to all users. in turn, ginsim is a tool providing a wide range of analysis methods, including efficient identification of the states of convergence of a given model (attractors). model reduction functionality can also be employed to facilitate the analysis of large-scale models. viral infection and immune response are complex processes that span many different scales, from molecular interactions to multicellular behaviour. the modelling and simulation of such complex scenarios require a dedicated multiscale computational architecture, where multiple models run in parallel and communicate among them to capture cellular behaviour and intercellular communications. multiscale agent-based models simulate processes taking place at different time scales, e.g., diffusion, cell mechanics, cell cycle, or signal transduction [294] , proposed also for covid-19 [295] . physiboss [296] allows such simulation of intracellular processes by combining the computational framework of physicell [297] with maboss [298] tool for stochastic simulation of logical models to study of transient effects and perturbations [299] . implementation of detailed covid-19 signalling models in the pysiboss framework may help to better understand complex dynamics of multi-scale processes as interactions and crosstalk between immune system components and the host cell in covid-19. in this case study, we combine computational approaches discussed above and present results derived from omics data analysis on the covid-19 disease maps diagrams. we measured the effect of covid-19 at the transcription factor (tf) activity level by applying viper [300] combined with dorothea regulons [283] on rna-seq datasets of the sars-cov-2 infected cell line [168] . then, we mapped the tfs normalised enrichment score (nes) on the interferon type i signalling pathway diagram of the covid-19 disease map using the sif files generated by casq (see section 2.3). as highlighted in figure 4 , our manually curated pathway included some of the most active tfs after sars-cov-2 infection, such as stat1, stat2 , irf9 and nfkb1. these genes are well known to be involved in cytokine signalling and first antiviral response [301, 302] . interestingly, they are located downstream of various viral proteins (e, s, nsp1, orf7a and orf3a) and members of the mapk pathway (mapk8, mapk14 and map3k7). sars-cov-2 infection is known to promote mapk activation, which mediates the cellular response to pathogenic infection and promotes the production of proinflammatory cytokines [281] . altogether, we identified signaling events that may capture the mechanistic response of the human cells to the viral infection. in this use case, the hipathia [289] algorithm was used to calculate the level of activity of the subpathways from the covid-19 apoptosis diagram, with the aim to evaluate whether covid-19 disease map diagrams can be used for pathway modelling approach. to this end, a public rna-seq dataset from human sars-cov-2 infected lung cells (geo gse147507) was used. first, the rna-seq gene expression data was normalized with the trimmed mean of m values (tmm) normalization [303] , then rescaled to range [0;1] for the calculation of the signal and normalised using quantile normalisation [304] . the normalised gene expression values were used to calculate the level of activation of the subpathways, then a case/control contrast with a wilcoxon test was used to assess differences in signaling activity between the two conditions. the activation levels have been calculated using transcriptional data from gse147507 and hipathia mechanistic pathway analysis algorithm. each node represents a gene (ellipse), a metabolite (circle) or a function (square). the pathway is composed of circuits from a receptor gene/metabolite to an effector gene/function, with interactions simplified to inhibitions or activations (see section 2.3, sif format). significantly deregulated circuits are highlighted by color arrows (red: activated in infected cells). the color of the node corresponds to the level of differential expression of each node in sars-cov-2 infected cells vs normal lung cells. blue: down-regulated elements, red: up-regulated elements, white: elements with not statistically significant differential expression. hipathia calculates the overall circuit activation, and can indicate deregulated interaction even if interacting elements are not individually differentially expressed. results of the apoptosis pathway analysis can be seen in figure 5 and supplementary material 5. importantly, hipathia calculates the overall activation of circuits (series of causally connected elements), and can indicate deregulated interactions resulting from a cumulative effect, even if interacting elements are not individually differentially expressed. when discussing differential activation, we refer to the circuits, while individual elements are mentioned as differentially expressed. the analysis shows an overactivation of several circuits, specifically the one ending in the effector protein bax. this overactivation seems to be led by the overexpression of the bad protein, inhibiting bcl2-mcl1-bcl2l1 complex, which in turn inhibits bax. indeed, sars-cov-2 infection can invoke caspase8-induced apoptosis [305] , where bax together with the ripoptosome/caspase-8 complex, may act as a pro-inflammatory checkpoint [306] . this result is supported by studies in sars-cov, showing bax overexpression following infection [121, 307] . overall, our findings recapitulate reported outcomes. with evolving contents of the covid-19 disease map and new omics data becoming available, new mechanism-based hypotheses can be formulated. in the covid19 disease map community we strive to produce interoperable content and seamless downstream analyses, translating the graphic representations of the molecular mechanisms to executable models. we are also aware of parallel efforts towards modelling of covid-19 mechanisms, which we plan to include as a part of our ecosystem. these efforts are not yet directly interoperable with the covid 19 disease map content as they use either different notation schemes or require parameters not covered by our biocuration guidelines at the same time, they provide a complementary source of information and the opportunity to create an even broader toolset to tackle the pandemic. the modified edinburgh pathway notation (mepn) scheme [308] allows for the detailed visual encoding of molecular processes using the yed platform but diagrams are constructed in such a way as to also function as petri nets. these can then be used directly for activity simulations using the biolayout network analysis tool [309] . the current mepn covid-19 model details the replication cycle of sars-cov-2, integrated with a range of host defence systems, e.g. type 1 interferon signalling, tlr receptors, oas systems, etc. simulations of altered gene expression, interactions with drug targets or changes to interaction kinetics can be represented by introducing relevant transitions or nodes directly in the diagram. currently, models constructed in mepn can be saved as sbgn.ml files, however is a loss of information and the features associated computationally are not compatible with other covid-19 disease map diagrams (not modelled as petri nets). the covid-19 disease map can support dynamic kinetic modelling to quantify the behaviour of different pathways and evaluate the dynamic effects of perturbations. however, it is necessary to assign a kinetic equation or a rate law to every reaction in the diagram to be analysed. this process is challenging because any given reaction depends on its cellular and physiological context, which makes it difficult to parameterise. software support of tools like sbmlsqueezer [21] and reaction kinetics databases like sabio-rk [310] are indispensable in this effort. nevertheless, the most critical factor is the availability of experimentally validated parameters that can be reliably applied in sars-cov-2 modelling scenarios. the covid-19 disease map is both a knowledgebase and a computational repository. on the one hand, it is a graphical, interactive representation of disease-relevant molecular mechanisms linking many knowledge sources. on the other hand, it is a computational resource of curated content for graph-based analyses and disease modelling. it offers a shared mental map for understanding the dynamic nature of the disease at the molecular level and also its dynamic propagation at a systemic level. thus, it provides a platform for a precise formulation of models, accurate data interpretation, monitoring of therapy, and potential for drug repositioning. the covid-19 disease map spans three platforms and assembles diagrams describing molecular mechanisms of covid-19. these diagrams are grounded in the relevant published sars-cov-2 research, completed where necessary by mechanisms discovered in related beta-coronaviruses. this unprecedented effort of community-driven biocuration resulted in over forty diagrams with molecular resolution constructed since march 2020. it demonstrates that expertise in biocuration, clear guidelines and text mining solutions can accelerate the passage from the published findings to a meaningful mechanistic representation of knowledge. the covid 19 disease map can provide the tipping point to shortcut research data generation and knowledge accumulation, creating a formalized and standardized streamline of well defined tasks. this approach to an emerging pandemic leveraged the capacity and expertise of an entire swath of the bioinformatics community, bringing them together to improve the way we build and share knowledge. by aligning our efforts, we strive to provide covid-19 specific pathway models, synchronize content with similar resources and encourage discussion and feedback at every stage of the curation process. with new results published every day, and with the active engagement of the research community, we envision the covid-19 disease map as an evolving and continuously updated knowledge base whose utility spans the entire research and development spectrum from basic science to pharmaceutical development and personalized medicine. moreover, our approach includes a large-scale effort to create interoperable tools and seamless downstream analysis pipelines to boost the applicability of established methodologies to the covid-19 disease map content. this includes harmonisation of formats, support of standards, and transparency in all steps to ensure wide use and content reusability. preliminary results of such efforts are presented in the case 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through caspase-8 activation bax/bak-induced apoptosis results in caspase-8-dependent il-1β maturation in macrophages over-expression of severe acute respiratory syndrome coronavirus 3b protein induces both apoptosis and necrosis in vero e6 cells the mepn scheme: an intuitive and flexible graphical system for rendering biological pathways a graphical and computational modeling platform for biological pathways sabio-rk: an updated resource for manually curated biochemical reaction kinetics key: cord-014462-11ggaqf1 authors: nan title: abstracts of the papers presented in the xix national conference of indian virological society, “recent trends in viral disease problems and management”, on 18–20 march, 2010, at s.v. university, tirupati, andhra pradesh date: 2011-04-21 journal: indian j virol doi: 10.1007/s13337-011-0027-2 sha: doc_id: 14462 cord_uid: 11ggaqf1 nan patients showed rashes on face, hand and foot. ev detection carried out in vesicular fluid, stool, serum and throat swab specimens by rt-pcr of 5 0 ncr gene. serotyping was carried out by using rt-pcr of viral protein of vp1/2a junction region followed by sequencing and phylogenetic analysis using neighbor-joining-algorithm and kimura-2 parameter model of mega-4 software. overall ev positivity detected in hfmd patients from kerala, tamil nadu, west bengal and orissa states was found to be 51.6%, 66.6%, 62.5% and 71.4% respectively. typing of vp1 gene sequences indicated presence of ca-6, ev-71, echo-9 strains in kerala and ca-16 in west bengal, orissa and tamil nadu. phylogenetic analysis indicated ca-6, ev-71, echo-9 strains showed 94.8-95.7% and 95-94.4% homology with japanese, australian and french strains. however, ca-16 strains were closer to malaysian strains with 91.2-95.6% nucleotide homology. the present study documents the association of multiple types of ev's i.e., ca-6, ev-71, echo-9 and ca-16 strains contributing as prime viral pathogens in hfmd epidemics in the reported regions with new emergence of ca-6 circulating strain in kerala, india. tasgaon september 2010. sera were collected from 162 suspected hepatitis cases and there contacts and tested for anti hev igm/igg antibodies (elisa) and liver enzymes like alanine aminotransferase (alt). anti hev igm antibodies were detected in 45.7% (74/162) of the suspected cases. the overall attack rate was 0.7%. male to female ratio was 2:1. majority (60.4%) of the cases were in the age group 20-40 years and recovered without any clinical complications. weekly distribution of cases showed that the majority (79.4%, 116/146) cases occurred between 2nd and 3rd week of june. dark urine (97.5%), jaundice (93.5%), fatigue (35.9%), abdominal pain (32.6%), anorexia (29.4%), vomiting (26.5%), fever (22.8%), giddiness (14.3%), diarrhoea (12.6%) and arthalgia (3.7%) were the prominent symptoms. sera collected from 73 antenatal cases (ancs) showed anti hev igm antibody in 3. affected pregnant women had a normal outcome. a death of 32 year, male hepatitis e case was reported during the outbreak period that had cirrhosis of liver with oesophageal varices. sanitary survey revealed that water pipelines were laid down in close proximity of sewerage system, and water posts were without tap. these are the likely sources of faecal contamination of water supplies. among 17 water samples collected from various places, 5 were found to be unfit for drinking based on the routine bacteriological tests conducted at state public health laboratory, pune. no case occurred after the pipelines were repaired. this typical outbreak of hepatitis e re-emphasizes need for proper water supply/sewage disposal pipelines and adequate maintenance measures. jayanthi shastri, nilima vaidya, sandhya sawant, umesh aigal department of molecular biology, kasturba hospital for infectious diseases, mumbai, india dengue and dengue haemorrhagic fever are amongst the most important challenges in tropical diseases due to their expanding geographic distribution, increasing outbreak frequency, hyperendemicity and evolution of virulence. the gobal prevalence of dengue has grown dramatically in recent decades. who estimates 50-100 million cases of dengue virus infections worldwide every year resulting in 250,000 to 500,000 cases of dhf and 24,000 deaths each year. public health laboratories require rapid diagnosis of dengue outbreaks for application of measures such as vector control. laboratory diagnosis of dengue virus infection can be made by the detection of specific virus, viral antigen, genomic sequence and/or antibodies. currently 3 basic methods used by laboratories for diagnosis of dengue virus infection are virus isolation and characterisation, detection of genomic sequence by nucleic acid amplification technology assay and detection of dengue virus specific antibodies/antigen. molecular diagnosis based on reverse transcription (rt)-pcr s.a. one step or nested pcr, nucleic acid sequence based amplification (nasba), or real time rt-pcr, has gradually replaced the virus isolation method as the new standard for the detection of dengue virus in acute phase serum samples. several pcr protocols for detection have been described that vary in the extraction method, genomic location of primers, specificity, sensitivity and the methods to determine the products and the serotype. pcr-based dengue tests, due to the specificity of amplification, enable a definitive diagnosis and serotyping of the virus. in addition dna sequencing of the amplification product enables the virus to be genotyped, providing important information on the sources of infection. more recently tests have incorporated flurogenic probe, so called taq man technology for the specific real time detection of dengue 1-4 amplicons. product is detected by a specific oligodeoxy nucleotide probe that is labelled with 6 carboxy-fluorescein (fam). this technology offers the advantage of being both rapid and potentially quantitative. second, the detection of product by hybridisation of flurochrome labelled probes increases specificity. third, as the product is detected without the need to open the reaction tube, the risk of contamination by product carry over is minimised. the advantages of speed, contamination minimisation and reduced turn around time justify application of this assay over the currently used nested pcr assay. during the period january 2007 to october 2009, molecular laboratory received 900 samples from patients presenting with acute onset fever for dengue .6%) samples were tested positive by this method. the disease peaks in the monsoon season with a percentage of 17.5%. rapid tests, igm and igg capture elisa are popularly used tests for diagnosis of dengue infection. its utility is limited for diagnosing dengue in convalescecce (8-14 days) . specificity is also compromised due to infections with flaviviruses: japanese encephalitis and chikungunya. dengue ns1 ag elisa with its cost effectiveness, specificity and sensitivity should be considered as the test of choice for diagnosing dengue in the acute phase of illness in the developing countries. molecular diagnosis enables confirmatory diagnosis of dengue in the acute phase of the illness and is suitable for further typing methods. assistant general manager and r&d coordinator, division of quality control and r&d, bharat immunologicals and biologicals corporation ltd., village chola, bulandshahr, up vaccine development in india, though slow to start, has progressed by leaps and bounds in the past 60 years. it was dependent on imported vaccines but now it is not only self-sufficient in the production of vaccines conforming to international standards with major supplier of the same to unicef. the role of drug authorities is to enhance the public health by assuring the availability of safe and effective a2 indian j. virol. (september 2010) 21(suppl. 1):a1-a58 vaccines, allergenic extracts, and other related products. vaccine development is tightly regulated by a hierarchy of regulatory bodies. guidelines provided by the indian council of medical research (icmr) set the rules of conduct for clinical trials from phase i to iv studies as well as studies on combination vaccines. these guidelines address ethical issues that arise during a vaccine study. a network of adverse drug reaction (adr) monitoring centers along with the adverse events following immunization (aefi) monitoring program provide the machinery for vaccine pharmacovigilance. genetic modifications have been developed to develop effective and cheaper vaccines by the use of recombinant technology. to ensure safety of consumers, producers, experimental animals and environment, governments all over the world are following regulatory mechanisms and guidelines for genetically modified products. as with other industrializing countries undergoing rapid shifts, india clearly recognizes the need to restructure its regulatory system so that its biopharmaceutical industry can compete in international markets. genetic engineering approval council (geac), recombinant dna advisory committee (rdac), review committee on genetic manipulation (rcgm), institutional biosafety committees (ibsc) are responsible for development, commitment for parameters and commercialization of recombinant vaccines. to centralize and coordinate the whole system, government has taken to form two agencies to regulate the regulation laws to develop recombinant pharmaceuticals products including vaccines. the first is the creation of the national biotechnology regulatory authority (nbra), under the department of biotechnology (dbt), as part of india's long-term biotech sector development strategy. the second major initiative will affect the entire indian pharmaceutical industry. this is the replacement of most state, district, and central drug regulatory agencies with a single, central, fda-style agency, the central drug authority (cda). the cda is expected to have separate, semi-autonomous departments for regulation, enforcement, legal, and consumer affairs; biotechnology products; pharmacovigilance and drugs safety; medical devices and diagnostics; imports; quality control; and traditional indian medicines. it will set up offices throughout india and will be paid for inspection, registration, and license fees. its enforcement powers will be strengthened by a new law increasing the criminal penalties for illegal clinical trials. in the manufacturing area, though, the country has been tightening the rules and enforcement. an amendment to the regulations, ''schedule m'' of the drug and cosmetics act, now specifies the good manufacturing practice (gmp) requirements for factory premises and materials. these requirements were modeled after us fda regulations, to improve regulatory coordination between indian and us regulators. india has realized the importance of regulations in pharmaceutical specially in vaccine field but it will take several years to implementation of these. india has coordinated some of its regulatory functions with western organizations. the us pharmacopoeia established an office in hyderabad in 2007. a representative of the indian pharmaceutical lobby also recently has expressed openness to an expansion of the fda's oversight of indian manufacturing. as india expands its global drug and biologicals production, us and europe, as the world's largest drug importers, will likely expand their regulatory support in the development of the country's regulatory systems. rapid diagnosis of japanese encephalitis virus (jev) infections is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. however, the speed and accuracy of diagnosis must be balanced against test cost and availability, especially in developing countries. an antigen capture enzyme-linked immunosorbent assay (elisa) for detection of circulating jev specific nonstructural protein 1 (ns1) was developed by using monoclonal antibodies (mabs) specific to recombinant (ns1). the applicability of this jev ns1 antigen capture elisa for early clinical diagnosis was evaluated with 200 acute phase serum/ cerebrospinal fluid (csf) specimens collected from different epidemics during [2007] [2008] [2009] . jev ns1 antigen was detected in circulation from day 1 to 18. the sensitivity and specificity of jev ns1 detection in serum/csf specimens with reference to reverse transcriptase pcr was 82%, and 98.9% respectively. no crossreactions with any of the other closely related members of the genus flaviviruses (dengue, westnile, yellow fever and saint louis encephalitis (sle) viruses) were observed when tested with either clinical specimens or virus cultures. these findings suggested that the reported jev specific mab-based ns1 antigen capture elisa will be a rapid and reliable tool for early confirmatory diagnosis as well as surveillance of je infections in developing countries. manmohan parida the recent emergence of a novel human influenza a virus (h1n1) poses a serious global health threat. the h1n1 virus has caused a considerable number of deaths within a short duration since its emergence. a two-step single tube accelerated rapid real-time and quantitative swine flu virus specific h1 rtlamp assay is reported by targeting the h1 gene of the novel h1n1 hybrid virus. the feasibility of swine flu h1 rtlamp for clinical diagnosis was validated with a panel of 239 suspected throat wash samples comprising 116 confirmed positive and 123 confirmed negative cases of ongoing epidemic. the comparative evaluation of h1 specific rtlamp assay with real-time rt-pcr demonstrated exceptionally higher sensitivity by picking up all the 116 h1n1 positive and 36 additional positive cases amongst the negatives that were sequence confirmed as h1n1. none of the real-time rtpcr positive samples were missed by rtlamp system. the comparative study revealed that rtlamp was 100-fold more sensitive than rtpcr with a detection limit of 1 copy number. these findings suggested that rtlamp assay is a valuable tool for rapid, real-time detection as well as quantification of h1n1 virus in acute phase throat swab samples without requiring any sophisticated equipments. because of its recurrent nature. despite considerable progress in understanding of the virus at cellular and molecular levels, the proper management of the disease in its different stages is still a dilemma particularly whether to use antiviral or steroids or both. the risk of using steroids with its attendant complications has to be weighed against the risk of progression of the disease if avoiding the use of steroids. this dilemma can be reduced to a considerable extent if basic principles of virology and pathogenesis are kept in mind. this article reviews current concepts of virological and clinical aspects of hsv keratitis to enable a broad understanding of the disease process. it is recognized several influential host factors including the fact that hsk is more common in men than women. it is observed that the ability of hsv to establish latent infection in sensory neurons and possibly cornea, but have as yet been unable to use this knowledge to prevent the disease limitations. acknowledging limitations may further stimulate application of laboratory knowledge in coping with hsk which constitutes to present major challenge in terms of management. mvo-10 study on effect of human bhsp90 in immunity of hcv core protein and hbv hbsag there are more than 500 million individuals with hepatitis b and c in the world. in spite of vaccination in the different areas there are several reports about patients who got vaccine before. also there is not efficient vaccine against of hepatitis c and one of the important problems in vaccine project is development of effective and suitable adjuvant in human vaccines. at present research we applied human bhsp90 protein as adjuvant and chaperon. this protein injected to balbc mice as adjuvant together with recombinant proteins of hcv core and hbv hbsag. then humoral and cellular immune systems of the mice were studied. core and hbsag genes were cloned into petduet-1 vector and thermal vector of pgp1-2 was used for human heat shock protein 90 expressions. the different combination of these three proteins was injected to mice and we evaluated the total igg and igg2a of mice serums after a week. two weeks after booster injection, we studied the proliferation and cytokine secretion of spleen, inguinal and popliteal lymph nodes lymphocytes in vitro and ex vivo conditions. so the core/hbsag + hsp and core + hbsag + hsp complexes induced total igg and igg2a secretion. the spleen lymphocytes proliferation were increased equal to serum igg2a level that was constant in second time bleeding with significant different to complexes with freund's adjuvant. at first il-4 and il-5 cytokines were increased and then decrease of il-4 meaned no hypersensitivity. the chaperon effect of hsp90 on structure of core and hbsag proteins was studied by cd and flourometer. it could fold the proteins after heating and unfolding. hepatitis b virus (hbv) infection is vaccine preventable global public health problem. all commercially available vaccines contain one or more of the recombinant hepatitis b envelope protein or surface antigen (hbsag). measurement of antigen responsible for immunogenicity of vaccine is central to quality assessment. the problems associated with the use of a polyclonal antibody in an assay with regard to its poorly defined nature and batch-to-batch variation has been mitigated by the use of mabs as described in this paper. the initial capture of hbsag by the mab could orientate it such that the same antibody could bind to it as a detection antibody after labeling with out steric hindrance. the development of an immuno-capture elisa (ic-elisa) to measure the hbsag content using a monoclonal antibody (mab) specific to determinant ''a'' of hbsag in the experimental vaccine formulations is being discussed. murine mabs developed against hbsag, subtype adw2 were found to cross-react with the other subtypes viz. ad and ay too. the mabs have been characterized following which, one mab hbs06 was chosen for developing ic-elisa format for the quantification of the hbsag in the final algel adsorbed vaccines. the unadsorbed hbsag was used to establish the standard curve of hbsag/a. the elisa had a sensitivity of 10 ng/ml of hbsag. the recovery rate of hbsag/a was found to be around 70% in the vaccines treated to desorb the antigen from algel. twenty seven experimental batches of monovalent hepatitis b vaccines were analyzed for the hbsag content, both by ic-elisa and a commercial kit (axsym kit, abbott laboratories, usa). the statistical analysis of ic-elisa results indicated that an experimental equation f(x) = 0.0062(x) + 0.184, could precisely estimate the amount of hbsag in the adsorbed vaccines. the amounts of hbsag recovered from the adsorbed vaccines as estimated by the ic-elisa format had a good correlation with the estimates derived from a commercial kit, which is being used by several vaccine manufacturers in india for the quality control of vaccine antigen. the varying amounts of vaccine antigens that could be recovered seemed to depend on the quality of the hbsag and the methods of hbsag adsorption to the alum gel during vaccine manufacture. epidemiology of the spread of h1n1 virus. children of school going age have become victim of this deadly virus as evident from the reporting data generated in the past few weeks. the mortality rate has also been slightly increased. the disease spread in wave pattern and presently the world is passing through the second wave of pandemic with more severity in young and otherwise health people with a predilection for lungs leading to viral pneumonia and respiratory failure. now the pandemic gained hold in the developing world affecting more severely as millions of people live under deprived conditions having multiple health problems, with little access to basic health care. current data about the pandemic from developed counties need to be very closely watched in relation to shift in virus sub type, shift of the highest death rate to younger populations, successive pandemic waves, higher transmissibility than seasonal influenza, and demographic differences etc. presently the world appears to be better prepared. vaccine is available in market in many countries. even vaccine trials are actively going on in indian population. effective antivirals are available. although till now h1n1 diagnostic centers worked with cdc/who recommended h1n1 specific primer, probes with taqman chemistry by real time pcr, efforts on the development of indigenous diagnostics, vaccines and chemoprophylaxis is going on to have a better combat against this highly infectious virus. were positive for rotavirus infection by either page or elisa methods. the available data highlights the importance of rotavirus as a cause of diarrhea in children, which is severe enough to deserve specialized care. the observed proportion of 25.5% of all diarrhea cases being associated with rotavirus falls within the range of values reported by other workers. the reported positivity varies from 10.5 to 70.7%. in our study a complete concordance of elisa and page results were observed in 194 (97%) of the 200 tested specimens. this finding closely correlates with the findings of other authors who found a 96.7-97.14% concordance results between elisa and page methods. some authors found rna-page method that is as sensitive and rapid as elisa for detecting rotavirus in stool samples of cases of diarrhea and some others proposed elisa is more sensitive than page method fond to be 100% specific. the remaining 6 (3%) samples showed conflicting results. in a lone sample in which the od value of elisa test was 0.195, this value was almost at the cutoff level, the possibility of this sample being positive by elisa test is doubtful. negative result of the same sample in page method is difficult to explain, the possibility of presence of lot of empty virus particles or due to low concentration of viral rna in the fecal specimen and insufficient extraction of viral rna could be possible. on the other hand, 5 of the samples which gave positive results by page method were negative by elisa test. these 5 samples had a typical 4-2-3-2 rna pattern. the reason for their being elisa negative thus remains unexplained, however blocking factors or the presence of inhibitory substance in stools might have been responsible. the samples containing predominantly complete particles can also give false negative results. since, the group antigen is not exposed. earlier studies have also reported page to be the most sensitive technique although some are of view that it is laborious procedure. how ever, the page system used in this study was very simple to perform and the results were available on the same day. the main requirement was of trained personnel and proper standardization of the technique. most reports states that the greatest advantage of page and silver stain method are its lack of ambiguity and the fact that it provides information about viral electropherotypes. the modified page system was thus found to be reliable, simple and rapid, no expensive reagents were required. locally available reagents from hi media were used. the cost of the chemical for page per specimen was rs. 24 approximately as compared to rs. 110 per test by confirmatory elisa. a locally produced slab gel electrophoresis system with power pack was the only equipment required. this method could be used for the routine diagnosis of rotavirus infection in the laboratory. vaccine, rapid diagnosis plays an important role in early management of patients. in this study a qc-rt-pcr assay was developed to quantify chikungunya virus rna by targeting the conserved region of e1 gene. a competitor molecule containing an internal insertion was generated, that provided a stringent control of the quantification process. the introduction of 10-fold serially diluted competitor in each reaction was further used to determine sensitivity. the applicability of this assay for quantification of chikungunya virus rna was evaluated with human clinical samples and the results were compared with real-time quantitative rt-pcr. the sensitivity of this assay was estimated to be 100 rna copies per reaction with a dynamic detection range of 10 2 to 10 10 copies. specificity was confirmed using closely related alpha and flaviviruses. the comparison of qc-rt-pcr result with real-time rt-pcr revealed 100% concordance. these findings demonstrated that the reported assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of chikungunya virus in acute-phase serum samples. in india, measles vaccine was introduced as part of expanded programme of immunization in 1985. measles, mumps and rubella (mmr) vaccine is still not part of the national immunization schedule of india. the indian association of paediatrics (iap) recommends measles vaccine at 9 months of age and mmr vaccine at 15-18 months. however, in a recent policy update, iap committee on immunisation opined that there is a need for a second dose of mmr vaccine for providing adequate immunity against mmr. the aim of the present study was to assess the extent of sero-protection against mmr at 4-6 years of age in children who have received one dose of mmr between 12 and 24 months of age. an attempt has also been made to assess the sero-response to the second dose of mmr vaccine in 4-6 years old children. a total of 106 consecutive children between the ages of 4-6 years who had received mmr vaccine between 12 and 24 months of age and attending the immunization clinic of gtb hospital, delhi were enrolled. the vaccination status, anthropometry and physical examination findings were recorded. three ml of venous sample was again withdrawn for estimation of post vaccination antibody titre. it was observed that 20.39%, 87.38% and 75.73% children were seroprotected for mmr respectively after 2.5-4.5 year of receiving first dose of mmr vaccine. seroprotection rose to 72.62%, 100% and 100% for mmr respectively after 4-6 weeks of receiving second dose of mmr vaccine. geometric mean concentration of antibody also rose significantly in all three diseases. in view of low seroprevalence of mmr and hence high susceptibility to infection at 4-6 years of age, who have already received mmr vaccine, there is need to boost the immune responses against these three diseases by giving a second dose of mmr vaccine. baseline information on the epidemiology of viral agents causing stis and types of risk behaviour of affected persons are essential for any meaningful targeted intervention. the present study documents the pattern of viral stis in patients attending a tertiary care hospital, correlating the syndromic approach and the laboratory investigations to determine the aetiology. three hundred consecutive patients attending the sti clinic were diagnosed and categorized according to the syndromic approach of the who along with detailed history and demographic data. majority of the patients were men (53.12%) with a mean age of 24 years. men received education up to middle school. half of the female subjects were illiterate. sixty percent of the patients were married and among these, 19% were regular condom users. first sexual contact at or before 18 years of age was more in men (31% vs. 22 .22% in women). promiscuity was more among male patients who had contact with csw. genital herpes was the commonest viral sti (86/300) followed by genital wart (60/300). concomitant infection with more than one virus was seen in 35% of patients. hiv was prevalent in 10.3% of sti patients. hepatitis b, hepatitis c, herpes simplex type 1 and molluscum contagiosum were the other viral agents seen in sti clinic attendees at our centre. this disease currently prevalent in more than 100 countries world wide and annually 50-100 million people are infected with dengue virus among which 2.5-5 lakhs cases were dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) which are serious forms of dengue virus infection and due to this condition 25,000 deaths might occur annually world wide and approximately 3 million children were hospitalized for the fast 3 decades. this disease is characterized by sudden onset of high fever with sever headache, pain in the back and limbs, lymphadenopathy macuolo-papulur rash over the skin and retro-bulbar pain. early diagnosis can be established with simple and rapid lgg/1gm antibodies detection in the blood samples of the patients based on the bi-directional immunoassay system for its management and control to reduce morbidity and mortality. details will be presented. myocarditis and dilated cardiomyopathy (dcm) are common causes of morbidity and mortality both in children and adults. the most common viruses involved in myocarditis are coxsackievirus b or adenovirus. recently, the coxsackievirus and adenovirus receptor (car), a common receptor for coxsackieviruses b3, b4 and adenoviruses 2, 5 has been identified. increased expression of car has been reported in patients with dcm suggesting utilization of car by these viruses for cell entry. the present study was designed to study the expression of car in myocardial tissue of patients with dcm. formalin fixed myocardial tissues were obtained from autopsy cases. a total of 26 cases of dcm and 20 cases of controls which included non-cardiac (group-a) and cardiac disease other than dcm (group-b) were included in the study. expression of car was studied by immunohistochemical staining of myocardial tissue using car specific rabbit polyclonal antibody and biotin conjugated secondary antibody. the tissue sections were considered positive when[25% of the cell showed brown color staining by immunohistochemistry (ihc). the car positivity in dcm cases was found to be 96% (25/ 26) as compared to 30% in control group a and 40% in control group b respectively. the car positivity was significantly higher in the test group as compared to both the control groups. further car positivity in all the cellular types (myocytes, endothelial cells and interstitial cells) was found significantly higher in test group as compared to both the control groups. the expression of car was significantly higher in myocytes as compared to both endothelial and interstitial cells in all the groups. however, no significant difference was observed in car positivity between endothelial and interstitial cells. the present study highlights the increased expression of car in dcm cases with further significance of car expression in myocytes and endothelial cells. this may help further in understanding the tropism of viruses or cellular susceptibility, which in turn will help in appropriate diagnostic and therapeutic approach in management of viral myocarditis and dcm cases. food security and safety vary widely around the world, and reaching these goals is one of the major challenges, raising public concern for the wellbeing of mankind, in particular. industrialized production and processing as well as improper environmental protection have clearly shown severe limitations such as worldwide contamination of the food chain and water. contaminated water and food during the processes of production, processing and handling are essentially responsible for food and water borne viral infections/diseases. the cases of viral food borne outbreaks are on the rise, creating a threat to human health. recent researches indicate that epidemiological studies are meager to focus the frequently contaminated foods and food borne viral diseases. current paper projects the etiology of select food borne viral diseases, probable reasons for non availability of appropriate methods to detect the viruses responsible for the diseases, routes of water and food borne transmission of enteric viral infections, currently available methods of detection of select viruses and bio safety measures to prevent food borne viral infections. dietary/nutritional management in food borne viral diseases is crucial to control weakness and gastro enteric intolerance due to disease condition and antibiotic therapy. it will principally improve food intake, resulting in better nutritional status leading to optimum immune response. food borne viruses are mainly belong to rotaviruses, enteropathogenic viruses, astroviruses, adenoviruses and caliciviruses, causes acute gastroenteritis (ag) which is an important health problem. the frequency of rotavirus as a cause of sporadic cases of ag ranges between 17.3% and 37.4%. astroviruses cause ag, with a frequency ranging between 2 and 26%: outbreaks have been described in schools and kindergartens, but also in adults and the elderly. the frequency of identification of adenoviruses 40 and 41 as causes of sporadic ag in non-immuno suppressed children ranges between 0.7% and 31.5%, although there is probably underreporting because the sensitivity of conventional techniques is low. caliciviruses are separated phylogenetically into two genera: norovirus and sapovirus. norovirus is frequently associated with food-and water-borne outbreaks of ag. it is estimated that 40% of cases of ag due to norovirus are food borne. in sweden and some regions of the united states, norovirus is the first cause of outbreaks of food borne diseases. sapovirus outbreaks due to person-to-person and food borne transmission affecting both children and adults have recently been reported in countries such as canada and japan. it has been predicted that the importance of diarrhoeal disease, mainly due to contaminated food and water, as a cause of death will decline worldwide. evidence for such a downward trend is limited. this prediction presumes that improvements in the production and retail of microbiologically safe food will be sustained in the developed world and, moreover, will be rolled out to those countries of the developing world increasingly producing food for a global market. sustaining food safety standards will depend on constant vigilance maintained by monitoring and surveillance but, with the rising importance of other food-related issues, such as food security, obesity and climate change, competition for resources in the future to enable this may be fierce. in addition the pathogen populations relevant to food safety are not static. food is an excellent vehicle by which many pathogens (bacteria, viruses/prions and parasites) can reach an appropriate colonization site in a new host. although food production practices change, the well-recognized food-borne pathogens, such as salmonella spp. and escherichia coli, seem able to evolve to exploit novel opportunities, for example fresh produce and even generate new public health challenges, for example antimicrobial resistance. in addition, previously unknown food-borne pathogens, many of which are zoonotic, are constantly emerging. awareness and surveillance of viral food-borne pathogens is generally poor but emphasis is placed on norovirus, hepatitis a, rotaviruses and newly emerging viruses such as sars. it is clear that one overall challenge is the generation and maintenance of constructive dialogue and collaboration between public health, veterinary and food safety experts, bringing together multidisciplinary skills and multi-pathogen expertise. such collaboration is essential to monitor changing trends in the well-recognized diseases and detect emerging pathogens. it is also necessary to understand the multiple interactions between these pathogens and their environments during transmission along the food chain in order to develop effective prevention and control strategies. to analyse the effectiveness of these sirnas targeting rabies virus l gene, the bhk-21 cells expressing sirnas in shrna form were produced by transduction of cells with radv-l. the transduced bhk-21 cells expressing sirna were infected with rabies virus pv-11 strain. there was reduction in rabies virus multiplication as analysed by reduction in fluorescent foci forming unit (ffu) count by 51.85% (70 ffu in bhk-21 cells expressing sirna-l compared to 135 ffu in bhk-21 cells expressing negative sirna). the expression of l gene mrna was reduced by 16.11fold in rabies virus infected radv-l transduced cells compared to radv-neg transduced cells (negative control) as detected using real-time pcr. after analyzing the effectiveness of radv-l in vitro, its effectiveness was also evaluated in vivo in mice after virulent rabies challenge. the mice were inoculated with 10 7 plaque forming units (pfu) of radv-l in masseter muscle (i/m route) and challenged with 15 ld 50 rabies virus challenge virus standard (cvs) strain. the results indicated 50% protection with improved median survival from 7 to 11 days compared with group of mice treated with radv-neg. the results of this study indicated that sirnas targeting rabies virus polymerase (l) gene delivered through adenoviral vector inhibited rabies virus multiplication in vitro and in vivo. and 4 were successfully produced and purified from the infected spodoptera frugiperda (sf-9) cells using these recombinant baculovirus. the morphology of the vlps was validated by electron microscopy in comparison to the authentic bt virions. the vlps produced here were stable and were highly immunogenic with intact outer layer which is rapidly lost during normal infection of btv. these btv-vlps elicited long lasting protective immunity in vaccinated sheep against virulent virus challenge. with the use of btv-vlps it was also possible to differentiate the infected and vaccinated animals (diva). vlp-based btv vaccine has potential advantages with regard to controlling the spread of btv with multiple serotypes. it is possible to produce milligram quantities of correctly folded and processed protein complexes using this baculovirus expression system and hence it is a more promising system for producing new generation vaccines like vlp subunit vaccine against any viral diseases in large scale. peste des petits ruminants (ppr), goatpox and orf are oie notifiable diseases of small ruminants especially goat and sheep. these diseases are economically important, in enzootic countries like india and cause significant loss and are major constraints in the productivity. considering the geographical distribution of ppr, goat pox and orf infections and prevalence of mixed infection, in the present study, safety and potency of the experimental triple vaccine comprising attenuated strains of thermostable-ppr virus (pprv jhansi, p-50) grown at 40°c, high passaged goat poxvirus (gtpv uttarkashi, p100) and attenuated orf virus (orfv mukteswar, p51) was evaluated in sub-himalayan local hill goats. goats simultaneously immunized with 1 ml of vaccine consisting of either 10 3 tcid 50 or 10 5 tcid 50 of each of pprv, gtpv and orfv were monitored for clinical and serological responses for a period of 3-4 weeks post-immunization (pi) and post challenge (pc). specific immune responses i.e., antibodies directed to pprv, gtpv and orfv could be demonstrated by ppr competitive elisa kit and capripox indirect elisa, snt, respectively following immunization. all the immunized animals resisted infections when challenged with virulent strains of either gtpv or pprv or orfv on day 28 dpi, while in contact control animals developed characteristic signs of respective disease. further, ppr viral antigen could be detected by using ppr sandwich elisa kit in the excretions (nasal, ocular and oral swab materials) of unvaccinated control animals after challenge but not from any of the immunized goats. triple vaccine was found safe at dose as higher as 10 5 tcid 50 and induced protective immune response even at lower dose (10 2 tcid 50 ) in goats, which was evident from sero-conversion as well as challenge studies. the study indicated that these viruses are compatible and did not interfere with each other in eliciting immune response, paving the feasibility of use of this triple vaccine in combating these infections simultaneously. toll like receptors (tlrs), primary sensors of microbial origin, plays a crucial role in the innate immunity. till now 13 mammalian tlrs have been identified, while there is no information available on tlrs of yak. this study is part of world bank funded-icar project. yak, named bos grunniens for its distinctive vocalization and relationship with cattle, is natural habitant of extremely cold environment. when these animals comes to a lower altitude grazing land, adjacent to villages, become susceptible to the diseases of cattle, buffalo etc. thus, present study was undertaken to with genetic characterization and evolutionary lineage analysis of yak tlrs. we worked on tlr7 gene, which plays an important role in recognition of ssrna viruses. total rna was extracted from mitogen stimulated pbmcs of yak. the rt-pcr conditions were standardized for full length amplification of tlr gene 7 using specific self designed primers. the expected amplicon of 3559bps was obtained. it was cloned in pgemt-easy vector followed by transformation in e. coli top10 strain. the recombinant clones were screened, picked up for plasmid isolation and release of tlr7 was confirmed by restriction digestion. the cloned tlr7 product was sequenced and analyzed for the nucleotide and deduced amino acid sequences, and 3d structure analysis. the results revealed that yak shows more than 98% sequence homology with other bos indicus breeds and bos taurus breeds. however, identity was less than 88% with other animal species (equine, murine, feline, canine etc.). the evolutionary lineage findings cluster yak more closely with bovine species. point mutations revealed changes at 25 nucleotide positions with corresponding amino acid change at 15 positions. smart analysis of yak protein domain architecture revealed toll-interleukin i receptor (tir), leucine rich repeats (lrr) and signal peptide region. the variations in yak mainly lie in the lrr region. homology modeling revealed horse shoe shaped structure with 5 alpha helix. the additional alpha helix present in bos indicus was not detected in yak. the present study shows existence of genetic variability in tlr7 gene of yak, in particular the lrr region, which plays an important role in the pathogen recognition and the evolutionary lineage analyses shows its closeness with other bovine species. a.p. aquaculture and fisheries, tirupati in this new millennium, aquatic animal health management strategies in asia expanded and adjusted to the current disease problems faced by the aquaculture sector. this presentation will briefly discuss some of the most serious trans-boundary pathogens affecting asian aquaculture including a newly emerging disease and highlight recent regional and national efforts on responsible health management for mitigating the risks associated with aquatic animal movement. a regional approach is fundamental since many countries share common social, economic, industrial, environmental, biological and geographical characteristics. capacity and awareness building on aquatic animal epidemiology, science-based risk analysis for aquatic animal transfers, surveillance and disease reporting, disease zoning and establishment of aquatic animal health information systems to support development of national disease control programs and emergency response to disease outbreaks are needed. molecular diagnostics with emphasis towards standardization and harmonization, inter-calibration exercises and quality assurance in laboratories, accreditation program and utilization of regional resource centres on aquatic animal health will also be needed. whilst most of these strategies are directed in support of government policies, implementation will require pro-active involvement, effective cooperation and strategic networking between governments, farmers, researchers, scientists, development and aid agencies, and relevant private sector stakeholders at all levels. their contributions are essential to the health management process. generally, aquaculture plays an important role in economy as harvests from natural waters have declined or, at best, remained static in most countries. fish and shrimp, the main aquaculture product sources, have gained the most attention. many factors can cause losses in yields of fish products and infectious disease in fish and shrimp is the biggest threat to the fishery industry. shrimp and fish aquaculture has grown rapidly over several decades to become a major global industry that serves the increasing consumer demand for seafood and has contributed significantly to socio-economic development in many poor coastal communities. however, the ecological disturbances and changes in patterns of trade associated with the development of shrimp and fish farming have presented many of the pre-conditions for the emergence and spread of disease. shrimp and fish are displaced from their natural environments, provided artificial or alternative feeds, stocked in high density, exposed to stress through changes in water quality and are transported nationally and internationally, either live or as frozen product. these practices have provided opportunities for increased pathogenicity of existing infections, exposure to new pathogens, and the rapid transmission and trans boundary spread of disease. not surprisingly, a succession of new viral diseases has devastated the production and livelihoods of farmers and their sustaining communities. this review examines the major viral pathogens of farmed shrimp and fish, the likely reasons for their emergence and spread, and the consequences for the structure and operation of the shrimp farming industry. in addition, this review discusses the health management strategies that have been introduced to combat the major pathogens and the reasons that disease continues to have an impact, particularly on poor, smallholder farmers in asia. btv isolates from the same geographic region have been termed as 'topotypes' and initial observation on segment 3 nucleotide sequences identified a correlation between topotypes and genetic information. later topotyping was proposed based on segment 10, on the premise that the encoding protein ns3, which is involved in virus egress from insect cells, would lead to evolutionary fitness in parallel with the geographic distribution of the different culicoides species. further studies attempted to extend this to nucleotide sequence homology in segments 7 and 10, but failed to identify clear cut correlations or any evidence for positive selection. for example, south african isolates were found not to cluster into separate african lineage. in this study, we carried out a more extensive analysis of segment 10 sequences. our analysis showed no segregation of isolates into topographically distinct groups. instead we observed topological clustering of the clades, and we attribute this to genetic bottleneck resulting in genetic drift and founder effect leading to homogenous gene pool in a geographical area. we hypothesize that when a new virus enters a geographical area where local btv strains are already circulating, the new genes/segments would enter into a bigger gene pool. consequently, the newer incursions into a heavily endemic area tend to get diluted and disappear from the population because the rate of drift is inversely proportional to the population size, unless they are positively selected. use of live attenuated vaccine in israel, europe, south africa and usa also led to more homogenous population similar to the vaccine strains due to continuous infusion of the vaccine type genes into the gene pool. we conclude that restriction of specific strains to certain geographical areas could generate uniquely imprinted genotypes which would not only indicate origin but also predict movement of viral strains to new areas. vvo-10 viral diseases of zoonotic importance: indian context k. prabhudas pd-admas, ivri, campus, hebbal, bangalore 24 zoonoses are generally defined as animal diseases that are transmissible to humans. they continue to represent an important health hazard in most parts of the world, where they cause considerable expenditure and losses for the health and agricultural sectors. the emergence of these zoonotic diseases are very distinct, hence their prevention and control will require unique strategies, apart from traditional approaches. such strategies require rebuilding a cadre of trained professionals of several medical and biologic sciences. the article discusses virus infections that have significant zoonotic implications for india. buffalopox is a contagious viral disease affecting milch buffaloes and rarely, cows, with a morbidity rate up to 80% in the affected herd. although the disease is not responsible for high mortality, it adversely affects the productivity of the animals, resulting in large economic losses. furthermore, the disease has zoonotic implications, as outbreaks are frequently associated with human infections, particularly in the milkers. the causative agent, buffalopox virus (bpxv), is closely related to vaccinia virus. the outbreaks of febrile rash illness among humans and buffaloes were investigated in the villages of districts solapur and kolhapur of western maharashtra. clinico-epidemiological investigations of humans and buffaloes were carried out and representative clinical samples were collected respectively. the samples include vesicular fluid, scab, and blood. laboratory investigations for buffalo-pox virus (bpxv) was done by pcr on blood samples, scabs and vesicular fluid. in vitro virus isolation attempts were carried out by using vero e-6 cells. negative staining electron microscopy was also employed for detection of virus particles. a total of 166 human cases with pox lesions on hand and other body parts from village kasegaon, district-solapur and 185 cases from 20 different villages of kolhapur district were reported. besides pox lesions patients were having fever, malaise, pain at site of lesion and axillary and inguinal lymphadenopathy. in kasegaon village, attack rate in human cases was 6.6% and in buffaloes 41.9% (231/551). whereas in kolhapur area attack rate in buffaloes was 11.75% (2633/22398). bpxv was confirmed in blood, vesicular fluid and scab specimens from human cases and scab specimen from buffalo by polymerase chain reaction (pcr) method. the bpxv was also isolated from 3 different clinical specimens and further identified by pcr and electron microscopy. clinical manifestation of the disease in buffaloes from solapur district was as reported earlier like common pox lesions on teats and udders whereas the buffaloes from kolhapur district had lesions on hairless parts of ears and on the eyelids with purulent discharge. bpxv from human and buffalo cases showed similarity. vaccines have been made against several diseases and used for controlling the afflictions. however a few of them were not effective for successfully controlling the disease. the reasons for the failure are many, the major being, either the pathogen is not completely cleared from the vaccinated animal or it reemerges after changing its antigenic structure, thus making the vaccination programme less effective. in addition to this, emergences of newer diseases such as hiv the development of suitable vaccines have become a challenging task. this is especially true in the case of viral diseases. these challenges have warned the researchers ''that protection by vaccination is not that simple and strait forward approach'', and lot need to be understood in terms of host virus interaction and role of environment in perpetuating the disease. so the immediate step that was considered was the environmental safety by way using non infectious materials as vaccines. with the understanding that has been developed in molecular immunology and molecular biology and with the availability of molecular tools that have been developed through recombinant dna technology the field of vaccinology has changed dramatically to emerge as modern vaccinology. this presentation deals with the modern approaches that are being used to produce effective vaccines in the case of foot and mouth disease of cloven footed animals. the similar approach may be worked out for other viral diseases also. despite the availability of an inactivated vaccine that is noted to provide solid immunity against the disease over a short period of time, the search for an ideal vaccine, the criteria for which are; safety of the vaccine for environment, easy in its preparation, does not require a cold chain for its storage, provides longer lasting immunity, economically viable and may be able to clear the virus in case of persistent infection is on. the advent of recombinant dna technology together with the information available on the molecular biology of viruses has enabled to design the development of newer vaccines that can induce strong cellular and humoral responses. the underlying principal in the present vaccine development strategy world over is the virus antigen gene has to be expressed in the tissue and the vaccine backbone has to trigger the immune system for eliciting desired immune response. bangalore campus of ivri has been vigorously pursuing research to develop ideal vaccines for foot and mouth disease keeping above principal in mind to achieve the previously mentioned criteria. the approaches selected are to see that the virus antigen/s replicate transiently in the host. the self replicating vaccines that have been developed are pox virus vectored vaccines, alpha virus replicase based vaccines and fmdv vectored vaccines. the approach and the result obtained so far will be discussed. silkworm, bombyx mori is affected with various diseases caused by viruses viz., nuclearpolyhedrosis (bmnpv), densosnucleosis (bmdnv) and infectious flacherie (bmifv). silkworm viral diseases form major constraints for the silk cocoon production in all the sericultural countries. the losses due to silkworm diseases is estimated about 20-40% and among them viral diseases are most common. in sericulture, prophylactic measures play a vital role in the management of silkworm diseases. these include disinfection of silkworm rearing house and appliances, rearing area, rearing surroundings, silkworm egg and body, and rearing bed disinfection associated with maintenance of general hygiene and personnel hygiene. all these activities are generally carried out as rituals by using general disinfectants often with partial success. recent trends in complete management of silkworm diseases include development of silkworm hybrids evolved from disease resistant/tolerant breeds, effective eco-and user-friendly disinfectants, anti-microbial feed-supplements and use of transgenic silkworms. biotechnological breakthrough in this regard is through rna interference (rnai) approach involving dsrna mediated nuclear polyhedrosis management and this is presently pursued by apssrdi, hindupur in collaboration with centre for dna fingerprinting and diagnostics (cdfd), hyderabad. nadu and karnataka. the disease appears to be more severe in rural flocks than organized farms. our investigations revealed the morbidity, mortality and case fatality rates among rural and organised farms as 9.34%, 2.69%, 28.84% and 6.22%, 0.47%, 7.63% respectively. higher morbidity and mortality in rural areas may be due to stress factors like poor nutrition, parasitic burden, fatigue due to long walks and non availability of veterinary aid. kulkarni et al. 1992 also reported the severe bt outbreaks in rural areas of maharashtra with overall morbidity, mortality and case fatality of 32%, 8% and 25% respectively. all the south indian sheep breeds were found to be susceptible and clinical farm of the disease is evident in all of them though saravanabava (1992) reported variations in susceptibility among the indigenous sheep. trichy black and ramnad white sheep were found to be more susceptible than the vambur and mecheri sheep of tamil nadu. prevalence of bluetongue in sheep, goat and cattle appears to be high in the region. serological surveys conducted in andhra pradesh during 1991 revealed the prevalence of btv antibodies in sheep (47.5%) goats (43.56%) cattle (33%) and buffaloe (20%). similar high prevalence of btv antibodies in sheep and goats were also reported from the other states in the region. clinical disease has not been recorded in kerala though btv antibodies were recorded in sheep (13.76%) and goats (7.10%) (ravi sankar 2003) . culicoides are the known biological vectors of btv. all the culicoides species are not capable of transmitting the btv. the occurrence of the disease is related to the presence of the competent vectors in the area. jain et al. (1988) established the involvement of the culicoides in transmitting the btv by isolating the virus from culicoides at haryana, the north indian state. c. imicola and c. oxystoma were found to be prevalent in andhra pradesh and tamil nadu. narladakar et al.(1993) reported the presence of c. schultzei, c. perigrinus and c. octoni in marathwada region of maharastra. culicoid vectors are significantly affected by the climate and annual variations in the climate reflects the outcome of the disease. the monsoon season (june to dec) with the temperature ranging from 21.2 to 35.6°c appears to be favourable period for the multiplication of culicoides. the maximum no of outbreaks were recorded during the north east monsoon period (oct-dec) followed by south west monsoon period (june to sep) in the region. however, details on the distribution of the competent vectors, feeding habits and their dynamics in the region is lacking multiple btv serotypes were found to be circulating in the region. (kulkarni and kulkarni 1984; janakiraman etal. 1991; mehrotra et al. 1996) a total of 10 serotypes viz. 1-4, 8, 9, 15, 16, 18 and 23 were identified based on the virus isolations. sreenivasulu et al. 1999 isolated btv serotype 2 from an outbreak of bt in native sheep of andhra pradesh. btv serotype 9, 15 and 21 were also isolated from the outbreaks occurred in andhra pradesh. some of the isolates need to be serotyped. deshmukh and gujar (1999) isolated btv type 1 from maharashtra. following is the summary of the distribution of btv serotypes in this region. clinical picture of bt in native sheep appears to be slightly different, the major difference being that swelling of lips and face was less conspicuous. mucocutaneous borders appeared to be very sensitive to touch and bleed easily upon handling. the classical signs of cyanosis of tongue and reddening of coronary band are not the common features of the disease in native sheep. the disease was also confirmed by the virus isolation and identification. clinical disease has not been reported in cattle, buffaloes and goats in spite of high seroprevalence. in conclusion bt is established in native sheep and causes severe economic losses to the farmers. the disease is concentrated in the southern peninsula of the country. the disease is seasonal and is associated with the rain fall. multiple serotypes appear to be circulating in this region. the btv serotypes were of virulent in nature as evident by severe outbreaks. s. janardana reddy*, d. c. reddy department of fishery science and aquaculture, sri venkateswara university, tirupati 517 502 in less than three decades, the penaeid shrimp culture industries of the world developed from their experimental beginnings into major industries providing hundreds of thousands of jobs, billions of u.s. dollars in revenue, and augmentation of the world's food supply with a high value crop. concomitant with the growth of the shrimp culture industry has been the recognition of the ever increasing importance of disease, especially those caused by infectious agents. in india viral diseases have become an important limiting factor for growth of shrimp aquaculture industry. although more than 30 different viral pathogens have been identified in different species of shrimp world wide, only a few viruses have identified which are causing disease problems in cultured tiger shrimps in india, east coast of andhra pradesh, in particular. diagnostic methods for these pathogens include the traditional methods of morphological pathology (direct light microscopy, histopathology, and transmission electron microscopy), enhancement and bioassay methods, traditional microbiology, and the application of serological methods. while tissue culture is considered to be a standard tool in medical and veterinary diagnostic labs, it has never been developed as a useable, routine diagnostic tool for shrimp pathogens. the need for rapid, sensitive diagnostic methods led to the application of modern biotechnology to penaeid shrimp disease. the industry now has modern diagnostic genomic probes with nonradioactive labels for viral pathogens like infectious hypodermal and hematopoietic necrosis (ihhnv), hepatopancreatic virus (hpv), taura syndrome virus (tsv), white spot syndrome virus (wssv), monodon baculo virus (mbv), and bp. highly sensitive detection methods for some pathogens that employ dna amplification methods based on the polymerase chain reaction (pcr) now exist, and more pcr methods are being developed for additional agents. these advanced molecular methods promise to provide badly needed diagnostic and research tools to an industry reeling from catastrophic epizootics and which must become poised to go on with the next phase of its development as an industry that must be better able to understand and manage disease. within this field, shrimp immunology is a key element in establishing strategies for the control of diseases in shrimp aquaculture. research needs to be directed towards the development of assays to evaluate and monitor the immune state of shrimp. the establishment of regular immune checkups will permit the detection of shrimp immunodeficiencies but also to help monitor and improve environment quality. for this, immune effectors must be first identified and characterised. in the end, however, the assumption may be made that the sustainability of aquaculture will depend on the selection of disease-resistant shrimp, i.e. to develop research in immunology and genetics at the same time. the development of strategies for prophylaxis and control of shrimp diseases could be aided by the establishment of a collaborative network to contribute to progress in basic knowledge of penaeid immunity. however, to improve efficiency, it appears essential also to open this network to complementary research areas related to shrimp pathology, physiology, genetics and environment. bluetongue is an important viral disease of sheep causing severe economic losses to the farmers. lack of effective vaccine is the major impediments in controlling the disease. multiple serotypes were found to be circulating in the state. attempts are being made to develop the vaccine employing the available serotypes to control the disease. hence, it is essential to identify the antigenic relationship among the serotypes to identify the candidate vaccine strains to be incorporated in the preparation of vaccine. reciprocal cross neutralization test was employed to find out the r% values between btv-2, -9 and -15 which indicated the extent of antigenic relationship between the serotypes. r% value between btv-2 and btv-9 was recorded as 2.8 r% value of 3.53 and 2.8 were observed between btv-2 and -15 and btv-9 and -15 respectively. the r% values recorded in the present study revealed a weak antigenic relationship between the btv serotypes. the extent of antigenic relationship between the btv serotypes was also determined by multiple sequence alignment of the nucleotide and amino acid sequences of the reference btv serotypes 2, 9 and 15. the sequence analysis of the vp2 gene revealed a homology of 47-53% and 29-41% at the nucleotide and amino acid levels respectively. r% values obtained using reciprocal cross neutralization test with the btv-2, 9 and 15 serotypes isolated in native sheep of andhra pradesh and the genomic analysis of the reference serotypes of btv-2, 9 and 15 revealed very weak antigenic relationship and were highly divergent. diseases especially those by viral pathogens cause greater economic losses in most horticultural crop species throughout the world as compared to agricultural crops. non-genetic methods of management of these diseases include quarantine measures, eradication of infected plants and weed hosts, crop rotation, use of certified virus-free seed or planting stock and use of pesticides to control insect vector populations implicated in transmission of viruses. however, none of these measures is likely to provide an enduring solution against these diseases especially those caused by viruses due sometimes to the huge expenditure involved, but mostly to the questionable effectiveness and reliability of those methods. as key control pesticides are getting increasingly abandoned, development of alternative methods to control diseases has been a felt-need in the recent past. though breeding for disease resistance generally provides a reliable security in a long run, introgression of host plant resistance did not materialise in most important crops. non-availability of an appropriate source of resistance in inter-fertile relatives, linkage to undesirable traits, or often times polygenic nature of such sources of resistance are the stumbling blocks in breeding programs. the limitations of conventional breeding and routine cultural practices prompted the need for the development of other approaches of virus control that could be fully incorporated into traditional methods. in this perspective, the concept of pathogen-derived resistance offers an attractive strategy to evolve newer methods of virus management, by transforming crop plants with nucleotide sequences derived from the pathogen's genome. an increasing number of molecular characterisation of plant virus genomes and the stable transformation of a number of horticultural crop species have in fact opened an avenue for molecular breeding against virus pathogens. successful field-testing of genetically modified crop cultivars renders proof of their supremacy over existing cultivars. it also contributes to demonstrate their capability with regard to environmental safety with a view to winning over public concern and scepticism. in general, the eventual commercialisation transgenic lines expressing virus resistance will rely upon a host of factors including their field performance, genetic stability, public acceptance and the resolution of environmental concerns and patent related issues. as such, elaborate field trials and allied studies are now required to adapt genetically engineered horticultural crops expressing virus resistance for their implementation into practical agriculture. a few examples from current research at tnau, in india or elsewhere will be discussed in this presentation. virology unit, division of plant pathology, iari, new delhi 12 in recent times there has been greater emphasis on vegetatively propagated crops in india to help diversify the indian agriculture. fruit, flower, spice and plantation crops are important vegetatively propagated horticultural crops, which have become a driving force for economic development in several parts of india. however, most of the vegetatively propagated crops are threatened by biotic stress caused by plant pathogens in general and plant viruses in particular. plant viruses produce specific and non specific symptoms and in some cases no symptoms are produced. correct identification and diagnosis of viral diseases is first step in the management of any disease including viral diseases. there have been two major breakthroughs in virus diagnostics during last four decades. the first one was serological assay using monoclonal or polyclonal antibodies in enzyme linked immunosorbent assay (elisa) and the other one was the use of in vitro amplification of dna in polymerase chain reaction (pcr). a significant development in serological assays has been its simplification in form of user's friendly quick strip/dip stick method. the one-step lateral-flow (lf) tests have been developed for the on-site detection and identification of several plant viruses. rapid advancement in virus genome characterization has led to the development of novel approaches of nucleic acid based diagnostics which include conventional pcr, real time pcr, multiplex pcr, micro/macro arrays and biochips. pcr protocols already exist for many plant viruses of citrus, banana, apple, papaya, vegetables, ornamental and spice crops. a further advancement has led to development of realtime pcr assay which is relatively easy but requires training for diagnosticians. in real-time pcr assays, results can be available within 20 min. the nucleic acid template preparation in pcr has been simplified. membrane based dna template protocol and co-isolation of nucleic acid template preparation are novel approaches in pcr detection of virus and virus like pathogens. since many of the horticultural crops are often infected by more than one virus, their individual detection by pcr is not only expensive but also time consuming. therefore, multiplex pcr has been developed where in genome of more than one virus could be amplified and detected in the same reaction mixture. development of nucleic acid based chip is now one of the fastest and recent growing areas in the field of pathogen detection. these nucleic acid based chips have been named as dna/rna chips, biochips, genechips, biosensors or dna arrays. when it comes to applications of microarray technology for plant viruses, it is not too difficult to see the value of a method that could potentially detect a whole range of viruses using a single test. however, microarrays are unlikely to become the only method in use in a diagnostic laboratory. processing of germplasm including transgenic planting material imported for research purposes into the country. during the last two decades, a total of 49,923 samples of wheat including transgenics were imported from cimmyt (mexico), icarda (syria) and many other countries. these were grown in post-entry quarantine nursery each year at nbpgr, new delhi and the transgenic samples were grown in national containment facility of level-4 (cl-4) since its inception to ensure that no viable biological material/pollen/pathogen enters or leaves the facility during quarantine processing of transgenics. in addition, post-entry quarantine inspections of the transgenic wheat grown by indenters are also undertaken by nbpgr quarantine scientists. virus-induced gene silencing (vigs) is a technique in which viral genomes are used, usually after appropriate modifications, for transient gene silencing in plants. the mechanism behind vigs is the phenomenon called rna-interference (rnai), which is widespread in many organisms and is believed to be form of inherent defence system against intracellular pathogens, such as viruses and transposons. double-stranded rna or rna containing strong secondary structures, commonly produced during viral infections, are believed to cause triggering of rnai, which employs a battery of proteins and nucleoprotein complexes to identify and degrade specific viral transcripts. in vigs, viral genomes not causing severe symptoms, but which can accumulate and spread efficiently in the host plant are used as vectors in which a host gene is cloned and introduced into the plant. upon replication, the viral vector triggers rnai response in the host plant, which also targets the host gene, leading to its silencing and subsequently, the silenced phenotype revealing gene function in vivo. vigs has been used extensively to study gene functions in dicot plants, such as tobacco, tomato, pea, soybean, etc., using vectors derived from reference genes are commonly used as an/the endogenous normalisation measure for the relative quantification of target genes. the expression (characteristics) of seven potential reference genes was evaluated in tissues of 180 healthy, physiologically stressed and barley yellow dwarf virus (bydv) infected cereal plants. these genes were tested by rt-qpcr and ranked according to the stability of their expression (characteristics) using three different methods (two-way anova, genorm and normfinder tools). in most cases, the expression (characteristics) of all genes did not depend on the abiotic stress conditions or on the virus infections. all the genes showed significant differences in expression (characteristics) among plant species. glyceraldehyde-3-phosphate dehydrogenase (gapdh), beta-tubulin (tubb) and 18s ribosomal rna (18s rrna) always ranked as the three most stable genes. on the other hand, elongation factor-1 alpha (ef1a), eukaryotic initiation factor 4a (eif4a), and 28s ribosomal rna (28s rrna) for barley and oat samples; and beta-tubulin (tubb) for wheat samples were consistently ranked as the less reliable controls. the bydv titre was determined in two oat varieties by rt-qpcr by three different quantification approaches. statistically, there were no significant differences between the absolute and the relative quantification, or between quantification using gapdh + tubb + tuba +18s rrna and ef1a + eif4a + 28s rrna. the geometric average of gapdh, 18s rrna, tuba and tubb is suitable for normalisation of bydv quantification in barley and oat tissues. for wheat samples, a combination of gapdh, 18s rrna, tubb, eif4a and e1fa is recommended. department of microbiology, yogi vemana university, vemanapuram, kadapa 516 003 large scale production and import of propagative material poses potential risk of introducing several destructive pathogens particularly viruses and mycoplasma like organisms in our country. this demands adequate quarantine safe guards such as growing them under approved post entry quarantine facility for specific period so as to facilitate virus detection, thereby curtailing risk. when such facilities are coupled with propagation by tissue culture will ensure virus free propagative plant material. the requirement of nationwide network of post entry quarantine facility working in close collaboration with crop institutions are very much emphasized for considering import of high risk plant genera for agriculture development. present paper discusses about virus disease of quarantine importance affecting ornamental and fruit plants such as chrysanthimum, dahlia, dianthus, rosabengalensis, cattleya, cymbidium, dendrobium, lilium, citrus, vitis etc. the paper also discusses on immunodiagnostic methods of detection and methods of obtaining virus free propagative material. rice tungro occurs as epidemics in regular cycles and has been reported in the last 50 years from all the major rice growing regions of india, especially prevalent in the southern and eastern states. development of the durable resistant varieties to tungro is crucial for the management of the disease. molecular breeding, involving the use of dna markers linked to the resistant gene(s) for selection, can overcome the difficulties encountered in conventional resistant breeding programs. for successful marker-assisted selection (mas), the identification of closely linked markers through the process of gene tagging and mapping is a prerequisite. attempts have been initiated for identification of tungro resistance genes through molecular mapping and their introgression into the target varieties using marker-assisted selection at drr, hyderabad. the inheritance of resistance to rice tungro virus disease was studied in seven resistant rice cultivars with field evaluation at hot spot locations. the microsatellite markers linked to rice tungro resistance in utri merah was studied and found that resistance genes were linked to rm 336 on chromosome 7. through molecular mapping two qtl were identified controlling rtv resistance on chromosomes 7 and 2 in 'utri rajapan' explaining 40.8% and 21.6% of the phenotypic variance. in variety 'vikramarya', another two qtl for rtv resistance were detected on chromosomes 7 and 1 explaining 18.7% and 16.4% of the phenotypic variance. the closely linked markers identified in this study flanking the gene of interest through mapping will improve the efficiency and precision of introgression programs in marker assisted breeding for rtv resistance. functional characterization of these qtl for rtv resistance is under progress. there is only a limited pool of natural virus resistance in cassava against cassava mosaic geminiviruses and cassava brown streak ipomovirus hence the development of transgenic resistance in this significant crop might present an option. rna mediated resistance through the expression of inverted-repeat dsrna sequences derived from the virus genome and the modification of plant microrna to produce antiviral artificial microrna are strategies that have recently been proven very effective for induction of virus resistance (immunity) against a number of rna viruses. results from rna interference strategies against geminiviruses never resulted in immunity of transgenes. however, it suggest that viral mrna are targets of rna silencing and that the success of the strategy depends on the relevance of the target gene in the systemic spread of the virus. we have generated a number of rna silencing constructs to induce resistance against cbsv and the indian cassava mosaic viruses icmv and slcmv. due to the serious problems inherent with transformation of cassava and subsequent resistance screening, these constructs were tested for efficiency either by transient-or by transgenic expression in n. benthamiana. complete immunity was reached in transgenic n. benthamiana against cbsv using inverted repeat or amirna constructs. using different species of cbsv for resistance screening, immunity was broken, to show the minimum context for broad spectrum resistance. similarly, highly specific resistance was reached in expression of amirna. in contrast, virus resistance against icmv/ slcmv using single amirna constructs was not successful. results from the experiments to generate virus resistance against cbsv and icmv/slcmv will be shown; methods to evaluate efficiency of rnai gene constructs by transient gene expression in n. benthamiana and strategies to develop efficient resistance against rna and dna viruses in cassava will be discussed. bitter gourd (momordica charantia l.) which is also called bitter melon, balsam apple and balsam pear belongs to family cucurbitaceae. it is an important traditional vegetable of nutritive and medicinal value that is cultivated in tropical and sub-tropical asia, but is considered as a weed host reservoir for viruses in jamaica. viral disease-like symptoms were observed occurring naturally on the crops of bitter gourd grown in the fields of northern india during 2007-2009. an incidence of 78.5% of diseased plants was recorded which showed chlorotic spots and mosaic ranging from mild mottling to green blisters along with leaf smalling, leaf and fruit deformations, bud necrosis and stunted growth whereas 20.2% plants exhibited leaf curling alone or in combination with mosaic-type disease. a reduction of 34.5% in fruit yield was recorded in mosaic-like disease which could be attributed to lesser fruit setting due to bud necrosis, smaller fruit size and stunted plant growth. such plants produced deformed, notched, irregularly shaped fruits wherein pre-mature yellowing and necrosis on the anterior and posteriors ends made 22.4% fruits unfit for marketability. the dwindling yield and production of unmarketable fruits posed a major constraint for profitable cultivation of this economically important crop, thus warranting for studies on etiology and management of these diseases. the mosaic-like disease was transmitted to healthy seedlings of bitter gourd at 2-leaves stage by sap inoculation as well as by aphid viz., myzus persicae sulz. and aphis gossypii glov. initially studies were carried out to optimize protocols for efficient plant regeneration and agrobacterium-mediated transformation for nagpur sweet orange, which is a popular and elite citrus cultivar in india. organogenesis was induced in etiolated epicotyl explants of one-month-old axenically raised polyembryonic seedlings by culturing them in mt medium supplemented with 30 g/l sucrose with varying concentrations of plant hormones. it was found that bap at 1 mg/l without auxin was best for efficient shoot regeneration in citrus using epicotyl explants. a 100% regeneration frequency was obtained and multiple shoot formation was obtained from both the cut ends of all the explants. an average of 8.24 well-differentiated shoots per explant were obtained, all of which rooted normally under the influence of 1 mg/l iba. this improved regeneration protocol was utilized in standardizing agrobacterium-mediated transformation of citrus using a. tumefaciens strain eha 105, containing binary plasmid pcambia 2301 that harbors gus reporter gene and npt-ii plant selection marker gene. one-month-old epicotyl explants infected with over-night grown agrobacterium (a 600 0.6-0.8) for 15 min and co-cultured for 3 days were found to be optimum for transformation as assessed on the basis of pcr analysis and gus activity displayed by the stem and leaf sections of putative transgenics. overall transformation frequency ranged from 38 to 48%. current study focuses on the generation of citrus transgenics for ctv resistance using a. tumefaciens strain eha 105 containing binary plasmid pbinar harboring portion of coat protein gene of ctv and npt-ii gene employing the standardized protocols. several putative transgenic shoots were recovered on selection medium and they are being utilized for molecular analyses and resistance against ctv. work is also in progress on the generation of citrus transformants using rnai construct harboring ctv cp and p23 genes, singly and in conjunction. our lab was also involved in developing rice transgenics for resistance against rice tungro disease, which is one of the most important and widespread virus diseases of rice in south and southeast asia, causing an annual estimated loss in crop yield of economic losses worth millions of rupees are caused due to these diseases annually. virus diseases are frequently less conspicuous than those caused by other plant pathogens and last for much longer. this is especially true for perennial crops and those that are vegetatively propagated. one further problem with attending to assess losses due to various diseases on a global basis is that what most of the data are from small comparative trials rather than wide scale comprehensive surveys, even the small trials do not necessarily give data that can be used for more global estimates of losses. this is for several reasons, including: (1) variation in losses by a particular crop from year to year; (2) variation from region to region and climatic zone to climatic zone: (3) differences in loss assessment methodologies; (4) identification of the viral etiology of the disease; 5 variation in the definition of the term 'losses' and (6) chilli is the major vegetable and spice crop grown in thar desert areas of rajasthan. leaf curl disease (chlcd) is one of the major constrains in chilli cultivation faced by farmers and cause yield loss up to 100%. a survey was conducted in major chilli growing areas of thar desert; bikaner, nagur, jodhpur and jalore districts of rajasthan during november, 2009 to understand the present status of leaf curl disease in chilli. among the four district surveyed for chlcd, the disease incidence was recorded maximum (up to 98%) in jodhpur district followed by jolore district (up to 88%). no relation was found between the disease incidence and varieties. the major varieties grown in these area are; mehsana, rch (mandoria), haripur raipur, mathania and local cultivars. the number of whitefly was also counted in top, middle and bottom leaf of chilli grown in these areas. the average number of whitefly per plant ranged from 0.0 to 4.0. more number of whitefly (4.0) was recorded in jodhpur district and lowest (1.8) in jalore district. total dna was extracted from three leaf curl infected samples from each district and tested for the presence of begomovirus using coat protein (cp) and dna-b specific primers. all the samples were positive for cp and dna-b amplifications by pcr. the cloning and sequencing of selected cp gene and dna-b fragments are in progress. the preliminary investigations shows that the leaf curl disease of chilli is widespread in the arid region of rajasthan and may be caused by begomovirus associated with satellite dna-b. bittergourd (momordica charantia) is an important vegetable crop of kerala. the crop is affected by several diseases of which mosaic is a prominent one. a field experiment was conducted to evaluate the efficacy of potentised resistance inducing substances (ris) viz., mosaic affected bittergourd plant tissue, ash of mosaic affected bittergourd plant tissue, plumbago and salicylic acid for control of bittergourd mosaic in march 2008. ris were applied as drench and foliar spray at three potency levels twice, before flowering of the crop. the experimental crop was grown as per the package of practice recommendations in split plot design with five replications per treatment. the disease incidence, disease severity and yield of the crop were recorded. the result of the experiment shows that spraying was more effective than drenching of treatments for reducing mosaic incidence and severity. among treatments, infected plant extract at 19 potency was the most effective one for reducing mosaic incidence and it showed the maximum incubation period and minimum disease severity. the spray application of treatments produced significantly higher yield than drenching. among the treatments, ash of infected plant at 19 and 309 potency and infected plant extract at 69 potency were on par and produced comparatively higher yield. elephant foot yam (amoprhophallus paeoniifolius), colocasia (colocasia esculenta) and tannia (xanthosoma sagittifolium) are the major edible aroids cultivated in india. the elephant foot yam cultivation is gaining importance due to its high production potential, nutritional and medicinal values and good economic returns. all these aroids are vegetatively propagated and viral diseases are spreading through planting materials. ctcri has the mandate of producing healthy planting materials of these edible aroids. accurate diagnosis and identification of the virus is essential for production of healthy planting material and effective management of the disease. though occurrences of viral diseases on edible aroids in india were known in 1960s, not much attention was given for detection and identification of the virus involved. in case of elephant foot yam 5-30% mosaic incidence was observed with varying symptoms of mosaic, puckering, filiformy etc. in colocasia and tannia, 5-10% incidence was noticed. rt-pcr amplification with potyvirus group specific primers and subsequent cloning and sequencing of the amplified product has confirmed the association of dasheen mosaic virus (dsmv) with all the three edible aroids cultivated in india. the complete full length coat protein gene of dsmv infecting elephant foot yam was cloned in pgem-t vector and sequenced. further sequence analysis revealed that the cp of dsmv consisted of 942 nucleotides and the 3 0 utr comprised of 260 nucleotides. blast and phylogenetic analysis showed highest similarity of 89% with that of dsmv isolate af048981, reported from usa. the deduced amino acid sequence of cp had 92.0-98.0% identity with other dsmv isolates. blast analysis of the partial cp gene sequences of colocasia and tannia also confirmed that the virus involved is dsmv. rt-pcr analysis of large number of samples from all the three crops confirmed that the potyvirus group specific primers (mj1 and mj2) are good for rapid detection of dsmv in these crops. dsmv specific biotinylated cdna and digoxigenin labelled crna probes were also prepared and dsmv in elephant foot yam was detected through nucleic acid spot hybridization. yellow leaf disease (yld) caused by sugarcane yellow leaf virus (scylv) is a recently recorded disease in india and is found wide spread throughout country. in popular varieties, the disease incidence varied from 0 to 75.0% and attained epidemic levels under field conditions. detailed studies on the impact of yld on sugarcane revealed that the virus infection significantly reduces various cane growth parameters, cane yield and juice quality. sequence comparisons of the coat protein (cp) and movement protein (mp) of 22 scylv isolates from india and database sequences showed a significant variation between indian isolates and the database sequences both at nt and aa level in the cp/mp coding regions. the significant variation in our isolates with the database isolates, even in the least variable region of the scylv genome showed that the population existing in india is different from rest of the world. further, comparison of partial sequences encoding for orf 1 and 2 revealed that yld in sugarcane in india is caused at least by three genotypes viz., cub, ind and bra-per, of which a majority of the samples were found infected with cuban genotype (cub). the genotype ind was identified as a new genotype and this was found to have significant variation with the reported genotypes. we have identified specific primers from cp region of the virus and optimized rt-pcr conditions to diagnose the virus. this assay has been found efficient in detecting the virus in asymptomatic plants and tissue culture derived seedlings. elimination of the virus through meristem culture has been demonstrated to purify the virus from the infected planting materials and this technique needs to be adopted to supply disease-free planting materials for effective management of the disease. studies are also in progress to identify the yld-resistant sources in sugarcane germplasm to initiate breeding for yld-resistance in sugarcane. mycoviruses are viruses that infect fungi. they have been identified in all major fungal families. in the present scenario, mycoviruses are the important means of biocontrol of plant fungal pathogens. most identified fungal viruses have double stranded rna genomes, often with more than one dsrna present per virus particle, and have been spherical in shape. these viruses are mostly vesicle bound, as other viruses have protein coatings. to be a true mycovirus, they must demonstrate an ability to be transmitted-in other words be able to infect other healthy fungi through anastomosis and spores. mycoviruses lead 'secret lives', reduce the ability of their fungal hosts to cause disease in plants. this property, known as hypovirulence (hypovirulence is a term used to describe reduced virulence found in strains of pathogens), this phenomenon was first observed in cryphonectria (endothia) parasitica (chestnut blight fungus) on european castanea sativa in italy, where naturally occuring hypovirulent strains were able to reduce the effect of virulent ones. these slower growing hypovirulent strains of c. parasitica contain a single cytoplasmic element of double-stranded rna (ds rna) similar to that found in mycoviruses that was transmitted by anastomosis in compatible strains through natural virulent populations of c. parasitica. hypovirulence has also been reported in many other fungal plant pathogens, including rhizoctonia solani, gaeumannomyces gramini var. tritici, ophiostoma ulmi, sclerotinia homoeocarpa, diaporthe ambigua alternaria alternata, and fusarium sp. etc. hypovirulence has attracted attention owing to the importance of fungal diseases in agriculture and the limited strategies that are available for the control of these diseases. it reduces the use of toxic fungicides which also affect the plant growth. the symptoms resulted by the mycoviruses are reduction in growth, reduction in pigmentation and sporulation, excessive sectoring and aerial mycelial collapse. these are the consequences of alteration in complex physiological and biochemical processes involving interaction between host and virus. cassava (manihot esculenta crantz.) is the major tuber crop in peninsular india, it is grown in an area of 2.4 lakh hectares with the annual production of 6.7 million tonnes both for direct consumption and the starch grain (sago) producing industries, mainly in the southern states of tamil nadu, kerala and andhra pradesh (fao 2005) . in tamil nadu, cassava primarily produced for sago producing industries where it is considered as an industrial crop rather than food crop, so the resource rich farmers are cultivating the cassava as irrigated crop in their fertile land and the poor farmers are raising the crop under rainfed conditions. in south india in addition to cassava there is a practice of intercropping important vegetable crops like, tomato, brinjal, legumes and gourds in cassava fields since all the above mentioned crops are short duration and are money spinners for the farmers. unfortunately, the major production constraint in these vegetable crops including cassava is the geminiviruses belonging to the family of in recent years there has been growing concern regarding the standard of scientific researches in india. the strengths, weaknesses, opportunities and threats (swot) analysis on indian scientific research reviewed the progress of science during the last six decades. although the 'strengths' were highlighted in good measure, it was the list of 'weaknesses' that called for attention to upgrade the standard of research and 'opportunities' that provide scope for overall scientific growth. a comparison between india and other countries in terms of research papers published revealed that india's contribution to science has come down enormously. what ails indian science? should we compare the growth of indian science with other developed countries? what criteria should be adopted to judge the quality and standard of scientific research? how to motivate the scientists to improve their scientific output? how do motivate the scientists to improve their scientific output? how do indian journals perform in maintaining quality? this paper analyses critically the scientific journals around the world, based on the scores allotted by the national academy of agriculture sciences (naas) in 2003 and 2007 for 1460 and 1608 journals respectively. in general, the indian journals performed poorly irrespective of the disciplines with only 25-30% in the high standard. the paper dealt with the reasons for low impact factor, the anomalies in the allotment of scores to wide spectrum of the journals and the disadvantages the scientists face with the scoring system. a case study was presented of an institute with over 50 scientists whose publications were analyzed to discuss the merits and demerits of the system. the performance of the journals published by prestigious academics, societies and councils was also projected. the paper concluded with the need for enhancing the image of the country through research publications in high standard journals and the role of various scientific bodies with shore and long term measures. poster session herpes simplex virus (hsv) keratitis is a leading cause of corneal blindness throughout the world. the infection can be diagnosed by clinical manifestations but in case of atypical ocular cases, laboratory diagnosis is more helpful in timely management of disease. collection of corneal scrapings in all cases of stromal and epithelial keratitis may not be possible, but collecting tear fluid is a convenient procedure causing less discomfort to the patients. therefore, the present study was intended to evaluate the suitability of tear specimens for detecting hsv by polymerase chain reaction (pcr) and immunofluorescence (ifa). tear fluid and corneal scrapings were collected from 134 patients of suspected herpetic keratitis. hsv-1 antigen was detected by ifa using rabbit anti-hsv antibodies. pcr was performed to amplify 111 bp region of thymidine kinase (tk) coding gene and 144 bp region from dna polymerase coding gene of hsv. out of 134 patients hsv antigen was detected in 25 (18.65%) of corneal scrapings and 15 (11.19%) of tear specimens and in 12 (8.95%) patients from both the specimens. hsv gene could be amplified in 44 (32.83%) of corneal scrapings and 16 (11.94%) of tear fluids and in 13 (9.71%) patients from both the specimens. although, corneal scraping seemed to be marginally superior material for detection of hsv, tear fluid may also serve as an appropriate alternative clinical specimen, due to ease of collection and least discomfort to the patients. in either cases pcr detected higher number of hsv cases than ifa. therefore if and when feasible, both ifa and pcr should be used simultaneously on each specimen to obtain best results. cytokines play a key role in the regulation of immune responses. in hepatitis c virus infection (hcv), the production of inappropriate cytokine levels appears to contribute to viral persistence and to affect response to therapy. il-6 is produced by a variety of cells including t cells, phagocytes and fibroblast. cytokine genes are polymorphic at specific sites, and certain mutations located within coding/regulatory regions have been shown to affect the overall expression and secretion of cytokines in patients with hcv infection. to correlate the serum levels and polymorphism of il-6 gene in chronic hepatitis c patients and healthy controls. forty patients positive for hcv rna attending the medicine out patient department and wards of lok nayak hospital, new delhi as well as forty healthy controls were enrolled for the study. the serum level of il-6 was detected by using elisa. genomic dna was extracted from whole blood of hcv infected patients and healthy controls by using accuprep genomic dna extraction kit according to manufacture's instruction. the genotyping of il-6 promoter (-174 variant) was carried out by pcr and direct sequencing using the method of patricia woo et al. 1998. the serum level of il-6 was significantly down regulated in hcv infected chronic patients as compared to the healthy controls. genotyping of -174 promoter variant of il-6 was performed by pcr and direct sequencing. il-6 polymorphism in the g/g, g/c and c/c allele was non significant when compared to hcv patients and healthy controls. the il-6 serum levels were significant among hcv infected patients when compared to healthy controls. the polymorphism in the promoter region of il-6 (-174) was found nonsignificantly associated in hcv patients compared to healthy controls. in conclusion, the present study suggests that the host il-6 polymorphism alone may not play a significant role in the outcome of hcv infection. acute gastroenteritis (age) is a global health problem and has been associated with multiple etiological agents, which include bacteria, protozoa and viruses. viral gastroenteritis is considered as the second most common illness in children after upper respiratory tract infection. among enteric viruses, rota, noro, enteric adeno, astro and enterovirus are found to be associated with gastroenteritis. although, association of enteric viruses has been established in children hospitalized for age no such data is available from hospitalized children other than enteric infections. to determine the prevalence of enteric viruses circulating in hospitalized children. fecal samples, n = 292 (177 symptomatic and 115 asymptomatic for age) were collected from children \5 year of age from three different hospitals across the city of pune from june 2008 to feb. 2009. detection of group a rotavirus was carried out by using antigen captured elisa. rt-pcr and pcr was carried out for the detection of norovirus, enterovirus, astrovirus and enteric adenovirus detection by using primers targeted to rdrp gene, 5 0 ncr gene and consevered gene for serine protease and hexon gene respectively. out of 177 fecal samples tested for enteric viruses in age cases, the prevalence of rota, entero, noro, enteric adeno and astrovirus were 33.3% (59), 14.7% (26), 6.2% (11), 2.8% (5) and 1.1% (2) respectively. however, the presence of these viruses in the asymptomatic cases (n = 115) was detected at 7.8% (9), 5.2% (6), 7.8% (9), 0.86% (1) and 1.7% (2) levels respectively. mixed infections of enterovirus and rotavirus were found in both symptomatic 1.6% (3) and asymptomatic cases 0.8% (1). however, mixed infection of enterovirus with adenovirus were found only in asymptomatic cases 0.8% (1). no marked difference was observed in the seasonal pattern of all viruses in the patients with or without gastroenteritis. the findings of this study document highest circulation of rotaviruses in patients symptomatic and asymptomatic for age. the entero and noroviruses remain second most important enteric viruses in these patients. influenza in humans is a major public health concern and the understanding of its evolution in the light of its ''antigenic drift'' helps prediction of epidemics and update of yearly influenza vaccine. to antigenically characterize influenza a (h3n2) isolates and study antigenic drift during 1990 to 2009 in pune city. patients with influenza like illness were identified using a strict case definition from dispensaries located in different areas in pune and clinical samples (ns/ts) were collected after obtaining informed consent. these clinical samples were processed in vivo (in fertile eggs) and in vitro ( overall, an additional 35 (39.7%) positive cases of dengue could be detected when ns1 antigen assay was also used in the study. highest ns1 antigen positivity was encountered among the samples collected on the 3rd day of fever whereas mac elisa for anti igm antibody was positive after 4th day and gradually there was an increase in the positivity towards the convalescent phase of the disease. the results of this study indicate that ns1 antigen based elisa test can be an useful tool to detect the dengue virus infection in patients during the early acute phase of disease since appearance of igm antibodies usually occur after fifth day of the infection. concurrent use of both diagnostic assays namely ns1 antigen as well as mac elisa will improve the overall detection of dengue infection. early detection of acute dengue virus infection is crucial to provide timely information for the management of patients. human parvovirus b19, a member of the parvoviridae family, is a pathogen associated with a wide variety of diseases. most commonly, it causes childhood rash erythema infectiosum, but in some cases more serious symptoms such as persistent arthropathy, critical failures of red cell production causing transient aplastic crisis, this infection in pregnancy causes hydrops fetalis and myocarditis. traditional immunosuppressive therapy being unsuccessful, anti-viral therapy might be worthy of consideration. functional annotation would provide role of viral proteome in its survival and pathogenic mechanisms. svmprot functional family annotations of vp2 protein had deciphered its zincbinding, coat protein, outer membrane, chlorophyll biosynthesis, dna repair and calcium-binding nature. vp2 protein is having a key role in viral assembly of b19 virus and being non-homologous to human proteome, it was identified as an attractive molecular target for structure based drug discovery. the vp2 protein crystal structure was energy minimized using charmm. a structure based virtual screening method was applied using ligandfit to identify potential inhibitors of vp2 protein from chembank database and ten potential human parvovirus b19 vp2 inhibitors were proposed. prism 310 genetic analyzer. the drafting of the sequences was performed using bioedit software and were submitted in genbank. for phylogenetic interpretation denv representing the full extent of genetic diversity in denv-1, denv-2 and denv-3 were collected from genbank. neighbor joining algorithm was implemented with bootstrap value of 10,000 replicates for phylogenetic inference using mega 4.0.2. the genomic region 134 to 644 (c-prm gene junction) of denv were amplified directly from patient serum. twelve of 72 samples were positive for dengue viral rna. of these 4 were dengue type 1, 1 was dengue type 2 and 7 were dengue type 3. for molecular epidemiological survey and genotyping of the sequences more than 100 sequences from different geographical areas including sequences form previously reported north indian isolates were compared with our present data set. the critical analysis of the sequences revealed: 4 dengue type 1 sequences were clustered within sub-type 2 of genotype iii and all the 7 sequences of den-3 clustered along with genotype iii. thus, among the dengue types 1, 2 and 3 currently circulating in north india, dengue type 3, genotype iii, being the predominant one followed by, genotype iii of dengue type 1. although there is no specific treatment or vaccine available currently, the confirmative rapid diagnosis based on detection of viral nucleic acid or igm antibodies in serum, an indication of recent infection, helps in epidemiological monitoring, symptomatic treatment of patients and determining prognosis. serological detection of anti-cgv igm antibodies was performed using rapid immuno-chromatographic assay (rica) and igm-antibody capture enzyme linked immunosorbant assay (mac-elisa). eighty convalescent sera were tested by rica and 60 of them were found positive for anti-cgv igm antibodies. twenty-five anti-cgv igm antibody rica positive sera were further assayed using mac-elisa. more sera from the patients are currently being tested to compare the sensitivity of these two serological assays in anti-cgv igm antibody based early serological diagnosis of cgv infection and the findings will be presented. thus the present study was designed to evaluate the utility of multiplex pcr (mpcr) for simultaneous and rapid detection of dengue and chikungunya viral infections. seventy-two acute phase blood samples from clinically suspected dengue cases were subjected for dengue and chikungunya uniplex pcr using dengue genus specific primers and e gene specific primers for chikungunya virus as well as multiplex pcr was developed for simultaneous detection of dengue and chikungunya infection. standard strains of dengue and chikungunya virus were used as controls. 13 of the 72 clinically suspected dengue samples were found to be positive for dengue viral rna by dengue uniplex pcr as well as dengue chikungunya mpcr whereas none of the samples were positive for chikungunya virus infection by both uniplex chikungunya pcr and dengue chikungunya mpcr. the result of dengue and chikungunya uniplex pcr was found to be 100% concordant with dengue chikungunya multiplex pcr. dengue chikungunya multiplex pcr was found to be a potential rapid test to detect dengue and chikungunya viral infections simultaneously in clinical samples. sheetal malhotra, neelam marwaha, karan saluja, ratti ram sharma department of transfusion medicine, pgimer, chandigarh 160012 transmission through blood and blood products can be reduced to a great extent by efficient and reliable testing of the blood. the newer fourth generation elisa assays simultaneously detect antibodies against hiv-1 and 2 and the presence of p24 antigen and thus shorten the window period to about 14 days, as compared to 22 days with third generation elisa. to compare the hiv seroprevalance among blood donors using fourth generation elisa (antigen-antibody) versus third generation elisa (antibody) assay. this was a prospective study involving 5100 blood donors of which 3400 were voluntary donors (1700 being students and 1700 being non students) and 1700 were replacement donors. sex workers are one of the core group for transmission of sti/hiv and as a ''bridge group'' to the general population. accordingly, highest priority is given to this group in targeted intervention for prevention of hiv/aids. here we are describing one such female sex worker who was harbouring 5 concomitant sti including 4 viral sti. a 25 year old female sex worker was brought to the sti clinic of a tertiary care hospital by ngo with complaint of genital discharge for 3 days. on per speculum examination, cervix was slightly erythematous, tender with mucopurulent discharge. there was no vaginal discharge or ulcer in anogenital area. however, there was a wart at lateral wall of vagina. as per naco syndromic management guideline, treatment was given for n. gonorrhoeae, c. trachomatis and hpv. cervical swab was taken and subjected to various microbiological investigation for the detection of sti viz n. gonorrhoeae, c. trachomatis, t. pallidum, candida spp., t.vaginalis, hsv-1, hsv-2, hiv, hbv, hcv, hpv and m. contagiosum. saline wet mount showed pus cells, but no yeast cells or trophozoite of trichomonas vaginalis. gram stained smear showed more than four polymorphonuclear leucocytes in the absence of gramnegative intracellular diplococci and a presumptive diagnosis of non gonococcal urethritis was made. no organism was isolated on any culture media after appropriate incubation. cervical swab was negative for antigen of c. trachomatis. serum was tested positive for hbv, hcv, hsv-2 and t. pallidum though it was seronegative for hiv. in the present case, the female sex worker was harbouring four viral sti viz hsv-2, hbv, hcv and hpv alongwith t. pallidum. however clinically she was diagnosed and treated accurately only for genital wart while cervical discharge due to hsv-2 was misdiagnosed. it is necessary to try to test alternative approaches such as periodic presumptive therapy of viral sti, because this will not only boost up the efforts of sti control in the target group but also help in hiv control. alternatively, regular clinical and laboratory screening for viral sti may be tried. densonucleosis viruses (dnv) belong to parvoviridae family. they are the etiological agents of insect's disease known as densonucleosis, which leads to death or loss of vital functions of the infected insect. densonucleosis virus of mosquitoes has generated lot of scientific interests because of its tremendous potential in biological control and its application as a transducing vector. earlier, we have reported the isolation and characterization of a dnv from aedes aegypti mosquitoes and its prevalence among different ae. aegypti populations from india. there are reports suggesting that when aedes albopictus mosquitoes co-infected with dengue-2 and dnv, the multiplication of den-2 is suppressed. the present study focus on the effect of coinfection of ae. aegypti mosquitoes with dnv and chikungunya virus (chik). the first instar mosquito larvae were infected with dnv and the emerging dnv infected females were then infected with chikv by oral feeding. thus obtained chik infected female mosquitoes were analyzed by real time pcr for both dnv and chikv on alternate days post-infection, up to the 14th day. the data showed no significant difference in the multiplication of either of the viruses after co-infection. results suggest that chikv neither stimulates the replication of dnv nor is its own replication suppressed due to co-infection. this study forms an initial step in understanding the role played by such endogenous viruses on the vector dynamics. chandipura virus pathogenesis is manifested as encephalitis in young children with a very high mortality rate. this damage could be due to direct replication of the virus in brain parenchymal tissue or immune system mediated. this study aims at elucidating the role of brain infiltrating lymphocytes in pathogenesis using mice as the model system. mice were inoculated intracerebrally with the virus and the perfused brain tissue was used to isolate the lymphocytes. control mice were inoculated with an equal amount of media. in order to standardize the procedure for isolation of lymphocytes from brain tissue, splenocytes were processed to isolate the lymphocytes using histopaque density gradient method. methods to isolate lymphocytes from brain tissue as described by earlier workers were tested for the ease and efficiency of procedure using known suspension of lymphocytes from spleen. percoll density gradient method provided optimum yield of lymphocytes with an ease of handling. in this, brain cell suspension used to prepare 30% percoll is layered over 70% percoll prepared using media in 1:2 ratio. density gradient centrifugation is carried out at 9009g for 20 min at 15°c to obtain lymphocyte layer at the interface. leishman staining was performed to analyze the morphological characteristics of isolated lymphocytes. normal lymphocytes showed dark blue stained nucleus. some bigger sized cells with diffused nucleus characteristic of atypical lymphocytes were observed and some of the cells were surrounded by hair like structures. phenotypic characterization was carried out using flow cytometry. the presence of cd4 + , cd8 + and cd19 + cells was observed. the percentages of cd8 + , cd4 + and cd19 + cells was found to be 7.60%, 35.14% and 34.32% respectively in the lymphocytes isolated from infected animal and 5.65%, 30.27% and 3.13% respectively from control animal. hence, cd19 + cells showed maximum infiltration after infection. (santosh et al. 2008; pradeep et al. 2008 ). in the present study chikv suspected blood samples were collected and the acute phase samples were subjected to rt-pcr for the presence of virus specific rna by using the primer pair dvrchk-f/dvrchk-r as described by us earlier (naresh kumar et al. 2007 ). the convalescent phase samples were screened for chikv specific antibodies by using sd bioline chikungunya igm rapid test. six sets of primers were designed to amplify the complete nsp4 and complete structural genes of chikungunya virus. the products were further gel purified, cloned in ptz57r/t vector and the recombinant clones were sequenced and submitted to the genbank. the complete ns4gene and structural genes were compared with other available sequences in the genbank. sequence analysis results will be presented. the present study discusses these aspects in detail. . some of these phages (viz. v953, v954) showed plaques at 42°c but not at 37°c. thus they seem to be lysogenic. for propagating and increasing the titre of all the above isolates, various previously described methods were attempted, but none of these methods were satisfactory. but when siliconized glassware and plastic-ware were used, propagation was successful. we showed that siliconization of glassware and plastic-ware was essential for the propagation of our mycobacteriophage isolates v951, v952, v953, v954 and v955. also, phage dilution medium (pdm) as described by chaterjee et al. (2000) was found to be effective for picking out of the plaques made by the phages. in this way, the phage isolates were propagated up to p 3 . the various passages of the phage isolates v951, v952, v953, v954 and v955 (i.e. original, p 1 , p 2 and p 3 ) were stored at -80°c. the four major routes of transmission are unsafe sex, contaminated needles, transmission from an infected mother to her baby at birth (vertical transmission) and breast milk. screening of blood products for hiv has largely eliminated transmission through blood transfusions or infected blood products in the developed world. in 2008, globally, about 2 million people died of aids, 33.4 million were living with hiv and 2.7 million people were newly infected with the virus. hiv infections and aids deaths are unevenly distributed geographically and the nature of the epidemics vary by region. more than 90% of people with hiv are living in the developing world. there is growing recognition that the virus does not discriminate by age, race, gender, ethnicity, socioeconomic status-everyone is susceptible. however, certain groups are at particular risk of hiv, including men who have sex with men (msm), injecting drug users (idus), and commercial sex workers (csws). the present study indicates the prevalence of hiv infection among the people residing in the northern region of india predominantly among the foothills of the himalayas. the study was carried out on the patients visiting herbertpur christian hospital (a unit of emmanuel hospital association) under the integrated counselling and testing centre scheme at the respective hospital during the 2009-2010. the study indicates the screening of people groups residing in the respective area through community health schemes. the diagnosis of the hiv infection is done by three types of assays namely. the tridot method which is the rapid method of diagnosis followed by the. hiv coombs test which involves the dot immunoassay principle. the third assay is the enzyme linked immunosorbent assay (elisa). the number of patients screened during the period of september 2009 to march 2010 is 635 which include patients coming from four different states namely haryana uttarakhand uttarpradesh and himachal pradesh. the number of people who were tested positive are 8 and the number of people who were tested negative are 627. the people tested positive are sent to the higher centre for other confirmatory tests such as pcr and western blot analysis. these patients are sent for treatment and prophylaxis at a respective recognised centre in dehradun. the present study determines a consistent community hiv screening and treatment approach through diagnostics counselling and awareness programmes. classical swine fever (csf) also known as hog cholera is a highly contagious and fatal disease of swine. csf became rapidly a major issue of pig industries. it still causes important economical losses worldwide. it is considered as a major health problem of swines in india. during the month of august to october 2009 there was an outbreak of classical swine fever in bihar. from three districts darbhanga, patna and supol, total 36 numbers of different infected tissue samples like kidney, spleen and lymphnode were collected from the dead morbid/pigs. total rna was isolated from 20% homogenate of infected tissues in sterile pbs by tri-reagent (sigma, usa) according to the manufacturer's instructions and cdna was prepared by using commercial available kit. the cdna was stored frozen at -20°c until used. for the molecular detection of classical swine fever virus specific nested pcr amplification of e2 and 5 0 ntr was done along with ns5b and e rns amplification. primarily these samples were found positive with these primers. further confirmation by sequencing was done by cloning of these pcr products in pgem-t easy vector. e2 and 5 0 ntr sequences were considered for phylogentic analysis along with 20 complete available sequences of csfv. nucleotide sequence alignments were carried out using the clustalw program (dnastar) and phylogenetic tree analysis (dnastar) showed that 5 0 ntr have close proximity with taiwan strain (accession no. ay568569) and e2 shows close proximity with chinese isolate csfv-39 (accession no. af407339). peste des petits ruminants (ppr) and sheeppox are oie notifiable diseases of small ruminants especially sheep and goat. both the diseases are economically important, in enzootic countries like india and are major constraints in the productivity of animals. considering the geographical distribution of both ppr and sheep pox infections and prevalence of mixed infection, in the present study, safety and potency of the experimental duel vaccine comprising attenuated strains of thermostable-ppr virus (pprv-revati, p-50) grown at 40°c and attenuated sheep poxvirus (sppv-srinagar, p40) was evaluated in local non-descript sheep. experimental animals were grouped into four groups and each group was comprising six animals, received 100 doses (10 5 tcid 50 ), 1 dose (10 3 tcid 50 ) and 1/10th dose of vaccines and normal saline as control in 1 ml volume subcutaneously, respectively. serum samples were collected on 0, 7, 14, 21 and 28th day post vaccination. sheep simultaneously immunized with 1 ml of vaccine consisting of either 100 or 1 doses of each of pprv and sppv were monitored for clinical and serological responses for a period of 3-4 weeks post-immunization (pi) and post challenge (pc). specific immune responses i.e., antibodies directed to both pprv and sppv could be demonstrated by ppr competitive elisa kit and capripox indirect elisa, respectively following immunization. all the immunized animals' resisted infection when challenged with virulent strain of sppv (srinagar isolate at p-6) on day 28 dpi, while in contact control animals developed characteristic signs of sheeppox. the challenge of the sheep against ppr was not carried out, however, the antibody titre after immunization determined by snt and elisa, indicated that protective titre, as per earlier report on the goats. dual vaccine was found safe at higher dose and induced protective immune response even at lower dose (10 2 tcid 50 ) in sheep, which was evident from sero-conversion as well as challenge study with sppv. the study indicated that both the viruses are compatible and did not interfere with each other in eliciting immune response, paving the feasibility of use of this dual vaccine in combating both infections simultaneously. goatpox is one of the highly contagious, oie notifiable and economically important viral diseases of goats. the disease is caused by goatpox virus (gtpv) is classified of the genus capripoxvirus in the family poxviridae. the disease incurs severe economic losses in terms of high morbidity in adults and heavy mortality in young kids and is a major constraint in goat farming in india. considering the enzotic nature and economic impact of the disease, it is all important to control the infection by developing an effective vaccine. recently, vero cell based a live attenuated goat pox vaccine; using gtpv uttarkashi isolate (p60) has been developed in authors' laboratory and evaluated in goats. the vaccine was found safe, potent and immunogenic experimentally and even at field trials. the vaccine has been evaluated at large-scale at different regions of the country and found suitable for mass vaccination. however, the longevity of potency was not evaluated. therefore, a long term potency trials were studied for a period of 4 years with annual challenge by using virulent goatpox virus and sero-monitoring. a sufficient number of hill goats has been vaccinated with 1 dose of vaccine (10 2.5 tcid 50 /ml) and monitored for clinical and serological response. every year, significant number of vaccinated (n = 5) and control animals (n = 2) were used for challenge with virulent strain (2 9 10 7.0 srd 50 /ml, gtpv mukteswar). sera of pre-and post-challenged (14 dpc) animals including controls have been collected and monitored for serological response in the form of specific antibody production by snt and indirect elisa. all the vaccinated animals were protected on challenge, whereas, all unvaccinated controls developed infections. the same has been reflected in sero monitoring of collected sera. so the developed live attenuated goat pox vaccine was found safe, immunogenic and potent for a period of 4 years of immunization and suitable for mass scale vaccination in control and eradication of goat pox along with a/are suitable diagnostic tool/s in goatpox enzootic country like india. rotavirus infection in avian species varies from subclinical infections to outbreaks of diarrhea. the economic significance of rotaviral enteritis to the poultry industry has not yet been defined, but by analogy to the situation in mammals, it is likely to be significant. unlike the extensive studies performed on rotavirus infection in humans and animals, limited studies have been carried out to determine the extent of exposure of poultry birds to rotaviruses. to determine the prevalence of avian rotavirus antibodies in commercial broiler chickens. a total of 120 chicken serum samples were collected from the lairage of a poultry slaughter house where birds from four different broiler farms in and around pune city were supplied to. the serum samples were tested by an igg antibody capture elisa wherein purified chicken rotavirus ch2 was used as coating antigen. sera from specific pathogen free (spf) chick (n = 20) served as negative control in the test. cut off was calculated as mean negative control ? 3sd (standard deviation). s/co (mean sample od 450 /cut off) values above 1 (1.113-4.445) in 60% (72/120) serum samples were indicating positivity to rotavirus antibodies. the result of the study indicates exposure of the birds to avian rotavirus or similar agent that is circulating in pune city. bluetongue has become established in south india causing regular outbreaks in sheep. btv serotypes 2, 9, 15 and 21 were isolated from native sheep of andhra pradesh. the other serotypes circulating in the state need to be identified. however the major constraint is the serotype identification. to overcome the difficulties of traditional serotyping methods (neutralization tests), nucleic acid based tests are being tried. rt-pcr for serotyping was standardized using primers specific to vp2 gene of btv-2, 9 and 15 serotypes. rt-pcr resulted in 653 bp product of btv-2, 1241 bp product of btv-9 which was defined by specific primers. however non specific amplification at two different sites i.e. 700 bp and 1500 bp was noticed for btv-15. specificity of rt-pcr was evaluated. btv-2 and btv-9 specific primers could amplify only btv-2 and btv-9 respectively where as btv-15 type specific primers amplified not only btv-15 but also btv-2 and btv-9. nucleic acid sequence data obtained from btv-2 pcr product and btv-9 cloned products were specific to vp2 gene of btv-2 and btv-9 respectively. however, 700 and 1500 bp products of btv-15 were identical to vp 4 gene of btv-2, 8, 10, 11, 13 and 18 and vp 1 gene of btv-2, 8 and 10 respectively, indicating the non specific amplification of btv-15. foot and mouth disease is the most contagions and highly economically impotent disease of cloven footed animals. the disease is controlled by regular vaccination using the vaccine produced from the virus grown in the cell culture. the vaccine strain used for vaccine production is selected from the field isolates based on the adaptability and growth kinetics in bhk21 cells and antigen coverage. however the field viruses need to be passaged several times to adapt in tissue culture. passage of field viruses in tissue culture may results in development of mutants whose genetic makeup may differ from the field samples also some of the field strains may fail to adapt or may grow poorly in the tissue culture, thus the efficiency of the vaccine gets affected. structural proteins of fmdv carry the sequences which determine the serotype specificity and immunogenicity. thus one may replace the gene coding for structural proteins from the full length cdna copy of the vaccine strain that has been adapted to the tissue culture with the poly-structural protein gene (pi) so that the chimeric virus gets the serotype specificity of the field strain besides retaining the other characteristics that are needed for a vaccine virus. we have made replication competent fmdv asia i full length genome and cloned under t7 and cmv promoter separately in plasmid vectors. bam h1 sites were created for inserting pi-2a gene of other field strains. the p1-2a of type 'o' vaccine strain was amplified directly from the cattle tongue material, cloned in plasmid vector and studied the specificity by sequence analysis and gene expression. we have introduced 'o' p1-2a gene into the full length construct devoid of asia 1 structural protein gene, p1-2a. the in vitro transcribed rna in case of t7 promotered construct and plasmid dna in case of cmv promotered construct were transfected into the bhk21 cells. after the passaging the virus obtained was studied for the speciality. this approach may be used not only for rapid selection of vaccine strain and also as a repository of the cdna copy of the virus. the p1 is composed of 1a, 1b, 1c and 1d (vp4, vp2, vp3, and vp1) respectively of which the vp1 is the most immunogenic and subunit vaccine produced with vp1 alone was able to induce high level of neutralising antibodies. thus to control the disease in india polyvalent vaccine consisting of the inactivated virus of all the three serotypes are in use. however the conventional vaccines have several drawbacks which include safety and temperature sensitivity. hence alternatively sub-unit vaccines consisting of vp1 protein has been tried. however this showed limited success due to the antigenic variations occurring in the field viruses thus escaping the neutralization from the antibodies generated from single cloned protein. hence the present study was undertaken with an objective to include all the neutralizing epitopes present in the three serotypes by linking vp1 (1d) genes and produce a poly valent protein for using as poly subunit vaccine. in this study we have constructed a cassette by linking the genes of three serotypes 'o' (622 bp), 'a' (640 bp) and 'asia 1' (622 bp). these genes were cloned individually in commercially pbsk vector and confirmed by sequence analysis before linking in pc dna vector. the linked gene construct was sub-cloned in pet32 expression vector. the expression of the protein gene from the pet vector was induced with iptg and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page). a fusion protein of size 72 kda was observed in page gels. since the protein contains 6 his residues from the vector at the n-terminal end, affinity purification was carried out using nickel nitrilo-tri-acetic-acid (ni-nta) agarose matrix. the immunoreactivity of the purified protein was assayed by western blot with the anti fmdv type 'o' and 'asia 1' specific sera. the may be used as a subunit vaccine. silkworm diseases caused by viruses, bacteria, fungi and protozoans form major constraints for the silk cocoon production in all the sericultural countries and among these silkworm viral diseases viz., nuclear polyhedrosis and infectious flacherie caused by bmnpv and bmifv cause severe crop loss. the traditional disease management strategies include prophylactic measures and use of disease free silkworm eggs. the prophylactic measures such as disinfection of silkworm rearing house and appliances, egg surface, silkworm bed disinfection and rearing surroundings. the disinfectants used presently in sericulture are either formaldehyde or chlorine based products, but these chemicals are neither eco-nor user-friendly. the awareness about health hazards caused by formaldehyde and environmental pollution caused by cl 2 necessitated the development of eco-and user-friendly disinfectant products for use in sericulture. these include alternative disinfectant products developed using biodegradable chemicals and plant based ingredients by apssrdi, hindupur and central silk board for the management of silkworm diseases in india. the ideal disinfectant for sericulture would be the one which can inactivate silkworm pathogens of diverse origin and economical for sericulture. the paper discusses on the disadvantages of hcho and cl 2 based disinfectants and advantages of eco-and user-friendly disinfectant for the management of silkworm diseases especially the ones caused by viruses. the baculovirus expression vector system (bevs) is widely used for the production of high levels of properly post-translationally modified, biologically active and functional recombinant proteins and has facilitated basic biomedical research on protein structure, function, drug discovery and the roles of various proteins in disease. bevs is based on the introduction of a foreign gene into nonessential for viral replication genome region via of homologous recombination with a transfer vector containing target gene. the resulting recombinant baculovirus lacks one of nonessential gene (polh, v-cath, chia etc.) replaced with foreign gene encoding heterologous protein which can be expressed in cultured insect cells and insect larvae. insect cell-bev system is widely used to produce recombinant proteins. bevs also eliminates concerns regarding pathogens that could potentially be transmitted to humans as it is non-infectious to vertebral animals. these features make silkworm system an ideal expression and delivery package for producing proteins of medicinal importance. the efficiency, low cost and large-scale production of proteins using bevs represents breakthrough technology that is facilitating highthroughput proteomic studies. the bevs has become a core technology for cloning and expression of genes for study of protein structure, processing and function; production of biochemical reagents; study of regulation of gene expression; commercial exploration, development and production of vaccines, therapeutics and diagnostics; drug discovery research; exploration and development of safer, more selective and environmentally compatible biopesticides. utilization of silkworm larvae and pupae as bioreactor with recombinant bmnpv producing foreign proteins extends the usages of silkworms. due to its large-size and high protein synthesis ability as well as the expediency in mass culture, silkworm is considered as good candidate for producing recombinant proteins. wssbv is the causative agent of a disease, which has recently caused high shrimp mortalities and severe damage to shrimp culture. wssbv has been found across different penaeid shrimp species. in order to develop a effective diagnostic tool, a wssbv genomic library was constructed by cloning wssbv genomic dna extracted from purified virions. in the present study wssv disease free (confirmed by pcr analysis) were collected from hatcheries from different areas of guntur and prakasam districts and analysed to study the effect of various physical parameters like temperature, p h , salinity and turbidity on the prevalence of above disease. the studies on the surface water temperature revealed fluctuations in the ponds ranging between 19 to 30.2°c in diseased ponds and 25.2 to 34.5°c in healthy ponds. these results show definite influence of temperature on the prevalence of wssv. present day strategy in vaccine development is to include marker facility that helps in distinguishing antibody response due to vaccination vis-à-vis infection in vaccinated animals. such information becomes relevant for effective disease control programmes especially when using inactivated virus vaccines like foot and mouth disease (fmd). the antibodies generated in the animals, only through vaccination, is the measure of vaccine efficacy and safety. presently inactivated fmd virus (fmdv) vaccines are used to control the disease in the endemic countries like india. the quality assurance of the vaccine depends on the efficacy of the vaccine in generating protective antibody without causing subclinical disease due to improper inactivation. since protective antibody response in vaccinated animals can not be distinguished from that of infected animals one needs to assay the antibody response against non structural proteins (nsps) and the vaccine must be free of contaminated nsps. production of vaccine free of nsps requires the cumbersome method of virus purification which adds to the cost of the vaccine. alternatively one may develop a positive marker vaccine by including a foreign protein or epitope which is not expected to be present in the vaccine and the antibodies generated against which helps in detecting the vaccine related response. here we report a molecular approach by which we introduced a immuno-dominant epitope of green fluorescent protein (gfp) into the structural protein gene of foot and mouth disease virus vaccine strain asia 1 (63/72). our laboratory has produced a mini-genome of fmdv asia 1 that lacks structural protein gene (p1-2a) coding for all the structural proteins (vp1-4) of fmdv asia 1 as a vector (pcfl dasia 1). the p1-2a of the asia 1 vaccine strain was cloned separately into a plasmid vector and by successive pcr mutagenesis and cloning we have introduced nucleotide sequence corresponding to 9 amino acid epitope of gfp into p1-2a gene. gfp epitope was inserted by replacement at n-terminal region of vp-2 which is not immunogenic. the modified p1-2a was expressed in e. coli and studied. the modified p1-2a gene with gfp epitope was inserted into the pcfl dasia 1 to get full length replication competent cdna cloned under cmv promoter in pcdna (pcflasiagfp). this can be used to produce synthetic virus with gfp epitope that can generate antibodies not only against neutralizing epitopes but also against gfp epitope. presence of antibody against gfp epitope in the vaccinated animal will reveal vaccine efficacy. elisa against gfp can be used as a companion test not only for safety evaluation but also for quick evaluation of efficacy. further absence of nsp antibodies in the serum may reveal the quality of the vaccine in respect of safety. self replicating dna vaccines are developed to achieve robust immune response through enhanced antigen production and gamma interferon expression in vaccinated animals. since self replicating dna vaccines induce gamma interferon expression which helps in viral clearance such vaccines are expected to be useful to cure even the carrier and persistently infected animals. understanding the events that help in the elicitation of both the arms of immune response in vaccinated animals is necessary to understand the effectiveness of the vaccine. the work presented here deals with the immunological evaluation of a sindbis virus replicase based dna vaccine carrying linked fmdv vp1 genes in vaccinated guinea pigs. we have constructed self replicating dna vaccine vector and to the down stream of a sub genomic promoter we have inserted secretory signal followed by linked-vp1 genes of 3fmdv serotypes (o-a-asia 1) with glycine and proline bridge in between. guinea pigs were vaccinated with the construct and the sera at 28 days post vaccination were evaluated both for cellular response by studying the cd8 levels and by mtt and cytokine profiles by real time assays. the humoral response was evaluated by studying cd4 levels in the whole blood by facs analysis and serum antibody levels by snt and elisa. the animals were challenged with 100 gp infective dose of fmdv type 'o' virus lesions were scored. further the replicative efficiency of the challenge virus was studied by 3ab elisa. the results showed that all the assays except antibodies against 3ab protein have positive correlation with the protection. as expected the titre of the antibodies against 3ab protein was lower indicating that the challenge virus replication was inhibited in the vaccinated animals. the limited studies conducted by us showed that self replicating vaccine has a potentiality to emerge as potent vaccine for fmd. ganjam virus (ganjv) belongs to the genus nairoviruses (family bunyaviridae). these viruses cause diseases in livestock. it has been isolated from different animal hosts and tick vectors from india. genus nairoviruses includes a total of 34 tick-borne viruses, classified into 7 serogroups. the important serogroups are crimean congo hemorrhagic fever (cchf) and the nairobi sheep disease (nsd). the main members of the nsd group are nsd and dubge viruses. their genome consists of three segments of single stranded rna, viz. s, m and l that encodes viral nucleocapsid protein, viral glycoprotein g1 and g2 and the viral polymerase respectively. ganjv is very closely related to (nsdv). nsdv is found in east and central africa, causes very high morbidity and mortality in livestock. the present study involves phylogenetic comparison of ganjv isolates from india with other nairoviruses based on complete n gene. it will help to understand the kind of nucleotide (nt) and amino acid (aa) changes that have occurred in ganjv strains from different geographical areas. eight strains of ganjv isolated at niv during 1954-2002 from different parts of india were used in this study. virus stocks were prepared in vero e6 cell line these were used as the source of viral rna. the n gene was amplified either as a complete gene in one reaction or in fragments whenever necessary. thus obtained sequences were analyzed; annotated to get a consensus sequence, aligned against the sequence of prototype strain of ganjv and other representative nairoviruses. the nt sequences were converted to aa sequences and analysis was done at both nucleotide and amino acid levels. based on what nt or aa phylogenetic tree was constructed and compared with other nairoviruses (cchf, dugv, hazv, kupv and nsdv) where complete s segment sequences were available gen-bank database (ncbi). the phylogenetic data at both the nt and aa levels showed that all the strains of ganjv form monophyletic lineage with the nsdv. cchfv and hazv together formed another clade, whereas dugv and kupv made a separate branch in the tree. the different ganjv strains showed 9-10% difference with nsdv at the nucleotide level and 3-4% difference at the amino acid level. hazv showed 37-38% difference at the nt level and 37% difference at the aa level with ganjv as well as nsdv. the present data obtained suggests that ganjv and nsdv are minor variants of the same virus. diarrhoeal syndrome is one of the major concerns of the livestock industry. most of the diarrheic cases in animals go unnoticed and limited attention is paid on viral etiology. presence of large amount of fecal matter in animal shed acts as a source of infection for calves via drinking water, feed, or contaminated soil. keeping this in view, investigation was planned to detect the association of rotaviruses with diarrhea in dairy calves and to observe the genomic diversity among the circulating viruses in tarai area of uttarakhand. a total of 63 diarrheic fecal samples collected from instructional dairy farm, nagla, pantnagar, uttarakhand were screened during the study. samples were collected from both cow calves and buffalo calves in 0-3 months of age. for the diagnosis of rotavirus, all the fecal samples were subjected to rna-electrophoresis after nucleic acid extraction. viral genome segments were visualized by silver staining. out of the total 63 samples tested, seven were found positive in rna-page showing typical 11 genome segments migration pattern of bovine rotavirus. in the given samples prevalence of bovine rotavirus was 11.32% and 10% in cow and buffalo calves, respectively. on the basis of migration patterns of rotavirus in rna-page, group a were identified with typical 4:2:3:2 pattern. variation within movement of various genome segments among isolates of bovine rotaviruses was observed during the study that may be indicative of emergence of mutants in the circulating isolates. the vp6 gene based group a specific rt-pcr was standardized and all the isolates in this area were confirmed to be of group a type. work is in progress to genotype the bovine rotaviruses of this region based on vp7 and vp4 genes. this study emphasizes the need to explore the prevalence of bovine group a rotaviruses in different places of uttarakhand and their genetic characterization which could help in selection of control strategies for rotavirus infections. foot-and-mouth disease (fmd) is endemic in india causing enormous economic loss to the animal keepers and trade embargo with fmd free countries in livestock and animal products. rapid diagnosis of fmd is of immense importance in prevention and control of the disease. fmd is initially diagnosed clinically and confirmed by laboratory tests. virus isolation in cell culture and sandwich elisa for antigen detection are commonly practiced in laboratories. the virus isolation though is very sensitive but it can be slow and analytical sensitivity of the elisa is lower and can not be used with certain sample types. the use of molecular techniques in the diagnostic laboratory has greatly increased the speed, specificity and sensitivity of fmd diagnostic tests. molecular techniques like rt-pcr, pcr-elsa and dot hybridization can be used with more success for detecting carrier animals and animals harboring sub-clinical infection and can be applied in a wide range of clinical sample types. these techniques can be used as genus and serotype specific test including detection of particular lineage/genotypes with in the serotype. multiplex pcr has been used to differentiate serotypes of fmdv and the technique is sensitive, experimentally simpler, cost effective and less time consuming. the assay can be used for serotyping on elisa negative samples. the molecular techniques not only help in diagnosis but also useful for epidemiological studies. lineage differentiating rt-pcr has been useful in identifying different lineages of serotype asia 1 (lineage b, c and d) before proceeding with sequencing of 1d region. similarly genotype differentiating rt-pcr has been developed and used in differentiating two different genotypes of serotype a (genotype vi and vii). these assays have the potential to be applied on clinical samples directly, thereby saving much time needed for sample processing and nucleotide sequencing. recent development of real time rt-pcr methodology has allowed the diagnostic potential of molecular assays to be realised. advancement in real time pcr technology made it possible to combine several assays within a single tube which is in the progress in our laboratory. integration of these assays onto automated high throughput platforms provides diagnostic laboratories with the capability to test large numbers of samples. microarray technology was provided greater screening capabilities for pathogen detection. the microarray allows the addition of large number of oligonucleotide probes for identification of mutant pathogen and also for subtype determination. the combined properties of high sensitivity and specificity, low contamination risk, and speed has made realtime pcr and microarray technology a highly attractive alternative to conventional methods in increasing percentage of outbreaks confirmed and analyzed and for tracing the origin of fmd virus responsible for outbreaks. dna vaccines are expected to elicit both humoral and cellular responses, cellular response being long lasting. however the approach has several limitations like poor stability of dna, poor expression and risk of integration. poor expression becomes the major limitation in the case of fmd as fmdv proteins are poor immunogens. also dna vaccine vectors carrying only eukaryotic promoters elicit strong cmi response and weak humoral response. the methodology to achieve humoral response involves the expression and secretion of the expressed protein so that the antigen presenting cells will be able to process the antigen and produce humoral response. in case of fmd humoral response is as important as cellular response. the present project aims at addressing these issues; achieving higher expression and getting the protein secreted out by constructing self replicating gene vaccines for fmd and studying their efficacy. the vector for humoral immune response contains eef1 promoter, sindbis virus polymerase gene and secretory and anchoring signals. the integrity of the vectors was confirmed by sequence analysis. the linked polyvalent protein genes of fmdv serotype a, o and asia 1 were cloned into the vectors and the presence of the insert was confirmed by restriction enzyme digestion. the functionality of the constructed dna vaccine vector (pvac self rep 990) was assayed by transfecting the dna into bhk 21 cell monolayer and studying the 35 s labeled proteins in immuno-precipitation assays. the studies showed high level of expression in case of constructed vector as compared to infected virus for the specific protein. the secretion of the expressed protein was assayed by immuno-fluorescence assay and found to be positive. encouraged with these studies the preliminary studies were conducted on vaccine efficacy studies in guinea pig model. the immunized guinea pigs showed high antibody titres by snt and elisa, as compared to conventional dna vaccines (pup3cd) even at 1/10th of the dose. this approach of constructing self replicating dna vaccine for humoral response is the first report. genetically engineered microorganisms are important sources of industrial and medicinal proteins. over the past decade, plant host system has been investigated as potential host system for expressing proteins of therapeutic and diagnostic use. however concerns regarding the stability and environmental safety need to be addressed. chloroplast engineering is expected to resolve some of these issues since, plastids/chloroplasts are inherited maternally and are not disseminated through pollen. this makes plastid transformation a valuable tool for transgenic creation besides offering biological containment. since foot and mouth disease (fmd) of cloven footed animals is a major concern in the world over. foot and mouth disease (fmd) is the most feared, viral disease of the cloven footed animals causing heavy losses to the live stock industry. the disease is enzootic in many parts of the world including asia. the conventional vaccines for fmd have several limitations which include safety, temperature sensitivity and duration of immunity. attempts have been made to overcome these limitations using recombinant dna technology. amongst the newer vaccines, edible vaccines are cost effective and easy to administer. since the stability of the gene of interest is the major concern in the case of plant transgenics, marker genes are used for regular selection. the detection methods based on the available marker proteins like b glucoronidase (gus) protein/antibiotic selection are cumbersome and cost intensive. however selection based on herbicide resistance is much simpler and easy. hence in the present study, the 5-enolpyruvylshikimate-3-phosphate synthase (epsp) gene was used as a marker along with the immunogen gene of fmdv. epsp is the key enzyme in the shikimate biosynthesis pathway necessary for the aromatic amino acids production. in order to investigate the mechanism of long term immunity and the effect of protective immunity induced by cationic plg micro particle coated dna vaccination. we constructed the expression plasmid containing a foot-and-mouth disease virus (fmdv) id gene sero type a. intramuscular vaccination of guinea pigs with the micro particles coated plasmid dna induced a strong antibody response and neutralization antibodies, cellular mediated immune response which lasted 1 year. we further analyzed the persistence and expression of id gene by polymerase chain reaction and reverse transcriptase polymerase chain reaction and quantitative pcr. the results showed that id gene was present and expressed in the muscle cells up to 1 year after days post vaccination. furthermore, guinea pigs vaccinated with micro particles coated plasmid dna were protected against a challenge with fmdv virus. therefore the micro particles coated plasmid dna vaccination dose induce a protective immunity and long term humoral, cellular immuno responses against fmdv, which could be maintained by persistent expression of id gene in muscle cells. foot and mouth disease virus (fmdv) causes a highly contagious viral disease of cloven hoofed animals, which has a considerable socioeconomic impact on the countries affected. interleukin-18 (il-18) enhances the il-12 driven th1 immune response that is important in immunity against intracellular pathogens. the multiple roles of il-18 in many physiological and pathological processes have generated a great deal of interest in recent years. antiviral effects of il-18 have been reported. we evaluated the effects interleukin-18 (il-18) on the replication of fmdv in vitro in bhk-21 cells. bovine il-18 mature protein coding sequence was amplified from the bovine pbmc cells and cloned into prokaryotic expression vector pet32a. protein expressed was purified and specificity was confirmed by immunoblotting. bhk-21 cells were treated with purified expressed il-18 protein with (2 lg/ml) 4 h prior to fmd infection. cell culture supernatants were collected at 24 h post infection were subjected for elisa and virus titration assay. rna extracted from the cells was subjected to real time pcr for viral rna quantification. 2 log titer reduction was observed in the fmd virus titer in il-18 treated cells compared to the untreated cells where as virus antigen quantified by elisa has shown a reduction of 60-folds. 69-fold reduction in the fmd viral rna copy number was observed in the il-18 treated cell compared to the untreated measured by qpcr. current study demonstrated the potent anti viral activity of il-18 on fmdv by inhibiting the viral replication. these results further suggests that il-18 has the potential role of il-18 as molecular adjuvant in fmd vaccine development and development of therapeutic for fmd. foot and mouth disease is the most contagious viral disease of farm animals. control of the disease in animals is by vaccination and slaughtering of infected animals. conventional oil adjuvant vaccine has its own limitation. alternate to this genetic vaccines where the dna encoding viral antigen may be a promising approach. naked dna vaccine has limitations like poor uptake of dna by cells and more importantly by nucleus. as a result delivery of naked dna through calcium phosphate nanoparticle was attempted. calcium phosphate nanoparticle is a potential delivery agent which proved to enhance the immune response. fmdv p1-3cd ''o'' vaccine gene constructs in pcdna3.1? entrapped by the nanoparticles was prepared by using different molarity of calcium chloride and disodium hydrogen orthophosphate. the nanoparticles entrapping fmdv p1-3cd ''o'' and naked dna were presented to the guinea pigs through intramuscular injection to study the mrna expression of antigen by rt-pcr. animals were sacrificed at defined time to collect different organs and total rna was extracted. each time blood was collected to analyse the fmdv specific serum antibodies. dna vaccines presented through calcium phosphate produced transcripts in the injected muscle up to 240 days whereas naked dna up to 120 days. serum antibody levels of naked dna vaccine showed antibody titre till 60 days. whereas nanoparticle injected animals showed serum antibody till 120 days. serum neutralization titres of 1.5 were observed in calcium phosphate dna vaccines at about 28-150 days, where as naked dna sn titers were observed for short period of 30-90 days. the study clearly showed calcium phosphate nanoparticle entrapping fmdv vaccine dna may be a better delivery system for dna vaccines as it confirms availability of the antigen and persistence of antibody for longer duration than naked dna. capripox is highly infectious, contagious, and oie notifiable disease of small ruminants, caused by sheeppox and goatpox viruses which are members of capripoxvirus genus of the family poxviridae. in the present study, we analyzed the partial gene sequences of p32 protein, an immunogenic envelope protein of capripox viruses (capv) to assess the genetic relationship among different sheep pox and goat pox virus isolates from different geographical areas of the country. product of this gene has been shown to be important in attachment of capv to host cell surface receptors during viral entry and host immune response. the following virus isolates have been used in the analysis: gtpv-uttarkashi, p60, vaccine virus; gtpv mukteswar, p10, challenge virus; gtpv (akola), gtpv bareilly/00, gtpv ladakh/01 and gtpv sambalpur/82, field isolates and sppv srinagar, p40; sppv ranipet, p50; sppv-rf, p50, vaccine viruses and sppv makdhoom/07, sppv cirg/08, sppv pune/08, sppv bareilly, sppv 183/03 and sppv 125/02, field isolates. in this study, all virus isolates were confirmed by pcr amplification and analysed in pcr-restriction fragment length polymorphism (pcr-rflp) using ecori enzyme to confirm their specificity. further, the amplicons were cloned and sequenced commercially. nucleotide and the deduced amino acid (aa) sequences were compared with published sequences of the members of the genus capripox virus. sequence analysis of partial 172 bp sequence has shown high sequence identity among all indian sppv and gtpv isolates at both nt and aa levels. it revealed a 99.4-100% and 98.2 for gtpv field isolates where as, 100% for sppv field isolates at both the nt and aa levels. in general, capv isolates in this study shown 98.3-98.8 and 96.5% homology between gtpv and sppv at nt and aa levels as reported earlier. further, it revealed a unique change of g120a in all gtpv isolates resulting in formation of drai site in place of ecori and possible development of restriction enzyme specific pcr-rflp for differentiation of sppv and gtpv from field isolates. orf or contagious ecthyma is considered as non-contagious, proliferative disease and is caused by orf virus of the genus parapox virus of the family poxviridae. it is reported most commonly in sheep and goats and also a zoonotic agent. camels are also infected by orf virus and reported in camel rearing countries as a mixed infection with camel pox, the later is caused by an orthopox virus. in india, there are few reports of the orf virus infection in camels and identified by clinical signs and pcr. in this study, we identified the presence of orf virus from clinical samples of suspected case of sporadic infection in camels by serological and molecular techniques. viral dna isolated from processed scabs used initially in nested polymerase chain reaction as diagnostic pcr which successfully amplified 235 bp fragments and also sequenced to check the fidelity of the product. after confirming the infection by pcr, some of the structural and non-structural genes were amplified for sequence analysis. out of the five genes characterized, the major important one selected for sequence and phylogenetic analysis is b2l gene which is homologous to a major envelope protein p37k of vaccinia virus. full open reading frame of 1137 bp from orf b2l was amplified by pcr, cloned and sequenced commercially. nucleotide and deduced amino acid sequences of b2l were compared with other published sequences of the members of the genus papapox virus. sequence analysis shows a maximum percent identity of 94.8 and 95 (indian orf virus isolates); 94.7 and 94.5 (other orf isolates); 98.8 and 98.7 (orf-camel/jodhpur/08); 85 and 82.8 (bovine popular stomatitis virus) and finally 97.4 and 97.6 (pseudo cowpox virus) respectively at nt and aa levels. phylogenetic analysis of the isolate was also performed using the neighbour joining method in mega 4 program to know the phylogeny relatedness of the virus, which revealed that the isolate is well grouped with the jodhpur isolate and closely related to pseudo cowpox virus. it warrants further analysis of other potential genes to confirm the causative agent of the contagious ecthyma in camels as pseudo cowpox virus. chikungunya an arboviral disease is transmitted through the bite of an infected aedes mosquito. it causes a self limited febrile illness along with arthralgia and myalgia. in some cases neurological and severe hemorrhagic manifestations has been observed. chikv epidemic has been reported in africa, india, south east asian countries and during the current out break imported cases of chikv has been encountered in most of the european countries. the causative agent belongs to the genus alphavirus family togaviridae. human beings serve as the chikungunya virus reservoir host during epidemic periods. outside these periods the main reservoirs are monkeys, rodents, birds, and other unidentified vertebrates. antibodies to chikv have been detected in domestic animals. in the present study we surveyed madanapalli, palamaner, b. kotta kota and tirupati and collected a total of 67 rodent samples, 75 bovine samples; 20 sheep samples and 15 canine samples. total rna was isolated from all these samples and subjected to rt-pcr using a primer pair dvrchk-f/dvrchk-r which could amplify a 330 bp e1 gene product specific to chikungunya virus (naresh kumar et al. 2007 ). all the serum samples were further screened for chikv specific igm antibodies using commercially available ctk biotech strips. none of the samples were found positive either for chikv specific rna or chikv specific igm antibodies. more number of samples from domestic animals as well as rodents are being screened to study their possible role if any in the maintenance of chikv in nature and during the inter epidemic periods. the present study discusses these aspects in detail. petunia hybrida is widely used as experimental host plant for begomovirus identification and its characterization. hitherto, natural infection of begomovirus on petunia has not been reported in india. recently, petunia hybrida grown in and around ludhiana were found to be depicting typical symptoms caused by begomovirus. the symptoms include severe reduction in leaf size, downward curling and distorted leaves. severely infected plant became bushy, stunted and produces no flower. total genomic dna was extracted from the plants showing symptoms of begomovirus, by ctab method. the presence of virus was confirmed by using degenerated primers, designed to identify all the begomovirus prevailing in the world. to identify the strain associated with the disease, the positive samples along with healthy control were tested against different strain specific primers of tomato leaf curl virus, so far reported in india i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus. among these, only tomato leaf curl new delhi virus specific primer was able to give the desired amplicon of *1180 bp. hence, it is confirmed that the leaf curl disease of petunia hybrida is associated with tomato leaf curl new delhi virus. this disease of petunia can become a sever production constraint in coming years. from last 2 years (2008 and 2009) it was observed that some varieties of brinjal grown in rainy season, showed typical leaf curl type of symptoms. the symptoms include upward curling of the leaves, cupping, vein thickening, reduction in leaf size and distortion of leaves. the severely infected plant remains stunted and bushy, became unproductive or produces only few fruits. the disease was experimentally transmitted from naturally infected brinjal to healthy seedlings by whiteflies (bemisia tabaci) and grafting, but not by mechanical or aphid transmission. to detect the begomovirus associated, total genomic dna was extracted from the plants showing disease symptoms. the presence of virus was confirmed by using pcr based begomovirus geneus-specific primers designed by deng et al., wyatt and brown and rojas et al. these degenerated primers give the expected product size of *530, *575 and *1280 bp, respectively. core coat protein (cp) gene and dna-b was also amplified in the samples using specific primers. to identify the strain associated with leaf curl virus, dna was subjected against primers of different indian tomato leaf curl virus strain i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus, using pcr. among these, only tomato leaf curl new delhi virus primer was able to show the desired product size of *1180 bp. therefore, it was confirmed that leaf curl disease of brinjal is caused by tomato leaf curl new delhi virus in association with satellite b-dna. to identify the strain associated with the disease, all samples were further subjected to the specific primers, designed to amplify all the tomato leaf curl virus strains, so far reported from india i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus, using pcr. among these, only tomato leaf curl palampur virus specific primer was able to give the expected product size of *900 bp. this shows the association of tomato leaf curl palampur virus with leaf curl disease of calendula and marigold. thus, calendula and marigold can act as a reservoir for the tomato leaf curl palampur virus and may cause severe constrain in the production of these important ornamental plants. groundnut bud necrosis virus (gbnv) belongs to serogroup iv of the genus tospovirus in bynayaviridae family and infects several economically important crops all over india. the nucleocapsid protein (np) encoded by the small rna of gbnv encapsidates the viral rnas. apart from this structural role, the np has also been implicated in the replication, transcription, maturation and cell to cell movement. with a view to study the structure and function, the np of gbnvtomato isolate from karnataka was over expressed in e. coli and purified by ni-nta chromatography. the purified np was present as ribonucleoprotein complex and as heterogeneous mixture containing monomers, tetramers and higher order multimers. in order to determine the regions involved in oligomerization and nucleic acid binding, mutational approach was taken. n-and c-terminal deletion clones were generated (n20np, n40np, c15np and c37np), over expressed in e. coli, and were purified by a procedure identical to that used for the wild type protein. initial studies on oligomeric status suggested that in addition to n-and c-terminal regions there may be additional regions or residues which contribute to multimerization of np. the amount of rna bound to the truncated proteins was reduced in case of n20np, n40np and c15np. interestingly removal of 37 amino acid residues (natively unfolded region) from the c terminus resulted in complete loss of nucleic acid binding suggesting that the rna binding domain was located in c-terminal region of np. further np was observed to get phosphorylated in in vitro kinase assays by a kinase present in the soluble fraction of tobacco plant sap. both atp and gtp were utilized as phosporyl donors and mn 2? was the preferred metal ion which suggests that np might be phosphorylated by a ck2-like protein kinase. phosphorylation studies with n-and c-terminal truncated proteins revealed that the site of phosphorylation lies within the amino acid residues 40-239. by mass spectrometric analysis of the protein threonine-84 and serine-202 were identified as possible phosphorylation sites. a naturally occurring isolate of virus infecting gherkin (cucumis anguira l.) showing mosaic symptoms of mosaic, leaf distortion and dark green islands in the lamina was identified in the export cultivars of gherkin grown in commercial fields of kuppam rural, chittoor district, andhra pradesh. the virus infection was deadly prevalent among the field that caused a lot of economic damage to the crop that resulted in yield losses and reduced quality of fruits meant for export. symptoms of the infected fruit included blistering and malformation of the fruit. the virus infected leaf samples were collected and initial host range tests were conducted with different cucurbit species showed that the host range include propagation hosts like cucumis anguira (gherkin), cucumis sativus, cucurbita pepo, cucumis melo, langeneria vulgaris, momordica charantia and local assay host like chenopodium amaranticolor. the virus host range was only restricted to cucurbit species and chenopodium. the virus was maintained for further studies on cucurbita pepo by sap or mechanical inoculation. the virus induced mosaic, vein clearing symptoms on pumpkin. electron microscopy of the leaf dip preparations stained with 2% uranyl acetate from the pumpkin leaves showing symptoms revealed the presence of a long flexuous filamentous particle measuring 750 9 12 nm. the virus positively reacted to the polyclonal antisera of papaya ringspot virus-w, potato virus y, tobacco etch virus and also strongly reacted with the polyclonal antiserum of zucchini yellow mosaic virus in direct antigen coated-enzyme linked immunosorbent assay (dac-elisa). because of very strong reaction to polyclonal antisera of zucchini yellow mosaic virus, we tried to amplify the partial nib and cp genes of the virus along with the 3 0 utr by using two primers zy2 5 0 gctccatacatagctgag acagc3 0 and zy3 5 0 taggctttttgcaaacggagtcta at c3 0 . total rna from gherkin infected leaves was isolated using trizol ls reagent (sigma). rt-pcr was performed to obtain an amplicon of *1.2kbp, cloned into fermentas ptz57r/t vector and sequenced at mwg biotech, bangalore. sequence analysis revealed that the virus was isolate of zucchini yellow mosaic virus and was showing 98% of homology to that of the zucchini yellow mosaic virus strain b genome ay188994 and zucchini yellow mosaic virus nat genome ef062582 which were strains reported from israel. the sequence of the present study was submitted to the genbank gq482976. the results state a suspicion that the virus could have been mobilized by some infected source brought by the commercial israeli based companies into india due to poor quarantine regulations as the gherkin cultivation in these regions is chiefly supported, purchased, exported and marketed by these private companies that are based from israel. this is the first report on molecular characterisation of zucchini yellow mosaic virus infecting cucumis anguira (gherkin) from india. they also exhibited synergism with other virus which was region specific. fifty percent of the total symptomatic plant population was found be positive only for carla while remaining showed mixed infection of carla with tospo in some regions while in others carla virus was found to be associated with cmv. presence of only carlavirus was up to 10-20% incidence, without association of tospo, cmv, poty or tobamo viruses was also observed in some fields. avijit tarafdar, raju ghosh, k. k. biswas plant virology unit, division of plant pathology, indian agricultural research institute, new delhi 110012 citrus tristeza virus (ctv), a brown citrus aphid (toxoptera citricidus) transmitted closterovirus under family closteroviridae, is one of the major limiting factors in cultivation of citrus worldwide. ctv is a longest known plant virus having flexuous particle of 2000 9 11 nm in size. ctv genome is a positive sense ssrna of about 20 kb nucleotide containing 13 open reading frames (orfs) encoding 17 proteins. several biological as well as genetic variants of ctv are reported in all the citrus growing countries in the world. ctv causes decline and death of millions of citrus trees in the world. in india, ctv is a century old problem, and has killed an estimated one million citrus trees till today. in molecular and genetic level, ctv isolates from india were not fully characterized. genetic diversity and sequence divergence in ctv isolates of india are not fully established. further, evidence of recombination and causes of evolution of ctv variants in india have not been studied till date. therefore, in the present study, effort has been made to characterize several indian ctv isolates in genetic level, examine their genetic diversity, identify recombination events and analyze evolution of divergent ctv. a total number of 73 ctv isolates from different regions of india (35 from darjeeling hills, five from bangalore, 15 from delhi and 18 from vidarbha) were under taken for genetic study. two genomic regions of ctv, i.e., entire cp gene (cpg) (672 nt) and a gene fragment of 5 0 orf1a (orf1a) (404 nt) were amplified, cloned, sequenced and nucleotides were analyzed. based on cpg, indian isolates shared 88-99% nucleotide identity, and based on orf1a they shared 82-99% identity, among them. incongruence of phylogenetic relationship was observed as on sequence analysis five phylogenetic clades based on cpg, and eight clades based on orf1a, were generated suggesting the recombination events have been occurred between the sequences of indian ctv isolates. thus, to identify the potential recombination events, and determine the parental sequences in ctv isolates, six recombination detecting algorithms, namely, rdp, genconv, bootscan, maxchi, chimera and siscan were used. out of 73 indian ctv, cpg of 18 and orf1a of 47 isolates of ctv showed recombination events suggesting orf1a was more prone and fragile to rna recombination as compared to cpg. this findings indicated that high degrees of genetic diversity and incongruent relationships of indian ctv isolates are due to genetic recombination occurred, which may be the important factors in driving evolution ctv variants in india, that was also supported by a splittree decomposition analysis. b. v. bhaskara reddy, y. sivaprasad, k. rekha rani, k. raja reddy department of plant pathology, regional agricultural research station, acharya n.g. ranga agricultural university, tirupati, andhra pradesh sunflower (helianthus annus l.) is one of the most important oil seed crops in the world which ranks third in area after soyabean and groundnut. the sunflower necrosis disease (snd) is characterized by necrosis of leaves, necrosis streaks on petioles, stem, floral parts and stunted growth. the causal agent of the disease has been identified as tobacco streak virus (tsv) which belongs to genus ilarvirus of the family bromoviridae. the suspected tsv infected sunflower samples collected from chittoor district in andhra pradesh were found positive for tsv-dac elisa. total rna was extracted from sunflower using rnaeasy isolation kit (qiagen). the tsv coat protein (cp) gene, movement protein (mp) gene and replicase (rep) gene were amplified by rt-pcr with specific primers, cloned in ptz57r/t vector, sequenced and deposited in genbank (gu355899, gu355900 and gu371445). the size of cloned cp gene was 717 bp and codes for 239 amino acids. the cp gene sequence analysis revealed that the tsv-tpt infecting sunflower has 98-100% homology at nucleotide level with soybean, tagietus-tpt and okra-tn isolates and 93-99% homology at amino acid level. the movement protein gene was 615 bp and codes for 205 amino acids. the mp gene sequence analysis showed that it has 94-97% homology at nucleotide level and 92-95% at aminoacid level. chilli (capsicum annuum), the important commercial vegetable/spice of himachal pradesh, is affected by several viral diseases; of them cucumo, tospo, poty and gemini viruses are the most common genera. however, these viruses are not identified clearly and characterized fully, which are foremost needed to formulate the management strategy. therefore, in the present study, effort has been made to identify and characterize the important viruses causing diseases in chilli. in this study, several farms in major chilli growing areas of bilaspur and kangra districts in himachal pradesh were surveyed and infected plant samples were collected randomly. virus infection in these samples were detected by das-elisa using antisera to cucumber mosaic virus (cmv) and potyvirus (group specific) and through slot-blot hybridization (sbh) using cmv, iris severe mosaic poty virus (ismv), tomato spotted wilt tospo virus (tswv) and chilli leaf curl gemini virus (clcuv). based on das-elisa and sbh, the incidence of disease was estimated and ranged from 18.2 to 21.8% by cmv and 3.5 to 5.4% by potyvirus. to detect tospo and geminivirus in the infected chilli, sbh test was carried out. infected samples showed maximum virus titer in both das-elisa and sbh test were further confirmed by pcr using specific primers. desired sizes of amplicons; *540 bp, *800 bp, *570 bp and *460 bp of cmv, poty, gemini and tospo viruses, respectively, were obtained. as the present study clearly indicated that cmv appeared as a major one among the viruses infecting chilli in the hilly region of himachal pradesh, two isolates of cmv were characterized in genetic level. thus the amplified products (*540 bp) of cmv, palampur 1 and palampur 2 were cloned in pgemt cloning vector, sequenced and the sequences were submitted to ncbi database (palampur 1: acc-fm209497 and palampur 2: acc-fm209498). the sequences were then analyzed and compared with other sequences available in the data base. based on sequence analysis, it was found that present cmv isolates shared 99% nucleotide identity between them, are closely related with australian cmv isolate cmv-ly (acc-af198103) by 98% nucleotide identity. in phylogenetic tree analysis, it was observed that indian cmv isolates formed same cluster along with cmv-ly. as it is known that cmv subgroup ii comprises cmv-ly, it is concluded that the cmvs of this hilly region of himachal pradesh belong to subgroup ii. chilli is essentially a crop of the tropics and grows better in hotter regions. chlii (capsicum annuum), a member of family solanaceae is an important vegetable and spice crop of immense commercial importance. the pungency in pepper is due to an alkaloid known as capsaicine and peppers are characterized as sweet, hot or mild depending on capsaicine content. the present investigation were conducted to find out the highly resistant cultivars of capsicum annuum against cmv and tylcv among ten cultivars of chilli in agroclimatic condition of aligarh. the highest (70 and 80) percentage of infection was observed in hc-201 and kalyanpur type-1 by showing the positive reaction to cmv by elisa test. no symptoms was recorded in case of bc-16, lca-235 and jca-154 and showed negative reaction to cmv by elisa. bc-16 and lca-235 also showed negative reaction to tylcv by elisa and these were symptomless. maximum infection (70 and 80) was registered in hc-201 and c 8 , cultivar. so, the bc-16, lca-235 and jca-154 has proved highly resistant varieties against cmv and tylcv and these may be used in breeding programmes against viruses. cotton leaf curl virus belongs to the family geminiviridae, genus begomovirus. the members of this family contain circular single stranded dna molecules as their genomes. there are two kinds of begomoviruses-bipartite viruses with genomes consisting of two dna molecules designated dna-a and dna-b and the monopartite viruses which contain only dna-a but not dna-b. in monopartite viruses, the dna-a is accompanied by a small circular dna molecule called dna-b which is essential for the development of typical disease symptoms. cotton leaf curl virus is a monopartite virus and causes the cotton leaf curl disease which has emerged as a major disease of cotton in the indian subcontinent. the non-structural protein ac4 of cotton leaf curl kokhran virus-dabawali isolate (clcukv-dab) was cloned into pgex5x2 vector and overexpressed in bl21(de3)plyss e. coli cells. the overexpressed gst-ac4 protein was purified by glutathione sepharose chromatography. the purified gst-ac4 protein was found to possess atpase activity. the optimum temperature and ph for the activity were 37°c and 7.4 respectively. the atpase activity was inhibited in presence of edta, showing that it is dependent on divalent metal ions. the activity was supported by magnesium, manganese and zinc ions but inhibited in presence of calcium ions. it was also inhibited by the non-hydrolyzable atp analogue adenosine-b, c-imido triphosphate and in the presence of other nucleotides like ctp and gtp. the k m and the v max of the reaction for atp as the substrate are 1.54 mm and 95.2 nmol/min/ mg of the protein respectively. the enzyme could also utilize gtp as the substrate. the fact that ac4 is specifically an ntpase and not a general phosphatase is revealed by the finding that it does not hydrolyze p-nitrophenyl phosphate to yield yellow colour while a similar reaction carried out in parallel with alkaline phosphatase readily yields the colour. it has been suggested earlier that ac4 may be involved in cell to cell movement of the virus (rojas et al. 2001) . it is possible that by its ability to hydrolyze atp, ac4 serves to power viral movement in the plant. thirteen sugarcane yellow leaf virus isolates causing yellow midrib and irregular yellow spot pattern from six states of india were characterized by rt-pcr assays. scylv-615f and scylv-615r primers were used as forward and reverse primer pairs and the amplified products were cloned and sequenced. comparative coat protein sequence analysis confirmed that all the scylv-indian isolates were clustered into two major groups confirming the existence of two strains of scylv affecting sugarcane crops of india. in a separate experiment, the member of both of the phylogenetic groups were found to be transmitted by the sugarcane aphid, melanaphis sacchari from infected to healthy sugarcane suggesting its secondary spread in nature. the symptoms produced by the virus causing cotton mosaic disease were little bit different in both sap inoculation and under natural field condition. in natural field condition it has shown clear chlorosis type of symptoms on major leaves of plants but in sap inoculated plants veinal chlorosis and mosaic type of symptoms are found to be common. in field conditions infected plants grows erect and have less boll formation. there is no effect found on seed shape or seed size. the initial symptoms produced on cotton leaves after inoculation were wonderful. local lesions observed in second week from inoculation and then they changes to chlorotic type of symptoms and some are necrotic symptoms also. the plants at early stage are found to be affected, has less lateral branch development and hence reduction in yield production. the naturally field infected plants showing good symptoms are also difficult to identify in lateral stage of plant. because they disappear with time. the virus is very easily sap transmissible. the virus is found to be transmitted by thrips palmi and thrips tobacci in persistent manner. no seed transmission is observed. virus showed same physical properties as it shows in stem necrosis of peanut or sunflower necrosis disease. the physical properties are found to be thermal inactivation point (tip) 55-60°c, dilution end point (dep) 10 -2 to 10 -3 and longevity in vitro (liv) 5 h, virus infecting nineteen different host plants are identified belonging to five different types of families viz. malvaceae, chenopodiaceae, compositae, leguminaceae and solanaceae. however they found to produce same types of symptoms as in most of the host that have been tested before. in elisa test report it is found that the virus showing positive test only with anti serum of tsv of a cowpea and cotton but negative reaction with pbnv of cowpea and cotton which clearly denied possibility of presence of pbnv in cotton producing these kinds of symptoms elisa report clearly shows that tsv antiserum of cowpea is showing positive results with clear chlorotic types of symptoms. a powerful approach to functional genomics, and an alternative to the massive generation of transgenic plants, is the use of the recently described virus induced gene silencing (vigs) process, which allows viral vectors to knock out the function of a gene-of-interest. vigs is based on a silencing mechanism that regulates gene expression by the specific degradation of rna. as a tool for reverse genetics, vigs has many advantages over other common ways to study gene function because of the ability of viruses to replicate and move systemically within a plant. vigs can generate a phenocopy of a mutant without all the troubles of traditional methods of mutagenesis. geminiviruses with their small dna genomes and ease of inoculation through agrobacterium, are excellent candidates for vigs vector development. as a first step, the geminivirus bhendi yellow vein mosaic virus, characterized in our lab (jose and usha, virology 305: [310] [311] [312] [313] [314] [315] [316] [317] 2003) has been chosen. the satellite b dna associated with this virus has a single open reading frame (bc1). bc1 is essential for symptom development but not for replication. therefore, bc1 has been replaced by a multiple cloning site harbouring sali, xbai, bamhi, bsrgi and xhoi, initially in a cloning vector and then in the binary vector containing the partial tandem repeat of the b dna. in the place of the bc1 orf, the plant phytoene desaturase gene has been cloned and the resulting construct was used for agroinfiltration along with the partial tandem repeat clone of the begomovirus (dna a component chilli (capsicum annuum l.) plants exhibiting prominent symptoms of begomovirus like: leaf curl, vein swelling, shortening of petioles, crowding of leaves and stunting of plants were collected from rorkee, uttarakhand and dhaulpur, rajasthan, india. total genomic dna was isolated from naturally infected chilli samples and pcr was carried out with coat protein (located in dna-a) gene specific primers. as expected to the primers, *800 bp dna fragments were amplified from the infected chilli samples. to know the bipartite nature of the virus isolates, nuclear shuttle protein (located in dna-b) gene specific primers were employed which also resulted in positive amplification of *850 bp dna bands with all the coat protein tested positive samples. to ascertain the association of dnab component with the virus isolates, a set of dna-b specific primers were used which resulted in positive amplification of full length (*1.3 kb) dna bands in the chilli samples collected from rorkee, uttarakhand, however, multiple sizes bands were resulted with the samples collected from dhaulpur, rajasthan. these findings confirmed that both the virus isolates under study are bipartite begomovirus associated with dna-b satellite. the sequencing of the pcr products is under progress which analysis will be discussed. groundnut bud necrosis virus (gbnv) belonging to the genus tospovirus, which is a unique member of the family bunyaviridae, infects several economically important crops. the virus has three genomic ssrna segments namely s (ambisense), m (ambisense) and l (negative sense). the s rna codes for nucleoprotein (np) and non-structural protein (nss) from viral complimentary and viral strands respectively. many viral nonstructural proteins such as ns3 of hepatitis c virus, yellow fever virus, dengue virus, sv40 large t antigen and cytoplasmic inclusion protein of tamarillo mosaic potyvirus are known to exhibit rna/dna stimulated ntpase, dntpase and helicase activity. nss of gbnv does not have any sequence similarity with any of the above mentioned viral rna/dna helicases but has a ntp binding domain. however, it has been implicated as suppressor of gene silencing in vivo. with a view to elucidate the mechanism by which nss could act as a suppressor of gene silencing and examine the other potential roles of nss in the life cycle of the virus, the gbnv (to) nss was over-expressed in e. coli and purified by ni-nta chromatography. in vitro studies with the purified rnss suggest that it exhibits an rna stimulated ntpase activity. many of the proteins that possess the rna/ dna stimulated ntpase and datpase activity, are also shown to have atp dependent nucleic acid unwinding activity. it was therefore of interest to examine whether nss has the nucleic acid unwinding activity. the helicase assays revealed that nss has dna/rna helicase activity. helicase activity of nss was absolutely dependent on atp and mg 2? ion. nss could unwind dsdna substrate with 5 0 overhang, or 3 0 overhang. mutation of the crucial lysine in walker motif a (k189) severely affected the unwinding activity where as mutation of aspartate residue in walker motif b (d159) resulted in only 20% loss of activity. in this regard, rnss is a unique enzyme which does not have the canonical helicase motifs but can catalyze dsdna/dsrna unwinding in an atp and mg 2? dependent manner. the rnss might act as a suppressor of by unwinding the dsrna, the substrate for dicer. in addition to being a suppressor of ptgs, nss may also regulate the viral replication and transcription by modulating the secondary structure of the viral genome. this new research finding on nss might pave way for further studies on its role in viral replication and transcription. yellow vein mosaic disease of pumpkin (cucurbita moschata) poses a serious threat to the cultivation of this crop in india. the disease was found to be associated with whitefly-transmitted bipartite begomoviruses were detected in varanasi field using polymerase chain reaction (pcr) with primer design through coat protein conserved region of begomoviruses from ncbi database. all plant samples showing symptoms were infected with begomovirus. the virus species were provisionally identified by sequencing *750 bp of the viral coat protein gene (av1 ageratum conyzoides is commonly known as billygoat-weed, chick weed, goatweed and whiteweed. in india it is popularly known as bill goat weed. it is an annual herbaceous plant with a long history of traditional medicinal uses in several countries of the world and also reputed to possess varied medicinal properties including the treatment of wounds and burns. in cameroon and congo, it is used traditionally to treat fever, rheumatism, headache, and colic. during survey in and around gorakhpur in 2009, ageratum plants were found affected with the symptoms of leaf curling, mosaic mottling and leaf yellows. the infected leaf samples were processed for virus identification and association with pcr assays. total dna was extracted and pcr were performed with begomovirus specific primers (tlcv-cp). a *800 bp band was consistently amplified on 1% agarose. the pcr products were directly sequenced and sequence was submitted in genbank with the accession no. gq412352. the blast search analysis showed highest similarity of 98% with the ageratum enation virus. vernonia cinerea leaves with yellow vein symptoms were collected around crop fields in madurai. a 550 bp product amplified from total dna extracted from symptomatic leaves with degenerate primers designed to amplify a part of the av1 gene from begomoviral dna a component was cloned and sequenced. based on the above sequences, specific primers were designed and the full length dna a of 2745 nucleotides with typical genome organization of begomoviral dna a was obtained and was submitted to embl data base (acc no: am182232). the sequence comparison with other begomoviruses revealed the closest identity (83%) with emilia yellow vein virus from china and less than 80% with all known begomoviruses. the international committee on taxonomy of viruses (ictv) has therefore recognized vernonia yellow vein virus (vyvv) as a distinct begomovirus species. conventional pcr could not amplify the dna b or dna b from the infected tissue. however, the b dna (1364 bp) associated with the disease was obtained (acc no: fn435836) by the rolling circle amplification-restriction fragment length polymorphism method (rca-rflp) using phi29 dna polymerase. sequence analysis shows that dna b of vyvv has the highest identity (81%) with dna b of ageratum leaf curl disease and 58-77% with the b dna associated with other begomoviruses. infectious clones of vyvv dna a and dna b as dimers were made using the products of rca-rflp. these infectious clones will be used for agroinfection of vernonia and the results will be discussed. this is the first report of the molecular characterization of vernonia yellow vein virus (vyvv) from vernonia cinerea in india. production of bulb and seed crop of onion (allium cepa l.) is hampered by onion yellow dwarf virus (oydv) and iris yellow spot virus (iysv) with an incidence of 83.22% and 89.97% in bulb crop and 90.65% and 89.58% in seed crop, respectively in the popularly grown cv. hisar-2. four symptom-based variants of oydv designated as grade a, b, c and d produced varied types of symptoms in onion crop incurring heavy losses in bulb and seed production. iysv caused tiny hay coloured spots of different shapes and sizes on leaves and scapes which later coalesced and led to drying and lodging of scapes. the plant height, bulb weight and bulb size were 37.7 cm, 75.5 g and 24.2 cm 2 in plants infected with oydv, 39.6 cm, 79.7 g and 25.5 cm 2 in iysv infection, 35.1 cm, 68.4 g and 22.1 cm 2 due to their combined infection, as compared to 40.6 cm, 88.4 g and 27.6 cm 2 respectively, in healthy plants of bulb crop. in plants infected with oydv grade a the plant height was minimum (90.33 cm) whereas the number of umbels was maximum (9.20 umbels/pl.) but other yield parameters viz., weight/umbel (2.32 g), number of seeds/umbel (209), seed weight/umbel (0.64 g) and seed yield/plant (5.88 g) were recorded to be the lowest. the minimum reduction in plant height (100.26 cm), weight/umbel (6.72 g), number of seeds/umbel (633), seed weight/umbel (2.36 g) and seed yield/plant (11.90 g) were recorded in oydv grade d. the plant height was 98.84 cm with 5.10 umbels per plant, 4.24 g weight/umbel, 428 seeds/umbel, 1.25 g seed weight/umbel and 6.37 g seed yield/plant in iysv infected plants. the plant height (96.26 cm), umbels/plant (5.97), weight/umbel (4.60 g), number of seeds/umbel (432), seed weight/umbel (1.42 g) and seed yield/plant (7.82 g) were found to be the lowest in combined infection of oydv and iysv diseases in comparison to higher values in healthy controls (104.50 cm, 4.90, 7.84 g, 677, 2.60 g, 12.74 g, respectively). a minimum reduction in the test weight, germination and seed vigour index were found (3.06 g, 75.68% and 926) due to oydv grade a infection, whereas these were 2.92 g, 70.42% and 788 in iysv disease infected plants and 2.62 g, 70.4% and 776 in combined infection of oydv and iysv diseases in comparison to 3.84 g, 88.67% and 1276 in healthy plants. the maximum hampering of seed vigour parameters was recorded due to iysv infection. lodging of scapes caused by this disease was responsible for heavy losses in seed production and seed quality. cotton leaf curl disease is one of the major threats to cotton cultivation from northern india. survey conducted during 2009, observed the disease incidence ranged from 70 to 90% from bhatinda, abohar, fazilka, sri ganganagar, hanumanghar. in order to study genetic variability in the virus, twelve clcuv isolates were partially characterized (700 bp common region, full length av2 gene and partial sequences of ac1 and av1 gene). full length characterization of representative isolates from bhatinda, abohar, fazilka, sri ganganagar, hanumanghar is under progress. partial sequence analysis of clcuv isolates revealed that, the virus isolates collected during 2009 cropping season are closely related to cotton leaf curl burewala virus from pakistan and results were discussed. pratibha singh, h. s. savithri department of biochemistry, indian institute of science, bangalore tospoviruses, belonging to the family bunyavirideae, infect economically important plants such as groundnut, tomato, watermelon etc. they have a tripartite genome, with l, m and s segments of rna, in pseudo circular (panhandle) form. the viral genomes encode four structural proteins (l, n, g1 and g2) in the antisense orientation, and two non structural proteins nss and nsm in the sense orientation. the nsm is the only protein unique to tospoviruseses that infect plants in the bunyaviridae family and hence is proposed to be important for cell to cell movement. ground nut bud necrosis virus (gbnv), a member of the tospovirus genus, is the most prevalent virus infecting several species of leguminosae and solanaceae plants in india. total rna was isolated from gbnv infected tomato leaves and rt-pcr was performed using appropriate primers to amplify the nsm gene. the pcr product was cloned in pgex5x2 vector. the recombinant nsm clone was transformed into bl21 (de3) e. coli cells and over-expressed by induction with 0.3 mm iptg. sds-page analysis of induced and uninduced fraction revealed the presence of overexpressed protein of expected size. the soluble gst-nsm was purified by gsh sepharose affinity chromatography. purified gst-nsm was shown to interact with in vitro transcribed rna transcript by electrophoretic mobility shift assay. further nsm was shown to interact with viral encoded proteins np and nss using elisa and yeast two hybrid system. nsm was also shown to be phosphorylated in vitro by pellet fraction of plant sap. thus the recombinant gbnv nsm possesses the characteristic features of a movement protein such as nucleic acid binding, interaction with nucleocapsid protein, and ability to undergo posttranslational modification. solanum melongena, commonly called as egg plant is one of the most important vegetable crop in the world. it is cultivated widely in the tropical and sub tropical regions. several viruses such as cucumber mosaic cucumo virus (cmv), potato virus-y (pvy), potato virus-x (pvx) and tobacco ring spot virus (trsv) infect egg plant under natural conditions. in india major crop losses due to cmv infection in brinjal is 57% (fao stat-2008) . in the present study the infected leaf samples were collected from local fields of ramapuram, chandamama palli, chandragiri, madanapalli, yadhamari, durgasamudram villages in and around tirupati, were tested for cmv infection by dac-elisa with cmv antisera. the resulting positive samples were further inoculated to the raised brinjal seedlings of selected varieties through mechanical sap inoculation. different varieties of brinjal like mullabadhine, ankhur, ravya, mattigulla, casper and easter egg were used for monitoring the susceptibility to cmv infection. the mosaic symptoms were observed after 2 weeks of inoculation in all varities of brinjal except mullabadhina. among all these susceptible varities ankhur variety is selected to study induced biochemical changes such as chlorophylls, carbohydrates, proteins, nucleic acids and polyphenol oxidases in cmv infected brinjal leaves. in the infected leaves considerable reduction in chlorophyll and starch and increase in total proteins, sugars, rna and polyphenol oxidases was observed when compared to healthy leaves. the amount of total starch, protein and dna decreased to about 25, 136 and 645 lg/g respectively in infected leaves, where as sugars (75 lg/g), rna content (754 lg/g) and polyphenol oxidase activity was increased as compared to healthy leaves. the above results suggests that there is an altered concentrations of chlorophyll, proteins, nucleic acids, carbohydrates and polyphenol oxidase activity in the brinjal leaves due to the effect of cucumber mosaic cucumo virus infection. leaf analysis was found to be used as widely accepted diagnostic tool to assess the nutritional status of the vegetables. the present study deals with these aspects in detail. the total rna and dna was isolated from infected leaf samples. rt-pcr assays were performed using sugarcane yellow leaf virus (scylv) specific primers (scylv-615f and scylv-615r). the infection of scylv was detected in all the collected samples, which showed the expected size (*610 bp) amplicon during rt-pcr. in another experiment with nested pcr analysis, a phytoplasma characteristic 1.2 kb rdna pcr product were amplified from dnas of all infected samples but not in healthy sugarcane plants tested using phytoplasma universal primer pairs p1/p7 and fu3/ru5. dna extracts from plants with yellow mid rib and leaf yellows produced products of 1250 bp, which gave typical phytoplasma profiles when digested with hae iii and hha i. no pcr amplifications were produced using dna from symptomless plants. our results suggest that the yellow mid rib and leaf yellows symptoms on sugarcane varieties in uttar pradesh and uttarakhand states of india exhibiting midrib yellowing and leaf yellows symptoms is mainly caused by mixed infection of scylv and scylp. the affected clumps showed reduction in stalk height as compared to healthy fields. thirty-one sugarcane mosaic isolates belonged to sugarcane mosaic virus (scmv) and sugarcane streak mosaic virus (scsmv were collected from china and india), confirmed in indirect elisa and rt-pcr amplification with scmv and scsmv-specific primers. the amplicons (0.8 kb) from the coding region of coat protein (cp) were cloned, sequenced and compared to each other as well as to the sequences of 15 scmv isolates from sugarcane (australia, usa, china, brazil, mexico and south africa), maize (australia, china, iranian) and one scsmv isolate from sugarcane (india) in genbank. maximum likelihood and maximum parsimony analyses robustly supported two major monophyletic groups that were correlated with the host of origin: the scmv subgroup that included 18 isolates from china and only 13 isolates from india, and the scsmv subgroup that contained all isolates from india. maize dwarf mosaic virus (mdmv) and johnsongrass mosaic virus (jgmv) were not detected in any of the samples tested. a strong correlation was observed between the sugarcane groups and the geographical origin of the scmv isolates. the 11 millable sugarcane samples from china contained a virus tentatively described as sorghum mosaic virus (srmv). three isolates from nine chewing canes in fujian, yunnan and guizhou provinces of china also contained srmv, and the other 12 samples including five isolates from india was found infected with scmv. no srmv infection has been detected in sugarcane mosaic samples from india. sequence comparisons and phylogenetic analysis indicated that srmv can be considered as the most common and prevalent potyvirus infecting sugarcane in china, however in india sugarcane streak mosaic virus is dominant in causing mosaic symptoms on sugarcane. dig-labeled dna probe complementary to coat protein (cp) region of tobacco streak virus (tsv) sunflower isolate was designed for the sensitive and broad-spectrum detection of tsv isolates, the most devastating virus in india. dot-blot and tissue print hybridizations with the digoxigenin labeled probe were performed for the tsv detection at field levels. here, dot-blot hybridization was used to check a wide number of tsv isolates with a single probe and sensitivity with different sample extraction methods. the probe with cp conserved region prepared from sunflower pcr amplicon was hybridized with the tsv field isolates of gherkin, pumpkin, sunflower, marigold and globe amaranth samples because of highly conserved with little variability in cp region. the sensitivity limits were decreased from total nucleic acid to partially purified and crude extract preparations. in particular, tissue blot hybridization offers a simple, reliable procedure as dot-blot, but requires no sample processing. because there is minimal sample preparation, tissue-print hybridization could be an important component of tsv management programs. thus, the above non-radioactive labeled probe techniques can facilitate in screening the samples during tsv outbreaks and in quarantine services. savita patil, rupali sawant*, k. banerjee virology group, agharkar research institute, macs, g.g. agarkar road, pune 411 004 two mycobacterium smegmatis strains (ari lab nos. v842 and v946) were employed for the isolation of mycobacteriophages from soil and sewage samples. mycobacteriophages were isolated from soil samples collected from an area surrounding the tuberculosis (tb) ward, naidu hospital, pune, against m. smegmatis strain v842. these were numbered as v942, v943 and v944 and were isolated by using washed-cell preparation method. the bacteriophages against the other m. smegmatis strain, i.e. v946, were isolated from soil samples (collected from around tb ward, sassoon hospital, pune). some of these phages (viz.v953, v954) showed plaques at 42°c but not at 37°c. thus they seem to be lysogenic. for propagating and increasing the titre of all the above isolates, various previously described methods were attempted, but none of these methods were satisfactory. but when siliconized glassware and plastic-ware were used, propagation was successful. we showed that siliconization of glassware and plastic-ware was essential for the propagation of our mycobacteriophage isolates v951, v952, v953, v954 and v955. also, phage dilution medium (pdm) as described by chaterjee et al. (2000) was found to be effective for picking out of the plaques made by the phages. in this way, the phage isolates were propagated up to p 3 . the various passages of the phage isolates v951, v952, v953, v954 and v955 (i.e. original, p 1 , p 2 and p 3 ) were stored at -80°c. pvp-29 effect on pigments due to geminivirus infection on cowpea (vigna unguiculata) shail pande*, naveen pandey, k. shukla mahatma gandhi p. g. college gorakhpur, d.d.u. gorakhpur university, gorakhpur geminiviruses are one of the most important group of viruses causing economic losses in tropics. the symptom produced are yellowing of leaves which directly affect the pigments of diseased plants it in turn affects productivity and yield of diseased plant. cowpea vigna unguiculata is one of the important crop cultivated throughout india for its green pods which are used as vegetables and seeds are used as pulse. cowpea is affected by many viruses amongst them geminiviruses are one of the important virus on the cowpea plant. in the present study total chlorophyll content was studied in leaf of cowpea of diseased and healthy plants using arnon's method. carotenoids were also studied using ikan's method. it was found that chlorophyll content in diseased plants were lower compared to healthy plant similar results were found with carotenoids so the geminivirruses infection lowers the chlorophyll and carotenoid content in diseased plants which reduces yield of diseased cowpea plant. shweta sharma 1 , amrita banerjee 2 , j. tarafdar 2 , r. rabindran 3 , indranil dasgupta 1 * 1 department of plant molecular biology, university of delhi, south campus, new delhi; 2 bidhan chandra krishi vishwavidayalaya, kalyani, nadia, west bengal 741235; 3 tamil nadu agricultural university, coimbatore, tamil nadu 641003 rice tungro disease is an important disease of rice, caused by a joint infection by two viruses: rice tungro spherical virus (rtsv) and rice tungro bacilliform virus (rtbv) in south and southeast asia. the complex of rtbv and rtsv is transmitted by an insect vector green leaf hopper (glh). previously we reported complete genomic sequences of two geographically distinct isolates of rtbv; rtbv-wb (west bengal) and rtbv-ap (andhra pradesh) collected from the field in mid-1990s. both the sequences showed high homology all along the genome but showed divergence from previously reported southeast asian isolate i.e. rtbv-phil (philippines). to check whether a time period of a decade has resulted into variability in the genomic sequence of different isolates of rtbv in india, we cloned and sequenced the complete genome of rtbv from two geographically distinct regions of india i.e. west bengal and kanyakumari collected from the field in 2008. the complete nucleotide sequence of the dna fragments covering the whole genome of rtbv was determined using universal primers m13f and m13r and by primer walking, without any ambiguities remaining. the nucleotide sequences of overlapping clones were assembled and analyzed using the dna analysis software generunner and blastn program of ncbi. homology search at the nucleotide and amino acid level were performed using the blastn and blastp (respectively) programs of ncbi. multiple sequence alignments were performed using clus-tal-w software. sequence analysis results thus obtained showed that both the recently obtained complete genomic sequences of rtbv from two geographically distinct regions of india i.e. west bengal and kanyakumari showed very high homology (both at the nucleotide and amino acid levels) with the two previously reported rtbv isolates from india i.e. rtbv-wb (west bengal) and rtbv-ap (andhra pradesh) all along the genome. as observed earlier both the sequences diverged significantly from the southeast asian isolates. this suggests that even after the spatial and temporal difference (a time gap of approx 10 years) between the two previously reported rtbv isolates and the recently reported one, there is very little sequence variability between them. this further strengthens the earlier reports that the rtbv genomes in india are highly conserved. homology search at the nucleotide level using blastn program with the previously existing rtbv isolates revealed a very high percentage identity of 99% with the rtbv west bengal isolate and 95% with the rtbv andhra pradesh isolate. this further strengthens the earlier reports that there is not much genetic variability in the rtbv genomes in indian subcontinent. complete genomic rna sequences of two geographically distinct isolates of rice tungro spherical virus (rtsv), a member of the genus waikavirus, family sequiviridae, were determined from india. out of the two previously reported sequences, the indian isolates were closer to the resistance breaking strain rtsv-[vt6] than rtsv[phila] . between them, the indian sequences showed nucleotide as well as amino acid identities of 96%. a moderate homology was observed between the leader peptide and a putative helper component protein involved in insect transmission of the maize chlorotic dwarf virus, a closely related waikavirus, indicating its possible transmission-related function. unlike rice tungro bacilliform virus, which causes rice tungro disease jointly with rtsv, and is significantly different between isolates from india and philippines, rtsv genomes were observed to be much more conserved between isolates from the two countries. rice tungro bacilliform virus (rtbv) are believed to be the joint causative agents for the devastating tungro disease of rice prevalent in south and southeast asia [11] . rice tungro disease has become the major cause of production losses in rice during last three decades in several rice growing states of india. here, we report, for the first time the complete sequence analysis of two geographically distinct indian isolates of rtsv. we analyze the deduced protein sequences and their phylogenetic relationship with the two complete rtsv sequences from philippines as well as with other members of sequiviridae family. we provide molecular evidence that the indian isolates of rtsv are closely related to those from the philippines. we had earlier reported that rtbv isolates between india and philippines differ significantly from each other [18] . this study was undertaken in order to see whether rtsv isolates from india also show similar difference from those reported from the philippines. frequent outbreaks of tungro were reported near kanyakumari in the last 2-3 years. the present work was undertaken to clone and sequence the full-length rtbv and rtsv genomes from the infected rice plants collected from above region and to analyze the similarity of its genetic material with the existing indian isolates of rtbv and rtsv. a 1.1 kb dna fragment encoding the reverse transcriptase gene of rtbv genome was amplified and cloned in t/a vector and was sequenced commercially. homology search at the nucleotide level using blastn program with the previously existing rtbv isolates revealed a very high percentage identity of 99% with the rtbv west bengal isolate and 95% with the rtbv andhra pradesh isolate. this further strengthens the earlier reports that there is not much genetic variability in the rtbv genomes in indian subcontinent. similarly, the cp3 region of rtsv was amplified by rt-pcr and was cloned in t/a vector. recently, rice tungro disease has been reported from kanyakumari district of tamil nadu. it is important to determine the genetic nature of this isolate in order to develop resistance strategies. it is thus necessary to clone and characterize the viruses from kanyakumari and to determine the mechanism of virus resistance in transgenic lines. rice tungro disease is an important viral disease of rice. rice tungro is caused by infection by two viruses: rice tungro bacilliform virus (rtbv) and rice tungro spherical virus (rtsv). rtsv is a plant picornavirus with a 12 kb single stranded rna genome. it belongs to genus waikavirus in the family sequiviridae and is necessary for transmission of the two viruses by the leafhopper vector nephotellix virescens. rtsv rna is translated to form a large polyprotein, which is then self cleaved to form the viral proteins, including the three coat proteins, replicase, protease. studies have been conducted on rtsv from philippines. correct information of sequence variability of viral isolates to check whether different geographical conditions like those present in india select for genotypically variable strain and to design for transgenic resistance strategy, information on rtsv from india is absolutely essential. the objective of this study was to clone rtsv isolates from india and compare the genetic diversity of indian isolates from other southeast asian isolates and amongst each other. also develop strategy to impair the attack of virus-complex on rice. the achieve this, complete genomes of two isolates from india were cloned by amplifying different genes by rt-pcr and subsequently cloned in ta vectors, followed by sequencing. subsequently constructs containing cp1-3, antisense replicase, sense replicase and double stranded replicase were cloned in plant transformation vector. these constructs were used to transform aromatic rice variety from indian-pusa basmati (pb1). pcr analysis of the above plants was done to check the stable insertion of insert in the transgenics. jatropha (jatropha curcas) of the family euphorbiaceae is being grown in india as a major commercial fuel (bio-diesel) crop. jatropha is cultivated in 200 districts of 19 potential states of india. unfortunately, the cultivation of jatropha is limited by the severe mosaic disease. recently, a severe mosaic disease with significant disease incidence was observed in 2006-2009 on j. curcas grown in experimental plots of nbri and j. gossypifolia, a weed growing road side around lucknow and kathaupahadi, madhya pradesh. the disease consisted of the symptoms of severe mosaic, blistering, leaf distortion and stunting of whole plant and no fruit/seed production in severely affected plants. symptomatology and whitefly population observed on them suggested the occurrence of begomovirus infection. to detect the begomovirus infection, the total dna from leaf samples of infected jatropha plants was extracted and polymerase chain reaction (pcr) were performed using three sets of begomovirus genus specific (cpit-i/cpit-t, paliv 1978/paric 496 and paliv 722/palic 1960) primers and the expected size *800 bp, 1.2 kb and 1.2 kb amplicons were obtained which confirmed the begomovirus infection. further to identify the begomovirus/es and investigate the genetic diversity among them exists if any, the *1.2 kb amplicons were cloned and sequenced. the sequence data were deposited in the genbank database under accession nos.: gq847545 and fj346232 (from j. curcas) and eu727086 and fj177030 (from j. gossypifolia). during blast analysis gq847545 and fj346232 shared highest 95% sequence identity with each other and 84-88%% with sri lankan cassava mosaic virus (aj579307, aj607394, aj890225, aj89 0229 and aj890224) and indian cassava mosaic virus from india (ay738105) therefore, designated as two strains of jatropha mosaic india virus-lucknow. blast analysis of eu727086 showed maximum 93% similarities with croton yellow vein mosaic virus (aj507777), 82% with tomato leaf curl new delhi virus (dq629102) and 80-79% with papaya leaf curl virus (aj436992 and y15934), therefore, identified as strain of croton yellow vein mosaic virus. blast analysis of the virus isolate (fj177030) showed highest 83% identities with tomato leaf curl virus-bangalore ii (tolcv-b ii-u38239) and 82-81% with tomato leaf curl karnataka virus (tol-ckv, ay754812, fj514798), therefore, considered as new begomovirus species ''jatropha yellow mosaic india virus''. the phylogenetic analysis of gq847545 and fj346232 (from j. curcas) and eu727086 and fj177030 (from j. gossypifolia) was performed along with some selected isolates of begomovirus which showed [90% sequence identities during blast analysis. the isolate eu727086 showed closest relationship with croton yellow vein mosaic virus while fj177030 showed separate clustering of all the four begomovirus from jatropha species. during phylogenetic analysis these isolates formed three separate clusters, therefore, they were considered as three distinct begomoviruses. the above data clearly show that some genetic diversity exists among the begomoviruses infecting jatropha species in india. bitter gourd (momordica charantia l.) of the family cucurbitaceae, also known as bitter melon is extensively cultivated in north eastern region of uttar pradesh, india. it is regarded as one of the world's major vegetable crops and has great economic importance. a severe yellow mosaic disease on bitter gourd (momordica charantia) with a significant disease incidence was observed during the survey of different locations of eastern up, india in the year 2007. the whitefly (bemisia tabaci) population was also observed in the vicinity. the characteristic disease symptoms and whitefly population indicated the possibility of begomovirus infection. total dna were isolated from infected as well as healthy leaf samples. two primer pair (tlcv-cp and roja's primer) were used to study, which resulted *800 bp with tlcv-cp in 3/3 samples and *1.3 kb amplicons with roja's primer in 3/4 samples. for further identification of the begomovirus, the pcr amplicons were cloned and sequenced (genbank accession no. eu439260 and eu888908, respectively). the blastn search analysis of eu439260 indicated 99-95% identity with several isolates of tomato leaf curl new delhi virus (tolcndv). the phylogenetic analysis also showed closest relationships of the isolate (eu439260) with tolcndv isolates. based on highest sequence identity and closed relationships with tolcndv the virus isolated from bitter gourd was considered as an isolate of tomato leaf curl new delhi virus. while, blastn search analysis of eu888908 isolate, shared highest 99-97% identites with pepper leaf curl bangladesh virus (peplcbv) isolates. the phylogenetic analysis of the virus isolate with selected begomovirus isolates revealed a closest relationship with peplcbv. these results confirmed the association of peplcbv on bitter gourd. study revealed the variability of viruses on bitter gourd in eastern up, india. tobacco streak virus groundnut isolate was characterized biologically by taking six cultivars (jl24, tmv2, k6, k7, k9) and one pre-release culture (k1271) using seedlings of 7-84 days old under glasshouse conditions. there were clear differences were observed among cultivars tested regarding incubation period, percent seedling wilt and time taken to death of seedlings. k-7 was least susceptible among all the cultivars tested and it supported least virus titer (a 405 nm: 0.11-1.23). both localized (necrotic lesions on leaf, veinal necrosis, leaf yellowing, wilting) and systemic (petiole necrosis, necrotic lesions on young leaves, death of top growing buds not only on main stem but also on all primaries (side shoots), followed by stem necrosis, stunted growth, axillary shoot proliferation with small leaves having general chlorosis, peg necrosis, pod necrosis, pod size reduction, wilt of plants) symptom were observed in all cultivars tested. biological differentiation of tsv and gbnv was made by sap inoculation of both viruses separately using susceptible groundnut cultivar jl24 under glasshouse conditions. there were certain similarities and differences were observed between these viruses infecting groundnut. seed infection of tsv ranged from 18.9 to 28.9% in seeds collected from naturally infected and sap inoculated groundnut cultivars/pre-releases (jl24, tmv2, k-6, k-7, k-9 and k-1271) belonging to spanish and virginia types. tsv was detected both in pod shell and seed testa from pod samples produced by sap inoculation under glasshouse conditions. however, seed transmission of tsv was not observed in groundnut. coat protein (cp) gene of three groundnut tsv isolates (gn-ap-1-00; gn-ap2-04; gn-ap3-07) were sequenced and all the three isolates contained a single open reading frame (orf) of 717 bp nucleotide and could potentially code for 238 amino acids (aa). cp gene of tsv isolates originating from different hosts shared high degree of sequence identity both at nucleotide (97.6-100%) and amino acid (95.7-100%) levels respectively. tones grown in an area of 3.83.430 ha (fao stat2007). in india papaya is grown in nearly 80,000 ha with an annual production of 7,00,000 tones (fao stat 2007) and occupies fourth place in the world. the crop is severely affected by a number of viruses. papaya ring spot virus (prsv-p) is the most important virus. the detection of virus infection in plants has traditionally involved either bioassay on indexing plants and or immunological methods (hill 1981, torrence and jones 1981) . use of nucleic acid probes has improved the detection and sensitivity of viruses. the most common non-radioactive probes are biotynilated probes, which are very specific and sensitive. papaya ring spot virus (prsv-p) is a positive sense ssrna virus belonging to the genus potyvirus family potyviridae and transmitted by aphids. prsv-p coat protein gene region was used as template cdna for probe preparation. dot-blot hybridization with the biotin labeled probe were performed for prsv-p detection. the clarified sap of healthy and infected plants were serially diluted and spotted onto the nitrocellulose membrane, hybridized to biotin labeled probe. biotin labeled rna's are employed as probes, with a subsequent detection based on streptavidin-alkaline phosphatase conjugates. the sensitivity for viral detection of the biotin labeled probe was found to be sensitive than enzyme linked immunosorbent assay (elisa). in recent years tospovirus is causing devastating damage to the yield of vegetables in india. it infects economically important crops viz., tomato, chilli, peppers, groundnut, watermelon and various legumes. now it is emerging as severe disease in brinjal also. in order to monitor the natural occurrence and distribution of tospovirus in vegetable, surveys were conducted in the predominant brinjal growing areas of gujarat, karnataka, maharashtra and andhra pradesh during 2008-2010 incidence ranging from 5 to 10%, 0 to 80%, 1 to 40%, and 0 to 55.78% respectively. samples collected from different places of india were found positive to pbnv in direct antigen coating-enzyme linked immunosorbent assay (dac-elisa). pbnv infected brinjal plants showed mosaic mottling of leaves with leaf distortion, longitudinal streaks on the stem and necrotic rings on leaves and fruits. early infection led to severe stunting and abnormal fruiting. biological and molecular characterization of pbnv-brinjal isolates were compared with other isolates and results are discussed. for identification of virus causing mosaic symptoms on soybean various host plants were tested. plants species belonging to the different families viz. caricaceae, graminae, leguminosae, malvaceae and solanaceae were tested. the virus produced symptoms on diagnostic plant species like chenopodium album, c. quinoa, helianthus anus, phaseolus vulgaris and vigna ungiculata. among tested families the leguminosae that were the host of virus included arachis hypogea, the virus causing mosaic symptoms in soybean is inactivated between 50 and 55°c and between dilution of 10 -4 to 10 -5 . all the inoculated plants of assay host showed the symptoms at 50°c but not at 55°c. similarly local lesions produced at 10 -4 but not at 10 -5 . the virus in crude sap was infectious up to 72 h but not at 96 h at room temperature. however, the percentage infectivity decreased progressively as the aging of the sap was increased at room temperature. on the basis of reactions on diagnostic hosts pvp-38 identification and characterization of potyvirus infected chilli (capsicul annum l the virus under study caused mild mosaic and severe mottling symptom in leaves of infected plants. the dilution end point (dep) of the virus was found to be 10 -3 to 10 -4 , longevity in vitro (liv) 1-3 days at room temperature (25°c), thermal inactivation point (tip) 50-55°c. electron microscopy of purified virus preparation revealed the presence of flexuous particle of size 780 nm long and 14 nm in width with characteristic cytoplasmic inclusions: pinwheels and scrolls. the virus was transmitted by sap and by aphid myzus persicae. the host range study revealed that the host species were restricted to family chenopodiaceae and solanaceae. on the basis of above characteristic, the virus under study was identified as potyvirus associated with mild mosaic and severe mottling symptom in capsicum. phytoplasma causing grassy shoot disease and sugarcane yellow leaf viruses are important pathogens of sugarcane. these pathogens are causing severe losses in sugarcane productivity. with a view to producing virus and phytoplasma free planting material of sugarcane, experiments were undertaken using infected varieties of sugarcane growing at the farms of sugarcane research institute. apical meristems measuring about 2 mm in length, were dissected out, surface sterilized and cultured on agar gelled murashige and skoog's (ms) medium containing growth regulators for shoot induction. the established shoot cultures were multiplied through repeated subcultures on fresh media at 10-12 days interval. elimination of gsd and scylv was confirmed through molecular analysis of regenerated plants using specific primers of scylv and gsd. results revealed that apical meristem culture technique is effective in eliminating the pathogens like scylv and phytoplasma (gsd) from the infected clones. this is probably the first report on elimination of grassy shoot disease in sugarcane through meristem culture. papaya ringspot virus (prsv), which causes the most widespread and devastating disease in papaya, isolates originating from different geographical regions in south india were collected and maintained on natural host papaya. the entire coat protein (cp) gene of papaya ringspot virus-p biotype (prsv-p) was amplified by reverse transcription-polymerase chain reaction (rt-pcr). the amplicon was inserted into pgem-t vector by t-a cloning method, sequenced and sub cloned into a bacterial expression vector prset-a using directional cloning strategy. the prsv coat protein was over expressed as fusion protein in e. coli. sds-page gel revealed that cp expressed as a *40 kda protein. the recombinant coat protein (rcp) fused with 69 his-tag was purified from e. coli using ni-nta resin. the antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified prsv. the purified rcp was used as an antigen to produce high titer prsv specific polyclonal antiserum. the resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (ic-rt-pcr) assay and compared its sensitivity levels with elisa based assays for detection of prsv isolates. ic-rt-pcr was shown to be the most sensitive test followed by dot-blot immunobinding assay (dbia) and plate trapped elisa. key: cord-270587-k56fze59 authors: scherbinina, sofya i.; toukach, philip v. title: three-dimensional structures of carbohydrates and where to find them date: 2020-10-18 journal: int j mol sci doi: 10.3390/ijms21207702 sha: doc_id: 270587 cord_uid: k56fze59 analysis and systematization of accumulated data on carbohydrate structural diversity is a subject of great interest for structural glycobiology. despite being a challenging task, development of computational methods for efficient treatment and management of spatial (3d) structural features of carbohydrates breaks new ground in modern glycoscience. this review is dedicated to approaches of chemoand glyco-informatics towards 3d structural data generation, deposition and processing in regard to carbohydrates and their derivatives. databases, molecular modeling and experimental data validation services, and structure visualization facilities developed for last five years are reviewed. knowledge of carbohydrate spatial (3d) structure is crucial for investigation of glycoconjugate biological activity [1, 2] , vaccine development [3, 4] , estimation of ligand-receptor interaction energy [5] [6] [7] studies of conformational mobility of macromolecules [8] , drug design [9] , studies of cell wall construction aspects [10] , glycosylation processes [11] , and many other aspects of carbohydrate chemistry and biology. therefore, providing information support for carbohydrate 3d structure is vital for the development of modern glycomics and glycoproteomics. as result of growing interest to glycoprofiling, glycan microarrays, carbohydrate active enzymes (cazy) and glycan-binding proteins (gbp) which are involved in biological processes, several major international projects (e.g., glyspace [12] , glycosmos [13] , glycomics@expasy [14] , glygen [15] , jcggdb [16] , glytoucan [17] , mirage [18] , cfg [19] , rings [20] , glic (https://glic.glycoinfo.org/), sysglyco (https://sysglyco.org/)) were launched to integrate variety of data produced by glycobiological research. the main goal of existing glycoinformatics projects is to provide versatile resources with user-friendly access helpful for disease diagnostics [21, 22] , glycobioinformatics studies [23] , glycosylation site prediction [24] , cazy activity prognosis [25, 26] and other applications. appending of structural repositories with 3d structural data opens the way for computational glycobiology and modeling of carbohydrate structures at atomic resolution. design of novel workflows and techniques to connect carbohydrate spatial structure modes and experimental data with verification, processing, analysis and deposition of associated data has gained increased popularity in glycoscience community [27] . a carbohydrate structure database (csdb, [28] ) module for carbohydrate 3d structure modeling is a demonstrative example of 3d structural data integration facilities (as a database) combined with dedicated interface (as a glycoinformatics project). further details on csdb 3d facilities are discussed below. herein we focus on the important aspects of carbohydrate 3d structure availability to researchers: structural repositories; glycoinformatics tools and workflows to assist structure building, modeling and erroneous molecular geometry data detection and remediation; carbohydrate 3d structure presentation and visualization methods. structural databases make significant contribution to bringing information technologies to glycoscience [29] . with no focus on spatial structure, glycan databases and online tools have been recently reviewed [30] [31] [32] . depositing huge number of carbohydrates with detailed data for each entry, databases are valuable sources of structural information, biological assignments, references and external links. structural data are often accompanied by original and sometimes assigned experimental observables: nmr spectra, hplc and ms profiles, etc. the services built on top of the databases can include 3d structure simulation, validation, and storage. a viewpoint of the authors at the ideal integration of data resources and services in glycoinformatics is summarized in figure 2 . a subject of this review is databases providing theoretical or empirical 3d structures of carbohydrates and related data-mining tools. herein we focus on the important aspects of carbohydrate 3d structure availability to researchers: structural repositories; glycoinformatics tools and workflows to assist structure building, modeling and erroneous molecular geometry data detection and remediation; carbohydrate 3d structure presentation and visualization methods. structural databases make significant contribution to bringing information technologies to glycoscience [29] . with no focus on spatial structure, glycan databases and online tools have been recently reviewed [30] [31] [32] . depositing huge number of carbohydrates with detailed data for each entry, databases are valuable sources of structural information, biological assignments, references and external links. structural data are often accompanied by original and sometimes assigned experimental observables: nmr spectra, hplc and ms profiles, etc. the services built on top of the databases can include 3d structure simulation, validation, and storage. a viewpoint of the authors at the ideal integration of data resources and services in glycoinformatics is summarized in figure 2 . a subject of this review is databases providing theoretical or empirical 3d structures of carbohydrates and related data-mining tools. networking between glycoinformatics projects and related services that promotes achievement of data integration in glycomics. reproduced with permission from [29] , © 2020 wiley-vch verlag gmbh & co. kgaa, weinheim. the majority of existing repositories for carbohydrate 3d structures offer open-access data via web interface. deposited datasets can be represented by glycoproteins, protein-carbohydrate complexes, poly-and oligosaccharides with 3d structure experimentally resolved or specified by means of nmr, x-ray crystallography, cryoem, small angle x-ray scattering, etc. [27] . several databases such as glycam-web, ek3d, 3dsdscar, glycomapsdb contain data from molecular dynamics simulations. we have also mentioned databases featuring information on protein structures involving carbohydrate moiety in terms of glycosylation (as post-translational modification, dbptm), carbohydrate active enzymes (cazy) and homology modeling (swiss-model). table 1 displays currently active structural databases maintaining three-dimensional data on carbohydrates. for table 1 , we have selected carbohydrate and related databases using the following criteria: • database can be freely accessed through web user interface; • database must contain experimentally confirmed and/or predicted 3d structures (preprocessed and/or generated on-the-fly from a primary structure input) of glycans, glycoproteins, or protein-carbohydrate complexes; • stored 3d structures must be deposited as atomic coordinates in pdb, mol, or other format, and the structures must contain a saccharide moiety; • databases with records linked to other large 3d data collections (e.g., rcsb pdb, pdbe, pdbj, pdbsum, uniprotkb etc.) are included in table 1 (as long as database entries contain carbohydrate moiety, e.g., as a part of a lectin or an antibody); • databases with derived carbohydrate 3d structural data (conformational maps, conformer energy minima, etc.) are included in table 1 even if they provide no atomic coordinates (e.g., glycomapsdb and gfdb). despite no fit to the criteria above, assistance of large structure repositories offering only glycan primary structures (e.g., glytoucan [17] (https://glytoucan.org/), unicarbkb [33] (http://www.unicarbkb.org/)) can be useful for cross-referencing of existing carbohydrate resources and serve as supplementation to 3d modeling pipelines. some out-of-date projects, such as complex carbohydrate structural database (ccsd) [34, 35] , eurocarbdb [33, 36] , glycomedb [36] [37] [38] , glycoconjugate data bank [39] , glycosuite [40, 41] are noteworthy as they had shaped the modern vision of structural glycoinformatics. networking between glycoinformatics projects and related services that promotes achievement of data integration in glycomics. reproduced with permission from [29] , © 2020 wiley-vch verlag gmbh & co. kgaa, weinheim. the majority of existing repositories for carbohydrate 3d structures offer open-access data via web interface. deposited datasets can be represented by glycoproteins, protein-carbohydrate complexes, poly-and oligosaccharides with 3d structure experimentally resolved or specified by means of nmr, x-ray crystallography, cryoem, small angle x-ray scattering, etc. [27] . several databases such as glycam-web, ek3d, 3dsdscar, glycomapsdb contain data from molecular dynamics simulations. we have also mentioned databases featuring information on protein structures involving carbohydrate moiety in terms of glycosylation (as post-translational modification, dbptm), carbohydrate active enzymes (cazy) and homology modeling (swiss-model). table 1 displays currently active structural databases maintaining three-dimensional data on carbohydrates. for table 1 , we have selected carbohydrate and related databases using the following criteria: • database can be freely accessed through web user interface; • database must contain experimentally confirmed and/or predicted 3d structures (preprocessed and/or generated on-the-fly from a primary structure input) of glycans, glycoproteins, or protein-carbohydrate complexes; • stored 3d structures must be deposited as atomic coordinates in pdb, mol, or other format, and the structures must contain a saccharide moiety; • databases with records linked to other large 3d data collections (e.g., rcsb pdb, pdbe, pdbj, pdbsum, uniprotkb etc.) are included in table 1 (as long as database entries contain carbohydrate moiety, e.g., as a part of a lectin or an antibody); • databases with derived carbohydrate 3d structural data (conformational maps, conformer energy minima, etc.) are included in table 1 even if they provide no atomic coordinates (e.g., glycomapsdb and gfdb). despite no fit to the criteria above, assistance of large structure repositories offering only glycan primary structures (e.g., glytoucan [17] (https://glytoucan.org/), unicarbkb [33] (http://www. unicarbkb.org/)) can be useful for cross-referencing of existing carbohydrate resources and serve as supplementation to 3d modeling pipelines. some out-of-date projects, such as complex carbohydrate structural database (ccsd) [34, 35] , eurocarbdb [33, 36] , glycomedb [36] [37] [38] , glycoconjugate data bank [39] , glycosuite [40, 41] are noteworthy as they had shaped the modern vision of structural glycoinformatics. • mammalian glycans • pre-built libraries of predicted 3d structures of common bioglycans • 3d structure models * • 3d-atomic coordinates generation (http://glycam.org/pre-builtlibraries.jsp) a where unknown, the year of the first publication is given. b database is marked as curated if manual verification of data was reported in the original publication or at the database web site. c published coverage data can be outdated; database interface provides no statistics on current coverage. * database provides no search facilities for indicated carbohydrate 3d structural data. methods to probe a 3d structure of carbohydrate-containing biomolecules has been developed for decades. nmr techniques (interatomic distances derived from noe, and torsion angles derived from coupling constants), x-ray crystallography, and electron cryo-microscopy (the two latter being atomic models built on the basis of electron density map) are among most demanded methods for 3d strucural elucidation. these methods have been reviewed [93] [94] [95] [96] and are beyond the scope of this review focused in information technologies. for use of instrumental methods for the validation of a simulated structure, please refer to section 5 "experimental data validation". structural investigation of large biological systems involving protein-glycan interactions requires leveraging more resources and employing more complex experimental techniques compared to solely oligo-and polysaccharides studies. advances in nmr methods hold great potential for direct spatial structure determination of carbohydrate-protein complexes in solution based on intermolecular noes which affords estimation of atomic contacts between a protein and a carbohydrate ligand [97, 98] . further extraction of noe-derived distance restraints for a saccharide molecule results in generation of representative conformational ensembles [99] [100] [101] . support of experimental data with computer simulations can significantly improve quality of 3d structures. quantum mechanics [100, [102] [103] [104] [105] [106] and molecular dynamics modeling [107] [108] [109] [110] [111] are commonly applied to conformation search and nmr signal prediction. to date, the following theoretical models and methods are applied for in silico design of carbohydrate three-dimensional structure [112] [113] [114] [115] [116] : based on scopus [135] article count we estimated the application rate for quantum mechanics (10759 publications) and molecular mechanics (14871 publications) methods applied for carbohydrate structure modeling for the recent five years (2015-2020). search queries included abundant carbohydrate terms, typical glycan moieties, and common modeling approaches (query details are given in supplementary table s1 ). in spite of growing interest to qm approaches in carbohydrate structure simulation, the major contribution to the statistics for such resource-intensive calculations is application of qm to relatively simple model compounds. for complex bioglycans in solution predominance of mm methods is more pronounced [6, 8] . molecular dynamics methods have achieved broad scope of application in terms of reasonable computer resource consumption. they fulfill advantageous compromise between calculation accuracy and performance, when applied to glycan molecules and their structural complexity (variety of known monomeric elements, presence of ionogenic groups), high bridge flexibility and stereo-electronic effects [112, 113, 136, 137] . in molecular mechanics simulations, newtonian mechanics principles are applied to calculate potential energy of a system using parameter set specific for a class of compounds under study (force field). particular features of carbohydrate moiety, e.g., ring puckering, rotational barriers, hydrogen bonds, must be taken into account to perform precise analysis of molecular behavior in vacuo or in solution [138] . molecular dynamics simulations consider newtonian motion equations to observe evolution of a system during a certain timespan. conformation ensemble generation occurs via calculation of molecular trajectory at given temperature. accuracy of calculation depends on the employed force field and sufficient conformational sampling. md simulations are commonly used for interpretation and analysis of the nmr and x-ray observables in the context of carbohydrate 3d structure [139] . enhanced molecular dynamics sampling technologies, such as replica-exchange md (remd) [140, 141] , hamiltonian replica-exchange md (hrex) [142] [143] [144] , multidimensional swarm-enhanced sampling md (msesmd) [145, 146] , gaussian accelerated md (gamd) [147, 148] have been reported. density maps or energy maps built for a set of the glycosidic torsion angles (ϕ, ψ, ω) are a typical way to report conformational preferences of a glycan provided by population analysis of its md trajectory. as a representative example, conformational characteristics of highly flexible branched oligosaccharide glc 1 man 9 glcnac 2 (gm9) were investigated by explicit-water remd study and validated using paramagnetism-assisted nmr spectroscopy [149] (figure 3a,b) . due to the structural complexity of gm9, adequate exploration of conformational space requires long-timescale simulations. regular md simulations of similar manno-oligosaccharides were reported to fail reproduction of experimental data [150] . replica-exchange approach implies running periodically swapped parallel replicas of the system at different temperatures. ensemble of gm9 conformers sampled by this method was consistent with the nmr observables. populated areas of density maps built for glycosidic linkages of glc 1 man 3 branch of gm9 ( figure 3c ) were close to crystallographic conformations of a linear glc 1 man 3 tetrasaccharide (a gm9 determinant recognized by lectins) from pdb. molecular dynamics simulations consider newtonian motion equations to observe evolution of a system during a certain timespan. conformation ensemble generation occurs via calculation of molecular trajectory at given temperature. accuracy of calculation depends on the employed force field and sufficient conformational sampling. md simulations are commonly used for interpretation and analysis of the nmr and x-ray observables in the context of carbohydrate 3d structure [139] . enhanced molecular dynamics sampling technologies, such as replica-exchange md (remd) [140, 141] , hamiltonian replica-exchange md (hrex) [142] [143] [144] , multidimensional swarm-enhanced sampling md (msesmd) [145, 146] , gaussian accelerated md (gamd) [147, 148] have been reported. density maps or energy maps built for a set of the glycosidic torsion angles (φ, ψ, ω) are a typical way to report conformational preferences of a glycan provided by population analysis of its md trajectory. as a representative example, conformational characteristics of highly flexible branched oligosaccharide glc1man9glcnac2 (gm9) were investigated by explicit-water remd study and validated using paramagnetism-assisted nmr spectroscopy [149] (figure 3a,b) . due to the structural complexity of gm9, adequate exploration of conformational space requires long-timescale simulations. regular md simulations of similar manno-oligosaccharides were reported to fail reproduction of experimental data [150] . replica-exchange approach implies running periodically swapped parallel replicas of the system at different temperatures. ensemble of gm9 conformers sampled by this method was consistent with the nmr observables. populated areas of density maps built for glycosidic linkages of glc1man3 branch of gm9 ( figure 3c ) were close to crystallographic conformations of a linear glc1man3 tetrasaccharide (a gm9 determinant recognized by lectins) from pdb. force field (or potential energy function) is represented by atomistic parameter set obtained for a considered compound class. potential energy value can be calculated as a sum of interaction potentials for bonded (covalent bond stretching, angle bending, proper torsions) and non-bonded (electrostatic and van der waals interactions) terms, and can include other terms (e.g., improper torsions, solvation, hydrogen bonds [151] , nonconventional hydrogen bonds [101] , for protein-carbohydrate complexes-ch-π stacking interactions [152] [153] [154] [155] , chi carbohydrate intrinsic (chi) energy contribution [156, 157] ). several force fields developed for general representation of wide range of organic compounds (e.g., allinger's mm2, mm3, mm4) can be applied to carbohydrate 3d modeling [151, 158, 159] . of them, despite being a universal force field, mm3 [160, 161] still exhibits good performance on glycans [162] [163] [164] (reviews), [165, 166] (exemplary articles). however, a number of force fields specially tuned for carbohydrates have been developed (figure 4 ). in supplementary table s2 , we provided citation metrics of articles reporting carbohydrate-dedicated and selected general force fields that could be applied to carbohydrate structure modeling. unfortunately, usage of general force fields could not be adequately estimated via number of citations. automated full-text analysis and retrieval of data, needed to confirm employment of force fields for carbohydrate molecules, is beyond the scope of this review. nevertheless, statistical data obtained for general force fields supported in popular md software packages (e.g., amber, charmm, gromacs, tinker) shows obsolescence of modern force fields above allinger's ones, and mm3 in particular (see more detailed data, references to original publications and absolute values in supplementary table s2 ). force field (or potential energy function) is represented by atomistic parameter set obtained for a considered compound class. potential energy value can be calculated as a sum of interaction potentials for bonded (covalent bond stretching, angle bending, proper torsions) and non-bonded (electrostatic and van der waals interactions) terms, and can include other terms (e.g., improper torsions, solvation, hydrogen bonds [151] , nonconventional hydrogen bonds [101] , for protein-carbohydrate complexes-ch-π stacking interactions [152] [153] [154] [155] , chi carbohydrate intrinsic (chi) energy contribution [156, 157] ). several force fields developed for general representation of wide range of organic compounds (e.g., allinger's mm2, mm3, mm4) can be applied to carbohydrate 3d modeling [151, 158, 159] . of them, despite being a universal force field, mm3 [160, 161] still exhibits good performance on glycans [162] [163] [164] (reviews), [165, 166] (exemplary articles). however, a number of force fields specially tuned for carbohydrates have been developed (figure 4 ). in supplementary table s2 , we provided citation metrics of articles reporting carbohydrate-dedicated and selected general force fields that could be applied to carbohydrate structure modeling. unfortunately, usage of general force fields could not be adequately estimated via number of citations. automated full-text analysis and retrieval of data, needed to confirm employment of force fields for carbohydrate molecules, is beyond the scope of this review. nevertheless, statistical data obtained for general force fields supported in popular md software packages (e.g., amber, charmm, gromacs, tinker) shows obsolescence of modern force fields above allinger's ones, and mm3 in particular (see more detailed data, references to original publications and absolute values in supplementary table s2 ). detailed comparisons of all-chemical and dedicated force fields in a context of glycan modeling have been published [114, 139, 151, 167] . charmm36, glycam06, gromos and opls-aa-sei were reported as commonly used force fields for handling carbohydrate or glycoconjugate molecules. more details are provided in figure 5 . charmm36 force field with modern carbohydrate parameter table (c36 [168] ) was derived from charmm all-atom biomolecular force field [169, 170] . currently, charmm36 parameterization features include monosaccharides in furanose [171] and pyranose [172] forms, glycosidic linkages between monosaccharides [171, 173] , complex carbohydrates and glycoproteins detailed comparisons of all-chemical and dedicated force fields in a context of glycan modeling have been published [114, 139, 151, 167] . charmm36, glycam06, gromos and opls-aa-sei were reported as commonly used force fields for handling carbohydrate or glycoconjugate molecules. more details are provided in figure 5 . charmm36 force field with modern carbohydrate parameter table (c36 [168] ) was derived from charmm all-atom biomolecular force field [169, 170] . currently, charmm36 parameterization features include monosaccharides in furanose [171] and pyranose [172] forms, glycosidic linkages between monosaccharides [171, 173] , complex carbohydrates and glycoproteins [174] , monosaccharide-linked sulfate and phosphate groups [175] , acyclic carbohydrates and alditols [171] , as well as carbohydrate simulations in aqueous solution [176] . [174] , monosaccharide-linked sulfate and phosphate groups [175] , acyclic carbohydrates and alditols [171] , as well as carbohydrate simulations in aqueous solution [176] . glycam06 force field is compatible with carbohydrates of all ring sizes and conformations for both mono-and oligosaccharides built of residues common for mammalian glycans, such as widespread aldoses, n-acetylated amino-sugars, sialic, glucuronic and galacturonic acids [177] . parameter set was extended to non-carbohydrate moieties such as lipids [178] , glycolipids [179, 180] , lipopolysaccharides [181] , proteins and nucleic acids. parameterization of glycam06 for glycosaminoglycans was reported [182] . gromos represents a broad family of carbohydrate force fields. having been a classic one since 2005, gromos 45a4 [183] parameter set is used for explicit-solvent simulation of hexopyranose-based saccharides. in the recent decade, several parameters of 45a4 were optimized in gromos 56acarbo [184] including lipopolysaccharides [185] . gromos 53a6glyc was improved for explicit-solvent simulations [186] and extended for glycoproteins [187] . gromos 56acarbo_r [188] was designed to improve description of ring conformational equilibria in hexopyranose-based saccharide chains as compared to the previous 56acarbo version. another modification of 56acarbo named 56acarbo_cht [189] was developed for chitosan and its derivatives. recently, extensions of gromos 56acarbo/carbo_r parameter set were adapted towards charged, protonated and esterified urinates [190] and furanose-based carbohydrates [191] . gromos96 43a1 was reported to have good performance on glycan structure simulation in glycoproteins [192, 193] . opls-aa scaling of electrostatic interactions (sei) force field [194] consists of improved parameters for conformational changes associated with φ-ψ dihedrals combined with enhanced accuracy of qm relative energy calculation in carbohydrate molecules refined for opls-aa biomolecular force field [195, 196] . additionally opls force field was improved for explicit-water simulations [197] . rapidly developing charmm drude polarizable force field for carbohydrates based on classical drude oscillator has to be mentioned. parameter sets obtained for hexapyranoses [198] and their aqueous solutions [199] , aldopentafuranoses and methyl-aldopentafuranosides [200] , glycam06 force field is compatible with carbohydrates of all ring sizes and conformations for both mono-and oligosaccharides built of residues common for mammalian glycans, such as widespread aldoses, n-acetylated amino-sugars, sialic, glucuronic and galacturonic acids [177] . parameter set was extended to non-carbohydrate moieties such as lipids [178] , glycolipids [179, 180] , lipopolysaccharides [181] , proteins and nucleic acids. parameterization of glycam06 for glycosaminoglycans was reported [182] . gromos represents a broad family of carbohydrate force fields. having been a classic one since 2005, gromos 45a4 [183] parameter set is used for explicit-solvent simulation of hexopyranose-based saccharides. in the recent decade, several parameters of 45a4 were optimized in gromos 56a carbo [184] including lipopolysaccharides [185] . gromos 53a6 glyc was improved for explicit-solvent simulations [186] and extended for glycoproteins [187] . gromos 56a carbo_r [188] was designed to improve description of ring conformational equilibria in hexopyranose-based saccharide chains as compared to the previous 56a carbo version. another modification of 56a carbo named 56a carbo_cht [189] was developed for chitosan and its derivatives. recently, extensions of gromos 56a carbo / carbo_r parameter set were adapted towards charged, protonated and esterified urinates [190] and furanose-based carbohydrates [191] . gromos96 43a1 was reported to have good performance on glycan structure simulation in glycoproteins [192, 193] . opls-aa scaling of electrostatic interactions (sei) force field [194] consists of improved parameters for conformational changes associated with ϕ-ψ dihedrals combined with enhanced accuracy of qm relative energy calculation in carbohydrate molecules refined for opls-aa biomolecular force field [195, 196] . additionally opls force field was improved for explicit-water simulations [197] . rapidly developing charmm drude polarizable force field for carbohydrates based on classical drude oscillator has to be mentioned. parameter sets obtained for hexapyranoses [198] and their aqueous solutions [199] , aldopentafuranoses and methyl-aldopentafuranosides [200] , carboxylate and n-acetylamine saccharide derivatives [201] , alditols [202] and glycosidic linkages [203] demonstrated significant improvement of qm data reproduction compared to charmm additive force field. martini coarse-grained (cg) force field [204] can be used alternatively to all-atom (aa) level simulations with advantage of modeling large carbohydrate systems (solutions of oligo-, polysaccharides, glycolipids [205] [206] [207] ) on a long time scale at reasonable computational cost. blocked ring puckering (only 4 c 1 conformation is allowed) and restrictions on the anomeric effect and glycosidic bond flexibility cumulatively provide reduction of available degrees of freedom. another cg model pitomba [208] for carbohydrate simulations was developed based on gromos 53a6 glyc force field. docking methods for carbohydrate ligands utilize molecular modeling approaches for protein-carbohydrate complexes for initial geometry generation, conformational sampling, grafting, active site mapping and binding affinity estimation [129, 137, [209] [210] [211] . accurate reproduction of experimental data requires application of particular scoring function parameterization (empirical, force fields or knowledge-based [212] ) and docking protocols, which depend on the interaction types present in a system (ch-π interactions, chi-energy, hydrogen bonding, solvent model, influence of solvent molecules inclusion effects, charged moiety etc.) [8, [213] [214] [215] [216] [217] [218] [219] . extension of several docking software packages to handle carbohydrate molecules was reported to improve modeling of biologically relevant systems such as lectin-glycan [220, 221] , gag-protein [222] [223] [224] , or antibody-carbohydrate [225] . currently available web-based tools along with standalone software packages were developed to facilitate work with carbohydrate 3d structure. versatile online services for in silico molecular modeling allow users to start from a user-friendly structure input, and to automatize further procedures (see table 2 for references). glycam-web provides tools for glycan structure prediction, glycosylated protein 3d model generation, grafting and docking. charmm-gui modeler offers options for 3d structure generation and modeling of glycans including n-/o-glycoproteins and glycolipids [226, 227] . biological membranes can be simulated with the assistance of charmm-gui membrane builder (by combining features of lps and glycolipid charmm-gui modelers) and gnomm (a tool for building lipopolysaccharide-rich membranes). noteworthy standalone programming frameworks for structure modeling are glycosylated (modeling of glycans, glycoproteins and glycosylation) and rosetta carbohydrate (loop modeling [228] , glycan-to-protein docking, and glycosylation modeling). to build diverse saccharide 3d models online, one can use such tools as restless and sweet-ii. doglycans standalone framework can be used for preparation of the atomistic models of glycopolymers, glycolipids and glycoproteins. complex polysaccharide 3d models can be generated via polys and carbbuilder. another special class of polysaccharide builders is dedicated to glycosaminoglycans (gags) which can be accessed using polys gag-builder and glycam-web gag-builder. recently, another approach for building gag molecules was reported [229] (exemplary data pipeline only). unfortunately, application scope of the majority of the existing structure building and modeling services is limited to rigidly defined set of supported sugar residues, and lacks non-carbohydrate moiety support. tools for locating and identification of a carbohydrate moiety (e.g., pdb2linucs, glyfinder, glycan reader) are useful for the atomic coordinate analysis and extraction of glycoproteins and protein-carbohydrate complexes deposited in protein data bank (pdb). automated molecular geometry processing facilities can be accessed via glycoinformatics tools designed for conformational data analysis (cat, bfmp), nuclear overhauser effect (noe) calculation (md2noe, distance mapping) and 3d structural data analysis related to glycan moieties from pdb (glytorsion, glyvicinity, gs-align). in table 2 , we summarized freely available tools for generation and processing carbohydrate 3d structural data and divided them into eight categories of application. a web-service implies an automated pipeline for running a specific software (e.g., molecular modeling, structure building, carbohydrate coordinate extraction, format conversion). it results in 3d structural data output starting from primary structure input or atomic coordinate file upload. web-tool is employed for 3d structural data processing and analysis without 3d structural data output; it is a simpler application designed primarily for statistics and visualization. other types are self-explanatory. vast variety of methods provide information about 3d structure of individual glycans and glycan moieties of glycoproteins and protein-carbohydrate complexes ( figure 6 ) [285, 286] . the following approaches are most utilized for 3d structural data validation [287] [288] [289] : • ccombination of carbohydrate simulated geometry data with x-ray crystallographic data analysis [225, 290] ; • analysis of inter-glycosidic nmr spin couplings, which depend on glycosidic bond torsions [114, 291, 292] vast variety of methods provide information about 3d structure of individual glycans and glycan moieties of glycoproteins and protein-carbohydrate complexes ( figure 6 ) [285, 286] . the following approaches are most utilized for 3d structural data validation [287] [288] [289] : • ccombination of carbohydrate simulated geometry data with x-ray crystallographic data analysis [225, 290] ; • analysis of inter-glycosidic nmr spin couplings, which depend on glycosidic bond torsions [114, 291, 292] ; • deriving nuclear overhauser effects (noes) from relative populations of the interatomic distances, with subsequent comparison to the experimental noes in solution [99, 293, 294] ; • purely informatic detection of errors, such as incompatible atomic coordinates originating from incorrect processing or simulation [295] [296] [297] [298] ; • simulation by other computational methods at higher levels of theory [102, 103, 105, 108] . unfortunately, most of the data obtained on the basis of crystallographic experiments can dramatically differ from glycan conformations in solution or have poor resolution which needs further adjustment [299, 300] . moreover, not all of the objects of interest can be obtained as a single crystal. electron cryo-microscopy gains popularity for carbohydrate 3d structural research [301] , however, this method requires additional refinement procedures due to resolution restrictions of the obtained density unfortunately, most of the data obtained on the basis of crystallographic experiments can dramatically differ from glycan conformations in solution or have poor resolution which needs further adjustment [299, 300] . moreover, not all of the objects of interest can be obtained as a single crystal. electron cryo-microscopy gains popularity for carbohydrate 3d structural research [301] , however, this method requires additional refinement procedures due to resolution restrictions of the obtained density maps [302] [303] [304] . recently, cryo-em data were used for the refinement of sars-cov-2 spike glycoprotein stucture using privateer (see table 3 for references) software [305, 306] . van beusekom et al., illustrated [295] quality improvement of the pdb glycan structure model with incorrect (1-6)-linked fucose annotation, poor fit to the electron density, and missing (1-3)-linked fucose (figure 7a ) with the help of pdb-redo ( figure 7b ) and carp (figure 7d ) tools (see table 3 for references). structure model obtained by pdb-redo treatment was further manually inspected ( figure 7c ): corrections were made for acetylamino group geometry, distorted (1-6)-linked fucose ring conformation, and (1-3)-linked fucose residue was added. despite successful automated resolution of residue annotation problem and poor electron density refinement, complete revision could not be achieved without manual intervention. maps [302] [303] [304] . recently, cryo-em data were used for the refinement of sars-cov-2 spike glycoprotein stucture using privateer (see table 3 for references) software [305, 306] . van beusekom et al., illustrated [295] quality improvement of the pdb glycan structure model with incorrect (1-6)-linked fucose annotation, poor fit to the electron density, and missing (1-3)-linked fucose (figure 7a ) with the help of pdb-redo ( figure 7b ) and carp (figure 7d ) tools (see table 3 for references). structure model obtained by pdb-redo treatment was further manually inspected (figure 7c ): corrections were made for acetylamino group geometry, distorted (1-6)-linked fucose ring conformation, and (1-3)-linked fucose residue was added. despite successful automated resolution of residue annotation problem and poor electron density refinement, complete revision could not be achieved without manual intervention. nmr techniques are a powerful approach to investigate conformational and dynamic behavior of carbohydrate moieties in biomolecules [307] [308] [309] [310] . however, the nature of noe enhancement factor has been hampering obtaining the sufficient number of distance restrains [99] . in the case of saccharides with their multiple rotatable bonds, the stable 3d structure was difficult to define, making molecular modeling essential for this class of compounds. adjustment of experimental conditions helped to overcome the mentioned limitation and to reproduce crystal structures of oligosaccharides by modeling with noe-derived distance restraints [100, 101] . nmr techniques are a powerful approach to investigate conformational and dynamic behavior of carbohydrate moieties in biomolecules [307] [308] [309] [310] . however, the nature of noe enhancement factor has been hampering obtaining the sufficient number of distance restrains [99] . in the case of saccharides with their multiple rotatable bonds, the stable 3d structure was difficult to define, making molecular modeling essential for this class of compounds. adjustment of experimental conditions helped to overcome the mentioned limitation and to reproduce crystal structures of oligosaccharides by modeling with noe-derived distance restraints [100, 101] . since there is no direct way to derive detailed three-dimensional representation from the observed noe intensities, additional molecular modeling protocols are required to establish comprehensive view of conformational space at the atomic level [311] [312] [313] . frank et al., demonstrated conformation filtering based on the observed noe obtained by molecular dynamics in explicit solvent [314] . as a representative example, figure 8 depicts 1 h-1 h spatial contacts and conformation selection criteria illustrated by moraxella catarrhalis lgt2∆ bacterium heptasaccharide, which adopts an unusual conformation. since there is no direct way to derive detailed three-dimensional representation from the observed noe intensities, additional molecular modeling protocols are required to establish comprehensive view of conformational space at the atomic level [311] [312] [313] . frank et al., demonstrated conformation filtering based on the observed noe obtained by molecular dynamics in explicit solvent [314] . as a representative example, figure 8 depicts 1 h-1 h spatial contacts and conformation selection criteria illustrated by moraxella catarrhalis lgt2δ bacterium heptasaccharide, which adopts an unusual conformation. protein data bank (pdb) [315] and cambridge structural database (csd) [316] are historically considered the main repositories of experimentally determined carbohydrate three-dimensional structures. csd is reported to deposit over 4000 crystal structures of oligosaccharides [93] . unlike cambridge structural database, protein data bank provides open access to the entire structural archive. carbohydrate moieties deposited in pdb are usually represented as covalently bound to protein or imply non-covalently bound protein-carbohydrate complex formation [302] . according to recent reports, as at november 18, 2019 protein data bank contained ~13500 carbohydrate structures representing ~9.4% of total database records [317] . despite being a valuable source of 3d structural data for glycoscientists, pdb lacks convenient search facilities for glycan structures. some projects have developed data-mining tools capable of retrieving bioglycan molecular geometry data from pdb: glycan reader (glycanstructure.org) [260, 261] (http://www.glycanstructure.org/), pdb2linucs (glycosciences.de) [47, 259, 318] (http://www.glycosciences.de/database/start.php?action=form_pdb_data), glyconavi tcarp [61] (https://glyconavi.org/tcarp/) (https://gitlab.com/glyconavi/pdb2glycan) and glyfinder (glycam-web) [257, 258] (https://dev.glycam.org/portal/gf_home/). another issue of concern related to protein data bank is large proportion of errors in deposited coordinates, leading to requirement for a thorough checkup and development of data remediation services [319] . commonly occurring problems associated with nomenclature, poor glycan geometry, linkage errors, missing or surplus atoms can seriously decline the quality of the obtained 3d structures [300, 320, 321] . using privateer software, it was discovered [299] , [301] that pdb deposits significant number of erroneous n-glycosylated structures with pyranose ring distortions, considering preferred adoption of 4 c1 conformation for d-sugars and 1 c4 conformation for l-sugars ( figure 9 ). in most cases, poor electron density of carbohydrate moiety results in anomalous high-energy pyranose ring conformations (envelopes, half-chairs, boats, skew boats, etc.). to obtain a reasonable structure model, experimental data refinement programs should be applied to derive geometric restraints for sugar monomers. notably, despite a cryo-em method has a resolution limit protein data bank (pdb) [315] and cambridge structural database (csd) [316] are historically considered the main repositories of experimentally determined carbohydrate three-dimensional structures. csd is reported to deposit over 4000 crystal structures of oligosaccharides [93] . unlike cambridge structural database, protein data bank provides open access to the entire structural archive. carbohydrate moieties deposited in pdb are usually represented as covalently bound to protein or imply non-covalently bound protein-carbohydrate complex formation [302] . according to recent reports, as at november 18, 2019 protein data bank contained~13500 carbohydrate structures representing~9.4% of total database records [317] . despite being a valuable source of 3d structural data for glycoscientists, pdb lacks convenient search facilities for glycan structures. some projects have developed data-mining tools capable of retrieving bioglycan molecular geometry data from pdb: glycan reader (glycanstructure.org) [260, 261] (http://www.glycanstructure.org/), pdb2linucs (glycosciences.de) [47, 259, 318] (http://www. glycosciences.de/database/start.php?action=form_pdb_data), glyconavi tcarp [61] (https://glyconavi. org/tcarp/) (https://gitlab.com/glyconavi/pdb2glycan) and glyfinder (glycam-web) [257, 258] (https://dev.glycam.org/portal/gf_home/). another issue of concern related to protein data bank is large proportion of errors in deposited coordinates, leading to requirement for a thorough checkup and development of data remediation services [319] . commonly occurring problems associated with nomenclature, poor glycan geometry, linkage errors, missing or surplus atoms can seriously decline the quality of the obtained 3d structures [300, 320, 321] . using privateer software, it was discovered [299] , [301] that pdb deposits significant number of erroneous n-glycosylated structures with pyranose ring distortions, considering preferred adoption of 4 c 1 conformation for d-sugars and 1 c 4 conformation for l-sugars ( figure 9 ). in most cases, poor electron density of carbohydrate moiety results in anomalous high-energy pyranose ring conformations (envelopes, half-chairs, boats, skew boats, etc.). to obtain a reasonable structure model, experimental data refinement programs should be applied to derive geometric restraints for sugar monomers. notably, despite a cryo-em method has a resolution limit disadvantage, observed results indicate larger content of atypical conformations solved by x-ray crystallography, as compared to cryo-em data. disadvantage, observed results indicate larger content of atypical conformations solved by x-ray crystallography, as compared to cryo-em data. exceptions for the relevancy of high-energy conformations were found in complexes involving carbohydrate-active enzymes, which force pyranose ring distortion enabling catalytic transformation of a carbohydrate substrate via transition states (e.g., glycosydic bond hydrolysis) [322] . fushinobu has performed glycosidic torsion analysis for a set of pdb entries of crystal structure complexes bound to ligands bearing lacto-n-biose i (lnb, both α-and β-anomers) disaccharide unit presented in type-1 antigens. the study was supported by glycomaps db (see table 1 for references) [323] . obtained φ-ψ data for lnbs bound to various proteins was plotted against corresponding free energy maps. distortion of the energetically favored ring conformation strongly depended on substrate catalytic and recognition mechanisms. to date, existing tools for carbohydrate structural error detection and correction in pdb files (table 3 ) cannot be used directly as an integral part of protein data bank. nevertheless, initiative aimed at improvement of quality at wwpdb was carried out via collaboration with glycoscience community in july 2020 [324] (https://www.wwpdb.org/documentation/carbohydrate-remediation). it includes data annotation and validation of carbohydrate-containing records. exceptions for the relevancy of high-energy conformations were found in complexes involving carbohydrate-active enzymes, which force pyranose ring distortion enabling catalytic transformation of a carbohydrate substrate via transition states (e.g., glycosydic bond hydrolysis) [322] . fushinobu has performed glycosidic torsion analysis for a set of pdb entries of crystal structure complexes bound to ligands bearing lacto-n-biose i (lnb, both αand β-anomers) disaccharide unit presented in type-1 antigens. the study was supported by glycomaps db (see table 1 for references) [323] . obtained ϕ-ψ data for lnbs bound to various proteins was plotted against corresponding free energy maps. distortion of the energetically favored ring conformation strongly depended on substrate catalytic and recognition mechanisms. to date, existing tools for carbohydrate structural error detection and correction in pdb files (table 3 ) cannot be used directly as an integral part of protein data bank. nevertheless, initiative aimed at improvement of quality at wwpdb was carried out via collaboration with glycoscience community in july 2020 [324] (https://www.wwpdb.org/documentation/carbohydrate-remediation). it includes data annotation and validation of carbohydrate-containing records. proportion of carbohydrate-containing structures in pdb has been recently reported in [302] . figure 10 presents our analysis of data published in the framework of protein data bank carbohydrate remediation project. 14117 pdb entries from carbohydrate remediation list (https://cdn.rcsb.org/ wwpdb/docs/documentation/carbohydrateremediation/pdb_carbohydrate_list.list) were sorted by release year and plotted against the growth of pdb structures released annually (https://www.rcsb.org/ stats/growth/growth-released-structures) (as on august 10, 2020; 167,327 pdb entries were available). obtained results indicated that~8.4% of pdb records contained a carbohydrate moiety. additionally, each pdbx/mmcif file corresponding to pdb id from carbohydrate remediation list was parsed to reveal the presence of n-or o-glycosylation site annotations, which resulted in~4.2% (7076 n-glycosylated entries) and 0.2% (362 o-glycosylated entries) of total database records. a few s-and c-glycans (24 entries in total) were neglected. statistics on glycans in protein data bank was reported [259, 302, 317, 325] , as well as tools that could facilitate collection of statistical data (glycan reader [70, 260, 261] , glyfinder [258] , pdb2linucs and pdb-care [326] proportion of carbohydrate-containing structures in pdb has been recently reported in [302] . figure 10 presents our analysis of data published in the framework of protein data bank carbohydrate remediation project. 14117 pdb entries from carbohydrate remediation list (https://cdn.rcsb.org/wwpdb/docs/documentation/carbohydrateremediation/pdb_carbohydrate_lis t.list) were sorted by release year and plotted against the growth of pdb structures released annually (https://www.rcsb.org/stats/growth/growth-released-structures) (as on august 10, 2020; 167,327 pdb entries were available). obtained results indicated that ~8.4% of pdb records contained a carbohydrate moiety. additionally, each pdbx/mmcif file corresponding to pdb id from carbohydrate remediation list was parsed to reveal the presence of n-or o-glycosylation site annotations, which resulted in ~4.2% (7076 n-glycosylated entries) and 0.2% (362 o-glycosylated entries) of total database records. a few s-and c-glycans (24 entries in total) were neglected. statistics on glycans in protein data bank was reported [259, 302, 317, 325] , as well as tools that could facilitate collection of statistical data (glycan reader [70, 260, 261] , glyfinder [258] , pdb2linucs and pdb-care [326] ). carbohydrate structure visualization in publications and computer interfaces is extremely important in terms of perception universality, unambiguity, and machine-readability. hence, carbohydrate input [335] [336] [337] and visualization [338, 339] tools are actively developing. feature comparison of glycan sketchers, builders and viewers (occasionally including 3d ones) was reported in a recently published review [340] . in our review, we gave more emphasis to 3d visualization approaches. being informative to represent glycan primary structure, most of graphical input tools such as glycanbuilder [341] , drawrings [342] , sugarsketcher [343] , drawglycan-snfg [344, 345] and glycoglyph [337] are inappropriate for obtaining 3d structural models and their visualization due to lack of underlying modeling and insufficient data conversion functionality. at present, glycan 3d molecular models can be built in user-friendly software allowing constructing glycans from individual saccharide components. free web-tools, such as glycam-web, charmm-gui, polys glycan builder, gag-builder, sweet-ii should be noted (more references are listed in table 2 ). a few commercial molecular modeling software is equipped with special plugins for glycan 3d structure building based on a list of predefined monosaccharide templates, e.g., sugar builder tool in hyperchem (http://www.hyper.com/?tabid=360) software [346] or azahar [235] plugin in pymol package (schrödinger software) (https://pymol.org/2/) [347] . to render 3d glycan structure and its conformational features, it should be recorded using a notation which includes atomic coordinates, such as mol [348] or pdb [349] . all-atom visualization based on atomic coordinates is supported by the majority of existing molecular modeling software. several carbohydrate structure databases utilize interactive 3d visualization using open-source software engines. as one of the pioneers, glycosciences.de portal developed pdb2multigif [350] (http: //www.glycosciences.de/modeling/pdb2mgif/) visualization pipeline which generates an animated image of 3d model from a pdb file using rasmol [351] (http://www.openrasmol.org/). rasmol visualization was included in w3-sweet [263] (ancestor of sweet-ii) pipeline developed by same project. nowadays, more advanced interactive visualization applications have been developed for carbohydrate 3d molecule presentation. jmol/jsmol [352] (http://www.openrasmol.org/) visualization applet is useful to display 3d models of carbohydrate molecules applied in numerous projects, such as csdb, glycosciences.de, glycam-web and ek3d (see references in table 1 ). ngl [353, 354] (http://nglviewer.org/), litemol [355] (https://www.litemol.org/) and mol* [356] (https://www.rcsb.org/ news?year=2020&article=5efe0f606378d876901146f8) (https://molstar.org/) 3d viewers are handy for processing macromolecular pdb data stored in glycoproteomics databases (unilectin3d, glycan binding site db, procarbdb, glyconavi, procaff, etc.; see references in table 1 ) and general proteomics repositories such as pdb [315] (http://www.wwpdb.org/), uniprotkb [357] (https://www.uniprot.org/) or swiss-model [90] (https://swissmodel.expasy.org/repository). ngl viewer was developed mainly for convenient protein macromolecule structure processing. it allows only ball-stick representation for small molecules or non-peptide fragments, such as saccharide residues. litemol (and its successor, mol*) viewer could be applied for the visualization of an arbitrary glycan with facility of highlighting carbohydrate fragments or displaying specific interactions in protein-carbohydrate complex structure. due to these features, it was implemented in multiple carbohydrate structure databases (e.g., csdb, glyco3d, matrixdb, and eps-db). despite the absence of the experimental 3d structural data, a number of carbohydrate databases have opportunity to simulate 3d atomic coordinates for deposited or inputted compounds from primary structure owing to tools developed by glycoinformatics community. csdb (restless api [265] ), glycosciences.de (sweet-ii [264, 350] ) and glycam-web (http://glycam.org/) portals make it possible to generate 3d atomic coordinates recorded in pdb (all) and mol (csdb) file formats. polys developed by glyco3d project enables the construction of polysaccharides in pdb format; it was introduced in matrixdb and eps-db databases. more details are provided in table 2 . atomic coordinates and all-atom molecular models have not been popular in publications due to a lack of human readability. first attempts [358, 359] of prof. kuttel et al., to visualize carbohydrate molecules in an efficient and simple way were made by developing paperchain and twister graphic algorithms as a part of carbohydra [360] and visual molecular dynamics [361] software packages. later, group of prof. pé rez suggested to restrict visualized molecule to skeletal atoms via conditional cycle plane coloring in accordance with the color code adopted in snfg [338] visualization scheme (sweetunitymol software [362] , figure 11a ). another unitymol visualization approach called umbrella visualization [363, 364] was tailored for n-glycan structures. azahar plugin for pymol [235] affords cartoon models with polygons and rods. several solutions for convenient visualization came up with the development of snfg notation [339] . thus, group of prof. woods proposed to combine molecular structure elements with 3d snfg icons (figure 12a ). such convenient visualization technique was integrated in litemol (figure 12b ) [365] and mol* (figure 12c) [324, 356] . 3d snfg visualization plugins are available via visual molecular dynamics platform [366] (http://glycam.org/docs/othertoolsservice/ 2016/06/03/3d-symbol-nomenclature-for-glycans-3d-snfg/) and ucsf chimera [367] visualization software tangram plugin (https://github.com/insilichem/tangram_snfg). designed as part of ccp4mg [368] molecular-graphics software, glycoblocks [369] representation of monosacchrides uses shapes and colors, identical to those in snfg (figure 12d ). available as pymol plugin developed by widmalm group (http://www.organ.su.se/gw/doku.php?id=3dcfg), 3d-cfg representation [370] based on cfg notation [371] (often referred to as a predecessor of snfg) should also be noted as earlier approach to interpretation of carbohydrate 3d structures based on a symbol library. considering efficiency and usability of 3d representation based on snfg concept, which grows popular among glycoscientists, the development of alternative solutions in carbohydrate 3d structure representations has a potential for application in glycoinformatics projects. support of colored residues in 3d structures implemented via jsmol on glycosciences.de portal was reported [47] (figure 11b) . similarly, csdb project has developed a 3d viewer (http://csdb.glycoscience.ru/database/core/show_ 3d.php?csdb=-3)admanp(1-3)[ac(1-2)?dglcpn(1-6)]bdgal?(1-) with carbohydrate residue coloring according to the snfg notation in the framework of a modeling module based on restless api. in this tool, user can visualize input structure with help of sticks, balls and sticks, or van der waals spheres (figure 11c ). options for aglycone moiety (white) and pseudo-atoms (polymeric repeats, blue caps) are supported (figure 11d ). int. j. mol. sci. 2020, 21, x for peer review 28 of 48 development of glycoinformatics resources makes great impact on treating enormous masses of data sets produced by glyco-related research. tools for carbohydrate 3d structural information retrieval provide a framework for experimental and computational data quality validation. data sources based on conformational ensemble generation and analysis assist structure-function and structure-activity relationship prediction of biologically relevant bioglycans and glycoconjugates. in this review, we have summarized existing facilities on working with glycan spatial features that can provide harmonious network of structural databases, web-services, tools and standalone software development of glycoinformatics resources makes great impact on treating enormous masses of data sets produced by glyco-related research. tools for carbohydrate 3d structural information retrieval provide a framework for experimental and computational data quality validation. data sources based on conformational ensemble generation and analysis assist structure-function and structure-activity relationship prediction of biologically relevant bioglycans and glycoconjugates. in this review, we have summarized existing facilities on working with glycan spatial features that can provide harmonious network of structural databases, web-services, tools and standalone software for modeling and processing structural data. further advances in this field will help building better understanding of glycan participation in biological processes and supply glycoscience community with user-friendly access to voluminous data collections. funding: the work with carbohydrate molecular modeling and pdb data was funded by russian foundation for basic research grant 18-04-00094. the work with structural databases, glycoinformatic tools and visualization was funded by russian science foundation grant 18-14-00098. the authors declare no conflict of interest. three structural aspects of carbohydrates and the relation with their biological 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predicting oligosaccharide conformational ensembles within glycoproteins an improved opls-aa force field for carbohydrates opls all-atom force field for carbohydrates development and testing of the opls all-atom force field on conformational energetics and properties of organic liquids optimizing nonbonded interactions of the opls force field for aqueous solutions of carbohydrates: how to capture both thermodynamics and dynamics polarizable empirical force field for hexopyranose monosaccharides based on the classical drude oscillator proper balance of solvent-solute and solute-solute interactions in the treatment of the diffusion of glucose using the drude polarizable force field charmm drude polarizable force field for aldopentofuranoses and methyl-aldopentofuranosides drude polarizable force field parametrization of carboxylate and n-acetyl amine carbohydrate derivatives polarizable empirical force field for acyclic polyalcohols based on the classical drude oscillator charmm drude polarizable force field for glycosidic linkages involving pyranoses and furanoses martini coarse-grained force field: extension to carbohydrates overcoming the limitations of the martini force field in simulations of polysaccharides extending the martini coarse-grained forcefield to n-glycans martini force field parameters for glycolipids pitomba: parameter interface for oligosaccharide molecules based on atoms modelling of carbohydrate-aromatic interactions: ab initio energeticsand force field performance stacking interactions between carbohydrate and protein quantified by combination of theoretical and experimental methods applying pose clustering and md simulations to eliminate false positives in molecular docking scoring functions and their evaluation methods for protein-ligand docking: recent advances and future directions aromatic-carbohydrate interactions: an nmr and computational study of model systems a gibbs free energy correlation for automated docking of carbohydrates protein-carbohydrate interactions docking glycosaminoglycans to proteins: analysis of solvent inclusion flexibility and explicit solvent in molecular-dynamics-based docking of protein-glycosaminoglycan systems mannobiose binding induces changes in hydrogen bonding and protonation states of acidic residues in concanavalin a as revealed by neutron crystallography improvements, trends, and new ideas in molecular docking: 2012-2013 in review recognition of selected monosaccharides by pseudomonas aeruginosa lectin ii analyzed by molecular dynamics and free energy calculations in silico mutagenesis and docking study of ralstonia solanacearum rsl lectin: performance of docking software to predict saccharide binding finding a needle in a haystack: development of a combinatorial virtual screening approach for identifying high specificity heparin/heparan sulfate sequence(s) computational analysis of interactions in structurally available protein-glycosaminoglycan complexes identification and characterization of a glycosaminoglycan binding site on interleukin-10 via molecular simulation methods a. in silico analysis of antibody-carbohydrate interactions and its application to xenoreactive antibodies charmm-gui input generator for namd, gromacs, amber, openmm, and charmm/openmm simulations using the charmm36 additive force field charmm-gui supports the amber force fields residue-centric modeling and design of saccharide and glycoconjugate structures efficient construction of atomic-resolution models of non-sulfated chondroitin glycosaminoglycan using molecular dynamics data charmm-gui glycan modeler for modeling and simulation of carbohydrates and glycoconjugates glycosylator: a python framework for the rapid modeling of glycans the rosetta all-atom energy function for macromolecular modeling and design novel sampling strategies and a coarse-grained score function for docking homomers, flexible heteromers, and oligosaccharides using rosetta in capri rounds 37-45 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screening of the human tf-glycome provides a structural definition for the specificity of anti-tumor antibody jaa-f11 presentation, presentation, presentation! molecular-level insight into linker effects on glycan array screening data recent advances in employing molecular modelling to determine the specificity of glycan-binding proteins gly-spec: a webtool for predicting glycan specificity by integrating glycan array screening data and 3d structure combining 3d structure with glycan array data provides insight into the origin of glycan specificity automated builder and database of protein/membrane complexes for molecular dynamics simulations membrane builder for mixed bilayers and its application to yeast membranes charmm-gui membrane builder toward realistic biological membrane simulations modeling and simulation of bacterial outer membranes with lipopolysaccharides and enterobacterial common antigen the gram-negative outer membrane modeler: automated building of lipopolysaccharide-rich bacterial outer membranes in four force fields maker: an online tool for generating equilibrated micelles as direct input for molecular dynamics simulations probabilistic identification of saccharide moieties in biomolecules and their protein complexes new online tools for locating and curating carbohydrate structures in wwpdb tools to find glycoproteins in the protein data bank and generate realistic 3d structures for them data mining the protein data bank: automatic detection and assignment of carbohydrate structures glycan reader: automated sugar identification and simulation preparation for carbohydrates and glycoproteins glycan reader is improved to recognize most sugar types and chemical modifications in the protein data bank vattulainen, i. doglycans-tools for preparing carbohydrate structures for atomistic simulations of glycoproteins, glycolipids, and carbohydrate polymers for gromacs sweet-www-based rapid 3d construction of oligo-and polysaccharides automated translation of glycan sequences from residue-based notation to smiles and atomic coordinates a molecular builder for carbohydrates: application to polysaccharides and complex carbohydrates 0: an open source software package for building three-dimensional structures of polysaccharides an adjustable tool for building 3d molecular structures of carbohydrates for molecular simulation software for building molecular models of complex oligoand polysaccharide structures a pipeline to translate glycosaminoglycan sequences into 3d models. application to the exploration of glycosaminoglycan conformational space a web-tool for modeling 3d structures of glycosaminoglycans slick-scoring and energy functions for protein−carbohydrate interactions balldock/slick: a new method for protein-carbohydrate docking the haddock2.2 web server: user-friendly integrative modeling of biomolecular complexes docking server for the identification of heparin binding sites on proteins so you think computational approaches to understanding glycosaminoglycan-protein interactions are too dry and too rigid? think again! predicting glycosaminoglycan surface protein interactions and implications for studying axonal growth improved docking of sulfated sugars using qm-derived scoring functions conformational analysis of oligosaccharides and polysaccharides using molecular dynamics simulations a method for discretizing and visualizing pyranose conformations direct noe simulation from long md trajectories gs-align for glycan structure alignment and similarity measurement analysis of carbohydrate 3d structures derived from the pdb statistical analysis of amino acids in the vicinity of carbohydrate residues performed by glyvicinity rules of engagement of protein-glycoconjugate interactions: a molecular view achievable by using nmr spectroscopy and molecular modeling conformational studies of oligosaccharides structure, conformation, and dynamics of bioactive oligosaccharides: theoretical approaches and experimental validations conformational studies of oligosaccharides and glycopeptides: complementarity of nmr, x-ray crystallography, and molecular modelling analysis and validation of carbohydrate three-dimensional structures an efficient use of x-ray information, homology modeling, molecular dynamics and knowledge-based docking techniques to predict protein-monosaccharide complexes chapter 3 developments in the karplus equation as they relate to the nmr coupling constants of carbohydrates a perspective on the primary and three-dimensional structures of carbohydrates nmr structure determination of a segmentally labeled glycoprotein using in vitro glycosylation nmr structural biology of sulfated glycans making glycoproteins a little bit sweeter with pdb-redo automatically fixing errors in glycoprotein structures with rosetta leveraging glycomics data in glycoprotein 3d structure validation with privateer current developments in coot for macromolecular model building of electron cryo-microscopy and crystallographic data carbohydrate anomalies in the pdb numerous severely twisted n-acetylglucosamine conformations found in the protein databank structural glycobiology in the age of electron cryo-microscopy strategies for carbohydrate model building, refinement and validation structures of ebola virus gp and sgp in complex with therapeutic antibodies cryo-em structure of a native, fully glycosylated, cleaved hiv-1 envelope trimer cross-neutralization of sars-cov-2 by a human monoclonal sars-cov antibody structure, function, and antigenicity of the sars-cov-2 spike glycoprotein nmr spectroscopy in the study of carbohydrates: characterizing the structural complexity recent advances in the application of nmr methods to uncover the conformation and recognition features of glycans. in carbohydrate chemistry the recognition of glycans by protein receptors. insights from nmr spectroscopy novel nmr avenues to explore the conformation and interactions of glycans delineating the conformational flexibility of trisaccharides from nmr spectroscopy experiments and computer simulations conformational flexibility of the pentasaccharide lnf-2 deduced from nmr spectroscopy and molecular dynamics simulations molecular conformations of di-, tri-, and tetra-α-(2→8)-linked sialic acid from nmr spectroscopy and md simulations an unusual carbohydrate conformation is evident in moraxella catarrhalis oligosaccharides protein data bank (pdb): the single global macromolecular structure archive the cambridge structural database current status of carbohydrates information in the protein data bank data mining the pdb for glyco-related data carbohydrate structure: the rocky road to automation building meaningful models of glycoproteins carbohydrate 3d structure validation dissecting conformational contributions to glycosidase catalysis and inhibition conformations of the type-1 lacto-n-biose i unit in protein complex structures collaborating with glycoscience community to improve data representation of carbohydrates in the protein data bank building and rebuilding n-glycans in protein structure models pdb-care (pdb carbohydrate residue check): a program to support annotation of complex carbohydrate structures in pdb files crystallography & nmr system: a new software suite for macromolecular structure determination version 1.2 of the crystallography and nmr system tools to assist determination and validation of carbohydrate 3d structure data compatible topologies and parameters for nmr structure determination of carbohydrates by simulated annealing structural analysis of glycoproteins: building n-linked glycans with coot software for the conformational validation of carbohydrate structures motivevalidator: interactive web-based validation of ligand and residue structure in biomolecular complexes database of up-to-date validation results for ligands and non-standard residues from the protein data bank a practical guide to using glycomics databases implementation of glycanbuilder to draw a wide variety of ambiguous glycans a glycan visualizing, drawing and naming application symbol nomenclature for graphical representations of glycans updates to the symbol nomenclature for glycans (snfg) guidelines computational tools for drawing, building and displaying carbohydrates: a visual guide the glycanbuilder and glycoworkbench glycoinformatics tools: updates and new developments the rings resource for glycome informatics analysis and data mining on the web sugarsketcher: quick and intuitive online glycan drawing drawglycan-snfg: a robust tool to render glycans and glycopeptides with fragmentation information drawglycan-snfg and gpannotate: rendering glycans and annotating glycopeptide mass spectra the pymol molecular graphics system description of several chemical structure file formats used by computer programs developed at molecular design limited protein data bank contents guide: atomic coordinate entry format description. brookhaven natl. lab a web tool to create animated images of molecules biomolecular graphics for all fast and scriptable molecular graphics in web browsers without java3d. nat. prec a web application for molecular visualization ngl viewer: web-based molecular graphics for large complexes litemol suite: interactive web-based visualization of large-scale macromolecular structure data mol: towards a common library and tools for web molecular graphics uniprot: a worldwide hub of protein knowledge eborn, i. techniques for visualization of carbohydrate molecules visualisation of cyclic and multi-branched molecules with vmd rendering carbohydrate cartoons vmd: visual molecular dynamics three-dimensional representations of complex carbohydrates and polysaccharides-sweetunitymol: a video game-based computer graphic software new visualization of dynamical flexibility of n-glycans: umbrella visualization in unitymol umbrella visualization: a method of analysis dedicated to glycan flexibility with unitymol rapidly display glycan symbols in 3d structures: 3d-snfg in litemol 3d implementation of the symbol nomenclature for graphical representation of glycans ucsf chimera-a visualization system for exploratory research and analysis presenting your structures: the ccp4mg molecular-graphics software glycoblocks: a schematic three-dimensional representation for glycans and their interactions glycan synthesis, structure, and dynamics: a selection symbol nomenclature for glycan representation development of carbohydrate nomenclature and representation. in a practical guide to using glycomics databases key: cord-266147-s8rxzm0t authors: burnouf, thierry title: modern plasma fractionation date: 2007-03-28 journal: transfus med rev doi: 10.1016/j.tmrv.2006.11.001 sha: doc_id: 266147 cord_uid: s8rxzm0t protein products fractionated from human plasma are an essential class of therapeutics used, often as the only available option, in the prevention, management, and treatment of life-threatening conditions resulting from trauma, congenital deficiencies, immunologic disorders, or infections. modern plasma product production technology remains largely based on the ethanol fractionation process, but much has evolved in the last few years to improve product purity, to enhance the recovery of immunoglobulin g, and to isolate new plasma proteins, such as α1-protease inhibitor, von willebrand factor, and protein c. because of the human origin of the starting material and the pooling of 10 000 to 50 000 donations required for industrial processing, the major risk associated to plasma products is the transmission of blood-borne infectious agents. a complete set of measures—and, most particularly, the use of dedicated viral inactivation and removal treatments—has been implemented throughout the production chain of fractionated plasma products over the last 20 years to ensure optimal safety, in particular, and not exclusively, against hiv, hepatitis b virus, and hepatitis c virus. in this review, we summarize the practices of the modern plasma fractionation industry from the collection of the raw plasma material to the industrial manufacture of fractionated products. we describe the quality requirements of plasma for fractionation and the various treatments applied for the inactivation and removal of blood-borne infectious agents and provide examples of methods used for the purification of the various classes of plasma protein therapies. we also highlight aspects of the good manufacturing practices and the regulatory environment that govern the whole chain of production. in a regulated and professional environment, fractionated plasma products manufactured by modern processes are certainly among the lowest-risk therapeutic biological products in use today. protein products fractionated from human plasma are an essential class of therapeutics used, often as the only available option, in the prevention, management, and treatment of life-threatening conditions resulting from trauma, congenital deficiencies, immunologic disorders, or infections. modern plasma product production technology remains largely based on the ethanol fractionation process, but much has evolved in the last few years to improve product purity, to enhance the recovery of immunoglobulin g, and to isolate new plasma proteins, such as a1protease inhibitor, von willebrand factor, and protein c. because of the human origin of the starting material and the pooling of 10 000 to 50 000 donations required for industrial processing, the major risk associated to plasma products is the transmission of blood-borne infectious agents. a complete set of measures-and, most particularly, the use of dedicated viral inactivation and removal treatments-has been implemented throughout the production chain of fractionated plasma products over the last 20 years to ensure optimal safety, in particular, and not exclusively, against hiv, hepatitis b virus, and hepatitis c virus. in this review, we summarize the practices of the modern plasma fractionation industry from the collection of the raw plasma material to the industrial manufacture of fractionated products. we describe the quality requirements of plasma for fractionation and the various treatments applied for the inactivation and removal of blood-borne infectious agents and provide examples of methods used for the purification of the various classes of plasma protein therapies. we also highlight aspects of the good manufacturing practices and the regulatory environment that govern the whole chain of production. in a regulated and professional environment, fractionated plasma products manufactured by modern processes are certainly among the lowest-risk therapeutic biological products in use today. a 2007 elsevier inc. all rights reserved. c ollected human plasma may be used as a therapeutic product (known as bclinical plasmaq or bfresh frozen plasmaq) or as source material for the production of pharmaceutical fractionated products (also called bplasma productsq or bplasma derivativesq). this complex biologic material contains hundreds of proteins covering a myriad of physiological functions. many components still have undiscovered roles. the most abundant proteins, albumin and immunoglobulin (ig) g, are present at about 35 and 10 g/l, respectively, representing about 80% of all plasma proteins. less abundant proteins include the protease inhibitors, like a1-antitrypsin (aat) (1.5 g/l) and antithrombin (at) (300 mg/l), and the coagulation factors such as factor viii (fviii) (a few ng/l), which exhibit potent physiologic activity. currently, about 20 different plasma protein therapeutics are used for treating life-threatening diseases or injuries associated to bleeding and thrombotic disorders, immunological diseases, infectious conditions, as well as tissue degenerating diseases, thus addressing the clinical needs of countless patients. an updated list of the major therapeutic applications of plasma protein products can be found elsewhere. 1 this industrial process used to isolate therapeutic plasma proteins is known as bfractionation.q over 23 to 28 million liters of human plasma are fractionated each year in the world, in batches of several thousand liters, in about 70 factories. modern plasma fractionation combines manufacturing steps to isolate, in a sequential and integrated manner, the crude fractions that are further purified into individual therapeutic products. validated dedicated steps inactivate and/or remove infectious agents potentially present in the starting plasma pool. this sophisticated industrial process is performed under highly hygienic conditions in licensed facilities (plasma fractionation plants) that are operated in compliance with good manufacturing practices and following quality assurance principles. over the years, plasma fractionation has evolved from a medical service activity mostly oriented toward the needs of local communities into a global manufacturing industry conforming to high regulatory standards. these strict requirements start from the collection of plasma for fractionation and include product manufacture and distribution steps. in this article, we review the most current practices encompassing the collection of plasma for fractionation, the core industrial plasma fractionation process, and the purification and pathogen reduction technologies of individual plasma products. the practices used for the collection of plasma for fractionation have direct influence on the safety profile of protein products since individual donations contribute to large plasma pools used to manufacture therapeutic preparations intended for hundreds or even thousands of patients. it is therefore logical that the production of plasma is regarded as an integral part of the manufacture of modern fractionated products. collection requirements of plasma for fractionation may differ from those relevant to fresh frozen plasma. in a regulated environment, plasma for fractionation is collected by licensed/registered blood establishments (blood centers and apheresis collection centers) that are inspected by the relevant national regulatory authorities (nras). compelled by the same safety concerns, the plasma fractionators conduct audits to verify that the contractual plasma collection and quality and safety measures, agreed upon with the plasma supplier, are met. areas of specific relevance include (a) procedures for donor screening and donation testing; (b) labeling, documentation, and traceability requirements; and (c) the handling of blood and plasma. such information is part of the marketing license of plasma products and, in europe, is assembled into a document called the bplasma master file.q various requirements for the collection of plasma for fractionation have been described in various guides, eg, from the pharmaceutical inspection convention and pharmaceutical inspection cooperation scheme (jointly referred to as pic/s), us food and drug administration (fda) and in recent world health organization recommendations. 1 they are summarized below. candidate donors are provided with educational materials and undergo a medical interview to establish the absence of risks or signs of infections and to prove compliance for a donation of plasma for fractionation (table 1) . potential donors presenting a health hazard are asked to exclude themselves. medical information of donors is acquired and archived. continuous epidemiologic surveillance of the donor population is being required in some jurisdictions. 2 it helps to establish the background level (prevalence and incidence) and trends of known infectious markers (eg, hiv 1 and 2 antibodies, hepatitis c virus [hcv] antibodies, and hepatitis b surface antigen [hbsag]) in the population. this is also of interest for the early detection of emerging diseases, allowing early implementation of counter measures (such as more stringent donor screening processes or requirements for additional testing procedures). donors eligible to donate plasma for fractionation are individuals who meet donation criteria (such as age and donation frequency), do not present risk factors of blood-born infectious agents, and comply with requirements defined by the plasma fractionator and the nras of the country of plasma collection and of use of the products. in most situations, eligibility of whole blood donors and apheresis donors overlap, apart from donation frequency which is higher for plasmapheresis donors. eligibility criteria take into account scientific information about the risks of transmission of infectious agents by pooled plasma products (which may differ from those by blood components). special criteria may exist for the collection of hyperimmune plasma (used to make hyperimmune igg preparations), such as procedures for donors' immunization and minimal antibody titer. 1 currently, about 35% of the plasma fractionated in the world is obtained by centrifugation of whole blood (brecoveredq plasma), and 65% is obtained by apheresis. blood/plasma collection, processing, and storage may affect plasma quality, as well as having an impact on the recovery of the most labile proteins such as fviii. in particular, risks of activation of the coagulation, complement, and fibrinolytic systems, which may lead to generation of plasma proteases, should be avoided. to better preserve the integrity of recovered plasma and limit risks of activation of the coagulation cascade and of cellular components, (a) good mixing of the blood with the anticoagulant solution (a sodium citrate based solution 1 ) should be ensured from the initiation till the end of the collection process; (b) the duration of the collection should not exceed 15 minutes; and (c) temperature variations of the blood should be avoided. a few hours after donation, whole blood is subjected to a centrifugation that separates the cellular elements (most specifically red cells) from plasma. the mean plasma volume obtained from one whole blood donation is about 220 ml but varies depending upon the volume of collected whole blood (most often 400-450 ml) and donor's hematocrit. apheresis plasma (also called bsource plasmaq) is collected from donors through a process where blood is removed from the donor, anticoagulated (generally with a 4% sodium citrate solution), 1 and immediately separated by physical means (centrifugation or filtration, or a combination of both) into components. 3 at minimum, the red cells are returned to the donor while plasma is retained and collected in a container (bag or plastic bottle). the duration of a typical plasmapheresis procedure depends on the number of cycles (and, hence, the volume of plasma collected) and lasts generally from 35 to 70 minutes. apheresis plasma volume may range from 450 to 880 ml, depending upon the country's regulations and collection protocol. apheresis plasma can also be prepared as a byproduct of plateletpheresis (bconcurrent plasmaq), a procedure used primarily for the collection of platelets. both recovered and apheresis plasmas are suitable for the manufacture of the whole range of fractionated plasma products. the mean content in coagulation factors, more particularly fviii, is lower in recovered than in apheresis plasma because of (a) longer processing time before freezing (whole blood must be further processed to separate cellular components and plasma), (b) higher ratio of anticoagulant and, possibly, (c) the higher level of cellular contamination that may release proteolytic enzymes affecting the stability of coagulation factors. apheresis plasma contains less igg when collected from frequent donors. protein content and quality of fractionated proteins is apparently not affected by the apheresis system used, although residual cell content differ based upon the type and configuration of the cell separation device. 4 plasma from membrane apheresis procedures, as does recovered plasma prepared from whole blood leukoreduced on positively charged filters, may 4performed by most fractionators. ymandatory in europe for hcv. zmay contribute to viral clearance but does not necessarily result in robust and consistent removal. §for small viruses, robust removal is achieved by narrow pore size membranes (v20 nm). texpected contribution based on experimental studies using spiked tse agents, in the absence of information of the biological nature of the tse-human plasma associated agent. contain more activated complement component 3 and 5 (c3a, c5a) anaphylatoxins, 5-7 but the impact on the quality or yield of fractionated products is unknown. various infectious agents have been identified as potential contaminants of human blood. 8 bacteria, parasites, and intracellular viruses are not transmitted by plasma products because they are destroyed by freeze-thaw steps or removed by the 0.2-to 1-lm filtration steps used during the processing of fractionated products. pathogenic plasma-borne viruses include hiv, hcv, hepatitis b virus (hbv), west nile virus (wnv), hepatitis a virus (hav) and parvovirus b19 (b19). the various complementary safety nets in place during the production chain of fractionated products, from donor selection to industrial product extraction, to optimize safety against these agents are summarized in table 1 . the importance of viral testing on the safety of plasma products has been reviewed. 1, 9, 10 the extent of viral testing of plasma for fractionation takes into account the ability of validated fractionation processes to eliminate viral risks. 11 some testing is performed by blood establishments, other by plasma fractionators (table 1) . individual plasma donations must be negative for anti-hiv 1 and 2, anti-hcv, and hbsag. genomic assays of plasma minipools for nonenveloped hav and b19 may be performed. 11,12 relevance of testing for the absence of hiv p24ag or wnv nucleic acid testing (nat), which may be justified for the safety of non-virally inactivated blood components, is arguable for plasma for fractionation subjected to robust viral reduction steps of enveloped viruses. the industrial manufacturing pool (usually the cryo-poor plasma that is the first homogeneous pooled plasma fraction) is also tested to confirm the absence of serologic and/or genomic viral markers of hiv, hbv, hcv, hav, and b19. in spite of the most rigorous donor screening and donation testing, infectious viruses may still be present in plasma fractionation pools. therefore, the viral inactivation-removal steps that have been deliberately introduced during plasma products manufacture-and that are described below-play a most critical role in ensuring safety. altogether, these overlapping tests should ensure that the viral load of the manufacturing should is both minimal and significantly below the viral reduction capacity of the manufacturing processes used. preserving fviii during blood/plasma collection and preparation is important for most fractionators preparing coagulation factor concentrates. apheresis plasma can generally be frozen quickly, ensuring optimal fviii preservation. by contrast, whole blood has to be centrifuged to separate the various components. when blood is cooled to 48c after collection, plasma should be separated and frozen within 6 to 8 hours to preserve fviii, but when cooled rapidly at constant 208c using devices like butanediol plates, coagulation factors are stable for up to 18 to 24 hours. plasma frozen within 72 hours is suitable for igg and albumin production. 1 after separation from cellular elements, plasma for fractionation should be frozen rapidly below à208c, à258c, or à308c, depending upon local regulations. 13 plasma used to manufacture only albumin and igg may be frozen below à20 8c within 72 hours of collection. 13 in the us code of federal regulations, apheresis plasma should be stored at à208c or colder immediately after collection. rapid plasma freezing, to ensure rapid ice front velocity and core temperature of à208c, preserves fviii and appears more important than the actual freezing temperature itself. 14 plasma for fractionation is stored at less than à20 8c, or colder, typically for several months or more. storage temperature should be as constant as possible, including the transportation to the fractionation facilities. cross-continent or intercontinent shipment of plasma for fractionation is frequent. production steps taking place at fractionation plants are summarized in figure 1 , and typical plasma protein downstream methods are presented in table 2 . physical compliance with shipping requirements is verified at delivery of the plasma frozen plasmas are expelled from the plastic containers and pooled for cryoprecipitation and further manufacturing steps, as described below. intermediate fractions generated during production may be stored for subsequent pooling and processing. purified sterile-filtered products are aseptically dispensed into final containers (glass vials or bottles). albumin bottles undergo terminal pasteurization. many products, but albumin and some igg preparations, are freeze-dried, typically for a duration of 3 to 6 days, depending upon physicochemical characteristics and filled volumes. batches are quarantined while quality controls and checks of production files take place. batches meeting specifications are labeled and packaged and subsequently boxed and shipped for distribution. in a few countries, product batches may be released by regulatory authorities. the production cycle of fractionated products takes a few weeks to several months. current core fractionation technology largely relies on a backbone process encompassing cryoprecipitation and cold ethanol precipitation steps, as developed in the 1940s by cohn et al 15 in the united states, or modified by kistler and nitschman 16 in europe. this process involves successive processing steps at defined ethanol concentrations, associated with shifts in ph, temperature, and osmolality that result in selective precipitation of proteins, most notably igg and albumin. precipitates are separated by centrifugation or filtration. in the last few years, the complexity of the fractionation process has increased by (a) the introduction of chromatography to isolate new proteins from existing fractions such as cryoprecipitate, cryo-poor plasma, and cohn fractions; (b) the integration of chromatography to the ethanol fractionation process to increase igg recovery; and (c) the implementation of dedicated viral inactivation or removal steps. chromatography was introduced in the 1960s; however, its application developed mostly in the mid/late 1980s. anion-exchange chromatography and affinity chromatography are frequently used to capture proteins at physiological ph and ionic strength, therefore best preserving functional activity. 17, 18 immobilized heparin and monoclonal antibodies are common affinity chromatography ligands. chromatography is used for 4 specific goals: (a) improvement of products purity, (b) extraction of trace labile proteins, (c) optimization of protein recovery, and (d) removal of viral inactivation agents. figure 2 illustrates a typical industrial fractionation scheme of standard plasma. plasma packs (typically for a batch of 2000-4000 l) are opened under hygienic conditions, and plasma is expelled from the containers and thawed at 18c to 48c. cryoprecipitate is isolated using refrigerated continuous centrifuges, recovered from the centrifugation bowls and frozen at à308c or colder for storage until further pooling and processing. the cryo-poor plasma is immediately processed for primary chromatographic capture of labile coagulation factors (such as the factor ix [fix] complex and its components) and protease inhibitors (such as at and c1 esterase inhibitor [c1-inh]). the prepurified intermediates may be stored frozen until further processing. the coagulation factors/ anticoagulant-depleted plasma undergoes sequential ethanol precipitation steps. this leads to successive precipitations of fibrinogen, igg and albumin fractions, and intermediates for extraction of other therapeutic proteins, such as aat (fraction iv-1), or igm (fraction iii). depth filtration is preferred to centrifugation to separate precipitates and improve protein recovery. the fractionation of hyperimmune plasma (eg, anti-rhesus) is usually performed on small plasma batch sizes, increas ingly using full chromatographic processes to optimize the recovery of igg. no documented transmission of hiv, hbv, or hcv by products subjected to dedicated viral inactivation treatments has been recorded since the end of the 1980s. 19 viral reduction treatments include inactivation steps (where viruses are bkilledq) and removal steps (where viruses and proteins partition into distinct fractions). use of one, or preferably two, distinct dedicated viral reduction treatments is the current bgold standardq for all plasma products. the first treatment is performed primarily to inactivate the most pathogenic viruses (hiv, hbv, and hcv), whereas the second reduction step targets nonenveloped viruses but also contributes to added safety against all agents. most viral reduction treatments are integrated with the protein fractionation process (bin-processq treatments), but some currently based on heat inactivation procedures are applied on products filled in their final container (terminal treatment). fractionators are required by regulatory authorities to conduct down-scale experimental validation studies using relevant model viruses to establish the efficacy and robustness of viral reduction procedures. the robustness of the viral reduction procedures in place is exemplified by the absence of transmission of wnv, an emerging virus. 20 similarly, the lipid-enveloped severe acute respiratory syndrome coronavirus has been shown to be inactivated by core viral inactivation treatments of plasma products. 21,22 it is also likely, but not proven by validation experiments yet, that avian flu and simian foamy viruses, which also have a lipid envelope, would be inactivated by current processes in place if present in plasma. details on viral validation procedures can be found elsewhere. 23, 24 the major characteristics of current viral reduction treatments are summarized in table 3 9,23 and discussed briefly here. in-process viral inactivation treatments. the solvent-detergent (sd) treatment, developed in the mid 1980s, 25 remains the most frequent core viral inactivation procedure of plasma products. protein solutions are incubated for 4 to 6 hours at 248c to 378c in the presence of 0.3% to 1% tri-n-butyl phosphate (tnbp) and 1% tween-80 or triton x-100. typically, lipid enveloped viruses are inacti-vated in a matter of minutes, and the functional activity of even the most labile plasma proteinswith the possible exception of some serine prolease inhibitors-is well preserved, but nonenveloped viruses (less pathogenic in most individuals) are not inactivated. the sd agents are removed down to a level of a few parts per million usually by chromatographic adsorption or specific precipitation of proteins, or selective adsorption on hydrophobic chromatographic support. pasteurization, another common viral inactivation procedure, is a heat treatment of protein solutions for 10 hours at 608c, a treatment that denatures viral proteins and inhibits virus replication. 26 pasteurization can inactivate both enveloped and nonenveloped viruses, but stabilizers, needed to limit loss of protein functionality, may decrease the rate and extent of viral inactivation. 27 stabilizers may be removed by ultrafiltration, protein precipitation, or chromatography. vapor heat has also been used by one company; extent of virus inactivation is influenced by the temperature, duration, and pressure during treatment. risk of neoantigen formation, which can enhance protein immunogenicity, should be considered when using heat-based inactivation processes. low ph incubation, usually at ph 4, at 308c to 378c for more than 20 hours, was introduced in the early 1980s to allow the intravenous infusion of igg. this form of treatment was subsequently found to inactivate most lipid-enveloped viruses. caprylic (octanoic) acid precipitation/incubation at ph below 6 is a recently introduced treatment of human igg that can inactivate lipid-enveloped viruses. 28, 29 terminal viral inactivation treatments. in modern plasma fractionation, heat treatment of lyophilized products (dry heat) is used, due to limitations, mostly as a secondary viral inactivation step rather than the core inactivation treatment. the treatment is applied to some coagulation factor concentrates. performed at 808c for 72 hours or at 1008c for 30 minutes, generally in the presence of protein stabilizers, it provides added safety against havand other heat-sensitive viruses but may not be sufficient to exclude b19 transmission. 23 terminal (liquid) pasteurization at 608c for 10 hours is the bgold standardq treatment of albumin preparations. the fatty acids, caprylate, and tryptophanate, which protect albumin from heat denaturation, are added at doses compatible with therapeutic use and, therefore, are not removed before product infusion. viral removal treatment. nanofiltration is a specific viral filtration process applied to protein solutions using 15-to 75-nm multi-layers membranes, or equivalent systems, to remove viruses mostly by a sieving mechanism. 30,31 introduced in the early to mid 1990s, it has reached wide acceptance as a robust viral removal step for essentially all products, apart from albumin. nanofiltration is used to complement the core viral inactivation treatment and to provide enhanced safety against nonenveloped viruses or other resistant infectious agents. virus removal can also incidentally take place during protein precipitation, chromatography, or filtration steps; these steps contribute to the lowering of virus load from the protein production stream; they are difficult to monitor; and therefore do not guarantee, as standalone procedures, sufficient safety margin. prion removal methods. variant creutzfeldt-jakob disease (vcjd) can be transmitted by red blood cell concentrates, but to date, transmission has not been identified from plasma or plasma products. because of its biological nature, the prion agent is thought to be resistant to current viral inactivation procedures used during plasma fractionation. the methods known to inactivate abnormal misfolded prion proteins associated with transmissible spongiform encephalopathies (prp tse ) (such as oxidation, treatment with strong base, chaotropic agents, and extreme heat) destroy plasma proteins and, therefore, cannot be used. still, modern processes of fractionated products would seem to ensure significant removal of prp tse , as suggested by experimental spiking studies. 32 as scale-down and experimental transmissible spongiform encephalopathy (tse) spiking models are developed, knowledge on the capacity of manufacturing processes of plasma proteins to remove prions is growing rapidly, although much uncertainty remains because the unknown biological nature of the human plasma associated infectious agent. 33 several processes for manufacturing fviii, fibrinogen, von willebrand factor (vwf), fix, igg, and albumin products have been shown to be capable of removing prions in a consistent and reproducible manner. [34] [35] [36] generally, multistep fractionation processes are thought to be a contributing factor to prp tse elimination. two to more than 5 log removals of spiked prions occur during standard protein purification steps such as precipitation with ethanol or polyethylene glycol (peg), anion-exchange chromatography, and depth filtration. 34, 35, 37 mechanism of removal that may encompass an adsorption mechanism is still not fully understood and appears to be influenced by ph and the concentration of the precipitating agent. the partitioning process may reflect some prion aggregation because of prion hydrophobicity and insolubility. the removal capacity of nanofiltration membranes with pore sizes less than 75 or 35 nm has been extensively investigated, 34, 38 and prion removal is likely due in part to molecular sieving. extent of removal is related to the pore size of the filters and, presumably, to the aggregation state of prp tse . removal is superior with membrane of pore sizes of 15 nm, compared to 35 nm. the nature and origin of the spiking agent used for prp tse clearance studies is of critical importance because various spikes differ in size and characteristics. brain homogenate or brain-derived microsomal fractions from infected animals have usually been used as the source of prp tse spiking material. the most reliable and quantitative detection method of prp tse is based on animal bioassays, which require many animals and a time frame generally longer than 9 months. in vitro immunochemical methods, such as a western blot assay, are being used, at least as a first marker of the presence of prp tse and associated infectivity, by detecting the protease-resistant fragment. 33 thus, the risk transmission of vcjd by human plasma products appears remote, but caution should prevail since the biochemical nature of the infectious agent in human blood is not known. technologies to extract coagulation factors, protease inhibitors, and igg have evolved considerably in the last 20 years, leading to the development of products with improved safety and purity profiles. a description of major manufacturing techniques is given below. factor viii. several generations of fviii preparations have been developed since the mid-1980s, providing (a) safety from hiv and then hcv and hbv, (b) improved purity, and (c) enhanced safety from hav and b19. current development efforts focus on establishing prion removal. 36 all currently licensed plasma-derived fviii concentrates are purified from cryoprecipitate. in a typical process, cryoprecipitate is subjected to a combination of aluminium hydroxide adsorption and precipitation, or precipitation only (eg, using glycine), to reduce the level of trace vitamin k coagulation factors (as they may activate fviii during the downstream purification steps) or load proteins such as fibrinogen. the purified cryoprecipitate extract usually undergoes viral inactivation typically by sd or pasteurization. many processes include subsequent chromatography by anion exchange, monoclonal antibody affinity (using anti-fviii or anti-vwf murine antibodies), or immobilized heparin affinity to remove protein contaminants (such as fibrinogen or fibronectin), most or part of the vwf, and the sd agents. 39 immunopurified fviii eluate is further purified by chromatography to remove murine igg ligands that may have leached. prior to formulation and sterile filtration, some fviii products are nanofiltered using membranes with a pore size of 35, 20, or even 15 nm if a partial dissociation of fviii and high-molecular-weight vwf multimers is initiated. alternatively, some freeze-dried preparations are subjected to heat treatment at 808c or 1008c to inactivate nonenveloped viruses like hav. recovery of fviii, as expressed per liter of plasma, is usually comprised between 100 and 200 iu (1 iu is defined as the physiological activity present in 1 ml of plasma). factors lowering fviii yield include cryoprecipitation (ca 30% loss), chromatographic purification (ca 20% to 30 %), and viral heat inactivation (15%-30%). current fviii concentrates have a specific activity between 10 and 250 iu/mg. some products are formulated with human plasma-derived albumin, whereas others contain copurified vwf that helps stabilize fviii. purity of fviii concentrates has not been convincingly demonstrated to enhance immunological safety. long-term clinical experience indicates that the alleged reduced immunosuppressive effects of immunopurified preparations compared to lowerpurity plasma-derived preparations, as claimed in the late 1990s, were probably unfounded. processes used that, in part, influence residual vwf, may have an impact on the immunogenicity of plasma-derived fviii products. retrospective studies of previously untreated patients show that an sd-treated, nanofiltered, ion-exchange purified fviii product containing vwf appears to be 2 times less likely to induce anti-fviii inhibitors in hemophilia a patients than 2 full-length recombinant fviii concentrates. 40 von willebrand factor. because fviii chromatographic purification removes all, or part, of vwf, fviii products effective in treating von willebrand disease (vwd) are generally low-purity (bintermediate purityq) preparations prepared from cryoprecipitate by precipitation steps coextracting vwf and fviii in a ratio of higher than 1. the low purity and the high protein content of these products technically restrict the choice of viral inactivation treatments to pasteurization or terminal dry heat at 808c for 72 hours. one highly purified vwf concentrate, largely devoid of fviii and, therefore, specific for vwd treatment, is prepared from cryoprecipitate by a 3-step chromatographic procedure (integrated with fviii and fibrinogen purification processes) using 2 anion exchangers and immobilized gelatin polishing (to remove fibronectin). 41 viral reduction is by sd, 35-nm nanofiltration, and terminal dry heat at 808c for 72 hours. 42 fibrinogen. there are 5 registered fibrinogen preparations available for treating a fibrinogenemia or hypofibrinogenemia. 43 traditional preparations are obtained by multiple precipitation steps of plasma or cryoprecipitate using ethanol and glycine, whereas other modern products are purified by chromatography. viral reduction is achieved by sd treatment, often complemented by 35-nm nanofiltration or terminal dry heat treatment. single-step pasteurization at 608c for 20 hours is used for 1 product. fibrin sealants. fibrin sealants (fibrin glues) comprise fibrinogen-rich and purified thrombin concentrates. when mixing the 2 components, a strong adhesive clot exhibiting hemostatic, sealing, and healing properties is formed almost instantaneously or within a few seconds, offering multiple topical surgical applications. fibrinogen is prepared by precipitation methods from cryoprecipitate, or from the cohn fraction i; the fraction may also contain fibronectin, vwf, or factor xiii (fxiii), which may confer other physiological functions. 44 fibrinogen fractions are virally inactivated by sd, pasteurization, vapor-heat treatment, and/or nanofiltration. the fibrinogen concentration is typically above 80 g/l and may be formulated in the presence of an antifibrinolytic agent. prothrombin complex. prothrombin complex concentrate (pcc) is a mixture of vitamin kdependent coagulation factors in which fix, factor ii, and factor x and proteins c and s have a low specific activity between 0.5 and 2 iu/mg. 45 a few products contain also factor vii (fvii), but usually at levels lower than that of fix. the manufacture is usually based on a 1960s method that involves diethylaminoethyl (deae) sephadex or deae cellulose adsorption of cryo-poor plasma, but downstream ethanol fractions can also be used as starting materials. 46 anion exchangers coextract proteins sharing the presence of gamma-carboxyglutamic acid residues, whereas bulk plasma proteins, such as albumin and igg or at and aat remain in the unbound fraction. precipitation with tricalcium phosphate is used for one product. viral reduction is most often achieved by sd (tnbp-tween 80) treatment, complemented by 35-or 15-nm nanofiltration or by terminal dry heat. pasteurization and vapor heat are applied to 2 products. viral inactivation treatment by sd requires one subsequent ionexchange chromatographic step for removal of the virus-inactivating agents. recovery is in the range of 250 to 380 iu of fix per liter of plasma. single fix. high-purity fix products were developed in late 1989, 47 leading to reduced risks of thromboembolism, compared to pcc, in hemophilia b patients. fix is isolated by chromatographic purification of the pcc using anion exchange combined with either immobilized heparin, metal chelate affinity, or monoclonal antibody. these processes yield fix concentrates with a mean specific activity in the range of 100 to 150 iu/mg and a yield between 200 and 300 iu/l of plasma. factor vii. three specific concentrates rich in fvii and with reduced amount of other vitamin kdependent clotting factors are currently licensed to control bleeding in deficient patients. the manufacturing process includes ion-exchange chromatography or aluminium hydroxide adsorption, following a downstream procedure similar to that of pcc and fix. viral inactivation is achieved by sd treatment, vapor heat, or dry heat. factor xi. two factor xi (fxi) concentrates are currently available for deficient patients. one, of low purity, is purified by chromatography of cryo-poor plasma on deae cellulose and immobilized heparin. after freeze-drying, the product is virally inactivated by dry heat. in the other product, fxi is captured by adsorptive filtration then highly purified by cation-exchange chromatography. 48 this product undergoes dual viral reduction processing by sd and 15-nm nanofiltration. factor xiii. factor xiii is a transglutaminase that catalyzes the final step in the coagulation cascade, cross-linking the loose fibrin polymer into a highly organized structure. the early generation of fxiii concentrates for the treatment of fxiiideficient patients was extracted from placenta, but 2 plasma-derived products have subsequently been developed. one is purified from a cold-ethanol fraction from cryoprecipitate supernatant, purified by precipitation with sodium citrate and removal of fibrinogen by heating. the product is pasteurized in sorbitol solution, ultrafiltered to remove sorbitol, adsorbed with bentonite, and freeze-dried. the other product is obtained by precipitation steps and is also pasteurized. factor xiii is also a component of some fibrin sealants. although still subject to discussion, the presence of fxiii is claimed to contribute to fibrin c-chain cross-linking and tensile strength, possibly improving the hemostatic effect. 49 activated coagulation factors. human thrombin concentrates are available so far only as components of fibrin sealant. thrombin is prepared by activation of the pcc, usually in the presence of calcium chloride, followed by viral inactivation treatment by sd, purification by cation-exchange chromatography, and viral removal by 15-nm nanofiltration. final thrombin concentration is usually between 300 and 1000 iu/ml, but preparations with lower potency are available when a slower speed for clot formation of the sealant is preferred for some surgical applications requiring longer time for tissue gluing. a procedure for large-scale production of a purified plasma-derived fviia has been developed in japan. 50 fvii is purified by anion exchange and immunoaffinity chromatography and converted to fviia by autoactivation on an anion-exchange resin and incubation in the presence of ca 2+ for 18 hours at 108c. this preparation is virally reduced by nanofiltration and dry-heating and is intended for the treatment of hemophiliacs with antibodies against fviii or fix. antithrombin. antithrombin concentrates were the first plasma products extracted by affinity chromatography. production from cryo-poor plasma usually comprises ion exchange chromatography to remove the pcc components, followed by capture of at on immobilized heparin. viral inactivation is traditionally achieved by pasteurization in the presence of sodium citrate or a combination of sucrose and glycine, although sd treatment is used as well. because heat treatment may partially denature at, a second adsorption step on immobilized heparin can be used to remove altered molecules. recovery is between 250 and 350 u/l of plasma. fraction iv-1 is an alternative starting material, but yield is significantly lower. a1-antitrypsin (a1-protease inhibitor). there are now several licensed aat concentrates, including 3 in the united states. a1-antitrypsin augmentation therapy is indicated for the treatment of patients with lung emphysema secondary to congenital aat deficiency. because aat shares many physicochemical properties, in particular, molecular weight and isoelectric point, with albumin, it has been difficult to design production methods for aat not affecting the existing production process for albumin. most preparations are recovered from fraction iv [1] [2] [3] [4] . purification from this waste fraction is rather cumbersome and involves peg precipitation and ion-exchange chromatography, considerably compromising recovery (~0.2 g/l). the first preparations developed in the 1990s were virally inactivated by pasteurization, but dual viral reduction steps using sd and nanofiltration are also now used. recent isoelectrofocusing studies have provided evidence indicating that new anodal aat variants are present in at least one of the fda-licensed product, suggesting alteration of some isoforms during purification. loss of a c-terminal positive charged lysine, secondary to carboxypeptidase activity, was proposed as an explanation for these isoelectrofocusing migration shifts. under circumstances when clinical demand for albumin decreases, extracting aat from upstream fractions, such as the supernatant ii+iii, 51 appears a logical trend, offering the possibility of a more effective production scheme, characterized by a recovery of 0.6 to 1 g/l. 51 protein c. there are 2 protein c concentrates manufactured in europe and 1 in japan. in one process, the pcc undergoes cascade purification on 3 ion exchangers, 52 whereas in the other, immunoaffinity and affinity chromatographic processes are combined with ion exchange. viral reduction is achieved by sd treatment, which can be combined with 15-nm nanofiltration or vapor-heat treatment. 53 c1-esterase inhibitor. c1-esterase inhibitor concentrates are used for the treatment of acute phases of angioedema, primarily in the oropharyngeal region and gastrointestinal tract in patients with congenital or acquired c1-inh deficiency. there are 3 products licensed in europe. products are generally purified by chromatography from the cryo-poor plasma after extraction of the pcc and, potentially, at. viral inactivation is achieved by pasteurization, vapor heat, or sd, possibly combined with nanofiltration. essentially all therapeutic albumin preparations are prepared by fractionation of cryo-poor (or pccpoor and/or at-poor and/or c1-inh-poor) plasma by ethanol fractionation. a critical upstream process step (precipitation ii+iii) separates the igg fraction. to optimize recovery, the precipitates generated during the ethanol fractionation process are separated by depth filtration. albumin recovery of 75% to 85% (25-28 g/l) and purity of 96% to 98% are typically obtained. some processes combine ethanol fractionation with a polishing ion exchange chromatography, which generally improves product purity to ca 99%, whereas in one production method, albumin is purified mostly by anion exchange, cation-exchange, and size-exclusion chromatography. the adjustment of the concentration of the purified fraction, typically from 4% to 25%, is achieved by ultrafiltration. the standard viral inactivation method is pasteurization, which, according to most pharmacopeias, should be performed in the final container rather than on the albumin batch before aseptic filling. current mean albumin yield is 24 to 26 g/l of plasma. polyvalent igg preparations, either for intramuscular or intravenous uses, are traditionally prepared from the fraction ii that is obtained by stepwise fractionation of cryo-poor plasma using cold ethanol at concentrations up to 25%. increasingly, igg products are extracted from up-streamed ethanol precipitated fractions, such as supernatant iii or precipitate ii+iii, to optimize recovery. intermediate igg fractions are subjected to ion-exchange chromatography, caprylic acid, or peg precipitations to remove protein contaminants, proteolytic enzymes, and/or aggregates. most current viral inactivation procedures are low ph incubation, pasteurization, or sd; the caprylic acid treatment, recently introduced in the manufacture of human igg products, is also a robust viral inactivation process of igg. dedicated viral removal by 15-to 35-nm nanofiltration is commonly used to increase the safety against nonenveloped viruses, especially in a situation when the core viral inactivation treatments target only lipid-enveloped viruses. the igg recovery has long been in the 2.7-to 3.2-g/l range when combining traditional ethanol fractionation processes and centrifugation. depth-filtration and/or chromatographic purification from upstream fractions have improved the mean recovery to the 3.5-to 4.5-g/l range, or more. total chromatographic procedures are increasingly used for the production of hyperimmune igg products because such processes are amenable to the fractionation of smaller plasma volumes and can optimize recovery. the manufacturing process includes at least 2 dedicated viral reduction treatments. other protein components have been fractionated at pilot-scale and subjected to experimental or human trials. inter-a-trypsin inhibitor (iti) is a kunitz-type serine proteinase inhibitor. its inhibitory capacity is carried by bikunin, a chondroitin 4-sulfate proteoglycan, which is covalently linked to its heavy chains h1 and h2, but can be released by proteolytic cleavage. an iti concentrate has been obtained by fractionation of the prothrombin complex on an anion exchanger followed by immobilized heparin and viral inactivation by sd. iti, as a reservoir of bikunin, may be involved in control of inflammatory processes. in a porcine model of endotoxin shock, iti improved the hemodynamic, oxygenation, and coagulation parameters. 54 administration of iti very early after the onset of sepsis or repeated injections at later time points (10 and 20 hours) maintains cardiovascular stability and significantly reduces mortality in a rat model. 55 transferrin is the major iron binding plasma protein which may prevent cytotoxic effects or predisposition to septic infection due to accumulated free non-transferrin-bound iron when normal iron use is hampered and/or apotransferrin production is decreased. a liquid apotransferrin concentrate has been obtained from cohn fraction iv by 2 ion exchange chromatographic steps and ultrafiltration. viral safety was ensured by sd treatment, nanofiltration, and peg precipitation. the product had intact iron binding capacity, and maintained the bacterial growth inhibitory effect in serum. in hematological stem cell transplant patients, the product prevented the appearance of non-transferrin-bound iron. apolipoprotein a-i is the principal protein component of the plasma high-density lipoproteins. it prevents the accumulation of cholesterol-loaded macrophages which deposit on the arterial wall as foam cells. apolipoprotein a-i inhibits hepatic lipase and lipoprotein lipase in vitro. a concentrate has been isolated by precipitation from cohn fraction iii. 56 intravenous injections in men were well tolerated in an early clinical trial; clinical observations were consistent with combined inhibition of hepatic lipase and lipoprotein lipase activities. possible clinical applications include the treatment of hypercholesterolemic patients and atherosclerosis. recombination with lecithin forms a high-density lipoprotein complex that could help limit inflammation, endotoxin-induced activation of coagulation, and fibrinolysis in septic conditions. mannan-binding lectin (mbl) is a component of the innate (aspecific) immune system that can bind repetitive structures of mannan groups, such as those on the surface of micro-organisms, activating the complement system and leading to the destruction of a large variety of micro-organisms. the relatively frequent congenital deficiency of mbl is associated to recurrent infections, especially in infants when the specific immune system has not yet matured. an mbl concentrate has been produced from cohn fraction iii. 57 plasmin is the major fibrinolytic enzyme in plasma. encouraging results as a new fibrinolytic agent have been obtained in animal models where plasmin was applied directly to the clot through a catheter to treat peripheral arterial occlusion. von willebrand factor cleaving protease (vwf-cp, adamts13) cleaves ultra-large multimers of vwf that enter the blood stream directly after biosynthesis by endothelial cells. if developed, a purified vwf-cp could be useful to treat, in place of plasma, patients with thrombotic thrombocytopenic purpura with congenital or acquired deficiency of vwf-cp. activated protein c can be of value for the treatment of sepsis, as demonstrated through the clinical use of a recombinant preparation. there is no therapeutic plasma-derived activated protein c products available yet for therapeutic use, although a process for a highly purified preparation, where cryo-poor plasma is purified by immunoaffinity and anion-exchange chromatographic steps, and sd viral inactivation has been described. 58 there is also research needed to investigate alternative indications for currently available products. potential clinical use of c1-inh, aimed at benefiting from its role as inhibitor or attenuator of the activation of complement and contact systems, include septicemia, myocardial infarction, capillary leak syndrome, pancreatitis, and organ transplantation. intravenous use of a fxiii concentrate was found to increase epidermal growth factor and transforming growth factor-b, suggesting that it may accelerate wound healing of anastomotic leaks and nonhealing fistulas. factor xiii was also found to have osteoinductive properties, suggesting use in bone tissue engineering. a review on plasma fractionation should also cover the role played by national and international regulatory authorities. over the last few years, assuring the safety of large-pool plasma products has posed formidable challenges to regulatory authorities and fractionators alike. the complexity of the field, encompassing the diversity in blood and plasma product types and manufacturing processes, made it difficult to enact balanced decisions toward ensuring both product safety and guarantee of supply. since the 1980s, the agencies regulating the plasma fractionation industry have developed a comprehensive set of measures to ensure the viral safety of plasma products. multiple layers of regulatory oversight of the plasma industry have been established to ensure overlapping safeguards against the risks of the transmission of bloodborne infectious agents. several regulations, guidances, position statements have been issued by agencies like the us fda and the european medicine evaluation agency, which are updated as needed. those cover important safety aspects required at all stages of the manufacturing chain, from activities at blood establishment preparing plasma for fractionation, extending to the manufacturing and distribution of plasma products. regulatory oversight includes epidemiologic surveillance of the donor population; donor deferral policies and screening practices; mandatory donation testing; testing of manufacturing plasma pools; validation of viral reduction procedures and other production steps; as well as the assessment of product quality, safety, and efficacy for marketing authorization. most of relevant information on plasma collection is assembled in europe in the plasma master file, which allows establishing key levels of information regarding the quality and safety of the plasma raw material. post marketing, reports on adverse reactions associated with plasma products should be transmitted to nras and could prompt emergency response procedures and product recalls. 10 some harmonization of the requirements for manufacture and supply of plasma products in the united states, europe, and japan is taking place under the auspices of the international conference on harmonisation, but much work remains to be done. such measures, nonetheless, provide the framework through which modern plasma products now exhibit a very high level of quality, safety, and efficacy. however, the rigidity of the regulatory system has been an impediment to more significant technological evolution of the plasma fractionation process because process changes are currently associated to major regulatory work. the cohn plasma fractionation method initially designed to obtain albumin has, over the years, developed rather successfully into a well-established industrial procedure isolating a wide range of clinically useful products. today, more than 20 different protein products, and more if one considers the variety of hyperimmune igg preparations, can be extracted through large-scale fractionation of human plasma. the soundness and large-scale adaptability of the technology, on the one hand, and the rigidity of the current regulatory framework, on the other, explains why this technology remains the main core method in use at industrial scale, although it implies suboptimal yield for most proteins, apart from albumin. the technology has increased in complexity over the years, with the greatest progresses in purification being, without doubt, associated with the use of chromatographic methods that have made possible the development of new protein therapeutics and impressive improvements in product purity and quality. the fractionation scheme has also changed dramatically through the introduction of in-process viral reduction treatments, which have required the addition of downstream techniques such as chromatography and ultrafiltration. the change in protein drivers, with the prominent clinical role now played by igg, and the requirements to increase protein recovery and optimize the fractionation process may crystallize the incentive for most fractionators to abandon relative technical conservatism and introduce significant technological changes in processing technology. the production of igg from precipitate (i)+ ii+iii, already implemented by some manufacturers and, possibly, that of aat from supernatant (i)+ ii+iii, are signs of a gradual evolution in the direction of total chromatographic processing. with so much gained, over the last few years, in the understanding of the key parameters building plasma product quality, safety, and efficacy, one can hope that the regulatory paths, in particular, with regard to clinical studies, to license known protein therapeutics prepared by improved, more efficient technology should be simplified. this would, in turn, contribute to an improved supply. as the plasma fractionation industry has so far been targeting mostly proteins that were obvious candidates for replacement therapy, it is also hoped that it will invest more into developing new products because plasma remains a unique source of potential therapeutic proteins. proactive research and development work should be encouraged to isolate and evaluate new therapies among the well characterized plasma proteins with still unknown function. finally, one should not forget that plasma protein therapies are expensive and largely inac-cessible to the developing world. the new market drivers in rich countries are likely to diminish economical interest in the manufacture of fviii that remain a major protein therapy in need in the developing world. the belief that decreased use of plasma-derived fviii or fix in developed economies (as hemophiliacs are switching to recombinant therapies) would increase the supply of products to developing countries may be not economically viable. rather, this switch to recombinant products in rich countries can make poor countries unable to afford the increased cost of products no longer subsidized by the premium price paid in rich countries. it therefore remains important to develop affordable viral inactivation and processing technologies gradually allowing developing countries to make use of local plasma resources in a safe manner. 59 guideline on epidemiological data on blood transmissible infections. for inclusion in the guideline on the scientific data requirements for a plasma master file current instrumentation for apheresis. apheresis: principles and practice assessment of complement activation during membrane-based plasmapheresis procedures the effect of leukocyte depletion on the quality of fresh-frozen plasma current safety of the blood supply in the united states farrugia a: guide for the assessment of clotting factor concentrates for the treatment of hemophilia. www.wfh.org. montreal, world federation of hemophilia preparation and properties of serum and plasma proteins. iv. a system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids eight years experience with the alcohol fractionation procedure of nitschmann, kistler and lergier tabor e: the epidemiology of virus transmission by plasma derivatives: clinical studies verifying the lack of transmission of hepatitis b and c viruses and hiv type 1 contribution and interpretation of studies validating the inactivation and removal of viruses (revised) inactivation of viruses in labile blood derivatives. i. disruption of lipidenveloped viruses by tri(n-butyl)phosphate detergent combinations pasteurization of antihemophilic factor and model virus inactivation studies enveloped virus inactivation by caprylate: a robust alternative to solventdetergent treatment in plasma derived intermediates evaluation de l'efficacité des procédés de purification des proteins plasmatiques à éliminer les agents transmissibles non conventionnels distribution of a bovine spongiform encephalopathy-derived agent over ionexchange chromatography used in the preparation of concentrates of fibrinogen and factor viii al: influence of the type of factor viii concentrate on the incidence of factor viii inhibitors in previously untreated patients with severe hemophilia a in vitro study of a triple-secured von willebrand factor concentrate fibrin sealant: scientific rationale, production methods, properties, and current clinical use properties of a highly purified human plasma factor ix:c therapeutic concentrate prepared by conventional chromatography large-scale production and properties of human plasma-derived activated factor vii concentrate effects of inter-alpha-inhibitor in experimental endotoxic shock and disseminated intravascular coagulation delayed administration of human inter-alpha inhibitor proteins reduces mortality in sepsis a minipool process for solvent-detergent treatment of cryoprecipitate at blood centres using a disposable bag system key: cord-270514-36k9xo7f authors: van der woude, roosmarijn; turner, hannah l.; tomris, ilhan; bouwman, kim m.; ward, andrew b.; de vries, robert p. title: drivers of recombinant soluble influenza a virus hemagglutinin and neuraminidase expression in mammalian cells date: 2020-08-14 journal: protein sci doi: 10.1002/pro.3918 sha: doc_id: 270514 cord_uid: 36k9xo7f recombinant soluble trimeric influenza a virus hemagglutinins (ha) and tetrameric neuraminidases (nas) have proven to be excellent tools to decipher biological properties. receptor binding and sialic acid cleavage by recombinant proteins correlate satisfactorily compared to whole viruses. expression of ha and na can be achieved in a plethora of different laboratory hosts. for immunological and receptor interaction studies however, insect and mammalian cell expressed proteins are preferred due to the presence of n‐linked glycosylation and disulfide bond formation. because mammalian‐cell expression is widely applied, an increased expression yield is an important goal. here we report that using codon‐optimized genes and sfgfp fusions, the expression yield of ha can be significantly improved. sfgfp also significantly increased expression yields when fused to the n‐terminus of na. in this study, a suite of different hemagglutinin and neuraminidase constructs are described, which can be valuable tools to study a wide array of different has, nas and their mutants. influenza a virus (iav) is a continuous burden for human and animal health, and its eradication is near impossible given the wild waterfowl reservoir. iav contains a negative-sense segmented rna genome that allows for rapid nucleotide changes and exchange of whole segments both of which contribute to high variability. iav hxnx subtypes are determined by antigenicity, however, several subtypes are under immune pressure, from which they can escape, resulting in drifted viruses. the two surface envelope proteins of iav have opposing functions; the trimeric hemagglutinin (ha) binds to sialic acid containing glycans to enable the virus to enter cells, 1,2 the tetrameric neuraminidase (na) cleaves sialic acids to release new viral particles from the membrane. 3, 4 na is also important for the cell entry process as it removes decoy receptors. 5, 6 both envelope proteins are therefore of great importance for the viral lifecycle and elicited antibodies impeding ha and na biological functions and are therefore protective. [7] [8] [9] elucidating antigenicity, receptor specificity and other biological phenotypes of these two envelope proteins have been aided by means of recombinant soluble multimeric proteins. also, in vaccine development and antiviral discovery, these proteins have proven to be excellent tools. [10] [11] [12] [13] the use of recombinant proteins eliminates the lengthy process of virus generation either by reverse genetics or growth of wild type viruses that in turn are prone to adaptation in eggs and/or cell culture. 14 lab adaptation is especially problematic for older strains of influenza due to multiple rounds of infection in eggs, vero and mdck cells. 15 in addition, contemporary h3n2 viruses adapt quickly to laboratory hosts. 16, 17 in addition, with recombinant proteins there is no need to work in bsl-ii or -iii environments. individually expressed ha and na proteins enable their functions, such as receptor specificity for ha or sialidase activity for na, to be analyzed in great detail. here we report our observations gleaned over a decade of recombinant ha and na protein expression in mammalian cells. 16 we demonstrate increased expression yields using codon-optimized sequences and genetic fusions of super folder gfp (sfgfp). [17] [18] [19] although codonoptimization might not sound surprising, sfgfp fusions are generally utillized to facilitate routine expression and purification techniques. however, we observed a significant increase in expression yields and determined that it reduced the use of expensive antibodies and provided an excellent handle, as well as an internal read out, of a glycan binding protein. for example, we used has of contemporary h1 and h3 vaccine strains, the latter have been increasingly difficult to express and crystallize, most likely due to an increased number of potential n-linked glycosylation sites that may result in an elongated retention time in the er and golgi. 20 furthermore, we applied the same principles to several na subtypes, n1, n2, and n9. the n-terminal sfgfp increases yields, maintains biological activity, structure and antigenicity, and aids protein quantitation during expression and purification. our results should be valuable for other labs interested in the use recombinant ha, na, and perhaps other viral envelope proteins. recombinant soluble ha was created with the use of an expression plasmid in which the open reading frame (orf) is preceded by a human cytomegalovirus (cmv) promoter, a cd5 derived signal peptide for efficient translation and transport to the cell culture medium (figure 1a ). the sfgfp is cleavable by a tobacco etch virus (tev) protease recognition and cleavage site sequence. all codon optimizations, ha, na, and sfgfp where performed by genscripts propieratary software, standard cloning sites in the open reading frame are removed. to demonstrate the utility of codon-optimization for protein expression we created plasmids with and without codon-optimized genes of contemporary h3n2 and h1n1 influenza a virus vaccine strains. we choose two h3n2 vaccine antigens, a/texas/2012 [a/tx/12], a/switzerland/2013 [a/ch/13], and the corresponding h1n1 a/michigan/15 [a/mi/15]. codon-optimization is known to increase expression yields, whereas sfgfp fusions are not, so both sequence versions were cloned into plasmids with and without sfgfp fusions. we determined the yields by western blot and measurement of fluorescence (figure 1b,c) . all orfs were efficiently expressed when fused to a c-terminal sfgfp and with codon optimization. while non-codon optimized and non-sfgfp fused variants are also observed in the cell culture supernatants, yields are less than 1 μg/ml. using three examples, it is clear that in some cases only the addition of sfgfp or codon optimization can be sufficient to increase yields. for the a/tx/13 h3 the sfgfp increases the band intensity and incorporating a codon optimized gene further improved expression efficiency. a/ch/13 h3 ha requires both a sfgfp and codon optimization to express at high yields, whereas for the 2015 h1 vaccine component, a/mi/15 h1n1 codon optimization by itself increased expression 10-fold, but the addition of a sfgfp fusion did not further increase expression. we observed these expression patterns for several ha orfs and decided to show these examples, as it is increasingly difficult to express heavily glycosylated h3 has. as another example we cloned the receptor binding domain of an avian coronavirus into plasmids with and without a c-terminal sfgfp. we again observed an increase of expression yield by twoto four-fold as determined by band intensity (figure 1d ). whereas ha is a type i membrane protein, na is type ii with its n-terminus proximal to the membrane. thus, we introduced the sfgfp fusion at the n-terminus. in many expression platforms the original stalk domain is omitted 21 as it appears to be unstable, therefore several different tetramerization domains are routinely included. 4 to properly compare the effect of different multimerization motifs, gcn4 versus tetrabrachion (tb), and sfgfp fusion on expression, folding, and enzymatic activity, we created three different constructs ( figure 2a) . 4, 22, 23 we tested the gcn4, tb, and sfgfp-tb-na proteins from two h3n2 viruses, nl/16, and nl/03. the resulting n2 proteins were analyzed by western blot where it was evident that the constructs fused to a tb domain maintain oligomerization on gel (figure 2b ). to observe monomers, we reduced the samples prior to gel loading. the gcn4 construct appeared as monomers and dimers on sds-page. the reduced monomers of sfgfp-tb, tb, and gcn4 migrated to different positions on gel that reflect their difference in molecular weight. in contrast to ha, we did not observe a large increase in expression yield of na using the sfgfp fusion. we analyzed the structural arrangements of our n2 proteins to ensure they were folded into the correct tetrameric native conformation using negative stain em, similar to our previously described sfgfp-ha proteins. 24, 25 the em data demonstrates that the n2 na assembles into a stable tetramer that resembled known na structures ( figure 2c) . initially, 57,505 individual particles were picked, placed into a stack, and submitted to reference free two-dimensional (2d) classification. from the initial 2d classes, particles that did not resemble na were removed, resulting are final particle stacks of 32,672 particles, which were then subject to relion 2d classification. all resultant classes demonstrated evident tetramerization and distinct na, gcn4, and tb motifs, and four sfgfp protein structures could be identified in the em images. to determine that sfgfp-na fusions are enzymatically, antigenically and structurally similar to their non-fused counterparts, we analyzed the gcn4, tb, and sfgfp-tb-n2 proteins with munana and na specific antibodies (figure 3 ). in the munana assay we determined enzymatic activity by measuring methylumberriferyl, which is released from sialic acid upon digestion, as previously described. 21, 26 the sfgfp-tb-n2 and gcn4-n2 had the highest enzymatic activity (figure 3a) , and although tb-n2 also displayed sialic acid cleavage, it was significantly less active. we also tested enzymatic activity of additional sfgfp-tb nas, two additional n2s (nl91 and nl19), an n1 from the 2009 pandemic (ca04) and the 2013 h7n9 na (figure 3b ). all nas tested displayed efficient sialic acid cleavage that can be inhibited by oseltamivir. to analyze antigenicity, we requested three monoclonal mouse antibodies to n2 raised against perth09 at the international reagent resource program (https://www. internationalreagentresource.org/). the monoclonal antibodies were conveniently designated as #56 through #58 and binding to coated n2 proteins was analyzed using elisa. we observed efficient binding of antibodies #56 and #57 for all sfgfp-n2s tested, with minimal differences for n2 protein derived from viruses isolated from 1991 to 2019 (figure 4a ). antibody #56 also bound to ca0409 n1 whereas antibody #57 did not. ab #58 failed to bind any na that we coated, perhaps this ab is restricted to the homologous perth09 n2, while a/nl/16 differs by 12 amino acids. however, 2 of the 3 monoclonal antibodies had a considerable breadth across n2 strains and even n1 recognition. finally, we tested a recently published broadly protective na antibody, 1g01, a kind gift of florian krammer, 23,27 both as a capturing as well as a detection antibody (figure 4b ). we hypothesized that direct coating of na could potentially block the enzymatic site, which can be overcome with the bigger tb tetramerization domain in which the sfgfp is helpful to present the enzymatic sites. we therefore also coated 1g01 as a capture ab, and applied the recombinant n2 proteins in a concentration dependent manner that where subsequently detected using the streptag. in both elisa variants 1g01 efficiently recognized all n2 proteins either as detecting and capture antibody. we observed hardly any differences between the differentially expressed n2 proteins, both as a detecting as well as capture the 1g01 antibody was significantly inhibited by oseltamivir. oseltamivir efficiently inhibited capture of n2 proteins, whereas the detection of coated n2 proteins was reduced for n2 with a sfgfp but less efficiently for gcn4 and tb only n2 proteins. confirming our inititial hypothesis that direct coating of na can shield the enzymatic site. in this report we describe several optimization techniques that can substantially increase expression yields of recombinant soluble multimeric ha and na proteins. these techniques can be extremely useful when large amounts of protein are needed, or a large number of mutants need to be expressed. we hope that our data will further enable the laboratories creating recombinant ha and na proteins f i g u r e 3 enzymatic analyzes sfgfp fused neuraminidases. (a) enzymatic activity of sfgfp-tb, tb, and gcn4 neuraminidases: na enzymatic activity of preparations containing different amounts of the sfgfp-tb, tb, or gcn4-n2 was determined using the munana fluorometric assay (rfu, relative fluorescent units). oseltamivir was added to inhibit enzymatic activity. the results shown are a representative example of three independent assay performed in triplicates. (b) enzymatic activity of sfgfp-tb neuraminidases of different strains and subtypes: na enzymatic activity of sfgfp-tb n2, n9, and n1 proteins using the munana fluorometric assay (rfu, relative fluorescent units). oseltamivir was added to inhibit enzymatic activity. the results shown are a representative example of three independent assay performed in triplicates to do so in a cost-efficient manner. we want to highlight that one of our main objectives was to minimize the expenses of expressing these complex proteins so that they can be used by labs with limited means. our optimization techniques ranged from a two-to five-fold increases in protein expression individually, and combinations thereof could further increase yields 10-fold. although our methods are generally applicable, we note that as with any approach for recombinant protein expression, results are ultimately protein dependent and can vary based on the ha or na subtype. for example, for efficient expression of h1, the fold increases were remarkably lower compared to h3 as described in this report. especially interesting for us was the increase in expression yield when ha or na was fused to a sfgfp, which is in accordance with glycosyltransferases. 19 nevertheless, sfgfp induced increases in expression yield, is protein dependent, as for coronavirus spike receptor binding domain the increase is only two-to fourfold, while no increase in yields were observed for hiv env. 28 f i g u r e 4 antigenic analyzes of sfgfp fused neuraminidases. (a) antigenicity of na: purified n2 proteins analyzed with three titrated mouse monoclonal antibodies. n2 proteins where coated on maxisorp 96-wells plates and serially diluted abs were detected using a rabbit anti-mouse. the results shown are a representative example of three independent assay performed in triplicates. (b) pan-na antibody 1go1 efficiently recognizes recombinant nas and is inhibited by oseltamivir: purified na proteins where either detected using 1go1 (left). 1go1 was also used capture titrated purified n2, which was detected using a mouseanti-streptag-hrp labelled antibody. no significant differences where observed between the differently produced na proteins. oseltamivir was used to inhibit the reaction 1go1-na binding. the results shown are a representative example of three independent assay performed in triplicates we have not changed a206v in the sfgfp that would result in a monomeric fluorescent protein, 25 apparently our trimerization and tetramerization domains overrule the tendency of sfgfp to dimerize. furthermore, we have not yet used the tev cleavage sites in these constructs, as we found that all biological properties where equal when comparing non-and sfgfp fused proteins. 26 another observation we made, is that in high salt containing buffers and elongated storage at 4 c the sfgfp dissociates, as indicated by a separate sfgfp band on gel. both ha and na structures have been available for decades and now all different subtypes have been crystallized with constructs that include the tb motif, whereas multimerization motifs are normally lacking. 5, [27] [28] [29] [30] for example some labs routinely use a vasp, which perfectly amendable for crystallization after cleavage. 31 crystallization is however not feasible for many labs, yet structural information is in many cases vital. low resolution structural information sufficient for mapping epitopes for antibodies can however be obtained using a relatively small amount (<10 micrograms) with the use of negative stain electron microscopy (em). high resolution cryo-em structure determination requires more protein (100 micrograms) but is sufficient to solve atomic structures such as a 3.1 angstrom resolution structure obtained for an n9 protein fused to gcn4, 29,32 that closely resembled the negative stain image presented here. finally the fusion of the sfgfp facilitates several improvements in transfection efficiency, protein production determination and has been extremely useful in analyzing biological activity for ha. 24, 25 we now show similar results for na. however, the improvement of yields for na can still necessitate expression in suspension cultures when milligram amounts are desirable. suspension cultures, however, need expensive shaking incubators and medium. ha and na expression in this manuscript were all done in adherent cells, with a routine milligram yield from 100 ml of supernatant for ha, making it amendable for labs with minimal means. for na however, a 100 ml supernatant results in 250 μg. in conclusion, we demonstrate several ways to increase multimeric soluble proteins expression in mammalian cells. which would help to increase workflow and decrease costs. both original (a kind gift of erhard van der vries) and codon-optimized sequences were cloned into the pcd5 expression as described previously. 18 the pcd5 expression vector was adapted so that the ha-encoding cdnas are cloned in frame with dna sequences coding for a signal sequence, gcn4 trimerization motif, a tev cleavage site, a sfgfp if indicated, 25, 28 and the twinstrep, iba, germany). ha encoding cdna, a/tx/50/12 (a/tx/12 genbank kc892248.1), a/ch/9715293/13 (a/ch/13) and a/mi/45/15, was synthesized by genscript (dna sequences available upon request), both original (a kind gift of erhard van der vries) and codon-optimized sequences were cloned into the pcd5 expression as described previously. 16 the pcd5 expression vector was adapted so that the ha-encoding cdnas are cloned in frame with dna sequences coding for a signal sequence, gcn4 trimerization motif, a tev cleavage site, a sfgfp if indicated, 24, 26 and the twinstrep, iba, germany). the codon optimized na genes were synthesized at genscript, after conventional restriction enzyme cloning, 21 the open reading frame was preceded by sequences successively coding a strep-tag ii and a gcn4 tetramerization domain. 33 additionally we cloned the na genes in a vector adapted as such to be preceded with a sfgfp and or tb tetramerization motif. 29 30 the open reading frame was preceded by sequences successively coding a strep-tag ii and a gcn4-pli tetramerization domain. 34 additionally we cloned the na genes in a vector adapted as such to be preceded with a sfgfp and or tetrabrachion tetramerization motif. 27 ha and na expression plasmids, endotoxin free, where transfected on 80% confluent hek293s gnti(−)cells using polyethyleneimine i (linear 25 kda, polysciences, inc, warrington, pa) (pei) at a ratio of 1:9 g/g, before applying the dna-pei mix buffered for 30 minutes in dulbeccos modified eagles medium (dmem), 30% of the medium is removed to increase surface tension. at 6 hr post transfection, the transfection mixture was replaced with 293 sfm ii expression medium (gibco), supplemented with sodium bicarbonate (3.7 g/l), glucose (2.0 g/l), primatone rl-uf (3.0 g/l), glutamax (gibco), valproic acid (2 mm), and dmso (1,5%). tissue culture supernatants were harvested 5-6 days post transfection. ha and na protein expression and purification was confirmed by western blotting using a strepmab-hrp classic antibody. whereas ha proteins disassociates during an sds-page run, na proteins need to be reduced to observe the monomeric fraction. additionally, we measure fluorescence in the cell culture supernatant when applicable using a polarstar fluorescent reader with excitation and emission wavelengths of 480 nm and 520 nm, respectively. proteins are purified using a single-step with streptactin sepharose in batch format. sfgfp-tb, tb and gcn4-pi na proteins in 10 mm tris, 150 mm nacl at 4 c was deposited on 400 mesh copper negative stain grids and stained with 2% uranyl formate. the grid was imaged on a 120 kev tecnai spirit electron microscope with a lab6 filament and a 4 k × 4 k temcam f416 camera. micrographs were collected using leginon 33 and then uploaded to appion 34 particles were picked using dogpicker, 35 stacked, and aligned using msa/mra. 36 further 2d and 3d processing was undertaken using relion. 4.6 | biological activity and antigenicity of na proteins na enzymatic activities were measured in 100 mm tris (ph 6.15), 150 mm nacl, 10 mm cacl 2 buffer, using the fluorescent substrate 2 0 -(4-methylumbelliferyl)-α-d-nacetylneuraminic acid (4-mu-nana) [79] with excitation and emission wavelengths of 365 and 450 nm, respectively. the reaction was conducted for 1 hr at 37 c in a total volume of 80 μl. the reactions were all performed in triplicate and were stopped by adding 80 μl of 1 m na 2 co 3 . elisas were coated with 1 μg/ml n2 protein in pbs on maxisorp 96-wells plates over night at 4 c. plates were blocked for 1 hr at rt with 1% bsa in pbs supplemented with 0.1% tween20. mouse monoclonal antibodies were serially diluted and incubated for 1 hr at rt. primary antibody binding was detected with a secondary rabbit anti-mouse hrp antibody (novus) at 1:2,000 for 1 hr at rt and as an hrp substrate, sigma fast odp tablets were used. a similar procedure was used when 1g01 abs were coated, after blocking, the recombinant na proteins were serially diluted and detected with a mouse anti-streptag hrp labelled antibody at a 1:2,000 dilution. the hrp reactions were stopped after 3 min using 2,5 m h 2 so 4 . optical density was measured at 490 nm, all assays were performed in triplicates and a representative result is shown from three independent biological repeats. virus recognition of glycan receptors the biology of influenza viruses. vaccine the specific enzyme of influenza virus and vibrio cholerae a generic system for the expression and purification of soluble and stable influenza neuraminidase influenza virus neuraminidase structure and functions a new role of neuraminidase (na) in the influenza virus life cycle: implication for developing na inhibitors with novel mechanism of action hemagglutinin stalk-reactive antibodies interfere with influenza virus neuraminidase activity by steric hindrance neuraminidase inhibition contributes to influenza a virus neutralization by antihemagglutinin stem antibodies influenza hemagglutinin and neuraminidase: yin(−)yang proteins coevolving to thwart immunity vaccination with a soluble recombinant hemagglutinin trimer protects pigs against a challenge with pandemic (h1n1) 2009 influenza virus a site of vulnerability on the influenza virus hemagglutinin head domain trimer interface universal influenza virus vaccines that target the conserved hemagglutinin stalk and conserved sites in the head domain drug susceptibility evaluation of an influenza a(h7n9) virus by analyzing recombinant neuraminidase proteins changes in influenza virus associated with adaptation to passage in chick embryos influenza passaging annotations: what they tell us and why we should listen the influenza a virus hemagglutinin glycosylation state affects receptor-binding specificity synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast, pichia pastoris better and faster: improvements and optimization for mammalian recombinant protein production expression system for structural and functional studies of human glycosylation enzymes mechanisms of protein retention in the golgi n-glycolylneuraminic acid as a receptor for influenza a viruses expression and characterization of recombinant, tetrameric and enzymatically active influenza neuraminidase for the setup of an enzyme-linked lectin-based assay broadly protective human antibodies that target the active site of influenza virus neuraminidase fluorescent trimeric hemagglutinins reveal multivalent receptor binding properties assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions engineering and characterization of a fluorescent native-like hiv-1 envelope glycoprotein trimer structure of an influenza a virus n9 neuraminidase with a tetrabrachiondomain stalk structure of influenza virus n7: the last piece of the neuraminidase "jigsaw" puzzle structural basis of protection against h7n9 influenza virus by human anti-n9 neuraminidase antibodies recombinant soluble, multimeric ha and na exhibit distinctive types of protection against pandemic swine-origin 2009 a(h1n1) influenza virus infection in ferrets structural characterization of the 1918 influenza virus h1n1 neuraminidase leginon: a system for fully automated acquisition of 1000 electron micrographs a day appion: an integrated, database-driven pipeline to facilitate em image processing a switch between two-, three-, and four-stranded coiled coils in gcn4 leucine zipper mutants dog picker and tiltpicker: software tools to facilitate particle selection in single particle electron microscopy topology representing network enables highly accurate classification of protein images taken by cryo electron-microscope without masking drivers of recombinant soluble influenza a virus hemagglutinin and neuraminidase expression in mammalian cells key: cord-279463-bli8hwda authors: lipp, joachim; dobberstein, bernhard title: the membrane-spanning segment of invariant chain (iγ) contains a potentially cleavable signal sequence date: 1986-09-26 journal: cell doi: 10.1016/0092-8674(86)90710-5 sha: doc_id: 279463 cord_uid: bli8hwda abstract the human invariant chain (iγ) of class ii histocompatibility antigens spans the membrane of the endoplasmic reticulum once. it exposes a small amino-terminal domain on the cytoplasmic side and a carboxyterminal, glycosylated domain on the exoplasmic side of the membrane. when the exoplasmic domain of iγ is replaced by the cytoplasmic protein chloramphenicol acetyltransferase (cat), cat becomes the exoplasmic, glycosylated domain of the resulting membrane protein iγcat∗. deletion of the hydrophilic cytoplasmic domain from iγcat gives rise to a secreted protein from which an amino-terminal segment is cleaved, most likely by signal peptidase. we conclude that the membrane-spanning region of iγ contains a signal sequence in its amino-terminal half and that hydrophilic residues at the amino-terminal end of a signal sequence can determine cleavage by signal peptidase. the human invariant chain (ly) of class ii histocompatibility antigens spans the membrane of the endoplasmic reticulum once. it exposes a small amino-terminal domain on the cytoplasmic side and a carboxyterminal, glycosylated domain on the exoplasmic side of the membrane. when the exoplasmic domain of ly is replaced by the cytoplasmic protein chloramphenicol acetyltransferase (cat), cat becomes the exoplasmic, glycosylated domain of the resulting membrane protein i$at*. deletion of the hydrophilic cytoplasmic domain from l$xt gives rise to a secreted protein from which an amino-terminal segment is cleaved, most likely by signal peptidase. we conclude that the membranespanning region of ly contains a signal sequence in its amino-terminal half and that hydrophilic residues at the amino-terminal end of a signal sequence can determine cleavage by signal peptldase. translocation of proteins across the membrane of the endoplasmic reticulum (er) requires signal sequences and specific receptors that recognize them (see recent reviews by hortsch and meyer, 1984; walter et al., 1984; rapoport and wiedmann, 1985; wickner and lodish, 1985) . signal sequences have been found at the amino-terminal end of precursors for secretory and transmembrane proteins. in many cases they are cleaved during their translocation across the membrane by a specific protease (signal peptidase). signal sequences are quite variable in length, ranging from 16 to more than 50 amino acid residues (von heijne, 1983) . they all have a central core of hydrophobic amino acid residues, and most of them have a positively charged amino-terminal segment (von heijne, 1985) . signal sequences on nascent polypeptides are recognized by the signal recognition particle (srp), a ribonucleoprotein complex that mediates the interaction with the membrane by the selective binding to docking protein (or srp receptor) (walter et al., 1981b; meyer et al., 1982; gilmore et al., 1982) . membrane proteins are also inserted into the er membrane by an srp-mediated mechanism (anderson et al., 1983; rottier et al., 1985; spiess and lodish, 1986; lipp and dobberstein, 1986) . those spanning the membrane once have either the carboxyl terminus (type i membrane proteins) or the amino terminus (type ii membrane proteins) exposed on the cytoplasmic side. membrane insertion of type i membrane proteins most likely proceeds in a manner very similar to that of secretory proteins (lingappa et al., 1978) . type i membrane proteins are usually synthesized with a cleavable signal sequence and, in contrast to secretory proteins, are held in the membrane by a "stop transfer" sequence. examples of type i membrane proteins are the vesicular stomatitis virus g protein and class i and class ii histocompatibility antigens (lingappa et al., 1978; dobberstein et al., 1979) . of the type ii membrane proteins so far investigated, all are synthesized without a cleavable signal sequence. the neuraminidase of influenzavirus (60s et al., 1984) , the invariant chain (ii or ly) of class ii histocompatibility antigens (claesson et al., 1983; strubin et al., 1984; long, 1985; lipp and dobberstein, 1986) , the transferrin receptor (schneider et al., 1984) , and the asialoglycoprotein receptor (chiacchia and drickamer, 1984; holland et al., 1984; spiess and lodish, 1986 ) all belong to this class of membrane proteins. some steps in their membrane insertion must be similar to that of secretory and type i membrane proteins, as an srp-and docking protein-dependent membrane insertion has been demonstrated for some of them (spiess and lodish, 1986; lipp and dobberstein, 1986) . membrane insertion might occur in a loop-like fashion as this scheme can most easily explain how the different membrane topologies of membrane proteins are achieved (engelman and steitz, 1981) . as type ii membrane proteins contain only a single stretch of hydrophobic amino acid residues, this might function as a signal for membrane insertion as well as a membrane anchor (markoff et al., 1984; spiess and lodish, 1986) . to identify and characterize this sequence, we tested membrane insertion of the human invariant chain (ly) and several deletion and fusion proteins derived from it in a cell-free membrane insertion system. ly is a typical type ii membrane protein (claesson et al., 1983; strubin et al., 1984; lipp and dobberstein, 1986) . it exposes 30 amino-terminal residues on the cytoplasmic side, spans the membrane between residues 30 and 60, and exposes a large carboxy-terminal domain on the exoplasmic side. this domain has two sites for the addition of n-linked carbohydrate units. membrane insertion of ly requires srp and docking protein (lipp and dobberstein, 1986) . as the amino-terminal, cytoplasmic domain is hydrophilic and shows no resemblance to a signal sequence, it has been proposed that the membrane-spanning region, or part of it, functions as an internal, uncleavable signal sequence (dobberstein et al., 1983; claesson et al., 1983; lipp and dobberstein, 1986) . we demonstrate here that the membrane-spanning region of ly is composed of a potentially cleavable signal sequence fused to part of a membrane anchor, which together with the cytoplasmic domain determine the orientation of ly in the er membrane. deletion of the cytoplasmic domain exposes the signal sequence at the amino terminus of the membrane-spanning region, resulting in cleavage of this otherwise uncleaved signal. claesson et al., 1993) . ply. the complete ly coding and all of its 3' noncoding sequence was cloned behind the t5 promoter (p) in the pds5 expression vector. plycat, the portion downstream of the pstl site in ply was replaced by the chloramphenicol acetyltransferase (cat) gene resulting in an in-frame fusion protein. pan-lycat, the segment between the sau3a and sstll sites of plycat coding for the cytoplasmic domain was deleted. a new atg initiation codon right in front of the membrane-spanning segment is provided by the vector (see figure 4a ). the regions coding for protein are boxed. the membrane-spanning region of ly is indicated by loops; the hydrophilic domains by dots. cat-derived sequences are indicated by slanted lines. the position of n-linked glycosylation sites in ly and the potential n-linked glycosylation site in cat protein are indicated by an asterisk. relevant cleavage sites for restriction endonucleases are also indicated. protein segments that perform a particular function can be identified by their deletion or addition to unrelated proteins. we used this approach to localize and characterize the region in ly that is responsible for membrane insertion. deletions and fusions were made at the dna level after cloning of ly cdna into an expression vector. messenger rna was transcribed from these plasmids and translated in a cell-free system. the resulting proteins were tested for their ability to insert into microsomal membranes (blobel and dobberstein, 1975; stueber et al., 1984) . plasmids ply, plycat, and pan-lycat we have shown previously that cdna sequences cloned behind the strong t5 promoter in pds5 can be transcribed very efficiently by e. coli rna polymerase (stueber et al., 1985) . when transcription is performed in the presence of the cap analog 7mgpppa, the resulting mrna can be translated efficiently in eukaryotic cell-free systems. we have observed, however, that a stretch of gc residues at the 5' end of a cdna negatively affects expression of the resulting rna (unpublished observation). the ly cdna construct (py-2) had been gc tailed and was inserted into the pstl site of pbr322 (claesson et al., 1983) . we deleted the 5' gc tail and cloned ly cdna, or part of it, into the polylinker site of pds5 or p6/5r (see experimental procedures for details). ply contains the entire l-y coding region behind the t5 promoter ( figure 1 ). plycat is an in-frame fusion between the 5' region of ly encoding the cytoplas, mic, membrane-spanning segment plus 12 amino acids of the exoplasmic portion of ly and the gene encoding the cytoplasmic protein chloramphenicol acetyltransferase (cat). the cat protein contains one potential site for the addition of n-linked oligosaccharide 36 amino acid residues downstream of its original initiator methionine. in an-iycat, the entire hydrophilic, cytoplasmic segment from ly was deleted. the new initiator methionine is provided by the vector and is located in front of the hydrophobic segment. in vitro translation and membrane insertion of ly when ply was transcribed by e. coli rna polymerase and the resulting mrna translated in the wheat germ cell-free system, a single polypeptide species of 27 kd was obtained ( figure 2 , lane 1). this is the expected molecular weight for nonglycosylated ly (claesson et al., 1983) . when rough microsomes (rm), derived from dog pancreas, were added to the translation system, a higher molecular weight species of 33 kd appeared. this increase of 6 kd in molecular weight is consistent with the addition of two oligosaccharides to the two n-glycosylation sites. the 33 kd form ly* was reduced in molecular weight by about 2 kd when proteinase k was used to remove the cytoplasmically exposed domain (figure 2 , lanes 2 and 3). when protease digestion was performed in the presence of the detergent np 40, ly' was digested. these data suggest that ly' is integrated into the membrane and exposes 20-30 amino acid residues on the cytoplasmic side and a 30 kd domain on the exoplasmic side of the membrane. the identity of ly and its glycosylated form was confirmed by immunoprecipitation with antibodies raised against the amino-terminal 72 (anti-iyn) or against the carboxy-terminal 144 (anti-iyc) residues of ly. as shown in figure 2 , lanes 5, 6, 8, and 9, these antibodies recognize glycosylated and nonglycosylated forms of ly. no protein could be precipitated with anti-iyn antibody when the cytoplasmic domain was removed from membrane-integrated ly' by protease digestion (figure 2, lane 7) . as the antibody is directed against the amino-terminal portion of ly, the data directly demonstrate that the amino terminus is located on the cytoplasmic side and is accessible to the protease. with anti-iyc antibody, the processed form of ly is readily detectable, demonstrating an exoplasmic location of the carboxy-terminal portion of ly ( figure 2 , lane 10). membrane insertion of iycat an analysis of membrane insertion was performed for ly-cat and cat as described above for ly. cat was expressed from pds5. iycat was synthesized in the absence of microsomal membranes as a 34 kd protein ( figure 3 , lane 1) and in the presence of microsomal membranes as a 37 kd protein called lycat* ( figure 3 , lane 2). 12 3 4 in vitro translation and membrane insertion of ly ply was transcribed in the presence of the cap analog 7mgpppa by e. coli rna polymerase. the resulting mrna was translated in the wheat germ cell-free system in the absence (lanes 1,5. and 8) or presence (lanes 2, 3, 4, 6, 7, 9. and 10) of rm. the membrane topology of ly was determined by treatment with proteinase k (pk) (lanes 3, 7, and 10) or pk and the detergent np40 (lane 4). proteins were separated by sds-page and visualized by autoradiography. lanes 1-4 show total protein synthesized. samples characterized in lanes 5-7 were immunoprecipitated with an antibody raised against the amino-terminal 72 amino acid residues of ly (anti-iyn); in lanes 8-10, with an antibody against the carboxy-terminal portion of ly (anti-iyc). the increase in molecular weight is consistent with the addition of one n-linked oligosaccharide to the cat-derived portion. there is one potential site for n-linked glycosylation in the cat protein. after protease digestion in the presence of microsomal membranes, lycat* is reduced in molecular weight by about 2 kd, suggesting that it exposes 20-30 amino acid residues on the cytoplasmic side ( figure 3 , lanes 2 and 3). cat protein obtained after transcription-translation from pds5 is not modified by the added microsomes. as expected, no shift in molecular weight can be seen (figure 3 , lanes 5 and 6). cat protein was very resistant to protease digestion even in the presence of np40 (figure 3, lanes 7 and 8). i-&at, in contrast, was very sensitive to added protease. this might reflect a difference in conformation between the free cat protein and the cat-derived portion in iycat. the location of cat outside of the membrane vesicles can be demonstrated by sedimenting the membranes by centrifugation. cat protein is then found in the supernatant (data not shown). we conclude from the data obtained with ircat and cat that the signal for membrane insertion must be located within the first 72 amino acid residues of ly. to localize this signal more precisely, we deleted the first 30 residues of iycat. catinsertion of k&at and cat protein rna derived from plycat or pds5 was translated in the wheat germ cell-free system in the absence or the presence of rm. membrane insertion was tested by treatment with proteinase k (pk) and np40. addition of rm, pk, and np40 is indicated at the bottom of each lane. of an+cat in all secretory proteins the cleavable signal for membrane translocation is located at the amino-terminal end of the precursor polypeptide. the main feature of this signal appears to be its hydrophobicity. in ly the only hydrophobic stretch of amino acid residues that resembles a signal sequence is located in the membrane spanning region about 30 amino acid residues away from the amino-terminal initiator methionine. we asked whether removal of the 30 amino-terminal residues in iycat would affect its membrane insertion and topology. the cytoplasmic domain of iycat was deleted and the initiator methionine was placed in front of the membranespanning segment. the amino-terminal sequences of ly-cat and an-i$at as deduced from the dna sequences are shown in figure 4a . when rna derived from panlycat was translated in the wheat germ cell-free system, a single polypeptide of 29 kd was synthesized, an-iycat ( figure 46 , lane 1). this was, as expected, about 3 kd smaller than the iycat protein ( figure 46 , lane 1). in the presence of microsomes, two new protein bands appeared, one about 1 kd smaller and one 2 kd larger than an-ircat both of these forms were resistant to proteinase k, indicating that they were inserted into or translocated across microsomal membranes (figure 48 , lanes 3 and 4). we suspected that the smaller molecular weight form was generated by signal peptidase cleavage without concomitant glycosylation and that the larger molecular weight form was glycosylated and cleaved by signal peptidase. these possibilities were tested. from pan-lycat was translated in the wheat germ cell-free system in the absence or presence of rm. membrane insertion and topology was tested by treatment with proteinase k (pk) and np40. components were added as indicated below the lanes. iycat translated in the wheat germ cell-free system is shown for comparison. processed and glycoeylated to detect the signal peptide cleavage of a glycosylated protein on a polyacrylamide gel it is necessary to block its glycosylation, but still allow membrane insertion to occur. addition of n-linked oligosaccharides onto nascent polypeptides can be blocked by including synthetic acceptor peptides in an in vitro membrane insertion assay (bause, 1983; lau et al., 1983) . iycat and an-iycat were translated in the presence of microsomes with and without the acceptor peptide asn-leu-thr. the size of iycat synthesized in the presence of rm and acceptor peptide was indistinguishable from that made in the absence of rm. when proteinase k was used to digest its cytoplasmically exposed domain, the size was reduced by about 2-3 kd ( figure 5a ). we can conclude that nonglycosylated iycat synthesized in the presence of rm and acceptor peptide is inserted into the membrane in the same way as its glycosylated form and that no signal sequence is cleaved during membrane translocation ( figure 5a figure 58, lanes 2 and 3) . an-iycat'was also found to be protected against exogenous proteinase k ( figure 56, lane 4) . this suggested to us that the larger form was glycosylated and proteolytically processed and that an-iycat' was generated by a proteolytic cleavage, most likely by signal peptidase. to determine the site of cleavage in the proteolytically processed forms of an-ircat, the positions of leucine in the amino-terminal regions of an-itcat and membraneinserted an-lrcat*' were determined. an-i$at was translated in the absence or presence of rm with [sh]leutine as label. as an-iycat is essentially the only protein synthesized from pan-lycat-derived mrna, the complete translation mixture was subjected to automated edman degradation. as seen in figure 6a , leucine residues are found at the positions 3, 10, 13, 14, and 15, as predicted from the sequence deduced from py-2 cdna (claesson et al., 1983) . the initiator methionine is probably removed during or shortly after translation (kozak, 1983) . the positions of leucine residues in the membranetranslocated forms of an-i$at were similarly determined. as rm in the in vitro assay do not translocate all chains, some cytoplasmic forms remained (see inserts in figures 6a and 6b ). leucine residues were found at positions 1,2,3, and 13 ( figure 66) . larger peaks at positions 3 and 10 are consistent with the presence of some unprocessed an-iycat (see insert in figure 6b ). taking into account the size reduction of about 1 kd by the processing 13, 14, and 15 in authentic an-i$at, we conclude that processing has occurred between amino acid residues 12 and 13 ( figures 6b and 6c ). proteolytically processed an-lycat is translocated into the lumen of microsomal vesicles with the proteolytic removal of 12 of the 30 hydrophobic amino acid residues in the membrane-spanning region of an-iycat, the question arose as to whether the processed protein was still anchored in the membrane or whether it was now released into the lumen of the microsomal vesicles as is the case for secretory proteins. we used the extractability with carbonate as a criterion for membrane integration. treatment of rm with carbonate at ph 11 releases proteins that are not integrated into the lipid bilayer as well as proteins present in the lumen of microsomal vesicles. an+cat was translated in the presence of rm. membranes were isolated by centrifugation through a sucrose cushion and resuspended in carbonate buffer. solubilized components were then separated from membranes by centrifugation. proteins in the membrane pellet and supernatant were analyzed by sds-page and autoradiography. membrane-spanning proteins, ly and iycat, and the secretory protein, mouse granulocyte-macrophage colony stimulating factor (gm-csf), were used as control (gough et al., 1985) . as is shown in figure 7 , ly" and i-&at*, as expected for membrane-spanning proteins, were found in the membrane fraction. both an-i$at' and the gm-csf' were found essentially in the soluble, carbonatereleased fraction. thus an-iycat' is released after the proteolytic processing into the lumen of the microsomal vesicles. proteolytic processing, as described above for an-iycat, was also obtained for an+, a protein that lacks the amino-terminal 30 residues of ly (data not shown). our results show that the membrane-spanning segment of the type ii membrane protein ly contains a potentially cleavable signal sequence. this signal sequence is located in the amino-terminal half of the membrane-spanning segment, and it is cleaved when the preceding cytoplasmic domain is removed. all properties known to identify a signal sequence and a cleavage by signal peptidase can be demonstrated. to restrict vertical mobility of the membranespanning segment. (6) an-itcat, during its initial stage of membrane insertion, also spans the membrane with its hydrophobic segment. however, as no charged amino acid residues are present at the extreme amino-terminal end, the hydrophobic segment has some freedom to change its topology across the membrane. part of the hydrophobic segment might now be pulled into the lumen of the er membrane, and a former cryptic site for signal peptidase cleavage might become accessible to the active center of signal peptidase. first, the cleavage occurs concomitant with insertion into the er membrane as is typical for cleavable signal sequences of presecretory proteins (blobel and dobberstein, 1975) . second, the cleaved segment is located at the aminoterminal end of the deletion protein an-iycat. it is 13 amino acid residues long and composed entirely of hydrophobic or uncharged residues. signal sequences can vary in length from about 15 to over 60 residues. the only structural element identified so far for a signal sequence is its hydrophobic core, usually 8-12 residues long. it is followed by a more polar region 5-7 residues long, which is thought to define the cleavage site for signal peptidase. thus, a "minimal" signal sequence would be composed of an 8 residue hydrophobic core followed by a 5 residue region conferring cleavage specificity (von heijne, 1983 (von heijne, , 1985 . the segment cleaved from protein an-iycat would be consistent with such a minimal length signal sequence. finally, the amino acid residues around the cleavage site in membrane-translocated an-lycat*' are consistent with cleavage by signal peptidase. based on a sequence comparison of 78 eukaryotic signal sequences, von heijne found that only small neutral residues are found at the site of cleavage (-1 position) and that only small neutral and uncharged ones are found at the -3 position, that is 3 amino acid residues in front of the signal peptidase cleavage site (von heijne, 1983) . in the segment cleaved from an-i$at, threonine, a small neutral amino acid, is found at the -1 position, and leucine, an uncharged amino acid, at the -3 position. both of these residues fulfill the above described criteria for a signal peptidase cleavage site. thus, place (rm) and time of cleavage (cotranslational), hydrophobic character of the cleaved segment, and property of the cleavage site demonstrate that an-i$at contains a signal sequence at its amino terminus which is cleaved upon membrane insertion by signal peptidase. how can we possibly explain how the deletion of the cytoplasmic, hydrophilic segment from iycat reveals a cleavable signal sequence in a formerly membranespanning region? to us the most plausible explanation is that the position of the hydrophobic segment in the membrane is different in iycat and an-i$at signal peptidase is known to be an integral membrane protein not exposed on the cytoplasmic side of rm (jackson and blobel, 1977; lively and walsh, 1983; evans et al., 1986) . as in many secretory proteins, the cleavage site for signal peptidase is surrounded on either side by 1 or even 2 charged amino acid residues. it is reasonable to assume that the active center of this enzyme is located close to the exoplasmic side of the er membrane, not within the membrane. we propose that the removal of the cytoplasmic, hydrophilic segment from iycat allows the hydrophobic segment to shift its position within the er membrane. most likely it positions itself more toward the exoplasmic side. hence, a potential signal peptidase cleavage site becomes accessible to the active center of signal peptidase (see figure 8b ). it has been noted previously in type i membrane proteins that a deletion of the charged amino acid residues flanking the membrane-spanning region does not affect the overall topology (zuninga and hood, 1986; cutler et al., 1986) . in the case of es glycoprotein of semliki forest virus, it has been shown that mutation of the basic amino acid residues at the cytoplasmic side of the membranespanning segment reduces the stability of the mutant protein in the membrane (cutler et al., 1986) . when the membrane-spanning regions of type i and type ii membrane proteins are compared, no obvious structural difference can be found. in both types of membrane proteins these regions comprise a stretch of 20 to 30 hydrophobic amino acid residues that is flanked on the cytoplasmic side by positively charged amino acid residues. in type i membrane proteins the segment spanning the membrane does not appear to participate in the initial stage of membrane insertion. type i membrane proteins usually have cleavable signal sequences that initiate the membrane translocation of the amino-terminal half of the protein. the membrane-spanning region, in its position close to the carboxy-terminal end, seems only to function in anchoring the protein in the membrane. yost et al. placed the membrane-spanning segment of the murine surface immunoglobulin heavy chain close to the amino-terminal end of a fusion protein (yost et al., 1983) . in this position the segment did not provide the signal function for membrane insertion. as, however, a hydrophilic segment of about 40 amino acid residues precedes the membrane-spanning segment, the question still remains as to whether a membrane-spanning region from a type i membrane protein, when placed into the appropriate surrounding, can also initiate translocation across the er membrane. it is well conceivable that certain hydrophilic sequences preceding a hydrophobic segment play a crucial role in exposing a potential signal for membrane insertion. up to now no special structural features, besides hydrophobicity, are known to be crucial for the function of a signal sequence. a common step has been proposed for the early stage of membrane insertion of secretory and membrane proteins (dobberstein et al., 1983; spiess and lodish, 1986; lipp and dobberstein, 1986) . this was based largely on the finding that both of these types of proteins require srp and docking protein for their membrane insertion. here, we show that a type ii membrane protein can be converted into a secretory protein by removal of the cytoplasmic segment. this directly demonstrates that the signal for membrane insertion of these two types of proteins can be the same. further deletion into the carboxy-terminal half of the ly hydrophobic segment is required to elucidate whether the cleaved signal sequence contains all the information for membrane insertion. it is conceivable that the functional signal sequence extends over the cleaved signal sequence into the adjacent hydrophobic part. for some secretory protein it has been observed that the cleavable signal sequence is not sufficient for membrane insertion. in the case of staphylococcal protein a, sequences of the amino-terminal part of the mature protein are required for membrane insertion and correct processing (abrahmsen et al., 1985) . srp can arrest elongation of presecretory and type ii membrane proteins after 70 or even more amino acid residues have been polymerized (walter and blobel, 1981a; meyer et al., 1982; lipp and dobberstein, 1986; lipp et al., unpublished data) . these domains are then inserted into the er membrane by a yet unknown mechanism. as the amino terminus of a type ii membrane protein has to remain on the cytoplasmic side, the formation of a loop during membrane insertion has been proposed. in the case of a secretory protein, signal peptidase would be able to act as soon as the loop appears on the exoplasmic side. an initial interaction of basic residues in a signal sequence with the phosphates of the membrane lipids was originally proposed by lnouye for the lipoprotein of e. coli (inouye et al., 1977) . our results rule out an essential role of these basic residues in er membrane insertion. the an-iycat protein does not contain any charged amino acid residues preceding the hydrophobic segment. it is nevertheless translocated across the er membrane and processed. the rules that define the cleavage site for signal peptidase in presecretory proteins are not yet fully understood. von heijne points out that the type of amino acids at the -1 and -3 position in front of the site of cleavage are im-portant in assigning a cleavage site. here we show that sequences at the very beginning of a signal sequence can also influence cleavage by signal peptidase. in the case of ly, these charged residues can prevent cleavage by signal peptidase. the variability in the length and in the amount of charged amino acid residues at the amino terminus of asignal sequence has not as yet been explained. mutation and deletion experiments have clearly shown that charged residues are not essential for membrane insertion. in the light of our findings, we propose that the charged amino acids at the amino terminus of signal sequences function in the alignment of signal sequences in the er membrane such that signal peptidase can cleave at a very specific site with high fidelity. our prediction is that removal of charged residues from the amino-terminal end of the signal sequences can lead to an altered or less specific signal peptidase cleavage. wheat germ was obtained from general mills, california. the acceptor peptide benzoyl-asn-leuthr-n-methylamide was a generous gift from e. bause, cologne. standard molecular cloning techniques, as described by maniatis et al. (1962) were used. the cdna clone py-2, containing the entire coding region of the human invariant chain cloned into the pstl site of pbr322, was obtained from p. a. peterson's laboratory, uppsala, sweden (claesson et al., 1963) . the expression plasmids pds5, pds6, and pds5/3 have been described previously (stueber et al., 1964) . they allow efficient transcription by e. coli rna polymerase of cdnas cloned behind the strong t5 promoter p25. figures 9a and 9b summarize the construction of the fusion and deletion plasmids described below. plycat py-2 was digested with pstl, and the 317 bp fragment containing the 5' end and the 660 bp fragment containing the 3' end of the ly coding region were isolated. the 317 bp fragment coding for the ly cytoplasmic domain, the membrane-spanning segment and 12 amino acid residues of the exoplasmic domain, was cleaved by sauda to remove the sgc tail. the 234 bp sau3a-pstl fragment was isolated and cloned into bamhiipstl-cut pds5. this results in an in-frame fusion of the 5' end of ly to the cat gene. ph initial attempts to clone the completely coding region into pds5 failed. when expressed, this region is probably lethal to the bacterium. to repress transcription from the t5 promoter/operator (p/o) in bacteria, we cloned the lac i repressor between the b/a gene and the t5 p/o. this plasmid is called pfllycat. for the construction of ply, prlycat was linearized by pstl and the 660 bp pstl fragment, coding for the carboxy-terminal domain of it, was ligated into this site. transformants containing the 660 bp fragment were screened for expression of immunoprecipitable ly chain after in vitro transcription-translation. to delete the cytoplasmic domain from ircat, the 950 bp sstll-xbal fragment from plycat was isolated and ligated at the xbal site of bamhllxbal cut p6/5r. the protruding ends at the bamhl and the sstll sites were blunted with sl nuclease and ligated. as a result, a new atg initiation codon is placed just in front of the membrane-spanning segment of ly. the construction was confirmed by dna and amino acid sequence analyses (see figure 4a ). p6lsr to repress transcription from the t5 promoter the lac i gene was inserted between the b/a gene and the t5 p/o region of pds5/3 (stueber et al., 1984) . against ly domains to raise antibodies against the amino-and the carboxy-terminal domains of ly, fusion proteins of b-galactosidase and parts of ly were produced in bacteria and used as antigens to raise antibodies in rabbits. from a pstl digest of w-2, the 317 bp fragment coding for the aminoterminal 72 amino acids of ly and the 860 bp fragment coding for the exoplasmic carboxy-terminal domain of ly were isolated. each of the fragments was inserted into the pstl site of the bacterial expression vector pex1 (stanley and luzio, 1984) . fusion proteins expressed in nfl bacteria were separated on preparative sds-polyacrylamide gels (7% acrylamide; laemmli, 1970) . protein bands were visualized by koac precipitation, and fusion proteins were eluted from gel slices. two rabbits were immunized with each of the two fusion proteins. antibodies against the amino terminus of ly (anti-iyn) and its carboxyl terminus (anti-iyc) were obtained. they reacted with authentic ly chains synthesized by human raji cells (data not shown). lmmunoprecipitations after translation and posttranslational assays, antigens in a 25 vi aliquot were solubilized by adding nonidet-p40 (np40) to 0.5%. then 1 ~1 of either anti-iyn or anti+c antiserum was added and the mixtures incubated for 15 min at 4oc. forty microliters of a 1:l slurry of protein a-sepharose (equilibrated in 0.2% np40,lo mm tris-hci [ph 7.51, 150 mm naci, and 2 mm edta) was added to each sample, and incubation continued for 60 min at 4°c. beads were sedimented by centrifugation and washed three times with 0.2% np40, 10 mm tris-hci (ph 7.5), 150 mm naci, and 2 mm edta, twice with 0.2% np40,lo mm tris-hci (ph 7.5), 500 mm naci, and 2 mm edta, and once with 10 mm tris-hci (ph 7.5). sample buffer for sds-page was added to the sedimented beads, and antigens were analyzed by sds-page and fluorography. in vitro transcription and translation plasmids were transcribed in vitro by e. coli rna polymerase, and the resulting mrna was translated in a wheat germ cell-free system as described by stueber et al. (1984) . to test for membrane translocation, rough microsomes from dog pancreas were included in the translation (blobel and dobberstein, 1975) . glycosylation onto asparagine residues was blocked by the addition of the acceptor peptide benzoylasn-leuthr-n-methylamide to a final concentration of 30 pm (lau et al., 1983; bause, 1983) . assays to test translocation of in vitro-synthesized proteins across, or their insertion into, the er membrane, accessibility to proteinase k was used. a 10 pl aliquot of a translation mixture containing rough microsomes was incubated for 10 min at 25oc with either 0.3 mg/ml of proteinase k or 0.3 mg/ml of proteinase k and 0.5% np40. further proteolysis was stopped by the addition of phenylmethylsulfonyl fluoride (pmsf) to 0.1 mglml, and the sample was further characterized by sds-page (laemmli, 1970) and fluorography or, where indicated in the figure, by immunoprecipitation. to remove secretory and peripheral membrane proteins, rough microsomes were subjected to a carbonate wash with 0.1 m na&os, ph 11 (fujiki et al., 1982) . peptide; h. gausepohl for performing automated amino acid analysis: m. t. haeuptle, i. ibrahimi, and d. meyer for critical reading of the manuscript, and annie steiner for expert typing. this work was supported by grant do 199/4-z from the deutsche forschungsgemeinschaft. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked "advertisement" in accordance with 18 usc. section 1734 solely to indicate this fact. received april 23, 1986; revised july 1, 1986. multiple mechanisms of protein insertion into and across membranes a stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems clonal variation in cell surface display of an h-2 protein lacking a cytoplasmic tail we thank p a. peterson and l. claesson, uppsala, for plasmid py-2; e. bause, cologne, for the acceptor abrahamsen, l., moks, t., nilsson, b., hellman, u., and uhlen, m. (1985) . analysis of signals for secretion in the staphylococcal protein a gene. embo j. 4, 3901-3906. adams, g. a., and rose, j. k. (1985) . structural requirements for a membrane-spanning domain for protein anchoring and cell surface transport.cell 47, looi-1015.anderson, d. j., mostov, k. e., and blob& g. (1983) . mechanisms of integration of de novo-synthesized polypeptides into membranes: signal recognition particle is required for integration into microsomal membranes of calcium atpase and of lens mp26 but not of cytochrome be. proc. natl. acad. sci. usa 80, 7249-7253. bause, e. (1983) . structural requirements of n-glycosylation of proteins. biochem. j. 209, 331-336. blobel, g., and dobberstein, b. (1975) lingappa, v. r., katz, f. n., lodish, h. f., and blobel, g. (1978) . a signal sequence for the insertion of a transmembrane glycoprotein. similarities to the signals of secretory proteins in primary structure and function. j. biol. chem. 253, 8667-8670.lipp, j., and dobberstein, b. (1986). signal recognition particle-dependent membrane insertion of mouse invariant chain: a membrane spanning protein with a cytoplasmically exposed amino-terminus. j. cell biol. 702, 2169 -2175 . long, e. 0. (1985 . in search of a function for the invariant chain associated with la antigens. surv. immunol. res. 4, 27-34. lively, m. o., and walsh, k. a. (1983) spiess. m., and lodish. h. f. (1986) . an internal signal sequence: the asialoglycoprotein receptor membrane anchor. cell 44, 177-165. stanley, k. k., and luzio, j. p (1984) . construction of a new family of high efficiency bacterial expression vectors: identification of cdna clones coding for human liver proteins. embo j. 3, 1429 -1434 . strubin, m., mach, b., and long, e. 0. (1984 . the complete sequence of the mrna for the hla-dr associated invariant chain reveals a polypeptide with an unusual transmembrane polarity. embo j. 3,869~872. key: cord-276988-bvsz5q6d authors: neu, carolin t.; gutschner, tony; haemmerle, monika title: post-transcriptional expression control in platelet biogenesis and function date: 2020-10-15 journal: int j mol sci doi: 10.3390/ijms21207614 sha: doc_id: 276988 cord_uid: bvsz5q6d platelets are highly abundant cell fragments of the peripheral blood that originate from megakaryocytes. beside their well-known role in wound healing and hemostasis, they are emerging mediators of the immune response and implicated in a variety of pathophysiological conditions including cancer. despite their anucleate nature, they harbor a diverse set of rnas, which are subject to an active sorting mechanism from megakaryocytes into proplatelets and affect platelet biogenesis and function. however, sorting mechanisms are poorly understood, but rna-binding proteins (rbps) have been suggested to play a crucial role. moreover, rbps may regulate rna translation and decay following platelet activation. in concert with other regulators, including micrornas, long non-coding and circular rnas, rbps control multiple steps of the platelet life cycle. in this review, we will highlight the different rna species within platelets and their impact on megakaryopoiesis, platelet biogenesis and platelet function. additionally, we will focus on the currently known concepts of post-transcriptional control mechanisms important for rna fate within platelets with a special emphasis on rbps. blood platelets-the major players in hemostasis-are small anucleate cell fragments with a characteristic discoid shape and a diameter of 1 to 3 µm that originate from megakaryocytes (mks). platelets are the second most abundant "cell" type in the peripheral blood and constitutive renewal ensures a normal platelet count in the blood, despite their relatively short life span of 7-10 days [1, 2] . platelets are also interesting from a clinical point of view. platelet counts, which range from 150,000 to 350,000/µl of whole blood in healthy subjects, can drastically increase and decrease in pathophysiological conditions, resulting in thrombocytosis or thrombocytopenia, respectively [3] . thus, it is not surprising that a wide range of human pathologies are associated with abnormal platelet counts and/or functions like inflammatory diseases [4] , rheumatoid arthritis [5] , diabetes [6] , pulmonary hypertension [7] , alzheimer's disease [8] , cardiovascular disease [9] , and cancer [10, 11] . the unique life cycle of platelets starts with proplatelet biogenesis from mks in response to certain stimuli. mks develop from hematopoietic stem cells (hscs) present in the bone marrow, yolk sac, fetal liver, and spleen [12] . as a prerequisite, mks initially have to grow and multiply their genetic information via endomitosis. these processes are highly dependent on thrombopoietin (tpo), a cytokine that promotes the growth and development of mks from their precursors and that associates with its mk-specific receptor, i.e., cluster of differentiation 110 (cd110), encoded by the cellular myeloproliferative leukemia virus (c-mpl) oncogene [13] [14] [15] . both c-mpl −/− and tpo −/− mice are thrombocytopenic and have reduced numbers of mks in the bone marrow [16] [17] [18] . however, although platelet and mk numbers were significantly reduced, the remaining mks and platelets were structurally normal and platelets were functional as they were still able to form clots and responded to agonists [19] . this suggests that other regulatory factors exist [20] . furthermore, endomitosis, a process during which mks accumulate dna content ranging from 2n to 128n in their big polylobulated nucleus, also contributes to platelet formation and the degree of mk ploidy was shown to influence platelet quantity and quality [21, 22] . mechanistically, endomitosis is a process by which mks become polyploid through repeated rounds of dna replication without cell division, which requires both coordination and restriction of cell cycle components [23] [24] [25] . importantly, endomitosis is triggered by tpo and is necessary for mks to proceed with their final maturation and subsequent proplatelet formation [22] . another purpose of endomitosis is to produce large amounts of proteins and lipids necessary for the mk demarcation membrane system (dms) that serves as a membrane reservoir for proplatelet formation. the origin of the dms likely involves invaginations of the mk plasma membrane, de novo membrane synthesis, as well as contributions from the golgi-derived membranes and close endoplasmic reticulum (er) contacts [26] . in the final step of platelet biogenesis, terminally differentiated mks remodel their cytoplasm to form long protrusions, so-called proplatelets, which extend into bone marrow sinusoids and are the progenitors of mature platelets [27] . however, terminal platelet formation might not end in the bone marrow sinusoids, but likely continues in the circulation and was shown to be substantial in the lung [28, 29] . of note, in an attempt to delineate the final steps of proplatelet maturation and platelet release, a large (2-10 µm) intermediate discoid stage in platelet production, the preplatelet, was identified [30] . preplatelets are anucleate discs that have a thick cortical microtubule coil and are able to convert back into barbell-shaped proplatelets. final platelet release is achieved through continued bidirectional polymerization of microtubules at each end of the proplatelet followed by a fission event. this multi-step biogenesis yields approximately 100 billion platelets each day in an adult human, but these rates can increase by a factor of 10 or more when the demand increases [31] . interestingly, in recent years ex vivo protocols have been developed to generate functional platelets from megakaryocytes, which are differentiated from progenitor/stem cells. these models are generally based on bioreactors with biocompatible porous barriers that support mk function and platelet release is supported by media flow obtained by electronic pumps. these tissue models are especially important because platelet transfusions are common, but supply does not always match demand [32, 33] . however, if cultured platelets are to become a considerable clinical alternative, they must match the quality and functionality of donor-derived platelets. therefore, a better understanding of the mechanisms responsible for mk maturation, platelet generation and release is necessary [34] . mature platelets were first discovered by bizzozero in the late 19th century and have since been ascribed a primarily hemostatic role. while circulating in an inactive state in the blood stream, platelets become activated upon exposure to subendothelial collagen to support thrombus formation, highlighting their outstanding role as first responders in wound healing and hemostasis [35] . normally, intact endothelium expresses and releases factors that prevent platelets from activation and aggregation and block fibrin clot formation. after endothelial injury, subendothelial collagen gets exposed to the blood and initiates activation and adhesion of platelets via von willebrand factor. expression of tissue factor (tf) finally activates a sequence of events to establish a stable clot that seals the injury site and prevents excessive blood loss [36] . in the last centuries, it has become evident that platelets are not only implicated in hemostasis and thrombosis, but also act as key players in infectious and sterile inflammatory responses as well as cancer [11, 37] . many of the non-hemostatic functions of platelets result from the storage and release of bioactive molecules. these molecules, be they platelet proteins, lipids, growth factors, or cytokines, are mainly found in alpha (α) and dense (δ) granules [38] . the biogenesis of these granules begins in the mk, but maturation continues in circulating platelets [39, 40] . initially, it was hypothesized that granule packaging and release is a random process, however, recent studies suggested that platelets contain heterogeneous populations of granules that differentially release their content upon different physiological stimuli [41, 42] . besides their protein content, platelets contain different nucleic acids including small and long non-coding rnas and protein-coding messenger rnas (mrnas). first thought to be incapable of regulated gene expression, researchers started to shed light on the translational machinery and its regulation in platelets in the absence of a nucleus. subsequent studies revealed that platelets possess a functional spliceosome, allowing for rapid adaptations to changing environmental cues and a change of phenotype [43] . in recent years, in-depth transcriptional analyses of platelets have been performed suggesting that as many as 3000-6000 mrnas are contained in platelets [44] [45] [46] . however, the most intriguing question is: how and where do platelets get their content from? while the precise mechanisms are still largely unknown, it is becoming evident that the inheritance of proteins and mrnas from the megakaryocytes is not a random but highly regulated process. this review will highlight the different classes of rna species enriched in platelets, will focus on the role of non-coding rnas in platelet biogenesis and function and will feature the role of post-transcriptional control mechanisms with a special emphasis on the role of rna-binding proteins. a dynamic transcriptome is a prerequisite for platelet function, enabling rapid responses to external stimuli. however, rna abundance in platelets is~1000 times less than in leukocytes, leading to challenges when it comes to transcriptome analysis of platelets, as high purity samples are crucial. despite these challenges, independent studies have found a diverse set of nucleic acids within platelets such as different classes of rnas [43, [47] [48] [49] [50] as well as mitochondrial dna (mtdna) [45] . interestingly, it has been observed that the content of total rna decreases over time and young, reticulated platelets show 20-40 times higher rna levels compared to older ones, which indicates either decay and/or release of rna during the lifespan of platelets [51] . the bulk of platelet rna is derived from megakaryocytes, probably involving active transport mechanisms for sorting into platelets. in addition, other cellular sources might exist as well given the intimate interaction of platelets with other cell-types in the circulation or within tissue microenvironments. figure 1 summarizes the plethora of rna molecules in platelets including their source and additionally highlights the importance of intercellular communication via granule release and microvesicle shedding. besides their protein content, platelets contain different nucleic acids including small and long non-coding rnas and protein-coding messenger rnas (mrnas). first thought to be incapable of regulated gene expression, researchers started to shed light on the translational machinery and its regulation in platelets in the absence of a nucleus. subsequent studies revealed that platelets possess a functional spliceosome, allowing for rapid adaptations to changing environmental cues and a change of phenotype [43] . in recent years, in-depth transcriptional analyses of platelets have been performed suggesting that as many as 3000-6000 mrnas are contained in platelets [44] [45] [46] . however, the most intriguing question is: how and where do platelets get their content from? while the precise mechanisms are still largely unknown, it is becoming evident that the inheritance of proteins and mrnas from the megakaryocytes is not a random but highly regulated process. this review will highlight the different classes of rna species enriched in platelets, will focus on the role of non-coding rnas in platelet biogenesis and function and will feature the role of posttranscriptional control mechanisms with a special emphasis on the role of rna-binding proteins. a dynamic transcriptome is a prerequisite for platelet function, enabling rapid responses to external stimuli. however, rna abundance in platelets is ~1000 times less than in leukocytes, leading to challenges when it comes to transcriptome analysis of platelets, as high purity samples are crucial. despite these challenges, independent studies have found a diverse set of nucleic acids within platelets such as different classes of rnas [43, [47] [48] [49] [50] as well as mitochondrial dna (mtdna) [45] . interestingly, it has been observed that the content of total rna decreases over time and young, reticulated platelets show 20-40 times higher rna levels compared to older ones, which indicates either decay and/or release of rna during the lifespan of platelets [51] . the bulk of platelet rna is derived from megakaryocytes, probably involving active transport mechanisms for sorting into platelets. in addition, other cellular sources might exist as well given the intimate interaction of platelets with other cell-types in the circulation or within tissue microenvironments. figure 1 summarizes the plethora of rna molecules in platelets including their source and additionally highlights the importance of intercellular communication via granule release and microvesicle shedding. which are inherited from megakaryocytes. additionally, communications with surrounding cells might also influence rna expression landscape. transport of rna from megakaryocytes to platelets is not well understood, however, is suggested to be-at least in part-mediated by rna-binding proteins (rbps). pre-mrna and pre-mirna are processed by a functional spliceosome and by the presence of the cytoplasmic mirna processing into translate mrna and mature mirna, respectively, which can be signal-dependent. platelets may also host infectious virus particles and mediate dissemination of viruses in infected individuals. by secreting rna and protein contents by platelet granules or microvesicles, platelets may regulate signaling pathways in various interacting cells, including tumor or endothelial cells. among the different types of rna, mrnas are the best investigated class in platelets. they present typical features of eukaryotic mrnas like a 7-methylguanosine (m 7 g) 5 cap followed by a 5 -untranslated region (utr) as well as a 3 -utr and poly(a)-tail at their 3 -end. the m 7 g cap and poly(a) tail promote mrna translation and stability, while the utrs expose sequences for rna-binding proteins (rbps) and regulatory sites for microrna (mirna)-mediated translational and degradation control [45, [52] [53] [54] . getting a complete picture of the (dynamic) nature and composition of the platelets transcriptome is of great interest and next generation sequencing (ngs) has proven to be one of the preferred techniques for such studies [55, 56] . bulk platelet analyses demonstrated that platelets harbor a diverse set of mrnas and showed an enrichment of genes functionally linked to coagulation, platelet degranulation, cytoskeletal dynamics, receptor binding, secretion, and g-protein signaling, all being a prerequisite for a plethora of signaling pathways. along these lines, it is worth mentioning that platelets were shown to contain also pre-mrnas as well as small nuclear rnas (snrnas), which are functionally linked to gene expression and involved in pre-mrna splicing. in fact, denis et al. could show that platelets contain a complete set of snrnas as well as other essential splicing factors and are capable of signal-dependent splicing. here, in response to integrin engagement and surface receptor activation, platelets precisely excised introns from interleukin (il)-1β pre-mrna, thereby generating a mature mrna transcript that was translated into protein [43] . until then, it has been thought that splicing events only occur within nuclear boundaries, which has been challenged by these findings. however, since the initial discovery of a functional spliceosome in platelets, several other studies confirmed the existence and functional importance of splicing for platelet activation. for example, splicing upon platelet stimulation has also been shown for platelet factor xi (fxi) pre-mrna and significantly higher amounts of pre-mrna were found in resting compared to thrombin-activated platelets [57] . moreover, activation of platelets by fibrinogen and thrombin induced splicing of tissue factor (tf) pre-mrna leading to an increased tf protein expression, procoagulant activity, and accelerated formation of clots. mechanistically, it was revealed that tf pre-mrna splicing was dependent on cdc2-like kinase (clk)1-mediated splicing factor 2 (sf2) phosphorylation [58] . furthermore, lipopolysaccharide (lps) was shown to induce splicing, translation, and secretion of mature il-1β through tlr4 [59] . similarly, lps stimulation of platelets initiated splicing of cyclooxygenase-2 (cox-2) pre-mrna and ultimately production of the corresponding protein. however, this effect was extremely variable and it appeared that lps stimulated platelets either produced very little or non-active cox-2 protein [60] . of note, signal-dependent splicing, but also mrna degradation and translation might explain controversial findings regarding correlation between mrna and protein expression in platelets with some studies suggesting a weak correlation of the platelet transcriptome and proteome [61] , while others report a rather strong correlation, opening up the question on regulatory mechanisms of mrna metabolism in platelets under physiological and pathological conditions [62] . another intriguing aspect of the platelet transcriptome is the fact that the rna content of platelets is significantly influenced by interactions with surrounding cells including cancer cells. for example, best et al. recently discovered that tumor cells are able to educate platelets and induce an rna expression signature within platelets that could be used for non-invasive cancer diagnostics [63] [64] [65] [66] . moreover, a standardized protocol was established to enable isolation and analysis of spliced platelet mrna, which allowed detection of cancer with high accuracy [63] . this method has the potential to become clinically relevant, as platelet isolation is a rather simple standard procedure, already established in hematology laboratories. importantly, small amounts of only 100-500 pg of total platelet rna were sufficient for diagnostics and allowed separation of cancer patients from healthy individuals with 96% accuracy using mrna sequencing [64] . additionally, platelet rna signatures helped to distinguish patients with kirsten rat sarcoma viral oncogene homolog (kras), epidermal growth factor receptor (egfr) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (pik3ca) mutant and wild-type tumors, as well as patients with human epidermal growth factor receptor 2 (her2)-amplified versus her2-negative breast cancer and mesenchymal epithelial transition tyrosine kinase (met) overexpressing versus met non-overexpressing lung cancers [64] . although these studies seem promising, there are still some obstacles to be overcome such as the distinction between primary and metastatic tumors and the evaluation of cancer progression. one should also be aware of the fact that non-cancerous systemic factors like inflammation and cardiovascular events could influence platelet rna profiles as well [67] . however, using platelet-based mrna expression signatures in various diseases including cancer could open up new diagnostic avenues. taken together, rna expression profiling of platelets increased our understanding of the quantity and quality of their rna content. however, future studies are needed to shed new light onto the functional roles of these mrnas and their encoded proteins or whether these mrnas are simply the result of an inflammatory and/or pro-oncogenic environment. another class of rnas present in platelets is micrornas (mirnas). these 21-24 nucleotide (nt) spanning micrornas (mirnas) are known key regulators of eukaryotic gene expression [68] . alteration of mirna expression signatures has been observed in cancer and might predict prognosis and aid in diagnosis [69] . additionally, mirnas are important regulators of mk differentiation from hematopoietic progenitor cells [70] . microarray and rna-sequencing (rna-seq) analysis identified up to 532 different mirnas in human platelets [71] . interestingly, post-transcriptional modifications like adenylation and uridylation that are known for their regulatory impact on mirna stability and decay, also seem to play a role in mirna biology in platelets [72] . the relevance of mirnas in platelets has been recognized more and more in recent years and was particularly fueled by the discovery of a functional mirna processing machinery in platelets [49] . in general, mirna biogenesis starts with the transcription of primary mirnas (pri-mirnas), which are subsequently processed into precursor mirnas (pre-mirnas). a prerequisite for these initial steps is the micro-processor complex, consisting of nuclear rnase iii drosha and the digeorge syndrome critical region 8 (dgcr8), which is localized in the nucleus of cells [73] . following nuclear export, the cytoplasmic rnase iii enzyme dicer and trans-activation responsive rna-binding protein 2 (tarbp2) cooperatively cleave the stem of pre-mirna substrates yielding mature mirnas [74] [75] [76] [77] . landry et al. were the first to describe the cytoplasmic pre-mirna processing machinery in human platelets, including dicer and argonaute 2 (ago2) proteins [49] . in contrast, the nuclear microprocessor components drosha and dgcr8 could not be detected, consistent with the anucleate nature of platelets. using rnase activity assays, it was demonstrated that platelet dicer was functional and able to convert pre-let-7a-3 into mature mirna. additionally, platelet mirnas were able to mediate rna silencing as exemplified by the regulation of the purinergic receptor p2y12, which is involved in platelet aggregation, through an ago2/mir-223 effector complex contained in platelets [49] . moreover, functional ago2/mir-223 effector complexes were packaged into microparticles and released by platelets upon thrombin stimulation and subsequently internalized by endothelial cells, where they regulated f-box and wd repeat domain containing 7 (fbxw7) and ephrin a1 (efna1) mrna and protein levels [78] . additionally, platelet-secreted mir-223 was shown to promote endothelial cell apoptosis induced by advanced glycation end products via targeting the insulin-like growth factor 1 receptor (igf1r) [79] . moreover, rna-seq analysis of platelet mirnas in patients with myocardial infarction revealed nine differentially expressed platelet mirnas compared to healthy controls, which were released upon platelet aggregation and taken up by endothelial cells via a vesicle-dependent mechanism [80] . importantly, microvesicle-mediated mirna transfer to endothelial cells altered endothelial cell behavior and did not only influence endothelial cell survival but also inhibited tube formation as it has been shown for platelet-derived mirna let-7a, which targeted thrombospondin-1 (thbs-1) 3 -utr and inhibited thbs-1 release from endothelial cells [81] . next to endothelial cells, cancer cells also are recipients of platelet mirnas. for example, mir-223 expression was found to be significantly increased in platelets and platelet-derived microvesicles from non-small cell lung cancer (nsclc) patients as well as from mice intravenously injected with lewis lung carcinoma cells. of note, platelet microvesicles effectively delivered mir-223 to lung cancer cells and promoted cancer cell invasion by reducing erythrocyte membrane protein band 4.1-like 3 (epb41l3) levels [82] . these examples highlight the broad range of cell-platelet communication via mirnas, not only in the context of cancer. in fact, the majority of circulating mirnas derived from platelets and altered signatures of circulating mirnas were identified in patients with type 2 diabetes mellitus and with future myocardial infarction [83] [84] [85] . antiplatelet therapy using aspirin and prasugrel significantly decreased the level of platelet mirnas suggesting that plasma mirna expression could be used as surrogate markers of platelet activation and efficacy of antiplatelet therapy [83] . interestingly, the potential of mirnas as biomarkers of platelet activity in antiplatelet therapy monitoring has been recently reviewed elsewhere [86] . in summary, platelets inherit a functional mirna pathway from their mother cells which enables a complex crosstalk and post-transcriptional gene expression control based on the regulatory nature of mirnas [49] . thus, platelet-secreted mirnas are important modulators of signaling pathways in target cells and could serve as disease biomarkers, or as activation and therapy response markers. long non-coding rnas (lncrnas), of which some were identified in platelets [52] , constitute yet an additional group of regulatory non-protein-coding rnas. lncrnas can be subdivided into sense-, antisense-, intronic-, bidirectional-, and long intergenic ncrnas and are characterized by a minimum length of >200 nt [87] [88] [89] . importantly, lncrnas have been shown to play important roles in several human diseases, especially in cancer [90] [91] [92] [93] . in nsclc, the expression of lncrnas was analyzed in platelets in order to identify novel biomarkers of lung cancer. a selection of five lncrnas was identified and further measurements revealed a differential expression of two lncrnas, namely, membrane-associated guanylate kinase inverted 2-antisense rna 3 (magi2-as3) and zinc finger nfx1-type containing 1 antisense rna 1 (zfas1), in platelets of nsclc patients compared to healthy subjects [94] . both lncrnas have been assigned tumor suppressive functions in human breast cancer [95] [96] [97] , whereas zfas1 seems to be a potent oncogenic lncrna in other tumor types [98] . in platelets and plasma of nsclc patients, magi2-as3 and zfas1 were downregulated and their expression significantly correlated with tumor stage. in addition, magi2-as3 expression significantly correlated with lymph-node and distant metastasis and the authors suggested that both platelet-derived lncrnas could be used as potential diagnostic biomarkers in nsclc [94] . however, the molecular function of these and other lncrnas in platelets is currently not well understood and it is tempting to speculate about a potential transfer of lncrnas from platelets to diverse other cell-types to modulate expression programs and phenotypes. circular rnas (circrnas) belong to a group of highly stable non-coding rna-species with largely unknown functional relevance in platelets. in general, circrnas can act as sponges to sequester mirna or rbps. moreover, they serve as scaffolds to mediate complex formation between specific enzymes and substrates and recruit proteins to specific locations (reviewed in [99] ). circrna biogenesis involves the action of the spliceosome which connects the 5 and downstream 3 ends of exons within the same transcript resulting in a circular conformation which comes along with increased stability [100] . interestingly, circrnas were found to be abundant and highly enriched in human platelets as demonstrated by an analysis of publicly available rna-seq datasets of human platelets [50] . here, the authors analyzed three total rna-seq datasets using a back-splice exon junction discovery pipeline and identified 33,829 distinct structures consistent with circrnas. this number is about 15 times higher as reported for other cells and 24,632 (~73%) of the structures were also present in at least one other encyclopedia of dna elements (encode) rna-seq datasets previously mined [101] . in order to identify genes significantly enriched for circrnas in platelets, the authors analyzed 8041 circrna producing genes and identified 3162 significantly enriched. for the vast majority of enriched genes, the contribution of circrna exons to total transcription was >60% in nucleated tissues but >80% in platelets, and for 15 genes it was 100% in all three samples. thus, for some genes, only rna-seq reads derived from circrna-producing exons could be detected within platelet samples. importantly, circrnas were also enriched in other anucleate cells, but not in cultured megakaryocytes and the authors suggested that this was mainly due to a higher degradation rate of linear rnas compared to circrnas in mature platelets leading to the strong enrichment of the latter over time. intriguingly, extensive mrna degradation could explain some conflicting results concerning the correlation between the platelet transcriptome and proteome [50] . nevertheless, the biological relevance of platelet circrnas, if any, remains unclear: do they modulate platelet functions or are they simply byproducts of megakaryocyte transcription and platelet mrna degradation? compelling evidence against a rather random role of circrnas in platelets comes from the finding that they are differentially sorted into platelet-derived microvesicles via a specific, yet unknown mechanism [102] . the selective release of circrnas might represent a novel way of transferring information as vesicles from platelets to recipient cells, e.g., endothelial cells thereby inducing inflammatory responses. moreover, the same study could show that platelet circrnas associated with protein complexes of distinct sizes, so called circrnps, as demonstrated for six highly abundant circrnas, including a platelet-specific circrna, namely, plt-circr4 [102] . however, the circrna-specific binding partners and the functional significance of these interactions remain to be clarified. in summary, circrnas are highly enriched in platelets, they associate with proteins and can be released from platelets. however, not much is known about their regulatory roles in or outside platelets. in addition, their clinical implications remain unclear and it would be interesting to know if the abundance and/or composition of platelet circrnas varies depending on the age and health status of individual subjects. a fascinating aspect of platelets is the appearance of viral rna in platelets and their ability to aid in viral replication and the spread of virus in infected individuals [103, 104] . although both dna-and rna-containing viruses associate with platelets, only rna viruses are able to replicate, as platelets are incapable of dna transcription due to their anucleate nature. a study on platelets isolated from human immunodeficiency virus (hiv)-infected patients on antiretroviral drug therapy (art) revealed the occurrence of cytosolic hiv rna and intact viral particles [105] . noteworthy, replication competent viruses were only observed within closed vacuoles and not on the platelet surface. during the course of platelet elimination by tissue macrophages, hiv was transmitted from platelets to macrophages via phagocytosis. this process was prohibited by blocking platelet-macrophage interactions with the platelet-specific anti-integrin α iib /β3 antibody abciximab, underpinning the fact that viral spread is supported by intercellular interactions. most interestingly, a low cd4 + t cell count was predictive for the presence of hiv-containing platelets in patients receiving art, which correlated with immunological failure in these individuals [105] . in contrast, a recent study found no correlation of hiv + platelets with cd4 + t-cell count but with viral load and number of hiv + platelets were significantly reduced after art. this study additionally confirmed the ability of platelets to promote hiv viral spread, which was dependent on platelet activation and subsequent platelet-cd4 + t-cell complex formation [106] . importantly, platelet-virus interactions are not unique to hiv, but many other viruses were shown to infect platelets. platelets have been shown to be permissive to dengue virus entry, further enabling translation of viral rna, replication of the viral genome, and assembly of infectious virus. this study was further able to identify heparan sulfate proteoglycan (hsp) and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (dc-sign) as the major receptors for virus-platelet interactions [107] . other viruses that have been shown to interact with platelets, mostly via surface integrins, and are able to enter these include different adenoviruses, influenza virus and encephalomyocarditis virus (reviewed in [103, 104] ). starting in december 2019, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infections became a global problem with unforeseen consequences. some corona virus disease 2019 (covid-19) patients show hypercoagulation and thrombosis, which contributes to organ failure and worsened outcomes [108] . these clinical observations led to the hypothesis that platelets may play a role in covid-19 progression. protein or rna expression of the main receptor for sars-cov-2 binding, angiotensin-converting enzyme 2 (ace2), was not detected in platelets. nevertheless, mrna from the sars-cov-2 n1 gene was found in platelets of some covid-19 patients suggesting an ace2-independent entry of the virus into platelets [109, 110] . in addition, platelets might be attracted by the loss of endothelial integrity caused by the sars-cov-2 entry into endothelial cells, which ultimately results in platelet activation and degranulation thereby leading to a cytokine storm. thus, platelets might not only facilitate the dissemination of sars-cov-2 in infected individuals but might also contribute to hazardous thromboinflammation by the release of pro-inflammatory cytokines. moreover, megakaryocytes from the lungs might be susceptible to sars-cov-2, thereby generating sars-cov-2 rna-containing platelets with an altered transcriptome. rna-seq analysis of platelets isolated from severely ill covid-19 patients compared to healthy donors displayed significant differences in rna expression patterns associated with changes in platelet activation and aggregation [110] . whether platelets constitute a target for covid-19 therapy to alleviate the course of disease in severe cases will be an interesting topic for future research. as described above, platelets contain a diverse collection of coding and non-coding as well as linear and circular rnas, posing the question of the origins of these transcripts and the underlying molecular mechanisms, if any, that are responsible for the sorting of these rnas into platelets. intuitively, mks seem to be the prime source of platelet rnas and, indeed, mks transcribe a wide set of mrnas, which they pass on to their progeny in a highly regulated manner [111, 112] . as proven by transcriptome analysis, platelets contain thousands of mk-derived mrnas [45, [113] [114] [115] [116] [117] . careful analysis of rna expression data on matrix metalloproteinase (mmp) and tissue inhibitor of metalloproteinase (timp) mrnas uncovered different proportions of expression profiles in platelets compared to their abundancy in mks [118] . excluding mrna degradation or instability, cecchetti et al. hypothesized that mks differentially sort mrnas into platelets. this hypothesis is supported by the observation that mks expressed 10 out of 24 mmps and three out of four timps, yet several of these mrnas were missing or barely detectable in platelets, including transcripts encoding mmps 2, 9, 14, and 17 and timp-3. moreover, the abundance of a transcript in mks did not uniformly predict its expression level in platelets. for example, timp-1 mrna is expressed at high levels in both mks and platelets while timp-3 mrna is abundantly expressed in mks only, suggesting a regulated transfer of individual mrnas [118] . however, the exact sorting mechanisms remain elusive, but it was suggested that mks, similar to neurons, leverage rbps that bind to certain nascent transcripts in the nucleus thereby forming a complex that translocates to the cytoplasm. upon association with a kinesin motor, this complex is transported through the proplatelet by microtubules, and is ultimately delivered to the nascent platelet [119] . in fact, cecchetti et al. could detect the mrnas of a selected group of rbps, which were previously shown to be involved in rna localization, both in mks and platelets [118] . however, the expression and localization of the corresponding proteins await further investigation. in addition to mks, platelets may also acquire rna species by either direct or indirect interaction with surrounding cells in the circulation or in tissue microenvironments. in glioblastoma patients, platelets sequestered tumor-specific egfr variant iii (egfrviii) mrna, which was also detected in corresponding plasma samples [94] . in platelets from nsclc patients, echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (eml4-alk) rearrangements were detected on transcript level. the transfer of this rna was likely mediated via exosome shedding of cancer cells, thus confirming cross talk between platelets and tumor cells. most interestingly, serial monitoring of eml4-alk rearrangements in platelets showed a loss of detectable eml4-alk rearrangements after clinical responds to crizotinib treatment [120] . another intriguing, although likely indirect form of transfer of genetic information from cells to platelets might be emperipolesis, a process, where intact cells are present within the cytoplasm of another cell. histological examination of bone marrow routinely identifies mks, which have engulfed neutrophils or other hematopoietic cells. hence, emperipolesis is a physiological process that can be increased in pathological conditions, e.g., in gray platelet syndrome or essential thrombocythemia [121, 122] . recent studies have shown that emperipolesis of intact neutrophils in mks is mediated in part through the β2-integrin/ intercellular adhesion molecule 1 (icam-1)/ezrin pathway. neutrophil membranes merged with the dms and thereby transferred membrane material to daughter platelets, which accelerated platelet production [123] . intriguingly, some circulating platelets thus represent hybrids carrying both mk and neutrophil components. however, the mechanisms and functions of this conserved cellular phenomenon are largely unknown and whether emperipolesis is relevant for the transfer of rna into platelets should be investigated in the future. megakaryopoiesis, megakaryocyte maturation, as well as platelet formation, were shown to be highly complex processes that are regulated on multiple levels including epigenetic, transcriptional as well as post-transcriptional gene expression control mechanisms. indeed, several mirnas, which fine-tune gene expression at the post-transcriptional level, were shown to affect mk differentiation and platelet formation. for example, an inhibitory role for mir-155 during megakaryopoiesis was described, which is in line with the observed downregulation of this mirna during the differentiation of cd34+ hematopoietic stem cells along the megakaryocyte lineage [124] . in contrast, mir-150 was shown to support normal megakaryocyte differentiation by targeting the transcription factor myb [125, 126] . additionally, mir-130 targets the maf bzip transcription factor b (mafb), which increases with terminal mk differentiation and induces activation of the integrin alpha 2b (itga2b) gene [127] . very recently, mir-22 has been shown to promote megakaryocyte differentiation through repression of its target growth factor independent 1 transcriptional repressor (gfi1) [128] . ex vivo, megakaryocyte differentiation from hematopoietic stem/progenitor cells (hspcs) and generation of platelets was also shown to be regulated by mirnas. specifically, mir-125b and mir-660 increased the percentage of megakaryocytes with high ploidy and upregulated platelet biogenesis compared to controls. in contrast, overexpression of the mir-23a/27a/24-2 cluster in hspcs reduced colony formation associated with diminished megakaryocyte maturation and platelet production [129] . the importance of mirnas in the regulation of development and function of mks and platelets also becomes apparent upon mk-specific deletion of the ribonuclease dicer1 that is required for mirna biogenesis. knockdown of dicer1 in an in vivo mouse model led to reduced levels of mirnas in platelets and hence to an altered platelet mrna expression profile. the resulting phenotype was characterized by enhanced platelet reactivity and hemostatic function, presumably due to the upregulation of the fibrinogen receptor subunits integrin α iib and β 3 on the platelet surface in the absence of a proper mirna machinery [130] . furthermore, a global approach to identify mirnas and their respective targets in platelets detected protein kinase camp-dependent type ii regulatory subunit beta (prkar2b) as a mir-200b target, which functionally controlled platelet activation and altered levels of prkar2b led to impaired platelet reactivity [131] . moreover, hyperreactive platelets were found to have elevated levels of vesicle-associated membrane protein 8 (vamp8), a critical protein for granule release, which is a target of mir-96. overexpression of mir-96 restored normal vamp8 protein levels and thus confirmed the mir-96-vamp8 interaction [132] . importantly, alterations of mirna expression patterns have been linked to malignancies related to the megakaryocytic lineage, including essential thrombocythemia (et) [133] . in detail, it was shown that the expression of mir-9 and mir-490 significantly increased in platelets of et patients, while others, including mir-409 and mir-126, were downregulated. mirna expression was correlated with platelet aggregation and expression of platelet surface receptors including cd63 and p-selectin, suggesting that mirna may play a role in et, platelet maturation and function [133] . however, controversy exists as to what extent platelet-specific mirnas are important for platelet biology itself. some of the skepticism comes from genetic knockout studies in mice in which loss of mir-223, a highly abundant well-studied mirna in human platelets [134] , was dispensable for platelet production, both in terms of number and size as well as platelet functionality including overall life-span and surface expression of platelet adhesion receptors important for platelet activation like the adenosine diphosphate (adp) receptor p2y12 [135] . however, the lack of an overt phenotype might be due compensatory mechanisms as well as redundant targeting and functional rescue by other mirnas. moreover, lack of regulation of well-known mir-223 target transcripts might be explained by the lack of binding sites in the 3 -utr of murine as compared to human genes as evidenced in the case of p2y12 [136] . next to mirnas, lncrnas have also been implicated in the control of megakaryopoiesis. in general, lncrnas can regulate several cellular processes including differentiation applying a broad range of epigenetic, transcriptional as well as post-transcriptional mechanisms [90, 137] . for example, the expression of the human rna binding motif protein 15 (rbm15) was shown be positively regulated by its antisense lncrna, as-rbm15, during megakaryopoiesis [138] . importantly, the rna-binding protein rbm15 is known for its role in promoting terminal megakaryocyte differentiation through its impact on the alternative splicing of key transcription factors [139] . intriguingly, rbm15 protein synthesis underlies a complex regulatory mechanism itself involving as-rbm15 and the transcription factor runx1 [138] . specifically, as-rbm15, which is transcribed in the opposite direction within exon 1 of rbm15, was shown to enhance 5 cap-dependent protein translation of rbm15. this might be achieved via putative complementary binding of exon 1 of as-rbm15 deep within the 5 -utr of rbm15 and its incorporation into the rbm15 mrna-containing polysomes. both as-rbm15 and rbm15 transcriptions were activated by the transcription factor runx1 and repressed by the leukemic fusion protein runx1-eto, suggesting that as-rbm15 is not only important for megakaryocyte differentiation, but might also be implicated in leukemogenesis [138] . additional, although indirect, evidence that indicated a functional role of lncrnas in mks is provided by a transcriptome analysis, which identified 1109 polyadenylated lncrnas expressed in erythroblasts, megakaryocytes, and megakaryocyte-erythroid precursors of mice, and 594 lncrnas expressed in human erythroblasts [140] . importantly, it was recently shown that megakaryocyte-enriched protein-coding genes, but not erythroid ones, are "primed" in hspcs, and that these genes are occupied by a group of seven transcription factors (tfs) that facilitate low-level expression in hspcs [141] . lineage-specific alterations in tf occupancy subsequently led to an activation of these genes during megakaryopoiesis and repression during erythropoiesis. intriguingly, similar observations could be made for lncrnas and the model that some megakaryocyte-enriched genes are specifically primed in hspcs could be extended to lncrna loci, emphasizing common regulatory mechanisms with coding genes [140] . however, the relevance of these lncrnas for mk differentiation and function besides their regulation remains unclear and requires further investigation. in contrast, more direct evidence for the relevance of lncrnas for mk and platelet function was recently presented in the context of type 2 diabetes. here, platelet hyperaggregation and hypercoagulation are often observed in patients with type 2 diabetes (t2d) leading to an increased thrombogenic risk [142] . however, t2d patients frequently fail to respond to commonly used adp receptor blocker such as clopidogrel and cangrelor that are used as antiplatelet therapy thus leaving residual platelet reactivity [143] . moreover, high activity of the adp receptor p2y12 is found in t2d patients, exposing such patients to a prothrombotic condition [144, 145] . in this context, the non-coding metallothionein 1 pseudogene 3 (mt1p3) was found to be significantly upregulated in mks from type 2 diabetes patients compared to healthy controls and, most importantly, had an impact on platelet activation and expression of the p2y12 by sponging mir-126. of note, depletion of mt1p3 by small interfering rna (sirna) reduced the expression of the adp receptor, thereby inhibiting platelet activation and aggregation in a murine diabetes model [146] . in summary, small and long non-coding rnas are crucial regulators of megakaryocyte and platelet biology, and changes in ncrna expression are associated with alterations in megakaryopoiesis, platelet biogenesis and platelet function. however, our current understanding of the underlying molecular mechanisms is still very limited and much more research, especially on the role of lncrnas, is needed. post-transcriptional control mechanisms are crucial for platelets to dynamically alter their proteome, phenotype and function (summarized in figure 2 ). newly formed, anucleate platelets seem to have a higher biosynthetic potential than older ones, indicating post-transcriptional regulatory circuits acting during the life cycle of platelets [147] . post-transcriptional gene expression control is mainly achieved through mirnas via binding to the 3 -utr of their target transcript thereby regulating rna degradation and protein translation as mentioned above. in addition to mirnas, a second major class of post-transcriptional regulators consists of rbps. rbps recognize specific sequence or structure elements often present in the utrs of their target rnas. interestingly, platelets were found to have, on average, much longer 3 -utr compared to nucleated cells (1047 nt versus 492 nt, respectively), indicating an elongated binding region for mirnas and rbps thereby allowing potentially more complex regulation of translational and rna stability [44] . intriguingly, the key regulators of eukaryotic protein synthesis, i.e., eukaryotic translation initiation factor (eif) 4e and eif2α, are highly abundant in platelets [148] . furthermore, key ribosomal components, including ribosomal protein s6 as well as 18s and 28s ribosomal rnas (rrnas), are present in platelets [112, 115] . however, translation in resting platelets is generally prohibited or reduced via binding of eif4e to the inhibitory protein 4e-binding protein 1 (4e-bp1), which prevents the assembly of the eif4f initiation complex. activation of the mechanistic target of rapamycin (mtor) signaling pathway leads to phosphorylation of 4e-bp1 and to its dissociation from eif4e, thereby enabling formation of the initiation complex and subsequent translation in platelets [114, 149] . of note, pharmacological inhibition of mtor via rapamycin has controversial effects on platelet activity. while collagen-induced platelet aggregation was significantly reduced [150] , platelet adhesion to endothelial cells was facilitated by rapamycin through the remodeling of the endothelial membrane [151] . furthermore, a study that investigated the long-term effects of mtor inhibitors in patients that received kidney transplants observed altered platelet functions. the side effects included reduced granule secretion and impaired platelet aggregation, because of biphasic time-dependent alterations in calcium homeostasis and function in platelets [152] . these findings can be, at least partially, explained by the observation that inhibition of mtor activity using rapamycin reduced translational events of certain, but not all transcripts indicating specificity of mtor signaling-dependent protein synthesis [153] . importantly, these examples underscore the relevance of translational control in platelets. this is further supported by the observation that some proteins are constitutively translated in platelets, whereas the translation of others is regulated in a signal-dependent manner as it has been shown for bcl3 transcription coactivator (bcl-3) and il-1β [114, 115, 149, 154] . mechanistically, platelet integrins have been proposed to transmit outside-in signaling cascades thereby modulating signal-dependent translation in platelets. for example, while quiescent platelets harbor mrna for the precursor il-1β cytokine (pre-il-1β) at the site of polysomes, its translation is induced upon β 3 integrin signaling [115] . moreover, integrin signaling can lead to a redistribution of eif4e to mrna-rich areas in aggregated platelets thereby initiating protein translation upon platelet activation [114] . similarly, translation of bcl-3 mrna is constitutively repressed in resting platelets. however, upon thrombin receptor-mediated activation of platelets, bcl-3 protein is rapidly synthesized within minutes and the translation may be modulated via engagement of integrin α iib β 3 [155] . rapamycin was shown to abrogate bcl-3 translation indicating a regulatory role of mtor in signal-dependent protein translation in platelets. importantly, signal-dependent translation in platelets is not exclusively regulated via integrins. early studies investigated the impact of highly polyunsaturated fatty acids of the n-3 family on the platelet antioxidant status and observed elevated enzyme activity of glutathione-dependent peroxidase (gpx), which counteracts oxidative stress. the enhanced enzyme activity could be explained by increased protein translation, since concomitant treatment with the protein synthesis inhibitor cycloheximide abolished the additional enzyme activity [156] . in summary, small and long non-coding rnas are crucial regulators of megakaryocyte and platelet biology, and changes in ncrna expression are associated with alterations in megakaryopoiesis, platelet biogenesis and platelet function. however, our current understanding of the underlying molecular mechanisms is still very limited and much more research, especially on the role of lncrnas, is needed. post-transcriptional control mechanisms are crucial for platelets to dynamically alter their proteome, phenotype and function (summarized in figure 2 ). newly formed, anucleate platelets seem to have a higher biosynthetic potential than older ones, indicating post-transcriptional regulatory circuits acting during the life cycle of platelets [147] . post-transcriptional gene expression control is mainly achieved through mirnas via binding to the 3′-utr of their target transcript thereby regulating rna degradation and protein translation as mentioned above. in addition to mirnas, a second major class of post-transcriptional regulators consists of rbps. rbps recognize specific sequence or structure elements often present in the utrs of their target rnas. interestingly, platelets were found to have, on average, much longer 3′-utr compared to nucleated cells (1047 nt versus 492 nt, respectively), indicating an elongated binding region for mirnas and rbps thereby allowing potentially more complex regulation of translational and rna stability [44] . receptor-mediated signaling may lead to splicing and translational events that are silenced in resting platelets. lipopolysaccharide (lps) binding to toll-like receptor 4 (tlr4) and integrin signaling induces the proteolytic processing of pre-il-1β to mature interleukin (il)-1β, resulting in pro-inflammatory signaling. integrin-and thrombin receptor mediated platelet activation induces reorganization of the translation initiation complex and translation of certain mrnas, including serpin family e member 1 (serpine1) and bcl3 transcription coactivator (bcl-3), partially in an mechanistic target of rapamycin (mtor)-dependent manner. posttranscriptional gene expression control is also guided by mirna-binding to the 3 -untranslated region (utr) of target genes regulating rna degradation or translation. an additional layer of posttranscriptional gene expression control is represented by rbps responsible for megakaryocyte mk maturation, platelet biogenesis, platelet responsiveness and platelet interaction with immune cells as well as rna transport from mks to proplatelets. lastly, posttranscriptional regulation is mediated by l1 reverse transcriptase activity, which induces formation of rna-dna hybrids, thereby regulating global protein synthesis. besides these examples of general mechanisms that contribute to translational control in platelets, also more specific regulations of individual mrna by certain rbps have been identified. of note, more than 1500 human proteins have been annotated as rbps based on experimental evidence and the presence of canonical rna-binding domains (rbds) [157] . rbps typically use rbds, such as the rna recognition motif (rrm), hnrnp k homology domain (kh) or dead box helicase (ddx) domain, in order to bind to their target rna and subsequently form ribonucleoprotein (rnp) complexes. however, additional rbps have been identified lacking these characteristic rbds [158] . importantly, rbps are crucial post-transcriptional regulators that are able to execute a plethora of control mechanisms, including regulation of rna translation, splicing, transport, decay and editing [157] . hence, rbps could potentially contribute to multiple aspects of mk differentiation, platelet formation as well as platelet function. in line with this, a recent study revealed that the rbp ataxin2 (atxn2) modulates the mk transcriptome and proteome and affects expression of platelet surface proteins [159] . atxn2 has been implicated in regulating global mrna stability and translation and directly binds to over 4000 transcripts as evaluated by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (par-clip) [160, 161] . in human megakaryoblasts, atxn2 was shown to associate with the poly(a)-binding protein (pabp) and the rna helicase ddx6, and knockdown of atxn2 in meg-01 cells yielded 454 differentially expressed rnas and 20 differentially expressed proteins [159] . however, the authors of this study did not identify direct rna targets of atxn2 in megakaryoblasts. intriguingly, atxn2 knockout mice exhibited a decrease in type iv megakaryoid cells, and αiibβ3 integrin-mediated platelet aggregation was impaired upon stimulation with phorbol myristate acetate (pma) or with aggretin a. this could be due to deregulated total expression of itgb3 and aberrant surface receptor expression of cd31 in conjunction with c6orf25, coagulation factor ii thrombin receptor (f2r) and vav1 dysregulation in atxn2 knockout mice [159] . in addition to atxn2, other rbps have been implicated in megakaryopoiesis and mk maturation. as mentioned earlier, rbm15 was shown to play a role in megakaryocytic differentiation of hematopoietic stem cells acting in concert with protein arginine methyltransferase 1 (prmt1) which methylates rbm15 and thereby regulates its stability via the e3 ubiquitin ligase cnot4 [139, 162] . rbm15 was shown to bind to intronic sequences and interact with the splicing factor 3b subunit 1 (sf3b1) thereby regulating alternative splicing of genes that are important for megakaryopoiesis such as gata1, runx1, t-cell acute lymphocytic leukemia 1 (tal1) and c-mpl [139] . another rbp that may influence megakaryocyte differentiation is insulin-like growth factor 2 mrna-binding protein 1 (igf2bp1). igf2bp1 has been found to target the ets variant transcription factor 6 (etv6)/runx1 fusion transcript and potentially regulates its stability in acute lymphatic leukemia (all) [163] . however, it is currently unknown, if igf2bp1 modulates runx1 expression in hscs or megakaryocytes. however, it could be shown that another member of the igf2bp family regulates the human fetal-adult megakaryocyte transition [164] . in detail, the oncofetal igf2bp3 is expressed significantly higher in neonatal hematopoietic cells, including neonatal megakaryocytes, but is completely absent in its adult counterparts. high expression of igf2bp3 restricted megakaryocyte morphogenesis and polyploidy by inhibiting the positive transcription elongation factor b (p-tefb) kinase complex by binding and stabilization of the 7sk snrna. consequently, high igf2bp3 expression in neonatal megakaryocytes affected platelet function leading to hyporesponsiveness in full-term and premature neonates, which could lead to thrombocytopenia and bleeding commonly observed in premature neonates [164] . another example of an oncofetal rbp with a role in regulating mk and platelet function is lin28b, which is highly expressed in fetal, but not in adult megakaryocytes. high lin28b expression in fetal megakaryocytes negatively regulated surface p-selectin expression in fetal platelets, which influenced their interaction with neutrophils in vitro and in vivo [165] . hence, differential rbp expression in developing mks as well as old and young platelets may play a crucial role in regulating platelet function. in addition, post-transcriptional regulation by rbps might also be vital in regulating platelet production and release from mks, which includes cytoskeletal rearrangements. myosin heavy chain 9 (myh9) is a major non-muscle myosin expressed in mks and platelets, associates with the actin cytoskeleton and enables morphogenesis. mutations in or knockout of myh9 is linked to a group of platelet disorders leading to macro-thrombocytopenia, prolonged bleeding and clot retraction deficiency [166] . in erythropoiesis, myh9 is crucial for erythroid cell enucleation. this process was shown to depend on the activity of heterogeneous nuclear ribonucleoprotein k (hnrnp k), an rbp that inhibits translation of myh9 mrna [167] . it would be interesting to evaluate the effects of hnrnp k on myh9-dependent megakaryocyte maturation and platelet biogenesis, which has not been investigated so far. in addition to regulating stability and translation of mrnas, rbps can also be involved in the localization and transport of their target rnas from megakaryocytes to platelets, which is suggested to occur in a controlled rather than random manner as mentioned above [118] . while the exact sorting mechanisms remain elusive, mks and platelets were shown to contain mrnas encoding for rbps that were previously implicated in mrna transport processes including cancer susceptibility candidate gene 3 (casc3), staufen double-stranded rna binding protein 1 and 2 (stau1 and stau2) as well as eif4a3 [118, 168] . hence, these rbps might be expressed in mks and platelets and may play a role in the regulated transport of coding and non-coding transcripts into platelets [169] . consequently, rbps have also been detected within platelets themselves where they modulate transcript stability and translation efficiency. among the identified rbps are t-cell internal antigen-1 (tia-i), tia-i-related protein (tia-r), and elav like rna binding protein 1 (elavl1, hur) [112] . tia-1 has been implicated in alternative splicing regulation of various pre-mrnas and was shown to suppress translation [170] . upon platelet activation by thrombin, tia-i dissociates from the serpin family e member 1 (serpine1) mrna, thereby lifting repressive effects and allowing translation and de novo synthesis of serpine1 protein, also known as plasminogen activator inhibitor 1 (pai-1). similar effects were found for ago2, where dissociation of ago2 protein upon thrombin stimulation of platelets was found for serpine1 mrna whereas ago2/mirna complexes or associations of ago2 with other mrnas known to be translated upon platelet activation, including prostaglandin-endoperoxide synthase 1 (ptgs1) and itgb3 mrnas, were not affected. on the other hand, association of serpine1 mrna with the rbp hur did not change upon platelet activation, which might indicate that hur acts as a stabilizing factor rather than a translational regulator under these conditions [171] . overall, these findings implicate some degree of specificity for the regulation of individual platelet mrnas during platelet stimulation, which is likely distinct following platelet activation by other known platelet activating factors besides thrombin. finally, yet importantly, it is worth mentioning that not only mirnas and rbps could regulate protein synthesis in mks and platelets, but also other post-transcriptional regulators might exist. indeed, schwertz et al. demonstrated that translational events in platelets could be controlled by endogenous long interspersed nuclear element-1 (line-1) reverse transcriptase activity (ert) [172] . intriguingly, inhibition of ert in vitro in isolated platelets from healthy individuals or in people with hiv treated with rt inhibitors enhanced global protein synthesis. moreover, platelet activation was induced promoting a pro-thrombotic functional response, which was likely mediated by the generation of rna-dna hybrids. these results present a novel, previously unrecognized translational regulatory mechanism, which could have clinical implications for hiv patients that are at an increased risk of thrombosis, which might be related to rt inhibitor-based antiretroviral therapies [173] . in summary, post-transcriptional regulatory pathways are crucial for rapid adaptations to changing environmental properties upon platelet activation. rbps expressed in megakaryocytes and platelets are critical for megakaryocyte differentiation, maturation, and platelet genesis. moreover, rbps might be important transport factors mediating the sorting of rnas from megakaryocytes into platelets and thereby influence signaling pathways in circulating platelets. however, several of the underlying mechanistic details are not well understood so far, which warrants further in-depth investigations in the future. platelets contain a plethora of rnas that are inherited from megakaryocytes or taken up from interacting cells or microorganisms. the functions of these transcripts, whether they are crucial for platelet function per se independent of their role in megakaryocytes and the mechanisms that control their sorting and uptake as well as their constitutive or signal-dependent translation and decay, are just beginning to be resolved. studying the contribution of rbps to these processes will allow us to gain a deeper understanding of the complex networks underlying megakaryopoiesis, platelet biogenesis and function. we apologize to all scientists whose 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stability and protein expression prmt1-rbm15 axis regulates megakaryocytic differentiation of human umbilical cord blood cd34(+) cells etv6/runx1 transcript is a target of rna-binding protein igf2bp1 in t(12;21)(p13;q22)-positive acute lymphoblastic leukemia neonatal expression of rna-binding protein igf2bp3 regulates the human fetal-adult megakaryocyte transition lin28b regulates age-dependent differences in murine platelet function megakaryocyte-restricted myh9 inactivation dramatically affects hemostasis while preserving platelet aggregation and secretion translational control mediated by hnrnp k links nmhc iia to erythroid enucleation dendritic mrna: transport, translation and function the role of rna binding proteins for local mrna translation: implications in neurological disorders structure, dynamics and rna binding of the multi-domain splicing factor tia-1 dissociation of serpine1 mrna from the translational repressor proteins ago2 and tia-1 upon platelet activation endogenous line-1 (long interspersed nuclear element-1) reverse transcriptase activity in platelets controls translational events through rna-dna hybrids venous and arsterial thromboembolic complications associated with hiv infection and highly active antiretroviral therapy key: cord-266617-z8uecyl6 authors: pavesi, angelo title: asymmetric evolution in viral overlapping genes is a source of selective protein adaptation date: 2019-04-03 journal: virology doi: 10.1016/j.virol.2019.03.017 sha: doc_id: 266617 cord_uid: z8uecyl6 overlapping genes represent an intriguing puzzle, as they encode two proteins whose ability to evolve is constrained by each other. overlapping genes can undergo “symmetric evolution” (similar selection pressures on the two proteins) or “asymmetric evolution” (significantly different selection pressures on the two proteins). by sequence analysis of 75 pairs of homologous viral overlapping genes, i evaluated their accordance with one or the other model. analysis of nucleotide and amino acid sequences revealed that half of overlaps undergo asymmetric evolution, as the protein from one frame shows a number of substitutions significantly higher than that of the protein from the other frame. interestingly, the most variable protein (often known to interact with the host proteins) appeared to be encoded by the de novo frame in all cases examined. these findings suggest that overlapping genes, besides to increase the coding ability of viruses, are also a source of selective protein adaptation. many viruses produce novel genes inside pre-existing genes by overprinting of a de novo frame onto an ancestral frame (atkins et al., 1979; keese and gibbs, 1992; rancurel et al., 2009; sabath et al., 2012) . the high prevalence of overlapping genes in viruses has been attributed to the advantage of maximizing the gene information content of small viral genomes (miyata and yasunaga, 1978; lamb and orvath, 1991; pavesi et al., 1997) . in detail, the gene-compression hypothesis states that the size of the viral capsid imposes a biophysical limit on the size of the viral genome, thus making overprinting the most adequate strategy to gain new function (chirico et al., 2010) . in alternative, the gene novelty hypothesis argues that the birth of overlapping genes is driven by selection pressures favoring evolutionary innovation (brandes and linial, 2016) . this hypothesis is supported by the finding that overlaps, thought for a long time to be restricted to viruses, also occur in the large genomes of prokaryotic (delaye et al., 2008; fellner et al., 2015) and eukaryotic organisms (szklarczyk et al., 2007; bergeron et al., 2013; vanderperre et al., 2013) . a particularly interesting feature of overlapping genes is that they represent an intriguing example of adaptive conflict. indeed, they simultaneously encode two proteins whose freedom to change is constrained by each other (sander and schulz, 1979; krakauer, 2000; peleg et al., 2004; allison et al., 2016) , which would be expected to reduce the adaptive ability of the virus (simon-loriere et al., 2013) . we would expect, in principle, that overlapping genes are subjected to strong evolutionary constraints, as a single nucleotide substitution can impair two proteins (see the codon position "21" in fig. 1) . a typical example of "constrained evolution" is that occurring in hepatitis b virus (hbv), whose short genome (3.2 kb) contains a high percentage (50%) of overlapping coding regions (mizokami et al., 1997; zhang et al., 2010) . however, overlapping genes can also show a less conservative pattern of change, because of a high rate of non-synonymous substitutions in one frame (positive adaptive selection) with concurrent dominance of synonymous substitutions in the other (negative purifying selection). examples of positive selection concern the overlapping genes that encode the tat and vpr proteins of simian immunodeficiency virus (hughes et al., 2001) , the p19 and p22 proteins of the tombusvirus family of plant viruses (allison et al., 2016) , and the orf2 and orf5 proteins of trichodysplasia spinulosa-associated polyomavirus (kazem et al., 2016) . we can hypothesize for overlapping genes a first evolutionary model in which the two proteins they encode are subjected to similar selection pressures. when selection is strong both proteins (or protein regions) are highly conserved (e.g. the rnase domain of polymerase and the amino-terminal half of the x protein in hbv; see fig. 4 in mizokami et al., 1997) . when selection is not too strong both proteins can vary considerably (e.g. the spacer domain of polymerase and the pres1/s2 domain of the surface protein in hbv; see fig. 4 in mizokami et al., 1997) . this model is named "symmetric evolution", because the number of amino acid substitutions of one protein is expected to be not significantly different from that of the other. it corresponds to the "shared model" by fernandes et al. (2016) . in alternative, we can hypothesize for overlapping genes an evolutionary model in which the two proteins they encode are subjected to significantly different selection pressures. support for this model, which implies adaptive selection on one frame and purifying selection on the other, was provided both by viral (hughes et al., 2001; fujii et al., 2001; guyader and ducray, 2002; stamenković et al., 2016) and mammalian overlapping genes (szklarczyk et al., 2007) . this model is named "asymmetric evolution", because the number of amino acid substitutions of one protein is expected to be significantly different from that of the other. it corresponds to the "segregated model" by fernandes et al. (2016) . we recently assembled a dataset of 80 viral overlapping genes whose expression is experimentally proven (pavesi et al., 2018) , with the aim to provide a useful benchmark for systematic studies. a first analysis of the dataset revealed that overlapping genes differ significantly from non-overlapping genes in their nucleotide and amino acid composition (pavesi et al., 2018) . we also found that the vast majority of the 80 overlaps of the dataset have one or more homologs, suggesting further comparative studies. in the present study, i investigated the evolution of viral overlapping genes by sequence analysis of 75 pairs of homologs. the first aim of the study was to determine which of the two evolutionary models described above is the prevailing one. the second aim was to identify the type of nucleotide substitution that significantly affects the pattern of symmetric/asymmetric evolution. finally, the third aim was to assess whether the most variable protein (in the case of asymmetric evolution) is that encoded by the ancestral or the de novo frame. 2.1. selection criteria for homologous overlapping genes i first extracted from the dataset of 80 overlapping genes experimentally proven (s1 dataset from pavesi et al., 2018) the amino acid sequence of the two proteins encoded by each overlap. for each protein, i searched for homologs against the non-redundant protein sequences ncbi database using blastp (altschul et al., 1997) . when blastp did not detect any homolog i used tblastn, which compared the protein query sequence against the nucleotide collection ncbi database translated in all reading frames. i used tblastn because the amino acid sequence of the protein encoded by one of the two overlapping frames (usually that discovered more recently) may not be reported in many viral genomes present in the ncbi database (pavesi et al., 2018) . the selection of homologous overlapping genes was based on three criteria. the first was an equal length of the homolog. it was met in the great majority of cases (72 out of 80). in the remaining cases, the homolog was only slightly shorter than the query sequence. the exception was the overlap capsid protein/assembly activating protein (aap) of adeno-associated virus-2, whose homolog encodes an aap 9 amino acids shorter in the amino-terminal region and 26 amino acids shorter in the carboxy-terminal region. the second criterion was a homolog yielding, for both the encoded proteins, an alignment with no insertion/deletion (indel) or with a minimal number of indels. in the latter case, i imposed the rule that indel(s) must be located at the same amino acid position in the alignments of the two pairs of proteins (see for example the overlap polymerase/2b protein of spinach latent virus, which is the first overlap in supplementary file s1). by imposing this rule, i could align the two homologous nucleotide sequences in full accordance with the corresponding protein sequences. the alignment of protein sequences was carried out with clustal omega (sievers and higgins, 2014) . the third criterion concerned the cases in which i found multiple homologs meeting the two criteria described above. in these cases, i selected the most distantly related homolog, with the aim to cover the largest evolutionary space. the choice to select only one homolog for each overlapping gene was due to the fact that collection of a larger sample of homologs is limited to a few overlaps, mainly those occurring in virus species that are human pathogens (e.g. influenza and hepatitis viruses or sars and ebola viruses). the search for homologs yielded a dataset of 80 pairs of homologous overlapping genes (supplementary file s1). thirty-seven homologs came from a different virus species, in accordance with the ictv taxonomy (king et al., 2018) (https://talk.ictvonline.org/taxonomy/). the mean nucleotide identity between overlaps and homologs was 70.7%, with a standard deviation (sd) of 9.4%. the remaining 43 homologs came from isolates belonging to the same virus species. in this case, the mean nucleotide identity between overlaps and homologs was 89.6% (sd = 7.1%). for each pair of homologous overlapping genes, the supplementary file s1 contains the following information: i) the nucleotide sequence of the upstream frame and that of the homolog; ii) the amino acid sequence of the protein encoded by the upstream frame (up1) and that of the protein encoded by the homolog (up2); iii) the nucleotide sequence of the downstream frame (shifted of one nucleotide 3' with respect to the upstream frame) and that of the homolog; iv) the amino acid sequence of the protein encoded by the downstream frame (down1) and that of the protein encoded by the homolog (down2); v) the alignment of up1 with up2 and the percent amino acid identity; vi) the alignment of down1 with down2 and the percent amino acid identity; vii) the chisquare analysis, which compared by a 2 x 2 contingency-table the number of the amino acid identities and differences in the up1-up2 alignment with that in the down1-down2 alignment (cut-off of significance = 3.84; 1 degree of freedom; p < 0.05). 3.2. half of overlapping genes evolve in accordance with the asymmetric model i carried out a preliminary analysis using the t-student test for paired data. for each pairs of homologous overlaps, i counted the number of amino acid identities between up1 and up2 and that between down1 and down2. i then calculated the absolute value of the difference between them. the null hypothesis was a mean difference orientation of overlapping genes, with the downstream frame having a shift of one nucleotide 3′ with respect to the upstream frame. there are 3 types of codon position (cp): cp13 (bold character), in which the first position of the upstream frame overlaps the third position of the downstream frame; cp21 (underlined character), in which the second position of the upstream frame overlaps the first position of the downstream frame; cp32 (italic character), in which the third position of the upstream frame overlaps the second position of the downstream frame. based on the genetic code, a nucleotide substitution at first codon position causes an amino acid change in 95.4% of cases, at second codon position in 100% of cases, and at third codon position in 28.4% of cases. thus, nucleotide substitutions at the codon positions "13" and "32" are usually non-synonymous in one frame and synonymous in the other. nucleotide substitutions at the codon position "21" are almost all non-synonymous in both frames. virology 532 (2019) [39] [40] [41] [42] [43] [44] [45] [46] [47] between paired observations close to zero, indicating that overlapping genes evolve in accordance with the symmetric model. the null hypothesis was rejected (t-student = 5.91; 79 degrees of freedom; p = 10 −5 ), indicating that overlapping genes can also evolve in accordance with the alternative asymmetric model. in order to identify which and how many overlapping genes undergo symmetric or asymmetric evolution, i then compared the amino acid diversity between up1 and up2 to that between down1 and down2. i used the contingency-table chi-square test (snedecor and cochran, 1967) with a cut-off value of 3.84 for 1 degree of freedom (p < 0.05). i classified a pair of homologous overlaps as a case of symmetric evolution, if the number of amino acid substitutions in the up1-up2 alignment did not significantly differ from that in the down1-down2 alignment (chi-square < 3.84). an example is given by the overlap ns1 protein/ns2 protein from dendrolimus punctatus densovirus. for the ns1 protein, i found 73 identities and 86 differences when compared to the homolog from hordeum marinum itera-like densovirus. for the ns2 protein, i found 71 identities and 88 differences, yielding a chi-square value (0.01) largely below the cut-off of significance. in alternative, i classified a pair of homologous overlaps as a case of asymmetric evolution, if the number of amino acid substitutions in the up1-up2 alignment was significantly different from that in the down1-down2 alignment (chi-square > 3.84). an example is given by the overlap movement protein/replicase from turnip yellow mosaic virus. for the movement protein, i found 302 identities and 323 differences when compared to the homolog from watercress white vein virus. for replicase, i found 454 identities and 171 differences, yielding a chisquare value (76.3) largely above the cut-off of significance. the chi-square test was highly sensitive. for example, i found that the overlap capsid protein/p31 protein from maize chlorotic mottle virus undergoes asymmetric evolution, in spite of a nucleotide identity with the homolog extremely high (96.7%). indeed, the number of amino acid differences between p31 and homolog (12 out of 149 sites) was significantly higher than that between capsid and homolog (2 out of 149 sites) (chi-square = 6.07; p = 0.01). based on this finding, i set the upper limit of sensitivity of the chi-square test to a nucleotide identity between overlap and homolog of 97%. this filter limited the analysis to 75 (out of 80) pairs of homologous overlaps. overall, i found that 38 overlapping genes evolve in accordance with the asymmetric model (significantly different selection pressures on the two proteins). the highest chi-square value (113.8) concerned the overlap from apple stem grooving virus, which encodes the 36kd movement protein and the polyprotein linker-domain. indeed, the amino acid diversity between linker-domain and homolog (39%; 125 differences and 195 identities) was ten-fold higher than that between movement protein and homolog (4%; 13 differences and 307 identities). i found that the remaining 37 overlapping genes evolve in accordance with the symmetric model (similar selection pressures on the two proteins). the occurrence of similar selection pressures can yield two highly conserved proteins. for example, analysis of the overlap 3a protein/3b protein from human sars coronavirus revealed that the amino acid diversity between 3a and homolog is remarkably low (5.3%; 6 differences and 108 identities), as well as that between 3b and homolog (8.8%; 10 differences and 104 identities). however, the occurrence of similar selection pressure can also yield two proteins with a remarkably less conserved pattern of change. this is the case of the overlap from spinach latent virus, which encodes the zincfinger domain of polymerase and the 2b protein. sequence analysis revealed that the amino acid diversity between zinc-finger domain and homolog is considerably high (47%; 47 differences and 54 identities), as well as that between 2b and homolog (44%; 44 differences and 57 identities). the analysis of amino acid diversity in the 75 pairs of homologous overlapping genes is summarized in fig. 2 . it shows, for each overlap, the percent amino acid (aa) identity of the two encoded proteins with those encoded by the homolog. the subset of the 37 overlapping genes under symmetric evolution ( fig. 2a) contains 31 overlaps in which both proteins have high conservation (aa identity > 50%), 5 overlaps in which both proteins have poor conservation (aa identity < 50%) and 1 overlap with a protein having an aa identity above 50% and the other below 50%. the subset of the 38 overlapping genes under asymmetric evolution (fig. 2b ) contains 24 overlaps in which both proteins have high conservation (aa identity > 50%), 1 overlap in which both proteins have poor conservation (aa identity < 50%) and 13 overlaps with a protein having an aa identity above 50% and the other below 50%. finally, a list of the 75 overlapping genes, classified in accordance with the symmetric or asymmetric model (37 and 38 cases, respectively), is given in supplementary table s1. 3.3. validation of the model of symmetric/asymmetric evolution by analysis of the pattern of nucleotide substitutions in homologous overlapping genes in accordance with wei and zhang (2014) , i first classified the nucleotide sites of each overlapping gene into four categories depending on the impact of potential mutations on the two encoded proteins. the four categories are referred as nn, sn, ns, and ss sites, respectively, where n stands for non-synonymous change and s stands for synonymous change. that is, if all potential mutations at a site cause nonsynonymous change in both proteins, it is a nn site, and so on. i then classified the nucleotide substitutions occurring in the homolog into four categories: nn, sn, ns, and ss. using the contingency-table chisquare test, i compared the number of sn and ns sites in each overlapping gene with the number of sn and ns substitutions in the homolog. under symmetric evolution, i would expect a chi-square value below the cut-off of significance (3.84; 1 degree of freedom), that is a full concordance between the number of sn and ns sites and that of sn and ns substitutions. for example, in the overlap orf4/orf5 from barley yellow striate mosaic virus i counted 49 sn sites and 51 ns sites. in the homolog from maize yellow striate virus, i classified 23 nucleotide substitutions into the sn category and 28 substitutions into the ns category. the chi-square test yielded a value (0.08) largely below the cut-off of significance. under asymmetric evolution, i would expect a chi-square above the cut-off of significance, that is a significant discordance between the number of sn and ns sites and that of sn and ns substitutions. for example, the overlap capsid protein/ns4 protein from bluetongue virus (serotype 10) has 56 sn sites and 46 ns sites. the homolog from bluetongue virus (serotype 16) has 5 nucleotide substitutions belonging to the sn category and 29 substitutions to the ns category. the chisquare test yielded a value (15.07) largely above the cut-off of significance. the analysis of the pattern of nucleotide substitutions in the 75 pairs of homologous overlaps revealed 39 and 36 cases of symmetric and asymmetric evolution, respectively (supplementary table s2 ). this result was in accordance with that obtained previously (from analysis of the amino acid diversity, see supplementary table s1) in the 87% of cases (65 out of 75). overall, i found a total of 33 overlaps under symmetric evolution (they are marked with a single asterisk in supplementary tables s2a) and a total of 32 overlaps under asymmetric evolution (they are marked with a double asterisk in supplementary table s2b ). a list of the 32 overlapping genes under asymmetric evolution is given in table 1 . these findings were not affected by the fact that some homologs came from a different virus species, while others from an isolate within the same virus species. under symmetric evolution, i found 14 and 19 overlaps with the homolog within and between species, respectively. under asymmetric evolution, i found 18 and 14 overlaps with the homolog within and between species, respectively. finally, a further validation of the model of symmetric/asymmetric a. pavesi virology 532 (2019) 39-47 evolution was provided by a correlation test between the chi-square value from analysis of amino acid substitutions and the distribution of nucleotide substitutions at the codon positions "32" and "13" (fig. 1) . given the orientation of overlapping genes in our dataset (fig. 1) , a substitution at the codon position "32" (cp32) is usually synonymous in the upstream frame and always non-synonymous in the downstream frame, while a substitution at the codon position "13" is almost always non-synonymous in the upstream frame and usually synonymous in the downstream frame. under symmetric evolution, the number of substitutions at the codon position "32" is expected to be close to that at the codon position "13", yielding a similar distribution of the amino acid substitutions in the two pairs of homologous proteins. under asymmetric evolution, the number of substitutions at the codon position "32" is expected to be significantly higher (or lower) than that at the codon position "13", yielding a different distribution of the amino acid substitutions in the two pairs of homologous proteins. by comparing the upstream frame of each overlap with that of the homolog, i calculated the absolute value (abs) of the difference between the percent frequency (%f) of substitutions at the codon position "32" (%f.cp32) and that at the codon position "13" (%f.cp13). i then carried out a correlation test between abs (%f.cp32 -%f.cp13) and the chi-square value from analysis of amino acid substitutions. as the chisquare test depends on the extent of the sample (here the length of the protein encoded by the overlap), i normalized the chi-square value in accordance with the cohen's rule (cohen, 1988) . normalization was the square root of the ratio between the chi-square value and the overall length of the two proteins encoded by the overlap (e.g. the highest chisquare value, 113.83, was converted into the highest normalized chi-square value, 0.42). i found a significantly positive correlation between abs (%f.cp32 -%f.cp13) and the normalized chi-square value (r = 0.88; t-student = 14.36; one tailed p < 0.00001; 63 degrees of freedom) (fig. 3) . as expected, this result indicates that asymmetric evolution is significantly affected by an unbalanced distribution of the nucleotide substitutions at the codon positions "32" and "13". to answer the question, i investigated the genealogy of the 32 overlapping genes under asymmetric evolution. identifying which gene is ancestral and which one is de novo (the genealogy of the overlap) can be done by examining their phylogenetic distribution, under the assumption that the gene with the most restricted distribution is the de novo one (rancurel et al., 2009) . this approach yielded a set of 34 overlapping genes with a reliably predicted genealogy (see table 1 in sabath et al., 2012 and table 1 in pavesi et al., 2013) . this set included 16 out of the 32 overlaps under asymmetric evolution. another approach to infer the genealogy of overlapping genes is the codon-usage method. it is based on the assumption that the ancestral gene, which has co-evolved over a long period of time with the other viral genes, has a distribution of synonymous codons significantly closer to that of the viral genome than the de novo gene (keese and gibbs, 1992; sabath et al., 2012; pavesi et al., 2013; willis and masel, 2018) . due to the shortness of most overlapping genes, the method has been improved, with the aim to evaluate the correlation between the codon-usage patterns of overlapping and non-overlapping genes with a fig. 2 . analysis of the amino acid diversity in the 75 pairs of homologous overlapping genes. each pair of columns shows: i) the percent amino acid identity between the protein encoded by the upstream frame of the overlap and that encoded by the homolog (dark column); ii) the percent amino acid identity between the protein encoded by the downstream frame of the overlap (shifted of one nucleotide 3′ with respect to the upstream frame) and that encoded by the homolog (gray column). the horizontal line separates well-conserved homologous pairs (aa identity > 50%) from not well-conserved homologous pairs (aa identity < 50%). (a) subset of the 37 overlapping genes under symmetric evolution. (b) subset of the 38 overlapping genes under asymmetric evolution. the numbering of overlapping genes is in accordance with that given in supplementary table s1 . the underlined numbers indicate the overlaps in which the pattern of symmetric evolution (4 cases out of 37) or that of asymmetric evolution (6 cases out of 38) was not confirmed by chi-square analysis of the nucleotide diversity. virology 532 (2019) 39-47 table 1 list of the 32 overlapping genes evolving in accordance with the asymmetric model. minimal loss of information (pavesi, 2015) . using the improved version of the codon-usage method (pavesi, 2015) , i could predict the genealogy of 18 out of the 32 overlapping genes under asymmetric evolution. in 11 cases, the prediction by codon-usage was concordant with that established by the phylogenetic method. in the remaining 7 cases, the prediction was provided only by the codon-usage method (supplementary table s3 ). the overlap p130/p104 of providence virus is notable, as the ancestral frame p104 was acquired from another viral genome by distant horizontal gene transfer (pavesi et al., 2013) , which makes the codon usage an unreliable predictor of the genealogy. the prediction yielded by phylogenetics is supported by the finding that p104, unlike p130, has a wide phylogenetic distribution (pavesi et al., 2013) . overall, i collected a set of 23 overlapping genes, all under asymmetric evolution and with known genealogy (15 overlaps with a shift of the de novo frame of one nucleotide 3′ with respect to the ancestral frame and 8 overlaps with a shift of two nucleotides 3'). interestingly, i found that in all cases the most variable protein is that encoded by the de novo gene (table 2) . 3.5. symmetric and asymmetric evolution in the same overlap: the case of the overlap polymerase/large envelope protein of hepatitis b virus (hbv) chi-square analysis indicated that the overlap polymerase/large envelope protein of hbv evolves in accordance with the symmetric model (supplementary tables s1 and s2 ). on the other hand, theoretical and experimental studies (pavesi, 2015; lauber et al., 2017) demonstrated that this long overlap (1167 nt) is subjected to modular evolution, as the spacer domain of polymerase and the s domain of the large envelope protein originated de novo by overprinting. thus, the overlap can be subdivided into two regions: a 5′ region (480 nt), in which the spacer domain of polymerase (de novo gene product) overlaps the pre-s domain of envelope (ancestral gene product), and a 3' region (687 nt), in which the reverse transcriptase domain of polymerase (ancestral gene product) overlaps the s domain of envelope (de novo gene product). i carried out a chi-square analysis of the 2 regions of the overlap independently, under the hypothesis that they may have been subject to different evolutionary pressures. this analysis revealed that the 5' region of the overlap undergoes asymmetric evolution, because the amino acid diversity of the spacer domain (33.7%; 54 differences and 106 identities) is significantly higher than that of the pre-s domain (19.4%; 31 differences and 129 identities) (chi-square = 7.75; p = 0.005). asymmetric evolution was confirmed by analysis of the pattern of nucleotide substitutions (chi-square = 10.13; p = 0.001). in addition, chi-square analysis revealed that the 3' region of the overlap undergoes symmetric evolution, as the amino acid diversity of the reverse transcriptase domain (7.4%; 17 differences and 212 identities) does not significantly differ from that of the s domain (11.8%; 27 differences and 202 identities) (chi-square = 2.04; p = 0.15). symmetric evolution was confirmed by analysis of the pattern of nucleotide substitutions (chi-square = 1.61; p = 0.20). with the aim to further validate these findings, i carried out a further analysis using, as homolog, the most distantly related overlap of woolly monkey hbv (79.9% of nucleotide identity). again, chi-square analysis of the amino acid and nucleotide diversity revealed asymmetric evolution in the 5′ region and symmetric evolution in the 3' region. details of both analyses are reported in the supplementary file s2. finally, the finding that the spacer domain of polymerase (de novo gene product) is significantly more variable than the pre-s domain (ancestral gene product) confirms that the most variable protein, under asymmetric evolution, is usually that encoded by the de novo gene. several researchers have developed methods for estimating the strength of selection pressure on overlapping genes (pedersen and jensen, 2001; sabath et al., 2008; de groot et al., 2008; mir and schober, 2014; wei and zhang, 2014) . all methods evaluate, in both overlapping frames, the ratio of non-synonymous nucleotide substitutions to synonymous nucleotide substitutions (dn/ds) by correctly taking into account the problem of the interdependence between sequences imposed by the overlap. the aim is to assess if there is neutral evolution or positive selection in one frame (dn/ds higher than 1) and purifying selection (strong constraints) in the other frame (dn/ds lower than 1). however, the only method having an accessible implementation is that by sabath et al. (2008) . yet, the method has some limitations, as it restricts the analysis to the homologous overlaps in which the two encoded proteins have both an amino acid diversity smaller than 50% or greater than 5%. in the dataset examined here (see the first 75 pairs of homologous overlaps in supplementary file s1), these limitations would have considerably reduced the size of the sample from 75 to 43 pairs of homologous overlaps. i thus chose an approach focused, at first instance, on the evaluation of the amino acid diversity of homologous overlapping proteins, which is the final result of the complex pattern of the interdependent nucleotide substitutions that occur in dual-coding regions. unlike previous studies, limited to a few virus species (sabath et al., 2012; zaaijer et al., 2007; liang et al., 2010; shukla and hilgenfeld, 2015; brayne et al., 2017) , i examined a large dataset of 75 overlaps from 59 virus species. a possible limitation of the study concerns the selection criteria for homologous overlapping genes. in particular, the first two stringent criteria (an equal length of the homolog and an alignment with a minimal number of indels) led to exclusion, for some overlaps, of highly divergent homologs. an example is given by the overlap p3n-pipo/ polyprotein of turnip mosaic virus, in which the length of the p3n-pipo protein is quite variable among the different potyvirus species, ranging from 60 to 115 amino acids (hillung et al., 2013) . thus, the dataset used in this study likely underestimates the sequence diversity of overlapping genes, as it was created mainly to ensure a high quality in the homologous relationship. the finding that 32 out of 65 overlapping genes (table 1 ) undergo asymmetric evolution is striking, as well as that the most variable protein is encoded by the de novo gene in all cases examined (table 3 ). in particular, i would point out the overlap orf3/orf4 from tobacco bushy top virus, which encodes two proteins entirely nested within each other. this peculiar arrangement is similar to that of the overlap p19/ p22 from tomato bushy stunt virus, in which the de novo p19 protein fig. 3 . correlation between the normalized chi-square value (from analysis of amino acid substitutions) and the absolute value (abs) of the difference between the percent frequency (%f) of nucleotide substitutions at the codon position "32" (%f.cp32) and that at the codon position "13" (%f.cp13). empty circles indicate the 33 overlapping genes under symmetric evolution. black circles indicate the 32 overlapping genes under asymmetric evolution. virology 532 (2019) 39-47 table 2 list of the 23 overlapping genes with known genealogy and evolving in accordance with the asymmetric model. shows a previously unknown structural fold an a previously unknown mechanism of binding to small interfering rnas (vargason et al., 2003; baulcombe and molnár, 2004; scholthof, 2006) . i believe that structural or functional studies on the de novo orf3 protein from tobacco bushy top virus could reveal new interesting features. in addition, i would point out the overlap polymerase (pb1 subunit)/pb1-f2 protein of human influenza a virus. it shows, when compared to the homolog from duck, a sixteen-fold increase of substitutions at the codon position "32" (89.2%) with respect to the codon position "13" (5.4%). this yields only 3 amino acid differences between the two pb1 subunits and as many as 35 differences between the two pb1-f2 proteins. interestingly, the de novo pb1-f2 protein has been shown to largely contribute to viral pathogenicity by a pleiotropic effect (chen et al., 2001; varga et al., 2011; yoshizumi et al., 2014) . several other de novo proteins under asymmetric evolution are known to play a role in viral pathogenicity. eight de novo proteins (arfp, vp5, l*, x, vf1, pb1-f2, p6, and nss) act as suppressor or antagonist of the interferon response by the host (park et al., 2016; lauksund et al., 2015; sorgeelos et al., 2013; wensman et al., 2013; mcfadden et al., 2011; varga et al., 2011; garcía-rosado et al., 2008; jääskeläinen et al., 2007) . four de novo proteins (p19, p69, ac4, and movement protein) act as suppressor of rna silencing (silhavy et al., 2002; chen et al., 2004; chellappan et al., 2005; yaegashi et al., 2008) . two de novo proteins (apoptin and pb1-f2) act as apoptosis factor (noteborn et al., 1994; chen et al., 2001) . finally, the de novo protein pa-x has the ability to selectively degrade the host rna-polymerase ii transcripts (khaperskyy et al., 2016) . however, another possible limitation of the study depends on the fact that the subset of overlapping genes evolving asymmetrically and with known genealogy (23 overlaps) is too small to conclude that the de novo protein is always the preferred target of selection. furthermore, overlapping genes are subjected to a variety of selection pressures that are independent of the orientation of the overlapping frames relative to one another. thus, it is hypothetically possible that an ancestral protein may be significantly more variable than a de novo protein under peculiar selective constraints. despite this limitation, our findings suggest that the birth of new overlapping genes, besides to increase the coding ability of small viral genomes (chirico et al., 2010) , is also a valuable source of selective protein adaptation. none. positive selection or free to vary? assessing the functional significance of sequence change using molecular dynamics gapped blast and psi-blast: a new generation of protein database search programs binding of mammalian ribosomes to ms2 phage rna reveals an overlapping gene encoding a lysis function crystal structure of p19-a universal suppressor of rna silencing an out-of-frame overlapping reading frame in the ataxi-1 coding sequence encodes a novel ataxin-1 interacting protein gene overlapping and size constraints in the viral world genotype specific evolution of hepatitis e virus 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acquisition of new protein domains by coronaviruses: analysis of overlapping genes coding for proteins n and 9b in sars coronavirus clustal omega, accurate alignment of very large numbers of sequences a viral protein suppresses rna silencing and binds silencing-generated, 21-to 25-nucleotide double-stranded rnas the effect of gene overlapping on the rate of rna virus evolution statistical methods. iowa state university press evasion of antiviral innate immunity by theiler's virus l* protein through direct inhibition of rnase l substitution rate and natural selection in parvovirus b19 rapid asymmetric evolution of a dual-coding tumor suppressor ink4a/arf locus contradicts its function direct detection of alternative open reading frames translation products in human significantly expands the proteome the influenza virus protein pb1-f2 inhibits the induction of type i interferon at the level of the mavs adaptor protein size selective recognition of sirna by an rna silencing suppressor a simple method for estimating the strength of natural selection on overlapping genes the x proteins of bornaviruses interfere with type i interferon signalling gene birth contributes to structural disorder encoded by overlapping genes inhibition of long-distance movement of rna silencing signals in nicotiana benthamiana by apple chlorotic leaf spot virus 50 kda movement protein influenza a virus protein pb1-f2 translocates into mitochondria via tom40 channels and impairs innate immunity independent evolution of overlapping polymerase and surface protein genes of hepatitis b virus evolutionary selection associated with the multi-function of overlapping genes in the hepatitis b virus the author is grateful to alessio peracchi (university of parma) and alberto vianelli (university of insubria) for helpful suggestions. special thanks to xinzhu wei (university of michigan) for valuable comments and suggestions and to gianmarco del vecchio for preparing the figures. the author thanks the anonymous referees and the editor alexander e. gorbalenya for their helpful feedback and suggestions. the study was financed by the miur (ministero dell'università e della ricerca). supplementary data to this article can be found online at https:// doi.org/10.1016/j.virol.2019.03.017. key: cord-260949-w2xuf15h authors: galluzzi, lorenzo; brenner, catherine; morselli, eugenia; touat, zahia; kroemer, guido title: viral control of mitochondrial apoptosis date: 2008-05-30 journal: plos pathog doi: 10.1371/journal.ppat.1000018 sha: doc_id: 260949 cord_uid: w2xuf15h throughout the process of pathogen–host co-evolution, viruses have developed a battery of distinct strategies to overcome biochemical and immunological defenses of the host. thus, viruses have acquired the capacity to subvert host cell apoptosis, control inflammatory responses, and evade immune reactions. since the elimination of infected cells via programmed cell death is one of the most ancestral defense mechanisms against infection, disabling host cell apoptosis might represent an almost obligate step in the viral life cycle. conversely, viruses may take advantage of stimulating apoptosis, either to kill uninfected cells from the immune system, or to induce the breakdown of infected cells, thereby favoring viral dissemination. several viral polypeptides are homologs of host-derived apoptosis-regulatory proteins, such as members of the bcl-2 family. moreover, viral factors with no homology to host proteins specifically target key components of the apoptotic machinery. here, we summarize the current knowledge on the viral modulation of mitochondrial apoptosis, by focusing in particular on the mechanisms by which viral proteins control the host cell death apparatus. the sacrifice, via programmed cell death (pcd), of infected cells represents the most primordial response of multicellular organisms to viruses. this response is common to all metazoan phyla, including plants (which lack an immune system based on mobile cells) (text s2, [s1]). in mammals, microbial invasion does not only trigger pcd of infected cells but also elicits an immune reaction. this is hierarchically organized in a first-line response provided by innate immune effectors (e.g., infiltrating phagocytes and natural killer cells) [s2], followed by the activation of adaptive immunity, mediated by t and b lymphocytes [s3] . importantly, other layers of defense exist to prevent viral replication and spread [s2] . for instance, in invertebrates like drosophyla melanogaster (as well as in plants), a prominent antiviral mechanism is provided by rna interference (rnai) [s4] . although the rnai pathway is preserved in mammals, it has presumably been superseded in its antiviral role by the extremely potent interferon system, as well as by a number of additional mechanisms [s5] . such a multivariate antiviral response is designed to recognize virions, virus-infected cells, and virus-induced signals of stress (including cell death) to eliminate the pathogen (together with the host cell) and to elicit immunological memory [s6] . thus, the co-evolution between host and virus has forced the latter to develop strategies for modulating host cell pcd and/or for avoiding immunogenic cell death. apoptosis is an active mode of pcd exhibiting a series of morphological and biochemical changes by which it can be distinguished from other cell death subroutines [s7] . at a morphological level, these modifications include a dramatic reduction in cell volume (cell shrinkage), nuclear pyknosis (chromatin condensation), and karyorrhexis (nuclear fragmenta-tion). eventually, dying cells break down into small, discrete bodies known as apoptotic bodies [s7] . the morphological appearance of apoptosis is accounted for by an ensemble of biochemical events that include, but are not limited to: (1) loss of the structural integrity and bioenergetic functions of mitochondria, (2) cascade activation of a specific set of catabolic enzymes (e.g., proteases of the caspase family, nucleases), (3) exposure of phosphatydylserine on the outer leaflet of plasma membrane and, finally, (4) loss of the barrier function of the plasma membrane [s7] . apoptosis can be triggered by two fundamentally distinct signaling cascades, namely the extrinsic and intrinsic (or mitochondrial) pathways (for a recent review, see [1] ) ( figure 1 ). the extrinsic pathway is started by the ligand-induced oligomerization of specific cell surface receptors, such as fas/cd95 and the tumor necrosis factor receptor (tnfr). this induces the intracellular assembly of the death-inducing signaling complex (disc), a molecular platform for the activation of the caspase cascade that emanates from caspase-8 and results in the activation of effector caspases and nucleases (e.g., caspase-3, -6, and -7, caspase-activated dnase) ( figure 1) [s8,s9]. in contrast, the intrinsic pathway is controlled by mitochondria, which collect and integrate pro-and antiapoptotic signals incoming from other organelles as well as from the extracellular microenvironment. notably, proapoptotic stimuli as diverse as dna damage, endoplasmic reticulum (er) stress, lysosomal stress, reactiveoxygen species (ros), and calcium (ca 2+ ) overload are able to activate the intrinsic pathway of apoptosis by favoring mitochondrial membrane permeabilization (mmp) (figure 1) [s10,s11]. in some cells, mitochondrial apoptosis may ensue the activation of death receptors, due to the mmp-promoting activity of the bh3only protein bid, which can be proteolytically activated by caspase-8 [s9,s12]. mmp culminates in the loss of mitochondrial transmembrane potential (dy m ), an arrest of mitochondrial bioenergetic and biosynthetic functions, and in the release of mitochondrial intermembrane space (ims) proteins into the cytosol. these proteins include caspase activators like cytochrome c (cyt c) [s13,s14] and smac/diablo [s15], as well as caspaseindependent death effectors such as apoptosis-inducing factor (aif) [s16,s17] and endonuclease g (endog) [s18]. thus, mmp activates caspase-dependent and/or -independent mechanisms to execute cell death [1] . impaired mmp is associated with multiple pathological conditions, including autoimmune diseases and cancer [s19]. conversely, unscheduled mmp contributes to the development of diseases characterized by an excess of cell death, such as ischemia/ reperfusion injuries, trauma, toxic/metabolic syndromes as well as chronic neurodegenerative conditions like amyotrophic lateral sclerosis or alzheimer, parkinson, and huntington diseases [s20]. mmp is regulated by a complex network of signaling pathways that involves both endogenous (e.g., pro-and antiapoptotic bcl during the last decade, major efforts have been dedicated to the elucidation of the mechanisms underlying mmp in health and disease. according to current beliefs, mmp is executed via either of two distinct, yet partially overlapping and non-mutually exclusive, mechanisms. these two routes to mmp are initiated at different mitochondrial subcompartments (notably at the mitochondrial outer or inner membrane, i.e., om and im) and each relies on a specific set of factors. nonetheless, they both lead to a functional and structural collapse of mitochondria that commits the cell to death [1] . notably, mmp is associated with dramatic changes in the mitochondrial network as well as in mitochondrial ultrastructure, concerning both matrix volume and cristae organization [s50-s52]. how these structural modifications of mitochondria might impact on viral infection, however, remains to be elucidated. the abundant presence of the voltage-dependent anion channel (vdac) renders om freely permeable to solutes and small metabolites up to approximately 5 kda. this cutoff ensures that soluble proteins are retained in the ims under normal circumstances. the apoptosis-associated drastic increase in om permeability may originate at the om itself by means of multiple mechanisms, including (1) the assembly of large homo-or heteromultimeric channels, allowing for the release of ims proteins, by proapoptotic pore-forming proteins of the bcl-2 family (e.g., bax, bak) [10,11;s53,s54] ; (2) the destabilization of the lipid bilayer mediated by proapoptotic bcl-2 family members (e.g., bax, figure 1 . the extrinsic and the intrinsic (mitochondrial) pathways of apoptosis. the extrinsic apoptotic pathway involves the activation of death receptors at the cell surface, followed by a caspase cascade that eventually leads to the execution of cell death. in contrast, different proapoptotic stimuli initiate the intrinsic pathway by triggering mitochondrial membrane permeabilization (mmp). following mmp, intermembrane space proteins are released into the cytosol, the mitochondrial transmembrane potential (dy m ) is dissipated, and the bioenergetic and redoxdetoxifying functions of mitochondria are compromised. the resulting bioenergetic and redox crises, associated with the activation of both caspasedependent and -independent executioner mechanisms, commit the cell to death. the two pathways are interconnected by the bh3-only protein bid, whose truncated form (tbid) is generated by caspase-8 and can target mitochondria to trigger mmp. for a more detailed description of the intrinsic and extrinsic pathways of apoptosis please refer to the introduction and to [1] . disc, death-inducing signaling complex; er, endoplasmic reticulum. doi:10.1371/journal.ppat.1000018.g001 truncated bid, i.e., tbid), which results in the priming of mitochondria for the release of ims proteins [s55-s58]; and (3) the induction of the so-called mitochondrial permeability transition (mpt) at the im, following the interaction between bax (or tbid) and components of the permeability transition pore complex (ptpc) at the om [12;s59-s63]. in this latter case, mmp begins and ends at the om, yet is mediated by an event taking place mainly at the im, i.e., mpt (see the section ''mmp regulation by the ptpc'' for further details). independently from the specific mechanisms that activate mmp, the bcl-2 family of proteins exerts a major regulation of this process [s64]. the bcl-2 family is composed of antiapoptotic multidomain members (e.g., bcl-2, bcl-x l , mcl-1), which contain four bcl-2 homology (bh) domains (bh1-4) [s65], proapoptotic multidomain proteins (e.g., bax, bak) [s66], which contain three bh domains (bh1-3), and pro-apoptotic bh3-only proteins (e.g., bid, bad) [13] . due to an additional c-terminal domain, some members of all the subgroups share the ability to insert into the om and other intracellular membranes (e.g., er) [1] .the specific set of bh domains contained in each bcl-2 family member determines its profile of activity [s67-s69]. in this context, early structure-function studies identified bh1, bh2, and bh4 as the major antiapoptotic determinants of bcl-2 [s67-s70]. conversely, the presence of the bh3 domain was found to suffice for apoptosis induction by bax (as well as for heterodimerization with bcl-2/bcl-x l ) [s71-s72]. later, numerous reports showed that the conserved transmembrane (tm) domain and less conserved, unstructured loops between bh domains also contribute to define the functional profile of bcl-2 proteins, either by acting as targeting signals for subcellular compartments (e.g., mitochondria, er) or by modulating the overall tertiary structure [s73-s77]. bcl-2/bcl-x l stabilizes mitochondrial membranes via multiple mechanisms, including (1) the sequestration into inactive complexes of its proapoptotic counterparts, bax, bak, and bh3only proteins (e.g., bid) (for review: [s65,s78]), (2) inhibitory interactions with ptpc constituents, in particular with vdac and the adenine nucleotide translocase (ant) [12, 14] , (3) an enhancement of cyt c oxidase activity and mitochondrial respiration [s79], and/or (4) indirect effects on intracellular ca 2+ stores of the er [s80,s81]. while bax/bak execute mmp by one (or more) of the aforementioned mechanisms, bh3-only proteins exhibit indirect proapoptotic effects [1;s66,s82]. thus, ''activator'' bh3-only proteins (e.g., bid) would directly interact and activate bax/bak, whereas the ''derepressors'' (e.g., bad) would rather disrupt bcl-2/bcl-x l inhibitory complexes, thus allowing for the release of bax/bak [15,s82] . in healthy cells, inactive bak is constitutively associated with mitochondria, while bax is found as a cytosolic monomer [s66]. upon apoptosis induction, both undergo a conformational modification to become activated, and bax translocates to om in the form of an active dimer [s83,s84]. when bax or bak induce mmp, the release of specific ims proteins occurs via large pores in the om and may precede dy m loss. in some instances of bax-mediated apoptosis, indeed, mitochondrial membrane permeabilization (momp) occurs without discernable dy m alterations [1] (figure 2 ). large pores formed by the oligomerization of proapoptotic bcl-2 proteins (e.g., bax, bak) and/or the voltage-dependent anion channel (vdac) may promote selectively mitochondrial outer membrane permeabilization (momp). in this case, specific intermembrane space (ims) proteins are liberated in the cytosol, but the mitochondrial transmembrane potential (dy m ) is (at least initially) retained (a,b). on the contrary, some proapoptotic stimuli, such as calcium (ca 2+ ) overload, reactive oxygen species (ros), and the lipid second messenger ceramide, favor mmp by inducing the permeabilization of the inner mitochondrial membrane (im) via the activation of the permeability transition pore complex (ptpc). when the ptpc opens, dy m is immediately lost and an unregulated entry of solutes and water into the mitochondrial matrix occurs. this results in the osmotic swelling of mitochondria, followed by rupture of both mitochondrial membranes and the unspecific release into the cytosol of ims proteins (a,c) (please refer to the sections ''mmp regulation by bcl-2 family proteins'' and ''mmp regulation by the ptpc'' for additional details). notably, antiapoptotic proteins from the bcl-2 family play a role in both models. aif, apoptosis-inducing factor; ant, adenine nucleotide translocase; ck, creatine kinase; cypd, cyclophilin d; cyt c, cytochrome c; hk, hexokinase; oxphos, oxidative phosphorylation complexes; pbr, peripheral-type benzodiazepine receptor. doi:10.1371/journal.ppat.1000018.g002 despite their structural similarity, each bh3-only protein presents a specific mechanism of activation (either at a transcriptional level or mediated by post-translational modifications), and acts as a sensor of a particular type of cell stress [15,s82] . for instance, the bh3-only proteins puma and noxa are activated by dna damage via p53dependent transactivation [16;s85] , bim and bmf are released from cytoskeletal structures upon c-jun n-terminal kinase (jnk)-mediated phosphorylation [s86-s88], and bid is proteolytically processed by caspase-8 following the activation of the extrinsic pathway of apoptosis [s9,s12]. following caspase-8-mediated cleavage, a glycine residue of tbid is exposed, allowing for post-translational (rather than usual cotranslational) n-myristoylation. this modification has been shown to act as an activating switch and to enhance tbid-induced cyt c release and cell death [s89] . interestingly, tbid mitochondrial targeting [s90] and proapoptotic activity [s91] have been associated with cardiolipin, a mitochondrial lipid particularly abundant in the im. tbid-cardiolipin interaction requires three ahelical domains (a4-a6) of tbid and occurs prominently at the contact sites between the om and the im [s92]. cardiolipin might also be implicated in the dissociation of bid fragments (tbid and nbid), which would rather occur during the targeting of tbid to mitochondria than immediately after caspase-8-mediated cleavage [s93] . although bcl-2 family proteins exert their apoptosismodulatory functions mainly at mitochondria, extra-mitochondrial activities contribute to their effects. for instance, bcl-2/bcl-x l localize at the er and decrease luminal ca 2+ concentration, thus protecting against ca 2+ -dependent death stimuli [s80,s94,s95]. conversely, bax/bak favor the transfer of ca 2+ from the er to mitochondria and cell death [s96,s97]. moreover, bax 2/2 /bak 2/2 mefs show an impaired mobilization of er ca 2+ following numerous proapoptotic stimuli, which can be partially restored by overexpressing the sarco/endoplasmic reticulum ca 2+ atp-ase (serca) [17] . taken altogether, these observations point to the bcl-2 system as a prominent pharmacological target for the modulation of mitochondrial apoptosis (for a review, see [18] ). mmp may originate at the im due to the activation of the ptpc, a large multiprotein structure assembled at the contact sites between om and im. this applies in particular to cell death models characterized by enhanced ca 2+ fluxes and disproportionate ros generation [s98] . ptpc activation provokes a sudden increase in the im permeability to solutes of low molecular weight (i.e., mpt), which leads to the unregulated entry of water and osmotic swelling of the mitochondrial matrix. in turn, this may result in the physical rupture of the om, because the surface area of the im (with its folded cristae) largely exceeds that of the om [s63,s98,s99]. in the context of mpt-derived mmp, dy m dissipates before om is permeabilized and ims are released ( figure 2) [s63]. although its exact molecular composition remains elusive, numerous independent studies suggest that the ptpc might result from the association of multiple proteins, including ant (in the im) and vdac (in the om), in the context of a dynamic interaction with mitochondrial matrix proteins (e.g., cyclophilin d [cypd]), ims proteins (e.g., creatine kinase [ck]), om proteins (e.g., peripheral-type benzodiazepine receptor [pbr]), as well as with cytosolic factors (e.g., hexokinase isoforms) (for recent reviews, see [s63,s100,s101]). nevertheless, genetic studies performed in the murine system suggest that all the aforementioned components of the ptpc, most of which exist in multiple isoforms, are either dispensable for cell death or preferentially participate in necrotic pathways (rather than in apoptosis) [19-21;s102 ]. in addition, controversial views remain about the mechanisms by which the ptpc promotes mpt and therefore mmp. some authors have proposed that in physiological conditions vdac would be found within the ptpc in a state of low conductance, rapidly switching between the open and closed conformations [s103]. in this configuration, the ptpc would ensure the normal exchange of metabolites between the mitochondrial matrix and the cytosol. following proapoptotic stimuli, a state of high conductance for vdac would be favored, resulting in longlasting openings of the ptpc and mpt [12;s59] . alternatively, it has been suggested that the high conductance state of vdac would serve to its physiological functions, whereas cell death would result from a closed conformation, favoring a transient hyperpolarization of the mitochondrial matrix, followed by osmotic imbalance, swelling, and eventually mmp [s104-s105]. several reports indicate ptpc components as targets for the apoptosis-modulatory activity of both pro-and antiapoptotic bcl-2 family members. in this context, it has been demonstrated that bax and bak accelerate the opening of vdac in reconstituted proteoliposomes, and that vdac-deficient mitochondria do not exhibit bax/bak-induced cyt c release and dy m dissipation occurring in vdac-proficient control mitochondria [12;s106]. in the same model, recombinant bcl-2/bcl-x l as well as synthetic peptides corresponding to their bh4 domains were shown to prevent vdac opening, cyt c release, and dy m dissipation [s107,s108]. in addition, the bh3-only proteins bid and bim have been reported to interact directly with vdac, the latter interaction being remarkably enhanced during apoptosis [s62,s109]. bax and bcl-2/bcl-x l also modulate ptpc activity by binding to ant. as demonstrated by the yeast two-hybrid system, coimmunoprecipitation assays, and in artificial lipid bilayers, ant and bax directly interact and cooperate to form a channel with distinct electrophysiological properties as compared to the channels formed by bax or ant alone [22;s110]. in artificial membranes, the presence of bcl-2 inhibited cooperative channel formation by bax and ant as well as the atractyloside-induced assembly of channels by ant alone, thus pointing to a direct interaction between ant and bcl-2 [s46,s110]. furthermore, bcl-2 promotes (and bax inhibits) adp/atp exchange in ant-containing proteoliposomes, isolated mitochondria, and mitoplasts [s111]. interestingly, in this system the bax-mediated inhibition of ant translocase activity could be separated from the formation of cooperative channels by bax and ant [s111]. as determined by co-immunoprecipitation and proteomics analysis, the interactome of ant undergoes major rearrangements in the course of the chemotherapy-induced apoptosis. thus, soon after the treatment with etoposide of a human tumor cell line (ht29 cells), the amount of bax contained within the ant interactome significantly augmented, whereas the quantity of bcl-2 was decreased [s112]. as previously mentioned in the section ''mmp regulation by bcl-2 family proteins'', bcl-2 family members are known to modulate luminal ca 2+ concentration and ca 2+ release at the er [17;s94,s95]. in doing so, they exert an additional indirect control on the ptpc, since cytosolic ca 2+ liberated from er stores (for instance upon the induction of the unfolded protein response) can accumulate in mitochondria and promote ptpc opening, mptdependent mmp, and cell death [s40]. thus, it appears that an intricate crosstalk for the modulation of mmp exists between mitochondria and the er, in which proteins from the bcl-2 family participate at the level of both organelles [s80,s81]. during the last decade, numerous viral proteins have been reported to modulate (either positively or negatively, either in a direct or indirect fashion) the apoptotic response of host cells to infection ( figure 3 , tables 1 and 2) [7;s42]. with regard to this, viral factors can be classified into one of the four following subgroups: proapoptotic proteins (1) that insert into mitochondrial membranes and hence trigger mmp through the action of amphipathic a-helical domains or (2) that promote mmp indirectly, through the activition of host-encoded factors (table 1) , and antiapoptotic modulators (3) that exhibit sequence and/or structural similarity to multidomain bh1-4 members of the bcl-2 family (so-called viral bcl-2 proteins [vbcl-2s]) or (4) that inhibit apoptosis via other mechanisms (table 2) . notably, some viral proteins exhibit mixed apoptosis-modulatory functions, and hence cannot be unambiguously classified into one of the aforementioned groups. several proteins encoded by the human immunodeficiency virus 1 (hiv-1) exert a proapoptotic activity, thereby contributing to the hiv-induced depletion of cd4 + lymphocytes (for a review, see [23] ). among these, the viral protein r (vpr) has direct mitochondrial effects in numerous cell types independently from its mode of delivery (viral infection, transfection of a vpr gene, or exogenous administration of recombinant vpr protein) [s113,s114]. the c-terminal moiety (aa 52-96) of vpr directly interacts with ant and vdac, thereby triggering mmp associated with dy m loss, ims proteins release, and caspase cascade activation [24;s41,s115]. when added in vitro to purified mouse liver mitochondria, a synthetic vpr-derived peptide (vpr 52-96 ) induced large amplitude swelling (an indicator of im permeabilization and ptpc pore opening) in less than 15 minutes. this effect could be prevented by bcl-2 as well as by pharmacological agents targeting ant (such as bongrekic acid [ba]) or vdac (such as 4,49-diisothiocyanatostilbene-2,29disulfonic acid [dids]) [24] . in lymphoid cells, vpr-mediated mmp and apoptosis is facilitated by bax, yet inhibited by overexpression of bcl-2 or addition of the ptpc inhibitor cyclosporine a (csa) [s41,s115]. recently, it has been proposed that two distinct domains of vpr (namely aa 27-51 and aa 71-82) would bind to a region encompassing the first ant ims loop and part of its second and third tm helices [s114]. this model may explain why vpr is able to convert ant into a non-specific pore, as this has been observed experimentally when adding vprderived peptides to ant-containing proteoliposomes [24] . importantly, one of the vpr arginine residues that is required for the interaction with ant (r77) [s41] is frequently mutated (r77q) in long-term non-progressors (i.e., hiv-1-carriers who fail to develop signs of cd4 + t cell depletion within 15 years after infection) [s116]. this points to the functional relevance of the ant-vpr interaction to the development of aids. nonetheless, vpr has been reported to modulate viral replication independently from its proapoptotic function, presumably via an interaction with host proteins other than ant [s117]. the minimal cytotoxic domain of vpr that binds to ant (aa 67-82) is structured as an amphipathic a-helix [24] . this short cytotoxic domain (vpr [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] has been recently conjugated with a tumor blood vessel rgd-like ''homing'' motif. this hybrid peptide is highly efficient in killing human endothelial cells, presumably because it binds to the cell surface, internalizes, and finally interacts with mitochondrial membranes to trigger mmp [s118]. in contrast with vpr 52-96, vpr 67-82 induces cell death independently from bax and bak, and efficiently overcomes bcl-2-mediated apoptosis resistance [s118]. the differences in the modes of action of vpr 52-96 and vpr 67-82 remain elusive. nonetheless, the design of cell type-targeted mitochondriotoxic peptides, such as those derived from vpr, has opened tantalizing possibilities for the therapeutic induction of apoptosis (reviewed in [9;s119,s120]). hepatitis b virus (hbv) is one of the most prominent etiological agents of chronic liver disease and persistent infection is associated with hepatocellular carcinoma [25;s121]. hbv x protein (hbx) is implicated in viral replication and exhibits an oncogenic potential in animal models [25;s121]. moreover, hbx sensitizes cells to tumor necrosis factor a (tnfa)-induced apoptosis [s122]. heterologous expression of hbx in cultured human hepatoma cells results in its mitochondrial accumulation, interaction with vdac3, and dy m dissipation, coupled to a perinuclear redistribution of mitochondria [s123,s124]. mutagenesis studies revealed that hbx localization to mitochondria and dy m dissipation do not depend on its transactivation domain [s125,s126], but require a putative tm region (aa 54-70) of the protein, while two additional amphipathic helical domains (aa 75-88 and 109-131) provide only marginal contributions [s127]. morevoer, hbx binds to the heat shock protein of 60 kda (hsp60), a mitochondrial matrix chaperon [s126]. interestingly, ultrastructural and functional impairment of mitochondria as well as vdac3 overexpression have been associated with chronic liver disease, further supporting an etiological role for hbx in this context [s128]. poliovirus (plv) infection causes paralytic poliomyelitis, an acute disease resulting in flaccid paralysis associated with caspasedependent apoptosis of motor neurons [s129]. similar to hbx, the plv viroporin 2b localizes to mitochondria, induces a perinuclear redistribution of these organelles and alters their morphology, suggesting that 2b might exert proapoptotic effects by directly promoting mmp [s130]. as discussed below, this is not the sole mechanism accounting for plv-induced neurodegeneration (see the section ''indirect mmp facilitators''). similar perinuclear clustering of mitochondria and dy m loss, followed by partial cyt c release and signs of apoptosis (e.g., phosphatidylserine exposure on the outer leaflet of the plasma membrane and chromatin condensation), is observed upon the overexpression of the orfc protein from the walleye dermal sarcoma virus (wdsv), a retrovirus causing benign tumors in fish characterized by seasonal regression [s131,s132]. orfc is a basic protein of 120 aa that localizes to mitochondria, although it does not possess any homology to known mitochondrial targeting sequence (mts) [s132]. interestingly, regressing tumors express orfc at high levels, pointing to the involvement of orfc-mediated apoptosis in this phenomenon [s131,s132]. human t lymphotropic virus 1 (htlv-1) infection is linked with a diverse range of neurodegenerative and lymphoproliferative disorders, notably acute t cell leukemia [26] . the genome of this complex retrovirus codes for typical structural and enzymatic proteins but also for unique regulatory and accessory factors that are involved in both htlv-1 viral cycle and pathogenesis [s133,s134]. among these, p13(ii) is an 87-aa protein that is targeted to mitochondria, where it promotes a rapid flux of k + and ca 2+ across im together with swelling, dy m dissipation, and fragmentation [27;s135]. p13(ii) n-terminus includes a short hydrophobic leader peptide, followed by an amphipathic a-helical mts. within this region, ten residues are sufficient to target a green fluorescent protein (gfp)-p13(ii) fusion to mitochondria [s136], and four arginines form a positively charged patch on one side of the a-helix, which is responsible for its amphipathic properties [s137]. p13(ii) expression has been shown to enhance caspase-dependent fas-and c2 ceramide-induced apoptosis [s138], and to suppress the proliferative and tumorigenic potential of cells transformed by the myc and ras oncogenes [s135]. the bovine leukemia virus (blv) is an htlv-1 homolog causing lymphoproliferative disorders in multiple species [s139]. similarly to p13(ii), blv accessory protein g4 is localized to both the nucleus and mitochondria, due to an mts consisting of a hydrophobic region and an amphipathic a-helix [s140]. while g4 is known to alter mitochondrial morphology [s140], its exact role in blv pathogenesis remains poorly understood. indeed, whereas g4 exhibits an oncogenic potential both in vitro and in vivo [s141,s142] and g4-deleted blv variants are characterized by reduced in vivo propagation [s143], blv-infected peripheral blood mononuclear cells are protected from apoptosis independently from g4 expression [s144]. as a possibility, g4 effects on cellular transformation may rather result from its interaction with farnesyl pyrophosphate synthetase (fpps) than from the direct modulation of mmp [s145]. pb1-f2 is an 87-aa protein encoded by influenza a virus (iav) that determines virulence in murine models of infection [28;s146]. while iavs genetically engineered to lack pb1-f2 replicated normally both in tissue cultures and in mouse lungs, their pathogenicity and associated mortality were greatly diminished as compared to wild-type strains [s146]. pb1-f2 inserts into mitochondrial membranes via a positively charged amphipathic ahelix located in its c-terminus [29] . there, pb1-f2 acquires poreforming properties similar to those displayed by proapoptotic bcl-2 family members (e.g., bax) [s147]. the domain necessary and sufficient for mitochondrial targeting has been mapped to residues 46 to 75 of pb1-f2 [s148]. this region comprises a short hydrophobic region including several basic residues followed by an amphipathic a-helix, and closely resembles the mts found in vpr, p13(ii) and, g4 [29] . as assessed by glutathione-s-transferase pulldown assays followed by mass spectrometry, the sole mitochondrial proteins interacting with pb1-f2 are the isoform 1 of vdac (vdac1) and the isoform 3 of ant (ant3) [s149]. accordingly, ptpc blockers such as ba and csa prevent pb1-f2induced mmp and apoptosis [s149]. however, pb1-f2 is capable of destabilizing lipid bilayers in the electric field, implying that the protein may promote mmp by acting directly on mitochondrial membranes [s147]. altogether, human papillomaviruses (hpvs) are responsible for a broad range of infectious diseases, ranging from anogenital warts (e.g., hpv-6 and -11) to progressive dysplastic-neoplastic lesions of the genital mucosa (e.g., hpv-16 and -18) [s150]. the hpv genome codes for a 90-aa protein encompassing part of the e1 and e4 open reading frames (orfs), called e1e4 [s151]. in human mature keratinocytes (the natural host for hpv infection), e1e4 binds to cytokeratins, thereby inducing the total collapse of the keratin (but not of tubulin and actin) cytoskeleton [30] . thereafter (or in cells lacking cytokeratins), e1e4 localizes to mitochondria, due to an n-terminal leucine-rich region [s152]. via a yet unidentified mechanism, e1e4 displaces mitochondria from microtubules, promoting the aggregation of the organelles in a large perinuclear cluster, followed by dy m dissipation and apoptosis [s152]. both the structural protein vp3 and the non structural protein 2c encoded by the avian encephalomyelitis virus (aev) promote cell death [s153,s154]. in different cell lines, vp3 localizes to mitochondria and sets off the caspase cascade leading to apoptosis [s153]. comparable effects result from the expression of 2c, which is a conserved protein of picornaviruses with a role in several steps of the viral life cycle [s154]. the proapoptotic function of 2c is associated with an n-terminal domain of 35 aa (from residues 46 to 80), which has a putative a-helical structure and lies in the proximity of a coiled-coiled domain [s154]. as a last example, the non structural protein 4a (ns4a) from hepatitis c virus (hcv) localizes (at least in part) at mitochondria, thereby causing mitochondrial damage (associated with dy m dissipation and cyt c release) and impairing the intracellular distribution of these organelles [s155]. notably, acute hcv infection often progresses to chronic hepatitis, cirrhosis and hepatocellular carcinoma [s156], and hence it is not surprising that the genome of hvc encodes for several other modulators of apoptosis (see also the sections ''indirect mmp facilitators'' and ''other antiapoptotic viral strategies'') [s156,s157]. multiple proteins encoded by the hiv-1 genome initiate mitochondrial apoptosis in an indirect fashion (table 1) , without a physical interaction with mitochondrial membrane proteins. thus, nef stimulates lysosomal membrane permeabilization resulting in the release of cathepsin d from the lysosomal lumen into the cytosol. this triggers the activation of bax (but not that of bak) and mmp-dependent cell death [s158,s159]. the hiv-1 envelope glycoprotein complex (env, constituted by gp140 and gp41) is expressed by infected cells and promotes cellto-cell fusion by interacting with its receptor/co-receptor complex (cd4/cxcr4 or cd4/ccr5) on the surface of uninfected cells. env-elicited syncytium formation is followed by mmp after a latency of at least 12 hours [31] . env triggers mmp through a complex signal transduction pathway that involves the sequential activation of cyclin-dependent kinase 1 (cdk1), mammalian target of rapamycin (mtor), p38 map kinase, phosphorylation of p53, and p53-dependent transactivation of puma and bax [32,33;s160]. tat, a powerful activator of hiv-1 gene expression, triggers apoptosis of infected and uninfected cells, thereby contributing to the hiv-1-induced neurodegeneration [34;s161-s162]. transfection with tat results in its accumulation in mitochondria followed by dy m loss, ros overproduction, and caspase activation [s163]. however, recombinant tat protein added to cultured cells fails to localize at mitochondria and primarily accumulates in the endosomal compartment, presumably due to its uptake via the endocytic pathway [s164]. tat associates with the tubulin network through a 4-aa subdomain of its conserved core region, thereby altering microtubule dynamics, promoting the proteasomal degradation of the microtubule-associated protein 2 (map2), and activating a mitochondrion-dependent apoptotic pathway [35;s162]. tat cytotoxicity relies (at least partially) on the proapoptotic activity of the bh3-only protein bim. in healthy cells, bim is inactivated through its association with the microtubule-associated dynein motor complex, and tat liberates bim from this inhibition [35;s86] . thus, bim 2/2 cells exhibit increased resistance against tat-induced cell death [35] . nevertheless, tat may influence mitochondrial apoptosis through other mechanisms, and contradictory reports suggest that tat might regulate p53 activity as well as the expression levels of bax and bcl-2 [s165-s167]. the hiv-1-encoded protease is required for the viral life cycle because it processes large polypeptide precursors into mature viral proteins. due to its degenerate substrate specificity, the viral protease promotes the proteolytic activation of caspase-8, as assessed both in vitro and in t cells, which leads to bid cleavage and mitochondrial apoptosis [36] . reportedly, the hiv-1 protease would also favor apoptosis and viral replication via the cleavage and inactivation of bcl-2, which would result in the oxidative stress-dependent activation of nf-kb, a transcription factor required for hiv-1 enhancer activation [s168-s169]. conversely, high bcl-2 levels protect cells in vitro and in vivo from the viral protease and prevent apoptosis induced by hiv-1 infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, as well as the secretion of tumor necrosis factor b (tnf b) [s169]. additional viral proteases have been implicated in the induction of apoptosis. thus, the hcv non structural protein 3 (ns3) participates in a protease complex [s170], and has been reported to induce caspase-8-mediated apoptosis independently from its enzymatic activity [s171]. upon expression either as single proteins or in combination, plv proteases 2a (2apro) and 3c (3cpro) activate caspase-dependent apoptosis [s172-s174]. however, other mechanisms are involved in the induction of apoptosis by pvs, including (1) modulation of antiapoptotic proteins of the bcl-2 family (e.g., bcl-x l ) [s175]; (2) jnk-mediated activation of bax [s176]; and (3) mmp promoted by viroporins 2b and 3a (see also the section ''direct inducers of mmp'') [s130]. following the infection with human adenoviruses (advs), cells exhibit an apoptotic response mediated by the expression of the viral e1a protein. this lethal response can be counteracted by the vbcl-2 e1b-19k (see also the section ''viral bcl-2 homologs'') [37] [38] [39] . notably, e1a was the first viral protein found to promote apoptosis [37] [38] [39] , via both p53-dependent and -independent mechanisms, the latter involving additional viral factors and in particular products of the e4 gene that are expressed upon e1amediated transactivation [s177-s182]. moreover, e1a sensitizes cells to apoptosis induced by multiple stimuli including death receptor agonists (e.g., fasl, tnfa, and tnf-related apoptosis inducing ligand [trail]) [s183-s185] and nitric oxide (no) [s186]. recently, a prominent role for bh3-only proteins (and in particular for bik) has been reported in adv-induced apoptosis [40;s187]. bik is upregulated at the transcriptional level after adv infection, in a p53-dependent fashion [s188]. moreover, during the viral life cycle, the proapoptotic activity of bik is enhanced as a result of an activating phosphorylation. accordingly, sirna-mediated depletion of bik has been shown to dramatically diminish adv-dependent cell death [s187]. e4orf6, a 34-kda protein encoded by the adenoviral gene e6, can promote apoptosis by inhibiting the protein phosphatase 2a (pp2a) [41] . pp2a inhibition prolongs the signal of dna damage emanating from phosphorylated histone h2ax (ch2ax), thereby leading to poly(adp-ribose) polymerase (parp) hyperactivation, aif nuclear translocation, and ultimately cell death [41] . moreover, pp2a is known to maintain mitochondrial bcl-2 in an hypophosphorylated form, which allows for its antiapoptotic function [s30] . in this context, pp2a inhibition would also favor the phosphorylationdependent inactivation of bcl-2. e6 and e7 contribute to the transforming properties of high-risk hpvs by targeting p53 to ubiquitin-mediated degradation [42;s189] and by inactivating the retinoblastoma (rb) protein [43;s190], respectively. e6 (alone or together with e7) has been reported to sensitize cells to different apoptotic stimuli [s191-s193], via mechanisms that may depend [s191,s193] or not [s192] on p53. similar to e6, e7 has been implicated in the sensitization of cells to cell death induced by growth factor withdrawal [s194], chemotherapeutic agents [s191,s195], and ultraviolet (uv) irradiation [s196]. the vesicular stomatitis virus (vsv) belongs to the family of rhabdoviruses, and its infection is associated with the development of neurological disorders characterized by enhanced neuronal apoptosis [s197,s198]. thus, vsv-infected cells exhibit an early activation of the mitochondrial pathway of apoptosis [s199], which does not depend on de novo viral protein synthesis nor on viral replication [s199,s200]. multiple pathways are involved in vsv-induced apoptosis, including (1) while the induction of host cell apoptosis may favor viral dissemination at late stages of infection, it is vital for viruses to inhibit pcd at early steps of the infectious cycle, thereby avoiding premature cell death and allowing the virus to replicate. thus, viruses have developed a battery of bcl-2 homologs by which they mimic the major antiapoptotic system of host cells (for a recent review, see [s215]). in some instances, such vbcl-2s fail to show significant sequence similarity with their mammalian counterparts, yet exhibit striking structural resemblance. finally, a number of viral factors inhibit apoptosis via other mechanisms, which do not directly involve the bcl-2 system (table 2 ). the ''founder'' of the viral bcl-2 homolog (vbcl-2) family is the 19-kda protein encoded by the adenoviral e1b gene (e1b-19k) [39;s216] . during adv infection, e1b-19k blocks host cell apoptosis, thereby sustaining viral replication [47] have been originally identified thanks to their interaction with e1b-19k. while e1b-19k and bcl-2 exhibit limited overall sequence similarity [51] , they share short homologous domains that can be exchanged between the two proteins without a significant loss in their antiapoptotic functions [s226,s227]. accordingly, bcl-2 and e1b-19k functionally complement each other, and in hela cells, bcl-2 overexpression is able to substitute for the absence of e1b-19k, thereby allowing for productive adv infection and inhibiting tnfr-and fas-mediated apoptosis [51] . human cytomegalovirus (cmv) encodes several proteins that subvert host cell functions in order to favor viral propagation [52] . one of the best characterized among these factors is the product of the cmv gene ul37 (pul37x1), known also as viral mitochondria-localized inhibitor of apoptosis (vmia). vmia, which is required for viral replication, has been shown to inhibit apoptosis triggered by different stimuli, including ligation of death receptors and exposure to cytotoxic agents, as well as infection with a mutant adv strain lacking the antiapoptotic modulator e1b-19k [s228]. although vmia does not share any obvious structural similarity with bcl-2 nor with its viral homologs (vbcl-2s), vmia exerts its antiapoptotic activity predominantly by inhibiting mmp at the level of mitochondria [52] . deletion mutagenesis studies revealed that an n-terminal mts is necessary and sufficient for vmia mitochondrial localization, but not for its antiapoptotic activity, which requires an additional c-terminal region of the protein [52;s46,s228]. vmia can physically interact with bax, recruit it to mitochondria, and neutralize it [53] . since vmia effects on apoptosis are lost in bax-deficient cells, it appears that vmia exerts its antiapoptotic functions solely by neutralizing bax [53] . the structure-function relationship of the bax/vmia interaction has been addressed by mutational and computational analyses, suggesting a model in which the overall fold of vmia closely resembles that of bcl-x l [s229]. in contrast to bcl-x l , however, it seems that vmia does not bind to the bh domain of bax but rather engages in electrostatic interactions involving a region of bax between its bh2 and bh3 domains [s229]. this region is not conserved in bak, explaining why vmia (in contrast to bcl-x l ) fails to interact with bak [53;s229]. besides its ability to neutralize bax, vmia exerts multiple functions, not all of which are directly linked to its mmp-modulatory role. in particular, vmia (1) interacts with ant and enhances its antiporter activity [54] ; (2) induces the fragmentation of the mitochondrial network, which might hamper the propagation of ca 2+ -dependent proapoptotic signals [s230]; (3) inhibits the atp synthasome via an interaction with the mitochondrial inorganic phosphate carrier (pic, an im protein), thus reducing mitochondrial atp generation [54] ; and (4) induces the release of er ca 2+ stores into the cytosol [s231]. this mobilization of er ca 2+ might have several consequences, including activation of the unfolded protein response, modulation of mitochondrial functions, induction of mitochondrial fission, and protection against proapoptotic signals, through an inhibition in the propagation of ca 2+ waves [s231]. poxviruses cause several diseases of humans and domestic animals, including smallpox, cowpox, sheeppox, fowlpox, and goatpox [s232]. the genome of many poxviruses codes for proteins that, despite the lack of sequence similarity, fold like bcl-2 and exert similar antiapoptotic properties, thereby belonging de facto to the vbcl-2 family. f1l from vaccinia virus (vacv) was first characterized following the observation that viral strains lacking the serpin crma (which acts as a direct inhibitor of caspases [55] ) maintained the ability to protect cells from apoptosis [56] . the c-terminus of f1l is composed by a 12-aa tm domain flanked by positively charged lysines and followed by an 8-aa hydrophilic tail. as assessed by mutagenesis studies, this domain (which exhibits slight homology to the c-terminus of bcl-2) is required for both the mitochondrial targeting and the antiapoptotic function of f1l [s233]. f1l directly interacts with the bh3 domain of proapoptotic bcl-2 family members, including bak [57;s234] and bim [s235], thereby inhibiting dy m dissipation and cyt c release induced by diverse stimuli (e.g., staurosporine, fas crosslinking) [56] . the region of f1l involved in this interaction encompasses aa 64-84 and has limited sequence similarity to known bh3-binding domains [s234]. while f1l has been shown to inhibit mitochondrial translocation and activation of bax, a direct interaction between f1l and bax has never been detected, suggesting that f1l acts upstream of bax activation [s235]. other poxviruses, including variola virus, monkeypoxvirus, and ectromelia virus, encode functional f1l orthologs, all of which display 95% sequence homology in the c-terminus of the protein [56] . myxoma virus (mxv) is a leporipoxvirus causing myxomatosis (a highly lethal disease) in the european rabbit. productive infection by mxv requires the expression of m11l [s236], a 166aa protein with no defined structural motifs except a putative cterminal tm domain [s237] . within this region, six positively charged residues flanking a hydrophobic trait followed by a short positively charged tail constitute an mts, which is responsible for the targeting of m11l to om during infection [s237]. this kind of mts resembles that of some members of the bcl-2 family [s237]. from its mitochondrial localization, m11l suppresses apoptosis induced by treatment with staurosporine [s237] and the pbr ligand protoporphyrin ix [58] , overexpression of bak [s238] , and viral infection [59] . these effects derive from the ability of m11l to constitutively interact with pbr [58] , bak [s238], and bax [59] , and hence to inhibit mpt-dependent mmp as well as bak/bax-mediated momp. bax-mediated apoptosis is blocked in mxv-infected cells lacking bak expression, suggesting that m11l interacts with bax independently from bak [59] . m11l has no obvious sequence homology with bcl-2 or bcl-x l , yet adopts a virtually identical three-dimensional fold [60] . this high level of structural homology allows m11l to associate with a peptide derived from the bh3 domain of bak with an affinity comparable to that of bcl-2 and bcl-x l for the same peptide [60] . thus, m11l represent a structural mimic of bcl-2 and hence a bona fide vbcl-2 [61] . n1l is the most potent virulence factor of vacv and is required for productive replication in vivo, as assessed in murine models of mucosal and brain infection [s239,s240]. preliminary studies indicated that n1l associates with several components of the i-kb kinase (ikk) complex, and hence can interfere with innate immunity signalling mediated by nf-kb and toll-like receptors (tlrs) [s241,s242]. however, cells infected with recombinant viruses with or without the n1l gene exhibited no difference in nf-kb-dependent gene expression [62] , suggesting that another function of n1l underlie its important role in vivo. indeed, during viral infection, n1l binds to endogenous proapoptotic bcl-2 family members such as bid, bak, and bax as well as to exogenous bad and bax (following transfectionenforced overexpression) [62] . similar to m11l, n1l shares no sequence homology with cellular proteins yet exhibits a threedimensional structure that closely mimics that of bcl-2 and bcl-x l [s243]. thus, the surface of n1l possesses a constitutively open groove common to other antiapoptotic members of the bcl-2 family, which mediates the interaction with bh3-containing proteins [62] . the examples provided by m11l and n1l illustrate the importance of the conservation of structure, rather than of primary amino acid sequence, for the maintenance of protein function along evolution. recently, two novel bcl-2-like inhibitors of apoptosis encoded by poxviruses have been discovered: fpv039 from fowlpox virus (fpv) [63] and orfv125 from parapoxvirus orf virus (ppvo) [64] . in human and chicken cells, fpv039 localizes at mitochondria and constitutively interacts with bak, thereby suppressing apoptosis induced by tnfa and bak overexpression. moreover, fpv039 is able to substitute for f1l in inhibiting bak activation and apoptosis triggered by staurosporine and vacv infection, confirming that fpv039 is a functional homolog of f1l [63] . in uv-irradiated hela cells, orfv125 fully inhibits dna fragmentation, caspase activation, and cyt c release, but not jnk activation, an event occurring upstream of mitochondria. the mitochondrial localization of orfv125 is determined by a distinctive c-terminal domain, and is required for its antiapoptotic function. as assessed by bioinformatic analyses, orfv125 shares sequence and structure similarities with antiapoptotic members of the bcl-2 family (i.e., bcl-2, bcl-x l , and bcl-w). accordingly, orfv125 inhibited the uv-induced activation of bax and bak, strongly corroborating the idea that orfv125 represents a novel bona fide member of the vbcl-2 family [64] . epstein-barr virus (ebv) is a prominent member of cherpesviruses, invading primary resting b lymphocytes to establish a latent infection that eventually culminates in cell transformation [s215] . the potent mitogenic effect of ebv is mediated by the coordinated expression of several gene products, including the apoptotic modulators balf1 and bhrf1 [s215,s244]. these two factors share sequence and structure homology with bcl-2 and belong de facto to the growing list of vbcl-2s [65;s245] . while bhrf1 clearly resembles bcl-2 in its antiapoptotic function [66] , the role of balf1 (which is able to interact with bax and bak, and has been proposed as a bhrf1 antagonist) is controversial [65;67] . balf1 is actively expressed in ebv-positive burkitt lymphoma's cell lines and nasopharyngeal carcinoma biopsies, and renders cells independent from serum [s246]. this points to a prominent antiapoptotic (rather than proapoptotic) function for balf1 during ebv-driven tumorigenesis [s246] . as true for other dna viruses, antiapoptotic proteins appear to be essential for the early phases of the herpesvirus life cycle. however, both proteins are neither expressed nor required once latent infection is established [s215], nor they are essential for in vitro viral replication and transformation of b cells [68;s247] . as assessed by immunoelectron microscopy studies, bhrf1 colocalizes with bcl-2 at the om [66, 69] . the c-terminal hydrophobic portion of bhrf1 (which is responsible for bhrf1 targeting to intracellular membranes) exhibits high levels of homology with several members of the bcl-2 family, in particular with bcl-2 (38%), bcl-x l (32%), and bax (34%) [69;s245] . moreover, the threedimensional structure of bhrf1 closely resembles that of bcl-2 in thus far that it contains two central hydrophobic a-helices that are surrounded by several amphipathic a-helices. in contrast to bcl-2/ bcl-x l , however, bhrf1 is not able to sequestrate and inhibit proapoptotic bh3-only proteins, presumably because it lacks an exposed, pre-formed bh3 binding groove [69;s245] [72;s262] . hhv-8 orf16 encodes the so-called kaposi sarcoma-associated bcl-2 (ksbcl-2), a polypeptide of 175 residues that shares limited (15%-20%) overall sequence identity with other bcl-2 homologs (including bcl-2, bcl-x l , bax, bak, and bhrf1) [71] . interestingly, significant amino acid identity is concentrated in the bh1 and bh2 (but not in the bh3) domains [71] . moreover, although ksbcl-2 exhibits an overall fold almost identical to that of bcl-2/bcl-x l , key differences exist in the lengths of helices and loops [73] . presumably, structure and sequence dissimilarity with bcl-2 account for the fact that ksbcl-2 neither homodimerizes nor heterodimerizes with other bcl-2 family members, suggesting that it may have evolved to escape any negative regulatory effects mediated by proapoptotic host proteins from the bcl-2 family [71, 73] . mutation of a conserved arginine and the two adjacent residues within the bh3 binding groove resulted in a correctly folded protein that failed to bind bax bh3 peptide and to inhibit bax toxicity in yeast. moreover, viruses harboring the same mutation exhibited impaired persistent replication and reactivation from latency in vivo [s267] . altogether, these studies point to a major role of m11 in vivo for the establishment of a persistent viral pool and chronic infection, rather than for viral replication and virulence during acute infection [74] . in contrast to ksbcl-2, hvs orf16 has been shown to interact with bax and bak to inhibit virus-induced apoptosis. while orf16 exhibits highly conserved bh1 and bh2 domains, it lacks the core sequence of the conserved bh3 domain, suggesting that this portion might be dispensable (at least in some proteins) for antiapoptotic functions [72] . similar to other vbcl-2s (i.e., bhrf1 and ksbcl-2), orf16 contains a stretch of conserved hydrophobic residues at its c-terminus, ending with basic amino acids, that may direct its (not yet demonstrated) targeting to intracellular membranes [71] . interestingly, several herpesvirus-encoded vbcl-2s cannot be converted into proapoptotic factors by activated caspases during pcd (as it occurs to their mammalian counterparts), and hence fail to display any latent proapoptotic activity [75] . in addition to ksbcl-2, hhv-8 codes for yet another protein with limited homology to bcl-2 [76] . thus, k7 is a 16-kda glycoprotein structurally related to a splice variant of human survivin (survivindex3), a mammalian antiapoptotic factor belonging to the inhibitor of apoptosis protein (iap) family [s268,s269]. both proteins contain an mts, an n-terminal region of a baculovirus iap repeat (bir) domain, and a putative bh2 domain [76] . the mts of k7 consists of a single tm hydrophobic region flanked by positively charged residues, and resembles that of m11l from mxv [s270]. k7 efficiently represses apoptosis induced by activation of death receptors (e.g., fas, tnfr), bax overexpression and thapsigargin-mediated er stress [76;s271] . similarly to other iaps, k7 binds to and hence inhibits caspase-3 via its bir domain. however, k7 antiapoptotic effects depend on its bh2 domain, which mediates the interaction of k7 with bcl-2. thus, it seems that k7 exerts its functions by bridging effector caspases and bcl-2, thereby enabling the latter to inhibit caspase activity [76] . interestingly, k7 has also been shown to modulate intracellular ca 2+ concentration and protein stability, by interacting with the cellular ca 2+ -modulating cyclophilin ligand (caml) and with a regulator of the ubiquitin system, respectively [s270,s271]. whereas mutational analyses showed that the interaction between caml and k7 is required for its antiapoptotic activity [s270], the significance of k7-mediated proteasome regulation remains to be established [s271]. due to its molecular structure, k7 can be considered either as a viral iap (viap) or as a vbcl-2 [76] . to avoid premature apoptosis of the host cell, asfv encodes multiple antiapoptotic proteins, including the vbcl-2 family member a179l (also known as 5-hl) [77;s272,s273]. a179l codes for a polypeptide of 21 kda that contains all known domains associated with bcl-2 structural and functional features, including those mediating protein-binding (i.e., homo-and heterodimerization) and regulating cell death [s272]. thus, a179l has been shown to suppress apoptosis induced by multiple stimuli, including growth factor deprivation [s272] and exposure to cytotoxic agents [s274]. viruses inhibit host cell apoptosis via a plethora of mechanisms other than vbcl-2s. for instance, the of ul36 gene of cmv encodes the viral inhibitor of caspase-8 activation (vica), which has been shown to inhibit fas/cd95-mediated apoptosis by binding to the pro-domain of caspase-8 and preventing its autoproteolytic processing [78] . although this effect regards in particular the pathway of apoptosis emanating from death receptors, it also avoids the activation of the intrinsic pathway occurring along the caspase-8 r bid axis [78;s275] . recently, another mechanism of cmv-mediated inhibition of mitochondrial apoptosis has been discovered [79] . during cmv infection, a 2.7-kilobase virally encoded rna (b2.7) interacts with the mitochondrial respiratory chain complex i (reduced nicotinamide adenine dinucleotide-ubiquinone oxidoreductase) and prevents the mitochondrial release (that normally would be induced in response to apoptotic stimuli) of the complex i subunit grim-19. this stabilizes dy m and results in continued atp production, hence improving the viability of infected cells and favoring the successful completion of the viral life cycle [79] . thus, cmv employs two distinct strategies to influence mitochondrial respiration of infected cells, namely vmia-mediated inhibition of the atp synthasome and stabilization of complex i by b2.7 rna. as a net result, this combined modulation should not lead to an increase in dy m , but to decreased mitochondrial atp generation, correlating with the documented glycolytic switch of cmvinfected cells [s276] . ebv-induced transformation of primary b lymphocytes into continuously proliferating lymphoblastoid cell lines involves proteins other than vbcl-2s [s277]. among these, the epstein-barr nuclear antigens (ebna) 3a and 3c (ebna3a and ebna3c) but not ebna3b may downregulate the bh3-only protein bim, and hence reduce the propensity of host cells to undergo apoptosis [s277]. moreover, the ebna leader protein (ebna-lp) has been shown to interfere not only with host cell transcription but also with the apoptotic machinery [80;s278]. interestingly, it has been suggested that ebna-lp would interact with bcl-2 though the hs1-associated protein x-1 (hax-1) [ , and k13 is required for the long term survival of infected cells [85] . thus, k13 promotes the activation of nf-kb via multiple, partially distinct molecular pathways [s289-s292], which in turn lead to (1) reduced sensitivity to death receptor-mediated [s293] and intrinsic apoptosis [86] ; in addition, rid has been shown to target tyrosine kinase receptors, such as the epidermal growth factor receptor (egfr) [s304] . both rid subunits are tm proteins oriented with their ctermini in the cytoplasm [s305,s306]. ridb contains a cterminal tyrosine residue that is required for receptor internalization and inhibition of fas-and trail-induced apoptosis [s307]. rida has been reported to undergo o-glycosylation [s308] and phosphorylation on serine residues [s309]. how rida posttranslational modifications might affect rid antiapoptotic function remains to be established. mutagenesis studies revealed that the extracellular domain of rida is important for the clearance of egfr from the cell surface, but not for the internalization of death receptors like fas [s310]. interestingly, e3-6.7k, another protein encoded by the e3 unit, is specifically implicated in ridmediated clearance of trail-r2 [s299]. this suggests that additional viral (or cellular) factors might cooperate with rid to determine its target specificity. baculoviruses infect insect cells, and possess at least two different classes of proteins by which they control the host apoptotic response [s311]. one of these is represented by p35, a potent inhibitor of metazoan caspases acting via a cleavage-dependent mechanism [89;s312]. p35 mechanism-based inhibition of caspases is the most broadly acting antiapoptotic system known recently, the viral mitochondrial antiapoptotic protein (vmap), encoded by the m8 gene of chv-68, has been shown to suppress intrinsic apoptosis via a completely novel, dual mechanism [99] . via its n-terminus, vmap is able to augment the recruitment of bcl-2 to mitochondria and to enhance its affinity for bh3-only proapoptotic proteins, thereby suppressing bax activation. morevoer, vmap interacts with vdac1 via two leucine-rich motifs located in the central and c-terminal parts of the protein, thus repressing staurosporine-induced cyt c release and apoptosis. interestingly, vmap is necessary for efficient chv-68 lytic replication in normal cells (with an intact apoptotic apparatus), but not in bax 2/2 /ba 2/2 cells, pointing to a crucial role for apoptosis inhibition during the early steps of the viral life cycle [99] . we have reviewed the cellular impact of viral infection on cell fate via modulation of mitochondrial apoptosis. while specific cellular and molecular mechanisms have been elucidated for a number of individual proteins (e.g., vpr, vmia), a clear scheme of the integrated effects resulting from the expression of whole virus genomes has only recently begun to emerge from transcriptomics and proteomics analyses (for a review, see [100] ). future studies will have to take into account the variability of the host cell and its microenvironmental context (e.g., local inflammation, oxidative stress) as key factors susceptible to modulating the response to specific pathogens. this will undoubtedly be instrumental for the prediction of the general consequences of viral infections, as well as for a more accurate identification of novel therapeutic targets designed to eradicate infectious diseases. a complete list of accession numbers (uniprotkb/swiss-prot knowledgebase, http://www.expasy.org/sprot/) for the proteins discussed in this manuscript can be found online in text s1. text s1 list of the accession numbers (uniprotkb/swiss-prot knowledgebase) of all the proteins described in this article. mitochondrial membrane permeabilization in cell death direct activation of bax by p53 mediates mitochondrial membrane permeabilization and apoptosis ca2+-induced apoptosis through calcineurin dephosphorylation of bad requirement for gd3 ganglioside in cd95-and ceramide-induced apoptosis disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex i of the electron transport chain cell biology mitochondrion-targeted apoptosis regulators of viral origin mitochondrial apoptosis induced by the hiv-1 envelope mitochondria as therapeutic targets for cancer chemotherapy proapoptotic bax and bak: a requisite gateway to mitochondrial dysfunction and death bid, bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel vdac distinct bh3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics the permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins differential targeting of prosurvival bcl-2 proteins by their bh3-only ligands allows complementary apoptotic function and drug-induced apoptotic responses mediated by bh3-only proteins puma and noxa bax and bak regulation of endoplasmic reticulum ca2+: a control point for apoptosis pharmacological manipulation of bcl-2 family members to control cell death the adp/ atp translocator is not essential for the mitochondrial permeability transition pore cyclophilin d-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death mitochondrial apoptosis without vdac bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis apoptosis as an hiv strategy to escape immune attack control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with hiv-1 viral protein rr and bcl-2 hbx gene of hepatitis b virus induces liver cancer in transgenic mice multiple viral strategies of htlv-1 for dysregulation of cell growth control the human t-cell leukemia virus type 1 p13ii protein: effects on mitochondrial function and cell growth a novel influenza a virus mitochondrial protein that induces cell death the influenza a virus pb1-f2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function specific interaction between hpv-16 e1-e4 and cytokeratins results in collapse of the epithelial cell intermediate filament network apoptosis control in syncytia induced by the hiv type 1-envelope glycoprotein complex: role of mitochondria and caspases human immunodeficiency virus 1 envelope glycoprotein complex-induced apoptosis involves mammalian target of rapamycin/fkbp12-rapamycin-associated protein-mediated p53 phosphorylation nf-kappab and p53 are the dominant apoptosis-inducing transcription factors elicited by the hiv-1 envelope induction of apoptosis in uninfected lymphocytes by hiv-1 tat protein hiv-1 tat targets microtubules to induce apoptosis, a process promoted by the pro-apoptotic bcl-2 relative bim hiv-1 protease processes procaspase 8 to cause mitochondrial release of cytochrome c, caspase cleavage and nuclear fragmentation the 19-kilodalton adenovirus e1b transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha the adenovirus e1a proteins induce apoptosis, which is inhibited by the e1b 19-kda and bcl-2 proteins adenovirus e1b 19-kilodalton protein overcomes the cytotoxicity of e1a proteins nbk/ bik antagonizes mcl-1 and bcl-xl and activates bak-mediated apoptosis in response to protein synthesis inhibition the adenoviral e4orf6 protein induces atypical apoptosis in response to dna damage the e6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53 complex formation of human papillomavirus e7 proteins with the retinoblastoma tumor suppressor gene product vesicular stomatitis viruses expressing wild-type or mutant m proteins activate apoptosis through distinct pathways west nile virus infection activates the unfolded protein response, leading to chop induction and apoptosis induction of apoptosis by the severe acute respiratory syndrome coronavirus 7a protein is dependent on its interaction with the bcl-xl protein viral homologs of bcl-2: role of apoptosis in the regulation of virus infection bcl-2, bcl-xl and adenovirus protein e1b19kd are functionally equivalent in their ability to inhibit cell death the e1b 19k protein blocks apoptosis by interacting with and inhibiting the p53-inducible and death-promoting bax protein cloning of a bcl-2 homologue by interaction with adenovirus e1b 19k functional complementation of the adenovirus e1b 19-kilodalton protein with bcl-2 in the inhibition of apoptosis in infected cells a cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to bcl-2 cytomegalovirus cell death suppressor vmia blocks bax-but not bak-mediated apoptosis by binding and sequestering bax at mitochondria cytopathic effects of the cytomegalovirus-encoded apoptosis inhibitory protein vmia viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis the vaccinia virus f1l protein interacts with the proapoptotic protein bak and inhibits bak activation the myxoma poxvirus protein, m11l, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore myxoma virus m11l blocks apoptosis through inhibition of conformational activation of bax at the mitochondria structure of m11l: a myxoma virus structural homolog of the apoptosis inhibitor, bcl-2 a structural viral mimic of prosurvival bcl-2: a pivotal role for sequestering proapoptotic bax and bak functional and structural studies of the vaccinia virus virulence factor n1 reveal a bcl-2-like anti-apoptotic protein fowlpox virus encodes a bcl-2 homologue that protects cells from apoptotic death through interaction with the proapoptotic protein bak a novel bcl-2-like inhibitor of apoptosis is encoded by the parapoxvirus orf virus epstein-barr virus encodes a novel homolog of the bcl-2 oncogene that inhibits apoptosis and associates with bax and bak epstein-barr virus-coded bhrf1 protein, a viral homologue of bcl-2, protects human b cells from programmed cell death epstein-barr virus balf1 is a bcl-2-like antagonist of the herpesvirus antiapoptotic bcl-2 proteins bhrf1 of epstein-barr virus, which is homologous to human proto-oncogene bcl2, is not essential for transformation of b cells or for virus replication in vitro ultrastructural localization of bhrf1: an epstein-barr virus gene product which has homology with bcl-2 epstein-barr virus bhrf1 protein protects against cell death induced by dna-damaging agents and heterologous viral infection a bcl-2 homolog encoded by kaposi sarcoma-associated virus, human herpesvirus 8, inhibits apoptosis but does not heterodimerize with bax or bak herpesvirus saimiri encodes a functional homolog of the human bcl-2 oncogene solution structure of a bcl-2 homolog from kaposi sarcoma virus identification of the in vivo role of a viral bcl-2 antiapoptotic herpesvirus bcl-2 homologs escape caspase-mediated conversion to proapoptotic proteins characterization of an anti-apoptotic glycoprotein encoded by kaposi's sarcomaassociated herpesvirus which resembles a spliced variant of human survivin african swine fever virus gene a179l, a viral homologue of bcl-2, protects cells from programmed cell death a cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation complex i binding by a virally encoded rna regulates mitochondria-induced cell death epstein-barr virus (ebv) nuclear antigen leader protein (ebna-lp) forms complexes with a cellular anti-apoptosis protein bcl-2 or its ebv counterpart bhrf1 through hs1-associated protein x-1 the hepatitis c virus ns2 protein is an inhibitor of cide-b-induced apoptosis e2 of hepatitis c virus inhibits apoptosis viral flice-inhibitory proteins (flips) prevent apoptosis induced by death receptors modulation of host gene expression by the k15 protein of kaposi's sarcomaassociated herpesvirus kshv vflip is essential for the survival of infected lymphoma cells the human herpes virus 8-encoded viral flice inhibitory protein protects against growth factor withdrawalinduced apoptosis via nf-kappa b activation mechanism for removal of tumor necrosis factor receptor 1 from the cell surface by the adenovirus ridalpha/beta complex the adenovirus e3/10.4k-14.5k proteins downmodulate the apoptosis receptor fas/apo-1 by inducing its internalization prevention of apoptosis by a baculovirus gene during infection of insect cells control of programmed cell death by the baculovirus genes p35 and iap baculovirus p35 prevents developmentally programmed cell death and rescues a ced-9 mutant in the nematode caenorhabditis elegans inhibition of the caenorhabditis elegans cell-death protease ced-3 by a ced-3 cleavage site in baculovirus p35 protein an apoptosis-inhibiting baculovirus gene with a zinc finger-like motif cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death iaps block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases african swine fever virus iap-like protein induces the activation of nuclear factor kappa b nuclear and cytoplasmic survivin: molecular mechanism, prognostic, and therapeutic potential a novel inhibitory mechanism of mitochondrion-dependent apoptosis by a herpesviral protein we apologize to our colleagues for not having been able to cite their work. key: cord-259505-7hiss0j3 authors: kong, qingming; xue, chunyi; ren, xiangpeng; zhang, chengwen; li, linlin; shu, dingming; bi, yingzuo; cao, yongchang title: proteomic analysis of purified coronavirus infectious bronchitis virus particles date: 2010-06-09 journal: proteome sci doi: 10.1186/1477-5956-8-29 sha: doc_id: 259505 cord_uid: 7hiss0j3 background: infectious bronchitis virus (ibv) is the coronavirus of domestic chickens causing major economic losses to the poultry industry. because of the complexity of the ibv life cycle and the small number of viral structural proteins, important virus-host relationships likely remain to be discovered. toward this goal, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified ibv particles. results: apart from the virus-encoded structural proteins, we detected 60 host proteins in the purified virions which can be grouped into several functional categories including intracellular trafficking proteins (20%), molecular chaperone (18%), macromolcular biosynthesis proteins (17%), cytoskeletal proteins (15%), signal transport proteins (15%), protein degradation (8%), chromosome associated proteins (2%), ribosomal proteins (2%), and other function proteins (3%). interestingly, 21 of the total host proteins have not been reported to be present in virions of other virus families, such as major vault protein, tenp protein, ovalbumin, and scavenger receptor protein. following identification of the host proteins by proteomic methods, the presence of 4 proteins in the purified ibv preparation was verified by western blotting and immunogold labeling detection. conclusions: the results present the first standard proteomic profile of ibv and may facilitate the understanding of the pathogenic mechanisms. infectious bronchitis virus (ibv), the coronavirus of domestic chickens that causes acute, highly contagious respiratory disease, is one of the most important causes of economic loss in the poultry industry. ibv is an enveloped virus with continuous, positive and single-stranded rna genome, which is the largest of any rna virus characterized [1] and encodes four types of structural proteins. the spike (s) glycoprotein, together with small envelope (e) protein and matrix (m) glycoprotein, consists of the viral envelope, whereas the nucleocapsid (n) protein interacts with genomic rna of the virus to form the viral nucleocapsid, in the invariable order 5'-s-e-m-n-3'. proteins s, e, and m have been studied for their important roles in receptor binding and virus budding. s mediates attachment to cellular receptors and entry by fusion with cell membranes, whereas m interacting with s and n proteins is an essential component of virion and plays pivotal roles in virion assembly, budding and maturation [2, 3] . in addition, s protein can inhibit host cell translation by interacting with eif3f [4] and the interaction between m and actin facilitates virion assembly and budding [5] . e is a poorly characterized small envelope protein present in low levels in the virions. the significance of the e protein appears to be critical for viral budding. another role for protein e is that it can promote apoptosis [6, 7] . viruses constantly adapt to and modulate the host environment during replication and propagation. to govern egress from the host cell and initiation of replication in the target cell, viruses will carry some of the host proteins when released from infected cells. enveloped viruses particularly encoding only small proteins have the capability of incorporating numerous host proteins into or onto the newly formed viruses. it is an important prerequisite for the functional studies to know the protein composition of the purified viral particles, as it allows the analysis of specific proteins and their roles during the virus life cycle, resulting in better understanding of the infection process and the pathogenesis of viruses. as a large number of virus complete genomes have been sequenced since 1980s, more and more host proteins in different enveloped viruses have been studied using viral proteomic approaches. herpesviruses have been the most extensively studied in this respect, such as kaposi's sarcoma-associated herpesvirus (kshv) [8, 9] , marek's disease virus (mdv) [10] , epstein-barr virus (ebv) [11] , human cyotomegalovirus (hcmv) [12] and murine cyotomegalovirus (mcmv) [13] . other doublestranded dna (dsdna) viruses including vacciniavirus have also contributed to a better understanding of this intriguing phenomenon [14] [15] [16] . furthermore, recent studies on identification of the incorporated host proteins in rna viruses have also been undertaken. for retrovirus, various studies in this research area have been performed on human immunodeficiency virus type 1 (hiv-1) [17] [18] [19] [20] and moloney murine leukemia virus (mmlv) [21] . for paramyxovirus, numerous host proteins have been found incorporated into avian influenza virus (aiv) particles and respiratory syncytial virus (rsv) particles [22] [23] [24] . to date, no study of the host proteins in the virions of coronavirus has been performed yet. in this study, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified ibv particles. our analysis resulted in the identification of 2 virus-encoded structural proteins and 60 incorporated host proteins. in addition, we also discussed the functional implications of some host proteins in ibv infection and pathogenesis. viral proteomic analysis requires a highly purified preparation of virions. there was no available permissive cell line capable of supporting productive replication of ibv. although primary chick embryo kidney cell (cek) and chick kidney cell (ck) are capable of supporting productive replication of ibv, their poor yields prohibit them from being used for producing large quantity of ibv. in order to obtain large quantity of ibv virions, this study selected 10-day-old spf embryonated chicken eggs for the growth of ibv strain h52. the af with enrichment of h52 was clarified by differential centrifugation in order to remove the contamination of nuclei, mitochondria, lysosomes, peroxisomes and so on from the chicken embryo. the virus was concentrated through a 20% (wt/vol) sucrose cushion before purified over a non-linear 20%-50% sucrose-tne (tris-buffered saline including 50 mm tris, 100 mm nacl, 1 mm edta, ph 7.4) gradient. two distinct types of ibv particles were isolated by sucrose density gradients. the higher density particles banded at 30%-40% sucrose-tne gradients while the less density particles banded at 20%-30% sucrose-tne gradients. the purity of ibv was confirmed by electron microscopy analysis following negative staining to ensure that the virions have normal viral morphology and to exclude the possible inclusions of vesicles, other cellular organelles and debris (fig. 1a ). an abundance of intact virions were observed without obvious contamination from host cellular materia. proteins in purified virions were separated on 12% sodium dodecylsulfate polyacrylamide gel electrophoresis (sds-page) and stained withc coomassie brilliant blue (fig. 1b ). there were also some lighter bands visible that may represent cellular proteins besides the conjectured major virus-encoded structural proteins. furthermore, the four virus-encoded structural proteins were confirmed by immunoblotting test (fig. 1c) . taken together, the best purification of the ibv was obtained after differential centrifugation to remove the cellular contamination and condensation through a 20% (wt/vol) sucrose cushion with a non-linear sucrose gradient. to obtain a detailed protein composition profile associated with the ibv particles, the proteins in purified ibv particles were extracted for 2-de experiments. to authenticate the results and to compensate the variability figure 1 analysis of purified avian infectious bronchitis virus preparations. a: specific pathogen free (spf) chick embryo-grown major particles h52 from 30%-40% sucrose density gradients, negatively stained with 2% potassium phosphotungstate, ph 6.5. b: sds-page separation of proteins in a purified h52 preparation. 8 μg of proteins were separated on an 5-17.5% polyacrylamide gel and stained with coomassie blue. c: western blotting of the purified h52 virions. viral proteins were separated on 12% polyacrylamide gel and analyzed by western blot with chicken polyclonal antibody against infectious bronchitis virus (massachusetts). the identified viral proteins are indicated. s: spike, n: nuclecapsid, m: mebrane, e: envelope. of gel electrophoresis, three independent experiments were performed with three replicate gels for each experiment. the viral protein profiles were analyzed by 2-d with 250 μg of protein. after the electrophoresis separation, gels were stained with silver and processed for image analysis. for ibv particle-associated proteins separated in the ph3-10 range, 88 protein spots were detected (fig. 2) . to identify the proteins associated with ibv particles, all protein spots detected in the gels were excised and in-gel digested with trypsin followed by maldi-tof/tof (matrix-assisted laser desorption/ionizationtime of flight mass spectrometry) analysis. database search analysis revealed that 2 virus-encoded structural proteins and 60 host proteins were successfully identified. detailed information of the full set of the identified proteins is listed in table 1 ; additional file 1. to better understand the host proteins incorporated with ibv virion and their roles played in ibv infection, these proteins were categorized with biological processes according to uniprot knowledge database (swiss-prof/ trembl) and gene ontology database. the identified 60 host proteins were comprised of cytoskeleton proteins, molecular chaperone, macromolecular biosynthesis proteins, signal transport proteins and glycolytic enzymes (table s1 ; additional file 1). these host proteins were located mainly in the cytoplasm, including cytoskeleton, cytosol and mitochondrion (fig. 3) . to confirm the presence of host proteins in the purified ibv particles after the identification of them by pro-teomic method, we performed immunoblotting experiments. ibv preparation purified from af was analyzed for the presence of n protein, actin, hsp90, annexin a2 and tubulin (fig. 4) . extracts from 10-day-old specific pathogen free (spf) embryonated chicken eggs were included as a positive control. when analyzing the results of virion proteomic studies, the challenge is to prove that the host proteins are really an part of the virion and that they are not just attached non-specifically to the outside of the virus. to address this question, the af from uninfected 10-day-old spf embryonated chicken eggs were parallelly subjected to our standard density centrifugation procedure and the protein extracts from 30%-40% sucrose gradient was used as negative control. gradientpurified virions and the control were separated on 12% sds-page gels, transferred to pvdf membranes, and probed with the appropriate antibodies. as shown in fig. 4 , actin, tubulin and annexin a2 were both found in the purified virions and positive control but not in the af extracts from uninfected 10-day-old spf embryonated chicken eggs. hsp90 is a member of the heat shock protein family which is upregulated in response to stress and has low abundance in unstressed cells. in present study, we detected it only in the purified virions but not in normal cells. it is an expectable result that we also detected actin and tubulin in the af extracts from uninfected 10day-old spf embryonated chicken eggs which resulted from their high concentrations in all eukaryotic cells and subcellular fractions. to provide additional evidence that the host proteins are not just derived from a microvesicle or exosome that co-purified with the virus, we used the bromelain protease protection assay which has been shown to efficiently remove microvesicles from ibv virion preparations [25] . protease treatment of the purified virus preparation strips proteins off any contaminating microvesicles and off the outside of virus particles, such as s protein. in doing so, the microvesicles become lighter than the virions and therefore the virions can be isolated by density centrifugation. proteins that are inside the virion are protected by the lipid envelope and therefore will remain after the protease treatment. and then immunogold labeling of the purified virions was performed ( fig. 5 ). virions were either mock treated or subjected to digestion with bromelain and then were incubated with antibodies against actin, annexin a2, hsp90, ibv massachusetts strain and secondary gold antibodies followed by negative staining. one or two gold particles located on the surface of a virion could be seen for hsp90. this was significantly less compared with the degree of other labelings which is consistent with the fact that there is most likely far more actin, annexin a2 present on the virions than hsp90. in addition, the abundance of actin detected in the 2-de gels is much higher than that of hsp90. virus exploits multiple host proteins during infection for successful entry, replication, egress, and evasion. this is especially true for rna viruses because they encode only little proteins. learning the protein composition profile of the infectious viral particle is prerequisite for studying the role of host proteins during infection. to our knowledge, incorporation of host proteins in the envelopedvirus family coronaviridae has not been investigated so far. in this study, we revealed the presence of virusencoded proteins in infectious bronchitis particles and for the first time confirmed the incorporation of host proteins. a total of 2 viral and 60 host proteins associated with purified ibv particles were identified. in the present study, we failed to obtain m and e protein while other two structural proteins n and s were identified successfully. n protein is easy to identify because it is the most abundant virus-derived protein produced throughout the process of the virus infection, whereas s protein as a major structural protein of ibv located on the surface of viral particles is also easy for identification because it is large (about 175 kda) and has many tryptic cleavage peak in the ms analysis. the identification of m and e protein of coronavirus by ms has been thought to be a difficult task due to their properties, especially in the case of e protein for the following reasons [26] . first, e protein is a very hydrophobic protein. second, it is low-abundant in the virions. third, e is a low molecular weight protein with the mass about 12 kda. fourth, e protein contains two cysteines, which may form disulfide bonds within itself or with other proteins and make e protein difficult to be reduced and subsequently digested. of the total host proteins, 39 have also been described to be present in virions of quite diverse virus families, such as herpesviruses, poxviruses, paramyxovirus and retroviruses [27, 28] . there are some explanations that the incorporated host proteins are common to other virus families. first, they are all enveloped viruses. enveloped viruses contain the viral genome and core proteins wrapped within one or more membranes which are acquired from the host cell during virus assembly and budding. these viruses all share some fundamental feature in the particular stage of their life-cycle and these host proteins are involved in the common process. second, these host proteins would be either highly abundant cytosolic proteins or enriched at the virus budding sites. several of the highly abundant cytosolic proteins found within both ibv and other viral particles are beta actin, tubulin, annexins and enolase. proteins enriched at the virus budding sites including hsp70, hsp90 and gapdh are also identified in ibv and other viruses [9, 11, 12, 14, 19, 24] . some host proteins may be specially incorporated into the virions. in this study, 21 of the total host proteins are reported for the first time. the identified host protein functions in diverse biological processes and some functional groups are analyzed. these proteins participate in a broad array of cellular functions and are involved in many processes in the viral life cycle. the potential roles of some of these proteins are discussed below in relation with ibv infection, pathogenesis and early host antiviral response. numerous viral proteins interact with cytoskeletal elements. many viruses, such as retroviruses, herpesviruses and picornaviruses, even contain the main cytoskeletal element actins in their infectious particles. the transport machinery of actins are proven to be critical at almost every step along the infectious cycle [29] . actin has been found in preparations from several types of retroviruses and paramyxoviruses. for coronavirus, an association of m with cytoskeletal elements has been reported [5] , which indicates an essential function of actin in the replication cycle of coronavirus ibv. in our studies, actin and tubulin were all present in the interior of infectious bronchitis particles and this observation most likely reflects their active participation in moving the viral components to assembly sites. actin and tubulin have been characterized as the major folding substrates for cct (chaperonin containing tcp-1, also termed tric). both cytoskeletal proteins require in vivo and in vitro the interaction with cct to fold to their native states [30] . cct is the most different and complicated protein of all group ii chaperonins in eukaryotic cytosolic chaperonins, which might be involved in the assistance of the folding of a small set of proteins. in addition to the already mentioned actin and tubulin, cct has been found to interact either in vitro or in vivo with other cytoskeletal proteins, cell division control protein 20, protein phosphatase type 2a, and guanine nucleotide-binding protein (g protein) beta subunit [31] , which are all found to be associated with infectious bronchitis particles in present study. it's pleasantly surprising to find that certain viral proteins such as the epstein barr virus-encoded nuclear protein (ebna-3), the hepatitis b virus capsid and the type d retrovirus gag polyprotein are also folded by cct [32] [33] [34] . thus, cct may have an important role in infectious bronchitis viral proteins assembly. other cytoskeletal proteins found to be associated with infectious bronchitis particles are actin-related proteins, wd repeat containing protein, destrin and annexin. several annexin family members (a2, a5 and a11) were identified in purified infectious bronchitis particles. annexins are a well-known multigene family of ca 2+ regulated phospholipid-binding and membrane binding proteins with diverse functions. the presence of annexin a2 is thought to support viral binding, fusion and replication [35] [36] [37] [38] [39] . annexin a5, which interacts with annexin a2, has the opposite effect by preventing fusion, which possibly indicates a potential regulatory role [38] . annexin a2 tightly binds to a member of the s100 family of calciumbinding proteins, s100a10 (p11). upon binding, annexin a2 and p11 form a heterotetramer which is capable of binding two membrane surfaces simultaneously, which potentially promotes fusion events and also plays a role in exocytosis [40] . the p11 protein was also detected by our analysis, suggesting that ibv is also incorporated this complex. other s100 family members such as s100a6 and s100a11 were also detected in viral samples and could play various roles in fusion and membrane organization [41, 42] . heat-shock proteins (hsps) have been known as multifunctional proteins. they facilitate the folding and unfolding of proteins, participate in vesicular transport processes, prevent protein aggregation in the densely packed cytosol and are involved in signaling processes. most, but not all, hsps are molecular chaperones. several viruses require host molecular chaperones for entry, replication, and assembly, as well as other steps in viral production [43, 44] . hsp70 and hsp90 have been found incorporated into ibv. hsp70 interacts with various viral proteins and may be involved in the assembly of adenovirus [45] , enterovirus [46] , vaccinia virus [47] and hantaan virus [48] . alternatively, upon entry into susceptible target cells, virion-associated hsp70 might participate in early events of infection. for example, hsp70 might actively uncoat the viral capsid in a manner similar to its role in the uncoating of clathrin cages [49] . hsp70 and hsp90 have been shown to interact with hepatitis b virus reverse transcriptase and to facilitate the initiation of viral dna synthesis from hepatitis b virus pregenomic rna [50, 51] . for sendai virus (sv), the viral proteins synethsis will be inhibited as long as hsp70 synthesis occurs [52] . thus, hsp70 in ibv virions might serve a similar function in the virus life cycle. the chaperone hsp90 has been identified as an essential factor in the folding and maturation of picornavirus capsid proteins [53] . the involvement of hsp90 in viral replication has also been reported for many viruses and it has been demonstrated that hsp90 inhibition blocks viral replication [54] . recently, a role for hsp90 in the control of hepatitis c, flock house and influenza virus polymerase function has been shown [55] [56] [57] [58] [59] [60] [61] and it has been proposed that hsp90 is a major host factor that is of central importance for viral replication for a wide spectrum of rna viruses [56] , which implies the crucial roles of hsp90 in ibv replication. the importance of hsp90 for the replication of multiple viruses opens up an interesting possibility for developing new antiviral therapies which have not yielded drug-resistant viruses [62] . some proteins involved in the glycolytic pathway were identified, such as aldehyde dehydrogenase 9 family, member a1 (aldh9a1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), alpha-enolase, which were identified in other viral particles, like hiv-1, mmlv, hcmv, kshv and aiv [8, 12, 19, 21, 24] . some studies have suggested that several glycolytic enzymes interact with microtubules and tubulin [63] [64] [65] and may also contribute to transcription of rna virus genomes. in higher eukaryotes, enolase is found as a dimer of subunits, α, β, or γ. all enolase isoforms from mammalian have been reported that are capable of stimulating transcription of svgenome [66] . gapdh is a well-characterized key enzyme in glycolysis, but recent evidence suggests it also has rna binding properties and binds to the untranslated rna sequences of several different viruses, including human parainfluenza virus type 3(hpiv3), japanese encephalitis virus (jev), hepatitis a virus (hav) and hepatitis b virus (hbv) as well as hepatitis c virus (hcv) [67] [68] [69] [70] [71] . in the case of hpiv3, gapdh has been reported to inhibit actin-dependent in vitro transcription and is also present in purified virions [67, 72] . in vitro data indicates that gapdh serves a negative regulatory role in hpiv3 transcription and in a phosphorylation-dependent on manner [72] . in addition to these host proteins associated with enveloped viruses, the roles of which in the virus life cycles have been studied well, we also identified 21 host proteins in purified infectious bronchitis particles, which have not been described to be present in other virions of quite diverse virus families, such as apolipoprotein a-i (apoa-i), fatty acid-bingding protein 3, ovalbumin, tenp protein, tumor protein translationally controlled-1, transthyretin and so on. apoa-i, a major constituent of highdensity lipoproteins, alters plasma membrane morphol-ogy by participating in the reverse transport of cholesterol binding with atp-binding cassette transporter a1 [73] , and activates the small gtp-binding protein cdc42associated signaling including apoa-i induced cholesterol efflux, protein kinases, and actin polymerization [74] . what important is that apo a-i can inhibit herpes simplex virus (hsv)-induced cell fusion at physiological concentrations. this function may be related to the structure of apoa-i and before long its amphipathic peptide analogue was also found to inhibit cell fusion, both in hiv-1infected t cells and in recombinant vaccinia-virusinfected cd4+ hela cells expressing hiv envelope protein on their surfaces [75] . the results indicate that amphipathic helices may be useful in designing novel antiviral agents that inhibit penetration and spreading of enveloped viruses. ovalbumin is the main protein found in egg white, making up 60-65% of the total protein. the chicken ovalbumin upstream promoter transcription factors (coup-tfs), members of the steroid/thyroid hormone receptor superfamily, binds to a negative regulatory region in the human immunodeficiency virus type 1 long terminal repeat (ltr). ltr contains a negative regulatory element which downregulates the rate of ltr-directed transcription and hiv-1 replication [76] . the interaction between ovalbumin and np from influenza a virus as well as glycoprotein c from the herpes simplex type i virus was reported long time ago [76] . the tenp protein from g. gallus, however, was isolated as a transiently expressed gene in neural precursor cells in retina and brain, and has been proposed to function in the transition to cell differentiation in neurogenesis. after expressed in chicken embryonic fibroblast cells, tenp was immunodetected in membrane fractions, implying that tenp might be a membrane protein as predicted by a computer analysis of its primary sequence [77] . to date, there have been no reports about tenp associated with virus, but it's an enriched and abundant protein identified in purified infectious bronchitis particles which suggests to us that it may be a requisite host protein in ibv life cycles. the present study 1) provides the first proteomic analysis of infectious bronchitis particles, 2) establishes the most comprehensive proteomic index of ibv and 3) shows that most of the virion incorporated host proteins have central roles in virus life cycle. although some proteins may be associated with virus biology, further investigation of the function of these host proteins may facilitate the understanding of the pathogenic mechanisms. the ibv strain h52 was obtained from qianyuanhao biological corporation limited (beijing, china). virus was propagated in 10-day-old specific pathogen free (spf) embryonated chicken eggs (beijing merial vital laboratory animal technology co, ltd, beijing, china) for 48 h at 37°c. the allantoic fluid (af) with enrichment of ibv h52 was clarified by differential centrifugation. af was first centrifugated at 3,000 × g for 30 min and then the supernatant was centrifugated at 12,000 × g for 30 min. clarification and all subsequent centrifugations were performed at 4°c. the virus was sedimented through 5.5 ml of 20% (wt/vol) sucrose in tne buffer (50 mm tris, 100 mm nacl, 1 mm edta, ph 7.4) by centrifugation in a 70ti rotor (beckman coulter, optima™ l-100xp preparative ultracentrifuge) at 75,000 × g for 1.5 h. condensed virions were then diluted with 1.0 ml tne buffer and centrifuged to equilibrium in 11.5 ml non-linear 20%-50% sucrose-tne gradients at 75,000 × g for 2.5 h in a sw41 rotor (beckman coulter, optima™ l-100xp preparative ultracentrifuge). purified virions were diluted with tne buffer and pelleted by sedimentation at 75,000 × g for 1.5 h in a sw41 rotor to remove the sucrose. the purified ibv pellets were stored at -80°c until use. the purified ibv particles were dissolved in about 300 μl lysis buffer (7 m urea, 2 m thiourea, 2% triton x-100, 65 mm dtt, 2% biolyte ph 3-10) and incubated for 60 min at 4°c. then the lysis solution was sonicated for 4 min (pulse durations of 2 s on and 3 s off ) in an ice bath sonicator. the viral protein samples were prepared when the indiscerptible sediments were wiped off by centrifugation at 12,000 × g for 30 min. the supernatant was collected and the concentration of the prepared protein samples was determined by the bio-rad protein assay kit ii according to the manufacturer's instructions. the samples were then aliquoted and stored at -80°c until used for further analysis. purified virus particles treated with bromelain (bb0243, bbi) at 0.2 mg/ml in 50 mm dtt (ph 7.2) in dulbecco's phosphate buffered saline (pbs) at 37°c for 15 min. after incubation, the treated virus was directly centrifuged to equilibrium in 11.5 ml non-linear 20%-50% sucrose-tne gradients at 75,000 × g for 2.5 h in a sw41 rotor (beckman coulter, optima™ l-100xp preparative ultracentrifuge). purified virions were diluted with tne buffer and pelleted by sedimentation at 75,000 × g for 1.5 h in a sw41 rotor to remove the sucrose and then subjected to immunogold labeling and electron microscopy analysis. two-dimentional gel electrophoresis analysis was performed using 18 cm immobile drystrip (ipg strips, ph 3-10 non-linear, ge healthcare). first, 100 μl samples containing 250 μg protein were added into 400 μl sample rehydration buffer (7 m urea, 2 m thiourea, 2% (w/v) chaps, 65 mm dtt, 0.2% bio-lyte ph 3-10) and incubated for 30 min at 37°c prior to their separation by isoelectric focusing (ief) in the first dimension. the ipg strips were rehydrated at 20°c for 12 h by a passive rehydration method. ief was carried out for a total of 45 kvh at 20°c on an ettan ipgphor iii electrophoresis unit (ge healthcare). second, ipg strips were further transferred onto the second dimension of gel electrophoresis. before this step the ipg strips were reduced and alkylated in a equilibration buffer containing 50 mm tris-hcl, ph 8.6, 6 m urea, 2% sds and 30% glycerol supplemented with 1% (w/v) dl-dithiothreitol (dtt) or 2.5% iodoacetamide (iaa) instead of dtt for 15 min. subsequently, the viral protein samples were separated at 140 v on linear 5%-17.5% sodium dodecyl sulfate gradient polyacrylamide gel (sds-page) in tris: glycine buffer (192 mm glycine, 25 mm tris, 0.1% sds, ph 8.3) for about 10 h. third, proteins in the gel were stained by the modified silver staining method compatible with ms [78] and the gels were scanned at a resolution of 600 dpi using imagescanner™ iii (ge healthcare). gel pieces (1.0 mm 3 ) containing the whole protein spots from the 2d gel were cut and washed three times with 50 mm carbonic acid, monoammonium salt (nh 4 hco 3 , amresco). these gel pieces were destained with 15 mm potassium ferricyanide (k 3 fe(cn) 6 , amresco) and 50 mm sodium thiosulfate (nas 2 o 3 , amresco) in 50 mm nh 4 hco 3 and dehydrated in 100% acetonitrile (acn, wako) until gel pieces turn to white. after dring in speedvac concentrator (thermo savant, usa) for about 100 min, gel pieces were incubated with 12.5 ng/μl trypsin (sequenceing grade, promega) to cover dry gel pieces completely at 37°c overnight. the gel pieces were then extracted three times in 50% acn water solution containing 5% trifluoroacetic acid (tfa, wako). the supernatant was pooled and dried thoroughly in speed-vac. protein digestion extracts were resuspended with 5 μl of 0.1% tfa and then the peptide samples were mixed (1:1) with a matrix consisting of a saturated solution of αcyano-4-hydroxycinnamic acid (α-cca, sigma) in 50% acn containing 0.1% tfa. 0.8 μl aliquot was spotted onto stainless steel target plates. peptide mass spectra were obtained on an applied biosystem/mds sciex 4800 maldi tof/tof plus mass spectrometer. data were acquired in positive ms reflector using a calmix5 standard to calibrate the instrument (abi4800 calibration mixture). mass spectra were obtained from each sample spot by accumulation of 900 laser shots in an 800-3500 mass range. for ms/ms spectra, the 5-10 most abundant precursor ions per sample were selected for subsequent fragmentation and 1200 laser shots were accumulated per precursor ion. both the ms and ms/ms data were interpreted and processed by gps explorer software (v3.6, applied biosystems), then those obtained ms and ms/ms spectra per spot were combined and submitted to mascot search engine (v2.1, matrix science, london, u.k.) by gps explorer software and searched with the following parameters: trypsin as the digestion enzyme, one missed cleavage site, partial modification of cysteine carboamidomethylated and methionine oxidized, none fixed modifications, ms tolerance of 60 ppm, ms/ms tolerance of 0.25 da. mascot protein score in ipi_chicken (v3.49) database (based on combined ms and ms/ms spectra) of greater than 57 (p ≤ 0.05) or in ncbinr database of greater than 67 (p ≤ 0.05) was accepted. mouse monoclonal antibodies against actin (mab1501) and hsp90 (05-594) were purchased from millipore. rabbit polyclonal antibodies against annexin a2 (ab40943) and tubulin alpha-1 (ab4074), and chicken polyclonal antibody against ibv (massachusetts) (ab31671) were purchased from abcam. mouse monoclonal antibody against nucleoprotein of ibv (3bn1) was purchased from hytest ltd. for control, the af from 10-day-old spf embryonated chicken egg performed with the same protocol as the purification of ibv particles and the protein extracted from the normal 10-day-old spf embryonated chicken eggs included for western blot analysis. samples were separated at 120 v on linear 5%-17.5% sds-page with 5% stacking gels in tris: glycine buffer for about 3 h. for purified virus, 10 μg of total proteins were used per lane. for the control, a total of 15 μg proteins were loaded. after separated by sds-page, the proteins were transferred to a polyvinylidene fluoride membrane (pvdf, p/n 66485, biotrace, pall corporation). the membrane was blocked in freshly prepared 5% bovine serum albumin (bsa) with 0.05% tween-20 for 2 h at room temperature with constant agitation. the pvdf membrane was washed three times with tris buffered saline plus 0.2% tween 20 (tbst) and then incubated with properly diluted primary antibodies for 2 h at room temperature or overnight with agitation at 4°c. anti-rabbit or anti-mouse immunoglobulin g antibody conjugated to horseradish peroxidase (hrp) (00001-14, proteintech group, inc) was used as the secondary antibody and the pvdf membrane was incubated in it for 1 h at room temperature. the chemiluminescence system (ar1022, boster bio-technology co. ltd) was used for detection of antibody-antigen complexes. rabbit polyclonal antibody against chicken igg (15 nm gold) (ab41500), goat polyclonal against rabbit igg (5 nm gold) (ab27235) and goat polyclonal against mouse igg (10 nm gold) (ab27241) were purchased from abcam. purified ibv particles were suspended in pbs (ph 7.4) and then were collected onto 230-mesh formwar-coated nickel grids and adsorbed on the grids for 5 min. the viruses were fixed in 2% paraformaldehyde for 5 min at rt and treated with triton x-100 (0.2%) in pbs (ph 7.4) for 5 min and then blocked with 5% bsa in pbs-tween 20 (ph 7.4) for 30 min at rt. all grids were then blocked with blocking buffer (5% bsa, 5% normal serum, 0.1% cold water skin gelatin, 10 mm phosphate buffer, 150 mm nacl, ph 7.4) for 30 min. after washing with pbs, immobilized virions were incubated for 1.5 h with 50 μg/ml primary antibody (in 1% bsa), and washed three times for 5 min in pbs/1% bsa. anti-rabbit or anti-mouse immunoglobulin g coupled to 10 nm colloidal gold particles was used as the secondary antibody and virions were incubated in it for 40 min at room temperature. the grids were then washed extensively with pbs, washed twice more with distilled water to remove excess salt and negatively stained with 2% sodium phosphotungstate for 1 min. negatively stained virions were examined on a scan and transmission electron microscope. abbreviations 2d: two-dimensional; 2-de: two-dimensional electrophoresis; sds-page: sodium dodecylsulfate polyacrylamide gel electrophoresis; ms: mass spectrometry; maldi-tof: matrix-assisted laser desorption/ionization time of flight mass spectrometry; spf: specific pathogen free; af: allantoic fluid; bsa: bovine serum albumin; dtt: dithiothreitol; iaa: iodoacetamide; acn: acetonitrile; tfa: trifluoroacetic acid; α-cca: α-cyano-4-hydroxycinnamic acid; tne: tris-buffered saline including 50 mm tris; 100 mm nacl; 1 mm edta: ph 7.4; pbs: phosphatebuffered saline; tbs: tris-buffered saline; tbst: tris buffered saline plus 0.2% tween 20; hrp: horseradish peroxidase; pi: isoelectric point; mw: molecular weight. coronavirus avian infectious bronchitis virus envelope glycoprotein interactions in coronavirus assembly characterization of the coronavirus m protein and nucleocapsid interaction in infected cells coronavirus spike protein inhibits host cell translation by interaction with eif3f interaction of the coronavirus infectious bronchitis virus membrane protein with betaactin and its implication in virion assembly and budding induction of apoptosis in murine coronavirusinfected 17cl-1 cells induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of e protein as an apoptosis inducer host and viral proteins in the virion of kaposi's sarcoma-associated herpesvirus virion proteins of kaposi's sarcomaassociated herpesvirus a mass spectrometry-based proteomic approach to study marek's disease virus gene expression proteins of purified epstein-barr virus identification of proteins in human cytomegalovirus (hcmv) particles: the hcmv proteome identification of proteins associated with murine cytomegalovirus virions vaccinia virus proteome: identification of proteins in vaccinia virus intracellular mature virion particles krijnse locker j: identification of the major membrane and core proteins of vaccinia virus by two-dimensional electrophoresis protein composition of the vaccinia virus mature virion specific incorporation of heat shock protein 70 family members into primate lentiviral virions cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages proteomic analysis of human immunodeficiency virus using liquid chromatography/tandem mass spectrometry effectively distinguishes specific incorporated host proteins identification of host proteins associated with retroviral vector particles by proteomic analysis of highly purified vector preparations immunochemical identification of viral and nonviral proteins of the respiratory syncytial virus virion identification of cellular interaction partners of the influenza virus ribonucleoprotein complex and polymerase complex using proteomic-based approaches cellular proteins in influenza virus particles polypeptides of the surface projections and the ribonucleoprotein of avian infectious bronchitis virus proteomic analysis of sars associated coronavirus using two-dimensional liquid chromatography mass spectrometry and onedimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by mass spectroemtric analysis plunder and stowaways: incorporation of cellular proteins by enveloped viruses viral proteomics interaction of epithelial ion channels with the actin-based cytoskeleton the tcomplex polypeptide 1 complex is a chaperonin for tubulin and actin in vivo systematic identification of protein complexes in saccharomyces cerevisiae by mass spectrometry epstein-barr virus-encoded nuclear protein ebna-3 interacts with the epsilon-subunit of the tcomplex protein 1 chaperonin complex a eukaryotic cytosolic chaperonin is associated with a high molecular weight intermediate in the assembly of hepatitis b virus capsid, a multimeric particle type d retrovirus gag polyprotein interacts with the cytosolic chaperonin tric annexin ii enhances cytomegalovirus binding and fusion to phospholipid membranes secretory leukocyte protease inhibitor binds to annexin ii, a cofactor for macrophage hiv-1 infection annexin ii incorporated into influenza virus particles supports virus replication by converting plasminogen into plasmin annexin 2-mediated enhancement of cytomegalovirus infection opposes inhibition by annexin 1 or annexin 5 annexin 2: a novel human immunodeficiency virus type 1 gag binding protein involved in replication in monocyte-derived macrophages annexins: linking ca2+ signalling to membrane dynamics intracellular and extracellular roles of s100 proteins s100-annexin complexes: some insights from structural studies recruitment of hsp70 chaperones: a crucial part of viral survival strategies synthesis and quality control of viral membrane proteins association of hsp70 with the adenovirus type 5 fiber protein in infected hep-2 cells association of heat shock protein 70 with enterovirus capsid precursor p1 in infected human cells vaccinia virus infection induces a stress response that leads to association of hsp70 with viral proteins increased expression of hsp70 and co-localization with nuclear protein in cells infected with the hantaan virus uncoating atpase is a member of the 70 kilodalton family of stress proteins hsp90 is required for the activity of a hepatitis b virus reverse transcriptase two-dimensional blue native/sds-page analysis reveals heat shock protein chaperone machinery involved in hepatitis b virus production in hepg2.2.15 cells selective inhibition of virus protein synthesis by prostaglandin a1: a translational block associated with hsp70 synthesis evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance molecular chaperone hsp90 is important for vaccinia virus growth in cells hsp90 inhibitors suppress hcv replication in replicon cells and humanized liver mice hepatitis c virus rna replication is regulated by fkbp8 and hsp90 the cellular chaperone heat shock protein 90 facilitates flock house virus rna replication in drosophila cells identification of hsp90 as a stimulatory host factor involved in influenza virus rna synthesis involvement of hsp90 in assembly and nuclear import of influenza virus rna polymerase subunits antiviral activity and rna polymerase degradation following hsp90 inhibition in a range of negative strand viruses herpes simplex virus type 1 dna polymerase requires the mammalian chaperone hsp90 for proper localization to the nucleus development and application of hsp90 inhibitors enhanced association of mutant triosephosphate isomerase to red cell membranes and to brain microtubules a glycolytic enzyme binding domain on tubulin glycolytic enzyme-tubulin interactions: role of tubulin carboxy terminals enolase, a cellular glycolytic enzyme, is required for efficient transcription of sendai virus genome specific interaction in vitro and in vivo of glyceraldehyde-3-phosphate dehydrogenase and la protein with cis-acting rnas of human parainfluenza virus type 3 human hepatic glyceraldehyde-3-phosphate dehydrogenase binds to the poly(u) tract of the 3' noncoding region of hepatitis c virus genomic rna identification of glyceraldehyde-3-phosphate dehydrogenase as a cellular protein that binds to the hepatitis b virus posttranscriptional regulatory element glyceraldehyde-3-phosphate dehydrogenase (gapdh) interaction with 3' ends of japanese encephalitis virus rna and colocalization with the viral ns5 protein functional significance of the interaction of hepatitis a virus rna with glyceraldehyde 3-phosphate dehydrogenase (gapdh): opposing effects of gapdh and polypyrimidine tract binding protein on internal ribosome entry site function specific phosphorylated forms of glyceraldehyde 3-phosphate dehydrogenase associate with human parainfluenza virus type 3 and inhibit viral transcription in vitro specific binding of apoa-i, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing abc1 apolipoprotein a-i activates cdc42 signaling through the abca1 transporter apolipoprotein a-i and its amphipathic helix peptide analogues inhibit human immunodeficiency virus-induced syncytium formation immunoprecipitation, with an antiserum to ovalbumin, of protein np from influenza a virus and of glycoprotein c from the herpes simplex type i virus identification and characterization of tenp, a gene transiently expressed before overt cell differentiation during neurogenesis a modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry proteomic analysis of purified coronavirus infectious bronchitis virus particles proteome science the authors declare that they have no competing interests. qk performed the main proteomic experiments and data analysis and drafted the manuscript. cx created the detailed experimental design. xr and cz contributed to the initial phase of the proteomic experiments. ll and ds assisted in the propagation and purification of ibv. yb and yc helped conceive the research. all authors read and approved the final manuscript. key: cord-260345-ugd8kkor authors: giles, ian g. title: a compendium of reviews in biochemistry and molecular biology published in the first half of 1992 date: 1992-12-31 journal: international journal of biochemistry doi: 10.1016/0020-711x(92)90283-7 sha: doc_id: 260345 cord_uid: ugd8kkor abstract 1. 1. a compendium of reviews and mini-reviews in biochemistry and molecular biology published in the first half of 1992 is presented. in all 499 titles are listed from 95 different publications. 2. 2. this compendium presents the references by journal name. keywords have been included with each reference to increase the value of the collection. keyword and author cross-reference indexes are not included but are available in the electronic database from which this version was constructed. should anyone wish to have this information in electronic form it can be distributed on ms-dos formatted flopppy disks in either reference manager or medline format. the author should be contacted for details of the number of preformatted floppy disks required. krasikov n., thompson k. and sekhon g.s. (1992) brief clinical report-monosomy 18q12.1+21.1-a recognizable aneuploidy syndrom~report of a patient and review of the literature. am. j. med. geti. 43. 531-534. verloes a., mulliez n.. gonzales m., laloux f.. hemutnnsle t., pierard g.e. and koulischer l. (1992) restrictive dermopathy, a lethal form of arthrogryposis multiplex with skin and bone dysplasiac3 new cases and review of the literature. am. j. med. genet. 43, 539547. aplasia cutis ccugenita; pyloric atresia, newborn; sibs. leonard c., huret j.l., imbert mc., lebouc y., selva j. and boulley a.m. (1992) trisomy-16p in a liveborn offspring due to maternal translocation t(16-21)(ql l-p1 1) and review of the literature. am. j. med. gene; . 43, [621] [622] [623] [624] [625] spontaneous abortions; handing teclm' tques; duplication 16~; infant; segregation. xie l.q.. markides k.e. and lee m.l. (1992) biomedical applications of analytical supercritical fluid separation techniques. anal. biuchem. u)o, [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] chtumatography-mass spectrometry; amino-acid derivatives; gas-chromatograph stationary phase; chargeexchange; silica-gel; extraction; glycosphingolipids; resolution; interface. hozier j.c. and davis l.m. (1992) review-cytogenetic approaches to genome mapping. anal. biochem. 208, 205217 . chaiken i., rose s. and karlsson r. (1992) quantitative analysis of protein interaction with ligands. (2) analysis of macromolecular interactions using immobilized ligands. anal. b&km. 201, [197] [198] [199] [200] [201] [202] [203] [204] [205] [206] [207] [208] [209] [210] . neurophysin self-association; affinity-chmmatography; subunit-exchange chromatography; biosynthetic precursor; equilibtium-carstants; peptide recognition; sense pcptides; binding; purification; elution. ichikawa y.. look g.c. and wong c.h. (1992) review-enzyme-catalyzed oligosaccharide synthesis. anal. b&hem. 202,215-238. gal-l3-1+3(4)glcnac a-2+3 sialyltransferase; acetylneuraminic acid synthetase; immobilized l3-galactosidase; gdp-l-fucose; sialic acids; esckrichia coli; rat-liver, glycoprotein oligosaccharides; carbohydrate antigens; determines expression. gabriel 0. and gersten d.m. (1992) staining for enzymatic activity after gel electrophoresis-review. anal. bbckm. 203, sodium dodecyl-sulfate.; ted blood-cells; phosphoenolpyruvate carboxylase activity; nucleotide-linked dehydrogenases; alkaline-phosphatase isoenzymes; pathogenesis-related proteins; cr-l-fucosidase; polyactylamide-gel; produce phosphate; general-method. wood p.j. (1992) the measurement of parathyroid hormone. ann. cfin. b&km. 29, [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] cyclic amp; immtmoradiomettic assay; primary hypatparathytoidism; humoral hypercalcemia; calcium huneostasis; intact parathytin; clinical utility; circadian-rhythm; chromogranin a; lung-cancer. newman d.j.. henneberry h. and price c.p. (1992) particle enhanced light scattering immunoassay. ann. clin. b&hem. 29, . c-reactive protein; human chorionic-gonadotropin; latex agglutination-test; cell-labeled antibodies; shell corn patticles; counting immunoassay; turbidimetric immunoassay; spectroscopic immunoassay; passive hemagglutinatiom luteinizing-hormone. thompson d.. milfordward a. and whither j.t. (1992) the value of acute phase protein measurements in clinical practice. ann. clin. c-reactive protein; erythrocyte sedimentation-rate; inflammatory bowel-disease; tumornecrosis factor, amyloid a protein; tlteumatoid atthritis; plasma-ptoteins; polymyalgia rheumatica; acute-leukemia; tissue-injury; acute phase. soldi s.j. (1992) drug receptor assays-quo vudis. ann. . allen j.f. (1992) protein phosphorylation in regulation of photosynthesis. biochln. bi@ys. ac& la275 335. light hamsting complex; dtlomphyll a; b protein; cyanobncterium syn&ococc~~ 6301; excitatiarcnergy distribution; absorptial cross-section; ii reaction center. thylakoid membrane pelypaptides; state-l state-2 tmnsiticns; amino-acid-sequence; randomixed chlomplast iamellae. anthony c. (1992) the c-type cytochromes of methylotrophic bacteria. b&him. biqhys. acta 1099, l-15. methylobacteriwn extorquetu am; electron-transport chain; blue copper proteins; oxidation mutant classes; ammo-acid sequence; sp strain aml; paracoccus dcni~rificons; melhylophilvs mdylotrophur; obligate metbylotmpb; m&and dehydrogenase. hoch f.l. (1992) cardi~lipins and biomembrane function. biochim. biophys. acta 1113, 71-133. rat-liver-mitochondtia; fatty-acid composition; brown-adipose-tissue cytochmme-c-oxidaset munbmne lipid-ccmpositiau adenine-nucleotide translocase; age-related-changes; skeletal-muscle mitochond~, chronic ethanol-consum@n; lateral proton conduction. bandekar j. (1992) amide modes and protein conformation. biochim. biophys. actu 1120. 123-143. transforfn-infrared-spsccpy; laser raman-spectroscopy; secondary-structure-analysis; liver ahohd-dehydmgenase; hydrogadeuteriutn exchange; a transmembrane channel; valyl-glycyl-glycine; iii spectral region; vibratiaral analysis, gramicidin-a. b&him. biophys. acta 1123, 231-238. acetyl-coa carboxylase; coenzyme-a reductase; hormone-sensitive l&se; dependent multipmtein kinase; rat-lives; 3-hydmxy-3-methylglutaryl coenxyme; enxymic activity; phosphorylationdephosphorylatiar; hydmxymethylgfutaryl coenxyme; reversible phosphotylation. w&t k. (1992) origins and fates of fatty acyl-coa esters. biochim. biophys. acfu 1124, 101-111. coenzyme a syntbetase; performance liquid-chromatography; rat-liver micmsomcs; acyltransfemse-cataly~ cleavage; dependent transacylation system; rabbit alveolar macropbages; pemxisomal &oxidation; amcbidonic-acid; brain micmsomes; sbott-chain. low-density~l&protein; high-performance liquid; chrcmatogmphy mass spectmmetry; chicken vitellogam gene; thin-layer chmmatography; apolipoprotein-vldl-ii; fatty-acid composition; laying turkey hens; egg-yolk; plasma-lipoproteins. erlansonalbertsson c. (1992) pancreatic colipase-structural and physiological aspects. biuchim. biophys. acta 1125. 1-7. gastric-inhibitory polypeptide, messenger-rna levels; diabetic rats; pro-colipase; co-lipase; tymsine residues; porcine colipase; sequence; taurodeoxycholrte; triglyceride. coleman r. and rahman k. (1992) wehle e. (1992) reiter r.j. (1992) the ageing pineal gland and its physiological conxequences. bioessays 14, 169-175. malatonin receptors; admoet@c-receptors; circadian variations; n-acetylscrotonin, plasma melatcoin, serum melatouin; hamsters; gerbil; brain; reduction; downward j. (1992) regulatory mechanisms for ras proteins. bioessap 14, 177-184. gtaase-activating protein; neurofibranatosis type-l gene; nucleotide exchange-reaction; gap-associated proteins; growth-factor; tymsine phosphorylation; ras-p21 gtpase; stimulation; p21; recepors. rusciano d. and burger m.m. (1992) adamo m., roberta ct. and lemith d. (1992) how distinct are the insulii and ~sul~-l~e growth factor? -signalling systems. biofwtors 3.151-157. human-skin fibroblests; igf binding-protein; messenger ribonucleic-acid; cooh-teminal truncation; monoclonal-antibody; endothelial-cells; factor receptoc amniotic-fluid; dna-synthesis; rat-heart. hehnreich e.j.m. (1992) how pyridoxal s-phosphate could function in glycogen phosphorylaae catalysis. biofbctors 3, 159-172. aron d.c. (1992) insulin-like growth factor-i and erythropoiesis. biofators 3, 211-216. factor-binding-protein; erythroid colony formation; cultured human-tibroblasts; factor messenger-rna, n-terminal sequence; fetal bovine serum; igf-i; somatomedin c, clinical-applicstians; stimulating factors. silver b.j. (1992) platelet-derived growth factor in human malignancy. biofmtors 3, 217-227. terminal coding region; c-sis; b-chain. bmis w.d. and durst r.w. (1992) bajpai p. and bajpai p.k. (1992) arachidonic acid production by microorganisms. biofechnol. appl. biockm. 15. l-10. bellomo m.j., parlier v., muhlematter d., grob j.p. and beris p. (1992) three new cases of chromosome-3 rearrangement in bandq21 and band-q26 with abnormal thrombopoiesis bring further evidence to the existence of a 3q21q26-syndrome. cancer genet. cytogenet. 59. 138-160. acute nonlymphocytic leukemia; chronic myelogcnous leukemia; chronic myeloid-leukemia; acute megakaryoblastic leukemia; chronic myelocytic-leukemia; acute myeloblastic-leukemia; british cooperative group; myelodysplastic syndromes; blast crisis; tmnslocation t(l -3). pedersen b. (1992) survival of patients with t(l-7)(pl l-p1 l&report of 2 cases and review of the literature. cancer genet. cytogenet. 60, 53-59. acute nonlymphocytic leukemir; trsnslccation i-7; myelodysplastic syndromes; chromosome analysis; mycloid disorders; secondary. nossal g.j.v. (1992) the molecular and cellular basis of affinity maturation in the antibody response. cell 68.1-2. mutation. thomas g. (1992) map kinase by any other name smells just as sweet. cell 68, 3-6. protein-kinsse; insulin; identification; muscle. teach r.e. (1992) type-2 astrocyte developme ciliaty netttotmphic factoc fibmblast growth-factor. retinoic acid xceptor; chick limb bud; tymsine kinaae m eatiy xatqus embryos; pmto-oncogene int-1; activin-a; w-locus. greenwald i. and rubin gm (1992) mellman i. and simons k. (1992) the golgi complex--in vitro verirus. cell 68. 829-840. asparsgine-linked oligosaccharides; cell-free system; rough endoplssmic-reticulum; vesicular stomatitis-tims; plasma-manbrane proteins; bmfeldin a; cis-golgi; successive compartments; intracellular-transport; n-acetylglucosamine. chao m.v. (1992) hall a. (1992) signal transduction through small gtpases-a tale of 2 gaps. cell 69, 389-391. proteins. wetr d.z., peles e.. cupples r., suggs s.v., bacus s.s., luo y., trail g., hu s.. silbiger s.m.. benlevy r.. koski r.a., lu h.s. and yarden y. (1992) neu differentiation factor-a transmembrane glycoprotein containing an egf domain and an immunoglobulin homology unit. cell 69, 559-572. epidennal growth-factor. human mterleukin-2 receptor. human-breast-carcinoma; human mammary-tumors; factor-a; nudeotide sequence; molecular cloning; prom-oncogene; tyrosine phosphorylation; signal transduction. travers a.a. (1992) the reprogramming of transcriptional competence. cell 69. 573-575. position effect variegation; drarophila; protein. helenius a. (1992) unpacking the incoming influenza virus. cell 69, 577-578. amantscline: protein, virions. jan l.y., jan y.n. and hughes h. (1992) tracing the roots of ion channels. cell 69. 715-718. protein. gauss p. and walther c. (1992) pax in development. cell 69, 719-722. genes; conservation; drosophila; nowchord, proteins; domain; box. varshavsky a. (1992) the n-end rule. cell 69, 725-735. shon-hved protein; dependent pmteolytic system; ubiquitin-conjugating enzyme; repair gene ra&, escherichia coli; transfer rna; sacclurroqces cercvisiue; endoplasmic-reticulum; cell-cycle; amino-acid. cell signailing iwashita s. and kobayashi m. (1992) signal transduction system for growth factor receptors asso&ed with tyrosine kinase activity-epidennal growth factor receptor signalling and its regulation. cell signal. 4. 123-132. vogt w. and nagel d. (1992) eriksson l.c. and andersson g.n. (1992) nucleotide-sequence; femo-cofacscr. iritani n. (1992) nutritional and hormonal regulation of lipogenic-enzyme gene expression in rat liver. eiu. j. fatty-acid aynthase; acetyl-coa cuboxylase; post-transcriptiaral regulation; chickembryo hepatocytes; messenger rna levels; malic enzyme; thymid hormone; posttmnscriptional regulation; molecular-clcming. gavel y. and vonheijne g. (1992) the distribution of charged amino acids in mitochondrial inner-membrane proteins suggests different modes of membrane integration for nuclearly and mitochondriaily encoded proteins. eur. j. biochem. 205, 1207-1215. cytochmme-c-oxidrse; adp-awcartier. beef-heart mitochondria; nicotinamide nucleotide tmnshydrogenase; brown fat mitochondtia; diffemnt genes cdna, imn-sulfur proteirx saccharanyces cerevisiae; unce@ieg potdn; escherichia coli. frrmcklyn c., musierforsyth k. and schimmel p. (1992) youn y.k., lalonde c. and demling r. (1992) haas a. and goebel w. (1992) defromentel c.c. and soussi t. (1992) mackay t.f.c., lyman r.f. and jackson ms. (1992) hpitan n.l.v. (1992) sobell j.l., heston l.l. and sommer s.s. (1992) delineation of genetic predisposition to multifactorial disease-a general approach on the threshold of feasibility. genomics 12, l-6. polymense chain-reaction; fragment length polymorphisms; dependent diabetes-mellitus; sickle-cell anemia; factor-m gene; enzymatic amplificatiat; genomic dna; mutations; sequence; diagnosis; predisposition; genetics. troy f.a. (1992) polysialylation-from bacteria to brains. glycobiology. 2, 5-23. cell-adhesion molecule; escherichia coli kl; rainbow-trout eggs; deaminated neuraminic acid; endo-n-acetylne.uramiidase; group b meningococci; long oligosaccharide segment: netno-blastoma cells; polysialic acid; sialic-acid. varki a. (1992) diversity in the sialic acids. glycobiology. 2, 25-40 . n-acetylneuramhtic acid, infhtenxa c virus; de-ortho-acetylation; performance liquid-chromatography; hungonuhu d&her antigen; liver golgi vesicles; melanoma-associated ganglioside; bombardment mass-spectrometry; human gastmintestinal-trac deaminated neuraminic acid. stanley p. (1992) glycosylation engineering. glycobiology. 2, 99-107. hamster ovary cells; mcombinant human erythropoietin; tissue plasminogen-activator. n-linked oligosaccharide, protein glycosylaticm; biological-activity; lysosomal-enzymes; insect cells; animal-cells; sugar chains. harvey d.j. (1992) the role of mass spectrometry in glycobiology. glycoconjugate j. 9, 1-12. fast-atom-bombardment; 6-o-methylghtcose pelysaccharide; collisional activation; gas-chromatography; laser desorptien; molecular mass; oligosacdtarides; ionization; proteins. aquit d.a. and b~~~ii~ii c.f. (1992) heat-shock proteins and immunopathology-an overview. heat-shock t-cell receptor; messenger-rna degradation; gamma-delta; stress proteins; antigen-receptor, lymphocytes-t; ~ycobactcrium fuberc&arir. mammalian-cells; cyto-toxicity; dna-binding. fenick d.a. and gemmellhori l. (1992) potential developmental role for self-reactive t cells bearing gamma-delta t cell receptors specific for heat-shock proteins. dendritic epidermal-cells; toxic lymphocytes-$ antigen receptor; intraepithclial lymphocytes; rheumatoid arthritis; athymic mice; mycobacrcritert lubercularis; immune-response; thymic ontogeny; a-chain; cell receptor. christmas s.e. (1992) cytokine production by lymphocytes-t bearing the gamma-delta t cell antigen receptor. antigen; antigen receptor. mceptor. tumor-necrosis-factor; bone-marrow transplantation; a-p+ lymphocytes; interferon-gamma; monoclonal-antibodies; peripheral-blood; cytotoxic activity; fetal thymocytes; different forms; human intestine. wintield j., jarjour w. and minota s. (1992) stress protein autoantibodies and the expression of stress proteins on the surface of human gamma-delta cells and other cells of the immune system. heat-shock proteins and gamma-delta t cells 53, 47-60. stress; autoantibodies; immune-system; heat-shock protein; rous-sarcoma virus; juvenile rheumatoid-arthritis; t-cells; synovial-fluid; lymphocytes-t, mycobucterium tuberculosis; borrelia bwgdorferi; lupus erythematosus; transforming protein; stmss protein. modlin r.l.. lewis j., uyemura k. and tigelaar r.e. (1992) lymphocytes-t bearing gamma-delta antigen receptors in skin. dendritic epidermaltells; heat-shock protein; mycobacterium-tuberculosis; intraepithelial lymphocytes; limited diversity; murine epidermis; concanavalin a; thy-l antigen; nude-mice; expression. hohlfeld r. and engel a.g. (1992) the role of gamma-delta lymphocytes-t in inflammatory muscle disease. monochmal-antibody analysis; natural-killer cells; mononuclear-cells; cyto-toxicity; receptor; myopathies; recognition; expmssiost; ptoteins; canplex. aquino d.a. and selmaj k. (1992) heat-shock proteins and gamma-delta t cell responses in the central nervous system. heat-shock proteins and gamma-delta t cells 53, 86-101. experimental autoimmune encephalomyelitis; experimental allergic encephalomyelitis; fibrillary acidic protein; myelin basic-protein multiple sclerosis: stress prcteim spinal-cord, insitu hybridixation; alexander's disease; praxosin treatment. mario t., nagasawa m. and yata j. (1992) gamma-delta t cells in patients with primary immunodeficiency syndrome-their function and a possible role in the pathogenesis. heat-shock proteins und gamma-delta t cells 53, 102-120. wiskott-aldrich syndrome; heat-shock proteins; receptor-delta; ataxia telangiectasia; transgenic mice; lymphocytes t; bearing cells; recognition; expression; incmase. reardon cl., bom w. and obrien r.l. (1992) murine gammadelta lymphocyte-t recognition of hsp60 a possible source for bacterial immunity or autoimmunity. he&& j.e. and whitelaw p.f. (1992) the role of cellular oncogenes in myogenesis and muscle cell hyperuqhy. int. j. bkxhem. 24, 193203. muscle; all; rous sarcoma vins; fibroblast growth-factor; c-fos expression; embtyonal caminoma-cel~, chicken skeletal-muscle; myc messatger-rna; proto-oncogene; geneexpnssion; dna-binding; src gmc. colacicchi s.. ferrari m. and sotgiu a. (1992) in vivo electron paramagnetic resonance spectroscopy imaging -first experiences, problems, and perspectives. bat. j. b&hem. 24.205-214. &oxide spin labels; loop-gap resonator. free-radicals; soluble nitroxides; metabolism; phannacokinetics; oxygen; cells; specttusn~, sensitivity. seyer r.. richoux j.p. and aumelas a. (1992) probing angiotensin receptors. iru. j. biochem. 24, 369-377. message-address concept; if onnplementaty rna, paraventricular nucleus; biological-activities; subfomical organ; binding-sites; amino-acid; rat-brain; antagonists; analogs. tuck m.t. (1992) the formation of internal 6-methylademne residues in eucaryotic messenger rna. fnt. j. biochem. 24, 379-386. rekharsky m.v., nemykina e.v. and erokhin a.s. (1992) thermochemistry of n-c bonds hydrolysis in amides, peptides, n-acetyl amino acids and high-energy n-c bonds hydrolysis in n-acetyl imidazole and urea. lnl. konopinska d., rosinski g. and sobotka w. (1992) insect peptide hormones, an overview of the present literature. amino-acid-sequence; bombardment mass-spectranetry; pigment-concentrating hormone; locust adipokinetic hormone; periphneta americana l; akh-rpch family; corpora cardiaca; lencophaea mademe; cotpus caniiaann; matuiuca swrta. dinarello c.a. (1992) the biology of interleukin-1. interleukinomolecular biology and immunology 51. l-32. tumor necrosis factor, colony-stimulating factor; smooth-muscle cells; human-immunodeficiency-vims; vascular endothelial-cells; blood mononuclear-cells; recombinant human interleukin-1; human monocyte interleukin-1; hepatic protein-synthesis; autocrine growth-factor. dower sk.. sims j.e., cerretti d.p. and bird t.a. (1992) the interleukin-1 system--receptors, ligands and signals. interleukin.+molecular biology and hmunology 51, 33-64. tumor necrosis factor, pmtein kinase c, growth-factor-receptor. nf-kappa-b; factor increase phosphorylation; prostaglandin e production; thaunatoid synovial-cells; high-affinity receptors; gtp-binding pmt+ human t-cells. ihle j.n. (1992) interleukin-3 and hematopoiesis. interleukim-44olecular biology ad i mmunology 51, 65-106. colony-stimulating factor. human granulocyte-macmphage; protein kinase-c; recombinant human interleukin-3; cell growth-factor, murine bone-marrow; express functional receptors; acute lymphocytic-leukemia; gtpase-activating pm&n; factor-independent growth. ascorbic-acid deficiency; ischemic-heart-disease; low-density-lipoprotein; eastern finnish men; diabetes melli~rcs. guinea-pigs; blood-pressure; oral-contraceptives; experimental atherosclerosis; plasma-cholesteml. mannella ca., forte m. and colombini m. (1992) toward the molecular structure of the mitochondrial channel, vdac. j. bioenerg. biometnbmne 24, 7-19. outer-membrane channel; neurarpora crassa mitochondria; hexokinase-binding protein; voltage-dependent channel, rat-liver mitochondrip; synthetic polyanion; electron-microscopy; pore protein; sequence; arrays; molecular-structure. depittt~ v. and pahnieri f. (1992) benz r. and brdiczka d. (1992) the cation-selective substate of the mitochondrial outer membrane pore-ainglechannel conductance and influence on intermembrane and peripheral kinases. j. bioenerg. biomembrane x33-39. rat-liver mitochondria; hexokinase-binding protein, contact sites; synthetic polyanicn; creatine-kinase; inhibition; transport; stae; brain. arora k.k., parry d.m. and pedersen p.l. (1992) khorana h.g. 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red-cells; gene; expmssion; cdna 144-149. skeletal-muscle; neural controh mcuse embryo molecular genetics of sex detetmination in c. efegunr. trench genet. 8, 164-168. pest-amlnyonic development; cycle contro1 proteins; dosage canpensation hemtaphrodim, mutaticaas; trri-1 trends gatct. 8.169-174. position+ffect variegatimx dna methyhuien; gene-expression; cpg island; mplmatia~ 174-180. mjugation tube formation; succharomyee~ cerevisioe; tremslia mesenterica; dna; gene the retinoblastoma protein and cell cycle regulation patterning of the drosophila nervous system-the achaete-scute gene complex. tr& gener. 8. 202-208. aensay m development mating-tupe; yeast; intexunpsicm; fusion; fission yeast; schizosaccharomyces pom6e; cell linuge; dna arank ho gene; cassetks; asymmetly ustilogo viohxu; nst !itlt@ts; t'&unce; pcpi&ition cti~ dynamics; ctisciw; @ttc; reproduction integrins and metastases-an overview. tumor bid 12.309-320. murine melanoma-cells; mconstituted basement-membrane; fibronectin-recepor complex tumor-cells; surface galactosyltransferase; adhesion molecules; breast-cancer. carbohydrate cha& leukucy~ integrins rat brain haxddnase; amino-acid-sequence; intraceuular-localization; terminal sequence; tumor-cells; membrane; glucose; particulate; form; phosphorylation.mcenery m.w. (1992) the mitochondrial benzodiazepine receptor-evidence for association with the voltage-dependent anion channel (vdac). j. bioenerg. biomembrane 24, 63-69.membrane contad sites; rat-liver mitochondria; central nervous-system; binding-sites; heafl-mitochcndria; mdogcnous ligands; calcium-channel; creatine-k&se; cyclosporin-a; localization.thinnes f.p. (1992) evidence for extra-mitochondrial localization of the vdac/porin channel in eucaryotic cells. j. bioenerg. biomembrane 24, 71-75. dependent anion channel; outer-membrane; chloride channels; escherichia coli; cystic-fibrosis; conductance; protein; identification; hexokinase; state.beavis a.d. (1992) properties of the inner membrane anion channel in intact mitochondria. rat-liver mitochondria; heart-mitochcndria; plant-mitochondria; k+ flux; dibutylchlonnnethyltin chloride; triphenyltin compounds; conducting channel; transport pathway; light-scattering;binding-sites. moran 0. and sorgato m.c. 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(1992) ftir difference spectroscopy ol bacteriorhodopsin-toward a molecular model. resonance raman-spectroscopy; carboxyl protonation changes; hydrogen-dcutcrium exchange; oriented purple membrane; retinal schiff-base; to-blue transition; vibrational spectroscopy; low-temperature; neutron-diffraction; differcncc spea*scopy.lanyi j.k. (1992) proton transfer and energy coupling in the bacteriorhodopsin photocycle. j. bioenerg. biomembrane 24, 169-179. resonance raman-spectra; solid-state "c. purple membrane; halobaclerium halobium: chromophore structure; photochanical cycle; schiff-base; retinal cimmophom: nbsor@ion-spectra; aspanic acid-96.oesterhelt d., tittor j. and bamberg e. (1992) segrest j.p.. jones m.k.. deloof h.. brouiiiette c.g., venkatachalapathi y.v. and anamharamaiah g.m. (1992) the amphipathic helix in the exchangeable apoiipoproteins-a review of secondary structure and function j. lipid res 33, 141-166. high-density-lipcpmtein; synthetic pa&de analogs; lccithiu-choleste.ml acyltransferaae; gradient gel&ctmphcresis; lipid-pmtaiu iute~; a-i; plasma-hpopmteins; monoclonal-antibodies; electron-miaorcopy; netstrut-scattering. hide wa., chw l. and li w.h. (1992) daliongeviile j., lussiercacan s. and davignon j. (1992) modulation of plasma triglyceride levels by apoe phenotype-a meta-analysis-review. j. lipid res 33, 447-454.levek apoiipopmmht-e poiymotphism; coronaty-anery disease; density-lipoprotein chdestemk measured genuype infonnaticn; amein-e polymorphism; amino-acid-sequence; iii hyparlipoproteinemia; e isoforms; quantitative phenotypes; v hyperlipoproteinemia. bjorkhem i. (1992) mechanism of degradation of the steroid side chain in the formation of bile acids-review. j.lipid res 33, 455-471. rat-liver peroxisomes; h-tic mitochondrial cytochrome-p-450; cerebmtendinous xanthomatosis; cholic-acid; 3-a,7-a.12-a-trihydroxy-s-@cholestanoic acid; 26-hydroxylase system; chenodeoxycholic acid; 12-a-trihydroxy-5-~-cholestanoic acid; cholesterol 7-a-hydroxylase; vitamin-d3 25hydroxylase.hofmann a.f. and mysels k.j. (1992) bile acid soiubiiity and precipitation in vitro and in v&-the role of conjugation, ph, and ca*+ ions. j. lipid res 33.617-626. guo z.s. and depamphilis m.l. (1992) a-protein; bacteriophage-~, purificatiat. trumbly r.j. (1992) glucose repression in the yeast saccharomyces cerevisiae. mol. microbiol. 6, 15-21. carbcn catabolite repression; snfl protein-kinase; invettase synthesis; molecular analysis; maltose fermentation; cytocbrome genes; nuclear-pmtcim gal genes; suc2 gene; mutants.bischoff d.s. and ordal g.w. (1992) okane d.j. and prasher d.c. 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(1992) biophysicai aspects of neutron scattering from vibrational modes of proteins. prog. biophys. mot. bid 57, 129-179. pancm& tqprin-inhibitor, low-frequency vibrations; time-of-flight liquid glass-transition; hinge-bending mode; biological functions; globular pmtein; temperaturedependence; allosteric transition; supercooled liquids. cinti d.l., cook l., nagi m.n. and suneja sk. (1992) the fatty acid chain elongation system of memmalirm endoplaamic reticulum. prog. lipid res 31, 1-51. rat-liver nli crosomu; swine cembral mictosanes; acyl-coenzyme a; very-latg-chaim mouse-brain miaoscnnu; enoyl-coa hydratase; pemxisomal bifunctioual protein; cultured skin fib&lasts; lipid-lowering agentr; ~-oxidationheth2ea.m. lysolecithitt-lysolecithin acyltmttsferue; rabbit alveolar macmphages; myocatdial lysophos+&mse-tran~~, aortic endothelial-cells: cultumd netuo-blastoma; heart-muscle mictosomes; precursor fatty-acids; rat-brain microsomes; guinea-pig heart; fish oil diet kaya k. (1992) chemistry and biochemistry of taurolipids. prog. lipid res 31, 87-108. phosphoenolpymvate carboxykinase gtp; diet-induced hypercholesterolemia promoter-regulatory region; tissue-specific expression; enhancer-binding-pn&tu pymvate-kinase gene; rat-liver, messenger-rna, 6phosphofmcto-2kinasefructose 2,6-bispbosphatase; transcriptional regulation. barber j. and andersson b. (1992) lermrd j. (1992) moms d. (1992) smtctura 1 and fum&mal relationships between ~~yl-~f~ rna syt&tases. biodem. sci 17, 159-164. 3dimatsiand structure; atp, mechanisms; resohstion; homology; ~ueteim tymsyl; site; ammoacyl-transfer-rna. key: cord-262748-v4xue7ha authors: xu, yongtao; yu, shui; zou, jian-wei; hu, guixiang; rahman, noorsaadah a. b. d.; othman, rozana binti; tao, xia; huang, meilan title: identification of peptide inhibitors of enveloped viruses using support vector machine date: 2015-12-04 journal: plos one doi: 10.1371/journal.pone.0144171 sha: doc_id: 262748 cord_uid: v4xue7ha the peptides derived from envelope proteins have been shown to inhibit the protein-protein interactions in the virus membrane fusion process and thus have a great potential to be developed into effective antiviral therapies. there are three types of envelope proteins each exhibiting distinct structure folds. although the exact fusion mechanism remains elusive, it was suggested that the three classes of viral fusion proteins share a similar mechanism of membrane fusion. the common mechanism of action makes it possible to correlate the properties of self-derived peptide inhibitors with their activities. here we developed a support vector machine model using sequence-based statistical scores of self-derived peptide inhibitors as input features to correlate with their activities. the model displayed 92% prediction accuracy with the matthew’s correlation coefficient of 0.84, obviously superior to those using physicochemical properties and amino acid decomposition as input. the predictive support vector machine model for selfderived peptides of envelope proteins would be useful in development of antiviral peptide inhibitors targeting the virus fusion process. fusion process is the initial step of viral infection, therefore targeting the fusion process represents a promising strategy in design of antiviral therapy [1] . the entry step involves fusion of the viral and the cellular receptor membranes, which is mediated by the viral envelope (e) proteins. there are three classes of envelope proteins [2] : class i e proteins include influenza virus (ifv) hemagglutinin and retrovirus human immunodeficiency virus 1 (hiv-1) gp41; class ii e proteins include a number of important human flavivirus pathogens such as dengue virus (denv), japanese encephalitis virus (jev), yellow fever virus (yfv), west nile virus (wnv), hepatitis c virus (hcv) and togaviridae virus such as alphavirus semliki forest virus (sfv); class iii e proteins include vesicular stomatitis virus (vsv), herpes simplex virus-1 (hsv-1) and human cytomegalovirus (hcmv). although the exact fusion mechanism remains elusive and the three classes of viral fusion proteins exhibit distinct structural folds, they may share a similar mechanism of membrane fusion [3] . a peptide derived from a protein-protein interface would inhibit the formation of that interface by mimicking the interactions with its partner proteins, and therefore may serve as a promising lead in drug discovery [4] . enfuvirtide (t20), a peptide that mimicks the hr2 region of class i hiv-1 gp41, is the first fda-approved hiv-1 fusion drug that inhibits the entry process of virus infection [5] [6] [7] . then peptides mimicking extended regions of the hiv-1 gp41 were also demonstrated as effective entry inhibitors [8, 9] . furthermore, peptides derived from a distinct region of gb virus c e2 protein were found to interfere with the very early events of the hiv-1 replication cycle [10] . other successful examples of class i peptide inhibitors include peptide inhibitors derived from sars-cov spike glycoprotein [11] [12] [13] and from pichinde virus (picv) envelope protein [14] . recently, a peptide derived from the fusion initiation region of the glycoprotein hemagglutinin (ha) in ifv, flufirvitide-3 (ff-3) has progressed into clinical trial [15] . the success of developing the class i peptide inhibitors into clinical use has triggered the interests in the design of inhibitors of the class ii and class iii e proteins. e.g. several hydrophobic peptides derived from the class ii denv and wnv e proteins exhibited potent inhibitory activities [16] [17] [18] [19] [20] . in addition, a potent peptide inhibitor derived from the domain iii of jev glycoprotein and a peptide inhibitor derived from the stem region of rift valley fever virus (rvfv) glycoprotein were reported [21, 22] . examples of the class ii peptide inhibitors of enveloped virus also include those derived from hcv e2 protein [23, 24] and from claudin-1, a critical host factor in hcv entry [25] . moreover, peptides derived from the class iii hsv-1 gb also exhibited antiviral activities [26] [27] [28] [29] [30] [31] , as well as those derived from hcmv gb [32] . computational informatics plays an important role in predicting the activities of the peptides generated from combinatorial libraries. in silico methods such as data mining, generic algorithm and vector-like analysis were reported to predict the antimicrobial activities of peptides [33] [34] [35] . in addition, quantitative structure-activity relationships (qsar) [36] [37] [38] [39] [40] and artificial neural networks (ann) were applied to predict the activities of peptides [41, 42] . recently, a support vector machine (svm) algorithm was employed to predict the antivirus activities using the physicochemical properties of general antiviral peptides [43] . however, the mechanism of action of antiviral peptides is different from antimicrobial peptides; in fact, various protein targets are involved in the virus infection. e.g. hiv-1 virus infection involves virus fusion, integration, reverse transcription and maturation, etc. thus it is difficult to retrieve the common features from general antiviral peptides to represent their antiviral activities. virus fusion is mediated by e proteins. although e proteins are highly divergent in sequence and structure, they share a common pathway of membrane fusion dynamics. i.e. e proteins experience significant conformational change to form a-trimer-of-hairpin, which drives the fusion of viral membrane and host membrane [44] . the antiviral peptides derived from enveloped proteins function by in situ binding to their respective accessory proteins, disrupting forming of the trimer-of-hairpin and membrane fusion, and therefore inhibiting the virus infection. in view of the important role of e proteins in virus fusion process and common mechanism of action of self-derived peptides, we developed a svm model to predict the antiviral activities of self-derived peptides using sequence-based statistical scores as input features. the sequencebased properties were calculated by a conditional probability discriminatory function which indicates the propensity of each amino acid for being active at a specific position. our model exhibited remarkably higher accuracy in predicting the activities of self-derived peptides, compared to the previous models developed for general antiviral peptides using classical physicochemical properties as descriptors [43] . the method would be useful in identification of entry inhibitors as a new generation of antiviral therapies. 202 peptide virus entry inhibitors of enveloped viruses were collected, among them, 101 are active peptides and 101 are non-active peptides. these peptides comprised the 75p+75n training set of svm models. the remaining 26 active peptides and 26 non-active peptides inhibitors were used as the test set. amino acid composition. amino acid composition is the fraction of each amino acid in a peptide. the fraction of the 20 amino acids was calculated using the following equation: fraction of amino acid x ¼ total number of x = peptide length five physicochemical properties were used in svm models. isoelectric point (pi), molecular weight (mw) and grand average of hydropathicity (gravy) [45] were calculated using the protparam tool implemented in expasy web server. solvent accessibility and secondary structure features were calculated using sspro and accpro packages implemented in the scratch protein predictor server [46] . sequence-based statistical scoring function. the knowledge-based statistical function is developed from the concept of residue-specific all-atom probability discriminatory function (rapdf) [47] . rapdf is a structure-based statistical scoring function. it is based on the assumption that averaging over different atom types in experimental conformations is an adequate representation of the random arrangements of these atom types in any compact conformation. here we developed a sequence-based statistical scoring function, where we presume that averaging over different amino acid sequences with experimental validated inhibitive activities is an adequate representation of the random amino acid sequences with any inhibitory activity. the basis of this assumption is that the peptides share a common mechanism of action, i.e. the peptides derived from e proteins bind competitively to their partner proteins, disrupt the forming of a-trimer-of-hairpin, and therefore inhibit the virus membrane fusion. the sequence-based scoring function is described in the following form: sðfq i a gþ ¼ àln here, q i a 2 factiveg. pðq i a jcþ is the probability of observing amino acid i in an active peptide sequence; pðq i a þ is the probability of observing amino acid i in any peptide sequence, active or nonactive. they are approximately estimated using the following forms: similarly, we employed a dataset of experimentally verified non-active peptides in developing the statistical function, where q i a 2 finactiveg. for a given amino acid sequence, 20 columns of input are generated, corresponding to the occurrence of twenty natural amino acids at each position. each column is assigned a value of n ã (−log-likelihood), where n is the number of amino acid and −log-likelihood is derived from the statistical function score. each of the features thus combines the propensity of the amino acid for being active or non-active with the corresponding amino acid composition. below is an example of calculating the statistical scores for a given peptide sequence: the amino acid order for svm input features is set as: acdefghiklmnpqrstvwy. if the amino acid sequence of an active peptide inhibitor is: svm models combined with radial basis function (rbf) kernel parameters were developed using the c-svc module in libsvm (version 3.1) [48, 49] and executed under the matlab interface. the performance of svm depends on two parameters, gamma -g and cost-c [50] . the default value is 1 for -c and 1/k for -g, where k is the number of input entries. various pairs of (c, g) values were converted to exponential values (i.e. 2 x ;2 y ) and optimized using cross-validation and the pair with the best cross-validation accuracy was selected. 5-fold cross validation was performed to evaluate the performance of svm models. in the evaluation process, dataset was partitioned randomly into five equally sized subsets. the training and testing were carried out five times, each time four distinct subsets being used as training sets and the remaining subset as test set. the results were averaged over all five rounds of validation. the following equations were used to evaluate the prediction quality of the svm models [48, 51] : in the above equations, tp is the number of true positives, tn is the number of true negatives, fp is the number of false positives and fn is the number of false negatives. matthew's correlation coefficient (mcc) reflects the performance of the model. it ranges between -1 to 1 and a larger mcc value indicates a better prediction. svm learning algorithm is a powerful machine learning method that has been widely used in pattern recognition and classification. svm trains a dataset of experimentally validated positive and negative samples and generates a classifier to classify unknown samples into two distinct categories (positive or negative). we performed an exhaustive literature search on self-derived peptide inhibitors of enveloped proteins and collected experimentally validated peptides derived from the three classes of e proteins. for those peptides with overlapping segments, only one peptide sequence was kept. 202 peptides were found, among them, 101 are active peptides and 101 are non-active peptides ( table 1) . 75 active peptide inhibitors and 75 non-active peptides (75p+75n) of e proteins were used as the training dataset in svm learning; the remaining 26 active and 26 non-active peptides (26p+26n) were used as the test set. svm input features. three svm models were developed using different features as input descriptors, namely physicochemical properties (denoted as eapphysico), amino acid composition (eapcompo) and statistical scoring function amino acid composition (eapscoring). knowledge-based statistical functions are rooted in the bayesian (conditional) probability formalism and derived directly from properties observed in the known folded proteins [52] [53] [54] . in knowledge-based scoring function, it was presumed that averaging over different atom types in experimental conformations is an adequate representation of the random arrangements of these atom types in any compact conformation [55] . because the three classes of e proteins have different structural folds, it is difficult to retrieve a structure-based feature that is relevant to their antiviral activities. generally speaking, any property associated with folded proteins can be converted into an energy function [56] . since amino acid sequence determines the structural folds and properties of proteins/peptides, we presumed that a sequence-based statistical scoring function averaging over different amino acid sequences exhibiting inhibitive activities is an adequate representation of the random combinations of all twenty amino acid exhibiting any activity. in this approach, a peptide sequence derived from e protein is represented by twenty features each corresponding to the propensity of observing each of the twenty natural amino acids to be either active or non-active. a vector space of twenty sequence-based statistical scores was used as the eapscoring input entries in the svm learning. we also built a svm model using physicochemical properties as input features. because of the feature of membrane fusion process, it was suggested that functional regions in glycoproteins need to be solvent accessible, hydrophobic and flexible [57] . actually the majority of known peptide entry inhibitors share a common physicochemical property of being hydrophobic and amphipathic with a propensity for binding to lipid membranes [58] . therefore, here the properties of e peptide inhibitors were described by five physicochemical parameters: pi, mw, gravy index (positive and negative gravy values indicate hydrophobic and hydrophilic peptides, respectively), solvent accessibility (exposed or buried) and secondary structure features (propensity for adopting α-helix, β-sheet or turn structure). these physicochemical features were calculated for each of the peptides and used as the eapphysico input entries in the svm learning. a third svm model eapcompo was also built where the fractions of amino acids in a peptide were used as input features in the machine learning process. svm training. the svm models were trained using the experimentally validated 75p+ 75n data sets. during 5-fold cross validation, the training set was randomly partitioned into four subsets with equal size of (15p+15n) and a remaining subset (15p+15n). three svm models were built using sequence-based statistical scores, physicochemical properties and amino acid composition, respectively. the performances of the three models are shown in table 2 . it can be seen that the eapscoring model performed best among the three models during 5-fold cross validation. a "grid-search" combined with cross-validation was adopted to search for the optimal parameters -c and -g in svm models [49] . the result of the grid search is shown in the support information (s1 file). it is shown that the performances of three eap models during 5-fold cross validation have been improved significantly using the optimized parameters ( table 2) . the performance of the svm models was evaluated using an independent dataset of experimentally validated peptides that were not contained in the learning dataset (table 1 ). in the eapphysico model where physicochemical properties of peptides were used as input features, an accuracy of 65% with a mcc value of 0.31 was observed (table 3 ). in the eapcompo model where amino acid composition features were used, the predictive accuracy and the mcc value are slightly higher. when the sequence-based statistical function scores were used as input in the eapscoring model, a remarkable accuracy of 92% was achieved with a mcc value of 0.84. thus the sequence-based statistical scores developed in the present research are predominantly superior to the conventional physicochemical properties or amino acid decomposition features in identifying active peptides derived from enveloped proteins. avppred is a web server for prediction of the activities of general antiviral peptides (avps) based on a number of experimentally validated positive and negative data sets [43] . the peptide inhibitors employed in avppred target a variety of biological targets involved in virus infection. in contrast, the self-derived peptides of enveloped proteins being studied in the present research competitively bind to e proteins so as to mediate the virus fusion process. because the self-derived peptides share similar mechanism of action, it is feasible to retrieve common features from them to build predictive svm models. in order to evaluate the performance in predicting peptide inhibitors of the enveloped virus, we compared the avppred models with our eappred models using an independent 26p+26n dataset as test set. the results are shown in table 3 . four different features were employed in the avppred models, namely conserved motif search using meme/mast, amino acid composition, sequence alignment using blast and physicochemical parameters including secondary structure, charge, size, hydrophobicity and amphiphilic character [43] . when the avpmotif model was used to predict the activities of the self-derived peptide inhibitors, it performed rather poorly with accuracy of 52% and mcc of 0.14. this is not surprising because avpmotif was developed based on 20 general antiviral peptide motifs. however, the self-derived peptide inhibitors may not share a conserved motif with the general antiviral peptides since the latter interact with various biological targets with different mechanisms of action. in the avpalign model, the peptide sequences were classified into active and non-active databases and the query peptide sequences were matched against the active and non-active databases using the blast program. compared with avpcompo and avpphysico, avpalign performed better with a predictive accuracy of 73% and mcc value of 0.52. fusion mechanism is highly conserved among related viruses and entry of viruses into host cells has been inhibited by peptides derived from various regions of envelope glycoproteins [59] . self-derived peptides would inhibit interactions of their original domain by mimicking its mode of binding to partner proteins [4] . because similar sequences are often associated with similar structure and function, the sequence-based property avpalign would account for the activities of the self-derived peptide inhibitors which regulate the virus fusion by mimicking the binding to e proteins. in the avpphysico model, 25 best performing physicochemical properties were selected out of the 544 properties to build the svm model [43] . antiviral peptide inhibitors are generally amphiphilic [60] and the activities of peptide entry inhibitors are dependent on their interfacial hydrophobicity [58] . therefore we only employed five physicochemical properties reflecting hydrophobicity, solvent accessibility and secondary structure features as svm input features. it was demonstrated that the accuracy and mcc of eapphysico is comparable to that of avpphysico model, indicating the five properties used in current modeling building are critical for their activities. the mcc value of the avpcompo models is 0.20, indicating that the antiviral activities of the peptides are related to amino acid composition. when the amino acid composition was used as input, the predictive accuracy of the eapcompo model was higher than that of the avpcompo model, indicating the peptide inhibitors of e proteins employed in the training set is sufficient to represent the contribution of amino acid composition to their inhibitive activities. in the eapcompo model, the preference of the amino acid composition was ranked as: p, r, q, d, f, w, e, l, t, i, n, h, y, c, a, s, m, v, k, g (fig 1) . the role of arginine-arginine pairing and its contribution to protein-protein interactions has been investigated by computational approaches [61] . the higher abundance of r at protein-protein interfaces compared to k may be attributed to the formation of cation-π-interactions and the greater capacity of the guanidinium group in r to form hydrogen bonds (compared to k) [62] [63] [64] . furthermore, it was suggested that the interface regions are enriched in aliphatic (l, v, i, m) and aromatic (h, f, y, w) residues and depleted in charged residues (d, e, k) with the exception of arginine [62, [65] [66] [67] [68] [69] . this is in agreement with our amino acid composition analysis, where higher population of aliphatic leu residue as well as aromatic residues trp and phe was observed, whereas positively charged lys was hardly observed. the predominant occurrence of proline and glutamine residues is characteristic for the unique protein-protein interactions for e proteins. e.g. a conserved proline-rich motif was suggested to be engaged in monomer-monomer interactions in dengue e proteins [70] . a conserved glutamine-rich layer is involved in the extensive hbond network in hiv-1 gp41 e proteins [71] . thus the preference of the amino acid composition identified from the eapcompo model is generally in accordance with the predominant residues involved in protein-protein interactions, manifesting the amino acid composition of the self-derived peptide inhibitors are closely related to their potential activities in mediating the protein-protein interactions in the virus fusion process. because the antiviral activities of peptides are dependent on amino acid composition, we presume amino acid composition discriminated by the propensity of their activities would be an intrinsic feature in the self-derived peptide inhibitors which share a common mechanism of action. when statistical function scores were employed in the svm model (eapscoring), a remarkable predictive accuracy of 92% with an ideal mcc value of 0.84 was achieved, significantly better than any avp models. the logarithm form of the discriminatory function (eq 1) can be deemed as the pseudo energy of the system. in our previous study, we suggested that the stability of proteins is related to their in situ binding potential to the partner regions [72] . the prominent performance of eapscoring model indicates the sequence-based stability feature of self-derived peptides may reflect their potential of binding to e proteins so as to regulate the virus entry process. we developed three svm models using physicochemical properties, amino acid composition and statistical discriminative function as input features. the prediction accuracy and the mcc value of the eapphysico model where five physicochemical properties were employed are comparable with the previous avpphysico model where 25 physicochemical properties were used. the avpcompo and eapcompo models demonstrated that the activities of antiviral peptides are dependent on amino acid composition. a sequence-based scoring function was developed for the self-derived peptide inhibitors of e proteins. the outperformance of the eapscoring models supports our hypothesis that an intrinsic feature, represented by the propensity of each amino acid for being active in self-derived peptides, is responsible for the activities of the peptides to regulate virus fusion by mimicking the binding to their accessory proteins. the sequence-based statistical scoring function would be useful in development of novel antiviral therapies to target the initial step of viral infection. supporting information s1 file. parameters optimization by grid-research combined with 5-fold cross validation. x-axis is log2 g , y is log2 c and z-axis represents accuracy(%) ( figure a targeting cell entry of enveloped viruses as an antiviral strategy. molecules class iii viral membrane fusion proteins virus membrane-fusion proteins: more than one way to make a hairpin can self-inhibitory peptides be derived from the interfaces of globular protein-protein interactions? characterization of a putative cellular receptor for hiv-1 transmembrane glycoprotein using synthetic peptides propensity for a leucine zipperlike domain of human immunodeficiency virus type 1 gp41 to form oligomers correlates with a role in virus-induced fusion rather than assembly of the glycoprotein complex a synthetic peptide from hiv-1 gp41 is a potent inhibitor of virusmediated cell-cell fusion hiv gp41 c-terminal heptad repeat contains multifunctional domains. relation to mechanisms of action of anti-hiv peptides inhibition of human immunodeficiency virus type 1 entry in cells expressing gp41-derived peptides peptides derived from a distinct region of gb virus c glycoprotein e2 mediate strain-specific hiv-1 entry inhibition inhibition of severe acute respiratory syndrome-associated coronavirus (sars-cov) infectivity by peptides analogous to the viral spike protein synthetic peptides outside the spike protein heptad repeat regions as potent inhibitors of sars-associated coronavirus suppression of sars-cov entry by peptides corresponding to heptad regions on spike glycoprotein design and characterization of glycoprotein-derived peptide inhibitors of arena virus infection autoimmune technologies, safety, tolerability, and pk of escalating doses of flufirvitide-3 dry powder for inhalation in healthy subjects peptide inhibitors of dengue virus and west nile virus infectivity structural optimization and de novo design of dengue virus entry inhibitory peptides. plos neglected tropical diseases antiviral peptides targeting the west nile virus envelope protein peptide inhibitors of dengue virus entry target a late-stage fusion intermediate inhibition of dengue virus entry into target cells using synthetic antiviral peptides inhibition of japanese encephalitis virus entry into the cells by the envelope glycoprotein domain iii (ediii) and the loop3 peptide derived from ediii a fusion-inhibiting peptide against rift valley fever virus inhibits multiple, diverse viruses a peptide derived from hepatitis c virus e2 envelope protein inhibits a post-binding step in hcv entry early events in hepatitis c virus infection: an interplay of viral entry human claudin-1-derived peptide inhibits hepatitis c virus entry peptides containing membraneinteracting motifs inhibit herpes simplex virus type 1 infectivity evidence for a role of the membrane-proximal region of herpes simplex virus type 1 glycoprotein h in membrane fusion and virus inhibition the identification and characterization of fusogenic domains in herpes virus glycoprotein b molecules analysis of synthetic peptides from heptad-repeat domains of herpes simplex virus type 1 glycoproteins h and b conformational modifications of gb from herpes simplex virus type 1 analyzed by synthetic peptides multiple peptides homologous to herpes simplex virus type 1 glycoprotein b inhibit viral infection peptide inhibition of human cytomegalovirus infection a theoretical approach to spot active regions in antimicrobial proteins optimization of antibacterial peptides by genetic algorithms and cheminformatics antibp2: improved version of antibacterial peptide prediction application of 'inductive' qsar descriptors forquantification of antibacterial activity of cationic polypeptides qsar analysis of antimicrobial and haemolytic effects of cyclic cationic antimicrobial peptides derived from protegrin-1 design of novispirin antimicrobial peptides by quantitative structure-activity relationship qsar modeling and computer-aided design of antimicrobial peptides identification of novel antibacterial peptides by chemoinformatics and machine learning de novo design of potent antimicrobial peptides evaluating different descriptors for model design of antimicrobial peptides with enhanced activity toward p. aeruginosa avppred: collection and prediction of highly effective antiviral peptides structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme a simple method for displaying the hydropathic character of a protein scratch: a protein structure and structural feature prediction server an all-atom distance-dependent conditional probability discriminatory function for protein structure prediction support-vector networks working set selection using second order information for training svm a practical guide to support vector classification. initial version comparison of the predicted and observed secondary structure of t4 phage lysozyme distance-scaled, finite ideal-gas reference state improves structure-derived potentials of mean force for structure selection and stability prediction a distance-dependent atomic knowledge-based potential for improved protein structure selection statistical potential for assessment and prediction of protein structures comparison of database potentials and molecular mechanics force fields scoring functions for de novo protein structure prediction revisited peptide inhibitors against herpes simplex virus infections peptide entry inhibitors of enveloped viruses: the importance of interfacial hydrophobicity structure-based design of inhibitors of protein-protein interactions: mimicking peptide binding epitopes broad-spectrum antivirals against viral fusion the molecular origin of like-charge arginine -arginine pairing in water residue frequencies and pairing preferences at proteinprotein interfaces dissection of specific and non-specific protein-protein interfaces dissecting protein-protein recognition sites principles of protein-protein interactions studies of protein-protein interfaces: a statistical analysis of the hydrophobic effect the atomic structure of protein-protein recognition sites genome-wide studies of protein-protein interaction the interface of protein-protein complexes: analysis of contacts and prediction of interactions prediction of protein-protein interactions in dengue virus coat proteins guided by low resolution cryoem structures the fusion activity of hiv-1 gp41 depends on interhelical interactions computational identification of self-inhibitory peptides from envelope proteins the authors are grateful for the computing resources from qub high performance computing centre. the authors declare no conflict of interest. key: cord-006860-a3b8hyyr authors: nan title: 40th annual meeting of the gth (gesellschaft für thromboseund hämostaseforschung) date: 1996 journal: ann hematol doi: 10.1007/bf00641048 sha: doc_id: 6860 cord_uid: a3b8hyyr nan the variable molecular weight (mw) of vwf is due to differences in the number of subunits comprising the protein. it is assumed that endothelial cells secrete large polymeric forms of vwf and that smaller species arise from proteolytic cleavage. vwf has two main properties: it stabilizes factor viii protecting it from inactivation by activated protein c or factor xa, and it mediates platelet adhesion to subendothelium of the damaged blood vessel wall. each vwf subunit contains binding sites for collagen and for platelet giycoproteins gp ib and gp iib/i~a. multiple interactions of the multivalent vwf lead to extremely strong binding of platelets to subendothelial surface, that is capable of resisting high wall shear rate in the circulating blood. only the largest multimers are hemostatically active. lack of the largest vwf multimers was observed in patients with yon wiuebrand disease type 2a. unusually large molecular forms of vwf were found in patients with thrombotic thrombocytopenlc purpura. proteolytic enzyme(s) may be involved in the physiologic regulation of the polymeric size of vwf and thus play an important role in the pathogenesis of vwf abnormalities in some patients with congenital or acquired disorders of hemostasis. we have purified (-10,000-fold) from human plasma a vwf degrading proteas¢ using affinity chromatography and gel filtration. the proteolytic activity was associated with a high mw protein (mr -300 kd). vwf was resistant against the protease in a physiologic buffer but became degraded at low salt concentration or in the presence of 1 m urea. proteolytic activity had a ph optimum at g-9 and was not inhibited by serine protease inhibitors or sulfl~ydryl reagents. inhibition by chelating agents was best reversed by barium ions, the observed properties of the vwf degrading enzyme differ from those of all hitherto described pretenses. analysis of cleaved vwf showed that the peptide bond 842tyr-g43met had been cleaved -the same bond that has been proposed to be cleaved in rive. the endothelium releases the vasodilator nitric oxide (no) and the vasoconstrictor endothelin (et)-i. no is formed from l-arginine via the activity of constitutive nitric oxide synthase (cnos or enos). an inducible form of nos (inos) is activated by cytokines. no activates guanylyl cyclase in vascular smooth muscle and platelets, leading to the formation of cgmp which induces relaxation or platelet inhibition, respectively. in vessels, no is responsible for endothelium-dependent relaxations; in vivo it exerts a vasodilator tone which can be enhanced by shear forces and receptor-operated agonists such as acetylcholine, bradykinin, thrombin, atp and adp. infusion of no-inhibitors in vivo leads to vasoconstriction and increases in blood pressure and oral administration to hypertension in the rat. within the endothelium, no inhibits et gene expression and release of the peptide via cgmp. hence, no-induced hypertension is associated with increased plasma et levels. et, a 21-amino acid peptide, has potent vasoconstrictor properties via eta-and in part etb-raceptors on vascular smooth muscle. in endothelial cells, et activates etb-receptors linked to no and prostacyclin formation. under basal conditions, little et is formed, but is increased by thrombin, angiotensin ii, arginine vasopressin, cytokines and ox-ldl. et antagonists have been developed and allow to study the effects of et in vivo. et and no most likely play an important role in disease states such as hypertension, atherosclerosis, coronary artery disease, heart failure, pulmonary hypertension and subarachnoid hemorrhage. clinical trials to further define their role in these disease states are now under way. in summary, the endothelium is an important regulator of vascular tone and structure in vitro and in vivo. in disease states, their interaction is imbalanced leading to enhanced vasoconstriction, thrombus formation and structural changes of the blood vessel wall. pharmacological tools aiming to inhibit those changes are now being developed. j.m. harlan, r.k. w/nn, s. sharar, and n. vedder universit3, of washington, seattle, washington ischemia-reperfusion injury has been implicated in the pathogenesis of a wide variety of efinical disorders. [n preclinical models, tissue damage .clearly occurs during ischemia, but, parado.,dcally, may be exacerbated during reperfusion. this reperfusion injury appears to involve activation of the intlammato~, cascade with generation of complement 'components, lipoxygenase products, and chemokines as proximal mediators and neutrophils as final effectors of vascular and tissue damage. we have examined the role of leukocyte adhesion in reperfusion injury in two models -the rabbit ear as a model of isolated organ injury and hemorrhagic shock and resuscitation in the rabbit and primate as a model of traumatic shock and multiple organ failure. data regarding the efficacy, timing, and safety of leukocyte adhesion blockade using selectin-or integrindirected reagents in these models w/ll be presented. the current status of anti-adhesion therapy in other pre-clinical models and early clinical trials will be re~4ewed. an amidolytic assay for the determination of activated protein c (apc)-resistant factor va (fva) has been developed. this assay measures the cofactor activity of fva in diluted plasma samples via the rate of thrombin formation. the apc response is calculated from two fv determinations: one performed in the presence (apc-fv) and one in the absence of recombinant apc. the apc-fv activity is expressed as percentage of the initial fv activity and indicates the sensitivity of fva to apc. normal ranges were established by analysing plasma samples of 100 healthy individuals and an apc-fv activity above 60% was found to be indicative for apc-resistance (apc-r). in a control group of 34 patients the apc-r assay gave abnormal results in 15 patients. dna analysis confirmed heterozygous fv r506q mutation in all 15 patients and confirmed the non-carrier status in all of the 19 patients yielding normal results. an aptt-based apc-r assay performed on the same group of patients showed abnormal results in two of the non-carrier patients. one of these patients was diagnosed as positive for lupus anticoagulant, whereas the reason for the wrong positive result in the second patient remains unclear. eleven patients were analyzed before start of oral anticoagulation and during oral anticoagulant treatment. comparison of the assay results demonstrate a correlation of 96% indicating that the assay is independent of the activities of vitamin k-dependent clotting factors. the apc-r amidolytic assays allows specific and sensitive detection of fva-resistant to apc. the assay is performable in plasma samples of all persons in whom the diagnosis of apc-r may be indicated. in patients treated with oral anticoagulants or showing other clotting abnormalities affecting the aptt the apc-r amidolytic assay is helpful to establish the diagnosis of apc-r. dept of pediatrics, university hospitals kiel and mtinster, germany resistance to activated protein c (apcr), in the majority of cases associated with the arg 506 gin point mutation in the factor v gene is present in more than 50 % of patients < 60 years of age with unexplained thrombophilia. to determine to what exteut this relativdy common gene mutation affects the risk of thromboembolie events in infants and children, its occurrence was investigated in a population of children with unexplained venous or arterial thromboernbotism: thrombosis of the central nervous system (cns, n=4), vena portae (n=4), deep vein thrombosis (n=4), vena caval occlusion (n=4), neonatal renal venous thrombosis (rvi'; n--3), neonatal stroke (n=14), stroke (n=3), arteria femoralis ocdusion (n=2). four ont of these 38 patients showed a positive history of unexplained familial thrombophilia. apcr was measured in an activated thromboplastin time (afit) according to dahlbtick. the results were expressed as apc-ratios: clotting time obtained using the apc/caci2 -solution divided by dotting time obtained with cac12. concerning the special properties of the childhood hemostatic system, infants and children with apcr < 2 were considered to be apc-resistent only when the results were confirmed in a 1: l 1 dilution with factor v deficient plasma (instrumentation laboratory munich, germany). plasma of 268 healthy children served as controls. the arg 506 gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mul i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. consistent with the ap'it based method 10 out of 19 children with venous (v) thrombosis and eight out of 19 patients with arterial (a) vascular insults showed the common factor v mutation. additional coagulation defects (autithrombin, protein c type i, enhanced antiphospbolipid igg, enhanced lipoprotein (a)) were found in 30% (v) and 28% (a). furthermore, we diagnosed exogenlc reasons (septicemia, postpastal asphyxia, fetopathia diabetica, central line and steroid/asparaginase administration) in six out of 10 (v) and three out of 8 (a) children with thrombosis and apcr. all four patients with a positive family history of thrombophilia (mothers only !) showed the common factor v mutation arg 506 gin. in the control group the prevalence of apcr was 5.1%. the high incidence of additional exengenic factors in children with apcr confirm literature data of previously described inherited coagulation disorders during infancy and childhood: an acquired risk of thromboembolic disorders masks the coagulation deficiency in the majority of patients with an inherited prethrombotic state. furthermore, the incidence of 42 % apc resistant children with arterial insults in this study challenge the view that apcr is associated with venous but not with arterial thrombo-st8. 12 activated protein c resistance and plasminogen deficiency in a family with thrombophilia m, zttger 1 , f. demarmels biasiutti 1, ch. mannhalter 2, m. furlan 1 , b.~e 1 1httmatologisches zentrallabor der universit~t, inselspital, ch-3010 bern 2klinisches institut fur medizinische und ehemische labordiagnostik, universit~t wien, a-1090 wien several hereditary defects of the proteins regulating blood coagulation have been associated with familial thrombophilia. since the recent discovery of activated protein c (apc) resistance due to the factor v rf06q mutation as a highly prevalent hereditary risk factor for venous thromboembolism (tel, evidence is accumulating that familial thrombophilia may be due to a combination of genetic defects. thus, protein c-or protein s-deficient patients having suffered from te seem to be more likely to carry the factor v r506q mutation than expected from its allelic frequency in the population. we report a family (see figure) in which plasminogen deficiency (0.60 u/ml) had been found in the propositus having had twice postoperative deep vein thrombosis (dvt) at ages of 29 and 31 yrs, respectively, as well as in 5 family members (0.53-0.69 u/ml). out of these 5 plasminogen deficient individuals, only the propositus' daughter had suffered from recurrent dvt at age <20yrs. reinvestigation of this family in 1995 showed factor v r506q mutation in the propositus, his daughter, an asymptomatic sister and a brother with postoperative pulmonary embolism (pe). his father had had postoperative pe; he is deceased and could not be examined. ~, [] plasminogen deficiency ~ ,rll factor v rf06q mutation /~ propositus ~¢ bistory of dvt and/or pe e superficial phlebitis j2~,,~ not investigated "1" deceased even though this family is small for establishing unequivocal association of te with known defects, the two most severely affected individuals with recurrent te at ages <30yrs had combined plasminogen deficiency and apc resistance whereas those with isolated plasminogen deficiency were asymptomatic. these data support the concept of multigenie interactions leading to familial thrombophilia. resistance to activated protein c (apc resistance) is the most common dsk factor for venous thrombosis (vt). in most cases apc resistance is caused by a single point mutation at position arg 506 in the factor v gene (factor v leiden). while ample data in hetarozygous patients have been published, reports in homozygous patients are limited. we studied 29 patients (12 males [m] , 17 females it]) in whom a homozygous mutation had been verified by dna analysis. the median age at the time of the study was 38.8 years (y) (range 9-83 y). twenty-five patients had experienced vt (10 m, 15 f). four patients were discovered dunng family studies and were asymptomatic, three were children (between 9 and 13 y) and one patient was a 62 y old man. in males the first thrombosis occurred at a median age of 38 y (range 21-82 y), in females this was at a significantly younger median age of 26 y (range 17-49 y). twelve of the 15 symptomatic females had taken oral contraceptives (oc, estregen content 0.02-0.ling) for 6 to 150 months (median 71 m) pdor to thrombosis. in 2 women vt occurred during pregnancy, in 1 female it was precipitated by hormone replacement therapy. in contrast, in 8,<10 males the thrombosis happened spontaneously, in 2 males it followed surgery. the sites of thrombosis were dvt in 9 males and 14 females, dvt and pulmonary embolism (pe) in 4 females and 1 male, dvt and caval vein thrombosis in 1 female and superficial thrombophlebitis in 4 males and 1 female. eight females had at least one pregnancy, in total 11 children and 5 abortions. two had thrombotic events during pregnancy and 2 after delivery. all homozygous patients showed apc ratios between 1.12 and 1.68 (mean 1.39 + 0.19). conclusion: patients with homozygous fv leiden have similar clinical symptoms as patients with deficiencies of antithrombin-, protein c or protein s deficiency. however, in contrast to these defects a very high dsk dudng oral contraceptive medication leading to an ealier manifestation in females can be observed. several synthetic (efegatran, argatroban, inogatran and napsagatran) and recombinant (hirudin, peg-hirudin and hirulog) antithrombin agents are in different stages of nlinical development for cardiovascular and thrombotic indications. while the specificity of these agents for thrombin is a concern, little has been done to study the effects of these agents on other serine proteases involved in coagulation and fibrinolytic processes. fihrinolytic compromise by site directed thrombin inhibitors has been reported recently (thromb res 74(3): 193-205, 1994) . while these agents have been shown to inhibit plasmin and related enzymes, little or no informatienentheir effects onthe generation and functionality of apc is available. sinceapc plays an increasingly important role both as an antienagulant enzyme, by inhibiting factors v and viii, end a pro-fibrinelytic enzyme, by stimulating the release of t-pa fi'om endothelial sites, an inhibition of apc may result in both pro-coagulant state and fibrinolytic deficit. representative thrombin inhibitors (dup 714 -a prototype boronic acid peptide derivative, efega~an, argatroban, hirulog, hirudin and feg-hirudin) have been compared for their ability to inhibit apc (american red cross). these bionhemically defined studies in which the remaining activity of apc after incubation with a thrombin inhibitor was determined speclrophotometrically with a ehromogenic substrate (s-2288, pharmacia, franklin, oh) , demonstrated that dup 714 and efegatran inhibit apc in a coneentrafinn dependent manner (ic~0=0.75 and 8,4 gm resp~tively), hirnlog inhibits apc weakly (60 p2¢i produced only 25% inhibition), while argatroban and ~ have no anti-apc activities, while hirulog, hirudin and argatroban produced no direct enti-apc activities, it is conceivable that they may inhibit thrombomodnlin-bound thrombin and thus prevent activation of protein c, resulting in a functional apc deficit and failure to improve clinical outcomes despite higher dosage. while initially it was thought that sole targeting of thrombin will provide monospacifin anticoagulant agents devoid of some of the adverse effects observed with heparin, the recent clinical trials clearly suggest that thrombin is not the only determinant of thrombogenesis. furthermore, potent antithrombin agents such as hirudin, hirulog and peptides, indirectly inhibit the generation of apc, by compromising thrombomodulin-bound thrornhin and such agents as efegalran and dup 714 also produce direct apc inhibition. endogenous inhibition of formed ape by thrombin inhibitors may therefore compromise the feedback regulatory funetiens of apc and may lead to thrombotic amplification in fully enticoagulated patients. these studies warrant prcelinlcal assessment of thrombin inhibitors to evaluate their relative inhibitory effects on apc. poor anticoagulant response to activated protein c (apc resistance) causes a significant portion of deep vein thrombosis (dvt) whereas its association with coronary artery disease (cad) and myocardial infarction (md is still controversial. therefore, we investigated 346 recently hospitalised patients suffering from cad with or without previous mi. the cad was proven by coronary angiography. apc resistance was analysed by using the ap'l-i'-based assay coatest apc resistance (chromogenix). eleven patients showed an apc sensitivity index below 2.0 viewed as apc resistance. using pcr technology, the factor v mutation causing apc resistance 11691 g "-) a) has been shown in nine of these eleven patients. this represents 2.6 % (9/346), compared to 8,5 % found in healthy blood donors (8/94). one homozygous carrier (male, age 76) was identified (apc sensitivity index 1.4) who suffered from dvt at age 59. recent angiography demonstrated diffuse cad, no thrombotic events were reported in his family. in contrast, multiple thrombotic manifestations (dvt, mi, stroke) occurred in the relatives of four heterozygous patients. we conclude that the prevalence of apc resistance is rather low in patients with cad. nevertheless, the natural history of coronary manifestation of apc resistance seems to vary, probably depending on the presence and severity of cardiovascular risk factors. resitanee to activated protein c (apc resistance) is the most cormnon hereditary cause of thrombophilia and significantly linked to factor v leiden pcr based methods are used to identify the crucial point mutation in the factor v g(me. we designed primers in order to identify factor v leiden by allele-specific pcr amplification. amplification specificity for factor v was ensured by the 3'primer fv1001, located at the introng/intronl0 border of the g~ae. one sense and two antisense primers were used in ~vo separate primer mixes specific for factor v arts06 (wildtypo) or factor v otn506 (factor v leiden) yielding 235bp products each. in each pcr reaction a pair of primers amplifying a fragment of the human growth hormone gene was included, fimctioning as an internal positive amplification control (429bp pcrfragment). after an initial denaturation step 10/.tl samples (100rig genomic dna) were subjected to 10 two-temperature cycles fouowed by 20 threetemperature cycles. for visualisation 8p3 of the amplification product were run on a 2% agarose gel presmined with ethidittm bromide. the presence or absence of specific pcr amplification allowed defiu/te allele assignment without the need for any postamplifieation specificity step. the in~ernal positive control primers it~cate a sucessf-u/pcr amplification, allowing the assignment of homozygosity. in a prospective study 126 p~e.ients with tlaromboembolic events were analysed using this technique and enmpared with pcr -rflp according to bertina et al. the concordance between these techniques was 100 %. in 27 patients a heterozygous factor v ohas06 mutation was detected, whereas one pa6ent with recurrent thrombcembolism was homoz-ygous. no false-positive or false-negative results worn observed in the homozygous as well as hcterozygous samples. in addition in 15 samples identified to carry the point mutation by al/ele-specifin pcr anxplification automatic :~equencing confirmed the heterozygous or homozygous point mutation. due to its time-and cost-saving features allele-specific amplification should be considered for screening of factor v leiden. background: an initial intravenous course of tmfractionated heparin ~ljasted on the basis of the activated partial thromboplastin time is the currmt standard treatment for most patients with venous thrombosis. low-molecularweight hqmrin pre~a~tious can be administered subcutaneously, once or twice daily, without laboratory monitoring. we compared the relative effic.~y and safety of low-molecular weight heparin versus anfractionated heparin for the initial treatment of deep venous thrombosis. methods: english-language reports of randomized trials vtta'e identified th~ a medline search (1984 through 1995) and a complementary extensive manual search. reasons for exclusion from the analysis were no hepada dosage adjustments, the lack of um of obj~tive tests for deep venous thrombosis, dos~ranging studies that used higher doses of low-molecularweight heparin than are ctareatly in use, and the failure to provide blind endpoint ~sossmeat. we assessed the incidence of symptomatic recurrent vinous thromboembolic disease, the incidence of clinically ii~t bleeding and mortality. results: twelve of the 21 identified trials satisfied the predetermined criteria. the relative risk reductions for symptomatic thromboembolic complicatious, clinically ~t bleeding, and mortality varied firom 20.60% aad were all statistically significantly in favor of low-molecular-wedght hqtmrin. coadusions: low-mol~ular--weight hoparim administered subcutaneously in fixed doses adjusted for body weight aml without laboratory nmaitori~ =re more effective and safea" tlum adjn~_-dose standard h~. sauce low molec~dar weight hqmrim vary in o~apositiou =ad pharma~ogical im)fil~ the benefits of each ~ shodd ~tabllsbcd separately. unfraetionated hcparin (uh) and low molecular weight heparin (lmwh) are widely used for the prevention and treatment of thrombotic disorders. uh and lmwh induce platelet aggregation in vitro. rgd peptides compete with fibrinogm for the binding to the glycoprotein receptor (gp lib-ilia) of platelets and inhibit platelet aggregation. to inhibit the heparin-indueed platelet ~tion and prolong the half-life in blood of rgd peptides, we linked ac-rgdv-ssggs-ahx-yk eovalently to lmwh in a ratio 1:1. the peptide is composed of three regions: a. rgd-gives the specificity for the receptor gp lib-ilia; b..ssggs-ahx-is the spacer between carrier and ligand, which should facilitate the intnraetion between the conjugate and the gp lib-ills receptor; c. -yk arc functional antino acids for iodination (y) and for covalent attachment (k) to the cattier lmwh. the aggregation achieved with different concentrations of lmwh, lmwh-eonjugate and lmwh/rgd-peptide mixture in a ratio 1:1 was mea.~ared after 20 rain.; maximum aggregation after platelet activation with 10 pm adp was set equal to 100%. platelet aggregation in normal human plateletrich eitrated plasma (prp; 220 000/p.l) was induced by lmwh in a dose ~ndent manner. heparin can induce antbodies which interact with platelets and endothelial cells. this causes thrombocytopenia and thromboembolic complications. hitpatients do need effective parenteral anticoagulation. we treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory prooven hit (hipa-test) with recombinant (r-)hirudin. as these patients had been preseleeted by their immunological response during heparin treatment and the treatment duration of the study was longer than in any other study using thlrudin, all patient samples were investigated for anti-r-hirudin antibodies himdin antibodies were screened by a sandwich elisa using r-hirudin fixed to the solid phase as antigen. all plasma samples were screened for anti-hirudin antibodies of the igg class, but solar only a suset of samples for lge anti-r-himdin antibodies. 38 of 82 patients (46.3%) developed anti-hirudin antibodies of the igg class. anti-hirudin antibodies were not detectable not before 6 days of r-hirudin administration solar no ige anti-hirudin antibodies were found. none of the patients devdoped thromboeytopenia or allergic symptoms. however, in a subset of patients the anti-hirudin antibodies enhanced the anticoagulatory effect of r-hirudin. in 5 patients the hirudin dosage had to be decreased by 2-3 fold to maintain a stable aptt level, in 3 patients, despite stable r-hirudin maintenance dose the aptt increased to values >100 see.. during the study 4 patients with anti-hirudin antibodies had to be reexposed to a second course of r-hirudin for parenteral anticoaguhtion none of these patients developed any allergic reaction. in conclusion we found a high proportion of anti-hirudin antibodies in hatpatients treated with r-hirudin for more than 7 days. these antibodies seem to have minor clinical relevance in regard to allergic reactions. however, one has to consider that these antibodies may influence the pharmacokinetics of rhimdin and thereby enhance its antieoagulatory potency. therefore, aptt must be monitored closely in patients receiying r-hirudin for more than 5 days a major concern in the use of hirndin, the most potent and specific thrombin inhibitor, is the risk of bleeding associated with the potential effect of this drug on hemostasis, particularly when the antithrombotic therapy is combined with invasivo procedures, fibrinolytic treatment, or patient's predisposition to abnormal bleeding. thus, availability of an antagonist to hirudin would be essential for instant neutralization of the antithrombotie action. however, thueh a hirudin antagonist is unknown in nature. to prepare an antagonist to hirudin, a mutant derivative of human prothrombin, in which active site aspartate at position 419 is replaced by an asparagine, has been designed, expressed in recombinant chinese hamster ovary cells, and purified to homogeneity. d419n-prothrombin was converted to the related molecules d419n-meizothrombin and d419n-thrombin by limited proteolysis by e. carinatue venom and o. scvutellatus venom, respectively. both d419n-thrombin and d419nmeizothrombin exhibited no thrombin activity and titration resulted no detection of the active site. however, binding to solid phase immobilized hirudin and fluorescence studies confirmed that the binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins, hi vitro examinations showed that d419n-thrombin and d419nmeizothrombin bind to immobilized hirudin, neutralize hirudin as well as in the purified system and in human blood plasma and re-activate the thrombin-hirudin complex. animal model studies confirmed that d419nthrombin and d419n-meizothromi.,in act as hirudin antagonist in blood cireulatlon without detectable effects on the coagulation system. while i.v. injections of hirudin in mice resulted in an increase in partial thromboplastin time, thrombin time and anti-thrombin potential, additional injections of d419n-thrombin and d419n-meizothrombin resulted in a normalization of these coagulation parameters. elevation of plasma homocysteine is a hereditary disorder of methionine metabolism associated with a high risk of arterial vascular disease. however, as yet relatively little attention has been directed towards the association between hyperhomocysteinemia and juvenile venous thromboembolism (vte). consequently the aim of our study was to evaluate the prevalence of hyperhomocysteinemia (hyper-hcys) and juvenile vte. patients: 85 patients (29 men, median age 42 ys; 56 women, median age 35 ys) who had at least one verified episode of vte before the age of 45 ys were investigated in regard to their total plasma hcys levels. none of the patients had renal or liver dysfunction or evidence of any autoimmune or neoplastic disease. methods: plasma total homocysteine levels were determined by hplc with fluorescence detection. hyperhomocysteinemia was defined as hcys levels exceeding the upper limit of the normal range obtained in our laboratory from 80 healthy control subjects (40 males, median age 25 ys, hcys 95% ci: 2.02-5.67 pmol/l; 40 females, median age 27.5 ys, hcys 95% ci: 2.99-5.40 ,gruel/l). resuits: 13 out of 85 patients had hyper-hcys, giving a prevalence of 15.3 %. of these 13 patients, 9 were male and 4 female, indicating that the relation between elevated plasma hcys levels and vte may not be as strong in woman as in men. discussion: according to previous reports, our study shows that there is a high prevalence of hyper-hcys in patients with juvenile vte. however, the mechanisms by which hyper-hcys can provoke vte and whether hcys is an exclusive risk factor or if it contributes to other existing predispositions, possibly working as a trigger factor is unknown yet. some authors suggest hcys-iaduced effect on factor v activation or inhibition of thrombomodalin-dependent protein c activation. in addition an influence on thrombocyte aggregation has been postulated. conclusion: measurement of hcys levels may be useful in the evaluation of patients with a history of juvenile venous thromboembolism and could be clinically important as hyper-hcys is easily corrected by vitamin supplementation. detailed determination of the pathogenesis of vte in patients with hyper-hcys should be the aim of further investigations. a deficiency of one of the coagulation inhibitors antithrombin (at), protein c (pc) or protein s (ps) and resistance to activated protein c (apc resistance) are established risk factors for venous thromboembolism (vte). in the majority of patients with apc resistance, the .tug 506 gin mutation (factor v leiden) is present. whereas deficiencies of one of the coagulation inhibitors are rare in the normal population, the allele frequency of factor v leiden is 2-7% in western europe. heterozygous individuals have a 3-7fold, homozygous an 80fold increased risk for vte the typical clinical features of all abnormalities are deep vein thrombosis, pulmonary embolism, superficial vein thrombosis and thrombosis at unusual sites, like mesenteric vein thrombosis or cerebral vein thrombosis. the thrombotic risk is low during childhood, but increases considerably after the 13th year of age. a retrospective study in adult patients out of families with a symptomatic deficiency of at, pc or ps revealed that around 30% of surgical interventions and traumas of the lower extremities were complicated by vte. therefore, these patients should receive thrombosis prophylaxis al~er surgery and trauma if their age is higher than 13 years. pregnancy is associated with a very high risk for vte in individuals with at deficiency and prophylaxis should be initiated already in the first trimester. after delivery, thrombosis prophylaxis is adviced for all females known to have an abnormality. oc increase the risk, especially in at deficient and in homozygous factor v leiden females and are therefore contraindicated in these individuals. oc do also increase the risk for vte in patients heterozygous for factor v leiden and females known to have this abnormality should be discouraged from taking oc or should at least be informed on their increased risk. university hospital-', jerusalem, israel, hospital bcan.iou ~. paris, france, increased frequency of thrombocmbolie events have been observed iu patients with b-thalassemia. our findings of shortened platelet survival and enhanced urinary excretion ofthmmboxanc a: metabolitcs (blood 77:1749 (blood 77: , 1991 suggested an increased platclet activation in tbese patients. we also fouud that isolated thalassemie rbc enhance prothronlbin activation, suggesting an increased membrane exposure of procoagulant phospholipids i.e, phasphatidylserine (am j. hematol. 44:33, 1993) . we now show that annoxin v, which has a high specificity and affinity for anionic phospholipids inhibits pmthrombm activation by factor xa, by binding to thalassemic rbc (ic~, = 0.32 nm). kerckhoff-klinik, bad nauheim ~, medizinische poliklinik bonn 2, institut for immunologie und transfusionsmedizin universit~ll greifswald a antibody-mediated intravascular platelet activation is believed to be the basis for both arterial and venous thrombosis in patients with hat. while the development of arterial thrombosis can explained sufficiently by intravascular platelet activation, it is a matter of discussion whether additional risk factors are involved in the pathogenesis of hat-related venous thrombosis. since resistance to activated protein c (apc) is the most common inherited risk factor for venous thrombosis described the frequency of apc resistance among a population of 68 hat patients has been studied. hat was diagnosed using the heparin-induced platelet aggregation assay and confirmed by the ~4c-serotoninrelease test. the diagnosis of apc resistance was established by two functional assays and genetic analysis. at time of diagnosis of hat, 38 patients showed venous thromboembolic complications. among these, 24 were found positive for apc-resistance. pulmonary embolism was diagnosed in 18 hat patients, 14 of them were apc resistance positive. none of the 18 hat patients who showed exclusively thrombocytopenia were apc resistance positive. early oral anticoagulation (oa) was initiated in 7 patients after the diagnosis of hat has been established. six of these patients developed serious thrombotic complications including skin necrosis. these results demonstrate that apc resistance is an additional and common risk factor for the development of hat-related venous thrombosis. early initiation of oa during an acute episode of hat dramatically increases the risk of thrombosis. therefore, oa in hat patients should be initiated only after platelet counts have been returned to baseline levels and effective parenteral anticoagulation is achieved. controlled trials for primary and secondary prevention of stroke g. de (3aetano, c. cerletti and v. bertel~ consorzio mario negri sud, santa maria imbaro, italy this presentation will review the antithrombotic treatments to prevent ischemic stroke that have been evaluated in controlled clinical trials. in two studies of aspirin therapy for pdmary prevention in male physicians there was no reduction in the incidence of stroke, while that of first myocardial infarction was significantly lowered. similar results were obtained in a prospective study in a large cohort of women taking aspirin daily. the incidence of vascular death was not modified by aspirin in any of these trials. this is possibly due .to an excess of strokes associated to aspirin treatment: indeed the four vascular events avoided in 1000 us physicians under aspirin prevention for five years would result from five myocardial infarction and one vascular death avoided and two additional strokes occurred. oral anticoagulant therapy decreases the relative risk of stroke in patients with non valvular atrial fibrillation. warfarin appears to be superior to aspirin, but the latter drug is a useful alternative when long-term anticoagulant therapy cannot be administered. a metanalysis of about 150 trials and over 100,000 patients with different vascular diseases treated with aspirin (at different doses) and/or other platelet inhibitors showed 25% overall reduction of vascular events including stroke. the optimal dose of aspirin for secondary stroke prevention could not be established. in patients with previous minor strokes or tia there was 22% reduction of vascular events and 23% of non fatal strokes. the avoidance of nine strokes of any cause among the 38 expected in 1000 patients at risk would result from the sum of 10 ischemic events avoided and a haemorrhagic one occurred in excess. ticlopidine was reported to reduce the risk of stroke in two large tdals (one in patients with major stroke), but there is no evidence that it is better or safer than aspirin. we compared the effect of the direct specific thrombin inhibitors, napsagatran (na) and rec. hirudin (rh) with unfractionated heparin (uh) on the further growth of preformed thrombi. as a model of thrombogenesls, an annular perfusion chamber exposing rabbit aortic subendothelium was perfused with native rabbit blood at an arterial wall shear rate (650/s). fibrin and platelet thrombi were allowed to form during a 10min perfusion period after which the test agents were given iv as a bolus and a continuous infusion (3 and 10pg/kg/min, n=6) and the perfusion continued for 20min. the control groups were perfused for 10 or 30 rain (n=6). fibrin deposited and platelet thrombi formed on subendothelium were evaluated by microscopic morphometry. the % surface coverage with fibrin was not reduced in the drug-treated groups since fibrin deposition was similar in the 10 and 30 min control groups (39+8% and 335:6%, respectively, mean:l:sem). platelet thrombus area (ta) in the control groups increased from 24+7pm2/pm after 10min to 97+32pm2/lim after 30rain perfusion. na at 1011g/kg/min reduced ta by 94% to values (6+_2ptm2/ ~m) lower than those of the 10min control group whereas rh at this dose reduced ta by 70% (30-j:141.tm2/i.tm). uh at both doses was ineffective. these findings show that in contrast to uh the direct thrombin inhibitors na and rh inhibit the growth of preexisting thrombi. these results could be explained by the higher potency of na and rh as compared to uh for inhibiting clotbound thrombin (gast et al., blood coagul fibrinol 1994, 5.' 879-87) and suggest that thrombus-bound thrombin is an important modulator of platelet thrombus growth and/or stability in this thrombosis model. platelet adhesion -the initial event of thrombosis -is believed to be completely prevented by intact endothelium. we challenged this theory by superfusing intact human umbilical vein endothelial monolayers with activated human platelet rich plasma utilizing the stagnation point flow adhesio-aggregometer (spaa). the spaa provides flow mediated contact of platelets with the superfused surface. heparinized (3.5 -4.0 u/ml) platelet rich plasma (prp) was obtained from healthy volunteers and activated by addition of adenosine diphosphate (adp 2"10 -6 m). platelet deposition was recorded on-line by video as well as by measuring scattered light. fixed samples were examined by phase contrast and electron microscopy, inhibition experiments were performed with either the tetrapeptide rgds, the non-peptide gpiib/llla-inhibitor ro-43-8857 or a monoclonal antibody directed against the gpilb/llla complex. stimulation with adp prompted platelets to adhere to intact endothelium single or as microaggregates of a diameter of up to 100 micrometer. adhesion was dependent upon convective transport resulting in platelet collision with the endothelial monolayer. infusion of rgds or ro-43-8857 into the flowing, adp-stimulated prp completely prevented platelet adhesion to the endothelium as well as subsequent aggregation. when the inhibitor inflow was stopped while adp stimulation persisted, adhesion and aggregation occurred immediately. re-establishing the inflow of the inhibitors -with still continued adp stimulation -led to disintegration of the adhering aggregates. when prp preincubated with the monoclonal antibody against gpllb/llla was superfused, platelet adhesion to the endothelium and aggregation were irreversibly blocked. our results suggest that convective transport and stimulation of platelets are prerequisites to overcome endothelial thromboresistance and that subsequent platelet adhesion to the endothelium is mediated via the platelet gpilb/llla receptor complex. prevent thrombus formation affer ptca i.p. 8tepanova t, g.v. bashkov 2, l.p.kapralova, 2 s.p. domogatsky ~ cardiology research center t and national haematology scientific cettter 2 russian academy of medical sciences, moscow, russia percutaneous transluminal coronary angioplasty (ptca) results in atheroselerotie plague rapture, vascular wall damage and thrombogenic collagen exposure. subendothelial collagen type i-lll is a very ~rong agonist of platelet-dependent thrombus formation in arteries. the anlithrombotic action of rabbit polyclonal antibodies to rat collagen type i-ill and their chemically synthetized conjugate with monoclonals to human recombinant two-chain/one-chain urokinase type plasminogen activator (rtcu-pa/rseu-pa), cross reacting with rat tcu-pa/scu-pa was studied both an in vifro and in vivo. anticollagen antibodies and bispecific conjugate inhibited human platelet adhesion, aggregation and formation of thrombi-like ~ructures induced by rat collagen immobilized with the polystiroi surface in a condition mimics the high shear rate in the large elastic-type arteries. the short-term treatment of the collagen-soaked silk thread by the collagen antibodies suppressed the platelet-dependent thrombus formation in the arterio-venous shunt in rats by 56_+4 % (p<0.001) as well as by the bispecific conjugate (44_+4%, p<0.001). the treatment of collagen-adsorbed conjugate by rtcu-pa did not increase the autithrombotic effect of bifunctional antibodies. the present date suggest, that the local administration of the anticollagen antibodies at the site of atherosclerotic plague rapture may tm the efficient tool for prophylaxis of platelet-dependent thrombus formation in arteries after ptca. increased levels of certain hemostatic factors have been shown to be related to an increased risk of cardiovascular events. hypercoagulability is suggested to predispose to arterial thrombosis and thereby to participate in atherogcncsis. we therefore assessed fibrinogen, prothrombin fragment 1+2 (fi+2) and yon willebrand factor (vwf) antigen in 80 consecutive patients (aged 59+5 years) with known coronary artery disease (cad) who all underwent coronary angiography. the extent of coronary artery disease was quantified according to modified criteria of the american heart association (total, proximal and distal "score"). furthermore the intima-media thickness (imt) was determined in the carotid and femoral arteries by standardized ultrasonographie measurement, vwf antigen was found to correlate positively with the total and proximal coronary score (r=029, p<0.05 and r=o.36, p< 0.01). while fi+2 showed no correlation with the coronary scores, it was significantly correlated with the imt values in the carotid arteries (r=0.27. p< 0.05). after differentiating tertiles of the parameters patients belonging to the upper tertile of fi+2 concentrations had significantly higher imt values of the carotid and femoral arteries (0.81_+0.11 mm vs. 0.72+_0.13 mm in the lower tertile, p<0.05:1.38_+0.44 mm vs. i. 15-+0.25 ram, p=0.05) whereas in patients belonging to the upper tertile of vwf antigen concentrations the proximal coronary artery score was significantly higher (!.71-+0.59 vs. 1.31+ 0.92 in the lower fertile, p<0.01). fro correlation of fibrinogen concentrations and extent of cad or imt values of the carotid and femoral arteries could be demonstrated. in conclusion procoagnlatory mechanisms as indicated by elevated concentrations of yon willehrand factor antigen and fi+2 may be contributing factors in atherogenesis. we have previously shown that pgei is a potent inhibitor of pdgf-ioducod proliferation of vascniar smooth muscle cells (vscm) and inhibits dna replication by a camp-related mechanism (grol~er et al, 1994) . the present study investigates of whether or not this aatimitogeni¢ activity of pget can be amplified by trapidil, a compound that has been shown recently to inhibit the incidence of restenosis of hmnan coronary arteries subsequenmt to ptca (maresta et al. 1994) , vsmc were prepared from coronary arteries of adult bovine hearts, passagod and kept under standard tissue culture conditions. cells of passage 4-9 wore incubated in serumfree medium for 24h in the presence of indomethacin (3 p.m). addition of pdgf-bb (10 ng/ml) under these conditions stimulated dna-replication as assessed from 'hthymidin lncm'poration, by 3.-4laid above control level. trapidil at 10 idvl caused a minor reduction of pdgf-induced mitogenesis whereas 10t) of the compound resulted in a marked reduction of dna replication by 69% (p < 0.05, n = 4). pgei at 0.3 nm diminished the incorporation rate by t 1% while the simultaneous administration of both pged and trapidil (100 idyll caused a significantly stronger response as seen from n reduction of ~h-thymidine incorporation rate by 82% (p < 0.05, n = 4). as a possible mechanism of action, trapidil might have inhibited phosphodiesterases. to establish this, we measured the camp-depcudont proteinkiaasc (pk) a activity in cell homogenates. trapfdil increased the basal fka-activity from 19% to 31% of the maximum response while the response to pget (10 am) amounted to 55%. coincubation of pgei with trapidil caused a 65% stimulation of pka activity, sugesting a small though detectable inhibition of vscm phosphodiesterases by trapidil at anttmitogenic concentrations. essentially similar results wore obtained when thrombin was used as the mitogenic agent. the data demonstrate a significant antimitogenic effect of trapidil at p.molar concentrations that are in the range of plasma levels after therapeutic administration of the compound in rive. at these concentratrations, pget induced inhibition of mitogenesis is markedly enhanced by trapidil. inc. i~enna, and ~cenlral itematnlogy laboroto~. , university hospital of bern pibrinogen (fg), yon willebrand factor antigen (vwf) and tissue-type plasminogeu activator antigen (l-pal have recently been shown in be independent risk factors for subsequent coronary events in patients with angina pectoris (nejm 1995; 332:635) although paul antigen has also been proposed as a risk factor, conclusive dam showing its predictive value is still lacking. furthermore, we have recently shown in a study investigating 200 survivors of myocardial infarction that not only are fg, t-pa and pai-i significantly increased in these patients when compared to a heahhy conlro[ group, but pci activity is also elevated (7hrornb. tfaemost. 1995;73:1137 abst.) , hi order to obtain cut.off points for the individual parameters, frequency histogram plotl; were transformed into straight line cumulative frequency (probit) plots (thromb i/aemost. 1995;74:718) . the cut-off valu~ for the four parameters were determined as follows: fg at 2.7 g/l, t-pa at 8.7 ng/ml, pal-i at 25 ng/ml and pc[ at 126% of a normal pooled plasma. utilising there cut.off points it was then possible to determine the accumulative discriminatow effectiveness of the parameters. when fg w;qs employed alone as the discriminatow factor, it was observed that 47% (941200) of the coronary heart dir, ease (chd) group eilher had the cul..off value or were below it aud 29% (29199) of the normal group were above the cut-off value, thus, resulting in 47% false ne$atives and 29% false positives. when a second additional risk factor, t-pa wa_~ introduced, the number of false negatives dropped to 21% [i.e. 79% (158/200) had two, risk factors elevated] and the number of false positives to 13% to investigate whether a third parameter could discriminate further, pai-i antigen was used to analyse the rcnudning false positives and negatives. an additional 4% could be detected, resuhing in 83% of the chd group having three risk factors elevated. similarly, the number of normal aubjecta with three parameters elevated dropped by 4% to 9% furthermore. when a fourth parameter was introduced, namely pci, it was round to discriminate a further 8% in the chd group, thereby increasing tile di~riminalion to 91%. the number of false positives dropped to 4%, additionally, determination of pci increased the discrimination of patienta having had multiple infarctions from 88°/= when thrce parameters were mcasured to 100%. from these results it can be concloded that determination of fibrinogen levels alone is not sumcicnt to separate patients from controls as t-pa adds significant discrimination. pai-i antigen which correlated strongly with t-pa did not significantly increase the discriminatory potential of both fg and i-pa. however, by employing pci as a fourth paramctcr, virtually complete separation between the chd and normal groups as well as rurthcr recoguitiou of' patients having had multiple infarctions could be obtained. to test the hypothesis that oral contraceptives (oc) enhance exercise-induced activation of blood coagulation we examined 11 women (27 + 3 (sd) years, bmi 20.4 + 2.0 kg/m 2, vozm.. 49 + 7 ml/kg/min) without oc between day 18 and 22 of the menstrual cycle and 10 women (24 + 2 (sd) years, bmi 20,6 ± 1,6 kg/m', vo2max 47 + 7 ml/kg/min) taking oc (150 mg desogestrel and 30 mg ethinylestradiol) between day 7 and 21 of drug intake. prothrombin fragment 1 +2 (ptf1 + 2) and fibrtnopeptide a (fpa) were measured before and after running for one hour on a treadmill at a speed corresponding to the anaerobic threshold. mean heart rate [191 ± 10 vs. 196 ± 12 min 1) and mean plasma lactate (3.3 ± 1.6 vs. 3.1 + 1.2 mmot/i) wera comparable during exercise between control and oc group, respectively. results for markers of thrombin and fibrin formation were: ptf1 +2 (nmol/i) fpa (ng/ml) control before 0,66 ± 0.19 1.0 + 0.2 after 0.73 + 0.23 2.2 + 1.2" oc before 0.82 + 0.31 1.0 + 0.2 after 1.11 + 0.48* + 5.8 -+ 6.0* + * p < 0.05 vs. baseline, + p < 0.05 between groups. we conclude that oral contraception with 150 mg desogestrel and 30 mg ethinylestradiol enhances exercise-induced thrombin and fibrin formation, our data suggest that exercise testing might be useful for evaluating the risk of thrombosis associated with different compositions of oc. a. haushofer +, wm. halbmayer +, j. radek +, m. dittel *, r. spiel *, h prachar *, j. mtczoch *, m. fischer + + zentraltaboratorium mit thrombose-und c~rinnungsambulanz -krankenhaus lai~: * 4. medizinische ab[eilung mtt ka~liolo$i¢ -krankenhaus lainz und ludwig bottzmann-lnstitut ftlr herzinfarktforsohung, wien fifty-one patients (age 61.6 ± 9.2a; 34 m / 171) implanted with 74 coronary stems 03 palm~-schatz, 27 gianturco-roubin, 14 micro stcnts) received a now antithrombotic treatment using a combination of ti¢lopidine (tic) 2 ×250 mg/d for 28 days and acetyl salicylic acid (asa) zoo mg/d for long-term treatment. patients (pat) only received 15000 tu standard hepartn as i.v. bolus immediately before stent implantation (day l ). side effects and changes in hematological (day i to 7, 14. 28 and 35 [= without t[cil liver and kidney parameters (day 7, 14, 28, 35) were monitored. thirty-eight pat (75%) came for the controls to our del~rtment and were additionally monitored by thromboelastograpy (teg) and bleeding time (bt) (day 2g and 35). the other pat were monitored externally, side effects were reported. thrombin geucration after stenting was monitored from day i to 7 by prothrombin fragment 1+2 (f 1+2) and thrombin-antithrombin-lll-comptex (tat). "k" of the "leg decreased (day 28 vs 35; p< 0.0l ). bt prolongation was negatively correlated with the bodysurf ace area (tic+asa: p< 0.05, asa: p< 0.0 l) and showed a reduction after withdrawal of tic (2 l0 sec, 180/ 300 so: [median, quartiles] vs. 120 sec, 881157 sec; p< 0.ix)01). f 1+2 and tat of day i (blood collection: 0, 2, 4, 6 h after intervention, f 1+2:0.84 nmol/i, 0.68/i.07 nmol/l: tat: 2.6 pg/1, 2.0/4.6 ilg/1) were lower compared to day 2 to 7 (f i +2: 1.31 nmol/l, ].08/i .62 nmol/l; tat: 4.8 pg/i, 3.2/7.2 ijg/1; p< 0.0001). tic scorns not to be a strong thrombin generation inhibitor. during stenting one pat (i.9%) sustained a non penetrating mci and one (1.9%) an ischaemic stroke. tic+asa were very effective, only with one pat (1.9%) stent thrombosis (acute) occurred. side effects: 8/15.7% gastrointestinal (one lead to hospitalization), 5/9.8% hematomas at the needle site in the groin (one surgical intervention), 5/9.8% leucopcnias (one agranulozytosis with hospitalization), 3/5.9% allergic skin reactions and 2/3.9% increased liver enzymes (got, gpt, "pgt, alkaline phosphatase; > 2 × of the j. ). with one pat with gastrointestinal disturbances and skin reactions tic had to be withdrawn and treatment was changed to oral anticoagulatlon + asa. one pat showed a combination of skin reactions, gastrointestinal distufl~aneas and on day 28 a heavy reaction of the liver enzymes ( j. after 5 weeks). a decrease of the white blood count (day 1:7.19 gh, 6.03/8.2 g/l, day 28:5.46 g/l, 4.63/6.47 g/l; p< 0.000 i) could be observed. the safety of the therapy with tic+asa should be elucidated and extensively discussed. the serpins c1 esterase inhibitor (cllnh), antithrombin iii (atiii), alantitrypsin (slat), and a2-antiplasmin (azap) are known inhibitors of coagulation factor xla (fxla). although initial studies suggested al at to be the main inhibitor of fxla, we recently demonstrated cllnh to be a predominant inhibitor of fxla in vitro in human plasma (wuillemin et el., blood 1995; 85:1517) . the present study was performed to investigate the plasma elimination kinetics of human fxla-fxla inhibitor complexes injected in rats. the amounts of complexes remaining in circulation were measured using elisas. the plasma tl/2 of clearance was 98 min for fxla-alat complexes, whereas it was 19, 1 8, and 1 5 min for fxla-cllnh, fxla-a2ap, and fxla-atiii complexes, respectively. thus, due to this different plasma tl/2, preferentially fxla-alat complexes may be detected in clinical samples. this was indeed shown in plasma samples from thirteen children with meningococcal septic shock (mss), a clinical syndrome which is complicated by activation of coagulation, fibrinolytic, and complement systems. fxla-fxla inhibitor complexes were assessed upon admittance to the intensive care unit. fxla-a 1at complexes were elevated in all patients, fxla-c11nh complexes in nine, fxla-atiii complexes in one patient, and no elevated fxla-a2ap complexes were found. we conclude from this study that, (1) although c1 inh is the predominant fxla inhibitor, fxla-alat complexes may be the best parameter to assess activation of fxi in clinical samples, (2) measuring fxla-fxla inhibitor complexes in patient samples may not help to clarify the relative contribution of the individual serpins to inactivation of fxla in rive, and (3) fxl is activated in patients with meningococcal septic shock. dudng the coagulation of plasma about 20 % of the (x2ap present is covalently crosslinked to fibrin by factor xiila (aoki und sakata 1980, thomb. res. 19: 149-155) . we investigated the binding of azap by factor xiila to soluble fibrin (desaabb-fibdno) whose polymerization was inhibited by an isolated fibrin ddomain named d=,,, (haverkate and tiemann 1977, thromb. res. 10: 803-812) . d==. is known to have an intact fibrin-polymerization site and is able to block the prolongation of the fibrin protofibrils at an early stage depending on its concentration. lateral association to fibrin fibers does not take place, since the inhibited protofibnls formed at the conditions used here do not reach a sufficient length (williams et el. 1981, biochem. j. 197: 661-668; hantgan et al. 1983, ann. n. y. acad sci. 408: 344-365) . material and method: soluble desaabb-fibrino was prepared by incubation of (lztl)-fibrinogen (0.48 mg/ml), d=1o (1.91 mg/ml; molar ratio d==o to fibrin 14:1) and 0.4 u/ml thmmbin for 45 min. then q2sl)-c~2ap (16 p.g/ml), faktor ×ill (2 ulml) and ca 2) (5 mmol/i) were added. the crosslinking reaction was stopped at different times of factor xiila-incubation by adding of urea/edtasolution. the suspension was analysed by ultracentrifugation on gradients containing saccharose, urea and sos. re~ultl: the elution profiles of the ultrecentifugation-gradients show the formation of cmsslinked fibrin oligomers of increasing size depending on the time of factor xiila-action. the crosslinked fibrin polymers contained about 80% of the fibrin initially added. although factor xiila acted well, crosslinking of azap in the fibrin oligomers could not be observed. conclusl0n: as we already demonstrated (kelach et el. 1994, ann, hematol. 70 (suppll) : a 60) the crosslinking of azap to fibnn clots depends on the structure of the fibdn network, especially on the degree of lateral association of the fibrin pmtofibdla. in desaabb-fibrino no lateral association of fibrin protofibnls takes place under the conditions chosen here. thus it is consistent with our theory that we did not observe any binding of aiap to the fibrin oligomers of desaabb-fibrino. human pci is a non-specific serpin that inhibits several proteases of the coagulation and fibrinolytic systems as well as tissue kallikrein and the sperm protease acrosin. it is synthesized in many organs including liver, pancreas, and testis. the physiological role of pci has not been defined yet. recently, we have cloned and sequenced the mouse pci gene (zechmeister-machhart etal., manuscript in prep.) . this enabled us to study pci gone expression in murino tissues using mouse pci edna and crna probes. by northern blot analysis, mouse pci tar.ha was exclusively found in the reproductive tract (testis, seminal vesicle, ovary), all other organs analyzed -including the liver were negative for pci mkna, indicating that in the mouse pci is not a plasma protein. to determine which cells of the reproductive tract synthesize pci, cellular localization was assessed by in situ hybridization of mouse testis and ovary sections. in testis, pci mrna was present in the spermstogonia layer and in leydig cells, while sertoli cells and peritubular myoid cells were negative. these results are consistent with the immunohistological localization of human pc1 (laurell et al,, 1992) . in the mouse ovary, stroma cells of the medulla and around the follicles were positive for pci mrna. no pci expression was detected in theca or granulosa cells. we also studied the regulation of mouse pci gone expression by steroid hormones in vivo. [n mature male mice castration caused an increase in pci mrna in seminal vesicles, which was reversible upon the administration of testosterone. in tissues of intact adult male and female mice, pci mrna levels decreased after injection of human chorionic gonedotropin (hcg), while in castrated male mice, hco had no effect on seminal vesicle pci mrna. progesterone and 17-b estradiol decreased ovarian pci mrna levels in immature female mice. these data suggest direct down-regulation of mouse pci by sex steroids. the different tissue specific pci-geoe expression in men and mice furthermore indicates a different biological role of this serpin in the two species. ctr. transgene technology, leuven "[' 1-tissue factor ('it) is a 47 kda glycoprotein mainly known a the primary cellular initiator of blood coagulation. whether tf expression may also play a role in development is unknown, but the lack of spontaneous viable mutations of the tf gene in rive leads to the speculation that its absence may not be compatible with normal embryonic development. to determine the significance of "if in ontogenesis, the pattern of tf expression in mouse development was examined and compared to the 'if distribution in human postlmplantation embryos and fetuses of corresponding gestational age. at early embryonic period of both murine (6.5 and 7.5 pc) and human (stage 5) development there is a strong tf expression in both ectodermal and entodermal cells. "if decoration was seen during ontogenetic development in tissues such as epidermis, myocardium, bronchial epithelium, and hepatocytes, which express "if in the adult organism. surprisingly, during renal development and in adult organism tf expression differs between men and mice. in humans maturing stage glomerali were "if positive whereas in mice glomeruli were negative and instead epithelia of tubular segments were tf positive. in ncuroepithelial cells there was a striking 'if expression indicating a possible role of'if in neumlation. moreover, there was a robust tf expression in tissues such as skeletal muscle, and pancreas, which do not express in adult. in contrast to tf, its physiologic ligand factor vii was not expressed in early stages of human embryogenesis, but was detectable in fetal liver, the temporal and spatial pattern of tf expression during murine and human development support the hypothesis, that 'if serves as an important mo~hojzenic factor darinz embrvozenesis. to serve as an anticoagulant, protein c (pc) must be activated by a complex formed between the enzyme thrombin (t) and its cofactor thrombomodulin (tm). therefore, downregulation of endothelial cell surface expressed tm, for example, triggered by an inflammatory stimulus, could become a critical factor in effective pc activation. in order to develop a recombinant (r) pc mutant which can be activated independently of the tkm-complex, a peptide sequence including p1-7 in the activation peptide of pc was modified to be identical to the factor xa (fxa)-cleavage site in prothrombin. the mutant was expressed in hu 293 cells, purified and its anticoagulant properties characterized. using purified fxa the mutant showed activation rates between 0.02 and 0.13 nmlmin at pc concentrations between 97 and 770 nm, while the rpc wild type was insensitive for fxa activation. the activation reaction is calcium-dependent reaching maximal activation rates at a calcium concentration of 3 mm and was enhanced to 3.8-fold by addition of anionic phospholipids (pl). in contrast to the wild type pc the rpc mutant was insensitive for activation by the t/i-m complex. addition of the mutant to normal human plasma induces a prolongation of tissue-factor and p-it-based clotting assays. using normal human plasma as a source for fxa the the activation rates of the mutant were found 5-fold higher than in the purified system if tissue factor was used to generate fxa. in conclusion, our data demonstrate that the rpc mutant is effectively activated by fxa in a purified as well as in a plasma system. interestingly, the activation rates are enhanced in the presence of pl and normal human plasma. fudher studies should clarify the potential use of this mutant as a novel anticoagulant. thrombln plays a pivotal role in thrombotic events. the time course of thrombln concentration in blood or plasma after activation is of special interest to answer a variety of questions. with a chromogenic assay developed by hemker et el. [thromb. haemostas, 70, 617, 1993] it became possible to measure the generation of thrombin in activated plasma continuously. inhibitors of clotting enzymes which are to be developed as anticoagulants should be able to inhibit thrombin generation or to immediately block generated thrombin. we have used a test based on hemker's thrombin generation assay to elucidate which potency and specificity an inhibitor of factor xa needs to efficiently block thrombin generation in human plasma. thrombin generation after extrinsic (tissue factor) or intrinsic (ellagic acid) activation was followed using the chromogenic substrate h-~ala-gly-arg-pna (pentapharm ltd.). a series of synthetic low molecular weight inhibitors as well as naturally occurring inhibitors of factor xa with different potency were investigated. because of the inhibition of activated factor x the generation of thrombin in plasma is delayed and the amount of the generated thrombin is reduced. the concentrations which cause a 50 % inhibition of thrombin generation (icso) correlate with the k~ values of the inhibitors. low molecular weight inhibitors with k~ values of about 20 nmol/i inhibit the generation of thrombin after extrinsic activation with icso in micromolar range. after activation of the intrinsic pathway tenfold lower concentrations are effective. the strongest inhibitory activity after extrinsic as well as intrinsic activation is shown by recombinant tick anticoagulant peptide (r-tap) with ic~o of 0.049 pmol/i (axtdqsic) and 0.037 pmo/i (intdnsic). in the compadson of synthetic low molecular weight inhibitors of thrombin end factor xa which have similar k= values for the inhibition of the respective enzyme (lowest i<1 20 nmol/i), factor xa inhibitors are less effective tn the thrombin generation assay. in contrast, the highly potent xa inhibitor r-tap shows a stronger inhibition of thrombin generation than the tight binding thrombin inhibitor hirudin. background: resistance to degradation of coagulation factor v by activated protein c is associated with a point mutation in which adenine is substituted for guanine at nucleotide 1691 in the gene coding for factor v. to date this specifc mutation appears to be the most common inherited abnormality which predisposes patients to venous thrombosis. for this reason a reliable, fast and automatable system for the diagnosis of the described point mutation is required. the conventional methods used to identify the mutation are based on allele-specific restriction enzyme site analysis or direct sequencing. these methods have disadvantages for a large scale dna diagnosis, which include the need for electrophoresis or a high cost and time consumption. methods: an alternative strategy of dna diagnosis, the allele-specific oligonucleotide ligation assay, was adapted for the diagnosis of tile point mutation of factor v. following pcr amplification of the target dna, tile procedure was performed completely automatically on a robotic workstation with an integrated elisa reader using a 96-well microtiter plate. allelespecific restriction enzyme site analysis was performed to confirm the genotypes. results: in 10 patients with the mutation and in 20 individuals without the mutation the genotypes determined with the conventional allele-specific restriction enzyme site analysis were in 100% concordance with the elisabased oligonucleotide ligation assay. discussion: the pck-oligonucleotide ligation assay applied as automated detection system for the identification of the coagul;mon factor v point mutation allows tile rapid, reliable, and large scale analysis of patients at risk for thrombosis. resistance to the asticoa=m~lant activity of activated protein c (apc resistance) has emerged as the most con'anon inherited thrombophilic state. patients lreterozygous for factor v leiden are more likely to suffer from thromboembolie events than controls. this risk is even more pronounced in homozygotes. due to the low sensitivity and speeifity of most coagulation tests some investigators suggest to examine patients for the presence of factor v leiden mutation by pcr-based methods. re e~tly we presented an aptt-based functional test (acceleria inactivation test ait): 1:20 diluted plasma (50bi) is mixed with factor v deficient plasma (50~tl) and aptt reagent (501.d), incubated at 37°c and then coagulation is induced by caci2 and a.pc (50~d). using a standard curve, the clotting time (see) is transferred in per cent accelerin inactivation (%ai). using this test, the widely used apc-ratio as well as pcr-based factor v leiden detection (confirmed by direct sequencing) we prospectively studied 172 consecutive patients with thromboembolic events. patients without the factor v mntation eonsitently showed more flazm 50% al with the exception of one patient with severe factor deficiencies (including factor v) due to hepatic failure and heterozygous for factor v-leiden resulting in 62*/. ai, there was a complete concordance between the pcrbased method and dysaseelerinemia detected by ait. due to these result a specifity and sensitivity of ait above 95 % was calculated. furthermore, a clear discrimination could be obsoved beween 34 heterozygotes (20%<ai<50%) and 8 (incl. 5 samples obtained from jeaa) homozygotes (ai<20%). in contrast a.pc-ratio resulted in a great overlap between patients homozygous for the wildtype , hcterozgous or homozygous for factor v leiden. in addition we confirmed thc di.~eulties nsing the apc-rauo for patients trader anticoagulation, or with different types of other d~rombophilie defects such as lupus anticoagulant or protein s deficiency. due to the time and cost-saving featttres the/kit should be considered for the rapid detection of apc resistance without the use of pcr-based techniques. in summary we found, well in accordance with previous reports, that apcr due to the fv-leiden mutation is • highly prevalent defect in vte pts. pts with the fv-leiden mutation were eharacterised by a higher incidence of vte's without recognised prothrombotic risk factors and more frequent positive family history; no higher incidence of recurrent vte's or other prothrombotie coagulation defects was found; the age at initial manifestation of thrombophitia was comparable. acquired a recently identified mechanism for famdial thrombophilia is characterized by a poor anticoagulant response to actavated protein c (apc), it is now welt recoginzed to be due to a genetic defect in the factor v gene, which results in the syn-,thesis of an abnormal factor v molecule and to be a frequent risk factor for thromb.osis, however apc resistenee can also be found in certain acquired conditions. since pregnancy ~s known to be associated with a hypercongulable state resulting in a prevalance of thrombosis up to 1% and thromboembolic events, we determined the prevalence of apc resistance during the course of pregnancy at the city hoslfital ruesselsbeim. germany. blood samples from pregnant woman were drawn at mmester 1, 2, 3 and at term. besides the routine coagulatton screening, protein c and viii:c, the apc resistance were evaluated. a modified aptt method utilizing human plasma fracnonated apc (amencan red cross. bethesda, maryland, ~usa) was used to assay apc resistance. this method relies on the determination of two aptt's in the patient plasma, one in the absence of apc and one in the presence of a fixed amount of apc. clotting tune was determined after mcubation using the aptt reagent ( we investigated the sensitivity of various species towards activated, human protein c (apc) and found an impaired response in the order monkey < horse < pig < dog < rabbit. we assume that this effect is mainly caused by differences in the apc cleavage region of factor v, as recently described for a factor v mutation in bumat~s: factor v-leiden. this was corroborated by the finding that addition of human factor v similar to the factor v added with the animal plasma had only minor influence on test outcome. coagulation assays dependent on apc, such as for protein c and protein s determination, in the standard apc assay, the more factor v-leiden specific apc assay and in the pcat, a new screening assay for the whole protein c system, are based on the inactivation of factor va via apc. thus, in all these assays, 5% of rabbit plasma added to human plasma mimicked a apc response as observed for heterozygous carriers of factor v-leiden and 20% as for homozygous carriers. we exploited this factor v-leiden like behaviour to prepare plasma standards for calibration of apc dependent assays by mixing rabbit plasma with human plasma~ as reference for these standards the 0% value was assigned to the clotting time in the absence of apc, while the 100% value was assigned to the clotting time in presence of apc as obtained with a human, normal plasma pool. then these calibrated standards were used for establishing reference curves. on automated coagulation analyzers, the results obtained were automatically reported in '% of normal' or '% sensitivity'• this not only eases evaluation, more importantly, this procedure also considerably improves the comparability of results obtained with different reagents and instruments. in order to establish the methods for activated protein c resistance and the faktor v leiden-mutation we investigated 204 healthy individuals (128 women, 76 men, median age 37 ys) using the assay "contest ® apc-resistance" (chromogenix, sweden). the mean apc-ratio was 2.65, the cut-off level for low response to apc was calculated as 2.15, 46/128 women were oral-contraceptive users. they showed a slight decreased apc-ratio (mean 2.46). 24 of the 204 individuals (11,8%) had a poor response to apc (mean ratio 1.94) as a apc-resistance. for detecting the factor v gene mutation we a used non commercial own modified easy to handle pcr-based test. in 14 of the 204 individuals (6.9%) the factor v-leiden mutation could be found as heterozygotes. in this group, the apc-ratio was significant lower (mean 1.87) as in the group with apc-resistanee and without deteetiun of the factor v mutation (mean 2.04). nevertheless in one individual with beterozygote factor v-leiden, the apc-ratio was found in the normal range (2.24) , also in a few individuals with very iow apc-ratio the factor v mutation could not he detected. finally this data suggests a high prevalence of apc-resistance and factor v-leiden mutation in the population of the southwest of germany. resistance to activated protein c (apc) has been reported to interfere in clotting assays for protein c (pc) and protein s (ps) from various manufacturers, resulting in decreased values or even apparent deficiency (type ii') of pc. or is. in our laboratory, when using functional, avit-based assay kits for pc., is, and apc sensitivity from behring (marburg, germany) on an automatic turbidimctric coagulation analyzer (fibrintiraer a, behring), the interference in the is assay has been observed in a very pronounced manner soon after the introduction of apc sensitivity testing into the standard laboratory procedure for patients with thrumbophilia. yet we were not aware of an interferenca caused by apc resistance in the pc test. we therefore evaluated our results for pc eed ps with respnet to apc sensitivity status in a retrospective analysis. exclusion criteria were: oral anticoagulant therapy with vitamin k antagonists, impaired liver function, borderline result in the apc sensitivity test, known analytical intedercnc* other than apc resistance (e.g. elevated factor viii:c). resu/ts: apparant pc activity was slightly (12 %) lower and apparent ps activity was grossly (45 %) lower in ape resistent patients than in patients with normal apc sensitivity: there was no case with a presumed du~l defect of both at~ rnsistaac¢ and pc deficiency. in addition, none of the few patients diagnosed with pc deficiency before the introduction of the apc sensitivity assay turned out to ix: apc resistent. much more of a problem is the known sensitivity of the pc test to an elevated factor viii concentration which is quite a common finding among hospital patients but rarely known in an individual case. by aeccelerating the clotting process, elevated factor viii can cause apparent pc deficiency that can not be confirmed in a chromogunie pc assay. ,clearly, when combined with apc resistance, this problem is exspacted to be more severe. on the other hand, the comhination of both apc resistance and apparent is deficiency was found on a regular basis with many decreased and some bordeflinc values for is but only few in the lower normal range and virtually none in the upper non~al range. also, several #eats previously diagnosed with is deficiency turned out to be pc resistent, and the diagnosis had to be corrected, but dual defects cannot be ruled out. conclusion: for, the interpretation of pc and/or 1:"3 values from clotting assays the apc sensitivity status must be considered. when apc resistance and an apparent deficiency of pc or is is found, a type i deficiency may be confirmed or ruled out using antigen assays, but at present, there seems to be no reliable way other than genetic analysis to address the question of dual defects with presumed type ii deficiency, especially in the case of ps. therefore, there is a clear need for analytical improvements and the development of functional assays that are less sensitive to interference by apc resistance. the most common inherited risk factor for venous thrombosis is the resistance to activated protein c (apc). the main reason for this resistance is a point mutation in the gene of dotting factor v (f v). a nueleotide exchange leads to a different amino acid sequence in the protein and thus to the loss of one of the cleavage sides which is important for the inactivation of the procoagulatory form off v. the imbalance in the coagalation cascade leads to a strong risk for venous thrombosis, which is thought to be inoreaced by factor 7 to 10 for heterocygotes and about 80 for homocygote individuals. the prevalence in patients with a history of thrombosis is about 20%, among apcresistant patients as high as 80%. prevalence-off v ,leiden" is reported to be 2% of the normal population in leiden/netherlands and as high as 15% in a certain city in the south of sweden. due to the obviously different geographic distribution and the highly increased risk of venous thrombosis in carriers of the mutation investigated the distribution of this mutation in the normal population of different regions in germany. first attempts to assess the prevalence off v mutation by molecular methods (polymerase chain reaction followed by restriction fi'agment length polymorphism analysis) revealed that in the area of wiirzburg 7.7% of blood donors carry the mutation, which is significantly different (fischer t-test, level of significance 0.05%) from published data observed in freiburg (3.4%). homburg/saar and harmover revealed 5.8% and 8.5%, respectively, and were not significantly different from wfwzborg. further investigations of blood somples fi:om other regions should give an overview of the geographic distribution of this mutation in germany and might finally help to draw a map era regional risk of venous thrombosis. the regular apc resistance test is not applicable to plasma from orally anticoagulated (oac) or heparinized patients due to decreased levels of vitamin k-dependent clotting factors and to thrombin inhibition by antithrombin, respectively. the former limitation is overcome by prediluting samples 1-1-4 with fv-defident plasma. for this purpose a special quality of fv-deficient plasma has been prepared, which also allows analysis of heparinized plasmas. this fv-deficient plasma has now been evaluated with the coatest apc resistance assay. a complete ape ratio discrimination for the fv q506 mutation is obtained for plasmas from oac and heparin patients as well as from non-treated individuals. ratios above 2.2 are obtained in normal genotype plasmas, whereas heterozygotes for the mutation yield ratios in the range 1.6-2.0. the basal apt times for 15 oacplasmas with widely spread inr-values were reestablished into the normal range. .the stability of the reconstituted fv-deficient plasma was found to be' at least 3 hours at room tempetature, indicating its suitability for .routin.e u.~. f .u~ .e~mv~., the influence of preanalytical procedures is rmm~ single or double cenuifugation as well as freezing of samples do not alter the apc ratios to any significant extent. our results prove the usefulness of the modified coatest apc resistance kit method as a reliable, simple and rapid tool when screening for the presence of the fv q506 mutation. hereditary resistance against the anticoagulatory action of activated protein c (apc-resistance, apcr) which was first described by dahlb~ick et al. (pnas, 90:1004 , 1993 was identified as a possible new thrombophilic factor in a high percentage (17-60%) of young adults with thrombotic events. a single missense mutation (r506q) due to a g/a transition (g1691a) in exon 10 of the factor v gene (bertina et al., nature, 369:64, 1994) is tightly linked with apcr and is regarded as the causative molecular defect. identification of this mutation by pcr-based methods is easy to perform and avoids preanalytie and analytic errors in the eoagulometric assay for apcr. since the impact of this mutation in children with thromboembolic disease has not been determined to date, we initiated a multi-center prevalence study on two pediatric populations, with and without thromboembolie events. we compared 125 pediatric patients with thrombosis, divided into 3 different age groups (0 to <0,5 years; >0,5 to <10 years; >10 to <18 years) with a normal population of 159 children. the mutation g1691a was found with an unexpected high prevalenee of 12% in our normal controls. however, the prevalence was significantly higher in the age groups: 0 to< 0,5 years (25%) and >10 to <18 years (30%). in patients between >0,5 to <10 years the overall prevalence was similar to the control (13%). however in patients of this age with spontaneous thrombosis apcr was also a significant risk factor (29%). our results emphasize the impact of apcr for thrombogenesis in children. however, the significance is agedependent and does possibly reflect the different physiology of haemostasis in our three age groups. activated protein c (apc)-resistanec is a newly reeognised risk factor for thrombosis. in at least 90% of the cases it is caused by a single point mutation in the factor v gene (g->a at nucleotide 1691), which predicts replacement of arginin 506 with ghitamin. one of the apc cleavage sites in factor va is located c-terminal of arginin 506, and mutated factor va (factor v leiden) is resistant to apc-mediated inactivation. from epidemiologic studies it is known, that this abnormality can be found in about one third of patients with thrombosis. apc-resistance is a major basis for venous thromboembolism and is prevalent in about 5.10% of the general caucasian population. recurrent spontaneous abortion (rsa) affects 1-5% of couples and represents a major concern for reproductive medicine. in spite of extensive endocrine, genetic, serologic and anatomic evaluation some 30-40% of rsa women remain unexplained. a frequent morphologic finding in placentae of aborted pregnancies is an increase of fibrin deposition within the intervitlous space. because of these findings we studied the prevalence of apc-resistance in women with rsa (more than 3 miscarriages) of unknown origin. in 2 of 35 cases we found a pathologic apc-resistance, both patients had a history of recurrent thrombosis and were heterozygous for factor v leiden. the prevalance of apc-resistance is 5,7% and thus equals the prevalence in the general population. our data do not support the hypothesis that apc-resistanee is a risk factor for recurrent spontaneous abortion. h~matologisches zentrallabor der universit~t, inselspital, 3010 bern resistance to activated protein c (apc) due to the mutation 506 arg --~ (]in of factor v (factor v leiden mutation) is the most frequent hereditary thrombophilic defect known today, with a prevalence of 20 -50 % in patients with idiopathic venous thromboembolism and of about 3 -5 % in the general population. with an allele frequency of 2 % the expected number of homozygous individuals is about 4 in 10000. homozygous and heterozygons individuals differ considerably with respect to the relative risk of thrombosis (80 -fold versus 7 -fold) as well as to the age of the first thrombotic event (31 versus 44 years). deficiency of the vitamin k dependent protein s (p$), an important cofactor of apc, is another hereditary thrombophilia which is, however, much rarer than apc resistance with a prevalence of 5 to 8 % in patients with venous thromboembolism. factor v leiden mutation as well as ps deficiency are associated with impaired anticoagulatory activity of apc, which is most pronounced in case of the combination of the two defects. the combination of ps deficiency (with an assumed prevalence similar to that of pc deficiency) with heterozygous or homozygous apc resistance can be expected with a probability of 1: ~ 5000 or 1: ~ 500000, respectively. it is well known that ps levels decrease towards the low normal or even subnormal range during pregnancy. moreovar, there is increasing evidence that the sensitivity of plasma to the antieoagulatory effect of apc decreases during pregnancy resulting in an acquired apc resistance. these pregnancy associated effects art obviously much more relevant in case of preexisting ps deficiency or apc resistance and should contribute to the elevated thrombotic risk during pregnancy in a subject with either of the two defects, and even more so for a woman who suffers from both defects. we describe a young woman with a combination of homozygens apc resistance ( apc ratio 1.10, normal range: 2.02 -3.73), pronounced ps deficiency (free ps 0.ll u/i, total ps 0.30 u/i, normal range: 0.55 -1.20 u/1 and 0.65 -lag u/i, respectively) and, moreover, impaired fibrinolysis (no change of euglobulin -lysis time after 10 rain venous occlusion) who developed deep vein thrombosis after cesarean section in her first pregnancy. examination of her familiy showed heterozygous apc resistance in her asymptomatie father (apc -ratio 1.99) , combination of heterozygous apc resistance (apc -ratio 1.60) and ps deficiency (free ps 0.30 u/i, total ps 0.58 u/i) in her nsymptomatic mother and no defect in her sister. considering the fact that the mother was still thrombosis free at the age of 49 one may assume that the thrombosis risk in the proposita was mainly influenced by the homozygnsity for apc resistance. s. ehrenforth, m. adam, b. zwinge, i. scharrer university hospital, dept. of angiology, frankfurt a.m., germany introduction: apc resistance has been shown to be the most commonly inherited defect which constitutes a risk factor for venous thrombosis (vt). however, most of the present epidemiological studies concerning apc-r prevalence in thrombophilia were derived from results of tests conducted onplasmas collected under various conditions. this may influence the great differences reported on the prevalence of apc-r among these patients. for example, it has been shown that freezing of plasma specimens prior to analysis of apc-r causes a significant decrease in the assay results.the aim of our study was to evaluate the influence of eentrifugation conditions on the results obtained with the chromogenic apc-r assay. patients and methods: blood was collected from 31 patients (t9 women, 12 men; fv gent.type: 13 r/r 506, 14 r/q 506, 4 q/q 506) through veinpuncture into trisodmm ciwat (1:9). platelet-rieh and platelet-poor plasma was obtained by immediately centrifugation at 4"c for 10, 20, 30, 40, 50, 60 rain at 1000, 2000, 3000, 4000, 5000 and 6000 rpm. additional, pnp obtained from 14 healthy individuals (7 male, 7 female without hormonal trealraent) was prepared equally. apc-response was determined within one hour after centrifugation using the coatest apc resistance kit from chromogenix. results: for both, pnp and sin/gle plasma samples, we observed continuous higher af'c-ratios after increasing cenwifugation intensity. for example, an increase from 1000 to 6000 rpm resulted m an increased apcratio from 2.7 to 3.26 (20 min), from 2.88 to 3.326 (60 rain) respectively. even though less distinctive, similar results were observed concerning the duration ol eentrifugation: when the duration was increased from 10 to 60 minutes we observed a continuous increase in apc-ratio, for example from 2.74 to 3.12 when using 2000 rpm and from 3.09 to 3.36 when using 4000 rpm. the decrease of the ratio after low eentrifugation is the eonse-9nence of the shortening of affft in the presence of apc, without a signhcant influence of basal al:rl~ without apc. conclusion: our results demonstrate that centrifugation conditions are important to consider for the interpretation of apc-r results. supporting our observations, recent studies from sidelmann et al. have shown that an increase in plasma platelet concentration, low eentrifugation respectively, causes a signficant decrease in the apc-response. however, so far the mechanism responsible for the significant effect of both on apc-r assay results is unknown. although technically simple, the biochemical cemplexitiy inherent in the chromogenic apc-r assay necessitates a standardized plasma handling procedure to secure a reproducible determination of apc-il compapjson of different assays for determination of apc-resistance with the geno'fyping factor v (arg -> glu) g. siegert*, s. gehrisch*, e. runge**. r. naumann**, r. kn6fler*** *institute of clinical chemistry, **clinic of internal medicine, *** childrens hospital resistance to apc diagnosed on the basis of prolongated clotting time in the aptt assay" is now considered a major cause of thrombophilia. in the majority apc resistance is ~ted with a point mutation in factor v molecule (arg506glu), but both are not synonym. protongated baseline aptt is a limitation of the assay. following the determination is not possible in risk groups of patients (factor)ill deficiency, lupus anticoagulan0 and in patients under anticoagulant therapy. in these causes a dilution of plasma in factor v deficient plasma is recommended. the immunochrom assay is based on the inactivation of factor villa by apc. the aim of the study was to compare different functional apc response assays with the result of the dna analysis. apc response was tested in 100 healthy probands, 81 thro~ patients and 16 family members using the lmmtmochrem assay, the contest (chromogeaix) and the contest with 1+ 4 dilution of the plasma in native factor v deficient plasma (immune). the dna analysis was performed as described by bertina. one patiem was homozygoas for factor v mutstion~ a hetemzygous result was obtained in 4 members of the control group, in 28 patients and in 6 family members. in all cases with factor v mutation the ratio of the immunochrom assay was lower than the laboratory own value, independent on anticoagulant therapy. pathological ratios in this assay were also obtained in one member of a family" with high thrombotic incidence (dna arg/arg) and in 17 patients under anticoagulant therapy ( two of this patients are one cloned twins). in the contest a ai~ response was diagnosed in all cases with factor v mutation without anticoagulant therapy and in 40 % of heterozygous patients under anticoaglant therapy. results of the test using the dilution in factor v deficient plasma showed a good agreement vath the results of the dna analysis but the method is obviously only sensitive for the factor v mutation. the reason for pathogical ratios in the lrnmunechrem assay in wildtype patients is unclear. the majority of this patients is treated with anticoagulants, a comparison with the contest is not possible. interestingly in one patient under heparin and low ratio in the immunochrom assay' after reduction of hepann the ratio of the coatest was also low. it seems necessary to investigate in which distance to the thrombotic events the apc resistance should be tested. following pathological ratios in ftmctional apc assays must be discussed: high levels of factor viii and or v wiuebrand antigen (acute phase reactien), other mutations in factor v and viii. the factor v dilution assay should be replaced by the dna analysis. due to their differing compositions, the "sensitivities" of various aptf reagealts differ not only with respect to factor depletions, heparin and fibrin-fibrinogen degradation products, but also with regard to pathological inhibitors. for lupus anticoagulants this means that "lupus-sensitive" reagents can be delineated from "lupus-insensitive" reagents. with a "lupus-insensitive" ai~ reagent there is no or only slight prolongation of the aptt in the plasma under investigation, whereas with a "lupus-sensitive" reagent marked prolongation is observed. for the meaninof~l use of aptr reagents it is necessary to know the extent to which they are influenced by lupus anticoagulants. the following 14 apti' reagents were tested: • ptt-reageaz, p'rta, ptta liquid, ptt-la, pti'-lt (boehringer/stago) • pathromtin, pathromfin sl, necthroratin (behring) • platelin s, piatehn excel ls (organon tekinka) • actin-fs, actin-fsl (dade) • aptt silica lye, aptt silica liquid (instntmentation laboratory) the material for investigation consisted of 20 plasmas from patients with lupus anticoagulants. a confirmatory test (lupus anticoagulant test, immune) was positive for all of the patients. measurements were made using the sta coagulation analyser (boehringer/stago). it can be seen from the results that in some instances very different prolongations were obtained in identical plasmas by using differing aptt reagents. low susceptibility to lupus anticoagulants was shown by actirt fs (dade), ptt-reagenz (bcehrlnger) and neothromfin (behring). high susceptibility was shown by platetin excel ls (organon teknika), ptt-la and pti'-lt (boehringer/stago). lupus anticoaguhant screening with the aptt reaction is promising when two aptr reagents differing as greatly as possible in their lupus anticoagulant sensitivity are used. the resistance to the anticoagulant response of activated protein c (apc) is a major cause of venous thrombosis. apcresistance is due to a single mutation in factor v gene, which predicts replacement of arg 560 in the apc-cleavage site with gln (factor v leiden mutation). in contrast to other known genetic risk factors for thrombosis, this factor v 1691 g-a mutation has a high prevalence in the common population of western europe (average 4-5 %). we have determined the prevalence of the factor v 1691 g-a mutation in a population of 700 probands of north-eastern part of germany. the mutation was found in 7 %. (heterozygoty were found in 48 subjects 1 person was homozygous.) the results are compared with our studies of populations from argentine and poland. me analysed the factor v 1691 g-a mutation in 71 patients with thrombosis from germany and hungary. this mutation has been found in about 60 % of these patients. in contrast, the frequency of this mutation was strongly reduced in a group of 47 patients with thrombosis and pulmonary embolism of argentine (3 heterozygotes in 47 patients; 6 %). the results of these different populations will be described and discussed. past medical history: venous thromboembolic events (re) at 18, 19 and 2i years; intermittent oral anticoagulation (oac) without te's. diagnosis of autoimmune disorder with elevated antinuelear-antibody-fiters and positive lupus-anticoagulant test. no other relevant illnesses; family history uneventful. two weeks prior to the referral to us -acute febrile illness with nausea, diarrhea, abdominal pain; hospitalisation, treatment with iv antibiotics and anticoagulation with fraetionated heparin; development of extensive deep vein thrombosis (dvt) of the right leg; initiation of full-dose unfractionated heparin; decline of platelet count from 121 to a nadir of 21 g/l; referral to our department. on admission an extensive coagulation screen yielded the following results (n/normal, t/elevated, i/reduced, +/positive, -/negative): pt t, aptt t, tr n, factor ii, v, viii n, factor vii, ix, xi, xii /,, fibrinogan t, atiii n, protein c, s *, activated protein c sensitivity ratio 1.92 ($), fv-leidenmutation pcr -, fibrinolytic system n, tat t, ft÷2 t, lupus anticoagulant +, heparin induced platelet antibodies +; no diagnosis of a specific autoimmuna disorder could be made. an immunosuppressive therapy with corticosteroids and anticoagulation with recombinant hirudin were init'~at~; no p~ogr~zsion of the dvt oeeured and normalisation of the platelet count was observed. during follow-up under oac ) and low-dose corticosteroids, the patient was well, the pathologic coagulat;.on results, including lupusanticoagulant and activated protein c resistance, have returned to normal; no further te's have been observed. in summary we present a case of a complex coagulation disorder as part of an autoimmune process, resulting in a clinically manifest prothrombotie dysbalance including lupus anticoagulant, acquired resistance against activated protein c and heparin induced thrombocytopenia (type ii), entering complete remission under combined immunosuppressive and anticoagulant therapy. in the last 30 years, a vast number of simplified analytical procedures have been developed for the diagnosis of haemostatic disorders. today the detection method have evolved from the mechanical hooking method or ball coagulometry to optical systems, which additionally can utilise chromogenic substrates or immunological methods. in these systems the clotting time is derived from algorithms (e.g. threshold or maximum of the first or second integral). we studied 50 healthy subjects, aged 21 to 65 years and 118 patients, aged 9 to 93 years using a new aptt reagent (pathromtin $l). the results were compared with those obtained with a routinely used reagent (pathromtin). the reference range, factor-, heparin-and lupus anticoagulant sensitivity were determined. analysis was performed using the behring fibrintimer a (bfa) with optomechanical clot detection, the behring coagulation timer (bct) with op-"dcal clot detection by threshold and the dw test and dw confirm for lupus anticoagulant diagnostic. our results showed that the new pathromtin sl reagent met the demands for a higher factor and lupus anticoagulant sensitivity. it is highly suitable for monitoring heparin therapy and gave comparable results with the optical and the optomechanical analyser systems, hence reagent c~n be used for both systems. restenosis following percutaneous transluminal angioplasty (pta) continous to be a major clinical problem. neoinfimal hyperplasia, being the major undedying cause, can not be sufficiently avoided. vadous plasmatic coagulation and fibrinolytic factors, have been associated with artedal restenosis. anticardiolipin antibodies (act_) have been established as dsk factors for venous or arterial thrombosis. methods: in a cohort of 75 patients (53 men and 22 women, age 68±13 years) undergoing pta of a peripheral artery we prospectively evaluated whether acl could influence 6 months restenosis rate. patients were clinically examined before, 3 and 6 months after pta. noninvasive grading of artedal stenosis was done by duplex scanning of jet peak velocities. restenosis was arbitrarily defined as more than 50% occlusion of the lumen at the site of dilatation 6 months after successful intervention. laboratory investigation at the same time included acl and other known atherosklerosis risk markers, such as fibdnogen (fbg), yon willebrand factor (vwf), homocystein (hcy), c-reactive protein (crp). thrombin generation markers, such as thrombin-antithrombin iii complexes and prothrombin fragments 1+2, as well as thrombomodulin (fm) as an endothelial activation marker, were also measured. results: 31/75 (41.3%) patients were considered to have developed restenosis after 6 months. 9/75 (12%) patients were found to have positive igg-(19-35 gpl) and/or igm-acl (14-103 mpl) at all three measurements. 1/75 was negative before but seroconverted (igm) 3 months after pta. 2/10 (20%) acl-positive and 29165 (44.6%) acl-negative developed restenesis at 6 months (chi-square p-value=0.14). all above mentioned coagulation parameters did not differ between acl-positive and -negative patients, measured before or 6 months after pta. some of them are shown below (values before pta): fbg ( basilar artery stenosis is a rare event in young children. risk factors are head or neck trauma with consecutive dissection of the vertebral artery, cardiac diseases or hypercoagulability. elevated lipoprotein (a) (lp(a)) serum levels in adults can mediate atherosclerosis. in addition, lp(a) might interfere with fibrinolysis. here we report on a 3 year old boy , who presented with acute brain stem symptoms. history revealed neither trauma nor infectious disease. conventional and mr angiography showed stenosis of basilar artery without ischemic lesions. laboratory findings were normal in routine blood and csf tests. global coagulation parameters as well as procoagulant and anticoagulant factors were normal. cardiac and autoimmune disease could be ruled out. lp(a) serum levels were significantly elevated to 104 mg/dl (normal range <30 mg/dl). analysis of other family members revealed a hereditary hyperlipoproteinemia (a) which might explain family history of an increased incidence of myocardial infarction and cve in elderly family members. clinically the patient recovered completely from brain stem symptoms after heparinization and subsequent oral anticoagulation with phenprocoumon. however, radiological signs of basilar artery stenosis were progredient. in a recently developed specific test, an elevated anti-phosphatidylserin antibody titer was detected one year after primary diagnosis. in conclusion, this is the first report on a child with stenosis of the basilar artery and elevated levels of lp (a). it is unclear, whether apa contributed to the onset of basilar artery stenosis or developed secondary due to endothelial defects after thrombosis and anticoagulation. apa, however, might increase the risk of further thrombotic events in this patient. in 110 patients with thrombotic events respectively patients with systemic lupus erythematodes antioardiolipin antibodies (aca) aund lupus anticoagulant (la) were ~ea~ured. for aca detecting we use the assays from elias for igg-and ig~}-antibodies. we use as sensitive methods for detecting la in our laboratory the testkits from diagnostlca stago (staclot la with hexagonal array of phospholipids, ,ptt-la a very sensitive pttmethod and staclot p~p-a platelet neutralization procedure) and the ptt from organon teknik~ (platelin excel ls with two incubation times, 1 and 10 minutes). i"~e results of this tests were compared with three new or~e on german market: specktin apot (aktlvated plasma clotting time), specktin aptt (aptt wlth purified soy extract) and 8pecktin la (phospholipid preparation in concentrations between 10 and 500 ~g/ml); all wak chemie. traditional aptt reagents were developed for the sensitive detection of factor vib an ix as a cause of hemorhage. high sensitivity against lupus anticoagulants, which also prolong aptt, was not required for this purpose, with increasing recognition of the importance of antiphospholipid antibodies as a risk factor for thrombembolism, more sensitive reagents were designed, which now reliable detect this condition. using such reagents as a screening test in a general hospital makes it necessary to distinguish both conditions quickly. we here report an algorhythm, by which we use an inhibitor (lupus anticoagulant) sensitive (sta aptt, boehringer) and an inhibitor insensitive reagent (actin fs, dade) to distinguish anticoagulants and factor deficiencies as a cause of prolonged aptt. citrate plasma from 33 patients with various diseases showed an unexpectedly abnormal inhibitor sensitive aptt (>40s). 13 plasmas with factor deficiencies remained abnormal with the insensitive aptt reagent. a regular correction of their defect occured on mixing with normal plasma. by measurement of single coagulation factors five patients with contact factor xii deficiency were found. this condition is associated with thrombosis and very rarly with bleeding. three patients with factor xi deficiency and two patient with factor ix deficiency were also identified. antiplatelets, of any kind, permits a secondary prevention of myocard ischemic lesions. there is no general consensus regarding secondary prevention of cerebral ischemic lesions. aspirin remains the most common substance, ticlopidlne also brings about prevention, but with important secondary effects. european stroke prevention study i has demonstrated that the combination of antiplatelets, in particular aspirin/dipyridamole (persantln), is also very active. to collect more information, esps 2 was organized and 6602 patients receiving either a placebo,either 50 mg aspirin,either 400 mg sustained release form of dipyridamole (persantin (r)), or the combination aspirin/dipyrldamole, were recruited. it ended march 31st 1995 with the following conclusions: i-aspirin, 50 mg a day, brings about a significant secondary reduction of stroke (18.z %), after a two year follow-up. notwithstanding the low dose of aspirin, haemorrhages remain important. 2-dipyridamole, at 400 mg a day, brings about a significant reduction of stroke (i6.3~), similar to that of aspirin. one could thus substitute 50 mg aspirin by 400 mg dipyridamole. 3-the combination of 50 mg aspirin and 400 mg dipyridamole brings about a significantly greater reduction of stroke (37.0 ~). esps 2 revealed that a low dosage of aspirin is active, that dipyridamole alone is also active, but that the combination of both gives far better results. the study of the primary end-points,the study of the survival curves, the factorial statistical analysis and the pairwise comparison analysis, led to these conclusions. the conclusions drawn from esp£ 2 underline that the combination aspirin/dipyridamole is a privileged choice for cerebral ischemia, the state of activation of circulating platelets in acute cerebral ischemia is controversial. activation of platelets on single cell level can be assessed by determining the shape change or the expression of antigens such as p-selectin (cd62). shape change is an early and rapidly reversible event in platelet activation whereas p-selectin is irreversibly expressed on the platelet surface upon stimulation. methods: we investigated 20 untreated patients within one day after cerebral ischemia, 20 patients months after stroke treated with warfarin, and 21 age and sex matched control subjects without vascular risk factors. venous blood was collected into a fixation solution blocking the metabolic processes in platelets within milliseconds. we determined the fraction of resting discoid platelets by phase contrast microscopy. the expression of p-selectin was measured by flowcytometry. results: the fraction of platelets expressing p-selectin was higher in patients with acute cerebral ischemia (7.8_+4.7%) than in control subjects (1.9_+1.1%; p<0.001, u-test). patients with stroke (n=15, 7.8+4.5%) and patients with transient ischemic attack (tia; n=5, 7.6-+6.1%) had similar values. patients months after stroke still had higher values (4.3+9,3 %, p<0.05) than control subjects. the rate of discoid platelets was not different between patients with acute ischemia (n=17, 85.6-+8.8 %), patients months after stroke (n=19, 85.7-+5.1%) and control subjects (n=21, 86.7_+5.8 %). platelet count was not significantly different between groups. conclusion: the elevated proportion of platelets expressing pselectin indicates strong platelet activation in acute cerebral ischemia and in a majority of patients months after stroke. assessment of pselectin revealed a higher sensitivity for platelet activation after stroke or tia than analysing the reversible shape change. further studies have to clarify if monitoring of platelet activation by flowcytometry is helpful as a prognostic tool and to evaluate therapeutic strategies after stroke. vascular smooth muscle cell (smc) proliferation and migration into neointima are the hallmarks of atherogenesis. the complexity of these processes and their concerted action and interaction of molecules are yet to be fully elucidated, one crucial molecule seems to be the urokinase-type plasminogen activator receptor (upar) recently also assigned as cd87 antigen, upar serves a dual function: (1) it directs upa proteolytic activity to a special location on the cell surface and (2) induces cellular signals leading to various phenotypic changes. we have investigated the signal-transducing capacity of upar in human smcs and provide here a molecular explanation for uparrelated cellular events. activation of these cells with upa (even with inactivated catalytic center) results in the induction of tyrosine phosphorylation, suggesting modulation of upar-associated protein tyrosine kinases (ptks) upon ligand binding. we obtained patterns of tyrosine-phosphorylated proteins with molecular masses of ~ 55-65 and 35-40 kd. using antibodies against different types of ptks as well as immunoprecipitation-and immunoblotting techniques the ptks involved in the upar-signalling complex were identified to be members of the src-ptk family. the cotocalization of upar and ptks at the cell surface of smcs was further confirmed by confocal microscopy studies. we conclude that the upar-ptk complex is most likely involved in this signal transduction pathway that provides the coordinated action of extracellular proteolysis, adhesion, and cell activation, which is required for cell migration. this mechanism may be crucial for the progression of atherosclerotic plaques. activation markers of haemostasis have been found elevated in relation to diabetic vascular lesions. simultaneous pancreas-and kidney transplantation (pkt) in type i diabetes has been shown to improve diabetic complications and long term survival. we measured haemostatic vascular risk factors and activation markers in plasma of 18 patients after successful pki', 17 patients after pkt and rejection of the pancreas graft and 5 patients after pkt and rejection of the renal graft. blood samples were taken during routine ambulatory visits, patients were free of any ongoing acute disorder or transplant rejection and under continuous immunosuppressive medication. despite individually adjusted insulin therapy hba1 plasma levels increased after pancreas rejection (5,37 vs 7,i2, p<0.001). platelet counts and plasma levels of fibrinogen, f1+2 fragment, tat-, app-complex and-fibrin monomer were found significantly elevated as compared to diabetic controls but not significantly different with respect to complete or partial successful pkt. one major reason of the increased activation state of haemostasis may be cyclosporin treatment given to all patients, t-pa and pal i plasma levels were within the normal range and significantly correlated to plasma triglyceddes (r.0.049; p<0.005). d-dimer plasma levels were significantly lowered after pancreas rejection (772(375) vs 324(232) nglml; mean(sem) p<0.005), which might reflect impaired fibrin degradation related to increased glycosylation of fibrinolytic factors. in conclusion, despite the marked improvement of glucose and lipid metabolism, plasma markers of activation of coagulation and flbrinolysis are not decreased to normal after simultaneous pancreas and kidney transplantation. according to the investigations of fowler et al. and pepe et al. the probability of an ards occurring with one risk factor is 5-8%, and in the presence of several risk factors, 25%. goris et al. and johnson et al. determined the level of severity with the aid of a fixed scale: the injury severity score. all these investigations are however not to be interpreted as typical following coronary surgery. these investigations demonstrated that the kallikrein and factor xii systems are of great importance as intraoperative risk factors. here the factor xii system plays a major role with direct or indirect activation of the kauikrein-kinin system with the splitting products alpha-factor xiia and bfactor xha respectively. all ards scores take the pmn-elastase into account. if the pmn-elastase values (1000 pg/l) are constantly high postoperatively then lung complications are to be expected. patients developing an ards displayed significantly lower alpha2-macroglobulin values. patients who developed a highly significantly raised kallikrein-like acdvity (>60 u/i) after the beginning of bypass and showed constantly high values during ecc are difficult to keep under control due to the blood pressure behaviour. the platelet pal also shows a significant rise and intraoperatively runs analogous to platelet factor 4, only antiparailel, since it attacks the endothelium. we were able to show that pai-1 is suitable as an indirect marker for a possibly developing restenosis. 85% of the patients investigated with lowered pai-1 values in the postoperative phase did not develop a restenosis. however, with patients showing significantly rising pa[-1 values from the 1 st. to 3rd. postoperative day 50% of all the cases had a restenosis. a further risk factor in this respect are significantly raised fibrinogen levels which lay over 800% at the end of surgery. if these fibrinogen values do not fall from the 1st. postoperative day onwards a raised risk of thrombosis must be reckoned with in the absence of therapeutic intervention. the following parameters represented haemostaseological risk parameters with significant behaviour within the framework of this study: 1) regards the blood pressure behaviour, the kallikrein-like activity (>60 u/i); 2) with regards to the lung complications, aipha2-macroglobulin and pmn-elastase (>900 g/i); 3) and final/y as a possible marker for a developing restenosis pai-1 and fibrinogen (>800%). resulting from numerous clinical studies homocysteinemia is found to be an almost independent risk factor of atherosclerosis including thrombotic complications as well as of venous thromboembolism. experimental investigations on the underlying mechanisms suggest endothelial cell damage accompartied by the development of an atherogenic and thrombogenic potential, increased platelet reactivity, oxidative modification of ldl, and enhanced affinity of lp(a) for fibrin. to our knowledge no results are published on the influence of homoeysteine on leukoeytes although these cells are deeply involved in pathological events within the vasculature. therefore, as a first approach different functional parameters of human polymorphonuclear leukozytes (pmnl) were followed under incubation with 60, 300, and 600 i.tm (final concentration) dl-homocysteine (hc) in isolated fractions or whole blood, respectively: l) spontaneous mobility of pmnl, measured as migration distance into micropore filters in a modified boyden-chamber, is found to be significantly enhanced by the two smaller hc concentrations. 2) chemotaxis induced by 0.1 i.tm formylmethionylleueylphenylaianine (tmlp) shows no significant differences. 3)monitoring of chemiluminescence signals (autolumat lb953, berthold) is complicated as hc influences the luminol-mediated indicator reaction. adjusting appropriate conditions the following results are obtained: spontaneous chemiluminescence and that induced by zymosane, tmlp, and the ca2+-ionophore a23187 are entranced by the two higher hc concentrations. there are, however, differences between the blood donors as a minority does not respond to hc in repeated measurements. with phorbol 12-myristate 13 acetate the signal is diminished by hc in all cases and with all concentrations. 4) phagoeytosis induced by zymosane (microscopic evaluation) as well as by opsonized e. coil (cytoflowmetric evaluation) is significantly increased by the two higher hc concentrations. conclusion: the activation of human pmnl is enhanced with respect to the majority of investigated stimuli by hc in concentrations reached under pathophysiological condititions. the effect of pysical exercise on hemostatic parameters was studied in 12 patients (male, mean age: 55 [range 36-68] yrs) with angiographically documented coronary artery disease (cad) and in 11 controls (male, 53 [43-62] yrs) both participating in an 1 hour group exercise session for cardiac rehabilitation. in each group relevant arteriosclerotic lesions in carotid, abdominal and leg arteries were excluded by doppler ultrasound examinations. patients were all under 6-blocking agents and aspirin. plasma levels of prothrombin fragment 1+2 (ptfi+2) and fibrinopeptide a (fpa) reflecting formation of thrombin and fibrin, respectively, were measured at rest and immediately after 1 hour of exercise consisting of jogging, light gymnastics and ball games. training intensity in both groups was comparable as indicated by the mean heart rate during exercise corresponding in patients to 80+6% (mean-+sd) and in controls to 79-+5% of the maximal heart rate previously determined on a bicycle ergometer. baseline values for ptf1 +2 were significantly lower in oatients (0.67-+0.15 nmol/i; mean-+sd) than in controls (1.01-+0.21; p<0.001 i. after exercise we found an increase of ptf1 +2 in controls to 1.14-+0.24 nmol/i (p<0.001) while in patients ptfi+2 remained unchanged (0.67-+ 0.14 after). accordingly, exercise induced r se of fpa was more pronounced in controls (from 0.62-+0.24 to 1.60-+0.62ng/mt; p<0.001) than in patients (from 0.63-+0.26 to 1.20+0.46ng/ml; p<0.00t). we conclude that in terms of thrombin and fibrin generation exercise training does not exert detrimental effects on hemostasis in patients with cad. lower baseline values and lack of exercise induced increases of ptf1 +2 in patients with cad might be attributed to medication with aspirin and/or b-blocking agents. periodontitis marginalis (pm) is an inflammatory oral disease that is caused by gram-negative bacteria and that has a high incidence in the second half of the life. clinical signs of pm are gingival bleeding, periodontal pockets, alveolar bone destruction and loss of teeth. recent epidemiologlcal studies have provided some evidence for an association between pm and atherosclerosis. in the present paper we will summarise some of the results that we have obtained in studies on patients with pm as well as on patients with hypercholesterolaemia (hc) and atherosclerosis. pm was frequently found to be associated with hc (90 % in rapidly progressive pm) and increased reactivity of peripheral blood neutrophils and platelets (e.g. generation of oxygen radicals and paf-induced aggregation). patients with hc and atherosclerosis had a higher frequency of severe pm when compared with data on the community periodontal health. the severity of pm was higher in patients with plasma cholesterol levels _> 6.5 mm when compared to those with plasma cholesterol < 6.5 mm. in patients with coronary atherosclerosis the severity of pm was significantly correlated with plasma cholesterol level, systolic blood pressure and the number of diseased coronary arteries. these results provide further evidence for an association between pm, hc and atherosclerosis. it can be speculated that hc is not only a risk factor for atheroscterosis but also a risk factor for pm and acts by increasing the reactivity of neutrophils and platelets. on the other hand, pm as a mild chronic inflammation could promote the development of atherosclerosis due to effects of endotoxins on vessel wall, blood cells and haemostatic factors. it has been also speculated that phagocyting leukocytes in the inflamed periodontal tissues could contribute to oxidative modification of ldl. so far, there is no evidence that atherosclerosis may contribute to the pathogenesis of pro protein z (pz) is a vitamin k dependant plasma protein synthesized in the liver. it promotes the association of thrombin with phosphorlipid surfaces. recently it has been shown that a deficiency of pz may lead to a bleeding tendency. in patients undergoing chronic hemodialysis, disorders of hemostasis are common. to examine if plasma levels of pz are altered in patients with end stage renal disease we determined pz in plasma of patients at the beginning of hemodialysis treatment. the results were compared with a group of 50 healthy controls. the difference of pz levels in plasma of patients with end stage renal disease with the control group was not significant. control group was 2900 + 487 ng/ml and in patient group was 3133 + -1394 ng/ml. one patient with marked bleeding tendency after hemodialysis pz was 1387 ng/ml. we concluded that patients with bleeding disorders pz determination may be helpful. the normal range of actin fs was reinvestigated in a multicentric approach. a protocol was developed which requests from each center to assess the aptt with one common and one variable lot of actin fs in 50 samples of suspected normals. inclusion and exclusion criteria based upon the results of clotting assays, liver enzymes and clinical data were defined. results: a total of 1056 results was obtained. the majority of centers in this study used the electra 1000 or 1600 c (mla). results for the electra group (n = 6) showed a precision for the common lot of actin fs with a common lot of a three level control from 1.5 % (level 1) to 1.1% (level 3) with an excellent accuracy between the 6 centers. clotting times with the variable lots of actin fs were very similar. the results from normals, however, showed a somewhat higher dispersion using the common lot of actin fs. 4 of 6 centers had almost identical mean values (range 26.6 to 27.2 sue) whereas one reported shorter and one longer clotting times (25.2 and 29.7 sec). results with the variable lots gave almost identical results as the common one. a total of 556 results of all lots gave a normal range of 23.5 to 31.7 (5 -95 % percentiles) on electra. mean values on acl (n = 200) were 26.3, on bct, 27.65 sec, on amga coagulometric, 29.4 sec, on amga turbidimetric, 26.6 sec (n = 100 each). all centers used sarstedt monovettes with 3.2 sodium citrate. discussion: the results of this study demonstrate the lot to lot consistency of all lots of reagents included in this study since the common and variable lots showed very consistent results. interestingly in the large group of electra users the normal ranges showed some differences, though the controls in all centers were almost identical. this confirms the recommendation that a normal range as stated from the manufacturer should be used for orientation only and that each laboratory should assess its own range. direct acting anticoagulant agents such as hirudin (r-h), argatroban (arg), efegatran (efe) and peg-hirudin (ph), represent specific and potent inhibitors of thrombin. blood samples collected in r-h (10 ~g/ml), arg (50 ~tg/ml), efe (25 ~tg/ml) and ph (10 ~tg/ml) do not clot for extended periods (>24 hours), thus allowing for the collection of plasma for analytical purposes. unlike heparin, these agents do not require any plasma cofactor for their anticoagulant effect. in contrast to citrate, oxalate, edta and heparin, these antithrombin agents do not alter the electrolyte or protein composition of blood. thus, blood collected in these agents may provide a physiologically intact (native) sample for clinical laboratory profiling. we have used all of these agents to prepare whole blood and plasma samples for various diuical laboratory measuroments. plasma samples collected with these agents are obviously not suitable for global clotting tests (pt, aptt, thrombin time, fibrinogen); however, these agents are optimal anticoagulants for the collection of samples for the molecular markers of hemostatie activation, such as fibrinogen/fibrin related degradation products, prothrombin fragment, protease cleavage products, tfpi, tnf and other protein mediators. electrolytes, blood gases, enzymes and protein profiling can also be satisfactorily measured on blood samples collected with these agents. antithrombin anticoagelatad blood used fur hematologic analysis showed equivalent blood count and differential results as that obtained with edta blood. unlike other anticoagulants, these agents do not interfere in the cell staining process. washed blood cells can also be prepared using antithrombin aents supplemented buffers for morphologic and fuuctional studies. thrombin inhibitors such as hirudin have also been used for flow cytometry and image analysis of blood cells and tissue exudates. our observations suggest that these anticoagulants can be used as suitable anticoagulants for clinical laboratory blood sampling. these agents can also be used as a flush anticoagnlant fur most automated instruments as these exhibit superior anticoagulant properties to heparin. furthermore, the hematologic parameters obtained in antithrombin anticeagnlated blood may be physiologically more relevant than those determined on blood collected in edta, citrate or heparin. antithrombin ul determination is one of the most popular method for in vitro diagnostic of number of different disorders. human fhrombin a~nity purified on heparin-rnodified silica-based sorbents was used for level of antithrombine lu determination by abilgaard method in blood of 40 patients with pregnancy pathology, acute leukemia, thrombocytopenia and anemia. it was founded, that antithrombin level is decreased to 50-60% of normal values in case of pregnancy pathology, to <50% -in case of acute leukemia and thrombocytopenia, to s0% -in case of anemia. obtained results show the strong relationships between named disorders and patient antifhrombin iii level. therefore anfifhrombine iii estimation may be used as simple and quick method for preliminary diagnosis of above named disorders. bm coasys 110 is a complete automated analyzer system for coagulation tests. it is well suited for routine coagulation testing in random access in a medium throughput laboratory environment. analytical performance and practicability were tested by a common evaluation program in five hospital laboratories. within run and day to day cv's were below 5 % in different samples (controls, patients) . comparison in different therapeutic ranges confirms the declaration of the isi-value for calculating inr-values. normal values for coagulation tests with results in pdmary units were checked in 390 samples and confirmed. due to the optical measuring principle of the bm coasys 110 there was a little tendency to shorter times with the thrombin reagent. in conclusion the performance of coagulation tests with the bm coasys 110 was rated as well or better compared to existing systems in the laboratories with advantages due to short timed familiarizing and easy handling. flexibility and stability of the system permit optimal integration and innovation into the w0rkflow of the routine laboratory. the purified thrombin and antithrombin iii (at iii) have a great interest in the clinical diagnostic and treatment practice, so their isolation methods are very important. molecules of these proteins have some fragments replying for interaction with native glycosaminoglycan, heparin. this interaction is used for isolation and purification of thrombin and at ul from native materials, blood plasma or its fractional products. we have done comparative studying these proteins purification on heparin sorbenfs, which contain heparin immobilized on sificagel, modified by glycidooxipropyl, gamma-aminopropyl and tosyl chloride groups, or on cellulose: heparin-epoxy-silica (1), heparin-gammapropyl-silica (2), heparjn-tozylsilica (3), and heparin-cellulose (4). we founded that thrombin binds with all sorbents, while at iii doesn't binds with sorbents 2 and 3. there wasn't any difference between silica and cellulose sorbents in thrombin desorbfion by t m naci. at iii binds more stronger with heparin-ceuulose t[~,~n with silica sorbents but specific activity and purity degree were approximately the same on both kinds of sorbents. thrombin specific activity and purity degree were approximately twice higher on sorbents 2 and 3 in comparison with sorbents ! and 4 (2250-3000 nih units/mg versus 1260-t350 nih unlts/mg). therefore, sorbents 2 and 3 can be used for isolation and purification of thrombin and sorbents t and 4 can be used for isolation and puriiication of at ii1. we used these sorbents for large scale purification of named proteins. purified thrombin was used for production of diagnostic kits for anfithrombine iii, fibrinogen, fibrin/fibrinogen degradation products and thrombin time determination. after an aerobic or anaerobic physical exercise various alterations of the hemostatic system were detected. numerous investigations of the hemostatic system exist of running and of bicycle ergometer exercise but not of swimming. young volunteers (n=54; median age 25 years) were investigeted~ there was an aerobic exercise (achieved heart rate 130 --10/min, lactate < 4 mmol; n= 27) and an anaerobic exercise (achieved heart rate 150 ~ lo/min, lactate > 4 mmol/1; n= 27). in both groups there was a significant shortening of the ptt. under anaerobic conditions hematocrit and quick significantly increased. factor viii activity rose significantly in both groups. indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f1+2 only under anaerobic conditions (tat from 2,5 to 5,4 pg/1; f1+2 from 0,87 to 0,93 nmol/1). indicating activation of fibrinolysis t-pa activity increased significantly in the anaerobic group (from 0,1 to 0,4 iu/ml) but not in the other group. this findings indicate that there is e balance in the hemostatic system by activation of clotting as well as of fibrinolytic system in young volunteers during exercise by swimming dependend on the degree of exercise load. membranes as well as compact, porous disks are successfully used for fast analytical separations of biopolymers. as far as capacity, speed and performance of separation are concerned, the supports are as effective as other recently developed fast media for the separation of biopolymers °). so far, technical difficulties have prevented the proper scaling-up of the processes and the use of membranes and compact disks for preparative separations. in this report, the use of a compact tube made of poly(glycidyl methacrylate) for fast preparative separations of proteins is shown as a possible solution of these problems. the units have yielded excellent results, regarding performance and speed of separation as well as capacity. the application of compact tubes made of poly(glycidyl methacrylate) for the preparative isolation of the coagulation factors viii and ix from human plasma shows that this method can even be used for the separation of very sensitive biopolymers. in terms of yield and purity of the isolated proteins, this method was comparable to preparative column chromatography. the period of time required for separation was five times shorter than with corresponding column chromatographical methods. our measurements showed an excellent correlation of the two systems (r=0,99). the maximum amplitudes on the roteg were on average 2.2% higher than on the hteg, corresponding to a slightly lower reverse momentum of the measuring system in comparison to the hteg. we report first results out of the evaluation of sta compact (boehringer mannheim/diagnostica stack)). sta compact is designed for automated analyses of routine and special coagulation (chronometfic, photometric [405 nm] and turbidimetric [540 run]) tests. in addition, it does measure ,,derived" fihrinogen. tests as follows were evaluated: prothrombin time (pt), partial thromboplastin time (aptt), fibrinogen (clauss method), thrombin time, at iii (chromogen), hepato quick, as well as the factors ii, v, vii, x, and viii. results: within run cvs of the clotting tests were below 2% (calculated on the basis of seconds) in most cases, day to day cvs below 4% (not measured for factors, yet). at iii yielded within run cvs below 3% in the decision range. measuring ranges: at iii: 20-140 %; fibrinogen: 1.3-9.0 g/l (plasma -dilution 1/20), after rerun with other dilutions: from 0.3 g/1 (dilution: 1/5) to 18 g[l (dilution: 1/40). method comparisons, using sta as reference, yielded slopes close to 1.00 and negligible intercepts. throughput: with routine clotting tests about 100 tests/h, in a sample selective access mode. we conclude, that sta compact allows precise measurement of routine and special coagulation tests. it is also a reliable system for photometric tests and well suited for intermediate workloads as well as stat analyses. we did evaluate ptt lt, a new liquid, silica based ptt reagent. special attention was given to reference interval and heparin sensitivity. the new reagent is well suited for the measurement of intrinsic clotting factors and is reported to have high sensitivity for lupus anticoagulants (higher sensitivity than sta aptt [boehringer mannheim = bm]). it is stable for 4 days in the cooled compartment of the sta analyzer. methods: all experiments were made on sta. for comparison, we used three other ptt reagents (a lab. routine, silica based aptt, as well as sta aptt and sta ptt kaolin from bm). in addition, thrombin time (3 u/ml thrombin, sta thrombin reagent) and heparin (chromogenic xa test, rotachrom heparin) were measured. results: within mn imprecision (n=21) was below 0.7 % cv in the normal range and in two controls (mean values: 35 s, 76 s), and 1.4 % in a heparin plasma (mean: 81 s). between day imprecision (d=10) was below 3% in two controls ( mean values: 34 s and 58 s). the upper limit of the reference range is 40 s (97.5 th perc., median: 32 s; 90 patients with normal coagulation status [routine aptt, fib., pt], median age: 54 years); almost identical reference ranges were obtained with sta aptt and the routine ptt reagent, while sta ptt kaolin showed significantly lower values (97.5th perc.: 33 s, median 28 s). method comparison study: good agreement using plasmas from patients without heparin: (y= 0a + 0.98 x, n= 198, range of(x) from 25 to 79 s, r = 0.97; x = sta aptt). the median values from 54 patients under high dose heparin were: routine ptt: 81 s, sta aptt: 75 s, ptt -lt: 82 s0 sta ptt kaolin 54 s, thrombin time 37 s and heparin 0.5 iu/ml in conclusion, results of the new reagent compare well to our routine ptt and to sta aptt system reagent. it allows sensitive monitoring of high dose heparin therapy and is well suited for detecting abnormalities of the intrinsic clotting factor pathway. is a standard technique since many years. the interpretation of the thrombelastograms has been widely based on phenomenologic observations, while there is a lack of exact information concerning the coagulation mechanisms leading to the teg amplitude (a're~). the aa'ec is a measure for the mechanical stiffness of the clot and depends on: a) fibrin formation and adequate polymerisatiun of a 3-dlmensional network: measurements with nonrecalcified citrated blood activated with adp or epinephrine (both n=10) did not show any clot formation in the teg this relies on the need for a mechanical coupling between the teg pin and cup over a distance of 1 mm, which is accomplished by the fibrin network therefore, teg can only be performed under thrombin formation and thus under thrombin-activation of the platelets in the sample. factors, which inhibit platelet aggregation but don't limit thrombin-activation of platelets, cannot be monitored by teg. b) the attachment of the dot on the surface of the teg pin and cup. according to recent literature we suggest that the attachment of the clot in the teg relies exclusively on fibrinogen/fibrin adsorption to the surfaces of the pin and cup. interruption of this attachment can result in lower amplitudes or the so-called ,,stairway" phenomenon. we could show a complete interruption of the clot attachment by dipping the pin for one second in 30% albumine solution (n=10). c) the fibrinogen concentration (fg) and platelet count (pc) of the sample. in 50 volunteers we found only a poor correlation of the maximum amplitude (ma) with fg alone (r=0.40) or pc respectively (r=0.50), while there was a very good nonlinear correlation to the product of fg and pc. we suggest that the fibrin network forms the main structure of the clot while the thrombocytes enhance its stiffness in a concentration-dependent manner. this effect of the ptatelets can be completely reversed by gplibfllla antagonists. d) adequate coagulation activation: in nonactivated teg even small amounts of inhibitors can lead to a significant reduction of the ateg. conclusion: alterations in teg measurements can be judged more properly when the underlying mechanisms are understood. the consideration of the limitations of the method allows a more specific interpretation of the results. as a response on a customer request we did investigate the sample stability of blood samples for the aptt. the study was set up in a way that simulated the conditions of a large private laboratory in which the samples arrive several hours after blood collection. blood was drawn from 10 donors into 3.2 % sodium citrate and mixed well before it was divided into several aliquots which were kept at room temperature. the aliquots were centrifuged after ~ 0,5, 11, 23 and 29 h after venopuncture and the plasma was analyzed immediately with 3 different reagents on electra 1000. results: there was a clear difference in the change of the apttover time with these reagents. also f viii (determined with a chromogenic assay with complete and standardized activation) change considerably. 2 reagent a: ellagic acid, plant phospholipid, reagent b: sulfatide/kaolin, phopholipids, reagent c: ¢llagi¢ acid, plant and rabbit brain phospholipids the increase of aptt was apparently not a function of the decrease of fviii because the in vitro f viii sensitivity of reagent b. was inferior to reagent a though reagent b showed more prolongation of the aptt than reagent a. reagent c, however, showed only minor changes in the aptt. discussion: these data show that the sample stabifity of the aptt is reagent dependent and that it is not simply a function off viii sensitivity. other factors such as the buffer system but also the sensitivitiy towards other factors than f viii seem to contribute. a comparison of the technical principle of the roteg coagulation analyser and conventional thrombelastographic systems an. calatzis, p. fritzsche. al calatzis, +m. kling, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology yechnische universit~t m0nchen thrombelastography (teg) was introduced by hartert in 1948 as a method for continous registration of the coagulation process. in 1995 we presented the roteg coagulation analyser, using a newly developed technical method. in teg systems according to i/artert the sample (blood or plasma) is placed in a cup which is alternately rotated to the right and left by 4,75 °. a cylindrical pin, which is suspended freely on a torsion wire, is lowered into the blood. when coagulation starts, the clot begins to transfer the rotation of the cup to the pin against the reverse momentum of the torsion wire. the angle of the pin is electromagnetically detected, transformed to the teg amplitude and continously recorded. in the roteg the pin is attached to a short axis, which is guided by a ball beating. thus all possible movement is limited to rpotation (r_oteg). the cup is stationary, and the pin is rotated alternately by 5 ° to the right and left by a feather system. when a clot is formed, it attaches to the surfaces of the pin and cup and starts preventing their relative movements against the reverse momentum of the feather. here the reduction of the rotation of the pin, which is detected optically, is tranformed to the teg amplitude. as can be shown by theoretical analysis and by control measurements, the roteg provides the same measuring capabilities as conventional teg systems. the main advantage is the solid guiding of the measuring system, which makes the roteg easily transportable and less susceptible to shock or vibration during measurement. yhrombelastography (teg) is a standard monitoring procedure for evaluation of coagulation. usually only nonactivated native blood teg measurements (nateg) are performed, which leads to a) a long time interval until coagulation and fibrinolysis parameters are available b) very high susceptibility of the measurement to inhibitors like heparin, which disturbes the judgement of other components of coagulation, c) unspecific results. our aim was to develop a coagulation monitoring system based on teg providing fast and specific information on the different components of coagulation. methods: the following measurements are performed in paralel using disposable pins/cups (haemoscope): a) extrinsic activated teg (exteg): 3551al whole blood (wb) + 5~tl innovin (recombinant thromboplastin reagent, dade). b) intrinsic activated teg (integ): 3551al wb + 5~tl kaolin (suspension 5g/l, behring). c) aprotinin teg (apteg): exteg + 20 kie aprotinin (trasylol, bayer). d) heparinase teg (hepteg) as decribed in (1). results: exteg and integ provide information on the extrinsic/intrinsic system within 5-10 min and information on the platelet/fibrinogen status within 10-20 min. because of the addition of potent activators integ and exteg can be performed when inhibitors like heparin are present in the circulation. fibrinolysis effects can be seen on exteg and integ and by comparison of exteg and apteg (apteg: invitro-fibrinolysis inhibition by aprotinin). if fibrinolysis is detected by exteg or integ and aprotinin-susceptibility is verified by apteg, aprotinin therapy will be initiated. heparin effects are revealed by hepteg. discussion: by the comparison of parallel teg measurements which have been differently activated, specific and fast information on the different aspects of the clinical coagulation status is provided. the presented tests can be easily performed bedside and only a small specimen of whole blood is needed (0,4-1,8 ml). introduction: a severely prolonged aptt (333s; normal: 40~os) was observed during preoperative screening for a planned splenectomy in a 71-year-old man with an 8 year history of osteomyelofibrosis. fellewing neer-normal~atien (71s) of the ap'ci" after 10 rain preincubation in a kaolin based aptt assay, pk deficiency was suspected and studies were performed to further investigate the nature of the pk deficiency as well as the mechanism underlying the normalization of the prolonged aptt by increasing the preincubation time. methods: the apl-r assay was peal'armed using kaolin/inesithin. high molecular weight kininogen clotting activitiy (hk:c), fxii:c end pk:c were measured by an aptt based assay using neothromtin ® (behnng) and 1 rain (pk:c) or 4 min (hk:c, fxli:c) preincubation. pk amidolytic activity (pk:am) was assayed using cosset pk ~ (chromogenix) and pk antigen (pk:ag) by quantitative immunoblotting. fxll and hk proteolysis dunng activation of plasma by kaolin (10mg/ml at 37=c) or ds (12.5~tglml at 4=c) was demonstrated by immunotilotting assays of fxii and hk following sds-page. assay pk:c pk:am pk:a~i fxfi: the propositus had pk:c<5%, pk:am=5% and pi~ag<2.5% as compared to normal pooled human p(asma (nhp). his son and two daughters had pk:c-50% and normal aptt values, incubation of the propositus' plasma with ds did not result in fxii or hk cleavage within 180 rain, whereas jn nhp detectable f×ii and hk proteolysis occurred after 5 rain and complete proteolysis was observed after -120 rain. in contrast, kaolin activation of propositus' plasma led to slow activation of fxii after 10 rain, presumably by autoactivation, and to fxlla-induced hk proteolysis. near-normalization of the propositus' aptt by prolongation of the preincubation time paralleled fxii autoactivation as evidenced by immunobletting. we describe a propositus with severely prolonged aptt due to hereditary, crm negative pk deficiency suffering from omf. activation with a particulate suspension of kaolin led to slow fxii autoactivation and hk proteolysis, whereas ds in solution did not induce fxii or hk cleavage. fxii autoactivation seems to be responsible for the normalization of the prolonged aptt in pk deficiency after prolonged preincubation times. in our study we compared a conventional bag with silicone tubing (a) for blood donation with 2 new ones (]3 from biotrans and c from baxter) with a newly developed y-shaped adapter. this adapter is integrated into the tubing and therefore provides the advantage for drawing blood samples in a closed system. the 3 systems were identical in amount and content of anticoagulant, i. e. 63 ml of cpd per bag resulting in approximately 14% of the final whole blood volume. the purpose of the study was to determine whether the different tubings can influence the quality of plasma products conceming the blood coagulation system. in plasma samples we measured several factors of the procoagnlatory and fibrinolytic systems. intralndividual control eitrated (.135 m) blood samples were initially drawn from the contralateral cubital vein from the same male donor (34 in each group). in all bag samples we found small but significantly higher levels of the global test parameters ap'it and ti" compared to controls, indicating a higher amount of anticoagulant. pt, however, revealed no differences, thus suggesting that factor activities were not altered (statistics according to mann-whimey). increase of procoagulatory activity measured as tat complexes showed elevated levels in bags a and c whereas prothrombin fragments fl+2 decreased only in a. conceming the fibrinolytic system, plasminogen a~tivators and pai-1 values were diminished in all three systems 03 < a < c) compared to controls. d-dimers were lowest in a followed by slightly higher values in c, controls and b. fibrin monomers did not reveal any significant differences: a < c < controls < b. in summary, the quality, of the 3 different blood sampling devices was comparable to the intraindividual controls as to factor activities measured by global tests. the activation of the procoagulatory and fibrinotytic systems was slightly but in most cases significantly higher in the two new devices than in the conventional one. all values, however, obtained from the plasma samples did not exceed the normal range of healthy blood donors. therefore we concluded that the two new closed blood drawing systems are favorable for blood donating procedures. in 20 patients with acute myocardial infarction (ami) and thromholytie therapy (13 patients with rt-pa, 6 patients with streptokinase and one with heparln) with ck, myoglobin and ekg criterions the patients were divided in two groups (reperfusion/no fellow two hours after starting the thrombolytic therapy) . blood samples were taken before, 30 rain, i h, 2 h, 4 h, 8 h,12 h after lysis and than every day till day 10. because of the central role of factor xii in activation of coagulation, fibrinolysis, kallikreln-kinin-system and complement cascade we investlgate the role of factor xila initiated by ami and the relation of factor xiia to the thrombolytie agent and reoeclusion rate. for the investigatlens we take the kits from shield diagnostics (xiia), behring diagnostica (c~-inactivator, pl~nogen, ~-antip]~n~n, pap), chromogefiilx ab (prekallikrein) and di~nostica stage (vile). the results: there is an increase of factor xiia immediately after starting the fihrinolysis (max. 30 rain after starting); the increase 5/i independently of the thrombolytie agent. parallel to factor xiia raises factor viia without significant changes of c1-1naotor and prekalllkrein. that means: activation of xiia and fibrinolytic pathway leads to relatively mild c.hanges in kallikrein system, hut to significant activation of extrinsic system by vila-tissue factor. in some patients is an additional rise in the system xiia -viia, when the fibrinolytic system is already in the normal range. there will need further investigations to define the risk of reocclusion as a result of activation of faktor viia by faktor >li ia. autoimmune thrombocytopenic purpura (aitp) is a frequent complication of chronic lymphocytic leukemia (cll] which developes on different stages of the disease and needs special treatment measure. mechanism of autoimmune disorders in cll remains uncleared. we investigated immunologic phenotype of blood lymphoid cells in 22 patients suffering from cll with aitp. in these patients we did not observe disorders in expression of b-lineage markers as compared with cll patients without immune complications (13 patients). but in the 1st group of the patients the greater number of b-celts expressed markers of activation. according to ig heavy chain expression, the lymphocytes in most cases of cll complicated by aitp had more mature phenotype. in all patients with k phenofype of cll lymphocytes we found immune disorders. the development of aitp was accompanied by lowered level of t cells and changed dis'flibution of their immunoreguiatory subsets: diminished number of cdz~cells and increased one of cd~'÷lymphocytes. the results of our investigations undirectly proved that malignant b-cells in cll are involved in production of autoantibodies against blood cells. dysbalance in t-cell system with functional disturbances of immunoregulation are significant in development of autoimmune complications in cll a24 in women with severe fvii deficency (<10%) hypermenorrhagia may cause life threatening blood loss. therefore, hysterectomy at a young age is reported frequently in the literature. a 12 year old girl without history for a bleeding disorder was transfered with hypermenorrhagia. the initial laboratory data revealed an abnormal quick-test of 30% due to fvll of 9,0%, normal platelet count and hemoglobin level of 7,2 g/dl. antifibrinolytic therapy (tranexamic acid 4x15mg/kg bw/d) and lynestrenol substitution were started to reduce the hemorrrhage. despite treatment the daily blood loss increased to a maximum of 290ml. therefore, substitution therapy with recombinant fvila (rfvila) (novonordisk) was started at a dose of 15 ilg/kg bw q 6 h. subsequently blood loss decreased to 30ml/d, but even with an increasing dose of rfvlla up to 35 i~g/kg bwq 4h (fvil activity max. 7400% 10 min after injection) and additional hormonal support with a lh-fsh-anatgonist some hemorrhage remained. a short .course of methergin was stopped due to severe pain. ultrasound of the uterus revealed a hypertrophic endometrium causing the persistent bleeding. it decreased slowly over several weeks and hemorrhage stopped completely after 40 d. the total rfvlla dose administered was 118 rag. no side effects were observed. no transfusions of blood products were necessary. currently, menstrual cycle is suppressed by estriosuccinate. conclusion: due to close cooperation with a specialised gynecologist, hypermenorrhagia was controlled and in this woman with severe fvll deficiency hysterectomy was avoided. in three male members aged between 27 and 52 years of a family suffering from inherited bleeding disorders the diagnosis of protein z deficiency was established. plasma protein z evaluated by elisa (asserachrom protein z, diagnostica stago, france) ranged between 200 and 300 ng/ml. the patients mostly suffered from moderate bleeding complications like prolonged bleeding secondary to trauma or invasive measures and also spontaneous hematuria. previous laboratory investigations revealed variable platelet function deficiencies and transitory boderline decrease of von-willebrand factor. spontaneous bleedings were rarely recognized, however, they occured more frequently when analgetics were taken. bleeding complications showed good response to hemostyptic measures and antifibrinolytic therapy. the use of pcc containing a high level of protein z in these patients is restrained to severe bleeding disorders or major surgery. defibrotide is a mammalian polydeoxyribonucleotide derived anti-ischemic and antithrombotic drug (crinos s.p.a., v"flla guardia, italy). while the drug is known to produce polytherapeutic effects owing to its multicomponent nature, the exact mechanisms of its anti-ischemic effects remain unknown at this time. since defibrotide is found to be effective in ischemic disorders such as paod, vod related occlusive disorders and related rnicroangiopathic conditions, we studied the effect of this drug on the contraction of dog and pig arterial strip/rings obtained from various sites. in vitro supplementation ofdefibrotide to the organ bath containing control dog and pig arterial rings did not modulate the serotonin and thromboxane (generated) contraction, however, tissues obtained from dogs treated with 10 mg/kg defibrotide iv exhibited a profound desensitization to the agonist induced contractile process. the time course of these effects was found to be much larger than the plasma half-life of defibrotide. this presentation will provide additional data on the effect of defibrotide on the contraction of vascular smooth muscles as a possible explanation for the anti-ischemic effects of defibrotide. a. wehmeier, a. popescu, w. schneider klinik for h,~matologie, onkologie und klinische immunologie der heinrich-heine-universit&t d0sseldorf in chronic myeloid leukemia (cml), evolution of blast crisis is the limiting factor of survival. however, as in other chronic myeloproliferative disorders, bleeding and thrombotic complications are a major source of morbidity but their incidence has rarely been analysed in larger patient groups. we retrospectively evaluated 182 patients with cml during chronic phase (170 cases), accelerated disease (58 cases), and blast cdsis (72 cases), and determined the incidence of thrombohemorrhagic complications in relation to the stage of the disease. in chronic phase, 28 patients had bleeding complications (8.4%/patient year) and 15 patients thrombotic episodes (4%/patient year). the incidence of bleeding increased significantly in accelerated disease (18 patients, 51.2%/patient year) and blast crisis (37 patients, 347%/patient year), and many patients had repeated complications. contrary to our expectations, the incidence of thrombotic complications also increased to 10.2%/patient year in accelerated phase and 39.8% /patient year in blast crisis, tn chronic phase, 3 patients died because of bleeding events. in accelerated phase, 5 patients died due to bleeding and 1 patient due to thrombotic complications. in blast crisis, bleeding was associated with 21 deaths, and pulmonary embolism with 2 deaths. analysis of the cause of thrombohemorrhagic complications revealed that in chronic phase, bleeding was often associated with uncontrolled busutfan therapy, whereas in blast crisis, severe bleeding occurred mainly when platelet counts were low and peripheral blasts increased. however, there was no obvious explanation for thrombotic complications. we conclude that bleeding and thrombotic complications are a major source of morbidity and mortality also in cml, and that the incidence of such complications increase in advanced stages of the disease. klinik for innere medizin °, klinikum schwerin patients suffering from primary or secondary amyloidosis may occasionally acquire a coagulation disorder characterised by isolated factor x deficiency. we report on a 60-years-old man who presented with lower gastrointestinal bleeding and prolonged prothrombin time (quick 50 %). amyloidosis was suspected and proven using biopsy of the rectum and histological analysis. in addition, a monoclonal gammopathy of undetermined significance was diagnosed by immunofixation (light chain, type x). detailed investigation of the prolonged prothrombin time led to the discovery of a pronounced factor x deficiency (residual activity 4 %). inhibitors of coagulation factors could not be demonstrated. the treatment of the patient consisted of red blood cell transfusion and infusion of prothrombin complex concentrates. due to the extremely rapid clearance of infused factor x, no increase of its activity was observed. chemotherapy of the monoclonal gammopathy was initiated (melphalan/ prednisone). over the following six months the frequency of major bleeding episodes gradually decreased. however, subclinical occult bleeding continued. the factor x activity was repeatedly found between 10 and 12 %. we support the suggestion from literature data that clinically relevant bleeding episodes are likely to occur in patients with amyloidosis-associated factor x deficiency if the residual activity is below 10 %. sepsis and septic shock is a disease entity which is characterized by inflammatory reactions (sirs), coagulation abnormalities (dic), organ failure (mof) and severe hemodynamic alteration frequently leading to death in a shock. the aim of our studies was to investigate the efficacy of antithrombin iii (kybernin ®) on ~he outcome of septic shock in a pig endotoxemic model. pigs, in this model respond to lps with elevated tnflevels, decreased leukocytes and platelets counts, increased tat and fibrin monomer levels, hypotension and in increase of the pulmonary arterial pressure (pap), indicating impaired lung function. a total number of 13 male castrated juvenile domestic pigs (25 -30 kg) were anaesthetized, ventilated mechanically and infused with saimonella abortus equi lipopolysaccharide (s. equ-lps) over three hours (0.5 ~g/kg * h). a swan-ganz-catheter was inserted into the pulmonary artery to measure the pap. animals were allocated to two groups,, the treatment group (n = 7) received antithrombin iii (at iii) according to the following regimen: 250 u/kg (t = 60 -30, i. v. infusion), 125 u/kg (i. v. bolus, t = 0) and 250 u/kg (t = 180 -240 rain, i. v. infusion). the placebo group ( n = 6) received the appropriate amount of human serum albumin: 50 -25 -50 mg/kg (same schedule as with at iii). main objective was defined as the mortality rate at six hours a_~er s. equ-lps infusion. whereas in the placebo group 4 out of 6 animal died (mortality rate: 66 %) all at iii-treated pigs survived the observation period of 6 hours (p < 0.05, x2-test). the at iii group was shown to have a lower pap than the control group, especially the second peak of hypertension was abolished by at iii. it is therefore concluded that at iii should be a useful tool for the treatment of severe sepsis and septic shock. in a nationwide monthly survey all childrens hospitals in germany (esped) were asked to clinical and therapeutical informations about children suffering from pmi. during july 94 till june 95 299 children were registered. from these, 87 had either ecchymoses and/or necroses related to an increased mordibity and mortality (20%), whereas 212 showed no bleeding signs except for petechiae. of these children one died. the therapeutic interventions concerning hemostasis are listed according to the defined two risk ~oups. from the patients with ecchymoses or necroses, 13/87 received combination therapy (compared to 5/212 with petechiae or no bleeding sign) of at iii, heparin and/or plasma. only t child received protein c concentrate. the data show that children with low risk did in part receive higher doses of heparin and/or at iii concentrate than did high risk patients, whereas plasma therapy was adjusted to severity of eoagnlopathy. furthermore, the wide range of given therapeutics allows no information about the different medications. therefore, controlled studies with respect to the different therapeutic interventions in children with high risk pmi is desirable. a fully automated procedure for the reptilase time assay y. schmitt (1) and h.j. kolde (2) (1) institute for laboratory medicine, st~dtisches klinikum, darmstadt, frg, (2) dade diagnostics, unterschlei6heim the reptilase time assay is a relatively simple technique for the detection of fibrinogen degradation products and fibrinogen deficiency or abnormality. the procedure is performed with citrated plasma and batroxobin reagent, a snake venom enzyme from bothrops atrox. this enzyme cleaves fibrinogen by releasing fibdno peptide a only but not fibdno peptide b. in contrast to the physiological enzyme thrombin that is readily neutralized by antithrombin iii and hepadn batroxobin is not inactivated by physiological inhibitors. at present this assay is mainly performed manually or on mechanical instruments. we have adapted this assay to the electra 1000 fully automated coagulation analyzer (medical laboratory automation, pleasantville, n.y.) using the thrombin clotting time procedure in the instrument software with batroxobin reagent (dade diathe clot formation is registrated turbidimetrically and the dotting time is pdnted. the within run precision (n= 10) of this procedure was tested with two plasmas from the daily routine and was between 2.8 and 3.4 %. in 25 normal samples we found clotting times from 10.5 to 12.8 sec. in 30 samples with liver disease (confirmed by pseudochlinesterase < 2000 u/ml) or on thrombolysis therapy with streptokinase or urokinase the fully automated assay on the electra was compared to the semiautomatic method using a kc 10 coagulometer (amelung, lemgo, germany) based on a rolling metal ball pdnciple and magnetic endpoint detection. the two assays agreed very well with a correlation coefficient of r = 0,948 and a regression line according to passing and bablok of y = 1.0 x + 1.7. these data show that the reptilase time can be performed with good precision and with good correlation to the manual technique on mechanical instruments on the electra 1000. introduction: disseminated intravasal coagulation (dic), due to a massive activation of the coagulation system, is frequently observed in intensive care patients suffering from severe underlying diseases. laboratory diagnosis of dic is based on different coagulation tests, but unfortunately the routine haemostaseological parameters react with latency in the course of acute dic objective: in four cases from a cohort of 43 patients with severe sepsis and dic we analysed special haemostaseological parameters (tat, f1-t2, d-dimers, human-leucocyte-elastese (file), catepsin g and heparin cofactor ii (hc ii)) and correlated them with a mof-score in order to test their predictability on the prognosis of these patients. results: all patients were substituted with at iii concentrate. l,1 the investigated patients median time of treatment with at iii concentrate was 8 (6-9) days and median time of dic-duration was 6 (4-8) days. none of the presented patients died during observation period. all analysed parameters, except d-dimers, showed a sufficient correlation with the evaluated mof-score (tat: r= 0,78; f1-f2: r= 0,84; hle: r= 0,71; catepsin g: r=-0,75; hc ii: 1"=-0,88). the d-dimers did not correlate with the mof-score, which is probably due to the delayed reactive hyperfibrinolysis in the course of dic. furthermore, the decrease of the tat-complexes, f1-f2, hle and catepsin g levels were followed by an increase of at hi and hc ii activity. conclusion: in general the analysed activation markers and coagulation parameters are sufficiently to describe the ongoing process of the dic. the hyperfibrinolytic activity of dic is sufficiently represented by the d-dimer test, but is of defered reactivity in the course of dic. unfortunately these parameters are not established in the routine monitoring of dic on intensive care units and therefore further studies are needed to investigate the practicability and reliability in the daily routine monitoring. we have previously reported that notoginsenoside r1 (ng-r1) has an effect on counteracting lipopolysaccharide (lps) induced upregulation of plasminogen activator inhibitor-1 and tissue factor expression in cultured human umbilical vein endothelial ceils in vitro and in mice in vivo [fibrinolysis 1994;8:(suppl 1)119]. in this study we investigated the effect of ng-r1 on prevention of lps induced lethal toxicity in mice. because mice are relatively resistant to lps when applied as a single agent, we sensitized them by simultaneous treatment with d-galactosamlne. the 80% lethality induced by lps (1.5 mg/mouse) plus d-galactosamine (8 mg/mouse) in c3hs-ie mice was reduced to 16% by simultaneous administration of ng-r1 (1.5 mg/mouse) with lps/galactosamine (p<0.05 by x 2 test). ng-r1 also significantly delayed lps/galactosamine induced lethal toxicity from 12 hours to 30 hours with all animals surviving beyond 30 hours. because lethality induced by lps involves the synergistic effect of multiple effector molecules such as tumor necrosis factor (tnf)-ct, interleukin (il)-i, interferon 3' etc., we also investigated the effect of ng-r1 on lps induced tnf-ct production from leukocytes in cultured human whole blood cells (hwbcs) ex vivo. the production of tnf--ct induced by lps (1 ng/ml for 24 hours) in the supernatant of hwbcs was inhibited by 46% and 22% respectively, when the cells were incubated 1 ng/ml or 10 ng/ml lps together with 100 i~g/ml ng-r1, respectively (tnf-ct concentration, 1 ng/ml lps treated cells: 297+192 pg/ml, i ng/ml lps plus 100 l.tg/ml ng-ri treated cells: 162+137 pg/ml, p<0.01; 10 ng/ml lps treated cells: 3094_+487 pg/ml, 10 ng/ml lps plus 100 pg/ml ng-r1 treated cells 2423+713 pg/ml, /'=-0.02). the present results suggest that ng-r1 can prevent the onset of lps toxicity as well as the lps induction of cytokines. therefor ng-ri may be effective in preventing the effects of septic shock in gram-negative infections. to elucidate the mechanisms by which coagulation is initiated in septic patients in vivo, coagulation measurements were prospectively evaluated in patients with severe chemotherapyinduced neutropenia. this group of patients was chosen because of their high risk of developing severe septic complications, thus allowing serial prospective coagulation testing prior to and during evolving sepsis or septic shock. 62 patients with febrile infectious events were accrued to the study. of these, 13 patients progressed to severe sepsis and an additional 13 patients to septic shock. at onset of fever, factor (f) vlla activity, f vii antigen and antithrombin iii (at iii) activity decreased from normal baseline revels and were significantly lower in the group of patients who progressed to septic shock compared to those that developed severe sepsis (medians: 0.3 versus 1.4 ng/ml, 21 versus 86 u/dl and 45 versus 95%; p < 0.001 ). the decrease of these variables in septic shock was accompanied by an increase in a marker of thrombin generation like prothrombin fragment 1 + 2 (medians: 3.6 versus 1.4 rim; p=o.05). these differences were sustained throughout the septic episode (p < 0.0001 ). f vlla and at ill levels of <0.8 ng/ml and <70%, respectively, at onset of fever predicted a lethal outcome with a sensitivity of 100 and 85%, and a specificity of 75 and 85%, respectively. in contrast, fxila-alpha antigen levels were not different between both groups at onset of fever and were only marginally higher further during the course of septic shock (p=o.001). thus, septic shock in neutropenia is associated with significant coagulation activation, presumably driven by the tissue factor pathway rather than the contact system. furthermore, in septicemia both f vlla and at iii measurements are sensitive markers of an unfavourable prognosis. hemostatic parameters in sepsis patients treated with anti-tnfct monoclonal antibodies c. salat 1, p. boekstegers 2, e. holler 1,3, b. reinhardt i, r. pihusch 1, k. werdan 2, m. kaul 4, t. beinert 2, e. hiller 1 med. klinik iii 1 und i 2, klinikum grosshadern der ludwig-maximilians-universitat monchen, h~imatologikum der gsf 3, knoll ag ludwigshafen 4 tumor necrosis factor et (tnfc~) is a central mediator in the pathogenesis of sepsis and septic shock. as administration of anti-tnfct monoclonal antibodies was able to protect animals from an otherwise lethal endotoxin challenge clinical studies were initiated in patients with sepis. tnfct exerts a procoagulant effect, e.g. by enhancing pai-i and activating thrombin as indicated by an increase in tat and pf 1/2 levels. therefore it may be involved in disseminated intravascular coagulation in sepsis. we determined tat, pf 1/2, d-dimers, tpa, upa, pai-i and vwf levels in 30 patients with sepsis or septic shock. 14 patients received the anti-tnfa monoclonal antibody mak 195f (knoll ag, ludwigshafen), whereas 16 patients served as controls. we found a significantly lower level ofupa in anti-tnfc~ treated patients. since the difference existed before onset of treatment it can not be attributed to tnfot antagonisation. all other parameters investigated did not differ significantly between the two groups throughout the study period. failure to detect modulation of hemostasis by anti-tnf~ might be explained by delayed initiation of treatment in clinical sepsis. in animal experiments it has been observed that the antibody prevented lethal endotoxin effects when given prophylactically or 30 minutes after endotoxin challenge, but not when it was administered 2.5 hours later. in addition, beneficial clinical and hemostatic effects of tnfet antagonisation might be observed only in subgroups of patients with hyperinflammatory sepsis. larger studies addressing this point are under way. protease receptors for thrombin and trypsin have been described for different cell lines. we investigated the ability of trypsin to activate human umbilical vein endothelial cells (huvec). cell activation was measured by the increase of intracellular free ca 2* (caff) with help of microscope fiuorometry (fura-2) and by the von willebrand factor release measured by a sandwich elisa. incubation of huvec with thrombin (1u/ml) or trypsin (10nm) showed a 2-10 fold increase of c~ff. a subsequent homologous stimulation after 80 s lead to a 2-5 fold lower concentration of ca~ 2÷ compared to the first stimulation. therefore cells have been desensitised by the first stimulation. inhibition of the proteolytical activity of trypsin by soybean trypsin inhibitor was followed by failure of trypsin inducing an increase of ca~ 2÷ concentration. in cross stimulation experiments with thrombin and trypsin, we could demonstrate, that cells first stimulated with thrombin showed a second maximal response by subsequent stimulation with trypsin. the same effect was measured with first stimulus trypsin and second stimulus thrombin. trypsin and thrombin induced a release of von willebrand factor (2-5 fold in comparison to unstimulated cells). we found a vwf release dependent on the concentration of trypsin similar to thrombin. an electrophoretic analysis of the released von willebrand factor showed a different multimeric composition of vwf between trypsin and thrombin stimulation. these results indicate, that there might be a protease receptor on huvec for trypsin being different from the thrombin receptor. clinical and laboratory findings of coagulopathy were investigated by an 1-year-survey to 320 children's hospitals. 291 meningococcal infections were evaluable. severe disease (characterized by need for mechanical ventilation, dialysis and/or catecholamines) was seen in 42 of these children; 29 of those survived and 13 died. clinical signs of severe coagulopathy were seen in 83 children: ecchymoses (n = 73) and skin necrosis (n = 36) were associated with increased mortality (16% and 20%, resp., compared to 4.5% overall mortality). five of 29 surviving children with skin necroses required surgical interventions (skin transplantation and/or amputations). petechiae were frequent (n = 156) and as isolated finding not related to severe disease or fatal outcome (6% mortaliy). platelet counts at admission were lower in non-survivors (10th-90th percentile: 30 -450.000/gl, median: 139.000/i.tl) than in survivors (10th-90th percentile: 140 -480.000/i.tl, median: 242.000/gl). at iii values showed no difference between survivors and non-survivors. protein c was available in few patients (n =14): in this subgroup, protein c was lowered in patients with limited disease (10th-90th percentile: 20 -105%, median: 48%) as well as severe disease (10th-90th percentile: 30 -75%, median: 60%). in conclusion, the findings "ecehymoses" and "skin necroses" were related to fatal outcome and therefore included in a prognostic score for severity of meningncoccal disease. the influence of irradiation on pai-i and vwf levels in human umbilical vein endothelial cell cultures k. fragiadaki, c. salat, r. pihusch, b. reinhardt, m penovici, e. hiller med. klinik iii, klinikum grosshadern der ludwig-maximilians-universitat monchen an elevation of pai-1 in bone marrow transplant recipients developing veno-occlusive disease (vod) of the liver has been described earlier. endothelial cell damage due to the preparative myeloablative radioehemotherapy is supposed to be an important step in the pathogenesis of the disease, which is characterized by an obstruction of small intrahepatic venules. in order to investigate a possible role of irradiation we studied the influence of several doses (0, 5, 15, 30 gy) on pai-1 and vwf levels in the supematant of human umbilical vein endothelial cell cultures (huvec). pai-1 antigen and vwf were determined by enzyme immunoassays. whereas pai-1 and vwf levels remained unchanged alter irradiation with 5 gy and in control cultures, a rise was observed one day after irradiation with 15 gy (mean day 0"-)day +1) in pai-1 (100,0% --)171,2 %) and vwf (100%--)159,7%) levels. the increase was more pronounced and reached levels of statistical significance after a dose of 30 cry (pai-1 100%--) 278,7% and vwf 100%--)168%). both pai-1 and vwf levels decreased on day 2 after irradiation with 15 and 30 gy. our results indicate that irradiation induces an increase of pal-1 and vwf in endothelial cells. nevertheless, this effect was observed only in doses above those ones used during conditioning when patients receive 3x4 gy. additional factors seem to be of significance. cytokines like tnfo~ enhance pai-1 and vwf in endothelial cell cultures and are known to be elevated in bmt-associated complications. it can be speculated that irradiation in concert with these factors may contribute to the development of veno-occlusive disease. disseminated intravascular coagulation is characterized by high consumption of coagulation factors, systemic elevation of fibrinolysis by tpa and concomitant elevation of pai-i secreted from inflamed endothelial cells. in an attempt to investigate the contribution of inflammatory cytokines, endothelial cells lines of microvascular origin were stimulated in vitro and pal-1 antigen was measured 2h, 4h and 24h after stimulation. in contrast to results published from experiments performed with macrovascular human umbilical vein cells (huve), our results obtained with 3 different microvascular endothelia isolated from skin, solid tumor tissue and bone marrow revealed that inflammatory cytokines reduced pal-1 antigen levels. in addition to tnf-a (25ng/ml) and lps (10pg/ml), we found that il-10 (100 u/ml) and gm-csf (100 u/rot) also reduced pai-i levels within the first 2h of incubation (from 120ng/ml to 80-110 ng/mll and the effect was even more pronounced after 4h and 24h (from 380 ng/ml to 250 ng/ml). il-1 (10 u/ml) and lps (10 pg/l) also reduced constitutive levels of pal-1 but the effect occured later than 4h after addition of the stimulator. the strongest synergistic effect was demonstrated with gm-csf plus il-1 resulting in pal-1 suppression of 50% after 2h and 30% after 24h. in contrast, g-csf (300 u/ml) induced the immediate (120 to 140 ng/ml after 2h and 380 to 420 ng/ml after 24h) upregulation of pal-1 antigen. stimulation of pat-1 levels was also observed with tgf-i~ (10 pg/ml), however not earlier than 18h of incubation. interestingly, both stimulatory cytokines, ie. g-csf and tgf-13, alone were able to counteract the decrease of pat-1 antigen by tnf-a but only a combination of g-csf plus tgf-g neutralized the effect by il-1. results indicate that inflammatory cytokines regulate pal-1 fibrinolysis in a synergistic and antagonistic fashion. we established the culture of human brain microvascular endothelial cells (hbmec) in order to investigate the pathophysiology of hu~man cerebral malada, which is still associated with a high mortality rate. it is widely accepted that among the reasons for the fatal outcome of cerebral malaria, the interaction of endothelial cells with cytokines and paras lites with subsequent changes in haemostaseological parameters is involved. the human microvascular endothelium may therefore play a deci §ive role in the pathophysiology of cerebral malaria. ery throcytes containing later stages of p. falciparum specifically bind to capillary ec in vivo (sequestration). tnf-cq il-1 and il-6 are considerably elevated in severe malaria. coagulation factors such as tissue factor and von willebrand factor are affected by malada suggesting the involvement of the hbmec in cerebral malada. so far, research on the involvement of the hbmec has been performed on ec cultured from human umblilical veins (huvec). the relevance of this model may be questioned on t, ,he grounds that the capillary endothelium probably plays a greater role than the endothelium of the large vessels. besides, some propertie.$ of the endothelioum seem to vary, upon the organ of origi/n. for the~ reasons, our laboratory has established the hbmec as a model to study the pathophysiology of human cerebral malaria. to demonstrate the relevance of this model in the context of malaria, hbmec were challenged with sera from different patients with severe p. falciparum malaria and with serum from a healthy donor. we can demonstrate that in cells challenged with malaria patient sera icam-1 and substance p were upregulated. on the other hand cells challenged with serum from a healthy donor expressed neither icam-1 nor substance p. these results strongly suggest the relevance of this model for vessel involvement in malaria. both, histamine and serotonin have been described as potent stimulators of yon willebrand factor (vwf) release from human umbilical vein endothelial cells (huvec). we performed experiments to differentiate the receptors for histamine and serotonin induced vwf release. absolutely unexpected we don't found any significant vwf release after the addition of serotonin to huvec or human artery endothelial cells (huaec) in concentrations from 0.1 ijm to 50 pm. in the case of histamine (0.1 pm -50 pm) we measured a vwf release 2-5 fold compared to unstimulated cells. this release was in the same order of magnitude as the release induced with 11u thrombin. to verify these results we measured the effect of histamine and serotonin on the intracellular ca 2÷ concentration (ca~ 2÷) in huvec and huaec. cells were labelled with fura-2 and the change in fluorescence after agonist addition was measured with a microscope fluorometer. using the same agonist concentrations as above we found an 5-10 fold increase of caj 2. with histamine or thrombin but no effect by addition of serotonin. this results indicate a similar activation of human endothelial cells by histamine and thrombin and that serotonin don't stimulate endothelial vwf release or increase of cay. activation and/or dysfunction of the endothelium can be triggered by cytokines (e.g. interleukin-2, tumor necrosis factor-alpha) or bacterial substances (e.g. endotoxins) and may contribute to shock and multi organ failure. pal-l and tm were assessed as parameters of activated endothelium following bsct in three to four days intervals from start of conditioning therapy through day +35. data were compared to the occurrence of sepsis, veno-occlusive disease (vod), capillary leakage syndrome (cls) and graftversus-host-disease (gvhd). patients with neither complication served as controls. no *days after stem cell tranplantation pai-1 and tm were increased in all patients with sepsis, cls~ vod and/or gvhd. pai-1 peaked at days 14 to 18 and the increase was highest in sepsis and lowest in cls. the increase in tm values was somewhat delayed (day +24) and was highest in vod and cls and lowest in gvhd. pai-1 and tm are sensitive markers of endothelial activation in sepsis, vod, cls, and/or gvhd, but they do not allow a differention between these complications. endothelin (et) is the most potent vasoconstrictor. it is known that et plasma concentration is correlated with a poor prognosis in patients with non ischemic cardiomyopathy (cm). the contribution of the heart to the production of et is still unknown. to investigate the pathogenetic mechanism in patients without coronary artery disease (cad), we examined 13 patients with hypertension ( . pulmonary capillary wedge pressure (pcwp) was measured in all patients. et and its precursor big-endothelin (bet) were determined at rest and after pharmacological stimulation with dipyridamole (0.5 mg/kg body weight), that increases coronary blood flow by factor 2 -4 on a non endothelial pathway. cardiac coronary et and bet concentrations were determined from the arterial blood samples, obtained from the aorta, and simultaneously from the coronary sinus (venous blood). blood samples were collected into ice chilled vacutainer tubes and stored after centrifugation at -70 *c. et and bet were analysed after extraction by a sepal< c 18 cartridge by radio immuno assay technique (immundiagnostik). it is concluded that et is increased with elevated filling pressures of the heart in patients with cm. it is not produced in considerable quantity by the heart neither at rest nor at increased blood flow. there4ore the lung has to be considered as the major organ for the production of et and bet in patients without cad. to characterize the incompatibility of blood with foreign surfaces valide in vitro methods especially in testing of platelet function are neceessary. it seems to be effective to use test systems which can also be helpful lateron in the clinic when foreign surfaces (e.g. venous catheters) are used and evaluated in so called phase-4-studies. we studied the influence of 21 reference polymers under standardized and controlled flow conditions on platelets in citrated blood specimen of healthy blood donors.the following tests were performed pre and post platelet-pol)aner contact: decrease of platelet count, platelet aggregation (wu-gmtemeyer index), analysis of platelet spreading capacity on standardized plastic surfaces by using a visual microscopic evaluation according to breddin and bfirck (1963) and an interactive computer-aided system (ibas, kontron gmbh, manchen, frg) by digitalizing the morphological picture of the platelet slides and area detection with a resolution of 512x512 pixels. results: platelet counts showed significant differences pre and post polymer contact, the wu-grotemeyer index demonstrated platelet activation only by blood contact with large volumes of polymeric material whereas both visual and computer-assisted evaluation of platelet spreading ability revealed a marked shift in the different classes of platelets: platelet activation results in a decrease of large structural elements and an increase of elements with spider threads. (pre contact (n=1000): 27:~-6 large forms of platelets, 700~-39 small forms and 275:l-41 spider forms; post contact (n=1000): 6-+-5 large forms, 510a:56 small forms and 484±58 platelets with spider threads). in some series there were significant differences between visual and computer-aided evaluation in the detection of small and spider forms. however, the relative increase of these nonspread spider forms could be stated with beth methods (wilcoxon test). we therefore conclude, that platelet morphometry with both methods is a sensitive and reliable ex vivo method to evaluate platelet interactions with artificial surfaces and can also be used lateron in phase-4-studies in patients. however, the ibas-system requires further maprovement in hard-and so,ware to reduce the high expenditure of this method. despite for the most part standardised methods such as hypothermia, cardioplegia the perioperative myocardial infartion rate is still high at approx. 6%. in cardiovascular surgery it is well known that various cardioplegic solutions are employed for myocardial protection during the ischemic phase. in order to evaluate the possible influence of these solutions we selected two of the most commonly used cardioplegic solutions for investigation in a randomised double-blind study: htk (group 1) and st. thomas (group 2). after randomisation each group consisted of 20 patients who had to undergo aortocoronary bypass surgery. aim of the investigation was to establish possible varying cellular changes during the reperfusion phase or in the early operative phase in order to be better able to apply reinforcing clinical measures. in the context of this study the classical enzyme-diagnostical methods ck,ck-mb and ldh as most useful, however not as convincing. still, we have in the meanwhile been able to show that the cardiac muscle troponin t proves a particularly sensitive parameter regards differentiated ischemic damage to the myocardium. ~his we were able to conflrm in extensive preliminary trials. cardiac troponin t was registered with a one-step lmmunoassay using two highly specific monoclonal antibodies directly via two different epitopes of cardiac troponin t. simultaneously the corresponding pre-and postoperative ecg was registered. further, within this context we investigated parameters that indicate cellular damage, such as platelet factor 4 (pf4), t-pa, interleukin-6 and pmn-elastase. in the reperfusion phase in group 2 there is a significant rise in tmponin t while in group 1 these values remain practically unchanged up to the 1st. postoperative day. of special importance is interleucin 6 since according to most recent studies the release of this substance leads to platelet activation via the arachidonic acid metabolism. this pathway must, further, be regarded within the context of free radical formation. on the 1st. postoperative day the 11 6 values in group 2 are significantly higher. the effects of membrane damage is also observed via pf4 and the pmn-elastase to be different in both groups. on the basis of this study we arrive at the conclusion that the htkcardioplegia is essentially less damaging than that of the st. thomas solution. (2) r. hetzer (2) (1) department of hematology and oncology, vimhow klinikum, humboldt university, berlin, germany (2) we investigated the influence of two different vad systems on these hemostatic changes. vads were implanted in 18 patients [11 bi-vad (berlin heart), 7 left vad (novacor n 100)] with end-stage heart disease who were awaiting heart transplantation. the following hemostatic parameters were measured during the first 51 days of bddging or until heart transplantation: thrombin-antithrembin iii (tat) complexes, prekallikrein, factor (f) xll, plasminogen, or2 -antiplasmin, and i?,thremboglobulin. results: during the first week of bridging, significantly higher tat levels were observed in novacor patients compared to berlin heart patients. prekallikrein activity levels were significantly lower in the berlin heart patients in the early bridging period. all other parameters were comparable in both groups throughout the entire observation period. differences in hemostatic parameters became apparent only in the early bridging period with more enhanced pmthrombin activation in the novacor group and more prominent contact activation in the berlin heart group. avoidance of the transmission of viral infections and saving in the use of blood products encouraged the use of apparatwe intraoperative autetransfusion techniques. patients and methods: arer randomization apparative intraoperative autotransfusion was performed in 5x7 patients during elective hip surgery using i-iaemonetics cell saver ill, haemonetics cell saver v, electromedics elmd, haemolite 3 and fresenius continuous autotransfnsion system (cats). at defined tmaes we detenmned a lab panel (clinical chemistry, lipids, proteolytic capacity, hemolysis, coagulation panel) at 9 determination points in the reservoir, the retransfused blood and in the patient. results: no significant differences concerning proteolytic capacity, prothrombin time, platelets, lipids, electrolytes. increased hemolysis (p<0.01) in the hcs iii group vs. the other groups (lo rain. after application of the retransfnsed blood). low heparin concentrations of retransfused blood in the hcs iii group( 0.32+-0.3 u/ml) vs. high concentrations in the cats group (0.47 +-0.3;p--0.01). parameters of thrombin generation were elevated in the hcs iii group vs. the other groups (p=0.02). conclusions: the use of 5 different apparative autotransfnsion systems dunng elective hip surgery results in dysturbances of hemocompatibility. the activation of the coagulation system during the collection and filtering is partly influenced by the elimination kinetics and the dose regime of heparin. however intraoperative autotransfusion must be roan~ged very carefully and possibly adverse effects of perioperadve heparin peak levels have to be considered. little information is available on the management of patients with factor viii deficiency who require cardiac surgery. we report the case of a 54 year old man with factor viii deficiency and combined severe aortic stenosis and incompetence and mitral incompetence who underwent a double valve replacement at our institution. he had a history of several bleeding episodes following minor surgery. previous factor viii levels were between 8 and 26%. using standard cardiopulmonary bypass, a double valve replacement with a 23 and 29 mm bileaflet prosthesis in aortic and mitral position, respectively, was performed. a high dose aprotinin regime was used (5.5 x 10 a iu). three doses of factor viii concentrate were given in the perioperative period, totalung 7000 1u until the 1st postoperative day. repeated measurements of the factor viii level were performed. the postoperative chest tube drainage was 350 rot. until the 4th postoperative day an additional dose of 3000 iu of factor viii was given to maintain a level of at least 30%. the obligatory anticoagulation was achieved initially with heparin i.v. in therapeutic dosage. due to a persistent 3rd degree av block a permanent pacemaker was inserted with additional 2000 iu of factor viii. on the 17th postoperative day warfarin was commenced aiming for an inr of 3.0 -3.5. the patient was discharged home therearer. he was trained to monitor his inr with a coagu chek device. no bleeding episode occurred during the first 3 months follow up. open heart surgery can be performed safely in patients with factor viii deficiency with the use of factor viii concentrates and monitoring of factor viii levels. coating of biomaterials was developed using synthetic polymers with incorporated anticoagulants. stents were coated with a thin layer consisting of a polylactide polymer containing peg-hirudin and a stable prostacyclin analogue. these materials were tested with a ,,human shunt model" using nonant/coagulated blood of healthy volunteers. within minutes uncoated stents were covered by fibrin and aggregated platelets, which could be seen macroscopically and by scanning electron microscopy; coated stents were free from coaguiation plugs. this observations were supported by analysis of coagulatiuon activation markers. unlike coated stents, uncoated stents revealed high levels (>detection limit) of tat complexes and prothrombin fragments (f1-2). in a series of experiments stents were tested in sheep. in 16 sheep stents (coated/uncoated patmaz-schatz stents) were ptaced by conventional techniques in the left anterior descending artery. anticoagulant therapy consisted of a heparin bolus and intravenously given aspirin before stent implantation. no ant/coagulation was given thereafter. existing data show hyperplasia in the area of uncoated stents which was reduced around coated stents (this study will be finished in january 1996). this coating technique with incorporated anticoagulants reduces thrombogenicity during the early and late phase of biomaterial implantation. studies concerning catheters, vascular prosthesis and oxygenators are in progress. the mechanical circulatory support (mcs) is a therapy for patients (pts) with endstage cardiac insufficiency. during mcs thrombeembolic events, due to the surface thrombogenicity of the implanted device, are feared complications. activated blood platelcts play a major role in this context. therefore, patient's platelet morphology was investigated. during the period of mcs, using the novacor left ventricular assist system n100, blood samples of 8 pts were observed by means of scanning electron microscopy (sem). blood was collected preoperatively and after implantation daily during the first week as well as weekly for the first 3 months. samples were drawn via an 18gauge cannula into caeodylic-acid buffered glutaraldehyde and platelets were prepared for morphological investigations. platelet alterations were classified as non activated, activated and aggregated, based on "shape change" morphology. additionally, the common blood coagulation parameters were evaluated. preoperatively, 15.0 + 4.6 % of activated platelets were found. within the first postoperative week, the mean level of activated platelets raised to 32.8 + 8.0 % (p<0.05). comparing short-(<30 days) vs. long-term (>30 days) mcs, a significant difference of activated ptatelets (overall mean values) could be seen (24.3 +_ 3.3 % vs. 34.8 _+ 3.4 %, p=0.004). during mcs a correlation between hemolysis and platelet aggregates, as well as the values of activated dotting time and activated platelets were observed. also, specific platelet deformations and damages appeared during mcs, which could not be found preoperatively. all pts with mcs showed alterations of their platelet morphology induced by the activation of the implanted synthetic material. with regard to the postoperative antithrombotic therapy, these observations should be taken into consideration. during extracorporeal circulation (ecc) the blood and its compenents are exposed to artificial surfaces and inflammatory respenses are activated, especially the complement, coagulation, fibrinolytic and kallikrein systems. furthermore leukocyte activation occurs and platelet function is impaired. these humoral and cellular systemic responses are known as the "pustperfusion syndrome" with clinical symptomes like lenkocytosis, increased capillary perraeability, accumulation of interstitial fluid and organ dysfunction. the impertance and even perhaps the existence of the damaging effects of cpb have been widely debated in the literature over the past 30 years. many efforts have been made to reduce traumatizing factors, e.g. the use of membrane instead of bubble oxygenators. recently, heparin-coated equipmen~ and tubings have been proposed to avoid excessive contact activation during cpb, the here presented study was designed to assess changes in coagulation and flbrinolytie activity in 20 patients undergoing cpb. in this regard we investigated coagulation parameters like fibrinogen, antithrombin, pmthrombin-fragments fl+2, thrombin-anthhmmhin complex, tissue-factor, fibrin-monomeres and parameters of the fibrinolytic system like tissue-plasminogen-activator, plasminantiplasmin-complex, d-dimers and plasminogen-activator inhibitor before, during and after cpb. the activation of the complement cascade was followed by measuring the concentration of c5a, c4 and c3c. the results demonstrate distinct alterations in above mentioned parameters. in spite of a high dose hepariulzation (act>450s) combined with an antifibrinolytic tw, atment an activation of the coagulation system was observed immediately after the onset of cpb followed by an activation of the fibrinolytic system. therefore further efforts should be done to develop new anticoagulatory regiments and improve the biocompatibility of materials used for cpb. during cardiopulmonary bypass blood is exposed to nonphysiologic conditions. the contact with artificial surfaces and mechanical stress result in a periopemtive response which includes activation of the complement, coagulation, fibrinolytic and kallikrein system, activation of nentrophils with degranulation and pmtease enzyme release, oxygen radical production and the synthesis of various proinflammatory cytokines. this so-called "pest-pump intlammatory response" has been linked to respiratory distress syndrome, renal failure and neurologic injmy. our goal was to investigate the time course of eytokine levels and the activation of leukozytes and platelets and to quantitate leucocyte subpepulatioas in 20 patients undergoing cpb. at different time points, pre, during and pest cpb, we determined the levels of interleukin (il) 113, il-2, il-4, il-6, il-8, il-10, tumor necrosis factor ¢z (tnf-a) and interferon "1' (ifn'--/) using elisa-techulques. lymphozyte subpepulations were characterized by flow cytometry and specific monoclonal antibodies against cd3 (pan t-cell marker), cd4 (surface antigen on t-helper cells), cd19 (surface antigen on b-cells), monocytes were determined by cd14 and platelets by cd41 (act. gpilb/llla) and cd42b (gp ib). single cell activation was analyzed using markers against cd25 (il-2 receptor), cd126 (il-6 receptor), hla-dr (mhc class ii), cd71 (transferrin receptor) and cd69 (activation inducer molecule), platelet activation was monitored with an antibody against cd62 (gmp-140). preliminary results revealed distinct increases in r,-6, il-8, and il-io following cpb whereas tnf-a and ifn--/levels were not significantly influenced. fttnhermore, activation of particular cell populations was observed. finally, our investigations should contribute to a better understanding of the complex humeral and cellular respenses induced by cpb and thus might help to develop new strategies to circumvent the negative impacts of cpb. optimal adjustment of anticoagulation in machine plasmapheresis is important for the quality of the prepared fresh frozen plasma (ffp) as well as for the safety of the donation. in the present study the suitability of prothrombin fragment ( ft+2 ) in the assessment of anticoagulation during plasmapheresis was investigated. matarlal and methods: 75 plasmapheresis procedures were performed on 25 donors (10 ~, 15o" ) using 3 different plasmapheresis machines (a 200, baxter; mcs 3p, haemonetics; pph 900, electromedics/medtronic). acid citrate dextrose formula a (acd-a) in a ratio to whole blood of 8 : 92 was used for anticoagulation. the concentration of fi+2 in the donor's blood was measured before and after plasmapheresis and in the prepared ffp. the actual acd-a volume used was also registered. results: there was a significant rise of the ft+2 -concentration in the donors blood after plasmapheresis with each of the three automatons: a 200:1.32 vs 1.14, p < 0.05; mcs 3p: 1.26 vs 0.98, p < 0.05; pph 900:1.20 vs 1.05, p < 0.05. the ffp prepared with each machine showed the following f~+2concentrations: 0.91± 0.18, 1.0:2 ± 0.17 and 0.93 ± 0.11 respectively. the difference between the groups was not significant. the elevation of the ft+2 -concentration in the donor's blood showed a negative correlation with the volume of the acd-a used. during 6 of the 75 procedures technical problems occurred (inadequate venous acces, occlusion of the citrate tube, reduced whole blood flow). after these procedures there was a marked elevation of f~+2 in the donors blood (2.74 ± 0.53), accompanied by an elevated f~+2 -concentration in the prepared ffp's. conclusion: these data show that ft+2 is a suitable parameter for the assessment of anticoagulation during plasmapheresis. several epidemiologic studies demonstrated that fibrinogen is an independent cardiovascular risk factor and should be considered for screening programs. prothrombin time derived fibrinogen (df) measurement combines the advantage of an established highly reproducible automated method with no additional reagents, except for calibration. several studies showed that the df values correspond well with the clanss method except in cases such as thrombolytic therapy in which the df results are higher. however, no results exist whether in patients with coronary heart disease with fibrinogen as a risk factor the df values are also comparable to the established clausss method. the aim of our study was to compare df values to clauss method results in cardiac patients, especially in patients before and after coronary bypass grafting (cab(]). measurements of df were performed on an acl 3000 (il) using the pt-fibrinogen-hs reagent. fibrinogen clanss method was done on the acl using fibrinogen c reagent (il) and on a kc4 (amelung) with fibrinogen a reagent (boehringer maanheim). for calibration we used the calibration plasma half volume (it.) with the fihrinogen concentration proposed by the manufacturer. plasma samples were obtained from 24 patients at admission before cabg and postoperatively up to 1 week, and from 23 healthy persons (staff). within assay imprecisious using normal and abnormal controls (il) were comparable with both methods showing cvs between 1.99 and 4.22 %. in normal healthy persons the medians of the df and the clanss method run on the acl were very similar (296 vs 302 rag/all), whereas kc4 values were about 10% lower (268 md/dl). in cabg patients at admission we found the same differences as in normals with the clanss method (acl: 363 vs kc4: 337rag/all), however the df values were siginficantly higher (median 418mg/dl). if we took a cutoff value of 320 mg/dl, as suggested by the results from the northwick park heart study, we would categorize into the high risk group 21 out of 24 patients using the df method, 20 with the clanss-acl method and 16 with the clanss-kc4 method, i.e. nearly 30% more patients were classified in the high risk group using the df method. postoperative samples showed the expected increases due to the acute phase response with the same magnitude of differences. because of its rapidity and reproducibility the df method is well suited for routine measurements, however, standardization remains an urgent task in order to avoid misinterpretation of results. for fibdnogen measurements in clinical laboratories, the two most widely used methods are the clotting time method according to clauss (cfib) and the sn called "derived" fibrinogen method (dfib) implemented in optical coagulometera with the fibrinogen concentration being derived flora the optical density of the fibrin clot in a standard prothrnmbin time (pt) assay. it is well known that under certain circumstances, e.g. in the presence of fibrin(ogen) degradation products (fdp), there is a discrepancy between the two methods with higher values for dfib than for cfib. yet the opposite discrepancy, i.e. fibrinogen values derived from the optical density of the clot grossly lower than values from dotting time assays, seems to be very rare and is poorly understood so far. the patient (male, 26 years) had ingested the esterase inhibitor parathion (e605) in an attempt o f suizide and was treated with high doses of atmpin. he had no clinical signs or history or family history of bleeding or thrombotic disorders. except for a very low pseudocholinesterase activity, all laboratory results were normal ineinding pt, afft, thrombin time, and factor xiii. pt and aptt did nnt differ between an optical coagulometer (electra 1000c, mla) and a mechanical one (kc..4, amelung). there was no evidence of disorders known to interfere with hemostasis like paraproteinemia or dyslipldemia. however, in all 7 blood samples received for dotting tests during a period of 7 days the macroscopic appearance of the fibrin clot was quite unusual (only slightly turbid/almost transparent) and there was a striking discrepancy between a very low or low dfib on the electra (pt reagent: thromboplastin is, dade) and a normal or high cfib (kc4; thrombin reagent, dade). on admission, values were 57 mgml (derived) vs. 275 mgldl (clauss). cfib rose to s42 mg]dl with dfib at 155 mg/dl in the last sample on day 7. ~ al! samples dfib was about 20 % (ls-23) of cf[b. when the patient's plasma was added m normal pooled plasma it caused, in a dose-dependent manner, values lower than predicted for dfib and values slightly higher than predicted for cfib. in the absence of data from additional (e.g. immunologic) methods the following principal possibilities (and combinations) have to be considered: 1) normal fibrinogen concentration and clot formation rate, but abnormal optical properties of the clot (cfib correct, dfib falsely tow); 2) normal optical properties of the clot, but accelerated clot formation and very low fibrinogen concentration (dfib correct, cfib falsely high). in either case, the molecular basis could be: a) a genetic or acquired molecular abnnrmality of fibrin/fibfinogen; b) an interfering substance. direct effects of the loxic agent parathion and/or the antidot drug atropin are not likely to be the cause since other patients, often with more severe parathion inmxicatian requiring higher doses of atmpin, showed normal optical density of the clot. we hope to perform a more in depth investigation of this abnormality in the future, including various methods, reagents, and instruments for fibrinogen measurement, a survey of the patient "s family, and studies of the molecular nature of the phenomenon. increased fibrinogen is known to be an independent predictor of subseqtmnt acut~ coronary syndromes. however. a multitude of methods for fibrinogen determination is available. there is a lack of standardisation among fibrinogen assays. in a family cohort study (patients'with combined hyperlipidaemia and f or hypemricaemia) fibrinogen was determined in plasma samples from 340 family members using a functional and an immunochemical assay. the fimctional assay according to clauss was performed on the analyser ca 5000 using the test fibrinogen a from boehringer. the immmmephelometric assay was performed on ~e behring nephelometer system using the reagent and standard from behring. a good similarity between both assays was obtained at low and high flbrinogen levels as well as in samples with increased c-reactive protein (crp). values obtained by both assays correlated similar with total cholesterol, ldl--cbelesterol and apolipeprntein b. the ratio functional fibrinagen / immlmochemial fibrinogen showed no dependence on cholesterol, t-pa, v wiuebrand factor and crp. release of two fibrinopeptides a from fibrinogen generates desaa-fibrin monomer, which rapidly aggregates, forming fibrin complexes. fibrin monomers can be detected in plasma samples after chemical desaggregation of fibrin complexes using thiocyanate by monoclonal antibody binding to the alpha-chain neo-n-termini generated by fibrinopeptide release. although postulated, an intermediate of fibrin formation, carrying one fibrinopeptide a and one fibrin alpha-chain neo-n-terminus has so far escaped analytical procedures. we have employed a monoctonal antibody specific for fibrin alpha-chain neo-n-terminus, mab 2b5, attached to magnetic microparticles, for isolation of fibrin-related material from plasma samples of patients with elevated soluble fibrin. the material was desorbed by sds-urea buffer and subjected to sds-page and immunoblotting. immunostaining with panspecific anti-fibrinogen and anti-fdp-e antisera showed a range of bands corresponding to fibrin monomers, and fibrin derivatives containing the fibrin e-domain. lmmunostaining with monoclonal anti-fibrinopeptide a antibody resulted in a doublet band corresponding in size to fibrin monomer. similar results were obtained with polyclonal antisera against fibrinopeptide a. for a more quantitative approach, desa-fibrin monomer was detected by an elisa procedure using mab 2b5 as capture and monoclonal anti-fibrinopeptide a antibody as tag. a sample with extremely high level of desaa-fibrin monomer, determined by elisa (enzymun®-test fm) was used for calibration, since reference material is not available. a correlation of r=o.g4 was found between desaa-fibrin monomer and relative desa-fibrin monomer levels. detection of desa-fibrin monomer required sample pretreatment with thiocyanate for desaggregadon of fibrin complexes. from these preliminary data it appears that desa-fibrin monomer accounts for a fairly constant proportion of soluble fibrin and is a polymerizing species. fibrinogen has been shown to be a major cardiovascular risk factor. especially for epidemiological studies, exact quantitation of fibrinogen in clinical plasma samples is of great imporance. fibrinogen levels are generally measured by clotting assay according to clauss, or by determination of derived fibrinogen values upon photometric measurement of prothrombin time (derfbg). the clotting assay has been shown to be influenced by high levels of soluble fibrin derivatives. the pt-derived fibrinogen levels appear rather convenient in clinical routine, since no additional reagents are needed. we have compared the clauss assay and derfbg with a turbidimetric fibrinogen assay using snake venom protease for fibrinopeptide release, performed in photometric autoanalyzers. d-direct antigen was measured in parallel using tinaqaant d-dimer lpia. results were correlated with total fibrinopeptide a release by thrombin, measured by elisa. a total of 484 samples were included, of which 29 samples (6 %) were recorded as above measurung range by derfbg. these samples encompassed a range of 5.90-10.40 g/l and 5.21-12.37 g/l in clauss, and turbidimetric assay, respectively. the range of values measured by derfbg assay was 0.72-9.14 g/i, corresponding to 0.26-11.00 gll and 0.24-10.48 g/1 in the clauss and turbidimetric assay, respectively. the correlation of derfbg with the clauss assay was re0.91, correlation with turbidimetric assay was r=0.92 for the values actually detected. the correlation between clauss and turbidimetric assay was r=0.93 for all values. there was no dependency of test results or inter-test variation upon d-direct. correlation graphs displayed a decreased test response of clauss assay in the high concentration range, resulting in an underestimation of fibrinogen concentration. the derfbg assay, in contrast, showed normal range values in samples from patients with fibrinotytic treatment and low fibrinogen levels in the other assays. correlation with fibrinopeptide a release was r=0.88 for clauss assay, r=0.89 for turbidimetric assay, and r=0.82 for derfbg. for clinical routine, derfbg appears to be applicable for all samples between 1.00 and 5.00 g/l with exclusion of samples from patients with fibrinolytic treatment or endogeneous hyperfibrinolysis. other samples may be analyzed by clotting assay or turbidimetric assay, although the latter appears to be more suited for measurement of high range samples. for inhibition of pk is 0.067 pmol/l the antifibrinolytic activity of the inhibitors was determined by measuring the lysis of radiolabelled human plasma clots• the compounds which inhibit plasmin and pk influence remarkably the streptokinase-induced clot lysis but not lysis induced by uk and tpa. surprisingly, inhibitors of uk and tpa do not influence clot lysis induced by uk or tpa. the structure-activity relationships for inhibition of ptasmin, uk, tpa and pk could help in the design of more potent inhibitors of fibrinotytic enzymes. uk inhibitors are of interest for the development of anti-invasiveness drugs, while plasmin/pk inhibitors could be prototypes of a "synthetic aprotinin". in the ecat angina pectoris study t-pa antigen was an indepcndem risk factor of subsequent acute coronary syndromes. pat indicates the risk bat depends on other known risk factors. it should be tested in 183 members of a family cohort study (patients with combined hyperlipidaemia and / or hyperuricaemia), if the active pal antigen or the whole pai antigen showed a stronger relation to t-pa and metabolic variables. the active pall antigen was determined using elisa actibind pat-1 (technoclone / lmmuno) , the whole pai-i antigen was measured using the f_lisa pat-1 (technoclone i immuno). t-pa activity was determined with the coaset t-pa from chromogenix, the tintelize tpa from biopool was the used test for determination of t-pa antigen. the active pat antigen showed a stronger correlation to t-pa activity and t-pa antigen than the whole pal antigen. circulating t-pa activity was influenced predominantly by the active pal antigen. both pat antigens were correlated in similar manner with metabolic variables, lipoproteins and b/vii. table: correlations of active and whole pal antigen (** p < 0,001) active pal antigen whole pal antigen active pat antigen 1,000 0,851 ** whole pal antigen 0,851 ** 1,000 t-pa activity -0,594 ** -0,492 ** bpa antigen 0,604 ** 0,497 ** body mass index 0,502 ** 0,462 ** triglycerides 0,452 ** 0,441 ** total cholesterol 0,252 ** 0,255 ** ldl-cholesterol 0,263 ** 0,264 ** hdl-cholesterol -0,357 ** -0,355 ** apolipoprotein b 0,428 ** 0,402 ** apolipoprotein a i -0,233 ** -0,211 ** the lower relationship of the whole pat antigen to t-pa is obviously caused by patient samples with high levels of whole pat antigen in contrast to normal values of active pat as well'as of t-pa. possibly, a high ratio of whole pai antigen / active pat antigen is caused by a raise of latent pal the main form of pat in the platelets. the clinical importance of an increased ratio whole pal antigen / active pal antigen remains under investigation. the cyclic antibiotics-polypeptides bacifracin a, bacilliquin from boci/lu~, licheniformis and gramicidin s from bocil/us brevis, var. (3. b., were used for investigation. we studied their influence on the fibrinoly~c and coagulation activity in vitro• me~hods. to solution of human plasmin (thrombin). containing 0.2 mg of protein (1 nih unff)/ml, the analyses' solution of antibiotics (0.1-8,0 mg) was added. then we defined the tlbrinolytlc activity of the mixes using azofibrin lysis, and fhrombin activity was determined according to the speed of fibrin clots formation from fibrinogen solution. results. in following table are submifled the results received in our laboratory {we also offer results of antibiotics influence on urokinase activity): ki, mm ki --the constant of inhibition. n. d. ~ in studied lirnils the inhibitor's activity was not observed. ---the inhibitor's activity was not define. i. --the inhibitor% activity was observed but ki not determined. +, +% +++ --effect of inhibffion (in rela*iive indexes). conc/us/on.~ the results received by us testify to the necessity of cautious approach to the use of antibiofics-polypeptides for various sorts of therapy in view of their possible influence on fibrinolytic and coagulation actlvlfy, of the organism. these results were used for preparation in our laboratory of biospeciflc sorbents containing c-ramicidin a, bacil}iquirt and gramicidin s.as ligands, they can reversibly bind thrombin, plasmin {plosminogen) and urokinase directly from crude exkacts. the enzymes are selectively eluted without substantial losses of specific activity in e yield of 60-90%. there is a great body of rather contradictory informations dealing with fibrinolysis in liver.. cirrhosis, which can be accelerated, normal or reduced, depending on the type of cirrhosis and investigation techniques (clot-lysis, fibrinolytic component measurements). our previous finding was, that in vitro plasma-clot lysis, induced by exogeneously added tpa or streptokinase proved to be reduced, and this had a good correlation with severity of the disease and the elevation of plasmatic yon willebrand factor levels. in vitro clo~/-[ lysis tests, induced by tpa were performed in 41 patients with alcoholic liver cirrhosis, utilising a microplate light-scattering assessment method. the tests were repeated using the same plasma samples in each patients with a microplate which was covered by cultured endothelial-cell monolayer (umbilical vein, huvec}. clot lysis speed proved to be 1.5-2 times slower with huvec milieu in the control group, while in the cirrhotic patients this inhibition was stronger and resulted in 5-fold reduction of lysis speed. our results suggest, that cirrhotic plasma is able to accelerate the release of fibrinolytic inhibitors from cultured endothelial cells, which phenomenon may also contribute to the complex alterations of in vivo fibrinolysis in cirrhotic patients. deep vein thrombosis (dvt) is a systemic disease with prolonged clinical manifectation. anticoagulation therapy in dvt is not completely effective. thrombolytic therapy may give rise to a systemic lytic state, the fibrinospesific agents (scu-pa and t-pa) have short half-lives in the circulation. we investigated the potency of the acylated plasminogen streptokinase activator complex (gbpg-sk) to deep vein clot dissolution as compared to well known sk and apsac both in v~tro and in vivo in the model of venous thrombosis in artherio-venous shunt in rats. it was shown in in vitro study that fibrinolytic activity of plasminogen activators mainly depends on their stability in plasma. stability studies carried out by incubating sk and pg-sk activator complexes in plasma with euglobulin precipitation . total fibrinolytic activity was measured by the fibrin plate method. gbpg-sk possessed the greatest stability in human plasma than apsac or sk because of its prolonged inactivation period (the deacylation half-life for gbpg-sk was 230 :e 21 rain in contrast with 73 -~ 6 min for apsac). the stability degree of two acylated thrombolytics (gbpg-sk and apsac) was in order to inverse proportion of their first order rate deacylation constants (2.9 • 10 -4 and 6.0 • 10-s sec-1 respectively). the fibrinolytic potency of sk, apsac and gbpg-sk was measured by 1251-labeled fibrin clot lysis in plasma and in vivo by lysis of the preliminary formed 1251-labeled fibrin clot inserted into the jugular vein. fibrinolytjc activity of acylated plasminogen activators gradually increased in time. under sk administration, the clot lysis came to the end by 2 hours while apsac and gbpg-sk haven't lost their activity for 5 -6 hours. gbpg-sk possessed significantly more prolonged fibrinolytic activity than apsac, the acyl-enzymes did not significantly influence on plasminogen,,.~2-anfiplasmin and fibrinogen levels in plasma according to their activity specific to fibrin-bound plasminogen. in opposite, sk produced a significant depletion of plasminogen, ~-2antiplasmin and fibdnogen levels in plasma. it seems, on the basis on in vitro and an animal experimentation, than apsac with its moderately fast deacylation rate is more suitable for rapid thrombolytic effect, but gbpg-sk with its slow deacylation rate is suitable for deep vein thrombosis, when the rapid thrombolysis is less critical. it's well known that the complete lysis of thrombi usually isn't observed at the thrombolytic therapy. at present study we have attempted to quantify the possible mechanism of fibrinolysis inhibition during the thrombolysis. 125i-labelled partially cross-linked fibrin clots of different volume (0.1-0.35 ml) were immersed in tris-hcl buffer (3 ml) containing plasmin (5-100 nm) at 37°c. the lysis rate was detected by counting of soluble fibrin degradation products (fdp). at all the eases lysis slowed down and stopped in 3 hs though clots dissolved up to only 60-85%. no irreversible inlaibition of plasmin caused by denaturation occur as was judged by the measurement of fibrinolytic activity at the diluted samples. however the increase of fdp concentration in surrounding buffer led to the reversible inhibition of fibrinolytic activity of plasmin up to 5% of baseline. the sds-page analysis under non-reduced conditions shown the acoumulation of high-molecular weight fdp at the surrounding buffer. the inhibition phenomenon could be connected with the specific binding of plasrnin with soluble fdp having exposed lysine residues and the subsequent removal of enzyme from fibrin surface. unexpectedly since the heterogeneous character of occurred reactions tile change of the clots surface area during lysis didn't affect the fibrinolysis kinetics in all the concentration intervals. to estimate the kinetic parameters the kinetic curves were linear in the coordinates [p1/t (l/t*ln(isl°{(lslo.lpi)). the obtained parameters were following: keat=l.36 min-l,km=l.33 ixm,kp=0.12 ~tm. the clinical trials have shown that fdp concentrations at the thrombolytic therapy of deep venous thrombosis and acute myocardial infarction usually was approximately in the range 0.05-0.2 ~tm. therefore the described phenomenon of fibrinolysis inhibition by formed fdp may take place during thrombolytic therapy. al. calatzis, an. calatzis, +m. klmg, +l. mielke, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology technische universit~.t monchen thrombelastography (teg) is an established method for the detection of fibrinolysis. fibfinolysis is usually determined when the teg amplitude decreases by more than 15% atter the maximum amplitude is reached. this takes a considerable amount of time (more than 30 minutes). our approach bases on the understanding of fibrinolysis as a process which runs in paraue[ to coagulation and is not exclusively subsidiary to it. the effect of fibrinolysis on the growing clot in the teg is shown by the comparison of two parallely performed teg measurements: exteg: teg measurement with standardised activation of the extrinsic system. apteg: exteg with in-vitro-fibrinolysis inhibition via aprotinin. exteg-reagent (ex): 1:2 dilution of innovin (recombinant thromboplastin reagent, dade) with aqua dest. apteg-reagent (ap): 5 parts innovin, 2 parts trasy[ol (aprotinin, bayer, i0.000 kie/ml), 3 parts aqua dest. test procedure: l0 p1 ex or ap + 300 ~l citrated blood (cb) + 50 lal cacl2-solution 0,15 m. the only difference of the two reagents is the addition of 20 kie aprotinin in the apteg, leading to an in-vitro fibrinolysis inhibition. the usage of disposable pins and cups (haemoscope, illinois, usa/e.m.s., vienna) is recommended for ensuring standardised conditions for both measurements. results and discussion: when there is a better clot formation in the apteg (corresponding to a lower so-cafled k-value) than in the exteg, fibdnolysis can be suspected. this technique requires only commercially available reagents and is easy to perform on conventional teg systems. due to the standardised coagulation activation with a thromboplastin reagent, fibrinolysis can be detected also when inhibitors like heparin are present in the circulation. according to our experience using this technique during liver transplantation, clinical relevant fibrinolysis can be detected as described in less than l0 minutes. many thromboembolic (massive pulmonary embolism, proximal deepvein thrombosis, etc.) and coronary diseases (infarction, acute phase, etc.) require fibrinolytic therapy to early recanalizafion. the application of the well-known or new thrombolytic agents needs the use of specific, simple and reproducible methods for the determination of fibdnolyfic activity. we suggest new methods for measuring the blood plasma concentrations of plasmin, plasminogen, antiplasmins, and urine urokinase activity. these methods involve the employment of chromogenic substrafe azofibrin (human fibrin, covalently labeled with p-diazobenzenesulfonic acid). method~. 0.2 ml of studied solution was added to 0,8 ml of azofibrin suspension in certain buffer (5-10 mg/ml) and the mixture incubated at 37 oc for 10-60 rain. after the end of incubation the mixture was filtered, the volume of solution brought up to 4 ml by 0.02 m naoh and the optical density was determined at 440 nm. resuffs. azofibrin can be used for quantitative determination of proteinases activity in search of new fibrinolytic means. for comparison the results of our studies fibrinolytic activity of some proteinases with the use of azofibrin are presented: activity. with an increase of pal and ldl-and a decrease of hdl-cholesterol concentrations k is concluded that the increased cardiovascular risk in diabetes meilitus was partly caused by a down regulation of the fibrinolytic system, increase of erythrocyte aggregation and plasma viscosity. also disturbances of lipid metabolism an abnormal whr seems to be of an additional atherogenous factor in dm. plasma concentrations of thrombin-anfithrombin-iii (tat), alpha-2antiplasmin-plasmin (app) complexes and ddimer were investigated in 50 patients treated with thrombolytic therapy for acute myocardial infarction (ami) either with streptokinase (n=24), urokinase (n=16) or recombinant t-pa (rt-pa, n=10). all patients received an intravenous heparin bolus of 5,000 iu on admission, which was followed at once by an infusion of 1,000 iu/hr for the next three days titrated to maintain the partial thromboplastin time at twice control value. tat, pap and ddimer were measured by enzyme immunoassay on admission, 1, 2, 4, 6, 8, 12, 24 hours and on day 3 and 7 after admission. groups did not differ significantly in regard to age, sex, delay and infarct location. on admission, no marker differed significantly between groups. thereafter, tat levels increased significantly exclusively in rtpa treated group. from 2 to 6 hours after admission, tat were significantly higher in rtpa treated patients than in streptokinase and urokinase treated group (p<0.02). however, during continous heparin infusion, which was started immediately after stop of thrombolytic therapy, in each group tat concentrations decreased below admission values. app were significantly higher only 1 hour after admission in the rt-pa group (p=0.03). ddimer did not differ signifieanfly between groups. our results demonstrate, that rtpa induces a hypercoagulable state, which may contribute to reocclusion after successful reopening of the infarctrelated coronary artery. the significant tat decrease during continous heparin infusion supports the concomitant use of thrombin inhibitors as adjunctive therapy with thrombolytlc treatment for ami. thus, in acute myocardial infarction patients, thrombin generation is markedly influenced by the thrombolytic agent used and concomitant heparin therapy. endothelium derived relaxing factor-no (edrf-no) plays a major role in regulation of vascular tonicity and also exerts platelet inhibitory action~ however, due to the chemical nature of edrf-no few is known about its production and activity as a general index or marker of vascular function in human diseases. one way to achieve this can be measurement of nitrate/nitrite excretion in the urine, which seems to reflect vascular edrf-no production. in this report a self-developed elisa method is described, which was used for this perpose. nitrate/nitrite urinary exretion proved to significantly decreased in insulin dependent and in non-insulin dependent diabetes mellitus as well after a comparison of the excretion values to other markers of angiopathy (yon willebrand factod soluble thrombomodulin, beta -thromboglobulin) it seems to be acceptable, that urinary nitrate/nitrite excretion can be a useful indicate of diabetic vascular disorders. two major concerns still accompany the application of prothrombin complex concentrates (pcc). viral safety has to be guaranteed and therefore several measures for virus inactivation or elimination are taken during the manufacturing process. the inherent risk of thrombo-embolic side effects has to be considered. to minimize these risks and to achieve good clinical efficiency the quality criteria for pcc's are under pending discussion. it is generally accepted that a modem pcc-preparation should contain all of the four coagulation factors in a well balanced proportion and that it should also contain protein c and protein s. additionally, the concentration of activated coagulation factors should be kept at a minimum. a present pcc-produedon process mainly consists of a qae-sephadex extraction of cryopeer plasma followed by a solvent/detergent virus inactivation step. further purification is achieved by subsequent chromatography on deae-sephamse. the aim of this study was to improve product quality by avoiding f viiactivation without implementing major changes to the production process. at the same time, a second virus eliminating step was added to the production process. it could be shown that speeding up the chromatographical process by switching the deae-sepharose-chromatography from a classical axial column to a radial chromatography resulted in a significant reduction of f viia-genemtion. mainly the reduction of contact time, resulting from the highest possible flow rates, leads to the wanted effect. the relation between f vii/f viia was 10 : 1 or more. in order to investigate the feasibility of virus filtration the eluate of the deae-sepharose column was filtered through a virus removing ultipor vffilter. the analysis of the solution before and after fillration showed that the filtration had no influence on coagulation factors activity, protein content, proteolytic activity etc. preliminary studies showed significant virus reduction values. in the past few years the problem of expediency of the treatment aimed at developing immunological tolerance in hemophil;a patients by way of complete removal of inhibitor with high doses of factor viii has been discussed in literature. we observed 121 patients with hemophilia. inhibitors to factor viii:c were revealed in 32.7 % of patients with hemophilia a and fo factor ix --in 1.6 % of patients with hemophilia b. the level of an inhibitor was not higher than 87 befhesda u/ml, that is those patients were not regarded as "high responders". a high incidence of inhibifors in young patients [from 7 to 26 years of age, 51.9 %) compared with older patients (from 27 to 40 years of age, 11.2 %) testifies to the probability of inhibitors development during treatment with modern concentrated preparation of factor viii, ix. inhibitor development in patients (40.5 %] in the course of antihemophilic concentrates transfusions is an evidence of alloimmunization of patients with proteins. the investigations show that in the course of transfusion therapy patients develop secondary immunodeficiency due to chronic antigenic stimulation of immune system with high doses of allogenic proteins. against the background of immunodeficiency patients with hemophilia develop complications of immune character: infections complications --53.9 %, aufoimmune processes --44.9 %, secondary tumours --1.2 %. plasmapheresis is the most rational method of removing inhibitor in patients with low level of inhibitor ("low responders", < 10 bu/ ml) and in patients with mean response. thus it should be noted that the treatment of patients aimed at developing immunological tolerance is not only expensive and economically unprofitable but also not indifferent fo the organism. in a recent multicenter study 73 previously untreated patientens (pups) with severe hemophilia a were treated with a recombinant factor viii concentrate (rfviii, recombinate©). during fviii treatment 21 (29%) developed inhibitors, 6 high titer (>5 bethesda units (bu)/ml), 4 low titer (<5 bu/ml) and 11 transient inhibitors. plasma samples from before treatment and during treatment but before inhibitor occurrence were available in 12 inhibitor patients. these plasma samples were analyzed by a highly sensitive immunoprecipitation (ip) assay for the presence of anti-tviii antibodies. in 9 (66%) a significant increase of anti-fv]]i antibodies was seen indicating the development of a clinical relevant inhibitor titer. this immune response occurred after 2 to 17 (median 5) exposure days (ed). in the same period only 3 out of 15 inhibitor patients showed a decreased in vivo recovery. in 16 pups who developed no inhibitors plasma samples from the entire treatment period were available. an immune response to rfviii treatment was seen in 7 pups after 2 to 43 ed (median 24 ed). the immune response was later and less pronounced in comparison to inhibitor pups before inhibitor occurrence. with the ip method the detection of an early immune response is possible which might be predictive for a later inhibitor development. the inclusion of the lip method should be considered for future multicenter pup studies. in the past anaphylactie reactions to plasma and plasma components have been a common complication of replacement therapy in patients with hemophilia a and b. we report on 3 severe bleeding episodes in 2 patients with hemophilia a and b, respectively. both patients had a history of life threatening anaphylactic reactions after exposure to different plasma derived clotting factor concentrations including intermediate purity factor viii-and factor ix-concentrate, respectively. high purity factor concentrates were tolerated well without any allergic side effects. a 67 years old patient with a moderate form of hemophilia a (f viii 4 %) had a history of severe immediate reactions with skin manifestations and bronchospasm after exposure to fresh frozen plasma, ctyoprecipitate and 3 different plasma derived factor viii-concentrates of intermediate purity. in all episodes pretreatment with corticosteroids and antihistamines was unsuccessfull in avoiding severe bronchospasm. replacement therapy with two different recombinant factor viii concentrates was tolerated well without any side effects. a 12 years old haemophiita b patient developed hypersensitivity reactions to prophylactic factor ix substitution, which could be overcome by using a factor ix .concentrate with improved purity. a recent recurrence of hypersensitmty under this treatment was finally overcome by the use of highly purified (monoclonal antibodies) factor ix concentrate. we conclude from these findings that high purity of factor concentrates, possibly due to the absence of soluble hla-antigens, are advantageous in patients disposed to allergic reactions. introduction: antibody formation against factor (f) viii remains one of the most severe complications of repeatedly transfused patients with haemophilia a. as reported previously in our study about the incidence of fviii inhibitors, we have observed a high incidence of fviii inhibitors among our haemophilia a patients. it is still not clear why certain haemophiliacs develop antibodies and others do not. a number of previous studies suggest that there is a genetic predisposition for the fviii inhibitor development. thus, the purpose of our study was to examine, if there is a correlation between fviii antibody-formation and genetically determined histoeompatibility antigen (hla) patterns in our haemophiliacs. patients and methods: hla-class i (a, b, c) and hla-class ii (dr, dq) typing was carried out for 51 respectively 44 multi-transfused paediatric haemophilia a patients (fviii:c activity < 5%), including 22 who had developed an antibody to fviii: 19 were high responders (> 5 bu), 3 were low responders (< 5 bu). hla-typing has been performed by a standurcl two-stage microlymphoc~.ftotoxicity procedure (drk frankfurt) using antisera with defiend hla-specifity (biotest diagnostica). results: we found an under-representation of hla-a2 in fviii inhibitor patients when compared with the subgroup without inhibitor. in regard to the hla-b and hla-c antigen frequencies there are no apparent differences between the groups. among the class ii antigens there were higher frequencies of dr1, drw52 and dqwl in the non-inhibitor group. however, the reduction in hla-a1, hla-cw5, hla-dqw3 respectively hla-dr4 frequency for inhibitor patients as reported previously could not be confirmed in our study. conclusion: so far it remains unclear if there is a significant association of a certain hla allels with the development of fviii antibodies. recombinant factor sq (r-viii sq, pharmacia) is a b-domain-deleted recombinant factor viii. it is formulated without albumin (hsa). the product has been shown to have in vitro and in vivo biochemical characteristics similar to a plasma derived full-length protein (p-viii). the international clinical trial programme was initiated in march 1993. pharmacokinetic studies have shown that the b-deleted r-viii sq should be given according to the same dosage principles as a full length p-viii. at present, the product is being tested in previously treated patients (ptps) and untreated patients (pups) with severe haemophilia a (viii:c < 2 %), both during long-term treatment (on demand therapy or prophylaxis) as well as during surgery. the long-term study in previously treated patients in germany was started in january 1994. thirteen patients have been included in 8 centers. all patients are still on treatment with r-viii sq, most of them receiving prophylactic treatment. global treatment efficacy has in general been considered excellent or good. no serious clinical adverse events related to the study product have been reported, nor have any inhibiting antibodies to factor viii or antibodies to mouse-lgg or cho-cell components developed in the patients. further results such as data on efficacy, half-life, recovery and safety will be presented in detail at the meeting. nowadays it is not sufficient to regard hemophilia only as hemorrhagic diafhesis of coagulation genesis, caused by deficiency or molecular anomalies of coagulation factor, without taking into account the immunity state. on examination of 125 patients (pts) (hemophilia a --110 pts, hemophilia b --11 pts, willebrandt's disease u 4 pfs) the development of immune complications was revealed in 34.4 %. chronic persistent hepatitis (3.2 %), chronic active hepatitis (3.2 %), herpes simplex (1.2 %), chlamidiosis (1.2 %), bacterial infection (7.2 %} were regarded as infectious complications. bacterial infections have a routine course due to preserved phagocytic function of neufrophils. and viral infections, whose ability to resistance is connected with t -cell link immunity, take on a chronic persistent course, mechanism of the development of autoimmune processes (autoimmune thrombocytopenic purpura --2.4 % of pts, immunocomplex disease --4.9 % of pts, the appearance of immune inhibifors --34.4 % of pts} is connected with the impairment of immunological surveillance over b -cells aufoimmune clones as a result of dysbalance in the system of t -lymphocyfes immunoregulatory subpopulations. lymphadenopathy and splenomegaly (4.9%) develop due fo benign proliferation of lymphoid tissue as a result of impairment of regulatory function of t -lymphocytes system, or they may be an evidence of virus infection. we observed one episode of acute leukemia. immune complications in hemophilia patients develop against the background of secondary immunodeficiency caused by chronic antigenic stimulation of patients' immune system with high doses of allogenic proteins, which plasma preparations contain. in immune complications hemophilia patients develop hemorrhages, whose pathogenesis is quite different from that caused by coagulation factor, so it should be taken into account in the course of treatment. control of hemophilia therapy classically was based on four parameters: life span expectancy of patients, orthopedic status (normal zero), pettersson score and social integration. oren, however, these parameters described an irreversible status with permanent damage particularly of the joints, especially when patients were grown-up. in order to establish risk-adapted therapy protocols to prevent hemophllic osteoarthropathies, quality control programs have to he set-up that allow for early adjustment of dosage and substitution frequency. here bleeding frequency is one the main parameters, being a clear hint for the possible development of a target joint. since 1988 we have established a computer database (haemopat) that contains data from all patients treated in our center. tables and graphs allow for early detection of increased bleeding tendency in a given joint, and accordingly for adjustment of therapy. the results of 8 years of measuring reasons of joint damage and not documenting the orthopathies as such will be demonstrated. parallelly a new program (haemopat win 1.0) will he introduced allowing for easier handling of data and their evaluation. this program will be used as of december 1995. in combination with a substitution calender to be filled in by all patients, in which factor concentrates, lot numbers, dosage, and date of administration will he constantly recorded, this program will extend our existing database in order to follow closely clinical and orthopedic parameters of each patient, and consequently acts as strict control of therapy quality. additionally, it provides sufficient data to fulfil any documentation needs, requested by medical authorities. the program will be available for all those interested free of charge. 2) kinderklinik der westf. wilhelms univ. mttuster 3-6) biotest pharma gmbh, dreieich haemoctin® sdh; the fviii sdh (sdh = solvent detergent and dry heat = 100 °c, 30 rain) from biotest pharma is a high purity (specific activity ~ 100) fviii concentrate manufactured from large human plasma pools. virus validation studies have shown virus inactivation/reduction (log 10) during the manufacturing process for lipid coated vints~ such as: h]v-1 > 16.2; psr > 16.8; vsv > 14,5; bvdv > 15.7; hcv > 4.5* and non enveloped vimsas such as: parvo** = 2.7; reo > 5.3*** and hav > 13.9. more than 50 hemophilia a patients (ptps = previously treated patients), baseline fviii activity < 1%, were included in an international drug monitoring study to follow their fviii inhibitur status. the hemophilia centers included were three centers from hungaria (helm pal children hospital and the national inst. of haematology, budapest and regional blood transfusion center, debrecen) and four centers from germany (two from berlin, one fraukfurffmain and one monster). patients were enrolled in the drug monitoring beginning aug. 1993. at the entry none of the patients had a detectable inhibitor. at the end of sept. 1995 there were no side effects or adverse events in connection with the use of haemuetin®. before the haemoctin drug monitoring study, the patients were treated with cryoprecipitate, or purified fviii products. inhibitor testing was done on patients plasma samples using the bethesda method. repeated fviii recovery determination at one time (between 12 to 24 hrs) after haemoctin® application demonstrated the expected recovery and normal half life time. none of the hemophilia a patients, treated with haemuetin® sdh developed a clinical relevant inhibitor. at the beginning of the stud)', the clinical efficacy of haemuetin® was studied in 16 hemophilia a patients and shown to give an in vivo recovery of 71 + 15 % by one stage assay and 77 + 17 % by a chromogenic assay. t ½ values were 13 + 2.8 and 12.7 + 3.2 hrs respectively. the study for the clinical efficacy of haemoctin® sdh was repeated in a group of 6 patients approximately two years later. although cd4 lymphocyte counts are known as reasonable predictors of prognosis in hiv infection, the cd4 count is not in all cases an infallible indicator of prognosis. therefore several serological markers are used to predict disease outcome, including beta-2 microglobulin (132m), immunoglobulin a (iga), lymphocyte counts (lymph) and others. in this study we followed a cohort of 23 haemophiliacs (19 with haemophilia a, 4 with haemophilia b) and 2 patients with severe von willebrands disease over a period of 28 months (mean, range: 22-34). testing for l~2m, igg, iga, igm, cd4 and cd8 cell counts (abs. and relat.), cd4/cd8 ratio, and absolute resp. relative leucocyte and lymphocyte counts was performed at least 3 times a year. at the same time clinical examinations and review of history were undertaken. mean of laboratory tests for every quarter of a year and significant changes during time of observation were calculated and correlated with clinical data. 1-4 5-8 9-12 13-16 17-20 21-24 cd41 440+956 344+924 278+925 302.-1:240 273.+.220 166+125 cd8 ~ 1165+474 1171+523 1236+1187 1017+439 930±412 873+478 1~2m z 2.0+0.6 3.0+1.0 3.0+1.2 3.0+1.1 3.5±1.0 3.5±1.3 lymph ~ 1.98+0.6 1.83+0.6 1.72+0.7 1.67±0.6 1.46±0.6 1.31±0.5 means/pl ± standard deviation means mg/l ± standard deviation during time ef observation we found significant changes of cd4 (abs. and relat.), abs. cd8 counts, cd4/cd8 ratio, f~2m, leucocytes and lymphocytes. the abs. cd4 and cd8 counts correlated clearly with lymphocytes und leucocytes counts but not with 1~2m. the prognostic value of the tested parameters is discussed by calculation of correlations with clinical data, anti-retroviral treatment and treatment of haemophilia. the availability of high purity factor concentrates has recently encouraged clinicians to use perioperative continuous infusion of fviii or fix to prevent or reduce bleeding in patients with haemophilia. in conliast to repeated highdose bolus injections, the continuous infusion trealment regime maintains constant coagulation factor activity at a level necessary for hemostasis, reducing the total cost of treatment by about 20% and preventing possible side effects of bolus doses. the new application mode, however, requires stable products which tolerate slow passage through an infusion device. our objective was to test in vitro the fviii concentrate immunate (stim plus) and the fix concentrate immi.ynine (stim plus) at room temperature, under conditions of long-term contact with polypropylene tubing in an infusion pump. infusion rates were chosen to mimic clinical situation. the control samples were not infused through the pump but were otherwise treated identically. test samples were drawn before and at 1, 4, 8, 24 and 48 hours after the onset of each infusion run. fviii (one-stage, two-stage and chromogenic assay) and fix (one-stage) activity were measured using immuno reagents. presence of activated factors were measured by napt'i', while flla, fxa, plasmin and pre-kallikrein activator were detected with specific chromogenic substrates. the data showed equivalent results between test and control samples with no loss of fviii or fix activity. the potencies of both immunate (stim plus) and immunine (stim plus) remained within 100 + 20% of labeued values within 48 hours after onset of infusion. in conclusion, immunate (stim plus) and immunine (st1m plus) are suitable for contiuous infusion when using automatic infusion device within applied test criteria. in htanans, circulating half-lives of asparaginase enzymes from e. coli and erwinia chrysanthemi vary within a wide range. moreover, half-lives differ not only among different e. coli strains but also among commercial e. coli preparations. to investigate the possible influence of two different sources of e. coil asparagmase (asn) preparations on the fibfinolytic system of leukemic children a prospective randomized study was performed correlating asn pharmacokiuetics (asn activity, asparagine depletion) with fibrinolytic parameters (plasminogen (plas), o.2-antiphismin (ct2ap), tissue-type plasminogen activator (t-pa), tissue type plasminogen activator inhibitor 1 (pal 1), d -i)imer (i)-d)). together with prednisono, vincristine and an anthracycline 20 children received i0000 iu-/m 2 asn medae r (originally purchased: kyowa hakko, kyogo japan) and 20 children 10000 iu/m 2 crasintin r (bayer, leverkusen, germany). blood samples for pharmacokinetic and coagulation analysis were drawn before the first asn administration and every third day whilst on medication. the results are shown in the 0.05 asn activity shows a negative correlation (spearman: rho/p) to plas (-,637/0.0003) and ct2ap (-, 751/0.0001). a positive correlation was found between asn activity and d -dimer formation (0.475/0.01). t-pa and pal 1 showed no relationship to asn activity. all children showed complete aspamgiue depletion at a detection limit of 0.1 um during the course of asn admiatstration. two thrombotic events occurred in the kyowa group, one of the distinctions between the two e. coli asn preparations administered ill this stndy is the absence of cystine in the kyowa asn, which also has a lower isoelectric point and a longer half-life than the bayer type a asn. with respect to this observations this may lead to longer inhibition of protein synthesis, which then may be the cause of a bigher rate of side effects. along with studies on asn pharmacokinoties dose recommendations need to be tailored to the specific asn preparation employed to ensure optimal antineoplastic efficacy while minimizing the hazard of complications. different types of coagulopathy in hepatic veno-occlusive disease (vod) and capillary leakage syn-drome (cls) after bone marrow transplantation w. ntimberger, s. eckhof-donovan, st. burdaeh and u. g6bel department for pediatric hematology and oncology, heinrich heine university medical center, diisseldoff, germany it is generally accepted, that cls, coagulation activation and refractoriness to platelet transfusions are part of the syndrome of hepatic vod. we assessed patients with either vod or cls or both vod and cls, in order to analyze the influence of either syndrome on different aspects of hemostasis. vod was diagnosed according to jones et al. [transplantation 44 (1987) 778]. diagnosis of cls was >_3% increase of body weight in the past 24 hours and non-responsiveness to furosemide [niirnberger et al., ann hematol 17 (1993) 67] . patients with vod, cls or both were compared to control patients without either diagnosis. eight patients suffered from both vod and cls, 5 patients only from vod, and 8 only from cls. 61 patients had neither syndrome and served as control population. activation of the coagulation system was assessed by increase of tat-complexes and/or increased consumption of at iil the hemostasis patterns were as follows: no. introduction: lung cancer goes along with coagulation activation and increased thromboembolic risk. acute phase reaction in cancer patients leads to elevated levels of c4b-binding protein (c4b-bp) followed by a shift from free to c4b-bp-bound protein s. we tried to find out whether there is a correlation between alterations of c4b-bp, protein c protein s system and interleukin 6 (il-6), which is one of the most potent inducers of hepatic acute phase reaction. patients: i. 25 patients with lung cancer; 2. control group: 11 patients in complete remission after lung cancer. methods: clotting methods: protein c and s activity; elisa tests: protein c antigen, tat-complexes, prothrombin fragments f i+2, il-6. electroimmuno-diffusion (laurell): free and total protein s, c4b-bp. results: tat-complexes and f i+2 were elevated in cancer patients. c4b-bp levels were slighthly increased (129±19 % of n.), protein s activity was 89±33 % of n. (control group: 108±23 % of n.). il-6 in lung cancer patients was 37.2252.9 pg/l (control: 27.2±8.2 pg/l). conclusion: one source of the hypercoagulable state in lung cancer patients is decreased protein s activity due to elevated c4b-bp levels. this is probably caused by hepatic acute phase reaction which is triggered by increased il-6 levels. these plasma levels correlate with levels of the tumor marker ca 125 and with the stage of the disease but correlations with patient outcome (disease recurrence and overall survival) have not previously been shown. plasma levels of d -dimer and ca 125 (determined by sandwich elisa assays} were measured prior to treatment in 36 women with figo stage t to iii ovarian cancer and correlated with tumor stage, relapse and overall survival over a mean follow -up period of 28 months (range 16 to 40 months). levels in 71 healthy women and 27 patients with benign ovarian disease served as controls. the occurrence of deep vein thrombosis in the cancer patients was also determined by impedance plethysmography that, when positive , was confirmed by contrast venography. preoperative d -dimer and ca i25 levels in ovarian cancer patients were statistically signfficantly higher than in controls. preoperative cut off values were calculated for the prediction of cancer relapse and survival for both measurements. d -dimer levels above a cut off level of 2060 ng/ml were statistically significantly associated with the rate of relapse but ca 125 levels were not. deep venous thrombosis occurred in 33 % of cases but there was no difference between properafive levels of d -dimer in patients who subsequently did versus did not develop deep vein thrombosis. high levels of d -dimer are associated with more advanced disease and with poor prognosis in patients with ovarian cancer. the high levels of d -dimer are a biologic feature of the malignancy itself that may be attributable, at least in part, to increased conversion of fibrinogen to fibrin in the tumor bed with subsequent degradation of fibrin by the fibrinolytic mechanism. thus d -dimer levels may serve as a marker for overall tumor burden as well as "disease activity". a high incidence of deep vein thrombosis exists in the course of the disease in ovarian cancer patients but preoperative levels of d -dimer are not predictive of this occurence. yon tempelhoff georg -friedrich, michael dietrich, dirk schneider, lothar heilmann. dept. obstet. gynecol. city hospital of ruesselsheim. -germany. an increase of plasminogen activator inhibitor activity (pai act.) in the plasma of cancer patients has been recently discribed. we have longitudinally investigated pai act. in 136 patients with primary breast cancer and compared the results with the outcome of malignancy. patients with untreated primary breast cancer and without proof of metastasis (t 1-4 n0-2 m0) were eligible for this study. in all patients coagulation tests including fibrinogen {method according to clauss), d -dimer (elisa} and pal act. (upa dependent inhibition test) were performed prior to primary operation, 6 months thereafter and at the time of cancer relapse. seventy -two healthy women and 43 patients with benign breast disease served as controls. during a mean follow -up of 32 + 16 months 34 patients (25 %) developed cancer recurrence and 13 (9.6 %) patients died. in all cancer patients preoperative levels of fibrinogen and pai act. were significantly higher compared to healthy women and to patients with benign breast disease. preoperatively only pal act. was significantly higher in patients with vs. without cancer recurrence (4.52 _+ 1.67 u/ml vs. 3.4 1 + 1.55 u/ml; p = 0.002). in patients with later recurrence pai a~t. significantly dropped 6 months after operation (p = 0.02) and was again significantly increased at the time of cancer recurrence (4.90 _+ 2.89; p = 0.001). a preoperative cut off value (calculated via cox model) of pai act. above 3.52 u/ml was significantly associated with the rate of relapse (tog rank: p = 0.0005) and in 70 % of patients who died of cancer preoperative pai act. were also above this cut off. impaired fibrinolysis in patients with breast cancer is significantly associated with the outcome of cancer. a monoclonal heparin antibody (mab) has been raised against native heparin using a heparin-bovine serum albumin conjugate prepared by reductive amination. for further analyses tyramine, which was covalently bound to low molecular mass heparin by endp0int attachment (malsch r et al: anal biochem 1994; 217: 255-264) , was labeled with 125-iodine at the aryl residue. the tracer antibody complex was immunoprecipitated by goat anti-mouse immunoglobuline igg. the mab recognized specifically intact heparin and heparin fractions. the lower detection limit of heparin preparations was 100 ng/ml. no cross reactivity of the mab occurred with other glycosaminoglycans such as heparan sulfate, dermatan sulfate, chondroitin sulfate a and c. oversulfated heparin showed lower affinity to the antibody hl.18 than 2-0-and 6-0-desulfated beparin. the method established for the purification of the mab was ammonium sulfate precipitation with followed dialysis. sds-page and high pressure capillary electrophoresis prooved the high purity of the received antibody. the biological activity of mab was tested by the chromogenic assay $2222 and remained stabile while purified. in conclusion, the present abstract describes an purified igg 1 monoclonal antibody directed against heparin and heparin fractions, which can be used for biological measurements. the concentration of heparin and dermatan sulfate in biological fluids is usually measured using radiolabeling. for this purpose aromatic compounds are usually used to insert radioactive iodine labeling at the saccharide backbone of the glycosaminoglycan. we developed methods for the specific labeling of hepann and dermatan sulfate at the terminal residue. tyramine was bound by reductive amination to the 2,5 anhydromannitosyl end of heparin, produced by nitrous acid degradation and confirmed by 13c.nm r spectroscopy. (anal biochem 217: 254-264, 1994) this method was also used to produce a low molecular mass dermatan sulfate (lmmd)derivative after partial deacatylation. in order to choose the proper method for evaluating the specific anticoagulant activity in the row of chitosan polysulphate (cp) samples with different degrees of pol3~merization and sulphation we applied to pharmaeapea article (a~) when assessing the ability of direct anticoagulants to depress the coagulability of recalcificated sheep blood (using the 3rd international heparin standard), and to measuring such acti¢ity as per pharmacokinetic model (a2). the model admits the "kinetics of cp elimination be linear in ease of intravenous injection to rabbits, as it is observed in heparin: ct=co exp(-i~ x t), where ct is cp concentration at the time moment t; co is cp concentration at the moment of injection; i~ is the elimination constant. besides, it is assumed that there is a linear approximation of the anticoagulant effect on the dose, which finally makes it possible to calculate the specific actidty a2 : t=kt ct + tin, where t is the time of clot formation at different tlme intervals after of cp injection; t~, is the time of clot formation prior to cp injection. t value was assessed in two tests: in blood coagulation time (bct) and in activated partial thromboplastin time (aptt). no correlation was observed between a1 and a2. at the same time the values of ifm and the period of semieliminatinn (tvz) with the use of the original method that were obtained with the help of the quantitative determination of cp in rabbit's blood taken at different time intervals after injection, showed a close correlation (1"=0,94 p<0,05) between the same parameters, obtained with the help of the of the pharmacokinetic model in bct test. thus, experimentally it was proved that the assumption of the linear elimination and the effect-dose dependence was true, which is necessary for a2 calculation. we recommend to use intravenous injection of the samples to animals with further assessment of the results according to the pliarmacokinetic model to calculate the specific anticoagulant activity in the row of chemically related potential direct anticoagulants. in this investigation we compared the biological activity of a low-molecular-heparm (lmw-heparin, mono embolcx®) after intravenous, subcutaneous and oral application in rats. sprague-dawly rats were anaesthetized by ketamine/diazepam and the blood samples were taken from the retro orbital sinuus. 150 axa u/kg body weight of the lmw-heparin were injected intravenously and subcutaneously to 10 rats each. between 3 minutes and 10 hours after injection serial blood samples were taken. 200 mg/kg (20.000 axa u/kg) body weight of the lmw-heparin were applicated orally using a stomach tube. blood samples were taken between 1 and 24 hours after oral application. the antifactor xa and antithrombin activities of the plasma samples were measured, using ehromogenic assays and the substances s 2222 and s 2238 (kabi vitmm). after i.v. injection the maximum axa and alia activities were 2.8 axa u/ml and 0.8 aiia u/nil respectively. after s.c. application the antifactor xa activity of the lmw-heparin showed a maximum of 0.5 axa u/ml atter 120 minutes. the antithrombin activity exhibited an eatiier maximum activity of 0.2 alia u/nil 60 minutes after injection. after the oral application no increase of the axa or alia activities was measured. the lmw-heparin has a high antifaetor xa and antithrombin activity after i.v. and s.c. injection. after oral application no activity of the lmw-heparin was measurable. these results implicate that fractionated heparin is not absorbed after oral application or is inactivated in the gastrointestinal tract. to improve the activity after oral application modified hepatins have to be synthesized. in an in vitro study the effect of various heparin derivatives (calciparin, fraxiparin, cy 222, cy 231, astenose, hexasaccharide, ssh 14) on thrombin-and adp-induced platelet aggregation as well as on adpmediated platelet activation in whole blood was investigated. all heparin derivatives caused a concentration-dependent inhibition of thrombin-induced aggregation of washed platelets. calciparin and astenose were found to be the most effective compounds with ic5o values of 0.67 and 1.2 p, mol/l, resp.; higher concentrations (5-30 times) were required for the other compounds. furthermore, the heparin derivatives were studied with regard to their potentiating effect on adp-induced platelet aggregation. in a concenwation range from 1 to 10 u/nil calciparin, fraxiparin, cy 222 and astenose led to a potentiation of the adpinduced aggregation whereas cy 231, hexasaccharide and ssh 14 did not show this effect. the increase in aggregation was associated with an increase in thromboxane a2 lbrmation. in addition, the effect of calciparin, fraxiparin, cy 222 and astenose on adp-induced platelet activation in whole blood was investigated by flow cytometric analysis using monoelonal antibodies to platelet surface receptors opiiia (cd-61) and p-selectin (cd-62). at concentrations that caused a maximum potentiation of adp-induced platelet aggregation these substances led to a strong increase of adp-mediated activation of platelets in whole blood. the effect was most pronounced when the blood was anticoagulated with calciparin and astenose, resp. in conclusion, the results suggest that the aggregation-promoting effect of heparin derivatives included in this study is dependent on the molecular weight and the degree of sulfation and is in part due to the generation of thromboxane. heparins are negatively charged polysaccharides and bind protamine forming a stable complex. here we report on the properties of microbeads (4.5 pro) coated by protamine. protamine chloride (0.16 ijm) was covalently bound to 0.5 mg paramagnetic tosyl..activated microbeads m-450 (dynal). the covalent binding of protamine was from 1.0 to 13,0 mg/g beads. protamine-dynabeads were produced in a phosphate buffer at different ph (7,0; 7,5; 8,0 and 8,5). the protamine-dynabeads produced ph 7.5 showed the best properties for flow cytometry analysis. in saline solution they bound lmm-heparin-tyramine-fitc (lmmh-tyr-fitc) dose dependently from 0.001 to 2 u/ml, whereas in plasma and blood they bound lmmh-tyr-fitc from 0.05 to 2 u/ml. dependent on the binding protocol, the microbeads also bind proteins unspecifically, i.e bovine serum albumine and protamine to a lower extent.the adsorbed proteins, however do not bind lmmh-tyr-fitc dose dependently. the saturation of the proteins on the beads was determined as their relative fluorescence intensity (rfi). in saline solution the saturation was measured at 380 rfi, in human plasma at 325 rfi and in whole blood at 252 rfi. using flow cytometry erythrocyctes, lymphocytes, monocytes and granulocytes were not bound to protamine dynabeads. these data demonstrate that protamine-dynabeads can be used to measure the concentration of lmmh-tyr-fitc in saline solution, plasma and blood because they do not bind to human blood cells. the present study was designed to investigate the anticoagulant action of inhaled low molecular weight (lmw)-heparin in healthy volunteers. 3,000 iu (group t), 9,000 iu (group 2), 27,000 iu (group 3) or 54,000 iu (group 4) lmw-heparin were given to 20 healthy volunteers each at 4 weeks intervals. in group 1 tissue iactor pathway inhibitor (tfpi) antigen and activity, 82222 chromogenic factor xa assay, heptest, aptt and thrombin clotting time (tot) remained unchanged during the 10 days observation period. in group 2 tfpi antigen and activity, aptt, tct and the $2222 method remained uneffected. heptest coagulation times were 18.7 + 2.0 before, 26.1 + 5.2 sec. 6 hrs and to 20.5 + 1.9 sec. 24 hrs after inhalation. in group 3 tfpi antigen increased from 74.1 + 13.9 to 80.5 + 14.2 ng/ml 3 hrs after inhalation. tfpi activity remained unchanged. $2222 method increased from 0.01 to 0.08 + 0.06 iu/ml 6 hrs after inhalation. heptest coagulation values were prolonged up to 42 _+ 7.6 s ec after 6 hrs and returned to normal within 72 hrs after inhalation. aptt and tct remained unchanged. after inhalation of 54,000 iu lmw-heparin, the following changes were observed: tfpi antigen increased to 103 +_. 17.9 ng/ml and normalized within 24 hrs. -i'fpi activity increased to 1.14 _+ 0.23 u 3 hrs after inhalation and was normal after 24 hrs. antifactor xa activity, as measured by s2222 method, increased to 0.343 + 0.196 u/ml after 6 hrs and was normal after 72 hrs. heptest coagulation values increased to 77.5 + 11.8 sec 6 hrs after inhalation and normalized after 144 hrs. aptt and tct did not change throughout the observation period. the data demonstrate a resorption of lmw-heparin by intrapulmonary route in man. no side effects were observed. recently we developed a tritium-labelled arachidonic acid ([3h]aa) release test with high sensitivity to membrane-toxic agents. the assay performed in u937 cells is intended to evaluate ehemicals, drugs and biomatefials with regard to their eytomembrane toxicity [kloeking et at. (1994) , toxicology in vitro 8, 775-777]. local irritation reactions are described in patients receiving therapeurieat dosages of lmw heparin. this fact prompted us to examine the following lmw hepafins and heparinoids for their membrane toxicity in u937 cells: reviparin-sodium, enoxaparine-sodium, mueopolysccharide polysulphate (mps), pentosan polysulfate sodium (pps), polysulfated bis-lactobionic acid amide derivatives lw10082 (aprosulate) and lw10086. for this purpose, [31--1]aa labelled u937 ceils were incubated with different concentrations of lmw heparins and heparinoids at 37°c for 1 hour. compared with untreated cells, the [~h]aa release of cells treated with 5 mg of the drugs was two times higher with reviparin sodium, three rimes higher with bis-lactobionic acid amide lw10086, five times higher with pentosan polysulfate, 20 times higher with ertoxaparine-sodlum, but it was equal to the control with mucopolysaccharide polysulphate. the rate of araehidonic acid release in response to a test chemical may therefore be used to assess the membrane-toxic effect of this substance and to predict its the inflammatory potential in the skin. semi-synthetic glyensaminoglycans (gags) with antithrombotic properties can be prepared from the e. coli k5 polysaecharide by coupled chemical and enzymatic methods. the molecular weight of these semi-synthetic gags can be adjusted to obtain products mimicking the molecular profile of a low molecular weight hepatm. in order to compare the biochemical and pharmacologic properties of a semi-synthetic gag (sr 80486a, sanofi/choay) with a commereiany available low molecular weight heparin, fraxiparine (sanofi, paris, france), valid biooheanical and pharmacologic methods were used. the molecular profile of this agent as determined by hplc exhibited a comparable distribution profile (mr=5.7 kda) in comparison to fraxiparine (ma=5.1 kda) . the anticoagulant properties of sr 80486a were comparable to fraxiparine in the aptt and heptest. however, in the usp assay, this agent showed slightly weaker activity. sr 80486a also exhibi~d comparable affinity to atffl and hcii. in comparison to fraxiparine, it produced a much weaker response in the hit screening system. in~ viv0 studies, sr 80486a preduecd strong dose-dependent antithrombotic actions in both the iv and sc studies in the rabbit jugular vein stasis thrombosis model (ed50=i 5-60 gg/kg). additionally, it also produced antithrombotic aefiorts in a rat jugular vein clamping model. the hemorrhagic effects of this agent were comparable to those of fraxipafine as measured in a rabbit ear blood loss model. intravenous administration of sr 80486a also revealed a comparable pharmaeokinetie behavior to fraxiparine. no abnomaiitias of the clinical chemistry (change in liver enzymes) and hematology profile (thrombocytopenia and lencecytosis, etc.) were noted in primates. at a dosage of i and 2.5 mg/kg iv, this agent also caused a release of functional tfpi which was comparable to the observed responses of other low molecular weight heparins. these studies suggest that sr 80486a is capable of producing similar pharmacologic effects as other low molecular weight heparms, however, additional optimization studies are required for demonslrating product equivalence. limited information on the comparative pharmacoldnetics of low molecular weight heparin (lmwh) is available on the data obtained from aptt, heptest, anti-xa and antmia assays. since these drugs are currently used for therapeutic indications using relatively high dosages and intravenous administration. aptt, heptest and antmia test may be valuable in the assessment of their effects. in order to investigate the relative pharmacokinetics of lmwh using apt'i', heptest, anti-xa and anti-iia methods, certoparin (sandoz, basel, switzerland) was administered to individual groups of healthy male volunteers (52-70 kg) via intravenous (30 mg) and subcutaneous (50 nag) routes in a crossover study. blood samples were drawn at 0, 5, 15, 30, 60, 90, 180, 360, 540 and 720 minutes. using a baseline pool plasma obtained from the same volunteers, calibration curves for each of the individual tests were constructed to extrapolate circulating levels of certoparin. a non-compartmental model using trapezoidal technique was used to obtain pharmacokinetic parameters such as t 1/2, vd, and clsys. in the intravenous studies, the t 1/2 was found to be dosedependent for aptt, heptest, anti-xa and antm]a. the auc, however, was significantly different for each test and was dose-dependent following the order: aptt<heptest<anti-xa<anti-iia. in the subcutaneous studies, 1the t 1/2 was both test and dose-dependent. the auc was also dose-dependent and followed the order: anti-xa>heptest>aptt>antmia. the clsys of the antma was much faster in comparison to the other tests. the clsys of the aptt and heptest was independent of dose. however, anti-xa clsys by this route was lower than other tests. the apparent vd followed the order aptt>antmia>heptest>anti-xa. the bioavailability of the certoparin as measured by various tests ranged from 81-119%. these studies suggest that beside providing pharmacokinetic data, aptt, heptest and anti-iia assays may provide useful data on thier safety and efficacy at high dosages. the immunological type of heparin associated thrombocytopenla (hat ii) is a severe complication of heparin treatment and is associated with arterial and venous thrombosis. only patients with absolute thrombocytopenia have prompted suspicion of hat in clinical practice. we report on a 44 year old male, who developed thromboembolic episodes after coronary angiography like reinfarction and thrombotic episodes of a. brachialis. fibrinolytic therapy combined with i.v. unffactionated heparin treatment was the therapy of choice and was followed by severe fua~er thromboembolic adverse effects. besides an impaired fibrinolytic response and elevated antiphospholipid anitbodies, we diagnosed hat type ii in hipa and elisa (stago-boehringer, marmheim). this special patient had platelet counts within a normal range, when developing the thromboembolic episodes. it appears that the normal platelet count during the thromboembolic episodes reflect a relative thrombocytopenia. from a clinical point of view we recommend the use of a lab panel to exclude hat type ii in patients with thromboembolic episodes under therapy with fractionated or unfractionated hepafin. platelet counts within a normal range are no absolute exclusion criterion for hat ii. low molecular weight heparins (lmwhs) are now commonly used for the prophylaxis of post-surgical thromboembolic complications. in this indication, lmwhs are administered as a single or twice a day subcutaneous regimen. usually these agents are administered at 30-40 mg total dose which is equal to 3000-4000 anti-xa (axa) iu. newer methods such as ehromogenic substrate based axa methods and the heptest clotting time can be used to determine the effects of lmwhs during the initial phases of prophylactic therapy. this may be useful in the elderly and weight compromised patients where a fixed dosage may not be optimal and may produce bleeding effects. similarly in the overweight patients, a fixed dose may not be efficacious. thus, monitoring of lmwhs in these patients may be useful in the optimization of their therapy. lmwhs are also used in the treatment of deep vein thrombosis using both intravenous and subcutaneous protocols. high dosages of up to 200 mg sc/day and infusions of up to 30 axa iu/kg/hr have been administered. in these conditions, the monitoring of the circulating lmwh levels may be useful in optimizing the dosage. we have modified the aca heparin (do_pont merck, wilmington, de) method to measure the lmwh levels in the plasma of patients treated with both the prophylactic and therapeutic dosage. owing to the required turnaround time, simple operation and reliable results, this method was found to be of value in the monitoring of these agents. this presentation provides an overview of the clinical application of various lmwhs with particular reference to the need of monitoring for their effects to optimize the clinical outcome. a double-blind, multicentric, controlled trial was performed in order to compare the antithrombotic efficacy and safety of single daily doses of 5000 ie anti-xa of low molecular weight heparin (lmwh) sandoz (certoparin) and 5000 ie unfractionated heparin (ufh) tid. in 288 patients undergoing elective total hip replacement blood samples were drawn before the first subcutaneous injection of lmwh or ufh resp., two hours after administration on the first and 7th postop, day and on the last day of prophylaxis (day 10-14), anti-xaactivity was measured by chromogenic substrate assay, heptest and aptt by clotting assays and tissue factor pathway inhibitor (tfpi) and heparin-pf4-antibodies by elisa techiques. as expected, the anti-xa-activity and the heptest values were significantly higher in the lmwh-group at all time points after administration of the drugs; the mean values of heptest were 35 sec in the ufh-and 75 sec in the lmwh-group respectively, the aptt was not different in both groups. at the end of prophylaxis positive antibodies to heparin-pf4complexes were detected ~n both groups; this however was not correlated with clinical thrombocytopenia. a detailed correlation between patients with deep vein thrombosis (dvt) and positive antibodies has still to be done (all patients were screened for asymptomatic dvt between day 10-14 by bilateral phlebography. tfpi was markedly increased in the lmwh-and only slightly elevated in the ufh-group; the differences are statistically significant. summarizing it can be concluded that antibodies to heparin-pf4complexes may occur without clinical symptoms of hepafin-induced thrombocytopenia type ii and that tfpi may play a sigificant role for the antithrombotic efficacy of ufh and lmwh. unfractienated heparin represents one of the most severe and frequent causes of drug-induced thrombooytopenia. heparin-indueed thrombocytopeala (hit) occurring early in therapy is often mild and serf-limited, appearing to be caused by a direct aggregant effect of heparin on platelets (hit type i). hit type ii, however, is immune-related an may result in absolute thrombocytopenia (platelet count <i50/~l) or a relative decrease in platelet count (?_50% reduction from baseline values). unlike most other immune thrombecytopenias in which bleeding complications often predominate, hit type ii may results in severe arterial and/or venous thromboses. the management of patients with hit type ii and thrombosis is often difficult. heparin must be stopped immediately. antithrombotic, fibfinolytic, or antiplatelet agents have been used in these situations, as have anticoagulants other than unfraetionated hepafirl plasmapheresis, used in other immune-related disorders, has also been occasionally reported. at our institution this intervention appears to provide significant elininal benefit to hit patients. several recent reports have noted that the heparin-dependent antibodies in hit type ii are directed against a complex of heparin bound to platelet factor 4 (pf4) and an elisa-based test system for hepafin-pf4 eomplexinduced antibodies (hpia; stago) has been develuped~ we report our experience in 3 patients with hit type ii in whom plasmapheresis was instituted and the antibody levels in blood were measured pre-and post-treatment to identify wkether circulating antibody titers decreased thus providing some explanation for the clinical benefit. the patients developed profound absolute thrombocytopenia (range, 3-21 k/~tl) and positive heparin-induced platetet aggregation studies following treamlent with uofractionated heparin. thromh0cytopenia induced by hepariu (hat-syndrome) typically appears several days after initiation of heparin therapy. this phenomenon is caused by antibodies of the lgg type which are induced by a heparin-pf 4-complex and which also activate platelets. in a recent study (n engl j med 1995; 332:1330-5) the hat-syndrom was diagnosed in 2.7 % (9 of 332) patients who had received unfractionated heparin (ufh) and in none of those 333 patients who had been treated with low-molecular-weight heparin (lmwh). 8 out of the 9 patients treated with ufh presented with at least one thrombotic event (7 times a venous, once an arterial event). the incidence of heparin-dependent igg antibodies (7.8 %) was higher in the ufh treated group than in patients treated with lmwh (2.2 %). our patient was a male caucasian white, 62 years of age, otherwise in good clinical condition with no severe diseases in his pmh. both parents, however, had suffered several thrombotic episodes. the patient was admitted to the hospital in september "95 with clinical signs of a deep vein thrombosis (dvt) of the right leg. the diagnosis was phiebographically confirmed. after having given informed consent the patient was treated with 5 cycles of r-tpa (loco-regional application) and 3 cycles of uhsk as part of a clinical trial (uhsk vs. rtpa, boehringer/m). lysis was successful with complete recanalisation of the veins. after completion of lysis the patient was further anticoagulated with coumarin overlapped by heparin (ufh). as a complication the patient suffered from a retroperitoneal and an upper gi4ract bleeding from esophagitis grade ii to hi while being on coumarin prophylaxis. therefore the patient was put on lmwh and then discharged with this prophylaxis. after less than two weeks the patient presented again to the hospital with a relapse of dvt in the same leg. phiebographically nearly all deep veins were occluded to a higher degree than before, i. e. also with concomitant pelvic vein affection and with hea~ 3, swelling of the whole leg. blood count showed a marked decrease of platelets to 27/nl. the platelet aggregation test demonstrated a hat-syndrome which was confirmed by the hipa-assay. the heparinoid orgaran® was not reactive in the assay. having seen a previous bleeding episode in this patient, we did not see an indication for another b'sis therapy. therefore we decided to put the patient on an intermediate dose of coumarin overlapped by hiradin (hat study, behring) instead of orgaran®, since hirudin allows a much more precise dose adjustment. after 10 days the patient was discharged with no further complications. risk factors for dvt have not been detected until now. to our knowledge this patient is one of the first cases of hat-syndrome induced by lmveh. under routine conditions aptt is the most frequently used test method for the surveillance of patients undergoing hepann therapy. in literature a 1,5 fold to 2,5 fold prolongation of the initial value is recommended for individual therapy a~ixatian. in contrast to the control of the enmarin therapy by means of prothrombin time measurement the individual hupafin sensitivity of the ~ reagents of this standard value hardly receives notice. commercially marketed ~ reagents differ from one another method and concentration of the activator as well as their phosphelipids. when choosing a reagent the user must only take into consideration the sensitivity against its factors, heparin and lupus but also the adalxability of the reagent towards the given measurement system ( software variability towards incubation time, sodimentalion problems in reagents with high kaolin concentration). reagents offering a high heparin sensitivity already are showing a prolongation of the coagulation time at low heparin dosage (0.1 -0.3 u/ml). in the therapeutic range (0.4 -0.8 u/ml) very often no linear relationship between prolongation of the aptr and heparin concentration level is found_ therefore very often coagulation times above the routine measuring range (180 s) can be observed in this range. moreover, various medical measuring devices differ in their ability to recogniso the retarded entry of coagulation. coagulation limes with different apq't reagents in correlation with the heparin concentration level can be found in the adjoined table. significantiy prolonged coagulation ti.~es of aptt lead to a reduction of the hel~ dose in clinical practice. due to the discrepancy between heparin concentmon level and the prolongation of the afrt in reagents with a non-linear heparin sensitivity a reduction of heparin dosage leads to an inmt~cient anticeagulalion and the risk of further thrombosis. therefore the producers should be asked to include precise directions on the heparin sensitivity of the aptt reagent in the therapeutic range in order to enable an optimal surveillance of the heparin therapy. a coordination between clinic and laboratory should be compulsory for the choosing of the aptt reagent. introduction: hat is a rare but sometimes fatal complication of therapy with unfractionated (ufh) and low molecular weight beparins (lmwh). we report on our experience with the clinical coume and management in 12 patients (pts). in all cases a severe decline of platelet count and in 11 cases a positive hipa test (= heparin-induced platelet antibodies) were documented. patients, clinical course and outcome: 8 female and 4 male patients (median age 68 years, range 22-78) were analysed in this retrospective study. 9 pts were referred from outside hospitals. all pts had been treated with ufh (5 pts additionally with lmwh) for prophylaxis (n = 9. in 2 pts pedoperatively) or treatment (n = 3) of venous thromboembolism (v'te). in 11 pts at least one vte occured during heparin therapy, in all cases prior to hat-diagnosis (vte: n = 13; putmonary embolism: n = 6; artenal embolism n = 1; thrombosis of sinus transversus n = 1). hat was suspected 16.5 days (median; range 12 -48) after first heparin application. hepadn therapy was discontinued immediately, at that time the median plateiet count was 35 g/i (range 10 -117). 9 pts were treated with orgaran r, followed by recombinant hirudin (r-hirudin, behdngwerke ag: clinical study) in 4 cases; 3 pts were treated exclusively with r-hirudin. plat~let count normalized in 9 pts within 3 days (median; range 1-10). one patient in the orgaran" group died on recurrent pulmonary embolism. 11 pts recovered without any further thromboembolic complication. conclusion: in our small series of 10ts hat was complicated with vte in 11/12 cases. in many pts hat was diagnosed strikingly late. for early diagnosis careful monitonng of platelet counts are mandatory dunng hepadn therapy, both orgaran ~ and r-hirudin seem to be safe and effective in preventing further thromboembolism, p 179 to prospectively evaluate coagulation or fibrinolytic activation in children long-term anticoagulation with lmwh was administered a longitudinal study was perfomed. within a 30 months period 48 children and adolescents (6 -19 years) suffering from malignant bone tumors (ewing's sarcoma or osteosarcoma) received enoxaparin (clexane r/rhone-poulenc rorer, france) for primary propylams. the majority of patients received 0.6 mg/kg bw enoxaparin 12 hours before major surgery (limb salavage). in addition, ten neonates and children received enoxaparin in the same dosage for secondary prophylaxis. in both groups therapy was adjusted to 0.2 -0.4 iu/ml anti xa activity. median (range) duration of therapy was performed for l0 (6 -24) weeks. before ('1"1), one (t2) and 3 weeks (t3) within the context of the present study an investigation on the physiology of clotting was conducted in three groups each consisting of 12 patients, who had to undergo a total end-prosthetic hip implantation (teph) for the first time. criterion for allocation to a particular group was type of intraoperative transfusion the patient received : infusion of whole blood, erythrocyte concentrate or fresh frozen plasma. at eight fixed times the activity or concentration of the components of the kallikrein-kinin and inhibitory systems was determined. the statistic evaluation ensued with the spss software package. for all three patient collectives an activation of both systems was to be observed with the teph-implantation whereby there were no statistically striking differences between the three groups. thus in the case of the kallikrein-like activity an intraoperative rise of over 37% was registered, while at the same point in time there was only a 4% reduction in the prekallikrein activity. postoperatively a 10% rise in the prekallikrein activity was to be observed. as a reserve inhibitor alpha 2-macroglobalin showed no significant change in activity during the period of observation whereas the acute phase proteins alpha 1-proteinase inhibitor and cl-esterase-inhibitor yielded a postoperative rise of 135% and up to 100%. the beta-factor xllainhibition showed a rise of 121% during the reconvalescent phase. the 65 year old female caucasian patient presented here, was admitted to a nearby district hospital in december '94 with the acute symptoms of pulmonary embolism. the source of the embolus could not be detected. high dose heparin was administered. pmi-i revealed a coronary artery disease and a peripheral artery occlusive disease of the left lower leg, yet blood pressure values of the lower ex~emities were only slightly below brachial pressures. the first episode of pulmonary embolism was followed by a second one two weeks later for which the patient received heparin again. following this treatment the platelet count decreased significantly to 10/ni. with an acute thrombosis of the lower caval vein and with leriche's syndrome, i. e. occlusion of the abdominal antra from the bifurcation down to the ileofemoral arteries bilaterally, the patient was transferred to our hospital. on initial examination we saw a patient in no acute distress, no dyspnea, blood pressure 120/80 mmhg, pulse 94/rain, no peripheral edema or disturbances of sensitivity. neither femoral, popliteal, nor pedic pulses were palpable on either side. a heparin associated thrombopenia was detected with the platelet aggregation assay and confirmed by the heparin induced platelet aggregation assay (hipaa). the autoantibodies targeting the underlying pf4-heparin complex were determined later by an elisa method in serum samples of the patient (asserachrom hia, diagnostica st,ago). a temporary anth&~r filter system was inserted transbmchially into the lower eaval vein and placed jnst above the thrombus. in the following days, 2 cycles of lysis therapy with 9 mil u streptokinase were administered taking six hours for each cycle. heparin was given in the mean time via the filter system in therapeutic dosages in order to prolong values 1.5 to 2 times the individual base line level. at the end of the second cycle, pulses were palpable inguinally, two hours later both popliteal and the right pedic pulse were present. the left leg showed better microperfusion than before. computer tumoglaphy did not reveal an)-thrombotic material in the descending aorta and the iliac arteries. for continuous anticoagulation after lysis we used the heparinoid orgaran® to overlap the period until full anticoagulation was achieved by coumarin. the heparinoid was tested before us non-reactlve in the hipaa. with this prophylaxis the platelet count increased to normal values. hat ii related thrombosis is the most severe complication of heparin therapy, especially in patients with pre-existing thromoembolic disorders (dvt, dic). we present to our knowledge the first case of hat ii during thrombolytic therapy (it) with simultaneous hepatin administration. at admission a 16 year old girl showed complete occlusion of the pelvic veins of the right leg (r common and external iliac v., common and proximal superficial femoral v.). as in children no prospective studies on the thrornbolysis of dvt with rt-pa are available, we started tt according to the frankfurt/m. regimen: rt-pa (actilyse) bolus injection 0.4 nag/kg bw followed by continuous infusion of 1 mg/kg bw/d; simultaneous low dose heparin 100 -200 u/kg bw/d. doppler ultrasonographie control on day nine after the beginning of tr revealed a further distal growth of the thrombus into the popliteal vein. at the same time at ir was decreased to 70% and there was a dramatic drop in platelet count to <50% of the baseline value (216 to 86 gpt/1). the diagnosis hat ]i was confirmed by positive hipa-test. the only heparin preparation not cross-reacting with the antibody was the lmw heparinoid orgaran. heparin therefore was replaced by orgaran: bolus injection 1,500 u/h followed by infusion of 200 u/h. rt-pa-therapy was not discontinued. at ri substitution was performed at levels <70%. under orgaran administration platelet count rose from 77 to 239 gpt/1 within 5 days. on day 12 complete reperfusion was achieved and rt-pa was discontinued ,on day 14. the patient was discharged from the hospital on prophylaxis with phenprocomnon in good general condition at admission thrombophilia parameters (protein c and s, apc) were within normal range. heparin induced thrombocgopenia (hit) is a serious but rare side effect of treatment with unfractionated heparin. patients with hit present with a rapid decrease in thrombocyte count and occurence of venous or arterial thrombosis under heparin treatment. patients with arterial occlusive disease who need surgical intervention are of high risk to develop rethrombosis especially during the first few postoperative days while they are under i.v. heparin-treatment. in our study we investigated prospectively all patients who were admitted to a surgical unit because of arterial occlusive disease and received surgical intervention. 15 patients were screened on the 3rd and 6th postoperative day for heparin-antiplatelet 4-antibodies, decrease in platelet count and thromboembolic complications. 3 patients developed antibodies against heparin-platelet factor 4. one of the 3 patients developed a venous thrombosis in the operated leg in combination with a decrease in thrombocyte count. we conclude that heparin-platelet factor 4-antibodies are often produced in patients under i.v. heparin treatment. only few of these patients actually suffer from clinically s3anptomatic hit. medizinische universit~itsklinik, institut far klinische biochemie und pathobiochemie, d-97080 wtirzburg, germany the important physiological and pathophysiological role of platetets in health and disease is well established. platelets are also the primary target of many vasoactive hormones, factors and drugs. in addition, platelets provide a very important model system for studying agonist-and antagonist-mediated signal transduction events (1,2). platelet activation by agonists is associated with shape change, extension of filopodia, generation of intracellular messengers, secondary agonists and activation of adhesion receptors and integrins including the fibrinogen receptor gp iib/iiia. activation of protein tyrosine ldnases and calcium-dependent protein kinases appear to be of critical importance for the mechanism of platelet activation. in contrast, platelet inhibition by vasoactive factors and drugs is often mediated by the camp and/or cgmp signal transduction cascades. recent data form our laboratory suggest that the focal adhesion protein vasp, a major target of the intracellular no/cgmp and prostacyclin/camp effector systems, is an important link between signal transduction pathways and elements controlling cell motility (3-6). the implications of the recent advances in platelet signal transduction research for understanding the physiology, pathophysiology and pharmacology of human platelets will be discussed. domain. however, in vivo truncated "i'po is much less potent than full length tpo due to its rapid clearance. this suggest that the c-terminal glycosylated domain acts to stabilize circulating tpo. tpo stimulates both proliferation of progenitor megakaryocytes and their maturation to platelet producing megakaryocytes in vitro. in vivo tpo induces dramatic increa~ses in megakaryocyte number and platelet production in animals, indicating that tpo regulates both thrombopoiesis and megakaryocytopoiesis and is the primary regulator of physiological platelet production. this later notion is supported by the phenotype of ti~ and cmpl knockout mice in which platelet levels are only 10-20% of normal while other blood lineages are unaffected. in animal models of myetosuppresion ,and marrow transplant, tpo reduces the platelet nadir seen in the~se models and significantly accelerates platelet recovery, tpo also has profound effects on red cell recovery in these models. these results suggest that tpo will be clinically u~ful for the treatment of thrombocytopenia in caucer patients who undergo chemotherapy or bone marrow transplant. the haparg-study ( .~a.emostatic parameters as risk factors in healthy volunteers) was sponsored by the german ministry of research and started in 1984. a previous study ( pard, intern. angiology 5, 181-195,1986 ) had shown that spontaneously enhanced platelet aggregation as measured with the pat-ill-test and a high fibrinogen level were significant risk factors for new vascular occlusions in diabetic patients. it was the aim of the haparg study to establish if spontaneous plateletsggregation and other hemostatic variables are independent risk factors for vascular occlusions also in healthy volunteers. employees of hschst ag, frankfurt aged 40-65 years and personnel of the university of mainz, aged 30-60 y. entered this prospective study. besides anamnestic data on smoking, hypertension and diabetes blood pressure, the ankle/arm doppler -index and an ecg were recorded and serum cholesterol, hdl, ldl, triglycerides, uric acid and glucose were measured. 1885 men and 988 women entered the study and were followed for 4-6 years. during this period 53 vascular occlusions ocoured ( 36 coronary and 9 cerebral events and 8 peripheral vascular occlusions). only three of these events occured in women. besides age and gender as expected risk factors the multivariate logistic stepwise regression analysis revealed as significant risk factors slightly elevated levels of blood glucose (p=0.001), smoking (p=0.0013), an elevated diastolic blood pressure (p=0,018),followed by spontaneous aggregation (p = 0.017) and higher hdl-levels (p=0.034) as significant risk factors for men. higher fibrinogen levels just missed significance in this analysis (p=0.059). none of the other hemostatic parameters showed any significant correlation with the vascular events.to our knowledge this has been the first prospective trial in a large population of healthy individuals in which a platelet function parameter has been studied together with other possible risk factors. spontaneously enhanced platelet aggregation seems to be an independent risk factor and may be an indicator of an ongoing active atherosclerotic process. mild bleeding symptoms -petechia, prolonged bleeding after tooth extraction -were observed in a patient with normal coagulation tests but slightly disturbed phtelet function: decreased aggregation in response to adp, pal: and thrombin receptor-derived peptide (trap-6). platelet count, agglutination with both ristocetin and botrocetin and thrombininduced aggregation were in the normal range. an abnormal membrane glycoprotein detected in electrophoretic studies of the patient's platelet proteins was shown to be related to gp llxx by immunoblotting. the variant gp differed even from the largest normal polymorphic gp iba form a by an increase in molecular mass, platelets of the patient contain the variant and normal c form in equal amounts. the quantity of gp ib as detected by flow cytometry is normal. lmmunoblotting studies of proteolytic fragments of the abnormal gp indicated that the defect is located in the c-terminal part of the glycocalicin fragment. measurement of the length of glycocalicin molecules after glycerol-spraying rotary shadowing electron microscopy demonstrated two populations of molecules in the patient sample: a normal on with an average size of 470 angstr6m and a fraction with an average size 0f650 angstr6n~ preliminary results of molecular analysis of the gp iba gene in the patient confirm the localization of the genetic defect by protein studies. in summary, the patient is heterozygous for a hitherto undescdbed molecular variant ofgp ~a. barnes and co-workers have shown that simple triple-helical collagenlike peptides, comprising basically a repeat gly-pro-hyp structure, are highly platelet reactive in polymeric form. they additionally have shown that the activity does not involve gpla/lla (integdn c~z~l). platelets adhere under static conditions to these fibrillar peptides mainly in a bivalent-cation independent manner; the bivalent cation dependent adhesion can be inhibited by an antibody against gpflb/llla (integfin m~d33). however, platelets of a patient with type i glanzmann's thrombasthenia secreted their ¢z-granule-and dense body content just like normal controls while activated with the triple-helical gly-pro-hyp peptide. the lack of an essential involvement of cd36 was shown with cd36deficient platelets, which respond to the peptides as readily as normal platelets when fibdnogen binding and cd62 and cd63 expression were measured. to study the involvement of the yon willebrand factor (vwf) platetets from a patient with severe type 3 vwd (vwf not detectable in platelets and plasma) were investigated. the vwf deficient ptatelets responded quite normally to the fibritlar collagen-like peptide. addition of pudfied vwf to the vwf-deficient platelets before adding the paptide raised the platelet respond slightly we conclude that neither ~3~ integrin, nor m~ integdn, nor cd36, nor vwf are the pdmary platelet receptor for the collagen-like peptide. there is evidence that native low density lipoprotein (ldl) can be oxidatively modified in vivo and the oxidized ldl may contribute to thrombogenesis and atherogenesis by damaging endothelial cells, eliciting vasocontractiola and stimulating blood platelets. by means of biochemical methods, electron microscopy, reflection contrast microscopy and computer image analysis, we find that oxidized ldl, compared to native ldl, affects platelets at least in the following five aspects. 1) oxidized ldl alters platelet functional morphology in a time and dose dependent manner. 2) oxidized ldl induces secretion of serotonin from both morphological resting platelets and shape changed platelets. 3) oxidized ldl advances, accelerates and enlarges platelet adhesion. 4) oxidized ldl causes cytodamage in platelets and cultured endothelial cells. 5) oxidized ldl stimulates platelets and endothelium, which leads to a formation of microthrombi on the monolayer of human umbilical vein endothelial cells. further experiments show that oxidized ldl inhibits the activity of ca2+-atpase in purified plasma membranes of platelets, and therefore increases the cytosolic calcium level in platelets. these results clearly indicate that calcium signaling pathway is intimately involved in the mechanism of platelet activation induced by oxidized ldl. since oxidized ldl, a relevant platelet agonist, has been confirmed to be present in vivo, further studies on its role in platelet activation and plateletendothelium-interaction can help for better understanding thrombogenesis and atherogenesis. recantly physical parameters such as sheer stress, hypo-or byperoxic conditions and hyperthermia have been shown to modulate endothelial cell dependent fibrinolysis. in this study we investigated whether yet another physie~ stimulus namely hypothermia might have an impact on the expression of components of the fibdnolytic system of human endothelial cells in culture. confluent cultures of human umbilical vein endothelial cells (hlivecs) were exposed to gc ° for 10, 20, 40, 80, or 160 rain, respectively. control cultures were exposed to 37°c for the same time periods. thereafter medium was removed, fresh medium was added and the cells were incubated for 24 hours at 37°c conditioned media (cm) were harvested and plasminogen activator inhibitor-i (pal-i) antigen was determined by a specific elisa. our data showed that huvecs responded to exposure to 8°c with a time-dependent increase of pai-i in cm. maximum induction of pal-! antigen was seen after 20 minutes exposure to 8°c (pal-! antigen: 754_+.83 ng/105 cells for treated cells; 4202_17 ng/105 cells for control cells). the inereese in pal-1 antigen was also reflected on the level of specific mrna synthesis as evidenced by northern blotting which showed a twofold increase in pai-i specific mrna in huvecs exposed to 8°c as compared to control cells. in conclusion, our data gives evidence that hypothermic stress like several other forms of physiological or pathological stress, results in activation of endothelial ceils as evidenced by an increased expression of pai-i. our findings provide ~rther evidence that such activation of endothelial cells can be induced not only by biochemical mediators but also by physical stimuli. nitric oxide (no) is formed from intracellular l-arginine by two no synthase (nos) isoforms: a ca+ +-dependent constitutive form (cnos) and a ca+÷-independent inducible form (inos) which is expressed after challenge of cells with immunologic or inflammatory stimuli. endothelial cell derived no is an important signaling molecule inducing smooth muscle cell relaxation and platelet inhibition, however its role in cell proliferation is poorly understood. the present study investigates the pattern of nos isoforms and no production in actively proliferating low density human umbilical vein endothelial cells (ld-huvec) and in high density monolayers of quiescent cells (hd-huvec). ld-huvec but not hd-huvec expressed inos (measured as ca ++independent citrulline formation from l-arginine in cell lysates) with a maximal activity of 30-40 pmoles/min/mg protein, cnos (ca +÷dependent citrulline formation) was demonstrated in hd-huvec (0.02-0.2 pmoles/min/mg) but not measurable in ld-huvec lysates. lmmunoprecipitation of cell lysate with a mab against murine macrophage inos decreased inos activity of supernatant by 70% in ld-huvec. immunoprecipitation with an anti-ecnos mab did not modify inos activity in ld-huvec but abolished cnos activity in hd-huvec. no release (measured as nitrite + nitrate production in an 18 h incubation in the presence of 1 mm l-arginine) was 1.9 nmol/mg cell protein in the supernatant of hd-huvec whereas it was t0fold higher with ld-huvec. measurement of intracellular cgmp after stimulation of cells with i-2 #m ionomycin (a sensitive index of enos activity) showed a 400-800% increase of basal levels in hd-huvec but only 50-150% increase in ld-huvec. on the contrary, sodium nitroprusside-induced cgmp increase was similar in non-confluent and confluent cells. the expression of inos and the high no production in ld-huvec suggest a role of endogenous no in mediating cell proliferation. indeed, the nos inhibitors l-nmma and l-canavanine inhibited [~h]thymidine incorporation in ld-huvec by 60%. on the ++ nt no r uctlon ar x r d m other hand enos and ca -depende p od " e e p esse " differentiated huvec and correspond to the ability of the cells to regulate vascular tone and platelet activation. the processes of replication, maturation and motility of progenitor cells of different lineages in the bone marrow are tightly regulated, particulady by various cellular contacts and their modulation involving pericellular proteolysis. while mononuclear phagocytes and related cell lines are well characterized for surface localized plasminogen activation by receptor bound urokinase, megakaryocytes are poorly described in this respect. in the present study the expression of mrna of urokinase receptor in megakaryoblastic cell lines chrf-280, dami and meg01 was found at a low basal level and increased 5-10 fold after pma-treatment. urokinase receptor mrna levels remained differentiation dependent elevated for up to 5 days. functional analysis of upar expression on the cell surface was documented immunologically by facs-anatysis and on the functional level by direct binding of radio-labeled amine-terminal fragment of urokjnase and mirrawed the increase in urakinase recptar mrna level. as expected, antibody and ligand binding was sensitive towards treatment with phosphatidyl-inositol-specific phospholipase c, since the urokinase receptor belongs to the group of gpi-anchored receptors. in cultered megakaryocytes derived from bone marrow urokinase receptor mrna was demonstrated by rt-pcr, as well. these results indicate a differentiation-dependent increase of urokinase receptor expression on cells of megakaryoblastic cell lineage suggesting the participation of urokinase dependent events during maturation process and intravasation of these platelet precurors. regulation of am gene expression may be coupled to oxidative stress through redox sensitive transcriptional regulatory factors. the aim of this study was to investigate the influence of selected antioxidants on tnfa (100 u/ml, 6 h)-induced surface expression of elam-i, icam-i and vcam-i in human umbilical vein endothelial cells. am expression was determined by quantitative flow cytometry and immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase method). the following antioxidants were tested: pyrrolidine dithiocarbamate (pdtc), n-acetylcysteine (nac), glutathionemonoethyl ester (gshmee), probucol and the estradiol-derived scavestrogens j824, j861, j901. additionally, 17fi-estradiol was examined. the used concentrations were 1-5 mm for nac and gshmee and 1-100 ~m for all other substances. according to their influence on tnf-induced am expression, the tested antioxidants can be divided into three groups: 1) pdtc, nac and gshmee inhibit the expression of all three am, 2) j861 and j824 reduce vcam-i and icam-i and increase elam-i, 3) j901 and probucol show no effect. 17fl-estradiol elicits a potentiation of tnfa-induced expression of all three am. tnfa-induced vcam-i expression was most effectively inhibited by ptdc and j861 (100 em: 68% and 78% inhibition, respectively). the best inhibitors of icam-i induction were pdtc (100 ~m: 51% inhibition) and j824 (100 t~m: 47 % inhibition). j824 and j861 most effectively increased the elam-t induction (100/~m: 85 % and 62 %, respectively). the results confirm that antioxidant-sensitive mechanisms play a role in tnfct-induced am upregulation. it is suggested that icam-1-and vcam-l-expression is regulated differently from elam-i. the diversity of antioxidant effects might be due to distinct levels of redox control of transcription with an antioxidant-inhibited and an antioxidant-promoted step. heamophiliacs who continue to produce tilers of f.v11i inhibitors no higher than 5 to 10 bethesda units (bu) when given continuous f,viii replacement therapy are classified as low responders. studies carried out on low responders to assess the effect of immune tole~lcc therapy flit) in these patients. it was found that elimination ofiahibitors in low responders could be achieved using f.vlii at 20 to too u/kg given every 2 to 3 days over a period of 6 weeks. we consider that 1tt is justified in this group of patients. prevents rebooster effects and reduces elimimfion time of f.viii inhibitors. it also reduces the enhanced mortality in patients with inhibitors and has been shown to be cost-effective. in view of the improved prognosis, we recontmend starting itt in low respor, ders soon after the diagnosis has been made in order to minimize f.vill exposure before isfi.tiating el'. itr in low responders has a better success rate an.,d requires shorter u'eatment than itt in high responders. w.schramm, med.klin~, innenstadt, lmu, mfinchen following replacement therapie~ in hemphilm t_be developmem of inhibimrs to the p*ficat's deficient factor is the most serious sequelae. the incidence of inhibitors varies between 15 and 35 % of persons with hemophilia a. inhibitor develop, mostly in paticm~ with sovere bemoph/lia a, early in life and afmr few exposure days (9 -12 days). using the bethesdamethod the inh~itom can be derided in low (< 5 bu) and high msponder (>5 bu) hemopb~iacs with high fitcrs have ~ually serious ~ problems. they are resistent to mg,,flary replacement therapy, the ~ goal in the treawnent is to control severn acum bleedin~ and to eradicate the inlu'bitor perrmnanfly and to induce tolea'ance. in the tream'tmt of acute blcedings in patients with hlhibitors factor viii inhibitor bypassing ag~ts like activated prothro~ complex concenuxtes (feiba) or prothrombin complex concentrates (pcc) arc mostly used. the meehani~n of aefiou of theses concentrates is net fully investigated. their effect is usually related to the high coment of activated clotting factors ~d phosphoupids. since some years acdwated recombinant factor vii (f vii a) is used to treat patients with inl'dbitocs successfully in several clinical situations including surgery. in addition porcine factor vii1 is widely used in particular in the uk for the treatment of factor v].ii inhibitor patients and could demonstrade good clir3cal results, in case of life threatening bleedings a temporary reducfic~ of inhihitors could be. ~hieved by using extem*,ivc plasma exchange (protein a adsorption) and immune suppression with cyclophosphamid (~alm6 protocol). follow~g the first description by h. bmc~'~mn some modifications for tlm induction of irmnune tolerance in hemophilia a patients have ~en propet'ed. these schedule, can be derided into high, intemxxfime and low dosage roglrmms di:ffea'jng in the dosage of factor viii infused. successful rates about 70 to go % can be obtained with ~ and high dose regimens. but is has to be co~sidered that the~ expensive trea.t~nt regimens have a great physical and p .syc.hosocial impact to the benx~-li~s and thch" farm~e& the different immu~ mler-a.~ze mg~-'~ predominantly used in high rcsponder inhibitor. most of the patients with low concentrations of inhibitors cm be managed with factor viii in increased dosage. this is in agreement with the consensus recorrnr~rdadons for u'eatlncnt hemophiliacs in germany fi'orn 1994. before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was 1:4000 both idiopathic and secondary types. since 1981, 4 nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early 1980. however, in a small number of eases, vk deficiency oceured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacental transport of vk in the perinatal period,following studies were carried out. t)hepaplastintest(normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)correlations were made between mothers'and babies'hepaplastin test values. 3)transplacental transport of vk 2 was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastin test value of less than 120% of the normal adult value, the value of the hepaplastintest was less than 30 % of normal adult value in the cord venous blood° we also demonstrated that vk passed through the placenta but only in small qualities. 61hiv-negative patients (median age 4]yrs, 13-70}, formerly treated with non-virusinactivated coagulation products, underwent hepatologic examination, including afp screening and sonography. 31.suffer from severe, 20 from moderate or mild haemophilia a or b, 10 from other severe coagulation factor deficiencies. 52 had been treated with products of the swiss red cross (src) only (28 with small pool cryoprecipitate}, 4 with foreign products only, 5 with both src and foreign products. treatment intensity was variable with>20'000 iu/yr in 26,< 20'000 in ]6, < 1 treatment episode/yr in ]0, a total of only 1-3 treatment courses in 6 patients. 3 afibrinogenemic patients had prophylactic replacement therapy. hcv serology was positive in 56/6] patients (92%), in 47 with detectable hcv rna (77%). the 5 persons who escaped hcv infection, with normal alt-levels and without sonographic alterations, had low intensity treatment with small pool src preparations only. alt-levels were elevated in 33/56 anti-hcv positive patients (59%). 26/56 had abnormal sonographic findings (46%). there was a clear correlation between elevated alt-levels and abnormal sonographies: of 33 patients with elevated alt 23 had abnormal sonography, of 23 with normal alt 3 had abnormal sonography. 8 patients had liver cirrhosis (6 with clinically overt hepatopathy), 4 (4/56 = 1%!) with hepatocellu]ar carcinoma (hcc) with elevated afp-leveis. of these 4 patients 2 had intraarterial embolization with ]ipiodol-epirubicin; in 2 patients hcc diagnosis was made in a late stage. i patient with advanced liver cirrhosis underwent successful liver transplantation. 2 of the 6 patients with hepatopathy had severe haemophilia with temporary high alcohol intake, 4 had mild coagulatlon disorder with few treatment episodes. possible precipitating factors were coinfection with hbv, high alcohol consumation and first exposure to hcv contaminated blood products in an advanced age, but not intensive replacement therapy. very similar results for f vlll and vwf. since the factor viii level is kept steady above the level where there is an increased risk of haemorrhage, continuous infusion is haemostatically safer and more efficacious than bolus injections, another advantage is a progressive decrease of clearence during the first days after surgery which leads to a substantial reduction of factor concentrate consumption by avoiding the innecessary peaks of bolus injections. 22 children with severe form of haemophilia a undergoing elective surgery received continuous infusions with different plasma-derlved and recombinant f viii concentrates. before surgery, patients got bolus injections to raise the factor viii levels to more than 80 %. during continuous infusion factor viii levels were measured two to three times a day and the infusion rate of 4 to 5 iu/kg/h could be reduced on the second or third day to 2-3 iu/kg/h. the clinical efficacy was excellent with no bleeding events. in 5 children with vwd also undergoing elective surgery continuous infusions with humate pr were performed in the same way. no bleeding events were observed in these patients. none of the patients developed postoperative wound infections. the overall doses of f vtll concentrate 'were about 20-30 % lower than those required during replacement therapy with bolus doses. lg8 factor x frankfurt i : molecular and functional characterisation of a hereditary factor x defect (gla +25 lys) huhmann i., holler b., krinninger b., turecek p.l, richter g., scharrer i., forberg e., watzke h. univ. klinik f0r inhere med.i, abteilung for h~tmatologie und h~mostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrombin to thrombin. the congenital fx-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a 24 year old patient presenting a mild bleeding tendency. his p'fi (36 sec) is within the normal range, the pt ( 73% of normal) is slightly reduced. the factor x antigen level is reduced to 55% of normal. molecular charactedsation of the genetic defect was performed by amplification of the eight exerts and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of +25 gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboll site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exert ii. fx was isolated from plasma of the propositus by monoq ion exchange chromatography. performing clotting assays with purified fx frankfurt i we determined an activity of 89% of normal fx upon activation with rw, 77% upon intdnsic activation (aptt) and 81% upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt 56%, ptt 55% and rw 57% of normal) when the reduced fx-ag-level of the plasma (55%) is taken into account. we therefore conclude that the substitution of gla +25 to lys results in a fx molecule which is severely defective in both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardiothoracic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardiopulmonary bypass surgery. blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, thrombin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= 109 patients were examined (age: 64__+9 y). they lost 750+__460 ml blood (mean+sd) into the drains within the first 18 hours after end of surgery. a severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding 12 mg/i at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificity of 96% and a diagnostic efficacy of 91%. in contrast, soluble fibrin which correlated well with fibrinopeptidea (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n = 109). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from bredbacka (1994). other parameters were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibrin for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrin will provide further insights into fibrin(ogen) metabolism. heparin induced thrombocytopenia represents a multicomponent syndrome associated with the use of heparin and related drugs resulting in not only thrombocytopenia, but also arterial thrombosis of varying magnitude. the initial diagnosis ofthis syndrome is usually made by clinical observation and a drop in platelet count. conventional diagnostic methods include platelet aggregation responses to patient's serum and ~4c serotonin release in response to patient's serum, aggregation/agglutination of patient's platdets in response to heparins and the detection of patients anti-heparin platelet factor 4 (hpf4-ab) ned-antibodies by using elisa methodology. several other individualized methods are also used to demonstrate platelet activation. to test the diagnostic validity of the platelet aggregation (pa) 14c serotonin release (sr) and the relevance of hpf~-ab 340 serum samples collected from patients with clinically eunfwmed eases of lilt syndrome were compared in parallel in various assay systems. the diagnostic efficacy of these tests varied from 60-74% with the pa test providing better results than others. when the pa test was compared with serotonin release, a poor correlation was noted (r=0.47). in contrast, the correlation between the pa and hp4-ab was somewhat better (r=0.66). in another study, blood samples collected from 50 patients treated with ahigh dose low molecular weight beparin for two weeks (80 mg o.d.) were tested. 20 of these patients showed a high titre of hpf4.ab without any decrease of platelet count. none of these patients were found to be positive in the 14c serotonin release assay. a third study included blood samples from dvt patients administered with iv heparin infusion, high dose sc lmw heparin (certoparin) and iv lmw heparin for the management of dvt. none of these patient groups (20-34) exhibited any hit responses, hmvever, the incidence of high hpf4-ab titre was found to be 53% in heparin, 36% in patients with lmw heparin iv and 26% in lmw heparin sc groups. pa and sr studies revealed 8% and 12% false positive ~ respeetively. these studies clearty suggest that the currently available ~ for laboratory diagnosis of hit syndrome are of limited value, and caution should be exercised in the interpretation of the results obtained with these tests. heparin-induced thrombocytopenia (hit) is one of the major severe side effects during treatment with heparin. in postoperative medicine clinical studies demonstrated the prevalence of hit with unfractionated over fractionated heparins. few data are available from the non-ope1"ative medicine and from patients without thmmboembolism before heparinization. in a controlled prospective randomized study the safety and efficacy of low-dose heparin was compared with a lowmolecular-weight (lmw) heparin over 10 days in bedridden medical inpatients (haemostasis, in press). 1968 patients were randomized and controlled for the development of thrombocytopenia. thrombocytopenia was defined as a platelet count below 40.000lid at day 10. 4 patients developed thrombocytopenia in the heparin group and no patient in the lmwheparin group (p<0.05). none of the patients with thrombocytopenia developed a thromboembolic complication. in a second prospective case control study 90 patients with side effects on anticoagulants were treated with lmw-heparin once daily subcutaneously for a period of 1 month to 9 years. platelet count was performed every 1 to 3 months. none of these patients developed thrombocytopenia during heparinization with lmw-heparin. it is concluded that hit is a very rare complication in nonoperated bedridden medical patients. a decrease of platetet count may occur in about 0.5% of patients receiving low-dose heparin. the incidence of hit with thrombosis during low-dose heparin and of hit during lmw-heparin in non-operated patients is manyfold lower and remains to be determined. terminology: instead of the term "hemorrhagic disease of the newborn (hdn)" the term vkdb should be used, since neonatal bleeding is often not due to vkdeficieacy and vkdb may occur after the neonatal period (i.e. after 4 weeks). definition: vkdb is a bleeding disorder caused by reduced activity of vkdependent coagulation factors which responds to vk. diagnnsis: in a bleeding infant a prolonged pt (inr > 3.5) together with normal fibrinogen and platelet count is almost diagnostic of vkdb. the diagnosis is proven, if vk shortens the pt (after only 30-60 minutes) and/or stops bleeding. classification: classification by age of onset into early (< 24 h~. classic fdav 2-7) and lale form (> i week <6 months), and by etiology into idionathic and ~ec0nd~'y. in secondary vkdb in addition to breast feeding other factors can be demonstrated, such as poor intake or absorption of vk and increased consumption of vk. vk-prophylaxis: benefits: oral and intramuscular (i.m) vk (one dose of i nag) prevents equally well the classic form of vkdb. lm. vk appears to be more effective in preventing the late form (times 15-> 50). the protection achieved by single oral prophylaxis (times 3-5) is improved by triple oral vk (times 15-30). risks: because of poten[ial ri~l~ associated with extremely high levels of vk and the possibility of injection injury, i.m. vk has been questioned as the prophylaxis of choice for normal neonates. since vk is involved not only in coagulation but 'also in carboxviation with multiple effects, excessive deviations from the low physiologic concentrations, which prevail in the fully breast-fed healthy mature infant should be avoided. proposal: repeated (daily or weekly) small oral doses of vk are closer to physiologic conditions than single i.m. bolus doses, which expose neonates to excessively high vk levels. the incidence of intracranial vkdb can be reduced if the grave significance of warning signs is recognized (i.e, icterns, failure to thrive, feeding problems, minor bleeding, disease with cholostasis). whether or not the more reliable absorption of the new mixed mieellar (mm~ nrenaral~i0n of vk can reduce the protective oral dose of vk-.prophylaxis has to be evaluated. before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was 1:4000 both idiopathic and secondary types. since 1981, 4 nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early 1980. however, in a small number of cases, vk deficiency occured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacentel transport of vk in the perlnatal period,followlng studies were carried out. 1)hepaplastlntest(normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)correlatlons were made between mothers'and babies'hepaplastin test values. 3)transplacental transport of vk 2 was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastln test value of less than 120% of the normal adult value, the value of the bepaplastlntest was less than 30 % of normal adult value in the cord venous blood. we also demonstrated that vk passed through the placenta but only in small qualities. the point mutation g to a at nt 449 in exon v of the factor x gene (gin 102 to lys) has previously been found in two independent kindreds with fx deficiency. it occured in both families in an heterozygote state and was associated with two other genetic defect in the fx gene. we have identified another familiy in which this mutation occurs in a homozygote state. in this family the mutation is associated with the previously reported mutation gla 14 to lys which also occurs in a homozygote state. the pt and ptt of the proposita and her siter are markedly prolonged. the fx activity is reduced to <1% in the extrinsic system, to 30% in the intrinsic system and to 18 % after activation with rvv. the fx antigen is reduced to 20%. the coagulation profile of this family thus is identical with that of fx vorarlberg despite the fact that the fx vorarlberg kindred is only heterozygous for the mutation glal02 to lys. haplotype analysis could not rule out consanquinity with the fx vorarlberg kindred. these data suggest that the mutation at nt 449 which leads to a fairly dramatic amino acid change from glu to lys would indeed represent a polymorphism. to further address this question we cloned the fx gene in an expression vector (pcep 4) for transient expression in the human embryonic kidney cell line 293 and introduced the mutation at nt 449 by site directed mutagenesis. hereditary deficiency of factor ixa, a key enzyme in blood coagulation, causes hemophilia b, a severe x-chromosomelinked bleeding disorder; clinical studies have identified nearly 500 deleterious variants. the x-ray structure of porcine factor ixa shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for fx activation by phospholipid-bound flxa and cofactor villa. the 3.0 a resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (egf)-like modules; the n-terminal gla module is partially disordered. the catalytic module, with covalent inhibitor d-phe-pro-arg chloromethyl ketone, most closely resembles fxa but differs significantly at several positions. particularly noteworthy is the strained conformation of glu-388, a residue strictly conserved in known fixa sequences but conserved as gly among other trypsin-like serine proteinase. flexibility apparent in electron density together with modelling studies suggests that this may cause incomplete active site formation, even after zymogen activation, and hence the low catalytic activity of fixa. most hemophilic mutation sites of surface fix residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary flxa-fvilla-fx complex structure: the stabilizing fvilla interactions force the catalytic modules together, completing flxa active site formation and catalytic enhancement. factor x frankfurt i molecular and functional characterisation of a hereditary factor x defect (gla +25 ---, lys) huhmann i., holler b., krinninger b., turecek pi., richter g., scharrer i., forberg e., watzke h.. univ. klinik ftlr innere medi, abteilung for h~matoiogie und hamostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrembin to thrombin. the congenital f×-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a 24 year old patient presenting a mild bleeding tendency. his ptt (36 sec) is within the normal range, the pt ( 73% of normal) is slightly reduced. the factor x antigen level is reduced to 55% of normal. molecular characterisauon of the genetic defect was performed by amplification of the eight exons and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of +25 gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboti site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exon i1. fx was isolated from plasma of the propositus by monoq ion exchange chromatogrephy. performing clotting assays with purified fx frankfurt i we determined an activity of 89% of normal fx upon activation with rw, 77% upon intrinsic activation (aptt) and 81% upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt 56%, ptt 55% and rw 57% of normal) when the reduced fx-ag-level of the plasma (55%) is taken into account_ we therefore conclude that the substitution of gla +25 to lys results in a fx molecule which is severely defective ip both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardioth~)racic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardlopulmonary bypass surgery. blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, throm. bin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= 109 patients were examined (age: 64+9 y). they lost 750+__460 ml blood (mean_+sd) into the drains within the first 18 hours after end of sur. gory. a severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding 12 mg/i at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificily of 96% and a diagnostic efficacy of 91%. in contrast, soluble fibrin which correlated well with fibrinopeptide a (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n= 109). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from bredbacka (1994). other paramelers were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibdn for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrln will provide further insighls into fibrin(ogen) metabolism. this study was conducted as a randomized parallel -group clinical trial comparing the safety and efficacy of a low molecular weight heparin {lmwh} -monoembolex sandoz and unfractionated standard heparin glfh) for the perioperative prevention of venous thromboembolie disease (dvt) following major surgms' in patients with gynecologic malignancy.. three hundred and twenty 4 women (six drop outsl werr randomized and received either 3 times daily 5000 [l" s.c. ul.'i-i (sandoz nuemberg germany] (n = 164) or once a day t5000 i~'v'. units s.c. monoembolex (n = 160) plus two placebo injections. heparin therapy was started the morning before opcrati(m and continued until the 7th postoperative day. up to the 10th poatop, day the incidence of dvt was 6.25 % (n = 10; incl. 7 pulmona~ embolisms pe) in the lmwh group and 6.10 % (n = 10; incl. 4 pe} in the ufh group. the overall incidence of clinically hemorrhagic wound complications was significantly decreased in the lmwh group 16.3 % (n = 2hi compared to the ufh group 26.8 % {n = 44; p < 0.0051. the incidence of major hemorrhagic episodes was 9.4 % in = 151 in the lmwh group and 14.0 %/n = 23) in the ufh group. this difference was not statisticauy significant. one case of fatal pe was observed in the lmwh -treated group. five women deaths in the lmwh group were observed during the study and 3 in the ufh group. this study demonstrates that the perioperative treaunent of low molecular weight heparins is more safety than standard heparins in gynecologic -oncologic patients undergoing major surge .ry. however, the incidence of thromboembohc complications is simmilar in both treatment regimes. to explore the effect of targeting an antithrombin to the surface of a thrombus, recombinant hirudin (hir) was covalently linked to the fab' fragment of fibrin-specific monoclonal antibody 59d8 (fab) resulting in a stable conjugate (hir-fab). in vitro, hir-fab was 9times more efficient than hir alone in inhibiting fibrin deposition on experimental clot surfaces in human or baboon plasma (p<0.01). to validate these results in vivo, hir-fab was compared to hir in a baboon model. the deposition of ill-in-labeled platelets onto a segment of dacron vascular graft present in an extracorporeal arteriovenous shunt was measured. blood flow rate was 40 ml/min. one hour local infusions of 4500 atu of either hir-fab or hir resulted in deposition of 0.16 x 109 and 2.17 x 109 plate!ets, respectively. equieffective dosages were 2000 atu hir-fab and 9000 atu hir resulting in deposition of 1.06 x 109 and 0.93 x 109 platelets, respectively. based on full dose response curves (n = 14), hir-fab was found to be > 4.5-fold more potent (based on activity) than hir. because of the small total amounts of antithrombins used and the short duration of these experiments, no significant systemic effects were observed. thus, fibrin-targeted recombinant hirudin prevents platelet deposition and thrombus formation more effectively than uncoupled hirudin in vitro and in an in vivo primate model. triabin, a 17 kda protein from the saliva of the assassin bug triatoma pallidipennis, is a new specific thrombin inhibitor (1). tt does not block the catalytic center but interferes with the anionbinding exosite of thrombin. the recombinant protein was produced with the baculovirus/insect cell system and used to study the inhibitory effect of triabin on thrombin-induced responses of human blood platelets and blood vessels. aggregation of platelets in tyrode's solution was measured turbidimetrically at 37°c. for the studies on blood vessels rings (2-3 mm) from small porcine pulmonary arteries were placed in organ baths for isometric tension recording. the integrity of the endothelium was assessed by the relaxant response to bradykinin. like hirudin, triabin inhibited the thrombin (0.1 u/ml)-induced aggregation of washed human platelets at nanomolar concentrations (ec50 = 2.6 nmol/l); whereas the adp-and collagen-induced aggregation were not suppressed. in pgf2c~-precontracted porcine pulmonary arteries, the thrombin (0.2 u/ml)-induced endothelium-dependent relaxation was inhibited by triabin in the same concentration range as found for inhibition of platelet aggregation. higher concentrations of triabin were required fo affect the contractile response of endothelium-denuded porcine pulmonary arteries to thrombin (1 u/ml). in all these assays, the inhibitory potency of triabin was dependent on the thrombin concentration used. these studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of the thrombin-mediated cellular effects. dept. of medicine, university hospital benjamin franklin, free university of berlin, dept. of medicine and dept. of surgery, heinrich-heine:university dusseldod after standardized training in home prothrombine estimation using the coaguchek system, 150 consecutive patients (p) who had st. jude medical aortic or mitral valve implantation were allocated to two random arms; 75 p were asked to control the inr themselves every third day. in the remaining 75 p anticoagulation was managed by the home physician without recommending an interval for these controls. all 150 p were monitored during the education period to a target therapeutic range of inr 3.5-4.0. p were asked to contact their home physician immediately if the inr was measured 0.5 below or above the target range (inr-corrider 3.0-4.5). all p had out-patient re-examinations every three months. thrombotic, thromboembelic and hemorrhagic complications were documented by the p using special documentation cards. the following findings were documented during the follow-up period: 4.49 0.9 0 the results of this randomized study demonstrate a significant improvement in the management of oral anticeagulation by home prothrombine estimation. significant (p<0.001) more inr measurements were found inside the target therapeutic range. moreover. bleeding and thromboembolic complications could be reduced (p = 0.038) in the study group with home prothrombine estimation. life-threatening thromboembolic and hemorrhagic complications were not observed in p who were on home prothrembine estimation, while three such events (2.72 %/year) were documented in group a. local vascular injury following ptca exposes circulating platelets to prodmmbogenic stimuli. by binding to platelet gp iiblliia fibrinogen crosslinks platelets, which represents the final common pathway of platelet aggregation. fradafiban (bibu52zw) is a non-peptide compound with effective, reversible inhibitory effects on fibrinogen binding to gp iib/ii/a on human platelets. in the first double-blinded, prospective phase ii study three escalating doses of bibu52zw as a continuous 24 h-i.v, infusion were tested in comparison to placebo in 65 patients with stable angina pectoris undergoing elective ptca. the mean receptor occupancy with 20rag, 40ms and 60ms per hour were 71.974, 84.5 % and 87.9% at 24 hours, respectively. as compared to placebo breeding time was significantly prolonged (7 vs 20rain) during fi-adaiiban infusion with a weak dose-dependency. platelet aggregation in platetet rich plasma ex vivo with collagen (2.0 and 4.0 gg/ml), adp (2.5 and 5.0 gmol/ml) or ca-ionophor a 23187 (2.5 and 5.0gg/ml) was significantly and dose-dependently inhibited as compared to placebo. using the two upper doses of fradafiban, we observed major bleeding complications in 8 patients requiring blood transfusions or vascular surreal repair. in these patients, too, maximal antiplatelet effects could be documented. these data sugest that bibu52zw is an effective fibrinogen receptor antagonist in patients. the requirement of ad hoc receptor occupancy determination or platelet function monitoring for safe and effective clinical use should be evaluated. in a placebo controlled interaction study 9 healthy volunteers were randomized to receive either a 24 hour infusion of peg-hirudin ( 0.02 mg/kg/h) after an i.v, bolus of 0.2 mg/kg + placebo, or 325 mg/day acetylsalicylic acid (asa) for three days followed by a placebo infusion or the peg-hirudin infusion + asa. each volunteer received all three treaments. there was a washout period of at least 14 days between the infusions. at short intervals aptt, activated clotting time (act), ecadntime (ect), alia-activity using the chromogenic substrate 2238, collagen-induced aggregation, platelet adhesion and platelet induced thrombin gene,ration time (pitt) were measured, bleeding time (simplate) was studied before drug administration, on day three before the infusion and 6 hours after start of the infusion.the infusion of peg-hirudin after 4 and 8 hours led to a mean hirudin plasma level of 1.8 pg/ml. asa markedly inhibited collagen induced aggregation as expected. the mean bleeding time was prolonged under the influence of peg-hirudin from 5.2 to 6.22 min, after asa from 5.8 -18.2 min and after the combination of peg-hirudin + asa from 5.4 -33.7 min. in each volunteer the bleeding time was longer under the combination than after asa alone. in two volunteers receiving peg-hirudin + asa the bleeding time measurement was stopped after 60 rain. none of the coagulation parameters or platelet function tests correlated with the prolongation of the bleeding time. however the bleeding time was excessively prolonged in those volunteers who had a marked prolongation under asa alone.the combination of hirudin at a higher dosage with asa probably is associated with a relative high risk of bleeding. either the hirudin dosage should be reduced if the combination seems feasabie or asa should be given after the end of hirudin treatment. fibrinogen with the sta/stago and the mla/dade systems correlated well, but neither system correlated well with the acl/il system. at iii, protein c, protein s, and anti-xa heparin assays using stago reagents performed as expected for normals and low abnormals on the sta. factor levels on the sta/stago system were less sensitive than factor levels obtained with the dade reagents on the mla or fibrometer. using the sta/stago system, thrombin time results correlated well with the aptt and heparin levels. the thrombin time was not associated with additional manipulation for assay preparation, nor any cross-contamination of reagent or sample, since on the sta reagents do not come into contact with tubing. the sta was not sensitive to hemolytic, icteric or lipernic samples for clotting assays artd showed the same sensitivity as the mla for chromogenic assays. the overall data comparisons, high throughput, minimal operator intervention for reagent/assay change and ease of operation warrant further evaluation of the sta hemostasis analyzer. a. wehmeier, d. s6hngen, c. rieth klinik for h#,matologie, onkologie und klinische immunologie der heinrich-heine-universit~it d0sseldorf hirudin selectively inhibits thrombin by direct interaction. because the effect of hirudin is independent of antithrombin iii and other factors, it seems an attractive alternative to current anticoagulants. however, it is uncertain whether hirudin influences plateletassociated thrombotic disorders and how it compares with conventional and lmw heparin. we investigated the effect of 2 recombinant hirudin preparations (rhein biotech, dt3sseldorf) on platelet function tests: in vitro bleeding time, adhesion to glass beads, aggregation in platelet-rich plasma and whole blood. hirudin was used in concentrations of 0.1-100 i.tg/mi, and was compared to trisodium citrate (0.38%), conventional heparin (50 iu/ml) or lmw heparin (fraxiparin, 500 iu/ml). both recombinant hirudins showed normal activity in thrombin neutralization tests, and prolongation of thrombin time and aptt. however, in vitro bleeding time was not prolonged by hirudin, but was more than doubled by addition of conventional and lmw heparins. platelet retention to glass bead columns was reduced by hirudin in a dose-dependent manner to about 40% but was more effectively reduced by both heparin preparations and citrate. hirudin had an inhibitory effect on p!atelet aggregation in prp induced by thrombin, collagen, and predominantly epinephrine but not adp and ristocetin. in whole blood, a small effect could only be observed with hirudin concentrations of >25 ~g/ml as compared to citrateanticoagulated blood. in summary, thrombin inhibition by recombinant hirudin has little effect on in vitro platelet function tests in comparison to heparins and calcium depletion. the role of endothelin (et), prostaglandins and the coagulation system in the pathogenesis of acute renal failure is still to be defined. in anaesthesized pigs the effects of i.v. infusion of et (3 /~g/kg) alone (group 1, n=6) and after pretreatment with the potent thrombin-inhibitor hirudin (0,5 mg/kg)(group 2, n=6) on haemodynamics, coagulation parameters (factor viii, antithrombin iii, precallicrein, fibrin monomers, aptt) and prostaglandins were investigated. plasma renin activity (pra)-, creatinine clearance-, urine volume-measurement and blood gas analysis were performed hourly. et-infusion caused an initial bp-reduction and marked hr-reduction followed by a transient bp-elevation and hr-reduction. activation of platelets can be directly measured by flow cytometry using monoclunal antibodies. in an in vitro study the effect of the thrombin inkibitors argatroban, efegatran, dup 714, recombinant hirudin and peghirudin on platelet activation induced by various agonists was studied in whole blood. blood was drawn from normal human volunteers using the double syringe technique without use of a tourniquet to avoid autoaggregatiun of platelets. for anticoagulation of blood the thrombin inhibitors mentioned above were used at a final concentration of 10 ~tg/ml each. blood samples were then incubated at 37°c either with saline, r-tissue factor (rtf), arachidonic acid (aa), adenosine diphosphate (adp) or collagen. at definite times (1, 2.5, 5, 10 rain) aliquots were taken and after various steps of fixative procedure the percentage of platelet activation was measured by means of fluorescent monoclonal antibodies to platelet surface receptors gpiiia (cd-61) and p-selectin (cd-62). the agunists used induced a platelet activation of 37.4 + 15.2 % (rtf), 65.1 + 12.1% (aa), 19.3 + 7.4 % (adp) and 27.1 + 12.8 % (collagen). flow cytometric analysis showed that all thrombin inlaibitors studied caused a nearly complete inhibition of r-tissue factor-mediated platelet activation. in contrast, after induction of platelet activation with the other agonists an increased percent cd-62 expression was found showing a strong platelet activation with a maximum at the same times as in non-anticoagulated blood. in conclusion, the results show that in whole blood thrombin inhibitors are effective in preventing platelet activation induced by r-tissue factor. the formation of active serine proteases including thrombin may be effectively inldbked by these agents. the observations further suggest that, while thrombin inkibitors may control serine proteases, these agents do not inhibit the activation ofplatelets mediated by other agonists. this work was supported by the grant bmft 07nbl01. animal experimental studies on the pharmacokinetics of peg-hirudin e. bucha, a. kossmehl, g. nowak max-pianck-gesellschaft e. v., arbeitsgruppe "pharmakologische h~imostaseologie", jena hirudin, when complexed with polyethylene glycol (peg), increases its molecular weight from 7 to 17 kda, thereby preventing extravasation of this drug. peg-hirudin is distributed almost only in the intravascular blood space. in addition, its increased molecular weight retards the renal elimination. the elimination half-life of hirudin in rats (58 + 12 min, as determined) is increased five-fold (244 ± 18 min). with the same hirudin dose applied, the blood level of hirudin is increased 19-fold, measured in the 13-elimination phase. in the urine of rats, 2 -3% of the hirudin activity were recovered following hirudin administration, but 48% could be detected after peg-hirudin had been applied. after subcutaneous administration of peg-hirudin, the trnaxwalue is reached at 380 rain (r-hirudin: 65 min); the cmax-value is increased 3-fold, compared to that of r-hirudin (2.5 pg/ml). 24 hours later, still one fifth of the maximum concentration (cma,) is present in the blood, and the renal elimination is still retarded. in the urine of rats, 12% of the hirudin activity applied were recovered in the 24-h urine sample. with intact renal function, following subcutaneous administration, peghirudin is abte to produce a constant blood level of hirudin over a long pedod. thrombin inkibitors such as r-hirudin (rh), argatroban (a), efegatran (e), and peghiradin (ph) are currently undergoing extensive clinical trials in such cardiovascular indications as ptca, ami, and treatment of unstable angina. a rapid assessment of the anticoagulant actions of these agents is, therefore, crucial to assure their efficacy and safety. currently, act and aptt are used to measure the anticoagulant effect of these agents. we have utilized a dry reagent technology based on the motion of paramagnetic iron oxide particles (plop) to measure the antithrombin effects of various thrombin inhibitors (cv diagnostics, raleight, nc). the heparin monitoring card has been modified to measure antithrombin agents in various anticoagulant ranges for (a)0 (e), (rh), and (ph). blood samples drawn from patients treated with (a) and (rh) have been evaluated and concentrations of these agents have been calculated using an external calibration curve. in the in vitro setting, citrated whole blood or citrated frozen plasma can be used to evaluate the anticoagulant effects of these agents. the results obtained are comparable to the act which is conventionally used for the monitoring of these agents. both (rh) and ( period. we would like to present a case of heparin-induced-thrombocytopenia (hit) in a 47 years old woman who underwend open heart surgery. she suffered from a combined aortic valve disease and leading stenosis. laboratory analysis showed constant low platelet counts (50/nl) without heparin application, so that an idiopathic thrombocytopenlc purpura was suspected. but platelets also decreased after heparin application. heparin-antibodies were found in the heparin induced platelet activation assay (hipaa). treatment with corticosteroids and immunoglobulines, respectively, showed no improvement but the patient unfortunately developed a pneumonia with legionetla pneumophila. therefore, the only suitable anticoagulant for the necessary aortic valve replacement was hirudin: a bolus injection of r-hirudin of 0,75 mg/kg b.w. was administered 10 min. bevore start of the extracorporal circulation (ecc), the heart-lung machine (hlm) was primed with 6 mg r-hirudin and another bolus of 5 mg of r-hirudin was administered. additionally 10 mg of r-hirudin was applicated to the cell-saver-reservoir. during the period of ecc ecarin clotting time and aptt values were taken every ten minutes for monitoring r-hirudin concentration. the postoperative anticoagulation was performed with a constant infusion of r-hirudin starting eight hours after the end of ecc and monitored by aptt. due to mechanical aortic valve the further anticoagulation was performed with phenprocoumon, starting 3 days postop. the therapy with hirudin showed no side-effects. hirudin, threrefore seems to be a suitable anticoagulant in patients with high risk for bleeding complications like this. doses fi:om 2-30 mg/kg gave similar post-op blood loss measurements without s dnseresponse (4-15 oc/kg) (less blood oozing than a historical heparin control but equivalent post-op blood loss; 10 q-3 ec/kg). doses >30 mg/kg showed more intra-op blood loss than the lowe~ doses, but equal post-.op blood loss. the bleeding time test was less elevated than for heparin. platelet counts and hematoerit did not vary except for hemodihition on pump. liver enzymes did not vary significantly pre-op to post. act values showed arg was eliminated (dose-dependently) by 1 hour post-op. dogs were hemodymamieally stable during the peri-operative period, and overall gave predictable responses to arg (as opposed to variable responses to heparin). in a substudy it was demonstrated that hypothermia did not affect the activity of arg, nor did varioos formnlations. this dose finding study strongly suggests that arg may be a safe and effective alternative to heparin for patients undergoing cpb. this is particularly important for the growing population of patients with hit who require cardiac surgery, for which no anticoagulant alternative is presently available. three recent clinical tdals with r-hirudin (timi 9, gusto 2 and hit) have shown that the risk of severe haemorrhagic side effects was strongly associated with high aptt-levels. the large intedndividual variability of the aptt and the lack of a linear dose-effect ratio, however, limits its value for reliable monitoring of the anticoagulant effect of hirudin since even severe overdosage due to impaired renal elimination may not be detected with this assay. we have therefore evaluated the ecadn clotting time (ect) as descdbed by nowak and bucha (thromb. haemost. 1993; 69: 1306) under conditions which allow conclusions on its reliability in the clinical situation.for this, citrated venous blood obtained from healthy volunteers, patients with unstable angina pectoris, and patients treated with marcumar was supplemented with different concentrations of peghirudin. measurements of aptt and ect were made in duplicate. in contrast to the aptt, the ect showed a close, linear relationship with peg-hirudin plasma concentrations in the range of 100 and 10 000 ng/ml. the lineadty of this relationship was not affected by the presence of unfractionated or low molecular weight hepadns in concentrations of up to 10 pg/ml. the ect was not affected by fibdnogen concentrations 60% below normal. a somewhat higher slope but no change in linearity was found in plasma from marcumar-patients with quick-values between 20 and 32%. no significant differences were found between values measured in citrated blood or plasma or using different coagulation timers. the most potent thrombin inhibitor containing a benzamidine moiety is napap (k i = 6 nmol/i). unfortunately, the pharmacokinetic properties (fast elimination by hepatic uptake and biliary excretion, poor enteral absorption) are unsuitable for the use of napap as an oral anticoagulant. the application of choice of a synthetic thrombin inhibitor would be the oral one, therefore, we looked for other lead structures. with the nc~-arylsulfonylated piperazides of 3-amidinophenylalanine we found a new group of derivatives which inhibit thrombin with ki-values in the nanomolar range. the piperazides exert anticoagulant activities with high selectivity, leaving activated protein c and components of the fibdnolytic system unaffected. in rats, the piperazides are rapidly eliminated from the circulation (tl/2 ~ 10 min) upon i.v. administration, too. after oral administration, the systemic bioavailability is low. upon intraduodenal administration of high doses widely varying blood levels were seen, depending on the mode of administration. to cladfy the importance of a possible hepatic first pass effect we studied in more detail the pharmacokinetics of the n~-(2naphthylsulfonyl)-3-amidinophenylalanine n'-acetylpiperazide in rats using hplc-analysis. like other benzamidines the piperazide is excreted via the bile to a high extent. enteral absorption rates of about 20 % are found after blocking the hepatic uptake and biliary excretion. hence, a hepatic first pass effect appears to be the main reason for low systemic bioavailability after orallenteral administration. at the same time, fast elimination from the circulation by hepatic uptake is the main problem for maintaining effective blood levels with benzamidines. therefore, the elucidation of the structural elements influencing the absorption and elimination processes of these types of inhibitors is necessary. the piperazides of 3-amidinophenylalanine bear the possibility to easily introduce a wide variety of substituents on the second nitrogen of the piperazine moiety. a 69-year-old female patient with diabetic nephropathy increasingly developed signs of allergisation combined with dyspnea, erythema, pruritus, and circulatory insufficiency two months after start of heparin-anticoagulated haemodialysis und initial surgical application of a double lumen venous catheter. in addition, growing thrombocytopenia was observed involving a drop in platelets by 50%, compared to the initial values. the haemodialytic efficiency was reduced by massive thrombosis of the dialyzer and subsequent repeated interruption of treatment. at the end of may 1995 heparin antibodies were detected and the hat diagnosis was confirmed. immediately afterwards, haemodialysis treatment was continued, applying hirudin as anticoagulant. using steam-stedlised haemophan dialyzers and 0.14 mg/kg r-hirudin (iketon, italy), the minimum therapeutic blood level of hirudin (0.4 pg/ml whole blood) was reached. this provided therapeutically relevant blood level conditions during a 4.5h haemodialysis. more than 80 regular haemodialyses were run without problems. in all hirudin-anticoagulated haemodialysis treatments the ecarin clotting time was used as the method of choice for bedside blood level and dosage control. after the 34th haemodialysis, the frequency was reduced from 3(4) to 2 haemodialyses a week. accordingly, the hirudin dose was increased to 0.2 mg/kg. the creatinine clearance increased continuously from initially 2.6 to 10.4 ml/min after the 13th week of hirudin-anticoagulated haemodialysis. platelet count and haemodialytic efficiency normalized. we could demonstrate that the regular use of hirudin as anticoagulant along with dialyzers impermeable to hirudin enables very good results in haemodialysis treatment in heparin-associated thrombocytopenia, hirudin is suited for use as anticoagulant in problem patients with hepadn-induced allergy when combined with a drug monitoring method fit for bedside use. capillary electrophoresis methods provide a fast measurement of proteins. thus we developed for pharmacokinetic measurements of r-hirudin and peg-hirudin capillary electrophoresis methods. for the measurement of r-hirudin we used fused silica capillary and a borate buffer. this buffer was used to detect r-hirudin, but could not be used to measure peg-hirudin. for simultaneous measurement we used a neutral capillary to prevent protein absorption to the capillary wall. the buffer was a 20 mm tricine buffer (ph = 8.0 field strength 500 v/cm). it resolved r-hirudin from peg-hirudin at 214 nm using reverse polarity. a linear correlation between the peak area and the concentration was found between 80 pgtml and 10 mg/ml for hirudin (r 2 = 0.99) and between 2,5 and 10 mg/ml for peg-hirudin (r2= 0.99) was found by coinspiking of human plasma and urine with r-hirudin and peghirudin the two proteins were completely resolved. a linear correlation between the peak area and the concentration was found. the method separates r-hirudin from peg-hirudin and may be applied to biological systems to measure the concentration of r-hirudin. triabin is a thrombin inhibitor from the saliva oft. pallidipennis structurally unrelated to any protease inhibitor known and which probably functions by an interaction with the anionbinding exosite of thrombin. we used sf9 insect cells infected with recombinant baculovirus to produce sufficient triabin for a detailed biochemical characterization. the activity of the protein purified from cell lysates was assessed in a fibrinogen clotting assay and was found to be similar to that of the natural protein. a 4-fold prolongation of thrombin-clotting time and aptt was achieved with 22 nm and 600 nm triabin, respectively. a kinetic analysis of the thrombin-catalyzed fibrinopeptide a release from fibrinogen showed that triabin is a tight-binding inhibitor. using the graphical method of dixon, the ki was determined to be 3 pm. introduction: thrombocytopenia is a common adverse effect of heparin therapy, in type ii hit platelet decrease induces severe complications. we here present two special cases of type ii hit. case report i: a 66 year old male patient with dvt of the left leg was treated with therapeutic doses of heparin. from the first to the 12th day of therapy, platelet count decreased from 122000 to 54000/ui. hit was confirmed by hipa-test, heparin therapy was s~opped and treatment with the heparinoid orgaran n was started. during the following days, arterial thromboses in the right a. femoralis occurred. several thrombe~tomies were not successful and although orgaran ~" was stopped because of suspected crossreactivity, amputation of the right leg could not be avoided. during the following days under hirudin-treatment platelet count normalized and no further complications occurred. case report 2: a 74 year old female patients suffering from hip fracture was treated by surgery with tep-operation and received prophylactic heparin treatment. after 6 days, platelet count decreased from initially 170000 to 8000/ul and dvt of the right leg was diagnosed. on the same day, severe bleeding into the left leg was observed and hemoglobin concentration was diminished to 7.8 g% (before surgery 16.0 g%). hit was confirmed by hipa te~t, heparin was stopped and treatment with orgaran started. thrombocyte count normalized and no further complications occured. conclusion: hit type ii can cause severe bleeding as well as thromboembolic complications. because of possible cross-reactivity between heparin and orgaran~,, hirudin should be given in hit patients. currently thrombin time (ti), aptt, activated clotting time (act) or anti ila -activity (alia), measured by a chromogenic substrate test are used to monitor hirudin treatment or prophylaxis. the "13" responds very sensitive to hirudin plasma levels end thus requires variable thrombin concentrations. aptt appears to be more adequate, however, it shows large interindividual variations and does not respond sensitive enough to higher hirudin concentrations. act is a simple whole blood clotting assay, but it is strongly influenced by the blood collection technique. the ecadn clotting time (ect) is a new clotting assay, recently described by nowak and bucha (thromb.haemost 1993, 69,1306) . it measures the clotting time of citrated blood or plasma after prothrombin activation by ecarin, a snake venom of echis carinatus. ec.t shows a linear dependence on different hirudin concentrations over a wide concentration range ( e.g. 0.1-5 pg/ml). in a clinical interaction study healthy volunteers were administered hirudin, asa or both. 15 male volunteers received an i.v. infusion of peg-hirudin (0.02 mg/kg/h) for 24 hours after an initial i.v. bolus of 0.2 mg/kg to compare the sensitivity and reliability of ect with aptt, l-r end act. the act was measured on the hemochron 801, usa, ect on a fibrin timer, aptf using the aptt lyophylized silica reagent by il and alia on an acl (il-milan) with the chromogenic substrate 2238. all tests were performed in duplicate. ect was more sensitive to different hirudin concentrations than aptt, or act. the ect results were better correlated with the alia-activity than ap'l"r and act. the lower detection range for ect is 0.05 pg/ml hirudin. ect is a very sensitive, simple and reliable test for the monitoring of hirudin treatment and prophylaxis. recombinant and synthetic inhibitors of thrombin such as hirudin, efegatran and argatroban are currently in various phases of clinical trials in several surgical and medical indications. the therapeutic effects of these agents are usually monitored by aptt whereas in cardiovascular indications, cefite act and hemotech® act are used. the reliability of both aptt and the act tests in predieting the safety of various thrombin inhibitors has been heavily debated. furthermore, some of these inkibiturs are administered simultaneously to heperinized or coumadinized patients and the obtained aptt and act results do not lady refleot the effects of these agents. fcafin is a snake venom derived fi'om echis carinatus which converts prothrombin into mesothrombin, targeting the arg~3-ile tm bond between the a and b chains of prothrombm. while thrombin inhibitors are capable of inhibiting mesothrombin, atiii/beparin complex does not have any effect. using purified ecarin, nowak and bucha (1993 thromb haemost 69:1306) proposed to assay hirudin. since thrombin inhibitors exhibit similar mechanisms of thrombin inhibition, ecarin clotting time (ect) was evaluated to test its diagnostic efficacy in various experimental and clinical settings. lyphilized eoarin was obtained from knoll ag, ludwigshafen, germany). concentration dependent clotting times for himdin, efegatran and argatroban were obtained in a range of 0-15 p.g/ml. all of the antithrombin agents produced a concentration dependent prolongation of ect and showed va~angpotendies inthe order ofefegatran> argatreban> hirudin, on a gravimetriebasis. on a molar basis, the anticoagulant order of potency was found to be hirudin> afegatran> argatroban. utilizing the ect, the effect of these inhibitors on patients undergoing bolus or infusion therapy, resulting in a concentration level of ~ 10 gt g/rnt, have been measured. unlike such global tests as pt and aptt, patients receiving simultaneous heparin or oral anticeagulants can be monitored for antithrombin specific prolongation ofthe ect. plasma samples from heparinized (aptt 45-90 sec) or coumadinized ('pt 15-25 see) patients, supplemented with argatroban or hiredin did not show any differences m the ect. a medified ecarm act comparable to the celite act has also been developed. initial results demonstrate that this test is not affected by aprotinin, heparin and reduction of the prothrorabin complexes in the inr range of 1.5 -3.0. these results indicate that ecarin based clotting times provide slx~etlie ~lts of circulating levels of thrombin inhibitors, which can provide reliable information to optimize their safe(y and efficacy. r-hirudin is a highly potent and selective inhibitor of the serine proteinase thrombin. after intravenous administration, r-hirudin is eliminated exclusively with the urine. its plasma half-life is very short, 1-2h. peg-hirudin is a derivative produced by coupling polyethylene glycol (peg) to a specially designed recombinant hirudin mutein. peg-coupling results in a considerable prolongation of the plasma half-life of peg-hirudin, compared to r-hirudin. after intravenous administration of r-hirudin into rats, a very small amount of ,,hirudin-like" activity (2 -4% of applied activity) was recovered in the urine. in contrast, after peg-hirudin had been administered, more than 30% of the applied activity could be recovered in rat urine. these results suggest differences in the renal metabolism of peg-hirudin and r-hirudin. within the scope of pharmacokinetie studies in rats we investigated the appearance of biologically active metabolites of peg-hirudin after kidney passage in urine. affinity chromatography on immobilised thrombin was used as a quick and gentle method in searching for biologically active hirudin metabolites in rat urine. but it had to be completed by anion-exchange and/or reversed-phase chromatography to ensure that all active metabolites were detected. the isolated biologically active metabolites were purified by reversed-phase hplc and were biochemieally characterized. in previously reported studies we found a hlrud n derivative consisting of the amino acids 1-50 as the main metabolite in rat urine following intravenous administration of r-hirudin. this metabolite was not detected in the urine after administration of peg-hirudin, confirming the suggestion of a different renal metabolism. carrageenans are high molecular weight sulfated polygalactans of plant origin (derived from red algae) with anticoagulant properties. in previous studies we investigated the anticoagulant activity of lambda-carrageenan, a highly sulfated type of carrageonans. unlike heparin, lambda-earrageenan exerts its anticoagulant activity primarily through direct inhibition of the serine proteinase thrombin. only a part of its antithrombin activity is indirectly mediated through antithrombin iii. to investigate relations between molecular weight and biological activities, tambda-carrageenan has been hydrolysed and fractionated. the molecular weight has been determined with the aid of size exclusion hplc using dextrans as molecular weight standards. the degree of sulfation has been determined by anion-exchange hplc. we have obtained low molecular weight lambdaearrageenans ranging from 10,000 dalton to 100,000 dalton with degrees of sulfation of 13 -17% and 33 -38%. the anticoagulant and antithrombin activity of low molecular weight carrageenans have been determined using coagulation assays and purified systems, and we have compared their activities with those of heparin and other sulfated polysaecharides. further, we have investigated the ability of lambda-carrageenan and its low molecular weight derivatives to inhibit the activity of human blood phagocytes. the activity has been determined by measuring the cellular chemiluminescence in a mieroplate himinometer using a himinol-dependent assay and zymosan as phagocytosis activating agent. we have used an assay in human whole blood and assays with isolated human mononuclear and polymorphnuclear cells. the anticoagulant activity and also the ability of carrageenans to inhibit the activity of human macrophages decrease with decreasing molecular weight and decreasing degree of sulfation. the natural ocouring, yellow pigment curcumin is the major component of tumeric and is commonly used as a spice and food-coloring agent. since curcumin has been reported to have anti-tumorpromoting, antithrombotic and anti-inflammatory properties, we studied, whether curcumin acts on the transcription factors ap-l(jun/fos) and nf-~:b in cultured endothelial cells (ec). when ec were cultured in the presence of curcumin, electrophoretic mobility shift assays (emsa) demonstrated, that binding of endogenous ap-1 to its dna recognition motif was suppressed. inhibition was due to direct interactions of curcumin with the dna-binding motif for ap-i. enhanced ap-1 binding, induced after tnfa stimulation of ec, was decreased in cells pretreated with curcumin. this resulted in reduced transcription and expression of tissue factor, known to be controlled by ap-f and nf-~b. nuclear run on assays proofed, that curcumin directly reduced the tnfa mediated transcription of genes, regulated by ap-1, as tf, endothelin-1 and c-jun. thus, curcumin did not only suppress apl(jun/fos)-binding, but also inhibited tnfa induced jun transcription, transient transfections with tissue factor promotor plasmids confirmed, that inhibition by curcumin was dependent on intact ap-i sites. beside its effect on ap-l-binding, curcumin reduced the radical dependent activation of nf-kb due to its antioxidant properties, however, this inhibition was indirect and less prominent. the relevance of the in vitro data was confirmed in vivo in mice bearing meth-a-sarcoma. when mice received curcumin before tnfa was injected, tumors showed reduced ap-1 activation. simultanously fibrin/fibrinogen deposition decreased, most probably due to reduced tissue factor expression. thus, curcumin inhibits ap-t activation and expression of endothelial genes controlled by ap-t in vitro and in vivo. (jung, 1991) . additionally, haemorhenlogical parameters (plasma viscosity, erythrocyte aggregation) were measured. in all patients aptt, bleeding time, platelet adhesiveness, von wiuebrand f~ctor and factor viii concentration and activity were determined. the patients with von willebrand disease showed characteristic morphological changes of capillary geometry. tortuosity of nailfold capillaries was markedly increased as well as the diameter of capillariez on the arterial and venous side. plasma viscosity was significantly low. multiple parameter analysis concerning to galen and gambino (1983) and using the parameters ,,plasma viscosity below 1.25 mpas", ,,torquation index higher than 5", ,,erythrocyte column diameter bigger than 16,9 gin" showed a positive predictive value of 100 %. capillary diameter and capillary tortuosity have a positive predictive value of 91,2 %. additionally, a reduction of the vasomotorie reserve and/or a decreased erythrocyte velocity in the capillaries below the reference range was found in most of the yon willebrand patients. it was quite remarkable, that 16 of 40 of the yon willebrand patients showed significant capillary bleedings. these findings confirm some former observations (e.g. o'brian 1950) and preliminary reports of our group (koscielny 1994). polymerase chain reaction (pcr)-based quantitation of mrna transcripts is an important tool in the investigation of the underlying molecular defects in inherited platelet disorders, such as the bernard-soulier syndrome. however, for the exact quantitation of mrna a number of methological requirements has to be met. first, a standard (s) mrna must be synthesized which is able to undergo the same processing as the target wild type (wt) mrna. secondly, the quantitation step following the pcr must differentially recognize standard and target dna, and thirdly, the assay must be precise with respect to both inter-and intraassay variability. in order to satisfy these requirements we constructed a s-gpib mrna which is identical to the wt-gpib mrna except a 13 bp long primer recognition site at its 5" end allowing differentiation between the pcr amplified wt-or s-gpib cdna through incorporation of a fluorescein or biotin labelled 5" primer. both standard and w[ gpib mrna showed identical amplification kinetics in the pcr reaction. the amplified dna was quantified using an dna binding assay. in this assay binding of amplified dna to gcn4 fusionprotein-coated microtiterplates is measured. since the gcn4 binding motif is incorporated into the wt-and s-gpib cdna through an identical 3" primer, competition between s-and wt-cdna during amplification has been analyzed. at a given concentration of 250 nm of gcn4. primer no competition between the sdna and wt-dna for the primer was observed during 25 pcr cycles. the sensitivity limit of the assay performed in this way was 250 amol wt-gpib~, dna, and intraassay variability reached from 1.66% to 6.72% calculated for 100 fmol and 5 fmol dna, respectively. to sum up, combination of rt-pcr with the amplified dna binding assay and usage of an internal standard mrna allows sensitive and accurate quantitation of gpiba mrna in human platelets. since upa and thrombin are main conrtibutors to the process of proliferation and migration of vascular smooth muscle cells (vsmc), which is part of the pathogenesis of atherosclerosis. we are currently assessing the role of spatial expression of upa and thrombin receptor (tr) on cells with human carotid artery plaques (n=10). we have used a double immunolabeling approach, combining anti-upa and anfi-tr antibodies. to identify the different cell types, we used the following antibodies: anti a-smooth muscle actin (a-sma) for smooth muscle cells, ulex europaeus agglutinin i (uea i) for endothelial cells, inflammation cell cocktail (cd68+cd45) for monocyte/macrophage and lymphocytes and an anti-proliferation cell nuclear antigen antibody (pcna) to stain proliferating cells. in the carotid atherosclerotic plaques, upa immunostaining was distributed focally, preferentially in the fibrous cap and some cells of the foam cell rich region (fcrr). it was present in distinct patterns: cytoplasmic staining. tr staining was distributed similar to upa staining. with double staining combining anti upa antibodies with anti-tr antibodies, cellular co-localisation of both upa and tr was demonstrated. these cells were identified as smooth muscle cells by -sma. inflammatory cells were mainly localized within the fcrr, they only stained for upa. in conclusion: our data demonstrates that upa and tr are coexpressed in vsmcs in human carotid artery atherosclerotic plaque tissue. we therefore conclude, that the mitogenic activity of upa is associated with the thrombin signalling pathway. in the proficiency test of the ,,deutsche gesellschaft flir klinische chemie" (dgkc) 1/95, 5 lyophilised plasma samples (immuno ag) were sent to participants: a normal plasma and 4 plasmas from persons under oral anticoagulation (oac-plasmas. inr 1.9 to 3.7). the participants (n=552) returned the pt times obtained and in most cases (n=355) also the isi value for the thromboplastin used (isi of pack insert). the inr was calculated using the pt of normal plasma and the isi of pack insert (method i). two additional methods for inr calculation were compared with method i. according to the concept of calibrated plasmas (houbouyan et al., t993), a calibration curve was constructed using the normal plasma and the 30ac-plasmas. the inr calculated using the pt •fn•rma¿ plasma and the laboratory-specific isi value given as 1/slope of the calibration curve (method ii) or was read off directly (method hi). for inr values, calculated by the 3 methods from the participants data (n=355), outlier elimination (2sd, iterative) was performed. the inr mean values for all 3 calculation models remain in a narrow range. using calibrated plasmas (method 1i and m), less outlier were eliminated and cv's obtained were smaller than using the conventional procedure ( i ). obviously, the inr inherited problems, such as accurate isi value, pt value of normal plasma and instrument/laboratory influences on isi, can be reduced using calibrated oac-plasmas. practical approach and educational considera-tions of home prothrombin time estimation a. bernardo, a. bernardo, c. halhuber herz-kreislauf-klinik, bad berleburg, germany specific training is necessary for the patient to achieve reliable and reproducible results in prolhrombin time measurement. the training scheme is based in many respects on experience with similar training courses for home control and management of diabetes and asthma. the education program is divided into a theoretical and a practical part. the theory part has group sessions of twenty patients of a time. the practical course is reduced to a maximum of five patients. the sessions are conducted by a medical doctor and by specialized medicaf/technical assistants. on average eight hours of theoretical education and two hours of practical training are sufficient. the contents of the theoretical lessons are: • need for anticoagulation after heart valve replacement, • potential interaction between anticoagulants and other medication, • accurate recording of the measured prothrombin time results, • techniques of prospective determination of the necessary amount of anticoagulant, • calculation of the individual doses, • potential pitfalls and mistakes, • corrections in case of over-and under-dosage, • early recognition of thromboembelic and/or bleeding complications. an alternative is a full-day intensive course which can be held during the weekend. our recently reported (1) observation that oral anticoagulant treatment causes an increase of heparin cofactor ii (hc ii) activity in plasma is now confirmed by a more extensive study. in 43 thrombophilic patients who were on vitamin k antagonist therapy (marcumar r) we found a median hc ii level of 142 % as compared to 119 % for 72 thrombophilic patients without any therapy (p < 0.002" ) and 104 % for 59 healthy controls (p < 0.001" ). moreover we observed that the increase of hc ii level was significantly correlated with increasing inr-values (r = 0.63, p < 0.001). follow-up observations on some patients showed, however, clear differences in the levels of hc ii activity after onset of vitamin k antagonist therapy. thus, some patients responded rapidly with a significant increase in activity ("strong responders") while others showed only slight changes ("weak responders"). in conclusion, the determination of hc ii activity may result in an improved estimation of the risk of bleeding, especially in high intensity treated patients (inr > 3.5). after intracoronary stent implantation an aggressive oral anticoagulation (oac) therapy is mandatory. to find out whether coagulation activation occurs after coronary stent implantation during high dose oac therapy markers of plasmatic coagulation and d-dimer were measured. patients 5 male patients (average age 57 years) were examined. blood samples were taken before and right after stent implantation and during the following week. patients got 30 mg phenprocoumon during the first three days and additionally heparin and acetylsalicylic acid (asa) were given. methods ptz, aptt, tz, protein c, tat-complexes, fi+2 and d-dimer were measured. results d-dimer levels increased steadily between day 0 and day 7. tatcomplexes showed a slight increase from day 0 (2.4 bg/i) to day 3 (15.3 ~tg/i). on day 7 tat levels were down again (2.0 p,g/l). fl+2 (day 0:1.0 ng/ml) also showed a slight increase on day 3 (1.3 ng/ml). protein c decreased steadily from day 0 (108%) to day 7 (15%). conclusion during the initial phase of oac therapy a coagulation activation is reported but no significant elevation of tat or fl+2 was found. this result shows that additional heparin and asa therapy was sufficient to avoid systemic coagulation activation. the increase of d-direct should be interpreted as a si~=m of local fibrinolytic reaction due to stent implantation. three methods for the determination of prothrombin time from capillary blood in patients under oral anticoagulation have been investigated. two methods were run on coaguchek® monitors (boehringer mannheim) from capillary whole blood. after fingerpuncture the first drop of blood was applied to the well of a coaguchek® test strip directly from the finger-tip, whereas the second drop was sucked into a non-anticoagulated plastic capillary (hirschmann) and immediately applied to the test strip -and vice versa to eliminate any influence of first and second drop of blood. the third method was hepato quick (boehringer mannheim) which was determined out of citrated capillary blood from an earlappuncture. 66 specimen of patients under oral anticoagulation were investigated. the method comparisons between each of the coaguchek® methods and the laboratory method show good results and the correlation between the coaguchek® methods is excellent. mean differences to the lab methods are -0.1 inr in both cases. no mean deviation was detectable between the coaguchek® methods. scattering of coaguchek® versus hepato quick was +/-0.6 inr in the range 1 to 4 inr except for three outliers and one patient with fluctuating results in the lab method which could not be resolved. introduction: haemorrhagic coumarin skin necrosis is a severe complication during initial phase of oral anticoagulant therapy. histological examination shows thrombotic occlusion of small vessels, but little is known concerning the pathophysiologic background of the bleeding component. recently, we described protein z deficiency in patients with bleeding complications of otherwise unknown origin. thus, we were prompted to measure protein z in patients with coumarin skin necrosis. patients: 4 patients (i man, 4 women; age: 35±10 years) suffering from haemorrhagic coumarin skin necrosis were examined. all patients had normal liver protein synthesis function, none was under oral anticoagulant treatment during this study. method: protein z antigen test, diagnostika stago, france. results: 4 out of the 5 patients examined had diminished protein z levels ( 700, 820, 1080, 1700 ug/l) in comparison to normals (2900 ug/l). in one of our patients, protein z was normal (3020 ug/l). conclusion: low protein z levels are additional risk factors for haemorrhagic coumarin skin necrosis. oral anticoagulant therapy is the treatment of choice in patients with need for long-term anticoagulation. since oral anticoagulants interfere with the function of vitamin k, it is not clear whether stable oral anticoagulation can be achieved in patients with need for continous substitution of fat-soluble vitamins including vitamin k. we report about a 59-year-old man who had experienced progressive hypertrophic obstructive cardiomyopathy over the preceeding 21 years. atrial fibrillation has been first diagnosed 18 years ago. latter on, recurrent ischemic attacks and embolism of the right arteria iliaca occurred. in 1993 the patient received extirpation of the ileum and subtotal amputation of the jejunum because of mesenteric infarction. the resulting short bowel syndrome requires continous substitution of fat-soluble vitamins. since vitamin k free preparations of fat-soluble vitamins for parenteral use are not available, prophylaxis of thrombosis has been performed with unfractionated hepadn. as a consequence of the longterm treatment with hepadn the patient developed severe osteoporosis. therefore, the decission 1:o discontinuate heparin therapy and initiate oral anticoagulation has been made. because of its shorter halflife warfarin (coumadin) was used instead of dicoumarol. over a 4 weeks lasting induction phase inr values were controlled daily. a dosage regime starting with '10 mg warfarin at the day of vitamin application (day 1) followed by 3.75 mg on day 2 and 1.25 mg on days 3, 5, and 6, respectively, was found to be optimal to maintain inr values within the target range (inr: 2.0-3.0). in order to minimize the risk of hemorrhage the vitamin administration was changed to the subcutaneous route. during an observation period of 6 months neither any bleeding or thrombotic complications nor a vitamin deficiency occurred. these data indicate that stable oral anticoagulation can be achieved despite extreme variation of vitamin k plasma levels. portable monitors for home monitoring of inr are well established for adults on oral anticoagulants. patient's compliance is improved as well as long term outcome. experience concerning accuracy of the procedure in children is limited. 32 inr determinations were performed in parallel from venous and capillaryblood samples of an infant on phenprocoumon, starting at the age of 4 months. the coaguchek® monitor from boehringer mannheim was used. choosing an arbitrary range of agreement of ,qnr 0.5 for both determinations, 81% of the measurements were within the defined range. 5/6 outliers were due to low inr resulting from difficulties in capillary blood sampling. the degree of agreement increased when the procedure was performed at least once a week. in conclusion: inr determination with a portable monitor may be helpful in home monitoring oral an.ticoagulant therapy in young children. a dose adjustment should be done only on the base of inr determination of venous blood -if it is considered the gold standard -to avoid over-anticoagulation. a stable anticoagulation is one of the most difficult tasks in attending patients with heart-valve-prosthesis. if prothrombin times are out of the therapeutic range, the risk of bleeding or thromboembolism increases disproportionately. for this reason any improvement in anticoagulant control and/or management can have far reaching consequences in decreasing complications, in extending longevity and in improving quality of life. for the first time a clinical trial was started in 1986 and continues until today at the cardiac rehabilitation center bad berleburg, germany with patients mainly after heart valve replacement. the patients were trained to measure their own prothrombin time and to adjust their own dosage of the oral anticoagulant. within six years 600 patients were trained: 216 patients could be followed up with regard to their selfdetermined prothrombin times. the results were within the therapeutic range in 83.1% of the measurements (n=14.812) taken by the patients themselves. on average, the patients who determine their prothrombin time themselves did so at a weekly interval. neither major bleeding nor thromboembolic complications could be observed in the 205 patient-years of home prothrombin estimation. it is to be hoped that the usual rate of complications can be reduced when patients determine their prothrombin time themselves at a close interval, resulting in more constant values in the therapeutic range and slight corrections of the anticoagulant dose. home prothrombin estimation promises better quality of life and has a considerable potential to achieve this goal. circulating plasma thrombomodulin (tm) is a novel endothelial cell marker, which may reflect endothelial injury. tm acts as thrombin receptor which neutralises the fibrin-forming effect of thrombin, and also accelerates the formation of the anticoagulant protein c/s pathway. tm therefore belongs to the anticoagulant defence system against thrombosis. increased tm levels have been described in various diseases such as ards, thromboemboembolic diseases, ttp, diabetes, le and cml reflecting alterations of the vascular system at the endothelial level. to find out to what extent cardiac catheterisation imtates vascular endothelium, tm concentrations (stago, asnieres, france: x 10 3 iu/ml) were investigated prospectively in 58 infants and children (three days -16 years). blood samples were drawn before the intervention, immediately at the end and 24 h later, snap frozen (-70 °c) and investigated serially in dublicate six weeks -3 months later. the results (median and range values) are shown in the enhanced tm concentrations immedately after the operative intervention, followed by normalisation within 24 h, indicates that cardiac catheterisation in pediatric patients rather leads to a short lasting irritation of the vascalar endothelium than to severe irreversible endothelial damage. recently in an al=wl" based method dahlb~ick et al described in vitro resistance to the anticoagulant effect of activated protein c (apc) in thrombophilic adult patients. apcr is in the majority of cases associated with the arg 506 gin point mutation in the factor v gene. concerning the special properties of the neonatal hemostatic system (low vitamin k dependent coagulation factors, physiological prolongation of the pt and aptf) we adjusted this ap'it based method (chromogenix, m~,lndal, sweden) to neonatal requirements: apcr was measured in 120 healthy infants according to dahlb~ck. the results were expressed as apc-ratios: clotting time obtained in a 1:1, 1:5 and 1:11 dilution with factor v deficient plasma (instrumentation laboratory munich. germany) using the apc/caci2solution divided by clotting time obtained with cac12 in the same i:1, 1:5 and 1:11 dilution. in addition, plasma of 24 neonates with septicaemia were investigated and data of 18 infants aged birth -three months with arg 506 gin +/-were shown. the arg 506 gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mnl i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. results are shown in the 1.6(1.4-1.95) neonates and infants were considered to be apcr when the aptt ratio was < or = 2. concerning the special properties of the neonatal hemostatic system, our data show concordance with the pcr method in neonates and infants only, when the aptt based method was performed in the i: 11 plasma dilution. case report: we report on an 8-year old boy with severe hemophilia b and frequent screaming at night. eeg showed spike wave activity, starting from the temporal lobe, but generalizing within seconds. complex partial seizures were diagnosed and therapy with carbamazepine was initiated. as no improvement was seen nmr was performed. this revealed lesions within the right frontal cortex. higher doses of carbamazepine were not succcssfull as was therapy with phenytoin and pfimidone respectevely. the patient is now treated with carbamazepine and valproate. he still suffers from one short seizure per day. because of his seizures we started prophylactic replacement therapy with 600 i.e. factor ix twice per week. discussion: in 1992 wilson et al. first detected brain abnormalities in 25 of 124 children and adolescents with hemophilia a or b who were negative for immunodeficieney virus (1). the most common findings (14/25 patients) were small, focal, nonhemorrhagic white matter lesions of high signal intensity on t2weighed images. similar lesions have been reported in children with sickle cell cerebral infarction (2) . only three of these 14 patients had seizures, all of those having a documented history of intracranial hemorrhage. our patient has similar lesions as those described by wilson et al. but no history of intracranial hemorrhage is documented. even if tuberous sclerosis might be a differential diagnosis, we think that the abnormalities are related to hemophili a or its treatment, because the patient has no further signs of this disorder. conclusions: 1. in patients with hemophilia and seizures nmr might be useful as a high sensitive method for the detection of gray and white matter changes. 2. further studies should be initiated to determine the prevalence of pathological conditions in the brain of hemophiliac patients. disseminated intravascular coagulation (dic) is a rare, but foudroyant disease occuring in gram-negative sepsis like meningococcal septicemia. despite the avallibility of potent antibiotics, mortality in mertingococcal disease remains high ( about 10 % ), rising to 40 % in patients presenting with severe shock and consecutive dic. as the clinical course and the severity of manifestations of systemic meningococcal infections varies there is a need for early diagnosis of the infection and stage of coagulopathy in order to reduce the high mortality rate. few and rapidly available parameters are needed to classify the wide spectrum of clinical and laboratory findings in patients with dic. the parameters include partial thmmboplastin time, pmthmmbin time, plasma levels of fibrinogen, fibrin monomers and dimers, fibrin degradation products and the thrombocyte count. monitoring the course of hemostaseologicai findings in 26 pediatric patients with systemic meningococeal infections we observed a change of coagulation parameters as early as in the first stages of the infection: a prolongation of partial thromboplastin time to an average of 69. 1 sec (range 22 -150 sec, norreal 30 -45 sec), a decrease of prothrombin time to 45.7 % (range 13 -71 %, normal 70 -100 %) and of antithrombin iii to an average level of 16. 8 u/ml (normal 20 -29 u/ml ) was found 1 to 4 (-6) hours after admission. the consecutive development of hemostaseological parameters mentioned above permitted to define the stage of coagulopathy and thus to induce a stage related therapy. primary treatment consisted in control of shock by liquid substitution, compensation of metabolic acidosis, correction of clotting disorders ( at iii and heparin in stage of pre-dic ; at iii and fresh frozen plasma in case of advanced dic ) and treatment with g-lactam antibiotics ( e. g. cefotaxime or ceftriaxone ). an early assessment of the coagulation disorders in meningococcal disease can be based on few coagulation parameters, thus an appropriate treatment may be arranged to prevenl the patient from a fatal outcome of meningococcai septicemia and protect him from the development of a waterhouse-friderichsen-syndrome. this study was designed to prospectivdy evaluate coagulation and flbrinolyfie activation in 60 children (neonate -16 years) during cardiac catheterisation with low dose flush heparin (10 iu/ml saline). aptt (instrumentation laboratory: see), anti xa activity (xa; chromogenix: iu/ml), prothrombin fragment ft.2 (f1.2; behring werkc marburg: nmol/l) and d -dimer formation (d-d; bnhring werke/vhrburg: ug/l) were investigated before (t1), at the end (t2) and 24 h after cardiac catheterisation (t3). in addition, to evaluate the influence of inherited thrombophilia in all patients resistance to activated protein c (apcr), protein c, protein s and antithrombin were investigated. during catheterisation median (range) hepadn was administered in a total dose of 60 (17-206) iu/kg bw. in addition infants < 6 months of age (arterial catheterisatiun only) or patients with known thrombophilia received 300 -400 iu/kg hepafin for fmther 24 hours. the results (median and range) are shown in the ft.2 was sigificanfly elevated above the pediatric boundary immediately after the intervcation and nearly reached baseline values 24 h later. in contrast no cfinically relevant fibrinolytic activation was seen: d -dimer formation increased within the pediatric boundary immediately after the catheter and returned to basdine levels 24 h later. three children showed resitance to apc. tn one child stroke occurred before. not knowing the result of apcr in the remaining two patients only one neonate received further prophylactic heparin. the third neonate without heparin prophylaxis suffered from venous occlusion within two days after the intervenfon~ in addition, no protein c, protein s or antithrombin deficiencies were found. although administration of low dose flush heparinisation during cardiac cathetefisation could not prevent short -term coagulation activation, no thrombotic events occurred in children without inherited thrombophilia. if fnrther prophylactic hepariuisation in children with a~r, protein c, protein s or antithrombin deficiencies may prevent vascular occlusion requires a more intensive study. a.sandvoss, w.eberl, m.b0rchert introduction: capillary leakage, edema and hypovolemia are common complications in preterm infants especially if birth weigth is below 1.500 g. septicemia, asphyxia and immaturity seem to be most important risk factors. to determine the influence of c 1-esterase inhibitor (cilna) in preventing contact phase and complement activation we investigated c11na concentrations in normal and symptomatic preterm infants. methods: activity of cilna were measured by chromogenic substrate method (behringwerke), cilna concentration with radial immunodiffusion (behringwerke,germany). results: cllna-activity in asymptomatic preterm infants (n= 14) was 65+/-15% of normal at birth. healthy newborns showed activities of 80+/-20%. cilna reached normal adult values 2-4 days after birth. preterm infants with respiratory distress syndrome(n = 14) showed lower activity on day 2-5, patients with additional septicemia (n=15) had decreasing c1 ina-activities in the first three days of life. individual course of cllna-activity and thrombocyte count correlated in the group with irds with and without septicemia. in children with capillary leakage onset of diuresis went parallel with raising cllna-activity. markers of contact phase (f xlla) and complement activation (c 5al were investigated in single cases and evidence for involvement of both systems was found. conclusion: contact activation and complement system play an important role in capillary leakage in preterm infants. cilna regulates both systems. activity of cilna correlates with clinical course, substitution therapy is possible and may improve outcome of these critical ill patients. antiphospholipid antibodies (apa) interfere with hemostasis probably by inhibition of protein c or prothrombinase complex. thereby, apa might lead to thrombosis or increased bleeding. however, incidence and clinical importance of apa has not yet been investigated in children. therefore, we assayed plasma samples of 220 children, aged 0,1 to 19 years (mean 7 years) by elisa detecting igg-and lgm-antibodies directed against eardiolipin, phosphatidyl serine and phosphatidic acid. in patients with increased bleeding, thrombophilia or prolonged clotting tests a detailed coagulation analysis was performed. according to their diagnosis children were devided into 5 groups: i. autoimmune diseases, ii. infections, iii. metabolic diseases, iv. other diseases, v. healthy children. results: apa were found in 69/220 patients. in the respective groups we demonstrated apa in the following proportions: 1. lgg-isotype: activitiy of c1 esterase inhibitor (c11na) is reduced in preterm infants especially if birth weigth is below 1.500 g and respiratory distress syndrome and/or septicemia is present. capillary leakage with generalized edema, hypovolemia and hypotension is resulting in imbalance between inhibition and activation of contact phase and complement system. iln four patients we investigated seven courses of substitution ;with commercial c1 esterase inhibitor preparation (berinertr,behringwerke), case reports are given. all patients had clinical symptoms of capillary leakage, all had septicemia accompanied by either respiratory distress, disiseminated intravascular coagulation or mutiple organ failure. jefficiacy of substitution therapy is dose related, supranormal iactivities of cilna are necessary, reflecting raised consumption of inhibitor in ongoing disease. clinical effects on diuresis, catecholamine need and especially on thrombocyte counts are demonstrated. or arterial thromboembolic event in children e. lenz, c. heller, w. schr6ter*, w. kreuz johann w. goethe-universit~itskinderklinlk, frankfurt a. main, germany * georg-augast-universit/itskinderidinik, g/3ttingen, germany venous thrombosis as well as arterial thrombo-occlusive events are rarely observed in childhood, but can lead to life-threatening situations and longterm sequelae in these patients. after the initial stage of treatment (thrembolysis or thrombectomy) the pediatrician has to decide how to efficiently prevent re-thrombosis in the individual patient. anticoagulation after venous thrombosis is generauy recommended for 6 months after the event; if an underlying thrombophilic condition has been detected in the patient anticoagulation has to be considered lifelong. when evaluating antithrombotic therapies for children it is of importance to consider whether the anticoagulatory effect is mainly necessary in the venous or arterial vessel system. the hemorrhagic risk and side effects of the different anticoagulatory preparations have to be taken into account, especially when treating small children. only limited experiences exist concerning the suitability of the preparations for long-term anticoagulation in children and general recommendations on the ideal dosage in pediatric patients are still missing. we want to disscuss different types of anticoagulants (such as coumarins, unfractionated heparin, low molecular weight heparin (lmwh) and inhibitors of platelet aggregation) their mode of action, their suitability for pediatric patients and their side effects and relevance of these side effects especially in children. from the experience in our own pediatric patients, we would like to report on the indications, which can be given to administer these different preparations, the dosage regimen we recommend and the laboratory tests to monitor save and efficient re-occlusion prophylaxis in our patients. in this context we would like to present our data on 8 patients with either thrombosis or arterial infarction due to a thrombophilic condition, who had all contraindicatioas to oral anticoagulation by coumarins. because prophylaxis for re-thrombosis was mandatory in these patients, lmwh was given for long-term anticoagulation in a dally subcutaneous dosage of 100-150 anti-xa u/kgbw. monitoring was done by anti-xa-test (0,4-0,8 anti-xa u/ml). under this regimen none of the patients developed re-thrombosis or bleeding complications. alopecia was seen as a side-effect. this study was designed to prospectively evaluate coagulation and fihrinolytic activation after cardiopulmonary bypass with aprotinin (2x17000 u/kg bw) in 42 infants and children aged 0.1 -15 years, and to correlate these findings to the clinical outcome. prothrombin fragment f 1.2 (f1.2; behring werke marburg: nmol/l), antithrombin-serinesterase -complex (atm; stago: ng/ml), d -dimer formation (d-d; behring werke marburg: ug/l), tissue-type-plasminogen activator ag (t-pa; chromogenix: ng/ml), plasminogen activator inhibitor 1 antigen (pai; chromogenix: ng/ml) and cl-inhibitor (c1; behring werke marburg: x 10-3 g/l) were investigated before the operation (t1), at the end of the operation (t2), and on postoperative days 1 (t3), 4-6 (t4) and 7-9 (t5), respectively. the results are shown in the table (median and median absolut deviation): t1 t2 t3 t4 "1"5 nv fi.2 0.9 +/-0.5 1.7 +/-0.9 1.4+/-1 1.8+/-0.8 1.6+/-0. the platelet (pl) function defect induced by thrombolytic agents has been attributed either to the degradation of pl surface receptors or to the anti-aggregatory effect of fgdps. in contrast to other plasminogen activators scu-pa is intimately inked with pl: they can rapidly incorporate exogenous seu-pa, release it upon stimulation and bind the proenzyme. recently we have reported that exposure of prp to recombinant scu-pa (2.5-t00 um) in timed interval 1-30 min resulted in dose-dependent inhibition of pl aggregation. timecourse changes of the process were followed by the biexpotential kinetics: a rapid initial inhibition during the first 3-5 rain with the moderate suppression of pl aggregation in the 30 min period. when tcu-pa (25-100 nm) was exposed to prp in the same conditions dose-and time-dependent inhibition of pl aggregation was also observed. since the effect was obtained no earlier than t0 min after exposure of tcu-pa to prp, and the threshold dose was higher. comparable inhibition of pl aggregation was obtained with 25nm of scu-pa versus 100nm of tcu-pa and the llbrinogen depletion by the end of the 30 min period was 2% and 30% respectively. it's likely that tcu-pa and its precursor have different mechanisms of action on the pl aggregatory function. in a recent study we have shown that recombinant rscu-pa inhibits platelet (pl) aggregation in prp. to exclude the possible influence of rscu-pa/plasma interfere on this process the aggregation of washed pls was under the investigation. pls were washed according to modified mustard's method, suspended in buffer and adjusted to 250,109/1. the resuspended pls were exposed to 5-100 nm of rscu-pa for 30 min at 370(;. at time points 3, 5, 15 and 30 min the aggregation with 0.6 iu/ml of thrombin was measured. it was found that the exposure of pls to rscu-pa (20-100 nm) for 3 man resulted in marked inhibition of their aggregation. since after 15-30 man of incubation with 20-50 nm of rscu-pa the inhibitory effect on pl aggregation became less pronounce or even disappeared. when 5 nm of rseu-pa was used the inhibition of pl aggregation became significant only by 15 rain of exposure period and didn't change for 30 man of investigation. the observed results may be cormeeted with uptake of rscu-pa by pls from surrounding buffer as well as with individual variations of pl response to the same concentration of rscu-pa. loss of glycosylation may result in a reduced platelet (p) survival and perhaps altered function. we analyzed the structural and functional effect of specific deglycosylation (combinations of n/o-glycosidase and neuraminidase treatment) of p and isolated p gpib. washed and formaldehyde-fixed p were digested as follows: 1) with neuraminidase (0.125u/ml) + o-glycosidase (3.1mu/ml) + n-glycosidase (1.25u/ml), 2) with neuraminidase alone (0.2u/ml), 3) with n-glycosidase (2u/rnl) and 4) with neuraminldase (0.2u/ml) + o-glycosidase (5mu/ml). all reactions were performed in the presence of protease inhibitors (pmsf, leupeptin, sbti), after washing x2 the p and identically treated controls were analyzed by flowcytometry with the antibodies 6di (mab: a-gpib), 7i-l2 0vlab: a-gpiiia), and the lectins wheat germ agglutinatinln (wga, for neunac) and peanut agglutinin (pna, for [3dgal(1-3)-galnac) which confirmed effective and specific deglycosylation by the respective enzymes (but gave only minor differences with 6di and 7h2). the botrocetin (13) and ristocetin (r)induced agglutinations showed arer treatment 1) (all enzymes) a full inhibition of r-induced agglutination but only a mildly reduced b-induced agglutination (70% of normal). treatment 2 and 3 (neuraminidase alone, and n-glycosidase alone) affected both agglutinations only mildly (70-80% of normal).treatrnent 4) (o-deglycosylation) however showed a major inhibition of r-agglutination down to 30%, while b-agglutination interestingly was almost fully retained. the results of the rotary shadowing electron microscopy of purified gpib suggested a collapse of the normally stretched, glycosylated, gplb, not only after the treatment with all three glycosidases, but also .after o-deglycosylation alone. we conclude that oglycosylation is most important for ristocetin-induced platelet-von willebrand factor-interaction and responsible for the typical stretched shape. the phenomenon of in vitro platelet aggregation and consequent pseudothrombocytopenia (ptcp) in the presence of calciumchelatization by na-edta and sodium-citrate was studied in blood samples of a patient. initial platelet counts electronically measured were 20000/ul blood anticoagulated with na-edta and sodium-citrate. normal platelet counts were found in heparin-anticoagulated blood and in capillary blood. immunoglobulines of the igg and igm subclass were identified in the patients plasma. by incubation of the patient's serum with platelets of healthy individuals, platelet-clumping occurred in the presence of na-edta and sodium-citrate but not in the presence of heparin. the platelet membrane glycoproteins (gp) hb/llia, ix and iiia/vnr g-chain were involved in the antigen antibody reaction as demonstrated by specific antibodies and flow-cytometry. on platelet surface permanent calcium-exchange and -replacement is dependent on external calcium concentration. calcium depletion induced by calcium chelators as na-edta and sodium-citrate might conformationally change platelet surfaces and induce formation of neoantigens. the decrease of gp llb/illa platelet surface antigen to 10% (normal >75%) indicated the important role of the gp iib/iiia receptor at ptcp. the saliva of tdatoma pallidipennis, a triatomine bug, was found to contain a protein called "pallidipin", that specifically inhibits collageninduced platelet aggregation but not adhesion or shape change. to investigate the mechanism of action of recombinant pallidipin the influence on platelet fibdnogen binding after activation by collagen type i in different concentrations was measured by flow cytometry. the same concentrations of pallidipin that inhibited the couagen-induced platelet aggregation completely did not cause any inhibitory effect on fibdnogen-binding in the prp from the same donor measured contemporaryly. collagen type i-induced platelet aggregation of cd36-deficient platelets from two different unrelated blood donors was inhibited by the same concentration of pallidipin that inhibited aggregation of control platelets. there was no inhibition of collagen-induced fibdnogen-binding in the cd36-deficient platelets as well. pallidipin did not cause inhibition of collagen-induced membrane expression of cd62 and cd63 of control and cd36-deflcient platetets as measured by flow cytometry. however eadier studies had shown an inhibition of collagen-induced atp and {3tg secretion by pallidipin. therefore we compared the effect of pallidipin in unstirred and stirred prp samples. while pallidipin had no effect in unstirred samples it showed strong inhibition of ptg secretion in stirred samples. we therefore conclude that pallidipin does not act on collagen-induced aggregation through cd36 and that the inhibition is a post fibdnogenbinding event. pallidipin does not influence the first steps in secretion, which are independent from cytoskeleton and platelet-platelet contact, but inhibits the following steps. 17-hydroxy-wortmannin does not inhibit the transport of 1nm-gold labelled fibrinogen in resting platelets. e. morgenstem, b. kehrel and k.j. clemetson medical biology, saarland univ., homburg, germany, haemostasis research, univ. muenster, germany and theedor-kocher-lnstitut, univ. bern, switzerland. wortmannin, an inhibitor of phosphoinositide 3-kinase and of myosin light chain kinase blocks reactions of the activated platelet. to obtain informations about the role of the contractile cytoskeleton in receptor-mediated transport of resting platelets, the effect of 17-hydroxy-wodmannin (hw) on the endocytosis of fibrinogen from the surface of resting platelets was studied. gel filtered platelets (gfp) were incubated for 10 min at 37°c with hw (3x10-6m) or with iloprost. controls and gfp preincubated with hw or ilopmst were incubated with 1.4nm-gold labelled fibrinogen molecules (fg-au; final concentration 40p.g/ml) at 37°c. the experiments were stopped after 5 or 30 min by rapid freezing. after freeze substitution in acetone with 4% osmiumtetroxide, sedal sections were prepared. the sections were examined after incubation with ascorbic acid (5% in h20) for 30 rain at 20°c (to reduce metallic osmium) and silver-enhancement using danscher's (1981) method (to visualize the fg-au). examination of adp stimulated platelets in the presence of 40fg/ml fg-au shows that the ligand is able to mediate aggregation. the examination reveals, that fg-au was present in a low density on the platelet surface, in higher density in the surface connected system (scs), in coated pits and vesicles and separated smooth vesicles (representing endosomes?) as well as in the matrix of alpha-granules. after 30rain, the number of labeled granules was increasing. labels on the surface and on the mentioned cytoplasmic membranes were observed during the whole period of incubation. hw or iloprost did not alter the resting gfp and the mentioned qualitative ultrastructural findings in both preparations did not show differences to the controls. we conclude from the results with hwthat the regular contractile function of the cytoskeleton is not necessary to transport the fg-au in resting platelets. methods: edta anticoagulated whole blood was incubated with thiazole orange and analyzed with a flow cytometer. young platelets were defined by having a high fluorescence from thiazole orange (normalized to platelet size). platelets were also incubated with fluorescent antibodies to gpib, gp lib/ilia and gmp-140 (two colour method). results: surface expression of gpib was the same in young and older platelets. results for gp lib/ilia and gmp-140 (in resting and activated platelets) will be presented. conclusion: young platelets can easily be detected using thiazole orange and flow cytometry. there is no differential expression for gpib. further results will be presented. the influence of erythrocyte and thrombocyte content on the release of atp by different agents in whole blood specimens was tested. the measurement had been performed in the lumi-aggregometer using the principle of the luciferin-luciferase reaction. altogether 39 blood samples were diluted gradually before induction of the release reaction by arachidonic acid (1,25 mmol/i final concentration), adp (30 ijmol/i) and collagen (1,0 and 5,0 tjg/ml). the peak of the obtained curves was transformed into percent values of the maximal deflection by the undiluted sample (= peak in relation) and into atp concentrations (= absolute peak) after testing the atp standard in parallel for each dilution step separately. the peak in relation increases by increasing dilution with all inducers. it was identic with the atp standard and with collagen, somewhat lower with arachidonic acid and much higher by adp. a luminescence-optical effect may influence all these results. the absolute peak decreases by dilution under arachidonic acid and collagen as it was expected by the decreasing thrombocyte content of the samples. under induction by adp no decrease of the absolute peaks by increasing dilution of the samples was abserved. this can be explained only by liberation of atp from the erythrocytes. the atp standard is essential for the quantification of the release reaction. adp doesn't suit for it. collagen with a final concentration of 1 pg/ml was proven as the best inducer. platelet aggregation induced by several agents has been photometrically investigated in disc shaped rotating cuvettes coated with vessel wall tissues obtained from human umbilical cord, either endothelium or smooth muscle cells or extracellular matrix or combinations of them. in addition, effects of endothelium incubated with several cytokines on platelet aggregation have been studied. endothelial cells strongly inhibited aggregation depending on their cell count and the concentration of the inducer. smooth muscle cells showed the same effect but very less marked. in presence of extracellnlar matrix spontaneous aggregation occured. endothelium could inhibit this spontaneous aggregation when present in the same cuvette, smooth muscle cell could not. incubation of endothelium with several cytokines increased its anti-thombotic properties. for example, at a platelet count of 3x105/id in the prp, 10 -6 m adp led to maximal aggregation in uncoated cuvettes, in presence of 5,5x106 endothelial cells aggregation was completely abolished, in presence of 2,75x10 "6 cells aggregation was decreased to 40%. smooth muscle cells diminished the aggregation effect of 0,1 nih thrombin to 67% when only one side of the cuvette was coated and to 63% when both sides were coated. endothelium could not inhibit aggregation induced by 2,5 x 10 -6 m adp but endothelium incubated with 500 u/ml tnf-a or 30 u/ml intedeukin-lfl or lmm l-nitro-arginin for 24 h did completely inhibit aggregation. platelets become sticky and adhere to surfaces or to another without contracting and secreting. during maturation of megakaryocytes finally platelets lost their genomic nuclear message. only mitochondrial dna of platelets can be identified. we focused our attention on the impact of mitochondrial dna and the mitochondrial transscriptive mechausisms during platelet activation in normals. materials and methods: leucocyte free (nagentte chamber, flow cytometric analysis) platelet rich plasma or platelet concentrates a_~er hemapheresis were filtered by pall 100 leucocyte filters. the influence of different anticoagulants (commercially available sarstedt tubes containing citrate, heparim edta and 500 atu/ml hirudin wacker) was examined. activation was due to a 60 nun. hemapheresis procedure ( 3-5fold increase of cd 62, cd 63) and ex rive stmaulation due to 4 niy u/ml thrombin, 0.025 m cac12 or combmatious. the guanidiurn method for total rna preparation were used according to t. brown: current protocols in molecular biology 4.21-4.9.14,1991. different primers of mitochondrial genome (e.g. cytochrome b and atpase) were prepared using pcr and mitochondrial transscription was examined using northern-blot-technique. results: 1., there is less activation of mitochondrias using hirudin anticoagnlation, but a 2fold increase of mitochoindrial rna content in heparinized samples. 2., stimulation with thrombin leas to an increase to 5.5 e-l0 rna btg/platelet, compared to 4.7 -4.8 e-10 rna ~tg/platelet under unstimulated conditions.. conclusion: there is evidence for the importance of platelets mitochondrial dna and mitochondrinl transsefiption in regulation of cytosceleton and platelet activation. thrombospondin-1 (tsp-1) is a large homotrimeric glycoprotein originally identified as a platelet alpha-granule component. the investigation of its putative role in a variety of pathophysiologies like haemostatic disturbance, malignancy and wound healing requires specific laboratory reagents. monoclonal antibodies are one of the most powerful of these reagents. therefore, we purified human tsp-1 from thrombin-stimulated platelets using affinity chromatography to generate monoclonal antibodies in mice. a subclass igg 1 monoclonal antibody designated 48.42 was purified from ascitic fluid and further characterised. western blot experiments demonstrated that this antibody reacted only with the unreduced molecule whereas the tsp-1 subunit chain was not recognised. no cross-reactivities with human fibrinogen, fibronectin, vitronectin and von willebrand factor were found. preliminary results indicate that the monoclonal antibody 48.42 can be used to investigate tsp-1 function in several assays including immunocytochemistry and cell adhesion as has been demonstrated for hl-60 cells. in addition, a sandwich enzyme immunoassay was developed using goat-antihuman tsp-1 igg and derivatised monoclonal antibody 48.42 (peroxidase, biotin) as a sensitive method for detection of tsp-1 in human body fluids. in the following study the expression of the platelet antigen (cd62p) and the leukocyte antigen (cdllb) were measured in whole blood, in addition to platelet-leukoeyte adhesion (rosette formation) by means of multicolour fluorescent labelling (cd45, cd14, cd42a). the measurements were carded out both in freshly drawn whole blood which had been antieoagulated with different agents, and in stirred samples of whole blood under controlled conditions (37°c, 1000 rpm, different stirring times). the results are presented as the percent positive events in each gate (platelets, leukocytes -pmnl, monocytes, lymphocytes and rosettes -plateletpositive events in the pmnl, monocyte and lymphocyte gates), whose mean fluorescence is given in addition to an index comprising the product of the percent positive events and their mean fluorescence. stirring (max 15 rain) induced an increase of cd62p on the platelet surface of ca. 10%, without any change in the mean fluorescence. under these conditions increased cdllb on pmnl and monoeytes could be detected. an increase in the rosette formation could also be measured (greater index), in that the percent of monocytes which were platelet-positive increased with no change in the mean fluorescence of the positive events, whereas pmnl showed an increased mean fluorescence, but not an increased number, of platelet-positive events. the time-dependent changes in rosette formation on stirring could be further increased by addition of adp. these results show that it is possible to measure rosette formation, and also the influence of effector agents (inhibitors or activators of platelets or leukocytes) on rosette formation, in whole blood using flow eytometry. 17 itp patients undergoing splenectomy were observed after 1-30 years following operation and divided into 2 groups. first group consisted of 8 patients with normal platelets count and absence of haemorrhagic syndrome. second group was formed of 9 itp-patienfs with episodes of thrombocytopenia recovery following certain time period after splenectomy. in the aim to study the cellular immunity there were carried out immunophenotypical investigations of blood samples using immunofluorescence method with monoclonal antibodies application. the increase of b-cells, expressing cd22, cd37, hla-dr-antigen has been revealed in the 2nd group. quantity of srfc, cd3 +, cd5 + cells in the blood of recovered patients was lower than in patients of the first group. this group was also characterized by statistically significantly increased level of cd4 + cells while the cd4/cd8 ratio was equal to 1.0 :i: 0.3 % (0,5 + 0,1% in patients of the second group, respectively, p>o,05}. also the relatively high expression of activating antigens in patients with thrombocytopenia recovery after splenectomy was stated. among infectious complications in all patients observed were predominantly found various types of throat infection, mainly with unsatisfactory treatment possibilities. we have observed the opsi-syndrome in 2 patients, being featured with marked tiredness, breath loss, intolerance of hard physical working, diminished ability to maintain physical activity. extracellular matrix (ecm) produced by human endothelial cells closely resembles the vascular subendothellal basal lamina in its organization and chemical composition. thus it contains collagens, fibroneetin, von witlebrand factor, thrombospondin, fibrinogen, vitronectin, laminin and heparin-sulphate. platelets carry different receptors on their membrane surface with specific binding capacities for one or more of these extracellular matrix proteins, such as glycoprotein (gp) iibiiia, gp ib/ix and gpiiib. incubation of platelets with ecm results in platelet adhesion, degranulation, prostaglandin synthesis and aggregation. we studied patients whose platelets showed either a receptor defect in gpiibiiia or gpiiib or a storage pool disease. adhesion experiments were performed using siliconised glass, collagen coated surfaces, immobilized fibrinogen as well as human subendothelial matrix. platelet adhesion of patients with thrombasthenia glanzmann (receptor defect of gpiibiiia) resulted in a total lack of binding to silieonised glass and immobilized fibfinogen. adhesion to collagen was almost normal in spite of the fact that only single platelets sticked to the surface and no microaggregates were observed. the adhesion to ecm was diminished and also no aggregates were detected. patients with a receptor defect in gpiiib showed normal platelet adhesion to siliconised glass and immobilized fibrinogen but binding to collagen and ecm was markedly reduced, while platelets with a storage pool defect sticked to siliconised glass but failed to adhere to ecm. by centrifugation of citrate blood (250 x g, 10 min) erythrocytes and leucocytes go to the bottom, whereas plasma and thrombocytes stream in the upper part of the probe. so the thrombocyte count doubbles in the platelet rich plasma in contrast to the platelet count in the whole blood volume. if the thrombocytes are more or less activated, they adhaere on erythrocytes, leucocytes or aggregate end are not able to stream upwards. the quotient between thrombocyte counts in prp and whole blood is a measure for thrombocyte activation. we chequed the value of this screening in different groups of patients with arterial occlusions disease (aod), chronical venous disease (cvd), diabetes mellitus (dm] and in healthy control persons (control). variation coefficient of the method is 3.7 (prp) and 4.4 (tc) respectively (coulter counter). differences to the control group are significant. changes in the patient groups in dispensaires follow up 5 years are also significant. nicardipin -induced immunthrombocytopenia p. eichler 1, c. hinrichs 2 , g greinacher l i.institut fur immunologic und transfusionsmedizin, ernst-moritz-arndt-universitat greifswald, 2. deister-s0ntel-klinik, bad m0nder drug-dependent immune-thrombocytopenias are a rare but clinically important variant of immune-thrombocytopenias. patients are at risk to suffer from severe bleeding complications. especially in patients receiving multiple drugs, diagnosis of drug-dependent immune-thromboeytopenia is often difficult. we report the case of a 71 year old male patient who received allopurinol, captopril, digitoxin, furosemid, and nieardipin. the patient presented with hematomas (pit. count < 10 g/l) and later developed bone marrow dysplasia. in an elisa using whole platelets and patient serum, a weak reactivity in the presence of furosemid, but a stronger reactivity in the presence of nicardipin (antagonil, ciba-geigy) could be demonstrated. the reaction pattern is given in the the enzyme-immunological determination of soluble fibrin (sf) proved to be highly sensitive and specific. this sf-elisa detected fibrin hacking fibrinopeptide a (fpa) via the monoclonal antibody 2t35 specific for the neoepitope generated on the aa-chain after the split of fpa. lill et al. recently introduced a new assay modification which utilizes the same antibody as the old one but takes advantage of a pretreatment of plasma specimens with kscn. this strong chaotropic ion is used to dissociate the various fibrin complexes possibly hiding fibrin epitopes. it was the aim of this study, therefore, to compare the two sf-elisa modifications (with and without kscn-pretreatment of specimens) . in order to examine the dynamics of thrombin-induced fibrin(ogen) metabolism we made course observations in patients with a certain form of septicemia. both assay modifications detected fibrin(ogen) derivatives which differed considerably in kinetics (n= 160 samples from 10 courses). the former sf-elisa (no kscn) correlated well with prothrombin fragments, thrombin-antithrombin !11 -complexes and with the release of fibrinopeptide a ( r > 0.96, n= 151). results of the new sf-elisa with kscn pretreatment of patients' plasma, however, correlated conspiciously well with d-dimer levels (r > 0.94) but distinctly less with the markers of thrombin generation (-0.12 < r < 0.29). this good correlation with d-dimer levels was unaccountable since the d-dimer maximum occured significantly later than the peak of markers of thrombin generation (p < 0.05). therefore, kscnpretreatment of fibrin specimens seems to lead to a change in the specificity of the fibrin assay despite usage of the same catching antibody. different half-iifes of differently composed fibrin complexes should be considered in trying to explain the findings. nevertheless, the results of the former assay without kscn-treatment correlated much better with the well-known dynamics of thrombin-induced fibrin generation during hemostasis activation than the data from the new assay modification. consequently, further examinations are necessary to specify the effect of kscn on soluble fibrin complexes and the resulting assay specificity. a rapid assay for the determination of the primary hemostasis potential (php) of whole blood has been developed (kundu et al, 1995) from the original method of kratzer and born. the new system employs a disposable test cartridge which holds the sample (citrated whole blood) and all components for the tests at the same time. the test procedure is very simple. the cartridge is loaded with -500 p.l citrated whole blood and is inserted into the platelet function analyzer (pfa 100aaw). the test is started automatically after a preincubation phase of 2.5 rain. the reaction starts with the contact of the whole blood and the capillary which is connected with a collagerdephinephrin coated membrane with a small aperture inside the test cartridge. under constant negative pressure the sample is aspirated and through the contact ofplatelets and vwf with collagen adherence and aggregation begins. the adhesion and aggregation process leads to the formation of a platelet plug which obstructs the flow through the aperture. the result of the php is reported as closure time (ct). additional parameters such as bleeding volumes are possible as well. first results show good reproducibility, normal values in the range of up to 150 sec. and a good discrimination of healthy donors from patients with congenital or acquired platelet dysfunctions. the system detects aspirin induced thrombocyte function defects and von willebrand disease. in ease of an abnormal result in the collagerdepinephrin system a second type of cartridge with a collagerdadp coating can be employed. in the majority of cases aspirin induced dysfunctions are normalized and could thus detect aspirin use. the proposed system may be a valuable tool for routine assessment of the primary hemostasis potential in a routine citrate blood sample laboratory. inducing mental stress in 20 young healthy male volunteers aged 20 to 40 ),ears with no previous history of thmmbophilia or a hemorrhagic diathesis was performed by a first time parachute descent from an altitude of 1000 meters. the purpose of this investigation was to find out whether there are any changes in the corpuscular and plasmatic fractions of peripheral blood. we were especially interested in elucidating changes in the procoagulatory and/or fibrinolysis systems. venous blood samples were obtained directly before and directly after the jump. flight time from the departure of the airplane to the landing of the parachutists was approximately 20 minutes. the maximum time that elapsed between the two blood withdrawals were 45 minutes. in a preliminary study with different voinnteem, certain fluid imbalances had been observed. absolute numbers of leukoeytes (6.9 vs. 9. l/n0, erythrocytes (4.6 vs. 5.1/pl), and platelets (246 vs.276/nl) significantly increased (p < .001), as well as the hemoglobin concentration from 145 to 156 g/l (p < .018). even though fluid imbalances before and after the jump had practically been excluded by measuring nearly identical hematoerit values (.41 vs..42), we noticed a marked drop in aptr (27 vs. 23 sec) and a significant increase in factor viii ~tivity. as a direct stress response, we found a rise in fibrinogen concentration (2.4 vs. 2.8 g/l) which is one of the shortest acting acute phase proteins. concerning reactive fibrinolysis, d-dimers showed an increase in concentration from 115 lag/l to still normal values of 192 lag/l, which was not significant due to low numbers of values (p = .086). we observed similar changes in fibrin monomers and prothrombin fragments fl+2. from other investigations on the kinetics of the activation of the procoagulatory system we know that maximum activil7 is not reached until 24 hours after initiation of activation.these investigations studied perioperative changes in different kind of operations which served as a control group concerning the degrees of tissue damage and resulting coagulation disturbances. to better understand these phenomena we plan to induce mental stress in a laboratoq' environment to further exclude unknow~a influences on the mechanisms which can activate the procoagulatory and fibrinolytic systems. triodena (t) 30/40/30 ug ee, 50/70/100 ug gestodene) were tested for their effect on hemostatic parameters. three groups (n=20) of healthy female volunteers were treated for 6 months with one of these oc. blood was taken before treatment (day 24-28 of pretreatment cycle, 0) and on days 18-22 of the 3 ~ (i) and 62 (ii) treatment cycle. indications of an activation of blood coagulation and fibrinolysis were detected as the plasma levels of prothrombin fragment f i+2 and of fibrin split product d-dimer and plasmin antiplasmin complexes were found elevated during treatment. the following main regulatory components of blood coagulation, activators and inhibitors, were investigated: factor vii antigen fviiag, fvii clotting activity fviie, circulating activated factor vii cfviia and antithrombin 3 at3 activity, total protein s antigen tps-ag, free protein s antigen fps-ag, protein s activity psact, circulating thrombomodulin etm fviiag, fviie and cfviia significantly increased during treatment; cfviia: 0: c 32.4 mu/ml a prethrombotic condition characterized by elevated levels of circulating soluble fibrin has been claimed to be a predisposing factor for accumulation of coronary thrombotic material in acute myocardial infarction. the present study includes 161 patients with clinical suspicion of myocardial infarction. blood samples were drawn by the primary care physician, upon arrival in the hospital, and after 2, 6, 12, and 24 hours of hospital stay. patients with myocardial infarction were identified by typical course in 12 lead ecg, and upon sequential determination of troponine t, myoglobin, ck, and ck-mb. patients with primary cpr were excluded from evaluation. soluble fibrin was measured by enzymun®-test fm (boehringer mannheim). patients with acute myocardial infarction display soluble fibrin levels within the normal range (< 5 ~tg/ml) during the initial two hours after onset of symptoms. there was no significant difference between patients with myocardial infarction and patients with coronary heart disease without myocardial infarction. slightly elevated levels were found in patients with atrial fibrillation, reflecting intracardiac fibrin formation. in patients without fibrinolytie treatment, a slight increase of soluble fibrin levels with a maximum after approximately 8 hours is observed. most patients with fibrinolytic treatment display a considerable increase in soluble fibrin, with maximum levels immediately after infusion of the fibrinolytic agent. four patients with pulmonary embolism showed soluble fibrin levels in the range of 40-300 [.tg/ml, which remained in the same range during the entire observation period. in conclusion, circulating soluble fibrin is not increased in patients with acute myocardial infarction and does not appear to be a predictor of acute coronary events. high levels of soluble fibrin in patients with fibrinolytic therapy may reflect release of fibrin from thrombotic material, but also de novo generation of fibrin due to release of active thrombin from thrombi not necessarily located in the coronary vessels. detection of elevated levels of soluble fibrin in patients with acute chest pain should result in careful examination for signs of pulmonary embolism or aortic aneurysm. the possibility to determine activated coagulation factors opens the question if data provide evidence of an activated coagulation or fibrinolysis and if this has a prospective value. we investigated patients with confirmed thrombosis, postsurgical septieaemia and also after liver transplantation. in all patients factor viia, xii, xiia and also the fibrinolytic parameters t-pa, pai-1, pap, plasminogen and a2-ap were determined. in addition, f1+2 and apc-resistance with heterocygote factor v-leiden-mutation and confirmed thrombosis. we found increased factor viia which showed partly also an increased fl+2. patients with other pathological results such as a reduced t-pa and/or increased pai-1 showed a low incidence of elevations in factor vii or f1+2. the activation of factor xii seems to be of minor importance in patients with thrombosis. a different picture is found in septic and transplanted patients. obviously factor xii-activation is of major importance in this group. a deterioration of the clinical symptoms is correlated with an increased factor xiia which is paralleled by a decrease of factor xiiactivity. the investigation of fibrinolysis parameters such as pai-1 and pap demonstrate a fibrinolytic disturbance of the balance. statistically significant are differences in septicaemic patients both in the surgical and in the internistical group in contrast to polytrauma patients. in patients with liver transplantations significant changes are apparently related to rejection of the transplanted organ together with a deterioration of the clinical picture. the possibility to detect activated coagulation factors may be a tool to detect changes in the hemostasis system at an early stage and to use this for an improved therapy. control of long-term oral anticoagulation is usually performed by serial determinations of the prothrombin time. however, the assessment of effective anticoagulation versus the potential risk of bleeding complication is difficult to achieve. molecular markers of blood coagulation activation might add valuable information in individual cases. we investigated 48 patients with thromboembolic manifestations (deep vein thrombosis n=22, pulmonary embolism n= 13, myocardial infarction n= 13) for one year beginning with admission to the hospital. tat, prothrombin fragments f 1 +2, d-dirner and fibrin monomer concentrations were analysed. all markers were significantly increased at the time of initiation of anticoagulant therapy thus reflecting a prethrombotic situation. patients suffering from venous thromboembolism demonstrated higher concentrations of tat and f 1 +2 in comparison to myocardial infarction (34.6 vs 12.3 pg/1, p=o.009; 2.8 vs 1.3 nmol/i, p=0.0025). f 1 +2, tat and d-dimer concentrations decreased gradually over the first 14 days of anticoagulant therapy reaching values within the established normal ranges in all cases. f 1 +2 and tat concentrations reflect the activity of the coagulation system during long-term anticoagulation whereas analysis of fibrin monomer yielded partly controversial results. we conclude that f 1 + 2 and tat appear to be superior to fibrin monomer for the individual control of oral anticoagulant therapy. the influence of thyroid failure on haemostasis is controversial. mainly hypoceagulable states have been described in clinically overt hypothyroidism. since hypothyroidism has been associated with an increased risk of atherosclerosis, we studied a wide range of haemostatic factors in untreated female patients with subclinical (b, n=42, age 59+13) or overt (c, n=8, age 55-zcj) hypothyroidism, as well as in hypothyroid women under 1"4 treatment (d, n=8, age 57+9) and euthyroid controls (a, n=80, age 50+14). simple screening tests (prothrombin time, activated partial thromboplastin time, fibdnogen), procoagulant factors (fvii, fviii, von willebrand factor), coagulation inhibitors (antithrombin ill, hepadn cofactor ii, protein c, protein s) and fibdnolytic factors (plasminogen, antiplasmin, plasminogen activator inhibitor, tissue plasminogen activator) were measured. results factor vii activity (vii:c), factor vii antigen (vii:ag) and their ratio were found increased in hypothyroid patients. factor viii activity showed the same tendency, whereas von willebrand factor ramained unchanged, as did all other parameters with exception of free protein s, which declined in overt hypothyroidism and in t4 treated subjects. these differences tended to diminish after exclusion of 26 women with estrogen replacement therapy for menopause, but the ratio vii:cnii:ag, as well as fvii:c still remained significantly higher in hypothyroid patients. conclusions: subclinical and overt hypothyroidism are associated with significantly higher levels of factor vii:c and vii:ag. the disproportionate increase in vii:c compared to vll:ag, as shown by their ratio, might reflect the presence of activated factor vii (vila), which in turn indicates a hypercoagulable state. this pattern becomes more pronounced with the concomitant estrogen replacement after menopause. exocytosis following platelet activation leads to translocation of cd62p (p-selectin), cd63, and thrombospondin, from cytoplasmic granules to the cell surface membrane, where these molecules, serving as activation markers, can be detected by flow cytometry. we here report detectability of these molecules preformedprior to platelet activation -inside the cytoplasm of resting platelets. two different methods are compared, i. e. using either methanol or the fix&perm kit (an der grub) for cell membrane permeabilization. in addition, interleukin(il)-ice is shown to be present in platelet cytoplasm after methanol treatment, but not after permeabilization using fix&perm. whenever cell surface positivity for a specific marker coincides with intracellular presence, blocking of the surface membrane sites prior to membrane permeabilization is required in order to obtain fluorescence intensity attributable to cytoplasmic staining. our data demonstrate the feasibility of the methods presented for the detection of intracellular platelet molecules. this technique should also provide a means for estimating the relative quantity of intracellular platelet antigens, provided the permeabilization procedure does not lead to antigen leakage or destruction. physical exercise activates the clotting as well as the fibrinolytic system as indicated in numerous investigations of exercise by running and by bicycle ergometer but not by swimming. the positive effect of an endurance training in coronary sport groups is induced also by influences on the hemostatic system. the influences are suppression of the clotting activation by the acute exercise and by an increased fibrinolysis response. different hemostatic parameters, therefore, were analyzed before and after swimming of male coronary patients (n=33; median ag~ 61 years, achieved heart rate: 68/min). indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f1+2 among the coronary patients (tat from 2,1 to 3,4 pg/1; fi+2 from 0,92 to 1,1 nmol/1). the degree of clotting activation among the coronary patients was less than that observed in a group of young volunteers in a former investigation. this must be explained by existence of the coronary heart disease or by the higher age in the patient group. indicating an activation of fibrinolysis t-pa activity increased significantly in coronary patients (from 0,14 to 0,5 iu/ml) resulting in an unchanged balance between coagulation and fibrinolysis. from this findings of the hemostatic systems no increased risk of the coronary patients by swimming can be derived. a prerequisite, however, are precautions l±ke to devoid exercise in the anaerobic range, exclusion of major heart failure and of cardiac arrhythmias before begirming of the swim training. the principle of the fontan operation consists in anastomosing the right atrium to the pulmonary arteria, thus bypassing the right ventricle and using the only functional single ventricle as a pump for the systemic circulation. there are only few data about the influence of the changes in hemodynamics on coagulation and fibrinolysis. we investigated the coagulation system in 20 children and young adults aged 4 to 21 years in a general examination 4 to 61 months after fontan procedure. besides other abnormalities of the coagulation system, there were significantly increased values for the thrombin-antithrombin-iii-complex (tat) in 12 patients (60%). as a marker for an activation of the fibdnolytic system we found elevated plasmin-alpha2-antiplasmin-(pap-) levels in 14 patients (70%). less frequently, the concentrations for the prothrombin-fragments 1 and 2 (f1 and 2) (7 patients, 35%) or the d-dimer (2 patients, 10%) were increased. we didn't find significant differences in a clot-lysis-assay between fontanoperated patients and an age-matched control group. there was no significant correlation between activation of coagulation and clinical situation or diameter of the pulmonary arteria. whether the present data can help to estimate the risk for a thrombo-embolic complication following fontan procedure, still has to be investigated. the results of the clot-lysis-assay suggest, that for lysis of thrombi the same dose of rt-pa should be used as for other patients. a 2nd generation functional protein s assay p. van dreden* and e. adema** * serbio, gennevilliers france, ** boehringer mannheim, tutzing germany a second generation protein s test was developed with improved sensitivity to protein s and better reagent stability. the test result was found to be unaffected by apc-resistence (10 patients, heterozygote for the mutation with a apti' + apc ratio between 1.4 and 1.9), heparin up to 2 iu/ml and f viii activity between 1 and 250%. in the test, diluted sample is mixed with protein s deficient plasma, activated factor v, activated protein c, phospholipids and an intrinsic pathway activator. this mixture is incubated for 3 minutes. during this time, the activated protein c inactivates part of the f va. the extend of f va inactivation depends on the protein s concentration. after 3 minutes caci2 is added and the time untill clot formation is measured. the clotting time is a linear function of the protein s concentration between 10 and 140% protein s. for the three preproduction lots the difference in dotting time between 10 and 100% protein s was 43-54 seconds. this compares to 30-40 seconds typically obtained with the old test. within run precision (n= i0 on sta) is cv= 2 -7% on the basis of protein s. day to day precision (n=10 on sta) was found to be cv= 4 -11%, again calculated on the basis of protein s concentration. the cv of 11% was obtained for an avk plasma with 13% protein s; it corresponds to a standard deviation of only 1.5% in protein s. the insensitivity to interferences, in particular apc-resistence and better precision and stability are expected to improve the quality/reliability of a protein s determination. in this study we evaluated the use of hormonal contraception on the parameters protein c, protein s and pal. samples from 71 women with, without hormonal contraception and in menopause were assayed by coagulometric (protein s clotting test (behdngwerke, marburg, frg) or chromogenic methods (protein c activity test and pal reagent from behringwerke, marburg, frg) in double determination and were compared with the reference ranges. in addition thromboplastin time (thromborel s reagent) and fibrinogen (multifibrin) from behringwerke, marburg, frg, and aptt (actin fs reagent from dade corp., unterschlei6heim, frg) were determined. in women using hormonal contraceptives (p<0,01) and in menopause (p<0,05) protein s activity was significantly reduced compared to other women (<45 years) while protein c acitivity did not change. in menopausal women a higher susceptibility to thrombosis was supported by an increase of aptt (p<0,05) and fibronogen (p<0,01). while there was no change for pal, plasminogen was significantly lower in women using hormonal contraceptives and in menopause (p<0,05). we could not observe a higher turnover of coagulation and fibdnolytjc system with hormonal contraception. noteworthy was the occurence of low (<200 mg/dl) and borderline fibrinogen (max. 220 mg/dl) in 40,9% of women res. in 22,8% of women (together with borderline aptt) who had an individuell risk for arterial disease. protein s protein c fibdno~en aptt plasminog~ without hcc 109,1-+13,6 78,3-+14,1 255,0-+14,0 36, [2] [3] [4] [5] 4 24, [0] [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] 2 with hcc 85, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] 2 78, 5±12, 0 253, 8.+24, 1 35, [2] [3] [4] 8 14, 9 menopause 90, 3+97, 6 87, 4±41, 4 307, 05:57, 6 39, [8] [9] [10] 0 12, 6 hcc= hormonal contraception hemostatic parameters in a patient undergoing bone marrow and subsequent liver transplantation due to veno-occlusive disease c. salat 1, , e. holler t,3, hi. kolbl, 3, b. reinhardt l, r. pihusch 1, p. g0hring 2, s. poley 2, e. hiller 1 l=med. klinik iii, 2 = institut flit klin. chemie, klinikum grosshadern der ludwig-maximilians-universit~tt mfinchen, 3=h~tmatologikum der gsf a 40 year old patient suffering from all received allogeneic bone marrow transplantation (bmt). after an uncomplicated early posttransplant period the patient was dismissed after 4 weeks. a bilirubin rise with subsequent liver failure was observed during the following weeks. according to biopsy proven hepatic veno-occlusive disease (vod) liver transplantation was performed on day 79. unfortunately the patient died on day 140 due to aspergillosis. we monitored levels of protein c (pc) and s (ps) as well as pall during the pre-and posttranspiant period. pal1 level was normal (<43 ng/ml) during the first 4 weeks after bmt but increased with the manifestation of vod (317.5 ng/ml on day 47). it reached its peak immediately before liver transplantation (547.6 ng/ml) and returned to normal levels within the next few days. pc levels which were normal before bmt decreased prior to clinical diagnosis of vod and were normal after liver transplantation. ps levels lay within the normal range at all timepoints. vwf was elevated before bmt (240%) and remained relatively stable during the whole investigatonal period ranging from 170 to 260%. it is assumed that vod is initiated by an endothelial cell injury -possibly due to radiochemotherapy -and subsequent hypercoagulability. our results indicate that the "endothelial cell marker" vwf is not helpful in predicting vod. the kinetics of the investigated parameters underline the significance of pc and pai-1 as described by others and our group earlier, whereas ps does not seem to play a role in the pathogenesis of vod. the budd-chiari syndrome (bcs) is characterized by hepatic venous outflow obstruction that may be caused by the precipitation of a thrombus. it frequently coseggregates with other major diseases like myoloproliferative diseases or defects in the haemostatic system (antiprotein c and protein s deficiencies e.g.). only recently, the factor v leiden mutation (fvlm) has also been associated with bcs. we hypothesized that defects in the thrombo-modelling associated anticoagulant pathways (tmaap) are a major risk factor for the precipitation of bcs. we screened our cohort of 27 patients (pts) with bcs for the presence of defects in the tmaap and identified 3 pts with protein s deficiency (psd). these pts were screened for the three point mutations in exon 1 (codon-25; ins t), exon 15 (codon 636; a-->t) and in intron 10 (g-->a + 5) of the ps alpha-gene that have been demonstrated by bertina et al to coseggregate with psd. restriction enzyme analysis and confirmation-sensitive gel electrophoresis for the detection of single-base differences in doublestranded pcr-products were employed. all living family members of the indicator pts were also screened for heterogeneties in the three point mutation as described. no single abnormality in these genes despite presence of pbd in those family members was found. in addition, pts and family members were also screened for fvlm. one pt and two of his family members, in addition to psd, were subject to fvlm. the other two lots and their family members were not subject to fvlm. in contrast to the first family, despite psd, those two pts suffered from morbus crohn and acute myeloid leukaemia as risk factors for bcs. we conclude: psd is one major risk factor for the precipitation of bcs. to precipitate this disease, one additional risk factor is required. psd may be caused by genomic defects in the protein s gene other than those described by bertina. only a few publications describe a thromboembolic disease due to dramatically reduced protein s levels being associated with viral or bacterial infections, autoimmune mechanisms are suspected but the aetiopathogenesis is still under discussion. we report on a 5 year old boy who developed purpura fulminans of the left leg during varicella infection. on the fourth day of infection the disease started with pain and haemorrhagic efflorescence localized at the left taft. on admission the boy suffered from a purpura fulminans with central necrosis measuring 15x8 era. suspecting a hereditary thrombophilic disease we started therapy with protein c concentrate and recombinant tissue type plasminogen activator. the fellowing coagulation investigation showed a severe deficiency of protein s (total protein s-antigen < 5 u/ml, free antigen not measurable) in combination with factor v leiden mutation. other thrombophilie and coagulation parameters did not show deviation from normal range. after 4 weeks we saw a slight improvement of the total protein s antigen up to 50 u/ml. the free protein s antigen was still undetectable. during the following weeks the patient recovered slowly and the protein s activity and antigen normalized. because of skin necrosis thromboembolie prophylaxis was initiated with low molecular weight heparin (fragmin®, 100 ie/kgbw/die) and continued for 6 months. under this therapy there were no further thromboembolic events. these results suggested an autoimmune protein s deficiency in a patient suffering from chickenpo×. an analyses of autoantibodies at the time of diagnosis showed a slight increase of the antieardiolipin antibodies (igg 16,1 iu/ml, igm 15,1 iu/ml) which normalized during hospitalisation. we suspect an antibody to protein s probably caused by similar presented viral antigens. we suppose that autoimmune mechanism during different infections in combination with a heterzygous apc-resistance may be a potential risk factor for developing thrombotic disease. in the central nervous system mrna encoding for prothrombin and thrombin receptor is present and astroglial cells in culture process and secrete thrombin. moreover, effects of thrombin on brain cells including change of neudte outgrowth and astrocyte shape are described, but the molecular mechanisms are unclear. we investigated the effects of human <z-thrombin and a six amino acid thrombin receptor activating peptide (trap-6, sfllrn) on [ca2+]i, phosphoinositide hydrolysis and protein kinase c activation in the rat glioma c 6 cell line which is a widely used in vitro model for studying glial functions. stimulation of c 6 cells with both c{-thrombin and trap-6 resulted in transient [ca2+]i mobilization, [3h]inositol phosphate response and activation of the pkc isoenzymes c{,i],7,5, and s. these results suggest that ~-thrombin and trap-6 activate at least partially the same intraceltular signaling pathways in rat glioma c 6 cells which is an evidence for involvement of "tethered ligand" receptor in thrombin induced signaling in glioma c 6 cells. further investigations in this field are in progress including both receptor binding studies and more detailed analysis of thrombin receptor mediated signaling network and the cellular response of thrombin induced transmembranal signal transduction pathways in c 6 glioma. thrombin is a potent mitogen in vascular smooth muscle cells (smc). the mitogenic effect of bovine thrombin and human thrombin receptor-activating peptide (hutrap-14) was investigated in bovine coronary artery smooth muscle cells. confluent cells (passage 4-8) were incubated for24 h in serum-free medium with thrombin (10 nm) or trap (10-100 pm). the mitogenic response was assessed using [sh]thymidine incorporation. results demonstrated that thrombin induced a three-to fourfold increase in [3h]thymidine incorporation compared to the control, thus being as active as the potent mitogen pdgf-bb (20 ng/ml). the mitogenic effect of thrombin was dependent on its proteolytic activity. when the active site of thrombin was irreversibly inhibited by d-phe-pro-arg-chloromethylketone (ppack) no increase in [°h]thymidine incorporation was observed even at the high concentration of 250 nm ppackthrombin. thrombin-induced [3h]thymidine incorporation was significantly reduced by the reversible direct thrombin inhibitor nc£-(2naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide (napap, lpm) . in contrast to thrombin trap-14 at a concentration up to 100 pm did not increase [3h]thymidine incorporation in the absence or presence of the aminopeptidase inhibitor amastatin (10 ijm). in order to demonstrate whether hutrap caused a stimulation of the receptor and signal transduction in bovine smc the activation of protein kinase c (pkc) was measured as translocation of the cytosolic to the membrane bound fraction. it was found that both thrombin (10 nm) and trap (10 pm) induced a translocation of pkc after 20 rain. in conclusion, thrembin and trap activated pkc in coronary smooth muscle cells but only thrombin acted as a mitogen. endothelial cells once stimulated with thrombin are resistant to subsequent stimulation directly after the first. but after a recovery period of about 90 min the cells are sensitised again for activation by thrombin. in our experiments this resensitisation couldn't be inhibited by actinomycin d or cycloheximide nor by the phosphatase inhibitor ocadaic acid. therefore an intracellularly pool of thrombin receptors has been proposed possibly co-localised with golgi vesicles. brefeldin a (bfa) has been used extensively to investigate the intracellular sorting of proteins because of its dramatic alteration of the structural and functional organisation of the golgi apparatus. because of this we examined the effects of bfa on the regeneration of the thrombin receptor response in human umbilical vein and artery cells. the vwf release from weibei-patade vesicles was used as physiological parameter of activation of thrombin receptors by thrombin. the addition of bfa (20 pg/ml -20 ng/ml) to endothelial cells blocked the receptor response regeneration, whereas the first stimulation was not influenced by bfa at the concentrations used. these results give further evidence that the proposed storage pool of thrombin receptors is iocalised in vesicles of the golgi apparatus. randomized comparison of double bolus reteplase (r-pa) and front-loaded alteplase (rt-pa) in patients with acute myocardial infarction (rapid ii) c. bode, rw. smalling, j. kalbfleisch, g. berg, dj. odenheimer, e. bshm, wd weaver and the rapid ii investigators. med. klinik iii, university of heidelberg, germany the study was undertaken to assess the efficacy of a double bolus regimen of 10 + 10 megaunits of reteplase, a recently developed deletion mutant of tissue-type plasminogen activator, relative to front-loaded (gusto-regimen) alteplase. there was no age limit and patients were recruited up to 12 hrs after onset of symptoms. the primary endpoint "patency at 90 minutes" was centrally assessed in a blinded fashion. infarct related coronary artery patency (timi grade 2 or 3) and complete patency (timi grade 3) at 90 min for reteplase vs. alteplase were 83.4% vs. 73.3% (p=0.03) and 59.9% vs. 45.2% (p=0.01), respectively. at 60 minutes, the incidence of both, patency and complete patency was also significantly higher in retep ase vs. aiteplase treated patients: 81.8% vs. 66.1% (p=0.03). and 51.2% vs. 37.4% (p=0.01). reteplase treated patients required fewer additional coronary interventions within the first 6 hrs of treatment (13.3% vs. 26.5%, p=0.01). there were no significant differences between reteplase vs. alteplase regimens in 35 day mortality (4.1% vs. 8.4%), severe bleedings (12.4% vs 9.7%) • or hemorrhagic stroke (1.2% vs. 1.8%). reteplase, when given as a double bolus of 10+10 mu to patients with acute myocardial infarction, achieves significantly higher rates of early patency and requires fewer acute coronary interventions than front-loaded alteplase without an apparent increased risk of complications. lysis therapy with uhsk of deep vein thrombosis (dvt) of the lower extremity commoniy results in a relatively high patency rate. in our patients, patency was achieved in approx. 70-80%. these results were supported by several studies. in dvt of the pelvic veins, however, this kind of aggressive therapy is excluded due to the risk of iatrogenically induced pulmonary embolism. on the other hand, the need for an effective therapy is evident because of the usually occtnring complete occlusion of the pelvic veins and the following severe clinical symptoms 0phlegnmsia cerulca dolens etc.). until september "95, 44 consecutive patients (16 males, mean age 41 ranging from 18 to 63 years) with dvt of pelvic veins were included in this study. after clinical and phiebographic/computer tomographic (ct) diagnosis, a temporary filter system was placed intravenously either by a wambmchial (34 pat.) access route, lransfemorally (6 pat_) or wansjngularly (4 pat.) under mdiological control. the open filter was positioned above the thrembus in the lower caval vein: 26 times an antbeor system, 11 times an anglocor and 10 times a cook filter were used. in the following three days up to 3 cycles of lysis therapy with 9 mli u of streptokinase were administered, each cycle lasted six hours. heparin was given simultanously via the filter system in therapeutic dosages in order to prolong alrit values 1.5 to 2 times the individual base line level. effective lysis was controlled by a second phlebography/ct, and the filter system removed through the introducer sheath valve. after lysis therapy all patients were put on coumarin with overlapping heparinization. successful lysis (complete recaanlisation without remaining thrombi and lysis of more than 50 per cent of the original thrombus) could be achieved in 62% (29 patients). in two cases a fatal pulmonary embolism occurred. 5 times great parts of a thrombus were captured by the filter and could be removed with the system. due to an insufficient boparin therapy we detected a newly developed thrombosis of the lower cavul vein twice after lysis. this complication could be resolved by additional administration of streptokinase. in nearly all cases we found extensive hematomas on the catheter insertion sites. 4 patients with transfemoral applications had to be transfitsed with red packed cells due to retroperitoneal hematoma. one filter system was broken after use. lysis of dvt of the pelvic veins with uhsk and with the protection of a removable temporary filter system situated in the lower caval vein enables us to carry out an effective therapy. the filter systems, however, have to be ameliorated in order to better prevent fatal pulmonary embolism. heparin should be administered at least in the remainder of the day of lysis. the insertion site should be the enbital vein because compression for bleeding complication is easily feasible at this location. we report about seven of our patients (pts.) with bcs treated by fibrinolytic therapy (5 female, 2 male, age 27, 6 years + 8). only one case was of unknown origin while in four pts. myeloproliferative syndromes, in one protein c deficiency and in another one morbus behcet was detected in five pts. chronical and in two acute bcs was diagnosed. as complications were found: in three pts• thrombosis of vena iv.) cava, in one ofv. porta and v. lienalis, in one more of v• porta and v• cava. we treated just one patient with urokinase (3 x 800.000 iu/d for 18 days). four pts. were treated with rt-pa in concentrations of 25 to 50 mg/d (for a period of up to 14 days) and continuous heparin infusion. high doses of rt-pa (80 to 120 mg/d for one or four days) were given in one case of acute and one of chronic bcs. partial (after high-dose rt-pa nearly complete) recanalisation of thrombotic hepaticel veins was only achieved in the two acute cases of bcs. no significant recanalisation was proven in hepatical veins of chronic bcs. three times complete fibrinolysis of v. cava thrombosis (two after highdose therapy) and one partial lysis could be attained. in one patient partial lysis of v. porta and in another one of v. lienalis was found. furthermore complete recanatisation of v. porta followed local rt-pa lysis during surgical intervention. in our two pts. with acute bcs significant improvement of hepatical venous flow could be measured after fibrinolytic therapy• in chronic bcs hepatical venous flow did not improve while the complication of v. cava thrombosis could eventually be treated successfully. probably due to fibrinolytic therapy one case of esophageal varix bleeding occurred. the benefit of flbrinolytic therapy of bcs seems to be influenced by the dur~ion of the disease• according to our results fibrinolytic therapy should be considered in cases of acute bcs as well as in the above mentioned thrombotic complications• recent studies suggest that transport of thrombolytic agents through the clot can play a major role in determining the reperfusion time needed for thrombolysis. as the mechanisms of this phenomena are rather unclear we studied fibrin (0.4-1.5 ~tm) clot lysis by plasmin (30 nm) presented outside or inside the clot in purified system (tris-hc1 buffer, 37°c). the clots was formed by addition of cac12 (30 mm), thromboplastin (1 u/ml) and human thrombin (6 nih/ml)t the lysis degree was determined either by counting of radiolabelled fibrin degradation products or by time of total lysis. obtained kinetic parameters of this reaction: kest=99.4 rain -1, km=i.9 ~tm (plasmin inside clot); kcat=l.36 rain -1, km=i.3 lzm (plasmin outside clot). this data indicate that eatalytie efficiency of plasmin from inside the clot is 50-fold higher than from outside as measured from kcat/km. furthermore we studied plasma clot (0.25 ml) lysis in original plasma (3 ml) containing sk (5-100 nm) and seu-pa (5-100 rim) in the final concentrations from inside or outside the clot. the results are presented in the the similar results have been got when the fibrin clots were formed with batroxobin instead of thrombin. it may be concluded that fibfinolysis is a surface-dependent process. it's likely that the higher level of plasminogen activators in blood would prevent thrombus formation, while physiological clots would stay resistant. for the improvement of the thrombolytic therapy in children more clinical data are needed. therefore we report on our experiences with rt-pa presenting the clinical course in 11 patients (age range: 7 days to 16 years) with venous thrombosis (n=9) and purpura fulminans by meningococcosis (n=2). the patients were treated with rt-pa (actilyse r) for 10,5 hours to 13 days. the venous thromboses were localized in lilac/femoral veins (n=4), brachiocephalic/jugular/subclavian veins (n=3) and the cava superior vein (n=l). central venous catheters were causative in 2 cases. the dosage range was between 0.2 to 0.5 mg/kg for the initial dose and 0.8 to 2.0 mg/kg/d for the subsequent continous infusion. ten patients received simultaneously heparin. complete clot dissolution occured in 6 cases, partial clot dissolution in 1 patient. in 4 patients with a history of the first clinical signs of thrombosis of 3 weeks or more the lysis was not successful. concerning the side effects only small bleeding episodes on former venipuncture sites were observed in 5 patients. systemic effects such as the decrease of the plasma flbrinogen concentration were rare too. in conclusion, the thrombelysis with rt-pa presents an effective and safe therapy in children at: the rt-pa dosage used. in case of a history of the thrombotic event of more than 3 weeks a thrombotytic therapy should not be carried out. d.dimer is widely used in clinical situations associated with thrombotic diseases. the most popular application is the exclusion diagnosis of deep-veinthrombosis and pulmonary-embolism and the assay is performed in emergency. d.dimer is also frequently measured for evaluating the thrombotic risk in the post-operative period. quantitative, flexible and sensitive assays are required. we have developed a new automatic method which works on the coagulation walk-away instrument, sta. this assay was developed with two monocloual antibodies specific for d.dimer coated on microlatex particles and it uses an immuno-turbidimetric technology. absorbance is measured at 540 nm and is a direct relationship of d.dimer concentrations. the assay works with 50 ~tl undiluted plasma and is performed in less than 10 minutes. the dynamic range is from 0.2 to 16 [~g/ml and no prozone effect is observed up to concentrations higher than 100 ~tg/ml. the detection threshold is of 0.2 [lg/ml. the intra-assay reproducibility is below 5% and the inter-assay reproducibility is below 6%. recovery studies of purified d.dimer added to normal plasma were between 94 and 105%. no interference of heparin, bflirubine, lipids or of rheumatoid factor up to concentrations of 100 u/ml is observed. there is no effect of high fibrinogen concentrations (> 10 g/l). when compared with conventional elisa techniques (asserachrom ddi), the assay demonstrated a correlation coefficient of 0.97 on 131 samples from normal individuals and hospitalised patients with elevated d.dimer concentrations. slope was of 0.97 and intercept was of -0.07. this new assay offers a full flexibility for individual testing as the calibration curve is stable for at least one week on the instrument. it is then well adapted for all the applications of d.dimer measurements in coagulation laboratories. 16 children between an age of 3 days and 11 months ( median 6 weeks ) with thrombotic or embolic occlusion of major vessels were treated with rt-pa for thrombolysis. the affected vessels were both sided renal veins or one sided renal vein and v. cava inf. in 8 cases, the v. cava superior in 3, the v. cava inf. plus renal veins plus aorta in 1, the left ventdcle in 1, the aorta in 1, the a. femoralis in 1 and the v. portae in 1 case. 10 out of 16 occlusions were associated with an indwelling catheter. underlying dieseases were sepsis (4), prematudty (3), vitiurn (2), asphyxia (1), short bowel syndrome (1), hus (1), diabetes (1), cmv (1), exsiccosis (1) and m. hirschsprung (1). thrombolysis was performed with an bolus of rt-pa (0.1-0.2 mg/kg) followed by continuous infusion (0.8-2.4 (-9) mg/kg/24h, median 1.8 mg/kg/24h). low dose hepadn (100 ie/kg/24h) was given dudng full dose hepadn (aptt 1,5-2 times normal) after the thrombolysis. in 5 pts. rt-pa was administered locally through the catheter and in 11 cases systemically. in 13 patients the vessels could be recanalised completely, in 2 partially, in 1 patient the therapy had to be discontinued. in 2 vessels a reocclusion occurred. bleedings were noted in three patients, all from recent venous puncture sites. the results encouraged us to start a multi-canter trial which has been approved by the ethical committee and is open for recrural. the aim is to compare efficacy and safety of rt-pa with urokinase, the only recommended standard in the management of critical major vessel obstruction in newborns and infants. the design is a randomised, notblinded trial with a cross-over option after three days in cases without success. study end points are recanalisations, major bleedings and number of cross-overs. inclusion criteria are age under 1 year, lifethreatening vessel obstruction, age of thrombus up to 10 days, no precaeding fibdnolytic therapy. exclusion cdteda are cerebral hemorrage, pedventricular leukomalacia, surgery dudng the last 7 days and cns injuries during the last 2 months. although our knowledge on inherited thrombotic coagulation disorders has greatly expanded within the last years, there are still man}, patients with recurrent venous thrombosis in whom no obvious predasposition can be identified.thus we decided to include also so-called rare defects associated with thrombosis in our routine thrombophilia screening programme, such as fxii deficiency. fxii is an important element m the intrinsic pathway of fibrinolysis and there is evidence for an insufficient fibrinolytic activity in fxii deficient pts..up to date only few and controversial data exist about the frequency of fxii deficiency in pts. with thrombophilia. cons~uently the aim of our study was to evaluate the association between fxii deficiency and juvenile venous thrombosis in a great population. patients and methods: 1554 pts. (851 female, 703 male, aged i to 61 ys, median age 38.2 ys) with venous thromboembolism before the age of 45 ys were studied. one-stage clotting activity assay of fxii (fxii:c) was performed on acl using fxii deficient plasma from instrumentation laborato~. fxii antigen concentration (fxii:ag) was measured by electroimmundiffusion using reagents from behfingwerke, enzym research respectively. the normal ranges are tl~. routine reference values obtained m our labratory from 80 healthy subjects (40 males, 40 females, median age 26.2 ys); 95% range: fxii:c 53-135%, fxii:ag 57-132%). results: 122/1554 pts.were classified as fxi1 deficient (f 60, m 62), giving a prevalence of 7.8%. severe fxii deficiencies with fxii:c below 1% were observed in 7 pts..ll5 pts= proved to have moderate fxii deficiency with fxihc ranging lrom 2 to 51% and fxii:ag ranging from 1 to 53%. in none of them inherited deficiencies of other well established thrombophila risk factors could be detected. none of the fxii deficient pts. had positive lupus anticoagulant tests. familial fxii deficiency was found m 9 cases. discussion and conclusion: the precedences of fxii deficiency amongpts, with venous thromboembolism was previously described to be 7.5-10%. supporting these data, we have shown a praevalence of fxii deficiency of 7.8 %. in comparison to the frequency of other well established thrombophila risk factors we consequently have observed a relatively high prevalence of fxii deficiency m our study group.these data, from the largest such study reported, strongly indicate that fxii deficiency may not be a rare deficiency and may be more frequently associated with thrombosis than currently suspected. we describe a family with an exceptionally rare, i.e. plasminogen, deficiency, combined with subnormal activities of coagulation factor xii (hageman factor). the first thromboembolic event, pulmonary embolism in the proposita was diagnosed at age 35. since that time, 'spontaneous' venous thromboembolic events verified by phlebography and perfusion/ventilation lung scan recurred once every year despite oral coumarin therapy, whose intensity varied over an exceptionally wide range despite tight control the patient was repeatedly given succesful thrombolytic therapy with streptokinase or recombinant tissue plasminogen activator. her plasma plasminogen chromogenic activity was 51-59 % compared to a normal plasma pool (reference range 70-130 %), plasminogen antigen was diminished to the same extent. the patient's factor xii exhibited only 28-55 % activity in a factor-deficient plasma assay as compared to a normal plasma pool. other known risk factors for recurrent venous thromboembolism were not present : no evidence of malignancy, no obvious precipitating events, normal values of antithrombin iii, protein c, protein s, fihrinogen, thrombin time, platelets, lupus-like anticoagulant, aptt prolongation after addition of activated protein c. the proposita's mother had died at age 60 from pulmonary embolisnt no coagulation studies are available. the proposita's sister was first diagnosed deep leg vein thrombosis at age 17, since that time recurrent episodes of venous thromboembolism have been diagnosed also in an other hospital. this sister's plasminogen activity was 120%, but factor xii activity was reduced to 55 %. three brothers of the proposita were examined, too, all in their 3rd decade of life. none of them recalled symptoms of or treatment for thromboembolic disease. in one brother, factor xii activity was normal (100-105 %), but plasminogen only about 50 %. in the 2nd brother, factor xii was very variable (64, 42 and 94 %), plasminogen was in the lower normal range, in the 3rd brother, factor xii was about 50 % (repeatedly), plasminogen was normal. current knowledge about the risk of thromboembolism with both enzymes is limited, the optimal management remains controversial. msrgit serbsn,maria cucuruz,dan madras,carmen petrescu, natalie rosiu,rodica costa iii rd psediatric clintc,universtt v of medicine, the unsatisfactory efficiency of entihepetitis b vaccination in our haemsphiliscs suggested the control of the immune status in 52 hiv negative patients,by establishing through flowcitomstrie with monoclonsl antibodies the lymphocyte subsets (cd3,cd4,cds,cd&/cd8 ratio and cd19) and by seric tmmunoglobulins levels; the immunological parameters have been correlated with the serological markers of hepatitis infections (hay, hbv,hcv ebd hdv) as well as on dependence with the treatment (blood,plasma,crysprecipitate,fector viii/ix concentrate) and the quantity of their consumption (ui/k9 weight/yesr).the interpretation of the results pointed out • significant lower level of cd3,cd& (p<o,o01) st the group of hsemophiliscs treated with blood,plasma and cryopraclpitate; sdditionsly,in this group of patients it was found s significant drop of the cd19 counts (p< 0,001) without an immunoglobulin-deficiency. the same behaviour was found at the group of haemophiliacs carriers of hb~ and hcv markers.the superpositisn of hepatitis infections on the treatment with blood and native blood products in most of the cases has let unanswered the question related to the mechanism of the immunodeficiency: the hepatitis b or c infections or the negative impact of the itarative important alloantigenlc load ? illumination with l~tm methylenblue (mb) is used for inactivation of enveloped viruses in human plasma. via a photooxidative mechanism viral structures as well as plasmaproteins especially fibrinogen can be damaged. fibrinogen is an important mediator in the cell-cell interaction of platelets. the four rgd sequences (arg-gly-asp) of fibrinogen can be recognized by the activated fibrinogenreceptor gphb/iiia . after binding to gpiib/iiia one molecule fibdnogen can link two platelets. the aim of this study was to investigate the influence of mb treatment on the fibrinogen activity in human plasma. furthermore we were interested to demonstrate if altered fibrinogen inhibit platelet aggregation by blocking the gpiib/]iia receptor. human plasma was treated with 1-50~m mb. after illumination fibdnogen was isolated by fl-alanin precipitation. purified fibdnogen was analysed by one-and twodimensional gelelectrophoresis. influence of modified fibrinogen on platetet aggregation was measured with gelfiltrated platelets. possible receptorblockade were examined in an 125j-fibrinogen competition assay. densitometric scanning of reduced sds-pages showed a decrease of the a-subunit as well as an increase of high molecular aggregates with increased mb concentrations. this effect was not detectable for fibrinogen isolated from plasma with 1bm mb. this results were confirmed by two-dimensional gelelectrophoresis. ibm mb treatment showed no receptor blockade in aggrement with normal platelet aggregation after stimulation with adp and fibrinogen. proposed validation of a quantitive quality-assured pcr method f. dorner, j. eibl, g. zerlauth immuno ag we have developed a highly sensitive and reliable quantitative method, based on the polymerase chain reaction (pcr) and on the reverse transcriptase polymerase chain reaction (rt-pcr), to screen for the nucleic acids of human immunodeficiency virus type 1 (hiv-i), hepatitis b virus (hbv) and hepatitis c virus (hcv) in plasma and plasma products. the detection of pcr products is carried out by laser induced fluorescence, the procedure is therefore termed "laser induced fluorescence pcr" (lif-pcr). the procedure allows the precise quantitation of genome equivalents due to the use of two calibrators that are co-amplified by the same set of primers as the target template in one and the same test tube. a high degree of reliability is achieved by co-precipitation of the extract, co-amplification and co-determination of the calibrators together with the genome equivalents to be determined. in this report we present validation data for the lif-pcr and discuss them in the light of the recently published ich-guidelines on the validation of analytical procedures. taking into account the pecularitles of the replacement therapy of haemcphiliacs in romania the study aimed at evaluating the dimension and profile of blood-born infections in these patients; 142 hsemophlllacs,elasslfled in 3 groups: a-ulth no exposure to blood or bloodproducts, b-treated ~ith factor-concentrates and c-ulth plasma,cryoprecipitate + factor-concentrates in their therapy have been investigated (elisa method) for serological markers of hepatitis a,b,c end d, cvtomegalovirus (cmv) and hiv i and hiu 9 infection. the infections profile of our haemophiliacs~hes some peculsrities. the hepatitis infection is the most extended,lnvolving more than 75% of the patients. the frequency is for hepatitis -bz,z~ , u -"/~,~ , g -bb,7% end o -24% : the multiple infection was diagnosed in more than 50% of cases. the prevalence uas significantly hlgher in the group c. we studied blood count and relevant coagulation system parameters of autologous blood donors. indications for autologous blood donation were divided in 4 categories: a group of diverse (a, n=13), reduction mammoplasty (b, n:5), prostatectomy (c, n=io) and total hip replacement (d, n=74). we performed bleod count (bc), thromboplastin time (tt), partial thromboplastin time (aptt), fibrinogen (fib), f vhi act, vwf rco, vwf ag, f xiii, at iii, d-dimer-fragments (dd) and fibrinmonomers (fm). all parameters were analyzed as initial value, bc, tt, aptt, fib, f xiii and at iii were measured additionally at each donation. the percentage of initial pathologic data was (1) were included in the study. the inclusion criterittm was: an endegen(y.~ red cell reserve (ercr) of less than 400 ml in men and less than 540 ml in women. ercr is the volume of blood that can be withdrawn up to tim hematocrit of 0.34. the selected dosages of rhepo (200, 400 and 800 iu/kg body weight biweekly over 4 weeks) were based on the calculated blood volume and the ercr. a unit of whole blood, separated into an autologens red cell concentrate and one unit of fresh frozen plasma, was collected when ]mnoglobin concentration was at least 11.5g/100 ml (or hematocrit was at least 0.34) up to an autologons predeposit of 800 ml red cell volume (rcv). additionaly, platelet concentrations and serum ferritin levels were measured~ results: at the initial clinical examination the ercr was 333 ml in women and 307 in men. this resulted in a weekly dosage of 2x32.000 iu rlaepo in women and 2x25.000 iu rhepo in men. 7 patients donated 5 red cell each (858 ml rcv on an average) within 19.1 days. the targeted rcv could not be reached in 3 patients because the operation was reschednled. the collection from one patient had to be aborted because of angina pcetoris complains. tic following side-cffczts occurred: episodic hypcrtejlsion (2 tim~), chest pain (5), fafigne (8), inflnenza-lihe syndrome (3). platelet count increased from initially 203 4-68 x 10'//d (i00 %) during therapy to a maximum level of 260 + 93 10v~ (128 %) without any clinical signs of thrombeembolism. in the control group (autologous blood donation weekly 3 or 4 times without erythrupoiedn stimulation, n=12) platelets increased from initially 230 __+ 73 10'/fi (100%) to 255 + 71 10v#i (110%) i1]aximlzm colldttsions: the adlninist/'ation of lhepo in the above described manner to autologous donors undergoing heart surgery is well tolerated. it allows to collect a sufficient number of autologous blood units and the preparation of a predeposit at short notice. essential thrombocythemia is a chronic myeloproliferative disease with a benign clinical course that is mainly complicated by microcirculatory or thrombotic vascular disorders. it is at present uncertain which patients need (lifelong) therapy and whether cytoreduction is necessary. we retrospectively evaluated vascular complications in 128 patients (33 men, 95 women) with a median age of 61 years in relation to therapy. three major treatment groups were analysed: 17 patients treated with acetylsalicylic acid (ass) alone, 29 patients given alkylating agents, and 62 patients treated with interferon alpha (ifn). there was no significant number of patients treated with hydroxyurea. ifn-treated patients were younger (median age 54 years) than patients in the other groups, but platelet counts before therapy were similar (median 0.84-1.13 x 1012/i). before therapy, 47% of patients treated with ass, 45% treated with alkylating drugs, and 32% treated with ifn had bleeding events, microcirculatory or thrombotic complications. dudng therapy, median platelet count was 0.9 x 1012/i in patients on ass, 0.68 x 1012/i in patients treated with alkylants, and 0.48 x 1012/i in ifn-teated patients. the rate of patients with complications during therapy was 18% (9.5%/patient year) for ass, 14% (6.3%/patient year) for patients on alkylating drugs, and 15% (6.2°/dpatient year) for ifn-treated patients. we conclude that all three treatment regimes seemed to have a beneficial effect on the incidence of vascular complications in essential thrombocythemia. the results give a rough estimate of complication rates to be expected and may form the basis for randomized clinical trials. adhesion of activated platelets to leukocyte* has been shown to be mediated by at least two molecules on the platelet surface: p-selectin (cd62) and fibrinogen (fg) bound to the gp iib/iiia complex. interaction of platelets with teukocytes via cd62 is believed to stimulate the production of reactive oxygen species (ros) in leukocyte*. the role of platetet-bound fg as a signalling molecule in platelet-leukocyte interaction is controversially discussed: stimulatory as well as inhibitory effects on leukocyte ros production has been described. in the present study we measured ros production in undiluted samples of citrated whole blood (wb) as well as in suspensions of erythrocytes and leukocytes in platelet-rich plasma (platelet-rich wb) or platelet-poor plasma (ptatelet-poor wb). ros production was determined by measuring luminol-enhanced chemiluminescence (cl). stirring wb samples at 1,000 rpm for 10 min resulted in an activation of leukocyte* (increase of cl, upregulation of cd1 lb) and platelets (platelet aggregation, upregulation of cd62). stirring-induced cl was significantly higher in platelet-poor wb when compared to platelet-rich wb. higher cl in plateletpoor wb than in platelet-rich wb was also found when adp (moderate stimulation of platelets and leukocyte*) or fmlp (strong stimulation of leukocyte*) but not when pma (strong platelet and leukocyte stimulation) was added. dextran sulphate that is known to block cd62-mediated interaction ofplatelets with leukocyte* caused a dose-dependent inhibition of stirring-induced cl in wb samples. in contrast, when binding of fg to the platelet surface was blocked by rgds or the fg receptor antagonist gr144053, stirring-induced cl, but not cl in unstirred samples, was significantly enhanced. gr144053 also reduced adhesion of platelets to neutrophils, but did not affect cd62 exposure. the results indicate that interaction of platelets with leukocyte* in whole blood via cd62 and fg may have opposite effects on leukocyte ros production: stimulation by cd62 and inhibition by platelet-bound fibrinogen. late reocclusive processes represent one of the most commonly seen serious adverse events after coronary angioplasty. the pathophysiology of both of these processes is multifunctional and represens complex time dependent pathophysiologic events involving both the humeral and cellular processes. to test the effects of low molecular weight heparin (certoparin, sandoz ag, numberg germany) against late occlusive processes, four groups (groups a-d) of rats (7-10) were anesthetized and the external common carotid arteries were exposed. all groups received a 200 u/kg of unfraetionated heparin through the femoral vein. a 2f balloon emboleetomy catheter was inserted into the left carotid artery and passed throug the left common carotid artery leading to the aortic arck vascdar injury was induced by repeatedly (x3) inflating the catheter balloon and withdrawing it to the bifurcation. following this procedure, two hours after, equlgravimetric amounts of various agents were administered via sc route to each of the individual groups. group a was administered with certoparin od for 21 days. group b received a lower low molecular weight heprin (mr 3.4 kda). group c received unfraetionated heparin and group d served as a control. on day 21, perfusion fixation was performed on all animals. the injured and contralateral carotid arteries were excised for histelogic examinatior~ in comparison to the saline control, varying degrees of the inhibition of myointimal proliferation following arterial injury was noted in all groups. however, the degree of inhibition followed the order: heparin<certopann<llmwh_ in contrast to heparin treated group (c), the other groups a, b, and d did not exhibit any hemorrhagic lesions. however, degree of perivascular fibrosis, hemorrhage and inflammation were observed at the infusion site. in comparison to heparin, certoparin and its lower low molecular weight derivatives exhibit better efficacy in reducing balloon angioplasty induced restenosis. the observed efficacy of these agents may be due to better bioavailability and sustained duration of action. in the present paper the binding of heparin to granulocytes of human and animal species was analyzed and compared with the binding to monocytes and lymphocytes. a low-molecularmass heparin (lmmh) was covalently bound to tyramine (tyr) and subsequently tagged with fluorescein-5-isothiocyanate (fitc) by endpoint attachmenc. binding to leukocytes was quantified using flow cytometry analysis. the leukocyte populations were identified by their light scatter properties in man by cd4, cd13, and cd14 antibodies, in mice by cd 4 and gr 1 antibodies, and in rat by cd 45, ed 9, and rp 3 antibodies. granulocytes, monocytes and lymphocytes of all species bound dose-dependently lmm-heparin. the binding of lmmh-tyr-fitc ~to granulocytes was higher in human, rat, rabbit, dog, and pig as compared to monocytes and lymphocytes. mouse and sheep granulocytes did not bind more heparin than monocytes or lymphocytes. binding of lmmh-tyr-fitc was reversible in the presence of unlabeled heparin or lmmh. more than 99% of human, rat, rabbit, dog and sheep granulocyte populations could be'distinguished from monocytes and lymphocytes by their fluorescence intensity after incubation with lmmh-tyr-fitc. this separation was not obtained for mouse and pig granulocytes. the data present evidence of a specific heparin binding to granulocytes of many species and indicate the significance of fluorescent labeled lmm-heparin for biological investigations. background and purpose: ptca is an effective treatment for restoring coronary artery pat~cy; however, acute thrombotic rencclusion is a drawback of the procedure. currem prevention of rencclusion relies on antiplatelet agmts (aspirin) and antithrombin agents 0aeparin). in the present study we examined the time course of changes in blood coagulation and fibrinolytic function following ptca in patients with ischaemic heart disease. 20 patients were randomly assigned to receive either standard therapy (250 nag aspirin p.o.; 5.000 iu hepafin i.e. bolus, and 5-10.000 iu heparin intra-proeadurally)(n=10), or adjunctive therapy (aspirin, hepann; 100 mg sodium pmtosan polysulphate, spp intra-cortmarily and subcutaneously) (n=10). the sensitive markers of thrombin activation (thrombin-amithrombin iii complex ('rat), prothrombin fragmem f 1+2 (pl+2), d-dimer) and of fibrinolytic activation (plasmin-antiplasmin complex (pap), tissue type plasminogan activator (t-pa) and plasminogen activator inhibitor (pal)-i activities) were measured before, immediately after, and 6 hs after ptca. results: we found elevated mean levels of tat and fl+2 in both groups during the procedure, while plasma coneentrafiuns of d-dimer and pap remained in normal range. activity of pai-i was not elevated compared to normal comrol. the t-pa activity obtained immediately post-ptca showed significant increase in patients given spp. primary angiologic success was achieved in 80% of both groups. one patient in each group had recurrent ischaemia and emergency interventions (angioplasty, stent, bypass surgery) were performed. one patient receiving adjunctive therapy died in cardiogenic shock within 24hs arer ptca. major bleeding was observed in one patient given spp. conclusion: no significant intracoronary haemostatic activation occurred in the majority of patients during and up to 6hs after ptca. treatment with spp was associated with enhanced fibrinolytic activity. the benefits and risks of adding spp to heparm and aspirin should be evaluated by further studies, in july 19,¢1 the paul ehdich institute became responsible for quality and safety of bt~d and blood products. since ~his time, batch m~se of ppsb and factor ix prapamtions were carded out regularly, whereas ihe obligatop i testlng of all other products derived frem pooled plasma will be starling in janualy 1996. the european pharmacopoeia does .or contain directions for the quality control of ppsb preparations. therefore, ifua ~ coneenning factor ix clotting concentrates are applied, ircludmg the non activated ptt and the classical tkrombln test these m~ods are not su'rlable for reliable exclusion of ituombogenk~b/ ,of pp,sfl, because vlla actlvry is not d~. a hllhedo unsolved problem is the high sensitivity of file classical thrombin test often not correlating with ~ data of thrombogenlci~. in ordm td es~abl~ an effective quality conlrd protocol, different methods for the qual~ative detennlna~on of ila, vlla. ixa, and xa (chrornogenic, fluorege~c clolfing reactions) wera instaued. in agreement with the [deralure, considerable differences were found among the ppsb pr~ons registered in germany. cedain amounts of activated do~ng racers may be well tole~ted in redp/ents. ~no are in a steady state condition. however, in severely ill patienls, eg. post operatively, tissue factor may be expressed which pofenllatesthe dsk of ~aclor vlta. in the case of a certain ppsb preparation hlgh ¢onceatratio~ of acthrated cto~ng factors, especially ~vated factor vii, cotmlaled with fatal comlffcal~ns, evaluating ~ expe~ concenlralbn limits for adjvated prothrombin co~np~.x factors as prerequisite for ~e batch r~ease have to be applied. aprotinin is used as a hemostatic agent in cardiopulmonary bypass surgery and other indications where hemostatic compromise results in hemorrhagic manifestations. the mechanism by which this agent produces hemostatic effects is not known, however, inhibition of serine proteasas such as plasmin, kallikrein and dastase and modulataion of platelet receptors have been considered as possible target sites. to determine the effect of aprotinin on tissue factor mediated activation of human platelets, whole blood samples were drawn at baseline and 24 hours aider aspirin (650 mg po) ingestion (n=5). blood samples were supplemented with saline and aprotinin (40 ug/ml) and tissue factor activation was studied at 200 pg/ral. platelet activation was investigated using gtycoprotein ffla and gmp 140 specific antibodies on a flow cytometer (epic coulter). tissue factor was found to produce the activation of both the baseline and asp' "mnized platelets. aprotinirt augmented the tissue factor activation of platelets in both the control and aspirin supplemented groups. however, the relative increase in the aspifinized group was much monger (25% of the baseline). these results suggest that aprotinin augments the tissue factor mediated activation of platelets in both the normal and aspirinized individuals. while these effects may result in hemostatic restoration in aspirin compromised patients, in normal individuals the clinical implications of this effect remain to be clarified. , 10-20 (group 2) or >20 years (group 3) duration. anticoagulated whole blood was incubated with fluorescent antibodies to gpib and gmp-140 (two colour method) and analyzed with a flow cytometer. thrombomodulin, f1+2, protein s, 13-thromboglobulin were measured according to standard procedures. results: surface expression of gmp-140 was not different in groups 1 to 3, however, there was a tendency to higher acitvation in group 1 (<10 years iddm). results for thrombomodulin, f1+2, protein s, 13-thromboglobulin will also be presented. conclusion: though it did not reach statistical significance, platelet acitvation seems to be more important during early diabetes. this wilt be correlated with endothelial and plasmatic activation markers. in our clinic four patients with hiv-related thrombocytopenia were treated with a lot of gammagard (93f21abllf), which later turned out to be hcv contaminated. before infusion all patients were negative for hcv antibodies and hcv rna. 2 to 8 months after infusion 2/4 patients, who suffered from arc at the time of hcv infection with cd4 counts >100/pl, seroconverted, whereas in the two other patients, who suffered from aids with cd4 counts below 100/pl, there was no seroconversion. in all cases hcv rna was found. genotyping with inno-lipa (innogenetice) showed hcv genotype l(b) in all patients. liver enzymes and hcv rna copies were measured repeatedly over a period of one year after infection. the 2 patients with arc showed a strong increase of hcv rna titre during the first 3 to 4 months after infection, followed by a rapid decrease within the next months. in the patients with aids hcv rna copies increased moderately within the first 4 to 6 months, followed by a slow decrease. elevation of liver enzymes was mild in the aids patients and seems to be independent from the hcv rna titre. in the arc patients liver enzymes changed parallel to hcv rna titers with a delay of 2 to 3 months. the course of hiv infection was only slightly influenced by the acute hepatitis c as measured by cd4 counts, i%2microglobulin and hiv rna copies. introduction:mechanisms underlying ischemia/reperfusion injury have been thoumughly investigated in experimental models. leucocytes appear to play a main role through production of cytokines and overexpresssion of adhesion molecules. in experimental animals, administration of monocional antibodies (mab) recognizing cd18 can reduce organ injury following ischemia/repedusion. no data, however, have been reported concerning clinical ischemia situations. patients and methods:we investigated expression of cdt8, cd1 la, cdf l b and cd1 lc in granulocytes, monocytes and lymphocytes from peripheral blood of five patients undergoing elective hand surgery. the tourniquet was applied on the upper arm and heparinized samples from cubital veins were obtained before and at the end of ischemia. control samples were drawn from the nonischemic contralataral arm with the same timing, duration ot ischemia ranged between sixty and one hundred minutes (80~16). whole blood samples were incubated with specific, fluorochmme labelled antibodies and analyzed by fluorocytometry (facscan, becton dickinson, san jose, ca). mean fluorescence intensity (mfi), quantitatively reflecting surface expression of the indicated markers was evaluated for the individual cell populations. data were compared by the paired student's t-test, p<0,05 was evaluated as significant. results:mfi for all markers was comparable in all cell populations in samples obtained before ischemia from both arms. in contrast, expression of cd18 was significantly enhanced in granulocytes (321_+50 vs. 189_+38), monocytes (653-+54 vs. 426+122) and lymphocytes (299_+45 vs. 228-+36) from samples derived from the ischamic arm, as compared with the nonischemic arm, as measured at end of ischemia. at the same time, an increase of cdf lb on granulocytes (500~_342 vs. 213+150) and monocytes (533+359 vs.237-+206) but not on lymphocytes was found, no modifications of cdlta and cdttc expression could be observed. there was no correlation between duration of ischemia and quantitative expression of these markers, conclusions:our data indicate that relatively short ischemia periods induce an increased expression of ~2" integrins adhesion molecules on leucocytes. these results suggest, at close similarity with findings from expodmental models, that overexpression of adhesion molecules might play an important role in the induction of ischemia/reperfusion injury, in humans. in patients suffering from chronic inflammatory bowel diseases, such as morbus crohn and colitis ulcerosa, we observe massive, sometimes barely staunchable bleedings. hereby, the deficiency of coagulation factors, especially of factor xiii in plasma is established. ttowever the influence of factor xiii on the pathomechanism of the underlying disease is still under discussion. therefore we studied the f xiii content in the intestinal mucosa. an immunohistochemicat method was developed using commercially available antibodies against f xiii subunit-a, the detection of mucosal factor xiii depends on the amount of chromogen bound to the antibody-horseradish-peroxidase complex. with this method, it is possible to locate but not to quantify f xili in the intestinal tissue. therefore we developed an elisa-metbod in homogenized intestinal tissue, using commercially available antibodies. its precision was validated using a standard curve with commercially available factor xiii preparations (fibrogemmin®). the detection limit of this method is > 0.05 i.u. f xiii/ml of tissue solution. freezed dried intestinal tissue (lmg) was homogenized in 1 ml buffer using a potter. specimens of the large bowel revealed f xiii values of 0,21 + 0,0038 i.u. (x __+ sd), tissue solution. with this method it is possible to quantify tissue-bound faxtor xiii. studies are in progress to elucidate the content of f xiii in the intestine of patient's suffering from infammatory bowel diseases in order to contribute data to the pathomechanisms of f xiii deficiency. in a previous double-blind, controlled trial we were able to show that aprotinin administration has significantly contributed to reduce periand postoperative bleeding complications without increasing the risk of thromboembotic complications. the question arises whether this beneficial effect may be associated with its effects on intraoperative fibrinolysis. therefore, 20 patients were treated with or without aprotinin (2 million kiu loading dose over 15 minutes followed by 500,000 kiu per hour), and citrated blood samples were obtained at the following time points: before operation, after induction of the anesthesia, at the beginning of operation, intraoperatively when the femur shaft was implanted, and 24 hours postoperatively. the determinations of plasmin/antiplasmin-complexes, d-dimers, thrombin/antithrombin iii-complexes, and prothrombinfragments 1 +2 were performed by means of test kits from behring, germany (enzygnostrpap micro, enzygnost r d-dimer testkit, enzygnost r tat micro and enzygnost r f 1 +2 respectively). -all markers of activated fibrinolysis and blood coagulation were significantly increased in the groups with and without aprotinin treatment, the highest activities to be seen when the femur shaft was implanted. however, the values of pap and d-directs of the aprotinin group were below the values of the control group until the end of operation. the markers of activated coagulation showed the opposite effect, however the differences between the two groups were not significant. as expected, the aptt was significantly prolonged in the aprotiningroup. the aprotinin treatment was also associated with a significantly lower blood loss in these patients. -concluding it can be said it is not clear whether the blood saving effect of aprotinin may be exclusively attributed to its antiplasmin activity since the differences of the fibrinolysis parameters were not statistically significant. further blood samples should be analysed between the implantation of the femur shaft and the end of operation. in our laboratory large amounts of human prothrombin are required (30-50 mg/week). as we try to produce meizothrombin and meizothrombin-des-fragment-1 from human prothrombin and to apply it as an antidote for hirudin, the classical adsorption to barium sulphate or aluminum hydroxide from human plasma cannot be used. commercially available human prothrombin is expensive and of an unacceptable quality for our applications. in most of these batches we found small amounts of factor x and prothrombin activation products. we now developed a procedure to isolate prothrombin from "prothrombin complex concentrates" (ppsb-250-bulk, drk-blutspendedienst nds.). the concentrate also contains fac-tor vii, factor ix, and factor x. the prothrombin had to be separated from these factors. the concentrate we used contained amounts of other proteins and activation products of prothrombin (e.g. prethrombin-1) as well. for the preparation of prothrombin from ppsb we used anion exchangechromatography (resource-q ®) on an fplc ®. we applied dissolved ppsb directly or after buffer exchange on sephadex g-25 onto the column at room temperature. the prothrombin was eluted with an naci-gradient in trisodium citrate buffer, ph 7.0. the buffer conditions are similar to the conditions used in the preparation of ppsb. the quality of the prothrombin so obtained was sufficient for most of our experiments. a second purification step on ion-exchange resulted in a 99% pure product devoid of contaminating factor activities and activation intermediates as examined with coomassie and silver stained sds-page electrophoresis and assays for factor x. this prothrombin contained full enzymatic activity and its activation by specific snake venom prothrombin activators showed the known activation products. we are now able to isolate the amounts of pure prothrombin required for preclinical investigations. most of the commercially available lmwhs such as enoxaparin, fraxiparin, and fragrnin are prepared by chemical methods which can result in desulfation and other chemical modifications of the internal structure leading to differences in the pharmacologic effects. on the other hand, tiactionated lmwhs retain their native characteristics and are structurally similar to heparin. in addition, the oligosaocharide sequence responsible for atiii binding is not modified. physical methods such as gamma irradiation (~co) have been used to fi'agment sulfated glycosaminoglyeans yielding fragraents without chemical modifications (deambrosi et at. in : biomedical and biotechnological advances in industrial polysaccharides, pp. 45-53). utilizing this technique, depolymerized heparius exhibiting different molecular weights can be obtained. this communication reports on the biochemical and pharmacologic effects of several such depolymerized heparins to demonstrate the molecular weight dependence on biologic activity. fragments exhibiting molecular weights of 5, 7, 8, and 9 kda were prepared by exposing concentrated heparin solutions to a rectilinear gamma ray beam at intermittent doses of 2.5 to 25 mrad under controlled temperatures. unlike the chemically depolymerized heparins, these fractions did not exhibit any decrease in charge density or atiii affinity. in routine assays for heparin, a clear cut molecular weight dependance on the anticoagulant and antiprotease actions was observed. on a gravimetric basis, these agents produce superior antithrombotic actions in comparison to chemically depolymerized derivatives. these studies suggest that gamma irradiation can be used to prepare lmwhs which retain their molecular integrity and therefore may prove to exhibit a more comparable biologic profile to hepari~ futthermore, lmwhs produced by gamma irradiation lack the usual double bond fommtion which requires the use of additives which can alter the product profile. university hospital, dept. of angiology, frankfurt a.m., germany introduction: thromboembolic disease constitutes a major clinical problem and among others a defective fibrinolytic system has been suggested as a predisposing factor for the development of thrombosis. the plasma fibrinolytic system can be impaired by inherited deficiencies of plasminogen defective release from the wessel wall tissue plasminogen activator (t-p'a) or by high ptusma levels of regulatory proteins, such as plasmino-8en. activator inhibilors (pal). the aim ....... of the present study w~s to eshmate the prevalence of decreased fibnnolyl~c actwlty m young pls. with thrombophilia. patients: a great population of 884 pts. (fenmle 478, male 406; age 21-61 ys median 39.8 ys) with venous thromtx~emolism before the age of 45 years were investigated in regard to their plasma fibrinolytie system. in none of them well established thrombophilia risk factors could be identified previously. methods: plasminogen ~behdngwerke), pai-1 activity (ehromogenic assay, biopool), pal-i anugen coneentration (elisa, biopool), t-pa activity (chromogenic assay, biopool) and antigen concentration (elisa, biopool) were measured before and after venous oeclusion.vo was performed z 12 month after the last thromboembolic epi~xle. 24 healthy subjects (median age 24.7 ys) served as controls. results." 24 pts.(2.7%) were classified as plasminogen deficiencies (activity and antigen). 142 pts.(16%) had significantly elevated levels of pal activity (up to 120 u/ml) and pal antigen (up to 90 ng/ml). none of the pts. with high pal levels had laboratory signs of acute phase reaction. low t-pa activity could be demonstrated and confirmed in 121 pts., aecordingto a prevalence of 13.6% (range: 0-2.7 u/ml; reference limils: 2.8 -21.8 u/ml). however, there was a significant negative correlation between t-pa activity and pal values. in 67 pts. (55.4%) the low t-pa activity was associated with increased pal levels whereas the t-pa antigen concentration was normal. a parallel reduction of t-pa activity and t-pa antigen (range: 0.35-3.5 ng/ml; reference limits: 3.6 -21.0 ng/ml) were determined repeatedly in 54 pts. (f 23, m 31, median age 39 ys). thus, the prevalence of a defective t-pa release was 6.1% in our study group. conclusion." in comparison to the frequency of inherited deficiencies of other well established thrombophila risk factors we have observed a relativel~ high prevalence of diminished t-pa activity, elevation of pal respectively in our study group. our data strongly indicate that besides t-pa and pal acuvity, antigen concentration for both parameters should be determined in pts. with thrombophilia. the antithrombotic and anticoagulant effect of the supersulfated low molecular weight heparin ssh 14 was studied after i.v. and s.c. administration in rats. thrombus formation in the jugular vein was induced by i.v. injection of activated human serum and following stasis for 20 rain and was assessed by a thrombus score ranging from 0 (no thrombus formation) until 3 (complete thrombus formation). ssh t4 injected either 10 min (i.v.) or 30 rain (s.c.) before thrombus induction caused a dose-dependent antithrombotic effect in a range from 0.25 to 2 mg/kg i.v. and 1 to 4 mg/kg s.c. there were clear differences in the antithromboric effectiveness between female and male animals, i.e, in female rats antithrombotically effective doses were lower than in male rats (edh0 after i.v. injection in females 0.35 mg/kg, in males 0.9 mg/kg). the sex differences were confirmed in studies on the time course of the antithrombotic effect. after i.v. injection of fully effective doses (2 mg/kg i.v. and 4 mg/kg s.c., resp.) the antithrombotic effect disappeared after 8 h in female or after 4 h in male rats. for studies on the anticoagulant action blood was drawn from the femoral artery and after centrifugation global clotting assays were performed in plasma. similar to its antithrombotic action ssh 14 also caused doseand sex-dependent anticoagulant effects. the most sensitive assays were the aptt and the heptest; thrombin time and prothrombin time were less or not influenced by ssh 14. in conclusion, ssh 14 was found to be an effective anticoagulant and antithrombotic agent in experimental studies in rats. at present there is no explanation for the clear sex differences found in this species. venous thromboembolic disease is the most frequent complication in patients undergoing total knee replacement therapy. patients and methods: after informed consent 3x30 patients were included in an open randomized clinical study and the incidence of venous thromboembolisrn was examined using different regimes for heparin prophylaxis (30 patients received fraxiparin 36 rag once daily, 30 patients clexane 40 once daily and 30 patients 7500 u calciparin twice daily). there were no differences between the groups concerning age, sex, body weight, risk factors, surgeons, decrease in hemoglobin~ and requirements for blood products. pre surgery, day 1, day 5-7 phiebograms were performed and also tat, dimers, fl+2 prothrombin fragments were examined. results: 1., dvt in 26 patients (28.9%). dvt in 5/30 patients under calciparm prophylaxis, 8/30 patients under fraxiparin and 13/30 patients under clexane treatment. 2., low speciflty (3.4%) of dimers and tat (24%) for detecting a dvt in these special patients undergoing knee replacement therapy, elevated fi+2 fragments in the dvt group at ti and t2 vs the patients without dvt (t1 dvt: 3.24+-1.8 vs. 1.6+-0.3 -p= 0.0042). 3, only 8/26 patients (31%) with dvt had clinical signs of thrombosis. conclusions: 1., there is an increase of thrombin gneeration measured by tat and dimers after knee replacement therapy. there are further studies with more patients necessary to confirm that fl+2 prothrombin fragments can discriminate between patients with and without dvt from a clinician's point of view. 2., phlebographicauy confimled dvt in almost 30% of our patients demonstrate the high thromboembolic risk in these patients. von willebrand's disease (vwd) type 2 is characterized by absence of high molecular weight muitimers. qualitative changes in the structure of the molecule might be associated with enhanced binding of von willebrand factor (vwf) to platelet glycoprotein lb. therefore in some patients vwd type 2 is associated with severe thrombocytopenia. here, we report on a 9 year old boy who presented with severe purpura and platelet counts about 20000/gl at the age of 2 years. thrombocytopenia did not respond to corticosteroids. a normalized platelet count of short duration was observed after high-dose immunoglobulins. in addition, increase of platelets was seen after anti-d treatment. thus, although platelet associated antibodies were not detected, thrombocytopenia seemed to be caused by an autoimmune mechanism. despite platelet counts above 50000/gl, the patient experienced severe bleedings with a significant decrease of hemoglobin levels. therefore, he needed several transfusions. coagulation analysis revealed vwd. application of ddavp lead to a normalization of partial thromboplastin time (ptt) and an increase of factor viii with subsequent cessation of bleeding symptoms. recently, vwd was typed 2 by lack of high molecular weight multimers. in conclusion, we report a case with vwd type 2 responding to ddavp. however it is unclear, whether thrombocytopenia is part of the vwd type 2 or of autoimmune origin. since autoimmune antibodies have not been detected, the effect of immunoglobulin treatment might be explained by blockade of enhanced binding of vwf to glycoprotein lb. von willebrad disease (vwd) with a prevalence of 0,8% (ruggeri 1994, rodeignere 1987) seems to be the most frequent inherited hemostatic disorder. • the diagnostic criteria for vwd are clinical picture, family hostory, laboratory findings: bleeding time, partial tromboplastine time (ptt), level of factor viii:e, vwf, vwf:ag, ristocetin induced platelets aggregation (ripa) and multim~-analysis.the diagnosis ofvwd is occasionally difficult, especially in early childhood because the laboratory data may vary due to time of investigation, as well as abnormalities may not be present in all sub-types the aim of this study was the evaluation of diagnostic approach to vwd in childhood and diagnostic reliability of all available laboratory tests. all previously mentioned laboratory tests have been done on our own material (51 child who satisfied all criteria for vwd, 23 boys and 28 girls, 1-9 years old) except mulfimer analysis which was unavailable in some cases. majority of laboratory tests proved to be highly specific and necessary for diagnosis. however, the diagnostic reliability of fviii:c and adhesion of platelets is much lower in mild cases in comparison to total sample, while ptt is an unvaiied test. the most specific screening test for vwd is vwf which diagnostic reliability is almost 1,00. the optimal strategy to establish general diagnosis of mild forms ofvwd is use of vwf and vwf:ag plus ripa if necessary and multimer analysis to classify variant types. we report on a new multimeric structural defect of vwf detected in a german family (two sisters and their three children): all members of the family who presented to our outpatient clinic had an increased spontanous bleeding tendency (moderate or strong hematoma, epistaxis, menorrhagia). prolonged bleeding could be observed after surgical procedures (adenotomia, tooth extraction) and after trauma (laceration). wound heeling was impaired in two cases. clotting assays showed slightly prolonged apti" and a mild decrease of f viii:c, vwf:ag and vwf:rcof levels. collagen binding activity was within normal ranges. bleeding time (simplate i) was slightly prolonged. the analysis of the multimeric structure in plasma showed quantitative and qualitative abnormalities: all multimers were detectable; the structure of vwf was reproducably abnormal in all family members so that the defect must be caused genetically. the thmmbocytic vwf showed neither qualitative nor quantitative alterations. minirin@ (ddavp) was administered as a test dose of 0,3 ~tg/kg bw in 100 ml 0,9% nacl-solution i.v. to evaluate efficacy and tolerance: clotting assays showed normalization of a_vrt, f viii:c, vwf:ag, vwf:rcof in plasma and shortening of bleeding time in three cases. an insufficient rise of vwf:ag and vwf:rcof levels could be observed in one case. one patient had no rise of f vm:c but a corrected bleeding time. multimeric analysis showed no structural change. the administration of ddavp was well tolcrated in all cases. the existance of all multimers in plasma and the normal collagen binding activity suggest that the structural abnormalities of vwf in this family does not cause functional defects so that the defect could be classified as a type i vwd. the response to ddavp was only partially effective. mild von willebrand disease (vwd) is far the most frequent congenital bleeding tendency. its diagnosis is very helpful in pre-operative check-up in order to avoid bleeding complications during surgery. following post-operative periods or monitoring the management of haemorrhagic episodes in vwd patients is also strongly recommended. current methods involve complex technologies, are time consuming and require large series. these assays lack the expected flexibility for rapid individual testing in patients. a new and flexible assay which works on the fully automatic walk-away coagulation instrument, sta, has been developed for these applications (liatest vwf). the technology is an immuno-turbidimetric method using mierolatex particles coated with rabbit polyelonal antibodies specific for vwf. the assay has a dynamic range from 2 to 420% yon willebrand factor (vwf) concentration, it works with a 2 fold dilution of tested plasma (50 td) and it offers a calibration established with the nibsc international standard. the total assay time is of less than 10 minutes and the detection threshold is of 2% there is no prozone effect up to concentrations higher than 1,000% vwf. intra-assay reproducibility is < 4% and inter-assay one < 5%. in dilution studies a mean recovery of 98% was obtained. in a study on 55 plasma samples from norma~ individuals, patients with high vwf concentrations, and vwd, comparison with the elisa technique demonstrated a correlation coefficient of 0.997 with a slope of 0.978 and an intercept of 3.30. in the low assay range too, a good agreement was obtained with the elisa. we conclude that liatest vwf is a reliable, flexible, sensitive, and rapid automated assay which fits well the vw'f assay applications in coagulation laboratories. fibrinolysis, the process during which the active enzyme plasmin is generated in a regulated and localised way, is -in a classical understanding -responsible for the dissolution of blood clots formed in a vessel. for this activity, t-pa is generally assumed to be the most important plasminogen activator and its activity, is regulated by enzyme kinetic mechanisms dependent on the presence of fibrin. with this background t-pa is used for thrembolytic therapy with great success. however, data from t-pa knockout mice indicate that t-pa might not be responsible for inhibiting the spontaneous development of intravsacular thrombi but only for dissolution of fibrin formed upon a coagulation challenge. in contrast, u-pa, generally assumed to be important for extravascular proteolytie activity on activated or tumour cells, seems to lead to the development of spontaneous fibrin formation in a mouse knockout model. on the other hand, the major plasminogea activator inhibitor pal-i seems not only to regulate intravascular fibrinolysis but seems to also be important for the progression of vascular diseases (neointima formation is e.g. increased in a pai-1 knockout model, but increased levels of pai-1 seem to predict reocclusion after angioplasty). in addition to their functioning as enzymes and inhibitors, components of the fibrinolytic system seem also to be involved in signalling processes in tumour and other cells. the u-pa/u-pa-receptor system could be shown to function as a chemotactic system and to elicit a migratory and mitogenle response in monoeytes and tumour ceils as well as in vascular cells. for such a response activation of tyrosine kinases of the sre-family might be responsible in some cell lines, but other signal transduction pathways e.g. involving caveolae and the starprotein can not be excluded. there seems to be a further important role of components of the fibrinolytic system which involves serine protease inhibitors (serpins): serpins have homologies to hormone binding proteins and cleavage of serpins by their target enzymes not only leads to inactivation of the enzyme but also to a possible release of bound hormones from the serpins. from these data clearly the relevance of any regulation of the fibrinolytie, system depends on the specific function of the system to be dealt with. in addition to "fibrin binding", "receptor mediated" and "genetic control" (e.g. 4g vs. 5g in the pai-i promotor) also "signal transduction" and "hormone delivery" are distinct functions of the system with specific regulation. plasmatic for both, healthy persons as well as for patients with angina pectoris it could be shown that increased values of plasma fibrinogen, factor viic and vwf:ag are significantly associated with the risk to suffer an acute myocardial infarction or cardiac sudden death. the same holds for tpa:ag. however, a group analysis in quintiles reveals that particularly low tpa:ag values are connected with a particularly low coronary risk. unexpectedly also the acute phase protein crp is positively associated with increased coronary risk. for clinical purposes these factors have already been included into coronary risk scores in order to improve the individual risk prediction in combination with lipids and other risk factors. the assessment of the pathophysiological significance of these observations remains at dispute. 4 pathways are discussed: 1. the assumption that increased plasma values of those factors indicate increased coagulation activity could so far not be established in prospective studies. 2. both vwf:ag and tpa:ag are produced in endothelial cells. an increase of their plasma level could therefore indicate increased endothelial cell functions which accompanies progressive atheromatosis. the risk association of the two acute phase proteins crp and fibrinogen could be interpreted analogously. 3. first prospective studies favour the assumption of a genetic determination to an increased production of coagulation proteins in persons at particular coronary risk. it could also be shown that there is a certain dependance of the gene-polymorphism for co-and 13-fibrinogen chains from the coronary risk. 4. even slightly elevated concentrations of fibrinogen and/or vwf:ag may influence the quality of a coronary thrombus both by increased physical stability and by reduced fibrinolytic lysibility. this could mean that an early coronary clot under these conditions could more readily develop to a stable, occlusive thrombus. a newborn with pronounced bleeding tendency had a prothrombin (prth) deficiency below 2.8% in a clotting assay. both parents had activities of 71% and 69%, respectively. however, the immunological determination ofprth by elisa revealed normal concentrations in all family members (93%-101%). furthermore, thrombin generation as investigated by a chromogenic assay using ecarin for activation of prth was normal as well. activation of prth by fxa was investigated by reealcificafion of the plasma samples and further analyzed for prth and its derivatives produced. although clotting times still were different, finally, normal levels of fl+2 and tat were generated as determined by elisa. western blot analysis using polyclonal (rabbit) antibodies to prth and a monclonal antibody specific to human thrombin, revealed different patterns of prth degradation products. tat was only weakly visible in the serum of the mother and nearly absent in the child.the mobility of prothrombin and thrombin was different compared to normals indicating a lower molecular weight. after reduction of disulfide bridges a higher molecular weight of thrombin was observed compared to normals indicating an insufficient cleavage ofprth and formation ofprethrombin 2. these observations let suggest that prothrombin marburg is a deletion mutant lacking the cleavage region arg 320-ile321. upon cleavage by factor xa only prethrombin 2 is formed under liberation of fl+2. this prethrombin 2 is able to cleave chromogenic substrates in the ecarin assay. probably, prethrombin 2 forms a complex with atiii which is detected by elisa, but unstable under denaturing conditions as in the western blot. as a major complication of haemophilia a treatment, up to 30% of the severely affected patients develop antibodies to substituted factor viii. investigating 133 patients and considering the data of further 231 patients of the haemophilia database, we could show, that risk of inhibitor developement depends on the patient's mutation type. patients with more severe gene defects, like intron 22 inversions, stop mutations or large deletions had a risk of about 35% for inhibitor developement, which was about 7 times higher than for missense mutations or small deletions. besides an influence of mutation type, we investigated other parameters e. g. immune response genes (i-ila-genotype) and clinical aspects (treatment onset and frequency, type of concentrate) that might also affect inhibitor formation. to exclude any effect of mutation type, we focussed on 72 patients with an intron 22 inversion. hla-typing showed that some t-ila-alleles (dqb0602, bt) occurred more otten and others (dqa103, dqb603, dr13, c2) less frequent in inhibitor patients. treatment onset, frequency and type of concentrate apparently do not affect inhibitor incidence. the results presented here, prove that inhibitor development is considerably influenced by the mutation type. this supports the hypothesis that patients with severe molecular defects have no endogenous factor viii protein and that substituted factor viii represents a foreign protein, leading to an immune response, e. g. the production of alloantibodies. in addition, the immune response seems to be modified by the hla-genotype. however oar findings (in terms of genotype and treatment parameters) can only explain part of the inhibitor pathogenesis. it is still unsolved why substituted factor viii does not lead to a recognizable immune response in 2/3 of the patients with severe molecular factor viii gene defects. consequently other factors, probably concerning the antenatal phase, must be involved. viia in the treatment of patients with inhibitors against factor viii or ix: a german/swiss/austrian multi~center trial d. ellbriiek*, i. scharrer**, j. dethling***, and the rfviia study group *section h~mostaseology, university ulm **dept. of angiology, jwg-university hospital frankfurt a.m. ***novo nordisk, mainz administration of activated recombinant factor vii (rfviia) can by-pass the fvnlwlx pathway and offers an alternative treatment for patients with antibodies (inhibitors) against these factors. from november 1994 to october 1995, a total of 25 bleeding episodes and 10 surgical interventions in 18 patients were treated with rfviia in a phase iiib multicenter trial. diagnosis was hemophilia a (n = 15) or b (n=l) with inhibitor, and acquired inhibitor against factor viii (n=2). various serious bleeds, from complicated joint and gingival bleeds to lifethreatening psoas bleeds, have been treated. operations have been tooth extractions, radiosynovectomy, implantation and explantadon of porth-acaths and one adenotomy. dose regimen was 90-120/zg/kg bw every two to three hours until clinical improvement, with subsequent dose reduction. results: for bleeding episodes, response to rfviia after 24 hours was effective in 72%, partially effective in 12 9"0, ineffective in 129"o and not evaluable in 1 (4%) of the patients. two of the three treatment failures were associated with very long dosage intervals of rfviia. the third patient was in a critical situation with artificial high pressure respiration and polytransfusion because of a hematothorax, and suffered a terminal intracerebral bleed. the efficacy of rfviia for surgery was very good. response to treatment was independent of antibody titer. no signs of dic or activation of coagulation were noted. conduslon: in our experience, rfviia is an efficient and safe treatment for inhibitor patients with acute bleeding episodes. it should be investigated, whether rfviia can be an alternative treatment also for the hometreatment situation. successful immunetolerance therapy of f vih-inhibitor in children after changing from high to intermediate purity f vih concentrate w. kreuz, j. joseph-$teiner, d. mentzer, g. auerswald*, t. beeg, s. becker zentrum der kinderheilkunde, j. w. goethe-universit~itj frankfurt am main *professor hess kinderklinik bremen introduction: inhibitor to f viii is the most severe complication in treatment of patients with haemophilia a. the incidence of f viii inhibitors is estimated to range between 15-33%. several authors reported that the immunetolerance therapy (itr) of f viii-inhibitors can be induced with high dose f viii concentrate. objective: this presentation will show data of four children with haemophilia a and f viii inhibitor (high responder), who had an unsuccessful lit with high dose f viii concentrate (high purity) in the first step. f viii concentrate was changed to an intermediate purity product (haemate hs®) in the subsequent course of h't. all patients received bleeding prophylaxis with an activated-prothrombin-complex-concentrate (feiba®). results: median age was 13 (9-18) months, when the inhibitor was first detected. in all four patients the f viii inhibitor titre increased under immunetolerance treatment with f viii concentrate (high purity) in the first step of therapy. after changing the f viii concentrate (intermediate purity) the inhibitor titres decreased continuously after a rebooster effect to 0be within months. median duration of f viii inhibitor elimination time (until first testing of 0 be) was 3 (2-5) months. in all patients the f viii inhibitor was successfully eliminated. until now all patients are under prophylactic treatment with f viii concentrate and had no positive inhibitor testing since. median observation time since the first testing of 0 be is 14 (4-60) months. conclusion: different studies concerning immunetolerance treatment have been successful with f viii concentrates of different purity. according to our experience in these four presented patients, we assume that probably not the purity of the f viii concentrate is important for the induction of immunetoleranee, rather than the type of f viii presentation in the used concentrate. the used preparation (haemate hs®) is a f viii concentrate with high concentration of vwf, which is known to be important for the protection of f viii against degradation by proteases. this may be a mechanism for a prolonged antigen presentation to the immunesystem and thus may have a positive impact on the outcome ot itr. long scale trials are needed to prove the above assumptions. thrombasthenia glanzmann is a disease affecting platelet function because of a partial or total lack of glycoprotein (gp) ilbllla expression or a modification of this complex. since the receptor dysfunction goes along with reduced or absent platelet aggregation and adhesion, it causes bleeding complications in case of injury. here we report about a 60 years old women, who suffered since early childhood from a severe bleeding disorder. life threating bleeding complications occured after tooth extraction and after abdominal surgery. analysis of the patients platelets revealed normal values for the platelet count, whereas their volume showed to be increased (11 fl). clot retraction was diminished to 17%. platelet adhesion to siliconised glass and human subendothelial matrix was reduced, as was the spreading of the platelets. adp (i#m) induced platelet aggregation was inhibited, while collagen-, ristocetin-and thrombin-induced aggregation showed to be normal. cross immunelectrophoresis resulted in an atypical peak of gpiibllla with reduced electrophoretic mobility. in the electroimmunoassay according to laurell 14% of gpiibllla was detected. moreover we observed a markedly diminished 125j-fibrinogen binding. sequence analysis of the gpiib and gpiila cdna after pcr amplification unraveled a g2508 --, a transition in gpiib, substituting gly805 --* glu. the structure/function relationship of this mutation has still to be investigated. we report two new abnormal fibrinogen variants, denoted as bem iv and milano xi, both having an exchange of arginine to histidine in position 16 of the ac~-chain. routine coagulation studies revealed prolonged thrombin and reptilase clotting times, low plasma fibrinogen concentrations determined by a functional assay but normal fibrinogen levels measured by the immunological assay. the onset of turbidity increase following addition ofthrombin to purified fibrinogen was markedly delayed in both variants. release of fibrinopeptide b by thrombin, measured by reversed phase hplc, was normal whereas only one half amount of normal fibrinopeptide a was released. in addition to normal fibrinopeptide a, an abnormal fibrinopeptide a* was cleaved from both dysfunctional fibrinogens. the structural defect was determined by asymmetric pcr and direct sequencing of a gene fragment coding for the nh2-terminus of the aachain. both variants were found to be heterozygous for the transition g to a at nucleotide position 1203, leading to the substitution actl6 arg-->his, resulting in a delayed fibrin polymerization. the simple assay permits detection of the most common amino acid substitutions occuring in the nh2-terminus of the ac~-chain of the functionally abnormal fibrinogen variants. protein c inhibitor (pci) a member of the serpin family is also known as plasminogen activator-3 (pal-3). pci was first described as a component of human plasma, regulating the activity of activated protein c and other sedne proteases of the human coagulation and fibdnolysis system. since then pci was found to be present in extra-plasmatic systems also. high concentrations of pci were detected in human seminal plasma suggesting a role for pci in human fertility. significant concentrations of pci mrna and antigen were located in lysosomes of proximal tubular kidney cells suggesting an intracellular function for pci in this environment. in this study we present evidence that pci is also present in human pancreas. rna from human pancreas was reverse transcribed and pcr amplified. the resulting pci cdna was identical with pci cdna from human liver. ~p labeled antisense rna probes used in in situ hybridization experiments with human pancreas tissue sections showed that pci rna was located in the acinar ceils. pancreatic fluid was analyzed by sds-page and immunoblotting. using monospecific antibodies directed against human plasma pci, a 57 000 mw protein band was observed which comigrated with purified human plasma pci. our results show that pancreas cells contain a significant concentration of pci mrna. this message is localized in the secretory acinar cells. therefore we conclude that pci antigen found in pancreatic fluid is likely to originate in the pancreas. the role of pancreatic pci is unknown at present. however, since thrombosis and systemic hypercoagulable states are known complications of pancreatic diseases our results and in vitro experiments by others showing that pci can inhibit pancreatic enzymes such as chymotrypsin and trypsin indicate that pci may be part of the inhibitor potential which protects pancreatic tissue from auto degradation. these inhibitors normally prevent the release of active pancreatic proteases into the vasculature or microcirculation where destabilization of the coagulation balance and subsequent thrombus formation could occur. institute for clinical chemistry and laboratory diagnostics and *clinic for cardiology, universi w of duesseldorf p-selectin (cd 62p, the former granule membrane protein 140 or gmp 140) is an integrated membrane protein of platelets and endothelial cells. under inactivated conditions it is stored in the alpha granules of platelets and in the weibei-palade bodies of endothelial cells. endothelial cells covering atherosclerotic plaques show an increased expression of p-selectin. 13-thromboglobulin (13-tg), which is also expressed from the alpha granules of platelets during adhesion or aggregation, is regarded as a marker of platelet activation in vivo. coronary thrombosis plays a central role in the pathogenesis of acute coronary syndromes. we therefore analysed cd 62p and 13-tg in acute coronary syndromes, healthy subjects (hs, n=l i), patients with stable angina pectoris (sap, n=20), unstable angina pectoris (uap, n=l 2) and acute myocardial infarction (ami, n= 12). plasma samples were obtained by using ctad vacutainer tubes (0.109 m na~-citrate, theophylline, adenosine dipyridamole). patients with cad showed significantly increased plasma concentrations of cd 62p (hs: 98+20 versus sap: 133+38ng/ml, p<0.05; versus uap: 128+28 ng/ml, p<0.01; versus ami: 144+72 ng/ml, p<0.05) independent of the severity of clinical symptoms. in comparison only patients with ami showed significant higher 8-tg concentrations compared with hs (hs: 30+20 versus ami: 39+14ng/ml, p<0.05). although the cd 62p plasma concentrations showed no relationship to the clinical severity, hence there was a positive correlation between cd 62p (r=0.47; p< 0.001; n=55) to the severity of cad classified as i, 2, 3 vessel disease. it is concluded that elevated cd 62p concentrations are correlated with the severity of cardiovascular disease. cd 62p is not suitable for differential diagnosis of acute coronary syndromes, because it is elevated independently of the clinical status of the patients. the involvement of platelets in the pathogenesis of acute myocardial infarction may be indicated by the increased 13-tg concentrations. iklinik nr herz-, thorax-und herznahe gef&schirurgie und 2institut x~tr klinische chemie und laberatodumsmedizin der universint regensburg an increased blood loss following surgery with extracorporeal circulation (ecc) contributes to the morbidity and mortality. postoperative haemorrhage following ecc has been related to a platelet function defect and the activation of the blood dotting and fibrinulytic system. we investigated platelet surface antigen expression and parameters indicating activation of the clotting and fibrinolytic cascade to assess the predictive potential of these variables for increased blood loss after ecc. g0 patients referred for coronary bypass gra~ing with no history of a bleeding disorder and normal routine clotting tests were included. on the day prior to surge~ and immediately upon arrival on the intensive care unit blood samples were drawn. the surface expression of glycoprotein (gp) lib-ilia, gp lb, and p-selectin was meamred with and without in vitro stimulation with adenosine diphosphate (adp) using whole blood flow cytomet~y. platelet counts and platelet factor 4 (pf4), as well as, routine clotting tests were performed. activation of the clotting and fibrinolytic system were judged from thrombin-antithrombin-iii complex fiat), fibrinogen fig), d-dimers (dd), cc2-antiplasmin (tz2a), prothrombin fragment 1+2 (fl+2),and tissue plasm~ activator (t-pa). blood loss fxom chest tubes was measured hourly until removal of drains. following ecc the levels of pf4, tat, dd, o~2a, fl+2, and dd were sigulticnatly increased (p<0.0001) compared to baseline values. gp iib-iila, gp ib, p-selectin, platelet count, and fg were significantly reduced (p<0.0001). analysis of variance (anova) revealed that postoperative values of gp ib (p<0.0001), dd (p<o.05), fg (p<0.01),and platelet count (10<0.05) had a significant influence on blood loss. none of the preoperative data was significantly associated with increased blood loss. a subgroup of patients who bled more than the mean + 1 sd (14 cases) was formed_ in logistic regression analysis gp ib expression (p=0.01) and dd (p=0.007) were of value in identifying bleeders. the sensitivity of the equation was 56%, the stx~ificity 97% and the positive predictive value 82%. although the reasons for an increased blood loss following ecc are multifactorial, we were able to identify a small number parameters with a significant infl~nco. the combination of the expression of yon willebrand receptor on the platelet su_rfaco and d-dimer levels in plasma was successful in predicling an increased blood loss following heart surgery. this may be helpful to define indications for platelet transfusion and flesh frozen plasma. the most widely distributed receptor for the fc-region of igg is the fcreceptor iia (fcyriia). for this receptor a functionally relevant polymorphism of amino acids argmhis is known. we previously showed hit-antibodies to bind to platelets via feyriia and now whether clinical manifestation of hit is associated with the fe'/riia polymorphism. therefore, we developed a rapid two step allelle-specific pcr (asp) method for genotyping the foyriia. with control samples the asp was compared with the time consuming and complicated allele-specific oligonucleofide hybridization technique, the phenotype expression of fctriia (using the moabs iv.3 and 41h16) and the monocyte stimulation assay. complete concordance between the assays was found. we used the asp to determine the allelic distribution of the inst. physiol. chem., marburg; *)univ. of virginia, charlottesville, the functional heterogeneity of blood platelets is clinically significant and may contribute to the pathogenesis of cardiovascular and atherosclerotic diseases l). in order to understand the biochemical basis of the functional heterogeneity of platelets, we have investigated the signal transducing pathway of platelet activation a) in terms of guanylate cyclase and phosphodiesterase activities and b) at the level of protein phosphorylation and the receptor-effector coupling via g i-and gs-proteins in functional heterogeneous blood platelet subpopulations separated according to their densities. furthermore, we analyzed the adp-ribosylation of rhoa, which is a lowmolecular weight gtp-binding protein and is thought to be involved in the platelet "shape change" reaction and aggregation. aggregation was enhanced in low-density platelets (sp i) and less inhibited when cgmp was increased by no, compared to high-density platelets (spiii). sp i possessed a lower basal level of cgmp and exhibited a smaller increase in cgmp after stimulation with no. cyclic amp-dependent phosphodiesterase activity was higher in sp i compared to spiii, whereas cgmp-dependent phosphodiesterase activity was similar in sp i and sp iii. sp i, when compared to sp iii platelets, showed a higher degree of prephosphorylation of proteins in the range of 47, 32-34, and 20-22 kda. after thrombin stimulation, the phosphorylation of these proteins was similar in the subpopulations. pertussis toxin,induced adp-ribosylation of the 40-41 kda gi-protein was highest in sp i, whereas cholera toxin induced adpribosylation of the gs-protein and botulinum c3 exoenzyme-induced adpfibosylation of rhoa were highest in sp iii. the findings suggest that a) the higher aggregability in sp i compared to sp iii may be caused by a poorer ability of guanylate cyclase to form cgmp and a higher activitiy of campdependent phosphodiesterase and that b) the adp-ribosylation of g-protein subunits and rhea may contribute to the functional heterogeneity in the subpopulation leading to the greater ability of sp i for aggregation. 1) opper et al., atherosclerosis, 113, 211-217, 1995. causes of accelerated atherogenesis in non-insulin-dependent diabetes mellitus (niddm), known to be associated with increased plasma activity of plasminogen activator inhibitor type 1 (pal-l) as well as with increased plasma concentrations of proinsulin and insulin, remain enigmatic. this study was designed to determine whether proinsulin and insulin increase pal-1 in vivo thereby potentially accelerating atherogenesis. to simulate hyper(pro)insulinemia seen with niddm, we infused equimolar concentrations of insulin (1 iu/kg), proinsulin, c-peptide, or placebo, all endotoxin-free, i.v. over 1 h in 53 conscious rabbits maintained euglycemic. plasma pal-1 did not increase with placebo (n=16) or c-peptide (n=4), but did with proinsulin and insulin peaking in 3 h at 3.5-and 3.7-fold (n=16 and n=17, p<0.o02 for each) in parallel with pal-1 protein quantified by reverse fibrin autography. the increases were specific as reflected by unchanged activities of o~-antiplasmin and t-pa. pat-1 gene expression (pal-1 mrna) increased with proinsulin and insulin by 2.1-and 2.1-fold in 3 h in aorta, 1.9-and 2.4-fold in liver (p<0.05 for each), but not in heart, lung, spleen, or kidney and returned to baseline in 24 h (n=4 each). in situ hybridization localized the increased pal-1 mrna in aorta to endothelium (n=3 each). thus, both proinsulin and insulin directly augment gene expression of pal-1 in arterial endothelium in vivo, thereby increasing plasma pal-1 activity, attenuating endogenous fibrinolysis, and potentially accelerating atherogenesis. the sarco-endoplasmic-retikulum ca+÷atpases (serca) that sequester calcium into intracellular stores, represent the most important mechanism to maintain cytosolic calcium levels low and to return to a resting level after activation. recent studies in platelets and rat endothelial cells have identified two forms belonging to the serca 2b and serca 3 types. the monoclonal antibody pl/im 430 raised to human platelet intracellular membranes has been shown to recognise the serca 3 ÷+ isoform and to inhibit ca sequestration in human platelets. the aim of this study was to identify ca atpase distribution in human umbilical vein endothelial cells (huvec) and human polymorphonuclear leukocytes (pmnl). polyclonal antibodies with known specificity towards serca 2b (r 2/88) and serca 3 (89/3) in addition to pl/im 430 were used in histochemical studies of permeabilised cells and in western blotting experiments using cell lysates and mixed membranes. using the alkaline phosphatase anti-alkaline phosphatase (apaap) method r 2/88 and 89/3 reacted strongly with proteins in permeabilised huvec and pmnl, however pl/im 430 showed good staining only with pmnl. in western blotting experiments r 2/88 recognised proteins with molecular sizes of 100 kda (known to be the parent ca++atpase) and 70 kda (which may represent antibody recognised degradation product), in both huvec and pmnl. 89/3 ÷+ recognised bands at 100 kda (parent ca atpase), 74 kda and 46 kda in both huvec and pmnl membranes. in agreement with histochemical results pl/im 430 recognised proteins (100, 74 and 46 45 + + kda) only in pmnl membranes. in ca transport studies pl/im 430 ÷+ inhibited ca uptake in membranes prepared from pmnl but not in huvec. the distinct reaction of pl/im 430 with pmnl and huvec suggests the possible presence of distinct sub-isoforms of serca 3 with the pmnl and platelet proteins being similar and different to the serca 3 isoform present in huvec. microvascular endothelial cell (ec) lines with a limited life span of 2-3 months in vitro were established from human bone marrow, skin or solid tumor tissue. homogeneity of the lines was verified by flow cytometric analysis and cytokine expression patterns. the cultured ec constitutively expressed hla-class i antigens, cd44, procollagen type i, cd62e, cd34, and receptors for granulocyte colony stimulating factor (g-csf). endothelial cells constitutively produced il-6 and il-8 as well as tpa and pai-i as determined by elisa. in the presence of 5 iu/ml of exogeneous il-la, g-csf was secreted at high amounts (lng/ml) and gm-csf at low amounts (15pg/ml) in confluent monolayers and during over night incubation. these ec potently bound to isolated neutrophilic granulocytes. when granulocytes were stimulated by either 10~ 4 or 10 .7 m formyl-methionyl-leucyl-phenylanaline (fmlp) to produce oxygen radicals, the preincubation with a single cell suspension of ec caused oxygen radical scavenging. a ratio of 1 : 100 ec to neutrophilic granulocytes resulted in a 50 __+ 10% inhibition of fmlp-induced and luminol-enhanced chemiluminescence in vitro. this effect was probably due to the constitutive nitrogen oxide (no) production by ec. unexpectedly, preincubation of ec with 50 iu/ml of interferon-y (ifn-y) to increase no-secretion did not alter the extent of the oxygen scavenging effect measured at different ratios of ec to neutrophilic granulocytes. according to the surface antigen expression pattern, identical ifn-f treatment upregulated adhesion receptors on ec. concomitantly, binding of neutrophils to ec was significantly increased. thus, the net oxygen-scavenging effect by ec appears to be stable under the condition of ifn-y stimulation which indicates maintenance of ec function and effective neutralizition of non-specific oxygen radical damage. moreover, the established test system can be useful to investigate the selective contribution of other inflammatory cytokines, adhesion molecules, macrophages and platelets on ec function in vitro. atherosclerosis is characterized by intimal migration and proliferation of smooth muscle cells (smcs), by macrophage infiltration and extracellular matrix remodeling. circumstantial evidence suggests that urokinasetype plasminogen activator (upa) may be relevant in these processes. this study examines upa expression and upa antigen localization in human coronary arteries with ,carious degrees of atherosclerotic lesions. representative segments of macroscopically normal areas (mnas), early lesions (els) and fibrous plaques (fps) were processed in parallel for in situ hybddization and immunohistochemical analysis. in mnas, upa was detected both in the luminal endothelium and in celocalization with ~-actin-positive smcs throughout the tunica media. in els, upa mrna and antigen were substantially reduced in the luminal endothelium. however, cellular upa expression was detected in the subendothelial area in colocalization with tissue-infiltrating cd68positive macrophages. in the deeper intimal and in the medial layers strong upa expression was observed in association with ~-actinpositive smcs, with most of the upa-positive cells being located in areas with a high proliferative activity as detected by pcna staining. in fps, upa mrna was widely expressed in macrophage-containing areas within the fibrous cap and at the periphery of the necrotic core of the lesions. in addition, upa antigen was identified in acellular areas of the necrotic core regions. compared to the diseased intima of these lesions, the adjacent media stained only weakly for upa. in conclusion, this study demonstrates an enhanced expression and synthesis of upa by intimal smcs and macrophages in advanced atherosclerotic lesions of human coronary artedes these findings suggest a role for upa in the progression of the coronary atherosclerosis. adenosine produced by vascular cells, platelets and other tissues has been proposed to be a vasodilator, inhibits platelet aggregation, stimulates proliferation and migration of endothelial cells as well as exhibits antiinflammatory effects. adenosine of endothelial origin intra-and extracellularly formed and highly concentrated at the luminal surface may constitute an important antiaggregatory mechanism, which is part of the well-known antithrombogenicity of an intact endothelial lining. it was the aim of this study to investigate whether adenosine could influence the fibfinolytic potential of endothelial cells. when confluent human umbilical vein endothelial cells were incubated with adenosine (0.01-10mm) a significant dose dependent decrease in plasminogen activator inhibitor type-1 (pai-i) antigen production by this cells was seen as compared to control. this effect by adenosine was also time dependent and seen after 2 hours of incubation with adenosine. as determined by elisa, pai-i antigen in conditioned media of endothelial cells incubated with 10mm adenosine decreased to 57% as compared to control. pai-i antigen in extracelullar matrix of these cells was also reduced by incubation with adenosine. these results were also reflected on the level of specific mrna as determined by northern blotting. pai-i mrna decreased in endothelial cells after incubation for 4 hours with adenosine (10mm) to 77% (2.2kb) and 85% (3.2kb) of control. in conclusion our data demonstrate that adenosine can downregulate the expression of pai-1 in cultured endothelial cells thereby increasing the profibrinolytic capacity of these cells. this effect of adenosine might contribute to its antithrombotic potential in addtion to its known antiaggregatory effect. sowohl die effizienz als such die komplikationen einer antikoagulantien-dauerbehandlung sind in erster linie yon der gore und engmaschigkeit der gednnungskontrellen abh~ngig seit 1986 wird die schulung zur gerinnungs-selbstkontrolte in der herz-kreistauf-klinik bad bedebarg in gruppen eingeobt. die theoretische und praktische anleitung erfolgt in 8 stunden durch eine in der patientenschuiung erfahrene arzthelfedn (oder krankenschwested-pfleger) und einen arzt. die praktische schulung wird durch einen patientennahen theoretischen tell erg~nzt. verglichen mit konventionell ~berwachten oral antikoagulierten patienten (ca. 50% der tpz-werte liegen im theapeutischen bereich) erreichten 216 quickwert-selbstbestimmer, daf$ 83,1% yon 14 812 tpz-werten im therapeutischen bereich lagen (tabelle 1 ). schwerwiegende thromboernbolische ereignisse oder blutungskomplikatienen wurden nicht beobachtet. durchschnittlich kontrolliert der quickwert-selbstbestimmer seine gerineungswerte w6chentlich, itmgstens im zweiwochen-abstand. ob sich blutungs-und thremboembolierisiko langfristig senken lassen bei gleichze~iger verbesserung yon prognose und lebensqualit~it, bedarf des nachweises im rahmen einer randomisierten, kontrollierten und prospektiven studie. tabelle i verteilun~l der selbstbestimrnten gednnur ~swerte zeitraum 1986 zeitraum -1988 zeitraum 1986 zeitraum -1990 zeitraum 1986 zeitraum -1992 to evaluate the potential benefit of an optimized oral anticoagulation management with inr measurements performed by the patients themselves, two prospective randomized studies have been completed. the first study (n=60) was performed to evaluate the accuracy of inr self-testing (inr-st) compared with standard laboratory controls as well as the improvement of anticoagutation monitoring by inr-st, e.g. the percentage of measurements found inside the target therapeutic inr-range. the 231 parallel measurements revealed a high accuracy of inr-st with a 4.5% median difference between parallel controls of single measurements. in addition, inr-st resulted in a significant increase of measurements inside the target therapeutic range (from 54 to 80%) in the entire group. however, inr-st performed every 4 weeks did not result in a significant improvement, while selfcontrols every week (from 44 to 84%) and every 2 days (from 51 to 84%) did. in a second study (150 patients) the incidence of bleeding and thromboembolic complications were evaluated in patients who either had standard anticoagulation measurements o[ performed inr-st. during a median follow-up of 1.5 years in the group with standard oral anticoagulation management an incidence for bleedings of 10.9% per year and for thromboembolic complications of 3.6% per year was found, while in the group of patients with inr-st bteedings occured with an incidence of 4.5% per year and thromboembolisms with 0.9% per year. it can be concluded that inr-st can be performed with high accuracy by the patients themselves. inr-st results in more frequent controls of the inr and consequently in significantly longer time periods where the patients are inside the optimal target therapeutic inr range. the more accurate anticoagulation management resulted in our study group in a significantly lower incidence of thromboembolic and hemorrhagic complications. the cell biology of tissue factor (thromboplastin) h. prydz, the biotechnology centre of oslo, norway tissue factor (tf,thromboplastin) is the most potent trigger of blood clotting knuwn.lts presence in tissue extracts has been known since before the turn of the century.erwin chargaff (of dna fame) first described that tf consisted of two parts (a protein and a lipid moiety), a fact that was independently rediscovered in the early sixties by deutsch and lechner in vienna and by myself in oslo. in 1973 tf was first purified and shown to be an integral membrane protein of mr about 50 kda (this was a slight overestimate,recent studies indicate 46-48 kda). in 1987 four groupes independently cloned tf edna, later also genomic dna. the structure of tf suggested that it was a membrane spanning receptor, with an extracellular part of about 219 amino acids folding into two domains remotely like the domains of the ig-superfamiliy, and more specifically the eytokine receptors. tf is synthesized to a varying degree in many tissues, but not normally in tissues in contact with blood. however, during acute inflammatory situations and under the influence of a number of agents (immune cnmplexcs, lectins, cytuklnes,bacterial components, complement components etc) monucytes and vascular endothelial cells are induced to synthesize tf. another possible way tu get tf into contact with blood is thruugh trauma, surgical ur otherwise. the fact that tf is inducible has created a lot uf interest in its cell biology. we reported recently that binding of factor viia tu tf un the cell surface elicited a marked increase in intracenular ca spikes, thus rendering the final proof that tf not only luoks like a receptor but really is one, since all the three essential criteria are fulfilled: tf has a transmembrane segment and a cytoplasmic tail tf has a high specificity/high affinity ligaud (factor vii and factor x) tf induces an intracellular signal upon ligand binding recent results regarding tf as a receptor and regarding its intracellular traffic will be reported. both f.ix and f.x are activated by the tf/f.viia complex. we have used a tf-initiated model system to examine the roles played by f.ixa and f.xa in initiating coagulation. the in vitro model contained a cellular tf source; plasma concentrations of f.ii, v, viii, ix and x, tissue factor pathway inhibitor and antithrombin iii. we tested the ability of exogenously added f.ixa or f.xa to substitute for tf/viia activity in initiating platelet activation and thrombin generation. f.xa was 10 times more potent than f.ixa in initiating platelet activation, but f.ixa was more potent in promoting iia generation. in the presence of tf/viia, zymogen f.x was required both for platelet activation and iia generation, while f.ix was only required for iia generation. thus, tf/viia-activated f.ixa and f.xa have distinct physiological roles. the main role of f.xa is to activate platelets by producing an initial small amount of iia. the process of platelet activation is much more efficient if this initial iia is produced on the tf-bearing surface. f.ixa, by contrast, enhances iia generation on platelets by providing f.xa for platelet surface prothrombinase assembly. we conclude that activation of both f.ix and f.x by tf/viia is essential for efficient initiation of coagulation. furthermore, f.x activated on the surface of a tf-bearing cell isnot functionally equi.valent to f.x activated on the platelet surface. therefore, f.x activation by tf/viia does not compensate for a lack of f.ix or viii in hemophilia because the f.xa activated by tf/viia is located on the wrong surface for efficient thrombin generation. our results emphasize that the location, of an activated coagulation factor is a critical determinant of its role in hemostasis. the x-ray crystallographic structure of the fviia-tf complex located the areas within fviia in contact with tf to the first egflike and the protease domains. high-affinity binding of fviia to tf depends on ca2+ and previous studies suggest that ca2+ binding to a site in the protease domain is required. however, using surface plasrnon resonance we have shown that removal of the n-terminal gla domain and hydrophobic stack (hs) from fviia results in a decreased affinity for tf. in search for the mechanism by which regions in fvua not in direct contact with tf affect the affinity, various techniques have been employed. a plausible model is one in which the gla domain and hs optimize the interaction with tf by affecting regions that mediate the association with tf. using gel filtration we demonstrated that the hydrodynamic radii of des(1-38)-fviia and, especially, fviia decreased upon ca2+ binding, whereas des(1-44)-fviia was virtually unaffected. this suggested that the gla domain and the hs interact in a ca2+-dependent manner with other regions in fviia, presumably the protease domain, making fviia more globular. using cd spectroscopy, we could detect ca2+-induced changes in the secondary structure of fviia, all assignable to an increased helical content of the gla domain. the amidolytic activity of fviia in the absence of tf has a ca2+ dependency that suggests involvement of the gla domain in obtaining full activity. this activity was very sensitive to guanidine hydrochloride (c50<0.5 m) and its quenching corresponded to unfolding of the ca2+-loaded gla domain as measured by changes in the trp fluorescence of fviia. based on these results it is conceivable that the gla domain and perhaps the hs of fviia interacts with the protease domain and that this globular form mediates the initial contact with "it. several studies have indicated a profound role for fvii(a) in arterial thrombogenesis. in the present study we quantified the inhibitory effect of dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone recombinant fviia (degr-rfviia) on acute thrombus formation at medium-sized artery blood flow conditions characterized by a wall shear rate of 650 s -1. native human blood was drawn directly from an antecubital vein by a pump into a heparin coated mixing device where degr-rfviia (0.9 -880 nmol/l final plasma concentration) or buffer were mixed homogeneously with the flowing blood. subsequently, the blood entered a parallel-plate perfusion chamber in which a thrombogenic surface consisting of immobilized tf and phospholipids was positioned. fibrin deposition, platelet-fibrin adhesion and platetet thrombus volume triggered by this surface were measured by morphometry. degr-rfviia inhibited these thrombus parameters in a dose dependent manner with ic50 values between 0.5 and 0.7 nmol/l the plasma levels of thrombin-antithrombin m complexes were inhibited at an ic50 of 0.6 nmot/l since the concentration of fvii and fviia in normal plasma is about 10 and 0.1 nmol/l, respectively, theic50 values indicate that degr-rfviia is a very efficient inhibitor of thrombus formation in this model. thus, inhibition of the extrinsic pathway of coagulation may represent and entirely new and effective arterial anti-thrombotic approach. the anticoagulant agent from medicinal leeches, hirudin, is presently gaining popularity as an antithrombotic drug. the design of recombinant forms of the minipretein and their availability led to the clinical use of this naturally occurring thrombin inhibitor. extensive studies in experimental animals have shown that due to its pronounced and specific antithrombin effect hirudin is an antithrombotic agent of high quality. apart from its anticoagulant effect, hirudin is pharmacodynamically inert and is well tolerated in vivo. brain abnormalities in male children and adoleescents with hemophilia: detection with mr imaging mri of sickle cell cerebral infarction in addition, portions of the preparations were incubated with 1.4nm-gold labelled fibrinegen molecules (fg-au; final concentration 4gp_g/ml) for 10 see at 37°c. the expenments were stopped al~er 3 or 10 rain by glutaraldehyde fixation. serial sections were prepared and examined after silver-enhancement using danscher's (1981) method to visualize the fg-au. hwdid not alter the resting gfp. after .3,rain, activated hw-platelets changed their shape moderately but in contrast to the controls did not aggregate. moreover, the conatdcting cytoskeleton that centralizes platelet organelles was not formed. fg-au was found on the surface of the control platelets, in high density in the contact spaces between the aggregated platelets and internalized in the surface connected system. only a very small number of hwtreated cells had fg-au on their surface and showed internalization. 10 rain after adp most of the platelets in both preparations had retumod to a discospherecytic shape it is confirmed that hw inhibits the platetet aggregation and in addition, 1) the formation of the contractile cytnskeleton as well as 2) the binding and interalization of the used ligand fibrinogen santoso 2 l.institute for immunology and transfusion medicine, emst-moritz-arndt-university greifswald; 2. institute for transplant vasculopathy in cardiac allografts is the leading limitation to long-term survival of cardiac transplant recipients. activation of the coagulation mechanism by adherent monocytes expressing tissue factor (tf) is expected to be involved in the development of transplant atherosclerosis. in this in vitro study the effect of the immunosuppressant cyclosporine a (csa) on monocyte tf expression and the regulation of the tf gene transcription was analysed. csa was shown to inhibit the induction of monocyte procoagulant activity (pca) by decreasing lipopolysaccharide (lps)-induced tissue factor (tf) expression in monocytes. csa exerted a dosedependent inhibitory effect on monocyte tf expression at concentrations (1,5 txmol/l csa: 50 % inhibition) not decreasing the cellular protein synthetic rate. addition of csa during lpsstimulation prevented activation of the nuclear factor (nf) ~b as demonstrated by electrophoretic mobility shift assay. western blot analysis confirmed reduced nuclear translocation of nf-~b in the presence of csa. inhibition of the lps-induced nf-~b activation by csa resulted in a reduced tf gene transcription as demonstrated by rt-pcr-analysis. thus, cyclosporine a prevents tf expression by inhibiting the induction of tf gene transcription in activated monocytes. csa may therefore provide a therapeutical tool acting not only as a direct immunomodulating agent, but also by influencing local coagulation activation. the n-glyean pattern of recombinant human coagulation factors ii (rf-i~ and ix (rf-ix), derived from both transfected chinese hamster ovary (cho) cells and african green monkey (vero) cells, and produced at industrial scale were analyzed by binding to carbohydrate specific lectins and were compared with the glycan structure of human plasma-derived coagulation factors. human plasma-derived coagulation factors 1i (hpf-i1) and ix (hpf-ix) exhibited complex-type glycan structures with carbohydrate chains capped with c~(2-6)sialic acid. terminal galactose-b(1-4)-n-acetylglucosamine units were detected in hpf-ix. both cho cell-derived rf-]i and rf-ix exhibited complot-type glycosylafion and contained ~2-3)-sialio acid in addition to terminal galactose-b(1-4)-n-acetylglucosamine. vero cell-derived if-ix exhibited a complex-type glycan structure sinailar to that of cho cell derived rf-ix. in contrast, rf-li produced by vero cells exhibited a glycan microheterogeneity composed of hybrid-type glycosylation containing "highmarmose" structures and complex-type glycosylation containing et(2-3)-sialie acid. galaetose-b(1-4)-n-acetylglucosamine structures and a low concentration of ct(2-6)-sialic acid were detected in both micro-heterogeneity fractions of veto cell-derived rf-li. although different in the'tr carbohydrate structures, coagulation factors ii and ix obtained recombinantly from both transformed cho-cells and vero-cells exhibited coagulation activities comparable to the plasma-derived proteins. exposure of tissue factor (tf) to flowing blood, as a consequence of vascular injury, leads to tf/f.viia-initiated coagulation which may play a key role in triggering intravascular thrombosis. in order to evaluate the potential advantages of tf pathway blockade over existing therapy, the antithrombotic and the anticoagulant .effects of a monoclonal anti-rabbit tf antibody (ap-1) and heparin were compared in a rabbit arterial thrombosis model. thrombosis was induced by a local injury to the carotid artery and the recurrence of thrombus formation was followed by monitoring the cyclic flow variations (cfv) of the arterial blood flow. rabbits received intravenously either ap-1, heparin or vehicle, and cfv were monitored on line for 120 min. activated partial thromboplastin time (aptt), and activated clotting time (act) were measured at the end of the experiment control ap-i heparin <lmg/kg* 0.2mg/kg/hr 0.8mg/kg/hr intraabdominallretroperitoneal, cns) on a named-patient basis. later also mild/moderate bleeds, predominantly joint bleeds were included.in 80-90% of the bleeds novoseven was found to be hemostatically efficacious. also in major surgery (hip-and knee-replacement including a bilateral total condylar arthroplasty, herniotomy) hemostatic efficacy has been found to be 94% since rfvlla is proteotytically active only in complex with tf the risk for systemic activation of the coagulation system should be minimal. also very few side-effects have been seen.doses between 75-120 tjg/kg have been used, which initially have to be given at 2 hours intervals. to achieve satisfactory hemostasis both an initial dose high enough to induce immediate hemostasis and an appropriate dose interval ensuring a plasma level of fvli:c well above the hemostatic lower level for a period of time, the length of which is dependent on the type of bleeding, are important. accordingly, the highel the initial dose the more robust with regard to dose interval will the treatment be. acute myocardial ischemia is usually triggered by coronary thrombosis. heparin (hep), along with aspirin, is commonly used for the treatment of unstable angina (ua) and myocardial infarction (mi). hirudin (hiq is a potent antitbrombin agent which may offer therapeutic advantages over standard hep for the treatment ua and mi. the oasis phase i pilot study conducted in 24 canadian centres, has evaluated the safety, feasibility and efficacy of hir (hbw 023, behringwerke ag, marburg) compared to hep in 601 patients with mi or suspected non q-wave mi. patients (95 % received aspirin) were randomized to a 72 hr infusion of either hep (5000 u bolus followed by 1200 u/hr; n=253), or low dose hir (0.2 mg/kg bolus then 0.10 mg/kg/hr; n=175) or medium dose lair (0.4 ms/ks bolus then 0.15 mg/kg/hr; n=173). at seven days of follow-up the incidence of major bleeds was 0.4 % in the heparin group, 0.6 % low dose hit, 1.2 % medium dose lair (p=ns), minor bleeds were 10.3 %, 17.1% and 19.1% respectively (p---0.008 hep vs med dose); there were no hemorrhagic strokes. the incidence of the primary outcome of cardiovascular death, new mi and severe recurrent ischemia (refractory or severe angina) was 17.0 %, 15.4 % and 8.7 % respectively (p---0.014 hep vs reed dose). there was also a trend towards lower rates of coronary artery bypass surgery in reed dose hir compared to hep. in a 200 patient substudy, levels of thrombin antithrombin complex, fibfinopeptide a and d-dimer were all suppressed to a greater extent by mr compared to hep. these results indicate that hirudin may be a more effective agent for the treatment of acute coronary syndromes, but more information on safety and efficacy is needed from larger randomized trials. full clinical and coagulation resuks will be presented. hat is caused by an immunologic response to heparin. affected patients need effective parenteral anticoagulation. we treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory confirmed hat (hipa-test) with r-hirudin (behringwcrke ag, marburg). treatment regimens were: ai acute hat: 0.4 mg/kg b.w., continuous infusion 0a5 mg/kg b.w/h (51 patients); a2 acute hat with thrombolysis: bolus 0.2 mg/kg b.w., continuous infusion 0.1 mg/kg b.w./h (5 patients); b: proph, treatment: continuous infusion 0. i mg/kg b.w.pa (18 patients); c: during cardio-pulmunary bypass. primary objective was: increase in platelet counts or maintenance of normal baseline values during effective anticoagulation. secondary objectives were the incidence of new thromboembolic complications (tecs), major hemorrhage, limb amputations and deaths. results were compared with a historical control of 120 ix-at patients, all confirmed by the hipa test, but treated before rhirudin was available (danaparoid, marcumar, no anticoagulation). primary efficacy analysis: respunders ai: 74%o, a2: 60%, b: 44%, c: 50%, total: 65%. all adverse events beside bleeding complications had been judged to be caused by the underlying disease and not directly related to r-hirndin. proportion of patients with event in the obse~wation period; death: a1 5.9%, a2 0%, b 16.774, c 0%, total: 7.3%, during rhirudin treatment: 1.2%; new tec: ai 5.9%, a2 0%, b 27.8%, c 0%, total 9.8%, during rhirudin: 2.4%; limb amputation: ai 3.9%, a2 0%, b 5.6°/0, c 0%, total: 3.7%, during rbirudin: 3.7% eaapoiat we conclude, that r-hirndin is an effective anticoagulant in hat typa ii patients allowing rapid re, coved" of platelet counts. compared to a historical control the r-hirudin group had a strongly reduced incidence of combined endpuint of mortality, limb amputation and new tecs. major bleedings occurred slightly more frequent than in the historical control. however, the dosage used in regimen b might have been too low, as flds group revealed an effective anticoagulatiun and increase in platelet counts in only 43% and concomitantly had the higl)est incidence of new tecs. key: cord-282604-xp71rkxc authors: nikolaev, en; indeykina, mi; brzhozovskiy, ag; bugrova, ae; kononikhin, as; starodubtseva, nl; petrotchenko, ev; kovalev, g; borchers, ch; sukhikh, gt title: mass spectrometric detection of sars-cov-2 virus in scrapings of the epithelium of the nasopharynx of infected patients via nucleocapsid n protein date: 2020-05-25 journal: biorxiv doi: 10.1101/2020.05.24.113043 sha: doc_id: 282604 cord_uid: xp71rkxc detection of viral rna by pcr is currently the main diagnostic tool for covid-19 [1]. the pcr-based test, however, shows limited sensitivity, especially at early and late stages of the disease development [2,3], and is relatively time consuming. fast and reliable complementary methods for detecting the viral infection would be of help in the current pandemia conditions. mass-spectrometry is one of such possibilities. we have developed a mass-spectrometry based method for the detection of the sars cov-2 virus in nasopharynx epithelial swabs, based on the detection of the viral nucleocapsid n protein. the n protein of the sars-cov-2 virus, the most abundant protein in the virion, is the best candidate for mass-spectrometric detection of the infection, and ms-based detection of several peptides from the sars-coov-2 nucleoprotein has been reported earlier by the sinz group [4]. our approach shows confident identification of the n protein in patient samples even with the lowest viral loads and a much simpler preparation procedure. our main protocol consists of virus inactivation by heating and adding of isopropanol, and tryptic digestion of the proteins sedimented from the swabs followed by ms analysis. a set of unique peptides, produced as a result of proteolysis of the nucleocapsid phosphoprotein of sars-cov-2, is detected. the obtained results can further be used to create fast parallel mass-spectrometric approaches for the detection of the virus in the nasopharyngeal mucosa, saliva, sputum and other physiological fluids. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the causative agent of coronavirus disease 2019 (covid-19). the virus unit consists of a single-stranded rna with nucleoproteins enclosed within a capsid containing matrix proteins. the genome of the sars-cov-2 virus has been sequenced [5] , and the polymerase chain reaction (pcr) method is currently the main diagnostics approach for covid-19 diagnostics. however, it has shown limited accuracy and sensitivity, giving a high frequency of false positive and false negative results [1] [2] [3] , and is relatively time consuming. the detection of viral proteins in body fluids by mass-spectrometry based methods could serve as a complementary diagnostic tool. in addition, alternative testing assays would allow us to better understand the biological activity of the virus and to suggest potential drug targets for patient treatment. sars-cov-2 proteins can be grouped into two major classes -structural and non-structural proteins. nonstructural proteins are encoded by the virus, but are present only in the infected host cells and include the various enzymes and transcription factors necessary for virus replication. these proteins are not incorporated into the virion and are not as highly expressed, thus are less likely to be detected. the four structural proteins incorporated in the virion particle of sars-cov-2 virus are known as the s (spike), e (envelope), m (membrane), and n (nucleocapsid) proteins. the n protein encapsulates and protects the rna genome, and the s, e, and m proteins together create the viral envelope. from the data previously obtained for sars-cov [6] , which has a structure similar to sars-cov-2 structure, it can be concluded that the number of copies of e, m, and n proteins is much larger than that of s, but the e and m are relatively short and tightly membrane-bound proteins, what makes them difficult to extract, detect, and identify. thus, the main target for mass-spectrometry based detection of sars-cov-2 is the n protein. the possibility of developing such methods using gargle solution samples of covid-19 patients have been reported [4] . we have performed a pilot study on nasopharynx epithelial swabs already collected from patients with codiv-19 for rt-qpcr and showed confident identification of the n protein of the sars cov-2 virus by mass-spectrometry with the use of a very basic sample preparation procedure. also results on the unique easily detectable peptides characteristic of the infection can further be used as targets for creating highly specific and sensitive prm based detection methods or fast parallel mass-spectrometric approaches based on immunoprecipitation and maldi analysis (imaldi), allowing very fast testing for the presence of the virus in nasopharyngeal mucosa, saliva, sputum and other physiological fluids. all procedures for collection, transport, and preparation of the samples were carried out according to the restrictions and protocols of sr 1.3.3118-13 «safety procedures for work with microorganisms of the i -ii groups of pathogenicity (hazard)». swabs of the mucosa of the lower part of the nasopharynx and posterior wall of the oropharynx were used for the study. the sample was collected via a sterile velor swab with a plastic applicator. the swab was introduced along the outer wall of the nose to a depth of 2-3 cm to the lower shell, and after performing a rotational movement, was removed along the outer wall of the nose. after obtaining the material, the swab (up to the place of breakage) was placed into a sterile disposable tube with transport medium, and the end of the probe was broken off. oropharyngeal swab samples were taken with a dry sterile viscose swab by rotational movements along the surface of the tonsils, palatine arches and posterior wall of the oropharynx. in order to increase the viral concentration both the nasopharyngeal and oropharyngeal swabs were placed into a single tube, which was then sealed and marked. the samples were collected from 5 patients with covid-19 infection confirmed by rt-qpcr. the negative control samples were collected from 3 healthy individuals. before the virus has been inactivated and the outside of the tubes has been disinfected, all work must be carried out in accordance with the rules of biological safety level 3 [7] . 1. transfer 0.25 ml of the specimen to a clean polypropylene 1.5-2.0 ml eppendorf tube. 2. inactivate by heating at 65°c for 30 minutes using a water bath or a thermostated block heater) [8]*. 3. after incubation, add 0.75 ml of 100% isopropanol to obtain a 75% solution and allow the sample to sit for 10 minutes at room temperature [9] . 4. treat the outer surfaces of the tubes with 70% isopropanol or 1% sodium hypochlorite: spill from a jet wash and let stand for 15 minutes without getting wet. after this procedure, the virus can be considered deactivated, and the surface of the tube safe to handle. note: * -it is important to note, that during heat treatment, all parts of the tube are heated, so that all of the virus, even on "inaccessible" surfaces, will be inactivated. protocol 1: «standard proteomics preparation procedure» inactivated samples were lyophilized and resuspended in 50mm ammonium bicarbonate buffer, containing 0.1% rapigest sf surfactant (waters). reduction was carried out by incubating for 30 minutes at 50°c in 20mm dtt, followed by alkylation with 50mm iodacetamide for 45 minutes at room temperature in the dark, and tryptic digestion for 4 hours at 37°c. the reaction was terminated by adding formic acid to a final concentration of 0.5%. inactivated samples were cooled down to -20°c for 2 hours and centrifuged at 20 000 x g for 20 minutes. the pellet was resuspended in 50mm ammonium bicarbonate buffer, containing 0.1% rapigest sf surfactant (waters) and subjected to tryptic digestion for 4 hours at 37°c. the reaction was terminated by adding formic acid to a final concentration of 0.5%. the tryptic peptides were analyzed in duplicate on a nano-hplc dionex ultimate3000 system (thermo fisher scientific, usa) coupled to a tims tof pro (bruker daltonics, usa) mass-spectrometer. the amount of sample loaded was 200 ng per injection. hplc separation (injection volume 2 µl) was carried out using a packed emitter column (c18, 25cm x 75µm 1.6µm) (ion optics, parkville, australia) [10] by gradient elution. mobile phase a was 0.1% formic acid in water; mobile phase b was 0.1% formic acid in acetonitrile. lc separation was achieved at a flow of 400 nl/min using a 40-min gradient from 4% to 90% of phase b. mass spectrometric measurements were carried out using the parallel accumulation serial fragmentation (pasef™) [11] acquisition method. the esi source settings were the following: 4500 v capillary voltage, 500 v end plate offset, 3.0 l/min of dry gas at temperature of 180oc. the measurements were carried out over the m/z range from 100 to 1700 th. the range of ion mobilities included values from 0.60-1.60 vs/cm2 (1/k0). the total cycle time was set at 1.16 sec and the number of pasef ms/ms scans was set to 10. for low sample amounts, the total cycle time was set to 1.88 sec. the obtained data was analyzed using peaks studio 8.5 and maxquant version 1.6.7.0 using the following parameters: parent mass error tolerance -20ppm; fragment mass error tolerance -0.03da. due to light denaturation conditions, the absence of reduction and alkylation steps in one of the sample preparation approaches and short hydrolysis time -up to 3 missed cleavages were allowed, but only peptides with both trypsin-specific ends were considered. oxidation of methionine and carbamidomethylation of cysteine residues were set as possible variable modifications and up to 3 variable modifications per peptide were allowed. the search was carried out using the swissprot sars-cov-2 database with the human one set as the contamination database. fdr thresholds for all stages were set to 0.01 (1%) or lower. approximately 1000-1500 proteins were identified in each sample, among which the p0dtc9|ncap_sars2 nucleoprotein of severe acute respiratory syndrome coronavirus 2 was registered ( figure 1 ). depending on the viral content in the samples, preparation protocol and processing software used 1-17 peptides of the n protein were reliably detected and identified in the covid-19 patient samples (tables 1 and 2) . the n protein, being the most abundant protein in the virion, is the best candidate for mass-spectrometric detection of the infection, and so its detection is expectable. also ms-based detection of several peptides from the sars-coov-2 nucleoprotein has been reported earlier by the sinz group [4] in the gargle solution samples of covid-19 patients. we have performed a pilot study on nasopharynx epithelial swabs already collected from patients with codiv-19 for rt-qpcr and showed confident identification of the n protein with the use of a very basic sample preparation procedure. more than that the express procedure allowed better detection of the n protein than the more thorough one, standardly used for proteomic analysis. this is probably due to the significantly lower amounts of peptides from the much more abundant host proteins, which require reduction, alkylation, deglycosylation and other preparative steps for good sequence coverage. also since an untargeted lc-ms/ms with data-dependent acquisition approach was used to identify as many proteins as possible, it is expected that the use of targeted approaches aimed at monitoring the presence of this exact protein will result in significantly lower detection limits. for example the use of prm approaches on triple-quadrupole mass-spectrometers basing on the peptides identified in this study may allow to go below the sensitivity of rt-qpcr, while application of immunoprecipitation methods with subsequent maldi analysis or even maldi detection of viral proteins directly on the immobilized antibodies (imaldi) will allow to shorten the processing times to as low as 1hour. the observation of over 1000 host proteins also suggests that this approach could be used for detecting and studying the changes caused by the viral infection in the proteome of host cells, as well as the response of the organism to these conditions. table 1 . peptides from the p0dtc9|ncap_sars2 nucleoprotein identified via peaks studio in different samples sample preparation protocol. samples 1-5 were collected from 5 patients with covid-19 (1-3 were prepared by protocol 1; 4-5 by protocol 2). samples 6-8 -negative control (healthy individuals). two lc ms/ms runs were performed for each sample. the numbers in the table correspond to spectral counts for each peptide. detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr. euro surveillance : bulletin europeen sur les maladies transmissibles katrin zwirglmaier, christian drosten & clemens wendtner, virological assessment of hospitalized patients with covid-2019 risk of reactivation or reinfection of novel coronavirus (covid-19) mass spectrometric identification of sars-cov-2 proteins from gargle solution samples of covid-19 patients a new coronavirus associated with human respiratory disease in china severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection who laboratory biosafety guidance for novel coronavirus (2019-ncov): interim recommendations evaluation of inactivation methods for severe acute respiratory syndrome coronavirus in noncellular blood products stability and inactivation of sars coronavirus simplified high-throughput methods for deep proteome analysis on the timstof pro online parallel accumulation-serial fragmentation (pasef) with a novel trapped ion mobility mass spectrometer key: cord-272260-88l9bq4i authors: han, l.y.; cai, c.z.; ji, z.l.; chen, y.z. title: prediction of functional class of novel viral proteins by a statistical learning method irrespective of sequence similarity date: 2005-01-05 journal: virology doi: 10.1016/j.virol.2004.10.020 sha: doc_id: 272260 cord_uid: 88l9bq4i the function of a substantial percentage of the putative protein-coding open reading frames (orfs) in viral genomes is unknown. as their sequence is not similar to that of proteins of known function, the function of these orfs cannot be assigned on the basis of sequence similarity. methods complement or in combination with sequence similarity-based approaches are being explored. the web-based software svmprot (http://jing.cz3.nus.edu.sg/cgi-bin/svmprot.cgi) to some extent assigns protein functional family irrespective of sequence similarity and has been found to be useful for studying distantly related proteins [cai, c.z., han, l.y., ji, z.l., chen, x., chen, y.z., 2003. svm-prot: web-based support vector machine software for functional classification of a protein from its primary sequence. nucleic acids res. 31(13): 3692–3697]. here 25 novel viral proteins are selected to test the capability of svmprot for functional family assignment of viral proteins whose function cannot be confidently predicted on by sequence similarity methods at present. these proteins are without a sequence homolog in the swissprot database, with its precise function provided in the literature, and not included in the training sets of svmprot. the predicted functional classes of 72% of these proteins match the literature-described function, which is compared to the overall accuracy of 87% for svmprot functional class assignment of 34 582 proteins. this suggests that svmprot to some extent is capable of functional class assignment irrespective of sequence similarity and it is potentially useful for facilitating functional study of novel viral proteins. the complete genomes of 1536 viruses have been sequenced (viral genomes at ncbi http://www.ncbi.nlm. nih.gov/genomes/static/vis.html). knowledge of these genomes has facilitated mechanistic study of viral infections and provided important clues for searching molecular targets of antiviral therapeutics (herniou et al., 2003; marra et al., 2003; miller et al., 2003) . the function of over 15% of the putative protein-coding open reading frames (orfs) in these viral genomes is unknown (herniou et al., 2003; marra et al., 2003; miller et al., 2003) . determination of the function of these unknown orfs is important for a more comprehensive understanding of the molecular mechanism of specific virus and for searching novel targets for antiviral drug development. the sequence of many of these unknown orfs has no significant similarity to proteins of known functions, and their functions are difficult to probe on the basis of sequence similarity. for instance, 50%, 100%, 20%, and 67% of the unknown orfs in the recently determined genomes of ferde-lance virus (makeyev and bamford, 2004) , grapevine fleck virus (sabanadzovic et al., 2001) , indian citrus ringspot virus (rustici et al., 2002) , and sars coronavirus (he et al., 2004) are without a homolog in swissprot database (boeckmann et al., 2003) based on blast search against all swissprot entries as of september 2004. this suggests that a significant percentage of new viral proteins are likely to have no known sequence homolog. it is thus desirable to explore alternative methods or combination of methods for providing useful hint about the function of unknown viral orfs. various alternative methods for probing protein function have been developed. these include evolutionary analysis (benner et al., 2000; eisen, 1998) , hidden markov models (fujiwara and asogawa, 2002) , structural consideration (di gennaro et al., 2001; teichmann et al., 2001) , protein/gene fusion (enright et al., 1999; marcotte et al., 1999) , proteinprotein interactions (bock and gough, 2001) , motifs (hodges and tsai, 2002) , family classification by sequence clustering (enright et al., 2002) , and functional family prediction by statistical learning methods (cai et al., 2003 han et al., 2004; jensen et al., 2002; karchin et al., 2002) . in the absence of clear sequence or structural similarities, the criteria for comparison of distantly related proteins become increasingly difficult to formulate (enright and ouzounis, 2000) . moreover, not all homologous proteins have analogous functions (benner et al., 2000) . the presence of shared domain within a group of proteins does not necessarily imply that these proteins perform the same function (henikoff et al., 1997) . therefore, careful evaluation is needed to determine which method or combination of methods is useful for facilitating functional study of novel proteins with no homology to proteins of known function. the web-based software svmprot (http://jing.cz3.nus. edu.sg/cgi-bin/svmprot.cgi) to some extent has shown some potential for assigning the functional class of distantly related proteins and homologous proteins of different functions as well as homologous proteins (cai et al., 2003 . it classifies proteins into functional classes defined from activities or physicochemical properties rather than sequence similarity (bock and gough, 2001; cai et al., 2003 cai et al., , 2004 han et al., 2004; karchin et al., 2002) . in developing svmprot, proteins in a training set, represented by their sequence-derived physicochemical properties, are projected onto a hyperspace where proteins in a class are separated from those outside the class by a hyperplane. by projecting a new sequence onto the same hyperspace, svmprot determines whether the corresponding protein is a member of that class based on its location with respect to the hyperplane. the accuracy of svmprot depends on the diversity of the protein samples, the quality of the representation of protein properties, and the efficiency of the statistical learning algorithm. to some extent, no sequence similarity is required per se. thus svmprot may be potentially explored for facilitating functional assignment of proteins whose function cannot be assigned on the basis of sequence similarity. this work evaluates the usefulness of svmprot for predicting the functional class of viral orfs of unknown function. it is assessed by using novel viral proteins that are without a single homolog in the swissprot database (boeckmann et al., 2003) , with their precise function described in the literature, and are not included in the training sets of svmprot. these proteins are collected from an unbiased search of medline (wheeler et al., 2003) and swissprot database (boeckmann et al., 2003) . the svmprot predicted functional classes of these proteins are compared with the function described in the literature and databases to evaluate to what extent svmprot are useful for functional class assignment of novel viral proteins. the prediction accuracy for assignment of these novel proteins is compared with the overall accuracy of the svmprot assignment of a large number of proteins to examine the level of sequence similarity independence of svmprot classification. table 1 gives svmprot ascribed functional classes for each of the 25 novel viral proteins together with literaturedescribed function. more than one class may be characterized by svmprot and the probability of correct prediction for each class is also given in table 1 . there are 18 proteins with the top hit of the svmprot assigned functional class matching the literature-described function, representing 72% of the novel viral proteins studied in this work. these proteins are mota protein of bacteriophage t4 (gerber and hinton, 1996) , outer capsid protein vp4 of bovine rotavirus (serotype 10/strain b223) (hardy et al., 1992) , adometase of bacteriophage t3 (hughes et al., 1987) , r.cviji of chlorella virus il3a (skowron et al., 1995) , exonuclease of bacteriophage lambda (sanger et al., 1982) , r.cviaii of paramecium bursaria chlorella virus 1 (zhang et al., 1992) , orf13 of haemophilus phage hp1 (esposito et al., 1996) , protein kinase of enterobacteria phage t7 (dunn and studier, 1983) , dna-directed rna polymerase of african swine fever virus (strain ba71v) (yanez et al., 1995) , agt (miller et al., 2003) , bgt (miller et al., 2003; tomaschewski et al., 1985) , dnk (broida and abelson, 1985) , endonuclease ii (sjoberg et al., 1986) , endonuclease v (valerie et al., 1984) , gp61.9 (valerie et al., 1986) , irf protein (chu et al., 1986) , and i-tevii (tomaschewski and ruger, 1987) of enterobacteria phage t4. mota protein of bacteriophage t4 has been found to be a transcription activator that binds to dna (gerber and hinton, 1996) and the far-c-terminal region of the sigma70 subunit of escherichia coli rna polymerase (pande et al., 2002) . the top hit of svmprot predicted functional class for this protein is the dna-binding, which matches with literature-described functions. bovine rotavirus is a double-stranded rna virus that is naked. thus, the outer capsid protein vp4 of bovine rotavirus (serotype 10/strain b223) is located at the viral surface acting as part of the viral coat (hardy et al., 1992) . this protein is predicted by svmprot as a coat protein that is consistent with literature-described function. the other 14 proteins are enzymes, and these are all correctly assigned by svmprot to the respective enzyme ec class. because these proteins have no homolog of known function in the swissprot entries of swissprot database based on psi-blast search, our study suggests that svmprot has certain level of capability for providing useful hint about the functional class of novel proteins with no or low homology to known proteins, and this capability is not based on sequence similarity or clustering. the overall accuracy of 72% for the assignment of the novel viral proteins is smaller, but not too far away, than that of 87% for svmprot functional class assignment of 34 582 proteins. this indicates certain level of the sequence-similarityindependent nature of svm protein classification. several factors may affect the accuracy of svmprot for functional characterization of novel plant proteins. one is the diversity of protein samples used for training svmprot. it is likely that not all possible types of proteins, particularly those of distantly related members, are adequately represented in some protein classes. this can be improved along with the availability of more protein data. not all distantly related proteins of the same function have similar structural and chemical features. there are cases in which different functional groups, unconserved with respect to position in the primary sequence, mediate the same mechanistic role, due to the flexibility at the active site (todd et al., 2002) . this plasticity is unlikely to be sufficiently described by the physicochemical descriptors currently used in svmprot. therefore, svmprot in the present form is not expected to be capable of classification of these types of distantly related enzymes. some of the svmprot functional classes are at the level of families and superfamilies that may include a broad spectrum of proteins. it has been shown that svm works not as well as hmm for distinguishing proteins in a superfamily, but may be more accurate with subfamily discrimination (karchin et al., 2002) . thus, the use of some large families and superfamilies as the basis for classification may affect the prediction accuracy of svmprot to some extent. svmprot prediction may be further improved by using protein subfamilies as the basis of classification, more comprehensive set of protein samples, and more refined protein descriptors. svmprot optimization procedure and feature vector selection algorithm may also be improved by adding additional constraints, and by incorporating independent component analysis and kernel pca in the preprocessing steps. svmprot shows certain level of capability for predicting functional class of a number of novel viral proteins. this suggests that svmprot is potentially useful to a certain extent for providing useful hint about the function of distantly related proteins in viruses as well as in other organisms. further improvements in protein functional family coverage, sample collections, and svm algorithm may enable the development of svmprot into a practical tool for facilitating functional study of unknown orfs in virus genomes and other genomes. the key words, bnovel protein virusq or bnovel viral proteinq, are used to search the medline (wheeler et al., 2003) and the swissprot database (boeckmann et al., 2003) for finding viral proteins that are both described as novel and with their precise function provided. as the search of the medline is confined to the abstracts, those proteins whose function is not explicitly hinted in an abstract are not selected. thus, the selected proteins likely account for a portion of the known novel viral proteins with available functional information. psi _ blast (altschul et al., 1997) sequence analysis is subsequently conducted on each of these novel viral proteins against all swissprot entries in the swissprot protein database (boeckmann et al., 2003) so that those with at least one sequence homolog of known function (including that of the same protein in different species) are removed. the commonly used criterion for homologs, the similarity score e-value b the inclusion threshold value of 0.005 (altschul et al., 1997) , is used in this work. finally, those proteins that are in the training sets of svmprot are removed. a total of 25 novel viral proteins are identified in this process, which together with their protein accession number and literature-described functional indications and related references are given in table 1 . svmprot is based on a statistical learning method support vector machines (svm) (burges, 1998) . in addition to the prediction of protein functional class (cai et al., 2003 han et al., 2004; karchin et al., 2002) , svm has also been used for a variety of protein classification problems including fold recognition (ding and dubchak, 2001) , analysis of solvent accessibility (yuan et al., 2002) , prediction of secondary structures (hua and sun, 2001) , and protein-protein interactions (bock and gough, 2001) . as a method that uses sequence-derived physicochemical properties of proteins as the basis for classification, svm may be particularly useful for functional classification of distantly related proteins and homologous proteins of different functions (cai et al., 2003 . there are 75 protein functional classes currently covered by svmprot. these include 46 enzyme families, 13 channel/transporter families, 4 rna-binding protein families, dna-binding proteins, g-protein-coupled receptors, nuclear receptors, tyrosine receptor kinases, cell adhesion proteins, coat proteins, envelope proteins, outer membrane human herpesvirus 6 chemokine like (luttichau et al., 2003) no function predicted nm (continued on next page) proteins, structural proteins, and growth factors. two broadly defined families of antigens and transmembrane proteins are also included. the majority of known types of viral proteins are included in these classes. representative proteins of a particular functional class (positive samples) and those do not belong to this class (negative samples) are needed to train a svmprot classifier for this class. the positive samples of a class are constructed by using all of the known distinct protein members in that class. because of the enormous number of proteins, the size of negative samples needs to be restricted to a manageable level by using a minimum set of representative proteins. one way for choosing representative proteins is to select one or a few proteins from each protein domain family. the negative samples of a class are selected from seed proteins of the 7316 curated protein families (domain-based) in the pfam database excluding those families that have at least one member belong to the functional class. pfam families are constructed on the basis of sequence similarity. the purpose of using pfam proteins is to ensure that the negative samples are evenly distributed in the protein space. sequence similarity is not required for selecting positive samples. in this sense, svmprot is to some extent independent of sequence similarity. the svmprot training system for each family is optimized and tested by using separate testing sets of both positive and negative samples. while possible, all the remaining distinct proteins in each functional family (not in the training set of that family) are used as positive samples and all the remaining representative seed proteins in pfam curated families are used to construct negative samples in a testing set. the performance of svmprot classification is further evaluated by using independent sets of both positive and negative samples. there is no duplicate protein in each training, testing, or independent evaluation set. data set construction can be demonstrated by an illustrative example of viral coat proteins. the key word bvirus coat proteinq is used to search the swissprot, which finds 3012 entries. these entries are checked to remove noncoat proteins, redundant entries, and putative proteins, which gives 848 positive samples. these positive samples cover 140 pfam families; thus, 14 758 seed proteins of the remaining 7176 pfam families are used as the negative samples. these positive and negative samples are further divided into 346 and 1474 training, 305 and 8370 testing, and 197 and 4914 independent evaluation sets using the procedure described above. not all of the svmprot classes are at the same hierarchical level. these classes are mixtures of subfamilies, families, and superfamilies. some classes, such as antigen, need to be more clearly defined into specific subclasses. while it is desirable to define all of the classes at the same level, this is not yet possible because of insufficient data for the subhierarchies of some families and superfamilies. effort is being made to collect sufficient data so that svmprot classification systems can be constructed on the basis of a more evenly distributed family structures. transferase (wilfred et al., 2002) no function predicted nm splt13 (np _ 258405) spltmnpv virus a noval envelope protein (yin et al., 2003) no function predicted nm trl10 (aal27474) human cytomegalovirus (hcmv) structural envelop glycoprotein (spaderna et al., 2002) transmembrane ( nonetheless, prediction on the basis of the current structures provides useful hint about the function of a protein. svmprot is trained for protein classification in the following manner. first, every protein sequence is represented by specific feature vector assembled from encoded representations of tabulated residue properties including amino acid composition, hydrophobicity, normalized van der waals volume, polarity, polarizability, charge, surface tension, secondary structure, and solvent accessibility for each residue in the sequence (cai et al., 2003) . the feature vectors of the positive and negative samples are used to train a svmprot classifier. the trained svmprot classifier can then be used to classify a protein into either the positive group (protein is predicted to be a member of the class) or the negative group (protein is predicted to not belong to the class). the theory of svm has been described in the literature (burges, 1998) . thus, only a brief description is given here. svm is based on the structural risk minimization (srm) principle from statistical learning theory (burges, 1998) . in linearly separable cases, svm constructs a hyperplane that separates two different groups of feature vectors with a maximum margin. a feature vector is represented by x i , with physicochemical descriptors of a protein as its components. the hyperplane is constructed by finding another vector w and a parameter b that minimizes twt 2 and satisfies the following conditions: where y i is the group index, w is a vector normal to the hyperplane, |b| / twt is the perpendicular distance from the hyperplane to the origin and twt 2 is the euclidean norm of w. after the determination of w and b, a given vector x can be classified by: in nonlinearly separable cases, svm maps the input variable into a high dimensional feature space using a kernel function k(x i , x j ). an example of a kernel function is the gaussian kernel that has been extensively used in different protein classification studies (bock and gough, 2001; burges, 1998; cai et al., 2002; ding and dubchak, 2001; hua and sun, 2001; karchin et al., 2002; yuan et al., 2002) : linear support vector machine is applied to this feature space and then the decision function is given by: where the coefficients a i 0 and b are determined by maximizing the following langrangian expression: under conditions: a i z 0 and x l ià1 a i y i ¼ 0 a positive or negative value from eq. (3) or eq. (5) indicates that the vector x belongs to the positive or negative group, respectively. to further reduce the complexity of parameter selection, hard margin svm with threshold instead of soft margin svm with threshold is used in svmprot. scoring of svm classification of proteins has been estimated by a reliability index and its usefulness has been demonstrated by statistical analysis (cai et al., 2003; hua and sun, 2001) . a slightly modified reliability score, r value, is used in svmprot: where d is the distance between the position of the vector of a classified protein and the optimal separating hyperplane in the hyperspace, d n 0 indicates the sample belongs to the positive group and d b 0 the negative group. there is a statistical correlation between r value and expected classification accuracy (probability of correct classification) (cai et al., 2003; hua and sun, 2001) . thus, another quantity, p value, is introduced to indicate the expected classification accuracy. p value is 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machines characterization of chlorella virus pbcv-1 cviaii restriction and modification system key: cord-268416-8hw80qx8 authors: grunewald, matthew e.; fehr, anthony r.; athmer, jeremiah; perlman, stanley title: the coronavirus nucleocapsid protein is adp-ribosylated date: 2018-04-01 journal: virology doi: 10.1016/j.virol.2017.11.020 sha: doc_id: 268416 cord_uid: 8hw80qx8 adp-ribosylation is a common post-translational modification, although how it modulates rna virus infection is not well understood. while screening for adp-ribosylated proteins during coronavirus (cov) infection, we detected a ~55 kda adp-ribosylated protein in mouse hepatitis virus (mhv)-infected cells and in virions, which we identified as the viral nucleocapsid (n) protein. the n proteins of porcine epidemic diarrhea virus (pedv), severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov were also adp-ribosylated. adp-ribosylation of n protein was also observed in cells exogenously expressing n protein by transduction using venezuelan equine encephalitis virus replicon particles (vrps). however, plasmid-derived n protein was not adp-ribosylated following transient transfection but was adp-ribosylated after mhv infection, indicating that this modification requires virus infection. in conclusion, we have identified a novel post-translation modification of the cov n protein that may play a regulatory role for this important structural protein. adp-ribosylation is the covalent attachment of adp-ribose (adpr) moieties to a protein, resulting in either mono-adpr (mar) or poly-adpr (par). adp-ribosylation is catalyzed by poly-adpr polymerases (parps), also known as adp-ribosyltransferases (artds). the parp family consists of 17 proteins in humans and 16 in mice, which utilize nad as the adpr donor [reviewed in (bock and chang, 2016) ]. removal of adpr from proteins (de-adp-ribosylation) is catalyzed by different cellular proteins including par glycohydrolase (parg) and macrodomain proteins (bernardi et al., 1997; jankevicius et al., 2013; rosenthal et al., 2013; sharifi et al., 2013) . detection of adp-ribosylated proteins on a proteomic level is difficult due to the reactive nature and short half-life of the modification in cells (cervantes-laurean et al., 1997; wielckens et al., 1982) . despite this, studies of individual parps and adp-ribosylated proteins have elucidated several physiological roles for adp-ribosylation, including dna damage and repair, regulation of rna transcription, cellular stress response, inflammation, differentiation, and apoptosis (bock and chang, 2016) . parps are well-established to have both proviral and antiviral properties. parp1 has been shown to facilitate epstein-barr virus replication and latency, simian virus 40 induction of cellular necrosis, and hiv integration (gordon-shaag et al., 2003; ha et al., 2001; lupey-green et al., 2017; tempera et al., 2010) . tiparp has been shown to inhibit interferon (ifn) production by adp-ribosylation of tbk-1, leading to enhanced replication of several viruses (yamada et al., 2016) . finally, adp-ribosylation of the adenovirus core protein has been implicated in aiding viral replication and modulating stability of viral chromatin-like structures (dery et al., 1986) . other data suggest that parps can also be antiviral. many parps are expressed following ifn stimulation and several parps show evidence of rapid evolution, suggesting a microbial "arms race" between parps and cellular pathogens (atasheva et al., 2012; daugherty et al., 2014; macdonald et al., 2007) . for example, parp7, parp10, and parp12 restrict venezuelan equine encephalitis virus (veev) replication and can block cellular translation when overexpressed (atasheva et al., 2012 (atasheva et al., , 2014 . one notable parp, the zinc-finger antiviral protein (zap) or parp13, has been demonstrated to inhibit replication of several different viruses, potentially by binding to viral rna and directing it to be degraded by the rna exosome (bick et al., 2003; gao et al., 2002; guo et al., 2004 guo et al., , 2007 liu et al., 2015; muller et al., 2007; zhu et al., 2011) . parp13 is enzymatically inactive, and thus its antiviral activity is independent of adp-ribosylation. in addition, liu et al. have described a novel mechanism in which an unknown parp adp-ribosylates two subunits of the influenza rna polymerase, allowing subsequent binding to zap and degradation of these subunits by the proteasome (liu et al., 2015) . three different virus families, hepeviridae, togaviridae, and coronaviridae, encode for a viral macrodomain, which has been shown to de-adp-ribosylate proteins in vitro (li et al., 2016; rosenthal et al., 2013) . these macrodomains have been proposed to counter the antiviral effects of adp-ribosylation during infection. our previous work has focused on the coronavirus (covs) macrodomain. covs are large, positive-sense, single-stranded rna viruses which include human pathogens such as the severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov as well as important veterinary pathogens such as bovine cov and porcine epidemic disease virus (pedv). all covs encode a macrodomain within non-structural protein 3 (nsp3) that can remove both mar and par from proteins (li et al., 2016) . covs lacking this enzymatic activity generally replicate normally in vitro but are highly attenuated in vivo and elicit an enhanced innate immune response (eriksson et al., 2008; fehr et al., 2015 fehr et al., , 2016 kuri et al., 2011) . to identify potential targets of the cov macrodomain, we analyzed infected cells for changes in adp-ribosylation patterns utilizing antibodies specific for adpr. we focused on cells infected with a murine cov, mouse hepatitis virus (mhv). mhv causes acute and chronic encephalomyelitis, hepatitis and gastroenteritis (bailey et al., 1949) . surprisingly, we found that the cov nucleocapsid (n) protein was adpribosylated in cells during infection with mhv as well as several other covs. 2.1. cell culture, plasmids and reagents delayed brain tumor (dbt) cells, 17cl-1 cells, vero cells, and hela cells expressing the mhv receptor carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1) (hela-mhvr) were grown in dulbecco's modified eagle medium with 10% fetal bovine serum as previously described (zhou and perlman, 2007) . codon-optimized mhv-a59 n protein was synthesized and cloned directly into pcdna3 (genscript). a tagged construct was synthesized by inserting a 3x-flag sequence to the c terminus of the n protein using overlapping primers and recombination by in-fusion (clontech). control plasmid pcdna3-gfp was described previously (fehr et al., 2016) . recombinant mouse hepatitis virus (mhv) strains a59 (yount et al., 2002) and jhmv (wild-type and n1347a) (fehr et al., 2015) were propagated on 17cl-1 cells, and titers were determined on hela-mhvr cells. sars-cov (ma15) was propagated and titered on vero e6 cells, and mers-cov (emc12) and pedv (isu13-19338e, a gift from dr. kyoung-jin yoon, iowa state university) were propagated on vero-81 cells. for virus infections, 17cl-1, dbt, calu-3, vero e6, or vero 81 cells were infected with virus at the indicated multiplicity of infection (moi) and collected at the indicated hours post-infection (hpi). all work with sars-cov or mers-cov infectious virus was performed in a biosafety level 3 laboratory according to the guidelines set forth by the university of iowa. dbt cells were infected with mhv-a59 at an moi of 0.5 pfu/cell, and supernatant was collected and filtered at 12 hpi. the filtrate was subjected to ultracentrifugation at 27,000 rpm for 4 h over a 30% sucrose cushion as described previously. pellets were resuspended in 100 mm nacl and 10 mm tris-cl (ph 7.2) and treated with or without proteinase k (new england biolabs) in the presence or absence of sds. the reaction was stopped by incubation at 65°c for 10 min. cells were transfected with polyjet in vitro transfection reagent (signagen labs) as per the manufacturer's instructions. 24 h after transfection, cells were either treated with or without 1000 u/ml of ifn-β (pbl) for 24 h or were infected with mhv-a59 at an moi of 1 pfu/cell for 12 h before collection. veev replicon particles (vrps) encoding either gfp or mers nucleocapsid protein were created and titered as previously reported (zhao et al., 2014 . the vrps were transduced into vero 81 cells at indicated mois and collected at 24 h post-transduction. sample buffer containing sds, β-mercaptoethanol, protease/phosphatase inhibitor cocktails (roche), pmsf, parp inhibitor 3-aminobenzamide (3-ab, tocris bioscience), parg inhibitor adenosine 5ʹ-diphosphate (hydroxymethyl)pyrrolidinediol (adp-hpd, calbiochem) and universal nuclease (thermofisher scientific) was used to collect cell lysates. proteins were resolved on an sds polyacrylamide gel and transferred to a polyvinylidene difluoride (pvdf) membrane. following binding with a primary antibody, blots were then visualized by using a peroxidase-conjugated secondary antibody (thermo fisher scientific) detected with a chemiluminescent substrate (thermo fisher scientific) or by using an infrared (ir) dye-conjugated secondary antibody detected with a li-cor odyssey imager (li-cor, lincoln, ne) . ir secondary antibodies of different wavelengths were used to obtain different signals for antibody bound proteins. images of α-adpror α-nstained immunoblots were merged using image studio software. primary antibodies used for immunoblotting and immunoprecipitation included polyclonal (pab) α-mhv rabbit serum (perlman et al., 1987) , monoclonal (mab) α-mhv n (collins et al., 1982) (mab 5b188.2, a kind gift from dr. m. buchmeier, university of california, irvine), pab α-sars-cov n (novus biologicals), pab α-nsp3 (gift from mark denison, vanderbilt university), mab α-pedv n (gift from dr. kyoung-jin yoon, iowa state university), pab α-mers-cov n , mouse mab α-adpr (10 h, millipore sigma), rabbit pab α-adpr (trevigen), chicken pab α-adpr (tulip biolabs inc.), α-flag (sigma), α-gapdh (poly6314, biolegend), and α-actin (ac15; abcam, inc.) antibodies. secondary antibodies used included horseradish peroxidase-conjugated α-rabbit or α-mouse (sigma #a0545/ a0168) antibodies or ir-conjugated α-rabbit, α-mouse, or α-chicken (li-cor, #926-68071/926-32210/925-32218) antibodies. dbt cells infected with mhv-a59 at an moi of 1 pfu/cell were collected at 12 hpi and pelleted by low-speed centrifugation. cell pellets were lysed with immunoprecipitation (ip) buffer (0.5% np-40, 300 mm nacl, 5% glycerol, and 50 mm tris ph 8.0) containing protease/phosphatase inhibitor cocktails, pmsf, parp inhibitor 3-ab, parg inhibitor adp-hpd, and a universal nuclease for 2 h at 4°c. nuclei were pelleted by centrifugation (16,000 g for 15 min at 4°c). one aliquot of cell lysate was saved as the input control and boiled in sds sample buffer described above. protein g magnetic beads were conjugated to α-adpr or α-n antibodies (described above) as per manufacturer's instructions (thermofisher scientific). protein g antibody-conjugated were mixed with cell lysates overnight at 4°c. beads were washed with pbs-tween before elution by boiling in sds sample buffer. to screen for changes in protein adp-ribosylation during cov infection, we infected dbt cells, an astrocytoma cell line, with the a59 strain of mhv. cells were collected throughout the infection, and cell lysates were immunoblotted with a mouse mab antibody to adpr (mab 10 h). the 10 h antibody has been described to bind preferentially to linear 20+-mers of par with no binding activity to dna, rna, or adenosine-monophosphate. however, more recent reports have demonstrated that mab 10 h also binds to auto-marylated proteins (eckei et al., 2017; goenka et al., 2007; kawamitsu et al., 1984; kleine et al., 2008) . while the adp-ribosylation status of most proteins did not change over the course of infection, we noted the appearance of a ~55 kda band at 8 h post-infection (hpi) which increased in abundance up to 12 hpi (fig. 1a) . immunoblotting with two other commercial α-adpr antibodies raised in two other species also detected a similar protein band (fig. 1a) . a negative control antibody (α-ha) did not bind to this protein, suggesting a specific interaction of the 55 kda protein with adpr antibodies. based on to the size and abundance of this protein, we hypothesized that it was the nucleocapsid (n) protein. staining with monoclonal α-n antibody produced a signal that completely overlapped the signal from the adpr antibody (fig. 1b, left) . to confirm that this overlap was consistent in other cell lines and mhv strains, we infected 17cl-1 cells (a fibroblast cell line) with mhv-a59 or dbt cells with mhv-jhm (jhmv), a neurotropic strain of mhv, and immunoblotted with α-mhv serum or α-adpr antibody. the results showed that in all cases the n protein completely overlapped with thẽ 55 kda adp-ribosylated protein, demonstrating that n protein adpribosylation is not mhv strain or cell type specific (fig. 1b, middle and right) . to confirm the identity of this protein as the n protein, we collected mhv-infected dbt cells at 12 hpi and immunoprecipitated proteins with either monoclonal α-adpr or α-n antibodies and immunoblotted with the reciprocal antibodies. as expected, this~55 kda protein could be stained with the α-adpr antibody after immunoprecipitation with α-n and stained with α-n after immunoprecipitation with α-adpr (fig. 1c) , confirming that the n protein is adp-ribosylated. previous reports have demonstrated that the nsp3 macrodomain removes both mono-and poly-adpr from auto-adp-ribosylated substrates in vitro (fehr et al., 2016; li et al., 2016) . because the n protein is known to associate with nsp3, we speculated that the nsp3 macrodomain may de-adp-ribosylate the n protein (hurst et al., 2013) . to test this, we infected dbt cells with mhv-a59 or mhv-jhm encoding either wild-type or a catalytically-deficient (jhmv-n1347a, a59-n1348a) nsp3 macrodomain and immunoblotted for n protein adpribosylation. if the macrodomain was indeed removing the adpr from the n protein, we would expect to see increased n protein adp-ribosylation in cells infected with mutant virus compared to wild-type infected cells. however, we found that the level of the adp-ribosylated n protein was the same in cells infected with either mhv strain (fig. 2) . these findings suggest that the nsp3 macrodomain does not affect n protein adp-ribosylation under these conditions. next, we tested whether n protein adp-ribosylation was conserved in cov genera and lineages other than mhv. we infected vero cells with pedv (⍺-cov), sars-cov (lineage b β-cov), or mers-cov (lineage c β-cov) and then collected the cells and analyzed whether the n proteins from these viruses were adp-ribosylated (fig. 3) . in all cases, adpr antibody staining overlapped with the n protein of each virus, lysates were collected at 12 and 18 hpi, respectively, and immunoblotted with indicated antibodies. (c) immunoprecipitation with α-adpr antibody confirms n protein is adp-ribosylated. dbt cells were mock infected (m) or infected with mhv-a59 at moi of 1 pfu/cell. at 12 hpi, cells were collected and the lysates were subjected to immunoprecipitation with mouse α-adpr or α-n (mab) bound to protein g beads. cell lysates and eluted proteins were analyzed by immunoblotting with indicated antibodies. asterisk indicates cellular adp-ribosylated protein that is not immunoprecipitated with α-n antibody. m.e. grunewald et al. virology 517 (2018) [62] [63] [64] [65] [66] [67] [68] demonstrating that this modification is conserved across multiple genera and lineages of covs. it has previously been shown that the n protein is phosphorylated and that some phosphorylated residues depend on whether the protein is intracellular or virion-associated (white et al., 2007) . specifically, mhv n protein at amino acid s197 is phosphorylated in infected cells, but this modification is absent on n protein in virions (wu et al., 2014) . to determine if n protein adp-ribosylation is maintained within the virion, we purified virions and analyzed whether n protein from the cov virion was adp-ribosylated by immunoblotting. to rule out the possibility of detecting of any residual n protein from non-virion sources, pelleted virions were treated with proteinase k with or without virion lysis by sds. both the n protein as well as the spike (s) protein were detectable with α-mhv serum in both cells and virions (fig. 4) . although the s protein was not completely eliminated upon proteinase k treatment, the abundance of the s protein was reduced, consistent with the protein being exposed on the surface of the virus. a co-sedimented nonspecific band of~70 kda was also degraded by proteinase k treatment. in contrast, n protein, located in the interior of the virion, was protected from proteinase k treatment unless sds was also added to lyse the viral envelope. importantly, immunoblotting with α-adpr antibody demonstrated that the n protein maintained the adp-ribose modification in virions. to determine if n protein expressed in the absence of cov infection could be adp-ribosylated in cell culture, we transduced vero cells with veev replicon particles (vrps) encoding the mers-cov n protein or control gfp at different mois . transduced cells were collected at 24 hpi, and immunoblotting of cell lysates showed that n protein expressed from an alphavirus replicon was adp-ribosylated ( fig. 5a) . because the vrp platform utilizes a virus infection to express exogenous proteins, we hypothesized that the adp-ribosylation of the n protein may require a virus infection. to examine this possibility, we transfected dbt cells with a plasmid encoding codon-optimized mhv-n protein or gfp and then tested whether the n protein was adp-ribosylated. because several parps are ifn-stimulated genes, we also treated cells with or without ifn-β (atasheva et al., 2012; macdonald et al., 2007) . importantly, we were unable to detect any adp-ribosylation of the exogenous transfected n protein (fig. 5b) . in contrast, a positive control of mhv-a59-infected cell lysate, normalized to total n protein, stained with α-adpr antibody. furthermore, ifn-β treatment did not rescue the modification, suggesting that other factors present during infection drove adp-ribosylation of n protein. to confirm that n protein requires an infection to be adp-ribosylated, we added a 3x-flag tag to our plasmid-expressed n (n-flag) protein. we then 4 . the n protein is adp-ribosylated within the mhv-a59 virion. supernatant from dbt cells infected with mhv-a59 at moi of 0.5 was collected at 12 h. whole virions were purified by filtration and ultracentrifugation with a 30% sucrose cushion. virions were then treated with proteinase k to degrade extramembranous protein or proteinase k in the presence of sds to degrade all viral proteins. proteinase k was then inactivated at 65°c, and proteins were blotted with indicated antibodies. the nucleocapsid (n), spike (s), and a nonspecific (ns) proteins are indicated with arrows. m.e. grunewald et al. virology 517 (2018) 62-68 transfected plasmid expressing gfp or n-flag into dbt cells and then mock infected or infected cells with mhv-a59. at 12 hpi, we collected cells and immunoblotted with α-adpr antibody. the n-flag protein was detectable with α-adpr antibody following infection with mhv-a59 but not in mock infected cells (fig. 5c ). this suggests that n protein is only adp-ribosylated within the context of virus infection. the n protein was initially identified as a major structural protein, binding directly to viral rna, providing stability to the bound rna, and self-oligomerizing into the virus nucleocapsid. n protein also binds to the viral membrane (m) protein to facilitate genome loading and viral assembly (narayanan et al., 2000) . it plays a prominent role in transcription and replication of the viral genome (hurst et al., 2010) . in fact, the addition of n protein from an exogenous plasmid is often utilized to initiate cellular infection from recombinant cov cdna (yount et al., 2002) . the n protein provides additional accessory functions, including the ability to promote cell cycle arrest, inhibit host translation, and block the ifn response during infection [reviewed in (mcbride et al., 2014) ]. many of these functions are regulated by posttranslational modifications, most notably phosphorylation by host proteins. for example, sars-cov n protein phosphorylation modulates n protein oligomerization, translation suppression, and localization to stress granules (peng et al., 2008) . furthermore, phosphorylation of the mhv n protein is implicated to function as a cellular switch to control transcription of either genomic or subgenomic rna (wu et al., 2014) . in this paper, we have identified an additional post-translational modification of the n protein, adp-ribosylation, which could also play a role in the regulation of n protein functions. we have found that the n protein of multiple covs was apd-ribosylated in vitro, using multiple antibodies to adpr (figs. 1 and 3) . the 10 h α-adpr antibody primarily used in this study binds preferentially to par but can be used to detect marylated proteins as well (eckei et al., 2017; goenka et al., 2007; kawamitsu et al., 1984; kleine et al., 2008) . because the n protein band stained with either α-adpr or α-n antibodies did not appear as a smear, which is seen with long-chain parylated proteins, it is likely that the n protein is either marylated or parylated with only a few monomers of adpr. this is further supported by the fact that adp-ribosylation did not alter migration of the n protein to a detectable level ( fig. 5b and c) . furthermore, our data indicate that the n protein adp-ribosylation was detectable during infection ( fig. 1, fig. 5a, fig. 5c ) but not following transfection alone (fig. 5b ). this could be due to a number of factors including, but not limited to, virus infection-dependent expression of an adp-ribosylating enzyme or the localization of the n protein to a distinct cellular compartment during infection. n protein in infected cells has been shown to localize both in the cytoplasm as well as the nucleus, and the sars n protein has been shown to localize to stress granules under stress conditions (hiscox et al., 2001; peng et al., 2008) . several parps are known to colocalize with stress granules, which could potential sites of n protein modification (leung et al., 2011) . future experiments are required to parse out the number of adpr monomers that are attached to the n protein, to identify the amino acid residue(s) that is modified, and to determine where this modification occurs. the fact that adp-ribosylation of n protein was conserved across multiple cov lineages (fig. 3) suggests that it is important either for viral replication or pathogenesis or as a host defense mechanism. individual parps have been identified to be either proviral or antiviral [reviewed in (kuny and sullivan, 2016) ]. because the n protein is the major structural protein of the cov nucleocapsid, it is tempting to speculate that adp-ribosylation is providing a regulatory role for the structure of the genome, similar to adp-ribosylation of histones or of the adenovirus core protein (dery et al., 1986; strickfaden et al., 2016) . it is also possible that adp-ribosylation of n protein could regulate one or more of the other functions of the n protein such as inhibition of cellular translation or of ifn expression (kopecky-bromberg et al., 2007) . on the other hand, adp-ribosylation of the n protein may be an antiviral defense mechanism, similar to adp-ribosylation of influenza a virus polymerase subunits (liu et al., 2015) . if this were the case, the virus must have evolved methods to combat this modification, possibly including removal of the adp-ribose moieties from the n protein to mitigate its antiviral 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demyelinating encephalomyelitis in mice infected with mhv-jhm in the presence of maternal antibody macrodomain-containing proteins are new mono-adp-ribosylhydrolases deficiency of terminal adp-ribose protein glycohydrolase targ1/c6orf130 in neurodegenerative disease poly(adp-ribosyl)ation-dependent transient chromatin decondensation and histone displacement following laser microirradiation regulation of epstein-barr virus orip replication by poly(adp-ribose) polymerase 1 identification of mouse hepatitis coronavirus a59 nucleocapsid protein phosphorylation sites dna fragmentation and nad depletion. their relation to the turnover of endogenous mono(adp-ribosyl) and poly(adp-ribosyl) proteins nucleocapsid phosphorylation and rna helicase ddx1 recruitment enables coronavirus transition from discontinuous to continuous transcription constitutive aryl hydrocarbon receptor signaling constrains type i interferon-mediated antiviral innate defense systematic assembly of a fulllength infectious cdna of mouse hepatitis virus strain a59 rapid generation of a mouse model for middle east respiratory syndrome airway memory cd4(+) t cells mediate protective immunity against emerging respiratory coronaviruses mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna zinc-finger antiviral protein inhibits hiv-1 infection by selectively targeting multiply spliced viral mrnas for degradation we thank the members of the perlman, wendy maury, and pat sinn laboratories for valuable discussion, kyoung-jin yoon for pedv virus and antibodies, and rudragouda channappanavar for critical reading of the manuscript.this study was supported by national institute of health grants to s.p. (p01 ai060699 and r01 ns36592). m.g. and j.a. were supported by institutional predoctoral nrsa training grants (t32 ai007511 and t32 ai007533, respectively). a.r.f was supported by institutional (t32-ai007260) and individual (f32-ai113973) nih nrsa grants. none. key: cord-286635-7aflpgxd authors: yang, yong-xin; zhao, xing-xu; zhang, yong title: comparative proteomic analysis of plasma from clinical healthy cows and mastitic cows date: 2009-10-31 journal: agricultural sciences in china doi: 10.1016/s1671-2927(08)60337-5 sha: doc_id: 286635 cord_uid: 7aflpgxd abstract the current research presents the protein changes in plasma from healthy dairy cows and clinical mastitic cows using two-dimensional gel electrophoresis (2-de). after staining with silver nitrate and coomassie blue, differential expression proteins were detected by pdquest 7.4 software, and then subjected to ion trap mass spectrometer equipped with a surveyor hplc system, differential spots of protein were identified. three protein spots that originated from preparation gels were identified to be two proteins. overall, haptoglobin precursor was up-regulated in cows infected with clinical mastitis and could be a mastitis-associated diagnostic marker, whereas scgb 2a1 (secretoglobin, family 2a, member 1) was down-regulated protein. plasma protein expression patterns were changed when cows were infected with mammary gland inflammation; it suggests that analysis of differential expression protein might be useful to clarify the mechanisms involved in the pathophysiology, and find new diagnostic markers of mastitis and potential protein targets for treatment. blood can be regarded as a special form of connective tissue with cells separated by liquid plasma. when the body is infected with the disease, the mediators are derived from the cells and tissues through either active secretion or leakage from blood cells or tissues, and then, enter the circulatory system to cause the changes of the blood composition. in particular, plasma protein is a heterogeneous mixture of a large number of constituents, involved in the organism's defense against infection and regulation of the osmotic pressure as well as small molecule transport, which have been functionally characterized and associated with disease processes. after the human plasma project, proteomics were widely used to investigate the protein biomarkers and potential protein targets for therapies in plasma for human diseases, including cancer (ahmed et al. 2005; huang et al. 2006) . to investigate the mechanisms of mammary gland secreted milk and infected disease, previous research workers studied protein expression patterns using 2-de and mass spectrometry in plasma from healthy cows, subsequently, the changes of the milk whey proteins in dairy cows affected with mastitic inflammation and from unaffected animals using proteomic approach, and subsequently some proteins were identified to associate with disease (baeker et al. 2002; hogarth et al. 2004; talamo et al. 2003) . these results suggested that protein expression patterns during clinical and subclinical milk change could be applied to search for new diagnostic marker of mastitis. up to the present, the researchers failed to consider the this paper is translated from its chinese version in scientia agricultura sinica. yang yong-xin, e-mail: yangyongxin66@yahoo.com.cn; correspondence zhao xing-xu, professor, tel: +86-931-7632482, e-mail: zhaoxx@gsau.edu.cn changes of plasma protein when cows were infected with clinical mastitis. this investigation was to detect the expressed proteins in plasma from clinical mastitic and healthy dairy cows by 2-de providing a platform for parallel analysis, and then, to identify differential proteins by ion trap mass spectrometer equipped with hplc system, in order to probe the pathogenesis of mastitis and find new biomarkers of mastitis-associated proteins for diagnosis and treatment. blood samples were obtained from 12 healthy cows and 12 dairy cows infected with clinical mastitis in a dairy farm by jugular venipuncture and were anticoagulated with 0.5% edta. clinical mastitis cases were identified on the basis of clinical symptoms of heat, pain, redness, and swelling of the udder or clots in the milk. healthy dairy cows were confirmed with the lmt (lanzhou mastitis test, lanzhou, china), which is similar to cmt (california mastitis test) method. the blood samples were centrifuged at 3 000 × g for 20 min at 4°c, the suspension was collected and pooled, and used as the 2-de sample that was assayed for protein concentration using 2-d quant kit (amersham bioscience, sweden). the remainder of protein samples were stored in aliquots at -75°c until further use. for the first dimension, isoelectric focusing (ief) was carried out using commercially dedicated apparatuses, protean ief cell (bio-rad, ca, usa). ipg strips were used according to the manufacturer's instructions. 3-5 l plasma protein mixed with 10% sds/2.3% dtt for 5 min at 95°c (steel et al. 2003) , containing 200 g protein for silver nitrate staining gels or up to 1.5 mg for colloidal coomassie blue staining gels, were diluted to 300 l with rehydration solution (7 mol l -1 urea, 2 mol l -1 thiourea, 4% chaps, 65 mmol l -1 dtt, 0.5% ph 3-10 ipg buffer, and trace bromophenol blue), and applied by passive rehydration for 12 h with a nonlinear ph 3-10 gradient strips. the ief steps were carried out at 20°c on the protean ief cell as previously described (yang et al. 2007 ), briefly, for 30 min at 250 v, 1 h at 1 000 v, and then, a gradient was applied from 1 000 to 10 000 v in 5 h, and focusing was continued at 10 000 v for 8 h to give a total of 80 kv h. after the ief, ipg strips were placed in reducing equilibration buffer that contained 6 mol l -1 urea, 2% sds, 20% glycerol, 50 mmol l -1 tris-hcl (ph 8.8), 0.01% (w/v) bromophenol blue, and 2% dtt for 15 min with shaking followed in alkylating equilibration buffer with 2.5% (w/v) iodoacetamide instead of 2.0% dtt for an additional 15 min. then, strips were transferred on to 12% t polyacrylamide gel, low melting agarose was overlaid that contained 0.1% sds, and 37.5 mmol l -1 tris (ph 8.8) was warmed to its melting point and was used to seal the surface of the ipg strips. separation in the second dimension was carried out using protean ii xi electrophoresis apparatus (bio-rad, ca, usa) and tris-glycine-sds (ph 8.3) as the electrode buffer. the gels were started with 50 v and continued with 200 v until the bromophenol blue dye marker had reached the bottom of the gel. protein spots were visualized with silver staining as previously described (mortz et al. 2001) , and micropreparative gels were stained with coomassie blue. stained gels were matched and detected with the pdquest software (ver. 7.4, bio-rad, usa). three independent samples were repeated for each pooled sample. gel images were conducted to remove backgrounds and to automatically detect protein spots. for comparisons of protein levels among gels, normalization was done against the total intensity of all spots present in the gel. differentially expressed protein spots were excised manually using pipette tips, which were clipped off to form an orifice with an inner diameter of 2 mm and put into 1.5 ml microtubes. the gel particles were washed three times with milliq water for 30 min and twice with milliq water/acetonitrile (1:1, v/v) for 15 min at room temperature, and then, gel pieces were shrunk in 100 l acetonitrile. subsequently, the dried particles were subjected to 50 mmol l -1 ammonium bicarbonate containing 0.01% sequence-grade trypsin incubated at 37°c overnight. the peptides were extracted with 50% acetonitrile and 0.2% formic acid, and applied one round of vortexing and sonication (20 min each), and subsequently, the supernatant was transferred to a clean microtube. this step was repeated. peptide mass was determined using ion trap mass spectrometer (lcq deca xp plus, thermo finnigan) equipped with a surveyor hplc system (thermo, usa). before injecting the peptide mixes, 150 mm × 0.18 mm biobasic-18 column (thermo, usa) was equilibrated at a flow rate of 120 l min -1 . mobile phase a consisted of water and 0.1% formic acid, and mobile phase b consisted of acetonitrile and 0.1% formic acid. peptides were eluted with 0.1% formic acid/ acetonitrile linear gradient in 120 min. in the full scan mode, ms spectra for all samples were measured with an overall mass/charge (m/z) range of 400-2 000, and ms/ms was performed in data-dependent mode. peptides were identified using sequest software (bioworks 2.0, thermo finnigan) to search against the publicly available ncbi nonredundant protein database (http://www.ncbi.nlm.nih.gov). the protein identification criteria were based on delta correlation score delta cn ( 0.1) and cross-correlation score xcorr (one charge 1.9, two charges 2.2, three charges 3.75). the experiments were repeated three times under the same experimental conditions. the gel images were automatically matched and analyzed using pdquest 7.4 software to determine protein spots that differentially expressed. although protein spots in region a rectangle from healthy cows plasma and region b rectangle from clinical mastitic cows presented significant difference in silver-stained 2-de maps, they were not different in coomassie blue gels; only three spots were detected to be differentially expressed with changes in the stain density of threefold or more in both stained 2-de maps, that is, spot 1 was a down-regulated protein, whereas spots 2 and 3 were up-regulated in plasma from cows infected with clinical mastitis (fig.1) . the differential spots were excised from the gels and tryptic peptides were extracted; then, the peptides were automatically analyzed by hplc equipped with ion trap mass spectrometer, and protein identification was carried out by peptide sequencing with the search programs of sequest. three protein spots were identified to be two proteins, listed in table. a representative tandem spectra of a tryptic peptide obtained from spot 3 was shown in fig.2 . the double-charged peptide ions with a m/z ratio of 726.62 was effectively fragmented to yield sufficient structural information for identification peptide, vetgseatadscpk. protein visualized with silver-stained methods is comkda monly used in analytical gels of comparative proteomics approach, because of their highly sensitive range of detection, typically 1-10 ng of protein per spot. however, silver-staining methods are hardly sensitive to visualization for some proteins, which have not provided compatible identification using mass spectrometry. colloidal coomassie is commonly used with preparative gels that is greatly compatible with mass spectrometry and has a detection of 8-50 ng (simpson 2003) . although spots in regions a and b rectangle between healthy cows and clinical mastitic cows plasma presented significant difference in silverstained 2-de maps, in this study, they were not different in coomassie blue gels, because protein samples in micropreparative gels had not effectively separated or as a result of the protein visualized with silver-stained and coomassie blue g-250 methods inherently had significant difference in sensitivity. haptoglobin was a major acute phase protein, which has host defense and antibacterial activities. it connects to free hemoglobin released from erythrocytes to form haptoglobin-haemoglobin complex, and thereby, inhibits its oxidative activity and sequesters the iron within hemoglobin, and furthermore preventing ironusing bacteria benefitting from hemolysis. recently, haptoglobin was an up-regulated one in hepatocellular carcinoma, severe acute respiratory syndrome, and ovarian cancer using proteomic approach, and regarded as a useful diagnostic marker (ahmed et al. 2005; ang et al. 2006; wan et al. 2006) . however, comparative analysis of breast cancer serum using 2-d differential gel electrophoresis combination with maldi-tof/tof found that haptoglobin was a down-regulated protein (huang et al. 2006) . interestingly, haptoglobin concentrations were increased in milk and serum from cows with experimentally induced acute and chronic staphylococcus aureus matitis and from cows with clinical mastitis, indicating that it is an acute phase protein (eckersall et al. 2001; eckersall et al. 2006; gronlund et al. 2003) . our data showed that the haptoglobin precursor also increased in plasma of cows infected with clinical mastitis, and it might play a significant role in the early response to invasion of mammary by pathogenic bacteria. recently, scgb 2a1 (also know as mammaglobin) is predicted to be a secreted protein and when mrna as lymph node micrometastases in breast cancer is expressed, there is an increase in the expression of mammaglobin in mammary gland of breast cancer patients, and it is also identified as a novel serum marker for detection of breast cancer (bernstein et al. 2005; ooka et al. 2000; watson et al. 1999 ). however, mammaglobin was found in human normal prostate tissue and absent in tumor samples without significant differences (tucker et al. 2005) . also, its mrna levels did not vary significantly between wild-type and estramustine-resistant cells in prostate and ovarian cancer cell lines. in our data, although mammaglobin is identified in serum and is a down-regulated protein when cows are infected with clinical mastitis, the function of the mammaglobin protein associated with intramammary inflammation in cows remains unknown. it might provide valuable information to clarify the mechanisms involved in the pathogenesis of mammary. to probe protein expression pattern changes in plasma from healthy dairy cows and clinical mastitic cows, this subject analyzed and identified differential protein spots using 2-de and ion trap mass spectrometer equipped with hplc system. three protein spots originated from preparation gels were identified to be two proteins, haptoglobin precursor was an up-regulated protein, while scgb 2a1 was a down-regulated protein in cows infected with clinical mastitis. proteomic approach analysis of protein changes in plasma might be useful to clarify the mechanisms involved in the pathophysiology of cows infected with clinical mastitis, to find new diagnostic markers of mastitis, and to find potential protein targets for treatment. proteomic tracking of serum protein isoforms as screening biomarkers of ovarian cancer study of serum haptoglobin and its glycoforms in the diagnosis of hepatocellular carcinoma: a glycoproteomic approach lipocalintype prostaglandin d synthase in milk: a new biomarker for bovine mastitis identification of mammaglobin as a novel serum marker for breast cancer acute phase proteins in serum and milk from dairy cows with clinical mastitis acute phase proteins in bovine milk in an experimental model of staphylococcus aureus subclinical mastitis haptoglobin and serum amyloid a in milk and serum during acute and chronic experimentally induced staphylococcus aureus mastitis differential protein composition of bovine whey: a comparison of whey from healthy animals and from those with clinical mastitis biomarker discovery in breast cancer serum using 2-d differential gel electrophoresis/maldi-tof/tof and data validation by routine clinical assays improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis selection of mrna markers for detection of lymph node micrometastases in breast cancer patients proteins and proteomics: a laboratory manual proteins from bovine tissues and biological fluids: defining a reference electrophoresis map for liver, kidney, muscle, plasma and red blood cells evaluation of lipophilins as determinants of tumor cell response to estramustine inflammation inhibitors were remarkably up-regulated in plasma of severe acute respiratory syndrome patients at progressive phase mammaglobin expression in primary, metastatic, and occult breast cancer the project was supported by the china international sci & tech cooperation project (2005 dfa30720). key: cord-274293-kzmch37j authors: yang, li; zhang, jia hao; zhang, xiao li; lao, guang jie; su, guan ming; wang, lei; li, yao lan; ye, wen cai; he, jun title: tandem mass tag-based quantitative proteomic analysis of lycorine treatment in highly pathogenic avian influenza h5n1 virus infection date: 2019-10-02 journal: peerj doi: 10.7717/peerj.7697 sha: doc_id: 274293 cord_uid: kzmch37j highly pathogenic h5n1 influenza viruses (hpaiv) cause rapid systemic illness and death in susceptible animals, leading to a disease with high morbidity and mortality rates. although vaccines and drugs are the best solution to prevent this threat, a more effective treatment for h5 strains of influenza has yet to be developed. therefore, the development of therapeutics/drugs that combat h5n1 influenza virus infection is becoming increasingly important. lycorine, the major component of amaryllidaceae alkaloids, exhibits better protective effects against a/ck/gd/178/04 (h5n1) (gd178) viruses than the commercial neuraminidase (na) inhibitor oseltamivir in our prior study. lycorine demonstrates outstanding antiviral activity because of its inhibitory activity against the export of viral ribonucleoprotein complexes (vrnps) from the nucleus. however, how lycorine affects the proteome of aiv infected cells is unknown. therefore, we performed a comparative proteomic analysis to identify changes in protein expression in aiv-infected madin-darby canine kidney cells treated with lycorine. three groups were designed: mock infection group (m), virus infection group (v), and virus infection and lycorine-treated after virus infection group (l). the multiplexed tandem mass tag (tmt) approach was employed to analyze protein level in this study. in total, 5,786 proteins were identified from the three groups of cells by using tmt proteomic analysis. in the v/m group, 1,101 proteins were identified, of which 340 differentially expressed proteins (deps) were determined during hpaiv infection; among the 1,059 proteins identified from the lycorine-treated group, 258 proteins presented significant change. here, 71 proteins showed significant upregulation or downregulation of expression in the virus-infected/mock and virus-infected/lycorine-treated comparisons, and the proteins in each fraction were functionally classified further. interestingly, lycorine treatment decreased the levels of the nuclear pore complex protein 93 (nup93, e2rsv7), which is associated with nuclear–cytoplasmic transport. in addition, western blot experiments confirmed that the expression of nup93 was significantly downregulated in lycorine treatment but induced after viral infection. our results may provide new insights into how lycorine may trap vrnps in the nucleus and suggest new potential therapeutic targets for influenza virus. highly pathogenic influenza a viruses resulting from routine seasonal epidemics and global pandemics continue to circulate in nature. it is an extremely contagious and aggressive disease that causes rapid systemic illness and death in susceptible birds. moreover, certain strains, such as h5n1 subtypes, are capable of cross-species transmission and thus can infect humans with high morbidity and mortality (sun et al., 2015) . the new subtypes have emerged through accumulation and antigenic shift (zhu, wang & wang, 2017) , which may result in rare influenza a virus pandemics. resistance and timeliness to clinical antiviral therapy are the major problems in curing these infections (blanton et al., 2017; hu et al., 2017) . m2 inhibitors, such as amantadine and ramantadine, cannot protect the host from virus infection because of its amino acid mutation (wang et al., 2013) . to date, oseltamivir is the only commercial drug effective in human, where it inhibits na activity. however, drug-resistant strains have emerged because of one single amino acid residue substitution (h274y in n1) (yusuf et al., 2016) . therefore, novel influenza inhibitors must be developed. virus-host interactions involve a complex interplay of host cellular and viral networks. the discovery of host factors that may regulate viral replication is a promising approach. in this respect, genomic and proteomic studies have provided abundant information on host genes and proteins associated with viral infection and pathogenesis (wang et al., 2014; zhang et al., 2018) . the mode of rnp exit of influenza viruses from the nucleus complements active crm1-dependent export mechanisms via the nuclear pore complex (npc) and ensures the efficient production of infectious virus progeny (muhlbauer et al., 2015) . some of the host factors involved in viral replication and/or pathogenesis could be targeted for the treatment of aiv infection without obvious side effects. traditional chinese medicines (tcms) contain effective components that deal with different types of diseases, such as malaria and influenza a virus (crunkhorn, 2016; nonaka et al., 2018) . lycorine is a tcm that exhibits acetyl-cholinesterase-inhibitory and butyryl-cholinesterase-inhibitory activities (wang et al., 2012) . in russia, it is clinically used as an expectorant to treat chronic and acute inflammatory processes in lungs and bronchial diseases (henry et al., 2016) . over the past years, lycorine has attracted great research interest owing to its superlative biological potential and pharmacological actions, including antiangiogenic, antiviral, antibacterial, antimalarial, anti-parasite, antioxidant, hepatoprotective, analgesic, anti-inflammatory activities, and inhibition of ascorbic acid synthesis (kim et al., 2008; lamoral-theys et al., 2009; cedron et al., 2010; giordani et al., 2011; citoglu et al., 2012; ye et al., 2012; wang et al., 2014; cembrowska-lech & kepczynski, 2016; bendaif et al., 2018) . different drugs may affect different molecular pathways that may be affected within disease models. for instance, several studies have shown that lycorine induces apoptosis to suppress the propagation of carcinoma cells (li et al., 2007; yu et al., 2017) . other studies have demonstrated that its antitumor effect is through inhibiting inflammatory factors and suppressing p38 and stat activation (kang et al., 2012) . although lycorine exhibits a wide range of biological activities, the mechanism underlying its effects remains unclear. in our previous study, the efficacy and inhibitory effects of lycorine (ec 90 = 0.52 µm) were determined in madin-darby canine kidney (mdck) cells. results showed that lycorine can completely prevent h5n1 infection as indicated by the absence of any cytopathic effect (he et al., 2013) . unlike the mechanism of oseltamivir, the vrnps are retained in the nucleus by lycorine treatment instead of directly targeting viral proteins and rna polymerase activity (he et al., 2013) . these results strongly suggest the potential of lycorine as an antiviral agent for h5n1 strain. however, the molecular mechanism of the antiviral/inhibitor action of lycorine remains unknown. therefore, we performed a comparative proteomic analysis to determine the effects of lycorine at the protein level in gd178-infected mdck cells to understand its mode of action. gd178 (n0. ay737296-737300) and mdck cells were obtained from the key laboratory of veterinary vaccine innovation of the ministry of agriculture, p. r. china. the mdck cells were cultured in dulbecco's modified eagle's medium (dmem, invitrogen) containing 10% (v/v) fetal bovine serum (fbs, gibco). a plaque assay was used to determine viral activity in mdck cells (he et al., 2013) . three separate biological cell cultures were made for the tandem mass tag (tmt) proteomic experiments. lycorine (fig. 1a ) was obtained as previously described (wang et al., 2009) . all experiments involving live h5n1 influenza virus were carried out in biosafety level-3 facilities. three groups were designed: (1) m, mock group (control); (2) v, virus-infected group. they were then inoculated at 3 multiplicity of infection (moi) for 1 h (v), removed, and inoculated with dmem containing 10% fbs until 12 h post-infection (h.p.i.); and (3) l, lycorine-treated post-viral infected group. after 1 h adsorption of 3 moi gd178, lycorine was added at 0.52 µm for 12 h.p.i. for each sample, the proteins were diluted in 300 µl of sdt buffer (containing 4% sds, 100 mm tris-hcl, and 100 mm dtt, ph 8.0) following a standardized protocol (wisniewski et al., 2009) , homogenized, heated at 100 • c for 3 min, and then centrifuged at 14,000× g for 30 min at room temperature. each sample (one µl) was taken and quantified using the bicinchoninic acid method. the remaining lysate was frozen at −80 • c until use. protein digestion was conducted using the fasp procedure (wisniewski et al., 2009) . in brief, 300 mg of proteins were loaded onto an ultrafiltration filter (30 kda cutoff; sartorius, cells from each group were harvested, underwent trypsin digestion, and then labeled by tandem mass tag (tmt)-10 plex, and high-ph reverse-phase liquid chromatography was used to fractionate the pooled tmt-labeled peptide mixtures before nano liquid chromatography-mass spectroscopy tandem (lc-ms/ms) analysis. all raw files were searched together using proteome discover 2.1. the quantification data matrix was further used for statistical trend analysis and visualization by trelliscope. finally, western blot experiments were conducted for trend verifications. full-size doi: 10.7717/peerj.7697/ fig-1 goettingen, germany) containing 200 µl of ua buffer (8 m urea, 150 mm tris-hcl, ph 8.0), centrifuged at 14,000× g for 30 min, and then washed with 200 µl of ua buffer. then, 100 µl of 50 mm iodoacetamide in ua buffer was subsequently added to the filter to block reduced cysteine residues. the samples were incubated for 30 min at room temperature in the dark and then centrifuged at 14,000× g for 30 min. the filters were washed three times with 100 µl of ua buffer and then centrifuged at 14,000× g for 30 min after each washing step. next, 100 µl of dissolution buffer (applied biosystems, foster city, ca, usa) was added to each filter and then centrifuged at 14,000× g for 30 min, which was repeated three times. the protein suspensions were then digested with 40 µl of trypsin (promega, madison, wi, usa) buffer (6 µg trypsin in 40 µl of dissolution buffer) at 37 • c for 18 h. finally, the filter unit was transferred to a new tube, and 40 µl of dissolution buffer was added followed by centrifugation at 14,000× g for 30 min. the resulting peptides were collected as a filtrate, and the peptide concentration was analyzed at od280. nine serial samples from each subject and the common reference sample were included in one tmt10 labeling experiment set as shown in fig. 1c . this labeling strategy avoids the potential for missing value issues resulting from data-dependent ms/ms acquisitions (zimmer et al., 2006) . tmt-labeled peptides were subjected to high-ph reversed-phase fractionation in 1100 series hplc value system (agilent) equipped with a gemini-nx (phenomenex, 00f-4453-e0) column (4.6 × 150 mm, 3 µm, 110 å) (wang et al., 2011; batth, francavilla & olsen, 2014) . the peptides were eluted at a flow rate of 0.8 ml/min. buffer a consisted of 10 mm ammonium acetate, ph 10.0, and buffer b consisted of 10 mm ammonium acetate, 90% v/v acetonitrile (acn), ph 10.0. both buffers were filter sterilized. the gradient to perform the separation consisted of several steps: (1) 100% buffer a for 40 min, 0%-5% buffer b for 3 min; (2) 5%-35% buffer b for 30 min; (3) 35%-70% buffer b for 10 min; (4) 70%-75% buffer b for 10 min; (5) 75%-100% buffer b for 7 min; (6) 100% buffer b for 15 min; and (7) 100% buffer a for 15 min. the elution process was monitored by measuring absorbance at 214 nm, and fractions were collected every 1.25 min. the collected fractions (approximately 40) were finally combined into 10 pools. each fraction was concentrated via vacuum centrifugation and reconstituted in 10 µl of 0.1% v/v formic acid. all samples were stored at −80 • c until lc-ms/ms analysis. the labeled samples were analyzed using the easy-nlc nanoflow hplc system connected to the thermo scientific tm orbitrap fusion tm tribrid tm mass spectrometer (thermo fisher scientific, san jose, ca, usa). a total of 1 µg of each sample was loaded onto the thermo scientific easy column (two columns) using an autosampler at a flow rate of 200 nl/min. sequential separation of peptides on the thermo scientific easy trap column (100 µm × 2 cm, 5 µm, 100 å, c18) and analytical column (75 µm × 25 cm, 5 µm, 100 å, c18) was accomplished using a segmented 60 min gradient from 5% to 28% solvent b (0.1% formic acid in 100% acn) for 40 min followed by 28%-90% solvent b for 2 min and then 90% solvent b for 18 min. the column was re-equilibrated to its initial highly aqueous solvent composition before each analysis. the mass spectrometer was operated in a positive ion mode, and ms spectra were acquired over a range of 375-1,500 m/z. the resolving powers of the ms and ms/ms scans at 200 m/z for the fusion were set as 120,000 and 50,000, respectively. the data-dependent mode was top speed, cycle time was 3 s, and ions were fragmented through high energy collisional dissociation. the maximum ion injection times were set at 50 ms for the survey scan and 105 ms for the ms/ms scans, and the automatic gain control target value was set to 4e5 for ms and to 1e5 for ms/ms. the dynamic exclusion duration was 40s. all raw files were analyzed using the proteome discoverer 2.1 software (thermo fisher scientific). a search for fragmentation spectra was performed using the mascot search engine (matrix science, london, uk; version 2.2) embedded in proteome discoverer against the uniprot canidae protein sequence database (released in november 2016, 31,596 sequences). several search parameters were used: (1) monoisotopic mass; (2) trypsin as the cleavage enzyme; (3) two missed cleavages; (4) tmt labeling; (5) cysteine carbamidomethylation as fixed modifications; (6) peptide charges of 2+, 3+, and 4+; and (7) methionine oxidation. the mass tolerance was set to 20 ppm for precursor ions and to 0.1 da for the fragment ions. peptide spectral matches were filtered to a 1% false discovery rate. the relative quantitative analysis of the sample proteins was based on tmt reporter ion ratios from all unique peptides representing each protein. this analysis was performed using proteome discoverer (version 2.1). the relative peak intensities of the tmt reporter ions released in each of the ms/ms spectra were used. the final ratios obtained from the relative protein quantifications were normalized based on the median average protein quantification ratio. only unique peptides obtained with a confidence percentage of >95% were included in the ratio ≥1.20, or ≤0.8333-fold cutoff value was used to identify upregulated and downregulated proteins with p < 0.05. the mass spectrometry proteomic data have been deposited to the proteomexchange consortium via the pride partner repository with the dataset identifier pxd lycorine (accession number pxd012936). protein-protein interaction (ppi) analysis of dep proteins string (search tool for the retrieval of interacting genes/proteins) version 11.0 was employed in this study for the potential ppi analysis of the deps proteins (http://string-db.org/) (szklarczyk et al., 2015; shannon et al., 2003) . the parameter for confidence score was set to 0.4, and the yield ppi results were visualized by cytoscape software (shannon et al., 2003) . the light blue nodes indicated upregulated-proteins and the light green nodes indicated downregulated-proteins. the edge thickness was proportional to the combined score of the proteins. bioinformatics and enrichment analysis was performed as previously described (hui bin huang, 2017) . cells were homogenized on ice for 20 min in 1× ripa buffer with cocktail protease inhibitors (roche) and then centrifuged at 15,000× g for 20 min at 4 • c. the supernatant was collected and then stored at −80 • c. for western blot analysis, equal amounts of proteins were separated using sds-page and subsequently transferred onto a polyvinyl fluoride membrane (bio-rad). the membrane was blocked with 5% bsa and then incubated with monoclonal antibody nup93 (ab168805) and monoclonal antibody antiβ-actin (cell signaling technologies) at 4 • c overnight. membranes were washed in phosphate-buffered saline with tween20 and then incubated for 2 h at rt with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2,000; cell signaling technologies). statistical analyses were conducted as previously described (hui bin huang, zhang & li, 2017) . the concentration and exposure time of lycorine were determined to avoid any cytotoxic effects on the host cells and to ensure that the proteome of the host cells reflected the true response to the presence of lycorine. a prior study found through cck-8 assay that 0.52 µm lycorine is not toxic to cells at 12 h post-infection (h.p.i.) (he et al., 2013) . in addition, the time of lycorine addition was optimized. results showed that the earlier lycorine was added, the better effect on viral titers. therefore, a single replication cycle of influenza virus (12 h.p.i.) within lycorine treatment at 0.52 µm was chosen because it reflects a single viral replication and is a non-toxic concentration of lycorine at the time addition assay. dose-effect relationship experiments showed that inhibition of lycorine of aiv becomes invalid within high-dose virus (jun he et al., 2012; he et al., 2013) . hence, we ensured that the response of the whole cell proteome is caused by early infection and that the activity of lycorine is effective. thus, 3 moi was selected as the minimum infectious dose for a single cycle of viral infection for studying the single viral replication cycle (peschel et al., 2013) . therefore, 3 moi of aiv infections, lycorine concentration of 0.52 µm, and 12 h of incubation time were selected as the optimal dose and exposure time for further experiments. in this study, we applied a tmt method, which has been widely applied for quantitative proteomic cell biology studies and many model organisms, to explore potent anti-viral agents and different signaling pathways (lombardi et al., 2015; burton et al., 2016) . a schematic of the experimental workflow and venn diagram of v/m, l/v, and l/m is shown in fig. 1c . each experiment group consisted of three biological replicates (n = 3). in total, 5,786 proteins were identified and relatively quantified at 0.01% fdr in the proteome using maxquant (table s1 ). among the statistically significant proteins detected by anova (p < 0.05), protein abundances that changed <1.2-fold, >0.8333-fold, and p > 0.05 were discarded. the proteome profiles of mock control and infected cells were compared (v/m) to determine the effects of aiv infection on mdck cells. among those protein groups, 1101 proteins were identified, of which 340 deps were determined in the v/m group. the proteome profiles of the infected cells and lycorine-treated infected cells were compared to determine the effects of lycorine treatment on the infected cells. a total of 1059 proteins were identified in the v/l group, of which 258 were deps. these results indicated wide differences of protein expression in the hpaiv infection and lycorine treatment. inoculating with hpaiv and lycorine resulted in 691 identified deps (76 + 86 + 42 + 54 + 189 + 159 + 85) among the three groups of mdck cells in response to hpaiv infection and lycorine treatment, as shown in the venn diagram (fig. 1b) . the proteome profiles of the gd178-treated mdck cells and mock control were compared (v/m) to determine the effects of gd178 influenza virus on mdck cells. the v/m group contained 123 upregulated (>1.2-fold) and 217 downregulated (to <0.8333-fold) proteins in the v/m group (table s2) . some of the proteins in the same network were direct interaction or through intermediate partner at the ppi level, as shown in fig. s1 . gene ontology analyses of the proteins upregulated and downregulated by gd178 were performed to map the genes involved in different cellular processes, including biological and functional events ( fig. 2a) . a large number of biological process go terms were identified. the deps were strongly represented by ''catabolism, metabolism, and physiological process''; the infected cells were also assigned to numerous molecular functions, of which ''binding, catalytic and molecular transducer activities'' were the main ones, and cellular components, of which ''extracellular region part, organelle and membrane'' were dominant. go enrichment analysis was conducted on cell periphery (90 proteins), plasma membrane (88 proteins), intrinsic component of membrane (59 proteins) and molecular transducer activity (39 proteins) to further study the impact of deps in cell physiological processes and discover their internal relations (fig. 2b) . several identified proteins have known interactions with one another and function in the same biochemical pathways. therefore, a kegg pathway-based enrichment analysis was applied to identify the main pathways that were potentially affected by the deps in the v/m group (fig. 2c) . the enriched pathways showed that the proteins were involved in pathway in cancer (path: ko05200), extracellular matrix (ecm)-receptor interaction (path: ko04512), focal adhesion (path: ko04510), mitogen-activated protein kinase signaling pathway (path: ko04010), and pi3k-akt signaling pathway (path: ko04151). these data indicated that there are many changes in the protein profile in response to influenza virus infection at 12 h.p.i. the proteome profiles of the infected cells and the infected cells within lycorine treatment were compared (v/l) to determine the effects of lycorine treatment on the infected cells. in total, 258 deps were found (table s3 ). the ppi network was performed to determine whether these deps interact with each other and form protein complexes (fig. s2) . twohundred six proteins with at least 1.2-fold upregulation between the lycorine-treated and virus control group were found (v/l), and 52 proteins were significantly downregulated. the go terms of deps were strongly represented by ''cellular process, single-organism process, and biological regulation'' in the biological process and ''binding, catalytic and molecular transducer activities'' in molecular functions; the lycorine-treated cells were also assigned to numerous cellular components, of which ''membrane-enclosed lumen, organelle and membrane'' were dominant (fig. 3a) . go enrichment analysis showed that the three main parts were nuclear division (15 proteins), chromosome segregation (14 proteins), and condensed chromosome (eight proteins) (fig. 3b) . the functional classification of deps was conducted by kegg enrichment analysis, and each protein was assigned to at least one of the following pathways: human t-lymphotropic virus-1 infection pathway (path: ko05166), cell adhesion molecules (cams) (path: ko04514), epidermal growth factor receptor tyrosine kinase inhibitor resistance (path: ko01521), janus kinase-stat signaling pathway (path: ko04630), and pancreatic cancer (path: ko05212) (fig. 3c) . these results showed differences between v/m and v/l, and further analyses of these genes that were co-changed in the two groups may shed light on the antiviral mechanism of lycorine. the proteins co-regulated in v/m and v/l were selected for further analysis. these 71 deps were investigated and annotated using go terms and subjected to go functional analysis (table s4) . fifty-four proteins were upregulated in the v group but downregulated in the l group; this finding indicated that some of the proteins may participate in viral infections and may be blocked by lycorine to avoid progeny virus budding. seventeen proteins were downregulated in the v group but induced in the l group, implying that these proteins were downregulated after influenza virus infection but increased after lycorine treatment. a total of 71 proteins showed upregulated or downregulated difference in abundance, and a heat map of these proteins was obtained (figs. 4a and 4c). all co-upregulated and co-downregulated proteins were further analyzed by go enrichment analysis, and each protein belonged to at least one term. among the upregulated proteins, the three main terms in the bp category were cellular process (40 proteins), metabolic process (29 proteins), and organic substance metabolic process (29 proteins). the main terms in the mf category were transcription factor activity (42 proteins) and nucleic acid binding transcription factor activity (33 proteins). the main terms in the cc category were membrane part (49 proteins) and cell junction (41 proteins). in the downregulated proteins, the main terms in the bp category were localization (15 proteins), immune system process (14 proteins), and reproduction (12 proteins). the main terms in the mf category were structural molecule activity (15 proteins) and binding (11 proteins). the main terms in the cc category were extracellular region (16 proteins) and membrane part (16 proteins) (figs. 4c and 4d ). the analysis suggested that these biological functions may be affected by the treatment of aiv-infected mdck cells with lycorine. in addition, pathway analysis showed that these proteins were involved in viral replication-related pathways. for instance, oligoadenylate cyclase (oasl1), serine/threonine protein kinase (stk10), and other proteins (8/71) were involved in adenosine and guanine triphosphatase (atp and gtp) activity-related pathways; other pathways such as nup93, mitochondrial translational initiation factor (mtif3), and other proteins (9/71) participated in host cell responses to lycorine-related transcription or translation processes, such as cellular responses to stimulus and oxoacid-related metabolic processes. this may or may not be related to the antiviral properties of lycorine. according to our prior study, vrnp is retained in the nucleus under conditions of lycorine treatment to stop the cycle of influenza virus, which leads us to search for the proteins involved in the vrnp export pathway (he et al., 2013) . for several pathways relevant to proteins targeting nuclear export were enriched in the ''biological process'' category. the proteins with the same trend in the v/m and v/l groups were our candidate proteins. fortunately, a gene directly related to nuclear transport, nup93, was upregulated by 1.29-fold, in contrast to the low expression level (0.8) found in lycorine treatment over controls (table s2) . from the go terms, nup93 is related with export of material from the nucleus, such as mrna, trna, viral material, and viral transcription (table s4) . given its possible role in viral inhibition (muhlbauer et al., 2015) , western blotting assay with specific antibodies was performed to determine if the expression regulation for these proteins is at the protein level. as shown in fig. 5 , the trends in nup93 level changes in the infected cells and lycorine treatment groups were similar to the changed patterns as was observed. moreover, the protein levels of nup93 in the uninfected cells but treated with the same concentrations of lycorine were not changed as such as the cells treated with lycorine after influenza virus infection. these results indicate that nup93 expression was induced after influenza virus infection but was dramatically decreased after lycorine treatment at 0.52 µm. this concentration did not exhibit toxicity to the uninfected cells, which was in accordance with the tmt results. as a result, aiv infection may induce nup93 to complete the viral cycle, and the process of protein targeting into nup93 after lycorine treatment may partly be explained by the blockage of vrnps in the host cellular nucleus. an effective drug for hpaiv treatment is still a worldwide problem (arai et al., 2018) . our previous study found that lycorine exhibits stronger anti-influenza activity against gd178 than oseltamivir and retains vrnp in the nucleus (he et al., 2013) . hence, a comprehensive description of changes in viral and cellular proteins during aiv infection and lycorine treatment based on multiplex tmt-based quantitation was carried out. the interaction of the host cellular proteins with lycorine and the factor of nucleus transport are two of our main research interests. proteomics is the large-scale study of molecules known as proteins, the critical building blocks of both host and viruses (drayman et al., 2017; kim et al., 2017; kleiner et al., 2017; vasilijevic et al., 2017) . influenza virus modifies and hijacks numerous processes and cell organelles during its replication cycle (de castro martin et al., 2017) ; it also regulates the subcellular localization of protein complexes. in the present study, these phenomena were observed in the hpaiv-infected results ( fig. 2 and table s2 ) in conjunction with the total number of significantly regulated proteins (340 proteins). on the basis of the numbers of proteins being significantly modulated and the pathways associated with those proteins, the gd178 influenza virus induced more profound responses to ecm-receptor interaction, focal adhesion, and pi3k-akt signaling pathway through kegg pathwaybased enrichment analysis. yek et al. (2016) showed that the major function modulated by myxovirus (influenza virus) resistance 1 infection is predominantly enriched in the ecm-receptor interaction and focal adhesion pathways. liu et al. (2012) showed that infection with a h5n1 hpaiv strain with an moi of 0.5 cannot infect much of the cells in their study. thus, 3 moi or higher infection doses are better for studying one single viral replication cycle. considering the dose effects and efficiency of lycorine and gd178 infection, we chose 3 moi for the single cycle. therefore, a suitable time point (12 h.p.) must be selected to harvest virus-infected cells that have been treated by lycorine and can be used for proteomic analysis. after endocytic cell entry, vrnps are released into the cytoplasm and enter the nucleus where viral mrna synthesis and replication occur. the viral genomes are encapsulated with nucleoproteins (nps), and they are associated with trimeric polymerase complexes (the viral proteins m1, nep/ns2, and np) and recognized by crm1 to the cytoplasm via the npc (flatt & greber, 2015) . npc is a highly conserved protein complex that is localized at the nuclear periphery and required for the import and export of proteins and rna (labade, karmodiya & sengupta, 2016) . the influenza virus could induce proteins and pathways that it uses on dependently, replicate its genome in the nucleus, and export it to the cytoplasm via npcs. the nuclear pores promote the nuclear export of newly synthesized rnps, which is an important process that regulates cellular functions and facilitates viral npc assembly. muhlbauer et al. (2015) proved that virus-induced cellular caspase activities cause a widening of nuclear pores, thereby facilitating nucleocytoplasmic translocation. however, they focused on another npc protein, nup153, which is correlated with the import and export pathway for influenza virus infection. in the present study, the expression of nup93 was upregulated by 1.29-fold after gd178 infection at 12 h. however, the different npc proteins and different functions render direct comparison with our results impossible. the mdck cells infected with gd178 exhibited tremendous changes in the levels of many proteins and pathways. interestingly, the npc protein nup93 was confirmed to be directly related to the export of mrna/trna from the nucleus. we will pay close attention to the changes in nup93 expression after lycorine treatment. lycorine, the main constituent from lycoris radiata bulbs, exhibits a wide range of biological activities, including antiviral (masi et al., 2016 ), antimalarial (cho et al., 2018 , antibacterial (bendaif et al., 2018) , anti-parasitic and anti-inflammatory (park, 2014) . the first reported activity of lycorine is the inhibition of the termination of protein synthesis in poliovirus infection (vrijsen et al., 1986) . subsequent studies found that lycorine exhibits antiviral activity toward herpes simplex virus (renard-nozaki et al., 1989) , hiv-1 (lin et al., 1995) , coronavirus (li et al., 2005) , poliovirus (hwang et al., 2008) , west nile virus, dengue and yellow fever viruses (zou et al., 2009) , enterovirus 71 (liu et al., 2011) , influenza virus (he et al., 2013) , hepatitis c virus (guo et al., 2016) , and adult zika virus (masi et al., 2016) . although lycorine is a compound with various antiviral activities, the molecular mechanism underlying the effects of lycorine is still unclear. compared with other pharmacological activity mechanisms, studies on anti-cancer activity have gained deep insights (lamoral-theys et al., 2010) . potential targets for lycorine action include bcl-2 family proteins bcl-2 and mcl-1, hdac, tnf-α, stat, and hmgb1. however, no specific target for lycorine-induced anticancer effect has been identified so far. in the present study, it is most obvious by examination of lycorine treatment after hpaiv-infected results ( fig. 3 and table s3 ). on the basis of 258 proteins being significantly modulated and the pathways associated with those proteins, the lycorinetreated cells induced more profound responses to cams, egfr-related pathway, and jak-stat signaling pathway through kegg pathway-based enrichment analysis. shen et al. authenticated that lycorine directly interacts with egfr and inhibits egfr activation (shen et al., 2018) . hu et al. (2015) and jin et al. (2016) showed that lycorine inactivates the jak-stat signaling pathway to inhibit the proliferation of cancer cells. furthermore, our present results agree with these specific signaling pathways. in addition, go enrichment analysis showed that 15 proteins involved in nuclear division were differently expressed upon lycorine administration. notably, nup93 expression was decreased upon lycorine treatment. the 71 deps that were co-upregulated or co-downregulated in both v/m and v/l groups were selected as candidates ( fig. 4 and table s4 ). among them, 54 candidate proteins were increased by gd178 infection but decreased by lycorine treatment. viral infection played a significant down-modulatory role to the 17 candidate proteins that were upregulated by lycorine. however, the deps detected in the current study hardly match those determined by silac analysis conducted in 2014 and published in 2017 (hui bin huang, 2017) by our group. this result might be due to the different instruments applied and databases used. the mass spectrometer q-exactive was applied for silac experiment in the previous study, whereas fusion-lumos instrument was used in the present study. at present, we found that nup 93 protein was inhibited by lycorine treatment, which aroused our great interest. the same topic will be the focus of our follow-up work. to explore how lycorine affects nucleus transport, we analyzed the protein levels of nup93 by western blot assay and found that nup93 levels were increased after hpaiv infection but lowered with lycorine treatment. additionally, nup93 had the same levels in both lycorine control group and mock group. lycorine significantly reduced nup93 expression after influenza virus infection, which may have affected nucleocytoplasmic transport instead of lycorine toxicity in the host cells. npcs are composed of approximately 30 proteins known as nucleoporins. nup93 is one of the major subcomplexes of the npc and is responsible for the correct assembly of the npc (sachdev et al., 2012) . given its time-of-addition effect, lycorine is involved in the early steps of influenza virus replication aside from virus binding and viral rnp activity (he et al., 2013) . nucleocytoplasmic transport is integral to the majority of the influenza virus explicative cycle and critical for the efficient replication of influenza virus. importantly, shuttling of specific proteins out of the nucleus is essential for the regulation of the basic functions of the host cells. the functions of nucleo-cytoplasmic transport in regulating tumor growth, cell cycle, and apoptosis have become the therapeutic target of cancer (gravina et al., 2014; hill et al., 2014; rosebeck et al., 2016) . recent data have indicated that viral genome transport through the npc is not always smooth sailing but can be blocked. this result may be the basis for host defense mechanisms that evoke an intrinsic antiviral response (flatt & greber, 2015; kirli et al., 2015) . furusawa, yamada & kawaoka (2018) showed that viral rna accumulates in the nucleus of nup93-depleted cells. this observation suggests that nup93 is involved in the nuclear export of viral rna of the viral life cycle. a deeper understanding of how aiv rna is exported from the nucleus via npcs will thus help in the development of new antiviral drugs. lycorine is an inhibitor of influenza virus that effectively inhibits aiv infection. comparative proteomic analysis revealed that treatment with lycorine alters the expression of a number of proteins in aiv-infected cells. this finding suggests that lycorine affects the protein expression in aiv-infected cells. among the 71 deps that were modulated in both v/c and v/l groups, candidate targets were predicted to collectively inhibit viral infection (pathway analysis). go and kegg pathway analyses revealed that nup93, as an important npc component that directs nucleocytoplasmic transport, was induced by virus infection but sharply decreased by lycorine treatment. therefore, nup93 may serve as a potential antiviral target for genetic manipulation. however, further functional characterization of nup93 is necessary before this possibility can be confirmed. for instance, nup93 knockdown or knockout experiments may be performed on these proteins individually to evaluate the functional effects on aiv replication. genetic compatibility of reassortants between avian h5n1 and h9n2 influenza viruses with higher pathogenicity in mammals off-line high-ph reversed-phase fractionation for in-depth phosphoproteomics antibacterial activity and virtual screening by molecular docking of lycorine from pancratium foetidum pom (moroccan endemic amaryllidaceae) alternative non-apoptotic death pathway nucleo-cytoplasmic transport as a therapeutic target of cancer a conserved inhibitory mechanism of a lycorine derivative against enterovirus and hepatitis c virus amaryllidaceae alkaloids inhibit nuclear-to-cytoplasmic export of ribonucleoprotein (rnp) complex of highly pathogenic avian influenza virus h5n1 5, 10b-ethanophenanthridine amaryllidaceae alkaloids inspire the discovery of novel bicyclic ring systems with activity against drug resistant cancer cells targeting nucleocytoplasmic transport in cancer therapy lycorine is a novel inhibitor of the growth and metastasis of hormone-refractory prostate cancer design and expeditious synthesis of organosilanes as potent antivirals targeting multidrug-resistant influenza a viruses functional proteomic studies of lycorine-treated mdck cells on highly pathogenic avian influenza h5n1 virus infection rapid identification of inhibitors that interfere with poliovirus replication using a cell-based assay lycorine induces cell death in mm by suppressing janus kinase/signal transducer and activator of transcription via inducing the expression of socs1 amaryllidaceae alkaloids exhibit anti-influenza activity in mdck cells, an investigation of amaryllidaceae alkaloids and mdck cells insight lycorine inhibits lipopolysaccharide-induced inos and cox-2 up-regulation 7 cells through suppressing p38 and stats activation and increases the survival rate of mice after lps challenge hepatitis c virus induces the localization of lipid rafts to autophagosomes for its rna replication anti-inflammatory activity of crinum asiaticum linne var. japonicum extract and its application as a cosmeceutical ingredient a deep proteomics perspective on crm1-mediated nuclear export and nucleocytoplasmic partitioning assessing species biomass contributions in microbial communities via metaproteomics hoxa repression is mediated by nucleoporin nup93 assisted by its interactors nup188 and nup205 lycorine, the main phenanthridine amaryllidaceae alkaloid, exhibits significant antitumor activity in cancer cells that display resistance to proapoptotic stimuli: an investigation of structure-activity relationship and mechanistic insight lycorine and its derivatives for anticancer drug design identification of natural compounds with antiviral activities against sars-associated coronavirus apoptosis induced by lycorine in km3 cells is associated with the g0/g1 cell cycle arrest lycorine alkaloids from hymenocallis littoralis identification of human host proteins contributing to h5n1 influenza virus propagation by membrane proteomics lycorine reduces mortality of human enterovirus 71-infected mice by inhibiting virus replication evaluation of phosphopeptide enrichment strategies for quantitative tmt analysis of complex network dynamics in cancer-associated cell signalling alkaloids with activity against the zika virus vector aedes aegypti (l.)-crinsarnine and sarniensinol, two new crinine and mesembrine type alkaloids isolated from the south african plant nerine sarniensis influenza virus-induced caspase-dependent enlargement of nuclear pores promotes nuclear export of viral ribonucleoprotein complexes screening of a library of traditional chinese medicines to identify anti-malarial compounds and extracts synthesis and characterization of norbelladine, a precursor of amaryllidaceae alkaloid, as an anti-inflammatory/anti-cox compound comparison of influenza virus yields and apoptosis-induction in an adherent and a suspension mdck cell line effect of alkaloids isolated from amaryllidaceae on herpes simplex virus synergistic myeloma cell death via novel intracellular activation of caspase-10-dependent apoptosis by carfilzomib and selinexor the c-terminal domain of nup93 is essential for assembly of the structural backbone of nuclear pore complexes cytoscape: a software environment for integrated models of biomolecular interaction networks we would like to thank prof. ming liao for providing bsl-3 facilities. we gratefully acknowledge shanghai omicsspace biotechnology co. ltd. for technical assistance with the peptide/protein identification. this work was financially supported the national natural science foundation of china (grant no. 81603165) and the natural science foundation of guangdong province (no. 2016a030310085). there was no additional external funding received for this study. the the following grant information was disclosed by the authors: national natural science foundation of china: 81603165. natural science foundation of guangdong province: 2016a030310085. the authors declare there are no competing interests. • li yang performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper.• jia hao zhang performed the experiments, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper.• xiao li zhang, guang jie lao and guan ming su performed the experiments.• lei wang and yao lan li contributed reagents/materials/analysis tools.• wen cai ye contributed reagents/materials/analysis tools, approved the final draft.• jun he conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the paper, approved the final draft. the following information was supplied regarding data availability: raw data is available at pxd (iprox): http://proteomecentral.proteomexchange.org/cgi/ getdataset?id=pxd012936. supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.7697#supplemental-information. key: cord-272986-ebgusf3o authors: cao, yipeng; yang, rui; wang, wei; lee, imshik; zhang, ruiping; zhang, wenwen; sun, jiana; xu, bo; meng, xiangfei title: computational study of ions and water permeation and transportation mechanisms of the sars-cov-2 pentameric e protein channel date: 2020-05-17 journal: biorxiv doi: 10.1101/2020.05.17.099143 sha: doc_id: 272986 cord_uid: ebgusf3o coronavirus disease 2019 (covid-19) is caused by a novel coronavirus (sars-cov-2) and represents the causative agent of a potentially fatal disease that is of public health emergency of international concern. coronaviruses, including sars-cov-2, encode an envelope (e) protein, which is a small, hydrophobic membrane protein; the e protein of sars-cov-2 has high homology with that of severe acute respiratory syndrome coronavirus. (sars-cov) in this study, we provide insights into the function of the sars-cov-2 e protein channel and the ion and water permeation mechanisms on the basis of combined in silico methods. our results suggest that the pentameric e protein promotes the penetration of monovalent ions through the channel. analysis of the potential mean force (pmf), pore radius and diffusion coefficient reveals that leu10 and phe19 are the hydrophobic gates of the channel. in addition, the pore demonstrated a clear wetting/dewetting transition with monovalent cation selectivity under transmembrane voltage, which indicates that it is a hydrophobic voltage-dependent channel. overall, these results provide structural-basis insights and molecular-dynamic information that are needed to understand the regulatory mechanisms of ion permeability in the pentameric sars-cov-2 e protein channel. covid-19 is a severe and highly contagious respiratory illness that was first reported in china in early december 2019. subsequently, the virus has spread worldwide. as of may 6, 2020, millions of cases have been confirmed, and hundreds of thousands have died. the world health organization (who) announced a global pandemic for covid-19 in march 2020. in addition to the hazards of the disease itself, it has also led to a severe turbulence in international financial markets and may cause serious consequences such as a financial crisis. covid-19 is a disease caused by a new coronavirus named sars-cov-2. it is speculated that it originated from bats and was transmitted to humans through an intermediate host (some kind of wildlife). its symptoms include fever, general malaise, dry cough, shortness of breath, and respiratory distress. comparing similar diseases, including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), which are also caused by coronaviruses, the mortality rates for sars and mers were 10% and 36%, respectively 1 . currently, covid-19 has a mortality rate of 1 to 10% in different countries but appears to be more contagious than sars and mers 2 3 4 5 . similar to other coronaviruses, sars-cov-2 is a long positive-sense, long single-stranded (30 kb) rna virus. the structure of different human coronaviruses (hcovs) is similar. the viral genome is packed by nucleocapsid (n) proteins, forming a helicoidal nucleocapsid protected by a lipid envelope 6 . several viral proteins, including the spike (s), envelope (e), and membrane (m) proteins, are embedded within a lipid envelope 7 8 . studies have shown that s, m, and n proteins play important roles in receptor binding and virion budding. for example, the m protein participates in virus germination and interacts with the n and s proteins. the s protein has immune recognition sites and can be used to design vaccines 9 . currently, the importance of the e protein has not been fully revealed. evidence suggests that the e protein maintains its morphology after virus assembly by interacting with the m protein 10 11 12 . when an e protein gene mutation occurs, it promotes apoptosis. recent studies have shown that coronaviruses have a viroporin that can self-assemble into a pentameric structure and have ion selectivity. when there is a transmembrane voltage, the ion channel characteristics of viroporins are more significant. in addition, the asn25ala and val18phe mutations could destroy ion channel activity 13 . this indicates that the e protein may play an important role in regulating the ion equilibrium inside and outside the viral envelope. the ion channel activity of the e protein can lead to increased levels of the inflammatory cytokines il-1β, tnf and il-6 in the lungs, leading to the occurrence of an "inflammatory storm" 14 . this plays a key role in the progression of the disease and may cause the patient's condition to suddenly deteriorate and lead to death. in addition, its evolutionary conservatism may be an important cause of viral cross-host infection 15 16 17 . although previous studies have suggested that the e protein of coronaviruses such as sars and mers oligomerizes and has ion permeability, the specific mechanism of ion permeability and channel properties remain to be explored due to the lack of a crystal structure for the e protein. in 2014, li et al 17 identified the sars-cov e protein monomer structure. in 2018, surya et al. extracted the pentamer structure of the sars e protein by nuclear magnetic resonance (nmr) 18 , which provided strong support for the study of the sars-cov-2 e protein pentamer ion permeability mechanism. in this study, we obtained the amino acid sequence of the sars-cov-2 e protein from the national center for biotechnology information (ncbi) database 19 . the e protein pentamer model of sars-cov-2 was built by using the homology modeling method, and the reasonableness of the model was evaluated. subsequently, µs-level molecular dynamics (md) simulations were performed to evaluate the pentamer's stability in the membrane environment. we tried to use potential mean force (pmf) to reveal the permeability of different physiological ions and water molecules in the pores of the e protein pentamer. the characteristics of the pentameric channel were analyzed by combining the channel diffusion coefficient and geometric properties. in addition, computational electrophysiology was applied for different transmembrane voltages of the system to reveal the effect of the voltage on the ion permeability of the pentameric e protein. overall, exploring the mechanisms of the pentameric sars-cov-2 e protein not only provides valuable insights into the conduction of the channel but also has important implications for our understanding of the difference between sars-cov-2 and other coronaviruses. sequence alignment and homology modeling figure 1a shows the sequence alignment between the e proteins of two human coronaviruses (sars-cov and sars-cov-2 sequence) made by clustal x software. the blue dotted rectangle in figure 1b the rampage online program 20 was used to evaluate the accuracy of the sars-cov-2 pentameric e protein model. more than 98.9% of the amino acids are within the acceptable range, suggesting that the sars-cov-2 e protein is similar to the sars e protein nmr model. subsequently, a 1000 ns (1 µs) md simulation was performed to evaluate the stability of pentameric e protein embedded in the membrane environment. the root-mean-square deviation (rmsd) of the model is shown in figure 2 , and the red and blue curves represent the whole protein and tm region, respectively. in the first 200 ns, the rmsd continued to rise, indicating that the model needed longer optimization (compared with other ion channels) to reach the pentameric structure equilibrium. during the last 800 ns, the curves plateaued. the rmsd of the whole protein and the tm region converged at ~0.4 and ~0.3 nm, respectively. there is a 0.1 nm difference between the whole protein and tm region. these findings are consistent with other ion channel data that show that the tm region has a higher stability than the other parts of the membrane protein 21 . the permeability of the sars-cov-2 pentameric e protein channel is very important for understanding the replication ability of viruses in cells and how they are secreted into the extracellular medium 22 . it is possible to examine a profile of the free energy of a single ion or water as a function of its position along the pore axis by calculation of pmf. in this simulation, the ions or water molecules are restrained to a continuous position along the z-axis and move freely in the pore's xy plane. moreover, other parts of the system (proteins, ions, water molecules, and lipids) can move freely and reach equilibrium. the pmf of important physiological ions (mg2 + ; ca 2+ ; cl-; k + and na + ) and the water molecules as a function of their position along the pore (z)-axis were calculated separately. figure 3a shows the pmf of ions and water molecules permeating through the sars-cov-2 e protein pentamer pore. z m is defined as the axial direction along the pore, which is from -3 ~ 3 nm (the length of umbrella sampling is 6 nm in total). from the pmf curve, we found that the energy barriers of monovalent and divalent ions have a significant difference. the maximum energy barriers of the two divalent ions ca 2+ and mg 2+ are 60 kj/mol and 70 kj/mol, respectively. in contrast, the pmfs of the monovalent ions na + and k + are ~ 12 kj/mol and ~20 kj/mol, respectively, while the cl-energy barrier is ~ 30 kj/mol, which is significantly smaller than that of ca 2+ and mg 2+ . from an energy perspective, the sars-cov-2 pentameric e protein is almost impermeable to divalent ions. this confirms the previous hypothesis that the sars-cov and mers-cov e protein channel is a monovalent cation channel 23 24 . the maximum energy barrier in descending order is as follows: na + <k + <cl-<mg 2+ <ca 2+ . additionally, the barrier of water molecules is only ~ 5 k/mol, which is similar to the energy threshold of free diffusion in liquid. interestingly, there is no obvious peak in the pmf curve for na + ions and water molecules. this result indicates that the e protein pentamer pores are not permeable to divalent ions. the difference in the energy barrier between the monovalent cations is large, suggesting that the channel has selective permeability for specific ions. to explore the permeability of the channel, we calculated the diffusion coefficient (d c ) using a single k + ion as a probe. by fitting the k + mean square displacement (msd) with the einstein equation (msd = 2d(c)t), we obtained the diffusion coefficient. as shown in figure 3b , the average diffusion coefficient of k + outside the pore is ~ 2.0×10 -5 ±0.4 cm 2 /s, which is consistent with the experimental data of k + in the liquid environment 25 . however, the d c decreases by approximately 70% when the k + probe is in the channel pore. the d c curve of the internal channel pore has a flat bottom and is similar to the pmf. the mid-value of d c = 0.35×10 -5 ±0.2 cm 2 /s indicates that there is a large obstacle for probe diffusion in the inner pore, showing a similar value to other narrow hydrophobic pores 26 27 . next, we used the solubility-diffusion equation (eq 1) 28 29 to evaluate the permeability coefficient (p) between two different ions. eq 1 in eq 1, pmf(z) and d(z) are functions of pmf and dc along the z-axis, z1 denotes a position on one side of the pore and z2 denotes a position on the other side. kb is the boltzmann constant, and t is the simulation temperature. the part of the pmf in the tm region is approximate to the graph of the quadratic function, so the pmf(z) uses quadratic function fitting. the linear fitting of the corresponding d c gives the function d(z). the ratios of the p for na + , k + and cl-in the maximum energy barrier region were simply evaluated. p na+ /p k+ = 22.42, p k+ /p cl-= 5.82, and p h2o /p na+ = 402.57. covid-19 is an unprecedented threat to humanity. to understand the infection, the replication mechanism of the virus in the human body is particularly important. currently, research on the pathogenic mechanism and infection route of covid-19 mainly focuses on the s protein 38 the sars-cov-2 e protein pentamer has been considered an unknown nature ion channel. there is significant similarity in the tm region sequences (reaching 91.8%) and high gene homology between the sars-cov and sars-cov-2 e proteins; five different residues are all located at the region of interaction between the protein and membrane, while the sequences of the tm region are completely identical. the highly conserved tm region also suggests similar e protein functions in different coronavirus subtypes. as the rmsd of the e protein pentamer during the 1 µs md simulation, the structure has a certain flexibility, showing that its geometric deformation can make the channel permeable to ions and water molecules. figure 2 shows that the tm region of the sars-cov-2 e protein remains relatively stable during the simulation, indicating the stability of the pentamer in the membrane environment, which is consistent with previous studies of sars-cov and mers-cov. the free energy calculation of the ions permeating through the sars-cov-2 pentameric e protein channel strongly suggests that the pore has selection permeability for monovalent ions. the maximum energy barriers of na + , k + , and clare 15 kj/mol, 20 kj/mol, and 30 kj/mol, respectively. the comparison of the p values for na + , k + and clshows that the p of na + is 22 times greater than that of k + , while the p of k + is 6 times greater than that of cl -. although the computational permeability coefficient is two times higher or lower than that observed experimentally for sars-cov, it still shows a very large gap in the permeation between ions and water molecules. additionally, there is no obvious peak in the pmf when na + and water molecules permeate through the channel, further supporting that na + and water molecules have less penetration resistance at the inner pore. this further confirmed the selectively for na + . the ion selectivity of sars-cov-2 is different from that of sars-cov 23 . because the maximum energy barrier of mg 2+ and ca 2+ is 2 ~ 3 times higher than that of the monovalent ions, divalent ions cannot overcome such a very large energy barrier under the simulation environment. the conformational analysis indicates that it is mainly caused by the geometric size of the pore (fig 6) . the importance of transmembrane voltage on viroporin has been found in many viruses, including hiv1 vpu oligomers and the hepatitis c virus (hcv) p7 protein, etc. 42 43 44 . although it has been observed in many experiments, this mechanism has not been revealed. because water molecules play an important role in hydrophobic the channel wetting/dewetting transitions appeared under different transmembrane voltages, and the radius of the pore increased. meanwhile, the capacity to contain water molecules also increased. further analysis of the trajectories revealed that the monovalent cations (na + and k + ) will permeate through the pore with water molecules when the transmembrane voltage is > 0.3 v. computational electrophysiology also explained that the transmembrane voltage led to an easier wetting transition for the channel. the range for keeping the channel open should be between 0.15 v and2 0.45 v. na + and k + could be cotransported during water permeation, which is very similar to that in many hydrophobic channels 45 46 47 48 . intriguingly, we did not observe clconduction, which may be because chloride ions could not overcome the energy barrier under the maximum transmembrane voltage. in addition, the change in the pore radius at different voltages highlights two important sites, where the geometric radius changes the most, corresponding to leu10 and phe19. phe19 contains a benzene ring group at the hydrophobic surface of the inner pore as a hydrophobic gate. the isopropyl group of leu10 at the bottom of the pore prevents the reverse penetration (fig 6a) . previous studies indicated that asn15val and val25phe mutations will make the channel dysfunctional. this may be due to the additional side chain groups in the pores caused by these mutations (especially the benzene ring in val25phe), making 1) the radius of the pores decrease and 2) the hydrophobicity of the pores increase. in short, these mutations may increase the energy barrier, negatively affecting the wetting transition and causing the channel to be functionally closed. therefore, the sars-cov-2 e protein pentamer is a voltage-dependent hydrophobic channel. we propose that the e protein may play an essential role in the virus infection and replication processes through the following mechanisms: 1) the monovalent selective permeation of the e protein pentamer ion channel may change the intracellular ph, providing a suitable microenvironment for virus replication. 2) selective permeation can form a transmembrane voltage, creating feedback regulation and maintaining an intracellular microenvironment that is suitable for viral growth. 3) the disintegration of the ion equilibrium of the intracellular area affect the charges coming from the cell through ion channels in the cell membrane, changing the ph and making it easier for the virus to fuse with the cell membrane. this is the infection mechanism of the avian coronavirus as well as some types of influenza viruses 49 50 . 4) it has a certain signal transduction function. although the pentameric sars-cov-2 e protein crystal structure is not yet available, preparation of the sars-cov-2 pentameric e protein-membrane simulation system the amino acids sequence of the human coronaviruses e proteins were downloaded from the national center for biotechnology information (ncbi to obtain the equilibrated pentameric sars-cov-2 e protein model, charmm36 all-atom force field 53 was chosen for md simulation. the md time step was set at 2 fs. electrostatic interactions were described using the particle mesh ewald (pme) algorithm 54 with a cut-off of 1.2 nm. the lincs algorithm 55 was used to constrain the bond lengths. the pressure was maintained semi-isotropically at 1 bar at both x and y directions using the perinello-rahman barostat algorithm 56 and the system temperature was maintained at 310 k by the nose-hoover thermostat 57 . then, a 1000 ns (1 µs) md simulations were performed for the e protein pentameric. umbrella sampling the initial system for umbrella sampling simulations was derived from the equilibrated pentameric sars-cov-2 e protein mentioned above. a single ion that maintain physiological activity (na + , k + , ca 2+ , mg 2+ , cl -) or water molecule was placed at successive positions along the central pore axis by using gromacs pull code. energy minimization was performed before simulation for optimizing the water and ions position. the reaction coordinate defined from z +3 to -3 nm with the mass center at z = 0 nm, with a spacing of 0.1 nm between successive windows, resulting in 60 umbrella sampling simulation systems. the probe ion or water molecules were harmonically restrained by a force constant of 2000 kj mol/nm 2 same with the pore direction. each window was performed a 5 ns total umbrella sampling simulation. the initial 2 ns for system equilibration, then a subsequent 3 ns was applied for analysis. the pmfs were computed by the weighted histogram analysis method (wham) 58 , and the profile was generated by gromacs protocol 'g_wham' 59 . bootstrap analysis (n = 50) was used to estimate statistical error and the '-cycl' parameter was used to make the value equal. the diffusion-coefficient was calculated using the method described by shirvanyants et al. 60 . we restrained the e-protein pentamer helix backbones and made the side chains, membrane, water and ions free to move. due to the ion selectivity is related to the side chain of inner pore 61 , the backbone restraint maintained the e-protein pentamer tertiary structure but the side-chain flexibility in the inner pore was not influenced. a k + as the probe ion, the calculation system was used in the same manner as for umbrella sampling. the ion mean-square displacement (msd) was calculated along the pore's z-axis. a total of 25 simulation systems were obtained, each widows interval distance of the k + being set as 0.24 nm. the umbrella restraint was used to maintain the k + ion's position on the x-y plane of the pore. the einstein equation msd=2d(z)t was used to calculate the diffusion coefficient by the protocol g_msd. the umbrella restraint can be disregarded for these analyses because of the restraint force was negligible compared to thermally induced rms fluctuations. computational electrophysiology (ce) is a good tool for simulating the ion conduction ability of a channel under transmembrane voltage. we established a sandwich structure including three parts (membrane-protein-water) as described by kutzner et al. 62 as shown in fig 4a, each water layer contained a different number of ions. due to the imbalance of the ion distribution of the water layer, the ions gradient will produce transmembrane voltage. specific transmembrane voltage can be applied to the simulation system by adjusting the number of ions between the water layers. in this study, we built the sars-cov-2 e protein sandwich system. the initial single layer system was equilibrium for 50 ns to make the structures stable. then the single-layer system was duplicated along the z direction. the resulting system had a size of around 11×11×22 nm 3 sars and mers: recent insights into emerging coronaviruses real estimates of mortality following covid-19 infection case-fatality rate and characteristics of patients dying in relation to covid-19 in italy coronavirus: covid-19 has killed more people than sars and mers combined, 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three influenza virus h3 subtypes swiss-model: modelling protein tertiary and quaternary structure using evolutionary information charmm-gui: a webbased graphical user interface for charmm charmm general force field (cgenff): a force field for drug-like molecules compatible with the charmm all-atom additive biological force fields particle mesh ewald: an n⋅ log (n) method for ewald sums in large systems lincs: a linear constraint solver for molecular simulations polymorphic transitions in single crystals: a new molecular dynamics method the nose-hoover thermostat the weighted histogram analysis method for free-energy calculations on biomolecules. i. the method a free weighted histogram analysis implementation including robust error and autocorrelation estimates pore dynamics and conductance of ryr1 transmembrane domain principles of selective ion transport in channels and pumps. science (80-. ) computational electrophysiology: the molecular dynamics of ion channel permeation and selectivity in atomistic detail vmd: visual molecular dynamics gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers this study was supported by the national natural science foundation of china all other authors declare no competing interests. key: cord-280429-4fota9rl authors: medvedev, kirill e.; kinch, lisa n.; grishin, nick v. title: functional and evolutionary analysis of viral proteins containing a rossmann‐like fold date: 2018-06-13 journal: protein science doi: 10.1002/pro.3438 sha: doc_id: 280429 cord_uid: 4fota9rl viruses are the most abundant life form and infect practically all organisms. consequently, these obligate parasites are a major cause of human suffering and economic loss. rossmann‐like fold is the most populated fold among α/β‐folds in the protein data bank and proteins containing rossmann‐like fold constitute 22% of all known proteins 3d structures. thus, analysis of viral proteins containing rossmann‐like domains could provide an understanding of viral biology and evolution as well as could propose possible targets for antiviral therapy. we provide functional and evolutionary analysis of viral proteins containing a rossmann‐like fold found in the evolutionary classification of protein domains (ecod) database developed in our lab. we identified 81 protein families of bacterial, archeal, and eukaryotic viruses in light of their evolution‐based ecod classification and pfam taxonomy. we defined their functional significance using enzymatic ec number assignments as well as domain‐level family annotations. rossmann-like fold 1,2 is the most populated fold among a/b-folds in the protein data bank. 3 it was first found in a wide range of nucleotide-binding proteins that utilize diphosphate-containing cofactors such as nad(h). these structures included two sets of b-a-b-a-b units (321456 topology), forming a single parallel sheet flanked of a three layer a/b/a sandwich. 4 an important structural feature of this fold includes a crossover observed between strands 3 and 4. this crossover creates a natural cavity that participates in the binding of the nucleotide ring. 5 we can therefore define a minimal rossmann-like motif as a three-layer a/b/a sandwich with at least three parallel b-strands and a crossover between the second and third strands. in fact, protein structures containing this minimal defined unit constitute 22% of the protein data bank (see "materials and methods"). rossmann domains are linked to a great variety of different catalytic domains and metabolic enzymes and can be found in different viruses. 6 as an abundant life form that infects practically all organisms, viruses are a major cause of human suffering and economic loss. previous studies showed that in marine, soil, and animal-associated environments, the number of virus particles is typically 10-100 times greater than the number of cells. 7 this so-called "virosphere" is probably inclusive of every environment on the earth, from the atmosphere to the deep biosphere. 8 the viromes of the three domains of cellular life (bacteria, archea, and eukaryotes) are fundamentally different. in prokaryotes, most have double-stranded dna genomes, with a substantial minority of single-stranded dna viruses and only limited presence of rna viruses. on the other hand, in eukaryotes, rna viruses account for the majority of the virome diversity, although ssdna and dsdna viruses are common as well. 9 although several families of dsdna viruses are represented in both bacteria and archea, no viruses are known to be shared by eukaryotes with any of the other two cellular domains, even at the family or order level. 10 however, structural analyses of virion architecture and coat protein topology have revealed unexpected similarities, not visible in sequence comparisons, suggesting a common origin for viruses that infect hosts residing in different domains of life. 11 given the prevalence of rossmann-like folds in nature, their analysis in the viral structure proteome could provide an understanding of viral biology and evolution as well as could propose possible targets for antiviral therapy. in this current work, we provide functional and evolutionary analysis of viral proteins containing a rossmann-like fold that can be found in the evolutionary classification of protein domains (ecod) database developed in our lab. 12 ecod is a hierarchical classification based on evolutionary concepts that consists of five levels: architecture (a), possible homology (x), homology (h), topology (t), and family (f). 13 we identified and described protein families of bacterial, archeal, and eukaryotic viruses in the light of their classification in ecod and defined their functional significance using enzymatic ec number assignments as well as domain-level family annotations. 81 protein families were defined as viral proteins containing a minimal rossmann fold motif. a few well-populated viral folds tend to distribute across multiple host kingdoms, including ploop domains-related (2004.1), rossmann-related (2003.1) , and udp-glycosyltransferase/glycogen phosphorylase (2111.1). alternatively, numerous fold types tend to be specific to their viral host kingdom, potentially explaining the difference in their viriomes and suggesting targets for therapy. using the definition of a minimal rossmann-like folding unit that contains a three-layer a/b/a sandwich with at least three parallel b-strands and a crossover between the second and third strands, structures of 1427 viral rossmann-like domains were detected in the ecod database. these domains were found in 512 (6.7% of all known viral protein structures) pdb structures and were assigned by ecod to 81 protein family groups and 24 homology groups (fig. 1) . the structures represented gene products from 21 viral taxonomical families with host ranges from all kingdoms of life (http://prodata.swmed.edu/rossmann_fold/viruses/). 4 of 21 viral taxonomical families infect bacteria, one infects archea and eighteen infect eukaryota ( fig. 1 -green, violet, and orange colors, respectively). the biggest taxonomical family in terms of amount phages are viruses that infect bacteria; their selfreplication depends on access to a bacterial host. the rising tide of antibiotic resistance coupled with the low rate of antibiotic discovery has revived interest in phages as antibacterial agents. 14 phages have been used not only to treat and prevent human bacterial infections but also to control plant diseases, detect pathogens, and assess food safety. e.coli bacteriophages are the most popular objects for these purposes, 14 which perhaps explains their relative abundance of structures (fig. 1 ). our analysis detected 14 different bacterial virus structure topology types defined by ecod t-groups that contain a rossmann-like fold (fig. 2, 12 topology groups shown). the largest "rossmann-related" homology group (2003.1) adopts a classical rossmann-like topology with additional b-strands at the c-terminal end for several families [ fig. 2(a,b) ]. the tubulin family [ fig. 2(b) ] of this homology group has a sheet topology 321456 representing a canonical gtpbinding domain. pseudomonas phikz-like bacteriophages encode a group of related tubulin/ftsz-like proteins believed to be essential for the correct centering of replicated bacteriophage virions within the bacterial host. 15 this phage protein classifies together with bacterial and archeal ftsz proteins as well as eukaryotic tubulin alpha, beta, and gamma chains. the similar protein sequence and structure, in addition to the gtp-dependent polymerization activities suggest all evolved from a common ancestor. one family from the "p-loop domains-related" [2004.1, fig. 2 (c,d)] homology group, phosphomevalonate kinase [p-mevalo_kinase, pmvk, fig. 2 (c)], contains a subfamily of phage t4 deoxynucleotide kinases that are somewhat distinct from the canonical animal enzymes catalyzing phosphorylation of 5phosphomevalonate into 5-diphosphomevalonate, an essential step in isoprenoid biosynthesis. given the idea that pmvk enzymes arose from nonorthologous gene displacement early in animal evolution, 16 perhaps the phage system t4 deoxynucleotide kinases played a role in their alternate evolutionary origins. another family member of this h-group, the atpase p4 from bacteriophage phi12 [ntpase_p4, fig. 2(d) ] contains motor proteins involved in the packaging of pseudomonas phage phi-12 genome into preformed capsids using atp to drive translocation. 17 the central p4 structure core, together with part of the cterminal region, forms a rossmann-type domain containing a twisted, eight-stranded b-sheet of mixed parallel and antiparallel topology flanked by five helices. although the p4 family is limited to phage proteins, their presumed homologous relationship to other p-loop structures suggests common evolutionary origin. 18 the toprim-like [toprim_2, fig. 2 (g)] family belongs to "had domain-related" homology group [2006.1, fig. 2 (e-g)] and is represented by dna primase-helicase proteins. 19 phage t7 encodes dna polymerase, primase, helicase, and single-stranded dna binding activities that catalyze the replication of double-stranded phage dna. primase and helicase activities reside in a bifunctional primase-helicase protein that assembles into ring-shaped hexamers. 20 given their essential role in phage replication, these viral toprim-like proteins are predominantly found in bacteriophages and nucleocytoplasmic large dna viruses. 21 two possible evolutionary scenarios have been proposed for primase-helicase proteins. the first involves fusion of primase and helicase genes, while the second considers the primase-helicase gene as an ancestor that underwent duplication and divergence followed by physical separation of primase and helicase functions. the phage tail sheath protein domain [phage_-sheath_1, fig. 2 (h)] with the following common sheet topology (231456) contains proteins that play crucial roles in contracting the tail of bacteriophage t4 through the host outer membrane during infection. 22 the phage tail sheath protein resembles the fold of the type vi secretion system protein (t6ss) vipa/vipb heterodimer that intertwines to adopt the rossmann-like architecture. 23 in fact, components of the t6ss system, which delivers effector proteins into a target cell, and the phage tail-associated complex are thought to have arisen from a common ancestor. 24 the t4-glucosyltransferase family [ fig. 2 (k)], limited to phage proteins, catalyzes the transfer of glucose from uridine diphosphoglucose to 5hydroxymethyl cytosine. the role of glycosylation in protecting the infecting viral dna from host restriction enzymes has been reviewed. 25 comparison of the glucosyltransferase family fold to other families showed that it is completely embedded in the structure of glycogen phosphorylase. all nine a-helices and 13 b-strands of b-glucosyltransferase match elements of glycogen phosphorylase in sequential order. 26 the significance of the match is further supported by the fact that the first and second domains of the common core are architecturally distinct despite topographical similarity to the classical nucleotide-binding fold. it appears paradoxical that the architecture of t4 b-glucosyltransferase is simpler and therefore appears more primitive than that of glycogen phosphorylase, which is the older enzyme by phylogenetic arguments. 26 the paradox may be resolved by either of two evolutionary models that differ in the point of divergence between glycogen phosphorylase and t4 bglucosyltransferase. the first model postulates that the gene for t4 b-glucosyltransferase diverged rather recently from a fully evolved glycogen phosphorylase and evolution in t4 rapidly simplified the structure of the protein down to the essential catalytic core. in the second model, both descend along separate lineages from a very ancient common ancestor. the n-acetylmuramoyl-l-alanine amidase (ami-dase_3) family contains viral proteins that exhibit high specificity towards the cell walls of their host bacteria. the core of these domains is formed by a twisted, six-stranded b-sheet flanked by six helices. b-strands 4 and 5 are antiparallel, which makes the catalytic domain unique among the known listeria phage endolysins. 27 based on their antimicrobial properties, endolysins from phages infecting grampositive pathogens have recently attracted attention as potential therapeutic agents. 28 initial studies were successfully carried out with oral streptococci in mice 29 as well as with bacillus anthracis in vitro. 30 also genetically modified lactic acid bacteria that are able to synthesize and secrete active listeria phage endolysin were constructed to protect food fermentation products. 31 cutinase [ fig. 2 (l)] belongs to the a/b-hydrolase h-group and is another big family that contains proteins from different domains of life. cutinase is a serine esterase with the classical ser, his, asp triad of serine hydrolases. the structure of a cutinase shows an a/b sandwich organization similar to lysin b (lysb) enzymes. viral lysb proteins are produced by mycobacteriophages to degrade the host peptidoglycan layer. the activity also circumvents a mycolic acid-rich outer membrane covalently attached to the arabinogalactan-peptidoglycan complex in the wall of mycobacterium. 32 the five closest related structures of lysb are all cutinases, although there is no greater than 21% amino acid sequence identity with any of them. the lysb catalytic mechanism is expected to be similar to that for other serine esterases. acquisition of lysb by mycobacteriophages throughout their evolution likely confers a substantial selective advantage over those without it by providing faster and more complete lysis. 32 like the bacterial and eukaryotic branches in the tree of life, the archea are host to a multitude of functional and evolutionary analysis of viral proteins viruses. however, compared to viruses infecting the domains eukaryota and bacteria, studies of viruses infecting the archea are still in their infancy. 33 the pdb database includes 305 domains in 55 structures of archeal-specific viral nonchimeric proteins and only three domains in three structures were defined as rossmann-like (fig. 3 ). in addition to these archea-specific viruses, all types of dsdna viruses known to infect bacteria can replicate in archea, including head-tailed viruses (families myoviridae, siphoviridae, and podoviridae), icosahedral viruses with internal lipid envelopes (families sphaerolipoviridae and turriviridae in archea; tectiviridae and corticoviridae in bacteria), and pleomorphic viruses (families pleolipoviridae in archea and plasmaviridae in bacteria). 34 one example of an archealinfecting viral protein is the b116 protein (duf1874 family) of sulfolobus turreted icosahedral virus (stiv). 35 while the 37 stiv open reading frames (orfs) generally lack sequence similarity to other genes orf b116 is common to the genomes of three additional hyperthermophilic archeal viral families, the rudiviridae, the lipothrixviridae and the bicaudaviridae. the b116 polypeptide folds to form a fivestranded, predominantly parallel b-sheet (topology 31452) lined on one side by three a-helices, with strand b5 running antiparallel to the remaining strands. interestingly, an intramolecular disulfide bond, contributed by cys33 and cys62, is also observed. this covalent link between the a1 and a2 helices is likely to enhance the thermostability of the b116 fold. two copies of the b116 polypeptide are found in the asymmetric unit, giving rise to the homodimer and forming a larger 10-stranded unclosed barrel, giving rise to a saddle shaped protein. authors suggested that this unclosed barrel could be a place of nonspecific dna binding in which the rossmann-like fold also could take part. 35 one more example from stiv is the a197 protein (euf08621 family). the structure of the a197 monomer reveals a six-stranded, predominantly parallel, a/b/a sandwich that is flanked by a fourstranded antiparallel b-sheet with an extended c terminus. structure similarity data identified members of the glycosyltransferase (gt-a) superfamily as the closest structural homologues of this protein. a197 is one of the smallest known glycosyltransferases, composed of only the core catalytic gt-a fold and lacking additional functional domains. thus, its structure may define the minimal components necessary for glycosyltransferase activity. 36 the third example is the b204 protein (aaa_22_like family). it is an atpase belonging to the p-loop containing nucleoside triphosphate hydrolases h-group that is thought to drive packaging of viral dna during the replication process. the structure of stiv b204 is represented by a central nine-stranded b-sheet decorated with seven a-helices. b204 contains a core rossmann-like fold (sheet topology 32451) followed by a b-meander with a helical hairpin stemming from one of the loops. related p-loop atpases with this identical topology also function to translocate dna; including the bacterial conjugation protein trwb that transfers bacterial dna across membranes and between cells 37 and the virb4 atpase of the bacterial type iv secretion system that mediates the transfer of proteins and dna across bacterial membranes. 38 in contrast to bacteria and archea, eukaryota hosts numerous, diverse rna viruses, retrotranscribing elements and retroviruses that typically integrate into the host genome. 39 the giant viruses of the family mimiviridae are associated with a distinct class of satellite viruses, the virophages, which reproduce within viral "factories" inside their host protist cells and which depend on the latter for their replication. 40 in our dataset mimiviridae encodes two protein families. the glucose-methanol-choline oxidoreductase family, represented by the r135 protein, contains an nterminal fad binding rossmann-related domain followed by a c-terminal substrate recognition domain. the r135 oxidoreductase might participate in degrading the cell walls of their normal hosts, which include some lignin-containing algae. 41 the minimal rossmann domain is composed of a five-stranded parallel b-sheet with the same b-strand topology typical of a nucleotide-binding fold. the second mimiviridae protein family is represented by tyrosyl trna synthetases (tyrrs). these proteins have sixstranded b-sheet topology ordered 432561, with an antiparallel first strand. tyrrs shares 30% identity over 340 residues with the tyrrs of the hyperthermophilic euryarchaeota pyrococcus horikoshii, its closest known structural homologue. trna synthetases are pivotal in determining how the genetic code is translated in amino acids and in providing the substrate for protein synthesis. the discovery of four aminoacyl-trna synthetases encoded in the genome of mimivirus together with a full set of translation initiation, elongation, and termination factors appeared to blur what was once a clear frontier between the cellular and viral world. 42 another eukaryotic infecting virus, vaccinia virus (poxviridae), causes smallpox. the poxviridae genome includes eight protein family groups with five different topology types. one of example is h3 envelop protein-an immunodominant antigen that is expressed late in infection and found as a membrane protein on the surface of virion particles. the nine-stranded b-sheet is made up of strands in the order 679584132 that is surrounded with helices on both sides, and all of the strands except 7 and 8 are oriented parallel to each other. the fold belongs to glycosyltransferases (gts) of the gt-a group. 43 h3 is involved in attachment to the host cell, contributes to viral morphogenesis, and plays a role in infection. 44 since h3 is a major immune system target that is recognized by neutralizing antibodies, h3 is an important viral protein to be included in new vaccines. 45 another example is subunit d12 of vaccinia virus capping enzyme that executes all three steps in m7gppprna synthesis. 46 the topology of the stimulatory subunit d12 reveals a class i n7methyl-transferase (mt) like core, however, with a truncated s-adenosyl-homocysteine-binding domain, consistent with its lack of mt activity. this enzyme has a completely unique mode of binding of the adenosine moiety of s-adenosyl-homocysteine, a feature that could be exploited for design of specific antipoxviral compound. 47 zika virus (flaviviridae) include only one family with a minimal rossmann structure, non-structural protein ns1. the protein fold and domain arrangement of ns1 is virtually identical to dengue virus denv2 protein and west nile virus ns1 protein. despite this overall similarity, the zika virus ns1 crystal structure provides important new information about a domain containing minimal rossmann motif flexible loop that is not visible in previous structures. 48 the structure of the loop reveals an expanded surface permitting ns1 to associate with membranes during replication, to associate with immature virions during particle morphogenesis and to facilitate the interactions necessary for formation of the hexameric lipoprotein complex. the zika virus, which has been implicated in an increase in neonatal microcephaly and guillain-barr e syndrome, has spread rapidly through tropical regions of the world. ns1 plays crucial role in the zika virus life cycle, being a multifunctional virulence factor. 48 reoviridae is a big viral family infecting fish, shellfish, crustacean species, insects, ruminants and human hosts. the reoviridae family includes structure representatives from nine different protein families. rotaviruses are the principal agents of infectious dehydrating diarrhea of infants and the cause of nearly a half-million childhood deaths per year. 49 they have a segmented, double stranded rna (dsrna) genome, packaged within a multishelled virus particle. the outer protein layer of the virion, the molecular machinery for host-cell binding and penetration, contains two protein components, vp4 and vp7. 50 the domain containing minimal rossmann motif of vp7 protein has five parallel bstrands and is assigned to "flavodoxin-like" possible homology group. rotavirus infection and parenteral immunization with virions both induce a strong vp7-specific neutralizing antibody response. this protein is a principal target of protective antibodies. 51 we examined taxonomic distribution of sequences best hits of each family representative in order to define possible evolutionary relationships between viral and host proteins from the same family, which could be the result of horizontal gene transfer (hgt) between virus and host organism. looking at the similarity of the rossmann motif ecod families' sequences to those in the nonredundant sequence database, all rossmann-like fold families were divided into six groups according to appearance of blast hits from four taxonomy groups: a-archea, b-bacteria, e-eukaryota and v-viruses. the first group -31% (25 out of 81) of all protein families under study has universal blast hits from archea, bacteria, eukaryota and viruses ("abev", see two last columns at the online table: http://prodata. swmed.edu/rossmann_fold/viruses/). half (13 out of 25, or 52%) of these families belong to doublestranded dna viruses (dsdna), which can affect bacteria. blast score distributions allow us to assume that horizontal gene transfer (hgt) took place between virus and host bacteria for 7 out of 13 protein families in this universal group (e3uj3x1, e3u5ze1, e2ocaa8, e2ia5b1, e1juva1, e4ieea2, e1xova2). this assumption is based on high-scoring bacterial hits being among viral hits in the blast functional and evolutionary analysis of viral proteins score distributions. the second half of protein families with "abev" hits (12 out of 25, or 48%) belong to viruses that affect eukaryotes, including doublestranded dna and positive single-stranded rna ((1)ssrna) viruses. only one protein family seems to have evidence for hgt between virus and eukaryote host-vaccinia virus thymidine kinase (e2j87a3). the second group-6% (5 out of 81) of rossmann-like fold proteins has blast hits from three taxonomical groups: archea, bacteria and viruses ("abv"). two protein families infect bacteria (dsdna) and three infect archea (dsrna). all these protein families except one (e4r2ia1) have strong evidences for hgt between virus and host. the third group-16% (13 out of 81) has blast hits from three taxonomical groups: bacteria, eukaryota, and viruses ("bev"). six protein families infect bacteria (dsdna), and four of them have evidences of hgt (e3bgwf1, e3hc7a1, e5hd9a1, e2ihna2). the rest infect eukaryotes and belong to double-stranded dna and positive single-stranded rna viruses. the fourth group-11% (9 out of 81) has blast hits from two taxonomical groups: bacteria and viruses ("bv"). of the seven families that infect bacteria (dsdna and dsrna viruses), six have evidences for hgt (e4cu5b1, e4cu2a1, e1deka1, e1y8zb2, e1y8zb1, e2ia5a2). the fifth smallest group-2.5% (2 out of 81) has blast hits from two taxonomical groups: eukaryota and viruses ("ev"). both families belong to human vaccinia dsdna virus with blast distributions having only low-scoring eukaryotic hits. the biggest group-33% (27 out of 81) has blast hits limited to viruses ("v"), with only four families infecting bacteria (dsdna and dsrna viruses) and the rest (23 families) infecting eukaryotes (dsrna, (1) ssrna, dsdna, and (-) ssrna viruses). given the relatively high number of viral protein families that contain a rossmann-like fold (81 families), we sought to examine their evolutionary distributions among folds from the three major host kingdoms. figure 4 highlights the protein family counts from bacteria (blue bar), archea (red bar), and eukaryota (green bar)-infecting viruses that fall within all distinct minimum rossmann fold types. importantly, each fold type includes protein families related by a common ancestor (ecod hgroup). the fold types represented by more than one host kingdom tend to display relatively high family counts. these well-populated folds include p-loop domains-related (2004.1), rossmann-related (2003.1), and udp-glycosyltransferase/glycogen phosphorylase (2111.1). most of the other fold types include only one family representative, with 8 from eukaryotic hosts, 6 from bacterial hosts, and one from archeal hosts. the fold type nucleotidediphospho-sugar transferases (2111.6) contains two families from eukaryote and one from archea, while the fold type sgnh hydrolase (2007.5) contains one family from bacteria and one from archea. this almost biphasic distribution, with one wellpopulated universal fold set and one less populated unique fold set, suggests that despite the fundamental differences in the viriomes from the three domains of cellular life, they survive using a common set of folds. furthermore, these well-populated folds extend across all life forms, perhaps suggesting an ancient origin. accordingly, the p-loop domainslike fold, whose nucleotide metabolic enzyme components are thought to form the origin of the protein world, 52 also represent the most populated fold among viral genomes. such examples of ancient viral proteins should be useful for understanding evolutionary relationships among members of this unique domain of life. alternately, the unique fold types likely drive some of the differences observed in virus infecting different cellular forms of life. for example, the cystovirus bacteriophage phi12 encodes a unique p7 protein with a minimal rossmann fold. phi12 p7 is classified as its own unique x-group in ecod, implying a lack of evidence for homology to existing folds. p7 serves as a putative virion assembly cofactor thought to bind the unique threesegmented double-stranded cystovirus rna genome. 53 the avian coronavirus that infects chickens encodes another example (ibv nsp2a) of a unique rossmann-like fold domain. the n-terminal domain of nsp2a adopts a rossmann-like sheet topology (2314), with b-strand 2 being an insert to the core that is antiparallel to the rest. although the function of nsp2a is unknown, its lack of sequence/ structure similarity to other proteins has suggested a role in host specificity. 54 traditionally defined, helicases use energy derived from ntp hydrolysis to unwind double-stranded nucleic acids. 55 as such, they play roles in numerous cellular processes involving nucleic acids; including dna replication and repair, transcription, translation, and rna splicing and maturation, among others. 56 mechanistically, helicases can bind singlestranded or double-stranded nucleic acid; unwind rna, dna or hybrids, and translocate in both directions (3 0 -5 0 or 5 0 -3 0 ). 57 despite these functional distinctions, all helicases bind ntp using two structural elements formed by signature sequence motifs: a phosphate-binding p-loop (motif i/walker a motif) and mg 21 cofactor binding loop (motif ii/ walker b). these motifs have helped classify p-loop helicases into superfamilies (sf1-sf5) based on sequence, with the last family including also nonhelicase ntpases. 58 modular accessory domains or subdomains, including terminal extensions and insertions within the core, can also regulate helicase activity. 58 by shuffling core and regulatory domains, nature has created a diverse range of cellular helicase machinery that also plays key roles in viral function. as such, the relative abundance of helicases in viral genomes has been used in part to assess picornaviral evolution. 59 their key roles in viral function also suggest helicases as novel targets for treating viral infections. 58 in humans, several debilitating inherited disorders are linked to genetic defects in helicase genes, including bloom's, werner's, and rothmund-thomson's syndromes. 60 the potential of helicases as antiviral drug targets has recently been reviewed. 61 among viral protein structures containing the minimal rossmann fold, 14 protein families are known helicases (http://prodata.swmed.edu/ross-mann_fold/viruses/). helicase domains fall into two different homologs folds (h-groups): p-loop domainsrelated (13 families) and had domain-related (1 family). 19 given the number of p-loop domainsrelated representatives and their potential for informing viral evolution, we constructed a structure-based tree of domains that contain p-loop figure 5 . a tree of viral helicases. structure-based distances between representative p-loop domains from ecod family viral helicases were estimated using daliz scores. nodes of the tree are labeled by pdb and colored according to superfamily. structures are colored in rainbow according to the core rossmann-like topology common to all viral helicase domains. terminal extentions (white) and insertions (pink) decorate the core fold. where present, active site molecules are in stick. functional and evolutionary analysis of viral proteins motifs (fig. 5) . the tree reproduces traditional sequence-based classification: 58 dividing the viral helicase domains into four superfamilies (sf1-sf4). those in the sf1 and sf2 include a rossmann-like fold duplication, with the second domain helping form the dna binding site and contributing a motif to the active site (i.e., motif iv in 3upua3, not included in the tree). all the catalytic viral helicase domains include a core topology of four strands (order 1432) sandwiched by a helix on one side and two helices on the other. the common core binds nucleotide using the walker a (following core strand 1) and walker b (following strand 3) motifs. the structure-based tree correctly defines superfamilies based on terminal extensions and insertions to the core, with sf1 having an insertion (fig. 6 , pink cartoon) that extends one side of the b-sheet by a strand and a c-terminal extension that extends the other side. sf2 domains have a longer insertion than sf1 that extends the sheet by two strands. sf3 have a similar c-terminal extension as sf1 but lack the insertion, while sf4 includes both a longer c-terminal extension that adopts a four-stranded b-meander and a longer insertion that extends the sheet and has an additional helical subdomain. given the ability of the viral p-loop helicase domains with minimal rossmann folds to recapitulate traditional sequencebased classification, these domains might provide useful for further analysis of viral evolution. viral genomes possess several additional types of p-loops domain-related homologs that do not function as helicases. their activities include terminase or viral nucleic acid packaging that couples ntp hydrolysis to directional motion along nucleic acids, thymidine and other deoxynucleotide kinases providing dna precursors for synthesis in the host cytoplasm, polynucleotide kinase functioning in nucleic acid repair, and a recombinase promoting strand exchange. these additional structures bring the total number of p-loop domains-related families to 27 (removed incorrect dynein domains), which represents almost one third of the existing rossmann-like fold domains in viruses. looking at similarity to known protein sequences, about one third of the rossmann motif ecod families are limited to viral sequences (27 out of 81, or 33%). most of these viral-specific protein families exhibit characteristics of fast evolution, with their sequences being distinct from homologs (24 out of 27, or 89%). interestingly, most of these fast evolving families infect eukaryotes (21 out of 24, or 87%), with many of these classified as diverse methyltransferase domains (12 families). viral methyltransferase domains tend to function in mrna 5' cap biosynthesis. one viral methyltransferase example that has not rapidly diverged from its ftsj counterparts in other kingdoms (i.e., "abve") illustrates a typical methyltransferase fold bound to adomet substrate and cap [ fig. 6(a) ]. alternately, a fast evolving structure of the vaccinia virus protein vp39 highlights the typical methyltransferase binding sites for adomet substrate [ fig. 6(b) , black stick] with diversity arising from extensions at the termini, a replacement of the c-terminal helix with a strand, and a unique cap binding pocket [ fig. 6(b) , magenta stick] that allows for sensing substrate methylation status. 62 evolution of viral methyltransferases has been discussed, with the unique sequence features arising from their invention of alternate capping pathways, their intimate interactions with additional viral enzymes functioning in the process, and their inactivation as methyltransferases. 63 such inactive domains have transformed into rna-binding modules 64 fig. 6(d,e) , respectively]. interestingly, classified viral methyltransferase domains from sars nsp15 and prrsv nsp11 endoribonucleases have a largely degraded nterminus where adomet substrate usually binds; however, the distinguishing c-terminal b-hairpin that marks the methyltransferase fold remains intact [ fig. 6(f) ]. the function of this domain, like other viral structure additions, appears to be in oligomerization. given the uniqueness of the folds with respect to host methyltransferases, the rapidly diverging viral methyltransferases might serve as targets for therapy. indeed, zika virus nsp5, which includes an ftsj family methyltransferase domain, shows promise for drug design. 66 alternatively, to the majority of fast evolving viral rossmann domains, only a few of them (3 out of 27, or 11%) appear as being unique to virus. these belong to virus with diverse hosts, with one infecting bacteria (pseudomonas phage core protein p7) and two infecting eukaryotic hosts (ibv nsp2a and zika virus ns1). the ibv nsp2a n-terminus contains a minimal rossmann-like motif with an a/b insertion following the first helix that forms an antiparallel interaction with the crossover strand 2 [ fig. 7(a) ]. while nsp2 is one of the first proteins to be translated and processed in the ibv life cycle, its function remains unknown. zika virus ns1 also adopts a minimal rossmann-like fold. however, ns1 replaces the c-terminal helix addition with an nterminal helix addition. it also has an insertion in the same position as nsp2a, but the b-strand forms a parallel interaction with strand 2 [ fig. 7(b) ]. the function of zika virus ns1 remains unclear. the unique properties of these apparent viral-specific proteins, which could have arisen from degradation of more complete rossmann domains, preclude functional inference from their structure. the remaining families include blast hits from other domains of life (54 out of 81, or 66%). these could either represent proteins of ancient origin or proteins that have been horizontally transferred (hgt) between viral genomes and their hosts. 51% (28 out of 54) of these families infect bacteria, 44% (23 out of 54) infect eukaryotes and 5% (3 out of 54) infect archea. this nearly equal distribution suggests the potential for viral protein families that derive from ancient origin, as the prevalence of hgt stems from bacterial origins. 67 although evidence does exist for viral acquisition of eukaryotic proteins 68 (i.e., from hgt, not ancient origins), these viral families of eukaryotic hosts that perform universal functions serve as a potential examples of viral proteins with ancient origins. many of these more universal proteins serve as helicases, which provide important functions to all domains of life. rossmann-like fold containing viral helicases are divided into four superfamilies (sf1-sf4), according to traditional sequence-based classification 58 and our classification of the p-loop containing domains (fig. 5) . the sf3 helicases, whose viral examples belong to universal families, are thought to have been present at the last universal common ancestor stage as in virus-like "selfish" replicons. 69 key role of these proteins in viral function also suggest helicases as novel targets for treating viral infections. only one family appears as forming a homology group that is unique and distant from the othersresolvase protein family (pf00239). resolvases or figure 7 . novel viral rossmann-like motifs. two viralspecific families retain minimal rossmann-like motifs colored in rainbow from n-terminus (blue) to c-terminus (red). (a) the n-terminal domain from ibv nsp2a [3ld1] has a unique b/a insertion (white) after the first helix forming an antiparallel interaction with strand 2 and an additional c-terminal helix (salmon). (b) the n-terminal domain from flavivirus ns1 includes an n-terminal helix (slate) and a different insertion (white) in the same position as in ibv nsp2a, but forming a parallel interaction with strand 2. functional and evolutionary analysis of viral proteins recombinases are proteins that cause conserved dna rearrangements, interact with short sequences in the dna, bring two sites together in a synapse and then catalyze strand exchange so that the dna is cleaved and religated to opposite partners. 70 there are several known types of recombinases but only serine recombinases or resolvase/invertase family proteins contain a rossmann fold motif. this family has emerged from studies of phages, prophages, and transposons from predominantly grampositive bacteria and consists of three groups. the structural and evolutionary differences imply that an ancestral catalytic domain has fused to unrelated sequences to result in a family of structurally and functionally diverse proteins. thus, the modular nature of the serine recombinases resembles that in other recombination enzymes, such as the tyrosine integrases and the dde superfamily of transposases. 71 interestingly, serine recombinases have significant sequence similarity to the poxvirus f16 protein whose function is still unclear, but it is proposed that this protein may affect signaling functions of the nucleoli and it is unlikely to have serine recombinase activity. 72 thus, the most parsimonious evolutionary scenario of these orthologs involves acquisition of a serine recombinase gene by the ancestor of poxviruses from a transposon or a bacteriophage. 72 the minimal rossmann fold motif was defined as a three-layer a/b/a sandwich motif with a crossover between elements iii and v, which contained three parallel b-strands as a middle layer and three variations of the crossover element iv (fig. 8) . element iv can be represented as a-helix [ fig. 8(a) ], bstrand [ fig. 8(b) ] or linker [ fig. 8(c) ]. element ii was represented only as a helix since it forms the active site of most of rossmann fold proteins. for the motif search, we used the prosmos program developed in our lab. 73 we generated a database of pdb domains (ecod database version: develop159/20161205) with each represented by a secondary structure element (sse) interaction matrix describing the interactions (parallel or antiparallel) and hydrogen-bonding between the secondary structure elements of the pdb structure. this database was generated using palsse. 74 the structure consensuses of minimal rossmann fold proteins were represented as query matrices. query matrices specified the number and types of secondary structure elements in the motif under consideration, the hydrogen bonding and parallel or antiparallel relationships between its elements and also minimum and maximum length of the three component bstrands. then we used query matrixes as input for prosmos program. false positives were removed by visual inspection. domains were considered to belong to rossmann-like fold only when minimal rossmann fold motif in them formed the structural core of the protein domain. a full list of protein families and their characteristics can be found online: http://prodata.swmed. edu/rossmann_fold/viruses/. we generated functional information for each pdb representative included in the online table from several databases. virus taxonomy is from the latest report of the international committee for taxonomy of viruses (ictv). topology and evolutionary information are from ecod. the protein name and structure are from the protein data bank. 3 general functional descriptions are from the pfam database. 75 finally, enzyme function in the form of an ec number-is from the kegg enzyme database. 76 the helicase tree was built from structure-based distances of ecod domains (2004.1.1) with helicase ec function using the fitch program from phy-lip package 77 (available from: http://evolution. genetics.washington.edu/phylip.html) with global rearrangements. distances between representative structures were estimated by transforming dali z scores 78 from pairwise superpositions using the for each pdb family representative sequence from the online table (http://prodata.swmed.edu/ross-mann_fold/viruses/), a search against the ncbi nonredundant protein sequence database (national center for biotechnology information, nih, bethesda, md) was performed using blast. 79 settings of blast search were used as follows: number of hits-5,000, e-value cutoff-0.01. the rest settings were set on default. all hits were sorted in four big taxonomical groups: a-archea, b-bacteria, e-eukaryota, v-viruses, and were plotted as distributions against blast score. these plots can be accessed through the online table (http://prodata. swmed.edu/rossmann_fold/viruses/), see column "blast distribution plot". the last column of the online table entitled "blast hits kingdoms" defines taxonomical groups from the distribution plot. monophyly of class 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glycosyltransferase exhibiting the gt-a fold the bacterial conjugation protein trwb resembles ring helicases and f1-atpase structure of the virb4 atpase, alone and bound to the core complex of a type iv secretion system polintons: a hotbed of eukaryotic virus, transposon and plasmid evolution virophages or satellite viruses? a mimivirus enzyme that participates in viral entry virus-encoded aminoacyl-trna synthetases: structural and functional characterization of mimivirus tyrrs and metrs the vaccinia virus h3 envelope protein, a major target of neutralizing antibodies, exhibits a glycosyltransferase fold and binds udp-glucose vaccinia virus envelope h3l protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo vaccinia virus h3l envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice crystal structure of vaccinia virus mrna capping enzyme provides insights into the mechanism and evolution of the capping apparatus structural insights into the mechanism and evolution of the vaccinia virus mrna cap n7 methyl-transferase extended surface for membrane association in zika virus ns1 structure rotavirus and severe childhood diarrhea atomic model of an infectious rotavirus particle structure of rotavirus outer-layer protein vp7 bound with a neutralizing fab the origin, evolution and structure of the protein world structure and dynamics of the p7 protein from the bacteriophage /12 purification, crystallization and preliminary x-ray analysis of nonstructural protein 2 (nsp2) from avian infectious bronchitis virus unwinding the 'gordian knot' of helicase action structure of adeno-associated virus type 2 rep40-adp complex: insight into nucleotide recognition and catalysis by superfamily 3 helicases viral and cellular rna helicases as antiviral targets helicases: amino acid sequence comparisons and structure-function relationships the big bang of picorna-like virus evolution antedates the radiation of eukaryotic supergroups recq family helicases: roles as tumor suppressor proteins current progress in antiviral strategies structural basis for sequence-nonspecific recognition of 5 0 -capped mrna by a cap-modifying enzyme rna methyltransferases involved in 5 0 cap biosynthesis structural and functional insights into alphavirus polyprotein processing and pathogenesis a putative atpase mediates rna transcription and capping in a dsrna virus structure and function of zika virus ns5 protein: perspectives for drug design horizontal gene transfer: essentiality and evolvability in prokaryotes, and roles in evolutionary transitions viral proteins acquired from a host converge to simplified domain architectures evolutionary history and higher order classification of aaa 1 atpases diversity in the serine recombinases integrating dna: transposases and retroviral integrases vaccinia virus f16 protein, a predicted catalytically inactive member of the prokaryotic serine recombinase superfamily, is targeted to nucleoli searching for three-dimensional secondary structural patterns in proteins with palsse: a program to delineate linear secondary structural elements from protein structures the pfam protein families database: towards a more sustainable future using the kegg database resource phylip (phylogeny inference package) version 3.69 dali server update gapped blast and psi-blast: a new generation of protein database search programs the authors declare that they have no conflicts of interest with the contents of this article. key: cord-259603-bh198xgl authors: snijder, e.j.; decroly, e.; ziebuhr, j. title: the nonstructural proteins directing coronavirus rna synthesis and processing date: 2016-09-14 journal: adv virus res doi: 10.1016/bs.aivir.2016.08.008 sha: doc_id: 259603 cord_uid: bh198xgl coronaviruses are animal and human pathogens that can cause lethal zoonotic infections like sars and mers. they have polycistronic plus-stranded rna genomes and belong to the order nidovirales, a diverse group of viruses for which common ancestry was inferred from the common principles underlying their genome organization and expression, and from the conservation of an array of core replicase domains, including key rna-synthesizing enzymes. coronavirus genomes (~ 26–32 kilobases) are the largest rna genomes known to date and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral rna polymerases. the primary functions that direct coronavirus rna synthesis and processing reside in nonstructural protein (nsp) 7 to nsp16, which are cleavage products of two large replicase polyproteins translated from the coronavirus genome. significant progress has now been made regarding their structural and functional characterization, stimulated by technical advances like improved methods for bioinformatics and structural biology, in vitro enzyme characterization, and site-directed mutagenesis of coronavirus genomes. coronavirus replicase functions include more or less universal activities of plus-stranded rna viruses, like an rna polymerase (nsp12) and helicase (nsp13), but also a number of rare or even unique domains involved in mrna capping (nsp14, nsp16) and fidelity control (nsp14). several smaller subunits (nsp7–nsp10) act as crucial cofactors of these enzymes and contribute to the emerging “nsp interactome.” understanding the structure, function, and interactions of the rna-synthesizing machinery of coronaviruses will be key to rationalizing their evolutionary success and the development of improved control strategies. coronaviruses (covs) are the best-known and best-studied clade of the order nidovirales, which is comprised of enveloped plus-stranded (+rna) viruses and currently also comprises the arteriviridae, roniviridae, and mesoniviridae families (de groot et al., 2012a,b; lauber et al., 2012) . in addition to including various highly pathogenic covs of livestock (saif, 2004) and four "established" human covs causing a large number of common colds (pyrc et al., 2007) , covs have attracted abundant attention due to their potential to cause lethal zoonotic infections (graham et al., 2013) . this was exemplified by the 2003 outbreak of severe acute respiratory syndrome-coronavirus (sars-cov) in southeast asia and the ongoing transmission-since 2012-of the middle east respiratory syndromecoronavirus (mers-cov), which causes $35% mortality among patients seeking medical attention. both these viruses are closely related to covs that are circulating in bats (ge et al., 2013; menachery et al., 2015) and other potential reservoir species. they may be transmitted to humans either directly or through intermediate hosts, like civet cats for sars-cov (song et al., 2005) and dromedary camels for mers-cov (reusken et al., 2013) . formally, the family coronaviridae now includes about 30 species, divided into the subfamilies torovirinae and coronavirinae, the latter being further subdivided in the genera alpha-, beta-, gamma-, and deltacoronavirus. sars-cov and mers-cov are betacoronaviruses, and the same holds true for one of the best-characterized animal cov models, murine hepatitis virus (mhv). this explains why the bulk of our current knowledge of cov molecular biology is betacoronavirus based, even more so for the replicative proteins that are the central theme of this review, which will mainly summarize data obtained studying sars-cov proteins. despite their unification in the same virus order, nidoviruses cover an unusually broad range of genome sizes, ranging from $13-16 kilobases (kb) for arteriviruses, via $20 kb for mesoniviruses, to $26-32 kb for covs (nga et al., 2011) . together with the genomes of roniviruses, which infect invertebrate hosts, cov genomes are the largest rna genomes known to date . the common ancestry of these extremely diverse virus lineages was inferred from their polycistronic genome structure, the common principles underlying the expression of these genomes, and-most importantly-the conservation of an array of "core replicase domains," including key enzymes required for rna synthesis. while retaining this conserved genomic and proteomic blueprint, nidovirus genomes are thought to have expanded gradually by gene duplication and acquisition of novel genes (lauber et al., 2013) , most likely by rna recombination. in addition to the high mutation rate that characterizes all rna viruses, these genomic innovations appear to have enabled nidoviruses to explore an unprecedented evolutionary space and adapt to a wide variety of host organisms, including mammals, birds, reptiles, fish, crustaceans, and insects. whereas the poor replication fidelity generally restricts rna virus genome sizes, it has been postulated that nidovirus genome expansion was enabled by the acquisition of specific replicative functions that counter the error rate of the rna polymerase (deng et al., 2014; eckerle et al., 2010; snijder et al., 2003) (discussed in more detail later). as in all nidoviruses, at least two-thirds of the cov genome capacity is occupied by the two large open reading frames (orfs) that together constitute the replicase gene, orf1a and orf1b (fig. 1 ). these orfs overlap by a few dozen nucleotides and are both translated from the viral genome, with expression of orf1b requiring a -1 ribosomal frameshift to occur just fig. 1 outline of the cov genome organization and expression strategy, based on sars-cov. the top panel depicts the sars-cov genome, including various regulatory rna elements, and the 5 0 -and 3 0 -coterminal nested set of subgenomic mrnas used to express the genes downstream of the replicase gene. utr, untranslated region; trs, transcription-regulatory sequence. below the rnas, the 14 open reading frames in the genome are indicated, i.e., the replicase orfs 1a and 1b, the four common cov structural protein genes (s, e, m, and n) and the orfs encoding "accessory proteins." the bottom panel explains the organization and proteolytic processing of the pp1a and pp1ab replicase polyproteins, the latter being produced by -1 ribosomal frameshifting. the nsp3 (pl pro ) and nsp5 (3cl pro ) proteases and their cleavage sites are indicated in matching colors. the resulting 16 cleavage products (nonstructural proteins (nsps)) are indicated, as are the conserved replicase domains that are relevant for this review. domain abbreviations and corresponding nsp numbers: pl pro , papain-like proteinase (nsp3); 3cl pro , 3c-like proteinase (nsp5); tm, transmembrane domain (nsp3, nsp4, and nsp6); niran, nidovirus rdrp-associated nucleotidyl transferase (nsp12); rdrp, rna-dependent rna polymerase (nsp12); zbd, zinc-binding domain (nsp13); hel1, superfamily 1 helicase (nsp13); exon, exoribonuclease (nsp14); n7-mt, n7-methyl transferase (nsp14); endou, uridylate-specific endoribonuclease (nsp15); 2 0 -o-mt, 2 0 -omethyl transferase (nsp16). upstream of the orf1a termination codon (brierley et al., 1989) . the efficiency of this highly conserved frameshift event, which may approach 50% in the case of covs (irigoyen et al., 2016) , is promoted by specific primary and higher-order rna structures. as a result, in cov-infected cells, the replicase subunits encoded in orf1a are overexpressed in a fixed ratio relative to the proteins encoded in orf1b. the primary translation products of the cov replicase are two huge polyproteins, the orf1a-encoded pp1a and the c-terminally extended pp1ab frameshift product (fig. 1) . the former is roughly 4000-4500 amino acids long, depending on the cov species analyzed. the size of the orf1b-encoded extension is more conserved (around 2700 residues), resulting in pp1ab sizes in the range of 6700-7200 amino acids. probably already during their synthesis, either two or three orf1aencoded proteases initiate the proteolytic cleavage of pp1a and pp1ab to release (sometimes) 15 or (mostly) 16 functional nonstructural proteins (nsps; fig. 1 ). the highly conserved nsp5 protease has a chymotrypsin-like fold (3clike protease, 3cl pro ) (anand et al., 2002 (anand et al., , 2003 gorbalenya et al., 1989) and is the viral "main protease" (therefore sometimes also referred to as m pro ). the 3cl pro cleaves the nsp4-nsp11 part of pp1a and the nsp4-nsp16 part of pp1ab at 7 and 11 conserved sites, respectively. these sites can be summarized with the p4-p2 0 consensus motif (small)-x-(l/i/v/f/m)-q#(s/a/g), where x is any amino acid and # represents the cleavage. the processing of three sites in the nsp1-nsp4 region is performed by one or two papain-like proteases (pl pro ) residing in the very large nsp3 subunit (mielech et al., 2014) . whereas alphacoronaviruses and most betacoronaviruses (though not sars-cov and mers-cov) have two pl pro domains in their nsp3, presumably the result of an ancient duplication event, gamma-and deltacoronaviruses have only a single pl pro . the cleavage sites (lxgg# or similar) resemble the c-terminal lrgg# motif of ubiquitin, which explains why cov pl pro domains were found capable to also act as deubiquitinases (ratia et al., 2006) . this secondary function has been implicated in the disruption of host innate immune signaling by removing ubiquitin from certain cellular substrates. more than any other cov-encoded enzyme, the cov 3cl pro and pl pro domains have been characterized in exquisite structural and biochemical detail, both in their capacity of critical regulators of nsp synthesis and as two of the primary drug targets for this virus family. space limitations unfortunately prevent us from summarizing these studies in more detail, but a variety of excellent reviews is available to compensate for this omission (baez-santos et al., 2015; hilgenfeld, 2014; mielech et al., 2014; steuber and hilgenfeld, 2010) . once released from pp1a and pp1ab, most covs nsps studied thus far assemble into a membrane-bound ribonucleoprotein complex that drives the synthesis of different forms of viral rna (see later) and is sometimes referred to as the replication and transcription complex (rtc) . while viral rna production takes off, peculiar convoluted membrane structures, spherules tethered to zippered endoplasmic reticulum, and doublemembrane vesicles begin to accumulate in cov-infected cells (gosert et al., 2002; knoops et al., 2008; maier et al., 2013) . as for other +rna viruses, they have been postulated to serve as scaffolds, or perhaps even suitable microenvironments, for viral rna synthesis. nevertheless, many questions on their biogenesis and function remain to be answered, and the exact location of the metabolically active rtc still has to be pinpointed "beyond reasonable doubt" for covs and other nidoviruses (hagemeijer et al., 2012; neuman et al., 2014a; van der hoeven et al., 2016) . three orf1a-encoded replicase subunits containing transmembrane domains (nsp3, nsp4, and nsp6; fig. 1 ) have been implicated in the formation of the membrane structures that are induced upon cov infection and with which the rtc is thought to be associated (angelini et al., 2013; hagemeijer et al., 2014) . in addition to actively engaging in host membrane remodeling, they may serve as membrane anchors for the rtc by binding the nsps that lack hydrophobic domains, like all of the orf1b-encoded enzymes. for more details, the reader is referred to the numerous recent reviews of the "replication organelles" of covs and other +rna viruses (den boon and ahlquist, 2010; hagemeijer et al., 2012; neuman et al., 2014a; romero-brey and bartenschlager, 2016; van der hoeven et al., 2016; xu and nagy, 2014) . the common ancestry of nidovirus replicases is not only reflected in their conserved core replicase domains but also in the synthesis of subgenomic (sg) mrnas that are used to express the genes located downstream of orf1b ( fig. 1) . although some nidoviruses (e.g., roniand mesoniviruses) have only a few of these genes, they are much more numerous in arteriviruses and covs, their number going up to about a dozen orfs for some covs. in addition to the standard set of four cov structural protein genes (encoding the spike (s), envelope (e), membrane (m), and nucleocapsid (n) protein), genomes in different cov clusters contain varying numbers of orfs encoding so-called "accessory proteins" narayanan et al., 2008) . the proteins they encode are often dispensable for the basic replicative cycle in cultured cells, but highly relevant for cov viability and pathogenesis in vivo, for example, because they enable the virus to interfere with the host's immune response. most of the genes downstream of orf1b are made accessible to ribosomes by positioning them at the 5 0 end of their own sg transcript. occasionally, two or even three genes are expressed from the same sg mrna, usually by employing ribosomal "leaky scanning" during translation initiation. nidoviral sg mrnas are 3 0 -coterminal with the viral genome, but in most nidovirus taxa, including covs, the sg transcripts also carry common 5 0 leader sequences ($65-95 nucleotides in covs), which are identical to the 5 0 -terminal sequence of the viral genome ( fig. 1) (pasternak et al., 2006; sawicki et al., 2007; sola et al., 2011) . the joining of common leader and different sg rna "body" sequences occurs during minus-strand rna synthesis (sawicki and sawicki, 1995; sethna et al., 1989) . this step can be either continuous, to produce the full-length minus strand required for genome replication, or interrupted (discontinuous) to produce a subgenome-length minus-strand rna that can subsequently serve as the template for the synthesis of one of the sg mrnas. the polymerase jumping that is the basis for leader-to-body joining occurs at specific "transcriptionregulatory sequences" (trss). these conserved sequence motifs are comprised of up to a dozen nucleotides, and are found in the genome at the 3 0 end of the leader sequence and at the 5 0 end of each of the sg mrna bodies. quite likely, also higher-order rna structure and transcription-specific protein factors play a role in the interruption of minus-strand rna synthesis at a body trs, after which the nascent minus strand (with a body trs complement at its 3 0 end) is translocated to the 5 0 -proximal part of the genomic template. guided by a base-pairing interaction with the leader trs, the synthesis of the subgenome-length minus-strand rna is resumed and completed with the addition of the complement of the genomic leader sequence. in this manner, a nested set of subgenome-length templates for sg mrna synthesis is produced, providing a mechanism to regulate the abundance of the different viral proteins by fine-tuning the level at which the corresponding sg mrna is generated (nedialkova et al., 2010) . the cov transcription strategy allows the rtc to use the same 3 0 -terminal recognition/initiation signals in both full-and subgenome-length templates of either polarity. moreover, the presence of the common 5 0 leader sequence may be important for mrna capping or other translation-related features. during the past two decades, studies on the cov enzyme complex that controls this elegant replication and transcription mechanism have been accelerated by four important developments. first, using bioinformatics, expression systems, and virus-infected cells, the replicase polyprotein processing scheme and the proteases involved were elucidated, thus defining the boundaries of the 16 mature nsps (fig. 1 ) that are working together during cov replication (ziebuhr et al., 2000) . second, using this information and promoted by rapidly advancing methods in structural biology, x-ray or nmr structures were obtained for numerous (recombinant) full-length cov nsps or domains thereof, in particular for sars-cov (neuman et al., 2014b) . third, multiple techniques for the targeted mutagenesis of cov genomes were developed and refined, which was a specific technical challenge due to the exceptionally large size of the cov rna genome (almazan et al., 2014) . by launching engineered mutant genomes in susceptible cells, the rna and protein players in the cov replication cycle can now be interrogated directly, to reveal their importance, function(s) and/or interactions in vivo. finally, in vitro biochemical assays were developed for a variety of cov replicative enzymes, including many of those involved in rna synthesis and processing. for the purpose of this review, we have chosen to focus on these latter functions, as performed by the cov nsp7 to nsp16 products nga et al., 2011; sevajol et al., 2014; subissi et al., 2014a) . these subunits include several replicative enzymes that are more or less universal among + rna viruses, such as rna polymerase (nsp12) and helicase (nsp13), but also a number of rare or even unique domains involved in, e.g., mrna capping, cap modification, and promoting the fidelity of cov rna synthesis. several smaller subunits, in particular nsp7 to nsp10, have been identified as crucial cofactors of these enzymes and contribute to the emerging cov "nsp interactome," which will likely need to be advanced considerably to achieve a more complete understanding of the intricacies of cov rna synthesis. making that step will obviously be key to understanding the evolutionary success of covs, and nidoviruses at large. moreover, this knowledge will lay the foundation for the development of improved strategies to combat current and future emerging covs, including targeted antiviral drug development. 2. coronavirus nsp7-10: small but critical regulatory subunits? the 3 0 -terminal part of orf1a, the approximately 1.7 kb separating the nsp6-coding sequence and the orf1a/1b ribosomal frameshift site, encodes a set of four small replicase subunits, named nsp7 to nsp10 (fig. 1) . although highly conserved among coronavirinae, these proteins seem to lack enzymatic functions. instead, they have emerged as (putative) interaction partners and modulators of orf1b-encoded core enzymes like nsp12 (rna-dependent rna polymerase, rdrp), nsp14 (exoribonuclease, exon), and nsp16 (ribose 2 0 -o-methyl transferase, 2 0 -o-mtase). furthermore, several of them have been predicted or shown to interact with rna. additionally, a fifth, very small cleavage product is assumed to be released from this region of pp1a: the nsp11 peptide resulting from cleavage of pp1a at the nsp10/11 junction (fig. 1) . in the pp1ab frameshift product, the n-terminal sequence of nsp11 (encoded between the nsp10/11 junction and orf1a/1b frameshift site) equals the n-terminal part of the nsp12 subunit. depending on the cov species, nsp11 consists of 13-23 residues and its actual release, function (if any), or fate in cov-infected cells have not been established. in cell culture models, for some (infectious bronchitis virus (ibv)) but not other (mhv) covs, the nsp10/11 and nsp10/12 cleavages were found to be dispensable for virus replication (deming et al., 2007; fang et al., 2008) , even though the conservation of this cleavage site suggests that it is generally required for full replicase functionality. processing of the nsp7-nsp10 region of pp1a/pp1ab has been studied in some detail for mhv (bost et al., 2000; deming et al., 2007) , human cov 229e (hcov-229e) (ziebuhr and siddell, 1999) , and ibv (ng et al., 2001) , confirming the release of these subunits in infected cells and the use of the predicted 3cl pro cleavage sites. processing at these sites was found to be critical for mhv replication, the exception being inactivation of the nsp9/10 cleavage site, which yielded a crippled mutant virus. depending on antibody availability, the subcellular localization of nsp7 to nsp10 has been studied for several covs using immunofluorescence microscopy. without exception, and in line with their role as interaction partner of key replicative enzymes, these subunits localize to the perinuclear region of infected cells (bost et al., 2000) , where the membranous replication organelles of covs accumulate (gosert et al., 2002; knoops et al., 2008; maier et al., 2013) . it should be noted, however, that these labeling techniques cannot distinguish between fully processed nsps and polyprotein precursors or processing intermediates. the structure of the 83-amino acid sars-cov nsp7 was determined using both nmr (peti et al., 2005) and x-ray crystallography (zhai et al., 2005) , with the latter study resolving the structure of a hexadecameric supercomplex consisting of recombinant nsp7 and nsp8 (see later; fig. 2 ). in both structures, the nsp7-fold includes four helices, but their position and spatial orientation is quite different, suggesting that the protein's conformation is strongly affected by the interaction with nsp8, in particular, where it concerns helix α4 (johnson et al., 2010) . reverse-genetics studies targeting specific residues in sars-cov nsp7 confirmed the protein's importance for virus replication (subissi et al., 2014b) , although the impact of single point mutations was smaller than anticipated on the basis of the biochemical characterization of the rna-binding properties of nsp7-containing protein complexes in vitro (see later). the $200-amino-acid-long nsp8 subunit initially took center stage due to two studies, the first describing a fascinating hexadecameric structure consisting of eight copies each of nsp7 and nsp8 (fig. 2) (zhai et al., 2005) , and the second reporting an nsp8-specific "secondary" rna polymerase fig. 2 crystal structure of the sars-cov nsp7-nsp8 hexadecamer (pdb 2ahm) (zhai et al., 2005) . purified recombinant sars-cov nsp7 and nsp8 were found to self-assemble into a supercomplex of which the structure was determined at 2.4 å resolution. (a) the complex forms a doughnut-shaped hollow structure of which the central channel is lined with positively charged side chains (in blue) and was postulated to mediate doublestranded rna binding. the outside of the structure is predominantly negatively charged (red) surface shading). (b and c) sars-cov nsp8 resembles a "golf club"-like shape that can adopt two conformations, as presented here in orange and green. these nsp8 conformations are integrated into a much larger, hexadecameric structure that is composed of eight nsp8 subunits and eight nsp7 subunits, of which one is shaded pink. in (b), the hexadecamer is depicted against the background of the surface plot presented in (a). activity (imbert et al., 2006) that was implicated in the mechanism of initiation of cov rna synthesis. this template-dependent activity was reported to depend on the presence of mn 2+ or mg 2+ and to typically generate products of up to six nucleotides (for more details, see section 3.2). around the same time, purified recombinant sars-cov nsp7 and nsp8 were found to self-assemble into the hexadecameric supercomplex of which the structure was determined at 2.4 å resolution (zhai et al., 2005) . the complex was described, and also visualized by electron microscopy, as a doughnut-shaped hollow structure of which the central channel is lined with positively charged side chains ( fig. 2a) . a combination of structural modeling, rna-binding studies, and site-directed mutagenesis led to the hypothesis that the complex may slide along the replicating viral rna together with other viral proteins, possibly as a processivity factor for the rdrp (nsp12; see later). within the nsp7-nsp8 hexadecamer, sars-cov nsp8 was found to adopt two different conformations ( fig. 2b and c). these were named "golf club" and "golf club with a bent shaft" (zhai et al., 2005) , with the globular head of the golf club being considered a new fold. although the structures of feline coronavirus (fcov) nsp7 and nsp8 were found to resemble their sars-cov equivalents, they were found to assemble into a quite different higher-order complex, with two copies of nsp7 and a single copy of nsp8 forming a heterotrimer (xiao et al., 2012) . biochemical and reverse-genetics studies pointed toward an important role in rna synthesis for sars-cov nsp8 residues k58, p183, and r190, whose replacement was lethal to sars-cov. of these residues, p183 and r190 were postulated to be involved in interactions with nsp12, whereas k58 may be critical for nsp8-rna interactions (subissi et al., 2014b) . reverse-genetics studies targeting the 3 0 -proximal rna replication signals in the mhv genome provided strong evidence for an interaction between nsp8 and these rna structures (a so-called "bulged stemloop" and rna pseudoknot). when making a particular 6-nucleotide insertion in the rna pseudoknot, which strongly affected mhv replication, multiple suppressor mutations evolved, of which several mapped to the genomic region encoding nsp8 and nsp9 (z€ ust et al., 2008) . these interactions were postulated to be part of a molecular switch that controls minus-strand rna synthesis, or its initiation from the 3 0 end of the viral genome (te velthuis et al., 2012; z€ ust et al., 2008) . using screening approaches based on yeast two-hybrid and glutathione s-transferase (gst) pull-down assays, sars-cov nsp8 was reported to be an interaction partner of many other viral proteins (including nsp2, nsp3, and nsp5 to nsp16), although most of these interactions remain to be verified in the infected cell (von brunn et al., 2007) . the cov nsp9 subunit is about 110 amino acids long and was the second replicase cleavage product, after nsp5, for which crystal structures were obtained (egloff et al., 2004; sutton et al., 2004) . the biologically active form of the protein is believed to be a dimer that is capable of binding nucleic acids in a nonsequence-specific manner, with an apparent preference for single-stranded rna (egloff et al., 2004; ponnusamy et al., 2008; sutton et al., 2004) . several nsp9 point mutations that block cov replication have now been described (chen et al., 2009a; miknis et al., 2009 ), but the protein's exact function has remained enigmatic thus far. the nsp9 monomer consists of a β-barrel, composed of seven β-strands, and a c-terminal domain formed by a single α-helix. the latter domain plays a key role in the formation of the parallel helix-helix dimer conformation that-based on sequence conservation, structural considerations, and experimental data (miknis et al., 2009 )-is thought to be the biologically most relevant state of sars-cov nsp9. nevertheless, multiple alternative structures were described, including a sars-cov form that is stabilized by β-sheet interactions (sutton et al., 2004) and, for hcov-229e nsp9, an antiparallel helix-helix dimer that is stabilized by a disulfide bond (ponnusamy et al., 2008) . replacement of the hcov-229e cys residue involved in dimerization (cys-69) resulted in conversion to the parallel helix-helix dimer described for sars-cov nsp9. whereas wild-type hcov-229e nsp9 is organized as a trimer of dimers, the cys-69 ! ala mutant and sars-cov nsp9 both form rod-like polymers (ponnusamy et al., 2008) . disulfide bonding of the latter protein could not be detected (miknis et al., 2009) . although sars-cov and other betacoronaviruses do contain an equivalent cys residue, the feature is not conserved in alphacoronaviruses that are much more closely related to hcov-229e. thus, it cannot be excluded that the disulfide-bonded form of hcov-229e nsp9 is an artifact of recombinant protein purification and crystallization, although it was suggested that oxidative stress due to viral infection may favor its formation in cov-infected cells (ponnusamy et al., 2008) . we are not aware of experiments directly addressing the existence of such a disulfide-linked nsp9 dimer in covinfected cells. the importance of nsp9 dimerization for sars-cov and ibv viability was demonstrated in reverse-genetics studies (chen et al., 2009a; miknis et al., 2009 ) that also independently confirmed the importance of dimerization of the α-helical domain and in particular a putative gxxxg proteinprotein interaction motif. although rna binding in vitro was not disrupted in dimerization-incompetent sars-cov nsp9 variants, their affinity for ssrna 20-mers was reduced by 5-to 12-fold compared to the wild-type protein (miknis et al., 2009) . replacement of some of the basic residues (e.g., lys-10, lys-51, and lys-90) in the β-barrel domain of ibv nsp9 also significantly reduced the protein's capability to bind rna in vitro, but these mutations only modestly affected virus replication upon reverse engineering (chen et al., 2009a) . it remains to be studied how nsp9 dimerization and mutagenesis may affect interactions with other replicase subunits, like nsp8 and nsp12-rdrp. these proteins were identified as nsp9 interaction partners using different technical approaches (brockway et al., 2003; sutton et al., 2004; von brunn et al., 2007) and colocalize with nsp9 on the membranous replication organelles (bost et al., 2000) . at present, the available data suggest that, for efficient cov replication, nsp9 homodimerization is a more critical feature than the protein's affinity for rna per se. alternatively, the correct positioning of rna on larger protein complexes consisting of (or containing) nsp9 may be important for the protein's correct functioning in viral rna synthesis (miknis et al., 2009) . currently, the fact that suppressor mutations arose in mhv nsp9 (and nsp8) after mutagenesis of 3 0 -proximal mhv replication signals (see earlier) is the most compelling evidence for the involvement of nsp9-rna interactions in a critical step of cov replication. the protein may be part of a molecular switch (z€ ust et al., 2008) and/or possess features that are relevant to viral pathogenesis, as mutations in nsp9 were found to contribute to increased sars-cov pathogenesis in an animal model employing young mice infected with a mouse-adapted virus strain (ma-15) (frieman et al., 2012) . the small nsp10 subunit (139 residues in the case of sars-cov) is among the more conserved cov proteins and is thought to serve as an important multifunctional cofactor in replication. using yeast two-hybrid assays, nsp10 was shown to interact with itself, as well as with nsp1, nsp7, nsp14, and nsp16. these interactions were confirmed by coimmunoprecipitation and/or gst pull-down assays (brockway et al., 2004; imbert et al., 2008; pan et al., 2008; von brunn et al., 2007) . the important role of nsp10 in replication was first inferred from the phenotype of temperature-sensitive mutants of mhv in which an nsp10 mutation was responsible for a defect in minus-strand rna synthesis (sawicki et al., 2005) . in addition, the protein was implicated in the regulation of polyprotein processing since an engineered mhv nsp10 double mutant (asp-47 and his-48 to ala) was partially impaired in the processing of the nsp4-nsp11 region (donaldson et al., 2007) . when nsp10 was characterized in biochemical and structural studies, the protein was found to bind two zn 2+ ions with high affinity, suggesting the presence of two zinc-finger motifs (matthes et al., 2006) . additionally, in in vitro assays, nsp10 displayed a weak affinity for single-and doublestranded rna and dna, although no obvious sequence specificity could be established, suggesting that the protein may function as part of a larger rna-binding complex. crystal structures of monomeric and dodecameric forms of sars-cov nsp10 were solved by different laboratories, but obvious structural rearrangements between the two forms were not detected (joseph et al., 2007; su et al., 2006) . the structures revealed a new fold in which the zn 2+ ions are coordinated in a unique conformation and in which a cluster of basic residues on the protein's surface probably contributes to the rna-binding properties of nsp10. more recent biochemical studies revealed that nsp10 interacts with nsp14 and nsp16 and regulates their respective exon and ribose-2 0 -o-mtase (2 0 -o-mtase) activities (bouvet et al., 2010 (bouvet et al., , 2012 . both these cofactor functions will be discussed in more detail later, in section 5. although a virus-encoded rdrp is at the hub of the replication of all rna viruses, special properties have long been attributed to the cov rdrp. these ideas find their origin in a combination of cov features, like the exceptionally long rna genome , the complex mechanism underlying subgenomic rna synthesis pasternak et al., 2006; sawicki et al., 2007; sola et al., 2011) , the reported high rna recombination frequency (graham and baric, 2010; lai and cavanagh, 1997) , and the size and positioning of the rdrpcontaining subunit, nsp12, within the replicase polyprotein. it remains to be elucidated to which extent features like polymerase processivity, fidelity, and template switching (during either genomic recombination or subgenome-length negative-strand rna synthesis) are determined by the properties of the nsp12-rdrp subunit itself or by some of its protein cofactors, such as nsp7 and nsp8 (see earlier). in fact, some cofactors have been studied more extensively than nsp12 itself, and the same holds true for some of the specific rna signals employed by the rdrp during, e.g., replication and subgenomic mrna synthesis. protein subunits of the larger rnasynthesizing complex, like nsp7-nsp8, the nsp13-helicase, and the nsp14-exon, likely exert a strong influence on rdrp behavior and performance. on the other hand, a recent study employing homology modeling and reverse genetics of the mhv rdrp domain described the first two nsp12 mutations that can induce resistance to a mutagen and reduce the mhv rdrp error rate during virus passaging (sexton et al., 2016) . so, not unexpectedly, also features within nsp12 itself contribute to properties like nucleotide selectivity and fidelity regulation. all of the currently identified nsp12 cofactors, and most other cov nsps, assemble into membrane-associated enzyme complexes (see earlier). the large number of viral subunits in these complexes (subissi et al., 2014a) , the likely requirement for host factors (van hemert et al., 2008) , and the concept of rna synthesis occurring in a dedicated microenvironment in the infected cell (knoops et al., 2008; v'kovski et al., 2015) complicate the straightforward characterization of the cov rdrp. to reconstitute the enzyme's activities in vitro, purified recombinant nsp12 is a key reagent but, for many years, such studies were hampered by poor nsp12 expression in escherichia coli. the first in vitro activity assays have only been developed recently (subissi et al., 2014b; te velthuis et al., 2010) , and the same technical issues with protein production explain the current lack of an nsp12 crystal structure. consequently, structural information is restricted to sequence comparisons and some homology-based structure models of the c-terminal rdrp domain of the $930-residue-long nsp12 (xu et al., 2003) . moreover, most of what we have learned so far is based on the characterization of a single nsp12 homolog only, that of the sars-cov. the nsp12-coding sequence includes the orf1a/1b ribosomal frameshift site and a programmed -1 frameshifting event directs orf1b translation to yield the pp1ab polyprotein that includes nsp12. the 3cl pro -driven cleavage required to release the n-terminus of nsp12 is the same that separates nsp10 and nsp11. about 925-940 amino acids downstream (932 in the case of sars-cov), the nsp12/nsp13 cleavage site separates the cov rdrp subunit from the helicase-containing cleavage product, whichuniquely among +rna viruses-resides downstream of the rdrp domain for reasons that are poorly understood thus far . nsp12 consists of at least two domains, the recently described n-terminal "nidovirus-wide conserved domain with nucleotidyl transferase activity" (nidovirus rdrp-associated nucleotidyltransferase (niran); see later) (lehmann et al., 2015a ) and the c-terminal canonical rdrp domain (gorbalenya et al., 1989) . the latter possesses the common motifs and structural features found in other rna polymerases, which are often summarized as a "cupped right hand" with subdomains called fingers, palm, and thumb each playing specific roles in binding of templates and ntps, initiation, and elongation (te velthuis, 2014; xu et al., 2003) . in simplified form, the reaction catalyzed by the rdrp comes down to selecting the appropriate ntp to match with the template and the formation of a phosphodiester bond to extend the 3 0 end of the nascent rna chain with this incoming nucleotide (ng et al., 2008; van dijk et al., 2004) . reconstituting these activities in vitro using a purified rdrp preparation can be relatively straightforward, but sometimes is a huge technical challenge depending-among other factors-on the efficiency of recombinant rdrp expression and purification, the existence of specific template requirements (e.g., recognition signals), and the need for protein cofactors. the initiation mechanism of the cov rdrp, primer dependent or de novo, continues to be a much-debated issue, with important implications for the question of how covs maintain the integrity of the crucial terminal sequences of their genome. compared to a de novo-initiating rdrp, the enzyme's active site, which is enclosed by the thumb and fingers domains, needs to be more accessible when a primer-template duplex has to be accommodated. de novo initiation, on the other hand, requires specific structural elements (so-called "priming loops") that serve to properly position the initiating ntps for catalysis, thus creating an initiation platform for rna synthesis. bioinformatics analyses grouped the cov rdrp with primer-dependent rdrps, as found in, e.g., picornaviruses and caliciviruses, in part based on the identification of a specific sequence motif (motif g) that is thought to mediate primer recognition ( fig. 3a ) (beerens et al., 2007; fig. 3 comparison of coronavirus nsp12 and arterivirus nsp9, containing the highly conserved niran and rdrp domains. (a) similarity density plot derived from a multiple sequence alignment including rdrp subunits from all nidovirus lineages. to highlight local deviations from the average, areas displaying conservation above and below the mean similarity are shaded in black and gray, respectively. conserved sequence motifs of niran (subscript n; see also b) and rdrp (subscript r) are labeled. domain boundaries used for bioinformatics analyses and uncertainty with respect to the niran/rdrp domain boundary are indicated with vertical and by dashed horizontal lines, respectively. below each plot, the predicted secondary structure elements are presented in gray for α-helices and black for β-strands. gorbalenya et al., 2002; xu et al., 2003) . this prediction appeared to be further supported by the identification of sars-cov nsp8 as a de novoinitiating second rna polymerase (see earlier), capable of synthesizing products of up to six nucleotides in length that could serve to prime rna synthesis by the nsp12-rdrp (imbert et al., 2006) . support for a direct interaction between nsp8 and nsp12 was obtained using different technical approaches subissi et al., 2014b; von brunn et al., 2007) . however, although a similar primer-independent rdrp activity was reported for the fcov nsp8 (xiao et al., 2012) , other studies have called into question this concept of a primase-main rdrp (i.e., nsp8/nsp12) tandem working in concert to achieve initiation of processive cov rna synthesis (see later). using recombinant sars-cov nsp12, preliminary evidence for primer-dependent rdrp activity on poly(a) templates was first obtained using a gst-nsp12 fusion protein, although these efforts were hampered by protein instability, which also led to the conclusion that the n-terminal domain of nsp12 is required for activity (cheng et al., 2005) . subsequently, a c-terminally his 6 -tagged sars-cov nsp12 was found to mediate homopolymeric rna synthesis in a primer-dependent manner (te velthuis et al., 2010) . both these activities must probably be considered relatively weak and nonprocessive compared to the activity observed when a sars-cov nsp12 rdrp assay was supplemented with nsp7 and nsp8 (subissi et al., 2014b) . however, at the same time, this study reinvigorated the debate on the initiation mechanism of the coronavirus rdrp, as the nsp7-8-12 tripartite complex displayed both primer-dependent and de novo initiation of rna synthesis, whereas no de novo-initiating rdrp activity could be detected for nsp8 or the nsp7-nsp8 complex alone (subissi et al., 2014b) . to add to the confusion, other studies reported de novo initiation by sars-cov nsp12 alone (ahn et al., 2012) and primer-dependent rdrp activity of sars-cov nsp8, when expressed without affinity tags commonly used to facilitate purification (te velthuis et al., 2012). technical differences between these studies and those summarized earlier (e.g., regarding expression constructs and templates used) may have contributed to the contradictory results obtained on the rdrp activities of nsp8 and nsp12. thus far, five different laboratories addressed the two (putative) coronavirus rdrps in seven independent studies, none of which succeeded in exactly reproducing the results of any of the other studies (ahn et al., 2012; cheng et al., 2005; imbert et al., 2006; subissi et al., 2014b; te velthuis et al., 2010 te velthuis et al., , 2012 xiao et al., 2012) . nidovirus rdrps appear to be technically challenging and sensitive proteins that may respond to minute changes in purification protocols or assay conditions. clearly, both the role of nsp8 (primase or processivity factor?) and the initiation mechanism employed by the nsp12-rdrp require further study. although the bioinformatics-based prediction that nsp12 uses a primer-dependent initiation mechanism is compelling, it lacks the direct support of an nsp12 crystal structure. at the same time, the question of the nature and source of the primer that would be used by nsp12 seems to be wide open again. as for other rna viruses, the nsp12-rdrp of covs is a primary drug target that may, in principle, be inhibited without major toxic side effects for the host cell. nucleoside analogs constitute an important class of antiviral drug candidates that can target viral rdrps, but efforts to use them to inhibit cov replication were not very successful thus far (chu et al., 2006; ikejiri et al., 2007) . moreover, it remains to be established that their target in the infected cell is indeed the nsp12-rdrp. the mismatch repair capabilities attributed to the nsp14-exon domain (see later) (bouvet et al., 2012) may pose an additional hurdle, as the efficacy of a nucleoside analogue with anticoronavirus activity may be determined by the balance between its propensity to be incorporated by the nsp12-rdrp and its tendency to resist excision by the mismatch repair mechanism mediated by nsp14-exon. similar considerations apply to ribavirin, a guanosine analog with broadspectrum antiviral activity that is used to treat patients infected with a variety of rna viruses. its mechanism of action appears to differ on a case-by-case basis, but may include the induction of lethal mutagenesis by increasing the rdrp error rate, inhibition of viral mrna capping, and reduction of viral rna synthesis by inhibition of the cellular enzyme inosine monophosphate dehydrogenase (impdh), which decreases the availability of intracellular gtp (crotty et al., 2000 (crotty et al., , 2002 smith et al., 2013 smith et al., , 2014 . although ribavirin was used to treat small numbers of sars and mers patients, high doses were used and the benefits of the treatment remained essentially unclear (zumla et al., 2016) . experiments with different covs in animal models falzarano et al., 2013) and infected cell cultures (ikejiri et al., 2007; pyrc et al., 2006) also established its poor activity and strongly suggested that ribavirin does not target the cov rdrp directly or is targeted (itself ) by the nsp14-exon activity (smith et al., 2013) . innovative nucleoside inhibitors continue to be identified or developed (peters et al., 2015; warren et al., 2014) and the recently described in vitro rdrp assay (subissi et al., 2014b) may prove very useful for establishing their mechanism of inhibition more precisely. a better understanding of nsp12-rdrp structure and function will also be required to design strategies that minimize the impact of drug resistance-inducing mutations, which are a common problem when targeting enzymes of rapidly evolving rna viruses. since the delineation of the borders of the cov rdrp-containing replicase cleavage product (boursnell et al., 1987; gorbalenya et al., 1989) , which is now known as nsp12, it had been clear that the protein must be a multidomain subunit, with the canonical rdrp domain roughly occupying its c-terminal half (fig. 3a ). only recently, first clues to some of the properties and possible functions of the n-terminal part of nsp12 were obtained (lehmann et al., 2015a) . a renewed bioinformatics analysis across the (still expanding) order nidovirales revealed that the nidoviral rdrp-containing replicase subunit contains a conserved n-terminal domain of 200-300 residues ($225 residues in cov nsp12; fig. 3b ). in cov nsp12, about 175 residues separate the niran and rdrp domains, leaving space for the presence of an additional domain between the two. based mainly on biochemical data obtained with the arterivirus homolog (see later), the n-terminal domain was concluded to possess an essential nucleotidylation activity and hence it was coined nidovirus rdrp-associated nucleotidyltransferase (niran) (lehmann et al., 2015a) . niran conservation was found to be lower than that of the downstream rdrp domain (fig. 3a) , but the analysis suggested that the evolutionary constraints on niran have been similar in different nidovirus lineages, which would be in line with a conserved function. gorbalenya and colleagues identified three key niran motifs (a-b-c) containing seven invariant residues (fig. 3b) , with domains b and c being most conserved (lehmann et al., 2015a) . the identification of the niran domain was further supported by the conservation of its predicted secondary structure elements in different nidovirus families (fig. 3a) . extensive database searches did not reveal potential niran homologs in either the viral or the cellular world, although it cannot be excluded that the domain has diverged from cellular ancestors to a level that prevents their identification with the currently available sequences and tools. nevertheless, its unique presence in nidoviruses and its association with the important rdrp domain suggest that niran may be a crucial regulator or interaction partner of the downstream rdrp domain that must have been acquired before the currently known nidovirus lineages diverged. niran and the zinc-binding domain (zbd) that is associated with the nsp13-helicase protein (see later) are the only unique genetic markers of the order nidovirales identified thus far. mainly due to the lack of sufficient amounts of recombinant cov nsp12, the preliminary biochemical characterization of niran was restricted to its arterivirus homolog, using recombinant nsp9 of equine arteritis virus (eav) (lehmann et al., 2015a) . for both eav and sars-cov, it could be shown that replacement of conserved niran residues can cripple or completely block virus replication in cultured cells. a combination of biochemical assays revealed that in vitro the niran domain exhibits a specific, mn 2+ -dependent enzymatic activity that results in the self-nucleotidylation of eav nsp9. the activity was abolished upon mutagenesis of conserved key residues in niran motifs a, b, and c. although utp was found to be the preferred substrate for niran's in vitro nucleotidylation activity, also gtp could be used, albeit less efficiently. the conserved lysine residue in motif a (the eav equivalent of lys-73 in sars-cov nsp12) was concluded to be the most likely target residue for nucleotidylation via formation of a phosphoamide bond. although the importance of the niran domains of arterivirus nsp9 and coronavirus nps12 was supported by the outcome of reverse-genetics studies (lehmann et al., 2015a) , the role of the produced protein-nucleoside adducts in viral replication remains unclear at present. in fact, the unique dual specificity for utp and gtp seems to argue against two initially considered potential niran functions (lehmann et al., 2015a) . the first of these was a role as an rna ligase, a type of activity however that commonly is atp dependent. the second was its involvement in synthesizing mrna cap structures. one of the four enzymes required for this process, the crucial guanylyl transferase (gtase), still remains to be identified for covs (see later). however, niran's substrate preference for utp over gtp is difficult to reconcile with this hypothesis and has not been observed for other gtases involved in mrna capping. the third hypothesis that was put forward links back to the open question of the initiation of coronavirus rna synthesis, which presumably is a primer-dependent step (see earlier). nsp12 nucleotidylation could be envisioned to play a role in protein-primed rna synthesis, a strategy used by, e.g., picornaviruses and their relatives, which covalently attach an oligonucleotide to a viral protein (called vpg in the case of picornaviruses) that subsequently mediates the initiation of rna synthesis (paul et al., 2000) . the first step in the synthesis of the "protein primer" is a nucleotidylation step during which a nucleotide monophosphate is covalently attached to the vpg. niran could be involved in a similar mechanism either directly or indirectly, by transferring the bound nucleotide to another protein player. although such a mechanism would definitely revolutionize the concept of the initiation of cov rna synthesis, it is clearly not very compatible with some of the currently available data, such as the reported presence of a 5 0 cap structure (rather than a vpg-like molecule) on cov mrnas. evidently, the further in-depth characterization of niran is needed to fill the current knowledge gaps, starting with the biochemical characterization of a cov niran domain, which may confirm and extend the features now deduced from the analysis of its distantly related arterivirus homolog. helicases are versatile ntp-dependent motor proteins that play a role in cellular nucleic acid metabolism in the broadest possible sense, including processes like dna replication, recombination and repair, transcription, translation, as well as rna processing. helicases are also encoded by all +rna viruses with a genome size exceeding 7 kb, suggesting they are required for the efficient replication of +rna viral genomes above this size threshold. given the large size of the genomes of covs and related nidoviruses, they may depend on the function(s) of a replicative helicase even more than other +rna virus taxa. however, despite their abundance and conservation, the specific role of helicases in +rna virus replication remains poorly understood. for an extensive recent review of nidovirus helicases, the reader is referred to lehmann et al. (2015c) . currently, helicases are classified into six superfamilies (sfs) (singleton et al., 2007) , with +rna viral helicases belonging to sf1 (e.g., alphaviruses and nidoviruses), sf2 (e.g., flaviviruses), or sf3 (e.g., picornaviruses). the presence of a sf1 helicase (hel1) domain in the cov replicase polyprotein was discovered upon the early in-depth analysis of the first full-length cov genome sequence that became available (ibv) (gorbalenya et al., 1989) . the hel1 domain maps to the c-terminal part of the replicase cleavage product that is now known as nsp13, which is about 600 residues long. the cov hel1 domain contains all characteristic sequence motifs of the sf1 superfamily. the n-terminal part of nsp13 is formed by a multinuclear zbd, one of the most conserved domains across the order nidovirales (gorbalenya, 2001; nga et al., 2011) . this qualification also applies to the helicase-containing subunit as a whole, despite considerable size differences between, e.g., cov nsp13 and its arterivirus homolog (designated nsp10) (lehmann et al., 2015c) . the zbd and hel1 domains occupy a conserved position downstream of the rdrp domain in all nidovirus replicase polyproteins studied so far. sf1 helicases contain at least a dozen conserved motifs that direct the binding of ntps and nucleic acids. of these, motifs i and ii (also known as the walker a and b boxes) are common to helicases of all sfs as well as ntpases. structurally, the catalytic core of sf1 helicases like the cov hel1 domain is formed by two reca-like domains, designated 1a and 2a (fig. 4) , that bind to nucleic acids through stacking interactions of aromatic residues with the bases of their nucleic acid substrates (velankar et al., 1999) . cyclic conformational changes of the reca-like domains mediate the conversion of the energy from hydrolysis of the phosphodiester bonds of ntps into directional movement along the nucleic acid substrate, with the so-called "inchworm" model now widely being considered as best supported by the available experimental data (lehmann et al., 2015c; velankar et al., 1999; yarranton and gefter, 1979) . additional domains, located upor downstream of 1a and 2a, or inserted internally, can mediate supplemental protein-protein and protein-nucleic acid interactions or enzymatic activities, thus contributing to the functional versatility and specificity of the enzyme (lehmann et al., 2015c; singleton et al., 2007) . within helicase sf1, the cov hel1 domain belongs to the upf1-like family (sf1b) which is characterized by moving in the 5 0 -to-3 0 direction along the nucleic acid strand to which they bind. upf1-like helicases may unwind either dna or rna and, in some cases, also both substrates without a clear preference, as was readily observed during the in vitro characterization of different nidovirus helicases. the cov hel1 activity was first demonstrated in vitro using recombinant hcov-229e nsp13 (seybert et al., 2000a) . bacterially expressed nsp13 from hcov-229e and sars-cov, and also the homologous helicase (nsp10) of the arterivirus eav, displayed 5 0 -to-3 0 unwinding activity on double-stranded rna or dna substrates containing single-stranded 5 0 overhangs (ivanov and ziebuhr, 2004; ivanov et al., 2004b; seybert et al., 2000a,b; tanner et al., 2003) . following the biochemical characterization of sars-cov nsp13, it was calculated that unwinding occurs in discrete steps of 9.3 base pairs each, with a catalytic rate of 30 steps per second (adedeji et al., 2012a) . the nsp13 ntpase activity can use all four natural ribonucleotides and nucleotides as substrate, with atp, datp, and gtp being hydrolyzed most efficiently, and utp being the least preferred substrate (ivanov and ziebuhr, 2004; ivanov et al., 2004b; tanner et al., 2003) . replacement of a conserved lys in motif i, the walker a box (walker et al., 1982) , kills the in vitro ntpase activity of all nidovirus helicases tested thus far and, when introduced by reverse genetics, this mutation also abolished replication of the arterivirus eav (seybert et al., 2000b) . the substrate preferences summarized earlier support a threedimensional model of the sars-cov hel1 core domains (1a and 2a) that was based on structural information available for multiple cellular helicases (hoffmann et al., 2006) . the model predicts both the existence of multiple hydrogen bonding interactions with the βand γ-phosphates of the ntp and a lack of specific interactions with the nucleobase. thus, the mere presence of a 5 0 triphosphate group appears to be the main determinant for ntp/dntp binding. since nidovirus helicases are presumed to unwind double-stranded rna intermediates that are formed during viral replication, considerable attention was given to the in vitro characterization of their nucleic acid substrate preferences (seybert et al., 2000a,b) . the hcov-229e and eav helicases could not unwind substrates with 3 0 single-stranded tails or blunt-ended substrates. in contrast, rna and dna substrates with one or two 5 0 single-stranded regions were unwound efficiently, suggesting that the nidovirus helicase must bind to a single-stranded region before initiating unwinding in the 5 0 -to-3 0 direction. however, the in vitro assays did not yield any clear indications for the preferred recognition of specific sequences or higher-order structures in the substrate (lehmann et al., 2015c) . also a more in-depth biochemical characterization, performed with sars-cov nsp13, confirmed that the cov helicase does not discriminate between rna and dna substrates (adedeji et al., 2012a) . consequently, it cannot be excluded that the enzyme, in addition to being engaged in viral rna synthesis, may also target host dna. nuclear translocation of nidovirus helicases has not been reported thus far, but the light microscopy techniques used to study the protein's subcellular distribution would not suffice to detect the nuclear import of only a small fraction of the protein. as a final caveat it should be stressed that the biochemical properties summarized earlier are all derived from in vitro studies using recombinant helicases, expressed in different systems and sometimes containing substantial foreign sequences. the in situ characterization of the helicase as one of the key enzymes of the nidovirus rna-synthesizing machinery remains to be addressed. in that context, sequence specificity, for example, could be conveyed by other subunits of the replicase complex, which may target the helicase to, e.g., the initiation sites for viral genome or antigenome synthesis, or to signals controlling the production of subgenomic mrnas. as summarized by lehmann et al. (2015c) , other important helicase features that could be dramatically different in the setting of the infected cell are (the need for) helicase oligomerization, cooperativity between multiple helicase molecules binding to the same substrate, and-consequentlythe overall processivity of the enzyme, which in vitro appeared to be quite low given the large cov genome size (adedeji et al., 2012a) . the nidovirus helicase subunit domain is unique among its +rna virus homologs in having a conserved n-terminal domain of 80-100 residues that contains 12 or 13 conserved cys/his residues (den boon et al., 1991; van dinten et al., 2000) . the domain was recognized as a potential zbd (gorbalenya et al., 1989) and early in vitro studies with the recombinant hcov-229e and eav helicases confirmed that zn 2+ ions are essential for retaining the protein's enzymatic activities, suggesting that zbd modulates nidovirus helicase function (seybert et al., 2005) . a recent structural study of the arterivirus nsp10-helicase (deng et al., 2014) will be discussed in more detail later. this first nidovirus helicase structure confirmed the binding of three zinc ions by the zbd, which adopts a unique fold that combines a ring-like module with a so-called "treble-clef" zinc finger. zbd and hel1 interact extensively (deng et al., 2014) but, in the helicase primary structure, they are separated by a variable and uncharacterized domain that essentially explains the size difference of about 130 residues between cov and arterivirus helicase subunits (seybert et al., 2005) . using the arterivirus prototype eav, the functional importance of zbd was probed extensively by combining biochemistry and reverse genetics (seybert et al., 2005; van dinten et al., 2000) . this yielded a variety of phenotypes for nsp10-zbd mutants, the most striking being mutants deficient in subgenomic mrna synthesis while remaining capable of (and even enhancing) viral genome replication (van dinten et al., 1997 (van dinten et al., , 2000 (see later). most replacements of conserved zbd cys and his residues profoundly impacted the helicase activity of eav nsp10, even when performed in a semiconservative manner that could preserve zinc binding. in reverse-genetics studies, most of these zbd mutations rendered the virus nonviable. recently, the impact of these mutations on zbd integrity and zbd-hel1 interactions could be rationalized with the help of the nsp10 crystal structure (deng et al., 2014) . despite its importance as a potential drug target, a cov nsp13 or hel1 crystal structure has not been obtained thus far due to technical complications with recombinant protein production and crystallization. instead, several cov helicase models have been described, mainly based on cellular helicase structures (bernini et al., 2006; hoffmann et al., 2006; lehmann et al., 2015c) . given this limitation, and despite the large evolutionary distance between the two enzymes, it is interesting to have a closer look at the recently published eav nsp10-helicase structure (deng et al., 2014) . the overall structure of eav nsp10 (fig. 4) consists of the n-terminal zbd, a new domain designated 1b, the two reca-like hel1 domains (1a and 2a), and a short c-terminal domain, which is not conserved among nidoviruses and needed to be deleted to allow nsp10 crystallization. this 65-amino-acid c-terminal truncation did not affect the helicase core domains and only modestly influenced levels of atpase and helicase activity compared to the full-length protein (deng et al., 2014) . compared to cellular representatives of the sf1 helicase superfamily, nsp10 is most similar to upf1 and its close homolog ighmbp2, which also contain an n-terminal zbd. moreover, the location and orientation of the newly discovered 1b domain of nsp10 (residues 83-137) resembles that of the domain with the same name found in the upf1-like helicase subfamily. the nsp10 zbd uses 12 conserved cys and his residues to coordinate three zinc ions and folds into two zinc-binding modules that are connected by a disordered region. the n-terminal ring-like structure of nsp10 coordinates two zinc ions and the closest similarity that was found for this module was with the n-terminal zinc-binding ch-domain of upf1. both proteins have a second zinc-binding module downstream (a so-called treble-clef zinc finger in the case of nsp10), but these are structurally different, suggesting that the nidoviral zbd represents a new kind of complex zinc-binding element. the previous suggestion of zbd codetermining hel1 function was strengthened by the presence of an extensive interface of 1019 å 2 that was proposed to be involved in intramolecular signaling (deng et al., 2014) . a second crystal structure was obtained for nsp10 in complex with a partially double-stranded dna substrate, revealing possible nucleic acid-binding clefts at the protein's surface that are formed by the zbd + 1b and zbd + 1a domains. although the exact path of the nucleic acid strands could not yet be determined, it became clear that the positively charged zbd, and in particular its n-terminal ring-like module, must be involved in nucleic acid binding. in line with the biochemical data summarized earlier, most of the nsp10-substrate contacts identified are not base-specific and occur with the nucleic acid backbone. whereas the hel1 core domains were found to be quite similar in the absence or presence of bound substrate, a remarkable 29 degree rotation was observed for domain 1b, enlarging the dimensions of the nucleic acid substrate channel formed by domains 1a and 1b, but not allowing it to accommodate a duplex substrate. consequently, it was postulated that an element near the entrance of the substrate channel may destabilize the duplex and facilitate the entrance of one of the strands into the channel. since the double-stranded region of the duplex could not be modeled, additional studies are needed to verify the existence and molecular details of this proposed unwinding mechanism (deng et al., 2014; lehmann et al., 2015c) . likewise, direct structural information on cov nsp13 is needed to be able to assess to which extent the structural observations made for arterivirus nsp10 can be translated to distantly related (and larger) nidovirus helicases (fig. 4) . in general, however, the analysis of nsp10 provided a clear basis for a model in which the function of the common reca-like core domains of nidovirus helicases is modulated by specific extension domains, presumably to facilitate the involvement of the nidovirus helicase in multiple processes in the infected cell (see later). as outlined earlier, the biochemical characterization of purified recombinant nidovirus helicases, the functional probing of (in particular) the eav nsp10-helicase using reverse genetics, the eav nsp10 structure, and advanced bioinformatics analyses together have painted a picture of an enzyme that is involved in multiple critical steps of the viral replicative cycle. space limitations do not allow an in-depth discussion of all of these (putative) functions, which-based on eav nsp10 studies-may include a poorly characterized role in virion biogenesis (van dinten et al., 2000) , not unlike what was uncovered for, e.g., the helicase-containing ns3 protein of flaviviruses (liu et al., 2002; ma et al., 2008) . likewise, we will not discuss the first reports on possible interactions with host proteins, such as the ddx5 helicase (for sars-cov nsp13; chen et al., 2009b) and polymerase δ (for ibv nsp13; xu et al., 2011) . instead, we will focus on the most significant findings related to nidovirus helicase functions in rna synthesis and processing, specifically (i) genome replication, (ii) transcription of subgenomic mrnas, (iii) mrna capping, and (iv) posttranscriptional mrna quality control. the presumed "default" function of +rna viral helicases is to cooperate with the viral rdrp to achieve the efficient amplification of the genome. in this context, helicases are presumed to promote rdrp processivity by opening up the double-stranded rna intermediates of viral replication, and possibly also by removing rna secondary structures in single-stranded template strands (kadare and haenni, 1997) . in this light, reports on molecular interactions between the cov rdrp (nsp12) and helicase (nsp13) were not unexpected von brunn et al., 2007) . the same holds true for the observation that sars-cov nsp12 can stimulate the in vitro helicase activity of nsp13 (adedeji et al., 2012a) and for the fact that both nsp12 and nsp13 (like most other cov replicase cleavage products) associate with the membranous replication organelles in nidovirus-infected cells (denison et al., 1999; ivanov et al., 2004b; knoops et al., 2008) . in spite of all these indications for rdrp-helicase interplay, the polarity of nidovirus helicase-mediated unwinding (5 0 -to-3 0 ) remains a major conundrum, as it is opposite to the polarities of the rdrp and many other +rna helicases, which move in a 3 0 -to-5 0 direction on the rna strand they initially bind to (seybert et al., 2000a) . this strongly suggests that the two enzymes cannot simply operate as a tandem that moves along the same template strand while copying it, a consideration that also applies to the sf1b helicase employed by alphaviruses. this problem could be resolved if rdrp and helicase would move along different strands of the same rna duplex, which might allow the helicase to separate the two strands and provide a single-stranded template to be copied by the rdrp (lehmann et al., 2015c) . also, it is tempting to speculate that the helicase, by trailing along the nascent strand (following the rdrp at a certain and, possibly, somewhat flexible distance), provides (i.e., leaves behind) a single-stranded template, thus facilitating initiation and elongation of rna synthesis by the next rtc. this, for example, could occur in cases when multiple rtcs act simultaneously/consecutively on the same template to produce multiple plus-strand rnas from the same minus-strand template, a process that is generally thought to add to the large excess of plus-over minus-strand rnas in nidovirus-infected cells. clearly, significantly more work is needed to explore this possibility. nidovirus sg mrnas are each produced from their own subgenomelength minus-strand template (see section 1). in the case of arteri-and coronaviruses, these derive from a process of discontinuous minus-strand rna synthesis and this unique mechanism likely requires specific functional interactions between (among others) rdrp and helicase. these interactions may contribute to maintaining a proper balance between continuous and discontinuous minus-strand rna synthesis, and thus between the production of new genomes and sg mrnas. the serendipitous identification of an arterivirus nsp10-zbd mutation (ser-59 ! pro) that essentially inactivated transcription while leaving replication unaffected was an early indication for the involvement of the nidovirus helicase in the control of sg mrna synthesis (van dinten et al., 1997) . such control functions could also be related to the recognition of trss (fig. 1) , the frequency with which each of the trss is used to produce a subgenome-length minus strand, or mechanistic aspects of the stalling and reinitiation of rna synthesis or the transfer of the nascent strand to an upstream position on the template (see earlier), which must occur during the discontinuous step in sg rna synthesis (lehmann et al., 2015c) . recently, the eav nsp10 ser-59 ! pro mutation, which selectively reduces transcription of all subgenomic mrnas to below 1% of their normal levels, was reanalyzed in the context of the nsp10 crystal structure. as postulated when this virus mutant was first described, its phenotype appears to be based on the special structural properties of the pro residue in combination with the position of residue 59 in a "hinge" region that connects zbd to the rest of the protein (deng et al., 2014) . although residue 59, located just downstream of the second zinc-binding module of the zbd, is fairly distant from the rna-binding surface, it resides in a region that connects the zbd treble-clef zinc finger to a helix that interacts with domains 1a and 1b and with the nucleic acid. thus, specific mutations affecting the flexibility of this hinge region may drastically influence the long-distance signaling within nsp10, apparently preventing the rnasynthesizing machinery to work in "transcription mode" and dedicating it exclusively to full-length minus-strand rna synthesis and genome amplification. since this kind of mutations barely affected nsp10s in vitro ntpase and helicase activities (seybert et al., 2005) , it may well be that changed interactions with specific protein partners will turn out to be the key to explaining the transcription-negative phenotype of the corresponding virus mutants (lehmann et al., 2015c) . for the coronavirus ibv, a point mutation in a somewhat comparable position of nsp13 (arg-132 ! pro; just downstream of zbd) was reported to cause a similar block of sg mrna synthesis (fang et al., 2007) but, thus far, this observation has not been followed up in more detail for ibv or confirmed for nsp13 of another cov. in addition to its role in rna synthesis, the nidovirus helicase is also assumed to be involved in the capping pathway of viral mrnas by exhibiting an rna 5 0 -triphosphatase (rtpase) activity that can remove the 5 0 -terminal triphosphate from the rna substrate. for sars-cov and hcov-229e nsp13, this first step in viral cap synthesis was shown to rely on the same ntpase active site that provides the energy for the protein's helicase activity (ivanov and ziebuhr, 2004; ivanov et al., 2004b) . the cov capping pathway is discussed in section 5 of this review. finally, the remarkable similarities between eav nsp10 and the cellular helicase upf1 (deng et al., 2014) have given rise to the intriguing but still speculative hypothesis that the nidovirus helicase may be involved in the posttranscriptional quality control of viral rna. common features of the two helicases include their 5 0 -to-3 0 polarity of unwinding, their lack of substrate specificity and striking similarities in terms of domain organization and fold (fig. 4) (lehmann et al., 2015c) . using several pathways, including nonsense-mediated mrna decay, upf1 mediates rna quality control in eukaryotic cells (cheng et al., 2007; clerici et al., 2009) , while its activity can be modulated through interactions of its n-terminal zbd. it was postulated that a similar function in nidovirus replication, e.g., detection and elimination of defective viral mrnas (including the genome), could explain the conservation of the unique zbd across the nidovirus order, which stands out for containing members with very large + rna genomes. such a function would prevent the synthesis of defective viral polypeptides, which might interfere with the proper functioning of full-length viral proteins. in this manner, not unlike the nsp14-exon domain (see earlier), the nidovirus helicase may have contributed to genome expansion by providing a form of compensation for the relatively poor fidelity of genome replication by the nidoviral rdrp (deng et al., 2014) . clearly, this is just one of the scenarios for the involvement of the helicase in the posttranscriptional fate of viral rna products. further experimental work will be needed to explore these possibilities in more detail, as they are compatible with the much broader realization that the functions of rna helicases can extend far beyond merely the unwinding of rna structure. due to its multifunctionality and involvement in several key processes in viral rna synthesis and processing, the nidovirus helicase is an important target for antiviral drug development, which was mainly explored for covs following the sars-cov outbreak. the highly conserved nature of the helicase offers the interesting perspective of developing inhibitors with a potential broad-spectrum activity. on the other hand, avoiding toxicity resulting from inhibition of the abundant cellular ntpases/helicases poses a serious challenge, which is why-as in the case of the rdrp-obtaining a crystal structure for a cov helicase should be considered a research priority. in theory, a variety of helicase properties may be targeted with specific inhibitors, ranging from the active and nucleic acid-binding sites of the enzyme to interaction surfaces for multimerization and modulation by protein cofactors (kwong et al., 2005) . several compound families were found to target the atpase of nsp13, and thus also its helicase activity. these include naturally occurring flavonoids (yu et al., 2012) , chromones (kim et al., 2011) , and bananins (kesel, 2005; tanner et al., 2005) , all exhibiting in vitro ic 50 values in the low-micromolar range. other compounds appear to target the helicase of nsp13 activity more specifically, like the triazole ssya10-001, which was found to inhibit the replication of multiple beta-covs (sars-cov, mers-cov, and mhv) in cell culture-based infection models (adedeji et al., 2012b (adedeji et al., , 2014 . the ic 50 value in in vitro helicase assays was about 5.5 μm, whereas ec 50 values in cell cultures assays were in the range of 7-25 μm, depending on the cov analyzed, suggesting that broad-spectrum activity may indeed be achieved. to our knowledge, the antiviral potential and toxicity of ssya10-001 in animal models remain to be tested. another interesting group of helicase-directed antiviral hits are bismuth complexes, which were postulated to inhibit the ntpase and helicase functions by competing for zinc ions with the zbd. in sars-cov-infected cell cultures the determined ec 50 and cc 50 values were 6 μm and 5 mm, respectively (yang et al., 2007) . nsp10-13-14-16 cap structures consists of a 7-methylguanosine ( m7 g) linked to the first nucleotide of the rna transcript through a 5 0 -5 0 triphosphate bridge (for a review, see decroly et al., 2012) . in eukaryotic cells, the synthesis of the cap structure is a multistep process that occurs in the nucleus and is coupled to rna pol ii-driven transcription (shatkin, 1976) . capping begins with the hydrolysis of the 5 0 γ-phosphate of the nascent rna transcript by an rna 5 0 -triphosphatase (rtpase). subsequently, a gmp molecule is transferred to the 5 0 -diphosphate of the rna by a gtase, leading to the formation of gpppn-rna. the cap structure is methylated at the n7 position of the guanosine by an (adomet)-dependent n7-mtase, yielding cap-0 ( m7 gpppn). the cap-0 structure is then converted into cap-1 ( m7 gpppn 2 0 -om ) or cap-2 by an adomet-dependent 2 0 -o-mtase that methylates the 2 0 -o position of the ribose of the first or first and second rna nucleotide, respectively. due to the cytoplasmic localization of their mrna synthesis, nidoviruses, and all other +rna viruses of eukaryotes, cannot rely on the standard capping pathway outlined earlier, which is executed by host cell enzymes in the nucleus. cap structures can protect viral mrnas from degradation by the cellular 5 0 -to-3 0 exoribonuclases involved in rna turnover (liu and kiledjian, 2006) . cap methylation is critical for mrna recognition by translation initiation factor eif4e (filipowicz et al., 1976; ohlmann et al., 1996) , and thus for viral translation and replication as a whole . in addition to promoting mrna stability and securing their recognition by host cell ribosomes, capping of viral mrnas also promotes escape from certain antiviral responses of the host cell. the retinoic acidinducible gene 1 and melanoma differentiation-associated protein 5 (mda5) were shown to detect either uncapped triphosphorylated rna or cap-0-containing rna (devarkar et al., 2016; hyde and diamond, 2015; hyde et al., 2014; schuberth-wagner et al., 2015; z€ ust et al., 2011) , resulting in the expression of antiviral interferon-stimulated genes (isgs) in infected and neighboring cells. interferon-induced proteins with a tetratricopeptide repeat 1 (ifit1/56) and ifit2/54 (ifit2) have been shown to recognize miscapped rnas, in order to restrict viral translation (daffis et al., 2010; pichlmair et al., 2011) . a subsequent study identified ifit1 as the only interferon-induced protein whose rna-binding affinity was affected by the ribose-2 0 -o methylation state of the 5 0 cap structure (cap-0/cap-1). the data support a model in which ifit1 efficiently binds and sequesters capped mrna that lacks a ribose-2 0 -o-methyl group. consistent with this, viral mrna translation was shown to be reduced in cells infected with 2 0 -o-mtase-deficient covs (habjan et al., 2013) . other studies suggested that 2 0 -o methylation of cap structures prevents or delays the mda5-dependent recognition of viral mrnas as "nonself." this mrna cap modification thus limits the antiviral response launched upon infection, thereby affecting viral pathogenesis (daffis et al., 2010; schuberth-wagner et al., 2015; z€ ust et al., 2011) . the genomic and sg mrnas of nidoviruses are presumed to be capped at their 5 0 end and polyadenylated at their 3 0 end (fig. 1) . the presence of a cap structure was first suggested based on studies using 32 p-labeled mhv rna that was digested with rnase t1 and t2 and subjected to deaecellulose chromatography (lai and stohlman, 1981) . the presence of a cap was further substantiated by immunoprecipitation experiments using a cap-specific monoclonal antibody that was shown to specifically bind to equine torovirus mrnas (van vliet et al., 2002) . although the (presumed) capping machinery of arteriviruses has remained essentially uncharacterized thus far (lehmann et al., 2015b) , three conserved putative capping enzymes were identified in the conserved orf1b-encoded part of the replicase of coronaviridae, roniviridae, and mesoniviridae, which all have substantially larger genomes. these enzymatic activities, which were proposed to participate in the synthesis of a cap-1 structure ( n7m gpppn 2 0 om ), are the following: (i) the nsp13 helicase/rtpase (ivanov and ziebuhr, 2004) , (ii) the nsp14 n7-mtase (chen et al., 2009c; ma et al., 2015) , and (iii) the nsp16 2 0 -o-mtase (bouvet et al., 2010; decroly et al., 2008; snijder et al., 2003; von grotthuss et al., 2003; zeng et al., 2016) . at present, structural and functional studies, which were mainly performed with purified recombinant sars-cov nsps, suggest that covs follow a capping pathway that is very similar to that of eukaryotic cells. cap synthesis is presumed to start by hydrolysis of the 5 0 end of a nascent rna by the rtpase function of nsp13 to yield pp-rna (ivanov and ziebuhr, 2004) . then, a still elusive gtase must transfer a gmp molecule onto the pp-rna to yield gppp-rna. the cap structure is then methylated at the n7 position by the n7-mtase domain of the bifunctional nsp14 (bouvet et al., 2010; chen et al., 2009c) . cap modification is completed by the conversion of the cap-0 into a cap-1 structure, which involves the nsp10/nsp16 complex (bouvet et al., 2010; chen et al., 2009c) in which nsp16 possesses 2 0 -o-mtase activity. in the following paragraphs, we will describe the different cov enzymes involved in mrna capping in more detail. as described earlier, the nsp13 helicase domain is thought to unwind double-stranded rna in a 5 0 -to-3 0 direction, an activity that energetically depends on the nucleotide triphosphatase (ntpase) function of the enzyme (ivanov and ziebuhr, 2004; ivanov et al., 2004b; seybert et al., 2000a) . additionally, using the same active site, the protein was found to exhibit rtpase activity (ivanov and ziebuhr, 2004) , which was found to be abolished if the conserved active-site lys residue of the walker a box motif was replaced with ala (ivanov and ziebuhr, 2004; ivanov et al., 2004b) . specific rtpase activity on viral mrna species would require specific recruitment of nsp13 to the 5 0 end of viral mrnas, which has not been demonstrated for cov helicases but may involve yet other factors. in this context, it remains to be studied if the common leader sequence present on cov mrnas contributes to the recruitment of nsp13 and/or other proteins involved in 5 0 capping reactions. several other +rna virus helicases were shown to possess an activity that can target the phosphodiester bond between the β and γ phosphate groups of the 5 0 -terminal ntp of diverse substrate rnas, suggesting that this dual function of helicase and capping rtpase is a common feature in this group of viruses ivanov and ziebuhr, 2004) . even though experimental evidence has been obtained to suggest an nsp13-associated 5 0 rtpase activity, coronavirus nsp13 homologs proved to be unable to transcomplement the yeast strain ybs20, which lacks the cet1 locus encoding the yeast rtpase involved in mrna capping (chen et al., 2009c) . the lack of trans-complementation by nsp13 could be due to technical reasons, such as inappropriate subcellular localization of nsp13, misfolding of the protein, or a functional mismatch with other players of the distantly related yeast capping machinery. alternatively, it could indicate specific substrate requirements for coronavirus nsp13-associated rtpase activities. in any case, a possible involvement of nsp13 in the first step of cov mrna capping remains to be corroborated in further studies, for example, by in vitro reconstitution experiments of the entire cov capping pathway. the cov nsp involved in the second step of rna capping, gtase, remains to be identified. bioinformatics analysis of cov genome sequences failed to identify replicase gene-encoded domains that may perform this activity. eukaryotic rna capping enzymes belong to the ligase family and have been shown to form a gtase-gmp adduct upon incubation with gtp . a substantial number of sars-cov nsps were expressed and purified (nsp7, nsp8, nsp10, and nsp12-16), but covalent linkage of gmp to any these proteins could not be demonstrated (jin et al., 2013) . in addition, nsps were screened for gtase activity in a yeast trans-complementation system using the ybs2 strain lacking the gene (ceg1) encoding the yeast gtase (chen et al., 2009c) , but also this powerful approach failed to identify the cov gtase. consequently, several hypotheses remain to be explored. first, it is possible that the n-terminal niran domain of nsp12 (see earlier) forms a covalent adduct with gtp, as observed for its arterivirus homolog nsp9 (lehmann et al., 2015a) . another possibility is that the cov capping pathway is unconventional and, for example, resembles that of alphaviruses in which the gtp molecule needs to be methylated at its n7 position before the gtase-m gmp adduct can be formed (ahola and ahlquist, 1999) . interestingly, this second possibility might explain nsp14s capability to convert gtp into m gtp (see later) (jin et al., 2013) . finally, the involvement of a host gtase remains an interesting possibility, in particular since cytoplasmic forms of cellular capping enzymes have been described recently (mukherjee et al., 2012; schoenberg and maquat, 2009 ). further work is needed to explore these various hypotheses and resolve this important question. coronavirus nsp14 is a bifunctional protein that plays a crucial role in viral rna synthesis. its n-terminal exonuclease (exon) domain (minskaia et al., 2006; snijder et al., 2003) , which is thought to promote the fidelity of cov rna synthesis, will be discussed in more detail in section 6. the c-terminal part of nsp14 carries an adomet-dependent guanosine n7-mtase activity. interestingly, as in the case of the gtase (see earlier), bioinformatics analyses of cov genome sequences failed to identify proteins or protein domains related to cellular and/or viral n7-mtases, again illustrating the significant divergence of nidoviruses from other viral and cellular systems. however, using a trans-complementation assay and a yeast strain lacking the abd1 (n7-mtase) gene, guo et al. discovered a sars-cov nsp14-associated n7-mtase activity (chen et al., 2009c) by demonstrating that the protein was able to restore the growth of the △abd1 yeast mutant. they also showed that a range of alphacoronavirus nsp14 homologs were able to complement the defect of the △abd1 yeast strain. the n7-mtase activity of nsp14 was subsequently confirmed and characterized using purified recombinant enzymes (bouvet et al., 2012; chen et al., 2009c) . the sars-cov n7-mtase was shown to methylate 5 0 cap structures in a sequenceindependent manner using a range of rnas and it also proved to be active on cap analogues and gtp (jin et al., 2013) , corroborating the trans-complementation experiments in yeast in which rescue required efficient methylation of a wide range of cellular rnas. in contrast to the exon activity, the in vitro n7-mtase activity was not found to be affected by interactions with nsp10 (bouvet et al., 2010) . the n7-mtase domain was further characterized by alanine scanning mutagenesis and key residues for enzymatic activity were identified (chen et al., 2009c) including 10 crucial residues distributed throughout the domain and two clusters of residues essential for mtase activity (fig. 5 ). the first cluster (nsp14 residues 331-336) corresponds to the dxgxpxa motif of the adomet-binding site. in cross-linking experiments, mutations in this motif strongly decreased the binding of 3 h-labeled adomet. the role of the second cluster, between residues 414 and 428, was revealed by x-ray structure analysis of a sars-cov nsp10/nsp14 complex expressed in e. coli (fig. 6) (ma et al., 2015) . these residues form a constricted pocket that holds the cap structure (gpppa) between two β-strands (β1 and β2) and helix 1, placing the n7 position of the guanine in close proximity of adomet and ready for methyl transfer using an in-line mechanism. , and ibv (genus gammacoronavirus). the alignment was generated using clustal omega (sievers et al., 2011) and rendered using espript version 3.0 (robert and gouet, 2014) . conserved exon motifs i, ii, and iii and clusters of residues involved in sam binding and n7-mtase activity (1 and 2) are highlighted in gray. catalytic residues of exon and residues involved in the formation of zinc fingers are indicated by asterisks and arrowheads, respectively. also shown are the secondary structure elements of sars-cov nsp14 (pdb 5c8s) and the border between the n-terminal exon and c-terminal n7-mt (nmt) domains. the structure also revealed that the nsp14 n7-mtase domain exhibits a noncanonical mtase fold. whereas the canonical fold contains a 7-strand β-sheet that is commonly present in the rossman fold, nsp14 contains only a 5-strand β-sheet and an insertion of a 3-strand antiparallel β-sheet between β5 and β6. in line with previous mutagenesis data (chen et al., 2009c) , the fig. 6 surface representation of the three-dimensional structure of the nsp10/nsp14 complex (pdb 5c8s). the nsp10 ribbon structure is shown with conserved residues colored in blue (using a scale from dark to light blue). the coloring of the nsp14 surface is based on the conservation of the respective residues among covs (using a scale from dark to light red). the upper panel shows the surface containing the exon catalytic site with one mg 2+ ion bound in the active site (green sphere). the lower panel shows the opposite side of the structure with the n7-mtase active site. the cap analog gpppa and sah are shown in stick representation. the figures were generated using ucsf chimera (pettersen et al., 2004) . the degree of conservation of specific residues was determined using an alignment of nsp10 and nsp14 sequences of eight coronaviruses representing the four genera of the coronavirinae subfamily (sars-cov, mers-cov, mhv, tgev, fcov, hcov-229e, ibv, and bulbul coronavirus hku11). nsp14 x-ray structure revealed functionally relevant interactions between the n-terminal (exon) and c-terminal (n7-mtase) domains, with three α-helices of exon stabilizing the base of the n7-mtase substrate-binding pocket. the specific role of n7-mtase activity in virus replication was supported by reverse genetics. nsp14 mutations were introduced in sars-cov rna replicons expressing a luciferase reporter gene. a d331a mutation in the adomet-binding site, which blocks the n7-mtase activity of nsp14, was shown to reduce the luciferase expression (by 90%), indicating that viral rna replication and/or transcription were impaired in this in vitro system (chen et al., 2009c) . the nsp14 n7-mtase is an attractive target for antiviral strategies, especially because it exhibits a range of features that are distinct from host cell mtases . the druggability of the enzyme was explored using a small set of previously documented mtase inhibitors. these in vitro assays revealed that adohcy (the coproduct of the methylation reaction), sinefungin (another adomet analog), and ata efficiently inhibited nsp14 n7-mtase activity with ic 50 values of 12 μm, 39.5 nm, and 2.1 μm, respectively (bouvet et al., 2010) . ata was also shown to limit sars-cov replication in infected cells (he et al., 2004) . in the yeast-△mtase trans-complementation assay mentioned earlier, micromolar concentrations of sinefungin were reported to effectively suppress the nsp14 n7-mtase activity of sars-cov, mhv, transmissible gastroenteritis virus (tgev), and ibv (sun et al., 2014) . however, other compounds, such as ata and adohcy, did not exert an inhibitory effect in the context of yeast cells. these discrepancies may reflect differences in cell penetration of the compounds between yeast and (virus-infected) mammalian cells. the yeast system was also applied to screen a library of 3000 natural product extracts, and three hits were obtained displaying potent inhibitory effects on the cov n7-mtase (sun et al., 2014) . further work is needed to optimize these hits and test their inhibitory activities in assays using cov-infected cells. the presence of a 2 0 -o-methyl transferase (2 0 -o-mtase) domain in cov nsp16 was first inferred using bioinformatics (snijder et al., 2003; von grotthuss et al., 2003) . computational threading produced a model containing a conserved k-d-k-e catalytic tetrad that is characteristic of adomet-dependent 2 0 -o-mtases and a conserved adomet-binding site ( fig. 7) (von grotthuss et al., 2003) . the 2 0 -o-mtase activity was then confirmed by in vitro biochemical assays using purified fcov nsp16 (decroly et al., 2008) . the recombinant protein was shown to specifically recognize short, cap-0-containing rnas and to transfer a methyl group from adomet to the 2 0 -o position of the first nucleotide of the n7-methylated substrate. in contrast, a recombinant form of the sars-cov nsp16 homolog was inactive under similar experimental conditions. since yeast two-hybrid experiments and gst pull-down assays had revealed that sars-cov nsp16 strongly interacts with nsp10 pan et al., 2008) , a possible involvement of the latter in regulating or supporting the 2 0 -o-mtase activity was tested. it was shown that purified nsp10 interacts with nsp16 in vitro and thereby triggers a robust 2 0 -o-mtase activity (bouvet et al., 2010) . effective methyl transfer was demonstrated using synthetic capped n7-methylated rna and longer rna mimicking the 5 0 end of the sars-cov genome (bouvet et al., 2010) . in contrast, rna with a nonmethylated cap structure (gppp-rna) was not bound by the nsp10/ nsp16 complex and, consequently, could not serve as a substrate. these observations suggest that sars-cov mrna capping follows a strict order of reaction steps: after gtp transfer by the still elusive gtase, the cap is methylated by the nsp14 n7-mtase at the guanosine-n7 position to produce a cap-0 structure that, in a subsequent reaction, is bound by the nsp10/ nsp16 complex and converted to a cap-1 structure employing the 2 0 -o-mtase activity of nsp16. mutagenesis of sars-cov nsp10 and nsp16 confirmed the importance of the catalytic tetrad of the nsp16 2 0 -o-mtase (decroly et al., 2011) and showed that the nsp10-nsp16 interaction is absolutely required for activity (decroly et al., 2011; lugari et al., 2010) . crystallographic studies of the nsp10/nsp16 complex revealed the molecular basis for the stimulation of the nsp16 2 0 -o-mtase activity by nsp10 (fig. 7) (chen et al., 2011; decroly et al., 2011) . the cov 2 0 -o-mtase belongs to the rrmj/fibrillarin superfamily of ribose 2 0 -o-methyl transferases (feder et al., 2003) which have a number of viral orthologs in flaviviruses, alphaviruses, and other nidoviruses. as mentioned earlier, this family contains a conserved k-d-k-e catalytic tetrad (fig. 7) that is located in close proximity to the substrate-binding pocket accommodating the rna substrate. the structure revealed that nsp10 is an allosteric regulator that stabilizes nsp16. moreover, structural and biochemical analyses indicated that nsp10 binding extends and narrows the rna-binding groove that accommodates the rna substrate, thereby promoting the rnaand adomet-binding capabilities of nsp16 (fig. 7) . ) , and ibv (genus gammacoronavirus). the alignment was generated using clustal omega (sievers et al., 2011) and rendered using espript version 3.0 (robert and gouet, 2014) . residues of the catalytic tetrad k-d-k-e are indicated by asterisks and secondary structure elements of sars-cov nsp16 (pdb 2xyv) are shown. (b) surface representation of the three-dimensional structure of the nsp10/nsp16 (continued) the functional relevance of 2 0 -o-mtase regulation by nsp10 for virus replication is not yet understood. the nsp16-associated 2 0 -o-mtase activity (itself ) is highly conserved across the coronaviridae family and its functional relevance has been supported by reverse-genetics studies using genetically engineered alpha-and betacoronavirus mutants that lack 2 0 -o-mtase activity (devarkar et al., 2016; hyde and diamond, 2015; schuberth-wagner et al., 2015; z€ ust et al., 2011) . furthermore, the phenotype of temperature-sensitive mhv mutants suggested nsp16 to play a role in rna synthesis or in the stability of plus-strand rna (sawicki et al., 2005 ). an early sars-cov study was able to show that the insertion of a stop codon immediately upstream of the nsp16-coding sequence blocked viral rna synthesis (almazan et al., 2006) . subsequently, for hcov-229e, mhv, and sars-cov, several mutants with reduced or ablated 2 0 -o-mtase activity were described that generally retained robust viral replication in cell culture (menachery et al., 2014; z€ ust et al., 2011) . the studies also revealed that the impact of nsp16 mutations may depend on the cell types used and that the lack of 2 0 -o-mtase activity causes more profound effects in primary cells and immune cells. the sars-cov nsp16 mutants were further characterized in infected mice and showed a robust attenuation as judged by viral titers, weight loss, lung histology, and respiratory function. the nsp16 mutants also displayed increased sensitivity to treatment with type i interferon. this was also observed for the corresponding mhv and hcov-229e nsp16 mutants (z€ ust et al., 2011) . however, in contrast to the latter study, the sars-cov nsp16 mutant was not found to induce type i interferons, either in vitro or in vivo (menachery et al., 2014) . this observation suggests that sars-cov may have a larger repertoire of functions for preventing induction of type i interferons following cellular sensing of "nonself" rnas, such as viral rnas with incompletely methylated 5 0 cap structures. together, these data established that the highly conserved nsp16 2 0 -o-mtase plays an important role in limiting the detection of viral fig. 7 -cont'd complex (pdb 2xyv). nsp10 is shown in ribbon representation with conserved residues colored in dark to light blue according to their conservation among covs. zinc molecules are shown as spheres and zinc-coordinating residues are shown in stick representation. the surface of nsp16 is colored in dark to light red according to the conservation of the respective residues among coronaviruses. sah is depicted in a stick model. the figure was generated using ucsf chimera (pettersen et al., 2004) . the degree of conservation of specific residues was determined using an alignment of nsp10 and nsp16 sequences of eight coronaviruses (see fig. 6 ). rna by the host's antiviral sensors, but that the specific role of this activity in escaping host innate immune responses may differ to some extent among covs. the mechanism underlying the induction of the antiviral immune response following infection with 2 0 -o-mtase knockout mutants was studied using specific knockout mice in which viral replication was found to be restored. the absence of mda5, a cap-0 sensor, restored mhv replication, whereas the nuclear translocation of irf3 and interferon induction were reduced (z€ ust et al., 2011) . this suggested mda5 to function in the primary recognition of the rna produced by cov 2 0 -o-mtase mutants and to initiate the isg cascade that restricts viral replication. among the isg products, the ifit family of proteins was shown to be critically involved in reducing the replication of cov nsp16 mutants. the replication of mhv and hcov-229e nsp16 mutants and wild-type controls was similar in ifit1à/à knockout mice (habjan et al., 2013; z€ ust et al., 2011) . likewise, replication of a sars-cov δnsp16 mutant was increased in both ifit1 and ifit2 knockdown mice (menachery et al., 2014) , suggesting that ifit family proteins mediate the primary attenuation of sars-cov 2 0 -o-mtase knockout mutants. the earlier results indicate that the nsp16 2 0 -o-mtase constitutes a new and attractive target for the development of antiviral drugs against sars-cov and hcov-229e, as well as newly emerging covs like mers-cov and porcine epidemic diarrhea virus (pedv). for example, the nsp10-nsp16 interface may be targeted to limit viral 2 0 -o-mtase activity and thus restore the antiviral responses mediated by mda5 and ifit1 (menachery and baric, 2013) . interestingly, the nsp10 residues involved in the nsp10/nsp16 interaction are quite conserved within the cov family and it was recently demonstrated that nsp10 of different covs (fcov, mhv, sars-cov, mers-cov) is functionally interchangeable in the stimulation of nsp16 2 0 -o-mtase activity . thus, molecules or peptides blocking this interface may have broad-spectrum anti-cov effects, a concept that was explored and supported using synthetic peptides that mimic the nsp10 interface and suppress nsp16 2 0 -o-mtase activity in vitro (ke et al., 2012; wang et al., 2015) . the antiviral effect of the mhv tp29 peptide, for example, was first demonstrated in mhv-infected cells and was subsequently confirmed to limit mhv replication in mice and to enhance the type i interferon response . the same peptide was also effective in blocking the replication of a sars-cov replicon. the cov exon domain was identified on the basis of comparative sequence analyses (snijder et al., 2003) that suggested a distant relationship of the nsp14 n-terminal domain (and equivalent polyprotein regions of other nidoviruses) with cellular dedd exonucleases, a large protein superfamily containing rna and dna exonucleases from all kingdoms of life (zuo and deutscher, 2001) . the designation "dedd" alludes to four invariant asp/glu residues that are part of three sequence motifs, i-iii, that are conserved in members of this superfamily (fig. 5) . the dedd superfamily is also referred to as dnaq-like family because it includes dnaq, the ε subunit of e. coli dna polymerase iii, a well-characterized proofreading enzyme scheuermann et al., 1983) . conservation of a fifth residue, his, located four positions upstream of the conserved asp in motif iii identifies exon as a member of the deddh subgroup, while members of the deddy exonucleases contain tyr at the equivalent position. the acidic residues are required to form two metal-binding sites. based on catalytic models initially developed for cellular exonucleases and catalytic rna (beese and steitz, 1991; steitz and steitz, 1993) , the conserved his and the site a metal ion are thought to activate a water molecule that launches a nucleophilic attack on the phosphorus group of the 3 0 -terminal phosphodiester, while the site b metal ion is thought to stabilize the transition state (derbyshire et al., 1991) . exon is conserved in covs and all other known nidoviruses with genome sizes of >20 kb minskaia et al., 2006; nga et al., 2011; snijder et al., 2003; zirkel et al., 2011) . the correlation between genome size and exon conservation suggests that, in nidoviruses with medium-and large-size genomes, the correct nucleotide selection and recognition of properly formed base pairs by the rdrp is not enough to accomplish the necessary replication fidelity and, therefore, requires additional functions suitable to detect and remove misincorporated nucleotides. recently, biochemical evidence has been provided to suggest that exon may have exactly this function (bouvet et al., 2012) . cov mutants that lack exon activity provided additional evidence for exon being involved in mechanisms that keep the cov mutation rate at a relatively low level (<10 à6 mutations per site per round of replication for mhv and sars-cov) (eckerle et al., 2007 (eckerle et al., , 2010 , while other rna viruses have much higher mutation rates, ranging from 10 à3 to 10 à5 mutations per site per round of replication (drake and holland, 1999; sanjuan et al., 2010) . exon knockout mutants of sars-cov and mhv were shown to display a mutator phenotype with significantly increased mutation frequencies approaching those of other rna viruses (eckerle et al., 2007 (eckerle et al., , 2010 . considering that these studies were performed under selection pressure favoring genotypes with high replication efficiency, the total number of mutations in rnas produced by exon-deficient viruses may be even higher than calculated in that study, especially in genome regions that are not subject to selection in in vitro cell culture systems. while inactivation of exon activity was tolerated by mhv and sars-cov (albeit with reductions in replication efficiency), stable exon-deficient mutants of the alphacoronaviruses hcov-229e and tgev could not be recovered (becares et al., 2016; minskaia et al., 2006) , supporting the critical role of this activity in cov replication. taken together, the available information provides compelling evidence for exon playing a key role in high-fidelity replication of covs. consistent with this hypothesis, genetically engineered exon knockout mutants were shown to be significantly more sensitive to rna mutagens such as ribavirin and 5-fluorouracil (up to 300-fold). furthermore, compared to wild-type virus, the exon knockout mutants were shown to accumulate a much higher number of mutations when propagated in the presence of mutagens (smith et al., 2013) . the lack of exon activity was also shown to have profound effects on viral replication and pathogenesis in vivo (graham et al., 2012) . exon-negative mutants displayed a stable mutator phenotype in a number of mouse models of human sars, providing a promising approach for the stable attenuation of highly pathogenic covs with important implications for vaccine development (graham et al., 2012) . coronavirus nsp14 is a bifunctional protein comprised of an n-terminal exon domain and a c-terminal n7-mtase domain. surprisingly, the latter domain is not conserved in the torovirinae (genera torovirus and bafinivirus), representing the other subfamily of the coronaviridae, but in other (more distantly related) nidovirus branches (lauber et al., 2013; nga et al., 2011) . this conservation pattern suggests that a common ancestor of the corona-, mesoni-, and roniviridae contained the two-domain exon/n7-mtase structure while some lineages lost the n7-mtase domain at a later stage. although nsp14 does not require other proteins for activity minskaia et al., 2006) , its ribonucleolytic (but not the n7-mtase) activity was shown to be stimulated significantly in the presence nsp10. in line with this, nsp10 variants carrying amino acid substitutions that prevent the interaction with nsp14 failed to stimulate exon activity (bouvet et al., 2012 (bouvet et al., , 2014 . to date, there is no evidence to suggest a direct role for nsp10 in catalysis. most likely, interactions between the nsp10 and the n-terminal domain of nsp14 stabilize the exon active site in a catalytically competent conformation. mutagenesis data and a recent x-ray structure analysis of a sars-cov nsp10/nsp14 complex (see fig. 6 ) revealed that the nsp10 surface required for interaction with nsp14 overlaps with the surface involved in the interaction and activation of the nsp16 2 0 -o-mtase activity (see earlier) (bouvet et al., 2014; ma et al., 2015) . coronavirus exon activities were first demonstrated using recombinant forms of sars-cov nsp14 expressed in e. coli (minskaia et al., 2006) . the protein was shown to require mg 2+ or mn 2+ ions for activity and to degrade a range of single-stranded (ss) synthetic rnas with 3 0 -to-5 0 directionality to yield reaction products of about 8-12 nucleotides. the data further suggested that rna secondary structure affects exon activity (minskaia et al., 2006) . mutational analysis of predicted active-site (asp/glu) residues confirmed their critical involvement in catalysis (minskaia et al., 2006) . in a subsequent study, using nsp10/nsp14 complexes, the substrate specificity was characterized in more detail and revealed dsrna with a terminal mismatch to be the preferred substrate for exon activity. excision efficiencies using different mismatched base pairs (a:g, a:a, a:c, u:g, u:c, u:u) were found to be similar, suggesting that the mismatch rather than the nature of the nucleotide misincorporated at the 3 0 end determines exon activity (bouvet et al., 2012) . in a recent study, the structures of unliganded, sam-bound, and sah-gpppa-bound sars-cov nsp10-nsp14 complexes were determined by x-ray crystallography (ma et al., 2015) . the structures provide important insight into the two-subdomain structure of nsp14, the two catalytic sites of exon and n7-mtase, critical substrate-binding residues, the contribution of nsp10 to enhancing exon activity, and the roles of as many as three zinc fingers present in nsp14 (fig. 6) . in the structure, one molecule of nsp14 was found to bind one molecule of nsp10. given that nsp10 tends to form multimers , it is tempting to speculate that, in infected cells, the nsp10-nsp14 complex may form even larger complexes, for example, by interacting with nsp10-nsp16 complexes that might be stabilized by nsp10-nsp10 interactions. in this way, consecutive methylation reactions of the 5 0 cap structure could be spatially coordinated. comparison of the nsp10 surfaces that interact with nsp14 and nsp16, respectively, revealed a significant overlap (bouvet et al., 2014) , with a substantially larger surface being involved in interactions between nsp10 and nsp14. buried solvent accessible areas for interactions with nsp14 and nsp16 were determined to be 2236 and 938 å 2 , respectively (decroly et al., 2011; ma et al., 2015) . the structure of the nsp10-nsp14 complex also helps to explain the observed stimulation of exon activity by nsp10 (see earlier). two regions of nsp10 interact extensively with different structural elements of the nsp14 n-terminal domain, most likely to maintain the structural integrity of the exon domain. interestingly, the observed interaction of n-terminal residues of nsp10 with nsp14 led to interpretable electron density for these residues that had not been observed in previous structures of nsp10 (joseph et al., 2007; su et al., 2006) or the nsp10-nsp16 complex (chen et al., 2011; decroly et al., 2011) , consistent with the proposed role of the n-terminal loop of nsp10 in stabilizing interactions with nsp14. the structure of the exon domain is essentially comprised of a twisted β-sheet that is formed by five β-strands and flanked by α-helices on either side. it resembles that of other dedd superfamily exonucleases, such as the ε subunit of e. coli dna polymerase iii, but also has unique features. these include two segments (residues 1-76 and 119-145) that are involved in the interaction with nsp10 and two zinc fingers in the exon domain (see fig. 5 ). the second zinc finger was found in close proximity to the catalytic site. both its position in the structure and mutagenesis data support a role for this zinc finger in catalysis (ma et al., 2015) . the other zinc finger appears to be required to maintain the structural integrity of nsp14. consistent with this, nsp14 variants containing substitutions in zinc finger 1 proved to be insoluble when expressed in e. coli. five residues predicted to coordinate two mg 2+ ions, , were found in the catalytic site, with one mg 2+ ion being coordinated by asp-90 (exon motif i) and glu-191 (motif ii) (fig. 5) . the second mg 2+ ion expected to be involved in the two-metalion-assisted catalytic mechanism of exon (beese and steitz, 1991; chen et al., 2007; ulferts and ziebuhr, 2011) was not identified, presumably due to the lack of an rna substrate and/or product in this structure. with one exception, metal ion-coordinating residues identified in the active site corresponded well to those identified in related cellular proofreading exonucleases (beese et al., 1993; hamdan et al., 2002) and previous predictions for nidovirus homologs (snijder et al., 2003) . the structure clearly revealed to be involved in catalysis, thus revising previous predictions on the identity of exon motif ii in the cov nsp14 primary structure and making nsp14 a "deed outlier" in the dedd superfamily of exonucleases. the combined structural and functional information obtained for nsp14 including its subdomains and the nsp10 cofactor provides an excellent basis for studies using even larger multisubunit complexes to obtain insight into the coordinated action of key replicative enzymes involved in rna synthesis, quality control, capping, methylation, and other functions (subissi et al., 2014a) . the nsp15-associated endou domain is one of the most conserved proteins among covs and related viruses (fig. 8) , suggesting important functions in the viral replicative cycle. already in the first sequence analyses of torovirus and arterivirus replicase genes published more than 25 years ago (den boon et al., 1991; snijder et al., 1990) , the identification of a conserved sequence in the 3 0 -terminal orf1b region, including the (at the time unknown) endou domain, was key to establishing phylogenetic relationships between corona-and toroviruses and, subsequently, also arteriviruses (cavanagh and horzinek, 1993; snijder et al., 1993) . outside the nidovirales, no viral homologs of endou have been identified to date. together with the helicase-associated zbd (see earlier), the nidoviral endou has therefore been proposed to be a unique and universally conserved genetic marker common to all nidoviruses (ivanov et al., 2004a; snijder et al., 2003) . only recently, with the identification of the first nidoviruses in insects (now classified in the family mesoniviridae) and reanalysis of the ronivirus replicase gene, it was found that endou is not conserved in those nidovirus branches that replicate in invertebrate hosts (mesoniviridae, roniviridae) (lauber et al., 2012 (lauber et al., , 2013 nga et al., 2011; zirkel et al., 2013) , suggesting specific roles in vertebrate hosts. to date, these functions and, more specifically, the biologically relevant substrates of endou have not been identified. characterization of mhv endou knockout mutants revealed only minor effects on viral rna synthesis in infected cells, with all rna species being equally affected, and caused a slight reduction in virus titers, which was most evident at later time points post infection (kang et al., 2007) . for the arterivirus eav, substitutions of several conserved residues in the endou domain were found to cause more profound effects, both in virus reproduction and viral rna accumulation, with sg rna being more affected than genome replication in several mutants. in some cases, reduction in virus titers by up to 5 log was observed (posthuma et al., 2006) . the (limited) information obtained in these studies suggests that endou activity is not strictly required for nidovirus rna synthesis, at least in cell culture. however, the strong conservation clearly suggests an important in vivo function that remains to be identified in suitable model systems. initial evidence for specific functions of nidovirus endou domains in infected cells was obtained in studies showing that sars-cov nsp15, but surprisingly not the homologous proteins from hcov-nl63 and hcov-hku1, counteracts mavs-induced apoptosis (lei et al., 2009) . further experiments will be necessary to confirm and assess the significance of these functions for virus replication and/or virus-host interactions. nidovirus-encoded endou activities have been characterized using recombinant forms of cov nsp15 (sars-cov, hcov-229e, mhv-a59, ibv) and arterivirus nsp11 (eav, prrsv) (bhardwaj et al., 2004; ivanov et al., 2004a; kang et al., 2007; nedialkova et al., 2009) . in a number of studies, recombinant nidovirus endous were shown (i) to have endonucleolytic activity, (ii) to cleave 3 0 of pyrimidines, preferring uridine over cytidine, and (iii) to release reaction products with 2 0 ,3 0 -cyclic phosphate and 5 0 -oh ends. using suitable test substrates, rna structural features were shown to affect endou cleavage efficiency, with unpaired pyrimidines being processed more efficiently (bhardwaj et al., 2006; nedialkova et al., 2009 ). the role of metal ions in endou activity is not entirely clear, with somewhat contradictory data being reported for different homologs. while the activities of cellular and cov homologs were shown to require (or to be significantly stimulated by) mn 2+ ions (bhardwaj et al., 2004; ivanov et al., 2004a; laneve et al., 2003 laneve et al., , 2008 , arterivirus endou activities proved to be less dependent on metal ions. low concentrations of mn 2+ were found to stimulate only marginally the arterivirus endou activities, whereas higher concentrations (previously shown to be required for optimal nucleolytic activities in cov and cellular homologs) inhibited the activities of eav and prrsv endous (nedialkova et al., 2009) . metal ion requirements are commonly used to distinguish between the two basic catalytic mechanisms employed by ribonucleases: the metal-independent mechanism that, for example, is employed by rnase a and results in products with 2 0 ,3 0cyclic phosphate ends (as described earlier for endou), and the metaldependent mechanism in which catalysis is aided by two divalent cations coordinated by conserved acidic residues and generates products with 3 0 -oh and 5 0 -phosphate ends (as described earlier for exon). the critical (or supportive) role of metal ions observed for several cellular and viral endou homologs is inconsistent with an rnase a-like (metal-independent) reaction mechanism. also, metal ions were not detected in any of the structures determined for coronavirus nsp15s or xendou, arguing against a direct role of metal ions in catalysis (renzi et al., 2006; ricagno et al., 2006) . however, mn 2+ ions were found to change the intrinsic tryptophan fluorescence of sars-cov nsp15, suggesting conformational changes that, potentially, may affect activity and were shown to be unrelated to protein multimerization (bhardwaj et al., 2004; guarino et al., 2005) . also, regarding the role of mn 2+ in rna binding, contradicting data have been reported, with rna binding by sars-cov nsp15 being enhanced in the presence of mn 2+ or not affected by metal ions in the case of xendou (bhardwaj et al., 2006; gioia et al., 2005) . structural information has been obtained by x-ray crystallography and cryoelectron microscopy studies for several cov and cellular endou homologs (bhardwaj et al., 2008; renzi et al., 2006; ricagno et al., 2006; xu et al., 2006) . sars-cov and mhv nsp15s were shown to form homohexamers comprised of a dimer of trimers. the nsp15 monomers have an α + β structure with three subdomains, a small n-terminal, an intermediate-sized middle, and a large c-terminal domain, the latter basically representing the "conserved domain" of the cov-like superfamily (see earlier). one side of this domain contains two β-sheets that line the positively charged active-site groove, while the other side is formed by five α-helices. the structures of the catalytic domains are largely conserved among cov endous and xendou, supporting the proposed common phylogeny of viral cellular members of this large endoribonuclease family (bhardwaj et al., 2008; renzi et al., 2006; snijder et al., 2003) . the endou hexamer reported for sars-cov nsp15 (ricagno et al., 2006) has dimensions of 80-96 â 110 å , forming a three-petal-shaped surface that surrounds a small, predominantly negatively charged central channel with an inner diameter of $15 å . the hexamer has six-independent active sites located on the surface of the molecule. interactions between individual protomers are predominantly mediated by residues located in the n-terminal and middle domains. database searches failed to identify closely related structural homologs, suggesting that endous diverged profoundly from other ribonucleases (renzi et al., 2006; ricagno et al., 2006) . nevertheless, the presumed endou catalytic residues (his/his/ lys, fig. 8 ) could be superimposed with the catalytic his/his/lys residues of bovine rnase a, the prototype of a large superfamily of pyrimidinespecific ribonucleases (ricagno et al., 2006; ulferts and ziebuhr, 2011) . the superposition also includes a number of conserved substrate-binding residues. a comparison of viral and cellular endou structures with that of rnase a and related nucleases using the pdbefold server revealed similarities between the structural cores of these enzymes that may be described as "interrelated by topological permutation," providing initial evidence for a common ancestry of the two endonuclease families (ulferts and ziebuhr, 2011) . further studies are required to substantiate this hypothesis (see later). the hexameric form is thought to be the fully active form of cov nsp15. this is supported by the exponential increase of activity with increased protein concentrations, the reduced activities determined for protein variants that do not multimerize and the increased rna-binding activities observed for hexameric forms of endou (bhardwaj et al., 2006; guarino et al., 2005; xu et al., 2006) . consistent with this, hexamerization has been confirmed for different cov endou homologs and residues confirmed to be involved in intersubunit interactions are highly conserved among covs. in the structure of a truncated, monomeric form of sars-cov nsp15 that lacks 28 n-terminal and 11 c-terminal residues, two loops of the catalytic domain were found to be displaced compared to their location in the hexamer, resulting in the destruction of the active site (joseph et al., 2007) . in hexameric structures, the two loops pack against each other and are stabilized by intermonomer interactions, suggesting that hexamerization may induce an allosteric switch. furthermore, cross-linking and cryoelectron microscopy studies support a specific role of hexamerization in rna binding (bhardwaj et al., 2006 (bhardwaj et al., , 2008 . in the structure model, the proposed active-site his and lys residues (fig. 8) identified by comparative sequence analysis and site-directed mutagenesis (bhardwaj et al., 2004; gioia et al., 2005; guarino et al., 2005; ivanov et al., 2004a; kang et al., 2007; snijder et al., 2003) were found to be embedded in a positively charged groove of the catalytic domain. other residues identified in the active site and proposed to be involved in binding to the substrate phosphate include the side chain of a conserved thr (fig. 8 ) and the main chain amide of a conserved gly (fig. 1) (bhardwaj et al., 2008; ricagno et al., 2006; xu et al., 2006) . the proposed functional role of the latter residues has also been corroborated by mutagenesis data for several endou homologs (kang et al., 2007; renzi et al., 2006; ricagno et al., 2006) . as mentioned earlier, endou and rnase a share a number of features in their active sites. in rnase a, pyrimidine binding primarily involves thr-45 and phe-120. while thr-45 forms hydrogen bonds with the pyrimidine base, phe-120 interacts with the base through stacking interactions (raines, 1998) . the ser-293/tyr-342 residues of sars-cov nsp15 and the thr-45/phe-120 residues of rnase a occupy similar positions in the active-site clefts of the two enzymes (ulferts and ziebuhr, 2011) . the ser/thr residue is conserved in viral and cellular domains, while tyr-342 is conserved in viral endou homologs while conservative substitutions (phe, trp) are occasionally found in cellular endou homologs (renzi et al., 2006; ricagno et al., 2006) . the role of sars-cov endou ser-293 and tyr-342 (and equivalent residues in related enzymes) in substrate binding received strong support by molecular modeling and site-directed mutagenesis data, with the conserved ser/thr residue being confirmed to have a critical role in the differential cleavage of uridine-and cytidinecontaining substrates, respectively (bhardwaj et al., 2008; nedialkova et al., 2009; ricagno et al., 2006) . similarities in their active-site structures and reaction products containing 2 0 ,3 0 -cyclic phosphate ends suggest that endous and rnase a-like endoribonucleases employ similar catalytic mechanisms (nedialkova et al., 2009; ricagno et al., 2006) . for rnase a, it has been established that two his residues in the active site act as general base and acid, respectively. the his residue that acts as a general base attracts a hydrogen from the ribose 2 0 -hydroxyl group that subsequently attacks the 5 0 p-o bond. the second his donates a hydrogen to the 5 0 -o, thus facilitating displacement of this group and subsequent product release (raines, 1998) . in a second step, the 2 0 ,3 0 -cyclic phosphate is hydrolyzed, resulting in a 3 0 -phosphomonoester product and recovery of the enzyme. the latter is essentially a reverse reaction of the transphosphorylation reaction, with the protonated his now acting as an acid and the other his acting as a base. in both reaction steps, lys interacts with the phosphate to stabilize the pentavalent reaction intermediate. although the reaction mechanisms employed by endous have not been studied in detail, the enzymes are thought to use an rnase a-like catalytic mechanism. this is supported by several lines of evidence, including (i) the conserved spatial positions in the structure and the critical functional role of two his and one lys residue(s) (ricagno et al., 2006) , and (ii) the release of 2 0 ,3 0 -cyclic phosphate-containing reaction products (bhardwaj et al., 2004; ivanov et al., 2004a) that are converted to products with 3 0 -hydroxyl ends (nedialkova et al., 2009 ). since the first in-depth analysis of a cov replicase in 1989 (gorbalenya et al., 1989) , significant progress has been made in terms of its structural and functional characterization. a multitude of enzymatic functions has been identified and characterized in vitro, although mainly using artificial substrates so far. protein structures were obtained for most of the subunits from the nsp7-16 region (neuman et al., 2014b) , but unfortunately two prominent remaining "blank spots" on this map concern two key enzymes in cov rna synthesis, rdrp and helicase. filling those gaps would constitute an important step forward, to address basic questions like the priming mechanism employed by the rdrp and the function of the niran domain, and to accelerate targeted drug discovery, for example, in the area of nucleoside inhibitors of cov rna synthesis, which has received little attention thus far. clearly, where cov mrna capping is concerned, identification of the elusive gtase remains a research priority (subissi et al., 2014a) . for other nsps, potential functions (nsp9, the n-terminal domain of nsp15) or substrates (nsp15-endou) remain to be found. as highlighted by several nsp-wide interaction screening studies pan et al., 2008; von brunn et al., 2007) and more specifically by the in vitro data on the interplay between nsp7-8-12-14 (subissi et al., 2014b) and nsp10-14-16 (bouvet et al., 2014) , cov nsps need to work together in many ways. the further characterization of the "nsp interactome," now also inside the cov-infected cell, will undoubtedly provide more clues as to how specific functions are switched on and off or modulated. likewise, attention should be given to defining the interactions of cov nsps with the specific rna signals for genome replication (madhugiri et al., 2014; yang and leibowitz, 2015) , discontinuous minus-strand rna synthesis (attenuation at body trss, nascent minus-strand transfer, and reinitiation; pasternak et al., 2006; sawicki et al., 2007; sola et al., 2011) , and the transcription, capping, and polyadenylation of sg mrnas (fig. 1) . these rna sequences, several of which may be cis-acting, could provide starting points for improved biochemical assays, ultimately paving the way for the complete in vitro reconstitution of some of these multi-nsp-driven processes. the analysis of cov rtc structure and function in the living infected cell remains an enormous technical challenge, requiring continued toolbox development. it is likely that several functional riddles can only be solved by studying infected cells. for example, the endoribonuclease activity of the nsp15 endou domain, a potential "suicide enzyme" for an rna virus, must be controlled tightly in the infected cell. whereas the enzyme is highly active and displays only very limited substrate specificity in vitro (ivanov et al., 2004a; nedialkova et al., 2009) , it may be confined to a specific compartment in the infected cell and/or its activity may be modulated by interactions with other nsps or host factors. such differences between in vitro and in vivo activities will surely emerge for other nsps as well, and they may be better understood following the further characterization (including their lipid composition) of the membranous replication organelles with which the metabolically active cov rtc presumably is associated (hagemeijer et al., 2012; neuman et al., 2014a; van der hoeven et al., 2016) . these studies should also answer the question of how both nsps and viral rna substrates are targeted to or recruited by the membrane-bound cov rtc, in particular also during the earlier stages of infection when viral rna synthesis appears to be taking off in the absence of the prominent membrane rearrangements observed later in infection. specific mutations in cov genomes can now be reverse engineered, but many of the functions encoded by nsp7-nsp12 are so basic that their inactivation will merely result in dead virus mutants that do not provide many deeper insights into nsp function. this in part explains why most progress thus far has been made for some of the functions that can-fortunatelybe inactivated without such lethal consequences, like the nsp14 exon and nsp16 2 0 -o-mtase enzymes (eckerle et al., 2007 (eckerle et al., , 2010 graham et al., 2012; menachery et al., 2014; smith et al., 2013; z€ ust et al., 2011) . for this reason, the field should continue to also employ "traditional" (forward) genetic methods to characterize (and produce more) conditionally defective covs, like temperature-sensitive mutants (sawicki et al., 2005) . thanks to the advent of next-generation sequencing technologies, tracing the evolution of crippled virus mutants and (pseudo) revertants has become much more straightforward than before, and this approach (letting the virus do the work) likely is among the most economical ones in uncovering previously unknown interactions between the protein and rna players in cov replication (z€ ust et al., 2008) . furthermore, it may be possible to develop cell-based assays for the analysis of cov nsp functions that do not rely on having a replication-competent virus to start with. unraveling the molecular mechanisms underlying the presumed mismatch excision function (bouvet et al., 2012) of the nsp14-exon, which is uniquely encoded by rna virus genomes larger than 20 kb (nga et al., 2011) , connects to the mechanisms driving the evolution of nidoviruses at large (lauber et al., 2013) . also, replicases from other nidovirus branches will need to be studied to fully understand the basic principles governing the profound divergence and genome expansion of this exceptional order of +rna viruses. the error rate and genomic plasticity of rna viruses are among their most fascinating features, and also form the basis for the many problems caused by rna virus mutation and adaptation, including successful zoonotic transfer. as exemplified by the viable cov mutants lacking the exon or 2 0 -o-mtase functions (graham et al., 2012; habjan et al., 2013; menachery et al., 2014; z€ ust et al., 2011) , the functional characterization of cov replicative enzymes can be key to the development of conceptually new live attenuated vaccine prototypes. likewise, it will contribute to the development of broad-spectrum and highly effective antiviral drugs targeting essential enzyme functions, critical interactions with nsp cofactors, or "nonessential" nsp functions that promote efficient viral replication and/or pathogenesis. as highlighted by the sars and mers outbreaks of the past 15 years, having such compounds available would definitely strengthen our first line of defense against cov infections in humans. with great respect, we acknowledge the many ground-breaking contributions of our longterm collaborator dr. alexander gorbalenya, founding father of the functional characterization of the replicase of coronaviruses and nidoviruses at large. we are also grateful for the scientific and technical contributions over many years by numerous past and present lab members and colleagues in leiden (e.j.s.), marseille (e.d.), and giessen/ belfast/w€ urzburg (j.z.). specifically, we would like to acknowledge the input of our long-term collaborators clara posthuma, bruno canard, bruno coutard, isabelle imbert, volker thiel, and stuart siddell. we thank aartjan te velthuis for his assistance with preparing fig. 2 . mechanism of nucleic acid unwinding by sars-cov helicase severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase evaluation of ssya10-001 as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and middle east respiratory syndrome coronaviruses. antimicrob biochemical characterization of a recombinant sars coronavirus nsp12 rna-dependent rna polymerase capable of copying viral rna templates putative rna capping activities encoded by brome mosaic virus: 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cord-264392-he1vekrt authors: lambeth, l. s.; yu, m.; anderson, d. e.; crameri, g.; eaton, b. t.; wang, l.-f. title: complete genome sequence of nariva virus, a rodent paramyxovirus date: 2008-12-23 journal: arch virol doi: 10.1007/s00705-008-0287-3 sha: doc_id: 264392 cord_uid: he1vekrt nariva virus (narpv) was isolated from forest rodents (zygodontomys b. brevicauda) in eastern trinidad in the early 1960s. initial classification within the family paramyxoviridae was based mainly on morphological observations including the structure of nucleocapsids and virion surface projections. here, we report the characterization of the complete genome sequence of narpv. the genome is 15,276 nucleotides in length, conforming to the rule-of-six, and has a genome organization typical of most members of the family, with six transcriptional units in the order 3′-n–p-m-f–h-l-5′. the gene junctions contain highly conserved gene start and stop signals and a tri-nucleotide intergenic sequence present in most members of the subfamily paramyxovirinae. sequence comparison studies indicate that narpv is most closely related to mossman virus, which was isolated from wild rats (rattus leucopus) in queensland, australia, in 1970. this study confirmed the classification of narpv as a member of the subfamily paramyxovirinae and established the close genome organization and sequence relationship between the two rodent paramyxoviruses isolated almost a decade apart and from two locations separated by more than 15,000 km. members of the family paramyxoviridae are pleomorphic enveloped viruses possessing a nonsegmented negativestrand (nns) rna genome [11] . they are divided into two subfamilies, paramyxovirinae and pneumovirinae. the subfamily paramyxovirinae is currently classified into five genera: respirovirus, morbillivirus, rubulavirus, avulavirus and henipavirus. the subfamily pneumovirinae consists of two genera: pneumovirus and metapneumovirus [10] . paramyxoviruses are well-known pathogens of humans (measles virus, mumps virus, etc.) and livestock animals (newcastle disease virus, rinderpest virus, etc.). the emergence of henipaviruses (hendra and nipah viruses) further highlighted the broad host range and the severity of the diseases that can be caused by novel paramyxoviruses [4, 24] . bats are increasingly recognized as an important reservoir of novel viruses, including paramyxoviruses, coronaviruses and, potentially, filoviruses [2] . at least five bat paramyxoviruses have been identified, which include hendra virus, nipah virus, tioman virus, menangle virus and mapuera virus. there is anecdotal evidence suggesting that porcine rubulavirus may also originate from bats [19, 26] , and indian bats may carry a paramyxovirus antigenically related to simian virus 41 and parainfluenza virus 2 [15] . while it is not clear why bats seem to be an ideal reservoir host for many different viruses, one suggested possibility is the direct result of their great species diversity and population abundance. if these are the main drivers for virus distribution and evolution, one would expect to find even more viruses in rodents, since they are the most diverse mammalian animals on earth [1] . sendai virus (sev), although first isolated from a human specimen, is believed to have rodents as its natural reservoir [5] and represents the first and best characterized paramyxovirus of rodent origin. since then, at least four other paramyxoviruses, mossman virus (mospv), j virus (jpv), beilong virus (beipv) and nariva virus (narpv) have been isolated from rodents. the complete genome sequences of the first three rodent viruses were determined previously by our group [7, 12, 13] . here, we report the full-length genome sequence of narpv to complete the molecular analysis of all known rodent paramyxoviruses. narpv was isolated from forest rodents, zygodontomys b. brevicauda, trapped in the nariva swamp in eastern trinidad in the early 1960s [8, 21] . the virus grew in suckling mouse brain and formed syncytia in vero and bhk cells. narpv was identified as a member of the family paramyxoviridae, mainly based the structure of its nucleocapsids (approximately 20 nm in diameter and mean length 1.8 lm) and its virion morphology, being enveloped, spherical and pleomorphic with surface projections [23] . the virus displayed no serological cross-reactivity with a range of known paramyxoviruses at the time of isolation, including parainfluenza virus types 1-4, mumps virus, newcastle disease virus and measles virus [8] . narpv resembles members of the genera respirovirus and rubulavirus in that it generates only cytoplasmic inclusion bodies in virus-infected cells [23] . in this respect, it differs from members of the genus morbillivirus that produce both cytoplasmic and nuclear inclusions in infected cells. however, haemagglutination and cell-binding studies performed on guinea pig and monkey red blood cells suggested that narpv, like measles virus, does not use sialic acid receptors on red blood cells as do respiroviruses and rubulaviruses [8] . the data presented in this study confirmed the classification of narpv as a member of the subfamily paramyxovirinae. although narpv could not be classified into any of the existing genera, it is clear that narpv is most closely related to mospv, confirming its rodent origin. analysis of the narpv h protein also revealed the lack of the consensus nrkscs sequence motif known to be important for sialic acid binding for the hn proteins of respiroviruses and rubulaviruses [14] . virus culture and rna isolation vero cells were infected with narpv or salem virus (salpv) [17] , which was used as a driver in the cdna subtraction experiment described below, and incubated at 37°c until the appearance of syncytia. upon reaching approximately 80% cpe, the medium was replaced with pbs, and cells were collected in pbs. cells were pelleted by centrifugation at 600 9 g for 10 min, and virus in the supernatant was then harvested by ultracentrifugation at 300,000 9 g for 30 min. rna extraction was performed using the rneasy mini kit (qiagen) according to the manufacturer's instructions. after elution from the column in rnase-free water, rna concentration was determined using a gene quantii (pharmacia). the rna concentration was adjusted to approximately 1 lg/ll for subsequent applications. genome characterization by cdna subtraction and gap-filling pcr a clontech pcr-select cdna subtraction kit (clontech, usa) was used to select virus-specific fragments as previously described by our group [3, 7, 13] . double-stranded cdna was made using random hexamer oligonucleotide primers and 4 lg each of total rna as prepared above from pelleted narpv (tester cdna) and salpv (driver cdna). digestion, adaptor ligation, hybridization and pcr reactions were then carried out as described in the instructions provided with the kit. nested pcr products from both subtractions were size-purified on a 1% agarose gel in three fractions (0.1-0.4, 0.4-0.8 and 0.8-2.0 kb) using the qia-quick pcr gel extraction kit (qiagen). the three purified fractions were cloned, and plasmids with insert sizes 100 bp or greater were randomly selected and sequenced. for filling the gaps that were not covered by fragments obtained from the cdna subtraction above, random cdna was synthesized using the omniscript rt kit (qiagen) with random hexamer primers. virus-specific primers were designed using either cdna subtraction-derived sequences or consensus sequences of published paramyxovirus genomes and made by a commercial provider (geneworks, australia). the platinum pcr supermix kit (invitrogen, usa) was used to perform pcr on the cdna template synthesized as described above. pcr products were visualized with ethidium bromide on 1-2% agarose gels and purified, using either the qiaquick pcr purification kit (qiagen) or the qiaquick gel purification kit (qiagen), prior to dna sequencing. the sequences of the 5 0 genome and 5 0 anti-genome termini were determined using a modified procedure from a previously published method [22] . virus growth and rna extraction were conducted as described above. total rna (containing both genome and anti-genome rna) was used for cdna synthesis using the thermoscript rt-pcr system kit (life technologies, usa) and virus-specific primers located within approximately 100-200 nt of the genome termini. reverse transcriptase reactions were incubated at 37°c for 60 min followed by rt inactivation at 85°c for 5 min and treatment with rnase h. the firststrand cdna was purified using the qiaquick pcr purification kit (qiagen) prior to ligation with the anchor oligonucleotide (5 0 -gaagagaaggtggaaatggcgt tttgg, 5 0 -phosphorylated and 3 0 -blocked) using t4 rna ligase (new england biolabs, usa). the ligated product was amplified by pcr using a virus-specific primer, nested with respect to the first primer used for cdna synthesis, and a 27-nt adaptor primer complementary in sequence to the adaptor. when required, a hemi-nested pcr, using the same adaptor primer and an additional (third) nested virusspecific primer, was also performed. the pcr products obtained were gel purified as described earlier and either sequenced directly or cloned before sequencing, in which case at least six individual clones were sequenced to ensure a reliable consensus sequence. purified pcr products or plasmid dna were sequenced using the bigdye ò terminator v1.0 kit (applied biosystems, usa) and an abi prism 377 dna sequencer (applied biosystems). every nucleotide in the genome was sequenced with a minimum of threefold redundancy, at least once in each sense and at least once directly from pcr products without cloning. the clone manager and align plus programs in the sci ed central software package (scientific and educational software, usa) were used for routine sequence data management and analysis. sequence similarity searches were conducted using the blast service at the national center for biotechnology information (ncbi). phylogenetic trees were constructed using the neighbour-joining algorithm with bootstrap values determined by 1,000 replicates in the mega4 software package [20] . the full-length genome sequence of narpv has been deposited into genbank under the accession number fj362497. accession numbers for other sequences used in this study are listed below. for viruses where full-length genome sequence was not available, individual protein sequences were used and are indicated by the abbreviated gene letter in parentheses following the accession number. the new naming convention for paramyxovirus abbreviations, as proposed in the 8th ictv report [10] , was used in this paper for those viruses which have not been formally classified. atlantic salmon paramyxovirus (asapv) eu156171; avian paramyxovirus type 6 (apmv6) characterization of the narpv genome similar to strategies used for characterization of paramyxovirus genomes used in our group, a cdna subtraction approach was used initially to obtain narpv-specific sequences. this was then followed by pcr to fill in the ''gaps'' using specific primers designed from narpv nariva virus genome 201 sequences obtained from the cdna subtraction or degenerate primers designed using highly conserved consensus sequences of known paramyxoviruses in the subfamily paramyxovirinae. finally, the genome terminal sequences were obtained using a modified 5 0 race technique as described in ''materials and methods''. as shown in fig. 1a , the narpv genome organisation is similar to those of most members of the subfamily paramyxovirinae, containing six transcriptional units in the order 3 0 -n-p/v/c-m-f-h-l-5 0 . at 15,276 nt, the narpv genome is at the lower end of the genome size spectrum known for members of the subfamily paramyxovirinae, varying from 15,178 nt for porcine rubulavirus [25] to 19,212 nt for beilong virus [12] . the genome length of narpv conforms to the rule-ofsix [11] , a characteristic known to be true for all sequenced members of the paramyxovirinae. as with all other members of the paramyxovirinae, narpv has a 3 0 leader of 55 nt. the 5 0 trailer of narpv is 42 nt long. the first and last 11-13 nt of paramyxovirus genomes are highly conserved and complementary in nature and are critical elements of the genome and antigenome viral rna polymerase promoter [11] . this is also true for narpv (fig. 2a) . when the 3 0 leader sequence was aligned with those of other subfamily members (fig. 2b) , it was evident that narpv's genome end sequence is most similar to that of mospv and least similar to those of rubulaviruses or avulaviruses. it is worth noting that the narpv genome has a c residue at the fifth position from both ends, which is unique among known members of the paramyxovirinae. this region contains the promoter for replication and transcription and has been shown by reverse genetics to be important for pathogenicity and attenuation of certain paramyxoviruses [11] . overall comparison of deduced sizes and amino acid sequences of all proteins indicated that narpv is most closely related to mospv (table 1) genome is significantly larger (16,650 nt) than that of narpv due to the longer untranslated regions (utrs) located at the 3 0 end of most genes ( fig. 1b and table 1 ). the narpv genome has a coding capacity of approximately 94%, which is higher than the average of 92% for members of the subfamily paramyxovirinae [25] , whereas the presence of longer utrs has resulted in a lower coding capacity of 87% for the mospv genome. transcription of individual genes of paramyxovirinae members is carried out by a stop-and-reinitiation mechanism controlled by conserved sequences at the gene borders [11] . members of genera respirovirus, morbillivirus and henipavirus have a conserved trinucleotide intergenic region (igr), whereas viruses in the genera rubulavirus and avulavirus have igr of variable length and sequence. the gene start, stop and igr sequences of narpv are listed in table 2 (shown as cdna sequence in the antigenome 5 0 ?3 0 sense). the sequence comparison suggests that the transcriptional regulatory elements of narpv are most similar to those of the mospv genome. the coding strategy of the multi-cistronic p gene of paramyxovirinae members is an important genomic feature used for classification. similar to most subfamily members, the non-edited mrna of the narpv p gene codes for the p protein, whereas the mrna with a single g-insertion at the rna editing site codes for the v protein. both mrna species have the coding capacity for a c protein, which is coded in an alternative reading frame. however, insertion of two g's results in an mrna containing a stop codon immediately after the editing site, suggesting that narpv does not code for a w-like protein present in certain subfamily members such as henipaviruses [11] . the narpv p gene has the editing site sequence 5 0 -actaaaaggg gca-3 0 , which is identical to that in the mospv genome [13] . a for the p/v/c gene of narpv, there are two different types of mrna made via rna editing, each encoding a different protein product, i.e., p (non-edited mrna) and v (?1g) b the putative c protein can be made from either of the two possible mrna species molecular features of deduced narpv structural proteins the n proteins of narpv and mospv share a 59% sequence identity. as for other paramyxovirus n proteins, the most variable region is located in the c-terminus. the sequence identity increases to 71% when the n-terminal 400-aa regions are compared. the region containing the highly conserved paramyxovirinae n-protein motif f-x4-y-x3-u-s-u-a-m-g (x = any aa; u = aromatic aa) has identical sequence between narpv and mospv, both having the sequence fapgnypllwsyamg. the p and v proteins of narpv are slightly acidic in nature, with pi values of 5.28 and 4.51, respectively. the sequence identities between these two proteins of narpv and mospv are much lower than that observed for their n proteins (table 1 ). it is interesting to note that the difference in size of the two p proteins is due to a 26-aa deletion in the narpv p protein (or a 26-aa insertion in the mospv p protein), present in the region upstream of the p/v editing site (fig. 3a) . since the ''deletion'' area was located close to the stop codon of the narpv c orf, the conservation of these proteins was largely unchanged as this resulted in only a 3-aa truncation in comparison to the mospv c protein (fig. 3b) . it is also interesting to note that, in overlapping coding regions, there are significantly higher amino acid sequence identities between the c proteins and v proteins of the two viruses than the p proteins. for the c and p common region, the percentage sequence identify is 51% for c and 39% for p. for the v and p common region, it is 61% for v and 36% for p. the m protein of narpv is 340 aa in length. the m proteins of narpv and mospv have the highest sequence identity among all the deduced proteins and a very similar pi, indicating the basic nature of the proteins (table 1) . m is also the only protein that has an identical size between the two viruses. the f protein of narpv, like the f proteins of other paramyxoviruses, is predicted to be a type i integral membrane protein. the predicted cleavage site of the a alignment of the full-length p protein sequences with identical residues shaded. the overlapping coding regions for the c proteins and v-specific polypeptide are indicated by the light grey letters c and v above. b alignment of the full-length c protein sequences. c alignment of the v-specific protein sequences narpv f protein, sgrnk, is dibasic and does not conform to the consensus sequence motif for cleavage by furin, r-x-k/r-r [6] . it is interesting to note that all of the rodent paramyxovirus f proteins characterized so far have the mono-or dibasic cleavage site instead of the more common multi-basic furin cleavage site observed in most other paramyxoviruses. their cleavage sites resemble those of the bat-derived henipaviruses and the fish-derived asapv (see fig. 4 ). the predicted cleavage site of the narpv f protein is immediately followed by a highly conserved 25-aa putative hydrophobic fusion peptide as for all known paramyxovirus f proteins. the mprpv f protein contains 6 potential sites for n-linked glycosylation. unusually, 4 are located in the f 2 region, and 3 of the 4 are also conserved in the mospv f 2 subunit. in contrast, only 2 are located within the f 1 subunit, and none of them were conserved in the mospv f 1 protein. like other members of the paramyxovirinae, the narpv h protein is predicted to be a type ii integral membrane protein with a hydrophobic domain located at the n-terminal region (aa 41-68) functioning both as the signal sequence and the transmembrane anchor. for certain paramyxoviruses, the attachment protein can be made in a soluble form by using an alternative in-frame atg codon within the signal/anchor sequence [18] . for narpv h protein, an in-frame atg codon was present at aa 56, which has the potential to produce a soluble h protein if translation initiates from this atg codon. the sequence surrounding this atg codon (tgcatgt) does not have a strong conservation to the kozak consensus sequence (accatgg). this is also true for the first atg codon of the predicted h orf, which has the sequence aaaatgg. it is therefore possible that both atg codons are used in vivo, albeit probably with different efficiencies. the 6-aa motif (nrkscs) present in the predicted neuraminidase active site found in respirovirus and rubulavirus hn proteins [14] is absent in the narpv h protein. it had the sequence aydgca at the corresponding site, with only the c residue conserved. the mospv g protein has the sequence vfdgcs in this region. there are three potential n-linked glycosylation sites predicted for the narpv h protein at n70, n177 and n575. although mospv g protein also has three predicted n-linked glycosylation sites at n155, n175 and n319, only one site at n177 for narpv and n175 for mospv is located at the same location in the protein. for narpv h protein, the n70 site is located very close to the transmembrane domain and is therefore unlikely to be glycosylated in vivo. the l proteins of narpv and mospv share more than 60% sequence identity. the six strongly conserved linear domains identified within the l proteins of nonsegmented negative-strand (nns) rna viruses by poch et al. [16] can also be identified within these two l proteins. it is interesting to note that in the most conserved domain iii, both l proteins contained the gdne sequence motif, which has only been found in hev, niv and tuppv, and is different from the highly conserved gdnq motif found in all other known viruses in the order mononegavirales [25] . phylogenetic trees were generated by neighbour-joining methods from sequence alignments of deduced aa sequences of the six major proteins of all known members of the subfamily paramyxovirinae. a representative tree based on the n protein aa sequences is shown in fig. 5 , which clearly shows the close relationship between narpv and mospv and indicates that these two viruses, together with jpv, beipv, tuppv and salpv, cannot be placed in any of the five existing genera in the subfamily. phylogenetic trees based on other proteins give similar results (data not shown). the discovery of hev about 15 years ago opened a new chapter in the genetic diversity of paramyxoviruses. it not only challenged the notion that paramyxoviruses have relatively uniform genome sizes, it also revealed several genetic features not observed in previously known paramyxoviruses, such as the lack of both haemagglutination and neuraminidase activities in the attachment protein, the lack of a multi-basic cleavage site in the f protein and the replacement of the highly conserved gdnq sequence by gdne in the l protein. interestingly, many of the newly discovered paramyxoviruses seemed to contain genome features that keep pushing the ''boundaries'' of known paramyxoviruses in terms of genome size, genome organization and gene sequences. this is best demonstrated by genomes of the jpv and beipv. they are much larger than most other paramyxovirus genomes, contain several novel genes and have an exceptionally large gene for the attachment protein [7, 12] . another interesting example is fdlpv [9] . at 15,378 nt, the fdlpv genome is relatively small among the known members of the subfamily paramyxovirinae but contains a seventh gene, u (for unknown), positioned between the n and p/v genes. although rodents represent the most diverse mammals on earth, sev remained the only known paramyxovirus of rodent origin until the recent characterization of mospv, jpv and beipv by our group [7, 12, 13] . in this context, we decided to determine the genome structure and sequence of narpv, another unknown rodent paramyxovirus. it is obvious that sev and the rest of rodent viruses characterized to date do not share a common ancestor virus. in contrast, jpv and beipv are closely related both in genome organization and size and in gene sequences. in this paper, we have shown that narpv and mospv also share many genetic features, not only confirming their rodent origin, but also suggesting that these two viruses evolved from a common progenitor virus. it is worth noting that although jpv/beipv and narpv/ mospv represent two quite different groups of rodent paramyxoviruses in terms of genome size and organization, their common proteins do share more sequence identity than any other known paramyxoviruses, as shown by the phylogenetic tree in fig. 5 . the only common feature we could identify among all known rodent paramyxoviruses was the lack of the multibasic cleavage site present in most other paramyxoviruses. the only other paramyxoviruses with a monobasic cleavage site are henipaviruses of bat origin and the only known paramyxovirus of fish origin, asapv (fig. 4) . the biological significance of this observation is not known at the present time. in conclusion, the characterization of the narpv complete genome sequence in this study further highlights the great genetic diversity present among the paramyxoviruses of wildlife origin, ranging through bats, rodents, reptiles and fish. further studies are required to determine whether these rodent viruses have the potential to infect and cause diseases in humans and livestock animals. the taxonomic status of these viruses is yet to be determined. due to their major differences in genome size, genome organization and deduced amino acid sequences, it is most likely that these viruses will be classified into new genera within the subfamily paramyxovirinae. molecular phylogeny and divergence time estimates for major rodent groups: evidence from multiple genes bats: important reservoir hosts of emerging viruses tioman virus, a novel paramyxovirus isolated from fruit bats in malaysia hendra and nipah viruses: different and dangerous sendai virus, the mouse parainfluenza type 1: a longstanding pathogen that remains up-to-date arg-arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway the complete genome sequence of j-virus reveals a unique genome structure in the family paramyxoviridae nariva virus: further studies, with particular reference to its hemadsorption and hemagglutinating properties complete genome sequence of fer-de-lance virus reveals a novel gene in reptilian paramyxoviruses family paramyxoviridae paramyxoviridae: the viruses and their replication beilong virus, a novel paramyxovirus with the largest genome of non-segmented negative-stranded rna viruses full-length genome sequence of mossman virus, a novel paramyxovirus isolated from rodents in australia site-directed mutagenesis of a conserved hexapeptide in the paramyxovirus hemagglutininneuraminidase glycoprotein: effects on antigenic structure and function isolation of a new parainfluenza virus from a frugivorous bat, rousettus leschenaulti, collected at poona, india sequence comparison of five polymerases (l proteins) of unsegmented negative-strand rna viruses: theoretical assignment of functional domains identification and phylogenetic comparison of salem virus, a novel paramyxovirus of horses the membrane-associated and secreted forms of the respiratory syncytial virus attachment glycoprotein g are synthesized from alternative initiation codons prevalence of rabies and lpm paramyxovirus antibody in nonhematophagous bats captured in the central pacific coast of mexico mega4: molecular evolutionary genetics analysis (mega) software version 4.0 nariva virus, a hitherto undescribed agent isolated from the trinidadian rat, zygodontomys b. brevicauda (j. a. allen & chapman) optimized rapid amplification of cdna ends (race) for mapping bacterial mrna transcripts electron microscopic evidence of nariva virus structure emerging paramyxoviruses genome diversity of emerging paramyxoviruses full-length genome sequence and genetic relationship of two paramyxoviruses isolated from bat and pigs in the americans nariva virus genome acknowledgments we thank dr. n. karabatsos for supplying the nariva virus stock, kaylene selleck and eric hansson for technical assistance, tony pye for providing the automated sequencing service, and drs. glenn marsh and jackie pallister for critical review of the manuscript. key: cord-263315-g7os15m1 authors: martins-da-silva, andrea; telleria, erich loza; batista, michel; marchini, fabricio klerynton; traub-csekö, yara maria; tempone, antonio jorge title: identification of secreted proteins involved in nonspecific dsrna-mediated lutzomyia longipalpis ll5 cell antiviral response date: 2018-01-18 journal: viruses doi: 10.3390/v10010043 sha: doc_id: 263315 cord_uid: g7os15m1 hematophagous insects transmit infectious diseases. sand flies are vectors of leishmaniasis, but can also transmit viruses. we have been studying immune responses of lutzomyia longipalpis, the main vector of visceral leishmaniasis in the americas. we identified a non-specific antiviral response in l. longipalpis ll5 embryonic cells when treated with non-specific double-stranded rnas (dsrnas). this response is reminiscent of interferon response in mammals. we are investigating putative effectors for this antiviral response. secreted molecules have been implicated in immune responses, including interferon-related responses. we conducted a mass spectrometry analysis of conditioned medium from ll5 cells 24 and 48 h after dsrna or mock treatment. we identified 304 proteins. at 24 h, 19 proteins had an abundance equal or greater than 2-fold change, while the levels of 17 proteins were reduced when compared to control cells. at the 48 h time point, these numbers were 33 and 71, respectively. the two most abundant secreted peptides at 24 h in the dsrna-transfected group were phospholipid scramblase, an interferon-inducible protein that mediates antiviral activity, and forskolin-binding protein (fkbp), a member of the immunophilin family, which mediates the effect of immunosuppressive drugs. the transcription profile of most candidates did not follow the pattern of secreted protein abundance. insect-borne diseases afflict billions of people throughout the world. these illnesses are caused by different pathogens such as helminths, protists, bacteria, and viruses, which are the etiologic agents of diseases such as filariasis, leishmaniasis, babesiosis and dengue. among the most important insect vectors are mosquitos and sand flies [1, 2] . phlebotomine sandflies from the genera phlebotomus and lutzomyia are the main vectors of leishmania in the old and new world, respectively. in the old world, these dipterans are also proven virus vectors of phlebovirus, vesiculovirus and orbivirus [3] [4] [5] . despite the lack of evidence proving the vectorial capacity of new world sandflies in the transmission of viruses to humans, the occurrence of diverse virus species naturally infecting lutzomyia spp. populations has been demonstrated [6] [7] [8] [9] . among insect transmitted diseases, the arboviruses are among the most devastating infectious illnesses in our planet. chikungunya virus (chikv), west nile virus (wnv) and dengue virus (denv) are three of a large number of human pathogenic arthropod-borne viruses [10] . also, the world recently witnessed the reemergence of zika virus (zikv), which is associated with severe human neurological pathologies in adults and fetuses [11] . the predominant strategy for controlling vector-borne diseases consists of targeting the vectors, mostly using insecticides. the deleterious side effects of insecticide usage are widely known. among these are consequences to ecosystems, animal and human health, and development of resistance, pointing to the urgency of developing new control strategies. the understanding of biological mechanisms involved in the relationship between pathogens and vectors can provide new approaches for the development of control strategies. the infection of aedes aegypti with wolbachia [12] is an excellent example of a novel arbovirus control strategy. our group has been studying the interaction and immune responses of the sand fly lutzomyia longipalpis (diptera: psychodidae: phlebotominae), the most important vector of american visceral leishmaniasis [13] , when infected with different pathogens [14] [15] [16] . the establishment of continuous insect cell lines has been an important tool for the study of insect biology. cells from drosophila melanogaster (s2), aedes albopictus (c6/36), and anopheles gambie (sua5b), among other insect cell lines, have been employed to scrutinize diverse features of insect immunity [17] [18] [19] . we have detected complex immune responses in the lutzomyia longipalpis ll5 embryonic cell line [20] when challenged by different pathogens [21] . we have also identified a non-specific antiviral response in these cells when transfected with non-specific double-stranded rna (dsrna) [22] , in a way similar to interferon response in mammals [23] . after dsrna transfection, regardless of nucleotide sequence, there was no replication of a west nile reporter virus that expresses luciferase and lacks structural genes, as indicated by a lack of luciferase production [24] . we are presently searching for molecules responsible for this response. secreted proteins have a key role in biological processes involving cell signaling. naïve cells can be modulated by viral or host components transferred from neighboring infected cells via the release of extracellular vesicles and secretion of soluble molecules, leading to activation of host immune response and changes in the secreted protein repertoire [25] [26] [27] . in order to investigate the effect of polyinosinic:polycytidylic acid (poly i:c) transfection on the protein secretion profile of l. longipalpis ll5 cells, we carried out mass spectrometry (ms) analyses comparing the protein composition of conditioned medium from poly (i:c) and mock-treated ll5 cells. l. longipalpis embryonic ll5 cells obtained from dr. robert b. tesh (university of texas medical branch, galveston, tx, usa) were grown at 30 • c in l15 medium (sigma, mendota heights, mi, usa) supplemented with 10% fetal bovine serum (fbs; hyclone, logan, ut, usa), 10% tryptose phosphate broth, and 1% antibiotics (penicillin 100 u/ml and streptomycin 100 mg/ml, sigma). for mock transfection, 4 ng/ml lipofectin transfection reagent (invitrogen, carlsbad, ca, usa) were added to l15 medium (leibovitz) containing 20% tryptose phosphate broth (tpb). the transfection mix contained the mock transfection mix added with 2 ng/ml of polyinosinic:polycytidilic acid (poly i:c), a synthetic analog of double-stranded rna (invitrogen). these mixtures were added to 8 × 10 7 ll5 cells for 24 h and supernatant was aspirated and stored. new medium was added and supernatant was aspirated and stored after 24 h. for rna preparation, transfected and mock-transfected cells were collected after 6, 12, 24, and 48 h incubations, as described above. cell viability was verified by trypan blue staining. more than 98% of cells were viable in all experiments. protease inhibitor cocktail 1× (sigma) was added to the media. dead cells and large debris were pelleted by centrifugation at 2000× g for 10 min. supernatants were centrifuged again at 10,000× g for 30 min to exclude remaining cell debris and microvesicles. the proteins in the supernatant were then precipitated using trichloroacetic acid (tca). one volume of tca 100% (sigma) was added to four volumes of conditioned medium and incubated for 20 min at −20 • c. the samples were centrifuged at 10,000× g for 10 min. the supernatant was removed and the protein pellet was washed with cold acetone, vortexed and centrifuged at 10,000 × g for 5 min. the material was submitted to mass spectrometry. the pellets were suspended in 6 m urea, 2 m thiourea, and 10 mm 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (hepes). the proteins were reduced with 1 mm dithiothreitol (dtt) and 50 mm ammonium bicarbonate (abc), and alkylated with 5.5 mm iodacetamide and 50 mm abc. before trypsinization, the samples were purified with detergent removal spin columns (thermo scientific). then, the samples were digested in 50 mm abc with trypsin (promega, cat. v5113, madison, wi, usa) in a 1:50 trypsin to protein mass ratio by incubation at 24 • c for 18 h. after trypsinization, trifluoroacetic acid (tfa) was added to a final concentration of 0.5%. peptides were desalted with homemade c18 spin columns. the peptides were analyzed in triplicate by liquid chromatography tandem-mass spectrometry (lc-ms/ms) in a thermo scientific easy-nlc 1000 system coupled to a ltq orbitrap xl etd (mass spectrometry facility rpt02h/carlos chagas institute-fiocruz, curitiba, pr, brazil). peptide separation was carried out in 15-cm (75-µm inner diameter) fused silica, in-house packed with reversed-phase reprosil-pur c18-aq 3-µm resin (dr. maisch gmbh, ammerbuch-entringen). chromatography runs were performed in a flow rate of 250 nl/min from 5 to 40% mecn in 0.1% formic acid in a 120-min gradient, and a voltage of 2.3 kv was applied for peptide ionization. the mass spectrometer operated in a data-dependent acquisition mode. survey full-scan ms spectra (at a 300-1650 m/z range) were acquired in the orbitrap analyzer with resolution of 60,000 at m/z 400 (after accumulation to a target value of 500,000 in the c-trap). the ten most intense ions were sequentially isolated and fragmented in the linear ion trap using collision-induced dissociation at a target value of 30,000. the "lock mass" option was enabled at 445.120025 m/z in all full scans to improve mass accuracy of precursor ions [28] . protein identification was performed with maxquant algorithm [29, 30] version 1.4.1.2. default parameters of the software were used for all analysis steps, unless stated otherwise. proteins were searched against an l. longipalpis protein sequence database (containing 10,110 protein sequences from the vectorbase protein database, downloaded on 9 december 2013) and common contaminants, besides their respective reverse sequences to estimate the false discovery rate (fdr). carbamidomethylation of cysteine was set as a fixed modification, while methionine oxidation and n-terminal acetylation (protein) were allowed as variable modifications. in addition, an fdr threshold of 0.01 was applied at both peptide and protein levels. protein quantification was performed using a label-free approach, where the peptide peaks were detected as three-dimensional features-retention time versus signal intensity (extracted ion chromatogram, xic) versus mass/charge-and were aligned and compared across the runs, as previously described [31] . the amino acid sequences of identified secreted proteins were subjected to bioinformatic analyses. the protein signal peptide (sp) from 304 proteins was estimated using the software tool prediction of signal peptide "predsi" at http://www.predisi.de./home.html [32] , under standard configuration. protein-protein interactions were performed using the search tool for the retrieval of interacting genes/proteins (string) database v 10.0 at http://string.embl.de/ [33] . three biological replicates of 8 × 10 7 cells were transfected. the supernatant was removed 6, 12, 24, and 48 h post-transfection and the cells, after being washed three times with pbs, were resuspended in 1 ml of trizol (ambion, waltham, ma, usa). the rna was extracted following the trizol manufacturer's instructions. total rna was precipitated with isopropanol, resuspended in water and stored at −70 • c. rna was treated with dnase i (promega) and cdna was synthesized from 5 µg of total rna using superscript iii first-strand synthesis (invitrogen). real-time polymerase chain reaction (qpcr) was performed using sybr green pcr master mix (applied biosystems, foster city, ca, usa) and the primers listed in table 1 . expression levels were determined through 2(-delta delta c(t)) method (ddct) [34] , normalized using rp49 gene expression [35] , yielding the relative expression value. statistical analyses were done using graphpad prism software, version 5.04 (graphpad software, inc., san diego, ca, usa). for comparison of the transfected and mock samples at different times after transfection, an unpaired two-tailed student's t test was performed. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. table 1 . list of primers used for qpcr. hemocytin basic transcription factor 3 btf3-f 5 agtgcataagcaggcaacacca3 btf3-r 5 tacgtcgccaccgaaatgagtt3 juvenile hormone-inducible protein jhip-f 5 ccttgctgagctccttgagaaact3 jhip-r 5 tacacatggccattcccatcttcc3 eukaryotic translation initiation factor 3 etif3-f 5 tcgataggcatctcacgtttccac3 etif3-r 5 tcttccccactgtatccaggatgt3 transmembrane protease serine 9-like repressor splicing factor rrm-f 5 aatttgggaagctcaata3 rrm-r 5 gaggatgtcgcaagccttct3 forskolin-binding protein proliferating cell nuclear antigen pcna-f 5 catgaatctcgaccaggagcactt3 pcna-r 5 tcacggcaaatgcgtgcaaa3 tubulin-specific chaperone a tbca-f 5 cgtacgaaaaggaagcagatcagca3 tbca-r 5 cttccttccggatcacgtgttcat3 ribosomal protein 49 rp49-f 5 gaccgatatgccaagctaaagca3 rp49-r 5 ggggagcatgtggcgtgtctt3 comparative analysis of expression values was performed to verify the correlation between mrna levels of ll5 cells (samples collected 6, 12, 24 and 48 h post transfection) evaluated by qpcr, and secreted protein levels (samples collected 24 or 48 h post transfection) evaluated by mass spectrometry. linear regression, pearson's correlation coefficient (r), and goodness of fit (r 2 ) were calculated using graphpad prism software (version 6.05). immune responses are triggered through the recognition of pathogen-associated molecular patterns (pamps) by host pattern recognition receptors (prrs). binding of pamps leads to the activation of signaling pathways: rnai, janus kinase/signal transducers and activators of transcription (jak/stat), immune deficiency (imd) and toll [36] [37] [38] [39] . the major insect innate immune response triggered by virus infection is normally the small interfering rna pathway. rnai is a molecular mechanism in which the presence of intracellular dsrna leads to the production of small rnas that suppress the expression of genes with matching sequences [40] . nevertheless, alternative mechanisms exist. in previous work, our group identified an antiviral response when the ll5 embryonic l. longipalpis cell lineage was transfected with dsrna, which included the synthetic poly i:c. after dsrna transfection, cells did not support wnv virus like particle (vlp) replication, as evidenced by lack of luciferase reporter gene expression. this block in replication may be due to a cellular antiviral response triggered by dsrna. [22] . a similar non-specific antiviral response was observed in the marine shrimps litopanaeus vannamei and penaeus monodon [41, 42] . more recently, the occurrence of dsrna-mediated non-specific antiviral response was also described in two members of the apidae family: the honey bee apis melifera, and the bumblebee bombus terrestris [43] [44] [45] . this non-specific response differs from what is seen in drosophila, where transfection with virus-specific dsrna triggers an rna interference (rnai)-mediated response against the virus, whereas non-related dsrnas fail to activate the antiviral response [46] . in vertebrates, intracellular dsrna is recognized by the prr retinoic acid-inducible gene 1 (rig-i) and the melanoma differentiation-associated protein (mda5). these are also known as rig-i-like receptors (rlrs) [47, 48] . these receptors recruit the mitochondrial antiviral-signaling (mavs) protein that mobilizes kinases and ubiquitin ligases, leading to the activation of transcription factors, such as interferon receptor factor 3 (irf-3) and nuclear factor κb (nf-κb) [49, 50] . the transcription factors induce type i interferon (ifn) and pro-inflammatory cytokine production. dsrna, including poly i:c, is also detected by toll-like receptor 3, initiating the type-i interferon (ifn-α, β) signaling pathway via a toll/interleukin-1 receptor (tir)-domain-containing adapter-inducing interferon-β (trif) signal, which activates interferon receptor factor 3 (irf-3) and nuclear factor κb (nf-κb), leading to ifn-β expression [51, 52] . to investigate variations in the exoproteome profile of dsrna-transfected l. longipalpis ll5 cells, we carried out mass spectrometry (ms) analyses comparing the protein composition of conditioned medium from poly i:c and mock-treated cell cultures at two time points (24 and 48 h); in total 304 proteins were identified (table s1) . only 17.6% (53) of all identified secreted proteins presented a signal peptide (sp). among proteins with ≥2-fold positive change variation, this percentage decreased to 11.53%, with only six proteins with sp: 2 at 24 h and 4 at 48 h (table s1 ). the lack of signal peptide in the majority of secreted proteins suggests that unconventional protein secretion pathways are the main secretory mechanisms in ll5 cells, as already seen in other systems [53] . this could be due at least partly to exosomal transport. we identified some of the known exosome markers among our secreted proteins, based on the exo carta exosome marker list (www.exocarta.org/exosome_markers). among these were glyceraldehyde-3-phosphate dehydrogenase (gapdh), enolase (eno), fructose-bisphosphate aldolase (aldoa), moesin (msn), phosphoglycerate kinase (pgk), cluster of differentiation (cds) 37, 48 and 63, the heat shock proteins (hsps) 70 and 90, and annexins (table s1 ). these were not differentially expressed with a greater than 2-fold change and were present in both mock and poly i:c transfection samples. we performed an interaction analysis of the ll5 dsrna-transfected exoproteome. the network analysis of 304 proteins presenting different abundance between mock and transfected groups (table s1) predicted that 271 proteins (table s2) were connected by 2184 edges ( figure 1a) forming an intricate network. the prevised protein interaction (ppi) enrichment p-value was 0.0, meaning that secreted proteins have more relations among themselves than what would be expected for a random set of proteins of similar size drawn from the genome. exoproteome functional enrichment analysis identified enhanced interactions related to specific biological functions. these interactions have a significant false discovery rate (fdr) of less than 0.05. among these gene ontology (go) categories and pathways, we can quote: biological process go:0006518 (peptide metabolic process) fdr: we also carried out string interaction analysis ( figure 1b ) of proteins with ≥2-fold change variation detected on mass spectrometry analysis. from the 51 protein sequences (table s2 ) shown in figure 2b ,c, 45 were identified in the string database (table s2 ) and from those, 26 (table s2 ) ( figure 1b) were predicted as connected at least to one protein. this interaction analysis displayed a significant ppi enrichment p-value of 4.51 × 10 −4 . in this interactome we can distinguish two major protein groups: the first one with 12 members is related to oxidative stress modulation and includes phospholipid scramblase (plscr1), thioredoxin (txn), glutaredoxin (glrx3), superoxide dismutase (sod1), and forskolin-binding protein 4 (fkbp4), among others. the second group is comprised of 10 proteins linked with peptide synthesis and cytoskeleton organization. among these are the riboproteins rps28 and rplp2, the eukaryotic translation initiation factor 3 (eif3d), moesin (msn), cortactin protein (cttn), and others. two biological functions were enhanced in the ≥2-fold change secreted protein cluster: cellular component: go: 0070062 (extracellular exosome) fdr: 5.58 × 10 −12 and molecular function: go.0003723 (rna binding) fdr: 4.41 × 10 −2 . these same biological function enrichments are found when we study the whole exoproteome. the complete fdr-filtered (p < 0.05) lists are shown in table s3 . the measurement of relative protein abundance revealed that the secretion of most proteins did not vary significantly between test and control supernatants (figure 2a ). only 6.95% (18) of all identified proteins at 24 h presented variation of ≥2-fold change, and 6.22% (17) presented variation of ≤2-fold change ( figure 2b ). these percentages change to 11.54% (33) and 24.83% (71) , respectively, at 48 h after dsrna transfection ( figure 2c ). we focused on the 51 proteins with ≥2-fold change variation, which represent 17.28% of the total identified proteins ( figure 2c ). the proteins with intensified secretion in the first 24 h had their relative amounts decreased at 48 h. most proteins more abundant at 48 h had a basal detection level at 24 h. interestingly only one protein, the enzyme carboxylesterase, presented secretion levels with ≥2-fold change at both time points. in insects this enzyme is related to detoxifying processes, including insecticide resistance [54] . several proteins with higher secretion at 24 h or 48 h are related to immune responses. among the proteins with higher secretion at 24 h we can highlight eight proteins related to immune response. phospholipid scramblase 1 (plscr1) had a positive variation with a greater than 8-fold change. scramblases are conserved in all eukaryotic organisms. they are multiple palmitoylated, endofacial membrane proteins originally identified based on their capacity to promote accelerated transbilayer phospholipid movement in response to ca 2+ [55] . plscr1 was identified as an interferon and growth factor-inducible protein necessary for the ifn-dependent induction of gene expression and antiviral activity [56] . in human and mouse plasmacytoid dendritic cells (pdcs), which are professional interferon-producing cells specialized in recognizing viral rna and dna through the endosomal toll-like receptors (tlrs) tlr7 and tlr9, respectively, plscr1 was described as a tlr9-binding protein that plays a significant role in type-1 interferon responses in pdcs by regulating tlr9 expression and trafficking [57] . the direct interaction of plscr1 with human t-cell leukemia virus type-1 (htlv-1) leads to a reduction of virus titers [58] . these reports demonstrate the significant, but not fully understood, role of phospholipid scramblase in antiviral response. another interesting protein with a greater than 5-fold increased secretion at 24 h was the hexameric chaperone complex prefoldin (pfd). subunits of prefoldin have been found to be associated with cellular response to viral infection. the pfdn1 expression was positively modulated in both vaccinia virus-infected hek 293 cells and in the rabv-infected n2a cells [59, 60] . pfdn2 was found directly bound to the hepatitis c virus (hcv) f protein [61] . the string interactome maps were generated using default settings (medium confidence of 0.4, with 7 criteria for linkage: neighborhood, genefusion, co-occurrence, co-expression, experimental evidence, existing databases, and textmining). proteins were represented as nodes and their functional links were defined by solid lines. the thickness of the lines signifies the level of confidence of the reported association. the non-connected nodes were suppressed. no additional interplay proteins were added to the networks. the protein symbols and node colors are listed in table s2 . still within the proteins with higher secretion at 24 h, we found forskolin-binding protein 59 (fkbp59), an immunophilin family member, with a secretion level increase of greater than 5-fold. this is a highly conserved group of proteins which binds to immunosuppressive drugs such as fk506, rapamycin, and cyclosporine [62, 63] . fkbps are involved in several biochemical processes and participate in the immune response against viral infection. mitochondrial fkbp51 was identified as a tumor necrosis factor (tnf) receptor-associated factor 3 (traf3) and tnf receptor-associated factor 6 (traf6) binding protein. binding of fkbp51 to traf proteins facilitates the type-i interferon response induced by dsrna transfection or newcastle disease virus (ndv) infection in murine fibroblasts. depletion of fkbp51 reduced the expression of type 1 inf in these cells [64] . one interesting protein with a greater than 5-fold enhanced secretion at 24 h is coatomer protein i (copi). copi-coated vesicles are associated with transport from the golgi to the endoplasmic reticulum [65] , endocytosis [66] , maturation of autophagic vacuoles [67] , expression of cell surface receptors, and modulation of the plasma membrane lipid composition [68] . studies demonstrated the involvement of copi subunits in diverse aspects of virus infection [69] [70] [71] . depletion of the copi β 2 subunit in human 293/ace2 cells led to a decrease in the severe acute respiratory syndromecoronavirus (sars-cov) replication [72] . the string interactome maps were generated using default settings (medium confidence of 0.4, with 7 criteria for linkage: neighborhood, genefusion, co-occurrence, co-expression, experimental evidence, existing databases, and textmining). proteins were represented as nodes and their functional links were defined by solid lines. the thickness of the lines signifies the level of confidence of the reported association. the non-connected nodes were suppressed. no additional interplay proteins were added to the networks. the protein symbols and node colors are listed in table s2 . still within the proteins with higher secretion at 24 h, we found forskolin-binding protein 59 (fkbp59), an immunophilin family member, with a secretion level increase of greater than 5-fold. this is a highly conserved group of proteins which binds to immunosuppressive drugs such as fk506, rapamycin, and cyclosporine [62, 63] . fkbps are involved in several biochemical processes and participate in the immune response against viral infection. mitochondrial fkbp51 was identified as a tumor necrosis factor (tnf) receptor-associated factor 3 (traf3) and tnf receptor-associated factor 6 (traf6) binding protein. binding of fkbp51 to traf proteins facilitates the type-i interferon response induced by dsrna transfection or newcastle disease virus (ndv) infection in murine fibroblasts. depletion of fkbp51 reduced the expression of type 1 inf in these cells [64] . one interesting protein with a greater than 5-fold enhanced secretion at 24 h is coatomer protein i (copi). copi-coated vesicles are associated with transport from the golgi to the endoplasmic reticulum [65] , endocytosis [66] , maturation of autophagic vacuoles [67] , expression of cell surface receptors, and modulation of the plasma membrane lipid composition [68] . studies demonstrated the involvement of copi subunits in diverse aspects of virus infection [69] [70] [71] . depletion of the copi β 2 subunit in human 293/ace2 cells led to a decrease in the severe acute respiratory syndrome-coronavirus (sars-cov) replication [72] . we identified two juvenile hormone-inducible proteins at 24 h with 4.43-and 2.12-fold increases. despite the absence of information on the possible involvement of juvenile hormone-inducible proteins in the insect antiviral response, the juvenile hormone itself and retinoic acid are terpenoids, with similar structure and biological effects in mammalian and insect cells [73] . retinoic acid-inducible genes are related to type-i ifn response in mammals [48, 74] . viruses 2017, 9, 377 8 of 16 we identified two juvenile hormone-inducible proteins at 24 h with 4.43-and 2.12-fold increases. despite the absence of information on the possible involvement of juvenile hormone-inducible proteins in the insect antiviral response, the juvenile hormone itself and retinoic acid are terpenoids, with similar structure and biological effects in mammalian and insect cells [73] . retinoic acidinducible genes are related to type-i ifn response in mammals [48, 74] . another virus infection-related protein found was kinesin, an atpase protein belonging to the class of motor proteins. kinesins move along microtubule (mt) filaments, and are power-driven by the hydrolysis of adenosine triphosphate (atp) [75] . recently this protein has been associated with the intracellular transport of different viruses. hiv-1 trafficking to the nucleus is kinesin-and dynein motor-dependent [76] . kinesin was also identified as a chandipura virus matrix binding protein [77] . kinesin from lepidopteran trichoplusia ni binds directly to nucleocapsid proteins of the autographa californica multiple nucleopolyhedrovirus (acmnpv) [78] , and the α herpersviral envelope protein pus9 uses kinesin 1 to promote the anterograde axonal transport of herpes simplex virus 1 (hsv-1) [79] . these results demonstrate that kinesin protein interacts directly with the virus particle. the insect protein canopy 4 is a homolog of the human protein associated with toll-like receptor (tlr) 4 (prat4). prat4 is required for cell surface expression of the toll family of receptors and for the intracellular nucleic acid-sensing tlr7/9. this protein is essential for tlr-dependent immune response [80] . the 2.7-fold increased secretion of this prat4 homolog signals the participation of toll-like receptors in the ll5 antiviral non-specific immune response. the barrier to autointegration factor (baf) dna binding protein had an increase of 2.65-fold at 24 h. this protein is described as a potent antiviral effector against poxyviruses, retroviruses and the herpes virus. the baf antiviral property is regulated by kinases and phosphatases, with dephosphorylation activating its antiviral properties [81] . notwithstanding the higher number of positively modulated secreted proteins at 48 h when compared with 24 h, the number of proteins described in immune response to viruses in interferon-like responses was smaller than what was seen at 24 h. among them we quote the protein hemocytin, with a secretion increase of 5.66-fold. hemocytin is an insect humoral lectin homologous to mammalian von willebrand factor, which is involved in platelet adhesion to subendothelium [82] . hemocytin expression is induced by pathogens. lectins are adhesive molecules which bind carbohydrates and among other functions are involved in hemolymph bacterial clearance [83] . also of interest is the occurrence of glutaredoxin (fold change of 7.96), thioredoxin (fold change of 4.95) and superoxide dismutase (fold change of 2.99), antioxidant enzymes involved in the control of oxidative stress at 48 h. reactive oxygen species (ros) production, generated due to microbial invasion, has long been known to exert an antimicrobial effect. the activation of the antiviral and inflammatory signaling pathways has also been linked with the production of ros [84, 85] . due to the high chemical reactivity of ros, cells possess scavenger antioxidant mechanisms that maintain redox homeostasis [86] . real time pcr (qpcr) analysis of 13 genes coding for significantly positive-or negatively-modulated secreted proteins revealed the existence of some genes with transcriptional profile correlated with the amount of protein detected in the supernatants ( figure 3a ). there were seven molecules that presented a correlation between mrna and secreted protein levels (represented in figure 3a ). three of them, hemocytin (hmc), lecithin cholesterol acyltransferase (lcat), and scramblase (scr), showed coordinated mrna and secreted protein levels. when mrna levels changed at 12 or 24 h post transfection (either by up-or down-regulation), we detected a corresponding change in secreted protein levels 12 or 24 h after the mrna level changes. hemocytin (hmc), an insect humoral lectin which plays an important role in a non-specific self-defense mechanism [82] , had increased mrna and secreted protein levels at 12 h and 24 h. this indicates that the transcription, translation and secretion of this molecule are correlated and the prolonged increase of its expression suggests this molecule may be important for the ll5 cells response to poly i:c transfection. two other molecules, lecithin cholesterol acyltransferase (lcat) (a plasma enzyme which esterifies cholesterol) [87] , and scramblase (scr) (a regulator of transbilayer lipid asymmetry in eukaryotic cell membranes) [55] , were reduced both in mrna and secreted protein levels at 12 or 24 h post-transfection. these two molecules are involved in lipid metabolism or membrane formation and the reduction of their expression may suggest that ll5 cells reduce vesicle formation 48 h after transfection challenge. three other molecules showed increased mrna levels at 12 or 24 h post transfection and increased secreted protein levels at 24 h, with posterior decrease at 48 h. one was the eukaryotic translation initiation factor 3 (etif3), which in humans is directly recruited by the hepatitis c virus (hcv) internal ribosome entry site (ires) to promote the translation of viral proteins [88] . in ll5 cells it showed increased mrna levels at 12 h and 24 h, and increased secreted protein levels 24 h after mrna increase. curiously, 48 h post transfection the secreted protein levels decreased drastically. juvenile hormone-inducible protein (jhip) showed increased mrna levels after 12 h and increased secreted protein levels 24 h after mrna increase. curiously, at 48 h post transfection the secreted protein levels decreased drastically regardless of changes in mrna levels prior to this timepoint. basal transcription factor 3 (btf3) has been reported to play a significant role in the transcriptional regulation related to eukaryote growth and development [89] . in ll5 cells it showed increased mrna levels at 12 h and increased secreted protein level 12 h after mrna increase. at 48 h post transfection the secreted protein levels decreased drastically regardless of changes in mrna levels prior to this time point. transketolase mrna levels increased at 48 h post transfection and the secreted protein levels also increased at this same time point, suggesting a late effect. curiously, regardless of the maintenance of mrna levels from 6 h to 24 h, the secreted protein levels were considerably low at 24 h post transfection. changes in transketolase activity are involved in a number of tumors and degenerative diseases [90] . based on the data presented in figure 3a , we observed that in the majority of the molecules (six out of seven) there was a correlated change of secreted protein levels 12 h to 24 h after changes in mrna levels. the remaining genes investigated showed no correlation between the mrna levels and the detection of proteins in the supernatants. ( figure 3b ). the linear regression of mrna levels plotted against secreted protein levels revealed a weak correlation (figure 4 ). the pearson correlation coefficient (r) and goodness of fit (r 2 ) calculated for mrna levels (6 h, 12 h, 24 h and 48 h) versus secreted protein (24 h and 48 h) are shown in figure 4 and table 2 . the non-correlation between transcription levels and amounts of secreted protein indicates that secretion is regulated by some not-yet disclosed mechanism, involving transcription, translation, storage, sorting, and transport, as already reported for other systems [91] . this non-correlation was also observed when the exoproteome of wrl-68 human hepatic cells infected with the chikungunya virus was analyzed [92] . we observed that the majority of analyzed genes were transcribed in the first 24 h ( figure 3a,b) . the results obtained with the signal peptide prediction, the secreted protein interaction network, the identified biological functions enriched pathways, and the relatively small number of proteins with significant abundance variation reveal the existence of finely-tuned protein secretion modulation in response to dsrna treatment. there is also a strong indication for the participation of the exosomal secretory pathway as the major ll5 cell secretion mechanism in this response. the increased secretion of proteins such as phospholipid scramblase, fkbp, juvenile hormone-inducible proteins, and protein canopy 4 in the first 24 h indicate that this non-specific antiviral response is in a way similar to an interferon-like response mediated by the toll pathway. this response occurs preferentially in the first hours after transfection. the increase of antioxidant enzymes at 48 h indicates that the cells are working to control the oxidative stress level in the culture. we are presently in the process of validating these findings by silencing specific genes with subsequent analysis of nonspecific antiviral response. (table 1) and samples collected at different time points posttransfection. quantification was normalized relative to the house keeping gene rp49, and relative gene expression expressed as fold change was calculated relative to the mock group. bars represent mean with standard error (sem) of two biological replicates. tables represent protein fold change corresponding to each gene name. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. (table 1) and samples collected at different time points post-transfection. quantification was normalized relative to the house keeping gene rp49, and relative gene expression expressed as fold change was calculated relative to the mock group. bars represent mean with standard error (sem) of two biological replicates. tables represent protein fold change corresponding to each gene name. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. biology of phlebotomine sand flies as vectors of disease agents a new arbovirus isolated in india from patients with febrile illness isolation of chandipura virus from sandflies in aurangabad the genus phlebovirus and its vectors carajas and maraba viruses, two new v esiculoviruses isolated from phlebotomine sand flies in brazil viremia and immune response with sequential phlebovirus infections ecuador paraiso escondido virus, a new flavivirus isolated from new world sand flies in ecuador, is the first representative of a novel clade in the genus flavivirus 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control of cystic fibrosis transmembrane conductance regulator membrane trafficking: not just from the endoplasmic reticulum to the golgi differential analysis of the secretome of wrl68 cells infected with the chikungunya virus the authors declare no conflict of interest. key: cord-017400-jxbhdch8 authors: koroleva, olga; mckeown, peter; pendle, alison; shaw, peter title: proteomic analysis of the plant nucleolus date: 2007 journal: plant proteomics doi: 10.1007/978-3-540-72617-3_16 sha: doc_id: 17400 cord_uid: jxbhdch8 the nucleolus is a prominent sub-nuclear structure found in all eukaryotes. it is where the ribosomal rna genes are transcribed and ribosomes are synthesised. however, much evidence has now accumulated that the nucleolus is involved in many other nuclear processes. nucleoli are of moderate protein complexity, comprising a few hundred proteins, and can be isolated for proteomic analysis. in this chapter we describe the purification and analysis of plant nucleoli by proteomic methods and summarise the current results. we also discuss more specific tagging methods that have been used to analyse individual protein complexes, as well as methods for analysing post-translational modifications of nucleolar proteins. finally we discuss the assessment of the reliability of such proteomic data, and the presentation and curation of this type of data. the nucleolus is a prominent structure found in the nuclei of all eukaryotes. it is where ribosomal rna genes are transcribed by rna polymerase i and where these transcripts are processed to form pre-ribosomes. like all such nuclear substructures, the nucleolus lacks any bounding membrane, and must therefore be held together by intermolecular interactions as well as by the continuity of the rdna within it with the rest of the nuclear dna. it has been suggested that the biochemistry of ribosome biogenesis itself is responsible for the existence of the nucleolus as a distinct compartment (melese and xue 1995; hernandez-verdun et al. 2002) and nucleolar volume correlates with transcription levels. however rrna transcription occurs without nucleolus formation in archaea (omer et al. 2000) , whereas nucleoli are formed in organisms lacking rna polymerase i ( conrad-webb and butow 1995) or multiple rrna repeats (matsuzaki et al. 2004) , suggesting that the formation of a nucleolus is not essential to rrna transcription, but is an adaptation to create a compartment in which pre-ribosomes may be formed with high efficiency (raska et al. 2006) . however, much evidence now suggests that there must be more to nucleolar structure and function than making ribosomes. in the first place, nucleolar structure varies considerably between species, between cell types and between individual cells. second, a number of unexpected, unconventional activities have been localised, at least in part, to the nucleolus (raska et al. 2006) . in most cases these unconventional activities have been investigated in only one or two species, so that it is not yet clear whether they are general nucleolar functions or specific adaptations. for example the nucleolus has been implicated in many other aspects of rna biology, including biogenesis of snrnas and snornas, of the signal recognition particle, of trnas and rnase p, and of telomerase, as well as in mrna surveillance. the nucleolus has also been linked to viral infections, to cell cycle regulation, to cancer and to stress responses (hiscox 2002; rubbi and milner 2003; maggi and weber 2005; mayer and grummt 2005; yuan et al. 2005) . in many cases the involvement of the nucleolus was discovered by localising factors involved in the various processes to the nucleolus. however, recent approaches using high throughput localisation studies and proteomic analysis of purified nucleoli have put these unconventional activities on a more systematic basis (scherl et al. 2002; andersen et al. 2005; pendle et al. 2005) . for the most part, proteins are recruited to the nucleolus as a consequence of their intermolecular interactions rather than by recognisable nucleolar targeting sequences. this has frustrated attempts to predict nucleolar localisation in silico. nucleolar localisation sequences (nols) exist in viral proteins, such as the plant umbravirus orf3 protein (kim et al. 2004 ) and both potato leaf-roll virus (plrv) capsid proteins (haupt et al. 2005) . indeed, the nucleolar location of one plrv capsid protein was predicted through nols identification (haupt et al. 2005) , and comparison of coronavirus n-protein sequences allowed similar predictions for components of the sars virus (reed et al. 2006 ). however, these reflect binding to existing nucleolar proteins (nucleolin, in the latter case) and the interacting sequences are not well conserved. similarly, a native nucleolar protein may include sequences important for localisation, as in the basic c-terminal domain of yeast nop25 (fujiwara et al. 2006 ) but even this is not widely conserved. hence, nucleolar localisation remains difficult to predict. without reliable prediction, direct determination of the composition of nucleoli is the best option, and it has recently become possible to use high-throughput proteomic techniques to identify large numbers of nucleolar proteins from complex mixtures derived from purified nucleoli. direct visualisation of subcellular localisation has also been used in mice to identify nucleolar proteins amongst other nuclear proteins via an enhancer-trap system, although this study took as a starting point a broad range of predicted nuclear proteins of which only 10% were exclusively nucleolar (sutherland et al. 2001) . in pilot screens aimed at systematically localising proteins in mammalian (simpson et al. 2000) and plant cells (cutler et al. 2000; escobar et al. 2003; tian et al. 2004; koroleva et al. 2005 ) by systematic green fluorescent protein (gfp) open reading frame fusion expression, 2-3% of gfp fusions were highly enriched within the nucleolus. as expected, these included many rna processing functions as well as protein kinases and phosphatases that may regulate various aspects of rrna metabolism (koroleva et al. 2005) . perhaps more surprising was the finding that certain transcription factor-like proteins were preferentially located in the nucleolus, whereas related family members were found mainly in the nucleoplasm (tian et al. 2004; koroleva et al. 2005) . the two techniques commonly used for proteomic purposes are 2d polyacrylamide gel electrophoresis (2-de) and liquid chromatography / mass spectrometry (lc/ms) methods. although 2d gel approaches do have some advantages (see chap. 2 by hurkman and tanaka, this volume), such as quick visualisation of the distribution of major protein groups, accessibility and relatively low basic costs, and ease of sample and data storage, they are applicable only to the major protein components of the proteome, and not suitable for proteins with extreme values of mr (relative molecular mass) or pi (isoelectric point), hydrophobic membrane components or low-abundance proteins. more advanced uses of this methodology, such as difference in gel electrophoresis (dige) (unlu et al. 1997; lilley and dupree 2006) significantly increase the costs -fluorescent dyes and image analysis software are expensive -but the dynamic resolution is still not very high. furthermore, since many plant proteins are extensively modified, plant protein mixtures do not generally resolve very well on 2-de gels. it has been estimated that single gel-based analyses allow the identification of approximately 5% of expressed cellular proteins (heazlewood and millar 2003) . only an estimated 120 nucleolar proteins had been identified by such techniques prior to the use of high throughput ms methods . therefore, there seems be more future in non-gel-based approaches for analysing the plant proteome, such as recently developed techniques like lc-maldi ms; lc esi ms/ms and mudpit (aebersold and mann 2003) . nevertheless, there are several examples where a combination of 2d gel separation with subsequent identification of proteins from excised spots have allowed analysis of the plant nuclear proteome. in arabidopsis, 500-700 spots were detected on 2d gels, and analysis using maldi-tof (matrix assisted laser desorption / ionisation-time of flight) ms led to the identification of 184 spots corresponding to 158 different proteins (bae et al. 2003) . in rice nuclei isolated from suspension culture cells, from a total of 549 proteins resolved on 2-de, 190 proteins were identified by maldi-tof ms from 257 major protein spots (khan and komatsu 2004) . maldi-tof is the most widespread type of ms analysis. this method simply measures the mass of each ionised peptide, providing a "fingerprint" of protein composition, with protein identification based on matches between measured masses of peptides and predicted masses of proteolytic cleavage products of the proteins present in databases. the caveat is that with the increasing length of the peptide chain, many peptide fragments with different sequences will have very similar masses. the same problem sometimes results from protein modifications. besides maldi, another common ionisation method is electro-spray ionisation (esi). the multiply charged ions generated by esi often produce ms/ms spectra that are cleaner and potentially easier to interpret (bodnar et al. 2003) . in general, thorough protein separation/purification is required for esi, since this technique is very intolerant of any contaminants. a more complex analytical approach is tandem mass spectrometry (ms/ms), which has two ms stages, often a combination of quadrupole and time-of-flight mass detectors (q-tof). in this technique, peptide fragments of a given mass are resolved by the first ms stage, then fragmented in a collision chamber and the mass fragmentation patterns are recorded by the second ms stage. the advantage of tandem ms over single ms fingerprinting is that the precise sequence of amino acids in each peptide can be determined, which allows much more reliable identification. an important principle for the preparation of samples for proteomic analysis is to reduce sample complexity by protein fractionation, therefore increasing the possibility of detecting proteins with lower abundance in the complex protein mixture. very commonly, lc separation precedes the ionisation and ms stages. maldi tandem mass spectrometers allow a preliminary lc separation "off-line", in which the eluted fractions are spotted onto a maldi sample plate for later ms analysis. therefore, lc-maldi techniques do not suffer from the time constraints imposed by the transient presence of peptides eluting from a column; if necessary, each sample can be analysed more than once. on the other hand, with lc-esi techniques the sample eluted from the lc is sprayed directly into the esi source and data is acquired immediately. because in lc-maldi the lc is decoupled from the ms analysis, the data is not available immediately, as it is with esi analyses, and chromatography problems can be present but undetected until too late. mudpit (multi-dimensional protein identification technology) is a development of an online lc approach, in which two sequential microcapillary columns (an ion exchange column followed by a reverse phase column) are used to separate very complex mixtures of peptides for ms/ms analysis. this type of analysis allows the identification of very large numbers of proteins/peptides from complex mixtures with the minimum of pre-treatment. nucleoli are not bounded by membranes and, since they contain rdna, they are connected by these dna strands to the rest of the nuclear chromatin. for these reasons, all methods for preparing nucleoli have relied on mechanical fragmentation procedures. in the case of plant cells, the cell wall must be digested by degrading enzymes, such as cellulases and pectinases, to produce protoplasts. this is necessary to avoid the nuclei and nucleoli being trapped inside the cell wall residues after cellular fragmentation. pendle et al (2005) used arabidopsis suspension cultures as a convenient, reproducible and abundant source of cells. the cells were protoplasted, and then the protoplasts were carefully disrupted by a small number of strokes of a stainless steel homogeniser, with a clearance between the piston and chamber of 25 µm, observing the state of the preparation after every few strokes. in general the first few strokes released mostly intact nuclei, while further homogenisation then disrupted the nuclei to release nucleoli. nucleoli constitute the densest cellular component with the exception of starch granules, which may be an unavoidable contaminant, and can be quickly and efficiently purified from the other cellular debris by differential centrifugation. one of the most critical factors is the mg 2+ concentration. magnesium ions causes chromatin to cross-link into an unworkable network, and centrifugation steps then bring down the chromatin with the nucleoli enmeshed in it. to solve this problem, pendle et al (2005) simply left mg 2+ out of the homogenisation buffer. the nucleoli could then be separated from the chromatin. however, a small concentration of mg 2+ was added immediately after centrifugation, since lack of mg 2+ caused a gradual disintegration of the nucleoli. similar methods have been used for the purification of nucleoli from human cell culture scherl et al. 2002) , although in this case nuclei were purified first, with sonication being necessary to release the nucleoli. this may indicate that human nucleoli are more tightly associated with the chromatin and the rest of the nucleoplasm. we have extracted nucleoli from arabidopsis seedlings on a pilot scale, although not so far on a preparative scale, by chopping tissue with a razor blade to release nuclei, which can them be homogenised in the same way as culture cells and nuclei. studies during the past few years using live cell imaging of fluorescently tagged proteins have caused a reappraisal of the dynamic nature of the nucleolus, and indeed all other nuclear compartments. it has become clear that virtually all nuclear and nucleolar proteins are rapidly exchanged with the nucleoplasm, and that what distinguishes 'nucleolar' components is a mean nucleolar residence time of the order of seconds rather than of fractions of a second (phair and misteli 2000; raska et al. 2006 ). this raises the interesting questions of how nucleoli can be isolated as distinct structures over the timescale of minutes or hours necessary for the purification, and how the structures isolated relate to nucleoli seen in living cells. it would be expected that most of the associated protein and other mobile factors would be lost to the medium during extraction and purification, leaving only the most strongly attached factors. the fact that most of the expected proteins are present in purified nucleolar fractions suggests that the situation is more complicated. one possibility is that, along with the rapidly exchanging population of proteins, there is a more stable population. another possibility is that the changes in medium, ionic strength, metabolite concentrations, etc., that occur during nuclear fragmentation and nucleolar purification cause the dynamic exchange of many of the proteins to stop, either because active processes necessary are halted or because changes in the medium cause an effective precipitation of nucleolar contents into a less soluble state. it is important to bear in mind that these processes may affect different nucleolar constituents to different extents, and that 'purified' nucleoli are unlikely to have exactly the same composition as their in vivo counterparts. however, extracted arabidopsis nucleoli remain transcriptionally competent (p. mckeown and p. shaw, unpublished data), which gives some confidence in the validity of the purified fractions. these approaches were successfully applied in the two first published proteomic analyses of human nucleoli purified from hela cells, in which 257 proteins and 210 proteins (scherl et al. 2002) , respectively, were identified. recently, a new study identified 667 proteins within nucleoli prepared from hela cells . so in total, the results obtained from the three independent analyses of hela nucleoli provided a list of 713 individual proteins ). the latest study by andersen et al (2005) employed both an lc ms/ms q-tof instrument and a linear ion trap fourier-transform ioncyclotron resonance mass spectrometer (ft-icr-ms), which provided very high resolution and mass accuracy. the development of hybrid mass spectrometers employing ft-icr potentially presents new opportunities for proteomics analysis of the nucleolus . the arabidopsis nucleolar preparation obtained as described above from suspension cell cultures was subjected to high throughput proteomic analysis as described by pendle et al (2005) , which identified 217 proteins. this has been subsequently increased to over 500 proteins by a mudpit approach using twostage microcapillary lc linked to a q-tof instrument (p. mckeown, p. shaw, a. bottrill, unpublished data). many of the proteins that were identified were expected: known nucleolar proteins, ribosomal proteins, proteins involved in rdna transcription, and other rna-interacting proteins involved in ribosome biogenesis. however, many unexpected proteins were also found in the nucleolus, including for example, spliceosomal proteins, small nuclear rnp (snrnp) proteins and translation factors (see fig. 16 .1). these results reinforce the results of several previous studies, implicating the nucleolus in a variety of functions in addition to ribosome biogenesis, including the biogenesis or transport of a range of rnas and rnps, and roles in mrna maturation, cell cycle control and stress responses (rubbi and milner 2003; andersen et al. 2005; olson and dundr 2005; pendle et al. 2005; pontes et al. 2006) . it could be argued that many of the proteins identified were contaminants -a large and poorly defined structure such as the nucleolus is likely to be impossible to fully separate from other nuclear and cellular components. nevertheless, the proteomic analysis has been confirmed by systematic protein expression studies using gfp-tagged proteins. for example, pendle et al (2005) showed that the vast majority (87%) of the proteins identified by proteomic analysis were also found to be nucleolar-located by the structural criterion of expressing gfp fusions of the identified proteins. many of the unexpected human proteins have also subsequently been confirmed as being in the nucleolus by structural methods such as gfp tagging and immunofluorescence . it is also notable that a large proportion of the nucleolar proteins identified in these proteomic analyses are currently completely uncharacterised, further supporting the view that we have as yet a very incomplete picture of the biochemistry that is taking place in the nucleolus. the following websites host current databases of identified nucleolar proteins: http://lamondlab.com/nopdb/; http://bioinf.scri.sari.ac.uk/cgi-bin/atnopdb/home; http://www.expasy.org/ch2d/. post-translational modifications (ptms) are very common in nucleolar proteins, and several hundred specific modifications, many of which are likely to be functionally important in nucleoli, are known. for example, arginine dimethylation has defined roles in nucleoplasmic shuttling, and unknown roles on the abundant nucleolar proteins fibrillarin, nucleolin and gar1 (lapeyre et al. 1986; najbauer et al. 1993; xu et al. 2003) . histone modifications are implicated in the control of rdna transcription (earley et al. 2006 ) and both ubiquitination and sumoylation pathways are active in the nucleolus (mo et al. 2002; song and wu 2005; panse et al. 2006) . modifications of histones include phosphorylations, methylations, acetylations and deaminations, and the complex patterns of histone modifications have been proposed to constitute a 'histone code' (nightingale et al. 2006) . a detailed analysis of nucleolar histone modifications could in principal explore the potential histone code as it applies to the rdna. it is also likely that ptms of other non-histone proteins have been underestimated, and will be revealed by detailed ms analyses. the use of tandem ms/ms processing to sequence covalently modified peptides was first reported by olsen and mann (2004) , and the method is amenable to highthroughput techniques by using a quadrupole as the first ms stage. as with identification of unmodified peptides, it is possible to study either a pre-selected group of proteins of interest, or to sample the entire proteome. more focussed approaches have been used in several organisms for different modifications, and may allow a greater level of detail. nevertheless, full determination of all modification sites is still technically difficult. for example, analysis of phosphorylation sites of the nucleolar rent complex in yeast failed to detect all known sites, even when several different techniques were focussed on just these proteins (chen et al. 2002) . when extracted arabidopsis nucleoli were studied with q-tof ms/ms, a number of modifications were detected, including a ribosomal protein found in an acetylated form (p. mckeown and p. shaw, unpublished work). in the same study, eef1a (1g07930) was found acetylated at two sites, and the mammalian equivalent has also been detected in an acetylated form (kim et al. 2006 ). the total number of acetylations found was fewer than in mouse, partly because the majority of acetylated proteins in mouse are mitochondrial or cytoplasmic, and many acetylated nuclear proteins are probably not enriched in the nucleolus (kim et al. 2006 ). kim et al. made use of affinity purification with an anti-acetyl lysine antibody, but were still unable to detect many known acetylations, especially those associated with rare transcription factors. combining fractionation with affinity tagging may increase the number of nucleolar covalent modifications detected, but restricts the number of modifications that can be searched. for example, our survey detected seven sites of methionine acetylation that would not have been detected had an acetyl lysine-specific antibody been used. care is needed, however, in the interpretation of this type of result. some small modifications have very similar masses: methylation and formylation are close enough in mr for methylated peptides to also be detected in a screen for formylations, although with lower confidence ratings, which may allow them to be rejected. dimethylation and citrullination are isobaric and hence cannot be distinguished by this method. additionally, it may not be possible to distinguish between the modifications of different residues within peptides, a particular problem with highly modified proteins such as histones. modifications have to be actively sought in search engines such as mascot, precluding the detection of novel modifications, although known modifications can be found on novel substrates or at positions not previously identified. in studies of large modifications such as ubiquitination or sumoylation, which themselves fragment, it has been found to be necessary to isolate the modified proteins before carrying out ms analysis. plasma membrane-bound proteins modified with glycosylphosphatidylinositol (gpi)-anchor proteins were identified by a 'shave and conquer' technique: isolated membranes were treated with phospholipase d to liberate any gpi-anchored protein, and this protein fraction was analysed (elortza et al. 2003) . covalent modifications have also been determined by silac (stable isotope labelling by amino acids in cell culture), with one population of hela cells fed with labelled tyrosine and the other fed with labelled arginine and lysine. again, this allowed identification of unknown gamma-phosphorylation sites, and could be applicable to nucleoli (amanchy et al. 2005 ). a typical proteomic analysis of a protein mixture produces a list of identified components but without any quantification of the relative amounts of the different proteins. the detection and successful identification of a particular peptide will depend on a several other factors apart from the relative abundance of the original protein in the mixture: first, on effective digestion by the proteolytic enzyme used to produce the peptide; second, on how easily the peptide is volatilised and ionised; third, on the way the peptide fragments in the collision cell; fourth, on the presence of modifications, which may not have been predicted. finally, it will also depend on a presence of the peptide sequence in the database or databases to be searched subsequently. the last factor is a particular problem for species without completely sequenced genomes, since mascot and similar search engines search for a sequence match that is based on accurate mass data and therefore cannot rely on homology. for many proteomic applications, for example, cataloguing a particular tissue/ organellar proteome or getting a list of binding partners in a protein complex, simple identification of proteins present in the sample is all that is required. on the other hand, quantitative analysis is essential for many other proteomic applications, where the research involves comparison between different tissues and/or developmental stages of an organism, specific conditions or treatments, mutants or transgenic organisms with over-expressed or silenced genes. therefore, methods have been developed for both relative and absolute quantification of protein amounts in proteomic analysis. most biological applications require relative analysis, to compare two or more datasets, and in this review we will consider current proteomics techniques for relative quantification. one possibility for quantification is 2-de comparison. although significant progress has been achieved recently with the development of fluorescent stains and the dige technique (unlu et al. 1997; lilley and dupree 2006) , 2-de still allows only a subset of the proteome to be analysed, as certain groups of proteins, such as membrane proteins, co-migrating, low abundance proteins and those with extreme values of molecular mass and pi, cannot be clearly separated and/or visualised. in our experience this means that the majority of plant nuclear and nucleolar proteins either do not run cleanly in 2d gels or do not enter the gel at all. the low dynamic range of dige is also a major limitation of this technique. protein identification from excised gel spots also has limitations because the spots are often contaminated with other proteins. alternative approaches, non-gel-based quantitative proteomic methods, are based on comparison of peptide abundance, determining the isotopic composition of one or more elements in a compound after stable isotope labelling of a sample, either in vivo or in vitro. we will describe here a selection of isotopic labelling techniques that have been used on plant material. the labels can either be incorporated in vivo as substituted metabolites such as amino acids, or after purification and digestion by reaction with the resulting peptides. silac uses the in vivo incorporation of isotopically substituted amino acids to shift the molecular masses of the resulting peptides. two or more cell cultures are grown in parallel using different substituted amino acid mixtures, and then the cultures are subjected to different conditions or treatments, such as drug treatments or stress. after treatment, the cultures are pooled and used for biochemical purification. at the ms stage the relative amounts of each peptide can be quantified, since the origin of the peptide can be determined by its isotopic composition. in general, isotopically substituted lysine and arginine are used together, since all tryptic peptides should then be labelled. it is usually necessary to grow the cell culture in the substituted amino acids for a few days to get a good level of incorporation. this approach was used by andersen et al (2005) to determine the effects of different drug treatments on human nucleoli. other typical applications are analysing dynamics events by adding isotopically substituted amino acids to a cell culture at a given time, and analysing samples during a time course (ong et al. 2002; blagoev et al. 2004) . a large scale proteomic study tested the feasibility and technical challenges associated with silac to uncover quantitative changes during apoptosis in the nuclear proteome, resulting in the identification and quantification of 1,174 putative nuclear proteins (hwang et al. 2006) . another recent example of the application of this technique is selective isotope labelling of proteins from arabidopsis cell cultures by growing cells in the presence of a single stable isotopically labelled amino acid (gruhler et al. 2005) . a potential problem in applying this method to plants is that plant cells are better at inter-converting amino acids than animal cells, and so there is a risk that other amino acids may eventually be labelled. in isotope coded affinity tagging (icat), free cysteines in a protein are reacted with a special affinity tag. labeled proteins are enzymatically digested and labelled peptides are separated from the bulk mixture, first using affinity chromatography and then, in a second round, using ion-exchange chromatography prior to ms. the tag has three functional elements: a biotin tag, used during affinity capture (avidin chromatography); the isotopically encoded linker chain (with either eight hydrogens or eight deuteriums); and the reactive group, which will bind to and modify cysteine residues of the protein (gygi et al. 1999; li et al. 2003) . the tag is marketed by applied biosystems (foster city, ca). however, icat has limitations, because it only labels cysteine-containing peptides; 10-15% of every genome codes for proteins with no cysteine, and many proteins contain only a single cysteine, providing only a single peptide that can be potentially quantified. localisation of organelle proteins by isotope tagging (lopit), a variation of the icat technique, was developed for discovering novel proteins in endomembrane organelles (dunkley et al. 2004b; lilley and dupree 2006) , and uses analytical centrifugation in combination with differential isotope labelling. the method involves partial separation of organelles by density gradient centrifugation, thus producing over-lapping fractions, followed by the analysis of protein distributions in the gradient by icat labelling (dunkley et al. 2004a (dunkley et al. , 2004b and ms. multivariate data analysis techniques are then used to group proteins. a good correlation was observed between identification lists of proteins clusters in lopit and previous experimental evidence of protein locations within sub-cellular structures, and it has been shown that the lopit technique can be used to discriminate golgi, endoplasmic reticulum (er), plasma membrane, and mitochondrial/plastid proteins (lilley and dupree 2006) . in the latter paper the more versatile itraq method (see below) was used instead of icat. isobaric tag for relative and absolute quantification (itraq) involves chemical tagging of the n-terminus of peptides generated from protein digests; the reagent used reacts with primary amines (zieske 2006) . fragmentation of the tag attached to the peptides generates a low mass unique reporter ion. there are four tags available, all with an identical mass of 145 da. the advantages of this approach are that all tryptic peptides are labelled, resulting in higher quality data; up to four labels can be used for multiple experiments; improved ms/ms fragmentation results in better confidence identification; and post translational modifications can be detected. the subsequent data analysis requires specialised software, and apart from the proquant software supplied by applied biosciences, non-commercial i-tracker software is also available for quantitative proteomics using itraq (shadforth et al. 2005) . disadvantages of itraq include the increased ms time required because of the increased number of peptides, and the fact that samples must be prepared according to strict guidelines. what is the best ms quantification technique to use? recently a comparative study was carried out on three proteomic quantitative methods, dige, icat, and itraq, using either 2-de or lc-maldi tof/tof (wu et al. 2006) . all three approaches yielded quantitative results with reasonable accuracy when the same protein mixture was used. in dige, accurate quantification was sometimes compromised due to the full or partial co-migration of proteins. the itraq method was more susceptible to errors in precursor ion isolation, especially with increasing sample complexity. the global-tagging itraq technique was more sensitive than the cysteine-specific icat method, which in turn was as sensitive as, if not more sensitive than, the dige technique. the tandem affinity purification (tap) method (rigaut et al. 1999 ) was developed to improve the purification of protein complexes, particularly for subsequent proteomic analysis. the most common previous purification methods for protein complexes used antibody pull-down, and this caused a high background of peptides arising from the antibodies used. the original tap tag consists of tandem protein a domains, linked via a tobacco etch-virus (tev) protease site to a calmodulinbinding protein (cbp) domain. in practice, the protein of interest is expressed as a tap-tagged fusion protein, and a cell extract is made after expression of the fusion. the tagged protein, along with complexed proteins, is absorbed on to igg beads via the protein a domains, washed, and then released by tev protease treatment. a second round of purification involves binding to calmodulin beads in the presence of ca 2+ , followed by washing and release by a buffer containing egta to remove the ca 2+ , and dissociate the calmodulin complex. subsequently, the tap tag has been made more general by removing a nuclear localisation signal, and has been adapted for use in plant cells by removing a cryptic splice site, polyadenylation sites and at-or gc-rich regions, and by the inclusion of castor bean catalase intron 1 for improved expression in plants ( rohila et al. 2004 rohila et al. , 2006 . two gateway-compatible binary vectors, ntapi and ctapi, were constructed and initially tested using agrobacterium-mediated transformation for transient expression in nicotiana benthamiana leaves. recently, in a project studying protein kinase signaling networks in cereal leaves, transgenic stable transformants expressing 41 tap-tagged rice protein kinases were produced and used for subsequent analysis of interacting proteins in rice plants (rohila et al. 2006) . in total, all 41 rice kinases were purified, and 23 of these were isolated as complexes with one or more interacting proteins (rohila et al. 2006) . however, the ntapi and ctapi tags also have some disadvantages. first, many plant proteins have cbds and therefore will be co-purified with the tagged protein (reddy et al. 2002) . we regularly observe co-purification of elongation factor (ef) 1-alpha proteins, from a small ef-tu/ef-1a subfamily of identical genes at1g07920, at1g07930, at1g07940, at5g60390 (an example present in table 16 .1), which have a cbd, in protein mixtures pulled down by a number of different ntapi-and ctapi-tagged proteins. two rice ef-homologous proteins were reported to be among the recurring cellular proteins in the tap procedure by rohila et al. (2006) . second, the proteolytic treatment by tev protease involves incubation at 16°c, which may lead to proteolysis by endogenous proteases, because at this stage general protease inhibitors cannot be added to the reaction buffer because they would inhibit the tev protease. the exception is e-64, but this inhibits only cysteine proteases. these problems have been addressed recently by development of an alternative tandem affinity purification tag for the isolation of plant protein complexes, called pc-tapa (rubio et al. 2005 ). this construct is also a gateway-compatible vector, allowing convenient recombination of orfs from pre-existing gateway entry clones. the pc-tapa tag consists of two protein a binding domains, a 3c hrv protease site, six histidine repeats (6-his) and nine myc epitope repeats (myc tag). an advantage of 3c hrv protease is that this enzyme is active at a wide range of temperatures from as low as 4°c (although this requires an increase of either the amount of the enzyme in the reaction mixture, or in incubation time, compared to the relatively fast cleavage at 16°c). the second purification step can use either the 6-his tag or the myc tag, but 6-his tag ni affinity chromatography has been reported to be more efficient than myc-tag epitope immunoprecipitation (rubio et al. 2005) . we have used ntapi-fusion for purification and proteomic analysis of proteins interacting with nuclear transport factor p15h2 (at1g27970) using transient expression in arabidopsis cell culture, as described previously (koroleva et al. 2005) . this produced relatively high levels of expression of this particular protein. another significant change made to the original protocol was using tca protein precipitation and tryptic digestion of the pellet, instead of separating proteins on a gel and cutting out bands with subsequent digestion. this modification of the procedure avoided losses of yield at the stage of gel separation and extraction of peptides from the gel, which can be 50% or more. it also minimised the time required for ms analysis, as 39 proteins (table 16 .1) were identified simultaneously during a single 1 h lc run followed by nano esi q-tof analysis. several fractions were set aside from the affinity purification steps for subsequent western blot analysis, which showed the molecular mass for the fusion protein band to be much higher than expected for this small 14 kda protein (fig 16.2 ). this suggested that the protein was isolated in its homo-or hetero-dimeric form. another nuclear transport factor, p15h3 (at1g27310), was present among the copurified proteins (table 16 .1), and so it is likely that these proteins form a stable hetero-dimer or higher order complex. half of the identified proteins (table 16 .1) were components of the large and small subunits of the ribosome, which would be expected for a nuclear transport factor. we expressed a gfp fusion of p15h2 orf in arabidopsis suspension culture and observed a striking pattern of localisation on the surface of nuclear envelope, which probably corresponds to nuclear pores with which p15h2 is associated (fig 16.3) . from our experience with several other tap-tagged proteins, the most essential factors for the successful isolation of protein complexes are the abundance and stability of the complex, a conclusion also drawn by rohila et al (2006) . fig. 16.2 purification steps of p15h2-ntapi and interacting proteins for proteomic analysis. western blot with rabbit anti-cbp primary antibody and anti-rabbit secondary antibody. lanes: 1 protein relative molecular mass (mr) markers, 2 initial cell extract, 3 10x extract after igg bead adsorption, 4 wash from igg beads, 5 tobacco etch-virus (tev) wash, 6 tev eluate, 7 wash through from calmodulin column, 8-11 fractions eluted from calmodulin column numerous small foci at the nuclear periphery are labelled, consistent with localisation to the nuclear pores and with the function of p15 in nuclear export. a a single optical section showing the foci labelled at the nuclear periphery. b 3d projection from the entire focal section stack through the nucleus shown in a proteomic analysis is an exciting new technique that can provide large data sets in short periods of time. however, when data from different large-scale projects are compared, certain groups of proteins appear to be ubiquitous despite pre-proteomic sample fractionation techniques. so an important challenge is how to set up criteria to distinguish between true positive identifications and false positives. a consortium has been established to develop general criteria for the publication and exchange of ms data and database search results (kaiser 2002) , and guidelines have been developed to assist researchers in the publication of protein identification from ms data (carr et al. 2004) . a method to estimate the rate of false positive identifications using a reverse database (peng et al. 2003) has provided a technique for reporting the stringency of the search parameters (cargile et al. 2004 ). this approach was applied to the analysis of nucleolar proteins in the most recent publication of the nucleolar proteome . the criterion used in this study was set up as at least two matching peptides per protein, a mass accuracy within 3 ppm, a mascot score for individual peptides of more than 20, and a delta score of more than 5. the threshold of statistical significance in mascot searches is a generally accepted criterion for protein identifications. however, researchers working with unsequenced or incompletely sequenced genomes have to perform searches against expressed sequence tag (est) databases and sometimes have to consider single peptide matches. although some aberrant proteins can be distinguished by the use of replicates, this is insufficient to deal with 'persistent contaminants' -high abundance proteins, or those with a tendency to aggregate with nucleoli during extraction (cytoplasmic metabolic enzymes seem especially prone to this). in fact, as far as proteomic analysis is concerned, these proteins are genuine components of the starting preparation and it is only from the cell biological point of view that they are contaminants. proteomic analysis is unlikely to resolve this problem, although comparison with other studies and databases of common contaminants can help to identify suspect proteins. the best solution is to use a completely different technique to confirm the sub-cellular location of proteins identified. the most direct technique is visualisation of cellular location by fluorescent tagging or immunofluorescence, either by sampling a small sample of proteins whose localisation is unconfirmed or by the use of high-throughput approaches such as the gateway system to analyse a large proportion of the proteins identified (pendle et al. 2005) . this also allows a distinction to be drawn between proteins that are largely nucleolar, and those that are also found in other cellular compartments. the great advantage of high-throughput techniques is that single sets of experiments produce large volumes of data, but this can be a mixed blessing, especially in the absence of generally agreed strategies for ensuring data quality. hence, the utility of proteomic data to the scientific community depends on curation of data into easily navigable databases, and the use of regularly updated formats that allow an assessment of the biological relevance of any given protein or proteins within a proteome. following the identification of the proteins from arabidopsis nucleoli, the results were classified by probable protein function and placed online at the arabidopsis nucleolar protein database (http://bioinf.scri.sari.ac.uk/cgi-bin/ atnopdb/home; brown et al. 2005) . potential human and yeast orthologues of the arabidopsis nucleolar proteins were identified by reciprocal blast search, and comparisons with the human nucleolar proteome were performed. a database of the human nucleolar proteome is also available, and is sufficiently well characterised for use as a test system for novel ms techniques (vollmer et al. 2006) . over 700 proteins are listed, 500 dynamically characterised, with orthologues annotated from yeast, drosophila and caenorhabditis elegans (http://www.lamondlab.com/ nopdb/; leung et al. 2003) . both databases are cross-referenced to the pubmed systems and the lamond nopdb database also includes raw data, i.e. sequenced peptides from tandem ms/ms. as coute et al. (2006) note, the data on the basis of which such identifications were made may need to be provided to ensure valid comparisons between different experiments or systems (such as the full list of peptides made available following recent analysis of the human acetylproteome; kim et al. 2006 ) in a manner analogous to the 'miame' standards (http://www.mged. org/workgroups/miame/miame_1.1.html), which allow direct comparisons between microarray data. proteomic analysis of isolated organelles cannot determine whether a protein is specific to the organelle in question, or whether it is also located in other parts of the cell. neither can it determine the distribution of that protein within the organelle. all of this information provides important guides to function. for these reasons, and also to assess the rate of false positives, about half of the proteins identified in our original arabidopsis nucleolar proteome (those for which we could obtain orfs at the time) were transiently expressed as gfp-fusions (pendle et al. 2005) . these data are also provided in the atnopdb database, including specificity for subnucleolar compartments and presence in other parts of the nucleus and cytoplasm. attempts to understand the complexities of protein localisation could be aided by the use of databases that compile and compare localisation data from different sources. unfortunately, neither the cross-species organelle db (http://organelledb. lsi.umich.edu; wiwatwattana and kumar 2005) nor the arabidopsis-specific gfp/ ms-based "sub-cellular location database for arabidopsis proteins (suba)" at www.suba.bcs.uwa.edu.au (heazlewood and millar 2003) or gfp-based database at http://aztec.standford.edu/gfp/ ) specify sub-nuclear regions. even after reciprocal blast search, 37 arabidopsis nucleolar proteins are of unknown function due to the lack of characterised orthologues. the presence of protein domains of known function within such proteins can help both in suggesting potential functions and in the broader study of the nucleolus in evolution. for example, an analysis of protein domains of the human nucleolar proteome concluded that the core functions of the nucleolus (i.e. those connected to ribosome biosynthesis) were of archaeal origin, but that the many eukaryote-specific domains suggested that the nucleolus had undergone a massive subsequent enlargement, driving the evolution of, for example, rna helicases (staub et al. 2004 ). accordingly, the human proteome database is searchable by protein domain, as well as by gene ontology. in the examples cited above, genome sequences can be used to identify the proteins detected by ms. this is not possible in organisms with as yet unsequenced genomes, such as wheat, and alternative strategies will be needed to present ms data in a useable manner. full use will have to be made of est databases, and it will be necessary to ensure that such libraries are as amenable to automated searching by mascot or related software as are genome databases. blast search against the genomes of sequenced relatives such as rice will also be important. although considered the 'model grass', rice is only distantly related to wheat (diverging 130-240 million years ago; crane et al. 1995) , and the proposed sequencing of the brachypodium genome may be important for searching for homologues of wheat proteins within a more closely related species. the presumed goal of proteomics is to catalogue all the different proteins that can be expressed in a given organism along with all the ptms of each protein, to quantify the expression and modification levels and to determine the way these levels change in different cell types and cell stages. cell biologists would add to this the determination of the sub-cellular and sub-organellar location and the dynamics of all the cell's proteins. we are a long way from achieving these ambitious goals, if indeed they are achievable or if they are still considered worth achieving when they become technically possible. one major problem is the sheer number of possibilities; most mammalian genes are subject to alternative splicing, and alternative splicing is likely to be much more widespread in plants than currently expected. each resulting polypeptide is then potentially subject to hundreds of ptms. in addition to the enormous number of possible molecular species arising from each gene, the dynamic range in concentrations of proteins is also enormous, ranging from as low as one or two molecules to many billions per cell. this is currently one of the most significant limitations in proteomics technology; low abundance species are simply swamped by the overpowering concentrations of common proteins. nevertheless, huge improvements have been made recently both in the experimental methods and in the instrumental flexibility and sensitivity for proteomics. each new generation of ms machines has greater sensitivity, speed and throughput. the result is that current technologies are proving highly productive for samples of medium complexity, such as nucleoli, which contain a few hundred different proteins. in many cases, these 'partial proteomes' seem more accessible than total cellular proteomes and, at least in the short term, the information that this type of analysis can provide seems more useful. many practical problems of displaying, interpreting and curating the information remain, as well as issues of reproducibility and control of artefacts, but these technical problems are likely to be alleviated as the technology matures. as with other types of large-scale bioinformatics data, accessibility and interoperability of different types of data is still a problem. in our experience, the power of proteomics analysis has to be seen in the context of the complementary cell biology techniques for localisation of the proteins identified. proteomics will clearly be most powerful when closely integrated with other techniques in multi-disciplinary approaches to biology. mass spectrometry-based proteomics phosphoproteome analysis of hela cells using stable isotope labeling with amino acids in cell culture (silac) directed proteomic analysis of the human nucleolus nucleolar proteome dynamics analysis of the arabidopsis nuclear proteome and its response to cold stress temporal analysis of phosphotyrosinedependent signaling networks by quantitative proteomics exploiting the complementary nature of lc/maldi/ms/ms and lc/esi/ms/ms for increased proteome coverage arabidopsis nucleolar protein database (atnopdb) potential for false positive identifications from large databases through tandem mass spectrometry the need for guidelines in publication of peptide and protein identification data: working group on publication guidelines for peptide and protein identification data mass spectrometrybased methods for phosphorylation site mapping of hyperphosphorylated proteins applied to net1, a regulator of exit from mitosis in yeast a polymerase switch in the synthesis of ribosomal-rna in saccharomyces cerevisiae deciphering the human nucleolar proteome the origin and early diversification of angiosperms random gfp::cdna fusions enable visualization of subcellular structures in cells of arabidopsis at a high frequency the use of isotope-coded affinity tags (icat) to study organelle proteomes in arabidopsis thaliana localization of organelle proteins by isotope tagging (lopit) erasure of histone acetylation by arabidopsis hda6 mediates large-scale gene silencing in nucleolar dominance proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins high-throughput viral expression of cdna-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata paraspeckles: a novel nuclear domain mapping a nucleolar targeting sequence of an rna binding nucleolar protein stable isotope labeling of arabidopsis thaliana cells and quantitative proteomics by mass spectrometry quantitative analysis of complex protein mixtures using isotope-coded affinity tags nucleolar localization of potato leafroll virus capsid proteins integrated plant proteomics -putting the green genomes to work emerging concepts of nucleolar assembly the nucleolus -a gateway to viral infection? systematic characterization of nuclear proteome during apoptosis: a quantitative proteomic study by differential extraction and stable isotope labeling proteomics. public-private group maps out initiatives rice proteomics: recent developments and analysis of nuclear proteins substrate and functional diversity of lysine acetylation revealed by a proteomics survey involvement of the nucleolus in plant virus systemic infection high-throughput protein localization in arabidopsis using agrobacterium-mediated transient expression of gfp-orf fusions protein and cdna sequence of a glycine-rich, dimethylarginine-containing region located near the corboxylterminal end of nucleolin (c23 and 100 kda) bioinformatic analysis of the nucleolus protein profiling with cleavable isotope-coded affinity tag (cicat) reagents: the yeast salinity stress response systematic analysis of arabidopsis organelles and a protein localization database for facilitating fluorescent tagging of full-length arabidopsis proteins methods of quantitative proteomics and their application to plant organelle characterization nucleolar adaptation in human cancer genome sequence of the ultrasmall unicellular red alga cyanidioschyzon merolae 10d cellular stress and nucleolar function the nucleolus -an organelle formed by the act of building a ribosome nucleolar delocalization of human topoisomerase i in response to topotecan correlates with sumoylation of the protein peptides with sequences similar to glycine, arginine-rich motifs in proteins interacting with rna are efficiently recognized by methyltransferase(s) modifying arginine in numerous proteins histone modifications: signalling receptors and potential elements of a heritable genetic code improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation the moving parts of the nucleolus homologs of small nucleolar rnas in archaea stable isotope labeling by amino acids in cell culture, silac, as a simple and accurate approach to expression proteomics formation and nuclear export of preribosomes are functionally linker to the small-ubiquitin-related modifier pathway proteomic analysis of the arabidopsis nucleolus suggests novel nucleolar functions evaluation of multidimensional chromatography coupled with tandem mass spectrometry (lc/lc-ms/ms) for large-scale protein analysis: the yeast proteome high mobility of proteins in the mammalian cell nucleus the arabidopsis chromatin-modifying nuclear sirna pathway involves a nucleolar rna processing center structure and function of the nucleolus in the spotlight genes encoding calmodulin-binding proteins in the arabidopsis genome delineation and modelling of a nucleolar retention signal in the coronavirus nucleocapsid protein a generic protein purification method for protein complex characterization and proteome exploration improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants protein-protein interactions of tandem affinity purification-tagged protein kinases in rice disruption of the nucleolus mediates stabilization of p53 in response to dna damage and other stresses an alternative tandem affinity purification strategy applied to arabidopsis protein complex isolation functional proteomic analysis of human nucleolus ) i-tracker: for quantitative proteomics using itraq systematic subcellular localization of novel proteins identified by large-scale cdna sequencing identification of a novel nucleolar localization signal and degradation signal in survivin-deltaex3: a potential link between nucleolus and degradation insights into the evolution of the nucleolus by an analysis of its protein domain repertoire large-scale identification of mammalian proteins localized to nuclear subcompartments high-throughput fluorescent tagging of full-length arabidopsis gene products in planta difference gel electrophoresis: a single gel method for detecting changes in protein extracts multidimensional hplums of the nucleolar proteome using hplc-chip/ms organelle db: a cross-species database of protein localization and function comparative study of three proteomic quantitative methods, dige, cicat, and itraq, using 2d gel-or lc-maldi tof/tof in vivo analysis of nucleolar proteins modified by the yeast arginine methyltransferase hmt1/rmt1p genetic inactivation of the transcription factor tif-i! leads to nucleolar disruption, cell cycle arrest, and p53-mediated apoptosis a perspective on the use of itraq reagent technology for protein complex and profiling studies we are grateful to andrew bottrill and mike naldrett (john innes centre) for their help with proteomic analysis. financial support for this research was provided by the uk biotechnology and biological sciences research council (bbsrc). key: cord-274393-1geyuxtk authors: xie, wenyan; wen, hongling; chu, fulu; yan, shaofeng; xie, wenli; lin, bin; chen, yuzhen; li, zhenmei; ren, guijie; song, yanyan; zhao, li; wang, zhiyu title: mutations in the leucine zipper-like motif of the human parainfluenza virus 3 fusion protein impair fusion activity date: 2015-12-23 journal: intervirology doi: 10.1159/000441978 sha: doc_id: 274393 cord_uid: 1geyuxtk objective: to investigate the effect of the leucine zipper-like motif between hra and hrb of the human parainfluenza virus 3 fusion protein on fusion activity. methods: site-directed mutagenesis was utilized to substitute the heptadic residues at 257, 264, 271, 278, 285, 292, and 299 in this motif with alanine. additionally, 3 middle heptadic leucine residues at 271, 278, and 285 were replaced with alanine singly or in combination. a vaccinia virus-t7 rna polymerase transient expression system was employed to express the wild-type or mutated fusion (f) proteins. three different types of membrane fusion assays were performed to analyze the fusogenic activity, fluorescence-activated cell sorting (facs) analysis was executed to examine the cell surface expression level, and a coimmunoprecipitation assay was conducted to probe the hemagglutinin-neuraminidase (hn)-f interaction at the cell surface. results: all of the substitutions in this motif exhibited diminished or even lost fusion activity in all stages of fusion, although they all had no effect on cell surface expression. in the coimmunoprecipitation assay, all mutants resulted in decreased detection of the hn-f complexes compared with that of the wild-type f protein. conclusions: this motif has an important influence on fusion activity, and its integrality is indispensable for membrane fusion. membranes, which results in delivery of the viral genome (a single-stranded, nonsegmented, negative-sense rna) into the cytoplasm of the target cell [4] . two types of virion-associated surface glycoproteins, i.e. the hemagglutinin-neuraminidase (hn) protein and the fusion (f) protein, cooperate to facilitate this process [5] . the hn glycoprotein is responsible for the initial binding between the virus and the target cells [6, 7] , whereas the f glycoprotein is involved in disruption of the host-cell plasma membrane and direct induction of membrane fusion [8] . the hpiv-3 f protein, which is classified as a type i integral membrane protein [9] , is synthesized as an inactive precursor, designated f 0 , that is then activated by proteolytic cleavage during transport through the golgi membranes into a disulfide-linked heterodimer of f 1 and f 2 [10] . several structural features of this protein are known to be important for its fusion activity. a stretch of hydrophobic amino acids at the amino terminus of the f 1 subunit, termed the 'fusion peptide', is considered to be responsible for insertion into the host cell membrane to initiate the fusion process [5, 11, 12] . in addition, 2 heptad repeat regions (i.e. hra and hrb) in the f 1 polypeptide are believed to have important roles in membrane fusion. hra is located at the carboxyl terminal of the fusion peptide, and hrb is located immediately upstream of the transmembrane region. upon triggering, these 2 domains undergo conformational changes to form a stable 6stranded coil that pulls the viral and target cell membranes together [9, 12, 13] . there is a large intervening region between hra and hrb, and sequence analysis indicates that it contains a leucine zipper-like motif. in this highly conserved motif, characterized as a periodic repeat of leucine or isoleucine residues every 7 amino acids, nonpolar residues are found at all first and most fourth positions when displayed on an α-helical wheel [14, 15] . previous studies of this motif in 3 different paramyxovirus systems have suggested that this region is important for fusion activity [16] [17] [18] . however, whether the leucine zipper-like motif located between hra and hrb of the hpiv-3 f protein plays an important role in fusion activity has not yet been determined. to investigate the effect of this domain on fusion activity, point mutations were introduced by using site-directed mutagenesis to substitute the heptadic residues at amino acids 257, 264, 271, 278, 285, 292, and 299 in this motif with an alanine residue. additionally, 3 highly conserved middle heptadic leucine residues (i.e. l271, l278, and l285) were replaced with alanine in various combinations. the effects on the fusion activity, cell surface expression, and hn-f interactions of such mutated f proteins were examined. our data showed that all of the mutants, whether individually or in combination, caused no reductions in the cell surface expression levels of f, but they led to a decreased fusogenic activity in all steps of membrane fusion. thus, it seems that the defective fusogenic activity of these mutated f proteins is related to decreased hn-f interactions at the cell surface. cell and viruses bhk-21 cells, obtained from the american type culture collection, were maintained in dulbecco's modified eagle's medium (dmem; gibco, usa) supplemented with 10% fetal calf serum (fcs). fresh human erythrocytes (rbc) were obtained from the physical examination center of qilu hospital of shandong university. this study was approved by the institutional review board of shandong university. written informed consent was obtained from all patients, and the hospital ethics committee approved the experiments. wild-type (wt) vaccinia virus was used to quantify cell fusion, and recombinant vaccinia virus vtf7-3, a generous gift from dr. bernard moss, was used to provide t7 rna polymerase in the vaccinia-t7 rna polymerase expression system [19] . viruses were maintained in bhk-21 cells. the hpiv-3 hn and f genes were cloned into pbluescript sk(+) (pbsk + ) at the bamh i site to generate pbsk-hn and pbsk-f, as previously described [20] . recombinant plasmids were purified from escherichia coli tg1 cells using an enza plasmid miniprep kit (omega bio-tek inc., norcross, ga., usa). the wt and all mutated f proteins were expressed in bhk-21 cells using the vaccinia virus-t7 (vtf7-3) rna polymerase expression system, as previously described [19] . in all experiments, except in the spectrofluorometric analysis of lipid mixing, bhk-21 cells were seeded in 6-well plates for 1 day prior to use at 4 × 10 5 cells/well. monolayer cells were infected with the recombinant vaccinia virus expressing vtf7-3 at a multiplicity of infection (moi) of 10 for 1 h at 37 ° c in a 5% co 2 incubator and transfection was performed as recommended by mirza et al. [21] , using lipofectamine tm 2000 (invitrogen) at 1 μg for each plasmid. site-directed mutagenesis was performed according to protocols previously described [22] . the hpiv-3 f recombinant plasmid vector was used as the template in each pcr mutagenesis to generate alanine codon substitutions. two pairs of reverse-complement mutagenesis primers (sangon biotech co. ltd., shanghai, china) were designed to only mutate the target amino acids. the primers were as follows (sequences were started with the 5 ′ nucleotide, and the mutant sites are underlined in the primer sequences): ′ -t tat gat cta gca ttt aca gaa t-3 ′ l257a-cp1: 5 ′ -tga ttc tgt aaa tgc tag atc ata a-3 ′ v264a-p2: 5 ′ -gaa tca ata aag gcg aga gtt ata g-3 ′ v264a-cp2: 5 ′ -c tat aac tct cgc ctt tat tga ttc the pbsk-f plasmid digested by kpn i was used as the template to generate one pcr fragment with primers l257a-p1 (complemented with l257a-cp1) and vp2 (complemented with vp1). the pbsk-f plasmid digested by not i was used as the template to generate the other pcr fragment with primers l257a-cp1 (complemented with l257a-p1) and vp1 (complemented with vp2). two pcr products with homologous ends were transformed into e. coli tg1 cells that were then selected for ampicillin resistance. identification of colonies carrying the mutated f genes was facilitated by screening for the presence of 2 unique restriction sites (i.e. the kpn i site and the not i site). finally, mutated f protein genes were sequenced in their entirety to verify the desired mutation(s) and that no other changes in the f protein were present. the other single mutants were obtained with the same procedures. double and triple mutants were made by sequential mutagenesis on previously mutagenized templates. all mutations, as verified by dna sequencing, were successfully introduced. monolayers of bhk-21 cells grown in 6-well plates were cotransfected with cdna encoding wt or mutated f proteins along with hpiv-3 hn proteins or empty vector alone, with a total amount of 1 μg of dna per well. at 36 h posttransfection, transfected monolayers were washed with phosphate-buffered saline (pbs) and fixed with methanol for 5 min. then, the cells were stained with giemsa accustain (sigma chemical co.) for syncytium observation [23] . in each field of view, all syncytia with 3 or more nuclei were counted. representative fields were captured with an inverted microscope (olympus ix71). quantification of syncytia was accomplished by measuring the syncytia-covered area in 3 random fields and comparing this with the total area of the field. areas were quantified using the analysis tool in adobe photoshop cs6 [24] . the content-mixing assay, based on the cytoplasmic activation of the reporter gene β-galactosidase, was essentially performed as previously described [22] , with slight modifications. one population of bhk-21 cells was infected with the recombinant vtf7-3 vaccinia virus as described above and cotransfected with the desired f and hpiv-3 hn genes. a second population of bhk-21 cells was infected with the wt vaccinia virus (moi = 10) and transfected with 1 μg per well of plasmid pg1nt7β-gal, which carries the β-galactosidase gene under the control of a t7 promoter. at 22 h posttransfection, the cells were removed from the wells by trypsinization (0.05% trypsin, 0.53 m m edta; gibco) and washed, pelleted, and resuspended in complete medium. equal numbers (1 × 10 5 ) of the 2 populations were mixed in triplicate in a 96-well microtiter plate. after incubation at 37 ° c for 5 h, the cells were lysed and the lysates were assayed for β-galactosidase activity according to the procedures of the high-sensitivity β-galactosidase assay kit (stratagene, la jolla, calif., usa). the extent of fusion was quantified by measurement of the absorbance at 590 nm with a plate reader (elx800; bio-tek instruments inc.), with background fusion obtained from cells transfected with comparable amounts of the vector alone subtracted [23] . the dye transfer assay was performed as previously described [22, 25] . fresh human rbc were washed, resuspended in cold pbs (1% hematocrit), and incubated with 15 μl octadecyl rhodamine b (r18; invitrogen) (1 mg/ml in ethanol) for 30 min at room temperature in the dark. three volumes of complete medium were added, and incubation continued for an additional 30 min. the rbc were washed and then resuspended in pbs containing 0.1 m m cacl 2 and 1 m m mgcl 2 (pbs-cm) (0.1% hematocrit) for downstream experiments. after 22 h of incubation, cell monolayers coexpressing wt or mutated f and hn proteins were washed and treated with 50 mu per ml of neuraminidase (sigma chemical co.) in dmem for 1 h at 37 ° c; then, the cells were washed again and overlaid with r18-labeled rbc at 4 ° c for 30 min with occasional gentle agitation. the cells were washed and then incubated for 60 min at 37 ° c. finally, the cells were washed and immediately visualized, counted, and photographed using a nikon fluorescence microscope. rbc were labeled with r18 in the same way as in the dye transfer assay. monolayers of bhk-21 cells grown in 6-cm-diameter dishes were infected with vtf7-3 at an moi of 10 per well for 1 h at 37 ° c. after being washed twice with dmem (gibco), cells were cotransfected with hpiv-3 wt or mutated f and hn genes. plasmids encoding the hn and f proteins were kept at 2.5 μg, and the total transfected dna was kept at 5 μg. at 22 h posttransfection, monolayers were washed with pbs and subjected to neuraminidase treatment as described above. then, the cells were washed twice and incubated with 3 ml r18-labeled rbc (0.1% hematocrit) for 30 min at 4 ° c. excess unbound rbc were removed, and the rbc-cell complexes were transferred from the dish into a new 2.0-ml eppendorf tube for incubation for 30 min at 4 ° c in a solution of pbs containing 50 m m edta. the rbc-cell complexes were washed and placed on ice until further use. fifty microliters of the r18-labeled rbc acceptor cell suspension was injected into a cuvette containing 3 ml pbs prewarmed to 37 ° c, and the fluorescence changes were continuously measured using an f-4500 spectrofluorometer (hitachi, tokyo, japan) with 5 s of time resolution at 560-and 590-nm excitation and emission wavelengths, respectively. to normalize the data, the percent fluorescence dequenching (% fdq) at each time point was calculated according to the following equation: % fdq = 100(f -f 0 /f t -f 0 ), where f 0 and f are the fluorescence intensities at time zero and at a given time point, respectively, and f t is the fluorescence intensity in the presence of 0.1% triton x-100 and taken as fluorescence at an infinite dilution of the probe [26, 27] . the cell surface expression of hpiv-3 wt and mutated f proteins was examined by flow cytometry as previously described [23] . bhk-21 cells were transfected with plasmids encoding the hpiv-3 wt or mutated f proteins. at 22 h posttransfection, the monolayers were detached, pelleted, washed twice with pbs-fcs (pbs containing 5% fcs), and then incubated with an anti-f monoclonal antibody (cd9-4-3; millipore) at a dilution of 1: 100 in pbs containing 1% bsa at room temperature for 1 h. after 3 washes with pbs-fcs to remove unbound antibodies, the cells were incubated with 1: 100 diluted alexa fluor 488-conjugated goat anti-mouse immunoglobulin g antibodies (proteintech). following 2 more washes, the cells were fixed in pbs containing 2% paraformaldehyde at 4 ° c for 10 min. after being washed 2 additional times, the cells were resuspended in 0.4 ml pbs and subjected to flow cytometry in a facscalibur flow cytometer (becton dickinson biosciences). cells transfected with vector alone were used as controls. the abilities of hpiv-3 wt or mutated f proteins to interact with hpiv-3 hn proteins at the surface of transfected bhk-21 cells were assayed using a modification of a coimmunoprecipitation assay as previously described [22, 28, 29] . bhk-21 monolayers were grown in 6-well plates ( ∼ 80% confluence) and transfected with cdna encoding the hn protein as well as wt or mutated f proteins as described above. color version available online on ice using cold pbs containing 1% triton x-100, 0.5% deoxycholate, and 1 m m phenylmethylsulfonyl fluoride. the cell lysates were clarified by centrifugation and the supernatants were collected into a 1.5-ml eppendorf tube containing 50 μl anti-hn antibody at a 1: 100 dilution that had been cross-linked to dynabeads protein a (invitrogen). the anti-hn antibody is a mixture of 2 monoclonal antibodies (i.e. m02122321 and m0301307) that were obtained from abcam. the mixes were incubated with rotation at room temperature for 30 min to form the dynabeads-hn ab-hn ag-f ag complexes. after being washed with pbs using a magnet, the bound proteins were released by boiling for 10 min, resuspended in pbs, and then incubated with streptavidin beads (pierce) overnight at 4 ° c. following 2 washes, the beads were resuspended in gel sample buffer. the cells expressing vector, hn, or f alone were used as controls to ensure that the f protein coimmunoprecipitated by the hn antibody occurred only through its interaction with the hn protein. proteins diluted in gel sample buffer in the presence of 1 m β-mercaptoethanol, were separated in 10% polyacrylamide gels [30] . after electrophoresis, the gels were equilibrated in transfer buffer and transferred to immobilon-p (millipore corp.) membranes. the membranes were blocked, washed, and incubated with the primary antibody (either a monoclonal antibody specific for the f protein or a mixture of 2 anti-hn monoclonal antibodies) diluted to 1: 1,000 in pbs-tween 20 and 0.5% nonfat milk overnight at 4 ° c. the anti-f antibody is a mouse monoclonal antibody and was purchased from chemicon international inc. (mab10207). after being washed, the blots were incubated with the secondary antibody (goat anti-mouse immunoglobulin g coupled to horseradish peroxidase; zsgb-bio, beijing, china) diluted to 1: 2,000 in pbs-tween 20 for 1 h at room temperature. following extensive washes, the protein bands were detected using an enhanced chemiluminescence western blotting detection reagent system (amersham biosciences). quantification of the signal was accomplished using a fluor-s imager (bio-rad). all results were expressed as means ± sd of at least 3 independent experiments. statistical analysis was performed using student's t test, and p < 0.05 was considered statistically significant. the leucine zipper-like motif of the hpiv-3 f protein is located between hra and hrb at amino acids 257 to 299 ( fig. 1 a) . the amino acid sequence in this region of the f protein is shown in figure 1 b. to investigate the role of the leucine zipper-like motif in f-mediated membrane fusion, alanine substitution mutagenesis on the heptadic residues (lvlllqi) was carried out. mutations were introduced both individually and in combination as indi-cated in figure 1 b. the reason we chose alanine rather than other amino acids as a substitute residue is that it has a short side chain that is believed to have a minimal disruptive impact on the potential protein structure. the locations of mutations on the α-helix are displayed in figure 1 c. to investigate the abilities of different mutated f proteins to induce cell-cell fusion in the presence of hn coexpression, 3 different types of membrane fusion assays, i.e. a syncytium formation assay, a content-mixing assay, and a lipid-mixing assay, were performed. first, to test the mutant f proteins for an overall level of cell-cell fusion, recombinant vaccinia virus-infected bhk-21 cells were monitored for syncytium formation. multinucleated giant cells were observed at 36 h posttransfection, and photomicrographs from a representative experiment exhibiting syncytia are shown in figure 2 a. cells expressing vector or hpiv-3 wt f protein alone, without coexpression of the hpiv-3 hn protein, were used as negative controls. in contrast to syncytia observed in cells coexpressing the hpiv-3 wt f and hn proteins, the syncytia formed in monolayer cells coexpressing mutated f and hn proteins were not only smaller but also fewer. quantification of syncytia, expressed as a percentage of those detected in cells coexpressing wt f and hn proteins, is shown in figure 2 b. three single mutants (l257a, l271a, and i299a) and 3 double mutants (l271a-l278a, l271a-l285a, and l278a-l285a) were severely debilitated for syncytium formation (less than 6.0% of that of the wt f and hn proteins), whereas the v264a, l285a, and q292a mutants displayed a slightly decreased extent of syncytium formation (64.7, 69.1, and 68.5% of the wt f and hn level, respectively). in addition, while the l278a mutant resulted in a decrease in syncytium formation, i.e. to 38.1% of the wt f and hn level, the triple mutant (l271a-l278a-l285a) lost its ability to form syncytia. these results suggest that in the presence of hpiv-3 hn coexpression all of these mutants mediated reduced levels of syncytium formation. to more accurately quantify the membrane fusion activity of the mutants, we performed a β-galactosidase reporter gene assay to measure the levels of content mixing. for this assay, cells coexpressing the wt or mutated f along with hpiv-3 hn proteins and cells transfected with a plasmid containing the β-galactosidase gene were used to analyze the fusion activity. the results are reported in figure 3 . three single mutants (l257a, l271a, and i299a) and 3 double mutants (l271a-l278a, l271a-l285a, and l278a-l285a) had a markedly diminished fusion activity, corresponding to a percentage of less than 16.0% of the wt f and hn level. whereas the v264a, l285a, and q292a mutants retained about 85% of the wt level in content mixing, the l278a mutant reduced the fusion activity to approximately 50% of the wt f level. additionally, only a background level of fusion activity (2.2% of wt f) was detected from the remaining triple mutant. these results recapitulate those obtained from the syncytium for-mation assay. these data demonstrate that alanine substitutions resulted in different levels of reductions in content mixing. membrane fusion is generally thought to include the following steps [31, 32] : hemifusion, pore formation, and pore expansion. because alanine substitutions in the leucine zipper-like motif of the hpiv-3 f protein weakened these mutants' abilities to form syncytia and induce content mixing to various degrees, we decided to test whether these mutants were capable of mediating mixing of the outer leaflets of the plasma membrane, often referred to a b with pbsk + , pbsk + -f, pbsk + -f and pbsk + -hn or pbsk + -hn and mutant f genes. at 36 h posttransfection, the monolayers were fixed with methanol and stained with giemsa for syncytium observation. a representative photomicrographs of syncytium formation. syncytia are indicated by arrows. b quantification of syncytia was accomplished by measuring the area covered by syncytia and comparing it with the total area of the field for 3 random fields. the data obtained for the mutated f proteins are expressed as percentages of the wt value. values are shown as means ± sd of 3 separate experiments. * p < 0.05, * * p < 0.01, * * * p < 0.001. intervirology 2015;58:297-309 doi: 10.1159/000441978 303 as hemifusion, which is assessed by transfer of the lipophilic r18 from the membrane of r18-labeled rbc to the membrane of cells coexpressing wt or mutated f and hn proteins. as demonstrated in figure 4 a, there was dye transfer from the labeled rbc into underlying cells coexpressing the wt f and hn proteins, but not when labeled rbc were incubated with cells expressing pbsk + or the wt f protein alone. the extent of dye transfer was expressed as the average number of dye transfer events in 6 microscopic fields. in the presence of hpiv-3 hn coexpression, the overall extent of the dye transfer promoted by the mutated f protein was lower than that of the wt f ( fig. 4 b) . three single mutants (l257a, l271a, and i299a) and all of the double mutants gave only a very small number of fusion events, almost similar to the negative control level, whereas the v264a, l285a, and q292a mutants reduced the dye transfer events to 84.1, 86.8, and 85.7% of the wt f level, respectively. the l278a mutant caused approximately half of the dye transfer of wt f, and no dye transfer event was observed from the triple mutant. therefore, the alanine substitutions resulted in the defective abilities of the mutated f proteins to mediate hemifusion. to obtain further quantitative information about the rate and extent of the initial fusion induced by hpiv-3 wt or mutated f along with hn proteins, the r18-labeled rbc were mixed with acceptor cells coexpressing hpiv-3 wt or mutated f and hn glycoproteins; the kinetics of fluorescence dequenching of the r18 probe were measured using a fluorometer. as shown in figure 5 a, when cells individually expressing pbsk + or the hpiv-3 f protein were mixed with r18-labeled rbc, no fluorescence dequenching was detected (blue curve or purple curve; colors refer to the online version only); however, with cells coexpressing wt f and its homologous hn proteins, fluorescence dequenching rapidly and significantly increased (red curve; color refers to the online version only). the rates of fluorescence dequenching were calculated from the maximum slopes of the curves, as shown in figure 5 a and b , and normalized to the maximum rate of fusion induced by the wt f and hn proteins ( fig. 4 c) . the maximum extent of fluorescence dequenching at 5 min was calculated from the kinetics curves, as shown in figure 5 a and b , and are expressed as the percent activity relative to the level of the wt f and hn proteins ( fig. 4 d) . when cells coexpressing 3 single mutant proteins (l257a, l271a, and i299a), 3 double mutant proteins (l271a-l278a, l271a-l285a, and l278a-l285a), or the triple mutant protein (l271a-l278a-l285a) along with hpiv-3 hn proteins were mixed with r18labeled rbc, almost no fluorescence dequenching was observed; both the rate and the extent of hemifusion were less than 9.5% of the wt f and hn level. the initial rate of fusion for the v264a, l285a, and q292a mutated f proteins coexpressed with hpiv-3 hn proteins was 88.8, 87.6, and 85.0%, respectively, and the extent of dequenching at 5 min was 85.5, 87.2, and 83.1%, respectively. additionally, the initial rate and extent of fusion of cells cotransfected with l278a and hpiv-3 hn genes were reduced to 52.8 and 60.6% of the wt level, with both values being lower than those of cells cotransfected with wt f and hn genes. consistent with the results obtained from the syncytium formation assay, the content-mixing assay, and the dye transfer assay, the results of spectrofluorometric analysis of lipid mixing showed that the mutated f proteins reduced the rate and extent of hemifusion. therefore, mutations in the leucine zipper-like motif of the hpiv-3 f protein, when coexpressed with their homologous hn proteins, diminished the fusion activity to various degrees in all stages of membrane fusion. to inspect whether the alanine substitutions in the leucine zipper-like motif diminished or even abolished results are expressed as means ± sd of 3 independent experiments. * p < 0.05, * * p < 0.01, * * * p < 0.001. fusion activity by affecting the expression levels of each mutated f protein on the surface of transiently transfected bhk-21 cells, fluorescence-activated cell sorting (facs) analysis was performed with an anti-f monoclonal antibody as described in materials and methods. the levels of mutated f proteins that were transported to the cell surface were indicated by the mean fluorescence intensity value for each mutant as a percentage of that of the wt f after subtracting the background. as shown in figure 6 , all of the mutated f proteins were expressed at the cell surface at levels quite comparable to that of the wt f protein. these results indicate that each of the mutated f proteins retained its ability to be efficiently trafficked to the cell surface. to probe whether the mutated f proteins in the leucine zipper-like motif weakened the membrane fusion activity by altering the interactions between the hn and f pro-a b fig. 4 . hemifusion directed by the wt or mutated f proteins when coexpressed with the hpiv-3 hn protein. dye transfer was detected as described in materials and methods by the spread of fluorescence from r18-labeled red blood cell membranes to underlying transfected bhk-21 cell membranes. images were monitored by fluorescent microscopy. a representative photomicrographs of dye transfer caused by hpiv-3 wt or mutated f proteins in the presence of hn; dye transfer events are indicated by arrows. b quantification of dye transfer caused by the mutated hpiv-3 f proteins. the extent of the dye transfer is expressed as the average number of dye transfer events in 6 microscopic fields. * p < 0.05, * * p < 0.01, * * * p < 0.001. intervirology 2015;58:297-309 doi: 10.1159/000441978 305 teins at the cell surface, a modified coimmunoprecipitation assay was used to determine the amount of f brought down from extracts of hn-f-cotransfected cells through its interaction with hn by a mixture of 2 anti-hn monoclonal antibodies. the f protein existing in the immunoprecipitates was detected by western blot analysis using an anti-f monoclonal antibody, while the hn protein was detected in a separate western blot analysis using 2 anti-hn monoclonal antibodies. critical controls for this assay are shown in figure 7 a. the first lane shows that neither protein can be immunoprecipitated from control cells transfected with empty vector. the second lane demon-strates that the f protein is not immunoprecipitated with a mixture of 2 anti-hn monoclonal antibodies from the extracts of cells individually expressing the f protein, and the third lane demonstrates that the protein that coimmunoprecipitates with hn is not present in cells that have not been transfected with the hpiv-3 f gene. these controls established the specificity of the coimmunoprecipitation of f by the anti-hn antibodies. the relative levels of coimmunoprecipitated f proteins from the extracts of cells coexpressing mutated f and hn proteins were expressed as a percentage of that coimmunoprecipitated from extracts containing the hpiv-3 wt f and hn proteins. there is no detectable coimmunoprecipitation of the f protein for the triple mutant (l271a-l278a-l285a), consistent with its lost fusogenic activity. three single mutant proteins (l257a, l271a, and i299a), which had a fusion activity below 16.0% of the wt level, were coimmunoprecipitated with anti-hn monoclonal antibodies below 30% of the wt f amount. three double mutant proteins (l271a-l278a, l271a-l285a, and l278a-l285a), which exhibited a fusion activity similar to that of l257a, coimmunoprecipitated less than half of the wt f protein. the v264a, l285a, and q292a mutant f proteins, which had no significant effect on fusion activity (retaining about 85% of the wt f level), could be coimmunoprecipitated at 63.3, 64.3, and 66.7% of the wt level, respectively. in addition, the l278a mutant fused at 53.1% of the wt level and coimmunoprecipitated at 65.1% of the wt f level. taken together, these results imply that the reduction in fusion activity correlates with the defective abilities of these mutated f proteins to interact with hpiv-3 hn proteins at the cell surface. the leucine zipper motif which contains a periodic repeat of leucine or isoleucine residues every 7 amino acids [33] was found to have an important effect on membrane fusion in paramyxoviruses [16, 17, 34] , and there is more than one leucine zipper motif in paramyxovirus fusion proteins. to analyze the role of this motif located between hra and hrb of the hpiv-3 f protein in fusogenic activity, we performed site-directed mutagenesis to substitute the heptadic leucine residues with the alanine residue, either singly or in combination. cell surface expression and the interaction between f and hn proteins, which may affect the fusion activity of the f protein, were also analyzed. interestingly, all of the mutants in this motif exhibited a decreased fusion activity. in this study, we used 3 different types of membrane fusion assays to quantitatively analyze the effect of mutations in this motif on the fusogenic activity of the f protein. our results indicated that amino acid residues l257 and i299, which are positioned at either end of the leucine zipper-like domain, displayed an extremely exhausted fusion activity in all stages of the fusion process when they were replaced by alanine. one nonexclusive possibility is that l257 and i299 are not only key residues in stabilizing the structure of this motif but they are also involved in fusion. while v264a and q292a mutants both retained about 85% of the fusion activity compared with the wt f protein, it appears that the 2 residues were much more tolerant of alanine substitution than the ones located at either end of this motif without significantly affecting its fusogenicity. as shown in figure 8 , comparison of the primary amino acid sequence of the leucine zipper-like motifs of different paramyxovirus f proteins suggested that the 3 middle heptadic leucine residues were highly conserved. analysis of these 3 heptadic leucines, which were replaced individually or in combination by alanine, revealed that single substitution was able to diminish the fusion activity of the protein, while replacement in combination could further reduce or even abrogate fusion activity. these data are in agreement with the results from the mutational analysis of the leucine zipper domain of the murine coronavirus spike (s) protein; substitution of at least 2 heptadic leucine residues is necessary to abrogate fusion activity [35] . since these mutants exhibited similar levels of cell surface expression with respect to the wt f protein, the lack of their fusion activity could not be accounted for by an insufficient amount of f proteins available on the cell surface. traditionally, membrane fusion can be divided into 3 sequential stages [30, 31] . hemifusion, the first step, is the merger of the outer leaflets of the target and effector lipid bilayers and can be measured by the r18 assay [36, 37] . pore formation follows hemifusion and results in mixing of both outer and inner leaflet membrane lipids with concomitant mixing of aqueous contents; it is measured by a reporter gene assay [26, 27] . pore expansion is the final stage, it leads to the formation of multinucleated giant cells, and it is measured by syncytium formation assay a c b d fig. 7 . coimmunoprecipitation of hpiv-3 wt or mutated f proteins with hpiv-3 attachment proteins. surface proteins of cells transfected with the desired plasmids were biotinylated, and then the cells were lysed. dynabeads-hn ab-hn ag-f ag immune complexes were formed with the dynabeads-cross-linked anti-hn antibody; after being washed sufficiently, they were resuspended in pbs and then incubated with streptavidin beads as described in materials and methods to precipitate the cell surface complexes. the coimmunoprecipitated f proteins in the complexes were detected by western blot analysis with the anti-f monoclonal antibody (top), while the hn proteins were detected using a mixture of anti-hn monoclonal antibodies (bottom). all proteins were electrophoresed in the presence of a reducing agent. a critical controls in this coimmunoprecipitation assay: neg.c = empty vector; hn only and f only = these plasmids alone; fwt = wt f protein and hn protein. b , c the results of a coimmunoprecipitation assay for each of the proteins carrying alanine substitutions in the leucine zipper-like motif. wt f protein or the mutants indicated were cotransfected with hpiv-3 hn and are indicated above the gels. d quantification analysis for b and c using the densitometry method from 3 independent experiments. data are shown as means ± sd. * * p < 0.01, * * * p < 0.001. [24, 38] . six of the mutated f proteins (l257a, l271a, i299a, l271a-l278a, l271a-l285a, and l278a-l285a), which nearly lost their ability to form syncytia, directed less than 16.0% of the levels of pore formation of the wt f protein and exhibited a hemifusion activity almost similar to the negative control level, with the rate and extent of initial fusion below 9.5% of the wt f level. the minimal levels of hemifusion, the low levels of pore formation, and the almost negative control levels of syncytium formation detected with these mutants suggest that the low levels of syncytium formation were likely due to slow fusion kinetics being unable to efficiently support pore expansion. in most paramyxovirus systems, including hpiv-3, membrane fusion requires hn and f proteins derived from homologous viruses [39] , which suggests that there is a virus-specific interaction between the 2 membrane glycoproteins that is crucial for the induction of cell fusion [5] . upon attachment of the hn protein to its sialic acid receptor, the hn protein undergoes a conformational change that stimulates the associated f protein refolding into its postfusion conformation. our study shows that all of the mutations in the leucine zipper-like motif of the hpiv-3 f protein not only diminished or even abrogated fusion activity but also reduced the amount of hn-f complexes. there seems to be a correlation between fusion deficiency and the decreased ability of f to interact with hn. this result seems to be in line with the findings of melanson and iorio [40] . thus, the most reasonable explanation for the defective fusion activity is that these mutant f proteins failed to form a sufficient amount of functional complexes containing the hpiv-3 hn protein, thereby blocking signal transmission between the hn and f proteins. these signals are presumably involved in modulation of the membrane fusion pro-cess. it has been reported that the domains m1, m2, and b of the piv5 f protein and the m1 region of the piv2 f protein are involved in the hn-f interaction [41, 42] ; the corresponding region of this motif of the hpiv-3 f protein in the piv5 and piv2 f proteins was identified as being included in these domains by amino acid sequence alignment. our present findings thus do not exclude the possibility that this motif per se was the constituent of its specific interaction domains with hn. in summary, the substitutions we have introduced into the leucine zipper-like motif located between hra and hrb of the hpiv-3 f protein resulted in these mutants having a defective fusion activity in all stages of the fusion process. the mutants formed by replacing the 3 highly conserved middle heptadic leucine residues (l271, l278, and l285) in combination, and 2 single mutants (l257a and i299a) at the end of the motif drastically diminished or even abrogated fusogenic activity. based on these findings, we propose that the leucine zipper-like motif of the hpiv-3 f glycoprotein has an important effect on its fusion activity, and the integrality of this motif is indispensable for membrane fusion. studying the influence of this motif of the hpiv-3 f protein on fusion activity could provide clues to develop antiviral vaccines and therapeutic drugs to prevent and cure paramyxovirus infection. epidemiology and clinical presentation of the four human parainfluenza virus types parainfluenza virus infection of young children: estimates of the population-based burden of hospitalization seasonal trends of human parainfluenza viral infections: united states proteinmediated membrane fusion paramyxovirus fusion: a hypothesis for changes crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase structure of the haemagglutinin-neuraminidase from human parainfluenza virus type iii the role of viral glycoproteins in adsorption, penetration, and pathogenicity of viruses paramyxovirus membrane fusion: lessons from the f and hn atomic structures mutations in the hpiv-3 f protein cause defects in membrane identification of biological activities of paramyxovirus glycoproteins: activation of cell fusion, hemolysis, and infectivity of proteolytic cleavage of an inactive precursor protein of sendai virus fusion peptides and the mechanism of viral fusion structural basis for paramyxovirus-mediated membrane fusion sixhelix bundle assembly and characterization of heptad repeat regions from the f protein of newcastle disease virus the leucine zipper: a hypothetical structure common to a new class of dna binding proteins evidence that the leucine zipper is a coiled coil a leucine zipper structure present in the measles virus fusion protein is not required for its tetramerization but is essential for fusion mutational analysis of the leucine zipper motif in the newcastle disease virus fusion protein a leucine zipper motif in the ectodomain of sendai virus fusion protein assembles in solution and in membranes and specifically binds biologically-active peptides and the virus eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase localization of a domain on the paramyxovirus attachment protein required for the promotion of cellular fusion by its homologous fusion protein spike site-directed mutagenesis of a conserved hexapeptide in the paramyxovirus hemagglutinin-neuraminidase glycoprotein: effects on antigenic structure and function role of n-linked glycosylation of the human parainfluenza virus type 3 hemagglutinin-neuraminidase protein effects of heptad repeat regions of f protein on the specific membrane fusion in paramyxoviruses entry of newcastle disease virus into the host cell: role of acidic ph and endocytosis kinetics of ph-dependent fusion between 3t3 fibroblasts expressing influenza hemagglutinin and red blood cells: measurement by dequenching of fluorescence membrane fusion promoted by increasing surface densities of the paramyxovirus f and hn proteins: comparison of fusion reactions mediated by simian virus 5 f, human parainfluenza virus type 3 f, and influenza virus ha quantitative measurement of paramyxovirus fusion: differences in requirements of glycoproteins between simian virus 5 and human parainfluenza virus 3 or newcastle disease virus base of the measles virus fusion trimer head receives the signal that triggers membrane fusion inhibition of receptor binding stabilizes newcastle disease virus hn and f protein-containing complexes morrison tg: the transmembrane domain sequence affects the structure and function of the newcastle disease virus fusion protein the many mechanisms of viral membrane fusion proteins virus-cell and cell-cell fusion leucine zipper motif extends peptides from conserved regions of paramyxovirus fusion (f) proteins are potent inhibitors of viral fusion amino acid substitutions within the leucine zipper domain of the murine coronavirus spike protein cause defects in oligomerization and the ability to induce cell-to-cell fusion thiol/ disulfide exchange is required for membrane fusion directed by the newcastle disease virus fusion protein lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion role of the simian virus 5 fusion protein n-terminal coiled-coil domain in folding and promotion of membrane fusion functional interactions between the fusion protein and hemagglutinin-neuraminidase of human parainfluenza viruses amino acid substitutions in the f-specific domain in the stalk of the newcastle disease virus hn protein modulate fusion and interfere with its interaction with the f protein identification of domains on the fusion (f) protein trimer that influence the hemagglutinin-neuraminidase specificity of the f protein in mediating cell-cell fusion identification of regions on the fusion protein of human parainfluenza virus type 2 which are required for haemagglutinin-neuraminidase proteins to promote cell fusion the authors thank dr. ronald iorio for providing the hpiv-3 f and hn genes, and dr. bernard moss for the recombinant vaccinia virus vtf7-3. thanks to dr. edward c. mignot (shandong university) for linguistic advice. this research was supported by grants from the national natural science foundation of china (no. 81271806) and the natural science foundation of shandong province (no. 2009zrb019vw). key: cord-261159-9pkg7mbh authors: regnier, fred e. title: chromatography of complex protein mixtures date: 1987-07-17 journal: journal of chromatography b: biomedical sciences and applications doi: 10.1016/0378-4347(87)80007-5 sha: doc_id: 261159 cord_uid: 9pkg7mbh abstract this review has shown that a variety of chromatographic techniques are available for fractionating proteins. fortunately, high-quality columns of every type described in this review are commercially available. most water-soluble proteins may be eluted from size-exclusion, hydrophobic-interaction, ion-exchange, metal chelate, and bioaffinity columns with ease. when this is the case, high recovery and retention of biological activity are the norm. the exception is reversed-phase chromatography where the organic solvents and acids used in polypeptide elution denature many proteins. when problems do occur, they are generally the result of unique structural features of the protein. very hydrophobic proteins have presented the biggest problem in that they are difficult to solubilize, particularly with retention of biological activity. it has been found that zwitterionic an non-ionic detergents are the most suitable solubilizing agents, but area has also been used in cases where hydrophobic interacts are not as strong. unfortunately, there is still an element of trial-and-error in selecting the most suitable solubilizing agent. heterogeneous glycosylation of proteins also presents a problem. both neutral and charged monosaccharides can be incorporated into proteins through multiple steps at several sites. thus, there is the potential in a sample for a large number of glycoprotein species which have the same polypeptide backbone and differing amounts of oligosaccharide. a problem arises when size-exclusion, ion-exchange, hydrophobic-interaction, reversed-phase and bioaffinity systems begin to discriminate between these very similar glycoprotein species. chromatographic peaks can become very load, due to incomplete fractionation, and the polypeptide chain of interest can be associated with multiple peaks. the separation of glycoproteins requires much more study before logical procedures can be suggested for column selection and operation. aggregated species are another class of proteins which present occasional problems. multimeric proteins are adsorbed to sorbents by a series of forces, among which are hydrogen bonding, hydrophobic interactions, and electrostatic forces. these forces are also responsible for the maintenance of quaternary structure in proteins. when the same forces dominate both retention of protein structure and adsorption at the sorbent surface, the quaternary structure of the protein can be disrupted during elution. very basic proteins also present a problem in some cases. columns with residual negative charges, such as a silica-based reversed-phase column, adsorb anionic species so strongly that they are difficult to elute. this is the case with ribosomal and nucleoproteins. the solution is to use exhaustively end-capped columns which diminish electrostatic interactions. able, the equally important task of optimizing their operation for protein separation was undertaken. this latter endeavor is the subject of this review. it is logical, in teaching protein chemistry, to emphasize the common features relating to the genetic regulation, biosynthesis, and structure of proteins. as a consequence, we often fail to appreciate the enormous difference between proteins and the implications of these differences in their fractionation. it is the development of strategies to deal with these differences that has driven the recent diversification of protein fractionation into so many areas of biochemistry. this review will examine the features that are unique in the chromatography of some protein-classes.* two criteria were used in the selection of classes of proteins for inclusion in this review. first, the body of literature had to be sufficiently large that general conclusions could be made. second, there had to be unique problems associated with the separation of the particular class of proteins. cells and often cellular organelles are enclosed within a lipid bilayer membrane. this non-polar membrane appears to provide two problems for the cell. first, the lipid layer would be a barrier for the transport of polar substances into and out of the cell. second, it would be difficult to anchor the proteins and carbohydrates necessary for cellular recognition and communication at a lipid bilayer. nature has solved these problems by developing special groups of proteins such as the peripheral membrane proteins that adsorb at the exterior of the membrane and the integral membrane proteins that penetrate into the membrane. it is probable that peripheral membrane proteins are adsorbed through electrostatic and hydrophobic interactions with both polar fucntional groups of the membrane lipids and portions of integral membrane proteins which protrude out of the membrane. 'internal membrane proteins are of three types: transmembrane proteins which completely span the membrane and are exposed to the aqueous environment on both sides; proteins which have both an exposed and membrane embedded .portion; and proteins that are completely embedded within the lipid of the membrane. peripheral membrane proteins are generally dissociated from the membrane by using salt and edta or changes in ph to disrupt the electrostatic forces holding them in place. the character.isticsof peripheral membrane proteins are not substantially different than those of other water-soluble proteins ( 21. for this reason they do not present unique problems in their chromatographic separation. in contrast, integral membrane proteins are much more challenging. portions of an integral membrane protein within the bilayer contain many non-polar amino *readers searching fordetailed irrformation rather than for generalizations in separation of individual protein categories are deferred to specialized chapters of this volume. acids which are strongly associated with the hydrocarbon segments of membrane lipids. releasing integral membrane proteins from the membrane requires a detergent which will both disrupt the membrane.and keep the protein in solution once the lipids have been removed. although sodium dodecyl sulfate (sds) and other powerful detergents easily disrupt membranes and solubilize proteins, the objective is often to try to maintain some of the original secondary and tertiary structure of the protein during purification. accomplishing this usually requires a detergent which is less harsh. two reviews on the solubilization of functional membrane-bound proteins [ 3, 4] are suggested for a discussion of this non-chromatographic portion of membrane protein purification. as a note of caution, the reader is reminded that detergents which contain an aromatic residue have substantial absorbance in the uv portion of the spectrum and present detection problems. purification of a protein that requires a detergent to keep it in solution presents a challenge to the chromatographer. detergents are not always compatible with chromatographic processes. fortunately, the use of detergents and solubilizing agents such as sds, zwitterionic detergents, non-ionic detergents, organic solvents, urea, and guanidinium hydrochloride has been examined in the sec of proteins [ 3-81. there is also a series of reports on detergent use in the sec of integral membrane proteins f g-151. although sec is of limited resolving power in the purification of membrane proteins [ 121, it has sufficient resolution to be used in the selection of detergents for solubilization [ 91, optimization of detergent concentration [ 111, and quick estimation of the purity of a sample purified by other techniques [ 131. sec, using sodium cholate, edta, dithiothreitol (dtt) , and glycerol in the mobile phase to preserve activity, has also been used to study the induction pattern of microsomal cytochrome p-450 [ lo] . still another application of sec has been to monitor the aggregation state of detergent-solubilized sarcoplasmic reticulum adenosine triphosphatase [ 161. when sec is used in the preparative mode it is usually in conjunction with another chromatographic step. the purification of spectrin subunits from erythrocytes by a combination of sec and rpc is an example [ 171. spectrin is composed of dissimilar 220-and 240-kd subunits which, through dimerization, form the major structural proteins in the cytoskeleton of red blood cells. iec has proven to be much more useful in the separation of integral membrane proteins. although large amounts of organic solvent are occasionally used in the mobile phase to solubilize proteins, as in the case of chloroplast proteins [ 181, most iec separations are carried out with either non-ionic detergents or the 3-[ ( 3-chloroamidopropyl) dimethylammonio] -l-trifluoroacetic acid ( chaps) zwitterion [ 24,251. there is strong evidence that the quality of the detergent as a solubilizing agent and its concentration has an impact on resolution in iec. using a series of polyethylene glycol alkyl ether (c,e,) detergents as a mobile phase additive it was possible to show that both alkyl chain length and the number of glycol residues influenced resolution of escherichiu cozi k-12 membrane proteins in anion-exchange separations [ 191 (c&e, was optimal) . with both c&es [ 191 and n-octyl-/3-d-glucopyranoside (octyl glucoside) [ 201, it was reported that resolution was better above the critical micelle concentration of the detergent. this was attributed to diminished protein aggregation on the column. the presence of edta in the extracting medium and mobile phase also increased resolution [ 201; apparently divalent cations are involved in aggregate formation. all three of these phenomena indicate that complete intermolecular dissociation of proteins is a prerequisite to good chromatographic resolution of membrane proteins. occasionally, it is even possible to partially purify peripheral membrane proteins on the basis of differential extraction. a receptor peptidoglycan on the outer portion of the e. coli k-12 membrane is solubilized with difficulty by any nonionic detergent but triton x-100. following exhaustive extraction by other detergents, iec of the triton x-100 extracted material indicates that the peptidoglycan is almost pure [ 191. based on the discussion above it is clear that the single greatest obstacle to the use of iec in the fractionation of membrane proteins is solute solubility. when effective conditions for solubilization have been selected that completely disrupt intermolecular interactions without disturbing the electrostatic interactions necessary in iec, it is possible to carry out iec separations. unfortunately, it appears that the optimum conditions for both solubilization and chromatography may be protein-specific and a general protocol cannot be provided. when maintenance of native structure is less important, rpc becomes a viable option for membrane protein fractionation. the problem in rpc is much the same as in iec and sec; the more hydrophobic membrane proteins are difficult to solubilize. although recovery is more of a problem in rpc of membrane proteins than water-soluble proteins [ 261, it is still widely used. khorana et al. [ 271 have made the observation that "the straightforward application of current methods for sequencing water-soluble proteins to large hydrophobic membrane proteins such as bacteriorhodopsin is not feasible". in addition, the extreme hydrophobicity of an rpc column makes elution of very hydrophobic proteins even more difficult. khorana et al. [ 271 and tahagahi et al. [ 281 have successfully solubilized proteins such as bacteriorhodopsin, cytochrome b6 and cytochrome b5 reductase with formic acid. elution of proteins and peptide fragments from rpc columns was achieved with mobile phases containing 5% formic acid. the fact that formic acid can cleave aspartyl-prolyl (asp-pro) bonds, deaminates asparagine and glutamine, aggregates cleavage fragments, slowly destroys tryptophan residues, and is viscous, is a concern. addition of ethanol to a final concentration of 70% in the extracting solvent diminished these problems. poliovirus coat proteins were even more difficult to solublize [ 29,301. in this case, it was necessary to use 60% formic acid and 2-propanol as a mobile phase (fig. 1 ). since one of the peptides being examined had three asp-pro pairs which are known to be labile in acid, the possibility of fragmentation was examined. with this particular peptide no cleavage occurred in 24 h when the samples were incubated with a mixture of formic acid-acetonitrile (60~20). again the addition of organic solvent diminished degradation. in all but a few cases, there was no change in proteins after 6 h of storage in the mobile phase as determined by electrophoresis and chromatography. antisera prepared against proteins which had been purified by rpc with the 60% formic acid mobile phase reacted with intact virus. this indicated that sufficient native structure was recovered when these proteins were returned to a physiological environment that antibodies could be produced which were functional against native viral proteins. although formic acid is a superior solubilizing agent, it should be appreciated that with silica-based sorbents substantial degradation, i.e. cleavage of the alkyl group from the support, occurred during its use. inferior performance with other mobile phases may be experienced after use with formic acid because large areas of underivatized silica surface would have been generated. the most popular mobile phase still contains trifluoroacetic acid (tfa) and either acetonitrile or an alcohol such as ethanol, 2-propanol, or butanol [ 31-341. it has also been noted in the tfa system that quite different selectivity can be obtained by using mixtures of acetonitrile and 1-propanol [ 331. selectivity varied almost continuously with the ratio of these two solvents. separations which could not be achieved at one solvent ratio were easily accomplished at another. pecoveries were typically greater than 90%. prior to the introduction of formic acid and tfa, acetate (ph 6.5 ) with chloroform-methanol was successfully employed in the isolation of mitochondrial sector proteins [ 351. although resolution is generally quite good and recovery high in rpc [ 331, this is not always the case. less than 50% of membrane proteins are recovered from an rpc column in some cases [ 14, 32, 341. the proteins are either too hydrophobic or insufficiently soluble in the mobile phase for elution from an rpc column. the introduction of sample heterogeneity by partial oxidation of cysteine during protein extraction has also been noted as a problem in the case of yeast cytochrome c oxidase [ 331. this problem may be overcome by-pretreating samples with a reducing agent such as dtt. sorbent pore diameter could also play a role in rpc of membrane proteins. in the case of red blood cell membrane proteins, the resolution of 1000 a pore diameter organic resin-based sorbents was superior to 300 a pore diameter silica-based materials when the columns were eluted with a tfa-acetonitrile mobile phase [ 261. apparently these pore diameter effects are eliminated with mobile phases containing formic acid [ 281, it is not clear whether this problem is due to differences in the pore diameter or to differences in the matrix and ligand density. there is also a report of the use of hydroxyapatite in the purification of membrane proteins [ 361. the coronavirus glycoprotein e2, which is responsible for virus attachment to cell receptors and virus-induced cell fusion, is composed of two distinct 90-kd subunits. although these two subunits have identical electrophoretic mobilities when analyzed by sds polyacrylamide gel electrophoresis (page), they could be widely separated by hydroxyapatite columns in the presence of sds. solutes were eluted in a 0.15-0.5 m sodium phosphate buffer gradient, ph 6.8, containing 0.1% sds. there was some difference in resolution between hydroxyapatite columns from various suppliers. to avoid column dissolution, it was recommended that the mobile phase contain calcium and phosphate at the solubility product levels of calcium phosphate. however, the formation of a precipitate in the presence of sds precluded the addition of calcium and phosphate to the mobile phase when sds was present. this reduced the lifetime of the column. conventional columns were discarded after a single use whereas hplc columns had a useful lifetime of twelve to fifteen runs. erosion of the chromatographic support was accompanied by a gradual increase in headspace and back-pressure. high-performance bioaffinity chromatography may also be useful in the future. the separation of human plasma membrane proteins of cultured human fibroblasts has been achieved by affinity chromatography on microparticulate silica derivatized with an affinity ligand [ 37 ] . isothiocyanatopropyl silane-derivatized silica was used to immobilize a series of nucleophilic ligands ranging from synthetic triazine dyes to concanavalin a. both the procion orange and mannan columns successfully retained and released plasma membrane proteins as determined by sds-page. ribosomes of a typical procaryote, such as e. cali, have a relative molecular mass approaching 2.3*106, of which 65% of the mass is nucleic acids and 35% is protein. e. coli ribosomes have been fractionated into two major components, a 50s component and a 30s component. the 50s component is known to contain 33 polypeptides ranging in size from 9 to 28 kd, some of which are present in multiple copies. the smaher 30s component contains 21 polypeptides which range in size from 10 to 28 kd with the exception of the 65kd sl protein. some of the ribosomal proteins are very basic and probably adsorb electrostatically to polynucleotides. although other proteins are associatd with the ribosome during protein synthesis, functional ribosomes may be reconstituted from the 2130s proteins and 33 50s poteins without addition of other proteins or enzymes. ribosomes of eucaryotes are larger and contain more polypeptides than those of procaryotes. a 40s subunit contains 30 polypeptides whereas a 60s subunit contains 40. the relative molcular masses of eucaryotic ribosomal proteins are also generally larger than those of their procaryotic counterparts. the classical chromatographic methods for purifying ribosomal proteins include sec and various types of iec. in addition to these methods, rpc has been found to be useful as will be shown below. due to the inherently low resolving power of sec, it provides the least resolution of ribosomal protein mixtures. at best, no more than seven peaks can be seen in an sec separation [ 38, 391. it is even doubtful that the separation is truly based on size exclusion. elution profiles are strongly dependent on the column manufacturer and the mobile phase. however, the fact that greater than 90% of the ribosomal proteins were recovered [ 391 makes the technique acceptable as a first step in fractionation. fractionation of ribosomal proteins by rpc is a relatively straightforward process. alkyl chain length does not seem to be a critical variable but tfa is almost universally selected as the mobile phase pairing agent [ 38-421. although both acetonitrile [ 381 and p-propanol [ 391 have been used as mobile phase displacing agents, it appears that acetonitrile gives lower recovery and allows the formation of protein complexes [ 391. for example, one of the 30s ribosomal proteins was found to complex and coelute in seven other protein peaks with acetonitrile. this problem precluded purification of the protein. in contrast, propanol prevented complex formation and the protein could be isolated in pure form. the rpc system purified 4 of the 21 proteins from the procaryotic 30s subunit to homogeneity, while an additional 15 were purified to a high degree [ 391. the 50s proteins were separated into 23 fractions (fig. 2) . recoveries ranged from 27 to 91% with the average being 70% [ 411. the most strongly retained proteins gave the lowest recovery. proteins non-specifically retained by the column were probably responsible for the "ghosting" phenomenon observed when a blank gradient was run after a ribosomal protein sample. residual materials were removed from the column by running a series of blank gradients. analytical columns were capable of fractionating 2-3 mg protein loads when used in the preparative mode [381. it is amazing that although ribosomal proteins are partially denatured in the process of purification by rpc, active ribosomal subunits may be reconstituted from these proteins [ 411. reconstitution resulted in the production of multiple supramolecular complexes which were fractionated by centrifugation. only the a a fig. 2 . rpc of tp50 (total protein from 505 ribosomal subunits) [41] . a solution of tp50 (700 pmol) in 60 d of 0.1% tfa was applied to a synchrom rp-p column and eluted with the following gradient: 17-34.9% ace&&rile in 20 min; 34.9-3636 acetonitrile in 10 min; 36% acetonitrhe for 10 min; 3645% acetonitrile in 10 min; 45-50% acetonitrile in 5 min; 50-75% acetonitrile in 5 min; 75%. acetonitrile for 5 min. absorbance at 214 nm was measured at 0.6 a.u.f.s. proteins l7 and l12 were present in low amounts in the 50s subunits from which these proteins were extracted and are not shown. 30s and 50s subunits isolated from these mixtures showed activity. reconstituted 30s subunits had 64% of the activity of the native subunit and an identical composition except for the s-3 protein. the 50s subunit had 52% of the activity of the native subunit and a nearly identical composition to the 33 ribosomal proteins. puromycin is known to be an inhibitor of protein synthesis. photoincorporation studies directed at determining which protein (a) in the ribosome interact with puromycin used rpc to determine that proteins l-23, s-7, and s-14 were labelled [ 411. this would imply that puromycin is bound to these proteins in some way during the course of inhibition. it has been suggested that by purifying these labelled proteins and incorporating them into the ribosome a clearer picture of puromycin inhibition could be obtained [ 411. eucaryotic ribosomal proteins have also been examined by rpc. in a study [ 421 directed at the purification of the s-6 ribosomal protein from rat liver, whole ribosomes were extracted with 6 m guanidinium hydrochloride and the rna precipitated by acidification., highly purified proteins were obtained in two chromatographic steps using cd, cs, and diphenyl columns. iec is equally effective in the purification of ribosomal proteins and in many ways complements rpc and sec [ 391. urea (5 m) has been found to be an essential mobile phase component [ 39, [43] [44] [45] due to the basic nature of most ribosomal proteins [ 38,391, cationexchange chromatography is the most useful. in the case of procaryotic ribosomes, proteins from the 30s subunit gave much broader peaks than those from the 50s subunit. the reason for this is not readily apparent. iec of the 30s ribosomal proteins on a weak cation-exchange (cm) column resolved, at least partially, 18 of the 231 proteins [ 431. the remaining five proteins eluted in two peaks. one peak contained proteins s-9, s-19, and s-15 while the other contained s-14 and s-18. through the use of very shallow gradients it was possible to partially resolve even these components. recovery ranged from 60 to 100% with the average being 90%. the chromatographic behavior of a strong cation-exchange (sp) column with e. coli ribosomal proteins was essentially the same as that of the weak ion-exchange (cm) column [ 441. recovery of many proteins approached 100% with the lowest being 80%. preparative separations on a 150 x 21.5 mm sp column fractionated 400 mg of protein per 5-h elution cycle. regeneration between cycles was achieved with a potassium hydroxide wash. iec was also useful in the fractionation of 50s proteins; a weak cation-exchange to resolve the ribosomal proteins in the 60s subunit from sacchuromyces cereuisiae [45] . there are four major classes of cereal proteins which are classified according to their solubility. albumins and globulins which may be extracted by water and dilute salt, respectively, may be treated in separation systems much like the cytoplasmic proteins of bacteria and animals. in contrast, the prolamines are much more hydrophobic, requiring 70% ethanol for extraction from cereals. the gliadins in wheat and zeins in corn belong in the prolamine class. these 30-40 kd storage proteins owe their solubilty properties to a high content of glutamine, proline, and hydrophobic amino acids. glutelins are the endosperm proteins remaining after the extraction of albumins, globulins and prolamines. extensive disulfide cross-linking, hydrogen bonding, and hydrophobic association between these high-molecular-mass proteins contributes to their insolubility. through the use of extremes in ph, detergents, denaturants, reducing agents, and alkylating agents, many of these proteins can be solubilized. rpc is by far the most successful of the hplc modes for the separation of cereal proteins [ 46 1. for example, a low-molecular-mass gliadin sample that could be resolved into 46 protein components by two-dimensional electrophoresis produced 36 components (fig. 3 ) in an rpc system using a loomin tfa-acetonitrile gradient at a flow-rate of 1 ml/min [ 471. using a higher flow-rate (3 ml/min) and column temperature of 70â°c it has been possible to reduce the separation time to lo-15 min [ 481. although several different organic solvents and pairing agents have been examined, they offered no distinct advantage over tfa-acetonitrile in these profiling studies. preparative separations using a 250 x 10 mm rpc column and a 25-mg sample load in conjunction with sec and cation-exchange chromatography have been used in the purification of gliadins [ 49,501. intercolumn variation is often a problem in rpc of proteins (see section 2.7). this was not found to be the case with cereal proteins [ 471, probably due to their lower content of basic amino acids. at the present time rpc has been applied to the identification of wheat varieties [ 491, prediction of bread quality [ 511, genetic studies of wheat [ 511, and the analysis of corn proteins [ 52 1. high-performance sec has also seen some use in the separation of cereal proteins. bietz et al. [ 531 have used sec in the separation of both native and reduced wheat gliadins. reducing conditions confirmed that high-molecular-mass gliadins are composed primarily of low-molecular-mass gliadins joined by disulfide bonds. sds solubilization of glutenins from single kernels of various wheat varieties provided samples which could be analyzed on sec columns eluted with a mobile phase containing sds [ 53, 541 . these separations revealed marked differences in the relative molecular mass and distribution of proteins among the varieties examined. high-performance iec has seen little use in the separation of cereal proteins. bietz [ 461 reported a separation of the water-soluble wheat albumins and globulins on a carboxymethyl high-performance cation-exchange column and batey [ 551 demonstrated that it was possible to identify wheat varieties using highperformance anion-exchange chromatography. it appears that hplc techniques will make it possible to correlate specific endosperm proteins with either functional or nutritional quality of cereal products which will in turn provide geneticists with an analytical tool to be used in breeding programs for cereal grain improvement. hemoglobin is a tetrameric protein composed of two sets of identical globin chains and four heme groups. fetal hemoglobin ( hbf) , consisting of cr-and y-chains, is normally present at birth. early in life hbf is replaced by hemoglobin & (hb&) which is composed of cy-andp-chains and is the dominant hemoglobin in adults. in some segments of the population, there are genetically transmitted variants of these hemoglobins which lead to serious health problems. for example, the substitution of valine for glutamine at the sixth residue in the pchain leads to the formation of hemoglobin s (hbs) which is responsible for sickle cell anemia. in other cases such as (x-andg-thalassemia, synthesis of either the cx-or p-globin chain is reduced or absent. homozygous states of any of the above hemoglobinopathies or combinations of any two abnormalities may result in clinical manifestations especially during pregnancy, surgery, or other noxious situations. glycosylated hemoglobins are also of clinical importance. they are minor con-,stituents of the hemoglobin pool in adults which arise from the non-enzymatic 'glycosylation of hba. one of these glycosylated hemoglobins, hba1,, has come into wide use as an indicator of glucose regulation in diabetics. hba1, is normally found in levels of 4-6%, but can be more than double this value in the blood of uncontrolled diabetics. the quantity of hba1, reflects the average glucose level in a patient's blood over the life span of the erythrocyte. the advantage of hba1, measurements over blood glucose levels for monitoring diabetics is that they circumvent the physical discomfort of glucose tolerance tests. in the past, electrophoresis and iec on carbohydrate columns have been used in hemoglobin determinations. it is shown below that hplc columns are equally capable of determining hemoglobinopathies with much greater speed and less manipulation. hemoglobin variants can be determined chromatographically in three ways: direct separation of the intact protein where possible; analysis of the globin chains; and analysis of a globin chain tryptic digest. since the thrust of this review is on complex proteins, the discussion will be restricted to the separation of intact hemoglobins. understandably, a subject of such clinical significance has an extensive literature. the objective here is only to discuss some of the more critical variables in hemoglobin separations that have been examined in the recent literature. although hemoglobins are easily eluted from sec columns, this technique is not discussed here because sec will not discriminate between the various hemogoblin species. both cation-exchange and anion-exchange chromatography are effective in the separation of hemoglobins. using a weak cation-exchange column it has been possible to completely separate hemoglobins bart's f, a,,, a2, s, c, d,e,g,sg, winnipeg, and sealy in a 30-min gradient elution [ 561. the gradient was constructed by varying both ph (from 6.5 to 6.8) and ionic strength. variant hemoglobins e, d, g, s, and c result from a single amino acid substitution in either the cy-or p-chain. they are detectable by iec because the mutation results in either the elimination of a charge, addition of a charge, or a switch in the sign of a charged group in the hemoglobin. a recent study on strong cation-exchange columns finds them to be equally effective under the same conditions used with the weak cation-exchange column [ 571. both of these iec systems are vastly superior to conventional electrophoretic and column iec methods. it should be noted, however, that there are small differences in selectivity between commer-cyl.c w. 40 60 :onds anion-exchange chromatography has also been widely used in the separation of variant hemoglobins [ 59-611. a good high-performance anion-exchange column can discriminate between hemoglobins ao, c, s, and f. separation speed, recovery, ease of sample manipulation, and column lifetime are all comparable to cation-exchange chromatography. however, it is the conclusion of most practitioners that cation-exchange chromatography is more useful because it discriminates between more variant hemoglobins. cation-exchange chromatography is by far the most suitable for hba1,. using gradient elution of either a weak [ 621 or strong [ 631 cation-exchange column it has been possible to determine the concentration of hba1, in a little over 6 min, even in the presence of elevated levels of hbf, hba1,, hbalb and hba2. allowing for column recycling and re-equilibration, it was possible to make four measurements per hour. full automation of the method gave a between-assay coefficient of variation of 2-3%. small differences in the selectivity between different manufacturers' columns may require modification of gradient shape to achieve maximum throughput on any particular column. although the speed of these separations seems remarkable, determination of the hba1, concentration in a serum sample has recently been achieved in less than 40 s (fig. 4) using a new non-porous cation-exchange column [ 641. the speed of non-porous particle chromatographic systems should have a major impact on the utilization of hplc in quality control and clinical environments. steroid hormones are known to penetrate the cell membrane and bind to cytoplasmic receptor proteins. it appears that for a single steroid there may even be multiple receptor proteins which vary with cellular differentiation [ 651. since receptor proteins do not have unique spectral properties, they are generally detected (or assayed) by their ability to bind labelled steroids. radiolabelling is the most widely used [ 65, , but fluorescent labelling has also been reported [ 661. the labelling of proteins with a steroid may be done before or after the separation [ 65-681 as shown in fig. 5 . post-separation detection is so time-consuming and labor-intensive that it is seldom used. although much quicker and more widely used, preseparation labelling presents two types of problems. many cytoplasmic proteins, in addition to specific receptor protein ( s ) , may bind steroids . this makes it difficult to differentiate specific steroid receptor protein (s) from those that bind steroid non-specifically. fortunately, steroid receptor proteins have a higher affinity for labelled steroid than those that adsorb the steroid non-specifically. this difference in affinity is the basis for the second problem. during passage through the column, weakly bound steroid will begin to dissociate from proteins and either migrate through the column as free steroid or be adsorbed by the sorbent matrix [ 651. if dissociation is incomplete, the labelling pattern in the column effluent will be misleading. the degree of dissociation is due in large part to the binding constant of the steroid and the rate of dissociation. one of the ways to overcome this problem is to separate labelled proteins in the presence of a large excess of non-labelled steroid [ 68, 691. in those cases where the labelled steroid is weakly associated with protein, the large excess of non-labelled steroid will displace the labelled steroid. estrogen receptor proteins have been examined with sec [ 66, 671, anionexchange chromatography [ 681, and high-performance chromatofocusing (hpcf) [ 65-681. hpcf is a form of iec in which a ph gradient is generated along the length of the column using mobile phase buffers and the buffering capacity of a weak anion-exchange sorbent [ 651. at least three isoforms of estrogen receptor protein have been identified with iec and hpcf [ 65-681. anion-exchange chromatography on 300,500, and 1000 a pore diameter sorbents indicated that each sorbent had slightly different properties, particularly in regard to the retention of free steroid [ 681. the reason for this was not determined. in a direct comparison with soft-gel chromatofocusing media, 300 and 500 a pore diameter hpcf sorbents were judged to be superior in resolution [ 681. it is interesting that none of the chromatofocusing columns behaved in exactly the same way. these differences were attributed to the buffering properties of the sorbents. although greater than 90% of the receptor activity was recovered when hpcf was carried out over the ph range of 8.3-4.5 in 60 min, there was some variability between samples. sodium molybdate was shown to produce an irreversible size and charge stabilization of the receptor [ 651. previous isoelectric focusing studies have indicated that the estrogen binding proteins have a pl of 7-8. it is interesting that none of the estrogen receptors eluted from the hpcf column in this ph range. this suggests surface charge heterogeneity in the estrogen receptor proteins. the human uterine progesterone receptor has also been studied with both anionexchange chromatography and hpcf [ 691 using three different tritiated progesterones and an axoanalogue of progesterone. the function of the analogue was to covalently label the steroid-specific binding site of the receptor. labelled cytosol components were separated on an anion-exchange column in both the presence and absence of a high molar excess of the respective unlabelled competitor steroids. after elution with a sodium chloride gradient, labelling was determined in each fraction and the elution profiles were superimposed. one specifically labelled and two non-specifically labelled peaks were located. the same specifically labelled peak was detected by chromatofocusing. when this covalently labelled peak was subjected to sds-page, 45-kd and 27-kd proteins were detected. immunoglobulins (ig) are produced by plasma cells derived form b-lymphocytes. on the basis of relative molecular mass and chemical properties, five major classes of immunoglobulins have been recognized igg (150 kd ) , igm (950 kd ) , iga (300 kd ) , .igd (160 kd) , and ige (190 kd) . within each major immunoglobulin class there can also be subclasses which are of similar relative molecular mass. igm and iga are sufficiently different in relative molecular mass that they may be differentiated from each other and the other immunoglobulins by sec [ 70,711 . -in contrast, igg, igd, and ige are sufficiently close in relative molecular mass that no real differentiation is to be expected with sec. the basic structural unit of all antibodies is composed of two light chains and two heavy chains joined by disulfide bridges. in the cases of igg, ige, and igd, the antibody is composed of a single structural unit, i.e. two heavy and two light chains. the light-chain pair may be of either the k-or l-type. although these two types of light chains are approximately equal in relative molecular mass, they differ chemically. the heavy-chain pair is identical within any antibody class but varies in relative molecular mass between classes. in the cases of igm and iga, the antibody is a multimer of the basic structural unit. the basic structural unit of an immunoglohulin has a "y" shape and is bivalent. igg is frequently cleaved with papain to produce two fab and an f, fragment. the fab and f, fragments are sufficiently different in molecular properties that they may be separated by either sec or iec. the fab fragment is essentially one of the arms of the y which contains an unmodified light chain, retains full immunological activity, and is monovalent. the f, fragment, in contrast, has no immunological activity but is that portion of the antibody which binds to the stuphylococcus aureus protein a. the use of protein a in antibody immobilization will be discussed below. reduction of the basic immunoglobulin structural unit with mercaptoethanol in the presence of 6 m urea produces a mixture of heavy and'light chains which vary two-fold or more in relative molecular mass and are separable by sec [ 711. of particular importance in chromatography is the fact that immunoglobulins are glycoproteins. structural studies indicate that fucose, mannose, gala&se, nacetylglucosamine, n-acetylgalactosamine, and sialic acid are attached in the f, region through serine, threonine, and asparagine residues. apparently carbohydrate groups are attached to the heavy chains in a stepwise manner at different subcellular sites during transport of the protein through plasma cells. this process is not always complete and accounts for the chromatographic heterogeneity in antibodies. six general types of high-performance columns have found application in the separation and study of immunoglobulins: size-exclusion, anion-exchange, cationexchange, hydrophobic-interaction, hydroxyapatite, and protein a bioaffinity columns. classical dye columns are also used in preliminary fractionations prior to high-performance separations. although sec is useful in the separation of (1) igm and iga from igg, igd, and ige [ 70,711, ( fig. 6 . iec of high sample load (25 mg protein) of ammonium sulfate-precipitated aecitee fluid [75] . the hatchedareae indicate antibody activity. the eeparation wae achieved on a gradient-eluted mono q hk 5/5 anion-exchange column (50x5 mm). mobile phase a was 0.02 mol/l trie-hcl (ph 8.0). mobile phase b was 0.02 mol/l trie-hcl (ph 8.0) containing 1.5 mol/l sodium chloride. the gradient was generated over 50 min at a flow-rate of 1 ml/min. body purification. the new high-performance iec columns are sufficiently similar to the old gel-type deae columns that they may be substituted in older purification procedures, such as in the fractionation of igg from serum proteins on deae cellulose [ 741. when the fractionation of ascites fluid on a high-performance anion-exchange column is preceded by either an ammonium sulfate precipitation [ 701 or dye-column step [ 751, monoclonal antibody (mab) collected from the anion-exchange column is pure. up to 25 mg of prefractionated sample per run was processed (fig. 6) on an analytical column [ 751. without-the ammonium sulfate precipitation step, both iec and hic columns are often required to obtain high purity. for example, direct fractionation of mouse mab igg, sample on an anion-exchange column produced an antibody with 7% transferrin contamination [ 761. anion-exchange columns may also be used in the fractionation of igg subclasses. although mouse igg, and igg,, coelute, igg,, and igg, may be resolved from the other antibodies. igm and igg from sheep and cattle [ 701 and fab and f, fragments from igg [ 761 have also been fractionated by anion-exchange chromatography. one of the difficult problems in the purification of some igg samples on an anion-exchange column is the elimination of albumin and transferrin. this problem has been circumvented with a strong cation-exchange column [ 711. contaminating proteins from the anion-exchange column often have a lower p1 than igg and elute much earlier on the strong cation-exchange column. there is also a report of the use of hpcf columns for the fractionation of igg and igm [ 771 .in addition, the technique was used in the fractionation of submilligram quantities of normal and abnormal igg of differing protein a bioaffinity chromatography is the second major method used in the purification of igg antibodies. with the recent commercial availability of highperformance protein a columns [ 781, a rapid and efficient tool has been added to the arsenal of high-performance columns available to the immunologist. when a protein a column is eluted with a ph gradient ranging from 8 to 2, the igg subclasses from mice elute in the order igg, < igg, < igg1. it has also been reported that protein a columns can separate the iggza and iggpb antibodies of mice [ 791. protein a columns have also been used to prepare immunosorbent matrices by cross-linking antibodies after adsorption [ 801. the principal limitation of protein a columns is that they do not bind all antibodies. in a direct comparison of iec, hic, and hydroxyapatite columns in the fractionation of mouse monoclonal antibodies directed against coagulation factor viii, pavlu et al. [ 751 reported that the resolution of both the iec and hic columns was superior to the hydroxyapatite. however, this is only one case. hydroxyapatite columns are widely used in the purification of antibodies where they generally eliminate greater than 95% of the non-immunoglobulin proteins and give 75% or greater recovery [ 81-831. as noted above, hic has also been used with success in the fractionation of immunoglobulins [ 73, 751 . i n view of the fact that ammonium sulfate fractionation has been used widely in antibody purification, it is possible that hic could accomplish this step with greater resolution. the principal limitation of hic today is that it has not been applied widely enough to make general conclusions concerning its broad utility. the chromosomes of eucaryotic cells contain a family of small, basic proteins which are tightly associated with dna and function to package it into nucleosome structural units. there are five major classes of histones which are designated hl, h2a, h3b, h3, and h4 and differ in both relative molecular mass and amino acid composition. for example, hl is a 21-kd protein containing 29% lysine wheras h2a and h3b have 14.5 and 13.7 kd, respectively, and are approximately equal in arginine and lysine content. these three histones also vary considerably among eucaryotic species, i.e. there is little sequence homology. in contrast, the 15.3-kd h3 histone and 11.3-kd h4 histone are arginine-rich and almost identical among species. a striking feature of the h4 histone is that the basic residues seem to be clustered at one end of the molecule where there is a net positive charge of 16. the fact that arginine and lysine constitute approximately one fourth of the amino acid residues in histones presents both an opportunity and challenge in their separation. fractionation of all the major classes of histones has been achieved by rpc using a cls or cn column [84-901. there are, however, some special requirements. the first is the use of end-capped columns. non-end-capped columns gave very poor recovery in the case of c1s, ce, and cn columns. since this is not always the case with proteins, it is probably due to the very basic nature of histones. the second requirement was the use of tfa in the mobile phase. using 0.3% tfa in an aqueous-to-acetonitrile gradient on a waters radial-pak pbondapak cl8 column it was even possible to split the h2a and h3 histones into less hydrophobic (lhp) and more hydrophobic (mhp) variants [ 851. when these two requirements are met, histones may be fractionated and recovered in greater than 95% yield. although the cn column will not separate (mph)h2a and h4 components, gurley et al. [ 861 favor this column for analytical work because histones elute at lower organic solvent concentration and better sensitivity is obtained. histones are known to undergo post-translational modifications such as acetylation. using labelled histones it was found that rpc was ineffective in resolving histones varying in degree of modification [ 871. a variety of cis, cs, and cn silica-based columns and the polystyrene-divinylbenzene-based prp-1 resin column were examined in the fractionation of histone mixtures [ 881. as in the case of cytosolic proteins, not all columns of the same type of bonded phase were equally effective. resolution of the (lhp) h2a, (mph) h2a, and h4 triplet seems to be the most sensitive (fig. 7) . in fact, there were even large interlot differences between columns. the exact reason for these variations between columns has not been determined. although of comparable selectivity, the prp-1 column gave poorer resolution of histones than the silica-based columns. resolution of the ( mhp) h2a and h4 doublet is also sensitive to mobile phase composition [89] . at 0.1% tfa there was little resolution of the doublet on any of the columns tested. however, at 0.3% tfa it was possible to resolve this pair on the radial-pak pbondapak cl8 column. although increasing tfa concentration increased histone retention on all columns, the retention difference between (mph) h2a and h4 increased to the point that they were resolved at 0.3% tfa. using a cn column and perchlorate-extracted proteins from the chromatin of chinese hamster cells, it has been possible to isolate two hl histone variants (hl and hl') and the high-mobility group (hmg) non-histone proteins. additional preparative studies on drosophila h2a histones produced a drosophila-specific d2 histone [ 901. through the use of column switching, these studies have shown that it is now possible to fractionate the total nuclear proteins of whole nuclei in one step. this will substantially diminish quantitation problems with small samples and aid in studies correlating histone concentrations with cellular changes. technically there are two types of lipoproteins: (1) those 'in which the lipid is covalently bonded to the protein and (2) those in which the lipid and protein form a non-covalently associating complex. there is only a single reference to aminoacylated peptides and proteins in which the acyl group (s) are long-chain fatty acids [ 911. in contrast, there is abundant literature on the separation of lipoproteins. excellent review articles on the separation of plasma lipoproteins [ 921 and apolipoproteins (apo) [ 931 have appeared recently. the function of the plasma lipoproteins is to transport phospholipids, neutral lipids, and cholesterol esters. since the density of triglyceride (tg) is lower than that of protein, the density of lipoproteins decreases as they are loaded with tg. this is the basis for classifying plasma lipoproteins. high-density lipoproteins ( hdls) contain approximately 3% tg and are of the highest density. the low-density lipoproteins ( ldls) are of intermediate density with 10% tg while the very-low-density lipoproteins (vldls) contain 60% tg. in addition to the lipoproteins, plasma contains chylomicrons. chylomicrons are particles of approximately 96% tg with a thin layer of protein coating the outer surface. the fact that the concentration ratio of lipoproteins in plasma may be related to atherosclerosis is the reason that so much work has been done on the separation of lipoproteins and determination of their lipid composition. the hplc analysis of serum lipoproteins is based primarily on sec and selective detection of lipids [ 921. at the present time all of the work reported has been done on the tsk columns of the polymer-based (pw) and silica-based (sw) series. the molecular mass range of chylomicrons and lipoproteins in a human serum sample is such that tandem sec columns, often of different pore sizes, must be used to gain sufficient resolution of the lipoprotein classes for quantitation [94-971. for example, three peaks are obtained on the g5000pw + g3000sw + g3000sw three-column set. chylomicrons and vldls elute first as a mixure followed by ldls in the second peak and hdlz and hdl3 in a partially resolved third peak. the g5000pw column alone is capable of partial resolution of chylomicrons, vldls and, ldls but incapable of resolving hdl2 from hdlb. in contrast, coelution of the first three components is experienced on the g3000sw columns with partial resolution of the hdl species. the three-column set noted above is a compromise to achieve the best, albeit incomplete, resolution of the mixture. lipoprotein detection was based on am while simultaneous selective detection of either total cholesterol, triacylglycerides, or phospholipids was achieved in a set of post-column enzymatic reactions. for example, total cholesterol was determined by mixing the column effluent with cholesterol e&erase, cholesterol oxidase, a substrate dye for peroxidase, and peroxidase. this mixture was then passed through a 20 000 x 0.4 mm ptfe reactor and the colored product determined at ass5 [ 981. triacylglycerides were determined in much the same way except that the glycerides were hydrolyzed with lipoprotein lipase and the glycerol oxidized with glycerol oxidase [ 991. hydrogen peroxide was determined by peroxidase, as above. choline-containing phospholipids were determined by mixing the effluent with phospholipase d and choline oxidase. hydrogen peroxide was determined with the peroxidase system noted in both cases above [ 100, 1011. excellent correlation was seen between these flow reactors and the more conventional techniques for determining lipid components [ 921. the lipid-free forms of serum lipoproteins are termed apolipoproteins. the apolipoproteins apoa-i and apoa-ii are obtained by the delipidation of hdls. in contrast, apoc-i, apoc-ii, and apoc-iii are produced by delipidation of either hdls or vldls. both the 28kd apoa-i and 17-kd apoa-ii have been purified by either sec in 6 m urea [ 1021, anion-exchange chromatography in 6 m urea [ 1031, or rpc using 1% triethylammonium phosphate at ph 3.2 [ 1041. with an extended gradient, the anion-exchange column shows that there are two isoforms of apoa-i. in contrast, the apoa-i isoforms cannot be resolved on an rpc column (fig. 8) [ 1051. the apoc apolipoproteins range from 6.6 kd (apoc-i) to 8.5 kd (apoa-ii) and 8.8 kd (apoa-iii). being smaller and more nearly the same molecular weight, sec is ineffective in their resolution. they may, however, be resolved both by anion-exchange chromatography in 6 m urea [ 1031 and rpc [ 1041. with the proper column and gradient conditions, apoc-iii was fractionated into three isoforms. there are two other major classes of apolipoproteins, apob and apoe. apob is the product of either vldl or ldl delipidation whereas apoe is derived from vldls. both apob and apoe have been chromatographed on sec columns in the presence of 4 m guanidinium hydrochloride [ 1061. it should be noted that the use of high concentrations of urea can lead to the carbamylation of lysine residues in a protein and the production of artificial isoforms [ 1071. if possible, this may be prevented by working at acidic conditions. it is common in nature to find different proteins which catalyze the same reaction. these different forms are often referred to as isoenzymes. three different types of isoenzymes will be discussed here: multimeric enzymes that vary in subunit composition; enzymes that are of the same primary structure but vary in degree of post-translational modification; and those that are of different primary structure. lactate dehydrogenase ( ldh) is a typical example of a multimeric isoenzyme. aggregation of the dissimilar 35&d [m] and [h] subunits into a 140-kd tetramer produces five isoenzyme species (ma, m3h, m2h2, mhb, and h4) which have been separated by high-performance anion-exchange chromatography [ 1081. with the addition of computer-aided data acquisition and analysis and a postcolumn reaction detector that allowed ldh activity to be monitored in the column effluent, it has been possible to build an automated high-performance anionexchange chromatographic system for the detection of myocardial infarction [ 109-1111. greater than 95% of the activity of all five isoenzymes was recovered on clinical serum samples with a 1% relative standard deviation. resolution apparently depends more on mobile phase ph than on the type of ion-exchange column. there was relatively little difference in resolution between weak and strong anion-exchange columns whereas increasing mobile phase ph from 7 to 8 increased resolution [ 1121. creatine phosphokinase (cpk) is a second isoenzyme system that has been used in monitoring myocardial infarction by high-performance anion-exchange chromatography [ lo&llo] . the dissimilar [m] and [b] subunits aggregate into a dimer to produce three cpk isoenzymes: mm,mb and bb.again a postcolumn reaction detector is used to monitor the activity of eluted enzymes and correlates elevations in the mb isoenzyme with myocardial infarction. a separation of cpk isoenzymes is shown in fig. 9 . hexokinase (hk) and alkaline phosphatase ( ap) isoenzymes from murine tissue extracts have also been separated by hplc [ 1131. weak anion-exchange chromatography resolved the pancreatic and duodenal forms of ap in the presence of 4 m urea. in the absence of urea, these isoenzymes eluted as an aggregate. it has recently been shown [ 1141 that the heterogeneity in duodenal ap is apparently due to differences in glycosylation. using a concanavalin a (con a) column, which discriminated on the basis of mannose content, two rat duodenal enzymes have been identified. it is probable that these isoenzymes are formed as a result of post-translational modification of the ap primary structure. canine plasma isoenzymes of ap have also been reported which can be separated by anion-exchange chromatography [ 1151. these enzymes differ in that one is corticosterone-inducible and stable to 65â°c while the other is more temperaturesensitive and non-inducible. malate dehydrogenase (mdh) occurs in almost all eucaryotic cells in mitochondrial ( m-mdh) and cytosolic (c-mdh ) forms. a rapid loo-fold purification of the beef heart mdh isoenzymes was achieved in a single pass through a strong cation-exchange column [ 1161. hic did equally well in a descending 30 to 0% ammonium sulfate gradient where c-mdh eluted first followed by m-mdh. additional successful applications of high.-performance anion-exchange chromatography in isoenzyme purification are the fractionation of b-n-acetylhexosaminidase from human liver into the a and b forms [ 1171, y-butyrobetaine hydroxylase from human kidney into three forms [ 1181 ,cyclic-amp-dependent protein kinase isoenzymes from rabbit gastric mucosa parietal cells into two forms [ 1191, and glutathione s-transferase (gst) from human heart, lung, and erythrocytes into a major and two minor isoforms [ 1201. although homogeneous by biospecific affinity chromatography and isoelectric focusing, the isoenzymes of gst apparently had sufficient charge asymmetry that they could be further fractionated by iec. glycoproteins are produced by post-translational modifications. as a consequence, they have a protein core to which various mono-and oligosaccharides ranging up to 50 units are attached in a stepwise manner at different positions in the primary structure. although glycoproteins may exist as a single pure species, it is common to find the same polypeptide with varying degrees of glycosylation. covalent coupling of a carbohydrate to the protein is either through an o-glycosidic linkage to serine or threonine or an n-glycosidic linkage to asparagine. as much as 50% of the mass of a glycoprotein may be carbohydrate. because of their contribution to chromatographic retention in iec, it is also important to note that glucosamine, galactosamine, and the sialic acids are constituents of glycoproteins. when glycosylation with these monosaccharides is incomplete, it means that a variety of charged species with the same polypeptide backbone will be found. there are many papers on the isolation of peptide cleavage fragments of glycoprotein by both sec and rpc. in view of the fact that this review is dealing with intact glycoprotein structure, papers on structure elucidation and the separation of cleavage fragments will not be examined. since so little work has been done on the retention of glycoproteins in the various modes of chromatography, a discussion of column selection must be based on some speculation. as a beginning, it can be said that column selection depends to a large extent on the objective of the research. when the objective is to study the protein portion of the glycoprotein, a technique should be chosen that shows little discrimination among all of the possible carbohydrate moieties coupled to the polypeptide. in contrast, when the objective is to study either glycosylation or the structure of the fully assembled glycoprotein or even perhaps the fate of a glycoprotein in the biological system, a technique should be chosen that gives maximum discrimination between all of the components of the glycoprotein. when the objective of the separation is to discriminate among differences in glycosylation, rpc should be chosen. unfortunately there are little data to support this conclusion except common logic and some work on glycopeptides. the retention behavior of glycosylated and non-glycosylated peptides of the same amino acid sequence showed very similar behavior in rpc [ 1211. however, glycosylated peptides eluted slightly earlier. when the amount of glycosylation was the same between two peptides, the one with multiple sites of glycosylation eluted first. this is to be expected. retention of polypeptides in rpc is based on the hydrophobic contact area between the solute and alkyl ligands on the sorbent [ 1221. hydrophilic portions of a molecule are directed outward toward the solution and do not participate directly in the adsorption process. their contribution is simply to decrease the partition coefficient by making the compound more soluble. differentiating among variations in the carboyhydrate portion of a glycoprotein is much more difficult. when the differences are the result of either steric shielding of charged groups or the addition or deletion of a charged species, i.e. an amino sugar or sialic acid, ion-exchange columns will be useful. for example, multiple glycosylated species of hemoglobin ( hba1,, hbai,,, hba1,, and hbaj and some antibodies can all be separated by iec [ 631. however, as the degree of complexity in the glycoprotein increases through the introduction of charge in multiple-oligosaccharide chains, heterogeneity of charge within any one oligosaccharide, and steric shielding of charge in the polypeptide backbone by the carbohydrate, the ability of the ion-exchange system to discriminate among these myriad species will be overwhelmed. it was noted above in the discussion of the fractionation of glycosylated immunoglobulins that hydroxyapatite columns provide selectivity that could not be obtained with ion-exchange columns [ 811. if any part of this discrimination was due to the carbohydrate portion of the molecule, then hydroxyapatite would be useful in the fractionation of glycoproteins when the objective is to discriminate between different carbohydrate moieties on the polypeptide. biospecific affinity chromatography of glycoproteins on lectin columns is another technique for discriminating between carbohydrate groups in a glycoprotein. for example, al-antitrypsin (cu,-protease inhibitor) has two major and three minor fractions with isolectric points between 4.4 and 4.7. the molecule consists of a single polypeptide chain with 394 amino acid residues and three carbohydrate chains, giving a relative molecular mass of 51000. con a-sepharose separates (fig. 10 ) these proteins into three fractions based on the mannose content of the oligosaccharides [ 1231. future use of lectin columns for the fractionation of glycoproteins will probably increase. the discussion above would indicate that hplc has attained a secure position in the arsenal of techniques available to the protein chemist for the purification and characterization of proteins. as in the past, further developments in the field will depend very much on the needs of both the life science research community and the biotechnology industry. at the present time there are several trends in protein chemistry and biotechnology which will have an impact on developments in hplc. protein characterization on an ever diminishing scale will continue to drive the development of microvolume columns and the equipment needed to drive them in the gradient elution mode. the fact that microvolume columns contain much less sorbent allows higher recovery of small samples and provides less sample dilution. diminished sample dilution is important because it enhances both detection sensitivity and solute recovery. continuing effort must also be made in eliminating non-specific adsorption on column frits and other surfaces in the instrument system itself. proteins synthesized in genetically engineered organisms are often contaminated with structural variants that result from expression errors during synthesis, incomplete post-translational modification, or chemical modification during isolation. these variants may be very similar in structure to the native species and be present in small quantities. the possibility that variant forms of a therapeutic protein could be harmful to a person during long-term consumption requires the development of systems for monitoring and eliminating non-native proteins from therapeutic materials. at the present time our understanding of the ability of liquid chromatography (lc) systems to discriminate between similar proteins is incomplete. this is based largely on a lack of understanding of the mechanism by which proteins are adsorbed at surfaces. much more data must be collected on the ability of a series of different types of lc columns to discriminate among mutant proteins before it will be possible to assess the utility of lc in this difficult task and to design higher-resolution separation systems. the scale-up in protein production initiated by biotechnology has had an impact on preparative separations. there is a need for larger systems that are economical and of high resolving power and throughput. it would seem that the most logical way to design preparative systems is to take all of the concepts and materials used so successfully in analytical separations and simply make a much larger analytical system. this approach will be very satisfactory if the columns are not overloaded and economics is not a serious question. however, when overloading and economics are inserted into the equation much of what we know in analytical separations begins to fail. questions of loading capacity, economics, and the kinetics of loading large quantities of solute were never considered when designing the current generation of high-performance sorbents. there is a strong possibility that a generation of true preparative packing materials will emerge in the next five years. it is likely that these materials will be mechanically stable to more than 100 bar, have 1.5 to 2 times the loading capacity of analytical materials, be .available at 20% of the cost of current hplc sorbents, have 'a dynamic loading capacity that is equal to 70% of that obtainable in the static mode, have good chemical stability from ph 2 to 11, and be easily cleaned after they have been fouled with protein. much more study will be devoted to understanding the chromatographic process in the overloaded state. an appreciation of displacement effects and even the use of displacement chromatography on the process scale is a distinct possibility, particularly with smaller $olypeptides. there is sufficient difference between preparative and analytical chromatography of proteins so that, with time,they will evolve as separate, but related fields, the large-scale commercialization of protein production requires that much more rapid and specific methods be developed for monitoring product purity at all stages of the purification process, monitoring the composition and purity of raw materials used in the manufacturing process, and circumventing lengthy biological assays. it is in these very-high-speed and specific systems that bioaffinity materials such as immunosorbents and receptor protein sorbents will be extremely useful. it is also likely that through the use of non-porous sorbents it will be possible to obtain the complete chromatographic analysis of a mixture in less than 30 s. since most of these separations require the generation of a gradient, this capability must be incorporated into new lc instruments. this review has shown that a variety of chromatographic techniques are available for fractionating proteins. fortunately, high-quality columns of every type described in this review are commercially available. most water-soluble proteins may be eluted from size-exclusion, hydrophobic-interaction, ion-exchange, metal chelate, and bioaffinity columns with ease. when this is the case, high recovery and retention of biological activity are the norm. the exception is reversed-phase chromatography where the organic solvents and acids used in polypeptide elution denature many proteins. when problems do occur, they are generally the result of unique structural features of the protein. very hydrophobic proteins have presented the biggest problem in that they are difficult to solubilize, particularly with retention of biological activity. it has been found that zwitterionic and non-ionic detergents are the most suitable solubilizing agents, but urea has also been used in cases where hydrophobic interacts are not as strong. unfortunately, there is still an element of trial-and-error in selecting the most suitable solublizing agent. heterogeneous glycosylation of proteins also presents a problem. both neutral and charged monosaccharides can be incorporated into proteins through multiple steps at several sites. thus, there is the potential in a sample for a large number of glycoprotein species which have the same polypeptide backbone and differing amounts of oligosaccharide. a problem arises when size-exclusion, ion-exchange, hydrophobic-interaction, reversed-phase and bioaffinity systems begin to discriminate between these very similar glycoprotein species. chromatographic peaks can become very broad, due to incomplete fractionation, and the polypeptide chain of interest can be associated with multiple peaks. the separation of glycoproteins requires much more study before logical procedures can be suggested for column selection and operation. aggregated species are another class of proteins which present occasional problems. multimeric proteins are adsorbed to sorbents by a series of forces, among which are hydrogen bonding, hydrophobic interactions, and electrostatic forces. these forces are also responsible for the maintenance of quaternary structure in proteins. when the same forces dominate both retention of protein structure and adsorption at the sorbent surface, the quaternary structure of the protein-can be disrupted during elution. very basic proteins also present a problem in some cases. columns with residual negative charges, such as a silica-based reversed-phase column, adsorb anionic species so strongly that they are difficult to elute. this is the case with ribosomal and nucleoproteins. the solution is to use exhaustively end-capped columns which diminish electrostatic interactions. receptor biochemistry and methodology key: cord-258489-pyfc7jde authors: lico, chiara; chen, qiang; santi, luca title: viral vectors for production of recombinant proteins in plants date: 2008-03-10 journal: j cell physiol doi: 10.1002/jcp.21423 sha: doc_id: 258489 cord_uid: pyfc7jde global demand for recombinant proteins has steadily accelerated for the last 20 years. these recombinant proteins have a wide range of important applications, including vaccines and therapeutics for human and animal health, industrial enzymes, new materials and components of novel nano‐particles for various applications. the majority of recombinant proteins are produced by traditional biological “factories,” that is, predominantly mammalian and microbial cell cultures along with yeast and insect cells. however, these traditional technologies cannot satisfy the increasing market demand due to prohibitive capital investment requirements. during the last two decades, plants have been under intensive investigation to provide an alternative system for cost‐effective, highly scalable, and safe production of recombinant proteins. although the genetic engineering of plant viral vectors for heterologous gene expression can be dated back to the early 1980s, recent understanding of plant virology and technical progress in molecular biology have allowed for significant improvements and fine tuning of these vectors. these breakthroughs enable the flourishing of a variety of new viral‐based expression systems and their wide application by academic and industry groups. in this review, we describe the principal plant viral‐based production strategies and the latest plant viral expression systems, with a particular focus on the variety of proteins produced and their applications. we will summarize the recent progress in the downstream processing of plant materials for efficient extraction and purification of recombinant proteins. j. cell. physiol. 216: 366–377, 2008. © 2008 wiley‐liss, inc. recombinant dna technology was initially used to express proteins that were difficult to produce in their native organisms. increasing efforts, however, have been focused on designing new molecules with more desirable characteristics and/or functionality. pharmaceuticals and industrial enzymes were the first recombinant biotech products on the world market and biopharmaceuticals were the majority of commercialized recombinant proteins (pavlou and reichert, 2004) . many protein-based drugs, similar to traditional small molecule pharmaceuticals, function as antagonists by binding to and thereby inhibiting the activity of their target, such as an enzyme or a receptor. classical protein antagonists include full monoclonal antibodies (mabs), their single-chain derivatives (scfv) and mab-fusion proteins. recent research programs have also focused on non-antibody antagonists that consist of a scaffold protein displaying the inserted affinity peptide (walsh, 2006) . recombinant dna technology also provided an excellent alternative for developing safer vaccines. subunit vaccines are based on immunodominant protein components of a pathogen, but do not contain its genetic material. consequently they cannot replicate, cause disease, or introduce pathogens into non-endemic regions. viral coat proteins are exceptional subunit vaccine candidates and in some cases are able to form virus-like particles (vlps) when expressed in heterologous systems. in fact, the only recombinant subunit vaccines presently available are based on vlps. they are highly immunogenic and able to induce both humoral and cellular responses (chackerian, 2007) . in addition to the pharmaceutical industry, many other fields are also relying intensely on recombinant proteins. areas as diverse as agro-food technology, chemistry, detergent production, bioremediation, biosensoring, petroleum, and paper industries all receive significant contribution from applications of recombinant proteins. for example, increasing needs for a diversity of food processing enzymes, for example, amylase, lipase, xylanase, pullulanase and pectin modifying enzymes, demand a substantial involvement of recombinant protein technology (olempska-beer et al., 2006) . in the coming years, there will be a significant increase in demand for high quality recombinant proteins. in response, biological systems used for the production of proteins must be scalable, cost-effective, safe and flexible enough to meet market requirements. current systems rely on ''bio-factories,'' that is, mammalian, insect, yeast, and microbial cell cultures. the majority of the recombinant proteins are currently produced in escherichia coli or mammalian cells with a few exceptions of yeast or insect cells (yin et al., 2007) . all of these bio-factories are based on fermentation technology of suspension cells in bioreactors, which requires an enormous upfront capital investment and, thereby, severely constrains their scalability. the use of plants as production systems for recombinant proteins has been actively investigated over the last two decades. plants are attractive as protein factories because they can produce large volumes of products efficiently and sustainably and, under certain conditions, can have significant advantages in decreasing manufacturing costs (hood et al., 1999; giddings, 2001) . plant systems are far less likely to harbor microbes pathogenic to humans than mammalian cells or whole transgenic animal systems. in addition, one of the major advantages of plants is that they possess an endomembrane system and secretory pathway that are similar to mammalian cells (vitale and pedrazzini, 2005) . thus, proteins are generally efficiently assembled with appropriate post-translational modifications. these cost, scale, and safety advantages make plant-made pharmaceuticals very promising for both commercial pharmaceutical production and for manufacturing products destined for the developing world. three approaches are commonly used to express heterologous proteins in plants: (1) stable transformation of the nuclear genome, (2) stable transformation of the chloroplastic genome, and (3) viral transient transformation. in stable transformation technology, an expression cassette harboring the exogenous gene of interest is integrated into the nuclear or plastid genome of plant cells, which results in the acquired character to be stably inherited over generations. these lines can be propagated vegetatively or by seed and thus, readily scaled up for protein production. stable nuclear transformation is often achieved using agrobacterium tumefaciens, which delivers fragments of dna into the plant cell nucleus at random positions. alternatively, the ''biolistic'' method (microprojectile bombardment) can be used for plant hosts that are difficult to transform by agrobacterium (hansen and wright, 1999) . due to their double membrane structure, chloroplast transformation can be achieved only by bombardment with tungsten or gold particles coated with fragments of dna. stable transformation of the chloroplast offers several distinct advantages in areas of transgene targeting, product yield, and regulatory compliance. since the chloroplast genome allows for homologous recombination, transgenes can be precisely targeted to a specific locus of the genome to avoiding positional effect or accidental gene knock-outs. each plant cell has several chloroplasts and each of these contains many circular genomes. therefore, the shear copy number of the transgene enables high-level expression of the target recombinant protein. since the chloroplast is inherited maternally, this technology reduces the risk of potential transgene escape by pollen dissemination. similar to bacterial cells, however, the chloroplast is unable to perform typical eukaryotic posttranslational modifications, such as glycosylation. as a result, this technology cannot be used to produce proteins when such modifications are essential for their function (bock, 2007) . the third strategy relies on replicating plant viruses. these viruses are small, can be easily manipulated, and their infection process is relatively simple. the above features make viral vectors an attractive alternative to stable transgenic systems for the expression of foreign proteins in plants. in this strategy, the gene of interest is inserted among viral replicating elements, episomically amplified, and subsequently translated in the plant cell cytosol. most of the transient expression systems are based on non-food, non-feed plants like tobacco, therefore, requiring further purification prior to application. production of recombinant proteins with stably or transiently transformed plants has been performed both in green-houses and in field. neither practice requires large investments in hardware or culture media, thus making scale-up more economical than fermentation cultures. the possibility of producing recombinant protein agents on an agricultural scale by ''molecular farming'' is extremely attractive. although the use of plants as expression systems for recombinant proteins was first conceived for oral delivery of antigenic proteins (walmsley and arntzen, 2000) , plant-made recombinant proteins have also been purified and used in many different applications. an enormous number of proteins different in size, structure, origin and biological function have been successfully expressed in stably transformed monocot and dicot plants such as maize (chikwamba et al., 2003) , rice (nochi et al., 2007) , wheat (brereton et al., 2007) , potato (youm et al., 2007) , tomato (huang et al., 2005) , tobacco (watson et al., 2004) , lettuce (sun et al., 2006) , alfalfa , lupin (smart et al., 2003) , carrots (marquet-blouin et al., 2003) , barley (joensuu et al., 2006) , soybean (moravec et al., 2007) , and thale cress (carrillo et al., 1998) . many antigenic monomeric proteins from viruses (webster et al., 2006) and prokaryotes (alvarez et al., 2006) have been produced and in a few cases, their immunogenic properties have been successfully evaluated in human volunteers during phase i clinical studies (tacket et al., 1998 (tacket et al., , 2000 thanavala et al., 2005) . complex oligomeric mabs were first expressed in stable transgenic plants by hiatt et al. (1989) . since then, the number and type of plant-expressed antibodies, such as secretory antibodies, antibody fragments and, more recently, immune complexes, have increased steadily. several commercial candidates of mabs have been developed, with five mabs under clinical evaluation, and two having reached phase ii clinical studies . in addition to pharmaceuticals, recombinant proteins for a variety of applications have also been expressed in transgenic plants. examples include alpha-amylase enzyme (pen et al., 1992) , the protein brazzein for commercial sweeteners (lamphear et al., 2005) , and the fungal enzyme manganese peroxidase (clough et al., 2006) . moreover the chicken avidin diagnostic protein (hood et al., 1999) and the bovine trypsin enzyme (woodard et al., 2003) from transgenic maize, and the human anti-microbic proteins lysozyme and lactoferrin (humphrey et al., 2002) from transgenic rice have been commercially produced and are currently marketed by sigma chemical company (st. louis, mo). the major challenges facing transgenic plant technology lay in increasing the quantity of protein, the optimization of expression systems, and improvement of downstream processing. in this review, we will focus on transient production strategies using plant viral expression systems, with a particular focus on the variety of proteins produced, and their applications. we will also discuss the latest developments in the downstream processing of plant materials for efficient extraction and purification of recombinant proteins. the history of genetic engineering and applied virology are intimately connected; the first recombinant molecule assembled was a chimeric sv40 containing genes from the bacteriophage l (jackson et al., 1972) . the unique properties of viruses such as ease of manipulation, high level amplification, site specific recombination, strong infectivity, enhanced translation and compact and repetitive morphological structure have enabled their broad application, from basic research to product development, including the generation of robust expression systems. viruses with a variety of host ranges such as bacteriophages, mammalian retroviruses, invertebrate infecting baculoviruses, and plant viruses have been genetically modified to express heterologous proteins. from the discovery of viruses in 1898 (tobacco mosaic virus, tmv) (bos, 1999) , to the first demonstration of rnas role in virus replication by turnip yellow mosaic virus (tymv) (matthews, 1989) , to the very recent discovery of gene silencing and its implication in host response to infection, gene regulation and transgene expression (baulcombe, 1999; lu et al., 2003; waterhouse and helliwell, 2003) , plant virology has played a crucial role in the understanding of the most fundamental concepts of modern biology. in addition, plant viral elements such as promoters, terminators, translational enhancers and various cis-regulatory sequences have been extensively used in plant biotechnology. plant viruses have been used to introduce foreign genes in plants since the early 1980s and technical advances in molecular biology and plant virology have allowed the generation of many improved expression systems. these recent systems provide many advantages including rapid, high-level transgene expression, and, in the case of movement defective systems, better transgene containment due to the lack of vertical and horizontal gene transfer. the earlier plant virus expression systems were based on the cauliflower mosaic virus (camv) of the caulimoviridae family, the only plant viruses with a double stranded dna genome. however, limited packaging capacity and the restricted amount of viral dna that can be removed without affecting essential functions hindered the application of these initial expression systems. fortunately, technical advances in molecular biology such as generating cdna from an rna template have enabled us to expand our search for expression systems into single stranded rna viruses, the most represented types of plant viruses in nature. another technical breakthrough was the use of agrobacterium tumefaciens to promote viral infection (grimsley et al., 1986; turpen et al., 1993) . generally, viral infection can be initiated by mechanical inoculation of infectious viral particles on leaves or alternatively by transfection of nucleic acids. the use of agrobacterium tumefaciens, known as ''agroinfection,'' allows the direct and efficient targeting of cdna to the plant cell nucleus (fig. 1) . the cdna constructs in the plant expression cassette would result in an infectious, autonomously replicating nucleic acid after host cell nuclear transcription and processing. moreover agroinfection allowed the use of viruses that in nature are not mechanically transmissible, but instead need a special insect vector to initiate the infection process. these viral vectors are commonly divided into gene substitution vectors, gene insertion vectors, modular or deconstructed vector systems and peptide display vectors. gene substitution or replacement vectors are based on the exchange of an endogenous viral sequence with a heterologous gene of interest. the first successful expression of a foreign gene ever achieved in plants was obtained using a substitution vector based on the camv. usually, the coat protein (cp) gene is the viral coding sequence of choice to be replaced. however, with a few exceptions, the cp is essential for cell to cell and/or systemic movement and infection. however, caution has to be taken if leaves of the whole plants are intended to be infected. for example, a cp replacement vector was developed using the tomato bushy stunt virus (tbsv). in this case the cp is not essential for systemic movement of the virus in certain species of nicotiana (scholthof et al., 1993) and it was indeed demonstrated that the insertion of a heterologous gene supports a systemic infection (scholthof et al., 1993) . in tmv-based substitution vectors, where the cp is necessary for systemic infection, a chloramphenicol acetyl transferase (cat)-cp gene replacement vector generated only local lesions and cat activity was confined on the inoculated leaf (takamatsu et al., 1987) . in another recently developed tmv-based gene substitution vector the cp gene was exchanged with the gene of interest fused to a thermostable carrier molecule, the beta-1,3-1,4-glucanase enzyme (lichenase) of clostridium thermocellum, to facilitate target expression, stability and purification. the heat tolerant property of the lichenase (658c) allows easy target proteins purification by heat treatment which precipitates up to 50% of contaminating plant proteins. the protein of interest, fused at the n-or c-terminus of lichenase, or inserted in the central loop of an engineered version of the enzyme, is protected from the degradation and thus purified. gene insertion vectors consist of complete functional viruses with the addition of an extra open reading frame (orf) for the target protein. they are capable of cell to cell and systemic movement and infection. viruses with both spherical and rod shaped virions have been investigated. viruses with rod-shaped particles have a better potential due to fewer constraints on the amount of nucleic acid inserted. however, there is still a limit on the genome size of the chimeric rod-shaped virion based vectors. it has been proven that chimeric vectors with genome size beyond certain limits resulted in unsustained virion assembly. two of the most famous gene insertion vectors have been derived from tmv and potato virus x (pvx). both viruses have a single stranded positive rna genome. they use subgenomic promoters and consequently subgenomic rnas to express some of their orfs and have a helical virion symmetry which results in rod-shaped particles. a chimeric tmv was constructed with the cat gene inserted between the movement protein (mp) gene and the cp gene; the expression was regulated by an additional copy of the subgenomic promoter of the cp gene that, as a result, was duplicated in the hybrid vector. the vector was able to replicate, to form subgenomic rnas, to assemble correctly, and to produce reporter gene activity. nevertheless the duplication of the subgenomic promoter sustained homologous recombination causing instability and the consequent loss of the exogenous gene (dawson et al., 1989) . this problem was solved by using a subgenomic cp promoter derived from a different virus belonging to the tobamovirus genus to prevent homologous recombination. similar problems and solutions of recombination between duplicated subgenomic promoters were reported in a recent insertion vector system based on the grapevine virus a (gva) for n. benthamiana (haviv et al., 2006) . analogous to tmv, a pvx-based vector was generated using the duplicated promoter of the cp gene. however in this case the vector was stable and able to retain the additional coding sequence (chapman et al., 1992) . nevertheless, recently it has been demonstrated that the size of the inserted gene of interest can play a crucial role in the stability of the modified pvx expression vector, with a positive correlation between the elimination rate of the gene of interest and its length (avesani et al., 2007) . recombination can involve homologous cp promoter sequences but also other mechanisms mediated by both the host plant and the virus (avesani et al., 2007) . another well established system is based on cowpea mosaic virus (cpmv) the type member of comoviridae whose bipartite genome is constituted by single stranded positive rna molecules. rna-1 encodes for proteins involved in replication while rna-2 carries the genetic information for the movement and structural proteins. the system is very versatile; it can be utilized in a totally transient fashion, co-infiltrating wild-type plants with two separate c-dna constructs representing rna-1 to sustain amplification and rna-2 harboring the gene of interest, or it can be utilized combining transgenic plants with virus-mediated transient expression. in this case the rna-2 is used for stable transformation and the rna-1, necessary to prime the active replication of rna-2 and of the heterologous gene, is supplemented by agro-infiltration. it is worth noticing that a deleted version of rna-2, unable to produce infectious clones, it has also been utilized to prevent possible environmental containment concerns (liu et al., 2005; sainsbury et al., 2008) . in conclusion it is possible to obtain an inducible expression system if rna-1 is supplemented by agroinfiltration and a constitutive stable expression system if it is supplemented by crossing with an rna-1 stable transgenic line (canizares et al., 2006) . modular or deconstructed vectors are a new generation of viral expression systems. their development was driven by the understanding that: (1) not all viral components are essential or beneficial for an expression vector and (2) viruses can be broken down into different genomic elements that would still operate together in the infection process as wild-type multipartite genome viruses. moreover, agroinfection provided the technical possibility to co-deliver multiple different components. hence in modular systems, viral components are separated into distinct portions and inserted into binary vectors contained in an agrobacterium strain. the strains are mixed together and co-infiltrated into plant leaves. this strategy allows the replicative portion, the so called replicon, to be reduced in size, down to minimal to accommodate the insertion of transgene, while other viral components necessary for the vector function can be provided in trans during the infection process. at present, one of the most famous and broadly used vector is the deconstructed system based on tmv and developed by icon genetics (halle, germany), recently acquired by bayer innovation gmbh. the vector has been engineered to divide the tmv genome into two major cdna modules: a 5 0 module which contains the viral rna dependent rna polymerase and the mp, and a 3 0 module that carries the gene of interest and the 3 0 untranslated region (utr) of the virus essential for the efficient replication and amplification of the vector. in this particular case, viral functions are not complemented in trans but the two modules actually assemble together in vivo by a site specific recombinase delivered by a third agrobacterium cell line. furthermore, different 5 0 modules carry different organelle targeting signals which, fuse in frame with the gene of interest after recombination and nuclear processing, allow a single construct to be combined pair wise with various targeting elements in separate constructs. this feature greatly facilitates the experimental procedure to test optimal expression in terms of accumulation in different sub-cellular compartments. an additional attribute of the vector, that permitted a dramatic enhancement of expression levels, was the introduction of several introns in the coding sequences of the 5 0 module (marillonnet et al., 2004 . levels of expression are impressive and can reach up to 5 mg of recombinant protein per gram of fresh weight (fw). the system was extensively tested with different genes, coding for antibodies and antibody-derivatives, interferons, growth hormones, bacterial and viral antigens, adjuvants and enzymes , confirming each time its versatility and robustness. another interesting modular system was developed using the bean yellow dwarf virus (beydv) of the geminiviridae family. geminivirus are single stranded circular dna viruses known to replicate through the rolling circle process, promoting high level of genome amplification in the nucleus of the infected cell. the beydv based system is depleted of the mp and cp gene and consequently the expression of the protein of interest is confined to the inoculation area. the basic version involves two modules, one containing the gene of interest inserted between the beydv long and short intragenic regions, lir and sir respectively. both intragenic regions are necessary to sustain rolling circle replication. the gene of interest is driven by a strong plant specific constitutive promoter. the second module is responsible for the expression of the rep protein which catalyzes various aspects of the rolling circle replication. initially, the system was used as an expression vector in tobacco plant cell culture (mor et al., 2003) but its use has recently been extended to whole leaves with agroinfection. the modular nature of this system also permits the co-expression of a variety of sequences of different viral or non-viral origin that are beneficial for replicon amplification and transgene expression. for example, genes of post-transcriptional gene silencing (ptgs) inhibiting proteins can be co-expressed to prevent ptgs and hence enhance target protein accumulation. display viral vectors have been extensively used to provide a molecular scaffolding for different kinds of peptides to be fused with the viral cp and, therefore, to be exposed and displayed on the surface of chimeric virus particles (cvps). coat protein genes of several plant viruses were genetically modified to support fusions with coding sequences of heterologous peptides in specific regions known to be well exposed on the virion surface (johnson et al., 1997; porta and lomonossoff, 1998) . therefore, the virus and the plant host would provide the expression system to produce large quantities of the chimeric fusion protein. clearly this is only feasible for well characterized viruses in terms of assembly, movement strategies and structure. fusions must be compatible with the normal assembly and viral fitness, and must avoid any possible steric hindrance or interference with virus movement. defining peptide features for their correct display on cvp is of particular interest among studies in this area (bendahmane et al., 1999; porta et al., 2003; lico et al., 2006) . two strategies have been developed to overcome assembly problems for long or difficult peptides. one strategy is to modify the 3 0 terminus of the cp gene so that the cp stop codon would function in a leaky fashion. this would cause most of the cp produced to be unfused, while a small portion would be fused to the peptide displayed on the surface (hamamoto et al., 1993; sugiyama et al., 1995; borovsky et al., 2006) . the other strategy employs the 2a peptide of the small foot and mouth disease virus (fmdv). this sequence, inserted between the epitope coding sequence and the 5 0 terminus of the cp gene, produces a ribosomal skip during the translational procedure (donnelly et al., 2001; de felipe et al., 2003) , with the result that only a small portion of the translational products will consist in a fusion between the heterologous sequence and the cp (fig. 2) . the use of these strategies, however, opens the question of the precise dose of the antigen in the vaccine formulation. many plant viruses have been used to display different kinds of peptides. the most widely used viruses for this purpose are the cpmv, the tmv and the pvx. extensive research regarding the symmetry, shape and structure at atomic level for these viruses have allowed the precise definition of specific sites for peptide insertion to ensure the successful display of fusion peptides (klug and caspar, 1960; lomonossoff and johnson, 1991; baratova et al., 1992a,b; johnson et al., 1997; parker et al., 2002) . major efforts are now directed to the development of new viral vector typologies in terms of both improvement of the expression strategies and virus choice. host plants have also been extended to a wider variety of species including herbaceous plants and cucurbitaceous family. besides the viruses mentioned above, other viruses have also been investigated as useful vectors, such as tomato mosaic virus (tomv) (dohi et al., 2006) ; maize streak virus (msv) (palmer and rybicki, 2001) ; bean pod mottle virus (bpmv) (zhang and ghabrial, 2006) ; beet necrotic yellow vein virus (bnyvv) (schmidlin et al., 2005) ; tomato golden mosaic virus (tgmv) (hayes et al., 1989) ; potato virus a (pva) (kelloniemi et al., 2006) ; zucchini yellow mosaic virus (zymv) (hsu et al., 2004) ; cucumber mosaic virus (cmv) (nuzzaci et al., 2007) ; and cucumber green mottle mosaic virus (cgmmv) (ooi et al., 2006) . antibodies are key molecules of the vertebrates' immune system. they are responsible for the recognition and binding of target antigens with high affinity and specificity. monoclonal antibodies (mabs) are used in a wide range of applications, including diagnosis, prevention and treatment of many diseases. for example, they are being successfully utilized as ''targeting vectors'' to direct drugs, radioisotopes and other therapeutic molecules to their target cells or tissues, as immunomodulators in pathology-related functions, as well as diagnostic tools to identify and localize molecular alterations. in addition to their pharmaceutical applications, mabs can also be used to modulate plant traits, generate disease or pest resistant crops (tavladoraki et al., 1993; nolke et al., 2004; peschen et al., 2004; villani et al., 2005) , and to study plant metabolic pathways (nolke et al., 2005) . antibodies are complex glycoproteins consisting of four polypeptides, linked by disulfide bridges and non-covalent bonds. despite their complexity, mabs and their derivatives can be easily expressed in heterologous systems including plants . however, mabs produced by heterologous systems may not share the precise structural and functional properties of the native molecules. for example, mabs produced by a bacterial expression system will lack post-translational modifications including glycosylation. non-mammalian eukaryotic expression systems could fail to use the appropriate glycans groups resulting in different glycosylation patterns. yeasts, for example, tend to hyperglycosylate (harashima, 1994; malissard et al., 1996) ; while plants preferentially introduce xylose and fucose residues and fail to use galactose and syalic acid (gomord and faye, 2004) . although proper glycosylation is required for functions of many mabs, it is to be noted that different glycosylation patterns do not always lead to loss of function in vitro or in vivo, or cause side-effects in humans (chargelegue et al., 2000) . different strategies have been adopted to circumvent these limitations such as targeting mabs to the endoplasmic reticulum, or abolishing glycosylation sites by mutagenesis to avoid inappropriate glycosylated products. in fact, it has been reported that the absence of glycans in some cases did not interfere with correct assembly and activity (rodriguez et al., 2005; nuttall et al., 2005) . in addition a fascinating approach that requires sophisticated genetic and metabolic engineering resides in the inactivation of endogenous glycosyltransferases and/or expression of heterologous mammalian specific enzymes in the host plant system (saint-joredupas et al., 2007) . complete antibodies and their derivatives have been produced with stable plant transformation technologies . thanks to the recent improvements of viral-based vectors, mabs have been produced with transient expression systems to quickly achieve much higher production levels along with other complex proteins. single chain fragments, that preserve antigen-recognition elements of the full length immunoglobulin, are often more effective as drugs or diagnostic tools than mabs due to their increased ability to penetrate target tissues, reduced immunogenicity, and more rapid clearance. a chimeric gene insertion tmv based vector was used for the expression of a human scfv for treatment of non-hodgkin's lymphoma (nhl) (mccormick et al., 1999 (mccormick et al., , 2003 . the plant-derived scfv generated an antibody response in vaccinated animals, and was an effective vaccine in a murine nhl tumor challenge model. the tmv strategy provides the speed and versatility required for the development of a patient specific treatment and this anti-idiotype vaccine is currently undergoing clinical trials for safety and immunogenicity evaluation in humans. another example is a scfv derivative (small immune protein, sip) to treat transmissible gastroenteritis virus (tgev), a porcine coronavirus. the sip was expressed by a pvx gene insertion vector and by a cpmv based display vector using the 2a expression strategy. the aim was to provide passive protection against enteric infections upon oral delivery in crude plant extracts . the sip folded correctly and preserved the ability to bind and neutralize tgev in tissue cultures. moreover, it provided in vivo protection against challenge with tgev in piglets vaccinated orally with crude extracts of scfv-expressing tobacco plants. the same researchers also expressed a full length tgev-specific iga by co-infecting plants with two separate pvx insertion vectors for the light chain (lc) and the heavy chain (hc) respectively . the iga was correctly assembled in plant tissue and provided in vivo protection against tgev in piglets after oral administration. a previous work (verch et al., 1998) also indicated the possibility to produce a full size mab in plants through the co-infection of two modified tmv based viral vectors. these results clearly indicated the possibility to obtain functional mabs through plant expression systems. nonetheless, expression of heterooligomeric proteins such as mabs might be inefficient due to the co-delivery of viral vectors built on the same virus backbone. in fact, this feature may result in early segregation and subsequent preferential amplification of one of the vectors in one cell. this problem has been recently overcome by utilizing two sets of vectors derived from non-competing tmv and pvx to express the two different mab chains . tmv and pvx may interact with different host proteins to sustain their replication and movement without interfering with each other. as a result, neither of the two vectors gains a replicative advantage over the other, allowing efficient co-expression of hc and lc in the same cells. the level of expression by each vector is independent within the same cell and can reach up to 0.5 mg/g fw of full assembled mab in few weeks. this strategy represents a useful platform for rapid large-scale manufacturing of mabs and other hetero-oligomeric proteins especially in situations requiring rapid responses such as pandemic threats and bioterrorism events. a vaccine is an antigenic preparation used to establish immunity to a disease. vaccination has been one of the greatest revolutions in medical science and has dramatically improved the quality and length of life expectancy. although vaccines are the most cost effective form of disease protection in healthcare, their cost is still too excessive for many people in the world, especially in developing countries. vaccines can be therapeutic, to overcome an infection already established, or prophylactic, to prevent a future infection. the aim of vaccination strategies is to obtain efficient and safe vaccine formulations to induce a long lasting immunity against various pathogens. to achieve this goal, the vaccine component should generate not only a neutralizing antibody response, a long-term memory b cells stimulation, but also a t cell mediated immunity to eliminate infected cells which represent the pathogen reservoirs. recombinant subunit vaccines, based on a single protein (antigen) or a single peptide (epitope) derived from the pathogen, are particularly attractive strategies when compared to classic inactivated, attenuated or live recombinant vaccines. recombinant subunit vaccines offer potentially equivalent efficacy but are much safer and in some cases easier to produce. plants offer unique advantages for the production of subunit vaccines in terms of scale, speed, costs, yield, and safety. the first work that describes the expression of the hepatitis b surface antigen in stable transgenic tobacco in 1992 marked the beginning for developing low cost vaccine formulations in plants (mason et al., 1992) . researchers focused on expressing their antigens in edible plant tissues as oral vaccines, opening a new prospect for vaccine delivery (mor et al., 1998) . since then, many research groups have adopted this system and carried out studies in the areas of improving target protein expression levels, analyzing the administration route and schedule, choosing the appropriate animal models, and exploring the use of possible adjuvants. the first report in this area described the expression of the fmdv structural protein vp1 with a tmv-based gene insertion vector (wigdorovitz et al., 1999) . a yield of approximately 0.15 mg/g fw was achieved and subsequently the antigen was administered to mice without further purification. all mice immunized intraperitoneally developed a protective immune response against experimental challenge with virulent fmdv. a great number of antigens have been produced in plants with different immunologic results (streatfield and howard, 2003) . altogether, these works demonstrated that plant-derived antigens retained their antigenicity and were able to induce active protective humoral and cell-mediated immune responses. the development of plant derived vaccines for human and veterinary uses against viral pathologies, tumors, allergies, bacterial infections, and diabetes-associated autoantigens has been extensively investigated (table 1) . yusibov et al., expressed the e7 oncoprotein from human papilloma virus (hpv) in n. benthamiana plants with a gene replacement vector. the antigen was subsequently purified from infected leaves using the thermotolerance properties of lichenase (massa et al., 2007) . mice, immunized subcutaneously with the plant-produced e7, showed a specific antibody and cytotoxic t-cell response. the dose also protected the animals against challenge with e7 tumor cells. moreover, the recombinant antigen was able to prevent tumor development when administered after virus challenge, supporting the ultimate goal to produce an anti-tumor vaccine with both therapeutic and prophylactic uses. a fundamental revolution within the last few years came from the use of modular deconstructed systems. a recent work using the magnicon tmv based system is worth noting. santi et al. (2006) focused on the production of antigens for agents of biological warfare, for example, the yersinia pestis bacterium, the causative agent of plague. in this work the protective efficacy of the fraction 1 capsular antigen (f1) and the low calcium response virulent antigen (v) expressed at levels up to 2 mg/g fw in n. benthamiana leaves was evaluated. guinea pigs, immunized with purified antigens, showed antigen-specific serum igg titers and, more importantly, 75% of animals were protected from an aerosolized challenge of virulent y. pestis. in a subsequent work mett et al. (2007) expressed the same y. pestis f1 and v antigens in n. benthamiana plants with an average yield of 0.38 mg/g fw with their lichenase optimized vector. the purified proteins were administered subcutaneously to non-human primates, cynomolgus macaques, producing antigen specific igg and iga responses which resulted in complete protection against lethal challenge with y. pestis. recent work show a particular interest on emerging and re-emerging diseases, as well as agents of biological warfare, and have extended plant-derived vaccine development to smallpox (golovkin et al., 2007) , anthrax , dengue virus (saejung et al., 2007) , and avian influenza a virus (nemchinov and natilla, 2007) . altogether, these data collectively show the establishment of a portfolio of well characterized and novel, improved strategies for the expression of effective vaccines in plants. journal of cellular physiology determined by the amino acid sequence, or conformational, determined by neighboring amino acids only in the tridimensional tertiary structure. an epitope alone could be poorly immunogenic and often possess a short half-life in the serum (lien and lowman, 2003) . as a result, extensive efforts are being directed to the development of new delivery strategies to increase the immunogenicity and half-life of epitope vaccines (purcell et al., 2007) . plant viral display vectors have the potential to play an important role in such strategic development. plant virus particles can structurally function as a scaffold to support, stabilize and display epitopic peptides. for example, a target epitope can be genetically fused to the cp protein. the chimeric virion will be formed by the cp-epitope fusion, representing the vaccine by itself. as a result, plant viruses can be used as a carrier to stabilize epitopes and to present them correctly to the immune system. strategies such as the use of the amber leaky stop codon and 2a peptide (described earlier) have been adopted successfully to display peptides (turpen et al., 1995; marconi et al., 2006) , as well as for full-length protein fusions (o'brien et al., 2000) . in addition, new strategies involving biotinylation of the capsid and the subsequent binding of streptavidin-conjugated target protein have been developed for displaying long sequences. the canine oral papillomavirus l2 protein, displayed on the tmv surface with this strategy, was significantly more immunogenic in animal models compared to the uncoupled antigen . with this strategy, it is also possible to combine helper epitopes and peptides known to facilitate cellular uptake or other additional t-cell targets to avoid the use of adjuvants. coexpression of melanoma-associated cytotoxic t lymphocytes (ctl) epitopes p15e and trp2 on a tmv scaffold stimulated effective tumor protection from challenge and showed a significant survival improvement over the single peptides alone (mccormick et al., 2006a) . the capability of cvps in inducing an antibody response specifically to the displayed epitope upon intranasal, intraperitoneal or oral administration, have been extensively demonstrated in different animal models (table 1) . other data show cvps ability to elicit a strong specific neutralizing immune response in the absence of adjuvants (yusibov et al., 1997; brennan et al., 1999; marusic et al., 2001) . oral delivery of spinach leaves infected with cvps elicited a strong antibody response to the displayed rabies virus peptide in mice and human volunteers (yusibov et al., 2002) , indicating a potential use of this system as a supplementary oral booster for rabies vaccinations. alfalfa mosaic virus (amv)-derived cvps were able to elicit t-and b-cell responses, in non-human primates (yusibov et al., 2005) . chimeric tmv particles was shown to be able to directly interact with and stimulate mammalian antigen presenting cells to induce a strong cellular anti-tumor immune response (mccormick et al., 2006b) . studies of body distribution for orally administered cvps also suggested their application as nanocapsules in novel drug delivery systems (rae et al., 2005; smith et al., 2007) . applications of peptide-displaying cvps extend far beyond the area of vaccine development. of particular interest are a killer peptide to confer broad-spectrum resistance to phytopathogens (donini et al., 2005) , a metal-binding peptide converting cvps into an artificial metal-adsorbing sink for metal tolerance and phytoremediation (shingu et al., 2006) , and a mosquito hormone-derived decapeptide as a larvicide to protect plants against agricultural insect pests and to control vector mosquitoes (borovsky et al., 2006) . finally werner et al. (2006) fused a fragment of protein a (133aa) to the tmv cp c-terminus via a 15-aa linker. this chimeric nanoparticle allowed a simple purification of mabs with 50% recovery yield and product purity of greater than 90%. this technology provides an inexpensive self-assembling matrix that could be used as industrial immuno-adsorbent for antibody purification. due to their simple macromolecular organization, assembly capabilities, structure stability, easy scalability and facile purification, plant viruses can offer a cheap source of biopolymers and nanoparticles. well characterized viruses with stable, highly ordered and repetitive structures, such as tmv and cpmv, are of particular interests. for example, cpmv, cowpea chlorotic mottle virus (ccmv) and other icosahedral viruses have been used as nucleation cages for the mineralization of materials (douglas and young, 1998) . another promising application is in noninvasive in vivo vascular imaging techniques (manchester and singh, 2006) . cpmv for example has been fluorescently labeled to visualize blood flow for periods of at least 72 h to identify vessels and to monitor tumor neovascularization (lewis et al., 2006) . moreover, cpmv can be modified to generate new surface properties for developing novel biomaterials, electrochemical biosensors, or nanoelectronic devices (wang et al., 2002; chatterji et al., 2004; steinmetz et al., 2006) . rod-shaped viruses, like tmv, have been extensively used in the synthesis of a variety of metals nanowires, magnetic materials and semiconductors. tmv can be used as organic templates for the controlled deposition of gold, silver and platinum to prepare 1-d arrays (dujardin et al., 2003) . self-assembled, modified cp monomers of tmv were also employed for the construction of photovoltaic components in a novel light-harvesting system (miller et al., 2007) . cvps can be also incorporated in liquid crystal systems or used to generate thin films and fibers (flynn et al., 2003) . these examples clearly demonstrate the broad application and emerging potential of plant viruses in nanotechnology fields. over the last decade, plant based production of pharmaceuticals has made remarkable progress as the expression of a diverse set of proteins has been demonstrated, several proteins have moved into clinical testing, and a plant-derived veterinary vaccine has been approved. recent developments in transient expression systems and production in a controlled green house environment are directly addressing the issues of low expression levels and under developed regulatory standards for plant made pharmaceuticals (pmps). in spite of this progress, barriers remain that prevent the broad adoption of this technology platform. one of these is the lack of translational research to bridge the gap between bench discoveries and their corresponding clinical products. in fact, many product failures during development are ultimately caused by problems of transition from laboratory prototype to industrial product, as stated by the fda critical path report (http://www.fda.gov/oc/initiatives/criticalpath/). product industrialization programs are routinely delayed or derailed by inadequate efforts in downstream manufacturing, scale-up development, and quality control. therefore, as tremendous progress has been realized in recombinant protein expression, research focus has gradually been shifted to improvements in downstream processes including extraction, purification and recovery of the final products. downstream processing is fundamentally important to the commercial viability of the specific pmps and the pmp technology in general. an optimized downstream process will not only provide the additional cost-saving measures for the overall product cost, it will also enable the large-scale production of the products to meet the market demands. in addition, downstream process development is an essential step in compliance with fda's current good manufacture practice (cgmp) regulations. the general downstream processing steps for extraction and purification of pharmaceutical proteins are similar to those of other systems. these steps include harvest and fractionation of the tissue containing the targeted protein, extraction of target protein into designed buffer, clarification of cell debris and particles from the extract, purification of the target protein, and vialing of the purified products into the proper container in the desired formulation buffer. the unique structure and biochemistry of plant cells and tissues present different challenges and opportunities for each of the above processing steps. in addition, the choice of host plant species, tissue and subcellular organelle targeting of the product will have profound impact on the strategy and outcome of downstream processing efforts. since this review concerns primarily viral vector based transient production of pmps, our discussion will focus on the downstream bioprocessing of proteins targeted to the cytoplasm of leafy plants. pmp production with seeds has been extensively reviewed by others (menkhaus et al., 2004; nikolov and woodard, 2004) . the first step of biomaterial processing is extraction and purification from tissue with a high concentration of the target recombinant protein. the selective harvest of the target tissue will reduce the volume of initial biomaterial, and in turn, reduce the unnecessary need to eliminate the extra host proteins introduced by the inclusion of non-target tissue, and the total operational cost. the selectivity will also allow the avoidance of tissues which are hard to process, prime to introducing environmental contaminants (e.g., roots), or rich in other undesirable molecules such as proteases, alkaloids, and phenolics. for transient expression with viral vectors, leaves are primarily the target tissue for protein accumulation. currently most of the biomaterial production with these vectors is accomplished in greens houses with a controlled environment. for example, 4-18 days after inoculation, n. benthamiana leaves will be selectively harvested away from roots and stems (werner et al., 2006) . for small scale preparation, some researchers perform further fractionation of leaves by manually separating the central vein from the rest of leaves. for largescale production, this kind of operation becomes impractical and whole leaves are processed thoroughly in the next step. if the biomaterial is produced in open fields, extra steps have to be performed to remove the diverse array of environmental contaminants. rinsing and light washing of plants, before harvesting the target tissue, is a common practice for this purpose. regardless of where the biomaterial is generated, it is important to lower the bioburden and other contaminants as much as possible at this step. this measure will prevent microorganisms and other contaminants from entering purification feed streams and thus, greatly simplify the subsequent purification process and ensure the regulatory compliance of the final product. one of critical factors in choosing plant expression platforms is the protein stability in the targeted tissue. with all the advantages of the viral based transient expression system, recombinant proteins generally do have less long-term stability in leaves due to higher water content in contrast to the seedbased stable transformed expression systems (doran, 2006) . however, some proteins did show relative long-term stability in leaves of up to 7 days at room temperature (fiedler et al., 1997) or even longer (12 weeks) in dry alfalfa tissue (khoudi et al., 1999) . depending on the nature of the target protein, some of them need to be further processed as fresh leaf tissue, while others can be stored up to months as frozen tissue without losing their recovery and/or biological activity (chen, unpublished work). the possibility of storing tissue at a cold temperature for certain proteins offers and expands the flexibility of this production platform. the major goal of this processing step is to release the target protein into a liquid buffer solution from the plant tissue. to achieve this goal in a leaf-based transient expression system, leaf tissue is first ground in the presence of a desired extraction buffer to break the tissue and cells. the tissue homogenate will then be further pressed to release the protein into the aqueous buffer. the crude extract will be clarified to separate the protein-containing buffer from plant debris, insoluble proteins (see below) and other particulate matters. in comparison to other production hosts, plants produce more solid debris (up to 30% of totally weight of the biomaterial (menkhaus et al., 2004) ). the larger and denser solids make it impossible to achieve effective clarification by filtration methods. as a result, continuous centrifugation remains the most effective and scalable technique for plant extract clarification. it should be noted that it is possible to extract small recombinant proteins (<50 kda) that are targeted through the endomembrane system to the apoplast without breaking the plant cells. for example, some of the smaller proteins can be released directly into the extraction buffer by simply rinsing the tissue or by a specialized centrifugation technique (lohaus et al., 2001) . the purification process for this class of proteins can be greatly simplified with the result of a reduction in host protein contaminants. the choice of extraction buffer has to be carefully determined based on the properties (e.g., pi, size, hydrophobicity, and stability) of the target protein and the major contaminating host molecules. for example, the major contaminating protein in plant leaves is ribulose 1,5bisphosphate carboxylase-oxygenase (rubisco). correspondingly, the choice of low ph buffers (ph 5.3) should be encouraged to keep rubisco insoluble and prevent it being extracted into the aqueous phase. at laboratory bench scale, protease inhibitors and antioxidants are routinely added to the extraction buffer to counter the denaturation, degradation and structural modification by proteases and phenolic compounds. due to regulatory restraints and low-cost requirement, these molecules should only be included when deemed absolutely necessary for large-scale production. both chromatographic and non-chromatographic methods have been employed to purify plant-derived pharmaceutical proteins. like proteins from other production hosts, purification strategies are formulated for each individual protein based on its solubility, size, pi, charge, hydrophobicity, and affinity to specific ligands and the parallel characteristics of host proteins. for mab-based pmps, while protein a or g-based chromatography provides a superb and convenient step (langone, 1982) , much effort is needed to eliminate plant host molecules which cause resin fouling or/and interfere with the binding of the target protein to protein a resin, and thus, reduce its capacity. non-chromatographic scalable processes such as aqueous two-phase partitioning systems (atps) are being developed to address this key issue (platis and labrou, 2006) . for other non-mab-based vaccines and therapeutics, a purification scheme has to be developed individually which is based on multiple steps of conventional chromatographic methods (menkhaus et al., 2004) . this time-consuming and challenging process calls for the need to develop more ''universal'' or versatile purification methods. the employment of affinity tags, particularly the tandem affinity tag purification (tap) strategy, provides possibilities for such versatile solution (lichty et al., 2005; arnau et al., 2006; tagwerker et al., 2006) . however, the search for both efficient and precise proteases for tag removal still presents serious challenges. in addition, the inclusion of proteases in the manufacturing process further complicates product purification, as well as raises cost and regulatory concerns (feeney et al., 2006; kenig et al., 2006) . several non-enzymatic affinity tag removal techniques have been explored (rais-beghdadi et al., 1998; wood et al., 1999) , but still require further optimization before becoming practical for product manufacturing. in addition to native proteins, carbohydrates and other host molecules, unique plant endogenous molecules such as phenolics needed to be removed during purification. phenolics tend to modify proteins by forming complexes, thus impeding purification if not removed earlier during purification (kusnadi et al., 1998; menkhaus et al., 2004) . some plants, such as tobacco, also produce a high-level of toxic alkaloids that have to be eliminated from final product . regarding agroinfection, the potential elevated-level of endotoxin (lipopolysaccharide, lps) from a gram-negative bacterium is another concern. specific purification strategies to lower the lps to acceptable levels must be incorporated into the overall scheme to ensure the safety of pmps produced using this platform (magalhaes et al., 2007) . the overall purification design has to be robust, scalable, cost-effective and compliant to cgmp regulations. ideally, no more than three chromatographic steps should be included to obtain a highly purified product. otherwise, recovery will drop to an impractical level. whenever possible, resins widely accepted by standard pharmaceutical industry should be considered first during process-development to minimize future regulatory expenditure. the requirement for an independent quality management system (qms) to govern the manufacture of pharmaceuticals also applies to the pmps. in addition to cgmp compliance during downstream processing, analytical tests for releasing the final purified products have to address plant specific contaminants in addition to the standard required assays. even though plants do not contain animal viruses and other infectious agents, it must be validated that lps, phenolics, and alkaloids, as well as herbicides and insecticides have been adequately removed from the final product. a significant increase in demand for high quality recombinant proteins is already on the horizon. new biological systems for the production of these proteins must be developed to meet market demands. plant expression systems based on viral vectors have the greatest potential to provide such technology. once optimized and implemented at a commercial scale, these expression systems should create a technology platform to produce recombinant proteins with scalability, speed, efficiency, cost-effectiveness and safety. use of virus vectors for the expression in plants of active full-length and single chain anti-coronavirus antibodies plant-made subunit vaccine against pneumonic and bubonic plague is orally immunogenic in mice current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins stability of potato virus x expression 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human enzyme virus-based nanoparticles (vnps): platform technologies for diagnostic imaging in planta production of two peptides of the classical swine fever virus (csfv) e2 glycoprotein fused to the coat protein of potato virus x in planta engineering of viral rna replicons: efficient assembly by recombination of dna modules delivered by agrobacterium systemic agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants neutralizing immunogenicity of transgenic carrot (daucus carota l.)-derived measles virus hemagglutinin chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1 expression of hepatitis b surface antigen in transgenic plants anti-cancer activity of plant-produced hpv16 e7 vaccine roy markham: pioneer in plant pathology rapid production of specific vaccines for lymphoma by expression of the tumorderived single-chain fv epitopes in tobacco 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nicotiana benthamiana coats pvx rods and also assembles into viruslike particles food-processing enzymes from recombinant microorganisms-a review the full-length clone of cucumber green mottle mosaic virus and its application as an expression system for hepatitis b surface antigen investigation of the potential of maize streak virus to act as an infectious gene vector in maize plants surface features of potato virus x from fiber diffraction recombinant protein therapeutics-success rates, market trends and values to2010 direct screening for high-level expression of an introduced alpha-amylase gene in plants bovine herpes virus gd protein produced in plants using a recombinant tobacco mosaic virus (tmv) vector possesses authentic antigenicity fusion proteins comprising a fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen inactivation and purification of cowpea mosaic virus-like particles displaying peptide antigens from bacillus anthracis development of an aqueous two-phase partitioning system for fractionating therapeutic proteins from tobacco extract scope for using plant viruses to present epitopes from animal pathogens development of cowpea mosaic virus as a high-yielding system for the presentation of foreign peptides cowpea mosaic virus-based chimaeras. effects of inserted peptides on the phenotype, host range, and transmissibility of the modified viruses more than one reason to rethink the use of peptides in vaccine design systemic trafficking of plant virus nanoparticles in mice via the oral route purification of recombinant proteins by chemical removal of the affinity tag transient expression in tobacco leaves of an aglycosylated recombinant antibody against the epidermal growth factor receptor production of dengue 2 envelope domain iii in plant using tmv-based vector system expression of multiple proteins using full-length and deleted versions of cowpea mosaic virus rna-2 from planta to pharma with 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hemagglutinin protein expressed in transgenic lettuce induces neutralising antibodies in mice following mucosal vaccination immunoabsorbent nanoparticles based on a tobamovirus displaying protein a protection of mice against challenge with foot and mouth disease virus (fmdv) by immunization with foliar extracts from plants infected with recombinant tobacco mosaic virus expressing the fmdv structural protein vp1 a genetic system yields self-cleaving inteins for bioseparations maize (zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes oral immunogenicity of potato-derived hbsag middle protein in balb/c mice antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and hiv-1 expression in plants and immunogenicity of plant virusbased experimental rabies vaccine peptide-based candidate vaccine against respiratory syncytial virus development of bean pod mottle virus-based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean in planta expression of hiv-1 p24 protein using an rna plant virus-based expression vector key: cord-279691-v5kpmk0b authors: hagemeijer, marne c.; rottier, peter j.m.; de haan, cornelis a.m. title: biogenesis and dynamics of the coronavirus replicative structures date: 2012-11-21 journal: viruses doi: 10.3390/v4113245 sha: doc_id: 279691 cord_uid: v5kpmk0b coronaviruses are positive-strand rna viruses that are important infectious agents of both animals and humans. a common feature among positive-strand rna viruses is their assembly of replication-transcription complexes in association with cytoplasmic membranes. upon infection, coronaviruses extensively rearrange cellular membranes into organelle-like replicative structures that consist of double-membrane vesicles and convoluted membranes to which the nonstructural proteins involved in rna synthesis localize. double-stranded rna, presumably functioning as replicative intermediate during viral rna synthesis, has been detected at the double-membrane vesicle interior. recent studies have provided new insights into the assembly and functioning of the coronavirus replicative structures. this review will summarize the current knowledge on the biogenesis of the replicative structures, the membrane anchoring of the replication-transcription complexes, and the location of viral rna synthesis, with particular focus on the dynamics of the coronavirus replicative structures and individual replication-associated proteins. positive-strand rna (+rna) viruses are the most abundant viruses in nature. many important pathogens belong to this category, including poliovirus (pv), hepatitis c virus (hcv) and the severe acute respiratory syndrome (sars)-coronavirus (cov). a distinctive common feature of +rna viruses is the replication of their genomes in the cytoplasm of the host cell in association with rearranged cellular membranes that are remodeled into organelle-like membranous structures to which the viral replication-transcription complexes (rtcs) localize. the various membrane rearrangements observed in +rna virus-infected cells range in size from 40 to 400 nm, contain lipids that are derived from various cellular compartments and demonstrate an impressively diverse plethora of morphologies that include, among others, clusters of vesicles for the picorna-and togaviridae, spherule-like invaginations for the bromoviridae and nodaviridae, and vesicle packets and membranous webs for the flaviviridae (reviewed in [1] [2] [3] ). these membrane rearrangements seem to be beneficial for (i) sequestering and concentrating all viral and cellular components necessary for viral rna synthesis and (ii) to provide a protective microenvironment against virus-elicited host defense mechanisms. also coronaviruses (covs), enveloped +rna viruses that belong to the family coronaviridae, extensively rearrange cellular membranes into organelle-like replicative structures during infection. these replicative structures consist of double membrane vesicles (dmvs) and convoluted membranes (cms). the viral replicase proteins involved in rna synthesis localize to both these structures, while double-stranded rna (dsrna), presumably functioning as replicative intermediate during viral rna synthesis, has been detected at the dmv interior [4, 5] . recent studies have provided new insights into the assembly and functioning of the cov replicative structures. this review will summarize the current knowledge on the biogenesis of the replicative structures, the membrane anchoring of the rtcs, and the location of viral rna synthesis, with particular focus on the dynamics of the coronavirus replicative structures and individual replication-associated proteins. the coronaviridae are a family of evolutionary related, enveloped +rna viruses that together with the arteriviridae and the roniviridae, belong to the order of the nidovirales. historically, covs have been recognized as important infectious agents for domestic livestock, poultry and companion animals. in contrast to the animal viruses, human covs (hcovs) have been associated with relatively mild upper and lower respiratory tract infections, including ordinary common colds. however, in 2002-2003 the outbreak of a novel hcov in china, causing severe fatal atypical pneumonia in infected individuals, demonstrated that hcovs are also able to induce severe life-threatening disease in humans. this virus was called the sars-cov [6, 7] and emerged in the human population from an animal reservoir, probably originating from bats, with palm civet cats acting as intermediate hosts [8, 9] . among the +rna viruses, covs clearly distinguish themselves by carrying the most complex and largest genomes, which range in size from ~26 to 32 kb [10] . despite the variation in size, the overall genome organization of the various covs is quite conserved. the genome contains all the genetic information necessary to direct both the synthesis of full-length genomic rna (replication) and the (discontinuous) production of subgenomic mrnas (transcription) [11] . the linear +rna genome of covs is 3' polyadenylated and has a 5' cap structure, thereby mimicking cellular mrnas. the 5' and 3' ends of the genome contain untranslated regions (utrs) with cis-acting elements that are important for replication and transcription. two-thirds of the genome consists of two large open reading frames (orfs), orf1a and orf1b. the remaining 3' one-third part encodes the structural proteins interspersed with sequences encoding some accessory proteins. a schematic picture of the prototype mouse hepatitis virus (mhv) genome is shown in figure 1a . the structural and the accessory proteins are expressed from a nested set of 3' coterminal subgenomic (sg) mrnas that are generated via discontinuous transcription during subgenome-length minus-strand rna synthesis [12, 13] . the rna-dependent rna polymerase (rdrp) copies the genomic positive-sense rna into a negative-sense template until it reaches a transcription-regulation sequence (trs). at this point, rna synthesis may either continue or the rdrp may relocate to the 5' end of the genome and complete the negative-sense sgrna. these negative-sense sgrnas serve as templates for the synthesis of the corresponding positive-sense sgrnas. as a result, the positive-sense sgrnas form a nested set of mrnas, which extend for different lengths from a common 3' terminus while also having a common 5' end, which is known as the leader sequence. generally, only the 5' first unique gene of each sgrna is translated (reviewed in [11] ). minus-and plus-strand rna synthesis is already detected at 75 to 90 min post infection (p.i.) [11] . minus-strand synthesis, of which the resulting rna species are mainly present as double-stranded intermediates because of their association with plus-strand rna molecules, peaks at 5 to 6 h p.i. after which the synthesis declines but does not stop [14] . the plus-stranded rnas are produced in a 50-to 100-fold excess over their minus-strand counterparts [11, 15] . the viral replicase is encoded by the two most 5' orfs of the genomic rna, orf1a and orf1b. translation of orf1a and orf1b of the genomic rna generates two very large replicase polyproteins (pp), pp1a and pp1ab. the latter is synthesized via a −1 ribosomal frameshift mechanism mediated by a pseudoknot structural element at the end of orf1a [16, 17] . these replicase polyproteins are extensively processed by viral proteinases (reviewed in [18] ), resulting in the generation of sixteen nonstructural proteins (nsps). a schematic representation of the cov replicase polyprotein is shown in figure 1b . the cov nsps form together with the nucleocapsid (n) protein, and presumably several host proteins, the membrane-associated rtcs. to generate the functional cov replication complexes, the replicase polyproteins pp1a and pp1ab have to be proteolytically processed to liberate the sixteen individual protein products. cleavage of pp1a and pp1ab is performed by two viral proteinases that reside in nsp3 and nsp5: the papain-like proteases (plpro1 and plpro2) located in nsp3 and the chymotrypsin-like cysteine proteinase (3clpro), or main protease (mpro), present in nsp5 (reviewed in [18] ). besides the mature nsps, also the intermediate and precursor polyproteins are likely to be functionally important. hence, the cleavage of these proteins may somehow be involved in the temporal regulation of plus and/or minus sense viral rna synthesis [18] [19] [20] . schematic representation of the +rna genome of mhv-a59. the coronavirus genome contains a 5' cap structure and a 3' poly(a) tail, together with untranslated regions (utrs). the first two-thirds of the genome consist of two large open-reading frames (orfs), orf1a and orf1b, which are translated into two large replicase polyproteins (pp1a and pp1ab). pp1ab is synthesized via a −1 ribosomal frameshift mechanism at the end of orf1a (rfs). the final one-third of the genome contains the canonical cov structural proteins-encoding genes (s, e, m and n), interspaced by several accessory genes (2a, he, 4, 5a); (b) a schematic representation of pp1ab is shown. the coronavirus polyproteins are processed by viral proteinases residing in nsp3 (plpro1 and plpro2; grey arrowheads indicate cleavage sites) and nsp5 (mpro; black arrowheads indicate cleavage sites), thereby generating 16 mature nsps. hydrophobic domains (tm1, tm2 and tm3) in nsp3, nsp4 and nsp6 are indicated, together with predicted and identified rna(-modifying) enzymes: the rna-dependent rna polymerase (rdrp; nsp12), the helicase (hel; nsp13), the exonuclease (exon; nsp14), the uridylate-specific endoribonuclease (n; nsp15), and the methyl transferase (mt; nsp16); (c). schematic representation of the topology of the coronavirus polyprotein. only the part of the polyprotein is shown that contains the hydrophobic domains (indicated by boxes) residing in nsp3, nsp4 and nsp6. nsp3 and nsp6 contain hydrophobic domains that do not span the lipid bilayer. mpro indicates the viral protease residing in nsp5, in between nsp4 and nsp6. the key enzyme involved in genome replication is the rna-dependent rna polymerase (rdrp), which is present in nsp12 [10] . the nsp12 protein is able to utilize both homo-and heteropolymeric rnas as template but its rdrp activity is dependent on primers to copy the viral rna [21] . these primers might be produced by a non-canonical rdrp activity that has been described for the nsp8-encoded 'rna primase', as this protein is able to produce short oligonucleotides complementary to the rna genome [22] . nsp8 has been shown to associate together with nsp7 into a hexadecameric complex, consisting of eight copies of each protein, thereby forming a channel that can harbor rna and may serve as a processivity factor for nsp12 [23] . nsp8 also interacts with nsp7 in a 1:2 ratio [24] and is able to synthesize much longer transcripts [24, 25] . the cov nsp13 protein contains a superfamily 1 helicase domain with an amino-terminal zincbinding domain that is important for the unwinding activity of duplex rna (and dna) in a 5'-to-3' direction [26] [27] [28] . the resulting single-strands probably serve as templates for rna synthesis. the multifunctional nsp13 protein additionally possesses nucleotide triphosphatase activity [27, 29, 30] and is likely to be involved in removal of one of the terminal phosphate groups at the 5' end of the positivesense rnas, which is the first step in the formation of the 5' cap structure. although the enzyme that subsequently adds the guanine to the terminal phosphates (guanylyl transferase) has not been identified yet, nsp14 has been shown to exert s-adenosyl-l-methionine (adomet)-dependent (guanine-n7)methyltransferase (n7-mtase) activity [31] . finally, the cap-1 structure is formed by the adometdependent (nucleoside-2'o)-methyltransferase (2'o-mtase) activity that is present in nsp16 [32] , and for which the latter needs to form a complex with nsp10 [33, 34] . covs possess the largest genomes among the +rna viruses [10] . they encode a large number of additional rna-modifying enzymes, which are often not present in other rna viruses. these additional enzymatic activities are probably required to ensure proper rna synthesis and might account for their large size. in addition to the cap n7-mtase activity, nsp14 has metal ion-dependent 3'-to-5' exoribonuclease (exon) activity [35] and contains a nidoviral uridylate-specific endoribonuclease (nendou; nsp15) [36] , both able to degrade rna and dsrna [36, 37] . while the function of the nendou activity in the cov infection cycle is not known, it appears that the exon activity is required to ensure high replication fidelity of the extremely large cov genome [38, 39] and that nsp14 may function as a proofreading enzyme [40] . covs encode three nsps that contain hydrophobic stretches that are predicted to function as transmembrane domains: nsp3, nsp4 and nsp6. consistently, membrane association has been demonstrated for the nsp3, nsp4 and nsp6 proteins of sars-cov [41] [42] [43] and mhv [42] [43] [44] [45] . both nsp3 and nsp4 become n-glycosylated upon insertion into the membranes of the endoplasmic reticulum (er) [42] [43] [44] . interestingly, transmembrane domain predictions based on the multiple alignment of 27 cov replicase polyprotein sequences revealed an uneven number of hydrophobic domains for both nsp3 and nsp6 [42] . this prediction is peculiar as it would separate the viral proteinases residing in nsp3 and nsp5 from their target sequences, implying that some of these hydrophobic domains might actually not span the membrane. in agreement herewith, and in contrast to the predictions, all three nsps were shown to have both their amino terminus and their carboxy terminus exposed in the cytoplasm. while all four hydrophobic domains of nsp4 span the lipid bilayer, this is the case for only two of the three hydrophobic domains in nsp3 and for six of the seven in nsp6 [42, 45] . this experimentally established topology model ( figure 1c ) makes more sense, as it positions all of the proteinase cleavage sites on the same side of the membrane as the viral proteinases themselves. proteolytic processing of the replicase polyproteins probably starts during translation and prior to membrane insertion. interestingly, the cleavage between nsp3 and nsp4 appears to be a rapid event [41, 46, 47] . this cleavage at the amino-terminus of the first hydrophobic/transmembrane domain of nsp4 probably facilitates the membrane insertion of nsp4 as it may enable the first hydrophobic domain to function as a signal peptide. the occurrence of conserved non-membrane spanning hydrophobic domains in nsp3 and nsp6, which are likely to be peripherally associated with the membrane, suggests an important function for these domains, possibly in the biogenesis of the cov replicative structures. in this respect it is of interest to mention that the seventh hydrophobic domain of nsp6 contains putative palmitoylation sites (our own predictions and [45] ). the addition of palmitic acid to this hydrophobic domain may stabilize its peripheral membrane association. in addition to the nsps mentioned above, other cov nsps are involved in rna binding (nsp9 and nsp10; [48, 49] ) or in evasion of the antiviral response of the host (nsp1 and nsp3; [50] [51] [52] [53] [54] [55] [56] [57] ). the function of nsp2 is not yet known, although this protein was shown not to be essential for virus replication [58, 59] . the reader is referred to several excellent reviews on this topic for more detailed insights [10, 60, 61] . upon infection of host cells, covs induce a variety of membranous structures of which some have been associated with viral rna synthesis. the first detectable membrane rearrangements in cov-infected cells are 200 to 350 nm organelle-like structures that have been described for both mhv [47, 62] and the sars-cov [5, 63] and consist of spherical vesicles containing double lipid bilayers, termed dmvs ( figure 2 ). in between the clusters of dmvs, reticular cms are characteristically present [4, 62, 64] . later in infection large virion-containing vesicles (lvcvs) [4, 5, 62, 65] , highly organized cubic membrane structures [5, 62] and condensed tubular bodies [62, 64] are formed. the latter two structures are likely a result of the overexpression of cov structural proteins during infection and do not seem to be involved in cov replication [62] . electron tomography studies demonstrated that in sars-cov infected cells the dmvs and cms form an interconnected membranous network that is also continuous with the er [4] . this latter observation is in agreement with previous reports describing dmvs in close proximity to the er or continuous with it [5, 47, 63] . moreover, (partial) colocalization of replicase proteins together with the er resident protein disulfide isomerase (pdi) has been reported [63] , while also the translocon subunit sec61α was found to be redistributed to the replicative structures upon sars-cov infection [66] . the combined data indicate that the er is the most likely membrane donor for the dmvs, despite the absence of most conventional er markers on these structures [43, 62, 63, 67] . the cov replicative structures, i.e., dmvs and cms (figure 2) , have been associated with viral rna synthesis, as the mhv and sars-cov nsps have been shown to localize to these structures [4, 5, 19, 43, 59, 62, 63, [68] [69] [70] [71] . in addition, antibodies recognizing dsrna, the presumed replicative intermediates, label the interior of the sars-cov-induced dmvs [4] . newly synthesized viral rna, visualized by 5-bromouridine 5'-triphosphate (brutp) labeling, was observed in mhvinfected cells in close proximity to the replicative structures by immunoelectron microscopy [47, 71] . remodeling of eukaryotic cellular membranes into the replicative structures is likely dependent on the combined effects of both viral and cellular proteins and probably also on the specific lipid composition of the membranes themselves. nevertheless, only few studies have been published addressing the involvement of cellular constituents in cov replication and the generation of the replicative structures. it is conceivable that covs hijack cellular pathways to meet the conditions that are required for their replication, consequently adopting intrinsic properties of the utilized pathways themselves. in agreement with the er being the most likely membrane donor of the dmvs, an intimate association between the early secretory pathway and cov replication has been demonstrated. rtc formation and replication in mhv-infected cells were inhibited when the secretory pathway was interfered with by blocking protein export at er exit sites by treatment with the kinase inhibitor h89 or by expression of a dominant active sar1 mutant [43] . also treatment with brefeldin a (bfa), an inhibitor of er-to-golgi trafficking, or knockdown of its target gbf1, inhibited mhv replication while reducing the number of dmvs [67] . similar results were published for sars-cov infected cells treated with bfa and it was noticed that the inner and outer membranes of the dmvs were separated in bfa-treated cells [66] , which may explain the observed inhibition of viral replication. by their main ultrastructural characteristic, the double lipid bilayer, cov dmvs very much resemble autophagosomes. this similarity prompted studies into the role of the autophagy machinery in cov replication. initial studies revealed a colocalization between the autophagosomal protein marker microtubule-associated protein light-chain 3 (lc3/atg8) with the replicative structures [68] ; moreover, viral replication was impaired and dmvs were not detected in the absence of the essential autophagy protein atg5 [72] . in other studies however, these colocalization data could not be reproduced [63] , while the absence of atg5 did not affect cov replication [73, 74] . yet, lc3 was found in association with the replicative structures by zhao and coworkers [73] . furthermore, others showed that, while the endogenous lc3 protein was recruited to the replicative structures, this was not the case for a gfp-tagged form of lc3 [69, 75, 76] that is often used as a marker for autophagosomes [77] . in agreement herewith, cov replication was shown to be unaffected in autophagy-deficient cells lacking atg7, although depletion of lc3 severely affected cov replication [69] . unlike autophagosomes, which may also be induced by expression of cov nsp6 [74] , cov replicative structures were shown to be decorated with the non-lipidated form of lc3. similar findings were also reported for edemosomes [78, 79] , er-derived vesicles that transport er chaperones to lysosomes. as the edemosome cargo proteins edem1 and os-9 were also detected in association with the cov replicative structures, it was proposed that covs hijack edemosomes for their replication [69, 80] . in agreement herewith, the transmembrane sel1l protein, which was recently shown to interact with lc3 and to have a critical function in edemosome biosynthesis, was also shown to colocalize with dsrna foci in cov-infected cells, while its depletion negatively affected cov replication [80] . the cov nonstructural membrane proteins, nsp3, 4 and 6 probably play an essential role in the membrane rearrangements required for the induction of the replicative structures and in the anchoring of the rtcs to these structures. these proteins were shown to be engaged in homo-and heterotypic interactions [81] . interestingly, co-expression of nsp4 with the c-terminal one-third part of nsp3 (nsp3 c ) resulted in the relocalization of these proteins from the er into discrete foci mostly localizing to the perinuclear region of the cell [81] . although nsp6 was not required for the observed relocalization, it was recruited to the perinuclear foci when co-expressed [82] , in agreement with this protein interacting with nsp4 [81] . ultrastructural analysis of cells co-expressing nsp4 and nsp3 c revealed that the membranes of the er exhibited more curvature, although these membranes did not resemble the dmvs observed in cov-infected cells [82] , which may not be surprising as only the cterminal part of nsp3 was used in these co-expressions with nsp4. similar membrane rearrangements were not observed when the nsp3 from mhv was co-expressed with nsp4 from sars-cov (or vice versa), while (deletion) mutagenesis studies indicated essential roles for the large luminal loops of nsp3 and nsp4 in the relocalization of these proteins [82] . while co-immunoprecipitation and immunofluorescence assays indicate that nsp3 and nsp4 of mhv interact, this interaction could not be confirmed using the venus protein-fragment complementation assay [81] . we hypothesize that the interactions between nsp3 and nsp4 mediate some kind of "zippering" of the lipid bilayers of the er, which ultimately leads to the formation of the dmvs. in this model nsp3 and nsp4 interact via their luminal loops in such a way that their interaction prevents reconstitution of a functional venus protein. several other studies also suggest an important role for nsp4 in the generation of the replicative structures. disruption of the nsp4 glycosylation sites present in the loop between the first and second hydrophobic region, leads to the formation of aberrant dmvs in which the inner and outer membranes are detached, while the number of cms is increased [83] . in agreement herewith, although the fourth hydrophobic domain of nsp4 is dispensable, the other three transmembrane regions are required for cov replication [84] . furthermore, co-expression of the counterparts of the cov nsp3 and nsp4 of the distantly-related equine arteritis virus (eav), resulted in the rearrangement of host cell membranes into dmvs, albeit with a morphology [85] differing from that observed in eav-infected cells [86] . mutations of cysteine residues present in the luminal loop of eav nsp3 (the eav counterpart of cov nsp4) resulted in altered morphologies of the dmvs, while the introduction of a n-glycosylation site in this loop also affected their morphology to some extent [87] . like covs, other +rna viruses also somehow induce membrane rearrangements that are required for their replication and transcription. for several of these viruses similar membrane rearrangements can be induced by the (co-)expression of nsps (for reviews see [1] [2] [3] ). these nsps are either integral transmembrane proteins, examples being the ns4a proteins of dengue virus (denv) [88] and kunjin virus [89] and the ns4b protein of hepatitis c virus (hcv) [90] , or alternatively the proteins are only peripherally associated to the lipid bilayer, such as the 1a protein of brome mosaic virus (bmv) [91] and the 2c protein of poliovirus (pv) [92] . strikingly, however, also for the integral membrane proteins the occurrence of hydrophobic/amphipathic regions that do not span the lipid bilayer but are peripherally associated with membranes, which has also been demonstrated for cov nsp3 and nsp6 [42, 44, 45] , appears to be a common feature [88, [93] [94] [95] . another similarity among the nsps of different +rna viruses appears to be their ability to assemble into larger protein complexes. such interactions have been observed not only between nsp3, nsp4 and nsp6 of covs, but also between the nsps of other +rna viruses, including the bmv 1a protein [96, 97] and the flavivirus ns4a and ns4b proteins [98, 99] . how are the nsps of covs able to induce the observed membrane rearrangements? we speculate that the similarities between the membrane-associated nsps of different +rna viruses relate to their common ability to remodel membranes. the induction of membrane curvature in lipid bilayers is critical when remodeling host cellular membranes. the membrane-associated viral proteins may act as multimeric scaffolds able to impose such curvature, for instance by acting as wedges by inserting their amphipatic helices partially into one side of the bilayer (reviewed in [100] [101] [102] ). the viral proteins may function similar to host cellular proteins known to induce membrane bending via a scaffold mechanism, as exemplified by the copi and copii complexes, and/or via the insertion of amphipathic domains into the lipid bilayer, as has been proposed for the small gtpase sar1 (reviewed in [100, 102] ). the last few decades have provided virologists with exciting new information regarding the functions and structures of individual nsps and the characterization and formation of the membranous structures induced by +rna viruses. these insights were obtained by classical biochemical, immunofluorescent and ultrastructural approaches. unfortunately, little information regarding the dynamics of the (viral) proteins present at the membranous replicative structures in living cells is known. such studies are important as classical approaches only provide static views of cellular processes and do not necessarily reflect the dynamics underlying virus replication in living cells. to date, only few studies on the dynamics of the replicative structures of plus-strand rna viruses and their associated proteins have been published [103] [104] [105] [106] [107] [108] [109] . by using a gfp-tagged version of nsp2, shown by immuno-electron microscopy (iem) to be recruited to the dmvs and cms, as a marker the dynamics of the cov replicative structures were studied using live cell imaging [110] . these studies showed for the first time that the cov replicative structures are moving through the cell. however, it appeared that the cov replicative structures consist of two classes that demonstrate different mobilities: large structures lacking any displacement and smaller structures with relatively high saltatory mobility. the smaller replicative structures were hypothesized to correspond to individual dmvs, while the larger ones supposedly represent the dmv/cm assemblies that have been observed in ultrastructural studies of cov-infected cells [4, 62] . in sars-cov-infected cells the dmvs are confined to a reticulovesicular network [4] . we therefore speculate that the small structures have not yet been 'captured' into this network. however, correlative light-electron microscopy studies will be required to solve this issue. live cell imaging studies of hcv-and semliki forest virus (sfv)-infected cells also reveal the presence of replicative structures that could be discriminated on the basis of their size and mobility. hcv induces the formation of a so-called membranous web, in which dmvs can be observed [90] . large hcv structures, probably representing membranous webs, exhibited limited movement, whereas smaller ones were mobile and could travel long distances throughout the cytoplasm [108] . sfv-induced vacuoles are assembled at the plasma membrane after which they are transported to modified lysosomes [106, 107] . sfv-infected cells also harbor large acidic immobile perinuclear vesicles and smaller acidic cytoplasmic vesicles that showed saltatory movements. in addition to these acidic vesicles, sfv-infected cells contain a class of non-acidic highly mobile vesicles that displayed multidirectional short-distance movements. moreover, fusion of the neutral mobile structures with the acidic mobile structures resulted in the formation of the large acidic structures [107] . such events of fusion of smaller replicative structures into larger ones have not been observed (yet) for the cov and hcv replicative structures [108, 110] . the calculated velocities of the saltatory movements of the smaller nsp2-positive structures in cov-infected cells [110] correspond to those measured for microtubule-mediated transport [111] . the observed association of these structures with microtubules and the inhibition of trafficking in the presence of a microtubule network-disturbing drug confirmed the transport of the smaller structures on microtubular tracks [110] . a role of the cytoskeleton in the movement of replicative structures has also been described for hcv, sfv, and pv [105, [107] [108] [109] . disruption of a functional microtubular network inhibited the movement of the small hcv and nascent pv replicative structures [108, 109] and the trafficking of sfv neutral vesicles to acidic organelles [107] , concomitant with dispersal of these structures throughout the cytoplasm. strikingly, inhibition of microtubule-dependent trafficking did not or only modestly affect the replication of these viruses [109, 110, 112] . disruption of the actin network also did not affect cov replication much, although the replicative structures again failed to accumulate in the perinuclear area [113] . collectively, these results show that the cytoskeleton is required for the perinuclear accumulation of the replicative structure rather than for replication per se. additional studies are required to clarify the role of the perinuclear targeting of the cov replicative structures during infection. up till now, only few studies have addressed the dynamics of individual viral proteins when present at the replicative structures. recently, we analyzed the dynamic properties of three replication-associated proteins, i.e., the soluble nsp2 and n proteins and the integral membrane protein nsp4, and demonstrated that these proteins display different diffusional mobilities when present at the replicative structures. nsp2, when expressed in trans is recruited to the replicative structures [110] . after recruitment, nsp2 was shown to be immobilized by using fluorescent recovery after photobleaching (frap) methodology. in other words, nsp2 already associated with the replicative structures was not exchanged by nsp2 present at other locations in the cell. similar results have been demonstrated for other +rna virus replication-associated proteins, although the published literature on this subject is limited. also in hcv-infected cells, ns5a-positive structures showed a static internal architecture when (part of) the ns5a fluorescent protein pool was bleached [108] . when expressed individually, ns5a is highly mobile [103] , similar to nsp2. apparently, in the context of a viral infection, when other viral proteins are present, mhv nsp2 and hcv ns5a are immobilized at the replicative structures presumably due to protein-rna or protein-protein interactions. in agreement herewith, large-scale protein-protein interaction studies demonstrated that nsp2 of mhv and sars-cov is engaged in a multitude of interactions with itself, nsp3, nsp4, nsp6, nsp7, nsp8, nsp11, nsp15 and nsp16 [82, [114] [115] [116] . these observations are remarkable in view of the dispensability of nsp2 during cov infection in vitro [59] . yet, nsp2 has to be somehow important in vivo as its coding sequence is maintained during evolution. in addition to its structural role in the coronavirion, i.e., in packaging of the genomic rna into the rnp complex, the structural n protein is also important in coronavirus replication and has been detected in the perinuclear region of infected cells colocalizing with markers for the replicative structures [62, 67, 117, 118] . the interaction of the n protein with nsp3 is presumably important for the initial recruitment of n to the replicative structures [119] , while n-n protein interactions were sufficient for recruitment of (mutant) n proteins in the presence of wild type n proteins [120] . in contrast to nsp2, the n protein is not immobilized at the cov replicative structures but is associated with it rather dynamically [120] . this may not be surprising, as the multifunctional n protein is both involved in viral replication [121] [122] [123] and virion assembly [124] . as the cov replicative structures and virion assembly sites appear to be spatially separated, the newly synthesized genomic viral rna needs to be transported from the replication sites to the assembly sites. the n protein presumably facilitates this transport, consistent with its dynamic behavior. when expressed in trans in infected cells, the nsp4-gfp fusion protein was detected at the er and at the replicative structures [43, 81] . by performing fluorescence loss in photobleaching (flip) experiments continuity was demonstrated between the membranes of the er and the replicative structures that harbor nsp4 [81] , in agreement with the model that the dmvs and cms form an interconnected network that is continuous with the er [4] . however, nsp4 displayed different diffusional mobilities at different subcellular locations [81] . it was more mobile in the er than at the replicative structures. this reduced mobility may be (partly) due to its engagement in interactions with the other transmembrane-containing nsps as well as with itself [81] . also the mobility of the hcv ns4b protein, which is an integral transmembrane protein as well, depends on its intracellular location. when expressed in the absence of other viral proteins, this protein is present at the er and at so-called membrane-associated foci (mafs) that are induced upon expression of this protein [104] . frap analysis showed that ns4b present at the mafs had a reduced mobility compared to ns4b at the er, which was suggested to result from ns4b being engaged in different interactions when present on mafs or the er [104] . currently, one of the most enigmatic issues regarding cov replication is the precise localization of the sites of active viral rna synthesis. although cov rna synthesis appears to be protected by membranes [125] it is still unclear whether synthesis of nascent viral rna occurs at sites of dsrna accumulation, as pores connecting the interior of the coronavirus dmvs with the cytoplasm have not been detected [4] , while the nsps localize to both dmvs and cms [4, 5, 19, 43, 59, 62, 63, [68] [69] [70] [71] . to identify sites of nascent viral rna synthesis, one has to define what actually constitutes the active viral replication complexes. although dsrna molecules function as intermediates of replication and transcription, their presence at certain sites per se does not imply (all) these structures to be actively involved in rna synthesis. likewise, the location of viral enzymes that are required for rna synthesis does not need to correlate with active rtcs. moreover, newly synthesized rnas are not necessarily located at their site of synthesis as they may diffuse or be transported away to other subcellular locations. in view of these considerations, sites active in rna synthesis are expected to contain at least three components: the rdrp, dsrna intermediates active in replication/transcription and nascent viral rna. newly synthesized viral rnas, visualized by 5-bromouridine 5'-triphosphate (brutp) labeling, were shown to colocalize with antibodies recognizing either nsp5 or the c-terminal part of pp1a [71] , and were observed in close proximity to the dmvs by immunoelectron microscopy in mhv-infected cells [47, 71] . recently, a new method was used to detect and visualize newly synthesized coronaviral rna by incorporation of an alkyne-modified uridine analog, 5-ethynyl uridine (eu), onto which an azide-derivatized fluophore was coupled via a copper (i)-catalyzed cycloaddition reaction (click chemistry). with this method, it was shown that throughout mhv infection foci of nascent rnas could be detected, which colocalize with the rdrp-containing nsp12, indicating that they correspond with sites of active coronaviral rna synthesis. the relationship between nascent rna and dsrna is, however, less clear. while early in infection nascent rnas colocalize at or adjacent to patches of dsrna dots, presumably corresponding to dmvs, this correlation is much less apparent at later times when the dsrna dots are spread throughout the cell. many dsrna dots are apparently not transcriptionally active as no eu labeling was associated with them, while many foci of eu labeling did not appear to colocalize with the dsrna dots [113] . different models can be put forward to explain these observations. in one model, dmvs function as the sites of active rna synthesis. at the later times in infection, many dmvs are no longer active, while the ones that are active may contain only little dsrna, resulting in less apparent colocalization of dsrna and eu labeling. in another model, dmvs are non-functional end-stage products. they are not actively involved in rna synthesis, but rather harbor dsrnas that are not (longer) functioning as intermediates in rna synthesis. in this model, which is in agreement with the presumed absence of pores in these structures [4] , the cms would be the only plausible alternative for the sites of active rna synthesis. in yet another model, dmvs may be the initial sites of active rna synthesis, particularly early in infection, while at later times the membranes become sealed, connections are lost and rna synthesis shifts to the cm assemblies. clearly, ultrastructural studies will be required, ideally (co)localizing nascent rnas as well as the rdrp, to definitely determine the precise localization of cov rna synthesis. for most other +rna viruses the identification of the sites of active rna synthesis appears less complicated. nascent rnas, as well as nsps, have been shown to label the spherules that are observed in bmv[126] , sfv[106] or flock house virus (fhv)[127] infected cells at the modified er, lysosomes and mitochondria, respectively, indicating that these structures correspond with the sites of active rna synthesis. nascent rnas were previously also shown to colocalize with dsrna in kunjin flavivirus-infected cells [128] . electron tomography of the membrane rearrangements observed in flavirus-infected cells revealed that the inner content of the dmvs, which contains nsps and dsrna, is connected to the cytoplasm via a pore [129, 130] , indicating that these dmvs are actually spherule-like invaginations (once) active in rna synthesis. cov replicative structures/rtcs are macromolecular assemblies, the components of which are engaged in a plethora of protein-protein, protein-rna, and protein-lipid interactions [114] [115] [116] 131] . currently, the exact composition of the replicative structures/rtcs is not known, let alone the full arsenal of interactions occurring within these structures. moreover, the replicative structures are likely to be subject to some form of maturation [14, 19, 20, 113] , as their composition appears to change during the course of infection as determined by biochemical and immunofluorescence analyses. thus, it will be of interest to confirm, extend and refine the previously published protein-protein interactome studies that have been published for sars-cov [114] [115] [116] , for example by investigating proteinprotein interactions of other covs using novel (large-scale) screening approaches. likewise, it will be of interest to get more insight into the involvement of host proteins in the formation of these structures, for example by screening for host proteins that interact with the cov nsps or by elucidating the protein content of purified replicative structures -preferably in time-using mass spectrometry. one of the difficulties associated with these studies is the complexity of discriminating whether host proteins are directly involved in the formation of the replicative structures themselves or in rna synthesis per se, as inhibition of either process will result in reduced rna synthesis, protein expression and dmv formation. therefore, assays are needed in which the formation of replicative structures can be studied independent of viral replication. such assays may be provided by co-expression of viral proteins [81, 85] that induce the rearrangement of cellular membranes. recently, by using such a replication-independent assay, reticulons, which form a family of er membraneshaping proteins, were implicated in the formation of the bmv-induced, er-derived spherules that are associated with viral rna synthesis [132] . reticulons are involved in the induction of er membrane curvature [133] [134] [135] and seem to be required by bmv for determining the size of the spherules and for stabilizing the spherule necks [132] . it will be of interest to study the putative role of these proteins in cov replication and in reshaping of the er membranes by cov nsps. other proteins playing a role in shaping and remodeling of the er are the atlastins and climp-63 [133, 136] . also these proteins are putative candidates involved in cov-induced membrane remodeling and would deserve to be investigated. in addition to the role of virus and host proteins in formation of the replicative structures, lipids may also be important in this process. dynamic alteration of the lipid composition can induce curvature of membranes to some extent [100, 102, 137, 138] , which is, however, unlikely to be sufficient to generate the organelle-like structures observed in +rna virus-infected cells. it is more likely that lipids, together with lipid-modifying enzymes, contribute to the formation of the replicative structures by providing a suitable microenvironment to which the viral (and cellular) membrane shaping proteins are recruited. even more, +rna viruses may specifically hijack lipid-modifying enzymes for their own advantage [139, 140] . this is underscored by several studies, which show that inhibition of lipid synthesis by using either drugs or small interfering rnas dramatically affects the replication of +rna viruses [139, [141] [142] [143] [144] . it will be of interest to study how and to what extent cov infection affects the lipid homeostasis in infected cells, for example by analyzing the lipid repertoire of infected cells by lipidomics techniques. finally, as all processes occurring in living cells are inherently dynamic in nature, it is also desirable to get more insight into the biogenesis and functioning of the replicative structures using various live cell imaging approaches. for example, photoactivatable fluorescent proteins can be used to investigate the formation of the replicative structures in real-time by 'optical pulse-labeling' in living cells, while the behavior of individual nsps associated with the rtcs can be studied by selectively 'switching-on' (sub)populations of proteins [145, 146] . furthermore, tagging of viral rna by genetic incorporation of specific rna sequences that bind fluorescently-tagged rna-binding proteins [147, 148] , hybridization 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c-terminal elements is required for the establishment of functional hepatitis c virus replication complexes subcellular localization and some biochemical properties of the flavivirus kunjin nonstructural proteins ns2a and ns4a membrane curvature and mechanisms of dynamic cell membrane remodelling mechanisms shaping the membranes of cellular organelles how proteins produce cellular membrane curvature mobility analysis of an ns5a-gfp fusion protein in cells actively replicating hepatitis c virus subgenomic rna mobility of the hepatitis c virus ns4b protein on the endoplasmic reticulum membrane and membrane-associated foci visualizing the dynamic behavior of poliovirus plus-strand rna in living host cells biogenesis of the semliki forest virus rna replication complex phosphatidylinositol 3-kinase-, actin-, and microtubule-dependent transport of semliki forest virus replication complexes from the plasma membrane to modified lysosomes a dynamic view of hepatitis c virus replication complexes intracellular location and translocation of silent and active poliovirus replication complexes dynamics of coronavirus replication-transcription complexes cytoplasmic dynein-associated structures move bidirectionally in vivo cytoskeletal requirements for hepatitis c virus (hcv) rna synthesis in the hcv replicon cell culture system visualizing coronavirus rna synthesis in time by using click chemistry the sars-coronavirus plnc domain of nsp3 as a replication/transcription scaffolding protein genome-wide analysis of protein-protein interactions and involvement of viral proteins in sars-cov replication analysis of intraviral protein-protein interactions of the sars coronavirus orfeome four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly the putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral rna synthesis an interaction between the nucleocapsid protein and a component of the replicase-transcriptase complex is crucial for the infectivity of coronavirus genomic rna the coronavirus nucleocapsid protein is dynamically associated with the replication-transcription complexes the nucleoprotein is required for efficient coronavirus genome replication in vitro replication of mouse hepatitis virus strain a59 selective replication of coronavirus genomes that express nucleocapsid protein molecular interactions in the assembly of coronaviruses sars-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro a positive-strand rna virus replication complex parallels form and function of retrovirus capsids three-dimensional analysis of a viral rna replication complex reveals a virus-induced mini-organelle nascent flavivirus rna colocalizedin situwith double-stranded rna in stable replication complexes the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex composition and three-dimensional architecture of the dengue virus replication and assembly sites rna-rna and rna-protein interactions in coronavirus replication and transcription membrane-shaping host reticulon proteins play crucial roles in viral rna replication compartment formation and function a class of dynamin-like gtpases involved in the generation of the tubular er network membrane proteins of the endoplasmic reticulum induce high-curvature tubules a class of membrane proteins shaping the tubular endoplasmic reticulum subdomain-specific localization of climp-63 (p63) in the endoplasmic reticulum is mediated by its luminal α-helical segment mechanisms of membrane deformation mechanisms determining the morphology of the peripheral er viral reorganization of the secretory pathway generates distinct organelles for rna replication recruitment and activation of a lipid kinase by hepatitis c virus ns5a is essential for integrity of the membranous replication compartment phospholipid biosynthesis and poliovirus genome replication, two coupled phenomena dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis synthesis of semliki forest virus rna requires continuous lipid synthesis dengue virus infection perturbs lipid homeostasis in infected mosquito cells photobleaching and photoactivation: following protein dynamics in living cells development and use of fluorescent protein markers in living cells single mrna molecules demonstrate probabilistic movement in living mammalian cells lambdan-gfp: an rna reporter system for live-cell imaging molecular beacons: probes that fluoresce upon hybridization copper-free click chemistry for dynamic in vivo imaging imaging intracellular fluorescent proteins at nanometer resolution sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (storm) breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy correlative light-electron microscopy (clem) combining live-cell imaging and immunolabeling of ultrathin cryosections we would like to thank oliver wicht and christine burkhard from the virology division, faculty of veterinary medicine, utrecht university for stimulating discussion and fulvio reggiori the authors declare no conflict of interest. key: cord-253466-7gpije5d authors: netherton, christopher; moffat, katy; brooks, elizabeth; wileman, thomas title: a guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication date: 2007-08-31 journal: adv virus res doi: 10.1016/s0065-3527(07)70004-0 sha: doc_id: 253466 cord_uid: 7gpije5d virus replication can cause extensive rearrangement of host cell cytoskeletal and membrane compartments leading to the “cytopathic effect” that has been the hallmark of virus infection in tissue culture for many years. recent studies are beginning to redefine these signs of viral infection in terms of specific effects of viruses on cellular processes. in this chapter, these concepts have been illustrated by describing the replication sites produced by many different viruses. in many cases, the cellular rearrangements caused during virus infection lead to the construction of sophisticated platforms in the cell that concentrate replicase proteins, virus genomes, and host proteins required for replication, and thereby increase the efficiency of replication. interestingly, these same structures, called virus factories, virus inclusions, or virosomes, can recruit host components that are associated with cellular defences against infection and cell stress. it is possible that cellular defence pathways can be subverted by viruses to generate sites of replication. the recruitment of cellular membranes and cytoskeleton to generate virus replication sites can also benefit viruses in other ways. disruption of cellular membranes can, for example, slow the transport of immunomodulatory proteins to the surface of infected cells and protect against innate and acquired immune responses, and rearrangements to cytoskeleton can facilitate virus release. viruses are obligate intracellular parasites. unlike their hosts, they cannot replicate by growth or division but use their genomes to redirect host cell processes to produce all the components needed to make new viruses. virus replication and assembly are often confined within specific intracellular compartments called virus factories, viroplasm, or viral inclusions. these are thought to provide a physical platform to concentrate new genomes and proteins involved in replication and assembly, and this is likely to increase the efficiency of virus production. the formation of specialized sites of replication can involve extensive reorganization of cellular cytoskeleton and membrane compartments. this can lead to cell rounding and swelling and a ''cytopathic effect'' that has been documented for many years (reissig et al., 1956; robbins et al., 1950) . recent advances in microscopy, such as live cell imaging and tomography, combined with the power of reverse genetics, are now allowing the cytopathic effect to be redefined in terms of specific effects of viral proteins on specific cellular processes rather than an overwhelming assault on the cell in preparation for cell lysis. there is considerable interest in understanding how virus infection leads to the large changes in cellular organization required to produce complex replication sites. in the simplest model, virus replication sites would form passively through self-association of viral components and exclusion of host organelles. viruses, however, require a considerable number of host proteins to facilitate replication, and there is increasing evidence that these are specifically transported to sites of replication. host proteins may move to replication sites because they are actively recruited by binding to specific viral proteins. alternatively, viruses may transport viral and host material to replication sites by subverting host defences against infection [reviewed by kirkegaard et al. (2004) and wileman (2006) ]. the large scale changes in cellular membrane and cytoskeletal organization, which occur during the formation of replication sites, can offer further benefit to viruses. rearrangement of the cytoskeleton can, for example, facilitate virus release, and the block in the secretory pathway seen during infection with positive-stranded rna viruses can reduce release of inflammatory mediators and protect against innate and acquired immune responses. this is a broad subject of considerable interest to virologists and cell biologists, and we have benefited from excellent reviews that have been published (mackenzie, 2005; novoa et al., 2005) . in writing this chapter, we have concentrated on describing sites of virus replication in the context of the cell in which its replication takes place. we have illustrated these concepts with reference to replication sites produced by many different viruses and, where possible, described how virus replication impacts on the functioning of the host cell. virus replication sites have been studied for many years and have evolved their own terminology. early studies of poxvirus replication (dales and siminovitch, 1961; morgan et al., 1954) describe electron-dense aggregates and amorphous material induced early during infection called viroplasm. viroplasm has also been used to describe similar structures induced during infection with poliovirus (dales et al., 1965a) . viroplasm is often concentrated within perinuclear areas that exclude host organelles. viroplasm is thought to indicate sites of virus replication, and concentrations of viroplasm have been called virosomes, or virus factories, to reflect an organelle involved in virus production. virus infection also produces inclusion bodies. as a working definition, these can be considered to form later during infection. they can form virus factories once virus production has peaked, and/or at other sites in the cell they probably arise from an accumulation of viral proteins that do not become incorporated into viruses. the positive-stranded rna viruses encode nonstructural proteins (nsp) that cause proliferation and modification of membranes of the host secretory pathway. the membranes are thought to provide a physical framework or ''replication complex'' that concentrates the cellular and viral components required for virus replication (bienz et al., 1987; egger et al., 2002; froshauer et al., 1988; gazina et al., 2002; magliano et al., 1998; schlegel et al., 1996; van der meer et al., 1998) . assembly of the replicase on membranes, rather than the cytosol, may also help viruses evade host defence pathways that monitor cells for double-stranded rna (dsrna) intermediates indicative of virus replication. the replicase complexes of all the positive-stranded rna viruses contain an rna-dependent rna polymerase (rdrp), a protein with ntpase and helicase activity, and in many cases a methyl transferase to cap viral rna. these proteins are generated from the viral polyproteins by viral proteases, and are then targeted to membranes in ways that differ depending on virus family (fig. 1 ). the replicase proteins of positive-stranded rna viruses are directed to membranes by nsp with membrane-targeting information. (a) picornavirus. the replication complex contains 3d, the rdrp (red), and 2c which has ntpase and helicase motifs (purple). the 3d polymerases do not have membrane-targeting information but are synthesized as a 3abcd precursor. 3abcd is processed to 3ab by the 3c protease (red triangle) and a hydrophobic domain in 3a targets 3ab to the cytoplasmic face of er membranes. 3ab binds directly to 3d and this targets the polymerase to the replication complex. the replication complex also requires 2bc and 2c proteins that are targeted to membranes via their own hydrophobic domains (black lines). (b) flavivirues. the replication complex is encoded at the c-terminus of a polyprotein that is processed by the ns2 protease (red triangle). ns5b is the rna-dependent polymerase (red), and ns3 acts as helicase (purple). ns4b is a polytopic membrane protein inserted into the er cotranslationally. ns4a, 5a, and 5b have hydrophobic domains (gray lines) that allow posttranslational insertion into the cytoplasmic face of the er membrane. ns3 is recruited into the complex by associating with ns4a. (c) alphavirus. the nsp1234 polyprotein is processed by a protease activity in the c-terminus of p2 (red triangle). the polyprotein is anchored to the cytoplasmic face of endosome and lysosome membranes membranes that lie between the er and the golgi apparatus called the er-golgi intermediate compartment (ergic), or tubulovesicular structures, and specific fusion with ergic membranes is determined by a complex of proteins called transport protein particle 1 (trapp1). trapp1 proteins tether the vesicles on ergic and golgi membranes, allowing interactions between vesicle and target snare (soluble n-ethylmaleimidesensitive factor attachment protein receptor) proteins to facilitate membrane fusion. the snare interactions are controlled by vesicle-specific small gtpases called rab proteins (fig. 2) . further sorting events in the ergic and early golgi involve a second complex of coat proteins called copi. the copi complex contains seven proteins (a, b, b 0 , g, d, e, and z cop proteins), which generate vesicles that take proteins from the ergic and golgi apparatus back to the er through a retrieval pathway (fig. 2) . the copi proteins are recruited from the cytosol by the arf1-gtpase. activation of arf1 requires binding to gtp and is facilitated by gtp exchange protein, arf-gef. arf1-gtp inititates coat assembly while hydrolysis of gtp by arf1 leads to coat disassembly. this disassembly is stimulated by an arf1-gtp-activating protein (arf-gap) that promotes gtp hydrolysis by arf1. a possible role for arf1 in the generation of vesicles during picornavirus replication has been the focus of much work following the observation that poliovirus replication is blocked by brefeldin-a (bfa), a drug that inhibits the recruitment of arf1 onto membranes (maynell et al., 1992) . membrane vesicles are also produced in cells in response to starvation. this pathway, known as autophagy, is used as a part of a quality control system that removes long-lived proteins and damaged organelles from the cytoplasm and has been shown to provide a defence against intracellular pathogens (deretic, 2005; kirkegaard et al., 2004; levine and klionsky, 2004; shintani and klionsky, 2004) . the origins of the membranes formed during autophagy are unclear but may be derived from the er (reggiori and klionsky, 2005) . autophagy is suppressed by the target of rapamycin (tor) kinase and is activated by conditions that lead to inactivation of tor. this leads to the production of membrane crescents in the cytoplasm, called isolation membranes, which mature into doublemembraned vesicles of 500-to 1000-nm diameter called autophagosomes. this maturation engulfs small quantities of cytoplasm, and any organelles or pathogens present at sites of autophagy become trapped within autophagosomes. the autophagosomes ultimately fuse with lysosomes resulting in degradation of their content. autophagosomes are of interest because infection of cells with picornaviruses and coronaviruses (covs) can generate double-membraned vesicles that may be related to autophagosomes. in addition to supplying membrane and proteins to the secretory pathway, the er acts as a major site of lipid synthesis. as a consequence, the er contains a large quantity of membrane, and this is organized into a complex reticulum made from tubular and lamella structures (borgese et al., 2006) . the smooth er increases in response to a buildup of er membrane proteins and can be organized into lamellae or concentric whorls called organized smooth er (oser). structures similar to oser are also seen during virus replication. b. picornavirus replication induces numerous membrane vesicles picornaviruses are nonenveloped positive-stranded rna viruses. the genome encodes a large polyprotein that is processed to generate capsid proteins from the p1 region and nonstructural replicase proteins from the p2 and p3 regions. picornavirus 3d contains the rdrp, while 2c has ntpase and helicase motifs. the 3d polymerase does not have membrane-targeting information but is synthesized as a 3abcd precursor. 3abcd is processed to 3ab by the 3c protease, and a hydrophobic domain in 3a targets 3ab to the cytoplasmic face of the er. 3d binds directly to 3ab, and this targets the polymerase to the replication complex. the 3d polymerase of poliovirus is believed to self-assemble into a large ordered array on membranes, which is critical for binding rna and rna elongation (lyle et al., 2002) . the replication complex also requires 2bc and 2c proteins that are targeted to membranes via their own hydrop hobic dom ains ( fig. 1a ). the accumulation of large numbers of densely packed membrane vesicles in the cytoplasm is characteristic of a picornavirus infection (bienz et al., 1983 (bienz et al., , 1987 cho et al., 1994; dales et al., 1965a; schlegel et al., 1996; stuart and fogh, 1961; suhy et al., 2000) . studies have suggested that vesicles induced by poliovirus are derived from the er, either from copii-coated vesicles or from er-derived autophagic double-membraned vacuoles (bienz et al., 1987; jackson et al., 2005; rust et al., 2001; schlegel et al., 1996; suhy et al., 2000) . however, the detection of er, golgi, and lysosomal markers in membranes induced at later stages of infection by poliovirus suggests that more than one organelle may contribute membranes to the replication complex (schlegel et al., 1996) . in interpreting these studies, it is important to consider if the vesicles observed are involved in replication, or if they represent a bystander response to virus infection. evidence for a role of specific membranes in replication is provided by the presence of replicase proteins, or better still dsrna or negative-stranded intermediate viral rna (egger and bienz, 2005) . examination of cells infected with poliovirus for the first appearance of negative-stranded rna suggests that this initial stage of replication starts on the er. this is consistent with high-resolution immunofluorescence microscopy (rust et al., 2001) showing the poliovirus 2b protein associated with eres containing the sec13-sec31p proteins of the copii complex. these sites exclude resident er proteins, suggesting colocalization of 2b with copii-coated transport vesicles. replication complexes containing negative-stranded rna then move on microtubules to a perinuclear area to initiate synthesis of positive-stranded rna (egger and bienz, 2005) . membrane rearrangements have been studied by expressing individual, or combinations, of picornavirus proteins in cells. most of this work has involved studies of poliovirus proteins, and membrane rearrangements are reported for the 2b, 2c, 2bc, 3a, and 3ab proteins. poliovirus 2b causes fragmentation of the golgi complex (sandoval and carrasco, 1997) . the 2bc and 2c proteins lead to vesiculation and tubulation and sometimes myelin-like swirls of er-derived membranes (aldabe et al., 1996; cho et al., 1994) . similar structures are induced by 2c and 2bc of hepatitis a virus (teterina et al., 1997) . expression of the poliovirus 3a protein causes swelling of er cisternae and the disappearance of vesicles budding from the er, while the 3ab protein also induces myelin-like swirls of er (egger et al., 2000) . the membrane rearrangements induced by expression of single proteins do not, however, mirror those observed in infected cells, and since myelin-like modifications to the er are also seen following overexpression of er proteins [reviewed by borgese et al. (2006) ], their relevance to viral replication is unclear. importantly for poliovirus, it is a combination of 2bc and 3a protein expression that induces membrane structures morphologically similar to those seen in infected cells (suhy et al., 2000) . gazina et al. (2002) have studied replication complexes formed by several different picornaviruses. encephalomyocarditis virus (emcv), parechovirus 1, and echovirus 11 induce clustered vesicles containing dsrna in the perinuclear region of the cell. the precise nature of the vesicles varied with virus. parechovirus 1 produced homogeneous vesicles of 70-100 nm, while membranes produced by emcv and echovirus 11 were heterogeneous but more compact and associated with electron-dense material. differences for parechovirus 1 have also been reported by krogerus et al. (2003) who suggest that replication may occur on membranes derived from the late golgi rather than early er and ergic compartments. all three viruses, however, cause loss of ribosomes from the er and lack of visible golgi apparatus. the copi coat protein b-cop was found to colocalize with echovirus 11 replication complexes, but not with replication complexes produced by emcv, again suggesting that vesicles produced by different picornaviruses may differ. infection with foot-andmouth disease virus (fmdv) also results in loss of ribosomes from the er and an accumulation of heterogeneous vesicles to one side of the nucleus . high-pressure freezing can be used to increase the preservation of cellular ultrastructure during processing for electron microscopy. such analysis of cells infected with poliovirus shows that the vesicles have two membranes suggestive of autophagosomes ( jackson et al., 2005; suhy et al., 2000) . double-membraned structures containing electron-dense material, and possibly viruses, were also revealed by the early work on poliovirus (dales et al., 1965a) . high-pressure freezing has been used to compare fmdv and bovine enterovirus (bev). bev produced heterogeneous membrane clusters similar to the rosettes described for poliovirus (egger et al., 1996) . many of the vesicle membranes have high electron density suggestive of double membranes and lie adjacent to accumulations of virus-like particles. clusters of fmd viruses were also associated with vesicles and electron-dense material, but there were fewer doublemembraned vesicles . immunofluorescence analysis of poliovirus vesicles shows colocalization of replicase protein 3a and autophagy marker lc3, suggesting assembly of the replicase on autophagosomes. similar work suggesting the use of autophagosomes during replication of covs will be described below. for poliovirus, expression of 3a and 2bc, which produces vesicles similar to those seen in infected cells (suhy et al., 2000) , can induce autophagy (jackson et al., 2005) , and inhibition of autophagy reduces yields of extracellular virus. the results suggest that the autophagy pathway may facilitate the release of poliovirus from cells, and it will be interesting to see if this is true for other enteroviruses that are resistant to the low ph and proteases present in lysosomes and autophagosomes. evidence that different members of the picornavirus family vary in the way that they interact with host membranes is provided by studies of virus sensitivity to bfa. bfa completely inhibits poliovirus and echovirus 11 replication (cuconati et al., 1998; gazina et al., 2002; irurzun et al., 1992; maynell et al., 1992) and partially inhibits parechovirus 1 replication (gazina et al., 2002) but not other picornaviruses such as emcv (gazina et al., 2002) or fmdv o'donnell et al., 2001) . bfa prevents assembly of copi coats and this has generated considerable interest in understanding how copi and copii coats contribute to formation of the replication complex, and how bfa inhibits picornavirus replication. in cells infected with the highly bfa-sensitive virus echovirus 11, b-cop was recruited into the replication complex; in contrast, the replication complex formed by the bfa-resistant emcv did not contain b-cop. this correlation suggests that bfa-sensitive viruses may require copi coats for replication (gazina et al., 2002; mackenzie, 2005) . since copii coats are resistant to bfa (lippincottschwartz et al., 2000; orci et al., 1993; , it is suggested that copii coats may provide the membranes for replication complexes formed by bfa-insensitive viruses. the observation that poliovirus replicase 2b protein is seen in eres containing copii proteins, but poliovirus is sensitive to bfa, can be reconciled if this association of 2b with eres is considered to be an early step in generation of membrane for the replication complex that precedes recruitment of copi coat proteins. this is supported by work showing the movement of poliovirus replication complexes containing negative-stranded rna from the er to perinuclear sites (egger and bienz, 2005) . direct evidence that copi coat proteins are required for picornavirus replication comes from studies of drosophila c virus (dcv). dcv is a positive-stranded rna dicistronic virus that is similar to poliovirus and replicates in a cytoplasmic compartment containing virus-induced membrane vesicles. a genome-wide rna silencing screen identified six (a, b, b 0 , g, d, and z) of the seven copi coat proteins as essential for virus replication. furthermore, the formation of virus-induced vesicles required b-cop, but not copii protein, sec23p. notably, small interfering rnas against a-cop, but not sec23p, also slowed poliovirus replication (cherry et al., 2006) . the formation of copi-coated vesicles is regulated by the arf1-gtpase. the observation that bfa inhibits the replication of enteroviruses such as poliovirus, and also inhibits the function of the arf1-gtpase, provides a second link between virus replication and copi coats. arf proteins are regulated by arf-gefs that facilitate binding of gtp by removing gdp, and by arf-gaps that increase hydrolysis of gtp by arfs. arf1-gefs are inhibited by bfa, and bfa therefore reduces levels of arf1-gtp in cells. the gefs affected by picornavirus infection are golgi-associated bfaresistant protein (gbf1) and bfa-inhibited protein (big1/2). work by belov et al. (2005 belov et al. ( , 2007 indicates that infection of cells with poliovirus increases intracellular arf-gtp levels fourfold, suggesting increased activity of arf1-gefs or inhibition of arf1-gap proteins. in the absence of virus, arf1 is concentrated in the golgi apparatus, but during infection with poliovirus arf1 staining fragments and colocalizes with replicase protein 2c. this suggests that infection leads to a redistribution of arf proteins from the golgi apparatus to the replication complex. the binding of arf proteins to membranes is dynamic, with arf-gdp being released from membranes following hydrolysis of gtp. cytosolic arf1-gdp would redistribute naturally to membranes enriched for the arf1-gefs that facilitate loading of new gtp. significantly, poliovirus infection causes enrichment of gefs in membranes containing replicase proteins, and this would provide a mechanism for increasing levels of arf1-gtp at sites of virus replication. translation of poliovirus rna on membranes in vitro provides an alternative means of studying the role of arf proteins in virus replication. replication is inhibited by bfa and peptides that function as competitive inhibitors of arf (cuconati et al., 1998) , and for the most part, the assay mimics what is observed in infected cells. translation in vitro leads to recruitment of arf3 and arf5 but not arf6 (belov et al., 2007) onto membranes. suitable antibodies recognizing the er-associated arf1 were not available for these experiments, so it is not known if arf1 is also recruited to membranes during translation. membrane recruitment of arf proteins can be reconstituted by translation and expression of poliovirus 3a or 3cd. poliovirus proteins do not show intrinsic gef activity, but 3a and 3cd will induce association of gbf1 and big1/2, respectively, with membranes in vitro. this raises the possibility that recruitment of 3a and 3cd to the replication complex during infection targets arf-gef to virus-induced membranes, which in turn increases local levels of arf1-gtp. this is thought to be necessary for replication because inhibition of arf1-gef by bfa blocks replication, and replication can be rescued by overexpression of gbf1 (belov et al., 2007) . high levels of arf1-gtp would also increase recruitment of copi proteins and be consistent with the work on dcv showing that copi proteins are required for replication and vesicle production (cherry et al., 2006) . a poliovirus 3a mutant with a serine insertion at position 16 is unable to cause translocation of arf to membranes (belov et al., 2005) . poliovirus carrying the 3a mutation does not, however, show defects in replication, suggesting that arf1-gef recruitment to membranes by 3a is not essential for replication. it is possible that during infection the defect in 3a is compensated for by 3cd. interestingly, a bfa-insensitive poliovirus with mutations in the 2c and 3a proteins (crotty et al., 2004) induces vesicles and dispersal of the golgi apparatus, which begs the questions, does this mutant use a different process for forming the replication complex, or do the mutations in 3a allow the proteins to compete with bfa for gbf1 recruitment? the role of arf proteins during coxsackievirus infection has also been studied. in common with poliovirus, coxsackieviruses are enteroviruses and their replication is inhibited by bfa. expression of coxsackievirus 3a causes loss of copii coats from eres, and an accumulation of 3a, copii and a model secreted protein in both the er, and tubular-vesicular post-er structures containing ergic marker proteins. these effects closely resemble the effects of adding bfa to cells, suggesting coxsackievirus 3a may affect the function of arf proteins. coxsackievirus 3a affects the regulation of arf proteins (wessels et al., 2006b) . interestingly, the process differs to that described by belov et al. (2005 belov et al. ( , 2007 for poliovirus 3a translated in vitro. expression of coxsackievirus 3a in cells caused loss of copi and arf1 from membranes, and there was redistribution of big1/2 and gbf1 from the golgi apparatus into the cytoplasm. this suggests that coxsackievirus 3a reduces, rather than enhances, levels of arf1-gtp. coxsackievirus 3a also caused redistribution of arf1-gap to punctate structures suggestive of the ergic. a block in arf1-gef activity, combined with recruitment of arf1-gap, would reduce the levels of arf-gtp and inhibit membrane recruitment of copi. wessels et al. (2006a) examined the effects of the 3a proteins of other picornaviruses and found that only the 3a proteins of enteroviruses bound gefs. intriguingly, wessels' work contrasts with belov in that they found the interaction of 3a with gefs lead to a loss of arf proteins from membranes. why these differences are seen is, as yet, unknown but may be due to differences in cell type/methods used or differences in levels of 3a protein expression. poliovirus and coxsackievirus slow protein movement through the secretory pathway (doedens and kirkegaard, 1995; wessels et al., 2005) . expression of 2b, 2bc, and 3a individually were all able to slow secretion (cornell et al., 2006; doedens and kirkegaard, 1995; doedens et al., 1997; van kuppeveld et al., 1997; wessels et al., 2005 wessels et al., , 2006a , but for both viruses the 3a protein was found to have the greatest impact on er-to-golgi transport. poliovirus infection, and the 3a protein expressed alone in cells, reduces surface expression of mhc class i, the tnf receptor, and secretion of b-ifn, il-6, and il-8 (choe et al., 2005; deitz et al., 2000; dodd et al., 2001; neznanov et al., 2001) , and this may offer an immune evasion strategy to the picornaviruses. this is consistent with the observation that the ability of the coxsackievirus 3a protein to slow secretion may be important for virulence (wessels et al., 2006b) and has led to studies of the mechanism of action of 3a in blocking er-to-golgi transport. deletion analysis has identified residues in the unstructured n-terminal region of poliovirus and coxsackievirus 3a as important for the block in host protein secretion (choe et al., 2005) . an n-terminal proline-rich region (particularly pro19) is important for coxsackievirus block in trafficking (wessels et al., 2005) . in poliovirus, lys9 appears important, and in the triple-proline motif (positions 16-18), only the pro18 is indispensable for inhibition of protein secretion (choe et al., 2005) . a serine insertion in 3a protein between thr14 and ser15, creating the 3a-2 mutant virus (berstein and baltimore, 1988) , was found to abolish the er-to-golgi inhibition of protein trafficking but has little effect on virus replication or membrane rearrangements (dodd et al., 2001; doedens et al., 1997) . this important observation shows that the ability of 3a to inhibit protein secretion is separate from its role in membrane rearrangements and viral replication. there is continuing interest in understanding how picornavirus proteins block secretion. poliovirus 3a and 3cd, and coxsackievirus 3a, can interact with arf-gef, but the downstream events are unclear. the recruitment of arf-gef by poliovirus 3a and 3cd would increase recruitment of arf-gtp to membranes of the replication complex. this would increase recruitment of copi coat proteins into sites of virus replication and reduce the pool of copi proteins available to the ergic and golgi apparatus. alternatively, inhibition of arf-gef and recruitment of arf-gap onto ergic membranes by enterovirus 3a would decrease membrane association of arf-gtp and again reduce recruitment of copi onto ergic and golgi membranes. both mechanisms would reduce the formation of copi vesicles, and as seen for bfa, block secretion. poliovirus 3a also binds and inactivates l1s1, a component of the dynein-dynactin motor complex (kondratova et al., 2005) , which is required to move copiiderived vesicles from eres to the ergic. as seen for expression of 3a, mutant l1s1 leads to disruption of the er-to-golgi traffic and reduction in plasma membrane receptors such as tnf receptor. it is possible that 3a may also slow er-to-golgi transport by binding l1s1. a. picornaviruses differ in the use of nonstructural proteins to block secretion the ability of 3a to inhibit er-to-golgi trafficking has not been conserved in all picornaviruses (choe et al., 2005; cornell et al., 2006; deitz et al., 2000; moffat et al., 2005) . for example fmdv infection leads to reduced surface expression of mhc class i (sanz-parra et al., 1998) , but the fmdv 3a protein does not inhibit er-to-golgi transport . a lack of inhibition of secretion has also been reported for 3a proteins of human rhinovirus, hepatitis a, theiler's virus, human enterovirus, and emcv (choe et al., 2005; wessels et al., 2006a) . the 3a protein of human rhinovirus is unable to bind gbf1, or inhibit copi recruitment to membranes, and this may explain its inability to slow secretion. importantly, studies on fmdv have shown that the 2bc protein, or a combination of the processed products, 2b and 2c, inhibits protein movement from the er to the golgi apparatus (moffat et al., , 2007 , and this may be similar for other picornaviruses with 3a proteins that do not block er-to-golgi transport. a lack of effect of fmdv 3a on secretion does not result from an inability to bind membranes. fmdv 3a is recovered from postnuclear membrane fractions, and when expressed alone in cells it colocalizes with resident er proteins. in common with 3a, picornavirus 2b, 2c, and 2bc proteins also contain membrane-binding sequences. sequence alignment of the 2b, 2c (2bc), and 3a proteins of different picornaviruses showed a high level of conservation between the 2c proteins, which contain an ntp-binding site and predicted helicase motifs (gorbalenya et al., 1990) but large variations in the sequences of the 2b and 3a proteins (choe et al., 2005; moffat et al., 2005) , and these may explain their different abilities to block secretion. the fmdv 3a protein is, for example, much longer than 3a of enteroviruses, such as poliovirus, and it does not contain the n-terminal sequences thought important for poliovirus 3a to block the secretory pathway. the 2b protein of fmdv also locates to er membranes but shows a more reticular pattern than the fmdv 3a protein and can be seen in punctate structures aligned along the er suggestive of eres (fig. 3) . this is similar to the 2b of poliovirus that colocates with both figure 3 subcellular location of foot-and-mouth disease nsp encoded in the p2 region of the fmdv genome. vero cells expressing fmdv 2b (top), 2bc (middle), or 2c (bottom) were fixed and permeabilized and processed for immunofluorescence. 2c and 2bc were located using antibodies specific for 2c (3f7) and 2b was located using an antibody raised against an epitope tag in 2b. cells were counterstained using antibodies against er luminal protein erp57 (top and middle panels), or copi protein b-cop (bottom). merged images are shown at higher magnification on the far left. see moffat et al. (2005) for more details. reprinted from moffat et al. (2005) with permission from american society for microbiology. sec13p and sec31p of the copii coat. as expected, fmdv 2c is also membrane associated. when expressed in cells, 2c produces faint er staining, but mainly locates to bright punctate structures in a perinuclear region close to b-cop, reminiscent of golgi staining. the b-cop staining is, however, fragmented suggesting dispersal of the golgi apparatus, and there is not complete colocalization since 2c structures negative for b-cop protein can also be seen (moffat et al., 2007) . a similar location of fmdv nsp within the area of the cell occupied by the golgi apparatus is seen in cells infected with fmdv, and again they do not colocalize with golgi markers . the 2bc protein of fmdv is also recovered in postnuclear membrane fractions, but when expressed in cells, 2bc staining differs from that seen for the processed products, 2b and 2c (fig. 3) . fmdv 2bc locates to punctate cytoplasmic structures and larger structures surrounding the nucleus that contain er markers suggesting swelling of the er. 2bc shows partial overlap with luminal er markers but, unlike poliovirus 2bc, does not colocate with the copii marker sec13p. the er markers also appeared punctate in cells expressing 2bc, suggesting disruption of the er . interestingly, coexpression of 2b and 2c blocks secretion within post-er compartments, similar to those containing 2c. the site of block therefore seems to be determined by the subcellular location of 2c (moffat et al., 2007) and is consistent with the observation that the block in the presence of 2b can be redirected to the er, if 2c is tethered to the er by an er retention sequence. sindbis virus (sbv) and semliki forest virus (sfv) are the best studied examples of alphavirus replication in mammalian cells [reviewed by salonen et al. (2005) ]. early electron microscopy studies showed that vesicular structures called cytopathic vacuoles between 600-and 2000-nm diameter, accumulated in infected cells. the vacuoles contained 50-nm-diameter vesicles called spherules, many of which were aligned along the inside face of the vacuole and attached by a neck to the limiting membrane. the neck was often seen connected to an electron-dense matrix extending into the cytoplasm. the observation that the cytopathic vacuoles contained nsps required for rna replication, cofractionated with lysosomal enzymes, and could be labeled with endocytic markers (froshauer et al., 1988) , led to the conclusion that they are sites of viral replication derived from endosomes and lysosomes. in many cases, the vacuoles were also connected to the rough er by filaments and granular material containing the rna polymerase. alp havirus ns ps are syn thesized in the cytop lasm and bind to endosom es and lysos omes to generate a replication compl ex. the rep licase pro teins are syn thesized as a polyprote in (p1234) . the p4 domai n is the rdrp wh ile p2 has ntpase and helicase activi ties, and p1 is the methytran sferase require d to cap rna ( fig. 1c) . the p12 34 poly protein locate s to endosome or lysosome membranes via an amphipathic peptide sequence in p1 (salonen et al., 2003) . at this stage the p4 polymerase is cleaved from the polyprotein and functions with the remaining p123 protein to generate negative-stranded rna. interestingly, once the p123 is processed to individual nsps, the polymerase preferentially produces positive-stranded rna. expression of individual nsps does not lead to the formation of a cytopathic vacuoles or spherules. formation of spherules requires interactions between nsp p1, p3, and p4 and the p123 polyprotein precursor complex (salonen et al., 2003) . rubella virus is a member of the togaviridae family within the alphavirus genus. cells infected with rubella virus also contain vacuoles containing spherules and these colocalize with lysosomal markers, suggesting use of lysosomes for replication. a fibrous material connects the vacuoles to the er (lee et al., 1994; magliano et al., 1998) , again suggesting strong similarities with sfv and sbv. members of the alphavirus superfamily share homologies between proteins required for rna replication, and this extends to plant viruses. alfalfa mosaic virus replicase proteins colocalize with the plant vacuole (van der heijden et al., 2001) , and turnip yellow mosaic virus uses the chloroplast outer envelope as a site for replication. replication of tobacco mosaic virus, a tobamovirus, is dependent on arabdopsis proteins tom1 and tom2a that are integral membrane proteins of the tonoplast (hagiwara et al., 2003) . the tonoplast is a membrane compartment within plants that surrounds the vacuole/lysosome, suggesting plant alphaviruses also use the endosome/lysosome system as a site of replication. infection of plants with alphavirus-like superfamily viruses can also induce the formation of spherules (prod'homme et al., 2001) . there is evidence that tobacco mosaic virus also uses the er as a site of replication because the replicase enzyme and viral rna are located on the er of infected cells, and infection causes major changes in er morphology (reichel and beachy, 1998) , including er aggregation and formation of lamella structures. flock house virus replicates in spherules in the outer membrane of mitochondria. the rna polymerase (protein a) of flock house virus is the only protein required for rna replication and is targeted directly to the mitochondrial outer membrane by hydrophobic amino acids at the n-terminus. this sequence contains a mitochondrial localization signal and transmembrane domain that leaves the bulk of the protein exposed to the cytoplasm (miller and ahlquist, 2002) . brome mosaic virus replicates in yeast and has been studied extensively. the 1a and 2a replicase proteins are produced from separate viral rnas. the 1a protein contains a c-terminal helicase domain and an n-terminus required for rna capping. 1a is targeted to the cytoplasmic face of er membranes and recruits the 2a polymerase to the replication complex (schwartz et al., 2002) . importantly, replication of brome mosaic virus on the cytoplasmic face of the er in yeast induces membrane invaginations of 50 nm that are very similar to the spherules produced in endosomes and lysosomes during alphavirus infection of mammalian cells. it has been suggested that the active formation of spherules to separate viral rna from host responses is analogous to the coordinated assembly of viral proteins, which leads to capsid assembly, genome packaging, and budding (ahlquist, 2006; schwartz et al., 2002) . the brome mosaic virus replication complex contains viral 1a and 2a pol proteins within spherules. expression of 1a alone produces a shell containing hundreds of copies of 1a on the inside of 50-nm spherules. in a capsid assembly model (schwartz et al., 2002) , vesicles of uniform size would arise if the 1a protein first made a planar lattice with hexameric symmetry on membranes and achieved curvature by localized rearrangement of 1a into pentamers. interestingly, the formation of spherules is dependent on the relative levels of 1a and 2a pol . when levels of 2a pol are high, the spherules are lost, and 1a and 2a pol assemble into flat lamella structures associated with the er (schwartz et al., 2004) . one explanation for a failure to achieve curvature is that high levels of 2a pol may interfere with this hexamer to pentamer transition. this is supported by the observation that when domains that allow association of 1a and 2a pol are deleted, the 2a pol is unable to alter the structure of spherules formed by 1a. the correct ratio of 1a and 2a pol is clearly important for replication complex assembly and may be maintained during infection through inhibition of translation initiation of the 2a rna. d. the flaviviridae replicate in vesicular packets and membraneous webs in the flaviviridae family, which includes the flavivirus, pestivirus, and hepacivirus genera, the rna genome encodes a polyprotein precursor that is cleaved by viral proteases to produce structural proteins from the n-terminal region. the replicase of the flaviviridae is made from nsps, ns5a, ns5b, ns4b, and ns3-4a, found at the c-terminus. wi th the exce ption of the polytop ic ns4b membr ane prote in, whic h is ins erted co transl ationall y into the er, the membr ane-an chored co mponent s of the compl ex are inserted into the cytopla smic face of the er after tran slation ( fig. 1b) . the ns 5b is the rdrp, and a c-termi nal stretch of 21 hydrophobic amino acids directs ns5b to the cytoplasmic face of the er (dubuisson et al., 2002; moradpour et al., 2004) . the ns3 protein has ntpase/helicase activity. ns3 is not a membrane protein but is recruited to the complex through association with membrane-anchored ns4a. ns5a is also membrane associated, and association is mediated via 31 amino acids at the n-terminus that form an amphipathic a-helix (brass et al., 2002; elazar et al., 2003) . replication of flaviviruses (e.g., dengue, west nile, and yellow fever viruses) takes place in membrane invaginations. for historical reasons, these are called vesicular packets [reviewed in mackenzie (2005) ]. they are larger (80-to 100-nm diameter) than the 50-nm alphavirus spherules, and form from the limiting membrane of the trans-golgi network (tgn) (uchil and satchidanandam, 2003; westaway et al., 1997b) . infection by kunjin virus leads to unique membrane structures thought to be derived from both the early and late secretory pathways. these include convoluted membranes and paracrystalline arrays derived from the rough er and ergic, and vesicle packets derived from the tgn (mackenzie et al., 1999; ng, 1987; roosendaal et al., 2006; westaway et al., 1997b) . the detection of dsrna and viral nsps (ns1, ns2a, ns3, and ns4a) within the vesicle packets points strongly to this being the site of rna replication (mackenzie et al., 1998; westaway et al., 1997b) . the vesicle packets associate closely with the convoluted membranes and paracrystalline arrays, which are thought to be the sites of proteolytic processing of ns3 and ns2b (westaway et al., 1997b) . these modified membranes are linked with the er, and ultrastructural studies have shown virions present in the er, cytoplasmic vesicles, golgi cisternae, and vacuoles. the results suggest that membranes containing the spherules responsible for replication may become associated with the er to facilitate delivery of genomes to viruses, budding into early compartments of the secretory pathway (mackenzie and westaway, 2001) . hepatitis c virus (hcv) is closely related to the flaviviruses, and its importance as a human pathogen has generated great interest in its mechanism of replication. until, recently infection models have not been available to study the replication complex of hcv, and the studies discussed here have focussed on the expression of the entire polyprotein from replicons gosert et al., 2003) . however, the recent production of a hcv that rep licates ef ficiently both in vivo and in cell culture (li ndenbac h et al. , 2006; wakita et al., 2005; zhong et al., 2005) will exp and the possi bilities for studying and understanding the viral replication cycle. hcv replication is thought to occur on membranes derived from the er as all studies of nsps have found them localized to this organelle (dubuisson et al., 2002; hugle et al., 2001; kim et al., 1999; wolk et al., 2000) . studies have also identified a ''membraneous web'' of membrane vesicles of $85-nm diameter associated with the er and a population of irregular doublemembraned vesicles. the web resembled the ''sponge-like inclusions'' seen in the liver of chimpanzees infected with hcv, suggesting it is physiologically relevant. interestingly, the great majority of the nsp synthesized by full-length genomes or subgenomic replicons may not be involved in rna replication (quinkert et al., 2005) . the bulk of the nsps associated with membranes isolated from cells expressing replicons is sensitive to protease, while in vitro replicase activity is resistant to protease and nuclease activity (el-hage and luo, 2003; quinkert et al., 2005) . the results suggest that replication of hcv takes place within membrane vesicles, rather than on the surface of the membraneous web. these vesicles may be associated with the membraneous web, but the similarity between hcv and the flaviviruses leaves open the possibility that the membrane invaginations responsible for replication may also form in the tgn but be closely associated with the er. studies have investigated which viral proteins are responsible for membrane rearrangements seen in cells infected with flaviviruses. the ns4a of kunjin virus induces the characteristic convoluted membranes and paracrystalline arrays seen in flavivirus infections. the ns4a-b protein also causes membrane rearrangement, but the highly condensed structures seen in infected cells are not produced until the ns2b-3 protease cleaves ns4a free from ns4b (roosendaal et al., 2006) . the ns4b then translocates to the nucleus (westaway et al., 1997a) . interestingly, this contrasts with hcv where ns4b (and ns4a-b) konan et al., 2003) rather than ns4a is able to induce the membranous structures. flaviviruses have been found to upregulate cell surface expression of mhc class i and ii in response to interferon (king and kesson, 1988; liu et al., 1989; lobigs et al., 2004) . this is not caused by effects of the ns4a or ns4b proteins on membrane traffic; instead flavivirus infection increases expression of the er peptide transporter, tap1. this increases the supply of peptides that are necessary for the folding and export of newly synth esized mhc proteins from the er. inc reased tap expression is media ted by increas ed transcrip tional activ ity of p53 and can be induc ed in liver hepg2 cells by express ion of the hcv core/cap sid pro tein alon e ( herzer et al., 20 03; mombu rg et al ., 2001 ) . whil e the cap sid/cor e prote in is able to in crease cell surface expression of mhc clas s i through increase expres sion of tap1, exp ression of the hc v polyp rotein has been sho wn to slow the moveme nt of prote ins thr ough the secretory pathway of hos t cells ( konan et al ., 2003 ) . the rate of delive ry of mhc cl ass i to the plasm a me mbrane in cells infected with hc v was reduced three-to five fold relative to cu red contr ol cells. exp ression of the pre cursor ns4a-b was fou nd to red uce er-to-g olgi traf fic two-to threefo ld (kon an et al., 2003 ) , while the ot her ns proteins of hc v inclu ding ns4a and ns4b, indiv idually or comb ined, were unabl e to interf ere with the traf ficking pathway . ns 4b a lone indu ces a memb raneo us web in ce lls , and both ns4a-b and ns4b indu ce, and locat e to, clust ered and aggregate d membr anes looking v ery similar to the me mbraneo us web seen in cells expre ssing rep licons. in addition to agg regated me mbrane s, ns4a/b also ind uces, but does not coloca lize with , swol len vesicul ar structure s. thes es swo llen vesicl es have a similar morphology to the vesicles induced by the 3a protein of p o li ov ir us , wh ich swe lls er memb ranes and blocks sec retio n betwee n the er and the golgi appar atus (doe dens et al., 1 997 ). konan et al . (2003) hypo thesize that the ns 4a/b could be func tioning in a sim ilar man ner to poliovi rus 3a. e. the nidovira les replicate in association with doub le-membraned vesicles 1. the nidovirus replicase is generated from two polyproteins the nidovi rales order comp rises the arterivi ridae, coron aviridae , and ron iviridae famil ies. the rep licase gene is co mposed of two ope n read ing fram es termed orf1a and orf1b. orf1b is gen erated from a fram eshift in 1a, and both reading frame s encode co mplex poly proteins pro cessed by viral prote ases (go rbalenya et al., 2006 ; ziebu hr, 2006 ) . the arter ivirus orf1b encode s nsps 9-12, incl uding the rdrp (nsp 9) and helicase (nsp 10). the orf1b, however , lacks hydroph obic dom ains able to target the rep licase to membr anes. intere stingl y, the hydroph obic domains necessar y for membr ane targetin g are enco ded by orf1a in nsp2, 3, and 5, sugge sting that orf1a pr oteins produc e a scaffold to locate the viral rep lication -transcr iption compl ex to membr anes ( fig. 1d ) ( pedersen et al., 1999; van der meer et al., 1998) . a similar strategy is used by cov, for example mouse hepatitis virus (mhv) and severe acute respiratory syndrome-cov (sars-cov) (prentice et al., 2004a,b) , where transmembrane domains are located in nsp3, 4, and 6, and helicase and polymerase proteins are nsp12 and 13, respectively, and nsp16 encodes the methytransferase. the nidovirales have the largest coding capacity of the single-stranded rna viruses, and not all the 16 nsps have been studied in detail. it is possible that other proteins encoded by orfs1a and 1b, such as rna processing enzymes, are incorporated into the replication complex. several studies have investigated the intracellular sites of replication of equine arterivirus (eav), mhv, and sars-cov. such studies are difficult because during nidovirus infection, the processes of replication and envelopment occur on different membranes, and these may merge during encapsidation. furthermore, late during infection cells infected with mhv can form syncitia. newly synthesized mhv viral rna has been found in perinuclear sites colocalized with the rdrp (shi et al., 1999) , and depending on whether human or murine cells were infected, these sites colocalized with golgi or er membranes, respectively. similar studies in mouse l cells report that the polymerase and newly synthesized rna locate to late endosomes and endocytic carrier vesicles . this discrepancy is in part reconciled by later work showing that the subcellular distribution of the replicase proteins can change during the course of infection, since replicase proteins move to sites of envelopment in the ergic (bost et al., 2001) . this is supported by the finding that individual replicase proteins distribute differently following cell membrane fractionation (sims et al., 2000) . membrane fractionation has also been carried out by gosert et al. (2002) , who showed that several proteins encoded by orf1a and b were associated with membranes, and when observed by immunogold electron microscopy, these were associated with rosettes of double-membraned vesicles 200-350 nm in diameter. the role of these vesicles in viral rna replication was confirmed by in situ hybridization of labeled riboprobes. double-membraned vesicles are also seen in cells infected with eav (pedersen et al., 1999) . eav replicase proteins accumulate in perinuclear regions containing ergic and er markers and colocalize with newly synthesized viral rna, again suggesting sites of genome replication. notably, similar structures can be produced by expression of arterivirus orf1a-encoded proteins nsp2-7, which contain the membrane proteins thought to tether the replicase to membranes. double-membraned vesicles are usually rare in cells but are induced during autophagy. a role for autophagy during mhv infection is suggested because autophagy is induced in cells infected with mhv. furthermore, in cells lacking atg5, a protein required for the formation of autophagosomes, there is a 99% reduction in virus yield and mhv fails to induce double-membraned vesicles (prentice et al., 2004a) . electron micrographs show that the double-membraned vesicles induced by sars-cov extend from the er and can be labeled with antibodies specific for replicase proteins. this suggests that, in common with mhv, the vesicles are a site of replication . even though all sars-cov replicase proteins tested colocalize to punctate structures that accumulate near the nucleus, there are conflicting reports about their relationship with autophagosomes. in monkey vero cells, the replicase proteins colocalize with autophagosomes identified using antibodies against lc3 (prentice et al., 2004a) . however, when autophagosomes are identified by expression of gfp-lc3, the replicase proteins do not colocalize with the gfp signal . the vesicles induced by sars-cov are smaller at 100-to 300-nm diameter than autophagosomes (500-1000 nm) and are labeled with er markers. this has lead snijder and colleagues to suggest that they are virus-induced extensions to the er, rather than bona fide autophagosomes (pedersen et al., 1999; snijder et al., 2006) . the precise origins of the membrane crescents that form at the start of autophagy are unclear, and a number of studies have suggested they may form from the er. this makes it possible that the double-membraned structures may be autophagosomes that have been modified by an accumulation of viral protein. determining if autophagy is beneficial to sars-cov replication will have to await studies in cells where key proteins in the autophagy pathway have been removed or suppressed by gene silencing. the asfiviruses, poxviruses, iridoviruses, and the phycodnaviruses are large dna viruses encoding hundreds of proteins from genomes ranging between 150 and 350 kbp. a comparison of protein sequences encoded by these viruses has suggested that they should be grouped together in a family of viruses called the nucleocytoplasmic large dna viruses (ncldv) (iyer et al., 2001) . sequence similarities are seen in the major capsid proteins, redox enzymes that maintain disulphide bonds in the cytosol, and proteins that regulate apoptosis; and the family has been extended to include the giant mimivirus isolated from the ameba acanthamoeba polyphaga (la scola et al., 2003) . even though these viruses infect a diverse range of hosts from different phyla, including vertebrates [poxviruses, african swine fever virus (asfv)], arthropods (entomopox, asfv, chloriridoviruses), amphibians and fish (ranavirus, megalocytivirus, and lymphocystivirus genera of the iridoviridae family), marine algae (phycodnaviruses), and protozoa (mimivirus), they all generate cytoplasmic factories as major sites of virus assembly and replication (illustrated in fig. 4 ). the factories share many similarities with one another, again suggesting that this diverse group of viruses may be related and that the need to produce a virus factory in the cytoplasm was generated early in virus evolution. 1. asfv factories form next to the microtubule organizing center asfv is the sole member of the asfivirus genus, family asfarviridae but shares striking icosahedral similarity with the iridoviruses, phycodnaviruses, and mimivirus. asfv is a large double-stranded dna (dsdna) virus with a genome size ranging from 170 to 190 kbp. gene expression is a regulated cascade and immediate early, early, early/late, intermediate, and true late gene types have been characterized to date. the virion has multiple concentric layers with an electron-dense core at the center that contains the viral genome. a protein matrix surrounds the core, which in turn is enclosed by a lipid bilayer. finally, the bilayer is surrounded by a protein capsid layer. asfv can gain a third envelope when it buds from the plasma membrane at the tip of actin-rich projections that resemble filopodia (jouvenet et al., 2006) . asfv probably enters cells by receptor-mediated endocytosis, but the steps following entry are poorly understood. it is possible that a viral core is delivered into the cytoplasm intact; alternatively, cores may dissociate in endosomes requiring some mechanism of genome delivery across the endosome membrane. genome replication occurs both in the nucleus and cytoplasmic factories. transfer to the nucleus may involve microtubule transport since late gene expression is inhibited by agents that depolymerize microtubules and the dominant-negative dynein motor protein p50-dynamitin (alonso et al., 2001; heath et al., 2001) . asfv does not produce nuclear inclusions analogous to those seen in herpesvirus and adenovirus infection, but there is evidence that small fragments of viral dna are synthesized in the nucleus. the major site of asfv dna replication is, however, the virus factory (rojo et al., 1999) . a. cytoplasmic factories formed during asfv infection are assembled at the microtubule organizing center asfv induces one principal factory in the cytoplasm during infection. electron microscopy shows that the virus factory excludes obvious cellular organelles and contains mostly viral dna, viral proteins, virus-induced membranes, and partially and fully assembled virions (table i; fig. 5a ; brookes et al., 1996; moura nunes et al., 1975; rouiller et al., 1998) . the mechanisms that target viral proteins, virus-induced membranes, and viral dna to the asfv factories are poorly understood. immunofluorescence staining for viral structural proteins generally reveals a strong signal at the factory and a weaker signal in the cytoplasm. the b602lp protein (cap80), which is a viral chaperone involved in folding and membrane recruitment of the major capsid protein, p73, is, for example, absent from the virus factories (cobbold et al., 2001; epifano et al., 2006) . this suggests that p73 is synthesized and folded in the cytoplasm and then recruited to factories. similarly, the viral dutpase, which is necessary for efficient replication, is excluded from the viral factory (oliveros et al., 1999) . since the bulk of viral dna synthesis occurs in the factory (garcía-beato et al., 1992) , it is not easy to explain how the viral dutpase edits uracil from progeny viral genomes, without being present at the site of viral dna synthesis and encapsidation. asfv factories disperse when cells are incubated with drugs that depolymerize microtubules (heath et al., 2001) suggesting their formation involves microtubule motors. this may involve dynein motor proteins since p50-dynamitin, a dominant-negative version of the dynein motor, prevents both late asfv gene expression (heath et al., 2001) and vimentin recruitment to factories (see below and stefanovic et al., 2005) . yeast-twohybrid screens and in vitro pull-down experiments show that one asfv structural protein, p54/j13lp, interacts with dynein (alonso et al., 2001) . while direct binding of p54/j13lp to the motor protein has not been observed in infected cells, it is possible that the protein is involved in transporting some viral proteins into factories. the protein locates to virus factories and deletion of the e183l gene encoding p54/j13lp generates factories that lack viral membranes, the major capsid protein p73, and the polyprotein precursors (pp220, and pp62) of the viral matrix (epifano et al., 2006; rodríguez et al., 2004) . p54/j13lp is a membrane protein with the bulk of the protein, including the dynein-binding motif, exposed to the cytosol. the p73 capsid protein and pp220 polyprotein associate with membranes before assembly into viruses (cobbold and wileman, 1998; cobbold et al., 1996; heath et al., 2003) . if these membranes contain p54/j13lp, it would provide a means of allowing recruitment to factories by retrograde transport along microtubules. the formation and morphology of asfv factories closely resemble the formation of aggresomes (heath et al., 2001) , a cellular response to accumulation of misfolded protein aggregates (johnston et al., 1998) . aggresomes are microtubule-dependent inclusions containing protein aggregates that human herpesvirus 6 induces nuclear tegusomes (t). herpesviruses induce cytoplasmic assembly sites where envelopment and some tegument are acquired (env) in human herpesvirus 5, these sites include electron-dense bodies (db). iridoviruses induce multiple cytoplasmic virus factories (vf) and crystalline arrays (ca), both of which associate with mitochondria. reoviruses also induce multiple cytoplasmic virus factories (vf) and crystalline arrays (ca) that are enclosed within lysosomal membranes. alcamí et al., 1993; alonso et al., 2001; andrés et al., 1997 andrés et al., , 2001 borca et al., 1996; brookes et al., 1998a,b; carrascosa et al., 1986; chacó n et al., 1995; cobbold et al., 1996; galindo et al., 2000; garcía-beato et al., 1992; heath et al., 2001; hingamp et al., 1992; jouvenet and wileman, 2005; jouvenet et al., 2004; martinez-pomares et al., 1997; moura nunes et al., 1975; rodríguez et al., 2006; rouiller et al., 1998; sanz et al., 1985; simón-mateo et al., 1997; sun et al., 1996; vigário et al., 1967 contents of cellular origin ubiquitin, hsp70 chaperone, g-tubulin, pericentrin, p21, mdm1 surrounded by: er membranes, vimentin, p230 golgin, mitochondria, and tubulin. granja et al., 2004; heath et al., 2001; hingamp et al., 1992; jouvenet and wileman, 2005; netherton et al., 2004 netherton et al., , 2006 rojo et al., 1998; rouiller et al., 1998; stefanovic et al., 2005 poxviridae almazán et al., 2001; beaud and beaud, 1997; betakova et al., 2000; chiu et al., 2005; cudmore et al., 1996; da fonseca et al., 2000; davis and mathews, 1993; de silva and moss, 2005; domi and beaud, 2000; krijnse-locker et al., 1996; murcia-nicolas et al., 1999; nerenberg et al., 2005; ojeda et al., 2006; palacios et al., 2005; pedersen et al., 2000; reckmann et al., 1997; resch et al., 2005; risco et al., 1999; roper, 2006; salmons et al., 1997; senkevich et al., 2002; sodeik et al., 1995; szajner et al., 2004a,b,c; tolonen et al., 2001; vanslyke and hruby, 1994; welsch et al., 2003; wolffe et al., 1995; yeh et al., 2000; yuwen et al., 1993 (continued) contents of cellular origin hmg20a viral genome binding protein, hsp90; transient association, ubiquitin, ying-yang 1 transcription factor, tbp transcription factor, sp1 transcription factor, rna polymerase ii, sumo-1, ergic-53 c surrounded by: vimentin and mitochondria. broyles et al., 1999; dales and siminovitch, 1961; hsiao et al., 2006; hung et al., 2002; husain and moss, 2003; nerenberg et al., 2005; oh and broyles, 2005; palacios et al., 2005; risco et al., 2002; wilton and dales, 1989 iridoviridae, ranavirus appearance and contents f electron lucent, virus, viral dna, 108k early protein, 57k, 55k major capsid protein (orf 90r in fv3), 38k, 17k, 16k rana grylio virus dutpase (orf 63r in fv3). surrounded by vimentin, rough er, mitochondria and polysomes. chinchar et al., 1984; darlington et al., 1966; huang et al., 2006; goorha, 1983, 1989; zhao et al., 2007 herpesviridae barnard et al ., 1997; de bruyn kops et al ., 1998; everett and maul, 1994; goodrich et al ., 1990; jahedi et al ., 1999; knipe et al., 1987; lamberti and weller, 1998; leopardi et al ., 1997; liptak et al., 1996; markovitz and roizman, 2000; olivo et al ., 1989; randall and dinwoodie, 1986; reynolds et al ., 2000; taus et al ., 1998; ward et al ., 1996 contents of cellular origin rna polymerase ii, eap ribosome component, proliferating cell antigen, retinoblastoma protein, p53, dna ligase 1, dna polymerase a, promyelocytic leukemia (pml), dna-pkcs, ku86 nonhomologous end joining, bloom syndrome gene product, breast cancer-associated gene 1 protein, msh2, rad50, wrn recq helicase family member, brg1 or brm-associated factor 155, brahma-related gene-1 protein, brahma protein, histone deacetylase 2, hsnf2h, msin3a, tata binding protein (tbp), tbp-associated factors. leopardi et al., 1997; quadt et al., 2006; taylor and knipe, 2004; wilcock and lane, 1991 nuclear sites of capsid assembly or assemblons contents of viral origin d ul7 (hhv-2), ul14 (hhv-2) tegument, ul16 capsid, ul19 icp5 major capsid protein, ul26.5 icp35 dna packaging, ul27 dna packaging, ul35 vp26 p12 capsid, ul38 vp19c capsid assembly, ul43.5, ul55. de bruyn kops et al., 1998; goshima et al., 1998; nalwanga et al., 1996; nozawa et al., 2002; wada et al., 1999; ward et al., 1996a,b; yamada et al., 1998 contents of cellular origin actin, myosin 5a actin motor feierbach et al., 2006 cytoplasmic assembly and envelopment site contents of viral origin d membranes, vacuoles, capsids and enveloped virus ul19 (hhv-2) vp5 major capsid protein ul27 (hhv-2) gb vp7 ul36 (hhv-2) icp1-2, tegument ul46 (hhv-2) tegument, ul48 (hhv-2) tegument kato et al., 2000; murata et al., 2000; nozawa et al., 2004; watanabe et al., 2000 contents of cellular origin mitochondria, g-tubulin, hsp40 chaperone, hsp70 chaperone, gm130 golgi marker murata et al., 2000; nozawa et al., 2004 (continued) becker et al., 2001 becker et al., , 2003 broering et al., 2004; cashdollar, 1994; dales et al., 1965b; miller et al., 2004; sharpe et al., 1982; silverstein and schur, 1970 reoviridae, rotavirus appearance and contents electron-dense viroplasm, assembling and complete double-shelled particles vp2, vp6, vp9, nsp2, nsp5, nsp6 altenburg et al., 1980; gonzález et al., 2000; petrie et al., 1982 petrie et al., , 1984 silvestri et al., 2004 silvestri et al., , 2005 a african swine fever virus gene nomenclature is based on that for the badajoz 1971 vero adapted strain with that of the malawi lil 20/1 strain in parentheses. b vaccinia virus gene nomenclature is based on that for the copenhagen strain with that of the western reserve strain in parentheses. c one report places in ergic-53 within the virosome (risco et al., 2002) , one report places it outside (husain and moss, 2003) . d open reading frames from human herpesvirus 1 (herpes simplex virus 1) unless specified otherwise. e open reading frames from human herpesvirus 5 (human cytomegalovirus) unless specified. f proteins specified by frog virus 3 unless indicated otherwise. form next to the microtubule-organizing center (mtoc). aggresomes recruit cellular components needed to deal with the problems associated with a buildup of aggregated misfolded protein. these include cellular chaperones and proteasomes to facilitate protein folding and/or degradation and mitochondria that may provide the atp required for folding and proteolysis. the most striking structural changes seen during aggresome formation are the collapse of the intermediate filament protein, vimentin, into a cage surrounding the protein aggregates and the gross fragmentation of the golgi apparatus. asfv factory formation shows many similarities with this response to protein aggregation. factory formation is preceded by clearance of cytoplasmic proteins from perinuclear areas around the mtoc. vimentin then concentrates at the mtoc where it forms an aster aligned along microtubules (stefanovic et al., 2005) . following the onset of virus dna replication and synthesis of late structural proteins, the vimentin aster is rearranged into a cage around the factory ( fig. 5b ; heath et al., 2001; monaghan et al., 2003; stefanovic et al., 2005) . during this period, mitochondria and cellular chaperones are recruited to the factory (heath et al., 2001; rojo et al., 1998) . formation of vimentin cages in asfv-infected cells is linked to phosphorylation of vimentin at serine 82 by calcium calmodulindependent protein kinase ii (camkinase ii) (stefanovic et al., 2005) , and drugs that inhibit camkinase ii activity block late gene expression and vimentin rearrangement. as will be discussed for poxviruses and iridoviruses, the vimentin cage may form a physical scaffold within the factory, or act as a cage to prevent movement of viral components into the cytoplasm. chaperones recruited to the factory may facilitate folding of viral structural proteins during assembly, as has been shown for other viruses. the proximity of mitochondria to viral factories may provide the atp that is required for asfv assembly (cobbold et al., 2000) or be indicative of an antiviral response as mitochondria are effectors of apoptosis. taken together these results suggest that a cellular response originally designed to deal with the buildup of protein aggregates in cells is used by asfv to generate a site specialized for virus assembly. as will be described later, similarities between aggresomes and virus assembly sites are also seen for the iridoviruses and poxviruses. following the onset of asfv dna replication, the microtubule network becomes disorganized. microtubules are partially excluded from virus factories and form bundles and concentric rings in the cytoplasm (jouvenet and wileman, 2005) . asfv infection leads to disassembly of g-tubulin and pericentrin from the centrosome, and the centrosome becomes less able to nucleate microtubules. at the same time microtubules are stabilized by acetylation (jouvenet et al., 2004) . since pericentrin and g-tubulin play key roles in microtubule organization and nucleation at the mtoc, their loss from the centrosome, coupled with acetlylation of tubulin, may explain the rearrangement of microtubules induced by asfv. the reasons for these profound effects on microtubules are not known but they may facilitate disruption of the virus factory allowing release of assembled viruses into the cytoplasm. c. membrane rearrangements caused by asfv infection perturb the secretory pathway current models for asfv envelopment in virus factories predict that viral membranes are obtained from the er. the major structural proteins are recruited from the cytoplasm onto the cytoplasmic face of the er, and after which protein-protein interactions between these, and possibly viral proteins targeted to the er lumen, lead to constriction of er cisternae and clearance of host proteins from the er lumen prior to envelopment (andrés et al., 1998; netherton et al., 2004 netherton et al., , 2006 rouiller et al., 1998) . this is consistent with low levels of er proteins observed at asfv assembly sites by immunoelectronmicroscopy (rouiller et al., 1998) and standard fluorescence microscopy where er proteins appear to be actively excluded from areas of viral replication (andrés et al., 1998; netherton et al., 2004) . in addition to effects on the er, asfv also affects the structure and function of later golgi compartments of the secretory pathway (mccrossan et al., 2001; netherton et al., 2006) . golgi structure is linked to microtubule organization and the changes seen during infection may in part be related to effects of asfv infection on centrosome and microtubule function listed above. asfv infection causes dispersal of ergic marker protein ergic-53, the peripheral golgi protein gm130, and late golgi protein galnac-t2 transferase, suggesting disruption of ergic and golgi membrane compartments. most striking is the complete loss of the tgn. tgn loss is dependent on microtubules and involves dispersal of the tgn into separate vesicle populations containing either peripheral golgi proteins or the integral membrane protein, tgn46. not surprisingly, this dispersal slows the transport of proteins through the secretory pathway. asfv slows the delivery of newly synthesized lysosomal enzymes to lysosomes (mccrossan et al., 2001) , and in macrophages reduces transport of newly synthesized mhc class i to the plasma membrane (netherton et al., 2006) . thus, in common with picornaviruses, disruption of the secretory pathway by asfv has the potential to slow the transport of important immunomodulatory proteins to the surface of infected cells and may mask them from immune surveillance. poxviruses are large dsdna viruses with genomes ranging from 130 to 375 kbp. poxvirus gene expression follows the regulated cascade of other large dsdna viruses with early, intermediate, and late transcripts described. poxvirus progeny genomes are replicated exclusively in the cytoplasm in virus factories. the virus encodes all the enzymes necessary for transcription and replication of its genome. genetic analysis has identified a minimum of five viral genes necessary for genome replication, these are a20r, b1r, d4r, d5r, and e9l encoding the dna polymerase processivity factor, serine/threonine protein kinase, uracil dna glycosylase, dna-independent nucleoside triphosphatase, and the dna polymerase, respectively (de silva and moss, 2005; evans et al., 1995; millns et al., 1994; punjabi et al., 2001; rempel et al., 1990; sridhar and condit, 1983) . only the product of the d4r gene, encoding the viral dna glycosylase, has been confirmed to localize to the site of genome synthesis (de silva and moss, 2005) , and it would be interesting to discover the subcellular location of the other members of the minimum replicase. when viewed by electron microscopy, infectious virions have a striking brick-shaped morphology, and different forms of virus are documented which vary in degree of complexity [for review, see condit et al. (2006) ]. the interior of all poxvirus particles contains the virus core which houses the viral genome. cores are enveloped in virus factories to produce the intracellular mature virus (imv), which is fully infectious. additional envelope layers gained at the tgn give rise to intracellular enveloped viruses (iev), which after budding through the plasma membrane form cell-associated and extracellular enveloped viruses (cev and eev). poxviruses induce two principal inclusions during infection, the a-type inclusion that is nonreplicative and the b-type inclusion where virus replication and assembly occur in the virus factory ( fig. 4 ; kato et al., 1959). a. poxvirus a-type inclusions contain the mature intracellular virus but not enveloped viruses a-type inclusions are cytoplasmic bodies of dense homogeneous matter that contain mature virus particles and are studded with polyribosomes (fig. 6a) (ichihashi et al., 1971) . a-type inclusions are extremely rare in vaccinia, variola, and rabbit pox infections but are prominent in cowpox, ectromelia, fowlpox, and canarypox infections where they are also referred to as downie, marchal, bollinger, and burnet bodies, respectively (kato et al., 1959) . the major component of a-type inclusions is the product of the a26l gene or its equivalents. in vaccinia, a26 is truncated and produces a protein of 92-94 kda whereas the fulllength gene in cowpox encodes a protein of 160 kda (patel et al., 1986) , both versions are myristylated (martin et al., 1999) . immunfluorescence analysis of cells infected with vaccinia virus with antibodies raised against a26 does reveal multiple a-type inclusions in the cytoplasm, but they are much smaller than those seen in cells infected with cowpox, and do not contain virus particles (patel et al., 1986) . in cells infected with wild-type cowpox, only imv particles were observed within a-type inclusions, but treatment with rifampicin, a drug that blocks poxvirus maturation at an early stage in morphogenesis, caused aberrant immature virus particles to integrate into the inclusions (ichihashi et al., 1971) . the factor necessary for occlusion of viral particles in a-type inclusions has been identified as the 4c core protein (mckelvey et al., 2002; shida et al., 1977; ulaeto et al., 1996) . it has been hypothesized that 4c retains vaccinia virions within the cell as imvs in a-type inclusions preventing their transport to the tgn for envelopment and maturation to the iev types of virion (mckelvey et al., 2002) . a-type inclusions are predicted to protect imvs during transport between hosts akin to that of the polyhedra that occlude entomopox and baculoviruses (rohrmann, 1986) . therefore, eevs may be important for cell-to-cell spread, while imvs (whether occluded or not) may be more important for host-to-host spread (mckelvey et al., 2002) . b. poxvirus b-type inclusions are factories and are the main sites of replication and assembly b-type inclusions originally called guarnieri bodies (guarnieri, 1893) are the primary replication centers of the poxviruses, now generally referred to as virosomes or virus factories (fig. 6c) . electron microscopic analysis of b-type inclusions revealed a granular matrix that was denser than the surrounding cellular material and in a defined area of the cytoplasm called viroplasm (dales and siminovitch, 1961; higashi, 1973) . the factories also contain viral crescents consisting of membrane and viral proteins associated with viroplasm, spherical immature virus, and imvs (dales and siminovitch, 1961) . factories are surrounded by mitochondria, increase in number and size during the replication cycle and can occupy the majority of the cytoplasm at late times of infection (dales and siminovitch, 1961) . the assembly and envelopment of vaccinia virus within virus factories has been the subject of many studies and is discussed in papers and reviews (griffiths et al., 2001; heuser, 2005; hollinshead et al., 1999; sodeik and krijnse-locker, 2002) . here, we will review some of the early steps that lead up to the start of genome replication and factory production. these have also been described in a review (schramm and krijnse-locker, 2005) . it is generally believed that infection results in the delivery of viral cores into the cytoplasm. cores are seen associated with microtubules (carter et al., 2003; mallardo et al., 2001; ploubidou et al., 2000) and may use microtubules to reach perinuclear sites that will eventually house the virus factories. viral cores can transcribe as many as 100 early mrnas before the onset of dna replication, and these early mrnas appear in discrete foci that associate with microtubules, contain polyribosomes and other translational machinery. it is unlikely that foci involved in transcribing early rnas mature into viral replication sites because they do not initiate dna synthesis (mallardo et al., 2002) . it is likely that each infecting virus can induce its own replication center (cairns, 1960) , but it is not clear where in the cell the cores initiate dna synthesis. it has been suggested that the onset of dna synthesis may occur at peripheral sites and therefore precedes delivery to the perinuclear region of the cell. when cells are incubated with hydroxyurea to prevent the onset of viral dna replication, it is possible to localize viral dna released into the cytoplasm. under these conditions, viral genomes are seen at several discrete sites that contain b1 protein kinase, e8 membrane protein, i3 ssdnabinding protein, and h5 late transcription factor (domi and beaud, 2000; welsch et al., 2003) . after removal of hydroxyurea, these foci begin to make new viral dna, showing that they are sites of dna replication. live cell imaging studies have shown that these initial sites of dna replication form in the cell periphery and then move toward the nucleus where they coalesce into large structures (schramm and krijnse-locker, 2005) . electron micrographs suggest that sites of dna release from cores are intimately associated with er membranes and become completely enclosed by them during the initial stages of dna replication (mallardo et al., 2002) . this process is likely facilitated by the e8r gene product which is a membrane protein localized to the er and early golgi membranes, has dna-binding activity, and is able to capture viral genomes tolonen et al., 2001) . these er-enclosed genomes are short-lived structures because they are not seen once viral crescents, iv and imvs, appear in factories (tolonen et al., 2001) . the sites of dna replication are also separate from the foci involved in transcribing early rnas, and it is interesting to consider how the cores are separated from newly transcribed rna. viral cores and sites of rna transcription both align on microtubules and partially colocalize with the l4 core dna-binding protein (mallardo et al., 2001) . the l4 protein is able to bind microtubules (ploubidou et al., 2000) and may be involved in separating rna from cores along microtubule tracks (mallardo et al., 2001) . inducible recombinants or temperature-sensitive mutants grown under nonpermissive conditions can give further insight into the early stages of inclusion formation. electron micrographic analysis of the factories formed under these conditions yield striking images of distinct inclusions of homogeneous electron-dense viroplasm next to empty spherical immature virions ( fig. 6b and c) (szajner et al., 2001 (szajner et al., , 2003 (szajner et al., , 2004a . a seven-protein complex comprising the gene products of the a15l, a30l, d2l, d3l, f10l g7l, and j1r open reading frames has been identified as being necessary for association of viral membranes with the viroplasm (szajner et al., 2004a) . consistent with this role, all of these proteins are known to localize to the virus factory except d2 and d3 (table i) ; however, these have been identified as core proteins (dyster and niles, 1991) so are likely to reside at viral assembly sites. localization of d13l to the virus factory is sensitive to the antibiotic rifampicin (miner and hruby, 1989) , and treatment with this drug induces irregular shaped viral membranes instead of the well-defined hemispherical viral crescents seen in natural infection (moss et al., 1969; pennington et al., 1970) . therefore, it was suggested that d13l may act as a scaffold on which the viral membrane is shaped, allowing correct association with the viroplasm (mohandas and dales, 1995) . deep etch electron microscopy has confirmed this role for d13l, as it forms the honeycomb lattice identified as the outer coat of the viral membrane of immature virions (heuser, 2005; szajner et al., 2005) . interestingly, d13l shares a structural similarity with structural proteins from many other virus families, including those of the other large dsdna viruses (benson et al., 2004) . it will be interesting to see if the structural similarities to d13l translate to functional similarities in the assembly strategies of other viruses. to the viral factory. ying-yang 1 (yy1), tbp, sp1 transcription factors, and rna polymerase ii are recruited from the nucleus to the factory (broyles et al., 1999; oh and broyles, 2005; wilton and dales, 1989) . yy1 is a nuclear transcription factor that can activate late viral promoters and although poxviruses encode most of the genes necessary for transcription, there is evidence that cellular factors may be required for intermediate and late gene expression (lackner and condit, 2000; rosales et al., 1994; wright et al., 2001) . the function of the other transcription factors in viral replication is unknown. they may be necessary for viral transcription like yy1, or perhaps they are sequestered into the factory to divert them from their normal roles in the nucleus, or their presence may represent an antiviral response by the cell. the presence of rna polymerase ii in the viral factory is a surprise because the virus encodes its own rna polymerase activity which accounts for at least 9 orfs and $7% of the genome capacity [western reserve (wr) strain]. another cellular protein recruited from the nucleus to the cytoplasm is the hmg20a protein. this protein can bind the viral genome and has been implicated in host range restriction of vaccinia virus in chinese hamster ovary cells (hsiao et al., 2006) . during unproductive infection by vaccinia virus, hmg20a is recruited from the nucleus to the factory where it binds viral dna. if the cowpox host range gene cp77 is artificially introduced into vaccinia virus then cp77 also enters the virus factory and binds to hmg20a; the cellular protein then dissociates from the viral genome and replication proceeds (hsiao et al., 2006) . as seen for iridovirus and asfv replication sites, vaccinia factories are surrounded by a vimentin cage (risco et al., 2002; schepis et al., 2006) and recruit molecular chaperones (hung et al., 2002) , suggesting similarity with aggresomes. many proteins targeted to aggresomes are ubiqutinated, and most poxviruses encode a ring protein that is both a functional ubiquitin ligase and a virulence factor (nerenberg et al., 2005) . exceptions to this are the two most common laboratory strains of vaccinia, copenhagen and wr. the ring protein from the ihd-w strain of vaccinia is capable of directing transfected tagged ubiquitin to wr virus factories (nerenberg et al., 2005) ; however, it is unknown if native ubiquitin is localized to wr factories. the product of the a40r gene of vaccinia is tagged with the ubiquitin-like protein sumo-1, and this modification is necessary for a40 targeting to viral factories, where it associates with er membranes and may play a role in the formation of i3 sites (palacios et al., 2005) . it is not known if movement of sumolyated a40 and ubiquitinated protein is directed along microtubules in a manner analogous to hdac6mediated targeting of misfolded proteins to aggresomes (kawaguchi et al., 2003) . as reported for asfv (see above) and cells infected with herpes simplex virus (avitabile et al., 1995) , infection of cells with vaccinia virus also leads to disruption of microtubule organization and centrosome function and dispersal of the golgi apparatus (ploubidou et al., 2000) . whether these are bystander effects of the production of virus factories close to the centrosome or induced deliberately to facilitate virus egress is not known. imv exit from the factory and transport to envelopment sites at the tgn is nonetheless dependent on microtubules (sanderson et al., 2000) and has been reported to be dependent on the a4l and a27l gene products (sanderson et al., 2000; ward, 2005) . following envelopment, the a35l and f12l gene products then regulate microtubule-dependent movement of intracellular enveloped viruses from the tgn to the plasma membrane (herrero-martínez et al., 2005; ward and moss, 2001 ). crystalline arrays a. iridoviruses iridoviruses are large dsdna viruses with genomes ranging from 100 to 210 kbp in length encoding between 100 and 230 proteins (williams et al., 2005) . much of the work on iridovirus replication has been carried out on the ranavirus frog virus 3 (fv3). fv3 genome synthesis occurs in the nucleus and cytoplasm. no nuclear inclusions have been reported during fv3 infection, and as such it is unclear how the nuclear replication stage is mediated. however, viral dna is initially synthesized as units that are 1-2 genomes in length and then transported to the cytoplasm where multiple length concatemers are produced (goorha, 1982) . b. cytoplasmic factories formed during iridovirus infection resemble aggresomes infection induces two cytoplasmic inclusions. viral factories form in the cytoplasm and become the major site of viral dna replication. fv3 also induces large crystalline arrays of viral particles which give rise to the iridescent coloring of purified virus, and hosts, that are characteristic of iridovirus infections. virus factories are electron lucent relative to the cytoplasm and contain viral membranes, partially assembled viruses, and are surrounded by rough er membranes and polysomes. fv3 factories also resemble aggresomes since they recruit intermediate filaments (fig. 7a) and mitochondria, some of which show darlington et al. (1966) with permission from elsevier. signs of damage (darlington et al., 1966; granoff et al., 1966; huang et al., 2006; tripier et al., 1977) . crystalline arrays of virus are associated with virus factories and can induce nuclear deformations that lead to kidneyshaped nuclei similar to those seen in asfv infection (fig. 7b ) and after aggresome formation (darlington et al., 1966; heath et al., 2001; johnston et al., 1998) . as seen for asfv and poxviruses, the intermediate filament vimentin plays an important role in replication (murti and goorha, 1983) . vimentin is phosphorylated during fv3 infection, prior to factory formation (chen et al., 1986; willis et al., 1979) , and temperature-sensitive mutants that are unable to phosphorylate vimentin do not form vimentin cages and are unable to proceed to late gene expression. drug treatment with taxol or colchicine (murti et al., 1988) showed that recruitment of vimentin to assembly sites requires dynamic, but not polymerizing microtubules, and microinjection of anti-vimentin antibody prevented recruitment of vimentin to factories. this allowed intrusion of cell components into assembly sites and reduced virus growth by 70-80% (murti et al., 1988) . vimentin may therefore provide a scaffold for iridovirus replication, maintaining a barrier between the cytoplasm and the contents of the virus factory. consistent with this hypothesis is the observation that during infection, polyribosomes and most newly synthesized viral proteins associate with intermediate filaments (murti and goorha, 1989) . fv3 factory formation may also be dependent on the early 108k protein, as it is recruited to factories in the absence of late protein synthesis (chinchar et al., 1984) . phycodnaviruses and the recently described giant virus mimivirus (la scola et al., 2003) induce replication complexes in the cytoplasm of infected ameba (meints et al., 1986; raoult et al., 2007) . the factories of the phycodnavirus paramecium bursaria chlorella virus 1 (pbcv-1) are electron translucent areas of the cytoplasm and contain viral membranes, electron-dense viroplasm, and assembling viruses. unlike many viral factories, a distinct order appears to be present in pbcv-1 virosomes. the assembling viruses are arranged at the periphery of the virosome/ factory, giving the appearance of a rosette (meints et al., 1986) . phycodnavirus replication and factory formation are not affected by a wide range of pharmacological disruptors of the cytoskeleton, including microtubule depolymerization by nocodazole and taxol, and depolymerization of actin by cytochalasin d (nietfeldt et al., 1992) . in this way, they differ from factories formed by large dna viruses such as asfv, vaccinia, and fv3. the successful cultivation of algae in the laboratory has allowed studies of the intracellular sites of replication of large icosahedral mclav-1 and hincv-1 viruses (wolf et al., 1998 (wolf et al., , 2000 . these viruses produce a latent infection that becomes apparent once the algae produce reproductive organs that become host to millions of virus particles. replication of these viruses begins in the nucleus, but the first evidence for virus assembly is provided by the appearance of electron-dense bodies next to the nucleus at sites of breakdown in the nuclear envelope. infection leads to stacking of er cisternae that may provide membranes for virus envelopment. the dense bodies remain next to the nucleus in large inclusions, and take on the angular shape characteristic of capsid assembly seen for iridoviruses and asfv. the nucleus eventually disintegrates, and the virus factory occupies most of the cytoplasm. herpesviruses are large dsdna viruses with genomes ranging in size from 120 to 250 kbp. herpesvirus genes are expressed in a regulated cascade starting with the immediate early a genes, then early b genes, and finally two subsets of late g genes, g 1 and g 2 . complete herpesvirus particles have four main layers, the core containing dna, an icosahedral capsid, a poorly defined layer of protein called tegument, and finally the viral envelope containing several glycoproteins. genome synthesis and packaging and capsid assembly occur in inclusions in the nucleus. nucleocapsids then obtain tegument in either the nucleus or the cytoplasm, or both, and the viral envelope is acquired exclusively in the cytoplasm [see mettenleiter (2002) and for more thorough analysis]. the transfer of virus from the nucleus to the cytoplasm and acquisition of tegument appears well defined for human herpesvirus 6 (hhv-6) (roffman et al., 1990) but is controversial for the alphaherpesviruses (campadelli-fiume and roizman, 2006; mettenleiter and minson, 2006) . the subcellular organization of herpesvirus replication complexes formed in the nucleus during the early stages of productive infection has been described in considerable detail. the inclusions function as sites of virus replication and contain the virally encoded proteins and host proteins needed for virus replication. interestingly, nuclear inclusions formed during herpes virus replication also contain cellular proteins involved in the control of dna damage and repair. these may be recruited into inclusions in response to virus genome replication, and whether they are beneficial or detrimental to virus replication is a subject of considerable interest [reviewed by everett (2006) ]. herpesviruses enter the cell by fusing their envelopes with the plasma membrane, whereon the naked nucleocapsids migrate to nuclear pores, possibly along microtubules (granzow et al., 1997; sodeik et al., 1997) [reviewed by smith and enquist (2002) ]. nuclear inclusions housing herpesvirus dna replication are globular and can occupy the majority of the nucleus (de bruyn kops and knipe, 1988; randall and dinwoodie, 1986; taylor et al., 2003) . they are identified through the presence of the viral dna-binding protein encoded by the ul29 gene, which is also known as infected cell protein 8 (icp8). a minimum set of seven genes, ul5, ul8, ul9, ul29, ul30, ul42, and ul52, has been identified as necessary for viral dna replication (challberg, 1991) . a plasmid transfection system has shown in vitro these can form globular nuclear compartments that are sites of 5-bromo-2 0 -deoxyuridine (brdu) incorporation and visually are similar to those formed during infection zhong and hayward, 1997) . nuclear inclusions organizing viral dna replication have been followed in real time by a recombinant virus expressing a gfp-icp8 fusion protein. small inclusions merge with adjacent replication complexes and increase in size to form globular replication complexes, which eventually fill most of the nucleus (randall and dinwoodie, 1986; taylor et al., 2003) . replication compartments are formed from a number of different discrete foci that are induced early in infection and whose interrelatedness is not fully understood. the initial stages of productive herpesvirus infection are, however, intimately linked with nuclear structures called nd10 bodies (illustrated in fig. 8 ) , maul et al. (1996) , review by borden (2002) ]. live cell studies have shown that the immediate early regulatory protein icp4, which binds viral dna, forms discrete foci as early as 30-min postinfection (fig. 8a) . these initially appear close to the nuclear envelope, possibly at sites where the genome first enters the nucleus following capsid disassembly at nuclear pores (everett and murray, 2005) , and are then seen throughout the nucleus (everett et al., 2004) . icp4 foci are seen juxtaposed to the nd10 marker promyelocytic leukemia protein (pml) some 60-min later. the early and late regulatory protein icp27 is recruited to icp4 foci 2-h postinfection and facilitates efficient early gene expression (everett et al., 2004) . during the same period, the immediate early regulatory protein, icp0, colocalizes with nd10 bodies, some of which are likely juxtaposed to icp4 bodies (everett et al., 2003) . icp0 mediates the ubiquitin and/or sumo-1-targeted proteasomal degradation of nd10 components (chelbi-alix and de everett, 2000; everett and maul, 1994; everett et al., 2004) . finally, parental genomes localize to icp4 foci (everett and murray, 2005) , and the icp4 foci enlarge into structures that resemble early icp8 replication compartments (everett and murray, 2005; everett et al., 2003) . formation of icp8 replication compartments (taylor and knipe, 2004) is also known to involve redistribution of nd10 bodies (burkham et al., 1998) . the relationship between the early icp4 structures associated with parental genome and the later icp8 compartments associated with replication and production of progeny genome is not clear; however, icp4 and icp8 both localize to late replication compartments (knipe et al., 1987) . a description of the relative and temporal distribution of the two proteins at early times awaits live cell studies following both proteins simultaneously. a second prominent nuclear inclusion induced by herpesvirus infection is the assemblon (ward et al., 1996b) . this is the site where capsid proteins accumulate and assemble into nucleocapsids (fig. 8b) . the assembly of herpesvirus nucleocapsids has been researched in great detail at the ultrastructural level facilitated by a cell-free system for reconstituting the particles (heymann et al., 2003; newcomb et al., 1994 newcomb et al., , 1996 . the mature herpesvirus capsid is icosahedral with a t ¼ 16 symmetry and is composed of 150 hexons and 11 pentons of the major capsid protein ul19. the place of the remaining penton is taken by a 12-mer of the portal protein ul6, which by analogy with bacteriophage may be the site of genome entry. nucleocapsids mature from fragile procapsids, through b capsids that lack dna and contain the internal scaffold protein ul26.5, to c capsids that contain the viral genome. the relationship between assemblons and sites of viral dna replication has been a topic of some controversy as some reports show direct colocalization (taus et al., 1998) , whereas others have shown a proximity (nalwanga et al., 1996; ward et al., 1996b) , similar to that seen between nd10 bodies and icp4 foci during the initial stages of infection. clearly, the dna has to reach the capsid in order to complete assembly, and it is likely that the different results are indicative of the dynamic interactions between different herpesvirus nuclear inclusions. the dna cleavage and packaging proteins encoded by the ul17 and ul32 genes are required for colocalization of viral dna and capsids (lamberti and weller, 1998; taus et al., 1998) . cells infected with a virus encoding a faulty ul32 gene exhibit nuclear localization of the capsid protein vp5 that is separate from replication sites (lamberti and weller, 1998) . similarly, in cells infected with mutants that lack functional ul17, the icp8 protein fails to colocalize with icp5 and icp35 (taus et al., 1998) . actin also plays an important role in the correct nuclear subcompartmentalization of viral proteins. infection with hhv-1 1 or suid herpesvirus-1 2 causes actin filaments to assemble in the nucleus, prior to the accumulation of capsid proteins (feierbach et al., 2006) . depolymerization of actin with latrunculin a inhibited correct nuclear compartmentalization of a representative capsid protein (vp26). vp26 colocalizes with the actin motor myosin va (feierbach et al., 2006) , and capsid movement within the nucleus is inhibited by the myosin motor inhibitor 2,3-butanedione monoxime (forest et al., 2005) . this suggests that the organization of nuclear inclusions involved in herpesvirus assembly is dependent on cellular actin filaments, and it will be interesting to see if the organization of inclusions housing viral dna replication sites is similarly dependent. other inclusion bodies have been reported in the nucleus of cells infected with herpesvirus. the tegument proteins vp22 and vp13/14 localize to inclusion bodies that align closely but do not overlap icp0/nd10/icp8 pre-replication complexes or assemblon inclusions (hutchinson et al., 2002) . ul55 also localizes to structures that overlap but are distinct from assemblons and dna replication complexes (fig. 8b ) . ul11 localizes to type iv and type v intranuclear dense bodies as well as virions and cytoplasmic ribbon structures (baines et al., 1995) . the alkaline dnase encoded by the ul12 gene localizes to discrete electron-dense bodies within the nucleus that also contain b-36 nucleolar protein (lopez-iglesias et al., 1988; puvion-dutilleul and pichard, 1986) . it is unknown whether these different structures are related to each other, whether they are homogenous accumulations of the individual herpesvirus protein(s), or if they are simply dead-end accumulations of protein. a large number and variety of cellular proteins accumulate at nuclear sites of herpesvirus replication and assembly. a comprehensive proteomic analysis of icp8 interacting proteins revealed more than 50 viral and cellular proteins that maybe recruited to dna replication sites (taylor and knipe, 2004) . a number of these interacting proteins were confirmed to localize to replication sites by microscopy experiments (taylor and knipe, 2004) , and these as well as proteins identified in other studies (leopardi et al., 1997; quadt et al., 2006; wilcock and lane, 1991) reveal that at least 23 cellular proteins are known to localize to nuclear inclusions involved in dna replication during herpesvirus infection (table i) . the functions of these proteins span the expected functions of nuclear genes, including dna replication, transcription, chromatin remodeling, dna repair, recombination, and nonhomologous end joining. of particular importance is the recruitment of rna polymerase ii, which is required to transcribe the viral genome. rna polymerase ii is phosphorylated during viral infection by icp22 and icp27, and the latter modification is required for targeting to replication complexes (dai-ju et al., 2006) . the role of all of these cellular genes in the viral replication cycle is poorly understood; however, cells deficient in wrn, a recq helicase family member, produced reduced virus yields while cells lacking ku86, part of a nonhomologous end-joining protein complex, produced increased yields of virus (taylor and knipe, 2004) . the implication therefore is that some cellular proteins may be actively recruited to replication complexes to aid viral replication, and some may be recruited by the cell as part of an antiviral response or sequestered by the virus in inclusions to subvert their antiviral nature. pml is induced by interferon, suggesting an antiviral role. many of the genes shown to be required for recruitment of pml to viral pre-replication sites are part of the minimal set of genes required to synthesize viral dna. recruitment of pml to viral replication sites is, for example, dependent on the viral dna polymerase (ul30), the origin binding protein (ul9 gene) and the helicase-primase complex (ul5, ul8, and ul52) (burkham et al., 2001) . recent evidence has suggested that this may be the reason why icp0 causes dispersal of pml early in infection. pml knockdown by short interfering rnas (sirna) facilitates productive replication of icp0 null mutants of herpesvirus (everett et al., 2004 ; moreover, icp0 null mutants are hypersensitive to interferon in a manner dependent on pml (chee et al., 2003) . this is of particular importance because icp0 plays a role in determining whether herpesvirus induces a quiescent or a productive, lytic infection (mossman and smiley, 2002) . the tegument layer of alphaherpesviruses is composed of at least 15 different proteins (mettenleiter, 2002) . us11, ul17, ul47, ul48, and ul49 are components of the tegument, and all are localized to the nucleus (if not exclusively) during the productive life cycle of the virus hutchinson et al., 2002; kopp et al., 2002; roller and roizman, 1992; taus et al., 1998) . ul48 may play a role in egress from the nucleus, though this has not been unequivocally established (mossman et al., 2000) . therefore, it is likely that some tegument proteins are acquired in or during viral egress from the nuclear inclusions. recently, cytoplasmic aggresome-like struct ure s have been desc ribed in ce lls infected with hhv-2. 1 these contain the major capsid protein, tegument proteins, envelope glycoproteins, and markers for the golgi complex (nozawa et al., 2004) . the latter finding is particularly interesting because herpesvirus envelopment involves membranes from the tgn turcotte et al., 2005) . hhv-5 3 is a betaherpesvirus and late during infection produces a juxtanuclear ''assembly compartment'' that again contains tegument proteins (pp150, pp28, and pp68), the major capsid protein, and viral envelope proteins (gb, gh, and gp65), suggesting a cytoplasmic site specialized for tegumentation and envelopment (fig. 8c) ; (adair et al., 2002; sanchez et al., 2000) . the precise role of the cytoplasmic assembly compartment is unclear. on the one hand, the concentration of glycoproteins and tegument proteins in one site may facilitate final stages of assembly prior to release from the cell. interestingly, in common with aggresomes induced by asfv and misfolded proteins, the cytoplasmic assembly compartment recruits chaperones and mitochondria and is dependent on microtubules and localizes to the microtubule organizing center. at present, the assembly compartments are not considered to be bona fide aggresomes because they are not surrounded by a collapsed cage of intermediate filaments (nozawa et al., 2004; sanchez et al., 2000) . it is nevertheless possible that these structures are related to aggresomes and are produced in response to a buildup of products resulting from nonproductive assembly pathways that occur late during infection. they may also contribute to the cytopathic effect seen in cells infected with hhv-5. hhv-5 infection results in cytomegaly characterized by increased cell size and intracellular water content. cytomegaly and virus replication are both dependent on the presence of extracellular na þ , and infection results in sequestration of the plasma membrane na-k-cl-cotransporter protein in large perinuclear structures that resemble the assembly compartment/ viral aggresome (maglova et al., 2004) . electron-dense bodies can be seen by electron microscopy within the cytoplasmic assembly compartments induced during hhv-5 infection (craighead et al., 1972) . dense bodies are enveloped and obtain viral glycoproteins but do not contain dna and are noninfectious. as can be seen in fig. 8c , dense bodies bud into membranes and appear as oversized enveloped viral particles without a dna containing core. dense bodies exit the cell to become extracellular dense bodies (craighead et al., 1972) . interestingly, hhv-5 immediate early ie1 proteins also become associated with extracellular dense bodies despite no reported localization to their intracellular relations (tsutsui and yamazaki, 1991) . purified extracellular dense bodies are mostly composed of ul83 but have a full complement of viral glycoproteins (irmiere and gibson, 1983) . the function of dense bodies remain unclear, and they may represent the end point of a nonproductive assembly pathway resulting from attempts to envelope capsids lacking genomes or may be used to deliver viral components to neighboring cells. interestingly, for human herpesvirus 6 (hhv-6), the tegument layer appears to be acquired within a dedicated structure that has been dubbed the tegusome (roffman et al., 1990) . this work is based on electron microscopy of cells infected with hhv-6 and shows tegusomes as intranuclear membrane compartments that abut the nuclear envelope (fig. 8d ). tegusomes may be cytoplasmic invaginations of the nuclear envelope into the nucleus because they appear to contain ribosomes and are sometimes in continuity with the cytoplasm. nucleocapsids appear to bud into the tegusome, capsids obtain a tegument layer, and then bud into cytoplasmic vacuoles where they acquire envelopes and exit the cell. adenovirus are medium-sized, nonenveloped dsdna viruses with genomes ranging from 26 to 45 kbp in length and virions of the order of 70-100 nm in diameter. like other dna viruses, they have an ordered cascade of transcripts, early, delayed early, and late types having been described. adenovirus transcripts are spliced to generate multiple transcripts from a given transcriptional unit. viral replication occurs in the nucleus, and adenovirus infection was utilized extensively as a model system for exploring different nuclear subcompartments. a productive infection of lytic adenovirus induces profound rearrangement of existing subcompartments and the induction of several new ones within the host nucleus. a study on the localization of the human adenovirus5 iva2 protein described 10 distinct nuclear and nucleolar subcompartments induced or associated with virus replication (lutz et al., 1996) , and these are listed in table i . earlier studies carried out before markers for specific nuclear subcompartments were available have described the structures in terms of shape and location (see table i ). during the initial stage of infection, viral rna , single-stranded dna (ssdna), and dsdna (puvion-dutilleul and pichard, 1992) are all synthesized in small fibrillar regions termed early replication sites. by the intermediate stage of replication, the ssdna is deposited in the center of these structures, while transcription and dsdna synthesis occur on the outside and begin to form an inclusion. the inclusion has a characteristic doughnut shape, and has been called the fibrogranular network. at late stages of infection, dsdna, viruses, and trace amounts of ssdna appear in large viral inclusions (besse and puvion-dutilleul, 1994; puvion-dutilleul and pichard, 1992) . targeting of the initial replicon is dependent on a dcmp modification of the preterminal protein (ptp), which enables ptp to form a complex with the dna polymerase and the genome (temperley and hay, 1992) . ptp mediates targeting of the heterotrimeric complex to the nuclear matrix (fredman and engler, 1993) , possibly through an interaction with cad (carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase) (angeletti and engler, 1998) . transcription and splicing are mediated by host proteins and viral rna, and non-snp rna splicing factor, hnrnp c proteins, and rna polymerase ii all colocalize with viral rna in nuclear inclusions. splicing small nuclear ribonucleoproteins (snrnps) colocalize with viral rna but not replication foci (pombo et al., 1994) , and snrnps then move to interchromatin granules late in infection, which is blocked by mutations in e4 (bridge et al., 2003) . a. rearrangement of host nuclear compartments during adenovirus replication like herpesvirus described above, adenovirus infection redistributes the components of nd10 bodies. prior to infection, pml is associated with interchromatin granules but is redistributed to the fibrillogranular matrix within the nucleus along with sp100, another nd10 component (carvalho et al., 1995) . later in infection, pml is redistributed once again from the fibrillogranular matrix to clear amorphous inclusions and protein crystals (puvion-dutilleul et al., 1995) . another study reported that sp100 and ndp55, but not pml, were relocated from nd10 bodies to viral inclusions (doucas et al., 1996) . while this is confusing, it is clear that adenovirus employs multiple mechanisms to reorganize pml. the initial movement of pml, sp100, and ndp55 to the fibrillogranular matrix occurs prior to viral dna synthesis and is dependent on the e4-orf3 11-kda protein (carvalho et al., 1995; doucas et al., 1996) . it may also be mediated by e1a proteins that colocalize with pml (carvalho et al., 1995) . e1b-55-kda protein also colocalizes with pml early on in infection, then associates with the periphery of replication centers; these interactions are mediated by the orf6 protein of the e4 transcriptional unit (lethbridge et al., 2003) . interestingly, e1b-55k and e4-orf3 target the mre11-rad50-nbs1 (mrn) complex to aggresomes for degradation (araujo et al., 2005; liu et al., 2005) . polyoma-and papillomaviruses are small double-stranded tumorigenic dna viruses with genomes of 5 and 8 kbp, respectively. replication and assembly of these two viruses follow similar strategies, and both involve nd10 bodies. the vp1 capsid protein of human polyomavirus jc is targeted to nd10 domains by vp2, vp3, and agnoprotein where they are assembled into virions (shishido-hara et al., 2004) . a similar process occurs during papillomavirus infection where the minor capsid protein, l2, is responsible for targeting capsomeres of the major capsid protein, l1, to nd10 domains (florin et al., 2002a) . this process involves l2-induced redistribution of nd10 bodies by targeting sp100 for proteasomal degradation. at this point the cellular daxx protein is recruited (florin et al., 2002b) . daax has multiple functions in the nucleus including transcriptional activation and modulating fas-mediated apoptosis [reviewed by salomoni and khelifi (2006) ]. its role in virus replication is at present unclear. one characteristic of papillomavirus infections is the appearance of nuclear and cytoplasmic inclusions in cells contained within warts. the size and number of inclusions is dependent on the type of papillomavirus and the site of infection. human papillomavirus 1 (hpv-1), for example, induces many small inclusions while hpv-4 induces one single inclusion that takes over most of the cytoplasm (croissant et al., 1985) . in vivo these structures label strongly with antiserum raised against e4 gene products which are the 17-kda e1 ∧ e4 and 16-kda e4 proteins (doorbar et al., 1986; rogel-gaillard et al., 1993) . inclusions can be induced in certain cell types in vitro by expressing e4 gene products. hpv-1 e4 staining reveals an initial association with the intermediate filament keratin and subsequent formation of inclusion bodies in the cytoplasm and nucleus (roberts et al., 2003; rogel-gaillard et al., 1993) . the hpv-1 cytoplasmic inclusions retain their association with keratin and appear to induce small cages surrounding e4 protein that are interconnected by keratin filaments (roberts et al., 2003) . the e4 gene gives rise to two proteins, the 17-kda e1 ∧ e4 which can induce cytoplasmic and nuclear inclusions whereas the 16-kda e4 can induce inclusions solely in the cytoplasm (rogel-gaillard et al., 1993) . interestingly, expression of e1 ∧ e4 gene product from hpv-16 induces the complete collapse of the keratin network, but not that of the microtubule or actin networks (doorbar et al., 1991) . it is unclear what the role of the inclusions is in viral replication or the pathology of infection. however, hpv-1 e4 expression induces the redistribution of nd10 to the periphery of nuclear inclusions in cells in culture, and similar signals are seen in vivo (roberts et al., 2003) . the temporal and functional connection between e4 and l1 redistribution of pml is unknown. members of the reoviridae family are dsrna viruses with segmented genomes and include the clinically important rotavirus and orbiviruses that cause diseases in human and animals. reoviruses are nonenveloped viruses with genome segments contained inside a virion $80 nm in diameter. the genome is encapsidated by two protein shells, an outer capsid and an inner core shell. the core contains the rdrp, capping enzymes, and the dsrna genome segments [reviewed in yue and shatkin (1998) and furuichi and shatkin (2000) ]. viruses are taken up by receptor-mediated endocytosis, the outer capsid is lost and the core is delivered into the cytoplasm. the core does not disassemble on entering cells and imports ribonucleoside triphosphates and s-adenosyl-l-methionine from the cytosol to synthesize and then export viral mrnas. in this way the core particle functions as a self-contained transcriptional unit and as such represents the replication complex. viral mrna transcribed in the cytoplasm make viral proteins that eventually form large perinuclear inclusions, called virus factories that function as sites of further virus replication and assembly. the reoviridae family contains 13 genera, and this chapter will concentrate on the two best characterized of these, the orthoreoviruses and rotaviruses. 1. formation of factories during orthoreoviruses replication and assembly a. the shape of orthoreovirus factories is determined by association with the cytoskeleton orthoreoviruses contain 10 genome segments which are classed by size and then numbered, that is l1 is large segment 1. large segments encode l genes, medium (m) segments encode m genes, and small (s) segments encode s genes. virus replication occurs in the cytoplasm in virus factories, and the majority of the virus-encoded proteins have been shown to localize completely or partially with factories (table i ). early observations revealed that different strains of orthoreoviruses induced factories with different appearances; orthoreovirus type 1 lang factories were filamentous while the factories of the dearing isolate of orthoreovirus type 3 were globular ( fig. 9a and b) . this difference maps precisely to a serine-proline switch at residue 208 of the m2 core protein . control of the localization of orthoreovirus factories reflects the degree of association m2 has with the microtubule network. filamentous virus m2 stabilizes microtubules to a greater relative degree than globular virus m2, and depolymerizing microtubules with nocodazole convert filamentous factories to globular ones. many of the events of orthoreovirus factory formation have been successfully reconstituted in vitro. a screen of orthoreovirus proteins revealed that mns, sns, and s3 were the first viral proteins to localize with viral mrna prior to the synthesis of progeny dsrna (antczak and joklik, 1992) . subsequently, it was discovered that expression of the mns protein of isolate dearing in the absence of other viral proteins induced a phasedense structure that was indistinguishable in appearance from that observed during wild-type infection . the shape of the artificial mns inclusion could be altered from globular to filamentous by coexpressing a m2 protein from a filamentous virus . similar experiments showed that coexpression of l1, l2, and s2 core surface proteins with mns caused them to localize to the mns inclusion (broering et al., 2004) . furthermore, the shape of the mns structure that the core proteins colocalized to could be altered to filamentous by coexpressing m2 from a filamentous virus (broering et al., 2004) . mns can also recruit sns, but not s3, to artificial inclusions (becker et al., 2003) , so other factors or conditions are necessary for complete assembly of an orthoreovirus factory. the precise domains involved in initiating factory formation are beginning to be elucidated. the minimal region of mns necessary for inclusion like body formation in vitro is the region composed of 250 c-terminal amino acids of the 721 residue proteins (broering et al., 2005) . residues 1-11 of sns are important for the interaction between sns and rna (gillian and nibert, 1998) , and treatment with rnase dissociates a proportion of mns from sns in coimmunoprecipiation experiments (miller et al., 2003) . interaction between mns and m2 is dependent on residues 1-40 or 41 of mns and residues 1-13 are necessary for interaction between mns and sns (miller et al., 2003) . it is likely that factory formation occurs through an interaction between mns and a sns-rna complex; this can then recruit m2 that will determine the globular or filamentous localization of the factory and hence the localization of the other viral proteins. orthoreovirus factories are clearly intimately associated with the microtubule network (fig. 9c) and have also been suggested to interact with intermediate filaments. orthoreovirus type 3 infection induces a redistribution of vimentin and viral inclusions reported to contain filamentous structures (sharpe et al., 1982) . it will be interesting to see if the in vitro factories induced by mns can also alter the distribution of the intermediate filament network. orthoreovirus factories are also ubiquitinated, and interestingly the nature of the factory determined the degree of ubiquitination; globular factories are prone to contain more ubiquitinated protein than filamentous ones . ubiquitination of orthoreovirus factories has been mapped to the m2 protein but is independent of the filamentous/globular factory determinate of m2, that is converting a filamentous factory to a globular factory does not lead to an increase in ubiquitinated m2. 2. formation of factories during rotavirus replication and assembly a. virus nonstructural proteins organize factory formation and virus assembly rotaviruses contain 11 genome segments of dsrna and like the orthoreoviruses replicate in cytoplasmic factories. rotavirus virions are composed of three protein layers. these are the core which contains the genome and polymerase, an inner capsid layer, and an outer capsid layer. the core and inner capsid layer comprise the double-layered particle (dlp), while the addition of the third capsid layer forms the mature triple-layered particle (tlp). the acquisition of the third capsid layer occurs after the virus buds into the er, and in doing so obtains a transient envelope. rotavirus factories are composed of electron-dense viroplasm often in proximity to membranes derived from the er (fig. 9d) (altenburg et al., 1980) . viroplasm contains high levels of nsp2 (fig. 9e ) and nsp5 which are thought to coordinate assembly of the factory and recruitment of structural proteins such as the inner core protein vp2 and viral polymerase vp1. the factory also contains double-layered rotaviruses, whereas the er membranes associated with the factory contain enveloped intermediates and tlp (arrowed in fig. 9d ). virus factories grow in size and decrease in number during the course of infection as neighboring factories merge (eichwald et al., 2004) . rotavirus factories appear to have an internal structure, as their centers occasionally appear more electron lucent than the periphery, giving a doughnut-like appearance (fig. 9e ). electron microscopy shows dlp at the periphery of the factory and this is (altenburg et al., 1980) consistent with fluorescent microscopy showing that the nonstructural protein nsp2 localizes to the center of the virus factory, whereas nsp5 and inner capsid protein vp6 localize to the periphery (eichwald et al., 2004; gonzález et al., 2000) . these different localizations could have functional relevance because vp6 binds the er-targeted nsp4 membrane protein and is implicated in the budding of dlps into er membranes associated with factories (silvestri et al., 2005) . therefore, a localization to the exterior of the factory may represent an organized progression of virus maturation from the interior of the viroplasm to the exterior. however, things are probably not that straightforward because vp6 is also part of the viral rna complex along with nsp2 (aponte et al., 1996) which, as noted above, is localized to the center of the viroplasm. virus factory-like structures can be introduced in vitro by coexpressing nsp2 and nsp5 (fabbretti et al., 1999) , and this is regulated by domains in the n-and c-termini (fabbretti et al., 1999) as well as the central portion of nsp5 (eichwald et al., 2002) . the process is also dependent on phosphorylation of nsp5, possibly by cellular casein kinase ii (eichwald et al., 2002) . structures similar to factories can also be induced by expressing the inner capsid protein vp6 in vitro (nilsson et al., 1998) . these structures look similar to factories in the electron microscope but lack electron-lucent areas and dlps. interestingly, expression of vp6 of group a simian rotavirus sa-11 induced globular structures, whereas expression of vp6 from group c porcine rotavirus cowden/amc-1 induced filamentous structures (nilsson et al., 1998) analogous to the difference between type 1 and type 3 orthoretroviruses. it is not clear if the difference in factory shape is solely determined by vp6 and if this involves differences in association of the factory components with microtubules. b. virus factories organize viral rna replication and translation the factory does provide the virus with a mechanism to organize viral rnas. positive-stranded viral rna is utilized as the template for synthesizing progeny dsrna genomes and as mrna for translating viral proteins. interestingly, sirna-targeted degradation of nsp1 rna blocks translation of the protein but does not block genome synthesis (silvestri et al., 2004) . furthermore, rna synthesis occurs in factories, but viral rna transcribed in vitro and introduced to infected cells after infection does not localize to factories. the implication of these experiments are that the factory enables rotavirus to sort viral rna into separate pools, one within the factory to act as a template for the rna polymerase and genome replication, and the other outside the factory where it translated on ribosomes to make viral proteins. it likely that this organization allows the virus factory to protect dsrna genomes from antiviral responses. arenaviruses are negative-stranded rna viruses that have two singlestranded genome segments which are packaged into 60-to 200-nmdiameter enveloped virions. lassa, junín, and manchupo viruses are responsible for emerging hemorraghic fevers in humans. arenaviruses induce moderately electron-dense inclusions in the cytoplasm that are composed of 20-to 25-nm-diameter granules identical to those seen within virus particles in the electron microscope (murphy et al., 1970) . the granules represent host ribosomes and between 2 and 10 are packaged into virions (pedersen, 1979) . the inclusions increase in size and density during infection until cytopathic effects are observed in cells (buckley, 1965; buckley and casals, 1970) and stain positive for viral antigens (young et al., 1987) ; however, it is unclear if they represent true virus factories. arenavirus replication is believed to occur in the cytoplasm but also requires a nuclear step as limited growth is observed in enucleated cells (banerjee et al., 1975) . the viral z protein may play a role in this as it is sufficient in vitro to shuttle pml from the nucleus to cytoplasmic inclusion bodies as occurs in vivo (borden et al., 1998) . n protein also localizes to discrete nuclear foci, as well as in the cytoplasm (young et al., 1987) , but the relationship to nd10 bodies and z protein is unknown. rabies virus is a neurotropic lyssavirus of the rhabdovirus family. rhabdovirus virions are bullet-shaped 180 â 75 nm 2 particles containing a single negative strand of rna. rabies induces two types of inclusion body in vitro, neither of which have been proven as replication sites. negri bodies are induced by street rabies viruses in infected neurons of the brain (negri, 1903) and are a good indicator for the presence of an infection site in tissue ( jackson et al., 2001) . different neuronal cell types appear to be more prone to negri bodies ( jackson et al., 2001) . negri bodies contain innerbodies (negri, 1909) and electron microscopic studies suggest the subcompartments may be cytoplasmic material engulfed by the coalescence of several smaller negri bodies (matsumoto, 1970) . the role of negri bodies in infection is poorly understood. initial em observations showed virions localized to some bodies in some cells (matsumoto et al., 1974) , and cytological staining show they contain genetic material, indicating they may be replication complexes. however, 3 h-thymidine or 3 h-uridine fail to label the structures, arguing against this conclusion (matsumoto, 1970) . fixed (brain-adapted laboratory strains) rabies can infect nonneuronal cell lines and in these cell types induce fuchsin-stained cytoplasmic structures (fcps) as well as negri-like bodies (ni et al., 1996) . fcps increase in size during infection that correlates with cytopathic effects and are composed of rabies glycoprotein and matrix protein, whereas negri bodies contain nucleocapsid (ni et al., 1996) . this chapter has described the changes to cell architecture that are induced during virus replication. we have focused on viruses that induce new cellular structures, such as inclusion bodies, virus factories, or replication complexes, to concentrate virus and host factors necessary for replication and assembly. much progress has been made in identifying which cellular components are used to generate these structures, and in some cases specific virus proteins have been identified that are able to induce them. virus inclusions often result in rearrangement of cellular membrane compartments and/or cytoskeleton. the functions of these organelles are carefully regulated in cells, and it is a challenge for the future to determine how viruses disrupt them for use as sites of replication and assembly. changes in cellular architecture may represent bystander responses to the stress associated with virus infection, and some viruses may replicate perfectly well without them. alternatively, viruses may have evolved to target key stages in the regulatory pathways that control organelle structure and function to generate sites that are essential for replication and assembly. given the coevolution of viruses with the cells that carry them, changes in cell structure induced during infection are likely to involve a combination of the two. it is also important to appreciate that many of the structures that have been studied to date have been generated by infecting tissue culture cells with attenuated viruses, often with disregard to the host range and tropism. it is possible that in the natural setting, changes in cell structure induced by viruses will be more subtle, particulary during persistent infections that occur without inflammation or cell lysis. the products of human cytomegalovirus genes ul23, ul24, ul43 and us22 are tegument components parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses mapping and sequence of the gene encoding the african swine fever virion protein of m r 11500 membrane permeabilization by poliovirus proteins 2b and 2bc the vaccinia virus superoxide dismutase-like protein (a45r) is a virion component that is nonessential for virus replication african swine fever virus protein p54 interacts with the microtubular motor complex through direct binding to light-chain dynein ultrastructural study of rotavirus replication in cultured cells assembly of african swine fever virus: role of polyprotein pp220 african swine fever virus is enveloped by a two-membraned collapsed cisterna derived from the endoplasmic reticulum african swine fever virus protease, a new viral member of the sumo-1-specific protease family adenovirus preterminal protein binds to the cad enzyme at active sites of viral dna replication on the nuclear matrix reovirus genome segment assortment into progeny genomes studied by the use of monoclonal antibodies directed against reovirus proteins recovery and characterization of a replicase complex in rotavirus-infected cells by using a monoclonal antibody against nsp2 adenovirus type 5 e4orf3 protein targets the mre11 complex to cytoplasmic aggresomes redistribution of microtubules and golgi-apparatus in herpes-simplex virusinfected cells and their role in viral exocytosis the herpes simplex virus 1 ul11 proteins are associated with cytoplasmic and nuclear membranes and with nuclear bodies of infected cells requirement of cell nucleus for the replication of an arenavirus expression and characterization of a novel structural protein of human cytomegalovirus, pul25 preferential virosomal location of underphosphorylated h5r protein synthesized in vaccinia virus-infected cells reovirus sns protein is required for nucleation of viral assembly complexes and formation of viral inclusions reovirus sns and mns proteins form cytoplasmic inclusion structures in the absence of viral infection poliovirus proteins induce membrane association of gtpase adp-ribosylation factor hijacking components of the secretory pathway for replication of poliovirus rna does common architecture reveal a viral lineage spanning all three domains of life? deletion mutants of the herpes simplex virus type 1 ul8 protein: effect on dna synthesis and ability to interact with and influence the intracellular localization of the ul5 and ul52 proteins poliovirus mutant that contains a cold-sensitive defect in viral rna synthesis high resolution localization of replicating viral genome in adenovirus-infected hela cells the vaccinia virus a14.5l gene encodes a hydrophobic 53-amino-acid virion membrane protein that enhances virulence in mice and is conserved among vertebrate poxviruses intracellular distribution of poliovirus proteins and the induction of virus-specific cytoplasmic structures association of polioviral proteins of the p2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography the mechanisms of vesicle budding and fusion a structural dna binding protein of african swine fever virus with similarity to bacterial histone-like proteins pondering the promyelocytic leukemia protein (pml) puzzle: possible functions for pml nuclear bodies an arenavirus ring (zincbinding) protein binds the oncoprotein promyelocyte leukemia protein (pml) and relocates pml nuclear bodies to the cytoplasm endoplasmic reticulum architecture: structures in flux nuclear factor i is specifically targeted to discrete subnuclear sites in adenovirus type 2-infected cells mouse hepatitis virus replicase protein complexes are translocated to sites of m protein accumulation in the ergic at late times of infection an amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis c virus nonstructural protein 5a adenovirus early region 4 promotes the localization of splicing factors and viral rna in late-phase interchromatin granule clusters mammalian reovirus nonstructural protein mns forms large inclusions and colocalizes with reovirus microtubule-associated protein m2 in transfected cells reovirus nonstructural protein mns recruits viral core surface proteins and entering core particles to factory-like inclusions carboxyl-proximal regions of reovirus nonstructural protein mns necessary and sufficient for forming factory-like inclusions assembly of african swine fever virus: quantitative ultrastructural analysis in vitro and in vivo intracellular virus dna distribution and the acquisition of the nucleoprotein core during african swine fever virus particle assembly: ultrastructural in situ hybridisation and dnase-gold labelling characterization of african swine fever virion proteins j5r and j13l: immuno-localization in virus particles and assembly transcription factor yy1 is a vaccinia virus late promoter activator junín and tacaribe work in hela cells lassa fever, a new virus disease of man from west africa. 3. isolation and characterization of the virus nd10 protein pml is recruited to herpes simplex virus type 1 prereplicative sites and replication compartments in the presence of viral dna polymerase interactions of herpes simplex virus type 1 with nd10 and recruitment of pml to replication compartments the initiation of vaccinia infection the egress of herpesviruses from cells: the unanswered questions localization of structural proteins in african swine fever virus particles by immunoelectron microscopy vaccinia virus cores are transported on microtubules targeting of adenovirus e1a and e4-orf3 proteins to nuclear matrix-associated pml bodies characterization and structural localization of the reovirus l3 protein the african swine fever virus iap homolg is a late structural polypeptide herpes simplex virus dna replication promyelocytic leukemia protein mediates interferon-based anti-herpes simplex virus 1 effects herpes virus induced proteasome-dependent degradation of the nuclear bodies-associated pml and sp100 proteins interaction of frog virus 3 with the cytomatrix. iv. phosphorylation of vimentin precedes the reorganization of intermediate filaments around the virus assembly sites copi activity coupled with fatty acid biosynthesis is required for viral replication early proteins are required for the formation of frog virus 3 assembly sites effects of a temperature sensitivity mutation in the j1r protein component of a complex required for vaccinia virus assembly membrane rearrangement and vesicle induction by recombinant poliovirus 2c and 2bc in human cells inhibition of cellular protein secretion by picornaviral 3a proteins the major structural protein of african swine fever virus, p73, is packaged into large structures, indicative of viral capsid or matrix precursors, on the endoplasmic reticulum involvement of the endoplasmic reticulum in the assembly and envelopment of african swine fever virus biochemical requirements of virus wrapping by the endoplasmic reticulum calcium store during envelopment of african swine fever virus a virally encoded chaperone specialized for folding of the major capsid protein of african swine fever virus in a nutshell: structure and assembly of the vaccinia virion inhibition of protein trafficking by coxsackievirus b3: multiple viral proteins target a single organelle nonviral microbodies with viral antigenicity produced in cytomegalovirus-infected cells specificity of cytopathic effect of cutaneous human papillomaviruses the poliovirus replication machinery can escape inhibition by an antiviral drug that targets a host cell protein brefeldin a inhibits cell-free, de novo synthesis of poliovirus a vaccinia virus core protein, p39, is membrane associated characterization of the vaccinia virus h3l envelope protein: topology and posttranslational membrane insertion via the c-terminal hydrophobic tail icp27 interacts with the c-terminal domain of rna polymerase ii and facilitates its recruitment to herpes simplex virus 1 transcription sites, where it undergoes proteasomal degradation during infection analysis of intracellular and intraviral localization of the human cytomegalovirus ul53 protein the development of vaccinia virus in earle's l strain cells as examined by electron microscopy electron microscopic study of the formation of poliovirus the uptake and development of reovirus in strain l cells followed with labeled viral ribonucleic acid and ferritin-antibody conjugates viruses and renal carcinoma of rana pipiens: ii. ultrastructural studies and sequential development of virus isolated from normal and tumor tissue acidic c terminus of vaccinia virus dnabinding protein interacts with ribonucleotide reductase formation of dna replication structures in herpes virus-infected cells requires a viral dna binding protein comparison of the intranuclear distributions of herpes simplex virus proteins involved in various viral functions origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors mhc i-dependent antigen presentation is inhibited by poliovirus protein 3a autophagy in innate and adaptive immunity poliovirus 3a protein limits interleukin-6 (il-6), il-8, and beta interferon secretion during viral infection inhibition of cellular protein secretion by poliovirus proteins 2b and 3a inhibition of endoplasmic reticulum-to-golgi traffic by poliovirus protein 3a: genetic and ultrastructural analysis the vaccinia virus e8r gene product: a viral membrane protein that is made early in infection and packaged into the virions' core the punctate sites of accumulation of vaccinia virus early proteins are precursors of sites of viral dna synthesis identification of the human papilloma virus-1a e4 gene products specific interaction between hpv-16 e1-e4 and cytokeratins results in collapse of the epithelial cell intermediate filament network adenovirus replication is coupled with the dynamic properties of the pml nuclear structure interaction of hepatitis c virus proteins with host cell membranes and lipids genetic and biochemical characterization of vaccinia virus genes d2l and d3r which encode virion structural proteins intracellular location and translocation of silent and active poliovirus replication complexes reversible dissociation of the poliovirus replication complex: functions and interactions of its components in viral rna synthesis formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral rna synthesis expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex rotavirus nsp5: mapping phosphorylation sites and kinase activation and viroplasm localization domains characterization of rotavirus nsp2/ nsp5 interactions and the dynamics of viroplasm formation replication of hepatitis c virus rna occurs in a membranebound replication complex containing nonstructural viral proteins and rna amphipathic helix-dependent localization of ns5a mediates hepatitis c virus rna replication the african swine fever virus non-structural protein pb602l is required for the formation of the icosahedral capsid of the virus particle the vaccinia virus d5 protein, which is required for dna replication, is a nucleic acid-independent nucleoside triphosphatase icp0 induces the accumulation of colocalizing conjugated ubiquitin interactions between dna viruses, nd10 and the dna damage response hsv-1 ie protein vmw110 causes redistribution of pml nd10 components relocate to sites associated with herpes simplex virus type 1 nucleoprotein complexes during virus infection recruitment of herpes simplex virus type 1 transcriptional regulatory protein icp4 into foci juxtaposed to nd10 in live, infected cells formation of nuclear foci of the herpes simplex virus type 1 regulatory protein icp4 at early times of infection: localization, dynamics, recruitment of icp27, and evidence for the de novo induction of nd10-like complexes pml contributes to a cellular mechanism of repression of herpes simplex virus type 1 infection that is inactivated by icp0 two non-structural rotavirus proteins, nsp2 and nsp5, form viroplasm-like structures in vivo alpha-herpesvirus infection induces the formation of nuclear actin filaments assembly and translocation of papillomavirus capsid proteins reorganization of nuclear domain 10 induced by papillomavirus capsid protein l2 active intranuclear movement of herpesvirus capsids adenovirus precursor to terminal protein interacts with the nuclear matrix in vivo and in vitro alphavirus rna replicase is located on the cytoplasmic surface of endosomes and lysosomes the ul48 tegument protein of pseudorabies virus is critical for intracytoplasmic assembly of infectious virions viral and cellular mrna capping: past and prospects characterization of the african swine fever virus protein p49: a new late structural polypeptide role of the host cell nucleus in the replication of african swine fever virus dna differential requirements for copi coats in formation of replication complexes among three genera of picornaviridae amino terminus of reovirus nonstructural protein sns is important for ssrna binding and nucleoprotein complex formation relative localization of viroplasmic and endoplasmic reticulum-resident rotavirus proteins in infected cells localization of the herpes simplex virus type 1 65-kilodalton dna-binding protein and dna polymerase in the presence and absence of viral dna synthesis frog virus 3 dna replication occurs in two stages a new superfamily of putative ntpbinding domains encoded by genomes of small dna and rna viruses nidovirales: evolving the largest rna virus genome rna replication of mouse hepatitis virus takes place at double-membrane vesicles identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons subcellular localization of the us3 protein kinase of herpes simplex virus type 2 modulation of p53 cellular function and cell death by african swine fever virus viruses and renal carcinoma of rana pipiens. i. the isolation and properties of virus from normal and tumor tissue ultrastructural analysis of the replication cycle of pseudorabies virus in cell culture: a reassessment structure and assembly of intracellular mature vaccinia virus: thin-section analyses recherches sur la pathologie et étiologie de l'infection vaccinique et varioleuse subcellular localization of host and viral proteins associated with tobamovirus rna replication aggresomes resemble sites specialized for virus assembly membrane association facilitates the correct processing of pp220 during production of the major matrix proteins of african swine fever virus vaccinia virus intracellular enveloped virions move to the cell periphery on microtubules in the absence of the a36r protein upregulation of major histocompatibility complex class i on liver cells by hepatitis c virus core protein via p53 and tap1 impairs natural killer cell cytotoxicity deep-etch em reveals that the early poxvirus envelope is a single membrane bilayer stabilized by a geodetic ''honeycomb'' surface coat dynamics of herpes simplex virus capsid maturation visualized by time-lapse cryo-electron microscopy electron microscopy of viruses in thin sections of cells grown in culture a ubiquitin conjugating enzyme encoded by african swine fever virus vaccinia virus intracellular mature virions contain only one lipid membrane a poxvirus host range protein, cp77, binds to a cellular protein, hmg20a, and regulates its dissociation from the vaccinia virus genome in cho-k1 cells electron microscopic examination of the viromatrix of rana grylio virus in a fish cell line the hepatitis c virus nonstructural protein 4b is an integral endoplasmic reticulum membrane protein molecular chaperone hsp90 is important for vaccinia virus growth in cells evidence against an essential role of copii-mediated cargo transport to the endoplasmic reticulum-golgi intermediate compartment in the formation of the primary membrane of vaccinia virus sequential localization of two herpes simplex virus tegument proteins to punctate nuclear dots adjacent to icp0 domains biogenesis of poxviruses: role of a-type inclusions and host cell membranes in virus dissemination isolation and characterization of a noninfectious virionlike particle released from cells infected with human strains of cytomegalovirus involvement of membrane traffic in the replication of poliovirus genomes: effects of brefeldin a the periphery of nuclear domain 10 (nd10) as site of dna virus deposition common origin of four diverse families of large eukaryotic dna viruses quantitative study of the infection in brain neurons in human rabies subversion of cellular autophagosomal machinery by rna viruses colocalization of the herpes simplex virus 1 ul4 protein with infected cell protein 22 in small, dense nuclear structures formed prior to onset of dna synthesis aggresomes: a cellular response to misfolded proteins african swine fever virus infection disrupts centrosome assembly and function transport of african swine fever virus from assembly sites to the plasma membrane is dependent on microtubules and conventional kinesin african swine fever virus induces filopodia-like projections at the plasma membrane a study on the morphological and cyto-immunological relationship between the inclusions of variola, cowpox, rabbitpox, vaccinia (variola origin) and vaccinia ihd and a consideration of the term ''guarnieri body synthesis, subcellular localization and vp16 interaction of the herpes simplex virus type 2 ul46 gene product the deacetylase hdac6 regulates aggresome formation and cell viability in response to misfolded protein stress subcellular localization of hepatitis c viral proteins in mammalian cells interferon-independent increases in class i major histocompatibility complex antigen expression follow flavivirus infection cellular autophagy: surrender, avoidance and subversion by microorganisms stages in the nuclear association of the herpes simplex virus transcriptional activator protein icp4 foot-and-mouth disease virus replication sites form next to the nucleus and close to the golgi apparatus, but exclude marker proteins associated with host membrane compartments nonstructural protein precursor ns4a/b from hepatitis c virus alters function and ultrastructure of host secretory apparatus poliovirus protein 3a binds and inactivates lis1, causing block of membrane protein trafficking and deregulation of cell division identification and characterization of the pseudorabies virus tegument proteins ul46 and ul47: role for ul47 in virion morphogenesis in the cytoplasm the role of a 21-kda viral membrane protein in the assembly of vaccinia virus from the intermediate compartment replication complex of human parechovirus 1 a giant virus in amoebae vaccinia virus gene a18r dna helicase is a transcript release factor the herpes simplex virus type 1 cleavage/packaging protein, ul32, is involved in efficient localization of capsids to replication compartments human cytomegalovirus structural components: intracellular and intraviral localization of p38 characterization of rubella virus replication complexes using antibodies to double-stranded rna association of herpes simplex virus regulatory protein icp22 with transcriptional complexes containing eap, icp4, rna polymerase ii, and viral dna requires posttranslational modification by the u(l)13 proteinkinase nuclear matrix localization and sumo-1 modification of adenovirus type 5 e1b 55k protein are controlled by e4 orf6 protein development by self-digestion: molecular mechanisms and biological functions of autophagy cell culture-grown hepatitis c virus is infectious in vivo and can be recultured in vitro secretory protein trafficking and organelle dynamics in living cells functional order of assembly of herpes simplex virus dna replication proteins into prereplicative site structures flavivirus infection up-regulates the expression of class i and class ii major histocompatibility antigens on and enhances t cell recognition of astrocytes in vitro adenovirus exploits the cellular aggresome response to accelerate inactivation of the mrn complex evidence that a mechanism for efficient flavivirus budding upregulates mhc class i herpes simplex virus type 1-induced modifications in the distribution of nucleolar b-36 protein formation of herpes simplex virus type 1 replication compartments by transfection: requirements and localization to nuclear domain 10 herpes simplex virus type 1 prereplicative sites are a heterogeneous population: only a subset are likely to be precursors to replication compartments nucleoplasmic and nucleolar distribution of the adenovirus iva2 gene product visualization and functional analysis of rna-dependent rna polymerase lattices wrapping things up about virus rna replication assembly and maturation of the flavivirus kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively subcellular localization and some biochemical properties of the flavivirus kunjin nonstructural proteins ns2a and ns4a markers for trans-golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells rubella virus replication complexes are virus-modified lysosomes perinuclear localization of na-k-clcotransporter protein after human cytomegalovirus infection microtubule-dependent organization of vaccinia virus core-derived early mrnas into distinct cytoplasmic structures relationship between vaccinia virus intracellular cores, early mrnas, and dna replication sites small dense nuclear bodies are the site of localization of herpes simplex virus 1 u(l)3 and u(l)4 proteins and of icp22 only when the latter protein is present novel acylation of poxvirus a-type inclusion proteins characterization of the african swine fever virus structural protein p14.5 a dna binding protein rabies virus further studies on the replication of rabies and rabies-like viruses in organized cultures of mammalian neural tissues nuclear domain 10 as preexisting potential replication start sites of herpes simplex virus type-1 inhibition of poliovirus rna synthesis by brefeldin a the trans golgi network is lost from cells infected with african swine fever virus identification of the orthopoxvirus p4c gene, which encodes a structural protein that directs intracellular mature virus particles into a-type inclusions assembly site of the virus pbcv-1 in a chlorella-like green alga: ultrastructural studies herpesvirus assembly and egress egress of alphaherpes viruses herpesvirus assembly: a tale of two membranes flock house virus rna polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion reovirus sns protein localizes to inclusions through an association requiring the mns amino terminus increased ubiquitination and other covariant phenotypes attributed to a strain-and temperature-dependent defect of reovirus core protein m2 the vaccinia virus-encoded uracil dna glycosylase has an essential role in viral dna replication rifampicin prevents virosome localization of l65, an essential vaccinia virus polypeptide effects of foot-and-mouth disease virus nonstructural proteins on the structure and function of the early secretory pathway: 2bc but not 3a blocks endoplasmic reticulum-to-golgi transport inhibition of the secretory pathway by the foot-and-mouth disease virus 2bc protein is reproduced by co-expression of 2b with 2c and the site of inhibition is determined by the subcellular location of 2c involvement of spicules in the formation of vaccinia virus envelopes elucidated by a conditional lethal mutant modulation of transporter associated with antigen processing (tap)-mediated peptide import into the endoplasmic reticulum by flavivirus infection high-pressure freezing in the study of animal pathogens the ultrastructure of the developing replication site in foot-and-mouth disease virus-infected bhk-38 cells membrane association of the rna-dependent rna polymerase is essential for hepatitis c virus rna replication structure and development of viruses observed in the electron microscope. ii. vaccinia and fowl pox viruses rifampicin: a specific inhibitor of vaccinia virus assembly herpes simplex virus icp0 and icp34.5 counteract distinct interferon-induced barriers to virus replication evidence that herpes simplex virus vp16 is required for viral egress downstream of the initial envelopment event ultrastructural study of african swine fever virus replication in cultures of swine bone marrow cells mitochondrial distribution and function in herpes simplex virusinfected cells identification by mass spectroscopy of three major early proteins associated with virosomes in vaccinia virusinfected cells arenoviruses in vero cells: ultrastructural studies interaction of frog virus-3 with the cytoskeleton. i. altered organization of microtubules, intermediate filaments, and microfilaments synthesis of frog virus 3 proteins occurs on intermediate filament-bound polyribosomes a functional role for intermediate filaments in the formation of frog virus 3 assembly sites localization of adenovirus-encoded dna replication proteins in the nucleus by immunogold electron microscopy the ul 16 gene product of herpes simplex virus 1 is a virion protein that colocalizes with intranuclear capsid proteins beitrag zum studium de aetiologie der tollwuth ü ber die morphologie und den entwicklungszyklus des parasiten der tollwut the poxviral ring protein p28 is a ubiquitin ligase that targets ubiquitin to viral replication factories the subcellular distribution of multigene family 110 proteins of african swine fever virus is determined by differences in c-terminal kdel endoplasmic reticulum retention motifs african swine fever virus causes microtubule dependent dispersal of the trans-golgi network and slows delivery of membrane protein to the plasma membrane cell-free assembly of the herpes simplex virus capsid assembly of the herpes simplex virus capsid: characterization of intermediates observed during cell-free capsid formation poliovirus protein 3a inhibits tumor necrosis factor (tnf)-induced apoptosis by eliminating the tnf receptor from the cell surface ultrastructural studies of kunjin virus-infected aedes albopictus cells studies on unusual cytoplasmic structures which contain rabies virus envelope proteins chlorella virus pbcv-1 replication is not affected by cytoskeletal disruptors assembly of viroplasm and virus-like particles of rotavirus by a semliki forest virus replicon virus factories: associations of cell organelles for viral replication and morphogenesis identification and characterization of the ul7 gene product of herpes simplex virus type 2 formation of aggresome-like structures in herpes simplex virus type 2-infected cells and a potential role in virus assembly subcellular distribution of the foot-and-mouth disease virus 3a protein in cells infected with viruses encoding wild-type and bovine-attenuated forms of 3a host cell nuclear proteins are recruited to cytoplasmic vaccinia virus replication complexes entry of vaccinia virus and cell-cell fusion require a highly conserved cysteine-rich membrane protein encoded by the a16l gene african swine fever virus dutpase is a highly specific enzyme required for efficient replication in swine macrophages herpes simplex virus 1 gene products required for dna replication: identification and overexpression bfa bodies'': a subcompartment of the endoplasmic reticulum quantitative sumo-1 modification of a vaccinia virus protein is required for its specific localization and prevents its self-association reovirus core protein m2 determines the filamentous morphology of viral inclusion bodies by interacting with and stabilizing microtubules isolation of cowpox virus a-type inclusions and characterization of their major protein component structural components and replication of arenaviruses open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex characterization of vaccinia virus intracellular cores: implications for viral uncoating and core structure events in vaccinia virus-infected cells following the reversal of the antiviral action of rifampicin localization of rotavirus antigens in infected cells by ultrastructural immunocytochemistry ultrastructural localization of rotavirus antigens using colloidal gold immunoelectron microscopy analysis of hcmv gpul73 (gn) localization vaccinia virus infection disrupts microtubule organization and centrosome function adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells coronavirus replication complex formation utilizes components of cellular autophagy identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins detection and subcellular localization of the turnip yellow mosaic virus 66k replication protein in infected cells clustered charge-to-alanine mutagenesis of the vaccinia virus a20 gene: temperaturesensitive mutants have a dna-minus phenotype and are defective in the production of processive dna polymerase activity simultaneous detection of highly phosphorylated proteins and viral major dna binding protein distribution in nuclei of adenovirus type 5-infected hela cells viral alkaline nuclease in intranuclear dense bodies induced by herpes simplex infection segregation of viral double-stranded and singlestranded dna molecules in nuclei of adenovirus infected cells as revealed by electron microscope in situ hybridization replicating single-stranded adenovirus type 5 dna molecules accumulate within well-delimited intranuclear areas of lytically infected hela cells distribution of viral rna molecules during the adenovirus type 5 infectious cycle in hela cells rearrangements of intranuclear structures involved in rna processing in response to adenovirus infection adenovirus infection induces rearrangements in the intranuclear distribution of the nuclear body-associated pml protein deletion of the fiber gene induces the 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a11r gene is required for formation of the virion membrane characterization of the ul33 gene product of herpes simplex virus 1 the vaccinia virus 39-kda protein forms a stable complex with the p4a/4a major core protein early in morphogenesis endoplasmic reticulum-golgi intermediate compartment membranes and vimentin filaments participate in vaccinia virus assembly cytopathogenic effect of poliomyelitis viruses 'in vitro' on human embryonic tissues the nd10 component promyelocytic leukemia protein relocates to human papillomavirus type 1 e4 intranuclear inclusion bodies in cultured keratinocytes and in warts african swine fever virus structural protein p54 is essential for the recruitment of envelope precursors to assembly sites african swine fever virus pb119l protein is a flavin adenine dinucelotide-linked sulfhydryl oxidase putative site for the acquisition of human herpesvirus 6 virion tegument cytopathic effect in human papillomavirus type 1-induced inclusion warts: in vitro 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vitro the coronavirus replicase: insights into a sophisticated enzyme machinery key: cord-277293-eo3bei9x authors: fondong, vincent n. title: geminivirus protein structure and function date: 2013-04-25 journal: molecular plant pathology doi: 10.1111/mpp.12032 sha: doc_id: 277293 cord_uid: eo3bei9x geminiviruses are a family of plant viruses that cause economically important plant diseases worldwide. these viruses have circular single‐stranded dna genomes and four to eight genes that are expressed from both strands of the double‐stranded dna replicative intermediate. the transcription of these genes occurs under the control of two bidirectional promoters and one monodirectional promoter. the viral proteins function to facilitate virus replication, virus movement, the assembly of virus‐specific nucleoprotein particles, vector transmission and to counteract plant host defence responses. recent research findings have provided new insights into the structure and function of these proteins and have identified numerous host interacting partners. most of the viral proteins have been shown to be multifunctional, participating in multiple events during the infection cycle and have, indeed, evolved coordinated interactions with host proteins to ensure a successful infection. here, an up‐to‐date review of viral protein structure and function is presented, and some areas requiring further research are identified. geminiviruses (family geminiviridae) are among the most devastating plant pathogens worldwide and, together with potyviruses (family potyviridae), constitute the two largest and most important plant virus families (gibbs and ohshima, 2010; scholthof et al., 2011) . by causing heavy losses on food and cash crops, such as cassava, tomatoes, grain legumes, vegetables, maize and cotton, geminiviruses represent a new threat to global food security and sustainability. during the last two decades, epidemics of re-emerging and newly emerging geminiviruses have caused huge crop losses and threatened crop production, particularly in the tropics and subtropics. because of their agricultural importance, geminiviruses have been characterized extensively at the molecular level. geminivirus genomes consist of one (monopartite) or two (bipartite) circular single-stranded dna (ssdna) molecules, which are packaged in icosahedral twinned particles (böttcher et al., 2004; krupovic et al., 2009; zhang w. et al., 2001) . these viruses are divided into four genera-begomovirus, curtovirus, topocuvirus and mastrevirus-based on genome organization, nucleotide sequence similarities and biological properties (brown et al., 2012) . begomoviruses (type species bean golden mosaic virus, bgmv) have either monopartite or bipartite genomes and are transmitted by whiteflies (bemisia tabaci) (brown, 1994) . the two genomic components of bipartite begomoviruses are designated dna-a and dna-b (fig. 1a) . dna-a has six open reading frames (orfs), two in the virion sense (av1 and av2) and four in the complementary sense (ac1, ac2 and ac3 and ac4). dna-b has two orfs, the virion-sense bv1 and complementary-sense bc1. the av2 orf is found in old world bipartite begomoviruses, but not in new world viruses. except for an approximately 200nucleotide segment of the 5′ intergenic region (ir), designated the common region (cr), the two genomic components of bipartite begomoviruses are different in sequence. the cr contains an origin of replication organized modularly, including a stem-loop structure containing the invariant nonanucleotide taatattac sequence, which is required for the cleavage and joining of the viral dna during replication (laufs et al., 1995) . the orfs of monopartite begomoviruses include the virion-sense v1 and v2 genes and the complementary-sense c1, c2, c3 and c4 genes (fig. 1b) . mastreviruses (type species maize streak virus, msv) are transmitted by leafhoppers (order hemiptera, family cicadellidae) and have a single genome component. except for a recent identification of mastreviruses in sweet potato in peru (kreuze et al., 2009) and in dragonflies (suborder anisoptera) in puerto rico (rosario et al., 2013) , these viruses have been found only in the old world, where they infect a range of monocotyledonous plants (reviewed in brown et al., 2012) and, from recent reports, dicotyledonous plants (hadfield et al., 2012; liu et al., 1999; nahid et al., 2008) . mastreviruses have four orfs, v1 and v2 on the virion-sense strand, and c1 and c2 on the complementary-sense strand, separated by two irs on opposite sides of the genome, designated as large (lir) and small (sir) (kammann et al., 1991) (fig. 1c) . the sir contains termination and polyadenylation sequences for both the complementary-sense and virion-sense transcripts, and is probably the origin of replication of the complementary-sense strand (hayes et al., 1988) . for mastreviruses, but not for viruses of other genera, an approximately 80-nucleotide primer is present in the virion and specifically anneals to the sir (kammann et al., 1991; shen and hohn, 1991) . curtoviruses (type species beet curly top virus, bctv) infect dicotyledonous hosts, are transmitted by the beet leafhopper (circulifer tenellus) and have monopartite genomes (fig. 1d) . the genome has seven orfs and a single ir (hormuzdi and bisaro, 1995; . the three virion-sense orfs are v1, v2 and v3. the four complementary-sense orfs are c1, c2, c3 and c4. like mastreviruses, curtovirus genomes contain a polyadenylation signal of approximately 20 nucleotides between the converging v1 and c3 orfs. the genus topocuvirus, which so far has only one member, tomato pseudo-curly top virus (tpctv), has a monopartite genome and is transmitted to dicotyledonous hosts by treehoppers (micrutalis malleifera) (briddon et al., 1996) . topocuviruses have six orfs (fig. 1e) : the virion-sense strand contains v1 and v2 and the complementary-sense strand contains c1, c2, c3 and c4, which are homologues of ac1, ac2, ac3 and ac4, respectively. previous comprehensive reviews (gutierrez et al., 2004; jeske, 2009; stanley, 2004) have focused on the biological and molecular properties of geminiviruses. in this article, a review of current information on the molecular biology of geminiviruses and their satellites is provided, with special emphasis on viral protein structure, function and interactions with host proteins. important areas needing additional research are also identified. the replication-associated protein (rep) encoded by the ac1 orf (also called al1) in bipartite geminiviruses and by c1 (also called l1) in monopartite geminiviruses (except mastreviruses) is conserved in sequence, position and function (hanley-bowdoin et al., 2004) and is expressed under the control of a bidirectional core promoter in the ir (hanley-bowdoin et al., 1999) . in the mastreviruses, the full-length rep is translated from a spliced transcript of the c1 and c2 orfs. the multifunctional nature of begomovirus reps and the functional domains have been characterized (heyraud-nitschke et al., 1995; orozco et al., 1997) (fig. 2) . rep is essential for rolling circle replication (rcr) and is involved in the modulation of gene expression (elmer et al., 1988; etessami et al., 1991; haley et al., 1992; hong and stanley, 1995; saunders et al., 1991) , analogous to some animal and bacterial ssdna viruses (stenger et al., 1991) and plasmids (oshima et al., 2001) , suggesting a strong evolutionary link between these proteins (ilyina and koonin, 1992) . the first step in geminivirus replication is the synthesis of the complementary strand from the genomic ssdna using a stillunknown mechanism that is thought to be catalysed entirely by host factors. rep, an early gene, confers virus-specific recognition of its cognate origin of replication (fontes et al., 1994) and initiates dna replication (laufs et al., 1995; lazarowitz et al., 1992; orozco et al., 1997) . the recognition mechanism appears to differ between mastreviruses and begomoviruses, based on the architecture of the origin of replication (gutierrez et al., 2004) . for mastreviruses, the origin consists of a large cis-acting region containing several rep binding sites (castellano et al., 1999; sanz-burgos and gutiérrez, 1998) , whereas the begomovirus origin contains a single rep binding site (fontes et al., 1992 (fontes et al., , 1994 lazarowitz et al., 1992; orozco and hanley-bowdoin, 1998) . in either case, however, the rep oligomerizes into a complex as illustrated for msv (horvath et al., 1998) , wheat dwarf virus (wdv) (castellano et al., 1999; sanz-burgos and gutiérrez, 1998) and tomato golden mosaic virus (tgmv) (orozco and hanley-bowdoin, 1998) from mutational analyses of the oligomerization domain. in planta rep oligomerization has been elucidated recently using abutilon mosaic virus (abmv) rep bimolecular fluorescence complementation (bifc) assay in nicotiana benthamiana plants, where rep oligomers accumulated in the nucleoplasm and replicated abmv dna-b (krenz et al., 2011) . to initiate the rcr, the begomovirus rep binds to the rep complex binding site, which contains a directly repeated sequence between the tata box and the transcription start site (fontes et al., 1992) . for the mastreviruses, the complex binds presumably to three sites located in the proximity of the two divergent tata boxes upstream and downstream of the dna replication initiation site and at the base of the conserved stem-loop (castellano et al., 1999; laufs et al., 1995) . on binding, a process which involves motif i (fig. 2a) , the rep complex cleaves the phosphodiester bond between the last t and a in the invariant loop sequence in a biochemical process mediated by motif ii, which is a metal binding site that may be involved in protein conformation (arguello-astorga et al., 2004; laufs et al., 1995; orozco and hanley-bowdoin, 1998) . a third sequence, motif iii, is the catalytic site that is required for dna cleavage and ligation (orozco et al., 1997) , thus providing the necessary 3′-oh for priming nascent viral dna synthesis by host dna polymerases. recently, nash et al. (2011) have identified a conserved motif, named the geminivirus rep sequence (grs) (fig. 2a) , and have shown that it is required for the initiation of virus replication. an artificial zincfinger protein designed to bind to the tomato yellow leaf curl virus (tylcv) rep binding site with high affinity has been shown recently to inhibit rep binding and tylcv replication (mori et al., 2012) . several rep-interacting proteins that are important in replication have been identified. thus, a family of proteins involved in the negative regulation of the cell cycle related to retinoblastoma, designated retinoblastoma-related proteins (rbr), interacts with reps of several geminiviruses (ach et al., 1997; kong et al., 2000; xie et al., 1996) . rbr represses cell cycle progression through its interactions with e2f (arguelloastorga et al., 2004) . accordingly, interaction between rep and rbr interferes with the interaction between rbr and e2f and frees the activator class of e2f to activate the expression of genes required for dna replication, including proliferating cell nuclear antigen (pcna) (arguello-astorga et al., 2004; egelkrout et al., 2001; kong et al., 2000) , which is permissive for viral dna replication. also critical to viral dna replication is the interaction between rep and replication factor c (rfc), which catalyses the loading of pcna, a sliding clamp for dna polymerase at the primer-template junction (pavlov et al., 2004) . interaction between rep and rfc is said to recruit rfc into the replication complex, where it facilitates the assembly of other replication factors (luque et al., 2002) . rep also binds pcna, which starts to accumulate in the g1 phase of the cell cycle, reaching the highest level during the s phase and decreasing during the g2 and m phases (gutierrez, 2000) . furthermore, tgmv rep has been shown recently to interact with small ubiquitin-related modifier (sumo)-conjugating enzyme (sce1) to presumably modulate the sumoylation level of host targets, thereby creating a suitable environment for virus replication (castillo et al., 2004; sánchez-durán et al., 2011) . although sumoylation plays an important role in human dna virus infection by altering the molecular interactions of the modified substrate, changing the localization, stability and/or activity (reviewed in wimmer et al., 2012) , definitive conclusions on the role of sumo modification during plant virus infection are yet to be determined. rep also interacts with the replication protein a (rpa) (singh et al., 2007 (singh et al., , 2008 , a key protein in the recruitment of important proteins of the replication apparatus, such as dna polymerase a, rfc and pcna (loor et al., 1997) . it is important to indicate that rpa is critical to other viral replication initiator proteins, including sv40 large t antigen (weisshart et al., 1998) , ebna1 proteins of epstein-barr virus (zhang et al., 1998) , e1 proteins of papilloma virus (loo and melendy, 2004) and ns1 proteins of parvovirus (christensen and tattersall, 2002) . furthermore, rep interacts with a kinesin-like motor protein (kong and hanley-bowdoin, 2002) to presumably prevent cell cycle progression through m and favour the occurrence of endocycles (desvoyes et al., 2006; jordan et al., 2007) , thus favouring viral dna replication. we have also found that rep interacts with b-tubulin6 (tub6) (v. n. fondong et al., unpublished data) , suggesting that rep movement may be microtubule dependent. in addition to replication, some geminivirus reps have been shown to be involved in viral gene regulation by repressing complementary-sense gene expression of mastreviruses (collin et al., 1996; hefferon et al., 2006) and begomoviruses (eagle et al., 1994; gröning et al., 1994; haley et al., 1992; shivaprasad et al., 2005) , presumably after binding to rep binding sites in the ir (fontes et al., 1992; frey et al., 2001) . however, rep appears not to repress virion-sense gene expression, as elucidated for mungbean yellow mosaic virus (mymv) rep . together, these findings have contributed significantly to our understanding of the role of rep in geminivirus replication and the mechanism of rcr; they have revealed the functional relationship between the geminivirus rep and replication initiation proteins of other ssdna viruses, as well as plasmids, and have supported the view that geminiviruses evolved from plasmids, even though, in a recent study based on sequence analysis, saccardo et al. (2011) rejected the hypothesis that geminiviruses originated from plasmids of phytoplasma. however, still to be fully determined are the mechanism of cellular factor recruitment and the biochemical events leading to rep binding to viral dna to initiate genome replication. in addition, it is not yet known whether a ssdna binding protein coats and stabilizes the geminivirus genomic ssdna prior to replicative form synthesis, as has long been known for ssdna phages that replicate via the rcr mechanism (arai and kornberg, 1979; geider et al., 1978) . the replication-associated protein a (repa), the protein product of orf c1, and rep are the two complementary-sense proteins of the mastreviruses (collin et al., 1996; gutierrez et al., 2004; hofer et al., 1992; liu et al., 1997) , and are both required for virus replication. the repa mrna accounts for most of the complementary-sense transcripts in monocot-infecting mastreviruses, including msv, wdv and digitaria streak virus (dsv) (dekker et al., 1991; mullineaux et al., 1990; wright et al., 1997) , as well as in the dicot-infecting mastreviruses (gutierrez, 2000; hefferon et al., 2006; munoz-martin et al., 2003) . repa and rep have identical primary sequences in their approximately 200 n-terminal amino acid residues (fig. 2b) . like rep, repa is multifunctional and, in several cases, performs the functions of bipartite geminivirus reps, including the transactivation of virus-sense orfs, as has been elucidated for msv, wdv and bean yellow dwarf virus (beydv) (hefferon et al., 2006; munoz-martin et al., 2003) . repa also represses its own expression and the expression of rep, as elucidated in beydv (hefferon et al., 2006) , by binding to a site in the lir. repa interaction with rbr has been mapped to the lxcxe motif in the mastreviruses msv, beydv and wdv (horvath et al., 1998; liu et al., 1999; xie et al., 1995) . however, the lxcxe motif is absent in some mastrevirus repas, including sugarcane streak virus (ssv), miscanthus streak virus (misv) (hughes et al., 1993) and beydv (hefferon et al., 2006) , suggesting that it is not an absolute requirement for mastreviruses. furthermore, lxcxe is absent in begomovirus reps, which bind to rbr through a novel, conserved a-helical region (arguello-astorga et al., 2004; hanley-bowdoin et al., 2004; kong et al., 2000) . other repa interacting partners include a host transcription factor, designated grab (geminivirus repa-binding), in the nac family [acronym for nam (for no apical meristem), ataf1-2 and cuc2 (for cup-shaped cotyledon)] (gutierrez et al., 2004; xie et al., 1999) . grab proteins, which are involved in plant development and senescence, impair geminivirus replication, suggesting that they modulate the viral replication cycle (lozano-duran et al., 2011; xie et al., 1999) . curiously, circoviruses, another group of circular ssdna viruses, which infect animals and replicate using the rcr mechanism, also have two replication initiation proteins, designated rep and rep'. rep and rep' (a spliced isoform of rep) are indispensable for circovirus replication (mankertz and hillenbrand, 2001) and have structural and functional similarities to rep/repa, suggesting a close evolutionary relationship between the two virus groups. however, whereas, in circoviruses, replication is terminated by a rep/rep'-catalysed nucleotidyl transfer reaction to cleave monomer units (steinfeldt et al., 2006) , geminivirus replication produces monomers and multimers, suggesting an inefficient repmediated monomer cleavage during rcr. the transcriptional activator protein (trap) is encoded by the ac2 (or al2) orf in bipartite begomoviruses and by c2 in monopartite begomoviruses. trap is a positional analogue of c2 (or l2) protein in curtoviruses and topocuviruses. ac2, together with ac3, is expressed from a dicistronic transcript driven by a strong monodirectional promoter located within the ac1 coding sequence . trap, which has been well characterized in begomoviruses, is a multifunctional protein. it is involved in gene activation (haley et al., 1992; shivaprasad et al., 2005; sunter and bisaro, 1992) , virus pathogenicity (hong et al., 1996) and suppression of gene silencing (chowda-reddy et al., 2009; trinks et al., 2005; vanitharani et al., 2005; voinnet et al., 1999; wang et al., 2005) . trap activates the transcription of the genes coding for coat protein (cp) and movement protein (mp) in a non-virus-specific manner (hanley-bowdoin et al., 1999; saunders and stanley, 1995; sunter et al., 1994) , and transactivation is dependent on its zinc-finger and c-terminal acidic domains ( fig. 3 ) (hartitz et al., 1999) , as well as on its ability to form multimers (yang et al., 2007) . trap regulates the tissue-specific expression of the cp promoter by overriding a putative host repressor in phloem tissues (sunter and bisaro, 1997) . cp promoter modulation is thought to occur through trap interaction with different components of the cellular transcriptional machinery which binds the viral sequences required for the regulation of cp promoters (lacatus and sunter, 2009 ). the arabidopsis transcription factor, peapod2, specifically binds to activating sequences in the cp promoter of tgmv and cabbage leaf curl virus (calcv) in mesophyll tissues, but not to sequences required for trap-mediated derepression in phloem tissues (lacatus and sunter, 2009 ). trap does not specifically bind to double-stranded dna (dsdna) and, instead, is thought to be directed to responsive promoters through interactions with cellular proteins (hartitz et al., 1999; lacatus and sunter, 2009 ). thus, trap is similar to the adenovirus e1a protein, a transactivational regulatory factor, which does not bind dna, but is necessary for transcriptional activation (avvakumov et al., 2002; zu et al., 1992) . conclusive evidence of transcriptional activation activity by a monopartite begomovirus c2 protein was reported for tomato leaf curl virus (tolcv) (dry et al., 2000) , and it is likely that other monopartite begomovirus c2 proteins are also transcriptional activators. in contrast, the curtovirus c2 does not activate transcription (hormuzdi and bisaro, 1995) . a possible mechanism of trap nuclear import for transcriptional activation is provided by the reported interaction between c2 of the begomovirus bhendi yellow vein mosaic virus and karyopherin a (chandran et al., 2012), a soluble receptor, which probably transports c2 through nuclear pore complexes into the nucleoplasm. trap is also a pathogenicity factor, and may counter a hypersensitive response (hr) in infected cells. the hr, a form of programmed cell death (pcd) associated with resistance to pathogens, is induced at infected sites or within defined areas surrounding the infected sites and limits pathogen growth (a review is provided in postel and kemmerling, 2009 and in coll et al., 2011) . however, the trap of the bipartite begomovirus tomato leaf curl new delhi virus (tolcndv) can overcome nuclear shuttle protein (nsp)-induced hr (discussed below) (hussain et al., 2007) . trap compromises the ability of cop9 signalosome (csn), a protein complex that functions in the ubiquitinproteasome pathway, to bind to cullin-1 (cul1), which is an essential component of the scf (skp1, cul1/cdc53, f box proteins) ubiquitin e3 ligase complex. trap-cop9 interaction alters the cellular processes regulated by scf complexes, including jasmonate signalling (lozano-duran et al., 2011) , thereby regulating host response to infection. most plant viruses contain suppressors of rna silencing, a phenomenon that regulates gene expression and protects plants from transposable elements and viruses (reviews are available in pantaleo, 2011 and in wang et al., 2012) . the bipartite geminivirus trap and c2 from monopartite geminiviruses have been shown to suppress rna silencing (chowda-reddy et al., 2009; dong et al., 2003; hamilton et al., 2002; trinks et al., 2005; vanitharani et al., 2004; voinnet et al., 1999; wang et al., 2005) . correspondingly, beet severe curly top virus (bsctv) c2 has been shown to decrease dna methylation, thus affecting the production of small interfering rnas (sirnas) which are required for the targeting and reinforcing of rna-directed dna methylation and rna silencing (yang l.p. et al., 2012) . consistent with these findings, the transient expression of tgmv trap and bctv c2 increases the susceptibility of tobacco to these geminiviruses (hao et al., 2003; sunter et al., 2001) . taken together, these findings establish the involvement of trap and c2 in gene regulation and virus pathogenicity, and emphasize the multifunctional nature of geminivirus proteins, which, together with overlapping genes, contribute to viral genetic economy. the replication enhancer protein (ren), which is absent in mastreviruses, is encoded by the ac3 (or al3) orf in bipartite begomoviruses and by the c3 (or l3) orf in curtoviruses and monopartite begomoviruses. like ac2, ac3 expression is driven by a strong monodirectional promoter located within the ac1 coding sequence . although not essential for virus replication, ren enhances viral dna accumulation and symptom development in plants infected by begomoviruses (sung and coutts, 1995; sunter et al., 1990) and curtoviruses . the role of ren in viral dna replication involves its interactions with rep and pcna. regions of ren responsible for these interactions were elucidated in two bipartite begomoviruses, tylcv and tomato yellow leaf curl sardinia virus (tylcsv) (castillo et al., 2003; settlage et al., 2005) (fig. 4) . like rep, ren does not contain an lxcxe motif, which mediates the binding of several mastrevirus repas to rbr, suggesting the existence of an alternative rbr interaction domain in ren (settlage et al., 2001) . like repa, ren also interacts with sinac1 (solanum lycopersicum nac1) (selth et al., 2005) . sinac1 levels were shown to be higher in tolcv-infected cells, suggesting that nac1 is involved in viral dna replication (selth et al., 2005) , possibly through an interaction with ren. recently, the tomato leaf curl kerala virus ren was shown to interact with rep and enhance the repmediated atpase activity (pasumarthy et al., 2010) , thus confirming a role for ren in viral dna replication. the orfs ac4 (or al4) in bipartite geminiviruses and c4 (or l4) in monopartite geminiviruses (except mastreviruses, where c4 is absent) are contained entirely within the ac1 orf, but in a different frame, and are the least conserved of all geminivirus proteins, with divergent biological functions in monopartite and bipartite geminiviruses. disruption of the c4 orf of tolcv (rigden et al., 1994) , tylcv (jupin et al., 1994) and bsctv (teng et al., 2010) caused a reduction in viral dna levels and symptom development, suggesting its involvement in symptom development and virus movement. c4 is also the major determinant of the characteristic vein swelling phenotype observed in plants infected by bctv (mills-lujan and deom, 2010; . expression of the bctv and bsctv c4 proteins in transgenic arabidopsis results in phenotypes that mimic the symptoms seen during viral infection (mills-lujan and deom, 2010) . similarly, the expression of bctv c4 in transgenic n. benthamiana results in ectopic cell division (latham et al., 1997; piroux et al., 2007) , implying that the vein swelling associated with bctv c4 is caused by abnormal cell division. consistent with this finding, lai et al. (2009) reported that bsctv c4 induced the expression of rkp, which may be involved in cell cycle regulation. according to a study by park et al. (2011) , infection by bsctv, as well as overexpression of bsctv c4, leads to the induction of expression of athb7 and athb12 in arabidopsis thaliana. these observations implicate c4 in infected cell development and differentiation. tgmv ac4 has been reported to be involved in virus movement , but is not critical for virus infection (elmer et al., 1988) , and disruption of the ac4 orf in african cassava mosaic virus (acmv) and east african cassava mosaic zanzibar virus (eacmzv) has no effect on the infection phenotype in n. benthamiana (bull et al., 2007; etessami et al., 1991) . similar observations were reported for ac4 encoded by bgmv (hoogstraten et al., 1996) and potato yellow mosaic virus (sung and coutts, 1995) . the ability of ac4 and c4 to suppress rna silencing is conserved for several bipartite and monopartite geminiviruses. for example, ac4 proteins of acmv and east african cassava mosaic virus (eacmv)-like viruses have been shown to suppress rna silencing (fondong et al., 2007; vanitharani et al., 2004) by binding micrornas (mirnas) and sirnas (chellappan et al., 2004) . the ability of east african cassava mosaic zanzibar virus (eacmzv) ac4 to suppress silencing depends on its n-terminal myristoylation sequence, in which acetylation occurs as a result of the attachment of a myristate and a palmitate in the n-terminus (fondong et al., 2007) . furthermore, tolcv-au c4 suppresses silencing by binding to shaggy-like kinase (slsk) through its c-terminus (dogra et al., 2009) . the geminivirus cp is a late gene and is the only structural protein of geminivirus particles (stanley and gay, 1983) . it is encoded by the av1 (also ar1) orf in bipartite geminiviruses and by the v1 orf in monopartite geminiviruses (zhang w. et al., 2001) . in addition to virus genome packaging, the cp has been associated with several other functions, including insect transmission (boulton et al., 1993; briddon et al., 1990; mullineaux et al., 1984) , shuttling of viral dna into and out of the nucleus in monopartite geminiviruses (liu et al., 1999) , and cell-to-cell and systemic spread of virus (boulton et al., 1989; liu et al., 1997; pitaksutheepong, 1999) . the cp binds ss-and dsdna (liu et al., 1997) and may participate indirectly in viral dna replication during rcr by binding and sequestering ssdna from the replication cycle (saunders et al., 1991; stenger et al., 1991) . although all begomovirus cps contain sequences that are highly conserved, they also contain regions that are variable and can be used to correlate phylogenetic inferences with biotic and geographic characteristics (padidam et al., 1995) . evidence that the cp plays a critical role in insect virus transmission was elucidated using the whitefly-transmitted acmv and the leafhopper-transmitted bctv. an acmv chimeric virus containing the bctv cp gene is transmitted by leafhoppers, which, in nature, transmit bctv but not acmv (briddon et al., 1990) . sequences important for insect transmission have been mapped to the central part of cp (hohnle et al., 2001; kheyr-pour et al., 2000; liu et al., 2001; noris et al., 1998; unseld et al., 2001) and may play a role in cp multimerization, which is necessary for virus capsid assembly and insect transmission (hallan and gafni, 2001; noris et al., 1998; zhang w. et al., 2001) . some monopartite geminivirus cps shuttle viral dna between the nucleus and the cytoplasm, as has been elucidated for mastreviruses (liu et al., 1999 (liu et al., , 2001 and for tylcv (kunik et al., 1998; rojas et al., 2001) . this is consistent with the fact that the cp of squash leaf curl virus (sqlcv), a bipartite begomovirus, can functionally replace nsp (encoded by bv1) (ingham et al., 1995; qin et al., 1998) . nuclear import and export appear to be dependent on ssdna binding to the n-terminal domain of the cp (pitaksutheepong et al., 2007) . the cp nuclear localization signal (nls) has been localized to the n-and c-termini and the central region of the protein (unseld et al., 2001) (fig. 5) . the bipartite begomovirus mymv cp interacts with importin a, a component of the nuclear pore-targeting complex (guerra-peraza et al., 2005) , and the n-terminal nls of tylcv cp binds to karyopherin a1 (kunik et al., 1999) for nucleocytoplasmic trafficking. the n-terminal nls also binds to the groel protein of arsenophonus, a bacterial endosymbiont found in the midgut of bemisia tabaci, presumably to protect the virions in the insect vector haemolymph (kunik et al., 1999; morin et al., 2000; rana et al., 2012; yaakov et al., 2011) . a leucine-rich nuclear export signal (nes) that mediates trafficking from the nucleus to the cytoplasm was mapped to the cp central region (ward and lazarowitz, 1999) . it is important to note that bipartite begomoviruses with mutated or replaced cp are infectious, albeit with delayed and attenuated symptoms (brough et al., 1988; sudarshana et al., 1998) , suggesting that other host proteins functionally replace the cp and import the genome to the nucleus on initial viral infection and prior to the synthesis of nsp. interestingly, geminivirus cps accumulate preferentially in the nucleolus, as exemplified by cps of the monopartite begomoviruses tomato leaf curl java virus (tolcjv) and tylcv (rojas et al., 2001) , and the bipartite begomovirus eacmcv (v. n. fondong et al., unpublished data) . it is worth noting that increasing numbers of key proteins from both animal rna and dna viruses have been shown to localize to the nucleolus and to play important roles in the virus infection cycle, including herpesvirus saimiri orf57 protein (boyne and whitehouse, 2006), coronavirus protein n (hiscox et al., 2001) , groundnut rosette virus orf3 (kim et al., 2007a) , potato leaf roll virus cp (kim et al., 2007a) and potato mop top virus triple gene block 1 protein (tgb1) (kim et al., 2007b) . these observations implicate the nucleolus in the virus infection cycle. indeed, it has been shown recently that nucleolar localization is required for alfalfa mosaic virus virion formation and systemic movement (herranz et al., 2012) . in addition to nuclear shuttling, cps of mastreviruses (liu et al., 2001) and monopartite begomoviruses (briddon et al., 1989; padidam et al., 1996; rigden et al., 1993) are involved in cell-to-cell movement and systemic spread of viral dna. movement is effected presumably through interaction with the mp (v2 orf), as demonstrated for tylcv (poornima et al., 2011) and cotton leaf curl kokhran virus (poornima et al., 2011) . although bipartite begomovirus cps may not be required for infection, they have been shown to enhance cell-to-cell and/or systemic spread of several viruses, including sqlcv (ingham et al., 1995) , acmv (stanley and townsend, 1986) , bean dwarf mosaic virus (bdmv) (seo et al., 2004) , pepper huasteco virus (guevara-gonzález et al., 1999) , bgmv ), calcv (carvalho et al., 2008b and tgmv (gillette et al., 1998; . these observations suggest that the bipartite begomovirus cps have not lost these functions during evolution from their monopartite progenitors. although not essential in virus replication, the absence of cps results in reduced levels of viral ssdna with or without a reduction in the level of dsdna of tylcv (padidam et al., 1996) , tgmv (brough et al., 1988; sunter et al., 1990) , bctv (briddon et al., (padidam et al., 1999) , sqlcv (qin et al., 1998) and beydv (hefferon and dugdale, 2003) . together, these findings provide further evidence that the evolution of multifunctional proteins compensates for small-genome viruses and that multifunctional proteins highlight the plasticity through which evolution brings together functional domains into a single polypeptide chain (walsh and mohr, 2006) . the av2 orf (v2 in monopartite geminiviruses), whose start codon precedes that of the av1 orf, is found in 'old world', but not in 'new world', bipartite begomoviruses (bull et al., 2007; padidam et al., 1996; rojas et al., 2001; selth et al., 2004) , and its exact function is uncertain. although acmv clones containing mutations in av2 are infectious in n. benthamiana (etessami et al., 1989) , recent findings showing that mungbean yellow mosaic india virus (mymiv) clones containing mutations in av2 reverted to wild-type (rouhibakhsh et al., 2011) raise the possibility that the progeny from mutant acmv clones might have reverted to wild-type, thereby masking the effect of the mutation. this is supported by evidence that tolcndv containing mutations in av2 accumulates low levels of viral dna in infected plant tissues (padidam et al., 1996) . correspondingly, v2 orfs of the monopartite begomovirus tylcv (wartig et al., 1997) and the curtovirus bctv (hormuzdi and bisaro, 1995; appear to be pathogenicity determinants, and mastrevirus and monopartite begomovirus v2 orfs encode mps, as discussed in the section on movement proteins below. plants infected with tolcv and tolcndv containing mutations in v2 and av2, respectively, resulted in low levels of mutant viral dna in infected plant tissues (padidam et al., 1996; rigden et al., 1993; selth et al., 2004) . similar results have been reported recently for papaya leaf curl virus and cotton leaf curl kokhran virus (mubin et al., 2010) . these findings were subsequently supported by studies showing that both av2 and v2 suppress rna silencing, as illustrated for eacmcv av2 (chowda-reddy et al., 2008) and v2 of the monopartite begomovirus ageratum yellow vein virus . similar observations have been made for tylcv v2, most probably as a result of its interaction with the suppressor of gene silencing3 protein (sgs3) (glick et al., 2008) . bv1 (also br1) is one of the two orfs found in the b component of bipartite begomoviruses. bv1 encodes the nsp (fig. 6 ) required for trafficking viral ssdna between the nucleus and the cytoplasm (noueiry et al., 1994) in the form of a viral dna-nsp complex (ward and lazarowitz, 1999) . evidence that nsp binds to dna in a sequence non-specific manner has been illustrated for sqlcv (pascal et al., 1994) , bdmv (rojas et al., 1998) and abmv (hehnle et al., 2004) . to achieve cell-to-cell and long-distance movement, the nsp-viral dna complex is trapped by the mp in the cytoplasm and redirected to and across the cell wall into adjacent cells, where nsp directs the viral genome to the nucleus for new cycles of replication (lazarowitz and beachy, 1999; noueiry et al., 1994; sanderfoot and lazarowitz, 1995; sanderfoot et al., 1996; ward et al., 1997) . it is important to note that nsp is consistently being shown not to be required for virus infectivity, presumably because the cp exhibits nuclear import and export functions as discussed above. this has led to the suggestion that both proteins have a common evolutionary origin and share redundant functions (zhou et al., 2007) , including the nucleocytoplasmic transport of viral dna (liu et al., 1997; rigden et al., 1993; saeed et al., 2007) . accordingly, both proteins localize in the nucleus, preferentially to the nucleolus (fig. 6) (rojas et al., 2001; sharma and ikegami, 2009; zhou et al., 2011) . the mechanism by which nsp shuttles viral dna between the nucleus and the cytoplasm has been elucidated using calcv nsp, which interacts with an arabidopsis protein, nuclear shuttle protein interactor (atnsi) (mcgarry et al., 2003) . this interaction presumably results in the acetylation of nsp and the segregation of ssdna for movement and encapsidation. several geminivirus nsps are also known to interact with a cytoplasmic gtpase to export the viral dna-nsp complex from the nucleus to the cytoplasm, where it is redirected to the cell surface to interact with the viral mp (carvalho et al., 2008a) . evidence from sqlcv nsp shows that the nuclear export of nascent viral dna from the nucleus to the cytoplasm is mediated by a leucine-rich nes. in addition to virus trafficking, nsp is an avirulence determinant in some hosts. thus, bdmv nsp has been reported to induce an hr in phaseolus vulgaris (garrido-ramirez et al., 2000; zhou et al., 2007) , as does tolcndv nsp in n. tabacum and tomato (solanum lycopersicum) (hussain et al., 2005 (hussain et al., , 2007 . the involvement of nsp in virus pathogenicity is supported by its interactions with membrane receptor kinases, including nsp-interacting kinase (nik), belonging to the receptor-like group of kinases implicated in a wide range of signal transduction pathways (fontes et al., 2004; rocha et al., 2008; sakamoto et al., 2012; santos et al., 2009) . nsp also interacts with proline-rich extensin-like receptor protein fig. 6 the nuclear shuttle protein (nsp). the nsp n-terminal half contains two nuclear localization signals (nls-a and nls-b), dna binding and sequences required for the induction of the hypersensitive response, whereas the nsp c-terminus contains the nuclear export signal (nes), movement protein (mp) binding site and the arabidopsis nuclear shuttle protein interactor (atnsi). kinase (perk), probably to phosphorylate and regulate nsp function (florentino et al., 2006; fontes et al., 2004) . furthermore, a recent study has shown that a host nucleoprotein, histone h3, interacts with bdmv nsp and mp, forming a complex of histone h3, nsp, mp and viral dna (zhou et al., 2011) , to presumably facilitate the export of nascent viral dna from the nucleus to the cytoplasm for cell-to-cell and long-distance movement. the mp is encoded by the bc1 (or bl1) orf on the dna-b components of bipartite begomoviruses. in monopartite begomoviruses, it is encoded by the v2 orf, which has no sequence identity with bv1. the mp is required for virus cell-to-cell and long-distance movement, through its cooperative interaction with the nsp in bipartite begomoviruses (noueiry et al., 1994; ward et al., 1997) . recent data have suggested that mp-nsp cooperation, leading to nascent viral dna export from the nucleus, follows a 'couple-skating' transport model (frischmuth et al., 2007; hehnle et al., 2004; zhang s.c. et al., 2001) , in which mp binds to nsp-dna complexes at the cytoplasmic face of plasma membranes or microsomal vesicles and enables transfer to adjacent cells (kleinow et al., 2009) . the central region of mp is required for the formation of the mp-dna-nsp complex (fig. 7) (frischmuth et al., 2007; sanderfoot and lazarowitz, 1995; zhang et al., 2002) . unlike nsp, some geminivirus mps do not appear to bind to dna, as elucidated for mps of sqlcv (pascal et al., 1994) and abmv (hehnle et al., 2004) . in contrast, mps of bdmv (rojas et al., 1998) and mymiv (radhakrishnan et al., 2008) bind both ss-and dsdna with high affinity. in monopartite begomoviruses and mastreviruses, the mp is encoded by the v2 orf (boulton et al., 1989 (boulton et al., , 1993 mullineaux et al., 1988; wartig et al., 1997) , whereas cp (encoded by v1) shuttles viral dna between the nucleus and cytoplasm, functionally replacing nsp. although the monopartite geminivirus cp is required for viral movement (boulton et al., 1989) , v1 and v2 share no significant sequence identity with bc1 or bv1 (rojas et al., 2001) . furthermore, there are functional differences between monopartite begomovirus mps and mps of mastreviruses. for instance, similar to bipartite begomovirus mps, monopartite begomovirus mps bind viral dna, whereas mastrevirus mps do not. furthermore, cell-to-cell movement in monopartite begomoviruses is mediated by c4 (krichevsky et al., 2006; rojas et al., 1998 rojas et al., , 2001 ; cp, mp and c4 are involved in the delivery of tylcv dna, as virions or as nucleoprotein complexes, to the plant cell periphery (rojas et al., 2001) . to move the complex from the cell periphery for cell-to-cell transport, mp interacts with plasmodesmata (rojas et al., 2001) and, because mastrevirus mps do not bind viral dna, virus movement is effected through interaction with the cp (kotlitzky et al., 2000; liu et al., 2001) . monopartite geminivirus mps presumably enhance cp-mediated nuclear export of viral dna and a nes was mapped to the n-terminus of tolcjv v2 . a model has been proposed, whereby mp diverts the cp-dna complex from the nucleus to the cell periphery, where it mediates movement through the pd (liu et al., 2001) . curtovirus movement has been much less studied compared with that of other geminiviruses. a genetic analysis of bctv genes showed that the v3 orf encodes the mp (hormuzdi and bisaro, 1995), which forms vesicle-like structures that co-localize with the endoplasmic reticulum (er) and are trafficked intracellularly from the nucleus to the cell periphery (soto, 2001) . the significance of these vesicles remains to be established. the er appears to play important, but differential, roles in mp intracellular trafficking. for instance, the mp of abmv, a phloemlimited virus, exploits the cellular membrane flow from the er to the plasma membrane (zhang et al., 2002) . conversely, in virusinfected pumpkin sink leaves, mps of sqlcv (a phloem-limited virus) and calcv (which infects all cell types) predominantly associate with er-derived tubules that penetrate the cell wall and are found only in undifferentiated vascular tissues (procambium) and in lesser amounts within the plasma membrane (ward et al., 1997) . furthermore, microtubules appear to play a role in mp-viral genome movement, as has been shown for euphorbia mosaic virus (kim and lee, 1992) and indian cassava mosaic virus (roberts, 1989) . in these cases, the mp-containing tubules appear to serve as conduits for the intercellular transport of viral complexes. several host proteins that interact with geminivirus mps have been identified, including an arabidopsis regulator of endocytosis, synaptotagmin (syta), which interacts with calcv mp at the plasma membrane and directs the mp onto early endosomes (lewis and lazarowitz, 2010) . these endosomes are thought to traffic the complex via a recapture pathway to dock at the pd for intercellular transport. a similar interaction occurs between syta and the mp of tobacco mosaic virus, an rna virus, suggesting a convergent evolutionary lineage between these mps, as there is no evidence that they share a common ancestry. synaptotagmins are calcium sensors, and their ability to regulate synaptic vesicle exo-/endocytosis has long been characterized in animals (fukuda, 2003) and, more recently, in plants (chapman, 2008) . recently, abmv mp has been reported to form a complex with the heat fig. 7 the movement protein (mp). the bc1 encoded mp contains a pilot domain required for localization to the cell periphery, a central anchor domain that also targets the protein to the cell periphery and a c-terminal domain that facilitates oligomerization. the nuclear shuttle protein (nsp) binding site is in the centre of mp. shock cognate 70-kda protein to facilitate viral movement along cellular plastids and stromules into a neighbouring cell (krenz et al., 2010 (krenz et al., , 2012 . these data suggest that geminivirus movement may be tubule guided and non-tubule guided. it is clear from these findings that geminiviruses, unlike most other virus groups, use different mechanisms to move within and between cells. there have been an increasing number of reports of monopartite begomoviruses occurring in association with circular ssdna satellites, referred to as alphasatellites (briddon et al., 2004; paprotka et al., 2010; romay et al., 2010) and betasatellites yang x. et al., 2012) . furthermore, a new class of satellite dnas has been found to occur in association with the bipartite sida golden yellow vein virus (fiallo-olivé et al., 2012) . these satellite dnas depend on the helper virus for encapsidation and systemic infection (briddon and stanley, 2006) . alphasatellites encode a rep and are capable of autonomous replication in host plant cells . betasatellites, which encode the bc1 protein, depend on the helper virus for replication (cui et al., 2005; saunders et al., 2008) , and may augment the accumulation and symptoms of their helper viruses (briddon et al., 2003; jyothsna et al., 2013; patil and fauquet, 2010) as a result of the interaction of bc1 with snf1-related kinase and with s-adenosyl homocysteine hydrolase to suppress rna silencing (yang x. et al., 2012) . because of their very small genomes, geminiviruses have only four to eight proteins, some of which, most notably rep/repa, trap, and cp have evolved into multifunctional proteins to compensate for the small genomes. protein multifunctionality is evidence of the plasticity through which evolution brings together functional domains into a single polypeptide chain; together with gene overlap it contributes to genetic economy exhibited by geminiviruses. in this review, current knowledge on the function of these proteins and their interactions with host proteins have been discussed. while many of these interactions and their biological functions have been elucidated, many are still poorly understood and more information will continue to be discovered so as to provide new opportunities in the design of virus control strategies. with the geminivirus nuclear shuttle protein and potentiates viral infection the consensus n-myristoylation motif of a geminivirus ac4 protein is required for membrane binding and pathogenicity the geminivirus nuclear shuttle protein is a virulence factor that suppresses transmembrane receptor kinase activity a geminivirus replication protein is a sequence-specific dna binding protein interaction between a geminivirus replication protein and origin dna is essential for viral replication simultaneous analysis of the bidirectional african cassava mosaic virus promoter activity using two different luciferase genes the movement protein bc1 promotes redirection of the nuclear shuttle protein bv1 of abutilon mosaic geminivirus to the plasma membrane in fission yeast molecular cloning, expression, and characterization of a novel class of synaptotagmin (syt xiv) conserved from drosophila to humans bean dwarf mosaic virus bv1 protein is a determinant of the hypersensitive response and avirulence in phaseolus vulgaris an rna transcribed from dna at the origin of phage fd single strand to replicative form conversion potyviruses and the digital revolution genetic determinants of host-specificity in bipartite geminivirus dna a components interaction with host sgs3 is required for suppression of rna silencing by tomato yellow leaf curl virus v2 protein simultaneous regulation of tomato golden mosaic virus coat protein and al1 gene expression: expression of the al4 gene may contribute to suppression of the al1 gene coat proteins of rice tungro bacilliform virus and mungbean yellow mosaic virus contain multiple nuclear-localization signals and interact with importin alpha complementation of coat protein mutants of pepper huasteco geminivirus in transgenic tobacco plants dna replication and cell cycle in plants: learning from 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replication protein a by the papillomavirus e1 protein and modulation by single-stranded dna identification of dna replication and cell cycle proteins that interact with pcna geminiviruses subvert ubiquitination by altering csn-mediated derubylation of scf e3 ligase complexes and inhibit jasmonate signaling in arabidopsis thaliana interaction of geminivirus rep protein with replication factor c and its potential role during geminivirus dna replication replication of porcine circovirus type 1 requires two proteins encoded by the viral rep gene a novel arabidopsis acetyltransferase interacts with the geminivirus movement protein nsp geminivirus c4 protein alters arabidopsis development inhibition of binding of tomato yellow leaf curl virus rep to its replication origin by artificial zinc-finger protein the groel protein of the whitefly bemisia tabaci interacts with the coat protein of transmissible and nontransmissible begomoviruses in the yeast two-hybrid system the hypersensitive 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for recognition of pathogenassociated molecular patterns the bipartite geminivirus coat protein aids br1 function in viral movement by affecting the accumulation of viral singlestranded dna dna recognition properties of the cell-to-cell movement protein (mp) of soybean isolate of mungbean yellow mosaic india virus (mymiv-sb) arsenophonus groel interacts with clcuv and is localized in midgut and salivary gland of whitefly b. tabaci mutagenesis of the virion-sense open reading frames of tomato leaf curl virus orf c4 of tomato leaf curl geminivirus is a determinant of symptom severity indian cassava mosaic virus: ultrastructure of infected cells the ribosomal protein l10/qm-like protein is a component of the nik-mediated antiviral signaling bean dwarf mosaic geminivirus movement proteins recognize dna in a form-and size-specific manner functional analysis of proteins involved in movement of the monopartite begomovirus, tomato yellow leaf curl virus association of an atypical 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subunit of replication protein a (rpa) participates in the dna replication of mung bean yellow mosaic india virus (mymiv) by interacting with the viral rep protein mymiv replication initiator protein (rep): roles at the initiation and elongation steps of mymiv dna replication intracellular distribution of the three virion-sense encoded proteins of beet curly top virus subviral dnas associated with geminivirus disease complexes nucleotide sequence of cassava latent virus dna a symptom variant of beet curly top geminivirus produced by mutation of open reading frame c4 infectious mutants of cassava latent virus generated in vivo from intact recombinant dna clones containing single copies of the genome the nucleotide sequence of an infectious clone of the geminivirus beet curly top virus mutational analysis of the monopartite geminivirus beet curly top virus demonstration of nicking/ joining activity at the origin of dna replication associated with the rep and rep' proteins of porcine 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with beet severe curly top virus functional modulation of the geminivirus al2 transcription factor and silencing suppressor by self-interaction suppression of methylation-mediated transcriptional gene silencing by bc1-sahh protein interaction during geminivirus-betasatellite infection human rpa (hssb) interacts with ebna1, the latent origin binding protein of epstein-barr virus movement proteins (bc1 and bv1) of abutilon mosaic geminivirus are cotransported in and between cells of sink but not of source leaves as detected by green fluorescent protein tagging subcellular targeting domains of abutilon mosaic geminivirus movement protein bc1 structure of the maize streak virus geminate particle histone h3 interacts and colocalizes with the nuclear shuttle protein and the movement protein of a geminivirus the n-terminus of the begomovirus nuclear shuttle protein (bv1) determines virulence or avirulence in phaseolus vulgaris transcriptional regulation by a point mutant of adenovirus-2 e1a product lacking dna binding activity i thank linda hanley-bowdoin for critical reading of the manuscript. this work was supported by the national science foundation award #0724083. geminivirus-induced gene silencing of the tobacco retinoblastoma-related key: cord-271470-j58mr9xk authors: zhu, feifei; li, dong; song, dandan; huo, shuhao; ma, shangshang; lü, peng; liu, xiaoyong; yao, qin; chen, keping title: glycoproteome in silkworm bombyx mori and alteration by bmcpv infection date: 2020-04-29 journal: j proteomics doi: 10.1016/j.jprot.2020.103802 sha: doc_id: 271470 cord_uid: j58mr9xk abstract the biological functions of protein glycosylation have been increasingly recognized but not yet been very well understood, especially in lower organisms. silkworm as a model lepidopteran insect and important economic insect, has been widely studied in life science, however, the current knowledge on the glycosylation status of its proteome is not satisfactory, and little is known about how pathogenic infections could affect the glycosylation status. this study performed large scale glycosite mapping for the silkworm bombyx mori p50 strain, and quantitatively compared with that infected with the bombyx mori cytoplasmic polyhedrosis virus (bmcpv). some 400 glycoproteins were mapped in the silkworm, including nand o-glycoproteins. upon virus infection, the glycosylation levels of 41 n-glycopeptides were significantly changed, some of them belonging to transmembrane glycoproteins. the o-glycosylation profiles were also affected. in addition, 4 bmcpv-encoded viral proteins were found to be glycosylated for the first time, including polyhedrin, p101, vp3, and the ns protein. this study drafted a silkworm protein glycosylation map and underlined the potential impact of virus infection on glycosylation. significance this study reveals the characteristics of the glycoproteome in the silkworm strain p50, and quantitatively compared to that infected by the virus bmcpv, which underlines the impact of virus infection on the alteration of protein glycosylation in invertebrate species. our findings add to the knowledge of the post translational modifications of this model organism, and also uncovered for the first time the glycosylation status of the viral proteins expressed by bmcpv. silkworm bombyx mori is an economically important lepidopteran insect. since its genome revealed in 2004 [1] , it has become an increasingly popular model organism for molecular biology [2] . however, compared to the current knowledge on the silkworm's genome and transcriptome, we do not know much at the proteomic level, especially the status of post translational modifications (ptms) of the proteome. glycosylation, being one of the most common and diverse ptms, has been widely acknowledged in vertebrates as an important indicator of their physiological or pathological state [3] , however, very little is known about how glycosylation are altered in lower organisms, like during a pathogen invasion. such investigations can provide clues to the interactions between glycosylation pathways, inflammatory responses and viral immune escape [4] . the initial steps of viral infections always involve the attachment of viral particles to the receptors on the host cell surface. an important class of the receptors is transmembrane glycoproteins, which play vital roles cell adhesion and receptor bindings [5] . therefore, glycosylation patterns of the host cell, especially on the cell surface, can influence the interactions between an infecting virus and the host, and identifying such molecules are critical to the understanding of viral infection mechanisms and host immune responses. once a virus enters the host cell, it hijacks the host's cellular machinery for viral replication, altering numerous biological pathways of the cell, which is evidenced by changes in the expression levels of many genes, transcripts, and proteins. although several studies have performed transcriptomic and proteomic comparisons on the silkworm upon virus infection, such as by the bombyx mori cytoplasmic polyhedrosis virus (bmcpv) [6, 7] , whether the glycosylation profiles are affected have not been reported. bmcpv is one of the earliest identified viruses infecting silkworms, and often results in significant economic loss to the sericulture industry. bmcpv is a double stranded rna virus belonging to the cypovirus genus, reoviridae family. its genome contains 10 discrete copacked rna segments [8] , but only a few of them has been cloned and characterized so far [9, 10] . the rna genome is wrapped by 5 structural proteins, namely vp1-5 [11] . the virus forms occlusion bodies in infected silkworm midgut, which is composed of the matrix protein polyhedrin that encloses many visions within the matrix [12] . in this study, we performed large scale glycosylation site mapping on the silkworm strain p50, and quantitatively compared to that infected by bmcpv. the mapping strategy, which was first introduced by zielinska et al. [13] , uses high precision mass spectrometer for detection of a +2.988 da mass shift after enzymatic deamidation of the n-glycosylated asparagine residue in o 18 water. taking advantage of the greatly reduced glycosylation complexity due to removal of the n-glycans, we were also able to simultaneously map the o-glycosites and their associated glycans by searching against an insect o-glycan panel. additionally, the glycosylation profile of the virus-encoded proteins were obtained. transmembrane glycoproteins and significantly regulated glycoproteins upon virus infection were marked, which provides clues for future investigation of the role of glycosylation during viral infection in invertebrate systems. the silkworm were reared on fresh mulberry leaves at 25±1 °c and 70-90% relative humidity. on the first day of 5th instar, the silkworm was fed with 5 µl of bmcpv virion at 10 8 polyhedra/ml in phosphate buffered saline (pbs), and the control larvae were fed with pbs. after 72 hours, silkworms were harvested and residual mulberry leaves were removed from the gut and then preserved at -80 °c. biological triplicates in each group were prepared for the following sample processing and analysis. to the silkworm sample, 1.5 ml sdt lysis buffer containing 4% sds, 100 mm tris/hcl, and 0.1 m dtt at ph 7.6 was added, and the sample was homogenized in a tissue lyser (shanghai jingxin co., shanghai, china) using three beating cycles at 120 hz, 60 s at 4 °c. the solution was then sonicated in an ice bath for 10 minutes, boiled for 15 minutes, and then centrifuged at 14000 g for 40 minutes. the supernatant was collected and the protein concentration was determined by bradford analysis (thermofisher scientific) and stored at -80 ° c for further use. the protein extract was aliquoted to a tube containing 400 µg of total protein and was boiled for 5 minutes, cooled to room temperature, and then mixed with 200 µl of ua buffer (8 m urea, 150 mm tris hcl, ph 8.0). the mixture was transferred to a 10 kd ultrafiltration tube (merck millipore) and centrifuged at 14000 g at 4 °c for 15 minutes. the filtrate was discarded and this step was repeated before 100 µl of 100 mm iodoacetamide (iaa) in ua buffer was added. after mixing at 600 rpm for 1 minute, the samples were incubated for 20 min in darkness. the filters were washed three times with ua buffer at 14000 g, 4 °c for 15 minutes, then 100 µl of 25 m m nh 4 hco 3 were added to the filter and the sample was centrifuged for 15 minutes under the same conditions. this step was repeated twice. trypsin was added at a ratio of trypsin: protein = 1:50 to the samples and gently mixed for 1 min, and then incubated at 37 °c for 16 j o u r n a l p r e -p r o o f hours. the filtrate was then collected by centrifugation. the filter was washed with 40 µl 25 mm nh 4 hco 3 and the filtrate was combined. the digested peptides was transferred to a new 10 kd filter and mixed with 100 μl lectin mixture containing 2.5 mg/ml con a, 2.5 mg/ml wga, and 0.8 mg/ml rca in buffer a (table s1 ) were set as the variable modifications to the threonine or serine residues. a maximum of 5 ptms were allowed per peptide. the results were further filtrated using the following parameters: de novo average localized score ≥ 50%, unique peptide ≥ 2, ptm ascore ≥ 20, and false discover rate ≤ 1%. for quantitative analysis, perseus software [15] was used for further data processing and visualization. briefly, the raw intensities obtained by peaks software were transformed using natural log, and those had at least two valid intensities among the triplicate in at least one group (control or infected) were retained, and the remaining missing values were replaced with normal distribution (width = 0.3; downshift = 2.6). student's t-test was performed to obtain statistically different (p<0.05) (glyco)peptides between the groups. gene ontology enrichment was performed using the shinygo webserver [16] . transmembrane topology was predicted using the phobius web server [17] . and serve as a protective shield from the host immune systems [18, 19] . alteration of the glycans on the virus particle can alter the virulence and change the host range [20] [21] [22] . therefore (table 1) . these viral glycoproteins were not found in the control p50 group. polyhedrin, encoded by the genome segment 10 of the bmcpv virus and organized in octameric or dodecameric forms [23] , is the major matrix protein surrounding the virus envelope and protects the virions against long term harsh environments. polyhedrin has typical molecular weight of 27-31 kda, but no significant sequence similarities were found between the polyhedrins derived from the cpvs of different host species [9, 24, 25] . while baculovirus polyhedrin has been found with glycosylation potential [23] , there has been controversy on whether the cypovirus polyhedrin is glycosylated or not. it has been previously suggested to be glycosylated based on positive color-reactions with acid-schiff reagent [11] , however, molecular cloning of this protein showed that polydedrin does not contain a transmembrane signal peptides which guide peptide glycosylation [9] . in this study, glycosite mapping showed that bmcpv polyhedrin is nglycosylated at the single site n77, confirming that it is a glycoprotein, and it is speculated that the sugar coating could further enhance the stability and protection of the virion. another glycoproteins p101, which is a 101 kda protein encoded by the j o u r n a l p r e -p r o o f genome segment 5, was identified with a single glycosylation site at n684, although this protein was predicted with three possible n-glycosylation sites based on motif analysis [26] . the other two viral glycoproteins were vp3, a capping protein [8] , which was glycosylated at n801 and the non-structural (ns) protein, which was mapped with three glycosylation sites at n69, n48, and n138. the functions of these viral proteins have not been clarified or experimentally validated so far, and even less is known about what their glycosylation does for these proteins during virus infections. therefore, it would be of great interest to investigate these glycosylated viral proteins and their roles during virus infection. a total of 762 n-glycopeptides belonging to 389 n-glycoproteins were identified in the p50 control and bmcpv-infected groups (table s2) , revealing a high level of glycosylation in this lepidopteran insect. more than 95% of the mapped glycoproteins were within 200 kda, and a significant portion of them were closely resided within 10-100 kda (figure 1a) . the n-glycoproteins were predominantly singly or doubly glycosylated, although 14 of them were mapped with more than 5 glycosites per protein ( figure 1b) . figure 1c showed that all the 762 identified glycosylation site has the consensus sequence n-x-t/s, demonstrating the highly conserved motif required for nglycosylation. among the 389 identified n-glycoproteins, 118 were predicted to be transmembrane glycoproteins (table s3) , which can be interesting candidates to explore as potential cell surface receptors to outside pathogens. the total glycoproteins identified in the silkworm were analyzed by gene ontology (go) enrichment in the category of biological process, indicating that the silkworm glycoproteome mainly involve in metabolic processes, cell adhesion, and proteolysis processes (figure 1d ). journal pre-proof [27, 28] . previous study indicated that it is a glycoprotein [29] , but the specific sites were not known. in this study, the storage protein is mapped with three n-glycosites at n208, n519, and n598. the second most abundant silkworm n-glycoprotein is a putative cuticle protein glycosylated at n66. although many insect cuticle proteins were found to be glycosylated [30] , the functions of these modifications are not yet clear. the protein containing the highest number of glycosite was h9jrt0, with 10 n-glycosites, and was the eighth most abundant n-glycoprotein. however, it has not been characterized at the protein level. as table 2 shows, a lot of the glycoproteins are "uncharacterized", thus continued effort are needed to investigate their structures and functions, including their glycosylation status. to understand how protein glycosylation status is affected by viral infection, the total glycoproteomes in the p50 control and bmcpv-infected groups were analyzed and compared. for reliable quantitative comparison between the glycoproteomes, the data were further filtered retaining those detected at least twice in the triplicate in at least one group. this yields 489 glycopeptides belonging to 287 glycoproteins (table s4) , which were then used for quantitative comparison between the control and bmcpv-infected group. the glycopeptide intensities within each group are highly replicable (figure 2a) , as demonstrated by low discrepancy in intensity between the replicates. majority of the glycopeptide intensities in the replicates were within 10% from each other. a heat map was constructed to compare the identified glycopeptides in the two groups ( figure s1 ), which showed that majority of the n-glycopeptides had a similar profile between the control and the virus-infected groups. statistically significant (p<0.05) glycopeptides between the two groups were shown in figure 2c and 2d. a total of 14 glycopeptides were significantly downregulated in the virus-infected group, and another 27 glycopeptides significantly upregulated. among them, 9 were predicted transmembrane proteins (table s3) physiological and pathological processes [32] . previous studies showed that silkworm integrin beta interacts with the bmcpv virion, and that silencing integrin beta gene could inhibit viral infection to the silkworm [33] . our data showed that integrin beta was upregulated during bmcpv infection, indicating its potential role as a cell adhesion receptor facilitating viral propagation. sepin, a glycosylated serin protease inhibitor, was previously found significantly upregulated in the bacteria-infected fat body of the silkworm [34] , similar to what we observed in the silkworm upon viral infection in this study. it is noted that the ecdysone oxidase and another glycoprotein (h9iui0) both contain two glycopeptides that were downregulated. however, another interesting observation is that while the glycopeptide (n181) of ssr was downregulated, its glycopeptide at n126 wass upregulated. the results indicate that while viral infection affects the level of protein glycosylation, it may or may not influence in the same way. therefore, to evaluate or compare the profiles of multiply glycosylated proteins, it is necessary to locate individual glycopeptides instead of the glycoprotein as a whole, because each glycosylation site might change independently from the rest of the glycosylation sites upon perturbation or stimuli. the o-glycosylation mapping results for the p50 control and virus-infected groups were shown in table 3 . only those identified at least twice in the triplicate were retained for comparison purposes. a total of 13 o-glycoproteins were mapped in the p50 and its bmcpv-infected strains. the most frequently occurred o-glycans were fucose (f) and hexnac (n), followed by hex1hexnac2 (h1n2), hexnac2 (n2), hexnac3 (n3) and hexhexnacfuc (h1n1f1). a lot of the o-glycoproteins are also n-glycosylated (table 3 ). it was noticed that the uncharacterized protein h9ixn5 contain a surprisingly large number of n-glycosite: a total of 20, in addition to the o-glycosite identified (table 3) . a protein coverage map with its supporting peptides for this unknown but interesting protein was shown in figure s2 , demonstrating a reliable glycosite mapping of this protein. myosin regulatory light chain (rlc), an important structural element of myosin, is critical to cell movement and muscle contractions. rlc is a well-known phosphoprotein and its dynamic phosphorylation state is tissue-specific and case-specific [35, 36] . however, it has not been reported to be glycosylated before. in this study, silkworm rlc was found to be o-fucosylated at s104, s121 or s124, depending on whether the silkworm was infected with the virus or not. the silkworm larval cuticle protein lcp-17 was found to be n-glycosylated at n52 and n97 in both groups but only o-glycosylated in the control group at site s104 (table 3) . on the other hand, the silkworm storage protein (h9jhm9) and the uncharacterized proteins h9jrt0, h9jty5, h9j9m0, h9jta0, and h9j609 were (table s5 ). the changes in glycosylation states, together with other molecular differences such as genetic mutations, may contribute to the different responses these strains develop to virus invasions. it has been well established that alteration of glycosylation is closely associated with changes in physiological and pathological states. our previous study also showed that baculovirus resistant and susceptible silkworm strains were differentially glycosylated [14] . combining the results from this study, which showed that viral infection altered protein glycosylation in the silkworm, the relationships between host glycosylation and virus/microbe infection can be intertwined: 1) host glycosylation changes lead to infection and inflammation, 2) infection and inflammation lead to host glycosylation changes, which leads to molecular and cellular functional differences. glycosylation modification is highly dynamic and can be affected by many cellular factors and processes. during bmcpv infection, the silkworm's cellular machinery was hijacked and many biological pathways were altered, including the glycosylation pathway. in this study, the total glycoproteome in the silkworm p50, including the n-and o additional data are provided as supplementary figures s1-2 a draft sequence for the genome of the domesticated silkworm (bombyx mori) silkworm: a promising model organism in life science glycosylation in 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(prote)omics data shinygo: a graphical enrichment tool for ani-mals and plants. biorxiv advantages of combined transmembrane topology and signal peptide prediction-the phobius web server glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy the epsteinbarr virus glycoprotein gp150 forms an immune-evasive glycan shield at the surface of infected cells removal of either n-glycan site from the envelope receptor binding domain of moloney and friend but not akv mouse ecotropic gammaretroviruses alters receptor usage role of n-linked glycosylation of the 5-ht2a receptor in jc virus infection alteration of the n-linked glycosylation condition in e1 glycoprotein of classical swine fever virus strain brescia alters virulence in swine how baculovirus polyhedra fit square pegs into round holes to robustly package viruses nucleotide sequence of the polyhedrin gene of euxoa scandens cytoplasmic polyhedrosis virus (escpv). virology analysis of the cpolyhedrin genes from different geographical isolates of a type 5 cytoplasmic polyhedrosis virus nucleotide sequence of genome segment 5 from bombyx mori cypovirus 1 vitellogenin receptor selectively endocytoses female-specific and highly-expressed hemolymph proteins in the silkworm, bombyx mori expressional analysis of the silkworm storage protein 1 and identification of its interacting proteins structural determination of the n-glycans of a lepidopteran arylphorin reveals the presence of a monoglucosylated oligosaccharide in the storage protein cuticle proteins of the boll weevil, anthonomus grandis, abdomen: structural similarities and glycosylation a signal sequence receptor in the endoplasmic reticulum membrane the integrins integrin beta and receptor for activated protein kinase c are involved in the cell entry of bombyx mori cypovirus serpin-5 regulates prophenoloxidase activation and antimicrobial peptide pathways in the silkworm, bombyx mori feifei zhu: conceptualization, investigation, formal analysis, funding acquisition dandan song: investigation, formal analysis shangshang ma: validation peng lü: resources, validation; xiaoyong liu & qin yao: review and editing keping chen: funding acquisition, supervision, project none.j o u r n a l p r e -p r o o f key: cord-287450-hydy874v authors: wendt, k ulrich; weiss, manfred s; cramer, patrick; heinz, dirk w title: structures and diseases date: 2008 journal: nat struct mol biol doi: 10.1038/nsmb0208-117 sha: doc_id: 287450 cord_uid: hydy874v structural biology is making significant contributions toward an understanding of molecular constituents and mechanisms underlying human diseases at an atomic resolution, as discussed at the international murnau conference on structural biology of disease mechanisms held in september 2007 in murnau, germany. from its very beginning, biostructural research has not only provided tremendous breakthroughs in basic biological processes but also significantly contributed to the understanding of the molecular mechanisms underlying human disease. this was first exemplified when max perutz and colleagues elucidated the molecular pathology of human hemoglobin mutations leading to sickle cell anemia 1 . more recently, three-dimensional structures of many human and pathogen proteins have served to guide drug design, with an increasing number of potential target structures determined in the context of structural genomics 2 . beyond singleprotein approaches, structural biology is now en route toward a high-resolution picture of the cell, assessing protein-protein and protein-nucleic acid complexes at steadily increasing levels of complexity. it is clear that an ever more integrated understanding of the molecular mechanisms of human disease will emerge from this path, although a major challenge lies in translating such knowledge into therapeutic strategies. in early september 2007, about 180 structural biologists and biochemists met in the picturesque town of murnau, located near staffelsee lake in the bavarian alpine upland, to reflect on these questions and discuss recent biostructural data on the molecular determinants of human diseases, including microbial and viral infections, protein misfolding diseases, cancer and metabolic disorders. these topics were addressed during five scientific sessions, two poster sessions and ample time for discussions. the scientific part of the meeting was complemented by a traditional bavarian-style social program. the meeting started out with the "murnau lecture" held by wim hol (university of washington, seattle), who gave an impressive overview of the mechanisms of maturation and activity of cholera toxin. with the example of the medical structural genomics of pathogenic protozoa (msgpp) program, he also demonstrated the power of integrating structural biology and biophysical screening approaches to quickly obtain new lead structures for potential drug targets 3 . the first session, on infectious diseases, was opened by gabriel waksman (university college london and birkbeck college), who investigates the assembly process of escherichia coli p pili, structures crucial for bacterial virulence. combining structural and biophysical methods, waksman and colleagues have shown that the donor strand-exchange mechanism of the p pilus assembly proceeds through a 'zip in-zip out' process, involving a transient intermediate complex with a key role in subunit ordering and biogenesis termination 4, 5 . this approach also led to the characterization of pilicides, small-molecule inhibitors of pilus formation that interrupt the interactions between the chaperone and the usher 6 , a noncooperative event in the cooperative assembly process. another surface structure involved in virulence of gram-negative pathogens such as shigella flexneri and yersinia pestis is the type iii secretion system (t3ss). using em, x-ray crystallography and molecular modeling, steven johnson (university of oxford) presented the first near-atomic model of a t3ss needle 7 and models of the proteins associated with the needle tip 8 , with the structural basis for the regulation of t3ss assembly under investigation. erec stebbins (rockefeller university) presented a wealth of structural data on the mechanisms of action of t3ss effectors, such as the yersinia protein kinase a (ypka), a guanine nucleotide dissociation inhibitor (gdi) for rac1 gtpase that disrupts the actin cytoskeleton of the host cell 9 . deshmukh gopaul (institute pasteur) presented data on integron integrases, enzymes that mediate recombination between short symmetric dna sequences and are thus involved in genetic information exchange between bacteria. the structure of such an enzyme from vibrio cholerae bound to dna shows that it recognizes dna structure rather than a specific sequence 10 . by exploring cell-wall biosynthesis, gunther kern and gautam sanyal (astrazeneca) showed that glutamate racemases are suitable targets for narrow-spectrum antimicrobial agents, which are sorely needed against hospitalacquired infections 11 . hartmut niemann (helmholtz centre for infection research and university of bielefeld) showed how the protein inlb from listeria monocytogenes exploits the signaling pathways of the receptor tyrosine kinase and protooncogene met to promote bacterial uptake by the host cell. they find that inlb functionally mimics the natural ligand hepatocyte growth factor/scatter factor (hgf/sf), albeit binding met at a different domain. the structure also provides insight into the activation mechanism of met, an important cancer drug target 12 . stephen matthews (imperial college london) presented the latest results on host-cell recognition by the protozoan parasite toxoplasma gondii, which secretes microneme proteins (mics) to attach to and penetrate host cells 13 . starting the session on viral diseases, rolf hilgenfeld (university of lübeck) reviewed the work from his laboratory on proteases of rna viruses, such as severe acute respiratory syndrome (sars) coronavirus and coxsackievirus b3, and also highlighted recent structural data on falcipain-2 from plasmodium falciparum, discussing implications for the design of active-site directed and allosteric inhibitors for these cysteine proteases 14 . young do kwon from peter kwong's group (us national institutes of health) shifted the focus to hiv-1, whose ability to evade the human immune system is a major obstacle for vaccine development. the binding site for human receptor cd4 on viral gp120 is accessible, but most antibodies directed to this site are not neutralizing. kwon compared the structure of gp120 in complex with non-neutralizing antibody f105 and with neutralizing antibody b12 (ref. 15 ; fig. 1 ), revealing that, upon cd4 binding, a hydrophobic surface in gp120 becomes exposed, to which the non-neutralizing antibody f105 binds. dennis bamford (university of helsinki) explored the architectural principles of capsids from viruses that infect various hosts from bacteria to humans to suggest that early cells were infected with many different viruses and only a limited number of folds have been selected to assemble viable virus coats 16 . eloise mastrangelo from martino bolognesi's group (university of milan) presented work on the ns3 proteasehelicase from kunjin virus, a flavivirus affecting livestock and man. small-angle x-ray scattering (saxs) data reveal that domain rearrangements upon rna binding may explain the unwinding efficiency of ns3 (ref. 17) . winfried weissenhorn (european molecular biology laboratory grenoble) presented the three-dimensional structure of the rabies virus nucleoprotein-rna complex at 3.5-å resolution, revealing how these viruses compact and protect their genomes 18 . various pathogens are efficiently cleared from the bloodstream by the complement system, an important part of innate immunity. the complement protein c3 in its activated form (c3b), the subject of recent hot debate among the protein crystallographic community, binds to pathogens and tags them for phagocytosis via the complement receptors (crig). christian wiesmann (genentech) presented the structure of the c3b-crig complex, revealing the dramatic structural rearrangements that take place during complement activation 19 . wiesmann and colleagues further demonstrated that crig inhibits alternative pathway convertases, a finding with implications for the development of therapeutics targeting the complement system, which is involved in various human diseases. the session on protein misfolding was started out by roland riek (salk institute), who recently returned to the swiss federal institute of technology (eth, zurich). combining solid-state nmr, em and hydrogen-deuterium exchange methods, riek has identified fibrillogenic sequences in various proteins, including the amyloid peptide aβ 1-42 implicated in alzheimer's disease 20 . studies with aβ 1-42 mutants figure 1 the site on hiv-1 gp120 (gray) of interaction with the cd4 receptor (yellow) represents a conserved accessible surface on hiv-1, and many commonly elicited antibodies compete with cd4 for binding to gp120. however, most of these are weakly neutralizing and relatively impotent against primary hiv-1 isolates. one exception is the b12 antibody: whereas all of the cd4 binding site ligands seem to have an extended loop tipped by a hydrophobic residue (red), b12 recognizes gp120 in a slightly different way than cd4 does. these structures might help design an immunogen that is able to elicit more b12-like antibodies. figure indicate that the toxicity of aβ 1-42 relates to the morphology of the aggregate formed. riek also discussed the use of amyloid fibrils in nanotechnology as a deposit form for the slow release of bioactive peptides. marcus fändrich (leibniz institute, jena) reported the three-dimensional em reconstruction of an aβ 1-40 amyloid fibril in collaboration with niko grigorieff (brandeis university). at 26-å resolution, the presented em map deviates significantly from previous models, with an entirely different fibril cross section. on the basis of fibril classification results, fändrich also demonstrated that aβ 1-40 fibrils can be vastly heterogeneous, which is important when considering the biological or structural properties of a given amyloid sample. luigi vitagliano (l' istituto di biostrutture e bioimmagini and consiglio nazionale delle ricerche, naples) presented insights on fibril models from molecular dynamics calculations, starting from the crystal structure of hexa-or heptapeptides from eisenberg's laboratory (see below) and showing that the minimally stable oligomer was a pentamer of hexapeptides, with one peptide significantly shielded by the other four 21 . christian betzel (university of hamburg) presented work on aggregated prion proteins, using a cellfree assay that converts cellular prp into an isoform similar to its infectious form, prpsc, by mimicking oxidative stress, with saxs analysis providing structural insights into the mechanism of oligomerization 22 . the session was concluded by a superb presentation by david eisenberg (university of california los angeles), who gave a brief overview of early fibril models derived from cross-β-diffraction images before moving on to recent work on amyloidogenic peptides. on the basis of about 30 crystal structures of amyloid peptides determined in the eisenberg laboratory 23 , computational approaches were developed to predict the amyloid propensity of peptide stretches in proteins, identifying lvealyl from human insulin as a potentially fibrillogenic sequence. in summary, this session gave a good overview of the recent achievements in this field. although it might be a long way until a drug against the disease discovered by aloys alzheimer is developed and available to patients, it is truly remarkable how much has been learned about a process that a few years ago was the subject of wild speculations. alan fersht (university of cambridge) opened the session on cancer, reporting on the tumor-suppressor protein p53, which is inactive in about half of all human cancers, in many cases because of mutations that lower the thermal stability of the core domain to below body temperature. fersht's group has designed compounds to rescue the function of such p53 mutants as a potential cancer therapeutic strategy 24 , with the crystal structures of oncogenic p53 mutants revealing cavities that could be appropriate targets for drugs designed to chemically rescue p53 function. finally, the combination of saxs, em and nmr studies, together with previous crystallographic data, resulted in the complete architecture of the p53 tetramer, which includes large intrinsically unstructured regions 25 . holger rehmann (university medical centre utrecht) reported on the structural basis of the regulation of the guanine nucleotide exchange factor epac by camp 26 . alfred wittinghofer (max-planck institute, dortmund) has applied the detailed understanding of the gtpase reaction of ras to devise potential strategies for cancer therapy. wittinghofer's group found a small molecule of undisclosed structure that could induce activity of an oncogenic inactive ras variant in vitro 27 . guillermo montoya (spanish national cancer center, madrid) reported the molecular basis of substrate recognition by polo-like kinase 1 (plk1) and its implications for centrosomal localization 28 . claus kuhn (gene center, munich) reported on the functional architecture of yeast rna polymerase i, which carries out the synthesis of ribosomal rna, a question that was unraveled by combining cryo-em, x-ray crystallography and homology modeling. biochemical and genetic experiments nicely complemented the structural data, showing how rna polymerase i deviates in structure and function from rna polymerase ii 29 . finally, titia sixma (netherlands cancer institute, amsterdam), out of a collaboration with andrea pichler (medical university vienna) and frauke melchior (university of göttingen), reported the crystal structure of a sumoylated e2, which showed that sumoylation affects the e2 enzymatic activity by modulating e2's interaction with e1 and that recognition of the sumoylation site depends on the surrounding structure of the site. finally, she showed that sumo can bind to different sites on ubc9, depending on whether a covalent thioester-linked complex, a modified lysine or a non-covalent complex is formed 30 . the final session started with structures of extracellular receptors involved in metabolic control and cardiovascular disease. michael lawrence (walter and eliza hall institute) presented the 3.8-å crystal structure of the insulin receptor ectodomain in complex with four fabs and an insulin mimetic peptide 31 , thereby providing the first view of the spatial arrangement of low-and high-affinity insulin binding sites. günter fritz (university of konstanz) presented the unpublished structure of the ligand binding domain of rage, a multiligand receptor for advanced glycation end products, s100 proteins, hmgb1 and amyloid-β, whose activation is key to numerous chronic diseases such as diabetes, inflammation, arteriosclerosis and neurodegeneration, making it a potential therapeutic target 32, 33 . armin ruf (hoffmann-la roche) provided a view on the structure-guided design of two newly identified classes of pparα/γ dual agonists whose profile seems well suited for addressing both hyperglycemia as well as the enhanced cardiovascular risk of diabetic patients 34 . annalisa pastore (medical research council, london) shifted the focus of the session to rare diseases, with her recent findings on the anomalous expansion of polyglutamine motifs as a basis for neurodegenerative misfolding diseases 35 . antti haapalainen (university of oulu) presented structural and functional studies of the human mitochondrial acetoacteyl-coa thiolase t2, whose loss-of-function mutations result in severe ketoacidosis 36 . markus wahl (max-planck institute, göttingen) combined structural and functional studies in search of a molecular basis for retinitis pigmentosa (rp). the data support a model in which prp8, a spliceosomal factor whose mutations cause the severe rp13 form, serves as a scaffold for the assembly of other factors such as the dead box protein brr2 and gtpase snu114, and show that viable rp13-related mutations weaken but do not abolish these interactions 37 . ryota kuroki (japan atomic energy agency) presented recent structural studies on the complex of human granulocyte colony-stimulating factor (gcsf), a cytokine used for treatment of granulopenia, with its receptor. although various stochiometries had been proposed for this assembly, kuroki's work underscores the relevance of the 2:2 complex, a result that is in line with thermodynamic and mutational analyses 38 . roger williams (medical research council, cambridge) closed the session with a comprehensive overview of recent structural results on the cellular escrt machinery 39 , which mediates the trafficking of monoubiquitinated proteins to lysosomes via the multivesicular budding (mvb) pathway. this process has a role in the downregulation of cell-surface receptors and the budding of hiv and other retroviruses. williams discussed the molecular understanding reached for many components of the mvb pathway, their molecular assemblies and the sorting signal ubiquitin. major challenges remain in understanding the higher-level molecular organization of the escrt lattice together with a molecular mechanism for vesicle budding. solution of these challenges will require the application of hybrid methods to close the resolution gap between x-ray structures and current em reconstructions. in summary, the 2007 murnau conference featured 31 presentations and 66 posters on many molecular aspects underlying human disease. several of the presentations demonstrate how an advanced molecular understanding of disease-relevant factors can open new strategies for both the design of interfering small molecules and screening procedures for such compounds. in other cases, the complexity of supramolecular assemblies solved by structural methods seems to overwhelm our current ability to translate the emerging higher-order molecular view into concrete options for therapeutic intervention. as demonstrated by hol, waksman, fersht, wittinghofer, riek and many others, this step can come within reach only when structural biology research is tightly integrated with biophysical, biochemical and cellular studies. this strategy of integrating functional and structural studies of ever more complex cellular processes in search of new therapeutic entry points was reflected in many presentations and discussions at murnau. for the upcoming meeting in 2010, it may be of interest to focus on the interface of structural and chemical biology, where structural information is used to reveal the action mechanism of new bioactive compounds for improved intervention strategies 40 . structural characterization of β-sheeted oligomers formed on the pathway of oxidative prion protein aggregation in vitro key: cord-272241-2fwz8z8n authors: kumar, amit; kumar, prateek; saumya, kumar udit; kapuganti, shivani k.; bhardwaj, taniya; giri, rajanish title: exploring the sars-cov-2 structural proteins for multi-epitope vaccine development: an in-silico approach date: 2020-09-09 journal: expert review of vaccines doi: 10.1080/14760584.2020.1813576 sha: doc_id: 272241 cord_uid: 2fwz8z8n introduction: the ongoing life-threatening pandemic of coronavirus disease 2019 (covid-19) has extensively affected the world. during this global health crisis, it is fundamentally crucial to find strategies to combat sars-cov-2. despite several efforts in this direction and continuing clinical trials, no vaccine has been approved for it yet. methods: to find a preventive measure, we have computationally designed a multi-epitopic subunit vaccine using immuno-informatic approaches. results: the structural proteins of sars-cov-2 involved in its survival and pathogenicity were used to predict antigenic epitopes. the antigenic epitopes were capable of eliciting a strong humoral as well as cell-mediated immune response, our predictions suggest. the final vaccine was constructed by joining the all epitopes with specific linkers and to enhance their stability and immunogenicity. the physicochemical property of the vaccine was assessed. the vaccine 3d structure prediction and validation were done and docked with the human tlr-3 receptor. furthermore, molecular dynamics simulations of the vaccine-tlr-3 receptor complex are employed to assess its dynamic motions and binding stability in-silico. conclusion: based on this study, we strongly suggest synthesizing this vaccine, which further can be tested in-vitro and in-vivo to check its potency in a cure for covid-19. severe acute respiratory syndrome coronavirus 2 (sars-cov -2) is the causative agent for the associated disease named as covid-19, which had spread throughout the world and declared as a pandemic [1, 2] . sars-cov-2 has been demonstrated to have a strong affinity for human respiratory receptors (hace2), which makes it more vulnerable to humans globally [3] . sars-coronavirus infections are commonly found to be associated with multiple disorders, such as respiratory, enteric, hepatic, and neurological complications [4] . in general, coronaviruses (cov) belong to a family coronaviridae, which consists of enveloped positive-sense, single-stranded rna viruses [5, 6] . among the four genera of covs, sars-cov, mers-cov, and sars-cov-2 belong to betacoronaviruses [7] . inside host cells, sars-cov-2 genome is translated into two large polyproteins: replicase polyprotein 1a (pp1a) and 1ab (pp1ab) [8] . the larger pp1ab comprises 16 non-structural proteins (nsp1-nsp16), which are responsible for constructing a viral replication-transcription complex [9] . alongside the viral rna transcribes subgenomic rnas, which encode four structural proteins; spike (s), envelope (e), membrane (m), and nucleocapsid (n) and several accessory proteins [10] . while accessory proteins are engaged in host immune suppression, including downregulation of the interferon pathway, structural proteins shape the virion, facilitating genome encapsulation, viral particle assembly, and release [11, 12] . recently, one health concept is used and hypothesized that the prior exposure of humans with pet animals lowers the adverse effect of sars-cov-2. the n and s protein epitopes in comparison with taxonomically related coronaviruses' epitopes share high sequence homology, which has high implications for vaccine designing against sars-cov-2 [13, 14] . additionally, there are repurposing of drugs are also important to cobat this disease [15, 16] . since the beginning of the 21 st century, two outbreaks of sars-cov and mers-cov have led the researchers into designing vaccines against sars coronaviruses. the third outbreak, wherein sars-cov-2 infects promiscuously to humans, has given the need for speedy research into developing a broad-range vaccine against these coronaviruses. evidence shows that humoral and cell-mediated immune response plays a protective role against coronavirus infections, specifically against s and n proteins [17] [18] [19] . although the effective antibody response is short-lived, the t cell responses are found to provide long-term protection [20, 21] . similarly, t c cell response against the structural proteins s and n are long-lasting [22] . previously, immunoinformatics approaches have been used to construct the vaccines against the zika virus nipah virus, and human papillomavirus, where in-vivo studies of the human papillomavirus showed promising results [23] [24] [25] . hence, in this study, we have used immunoinformatic approaches to predict highly antigenic epitopes from sars-cov-2 structural proteins that would evoke a strong immune response in humans. for this purpose, we have used the structural proteins: spike, envelope, and nucleocapsid to predict b-cell, cytotoxic t lymphocyte (ctl) and helper t lymphocyte (htl) epitopes for construction of vaccine. thus, the multi-epitopic subunit vaccine would be capable of eliciting humoral as well as cellmediated immune responses. we have also performed the docking and molecular dynamic simulations (mds) between the vaccine and human toll-like receptor-3 (tlr-3) to study their binding stability. the outcome of the present study will result in a novel and immunogenic vaccine, which may be further accessed against sars-cov-2. the iedb tools (http://tools.iedb.org/bcell/) were used to identify the b cell epitope and for verifying antigenicity, bepipred linear epitope analysis was performed. according to the bepipred linear epitope prediction method, residues with scores above the threshold (default value 0.35) are predicted to be part of an epitope having 49% sensitivity and 75% specificity [26] . the iedb server (http://tools.iedb.org/mhcii/) was used for the prediction of mhc ii antigenic binders in translated sequences. top 10 htl epitopes were sorted based on their percentile rank and ic 50 value. epitopes with the lowest percentile rank were considered an excellent binding affinity, whereas, peptides with ic 50 values in lower (<50 nm), middle (<500 nm), and higher (<5000 nm) range corresponds to the highest, intermediate, and lowest binding affinity, for the t-cell, respectively [27] . mhc class i binding/ctl epitopes for supertype a3 were predicted for all three proteins by using an online server netctl 1.2 (http://www.cbs.dtu.dk/services/netctl/) [28] . default threshold of 0.75 was chosen for vaccine construction in this study (>1.25 corresponds to 54% sensitivity and 99.30% specificity; >1.00 shows 70% sensitivity and 98.5% specificity; >0.90 shows 74% sensitivity and 98% specificity). mhc class i binding and proteasomal cleavage prediction was performed using artificial neural networks. accordingly, tap transport efficiency was predicted using a weight matrix. mhc class ii binder epitopes/htl, with the ability to induce cell-mediated immunity, were identified using the ifnepitope server (http://crdd.osdd.net/raghava/ifnepitope/). the prediction was performed by a motif and support vector machine (svm) hybrid approach [29] . all the positive epitopes able to induce interferon-γ (ifn-γ) were selected for vaccine construct. selected high-scoring ctls, high-affinity htls, and b-cell epitopes were used to generate the vaccine sequence. the different epitopes were linked together using linkers: ctl linker (aay), htl linker (gpgpg), and b epitope linker (kk). to improve the immunogenicity of the vaccine, β-defensin 1 (uniprot id: p60022) as an adjuvant was added through an eaaak linker at the n-terminal of the construct. the servers antigenpro (http://scratch.proteomics.ics.uci.edu/ ) and vaxijen v2.0 (http://www.ddg-pharmfac.net/vaxijen/ vaxijen/vaxijen.html) were used for the antigenicity evaluation of the final vaccine construct [30, 31] . the vaxijen 2.0 server predicts the antigenicity of the multi-epitope vaccine peptide based on the physicochemical properties of the input protein. whereas, antigenpro server predicts the antigenicity of the multi-epitopic vaccine based on the protein microarray data analysis of the target organism. the server allertop v2.0 (http://www.ddg-pharmfac.net/ allertop) and allergenfp were used to predict the allergenicity of multi-epitopic vaccine [32, 33] . allertop v2.0 classifies the vaccine protein sequence by machine learning methods for the classification of allergens. allergenfp is an alignmentfree online server that uses svm module to predict the allergenic and non-allergenic nature of proteins. vaccine construct was accessed for its physicochemical properties using the protparam server (http://web.expasy.org/prot param/) that predicts the theoretical pi, instability index, halflife, stability profiling, aliphatic index, and grand average of hydropathy of the sequence [34] . protein secondary structure was determined using psipred, a web-based freely accessible online server (http://bioinf.cs. ucl.ac.uk/psipred/) [35] which also gives information of contact analysis, fold recognition, structure modeling, function prediction, protein intrinsic disorder prediction and domain prediction of a query sequence [36] . the disorder profile of the vaccine construct was generated using pondr pool of four predictors (http://original.disprot. org/metapredictor.php) [37] [38] [39] and iupred 2a predictor [40] as described in our previous reports [41] [42] [43] . the tertiary structure of the vaccine construct was generated using the i-tasser web server [44] . it provides an energy minimized model through iterative template-based fragment assembly simulations. the structure was further optimized upon adding hydrogen and missing side-chain atoms followed by minimization in schrodinger's protein preparation wizard using opls 2005 forcefield [45] . this produced a high-quality model for protein-protein docking and molecular simulations. rampage [mordred.bioc.cam.ac.uk/~rapper/rampage.php] and procheck [46] web servers were used for evaluation of the vaccine model based on the distribution of amino acids in the ramachandran plot. piper program embedded in the bioluminate module of schrodinger for protein-protein docking was implemented for docking of vaccine model and tlr-3 (pdb id: 20az) and tlr-5 (pdb id: 3j0a) receptors [47, 48] . it is an efficient tool that reduces false positive poses and performs a global search with fast-fourier transform (fft) approach. using 1000 conformations of input structures, the top 50 clusters were selected with cluster radius 9 å, which then centralized, and the outcomes of the docking based on cluster size were evaluated. with the largest cluster size, the docked complex out of 10 complexes was selected for molecular dynamics simulation. the ~86kda vaccine model in complex with tlr-3 receptor was evaluated for its binding stability in an aqueous environment for 20ns. with a total of 6,66,607 molecules of tip3p water model, 60 neutralizing cl − ions, and 0.15 m concentration of salt, the complex was incorporated in a cubic box. recently updated forcefield charmm36 was implemented to generate the topologies of the protein-protein complex. the generated system was minimized for 50,000 steps of the steepest descent algorithm. further, the equilibration process under npt and nvt conditions for 1ns was done with parrinello-rahman and v-rescale methods for coupling of pressure and temperature, respectively. lastly, production md run for all three systems was performed in periodic boundary conditions for 20ns. lincs algorithm was used for calculating bond parameters. particle mesh ewald (pme) for long-range electrostatics with fourier spacing of 0.16 was used for production md. an analysis of md trajectory, root mean square deviation (rmsd), and root mean square fluctuation (rmsf) was calculated with gmx rms, and rmsf commands. for the production of final vaccine in large quantity, java codon adaptation tool (http://www.jcat.de/) was used for codon optimization and attached with expression vector. the optimized codon sequence includes codon adaption index (value 1.0) and %gc content (30-70%), which denotes the high level of protein expression and dna stability. finally, restriction cloning was performed using tool snapgene v3.3.4. the restriction sites ndei and xhoi were added to the n and c terminals of the optimized complementary dna sequences followed by their insertion within the pet-21a(+) vector to ensure the polyprotein synthesis within the e coli expression system. htl epitopes for all structural proteins were predicted using iedb server for human mhc-ii alleles. among these, top 10 epitopes based on their least percentile rank depicting their high affinity were selected. the final selection of epitopes was made on the basis of overlapping sequences, which were further assessed for their ifn inducing potential. epitopes with positive ifn inducing potential were finally selected for vaccine construction (table 1) . for each structural protein, ctl epitopes for supertype a3 were predicted using an online server netctl 1.2. a total of 40, 5, and 4 epitopes were predicted for s, n, and e proteins, respectively (table 2) . linear b-cell epitopes in all three structural proteins were predicted using another online server bcpreds. epitopes selected on the basis of prediction scores were further screened for their non-allergic and antigenicity potential. two epitopes each for s and n with high antigenicity scores were selected for final vaccine construction are tabulated in table 3 . however, no b cell epitopes were found for the e protein. the designed construct comprising of 48 ctl epitopes, 4 htl epitopes, and 4 b-cell epitopes were then joined using linkers. additionally, in order to improve the immunogenicity of multi-epitope vaccine, the construct was tagged with an adjuvant (β-defensin-tlr-3 agonist) at n terminal region, which enhances the natural immune response. the vaccine must be antigenic and non-allergic in nature and also induce humoral as well as cell-mediated immune responses against the targeted virus. our vaccine was observed to be antigenic with a respective probability score of 0.566 and 0.845 predicted by vaxijen v2.0 and antigenpro servers. the construct was also detected as a non-allergen, checked using the allertop v2.0 server. the vaccine construct is composed of 799 amino acids and has a molecular weight of 86.35 kda. it is basic in nature (theoretical pi 9.70) and has a half-life of 30 hrs in mammalian reticulocytes (in vitro). the instability index is estimated to be 32.21, which denotes the stability of the protein. the aliphatic index (68.42) showed that the protein is thermostable, whereas grand average of hydropathicity is observed to be negative (−0.084), indicating the hydrophilic nature of vaccine. the secondary structure was predicted using pesipred 4.0 server. as shown in figure 1 , the vaccine contains several αhelices together with many the β-strands. six long helices from residues 3-19, 65-102, 119-142, 433-448, 475-541, and 597-614 with a long β-strands of 14 amino acids from residues 401-414 can be observed. the 799-residue long vaccine construct was modeled using i-tasser. the lowest energy predicted model was selected for further refinement and docking studies. however, the validation on ramachandran plot by procheck server suggested that 88% residues of the constructed vaccine model lies in the favored and allowed regions while the rest 12% are a part of generously allowed and disallowed regions (figure 2(b) ). similarly, rampage server also mapped nearly 90.7% residues in favored and allowed regions, while 9.3% residues were present in coordinates portraying the outlier region (figure 2(a) ). as predicted by secondary structure and disorder-based structure analysis, the tertiary structure of the construct has a high number of unstructured regions. based on disorder prediction data, it is largely disordered at its terminals. from the piper program, with highest cluster size 28, out of 10 complexes ranging till 15, the complex 1, also with greatest minimal local energy, was chosen for further analysis. the vaccine model interacts with the tlr-3 receptor (pdb: 20az) through its n-terminal and the middle regions (figure 3 ). the resultant interacting residues of vaccine-tlr-3 complex are shown in table 4 . at the interface of docked complex, asn617 of vaccine is observed to form a h-bond with asp153 residue of the receptor. additionally, a few more aromatic h-bonds between val621, leu620 residues of vaccine, and his60 of tlr-3 are identified. the complex if further stabilized by a pi-cation interaction between lys595 of vaccine and tyr283 of tlr-3 (table 4 ). figure 4 depicts a close view of vaccine-tlr-3 interface. several amino acids (red) of the receptor (green) are found engaged with vaccine residues (blue). additionally, we have also performed docking with another toll-like receptor (tlr-5) which is known to recognize the flagellin of bacteria. similar to tlr-3, the vaccine model has shown significant interactions with tlr-5 through its n-terminal as well as residues of middle region where the n-terminal folds in the constructed 1 . psipred graphical result of secondary structure prediction of vaccine is represented by different colors: α-helix (pink), β-sheet (yellow), and coil (gray). expert review of vaccines model, as shown in figure 5 . residues such as tyr5, glu17 from n-terminal, and thr511, lys514 from middle regions of vaccine construct interacts with residues asp607, ala674, cys585, and glu584 of tlr-5, respectively, through aromatic hydrogen bonds, salt bridge, and hydrogen bonds. to analyze the stability and dynamic motions of docked complex, we performed a 20ns long md simulation using gromacs v5.1. the simulation system comprised ~7 lacs atoms in a cubic box with proteins, solvent, and ions. based on the rmsd data, the complex was found to have an upward trend ranging from ~1 to 2.5 nm till 20ns with small fractions of stability in between ( figure 6(a) ). however, according to rmsf values depicted in the graph of figure 6 comparison with the other residues. overall, the average fluctuation in the residues of the vaccine model was near ~1.2 nm. moreover, the regions interacting with tlr-3 receptor are not disordered as per our analysis in figure 1(b) and had stability throughout the simulation. as calculated in visual molecular dynamics, the average h-bonds were found to be 200 in vaccine-tlr-3 complex during md simulations (figure 6(c) ). additionally, disordered regions based on the sequence of vaccine were also predicted. the graph in figure 6 (d) represents the moderate amount of disorder (~20% calculated as the mean of all predictors) within residues 50-100 and 650-799 near n-and c-terminal regions. the regions interacting with tlr-3 receptor are not disordered as per our analysis in figure 6 (d) and had stability throughout the simulation. the cai improved value for codon optimized sequence was 1.0, while gc content obtained for our vaccine construct was 49.56%. overall, the vaccine sequence lies in a suitable category for its expression in e coli. finally, the vaccine construct was cloned with restriction sites ndei and xhoi in the e coli pet-21a(+) vectors. finally, a cloned construct was generated, having a sequence length of 7767 base pairs (figure 7 ). sars-cov-2 is highly pathogenic, which has caused more than 4 lakh deaths globally until the writing of this manuscript. particularly among all, coronaviridae structural proteins s, m, e, and n are the main structural proteins and can be employed for protein/peptide-based vaccines. different human infecting coronaviruses like oc43, hku1, and mers-cov use a diverse set of lipids and receptors to enter into target cells [49, 50] . in sars-cov, the spike glycoprotein interacts with ace-2 receptor and facilitates the fusion of viral membrane with the lungs membrane [11, 51, 52] . e is an integral membrane protein with n-terminal region, a transmembrane domain (tmd), and a flexible hydrophilic c-terminal tail containing a pzdbinding motif in the last four amino acids [53, 54] . envelope protein forms pentamer to lead viroporin like structure that exhibits a membrane-destabilizing ion-channel activity on the host membrane [55] . cov envelope, which plays a vital role during virion assembly, consists mainly of m protein and only a small fraction of e protein [56] . whereas n protein shows interaction with m protein in lipid membrane of infected cells forming a double-layered vesicular structure in a secreted form that further interacts with host proteins [57, 58] . multiple vaccines are in clinical trial, but till date, no vaccine is approved against the sars-cov-2. in a recent in-silico report, the vaccine model using spike glycoprotein has been constructed but limited to the docking with tlr 5 receptor [59, 60] . it was reported that human coronaviruses oc43 recognize the mhc class i supertype a3 [61] . based on these reports, we have chosen the a3 super type for epitope prediction, which will help the vaccine construct to act significantly. according to information obtained from the world health organization (who), one vaccine is in phase 2, and two vaccines (dna and rna based) are in phase 12 clinical trial. moreover, 67 vaccines are in pre-clinical trials [62] . previously, studies showed that the whole virus vaccine and virus-like particle vaccine against sars-cov are able to cure infection but leads to the pulmonary immunopathologic type lung disease [63] . therefore, it is needed that the vaccine is selected on the basis of multi factors, i.e., antigenicity, immunogenicity, toxicity, allergenicity. in the present study, we have designed the vaccine construct by using immune-informatics approaches. three structural proteins (spike glycoprotein, nucleocapsid, and envelope) were selected to construct a multi-epitope vaccine, which is capable of eliciting the humoral and cell-mediated immune response. furthermore, the vaccine is antigenic, non-allergen, nontoxic, immunogenic in nature, and capable of ifn-γ production. the vaccine epitopes were joined with the help of specific linkers to enhance stability and immunogenicity. the vaccine has a molecular weight of 86.35 kda, basic in nature, and half-life of 30 hrs in mammalian reticulocytes (in-vitro). moreover, vaccine was found to be highly stable and hydrophilic in nature. finally, the build vaccine construct was found to be moderately disordered, containing few disordered residues at n-terminal and relatively a vast region at c-terminus. as previously known, the vaccine candidates often have an ample amount of protein intrinsic disorder as they feature in the immune system [64] . further, we also checked the interaction of vaccine construct through a modeled structure docked with two toll-like receptors (tlr-3 and tlr-5) structure. the adjuvant βdefensin 1 showed interaction with tlr-3 receptor and few residues from the same region also showed interaction with tlr-5. further, the stability of the vaccine-tlr-3 complex was monitored by implementing molecular dynamics simulations till 20ns. several residues at the n-terminal and middle region of vaccine construct were found to form stable interactions. finally, the cloned sequence vector was generated in-silico for the expression of the vaccine. newly emerged sars-cov-2 is declared as pandemic and scientists all over the world running a race against the time to find vaccine/drugs against covid-19. despite that, many obstacles like allergenicity, immunogenicity, toxicity, etc. limit the progress of vaccine development. in recent times, advancement in immunoinformatics approaches led researchers to speed up vaccine development. in this context, by precise prediction and selection of multi-epitope, we strongly suggest to experimentaly validate this multi-subunit vaccine, in-vitro and in-vivo. ak, pk, kus, sk, tb: acquisition and interpretation of data, writing, and editing of the manuscript. features, evaluation and treatment coronavirus (covid-19). statpearls understanding covid-19 via comparative analysis of dark proteomes of sars-cov-2, human sars and bat sars-like coronaviruses clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series sars-cov-2 causing pneumonia-associated respiratory disorder (covid-19): diagnostic and proposed therapeutic options is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic? coronaviruses -drug discovery and therapeutic options this important review on coronavirus details about drug discovery coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 sars and mers: recent insights into emerging coronaviruses coronaviruses: an overview of their replication and pathogenesis emerging coronaviruses: genome structure, replication, and pathogenesis structure, function, and evolution of coronavirus spike proteins a new coronavirus associated with human respiratory disease in china comparative computational analysis of sars-cov-2 nucleocapsid protein epitopes in taxonomically related coronaviruses. microbes infect molecular basis of covid-19 relationships in different species: a one health perspective structural and evolutionary analysis indicate that the sars-cov-2 mpro is a challenging target for small-molecule inhibitor design probable molecular mechanism of remdesivir for the treatment of covid-19: need to know more preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies a dna vaccine induces sars coronavirus neutralization and protective immunity in mice this important paper detail about vaccine development identification of an epitope of sars-coronavirus nucleocapsid protein long-lived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sars-recovered patients lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study virus-specific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection designing b-and t-cell multi-epitope based subunit vaccine using immunoinformatics approach to control zika virus infection strategic development of a next-generation multi-epitope vaccine to prevent nipah virus zoonotic infection in silico/in vivo analysis of high-risk papillomavirus l1 and l2 conserved sequences for development of cross-subtype prophylactic vaccine this important paper detail about insilico vaccine development followed by in vivo studies improved method for predicting linear b-cell epitopes nn-align. an artificial neural network-based alignment algorithm for mhc class ii peptide binding prediction large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction designing of interferon-gamma inducing mhc class-ii binders vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines high-throughput prediction of protein antigenicity using protein microarray data allertop -a server for in silico prediction of allergens allergenfp: allergenicity prediction by descriptor fingerprints protein identification and analysis tools on the expasy server protein secondary structure prediction based on position-specific scoring matrices the psipred protein analysis workbench: 20 years on exploiting heterogeneous sequence properties improves prediction of protein disorder predicting intrinsic disorder from amino acid sequence pondr-fit: a meta-predictor of intrinsically disordered amino acids iupred2a: context-dependent prediction of protein disorder as a function of redox state and protein binding the dark side of alzheimer's disease: unstructured biology of proteins from the amyloid cascade signaling pathway folding and structural polymorphism of p53 c-terminal domain: one peptide with many conformations understanding the penetrance of intrinsic protein disorder in rotavirus proteome the i-tasser suite: protein structure and function prediction protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments procheck: a program to check the stereochemical quality of protein structures decoys as the reference state) potentials for protein-protein docking piper: an fft-based protein docking program with pairwise potentials human coronaviruses oc43 and hku1 bind to 9-o-acetylated sialic acids via a conserved receptor-binding site in spike protein domain a identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein mechanisms of coronavirus cell entry mediated by the viral spike protein cooperative involvement of the s1 and s2 subunits of the murine coronavirus spike protein in receptor binding and extended host range biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein the sars coronavirus e protein interacts with pals1 and alters tight junction formation and epithelial morphogenesis this imporatant paper detail about envelope protein and their role in pathogenesis sars coronavirus e protein forms cation-selective ion channels nucleocapsidindependent assembly of coronavirus-like particles by co-expression of viral envelope protein genes analysis of multimerization of the sars coronavirus nucleocapsid protein analyses of coronavirus assembly interactions with interspecies membrane and nucleocapsid protein chimeras development of epitope-based peptide vaccine against novel coronavirus 2019 (sars-cov-2): immunoinformatics approach consider tlr5 for new therapeutic development against covid-19 human coronavirus oc43 interacts with major histocompatibility complex class i molecules at the cell surface to establish infection who. who | coronavirus disease world health organization immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus insights into the immunological properties of intrinsically disordered malaria proteins using proteome scale predictions the authors would like to thank iit mandi for research facilities. rg is thankful to dbt, govt. of india (bt/11/iyba/2018/06) to rg. ak is supported from dbt, govt. of india grant (bt/11/iyba/2018/06). pk and sk were supported by mhrd-india for their funding. kus and tb are grateful to the icmr srf and dst inspire phd fellowships, respectively. key: cord-271091-ffn59sgf authors: galao, rui p; scheller, nicoletta; alves-rodrigues, isabel; breinig, tanja; meyerhans, andreas; díez, juana title: saccharomyces cerevisiae: a versatile eukaryotic system in virology date: 2007-10-10 journal: microb cell fact doi: 10.1186/1475-2859-6-32 sha: doc_id: 271091 cord_uid: ffn59sgf the yeast saccharomyces cerevisiae is a well-established model system for understanding fundamental cellular processes relevant to higher eukaryotic organisms. less known is its value for virus research, an area in which saccharomyces cerevisiae has proven to be very fruitful as well. the present review will discuss the main achievements of yeast-based studies in basic and applied virus research. these include the analysis of the function of individual proteins from important pathogenic viruses, the elucidation of key processes in viral replication through the development of systems that allow the replication of higher eukayotic viruses in yeast, and the use of yeast in antiviral drug development and vaccine production. the yeast saccahromyces cerevisiae has been successfully used for many years as a model organism to unravel biological processes in higher eukaryotes. because it is easy to grow and to manipulate genetically it has been always at the forefront of technical advances. for example, s. cerevisiae was the first eukayotic organism whose genome was sequenced. the complete yeast genome is known since 1996 and comprises 6000 genes, from which more than 60% have an assigned function. remarkably, comparative genomic analysis have shown that approximately 40% of yeast genes share conserved amino acid sequences with at least one known or predicted human protein [1] . moreover, 30% of human genes with a recognized involvement in human diseases have orthologs in yeast [2] . due to this notable gene homology and the high conservation of fundamental biochemical pathways, studies in yeast have been essential for understanding fundamental cellular processes such as mrna translation and degradation [3, 4] , dna repair mechanisms [5] and the cell cycle [6] . many of these studies were carried out using classical genetic tools in which a few number of genes can be analyzed at a time. however new technological platforms and tools have been recently created that allow large-scale functional analysis in yeast. these platforms and tools are commercially available and comprise (i) gene-deletion mutant collections that include ~6000 heterozygous diploid strains, each of which contains a deletion of a single copy of one specific gene, and ~5000 homozygous diploid and haploid strains in which each of the non-essential yeast genes is deleted [7, 8] , (ii) essential gene mutant collections that include an extensive set of promoter-shutoff strains in which the essential genes are placed under the control of a tetracycline(tet)-repressible promoter [9] , and a set of heat inducible shutoff strains to generate ts alleles of essential genes [10] . both systems allow the study of the ~1000 essential genes in a homozygous background; (iii) geneexpression libraries that include plasmids in which each yeast gene is cloned under the control of the galactoseinducible gal1 promoter. an additional set of plasmids have been created in which the yeast proteins are fused with different tags such as the flag epitope, the glutathione s. transferase (gst) or the histidine 6 repeat (his6) [11] [12] [13] [14] ; (iv) dna microarrays have been created that allow to assess the level of expression of thousands of yeast genes in parallel [15, 16] , and (v) protein chips containing approximately 5800 yeast proteins fused to gst-his6 [14] . all these tools have opened new avenues in medical research in which systematic genome-wide screenings can be used to characterize disease-related molecular events and to discover novel medical compounds [17] [18] [19] . viruses remain a major thread for human and animal health as well as in agriculture. nowadays, there is still no vaccine or curative therapy available for main virus pathogens such as the human immunodeficiency virus (hiv) and the hepatitis c virus (hcv). in this respect, a detailed understanding of the biology of pathogenic viruses together with new and systematic screening tools for novel antiviral compounds will be most helpful. as for the cellular biology studies mentioned above, virologist have turned to the use of yeast as a model system to approach fundamental issues in basic and applied research of higher eukaryotic viruses. these studies have exploited the classical yeast genetics and also the recent yeast technological platforms that allow to apply a system biology approach to fundamental questions in virus biology. the present review will discuss first the expression of individual proteins from important pathogenic viruses in yeast to elucidate their function. the vast literature on the yeast two-hybrid system as a method to explore protein-protein interactions however is not a subject of this review. second, the establishment of yeast/virus systems that allow the replication of higher eukayotic viruses in yeast. these systems have made groundbreaking contributions in the dissection of the life cycle of viruses and the host factors involved. finally, the third and fourth sections will focus on recent advances in applied virus research, the use of yeast as a tool for antiviral drug development and as a vaccine vehicle, respectively. although the expression of viral proteins in yeast not always necessarily reflects their role in higher eukaryotes, here we selected some examples in which the analysis in yeast of their effect on highly conserved cellular processes such as cell cycle control, apoptosis or mrna degradation have contributed to the current understanding of the pathogenesis of important viral pathogens such as hiv-1 and hcv. with around 40 million people infected worldwide and no effective vaccine or curative therapy, hiv remains a major human thread. a better understanding of the hiv replication cycle and the viral and host factors involved are essential to develop new therapy options. yeast-based studies have brought to light key aspects of the function of three hiv proteins, the viral protein r (vpr), the protease (pr) and the regulatory viral protein (rev). the vpr protein plays a pivotal role in the pathogenesis of hiv-1. it is involved in the suppression of immune functions and the depletion of infected cd4 + t cells, which finally leads to the progression to aids [20] . however, the mechanism underlying its effects on pathogenesis was unknown. observations initially made in yeast and then corroborated in mammalian cells demonstrated that vpr acts as an inducer of g2 arrest and cd4 + t cell killing by induction of mitochondrial-dependent apoptosis [21] . the hiv protease is needed to cleave the precursor pr55 gag to the mature proteins matrix (p17), capsid (p24), nucleocapsid (p7) and p6 proteins [22] . studies in s. cerevisiae demonstrated that the pr specifically arrested cell growth and induced cell lysis mediated by the loss of membrane integrity [23] . the contribution of hiv-1 pr to hivinduced necrosis was later confirmed with a cd4 + human t cell line, where a pr-specific inhibitor led to prevention of virus-induced cell lysis [24] . these results highlight the importance of pr-inhibitors to prevent cd4 + t cell loss during infection. finally, the rev protein plays an essential role in the hiv replication cycle. rev mediates the export of unspliced and incompletely spliced hiv mrnas to the cytoplasm and thus, enables the production of hiv-1 particles. the important finding showing that the rev protein mediates the export of hiv pre-mrna by interacting with it via cis-acting rev responsive elements (rre) in the context of the spliceosome was first shown in yeast [25] . these observations led to the identification in s. cerevisiae of the small-nucleoporin-like protein rip1p as the responsible cellular component for rev-mediated export [26] and of the crm1p protein which mediates the interaction between rip1p and rev [27] . hcv has chronically infected more than 170 million people worldwide and is a major cause of liver cirrhosis and hepatocarcinoma. in spite of the great advances accomplished since its identification in 1989, there are still limited therapy options and no vaccine. the expression in s. cerevisiae of two hcv proteins, the non-structural protein 5a (ns5a) and the core protein, together with the construction of hcv chimeras to study hcv rna translation have made yeast a valuable tool in hcv research. in yeast and human cells, the ns5a protein predominantly associates with the er membrane through the highly conserved amphiphatic alpha-helix in its n-terminus [28, 29] . this is consistent with its proposed role as a key regulator for membrane-associated viral replication. moreover, studies in yeast and human cells showed that n-terminally truncated ns5a mutants were transported to the nucleus due to a functional nls and exhibited trans-activating properties [29] [30] [31] [32] . interestingly, the caspase-mediated cleavage of ns5a, occurring during apoptosis or after interaction of ns5a with the core protein during infection, resulted in similar truncations which translocated to the nucleus [33] [34] [35] . however, in which way the ns5a-dependent transactivation contributes to hcv pathogenesis still remains to be elucidated. the hcv core protein interacts with ns5a and is linked to many processes in hcv pathology. from mammalian studies it is known that the hcv core protein influences the transcription of cellular and viral promotors [36] , however the mechanisms involved were mainly unknown. studies in yeast demonstrated the effect of the core protein on the transcription factor ap1-like. the core protein is processed in yeast as in mammalian cells and localizes in the nucleus and the cytoplasm [37] [38] [39] [40] . the transport of the mature core protein to the nucleus was shown to be mediated by direct interaction with the ortholog of human importin 4 (kap123). this interaction in turn abrogates the transport of the ap1-like transcription factor (yap-1) into the nucleus [37, 41] and consequently contributes to the regulation of transcription by the core protein. a bicistronic vector has been recently designed to study hcv rna translation in yeast [42] . the hcv rna is not capped and thus translation depends on an internal ribosomal entry site (ires) located at the 5'non-translated region (ntr). hcv ires-mediated translation is modified by the 3'-ntr and several viral and probably also host proteins bind to the 5'-ntr of hcv [43] . since hcv ires is functional in yeast [44, 45] the bicistronic construct generated will allow to fully explore the effect of viral and cellular factors on hcv translation. an understanding of the fundamental steps of virus life cycles including virus-host interactions is essential for the design of novel effective antiviral strategies. such understanding has been hampered by the complexity of higher eukaryotic host organisms. to overcome these experimental difficulties, systems have been developed that allow the replication of higher eukaryotic viruses in s. cerevisiae. the first higher eukaryotic virus shown to replicate and encapsidate its genome in s. cerevisiae was the bromo mosaic virus (bmv), a positive strand rna ((+)rna) virus from plants [46, 47] . this pioneer work opened new avenues in virus research and allowed to apply the versatile yeast genetic tools in the elucidation of fundamental steps in virus replication. the list of higher eukaryotic viruses shown to replicate in yeast has been expanded ever since and include (i) other viruses with rna genomes that infect plants (carnation italian ringspot virus (cirv), tomato bushy stunt virus (tbsv) and cymbidium ringspot virus (cymrsv)) or animals (flock house virus (fhv) and nodamura virus (nov)), and (ii) viruses with dna genomes that infect humans (human papillomavirus), animals (bovine papillomavirus) or plants (mungbean yellow mosaic india virus (mymiv)) [48] [49] [50] [51] [52] [53] . importantly, these yeast systems reproduce the known features of virus replication in their corresponding natural hosts. since a detailed description of each of these systems and contributions has been recently reviewed [54, 55] , we will only highlight some main achievements in virus research. in particular, we will focus on studies carried out with yeast/ rna virus systems since they are the most advanced. all rna viruses that have been shown to replicate in yeast belong to the group of (+)rna viruses. this large virus group includes many important plant, animal and human pathogens such as hcv and the severe acute respiratory syndrome coronavirus (sars). all (+)rna viruses share common features in their replication cycles. first, the genomic rna serves as messenger rna (mrna) for translation of the viral proteins and as template for replication. since these two functions are mutually exclusive, the transfer of the genomic rna from the cellular translation machinery to the replication complex must be highly regulated. second, viral replication complexes are associated with intracellular host membranes which proliferate and rearrange in response to the expression of viral protein. and third, host factors are required at multiple steps of the viral replication cycle [56] [57] [58] . all the yeast/(+)rna virus systems developed show common characteristics. (i) the viral rna-dependent rna polymerase (rdrp) and additional proteins required for viral replication are expressed in trans from yeast plasmids, (ii) the genomic rna with authentic 5' and 3' ends is also transcribed from a yeast plasmid or transfected directly into the cell, and (iii) replication is monitored by detecting viral rna via northern blot analysis or by measuring the expression of a reporter gene inserted into the viral genome, such that its expression depends on viral rna replication. the fact that in these systems the individual viral proteins are given in trans from separate plasmids is of great advantage since it allows simplifying and controlling functional studies of each player on viral replication. with this, important aspects of the viral life cycles were studied like translation of viral proteins, the process of template selection, formation of the replication com-plex, viral rna replication, encapsidation and recombination (reviewed in detailed in [54, 55, 59] . these studies have resulted in an impressive progress in the understanding of these fundamental steps of viral rna replication and the viral proteins involved. furthermore, due to the common replication strategy of (+)rna viruses, the results obtained go much beyond the specific virus studied. the identification of the host factors involved in viral rna replication is a priority area of research in virology because it can provide new targets for antiviral drug development. the use of traditional yeast genetics and genomewide screenings have resulted in the groundbreaking identification of multiple host factors required for viral rna replication and recombination. the gene-deletion mutant collection in which all the non-essential yeast genes are deleted was used to carry out genome-wide screenings of host factors affecting the replication of bmv and tbsv [60, 61] . in the bmv studies each of the single deletion strains was transformed with plasmids expressing the bmv replicases (the polymerase 2a and the helicase 1a) and a bmv rna replication template harbouring a luciferase reporter gene which expression was screened as a marker for bmv rna replication. in the tbsv studies, a similar approach was applied; however, effects on replication were followed by direct visualization in ethidiumbromide-stained gels of a shorter form of the tbsv rna, which accumulates at a very high level in the wild-type strain. these analyses revealed that from the approximately 5000 non-essential yeast genes analyzed, around 100 were found to affect the replication of each virus. these results uncovered the involvement in viral replication of previously unconsidered cellular pathways, such as mrna turnover, stress response and the ubiquitin pathway of protein degradation. surprisingly and against all previous predictions, only 4 genes were found to be common in both studies. this was discussed to be possibly related to the fact that bmv and tbsv replicate in association with cellular membranes from different compartments. in spite of the impressive achievement in identifying essential host facors for replication, the above mentioned approach has the limitation that only the non-essential genes can be screened. to overcome this, an additional genome-wide screening was performed in the yeast tet promoter hughes collection (ythc) with tbsv [62] . in the ythc, which covers 800 of the ~1000 essential genes, each essential gene is under the control of a tet-titratable promoter in the genome [9] . in this way, the expression of the essential gene can be turned off by the addition of doxycycline to the yeast growth medium. with this approach 30 additional cellular genes were identified that are involved in the replication of tbsv [62] . tbsv not only replicates in yeast but also undergoes recombination [49] . to elucidate the eventual role of host factors in the viral rna recombination process, genomewide screenings were performed in both yeast collections mentioned above [63, 64] . since the recombinant and non-recombinant tbsv forms vary in length, a screening was performed by direct visualization of tbsv rna in agarose gels in combination with northern blot analysis and rt-pcr sequencing. the genetic screens identified 11 nonessential and 16 essential genes that affected tbsv recombination. the exciting observation that host factors can influence viral rna recombination and thereby evolution has brought a totally new perspective to the field. major viral pathogens still remain without effective vaccine or therapy options, thus safer and more effective antiviral drugs are urgently needed. for this, improved screening processes that enable to assess the mode-ofaction of the tested compounds are essential. in this section we will discuss the exciting contributions of yeast genetics and high-throughput screenings to drug development. we will present first the principal characteristics of these global approaches and then their application to the antiviral research field. the process that leads to the discovery and development of new drugs is long and costly. two approaches have been traditionally used. if the target is already known, the drug discovery search classically starts with in vitro assays to identify small molecule inhibitors. these assays give only a partial view of the effect of the compounds since, for instance, they cannot fully explore drug effects on other additional targets, which can be the source of undesirable side effects. putative toxic effects will be tested subsequently in cell culture and in animal models. however, if toxicity is detected, the underlying mechanism will still be unknown and no rational approach is possible to modify the drug to improve safety. an alternative drug discovery approach directly performs cell-based screens for molecules that produce a specific desired effect. this has the advantage that the molecule encounters natural physiological conditions and allows selecting those molecules that are stable within the metabolic environment and discarding those that show toxic effects. however, also with this approach the exact mechanism of action of the selected drug remains unknown. this is a great disadvantage, since its elucidation could lead to the design of new compounds with improved safety and efficacy profiles. both above approaches will benefit from the large-scale chemical and genetic tools developed in s. cerevisiae that allow to systematically screen putative targets and effects of the selected drugs [17, 19] . since the effect of a drug on every yeast gene is analyzed, a broad view of all the proteins and related metabolic pathways affected can be accomplished. thus, including yeast-based screenings at an early stage in drug development programmes, crucial information will be rapidly obtained that can be used to discard or to improve the tested compounds. the large-scale chemical and genetic yeast screens use the mutant strain collections and gene expression plasmids described above. there are five main screening approaches [19] . first, the drug-induced haploinsufficiency approach uses the heterozygous mutant collection and is based on the finding that reducing the copy number of a drug target gene from two to one copy can significantly sensitize a diploid cell to that drug [65] [66] [67] . the second approach uses the haploid or the homozygous diploid deletion collection and is based on the principle that the deletion of a gene that renders cell-hypersensitivity to a specific drug identifies pathways that buffer the cell against the toxic effect of the drug and thus provides clues about its mode of action [68, 69] . the third approach uses gene expression libraries and is based on the principle that increasing the concentration of a protein that is the target of an inhibitory drug should increase resistance to the drug. these three approaches can be performed in two different ways, (i) by growth measurement of arrayed strains, and (ii) by competitively growing pooled strains. the fourth approach is based on gene expression profiling and consists of comparing changes in mrna expression elicited by a drug of interest with those induced by other drugs or by gene deletions. finally, the plasmid collection of all yeast proteins expressed as gst-fusions was used to create protein chips that allow the screening of direct drug-protein interactions. the combination of all the five powerful techniques together with the rapidly growing knowledge of yeast genetic networks [70] has made yeast a recognized system in drug development that until today has not been fully exploited in antiviral research. after more than two decades of searching for antiviral drugs, a rather limited number of compounds is on the market. from these, almost all focus on hiv and herpesviruses. furthermore, most antivirals are not curative and produce major side effects. thus, there is an urgent need of novel and high-quality targets and drugs together with improved screening systems to identify them. two kinds of targets can be used in antiviral research, viral factors or host factors. classically, viral proteins have been used as targets for therapeutic interventions because the developed inhibitor molecules were expected not to produce significant side effects. however, this does not seem to be the case and, for instance, all the antiretrovirals currently available produce important side effects. since drug-resistant mutants are rather easily selected, researchers have shown again interest in host factors. they have the advan-tage of being genetically stable and may even be efficient as targets against groups of viruses. the above mentioned screening options could be helpful to select those host factors that when targeted by drugs will be lethal for the virus but have minor effects on the cell. two yeast cell-based assays have been established to screen for viral inhibitors. the protein m2 of influenza virus has a fundamental role in the disassembly of the influenza virion. in fact, some of the commercially available drugs target this protein. since expression of the m2 protein in yeast produced a slower growth rate, a screening procedure was set up based on the rescue of wt cell growth in the presence of test compounds [71] . more recently, a parallel approach has been applied to the serine protease encoded by the human cytomegalovirus (hcmv) [72] . this protease is essential for the virus and recognizes a 40 amino acid sequence, which was inserted into the coding sequence of a yeast enzyme involved in the biosynthesis of tryptophan. coexpression of the hcvm protease with the engineered enzyme resulted in an arrest of cell growth in media without tryptophan that could be rescued in the presence of protease inhibitors. this system provides the basis for the high-throughput screening of inhibitory molecules. related to the analysis of host factors as antiviral targets, the genome-wide screenings in the yeast/virus systems described in the previous section have provided many new candidates for which the therapeutic potential remains to be explored. few studies in antiviral research make fully use of the yeast platforms and tools available. because of their innovation in the field, we would like to point out two of them that explore the mode of action of antivirals plus other therapeutic compounds. in the first study the heterozygote yeast deletion collection was used to asses the cellular effects of 78 different compounds [67] . it is important to mention that in the construction of this collection, each yeast gene was replaced with a cassette containing the selectable marker gene kanmx plus two unique tag sequences located up-and down-stream of the marker gene. these tag sequences enabled rapid analysis of strains in a pool by using hybridization to high-density nucleotide arrays. importantly, the tag sequences were flanked with universal primer sets for polymerase chain reaction amplification [8] . thus, a pool of ~3500 heterozygous deletion strains were competitively grown for 20 generation in media containing the selected compounds. then, dna was extracted from the cell culture at generation 0 and 20 and amplified by pcr using universal primers and cy3 (green) or cy5 (red) labels, respectively. the amplified dna was hybridized to microarrays containing each specific tag sequence. since signal intensities from micro-array hybridizations are proportional to the relative tag abundance in the pool population, for each yeast strains red, green or yellow colours in the array will correlate with an enrichment, depletion or unchanged representation of the strain in the population. in this way the effect of a drug on each of the yeast deletion strains could be rapidly analyzed [67] . in a second study a complementary approach was followed. the ~5000 yeast haploid deletion mutant collection was tested for hypersensitivity to 82 different compounds including some molecules with antiviral activity [69] . since it is considered that compounds with similar biological effects lead to similar chemical profiles, clustering of the obtained results provided a very useful data set that allowed the organization of the compounds into functionally relevant groups. the results obtained with both studies helped to identify new modes of action of the tested compounds and revealed numerous insights into the cellular pathways involved, yeast has been utilized in vaccine development. the classical example is the recombinantly expressed hepatitis b surface antigen that has become a safe and efficient prophylactic vaccine worldwide [73] . however, in this section we will focus on its use not as a mere production tool for recombinant antigens but as a vaccine vehicle itself. immunization against bacterial pathogens and viruses is an attractive strategy in combating infectious diseases by stimulation of the immune system. traditional vaccines like soluble antigens formulated with adjuvants efficiently activate the humoral immune response. the production of neutralizing antibodies that bind to invading microorganisms inhibits their entry into target cells, thereby preventing an infection. however, cell-mediated immunity, not humoral immunity alone, is required to control infections with intracellular pathogens, especially viruses like hiv and hcv. in the classical view of cellular immunity, antigen-presenting cells (apc) present peptides from endogenously synthesized pathogen proteins in the context with mhc class i molecules on their cell surface. antigen-specific cd8-positive t lymphocytes become activated by recognizing these peptides and subsequently differentiate into a mature effector cell population. these cytotoxic t cells (ctl) are able to recognize and kill the respective infected cells. additionally, dendritic cells (dc) which are the most potent apc use the mechanism of cross-priming for antigen presentation, wherein peptides from exogenous antigens are also presented via mhc class i molecules [74] . "danger" signals as given by the interaction of pathogen components (e.g. cell wall components of yeast) with pattern recognition receptors on the surface of apc, such as toll-like receptors (tlr), mannose or glu-can receptors, leading to the maturation of the apc and an efficient antigen presentation [75, 76] . whole recombinant yeast cells, known as tarmogens™ (targeted molecular immunogens) represent a novel vaccination tool for the induction of potent cell-mediated immune responses against target antigens [77] . in most cases, the non-pathogenic baker's yeast saccharomyces cerevisiae has been used as antigen carrier because this genus combines several advantages. (1) products with s. cerevisiae, e.g. bread and beer, are consumed in large amounts worldwide and healthy individuals show only moderate t cell responses against s. cerevisiae, probably due to mechanisms of oral tolerance [78] . (2) important for primeboost immunization strategies is the observation that there is no neutralization of yeast vectors by the host immune system following multiple immunizations in mice and rabbits [79, 80] . remarkable low titres of antiyeast antibodies were found after repeated injections of either live or heat-killed antigen-expressing s. cerevisiae [77] . (3) yeast cells themselves have potent adjuvant effect via "danger" signals that lead to the maturation of dc without being dangerous, so no exogenous adjuvant is needed [80, 81] . (4) facilitated by the particulate structure of yeast, antigens are efficiently presented via the mhc class i pathway resulting in the activation of antigen-specific cd8 t lymphocytes [81] [82] [83] . stubbs et al. showed for the first time that recombinant yeasts expressing hiv-1 antigens and tumour antigens potently elicit antigen-specific ctl responses, including those mediating tumour protections in mice [80] . further studies with the animal tumour challenge model could demonstrate that yeast-based immunotherapy using mutated ras oncoprotein as target antigen caused a mutant-selective tumour remission in animals [77, 79] . this concept is now being tested in a phase i clinical trial in patients with colorectal, pancreatic and non-small cell lung cancers [77] . most recently, the same group has evaluated a recombinant yeast cell vaccine that expresses the hcv ns3-core fusion protein as an immunotherapeutic vaccine in chronic hcv-infected individuals [84] . one advantage of using whole recombinant yeasts as antigen carriers is the oral application route. feeding mice with s. cerevisiae expressing actinobacillus pleuropneumoniae antigen led to the induction of protective systemic and mucosal immune responses [85] . the fission yeast schizosaccharomyces pombe represents, like s. cerevisiae, a physiologically and genetically well characterized eukaryote that has been used in africa for hundreds of years in brewing of millet beer, from which it was first isolated. recombinant sz. pombe cells expressing the human cytomegalovirus pp65 protein were able to stimulate cd4-and cd8-positive memory t cells in human blood [82] . instead of using recombinant yeast expressing an antigen of choice as a vaccine, attempts have been made to express mhc proteins plus the desired antigenic peptide on the yeast surface and to use the modified cell as a kind of apc. brophy and colleagues succeeded in expressing mouse mhc i protein heavy chain, beta2-microglobulin plus an antigenic peptide from a single gene cassette. the resulting complex assembled in a functional conformation on the cell surface and even induced the activation of naïve t cells [86] . however whether this approach will be of practical use in vaccination has yet to be demonstrated. the strategy to use yeast cells for vaccination could also be used to elicit protective immune responses against human pathogenic yeasts. for example, the genus candida is able to cause a variety of infections from mucosal candidiasis to life-threatening disseminated and bloodstream infections, especially in immunocompromised individuals. unfortunately, the interplay between protective and inhibitory antibodies dictates the outcome of experimentally disseminated candidiasis in mice receiving a heatinactivated whole-cell c. albicans vaccine [87] . this observation may explain why subjects with elevated anti-candida antibody titers remain nonetheless susceptible to invasive candidiasis [87] . however, ibrahim and colleagues could show that vaccination with the recombinant n-terminal domain of the adhesin als1p improves survival during murine disseminated candidiasis by enhancing cell-mediated, not humoral, immunity in both, immunocompetent and immunocompromised mice [88, 89] . in conclusion, the yeast system has been extremely valuable in virus research. however, the full potential from the remarkable progress in systemic yeast biology over the last years has yet to be exploited. with the increasing options for analysing protein interaction networks at an unprecedented level of complexity and the rising number of viral replication systems in yeast that correctly mimic the fundamental properties of the virus life cycle in higher eukaryots, the era of system virology is expected to become a new focus in virus research. subsequently, the better understanding of the virus protein/host cell protein interaction networks will allow the search for and development of more efficient and safer antiviral 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responses targeting hcv ns3 and core proteins induction of antigen-specific immune responses by oral vaccination with saccharomyces cerevisiae expressing actinobacillus pleuropneumoniae apxiia a yeast display system for engineering functional peptide-mhc complexes interplay between protective and inhibitory antibodies dictates the outcome of experimentally disseminated candidiasis in recipients of a candida albicans vaccine vaccination with recombinant n-terminal domain of als1p improves survival during murine disseminated candidiasis by enhancing cell-mediated, not humoral, immunity the anti-candida albicans vaccine composed of the recombinant n terminus of als1p reduces fungal burden and improves survival in both immunocompetent and immunocompromised mice this work was supported by the spanish ministerio de educación y ciencia (grants bfu2004-00654 and ha2006-0110), and the daad. i. a-r was supported by fundação para a ciência e tecnología (sfrh/bd/9630/2002), portugal. the author(s) declare that they have no competing interests. all authors have contributed to the content and structure of the present review. key: cord-274056-9t3kneoo authors: abd elwahaab, marwa a.; abo-elkhier, mervat m.; abo el maaty, moheb i. title: a statistical similarity/dissimilarity analysis of protein sequences based on a novel group representative vector date: 2019-05-08 journal: biomed res int doi: 10.1155/2019/8702968 sha: doc_id: 274056 cord_uid: 9t3kneoo similarity/dissimilarity analysis is a key way of understanding the biology of an organism by knowing the origin of the new genes/sequences. sequence data are grouped in terms of biological relationships. the number of sequences related to any group is susceptible to be increased every day. all the present alignment-free methods approve the utility of their approaches by producing a similarity/dissimilarity matrix. although this matrix is clear, it measures the degree of similarity among sequences individually. in our work, a representative of each of three groups of protein sequences is introduced. a similarity/dissimilarity vector is evaluated instead of the ordinary similarity/dissimilarity matrix based on the group representative. the approach is applied on three selected groups of protein sequences: beta globin, nadh dehydrogenase subunit 5 (nd5), and spike protein sequences. a cross-grouping comparison is produced to ensure the singularity of each group. a qualitative comparison between our approach, previous articles, and the phylogenetic tree of these protein sequences proved the utility of our approach. sequence comparison is used to study structural and functional conservation and evolutionary relations among the sequences. the importance of similarity/dissimilarity of biological sequences returns to its relationship with the structures and functions. proteins with similar sequences usually have similar structures. the rate of addition of new sequences to the databases is increasing exponentially [1] . comparing these new sequences to those with known functions is a key way of understanding the biology of an organism. thus, sequence analysis can be used to assign function to genes and proteins by the study of the similarities between the compared sequences. there are many tools and techniques that provide the sequence comparisons. sequence comparison can be classified into alignmentbased methods and alignment-free methods [2, 3] . alignment-based methods assign scores to different possible alignments, picking the alignment with the highest score. some algorithms do global alignment or local alignment [4] [5] [6] . blast [7] and fasta [8] are the most widely used applications. alignment-based methods are computationally difficult with multiple sequence alignments at the same time. a wide range of scoring systems has been proposed such as amino acid substitution scoring matrices pam and blosum for protein alignment [9] . alignment-free approaches overcome the limitations of alignment-based methods. graphical representation approaches are one of them. graphical representations are usually accompanied by numerical characterization and then a descriptor to describe each protein sequence. a similarity/dissimilarity analysis is then done using these descriptors by evaluating euclidean distance or correlation angle among them. the smallest euclidean distance or correlation angle is the more similar. many graphical representations of dna and protein primary sequences have been proposed. some other approaches characterize numerically protein sequences without previous graphical representation and nongraphical representation methods [10, 11] . in this article, an alignment-free method is introduced. it is considered a nongraphical representation method. three groups of protein sequences are selected to illustrate our approach. they are beta globin, nadh dehydrogenase subunit 5 (nd5), and spike protein sequences. they are selected as each group has sequences of similar range of lengths. the 1 human aaa16334 147 2 chimpanzee caa26204 125 3 gorilla caa43421 121 4 mouse caa24101 147 5 rat caa29887 147 6 gallus caa23700 147 7 opossum aaa30976 147 opossum np 007105 602 most common sequences of each group are selected. the selected sample is assumed to be unbiased and the population distribution of each group is normal. therefore, the selected sample represents the group. statistics can be used to estimate the population's parameters. the adjacency vector is introduced as a novel descriptor for protein sequences. it is computed for each sequence in the selected sample of three groups. a reference vector is then computed for each group. this vector acts as a representative of the group. each sequence's degree of similarity in each group is measured according to its group's representative vector. so, a similarity/dissimilarity vector is constructed instead of ordinary similarity/dissimilarity matrix. our approach is independent of the protein sequence length. it does not require any previous graphical representation. it is a mathematically simple approach. the protein sequences used in this article are listed in tables 1, 2 , and 3. the sequences are downloaded from the national center for biotechnology information (ncbi) "https://www.ncbi.nlm.nih.gov/" as fasta files. these fasta files are imported into wolfram mathematica 8 where all the results and figures are produced. the phylogenetic tree of these protein sequences is also created by the basic local alignment search tool (blast) "https://blast.ncbi.nlm.nih .gov/blast.cgi". table 1 shows the 1 st sample set that consists of seven species of beta globin protein sequences. their range of lengths is from 121 to 147. this sample set is applied before in [12] . table 2 shows the 2 nd sample set which consists of nine nd5 protein sequences. their range of lengths is from 602 to 610. this sample set is applied before in [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . table 3 shows the 3 rd sample set which consists of 29 spike protein sequences. their range of lengths is from 1162 to 1447. these viruses are coronavirus. they are classified into four classes: class i that includes the porcine epidemic diarrhea virus (pedv) and the transmissible gastroenteritis virus (tgev). class ii includes the bovine coronavirus (bcov), human coronavirus oc43 (hcov-oc43), and the murine hepatitis virus (mhv). class iii contains the infectious bronchitis virus (ibv). the others are severe acute respiratory syndrome coronaviruses (sars-cov). this sample set is applied before in [26] . in this approach, a new vector is suggested to be a descriptor of a protein sequence. this vector is called the adjacency vector ( ); x refers to the species' protein sequence and y refers to its related group. it counts the occurrence of all possible pairwise adjacencies obtained by reading the protein primary sequence from left to right. the protein sequence table 4 aa ar an ad ac aq ae ag ah ai al ak am af ap as at aw ay av 1 0 1 0 0 0 0 1 4 0 3 0 0 1 0 0 1 0 1 2 table 5 va table 6 aa ar table 7 va is composed of 20 common different amino acids which are "a," "r," "n," "d," "c," "q," "e," "g," "h," "i," "l," "k," "m," "f," "p," "s," "t," "w," "y," and "v" as ordered alphabetically according to 1 st letter code. therefore, the adjacency vector (a xy ) consists of 400 elements. every 20 elements are related to each amino acid. the first 20 elements are related to "a" amino acid. the second 20 elements are related to "r" amino acid. the third 20 elements are related to "n" amino acid and so on by the same order which is illustrated previously according to 1 st letter code. we borrow our idea from the 20 ×20 adjacency matrix [27] . the adjacency vector counts the possibilities of each pair. in other words, it counts the number of times that each pair is repeated along the sequence length. if the pair does not exist, its value in the adjacency vector is zero. for example, to evaluate the adjacency vector of the two short segments of "yeast saccharomyces cerevisiae" protein [16, 19, [22] [23] [24] 28] protein i: "wtfesrndpakdpvilwlnggpgcssltgl" protein ii: "wffesrndpandpiilwlnggpgcssftgl" the two protein sequences are composed of 30 amino acids. protein i is converted to 29 adjacent pairs that are wt, tf, fe, es, sr, rn, nd, dp, pa, ak, kd, dp, pv, vi, il, lw, wl, ln, ng, gg, gp, pg, gc, cs, ss, sl, lt, tg, gl as reading sequence from left to right. protein ii is converted to 29 adjacent pairs that are wf, ff, fe, es, sr, rn, nd, dp, pa, an, nd, dp, pi, ii, il, lw, wl, ln, ng, gg, gp, pg, gc, cs, ss, sf, ft, tg, gl as reading sequence from left to right. for example, "nd" pair has a count one in protein i and two in protein ii. "dp" pair has a count two in both protein i and protein ii. "sl" and "lt" pairs have a count one in protein i and zero in protein ii. our approach is applied on three selected groups of protein sequences. the groups are beta globin, nd5, and spike protein sequences as illustrated in tables 1, 2 , and 3, respectively. the most common protein sequences are selected in each group. the selected sample is assumed to be unbiased and the population distribution of each group is normal. therefore, the selected three samples can represent the three groups. the samples consist of seven beta globin, nine nd5, and 29 spike protein sequences. seven adjacency vectors for beta globin proteins, nine adjacency vectors for nd5 protein sequences, and 29 adjacency vectors for spike proteins are evaluated. for example: (1) human (beta globin) protein sequence's first 20 elements of its adjacency vector (a human beta globin ) are as shown in table 4 . (2) gorilla (nd5) protein sequence's last 20 elements of its adjacency vector (a gorilla nd5 ) are as shown in table 5 . the adjacency vector is used to describe each protein sequence individually in its corresponding group. this article provides a descriptor to the group itself. the median vector is selected to play the role of the group representative (gr y ); y refers to its group. it acts as a reference vector for each group. the median is a better measure of central tendency. it separates the higher half from the lower half of the sample's data. it is not sensitive to extreme values like average. the suggested group representative vector (gr y ) is a vector which is composed of also 400 elements. each element of 400 is the median of the corresponding elements in all adjacency vectors related to its sample that represents the group. beta globin, nd5, and spike protein sequences' representative vectors are computed. for example: (1) beta globin representative vector's (gr beta globin ) 1 st 20 elements are as shown in table 6 . (2) nd5 representative vector's (gr nd5 ) last 20 elements are as shown in table 7 . (3) spike proteins representative vector's (gr spike proteins ) 1 st 20 elements are as shown in table 8 . biomed research international table 8 aa ar an ad ac aq ae ag ah ai al ak am af ap as at aw ay av 9 1 3 5 2 4 3 6 0 7 6 2 1 3 6 4 7 1 5 3 a similarity/dissimilarity vector is introduced instead of the regular similarity/dissimilarity matrix [10, 11] . the similarity/dissimilarity matrix is a square symmetric matrix with zeros in its main diagonal. in order to evaluate this matrix, it is required to measure the degree of similarity between each protein sequence and others in the same group. if the 1 st row represents human and the 2 nd row represents gorilla, the similarity of all species according to human in 1 st row is measured. then the similarity is measured again of all species in 2 nd row according to gorilla and so on. the calculations' number of this matrix equals ∑ 1 = (k − 1)/2 where n is the number of compared species. the similarity/dissimilarity vector is suggested to save time and number of calculations. it is a vector that has a number of elements equal to the number of protein sequences in the selected sample of each group. it measures the degree of similarity between each protein sequence's adjacency vector and the group representative vector. in other words, it measures the degree of similarity between each protein's descriptor and the "group representative." it is simpler than previous matrix. it is calculated only one time for each sequence. the calculations' number of this vector equals n where n is the number of compared species. to measure the degree of similarity, we suggest two methods: (ii) e nd method. compute the angle between each sequence's adjacency vector (a xy ) and the group representative vector (gr y ) in radians by for beta globin protein sequences, seven species are selected in our sample set: human, chimpanzee, gorilla, mouse, rat, gallus, and opossum, as illustrated in table 1 . there are seven adjacency vectors corresponding to them. the group representative gr beta globin is evaluated based on these seven adjacency vectors. therefore, the similarity/dissimilarity vector has seven elements. the 1 st element corresponds to human, 2 nd element corresponds to chimpanzee, and so on, by the same order as in table 1 . in the tables 2 and 3 , respectively. the similarity/dissimilarity vectors that are corresponding to beta globin, nd5, and spike protein sequences are illustrated in tables 9, 10, and 11, respectively, based on the two methods discussed before. the results in table 9 show that the magnitude ( , where x: species) cannot measure the similarity/dissimilarity degree well among all beta globin sequences. the human, chimpanzee, and gorilla have the same value that is equal to 0.5568, while the similarity is well measured between mouse and rat. also, the dissimilarity between opossum and human is very clear. the angle ( ) is successfully measured similarity/dissimilarity among all the species as shown in figure 1 . the closest values of both and mean more similarity. the results in table 10 show that both the magnitude ( 5 ) and the angle ( 5 ) can measure similarity/dissimilarity degree well among nd5 protein sequences as shown in figure 2 . it is obvious that pigmy chimpanzee, common chimpanzee, human, and gorilla are very similar. also it shows the similarity of the blue whale, fin whale, and the mouse and rat as pairs and the dissimilarity between human and opossum. these results are satisfied with [13, 14, 16, 18, 19, [21] [22] [23] [24] [25] . the results in table 11 show that both and classified the 3 classes of viruses and sars covs well each as a single coherent class except only the "mhvjhm" virus. this virus belongs to class ii but our approach cannot classify it well. the classification of 29 spike proteins into classes by our approach is illustrated in figure 3 . the mhvjhm virus is the only wrong classified sequence. it is colored red. despite the wrong classification of mhvjhm virus, our approach corrects the broken classification of class i in [26] . according to the results in tables 9, 10 , and 11, the angle is preferred to be used as shown in figures 1, 2 , and 3. the group representative vector ( ) carries the information of its group. a cross-group comparison is done to prove the singularity of each group. tables 9, 10, and 11 are evaluated based on the group's sample set of protein sequences related to their corresponding group representative vector. tables 12, 13, 14, and 15 are evaluated based on each group sample set of protein sequences with another group representative vector. the similarity/dissimilarity analysis among the seven beta globin sequences measured according to ( 5 ) is illustrated in table 12 and shown in figure 4 . the similarity/dissimilarity analysis among the nd5 sequences measured according to ( ) is illustrated in table 13 and shown in figure 5 . the similarity/dissimilarity analysis among the beta globin sequences measured according to (gr spike ) is illustrated in table 14 and shown in figure 6 . the similarity/dissimilarity analysis among the nd5 sequences table 15 and shown in figure 7 . the results show a big distortion that ensures the individuality of each group. the phylogenetic tree is a branching diagram showing the evolutionary relationships among various biological species based upon similarities and differences in their sequences. a qualitative comparison between our results and the phylogenetic tree of protein sequences is used to prove the utility of our approach. the matching between the results and phylogenetic trees means matching with the naïve measure of sequence similarity (sequence homology). the basic local alignment tool (blast) is used to draw the phylogenetic trees. the phylogenetic trees of beta globin's seven species, nd5 nine species, and 29 spike protein sequences are illustrated in figures 8, 9 , and 10, respectively. the qualitative comparison of the results of tables 9, 10, and 11 and figures 8, 9 , and 10 shows the utility of our work especially the angle results. the proposed method is an alignment-independent method. an adjacency vector is suggested as a descriptor of any protein sequence. it does not require any graphical representation. a group representative vector is introduced to represent each group of protein sequences. a similarity/dissimilarity vector is produced instead of the regular similarity/dissimilarity matrix. the similarity/dissimilarity analysis is done by two methods. our approach is applied on three sample sets of three groups of protein sequences. each sample has a different range of lengths than the others. our approach does not depend on protein sequence length. it successfully measured similarity/dissimilarity among different lengths. it is very mathematically simple. a cross-grouping comparison is introduced to prove the singularity of each group. the results approved the utility of our approach compared with previous articles and phylogenetic tree obtained by blast program. we hope to make the method available to include ambiguous amino acid residues and nonstandard amino acids. we hope also to include the analyses of partial or gapped sequences. all data is mentioned clearly in the manuscript in section 2 under the title "dataset." in this section, we illustrate the data in three tables: tables 1, 2, and 3. we also mention in the 1st paragraph of dataset that data are downloaded from "gene bank." all data files are with extension ". fasta". the authors declare that they have no conflicts of interest. dna sequence comparison by a novel probabilistic method linear regression model of short kword: a similarity distance suitable for biological sequences with various lengths sequence comparison via polar coordinates representation and curve tree a general method applicable to the search for similarities in the amino acid sequence of two proteins identification of common molecular subsequences an improved algorithm for matching biological sequences basic local alignment search tool rapid and sensitive protein similarity searches amino acid substitution matrices from protein blocks graphical representation of proteins similarity/dissimilarity calculation methods of dna sequences: a survey 3-d maps and coupling numbers for protein sequences a novel descriptor for protein similarity analysis similarity analysis of protein sequences based on 2d and 3d amino acid adjacency matrices a new method to analyze protein sequence similarity using dynamic time warping a 2d graphical representation of protein sequence and its numerical characterization graphical representation and similarity analysis of protein sequences based on fractal interpolation adld: a novel graphical representation of protein sequences and its application comparative analysis of protein primary sequences with graph energy uc-curve: a highly compact 2d graphical representation of protein sequences the graphical representation of protein sequences based on the physicochemical properties and its applications f-curve, a graphical representation of protein sequences for similarity analysis based on physicochemical properties of amino acids a novel method of 2d graphical representation for proteins and its application 3d graphical representation of protein sequences and their statistical characterization novel numerical characterization of protein sequences based on individual amino acid and its application similarities/dissimilarities analysis of protein sequences based on pca-fft on novel representation of proteins based on amino acid adjacency matrix a sequence-segmented method applied to the similarity analysis of long protein sequence it is a figure which summarizes our approach. it is submitted under the name of graphical abstract. (supplementary materials) key: cord-281124-4nhy35xn authors: soowannayan, chumporn; cowley, jeff a.; michalski, wojtek p.; walker, peter j. title: rna-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp date: 2011-08-03 journal: plos one doi: 10.1371/journal.pone.0022156 sha: doc_id: 281124 cord_uid: 4nhy35xn gill-associated virus (gav) infects penaeus monodon shrimp and is the type species okavirus in the roniviridae, the only invertebrate nidoviruses known currently. electrophoretic mobility shift assays (emsas) using his(6)-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the gav nucleocapsid (n) protein in vitro. the emsas showed full-length n protein to bind to all synthetic single-stranded (ss)rnas tested independent of their sequence. the ssrnas included (+) and (−) sense regions of the gav genome as well as a (+) sense region of the m rna segment of mourilyan virus, a crustacean bunya-like virus. gav n protein also bound to double-stranded (ds)rnas prepared to gav orf1b gene regions and to bacteriophage m13 genomic ssdna. emsas using the five n protein constructs with variable-length n-terminal and/or c-terminal truncations localized the rna binding domain to a 50 amino acid (aa) n-terminal sequence spanning met(11) to arg(60). similarly to other rna binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. to examine this domain in more detail, the 18 aa peptide (m(11)pvrrplppqpprnarli(29)) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length n protein in emsas. the data indicate a fundamental role for the gav n protein proline/arginine-rich domain in nucleating genomic ssrna to form nucleocapsids. moreover, as the synthetic peptide formed higher-order complexes in the presence of rna, the domain might also play some role in protein/protein interactions stabilizing the helical structure of gav nucleocapsids. gill-associated virus (gav) infects penaeus monodon shrimp and is the type species of the genus okavirus in the roniviridae, the only currently known invertebrate nidoviruses. in all other nidoviruses, the nucleocapsid (n) protein is encoded by a gene located near to the genome 39-terminus and downstream of genes encoding other virion structural proteins [1] . however, in gav and other genotypic variants in the yellow head virus (yhv) complex, the n protein is encoded in the orf2 gene which resides immediately downstream of the 20 kb 59-terminal orf1a/1b replicase gene [2, 3] . the deduced molecular masses of the 144-146 amino acid (aa) n proteins of gav and yhv (16.0-16.3 kda) are lower than those estimated by sds-page (20) (21) (22) , which for yhv has been reported to be due to a c-terminal cluster of acidic residues [3, 4] . immuno-electron microscopy has confirmed that the n protein is the primary structural protein component of okavirus nucleocapsids [2, 5] . amongst strains of genotypes 1 (yhv), 2 (gav), 3, 4 and 5 in the yhv complex [6] , most amino acid variations (up to 17.2%) in the deduced n protein sequence occur in the highly charged nand c-terminal domains [3, 6] . nonetheless, the n proteins of gav and yhv share common antigenic sites as evidenced by their cross-reactivity for a yhv n protein monoclonal antibody [5, 7] and polyclonal antiserum to a synthetic peptide designed to a c-terminal sequence of the gav n protein [2] . as with the n proteins of coronaviruses and toroviruses [8, 9] , the n proteins of gav and yhv lack cysteine residues and are highly basic [2, 3] . it also possesses proline-rich and basic residue-rich domains likely to facilitate rna binding as hypothesized for similar sequences in the n protein of toroviruses [2, 10] . the n protein length in okaviruses is intermediate to the corresponding proteins of arteriviruses (110-128 aa) and toroviruses (160-167 aa), and much shorter than the n protein of coronaviruses (377-454 aa) [2, 11, 12, 13, 14] . the process by which n proteins encapsidate genomic rna to form nucleocapsids has been examined in many rna viruses [8, 15, 16, 17] and in coronaviruses, as an example, the n protein interaction with rna shows no preference for sequence, indicating that the specific nucleation of viral rna likely requires additional factors [8, 18] . here we have examined recombinant gav n protein constructs in electrophoretic mobility shift assays (emsas) to identify its nucleic acid binding specificities in vitro, and its rna binding domain, as initial steps to understanding the process by which nucleocapsids form in okaviruses. the n protein was found to bind ssrna, dsrna as well as ssdna in a sequence independent manner and the rna binding site was localized to an 18 aa proline/arginine-rich sequence near to its n-terminus. a full-length orf2 gene and five constructs designed to produce n proteins with n-and/or c-terminal truncations (fig. 1a) were cloned into pqe10 and used to express recombinant his 6 -n fusion proteins in e. coli. the purity of the expressed proteins following ni 2+ -nta affinity chromatography and assessed by sds-page and cbb staining is shown in fig. 1b . migration of the full-length his 6 -n protein (qe10-n2; 144 aa, 16.0 kda deduced mass) trailed the 20.1 kda marker protein slightly, consistent with an elevated mass estimated previously [4] plus the additional ,1 kda mass of the n-terminal his 6 sequence. the his 6 -n protein construct qe10-n1 (134 aa, 14.8 kda) with a 10 aa n-terminal truncation migrated slightly below qe10-n2 and constructs qe10-n14 (84 aa, 9.3 kda) and qe10-n38 (51 aa, 5.6 kda) with 60 aa and 93 aa n-terminal truncations, respectively, migrated at lower masses of ,16.5 and ,15.5 kda, respectively. construct qe10-n22 (93 aa, 10.4 kda) with a 51 aa c-terminal truncation migrated at ,15 kda, and protein qe10-n25 (50 aa, 5.52 kda) with 10 aa and 60 aa n-and c-terminal truncations, respectively, migrated at ,10 kda. in addition to using cbb-stained gels (fig. 1b) , the fidelity of the recombinant his 6 -n protein constructs was confirmed in western blots using horseradish peroxidase (hrp) labeled-ni 2+ to detect the his 6 -tag on each protein (fig. 1c) . except for qe10-n14, all his 6tagged proteins were detected and were estimated to be .90% pure, with single bands detected for all but qe10-n1 and qe10-n38 by both methods. the qe10-n1 protein preparation contained additional minor bands of ,22 kda and ,29 kda. the qe10-n38 preparation contained a minor band (,28 kda) about twice the size of the primary protein suggesting that it might be a protein dimer rather than a contaminant. the reason why the recombinant qe10-n14 protein with a 61 aa n-terminal truncation failed to bind the ni 2+ -hrp probe following sds-page and transfer to a nitrocellulose membrane is not known, but possibly it was due to some refolding of the protein that rendered the nterminal his 6 -tag inaccessible [19] . yields of the purified proteins were estimated to be in the order of 10-30 mg/ml of bacterial culture. moreover, the identity of each recombinant n protein was confirmed by matrix-assisted laser desorption ionization time-offlight (maldi-tof) mass spectrometry analyses of peptides generated following tryptic digestion (data not shown). to assess the ability of the recombinant gav n proteins to bind rna in vitro, a total of 15 (+) and (2) sense ssrnas ( fig. 2a) synthesized to different regions of the gav genome (supplementary table s1 ) were examined in electrophoretic mobility shift assays (emsas). gav genome regions selected for analysis included the 59-and 39-terminal sequences, intergenic regions, and sequences in orf1a, orf1b, orf2, orf3 and orf4. of the 15 synthetic ssrnas, all but one (ssrna-4) resolved as a single band following electrophoresis in a non-denaturing agarose gel. emsa binding reactions (20 ml) were conducted at ph 8.0 in the presence of 100 mm nacl and contained 300 ng each synthetic ssrna and 1 mg full-length gav his 6 -n protein (qe10-n2) equivalent to rna:protein molar ratios ranging from 1:17 to 1:71 for the longest to shortest ssrnas. using these binding conditions, emsas showed that the gav n protein could bind to all ssrnas examined irrespective of sequence or polarity (fig. 2b) . migration shifts were smaller with the shorter ssrnas (i.e., ssrna-12, -13, -14 and -15), with protein-bound ssrna complexes migrating as smears slightly behind free ssrna. the likely reason for this was the rna:n protein molar ratios of the shorter ssrnas (approximately 1:17 for ssrna-12 and -13, and 1:27 for ssrna-14 and -15), which were considerably lower than for the longer ssrnas. in the presence of the full-length gav n protein, migration of a 849 nt ssrna synthesized to the mourilyan virus (mov) m rna segment was retarded similarly to equivalent-sized gav ssrnas (fig. 3a) . gav n protein binding to two dsrnas was also assessed (fig. 3b) . for dsrnas prepared from (+) ssrna-1 and (2) ssrna-3 to the genome 59-terminus and from (+) ssrna-6 and (2) ssrna-8 to the orf2 gene, migration in the presence of the full-length n protein was retarded as evidenced by a trailing smear above the primary dsrna band suggestive of variability in n protein binding amounts. gav n protein binding to dsdna and ssdna was also assessed (figs. 3c and 3d). using dsdnas comprising pcr products used as templates to synthesize gav ssrna-1 and ssrna-2 and the same reaction conditions used to assess ssrna binding, no evidence was obtained for the migration of either dsdna being retarded, although some smeared material that migrated more slowly than the primary band was observed (fig. 3c) . for the 6.4 kb genomic ssdna of bacteriophage m13, migration was clearly retarded in the presence of full-length n protein (fig. 3d) . to investigate interactions between the gav n protein and ssrna in more detail, emsas were undertaken using varied molar ratios of full-length n protein and ssrna-1 (fig. 4) . using a constant amount of ssrna in the presence of increasing amounts of n protein (ssrna:protein molar ratios of 1:10 to 1:100), ssrna migration was clearly retarded at a ratio of 1:20 and at ratios of 1:30 or greater, little if any ssrna with unaltered migration was evident (fig. 4a ). starting with a ssrna:protein ratio of 1:100, ssrna amounts were then increased progressively to return the ssrna:protein molar ratio to 1:10, and ssrna migration was observed to be retarded less as ssrna amounts increased (fig. 4b) . from either data set, it was evident the ssrna migration was retarded to similar extents at comparable ssrna:protein molar ratios (figs. 4a and 4b). to identify the gav n protein domain responsible for rna binding, emsas were undertaken using his 6 -n protein constructs with n-or c-terminal deletions or both (fig. 1a) and gav ssrna-1, -2 and -6 (supplementary table s3 ). as shown in fig. 5 , the migration of all three ssrnas was retarded in the presence of the full-length n protein and the constructs qe10-n1 and qe10-n22 containing 10 aa n-terminal and 51 aa c-terminal deletions, respectively. in the presence of n protein construct qe10-n25 containing both a 10 aa n-terminal and a 84 aa cterminal deletion, the amounts of ssrna-1 and -2 detected in the gel were low and ssrna-6 failed to enter the gel altogether. however, the migration of ssrna-1 and -2 that entered the gel was clearly retarded. migration of none of the three ssrnas was retarded in the presence of the n protein constructs qe10-n14 and qe10-n38 containing n-terminal truncations of 60 aa and 95 aa, respectively. as these data indicated that the n-terminus of the gav n protein was essential for ssrna binding, an 18 aa synthetic peptide m 11 pvrrplppqpprnarli 29 corresponding to a proline/arginine-rich domain in this region was tested in emsas for its ability to bind to various ssrnas (fig. 6a) . using ssrna-1, -2, -3, -6, -7 and -8 at ssrna:peptide molar ratios of approximately 1:319, 1:277, 1:319, 1:268, 1:221 and 1:268, respectively, the migration of each ssrna was retarded substantially. trailing ladder-like patterns were also evident particularly for ssrna-1 and ssrna-2, possibly due to the ssrna-peptide complexes aggregating or forming multimers. for ssrna-3 and ssrna-8, ssrnas were so aggregated in the presence of peptide that little ssrna was able to enter the gel. similarly when peptide binding to bacteriophage m13 ssdna was assessed, ssdnapeptide complexes or aggregates formed that precluded any ssdna from migrating into the gel (fig. 6b) . the same effect was observed when dsdna pcr products used to prepare ssrna-1 and ssrna-2 were incubated with the peptide, although small quantities of non-aggregated dsdna with unaffected migration were also evident in the gel (fig. 6b ). here we have characterized the rna binding properties and identified the rna binding domain of the gav n protein using agarose gel emsa analysis of various synthetic rnas reacted with various recombinant n protein constructs expressed in bacteria as well as a synthetic peptide. purified full-length gav n protein bound readily to synthetic (+) and (2) sense ssrnas prepared to various gav genome regions. the n protein also bound to a (+) sense ssrna prepared to the m rna segment of mourilyan virus (mov), a shrimp bunya-like virus, to dsrnas prepared from (+) and (2) sense gav ssrnas, and to genomic ssdna of bacteriophage m13 but not to dsdna amplified by rt-pcr from two gav genome regions. the ability to bind rna was expected due to the structural role of the n protein in okavirus nucleocapsids [2, 3, 5, 20] . the fact that rnas were bound irrespective of their sequence or polarity indicates that specific nucleation sequences, rna folding structures or other factors might be needed to direct the gav n protein to encapsidate genomic (+) ssrna, as with the n proteins of many viruses, including coronaviruses [8, 21] , which share the ability to bind rna in non-sequence specific manner. this may be due to the basic charge properties of these n proteins. as the gav n protein possesses more basic amino acids (20/144 = 13.9%, pi 9.84) than acidic amino acids (13/144 = 9.0%) [2] , its net positive charge is likely to promote association with polyanions such as rna soon after its synthesis. however, the processes directing specific binding of okavirus n protein to genomic rna to form nucleocapsids in the potential presence of other rnas also capable of binding clearly need to be investigated further, and experiments to examine this would be assisted greatly by shrimp cell culture systems capable supporting virus replication becoming available. to localize the nucleic acid binding domain, recombinant his 6tagged gav n proteins were expressed in e. coli from various plasmid constructs containing the full-length orf2 gene or five gene fragments designed to generate variable-length n-and/or cterminal truncations. the presence of the his 6 -tag allowed the proteins to be purified by ni 2+ -affinity chromatography and in sds-page analyses, all six purified recombinant n proteins possessed masses equivalent to those predicted from the length of truncations, the addition of the n-terminal his 6 -sequence, and knowledge that the masses of the gav and yhv n proteins estimated by sds-page are higher than those deduced from their amino acid sequences [3, 4, 22] . emsa data generated using three different ssrnas reacted with these truncated gav n protein constructs localized the rna binding domain to a 50 aa n-terminal sequence spanning met 11 -arg 60 . in this region, the gav n protein sequence is quite basic (theoretical pi = 11.72) due to the presence of eight positively-charged residues and only two negatively-charged residues. database searches undertaken using the interproscan program (http://www.ebi.ac.uk/interproscan/) [23] were unable to identify sequences close in similarity to other better characterized rna-binding or rna-recognition motifs. however, some general sequence similarities, mainly relating to the relatively high proportion of arginine residues, were noted with the short arginine-rich motifs (arms) that mediate rna binding of the rev and tat proteins of human immunodeficiency virus (hiv) [24] , and of the anti-terminator n protein of bacteriophages q21 and p22 [25] . (table 1) the propensity of the met 11 -arg 60 region of the gav n protein to bind nucleic acids was confirmed in emsa analyses of aproline-and arginine-rich peptide sequence (m 11 pvrrplppqpprnarli 29 ) synthesized to the first 18 aa portion of region, and which was found to bind strongly to ssrna as well as ssdna and dsdna. proline-rich protein sequences are known to primarily form helices rather than b-sheets, and in the p33 replicase protein of tomato bushy stunt virus (tbsv), for example, an arginine/proline-rich rprrrp motif has been shown to bind ssrna in preference to dsrna or dsdna [26] . however, as the n protein peptide displayed little obvious binding preference for ssrna compared to dsdna, additional analyses using variant peptide sequences will be needed to identify which amino acids are responsible for conferring this broad nucleic acid binding capability. the n protein of the coronavirus mouse hepatitis virus (mhv) has also been found to possess an ability to bind both genomic and sub-genomic viral rnas as well as cellular rnas to form ribonucleoprotein (rnp) complexes [27] . however, during the encapsidation process, association of the viral membrane (m) protein confers selective binding of genomic-length (+) ssrna directed by a 190 nt packaging signal positioned toward the 39-end of the orf1b gene [27] . in a preliminary attempt to identify an rna packaging signal in the gav genome, emsas were performed using ssrnas synthesized to various genome regions including (i) an orf1b gene 39-region spanning the relative position to the genome packaging signal identified in mhv [28] , (ii) a 39-terminal genome region corresponding in position to the region in the infectious bronchitis virus (ibv) genome reported to contain an rna binding domain [29] and (iii) the 59-genomic rna terminus which, in coronaviruses, has also been reported to interact specifically with n protein [30] . however, recombinant gav n protein bound all synthetic ssrnas tested and whilst it is possible that its binding affinity and/or interaction kinetics varied amongst the rnas, these could not be quantified using the emsa method. thus, based on this uniformity of binding, other experimental approaches will be needed to identify what sequence, if any, in the gav genome acts as a packaging signal or confers specificity for n protein binding. as in mhv, the identification of such rna packaging signals will likely require establishment of cell culture systems to allow the characterization of sequences of mutant defective-interfering genomic rnas packaged into virions [28, 31] . in the emsas, increasing the molar ratio of n protein retarded ssrna migration more significantly, suggesting the binding of multiple n protein molecules to each rna and/or interactions occurring between n protein-ssrna complexes. based on molar ratios examined using a 626 nt ssrna, migration was visibly retarded more when the estimated molar n protein:ssrna ratio was increased from 20:1 to 30:1, and whilst no additional retardation was evident at a ratio of 50:1, it increased again at a ratio of 100:1. this apparent biphasic n protein ssrna interaction suggests that there is some point at which n protein binding becomes saturated, but once present in vast excess, either more than one complex type can form or unit complexes aggregate. a similar phenomenon occurs in prunus necrotic ringspot virus, a plant ilarvirus containing a tripartite (+)ssrna genome, in which two different complex types can form in a nonsequence-specific manner between the 32 kda movement protein (mp) and ssrna4 [32] . one ssrna complex forming in the presence of high mp amounts (mp:ssrna molar ratio 400:1) was observed to enter an emsa gel, whereas another complex type that formed at a lower molar ratio (120:1) did not. moreover, urea denaturation had little in any effect on the type of ssrna:mp complex formed at higher mp amounts but disaggregated the complex type formed in lower mp amounts, suggested that the latter might comprise rod-like structures restricting gel entry and that excess mp might promote increased protein-protein interactions resulted in a more compact globular ssrna-mp complex capable of entering the gel matrix [32] . although something similar might be the reason for the biphasic interaction of the gav n protein with ssrna, delineation of the nature of the various complexes that can form will require further investigation. based on its structural role in nucleocapsids [4, 5] , it was expected that the gav n protein would be capable of binding ssrna. indeed such ssrna binding activity was confirmed in emsa analyses of various rnas and recombinant n protein constructs. moreover, analyses with variably truncated n protein constructs as well as a synthetic peptide showed that this activity was not dictated by any specific sequence constraints and was localized to a short, highly-charged n-terminal motif rich in arginines and prolines. whilst these findings advance our understanding of the rna binding capabilities of the n protein of the crustacean okaviruses, additional studies are now needed to determine the mechanism by which genomic ssrna is nucleated either specifically or preferentially. cloning and expression of full-length and truncated gav his 6 -tagged n proteins the gav orf2 gene and sequences with variable 59-and/or 39-terminal truncations (fig. 1a) were amplified by pcr using various primers containing bam hi sites (supplementary table s2 ) to allow in-frame insertion with the n-terminal his 6 -tag of pqe10 (qiagen). a plasmid containing the entire orf2 gene [2] was used as template for all pcrs. amplified dna products were digested with bam hi and ligated into pqe10. plasmid dna was transformed into competent dh5a or m13[prep4] e. coli host strains and clones were grown on luria broth (lb) agar plates containing 100 mg/ml ampicillin or 100 mg/ml ampicillin plus 25 mg/ml kanamycin. to screen clones for orf2 inserts in the correct orientation, rapid colony pcrs were performed using the pqe-specific primer 59-cccgaaaagtgccacctg-39 (qia-gen) in combination with various orf2 gene-specific anti-sense primers (supplementary table s2 ). plasmid dna prepared to each clone selected for analysis was sequenced using the pqespecific primer and clone specific reverse primers to confirm the fidelity of the dna insert. two plasmid clones containing the inserts were each inoculated into 2 ml lb medium containing 100 mg/ml ampicillin (plus 25 mg/ml kanamycin for m13[prep4] cells) and grown overnight at 37uc in a shaking incubator. a portion (500 ml) of each culture was inoculated into 10 ml pre-warmed lb medium containing antibiotics and the culture was shaken vigorously at 37uc until the optical density (od) at 600 nm had reached 0.5. protein expression was induced by the addition of 0.1 to 1.0 mm final concentration of isopropyl-b-d-1-thiogalactopyranoside (iptg) and incubation at 37uc was continued overnight. cells were collected by centrifugation (8,0006g, 10 min, 4uc), disrupted by sonication in lysis buffer (300 mm nacl, 10 mm imidazole, 50 mm nah 2 po 4 ph 8.0) for non-denatured protein purification and the orf2 his 6 -fusion proteins were purified using ni +2 -nta agarose beads as described in the qiaexpressionist tm instruction manual (qiagen). the proteins were stored at 280uc until used in emsas. for some orf2 his 6 -fusion proteins, e. coli cells were lysed in 8 m urea and protein was purified under denaturing conditions as described in the qiaexpressionist tm manual. purified denatured proteins (500 ml each) were refolded by dialysis overnight at 4uc in 200 ml refolding buffer (3 m urea, 500 mm nacl, 10 mm reduced glutathione, 1 mm oxidized glutathione, 1 mm edta, 20 mm tris-hcl ph 7.1). the dialysis buffer was then replaced with 200 ml pbs and dialysis was continued for 24 h at 4uc. purified his 6 -orf2 fusion proteins were concentrated by centrifugation using either a ym3 or ym10 centricon tm membrane concentrator (millipore) and protein yields were quantified using a bca protein assay kit (pierce) and also estimated visually in comparison to protein standards resolved by sds-page and stained with coomassie brilliant blue (cbb) r250. yields of purified proteins were in the order of ,4 mg each. an aliquot of each purified recombinant his 6 -orf2 protein was separated by sds-page using a 15% polyacrylamide gel run at 120 v for 90 min. a pair of identically loaded gels was separated simultaneously. proteins in one gel were stained with cbb ( fig 1b) and proteins in the other were electro-transferred onto a hybond tm -c nitrocellulose membrane (amersham) using towbin transfer buffer (25 mm tris, 192 mm glycine, 20% methanol and 0.1% sds) and a hoefer semi-dry protein transfer system run at 225 ma for 90 min. the membrane was blocked with 5% skim milk powder in tris-buffered saline (tbs; 0.9% nacl in 100 mm tris-hcl ph 7.5) for 15 min. the blocked membrane was washed with three changes of tbs for 10 min each before being incubated for 35 min in ni 2+ -hrp (sigma-aldrich) diluted 1:1,000 in tbs. the membrane was washed with three changes of tbs for 10 min each and incubated in developing solution (30 mg 4-chloronaphtol, 2.5 ml ice-cold methanol and 20 ml hydrogen peroxide in tbs, 50 ml) for 30 s to 3 min to detect color signal. once a positive signal was observed, the reaction was stopped by incubation in tbs for 10 min. images on membranes (fig. 1c) and cbb-stained gels were captured using an epson perfection v200 photograph scanner. the synthetic peptide (mpvrrplppqpprnarli) was purchased from shanghai science peptide biological technology co. ltd., china. the lyophilized peptide, reported by the manufacturer to be 99.2% pure as determined by high performance liquid chromatography (hplc), was dissolved to appropriate concentration in double-distilled water. plus-and minus-sense ssrnas were synthesized from pcr products amplified from plasmids containing cdna inserts corresponding to various regions of the gav genome ( fig. 2a) . the plasmids included: (i) pgav22, containing a 623 nt orf1a gene sequence extending to the 59-terminus of the genome; (ii) pgav16, containing a 4282 nt sequence beginning 509 nt upstream of the orf1b gene 39-terminus and extending to a position 3773 nt downstream of the orf3 gene 59-terminus; and (iii) pgav12.1, containing a ,2.7 kb insert extending from a region within the orf3 gene to the gav genome 39-polya tail (supplementary table s1 ). sequences of pcr primers used to amplify dna products are shown in supplementary table s3 . in each pcr, either the sense or the anti-sense gav-specific primer included a 59-terminal t7 rna polymerase promoter sequence. each pcr contained ,100 ng plasmid dna, 25 pmol of each primer, 5 ml 106pcr buffer (670 mm tris-hcl ph 8.8), 3 ml 25 mm mgcl 2 , 1 ml 10 mm dntp mix, 0.5 ml 5.5 u/ml taq dna polymerase (promega) and dnase/rnase-free water was added to final volume of 50 l. thermal cycling conditions used in the pcrs were 95uc for 4 min, 35 cycles of 95uc for 30 s, 56uc for 30 s, and 72uc for 120 min, followed by 72uc for 7 min. pcr products were purified using a qiaquick tm gel purification kit (qiagen) according to the manufacturer's instructions. purified dna products were treated with klenow dna polymerase to remove 39-adenosine overhangs and end-filled using taq dna polymerase prior to rna synthesis. to synthesize rna, 1 mg purified dna was added to a 20 ml reaction prepared using the megascript tm high yield in vitro rna transcription kit (ambion), and rna synthesis and dna removal were performed according to the kit instructions. synthetic rna was purified using a megaclear tm rna purification column (ambion), eluted in 100 ml elution buffer (10 mm tris-hcl ph 8.0) and stored at 280uc. mourilyan virus (mov) rna was synthesized using 1 mg pst i-linearized pmov4.1 dna containing a 849 nt cdna corresponding to the m (membrane glycoproteins g1/g2) ssrna genome segment [33, 34] as a template for in vitro t7 rna transcription. double-stranded (ds)rnas corresponding to the gav genome 59-terminus and a sequence including the intergenic region downstream of orf1b to the end of orf2 were prepared by adding together equimolar amounts of plus-and minus-sense ssrnas in megaclear tm (ambion) elution buffer, heating at 95uc for 10 min and then annealing at room temperature for 30 min. the formation of dsrna was confirmed by comparing its migration to each ssrna in a 1% agarose-tae (40 mm trisacetate ph 8.0, 1 mm edta) gel. three pcr products selected randomly were used to assess nprotein binding to dsdna in emsas. single-stranded dna extracted from bacteriophage m13 using phenol chloroform extraction was kindly provided by dr andrew mcdevitt, csiro livestock industries, australia. an electrophoretic mobility shift assay (emsa) was used to identify n-protein binding to nucleic acid [32, 35] . in each assay, 300 ng synthetic ssrna/ssdna/dsdna in megaclear tm elution buffer (ambion) was heated at 85uc for 5 min and cooled at room temperature for 15 min before addition of gav his 6 -n fusion protein or synthetic peptide (1 mg each), 10 ml binding buffer (100 mm nacl, 50% glycerol in 10 mm tris-hcl ph 8.0) and 2 units of ribolock tm rnase inhibitor (fermentas). the reaction volume was adjusted to 20 ml with diethyl pyrocarbonate (depc)-treated water and it was incubated at room temperature for 30 min. following the addition of 2 ml 1% bromophenol blue tracking dye, the rna-protein sample was separated by electrophoresis in a 1% agarose-tae gel containing 0.5 mg/ml ethidium bromide. the relative migration of free nucleic acid and nucleic acid-n protein complexes was visualized using a uv transilluminator and gel images were recorded using a gel-doc documentation system (biorad). the molar ratio of ssrna-1 to full-length his 6 -n protein (pqe10-n2) in the reaction was 1:42. to study the effect of relative rna and n protein concentrations on binding, varying molar ratios of rna:n protein (1:0, 1:10, 1:20, 1:25, 1:30, 1:50, 1:100) were also tested. table s1 details of each gav synthetic ssrna examined including positions in gav genome, polarities and length and the plasmid dna used as template for pcr. (doc) table s2 pcr primers used to amplify full-length and truncated orf2 gene coding sequences to construct pqe10 gav n protein expression vectors and also used in colony pcrs to determine insert orientations following cloning. (doc) nidovirales: evolving the largest rna virus genome the gene encoding the nucleocapsid protein of gill-associated nidovirus of penaeus monodon prawns is located upstream of the glycoprotein gene structural and antigenic analysis of the yellow head virus nucleocapsid protein p20 multiplex rt-nested pcr differentiation of gill-associated virus (australia) from yellow head virus (thailand) of penaeus monodon detection and differentiation of yellow head complex viruses using monoclonal antibodies genetic diversity in the yellow head nidovirus complex monoclonal antibodies specific to yellow-head virus (yhv) of penaeus monodon background paper. functions of the coronavirus nucleocapsid protein identification and primary structure of the gene encoding the berne virus nucleocapsid protein identification and characterization of a porcine torovirus antigenic structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus bovine torovirus: sequencing of the structural genes and expression of the nucleocapsid protein of breda virus sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes comparative analyses of the nucleocapsid genes of several strains of infectious bronchitis virus and other coronaviruses interactions amongst rabies virus nucleoprotein, phosphoprotein and genomic rna in virus-infected and transfected cells rabies virus chaperone: identification of the phosphoprotein peptide that keeps nucleoprotein soluble and free from non-specific rna localization of an rna-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus coronavirus nucleocapsid protein is an rna chaperone gene expression response to misfolded protein as a screen for soluble recombinant protein gill-associated virus of penaeus monodon prawns -molecular evidencefor the first invertebrate nidovirus. nidoviruses (coronaviruses and arteriviruses) in vitro assembly of an infectious cdna clone of infectious bronchitis virus and its application as a gene transfer vector yellow-head virus: a rhabdovirus-like pathogen of penaeid shrimp interproscan -an integration platform for the signature-recognition methods in interpro structural variety of arginine-rich rna-binding peptides rna binding specificity of hnrnp a1: significance of hnrnp a1 high-affinity binding sites in pre-mrna splicing characterization of the rna-binding domains in the replicase proteins of tomato bushy stunt virus cooperation of an rna packaging signal and a viral envelope protein in coronavirus rna packaging analysis of efficiently packaged defective interfering rnas of murine coronavirus -localization of a possible rna-packaging signal the infectious bronchitis virus nucleocapsid protein binds rna sequences in the 39 terminus of the genome specific interaction between coronavirus leader rna and nucleocapsid protein identification and characterization of a coronavirus packaging signal rna-binding properties and mapping of the rna-binding domain from the movement protein of prunus necrotic ringspot virus rt-nested pcr detection of mourilyan virus in australian penaeus monodon and its tissue distribution in healthy and moribund prawns preliminary molecular and biological characterisation of mourilyan virus (mov): a new bunya-related virus of penaeid prawns gel retardation conserved structures and diversity of functions of rna-binding proteins the authors would like to thank dr. andrew mcdevitt of csiro livestock industries, australia for providing bacteriophage m13 dna. key: cord-279106-3ffa9djf authors: syatila ab ghani, nur; emrizal, reeki; makmur, haslina; firdaus-raih, mohd title: side chain similarity comparisons for integrated drug repositioning and potential toxicity assessments in epidemic response scenarios: the case for covid-19 date: 2020-10-21 journal: comput struct biotechnol j doi: 10.1016/j.csbj.2020.10.013 sha: doc_id: 279106 cord_uid: 3ffa9djf structures of protein-drug-complexes provide an atomic level profile of drug-target interactions. in this work, the three-dimensional arrangements of amino acid side chains in known drug binding sites (substructures) were used to search for similarly arranged sites in sars-cov-2 protein structures in the protein data bank for the potential repositioning of approved compounds. we were able to identify 22 target sites for the repositioning of 16 approved drug compounds as potential therapeutics for covid-19. using the same approach, we were also able to investigate the potentially promiscuous binding of the 16 compounds to off-target sites that could be implicated in toxicity and side effects that had not been provided by any previous studies. the investigations of binding properties in disease-related proteins derived from the comparison of amino acid substructure arrangements allows for effective mechanism driven decision making to rank and select only the compounds with the highest potential for success and safety to be prioritized for clinical trials or treatments. the intention of this work is not to explicitly identify candidate compounds but to present how an integrated drug repositioning and potential toxicity pipeline using side chain similarity searching algorithms are of great utility in epidemic scenarios involving novel pathogens. in the case of the covid-19 pandemic caused by the sars-cov-2 virus, we demonstrate that the pipeline can identify candidate compounds quickly and sustainably in combination with associated risk factors derived from the analysis of potential off-target site binding by the compounds to be repurposed. epidemics caused by novel infectious agents result in situations where no known treatment regimens are 29 in practice. case management would therefore first rely on treating and alleviating the symptoms. the focus 30 of the treatment would then move on to eradication of the infectious agent from the host and more in-depth 31 therapeutic management. such an epidemic scenario presented itself in the city of wuhan, hubei province, 32 china in late 2019 [1] . the causative pathogen for the observed acute respiratory distress was later identified 33 to be a novel human coronavirus (ncov19) named as severe acute respiratory syndrome coronavirus 2 1 (sars-cov-2) [2] . although, many coronaviruses are found in bat reservoirs, it is probable that sars-cov-2 2 also has intermediate hosts such as pangolins and snakes [3] . 3 three months after it was first reported, the disease, named coronavirus disease 2019 (covid19) , had 4 progressed into a global pandemic. the fast spread of the disease was however paralleled by the speed that 5 data regarding the disease and its causative agent were generated. in mid-january 2020, the first genome 6 sequence was deposited into genbank (https://www.ncbi.nlm.nih.gov/genbank/); by mid-july 2020, more 7 than 40,000 complete genomes with high coverage from samples throughout the world had been deposited in 8 the gisaid database (http://www.gisaid.org/; http://epicov.org). while the rate of genome sequencing and 9 data sharing is unprecedented, the rapid availability of structure data has also been equally impressive. in late 10 september 2020, more than 400 structures of sars-cov-2 proteins had been deposited in the protein data 11 bank (pdb) [4] . 12 despite the number of confirmed cases passing 32.9 million with more than 1 million fatalities 13 worldwide in early october 2020, treatment options are still lacking for covid-19 although several vaccines 14 have recently started their trials in july 2020. this dire but data-rich scenario has led investigators to resort to 15 drug repurposing strategies. although such efforts to reposition approved drugs to a new target can be 16 explored in a clinical setting, we focus specifically on how computational approaches can feature prominently 17 in the identification of the candidate compounds. 18 various approaches have been deployed to explore the repertoire of known and approved compounds for 19 covid-19. zhou et al. utilized network-based analyses of drug targets and the virus-host interactions in the 20 human interactome to list 16 potential drugs to prioritize for repurposing [5] . an even larger effort that 21 generated a sars-cov-2-human protein-protein interaction map was able to identify 66 druggable human 22 proteins that could be targeted by 69 currently available fda approved compounds to be used as covid-19 23 treatments [6] . 24 side chain similarity comparisons [7, 8] have been reported to be a potential starting point in drug 25 repurposing efforts [9] . for such an approach, the 3d arrangements of known drug binding sites are collected 26 as a search database to identify similar sites in non-homologous structures thus implying the capacity to bind 27 similar ligands. drug-target interaction prediction using structural data has remained a largely unexplored 28 niche [10] . the identification of possible alternative binding sites for an approved drug can also provide 29 insights into their possible off-target effects. there is a clear urgency to discover and deploy suitable 30 candidates that can be repositioned against targets associated with covid-19. nevertheless, it is prudent to 31 steer clear of adverse effects resulting from the poly-pharmacological actions of promiscuous drugs with the 32 ability to bind to other targets [11] . 33 in this work, amino acid side chain similarity searching was utilized to propose alternative target sites in 34 sars-cov-2 protein structures for drug repositioning. these searches were based on the premise that if a 35 known drug binding site could be found in a sars-cov-2 protein, then that protein could also serve as an 36 alternative target for the same drug. this same principle was then used to identify off-target sites that could 37 present as side effects or result in some form of toxicity. the list of potential drugs derived from the side 38 chain arrangements similarity searches was then used to propose structurally similar compounds that could 39 also target the sites already identified for repositioning. our approach differs significantly from that reported 40 by zhou et al. [5] and gordon et al. [6] which can serve as additional confirmatory analysis and complement 41 the gaps in existing work. the details of these differences will be discussed in a later section. the downloaded sequences were clustered at 90% sequence similarity cut-off using the cd-hit program 3 [12]. members of individual clusters were sorted according to the x-ray crystallography resolution; the 4 sars-cov-2 protein sequence with the higher resolution structure was selected as the cluster's 5 representative. the pdb structures containing representative sequences were compiled together for further 6 similarity searches against the dataset of known drug binding sites derived from protein-drug complexes in 7 the pdb. 8 for the selection of drug compounds, we further selected drug compounds that are: (i) currently 9 undergoing clinical trials for covid -19, and (ii) protein was used as the receptor structure for docking. the python script contains all the necessary commands 28 that will be executed in the ucsf chimera command line to automatically pre-process structures and perform 29 blind molecular docking. the pre-processing steps of the ligand and receptor structures include the removal of 30 water molecules and ligands, assigning the partial charges for both standard and non-standard residues, as 31 well as an additional energy-minimization step. the atomic partial charges for standard residues including 32 standard amino acids, water and know ligands, as well as non-standard residues were assigned based on the 33 amber ff14sb force field (default), while the partial charges for non-standard residues were calculated 34 using the antechamber module based on the am1-bbc method. in the case of residues with missing side 35 chains, the amino acid side chains were replaced based on information from a rotamer library. energy 36 minimization was performed with steps of steepest descent minimization set to 100. molecular docking was 37 carried out using a local installation of autodock vina and linked for use in ucsf chimera. 38 blind docking was carried out instead of using the binding site as a reference point. therefore, a 39 whole protein structure target was exhaustively searched for potential binding poses using the default settings 40 for parameters such as exhaustiveness value (set to 8) and maximum number of binding modes (set to 9). the 41 default box size was used to sample the ligand orientation where it automatically covers the entire protein 42 receptor thus allowing for matches of binding poses to not only known binding sites, but also to other putative 43 sites that have not been reported elsewhere. 44 upon completion of the docking run using the python script, ucsf chimera loads a selection of 45 docking poses for visualization where the docking poses are ranked according to the docking scores reported 46 in kcal/mol with more negative values indicating better binding. the sites found from the sub-structural 47 similarity search is also visualized. the ucsf chimera session for individual script runs were saved for 48 further curation and analysis. the sites from the sub-structural similarity search were compared against the 1 sites in pre-computed binding poses from molecular docking, where an overlap of at least three matched 2 residues with poses of docking scores more negative than -6.5 kcal/mol selected for further analyses. 3 4 2.3. searching for potential off-targets from human for selected drugs proposed for covid-19 5 6 potential off-targets for selected drugs proposed for covid-19 were identified using three different 7 methods. first, known human proteins bound to the selected drug compounds were obtained from the pdb 8 through the 'advanced search' interface in the rcsb using the ligand pdb id as a query. the list of pdb 9 structures retrieved were filtered to only contain pdb with organism denoted as 'homo sapiens'. second, 10 human proteins with similarly arranged sites to drug binding sites for the selected drugs were retrieved from 11 pre-compiled results for sub-structural similarity searches in drug reposer web server. third, human 12 proteins with more than 30% sequence similarity to individual sars-cov-2 protein structures were retrieved 13 from blastp searches against the pdb. the list of proteins retrieved was filtered to only contain proteins with 14 sequences more than 30% sequence identity to the query sars-cov-2 protein. 15 these human structures were then used for molecular docking against the selected compounds. 16 molecular docking runs were conducted based on the above-mentioned protocol using python scripts 17 executed in ucsf chimera. a compound's involvement in specific biological mechanisms and potential 18 adverse effects upon interaction with the selected compounds were manually assessed and extracted from 19 information queried and similar ligands were structurally aligned in the ucsf chimera interface [15] . 28 the queried and the similar ligands were individually searched against the drug reposer 29 application database to retrieve results for sub-sructural similarity searches. both sets of results were 30 compared and shared sars-cov-2 protein targets from the list of proteins (proteins containing sites similar 31 to binding sites for both queried and similar ligands) were obtained for molecular docking against the 32 corresponding ligand molecules with autodock vina using the above-mentioned protocol [15] . 33 34 3. results and discussion 35 in this study, sub-structural similarity searches and docking analyses were carried out to: (i) identify 36 potential targets and drug binding sites in sars-cov-2 proteins; (ii) identify off-targets for proposed drug 37 compounds for covid-19; (iii) identify other approved drugs with similar structure to proposed drugs that 38 are potentially useful for covid-19 treatment. a total of 351 sars-cov-2 proteins were obtained from the 39 pdb that included the following proteins; adp ribose phosphatase (pdbid: 6w02), spike protein (pdbid: 40 6vsb), main protease (pdbid: 6lu7), nucleocapsid (pdbid: 6m3m), nsp7-nsp8 complex (pdbid: 6yhu), 41 nsp9 replicase (pdbid: 6w4b), nsp10-nsp16 complex (pdbid: 6w4h), nsp15 (pdbid: 6w01), orf7a 42 encoded accessory protein (pdbid: 6w37) and rna-dependent rna polymerase or nsp12 (pdbid: 6m71). 43 the substructure similarity searching used in this work utilized the assam computer program which 44 solves a maximal common subgraph problem to match similar 3d arrangements of amino acids in a dataset 45 of protein structures [7] . the arrangements of amino acids in 3d space are represented as graphs, where the 46 graph nodes are the pseudo-atoms representing side chain groups and the graph edges are distances between 47 the side chain groups. using this scheme, it is possible to match similar 3d arrangements, such as catalytic 48 sites and ligand binding sites, in non-homologous structures. drug reposer is an extended application of the 1 assam program that focuses on sub-structures that constitute the binding sites for approved drug molecules 2 [9]. 3 at the time of writing, approximately a third of the proteins encoded in the sars-cov-2 genome have 4 corresponding pdb structures. in anticipation that more structures will be deposited, we have enabled the 5 analysis pipeline to be deployed to process new structures as and when they become available, based on the 6 clustering of protein sequences and comparison to readily available structures. the results from the analyses 7 reported in this work and those that will be carried out by the pipeline for new structures will be made 8 accessible via a dedicated module of the drug reposer web application -9 http://mfrlab.org/drugreposer/covid19/. the list of pdb ids with pre-computed results from sub-structural 10 similarity searches and the sequence clusters are also available at the same resource. 11 the search for covid-19 treatments has resulted in the registration of more than 3000 clinical trials in 12 the clinicaltrials.gov database to explore the repurposing of more than twenty readily available drugs 13 ( searching for sites in the sars-cov-2 protein structures (hit sites) that are geometrically similar to 22 sites for approved drug compounds (query sites) using the drug reposer application [9] had identified 23 matches that included 22 sites from protein-drug complexes with sequence identities lesser than 30% to the 24 corresponding sars-cov-2 proteins (table 1 ). these results show that the computational approach adopted 25 in this study is able to find similarly arranged sites in unrelated proteins which could be an advantage when 26 there are limited numbers of homologous structural models to be used for comparison of binding sites. in 27 addition, the selection of matches to proteins with lesser than 30% sequence similarity could be indicative of 28 function differences, thus potentially distinct pathways where the bound drugs could be repurposed to. 29 the sites identified in the sars-cov-2 proteins were then docked with their corresponding drug 30 compounds derived from the protein-drug complex data. molecular docking runs resulted in the identification 31 of several poses with docking scores ranging from -6.0 kcal/mol up to -17.6 kcal/mol, which are congruent 32 with the results of the drug reposer searches (table 1, figure 1 ). of these 22 potential interactions, six have 33 been reported in other studies [18] [19] [20] . 34 the sub-structural similarity searches carried out revealed that six of the nine analysed sars-cov-2 35 contain multiple potential alternative binding sites for different compounds. for example, the nucleocapsid 36 protein contains potential sites for losartan, ritonavir, darunavir and aspirin ( the hiv protease inhibitors -darunavir (017), ritonavir (rit) and lopinavir (ab1) -inhibit the hiv 18 aspartyl protease and prevents the cleavage of gag and pol proteins into their subsequent protein components 19 [22] . the potential antiviral activity of such inhibitors against coronaviruses had been previously studied; 20 nelfinavir for example, had been reported to inhibit the replication of sars-cov and prevent cytopathic 21 effects [23]. lopinavir and ritonavir had been shown to improve clinical outcomes from sars-cov 22 infections and are hypothesized to bind to the 3-chymotrypsin-like protein (3clpro) or main protease [24] . 23 our analysis also demonstrated the potential ability of lopinavir (pdbid: 2qhc) to bind to adp ribose 1 phosphatase (pdbid: 6w02) with a docking score of -17.6 kcal/mol at a position close to the known substrate 2 binding site ( figure 1a ). 3 we found that the nsp10-16 complex (pdbid: 6w75) may potentially bind to ritonavir (rit) in a 4 manner similar to that observed in the hiv protease (pdbid: 1sh9) while adp ribose phosphatase (pdbid: 5 6w02) could potentially bind to lopinavir (pdbid: 2qhc) with a high docking score (-17.6 kcal/mol) ( figure 6 1a). a potential site for folic acid (fol) binding that is similar to the arrangement found in dihydrofolate 7 reductase (pdbid: 4i13) was also found at the interaction site between domain iii (residue 201-303) of two 8 monomers, where dimerization is crucial for protease function took place (pdbid: 6y2g). we also found that 9 the nucleocapsid might bind to losartan, darunavir and aspirin at the dimerization site between two monomers 10 in a similar manner to the sars-cov-2 main protease. 11 the drug reposer searches also identified similarly arranged sites between the indomethacin-bound 12 prostaglandin e synthase 2 (pdbid: 1z9h) and the nsp10 protein (pdbid: 6w4h) ( figure 1b ). an 13 arrangement of amino acid residues that make up the indomethacin binding site in cyclooxygenase-2 (cox-14 2), also known as prostaglandin synthase 2 (pdbid: 4cox), was also found to be similar to residues at the 15 vicinity of docked indomethacin in the nsp7-nsp8 complex (pdbid: 6yhu) ( response. in this context, the binding of indomethacin to these protein structures (nsp7/nsp8 or nsp10) may 30 also prevent potential inflammatory events. the same mechanism could be adopted by other nsaids like 31 naproxen (nps), that might recognize similar sites from cox-2 (pdbid: 3nt1) in the adp ribose 32 phosphatase (pdbid: 6w6y), as indicated from the sub-structural similarity we have uncovered. these sub-33 structural similarities to a known indomethacin binding site may explain the mechanism for studies that have 34 reported the ability of nsaids to bind to sars-cov-2 proteins [29] although the atomic level details of such 35 interactions have not yet been reported. 36 37 3.2. adp ribose phosphatase of nsp3 as potential target in sars-cov-2 38 39 our analysis revealed that the adp ribose phosphatase of nsp3 from sars-cov-2 has the most 40 number of 3d residue arrangements that are similar to the binding sites in known drug targets compared to 41 other sars-cov-2 proteins (table 1, figure 2 ). all the identified sites are within the substrate binding sites 42 with the docking scores for the different poses ranging from -6.7 to -17.0. in this case, the known adp ribose 43 phosphatase -apr complex was used as a control to obtain reasonable docking scores that could be 44 considered acceptable based on predicted binding poses between the adp ribose phosphatase and the 45 substrate, apr. the molecular docking with energy minimization steps resulted in several binding poses with 46 docking scores ranging from -7.9 to -9.8, with all sites located within the actual binding site for apr. 47 the adp ribose phosphatase of non-structural protein 3 (nsp3) is likely to be targeted by anti-48 retrovirals and several other drugs more than any other sars-cov-2 structures, particularly at the active site 1 of the structure (figure 2a ). this finding is in agreement with recent computational screening for the drug 2 binding ability of sars-cov-2 proteins which highlighted the promiscuity of nsp3 in binding to other 3 molecules at the adp ribose binding site [21,28]. the de-adp ribosylation activity of nsp3 suppresses the 4 expression of host innate immunity genes such as interferon and interleukin related genes [30] . disruption of 5 nsp3 function will allow for the host immune system to respond normally to the infection. 6 7 8 9 sub-structural similarity searches and molecular docking runs have revealed the potential binding sites 2 for darunavir (017) that originally targeted hiv protease (pdbid: 6dh3), as well as chloroquine (clq) that 3 originally targeted quinone reductase 2 (pdbid: 4fgl) and indicated for malaria and rheumatoid arthritis, onto 4 the adp ribosylation site of nsp3 (pdbid: 6w02) (table 1, figure 2 ). despite the similarity of these sites in 5 terms of their 3d arrangements, the similarity of their molecular functions is unlikely to be related. 6 the docking results indicate that hiv protease inhibitors and nsaids are among the existing drugs that 7 could potentially be repositioned against adp ribose phosphatase and several non-structural proteins for 8 treatment of covid-19. the similarly arranged residue patterns observed between the binding poses in 9 sars-cov-2 proteins from docking simulations and those from available drug-bound protein complexes 10 allow us to infer the similarities of the binding mechanisms shared by these proteins despite the lack of 11 sequence similarities. 12 13 3.3. potential off-targets of approved drugs proposed for covid-19 14 15 the binding of drug compounds to off-target sites in proteins other than their intended targets can lead to 16 unexpected pharmacological outcomes including the activation or disruption of molecular functions that cause 17 adverse effects or other unexpected conditions [11,31]. however, off-target effects are not necessarily 18 negative and it is this same concept that is in use to repurpose approved compounds for alternative indications 19 based on the availability of similar of binding sites shared among proteins involved in distinct disease 20 pathways [11, 31] . we deployed the same substructure searching methodology to identify off-target sites for 21 the drugs being explored as covid-19 treatments. 22 23 an assam search of the human protein structures in the pdb using the drug binding sites we have 26 identified was used as a means to investigate whether the use of these drugs could alter other pathways. the 27 searches led us to a compilation of potential off-target sites and/or effects for eleven approved compounds 28 (table 2 ). 29 30 the substructural similarity searches for potential off-target sites in human proteins using the drug 7 reposer application was able to identify several proteins that have similar geometry to the binding site of a 8 drug proposed for repositioning against sars-cov-2 targets (section 3.3.2). the same data also allowed us 9 to compile potential repurposing opportunities of these drugs for other indications including covid-19 10 ( table 2 and table 3 ). 11 non-homologous proteins that share similarly arranged sites for a particular drug molecule are more 12 likely to be considered as off-targets because they may have different molecular function and are involved in 13 distinct pathways that may not be associated with the original target disease. recent computational studies 14 have proposed several hiv protease inhibitors [18, 20, 21] , nsaids [29] , and losartan [41] as potential 15 therapeutic agents for covid-19. although we can confirm the presence of potential binding sites to these 16 drugs on sars-cov-2 proteins, we were also able to identify potential off-target sites where these drugs may 17 alternatively bind in the structures of human proteins (table 3) . 18 19 (table 3 ). peripheral neuropathy due to the neurotoxicity of 6 hiv protease inhibitors have been reported as complications resulting from anti-hiv treatment [53] . one 7 potential off-target protein that might cause such symptoms is the herc2 protein (pdbid: 3kci). disruption 8 of this protein causes reduction of e6ap activity that has been implicated in neurodevelopment disorders such 9 as angelman syndrome and autism [57] . 10 losartan targets the angiotensin type ii receptor, however, it may also bind to the drug metabolizing 11 cytochrome p450 (pdbid: 5x24) that has a similarly arranged site in ceruloplasmin (pdbid: 1kcw) ( table 3 , 12 figure 3b ). ceruloplasmin has been implicated with parkinson's disease where disruption of the oxidative 13 activity by ceruloplasmin causes increased iron levels in the brain that is correlated to parkinson's [47, 56] . on 14 the other hand, it was also reported that losartan could be useful for parkinson's where it might be able to 15 reduce oxidative stress and neurodegeneration [58] thus warranting further investigations regarding the 16 neuroprotective benefits of losartan. 17 the function inhibition of certain off-target proteins may provide coincidental antiviral effects (table 3) . 18 other than potentially targeting the sars-cov-2 proteins, nsaids such as naproxen (nps), indomethacin 19 (imn) and aspirin (ain) may also interact with host proteins involved in mounting the defense against viral 20 infections. for example, we found that naproxen might be able to bind polypyrimidine tract-binding protein 1 21 (ptbp1) (pdbid: 1qm9) based on the similarity of the binding site for naproxen in serum albumin (pdbid: 22 4po0) (table 4, figure 3c ). 23 the ptbp1 protein had been shown to activate the replication of picornaviruses and coronaviruses 24 through binding to its rna binding domain [49, 59] , thus binding of naproxen to its binding site could 25 potentially block viral replication. other nsaids like the indomethacin and aspirin might also induce 26 antiviral properties by binding to myeloperoxidase, which is a part of host defense system (table 3) . the 27 protein acts as tissue damage factor that induces secondary bacterial lung infections causing the acute 28 respiratory distress syndrome seen in influenza [51] . decreased function of myeloperoxidase had been shown 29 to potentially decrease inflammatory damage and lung viral load [51]. sars-cov-2 proteins that may share a similar fold to human proteins were also considered as potential 14 off-targets. in this case, sars-cov-2 proteins that retrieved a human protein by blastp alignment with more 15 than 30% sequence identity is a possible indication of fold similarity. these protein structures were then 16 analyzed to ascertain whether they contained a similar sub-structure arrangement as the sars-cov-2 protein 17 that is being targeted for drug repositioning (table 4) . 18 19 table 4 20 human proteins with more than 30% sequence identity to sars-cov-2 proteins retrieved by a blastp search of the 21 pdb database. the sars-cov-2 adp-ribose phosphatase from nsp3 has a similar sequence to human adp-ribose 14 binding protein and both share a similar mechanism of adp ribose binding (figure 4) . the sars-cov-2 15 spike protein is found have sequence similarities to the irap protein with both being associated to the renin-16 angiotensin pathway. no human sequences with possible fold similarities were detected for the main protease 17 and non-structural proteins that include the nsp7, nsp8 and nsp10 which are conserved in viruses. the binding of approved drugs or inhibitors to interleukin 17a and mineralocorticoid receptors that have 2 similar sequences to the nucleocapsid and nsp15 respectively, could prevent inflammation by the immune 3 response [65,66], a known complication of covid-19 (table 3 ). this would mean that such drugs could 4 target both the virus and the host in parallel with potentially therapeutic results. 5 6 3.4. other compounds with potential as covid-19 therapeutics based on ligand structure similarity 7 8 it is known that similar drugs may require a similar binding environmentand can have similar inhibitory 9 effects, thus making it possible that a target protein can interact with a set of drug molecules with similar 10 structures [67] . with this premise, the structures of the drugs proposed for repositioning against sars-cov-2 11 targets (proposed drugs) were used as a reference point to find other drug molecules with similar structures 12 (matched drugs). this was carried out using the ligand search interface in the pdb that is based on the 13 comparison of pharmacophores. the search identified 6 matches with similar structures to the input 14 queries -quinacrine, vardenafil, lenalidomide, pomalidomide, amprenavir and methotrexate ( table 15 5). with the exceptions of methotrexate, which has structural similarities to folic acid 16 (clinicaltrials.gov ids: nct04352465 and nct04434118), and lenalidomide, which is related to 17 thalidomide (clinicaltrials.gov id: nct04361643), none of these compounds are involved in any 18 known clinical trials for covid-19 at the time of writing. molecular docking targeting the sars-cov-2 19 proteins using both the proposed and matched drugs (shared sars-cov-2 protein targets) resulted in several 20 binding poses that is indicative that the matched drugs can potentially bind to sars-cov-2 proteins in a 21 similar manner as the proposed drugs (table 5, figure 5 ). 22 23 our analyses found that both darunavir and amprenavir can potentially bind to the same sars-cov-2 4 site (p125, g130, i131, v155, and d157) in nsp3 ( figure 4e , table 5 ). darunavir, when docked on nsp3, 5 has a molecular binding affinity of -9.4 kcal/mol. amprenavir, when docked at the similar site ( figure 4e ), 6 also has a molecular binding affinity of -9.4 kcal/mol (table 5) . 7 some structurally similar drug molecules are intended for similar indications. both darunavir and 8 amprenavir ( figure 4e ) are protease inhibitors that have been used for the treatment of hiv. however, 9 amprenavir is useful against infections that exhibit resistance to other protease inhibitors used in hiv 10 treatment [68] . thus, amprenavir might confer an advantage in a scenario where the protein target from 11 sars-cov-2 develops resistance towards darunavir. both chloroquine and quinacrine ( figure 4a ) have been 12 indicated for the treatment of systemic lupus erythematosus as well as other diseases [69] . 13 a study comparing the oculotoxicity of chloroquine and quinacrine in the management of lupus 14 erythematosus found that quinacrine exhibits less oculotoxicity compared to chloroquine if taken at low doses 15 [70]. thus, quinacrine might be a less toxic alternative compared to chloroquine with regard to any 16 ophthalmologic side effects. 17 sildenafil and vardenafil ( figure 4d) vardenafil, amprenavir and methotrexate had been reported to potentially bind to sars-cov-2 proteins 32 through structural analyses [19, 78] . to our knowledge, the potential use of quinacrine, lenalidomide, and 33 pomalidomide for covid-19 have not been reported elsewhere in the context of binding ability through 1 structural analyses. furthermore, the finding that quinacrine is a readily available compound that has yet to be 2 explored or proposed for covid-19 is novel to this work. should the current candidate drug molecules 3 proposed for covid-19 clinical trials fail at any stage of the process, these structurally similar drug 4 molecules can be investigated as potential alternatives. it is not unexpected that the use of these structurally 5 similar compounds could be used in concert as a cocktail for more effective therapy [79]. 6 7 8 indicate regions containing residues within less than 4.0ã� to docked drug molecules. the orange shaded areas 10 indicate regions containing residues that form the binding sites identified by drug reposer. 3.5 distinction from other covid-19 drug repurposing efforts and future directions 13 in this work, all drugs that have been proposed for clinical trials were analyzed using the drug reposer 14 pipeline to find their potential binding sites in any sars-cov-2 protein by virtue of having similar 3d 15 arrangements of amino acid residues to the known target sites. it is not unexpected that our results will 16 overlap or have parallels with the outcomes of other studies that have been recently published or are ongoing. 17 however, the results presented here and in the covid-19 drug reposer resource, will also provide the 18 relevant supporting insights regarding why or how a particular drug may be effective while at the same time, 19 have the added advantage of presenting the potential capacity for off-target interactions that may cause or 20 explain any side effects upon administration. 21 the human proteins based on the analysis of association networks between human and sars-cov-2 proteins [6] . 30 in comparison to our analyses, there are two drugs that overlap with our results, chloroquine (targeting 31 sigma1-receptor:nsp6) and indomethacin (targeting ptges2:nsp7). 32 this study was intended to develop a pipeline to identify drug compounds that could be repositioned 33 against sars-cov-2 targets using the available structural information in the pdb. this pipeline was also 34 able to identify potential side effects or toxicity associated with those compounds that arose from off-target 35 binding. integrating the data to pharmacophore matching tools allowed other similarly structured drug 36 compounds to be identified that also had the potential to be repositioned against sars-cov-2 targets. the 37 information derived from such analyses could be used as a means of decision making to prioritize down-38 stream experimental validation and assays. this study does not provide any experimental evidence validating 39 the binding of the proposed repositioned drugs to sars-cov-2 proteins. the results of this study should not 40 be regarded as an explicit treatment recommendation or protocol for covid-19. 41 a limited set of existing drugs extracted from lists of those currently undergoing or planned for covid42 19 trials was used in this work. the analyses reported only utilized data of compounds that were structurally 43 present as a standalone ligand in the pdb. both these factors restricted the number of potential candidates that 44 could be proposed for repurposing. despite these limitations, our analyses yielded 22 target sites for 45 repurposing of which only 6 had been mentioned in other studies. it is clear that the work reported here could 46 be extended to include all known drug binding sites in the pdb. although the current targets for repositioning 47 in this study considers only sars-cov-2 proteins, the pipeline can be integrated to network analyses 48 methods to identify human proteins that could also yield therapeutic effects for covid-19. furthermore, this 49 study can also be extended to include other sars-cov-2 structures as and when they become available. such 50 data will be updated via the specific drug reposer resource for covid-19. the fastest and safest route to providing drug treatments for covid-19 would be to reposition approved 4 compounds against targets from this newly described disease. at the time of writing, the search for effective 5 covid-19 treatments is still ongoing. despite being subject to the availability of associated protein 6 coordinate structure data in the pdb, the use of amino acid 3d side chain based sub-structure comparisons 7 have proven to be a feasible means of identifying candidate compounds to be repositioned for covid-19. 8 our analyses yielded 22 potential sites in sars-cov-2 proteins and 16 drug compounds that could be 9 repurposed for covid-19. it is clear that the use of structural data from the pdb is able to provide high 10 quality mechanistic level details for strategizing the selection of candidate compounds to be repurposed. the 11 capacity to not only identify new target sites, but also identify potential off-target sites, provide a deeper level 12 of context for the decision making process to safely proceed with exploring specific compounds to be 13 repurposed for the new disease. 14 15 acknowledgments 16 17 we thank the malaysia genome institute for the use of computational resources. this the authors have no affiliation with any organization with a direct or indirect financial interest 2 in the subject matter discussed in the manuscript 3 clinical features of patients infected with 2019 27 novel coronavirus in wuhan, china the species 30 severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars recent progress and challenges in drug development against covid-33 19 coronavirus (sars-cov-2) -an update on the status protein data bank: the 36 single global archive for 3d macromolecular structure data network-based drug repurposing for novel 39 coronavirus 2019-ncov/sars-cov-2 a sars-cov-2 protein 42 interaction map reveals targets for drug repurposing sprite and assam: web servers 44 for side chain 3d-motif searching in protein structures imaaagine: a webserver for searching 47 hypothetical 3d amino acid side chain arrangements in the protein data bank drug reposer: a web server for predicting similar amino 3 acid arrangements to known drug binding interfaces for potential drug repositioning drug repositioning or 6 target repositioning: a structural perspective of drug-target-indication relationship for available 7 repurposed drugs drug promiscuity in pdb: protein binding site similarity is 10 key cd-hit suite: a web server for clustering and comparing 12 biological sequences firdaus-raih m. sprite and assam : web servers 14 for side chain 3d-motif searching in protein structures autodock vina: improving the speed and accuracy of docking with a new scoring 17 function, efficient optimization, and multithreading ucsf chimera-20 a visualization system for exploratory research and analysis uniprotkb/swiss-prot trial reporting in clinicaltrials.gov -the final rule structure based drug discovery 27 by virtual screening of 3699 compounds against the crystal 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methotrexate adverse drug reaction to methotrexate: 1 pharmacogenetic origin potential covalent drugs targeting the main protease of the sars-cov-2 3 coronavirus nanoparticles for combination drug therapy key: cord-281528-xy8j5jiv authors: di paola, luisa; hadi-alijanvand, hamid; song, xingyu; hu, guang; giuliani, alessandro title: the discovery of a putative allosteric site in the sars-cov-2 spike protein using an integrated structural/dynamic approach date: 2020-06-17 journal: j proteome res doi: 10.1021/acs.jproteome.0c00273 sha: doc_id: 281528 cord_uid: xy8j5jiv [image: see text] sars-cov-2 has caused the largest pandemic of the twenty-first century (covid-19), threatening the life and economy of all countries in the world. the identification of novel therapies and vaccines that can mitigate or control this global health threat is among the most important challenges facing biomedical sciences. to construct a long-term strategy to fight both sars-cov-2 and other possible future threats from coronaviruses, it is critical to understand the molecular mechanisms underlying the virus action. the viral entry and associated infectivity stems from the formation of the sars-cov-2 spike protein complex with angiotensin-converting enzyme 2 (ace2). the detection of putative allosteric sites on the viral spike protein molecule can be used to elucidate the molecular pathways that can be targeted with allosteric drugs to weaken the spike-ace2 interaction and, thus, reduce viral infectivity. in this study, we present the results of the application of different computational methods aimed at detecting allosteric sites on the sars-cov-2 spike protein. the adopted tools consisted of the protein contact networks (pcns), sepas (affinity by flexibility), and perturbation response scanning (prs) based on elastic network modes. all of these methods were applied to the ace2 complex with both the sars-cov2 and sars-cov spike proteins. all of the adopted analyses converged toward a specific region (allosteric modulation region [amr]), present in both complexes and predicted to act as an allosteric site modulating the binding of the spike protein with ace2. preliminary results on hepcidin (a molecule with strong structural and sequence with amr) indicated an inhibitory effect on the binding affinity of the spike protein toward the ace2 protein. the recent pandemic caused by sars-cov-2 (covid-19) has posed the most challenging global health and sanitation concern of the twenty-first century, with 6,663,304 confirmed cases of infected and 392,802 confirmed fatalities (world health organization, updated on june 6th, 2020 1 ). moreover, this pandemic has caused a widespread lockdown, affecting more than 1 in 2 individuals in the world. the virus responsible for covid-19, sars-cov-2, emerged in late 2019, in the region of wuhan, china. 2 sars-cov-2 belongs to the family coronaviridae, which is characterized by large, enveloped, positive-stranded rna viruses that can potentially cause gastrointestinal, nervous system, and respiratory distress. 3 this family also includes sars-cov and mers-cov, 4 which, despite several similarities, present some interesting differences from sars-cov2. 4, 5 sars-cov-2, mers-cov, and sars-cov use an envelope protein, termed the spike (s) protein, to gain access to host cells. 4, 6 the s protein displays the highest degree of genetic variability in the virus genome. 6 additionally, the sars-cov-2 s protein exhibits a greater affinity for the ace2 receptor with respect to analogs in sars-cov and mers-cov. 7, 8 the sars-cov-2 s protein shares a 96.2% sequence similarity with the bat sars-like ratg13 s protein, indicating the natural origin of the virus. 5, 9 due to its crucial role in the very first stage of viral infection, several studies 7,10−14 point to the s protein as an elective target to develop coronaviruses therapies and vaccines (the s protein is also the main target of neutralizing antibodies 15−17 ) . the s protein belongs to the viral fusion class i proteins, 7 has a trimeric, crown-like shape, protrudes from the virus envelope, and targets diverse host cell receptors in different species (see table 1 in ref 4) . both sars and sars-like (mers and sars-cov-2) coronaviruses target the angiotensin-convertingenzyme 2 (ace2) present in pneumocytes and enterocytes in the respiratory system and other susceptible cells (e.g., intestinal mucosal, renal tubular epithelial, cerebral neurons, and immune cells). 18 the s-ace2 interaction occurs at the very first stage of viral entry to initiate viral fusion with the host cell. 19, 20 the hallmark of sars-cov-2 is that its s protein is optimized for ace2 binding. 9 the s protein comprises two subunits, termed s1 and s2, which are cleaved during the first stage of viral infection. the s1 subunit is responsible for the interaction of the s protein with the host receptor (ace2), while s2 promotes viral fusion. 21 the receptor binding domain (rbd) resides between residues 318 and 513 22 in sars-cov-2 and 318 and 510 in sars-cov. 13 the development of therapeutics targeting the s-ace2 complex requires a comprehensive study of this complex interface and its associated dynamics. 23 thus, investigating the allosteric nature of the s-ace2 complex will help to understand and quantify the extent by which the s protein binds to ace2, and therefore the molecular basis of virus infectivity. the identification of allosteric modulation regions (amrs) allows for the design and testing of allosteric drugs (i.e., drugs targeting a different site from the complex interface) with enhanced bioactivity with respect to orthosteric drugs targeting the protein−protein interaction interface, 24 which are often tightly packed and scarcely accessible. the field of allosteric drugs is a rapidly developing field in drug discovery and may play a crucial role in the therapeutic strategy for sars-cov-2. 25−28 in this study, we adopted a multifaceted computational approach to identify promising "druggable" allosteric sites of viral s protein. we first applied the method of protein contact networks (pcns) to the s-ace2 complex in sars-cov-2 and sars-cov. in previous studies, we adopted this method to investigate both protein−protein interactions 29−31 and the allosteric nature of binding. 32, 33 in this work, the application of pcns clearly highlighted a region in the sars-cov and sars-cov-2 s protein, which acts as the putative locus for allosteric modulation (amr). amr was further investigated in terms of its relevance in the stability of the complex and ability to transmit modulating signals to the binding site by means of independent computational approaches. specifically, we adopted a monomer-based approach 34 which can predict the affinity of the s protein to its ligand. in this step, the ensembles for the ground state of the structures have been produced using the anisotropic network model (anm) approach. 35 moreover, we adopted an unsupervised blind procedure to predict possible interaction sites on s proteins and their affinities for tentative partners using a softness-based prediction of intersubunits affinity (sepas). in addition, the dynamical difference in rbd between the s proteins of the two virus strains suggested that they may have different binding and allosteric properties. we also provided biophysical evidence based on the elastic network modeling (enm) approach, combined with perturbation-response scanning (prs) 36 that amrs in both viruses acted as a mediator of intermolecular allostery between the s protein and ace2. "the three methods converged in allosteric character for residues in amr in both complexes. this allowed us to state that residues in amr for sars-cov-2 amr are more susceptible to allosteric drug targeting than for sars-cov. a recent study 37 suggested that the many residues in the amr may be one of the most efficient epitopes for antibody recognition. preliminary docking studies individuated some molecules that bind to amr (hepcidin), which were independently shown to exhibit strong sequence similarities with the amr. 38 the analyzed structures consist of the following: complex sarsspike glycoprotein-human ace2 complex (stabilized variant, all ace2-bound particles, pdb code 6cs2, 39 termed the sars-cov s/ace2 complex) and the sars-cov-2 analogous spike glycoprotein-human ace2 complex 40 . purposed software was used to transform the full structural information in the pdb files into a protein contact network (pcn): the network nodes are the amino acid residues represented by α-carbons. links between nodes (residues) exist if the mutual distance of the residues (centered on αcarbons) were in the range between 4 and 8 å, thus including only significant (<8 å) noncovalent (>4 å) bonds, while discarding "obliged" contacts due to proximity along the sequence. the adjacency matrix a mathematically represented the pcn in terms of undirected, unweighted network and is defined as the topological role of nodes (residues) addressed the functional role at the corresponding residues, based on the value of network descriptors. the method is widely discussed elsewhere. 41 the basic network descriptor was the node degree k i , defined as to detect allosteric sites and, more generally, the functional regions activating upon binding, we adopted a method based on network spectral clustering. 42, 43 spectral clustering was based on the laplacian matrix spectral decomposition, where the laplacian matrix l is defined as where d is the degree matrix (i.e., a diagonal matrix whose diagonal is the degree vector). the spectral clustering was based on the eigenvalue decomposition of laplacian l. the sign of the fiedler vector (the eigenvector corresponding to the second minor eigenvalue) was used for binary partition (each network was parted into two clusters, which in turn could be parted into four, and so on), as outlined in refs 43 and 44. we previously demonstrated that network clusters (group of residues) correspond to functional regions in the protein. 43 once network nodes (again, protein residues) were parted into the given number of clusters (an independent user-defined parameter for the clustering software), it was possible to define the participation coefficient p where k si is the degree of the ith residue computed in the cluster it belongs (i.e., accounting only for links with nodes pertaining to the same cluster). this network descriptor has been demonstrated to represent the functional role of residues in binding and stability. 33, 41, 43, 45 residues with a high p value are responsible for the communication between clusters (functional regions) and are thus addressed via the role in allosteric communication. 46 in this study, the participation coefficient maps were projected onto ribbon protein structures (as heat maps), to highlight activating hotspots in the spike-ace2 complex. participation coefficient maps are visualized by pymol (https://pymol.org/2/). we characterized the spike protein/ace2 interface by means of descriptors stemming from the structural information and the network analysis described above: • the interchain degree k i ic was defined as the node degree, but was only computed over contacts between nodes (residues) belonging to different chains the interface roughness q/r 29 was defined for each chain participating in an interface, q was the number of chain residues in the interface and r, the sequence range of chain residues in the interface. • the interface amino acid range 29 iar = r/n, where n was the total number of residues in the chain • the interface energy matrix, e, defined as elastic network models (enms) provide efficient methods for investigating the intrinsic dynamics and allosteric communication pathways in proteins. we adopted two elastic network models: (1) anisotropic network model (anm); and (2) gaussian network model (gnm). in anm, 47 the interaction potential for a protein of n residues was: in anm, the 3n × 3n hessian matrix, h, determined the systems dynamics. where x i , y i , and z i represented the cartesian components of the ith residue, v was the potential energy of the system. we selected r c = 13 å. anms provide information about the amplitudes, as well as the direction of residue fluctuations. the similarity between two anm modes, u k and v l , evaluated for proteins with two different conformations, can be quantified in terms of the inner product of their eigenvectors: gnm 36 was based on the description of the protein structure as a network of c α connected by springs of uniform force constant γ if they were located within cutoff distance r c . in gnm, the interaction potential for a protein of n residues was where r ij 0 and r ij were the equilibrium and instantaneous distance between residues i and j, and γ was the n × n kirchhoff matrix: thus, the square fluctuations were ( 3 ) the normal modes were extracted by eigenvalue decomposition of the matrix γ = uλu t , with u being the orthogonal matrix whose kth column u k was the kth mode eigenvector. λ was the diagonal matrix of eigenvalues λ k . the perturbation response scanning (prs) approach was based on the linear response theory and allowed evaluation of residue displacements in response to external forces. 48 our study performed a prs analysis based on gnm by constructing the inverse laplacian matrix, γ −1 . the n-dimensional vector δr of node displacements in response to the application of a perturbation (a n-dimensional force vector f) obeys hooke's law (f = h • δr). the purpose of prs was to exert a force of a given magnitude on the network, one residue at a time, and f for other residues not being perturbed, equal to zero. thus, the force exerted on residue i was expressed as 12) and the resulting response was δr (i) was an n-dimensional vector that described the deformation of all the residues in response to f (i) . a metric for the response of residue k was the magnitude ⟨∥δr k (i) ∥ 2 ⟩ of the kth block of δr (i) averaged over multiple f (i) , expressed as the ikth element of the n × n prs matrix, s prs . the elements of s prs referred to the unit (or uniform) perturbing force. the response to unit deformation at each perturbation site was obtained by dividing each row by its diagonal value i k j j j j j j j j j j j y { z z z z z z z z z z z the average effect of the perturbed effector site i on all other residues was computed by averaging over all sensors (receivers) residues j and can be expressed as ⟨(δr i ) 2 ⟩ sensor . the effector profile ⟨(δr i ) 2 ⟩ effector described the average effect that the local perturbation in the effector site i had on all other residues. the maxima along the effector and sensor profiles corresponded to the functional mobile residues that underwent allosteric structural change. because the crystallized structure presented one snapshot of the protein dynamic life at the bottom of a potential well, we created a trajectory of protein dynamics in its ground state by setting the rmsd cutoff to 5 å using the anm 36,47 approach implemented in prody. 49 to predict the most possible protein binding patches (pbps) on spike proteins, we utilized ispred to predict the interface residues. 50 the ispred-predicted interface residues acted as a seed to create a possible pbp based on the procedure as previously described. 34 the predictions of monomer-based affinities based on the mechanical softness of pbps were performed by utilizing sepas ver. 1. 34 we introduced ala mutations into the covid-spike using foldx suite. 51 we computed the general interface properties of the spike/ ace2 complex using pisa software, 52 figure 1b ). the chains are also shown to highlight which chains participate in the two clusters ( figure 1a ). the interface between the fusion peptide and the ace2 ectodomain was so strong (in terms of number of contacts between viral and host proteins) that the clustering partition algorithm recognized the fusion+ace2 region as a single cluster, even though it comprised sequences belonging to different chains. the map of the participation coefficient projected onto the ribbon structure of the sars-cov/ace2 complex ( figure 1c ) shows an active region (p > 0) in the junction between the fusion peptide and the trimeric bulk phase of the spike protein. active residues are shown more in greater detail in red in figure 1d . as previously demonstrated, the participation coefficient describes the attitude of residues toward participating in the intercommunication between clusters; thus, it was a putative score of the involvement of the residues in allosteric communication. therefore, this region was a good candidate to intervene in the allosteric regulation of complex formation and there was value in investigating allosteric drugs that target this portion of the protein. figure 2 refers to the analogous analysis for the sars-cov2 s/ace2 complex. a similar active region appeared at the junction of the fusion peptide to the body of the spike protein; however, the active region in the sars cov s/ace2 was more compact than that of sars-cov2 s/ace2. active regions in both complexes were characterized by two β-sheets and unfolded traits, and could thus be targeted to peptides due to flexibility of the region and absence of a pocket to bind small organic molecules. 53 table 1 reports the values of the participation coefficient p in residues with p > 0, all of which were located in the chain carrying the fusion peptides of the sars-cov s/ace2 (chain b, in cyan in figure 1a ) and sars-cov2 s/ace2 (chain c, in magenta in figure 1a) complexes. the sars cov s/ace2 and sars-cov2 s/ace2 complexes accounted for 21 and 22 active residues, respectively. the average value was similar for the two distributions (0.47 for sars cov s/ace2, 0.48 for sars-cov2 s/ace2). however, the absolute values of higher p residues (>0.6) were higher for covid19 s/ace2, indicative of a corresponding larger reactivity of the active region. characterization of the s-ace2 interface table 2 reports the properties of interface s/ace2 in the two complexes. the interface area and the absolute value of energy of the sars cov s/ace2 complex were higher than the corresponding area for the sars-cov-2 s/ace2 complex; however, the specific values for the unit area and single residues were higher for sars-cov-2 s/ace2, indicating a more efficient yet less stable interface. this implied an optimized complex formation with the ace2 of sars-cov-2, as observed in ref 9 . this helped to explain the more rapid kinetics of infection, mediated by a faster complex formation with main receptor, ace2. the rbds for the two proteins are reported as a wide peptide, which comprised approximately 200 residues for both structures; however, a more limited core region, termed the receptor binding motif (rbm), was claimed to actively participate in the complex formation of the spike protein with its receptor, ace2. we independently identified the residues in the spike protein for sars-cov and sars-cov2 using the method of pcns. the position and the value of the interchain degree are reported in table 3 for both complexes. the two residues in the two structures with the highest value of interface degree were 488g in sars-cov and 502g in sars-cov-2, respectively. these two residues could be considered the hotspots of the ppi interface in the s/ace2 complex; both are glycine residues, likely due to the high steric adaptability of the small glycine residues in the most tightly packed region of the s-ace2 interface. this confirmed the high density of the interface and its extremely scarce accessibility to small molecules targeting the region, which also suggested the need to interfere with the s/ ace2 interface through allosteric regulation. in the previous sections, the amrs of the s/ace2 complexes have been identified for sars-cov-2 and sars-cov using the pcns approach ( table 1 ). the amr is accessible for binding, which is at odds with the pbd. one critical property of the protein complexes was the intersubunit affinity. therefore, besides the s/ace2 stability, we predicted the affinity between the amr and incomingdesigned molecule (peptide molecules), which will be designed to affect the function of amr. this was a crucial step that will provide an independent proofof-concept of both the allosteric features of amr (by definition, an allosteric site must "sense" the microenvironment) and the druggability of the predicted amr. we reviewed the need for complete 3d knowledge of the s/ ace2 complex by means of a recently introduced method to predict the intersubunit affinity of protein complexes using protein binding patch (pbp) on a single subunit by computing the pbp mechanical softness. 34 to alleviate the static presentation of the protein structure in a crystallized state, we created ensembles of protein complexes in their ground state using the anm approach. in the first step, we predicted the affinity of the sars-cov-2 and sars-cov s proteins for ace2. we compared the predicted affinity between the s protein and ace2 when the s protein was in its monomeric or trimeric state to obtain some hints about the effect of adjacent subunits on the affinity between spike and its ligand (figure 3 ). subunits (chains) are denoted as sa, sb, and sc. to manage the potential states of the spike protein, we propose different forms of pbps on the s protein: • the sa subunit is alone and waiting for its partner (left column, figure 3 ). we suppose that sa may interact with ace2 using pbps similar to those in the ensemble of s/ ace2 in its dimeric state (the first row, figure 3 ) or by using pbps similar to ones sa displays in the trimer spike and ace2 ensemble (second row, figure 3 ) • subunit sa finds ace2 and binds it directly (right column, figure 3 ). sa binds to ace2 via the pbps that it displays, either when no sb subunit is available (the first row, figure 3 ) or in association with sb monomers (second row, figure 3 ). our computations suggested that sars-cov-2 sa (dark blue curves, figure 3 ) was more sensitive to the presence of the sb subunit than sars-cov sa (red curves, figure 3 ). this result suggested approaches that potentially disrupted the sa-sb association may provide an efficient therapeutic route. we observed a higher affinity between s and ace2 in sars-cov-2 in sars-cov. this observation may explain the higher infectivity of sars-cov-2. we are interested in predicting the affinities between possible pbps on spike proteins and tentative protein partners. in this case, we did not know the pbps on spike proteins because we did not have the resolved 3d structures of all possible protein complexes between the spike protein and other natural or synthesized partners. we predicted the seed interface residues on the spike surface using the ispred approach. 50 by expanding the selection radius for defining the pbp region, 34 many possible pbps were generated for both sars-cov-2 and sars-cov spike proteins. using sepas, the affinities between the generated pbps and unknown partners were predicted. the affinities averaged over the whole are presented in figure 4a and b (for sars-cov-2 and sars-cov spikes, respectively); the average affinities mapped on the surface of the spike proteins are reported in supporting information figures 1 and 2 . some of the predicted regions with an affinity < −5 kcal/mol resided in the interface region of the spike subunits. there were some residues in the proposed pbps that belonged to amr (indicated as red triangles in figure 4 ). this meant that amr could act as a distinct pbp for some partners, suggesting that targeting amr may be a good choice for drug design. thus, in considering amr as distinct pbp, we were able to 1. predict the affinities between amr and unknown druglike protein partners, simply by using a 3d structure of amr on the spike subunit in different ensembles (monomer, case "c" in figure 5a ) 2. account for the effect of ace2 binding on amr affinity for its possible partners. we assumed the spike monomer in association with ace2 then predicted the affinity between amr and its possible partners (dimer, case "dc" in figure 5a ) 3. address the effect of the sb spike chains on the affinity of amr in the sa spike chain for its partners, feeding sepas with the trimeric state of spikes (trimer, case "abc" in figure 5a ) 4. analyze the condition of a trimer spike and ace2 in terms of a holo-complex (s/ace2 complex, case "abcd" in figure 5a ) these results indicated that the amr of sars-cov-2 spike exhibited a higher affinity for generic peptides than the amr of sars-cov spike. interestingly, the affinity of amr for their partners was increased upon binding to ace2, which suggested an interplay between the two distant regions, the ace2-binding site of spike and amr. this last result could be considered to be another proof-of-concept of the allosteric character of the amr. . predicted affinities of spikes to ace2 are presented. the affinity of sars-cov 2 spike (blue curve) and sars-cov spike (red curve) to ace2 is predicted using sars-cov 2/sars_x_c/b_y summarized the state of spike proteins in affinity prediction: x denotes number of chain in predictions. x = 1 means s1 protein in monomer form is considered, x = 2 means s1 + ace2 complex, and x = 4 means whole spike complex + ace2. y notes the considered pbp in computations: y = 2 means the pbp's residues of s1 in s1-ace2 complex is used to predict the affinity and y = 4 means the pbp's residues of s1 in whole spike proteins-ace2 complex is used to predict the affinity between s1 and ace2. horizontal axis presents predicted affinity (kcal/mol). normalized density denotes in y-axis. journal of proteome research pubs.acs.org/jpr article to further investigate the effect of the induced structural perturbation in amr on the binding affinity of the spike protein to ace2, we introduced two sets of ala mutations in amr. in one set, the amr residues that play a major role in allosteric modulation (p > 0.5 in table 1 ) were mutated to ala, and in the other set, all residues of amr on sars-cov-2 sa were mutated to ala. to simulate an sa perturbation, we used the ensemble of structures generated using the anm approach after setting the rmsd cutoff to 8 å for selecting normal mode states. such global perturbation only distorts the ace2-binding site of sa to less than 1.5 å. in figure 5b , we reported that wild-type sa exhibits a shoulder in its affinity density curve at a high affinity region. our observations suggested that the structural perturbation of amr due to the ala mutation of native residues decreased the affinity of sa for ace2. this was further confirmation of the allosteric properties of the amr. in order to explore amr druggability, we started by recognizing the strict structural similarity of the amr region with hepcidin, a crucial protein for iron regulation. moreover, a recent preprint reported a distant (albeit relevant) sequence similarity between hepcidin and the sars-cov-2 spike protein. 38 it has been established that there was some proof of the similarity between the hydrophobicity spectra of ligand− receptor pairs, which fostered the observed sequence similarity between hepcidin and the sars-cov-2 s-protein. 55, 56 moreover, hepcidin is strictly involved in interleukin 6 (il-6) regulation, 57 given that il-6 is the main actor associated with the "cytokine storm" that is linked to fatalities provoked by sars-cov-2 infection. 58 a preliminary docking analysis showed a perfect fit between hepcidin and the amr region; however, this now must only be considered as a first step with which to prioritize different peptides as allosteric effectors. in addition to the characterization of the s/ace2 interface, a comparison of rbd dynamics between sars-cov and sars-cov-2 was also important to understand the functional mechanism of the s/ace2 interaction. to this aim, the overlapping values between the slowest anm modes (eigenvectors) were calculated to compare the intrinsic dynamics of rbds in both s proteins (eq 8 in materials and methods). in figure 6a , the dot products (overlap values) were plotted in the map for the rbds in sars-cov versus sars-cov-2 for the first 10 anm modes. in this map, the upper limit of 1 indicated a perfect overlap (red) correspondent to a perfectly shared dynamics, while 0 indicated no overlap (blue), figure 4 . affinity of possible pbps on the s1 subunit of the spike to tentative proteins are predicted. possible pbps are defined for the s1 subunit. the affinity of the pbps on sars-cov2 (a) and sars-cov (b) s1 protein for their possible partners is presented. left vertical axis points to the averaged predicted affinity (kcal/mol) of the proposed pbps. right vertical axis denotes the p metric (table 1 ) of the amr participat residues. x-axis notes the residue of the pbp's central residue. red triangles represent the position of amr residues on the s1 protein sequence. figure 5 . affinity of amr for tentative protein partners and the effect of amr perturbation on ace2 affinity are presented. (a) affinity (kcal/ mol) of amr to possible partners are predicted when s1 is a monomer (state c), s1 is associated with ace2 (stade dc), s1 is accompanied by two s2 subunits (state abc), and when the whole spike binds to ace2 (state abcd). (b) affinity of s1 for ace2 is predicted when amr is intact (wt), residues of amr with p > 0.5 are mutated to ala (p > 0.5), or all amr residues are mutated to ala (p > 0). the affected region of affinity density curve is marked with vertical gray arrow. x-axis notes predicted affinity (kcal/mol) and vertical axis presents normalized density of affinities. indicating that they showed independent dynamics. although rbds in the two complexes showed very high structural similarity with an rmsd of 0.68 å, the overlap between the first 10 anm modes of two rbds exhibited some differences. overlap values with ∼0.6 for the first three modes indicated a weaker dynamical overlap, even at the lowest frequencies, which were those endowed with most important functional meaning. to further understand the relative motions and dynamics of the interface structures, the anm motions of the first mode for two rbds are shown in figure 6b and c. in anm 1, most of the interfacial residues were very stable (blue), while the peripheral loop exhibited the highest fluctuation (red). the most relevant dynamical difference between the two systems referred to this loop motion. for sars-cov rbd, the highest fluctuations point to residues 463d and 465 k, while the sars-cov-2 rbd displayed higher flexibilities at 476g to 479p (orange beads). this loop was found to be located at the c terminal of the α1 helix of the rbd, interacting with ace2 through van der waals forces, hereinafter referred to as an "interfacial c loop". we suppose that the difference in the dynamics of such an interfacial c loop may be caused by the amino acid mutations in their respective interfaces with ace2 (i.e., 442y to 455l, 443l to f, 460f to 473y, and 479n to 493q) from sars-cov-rbd to sars-cov-2-rbd. 59 thus, we further mapped these interfacial mutations onto the structures (yellow beads). thus, the lower fluctuations of these mutations in sars-cov-2-rbd may account for the higher affinity of such interface. accordingly, despite the overall structural similarity, rbds in the two s proteins showed different dynamics, especially for the s-ace2 interfaces. these differences in dynamics may also indicate that these two s-ace2 interfaces achieved their activities, including substrate binding and allosteric regulation. we performed perturbation response scanning (prs) based on gnm to quantify the allosteric properties of amrs and identify the key residues for which perturbation provokes structural dynamical changes at a long distance. first, the prs map provided information regarding the sensitivity and effectiveness of a given residue in transmitting signals. the column of the matrix corresponding to a single residue (e.g., for r313v) described how well-connected that residue was within the amr region and its likely relevance in transmitting allosteric signals to distal parts of the s-ace2 complex. here, we adapted this method to probe the allosteric response of the amr region upon perturbation on residues with p > 0.5 from the pcn prediction (table 1 ). for the s/ace2 complex in sars-cov, the dynamical changes of the other residues in amr (red arrows in figure 7a ) have been caught due to their close distance in space. in addition, all amr residues have allosteric effects on the interfacial c loop with high fluctuation. because the effectiveness of the three residues with p > 0.5 was almost identical, we only reported in figure 7a for the case of the perturbation of 568s that had the highest p value (0.64). it is appreciated that three residues (313v, 529n, and 568s) displayed long-range allosteric effects on some particular regions in ace2 through the s/ace interface. the residues relative to the amr in sars-cov-2 could be classified into three types according to their allosteric character. the first class included most residues, including 320q, 321p, 322t, 323e, 539n, 548t, and 578p. upon perturbation, these residues exerted a moderate dynamical effect on other amr residues, and a relatively weak but sensible intramolecular allosteric effect on residues of the s/ace2 interface, especially on the highly flexible c loop. the perturbation of these amr residues largely affected the dynamics of ace2 through stronger long-range allosteric communications. the perturbation of the second type of (531n and 532l) residues provokes a large intermolecular allosteric effect in the rbd, including some peaks at the interfacial c loop (figure 7c ). in addition, the perturbation weakened the intermolecular communications from amr to ace2. the last type only included 580t, which exhibited similar dynamical effects on three regions (amr, rbd, and ace2) via long-range communications. accordingly, our prs analysis provided important insight into amr allostery. among other findings, 3 residues in sars-cov amr and 10 residues in sars-cov-2 amr were predicted to have wide-ranging allosteric effects on the ace2 protein. this result in conjunction with the statistically significant higher effectiveness of the sars-cov-2 amr perturbation (si figure 3) , suggested that its allosteric character was stronger than that of amr in sars-cov. notably, in both complexes, the interfacial c loop formed a bridge to transmit the signal from amr to ace2. the reported rbd-ace2 interfacial mutations from sars-cov to sars-cov-2 were close to the interfacial c loop. therefore, we propose that the different allosteric properties of amr residues were due to mutations in the s-ace2 interface. in figure s3 , the box plot shows that amr in sars-cov-2 had a statistically higher effectiveness than that of sars-cov (under the p-value of 7.4 × 10 −5 by wilcoxon signed-ranked test). the application of pcns, sepas (affinity by flexibility) and prs based on elastic network modes to two complexes of the spike protein with its receptor, ace2, generated highly consistent results. the integrated computational approach disclosed active zones in the sars-cov and sars-cov2 complexes with the ace2 ectodomain. these active zones, as demonstrated in previous studies 41, 60, 61 and in the present research by the convergence among different approaches, have an extremely high probability of acting as allosteric sites, which can modulate spike/ace2 protein binding. these regions are worthy of further investigation from the perspective of drug or vaccine development as indicated by a preliminary docking simulation. from a methodological perspective, our results highlighted the need to identify convergence of different simulation methods arising from independent theoretical premises, in order to obtain a reliable picture of the associated biomolecular systems. the supporting information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jproteome.0c00273. figure s1 : affinity for the sars-cov s/ace2 complex. figure s2 : affinity for the sars-cov2 s/ace2 complex. figure s3 : wilcox test for effectiveness (pdf) coronavirus disease covid-2019 (in russ.). safety 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recognition of the sars-cov-2 by full-length human ace2 characterization of protein-protein interfaces through a protein contact network approach the authors declare no competing financial interest. the authors thank the encouraging support and the fruitful discussion from prof. c. serangeli. the project was supported by national natural science foundation of china (31872723) and the priority academic program development (papd) of jiangsu higher education institutions. key: cord-286970-4pl95r0o authors: mamipour, mina; yousefi, mohammadreza; hasanzadeh, mohammad title: an overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding date: 2017-04-12 journal: int j biol macromol doi: 10.1016/j.ijbiomac.2017.04.025 sha: doc_id: 286970 cord_uid: 4pl95r0o the majority of research topics declared that most of the recombinant proteins have been expressed by escherichia coli in basic investigations. but the majority of high expressed proteins formed as inactive recombinant proteins that are called inclusion body. to overcome this problem, several methods have been used including suitable promoter, environmental factors, ladder tag to secretion of proteins into the periplasm, gene protein optimization, chemical chaperones and molecular chaperones sets. co-expression of the interest protein with molecular chaperones is one of the common methods the chaperones are a group of proteins, which are involved in making correct folding of recombinant proteins. chaperones are divided two groups including; cytoplasmic and periplasmic chaperones. moreover, periplasmic chaperones and proteases can be manipulated to increase the yields of secreted proteins. in this article, we attempted to review cytoplasmic chaperones such as hsp families and periplasmic chaperones including; generic chaperones, specialized chaperones, ppiases, and proteins involved in disulfide bond formation. stanley cohen and herbert boyer were applied cloning and expression methods to produce recombinant proteins in different organisms [1] . at the beginning of the recombinant protein expression systems, escherichia coli (e. coli) is a remarkable hosts due to its rapid growth rate, requirement for inexpensive carbon sources, low costs and well-characterized genetic structure [2, 3] . however, e. coli has some defects such as inability for posttranslational modifications, ineffective cleavage of the amino terminal methionine, inability to produce proteins containing complex disulphide bonds, and expression of proteins as insoluble inclusion bodies (ibs) (ibs) [4, 5] . ibs, are inactive form of proteins, which are formed in both prokaryotic and eukaryotic cells that have been miss folded and are not in proper intra-molecular interaction [6] . the natural ibs in some bacteria is like ␤-lactamase which is a secretion protein [7] . the reasons of ibs formation in protein expression are amount and level of their expression that high level expression of recombinant protein led to ibs formation [8] . the microscopic analysis showed that the size and shape of ibs were different in cytoplasmic and periplasmic spaces [8] . ibs have been cylindrical or ovoid forms [9] and diameter of the shapes changed from 0.5 to 1.3 m [10] . moreover, ibs are highly hydrated and have spongy structures that can be simply separated by high speed centrifugation [11, 12] . the studies showed that ibs have dynamic, reversible, and kinetic structure that is consequent of unbalance between soluble and insoluble proteins [9, 13] . also, shifting of ib proteins in to active forms in in vitro is really hard and need to several approaches containing isolation, solubilisation, and refolding of ibs, which has low efficiency [12, 14] . the solubilisation of ibs are mostly dependent on denaturant agent such as urea, hcl, and guanidine hydrochloride which stimulate disruption of intramolecular interactions [13] . in production of recombinant proteins, several procedures have been used for soluble expression and preventing of ib formation such as using molecular chaperones, low temperature, suitable promoter, ladder tag to secretion of proteins into the periplasm, gene protein optimization and chemical chaperones [5, 15] . cells normally have molecular chaperones and other factors to correct folding of proteins [16] . numerous studies demonstrate positive effects of molecular chaperons on correct folding formation of recombinant protein and prevention of ibs formation in the cytoplasm and periplasm [17, 18] . also molecular chaperons have clinical and experimental application such as immunomodulatory [19] , treating neurodegenerative diseases [20] , and morbidity of chronic illnesses [20, 21] . in this review we focused on cytoplasmic and periplasmic chaperones category and their applications in recombinant protein expression with correct folding. molecular chaperones are proteins existing in bacteria and eukaryotic cells which have an important roles to assist cellular homeostasis under normal and detrimental growth situation and cell defences to inhibit aggregation of mis-folded proteins [22, 23] . various genetic sources have molecular chaperones because these agents have critical roles in cell such as prevention of accumulation and mis-folding protein, and also, help to preparing correct folding of protein [24, 25] . chaperones do not have steric information of target protein structure to determine exact folding. so, they prevent improper interactions within and between other polypeptides by enhancing the yield of correct folded proteins. however, they don't increase the amount of folding reactions. unfolded proteins have hydrophobic residues, which are abnormally exposed to the cell solution that own to form stable inactive accumulations of these proteins. molecular chaperones are able to bind to hydrophobic residues of unfolded proteins and assist to create the correct folding of protein [22, 26] . molecular chaperones exist in the cytoplasm and organelles of eukaryotic cells like nucleus, mitochondria, endoplasmic reticulum, chloroplast, and periplasmic space of prokaryotic cells [27] . molecular chaperones are divided in cytoplasmic and periplasmic categories that have been reviewed (table 1) . most of cytoplasmic chaperones are mainly expressed under stress conditions such as high temperature [28] . significantly, the chaperones bind to misfolded proteins to unfold them and in turn substrates are released from the chaperones to provide a new chance for unfolded protein to gain correct folding [16] . these chaperones can be categorized into five families based on their molecular weight including heat shock protein100 (hsp100), hsp90, hsp70, hsp60, and small hsps (shsps) that have molecular weight between 12 and 43 kd [19] . some type of cytoplasmic chaperones have an atp-dependant mode of substrates interactions (hsp100, hsp90, hsp70, hsp60) to have functional system, but in contrast, majority of shsps don't need atp consumption [29] . among them, hsp90 and hsp70 are the main chaperones responsible for protein holding to stop association of target protein with another protein [30] . cytoplasmic chaperones based on their function and mechanisms are divided to three sub-classes including disaggregate chaperones that enhanced the solubilisation of stressinduced accumulated proteins [31] , holding chaperones that keep and hold the folded proteins on their surface [32, 33] , and folding chaperones that interfere the refolding of protein and enhance the proteolysis of mis-folded proteins [34, 35] . in this section, we focused on the cytoplasmic chaperones based on their molecular weights. hsp100 or (clp) family are hsps with molecular weight of 100-104 kda, which give the organisms tolerate excessive stress and have variety of proteolysis functions [36] . also, hsp100 is responsible for protein disaggregation and degradation that lead to omitting non-functional and harmful polypeptides that is vital for the maintenance of cellular homeostasis [37] . unlike the other hsps, hsp100 family solubilize accumulated proteins, thermally [38] . at first the members of this family were recognized as components of the two subunit bacterial clp protease system, which contains regulatory atpase/chaperones such as (clpa and clpx) and proteolytic (clpp) subunits [39] . then this family separated to two classes and eight individual subfamilies within this classes [40] . furthermore, hsp100 family cooperate with hsp70, which is another atp-dependant chaperone system for saving proteins from aggregation. the hsp70 system gets the solubilized proteins from hsp100 and refolds them [41] . the hsp 100 is a member of aaa + superfamily (atpases associated with various cellular activities) proteins, which get their energy from hydrolysis of atp to stimulate creation of correct folding in target proteins [42] . all aaa + superfamily members have aaa domain which have two motifs for nucleotide binding and hydrolysis. moreover, the aaa domain is responsible for protein oligomerization that lead to made of hexameric structures with a central pore [43] . hsp100 chaperones are differ from each other based on the number of aaa domains which are one or two in each protomer and the existence of extra domains [43] . the hsp90 family are highly conserved and proteins, which exist in all organisms from bacteria to humans and their expressions rises in response to the stress conditions in prokaryotic and eukaryotic lola (b,c,d,e) periplasm space, inner membrane and outer membrane rapid transfer of associated lipoproteins atp (+) [108] papd and its family periplasm space and outer membrane biogenesis of pilus (−) [163] fimc periplasm space interacts with each pilus subunit (−) [164] cells. this family is often found with other chaperones that they have various functions depend on atp [44] . in human, two homologous isoforms of hsp90 are exist in equal amounts known as ␣ and ␤ isoforms [45] . hsp90 is a constitutive homodimer, which has main inter subunit in association with the cooh-terminal 190 residues [46] , the highly conserved 25 kda nh 2 -terminal domain that is the atp binding site [47] and middle domain, which is binding site of proteins and co-chaperones [48, 49] . most of the hsp90 substrates are signal transduction proteins such as steroid hormone receptors and signalling kinases [50] . forasmuch as, cancer cells are so sensitive to hsp90 inhibition, the hsp90 active form in these cells are in high level [51, 52] . therefore, hsp90 molecule is an attractive target for cancer targeted therapy [53] . the most important function of hsp90 is to manage protein folding and inhibit protein aggregation [54] . furthermore, this chaperone has roles in signaltransduction networks, cell-cycle control, protein degradation and protein trafficking [55] . despite hsp90, which are the most numerous chaperones in the cell but its functions in in-vitro is poorly understood and it has been proved that have functions with contribution of several cofactors [44] . the hsp70 family are highly protected class of the heat shock proteins and the most of the organisms contain several members of them. this family have some function in the cells including; binding strongly to partially synthesized peptide sequences, keeping translocation competent structures of er and mitochondrial precursors in the cytosol, and simplify translocation from inside the target compartment [56] . actually, the particular roles of hsp70 proteins are confirmed by their location in diverse subcellular sections [54] , the differential expression of hsp70 at dissimilar phases of growth [57] and their communication with specific groups of hsp70-associated proteins [58] . hsp70 family consist of two domains; the 25kda cooh-terminal domain, which is needed for polypeptide binding and the 44kda n-terminal highly protected atpase domain, which is binds to adp and atp in the existence of k + and mg 2+ that lead to hydrolyse atp. correct folding of protein is taken by cooperation of both domains [59, 60] . the atpase domain had similar structure to actin and hexokinase which contain four sub-domains [61] . hsp70 obtain two conformational conditions based on the binding to atp or adp [62] . the atp-bound form in comparison to adp-bound form showed high association and dissociation rate constants for substrates. attachment of substrate to hsp70-atp is hastening atp hydrolysis by hsp70 which lead to change in folding of substrate, after that adp replaced with atp result in releasing of substrate from hsp70 [63] . hsp70 known as dnak that expressed in prokaryotes which participate with dnaj and grpe proteins in reaction cycle of dnak [64] . in addition grpe assists in the releasing of the bound nucleotide from hsp70 and dnaj motivates atp hydrolysis by hsp70. furthermore, dnaj itself acts as a molecular chaperone by binding to unfolded proteins and probably control affinity of hsp70 for substrate proteins [65] . this family has several names such as groel family or tcp-1. most of these family members are known as chaperonins. x-ray crystallographic studies showed that groel (e. coli chaperone) has 14 identical subunits in two stacked heptameric rings, each containing central hole which bind to unfolded proteins [66] . groel simplifies protein folding by cooperation atp and its co-chaperonin known as groes [67] . groes has a heptameric ring structure that was made by identical 10 kda subunits that attached to one or both ends of groel cylinder [68] . groel chaperone have three different functions: first, groel form complexes with non-native polypeptides and inhibits their aggregations, so it will reduce the concentration of misfolding protein [69] . second function is folding substrate that bind to central cavity of groel complex, a protected environment without intramolecular interactions [70] . finally, groel seems to be capable to unfold trapped misfolding intermediates, so they are able to have a new chance for correct folding [71] . members of the hsp60 family are involved in construction of multiprotein complexes such as rubisco [72] . furthermore, sonoda et al. reported that co-expression of groel/groes with scfv lead to improve soluble expression that the resultant scfv demonstrated a 4.6 fold rise in antigen-binding activity [73] . also moeng et al. expressed five set of molecular chaperones with anti-bnp scfv. they reported that co-expression of groel/groes with anti-bnp scfv showed more effective than other set of chaperones in soluble expression of the scfv [74] . in prokaryotic and eukaryotic cells, majority of the small hsps (shsps) are constructed in stress conditions and some small hsps are expressed throughout certain developmental phases [27] . in stress conditions the small hsps bind to the denatured proteins that finally led to prevent its accumulation in an atp-independent manner [75, 76] . small hsps are in relation with nucleus, cytoskeleton, and membranes. also, this family consist of 12-43 kda chaperons that they bind to large multimeric structures [77] . the shsps endure dynamic assembly into mono-and poly-disperse oligomers that the rate of disassembly influence chaperoning [78] . the structure of the shsps include a conserved ␣-crystallin domain, which is enriched in ␤ strands formed in ␤ sheet that is in charge of dimer formation [79] . most of the shsps oligomerization and chaperoning are affected by the amino-terminus because the amino terminal domain is sensitive to phosphorylation and has slack structure [80, 81] . moreover, shsps have an important role in refolding by cooperation of atp-dependant chaperones such as dnak system [82] . the space between cytoplasmic and outer membrane of the gram-negative bacteria is called periplasmic space, which has total volume of 20-40% of the whole cell. it is an aqueous space that has numerous molecules such as membrane derived oligosaccharides, amino acids, peptides, and etc. [83] . the proteins in the periplasm are classified to three classes based on their function: enzymes, chaperones, and high affinity binding proteins for vitally important substrates which are essential for the transport across the cytoplasmic membrane [84] . the periplasm is an oxidizing region of bacterial cells that disulfide bonds formed in protein folding process. thus, many secreted proteins containing disulfide bonds are in the periplasmic space [85] . protein folding in the periplasmic space can be catalysed by two groups of overexpressing enzymes including protein disulfide isomerases (pdi), which accelerated the oxidation of disulfide bonds and peptidylprolyl-cis-trans isomerase (ppi) that are member of periplasmic chaperones [86] . periplasmic chaperones are divided to four classes including generic chaperones, specialized chaperones, ppiases, and proteins involved in disulfide bond formation. the members of generic chaperones are homodimers or homotrimer, which hold unfolded proteins and help to its correct folding formation [87] . in result, they have application to periplasmic expression and phage display of recombinant protein [88] . these chaperones can be active in presence and absent of atp condition in periplasmic space, and have the most generic folding activity [89, 90] . generic chaperones are including; skp and fkpa. skp is an 18 kda periplasmic protein that has significant role in folding and gathering of outer membrane proteins (omp) of e. coli, which it means that skp binds to denatured omps [91] . skp simplifies proper folding of expressed proteins that increased the solubility and affinity of proteins [92] . for instance, ow et al. expressed skp and scfv to achieve high soluble expression. in result, they observed high scfv soluble expression [93] . moreover, ute schafer et al. demonstrated that skp is involved in making and keeping the solubility of early folding intermediates of outer membrane proteins in the periplasmic space of gram negative bacteria [87] . fkpa is v-shaped homodimeric chaperone with two domains which exhibits like pplase activity [16] . the n-terminal domain, which has ␣-helixes structure and responsible for dimerization and activity of fkpa [94] . however, the c-terminal domain is capable to catalyse slow refolding cis-trans isomerization reactions in proline containing proteins [95] . in addition, zhang et al. evaluated the co-expression of fkpa with scfv, which the results demonstrated that co-expression of fkp was suitable for soluble expression of recombinant antibodies in e. coli [96] . furthermore, bothmann and plückthun showed that interacting fkpa with early folding intermediates inhibit their aggregation. also fkpa binds to partially unfolded proteins and reactivate them [95] . ow et al. demonstrated that the chaperone activity of fkpa, rather than its pplase activity. this activity is more critical in overcoming metabolic stresses from misfolded proteins [93] . the members of specialized chaperones have two ppiase domains, which have chaperone activity to prepare the maturation of three outer membrane proteins including ompa, ompf, lamb [97] . also, they stimulated folding of unstable proteins and pre-aggregated proteins. specialized chaperones include sura, lola, papd and fimc. sura is one of the vital proteins that is required to cell survival during stationary phase [98] . the sura has four domains consist of n-terminal domain that has approximately 150 amino acid, two parvulin-like pplase domains that have approximately 100 residues and a c-terminal domain that has approximately 40 amino acid [99] . three of these domains formed core shape of the complex protein and the second pplase domain has exhibit chaperone function [100] . sura is one of the first chaperones for omp folding that detects ar-x-ar motif in unfolded omps [101] . also, hennecke et al. reported two characteristics of sura that are essential for peptide binding such as particular pattern of aromatic residues and the direction of their side chains, which are founded mostly in integral outer membrane proteins. in addition, sura-peptide binding needs neither an active pplase domain nor the existence of proline, in result this data demonstrate the surapeptide binding depends on substrate specificity [102] . bitto and mckay realized that sura detects a peptide motif which is features of integral outer membrane proteins [103] . lol chaperon family consist of five members that by cooperation of atp-binding transporter lolcde complex, lola attached to the bacterial lipoproteins, then the lola-lipoproteins complex released from inner membrane to transfer of lipoproteins to lolb in the outer membrane [104, 105] . the stage of attaching lola to lipoproteins just needs atp [106] . lola and lolb have a similar structures and have function in monomeric form despite different sequences (fig. 1a) [106] . tajime et al. indicated disruption of the chromosomal lola gene was mortal for the strains without lipoproteins which lola is generally vital for the outer membrane localization of lipoproteins [107] . okuda and tokuda revealed that shifting lipoproteins via lol proteins happens in a mouth to mouth manner, which means their hydrophobic holes were found to bind lipoproteins inside [108] . papd is a 28.5 kda periplasmic protein from a family with 30 members, which has two domains with an immunoglobulin (ig) like structure [109] . three important performance of papd in bacterial cell are attaching and covering pilus subunits to avoid non-specific interactions [110, 111] , simplifying transfer of these subunits in the periplasm [112] and assistance to the folding of subunit [111] . as it is represented in the legend to (fig. 1b) periplasmic chaperones help in folding pilus subunits and attacking them to the om usher. at first, transferring fiber subunits to the periplasmic space done by sec system (yeg). then the chaperones in the periplasm has function with stop aggregation and degradation of pilus subunits by binding their interfacing regions and facilitating correct protein folding before bringing them to the usher/fimbriae complex. jacob-dubuisson et al. show that papd is responsible to made of native-like conformation in pilus subunits [113] . fimc is one of the periplasmic proteins has similar structure, function and the mechanism of action to papd. pilus chaperones like fimc identify specific carboxyl terminal motifs on pilus subunits and creating chaperonesubunit complexes [114] . also, jones et al. indicated that fimc has a high degree of homology to papd and it is in accordance with sequence of periplasmic chaperones [115] . peptidyl-prolyl isomerases (ppls) are housekeeping proteins and highly conserved in prokaryotic and eukaryotic cells. they exist in the periplasm of e. coli and in eukaryotic cell organelles such as cytosol, mitochondria, endoplasmic reticulum [116, 117] . the pplases are enzymes that facilitate the slow cis-trans isomerization of peptidyl-prolyl bonds in folding of target proteins (fig. 2) [118] . the first discoveries showed that immunosuppressant drugs attach to ppls and prevent the ppl activity, so ppls are necessary for immune system function [119] . in immune system they act by separating calcineurin, which is a calcium/calmodulindependent protein phosphatase. pplases are divided to three families based on drug specificity and primary sequence homology: 1) the cyclosporine a (csa)-binding proteins and cyclophillins, 2) rapamycin binding proteins, binding proteins fk506 and fkbps 3) parvulins, which do not attach immunosuppressant drugs [120, 121] . the sura, ppid, fkpa, and ppia are the members of pplase family. for example, juctice et al. show that the four known periplasmic cis-trans prolyl isomerases simplify suitable protein folding by raising the rate of transferring of proline residues between cis-trans state. also, pplases are not necessary for cell growth but they have great roles in survival in environmental and pathogenic positions [122] . sura, as it is mentioned above is specialized for the biogenesis of omps and also it has a peptidyl-prolyl cis-trans isomerise activity [99, 100] . ppid have a single parvulin domain and attached to the inner membrane by an n-terminal transmembrane section. the study showed that n-terminal section of ppid and sura have similar structure and have overlapping role in omp biogenesis [123] [124] [125] . loss of ppid in the cells own to modified outer membrane configuration which has negative effects on protein folding process [84, 123] . also, findings demonstrated that ppid has chapronic function to help in the early periplasmic folding of proteins [124] . dartigalongue and raina showed that a null mutation in ppid own to completely decreasing in the level and folding of omps and to the stimulation of the periplasmic stress response. null mutation in ppid and sura led to death of cells [123] . ppia has two functions including peptidyl-prolyl cis-trans isomerase function and chaperonic function. ppia is vital in viral infections like hiv, influenza a virus, sars coronavirus, vaccinia virus which it might have a role in life cycle of viruses [126, 127] . tremillon et al. indicated that ppia gene exists in lactococcus lactis which expressed under normal and stress conditions. in normal conditions, ppia protein expressed and released from cell with added protease then exposed at the cell surface. a recombinant soluble form of ppia was produced and secreted from l.lactis that showed both pplase and chaperone activities [128] . these chaperones known as disulphide bonds proteins (dsb proteins) have highly conserved motifs which are necessary for disulphide oxido-reductase activity. in numerous secreted proteins, disulphide bonds formation is essential for their folding and stability because disulphide bonds generally stabilized the tertiary structure of the protein. the dsb proteins reorganized primary disulphide bonds and made new disulphide bonds in target protein. in e. coli the disulphide bonds are made by the dsb proteins in the periplasm space. in periplasmic spaces dsb proteins increase the solubility of recombinant proteins and the secretion efficiency [129, 130] . dsb proteins are including dsba, dsbb, dsbc, dsbd, dsbg, dsbe, ccmh. (fig. 3) . dsba(thioredoxin-like thiol-disulphide oxido-reductase) and dsbb(cytoplasmic membrane protein) are two enzymes that help to disulphide bonds formation in periplasmic proteins [131, 132] . the dsba is a small 21 kda polypeptide, which its active site contains the motif known as cys30-pro31-his32-cys33 has high oxidizing potential activity. in the oxidative process, disulphide bond formed between two cysteines by dsba which dsba need re-oxidized by fig. 3 . the mechanisms of disulfide bond formation in the periplasm. one of the disulfide bond donors in the periplasm is dsba, which is oxidized by dsbb and this oxidizing reaction needs respiratory quinone component. whenever there is more than two cysteine, the incorrect disulfide bond may occur. for preventing that, the disulfide bond isomerase dsbc comes to the rescue and it is oxidized by dsbd. dsbb [133, 134] . kishigami et al. recently suggested that synthetized dsba is quickly oxidized by pre-existing dsba, whereas oxidation of functional dsba needs dsbb, which has opposite action with dsba-reducing enzymes [135] . moreover, zhuo et al. were co-expressed dsba/dsbc proteins and recombinant reteplase in e coli. as result, the co-expression system improved soluble expression of recombinant reteplase, and high yield of soluble reteplase achieved especially with the dsbc co-expression system [136] . the dsbb is a 20 kd periplasmic protein that has four transmembrane (tm) fragments and two periplasmic loops that each loop contains a pair of cysteines [137] . kishigami and ito suggested that dsbb stimulated recycling dsba by disulfide interaction between cys30 of dsba and cys104 of dsbb [137] . dsbc(periplasmic oxido reductase) and dsbd(cytoplasmic membrane protein) have roles in isomerization of non-native disulphide bonds [131, 132] . dsbc is 23 kda homodimeric soluble protein containing four cysteines residue which these residue act as a catalyst for reorganization of disulfide bonds in protein with multiple disulfide bonds. dsbc deficiency can lead to misfolding of proteins with numerous disulphide bonds [138] . the active site cysteines of dsbc are found in dithiol form in isomerization activity which dsbc reduced by dsbd in this activity [139] . also dsbc has a reductase activity to proteins with false disulfide bonds and then re-oxidize by dsba. studies showed that the periplasmic proteins like rnasei and mepa needs to dsbc for their stability [140] . in addition, missiakas et al. indicated that overexpression of dsbc can functionally substitute for dsba loss [141] . for example purified dsbc protein was capable to catalysed insulin oxidation in a dithiothreitol dependant man-ner [142] . heo et al. showed that production of anti-c-met scfv could be more increased by co-expressing molecular chaperones such as groel and dsbc with scfv. in e. coli trxb/gor mutant, coexpression of dsbc enhanced the efficiency of functional anti-c-met scfv about 2.5 fold [143] . dsbd has three domains including amino terminal periplasmic domain, hydrophobic core domain containing eight transmembrane fragments and carboxy terminal periplasmic domain [144] . each of these domains has a pair of cysteines that have necessary role for electron transfer from the cytoplasm into the periplasm space [145] . kurokawa and his colleagues represented that overexpression of dsbd proteins are responsible for formation and isomerization of disulfide bonds that can significantly increase periplasmic production of human nerve growth factor b (ngf) [129] . dsbd is required for the reduction of dsbg. also it is needed for the biosynthesis of c-type cytochromes [146] . dsbg has negative charged surface, which is appropriate to interact with folded proteins [147, 148] . it can act as a molecular chaperone and its activity is not based on its active site cysteines [149] . also, andersen et al. showed that dsbg acts mainly as an oxidant agent during protein disulfide bond formation. in addition, the substrate range of dsbg could be slighter than the other periplasmic oxidative enzymes such as dsba and dsbc [150] . dsbe is known as ccmg because implicated in maturation of cytochrome c [151] . ccmg selectively gives electron to apocytochrome c cysteines via ccmh, which is another periplasmic cytochrome c maturation factor with a redox activity cys-x-x-cys motif [152] . this proposes that ccmg is capable to identify and selectively cooperate with dsbd and ccmh [151] . also dsbd is responsible for keeping dsbe in reduced form in the periplasm [145] . the eventual objective of amplify production of desired recombinant protein in an e. coli host cell depends on some factors that lead to outstanding out come in production of recombinant protein. as usual, recombinant proteins were expressed as inclusion body in the e. coli. for understanding of some problem we described the details to find suitable ways for enhancing soluble expression of recombinant proteins with correct folding that several approaches are used including; chemical chaperones, gene protein optimization, signal peptide sequences, molecular chaperone, and etc. molecular chaperones either cytoplasmic or periplasmic are more efficient in production of recombinant proteins with appropriate folding. cytoplasmic chaperones hsp100 (clp), hsp90, hsp70 (dnak/dnaj/grpe), hsp60 (groel/groes) and small hsps bind to aggregated and misfolded recombinant proteins to unfold them and finally the recombinant proteins acquire their correct folding. the hsp60, hsp70 and their co chaperones used atp to solubilized and refolded aggregated recombinant protein. periplasmic chaperones, located in periplasm space, were fold the recombinant protein correctly via formation of disulfide bonds, which finally produce soluble form of recombinant proteins. efficiency of folding process mediated by periplasmic chaperones depends on type of recombinant protein. some factors including; enhance the level of some periplasmic chaperones efficiency such as fkpa, and sura, which each of them facilitate the slow cis-trans isomerization of peptidylprolyl bonds in folding of target proteins and dsba and dsbc which help to form disulphide bridge. these activities have more important roles in overcoming to production of misfolded proteins. the authors declare that they have no competing interests. protein expression technologies: current status and future trends microbial cell fact inclusion bodies and purification of proteins in biologically active forms the biology of heat shock proteins and molecular chaperones proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci molecular chaperones in the life cycle of proteins, marcel dekker proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci drug target insights proc. natl. acad. sci molecular chaperones and protein folding in plants, in: post-transcriptional control of gene expression in plants proc. natl. acad. sci. 96 the authors hope the best for all contributors in tabriz university of medical science and college of rab-rashidi. key: cord-016095-jop2rx61 authors: vignais, pierre v.; vignais, paulette m. title: challenges for experimentation on living beings at the dawn of the 21(st) century date: 2010-06-08 journal: discovering life, manufacturing life doi: 10.1007/978-90-481-3767-1_5 sha: doc_id: 16095 cord_uid: jop2rx61 “we can talk endlessly about moral progress, about social progress, about poetic progress, about progress made in happiness; nevertheless, there is a type of progress that defies any discussion, and that is scientific progress, as soon as we judge it within the hierarchy of knowledge, from a specifically intellectual point of view.” introduction of new exploratory methods such as biocomputing or bioinformatics and high-throughput screening, which involves the simultaneous processing of hundreds or even thousands of samples. this approach is in contrast with traditional biology, in which the research strategy is based upon the observation of effects obtained as a function of experimental parameters that are modified one by one. another aspect of modern times is that, with the irresistible trend in genetic manipulation towards a focus on human beings, certain areas of fundamental research are finding themselves locked into philosophical dilemmas that are matter for ethical and sociocultural consideration, and the subjects of fierce debate. instead of setting out to discover unknown mechanisms by analyzing effects that are dependent on specific causes, with some uncertainty as to the possible success of the enterprise being undertaken, which is the foundation stone of the bernardian paradigm of the experimental method, many current research projects give themselves achievable and programmable objectives that depend upon the means available to them: sequencing of genomes with a view to comparing them, recognition of sequence similarities in proteins coded for by genes belonging to different species, with the aim of putting together phylogenetic trees, synthesis of interesting proteins in transgenic animals and plants, analysis of the three-dimensional structure of proteins, in order to find sites that are likely to fix medicinal substances, and synthesis of molecular species able to recognize pathogenic targets. the facilities that are called into play include instruments that are often sophisticated, the performance of which, in terms of miniaturization, computerization and robotization, is far beyond that of apparatus that was in use a few decades ago. these facilities, applied to research into living beings, have entered the framework of a methodology that has been given the label biotechnology. proceeding handin-hand with applications that have become more and more meaningful in the domains of medicine, pharmacology, agronomy and animal husbandry, the biotechnological process has come to the fore as a new paradigm for the experimental method as applied to living beings. in addition to new discoveries, the driving forces behind biotechnologies are related to economic imperatives as well as the interest and support they receive from the political powers-that-be. the academic spirit that presides over fundamental science gives way to the entrepreneurial spirit that implements a rational programming of facilities and an efficient organization of scientific collaborations. as an example, the sequencing of the human genome, which includes three billion nucleotide base pairs, required the coordination of several dozen scientific teams around the world and the matching of several tens of thousands of results. research on dna provides a typical illustration of the way in which research has become divided, over the last few decades, between an approach and an interest that had previously been purely academic, and the increasing role of technology, which can be justified by the results that arise in the life of society at large, but which, because of these results, also gives rise to questions concerning how wellfounded some of these results are, particularly in the health domain. the experimental method, which had been confined to the laboratory, is now a matter for public debate. before it won acclaim, dna, which was isolated under the name of nuclein by johann friedrich miescher (1844 -1895), at the end of the 19 th century, had to undergo a series of structural evaluation tests that were spread out over the first five decades of the 20 th century. an overall conclusion then came to the fore. dna is a polydeoxyribonucleotide that carries four cyclic bases, adenine, thymine, cytosine and guanine. each base is involved in the structure of a mononucleotide where it is itself associated with a sugar, deoxyribose, which is associated with a phosphate residue. dna was compared to a ladder, the rungs of which (mononucleotides) were linked by ester bonds between an acid group of a phosphate residue of a nucleotide and the free hydroxyl group of the deoxyribose of the following nucleotide. research committed them to this path. it was the curiosity of each, a new way of considering old problems, that led a few men and women to solve the problems of heredity." the middle of the 20 th century saw an accumulation of experimental evidence showing that dna carries genetic information, and because of this, that it controls the transmission of hereditary characteristics: the proof provided in 1944 by oswald avery (1877 -1955) , colin macleod (1909 -1972 and maclyn mccarthy (1911 -2005 of the transforming power of dna in pneumococcus, the highlighting by alfred hershey (1908 -1997) and martha chase (1927 -2003 , in 1952, of the role played by bacteriophage dna as an infectious agent for bacteria, the revelation by erwin chargaff at the beginning of the 1950s of the equivalence of molar concentrations of adenine (a) and thymine (t), on the one hand, and of cytosine (c) and guanine (g), on the other hand, in dnas arising from a multitude of sources, animal, plant and microbial, thus suggesting a complementary pairing of adenine and thymine, and cytosine and guanine. based on the pairing of a/t and c/g bases, the model of the double helix structure of dna, formulated in 1953 by james watson and francis crick, made it possible to understand the identical synthesis of double strands of dna by replication during cell division (figure iv.1 ) and, as a consequence, the conservation of hereditary characteristics in descendants. afterwards, it was found that the information contained in the dna base sequence determines the amino acid sequence in proteins. then the roles played by messenger rna and transfer rnas were elucidated, the former acting as a carrier of information between dna and the proteins being synthesized and the latter acting as double-headed adaptors, able to recognize nucleotide triplets (codons) in messenger rna and to specifically fix amino acids in order to position them on the ribosomes, the final result being the synthesis of a protein chain. in 1966, the genetic code was deciphered. the veil of mystery that had covered the mechanism of the synthesis of proteins was lifted, and the decisive role played by nucleic acids in this synthesis was shown. later on, there were a few adjustments. although, in bacteria, proteins are coded for by a continuous sequence of nucleotide triplets in dna, in the 1970s the surprising discovery was made that in eukaryotic organisms, genes are discontinuous and made up of coding dna sequences (exons) interrupted by non-coding sequences (introns). from the end of the 1950s, françois jacob and jacques monod had postulated the existence of a dual determinism for protein synthesis and shown that, next to structural genes expressed as proteins, there are regulatory genes able to control the expression of the structural genes. the importance of the differential regulation of gene expression in cell differentiation in higher organisms was quickly recognized. from this point on it was possible to explain why a particular species of protein is more specifically expressed in a given tissue and another species of protein is more particularly expressed in another tissue, each type of tissue finding its specificity in its molecular components. this fantastic framework of knowledge, which was built up over a couple of decades, has been used as the foundation stone for the so-called central dogma of molecular biology, which explains the transcription of dna sequences into messenger rnas and the translation of messenger rnas into proteins and which, with only a few variants, is the same throughout the living world (figure iv.1) 1 . 1 the fascinating history of molecular biology was well described by james d. watson in molecular biology of the gene (3 rd edition 1976). there are now many works concerning this subject and how it has progressed, including two well-documented books in french: the secrets of the gene by françois gros (1986) and histoire de la biologie moléculaire by michel morange (1994) . new strand a -double helix structure of dna and simplified representation of its self-replication. each strand of the parent molecule of dna acts as a matrix for the synthesis of a daughter molecule of complementary dna, in conformity with the rules of pairing: adenine (a) with thymine (t) and guanine (g) with cytosine (c). the double strands that appear are identical to each other and identical to the parent dna molecule. b -transcription of dna into messenger rna and its translation into an amino acid chain. diagram of gene expression in a eukaryotic cell. one of the strands of dna (the coding strand) has coding sequences (exons) and non-coding sequences (introns). it is said that the gene is split. the transcription of the exons, accompanied by their splicing leads to the formation of a messenger rna, the codons (nucleotide triplets) of which are translated into amino acids that are linked to each other by covalent bonds in order to form a protein chain. in prokaryotic cells (bacteria), the genes do not contain introns and are not split. a: adenine; c: cytosine; g: guanine; t: thymine; u: uracil. met: methionine; his: histidine; tyr: tyrosine; gly: glycine; phe: phenylalanine. interlinked with the epic rise of molecular biology, there was a succession of technical innovations that led to the synthesis of dna by chemical or enzymatic means, and to its being cleaved at specific locations, with the pieces that were obtained being joined together again 2 . in 1962, in geneva, werner arber (b. 1929) 3 and daisy dussoix highlighted the restriction phenomenon, which involves the degradation of bacteriophage dna by a recipient bacterium. they discovered that an extract of e. coli has a restriction activity, and that this activity is of an enzymatic nature, caused by a nuclease that breaks the phosphodiester bonds in dna. in 1970, the americans hamilton smith (b. 1931) and kent wilcox 4 purified the first restriction enzyme from a strain of haemophilus influenzae. in 1971 , daniel nathans (1928 -1999 and kathleen danna 5 (b. 1945) at johns hopkins university in baltimore (usa) drew up the first restriction map based on the circular dna of the monkey sv40 virus, using a restriction enzyme that was named hindiii and a follow-up of the sequential appearance of shorter and shorter fragments resulting from the partial digestion of dna. in the following years, dozens of restriction enzymes were isolated, all of them endowed with a surprising specificity with respect to specific base sequences in dna ( figure iv .2). these enzymes were to be indispensable tools in genetic recombination experiments. the transformation of rna back into dna was observed by howard temin (1934 -1994) and s. mizutani 6 , in experiments on the rous sarcoma virus, a virus with rna that, when it proliferates in host cells, is able to synthesize a dna that is complementary to its rna. the enzyme responsible, reverse transcriptase, was purified by both h. temin and david baltimore (b. 1938) 7 . starting with a determined messenger rna, it then became possible to work back to the dna, i.e., to the gene, by a simple enzymatic reverse transcription operation. dna that has been synthesized in this way is called complementary dna (cdna). in eukaryotic organisms, reverse transcription has proved to be all the more useful as a technique in that all cdna is coding, unlike the situation in vivo in which the genes are divided up into portions that are coding (exons) and portions that are non-coding (introns). the ability to cleave dna and to join together the fragments obtained in a deliberately chosen order, or, in other words, to manufacture previously unseen dna sequences by making new combinations, led to the dawning of recombinant dna technology and caused scientists to come to the sudden realization that the pandora's box that contains the secrets of life had been opened, that uncontrollable catastrophes might arise from this, and that there was a potential danger of causing tumorigenic viruses to reproduce in commensal bacteria such as the enterobacterium escherichia coli. in 1975, around a hundred molecular biologists gathered together at the asilomar conference center 8 near monterey in california, in order to discuss the dangers of the new dna technology. they proposed strict regulation to govern genetic manipulation. time and experience have shown that the risks being run were very low. in 1977, dna sequencing methods were published. one of them made use of chemical techniques 9 , the other made use of enzymatic techniques 10 . applications were not slow in appearing. from 1977, the team led by frederick sanger (b. 1918) in cambridge (uk) determined the first sequence of a genome, that of bacteriophage phix174, which is 5 375 nucleotides long. this was the beginning of an audacious adventure, the apparently senseless challenge being met with unbelievable rapidity thanks to the innovative methods of bioengineering, resulting, during the first years of the 21 st century, in the sequencing of the human genome. analysis of the human dna sequence involved the participation of two rival groups, one of them being academic, coordinated by francis collins (b. 1950) , and bringing together dozens of laboratories around the world, and the other being a private californian company directed by craig venter (b. 1946) . at the beginning of the 1980s, when everyone was persuaded that the rnas could be placed into three well defined categories, messenger rnas, transfer rnas and ribosomal rnas, it was with great surprise that it was learned that there were rnas that have catalytic properties (see thomas cech 11 [b. 1947] ). these rnas, called ribozymes, have, in keeping with enzymatic proteins, structured catalytic sites that are able to catalyze rna or dna cleavage or ligation reactions. recently, engineering techniques have been used to obtain artificial ribozymes that have been found to be able to catalyze reactions as varied as oxidations or the synthesis of peptides and nucleotides, thus opening up wide-ranging possibilities of applications in molecular therapeutics, and, in addition, reinforcing the famous theory of the "world of rna" at the beginning of the appearance of life on earth 12 . another discovery of the 1980s was the role of methylation of dna bases, cytosine and adenine, and its deregulation in a certain number of pathologies: fragile x syndrome, scapulohumeral dystrophy, certain forms of cancer 13 … in the past decade or so, basic proteins known as histones that are associated with the nuclear dna of eukaryotes in the form of a complex called chromatin and which had previously been assigned a structural role, have now acquired the status of functional partners. thanks to specific modifications of certain amino acids (acetylation, methylation, phosphorylation), histones control the state of condensation of the chromatin and the efficacy of transcription of dna contained in the chromatin, to such an extent that we now speak of the "histone code". the development of our understanding of histones is a good illustration of the complexification of a concept, the dna code, into an entity that comes closer to living reality, the dna code in partnership with the histone code. there has also been the discovery of interfering micrornas, small polymers made up of around twenty nucleotide units, the role of which is to control protein synthesis (chapter iv-1.2.2). methylation of dna, structural modifications of the chromatin histones, and blocking of transcriptional activity by interfering micrornas are a few of the major areas of research in a scientific domain that is in full expansion, epigenetics, which could be said to have "pipped the science of genetics at the post," and which explains the plasticity of the functions of living beings. with the arrival of restriction enzymes and reverse transcription, the foundation stones of genetic engineering have now been laid, and are ready to be used, this being all the easier in that synthetic chemistry is now able to manufacture dna chains that are several hundreds of nucleotides long, and progress in robotics and computing techniques made it possible for chemists to avoid carrying out tedious routine tasks by using completely-programmable machines. the hope that it would be possible to experiment on living beings by means of the manipulation of dna became a reality when the american researchers paul in the first work carried out on the expression of foreign genes, the use of plasmids as vectors was preferred, particularly that of plasmid pbr322, because of its considerable replication capacity. in 1978, a first success was obtained by herbert boyer and his co-workers, with the expression of the gene for somatostatin, a peptide hormone comprising twelve amino acids that negatively regulates the secretion of growth hormone, in the bacterium e. coli. because of its small size, the somatostatin gene was synthesized by chemical means. the expression of somatostatin in e. coli was verified using immunological and physiological criteria, thus demonstrating the validity of the procedure that was used. the following year, human insulin was produced in e. coli. fairly soon, yeast was substituted for this bacterium because, as a eukaryotic organism, it has enzyme systems that are able to carry out chemical finishing operations on neosynthesized proteins that bacteria are unable to do, for example the formation of disulfide bridges in insulin. genetic recombination is used in order to cause bacteria to manufacture a foreign protein of animal or plant origin. this involves the insertion of the fragment of animal or plant dna that codes for this foreign protein into a plasmid. the plasmid, a small ring of bacterial dna, acts as a vector for the foreign dna. in order to carry out insertion, plasmid dna is cleaved by an appropriate restriction enzyme. the foreign dna is obtained by reverse transcription from a useful messenger rna. its duplication is catalyzed by a dna polymerase. the s1 nuclease makes it possible to break the covalent bond between two strands of dna. in the following step, a terminal transferase is used to add four nucleotides for which the base is a cytosine (c) to each of the two dna strands. the same lengthening operation is carried out on the bacterial plasmid, but, in this case, the addition involves a sequence of four nucleotides for which the base is a guanine (g) (complementary to the cytosine c). the bacterial plasmid is hybridized in vitro with foreign animal or plant dna and then introduced into the bacterium which, using its own machinery, perfects the junction between the integrated dna and the plasmid dna. a very small volume of dna (2.10 -9 ml) is injected under the microscope into eukaryotic cells (hela cells in inset) using a micropipette with a very fine end that pierces the cell membrane. the swelling of the cells at the moment of injection can be seen (inset). supported by these successes, genetic engineering started to come to the fore as an application-oriented discipline. levels of performance that would never have been imagined half a century before were achieved, such as the production of growth hormone, interferons, blood coagulation factors and vaccines. in the final decades of the 20 th century, phenotype transformations using genetic modifications that had previously been carried out in bacteria and yeasts were successfully attempted in animals and plants. it was observed that a mutated dna integrated into a plasmid and introduced into a fertilized mouse egg (by micromanipulation) modifies the mouse's genetic inheritance, which affects first the embryo and then the adult mouse with phenotype modifications. such mice, which are said to be transgenic because of the stable integration of a foreign dna into their genome, are now widely used as animal models in studies that aim to understand the mechanisms involved in high-incidence human pathologies such as cancer, diabetes, and rheumatoid conditions. in 1982, two american researchers 16 , ralph brinster (b. 1932) and richard palmiter (b. 1942) carried out a spectacular transgenesis experiment in mice. using microinjection, they introduced the growth hormone gene (obtained from the rat) into oocytes of mice from a "little" germ line. once the transgeneic mice had reached adulthood, they were giants. at present, the transgenesis technique is being applied both to the animal kingdom and to the plant kingdom. in the 1980s, kary mullis 17 (b. 1944) perfected an ingenious technique, the polymerase chain reaction (pcr), which makes it possible to produce several tens of thousands of copies of a fragment of dna. using this technique, it is possible to detect traces of a fragment of dna of a given sequence down to an attomolar concentration, i.e., one billion billion (10 -18 ) times smaller than molar concentration. by the end of the 20 th century, genetic engineering had become well-established and wide-spread, thanks to a mastery of techniques involving the manipulation of dna such as the accurate cleavage of a gene into fragments using commercially available restriction enzymes, the covalent assembly of two fragments of dna by ligases, the automated chemical synthesis of fragments of dna of more than one hundred nucleotides and the possibility of manufacturing a complementary dna (cdna) from a messenger rna by using a reverse transcriptase and automated dna sequencing. given this particularly well-equipped toolbox, the molecular biologist is now able to manipulate dna, that is to say, the chemical material that contains the information that is central to the functioning of living structures (microorganisms, plant and animal organisms), and thus to modify, at will, the genotype of these structures that the selective pressure of evolution has previously favored. genomics has produced enormous quantities of data that are stored in databanks. automated procedures have been invented to make the information contained in these data intelligible, these procedures forming the basis for a new discipline, biocomputing, or bioinformatics, which develops programs, or algorithm-based strategies, that are able to solve specific problems, of which the annotation of genomes, i.e., the identification of coding and non-coding sequences. while the annotation of the prokaryotic genomes is relatively easy, because of the absence of introns, that of the eukaryotic genomes is considerably more difficult because of the alternating exons and introns and the small proportion of coding exons (fewer than 2% in the case of the human genome). this explains why, at the time of writing, several hundred genomes of prokaryotes (around 300 at the beginning of 2006) have been sequenced, as opposed to only a few dozen genomes of eukaryotes. annotation was carried out manually at first, but it has become automated and it is now possible to analyze thousands of items of genomic data. the comparison of nucleotide base sequences in dnas and of amino acids in proteins of different origins involves biocomputing. the identification of similar or identical regions that provide information about functional similarities and phylogenetic proximity involves the use of alignment methods. one of these methods, which is in current use, is called blast (base local alignment search tool). comparison of protein sequences has been particularly instructive in the science of evolution. it has highlighted evolutive processes in the phylogenesis of proteins and linked these processes to precise functions. it has been possible to deduce that, over time, different families of proteins with similar functions appeared independently and evolved along different routes. this is the case for membrane proteins whose polypeptide chain crosses the thickness of the membrane six or twelve times; thus, the mitochondrial membrane proteins that transport metabolites are formed by triplication of an element with two transmembrane segments, while proteins located in other membranes of the cell are derived from duplication of an element with three transmembrane segments. from this academic context arose the study of paleogenetics, a new discipline that compares dna sequences extracted from fossils and amplified by pcr with dna sequences of current species. in addition to being of immense interest to fundamental biology, genetic bioengineering has led to innumerable industrial applications making use of genetically modified microorganisms that are able to synthesize molecules with a high added value that can also be used in xenobiotic depollution operations. so much data has already been deposited, equivalent to the sequencing of more than one hundred billion nucleotides, that it is inevitable that there have been errors, some of which might prove prejudicial for future use (comparison of sequences, screening of drugs…). nevertheless, the ever-increasing numbers of genome sequencing projects for animal, plant and microbe species show the interest that is shown in understanding the genetic information present in different types of cells, in order to be able to exploit their potential. dna chips appeared in the last decade of the 20 th century, and came to the fore as part of a new technical revolution, the "high throughput" revolution ( figure iv.5 ). an article written by the group headed by ronald davis and patrick brown (b. 1954 ) at the university of stanford 18 gives a precise description of the hybridization technique used in dna chips. thus, around a hundred short dna strands, corresponding to portions of genes of the plant arabidopsis thaliana, commonly known as mouse-ear cress, a small plant in the brassicaceae (formerly cruciferae) family, are synthesized. a robot is used to deposit microquantities of these dnas in solution in a dot pattern on a small glass slide coated with poly-l-lysine, thus comprising a "chip" on which the covalently fixed dnas act as probes for specific molecules. a later step involves both the use of reverse transcription to produce complementary dnas (cdnas) from messenger rnas arising from the expression of genes in the same plant and the labeling of these cdnas with fluorescent ligands for use in screening. once these fluorescent cdnas have been denatured, i.e., after separation into single strands, they are brought into contact with the dna chip. the unhybridized molecules are removed by washing. a -the term dna chip corresponds to a small, chemically-treated glass (or sometimes silicon) plate on which a robot has deposited dna strands of known sequence in a pre-determined order. b -the dna chip may be used for different types of diagnostic procedures. in the differential diagnosis experiment represented here, messenger rnas are prepared from two cell samples that have been treated in parallel, the control sample (normal cell) and the experimental sample (pathological cell). these messenger rnas are reverse transcribed into complementary dnas (cdnas) by means of a reverse transcriptase. each of these two types of cdna, corresponding to the two types of messenger rna, is labeled by a chemical reaction with a specific fluorescent ligand (cy3, which emits at 568 nm and cy5, which emits at 667 nm). they are then hybridized with chip dna strands. after hybridization and then washing, the fluorescence emitted at 568 nm and 667 nm under laser irradiation are analyzed using an appropriate detection system. the differential expression of the genes in the control cell sample and the experimental sample (shown by a color difference) can thus be analyzed. hybridization between the fluorescent dnas called targets and the complementary nucleotide probes fixed to the dna chip is detected by means of an automated fluorescence detection system. at the beginning of the 1990s, stephen fodor 19 and his group, who were working at the affymax research institute (palo alto, california), developed an ingenious procedure for microphotolithography that led to the synthesis of a network of a thousand peptides on chemically pre-treated glass microscope slides. the resolution of the network was shown by epifluorescence microscopy after fixation of specific antibodies labeled with fluorescent probes. soon after this, microphotolithography was used for the manufacture of dna molecule networks on solid supports. from then on, two competing techniques for the preparation of dna chips became well-established, either the depositing on a solid support of cdna obtained by gene amplification (technique used by davis and brown), or the synthesis in situ of oligonucleotides carried out directly on a solid support (technique used by the american affymetrix company, arising from affymax). one considerable advantage of dna chip technology is that it provides information on the level of transcription of thousands of genes into messenger rnas (mrnas), in a simultaneous manner and in a relatively short lapse of time. experiments that previously required weeks, months or even years to be completed can now be carried out in a matter of hours. we therefore have a sort of instantaneous, precise, freeze-frame picture of the state of a cell at a given moment, with a great number of parameters explored in a semi-quantitative manner. dna chips are a typical example of the application of high throughput technology to the study of living beings. the panoply of mrnas produced by the transcription of dna is called the transcriptome. the method by which transcriptomes are obtained is called transcriptomics. it should be added here that there is not necessarily a correlation between the abundance of a mrna, evaluated on a dna chip, and the functionality of the corresponding protein, which depends on multiple factors that particularly involve post-translational modifications: phosphorylation, glycosylation, hydroxylation, and so on. at the end of the 1990s, dna chips were being used extensively in the research programs of many biology laboratories. they are used in a variety of domains: human pathology, to differentiate between forms of cancer linked to multiform mutations; microbiology, to identify pathogenic germs; comparative genomics, to look at model eukaryotic organisms that have in common a certain number of genes; or even in populations genetics, to detect polymorphisms linked to a change in a single base in a dna sequence. as a complement to the dna chip technique, the fish (fluorescent in situ hybridization) method holds a key position in the study of cytogenetics. this method is based on hybridization between fluorescent nuclear probes of known nuclear sequence and of complementary motifs located in the dna of the chromosomes. it allows the detection of chromosomal modifications with a gain or loss of genetic material, such as those that are found in certain tumors. it is used in prenatal diagnostics to diagnose such modifications. protein chips, which are used to characterize the reactivity of proteins with respect to specific ligands, are another example of the application of high throughput technology. dozens of proteins of different types (antibodies, for example), as well as derivatives of nucleic acids or even molecules capable of being ligands of proteins that might arise from combinatorial chemistry (chapter iv-3.3), are arranged in a network on small glass plates that are chemically treated to act as hooks to entrap specific proteins present in a tissue extract or serum ( figure iv .6a). this procedure, which is essentially analytical in nature, is complemented by a functional study in which proteins that have been isolated in their native form, i.e., those that are capable of expressing the same functions that they posses in vivo, are deposited on a glass microplate. this type of biochip makes it possible to analyze the reactivity of the proteins that are fixed to it with respect to a multitude of targets (proteins, nucleic acids or pharmaceutical substances) (figure iv.6b). antigens peptides a -biochip for the identification of proteins. different types of ligands, antibodies, antigens, dna or rna, small molecules with a high affinity and specificity, are deposited on a reactive surface. these biochips can be used to determine the level of expression of proteins and the type of proteins expressed in cell extracts. they can be used for clinical diagnostics. b -biochip for the functional study of proteins. native proteins or peptides are arranged in micronetworks on a reactive medium. biochips produced in this way are used to analyze the activities of proteins and their affinities as a function of posttranslational modifications. they are useful for identifying drug targets. analytical proteomics, which was still in its infancy in the last decades of the 20 th century (chapter iii-6.2.3) has become a vigorous discipline. the association of liquid nanochromatography and of mass spectrometry allows the identification of peptides obtained by the trypsin hydrolysis of samples of proteins of around one picomole in size. applied to fundamental and pathological cell biology, the aim of analytical proteomics is not only to decipher the list of proteins on the scale of the cell, but also to highlight variations in the abundance of synthesized proteins as a function of environmental conditions. it also aims to determine the posttranslational modifications that are undergone by the proteins inside the cells, which, for a large part, control the specificity of their operation. in parallel with the study of proteomics, the study of peptidomics, or the study of all of the peptides (peptidome) present in animal and plant cells and in the fluids that bathe these cells, has developed. for example, several hundred different peptides have been found in the cerebrospinal fluid. structural proteomics, which deals with the three-dimensional structure of proteins, has also become a domain in which the activity has been increasingly dominated by high-throughput techniques. this is partly due to the fact that the pharmaceutical industry has given considerable, sustained attention to the understanding of the structure of proteins that could play the role of therapeutic targets. this is the case for protein kinases that catalyze, via atp, the phosphorylation of endocellular proteins, of proteases involved in hydrolysis reactions, or even of cell surface receptors that are able to combine with ligands such as hormones. the use of automated crystallization systems has become common in structural biology. from the classical technique of the hanging drop, in plates comprising 24 wells, we have moved on to plates with 96, 384 and, recently, 1536 wells. obviously, this increase in dimensions requires the use of an automated system that includes a robot in charge of transferring microaliquots of the protein solution into the wells and adding media that differ according to their ph, ionic force and molecular composition to these wells. the crystallization process is followed by an automated microscopic examination coupled with video photography. making use of the recent development of genomics and of proteomics, and a detailed inventory of the structures and functions of the different protein species of living beings, contemporary biology is now able to sketch out a scheme of molecular systematics, including a classification into phylla, families, and classes that echoes those of the zoological and botanical systematics of the 17 th and 18 th centuries. however, modern systematics does not tell us how protein macromolecules interact within dynamic networks. there is still an enormous amount of work to be done in order to achieve an understanding of the meaning of the dialogue between macromolecules in a normal or pathological cell context. this work will require a detailed analysis of metabolic pathways and of how they are controlled, and their evaluation in kinetic and thermodynamic terms. it will be accompanied by modeling (chapter iv-4). there is no doubt that it will be successful. making use of subtle differences in the qualitative and quantitative expression of genes, it will become possible to understand the molecular principles that modulate differences in morphology and in function between neighboring animal, plant or microbial species. the science of evolution should benefit from this. in medicine, the forecasting of predispositions for certain diseases should be made easier (chapter iv-3.2), opening up the perspective of prevention strategies. using recombinant dna technology, metabolic engineering applied to microorganisms and plants should make it possible to improve the production of molecules that are of economic interest or can be used for drugs. the diversity of bacteria is amazing, much greater than might be supposed by looking at the number of bacterial species identified by culturing on appropriate media. in fact, the number of bacterial species that can be cultivated only represents 1% of the total number of existing bacterial species on the surface of the earth. there are two major reasons for this: we do not know the appropriate conditions for culturing these bacteria; a certain number of environmental bacteria live in symbiosis, acting as commensal organisms that benefit from the products secreted by other organisms. nevertheless, the study of the bacterial genome, without any clonal culture, has been carried out, and comprises a branch of genomics known as metagenomics. instead of looking at an isolated, well-identified bacterial species, in order to analyze the sequence of its dna, as has been done traditionally, researchers look at a heterogeneous bacterial sample from which the dna is extracted, amplified, and then sequenced by high throughput methods. computer processing of the data provides information about individual germs. craig venter, who had already gained notoriety with the sequencing of the human genome, recently applied "metagenomic" procedures to the study of the sequence of the "metagenome" of the bacterial species of the sargasso sea 20 . he came up with nucleotide sequences corresponding to approximately 1 million kilobases of non-redundant nucleotides, attributable to more than two thousand different genomes. the challenge to be met by metagenomics is to connect a function to its phylogenetic source and to extend this information to specific species within a bacterial community. group 21 . in human biology, a metagenomics approach has been applied to the study of the population of bacteriophages present in the intestinal flora. approximately 1 200 genotypes have been identified, a number that greatly exceeds the 400 bacterial species of this flora 22 . this result leads us to think that the luxuriant community of bacteriophages which cohabits with that of the intestinal bacteria may influence the diversity of the latter by selective bacterial lysis and also by promoting the exchange of genes between bacteria. a rapid overview of the history of the exploration of genomic dna over the last fifty years shows the rapidity with which a traditional experimental paradigm can move thanks to modern computing and robotics procedures. in less than twenty years, we have moved from the manual sequencing of dna that was developed at the end of the 1970s to automated high throughput sequencing. at the turn of the 21 st century, the sequencing of communities of genomes (metagenomics) has been substituted for the sequencing of individual genomes. dna and protein chips have become objects of everyday use in fundamental and applied biology. transgenesis is widely practiced. dna, a molecule that remained mysterious for a long time after it was discovered, delivered some of its secrets during the second half of the 20 th century. the purpose of the first experiments on dna was to understand how dna, detector of the genetic code, transmitted its message. after having questioned dna, researchers moved on to manipulating it. the current aim is to use oligonucleotides to build nanoscale constructions with original and, if possible, useful, properties. in addition, the possibility that has recently become available of being able to interfere with the expression of the genome in living cells, with the intervention of small rna molecules, allows the programmed manipulation of the genome. another challenge, the extending of the coding power of the genetic code, now appears to be achievable. from the fact that each of the strands of the double helix overhangs in one direction, and in the other the strand with which it is paired, thus leaving a few bases free ( figure iv.7) . if two strands of dna with sticky ends are brought into contact, when the bases of these ends are complementary, a branched structure will appear spontaneously. using this principle as a basis, cube-shaped nanometric constructions that make it possible to encage molecules of interest have been built. the opening of the cage by appropriate devices liberates the encaged molecules, which can act as substrates in specific reactions. the cutting of a double strand of dna using restriction enzymes able to create fragments with cohesive ends (a) has been used to "build" an artificial construction (b), which, in this case, is a cube (c), but which could be an object of a different geometrical type. a dna nanomachine that is capable of movement is becoming a reality. one dna nanomachine, which is admittedly still rudimentary, has been put together based on the structural difference that exists between b-dna, the classical double helix that twists to the right, and z-dna, a double helix that twists to the left. a propensity to adopt the z-form is triggered when there is an alternating sequence of cytosine (c) and guanine (g) (cg sequence) in the dna. the experiment illustrated in figure iv.8 makes use of a duplex formed of b-double helices. the dna nanomachine constructed by n.c. seeman comprised a duplex of double strands of dna. one of the double strands, of the classical b-form of dna (right-hand twist), has been cleaved in such a way as to fix fluorescent molecular probes onto the cleavage zones. facing this cleavage zone, a short nucleotide sequence, in which the cytosine (c) guanine (g) motif is repeated, can be found in the other dna double strand, which is also of b-form. the addition of cobaltihexammine induces the transition of the cg segment from a right-hand twist (b-dna) to a left-hand twist (z-dna), which leads to a rotation of this segment and to a rotation of the assembly, which can be detected using fret (fluorescence resonance energy transfer) spectroscopy. one of the double helices has a short cg segment. facing the cg segment, the other double helix is interrupted, and its ends, where the interruption is, carry fluorescent molecular probes. the simple fact of adding a cationic substance such as cobaltihexammine, which neutralizes the negative charges of the phosphate groups, triggers a conformational transition, with the cg segment taking the z-form, causing a rotational movement of the assembly that is detected by the movement of the probes. there is no doubt that the use of dna strands in order to build nanomolecular constructions that are capable of programmed movement marks the beginning of an adventure that we may imagine will be rich in outlets for domains such as computer technology, nanomechanics and even the life sciences. in addition, the discovery that dna conducts electrical current gives rise to dreams of a revolutionary technology in which dna may be used in the design of electrical circuits, in competition with classical electronics 24 . interfering rnas are non-coding rnas of around twenty nucleotides that control gene expression at post-transcriptional level. as with many discoveries, that of rna interference was the result of serendipity. it began during the 1980s with observations made by two american research groups, that of victor ambros 25 now at darmouth medical school, hanover, and that of gary ruvkun 26 (b. 1951) at boston's massachusetts general hospital, that a gene named lin-4, which is involved in the post-embryonic development of the nematode c. elegans, did not code for a protein, but for a small size rna that played an antisense role. this odd discovery was supported and made more explicit a few years later by the research groups of andrew fire (b. 1959) at baltimore's carnegie institute and craig c. mello (b. 1960 ) at the university of massachusetts in worcester 27 . in order to block the production of certain proteins in the nematode c. elegans, the researchers used synthetic antisense rnas. the control involved the use of sense rnas according to a classical protocol. unexpectedly, protein synthesis was blocked in both cases, suggesting that a contaminant was present in the sense and antisense rna preparations. this contaminant was identified as a double strand rna (dsrna -double strand) that is, an rna that is folded back on itself in a "hairpin" loop because of the pairing of complementary bases (adenine vs uracil and guanine vs cytosine). in order to verify the mechanism by which the translation of messenger rnas into proteins is silenced, the nematode c. elegans was injected with a synthetic dsrna, part of the sequence of which was complementary to that of the gene unc-22, known to code for a protein involved in muscular contraction. within a few hours, the worm was making disordered movements, suggesting that the dsrna interferes with the production of proteins in the process of muscular contraction. the mechanism of action of dsrna was quickly unraveled: dsrna gives rise to two single strand rnas after cleavage by a specific enzymatic mechanism. one of the single strand rnas (sirna -small interfering rna) is paired thanks to a complementarity of bases with a short sequence of messenger rna transcribed from the gene unc-22. the result is a blockage of the translation of messenger rna into a protein, followed by the destruction of messenger rna. this phenomenon was named rna interference ( figure iv the dicer cleavage enzyme, which has a ribonuclease activity, cuts the double strand rna into two strands. in the presence of the risc (rna-induced silencing complex) protein complex, one of the rna strands finds a complementary nucleotide sequence in a messenger rna (mrna) and associates itself with this rna, making it unable to be translated into protein. we now know that eukaryotic cells from animals and plants produce and host interfering rnas that are said to be "natural" 28 . natural interfering rnas, of around twenty nucleotides, are called micrornas (mirnas). although a few details differ between the modes of formation and action of natural interfering rnas and those of synthetic interfering rnas, in particular the fact that messenger rnas are not destroyed by mirnas but blocked in their translation, the effect of negative regulation on the production of specific proteins comes to the same thing ( figure iv.9 ). there is a far from negligible number of genes that code for mirnas. already, several hundred mirnas have been identified in the genomes of plants and animals. the amount of interest that they arouse, and the feverishness of the research being carried out on them, are in keeping with the major mechanisms that they control: embryogenesis, hematopoiesis, neuronal differentiation, etc. given an understanding of the genome sequence in man, the rat and the mouse, trials have begun that aim to achieve an understanding of how the expression of mammal genes of known sequence might be manipulated by the interplay of interfering rnas (chapter iv-3.1). the treatment of viral infections such as aids or hepatitis b, which are worrying public health problems, could benefit from this new technology. it appears that interfering rnas have much more to give to us in the near future than they have taught us up until now. the deciphering of the genetic code in the middle of the 1960s was the end of a first step in elucidating the mechanism by which a sequence of nucleotides in dna is translated into a sequence of amino acids in a protein (chapter iv-1.1). during the years that followed, the subtleties of the transcription of dna into messenger rna and of the translation of messenger rna into protein via transfer rnas were explored in hundreds of laboratories around the world. particular attention was paid to the understanding of how a given amino acid is activated and bound to a transfer rna (trna) after being picked up by an aminoacyl-trna synthetase. nevertheless, the idea remained of a code in which triplets of purine and pyrimidine bases of messenger rnas are translated into natural amino acids. recently, methods have been developed that give more flexibility to the action of the aminoacyl-trna synthetases, or, in other terms, relax their specificity 29 . synthetases that have been manipulated in this way are able to recognize non-natural amino acids and to incorporate them into proteins by working together with the ribosomal machinery. it is in this way that, at the time of writing, around thirty non-natural amino acids, obtained by insertion of different types of chemical residue (photoactivable, fluorescent or radioactive residues capable of acting as probes for structural and functional analyses) (figure iv.10) have been incorporated into protein structures. with such an innovation, an unexpected field of exploration has opened up to research in domains as far apart as pharmacology and the science of evolution, giving rise to burning questions: could such non-natural proteins have therapeutic properties? could they give a selective advantage to the organisms that host them? with the addition of non-natural amino acids to the genetic code and the demonstration that proteins containing such amino acids can function in living cells, in sum, with the transgression of the potentialities of the natural genetic code, the experimental method appears to challenge the order of living beings. the triumph of genetic engineering via the study of dna is not unique to biology. many other sectors are undergoing changes in their type of experimental approach, dictated by the technosciences and making use of computer sciences, robotics and high-throughput screening. however, given the many questions that its operation continues to raise, and its central position at the heart of scientific ethics, the study of dna remains a typical example of the way in which the experimental life sciences and the techniques that underlie them are evolving nowadays. "i perfectly agree that when physiology is sufficiently advanced, the physiologist will be able to make new animals or plants, as the chemists produces substances that have potential, but do not exist in the natural order of things." more than a century after claude bernard predicted a genetically manipulated world, it has come to pass. the molecular biologist, having original, highperformance methods for "tinkering" with dna, has moved on to the application and use of his technical expertise for utilitarian ends. during the 1970s, with transgenesis, research on bacteria (chapter iv-1.1.2) opened up a new biological domain, that of genetically modified organisms (gmos). results led to predictions that it would be possible to transfer a fragment of dna corresponding to a gene of a certain species into the genome of another species and have this foreign gene express itself as a protein in the host cell. in 1983, the successful trans-genesis of a gene for resistance to an antibiotic, kanamycin, in tobacco plants, signaled the beginning of the technology of the first plant-type genetically modified organisms, still called gmps (genetically modified plants). in 1996, the birth of dolly the ewe unveiled the era of reproductive cloning in mammals, i.e., the identical reproduction of an already-existing organism. an additional step was taken with the first tests on the differentiation of embryonic stem cells towards different types of lines that are characteristic of well-defined tissues, such as nerve tissue, cardiac tissue or the hepatic parenchyma, thus opening up promising perspectives in regenerative medicine. the frontiers of the experimental method continue to be pushed back to the limit of what is feasible and sometimes into the realm of fiction, as in immunology, for example, with the idea of xenotransplantation, using "humanized" animal organs. given the universality of the genetic code, any gene that is introduced into the genome of a plant, whether that gene is of animal, plant or microbial origin, is able to replicate itself and be expressed as a specific protein. thus plant gmos or genetically modified plants (gmps) are able to express specific foreign proteins from another plant, a bacterial microorganism or an animal organism. in the 1990s, a short time after fundamental research had revealed the feasibility of plant transgenesis, the first transgenic plant, the flavr savr tomato, was marketed in the usa. since this time, numerous other plant gmos have been cultivated on a large scale and become available on the world market, including corn, soya, rice, cotton and the poplar. one of the desired aims is to produce modified plants that are able to resist destruction by the herbicides that are commonly used to eliminate weeds, while another is to prevent predation by harmful insects. in the first case, transgenesis involves the insertion of a herbicide-resistant gene, and in the second case, the inserted gene codes for an insecticidal toxin. recently, plant gmos that produce proteins with a therapeutic effect have appeared, ranging from antibiotic peptides to antibodies or proteins as unexpected as human hemoglobin. current projects aim to create plants that are resistant to adverse conditions such as the dryness of arid climate zones. the preferred procedure for producing a plant gmo is to use a bacterium, agrobacterium tumefaciens, a microorganism that is able to insert fragments of its own dna into plant cells ( figure iv .11). the useful gene that we wish to transfer into the plant may be a gene for resistance to a pesticide such as glyphosate, marketed under the name of roundup, phosphinothricin (basta) or glufosinate (liberty). the plasmid is reintegrated into a. tumefaciens. during infection of a plant cell by a. tumefaciens the t-dna carrying the gene of interest is inserted into one of the chromosomes of this cell. the ti plasmid is isolated and cut using a restriction enzyme. a foreign gene, known as a gene of interest, is inserted into the t-dna of the plasmid. a plant is generated from a modified clone. all of its cells carry the foreign gene. transgene of interest in the case of the fight against insect predators, the useful gene is carried by a fragment of dna contained in the genome of the bacterium bacillus thuringiensis. this gene, called bt, expresses a toxin responsible for the insecticidal capability of b. thuringiensis. a current application involves the protection of bt corn with respect to the corn-borer, a devastating insect whose caterpillars are particularly destructive. another, more direct, gene transfer method, known as biolistics, involves bombarding plant cells with tungsten microbeads covered with modified dna. with the implementation of large-surface-area experimental fields and the first marketing of gm soya, in 1996, the question of whether or not the advantages achieved with respect to crop yields are counter-balanced by risks for the environment and for consumers came to the fore. food risks could arise from the toxicity or allergenic power of artificially synthesized proteins. at the time of writing, this question remains unanswered, due to the lack of epidemiological studies carried out rationally over several years. when the first creations of gmos took place, the transfer of the gene of interest was carried out by means of the co-transfer of an antibiotic resistance gene. the transformed cells were selected according to the criterion of their resistance to this antibiotic, which involved a risk of dissemination of the resistance gene. this selection technique has been abandoned. in practice, it is difficult to evaluate the theoretical ecological risk of wild plants being invaded by genes that have been inserted artificially into gmos. as a precaution, zones used for experimentation of plant gmos are now surrounded by refuge zones, i.e., fields in which the same species of plants, in non-gmo form, are cultivated. there has been a much fiercer and completely legitimate debate concerning the presence of the terminator gene in seed from the first gmos marketed by the monsanto company in the usa. the terminator gene blocked germination of the seed from the cultivated plant, so it was necessary for the farmer to buy more seed from the company each season, thus creating a state of dependency. this technique is no longer in use, but the fact remains that most transgenic seed is patented, and therefore farmers who use such seed are dependent on the companies that posses this genetic know-how. the culture of plant gmos has spread around the world, covering more than a billion hectares of our planet, more than half of which are in the united states of america. this type of culture is used on a large scale for soya and in a less extensive way for corn, rape seed and cotton, but there are many other applications of plant transgenesis. among the countries that are actively involved we may mention argentina, brazil, canada and china, and more recently india, paraguay and south africa. while the policies of these countries are based on the fact that gmo products do not differ fundamentally from non-gmo products with respect to checks carried out a posteriori, and that there is thus no reason to prohibit them, european policy has taken refuge behind a principle of precaution, and it remains basically restrictive. although the moratorium on the culture and marketing of plant gmos that was put in place in 1999 was lifted in 2004, mandatory labeling for any consumable product containing more than 0.9% gmo remains dissuasive. the united states of america has refused to use such labeling. the worries that are aroused by plant transgenesis, which are often exacerbated by the diktats of ecology groups, must be analyzed in a reasoned manner. common sense and lucid thought dictate that the debate should be situated within a scientific perspective in which the main role is played by the experimental method in long-term applications. simple reflection leads us to think that with time parasites and self-propagating plants will develop a resistance to the most drastic treatments, as was the case for bacteria confronted with antibiotics. the perspective of an acquisition of uncontrolled resistances, which gives rise to so much passionate debate, is, in fact, only the first stage of a technology with promising applications. the mastery of plant transgenesis that was acquired through the first experimentations should, in fact, allow the emergence of plant gmos that are assigned to the production of molecules with therapeutic effects (drugs, vaccines, human proteins, vitamins…). in this domain, there have already been creations that include golden rice, which carries β-carotene, the precursor of vitamin a, banana plants that express a vaccine against hepatitis b and tobacco that produces human lactotransferrin and hemoglobin. if we just look at the production of golden rice as a palliative for vitamin a deficiency, it should be remembered that, in certain countries of our planet, this deficiency affects people's sight and is a frequent cause of blindness, that it generates problems with development and the immune response to infections, that it affects more than a hundred million children around the world and that it is responsible for the death of three million of them each year. if these plants are considered to be a material of choice for the production of proteins with a therapeutic effect, this is partly due to the yield of such crops over large surface areas, and also partly due to the low risk of transmission of viral pathogens to man, because of the species barrier, a risk that is less negligible when animal productions are involved. genetically modified plants are also potential factories for the manufacture of chemical products with an industrial impact, for example lubricants, perfumes and aromas. given the unpredictable outlets that plant gmos may have in human medicine and the different domains of the economy, plant gmo technology should be considered in a manner that is free of any pressure or passion, and, as far as the political authorities are concerned, it should be subject to appropriate measures to surround and protect certain strategic experiments. when looking at the worries being expressed by the european society, it should be remembered that the genetic inheritance of plants has never ceased changing, not only in the most of natural of manners, over millions of years, particularly with the mobility of transposable elements located in the genome, but also artificially, at the hands of farmers from ancient times onwards, with their methods of hybridization and selection. the nervousness of european authorities, showing an ignorance of basic scientific ideas, with the pretext of a principle of precaution, and sometimes political compromises that are exemplified by fluctuating and contradictory positions, runs the risk, in the short term, of causing their countries to lag disadvantageously behind the united states of america, which holds the majority of plant biotechnology patents. the principle of gene therapy is simple: the introduction of an appropriate gene into the cells of a patient who carries a mutation can correct the phenotypical consequences of this mutation, or, in other terms, cure the disease affecting the patient, or at least slow down its evolution. the technical difficulty involved in gene therapy is that of finding an appropriate vehicle or vector for the transfer of the gene and addressing it to an appropriate location in the genome of the host cell. the most commonly used vectors in human gene therapy are viral. a certain number of criteria are necessary for a transfer to be efficacious, including a high concentration of viral particles carrying the gene to be transferred (more than a billion viral particles per milliliter) and a good capability on the part of the foreign gene to be integrated into the host's genome. the patient's immune response remains a major worry in the use of viral vectors: at cell level it often leads to a proliferation of cytotoxic lymphocytes and, especially at humoral level, to the synthesis of antibodies directed against the viral proteins. in order to minimize its immune response, the genetic material of the viral vectors is modified. for ethical reasons, gene therapy is currently only applied to somatic cells, germinal gene therapy being rejected. somatic gene therapy has been experimented in the treatment of hereditary illnesses linked to hematopoiesis. one of the technical reasons for this choice is easy access to the progenitor cells of the bone marrow, with the aim of transfection. it was with this in mind that mouse gene therapy models were developed a few years ago. the sickle cell mouse is one of these models. human drepanocytosis (sickle cell anemia) is a serious disease that is caused by a mutation in the β protein chain of normal human hemoglobin a. the molecules of sickle cell hemoglobin s tend to aggregate and form fibers that obstruct the blood capillaries of the microcirculation. somatic gene therapy has been applied to these sickle cell mice. this involves an autograft of bone marrow hematopoietic cells transfected with a retrovirus hosting the gene coding for the β subunit of normal hemoglobin. encouraging results have shown the validity of this approach. in 2000, a gene therapy protocol that had been applied with success to man was described by the group of alain fischer (b. 1949) and marina cavazzana-calvo at the necker hospital in paris 30 (science, vol. 288, pp. 669-672). the purpose of this therapy was to bring about a long term remission in the case of an immune disease known as scid-x1 (severe combined immunodeficiency linked to a mutation on the x chromosome). because of their susceptibility to microbial and viral infections, babies who are affected can only survive in sterile rooms. they are known as bubble babies. in this illness, the hematopoietic progenitor cells of the bone marrow are unable to differentiate into t and nk (natural killer) lymphocytes because of a mutation that affects a cytokine receptor. previous experiments carried out on model mice show that scid can be corrected by in vivo transfer of the cytokine receptor gene into hematopoietic progenitors. the transfer of the gene of interest paired with a retroviral vector was carried out first in march 1999, in two babies, one of them eleven months and the other two months old. progenitor cells from their own bone marrow, cultured and modified genetically, were injected into them. these were therefore autografts, without any risk of immune rejection. a remission of symptoms over a period of nearly a year, shown by the almost normal behavior of the babies' immune cells, encouraged the application of the same therapy to other babies. in total, ten babies were given this therapy. the enthusiasm that greeted the successes that were recorded was nevertheless tempered by fact that in the spring of 2002, and again in the following year, a child who had undergone the gene therapy developed a leukemia characterized by an anarchical proliferation of lymphocytes, necessitating chemotherapy. these two occurrences were explained by the random character of the insertion of the gene of interest into the patients' genomes: insertion into a site close to a proto-oncogene had led to activation of this proto-oncogene and the proliferation of the lymphocytes. while the trial carried out at the necker hospital gave rise to great hopes, it nevertheless showed that there is still a long way to go before we achieve a targeted transfection of genes so that no undesirable consequences follow. here we have a typical example of the limits of an experimental method that is based on an in-depth technological know-how, but also on a still imperfect understanding of the complex arcana of the mechanisms that regulate the positioning and interaction of genes in the chromosomes of eukaryotic cells. this example highlights a harrowing ethical dilemma: should we not treat a patient whose illness is likely to be fatal, or attempt a therapy that may save the patient, without having any formal assurance of its success? an experimental medicine that has the power to modify the human organism via its genetic material is now able to take over from the experimental method that up until now operated on animals and plants. we can easily understand, given the progress that has already been accomplished and that which is to come in the domain of gene therapy, that the temptation will be great, in the future, to consider manipulations of the human germ cell genome as being licit, insofar as such manipulations make it possible to eradicate a handicapping defect in our descendents. at present, the idea of any attack on the germinal genetic inheritance has been rejected unconditionally on the basis of ethical considerations. nevertheless, the history of science shows that prohibitions that were once considered to be untouchable finish by being contravened. this was the case for abortion. in a text entitled why genetic engineering should continue its battle 31 , james watson writes of his confusion when faced with a choice that is likely to become more and more insistent over the years: "dare we be entrusted with improving on the results of several million years of darwinian natural selection? or do the human germ cells represent on the contrary rubicons that geneticists will never dare to cross?" a mastery of the differentiation of stem cells and of cloning are two essential weapons in the biotechnological arsenal, the use of which for utilitarian ends, particularly in human medicine, gives rise to hope and disquiet, agreement and disapproval. at the beginning of the 1960s, experiments carried out by the canadian biologists ernest mcculloch (b. 1926) and james till (b. 1931) attracted attention to the particular properties of cells in the bone marrow, the stem cells, which would subsequently be found in other tissues 32 . the experimental protocol is simple. bone marrow cells from a mouse are injected into another mouse that has previously been irradiated in order to destroy its stem cells. the injected cells go to the spleen where they divide and form colonies that take the form of nodules of different sizes. the researchers realized that the cells of these nodules present differences in their potential for renewal, which is more or less rapid. they reinjected the nodule cells into mice from a second batch. the reinjected cells showed themselves capable of multiplying and generating several types of blood line. these observations suggest the presence in the nodules of progenitor cells that have a strong potential for self-renewal and self-differentiation. in the following years, these observations were confirmed and explained by two characteristic criteria of stem cells; self-renewal and differentiation into multiples cell lines with specific characteristics. from this point on, it was possible to understand the enigma of the amputated hydra in the experiments carried out by trembley, two centuries beforehand (chapter ii-3.4.2). we now understand why, like the hydra, organisms like the flatworm, the salamander, the starfish and the zebrafish are able to recreate an amputated or damaged part of their bodies. the hydra mobilizes stem cells that it has preserved since its birth. in the case of the salamander, regeneration involves the reprogramming of cells that have already been differentiated. like all stem cells, embryonic stem cells (or es cells) are able to self-renew and differentiate into the different types of known adult cell line, giving rise to different types of cell such as neurons, cardiac cells that are able to contract, or hepatocytes ( figure iv .12). this potential has led to the hope that es cells could be used in regenerative medicine. in a fertilized egg that has developed to the blastocyst stage, it is possible to distinguish a cell mass (inner cell mass, icm) which protrudes inside the blastocyst. the icm cells are removed and placed on a mat of irradiated (and thus unable to divide) fibroblasts that provide them with a support and nutrients (steps 1 and 2) so that they can proliferate. the stem cells arising from the icm cells, placed in a medium that has been specifically conditioned to provide cytokines and other biomolecules, are able to differentiate into various cell types (step 3). at what stage of embryo development is it possible to remove es cells for experimental purposes? after fertilization by a sperm cell, the ovum undergoes a series of divisions that give rise to a microstructure, the blastocyst, the cells of which are called blastomeres. each isolated blastomere remains capable of producing an entire organism of fetus and placenta, by division and differentiation. at this stage, blastomeres are totipotent. five days after fertilization, the embryo has the form of a hollow sphere. an external layer of cells, the trophoectoderm, surrounds a cavity, the blastocele, inside which a small mass of cells, the inner cell mass, protrudes. from the beginning of the implantation of the blastocyt in the uterus, the trophoectoderm evolves to form the placenta. the cells of the inner cell mass take part in the process of differentiation that generates all of the tissues of the future adult organism. these are called embryonic stem cells (es cells). es cells are said to be pluripotent. isolated, they have lost their ability to give rise to a complete individual, but they have maintained the possibility of differentiating, according to their environment, into any of the two hundred cell types that make up animal tissues. during their division, es cells evolve from a stage of being pluripotent to a stage of being unipotent, passing through a stage of multipotency beyond a hundred cells. a state of multipotency characterizes cells that give rise to a restricted number of cell lines in the tissues in which they nest. this is the case for of the hematopoietic stem cells of the bone marrow that form the red blood cells and the white blood cells. the term unipotent refers to the progenitors, which give rise to a single type of cell, for example the hepatocyte of the liver or the cardiomyocyte of the heart. when es cells are cultivated for 4 to 7 days in a conventional nutritive medium, they multiply and aggregate. if the culture medium is supplemented with certain biomolecules such as insulin, retinoic acid, transferrin or fibronectin, the differentiation of the es cells is oriented towards cells of different types, such as neuron cells, glial cells or muscle cells. there are many publications about experiments concerning the grafting of differentiated stem cells in the mouse or the rat. for example, neuron precursors derived from the spinal cord or the brain are grafted into rats whose spinal chords have been injured. five weeks after the grafts are carried out, the transplanted cells have filled the area of the injury and differentiated into oligodendrocytes, astrocytes and neurons. what is more, after about twelve weeks, locomotive function has been partially restoredirradiated 33 . other experiments involving the grafting of differentiated stem cells have been carried out on rats in which the dopaminergic neurons of the "substantia nigra" of the brain that secrete the neurotransmitter dopamine have been selectively destroyed by injection of 6-hydroxydopamine. the problems found in the rat as a result of this neuronal degenerescence mimic those found in man in patients suffering from parkinson's disease. dopaminergic neurons obtained by the differentiation of mouse es cells are grafted into the striatum of each of these rats, a region of the brain whose neurons communicate with those of the substantia nigra and play a fundamental role in the control of movement. this results in a significant improvement in the motor deficit, coupled with the establishment of functional synapses between the injected neurons and those of the host 34,35 . a recent publication bringing together the results of two french research teams, that of michel pucéat (b. 1961) in montpellier and that of philippe menasché (b. 1950) in paris, provides interesting information about how mouse embryonic stem cells, grafted into sheep cardiac tissue where an infarctus has been artificially induced, are able to colonize the infarct zone and regenerate cardiac contraction in a functional manner. moving from the mouse to the sheep constitutes a considerable species leap, and the absence of any immune rejection leads us to say that embryonic stem cells have an "immune privilege" 36 . the use of es cells in regenerative medicine necessarily requires that their differentiation be regulated in an exhaustive manner into well-defined pathways, in order to produce homogeneous cell lines with a view to implanting them in damaged tissues. in fact, contamination with non-differentiated es cells is likely to cause tumors (teratomas) over the long term. the mastering of the use of es cell culture and differentiation, as well as of cloning, in such a way as to overcome problems of histocompatibility, is still in its infancy. for a long time, the mouse was the preferred animal model for experimental studies on the differentiation of es cells. in 1981, the first es cells from mouse blastocysts were isolated and successfully cultured by two groups of researchers in great britain and the usa. it was only in 1998 that human embryonic stem cells (hes) were isolated for the first time 37 and held in culture, on a nutritive layer of fibroblasts from irradiated mice. this delay with respect to the ability to culture animal es cells can be explained by the fact that the molecular machinery that activates replication and cell differentiation programs is not completely identical in man and the mouse 38 . for example, a cytokine called lif (leukemia inhibitory factor), which is indispensable for the renewal of es cells in an undifferentiated state in the mouse, has no effect on human es cells. there are several other differences concerning the control of proliferation and differentiation in human and murine es cells by growth factors. briefly, the conclusions obtained from experiments carried while there is a highly promising future for the use of es cells, this future is littered with obstacles, and rigorous checks and balances need to be put in place. nevertheless, research on such cells is mandatory if we wish to move on to a regenerative medicine that aims to be a new frontier in the art of healing. after specific differentiation, hes cells could provide unlimited quantities of the tissues needed to replace damaged tissues responsible for handicapping illnesses (dopaminergic neurons in parkinson's disease, cardiomyocytes in myocardial infarction, pancreatic islets of langerhans cells in diabetes, fibroblasts in skin grafts, chondrocytes in rhumatoid arthritis). in addition, metabolic analysis of hes cells carrying defective genes whose phenotypical expression is known in human pathology should improve our understanding of the perturbed mechanisms, and could lead to pharmacological advances. as well as the technical difficulties involved, which have not yet been adequately overcome, the handling of hes cells is subject to much ethical debate in many countries, with those who object to it holding to their prejudices, which are linked to religious or cultural traditions. this is the case in france, where, nevertheless, a few timid dispensations had begun to appear at the time of writing. in contrast, in great britain, the law authorizes the isolation of hes cells for therapeutic purposes, using embryos of less than one hundred cells, produced by in vitro fertilization, and surplus to requirements. the british response to the burning question of whether an isolated hes cell may be considered as a potential human embryo is clearly "no", for, in order to be able to develop in utero, such hes cells would need to have the placental progenitor cells. an alternative to the use of es cells is to make use of adult stem cells. however, the proliferation capacity of adult stem cells is considerably lower than that of their embryonic homologues. the hematopoietic stem cell is the paradigm of the adult stem cell. it can differentiate into all known types of cells. in the last decade of the 20 th century, several publications concerning the plasticity of the adult stem cell awakened a hope that these cells could transform the treatment of degenerative illnesses. certain of these publications stated that adult bone marrow stem cells, implanted into different types of tissues, differentiate into hepatocytes, cardiomyocytes or neurons, depending on the specific environment. careful re-examination of the techniques used revealed that, in certain cases, interpretation of the results as showing cell transdifferentiation was an erroneous one, and that the fusion of the bone marrow stem cells with cells from other tissues was a more plausible explanation. in any case, while not ignoring the use of adult stem cells, experimentation on hes cells remains a judicious choice, given our current state of understanding. in france, the 2004 law application decree that was issued on the 7 th of february 2006, revising the restrictive bioethical standards of 1994, opens up the possibility of using human embryonic stem cells for scientific purposes, with certain ethical reserves being maintained. one of the obstacles to the stabilization over time of a stem cell graft in a receiver involves the phenomenon of rejection for reasons of histocompatibility. considered to be foreign by the receiver (host), grafted stem cells coming from a donor are rejected. this obstacle could be overcome by using the technique of cloning. based on experiments on several animal species, it is now accepted that the transfer of the nucleus of an adult somatic cell from a host into an enucleated oocyte makes it possible to obtain from this oocyte, which is once again nucleated, and which is the equivalent of a zygote and able to divide, es cells whose genome is identical to that of the host. because of this, the es cells are immunologically compatible with the tissues of the host. in man, such cells could be directed by differentiation towards stable cell lines creating well-defined tissues and organs (liver, muscle…) that could be used in regenerative medicine. this is the principle of therapeutic cloning. in march 2004, korean veterinary researcher woo suk hwang (b. 1953) and his co-workers, who were recognized experts in animal cloning, announced in the american review science 39 that they had succeeded for the first time in obtaining around thirty human blastocysts by cloning, i.e., by the transfer of nuclei of somatic cells into enucleated ova. this first experiment involved autologous cloning (ovum nuclei and enucleated somatic cells taken from the same woman). hwang and his team used 176 ova, and the yield from the experiment was close to that obtained at that time for the cloning of mammals. using the inner cell mass of one of the blastocysts, they isolated a line of embryonic stem cells able to maintain a normal karyotype after several dozen divisions. this publication, which appeared in a highly prestigious scientific review, triggered an enthusiasm in the media that was in keeping with the spectacular nature of the team's exploit, tempered here and there by a few comments that were mainly linked to questions of medical ethics. in 2005, there were numerous other articles by the same team on the same subject, reinforcing the first results with a heterologous cloning technique (ovum nuclei and enucleated somatic cells taken from different people), thus giving rise to great hopes that the era of regenerative medicine was near. at the beginning of 2006, professor hwang's retractation of all his work, and a public confession of a spectacular fraud, were even more dramatic, offering certain media an occasion for a disproportionate level of fury against therapeutic cloning. however, despite such rear-guard combats, it is obvious that one day these technical difficulties will be overcome. human cloning, in order to obtain stem cells for therapeutic purposes, cannot escape the future. once this aim has been achieved, it will be spoken of as the outcome of a long story. the adventure of animal reproductive cloning began in 1960. in developmental biology 40 , two american researchers, robert briggs (1911 -1983) and thomas king (1921 -2000 described experiments involving the transfer of cell nuclei of embryos from a frog (rana pipiens), at the blastula and gastrula stages, into enucleated eggs of the same species. a high percentage of the clones obtained in this way were able to reach the tadpole stage when the transferred nuclei came from the early blastula stage, but only mediocre success was achieved when the nuclei came from the later gastrula stage. these experiments emphasized both the totipotency of the embryo somatic cells and the equivalency of the somatic cell nucleus and the nucleus of the fertilized egg in cell division and differentiation. briggs and king's publication did not arouse any particular interest. it is true that the 1960s were dominated by the saga of molecular biology, which would reach its culmination in the deciphering of the genetic code. from the 1980s onward, the first attempts to clone mammals (rat, mouse, pig) began. moving from the amphibian egg, which was a millimeter wide, to a mammal egg that was one hundred times smaller, presented a technical difficulty that would be overcome by a technique of cell-to-cell electrofusion. cloned embryos were thus obtained by nuclear transfer and then implanted into the uterus of a surrogate female. however, in all cases, the nucleus came from embryo cells. in february 1997, the announcement made by ian wilmut (b. 1944), keith h. campbell (b. 1954) and their collaborators at edinburgh's roslin institute 41 of the birth of the cloned lamb dolly had an immediate effect in the media. in fact, this was not only the cloning of a higher mammal, but, above all, the cloning by insertion of an adult somatic cell, in this case a mammary tissue cell, into an enucleated oocyte. this went far further than the experiment carried out by briggs and king, which essentially involved the transfer of embryo cell nuclei into enucleated frog eggs. the trick that gave wilmut and campbell their success was to bring the cells providing the nuclei to a quiescent state corresponding to the interphase stage of the cell cycle, by impoverishing their culture medium, before electrofusion with enucleated oocytes. although we should be aware that 434 attempts were made before a positive result was achieved, this does not make it any less astonishing that the nucleus of a cell in its adult state, i.e., completely differentiated, was able to behave as if it were totipotent. despite being committed to a program of differentiation that is considered to be more-or-less irreversible, and which will give it a specific identity, the nucleus of an adult cell can be reprogrammed and become totipotent. since dolly, many other mammals have been cloned from nuclei of adult cells; mice, cows, goats, pigs, rabbits, cats, dogs, rats and horses. as far as ethical discussion about cloning is concerned (chapter iv.5), it is essential to note that the demarcation line between reproductive cloning and therapeutic cloning is situated where decisions are made concerning the destiny of the cloned blastocyst ( figure iv .13). the transfer of the nucleus of a somatic cell (liver, epidermis, muscle) containing 2n chromosomes into an enucleated oocyte gives rise to an egg (2n chromosomes) that is able to divide and to produce a blastocyst. the cells of the blastocyst inner cell mass (icm) can be used as stem cells that can differentiate into different types of cell line (therapeutic cloning). on the other hand, if the whole blastocyst is implanted into a uterus, it will produce an embryo which, after birth, will grow into an adult animal (reproductive cloning). reproductive cloning and therapeutic cloning therefore differ because of the fact that in reproductive cloning, the whole blastocyst is used, while in therapeutic cloning, only certain cells, corresponding to the inner cell mass (icm) of the blastocyst, are used. the structural and functional identity of the cells of a given tissue in an adult organism involves a basic mechanism: while each cell has the same set of genes, only some of the genes are expressed as proteins and the genes that are expressed differ according to the tissue involved. the key to the mechanism responsible is in the epigenetic type chemical modifications of cell dna, for example methylations, which repress the expression of certain genes without altering the expression of others. these modifications of the dna, which control cellular specificity (muscle, liver, brain…) are not very reversible, but, in certain circumstances, they can become so. this is what happens from time to time when the nucleus of an adult cell is inserted into an enucleated oocyte. we are thus able to assume that in the molecular arsenal of the oocyte cytoplasm there are substances that can cancel the epigenetic modifications of the dna present in the nucleus of an adult somatic cell and recreate a state of pluripotency in this nucleus, or, in other words, provoke the reprogramming of the somatic cell nucleus. in the long term, it is to be hoped that biochemical technology will be able to find and purify the molecules responsible for the nuclear reprogramming of somatic cells. the use of human oocytes for the purpose of therapeutic cloning is still subject to severe criticism. certain groups wish it to be prohibited, because of a fear of a drift towards reproductive cloning. to obviate this risk, the idea has been to make use not of human oocytes but of those of animals, transferring the nuclei of human somatic cells into them. even supposing that the technical difficulties involved could be overcome, the cells that would result, a sort of man-animal chimera, would also be the subject of an ethical debate, even if the purpose of this type of cloning were to be solely therapeutic. some japanese researchers 42 have succeeded in creating mice according to a parthenogenetic process that involves adding the nucleus of an oocyte that is haploid (1n chromosomes) to another haploid oocyte, the result being the equivalent of a fertilized egg (2n chromosomes). this exploit is achieved by the invalidation of one of the genes (h19) involved in the control of the parental imprint. it is known that sexual reproduction in mammals involves a phenomenon called the parental imprint, which, by means of the methylation of dna and perhaps also of histones, allows the expressing or silencing of certain genes in male and female gametes. a single copy of a given gene, originating either from the oocyte or from the sperm cell, is therefore expressed, while the other is inactive. in the japanese experiment, if the mouse h19 gene had not been invalidated, the result would have been an anarchical development of the responsible genes involved in the parental imprint with overexpression in the case of some and an absence of expression in the case of others. these disturbances would have been incompatible with the viability of the embryo. however limited its application might be, the manipulation of the germinal genome poses the problem of the mechanism by which the parental imprint intervenes in the viability of the egg, a parameter that at the time of writing is still not completely understood, but is being actively explored. in boston, massachusetts, in 1954, a kidney was transplanted from a healthy boy into his twin brother, who was suffering from a fatal renal anomaly. the success of this graft ushered in the era of transplantations of such organs as the heart, liver and kidney in man. in order to try to prevent the rejection of grafts, caused by an immune incompatibility between the receiver and the graft from the donor, different immunosuppressing treatments were tried, one by one, involving corticosteroids or cytostatic agents such as 6-mercaptopurine. in the 1980s, a decisive step forward was made with the fortuitous discovery of the powerful immunosuppressive effect of the cyclosporin a, a cyclic polypeptide isolated from the mold tolypocladium inflatum. each year, human organ transplants into patients make it possible to save many lives. however, for some time now, organ transplantation has been suffering from penury of donors. one alternative to the homograft is the grafting of animal organs, or the xenograft, and, at the dawn of the 21 st century, this type of graft has entered an active, promising phase, with the creation of pigs that have been partially "humanized" and are thus, as a consequence, immunocompatible. for reasons of genetic and physiological similarity, the first choice for such grafts was to use apes or monkeys. however, this idea was quickly abandoned, for several reasons; a non-negligible risk of viral infection due to the phylogenetic kinship of human and simian species; a slow growth rate; a low reproduction rate and, finally, laws that protect primates. these disadvantages are not found, or are at least minimized, in the pig: the risk of a viral infection passing from the pig to man should be low because of the species barrier (but nevertheless, it ought to be evaluated), the pig growth rate is relatively rapid, pig litters are large and pig organs are of a size close to those of man. hyperacute rejection of grafts is the critical obstacle that must be overcome before it is possible even to envisage the feasibility of xenotransplantation. hyperacute rejection is caused by the presence in man of natural antibodies (xenoantibodies) that accumulate throughout a lifetime and are directed against antigenic motifs carried by the products of the digestion of food or dust that is breathed in. xenoantibodies are mobilized when a xenograft occurs, and when they combine with xenoantigens brought by the graft this activates immune proteins such as the complement proteins. the catastrophic effect of this xenoantibody/xenoantigen combination is a vascular thrombosis followed by necrosis and rejection of the graft. the pig xenoantigen that is considered to be the one mainly responsible for the phenomenon of rejection in man is a sugar molecule, galactose α-1,3-galactose, located on the plasma membrane of endothelial cells. synthesis of this molecule requires the enzyme α-1,3-galactosyltransferase, which is present in most mammals, but absent in man and the primates. this enzyme disappeared in man around twenty million years ago, following a double mutation of the gene. in 2002, cloning by nuclear transfer, associated with the invalidation of the gene coding for galactosyltransferase, made it possible to create pigs without galactose α-1,3-galactose 43 . this performance shows that the xenotransplantation objective, although it can only be envisaged over the long term, is not based on false hopes. plant gmos, gene therapy, embryonic stem cells, therapeutic cloning, and xenotransplantation are a few of the many examples that show how far experimentation on living beings has progressed in just a few decades, from inquiries into the operating mechanism of an organ or a cell, in the interests of pure understanding, to a programmed process, planned with an objective in mind, the chances of success of this objective being analyzed and counted in terms of impact and cost-effectiveness. during the renaissance, ecclesiastical authorities, worried by the libertarian forces that were assailing them, applied the brakes to audacious questioning of dogma such as the circumterrestrial revolution of the sun that had, since ancient times, placed man at the center of the universe. nowadays, civil authorities, conscious of the potential but also of the possible misuses of genetic manipulation, insist on having the right to oversee such procedures. in truth, since the 19 th century, governments have been interesting themselves in research on living beings and encouraging it, as long as its applications have allowed improvements in human health. this has been the case for vaccinations against infectious diseases or for prevention of microbial infections by means of aseptic or antiseptic methods. with the breakthroughs made in genetic manipulation at the end of the 20 th century, it was more than just the results of experiments on living beings that attracted the attention of the political authorities, it was, above all, the manner in which the experimental method, with all its hazards, made use of living material, sometimes of human origin, in order to unlock mysteries. conscious of the social impact of emerging discoveries that are subject to considerable media coverage and are sensationalized in both the written and audiovisual media, the state, with the help of researchers and philosophers, has laid down a code of bioethics, applied through strict or even restrictive legislation. it remains to be seen whether the rules of this code will continue to be an inviolable absolute or will be modified according to the evolution of the moral codes and the cultures of nations. university teaching and the education of a society must now take into account not only the content of successive discoveries, but also the fallout of these discoveries, insofar as they concern man, and even the ethical justification of the methods that have allowed these discoveries. in "remaking" living beings according to imposed norms, and in scheduling, in a certain fashion, the manufacture of life according to new codes, certain questions move from the "how" to the "why", i.e., from the scientific domain that is accessible to human thought to the metaphysical sphere, with its problems of the limit of what is surmountable and tolerable in terms of ethics. in his birth of predictive medicine, jacques ruffié (1921 -2004) reminds us that medicine has evolved through three stages over the course of time: curative medicine, which has been practiced since ancient times and is still being practiced; preventive medicine, which is more recent, and is designed to prevent people from falling ill, either by vaccinating them, in the case of infectious diseases, or by recommending an appropriate diet and medication in the case of metabolic disorders such as diabetes or arterial hypertension that have been detected by means of systematic examination; and, finally, predictive medicine, a branch of medicine that is still in its early phases, and which is based on modern technology and is able to predict situations of risk because of anomalies detected in the genetic inheritance or because of exposure to environments that are reputed to be dangerous (for example, carcinogenic smoke, asbestos). about one and a half centuries ago, the publication of the introduction to the study of experimental medicine (1865) provided proof, based on scientific arguments, that the time had come to transfer the experience that had been acquired through the experimental method practiced on animal models to the ill person. after claude bernard, attentive to the progress made by ideas and techniques in the physical and chemical sciences, and making use of its own advances in the understanding of the living cell, both normal and pathological, experimental medicine was to live through a development that was without precedent in the history of humanity. to understand the causes of epidemics, nutritional deficiencies, metabolic deviations of hereditary origin and degenerative illnesses, and then to translate these causes in cellular and molecular terms, this was the process undertaken by medicine once it began to use the experimental method. in fact, for several decades, from the beginning of the 19 th century, medicine had already undergone some major revisions of outdated practices and had inaugurated a new era in diagnosis. for example, the differential diagnosis of pulmonary ailments became possible because of the invention of the stethoscope by rené laennec (1781 -1826) and the practice of auscultation and percussion by joseph skoda (1805 -1881), the uncontested master of the vienna school. in france, pierre louis (1787 -1872) used statistical methods to evaluate the efficacy of different treatments. armand trousseau (1801 -1867), a pupil of pierre bretonneau (1787 -1862) wrote a famous treatise on the hôtel-dieu medical clinic in paris. in great britain, chronic nephritis, with its identifying symptoms, was described by richard bright (1789 -1858), paralysis agitans by james parkinson (1755 -1824) and addison's disease, which affects the adrenal glands, by thomas addison (1793 -1860). during the 19 th century, many other famous names signaled the arrival of a medicine that was resolutely anatomoclinical in nature, in line with bernardian doctrine. "experimental medicine is thus a medicine that claims to understand the laws of the organism in sickness and in health, in such a way that it not only predicts phenomena, but also in such a way that it can regulate and modify them, within certain limits." in the introduction to the study of experimental medicine, claude bernard stigmatizes the relics of an empirical medicine that was still being practiced in his day and was forgetful of rationalism. the terms that he uses are without leniency: "i have often heard doctors who, when asked the reason for a diagnosis, reply that they don't know how they recognize such a case, but it is obvious, or who, when asked why they administer certain remedies, reply that they don't really know how to put it exactly, and that anyway they are not required to give a reason, because it is their medical tact and their intuition that guides them. it is easy to understand that doctors who reason in this way are denying science. what is more, it is impossible to be too forceful in rising up against such ideas, which are bad not only because they stifle any scientific seed in the young, but also, above all, because they favor laziness, ignorance and charlatanism." in order to evaluate the meaning of these words, it should be remembered that in claude bernard's time, the medical profession was far from considering the microscope as a useful instrument for the study of cell structures and that the cause and effect relationship between bacterial germs and infections was still to be shown. with the development of increasingly effective instruments for exploration, and of methods for microanalyses concerning a wide range of blood and humoral constants, throughout the 20 th century, medicine, which was once empirical, has now become scientific. claude bernard's dream, experimental medicine, is now operative. this medicine is no longer content simply to determine the cause of an illness and to locate the affected organ, which was the major objective of clinical medicine, but it seeks to detect the mechanisms of pathological processes by means of histological and physicochemical explorations. this medicine is no longer willing to passively monitor the evolution of an infectious disease. after having identified the responsible germ, it tries to target this germ with the chemical weapon that is able to selectively destroy it. this medicine is no longer content simply to find remedies, it aims to understand the mode of action. it sets itself the goal of meeting challenges such as finding the genetic cause of degenerative illnesses or of cancers and developing appropriate therapies. it is supported by statistical data. when a new drug is implemented, the results are now evaluated by the double blind method: neither any of the patients (treated and non-treated) nor any of the investigators are aware of who has been administered with the drug and who has been administered with a placebo. in the surgical domain, audacious techniques have also led to considerable progress, particularly in neurosurgery and in cardiovascular surgery. thanks to robotics and to computer technology, remote surgery or telesurgery has become practicable, although up until not that long ago, it was only to be found in fiction. faced with emerging problems in public health, the task undertaken by experimental medicine is immense. in the middle of the 20 th century, the spectacular recovery from high-incidence infectious diseases such as pneumococcal pneumonia, meningococcal meningitis or acute forms of tuberculosis, which that was brought about by antibiotics, gave rise to the idea that medicine had won a battle against the microbial world and that, from then on, it would be able to control the evolution of infectious diseases and to offer rational treatments. the gradual appearance of a microbial resistance to antibiotics has brought an end to this euphoric era. penicillin, for example, which was put on the market at the end of the 1940s, was active on practically all strains of staphylococcus aureus. sixty years later, more than 90% of the strains of this same microbe are resistant to penicillin. the incidence of nosocomial infections, which are contracted in health care facilities, never ceases to rise. at present, around 10% of the hospitalizations that take place are complicated by the patient developing a nosocomial infection. equally worrying are the re-emergence of diseases that were once considered to be under control, such as tuberculosis or poliomyelitis in africa, and the emergence of new diseases such as aids, whose hiv virus (human immunodeficiency virus), which was identified at the beginning of the 1980s, has generated a pandemic that has spread throughout the planet. infectious diseases are currently responsible for more than a quarter of human deaths. the koch bacillus responsible for tuberculosis and the pneumococcus kill three to four million people a year, around the world. in 2004, hiv killed more than three million people, and more than forty million people are infected. one person is infected every 30 seconds. in viral diseases, the role of vectors (insects, various animals) as well as the notions of contagiousness and aggressivity have been emphasized. we have only to remember the dreadful contagiousness and aggressivity of the spanish flu virus (influenzavirus ah1n1) which, in 1918 -1919, killed more human beings around the world than the first world war that preceded it. in contrast, the sars (severe acute respiratory syndrome) epidemic of 2003, the vector of which was doubtless the civet, a small carnivore raised in china and desired for its meat, was rapidly contained because of its low contagiousness and also because of the isolation measures that were taken. human behavior is not without its effect on the emergence of viral diseases. the growth in intercontinental travel and human migration, as well as intensive deforestation in africa and south america, which bring virus vectors into contact with man, are factors concerned in the emergence of viral diseases that risk being explosive and devastating. in this context, the history of the ebola virus and of the marburg virus, which cause violent hemorrhaging, is edifying. in 1967, in the german village of marburg, an epidemic of unknown origin broke out, the illness manifesting itself with brutal suddenness by vomiting, diarrhea, a high fever and an increased tendency to bleed. this pathology, which was contained rapidly by means of drastic isolation measures, was found to be of viral origin. the pathogen concerned was a filovirus (filiform virus). a brief enquiry showed that the origin of the epidemic was contact between technicians of a pharmaceutical company and monkeys that had been imported from uganda and that were carrying the virus. in 1976, two other epidemics, characterized by severe and often fatal hemorrhagic fevers, were reported in the sudan and the republic of the congo. here again, the illness was caused by a filovirus, the ebola virus. at the time of writing, only public health organizations, including the nih (national institutes of health) in the usa, have attempted to set up vaccination and therapeutic strategies. research on these dangerous viruses requires high security installations that are particularly costly, so that private companies are reticent about investing in work that is only targeted on poor regions and which concerns epidemics that have so far been contained successfully, although one day the ebola and the marburg virus could quite well escape their african niches. experimental medicine must also understand the colossal challenge of the five thousand hereditary diseases that are currently listed, the most handicapping of which are myopathies and neuropathies. given the means that are available to the contemporary clinician in order to assign each of these diseases to a genetic defect, one can only be amazed by the mass of information about them that has accumulated over a century, since the first diagnosis of a hereditary disease, alcaptonuria, which was made in 1902 by archibald garrod (1857 -1936), a doctor at london's st bartholomew's hospital. alcaptonuria is a non-serious genetic flaw that can be detected easily by a blackening of the urine. it is the result of a blockage caused by the mutation of an enzyme involved in the catabolism of an amino acid, tyrosine, this blockage leading to the accumulation of homogentisic acid, the polymerization of which gives rise to a brownish color. the patient examined by garrod was a young boy. investigation of the family history revealed that transmission of the flaw was correlated to cross-cousin marriages and followed mendel's laws for recessive traits. garrod demonstrated other hereditary-type anomalies, cystinuria, porphyria and pentosuria. in 1909, these observations were published in a work that became a classic: inborn errors of metabolism. in 1956, the specific molecular defect of a metabolic anomaly linked to a mutation was identified for the first time by the german-born british biochemist vernon ingram. this was the hemoglobin defect responsible for drepanocytosis or sickle cell anemia: a glutamic acid in the β chain is replaced by a valine. the consequence of this simple change is a modification of the structure of the hemoglobin, leading to a sickle-shaped deformation of the red blood cells, the increased fragility of these cells and also a tendency towards cell lysis. this discovery made use of the electrophoresis and chromatography techniques that had just been introduced in biochemistry (chapter iii-6.2.2): such a discovery would not have been possible without these techniques. because of the progress made in molecular biology, the nosological framework of hereditary diseases has been greatly enriched over the last twenty years. for example, at present, more than one hundred hereditary-type myopathies have been identified by accurately locating molecular lesions in the genomic dna and characterizing the structural and functional modifications of the mutated proteins. certain health problems present real challenges for experimental research. this is the case for the spongiform encephalopathy caused by a prion (proteinaceous infectious particle) , which has all the more impact on the imagination because its etiology remains a mystery. it is also the case for degenerescence of the central nervous system correlated with aging, alzheimer's disease being a striking example, although, as far as familial forms of this illness are concerned, i.e., those of the hereditary type, it has been possible to link the invasion of the brain by a so-called amyloid peptide, which accumulates in plaques, on the one hand, and, on the other hand, the absence, due to a mutation, of an enzyme, a peptidase, which normally degrades the amyloid peptide. contemporary scientific medicine sometimes acquires a revolutionary aspect. here again, as with other disciplines involved in the study of living beings, it arises from discoveries resulting from the principle of serendipity (chapter iii-2.2.3). this was the case when, in january 1987, a team in grenoble, france 44 , led by the neurosurgeon alim-louis benabid (b. 1942) and the neurologist pierre pollack (b. 1950) discovered by accident that in patients affected by parkinson's disease a beneficial effect was achieved by deep, high-frequency electrical stimulation of the brain. the three major symptoms of parkinson's disease are muscular rigidity, a tremor when at rest and a slowing down of the execution of movements. in the 1960s, the swedish team of arvid carlsson (b. 1923) , who won the nobel prize for physiology and medicine, demonstrated a relationship between the parkinson syndrome and a deficit in the secretion of a neurotransmitter, dopamine. a group of neurons that is limited to half a million (of the 100 billion contained in the brain) produces this neurotransmitter in a small structure located in the midbrain, called the substantia nigra. the neurons of the substantia nigra have elongations that interact with different nerve formations (called nuclei) including the subthalamic nucleus. in 1990, bergman et al. 45 published an article that describes a curious relationship between a provoked lesion of the subthalamic nucleus and the disappearance of the signs of parkinson's disease in a monkey which had been made parkinsonian by chemical treatment. this publication led the team in grenoble to target their electrical stimulation on the subthalamic nucleus. this was completely successful. this electrical stimulation procedure, which is now well-codified, involves using stereotactic neurosurgical techniques, controlled by magnetic resonance imaging, to implant an electrode into the subthalamic nucleus. the electrode is connected to a generator that is implanted under the patient's clavicle. the generator sends brief electrical impulses of frequencies from 100 to 200 hz. under the effect of this stimulation, the characteristic symptoms of the illness, particularly the static tremor and bradykinesis, regress in a spectacular manner. the mechanism by which this stimulation acts is not yet understood. no doubt this has to do with complex phenomena involving the inhibition of certain neuron relays near to the substantia nigra, which remain to be deciphered. here we have a typical case of a progression from an experimental fact, discovered by accident, towards the analysis of its cause. from the point of view of the experimental method, it is interesting to make parallels between this discovery by serendipitous means of the beneficial role of electrical stimulation of the midbrain in parkinson's disease and cartesian style programmed research that aims to graft into the brain of parkinson's disease sufferers embryonic stem cells differentiated into dopaminergic neurons 46 . civilian society and its armed force, the political authorities, have understood that experimental science has the tools, the method and the thought processes necessary to develop strategies for prevention and healing. immune cells of this person and induces the synthesis of immune proteins. finally, it seems relatively certain that hopes concerning gene therapy for hereditary diseases will be fulfilled within before too long (chapter iv-2.3.1). one of the traits that is characteristic of the period we live in, and which arises partly from the economic stakes involved, is the shortening of the time that elapses between a discovery being made and the application of that discovery. for example, interfering rnas, which were discovered in the 1990s (chapter iv-1.2.2) are already the subject of therapeutic investigation. more than a hundred biopharmaceutical companies around the world are using them with a view to producing drugs from them 47 . in mice, a certain number of synthetic interfering rnas have proved their efficacy in silencing genes which, following mutation, have acquired carcinogenic potential. however, the use of interfering rnas as therapeutic agents requires them to be stabilized, because they are fragile molecules. the group headed by achim aigner 48 (b. 1965 ), at the school of medicine in marburg, germany, managed to stabilize a synthetic interfering rna by complexing it with polyethyleneimine, and this interfering rna is able to block the expression of a receptor involved in cancerization (c-erbb2/neu(her-2) receptor). used in mice, such a drug appears promising. despite the undeniable progress that has been made, experimental medicine is still some way from finding solutions to some of the enigmas that it meets along the way, and which underline the complexity of living beings. some time ago, it was thought that after having invalidated a gene coding for a protein that is indispensable to a function, we would discover the secret of a cause-and-effect relationship. experimental practice has shown that, generally, this is far from being the case. another example of the complex relationships that exist in living beings is the interference of the mental and the organic. one experiment that suggests this interference was carried out on mice who had acquired a form of pathology similar to huntington's chorea, by transgenesis. mice from the same line were separated into two batches, one acting as a control, and the other being subjected to daily mental stimulation, including memorization tests. unexpectedly, the appearance of symptoms was noticeably slowed down in mice who had been subjected to mental gymnastics 49 , as if the brain, by intentionally mobilizing its neuron activity, was able to secrete substances able to alleviate its own defects. in short, by means of possible retroactive mechanisms that are called upon by the mind, the brain appears to act as actor and spectator. at the turn of the 21 st century, experimental medicine was being nourished by techniques inherited from experimental physics, chemistry, and even mathematics and computer technology, in the same way as the other sciences of living beings. the progress made in medical imaging techniques has been particularly impressive since the time, at the end of the 19 th century, when the x-rays discovered by wilhelm röntgen made it possible to view the structure of the human skeleton. the saga of x-radiation continued through the 20 th century (chapter iii-2.6.1). for the last few decades, new imaging techniques have come to the fore. they have spread rapidly, and been refined. ultrasound imaging, which is based on the principle of the reflection of ultrasound waves off of different kinds of surfaces, has become an everyday technique for viewing blood flow in blood vessels and the heart. however, it is mainly in the study of the brain that medical imaging has benefited from technical advances in the domains of physics and computer technology, and it has been innovative in assigning cognitive activities to well-identified anatomical structures. this functional neuroanatomy makes it possible, in a non-trauma-inducing manner, to monitor and locate the operation of neuron networks with great temporal and spatial precision, during various cognitive tasks such as reading and the written or oral expression of thought. the middle of the 20 th century saw the gradual development of two methods for exploring zones of cerebral activity, electroencephalography and magnetoencephalography. at present, these techniques are being taken over by mri (magnetic resonance imaging) ( figure iv.14) . the principle of mri is based on the detection of hydrogen nuclei and their differentiation according to their environment. functional mri leads to the location of the areas of the brain that are active during calculation exercises, the perception of sounds, language and objects, and memorization, with a resolution of just a few millimeters. its power of exploration is such that it has been possible to analyze the brain response, in sleeping or awake babies who are only three months old, to auditory stimuli from language that either makes sense or does not make sense 50 . the response, located in the left hemisphere and the prefrontal cortex, leads to the conclusion that, from the first months of life, there are zones of the brain that are potentially active before the first attempts at language appear. both in france (cea-saclay and the frederic joliot hospital at orsay) and abroad, recent mri performance has encouraged projects concerning the manufacture of instruments able to produce magnetic fields of around ten teslas, which allows an unequaled definition in the identification of areas of the brain assigned to specific cognitive functions and in the highly accurate determination of the location of pathological lesions. a technique that is complementary to mri is positron emission tomography (pet). this generally uses water labeled with oxygen 15 ( 15 o), a radioactive isotope of natural oxygen that has a very short lifetime (123 s), produced extemporaneously in a cyclotron by bombardment of an 14 n target with protons. the radiolabeled water is injected into the blood flow of the patient. it is found in greater concentration in the zones that are the most irrigated by blood capillaries. the positrons that it emits collide with the surrounding electrons and give rise to photons that can be detected by the appropriate apparatus. affected by a stimulus (whether this stimulus results from talking, writing or listening), the blood irrigation of the zones of the brain that have been specifically excited increases noticeably. the location of the positron emission provides information about the location of these zones. within a few dozen minutes, it is possible to locate a highly vascularized cerebral tumor. pet can use molecules other than water, such as organic molecules labeled with positron-emitting atoms, ( 18 f) fluorine (half life 110 min) and ( 11 c) carbon (half life 20 min). around twenty years ago, in canada, an analogue of l-dopa, the precursor of dopamine in the brain, 18 f-6-l-fluorodopa, was synthesized, and was found to be an excellent probe for determining the capture capability of the endings of the dopaminergic neurons in the striatum. in patients suffering from parkinson's disease, this capture capability is noticeably reduced. at present, pet involving 18 f-6-l-fluorodopa is being used to evaluate the survival of dopaminergic cells grafted into the striata of parkinson's disease sufferers 51,52 . nowadays, brain imaging techniques can be used to explore the electromagnetic anomalies of neurological or neuropsychiatric illnesses such as huntington's chorea, the different forms of alzheimer's disease or even autism, the genetic origin of which is in the process of being deciphered. a bridge has now been built between the molecular defects identified by genetics and the electromagnetic anomalies that result, analyzed by functional cerebral imaging. it was not so long ago that descartes considered that human thought was unconnected to a material support (chapter ii-3.4.3). we are not far from the era when broca located the language area in a specific zone of the brain after the autopsy of an aphasic patient (chapter iii-3.1), thus opening the door to another scientific domain, neuropsychology, which had previously only been the subject of speculation. the consequences, from the societal point of view, were far from being insignificant. thus autism, which was once suspected of being caused by errors in the mother's behavior with respect to her child, has been shown to be a disturbance in the development of the fetal nervous system, in the temporooccipital region. while the neurosciences occupy a preponderant position in the medicine of the beginning of the 21 st century, because of the development of techniques that aim to analyze even the functions of thought, emerging methodologies of another order, such as gene therapy (chapter iv-2.2), are in the process of completely modifying our ways of treating and curing a range of previously incurable human diseases, from incapacitating immune disorders to cardiovascular diseases and cancer. "it is in the domain of thought about the future that man is singled out. we are beings who have an imagination. not content to live in the present, to profit from past experience, we remain haunted by a future that we are conscious of constantly entering. this obsession with the future has been a powerful driving force in cultural evolution. we seek to predict in order to avoid the worst and to better prepare for our tomorrows." by predicting potential dangers in subjects who are in good health, predictive medicine aims to provide the means of avoiding these dangers. these dangers can be intrinsic in nature, being written, for example, into a certain genome dna sequence, or they can be extrinsic in nature, linked to an unsuspectedly deleterious environment. in each generation, mutations occur, certain of which can lead to so-called genetic diseases; between 3 and 4% of newborns are affected. besides these spontaneous mutations, there are also mutations arising from the genetic inheritance of the parents. the purpose of genetic counseling is to warn parents when the existence of a potentially serious genetic flaw is suspected. the highlighting of genes that give a predisposition for cancer (proto-oncogenes) is a convincing illustration of the power of predictive medicine. this involves genes that control the synthesis of growth factors, the activity of which is essential to embryogenesis and to the repair of damaged tissue. while they are normally subject to strict control by anti-oncogenes, proto-oncogenes are able to become active in an anarchical manner, under different influences, and to transform themselves into cancer-generating oncogenes. recently, mutations have been found in two genes, brca1 and brca2, these mutations giving a predisposition for cancers of the breast and of the ovary. thanks to genetic exploration, it will soon be possible to predict whether a cancer of the breast will have a rapid progression leading to uncontrollable metastases or a slow progression. depending on the case patients will be subject to heavy chemotherapy or to a less aggressive treatment. in this context, targeted therapy with monoclonal antibodies is a source of great hope. while genetic inheritance has a role in cancer, the environment plays a notinsignificant role as well. this is the case, for example, in lung cancer sufferers who smoke tobacco, cancer of oesophagus in those who drink alcohol and job-related cancers in those working in factories producing colorants or materials derived from asbestos or tars. cardiovascular diseases are the primary cause of death in the more developed countries, involving either an infarctus, or a stroke. many risk factors for these diseases are known, i.e., metabolic deviations affecting cholesterol or the blood serum proteins involved in the transport of lipids. these metabolic anomalies result in a syndrome known as atherosclerosis, which is characterized anatomically by the deposit of fats in the form plaques in the arteries. while genetic factors are at the origin of these metabolic problems, the latter are clearly amplified by an inappropriate diet. the role of predictive medicine is to recognize the genes that are responsible, warn individuals of the risks they are running and to advise them about the types of lifestyle and diet that do not increase these risks. being able to predict, predictive medicine should be able to prevent by means of targeted drugs. within this context, it gives rise to reflection upon polymorphism linked to variation in a single nucleotide in the dna of the genome of an individual. known as snp (single nucleotide polymorphism), this polymorphism has proved to be a very useful auxiliary in molecular medicine. hundreds of thousands of snps are present in the human genome and several tens of thousands in genes coding for the proteins. where they are located differs according to ethnic backgrounds. among these snps, some appear to be linked to certain pathologies, such as certain forms of cancer or degenerative illnesses such as alzheimer's disease. in addition, in a small number of patients, the location of certain snps has been connected with previously-inexplicable drug incompatibilities. in line with these observations, pharmacogenomics, a branch of pharmacology that deals directly with genome sequence data, is trying to evaluate the impact of "snp variants" on the efficacy and toxicity of drugs and to understand the genetic bases that explain the differences that are observed in the responses of different individuals to the same medication 53 . rather than using a standard drug that is not very efficacious or causes adverse side effects, it might be possible, depending on the genetic profile of the patient, to use a drug that is more appropriate to his or her genetic map. it is doubtless not just a fantasy to imagine that, in 20 or 30 years' time, a patient visiting the doctor will be offered a genetic map thanks to cells taken from the buccal mucosa. finding snp variants that are known to be responsible for drug incompatibilities in such a map will make a targeted prescription possible. it will allow the detection of genes for susceptibility to an illness, at the same time uncovering targets for new drugs. pharmacogenomics, which is still called new pharmacogenetics, contrasts with old pharmacogenetics in which, having found an adverse clinical response to a certain therapy, an attempt was made to identify the protein target of the incriminated drug, and then to go back to the gene coding for this protein, and to look for the mutation responsible for the aberrant response to the drug. the existence of customized predictive medicine, which would read the destinies of individuals in their genes, would not be without its consequences in the life of a citizen. by registering each citizen with a genetic map, matched with a named identity card, predictive medicine might begin to take on the aspect of a janus, with his beneficent face warning subjects of potential risks of metabolic problems, and guiding them towards the actions to be taken to lower the risks, but also with his evil face delivering each individual's intimate details to the indiscrete inquisitiveness of investigators who are operating towards their own ends (insurance companies, employers…). no less worrying would be the sly but predictable transformation of the individuality of the repaired or even doped human being within a system of imposed, docilely-accepted assistance. in the 19 th and 20 th centuries, the methodology for biological experimentation underwent a revolution caused by the progress made in the domain of chemistry, both analytical chemistry, with the deciphering of increasingly complex molecular structures, and also in synthetic chemistry, with the large-scale production of tens of thousands of new molecules. the effects of these molecules, which might eventually be used as drugs, were tested directly on animals. it was thus that in 1910 the german chemist paul ehrlich discovered salvarsan, a derivative of arsenic, which was active against a type of treponeme, the agent of syphilis. this was the result of a systematic analysis of the effect of synthetic products, aromatic derivatives of arsenic acid, on syphilis in rabbits. salvarsan was the 606 th derivative that was tested, and this is why it was called 606 for a long time before it was given the name salvarsan. sometimes, lucky chance shows surprising and unexpected properties in synthetic molecules. this was the case for chlorpromazine, which was initially used as an antihistamine. it was luck that led to its antipsychotic activity being discovered in 1950. a new era opened up in psychiatry with the arrival of synthetic narcoleptics like chlorpromazine. a new chemical science known as combinatory chemistry, which dates from the 1990s, has aroused an increasing amount of interest in pharmacology. this involves making two or more species of organic molecules that carry reactive functional residues react in solution or in the solid phase in such a way as to synthesize, by means of all possible combinations, a number of final and intermediary products that is situated in the hundreds or even the thousands, and which makes up chemical library or drug library. we can directly test all of the products formed on a sample of eukaryotic cells, in order to verify their effects (for example the inhibition of an anarchical proliferation of cancerous cells), or on microorganisms in order to evaluate an antibiotic capability. we can also proceed straight away with the fractioning of the reaction products and the testing of each of the fractions. if the response is positive, fractioning is continued until the molecular species responsible for the desired effect is obtained. other evaluation parameters for this molecule, such as its absorption, its toxicity and its metabolic future (distribution in the organs, chemical modifications and excretion) are then explored, first in cells, and then in animals (rats, mice), thus comprising pre-clinical tests. these screening operations, which are said to be high-throughput, require automation and robotization aided by powerful computer technology. each year, pharmaceutical companies screen tens of thousands of different molecules on hundreds of targets. complementary to combinatory chemistry, in silico chemistry works by molecular modeling and uses computer programs for the rational design of new drugs that are able to fix onto specific protein targets. the purpose of this is to provide a virtual follow up to modifications in the reactivity of a given drug molecule as a function of the modifications imposed on its structure, for example, the addition of residues that differ according to their electrophilic or hydrophilic properties, or according to the length of their side-chain. provided there is a chemical library and we know the three-dimensional structure of a macromolecule, for example an enzyme, as well as the nature of the residues that define its active site, we can hope to select and chemically modify a substance that is able recognize the active site of this enzyme and to make an almost perfect ligand out of it which is able to efficiently block the operation of the target enzyme. this method, which is based on computer-aided chemistry, is called "structure-based drug design", and has had some notable successes. it has made it possible to develop an inhibitor capable of blocking a protease involved in the replication of the aids virus. however, both in combinatory chemistry and in molecular modeling, the many successes that have been achieved remain modest in number compared to the means that have been deployed to achieve them. in terms of statistics, out of ten thousand molecules that are recognized as being efficacious for a given target in vitro, around one hundred are chosen for preclinical trials on animals, around ten are chosen for preclinical trials in man and only one will come out as a drug. the financial and economic effect is far from being negligible. it has even become a preoccupation in a system where merciless competition is the rule. in addition to synthetic chemistry, preparative chemistry, which is based on the isolation of natural molecules, is now the subject of renewed interest, due to the introduction of high-throughput techniques. high-throughput screening, which is an essential tool in combinatory chemistry, is also carried out to ensure the systematic detection and isolation of natural substances having interesting pharmacological activities such as antibiotic activities or anti-cancer activities, based on marine animals, microscopic fungi, prokaryotic organisms and various plants. for example, among the substances that have been isolated recently are cibrostatin, a specific cytostatic of melanoma cells, from a marine sponge, mannopeptimycin, a bacterial antibiotic from an actinobacterium streptomyces hydroscopicus and a whole set of alkaloids with a cytostatic activity with respect to human tumor cells from an exotic plant of the genus daphniphyllum. the molecular diversity of the living world is such that the reserves of natural products having pharmacological activities are far from being exhausted. so far, only a small percentage of the microbial species populating the earth have been listed. the depths of the oceans harbor many unknown species. thousands of insect species remain to be discovered in the canopies of tropical rainforests. exploration of the plant kingdom is far from being complete. the listing of natural molecules having a therapeutic activity has only just begun. the hunt promises to be a fruitful one, all the more so because the highthroughput screening methods that can now be used greatly increase the efficiency of the search. high-throughput screening, applied to natural molecules, has overturned the methodological procedures that were in use until recently, which progress through logical steps, using relatively simple artisanal analytical methods, from observation, often resulting from serendipity, to the isolation of the active substance. thus, in the 19 th century, using inherited traditional knowledge that a decoction of cinchona officinalis bark calms malaria crises, pierre joseph pelletier (1788 -1842) and joseph bienaimé caventou (1795 -1877) decided to isolate the active substance of this bark. from the raw extract, they purified an alkaloid, quinine, which proved to be the anti-malarial substance they were looking for. more recently, the starting point of florey and chain's isolation of penicillin from the microscopic fungus penicillium notatum was the fortuitous observation made by fleming that this penicillium secretes an antibiotic factor (chapter iii-2.2.5). there are many examples in which serendipity has been the principle factor involved in the discovery of a drug, and this will no doubt continue to be the case. the appearance of a lucky chance, after all, is not incompatible with highthroughput practices. also, it is not impossible that in the future there will be a conjugation of the discovery of new natural substances and the use of combinatory chemistry, with the aim of manufacturing derivatives having a much greater power of action and quality of specificity from these substances 54 . to sum up, the experimental method has caused contemporary medicine to take a giant leap forward, with the discovery of increasingly high-performance functional exploration techniques, the development of therapies using molecules that are already present in nature or are manufactured by synthesis and the more and more advanced understanding of molecular mechanisms that takes into account the basic idea of the pioneers of molecular biology was that the function of a macromolecule depended on its structure. thus, perutz's elucidation of the tetrameric three-dimensional structure of hemoglobin, and of its modifications depending on the degree of oxygenation, shed a considerable amount of light on the cooperative mechanism of the transition from the hemoglobin state to the oxyhemoglobin state (chapter iii-6.2.1). in the same way, an understanding of the structure of many enzymes, receptors and transporters of metabolites has shed light on their mechanisms. in a parallel manner to the exploration of the structures and functions of proteins, that of genomes has made remarkable progress. the subtle entanglements of genomics and proteomics that have become accessible to the experimental method are the order of the day. one major challenge for post-genomics is to understand how proteins, expressed by genes, interact with one another to generate functions that characterize cellular specificity. even more ambitious are attempts to understand the operation of organs or even of living organisms, based on mechanisms that are implemented at molecular level. these attempts lead straight to an integrated biology, that is, a biology that aims to understand the overall functioning of living beings. taking as its purpose the access to emerging functions, resulting from interactions between macromolecules, integrated biology first tries to invent methods that make it possible to detect these interactions. strengthened by the information obtained, it tries to integrate this information with a mathematized language into modules that attempt to simulate living beings. from the simplistic procedures of the middle of the 20 th century, which were justifiable within the reductionist context of this period, and which involved considering each species of proteins as an autonomous functional entity, we have moved on to the idea that the different species of protein that inhabit a cell have a dialogue with one other, and that they may move from one endocellular organelle to another, depending on post-translational modifications (for example, phosphorylations) that change their conformation and, at the same time, their reactivity and their behavior. thus, an enzyme protein is not only defined by its catalytic performance with respect to a given substrate, but also by its place in a metabolic network where it interacts in a dynamic and transitory manner with a multitude of other protein species (figure iv.15a) . the concept of cell signaling has also evolved. instead of considering that a cell membrane receptor, activated by fixation of an extracellular ligand (a hormone, for example), addresses the information received to an endocellular effector protein via a linear cascade of individual proteins, it has come to be postulated that communication between an activated receptor and its effector is mediated by proteins organized into interactive networks ( figure iv.15b) . this machinery provides more flexibility in the addressing of messages to effectors. the diagram on the right shows that besides its catalytic function, protein a interacts with other proteins in the cell. b -case of the transduction of a signal that is external to the cell (a hormone, for example). the diagram on the left refers to the classical idea of signaling from a receptor r according to a linear cascade of protein-protein interactions inside the cell, leading to an effector z. the diagram on the right shows that the signal is spread through proteins organized into interactive networks. another subject to be considered is the density of macromolecules of all types, such as proteins, nucleic acids, lipids and polysaccharides, contained by a microorganism or a eukaryotic cell, which reaches values of 300 to 500 g/liter, denoting a semi-solid state or a considerable degree of compacting. however, for technical reasons, kinetic studies carried out in vitro on isolated enzymes have been carried out with solutions that are 1 000 or 100 000 times more dilute. conscious of this difference in scale between information obtained from in vitro studies and the in vivo reality, the biology of today is trying to re-evaluate molecular dynamics within the context of a cell. this is why we are seeing the birth of an integrated (or integrative) biology of functions, which, using modeling procedures, aims to achieve an understanding of the temperospatial dynamics of the interactive components inside cells. this holistic conception of biological systems ("systems biology ") has been made possible by progress in technological expertise in domains as varied as biochemistry, molecular biology, physical optics, electronics, nanomechanics, physical and mathematical modeling and computer technology. it is a necessary complement to the classical experimental method based on bernardian determinism which, in order to connect an effect with a cause, explores living beings in a manner that is often monoparametric and is inevitably reductionist. this signals a change in paradigm in the experimental approach to living beings. a particularly effective investigative method used to explore the dialogue between proteins is the double-hybrid by genetic construction, two proteins, p 1 and p 2 , whose interaction is to be tested, are expressed in the form of fusion proteins in yeast. protein p 1 is fused with the binding domain (gal4-bd) to the dna of gal4, a protein that regulates the transcription of the β-galactosidase gene. protein p 2 is fused with the activation domain of gal4 (gal4-ad). insofar as p 1 interacts with p 2 (b), the gal4 transcription regulation activity is re-established, which is verified by the transcription of the reporter gene. if the opposite occurs, i.e., in the absence of any interaction between the two domains of gal4 (a), the reporter gene is not transcribed. the principle of this method is based on the modular nature of numerous transcription factors in eukaryotes. these factors contain both a dna-binding domain that includes a specific dna-binding site and a transcription activation domain that starts up the machinery for transcribing dna into messenger rna. these two domains can be dissociated and then re-associated in a functional manner, by forming hybrids with interacting proteins. a first protein, p1, is fused with the dna-binding domain of a transcription factor by genetic manipulation, and a second protein, p 2 , is fused with the activation domain of the same transcription factor. if p 1 is able to interact with p 2 , the transcription factor is reconstituted and the reporter gene upon which it depends can be expressed. the trapping technique, which is complementary to the double-hybrid system, was developed to make it possible to identify a set of interactive proteins within a cell. a protein that is included in this set (protein of interest) is fused by genetic engineering techniques to a short polyhistidine chain (called a tag). using this tag, the protein of interest is fixed to a solid medium containing nickel ions, a material that is reactive with respect to the polyhistidine chain. in the presence of a soluble cell extract, the protein of interest binds the cognate proteins of this extract, making it possible to retrieve a complex whose components, corresponding to interactive proteins, can be resolved after denaturing gel electrophoresis and then characterized ( figure iv .17). the techniques described above are backed up by cell imaging techniques that make use of confocal microscopy, which is more directly in keeping with living reality. the optical performance level of confocal microscopes has improved lately, with the arrival of biphotonic and multiphotonic lasers that illuminate precise points of the cell. as we have seen previously (chapter iii-6.2.1), it is possible to create a protein chimera made up of a protein of interest fused with a fluorescent protein, in this case gfp (green fluorescent protein), inside a cell. there are currently several variants of gfp that are able to emit fluorescent light at different wavelengths. this has allowed the development of a technique known as fret (fluorescence resonance energy transfer ) which explores the interaction between two fluorescent proteins. in practice, two gfp variants that have neighboring emission spectra are fused, by genetic engineering inside the cell, to two proteins of interest, p 1 and p 2 , that are suspected of being interactive. if this is the case, the fluorochromes that they carry are sufficiently close that the result is a modification in the intensity of the emission fluorescence of the donor fluorochrome (decrease) and of the receiver fluorochrome (increase), which is readily detectable. all of these studies, taken together, have given rise to the idea that endocellular proteins are organized into networks, that these networks are interactive and that their location in defined compartments of the cell is dependent on epigenetic events such as phosphorylations. two attributes can be found in integrated systems: firstly, the presence of modules, i.e., interactive motifs, which, like the pieces of a jigsaw puzzle, fit together to produce a complex, coherent structure, and, secondly, the emergence of functional properties due to the newly created interactions. the protein of interest p is expressed in the form of a protein fused to a protein "tag" t that is able to bind to a solid support with a specific affinity. the assembly is brought into contact with a cell extract. certain proteins of this extract, a, b and c, which are able to interact with the protein p, become fixed to the latter. in a second step, the tag t is freed from its attachment to protein p by means of a specific cleavage enzyme. the pabc complex that is recovered from the solid medium in soluble form is subjected to polyacrylamide gel electrophoresis, in order to separate and identify its components. given an analytical description of the basic building blocks that are used to construct living systems, and an understanding of their modes of association in defined circumstances, it is normal to try to reconstruct, in their entirety, mechanisms that show the functioning of these systems. this idea was first applied to the yeast saccharomyces cerevisiae for different reasons, such as cell homogeneity (in principle, and, in any case, statistically speaking, all yeast cells have the same genome and the same proteome), an in-depth understanding of the genome and the proteome and the presence of a vast directory of well-characterized mutants. the use of techniques for the detection of interactions between proteins has revealed the existence of a potential dialogue of unexpected richness between a multitude of proteins ( figure iv.18) , in the yeast cell. it is necessary to reflect upon this evidence, which leads to the postulate that, for a given protein, there are mechanisms that restrict and select the many partners able to react with it at a precise moment.faced with a situation in which chance has the upper hand, leading to uncontrollable anarchy, it is necessary to have regulation, which is underpinned by darwinian logic. example of an interaction network involving the yeast sup45 prion protein. the line of dashes refers to experimental data concerning the interaction of sup45 with another protein, sup35. the lines in bold refer to interactions taken from experimental data; while the fine lines refer to predictions, particularly phylogenetic ones. this logic arises from a choice of the most efficient reaction path, which is first of all dictated by the speed constants involved in the association and dissociation of molecular partners, without, however, neglecting any stochastic events that may arise. chemical modifications of proteins participate in this regulation, such as phosphorylation, glycosylation and acylation. the result at cell level is a coherent channeling of the information that is carried from a molecular signal. thus, fixation of a hormone onto a receptor induces a series of modifications to the intracellular proteins that channel information towards an effector terminal, for example an enzyme responsible for the production of a metabolite with a strategic function. how can the sum of the scattered experimental data that we have concerning the catalytic capabilities of a multitude of enzymes of cellular origin be integrated into the operation of a cell? how can we envisage the gene-enzyme relationship according to current evidence concerning the complexity of the genetic message? biocomputing, or bioinformatics, a science that emerged towards the end of the 20 th century, proposes to try to answer these questions. at the turn of the 21 th century, with the development of increasingly powerful computer microprocessors that are able to carry out complex operations with amazing rapidity, the hope arose that it would be possible to simulate processes as varied as the regulation of the cell cycle, molecular flow in metabolic pathways and the reception of molecular signals, for example from hormones by living cells, as well as the transmission of the messages that result. the dream of an in silico virtual biology has become achievable. the first mathematical theory of simple enzyme reaction kinetics was put forward approximately a century ago, by victor henri (1872 -1940). born in marseilles to russian parents, victor henri studied in saint petersburg and then spent time at the universities of göttingen and leipzig before becoming established in paris. having an eclectic mind, studying both psychology and physicochemistry, he had the wonderful intuition that enzyme catalysis arises from a specific mechanism, different from that implemented in a chemical reaction. the study carried out by henri concerned the cleavage of sucrose (table sugar) into fructose and glucose by the action of an enzyme called invertase (sucrase). the term invertase was used because during reaction there was a change in the rotatory power of the sucrose solution, shown by a polarimeter. analysis of the reaction suggested that an enzyme-substrate complex is formed, which breaks down to regenerate the enzyme and liberate the product of the reaction. this analysis gave rise to an equation for the speed of the enzyme reaction as a function of substrate concentration. henri published the results of his experiments both in his thesis, which he presented to the paris faculty of sciences in 1902, and in two articles that appeared in the reports of the academy of sciences 56,57 . in 1913, in biochemische zeitschrift (vol. 49, pp. 333-369), leonor michaëlis and maud menten (1879 -1960) confirmed the results of victor henri and formulated an equation that became a classic, describing the speed of formation of a product from a substrate in enzyme catalysis. since the period of these first studies, the concepts involved in enzyme kinetics have evolved considerably. the first metabolic pathway be deciphered was that of the degradation of glucose (glycolysis) , either into ethanol in yeast, or into lactic acid in muscle tissue. after this, researchers became aware that the activity of enzymes could be modulated as a function of covalent modifications of amino acid residues of their protein chain (phosphorylation, dephosphorylation, acylation…). metabolic flow analysis led to the idea of the limiting reaction. in the 1970s, the idea that there is a single limiting reaction in a chain or a cycle of reactions gave way to the idea that metabolic control is distributed over several reactions, and that each reaction has its own, more-or-less intense control force. another complexity factor came to light with the discovery of allostery 58 . allosteric enzymes have the particularity that they can fix reversibly onto a site that is different from the active site (allosteric site) molecules that are often the terminal products of a chain of reactions: the consequence of this is a conformational modification of the structure of the enzyme that has repercussions on the geometry of the active site and modifies its reactivity with respect to the substrate. in the 1980s, faced with the complexity of the tangle of listed metabolic and signaling networks, attempts were made to use mathematical modeling to show the progress of the traffic of molecules inside a cell in relatively simple metabolic pathways such as glycolysis. in the modeling procedure, the concentrations of the different molecular species are considered to be variables whose variations over time depend on their speed of production and their speed of disappearance, which leads to a set of paired differential equations. with this procedure, we entered the domain of virtual biology. thanks to the creation of increasingly powerful software, the aim of virtual biology is to simulate signaling and metabolic pathways. in the longer term, the aim is to understand the molecular and cellular processes that direct embryo development, or to test the effects of drugs of known target on the metabolic behavior of the cell. metabolic engineering (which is already well developed) comprises two types of models, stoichiometric models and kinetic models. stoichiometric models describe metabolic networks in the stationary state, based on analytical data. kinetic models combine stoichiometric information and that concerning the catalytic capabilities of the enzymes in a metabolic network. in canada, the cyber-cell project plans to model the overall functioning of the machinery which, in the bacterium, includes its metabolism and its proliferation. the aim of the afcs (alliance for cellular signaling), which was launched in the usa, is to understand how signaling occurs in cells such as the b lymphocyte, the macrophage or the cardiac cell in response to different types of stress. the techniques that are used range from identification of all signaling network proteins to the evaluation of the flow of circulating information and to the integration of the data acquired into theoretical models. the european nerve synapse project makes use of similar procedures, with its long-term hope of linking the functioning of nerve cells with the cognitive and behavioral functions of living beings. this is a sizable challenge. in fact, there is far from being a real consensus concerning the principle of a demarcation between, on the one hand, cognitive functions such as language or memory, which are located in precise zones of the brain, and which could be reduced to physicochemical processes, and, on the other hand, forms reflective thought that are expressed through the creative imagination or by judgements concerning ethics or esthetics, the notion of personal responsibility, or even pictorial, architectural or musical beauty. should we see the human soul as the programmer of a superb computer that never ceases to develop from the embryonic state onwards, like john eccles (1903 -1997) and others, or should we admit, like jean-pierre changeux (b. 1936), stanislas dehaene (b. 1965), daniel dennett (b. 1942) and others, that thought is not transcendent, and that it is intrinsically dependent on the brain, which is considered to be a neurochemical system, and thus look for the secret of the individuation of the human being in brain information storage mechanisms with retrocontrol loops associated with subtle neuron architectures, or, in short, refer to a sort of turing machine? whatever the case, in this domain, as in others, simple animal models are used in order to identify elementary processes that are able to explain easily-tested functions such as the memory in anatomical and physiological terms. this is the case for the sea slug or sea hare (chapter iii-6.1) which, despite its rudimentary cognitive capabilities, provides information that can be used to reconstruct higher cognitive functions, present in the brains of mammals. it is clear that the cognitive sciences have reached a stage in which they are emerging from their infancy (chapter iv-3.2). now, ingenious computing methods and a basis for reflection that has spread beyond the confines of psychology and philosophy, are available to them. they have set themselves the goal of producing an artificial intelligence, using ultrarapid computers as well as software that is able to model the operation of neural networks and to come close to the performance of human intelligence in terms of the power of their reactivity and their memorization. at present, many other biological systems are being subjected to multiparametric exploration, with the aim of producing models. this is the case, for example, with the program of the differentiation of certain white blood cells, the neutrophils (chapter iii-2.2.4), from precursors located in the bone marrow, a differentiation that leads to the emergence of functions such as phagocytosis that are implemented in the fight against microbial infections 60 . in a domain that is closer to mechanical science, hydrodynamics, the digital simulation of the cardiovascular system has already made it possible to represent the physical phenomena associated with the propagation of a wave in deformable arteries during a cardiac contraction, in the form of equations, with a good approximation 61 . in short, from a monoparametric approach that often began as being essentially and necessarily reductionist, the experimental method, applied to living beings, has become a "globalized", or synthetic, multiparametric approach, the aim of which is to understand the dynamics of molecular interactions in defined biological systems. making use of data obtained, the hope is to use mathematical processing to simulate the overall functioning of a cell, organ or organism. this new paradigm of the experimental method ("systems biology" 62 ) is not limited to a simple accumulation of observations concerning a given biological system and their abstraction in mathematical form. the originality of this approach is that it formulates predictions of changes in the behavior of a system as a function of the manipulation of parameters such as substrate concentration, the presence of inhibitors, and so on. the mathematical processing of experimental data, with a view to learning about the functioning of complex systems by modeling, is supported by the technosciences, particularly biocomputing. it is linked not only to the enormous sum of accumulated knowledge concerning the structures and functions of living beings in the post-genomic era, and to the notion that the life of a cell depends on multiple networks of molecular interactions and thousands of enzyme reactions located in its different organelles, but also to the information that comes to it from its environment. a first type of modeling is based on observations made or experiments carried out on an easy-to-study model system. laws are drawn up from this. this so-called "bottom-up" (or synthetic) procedure, which proceeds from the simple to the complex, makes it necessary to have a set of very precise biochemical data. this great precision is all the more imperative in that any deviation, even a minimal one, in the integration of an experimental result can generate a mathematical model that is apparently plausible but which is unconnected to the living reality. the reverse, "top-down" (analytic) procedure proceeds from the overall operation of an organ and its theoretical analysis towards the specific mechanisms of its components. it takes into account the functioning of complex integrated systems such as the nerve and endocrine systems, immune and reproduction systems and the system controlling homeostasis, descending in stages towards the cellular, molecular and genetic levels. in the end, an understanding of living beings involves the management of an amazing capital of experimental data. this makes it necessary to consider all of the genes (genome), all of the transcripts coding for the proteins (transcriptome), all non-coding rnas (rnaome), all proteins expressed in a particular cell type (proteome) and all metabolites (metabolome) as a function of the enzyme catalyzers expressed and the energetics that underlie the catalyzed reactions, and, finally, to connect upstream events (mutations of genes and interference of messenger rnas, chemical modifications to amino acid residues in the proteins) to phenotypical modifications on the scale of the whole organism (phenome) (figure iv.19) . the goal of integrated or integrative biology ("systems biology") is therefore to put living beings into equations, that is, to represent them in virtual systems for which the behavior, accessible by means of calculation, can be predicted as a function of modifying parameters. in addition to the possibilities that are opened up in terms of a deeper understanding of physiological mechanisms, such virtual systems could be used for the design of new drugs or for the manufacture of economically valuable biomolecules. the diagram illustrates the different levels of complexity in the pathway that goes from all the genes together (genome) to all of the expressed characteristics (phenome) in the living being, passing via coding rnas (transcriptome) and non-coding rnas (non-coding rnaome), all the proteins (proteome), all addressing systems in the cell compartments (localisome) and all of the metabolic pathways (metabolome). at a scientific meeting held in sheffield, england, in january of 2005 63 , with the theme systems biology: will it work?, an argumentative discussion of the advantages as well as the disadvantages of an integrated, mathematized biology was useful in that it included a reminder that most of the parameters used in "systems biology" come from studies that are carried out in vitro on purified enzymes, and that it is not sufficient to know the value of the michaelian parameters (v max and k m ) in order to reach biological reality. in fact, in vivo, many enzymes record variations in activity that are hard to control due to allosteric type regulation or interenzyme contact; several enzymes of a metabolic pathway being able to interact to form a metabolon. however, by compacting several enzymes that catalyze contiguous reactions in a metabolic pathway, a metabolon considerably increases the catalytic efficiency of this pathway. another element of uncertainty arises from the protein density of the cell medium, and also from the fact that covalent modifications of enzymes can introduce a change in endocellular location (nucleus, organelles of the cytoplasm…). nevertheless, an approximative approach could limit itself to dealing with biological systems in modular terms, i.e., to considering them as being made up of a number of black boxes, each black box containing a series of reactions being processed mathematically together, with an input and an output. there is still a long way to go if we place ourselves on the cellular scale, but the end of the pathway seems even further away if we envisage the organism as a whole, taking into account the remote interactions between organs involving the interplay of chemical mediators. the brain plays a critical role in the dialogue between different organs, and in the regulation of the energy equilibrium in higher animals. this equilibrium can be disturbed by fasting or intense, prolonged muscular activity, or by an overabundant diet. the corrective response comes from a deep region of the brain, the hypothalamus, via the secretion of different types of peptides, some of which stimulate the appetite and others of which suppress it 64 . while taking into account the multitude of parameters that affect the complexity of living beings on an individual level, the theoretical approach to the study of cell function by modeling has the advantage that it produces predictions and provides information about the validity of conclusions and of theories based on experiments that are old and accepted in the absence of contradictory elements. this was the case for the theory that stated that the state of activation of a gene is determined only by the presence in its environment of transcription factors. recent studies concerning the level of gene transcription in isolated cells have shown that there are probabilistic-type factors which mean that a given gene in a given cell can be activated at any moment. a review which came out in 2005 65 sums up this subject. in this review, the authors use modeling to analyze the behavior of cells in the process of differentiation during embryogenesis. their darwinian model, which associates contingency and selectivity, competes advantageously with the determinist (or instructive) model, based on an all-or-nothing logic, that has been implicitly accepted up until now. the darwinian model takes into account the occurrence of stochastic events at gene expression level, events that are partially linked to the structural modifications to the chromatin that depend on covalent modifications of an epigenetic nature (phosphorylation, methylation…). by basing itself on the existence of mutational fluctuations that arise by chance, associated with a selfregulation of gene expression, the model that is obtained shows that during embryogenesis a cell has a choice either to differentiate into another cell type or to remain in its initial state. differentiated cells stabilize their own phenotype and, in their surroundings, stimulate the proliferation of foreign cell phenotypes. a harmonious equilibrium between these two processes is the necessary condition for the setting up of the steps that lead to the arrangement of different cell types during organogenesis, which take place in an apparently inescapable order, in the absence of disturbances. a break in this equilibrium leads to an anarchical cell proliferation. generally speaking, from the point of view of experimental science, the lesson that can be drawn from current modeling experiments is that the bernardian determinism that has prevailed as the essential foundation stone of the methodology applied to the study of living beings may find itself being requalified by the taking into account of stochastic phenomena. this is the case when the number of reacting molecules is low and the probability of stochastic events is non negligible. the modeling of such systems necessitates having recourse to a complex mathematical formalism. it remains true that determinist models for simulation of the dynamics of living beings, represented by classical differential equations, are more-or-less valid when the number of reacting molecules involved is high and the reactions supposedly take place in a homogeneous medium. should "systems biology" be regarded as a resurgence of a physiology that has been somewhat neglected over the last few decades, but has been reinvigorated by a salutary hybridization of biologists and model-makers? in any case, this is the intention of the "physiome" project 66 which has recently been launched on an international scale. it is also doubtless due to this state of mind that a trend which had gone out of fashion, involving the simulation of the performance of living beings by very elaborate concrete models, robots, is being reborn. an immense distance has been covered in just over two centuries, since the time when vaucanson presented automata in the forms of human figures, moved by ingenious springs and cogs, and giving the illusion that their movements were controlled by an intelligence, to a marveling public (chapter ii-6.4). in the last decades of the 20 th century, considerable progress was made in the understanding of the operation of the nervous system and in the development of technologies in which miniaturized electronics have come to the aid of already high-performance micromechanics. the brain being considered as an information processing machine, the aim is to understand the logic of this information machine by means of simulations on computers and, based on the results obtained, to construct robots whose electrical circuits take their inspiration from the operation of animal neurons. these robots are called biorobots or animats. insects have been chosen as a reference for the construction of such creatures because of the relative simplicity of their nervous systems: several hundred thousand neurons, in comparison with the billions of neurons present in mammals (100 billion in man). the fly's system of vision has been favored as a subject of study because of the possibility it offers of being able to record the electrical response of neurons that can be identified one by one. in the middle of the 1980s, in france, this inspired the pioneering work in biorobotics carried out by nicolas franceschini (b. 1942) and his team 67,68 (figure iv.20) . their objective was to study how an animal can avoid obstacles by means of its ocular perception and its movement-detecting neurons, the operation of which the team just analyzed using microelectrodes and a microscope-telescope specially built for the purpose. the fly's composite eye has 3 000 elementary units or ommatidia, each carrying eight light receptor neurons. the electrical signals emitted by these neurons in response to captured light (at most a few dozen millivolts) are sent to subjacent neurons that are organized into three levels that correspond to the optical ganglions called the "lamina", "medulla" and "lobula". the lobula is a strategic decoding center which, because of the small number of neurons contained in it (sixty), has been the subject of in-depth electrophysiological investigation. each of the sixty neurons of the lobula operates as a signal integrator. the neurons of the lobula send their messages to motor neurons involved in the contraction of small muscles that control the guidance and stabilization of the fly's flight. based on an exhaustive study of the neuron wiring of the fly's eye, franceschini and his colleagues were able to reconstruct a facetted artificial eye that can retranscribe the light signals received optoelectronically. this artificial eye, the electronic components of which correspond to around one hundred movement detectors in the fly, was incorporated into the head of a robot. the recorded light signals were transmitted to the moving components of the robot. a -head of the blowfly, calliphora, seen from the front, showing the two compound eyes with their multifacetted array. each eye hides 40,000 photoreceptors that drive various image processors based on a few hundred thousand neurons. b -"elementary motion detector" (emd) neuron and its evolution over fifteen years: on the left, first generation (1989), using surface mounted device (smd) technology, compared to a one franc coin from that period; on the right, the 2003 version of the highly-miniaturized hybrid (analog + digital) emd circuit (mass 0.8 grams), compared with a one euro coin. c -autonomous vehicle (12 kg) able to move around in a field of obstacles that it does not know about in advance. its vision is based on a genuine compound eye, whose circuits are inspired by those of the fly. it includes a network of 114 "motion detecting neurons", transcribed electronically according to the principle analyzed in the fly's eye by means of microelectrodes and a specially-constructed microscope-telescope. this network is arranged around a ring that is about thirty centimeters in diameter. the recently-constructed roboflies, oscar and octave, only weigh around one hundred grams. d -routing of the electronic components (resistances, condensers, diodes and amplifiers that operate in their thousands) soldered onto the six-layer printed circuit-board that provides the connection between the sensors and the steering motor on board the autonomous mobile robot shown in (c). figure iv .20 illustrates the neuromimetic biorobofly constructed according to this principle. completely autonomous because of its on-board power supply, this robot was able to move around at high speed (50 cm/s) in a cluttered area, avoiding the obstacles. this first "terrestrial" robofly, which was completed in 1991, was followed by several much lighter brothers and sisters: fania, oscar and octave are aerial roboflys 69 . constructed in 1999, oscar is a captive robot that weighs around one hundred grams. it is equipped with an eye that reproduces the retinal microscanning of the fly's eye discovered by franceschini, oscar is able to rotate around a vertical axis because of its two diametrically opposed helices and can thus orient its view towards an object. if this object moves, oscar follows it with its eye, up to an angular speed comparable to the tracking speed of the human eye. produced in 2003, octave is another aerial robofly that is able not only to take off and distinguish a relief, but also to land automatically and to react sensibly to a contrary wind in a turbulent atmosphere. on board, it has an electronic visuomotive self-regulation system, the operation of which is based on the signal processing operations that, in the insect, carry out the automatic pilot functions 70 . the age of biorobotics, in which robots take their inspiration from animals, has only just begun 71 . if specimens are still so rare, this is because behaviors for which we have a good understanding of the underlying neuronal bases are also rare. at the time of writing, a robot rat named psikharpax, with artificial muscles and a vision system that enables it to perceive objects in three-dimensional space, is being developed at the university of paris vi. almost in the realm of science fiction, we find hybrid robots obtained by hybridization of the living and the non-living. this is the case for the hybrid robot produced by japanese researchers, based on the silkworm moth. control of the nervous system of this insect is spread throughout its body. if its head is cut off, it continues to fly, which gave rise to the idea of replacing the head with an electronic transistor system 72 . using a remote measurement device, it was possible to explore certain behavioral aspects of the insect. although the construction of hybrid robots may raise ethical objections, such technology is capable of giving rise to spectacular applications in the domain of prostheses. the neurological prostheses of the future will nevertheless require that a contact be made between living neurons and the electronic chips that are able to improve the inadequate processing of the physiological signal. such a contact was produced recently in a german laboratory directed by peter fromherz 73 (b. 1942) . a small network of snail neurons, chosen because of their large size, was cultured on the surface of a silicon chip. a signal emitted at one location of the chip was able to be transmitted to another location via the synapse connection between two neurons 73 ( figure iv.21) . on the molecular scale, mitochondrial atpase or atp synthase, with a size of around ten nanometers (chapter iii-6.2.1) was used recently for the manufacture of a biorobot that made its mark in the media as the smallest known rotating molecular motor. the membrane-type enzyme catalyzes the reversible reaction atp + h 2 o adp + pi. this enzyme therefore has a double function; hydrolysis and synthesis. for this reason it is called atpase or atp synthase depending on the physiological context in which it is involved. in the mitochondria that oxidize metabolites, the enzyme operates like atp synthase. it catalyzes the synthesis of atp coupled to oxidation reactions. in the absence of respiration or of oxidizable substrates, the enzyme operates like atpase; it catalyzes the hydrolysis of atp. for ease of language, the enzyme will be designated here by the term atpase. it should be remembered that mitochondrial atpase includes two sectors, a hydrophobic sector, fo, characterized as a proton channel located inside the mitochondrial membrane, and a hydrophilic sector, f1, carrying catalytic subunits that are arranged as if they were on a turret (see figure iii .18c). fo contains two master parts of the atpase motor, i.e., a rotor comprising an assembly of around ten so-called "c" subunits and a stator that corresponds to the "a" subunit. the "c" subunit assembly is attached to the "γ" subunit of the catalytic sector f1, which thus functions as a rotor. in 1961, the british biochemist peter mitchell (1920 -1992) showed that phosphorylative oxidation in the mitochondria is associated with a transmembrane transfer of protons. the mechanism involved is said to be chemiosmotic. the most important experiment involved an almost serendipitous observation, carried out with a simple ph meter. when a current of oxygen was passed through a suspension of mitochondria in an unbuffered saline medium, in the absence of adp and phosphate, an instantaneous acidification of the extramitochondrial medium occurred, shown by means of the ph meter electrode immersed in this medium. it was concluded that the sudden switch from anaerobiosis to aerobiosis, i.e., the start up of respiration, is correlated with an ejection of protons from the mitochondrial matrix to the extramitochondrial medium. afterwards, this fact was linked with several others, the whole leading to the formulation of the chemiosmotic theory. briefly, mitochondrial respiration generates a vectorial movement of protons from the interior to the exterior of the mitochondrion. because of this, a proton concentration difference is established on either side of the mitochondrial membrane. the electrical potential that is created in this way is used by the mitochondrial atpase in order to synthesize atp from adp and mineral phosphate. this process involves two correlated events: return movement of protons towards the inside of the mitochondrion across the fo sector of the atpase; rotation of the assembly of c subunits and the γ subunit that is interdependent with it. we have therefore moved from electrical to mechanical energy. during its rotational movement, the γ subunit establishes contacts with the three catalytic subunits of the f1 sector, in succession. one after the other, each of the three catalytic subunits in contact with the γ subunit undergoes a change in the conformation of its active site, which is at the origin of the synthesis of atp. in the absence of mitochondrial respiration, the reverse process occurs. the atp is hydrolyzed into adp and mineral phosphate, and the energy released at each of the three catalytic subunits is used to rotate the γ subunit in the reverse direction to that which accompanies the synthesis of atp. the existence of a rotational movement of mitochondrial atpase, which had been suggested on the basis of biochemical arguments 74 and of structural data 75 was authenticated by masasuke yoshida and his co-workers in japan in 1997, thanks to an imaging technique 76 . in a first step, the molecular system was simplified by being limited to the catalytic f1 sector of the enzyme. a methodological trick was employed: genetic engineering was used to modify the α and β subunits of this sector by fixing polyhistidine chains to them. because of the strong affinity between polyhistidine and nickel ions, the f1 sector α and β subunits were immobilized on a medium covered with nickel ions (carried by an organic molecule). an actin filament labeled with a fluorescent ligand was attached to the end of the f1 sector γ subunit. this assembly made it possible, under a fluorescence microscope, to display a rotational movement of the actin arm carried by the g subunit affected by the addition of atp and by its hydrolysis into adp and mineral phosphate. a similar rotational movement of the γ subunit carrying a metal microbar was observed by an american research group 77 . remarkably, in 2004, after having fixed a metal microbead onto the γ subunit, the japanese researchers 78 demonstrated synthesis of atp from adp and mineral phosphate by rotating the γ subunit by means of rotation of the magnetic bead, induced by magnets. thus, the experimental coupling of a mechanical force and a chemical synthesis was demonstrated. in 2005, the japanese research team 79 succeeded in photographing the rotational movement of the enzyme powered by atp under the microscope, this time looking at the entire atpase complex, f1fo. after having attached a gold microbead onto the "c" subunits of the fo sector, to act as a probe, the researchers were able to confirm that the rotational movement of these subunits depended on the hydrolysis of atp into adp and phosphate ( figure iv.22) . the whole of the mitochondrial atpase (atp synthase) does, in fact, function as a molecular rotational motor powered by a proton flow, rather like an industrial rotational motor powered by a fossil fuel or electricity. the analogy is a striking one; the γ subunit of the enzyme corresponds to the motor driveshaft and the "c" subunits correspond to the motor itself. because of its association with non-living structures, for example metal bars or gold beads, which are carried along in the rotational movement of the enzyme, it is possible to speak of molecular biorobots. this domain, in which nanomachines use macromolecules from the living world, has only just opened up, but its future is full of promise. the story of scientific progress made with respect to the mechanisms of phosphorylative oxidation via the functioning of mitochondrial atpase, from the time of mitchell's experiment with the ph meter until the time of the manufacture of yoshida's biorobots, is an exemplary one. it is typical of the way in which a mode of thought evolves over time, from a primary discovery resulting from serendipity or an experiment "to see what happens", leading to the proposal of the existence of a mechanism, to a carefully programmed project which, because of its inventive technicity, shows the validity of the proposed mechanism, and, in addition, demonstrates its future utilitarian value. nowadays, certain biotechnologists dream of being able to "synthesize life" 80 in terms of cells that are able to imitate the performance of living cells. the concept of the "lab-in-a-cell " is coming to the fore 81,82 . nevertheless, it would be necessary to design an artificial cell that is an authentic replica of a living cell, and which benefits from all the attributes of a living cell. this is not achievable at the moment. thus, the current aim of nanobiotechnology is limited to scheduling the construction of artificial cells that are relatively simple both in composition and in function, for example, a microvesicle edged with a lipid membrane, containing a system of protein synthesis expressed from a short sequence of dna, as well as a system of atp synthesis able to supply the energy necessary for this protein synthesis. demonstration of a rotational movement of f1fo mitochondrial atpase (atp synthase) , induced by atp. atpase or atp synthase (reversible catalysis enzyme that hydrolyzes or synthesizes atp) has two sectors (see figure iii .18). the membrane sector, fo, comprises an assembly of a dozen so-called c (rotor) subunits and an a (stator) subunit. the other, extra-membrane sector, f1, is catalytic. it comprises three β catalytic subunits and three α non-catalytic subunits arranged in a ring, in alternating order. at the center of the ring is the γ subunit which is attached to the c subunits of the fo sector. subunits δ, ε and b stabilize the whole of the molecular complex. in the experiment illustrated in this figure, subunits α and β of the f1 sector of the enzyme have been genetically modified to include polyhistidine chains (his-tag, artificial ligand). due to the interaction of these chains with nickel ions (linked to an organic molecule) covering a solid support medium, the α and β subunits are immobilized. in addition, a gold microbead is fixed onto the ring of the fo sector c subunits by means of a chemical device (streptavidin molecule, artificial ligand). following the addition of atp, rotation of the microbead attached to the ring of fo sector c subunits is observed by microscopy on a black background. this rotation is dependent on (and at the same time an indicator of) the rotation of the c subunits, itself led by the rotation of the γ subunit in contact with the catalytic β subunits. note the ejection of protons. when the enzyme functions as atp synthase, the proton movement takes place in the opposite direction. "progress in biology is possibly mainly tributary to the drawing up of concepts or principles […] . in the process of elaborating concepts, which marks scientific progress in biology, there is sometimes a crucial step, when we realise that a more-or-less technical term that we had previously considered to cover a given concept, in fact covers a mixture of two (or more) concepts." ernst mayr translated from a french translation entitled history of biology. diversity, evolution and heredity -1989 on the margins of the modeling and the difference in mathematized systems that comprise theoretical biology, particularly in silico biology, concepts are mental representations, often image-filled and idealized ones, of fundamental mechanisms that are deduced on the basis of experimental results. from the imaginary domain of the probable, they extrapolate constructions of the mind that are in phase with the facts and experimental data, within a reflective projection that gives them their meaning and makes it possible to make certain predictions. there are premonitory concepts. this was the case for the concept of the reflex arc that associates movement with sensation. this concept was already present in the ideas of descartes (chapter ii-3.4), but it took more than a century before the theory of the existence of a reflex arc was supported by bell and magendie's demonstration of the existence of relay centers for sensory and motor nerves in the spinal chord (chapter iii-1). there have been premonitory concepts that, while they were demolished at the time they were first proposed, were shown to be completely accurate a few decades later. in the middle of the 19 th century, the german pathologist jacob henle (1809 -1885) needed a healthy dose of imagination and audacity in order to oppose the theory of the "miasma", a theory that was taught as a dogma, with a new theory that not only explained the spread of contagious diseases by microscopic beings, but also formulated the criteria for validating this theory, i.e., isolation of the pathogenic agent and its development in culture away from the diseased organism, then reproduction of the original pathology after injection of the pathogenic agent, which has been isolated, characterized and multiplied in a culture, into a model animal. thirty years would go by before the formulation of koch's postulates, based on experimental evidence (chapter iii-4). we may ask ourselves whether or not history is currently repeating itself in the case of spongiform encephalopathies that affect humans and animals, for which, according to the thesis of stanley prusiner 83 (b. 1942) , the prion, as an infectious protein, is responsible. other evocative concepts hold the keys that open doors to domains that are unknown, but are potentially rich in information. it is thus that the double helix dna structure proposed by crick and watson, based on the complementarity of adenine-thymine and cytosine-guanine bases (chapter iv-1.1.1), led to the concept of dna replication with reconstruction of a double strand that is identical to the original double strand. the concept of dna replication spurred on matthew meselson (b. 1930) and franklin stahl (b. 1929) to develop an experimental protocol based on the labeling of the dna nucleotide bases of the enterobacterium e. coli with a heavy isotope of nitrogen, 15 n, and on the differentiation of monocatenary dna strands in the process of synthesis by measurement of their density, as analyzed by centrifugation in cesium chloride gradients. in the same vein, jacob and monod's discovery of regulatory genes (chapter iv-1.1.1) gave rise to the concept of the operon which, in the bacterium, defines a genetic unit comprising structural genes and regulatory genes. the concept of the regulation of gene expression, extended to higher eukaryotes, makes it possible to explain the phenomenon of differentiation in cells with specific activities (muscle cells, nerve cells, epithelial cells…) by the silencing of certain genes and the activation of others. within the framework of bioenergetics, the chemiosmotic theory put forward by mitchell, in order to explain the coupling of mitochondrial respiration with atp synthesis (chapter iv-4.3), gave rise to consideration of the concepts of transmembrane transport of metabolites and of vectorial metabolism. some generalizing concepts that carry a unifying virtue within them are known. one such is the concept of compartmentation. the cell is no longer considered to be a bag of enzymes, as used to be the case. it is now considered to be a compartmented structure in which each type of compartment corresponds to a type of organelle delimited by a membrane and characterized by specific functions. thus, because of the genetic material that is present in it, the nucleus of the cell holds the information necessary for the manufacture of proteins. the mitochondria, which are called cell power plants, are in charge of oxidizing the products of cell catabolism and using the resulting energy for the synthesis of atp from adp and mineral phosphate. the lysosomes are the garbage collectors of the cell. among the functions carried out by peroxisomes is the partial breakdown of very long chain fatty acids. the endoplasmic reticulum and the golgi apparatus are involved in the maturation and the secretion of proteins. the ribosomes represent the machinery upon which messenger rnas are displayed in order to be decoded into proteins. a sign of the extreme sophistication of this setup is that the membranes of the endocellular compartments are not sealed common walls. they contain proteins that act as selective transporters of metabolites or highly specific ion channels, allowing the exchange of messages throughout the cell. thus, each organelle, informed of the condition of the others, is able to adjust its own activity to ensure the greatest harmony of the whole. this conditioned compartmentation at cell level may be compared to the socialization of human communities. while endocellular organelles are compartments delimited by membranes, there are non-membrane-bound compartments in the cell, such as protein complexes in which two, three or even more proteins are closely linked. often, these are enzymes that catalyze reactions that are contiguous in a metabolic pathway. being compacted into a complex known as a metabolon results in an increased efficiency of the flow of metabolites by facilitating the channeling of this flow. concepts evolve, often adjusting their representations according to accumulated knowledge. a good example of this is the evolution of the concept of the gene since its formulation at the beginning of the 20 th century. the term "genetics" was created in 1906 by the english naturalist william bateson (1861 -1926) . the term "gene" was introduced three years later by the dane wilhelm johannsen. this term designated a principle which, in the chromosomes of fertilized egg, and in an intentionally vague manner, was supposed to have an influence on the phenotype of the progeniture. during the same period, the term "locus" appeared out of the experiments carried out by the american thomas hunt morgan on the drosophila, a locus being defined as a region of a chromosome which, when altered by a mutation, leads to a modification of the phenotype of the living organism. based on cross-breeding experiments carried out on hundreds of drosophila mutants, morgan and his co-workers drew up the first genetic maps. by chance, the salivary glands of the drosophila have a particular characteristic; the nuclei of their cells contain giant chromosomes called polytenes, which result from the association of a hundred replicate copies of chromosomes that, after staining, are visible under the optical microscope. on these chromosomes, it is possible to distinguish colored bands separated by clear bands. it was observed that specific mutations had specific effects on the arrangement and number of these bands. the material contained in the bands was therefore the site of mutations. in the middle of the 1930s, the listing of more than 3 500 bands made it possible to construct a cytological map that was already highly detailed. the concept of the gene, the material basis of inheritance, took root. the sporadic mutagenic effect of x-radiation in the drosophila, which was shown by the geneticist and biophysicist hermann müller (1890 -1967), led the austrian physicist erwin schrödinger to question the sporadic event which, at the level of a target of a few dozen atoms, determines a mutation. he postulated that the target is located in the chromatin of the chromosomes, organized as an aperiodic crystal. the chemical nature of this target was identified with dna, following bacterial transformation experiments (avery, macleod and mccarthy, 1944) and experiments concerning bacterial infection by the bacteriophage (hershey and chase, 1952) (chapter iv-1.1.1). this is how the idea that gene = dna was born. the simple and reassuring idea that one gene → one enzyme, which was deduced from mutation experiments carried out by beadle and tatum on the mold neurospora crassa (chapter iii-6.1), had only a limited lifetime. a first stumbling block appeared when it was shown that the activity of a gene, and in consequence its contribution to the phenotype, depends on nucleic elements outside the gene. the definition of the term "gene" was then extended to include promoting and regulatory sequences. in the case of the lac operon of escherichia coli, these sequences are located just upstream of the site where transcription begins. however, in eukaryotes, a regulatory sequence may be distant from the gene that must be transcribed and sometimes it may be involved in the regulation of several genes (chapter iv-1.1.1). in the 1970s, the idea of the existence of the mosaic gene in eukaryotes appeared. a gene was now thought of as an assembly of several exons that originally in the chromosome are separated by introns. the alternative splicing of these pieces of genes gives rise to numerous possibilities for reconstitution, i.e., many messages coding for many different proteins. thus, however useful the concept of the gene has been with respect to its ability to generate discussion and to provoke experimentation concerning the molecular machinery responsible for the transmission of the hereditary characteristics, we can see that the term itself has not ceased to be the subject of readjustments, since the time it was first formulated. certain concepts are matched with metaphors. while some metaphorical concepts, particularly those that make use of images designed to grab the imagination, and to be easy to understand, tend to take liberties with the realities of living beings, they can also shed light on unsuspected mechanisms in sectors that have been neglected. metaphorical concepts are not a current fashion. it should be remembered that in his passions of the soul (1649), descartes, when asked "how limbs can be moved by objects of the senses and by the mind without the help of the soul," responds that this takes place "in the same way as the movement of a watch is produced only by the force of its spring and the arrangement of its cogs." later on, with lavoisier's clear vision of the vital role of oxygen, and his comparison of respiration with combustion, the concept of the chemistry of life, combined with that of bioenergetics, came to the fore, and was at the heart of studies on the metabolism. chemical reactions that liberate and absorb heat were substituted for the cogs of cartesian mechanics. the second half of the 20 th century saw the birth and development of the concept of the program, a concept with computer technology connotations, which was destined to explain the phenomena of inheritance. this concept began to fill out from the moment when it became certain that, in its nucleotide sequence, dna contains the necessary information for the construction of the protein material of cells. for a certain period of time, the passion for molecular genetics eclipsed the interest that had previously been given to metabolic chemistry. the powerfulness of the metaphorical concept may be measured according to the effect it has in pushing scientific research in particular directions, with the results this has on society. thus, during the 17 th and 18 th centuries, the study of human pathology was impregnated with a strong iatromechanical current. physiological chemistry and its corollary, pathological chemistry, which emerged as disciplines in their own right in the 19 th century and achieved full expansion in the 20 th century, are our inheritance from lavoisier and the concept of discussion about concepts necessarily leads to a brief discussion of scientific semantics, as shown by the few examples given in the previous pages. as we have just seen, the word gene that was put forward by johannsen around one century ago did not have the same meaning at that time as it has now, a meaning that still remains fluid. the gmo, an acronym meaning genetically modified organism, which has been the subject of vehement diatribes over the last few years, becomes much less of an object of passion if it is considered within the context of evolution. after all, for the last two to three billion years, living organisms have been genetically modified constantly by spontaneous mutations, which is why the human beings that we are today are able to discuss them! the term cloning is another example of a semantic misunderstanding that leads to inaccurate interpretation and arouses the passions. the primary meaning of the term cloning is the multiplication and the identical reproduction of a living cell. the simplest and most unambiguous example is that of bacterial cloning, a bacterial cell producing millions of cells that are identical to the original cell by its multiplication in a nutritive medium. the term animal reproductive cloning does not carry exactly the same semantic weight. it should be remembered that, in eukaryotes, the preliminary act of the cloning procedure involves the injection of the nucleus (with 2n chromosomes) from a somatic cell into an enucleated oocyte (chapter iv-2.3.2; see also chapter iv-6.1). the somatic cell nucleus, by providing its genetic equipment, gives the being that will develop in the uterus a phenotype that is practically identical to that of the somatic cell donor, but nevertheless not completely identical, as the cytoplasm of the enucleated ovum, with its mitochondria, provides a small but non-negligible fraction of genes, the mitochondrial genes. as for therapeutic cloning (in the absence of uterine implantation), this is used for the manufacture of differentiated cells that may be grafted into the individual who has donated the original somatic cell, with no immune-related rejection occurring. this is non-reproductive cloning. the passionate argument that has arisen because the term cloning is bandied about in an ill-considered fashion illustrates the confusion that can result from a lack of precision in the use of certain terms with a high level of media impact. "all the major problems of the relations between society and science lie in the same area. when the scientist is told that he must be more responsible for his effects on society, it is the applications of science that are referred to […] . no government has the right to decide on the truth of scientific principles, nor to prescribe in any way the character of the questions investigated." the progress of science is linked to that of civilization. it is in keeping with the state of mind, the beliefs, the lifestyle and the thought patterns of societies. in ancient greece, where manual work was considered to be servile, science remained essentially theoretical, confined to logic and dialectics, and strongly attached to questions of philosophy. the birth of experimental science in the 16 th and 17 th centuries went hand-in-hand with the rehabilitation of manual work. the technical side dominates in modern biology, which seeks to solve problems concerning the "how", rather than to address philosophical problems concerning the "why". as ian hacking (b. 1936) says in representing and intervening (1983), nowadays engineering, and not theorizing, is the greatest proof of scientific realism, which leads to the minimization of philosophical thought. in a skeptical biochemist (1992) , the polish-born american biochemist joseph fruton (1912 fruton ( -2007 emphasizes the contrast between the 19 th century and the first half of the 20 th century, when eminent scientists were still interested in the ideas of the professional philosophers of the history of the sciences concerning the progress of experimental research and, in contrast, the end of the 20 th century, when philosophy and the experimental sciences pretended to ignore one another. this is doubtless partly because the history of biology has become the history of biotechnologies to such an extent that, according to some, the objects being explored are so familiar that they are now part of the life of society. in pandora's hope (1999), bruno latour (b. 1947) considers that the current confrontation between subject and object, in which the researcher-subject explores the structure and function of the object, is being transformed into a human-nonhuman dialogue, in which the nonhuman-object becomes "socialized". taking yeast as an example, latour writes that it has been "working for millenia in the brewing industry, but now it works in a network of thirty laboratories where its genome is mapped, humanized and socialized like a code, a book, or a program of action that is compatible with our ways of coding, counting and reading […] . non-humans have become automatons, admittedly without rights, but much more complex than material entities." latour visualizes the human-nonhuman associations in the form of collectives that are organized into strata that implement the technical, the political, the social, the ethical, the ecological… the technosciences correspond to one of these strata, the sociotechnical stratum that is directly linked to the stratum of political ecology. in the same spirit, the belgian philosopher gilbert hottois (b. 1946), in his philosophies of the sciences, philosophies of techniques (2004) remarks that "laboratories produce things that go off to live their lives in society and in nature." thus, bacteria, yeasts or genetically modified plants are able to produce drugs such as insulin, growth hormone and vaccines for human medicine. these drugs become part of and indispensable to life in society. they are evaluated according to their market value by the companies that patent, manufacture and sell them, and according to the comfort they bring to the patients to whom they are administered. the financial management that results from their consumption becomes a worry for those responsible for public health, while their manufacture by specialized companies generates industrial activity and economic growth which may be measured according to how fashionable they are and how they sell. for a long time, society, while benefiting from scientific progress, remained indifferent to the experimental method, that is to say, the way in which knowledge progresses. in the last decades of the 20 th century, society became aware, via information concerning the occasionally demonized exploits of genetic engineering, that science can "take liberties" with the human being. populations were well informed about the effects that genetic engineering could have on the mortality rates of pathologies such as cancer and diabetes or on degenerative illnesses of the nervous system, and about the closeness of possible solutions. however, they were also warned about the risks to which science was exposing humankind. remembering certain tragic episodes concerning hiv-contaminated blood transfusions, growth hormone and mad cow disease, and certain cassandra-like predictions, such as a catastrophic epidemic of spongiform encephalopathy that has happily yet to appear, society shows reservations when the media inform its members of new feats of modern technology. political authorities, for their part, afraid of potential problems, tend to follow the principle of precaution, which in fact hides a fear of risk. however, evaluating risk involves not being afraid of it but understanding it in a lucid and courageous fashion. informed by the media, which often use sensationalism, the citizen is increasingly calling into question whether certain practices involving the biosciences, such as cloning, or certain mercantile transactions such as the taking out of patents concerning gene sequences, or even experimentation on live animals, are well-founded. "the problem of experimentation on man is no longer a simple problem of technique. it is a problem of value. from the moment that biology concerns man no longer simply as a problem, but as instrumental to the search for solutions concerning him, the question arises of deciding whether the price of knowledge is such that the subject of the knowledge is able to consent to become the object of his or her own knowledge. we have no difficulty here in recognizing the still open debate concerning man as a means or an end; an object or a person. this is to say that human biology does not contain in and of itself the answer to questions concerning its nature and its significance." knowledge of life -1965 written at a time when people were far from imagining how molecular biology was going to expand, the prophetic words of georges canguilhem (1904 -1995) have maintained their philosophical validity. manipulation of the human embryo, whether this involves its creation by cloning or the modification of its genetic inheritance, obviously leads to the need to consider the societal, religious and political points that arise from the domain of bioethics and are a reflection of the period in which we are living. until recently, advances made in biology left moralists indifferent. this ceased to be the case when scientific experimentation began to look at the human embryo with a view to utilitarian ends in the health domain. the specter of cloning was brandished without any clear distinction being made between reproductive cloning and therapeutic cloning. biology became demonized. however, as biologist pierre chambon (b. 1931) said in an interview in the french journal biofutur: "in absolute terms, biology is unable to tell us whether the cloning of a human being is moral or immoral, it simply tells us whether it is biologically possible." the birth dolly the sheep in 1997 (chapter iv-2.3.2) triggered a virulent debate because now that the cloning of an animal had become possible, that of a human being became envisageable. the media sensationalized this debate all the more in that it was exacerbated by debate concerning gmos (chapter iv-2.1). the dolly affair became a problem of society. up until then, the biosciences had been happy just to try and understand the mechanisms that explained the functions of living beings, but now, with the advent of gmos and cloning, it became obvious that a forbidden barrier had been crossed and that man had the power not only to transform but also invent himself. faced with this desacralisation of nature, the need arose for some philosophical reflection. this was given the name of bioethics, which is the title of the book, bioethics, a bridge to the future, which was written by the american biologist van rensselaer potter (1911 -2001) in 1971. the term bioethics covers philosophical considerations that range from the biosphere to the human person. bioethics tries to give a wider meaning to the moral codes which, in human societies, depend on ancestral traditions. it aims to prescribe that which is desirable according to the kantian maxim of the categorical imperative. in his what is bioethics? (2004), the belgian historian gilbert hottois reminds us that bioethics are above traditional morals, the latter being a set of norms that are most often spontaneously respected as good habits, without any critical reflection being involved, while bioethics, on the other hand, arises out of critical thought, analysis, discussion and the evaluation of established mores. over the last few years, the problems that are targeted by bioethics have moved towards today's burning issues. human cloning is an example. while allegations of the transcendence of man in nature may lead to human reproductive cloning being considered as a crime, strictly scientific considerations lead to an emphasis on the lack of responsibility shown by a few zealots, given the hazards involved in cloning in animals, such as the need to use a large number of oocytes in order to achieve success in cloning, the very low viability of the cloned embryos and the development of serious functional anomalies in the clones that survive. even supposing that scientific progress will one day overcome these difficulties, human reproductive cloning will come up against an insurmountable obstacle, the cloned subject's fear of finding that he or she is identical to the relative from whom his or her genetic inheritance comes. after all, the notion of manipulation of the human ovule with the aim of serial reproduction has often haunted science fiction stories. in brave new world (1932), aldous huxley (1894 -1963) gives an apocalyptic vision of the budding of human eggs that produce hundreds of identical twins which are conditioned into classes and subclasses while being raised in jars, depending on the quality of the nutritive substances they are given. in the artificial uterus (2005), henri atlan (b. 1931) predicts that the raising of human fetuses in jars could well become an alternative to uterine gestation in a distant future. let it be understood that human reproductive cloning, which is no longer part of the domain of science fiction, as it has become feasible, must be considered as being reprehensible because it goes beyond the limits of reason, and is a denial of human transcendence. man as subject cannot be considered as an object. the problem of therapeutic cloning is quite different, although it leads to reticence and prohibition because the demarcation between therapeutic and reproductive cloning depends mainly on whether a cloned embryo is implanted in a uterus. while, at the time of writing, therapeutic cloning has been prohibited in france, germany and other countries, it is tolerated in great britain. in the usa, the prohibition only applies to publicly-financed researched, while each state has its own legislation, which is relatively flexible. the objective of therapeutic cloning is to provide patients with tissues that arise from their own selves, and are therefore immunocompatible and able to be grafted without there being any risk of rejection (chapter iv-2.3.2). it is based on the removal of somatic cells from the subject to receive the graft and the transfer of the nuclei of these cells into enucleated oocytes. the stem cells that are obtained after the first divisions are stimulated using appropriate growth factors. depending on the factor used, the stem cells differentiate to form a type of tissue (hepatic, muscular, nerve…) that can be used as a graft. such a procedure may be envisaged for patients who have suffered a serious, invalidating trauma, for example section of the spinal chord. a graft of immunocompatible nerve cells might make it possible to re-establish nerve continuity. a similar type of therapy has been considered for parkinson's disease, the cause of which is a degenerescence of certain cells of the encephalon (chapter iv-2.3.1). given the hopes that are raised by the possibility of such therapies, and the fact that, after all, such therapeutic cloning is the equivalent to an autograft, even if the ways in which the graft is obtained are slightly tortuous, the demonization and rejection of such practices should be reconsidered, calmly and coolly. another option for therapeutic cloning is the correction of mutations identified in the mitochondrial genome of a woman wishing to have children. it is, in fact, the mother's ovum that provides the fertilized egg with its complement of mitochondria that are indispensable for its viability. the manipulation involves inserting the nucleus of a fertilized ovum from the mother, obtained by artificial insemination, into an enucleated oocyte taken from a woman who is not suffering from the mitochondrial defect. the cytoplasm of the enucleated oocyte provides the stock of functional mitochondria that are indispensable to normal cell function in the future embryo. in a domain of the bioethics, in which rational objectivity comes up against deliberately technophobic religious and cultural considerations, it is useful to remember certain legal and legislative paradoxes. thus, in france, after having been considered to be a criminal act that was subjected to severe repression by the law up until 1975, the right to have an abortion before the end of the third month of pregnancy became not only authorized but also protected by law. it is interesting to note that in the 13 th century, thomas aquinas, the father of the church, had acknowledged that a fetus only becomes "animated" by the implantation of the soul by holy will in the third month after fertilization. another subject to be considered is pre-implantation genetic diagnosis (pgd), in which human embryos that have been fertilized in vitro are sorted in order to find those that are without defects, a practice which is on the verge of being a deviation in the direction of eugenics. nevertheless, pgd is the basis of a practice that is either already legalized or is in the process of being so in several european countries, the creation of so-called designer babies. a typical example is that of a designer baby arising from an embryo whose immune profile to that of an older sibling who is suffering from leukemia. in this case, there is good reason to hope that a graft of immunocompatible blood cells from the designer baby into the sibling who is suffering from leukemia will save the latter from death. out of the disharmony of opinions that arising from cultural tradition, religious conviction or simply scientific pragmatism, the american biologist and philosopher h. tristram engelhardt (b. 1941) , in the foundations of bioethics (1996) proposes a lay bioethics that is based upon the principle of permission. lay bioethics advocates tolerance while admitting that this tolerance in no way prevents anyone from taking up a personal position; it means that each human being has a moral sensitivity as well as the ability to reason and to choose within a defined limit of non-harmfulness and of justice. the individual is free to modify his or her destiny, or to manipulate his or her nature by genetic interventions because, adds engelhardt, "there is no lay moral foundation to prohibit such an intervention." when a researcher or the research organization that the researcher belongs to files for a patent for an invention with a patent office, it is necessary to demonstrate the novel and utilitarian nature of this invention. if a patent is accepted, this gives the person or body that filed it the exclusive right to make use of the invention over a pre-determined period of time, generally 20 years, which is a means of protection, or, if desired, to allow others to make use of the invention by issuing a license to do so. in the domain of living beings, there has sometimes been confusion between invention and discovery. in 1991, craig venter, known for his participation in the sequencing of the human genome, filed a demand for a patent covering the sequences of 2 700 fragments of recombinant dna (cdna) called est (expressed sequence tags) that are obtained by reverse transcription from human brain messenger rnas, in the name of the nih (national institutes of health) at bethesda (usa). the patent specified that ests could be used as probes to characterize genes that are potentially involved in neurological ailments. the resulting outcry led the nih to withdraw its patent demand. in fact, the patenting of living beings has a long history that goes back to the patent that was filed in 1895 in france by louis pasteur, and then in 1873 in the usa, for the use, in brewing, of a yeast culture that was free from pathogenic bacteria. from this historical perspective, the case of ananda chakrabarty (b. 1938) set a legal precedent. in 1994, chakrabarty filed a demand with the us patent office for a patent relating to a pseudomonas type bacterium which, by genetic modification, had acquired the ability to digest crude oil. his demand was refused. after an appeal and many legal battles, the united states supreme court overturned the patent demand refusal, the basis of the judgement being that any modified microorganism is a product of human ingenuity and has a specific name, characteristics and use. thus, from 1980 onwards, the arrival of an era of patents derived from genetic engineering was indicative of how this discipline was growing. in december of that year, stanley cohen and herbert boyer, acting on behalf of the university of stanford, patented a nucleic chimera comprising a recombinant dna carried by a vector. in 1982, a patent concerning the growth hormone gene was awarded to the university of san francisco. in 1984, the university of california at berkeley obtained a patent for the human insulin gene. in 1985, the american company pioneer hi-bred succeeded in patenting a variety of corn in which genetic modification has led to an increased synthesis of tryptophan, an amino acid that is indispensable for animal feed. in 1988, the genentech company acquired a patent for the gene coding for human gamma interferon. this was followed in japan by a patent for the gene coding for beta interferon. in the same year harvard university patented the oncomouse, a transgenic mouse whose susceptibility to cancer is greatly increased. after this, several species of transgenic animals were patented for utilitarian purposes, such as the production of human alpha-1-antitrypsin taken from the milk of transgenic goats and used for the treatment of cystic fibrosis. the frenetic patenting of living beings has reached the domain of natural products arising from the plant world in tropical regions, the immensely varied essences arising from these plants being full of pharmacological potential. the potential for producing drugs of a considerable commercial value from such plants is very high. here we return to the problem of the patenting of genetically modified, cultivatable plants (gmps) (chapter iv-2.1). thus, the experimental method, the principle of which is to acquire pure knowledge, finds itself led astray in its applications. whatever the motives that are given, particularly for manipulations that give rise to the manufacture of marketable products, the patenting of genomes for mercantile ends shows the regrettable, but unfortunately inevitable, direction in which the very spirit of a science, molecular biology, which half a century ago wished to be at the heart of an understanding of living beings, has drifted. the suffering of animals that are being experimented upon gives rise to a moral problem. the end of the 19 th century saw large-scale demonstrations against vivisection and repeated demands for it to be abolished. today, there is renewed vigor in the call for the abolition of vivisection, without any real coherent basis. this desire to stop experimentation on animals ignores the imperatives of contemporary medicine, which must meet the challenge of pathologies whose increasing incidence is worrying, such as cardiovascular diseases, diabetes, cancer, and the degenerative illnesses that are linked with aging or are of genetic origin. it is true that animal experimentation inevitably leads to questions. are the stakes involved in a particular experiment, in terms of the acquisition of new knowledge, worth the suffering of an animal used in that experiment? is it not necessary to ensure that the experimental protocol is well-documented, that it is not redundant, or even that it has been the subject of previous studies carried out on cells in culture? it is easy to see the size of the methodological chasm that separates contemporary physiology from that of the time of claude bernard, when cell culture techniques were not yet being used, when the main instrument used was the scalpel and the researcher, using his or her imagination and creativity, had to develop specific protocols that were able to validate or refute a working hypothesis. each period in history operates in its own way according to its moral laws and its technical capabilities. the bloody operations carried out by magendie and by claude bernard in the 19 th century, which were tolerated at this time despite criticisms from antivivisectionists, would not be permitted today. nevertheless, it is true that the physiologists of the 19 th century, by means of the results of their experiments, wove a tapestry of new knowledge on which contemporary biologists were going to work and without which the level of understanding the modern science would be much lower than it is. animal experimentation remains indispensable in many areas of physiological investigation, in genomics, in toxicology and in pharmacology. it is a precondition for clinical trials of any new drug, being used to test for the drug's efficacy, its metabolism and any toxicity. however, not all data arising from animal experi-mentation can be extrapolated to man. the margin of uncertainty can be reduced by means of comparative trials on several animal species. because of their phylogenetic proximity to man, primates may seem to be the solution for experimentation prior to the application of a drug in man. this was the case for the development of a vaccine against hepatitis b. it has been proposed the grafting of stem cells in man should be preceded by experimentation in apes, in order to ensure the absence of tumorization over the long term. however, the researcher is confronted with a dilemma: should he or she ensure the safety of man with respect to possible deleterious effects or respond to ethical demands that recognize the very great genomic similarities between man and the chimpanzee. a consideration of cloning, patenting and animal experimentation practices illustrates the excesses of the experimental method in domains where political authorities consider themselves able to legislate. administrative decisions, often made in the absence of any dialogue with scientific authorities, can have serious consequences. thus, given the pretext of strict obedience to the principles of bioethics, which are a matter of tradition, and while certainly respectable, are nevertheless arguable, and also given the pretext of a sickly and unconsidered fear of the risk involved in certain experimental practices, and the absence of an intelligent evaluation of this risk, research, which until recently took place in a motivating atmosphere of liberty, may, over the long term, be weighed down with a highly prejudicial handicap and a limitless sense of discouragement. in the 17 th and 18 th centuries, experimental research, which was still in an emergent phase, was mainly artisanal, and in the hands of rare scholars. it took form during the 19 th century in the west, particularly actively in germany, and became operational in the 20 th century, under the aegis of governmental authorities, with the creation of institutes, the programmed recruitment of researchers and the allocation of renewable budgets. modern science, based on the principles of the experimental method, came to the fore much later in the east than in the west. the globalization of knowledge has meant that at present experimental science, in all domains, including that of the life sciences, has spread throughout the world, with even those countries that had become relatively backward in these domains because of their isolation catching up rapidly. nevertheless, it is true that the progress of the experimental sciences in the usa and in the united kingdom has been distinguished by pragmatic management of these countries' science policies, based on the excellence and the high degree of autonomy of their universities and research institutes with respect to recruitment and choice of subjects of study. the efficacy of this policy in the life sciences may be judged by the number of researchers who have won nobel prizes since the second world war (at the time of writing, more than 80 in the usa and twenty or so in great britain as opposed to only 4 in france). in france, research on living beings is carried out in the laboratories of universities, in institutes connected with higher education and in laboratories that are run by large organizations such as the national scientific research center (cnrs), the national institute of health and medical research (inserm), the national institute of agronomic research (inra), the atomic energy commission (cea), the national institute of research in computer processing and automation (inria), the national center for space studies (cnes) and the french institute of research on the seas and oceans (ifremer). equivalent bodies exist in countries other than france, some of them being institutes that are dedicated solely to research, and some being university laboratories that associate research and teaching. at the beginning of the 20 th century, the function of researcher was most often associated with that of a professor occupying a chair at a university, surrounded by a few assistants, the professor directing the research work in his area of specialization. now, within a period of a few decades, the status of researcher has been modified greatly. today we talk of research careers classified according to level of expertise and technicality. management, or the supervision of career paths and the control of financing, is carried out by an administration that is itself highly hierarchical. the scientific process has undergone a metamorphosis, shown by changes in the behavior of researchers not only within the institutions in which they work but also in their relationships with the media, the political sphere and society. the teaching of the life sciences needs to take this into account. "long ago, there was a time when scientists recounted the exact circumstances of their discoveries, without shame, even when their recital showed up the fragility of their forecasts or an indecent collaboration on the part of every bit of luck. such times are past, and the researchers of today often like to make us believe that they only find what they are looking for. the thousands of pages pasteur's lab books provide an opportune reminder to us (and to program-makers or impatient users) that it is just as difficult to ask a question as to answer it, that a scientific discovery often occurs after a long, winding path, that rather than following the fashion, it is preferable to follow one's ideas, particularly if they are good ones, and are in advance of the fashion." jean jacques molecular dissymmetry, in "pasteur, workbooks of a scholar" -1995 current technological progress, the accumulation of the scientific knowledge, the institutionalization of the public research and many other factors are disrupting a ritual of the experimental process that had survived until the middle of the 20 th century, and even beyond. the experimental life sciences of the 21 st century will necessarily see themselves remodeled with respect to their objectives and procedures. faced as it is by an increasingly tough international competition, the scientific community is also subject to restrictions in terms of operation and prospectives. an organization into small teams of a few researchers gathered around a boss, working in friendly interaction, is increasingly giving way to large groupings that sometimes seem like consortiums. focused on research subjects that are deemed to be "cost-effective", these superstructures are encouraged, or even imposed, in the sadly illusive hope that the will lead to greater efficacy. the person in charge of such large groups is taken up with everyday management tasks and by maintaining good relations with the administrative bodies on which his or her organization's survival depends. he or she may become distanced from the experimentation and forget the intellectual motivations that in the past caused his or her competence to be recognized. it should be emphasized that the secret of future successes lies in situations where young researchers are in direct contact with their bosses, and where friendly interaction with a known master teaches the apprentice researcher how to learn, how to think and how to experiment in a critical fashion. preoccupied by the rapid expansion of the scientific population, accompanied by the creation of laboratories whose operation necessarily requires financing, often on a large scale, political authorities, giving way to the requirements of media-fed public opinion, are interfering more and more, via administrative relays, in the control of the objectives of experimental research. short-term objectives, considered to be "visible", are favored. a priori, the viability of a project is judged according to the scientific context of the period and its impact on society, insofar as the project looks at health problems with a high degree of media coverage (cancer, degenerative illnesses, viral infections…) and often in agreement with a consensus that avoids going against the orthodoxy of the moment. this leads to a rigid management of projects that are financed and controlled according to objectives that have been fixed in advance, and that are all the more easily accepted by state authorities when they are somewhat fantastic in character. however, fundamental research proceeds from a playful activity, and for this reason, its efficacy is dependent on the passion of the researcher for the problem that he or she is studying. in contrast to what is believed by the narrow-minded, the effectiveness of a researcher in terms of discoveries depends upon the liberty that is given to this researcher, assuming, of course, that this liberty is underpinned by criteria of confidence such as the researcher's scientific past, his or her motivation, and judgements made concerning the researcher by impartial peers. it should not be forgotten that the determination of the three-dimensional structure of hemoglobin by max perutz (chapter iii-6.2.1) took around twenty years of solitary, uninterrupted and untiring labor. the theoretical and technical tricks that led to this success helped to open up the domain of the structures of giant macromolecules, several dozen kilodaltons in size, which no-one had dared study before. anyone who uses the experimental method realizes that while fundamental research must be organized, it cannot be scheduled. such a person knows that the pathways to discovery are convoluted, and that an inexplicable observation that appears unexpectedly during an experiment can sometimes, if the researcher is sufficiently perspicacious, be the beginning of an adventure that leads to a discovery. it was to just such a convoluted path that the belgian biologist christian de duve (b. 1917) alluded in his speech when he received the nobel prize for medicine and physiology in 1974. after working at the university of saint louis in the usa, de duve, who had taken up a post at the university of louvain, belgium, decided to look at a research theme that had received a great deal of media coverage, diabetes and insulin. it was while operating on one of the subcellular fractions obtained from ground rat's liver, and analyzing certain of the enzyme activities of these fractions, that he was surprised to find, in one of them, enriched with mitochondria, a phosphatase activity that, paradoxically, increased with time, while the enzyme activities specific to the mitochondria declined. this was an activity belonging to organelles that were contaminating the mitochondria. dropping all research on diabetes, de duve set out to identify and characterize these unknown organelles. he discovered that they were involved in the breakdown (lysis) of molecules that are undesired by the cell and, for this reason, he called them lysosomes. the discovery of lysosomes helped to open a new chapter in cell biology and to attribute a molecular cause to diseases with serious prognoses whose etiology had remained a mystery up until then. these diseases were given the label lysosomal diseases. these diseases result from the absence of a lysosomal enzyme that is responsible for the breakdown of a given metabolite. the accumulation of this non-broken-down metabolite in the lysosomes leads to cell malfunction, which causes the lysosomal disease. as de duve said jokingly, if he had carefully followed the experimental process laid down in his diabetes research project, and if he had not given way to the temptation of "playing hooky" or "playing truant" he would never have mounted the podium in stockholm. in the same way, henri-gèry hers 84 (1923 -2008), a cell pathologist at the internationally renowned louvain school, remarked in an article published in the review médecine/sciences: "i believe we would obtain maximum value for the money devoted to research if we were willing to distribute it to those who have been shown to be productive, according to their needs, and without asking them for a program." hers concluded, in a tone that was deliberately playful, but thought-provoking, "such a simple system would lead to unemployment for a large number of administrators, which is why i suspect that it will never be adopted." research has its own set of ethics, driven by anticonformity and the creative imagination, capable of shaking up firmly-anchored ways of thinking and established hierarchies, and of leaving the researcher the freedom to express him or herself and to experiment off the beaten paths. as eccles says in evolution of the brain and creation of the conscience, it is important to distinguish between intelligence and imagination. intelligence is measured according to the rapidity and depth of understanding and clearness of expression. it may be measured and even given a numerical value. the same is not true for the imagination, a more subtle, unmeasurable phenomenon that cannot be learned. the imagination is one of the levers that is able to lift the boulder that hides scientific truth. the imagination is the ultimate weapon of research, which shakes up the knowledge acquired by the intelligence. nevertheless, the imagination must be tempered by a good critical sense that is able to perceive potential sources of artifacts, both in sophisticated instruments that act as so many black boxes from which already manufactured information emerges and in genetic or chemical cell exploration methods whose specificity must be carefully checked. the benefits that can sometimes be gained from prospective research that is far from dogma that is rooted in sterilizing tradition, the way in which knowledge progresses, most often by moving away from any orthodoxy, the way discoveries appear unexpectedly on the fringes of carefully put together projects, all of these points are matters for reflection for those in power in the worlds of politics, economics and industry. publication is an essential tool for communicating scientific knowledge, and is the judgement criterion for committees in charge of evaluating the creativity of a researcher. in order to have meaning, a publication must provide information that is sufficiently innovative with respect to parallel work carried out in other laboratories. here again, media coverage has quietly infiltrated the scene. its role is all the more perverse in that the rating of a publication is estimated according to its impact index, or, roughly speaking, the renown of the scientific journal in which it is published. curiously, it has happened that articles that would later be considered to be of primary importance have been rejected by highly prestigious journals, simply because the facts mentioned in the article and the conclusions made have not coincided with the orthodox opinions of the period and the traditionalist spirit of the journal's editorial committee. this was the case for an article which the biochemist hans krebs (1900 krebs ( -1980 submitted to the british journal nature in 1937. in this article krebs described a series of experiments showing that an endocellular metabolite, pyruvate, product of glycolysis, is completely degraded during a cycle of enzyme reactions. this degradation cycle would later be recognized as the central pivot of the intermediate metabolism. called upon to judge revolutionary scientific considerations, and unable to perceive their importance, nature's editorial committee rejected the article. krebs then sent his article to a journal with a relatively restricted audience, enzymologia. it was accepted and published in the two months that followed. the importance of the concept that was put forward in the article ensured that its author gained international recognition, leading to his winning the nobel prize for physiology and medicine in 1953. for the researcher, publication is a way of making his or her work known. it is also the way in which the researcher learns about the work of others. while the rhythm at which publications in the life sciences appeared increased slightly in the first half of the 20 th century, the second half of that century saw a great acceleration in this rhythm, leading to a difficult-to-manage proliferation of reviews and books. it has been estimated that in the last thirty years the volume of publications in the biological domain has increased five-fold; in the preceding twenty years it had already doubled. this accumulation of publications makes it harder for the researcher to judge the quality of the huge mass of published articles, even in the highly targeted domains that are within his or her area of expertise. the researcher, therefore, will deliberately choose a particular article according to the prestige of the journal in which it is published, which is not an inviolable criterion of quality. in addition, any judgement concerning the pertinence of a scientific article necessitates a dissection of the subtleties of the methodology, the well-groundedness of the experimental protocol and the validity of the results, by means of a careful examination of tables of results and graphs, and, finally, the logic of the discussion. this restrictive yet absolutely necessary requirement limits the number of articles that are likely to be screened. however, this is not the worse fault of publication today. there is another problem that is much more worrying. many documentation centers have reacted to this inflation in the scientific press by equipping themselves with computing facilities that are able to find, in data banks, articles that have been selected on the basis of a key word index, and to display them on screens. while acknowledging that this constitutes an inescapable change in the transmission of scientific know-how, it should be recognized that in browsing through the pages of a highquality scientific review, it is possible to come across an article containing an innovative idea or a useful technique, an advantage that is less available when using the on-line system of scientific publication that is most prevalent nowadays. mention should also be made of the requirement to publish frequently and within short time frames, for reasons of competitivity, when aspiring to obtain jobs or promotions, or even just to obtain recognition, this requirement being another factor that is prejudicial to fundamental research. it is the cause of worrying excesses, such as experiments that are hastily published and non-reproducible, or even the falsification of experimental results, occasionally within a context of considerable media coverage. although such practices, which are the exception rather than the rule, are rapidly detected and condemned in a scientific culture where information circulates freely, the publicity that they incite, which reaches society at large via the media, leads to an overall discrediting of experimental research. at present, one of the most noticeable trends in scientific publication is that of collectivism. while, in the 19 th century, scientific articles were usually published in the name of a single author, occasionally two authors, and very rarely more than two, nowadays publications are often co-authored by several people, and when the work involves the analysis of structures, or the sequencing of genomes, several dozen researchers may be co-authors. from being the work of individuals, research has become collective. in domains whose complexity requires a wide selection of techniques that may range from physics to genetics, the hybridization of specific areas of expertise is certainly indispensable, and this requires the collaboration on a particular project of researchers who are sometimes physically remote from one another. the downside for the researcher, particularly one who is young, is that this requires him or her to abandon individuality and creativity. both collectivism and inflation in scientific publication are facts that are an integral part of contemporary science, facts which reflect an irreversible trend that it would be difficult to obviate. over the last few years, scientific publication has been subject to a type of restraint, in that certain "sensitive" data in the domain of molecular biology might be used for the manufacture of biological weapons in a form of terrorism known as bioterrorism. thus, the means of synthesizing de novo viruses (influenza virus, poliomyelitis) and the possibility of modifying their tropism by "directed molecular evolution" (change from a sexual tropism to a respiratory tropism for the aids virus) have been the subject of publications in prestigious journals. given sufficient means, terrorist pharmacists could well make use of such data in order to carry out malicious actions with catastrophic consequences 85 . in order to please a public that is eager for progress and the sensational, politicians favor, by means of targeted financing, the types of organization that appeal to their sensibilities, such as the technological platforms. while recognizing that such platforms are now an integral part of the landscape of research on living beings, and that they must therefore be taken into account, and while acknowledging that projects which implement the latest technologies in different domains need to be federated, it is nonetheless vital not to underestimate the potential creativity of small groups of researchers, a point that was expressed by one of the greatest of contemporary biologists, arthur kornberg (1918 kornberg ( -2007 , winner of the nobel prize for physiology and of medicine, in a speech given in 1997: "as i view the steady growth of collective science and big science, the greatest danger i see is a dampen-ing of individual creativity and reversion to the old politics -the inevitable local politics that infects every group and institution." however, conscious of the metamorphosis that is occurring in the experimental method, and faced with a particularly inventive and all-conquering technology, fundamental research in the life sciences must come to terms. a century ago, fundamental research and technological research interacted all the more directly because they were both in their infancy. this is no longer the case. management of the ever-increasing amount of knowledge in the life sciences, and the degree of sophistication achieved by bioengineering techniques and instruments, is widening a gap that makes dialogue increasingly laborious. however, dialogue appears to be a guarantee of future progress. the solution can only come from an increase in cross-disciplinarity, which should begin with university teaching and the establishment of a recruitment policy that advocates the cohabitation of talents from different educational backgrounds in the same laboratory. fortified by such hybrid expertise, while maintaining its share of originality and liberty in the choice of problems to be studied, fundamental research on living beings can only be enriched by a marriage of reason with biotechnology. convinced of the necessity for such a marriage, stanley fields, the inventor of the double hybrid method (chapter iv-4.1), in an article entitled "the interplay of biology and technology" (proceedings of the national academy of sciences, usa, 2001, vol. 98, pp. 10051-10054), concludes,: "it is at the interfaces of biology and other sciences that many of the future discoveries will be made, at the interfaces of biology and engineering that these discoveries will come to be exploited, and at the interfaces of biology and ethics and law that their consequences for society will be decided." the desired dialogue between biology and technology also implies the breaking down of barriers that too often isolate fundamental research and so-called applied research, and the facilitating of consistent interaction between the discoveries made in the academic institutions and their application for utilitarian ends in private companies. this is where the twin demons of money and power raise their heads. already, at the turn of the 1980s, a. bartlett giamatti 86 (1938 -1989) , who was then president of yale university in the usa, commenting on american university policies, spoke of a "ballet of antagonisms" between, on the one hand, commercial companies that are interested in the rapid cost-effectiveness of any new therapeutic advance and, on the other hand, non-profit university laboratories. recently, james j. duderstadt 87 (b. 1973), emeritus president of the university of the michigan, argued that the university is a "counter-hierarchical" organism. in fact, its members are free to carry out the research that pleases them and to think in the ways that they wish to think, in any case within an academic norm that considers itself as being free from the constraints dictated by private interest groups. until recently, such behavior was considered as a sort of ethic which arose out of the university conscience and dignity. the crumbling away of this ethic in the final decades of the 20 th century coincided with the rise of biotechnologies and the large-scale filing of patents relating to molecular genetics techniques that could be applied to the manipulation of living beings, by researchers in the public sector. the intrusion of the american private sector into public research laboratories, in the form of collaborations with transfer of "sensitive" information from the public to the private, has become such a worrying problem that drastic control measures have had to be taken. within this context, the american federal government, in february 2005, issued a certain number of prohibitions targeting the national institutes of health (nih) of bethesda, particularly with respect to the retribution of researchers for services rendered to industry 88 . these stands call for thought concerning the place that is currently held in universities with respect to fundamental research. without arguing against the efficacy of major research institutes, it is nevertheless necessary to remember the part played by the university in this domain. the university is not only the place where knowledge, both as it is now, in its current state of advancement, and as it has been, it is also the place where knowledge must be created by fundamental research. for the last few decades, under pressure from state policies, and also as a function of an improvement in social status, the world of the university has opened up to a wider public, leading to an influx of students that is sometimes so enormous that the task of teaching them has become overwhelming. because of this, the share of their time that university researchers can, in practice, devote to their research tasks has shrunk. this situation is highly prejudicial to the mission to innovate, which should be a priority. it is, in fact, during their university studies that the thought patterns of young students are forged by contact with teachers who not only instruct them, but also educate them by inspiring in them a motivation and an enthusiasm that gives rise to hope. how could this be true if the teaching faculty did not itself participate in scientific creation? "what can teaching, ex cathedra, do to guide the researcher? nothing, obviously. the researcher is trained in the laboratory. and the first stroke of genius on the part of a future researcher is to find a good boss. such a find will open up the royal road to success. the road will be opened -but the researcher must travel along it. a researcher may be taught many things. he or she can become familiar with techniques and with equipment. she or he can be assigned a problem to resolve. however, what is essential for the researcher is to know how to understand relationships between phenomena that seem unrelated, and to be able to progress from the particular to the general. a boss may develop such qualities in a gifted young researcher, but intuition is a gift; it cannot be taught." while the bernardian style experimental method, based on a working hypothesis aroused by an observation, followed by implementation of an experimental protocol, is still extant in the life sciences, and while "serendipity" is still the origin of great discoveries, "big science" , underpinned by sophisticated biocomputing or bioinformatics procedures, is intruding more and more, while genomics and proteomics are not far behind. the methods and instruments developed by the biotechnosciences have led to profound modifications in the ways that the structures and functions of living beings are investigated. for example, by varying multiple parameters in dna chips or protein chips, at the same time, the experimenter is able to ask questions that lead to grouped all-or-nothing answers (chapter iv-1.1.3). in combinatory chemistry, screening makes it possible to detect a molecule that is active for a given pathology from among a multitude of molecules (chapter iv-3.4). the mathematical simulation of metabolic networks or of signaling chains is already well under way (chapter iv-4.2). given this new technological outlook and the hope that it can provide rapid solutions to health problems subject to considerable media coverage, the teaching of biology in universities must not be limited to a description of current advances, no matter how brilliant and promising they may be. this teaching should return to its origins, be a reminder of history, and should not hesitate to use examples to illustrate how a major discovery can arise from a long period of wandering in the wilderness. in practical terms, while being conscious of the extraordinary complexity of living nature, and carefully avoiding the dangers of simplification, it is important to remember that the reductionist method was a necessary path to an understanding of the integrated, modelized biology that is emerging nowadays. at present, certain people call reductionism naive, but this is only the case insofar as we have faith in recent advances in integrated biology 89 . with this in mind, it should be noted that the deciphering of the protein synthesis mechanism in prokaryotic microorganisms (chapter iv-1.1.1) was, along with the discovery of the genetic code, a jumping-off point for an inventory of similar, but noticeably more sophisticated, mechanisms in eukaryotic organisms. the reductionist "one gene, one enzyme" dogma, formulated on the basis of beadle and tatum's experiments on the mold neurospora crassa (chapter iii-6.1) was a necessary prerequisite to a considerably more elaborate understanding of the relationship between the genotype and the phenotype. the way in which the nucleic acid and protein units in the tobacco mosaic virus spontaneously organize themselves (chapter iii-7.3) acted as a basis for thought concerning the self-organization of macromolecular complexes in the cell. these few examples underline the fact that it is difficult to comprehend the scientific research process if we only refer to experiments carried out in the present, and if we do not have a clear idea not only of the way in which hypotheses, even false ones, were once formulated, but also of the way in which experimental work, which may have led to failures, was once carried out, or, in brief, if we do not look back at the past. let us add that it is occasionally good for us to show some humility when we take the trouble to examine the past. thus, the processes involved in the phagocytosis of bacteria by innate immune cells (neutrophils, macrophages), which are today studied in the greatest detail with particularly refined technical facilities, had already been perceived more than a century ago by metchnikoff, and even analyzed, admittedly with the clumsy means at his disposal, but with such accuracy that none of the conclusions formulated at that time have yet been disproved (chapters iii-2.2.4 and iii-6.2.5). the experimental method applied to the life sciences, the history of its birth and of its development, the way in which it is regarded by political and societal authorities, and, finally, the dependencies that are developing at present between the technosciences, human medicine and the different branches of the economic sector, all of these aspects should be covered by university teaching that includes not only the pure sciences, but also the human, political and economic sciences, as well as philosophy. the student should not be saturated with book-learning, but he or she should be taught to reason, to imagine and to criticize, not to accumulate knowledge in an indigestible catalogue, but to ask questions about the way in which certain, carefully chosen, items of knowledge have been acquired, and not to deliberately accept science in its current state without knowing what it was like in the past. he or she should understand what pathways of thought led to dogmas that were established and taught as truths being refuted, and favor experimentation, with its risks and questions, rather than well-smoothed, abstract theoretical presentations without rough edges. these should be the principles of teaching that is designed to open up young minds to creativity. in anglo-saxon countries, the worlds of industry and research that welcome the graduate manage to communicate with one another, but these worlds ignore one another in france, or at least remain reserved, a situation which is prejudicial from the economic point of view. if we look at the pharmaceutical industry in particular, we see that only half a century ago the pharmacopeia was limited to plant extracts or active agents isolated from these plants, with antibiotics quietly beginning to make their appearance. in the last decades of the 20 th century, a great technological leap forward was made, with completely new methods in bioengineering, combinatory chemistry, and the finding of therapeutic targets in macromolecules, and this created a hiatus that severely handicapped countries that were unprepared for it. france, with its biological fundamental research training that is out of phase with that of the anglo-saxon countries, fell behind, and continues to be behind, a situation that is prejudicial for its economy. the remedy for this does not lie in incantatory speeches. it requires a volontarist policy for the management of experimental research. generally speaking, the fact that the major engineering schools in france, which recruit the scientific intellectual elite, students being chosen by competitive exams that select for intelligence rather than imagination, are unable to impose upon their students an end-of-course thesis that would authenticate their engineering degree, should not be tolerated. in contrast to other countries, in france only a small percentage of engineers have received doctoral training or had to present a thesis before entering their careers. the french dual system of major engineering schools and universities, which, a century ago, made sense for the economy of that period, has become completely obsolete, and deserves a courageous revision. "there is a question, much older than modern science, which has never ceased haunting certain men of science: that of the conclusions that the existence of science and the contents of scientific theories can lead to concerning the relationships that humankind has with the natural world. such conclusions cannot be imposed by science as is, but they are an integral part of the metamorphosis of this science." the new alliance. metamorphose of science -1986 (2 nd edition) in the 1950s -1960s, the hybridization of the techniques of genetics, biochemistry and biophysics gave birth to molecular biology. with the resolution of the double helix structure of dna, the demonstration of its replication, the elucidation of the mode of expression of its nucleotide sequence as a sequence of amino acids in proteins and finally the deciphering of the genetic code, biology underwent a revolution of an amplitude similar to that which, at the end of the 19 th century, saw a blossoming of the seeds of cell biology. the last decades of the 20 th century represented the utilitarian era of molecular biology. the introduction of genetic engineering into biological experimentation dates to the beginning of the 1970s. it was at this time that techniques were developed that made it possible to transfer a fragment of genomic dna from one species into the genome of another species. genetic engineering now fills a predominant position in the life sciences, supported by increasingly effective biocomputing or bioinformatics techniques. it is easy to understand that expertise and a high degree of knowledge about fundamental research is necessary in order to be able to master or even invent the genetic engineering techniques that are indispensable if we are going to produce biomolecules with a therapeutic impact, such as those that are currently being used in the pharmaceutical domain: insulin, growth hormone, blood coagulation factors, vaccines, etc. the engineering sciences that make up the greater part of contemporary biotechnology have now come to the fore in many domains of the life sciences. it is thus that a modernistic and original way of investigating nature has come into being. a multiparametric model, in which biocomputing or bioinformatics and high-throughput screening reign, is added to, or even substituted for, the bernardian model for the experimental method, based on observation, an a priori hypothesis, and experimentation to verify this hypothesis by varying a single parameter at a time. the aim of this globalized approach is to integrate the multiple reactions that take place almost simultaneously in different locations of a cell into a coherent whole, to rationalize the interpretation of the dialogue that operates between the different endocellular organelles, and finally to discover how the exchanges of information between cells in an organ and between organs in multicellular organisms are set up. we are therefore witness to the emergence of an integrated biology that has been labeled "systems biology". its long-term objective is to model the functioning of living beings and to theorize them. its development is encouraged by the perspective of consequences that could revolutionize certain sectors of the human economy and of public health. today, concrete, mechanical models, in the form of biorobots and hybrid robots, and, very recently, molecular motors are added to abstract models that are based on the logic of mathematics and algorithms, ushering in the era of nanobiomachines. becoming more utilitarian, the life sciences are imperceptibly detaching themselves from traditional philosophical concepts that try to explain the modes of reasoning of the researcher, or even to impose a framework for thought that is likely to orient his or her way of doing research. looking at genetic inheritance, contemporary experimentation has shown that at all levels of the tree of nature, including man, this inheritance can be modified. aware of his or her ability to influence the functioning and the destiny of living beings, the researcher is confronted with the dilemma of a desire for knowledge versus a questioning of the use to which discoveries may be put. there has never been such a real divorce between the world of phenomena that are understood by the experimenter and the world of noumena whose intelligibility is foreign to our senses. there has never been such a wide gap between the biotechnosciences, whose possibilities are coming to be seen as limitless, and a reflective analysis of thought, which wanders between freedom of action and prohibition. as society becomes aware of the potential applications of discoveries made concerning living beings, problems of bioethics, particularly those involving reproduction, have become problems of public interest. cloning and the production of stem cells are subjects that give rise to diatribes and passions. in the near future, genotyping, which is the result of progress in pharmacogenetics, could usher in a new form of customized medicine. elsewhere, the cognitive sciences that are bringing together philosophy and psychology in the domains of computer technology and artificial intelligence, and which are tackling the processes of thought, the creative imagination and memory, will no doubt be the subject of the considerable questioning concerning research on living beings with which the experimental method will be confronted in the 21 st century. when faced with the way in which biotechnologies have erupted into the life of society, the mind travels back to the allegorical illustration that embellishes francis bacon's novum organum (see figure ii.19) , showing vessels returning from unknown lands, loaded with precious cargoes and returning to port having sailed past the pillars of hercules. at present, the challenge has been partially met, but a great deal remains to be done. innumerable cargoes have already reached port, but what will be the destiny of this precious merchandise? after all, the seeds of the idea of technoscience were already in place in the 17 th century, in the philosophy of francis bacon and robert boyle (chapter ii-6). bacon recommended that the governments of the time promote experimental science by the creation of laboratories equipped with high-performance instruments and libraries, by the organization of researchers into teams and by appropriate financing. the utilitarian ends of scientific research were underlined. boyle imagined a situation in which laboratories were open to society and researchers were able to accept criticism. given innovations that upset tradition, protestations arose. the pneumatic machine or vacuum pump was the subject of the fameuse diatribe between boyle and the philosopher hobbes (chapter ii-6.2). hobbes criticized the validity of boyle's conclusions, drawn from experiments that he qualified as doubtful. following his words, he came to see in the discoveries of experimental science a possible threat to the power of governments and the hierarchical layout of society. such overcautious opposition to the pursuit of knowledge is in no way anecdotal, it is still a reality, with the uprooting of genetically modified plants and the veto that has been placed in certain areas on stem cell research. this type of opposition is also shown when pressures or even vetoes are in operation that take into account more the opportunism of the moment than an in-depth understanding of science and of its history and that forget that freedom of the mind is a guarantee of its creativity, because, just as in the world of arts and letters, the world of scientific research is situated outside those norms that can be modulated by state decrees. the creativity of the researcher cannot be manufactured on demand. where it exists, it still needs to be detected and encouraged. the atp-synthase -a splendid molecular machine structure at 2.8 å of f1-atpase from beef heart mitochondria direct observation of the rotation of f1-atpase powering an inorganic nanodevice with a biomolecular motor mechanically driven atp synthesis by f1-atpase atp-driven stepwise rotation of fo-f1 atp synthase key: cord-020664-m47ejlsn authors: schlüter, klaus-dieter; ross, günter title: intrakrine, parakrine und autokrine funktionen des pth/pthrp-systems date: 2006 journal: molekularmedizinische grundlagen von paraund autokrinen regulationsst&#x000f6;rungen doi: 10.1007/3-540-28782-5_6 sha: doc_id: 20664 cord_uid: m47ejlsn nan im vorliegenden kapitel werden intrakrine, autokrine und parakrine wirkungen des pth/pthrp-systems beschrieben. dabei handelt es sich um eine peptidfamilie, der derzeit drei peptide/proteine zugerechnet werden. dies sind das hormon der nebenschilddrçse, parathormon (¹parathyroid hormoneª, pth), das pth-verwandte protein (¹parathyroid hormone-related proteinª, pthrp) und das tuberoinfundibularpeptid bestehend aus 39 aminosåuren (tip39). die peptide dieser familie haben zwar strukturelle gemeinsamkeiten, çben aber im kaerper spezifische funktionen aus. ein groûer unterschied zwischen dem namensgebenden peptid der familie, pth, und den beiden strukturverwandten proteinen pthrp und tip39 ist, dass pthrp und tip39 hauptsåchlich auto-, para-oder intrakrin wirken, wohingegen die pth-effekte vorwiegend endokriner natur sind. es versteht sich deshalb von allein, dass ein beitrag çber parakrine und au-tokrine wirkungen im wesentlichen das pthrp behandelt, welches nahezu ubiquitår exprimiert wird. ûber tip39, ein neuropeptid, welches 1999 erstmals aus dem hypothalamus von rindern isoliert wurde, ist zur zeit noch recht wenig bekannt. es ist im zentralen nervensystem an der steuerung der hypothalamisch-hypophysalen achse und an der nozizeption beteiligt (usdin et al. 2003) . des weiteren wird es als lokaler vasodilatator im gefåûbett der niere (eichinger et al. 2002) und als modulator der kontraktilitåt des herzens beschrieben (ross et al. 2005) . da bisher wenig çber dieses neue peptid bekannt ist und vor allem seine klinische relevanz noch erforscht werden muss, wird im weiteren auf tip39 nicht ausfçhrlich eingegangen werden. in den 20er jahren des vergangenen jahrhunderts wurde erstmals eine hyperkalzåmie beschrieben, welche bei patienten im zusammenhang mit tu-morerkrankungen auftrat (zondek et al. 1924; gutman et al. 1936) . dieses krankheitsbild, genannt maligne hyperkalzåmie, ist eines der håufigsten endokrinen paraneoplastischen syndrome. etwa 40% aller auftretenden hyperkalzåmien beruhen darauf (meyer-heim u. ståubli 2002) . die maligne hyperkalzåmie wurde ursprçnglich auf lokale osteolytische prozesse zurçckgefçhrt, welche bei knochenmetastasen durch osteoklastenaktivierende zytokine ausgelaest werden (¹local osteolytic hypercalcaemiaª; loh). gegen diese annahme sprachen allerdings das auftreten von malignen hyperkalzåmien auch bei patienten, welche keine knochenmetastasen ausgebildet hatten (gellhorn u. plimpton 1956) , und das verschwinden der symptomatik nach entfernung eines knochenfernen primårtumors. eine weitere hypothese ging davon aus, dass dem krankheitsbild der malignen hyperkalzåmie ursåchlich ein ektoper hyperparathyroidismus zugrunde liegt (fuller 1941) und es sich bei dem humoralen faktor um pth handeln wçrde. diese these wurde von untersuchungen gestçtzt, bei welchen extrakte aus den tumoren, die mit maligner hyperkalzåmie einhergehen, in einer pth-sensitiven zelllinie die biologischen effekte des pth imitierten (stewart et al. 1983 ). im selben jahr wurde die expression von pth-mrna in tumoren mit maligner hyperkalzåmie untersucht. es konnte jedoch keine pth-expression nachgewiesen werden (simpson et al. 1983 ). damit konnte eine beteilung von pth an der malignen hyperkalzåmie ausgeschlossen werden. im jahre 1987 gelang es schlieûlich drei arbeitsgruppen unabhångig voneinander, aus verschiedenen tumorzelllinien ein protein zu isolieren, welches die pth-effekte an niere und knochen zu imitieren vermochte (burtis et al. 1987 , moseley et al. 1987 , strewler et al. 1987 . ursprçnglich wurden verschiedene bezeichnungen fçr diese proteine verwendet, wie ¹human adenylate cyclase-stimulating proteinª, ¹humoral hypercalcaemia of malignancy-(hhm)-factorª oder ¹parathyroid hormone-like protein (pth-lp)ª. seine eigenschaften und die hohe strukturelle homologie im n-terminalen bereich des proteins gegençber pth fçhrten schlieûlich zu der heute verwendeten namensgebung. diese peptide werden nunmehr als ¹parathyroid hormone-related proteinª (pthrp) bezeichnet. wir verwenden in diesem kapitel die bezeichnung pthrp und schlagen vor, dies auch zur vermeidung von missverståndnissen in der deutschsprachigen literatur so zu çbernehmen. wie im folgenden eingehender dargestellt, wird pthrp zum teil in form von proteolytischen teil-peptiden mit biologischer aktivitåt freigesetzt. fçr das c-terminale peptid (aminosåuren: 107±139) wird auch das synonym osteostatin verwendet, aufgrund der stimulierenden wirkung auf die knochenresorption. inzwischen konnten die gene, die beim menschen, der ratte und der maus fçr pthrp kodieren, identifiziert werden (hendy et al. 1988 (hendy et al. , 1990 suva et al. 1989 sequenz fçhrt bei anderen mrna zu einem beschleunigten abbau. fçr die pthrp-mrna kann eine halbwertszeit von 30 min bis zu 3 h angegeben werden. diese groûe zeitspanne kann dadurch erklårt werden, dass einige faktoren, wie beispielsweise ¹transforming growth factor b1ª (tgf-b1), die halbwertszeit der pthrp-mrna beeinflussen (sellers et al. 2004 b) . das pthrp-gen beinhaltet drei unterschiedliche promoterregionen (p1±p3). p1 und p3 besitzen tata-box-åhnliche sequenzen, wåhrend es sich bei p2 um einen gc-reichen promoter handelt (southby et al. 1995; richard et al. 2003) . eine strenge korrelation zwischen dem verwendeten promotor und der entstehenden pthrp-isoform scheint jedoch nicht zu existieren. wahrscheinlich spielt hier die rekrutierung verschiedener transkriptionsfaktoren eine wichtigere rolle als die verwendung der unterschiedlichen promotoren. die genaue regulation der pthrp-mrna-expression wird zur zeit noch intensiv erforscht. in vielen zellen scheint tgf-b1 daran beteiligt zu sein. in koronaren endothelzellen reguliert tgf-b1 die expression herab (wenzel et al. 2001) , dagegen wird die expression in keratinozyten, sich entwickelnden chondrozyten oder beim ovarialkarzinomen durch tgf-b1 heraufreguliert (pateda et al. 2000; werkmeister et al. 1998; yasui et al. 1997 casey et al. 1993; groh et al. 2004 interleukin-2 ikeda et al. 1993 prolaktin thiede 1989 mechanische dehnung schordan et al. 2004 yamamoto et al. 1992 tgf-b1 pateda et al. 2000 werkmeister et al., 1998; yasui et al. 1997 inhibitorisch tgf-b1 wenzel et al. 2001 law et al. 1994 testosteron liu et al. 1993 glukokortikoide ikeda et al. 1989 glatz et al. 1994 1,25-dihydroxyvitamin d 3 ikeda et al. 1989 medroxy-progesteron-acetat kurebayashi et al 2003 kreas, der parathyroidea oder aber der hypophyse, wird seine freisetzung hauptsåchlich durch bestimmte stimuli induziert. aber auch in nichtneuroendokrinen geweben kann seine freisetzung induziert werden, beispielsweise in koronaren endothelzellen, bei denen mechanische stimuli oder ein absinken des lokalen po 2 zu einer freisetzung fçhren (degenhardt et al. 2002; schlçter et al. 2000) . (tucci et al. 1996; aya et al. 1999; dunbar u. wylsolmerski 1999; torday et al. 1998; maclean et al. 2004; calvi et al. 2004; bui et al. 1993; mekaapiruk et al. 2002; lee et al. 1995) . rçckschlçsse auf die bedeutung des pthrp wåhrend der embryonalentwicklung ergeben sich vor allen dingen aus untersuchungen an transgenen tieren (abschn. 1.6.1.5). bis heute sind drei pth/pthrp-rezeptoren bekannt: der pth1-rezeptor (pth1r), der pth2-rezeptor (pth2r) und der pth3-rezeptor (pth3r). auûerdem gibt es hinweise fçr die existenz eines c-terminalen rezeptors (cpthr, divieti et al. 2004) . bisher konnte allerdings noch kein rezeptor fçr die mittregionalen abschnitte identifiziert werden. ono et al. 1997; yamamoto et al 1997 blutdruckregulation 1990 mannstadt et al. 1999) . das n-terminale ende beider peptide ist fçr die aktivierung des rezeptors entscheidend, so bindet beispielsweise das synthetische peptid pth(7±34) an den rezeptor, aktiviert diesen jedoch nicht. n-terminal deletierte teilpeptide des pth oder pthrp werden håufig als kompetitive inhibitoren eingesetzt. der pth1-rezeptor gehaert zu einer untergruppe g-protein-gekoppelter, heptahelicaler rezeptoren. dieser untergruppe, genannt klasse-ii-rezeptoren, gehaeren auch die rezeptoren fçr sekretin, calcitonin, vasoaktives intestinales peptid (vip) und glukagon an . die expression des pth1r ist in der niere und im knochen besonders hoch. in diesen geweben vermittelt er den klassischen endokrinen pth-effekt, nåmlich die kalziotrope wirkung. des weiteren wird der pth1r aber auch in vielen anderen organen exprimiert. dort vermittelt er verschiedene effekte, die durch lokal gebildetes pthrp ausgelaest werden (tabelle 1.6.2). der pth1r ist als target fçr die entwicklung neuer pharmaka sehr interessant, da er an der vermittlung einiger schwerer krankheiten beteiligt ist, wie z. b. dem hyperparathyroidismus, der malignen hyperkalzåmie und der osteoporose. der pth1r kann an unterschiedliche g-proteine angekoppelt sein. so aktiviert er çber das ga s -protein den cyclo-amp/proteinkinase-a(camp/ pka)-signal-weg und çber das g q/11 -protein den plc/pkc-signal-weg (abb. 1.6.3). die meisten bislang identifizierten effekte, die durch eine aktivierung des pth1r ausgelaest werden, sind çber eine aktivierung des camp/pka-signal-wegs vermittelt. die strukturfunktionsbeziehung zwischen ligand und rezeptor zeigt, dass die entscheidenden aminosåuren fçr die aktivierung an position 2, 3 und 6 liegen. die genaue position fçr die aktivierungssequenz fçr den phospholipase-c/proteinkinase-c(plc/pkc)-signal-weg ist dagegen umstritten. so induziert beispielsweise das tetrapeptid pth(29±32) in verschiedenen zelllinien den pkc-signal-weg (jouishomme et al. 1992) . in diesem abschnitt besitzen pth und pthrp zwar eine unterschiedliche primårstruktur, aber eine åhnliche sekundårstruktur. demgegençber gibt es aber auch untersuchungen, die zeigen, dass in der n-terminalen region von pth und pthrp ebenfalls sequenzen vorhanden sind, die den plc/pkc-signal-weg aktivieren kaennen (takasu et al. 1999 (vilardaga et al. 2001) . fçr die internalisierung selbst scheint auch eine aktivierung des rezeptors benaetigt zu werden. das bedeutet eine aktivierung des pkcund des pka-signal-wegs (sneddon et al. 2004) . nicht in allen zellen scheint eine aktivierung des rezeptors fçr die endozytose des rezeptors naetig zu sein. des weiteren weisen ergebnisse mit dem synthetischen teilpeptid pth(1±31), welches beide signalwege aktiviert, aber keine internalisierung des rezeptors induziert, daraufhin, dass aktivierung und internalisierung vollkommen unabhångig voneinander sein kaennen (sneddon et al. 2004) . maeglicherweise kaennte diese kontroverse dadurch aufgeklårt werden, dass der vorgang zellspezifisch ist. so konnte gezeigt werden, dass pth(7±34), welches an den rezeptor zwar hochaffin bindet, diesen jedoch nicht aktiviert, zelltypabhångig eine internalisierung induziert. der beginn der internalisierung ist etwa 6±7 min nach zugabe des agonisten zu beobachten. nach 15 min sind 50% aller rezeptoren internalisiert. der pth2-rezeptor wurde 1995 von der arbeitsgruppe um ted usdin auf der suche nach einem neuen klasse-ii-g-protein-gekoppelten rezeptor im zentralnervensystem entdeckt. dieser rezeptor besitzt in seiner primårstruktur eine 51%ige homologie zu pth1r. wie auch pth1r wird er in einer vielzahl von geweben bzw. organen exprimiert (usdin et al. 1996) , wobei seine expression im zentralnervensystem besonders hoch ist. pthrp kann an diesen rezeptor weder binden noch ihn aktivieren. pth kann zwar an den humanen pth2-rezeptor binden und diesen auch aktivieren, doch scheint sein natçrlicher ligand tip39 zu sein. dafçr spricht, dass tip39 die rezeptorvermittelte camp-produktion wesentlich potenter stimulieren kann als pth. den pth2-rezeptor der ratte vermag pth sogar kaum zu aktivieren (hoare et al. 1999) . des weiteren wird der pth2-rezeptor im zentralnervensystem in vielen gebieten exprimiert, aufgrund der blut-hirn-schranke ist dort allerdings kaum pth zu finden. auch dies gilt als indiz dafçr, dass tip39 der natçrliche ligand des pth2r ist. studien zeigten, dass die aminosåure an position 5 fçr die ligan-denselektivitåt des humanen pth2r sorgt. an dieser position steht ein histidin beim pthrp, aber ein isoleuzin beim pth (behar et al. 1996) . tip39 weist im vergleich von pth zu pthrp eine wesentlich geringere homologie in seiner aminosåuresequenz zu diesen beiden peptiden auf. so sind nur 5 aminosåuren zwischen tip39 und den peptiden pth(1±40) und pthrp(1±39) identisch. fçr diesen rezeptor ist neben einer stimulierung des camp-signal-wegs, auch ein rezeptorvermittelter anstieg der zytoplasmatischen ca 2+ -konzentration und eine aktivierung des plc/pkc-signal-wegs beschrieben (della penna et al. 2003) . im weiteren soll auf diesen rezeptor nicht weiter eingegangen werden, da dieser nicht von pthrp aktiviert werden kann und pathophysiologische dysregulationen zur zeit nicht bekannt sind. fçr weiter gehende informationen siehe usdin et al. 2002 . ein dritter pth-rezeptor konnte bisher nur beim zebrafisch nachgewiesen werden (rubin u. juppner 1999) . dieser rezeptor besitzt eine 69%ige homologie zu dem pth1r des zebrafisches. pthrp kann çber den pth3r den camp-signal-weg wesentlich potenter aktivieren als pth. eine aktivierung des plc-signal-wegs ist fçr den pth3r bisher noch nicht beschrieben. obwohl pthrp an zielzellen von såugetieren spezifische effekte ausçbt, die nicht von pth imitiert werden kaennen, also ein ligandenspezifisches profil zeigten, das dem des pth3r entspricht, konnte eine expression dieses rezeptors in såugetieren nie nachgewiesen werden. in seren vieler verschiedener spezies konnte man relativ hohe konzentrationen von c-terminalen pth-fragmenten nachweisen, die jedoch nicht mit dem pth1r interagieren. fçr dieses c-terminale pthrp-peptid wird auch das synonym osteostatin verwandt. osteostatin ist an der regulation der osteoklastenvermittelten knochenresorption beteiligt. zur bindung an den skelettalen cpthr scheinen drei verschiedene sequenzabschnitte von bedeutung zu sein, die aminosåurereste 25±27, 53+54 sowie 57,65+72 (divieti et al. 2004 ). diese reste sind innerhalb der pth-sequenz der såugetiere sehr stark konserviert. neben diesem skelettalen cpthr scheinen auch noch andere, eventuell organspezifische c-terminal-spezifische pth-rezep-a 1.6 intrakrine, parakrine und autokrine funktionen des pth/pthrp-systems toren zu existieren. ûber den strukturellen aufbau dieser rezeptoren und ihre angekoppelten signalwege ist bisher noch sehr wenig bekannt. in-vitro-daten lassen vermuten, dass innerhalb des knochenstoffwechsels der klassische pth1r und der cpthr gegensåtzliche wirkungen besitzen. aufgrund des vorkommens von biologisch aktiven mittregionalen pthrp-fragmenten kann vermutet werden, dass fçr diese teilpeptide auch entsprechende rezeptoren existieren, da diese peptide nicht in der lage sind, mit dem klassischen pth1r zu interagieren. beispielsweise scheinen mittregionale peptide an der regulation des kalziumstoffwechsels des fetus beteiligt zu sein (care et al. 1990 ). allerdings sind bisher noch keine rezeptoren fçr dieses fragment bekannt. dies gilt auch fçr rezeptoren, die wirkungen des pth/ pthrp im zentralen nervensystem vermitteln (yamamoto et al. 1997 ). unter physiologischen bedingungen ist kaum pthrp im blut nachweisbar (< 1,3 pmol/l). eine ausnahme stellt die laktationsphase dar, da wåhrend dieser auch haehere pthrp-blutspiegel detektierbar sind. ansonsten sind systemisch erhaehte pthrp-spiegel anzeichen pathophysiologischer ereignisse. da, wie oben bereits erwåhnt, pthrp als zirkulierendes agens kaum eine rolle spielt, entfaltet pthrp seine rezeptorvermittelten effekte hauptsåchlich durch eine lokal beschrånkte freisetzung. nach der sekretion bindet pthrp an den jeweiligen rezeptor und aktiviert verschiedene signalwege. durch internalisierung des rezeptors gelangt pthrp aber auch ins zytosol und kann dann in den kern transportiert werden (abb. 1.6.4). auch endogen gebildetes pthrp kann ohne vorherige sekretion eine intrakrine wirkung entfalten. innerhalb der letzten dekade wurden biologische pthrp-wirkungen nachgewiesen, welche nicht çber einen rezeptor an der zelloberflåche vermittelt werden, sondern auf intrakrinen mechanismen vermittelt durch eine kerntranslokalisation des pthrp beruhen. so konnte gezeigt werden, dass nukleåres pthrp in chondrozyten das auftreten von apoptose verzaegert (henderson et al. 1995) . pthrp besitzt zwei kernlokalisationsseabb. 1.6.4. kernlokalisation von pthrp in mikrovaskulåren endothelzellen (gelbe pfeile). in rot dargestellt die immunreaktion mit einem pthrp-antikaerper, in blau dargestellt die kernanfårbung. in den unteren bildern ist der primårantikaerper vor der histochemie mit dem liganden abgesåttigt worden. dies dient dem nachweis der spezifitåt des antikaerpers quenzen (¹nuclear localization sequenceª, nls). diese werden von den aminosåureresten 88±91 und 89±106 gebildet (henderson et al. 1995; massfelder et al. 1997) . des weiteren scheint die phosphorylierung der aminosåure threonin an position 85 fçr den nls-vermittelten transport sehr wichtig zu sein. kçrzlich wurde ein rezeptorvermittelter transport von pthrp in den zellkern, unter bindung an spezielle kernimportrezeptoren, sog. importine, beschrieben. hierbei scheint pthrp hauptsåchlich an importin-b 1 zu binden. fçr die bindung sind die aminosåureabschnitte 83±93 und 71±82 verantwortlich (lam et al. 2002) . im kern selbst kann pthrp drei maegliche zielmolekçle haben: rna, dna oder proteine. eine bindung von pthrp an die rna wurde in vitro nachgewiesen (aarts et al. 1999) . eventuell kaennte pthrp hierbei als transporter fçr mrna ins zytosol agieren. aufgrund der groûen homologie der nls von pthrp zu anderen transkriptionsfaktoren, wie beispielsweise c-jun, c-fos oder p53, låsst sich vermuten, dass auch pthrp die maeglichkeit besitzt, direkt an die dna zu binden und somit als transkriptionsfaktor zu fungieren. eine bindung von pthrp an nukleåre bzw. zytosolische proteine zur induzierung seiner zellulåren effekte kann ebenso nicht ausgeschlossen werden. verschiedene studien deuten daraufhin, dass pthrp durch seine nukleåre funktion hauptsåchlich als wachstumsfaktor, sowohl bei physiologischen als auch bei pathologischen prozessen, fungiert oder die apoptosetoleranz der zellen modifiziert. die zentrale rolle des pthrp und des pth1r bei der kalziumhomaeostase und als wachstumsregulator konnte durch eine reihe von transgenen tieren belegt werden. homozygote pthrp-knock-out måuse (pthrp ±/± ) versterben kurz nach der geburt. diese zeigten die symptome einer asphyxie und einer staerung der enchondralen ossifikation (karaplis et al. 1994) . morphologisch weisen die måuse einen verkleinerten brustkorb mit verminderter dehnbarkeit auf. dies wird fçr das postnatale versterben der måuse verantwortlich gemacht. des weiteren weisen diese knock-outs hypoplastische lungen mit einer gestaerten lungenentwicklung sowie einer mangelnden ausdifferenzierung der alveolarepithelzellen vom typ ii auf und daraus resultierend eine mangelnde surfactant-sekretion (rubin et al. 2004 ). die frçhzeitige mineralisierung und ossifikation, welche bei den pthrp ±/± -måuse zu beobachten ist, scheinen auf einem verfrçhten ûbergang der chondrozyten vom proliferativen zum hypertrophen stadium in den fetalen wachstumsfugen zu beruhen. des weiteren weisen die pthrp ±/± -knock-outs eine gestaerte zahnentwicklung auf (kitahara et al. 2004 ). knock-out-måuse fçr den pth1r (pth1r ±/± ) zeigen einen anderen phånotyp als die bereits oben erwåhnten pthrp ±/± -knock-outs. der phånotyp dieser knock-outs ist vergleichbar mit dem blomstrand-syndrom (abschn. 1.6.3.6). der unterschiedliche phånotyp von pthrp ±/± -und pth1r ±/± -måusen kaennte darauf beruhen, dass bei den pthrp ±/± -måusen pth teilweise die aufgaben von pthrp çbernimmt (lanske et al. 1999) . transgene måuse, die entweder pthrp oder aber pth1r in den glattmuskelzellen der gefåûe çberexprimieren, sind hypotensiv und weisen eine gestaerte reaktion bei applikation von vasodilatoren auf qian et al. 1999) . diese untersuchungen zeigen, welche zentrale rolle pthrp bei der lokalen tonusregulation im gefåûsystem zu spielen scheint, da die vielzahl von kaerpereigenen gegenregulationsmechanismen nicht in der lage ist, bei diesen tieren einen normalen blutdruck einzustellen. die transgenen måuse dieser zuchtlinie zeigten håufig sowohl missbildungen im bereich des herzens als auch des skelettsystems, des weiteren zeichnen sie sich durch eine hohe sterblichkeit post natum aus. dies zeigt auf, welche folgen eine ûberexpression von pthrp hat. eine ûberexpression sowohl von pthrp als auch pth1r ist auf jeden fall letal, doppelt transgene måuseembryonen sterben schon im uterus und weisen entwicklungsdefekte im herz-kreislauf-system auf (symons et al. 1985) . eine direkte hypertrophe wirkung von pth auf herzmuskelzellen ist nachgewiesen worden und beruht auf einer aktivierung der pkc (schlçter und piper 1992 (kitazawa et al. 2002) , wurde eine funktionelle relevanz der koexpression des pthrp postuliert. die zuvor be-schriebene wachstumskontrolle der zellen der nebenschilddrçse durch pthrp kaennte eine solche rolle sein (matsushita et al. 1999) . in seltenen fållen fanden sich erhaehte pth-spiegel auch ohne hyperparathyroidismus. in einem fallbeispiel çber eine juvenile akute lymphatische leukåmie wurde kçrzlich gezeigt, dass die leukoblasten pth freisetzen (lankisch et al. 2004) . die pthrp-konzentrationen waren hingegen im normbereich. im gegensatz zu erhaehten pthrp-konzentrationen war in diesem fall das klinische erscheinungsbild durch eine nur moderate nierenfunktionsstaerung charakterisiert. øhnlich kann beim pathophysiologischen erscheinungsbild des pseudohypoparathyroidismus davon ausgegangen werden, dass hier autokrine/ parakrine prozesse zumindest mitbeteiligt sind. beim pseudohypoparathyroidismus handelt es sich um mutationen des postrezeptoriell wichtigen ga s -proteins, das fçr die ûbertragung des initialen signals in ein intrazellulåres signal, in diesem fall camp, verantwortlich ist (patten et al. 1990 (sugihara et al. 1998 ). im gegensatz zur expression von il-1a und il-1b kann unter endokriner therapie eine reduktion der pthrp-bildung nicht erreicht werden. pthrp wirkt nicht nur endokrin, indem es die knochenmetastasierung unterstçtzt, sondern auch intrakrin. dabei steigert es durch einen noch nicht genau identifizierten mechanismus die expression von il-8 (lehrer et al. 2004 ). bei der entstehung der knochenmetastasen beim prostatakrebs kommt dem zytokin tgf-b eine dominierende rolle zu, weil tgf-b die expression und sekretion von pthrp in prostatakrebszellen steigert (sellers et al. 2004 a) . in form einer positiven rçckkopplung fçhrt die freisetzung von pthrp zur hyperkalzåmie (abb. schlieûlich lassen sich auch in einigen gliatumoren unterschiedlich starke expressionen an pthrp nachweisen. die stårke der pthrp-expression scheint dabei mit einem geringeren grad an differenzierung dieser zellen einherzugehen, was andererseits aggressivere tumoren bedeutet. folglich haben patienten mit hohen pthrp-expression auch geringere progressionsfreie zeiten und insgesamt eine kçrzere ûberlebensrate (pardo et al. 2004) . zusammenfassend låsst sich festhalten, dass verschiedene tumorformen, die sich besonders bei formen von brustkrebs, lungenkrebs oder prostatakrebs finden lassen, erhebliche mengen an pthrp produzieren, die dann lokal parakrine signalwege am knochen modulieren und zu einer gesteigerten osteoklastenaktivitåt, der mobilisierung von kalzium und letztendlich auch der entstehung 144 k.-d. schlçter und g. ross abb. 1.6.5. rolle des pthrp bei der osteolytischen aktivitåt von prostatakrebs und der entstehung von knochenmetastasen. prostatakrebszellen bilden pthrp, das çber die blutbahn an knochenzellen bindet und dort eine osteoklastenaktivierung auslaest. das fçhrt zu einer erhaehung der plasmakalziumkonzentration (maligne hyperkalzåmie). kalzium verstårkt die pthrp-bildung durch die tumorzellen weiter und unterstçtzt die metastasierung von tumorzellen im knochen von knochenmetastasen beitragen. insbesondere die bildung pthrp-produzierender knochenmetastasen ist von groûer klinischer relevanz. unter der standardtherapie von brust-und prostatakrebs kommt es nach wie vor zu einem verlust der knochenmasse. ûber einen noch nicht vollståndig verstandenen mechanismus fçhrt dies wiederum zu einer verstårkten neigung der knochenmetastasierung. nachdem sich die knochenmetastasen erst ausgebildet haben, sind die patienten praktisch nicht mehr kurierbar (mundy 2002) . eine frçhzeitige unterdrçckung der pthrp-bildung als initiales ereignis der kausalkette ist deshalb von herausragendem interesse. die applikation von neutralisierenden pthrp-antikaerpern ist in der klinischen erprobung (chirgwin et al. 2004) . die hier angesprochenen pthrp-produzierenden tumoren stellen nur eine auswahl besonders gut charakterisierter tumoren dar. pthrp-bildung findet sich aber auch bei anderen krebsformen, so am pankreas (grzseiak et al. 2004) , an der leber (pun u. tam 1995) , in kardialen chondrosarkomen (kase et al. 2004 ) oder tumoren des gastrointestinaltraktes (yoshizaki et al. 2004 pthrp wird sowohl in der normalen als auch in der malignen lunge konstitutiv exprimiert und beeinflusst lungenreifung, lungenfunktion und die pathophysiologischen verånderungen bei lungenerkrankungen und lungenkrebs (zur ûbersicht siehe hastings 2004). pthrp wird wåhrend der embryonalentwicklung (7.±12. woche) frçhzeitig in der lunge exprimiert. die expression ist beschrånkt auf epithelzellen, wåhrend der korrespondierende rezeptor in den benachbarten mesenchylmalen geweben exprimiert wird. dieses expressionsmuster weist bereits auf eine parakrine rolle bei der lungenentwicklung hin. in transgenen pthrp-defizienten måusen weist eine ver-minderte oder fehlende produktion von surfactant-proteinen auf die elementare bedeutung des pthrp fçr die entwicklung einer funktionellen lunge hin (rubin et al. 2004 im klinischen interesse stehen allerdings weniger die dilatierenden eigenschaften des pth und/ oder pthrp. im vordergrund steht die beobachtung, dass pthrp als proinflammatorisches peptid einen eigenståndigen beitrag wåhrend arterosklerotischer prozesse ausçben kaennte. pthrp kann offenbar von inflammatorischen zellen gebildet werden. diese finden sich in der kappenstruktur der plaques. von dort freigesetztes pthrp stimuliert die bildung des ¹monocyte chemoattractant protein 1ª (mcp-1) in zellen der glatten muskulatur, die wiederum plaques destabilisieren (martin-ventura et al 2003) . neben den makrophagen sind die glattmuskelzellen selbst ein wesentlicher bildungsort des pthrp. die wirkung von pthrp auf zellen der glatten muskulatur ist nicht beschrånkt auf die induktion von mcp-1. pthrp scheint das wachstum der zellen zu inhibieren und verhindert somit die entstehung einer neointima nach arterosklerotischen låsionen (ishikawa et al. 2000) . da hierbei die expression in den glattmuskelzellen selbst erheblich ansteigt, muss von einem autokrinen prozess ausgegangen werden (nakayama et al. 1994) . eine strenge korrelation zwischen schweregrad und progression der arterosklerose und dem ausmaû der pthrp-expression spricht ebenfalls fçr eine kausalbeziehung. in arterosklerotischen plaques scheint pthrp einmal plaques zu destabilisieren und andererseits die neointimabildung zu blockieren. die gemeinsamkeiten zwischen inflammatorischen eigenschaften des pthrp und anderen inflammatorischen peptiden hat zu der vermutung gefçhrt, dass pthrp auch bei rheumatischen erkrankungen beteiligt sein kaennte (funk et al. 2002) . im gegensatz zu den glattmuskelzellen exprimieren gefåûendothelzellen keinen pth1r (rian et al. 1994) . sie sind aber andererseits ein wichtiger bildungsort des peptids. direkte wirkung auf die endothelzelle entfaltet pthrp in erster linie durch eine intrakrine wirkung. diese versetzt die endothelzelle in die lage, eine haehere apoptosetoleranz zu entwickeln (schorr et al. 2003) . mechanistisch ist dies durch eine verstårkte expression von bcl-2, einem antiapoptotischen gen, vermittelt. wie pthrp im zellkern die expression von genen veråndert, vorzugsweise solchen, die in die apoptosetoleranz der zelle eingreifen, ist noch gegenstand eingehender untersuchungen. eine verminderte expression von pthrp, wie sie sich beispielsweise im chronisch druckbelasteten herzen wiederfindet, kaennte aber die apoptosesuszeptibilitåt der endothelzellen erhaehen und damit kausal ein erhaehtes risiko fçr die bildung von arterosklerotischen plaques darstellen. neben der intrakrinen wirkung von pthrp ist die parakrine wirkung zu berçcksichtigen. pthrp wird sowohl im zellkulturexperiment als auch unter klinisch relevanten bedingungen lokal und mechanosensitiv freigesetzt. wie bereits weiter oben ausgefçhrt, wirkt pthrp vasodilatierend. das bedeutet, dass die mechanosensitive freisetzung des pthrp einen zusåtzlichen mechanismus darstellt, çber den das gefåû auf drucksteigerung dilatierend reagieren kann. eine solche mechanosensitive freisetzung fand sich auch bei patienten mit pulmonalem hochdruck, die erhaehte pthrp-konzentrationen in abhångigkeit von der druckbelastung zeigten (abb. 1.6.7). keine solche druckinduzierte freisetzung zeigen jedoch kinder mit endothelialer dysfunktion. somit ist in gefåûen mit endothelialer dysfunktion nicht nur die no-bildung gestaert, sondern auch die pthrp-freisetzung. beides kann zur lokalen perfusionsstaerung beitragen. als dritte wesentliche zielzelle fçr pth und/ oder pthrp im kardiovaskulåren system muss schlieûlich die ventrikulåre herzmuskelzelle angesehen werden. auch hier ergaben sich historisch gesehen erste hinweise aus der wirkung von exzessiven pth-konzentrationen. hyperparathyroi-a 1.6 intrakrine, parakrine und autokrine funktionen des pth/pthrp-systems dismus geht håufig einher mit hypertrophem zellwachstum des herzmuskels und der manifestation einer myokardhypertrophie. sowohl pth als auch pthrp vermag die proteinsyntheseleistung der herzmuskelzelle zu steigern (schlçter u. piper 1992; schlçter et al. 1997) . auch wenn keine gesicherten klinischen daten vorliegen, kann doch aus tierexperimentellen untersuchungen hergeleitet werden, dass im chronisch druckbelasteten herzen zunåchst die pthrp-konzentration lokal ansteigt, um dann langfristig zu fallen (ogino et al. 2002 der fetale anteil der plazenta stellt eine der hauptquellen der endogenen pthrp-bildung dar, die sonst nur von tumoren çbertroffen wird. als hauptbildungsort kann das amnion angesehen werden. die konzentration an pthrp in der amnionflçssigkeit çbersteigt diejenige im fetalen oder maternalen plasma um das mehr als zehnfache (farrugia et al. 2000) . die pthrp-konzentration im fetalen plasma çbersteigt dabei diejenige des mçtterlichen plasmas. pthrp ist das wichtigste kalziotrope hormon des faeten. die zellulåre wirkung des pthrp ist extrem gewebsspezifisch. im uterus reguliert pthrp den blutfluss und senkt den myometralen tonus und verhindert so eine zu frçhe myometrale kontraktile aktivitåt. kurz vor der geburt sinken die lokalen pthrp-konzentrationen und erlauben so einen anstieg der kontraktilen aktivitåt. daneben sind die rolle des pthrp fçr den plazentalen kalziumtransport und den gefåûtonus der plazenta gut charakterisiert. eine besondere bedeutung scheint dem pthrp bei der regulation der trophoblasteneinwanderung und dem apoptotischen abbau dieser zellen zuzukommen. wie oben beschrieben, beruht pråeklampsie initial auf einer fehlerhaften plazentation. diese erfolgt durch die verstårkte bildung von matrixmetalloproteinasen (mmp). in diesem zusammenhang ist es von wichtigkeit, dass pthrp die expression von mmp-isoformen in verschiedenen zielzellen induzieren kann (kawashima-ohya et al. 1998; uchida et al. 2001; maioli et al. 2002) . obwohl eine kausalbeziehung noch nicht eindeutig belegt werden konnte, ist es anzunehmen, dass eine solche hier wahrscheinlich vorliegt. die entwicklung und differenzierung der trophoblasten beruht auf einer ausgewogenen balance zwischen proliferation und apoptose. pthrp abb. 1.6.7. plasmakonzentration des pthrp in der pulmonalarterie (pa) normiert auf die konzentration in der a. femoralis (sa) in abhångigkeit von der druckbelastung in der pulmonalstrombahn bei kindern mit pulmonaler hypertension. zu erkennen ist eine lokale, durckabhångige freisetzung des pthrp scheint insbesondere die apoptoseanfålligkeit verschiedener zelltypen zu beeinflussen. durch den transfer des peptids in den zellkern hinein (intrakrine wirkung) werden chondrozyten, endothelzellen und glatte muskelzellen der gefåûwand weniger anfållig gegençber apoptose. pthrp ist ein integraler bestandteil der normalen epidermalen erneuerung, die fortwåhrend erfolgt. diese besteht aus einer andauernden proliferation von keratinozyten auf der basalmembran, die sich nachfolgend von der basalmembran laesen und zu keratinbepackten sog. korneozyten konvertieren. aufgrund ihrer schnellen proliferationsrate werden die sich ablaesenden keratinozyten auch als ta(¹transit amplifierª)-keratinozyten bezeichnet. diese bilden einen untypischen und molekular bislang nicht identifizierten pth/pthrp-rezeptor aus, der nicht an die adenylatcyclase koppeln kann. wenn die zellen erst einmal aufhaeren zu proliferieren und sich von der basalmembran laesen, synthetisieren sie selbst pthrp (abb. 1.6.8). induziert wird die expression von pthrp vermutlich durch mitglieder der egf-familie (cho et al. 2004) . vermutlich stellt diese pthrp-bildung ein signal fçr die anzahl suprabasaler zellen dar, welches die reifung und proliferation der keratinozyten auf der basalmembran hemmt. pth-antagonisten, die auch die lokale pthrp-wirkung antagonisieren, stimulieren die epidermale proliferati-on (holick et al. 1994 sen worden und beruht auf einer deletion im c-terminalen zytoplasmatischen teil des rezeptors (duchatelet et al. 2005) . in allen genannten fållen liegt der defekt auf der ebene des pth1r. dies bedeutet, dass unabhångig von messbaren oder hohen systemischen pthrp-konzentrationen dem pthrp eine wichtige und entscheidende rolle in wachstum und regulation zukommt, die durch parakrine oder autokrine wirkungen vermittelt wird. da bislang keine genetischen erkrankungen gefunden wurden, die auf eine mutation im pthrp-gen beruhen, kann auch vermutet werden, dass solche verånderungen wohl letal sind. darçber hinaus bleibt festzustellen, dass formen eines defekten pth1r schwerwiegende symptomatiken zeigen. aus dem fehlen von pthrp-mutationen und dem nachweis von pth1r-mutationen kann man entweder folgern, dass einige funktionen des pthrp çber einen bislang nicht identifizierten rezeptor vermittelt werden oder aber dass der ausfall der intrakrinen rezeptorunabhångigen wirkungen bei fehlen des pthrp schwerwiegender ist. eine betrachtung der oben aufgefçhrten klinischen erscheinungsformen, die sich entweder aus einer verminderten lokalen verfçgbarkeit des pthrp ergeben oder aus der massiv erhaehten konzentration des pthrp resultieren, låsst umgekehrt den schluss zu, dass eine enge balance zwischen pthrp-konzentration und -wirkung notwendig ist. hinsichtlich jener fålle, in denen entweder die pthrp-produktion zu niedrig ist, z. b. bei der pråeklampsie, ist es schwer, regional die pthrp-konzentrationen zu normalisieren. eine ausnahme bildet die oben genannte schuppenflechte, weil dort lokal von auûen auf die haut aufgetragen pth direkt appliziert werden kann (holick et al. 2003) . in den håufigen fållen einer massiven ûberproduktion des pthrp mit systemisch relevanten konzentrationen im plasma ist andererseits aber eine inhibierung der pthrp-wirkung durch applikation von pthrp-antagonisten maeglich. beispiele fçr einen viel versprechenden ansatz finden sich durch verwendung neutralisierender antikaerper. hier konnte gezeigt werden, dass solche antikaerper in der tat die pthrp-wirkung praktisch vollståndig unterdrçcken (onuma et al. 2004; miki et al. 2004 ). in vivo konnte gezeigt werden, dass die antikaerper signifikant die konzentration an ionisiertem blutkalzium in måusen senken, die einen pthrp-produzierenden tumor trugen. øhnliche ergebnisse mit neutralisierenden antikaerpern fanden sich auch in einem anderen mausmodell mit knochenmetastasenbildung. vergleichbar vielversprechend scheinen ansåtze zu sein, in denen statt pthrp das distal dem pthrp agierende il-11 neutralisiert wird. dies belegt eindrucksvoll, dass pthrp çber die induktion von il-11 seine osteolytisches potential entwickelt. schlieûlich setzten neue, in der tierexperimentellen phase befindliche verfahren, auch oberhalb des pthrp an. prostatatumoren scheinen pthrp primår durch die wirkung eines prostatasekretorischen proteins (psp-40) zu entfalten, das wiederum die pthrp-expression steigert. dessen wirkung låsst sich durch synthetische peptide blockieren und somit die wirkung des pthrp bei der knochenmetastasierung des prostatakrebses einschrånken. dabei besteht allerdings immer die gefahr der immunisierung gegen solche peptide. einen weiteren vielversprechenden ansatz zur reduktion der pthrp-expression durch tumoren zeigten gallwitz und mitarbeiter (2002) auf. sie konnten im tierexperiment zeigen, dass durch gabe von guanin-nukleotid-analoga die expression von pthrp spezifisch reprimiert wurde. das pthrp-peptid exponiert antigene oberflåchen, die teilweise zur aktivierung von peptidspezifischen zytotoxischen t-lymphozyten beitragen. so fanden yao et al. (2005) in patienten mit prostatakrebs immunglobuline (igg), die reaktiv gegençber pthrp(42±51) sind. die peptidspezifische aktivierung der zytotoxischen t-lymphozyten fçhrt zu einer spezifischen abwehr der prostatakrebszellen. eine spezifische immuntherapie mittels solcher pthrp-teilpeptide kaennte ein vielversprechender neuer ansatz sein, um patienten mit prostatakrebs zu behandeln und die progression der knochenmetastasierung zu unterdrçcken. fçr die diagnose stehen heute geeignete elisa-verfahren zur verfçgung, welche eine bestimmung der plasmakonzentrationen an pthrp ermaeglichen. von bedeutung ist dies im hinblick auf die allgemein schlechte prognose bei tumorpatienten mit hohen pthrp-spiegeln. neben der oben nåher erlåuterten wechselwirkung des pthrp fçr die a 1.6 intrakrine, parakrine und autokrine funktionen des pth/pthrp-systems metastasierung von tumoren steht hier eine hohe pthrp-konzentration immer auch fçr eine hohe tumoraktivitåt. die normalen und niedrigen pthrp-konzentrationen (5±21 pmol/l) kaennen nach blind et al. (1993) in gegenwart von pthrpbildenden tumoren um mehr als das zehnfache ansteigen (22±333 pmol/l). die bestimmung der pthrp-konzentrationen unterliegt keinem normalisierten verfahren, so dass unterschiedliche werte in der literatur genannt werden. dies hångt zum einen mit der verwendung unterschiedlicher antikaerper zusammen und zum anderen mit dem nachweis verschieden strukturierter teilpeptide, die durch glykosylierung in der affinitåt gegençber antikaerpern zusåtzlich variieren. so konnten wir beispielsweise im plasma von kindern mit pulmonalem hochdruck durch verwendung zweier unterschiedlicher primårantikaerper, die gegen n-terminales pthrp oder mittregionales pthrp gerichtet waren, haechst unterschiedliche konzentrationen in ein und der selben plasmaprobe bestimmen (tabelle 1.6.3). auch hångt die absolute konzentration von der referenzgraeûe ab (synthetisches teilpeptid, biologische aktivitåt oder authentisches peptid). fçr aussagen zur verånderung der lokalen, parakrinen, autokrinen oder intrakrinen wirkung von pthrp sind diese verfahren nicht geeignet. in den vergangenen jahren konnte pthrp als ubiquitår exprimiertes peptid mit seinen zahlreichen intrakrinen, autokrinen und parakrinen wirkungen unter physiologischen bedingungen identifiziert werden. dies umfasst praktisch alle organe. um die bedeutung des pthrp fçr die entstehung verschiedenster krankheitsformen zu verstehen, bedarf es einer extensiven erforschung der zellspezifischen regulation der expression, seines zellspezifischen freisetzungsmechanismus und der wirkungsweise des peptids. hier steht die forschung leider noch ganz am anfang. von entscheidender bedeutung wird dabei ein besseres verståndnis der rezeptoren und der liganden-rezeptor-interaktion sein. es ist bislang vaellig unklar, ob ein rezeptor verschieden ausgeprågte ligandeninteraktionen eingeht oder ob andere bislang nicht identifizierte rezeptoren, pleiotrope wirkungsweisen des pthrp vermitteln kaennen. entsprechend vage sind derzeit alle versuche, durch geeignete rezeptorantagonisten therapeutisch einzugreifen. expression of parathyroid hormone-related peptide messenger ribonucleic acid in developing kidney. kidney int 55: 1696±1703 behar v, nakamoto c, greenberg z et al. 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(2004) key: cord-286217-3uklf2u2 authors: jiang, he-wei; li, yang; zhang, hai-nan; wang, wei; yang, xiao; qi, huan; li, hua; men, dong; zhou, jie; tao, sheng-ce title: sars-cov-2 proteome microarray for global profiling of covid-19 specific igg and igm responses date: 2020-07-14 journal: nat commun doi: 10.1038/s41467-020-17488-8 sha: doc_id: 286217 cord_uid: 3uklf2u2 we still know very little about how the human immune system responds to sars-cov-2. here we construct a sars-cov-2 proteome microarray containing 18 out of the 28 predicted proteins and apply it to the characterization of the igg and igm antibodies responses in the sera from 29 convalescent patients. we find that all these patients had igg and igm antibodies that specifically bind sars-cov-2 proteins, particularly the n protein and s1 protein. besides these proteins, significant antibody responses to orf9b and nsp5 are also identified. we show that the s1 specific igg signal positively correlates with age and the level of lactate dehydrogenase (ldh) and negatively correlates with lymphocyte percentage. overall, this study presents a systemic view of the sars-cov-2 specific igg and igm responses and provides insights to aid the development of effective diagnostic, therapeutic and vaccination strategies. c ovid-19 is caused by the coronavirus sars-cov-2 1,2 . it is presently recognized by the world health organization as a global pandemic, and as of june 28, 2020, 9,653 ,066 diagnosed cases have been reported from 214 countries, areas or territories (http://2019ncov.chinacdc.cn/2019-ncov/). sequence analysis suggested that sars-cov-2 is most closely related to the batcov ratg13 and belongs to the subgenus, sarbecovirus, of the beta coronaviruses, together with the bat-sars-like coronavirus and the sars coronavirus 1, 2 . by comparing sars-cov to the other related coronaviruses, it was predicted that there are 28 proteins encoded in the genome of sars-cov-2 3 . further, such comparisons suggested that sars-cov-2 might utilize the same mechanism to enter the host cells, namely via high-affinity binding between the receptor-binding domain (rbd) of the spike protein (s protein) and angiotensin converting enzyme 2 (ace2) [4] [5] [6] [7] [8] [9] . though there is presently tremendous worldwide effort to identify and develop effective therapeutic approaches against this virus, none of this work has been successful at the moment. one possible approach that has shown some positive results is by treating infected patients with the plasma collected from convalescent covid-19 patients 10, 11 . here, it is believed that the humoral antibody response in these convalescent patients played an important role in their recovery, and so might likewise prove effective in other, presently infected patients. indeed, it is known that in combating many viral infections, including sars-cov and mers-cov, igg, and igm antibodies play many critical roles [12] [13] [14] [15] . however, because sars-cov-2 is a newly emerged pathogen, the precise igg and igm responses in the covid-19 patients are very poorly understood. indeed, in this regard, there are many important questions that need to be experimentally addressed: (1) what is the variation among different patients, especially for antibodies against the nucleocapsid protein (n protein) and s protein? (2) are there any other viral proteins that could trigger significant antibody responses in at least some of the patients? (3) is it possible to link the magnitude of the overall igg and igm response to the severity of the disease in patients? resolution of these questions is fundamental to the development of an understanding of the global igg and igm responses against sars-cov-2 and for the possibility to use this material in the development of effective therapeutic or diagnostic approaches. conventional techniques for studying patient igg and igm responses include elisa [16] [17] [18] and the immune-colloidal gold strip assay 17, 19, 20 . however, these techniques usually can only test a single target protein or antibody in a single reaction. by contrast, protein microarrays enable proteome-wide characterization of antibody responses in a high-throughput format, providing a more systemic description of these vital antibody responses. indeed, a variety of protein microarrays have already been constructed and successfully applied to serum antibody profiling, such as the mycobacterium tuberculosis proteome microarray 21 , the sars-cov protein microarray 12 , the dengue virus protein microarray 22 and the influenza virus protein microarray 23 . here, we describe the construction of the sars-cov-2 proteome microarray and its application in the characterization of the global igg and igm responses from 29 covid-19 convalescent patients. in this way, we provide a systemic view of these responses, revealing both common and unique features of these patients, which may aid future diagnostic and therapeutic efforts against this virus. schematic diagram and workflow. the genome of sars-cov-2 is~29.8 kb and is predicted to encode for 28 proteins 3 : 5 structural proteins (treating the s protein as two separate proteins, s1 and s2), 8 accessory proteins, and 15 non-structural proteins (nsp) (fig. 1a) . the corresponding nucleotide sequences of all of these proteins and the receptor-binding domain (rbd) of the s1 protein were synthesized and cloned into appropriate vectors for expression in e. coli, and the expressed proteins were purified by affinity chromatography. to obtain any even broader range of proteins that were produced from different prokaryotic and eukaryotic systems, we also acquired a number of recombinant sars-cov-2 proteins from commercial sources (supplementary data 1). after evaluating the proteins for quality control, these proteins were printed on appropriate substrate slides. convalescent sera were collected from 29 patients on the day of their discharge and were applied to the proteome microarray. we detected the sars-cov-2-specific igg and igm proteins bound to the array using fluorescent-labeled anti-human antibodies, thereby generating a global assessment of each patient's humoral antibody response. generation of the predicted sars-cov-2 proteins. to produce the recombinant proteins of sars-cov-2 for the microarray, we first determined the amino-acid sequences of the predicted proteins 3 based on the reference genome (genbank accession no. mn908947.3). we split s protein into s1 and s2, as suggested previously 3 , to enable a more precise analysis and also included the rbd alone owing to its critical role during the entry of sars-cov-2 into the cells. the protein sequences were converted to the corresponding nucleotide sequences, followed by optimization of the sequences, and then insertion of the sequences into expression vectors (pet32a or pgex-4t-1). the final expression library included 31 clones. further information about these clones is included in supplementary data 1. after several rounds of optimization, we successfully purified 17 of these proteins (supplementary fig. 1 ). western blotting with an anti-6xhis antibody and coomassie staining showed that most of the sars-cov-2 proteins exhibit clear bands with the expected size (±10 kda) and good purity. to cover the proteome of sars-cov-2 as complete as possible, and to include proteins with post-translational modifications (ptm), especially glycosylation, we also acquired recombinant sars-cov-2 proteins produced using yeast cell-free systems or mammalian cell expression systems from a variety of commercial sources ( supplementary fig. 1 ). among the collected proteins, there are several different full length and fragmented versions of the s and n proteins (supplementary data 1). in this way, we finally obtained 37 proteins from different sources, covering 18 out of the 28 predicted proteins of sars-cov-2, that were of suitable concentration and purity for microarray construction. fabrication of the sars-cov-2 protein microarray. a total of 37 proteins along with positive and negative controls were printed on the microarray slide (fig. 2a) . since most of the proteins were tagged with the 6xhis peptide, we examined the overall quality of the microarray by probing with an anti-6xhis antibody, which revealed uniform, spot-limited labeling across the entire microarray, thus attesting to the quality of the array (fig. 2a) . in addition, we noticed that nsp7 was contaminated during the microarray manufacturing process. thus, we decided not to include nsp7 for further analysis. when probed with convalescent sera from covid-19 patients, we generally observed high, multi-spot antibody responses, which were not observed with the control sera (fig. 2b) . to prevent or largely decrease nonspecific signals generated from the background of the expression system and minimize any influence from possible protein impurity, e. coli lysates and egfp were added during the incubation with serum samples, which significantly reduced nonspecific signals ( supplementary fig. 2a) . to test the experimental reproducibility of the serum profiling using the microarray, we randomly selected two covid-19 convalescent sera and probed them on three separate microarrays. the pearson correlation coefficients from the measured intensities over the entire array between two samples were 0.988 and 0.981 for igg and igm, respectively. further, the overall fluorescence intensity ranges of the repeated experiments were quite similar, demonstrating a high reproducibility of the microarray-based serum profiling both for igg and igm ( fig. 2c-e) . sars-cov-2-specific serum antibody profiles revealed by proteome microarray. to globally profile the antibody response against the sars-cov-2 proteins from the serum of covid-19 patients, we screened sera from 29 convalescent patients, along with 21 controls, using the sars-cov-2 proteome microarray. the patients were hospitalized in foshan fourth hospital in china from 2020-1-25 to 2020-2-27 for various durations. patient information is summarized in table 1 . serum from each patient was collected on the day of hospital discharge when standard criteria were met. all of the samples and the controls were probed on the proteome microarray, and after data filtering and normalization, we constructed the igg and igm profile for each serum and performed clustering analysis to generate heatmaps (figs. [3] [4] . the patients and controls formed clearly separate clusters for both igg and igm data. as expected, the n and s1 proteins elicited high antibody responses in almost all patients but were associated with only weak signals in control groups, confirming the efficacy of these two proteins for diagnosis. interestingly, we also found that in some cases, proteins such as orf9b or nsp5 can generate significantly high signals compared with that in the control groups. to further prove the specificity, we performed an immunoblotting-based serum analysis. as expected, the serum specifically recognized orf9b, s proteins and n proteins (supplementary fig. 2b ). strong antibody responses against s and n proteins. since s and n proteins have been widely used as antigens for diagnosis of covid-19, we next characterized the serum antibody responses against these two proteins in more detail. with the present cohort, the signals from both the n and s proteins, except for the s1-4 fragment, exhibited strong discriminatory ability between the covid-19 patients and controls using either igg or igm response ( fig. 5a , b, supplementary fig. 6a, b) . notably, two sera from the control group exhibited a significant igg antibody response to the n proteins, with one to n-nter and the other to n-cter (fig. 5g) , suggesting that the n protein might generate a higher false-positive measurement than the s protein, especially the s1 protein. to investigate the consistency of signal intensities fig. 5f ) using data of the convalescent sera. high correlations were observed among different concentrations of the same proteins as well as the same protein from different sources ( fig. 5d , h, supplementary figs. 5a-c, g and 6d, g), although the n protein at high concentrations generated almost saturated igg signals ( supplementary fig. 5g ). in particular, for the full-length s1 proteins from different sources, whether from e. coli (s1_t) or 293 t (s1_b and s1_s) expression systems, a high correlation between these proteins were observed ( fig. 5c fig. 2 sars-cov-2 proteome microarray layout and quality control. a there are 14 identical subarrays on a single microarray. a microarray was incubated with an anti-6xhis antibody to demonstrate the overall microarray quality (green). one subarray was shown. the proteins were printed in quadruplicate. the triangles indicate dilution titers of the same proteins. b representative subarrays probed with sera of a covid-19 convalescent and healthy control. the igg and igm responses were shown in green and red, respectively. c, d the correlations of the overall igg and igm signal intensities between two repeats probed with the same serum. proteins (n = 93) on the microarray were examined. e statistics of the pearson correlation confidence among repeats probed with the same serum. two serum samples from the convalescent group was examined in three independent experiments. nc negative control, pc positive control; 0.1, 0.2, 0.25, and 0.5 indicate the concentration of these proteins for microarray printing. t tao lab, b hangzhou bioeast biotech. co.,ltd., k healthcode co., ltd., s sanyou biopharmaceuticals co.,ltd., w vacure l biotechnology co.,ltd., y sino biological co.,ltd. expression system: (1) e. coli: all proteins from tao lab (t), n protein _s, n protein_w; (2) cell-free: all proteins from healthcode co., ltd. (k), (3) mammalian: s1_b, s1_s, s-rbd_s, s-rbd_y. fig. 6c, d) , indicating that the s1 proteins from different sources that we have tested are all similarly effective for detection. however, the background signals in the control group were much lower for proteins purified from mammalian cells (such as 293 t) (fig. 5a, supplementary fig. 6a ), suggesting that these samples might possess a higher specificity and could serve as better reagents for developing immune diagnostics. the signals of the full-length s1 protein were highly correlated with that of the s-rbd (fig. 5e, supplementary fig. 6e ) but with much stronger signals. in contrast, the correlation levels of the s1-4 fragment with the full-length s1 or rbd were lower (fig. 5c , supplementary fig. 5d) . also, the s1 signals were poorly correlated with s2 proteins, although significant s2 signals were observed for many of the patients (fig. 5f, supplementary figs. 5e and 6f) . these data might reflect a difference in the immunogenicity of different regions of the s protein, which could be resolved in the future with more refined epitope mapping. similar results were also observed for the n proteins ( supplementary fig. 5h, i) . interestingly, a moderate but significant linear correlation was observed between the igg responses against the n and s1 proteins ( fig. 5i) but not the igm responses ( supplementary fig. 6h) , while the correlations between the igg and igm signals for the same protein were low (fig. 5j, k) . this might partially be a consequence of overall lower igm signals than the igg signals ( supplementary fig. 6a, b) at the convalescent stage. antibody responses against other proteins. to statistically analyze the igg responses against sars-cov-2 proteins, we calculated the p-values followed by multiple testing correction (or q-values), and applied significant analysis of microarray (sam) to identify significant positive proteins (supplementary fig. 7 and data 2). besides s and n proteins, orf9b and nsp5 also had significant positive responses. particularly, 44.8% (13/29) and 10.3% (3/29) patients exhibited a "positive" igg antibody signal to orf9b and nsp5, respectively (fig. 6a-c) . although e protein and orf7b were statistically positive, the fluorescent intensities in both patient and control groups were too low, further verification is needed for these two proteins. to investigate if the igg responses against orf9b or nsp5 depended on the igg responses to the n or s proteins, we calculated the correlations between these measurements. we observed no obvious correlation between the igg signals to orf9b or nsp5 and the igg fig. 3 the overall sars-cov-2-specific igg profiles of the 29 convalescent sera against the proteins. each square indicates the igg antibody response against the protein (row) in the serum (column). proteins were shown with names along with concentrations (μg ml −1 ) and sources. sera were shown with group information and serum number. ncp novel coronavirus patients or covid-19 patients, lc lung cancer, nc normal control. blank means no serum. three repeats were performed for serum p534 and p535. fi fluorescence intensity. (fig. 6d, e) , suggesting these two proteins might provide complementary information to that generated from the n or s proteins, either for diagnosis or efforts to understand the specific immune response to this virus. igg responses were correlated with age, ldh, and lymphocyte percentage. it is known that the immune response is closely related to the development of the disease in individual patients. to study the relationship between the antibody response and the course of the disease, we examined the correlations between the s1 igg responses to various proteins with clinical characteristics. not surprisingly, the time after disease onset correlated with the igg response against the s1 (fig. 7a) as the igg response usually increases over time and reaches a maximum several weeks after disease onset, as observed in other studies 24 and sars patients 25 . we also found that age also correlated with the igg response to the s1 (fig. 7b) . we also found that the igg responses against s1 protein were positively correlated with peak lactate dehydrogenase (ldh) levels and inversely correlated with percentage of lymphocyte (ly %) (fig. 7c, d) . it was also demonstrated that the igg responses were slightly different between male and female patients (fig. 7e) . we further performed multiple linear regression to investigate the relationship among s1 igg level, age, gender, days after onset, peak ldh and ly% (fig. 7f) . consistent with above correlation analysis, age and peak ldh were statistically significant (both with p-values <0.05) and gender showed marginally significance (p = 0.059). as expected, days after onset, identified as a confounding factor, showed no statistically significant difference (p = 0.514) and was removed from the regression. ly% was still kept in the model as its low significant (p = 0.106) was probably due to the small sample size. the final equation (adjusted r-squared = 0.60, p-value < 0.001) is as follows: y = 1318 + 6155*x 1 + 2166*x 2 -4548*x 3 + 6842*x 4 , where y represents s1 igg level and x 1 , x 2 , x 3 , x 4 represents the normalized values (between 0 and 1) of age, gender, ly%, and peak ldh, respectively. to profile the sars-cov-2-specific igg and igm responses, we have constructed a sars-cov-2 proteome microarray with 18 of the 28 predicted proteins. a set of 29 convalescent sera were analyzed on the microarray, global igg and igm profile were obtained simultaneously through a dual color strategy. our data clearly showed that both n protein and s1 were suitable for diagnostics, while s1 purified from the mammalian cell might possess better specificity. when we were preparing this work, a preprint also found better specificity with mammalian versus insect cell expressed proteins 26 . meanwhile, significant antibody responses were identified for orf9b and nsp5. we further showed that the level of s1 igg positively correlated to age and the level of ldh while negatively correlated to ly%. it is well known that s1 and n proteins are the dominant antigens of sars-cov and sars-cov-2 that elicit both igg and igm antibodies, and antibody response against n protein is usually stronger. however, we found for two of the control sera, strong igg bindings were observed for n protein, and specifically, one control recognizing n protein at the n-terminal while the other at the c-terminal. this may be due to the high conserved n protein sequences across the coronavirus species. this indicating we should be aware of the false-positive when applying n protein for diagnosis. in contrast, s1 protein demonstrated a higher specificity. thus, an ideal choice of developing immunodiagnostics might be the combining of both n protein and s1. we also compared the antibody responses against a variety version of s1, including the full length, the rbd domain, the n-terminal, and the c-terminal. the antibody response to the rbd region was highly correlated with that to full-length protein but with weaker signals which is consistent with a recent study 26 , however, the correlations among other s1 versions were not significant, suggesting dominant epitopes that elicit antibodies might differ among individuals. further study of detailed epitope mapping might give us a clear answer. in this study, we also found the significant presence of igg and igm against orf9b (13 out of 29 cases) and nsp5 (3 out of 29 cases). orf9b is predicted as an accessory protein, exhibiting high overall sequence similarity to sars and sars-like covs orf9b (v23i) 3 , and is likely to be a lipid-binding protein 27 . previous studies showed that sars orf9b suppressed innate immunity by targeting mitochondria 28 . two previous studies have found antibodies against sars orf9b presented in the sera of patients recovering from sars 29, 30 . our study also demonstrates the potential of antibodies against orf9b for the detection of convalescent covid-19 patients. covid-19 nsp5 is also highly homologous to sars nsp5 (96% identity, 98% similarity). its homologous proteins in a variety of coronaviruses have been proven to impair ifn response [31] [32] [33] . our study provide experimental evidence to show the existence of nsp5-specific antibodies in convalescents. since nsp5 is a non-structural protein, theoretically, it should present only in the infected cells but not in nature communications | https://doi.org/10.1038/s41467-020-17488-8 article nature communications | (2020) 11:3581 | https://doi.org/10.1038/s41467-020-17488-8 | www.nature.com/naturecommunications virions. hence, antibody against nsp5 has the potential to be applied to distinguish between covid-19 patients and healthy people immunized with the inactivated virus. we have analyzed the correlations between the covid-19 specific igg responses with clinical characteristics as well. it is expected that igg responses improve over time within one or two months after onset 24, 34, 35 and we indeed have observed a significant correlation between igg signals with days after onset. we also found peak ldh was highly correlated with igg response, especially for female patients. as many studies reported, ldh tends to have a higher level in severe covid-19 patients and could be an indicator of severity 25, 36 . in fact, it has been observed in sars patients that more severe sars is associated with more robust serological response 25,37 , a similar association was confirmed in covid-19 patients. there are some limitations to the current sars-cov-2 proteome microarray. firstly, due to the difficulty of protein expression and purification, there are still 10 proteins missing 3 . we will try to obtain these proteins through vigorous optimization or other sources. an interesting finding is anticipated in the near future for these missing proteins. secondly, most of the proteins on the microarray are not expressed in mammalian cells, critical post-translational modifications, such as glycosylation is absent. it is known that there are 23 n-glycosylation sites on s protein, which is heavily glycosylated, and the glycosylation may play critical roles in antibody-antigen recognition 5, 38 . only a few fig. 6 igg response to other sars-cov-2 proteins. a other sars-cov-2 proteins that were recognized by igg from the convalescent sera, in comparison to that of the controls. b, c anti-orf9b igg (b) or anti-nsp5 igg (c) in the patient and control group. for b, c, each dot indicates one serum sample either from the convalescent group (n = 29) or the control group (n = 21). data are presented as mean values ± sd. the dashed line indicates cutoff value calculated as mean + 3x sd of the control group. p-values were calculated by the two-sided t-test and q-values were adjusted p-values using bh method. d, e correlations of the overall igg responses for n or s1 protein vs. orf9b (d) or nsp5 (e). for d, e, each dot indicates one serum sample from the convalescent group (n = 29) and p-values were calculated by the two-sided t-test. fig. 5 igg responses to s and n proteins. a box plots of igg response for s1 and s2 proteins. the proteins labeled with bold and red were overexpression in mammalian cell lines. b box plots of igg response for n proteins. for a, b, each dot indicates one serum sample either from the convalescent group (green, n = 29) or the control group (brown, n = 21). data are represented as boxplots where the middle line is the mean value. the upper and lower hinges are mean values ± sd. p values were calculated by the two-sided t-test. q values were adjusted p-values using bh method. ***q < 0.001. the exact p-values were shown in supplementary data 2. c pearson correlation coefficient matrix of igg responses among different s1 and s2 proteins. d-f correlations of overall igg responses among different s1 proteins (d), s1 vs. rbd (e) and s1 vs. s2 (f). g one part of a sub-microarray showed the igg responses of two controls, i.e., lc169 and nc96 against n proteins, n-cter and n-nter indicates the c-terminal and n-terminal of n protein, respectively. h, i correlations of the overall igg responses among different n proteins (h) and n protein vs. s protein (i). j statistics of the pearson correlation coefficients between igg and igm profile against constructs of s1 (n = 7), s-rbd (n = 2), s2 (n = 2), and n (n = 9). data are presented as mean values ± sd. k correlations between igg and igm profile against s1_0.1_w. for d-f, h-k, each dot indicates one serum sample from the convalescent group (n = 29). for f and i, p-values were calculated by the two-sided t-test. proteins on the current protein microarray were prepared using mammalian cell systems. we are trying the rest of the proteins. once the microarray is upgraded with many or all proteins purified from mammalian cells, ptm-specific igg and igm response may be better elicited. thirdly, only 29 samples at collected at a single time point were analyzed. though there are some interesting findings, we believe some of the current conclusions could be strengthened by including more samples. furthermore, longitudinal samples 39,40 collected at different time points from the same individual after diagnosis or even after cured may enable us to reveal the dynamics of the sars-cov-2specific igg and igm responses. the data may be further linked to the severity of covid-19 among different patients. the application of the sars-cov-2 proteome microarray is not limited to serum profiling. it could also be explored for host-pathogen interaction 41 , drug or small molecule target identification 42, 43 , and antibody specificity assessment 44 . through the same construction procedure, we could easily expand the microarray to a pan-human coronavirus proteome microarray by including the other two severe coronaviruses, i.e., sars-cov 12, 45, 46 and mers-cov 45 , as well as the four known mild human coronaviruses 47, 48 , i.e., cov 229e, cov oc43, cov hku-1, and cov nl63. by applying this microarray, we can assess the immune response to coronavirus at a system level, and the possible cross-reactivity could be easily judged. taken together, we have constructed the sars-cov-2 proteome microarray, this microarray could be applied for a variety of applications, including but not limited to in-depth igg and igm response profiling. through the analysis of convalescent sera on the microarray, we obtained the overall picture of the sars-cov-2-specific igg and igm profile. we believe that the findings in this study will shed light on the development of the more precise diagnostic kit, more appropriate treatment and effective vaccine for combating the global crisis that we are facing now. construction of expression vectors. the protein sequences of sars-cov-2 were downloaded from genbank (accession number: mn908947.3). according to the optimized genetic algorithm 49 , the amino-acid sequences were converted into e. coli codon-optimized gene sequences. subsequently, the sequences of optimized genes were synthesized by sangon biotech. (shanghai, china). the synthesized genes were cloned into pet32a or pgex-4t-1 and transformed into e. coli bl21 strain to construct the transformants. detailed information (the dna sequence, the protein sequence, the size of the protein, the system for protein expression, and etc.) of the clones constructed in this study was given in supplementary data 1. protein preparation. the recombinant proteins were expressed in e. coli bl21 by growing cells in 200 ml lb medium to an a600 of 0.6 at 37°c. protein expression was induced by the addition of 0.2 mm isopropyl-β-d-thiogalactoside (iptg) before incubating cells overnight at 16°c. for the purification of 6xhis-tagged proteins, cell pellets were re-suspended in lysis buffer containing 50 mm tris-hcl ph 8.0, 500 mm nacl, 20 mm imidazole (ph 8.0), then lysed by a high-pressure cell cracker (union-biotech, shanghai, china). cell lysates were centrifuged at 12,000 × g for 20 min at 4°c. supernatants were purified with ni 2+ sepharose beads (senhui microsphere technology, suzhou, china), then washed with lysis buffer and eluted with buffer containing 50 mm tris-hcl ph 8.0, 500 mm nacl and 300 mm imidazole ph 8.0. for the purification of gst-tagged proteins, cells were harvested and lysed by a high-pressure cell cracker in lysis buffer containing 50 mm tris-hcl, ph 8.0, 500 mm nacl, 1 mm dtt. after centrifugation, the supernatant was incubated with gst-sepharose beads (senhui microsphere technology, suzhou, china). the target proteins were washed with lysis buffer and eluted with 50 mm tris-hcl, ph 8.0, 500 mm nacl, 1 mm dtt, 40 mm glutathione. the purified proteins were analyzed by sds-page followed by western blotting using an anti-his antibody (merck millipore, usa, cat#05-949) and coomassie brilliant blue staining. recombinant sars-cov-2 proteins were also collected from commercial sources. detailed information on the recombinant proteins prepared in this study was given in supplementary data 1. protein microarray fabrication. the proteins, along with the negative (bsa) and positive controls (anti-human igg, cat#i2136 and igm antibody, cat#i2386), were printed in quadruplicate on path substrate slide (grace bio-labs, oregon, usa) to generate identical arrays in a 2 × 7 subarray format using super marathon printer (arrayjet, uk). protein microarrays were stored at −80°c until use. . e s1 igg responses in male (m, n = 13) and female (f, n = 16) groups. t-test data are presented as mean values ± sd. for a-e, p-values were calculated by two-sided t-test. f multiple linear regression model for s1 igg. p-values for coefficient was calculated by two-sided t-test and p-value for regression model was calculated by one-sided f-test. *p < 0.05. patients and samples. the institutional ethics review committee of foshan fourth hospital, foshan, china approved this study and the written informed consent was obtained from each patient. covid-19 patients were hospitalized and received treatment in foshan forth hospital during the period from 2020-1-25 to 2020-2-27 with variable stay time (table 1) . serum from each patient was collected on the day of hospital discharge when the standard criteria were met according to diagnosis 50 . briefly, the key points of the discharge criteria are: (1) body temperature is back to normal for more than three days; (2) respiratory symptoms improve obviously; (3) pulmonary imaging shows obvious absorption of inflammation; (4) nuclei acid tests negative twice consecutively on respiratory tract samples such as sputum and nasopharyngeal swabs (sampling interval being at least 24 h). sera of the control group from lung cancer patients and healthy controls were collected from ruijin hospital, shanghai, china. all sera were stored at −80°c until use. microarray-based serum analysis. a 14-chamber rubber gasket was mounted onto each slide to create individual chambers for the 14 identical subarrays. the microarray was used for serum profiling as li, y. et al. 39 with minor modifications. briefly, the arrays stored at −80°c were warmed to room temperature and then incubated in blocking buffer (3% bsa in 1 × pbs buffer with 0.1% tween 20) for 3 h. serum samples were diluted 1:200 in pbs containing 0.1% tween 20, added with 0.1 mg ml −1 egfp purified in the same manner as the egfp tagged proteins and 0.5 mg ml −1 total e. coli lysate. a total of 200 μl of diluted serum or buffer only was incubated with each subarray overnight at 4°c. the arrays were washed with 1 × pbst and bound antibodies were detected by incubating with cy3-conjugated goat anti-human igg and alexa fluor 647-conjugated donkey anti-human igm (jackson immunoresearch, pa, usa, cat#109-165-008 and cat#709-605-073 respectively), which were diluted 1: 1000 in 1 × pbst and incubated at room temperature for 1 h. the microarrays were then washed with 1 × pbst and dried by centrifugation at room temperature and scanned by luxscan 10k-a (capitalbio corporation, beijing, china) with the parameters set as 95% laser power/pmt 550 and 95% laser power/ pmt 480 for igm and igg, respectively. the fluorescent intensity data was extracted by genepix pro 6.0 software (molecular devices, ca, usa). immunoblotting-based serum analysis. the selected proteins were analyzed by sds-page followed by western blotting using a serum overnight at 4°c. to assure the quality of the proteins, an anti-his antibody (merck millipore, usa, cat#05-949) was also blotted. the serum for immunoblotting was diluted 1:200 in pbs containing 0.1% tween 20, with the addition of 0.1 mg ml −1 egfp purified in the same manner as the egfp tagged proteins and 0.5 mg ml −1 total e. coli lysate as mentioned before. statistics. signal intensity was defined as the median of the foreground subtracted by the median of background for each spot and then averaged the quadruplicate spots for each protein. igg and igm data were analyzed separately. before processing, data from some spots, such as nsp7_0.1_t and nsp9_k, were excluded for probably printing contamination. pearson correlation coefficient between two proteins or indicators and the corresponding p-value was calculated by spss software under the default parameters. cluster analysis was performed by pheatmap package in r 51 . p-values for statistical analysis were calculated by two-way t-test and q-values or adjusted p-values were obtained using bh (benjamini and hochberg) method. significant analysis of microarray (sam) was performed by "samr" package of the r language with default parameters 52 . to calculate the positive rate of antibody response for each protein, mean signal + 3* standard deviation (sd) of the control sera were used to set the threshold. the multiple linear regression was perfomed with the function "lm" from the "stats" package of the r language. to make the cofficients in the regression model more comparable with each other, the values of all predictor vairables (x) have been normalized as follows: 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coronavirus pneumonia (trial version 7 pretty heatmaps significance analysis of microarrays applied to the ionizing radiation response reporting summary. further information on research design is available in the nature research reporting summary linked to this article. the protein sequences of sars-cov-2 were downloaded from genbank (accession number: mn908947.3). the sars-cov-2 proteome microarray data are deposited on protein microarray database under the accession number pmde241 (http://www.proteinmicroarray. cn/index.php?option=com_experiment&view=detail&experiment_id=241). additional data related to this paper may be requested from the authors.received: 1 april 2020; accepted: 30 june 2020; we thank dr. daniel m. czajkowsky for english editing and critical comments. we thank dr. min guo of healthcode co., ltd. for providing affinity-purified proteins. we thank dr. guo-jun lang of sanyou biopharmaceuticals co., ltd. for providing proteins and antibodies. we also thank dr. jie wang of vacure l biotechnology co., ltd., dr. yin-lai li of hangzhou bioeast biotech. co., ltd., and sino biological co., ltd. for providing the proteins. this work was partially supported by the national the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-17488-8.correspondence and requests for materials should be addressed to d.m., j.z. or s.-c.t.peer review information nature communications thanks martijn van hemert and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. peer reviewer reports are available.reprints and permission information is available at http://www.nature.com/reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-259260-qcfgigga authors: ibrahim, ibrahim m.; abdelmalek, doaa h.; elfiky, abdo a. title: grp78: a cell's response to stress date: 2019-06-01 journal: life sciences doi: 10.1016/j.lfs.2019.04.022 sha: doc_id: 259260 cord_uid: qcfgigga abstract background glucose-regulated protein 78 (grp78) is a chaperone heat shock protein that has been intensely studied in the last two decades. grp78 is the master of the unfolded protein response (ubr) in the endoplasmic reticulum (er) in normal cells. grp78 force the unfolded proteins to refold or degrade using cellular degradation mechanisms. scope under stress, the overexpression of grp78 on the cell membrane mediates the vast amount of disordered proteins. unfortunately, this makes it a tool for pathogens (bacterial, fungal and viral) to enter the cell and to start different pathways leading to pathogenesis. additionally, grp78 is overexpressed on the membranes of various cancer cells and increase the aggressiveness of the disease. major conclusions the current review summarizes structure, function, and different mechanisms grp78 mediate in response to normal or stress conditions. general significance grp78 targeting and possible inhibition mechanisms are also covered in the present review aiming to prevent the virulence of pathogens and cancer. glucose-regulated protein 78 (grp78) or immunoglobulin heavy chain binding protein (bip) is a member of the heat shock protein 70 (hsp70) family. it is found in all eukaryotes on the membrane of endoplasmic reticulum (er) [1] . the 654 amino acid protein, grp78, corrects the folding and assembly and prevents the transport of proteins or protein subunits that are folded incorrectly [2] [3] [4] . grp78 expression is increased in cases of er stressors like when the cell is abridged from sugar, treated with reagents that inhibit the process of protein glycosylation or disturb the intercellular calcium storage [5] . it is a watersoluble protein, and only small patches are reported to be hydrophobic. these hydrophobic patches are essential for its function as it recognizes the unfolded proteins that are directed either to the degradation or refolding mechanisms [6] . grp78 structure is divided into two domains, atp binding domain (abd) (or nucleotide binding domain nbd), at the amino terminal and substrate binding domain (sbd) at the carboxyl-terminal [7] . fig. 1 shows the x-ray apo form of grp78 solved structure (pdb id 6eoc) at 1.67 å resolution and abd domain with bound atp molecule (pdb id 5f1x) at 1.90 å resolution. grp78 shares 60% homology with the hsp70 family, with the conservation of abd and sbd domains. abd domain shows the most considerable sequence conservation over the hsp70 family [1, 7, 8] . although grp78 is an hsp70 family a member and shares the properties of abnormal protein binding under conditions of stress, it differs in protein expression regulation. grp78, but hsp70, is sensitive to the protein synthesis inhibitor cycloheximide [9, 10] , which is a protein synthesis inhibitor in eukaryotic cells [11] . the most potent induction reasons for grp78 expression, like calcium ionophore a23187 and β-mercaptoethanol, doesn't affect the expression level of hsp70 proteins [12] [13] [14] . treating cells with such inducers increases the grp78 gene expression up to 10-25 folds in 5 h [12, 15] . grp78 expression appears to be in a direct correlation with the activity of the er. this means that intermediate molecules must exist and travel between both membranes of the er and the nucleus to reach the grp78 gene and communicate the signal. the way between the two membranes is not tricky as the er membrane is associated with the prenuclear membrane. grp78 is regulated over the transcriptional level [8] . in human, the gene responsible for grp78 encoding is on chromosome number 9 with a length of 4532 nucleotides [1, 2] . fluorescence in situ hybridization with a 24,000 bases genome phage clone that contains the whole coding region of grp78 gene is used to prove the positive signals of hybridization at the distal end of the long arm of chromosome 9, at band 9q34 [2] . in human there are two genes sequences are found at highly hydrophilic domains at the carboxyl third of the protein [6, 16, 17] . a deletion of 12 nucleotides near the amino-terminal and insertion of four nucleotides within exon number 5 was found in pseudogene compared to the functional gene. grp78 pseudogene is surrounded by gaaaattaaacaa sequence. upstream from the pseudogene, 100 nucleotides are 66% a-t rich while this ratio is 75% for the 200 downstream nucleotides [6] . in human, the 5′ flanking region is 1650 nucleotides. when this region is reduced to 358 nucleotides, no changes are observed in the basal or induced activity. on the other hand, the reduction of this region to 170 nucleotides results in two folds decline in both basal and induced activity [6, 8] . analysis of the human grp78 gene reveals that grp78 promotor contains a region that is sensitive to stress. this region is within the 170 nucleotides upstream from the transcription initiation site [6] . grp78 function is to work as a molecular chaperone. it binds to misfolded proteins and unassembled complexes and initiates erfig. 1. a) shows the x-ray apo form of grp78 solved structure (pdb id 6eoc) at 1.67 å resolution. the protein is represented by cartoon colored green (nbd), red (sbd) and purple (linker). grp78 sequence is represented also with the same color as the protein domains. b) atp binding domain with pound atp molecule (pdb id 5f1x) at 1.90 å resolution represented as the rainbow-colored cartoon. the binding site with atp is enlarged to show the amino acids involved in the interaction. pymol software is used to represent the entire figure [127] . (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) associated degradation (erad) responsible for upr regulation [18] [19] [20] . it binds to unfolded peptides with the sbd and gets the energy to prevent aggregation by the mean of atp hydrolysis in the (nbd) [21] . in the standard conditions of balance in the cell (homeostasis) grp78 is bounded in an inactive form to activating transcription factor 6 (atf6), protein kinase rna-like endoplasmic reticulum kinase (perk), and inositol-requiring enzyme 1 (ire1) which are upr transmembrane stress sensors. when the cell is exposed to unfolded proteins that are accumulated in the er, this stress can be revealed when grp78 is released from the upr sensors. the active form of upr then decreases the translation of protein and enhance the correct folding [19] (see fig. 2 ). perk, when activated, phosphorylates the alpha (α) subunit of eukaryotic translation initiation factor (eif2α). the phosphorylated eif2α then can inhibit the initiation of translation and inhibit protein synthesis reducing the influx of nascent proteins into the lumen of the er [20, 22] . the activated form of atf6 translocates to the golgi apparatus where it is cleaved. after that, the cleaved atf6 moves to the nucleus and works as an active transcription factor and upregulate the transcription of proteins that increase the folding capacity of the er like grp78. the ire1 active form has an endoribonuclease activity. it breaks a 24-base segment of mrna intron and encodes x-box binding protein 1 (xbp1). xbp1 target genes which are responsible for protein folding and erad [2] . at this point, if the homeostasis of the er could not be restored, the unfolded protein response can induce apoptosis [19] . in its basal level of expression, grp78 plays a role in the development of the embryo. it was found that homozygous mice, with grp78 gene, knocked down, witnessed fatality at day 3.5 of embryogenesis with a massive apoptotic inner cell mass death [20] . in human, when grp78 is knocked down, the expression of glucose-regulated protein 94 (grp94), ccaat-enhancer-binding protein homologous protein (chop), c-jun n-terminal kinase (jnk), and x-box binding protein 1 (xbp1) are spontaneously increased. as grp78 is suppressed down, the upr sensors are activated. grp94 expression is improved, but this increment does not compensate for the grp78 absence. this result shows that grp78 has an anti-apoptotic role and it is essential for cell viability. grp78 binds to caspase 7 and caspase 12 which are the main cytosolic implementers for apoptosis, despite there is no binding to caspase 3 [23] . there is a relation between aging and er stress. aging causes a decrease in protein expression and a decrease in the activity of several er chaperones such as grp78. this decline in the expression increases the tendency to er stress. this unmitigated er stress leads to age-related diseases [19] . grp78 has a non-chaperon function as it is the primary binding protein of ca +2 in the er and maintain its balance in the lumen. there is no specified domain for ca +2 binding, but it binds to the anionic amino acid residues. one hundred eleven anionic residues, from which 19 only paired, are found in grp78 leaving many residues to be favorable for ca +2 binding. this binding capacity still less than other er proteins [24] . grp78 can also be found on the cell surface. the last four amino acids in the grp78 protein (the motif kdel) which are responsible for er retention, is present in the cell surface grp78 (cs grp78). thus, the cleavage of this motif sequence is not required in surface expression. when grp78 is overexpressed, a fraction of it can escape the motif retention and translocate to the cell surface [20, 25] . as when the grp78 is overexpressed, the kdel motif receptors are fully saturated, and then grp78 can escape the retention. another thing is kdel receptor can be downregulated under some circumstances. there is a theory saying that it could be another protein that helps grp78 to escape retention by masking kdel sequence, but it has yet to be determined [25] . when grp78 moves to the surface of the cell, it can interact with plenty of ligands or other proteins on the cell surface and works as a multifunctional receptor. cs-grp78 plays an essential role as in cellular signaling, proliferation, migration, invasion, apoptosis, inflammation, and immunity. according to the ligand or the peptide that bind to cs-grp78, it will be activated in a defined signaling pathway that affects besides, if the cell is cancerous, cs grp78 will induce resistance to chemotherapy. the cell in different ways. when grp78 is on the cell surface, autoantibodies are secreted. antibodies for the n-terminal of the protein induces cell proliferation while antibodies bind to the c-terminal causes the induction of cell apoptosis [26] . ligand α2 macroglobulin (α2m), a plasma proteinase inhibitor, affects promoting cell proliferation and viability. cs-grp78 has a high affinity to α2m. when isthmin, 60 kda protein, binds to cs-grp78, it works as a proapoptotic ligand by induction of mitochondrial dysfunction [27] . par-4, which is prostate apoptosis response 4, can interact with cs-grp78 and induce apoptosis through er stress and the activation of the fadd/caspase-8/caspase-3 pathway. another way of cell apoptosis induction is by plasminogen kringle 5 which requires cs-grp78 to induce apoptosis of dermal microvessel endothelial cells [26] . as we remarked before hypoxia, glucose starvation or tumors cause er stress. hypoxia induces cs-grp78 levels to be increased over fourfold. besides, levels of cell surface grp78 is correlated to several pathological conditions like many cancers, arthrosclerosis, and rheumatoid arthritis. the presence of grp78 on the cell surface caused the immune system to generate an autoimmune response and circulating autoantibodies to cs-grp78 expressed cells [24] . mucorales, which are thermotolerant fungi, can be found in decaying organic material [28] . these fungi can cause mucormycosis disease which is considered as the third common invasive fungal infection in patients who undergo organ transplantations or having hematologic malignant tumors [29] . although there are treatment options for mucormycosis, this disease is characterized by high mortality rates often higher than 40% and in some cases can reach 100% [30, 31] . some of the risk factors that mediate the infection are hematologic malignancy, organ transplantation, and uncontrolled diabetes mellitus [31] . all mucorales can cause blood vessel thrombosis leading to tissue necrosis. therefore, the interaction of mucorales with the endothelial cells of blood vessels is responsible for their pathogenesis [31] . one of the most common fungi belonging to mucorales order is rhizopus oryzae (r. oryzae) which is responsible for 70% of reported mucormycosis cases [31, 32] . grp78 is found to be the receptor mediating the binding of r. oryzae during cell invasion [33] . the ligands on the surface of the r. oryzae that bind to grp78 are spore coat protein homolog (coth) cell surface proteins [32] . there are 3 of these surface proteins named (coth1, coth2, and coth3) surface proteins. the expression of coth2 and coth3 is increased 4-and 16-fold, respectively, when r. oryzae germlings were incubated on endothelial cells [32] . coth2 and, to a greater extent, coth3 surface proteins bind to grp78 and are responsible for adherence and invasion (endocytosis) of r. oryzae. both coth2 and coth3 proteins bind to the same alpha-helical bundle domain of grp78, in silico [32] . grp78 has an essential role in the development of some viruses' envelope proteins such as sindbis virus, hepatitis c virus, vesicular stomatitis virus, and influenza a virus [34] [35] [36] [37] . for some viruses to enter a cell, they need first to attach to a specific cell surface molecule, then uses another molecule that mediates their internalization [38] . this complex multi-step process is essential for the viruses to survive the hostile environment of the host immunity [39] . one of the deadliest re-emerging viruses is filoviruses ebola virus (ebov). human infection by ebov causes hemorrhagic fever with case fatality rates in some outbreaks that reached 90% [40] . the epidemic of ebov requires the utilization of host factors in the different stages of the viral life cycle [41] . epigallocatechin gallate (egcg) can inhibit the ability of grp78 to bind atp by binding to the atp-binding site [42] . using this compound, shurtleff et al. found that egcg can inhibit the ebov infection, demonstrating that the atpase activity of grp78 is required for the infection [43] . besides, they found that using grp78 sirna caused a decrease of viral transcript production [43] . also, the infection by ebov results in an increase of hspa5 expression due to the accumulation of ebov glycoproteins 1 and 2 (gp1 and gp2) in the endoplasmic reticulum causing a stress response [44] . although grp78 is involved in the entry of some viruses [38] , knockdown of grp78 did not affect the entry of ebov [43] . grp78 has a role in the budding of vp40 protein [43] . this interaction was suggested earlier by yamayoshi et al. [45] . one of the emerging infectious viruses is zika virus (zikv). zikv is a flavivirus related to microcephaly, a neurodegenerative disorder, and guillain-barré syndrome which is an autoimmune disorder [46, 47] . two possible routes of transmission are through aedes mosquitos, as they are the major species of mosquitos responsible for the transmission of the virus, or through sexual intercourse, though it is not significant [48, 49] . the genome of zikv is nearly 11 kb long with a single open reading frame (orf) flanked by noncoding regions at the two ends [50] . the end products of orf are capsid, the precursor of membrane, envelope, and seven non-structural proteins [51] . the envelope protein is responsible for cellular attachment, entry, and fusion [52] . one of the receptors responsible for the endocytosis of the virus is grp78 [53, 54] . another mosquito-borne virus belonging to the flavivirus genus is dengue virus (denv) [55, 56] . its genome is positive-stranded rna, which encodes, after the processing of the polyprotein produced from the rna, three structural and seven non-structural proteins. it has four serotypes (denv1-4) [57] . this virus can cause a wide range of diseases such as; dengue fever (benign self -limited febrile illness), dengue hemorrhagic fever, or dengue shock syndrome [55] . grp78 is identified as a receptor for dengue virus serotype 2 in hepatoma cells (hepg2) [58] . jindadamrongwech et al. found that using an antibody against n-terminal of grp78 inhibited binding and infection while using antibodies against the c-terminal of grp78 increase the infection and, to a less noticeable degree, the binding of the virus to the receptor. this increase is related to the concentration of antibodies [58] . this may be due to the conformational changes produced from the c-terminal antibody binding to grp78, enhancing the binding ability of the virus to the protein [58] . japanese encephalitis virus (jev) is a member of the flaviviridae family. other mosquito-borne, and medically important pathogens viruses belonging to this family are denv and west nile virus [59] . jev is a neurotropic virus which causes encephalitis in humans and has a mortality rate ranging from 25% to 30% [59] . for the jev to enter a cell, it needs first to attach to the cell membrane through the help of specific receptors such as heparan sulfate proteoglycans (hspgs) and glycosaminoglycans (gags) [60, 61] . nain et al. identified grp78 as a receptor for jev envelope domain iii (ed3) [62] . they checked the interaction between grp78 and jev ed3 by using firefly luciferase gene which showed a substantial activity, confirming this interaction [62] . using n-terminal grp78 antibody, the interaction is revealed to be between the n-terminal grp78 and jev ed3 as the binding between grp78 and jev ed3 reduced by nearly 38% [62] . besides, they confirmed the role of grp78 in the internalization of jev by using grp78 sirna, then measuring the jev rna 2 h post infection, at this time the level of rna is an indication of the viral entry only. this test shows a significant reduction in jev rna in cells by nearly 60% [62] . grp78 also has a role in the replication of jev [62] . this role is demonstrated by using subtilase cytotoxin which can break grp78 into its c-and nterminals leading to its deactivation [63] . a 90% reduction in the viral rna in all cells was reported, indicating the significant role of grp78 jev replication [62] . coronaviruses are enveloped single-stranded positive-sense rna viruses that can infect a wide range of species including humans [64] . they are classified into alpha-, beta-, gamma-, and delta coronaviruses [64] . one of the viruses belonging to betacoronaviruses is middle east respiratory syndrome coronavirus (mers-cov) which causes severe lower respiratory tract infection with a high mortality rate reaching 35% [64, 65] . the mers-cov spike protein utilizes dipeptidyl peptidase 4 (dpp4) as its functional receptor for host cell entry [66] . in addition to its functional receptor, mers-cov can recognize other molecules to facilitate their attachment and entry, for example, carcinoembryonic antigen-related cell adhesion molecule 5 (ceacam5), and tetraspanin cd9 are identified as a factor promoting the virus entry in permissive cells [67, 68] . recently grp78 was recognized as an attachment factor for mers-cov spike protein that enhances the virus entry in the presence of dpp4 [65] . in addition to grp78 role in the entrance of the virus, it has a role in the replication of mers-cov [65] . cs grp78 is upregulated after the entry of mers-cov, which helps in the attachment of the virus [65] . although knockdown of grp78 led to a decrease in the replication of the virus, this decrease is lesser compared to the reduction in the replication of the virus when dpp4 is reduced [65] . coxsackievirus a9 (cav9) is a non-enveloped rna virus that can cause flaccid paralysis, and chronic dilated cardiomyopathy [69] . besides, it is involved in autoimmune episodes that result in insulin-dependent diabetes mellitus [70, 71] . integrin alpha-v beta 3(αvβ3) is a receptor that can be used by cav9 [72] [73] [74] , but its utilization is not sufficient for its infection [72] . grp78 was identified by triantafilou et al. to be utilized by cav9 for infection [38] . grp78 delivers viral peptides to mhc-i [75] , and it is found that grp78 is associated with mhc-i molecules on the cell membrane [38] . after cav9 binds to grp78, it causes an increase in the clustering of grp78 and mhc-1 molecules [38] . triantafilou et al. used anti-mhc-i antibodies (w6/32, mca1115) to determine whether mhc-i helps in internalization of the cav9 virus [38] . although there is no change in binding, they found that the internalization is inhibited by 85% using these antibodies [38] . they suggested a model in which cav9 attach to the cell by binding to grp78 and integrin αvβ3, then uses mhc-i which is associated with grp78 for entry so it can use the cell machinery [38] . one of the viruses that belong to bornaviridae is borna disease virus (bdv) [76] . bdv is non-segmented, negative-strand rna virus with high neurotropic and non-cytopathic infection [76] . this virus infects many animals and causes central nervous system diseases which are associated with behavioral disturbances [77] . virus entry is mediated by endocytosis after the viral envelope glycoprotein (g) attach to the cell receptor [78, 79] . the n terminal in g protein is responsible for the attachment to the cell receptor [78] . one of these receptors is grp78 as demonstrated by honda et al. [80] . they used grp78 antibody and found that binding of the g protein to grp78 reduced to 40%. the grp78 domain that binds to g protein is the atp-binding domain [80] . grp78 has a crucial role in proliferation, invasion, and metastasis of many cancer cells such as renal cell [81] , endometrial [82] , gastric [83] , and prostate cancer [84] . the most common cancer in females worldwide is breast cancer [85] . the treatment of advanced breast cancer patients starts with gemcitabine [86] . gemcitabine is a pro-drug cytotoxic chemotherapeutic agent similar to cytarabine. its anticancer effects are manifested by its phosphorylated active metabolites (gemcitabine di-and triphosphate) [87] . these metabolites are combined with dna to stop replication and cell growth leading to apoptosis [87] . the major problem that affects therapy is drug resistance [88] . grp78 was demonstrated to promote drug resistance in breast cancer against doxorubicin [89] . usually, apoptosis has two pathways (extrinsic or intrinsic) [90] . extrinsic pathway requires that death receptors on the cell surface be activated. on the other hand, the intrinsic pathway involves a series of events that are processed in the mitochondria [90] . caspase 9 is triggered by the intrinsic pathway [91] and is found to be responsible for gemcitabine resistance and grp78-regulated chemosensitivity [88] . xie et al. show that overexpression of grp78 reduced the sensitivity of gemcitabine so it may have a critical role in the resistance of breast cancer cells. this reduction in sensitivity is caused by apoptosis inhibition [88] . the same group shows that caspase 9 and its phosphorylated form p37 were excessively down-regulated in grp78-overexpressed breast cancer cells and was markedly increased in grp78downregulated breast cancer cells [88] . the levels of anti-apoptosis protein bcl-2 is found to be high in grp78-overexpressing breast cancer cells, on the other hand, the levels of pro-apoptosis proteins, bax and bim are low, and vice versa [88] . akt can affect mitochondrial apoptosis by either targeting the pro-apoptotic protein bad or by inhibition of pro-apoptotic signals produced by transcription factors such as foxo [88] . akt is found to be markedly increased in gemcitabine resistance breast cancer and is related to the expression of grp78, so an increase in grp78 leads to akt increase. if akt expression is reduced in grp78overexpressing breast cancer cells, their sensitivity to gemcitabine increases and apoptosis also increases [88] . these results are in agreement with other types of cancers such as colon cancer [92] , and prostate cancer [93] . the fourth common cause of death in women is ovarian carcinoma which represents the most lethal type of gynecological malignancies [94] . epithelial ovarian carcinoma has only 30% 5-year survival rate because of the difficulty of early detection due to unclear symptoms [95] . grp78 overexpression in cancer cells and human tumors resulted in cancer malignancy and increased survival of cancer cells due to treatment resistance [96, 97] . the accumulation of polypeptides in endoplasmic reticulum due to the elevated metabolism of ovarian cancer cells leads to the overexpression of grp78 [98] . although the use of taxane and platinum-based chemotherapy in the treatment of ovary cancer results initially in high response rates, many of the patients suffer a relapse and develop resistance [99] . paclitaxel can lead to apoptosis by preventing microtubule formation leading to a mitotic block of the cell [100] and partly to endoplasmic reticulum unfolded protein response [101] . zhang et al. [97] demonstrated that treatment of three groups of ho-8910 cells with paclitaxel after transfection with grp78 sirna for one group, nonspecific sirna for the second group, or untreated ho-8910 cells led to a different decrease in survival rates of the three groups to paclitaxel. the survival rates of the grp78 sirna treated group is significantly lower than the other two groups which indicate the higher sensitivity of the treated group to paclitaxel treatment after the inhibition of grp78 protein [97] . in addition to the protective role of grp78 to paclitaxel, the apoptosis ratio in the sirna grp78 treated group reaches 56.92 ± 0.46% after 72 h of transfection, which corresponds to a previous study by martin et al. [102] . the increase in apoptosis may be due to the prevention of the interaction between grp78 and apoptosis pathway compounds such as caspase-7 [103] . chen and xu [104] studied the effect of cisplatin, which is a platinum-based chemotherapy reagent [105] , on ovarian cancer cells. in their study, they showed that grp78 overexpression in ovarian chronic cisplatin-treated cells leads to chemoresistance, which reduces the effectiveness of the treatment [98] . one of the most aggressive types of malignant tumors is pancreatic ductal adenocarcinoma (pdac) [106] . the only cure for pdac is surgery; however, the 5-year survival rate of patients after removal of the pancreatic cancer is approximately 20-25% [81, 107] . because of this, the identification of specific new factors that can increase the survivability of patients is critical [81] . zheyu et al. [108] found a strong relationship between the expression of grp78 and tumor stage indicating a potential role of grp78 in pdac progression. they show that low overall survival of patients was strongly related to the overexpression of grp78 on tumor cells [108] . overexpression of grp78 affects proliferation, cell cycle, invasion, and migration of pdac cells. such that overexpression increases proliferation, invasion, and migration, and increases the percentage of cells in s phase of the cell cycle [108] . the underlying mechanism of invasion of pancreatic cancer cell was demonstrated by yuan et al. [109] . he showed that the overexpression of grp78 caused an elevation of matrix metalloproteinase-2 and 9(mmp-2 and mmp-9) secretion and activity [109] . although mmps are degrading proteases, they have a vital role in invasion and metastasis [109] . dynamics of actin filaments, especially cytoskeletal f-actin stress fibers, is increased in response to knockdown to grp78 [109] . ras homolog gene family, member a (rhoa) and rac have an essential role in cytoskeleton dynamics and act as a downstream of focal adhesion kinase (fak) [110] , which have a crucial role in cancer invasion [109, 111] . the activity of rac and rhoa are related to the levels of grp78. overexpression of grp78 increases the activity of rac but decreases rhoa activity [109] . fak activity is regulated by the expression of grp78 such that, overexpression of grp78 increase the phosphorylation of fak y397 [109] . the c-jun n-terminal kinase (jnk), has a critical role in the invasion of many epithelial cancers. its activity is related to the expression of grp78 in a direct way [109] . colorectal cancer is the third cancer type according to incidence but is considered to be the second for its mortality [112] . this cancer can be treated by surgical intervention if it is still in the primary site; however, metastasis is the main factor leading to death from colorectal cancer patients [113] . the expression of grp78 was found to be high in colon cancer cells [114] , and its downregulation led to an increase of epirubicin-induced apoptosis, which is due to an increase in the nuclear factor erythroid 2-related factor 2 (nrf-2) expression [115] . moreover, silencing of grp78 resulted in a suppression of colon cancer growth by the downregulation of vascular endothelial growth factor/vascular endothelial growth factor receptor 2 (vegf/vegfr2) pathway [115] . high surface expression of grp78 in colon cancer shows a reduction in tumor proliferation and growth [116] , though, an increase in the invasiveness is observed with high surface grp78 expression [117] . the downregulation of grp78 increased colon cancer metastasis, due to a rise in nrf-2 and heme oxygenase -1 (ho1) level and change in the epithelial-to-mesenchymal transition (emt) biomarker expression [113] . nrf-2 has a function in cell migration ability [118] . the migration ability of colon cancer is increased after the downregulation of grp78 [113] . the raised migration is caused by an increase in vimentin and decrease in e-cadherin [113] . vimentin is an intermediate filament protein [119] , one of the emt markers, and considered to be important in emt induction [120] . e-cadherin is essential in cell polarity and organization of epithelium and can be regarded as a significant epithelial marker [121] . its level decreased in some cases such as tumor metastasis, and progression from adenoma to carcinoma [122] . usually treatment of cancer is done by chemotherapy; however, it is limited by its severe side effects. therefore, it is inevitable to develop new methods for cancer treatment [123, 124] . one of these new methods utilizes peptidic ligands as they can specifically target the tumor cells and deliver drugs [125] . one of these peptides is pep42 which is a cyclic 13-mer ctvalpggyvrvc [126] . the internalization of this peptide is studied after it was mutated at position 12 (valine to lysine) (mut42) for fluorescein isothiocyanate coupling (fitc), and it is found that this mutation doesn't affect pep42 internalization negatively [126] . pep42 can enter the cell through a receptor-mediated pathway, this receptor was identified to be grp78, and it is found on the surface of human melanoma cells (me6652/4) [126] . after internalization, mut42 was found to be colocalized with the er but not lysosomes. after using different concentrations of monoclonal antibodies against grp78, the uptake of mut42 is reduced according to the level of the antibodies. moreover, overexpression of grp78 increases the entry of mut42 which indicates the specificity of pep42 to grp78. mut42 is used with taxol on human melanoma cells, and 92.1% of the cells are found to be in the late stages of apoptosis [126] . the cyclic shape of this peptide is vital for internalization as reported by kim et al. [126] . besides, the peptide is mainly hydrophobic supporting the selectivity of the peptide to bind to grp78, which has the affinity to attach to the hydrophobic clusters of unfolded proteins under stress conditions. these are two crucial points for consideration when studying the interaction between cs grp78 and pathogenic proteins (envelope and spore coat proteins). also, targeting such binding site on the cs grp78 with cyclic peptides may be the future preventive routine for patients living with diabetes or cancers. hsp70 genes in the human genome: conservation and differentiation patterns predict a wide array of overlapping and specialized functions localization of the gene encoding human bip/grp78, the endoplasmic reticulum cognate of the hsp70 family, to chromosome 9q34 bip-a heat shock protein involved in immunoglobulin chain assembly protein folding in the cell coordinated regulation of a set of genes by glucose and calcium ionophores in mammalian cells human gene encoding the 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signal transduction biology the endoplasmic reticulum chaperone grp78 also functions as a cell surface signaling receptor, cell surface grp78, a new paradigm in cell surface grp78: anchoring and translocation mechanisms and therapeutic potential in cancer, cell surface grp78, a new paradigm in epidemiology and clinical manifestations of mucormycosis molecular mechanisms of mucormycosis-the bitter and the sweet recent advances in the management of mucormycosis: from bench to bedside mucormycosis and entomophthoramycosis (zygomycosis) coth3 mediates fungal invasion of host cells during mucormycosis the endothelial cell receptor grp78 is required for mucormycosis pathogenesis in diabetic mice quality control in the endoplasmic reticulum intracellular transport of soluble and membrane-bound glycoproteins: folding, assembly and secretion of anchorfree influenza hemagglutinin involvement of the molecular chaperone bip in maturation of sindbis virus envelope glycoproteins involvement of 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virus bovine lactoferrin inhibits japanese encephalitis virus by binding to heparan sulfate and receptor for low density lipoprotein fitness of japanese encephalitis virus to neuro-2a cells is determined by interactions of the viral envelope protein with highly sulfated glycosaminoglycans on the cell surface grp78 is an important host-factor for japanese encephalitis virus entry and replication in mammalian cells ab 5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone bip coronavirus host range expansion and middle east respiratory syndrome coronavirus emergence: biochemical mechanisms and evolutionary perspectives middle east respiratory syndrome coronavirus and bat coronavirus hku9 both can utilize grp78 for attachment onto host cells middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sarslike disease carcinoembryonic antigen-related cell adhesion molecule 5 (ceacam5) is an important surface attachment factor facilitating the entry 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metastasis via sp-1, fak inhibition, and ecadherin upregulation in colorectal cancer global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries glucose-regulated protein 78 (grp78) regulates colon cancer metastasis through emt biomarkers and the nrf-2/ho-1 pathway overexpression of glucose-regulated protein 78 in colon cancer grp78 knockdown enhances apoptosis via the down-regulation of oxidative stress and akt pathway after epirubicin treatment in colon cancer dld-1 cells colon cancer cells expressing cell surface grp78 as a marker for reduced tumorigenicity cell-surface grp78 facilitates colorectal cancer cell migration and invasion the role of nrf2 in migration and invasion of human glioma cell u251 introducing intermediate filaments: from discovery to disease vimentin: central hub in emt induction? sticky business: orchestrating cellular signals at adherens junctions a causal role for ecadherin in the transition from adenoma to carcinoma role of chemotherapy dose intensification in the treatment of advanced ovarian cancer metastatic melanoma: is biochemotherapy the future combinatorial discovery of tumor targeting peptides using phage display felding-habermann, targeting heat shock proteins on cancer cells: selection, characterization, and cell-penetrating properties of a peptidic grp78 ligand the pymol molecular graphics system mohamed ehab and amira abdulfatah are appreciated for their help and discussions. key: cord-268326-sbz3uk5h authors: bonam, srinivasa reddy; wang, fengjuan; muller, sylviane title: lysosomes as a therapeutic target date: 2019-09-02 journal: nat rev drug discov doi: 10.1038/s41573-019-0036-1 sha: doc_id: 268326 cord_uid: sbz3uk5h lysosomes are membrane-bound organelles with roles in processes involved in degrading and recycling cellular waste, cellular signalling and energy metabolism. defects in genes encoding lysosomal proteins cause lysosomal storage disorders, in which enzyme replacement therapy has proved successful. growing evidence also implicates roles for lysosomal dysfunction in more common diseases including inflammatory and autoimmune disorders, neurodegenerative diseases, cancer and metabolic disorders. with a focus on lysosomal dysfunction in autoimmune disorders and neurodegenerative diseases — including lupus, rheumatoid arthritis, multiple sclerosis, alzheimer disease and parkinson disease — this review critically analyses progress and opportunities for therapeutically targeting lysosomal proteins and processes, particularly with small molecules and peptide drugs. discovered in the 1950s by christian de duve, lyso somes are membrane bound vesicles containing numerous hydrolytic enzymes that can break down biological polymers such as proteins, lipids, nucleic acids and polysaccharides 1,2 . lysosomes have long been known to have a key role in the degradation and recycling of extracellular material via endocytosis and phagocytosis, and intracellular material via autophagy (reviewed elsewhere 2-5 ) (fig. 1 ). the products of lyso somal degradation through these processes can be trafficked to the golgi apparatus for reuse or for release from the cell through lysosomal exocytosis, which is important in immune system processes. in addition, it has become clear more recently that lysosomes have an important role in other cellular processes including nutrient sensing and the control of energy metabolism 3,5-7 (fig. 1) . alterations in lysosomal functions, either in the fusion processes involved in the general pathways mentioned above or related to the function of lyso somal enzymes and non enzymatic proteins, can result in broad detrimental effects, including failure to clear potentially toxic cellular waste, inflammation, apopto sis and dysregulation of cellular signalling 8 . such defects have been implicated in many diseases, ranging from rare lysosomal storage disorders (lsds), which are caused by the dysfunction of particular lysosomal proteins, to more common autoimmune and neurodegenerative dis orders 5, 9, 10 . despite some limitations, impressive results have been achieved in treating several lsds through enzyme replacement therapy (ert). in addition, sub stantial efforts have been focused on therapeutically targeting the autophagy processes upstream of lyso somes [11] [12] [13] [14] . however, there has so far been less attention on investigating the potential to directly target lysosomes with small molecules and peptide drugs. nevertheless, with recent advances in understand ing of lysosomal function and dysfunction in dis eases, promising novel opportunities for therapeutic intervention through targeting lysosomes specifically are beginning to emerge. this review will provide a brief overview of lysosomal biogenesis, structure and function, and describe the role of lysosomal dysfunc tion in lsds as well as other, more common diseases. specifically, the article will focus on organ specific and non organspecific autoimmune diseases, including lupus, rheumatoid arthritis (ra) and multiple sclerosis (ms), as these have not been extensively reviewed elsewhere, but will also briefly highlight neurodegen erative disorders such as alzheimer disease (ad) and parkinson disease (pd), to further illustrate the breadth and nature of the emerging therapeutic opportunities. the current 'toolbox' of pharmacological agents that modulate lysosomal functions and emerging novel tar gets and strategies in this set of indications will be high lighted. it should be noted that therapeutic approaches to treat inflammatory and autoimmune diseases aim to inhibit the deleterious excessive lysosomal activity, whereas lysosomal activation would be the goal in the treatment of neurodegenerative diseases. although beyond the scope of this review, such approaches may have applications in other diseases in which lysosomes may play a role, including cancer, metabolic diseases and ageing (reviewed elsewhere 15, 16 ). the formation of mature lysosomes is a complex process, which involves the fusion of late endosomes that contain material taken up at the cell surface with transport ves icles that bud from the trans golgi network 5, 8, 17 . these vesicles contain nearly 60 different hydrolytic enzymes (grouped into nucleases, proteases, phosphatases, lipases, endocytosis a vesicle-mediated process by which cells engulf membrane and extracellular materials. several endocytic pathwaysphagocytosis, pinocytosis and receptor-mediated endocytosis -utilize different mechanisms to internalize material. clathrinmediated endocytosis is the major endocytic pathway in mammalian cells. fig. 1 | the central position of lysosomes at the crossroads of major autophagic pathways. a | functional lysosomes are involved in the degradation (endocytic and autophagic) and regulation of exogenous and endogenous cellular material, including recycling processes. extracellular material endocytosed by the endosomes and intracellular cargo internalized by the autophagosomes fuse with lysosomes for degradation, which produces energy (atp production) and source molecules for the macromolecules. mechanistic target of rapamycin complex 1 (mtorc1) plays a key role in lysosomal nutrient sensing signals (lysosome-to-nucleus axis) to regulate energy metabolism. factors such as energy levels, type of ph, ion channel regulation and others decide the fate of the catabolic process. during lysosomal exocytosis, the lysosomal content favours plasma membrane (pm) repair, bone resorption, immune response and elimination of pathogenic stores. b | the lysosome is the ultimate cell compartment that digests unwanted protein materials generated by macroautophagy , microautophagy (pathways during which the cytoplasmic material is trapped in the lysosome by a process of membrane invagination) and chaperone-mediated autophagy (cma). in general, lipid droplets (lds) are degraded by lipophagy , a subtype of macroautophagy , which is activated by cytosolic lipases. cma has also been demonstrated to participate in the degradation of lds in which perilipin (plin2/3) proteins are phosphorylated (p) by amp-activated protein kinase (ampk) with the help of the hspa8 chaperone. mechanistic target of rapamycin complex 2 (mtorc2) and akt (also known as protein kinase b) are negative regulators of cma , where they exert their effect on the translocation complex of cma. in situations of starvation, negative regulators are controlled by pleckstrin homology domain and leucine-rich repeat protein phosphatase (phlpp). lysosomal stability effects the transcription factor eb (tfeb) translation to the nucleus in which tfeb binds to the coordinated lysosomal expression and regulation (clear) motifs to regulate the transcription of genes. ef1a, elongation factor 1a; lys, lysosome; rac1, ras-related c3 botulinum toxin substrate 1. an endocytic process by which certain cells called phagocytes (for example, macrophages) internalize large particles (>0.5 µm) such as bacteria, other microorganisms, foreign particles or aged red blood cells, for example, to form a phagosome. a vital, finely-regulated and evolutionarily-conserved intracellular pathway that continuously degrades, recycles and clears unnecessary or dysfunctional cellular components. autophagy is crucial for cell adaptation to the environment and to maintain cell homeostasis, especially under stress conditions. cytosolic apparatus, meant for the regulation of proteins (modification, storing and transportation) and some forms of lipids to the other cytosolic compartments via the trans-golgi network or outside the cell. a process of the secretory pathway in which lysosomes are fused with the plasma membrane and empty their contents outside the cell. this process plays an important role in plasma membrane repair, bone resorption, immune response and elimination of pathogenic stores (mainly in lysosomal storage disorders). (lsds) . a group of heterogeneous disorders caused by defects in the lysosomal enzymes leading to the accumulation of unmodified or unprocessed components in the lysosomes, which ultimately influence other vital pathways in the cells. lsds implicate various vital systems of the human body including the skeleton, brain, skin, heart and central nervous system, which are connected with different metabolic pathways. sulfatases and others), which are synthesized in the endo plasmic reticulum and delivered to the transport vesicles via diverse systems, such as mannose6phosphate tags that are recognized by mannose6phosphate receptors (mprs) at the membrane 8, 18 or glucocerebrosidase (gcase) that is transported to lysosomes by lysosomal integral membrane protein2, an ubiquitously expressed type iii transmembrane glycoprotein mainly located in endosomes and lysosomes 19 . mature lysosomes have an acidic internal ph, at which the lysosomal hydrolases are active, and a lin ing known as a glycocalyx that protects the internal lysosomal perimeter from the acidic environment of the lumen 5, 8, 20 . this acidic environment is maintained through the activity of a vacuolar type proton adeno sine triphosphatase (v atpase), which harnesses energy from hydrolysing atp to drive the translocation of protons through a v 0 membrane domain (reviewed elsewhere 5, 21 ). other key lysosomal proteins include struc tural proteins such as lysosomeassociated membrane protein 1 (lamp1); proteins involved in trafficking and fusion, such as soluble n ethylmaleimidesensitive factor attachment protein receptors (snares) and rab gtpases; transporters such as lamp2a, which has a key role in chaperone-mediated autophagy (cma); and ion channels such as the chloride channel clc7 and the cation channel mucolipin 1, a member of the transient receptor potential (trp) family that is also known as trpml1 (refs 22, 23 ). most of the proteins are deliv ered through the clathrin adaptor protein 3alkaline phosphatase (alp) pathway, but some proteins are translocated through the lysosome associatedprotein transmembrane5, a protein that is preferentially expressed in immune cells 3, 24 . although the concept still remains controversial, two lysosome species -conventional or secretoryare often distinguished based on their physical, bio chemical and functional properties. catabolism is the main function of conventional lysosomes, and several other lysosome related organelles (lros), such as mel anosomes, the late endosomal major histocompatibility complex class ii (mhcii) compartment (miic), lytic granules from neutrophils, eosinophils, basophils, mast cells, cd8 + t cells and platelets, complement these functions 8, [25] [26] [27] [28] [29] . many of the lros act as professional secretory organelles. lros share with lysosomes the majority of typical characteristics (acidic environment, lysosomal transmembrane proteins, fusion property to phagosomes and others), in addition to particular properties resulting from their specific cargoes (for example, melanosomes contain melanosome specific transmembrane glycoprotein, and natural killer cells and cd8 + t cells contain perforins and granzymes). the detailed mechanisms of biogenesis and secretion of lros remain unclear, although it is known that genetic defects in lros are involved in rare autosomal recessive disorders characterized by reduced pigmentation, such as chediak-higashi disease and hermansky-pudlak syndrome 30 . secretory lysosomes contain many more proteins in addition to those contained in conventional lysosomes, and they participate in multiple cell func tions such as plasma membrane repair, tissue and bone regeneration, apoptotic cell death, cholesterol homeostasis, pathogen defence and cell signalling 8 . lysosomal biogenesis and function are regulated by the basic helix-loop-helix leucine zipper transcription factor eb (tfeb) and the coordinated lysosomal expres sion and regulation (clear) network 4,31,32 (fig. 2) . for example, autophagy, a crucial process in immunity and autoimmunity 33 , is transcriptionally regulated by tfeb 31 . interestingly, lysosomal exocytosis, which is important in many immune functions, also depends on tfeb activation 31, 32 . moreover, it has been demonstrated that tfeb orchestrates lysosomal ca 2+ signalling 34 . the fact that multiple lysosomal processes are dependent on tfeb activation strengthens its role as a master regulator in lysosomal functions. like other transcription factors, tfeb undergoes phosphorylation and dephosphory lation via different cytosolic and lysosomal pathways (fig. 2) , processes regulated by mechanistic target of rapamycin complex 1 (mtorc1), a master controller of cell growth 35, 36 . lysosomes are at the crossroads of various degrada tive pathways, including endocytosis (phagocytosis) and autophagy (fig. 1 ). three main forms of autophagy have been described: macroautophagy (the most extensively characterized form), microautophagy and cma. at the initiation of macroautophagy, a double membrane sequestering compartment termed the phagophore, which contains cytoplasmic material, is formed and matures into a vesicle called the autophagosome. the cargo is degraded into vacuoles issued from the fusion of autophagic vesicles and lysosomes (called autolyso somes), and the resulting short products are released back into the cytosol for reuse or, according to some times contested observations, possibly dispatched into the miic for ultimate processing and mhcii molecule loading for presentation to cd4 + t cells 37, 38 . in contrast to macroautophagy, microautophagy is characterized by direct lysosomal engulfment of cytosolic material into lysosomes, via the formation of characteristic invag inations of the lysosomal membrane. the third major form of autophagy is cma, which involves the recog nition of substrate proteins containing a kferq like motif by a hspa8/hsc70containing complex (fig. 1b) . in cma, two proteins have a key role: hspa8 ensures the selectivity of proteins, which will be degraded via the cma pathway; and lamp2a translocates the tar geted cytosolic proteins across the lysosomal mem brane (reviewed elsewhere 7 ). the terminal step of autophagy is called autophagic lysosome reformation, in which tubular proto lysosomes are extruded from autolysosomes (containing lysosomal membrane com ponents) and mature into functional lysosomes 39 . this step is not solely a lysosomal biogenesis process; it also includes a series of elements that are tightly correlated with the regulation of autophagy 40 . in combination with autophagy, lysosomes are involved in both innate and adaptive immune func tions, including foreign material recognition (bacterial, parasitic and viral), activation of pattern recognition receptors (such as toll like receptors (tlrs) and nucleo tide oligomerization domain like receptor), antigen processing and presentation, especially in the context multiple sclerosis (ms) . a demyelinating disease in which the myelin sheaths wrapped around nerve fibres in the central nervous system are progressively destroyed by immune cells and possibly also by autoantibodies. parkinson disease (pd) . a neurodegenerative disorder with symptoms including slowness of movement and a loss of fine motor control, owing to the degeneration of dopamineproducing neurons in the substantia nigra. (cma). a selective autophagy pathway in which proteins that contain a signal kferq-like sequence are targeted by hsap8/hsc70 chaperones and translocated into lysosomes via lamp2a. transcription factor eb (tfeb). a protein that plays a pivotal role in the regulation of basic cellular processes, such as lysosomal biogenesis and autophagy. it controls lysosomal function via the coordinated lysosomal expression and regulation (clear) gene network (including genes coding for hydrolases, lysosomal membrane proteins and the proton pump v-atpase complex), and additional lysosome-related processes such as autophagy, endocytosis and exocytosis. a finely-regulated process during which the cell forms a double-membrane sequestering compartment named the phagophore, which matures into the autophagosome. a double membrane-bound vesicle, which encloses cellular constituents and fuses with lysosomes to form phagolysosomes where the engulfed material is digested or degraded and either released extracellularly via exocytosis or released intracellularly to undergo further processing. of mhcii molecules, t cell homeostasis, antibody pro duction and induction of various immune signals (co stimulation and cytokine secretion) 41 . besides being a degradative organelle, the lysosome has recently been recognized as a cellular signalling platform 3, 42 . it plays an important role in nutrient sensing through mtorc1 and other additional protein complexes, or the so called 'lysosome nutrient sensing machinery' . the discovery of a stress induced lysosome tonucleus signalling mech anism through tfeb further supports the key role of lysosomes in cellular signalling 36 . the lysosome occupies a central position in the main tenance of cellular homeostasis, being involved in the exclusion of infectious agents from penetrating host tis sue and concomitantly promoting immune regulation. lysosomes must therefore be able to respond quickly, with increased or decreased functions, to various metabolic conditions aimed at protecting cells from death or damage. lysosomes are very diverse in size and shape. for reasons that are not totally understood -pos sibly according to their position in the cytosol 43 and/or their composition -some lysosomes in a single cell are more prone to act and defend cells. given the wide range of functions of lysosomes in all metabolic compartments of the cell, any dysregulation of their activity could lead to the impairment of various elements of the cellular metabolic machinery (including the transport and bio genesis of sugar (glycolysis), lipids, proteins and nucleic acids) and of metabolic pathways, phagocytosis, endo cytosis and autophagy. although the underlying mech anisms are far from being fully deciphered, it has been seen that lysosomal dysfunction or defects in fusion with vesicles containing cargo are commonly observed abnormalities in proteinopathic neurodegenerative dis eases. dysfunctions of lysosomes can affect the proper activity of other organelles such as peroxisomes and after their synthesis in the rough endoplasmic reticulum (rer), the substrates (cargo) that are intended to be degraded through the endo-lysosomal pathway are transported to lysosomes via the trans-golgi network (tgn). among the key enzymatic systems that are involved in the lysosomal enzyme transportation of cargos from golgi to lysosomes, the best studied is the mannose-6-phosphate (m6p) receptor (mpr) system, which binds newly synthesized lysosomal hydrolases in the tgn and delivers them to pre-lysosomal compartments. a few components synthesized in the late golgi compartment are delivered directly to lysosomes via the 3-alkaline phosphatase (alp) pathway. lysosomal components, such as enzymes (lytic enzymes and kinases), membrane-bound proteins/complexes (mechanistic target of rapamycin (mtor)), transporters and ion channels (vacuolar-type proton adenosine triphosphatase (v-atpase), trpml1 and osteopetrosis associated transmembrane protein 1 (ostm1)) and chaperone-mediated transportation are the best-known targeting sites for lysosomal dysfunction. as depicted in the figure, many pharmacological antagonists and agonists exert activities that potentially correct lysosomal dysfunction and therefore represent potential effective pharmacological tools. clear , coordinated lysosomal expression and regulation; cq, chloroquine; hcq, hydroxychloroquine; mtorc1, mtor complex 1; ptdins(3,5)p2, phosphatidylinositol-3,5-bisphosphate; raptor , regulatory-associated protein of mtor; ser , smooth endoplasmic reticulum; tfeb, transcription factor eb. www.nature.com/nrd mitochondria, leading to excessive production of reac tive oxygen species with pathological features associated with ageing, cancer, chronic inflammation, neurological diseases, male infertility and infections. such dysregulation is thus central to lsds, and also implicated in a wide range of other disorders, includ ing autoimmune and neurological disorders, in which the autophagy-lysosomal network under the control of tfeb has attracted considerable attention. lsds are a heterogeneous group of about 50 inherited metabolic disorders, which have an incidence of ~1 in 5,000 live births 44 . these disorders and their treatment have been reviewed extensively elsewhere 45, 46 , and so will only be covered relatively briefly here. the mutations responsible for most lsds have been largely elucidated (tables 1,2), and many result in the dysfunction of a particular lysosomal hydrolase, leading to the accumu lation of the substrate of that hydrolase. for example, in gaucher disease, the sphingolipid glucocerebroside accumulates in cells (particularly macrophages) and organs, including the liver and spleen, owing to defi ciency in the enzyme gcase 24, 66 . in certain lsds, the resultant pathology can be explained by the nature of molecules that accumulate (tables 1,2). thus, the abun dance of cerebrosides and gangliosides that deposit in the central nervous system (cns) of patients with sphingo lipid storage disorders, such as type ii (acute infantile neuronopathic) gaucher disease, underlies the severe neurological symptoms of such disorders 67, 68 . in patients with pompe disease, which is caused by α glucosidase deficiency, the high levels of non degraded glycogen that accumulate in muscles could explain the observed myopathy 69, 70 . however, how the undegraded material accumulates and causes the observed cellular and organ pathology in many other lsds remains unclear. the accumulation of such undigested macromol ecules or monomers in lsds instigates the formation of secondary products, which ultimately escape from the endosomal-autophagic-lysosomal pathways 9,71 and lead to multiple consequences that affect most organs, including the brain, liver, spleen, heart, eyes, muscles and bone (table 2) . most, if not all, organelles are altered in lsds, including endosomes, autophagosomes and lysosomes, and their functions in lysosomal formation/ reformation and fusion of endosomes or autophago somes to lysosomes are abnormal. alterations in several autophagy processes have also been described in lsds. thus, deregulated mitophagy, which results in the accu mulation of damaged mitochondria, occurs in lsds, leading to major inflammatory consequences in specific tissues 67, 72 . perturbations in mitochondrial dynamics are frequently observed, which have been linked to the increased production of reactive oxygen species, atp production and ca 2+ imbalance. in lsds, reduced macro autophagy activity (with a decreased autophagic flux) rather than hyperactive autophagy processes, as seen in numerous autoimmune diseases, seems to be responsible for the accumulation of non degraded cytoplasmic pro teins such as α synuclein, huntingtin (htt) and others 73 . mucolipidosis type iv (table 2) , a disease characterized by severe neurological and ophthalmological abnormal ities, is caused by mutations in the mcoln1 gene and is inherited in an autosomal recessive manner. this gene encodes a non selective cation channel, mucolipin 1, which has recently been shown to be required for effi cient fusion of both late endosomes and autophagosomes with lysosomes 74, 75 . impaired autophagosome degrada tion results in the accumulation of autophagosomes in lsds 76 . microautophagy processes that do not involve de novo synthesis of nascent vacuoles also appear to be impaired in lsds, and were notably revealed in primary myoblasts from patients with the muscle wasting con dition pompe disease 77 . finally, defective cma compo nents, such as lamp2a, could also lead to lysosomal dysfunction. for example, mutations in the lamp2 gene have been claimed to cause danon disease (inherited in an x linked dominant pattern) 51 . further investigations are needed to support this assertion. lysosomes are involved in pathways central to the immune system, including the degradation of intra cellular and extracellular material, plasma membrane repair, cell death signalling, cell homeostasis and death. although the direct involvement of lysosomes in immunity is far from fully understood, it has long been expected that lysosome dysfunction will have a major impact in immune diseases (table 2) . strikingly, however, this field has not been extensively explored. however, elevated levels of lysosomal enzyme activity have been reported to occur in several autoimmune diseases, such as ra, systemic lupus erythematosus (sle), dermatomyositis and psoriasis 3, 14, 17, 18, [20] [21] [22] [23] . as discussed, autophagosomes formed during the autophagy process must fuse with lysosomes to generate mitophagy a key process that selectively disrupts damaged mitochondria by autolysosomal degradation, preventing excessive reactive oxygen species and activation of cell death. (htt). discovered in 1993, htt is a protein of 348 kda that is widely expressed within the central nervous system. its structure has been elucidated recently by cryo-electron microscopy. the protein is essential for embryonic development and neurogenesis. it is involved in transcription, vesicle transport, protein trafficking, endocytosis and autophagy. (sle). a chronic, relapsingremitting autoinflammatory syndrome that has multiple and heterogeneous symptoms, including arthralgia, swollen joints, fever, fatigue, chest pain, kidney inflammation, cardiovascular disease and neuropsychiatric complications. its aetiology is mostly unknown. reduction of α-d-mannosidases causes reduced lysosomal breakdown of mannose-based oligosaccharides in many tissues 47 inherited lsd characterized by immune deficiency (susceptibility to infections including pulmonary infections), facial and skeletal abnormalities, hearing impairment and intellectual deficit 47 fabry disease α-galactosidase reduced lysosomal metabolism of α-galactosyl lipids, globotriaosylceramides, causes vascular diseases (cardio, cerebro and renal diseases) in patients 45, 46 gaucher disease (types 1, 2 and 3) β-gcase accumulation of glucosylceramides in leukocytes (especially in macrophages) leads to abnormalities in the visceral organs (type 1) and neurological defects in both children and adults (types 2 and 3) 45, 46 gm1 gangliosidosis β-galactosidase abnormal lysosomal storage of gm1-ganglioside (oligosaccharides) causes skeletal manifestations and neurological impairment in humans 45, 46 krabbe disease (globoid cell leukodystrophy) defects in the galactocerebrosidase provoke accumulation of galactosylceramide and galactosylsphingosine (psychosine). patients' brain histology shows myelin loss, neuroinflammation and axonal degeneration 48 arylsulfatase a or saposin-b (activator protein; rare cases) defects in the enzymes lead to the accumulation of sulfogalactosylceramide in major organs. it affects the different age groups of humans with development signs and symptoms of the disease 45, 46 mucopolysaccharidoses enzymes involved in mucopolysaccharide catabolism accumulation of mucopolysaccharides within lysosomes leads to skeletal and joint abnormalities in humans 45, 46 multiple sulfatase deficiency sumf1 (formylglycinegenerating enzyme needed to activate sulfatases) abnormal accumulation of multiple, including sulfated, glycosaminoglycans causes neurodegeneration and psychomotor retardation in humans 49 pompe disease α-glucosidase accumulated undegraded glycogen in the muscles and peripheral nerves was observed in humans 45, 46 sandhoff disease β-hexosaminidase a and b enzyme defects cause gm2-ganglioside accumulation in lysosomes, which induces nervous system damage in humans 45, 46 mucolipidosis (type ii and iii) n-acetyl glucosamine phosphoryl transferase α/β enzyme deficiency results in accumulation of unphosphorylated glycoproteins, which causes motor function and neurological disorders in humans 45 mucolipidosis iv mucolipin-i defects in this lysosomal membrane protein (ca 2+ channel) cause accumulation of mucopolysaccharides and lipids, thereby resulting in hepatosplenomegaly , dysmorphic features and neurological disorders in humans 45 cystinosis cystinosin (cysteine transporter) defects in this lysosomal transporter, cystinosin, cause accumulation of cystine in different organs, first in kidneys and later in other organs in humans 45, 46 danon disease l amp2 defects in l amp2 (especially l amp2b) cause accumulation of glycogen and other autophagic components in cardiomyocytes of humans, which results in cardiac diseases 50 l amp2b is highly expressed in the brain, cardiac and skeletal muscles 51 defective autophagy processes observed in sgs of mrl/lpr mice 56 crohn's disease abnormal lysosomal ph deregulation of proton-sensing g protein-coupled receptor (gpr65) was observed in both mice and human 57 www.nature.com/nrd peptide epitopes for further processing, clear possibly deleterious apoptotic debris, fuel the amino acid pool and produce energy (fig. 1 ). any deviation in this com plex processing will affect crucial immune cell functions, such as the control of cytokine release, autoimmune cell anergy and programmed cell death of type i (apop tosis) and type ii (autophagy). secretory lysosomes regulate the release of both pro inflammatory and anti inflammatory cytokines, in a process that is depen dent on the type of stimulation. in addition, lysosomes degrade glucocorticoid receptors, which are essential to bind glucocorticoids, although the reasons are not known 78 . in this complex system, lysosomes execute anti inflammatory action via the phospholipase a2 and cyclooxygenase2 pathways, and also induce inflam mation through the il1β-caspase1 pathway. in both conditions (pro inflammatory and anti inflammatory), lysosomes act as indirect precursors for autoimmunity. however, induction and suppression of inflammatory signals are stimulus dependent 78 . lysosomal cathepsins have a central role in degrading biological macromolecules in the lysosomes and in the immune response. there are approximately 12 members in this large protease family, most of which are endo peptidases that can cleave peptide bonds of their protein substrates 79, 80 . cathepsins a and g are serine proteases, cathepsins d and e are aspartic proteases and cathep sins b, c, f, h, k, l, o, s, v, x and w are cysteine pro teases. for example, cathepsin s is responsible for the degradation of antigens (and autoantigens) in antigen presenting cells (dendritic cells, macrophages and b cells), and is therefore involved at an upstream level in the presentation of mhcii-(auto)antigenic peptide complexes to cd4 + t cells 81 . cathepsin l preferentially cleaves peptide bonds with aromatic residues in the p2 position and hydrophobic residues in the p3 position. it is central in antigen processing, bone resorption, tumour invasion and metastasis, and turnover of intra cellular and secreted proteins involved in growth regu lation. cathepsin l deficient mice display less adipose tissue, lower serum glucose and insulin levels, more insulin receptor subunits, more glucose transporter type 4 and more fibronectin than wild type controls 82 . cathepsin g is primarily known for its function in killing and digestion of engulfed pathogens 83 . it is also involved in connective tissue remodelling at sites of inflammation 84 . anti neutrophil cytoplasmic antibodies reacting with cathepsin g have been identified in some patients with sle 85 . abnormal antigen processing and presentation is known to be one of the upstream events that perturb immune responses in sle 86 . because this process is mediated through lysosomes, it was rational to speculate that lyso somal functions could be altered in lupus. interestingly, hypotheses were raised in the 1960s on the 'lysosomal fragility' in lupus, but without much further pursuit 87 . the composition and fluidity of the lysosomal mem brane are effectively crucial in the regulation of lyso somal fusion with other vesicular organelles and for lysosomal uptake of macromolecules. the integrity of the lysosomal membrane also ensures the prevention of release of lysosomal enzymes into the cytoplasm. some lysosomal enzymes released from 'fragile' lysosomes were regarded potentially harmful in lupus 88 . autoimmune diseases (cont.) lysosomes are abnormal in splenic b cells from fas deficient murphy roths large (mrl)/lpr mice, a mouse model of lupus, compared with b cells from healthy cba/j mice 89 . tfeb expression was increased, indicating an enhanced biogenesis of lysosomes, and the lysosomal volume was raised. the expression levels of lamp1 and cathepsin d were also increased. these results reinforce previous data showing that the expres sion and activity of some lysosomal enzymes (such as cathepsins s, l and b) that play important roles in antigen processing are altered in lupus and other autoimmune diseases 90, 91 . substantial variations of the acidic endo lysosomal ph also occur in mrl/lpr mice, being raised by 2 ph units in splenic b cells 53, 92 . this ph change could dramat ically influence the activity of soluble lysosomal hydro lases (such as cathepsins) as well as lysosomal membrane proteins (such as lamps) that are critical for lysosome activity. ph may also affect the elimination of immune complexes that accumulate in lupus as a result of deficits in complement, lower expression of scavenger receptors, increased expression of fcγ receptors and other rea sons 93 . these immune complexes, which contain non selective igg antibodies or autoantibodies associated with autoantigen (including some apoptotic debris), can initiate inflammation of tissues once deposited (for example, in the kidneys and the skin) and generate a cas cade of deleterious effects, such as the release of harmful cytokines and chemokines 54 . recent studies have highlighted the key role of mam malian target of rapamycin complex 2 (mtorc2) in the disruption of lysosome acidification that occurs in this process 94 . in normal conditions, the regulation of lyso somal acidification requires cleavage of the rab small gtpase rab39a, occurring on the surface of phago cytic vesicles by locally activated caspase1 94 . this finely regu lated process requires the association of cofilin with actin that surrounds the vesicle and recruits caspase11, which then activates caspase1 (ref. 94 ). in lupus prone macro phages, chronically active mtorc2 enhances cofilin phosphorylation, thereby hampering its association with actin and affecting the downstream cascade of events leading to the appropriate acidification of lysosomes 94 . the importance of mtorc1 and mtorc2 has been established earlier in lupus t cells, and in particular, in this context, mtorc1 activity was increased whereas mtorc2 activity was reduced 95 . in addition, lysosomal cathepsin k was seen to contrib ute to the pathological events that develop in fas lpr mice, another model of lupus disease, in part through its activity in tlr7 proteolytic processing and subsequent effects on regulatory t cells. cathepsin k deficiency in fas lpr mice reduced all kidney pathological manifesta tions (glomerulus and tubulointerstitial scores, glo merulus complement c3 fraction and igg deposition, chemokine expression and macrophage infiltration) and decreased the levels of potentially pathogenic serum autoantibodies 96 . in line with these internal alterations of lysosomes, notably those related to cathepsin functioning, deregu lation of autophagy has been reported to contribute to lupus pathology 92,97-100 . autophagy failures have been described in the lymphocytes of mrl/lpr mice and (nzbxnzw)f1 mice 56, 92, 97, 101 (two spontaneous murine models of systemic autoimmunity of distinct genetic ori gins and that display different mhc haplotypes) as well as in t and b lymphocytes of patients with sle 97,98,100 . murine and human t cells from the peripheral blood showed a significant accumulation of autophagic vac uoles compared with normal 97 . the underlying reasons for the dysfunctions in autophagy observed in lupus are not clearly understood, but several indepen dent investigations have identified risk loci spanning autophagy linked genes in patients with lupus 102-106 . recent studies have demonstrated an increase in the level of macroautophagy in salivary gland t lymphocytes and in tears and conjunctival epithelial cells of patients with primary sjögren's syndrome (sjs) 107, 108 . alteration of cma activity was also recently found to occur in the salivary glands of mrl/lpr mice that develop a second ary sjs like disease 56 . lysosomes, which as discussed are mechanistically involved at the downstream level of both macroautophagy and cma, were found to be altered in salivary glands. flow cytometry analyses revealed that the mean ph of acidic vesicles in mrl/ lpr salivary glands was significantly higher compared with those in mouse control glands and the atp con tent was significantly diminished in mrl/lpr salivary gland cells 56 . furthermore, amounts of several leukocyte glycosidases and proteases were revealed to be increased in leukocytes of patients with sjs in comparison with healthy controls 55 . notably, raised levels of the lyso somal enzymes glucosidase, β glucuronidase and dipeptidyl peptidase i are involved in the tissue injury in sjs 55 . increased expression of lacrimal gland cathepsin s was also reported, which may have application as a diag nostic tool in sjs 91 . two members of the ras oncogene family, rab3d and rab27, were found to be implicated in the regulation of cathepsin s secretion levels in sjs 109 . in vitro studies on lacrimal gland acinar cells suggested further that secreted ifnγ from acinar cells increases cathepsin s expression and that ifnγ stimulated the mhcii mediated antigen presentation in ocular pathogenesis of sjs 110 . lysosomal cathepsins have important roles in the induc tion and diagnosis of ra, and levels of several cathepsins (b, d, g, k, l and s) that are present in the serum and synovial fluid of patients have been proposed as a basis for ra diagnosis [111] [112] [113] [114] [115] [116] . cathepsin s and cathepsin l are highly expressed in synovial macrophages and thymic cortical cells. they each exert essential roles in the posi tive selection of t cells and antigen presentation, respec tively, and participate in the local inflammation and matrix degradation that occurs in joints 116 . cathepsin b is involved in collagen degradation, which leads to joint destruction in ra 112, 117 . expression of cathepsin g, which participates in joint inflammation through its chemoattractant activity, has been shown to be raised in the synovial fluid of patients with ra when compared with individuals with osteoarthritis 115 . autoantibodies www.nature.com/nrd reacting with cathepsin g were also identified in patients with ra 85 . compared with patients with osteoarthritis, cathepsin k expression was found to be elevated in ra 113 , and genetic deletion of this particular cathepsin was shown to reduce inflammation and bone erosion in ra conditions via tlr mediation 118 . neurological autoimmune diseases ms, myasthenia gravis, guillain-barré syndrome, chronic inflammatory demyelinating polyneuropathy (cidp), neuromyelitis optica and neuropsychiatric lupus are neurological diseases induced by abnormal auto immunity 62, [119] [120] [121] [122] [123] . neurological autoimmunity against various proteins, such as myelin in ms or n methyl d aspartate receptor in neuropsychiatric lupus 62, 123, 124 , can affect various structures within the cns and peripheral nervous system, with diverse consequences. although the exact cause of amyotrophic lateral sclerosis (als) still remains unknown, studies support the existence of auto immune mechanisms, and als is therefore also included in this section. indeed, autoantibodies against ganglio side gm1 and gd1a, sulfoglucuronylparagloboside, neurofilament proteins, fas/cd95 and voltage gated ca 2+ channels have all been reported in patients with als (reviewed elsewhere 125 ) . in general, the origin of the breakdown in immune tolerance that occurs in this set of neurological diseases is not known. only recently have investigations discov ered that autophagy processes are altered in some of these diseases 59, 62, [126] [127] [128] [129] [130] . in ms and in experimental auto immune encephalomyelitis, an experimental model of ms, upregulation of the protein kinase mtor has been described, and treatment with rapamycin/sirolimus (an immunosuppressant that inhibits mtor and conse quently stimulates macroautophagy) ameliorates some clinical and histological signs of the disease 131 . increased levels of macroautophagy markers were measured in the blood and brain of patients with ms 122,132 . however, impaired macroautophagy was found in the spinal cord of experimental autoimmune encephalomyelitis mice 133 . in a rat model mimicking human cidp, both macro autophagy and cma processes were found to be hyper activated in lymphatic system cells and non neuronal cells (sciatic nerves) of peripheral nervous system cells 59 . in als, current data are conflicted 62 . some data suggest an activation of macroautophagy processes with an accumulation of autophagosomes in brain tissues of patients with als, or an increase of autophagic vacuoles, aggregated ubiquitin and sod1 proteins associated with map1lc3b ii in motor neurons of mice developing an als like disease 134, 135 . in contrast, other data suggest a reduction of autophagy activity 136, 137 . mutations in sqstm1, valosin containing protein, dynactin (a pro tein complex that activates the dynein motor protein, enabling intracellular transport) and rab7 (a member of small gtpases that is important in the process of endosomes and autophagosomes maturation) have also been described in als [138] [139] [140] [141] . further studies are required to better understand the type and extent of autophagy dysfunction in this family of complex diseases. there are only a few published studies on lyso somal dys function in neurological autoimmune diseases (table 2) . these notably include lysosome fragility, which was observed in patients with ms in the white matter of cerebral tissue, an area of the cns that is mainly made up of myelinated axons 142 . lysosome fragility was also suspected in sle (see above) and other rheumatic auto immune diseases, albeit in other organs 53, 58, 92 . as noted above, significant variations in lysosomal ph have been measured in autoimmune conditions such as lupus and sjs, but to our knowledge such studies conducted in the brain or elements of the peripheral nervous system of patients or animal models with neurological autoimmune diseases have not been published 78 . in cidp, it has been shown that schwann cells dedif ferentiate into immature states and that these dediffer entiated cells activate lysosomal and proteasomal protein degradation systems 143, 144 . based on these observations, schwann cells have been claimed to actively participate in demyelinating processes via this dedifferentiation process, but the mechanism involved remains unde fined 145 . in the rat model of cidp mentioned above, it was shown that lamp2a expression was drastically increased in the sciatic nerve macrophages and reduced macroautophagy was observed in schwann cells and macrophages 59 . in ms, studies conducted on white matter demon strated that lysosomes are involved in myelin sheath degeneration as well as in the fragmented protein forma tion. lysosomal swelling was observed near the degen erated materials of astrocytes 146 , and an accumulation of lipids was found 60 . it has been hypothesized that lyso somal swelling/permeabilization might cause the release of hydrolases in the cytosol, where they affect native proteins 147 . in als, patients also show dysfunctions in the endo/ lysosomal pathways, which affect both lower and upper motor neurons (table 2) . cathepsin b was particularly found to be involved in the motor neuron degeneration, whereas cathepsins h, l and d were not significantly affected 148 . a cdna microarray analysis on post mortem spinal cord specimens of four sporadic patients with als revealed major changes in the expression of mrna in 60 genes including increased expression of cathepsins b and d 149 . several disease causing mutations in genes related to autophagy have been identified, such as sod1, tdp643, fus, ubqln2, optn, sqstm1 and c9orf72 (refs 61,150 ), but none of them code for lyso somal proteins. so, a crucial remaining issue is to clearly determine whether the lysosomal abnormalities that are observed are linked to intrinsic defaults of lysosomes or result from upstream dysregulation in autophagosome formation and fusion 61, 62, 151 . insufficient clearance of neurotoxic proteins by the autophagy-lysosomal network has been implicated in numerous neurodegenerative disorders 152 . in disorders such as ad, huntington disease (hd) and pd, modi fied or misfolded proteins abnormally accumulate in specific regions of the brain. accumulation of aggregated proteins is also seen in als (see above). these abnor mal proteins form deposits in intracellular inclusions or extracellular aggregates, which are characteristic for caused by antibodies targeting the muscle acetylcholine receptor or other neuromuscular junction proteins such as musclespecific kinase. these antibodies compromise communication between nerves and muscles, leading to muscular weakness and fatigue. chronic inflammatory demyelinating polyneuropathy (cidp). a progressive autoimmune disorder in which peripheral nerves (roots and trunks) and brachial plexuses are damaged owing to demyelination. it causes muscle weakness, sensory loss and reduced reflexes. also known as devic's syndrome, this disease is characterized by an inflammation and demyelination of the optic nerve (optic neuritis) and the spinal cord (myelitis). antibodies reacting with aquaporin-4 water channels in the brains of patients are implicated in neuromyelitis optica. (als). also known as motor neuron disease, this disease generally starts with muscle twitching and weakness in a limb, or slurred speech. it can affect control of the muscles needed to move, speak, eat and breathe, and can be fatal. nature reviews | drug discovery each disease [153] [154] [155] . although there has been substantial research in this field, it is still unclear why sophisti cated 'quality control' systems, such as the lysosomeautophagosome system in particular, fail in certain cir cumstances to protect the brain against such protein accumulation 156 . in ad, one of the most common neurodegenerative disorders, some alterations in the endo/lysosomal path ways have been described (reviewed elsewhere 157, 158 ). the amyloid precursor protein (app) is cleaved by β and γ secretases into amyloid β peptide (aβ) fragments, particularly aβ40 and aβ42 (ref. 159 ). these fragments are found in the amyloid plaques that are one of the hallmarks of ad (the other being neurofibrillary tangles containing phosphorylated tau), and have been widely considered to have an important role in ad pathogen esis 159, 160 . cell based experiments have demonstrated that lysosomal cathepsins have a role in the generation of aβ peptides (through cathepsins d and e) and the degrada tion of aβ peptides (by cathepsin b) 161 . lysosomal dys function has been observed in patients with ad 162, 163 , and accumulation of the aβ42 fragment in neuronal cells was shown to lead to lysosomal membrane alter ations, which cause neuronal cell death 63 . in this context, it is noteworthy that inhibition of cathepsin d, which is involved in the lysosomal dysfunction and notably in the cleavage of the tau protein into tangle like fragments, diminishes its hyperphosphorylation in the brain of patients with ad 164 . in addition, patients with ad with an inherited form of the disease may carry mutations in the presenilin proteins (psen1 and psen2), app or apolipoprotein e, resulting in increased production of the longer form of the aβ fragment (reviewed else where 165 ). mutation of psen1, for instance, leads to direct disruption of the lysosomal acidification due to impaired delivery of the v0a1 subunit of v atpase, a proton pump responsible for controlling the intracellu lar and extracellular ph of cells. the acidification defi cit causes excessive release of lysosomal ca 2+ through trpml1 channels, which has numerous deleterious effects 166 . these findings strongly support the hypothesis that dysfunction of endo/lysosomal pathways is pivotal in ad. approximately 15% of patients with pd have a family history of the disorder, although the underlying molec ular mechanisms remain unclear. in the context of lysosomal dysfunction, it is notable that the most com mon of the known pd genetic mutations are in gba1 (encoding the lysosomal β gcase) -the same gene that underlies gaucher disease -which are present in up to 10% of patients with pd in the united states 167 . gba1 mutations are also associated with dementia with lewy bodies 167 . several other genes linked to pd are directly or indirectly related to the endo/lysosomal machinery, such as mutations in snca (coding for α synuclein) 63, 168 . a hallmark of pd is the presence in neurons of protein inclusions called lewy bodies, which are mainly com posed of fibrillar α synuclein. the α synuclein protein is normally degraded by the lysosomes through the cma pathway, but macro aggregates of α synuclein mutants, which display a longer half life compared with the non aggregated wild type protein, are not degraded by this pathway and, rather, would be degraded via the macro autophagy pathway [169] [170] [171] [172] . it was further shown that the mutant proteins bind to lamp2a and inhibit the translocation of other substrates and, therefore, their final degradation 170 . biochemical analyses suggest that α synuclein is mainly degraded by lysosomal proteases and notably by cathepsin d, rather than by non lysosomal proteases (for example, calpain i) 173, 174 . accumulation of α synuclein was observed in cathepsin d deficient mice, whereas, conversely, the accumulation of α synuclein aggregates was reduced in transgenic mice that over expressed this cathepsin, resulting in protection of dopaminergic neuronal cells from damage 175 . hd is a rare autosomal dominant neurodegener ative disease caused by an aberrant expansion of cag trinucleotide repeats within exon 1 of the htt gene, which results in the production of aggregation prone htt mutants (mhtt) that are detrimental to neu rons 176, 177 . whereas htt has a protective role against neuronal apoptosis, accumulation of mhtt, however, induces pathophysiological consequences including lysosomal and autophagy dysfunctions. thus, mhtt perturbs post golgi trafficking to lysosomal compart ments by delocalizing the optineurin/rab8 complex, which, in turn, affects lysosomal function 177 . excessive mhtt induces accumulation of clathrin adaptor com plex 1 in the golgi and an increase of clathrin coated vesicles in the vicinity of golgi cisternae 177 . the activity of several cathepsins such as b, d, e, l and z has also been linked to hd 63, 80, 174, [177] [178] [179] . cathepsin d is respon sible for full degradation of htt but is less efficient at degrading mhtt, which is processed by cathepsin l 180, 181 . cathepsin z also cleaves htt and elongated poly glutamine tracts 182, 183 . thus, lysosomal modulators acting on cathepsin activity might have beneficial effects in the treatment of hd. notably, hyperexpression of cathepsin d (and cathepsin b) was shown to protect primary neurons against mhtt toxicity 179 . alterations in macro autophagy, mitophagy and cma have also been implicated in hd 184, 185 . cma activity was increased in response to macroautophagy failure in the early stages of hd 186 , a result supported by the findings that hspa8 and lamp2a have important roles in the clearance of htt 187 and that shrna mediated silencing of lamp2a increased the aggregation of mhtt 188 . other studies focusing on the htt secretory pathway revealed that mhtt secretion is mediated by the ca 2+ dependent lyso somal exostosis mechanism via the synaptotagmin 7 sensor in neuro2a cells 189 . the extracellular release of mhtt was efficiently inhibited by the phosphoinositide 3 kinase and sphingomyelinase inhibitors ly294002 and gw4869. hd dependent perinuclear localization of lysosomes was also demonstrated 190 . increasing evidence thus implicates lysosomal (and autophagy) dysfunction in the pathogenesis of neuro degenerative disorders 62, 63, 127, 128, 130, 191, 192 . tfeb has received particular attention in this regard [193] [194] [195] , with recent data suggesting that tfeb is selectively lost in patients with ad (as well as als) 196 . increasing tfeb activity might therefore prevent neuronal death and restore neuro nal function in certain neurodegenerative diseases, including pd 194 . a major microtubuleassociated protein of a mature neuron. hyperphosphorylated tau accumulates with ubiquitin in ageing neurons as the neurofibrillary tangles that were identified in numerous neurodegenerative diseases called tauopathies that include alzheimer disease. lysosomes as therapeutic targets given the evidence discussed above, the various lyso somal pathways and their components could represent potential pharmacological targets for a wide range of diseases. when considering lysosomes as targets, it is important to note the need for specificity; that is, agents that will not target all lysosomes, but will specifically tar get those lysosomes/lysosomal proteins that are defective in certain organs, tissues or cells. in addition, inhibitors or activators of lysosomal components may be required, depending on the disease context. there has been considerable interest in therapeuti cally targeting different autophagy pathways, including lysosome dependent pathways, and progress in the discovery and development of small molecules and biologics that target these processes has been reviewed extensively 11, [119] [120] [121] [122] 197, 198 . however, very few therapies that specifically target lysosomal components have so far been generated and found to be effective in clinical trials, with one general exception -the development of erts and small molecule drugs for lsds (box 1). this topic has recently been comprehensively reviewed 46 and so will not be discussed in depth here. it is important to target lysosomes and not the whole autophagy process for several reasons. first, regard ing safety, the integral role of lysosomes in several key physio logical processes means that therapeutic windows for pharmacological intervention with unacceptable side effects may be limited. for example, azithromycin, an antibiotic with anti inflammatory properties that is used in the treatment of patients with chronic inflammatory lung diseases such as cystic fibrosis, was found to block autophagy in macrophages, inhibiting intracellular kill ing of mycobacteria within them and, thereby, increasing the risk of mycobacterial infection 204 . second, in some diseases, autophagy may be enhanced in certain tissues or organs but compromised in others, for example in the spleen and salivary glands of mrl/lpr mice 56 . this phe nomenon makes it highly challenging to identify a single drug able to correct a failure, unless a cell specific target ing molecule could be incorporated into the autophagy activator/inhibitor to enable tissue specificity 205 . again, the precise targeting of lysosomes in specialized cells may circumvent the complexity of dysregulation mecha nisms of autophagy processes in pathophysiological settings 14, 56, 206, 207 . as indicated, the current arsenal of lysosome specific targeted drugs is small. in fact, many drugs claimed to target lysosomal components have also been found to be capable of interacting with several non lysosomal receptors, limiting their efficacy and safety 12 . one exam ple is provided by chloroquine (cq), a 4aminoquino line compound, and its derivative hydroxychloroquine (hcq), which are widely prescribed to patients with rheumatic diseases, and historically also for the prophy laxis and treatment of malaria (fig. 3) . cq and hcq are lysosomotropic agents and as such they raise intralyso somal ph, thereby affecting overall lysosomal function and impairing autophagic protein degradation (fig. 2) . although the mechanism of action of these agents is not fully elucidated, it is well established that cq and hcq display pleiotropic activity [208] [209] [210] and have impor tant deleterious properties. in certain settings, they have been claimed to operate by interacting directly with tlr ligands and not through an effect on the lysosomal ph, for example 211 . toxicity of cq/hcq, in particular in the eye (cornea and macula) and the occurrence of cardio myopathies 212 , remains a major limitation. the observed ocular toxicity is related to the total cumulative dose rather than the daily dose; therefore, it becomes a serious potential problem in the cases of long term use. several hcq analogues and mimics have been designed that aim to retain the therapeutic activity without secondary effects 213, 214 . furthermore, most, if not all, of the small molecules that have so far been identified and investigated as mod ulators of autophagy and/or lysosomal functions exhibit complex pleiotropic properties affecting the overall function of lysosomes, and also different autophagy pathways (for example, mtor dependent and mtor independent pathways), as well as other quality control mechanisms that affect the cell life/death balance. as dis cussed below, several widely used molecules exert dual, sometimes opposite, effects on upstream and down stream molecular events of the autophagy-lysosomal network. several robust assays to characterize autophagy acti vators and inhibitors, as well as lysosomal effectors, are currently available and validated (table 3) . however, each assay has inherent biases, and so it is necessary to use several independent, in vitro and in vivo approaches to ascertain the reactivity and specificity of novel molecules able to modulate these pathways (box 2). in this regard, the tremendous work in recent years to establish international guidelines for standardizing research in autophagy -and, in particular, to propose relevant methodologies for monitoring autophagy that are accepted by the whole community -is unique 231, 232 . a better definition of terms and concepts has also box 1 | enzyme replacement therapies for lysosomal storage disorders enzyme replacement therapy (ert) for lysosomal storage disorders (lsd) involves administration of a functional version of the defective enzyme in the particular lsd. following administration, the enzyme is delivered to the target cells (typically mediated by mannose or mannose-6-phosphate receptors), where it breaks down its substrate in lysosomes, thereby ameliorating the lsd 46 . the approach was pioneered with the use of glucocerebrosidase (gcase) purified from placentae in the 1980s to treat patients with gaucher disease, and a recombinant version of gcase was then introduced in the 1990s 199 . following the success of this approach in treating gaucher disease, other recombinant enzymes have been approved for other lsds, including fabry disease, mucopolysaccharidosis (mps) i, mps ii, mps vi and pompe disease (table 1) , and many further erts for other lsds are in clinical trials 200 . although ert has provided an effective treatment for patients with some lsds, it has limitations. recombinant enzymes administered by intravenous injection are not able to cross the blood-brain barrier, and so are not effective for central nervous system manifestations of lsds 201 . low expression of the receptors that mediate delivery on the cell surface of target cells can also be a challenge for the effectiveness of ert for some lsds 46 . for example, in pompe disease, the level of expression of mannose receptors on skeletal muscle cells is low, necessitating high doses of ert to achieve a therapeutic effect 202 . numerous developments are being studied to address such limitations, with a focus on enzyme modifications that enable better access of enzymes to their receptors and on nanomaterials that enable safe and efficient delivery of enzymes via intra-cerebroventricular/intrathecal administration 10, 46, 200, 203 . been adopted by the community, leading to much eas ier understanding between researchers worldwide 233 . these guidelines and definitions should be used by investigators evaluating new molecules designed to selectively target key steps of autophagy or developing new high throughput screening methods for autophagy modulating pharmacological molecules. however, even the more sophisticated and detailed assays will not reca pitulate the full complexity of integrated living systems, which can only be established in clinical trials. the pipeline of specific agonists and antagonists of autophagic activity is currently small, particularly for cma (tables 4,5; figs 2,3). however, high throughput screening programmes to identify such small molecules are ongoing, which should yield additional therapeutic targets and useful tools. small molecules that specifically target lysosomes are even rarer (table 4; fig. 2 ). small molecule drugs developed specifically for particular lsds, including substrate reduction therapies and small molecule chaperones, have reached the market, but other small molecule candidates for more common diseases are at an earlier stage of development. these molecules that more specifically act on lysosomes, some of which have been discovered by high throughput screening, mostly target lamp2a, various lysosomal enzymes such as cathepsins, acid sphingomyelinase, α galactosidase a and acid β glucocerebrosidase, and chaperones such as hspa8 and β nacetyl hexosaminidase. although not solely present in lysosomes, v atpase, a proton pump responsible for controlling the intracellular and extracel lular ph of cells, and trpml1, a cation channel located within endosomal and lysosomal membranes, are also pertinent targets. below and in table 4, we summarize the availabil ity of pharmacological tool compounds and progress in drug development, where applicable, for each broad target class. in addition to erts for lsds (box 1), drug discovery programmes have also focused on alter native small molecule based approaches, which may be particularly relevant for lsds that affect the cns, due to the lack of blood-brain barrier penetration by erts 283 . small molecules used in substrate reduction thera pies prevent the accumulation of substrates of the defec tive enzymes in lsds by inhibiting enzymes involved in substrate production 284 . miglustat was the first such drug to be approved in the early 2000s by the us food and drug administration and the european medicines agency for gaucher disease and in 2009 for niemann-pick disease type c in europe. this iminosugar inhibits glucosylceramide synthase (gcs), which catalyses the initial step in formation of many glycosphingolipids. within cells, glycosphingolipids tend to localize to the outer leaflet of the plasma membrane; they cycle within the cell through endocytic pathways that involve the lysosome. inhibition of gcs therefore reduces the deleterious accumulation of glycosphingolipids within lysosomes with potential therapeutic benefits in dis eases like lsds. miglustat also inhibits disaccharidases in the gastrointestinal tract, resulting in diarrhoea as a side effect 285 . eliglustat, another gcs inhibitor that does not penetrate the cns, was also approved for gaucher disease in 2014. other gcs inhibitors in clinical devel opment include lucerastat, a miglustat analogue with an improved safety profile that is currently in a phase iii trial for fabry disease (fd) 236, 286 , and ibiglustat, which penetrates the cns. the latter is in clinical development for fd (phase ii), for gaucher disease type 3 (phase ii) and for patients with pd who carry a mutation in gba (phase ii). recent findings generated in a small number of patients have suggested a possible link between pd and fd 287 , which also exists between patients with pd and gaucher disease who have gba mutations (see above). finally, genistein, a pleotropic natural product that inhibits kinases involved in the regulation of proteo glycan biosynthesis and also affects tfeb function, is in a phase iii trial for sanfilippo syndrome 288 . substrate mimetics that inhibit lysosomal enzymes have also been found to stabilize mutated enzymes in lsds, thereby leading to restoration of some enzyme activity when suitable subinhibitory concentrations are used, as the enzyme remains stable and functional after dissociation of the inhibitor 46, 283 . the pioneering exam ple of this approach is migalastat, described above, that binds to the active site of α galactosidase a, which is mutated in fd, and stabilizes the mutant enzyme. other examples of this strategy include afegostat in gaucher disease (which failed in a phase ii clinical trial in 2009 due to lack of efficacy), pyrimethamine in sandhoff disease and tay-sachs disease, and ambroxol in gaucher disease with neurological symptoms (table 4). agents that are at earlier developmental stages include n octylβ valienamine, a competitive inhibitor of βglucosidase, for gaucher disease; n acetylcysteine for pompe disease; α lobeline, 3,4,7trihydroxyisoflavone and azasugar in krabbe disease; and n octyl4epi β valienamine and 5n,6s(n′butyliminomethylidene) 6thio1deoxygalactonojirimycin indicated in gm1 gangliosidosis 289 . the chemical structures of these pharmacological chaperones have been described recently 290, 291 . finally, an alternative strategy for stabiliz ing mutant enzymes, by binding away from the active site, is also being investigated. a promising example of this approach is ncgc607, a non inhibitory small molecule chaperone of gcase discovered by screening for molecules that improved the activity of the mutant enzyme 46, 250 . treatment with ncgc607 reduced lyso somal substrate storage and αsynuclein levels in dopamin ergic neurons derived from induced pluripotent stem cells from patients with gaucher disease with parkinson ism 46, 250 . further testing of ncgc607 in patients with pd and gba mutations is awaited. although promis ing, conflicting viewpoints still remain on the strength of such small molecule based approaches, primarily because these compounds bind to the catalytic site of enzymes, which may be a risk at high concentrations if they inhibit rather than increase activity 291, 292 . more clinical trials are therefore required in order to analyse the robustness of this approach. cathepsin modulators. robust genetic and pharma cological preclinical investigations have consistently showed that regulating cathepsin activity can favour ably improve pathological features in certain auto immune and inflammatory diseases. inhibitors of several cathepsins (b, d, l, k and s) have been described 174, 293 and their activity has been evaluated in rheumatic auto immune diseases (such as sle, ra and sjs) and neuro degenerative disorders, notably in ad 294 (table 4) . selective inhibition of cathepsin s with a potent active site inhibitor known as ro5461111 (roche) mitigated disease in mrl/lpr lupus prone mice, by reducing prim ing of t and b cells by dendritic cells, and plasma cell generation 262 . promising data have also been generated in murine models, in the context of diabetic nephrop athy and cardiovascular diseases 295 . further studies based on cathepsin s inhibitors should evaluate the clinical safety and utility of treating patients affected by autoimmune and inflammatory diseases 295 . cathepsin k, which is highly expressed by osteoclasts and very effi ciently degrades type i collagen, the major component of the organic bone matrix, is also a potential target for modulating lysosomal dysfunction in some of the dis orders discussed above, such as sle 96 . yet further investi gations with selective cathepsin k inhibitors are required to determine whether this targeted strategy might apply in sle and other inflammatory conditions in which articular manifestations are a major component (ra, ankylosing spondylitis, psoriatic arthritis and others). it should be noted, however, that various cathepsin k inhibitors have been pursued for postmenopausal osteo porosis, including odanacatib (merck) which reached phase iii trials 296 . although odanacatib was effective, its development was discontinued in 2016 due to an increased risk of stroke in treated patients. other cathep sin inhibitors and their context of clinical evaluation are listed in table 4. despite multiple efforts to develop selective pharma cologic cathepsin modulators, important concerns still fluorescence measurement (flow cytometry or fluorescence microscopy) of cellular staining of acidotropic dyes, such as lysotracker dyes 92, 215 simple to use but is not quantitative as stated by the manufacturer; can be adapted to clinical trial settings western blot and fluorescence imaging of lysosomal markers such as l amp1, l amp2 etc. 216, 217 simple but does not provide information on subcell populations 89 limited usage in primary cells as they are hard to transfect bsa , bovine serum albumin; l amp, lysosome-associated membrane protein; n/a , not available; qpcr , quantitative pcr; tfeb, transcription factor eb. www.nature.com/nrd remain with regard to off target effects due to activity against other cathepsins or towards cathepsins present at non relevant or unwanted sites. nonetheless, the under lying biology and clinical effects of certain cathepsin inhibitors or activators remain of considerable interest and could guide future therapeutic approaches. as reported below, v atpase, a multisubunit atp driven proton pump, is best known for its role in acidification of endosomes and lysosomes. regulating the function of v atpase may impact lyso somal activity and, hence, the acidification of spe cialized cells and diverse signalling pathways, such as autophagy. v atpase inhibitors like bafilomycin a1 and concanamycin a are non selective compounds (table 4; fig. 3 ) that inhibit both mammalian and non mammalian v atpases, which control the lysosomal ph of acidic vesicles via a manner that is not fully under stood (fig. 2) . through this mechanism, bafilomycin a1 inhibits autophagic flux by preventing the acidifi cation of endosomes and lysosomes 297 . bafilomycin and cq also affect mitochondrial functions, as discovered recently using intact neurons 298 . benzolactoneenamides (salicylihalamide a, lobatamides and oximidines; 299 than bafilomycin a1 and concanamycin a, but also much less potent. further investigations into v atpase regulation of signalling pathways are needed to identify specific and safe molecules that regulate this vital proton pump 300 . ion channel modulators. as discussed above, lysosomal ion channels are master elements of lysosome activity and, thereby, of cell homeostasis. in the family of trp channels, trml1 is essential, being widely expressed in late endosomes and lysosomes, and preferentially associ ates with lamp1 in the lysosomal membrane 22, 301, 302 . genetic mutations leading to inactivation of trpml1 cause a rare genetic disorder called mucolipidosis type iv (mliv). pharmacological activation of trpml1 ameliorated some lysosomal functions that are clas sically associated with mliv, npcs and certain lsds (tables 2,4; fig. 2 ). thus, the small molecule sf22 (fig. 3) , which was identified in a screen for trpml3 acti vators, was defined as an activator of both trpml3 and trpml1 (ref. 274 ), and displayed an additive effect in combination with the endogenous activator phospha tidylinositol3,5bisphosphate (ptdins(3,5)p2) 274, 303 . an analogue of sf22, in which chlorine on the thiophene had been replaced by a methyl group, showed greater efficacy on trpml1 activation 303, 304 . another molecule called ml sa1 (figs 2,3) , acting as a mucolipin synthetic agonist, also showed an additive effect with endogenous ptdins(3,5)p2 on trpml1 channels 305 . it is important to note that in neurological diseases, as well as in other indications in which lysosomal acidification is defec tive (see above), interfering with trml1 may have contraindications. a central modulator of lysosomes is the lipid kinase fyve finger containing phosphoinositide kinase (pikfyve), which converts phosphatidylinositol3phosphate into ptdins(3,5)p2. the latter regulates ca 2+ release from the lysosome lumen and is required for acidification by v atpase. inactivation of pikfyve leads to many patho physiological problems including neurodegeneration and immune dysfunction, mostly related to impaired autophagic flux and alteration of lysosomes (trafficking, ca 2+ transport, biogenesis and swelling) 306 . the small molecule apilimod ( fig. 3; table 4 ) was originally identi fied as an inhibitor of tlr induced il12 and il23, and later found to be a highly specific inhibitor of pikfyve 276 . apilimod was evaluated in clinical trials involving sev eral hundred patients with t helper 1 and t helper 17 cell mediated inflammatory diseases such as crohn's dis ease, ra and psoriasis 277, 278 . it was well tolerated in more than 700 human subjects (normal healthy volunteers and patients with inflammatory disease), but the clinical trials did not meet their primary endpoints and further development was abandoned. apilimod is currently being evaluated in a clinical trial (nct02594384) aimed at defining a maximum tolerated dose in patients with b cell non hodgkin lymphoma and monitoring safety, pharmacokinetics, pharmacodynamics and prelimi nary efficacy 307 . ym201636 is another selective inhibi tor of pikfyve (table 4; fig. 3 ). this inhibitor contains a fyve type zinc finger domain. ym201636 was found to significantly reduce the survival of primary mouse hippo campal neurons in culture and reversibly impair endo somal trafficking in nih3t3 cells, mimicking the effect produced by depleting pikfyve with small interfering rna. it was also found to block retroviral exit by budding several parameters have been used to evaluate lysosomal functions (table 2) . alteration of lysosomal volume is an important sign of lysosomal dysfunction; it has been observed in various diseases, such as autoimmune syndromes, cancers and lysosomal storage diseases 215 . it can be measured by staining cells with acidotropic dyes such as lysotracker dyes and immunoblot of lysosomal membrane proteins such as lysosome-associated membrane protein 1 (lamp1). variation of lysosomal volume is often related to changes in lysosomal biogenesis, which can be assessed by the expression level and cellular location of transcription factor eb (tfeb). however, precise determination of lysosomal functions relies on measurement of lysosomal luminal ph and degradation activity. several fluorescence probes that measure lysosomal ph (table 2) are commercially available. abnormal lysosomal ph affects lysosomal degradation activity, which can be followed, for example, by detecting the degradation of endocytosed fluorescence dq-bsa 57 . in complement, the activity of specific enzymes, such as cathepsins b, d and l, can be tested using commercially available kits. other lysosomal parameters can be evaluated to deepen the examination of lysosomal status, including lysosomal membrane stability and integrity and lysosomal ca 2+ ion signalling, for example (table 2) . lysosomal function is essentially linked with autophagy activity as autophagy is a lysosomal-dependent degradation pathway. thus, a series of methods routinely applied for assessing macroautophagy in mouse models and patients with autoimmune diseases is summarized 89 . to ascertain the extent of autophagy defects, a combination of techniques, such as western blot and flow cytometry, measurement of autophagy makers, fluorescent imaging and electron microscopy, in the presence and absence of lysosomal protease inhibitors, is recommended. several review articles have described reliable methods dedicated to the measurement of chaperone-mediated autophagy (cma) activity [228] [229] [230] . increased expression levels of lamp2a and hspa8, two key players in cma, have been shown to occur in a mouse model of lupus 92 . however, it should be noted that increased expression levels of hspa8 and lamp2a starting from a total lysate is only indicative of cma upregulation; this test is not sufficient to allow any firm conclusion, and it is necessary to examine their expression levels in purified lysosomes or in lysosome-enriched fractions. nature reviews | drug discovery chaperone modulators. molecules targeting chaperone proteins involved in lysosomal function have also been designed for potential therapeutic applications. one of these molecules is ver155008, a small molecule inhib itor of hspa8, a key element of cma 308, 309 . ver155008 binds to the nucleotide binding domain of hspa8 and hsp70, and acts as an atp competitive inhibitor of atpase and chaperone activity. in a mouse model of ad (5xfad mice), intraperitoneal treatment with ver155008 reduced the two main pathological fea tures of ad (amyloid plaques and paired helical filament tau accumulation) and improved object recognition, location and episodic like memory 280 . another molecule, the 21mer phosphopeptide p140 (table 4; fig. 3 ), was also shown to interact with hspa8 (fig. 2) 310 and lodge in the hspa8 nucleotide binding domain 92, 311 . p140 and ver155008, however, do not have the same mechanism of action, and their effects were not additive 312 . p140 is a phosphorylated analogue of a nominal peptide that was initially spotted in a cellular screening assay using overlapping peptides covering the whole spliceosomal u170k protein and cd4 + t cells collected from the lymph nodes of lupus prone mrl/lpr mice 313 . p140 peptide enters b cells via a clathrin coat dependent endocytosis process to reach early endosomes and then late endosomes/lysosomes 92 . it affects cma that is hyperactivated in lupus, likely by hampering the cma mediating chaperone hspa8 (ref. 101 ). p140 peptide reduces the excessive expression of hspa8 and lamp2a observed in lupus mice, alters the (auto)antigen presentation by mhcii molecules in the miic compartment and, consequently, attenuates the activation of autoreactive t cells 92 . a significant diminution of mhc molecule expression at the surface of antigen presenting cells was measured in mice that received the p140 peptide intravenously and on patient's peripheral cells treated ex vivo with the peptide 92,101,314 . as a downstream consequence, the activation of auto reactive b cells and their differentiation into autoantibody secreting cells is repressed 101, 314 . t cells from patients with lupus are no longer responders ex vivo to peptides encompassing cd4 + t cell epitopes 315 . the effect of p140 on cma was demonstrated in vitro, using a fibroblast cell line that stably expresses a cma reporter 53, 92 . p140, which selectively targets the cma/lysosome process and has no effect on mitophagy 316 , has been evaluated in murine models mimicking other rheumatic diseases with very promising results, notably in mice developing sjs features 56 , in mice with neuropsychiatric lupus symp toms 62 and in rats that develop a cidp like disease with disturbance of both cma and macroautophagy in sciatic nerves 59 . in clinical trials that included patients with sle, p140 formulated in mannitol was found to be safe and non immunogenic after several subcutaneous admin istrations of peptide 312, 317, 318 . p140 showed significant efficacy in a multicentre, double blind, phase ii trial 317 . this peptide is currently being evaluated in phase iii trials in the united states, europe and mauritius. in con tinuation, an open label trial including several hundred patients with lupus worldwide is planned. another peptide has been discovered that, in con trast to p140, activates cma 319 . this 24mer peptide called humanin was originally identified from sur viving neurons in patients with ad, and was found to directly enhance cma activity by increasing substrate binding and translocation into lysosomes. humanin interacts with hsp90 and stabilizes the binding of this chaperone to cma cargos as they bind to the lyso somal membrane. these results are important as humanin had been shown to possess some cardioprotective and neuro protective properties in diseases such as ad, cardio vascular disease, stroke, myocardial infarction, diabetes and cancer 320 . in addition to the targets discussed above, there are a few emerging potential lysosomal therapeutic targets for which there is strong biological validation, but not yet any small molecules in development that target them. an example with likely pharmacological tractability is a lysosomal k + channel called tmem175, which is impor tant for maintaining the membrane potential and ph stability in lysosomes 321 . deficiency in tmem175 may play a critical role in pd pathogenicity 322 . importantly, the structure of tmem175 has been recently refined 323 . another target for which ligands have not yet been validated is the kcnq2/3 channel (also named m channel or kv7.2/7.3 channel). it has been shown in npc1 disease that reduced cholesterol efflux from lyso somes aberrantly modifies neuronal firing patterns 324 . this disruption of lysosomal cholesterol efflux with decreases in ptdins(4,5)p2dependent kcnq2/3 chan nel activity may lead to the aberrant neuronal activity. the cholesterol transporter and ptdins(4,5)p2 floppase, abca1, is responsible for the decline in ptdins(4,5)p2 that consequently modifies the electrical properties of npc1 disease neurons. dysfunction in the activity of kcnq2/3 or altered levels of ptdins(4,5)p2, due notably to genetic mutations, might also be involved in other neuropathies (for example, some forms of epilepsy, hd, pd, ad, als and friedrich ataxia). although further experiments are needed to validate the link discovered between hyperexcitability and cell death in npc1 disease and other neurodegenerative diseases, small molecules such as retigabine, an anti convulsant drug that keeps kcnq2/3 channels open, might rep resent important therapeutic tools 324, 325 . other channel opener ligands of kcnq2/q3 include ica069673 and its derivatives. another promising therapeutic target is sphingo myelin phosphodiesterase 1 (smpd1). defects in the gene encoding smpd1 cause niemann-pick disease type a and type b. smpd1 converts sphingomyelin to ceramide, and also has phospholipase c activity. reduced activity of acid sphingomyelinase, associated with a marked decrease in lysosomal stability, has been described in patients with niemann-pick disease, a phenotype that was corrected by treating cells with recombinant hsp70 326 . finally, as lamp2a, a specific lysosomal protein that displays a decisive role in cma, has been shown to be overexpressed in certain pathological settings such as certain cancers and inflammatory diseases (autoimmune or non autoimmune), downregulating its expression www.nature.com/nrd might be therapeutically beneficial 53, 327 . as mentioned above, however, in other indications there is a defect in lamp2a. the latter can be due to reduced stabil ity of the cma receptor and not to decreased de novo synthesis (for example, in ageing) 328 or can result from aggregation to the lysosomal membrane of pathogenic proteins such as α synuclein, ubiquitin carboxy terminal hydrolase l1 (a deubiquitinating enzyme) and mutant tau, known to amass in neurodegenerative disorders (see above). targeting lamp2a therefore remains a challenge, although several strategies may be envisaged, for example by controlling de novo synthesis, by ham pering its multimerization into lysosomes (possibly via hsp90 and/or other chaperones) or by regulating the degradation rate of lamp2a monomers (for reuse) into lysosomes. challenges and outlook current research into lysosomal function and dysfunc tion is revealing novel roles of lysosomes in disease pathogenesis and highlighting new opportunities to treat such lysosomal and autophagy related diseases. as in the case of autophagy modulation 14, 56, 207 , lysosomal activation or inhibition must be investigated with cau tion, as lysosomal activity can be abnormally reduced or enhanced in some organs or tissues and not in others, and, at another scale, lysosome activity can be altered in certain lysosomes and not in others within the same cell. biodistribution studies in vivo must be undertaken to avoid accumulation of pharmaceuticals in healthy organs or tissues. there is an obvious requirement for safety, to ensure that a drug used as a lysosome modu lator for a particular type of lysosomal disease does not increase vulnerability to another disease. there is still much to be learned about the intimate working of lysosomes. this is due to the abundance of constitutive elements that comprise these vesicles, the added complexity resulting from their plasticity (ion channels and transporters, acidification and swell ing) and the vast amount of proteins and peptides that are translocated into lysosomes and digested by lytic enzymes. sensitive analysis methods have allowed important information to be generated about lyso somal membrane proteins, a large majority of which are transporters 8 . however, many questions remain related to how their expression is regulated and how they reg ulate their translocator and chaperoning activities. for example, certain cells only contain so called secretory lysosomes (as in cytotoxic t cells), whereas other cell subsets contain both conventional and secretory lyso somes (as in platelets). considering the large family of endo lysosomal vesicles, the whole notion of 'secretory' and 'conventional' lysosomes remains a matter of debate. in many instances, lysosomes act as a basal cell metab olism organelle; whereas in other cases, they assist in the regulation of homeostasis through unconventional secretory pathways, known as lysosomal exocytosis, and different signalling mechanisms. although several assays used to measure the activ ity of lysosomes have been validated worldwide (box 2; table 3), they have their limitations, including issues associated with reliability, performance and sensitivity, notably in vivo. another level of complexity comes from the inherent organelle heterogeneity, which is an issue of tremendous importance. unfortunately, with the tools and equipment we have in hand today, it is virtually impossible to examine what happens in the lysosomes of an individual patient. the introduction of micro fluidic single cell analysis technologies has enabled cellular populations to be characterized and huge advances to be performed. however, the level of precision has not yet been achieved at the level of lysosomes (0.2-0.5 μm). we know that lysosomes are heterogeneous in nature, composition and activity even in 'normal' settings; they are not all equally competent for autophagy or any other types of activity. currently, this is obviously the focus of intense research. although a certain number of preclinical studies involving lysosomal regulators have been conducted over the years, only a small number of lysosome targeted therapeutics have so far moved into clinical develop ment. one of the biggest advances in developing such strategies would be the identification of a genetic sig nature that would allow those patients most likely to respond to a specific therapy to be selected. however, at this stage of our knowledge of specific lysosome directed drugs and intrinsic lysosomal failures, genetic features that might predict potential responders are still lacking (with the exception of lsds). further investi gations are required to achieve this level of knowledge, which obviously will also depend on the type of disease, heterogeneity and frequency. another issue associated with the development of lysosometargeted therapeutics relates to delivery. the use of nanovectors represents an attractive delivery method, owing, in particular, to their unique ability to penetrate across cell barriers and, via the endo lysosomal pathway, to preferentially home in on orga nelles such as lysosomes. several nanoscale galenic forms have been developed to serve as vectors or car riers of proteins, peptides or nucleic acids, and a vast literature describes the many advantages of using such nano structures in nanomedicine. however, safety is a concern as some carbon nanostructures have been claimed to induce nanotoxicity, accompanied by the induction of autophagy and lysosomal dysfunction [329] [330] [331] [332] (reviewed elsewhere [333] [334] [335] [336] . the purpose of this review is to gain awareness of the importance of lysosomes in disease, and to encour age the development of novel lysosomal targeted drugs. however, more research is needed to characterize com ponents that are specifically linked to the lysosome, such as lamp2a and hspa8, and to more clearly define their specific involvement in lysosome biogenesis and metabolism. special attention should be given to the mode of administration of lysosome targeted medica tions in order to minimize toxicity and promote specific targeting. it is our hope that a large field of therapeutic applications could emerge from such investigations, encompassing rare and common autoimmune, neuro degenerative and metabolic diseases, as well as cancer, senescence and ageing. nature reviews | drug discovery tissue fractionation studies. 6. intracellular distribution patterns of enzymes in rat-liver tissue lysosomal membrane permeabilization and cell 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function tmem175 deficiency impairs lysosomal and mitochondrial function and increases α-synuclein aggregation the lysosomal potassium channel tmem175 adopts a novel tetrameric architecture niemann-pick type c disease reveals a link between lysosomal cholesterol and ptdins(4,5)p2 that regulates neuronal excitability functional upregulation of the m-current by retigabine contrasts hyperexcitability and excitotoxicity on rat hypoglossal motoneurons hsp70 stabilizes lysosomes and reverts niemann-pick disease-associated lysosomal pathology chaperone-mediated autophagy is required for tumor growth restoration of chaperonemediated autophagy in aging liver improves cellular maintenance and hepatic function role for nanomaterialautophagy interaction in neurodegenerative disease a functionalized single-walled carbon nanotube-induced autophagic cell death in human lung cells through akt-tsc2-mtor signaling tuning cell autophagy by diversifying carbon nanotube surface chemistry silica nanoparticles enhance autophagic activity, disturb endothelial cell homeostasis and impair angiogenesis autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity exploiting intrinsic nanoparticle toxicity: the pros and cons of nanoparticle-induced autophagy in biomedical research berberine as a potential autophagy modulator the authors apologize to all those whose work is not cited due to space limitations. they gratefully acknowledge hélène jeltsch-david for critically reading the manuscript. this research was funded by the french centre national de la recherche scientifique, région alsace, the laboratory of excellence medalis (anr-10-labx-0034), initiative of excellence (idex), strasbourg university, and immupharma france. s.m. is grateful to the university of strasbourg institute for advanced study (usias) for funding f.w., and acknowledges the support of the transautophagy cost action (ca15138), the club francophone de l'autophagie (cfatg) and the european regional development fund of the european union in the framework of the interreg v upper rhine programme. all authors made substantial, direct and intellectual contribution to the work and approved it for publication. s.m. discloses the following conflicts of interest: research funding (paid to institution) and a past consultant for immupharma; co-inventor of cnrs-immupharma patents on p140 peptide; owns immupharma shares. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. s.r.b. and f.w. declare no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.www.nature.com/nrd key: cord-281101-gv1sgbk1 authors: shin, gu-choul; chung, yoon-seok; kim, in-soo; cho, hae-wol; kang, chun title: preparation and characterization of a novel monoclonal antibody specific to severe acute respiratory syndrome-coronavirus nucleocapsid protein date: 2006-08-30 journal: virus res doi: 10.1016/j.virusres.2006.07.004 sha: doc_id: 281101 cord_uid: gv1sgbk1 severe acute respiratory syndrome-coronavirus nucleocapsid (sars-cov n) protein has been found to be important to the processes related to viral pathogenesis, such as virus replication, interference of the cell process and modulation of host immune response; detection of the antigen has been used for the early diagnosis of infection. we have used recombinant n protein expressed in insect cells to generate 17 mabs directed against this protein. we selected five mabs that could be used in various diagnostic assays, and all of these mabs recognized linear epitopes. three igg(2b) mabs were recognized within the n-terminus of n protein, whereas the epitope of two igg(1) mabs localized within the c-terminus. these mabs were found to have significant reactivity with both non-phosphorylated and phosphorylated n proteins, which resulted in high reactivity with native n protein in virus-infected cells; however, they did not show cross-reactivity with human coronavirus. therefore, these results suggested that these mabs would be useful in the development of various diagnostic kits and in future studies of sars-cov pathology. severe acute respiratory syndrome (sars), which is caused by the sars coronavirus (sars-cov), is a newly emerging disease. sars-cov presented with high virulence and mortality, and affected 30 countries, with more than 8000 cases and over 750 deaths. indeed, as the clinical symptoms of sars are nonspecific compared to those of other respiratory viruses, diagnosis relies largely on laboratory tests . thus, the development of diagnostic laboratory tests for specific and early detection of sars-cov infection is of great importance for both rapid treatment of patients and control of sars outbreaks. thus far, virus isolation methods have generally been performed to determine the presence of infectious virus in samples; this process is relatively time-consuming and inefficient (keyaerts et al., 2005; yamashita et al., 2005) . rt-pcr and real time-pcr for direct detection of sars-cov rna is expen-sive and labor-intensive, relies on the availability of expertise, and may produce false-positive results due to contamination (keightley et al., 2005; huang et al., 2005) . serological methods, such as immunofluorescence assay and enzyme-linked immunosorbent assay (elisa), are not adequate for the early diagnosis of sars, because the median time to seroconversion in sars patients is 17-20 days after the onset of symptoms . therefore, the generation of monoclonal antibodies (mabs) against sars-cov antigens may provide possible diagnostic tools for early diagnosis of sars, because sars-cov can be specifically detected in the respiratory specimens, blood and stool much earlier than antibodies can be used for detection (lau et al., 2004; di et al., 2005) . the sars-cov nucleocapsid (sars-n) protein is a phosphoprotein of 48 kda, and performs multiple functions in viral pathogenesis, such as providing a nuclear-import signal, interfering in the cell process, participating in virus replication and packaging rna (egloff et al., 2004; yan et al., 2004; surjit et al., 2004 surjit et al., , 2005 . thus, this protein may have important roles in the pathogenesis of sars. furthermore, this protein is the most abundantly expressed structural protein during infection and is highly detectable in sars patients lau et al., 2004; di et al., 2005) . therefore, this protein may serve as one of the immunodominant antigens in the early diagnosis of infection. furthermore, some researchers have suggested that antibody against the n protein could modulate cytokine responses such as il 11; non-neutralizing antibodies against n protein were found to protect mice against lethal infection (nakanaga et al., 1986; cheng et al., 2005) . therefore, the development of mabs against sars-n protein may be critical in the development of drugs to treat sars-cov infection, and for further study of the pathogenesis of sars, as well as early diagnosis. here, we report the production of 17 mabs against sars-n and the properties of the mabs, which were determined by isotyping, affinity assay, epitope mapping and reactivity with various isoforms of sars-n protein. we also suggested the applicability of the mabs in various analytical methods, such as ifa, immunoblot and antigen-capture elisa, for diagnosis and functional study of sars-cov n protein. sp2/0 myeloma cells were kindly provided by metabolab inc. (seoul, republic of korea), and mrc-5 cells (atcc, ccl-171) were obtained from american type culture collection. sars-cov stock was provided from the center for disease control and prevention, and this virus was maintained in biosafety level-3 (bsl-3) containment laboratories in the national institute of health, korea center for disease control and prevention. the sars-cov titer was determined to be 1 × 10 4 50% tissue culture infectious doses/ml (tcid 50 /ml). the virus culture supernatants were inactivated by heating at 56 • c for 60 min prior to use. human coronavirus oc43 (atcc, vr-1558; hcov oc43), which was tested to determine cross-reactivity with sars-n mab, was prepared from mrc-5 cells. replication of these viruses was confirmed by rt-pcr and immunofluorescence assay using mab against nucleocapsid protein of human coronavirus oc43 (hcov mab). the hcov oc43 titer was determined by hemagglutination assay (ha) using human red blood cells (rbcs). the complete coding sequence for the n protein (urbani strain, genbank accession no. ay278741, 28,120-29,388 bp) was amplified from the sars-cov genomic rna using pcr. the amplified product was digested with ecori and bamhi, and then inserted into his-tagged pentr bhrnx vector (neurogenex, seoul, republic of korea) to create pentr np7. recombinant baculovirus was generated by co-transfection of sf21 cells with pentr np7 and linearized baculovirus dna using the baculogoldtm system (bd biosciences, san jose, ca). recombinant baculoviruses were harvested from sf21 cell culture medium 48 h post-transfection and broken by three cycles of freezing-thawing. the 6× histidine-tagged recombinant n protein (brsars-n) was purified by metal-chelating affinity chromatography (merck bioscience, darmstadt, germany). the purified protein was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page), as described previously (laemmli, 1970) . to examine the reactivity of sars-n mabs against the phosphorylated protein and non-phosphorylated protein, rsars-n protein expressed in escherichia coli (ersars-n) was purchased as nonphosphorylated protein from biovendor laboratory medicine, inc. (heidelberg, germany). balb/c mice (samtako inc., republic of korea; 9 weeks) were intraperitoneally injected with a mixture containing 50 g purified brsars-n proteins in 100 l phosphate buffered saline (pbs) and an equal volume of freund's complete adjuvant. a boost injection with the same amount of antigen in freund's incomplete adjuvant was administered at 2-week intervals. hybridoma fusion was performed using a method similar to that originally described (kohler and milstein, 1975) , with the following modifications. in brief, the splenocytes were harvested from immunized mice, mixed with sp2/0 cells at a 5:1 ratio, and fusion was carried out with 40% polyethylene glycol-1500 (roche, indianapolis, in). the fused cells were collected by centrifugation at 800 × g for 5 min and the cell pellet was resuspended in dmem (invitrogen, carlsbad, ca) containing 20% fbs and hat supplement (sigma-aldrich korea co., seoul, republic of korea). the cells were seeded in 96-well plates at 200 l/well (2 × 10 5 cells/well) and cultured in a co 2 incubator. antibody produced in medium was measured by indirect enzyme-linked immunosorbent assay (indirect elisa) as described below. a limiting dilution of hybridoma was carried out from putative positive individual wells, and the screening was repeated until hybridoma clones producing a strongly reactive sars-n mab were observed. selected hybridoma clones were maintained in dmem containing 10% fbs, 1× ht supplement (sigma-aldrich korea) and exchanged with fresh media once every 3 or 4 days. the sars-n mab of a selected hybridoma was purified using the immunopure (g) igg purification kit (pierce biotechnology inc., rochford, il) and isotyped with an immunopure monoclonal antibody isotyping kit ii (pierce biotechnology inc.), used according to the instructions of the manufacturer. the indirect elisa was carried out on a maxisorp plate (nalgen nunc international, rochester, ny), which had been coated with 1 g of recombinant sars-ns diluted in 50 mm carbonate buffer (ph 9.6) and incubated overnight at room temperature. non-specific protein binding sites were blocked with 1% bovine serum albumin (bsa) in pbs for 1 h at 37 • c. plates were washed with pbs containing 0.05% tween 20 (pbst). hybridoma supernatants, sars-n mab and anti-sars serum obtained from mice immunized with heat-inactivated sars-cov, or normal mouse serum as negative serum, were then added and incubated for 60 min at 37 • c. after washing with pbst, a 1:1000 dilution of alkaline phosphatase (ap)-conjugated goat anti-mouse igg+ iga+ igm antibody (abcam) in pbst containing 1% bsa was added to all wells and incubated for 60 min at 37 • c. after a final wash, p-nitrophenyl phosphate disodium salt (pnpp, pierce biotechnology inc.) solution was added, and the plates were further incubated for 30 min at room temperature. the color intensity was measured as the absorbance value at 405 nm (od 405 ) in an el340 elisa reader (bio-tek intstruments inc., winooski, vt). isotyping was performed using immunopure monoclonal antibody isotyping kit ii (pierce biotechnology inc.), used according to the instructions of the manufacturer. to examine whether sars-n mab recognizes the linear epitope of sars-n protein, we performed an immunoblot assay in denaturing conditions of the sars-n protein. the purified rsars-n protein were resolved by 10% sds-page, and were then electrotransferred onto a pvdf membrane (bio-rad, hercules, ca) and blocked with 1% bsa in pbst. membrane was incubated with sars-n mabs (1 g/ml) or anti-his tag mab (1:1000 dilution, abcam), as positive control, for 1 h, and non-specific adsorption was washed away by pbst. the bound antibodies were detected by horseradish peroxidaseconjugated goat anti-mouse igg+ iga+ igm secondary antibody (1:2000 dilution, abcam), followed by dab substrate solution (sigma-aldrich korea). a total of 19 linear peptides ranging in size from 11 to 26 amino acid residues were synthesized on the basis of the fulllength nucleocapsid protein sequences of sars-cov urbani strain, genebank aap13445 (cosmo co., seoul, republic of korea). the synthesized peptides were characterized by hplc and mass spectrometry. to identify the epitopes in sars-cov n protein that are targeted by sars-n mabs, competition elisa was performed as described previously (chiang et al., 2005) . the sars-n mabs (10 g/ml) were incubated with the competitor peptide or brsars-n protein (10 g/ml) for 1 h at 4 • c. the mabs were then transferred into the wells of a maxisorp plate that had been coated with 0.5 g/well of sars-n protein. the following steps were then accomplished as described in the indirect elisa procedure. affinity analysis was performed by non-competitive elisa as described previously (huang et al., 2005) , with the following modifications. in brief, maxisorp plate was coated overnight with 1 g/well of brsars-n, as described in the indirect elisa procedure. the plates were blocked with 1% bsa, followed by incubation of sars-n mab with serial dilution (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39 and 0.3 nm). after washing with pbst, ap-conjugated goat anti-mouse igg+ iga+ igm antibody (abcam) in pbst containing 1% bsa was added to all wells and incubated for 30 min at 37 • c. the following steps were accomplished in the same manner as that used for indirect elisa, described above. the affinity constants were presented as the concentrations (nm) of mab at 1.000 of od 405. reactivity of sars-n mabs with sars-cov infected cells was determined by immunofluorescence assay, performed according to the instructions of the manufacturer (euroimmun, germany). the sars-n mab (10 g/ml), anti-sars serum (1:100 dilution) and negative serum were added to each well, and the slides were incubated at 37 • c for 30 min. after washing three times with pbs, fluorescein isothiocyanate-conjugated goat anti-mouse igg+ iga+ igm antibody (icn biomedical, 1:100 dilution) was added, and the slides were then incubated at 37 • c for 30 min. the slides were then washed three times with pbs and one time with ultrapure water and observed by fluorescence microscopy. to prepare detector mabs, the purified mabs were labeled with biotin using the ez-link sulfo-nhs-lc-biotinylation kit (pierce biotechnology inc.) according to the instructions of the manufacturer. the purified mab for the antigen-capture was immobilized on the hi-bind microplate (costar, coring incorporated, ny) by incubating 5 g/ml antibody in 50 mm carbonate buffer (ph 9.6) at 4 • c overnight. the wells were then washed twice with pbs, followed by blocking with 2% bsa in pbs, ph 7.4, for 4 h at room temperature. after removing the blocking reagent, the wells were dried and stored at 4 • c prior to use. virus culture supernatant or recombinant n protein diluted in 1% bsa in pbst or virus culture medium was added to the wells (100 l/well) and incubated for 2 h at 37 • c. after washing, the wells were incubated for 1 h at 37 • c with 100 l per well of biotin-conjugated detector mab (diluted 1/200 in pbst with 1% bsa or virus culture medium). after washing, the wells were incubated for 1 h at 37 • c with 100 l per well of ap-conjugated goat anti-biotin antibody (diluted 1/500 in pbst with 1% bsa or virus culture medium, abcam). after washing, 100 l of pnpp was added to each well. the color reaction was stopped after 30 min with 100 l of 1n naoh to each well, and the plates were examined at 405 nm in an el340 elisa reader (bio-tek instruments inc.). the experiments involving the use of the inactivated sars-cov were performed in a bsl-2 laboratory. for the immunofluorescence assay to assess the crossreactivity of mabs with human coronavirus, spot slides were prepared by applying a suspension of cells infected with uvc-irradiated human coronavirus oc43 or a suspension of uninfected mrc-5 cells to the wells of teflon-coated slides. slides were allowed to air-dry before they were fixed in methanol. slides were stored at −70 • c until used for indirect fluorescence assay. the hcov mab (1:100 dilution, chemicon international inc., temecula, ca) as positive control, normal mouse serum as negative control, and sars-n mabs (100 g/ml) were added to the slides, and the slides were incubated at 37 • c for 30 min. the following steps were accomplished in the same manner as that described above for sars-cov. for antigen-capture elisa, human coronavirus oc43 (256 ha unit) supernatant that had been cultured from mrc-5 cells was heat-inactivated for 30 min at 56 • c and serially diluted two-fold. the protein samples were then extracted by three cycles of freezing-thawing before centrifugation at 14,000 rpm for 10 min and dilution in pbs with 1% bsa. the preceding steps were performed in the same manner as that used for sars-cov, described above. full-length sars-n protein was produced in a recombinant baculovirus system and used in the immunization of mice. two weeks after each antigen boost dose, immunized mice were screened for sars-n specific antibody response by indirect elisa. splenocytes isolated from the mice were fused with sp2/0 myeloma cells, resulting in ∼1000 proliferating hybridomas. subsequent screening of these hybridomas and single cell cloning yielded 17 positive clones that constitutively secreted mabs that reacted to sars-n protein by indirect elisa (data not shown). further isotyping of these mabs were determined in order to facilitate future utilization. for the heavy chain subclasses, most of the mabs were found to belong to the igm subtype, two of the mabs belonged to igg 1 , and three of the mabs belonged to igg 2b . the light chain of all of these mabs was of the kappa isotype (table 1) . five of the 17 mabs determined to be of the igg subclass were characterized by further analysis, because these mabs may be suitable for use as diagnostic mabs. immunoblotting was performed with brsars-n protein in order to analyze the reactivity of sars-n mabs against sars-n protein under denaturing conditions and to determine whether these mabs recognize the conformational epitope of sars-n table 1 isotypes of the sars-n mabs generated in this study these isoforms were also observed in immunoblot analysis of total proteins extracted from baculovirus-infected cells and sars-cov-infected cells (data not shown). thus, these isoforms may be fragment cleaved by intracellular protease. the fragment of 25-kda that was not observed in originally purified brsars-n protein could be obtained by freezing and thawing procedures of purified protein. thus, this result indicated that the epitopes of all of the sars-n mabs are linear, and the epitopes of igg 2b and igg 1 mabs are located in the n-terminus and c-terminus of sars-n protein, respectively. to more precisely analyze the epitope of sars-n protein recognized by these sars-n mabs, competitive elisa was conducted using the 19 synthetic peptides covering the full-length sars-n protein sequence. among these synthetic peptides, the n135 peptide (ategalntpkdhigtr; at position 135-150 of sars-n protein) and n17 peptide (tfggptd-stdnnqngg; at position 17-32) could effectively compete in the binding of all of the igg 2b mabs with the sars-n protein, and the n117 peptide (gpeaslpygankegiv; at position 117-132) seems to have slightly weak activity, while the other peptides did not exhibit any effect in this assay ( fig. 2a) . the n17 and n135 peptides contain no apparent common epitopes, which revealed that these mabs are mixed antibodies. the n215 peptide (ggetalalllldrlnqleskvsgkg; at position 215-239) resulted in complete inhibition of the binding activity of all of the igg 1 mabs with the sars-n protein, and the n245 peptide (qtvtkksaaeaskkprqkrtatkq; at position 245-268) seems to have slightly weak activity (fig. 2b ). according to the results of the immunoblot and competitive elisa, the epitopes of the igg 2b mabs is located in the nterminal region at aa 17-32 and 117-150, whereas that of the igg 1 mabs is located in the middle region at aa 215-268 (fig. 2c ). to examine the reactivity of the sars-n mabs against sars-cov n protein under non-denaturing conditions, the indirect elisa was performed with brsars-n protein. four of the selected mabs (22-05-03, 07-19-11, 07-19-21 and 21-10-06) bound slightly better to sars-n protein than 21-10-11, as shown in table 2 . to confirm the reactivity of mabs against sars-n protein, as estimated by indirect elisa, the affinity constants of five mabs were measured by non-competitive elisa, as described in section 2. the affinity constants of 22-05-03, 07-19-11, 07-19-21 and 21-10-06 were significantly higher than that of 21-10-11 (table 2) . these results were similar to those of indirect elisa and indicated that the affinity levels paralleled the reactivity estimated by indirect elisa, and all sars-n mabs, except for 21-10-11 mab, could be suitable for the development of sensitive methods for sars-cov diagnosis. since these mabs were generated using recombinant sars-n protein expressed in insect cells that contained phosphorylated n protein (data not shown), indirect elisa was performed to further examine the reactivity of sars-n mabs with the ersars-n expressed in e. coli as non-phosphorylated protein. although sars-n mabs showed slightly higher reactivity with the brsars-n protein than with the ersars-n protein, all of these mabs showed significant reactivity with the ersars-n protein compared with the reaction of negative serum (fig. 3) . this result revealed that all sars-n mabs were effectively bound with both phosphorylated and non-phosphorylated n protein. immunofluorescence assay was performed on sars-cov infected vero cells to further assess whether the sars-n mabs recognize the native-form of endogenously synthesized n protein in sars-cov infected cells. both the negative serum and the five mabs did not show non-specific reactions with uninfected cells. all five sars-n mabs strongly reacted with sars-cov infected cells, whereas negative serum showed no reaction (fig. 4) . however, 21-10-11 mab showed a significantly weak reaction in affinity constants, but reacted strongly in the immunofluorescence assay; the reason for this result is unclear. the fluorescence signals of the mabs were predominantly shown in the cytoplasm of sars-cov infected cells. this indicated that all mabs were able to detect native-form n protein in sars-cov infected cells. in order to establish a sensitive and less time-consuming antigen-capture elisa for the sars-n protein, we tested each of the pairs of mabs from the five selected mabs; this allowed us to determine the highest detection sensitivity for recombinant n protein and sars-cov culture supernatant. we found that the immobilization of a mixture of 21-10-06 and 07-19-11 mab as capture antibody on the elisa plate, followed by the detection with biotin-conjugated 22-05-03 mab, gave the best result (data not shown). to determine the sensitivity of antigencapture elisa, a serial dilution of the recombinant n protein was used to determine a standard curve (fig. 5) . normal vero cell culture media were used to determine the baseline for antigencapture elisa at an optical density of 0.230 at 405 nm (od 405 ). therefore, the cut-off value for detection of viruses in cell culture was set to be 0.300, which is equal to the mean + 3s.d. of the od 405 for normal cell culture media. according to the cutfig. 4 . detection of sars-n protein in sars-cov-infected cells by immunofluorescence assay. the immunofluorescence assay was performed using the sars ifa kit according to the instructions of the manufacturer. anti-sars indicated positive serum obtained from mice immunized with heatinactivated sars-cov and negative indicated normal mouse serum. these mabs reacted with sars-n in virus-infected vero cells, whereas they did not react with uninfected cells. off threshold (0.300), a 10 −3 dilution of recombinant n protein and a 10 −2 dilution of the virus culture supernatant were considered positive (fig. 5) . thus, it was deduced that as little as 100 pg of recombinant n protein and 10 tcid 50 of virus culture supernatant could be detected. this result revealed that the sars-n mabs could be useful for detecting the n protein in virus culture supernatant and respiratory specimens from sars patients. to determine the specificity of the five sars-n mabs, immunofluorescence assays were performed in human coronavirus-infected mrc-5 cells. all five mabs showed no cross-reactivity with the antigens of the human coronavirus (fig. 6a) ; uninfected mrc-5 cells also showed no crossreactivity (data not shown). to further assess the specificity of the mabs, antigen-capture elisa was performed with human coronavirus-infected cell lysates and brsars-n protein as positive control (fig. 6b) . all of the mabs reacted only with sars-n protein, and did not react with human coronavirus, as shown by the results of immunofluorescence assays. this revealed that all of the sars-n mabs could specifically recognize the n protein of sars-cov. sars-cov is an etiological agent that causes severe acute respiratory syndrome, an infectious disease that has only recently emerged . therefore, there is an intense need for the development of sensitive and specific detection methods for sars-cov infection. many methods have been employed recently for the detection of sars-cov infection (keyaerts et al., 2005; yamashita et al., 2005; keightley et al., 2005; huang et al., 2005) . of these diagnostic methods, rt-pcr and real time-pcr have been the most widely used. however, these methods possess some general problems, as they are time-consuming and labor-intensive, require sophisticated instruments, and have high rates of false positivity. on fig. 6 . cross-reactivity of sars-n mabs with human coronavirus antigens. (a) the immunofluorescence assay was performed by using sars-n mabs on human coronavirus oc43-infected mrc-5 cells. as a positive control, anti-hcov was used as the mab against human coronavirus oc43 n protein, and normal mouse serum was used as the negative control. fm and lm indicate fluorescence and light imagery. none of these mabs showed cross-reactivity with human coronavirus. (b) cross-reactivity of sars-n mabs was examined by antigen-capture elisa using human coronavirus oc43 lysates (256 ha unit), brsars-n protein (500 ng/well) and pbst buffer with 1% bsa as control. the other hand, laboratory methods detecting viral antigen by mabs, including antigen-capture elisa, provide more rapid, less labor-intensive, and more convenient alternatives (lau et al., 2004; di et al., 2005) . in this study, we generated five positive clones secreting specific and highly reactive antibodies against sars-cov n protein in order to develop diagnostic methods. these mabs were available for use in detecting sars-cov n protein by various diagnostic methods, such as immunoblot assay, immunofluorescence assay and antigen-capture elisa (table 3) . we also revealed the availability of these mabs in the quantification of sars-n protein by antigen-capture elisa. the detection limit of this test is 100 pg of recombinant protein and 10 tcid 50 of sars-cov. this sensitivity is consistent with previous studies of sensitivity in other antigen-capture elisas (che et al., 2004; di et al., 2005) . therefore, these five mabs may be employed in the construction of various diagnostic methods for the detection of sars-cov and in quantitative analysis of viral antigen and virus titer. the major antigens of sars-cov structure proteins are the spike (s) protein and n protein (lau et al., 2004; di et al., 2005; lu et al., 2004 lu et al., , 2005 . however, recent reports have demonstrated that, because the s protein is expressed at very low levels in vivo and in cultured cells (zeng et al., 2004; , it is difficult to directly detect the soluble s protein from sars patients. thus, the s protein may not be suitable for use as a practical diagnostic antigen. in contrast, the n protein can be detected at significant levels in patient serum, as well as in respiratory tract samples at early stages of sars-cov infection (lau et al., 2004; di et al., 2005) . these previous reports support that the development of mabs against sars-n protein is an adequate approach for the diagnosis of sars. therefore, we generated mabs directed against sars-cov n protein and demonstrated that these sars-n mabs can successfully detect the n protein in sars-cov-infected cells; this is very useful in diagnosing sars patients. sars n proteins exist as phosphorylated forms in mature viral particles, whereas, in host cells, this protein exists in both the dephosphorylated form and the phosphorylated form (kalicharran and dales, 1995; surjit et al., 2005) . therefore, the mabs available for developing sensitive diagnostic methods have to recognize the non-phosphorylated protein as well as phosphorylated protein. a previous report suggested that, because of conformational differences between proteins, the mabs recognizing a protein expressed in insect cells cannot recognize the protein of same cdna constructs expressed in e. coli (vapalahti et al., 1996) . therefore, the sars-n mabs obtained in the present study were generated using recombinant sars-n protein expressed in insect cells; these mabs may not recognize the n protein expressed in e. coli. however, all of the sars-n mabs reacted significantly with the ersars-n expressed in e. coli, as well as the phosphorylated form of the brsars-n protein. these results demonstrate that these mabs can effectively detect the non-phosphorylated n protein that exists in host cells during viral replication, as well as the phosphorylated n protein in host cells and viral particles. all of these mabs could successfully detect native-form n protein in infected cells, as well as in viral particles. thus, these mabs may be useful in the development of sensitive methods used for the diagnosis of sars. epitope mapping studies of the sars-n mabs demonstrated that one of the three epitopes that were originally reported to be located in the highly immunodominant region (chen et al., 2004; he et al., 2004) is located at the middle region of sars-n protein (aa 215-239; igg 1 subclass mabs). the others were newly identified at the n-terminus, which is shared with an rna binding domain (aa 17-32 and 135-150; igg 2b subclass mabs) that is the minor b cell epitope (he et al., 2004) . furthermore, all of these sars-n mabs were reactive in immunoblotting, which suggests that they recognized linear epitopes in the n protein. a recent report demonstrates that the n protein is easily degraded into various isoforms in the lysates of sars-cov-infected cells (zeng et al., 2004) . we can also suggest, as previously described, that various isoforms existed in sars-n protein expressed in insect cells and could be detected by immunoblot assay using these mabs. therefore, the blend of two mabs against the different epitopes can be used to detect various fragments from sars-n protein and enhance the sensitivity of diagnostic tools. although the sars-n protein shares low homology (approximately 20-30%) with n proteins of other hcovs, a previous report has described that the sars-n protein has strong crossreactivity with sera against hcovs (sun and meng, 2004) . hence, anti-sera against sars-cov may be cross-reactive with other hcovs. however, previous reports support the idea that the sars-n mabs did not recognize the n proteins of other hcovs (che et al., 2004) . therefore, the issues of cross-reactivity during the detection of sars-n protein with polyclonal anti-sera can potentially be overcome by the use of a specific mab against sars-cov. in the present study, the sars-n mabs did not show cross-reactivity with n proteins of hcov, as determined by immunofluorescence assay and antigen-capture elisa. therefore, all of these sars-n mabs will be useful as mabs for the development of specific diagnostic methods to discriminate sars-cov infection from hcovs infection. sars-n protein is currently assumed to play an important role in the pathogenesis of sars, as well as in viral transcription and replication. for example, the n protein can modulate numerous intracellular mechanisms involved in apoptotic signal transduction pathways, cell cycle regulatory pathways and cellular immune response and inflammation (egloff et al., 2004; yan et al., 2004 yan et al., , 2006 liao et al., 2005; surjit et al., 2004 surjit et al., , 2006 surjit et al., , 2005 chang et al., 2006) . furthermore, sars-n protein is released as a soluble antigen in infected cells, and as high levels of n protein circulating in the blood vessels (che et al., 2004; di et al., 2005) . thus, sars-n protein in its soluble form may play an important role in extracellular pathogenesis of sars. however, it is totally unclear which of the intracellular and extracellular mechanisms are involved in viral replication and pathogenesis, and how the intracellular mechanisms are regulated by the n protein. therefore, these sars-n mabs may be extremely useful in providing further insight into the mechanisms of the n protein 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b and ccaat/enhancer binding protein proteomic analysis of sars associated coronavirus using two-dimensional liquid chromatography mass spectrometry and one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by mass spectrometric analysis we gratefully acknowledge w. bellini from the centers for disease control and prevention for kindly providing of the sars coronavirus. we also thank neurogenex co. for the technical assistance in the production of recombinant baculovirus and cosmo inc. for the technical assistance in the production of the sars mabs.this work was supported by the intramural research fund from the korea centers for disease control and prevention. key: cord-262585-5vjqrnwh authors: hraber, peter; o’maille, paul e.; silberfarb, andrew; davis-anderson, katie; generous, nicholas; mcmahon, benjamin h.; fair, jeanne m. title: resources to discover and use short linear motifs in viral proteins date: 2019-08-16 journal: trends biotechnol doi: 10.1016/j.tibtech.2019.07.004 sha: doc_id: 262585 cord_uid: 5vjqrnwh viral proteins evade host immune function by molecular mimicry, often achieved by short linear motifs (slims) of three to ten consecutive amino acids (aas). motif mimicry tolerates mutations, evolves quickly to modify interactions with the host, and enables modular interactions with protein complexes. host cells cannot easily coordinate changes to conserved motif recognition and binding interfaces under selective pressure to maintain critical signaling pathways. slims offer potential for use in synthetic biology, such as better immunogens and therapies, but may also present biosecurity challenges. we survey viral uses of slims to mimic host proteins, and information resources available for motif discovery. as the number of examples continues to grow, knowledge management tools are essential to help organize and compare new findings. viruses exploit host cellular processes to replicate, and have developed myriad ways to subvert host immune defenses. molecular mimicry (see glossary) is a common and effective strategy, enabling a pathogen to usurp host protein function by resemblance [1, 2] . molecular mimicry varies over a continuum, from one extreme that includes sequence and structural similarity (i.e., orthologs) of entire proteins, to another extreme of chemical similarity at only a few localized sites, as is the case for short linear motifs (slims). the growing body of literature on slims indicates that some important virushost interactions can be attributed to a few well-chosen aas [3] [4] [5] [6] [7] . rather than devote entire proteins to one function, slims enable multifunctional viral proteins. interactions between globular virus and host proteins have picomolar affinities, while slims have micromolar binding affinities with globular host proteins [8] . moderate binding affinity of slims facilitates disruption of signaling interactions, rather than competing for stable formation of persistent protein complexes. synthetic biology practitioners can benefit from an introduction to how slims enable viral interference with host cell functions and computational resources available for slim analysis. viral slims are potentially useful in synthetic biology, to provide a toolkit for new functions, for example, to modulate immune responses or to complement and interact with newly developed adjuvants in a synergistic manner [9] . research efforts to develop broad-spectrum antiviral compounds or design broadly cross-protective vaccine immunogens benefit directly from knowledge of gene products, protein functions, and motifs involved with viral immune interference. n-linked glycosylation of the png sequon is a well-known example used by viral glycoproteins as camouflage against immune recognition [10, 11] . the distribution of n-linked glycosylation sites has recently been recognized as essential for the design of immunogens to induce broadly cross-reactive immune protection against such challenging viruses as hiv-1 [12, 13] . motifs associated with cellular trafficking (localization, transport, secretion, and sequestration) are readily edited to modify where expression products go, and change interaction profiles with other proteins [14] . in addition to motifs that stabilize the structure of immunogens, such as trimerization ('foldon') [15] [16] [17] [18] and dimerization [19, 20] domains, motifs that interact with cellular processes for innate antiviral pathways could be used to enhance immunogenicity. while slims in eukaryotic proteins have been discussed extensively, slim involvement in viral immunomodulation remains less thoroughly explored, and suggests new opportunities for use in engineered biotechnology applications. the ability to transfer genetic components across species, or to introduce such components de novo, enables new functions. while such functions are generally well intended, some risk also exists for harmful effects. subject to the technical advances of synthetic biology, such effects are not short linear motifs (slims) are patterns of three to ten consecutive aas used by eukaryotic cells for tasks that include: signaling, localization, degradation, and proteolytic cleavage. viruses use slims to their advantage, including interference with antiviral innate immune pathways. viral slims can tolerate mutations, evolve quickly to modify host interactions, and co-occur in a modular manner or involve multiprotein complexes. slims are useful in synthetic biology, where minor edits can alter target specificity, modulate persistence, reprogram interactions with cell-signaling domains, and alter protein function in myriad other ways. aside from possible beneficial uses, for example, to produce better immunogens and develop therapeutic interventions against infectious disease, slims may help characterize new and emerging threats to global health. necessarily a taxonomically relevant property. it may be necessary to evaluate risks of new functions by other means than taxonomy or even protein functional evaluations. instead, new methods are needed that assess functions at a finer resolution than the gene, whether by computational analysis or functional phenotypic assessments [21] [22] [23] . slim analysis might help with such assessments. viral proteins can modulate immunity in several ways, which include: shutdown of host macromolecular synthesis, inhibiting antigen production or apoptosis, and interference with such processes as antigen presentation by mhc, natural killer (nk) cell function, antiviral cytokines, or interferon responses. each of these processes involves coordination among multiple components in host cells. viral interference with these functions is frequently attributed to entire proteins, but in some important cases has been localized to slims. because of their compact size, slims are modular, rapidly evolvable sequence elements. different instances of a given slim can vary in sequence while maintaining the overall functional profile, that is, the regular expression for the sequence motif, where a few positions are invariant while other positions tolerate numerous substitutions. thus, partial sequence matches are sufficient for transient binding interactions with target domains, for example, signal transduction proteins. this observation led to the proposal of ex nihilo slim evolution -the evolution of a novel slim 'from nothing' -the appearance of a new functional module from a previously nonfunctional region of protein sequence [24] . because hosts' interaction networks are often conserved, slims represent a significant vulnerability for opportunistic exploitation. these properties enable pathogens to acquire host-like slims rapidly through ex nihilo convergent evolution, to rewire host interaction networks, and to acquire tropism and virulence traits needed for successful adaptation and propagation [25] . over 200 motifs are known, with 2,400 validated instances, and many more motifs may await discovery [3, 26] . focus on viral motifs may reveal practical utility, to broaden the repertoire of tools available to reprogram molecular function in synthetic biology. one example of how viral proteins use slims to subvert host cell function is illustrated by epstein-barr virus (ebv), which persists in resting memory b cells of nearly all (>95%) individuals throughout their adult lives [5] . latent membrane protein 1 (lmp1) is central to ebv persistence. the cytoplasmic tail of this membrane-bound protein includes pxxpxp and pxqxt motifs that recruit signaling proteins (jak3, that is, janus kinase 3, and several trafs, tumor necrosis factor receptor-associated factors, respectively) [5] . together, the motifs mimic the cytoplasmic domain of cd40 to activate nuclear factor-kb via intermediates, including a third motif yyd$ (where $ denotes the c terminus), the tradd (tumor necrosis factor receptor-associated death domain) binding domain. the overall result is that lmp1 inhibits apoptosis and infected b cell proliferation, to confer viral persistence [5] . other examples of viral slim contributions to motif mimicry involved with immune function include: protein degradation, transcription, translation, and transport into and out of the nucleus [5] . given the continued growth of this field [27] , established frameworks can manage and exploit this knowledge beyond catalogues of currently known motifs [5, 7, 28] or details on contributions of one viral protein (e.g., [14, 29] ). work to use slims in bioengineering can benefit from understanding viral protein function. this information is organized in viral knowledge bases, such as viralzone (box 1). ontologies describe systematically the many different functional roles of viral proteins, including immune evasion. by promoting use of standard terms for relationships between concepts, an ontology arranges concepts into a framework that can be updated as knowledge grows. protein function is captured broadly in such a framework, though the nuanced details of interactions with other molecules are not localized to domains or motifs. go is an authoritative resource for annotating functions of gene sequences [30, 31] . an example of interest is 'evasion or tolerance by virus of host immune response' [32] (www.ebi.ac.uk/quickgo/ glossary adjuvant: an additive to a vaccine that promotes nonspecific immune responses. when administered together with an antigen, it induces more potent responses than the antigen alone. autophagy: an evolutionarily conserved degradation system for maintaining cellular homeostasis and innate immunity to clear pathogens from cells. domain: structure-based modular subunit of a protein, often with a specific function. domains are generally larger and associated with structural subunits, while motifs are shorter and associated with intrinsically disordered protein regions. elm: the eukaryotic linear motif resource (elm.eu.org), a repository of known slims; includes annotation from primary literature and information about experimental assays used. glycosylation: post-translational modification by host transferases linking sugar molecules to side chains of either nitrogen (nlinked) in asparagine or oxygen (o-linked) in serine or threonine. viral proteins like hiv-1 envelope can be heavily glycosylated, providing camouflage against immune recognition, shifting as the png sequon mutates. go: gene ontology, an ontology for genetic products of any and all organisms, providing a framework to annotate gene and protein functions in genetic sequence databases and bioinformatics analysis procedures. go includes multiple dimensions to capture biological complexity in adequate depth: molecular function, cellular component, and biological process. go development is conducted by a consortium of research communities and databases, which regularly solicits input and feedback from the broader research community. immune modulation: interference with an immune-related process by a pathogen. intrinsically disordered protein/ domain: regions of a protein sequence that are predicted or experimentally shown not to form consistent structure, e.g., a helices or b sheets. such regions tend to be more accessible for interactions with other proteins. term/go:0030683, figure 1 ). concepts are hierarchically organized, and include a definition, synonyms, and lists of parents and children. functional annotation in go reflects the diverse effects of viral proteins on immune interference. modulating autophagy is an example of recent advances in this research area [33, 34] . a growing number of reports describe how virus proteins and slims therein modulate autophagy to promote various aspects of their life cycle [34, 35] . both go and viralzone have developed concepts to detail molecular mimicry: structural similarity that enables repurposing or hijacking of molecular function by pathogens, such as viruses. molecular mimicry varies in extent from entire globular proteins, to localized domains, down to short linear motifs. motif class: a regular expression that summarizes known variants to define a sequence motif. motif instance: a particular motif as found in a protein or translated genetic sequence from a specific organism, strain, or isolate. ontology: a representation of domain-specific knowledge organized as concepts, their properties, and their relationships. png sequon: three aa motif n [^p] [st] , that is, asparagine, then any aa except proline, then serine or threonine, recognized by glycosyltransferase as a potential nlinked glycosylation site. regular expression: a string of characters that concisely represents many alternative sequence variants; may include wildcards to represent any character, groupings of possible characters, repetition, negation, start and end of sequence, etc. short linear motif (slim): also known as minimotifs or morfs (molecular recognition features). frequently represented as regular expressions, typically three to te-naas long. viralzone: a knowledge base (https://viralzone.expasy.org) that documents viral families, genome architectures, proteins, hostprotein interactions, and an ontology for the functions of viral proteins. a bridge that links literature reports to go term annotation, viralzone is an online knowledge base that contains 'textbook' information about viral taxonomy, replication, genome organization, and virion structure, and provides links to viral sequence data [62, 63] . importantly, viralzone staff collaborate with the go consortium to define entries for virus-specific molecular functions [63] [64] [65] [66] . viralzone cross-references its keywords with go consortium terms and uniprot [67] identifiers. this makes it possible to search for viral proteins by their functional role. viralzone staff have developed go concepts specifically for viruses, to represent the diversity of viral replication and processes involved with viral entry, replication, and egress [66] . viralzone staff have also developed a detailed listing of virus-host interactions, with entries for 65 functions and 57 go terms [64, 65] . each entry ('keyword') has a unique identifier. unlike enzyme commission (ec) numbers [68] , viralzone ids are arbitrary numbers and do not indicate position in the concept hierarchy. instead, organization of the keyword hierarchy is provided online. the web address https://viralzone.expasy.org/886 is an entry point into the viralzone concept space ( figure i) . blue text indicates a link to more detail. shown is the vertebrate host-virus interactions page [64] . also available are summaries for invertebrate, plant, and bacterial host-virus interactions. adapted from www.ebi.ac.uk/quickgo/term/go:0030683 [32] . most arrows indicate 'is a' relations, where the hierarchy is refined by specialization. blue arrows indicate 'part of' relations, which relate to the symbiont process as parts to a whole. autophagy processes, including positive and negative regulation of xenophagy, the selective autophagy of pathogens [33] [34] [35] . understanding the functional roles of slims can help identify related mechanisms or processes, or possibly identify knowledge gaps where slims may be posited but not yet identified. an overview of slim functions may also help to prioritize which are of greatest potential for use or abuse when artificially added to modify protein function. databases and discovery tools are also useful to identify known and new slims. identification of shared structural features across divergent protein families led to analysis and identification of modular protein domains. protein domains are used to categorize protein function, and the interpro database [36] (www.ebi.ac.uk/interpro) aggregates information at this within-protein level. the identification of protein domains led to recognition of slims as compact, small-scale functional modules [37] . elm is a database of eukaryotic motifs (box 2), though its representation of viral-host interactions is not fully developed. at present, 264 elm entries map to 648 go terms. most motifs map to multiple go terms; the median is seven go terms per motif and the maximum is 29 (mod_plk_1). immuneassociated function of the lig_irf3_lxis_1 motif is involved in signal transduction responses to pathogen-associated molecular patterns; this motif maps to 25 go terms, but none occur in the viralzone vocabulary. in total, only three elm entries utilize go terms from viralzone: lig_bh_bh3_1, lig_hcf-1_hbm_1, and lig_rb_pabgroove_1. these three motifs all map to the most general (ii) deg, degradation sites, part of polyubiquitination; (iii) doc, docking sites, involved in protein recruitment but not directly targeted by an active site; (iv) lig, ligand binding sites, primarily for protein-protein interactions; (v) mod, post-translational modification sites; and (vi) trg, targeting sites for subcellular localization. elm has also spun-off several specialized databases: phospho.elm for phosphorylation sites with experimental evidence [71] , switches.elm for conditional molecular switches, such as requiring that a site be modified [72] , and ielm, with an emphasis on protein-protein interactions [73] . a detailed tutorial provides orientation for elm use [74] . elm documents each motif class with a concise description of its function. for example, one type of nuclear localization signal (nls), trg_nls_bipartite_1 the 'classic bipartite nls', which binds to importin-a for nuclear pore transfer and is utilized by the pb2 protein of influenza a, is documented here: elm.eu.org/elms/ trg_nls_bipartite_1. the abstract and functional site descriptions summarize what is known about the motif. go term, go:0019048 ('modulation by virus of host morphology or physiology', a synonym of 'virushost interaction'). this underscores the prevalent mode of elm motif discovery and annotation does not emphasize host-virus interactions, but rather systems-level interactions within eukaryotic cells. thus, better integration of viralzone-go-term vocabulary with elm or another domain-level representation of viral slims is needed to promote potential utility for biotechnology. despite not using the viralzone ontology, elm documents other motif classes with go terms that refer to 'viral', 'virus', 'immune', or 'immunity' (table 1) . because the focus is motif function in the host cell context, elm does not directly indicate how viral immune interference results. further, the relative lack of viral motifs in elm does not indicate their absence in vivo, but rather the evidence-based requirement for elm inclusion. related to the earlier observation, a review of how viruses use slims to interfere with host cells [5] lists 52 examples that represent viral mimicry of host slims (table 2 ). only 70% of these have corresponding elm entries, though the slims are known. the remaining 30% indicate that elm does not fully capture all known viral motifs. this strong requirement by elm for evidence-based motif classes and instances is not strictly a drawback. indeed, the elm creators are very aware that computational analysis alone is error prone and can yield misleading outcomes. in [38] , they discuss this issue in depth, and recommend a workflow for slim discovery that culminates in experimental validation, whether in vivo or in vitro. working with viral-host systems adds layers of difficulty to experimental motif validation, so it should not be surprising or to the detriment of available information resources that viral slims are less thoroughly documented. searching arbitrary sequences for motif instances is computationally straightforward. box 3 provides an example of motif searching with elm, which might facilitate comparative analysis of two related proteins from different species of human herpesvirus (hsv). resources such as interpro and uniprot are able to perform similar assessments, but give broader, domain-level representations with less functional detail than the slim searches enabled by elm. reports in the primary literature take a different approach, by marking slims in a protein alignment, which includes orthologues to mark conservation (e.g., figure 3 in [39] ). the elm-generated report combines predicted slims with information from annotated domains and local disorder predictions, for a perspective that complements the other approaches. clearly, false positives are inevitable among slim search results. this makes it necessary to filter for the most significant and informative outcomes. this leads to consideration of in silico (computational) methods for slim evaluation. a highly recommended, authoritative review of slim discovery techniques, from an author of the slimsuite software package, discusses motif identification techniques in depth [40] . methods for slim discovery can be divided into two broad classes: (i) de novo discovery of new slims, and (ii) instance prediction to find new occurrences of known slims. there are currently at least eight software packages available to discover new slims and 40 packages (25 stand-alone programs or servers and two software suites that consist of multiple tools: slimsuite [40] , which includes ten utilities, and meme [41] , which consists of five tools for slim instance detection). though meme was developed for discovery of dna sequence motifs, it generates ungapped, profile-based motifs using the expectation-maximization (em) algorithm. no single method is inherently better than the rest, but the choice of which to use depends on several factors, such as the input sequence data, whether one sequence, an alignment, or a collection of nonhomologous sequences [40] . to illustrate the diversity of motif discovery methods, this section mentions only a handful of the software tools available (table 3) . readers seeking to learn more about the full set of alternatives are strongly encouraged to consult [40] , particularly tables 1, 4, and 5 therein. another helpful resource ( table 1 in [38] ) lists online motif discovery bioinformatics services. several alternative approaches for discovery of new motifs have been advanced. edwards and palopoli [40] review the alternatives in depth, discussing their merits and drawbacks. briefly, they can be divided into alignment-based and alignment-free methods. an alignment-based approach looks for conserved sites among homologous sequences, but can be misled by high sequence conservation in globular domains. a program called slimprints works around this with a specialized approach to model substitutions [42] . slimprints uses a statistical model of relative local conservation, which looks for clusters of overly constrained sites in a window of about 30 aas, using iupred scores (intrinsically unordered prediction; see later) to weigh sites in intrinsically disordered protein regions more heavily than sites in globular (ordered) regimes [42] . in contrast, alignment-free methods look for enrichment of amino acid patterns in proteins that are expected by other means to perform similar motif-related roles, for example, by go category annotations or protein-protein interaction (ppi) data, that is, via databases that capture experimental evidence for protein colocalization and functional interactions. an important caveat is that to assume such sequences are independent could yield spurious enrichment of shared patterns, so alignment-free methods need to compensate for evolutionary constraints at the domain level, rather than for full-length homologous proteins. the development of such corrections and their relative advantages are detailed in [40] . some programs (e.g., slimdisc [43] , slimfinder [44, 45] , and dilimot [46, 47] ) produce regular expressions that compensate for phylogenetic relatedness, while others (meme suite, glam2 [48] , and nestedmica [49, 50] ) produce probabilistic profiles. for more discussion of these and issues of concern for computational motif discovery, see [40] . filtering methods control high false positive rates from slim instance detection. structural information, whether known or predicted, can be used for filtering. box 3 illustrates how elm filters results to hsv-1 and hsv-2 virulence factor icp34.5 assists in viral immune evasion by molecular mimicry. hsv-1 neurovirulence protein icp34.5, encoded by the g34.5 gene, initiates immune interference by binding and sequestering cellular proteins that would stimulate autophagy, translational arrest, and type i interferon responses. hsv-1 icp34.5 binds tank-binding kinase 1 (tbk1) to prevent type i interferon induction [75] , beclin-1 to prevent autophagy [76] , and both pp1a and eif2a to overcome translational arrest [77] . hsv-2 g34.5 contains an intron not present in hsv-1, and up to four isoforms of hsv-2 icp34.5 are known [78] . full-length hsv-2 icp34.5 has conserved pp1a and eif2a-binding domains, but lacks tbk1 and beclin-1 binding domains [79] . additional hsv-1 motifs influence intracellular localization [80] , virion maturation, and egress [81] , not yet characterized in hsv-2. hsv-2 is recognized as more virulent than hsv-1, but both can cause neuropathology, including viral encephalitis and meningitis [82] . to attenuate virulence, icp34.5 is routinely deleted or inactivated when making hsv-1 constructs for oncolytic therapy [82] . both are the same length and share domain structures, and partially share slim compositions ( figure i ). identifying differences in slims from each could provide clues for more detailed experimental investigations to understand icp34.5 virulence determinants and host protein targets. exclude a region predicted to fold as globular protein. these predictions were made by smart (simple modular architecture research tool) [51] and pfam [52] domain matches, corroborated by globplot [53] . another widely used approach is to identify regions of local disorder, where protein structure is not clearly defined, making that region accessible to interact with other proteins. iupred [54] is commonly used for this task, though the choice of parameter settings and how to interpret results varies. elm results include an iupred disorder score and a simple cutoff of 0.5 to define the disorder transition. above this value, local protein structure is considered accessible for interaction with other proteins. scoring schemes filter for statistical enrichment of motif instances. an approach of filtering by homology [55] seems inappropriate for use to detect virus interactions with host proteins, as it may exclude nonhomologous regions with motifs that do interact, yielding false negatives. regardless, failure to consider evolutionary relatedness among sequences being searched could introduce bias due to common ancestry, rather than independence, among sequences. a simple approach to instance prediction is a stand-alone program called shettimotif [56] . it was used to scan 2251 protein sequences from 11 poxviridae genomes (an average of 205 proteins per poxvirus) for low-complexity regions and regular expressions defined by prosite. the approach compared numbers of proteins per genome that carry each motif, and doubtlessly includes many motif instances that are not functional as slims. also, shorter motifs occur more frequently than longer motifs [3, 27] , partly due to chance alone. regardless, systematic error may be considered a source of background noise across the large number of proteins in 11 viral proteomes, each having different host specificities, to enable somewhat meaningful comparisons, in such a 'statistical genomics' approach [3] . the comparisons could be more meaningful if false positive motif instances were reduced. becerra et al. developed another approach to instance counting [57] , which involves comparison with a null distribution from permuting primary sequence and testing for presence of the motif in the permuted sequences. a motif is considered rare and therefore significantly unlikely to occur by chance if it is present at or below some cutoff frequency. restricting the sequence region that is used for permutation testing, such as by use of structural considerations, can further focus the search. indeed, such a hybrid filtering approach was described recently and evaluated on the hiv-1 proteome [57] . following methods described in an earlier study [58] , becerra et al. used iupred with a modified, window-based scoring procedure to identify intrinsically disordered protein regions, and tested for statistical rarity below 1% of 1000 shuffled variants. the approach further considered conservation above 70% in a set of aligned sequences, though combining three filtering criteria was too stringent and excluded all motif candidates [57] . while algorithmic approaches seek to identify a broad range of slim types, more specialized resources have emerged to track the distribution of a particular slim in viral proteins. for example, ilir@viral is a web resource dedicated to detecting lir motif-containing proteins in viruses [59] . lc3interacting regions (lir motifs) are slims that mediate protein-protein interactions involved in autophagy, as used by influenza a virus m2 protein to subvert autophagy and maintain virion stability [60] . using curated text mining analysis and position-specific scoring matrices, ilir@viral analyzed 16 609 reviewed viral sequences available from uniprot across 2569 individual viral species and found that 15 589 viral sequences contain lir motifs. while many predicted instances may represent false positives, the enrichment of lir motifs in viral sequences is consistent with viral adaptation to host xenophagy [35] . curiously, elm currently lists the lir motif as a candidate, rather than an accepted motif class. embedding slims into engineered constructs may enable specific effects on cellular immune processes, for applications that include targeted drug delivery, pathogen-specific adjuvants, potent and broadly effective immunogens, transformational medical countermeasures, and improved design of vectors for gene therapy. slim modularity may enable easy ways to reprogram protein function with a few localized modifications. to realize the potential utility of slims in synthetic biology, more research is needed to expand and integrate our collection of knowledge on viral slims (see outstanding questions). detecting slims in variant sequences may help to identify functional innovation or changes in virulence, in a manner that does not rely strictly on functional assessment at the whole-gene level, to identify how sequence-specific variation may interact with host responses. this may be particularly useful and important to understand new variants and assess the risk that they may spread and cause harmful effects on human health or agricultural interests. such knowledge is needed in an era where synthetic biology may introduce new risks for biological error and biological terror. detecting and understanding slim variants can help to reduce such risks and identify newly emerging threats to global health and security because watch lists for harmful organisms to ensure public safety by preventing access to select known risks may be inadequate [21] [22] [23] . slims in viral proteins can interact in many different ways with host proteins to modulate immune responses. a motif may be necessary but not sufficient for any inferred function. the simplest case is where a viral slim interacts directly with a host protein to yield an immunomodulated phenotype. more elaborate cases are known, such as the multifunctional proteins e1a (ebv), nef (hiv-1), and icp34.5 (hsv). computational prediction of slim classes and new instances is a process, which involves experimental confirmation and validation. high-throughput methods for experimental assessment of protein interactions are useful to validate computational predictions [38, 61] , and more assays are needed to evaluate functional and phenotypic effects of adding or deleting slims. resolved map of human-virus protein-protein interaction networks. plos pathog. 9, e1003778 what specific constraints limit slim evolvability? what strategies are most effective to advance knowledge of viral immunomodulatory slims in the design of vaccines and therapies to promote global health? for example, can some viral peptides be useful as adjuvants? signatures of pleiotropy, economy and convergent evolution in a domain pathogen mimicry of host protein-protein interfaces modulates immunity use of host-like peptide motifs in viral proteins is a prevalent strategy in host-virus interactions slimsearch 2.0: biological context for short linear motifs in proteins how viruses hijack cell regulation attributes of short linear motifs how pathogens use linear motifs to perturb host cell networks short linear motifs: ubiquitous and functionally diverse protein interaction modules directing 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short linear motif-mediated interactions exploring short linear motifs using the elm database and tools control of tank-binding kinase 1-mediated signaling by the g 1 34.5 protein of herpes simplex virus 1 hsv-1 icp34.5 confers neurovirulence by targeting the beclin 1 autophagy protein a conserved domain of herpes simplex virus icp34.5 regulates protein phosphatase complex in mammalian cells up to four distinct polypeptides are produced from the g34.5 open reading frame of herpes simplex virus 2 herpes simplex virus 2 icp34.5 confers neurovirulence by regulating the type i interferon response an n-terminal arginine-rich cluster and a proline-alanine-threonine repeat region determine the cellular localization of the herpes simplex virus type 1 icp34.5 protein and its ligand, protein phosphatase 1 replication of herpes simplex virus 1 depends on the g 1 134.5 functions that facilitate virus response to interferon and egress in the different stages of productive infection the herpes simplex virus neurovirulence factor g34. 5: revealing virus-host interactions elm: the status of the 2010 eukaryotic linear motif resource key: cord-264996-og3sg0qw authors: howell, gareth j.; holloway, zoe g.; cobbold, christian; monaco, anthony p.; ponnambalam, sreenivasan title: cell biology of membrane trafficking in human disease date: 2006-09-17 journal: int rev cytol doi: 10.1016/s0074-7696(06)52005-4 sha: doc_id: 264996 cord_uid: og3sg0qw understanding the molecular and cellular mechanisms underlying membrane traffic pathways is crucial to the treatment and cure of human disease. various human diseases caused by changes in cellular homeostasis arise through a single gene mutation(s) resulting in compromised membrane trafficking. many pathogenic agents such as viruses, bacteria, or parasites have evolved mechanisms to subvert the host cell response to infection, or have hijacked cellular mechanisms to proliferate and ensure pathogen survival. understanding the consequence of genetic mutations or pathogenic infection on membrane traffic has also enabled greater understanding of the interactions between organisms and the surrounding environment. this review focuses on human genetic defects and molecular mechanisms that underlie eukaryote exocytosis and endocytosis and current and future prospects for alleviation of a variety of human diseases. the human cell is a complex network of membranes and protein enclosed in a membrane lipid bilayer. the interactions within and associated with such biomembrane bilayers have profound consequences for the organism as a whole; a single defect in just 1 of the potential 30-40,000 gene products made by each cell can cause devastating, if not fatal, evects for the whole organism. in addition to this, humans pass genetic information onto their ovspring and, with it, any genetic mutations or polymorphisms. it is believed that at least 1 in 10 people have, or will eventually develop, a disease caused by mutation or variation at the gene level. understanding how genetic mutations increase risk for human disease is critical in our understanding and treatment of the majority of human ailments that are caused by interactions between the organism and the environment. this review focuses on the research undertaken in the past 30 years relating to the molecular mechanisms that underlie membrane traycking within eukaryotic cells. we address mechanisms and factors that control protein progression through the secretory and internalization pathways and highlight key human diseases that illuminate mechanisms of membrane traycking. in addition, current and future strategies for therapeutic intervention in such genetic disorders are considered. common to all eukaryotic cells is the presence of multiple biomembrane lipid bilayer compartments, or organelles, which are maintained by specific protein-protein and protein-lipid interactions. such interactions are maintained within each compartment in spite of continuous traycking of membrane-bound and soluble components to diverent intracellular locations, and for secretion from the cell. in the majority of cases, this transfer of material occurs through vesicular movement: fission, docking, and fusion of membrane bilayer-enclosed intermediates occurs between donor and acceptor compartments (palade, 1975) . proteins, including membrane-bound receptors, secreted enzymes, and antibodies, begin their journey by entering the early secretory pathway at the endoplasmic reticulum (er). from here they are transported through the golgi apparatus and finally distributed to their final destination such as other intracellular organelles, the plasma membrane, or the extracellular environment. but how does a specific protein ''know'' how to reach a specific cellular destination when hundreds of newly synthesized, diverent molecules require 2 specific transport and targeting? many of these transport intermediates or vesicles, whether derived from the er, other internal organelles, or the plasma membrane, are ''coated'' with unique protein complexes, tethering factors, and regulatory factors that ensure correct targeting to an acceptor compartment. vesicle coat proteins, such as the clathrin or coat protein (cop) complexes, are relatively well studied. such complexes are assembled onto the cytoplasmic face of donor compartments to facilitate the fission of transport intermediates. allied with these coat proteins are diverent molecules that mediate recognition of cytoplasmic motifs in cargo proteins either directly (e.g., transmembrane proteins) or indirectly (e.g., soluble secreted enzymes). the snare hypothesis is central to our understanding of vesicular targeting to intracellular compartments (rothman, 1994; sollner et al., 1993) . initially uncovered in a screen for intra-golgi transport docking and fusion regulators, the snare (soluble n-ethylmaleimide-sensitive fusion attachment protein receptor) proteins have been found to regulate diverent membrane interactions in all eukaryotes via a highly conserved mechanism for membrane traycking based on accessory docking and fusion regulators. snare proteins are present on both the vesicle (vesicular or v-snare) and the acceptor (target or t-snare) and comprise coiled-coil domains that assemble to facilitate vesicle docking and membrane fusion (bennett, 1995; pelham, 2001) . in conjunction with snare proteins, small ras-related rab gtpases are implicated in further ensuring the fidelity of vesicle docking and fusion (olkkonen and stenmark, 1997) . these 20-to 25-kda proteins are gtphydrolyzing enzymes that act to recruit diverent proteins or evectors to membranes in a gtp/gdp-regulated manner (collins, 2003) . rab gtpase activity and protein conformation are regulated by interaction with soluble and membrane-bound proteins; such regulators can also tether vesicles to acceptor membranes and mediate intracellular signaling. a. early secretory pathway the endoplasmic reticulum (er) is the first stage of quality control along the secretory pathway. proteins destined for secretion (e.g., hormones), the plasma membrane (e.g., membrane-bound receptors), or other intracellular membrane compartments such as the lysosome (e.g., lysosomal proteases) emphysema and liver cirrhosis (perlmutter, 2004) 107400 endophillin ii clathrin-coated pit formation leukemia (dreyling et al., 1996; jones et al., 2001; narita et al., 1999; tebar et al., 1999) alzheimer's disease presenilin 1 presenilin 1-involved in cleavage and trafficking of amyloid precursor protein to plasma membrane neurodegenerative disorder (uemura et al., 2004) tau tau -microtubular stability through formation of aggregates autosomal dominant polycystic kidney disease (adpkd) polycystin-1 or 2 causes a defect in e-cadherin assembly and basolateral trafficking renal cysts in kidney and other tissues leading to endstage renal failure (charron et al., 2000) 173900 (continued ) autosomal dominant retinitis pigmentosa rhodopsin inhibited interaction of rhodopsin and arf4, leading to inhibited post-golgi delivery to rod outer segment narrowing of visual fields, night blindness (deretic et al., 2005) 180380 autosomal dominant ventricular tachycardia cardiac arrhythmia, hyperthermia (yano et al., 2005) 604722 autosomal recessive primary hyperoxaluria mistargeting of peroxisomal proteins to mitochondria kidney disease (danpure, 1998 ) 259900 ab-lipoproteinaemia mtp er retention thus preventing apob secretion vascular disease (sharp et al., 1993) 200100 batten's disease cln1-cln8 group of gene products implicated in regulating the processing and targeting of lysosomal and synaptic proteins neurological disease (pearce, 2000) 204200 breast cancer caveolin-1 deletion or dominant negative mutation of caveolin-1 promotes tumor progression breast cancer (bouras et al., 2004; williams and lisanti, 2005) (hayasaka et al., 1993; matsuyama et al., 2002) chediak-higashi syndrome (chs) chs1/lyst lyst involved in regulation of protein secretion from lysosomes -enlarged lysosomes partial albinism, recurrent bacterial infections, impaired chemotaxis and abnormal natural killer cell function (shiflett et al., 2002; ward et al., 2003) 214500 choroideremia (chm) rab escort protein 1 (rep1) rab27a remains cytosolic due to defective geranylgeranyl modification in chm lymphoblasts x-linked form of retinal degeneration 303100 combined factors v and viii deficiency ergic-53/p58 c-type lectin er retention and defective secretion of factors v and viii blood disease 227300 congenital finnish nephritic syndrome nephrin (nphs1), podocin (nphs2) er retention kidney inflammation (kestila et al., 1998; kramer -zucker et al., 2005) 256300 600995 pancreatic atp-sensitive potassium channel (k-atp) er or golgi retention of k-atp due to mutations in its sulfonylurea-1 (sur1) subunit excess insulin leading to hypoglycaemia (dunne et al., 2004; yan et al., 2004) congenital hypothyroid goiter thyroglobulin er storage disease. thyroglobulin is misfolded and accumulates in er constipation, large tongue, swelling around the eyes, failure to suckle, mental retardation (hishinuma et al., 1998; kim and arvan, 1998) (fan et al., 1999; garman and garboczi, 2002) familial hemophagocytic lymphoschistiocytosis (fhl) perforin -defective ctl (cytotoxic t lymphocytes) mediated killing immunodeficiency (feldmann et al., 2003; stepp et al., 1999) munc (hogg et al ., 1999; mathew et al., 2000) three forms of menkes disease can arise from diverent mutations in the atp7a gene: premature stop codons, deletions, or splicing defects. these can prevent atp7a function and/or traycking. classical menkes disease is the most common and fatality usually results by the age of 3 years. in two other nonfatal forms of menkes, mild and occipital horn syndrome, atp7a maintains the ability to transport copper ions across intracellular membranes, although traycking to the plasma membrane can be compromised (la fontaine et al., 1999) . atp7a is ubiquitously expressed and is the major copper transporter in cells of the intestine, kidney, and brain. in the liver, however, the major copper transporter is the wilson's disease protein, atp7b (bull et al., 1993) . this second p-type atpase shares strong similarity with atp7a and also translocates copper ions across membranes. although these gene products share functional similarities, mutations in atp7b result in copper accumulation in the liver and brain. familial hypercholesterolemia is an autosomal dominantly inherited disease caused by mutations in the low-density lipoprotein receptor (ldlr), leading to premature atherosclerosis and coronary heart disease. in healthy individuals, the ldlr is expressed on the surface of cells, where it binds circulating ldl particles and promotes uptake and cellular metabolism of its constituents, which includes cholesterol. in these patients, ldlr alleles display amino acid substitutions (cassanelli et al., 1998; jensen et al., 1997) , truncations (lehrm an et al ., 1987) , or mis sense mutat ions ( leiter sdorf et al ., 1993) , which can result in er retention and degradation. the point mutation at residue 209 of the insulin receptor compromises the ability of the receptor to dimerize correctly within the er, therefore leading to er retention. decreased plasma membrane levels of insulin receptor cause inhibited insulin binding after stimulus by a meal, and subsequent elevations in plasma glucose levels. this then leads to type ii diabetes mellitus (kadowaki et al., 1991) . a number of human diseases can induce the er stress response. here, the mutant protein is retained within the er, resulting in either dilation of the organelle, such as in congenital hyperthyroidism (medeiros-neto et al., 1996) and hypofibrinogenemia (callea et al., 1992) , or chronic er stress as is the case for hereditary emphysema (perlmutter, 2003) . in pelizaeus-merzbacher disease, an x-linked leukodystrophy disease, er accumulation of proteolipid protein (plp) results in oligodendrocyte apoptosis (gow et al., 1998) and the subsequent disruption of white matter formation in the brain observed in humans and mouse models. plp is a central nervous system protein that is the major component of myelin and, when expressed in cultured fibroblasts, is localized to the plasma membrane (gow et al., 1994) . the link between plp and the er stress response provides a tool for elucidating the cellular response to misfolded protein accumulation . accumulation of proteins within the er, leading to blockage of protein secretion, is an unwanted cellular property and mechanisms have evolved to overcome such events. this disposal of unwanted proteins is termed er-associated degradation (erad) (fig. 1) . as recently as the early 1990s, it was still believed that aberrant proteins were degraded within the er (fra and sitia, 1993) ; however, current models suggest that aberrant er-retained proteins actually undergo retrotranslocation and subsequent degradation in the cytoplasm. retrotranslocation has been proposed to occur through the same ''pore'' used to translocate nascent proteins into the er lumen during translation, namely the sec61 translocon (biederer et al., 1996; rö misch, 1999) . various yeast and mammalian proteins have been shown to be retrotranslocated from the er and degraded within the cytoplasm in a proteasomedependent manner, including the budding yeast proteins carboxypeptidase y, and a mutant pro-a-factor. when a mammalian protein such as cftr is expressed in budding yeast, it matures relatively slowly within the yeast er, leading to retrotranslocation to the cytoplasm and degradation (ward et al., 1995) . a further example is that of a 1 -antitrypsin deficiency. a 1 -antitrypsin is responsible for inactivating the enzyme elastase produced by lung neutrophils. in this inherited disease, a mutated form of a 1 -antitrypsin is retrotranslocated and degraded in proteasomes, leading to retention of active elastase in lung tissues and thus is a cause of lung emphysema (rutishauser and spiess, 2002) . however, retrotranslocation and proteasomal degradation may not be functionally coupled processes. pharmacological inhibitors that cause proteasome inactivation lead to egress of molecules such as mhc class i (wiertz et al., 1996a,b) , ribophorin (de virgilio et al., 1998) , and carboxypeptidase y (biederer et al., 1997) from the er to the cell cytoplasm. in contrast, inhibition of protein ubiquitination results in the retention of such molecules within the er. schmitz et al. (2004) suggest that two distinct proteasome-regulated pathways mediate degradation of retrotranslocated b-amyloid precursor protein. interestingly, endocytosed toxins that target key cytosolic factors appear to use the erad pathway to move out of the er and into the cytosol (deeks et al., 2002; hazes and read, 1997) . cholera and ricin toxins are routed from the cell surface through the golgi apparatus and to the er before being retrotranslocated into the cell cytosol. it is believed that the unusually low lysine content of these protein toxins prevents subsequent er-associated ubiquitination for degradation by the cytosolic proteasome. protein cargo is shuttled between the er and golgi within vesicular intermediates or 50-nm-diameter spherical vesicles containing coat protein complexes fig. 1 quality control of protein assembly within the endoplasmic reticulum. proteins destined for the secretory pathway (this example shows a transmembrane protein) are cotranslationally translocated from the ribosome into the lumen of the endoplasmic reticulum (er) through a portal referred to as the sec61 translocon. as the newly synthesized protein enters the er, quality control mechanisms in the form of protein chaperones bind to it and fold it to its correct conformation. further processing occurs through interactions with other chaperones before the successfully folded protein is loaded into copii-coated vesicles and shuttled from the er to the golgi apparatus. however, if the protein carries a mutation that causes it to take on an aberrant conformation the er chaperones will trigger a misfolded protein response. this has two outcomes: either the chaperones will remain bound to the misfolded protein, preventing its escape from the organelle (er retention), or the protein will be ubiquitinated and retrotranslocated through the sec61 complex for proteasomal degradation in the cell cytoplasm. a number of human genetic diseases are a result of key proteins failing to trayc through the secretory pathway and as a consequence are retained or degraded in this manner. such as copi or copii. initially discovered in mammals and yeast (kaiser and schekman, 1990; malhotra et al., 1989; novick et al., 1980; rothman and wieland, 1996) , cop complexes are required for the formation of vesicles at the er, er-golgi intermediate compartment (ergic), and golgi apparatus. cop recruitment to membranes facilitates the specific capture, packaging, transport, and delivery of membrane-bound and soluble protein cargo to an acceptor compartment. copii recruitment to sites on the smooth er initiates the formation of anterograde (forward) transport vesicles. these copii vesicles move from the er to the ergic, or vesicular tubular clusters (vtcs). from here, copi-coated vesicles are thought to mediate the continued anterograde movement from the ergic to the cis face of the golgi apparatus (scales et al., 1997) . the sar1p gtpase regulates copii vesicle formation via interaction with the sec12p guanine exchange factor (gef). sec12p-mediated activation of sar1p to a gtp-bound form leads to recruitment of the sec23p-sec24p heterodimer to membranes; this also initiates protein cargo selection within the er and recruitment of v-snares such as bet1p and bos1p. binding of sec23p-sec24p mediates further recruitment of the sec13p-sec31p complex. this copii complex then acts as a protein scavold that causes deformation of the membrane, resulting in vesicular fission, with anterograde movement of protein cargo-containing copii vesicles to the ergic. copii docking at an acceptor compartment is thought to trigger sec23p function, causing a conformational change in sar1p and gtp hydrolysis and dissociation or uncoating of the copii complex. thus copii vesicle docking and fusion with an acceptor compartment are mediated by cognate v-snare/ t-snare interactions (kirchhausen, 2000; kuehn et al., 1998; matsuoka et al., 1998; tang et al., 2005) . a severe hereditary bleeding disorder called combined deficiency of factor v factor viii (f5f8d) highlights the functional importance of traycking between the er and ergic. some f5f8d patients are deficient in the ergic-localized ergic-53 (lman1) protein and display defective secretion of the factor v and viii clotting factors. ergic-53 is a mannosebinding lectin that acts as a ''cargo receptor'' and recycles between the er and ergic (neerman-arbez et al., 1999; . however, 30% of f5f8d patients show normal levels of ergic-53/lman1, but are deficient in an associated protein, mcfd2, another ergic resident that interacts with ergic-53/lman1 in a calcium-dependent manner . small intestinal cells called enterocytes absorb fats and fat-soluble vitamins from food in the form of fatty acids and monoglycerides. the fats enter the lumenal surface of absorptive enterocytes by free divusion across their membranes, and emerge from the basolateral surface as particulate structures referred to as chylomicrons. formation of chylomicrons occurs within the er and golgi apparatus by vesicular transport before being traycked from the golgi to the plasma membrane. chylomicron retention disease (cmrd), anderson disease, and a neuromuscular disorder, cmrd associated with marinesco-sjö gren syndrome (cmrd-mss) , are examples of inherited diseases that result in compromised fat absorption, low blood cholesterol, and severely depleted blood chylomicron levels. jones et al. (2003) identified eight mutations in the sar1p gene product and copii component associated with these lipid absorption diseases, thus strongly implicating a role for the copii vesicular transport system in the movement of dietary fats from the intestine to the circulating bloodstream. copii mediates anterograde trayc from the er to the golgi apparatus; however, copi vesicles appear to function primarily in the retrograde (backward) transfer of proteins from the golgi and ergic back to the er. this retrograde trayc is necessary for recovering escaped er resident proteins, coat and snare proteins that have arrived at the ergic and golgi from copii vesicles, or glycosylation enzymes that have been incorrectly modified (duden, 2003; lee et al., 2004) . the golgi-associated copi coatomer is a complex of seven polypeptides: a-, b-, b 0 -, g-, d-, e-, and z-cop gene products, which interact with the donor membrane to form copi vesicles. vesicle formation is triggered by the gtpase adp-ribosylation factor 1 (arf1), which recruits copi coatomer to the donor membrane. transmembrane proteins containing cytoplasmic lysine-based motifs such as kkxx or kxkxx, or soluble proteins containing the c-terminal kdel motif, are recycled by copi-coated vesicles from the golgi apparatus back to the er. the kdel motif, present in soluble er chaperones such as bip and protein disulfide isomerase, is recognized by the membrane-bound kdel receptor (majoul et al., 2001) . in both cases, cytoplasmic motifs in these transmembrane proteins are recognized and bound by copi coatomer, promoting inclusion into vesicles destined for the er. actin microfilaments are also involved in this retrograde transport step (valderrama et al., 2001) . this golgi-er step is regulated by the gtpase cdc42 and n-wasp protein (luna et al., 2002) , factors previously implicated in actin-linked processes at the plasma membrane. live imaging of cells expressing an engineered fluorescent and temperaturesensitive vesicular stomatitis virus g-glycoprotein (ts045vsvg) demonstrated sequential action of copii-and copi-coated vesicles (scales et al., 1997) . vsvg accumulated in structures close to the er that contained intermediate compartment resident proteins. these structures then matured into vesicles that contained copi proteins. stephens et al. (2000) showed that this ''segregation'' between copii and copi vesicles occurred at a location in close proximity to exit sites on er membranes. a cop-independent mechanism has also been implicated in retrograde trayc between the golgi apparatus and the er. the rab6 gtpase is implicated in regulating the movement of bacterial shiga toxin b fragment (stb) via a retrograde step from the golgi apparatus to the er. expression of a dominant-negative gdp-bound form of rab6 inhibited stb retrograde movement, whereas copi transport was unavected (white et al., 1999) . the golgi apparatus is composed of flattened cisternae and membrane compartments that are closely juxtaposed in a stack-like appearance. in mammalian cells these stacks are positioned end-to-end, forming a ribbonlike structure near the nucleus (barr and warren, 1996) . the golgi apparatus is a highly dynamic organelle sited at the hub of the secretory pathway with key processing and sorting functions. the golgi is a polarized structure with proteins and lipids from the er received at the cis side, followed by the medial and trans subcompartments, where further glycosylation modifications occur; the trans-golgi network (tgn) is the final subcompartment where sorting and packaging events take place. the golgi apparatus also sorts proteins and lipids bound on a retrograde pathway from the cis-golgi back to the er. in addition, proteins can also return to the tgn from the endomembrane/lysosomal system (fig. 2) . controversy exists regarding the mechanism for anterograde movement of cargo proteins within the golgi apparatus. the golgi apparatus contains secretory proteins that can vary in physical size, from relatively small polypeptides to large, bulky multisubunit complexes; all need to reach the tgn for final sorting into transport intermediates. there are also resident glycosylation enzymes that have spatially restricted functions within the golgi, that is, enzymes that function within specific subcompartments to ensure the correct addition or trimming of n-and o-linked sugars on secreted proteins as they progress through the pathway. this raises a key question: how do protein and lipid cargo move through the golgi apparatus while resident enzymes retain their localization? we know that many golgi enzymes contain transmembrane golgi localization signals that mediate targeting to a specific compartment (munro, 1998) . two models have been proposed: the cisternal maturation model and the vesicular transport model (elsner et al., 2003; storrie et al., 2000) . briefly, the cisternal maturation model suggests that large proteins or aggregates remain within a single golgi cisterna, which matures through the retrograde transfer of resident enzymes via copi vesicles. in contrast, the vesicular transport model proposes that newly synthesized protein is traycked from cisterna to cisterna via copi-coated vesicles that sequentially bud ov membranes and fuse with the next subcompartment. in either case, copi-coated vesicles play a central role in intra-golgi the secretory pathway and vesicular traycking. protein enters the secretory pathway at the endoplasmic reticulum (er) and is traycked in copii-coated vesicular structures to the intermediate compartment (ergic/vtc), from which copi-coated vesicles carry it to the cis face of the golgi. cargo protein (c) continues along the secretory pathway through the golgi apparatus to the trans-golgi network (tgn). retention signals in er resident proteins (r) ensure they undergo retrograde traycking from the golgi in copi vesicles. retrograde transport of 20 transport. a number of snare proteins, such as membrin, rbet1, gs27, and syntaxin-5, have also been localized to the golgi apparatus and are required for intra-golgi transport and homeostasis (nichols and pelham, 1998) . golgi-tethering molecules called golgins and golgi reassembly stacking proteins (grasps) belong to a family of regulatory factors involved in golgi maintenance and vesicular transport. the reader is pointed to an indepth review that covers golgins in more detail (short et al., 2005) . in brief, the golgins can be anchored to golgi membranes through various mechanisms and contain characteristic coiled-coil domains that extend from the membranes as a rod-like structure (burkhard et al., 2001) . golgins such as giantin and golgin-84 are securely anchored to the membrane via a transmembrane domain near their c terminus. electrostatic or ionic interactions mediate the attachment of other golgins to membranes. for example, proteins of the grasp family (grasp65 and grasp55) bind to gm130 and golgin-45 to recruit these factors to the cis and medial golgi membranes, respectively. moreover, a large number of golgins are recruited to membranes via interactions with the rab, arf, and arl (arf-like) gtpases. vesicular and cis-golgi membrane recruitment of golgin p115 is regulated by rab1, whereas membrane attachment of yeast golgin rud3p is regulated by arf1p. golgin-97 binds to membranes by interaction with arl1p, a member of a new class of arf-like gtpases termed arls (short et al., 2005) . interestingly, autoantibodies directed against giantin, golgin-245, golgin-160, gm130, and golgin-97 golgins and grasps are present in patients with autoimmune conditions such as sjö gren's syndrome and systemic lupus erythematosus. in sjö gren's syndrome, moisture-producing glands are targeted by the autoimmune response, resulting in dry eyes and mouth (lichtenfeld et al., 1976) . systemic lupus erythematosus is a chronic rheumatic condition that avects joints and muscles, causing skin rash and kidney problems. sjö gren's syndrome patients can also simultaneously display both rheumatoid arthritis and systemic lupus erythematosus. golgi biogenesis requires golgin function at diverent stages during cell division. mammalian p115 is crucial for maintenance of the stacked nature of the golgi cisternae (puthenveedu and linstedt, 2004) . during mitosis, the golgi stack disperses into clustered vesicles. these vesicles then fuse in the daughter cells to form new cisternae, alignment and stacking of which result in the formation of a fully functional organelle. grasp65 tethers have been proposed to hold cisternae in close proximity through interactions with p115 and gm130 (shorter and warren, 1999) . the golgin p115 is also involved in tethering copi vesicles to golgi membranes (sonnichsen et al., 1998) and may be needed for snare complex assembly (shorter et al., 2002) . the budding yeast p115 homolog (uso1p) tethers copii-coated vesicles to golgi membranes during anterograde transport from er exit sites to the cis-golgi (barlowe, 1997; cao et al., 1998; sapperstein et al., 1996) . mammalian p115 is also essential for the tethering of transport vesicles to the cis-golgi (alvarez et al., 2001) and during intra-golgi transport (seemann et al., 2000; waters et al., 1992) . golgins such as golgin-84 are implicated in the regulation of golgi structure and the formation of the golgi ribbon (diao et al., 2003) . golgin-97 may function as a tethering molecule in retrograde trayc from the endosome to the tgn (lu et al., 2004) . moreover golgins are also implicated as tethering components between the cytoskeleton and the golgi apparatus (short et al., 2005) . the trans-golgi network (tgn) is the final golgi subcompartment where secreted proteins are sorted, packaged, and directed to their final destination. traycking from the tgn can occur in either a constitutive or regulated manner. constitutive transport is the continuous release of protein from the trans-golgi network. regulated secretion occurs in response to extracellular stimuli such as secretagogues, metal ions, hormones, or growth factors, which trigger the docking and fusion of secretory granules or vesicles with the plasma membrane. various mechanisms control the traycking of proteins from the tgn by the formation and delivery of membrane-derived transport vesicles to the plasma membrane, endosomes, or lysosomal structures (ponnambalam and baldwin, 2003) . the expression of inactive (dominant-negative) protein kinase d isoforms in tumor lines (liljedahl et al., 2001) , polarized canine kidney cells (yeaman et al., 2004) , and mouse fibroblasts (prigozhina and waterman-storer, 2004) has been shown to inhibit vesicle fission (release) from the tgn. vesicle release is modulated by this family of kinases in response to cellular diacylglycerol (baron and malhotra, 2002) and binding to an as yet unknown evector protein on the cytoplasmic face of the tgn (van lint et al., 2002) . in addition, the cdc42 gtpase is linked to actin remodeling and has been shown to inhibit the exit of basolateral targeted proteins in polarized cells (kroschewski et al., 1999; musch et al., 2001) and copper-regulated protein transport (cobbold et al., 2002) . copper is an essential element and cofactor required for functionality of many secreted enzymes (cuproenzymes). at steady state, atp7a (menkes 22 howell et al. disease copper transporter; section iii.a.1) resides in the tgn, where it provides newly synthesized cuproenzymes such as lysyl oxidase with copper ions as they traverse the secretory pathway. when intracellular copper ion levels rise, atp7a responds to this environmental danger by redistributing to the plasma membrane in a cdc42-regulated manner (cobbold et al., 2002) . here, atp7a acts as a copper ezux pump to remove copper ions from the cytoplasm to maintain homeostatic function and prevent toxicity. when copper levels are reduced, atp7a recycles back to the tgn. this endocytic internalization and sorting event is independent of both clathrin and caveolae (cobbold et al., 2003) , although relying on a cytoplasmic dileucine motif present in the atp7a c-terminus petris and mercer, 1999) . dent's disease, an x-linked kidney disorder that presents with hypercalciuria, nephrocalcinosis (kidney stone formation), and progressive renal failure, is caused by missense, nonsense, and deletion mutations within the endosomal clc-5 voltage-gated chloride channel. clc-5 is a member of a large family of voltage-gated chloride channels that have a diverse array of cellular functions including membrane excitability, transepithelial ion transport, and cell volume regulation (thakker, 1997) . when expressed in xenopus oocytes, a number of missense mutations in the clc-5 gene localized the channel to the golgi apparatus and showed reduced conductance and significantly reduced plasma membrane (pm) localization (ludwig et al., 2005) . similarly, expression of mutant clc-5 alleles in cultured cells revealed an approximate 5-fold increase in golgi retention (carr et al., 2003) . a. receptor-mediated endocytosis clathrin-coated vesicles (ccvs) are a route for protein internalization conserved from yeast to humans. roth and porter (1964) first observed this process in mosquito oocytes and these vesicles have subsequently become one of the best characterized membrane transport steps in eukaryotes. clathrin is one of the principal proteins involved in this transport step and, in combination with more than 25 clathrin-associated factors, this unique structural component forms transport vesicles on the cytoplasmic face of the tgn, endosomes, and the plasma membrane. clathrin-coated vesicles bud from their donor membranes and are directed to target membranes by associated proteins and factors. this highly conserved 600-kda clathrin complex comprises heavy (180 kda) and light (25 kda) chain proteins that are assembled into a three-legged structure called a triskelion. triskelions can be polymerized by accessory factors into striking lattice-like ''cages'' comprising pentagons and hexagons, resembling a soccer ball structure or buckminsterfullerene. clathrin cages are 70-120 nm in diameter; significantly larger than copi or copii vesicles (ccvs). clathrin-coated vesicles are believed to assemble through a sequence of events that can be designated as activation, cargo capture, coat assembly, scission, movement, and vesicle uncoating (kirchhausen, 2000) . members of a class of clathrin-associated factor termed adaptor protein (ap) complexes are recruited to donor membranes through interactions with a docking complex, which then further interacts with motifs within the cytoplasmic tail of cargo proteins, resulting in ''cargo capture.'' this leads to clathrin cage assembly and the concomitant polymerization of the clathrin triskelion and resultant deformation of the donor membrane. scission, or vesicle release from the plasma membrane, is believed to occur through the action of the gtpase dynamin and other accessory proteins, such as amphiphysin (wigge et al., 1997) . in the fruit fly drosophila melanogaster, a dynamin gene mutation (shibire) causes temperature-sensitive paralysis. this is likely due to a block in the endocytic uptake of synaptic vesicle proteins at the plasma membrane, leading to a block in recycling and reformation of competent synaptic vesicles at nerve terminals (chen et al., 1991; koenig and ikeda, 1989; kosaka and ikeda, 1983; van der bliek and meyerowitz, 1991) . the expression of a dominant-negative gdp-bound dynamin mutant, k44a, results in compromised ccv formation (herskovits et al., 1993; van der bliek et al., 1993) and inhibition of clathrin-mediated internalization of the glucose transporter glut4 (al-hasani et al., 1998) , human immunodeficiency virus (hiv) (daecke et al., 2005) , and influenza virus (roy et al., 2000) . the scission function of dynamin is assisted by specific lipid-modifying enzymes such as endophilin, synaptojanin, and phospholipase d (bi et al., 1997; havner et al., 1997; ringstad et al., 1999; schmidt et al., 1999; woscholski et al., 1997) . finally, ccv uncoating at the target membrane occurs through the actions of the heat shock protein hsc70 (schlossman et al., 1984) and auxilin (ungewickell et al., 1995) . sorting of proteins from donor to target membranes involves the recognition of cytoplasmic sequences in membrane proteins by clathrin-associated ap complexes. four adaptor protein complexes (ap1-ap4), each comprising four diverent subunits, have been identified (robinson, 2004) . the ap1 complex is involved in clathrin-coated vesicle formation at the tgn for transport to late endosomes; evidence has also implicated a role for this complex in a tgn-to-plasma membrane step (folsch et al., 2003) . ap2 is the best-studied of the four complexes and mediates internalization of transmembrane 24 howell et al. receptors at the plasma membrane via clathrin-coated vesicles. the ap3 complex is involved in traycking from early endosomes to either late endosomes or lysosome-related organelles such as melanosomes, platelet-dense bodies, and antigen-processing compartments. finally, the ap4 complex was the last to be cloned (dell'angelica et al., 1999a; hirst et al., 1999; ) . in contrast to ap1-ap3, ap4 does not possess the b ''ear'' domain (see below), which allows interaction with clathrin and other cytosolic factors such as eps15 and auxilin 2 (lundmark and carlsson, 2002) . by electron microscopy, ap4 has been localized to vesicles at the tgn, plasma membrane, and early endosomes, although there is debate as to whether these vesicles are clathrin-coated (barois and bakke, 2005; hirst et al., 1999) . interestingly, ap3 and ap4 may function independently of clathrin (hirst et al., 1999; vowels and payne, 1998) , suggesting the existence of another, as yet unidentified, coat protein that is analogous to clathrin. all four ap complexes comprise two large 100-kda subunits: a b subunit (b1-b4) plus a g (ap1), a (ap2), d (ap3), or e (ap4) subunit. in addition, each ap complex contains a 50-kda subunit (m1-m4) and a small 20-kda subunit (s1-s4). ap1, -2, and -3 contain two carboxyl ''ear'' domains connected to the head of each large 100-kda subunit by a flexible hinge of approximately 20-30 residues. importantly, the ear domain of the b subunit and the hinge domains of the g and a subunits have been shown to bind clathrin (goodman and keen, 1995; morgan et al., 2000; owen et al., 2000) , and consensus sequences in the hinge domains of b1 and b2 have clathrin-binding properties (dell'angelica et al., 1998) . the b and m subunits of the ap complex interact with motifs present in the cytoplasmic domains of transmembrane proteins to mediate cargo recruitment into clathrin-coated vesicles. such motifs include npxy, yxxø, and dileucine-based sequences (ø represents a bulky hydrophobic amino acid). one such motif, npxy, is present in key cellular receptors such as low density lipoprotein receptor (ldlr), epidermal growth factor receptor (egfr or erb1), and insulin receptor, and mediates endocytosis and sorting. importantly, the jd mutation (y807c) in ldlr lies within this key motif and causes familial hypercholesterolemia (knoblauch et al., 2000) . the second tyrosinebased motif, yxxø, mediates plasma membrane internalization, lysosomal targeting, and basolateral targeting of cargo. this motif is found in lysosomal residents such as lamp-1 and -2, cd63, the recycling transferrin receptor (tfr), and tgn-associated recycling membrane proteins, furin and tgn38. di-leucine motifs present on transmembrane transporters such as glut4 (glucose transporter), atp7a, and mannose-6-phosphate receptors (m6pr) can fall into two categories: [de]xxx[li] and dxxll related motifs. the [de]xxx[li] motif is associated with proteins internalized from the plasma membrane and targeted to lysosomes, while dxxll motif is found in transmembrane proteins that shuttle between the tgn and endosomal system (bonifacino and traub, 2003) . another class of clathrin-associated factor is the golgi-localized, g-earcontaining, arf-binding proteins (ggas) found on the tgn and postulated to interact with ap1 to mediate transport of m6pr (section v.b) to endosomes (doray et al., 2002) . ggas can act as multifunctional adaptors that link transmembrane proteins, arf gtpases, clathrin and accessory proteins at sites of ccv formation (robinson and bonifacino, 2001) . the disease oculocerebrorenal syndrome of lowe (ocrl) is an x-linked disorder caused by mutations in the ocrl1 gene (lowe, 2005) . the gene product is an inositol 5 0 -phosphatase that catalyzes the removal of the phosphate from this position on the inositol moiety. the preferred ocrl1 substrate is pi(4,5)p 2 , a phosphoinositide shown to be important in endocytosis because of its central role in recruiting accessory proteins to ccvs (padron et al., 2003) . ocrl1 has been localized to clathrin-coated vesicles associated with endosomal and tgn membranes (choudhury et al., 2005) . this is not surprising as ocrl1 interacts with clathrin and promotes its assembly into clathrin lattices and cages (choudhury et al., 2005; ungewickell et al., 2004) . ocrl1 also interacts with the rac1 gtpase that regulates actin dynamics, possibly via a gtpase activation domain to accelerate gtp hydrolysis (faucherre et al., 2003) . although the exact function of ocrl1 is still unclear, the disease phenotype hints to ocrl1 function in membrane traycking. ocrl1 mutations can cause loss of protein expression and phosphatase activity. rnai-mediated inhibition of ocrl1 expression in cultured human cells results in partial redistribution of a cation-independent mannose-6-phosphate receptor and a tgn recycling protein (tgn46) to early endosomes (choudhury et al., 2005) . this suggests that loss of ocrl1 perturbs endosome-to-tgn vesicle transport, suggesting a functional requirement for this membrane trayc step. it is possible that ocrl1 plays a role in anterograde traycking from the tgn-to-endosomes as well, since ocrl1 is abundantly present on tgn-associated clathrin buds destined for the endocytic pathway. ocrl disease symptoms include congenital cataracts, mental retardation, and renal tubular dysfunction (lowe et al., 1952) . renal failure in ocrl patients is probably partly caused by defects in solute and protein readsorption in kidney proximal tubules. this is likely due to missorting of megalin and cubilin, cell surface receptors involved in kidney solute uptake. in ocrl1 patients plasma membrane shedding of these receptors is reduced (norden et al., 2002) , indicating ocrl1 regulation of either receptor traycking from the tgn-to-plasma membrane or recycling from plasma membrane-to-tgn. paraneoplastic stiv-person syndrome (sps) is a neurological autoimmune disease characterized by severe muscle stivness and spasms, and often has secondary symptoms including diabetes, epilepsy, and breast cancer. autoantibodies are produced against the clathrin-associated regulator, amphiphysin i 26 (de camilli et al., 1993) , a protein shown to bind dynamin in nerve terminals (david et al., 1996) and which is implicated in regulating the endocytosis of neuronal synaptic vesicle components (burns, 2005) . in support of this hypothesis, sommer et al. (2005) showed that sps-like symptoms could be triggered in rats injected with anti-amphiphysin antibodies from a human sps patient. genetic translocations leading to the formation of hybrid clathrin-accessory proteins can lead to other forms of acute myeloid leukemia, lymphoblastic leukemia and acute megakaryoblastic leukemia (dreyling et al., 1996; jones et al., 2001; narita et al., 1999; tebar et al., 1999) . in these diseases, an aberrant hybrid protein consisting of the putative transcription factor af10 and the clathrin accessory protein calm (clathrin assembly lymphoid myeloid leukemia protein) is formed because of a partial inversion of the af10 gene on chromosome 11 (salmon-nguyen et al., 2000) . finally, in hermansky-pudlak syndrome (hps) type 2, a condition that results in partial albinism and prolonged bleeding, mutations have been found in the b3a gene that encodes a subunit of the ap3 adaptor complex (dell'angelica et al., 1999b) . hps is discussed in more detail in section v.c. originally identified more than 50 years ago (palade, 1953; yamada, 1955) , caveolae are flask-shaped invaginations of approximately 50-100 nm in diameter at the plasma membrane. these plasma membrane profiles are related to lipid rafts and contain unique mixtures of gpi-anchored proteins, transmembrane proteins, signaling factors and lipids, such as cholesterol. caveolae are believed to mediate the uptake of small solutes, regulate protein traycking (hommelgaard et al., 2005; tagawa et al., 2005) , transcytosis (transport across endothelial cells) (simionescu et al., 2002) , signal transduction (insel et al., 2005; lisanti et al., 1994; ostrom and insel, 2004) and cholesterol homeostasis (fielding and fielding, 2001) . however, their exact role in the internalization of membrane proteins and soluble protein ligands is controversial. caveolin-1, also known as vip21, is a structural component essential for the formation and stability of caveolae (kurzchalia et al., 1992; rothberg et al., 1992) . of the three members of the caveolin gene family (caveolin-1, -2, and -3) tang et al., 1996) , caveolin-1 and -2 are abundant in a wide variety of cell types including endothelial cells, adipocytes, alveolar type i pneumocytes, and smooth muscle cells (williams and lisanti, 2004) , whereas caveolin-3 is a muscle-specific isoform expressed in striated muscle cells such as cardiac and skeletal myocytes (cohen et al., 2004; tang et al., 1996) . caveolin-1 and -3 are both able to induce formation of caveolae at the plasma membrane (galbiati et al., 2001; li et al., 1996) . however, caveolin-2 requires the presence of caveolin-1 for expression, membrane localization, and formation of caveolae (razani et al., 2002) . caveolae are absent from cells that lack caveolin-1 but can be induced by ectopic expression of the gene (fra et al., 1995) . caveolins adopt a hairpinlike structure that inserts into the membrane such that the n and c termini are cytoplasmic. caveolins can polymerize to form a striated coat surrounding an invagination site . caveolin-1 can bind cholesterol (murata et al., 1995) , which is enriched within both caveolae and lipid rafts (sargiacomo et al., 1993) ; this may explain why caveolae have been considered a subset of lipid rafts. however, caveolae and lipid rafts are considered to be independent entities as some proteins can be found in one but not the other (liu et al., 1997) . certain ligands can internalize via a lipid raftdependent but clathrin-independent mechanism in cells that lack caveolae (lamaze et al., 2001) . a large pool of the plasma membrane caveolar vesicles cluster into dense grape-like structures where individual caveolae appear stacked on top of each another (thomsen et al., 2002) . these structures are intimately associated with the actin cytoskeleton (stahlhut and van deurs, 2000) ; caveolaassociated proteins are also implicated in regulating plasma membrane dynamics and cellular movement. a small pool of ''transport-competent'' caveolar vesicles may undergo short-range constitutive fusion and budding cycles just under the plasma membrane . caveolae and caveolins can also be detected at the tgn (dupree et al., 1993; kurzchalia et al., 1992) and may form stable ''platforms'' for the movement of proteins and lipids from the tgn to the plasma membrane (tagawa et al., 2005) . the caveolar pathway can be hijacked and used by pathogens or toxins to gain entry into the cell. viruses such as polyomavirus, echovirus 1, and simian virus 40 (sv40) use caveolae to internalize viral particles. these viruses cluster lipid rafts and sequester them into caveolae through interactions with raft components such as integrins and glycosphingolipids ; in the case of sv40, the virus binds to the raft component ganglioside gm 1 (tsai et al., 2003) . tagawa et al. (2005) have shown that sv40 can trigger the long-range movement of transport-competent caveolar vesicles. moreover, cell infection with sv40 more than doubles the number of caveolae capable of undergoing viral internalization and long-range traycking. caveolae contain much of the molecular machinery required for ''classical'' vesicle fission, docking, and fusion, for example, snare proteins, monomeric and trimeric gtpases, annexins ii and vi, n-ethylmaleimide (nem)sensitive fusion protein (nsf), and atpases (schnitzer et al., 1995) . caveolae also contain the dynamin gtpases, which can be transiently recruited to 28 sv40-loaded caveolae and implicated in membrane scission (henley et al., 1998; oh et al., 1998; pelkmans and helenius, 2002) . internalized caveoladerived vesicles move to an endocytic compartment termed the ''caveosome'' and eventually arrive at the early endosome. after fusion with the target compartment, caveolae do not disassemble but maintain their integrity in the membrane, preserving their compartmentalization and retaining their lipid and protein components (pelkmans et al., 2004) . the fate of internalized sv40 viruses after reaching the caveosome eventually results in arrival at the smooth er (pelkmans et al., 2001) . interestingly, mutations in caveolin have been implicated in muscular dystrophy and cardiovascular disease, and mutations causing the downregulation of caveolin have been linked to the progression of various human carcinomas; it is therefore possible that caveolins may have a tumor suppressor role. the caveolin-1 and caveolin-2 genes are located on human 7q31.1 near the microsatellite repeat marker d7s522. this region is commonly deleted in various cancers (engelman et al., 1998) , hinting that caveolin gene deletion may be advantageous for tumor progression. in one report, the caveolin-1 p132l mutation was present in 16% of breast cancer patients studied (hayashi et al., 2001) . the p132l mutation was also linked to the metastatic potential of tumors and disease prognosis. the caveolin-1 p132l mutation also conferred increased cell migration and altered morphology. caveolin-1 protein levels can be reduced or absent from a number of human breast cancer cell lines compared with normal mammary cells (lee et al., 1998) . similarly, silent and missense mutations in caveolin-1 have also been associated with oral carcinomas (han et al., 2004) . caveolin-1, and to a lesser extent caveolin-2, gene expression is downregulated in some cases of thyroid carcinoma (aldred et al., 2003) . although it remains unclear as to why the loss of caveolin causes cell proliferation diseases such as cancer, one can speculate on the role of caveolin in regulating signaling pathways. in endothelial cells, which have a high abundance of caveolin, the key vascular endothelial growth factor receptor 2 (vegfr2) has been shown to be inactive when localized to caveolae (labrecque et al., 2003) . this receptor tyrosine kinase modulates the endothelial response to the key vegf-a cytokine and controls angiogenesis and new blood vessel formation, thus regulating neovascularization and tumor growth (neufeld et al., 1999) . similarly, platelet-derived growth factor (pdgf) receptor tyrosine kinase activity is reduced when associated with caveolae (yamamoto et al., 1999) . in addition to vegfr2 and pdgfr, a number of g protein-coupled receptors (gpcrs) have been shown to interact with caveola-associated factors (insel et al., 2005) . gpcrs are a large family of transmembrane receptors involved in a variety of signal transduction events. these receptors are activated by a range of ligands, including hormones and peptides, and have been linked to a number of cancers such as cell biology of membrane trafficking in human disease thyroid, lung, and gastric. the presence of a number of gpcrs in caveolae suggests that these plasma membrane structures may interact with gpcrs and modulate their signaling potential. lisanti and others (li et al., 1995a) have shown that caveolin-1 interacts solely with inactive forms of g-protein a subunits, lending credence to the negative regulation hypothesis caused by the association of caveolae with transmembrane signaling receptors. a number of mutations in muscle-specific caveolin-3 have been associated with four distinct but related autosomal dominant muscle disease phenotypes (woodman et al., 2004) : limb girdle muscular dystrophy type 1c (minetti et al., 1998) , rippling muscle disease, hyperckemia (persistently elevated levels of serum creatine kinase), and distal myopathy. some mutations cause aberrant retention of caveolin-3 in the golgi and subsequent degradation; other mutations may cause mutant caveolin-3 to act in a dominant-negative manner by forming unstable aggregates with wild-type caveolin-3 (galbiati et al., 1999; sotgia et al., 2003a,b) . hypertrophic cardiomyopathy (hcm) patients have a caveolin-3 t63s mutation that reduces plasma membrane levels (hayashi et al., 2004) . caveolin gene knockout mice are providing insights into protein function in diverent human diseases. for example, lack of caveolins can cause diabetes, atherosclerosis, and cardiomyopathies in mouse models (cohen et al., 2004; williams and lisanti, 2004) . however, such phenotypes have yet to be linked to caveolin dysfunction in humans. phagocytosis is a process used by white blood cells such as macrophages, neutrophils, and dendrites to ingest large particulate material into specialized vesicles called phagosomes. these professional phagocytes are paramount in the defense against infection as they engulf and ingest whole microorganisms such as bacteria. they also use this route for ''mopping up'' apoptotic debris or senescent cells from tissues. in contrast to constitutive pinocytic transport, phagocytosis is regulated by cell surface-localized fc receptor (fcr) contact or interaction with complement-or antibody-coated particles which results in clustering of fcr on the cell surface, a step important for subsequent intracellular signaling and cellular activation (daeron, 1997) . polymorphisms in leukocyte-specific fcg receptors may contribute to autoimmune diseases such as guillain-barré syndrome or rheumatoid arthritis, and enhanced susceptibility to infection (van sorge et al., 2003) . fc-mediated binding can trigger a complex signaling response involving extrusion of fine plasma membrane projections (pseudopodia) from the macrophage to surround and engulf the pathogen, forming a phagosome. the signaling response is reviewed in greater detail elsewhere (bokoch, 2005; chimini and chavrier, 2000; niedergang and chavrier, 2005) . in brief, the activation of tyrosine kinases and rho gtpases is triggered through fcr signaling. the rac and cdc42 gtpases, in conjunction with the downstream evector wasp, mediate remodeling of the actin cytoskeleton, leading to pseudopodium formation and phagosome closure (castellano et al., 2001; chimini and chavrier, 2000) . in contrast, the complement mediated-uptake of opsonized particles divers such that they appear to ''fall'' into the cell in a process that requires rho, but not rac or cdc42 (bokoch, 2005) . phagocytosis, although designed to destroy pathogens, can paradoxically be used as a route of entry by pathogens such as mycobacterium (m. leprae and m. tuberculosis) or leishmania (nguyen and pieters, 2005; scott et al., 2003) . normally, internalized pathogens are destroyed successfully through phagosome maturation into lysosomes and subsequent degradation. mycobacterium can evade host degradation by secreting a soluble serine/threonine protein kinase g molecule into the phagosome. this molecule initiates a signaling response that interferes with phagosome-lysosome fusion, and promotes intracellular pathogen survival (walburger et al., 2004) . furthermore, phagosome maturation is compromised by a pathogen induced block of p38 map (mitogen-activated protein) kinase recruitment to the tethering molecule early endosome antigen 1 (fratti et al., 2003) . the leishmania protozoan parasite, which is transmitted to humans by sand flies, produces a membrane molecule called a lipophosphoglycan, which is inserted into the lipid bilayer of the phagosome in infected macrophages. this lipophosphoglycan is thought to modulate intracellular signaling pathways, resulting in a less fusogenic phagosome and preventing maturation; this would facilitate pathogen replication and disease progression (lodge and descoteaux, 2005) . molecules internalized from the cell surface by receptor-mediated endocytosis and clathrin-coated vesicles are delivered to the early endosome for sorting. molecules such as low-density lipoprotein receptor (ldlr) and transferrin receptor (tfr) are eyciently recycled between the early endosome and the plasma membrane. however, after ligand-mediated activation (fig. 3) , receptor tyrosine kinases such as epidermal growth factor receptor (egfr) are sorted along the endocytic pathway for degradation. early endosomes are thought to be formed through the fusion of internalized vesicles and recruitment of specific proteins and lipids. one key 3 protein traycking through the endosomal-lysosomal system. cell surface receptors are internalized through clathrin-coated vesicles (ccvs) at the plasma membrane. in the cell cytoplasm, ccvs shed their coat components and fuse to produce endosomes. internalized receptors are either recycled from sorting endosomes (housekeeping receptors, e.g., transferrin receptor) or targeted for degradation within the lysosome (signaling receptors, e.g., growth factor receptors) after movement through the late endosome and multivesicular body (mvb) compartments. 32 endosomal regulator is the ubiquitously expressed rab5a gtpase. rab5a is present on the cytosolic face of the plasma membrane, vesicles, and tubular endosomal profiles (chavrier et al., 1990) . a number of rab5a-associated evector proteins regulate endosomal fusion and mediate protein cargo movement and endosomal sorting (zerial and mcbride, 2001) . such evector proteins, including , are clustered on the cytosolic face of the early endosome and stabilize the gtp-bound rab5a in an activated state (horiuchi et al., 1997) . gtp-bound rab5a directly binds to early endosome antigen eea1 to regulate vesicular and endosomal tethering. eea1 contains a c-terminal rab5a-binding domain, and a phosphatidylinositol 3-phosphate-binding zinc finger domain referred to as an fyve (conserved in fab1, yotb, vac1, and eea1) domain (gaullier et al., 1998; stenmark et al., 1996) . overexpression of wild-type rab5a, or a constitutively active rab5a mutant, causes endosome enlargement and defective traycking through this compartment, whereas expression of a constitutively inactive rab5a mutant leads to formation of small endosomes and decreased endocytosis (bucci et al., 1992) . a family of evector proteins that accelerate gtpase hydrolysis (rabgaps) have been identified: rabgap-5 binds to rab5a and regulates traycking through the endocytic pathway . the importance of rab5a activity is further illustrated in the genetic disorder tuberous sclerosis (ts), a disease that causes tumors in the brain, eyes, heart, kidneys, lungs, and skin. ts arises when the tumor suppressor gene, tuberous sclerosis complex (tsc), is absent; introduction of the wild-type tsc2 gene into an animal model or cultured cells results in tumor suppression and reduced cellular proliferation (kobayashi et al., 1995; yeung et al., 1994) . interestingly, the tsc2 gene product (tuberin) is implicated in regulating gtp/gdp exchange on rab5a, thus regulating traycking through this endosome system (xiao et al., 1997) . in chronic myelomonocytic leukemia (cmml) a genetic translocation causes fusion of rab5a evector rabaptin-5 and the pdgfbr (magnusson et al., 2001) . this chromosomal translocation results in enhanced cellular proliferation by compromising endosomal fusion and traycking, and thus regulation of growth factor degradation. it is likely that this aberrant gene product is not degraded and triggers sustained intracellular signaling, leading to cell proliferation and tumor progression in a subset of lymphoid cells. recycling from endosomes back to the cell surface is often used by receptors that internalize nutrients such as lipoproteins and ions. receptor recycling rather than degradation conserves receptor functionality and nutrient uptake and reduces energy expenditure in the synthesis of new receptors (mukherjee et al., 1997) . genetic screens in the nematode caenorhabditis elegans identified rme-1 and delineated a new family of conserved class of eps15 homology (eh) domain proteins . both the worm and mouse homologs of rme-1 are associated with the endosomal compartment: a dominant-negative rme-1 g429r mutant had little evect on receptor-mediated endocytosis but had a substantial evect on endosomal recycling, suggesting a functional role in this step . although information is currently limited, a number of neurological diseases are associated with dysfunction of early endosomal proteins. in some cases of demyelinating polyneuropathy, characterized by progressive weakening and sensory dysfunction of the legs and arms, eea1 autoantibodies have been detected (selak et al., 1999) . a number of disorders, from muscular dystrophy to rheumatoid arthritis, reveal the presence of circulating anti-eea1 antibodies. interestingly, eea1 epitopes recognized by such autoantibodies varied from patient to patient (selak et al., 2003) . autoantibodies against eea1 have also been detected in cases of subacute cutaneous systemic lupus erythematosus (scle), characterized by the appearance of an unsightly red rash, often occurring after sun exposure (mu et al., 1995) . lysosomes are terminal, membrane-enclosed degradative compartments that interact with other organelles through vesicular transport originating from the secretory, endocytic, and autophagic pathways. this organelle stores various proteases, lipases, hydrolases, and degradative enzymes within an acidic environment that maximizes enzymatic activity and degradation. resident lysosomal membrane proteins, integral proteins, and glycoproteins are targeted to the organelle via the endosome. lysosomal proteases such as cathepsin d are processed in the golgi apparatus to add a mannose 6-phosphate (m6p) moiety to n-linked sugars. the m6p moiety is recognized by plasma membrane or tgn-resident mannose 6-phosphate receptors (m6prs) and sorted to the late endosome and eventually the lysosome. here, the acidic ph (ph < 5.5) results in receptor-ligand disassociation and recycling of the m6pr to the tgn. fusion between the endosome and preexisting primary lysosomes allows the delivery of lysosomal resident proteins. the importance of m6p-mediated targeting of lysosomal proteins is highlighted in the human neurological disorder, i-cell disease (mucolipidosis ii), where lysosomal enzymes are secreted from cells rather than targeted to the lysosome. the defect in i-cell disease involves lack of m6p moiety addition as a result of mutations to the n-acetylglucosamine-1-phosphotransferase enzyme usually present within the golgi apparatus (ben-yoseph et al., 1987) . how lysosomes are formed is still unclear (luzio et al., 2003) . three mechanisms have been proposed to explain lysosomal biogenesis: vesicular transport between late endosomes and preformed primary lysosomes (griyths and 34 gruenberg, 1991), early endosomal ''maturation'' to lysosomes (murphy, 1991) , or the current favored model of ''kiss-and-run,'' in which transient interactions between endosomes and lysosomes transfer endosomal contents to the latter compartment (duclos et al., 2003; storrie and desjardins, 1996) . late endosome and lysosome interactions in the kiss-and-run model are thought to be regulated by the rab7 gtpase, which is present on late endosomes; a vps complex, homologous to budding yeast vacuole fusion regulators, is also implicated in sorting and delivery to lysosomes (seals et al., 2000) . the mammalian form of the vps complex interacts with syntaxin-7, a t-snare that is concerned in regulating membrane dynamics along this route (kim et al., 2001) . danon disease is caused by point mutations in, or complete absence of lysosome-associated membrane protein 2 (lamp2) or complete absence of this protein: changes which result in cardiomyopathy, myopathy, and mental retardation. in danon disease patients and lamp2-deficient mice, autophagic vacuoles accumulate within the cytoplasm; these vacuoles arise via intracellular engulfment of old membranes to form an autophagosome, thus sequestering membranes and proteins for eventual degradation (shintani and klionsky, 2004) . autophagosomes fuse with lysosomes, leading to degradation for provision of molecules for cellular homeostasis. the accumulation of autophagic vacuoles in lamp2-deficient cells suggests that lamp2 mediates interactions between autophagosomes and lysosomes. this pathway is commonly activated during conditions of cellular stress such as starvation or pathogenic infection (kirkegaard et al., 2004) . lysosomal storage diseases are caused through insufficient degradation of targeted components within lysosomes, leading to substrate accumulation and lysosome enlargement. more than 40 lysosomal storage diseases have been documented and generally manifest themselves as neurological disorders; disease severity correlates with the levels of lysosomal enzyme activity. niemann-pick disease is a neurodegenerative condition caused by sphingomyelin accumulation in reticuloendothelial cells and ganglion neurons, leading to cell death. it is classified into five types (a-e), each distinguished by either clinical severity or age-related disease phenotype. niemann-pick type a (npa) is most common, with death occurring before 3 years of age. npa patients have point mutations in the smpd1 gene that encodes a lysosomal sphingomyelinase (levran et al., 1991; takahashi et al., 1992) . interestingly, in npc patients, endocytosed ldl particles are not fully degraded in lysosomes, leading to defects in cholesterol metabolism (li et al., 2005a) . the npc disease is caused by mutations in the npc1 gene, which encodes a lysosomal resident protein with similarity to sterol-sensing enzymes and proteins (scott and ioannou, 2004) . fabry disease is an x-linked condition caused by changes in lysosomal a-galactosidase activity resulting in glycosphingolipid accumulation within vascular endothelial lysosomes. this leads to angiokeratomas (a wart-like thickening of the skin), progressive renal impairment, cardiomyopathy, and cerebrovascular disease. mutations in the a-galactosidase a gene can also show reduced enzymatic activity of the encoded protein and retention within the endoplasmic reticulum (yasuda et al., 2003) . receptor tyrosine kinases such as epidermal growth factor receptor (egfr) are degraded by lysosomes after ligand binding and receptor activation. egfr lysosomal targeting is dependent on ligand-stimulated ubiquitination of the cytoplasmic domain. binding of egf to egfr causes downstream signaling, clathrin-mediated internalization, and traycking to endosomes. internalized receptor-ligand complexes are sorted to the late endosome or multivesicular bodies (mvbs), which eventually deliver their contents to the lysosome (katzmann et al., 2002) . whereas other receptors such as tfr are recycled to the plasma membrane, egfr is moved through the endosome-lysosome system by a ubiquitin-dependent sorting and recognition system. these include the hrs/stam heterodimer and tsg101 (bilodeau et al., 2002) present on endosomal membranes. the tsg101 tumor suppressor gene is mutated in nearly 50% of breast cancer patients and encodes a membrane-associated protein (lee and feinberg, 1997) . this factor participates in the sorting of ubiquitinated proteins on the endosome, but its exact function is not clear. in some specialized cells, such as cytotoxic t lymphocytes (ctls), platelets, and melanocytes, regulated secretion can be routed through compartments other than the tgn. such cells have evolved mechanisms whereby modified or secretory lysosomes release their contents at the plasma membrane in response to extracellular stimuli. these secretory lysosomes (sls) share lysosomal characteristics such as acid ph and lamp (lysosome-associated membrane proteins) residents but also contain unique markers such as tyrosinase, present in melanosomes. the ctl secretory lysosomes contain unique components such as perforin and granzymes required for triggering apoptosis in target cells. on ctl contact with a target cell, sls trayc toward the immunological synapse formed between the ctl and target cell. a signal then causes sl fusion with the ctl plasma membrane (stinchcombe et al., 2001) , and release of sl contents and subsequent target cell death. a number of autosomal genetic diseases causing immunodeficiency and albinism involve defects in regulated lysosomal secretion (stinchcombe et al., 2004) . in the rare, fatal disease familial hemophagocytic lymphohistiocytosis (fhl) sls congregate at the plasma membrane in ctls, where they can dock but cannot fuse with the membrane. in one group of fhl patients, this disease is due to a mutation in the gene encoding munc13-4; this is closely 36 related to the neuronal munc13-1 gene product that is involved in snare complex formation in neuronal cells (feldmann et al., 2003) . assembly of this neuronal syntaxin-1, snap-25, and synaptobrevin complex is regulated by munc18-1, which binds and locks syntaxin-1 (t-snare) in a closed, inactive conformation, thus preventing it from interacting with snap-25 (yang et al., 2000) . however, munc13-1 (sassa et al., 1999) and rim (rab3a-interacting protein evector) (koushika et al., 2001) can compete with munc18-1 and displace it from syntaxin-1. this reinforces the syntaxin-1 open conformation and allows snare complex formation to occur. munc13-1 may act as a conformational switch to promote t-snare into an ''open'' state, thus allowing formation of the snare complex that mediates synaptic vesicle docking and fusion. from observations of fhl patients, one speculation is that munc13-4 has a role similar to that of munc13-1 in regulating snare complex formation for sl docking and fusion in ctls (yang et al., 2000) . chediak-higashi syndrome (chs) is a key example of a disease avecting sl function in ctls, with patients displaying hypopigmentation (stinchcombe et al., 2004) . chs patients have genetic mutations in the lyst or chs1 gene (barbosa et al., 1996; perou et al., 1996) and produce ctls containing strikingly enlarged sls that are able to polarize to the immunological synapse but are unable to fuse with the pm. this suggests a role for the chs1 gene product in regulating membrane docking and fusion . overexpression of chs1 leads to the presence of small lysosomes, indicating increased lysosomal fission (ward et al., 2003) . in addition, chs1 interacts with snare proteins, further indicating a role in sl fusion (tchernev et al., 2002) . griscelli syndrome patients also display defects in sl dynamics within ctls and exhibit hypopigmentation and silvery hair. in melanocytes, cells responsible for pigment storage and production, rab27a is required to recruit melanophilin to pigment granules called melanosomes (sls). melanophilin binds the myosin motor protein myosin va and regulates melanosome movement along actin cables to the plasma membrane strom et al., 2002; wu et al., 2002) . in type 1 griscelli syndrome patients, rab27a gtpase is missing or defective, whereas in type 2 griscelli syndrome patients the myosin va motor protein is absent. these defects are also evident in mouse models such as ashen (rab27a defective), dilate (myosin va defective), and leaden (melanophilin defective). in both the human griscelli syndromes and the mouse models, melanosomes are clustered in a perinuclear location, a defect attributed to rab27a dysfunction (wilson et al., 2000; wu et al., 2001) . interestingly, ctls isolated from type 1 griscelli syndrome patients and ashen mice (rab27a deficient) are unable to kill target cells, whereas type 2 griscelli syndrome patient and dilate mouse ctls are functional. this suggests that rab27a interacts with diverent evectors to induce sl fusion with the plasma membrane in diverent cell types (haddad et al., 2001) . hermansky-pudlak syndrome (hps) is a fourth example of sl dysfunction and is characterized by oculocutaneous albinism, ceroid deposition, and excessive prolonged bleeding (hermansky and pudlak, 1959; swank et al., 2000) . however, hps cannot be viewed as a single disease but a group of at least seven autosomal genetic disorders. each of the seven subgroups (hps1-7) is due to mutations in individual genes, most of which encode components of multisubunit protein complexes involved in vesicle traycking , whereas the function of others remains unclear. three of these complexes, termed blocs (biogenesis of lysosome-related organelle complexes), play a role in regulating traycking involved in platelet and melanosome secretion, but their exact functions are unclear (di pietro and dell'angelica, 2005) . in hps2 patients a nonsense mutation in the gene encoding the b3a subunit of the ap3 adaptor protein prevents expression of this subunit (huizing et al., 2002) . as previously mentioned, ap3 is involved in the recruitment of transmembrane proteins into vesicles at the early endosome for delivery to the lysosome (peden et al., 2004) . in melanocytes derived from hps2 patients, the tyrosinase that catalyzes the formation of melanin is not transported to maturing melanosomes (huizing et al., 2001) . this leads to the characteristic pattern of albinism seen in patients with this condition. furthermore, patients with hps2 display an impaired ctl response and immune response. ctls from hps2 patients have lytic granules that cannot move in an oriented fashion toward the microtubule-organizing center; therefore, when ctls are stimulated by contact with target cells, the lytic granules are not targeted to the immunological synapse for cell killing . studies on the cell biology of hiv infection have suggested the existence of a viral secretory compartment. work by marsh and others pelchen-matthews et al., 2003) has localized viral envelope (gp120) and matrix proteins (p17) to tetraspanin-positive endosome-related organelles in infected macrophages and dendritic cells. these viral secretory compartments move from an intracellular localization to an infectious synapse when infected macrophages or dendritic cells form an immunological synapse with activated t cells. this may be one mechanism for subsequent viral infection of cd4-positive t cells, thus causing the impaired immune response seen in patients with acquired immunodeficiency syndrome (aids). cells require a highly organized framework or cytoskeleton to station and move membrane organelles within three-dimensional space. components of the cytoskeleton can guide organelles or vesicles to specific destinations within the cell. the microtubule cytoskeleton is commonly associated with 38 the directional movement of intracellular transport vesicles or intermediates. in contrast, actin has been envisioned to have a structural role in determining cell shape, plasma membrane dynamics, and cell locomotion. however, evidence points to a role for actin in regulated traycking from the tgn (allan et al., 2002; badizadegan et al., 2004; cobbold et al., 2004) and endocytosis (ascough, 2004; engqvist-goldstein and drubin, 2003) . the cytoskeleton is a dynamic structure likened to a collapsible scavold that can be rapidly disassembled and reconstituted depending on cellular requirements. actin or tubulin polymerization (elongation) and depolymerization (breakdown) rely on the controlled addition or removal of monomers in a polarized and energy-dependent manner. protofilaments in either structure are both polarized, with the plus end growing at a faster rate. actin cables are each composed of two parallel protofilaments that twist around each other, whereas microtubules are composed of a hollow cylindrical structure comprising 13 parallel protofilaments. actin nucleation is an initial step required for elongation involving formation of a stable trimer subunit base for protofilament elongation. a heptameric complex termed arp2/3 (actin-related protein) binds to the ends and sides of actin filaments to nucleate and further accelerate the growth of the actin network (millard et al., 2004) . the function of the arp2/3 complex can be regulated by membrane-associated rho gtpases. these regulators, which include cdc42 and various rac isoforms, act as molecular switches that cycle between an active gtp-bound state and an inactive gdp-bound state. cdc42 regulates arp2/3 indirectly through its downstream target wiskott-aldrich syndrome protein (wasp), which binds directly to the arp2/3 complex (jave and hall, 2005) . patients with x-linked wiskott-aldrich syndrome display mutations in the wasp gene and have thrombocytopenia (reduced platelet count), eczema, recurrent infections, hematologic malignancy, and autoimmune disorders (lemahieu et al., 1999) . approximately 300 disease mutations in wasp have been reported, which lead to defective control of wasp in actin polymerization and severe disease phenotypes (burns et al., 2004) . wasp expression is restricted to hematopoietic cells, although the ubiquitously expressed n-wasp is present in various cells and tissues (burns et al., 2004) . the actin network is important for the formation of immunological synapses between cytotoxic t lymphocytes (ctls) and their targets, as well as t lymphocytes and antigen-presenting cells such as macrophages. the formation of the immunological synapse in ctls is essential for the transport, docking, and fusion of sls and subsequent destruction of the target cell as described above. defective wasp inhibits the formation of the immunological synapse and t cell activation (notarangelo and ochs, 2003) , probably causing the immunological deficiencies observed in wiskott-aldrich syndrome patients. wasp deficiency in t lymphocytes also avects the regulation and composition of lipid rafts (dupre et al., 2002) , indicating that the cell biology of membrane trafficking in human disease formation of the immunological synapse is dependent on lipid rafts, wasp, and actin dynamics. motor proteins provide the physical force to move membrane vesicles along the polymerized cytoskeletal filaments via atp-dependent hydrolysis. actinbased motor proteins belong to the myosin superfamily. the myosin va gene is mutated in a small number of patients with griscelli syndrome (bahadoran et al., 2003; pastural et al., 2000) (other traycking mutations contributing to griscelli syndrome are discussed in section v.c). mutations in the myosin viia gene can cause usher's syndrome, resulting in blindness and deafness. intracellular transport is probably compromised in usher syndrome patients; the mouse shaker model has a mutant myosin viia gene, displaying defective melanosome transport in retinal pigment epithelial cells (liu et al., 1998) and altered distribution in photoreceptor cells (richardson et al., 1997) . microtubule motor proteins, which actively move vesicles along the microtubules, and microtubule-associated proteins (maps), serve as docking molecules to bind cargo to motor proteins (gerdes and katsanis, 2005) . microtubule motor proteins belong to either the kinesin or dynein families. with the exception of the c-terminal kinesins, kinesin-based motors generally transport cargo toward the plus end of the microtubule, whereas the dyneins are minus end-directed motors. long-range vesicular transport is particularly important in neurons, where axons can reach up to 1 m in length. newly synthesized lipids, and secreted or membrane-associated proteins, are made in the cell body; long-range and directional transport is crucial for replenishing the constituents of the presynaptic cleft (at the terminal end of the axon) with synaptic vesicles and plasma membrane receptors (holzbaur, 2004) . a number of human neurological diseases are linked to mutations in microtubule motors and associated proteins. lissencephaly, a greek term meaning ''smooth brain,'' causes severe brain malformation resulting in mental retardation and epilepsy. one of the genes mutated in the disease is lis1 (originally identified in miller-dieker syndrome patients with lissencephaly) (reiner et al., 1993) . the lis1 protein regulates microtubule motor function by binding dynein1 and p150 glued , a component of the dynactin complex that binds to and activates dynein (smith et al., 2000) . it is proposed that lis1 regulates retrograde axonal transport. another gene mutated in some patients with lissencephaly is doublecortin, a microtubule-associated protein that binds tubulin and stabilizes microtubules (horesh et al., 1999; moores et al., 2004) . the kif1b kinesin regulates transport of synaptic vesicle precursors along the neuronal axon. patients with charcot-marie-tooth disease type 2a display neuronal axonal degeneration due to a loss-of-function mutation in the motor domain of kif1b . in alzheimer's disease, a classical sign is hyperphosphorylated aggregates of the microtubule-associated 40 protein, tau, in neuronal cells and tissues. tau protein can influence vesicular transport (ebneth et al., 1998) by regulating the attachment of motors to microtubules (trinczek et al., 1999) . one theory is that the tau protein can interfere with kinesin-dependent transport by blocking motor access to microtubules, thus slowing or preventing vesicle movement along axons (mandelkow et al., 2003) . moreover, an early sign of alzheimer's disease is the loss of synapses and retrograde degeneration of neurons, complemented by a breakdown in intracellular transport. the disruption of microtubule-mediated vesicular traycking may also be a causative factor of the neurodegenerative phenotype of huntington's disease. this disease is caused by expansion of polyglutamine repeats occurring in the brain-enriched protein huntingtin (htt). it has been demonstrated that htt enhances vesicular transport of brain-derived neurotrophic factor (bdnf) along microtubules (gauthier et al., 2004) . htt is localized in the cytoplasm and is associated with vesicular and microtubule-based trayc through its ability to bind huntingtin-associated protein 1 (hap1) (li et al., 1995b) , a protein that has aynity for the dynactin p150 glued subunit (engelender et al., 1997) . dysfunctional polyq-htt associated with the disease state may disrupt the transport of bdnf by binding and blocking the hap1/dynactinmediated delivery of bdnf vesicles along microtubules (gauthier et al., 2004) . this is further supported by the finding that bdnf levels are decreased in brains of huntington's disease patients (ferrer et al., 2000) . the current treatment of genetic disorders involves addressing the symptoms rather than the cause. to that end, many mild forms of disorders such as niemann-pick disease, and familial hypercholesterolemia, can be controlled by diet regimens and lifestyle changes. in contrast, a life-threatening disease such as cystic fibrosis requires extensive physiotherapy and pulmonary exercise to loosen and prevent mucus accumulation within the lungs. new antidementia drugs are increasingly successful in treating neurological disorders such as alzheimer's disease. drugs such as galantamine, donepezil, rivastigmine, and memantine target the posttranslational processing of bapp to reduce amyloid deposits (prasher, 2004) . in familial hypercholesterolemia, statin treatment is a common strategy for reducing plasma ldl and cholesterol levels by targeting hmg-coa reductase, the rate-limiting enzyme in cellular cholesterol biosynthesis. furthermore, less commonly used ldl-lowering drugs such as probucol have shown some success in (buckley et al., 1989) . the administration of adrenalin receptor antagonists (b-blockers) to patients with the cardiac condition long-qt syndrome reduces arrhythmia risk. enzyme replacement therapy (ert) has been carried out in patients with fabry's disease, a lysosomal storage disease. patients are given recombinant lysosomal a-galactosidase (mignani and cagnoli, 2004) to reduce the risks of strokes and kidney failure associated with the condition. finally, organ transplantation is occasionally carried out for some disease states: for example, bone marrow transplants for chediak-higashi syndrome patients (liang et al., 2000) and rab27a-defective patients with griscelli syndrome (schuster et al., 2001) and wiskott-aldrich syndrome (filipovich et al., 2001) . however, although transplant operations can be successful in alleviating the immunological issues associated with these diseases, it does not address problems associated with the nervous system or pigmentation. b. gene therapy: the next generation of medical treatment? completion of the human genome sequencing project has given science the ability to track gene(s) responsible for potentially any genetic disorder and, as a consequence, to allow these genes to be corrected in patients. this is the goal of gene therapy research. of course, gene therapy has a fundamental limitation: it is only really suitable for single-gene defect diseases, and multigenic or chromosomal defects will be beyond the ability of the technique because of the complex nature of the disease. however, there are more than 2500 single gene defects that cause human disease, so there are many diseases requiring such approaches. the history of gene therapy is discussed in more detail by russell (1997) and scollay (2001) . much evort has been made in developing techniques that allow successful replacement or augmentation of defective genes. gene therapy is performed by introducing a gene vehicle directly into the patient (in vivo) or by removing cells from a patient, introducing the gene into these cells in culture, and replacing the cells back in the patient (ex vivo). most studies have focused on the use of viral vectors as delivery vehicles. retroviruses are potentially the best gene delivery system (kurian et al., 2000) . these rna viruses are able to infect a great many cell types and replicate by inserting their viral genes into the genome of the host. the host cellular machinery is then modulated to produce and assemble viral particles. in gene therapy, retroviruses could be used to express the target gene to be replaced but be modified to prevent viral disease (hiv, which is the causative agent of aids, is also a retrovirus). the principal drawback of a retrovirus vector is the possibility that genomic integration could elevate oncogene expression, thus causing cancer. therefore, the majority of clinical trials using retrovirus vectors have been performed ex vivo. a ''successful'' gene therapy experiment was exemplified in the case of a 4-year-old female patient lacking adenosine deaminase (ada), which results in severely compromised immunodeficiency (ada-scid) and dysfunctional t cells (blaese et al., 1995) . in this case, a retroviral vector was used to deliver the coding sequence for ada into cells, resulting in successful expression of this enzyme in hitherto defective cells. although successful, it is uncertain whether enzyme replacement treatment (recombinant ada injections) also influenced the patient outcome. adenovirus (adv) (mcconnell and imperiale, 2004) is a dna virus and key gene therapy vehicle that maintains the viral genome as a separate transmissible episome within the nuclei of infected host cells. the use of attenuated or inactivated adv for human gene delivery has attracted much interest. the advantages of adv gene transfer are that its genome can easily be manipulated and recombinant virus can be grown to high titers in vitro with eycient transduction of target cells in vitro or in vivo. as adv can evectively infect nondividing cells such as lung pulmonary tissues it is a popular vector of choice for gene therapy to treat cystic fibrosis (cf) patients. although there are promising studies (zabner et al., 1993) , failures have also been noted (knowles et al., 1995) . a major disadvantage of an adv-based approach is the triggering of a strong host immune response to the virus, which becomes a serious problem in subsequent long-term delivery of recombinant virus for disease alleviation. one approach to circumventing such an issue is to use a viral delivery system that produces a low host immune response such as the adeno-associated virus (aav) (flotte, 2005) . aav is a nonpathogenic virus that requires coinfection with a helper virus to replicate. however, aav has broad host cell specificity and is diycult to grow in large quantities, probably because of its reliance on a helper virus. finally, nonviral methods are increasingly available for the delivery of dna constructs directly into cells and tissues. these are often lipid-based reagents (e.g., liposomes) that bind to the plasmid dna and fuse with the plasma membrane, thus enabling cytosolic delivery of the gene. the plasmid dna would then be transported into the host nucleus by endogenous cellular machinery. this type of gene delivery can only be performed ex vivo and can be limited by the poor dna transfection eyciency of primary cells or tissues. this type of method, however, is a potentially useful method for delivering genes into progenitors or precursors (e.g., stem cells) before cellular cell biology of membrane trafficking in human disease diverentiation and tissue formation within a particular microenvironment in the body (mendell et al., 1995) . numerous disease states are caused by protein misfolding within the er, leading to degradat ion (table i ; section s iii. a.1 an d iii.a.2 ). one strategy would be to promote the correct protein conformation in a mutant gene product either chemically or pharmacologically. a number of membrane-divusible chemical and pharmacological ''chaperones'' have been identified that could avect protein folding in cells. chemical chaperones such as glycerol and trimethylamine n-oxide (tmao) can restore the wild-type traycking and activity of cftráf508 in cultured epithelial cells (brown et al., 1996) , and porcine kidney epithelial cells expressing cftráf508 and treated with dimethyl sulfoxide (dmso) increased plasma membrane levels of the channel protein (bebok et al., 1998) . loo et al. (2005) have demonstrated that a novel quinazoline derivative specific for cftr will rescue the defective traycking of cftráf508 in cultured cells. cell surface levels of a water channel, aquaporin-2, can be enhanced with dmso (tamarappoo and verkman, 1998) . defects in this gene can result in x-linked nephrogenic diabetes insipidus, a condition in which patients are unable to concentrate their urine because of an inability to reabsorb water from the kidneys into the blood. although chemical chaperones are somewhat nonspecific in their action (the protein folding of the whole cell is avected and not just the target protein), pharmacological chaperones can be tailored to individual proteins. for example, the compound sr121463a is a nonpeptide vasopressin v2 receptor antagonist (morello et al., 2000; robert et al., 2005) . patients with a mutant vasopressin v2 receptor can also display nephrogenic diabetes insipidus. on treatment, the cell-permeant sr121463a compound would act as a chaperone and accompany the mutant v2 receptor to the cell surface to rescue correct functionality. geldanamycin, a naturally occurring antifungal agent, has potential as an anticancer drug (beliakov and whitesell, 2004; miyata, 2005) . geldanamycin interacts with and inhibits activity of the heat shock protein and chaperone hsp90, a cytosolic cellular stress protein that supports the correct folding, stability, and function of ''client'' proteins. many hsp90 client proteins are implicated in cell cycle progression, proliferation, and angiogenesis (whitesell and lindquist, 2005) . the erbb2 tyrosine kinase complex is implicated in regulation and development of epithelial breast tumors and is an hsp90 44 client. inhibition of hsp90 action by geldanamycin results in degradation of both erbb2 and downstream signaling evectors, resulting in reduced cellular growth and tumor formation (citri et al., 2004) . a number of cell-permeable peptide sequences found in viruses and host proteins have been discovered that mediate the delivery of cargo (proteins, drugs, plasmid dna, oligonucleotides) directly into cells (brooks et al., 2005; gupta et al., 2005; schwartz and zhang, 2000) . such peptide sequences could be fused or attached to recombinant or engineered proteins and administered to patients to complement defects of a particular gene product. for example, the drosophila melanogaster antennapedia homeodomain (antp) transcription factor contains a short 16-residue sequence that mediates protein translocation across biological membrane bilayers in an energy-independent manner (derossi et al., 1994; joliot et al., 1991) . other sources of cell membrane-permeable proteins have been uncovered in viruses. the hiv-1 replication protein tat contains a basic, arginine-and lysine-rich peptide sequence (residues 47-57) that modulates the translocation of exogenous tat across the plasma membrane in a number of cell types, and is able to activate intracellular genes controlled by an hiv promoter (frankel and pabo, 1988; mann and frankel, 1991) . this basic 10-residue sequence can internalize conjugated b-galactosidase and horseradish peroxidase (fawell et al., 1994) as well as a fab antibody fragment (anderson et al., 1993) . the major structural protein of herpesvirus (hsv-1), vp22, can trayc between cells (elliott and o'hare, 1997) , whereas the pres-2 domain of hepatitis b virus surface antigen acts as a shuttle for peptides and functional proteins (such as egfp) in hepatocytes and other cells (oess and hildt, 2000) , suggesting further the existence of naturally occurring peptide sequences that may act as drug delivery vectors. finally, a ''synthetic'' amphipathic peptide, fluos-klalklalkalkaalkla-nh 2 , has been shown to be internalized in mast and endothelial cells (oehlke et al., 1998) . the employment of small molecular inhibitors as a method of treating human disease has moved at exponential pace. a number of compounds have been synthesized or isolated from nonhuman organisms that directly avect cellular function and have been used in research on a variety of diseases. plant-and microorganism-derived polyhydroxylated alkaloids referred to as iminosugars have been used in the treatment of patients with gaucher disease (cox et al., 2000) . gaucher disease type i and type ii is a lysosomal storage disorder caused by a mutation in the gene encoding the cell biology of membrane trafficking in human disease acid b-glucosidase (gba) enzyme and results in the accumulation of toxic glucosylceramide in a patient's spleen, liver, and bones; manifesting itself in enlargement of these organs, as well as heart and lung disease. iminosugars act on glycosylating enzymes present within the er and golgi and inhibit their ability to transfer sugar moieties onto proteins. one member of the iminosugar family, n-butyldeoxynojirimycin (nb-dnj; also called miglustat or zavesca) inhibits the enzyme important in the maturation of the gba substrate glucocerebroside, namely ceramide glucosyltransferase (cgt) (butters et al., 2003) . inhibition of cgt has resulted in significantly reduced levels of glucocerebroside in the liver and spleen of patient in clinical trials (cox et al., 2000) . however, nearly 80% of patients in the trials displayed osmotic diarrhea as a side evect of the treatment. in a mouse model for human tay-sachs disease, which is caused by a mutation in the gene encoding hexosamidase a, levels of the harmful glycophospholipid gm 2 were significantly reduced on treatment with nb-dnj (platt et al., 1997) . in addition to the treatment of lysosomal storage diseases, an nb-dnj derivative called miglitol has been used in the treatment of diabetes mellitus, resulting in reduced activity of the sucrose-isomaltase enzyme complex and reduction of carbohydrate digestion (mitrakou et al., 1998) . a major aspect of human disease is the production and subsequent degradation of misfolded proteins, either by the proteasome or within the lysosome. lysosomotropic agents such as chloroquine cause an increase in the intralumenal ph of endosomes and lysosomes, reducing lysosomal protease activities and the traycking of proteins through the endosome-lysosome system. a number of proteasome inhibitors such as mg132, lactacystin, and alln can specifically inhibit the activity of a range of serine and cysteine proteases and chymotrypsin-like enzymes (kisselev and goldberg, 2001) . proteasome inhibition has been linked with a number of aspects of human disease. treatment of endothelial cells with proteasome inhibitors resulted in apoptosis of proliferating cells (drexler et al., 2000) and inhibition of plasminogen activator levels; this factor promotes angiogenesis and new blood vessel sprouting (oikawa et al., 1998) . however, inhibiting proteasome function has broad cytotoxic and apoptotic evects in cells and tissues. chemotherapeutic agents targeting signaling pathways are currently of much interest in relation to cancer therapeutics. cellular proliferation can be regulated by growth factor binding to a cell surface receptor and signaling through either the mitogen-activated protein kinase (mapk) or phosphoinositide-3-kinase (pi3k) cascades. activation of these pathways induces the expression of oncogenes such as c-jun and c-fos and inhibits apoptosis through a sequence of protein phosphorylation events. shelton et al. (2003) demonstrated that inhibition of the mapk pathway with small molecule inhibitors specific for raf-1 or mek reduced cell proliferation and induced apoptosis in conditionally transformed hematopoietic cells. however, 46 such pathways also regulate other cellular functions besides proliferation or apoptosis and there are likely to be consequences for cellular homeostasis. structural studies are important in the development of new small molecule inhibitors that target specific enzymes and regulators. c-akt (pkb) is a serine/ threonine protein kinase required for survival and proliferation in many human cancers and its structure has been elucidated (kumar and madison, 2005 ; yang et al ., 2002; and refer ences therei n). chem otherape utic agents have been consequently designed that inhibit c-akt activity; molecules such as h-89 target the atp-binding pocket in c-akt (kumar and madison, 2005) . compounds that bind specifically to c-akt isoforms or target specific domains within the kinase have been reported (barnett et al., 2005) , but there are no reports of clinical trials with such compounds (kumar and madison, 2005) . finally, small molecule inhibitors are being developed to target the posttranslational processing of proteins or peptides implicated in human disease. the enzyme that catalyzes the initial steps in b-amyloid synthesis, g-secretase, is an attractive target for prevention of amyloid deposits in alzheimer's disease patients (churcher and beher, 2005) . such small molecule inhibitors could also be used to treat pathogenic infections such as those caused by severe acute respiratory syndrome (sars), influenza, hiv, or hepatitis c viruses. attractive targets are virus-encoded or host proteases required for processing of viral proteins to generate infectious virus particles from the host cell. in the case of the sars virus, a viral chymotrypsin-like cysteine protease is responsible for processing sars viral proteins required for viral replication. inhibition of this protease would evectively inhibit viral replication. a molecule referred to as cs11 was found to inhibit the replication of human sars with no toxic evect on normal cells (dooley et al., 2006) . much work is also being carried out in targeting host proteases required for the processing of hiv envelope glycoproteins by the biosynthetic secretory pathway to generate viral gp120 and gp41 polypeptides. the completion of the human genome sequencing project has led to the prediction that a large number of diseases will be identified and understood at the gene level . as noted in this review, a number of examples exist in which a single gene mutation can have devastating evects on human function. at present, the symptoms of some mild forms of genetic diseases can be modulated through diet or drug regimens, and some success has been achieved with organ transplantation. gene therapy has attracted much attention but has suvered setbacks due to viral toxicity issues. an alternative strategy is the use of small molecule therapeutics, which cell biology of membrane trafficking in human disease may override specific defects or target specific pathways to compensate for gene defect(s). in addition, our understanding of how we respond at a genetic level to pathological infection will enable us to design evective drug strategies to presently chronic infections. in essence, understanding the cell biological basis for human diseases will enable us to design evective methods to deliver therapeutic strategies to patients. caveolin-1 and caveolin-2, together with three bone morphogenetic protein-related genes, may encode novel tumor suppressors down-regulated in sporadic follicular thyroid carcinogenesis endocytosis of the glucose transporter glut4 is mediated by the gtpase dynamin motoring around the golgi the p115-interactive proteins gm130 and giantin participate in endoplasmic reticulum-golgi trayc cftr and chaperones: processing and degradation tumor cell retention of antibody fab fragments is enhanced by an attached hiv tat protein-derived peptide endocytosis: actin in the driving seat traycking of cholera toxin-ganglioside gm1 complex into golgi and induction of toxicity depend on actin cytoskeleton comment on elejalde syndrome and relationship with griscelli syndrome identification of the homologous beige and chediak-higashi syndrome genes coupled er to golgi transport reconstituted with purified cytosolic proteins the akt/pkb family of protein kinases: a review of small molecule inhibitors and progress towards target validation the adaptor protein ap-4 as a component of the clathrin coat machinery: a morphological study role of diacylglycerol in pkd recruitment to the tgn and protein transport to the plasma membrane loss of function associated with novel mutations of the scn5a gene in patients with brugada syndrome disassembly and reassembly of the golgi apparatus activation of deltaf508 cftr in an epithelial monolayer hsp90: an emerging target for breast cancer therapy snares and the specificity of transport vesicle targeting mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase altered molecular size of n-acetylglucosamine 1-phosphotransferase in i-cell disease and pseudo-hurler polydystrophy congenital and acquired neutropenia the role of regulated cftr traycking in epithelial secretion phosphatidic acid formation by phospholipase d is required for transport from the endoplasmic reticulum to the golgi complex degradation of subunits of the sec61p complex, an integral component of the er membrane, by the ubiquitin-proteasome pathway role of cue1p in ubiquitination and degradation at the er surface the vps27p hse1p complex binds ubiquitin and mediates endosomal protein sorting t lymphocyte-directed gene therapy for ada-scid: initial trial results after 4 years regulation of innate immunity by rho gtpases signals for sorting of transmembrane proteins to endosomes and lysosomes caveolin-1 in breast cancer tat peptide-mediated cellular delivery: back to basics chemical chaperones correct the mutant phenotype of the delta f508 cystic fibrosis transmembrane conductance regulator protein the small gtpase rab5 functions as a regulatory factor in the early endocytic pathway probucol. a reappraisal of its pharmacological properties and therapeutic use in hypercholesterolaemia the wilson disease gene is a putative copper transporting p-type atpase similar to the menkes gene coiled coils: a highly versatile protein folding motif mechanisms of waspmediated hematologic and immunologic disease a step forward for stiv-person syndrome small-molecule therapeutics for the treatment of glycolipid lysosomal storage disorders hepatic endoplasmic reticulum storage diseases initial docking of er-derived vesicles requires uso1p and ypt1p but is independent of snare proteins a role for cbs domain 2 in traycking of chloride channel clc-5 a ''de novo'' point mutation of the low-density lipoprotein receptor gene in an italian subject with primary hypercholesterolemia actin dynamics during phagocytosis adpkd: a human disease altering golgi function and basolateral exocytosis in renal epithelia localization of low molecular weight gtp binding proteins to exocytic and endocytic compartments multiple forms of dynamin are encoded by shibire, a drosophila gene involved in endocytosis function of rho family proteins in actin dynamics during phagocytosis and engulfment lowe syndrome protein ocrl1 interacts with clathrin and regulates protein traycking between endosomes and the trans-golgi network gamma-secretase as a therapeutic target for the treatment of alzheimer's disease the achilles heel of erbb-2/her2: regulation by the hsp90 chaperone machine and potential for pharmacological intervention lytic granules, secretory lysosomes and disease adaptor protein 3-dependent microtubule-mediated movement of lytic granules to the immunological synapse novel membrane trayc steps regulate the exocytosis of the menkes disease atpase the menkes disease atpase (atp7a) is internalized via a rac1-regulated, clathrin-and caveolae-independent pathway actin and microtubule regulation of trans-golgi network architecture, and copper-dependent protein transport to the cell surface role of caveolae and caveolins in health and disease the human genome project: lessons from large-scale biology rab and arf gtpase regulation of exocytosis novel oral treatment of gaucher's disease with n-butyldeoxynojirimycin (ogt 918) to decrease substrate biosynthesis copper transporting p-type atpases and human disease defective intracellular transport and processing of oa1 is a major cause of ocular albinism type 1 involvement of clathrin-mediated endocytosis in human immunodeficiency virus type 1 entry fc receptor biology the molecular basis of alanine: glyoxylate aminotransferase mistargeting: the most common single cause of primary hyperoxaluria type 1 a role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals the synaptic vesicle-associated protein amphiphysin is the 128-kd autoantigen of stiv-man syndrome with breast cancer a novel form of hereditary myeloperoxidase deficiency linked to endoplasmic reticulum/proteasome degradation the low lysine content of ricin a chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol low-density lipoprotein receptor-its structure, function, and mutations association of the ap-3 adaptor complex with clathrin ap-4, a novel protein complex related to clathrin adaptors altered traycking of lysosomal proteins in hermansky-pudlak syndrome due to mutations in the beta 3a subunit of the ap-3 adaptor rhodopsin c terminus, the site of mutations causing retinal disease, regulates traycking by binding to adp-ribosylation factor 4 (arf4) the third helix of the antennapedia homeodomain translocates through biological membranes rab geranylgeranyl transferase alpha mutation in the gunmetal mouse reduces rab prenylation and platelet synthesis ubiquitination is required for the retrotranslocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome mutations in the mdr3 gene cause progressive familial intrahepatic cholestasis the coiled-coil membrane protein golgin-84 is a novel rab evector required for golgi ribbon formation the cell biology of hermansky-pudlak syndrome: recent advances from genome to drug lead: identification of a small-molecule inhibitor of the sars virus cooperation of ggas and ap-1 in packaging mprs at the trans-golgi network inhibition of proteasome function induces programmed cell death in proliferating endothelial cells the t(10;11)(p13;q14) in the u937 cell line results in the fusion of the af10 gene and calm, encoding a new member of the ap-3 clathrin assembly protein family remodeling of endosomes during lysosome biogenesis involves ''kiss and run'' fusion events regulated by rab5 er-to-golgi transport: cop i and cop ii function (review) hyperinsulinism in infancy: from basic science to clinical disease wiskott-aldrich syndrome protein regulates lipid raft dynamics during immunological synapse formation caveolae and sorting in the trans-golgi network of epithelial cells overexpression of tau protein inhibits kinesin-dependent traycking of vesicles, mitochondria, and endoplasmic reticulum: implications for alzheimer's disease a novel gene for autosomal dominant stargardt-like macular dystrophy with homology to the sur4 protein family intercellular traycking and protein delivery by a herpesvirus structural protein cisternal maturation and vesicle transport: join the band wagon! (review) huntingtin-associated protein 1 (hap1) interacts with the p150glued subunit of dynactin genes encoding human caveolin-1 and -2 are co-localized to the d7s522 locus (7q31.1), a known fragile site (fra7g) that is frequently deleted in human cancers actin assembly and endocytosis: from yeast to mammals accelerated transport and maturation of lysosomal alpha-galactosidase a in fabry lymphoblasts by an enzyme inhibitor lowe syndrome protein ocrl1 interacts with rac gtpase in the trans-golgi network tat-mediated delivery of heterologous proteins into cells munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (fhl3) brain-derived neurotrophic factor in huntington disease caveolae and intracellular traycking of cholesterol impact of donor type on outcome of bone marrow transplantation for wiskott-aldrich syndrome: collaborative study of the international bone marrow transplant registry and the national marrow donor program adeno-associated virus-mediated gene transfer for lung diseases the ap-1a and ap-1b clathrin adaptor complexes define biochemically and functionally distinct membrane domains the endoplasmic reticulum as a site of protein degradation de novo formation of caveolae in lymphocytes by expression of vip21-caveolin identification of a di-leucine motif within the c terminus domain of the menkes disease protein that mediates endocytosis from the plasma membrane cellular uptake of the tat protein from human immunodeficiency virus induction of p38 mitogen-activated protein kinase reduces early endosome autoantigen 1 (eea1) recruitment to phagosomal membranes caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and t-tubule abnormalities phenotypic behavior of caveolin-3 mutations that cause autosomal dominant limb girdle muscular dystrophy (lgmd-1c). retention of lgmd-1c caveolin-3 mutants within the golgi complex hiv-1 traycking to the dendritic cell-t-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse structural basis of fabry disease fyve fingers bind ptdins huntingtin controls neurotrophic support and survival of neurons by enhancing bdnf vesicular transport along microtubules microtubule transport defects in neurological and ciliary disease wilson disease the alpha chain of the ap-2 adaptor is a clathrin binding subunit intracellular transport and sorting of the oligodendrocyte transmembrane proteolipid protein disrupted proteolipid protein traycking results in oligodendrocyte apoptosis in an animal model of pelizaeus-merzbacher disease evidence that rme-1, a conserved c. elegans eh-domain protein, functions in endocytic recycling the arguments for pre-existing early and late endosomes intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides a gtpase-activating protein controls rab5 function in endocytic traycking defective granule exocytosis in rab27a-deficient lymphocytes from ashen mice synaptojanin 1: localization on coated endocytic intermediates in nerve terminals and interaction of its 170 kda isoform with eps15 mutation and aberrant expression of caveolin-1 in human oral squamous cell carcinomas and oral cancer cell lines traycking, turnover and membrane topology of prp charcot-marie-tooth neuropathy type 1b is associated with mutations of the myelin p0 gene invasion activating caveolin-1 mutation in human scirrhous breast cancers identification and functional analysis of a caveolin-3 mutation associated with familial hypertrophic cardiomyopathy accumulating evidence suggests that several ab-toxins subvert the endoplasmic reticulum-associated protein degradation pathway to enter target cells the delta f508 mutation shortens the biochemical half-life of plasma membrane cftr in polarized epithelial cells dynaminmediated internalization of caveolae albinism associated with hemorrhagic diathesis and unusual pigmented reticular cells in the bone marrow: report of two cases with histochemical studies evects of mutant rat dynamin on endocytosis characterization of a fourth adaptor-related protein complex missense mutation (c1263r) in the thyroglobulin gene causes congenital goiter with mild hypothyroidism by impaired intracellular transport a novel leukocyte adhesion deficiency caused by expressed but nonfunctional beta2 integrins mac-1 and lfa-1 caveolae: stable membrane domains with a potential for internalization doublecortin, a stabilizer of microtubules a novel rab5 gdp/gtp exchange factor complexed to rabaptin-5 links nucleotide exchange to evector recruitment and function hereditary neutropenia: dogs explain human neutrophil elastase mutations ap-3 mediates tyrosinase but not trp-1 traycking in human melanocytes nonsense mutations in adtb3a cause complete deficiency of the {beta} 3a subunit of adaptor complex-3 and severe hermansky-pudlak syndrome type 2 the leaden gene product is required with rab27a to recruit myosin va to melanosomes in melanocytes caveolae and lipid rafts: g protein-coupled receptor signaling microdomains in cardiac myocytes rho gtpases: biochemistry and biology a common w556s mutation in the ldl receptor gene of danish patients with familial hypercholesterolemia encodes a transport-defective protein antennapedia homeobox peptide regulates neural morphogenesis identification and molecular characterisation of a calm-af10 fusion in acute megakaryoblastic leukaemia mutations in a sar1 gtpase of copii vesicles are associated with lipid absorption disorders substitution of arginine for histidine at position 209 in the alpha-subunit of the human insulin receptor. a mutation that impairs receptor dimerization and transport of receptors to the cell surface distinct sets of sec genes govern transport vesicle formation and fusion early in the secretory pathway metabolic and molecular bases of menkes disease and occipital horn syndrome receptor downregulation and multivesicular-body sorting stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls positionally cloned gene for a novel glomerular protein-nephrin-is mutated in congenital nephrotic syndrome endocrinopathies in the family of endoplasmic reticulum (er) storage diseases: disorders of protein traycking and the role of er molecular chaperones a conditional mutation avecting localization of the menkes disease copper atpase. suppression by copper supplementation molecular characterization of mammalian homologues of class c vps proteins that interact with syntaxin-7 three ways to make a vesicle cellular autophagy: surrender, avoidance and subversion by microorganisms proteasome inhibitors: from research tools to drug candidates a cholesterol-lowering gene maps to chromosome 13q a controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis a germline insertion in the tuberous sclerosis (tsc2) gene gives rise to the eker rat model of dominantly inherited cancer disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in drosophila a post-docking role for active zone protein rim hiv interaction with endosomes in macrophages and dendritic cells organization of the pronephric filtration apparatus in zebrafish requires nephrin, podocin and the ferm domain protein mosaic eyes cdc42 controls secretory and endocytic transport to the basolateral plasma membrane of mdck cells copii-cargo interactions direct protein sorting into er-derived transport vesicles akt crystal structure and akt-specific inhibitors defective human ether-a-go-go-related gene traycking linked to an endoplasmic reticulum retention signal in the c terminus retroviral vectors vip21, a 21-kd membrane protein is an integral component of trans-golginetwork-derived transport vesicles regulation of vascular endothelial growth factor receptor-2 activity by caveolin-1 and plasma membrane cholesterol intracellular localization and loss of copper responsiveness of mnk, the murine homologue of the menkes protein, in cells from blotchy (mo blo) and brindled (mo br) mouse mutants interleukin 2 receptors and detergent-resistant membrane domains define a clathrinindependent endocytic pathway aberrant splicing but not mutations of tsg101 in human breast cancer tumor cell growth inhibition by caveolin re-expression in human breast cancer cells bi-directional protein transport between the er and golgi a missense mutation in the low density lipoprotein receptor gene causes familial hypercholesterolemia in sephardic jews novel mutations in the wiskott-aldrich syndrome protein gene and their evects on transcriptional, translational, and clinical phenotypes niemann-pick disease: a frequent missense mutation in the acid sphingomyelinase gene of ashkenazi jewish type a and b patients evidence for a regulated interaction between heterotrimeric g proteins and caveolin a huntingtin-associated protein enriched in brain with implications for pathology expression and characterization of recombinant caveolin hermansky-pudlak syndrome type 7 (hps-7) results from mutant dysbindin molecular, anatomical, and biochemical events associated with neurodegeneration in mice with niemann-pick type c disease the listeria protein internalin b mimics hepatocyte growth factor-induced receptor traycking bone marrow transplantation from an hla-matched unrelated donor for treatment of chediak-higashi syndrome familial sjogren's syndrome with associated primary salivary gland lymphoma protein kinase d regulates the fission of cell surface destined transport carriers from the trans-golgi network rme-1 regulates the distribution and function of the endocytic recycling compartment in mammalian cells caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis organized endothelial cell surface signal transduction in caveolae distinct from glycosylphosphatidylinositol-anchored protein microdomains mutant myosin viia causes defective melanosome distribution in the rpe of shaker-1 mice rescue of deltaf508 and other misprocessed cftr mutants by a novel quinazoline compound organic-aciduria, decreased renal ammonia production, hydrophthalmos, and mental retardation; a clinical entity structure and function of the lowe syndrome protein ocrl1 autoantigen golgin-97, an evector of arl1 gtpase, participates in trayc from the endosome to the trans-golgi network functional evaluation of dent's disease-causing mutations: implications for clc-5 channel traycking and internalization regulation of protein transport from the golgi complex to the endoplasmic reticulum by cdc42 and n-wasp the beta-appendages of the four adaptor-protein (ap) complexes: structure and binding properties, and identification of sorting nexin 9 as an accessory protein to ap-2 function and regulation of the mammalian coppertransporting atpases: insights from biochemical and cell biological approaches membrane dynamics and the biogenesis of lysosomes rabaptin-5 is a novel fusion partner to platelet-derived growth factor beta receptor in chronic myelomonocytic leukemia kdel-cargo regulates interactions between proteins involved in copi vesicle trayc: measurements in living cells using fret purification of a novel class of coated vesicles mediating biosynthetic protein transport through the golgi stack clogging of axons by tau, inhibition of axonal trayc and starvation of synapses endocytosis and targeting of exogenous hiv-1 tat protein a novel point mutation in cd18 causing the expression of dysfunctional cd11/cd18 leucocyte integrins in a patient with leucocyte adhesion deficiency (lad) copii-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes altered traycking and adhesion function of mpz mutations and phenotypes of charcot-marie-tooth disease 1b biology of adenovirus and its use as a vector for gene therapy the putative tumor suppressors ext1 and ext2 form a stable complex that accumulates in the golgi apparatus and catalyzes the synthesis of heparan sulfate congenital hypothyroid goiter with deficient thyroglobulin. identification of an endoplasmic reticulum storage disease with induction of molecular chaperones mutations in rab27a cause griscelli syndrome associated with haemophagocytic syndrome myoblast transfer in the treatment of duchenne's muscular dystrophy enzyme replacement therapy in fabry's disease: recent advances and clinical applications signalling to actin assembly via the wasp (wiskott-aldrich syndrome protein)-family proteins and the arp2/3 complex mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy long-term evectiveness of a new alpha-glucosidase inhibitor (bay m1099-miglitol) in insulin-treated type 2 diabetes mellitus genetic disorders avecting proteins of iron and copper metabolism: clinical implications hsp90 inhibitor geldanamycin and its derivatives as novel cancer chemotherapeutic agents mechanism of microtubule stabilization by doublecortin identification and functional analysis of two novel mutations in the multidrug resistance protein 2 gene in israeli patients with dubin-johnson syndrome pharmacological chaperones: a new twist on receptor folding a conserved clathrin assembly motif essential for synaptic vesicle endocytosis eea1, an early endosome-associated protein. eea1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine ''fingers'' and contains a calmodulin-binding iq motif localization of proteins to the golgi apparatus vip21/caveolin is a cholesterol-binding protein maturation models for endosome and lysosome biogenesis cdc42 regulates the exit of apical and basolateral proteins from the trans-golgi network consistent detection of calm-af10 chimaeric transcripts in haematological malignancies with t(10;11)(p13;q14) and identification of novel transcripts molecular analysis of the ergic-53 gene in 35 families with combined factor v-factor viii deficiency vascular endothelial growth factor (vegf) and its receptors the trojan horse: survival tactics of pathogenic mycobacteria in macrophages snares and membrane fusion in the golgi apparatus mutations in the er-golgi intermediate compartment protein ergic-53 cause combined deficiency of coagulation factors v and viii regulation of phagocytosis by rho gtpases urinary megalin deficiency implicates abnormal tubular endocytic function in fanconi syndrome wiskott-aldrich syndrome: a model for defective actin reorganization, cell traycking and synapse formation identification of 23 complementation groups required for post-translational events in the yeast secretory pathway cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically novel cell permeable motif derived from the pres2-domain of hepatitis-b virus surface antigens dynamin at the neck of caveolae mediates their budding to form transport vesicles by gtp-driven fission from the plasma membrane of endothelium the proteasome is involved in angiogenesis role of rab gtpases in membrane trayc the evolving role of lipid rafts and caveolae in g proteincoupled receptor signaling: implications for molecular pharmacology the structure and function of the beta 2-adaptin appendage domain phosphatidylinositol phosphate 5-kinase i{beta} recruits ap-2 to the plasma membrane and regulates rates of constitutive endocytosis fine structure of blood capillaries intracellular aspects of the process of protein synthesis two genes are responsible for griscelli syndrome at the same 15q21 locus localization and processing of cln3, the protein associated to batten disease: where is it and what does it do? localization of the ap-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins infectious hiv-1 assembles in late endosomes in primary macrophages snares and the specificity of transport vesicle targeting caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the er caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane trayc alpha1-antitrypsin deficiency: liver disease associated with retention of a mutant secretory glycoprotein in the endoplasmic reticulum alpha-1-antitrypsin deficiency: diagnosis and treatment identification of the murine beige gene by yac complementation and positional cloning the menkes protein (atp7a; mnk) cycles via the plasma membrane both in basal and elevated extracellular copper using a c-terminal dileucine endocytic signal participation of the endoplasmic reticulum chaperone calnexin (p88, ip90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator prevention of lysosomal storage in tay-sachs mice treated with n-butyldeoxynojirimycin correction of a mineralization defect by overexpression of a wild-type cdna for col1a1 in marrow stromal cells (mscs) from a patient with osteogenesis imperfecta: a strategy for rescuing mutations that produce dominant-negative protein defects constitutive protein secretion from the trans-golgi network to the plasma membrane review of donepezil, rivastigmine, galantamine and memantine for the treatment of dementia in alzheimer's disease in adults with down syndrome: implications for the intellectual disability population protein kinase d-mediated anterograde membrane traycking is required for fibroblast motility gene replacement reveals that p115/snare interactions are essential for golgi biogenesis constitutive skipping of alternatively spliced exon 10 in the atp7a gene abolishes golgi localization of the menkes protein and produces the occipital horn syndrome traycking and folding defects in hereditary spherocytosis mutants of the human red cell anion exchanger caveolin-2-deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae isolation of a miller-dicker lissencephaly gene containing g protein [beta]-subunit-like repeats myosin viia is required for aminoglycoside accumulation in cochlear hair cells endophilin/sh3p4 is required for the transition from early to late stages in clathrinmediated synaptic vesicle endocytosis mechanisms of cell-surface rerouting of an endoplasmic reticulum-retained mutant of the vasopressin v1b/v3 receptor by a pharmacological chaperone adaptor-related proteins adaptable adaptors for coated vesicles surfing the sec61 channel: bidirectional protein translocation across the er membrane yolk protein uptake in the oocyte of the mosquito aedes aegypti caveolin, a protein component of caveolae membrane coats mechanisms of intracellular protein transport protein sorting by transport vesicles early stages of influenza virus entry into mv-1 lung cells: involvement of dynamin science medicine, and the future. gene therapy endoplasmic reticulum storage diseases calm-af10 fusion gene in leukemias: simple and inversion-associated translocation (10;11) assembly of the er to golgi snare complex requires uso1p signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in mdck cells regulation of the unc-18-caenorhabditis elegans syntaxin complex by unc-13 visualization of er-to-golgi transport in living cells reveals a sequential mode of action for copii and copi identification, sequence, and expression of caveolin-2 defines a caveolin gene family an enzyme that removes clathrin coats: purification of an uncoating atpase endophilin i mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid endoplasmic reticulumlocalized amyloid beta-peptide is degraded in the cytosol by two distinct degradation pathways endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including vamp, nsf, snap, annexins, and gtpases griscelli syndrome: report of the first peripheral blood stem cell transplant and the role of mutations in the rab27a gene as an indication for bmt peptide-mediated cellular delivery gene therapy: a brief overview of the past, present, and future phagosome maturation: a few bugs in the system the npc1 protein: structure implies function rab gtpases, intracellular trayc and disease a ypt/rab evector complex containing the sec1 homolog vps33p is required for homotypic vacuole fusion the role of the tethering proteins p115 and gm130 in transport through the golgi apparatus in vivo early endosome antigen. 1: an autoantigen associated with neurological diseases identification of the b-cell epitopes of the early endosome antigen 1 (eea1) cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia diverential evects of kinase cascade inhibitors on neoplastic and cytokine-mediated cell proliferation chediak-higashi syndrome: a rare disorder of lysosomes and lysosome related organelles autophagy in health and disease: a double-edged sword golgins and gtpases, giving identity and structure to the golgi apparatus a role for the vesicle tethering protein, p115, in the postmitotic stacking of reassembling golgi cisternae in a cell-free system sequential tethering of golgins and catalysis of snarepin assembly by the vesicle-tethering protein p115 transcytosis of plasma macromolecules in endothelial cells: a cell biological survey regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian lis1 snap receptors implicated in vesicle targeting and fusion paraneoplastic stiv-person syndrome: passive transfer to rats by means of igg antibodies to amphiphysin a role for giantin in docking copi vesicles to golgi membranes phenotypic behavior of caveolin-3 r26q, a mutant associated with hyperckemia, distal myopathy, and rippling muscle disease phosphofructokinase muscle-specific isoform requires caveolin-3 expression for plasma membrane recruitment and caveolar targeting: implications for the pathogenesis of caveolin-related muscle diseases identification of filamin as a novel ligand for caveolin-1: evidence for the organization of caveolin-1-associated membrane domains by the actin cytoskeleton endosomal localization of the autoantigen eea1 is mediated by a zinc-binding fyve finger perforin gene defects in familial hemophagocytic lymphohistiocytosis copicoated er-to-golgi transport complexes segregate from copii in close proximity to er exit sites the immunological synapse of ctl contains a secretory domain and membrane bridges linking albinism and immunity: the secrets of secretory lysosomes the biogenesis of lysosomes: is it a kiss and run, continuous fusion and fission process? breaking the copi monopoly on golgi recycling a family of rab27-binding proteins. melanophilin links rab27a and myosin va function in melanosome transport retinal stimulates atp hydrolysis by purified and reconstituted abcr, the photoreceptor-specific atp-binding cassette transporter responsible for stargardt disease abnormal vesicular traycking in mouse models of hermansky-pudlak syndrome protein folding and translocation across the endoplasmic reticulum membrane role of calnexin in the glycan-independent quality control of proteolipid protein assembly and traycking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters identification and expression of five mutations in the human acid sphingomyelinase gene causing types a and b niemann-pick disease. molecular evidence for genetic heterogeneity in the neuronopathic and non-neuronopathic forms defective aquaporin-2 traycking in nephrogenic diabetes insipidus and correction by chemical chaperones misfolding of mutant aquaporin-2 water channels in nephrogenic diabetes insipidus molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle copii and exit from the endoplasmic reticulum identification of a familial hyperinsulinism-causing mutation in the sulfonylurea receptor 1 that prevents normal traycking and function of katp channels mutations of the pds gene, encoding pendrin, are associated with protein mislocalization and loss of iodide ezux: implications for thyroid dysfunction in pendred syndrome the chediak-higashi protein interacts with snare complex and signal transduction proteins clathrin assembly lymphoid myeloid leukemia (calm) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated trayc how peroxisomes arise chloride channels cough up caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic traycking tau regulates the attachment/detachment but not the speed of motors in microtubule-dependent transport of single vesicles and organelles gangliosides are receptors for murine polyoma virus and sv40 protein traycking and alzheimer's disease role of auxilin in uncoating clathrincoated vesicles the inositol polyphosphate 5-phosphatase ocrl associates with endosomes that are partially coated with clathrin actin microfilaments facilitate the retrograde transport from the golgi complex to the endoplasmic reticulum in mammalian cells dynamin-like protein encoded by the drosophila shibire gene associated with vesicular trayc protein kinase d: an intracellular trayc regulator on the move fcgammar polymorphisms: implications for function, disease susceptibility and immunotherapy a dileucine-like sorting signal directs transport into an ap-3-dependent, clathrin-independent pathway to the yeast vacuole protein kinase g from pathogenic mycobacteria promotes survival within macrophages degradation of cftr by the ubiquitinproteasome pathway use of expression constructs to dissect the functional domains of the chs/beige protein: identification of multiple phenotypes a novel 115-kd peripheral membrane protein is required for intercisternal transport in the golgi stack rab6 coordinates a novel golgi to er retrograde transport pathway in live cells hsp90 and the chaperoning of cancer sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction the human cytomegalovirus us11 gene product dislocates mhc class i heavy chains from the endoplasmic reticulum to the cytosol amphiphysin heterodimers: potential role in clathrin-mediated endocytosis the caveolin genes: from cell biology to medicine a mutation in rab27a causes the vesicle transport defects observed in ashen mice caveolinopathies: mutations in caveolin-3 cause four distinct autosomal dominant muscle diseases four contiguous amino acid substitutions, identified in patients with laron syndrome, diverently avect the binding aynity and intracellular traycking of the growth hormone receptor synaptojanin is the major constitutively active phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase in rodent brain rab27a enables myosin va-dependent melanosome capture by recruiting the myosin to the organelle identification of an organelle receptor for myosin-va the tuberous sclerosis 2 gene product, tuberin, functions as a rab5 gtpase activating protein (gap) in modulating endocytosis the fine structure of the gall bladder epithelium of the mouse caveolin is an inhibitor of platelet-derived growth factor receptor signaling sulfonylureas correct traycking defects of atp-sensitive potassium channels caused by mutations in the sulfonylurea receptor nsec1 binds a closed conformation of syntaxin1a abnormal ryanodine receptor function in heart failure fabry disease: characterization of alpha-galactosidase a double mutations and the d313y plasma enzyme pseudodeficiency allele protein kinase d regulates basolateral membrane protein exit from trans-golgi network predisposition to renal carcinoma in the eker rat is determined by germ-line mutation of the tuberous sclerosis 2 (tsc2) gene adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis rab proteins as membrane organizers bleeding due to disruption of a cargo-specific er-to-golgi transport complex charcot-marie-tooth disease type 2a caused by mutation in a microtubule motor kif1b the lebanese allele at the low density lipoprotein receptor locus. nonsense mutation produces truncated receptor that is retained in endoplasmic reticulum sucrase-isomaltase deficiency in humans. diverent mutations disrupt intracellular transport, processing, and function of an intestinal brush border enzyme surfing the sec61 channel: bidirectional protein translocation across the er membrane crystal structure of an activated akt/protein kinase b ternary complex with gsk3-peptide and amp-pnp key: cord-280679-jj3wzojy authors: marblestone, jeffrey g.; edavettal, suzanne c.; lim, yiting; lim, peter; zuo, xun; butt, tauseef r. title: comparison of sumo fusion technology with traditional gene fusion systems: enhanced expression and solubility with sumo date: 2006-01-01 journal: protein science doi: 10.1110/ps.051812706 sha: doc_id: 280679 cord_uid: jj3wzojy despite the availability of numerous gene fusion systems, recombinant protein expression in escherichia coli remains difficult. establishing the best fusion partner for difficult-to-express proteins remains empirical. to determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described sumo fusion system with a variety of commonly used fusion systems was completed. for this study, three model proteins, enhanced green florescent protein (egfp), matrix metalloprotease-13 (mmp13), and myostatin (growth differentiating factor-8, gdf8), were fused to the c termini of maltose-binding protein (mbp), glutathione s-transferase (gst), thioredoxin (trx), nus a, ubiquitin (ub), and sumo tags. these constructswere expressed in e. coli and evaluated for expression and solubility. as expected, the fusion tags varied in their ability to produce tractable quantities of soluble egfp, mmp13, and gdf8. sumo and nus a fusions enhanced expression and solubility of recombinant proteins most dramatically. the ease at which sumo and nus a fusion tags were removed from their partner proteins was then determined. sumo fusions are cleaved by the natural sumo protease, while an actev protease site had to be engineered between nus a and its partner protein. a kinetic analysis showed that the sumo and actev proteases had similark(m) values, but sumoprotease had a 25-fold higher k(cat) than actev protease, indicating a more catalytically efficient enzyme. taken together, these results demonstrate that sumo is superior to commonly used fusion tags in enhancing expression and solubility with the distinction of generating recombinant protein with native sequences. the lack of efficient methods to express structurally diverse proteins in escherichia coli is a major obstacle in structural genomics. in fact, the southeast collaboratory for structural genomics (secsg) reports that only 22.9% of proteins they have expressed in e. coli have been soluble (1463 soluble proteins of 6397 expressed; as published on the secsg web site 08/11/2005). numerous technological advancements have vastly improved recombinant protein expression in e. coli, including the development of strong promoters (studier and moffatt 1986) , coexpression with chaperones and foldases (ikura et al. 2002) , and the use of protein fusions. protein fusions have been particularly successful at enhancing the expression and solubility of recombinant proteins. fusion systems are characterized by their ability to enhance protein expression, reduce proteolytic degradation of the recombinant protein, improve protein folding, solubility, and simplify purification and detection. a variety of structures have been used as fusion motifs, including maltose-binding protein (mbp), glutathione s-transferase (gst), thioredoxin (trx), nus a, ubiquitin (ub), and sumo (table 1 ; pryor and leiting 1997; wang et al. 1999; de marco et al. 2004 ). there is nothing in common among these fusion proteins in terms of molecular weight, structure, or function, with the exception of ub and sumo, which share a common structure (bayer et al. 1998 ). as such, predicting which fusion tag will enhance the solubility of a difficult-to-express protein remains empirical. some comparison studies have been completed; however, none have examined the ability of the newly described sumo fusion system to enhance expression and solubility in comparison to other commonly used tags (davis et al. 1999; wang et al. 1999; de marco et al. 2004) . sumo (small ubiquitin-related modifier) , an ,100residue protein, modulates protein structure and function by covalent modification of target proteins in eukaryotes (johnson and blobel 1999; melchior 2000; tatham et al. 2001) . the sumo pathway is highly conserved in eukaryotes and notably absent in prokaryotes. yeast has a single sumo gene (smt3), while three genes have been described in vertebrates (sumo-1, sumo-2, and sumo-3) (kawabe et al. 2000) . the three human sumos are highly homologous, with human sumo-1 sharing 50% sequence identity with human sumo-2 and sumo-3 (muller et al. 1998) , and human sumo-2 and sumo-3 sharing 87% sequence identity with each other (melchior 2000) . sumo shares 47% sequence identity with human sumo-1. although the overall sequence identity between sumos and ub is ,18%, structure determination reveals that they share a common three-dimensional structure that is characterized by a tightly packed globular fold with b-sheets wrapped around one a-helix (bayer et al. 1998) . the conjugation of sumo to target proteins is a highly regulated and dynamic process, similar to ub. the enzymes involved in cleaving sumo fusions, the sumo proteases, have been extensively studied. hochstrasser and li demonstrated that the yeast sumo proteases, ulp1 and ulp2, remove sumo from proteins and play a role in progression through the g 2 /m phase and recovery of cells from check point arrest, respectively hochstrasser 1999, 2000) . ulp1 and ulp2 cleave the c-termini of sumo (-ggaty) to form a mature sumo (-gg) and also deconjugate it from the side chains of lysines within modified proteins. the sequence similarity of the two enzymes is restricted to a 200-amino-acid sequence called ulp domain that contains the catalytically active region. the three-dimensional structure of the ulp domain from ulp1 has been determined in a binary complex with sumo (mossessova and lima 2000) . it is interesting to note that sumo proteases are not related to the deubiquinating enzymes (dubs), but are distantly related to adenoviral processing protease hochstrasser 1999, 2000) . recently, a sumo fusion system that facilitates efficient expression of recombinant proteins in e. coli has been described (malakhov et al. 2004) . several proteins, including severe acute respiratory syndrome coronavirus (sars-cov) 3cl protease, nucleocapsid, and membrane proteins, have been recombinantly expressed using the sumo fusion system (zuo et al. 2005b ). the sumo fusion tag has lead to enhanced expression and solubility. a hexahistidine sumo fusion construct has been shown to enhance expression and facilitate purification with ni-nta chromatography (zuo et al. 2005a) . one distinguishing feature of the sumo fusion system is the ability of its associated sumo protease to cleave a variety of fusion partners with remarkable fidelity and efficiency (malakhov et al. 2004) . traditional gene fusion systems require engineered cleavage sites, which are recognized by the proteases and are positioned between the fusion tag and the protein target. proteases that have been used to cleave fusion tags include tobacco etch virus (tev) protease (carrington et al. 1989 ), factor xa, or thrombin protease (jenny et al. 2003) . a major drawback to the use of engineered cleavage sites and traditional proteases is the generation of non-native n-terminal amino acids. many structural and therapeutic proteins require specific n-terminal amino acids for biological activity (e.g., chemokines). cleavage by traditional proteases results in the retention of several amino acids, which are downstream from the cleavage site and required for protease recognition. for example, thrombin will cleave the sequence lvprgs at the arginine residue, resulting in an n-terminal extension of the target protein by two amino acids (gs) (jenny et al. 2003) . those proteins that require a specific n terminus for biological activity, half-life, or structural stability, will not be successfully expressed using gene fusions with traditional proteases. however, direct fusion of the recombinant protein to the c terminus of sumo results in the production of protein with the desired n-terminal amino acid. in addition, when using traditional gene fusion systems, if the target protein or fusion tag contains the cleavage recognition sequence, the target protein will also be cleaved (e.g., erroneous cleavage of the nus a tag has been observed when using factor xa) (davis et al. 1999) . the sumo protease recognizes the tertiary structure of sumo, and as such, does not cleave erroneously within the target protein. previous experience with the sumo fusion system suggests that it represents a technological advancement in recombinant protein expression, as this system enhances expression and solubility and utilizes a highly specific protease capable of generating native n-terminal amino acids. the aim of this study is to provide a direct comparison of sumo with other fusion systems to determine whether it truly represents such advancement. three candidate proteins, enhanced green fluorescent protein (egfp), and two previously described difficult-to-express proteins, matrix metalloprotease-13 (mmp13) and myostatin (growth differentiating factor-8, gdf8), were expressed as fusions with maltose-binding protein (mbp), glutathione s-transferase (gst), thioredoxin (trx), nus a, ubiquitin (ub), and sumo. these constructs were expressed in e. coli and evaluated for expression and solubility. in addition, the catalytic efficiency of the sumo and commonly used actev proteases were evaluated. the ability of commonly used fusion tags to enhance protein expression and solubility was investigated using three candidate proteins (egfp, mmp13, and gdf8) and six fusion tags (sumo, ub, mbp, gst, trx, and nus a). vectors were transformed into e. coli and cultures were induced for protein expression under the control of the t7 promoter with iptg. culture inductions were conducted at 20°c overnight for optimal expression levels and solubility. cell lysates from uninduced (ui) and induced (i) cultures, plus the soluble (s) and insoluble (ib) fractions from the induced cultures were analyzed by sds-page (figs. 1-3 ). there was a clear difference among the fusion tags with respect to their ability to enhance expression and solubility. the impact of different gene fusions on the expression and solubility of egfp following induction at 20°c overnight are shown in figure 1 . the his 6 -egfp construct was observed to have very low expression, while the sumo-, trx-, and nus a-egfp constructs had the greatest enhancement of soluble expression. the sumo and nus a fusions resulted in the best soluble expression of egfp (,90% and 100% soluble, respectively). while trx fusion resulted in greatly enhanced expression, only ,50% of the expressed protein was soluble. the ub, mbp, and gst fusions enhanced egfp expression equivocally, relative to each other. the difficult-to-express mmp13 (residues 20-274) was then investigated for enhanced solubility and expression with the various gene fusions (fig. 2) . as expected, the his 6 -mmp13 fusion did not generate any mmp13. sumo, nus a, and trx fusions produced the greatest degree of expression enhancement, as was observed for egfp. also consistent with the results from the egfp study, the sumo and nus a fusions resulted in enhanced soluble expression (,40% and 80% fusion mmp13 in the soluble fraction, respectively). in contrast to egfp, the trx-mmp13 fusion solely generated insoluble protein. the ub-mmp13 fusion was observed to have a moderate amount of expression; however, the majority of the protein produced was insoluble (,90%). again, the gst fusions only enhanced expression mildly, and the mbp fusion failed to enhance expression. gdf8 (mature), also considered to be a difficult-toexpress protein, was evaluated for enhanced expression and solubility with the various gene fusions (fig. 3) . the ub-and gst-gdf8 fusions seem to have enhanced expression levels, but the majority of the recombinant protein was found in the inclusion body, with little in the soluble fraction. similarly, the trx-gdf8 fusion yielded high levels of expression; however, this fusion was exclusively insoluble. the sumo-and nus a-gdf8 fusions were both observed to enhance expression at high levels, with the sumo and nus a fusions being the only tags that were able to preserve solubility. as was observed for the mmp13 and egfp constructs, nus a produced almost entirely soluble protein (,95%) with the sumo fusion less successful at retaining recombinant protein solubility (,50%). the mbp and his 6 fusions were observed to have very little enhanced expression of gdf8. the sumo-and nus a-tags produced the highest expression levels and solubility among the fusion proteins tested. a comparison of their respective proteases was then conducted to determine the catalytic efficiency of removing the fusion tag ( fig. 4 ; table 2 ). the commonly used actev protease was used to cleave the nus a tag, and an actev recognition sequence (enlyfq'gxx) was engineered between the nus a tag and the fusion partner. the kinetic parameters k m and k cat for actev and sumo protease were determined using the nus a-egfp and sumo-egfp substrates, respectively ( table 2 ). the generated data were fit to the michaelis-menton equation using nonlinear regression (r 2 values were equal to 0.97 and 0.99 for the sumo protease and actev plots, respectively) (fig. 4) . the apparent k m of actev for the nus a-egfp fusion (6.3 mm) is very similar to that of sumo protease for the sumo-egfp fusion (3.3 mm), but its k cat (0.028 sec -1 ) is ,25-fold less than that of sumo protease (0.782 sec -1 ), resulting in an enzyme with ,25 times lower catalytic efficiency. the k m and k cat values obtained for actev are not similar to what has been previously reported (nallamsetty et al. 2004 ). this is most likely due to the choice of substrates, as previous studies have been completed using peptide substrates with canonical recognition sites. the sumo protease does not recognize a linear sequence like actev, and as such, a full fusion construct had to be utilized for comparison purposes. it is expected that lower k m and higher k cat values would be obtained for the peptide substrate than for the full fusion construct. the preferred host for heterologous protein expression is e. coli, as this host system provides a simple and inexpensive means to produce recombinant protein. many obstacles are encountered, however, when using e. coli as a host system. gene fusion technology has been very successful at overcoming most of these obstacles and has increased the success of heterologous expression in e. coli. ideally, a fusion tag should enhance expression and solubility of a wide variety of proteins. while several studies have compared the effectiveness of commonly used gene fusions in enhancing the expression and solubility of difficult-to-express proteins, none have included the newly described sumo fusion system (davis et al. 1999; wang et al. 1999; de marco et al. 2004) . three candidate proteins were chosen for this study, egfp, gdf8, and mmp13. egfp was chosen due to its common use in biochemical laboratories and the ease at which it can be assayed. gdf8 is essential for proper regulation of human skeletal muscle mass (lee and mcpherron 2001) . gdf8 was chosen, as recombinant expression in e. coli would greatly facilitate research efforts aimed at exploiting this potentially useful therapeutic target. previous attempts to express gdf8 in e. coli have been hampered by poor yields, solubility, and lack of biological activity, and therefore, gdf8 is considered to be a difficult-to-express protein (thomas et al. 2000; taylor et al. 2001) . similarly, mmp13 is generally considered a potential therapeutic target and a difficultto-express protein (pathak et al. 1998; hardern et al. 2000) . although only a limited number of proteins were assayed for this study, patterns do emerge as to which tags are best for enhanced expression and solubility, especially for the difficult-to-express proteins used. in general, his 6 tag afforded little to no enhanced expression. mbp was also observed to have little impact on expression, and did not enhance expression at all for the difficult-to-express proteins in this study (mmp13 and gdf8). both the ub and gst tags did provide mild enhancement of expression; however, the recombinant protein was mostly contained within the inclusion bodies. sumo, trx, and nus a were the best tags at enhancing expression. the trx tag did not enhance solubility for the difficult-toexpress proteins, while both sumo and nus a had pronounced soluble fusion protein expression. for all polypeptides, there is a competition between aggregation and folding that rely on similar molecular interactions. fusion partners have been shown to act as solubility enhancers, although the exact mechanism by which they improve solubility has not been described. it is speculated that fusion partners are able to keep the target protein in solution long enough for it to undergo its natural folding process, otherwise the target protein aggregates before it has sufficient opportunity to fold. a soluble fusion partner that slows the aggregation process curve fitting to the michelais-menton equation was completed using nonlinear regression (r 2 as shown on each graph), and used to calculate k m and k cat (table 2 ). (b) an sds gel depicting the purification of gfp using the sumo fusion tag. soluble cell lysate was passed over a ni-nta column, washed with 20 mm imidazole, and eluted with 300 mm imidazole (affinity purified). cleavage with the sumo protease was conducted under standard conditions, and the sample was passed over another ni-nta column to remove the sumo protease and tag (subtracted). these various steps of a typical purification of sumo-gfp were analyzed in a 15% sds-polyacrylamide gel. proteins were visualized using coommassie blue staining. arrowheads indicate expected/observed positions of respective protein bands. inadvertently shifts the fusion protein to a folding pathway, increasing the amount of soluble protein. fusion tags have also been hypothesized to enhance the solubility of the protein target by acting as a nucleus of folding ("molten globule hypothesis") (creighton 1997; englander 2000) . this theory suggests that a fusion tag acts as a nucleation site for the folding of the target protein. attachment of sumo to the n terminus of a partner protein has been observed to promote correct folding and solubility. sumo and ub have highly homologous, rapidly folding structures (khorasanizadeh et al. 1996) . it may be that the tight, rapidly folding soluble structure of sumo provides a nucleation site for the proper folding of c-terminally fused partner proteins. indeed, expression of alternative configurations of the fusion (i.e., egfp-sumo) was not enhanced in e. coli (data not shown). the 55-kda nus a fusion tag also facilitates solubility of partner proteins. nus a, a hydrophilic tag, was identified in a screen for e. coli proteins that have the highest potential for solubility when overexpressed (davis et al. 1999) . protein expression levels are dependent on the stability of their mrna, where degradation plays an important role controlling the levels of unstable transcripts (arechaga et al. 2003) . both nus a and sumo were observed to enhance expression, but the role they play in stabilizing the mrna transcript is unknown. whereas nus a levels of expression have been well documented, the novel sumo tag has had modest published results. table 3 represents a variety of fusion proteins that have been successfully expressed using the sumo system. these sumo fusion proteins have established a 5-25-fold increase in expression levels compared with their unfused counterparts (t.r. butt, pers. comm.). in this study, neither of the commonly used tags, gst or mbp, dramatically enhanced expression or solubility. while for the purpose of the present study mbp was fused solely to the n terminus of partner proteins, successful expression has also been achieved when mbp is fused to the c terminus of a protein (sachdev and chirgwin 2000) . in fact, it has been found that n-terminal mbp fusions can reduce the efficiency of translation, which may explain the low yield in these experiments with mbp (hamilton et al. 2002; podmore and reynolds 2002) . these studies underscore the need for a comprehensive analysis of the fusion tags for the expression of various protein families. if the present study is confirmed, it would appear that commonly used fusions for enhanced soluble protein expression are not the best fusion tags available. removal of the chosen fusion tag can also present a formidable challenge. in the present study, the best tags for enhanced expression and solubility (sumo and nus a) were further examined for the ease at which they are removed by their respective proteases (sumo and actev). both actev and sumo proteases had similar affinities to their substrates. however, they differed dramatically in terms of catalytic efficiency (k cat ), with sumo protease being ,25 times more efficient than actev. in addition to being more catalytically efficient, sumo protease also has the advantage of recognizing the tertiary structure of sumo and not a linear amino acid sequence like actev. this characteristic prevents the sumo protease from erroneously cleaving within the target protein, and thus, sumo protease behaves as a universal protease, suitable for nearly every substrate. in addition, direct fusion of the target protein to sumo allows for the generation of recombinant protein with native n-terminal sequences. taken together, these data suggest that the sumo fusion system may be ideal for the soluble expression of proteins that have been impossible using traditional gene fusions. all plasmids were constructed by standard methods. e. coli strains dh5a and top10 (invitrogen) were used for plasmid construction and manipulation, and pet24d (novagen) was used as the backbone. n-terminal or c-terminal hexahistidinetags were added to all fusions except for gst. in addition, all of the fusion tags except sumo contained a cleavage site for actev protease to facilitate the proper removal of the fusion tag upon purification. actev was chosen in this study based on its high cleavage activity and it is commonly used in fusion technology. the cloning strategy used ncoi and bamhi sites upstream and downstream, respectively, of the fusion tag sequence, which was ligated pet24d vector. a bsai restriction site was introduced by pcr directly downstream of the fusion tag sequence, upstream of the bamhi site in the reverse primer to ensure a consistent cloning strategy. the cloning strategy to express the fusion proteins used bsai in the forward primer directly upstream of egfp (clontech), mmp13 (residues 20-274), or gdf8 (mature) with either a hindiii or ecori restriction site downstream of the gene sequence in the reverse primer. these primers allowed the dna fragment to be cloned in frame with the fusion tags. all plasmids were sequenced routinely (dna clinic inc.). in a typical experiment, a single colony of transformed rosetta (de3) plyss (novagen) was inoculated into 2 ml of lb-broth with chloramphenicol (25 mg/ml) and kanamycin (30 mg/ml) and grown overnight at 37°c with shaking. a total of 1 ml of this overnight culture was inoculated into 100 ml of lb (25 mg/ ml cm and 30 mg/ml kan), and the culture was grown to od 600 = 0.6 at 37°c with shaking. the culture was then cooled on ice, induced with 1 mm iptg, and incubated at 20°c overnight with shaking. cells were isolated by centrifugation at 10,000g for 20 min and resuspended in 3 ml of pbs (ph 8.0), 300 mm nacl, 10 mm imidazole. cells were lysed by mild sonication. triton x-100 (sigma) was added to 1% (v/v) and incubated at 4°c for 1 h, with shaking. the culture was centrifuged at 10,000g for 30 min, and the supernatant was decanted and stored at 4°c (soluble protein sample). inclusion body samples were prepared by resuspending the insoluble material in 3 ml of solubilization buffer (50 mm caps at ph 11, 0.3 m nacl, 0.3% n-lauryl sarcosine, and 1 mm dtt), and allowed to shake for 20 min at room temperature. the ib fraction was then centrifuged 10,000g for 20 min, and the supernatant removed and stored at 4°c (inclusion body protein sample). sumo-egfp and nus a-egfp, the substrates used for kinetic analysis, were purified from soluble fractions prepared as described above. both fusion constructs contained an nterminal his 6 tag and were purified by ni-nta superflow resin (qiagen) using the biologic duo-flow fplc (bio-rad) as described previously (zuo et al. 2005a) . briefly, the soluble cellular lysate was loaded onto a ni-nta (5 ml resin vol) column (20 cm · 1.6 cm) at 1 ml/min and the resin was washed extensively with 40 ml wash buffer (pbs [ph 8.0], 20 mm imidazole, 150 mm nacl) at 2 ml/min. purified protein was then eluted in 15 ml of pbs (ph 8.0), 150 mm nacl, 300 mm imidazole at 2 ml/min. the purified protein eluted typically as an isolated, symmetrical uv peak. peak fractions were pooled (10 ml) and dialyzed using 3.5 kda mwco dialysis tubing against pbs (ph 7.5) at 4°c for 24 h. the sumo protease assays were initiated by mixing 6 mm sumo protease (lifesensors) with purified sumo-egfp (12, 6, 3, 1.5, 0.75, and 0.375 mm) in pbs (ph 7.5), 1 mm dtt. the reactions were incubated at 30°c for 1 h and stopped by addition of sds-loading buffer. samples were then heated at 95°c for 3 min and loaded onto a 15% sds-polyacrylamide gel for analysis. the actev protease assays were initiated by mixing 0.48 mm actev protease (invitrogen) with purified nus a-egfp (24, 12, 6, 3, 1.5, 0.75 mm) in pbs (ph 7.5), 1 mm dtt. the reactions were incubated at 30°c for 2 h and stopped by addition of sds-loading buffer. samples were then heated at 95°c for 3 min and loaded onto a 15% sds-polyacrylamide gel for analysis. time course experiments concluded that 1 and 2 h were well within the initial velocity rates for sumo protease and actev protease, respectively (that is, the rate of reaction was within in the linear region [data not shown]). the sds-polyacrylamide gels were stained with coomassie blue and scanned such that each band could be quantified using the software scion image version beta 4.0.2 (scion corporation). densitometry analyses were performed, and when compared with a loading standard (known amount of product) used to determine the amount of product generated per unit time. the initial velocity values used for curve fitting were the mean of three independent experiments. these initial velocity measurements were plotted against the substrate concentration and fit to the michaelis-menton equation using kaleidagraph 3.5 (synergy corporation). the k cat values were calculated by assuming 100% activity for the enzyme. overexpression of escherichia coli f1f(o)-atpase subunit a is inhibited by instability of the uncb gene transcript structure determination of the small ubiquitinrelated modifier sumo-1 a second proteinase encoded by a plant potyvirus genome how important is the molten globule for correct protein folding? new fusion protein systems designed to give soluble expression in escherichia coli the solubility and stability of recombinant proteins are increased by their fusion to nusa protein folding intermediates and pathways studied by hydrogen exchange ampactivated protein kinase kinase: detection with recombinant ampk a1 subunit an analysis of two refolding routes for a cterminally truncated human collagenase-3 expressed in escherichia coli co-overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase in escherichia coli a critical review of the methods for cleavage of fusion proteins with thrombin and factor xa cell cycle-regulated attachment of the ubiquitin-related protein sumo to the yeast septins covalent modification of the werner's syndrome gene product with the ubiquitin-related protein, sumo-1 evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues regulation of myostatin activity and muscle growth the yeast ulp2 (smt4) gene encodes a novel protease specific for the ubiquitin-like smt3 protein sumo fusions and sumo-specific protease for efficient expression and purification of proteins sumo-nonclassical ubiquitin ulp1-sumo crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast conjugation with the ubiquitin-related modifier sumo-1 regulates the partitioning of pml within the nucleus efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro the expression, refolding, and purification of the catalytic domain of human collagenase-3 (mmp-13) purification and characterization of vanxy(c), a d, d-dipeptidase/d, d-carboxypeptidase in vancomycinresistant enterococcus gallinarum bm4174 high-level expression of soluble protein in escherichia coli using a his6-tag and maltose-binding-protein double-affinity fusion system fusions to maltose-binding protein: control of folding and solubility in protein purification use of bacteriophage t7 rna polymerase to direct selective high-level expression of cloned genes polymeric chains of sumo-2 and sumo-3 are conjugated to protein substrates by sae1/sae2 and ubc9 myostatin inhibits cell proliferation and protein synthesis in c2c12 muscle cells myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions to glutathione s-transferase, thioredoxin and maltose-binding protein enhanced expression and purification of membrane proteins by sumo fusion in e. coli expression and purification of sars coronavirus proteins using sumo fusions we thank drs. stefan masure, susan weiss, and hiep tran for their help with the sumo fusion project. we also thank dr. tejvir s. khurana and his colleagues at the university of pennsylvania medical school for help in expression and analysis of the gdf8 project. numerous colleagues in the protein expression field have kindly shared with us their problems and ideas, for which we are most thankful. research supported in part by grant numbers gm 068404-01, ai51752-01/02, and hl69744 awarded to t.r.b. by the nih. key: cord-290290-wyx9ib7s authors: sinegubova, maria v.; orlova, nadezhda a.; kovnir, sergey v.; dayanova, lutsia k.; vorobiev, ivan i title: high-level expression of the monomeric sars-cov-2 s protein rbd 320-537 in stably transfected cho cells by the eef1a1-based plasmid vector date: 2020-11-05 journal: biorxiv doi: 10.1101/2020.11.04.368092 sha: doc_id: 290290 cord_uid: wyx9ib7s the spike (s) protein is one of the three proteins forming the coronaviruses’ viral envelope. the s protein of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has a spatial structure similar to the s proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (rbd), which is a significant inducer of host immune response. recombinant sars-cov-2 rbd is widely used as a highly specific minimal antigen for serological tests. correct exposure of antigenic determinants has a significant impact on the accuracy of such tests – the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native s protein. based on the previously developed p1.1 vector, containing the regulatory sequences of the eukaryotic translation elongation factor 1 alpha gene (eef1a1) from chinese hamster, we created two expression constructs encoding sars-cov-2 rbd with c-terminal c-myc and polyhistidine tags. rbdv1 contained a native viral signal peptide, rbdv2 – human tpa signal peptide. we transfected a cho dg44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. for the rbdv2 variant, a high-yield clonal producer cell line was obtained. we developed a simple purification scheme that consistently yielded up to 30 mg of rbd protein per liter of the simple shake flask cell culture. purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for rbdv2 protein, the monomeric form content exceeded 90% for several series. deglycosylation with pngase f and mass spectrometry confirmed the presence of n-glycosylation. the antigen produced by the described technique is suitable for serological tests and similar applications. humanity is faced with an unprecedented challenge -the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which causes a severe respiratory illness -coronavirus disease 2019 (covid19) pandemic. countries were sent to lockdown; people could not make informed decisions about the possibility of social contacts; the need for diagnostic tests is very high. existing tests for sars-cov2 are reviewed in [1] . at the beginning of the pandemic, pcr testing methods dominated since such test systems can be developed urgently, soon after the emergence of a new virus in the population. among the disadvantages of pcr-tests is a high sensitivity to contamination and dependence on sampling's correctness, a high proportion of falsepositive signals. unlike pcr diagnostics, serological testing gives positive results long after the event of infection, at least for several months. this testing method makes it possible to reliably determine whether a person is infected with the sars-cov-2, even in the absence of disease symptoms. we need serological tests, both in express format and screening tests based on elisa. serologic tests are also needed to detect convalescent plasma of therapeutic interest and assess emerging vaccines' effectiveness. in order for serological testing to have a more significant predictive value, mapping of the epitopes to which neutralizing antibodies appear should be carried out, as was done for sars-cov1 [2] , аnd convalescent or postvaccinal sera should be massively tested for the presence of neutralizing antibodies, for example, with a surrogate virus neutralization test based on antibody-mediated blockage of ace2-spike protein-protein interaction [3] or another that can be carried out on a relatively large scale. the use of highly specific and high-affinity viral antigens is already a big step towards improving diagnostic accuracy. the immunodominant antigen of sars-cov2 is the rbd domain of the spike protein [4] . another antigen widely used for diagnostics -the nucleocapsid (n) protein -combines high sensitivity and low specificity; therefore, it needs accurate antigen mutagenesis to remove highly conserved areas without compromising affinity. cases are described for sars-cov when the results of testing with n-protein were clarified using two subunits of spike protein [5] . the coronaviruses' spike (s) protein forms large coronal-like protrusions on the virions surface, hence the name of the family coronaviridae. the s protein plays a crucial role in receptor recognition, cell membrane fusion, internalization of viruses, and their exit from the endosomes. it is described in detail in the review [6] . it consists of s1 and s2 subunits and, in the case of the sars-cov-2 virus, has 1260 amino acids [7] . the s protein is co-translationally incorporated into the rough endoplasmic reticulum (er) and is glycosylated by n-linked glycans. glycosylation is essential for proper folding and transport of the s protein. the s protein trimer is transported from the er. interacting with the m and e proteins s protein trimer is transported to the virus's assembly site. s protein is required for cell entry but not necessary for virus assembly [8] . during their intracellular processing, s proteins of many types of coronaviruses, including sars-cov-2 and mers-cov, but not sars-cov, undergo partial proteolytic degradation at the furin signal protease recognition site with the formation of two subunits s1 and s2. apparently, most of the s protein copies on the membrane of sars-cov-2 viral particles are trimers of s2 subunits that are incapable of interacting with the receptor. the full-length s protein trimer on the viral particle's surface also undergoes complex conformational rearrangements during the formation of the rbd-receptor complex and the virus's penetration into the cell. the s protein homotrimer binds to the ace2 dimer, detailed study of this interaction is available here [9] . as part of the trimer, the spike protein's monomer "moves its head" -the s1 subunit can form the open or closed conformation; that is, it can have a raised fragment or a lowered rbd domain, this can influence the affinity of antibodies targeted to it. the s protein of sars-cov-2 amino acid sequence is variable, with more than 200 and 18 relatively frequent s protein amino acid variations. a glycan shield is formed by n-linked glycans on the s protein surface, which is likely to help viral immune escape. in a comparative study of genome-wide sequencing data of natural isolates of sars-cov-2 [10] for the detected 228 variants of the s protein, all 22 potential nglycosylation sites within the s protein's ectodomain were completely conserved, which confirms the importance of each of these sites for maintaining the integrity of the s protein oligosaccharide envelope. it should be noted that not all of the 22 potential n-glycosylation sites are occupied, for s1 and s2 subunits, obtained from transiently transfected hek293 cells [11] n-glycosylation events were experimentally confirmed only for 17 out of 22 sites, also at least one o-glycosylation site was experimentally found inside the rbd-domain area of the s1 subunit with the mucin-like structures. non-vertebrate cells may be used to produce the s protein or its fragments; in this case, n-glycans are present mostly in the form of bulky high mannose or paucimannose structures, possibly blocking the interaction of antibodies with the folded s protein [12] . computational modeling of the glycan shield, performed for the hek293-derived s protein, revealed that in the case of human cells, around 40% of the protein's surface is effectively shielded from igg antibodies [13] . the use of full-length s protein for practical serological testing is nearly impossible due to its insolubility, caused by the presence of transmembrane domain. an artificial trimer of its ectodomain has been successfully used as an antigen in serological tests; however, such complex protein cannot be obtained in large quantities in mammalian cells, apparently due to the limitation on the folding of the trimerized abundantly glycosylated protein and subsequent difficulties in its isolation and purification. it is generally believed that the sars-cov-2 s protein receptor-binding domain is a minimal proteinaceous antigen, adequately resembling the immunogenicity of the whole spike protein. this domain contains only two occupied n-glycosylation sites [11] and 1-2 occupied o-glycosylation sites. it does not contribute to the trimer formation, and its surface is mostly unshielded. isolated rbd's of the s proteins of beta-coronaviruses were produced in various expression systems. bacterial expression of the rbd from mers-cov produced no soluble target protein, refolding attempts also were unsuccessful [14] . budding yeasts pichia pastoris were the suitable host for the secretion of mers-cov rbd with at least two (from three) n-linked glycosylation sites present. similar data were obtained for the rbd from sars-cov virus -removal of all n-glycosylation sites resulted in the sharp drop of protein secretion rate in the p. pastoris yeast, in the case of full rbd domain (residues 318-536), secretion of the unglycosylated target protein was stopped completely [15] . it may be proposed that the addition of n-glycans in these sites is needed for correct folding of the rbd in the er of eukaryotic cells. the sars-cov-2 s protein rbd, expressed in e.coli, also was detected only as inclusion bodies and was found to be unreactive even on blotting [16] . hyperglycosylated yeast-derived sars-cov-2 rbd was obtained in reasonable quantities (50 mg/l in bioreactor culture) by the p. pastoris expression system and successfully used for mice immunization [17] . unfortunately, yeast-derived glycosylated proteins contain immunogenic glycans and cannot be used for immune assays with human antibodies. similarly, sars-cov-2 rbd may be produced in the nicotiana benthamiana plant, resulting in non-vertebrate n-glycans addition, potentially reactive with human antibodies [18] . most early preprints and peer-reviewed articles describing the sars-cov-2 s protein and its rbd domain production methods were focused on transient transfection of hek293 cells [11] [19] and purification of small protein lots in a very short time. for example, d. stadlbauer [20] reports more than 20 mg/l target protein titer in transiently transfected hek-293 cells. simultaneously, the scalability of transiently transfected cell lines cultivation is still questionable, and gram quantities of rbd, needed for large scale in vitro diagnostic activity, may be produced only by stably transfected cell lines. previously we have developed the plasmid vector p1.1, containing large fragments of non-coding dna from the eef1a1 gene of the chinese hamster and fragment of the epstein-barr virus long terminal repeat concatemer [21] and employed it for unusually high-level expression of various proteins in cho cells, including blood clotting factors viii [22] , ix [23] , and heterodimeric follicle-stimulating hormone [24] . cho cells were successfully used for transient sars-cov rbd expression at 10 mg/l secretion level [25] . we have proposed that sars-cov-2 rbd, suitable for in vitro diagnostics use, may be expressed in large quantities by stably transfected cho cells, bearing the eef1a1-based plasmid. p1.1-tr2-rbdv1 construction. the rbd coding sequence was synthesized according to [26] . the dna fragment encoding the rbdv1 orf with kozak consensus sequence and c-terminal c-myc and 6xhis tags were obtained by pcr using primers ad-cov-absf and ad-rbd-myc6hnher (listed in table 1 the resulting ptm vector was sequenced as described above, available from addgene, plasmid # 162783. ptm-rbdv2 construction. rbd orf was amplified using adaptor primers ad-sfr2-nhef and ad-sfr2-xmar restricted by nhei and xmai (sibenzyme, novosibirsk, russia) and cloned into ptm vector, restricted by nhei and asigi (sibenzyme, novosibirsk, russia). the resulting construct was sequenced using sq-5ch6-f and sq-mych-r primers. ptm-rbdv2 is available from addgene, plasmid # 162785 plasmids for cell transfections were purified by the plasmid midiprep kit (evrogen, moscow, russia) and concentrated by ethanol precipitation in sterile conditions. the transgene copy number in the cho genome was determined by the quantitative real-time-pcr (qpcr) as described in [22, 24] . serial dilutions of p1.1-egfp [21] or pgem-rab1 plasmids were used for calibration curves generation. the weight of one cho haploid genome was taken as 3 pg, according to [27] . genomic chinese hamster ovary dg-44 cells (thermo fischer scientific) were cultured in the procho 5 medium (lonza, switzerland), supplemented by 4 mm glutamine, 4 mm alanyl-glutamine and hypoxanthinethymidine supplement (ht) (paneco, moscow, russia). cells were grown as a suspension culture in sterile 125 ml erlenmeyer flasks with vented caps, routinely passaged 3 to 4 days with centrifugation (300 g, 5 min) and seeding density 3-4*10 5 cells/ml. the 50-80 µg of each plasmid were precipitated by the addition of 96% ethanol and 3m sodium acetate, washed with 70% ethanol, dried, and resuspended in 100 µl of sterile r-buffer, neon transfection kit (thermo seeding cell culture was grown in 125 ml erlenmeyer shake flasks with 30 ml of lonza procho 5 medium, supplemented with 4 mm glutamine, 4 mm alanyl-glutamine and 2-8 µm mtx until cell concentration exceeds 1-1.5 mln cells/ml. cell suspension was transferred to four 250 ml erlenmeyer flasks, each containing 60 ml of culture medium, and grown to the same cell density. the entire cell suspension was transferred to a single 2 l erlenmeyer flask with 1 l culture medium, final seeding density 3-4*10 5 cells/ml. cells were cultured for three days, on the fourth day of culture, daily glucose measurements were started. glucose concentration in the cell supernatant was measured by the accutrend plus system (roche, switzerland); if glucose level was below 20 mm, it was added up to 50 mm as the sterile 45% solution. the culture in 2 l flask was grown for 6 to 8 days until the cell viability, measured by trypan blue exclusion, dropped below 50%. the clonal cell line was obtained by the limiting dilution method from the cell population, cultured in 8 µm mtx. methotrexate was omitted in the culture medium for two 3 d passage before cloning. cells were additionally split by 1:1 dilution 24 hours before the cloning procedure. cells were diluted in excell-cho (merck, germany) culture medium supplemented with 4 mm glutamine, 4 mm alanyl-glutamine, ht and 10% of untransfected cho dg 44 conditioned medium resulting in seeding density 0.5 cell/well, and the suspension was seeded into 96-well plates (200 μl/well). plates were left undisturbed for 14 days at 37°c, 5% co2 atmosphere. wells with single colonies were screened by microscopy; well grown colonies were detached by pipetting and transferred to the wells of 12-well plate, containing 4 ml of the excell-cho, supplemented as described above and grown for 7 days undisturbed. product titer was measured by elisa, as described below, 6 wells with highest rbdv2 titer were used for further cultivation. best-producing clonal cell lines were transferred to 125 ml erlenmeyer flasks with the procho 5 culture medium supplemented with 4 mm glutamine, 4 mm alanyl-glutamine and 8 µm mtx and after 5 days in suspension culture, the best producing clone was determined by measuring the product titer and cell concentration. sds-page was performed with the 12.5% acrylamide in the separating gel, in reducing conditions, if not stated otherwise, with the pageruler prestained marker, 5 µl/lane (thermofisher scientific). gels were stained by the colloidal coomassie blue according to [28] , scanned by the conventional flatbed scanner in the transparent mode as 16-bit grayscale images and analyzed by the totallab tl120 gel densitometry software (nonlinear dynamics, uk). sds-page was performed as described above, protein transfer, blocking, hybridization and color development were done according to [29] using nitrocellulose transfer membrane (gvs group, bologna, italy) and towbin buffer with methanol. primary anti-c-myc antibody (sci store, moscow, russia #psm103-100) was used at the 1:2000 dilution, anti-mouse-hrp conjugate (abcam, cambridge, uk, ab6789) was used at 1:2000 dilution; membrane was developed by the dab-metal substrate and scanned by the flatbed scanner in the reflection mode. multimeric forms of the rbd were quantified by size exclusion chromatography, utilizing waters extracts were vacuum-dried and redissolved in the 0.5% trifluoroacetic acid (tfa), 3% acn solution. prepared solutions were mixed at 3:1 ratio with 20% α-cyano-4-hydroxycinnamic acid (merck) solution in 20% acn, 0.5% tfa on the target plate. solutions of intact and deglycosylated proteins were passed through the ziptip c18 microcolumns (millipore), washed and eluted according to manufacturer protocol. one and a half µl of protein solutions were mixed on the target plate with 0.5 µl of the 20% 2,5-dihydroxybenzoic acid (merck) solution in 20% acn, 0.5% tfa. mass spectra were obtained by the maldi-tof mass spectrometer ultraflextreme peptides identification was performed by the gpmaw 4.0 software (lighthouse data, denmark) and by the mascot server (matrix science, boston, usa). glycopeptides mass assignment was performed by the glycomod online software tool [30] . sandwich elisa with anti-s protein antibodies was performed using a prototype of the sars-cov-2 antigen detection kit (xema co., ltd., moscow, russia, a generous gift of dr. yuri lebedin). pre-covid-19 normal human plasma sample (renam, moscow, russia) was used for preparation of the sars-cov-2 negative serum sample. serum samples of five patients with the pcr-confirmed sars-cov-2 infection were pooled for testing and one serum sample with the borderline igg titer level was tested separately. the blood sampling protocol conformed to the local hospital human ethics committee guidelines. antibody capture elisa with human serum samples was performed according to [29] at the 100 ng per well antigens load. antigens were applied on elisa 96-well plates (corning, usa) overnight at + 4oc, in pbs, the t-test was performed using the graphpad quickcalcs web site: https://www.graphpad.com/quickcalcs/ttest1.cfm (accessed november 2020). the native n-terminal signal peptide of sars-cov-2 s protein (amino acid sequence mfvflvllplvssq) was fused to the rbd sequence (319 -541, according to yp_009724390.1) and joined with a c-terminal c-myc epitope (eqkliseedl), short linker sequence, and hexahistidine tag. n-terminal part of the rbdv1 gene was constructed according to [26] , utilizing the optimized codon usage gene structure. c-terminal tags were not optimized for codon usage frequencies. the resulting synthetic gene was cloned into the p1.1-tr2 vector plasmid, a shortened derivative of the p1.1 plasmid [21] , and used for transfection of dhfr-deficient cho dg44 cells. the resulting expression plasmid p1.1-tr2-rbdv1 [genbank: mw187858] is shown on fig 1a. the stably transfected cell population was obtained by selection in the presence of 200 nm of dhfr inhibitor methotrexate, rbd titer 0.33 mg/l was detected for 3-days culture (fig. s1 ). one-step target gene amplification was performed by increasing the mtx concentration tenfold and maintaining the cell culture for 17 days until cell viability restored to more than 85%; the resulting polyclonal cell population could secrete up to 3,0 mg/l rbd in the 3-days culture. the target protein was purified by a single imac chromatography step, utilizing the ida-based resin chelating sepharose fast flow (cytiva), ni2+ ions, and step elution by increasing imidazole concentrations (fig 1b, fig 1c) . the resulting protein production method was found to be sub-optimal due to unexpectedly low secretion rate, signs of cellular toxicity of the target gene -33 h cell duplication time, maximal cell density in shake flask of 2.3 mln сells/ml (fig s2) , and unacceptable level of contaminant proteins co-eluting with the rbdv1. at the same time, the rbdv1 protein was stable in the culture medium during the extended batch cultivation of cells for at least 7 days (fig 1d) , making the long-term feed batch cultivations a viable option for its production in large quantities. we proposed that target protein secretion rate and its purity after one-step purification could be significantly improved by a simultaneous shift of the rbd domain boundaries, exchange of the sars-cov-2 s protein native signal peptide to the signal peptide of more abundantly expressed protein, two-step genome amplification and switch from ida-based resin to the nta-based one (fig 1e) . human tissue plasminogen activator signal peptide (htpa sp, amino acid sequence mdamkrglccvlllcgavfvsas) is commonly used for heterologous protein expression in mammalian cells. it was successfully used for the expression of sars-cov s protein in the form of dna vaccine [31] and envelope viral protein gp120 [32] . in the case of mers-cov s protein rbd -fc fusion protein, various heterologous signal peptides modulate target protein secretion rate by the factor of two [14] . corrected boundaries of the sars-cov-2 rbd were determined according to the cryo-em data [pdb id: 6vxx] [33] obtained for the trimeric sars-cov-2 s protein ectodomain. initially used 319 -541 coordinates, described in the [26] include one unpaired cys residue originated from the n-terminal part of the next domain sd1 (structural domain 1), so we excluded lys319 from the n-terminus of the mature rbd protein, aiming at the maximization of signal peptide processing, and removed c-terminal aminoacids c 538 vnf 541 , which form the structure of the sd1 domain. both linker areas surrounding the folded rbd domain core remain present in the rbdv2 protein (320 -537, according to yp_009724390.1). additionally, we redesigned c-terminal tags by introducing the pro residue immediately upstream of the c-myc tag, adding the short linker sequence sagg between the c-myc tag and polyhistidine tag, and extending the polyhistidine tag up to 10 residues. we expected this structure to expose the c-myc tag properly on the protein globule's surface and move the decahistidine tag away from possible masking negatively charged protein surface areas. we constructed an expression vector ptm [genbank: mw187855], where consensus kozak sequence, htpa sp and c-myc and 10-histidine tags are coded in the polylinker. rbd coding fragment was cloned in-frame, resulting ptm-rbdv2 expression plasmid [genbank: mw187856] is shown on fig.2a . cho dg44 cells were transfected by the ptm-rbdv2 plasmid, stably transfected cell population was established at the 200 nm mtx selection pressure. target protein titer was similar to the previous plasmid design -0.9 mg/l for 3-days culture, but after one step of the mtx-driven genome amplification, it increased eleven-fold to 9.7 mg/l at 2 µm mtx ( fig 2b) and then increased by a factor of 2.5 after second amplification step at 8 µm mtx, resulting titer was 24.6 mg/l for 3-days culture (fig 3a) . a steady increase of the target protein titer was detected for the extended batch cultivation of polyclonal cell population obtained at 8 µm mtx, peaking at 50 mg/l at 8 days of cultivation in the 2 l shake flask (fig 3d, 3e) . a similar ratio of product titer increase after multi-step mtx-driven genome amplification was described for the mers-cov rbd -40-fold increase after 9 steps of consecutive increments of mtx concentration, overall amplification period length was 60 days [14] . vector plasmid ptm, used in this study, allowed a much more rapid amplification course -a 27-fold titer increase in two steps, 33 days total. this all cell populations, secreting rbd proteins, were analyzed by the quantitative pcr and it was found, that increased productivity of populations, adapted to higher concentrations of mtx corresponds to higher copy numbers of target gene (fig 3c) . higher cell productivity in the case of rbdv2 protein was not due to higher target gene copy numbers, then in the case of rbdv1. cell culture medium pro cho5 (lonza), utilized in this study, contains unknown components, blocking histagged rbd protein's interaction with the ni-nta chromatography resin. clarified conditioned medium, used for protein purification, was concentrated approximately tenfold by tangential flow ultrafiltration on the 5 kda mwco cassettes and completely desalted by diafiltration, 20 diafiltration volumes of the 10 mm imidazole-hcl, ph 8.0 solution. rbdv1 and rbdv2 proteins were purified by imac utilizing ni-nta agarose (thermo fischer scientific, usa) in the same conditions. desalted conditioned medium was applied onto the column in the presence of 10 mm imidazole; the column was washed by the solution containing elution was performed by the 300 mm imidazole solution; further column strip by the 50 mm edta-na solution revealed no detectable target protein rbdv2 in the eluate (fig 2c) . purified proteins were desalted by another round of ultrafiltration/diafiltration on the centrifugal concentrators with 5 kda mwco membranes; diafiltration solution was pbs; final concentration 3-7 mg/ml. purified proteins were flashfrozen in liquid nitrogen and stored frozen in aliquots. overall protein yield for rbdv2 was 64%, 32 mg of purified rbdv2 were obtained from 1 l shake flask culture. the apparent molecular weight of intact rbdv1 was determined as 35.3 kda, deglycosylated rbdv1 -26.1 kda, theoretical molecular weight -27647 da. rbdv2 molecular weight was determined as 35.7 kda for the intact protein, deglycosylated protein -28.5 kda, theoretical molecular mass -27459 da (fig 4a) . both protein variants possess two distinct forms of intramolecular disulfide bonds sets, visible as two closely adjacent bands in non-reducing conditions and complete absence of such band pattern in reducing conditions. previously it was reported that sars-cov-2 rbd 319-541, expressed transiently in hek-293 cells, tends to form a covalent dimer, around 30% from the total, visible as the 60 kda band on the denaturing gel in nonreducing conditions [19] . we confirmed this observation; in the case of stably transfected cho cells, covalent dimerization was also 31% according to gel densitometry data. at the same time, it should be noted that the rbdv2 protein, redesigned explicitly for mitigation of this unwanted dimerization and containing an even number of cys residues, still forms 6 % of the covalent dimer. purified rbdv2 was tested by size exclusion chromatography. the major monomer form's apparent molecular weight was determined as 32.4 kda (fig s3) , admixtures peaks apparent molecular masses corresponded well to rbd dimer, tetramer, and two high molecular mass oligomers accounting for 6% of all peak areas (fig 4b) . mass-spectrometry analysis of rbdv1 and rbdv2 revealed that both proteins' molecular masses diminished ( fig s5, s6 ). this long peptide was completely absent in both spectra of de-glycosylated proteins (table s1 -s4) . a more detailed analysis of this area of the rbd protein may be of some interest for the s protein structure-function investigation but is out of scope for the present study. purified rbd variants were used as antigens for microplates coating and subsequent direct elisa with pooled sera obtained from patients with the rt-pcr-confirmed covid-19 diagnosis, 1 weakly positive serum sample from the rt-pcr-confirmed covid-19 patient, and serum sample obtained from a healthy volunteer before december 2019 (fig 4e) . both rbd variants perform equally -all serum samples produce highly similar od readings for all dilutions tested with both antigens. here we describe a method of generating stably transfected cho cell lines, secreting large quantities of monomeric sars-cov-2 rbd, suitable for serological assays. at present, serological assays for detection of seroconversion upon sars-cov-2 infection are mostly based on two viral antigens -nucleoprotein (np) and s protein or fragments of the s protein, including the rbd. there are various reports on the specificity and sensitivity of assays based on these two antigens. in some cases, the sensitivity of clinically approved npbased assays was challenged by direct re-testing of np-negative serum samples by the rbd-based assays [34] . other studies question the specificity of np-based elisa tests, demonstrating a significant level of false-positive results for the full-length sars-cov-2 np [35] . it may be proposed that testing of serum samples with both sars-cov-2 antigens will produce the most accurate results, as was done, for example, in the south-east england population study [36] ; this conclusion was made in the microarray study of a limited number of patients serum samples [37] . it is unclear yet, which part of the s protein is the optimal antigen for serological assays; microarray analysis revealed that s2 fragment generates more false-positive results than s1 or rbd antigen variants [37] in the case of igg detection, at the same time the rbd protein generated much lower signals on covid-19 patients serum samples then s1 or s1+s2 antigens. in another microarray study it was found that igg response toward the rbd domain in the convalescent plasma samples correlates well with the response toward full-length soluble s protein [38] . in the conventional elisa test format, rbd demonstrated nearly 100% specificity and sensitivity on a limited number of sars-cov-2 patients and control serum samples [4] . as of 26.10.20, at least 104 various immunoassays for sars-cov-2 antibodies were authorized for in vitro diagnostic use in the eu [39], many of them use rbd as the antigen. a simple elisa screening test with the 96-well microplate will consume around 10 µg of the rbd antigen for 40 test samples, so even one million tests will require 250 mg of the purified rbd protein, making the antigen supply a critical step in the production of such tests. method of the generation of highly productive stably transfected cho cell line, secreting the rbd protein, may be important for ivd test manufacturers in securing the sources of rbd antigen with highly predictable properties. although the rbd fragment of the s protein from sars-cov-2 is not the most popular antigen variant in the current efforts of anti-sars-cov-2 vaccine development [40] , it may be considered as the viable candidate for a simple subunit vaccine. it demonstrated the significant protective immune response development in rodents, without signs of ade effect [41] and some rbd-based protein subunit vaccine have advanced to phase ii clinical trials. cultured cho cells are the reliable source of rbd protein for this kind of vaccines; at the productivity level achieved in our study, only 30 m 3 of cell culture supernatant will provide enough antigen material for 100 mln of typical 10 µg/vial vaccine doses. с, d -protein sequence coverage by tryptic peptides, maldi-tof analysis. glycosylated peptides found are not pictured, signal peptides are yellow, detected tryptic peptides -violet, experimentally obtained masses, [m+h]+, are stated in the boxes. e -immunoreactivity of rbdv1 and rbdv2 by elisa with pooled serum samples from pcr-positive patients -(+)pooled, single serum sample from pcr-positive patient (+) and pre-covid-19 pooled sera (-). all sera samples were analyzed in duplicates, data are mean. supporting figure s1 . cell growth and viability dynamics of initial selection and mtx-driven target gene amplification. supporting figure s2 . cell growth curve for the extended batch cultivation of rbdv1 and rbdv2producing cell populations, 2 um mtx selection pressure. supporting figure s3 . size exclusion chromatography trace of molecular mass calibrators and molecular mass calibration curve. supporting figure s4 . maldi-tof spectra traces of intact proteins in glycosylated and deglycosylated forms. supporting figure s5 . maldi-tof spectra traces of tryptic peptides mxtures from intact and deglycosylated rbdv1. supporting figure s6 . maldi-tof spectra traces of tryptic peptides mxtures from intact and deglycosylated rbdv2. supporting table s1 . peptides mass list of the rbdv1 intact protein, 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vectors a 219-mer cho-expressing receptor-binding domain of sars-cov s protein induces potent immune responses and protective immunity a serological assay to detect sars-cov-2 seroconversion in humans eukaryotic genome size databases highly sensitive and fast protein detection with coomassie brilliant blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis antibodies : a laboratory manual glycomod--a software tool for determining glycosylation compositions from mass spectrometric data identification of two neutralizing regions on the severe acute respiratory syndrome coronavirus spike glycoprotein produced from the mammalian expression system extracellular matrix proteins mediate hiv-1 gp120 interactions with alpha4beta7 structure, function, and antigenicity of the sars-cov-2 spike glycoprotein testing for responses to the wrong sars-cov-2 antigen? whole nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 may cause false-positive results in serological assays estimates of the rate of infection and asymptomatic covid-19 disease in a population sample from se england analysis of sars-cov-2 antibodies in covid-19 convalescent blood using a ): p. 3581. 39. database. foundation for innovative new diagnostics. sars-cov-2 diagnostic pipeline a systematic review of sars-cov-2 vaccine candidates the sars-cov-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement we thank mr. arthur isaev (genetico, moscow, russia) and dr. alexander ivanov (institute of molecular biology russian academy of sciences, moscow, russia) for valuable comments and early access to the sars-cov-2 s protein sequence data, dr. yuri lebedin, eugenia kostrikina and xema co., ltd., for providing anti-rbd mabs and conjugates.the measurements were carried out on the equipment of the shared-access equipment centre "industrial biotechnology" the research center of biotechnology of the russian academy of sciences. dna sequencing was carried out in the inter-institutional center for collective use "genome" imb ras, organized with the support of the russian foundation of basic research.the authors would like to acknowledge all the doctors who diagnose and treat patients during the covid-19 pandemic. primers for rbdv1 cloning, restriction sites are underlined ad-cov-absf aacctcgaggccgccaccatgttcatgccttctt ad-rbd-myc6hnher gctagcctaatggtgatggtgatgatgaccggtatgcatat tcagatcctcttctgagatgagtttttgttcgaagttcacgc atttgtt primers for ptm construction, sticky ends of annealed pairs are underlinedctagtgatggtgatggtgatggtgatggtgatgaccgcctg cagacagatcctcttcgctgatcagtttttgttcaccggta primers for rbdv2 cloning, restriction sites are underlined ad-sfr2-nhef gctagcgtgcagcccaccgaatcc ad-sfr2-xmar cccgggtttgttcttcacgagattggt sequencing primers sq-5ch6-f gccgctgcttcctgtgac iresa rev aggtttccgggccctcacattg sq-mych-r gatgaccgcctgcagac key: cord-004584-bcw90f5b authors: nan title: abstracts: 8th ebsa european biophysics congress, august 23rd–27th 2011, budapest, hungary date: 2011-08-06 journal: eur biophys j doi: 10.1007/s00249-011-0734-z sha: doc_id: 4584 cord_uid: bcw90f5b nan o-003 structure determination of dynamic macromolecular complexes by single particle cryo-em holger stark max-planck-institute for biophysical chemistry, goettingen, germany macromolecular complexes are at the heart of central regulatory processes of the cell including translation, transcription, splicing, rna processing, silencing, cell cycle regulation and repair of genes. detailed understanding of such processes at a molecular level requires structural insights into large macromolecular assemblies consisting of many components such as proteins, rna and dna. single-particle electron cryomicroscopy is a powerful method for threedimensional structure determination of macromolecular assemblies involved in these essential cellular processes. it is very often the only available technique to determine the 3d structure because of the challenges in purification of complexes in the amounts and quality required for x-ray crystallographic studies. in recent years it was shown in a number of publications that it is possible to obtain near-atomic resolution structures of large and rigid macromolecules such as icosahedral viruses. due to a number of methodological advances there are now also great perspectives for high-resolution single particle cryo-em studies of large and dynamic macromolecules. successful high-resolution structure determination of dynamic complexes requires new biochemical purification strategies and protocols as well as state of the art electron microscopes and high-performance computing. in the future cryo-em will thus be able to provide structures at near-atomic resolution and information about the dynamic behavior of macromolecules simultaneously. detection and rapid manipulation of phosphoinositides with engineered molecular tools tamas balla section on molecular signal transduction, program for developmental neuroscience, nichd, nih, bethesda, md 20892, usa polyphosphoinositides (ppis) are ubiquitous lipid regulators of a variety of cellular processes serving as docking sites and conformational switches for a large number of signaling proteins. the localization and dynamic changes in ppis in live cells have been followed with the use of protein domain gfp chimeras. in this presentation we will show experimental systems that allow rapid manipulation of the levels of ppis in specific membrane compartments. we are also actively pursuing strategies that will allow us to map the distribution and possible functional diversity of the phosphatidylinositol (ptdins) pools within intact cells since they are the precursors of ppis. we will show our most recent progress in addressing this question: the use of a ptdins specific plc enzyme isolated from listeria monocytogenes together with a highly sensitive diacylglycerol sensor to determine the distribution and also to alter the level of ptdins in living cells. these studies reveal that a significant metabolically highly active ptdins pool exists associated with tiny mobile structures within the cytoplasm in addition to the known er and pm ptdins pools. we will show our most recent data on the consequences of ptdins depletion within the various ptdins pools on ppi production and on the morphology and functions of various organelles. the functionality of proteins is known to be intimately related to the motion of their constituents on the atomic/molecular level. the study of microscopic motion in complex matter is often reduced to the observation of some average mean square atomic displacement, a first, very partial characterization of the dynamics. the marked crossover in the temperature dependence of such quantities in hydrated proteins around 200 k, the so called ''dynamic transition'' has been originally observed a quarter of century ago. the origin, nature and the key characteristics of the atomic motions behind this remarkable evolution of the mean square displacement in proteins remained controversial over the past decades. recent analysis of mö ssbauer, dielectric relaxation and neutron scattering spectroscopic data provide unambiguous evidence that this phenomenon is caused by the temperature dependence of a relaxation process spread over several orders of magnitude in the time domain, similarly to the b relaxation process observed in glasses. the review and critical analysis of the available data highlights the inherent ambiguities of commonly used data fitting approaches. emerging evidence from model independent observations tend to exclude some of the proposed mechanisms. microbial rhodopsins: light-gated ion channels and pumps as optogenetic tools in neuro-and cell biology e. bamberg, c. bamann, r.e. dempski, k. feldbauer, s. kleinlogel, u. terpitz, p. wood department of biophysical chemistry, max-planck-institute of biophysics, frankfurt, germany microbial rhodopsins are widely used in these days as optogenetic tools in neuro and cell biology. we were able to show that rhodopsins from the unicellar alga chlamydomonas reinhardtii with the 7 transmembrane helix motif act as light-gated ion channels, which we named channelrhodopsins(chr1,2). together with the light driven clpump halorhodopsin chr2 is used for the non-invasive manipulation of excitable cells and living animals by light with high temporal resolution and more important with extremely high spatial resolution the functional and structural description of this new class of ion channels is given (electrophysiology, noise analysis,flash photolysis and 2d crystallography). new tools with increased spatial resolution and extremely enhanced light sensitivity in neurons are presented. a perspective for basic neurobiology and for medical applications is given. extracellular signals consists of the induction of specific gene expression patterns and the re-organization in space and time of stereo-specific macromolecular interactions that endow the cell with its specific morphology. we develop quantitative experimental and computational approaches to derive and conceptualize physical principles that underlie these dynamics of signal processing and cellular organization. we have an experimental emphasis on functional microscopic imaging approaches at multiple resolutions to study the localization and dynamics of protein reactions/ interactions, maintaining the inherent spatial organization of the cell. we have a strong recursion between computation of molecular dynamics in realistic cell geometries as sampled by microscopy, and experiments that reveal the dynamic properties of networks in living cells. we investigate the cellular topography of activities that transmit signals from receptors at the cell surface. here we ask, how spatial partitioning of intracellular signalling activities is achieved by the causality structure of the signalling network, and how this partitioning affects signal response. this entails the experimental elucidation of connections between reactions and the determination of enzyme kinetic parameters in living cells. o-008 molecular photovoltaics mimic photosynthesis michael grä tzel laboratory of photonics and interfaces, institute of chemical science and engineering, station 6, ecole polytechnique fé dé rale, ch-1015 lausanne, switzerland e-mail: michael.graetzel@epfl.ch the field of photovoltaic cells has been dominated so far by solid state p-n junction devices made e.g. of crystalline or amorphous silicon, profiting from the experience and material availability of the semiconductor industry. however, there is an increasing awareness of the possible advantages of devices referred to as ''bulk'' junctions due to their interconnected three-dimensional structure. their embodiment departs completely from the conventional flat p-n junction solid-state cells, replacing them by interpenetrating networks. this lecture focuses on dye sensitized mesoscopic solar cells (dscs), which have been developed in our laboratory. imitating natural photosynthesis, this cell is the only photovoltaic device that uses a molecular chromophore to generate electric charges from sunlight and that accomplishes the separation of the optical absorption from the charge separation and carrier transport processes. it does so by associating the molecular dye with a film constituted of tiny particles of the white pigment titanium dioxide. the dsc has made phenomenal progress, present conversion efficiencies being over 12 percent for single junction and 16 percent for tandem cells, rendering the dsc a credible alternative to conventional p-n junction devices. molecularly single-molecule imaging and tracking techniques that are applicable to living cells are revolutionizing our understanding of the plasma membrane dynamics, structure, and signal transduction functions. the plasma membrane is considered the quasi-2d non-ideal fluid that is associated with the actinbased membrane-skeleton meshwork, and its functions are likely made possible by the mechanisms based on such a unique dynamic structure, which i call membrane mechanisms. my group is largely responsible for advancing highspeed single molecule tracking, and based on the observations made by this approach, i propose a hierarchical architecture of three-tiered meso-scale (2-300 nm) domains as fundamental organizing principles of the plasma membrane. the three tiers i propose are the following. [tier 1] 30-200 nm compartments made by partitioning the entire plasma membrane by the membrane-associated actinbased meshwork (membrane skeleton: fences) and its associated transmembrane proteins (pickets). since the entire plasma membrane is partitioned by these structures, and the membrane skeleton provides important platforms for the molecular interactions and pools, membrane compartments are the most basic tier for the plasma membrane organization. [tier 2] meta-stable 1-5 nm raft domains that can be turned into stable *10-20-nm domains (receptor-cluster rafts), based on ligand-induced homo-dimers of glycosylphosphatidylinositol (gpi)-anchored receptors (coupling with [tier 3]) and facilitated by raft-lipid interactions. [tier 3] protein complexes of various sizes (3-10 nm) and lifetimes. i will also talk about how domains of tiers 2 and 3 are coupled to the membrane partitioning (tier 1). the concept of the three-tiered domain architecture of the plasma membrane and the cooperative interactions of different tiers provides a good perspective for understanding the mechanisms for signal transduction and many other functions of the plasma membrane. introduction: in the present study we investigate the effects of electromagnetic fields (emf) on the binding of norfloxacin (nrf) to human serum albumin (hsa) by fluorescence, three-dimensional fluorescence and uv-visible spectroscopic approaches. hsa is the most abundant protein in human blood plasma which works as a carrier that transports different materials in the body. nrf is used to treat variety of bacterial infections. it works by stopping the bacterial growth. methods: hsa, nrf and potassium phosphate buffer were purchased from sigma. fluorescence spectrofluorometer, uv-vis spectrophotometer, three-dimensional fluorescence and a home-built emf generator apparatuses were used. results: results obtained from this study indicated that nrf has a strong ability to quench hsa in 280 nm. in addition, there was a slight blue shift, which suggested that the microenvironment of protein became more hydrophobic after addition of nrf. moreover, synchronous fluorescence demonstrated that the microenvironment around tyrosine (tyr) had a trivial increase. these, and the results of hsa-nrf in the presence of emf with 1 khz, illustrates the same results inferred from quenching and blue shift. however, there was a significant decrease in k sv of nrf with hsa in presence of emf exposure. moreover, the binding parameters including the number of binding sites and the binding constant were calculated form hill equation. conclusion: it was shown that nrf could induce conformational changes in hsa both in the absence and presence of emf with no significant difference. yet, the affinity is decrease significantly in the presence of emf. the clinical implications are discussed in detail. characterization of the biochemical properties and biological function of the formin homology domains of drosophila we characterised the properties of drosophila melanogaster daam-fh2 and daam-fh1-fh2 fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. the results show that while the daam-fh2 fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the daam-fh1-fh2 fragment a profilindependent increase in the formation of actin structures is observed. the trachea specific expression of daam-fh1-fh2 also induces phenotypic effects leading to the collapse of the tracheal tube and lethality in the larval stages. in vitro, both daam fragments catalyze actin nucleation but severely decrease both the elongation and depolymerisation rate of the filaments. profilin acts as a molecular switch in daam function. daam-fh1-fh2, remaining bound to barbed ends drives processive assembly of profilin-actin, while daam-fh2 forms an abortive complex with barbed ends that does not support profilinactin assembly. both daam fragments also bind to the sides of the actin filaments and induce actin bundling. these observations show that the drosophila melanogaster daam formin represents an extreme class of barbed end regulators gated by profilin. electron spin echo studies of free chain-labelled stearic acids interacting with b-lactoglobulin rita guzzi, luigi sportelli, rosa bartucci dipartimento di fisica, università della calabria, 87036 rende (cs), italy b-lactoglobulin (blg) binds non-covalently fatty acids within its central calyx, a cavity in the barrel formed by the strands ba-bh. we present results of pulsed electron paramagnetic resonance (epr) spectroscopy on the interaction of blg with stearic acids spin-labelled at selected positions, n, along the acyl chain (n-sasl, n = 5,7,10,12,16). d 2 o-electron spin echo envelope modulation (eseem) fourier transform spectra indicate that all segments of the bound chains in the protein binding site are accessible to the solvent. the extent of water penetration decreases progressively on moving from the first segments toward the terminal methyl end of the chain. about 50% of the nitroxides in the upper part of the chain (n = 5,7) are h-bonded by a single water molecule and this fraction reduces to 30% at the chain terminus (n = 12,16). a lower fraction of the nitroxides are h-bonded by two water molecules, and it decreases from about 15% to a vanishingly small value on going down the chain. echo-detected ed-epr spectra reveal subnanosecond librational motion of small amplitude for both 5-and 16-sasl in the protein cavity. the temperature dependence of the librations is more marked for 16-sasl and it arises mainly from an increase in librational amplitude with increasing temperature. fusion peptides (fp) pertaining to the spike glycoprotein from severe acute respiratory syndrome (sars) coronavirus are essential for the fusion between viral and host cellular membranes. here we report a biophysical characterization of the interaction of two putative fps with model membranes. fluorescence and dsc experiments showed that both peptides bind stronger to anionic than to zwitterionic lipid membranes. esr spectra showed that toac-sars ifp rotational dynamics is modulated by lipid composition and ph as compared to the spectrum of this peptide in solution. however, stearic acid spin labels reported no changes on the dynamic structure of zwitterionic micelles, whereas the whole chain of anionic surfactants was perturbed by the peptides. finally, cd data revealed a predominant b-strand structure for sars fp and an a-helix for sars ifp in the presence of micelles, in contrast to their disordered structures in buffer. overall the results point out that electrostatic and hydrophobic interactions are both important to the energetic behavior of peptide membrane interaction. these findings might provide a useful rationale for the elucidation of one of the steps involved in the fusion process, and thus help understanding the more general way of action of fps at a molecular level. interaction of filamentous actin and ezrin within surface modified cylindrical nanopores daniela behn 1,2 and claudia steinem 1 1 institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße 2, 37077 gö ttingen, germany, 2 ezrin is a member of the ezrin-radixin-moesin (erm) protein family that acts as a dynamic linker between the plasma membrane and the actin cytoskeleton and is hence involved in membrane organization, determination of shape and surface structures and other cellular processes. the protein is highly enriched in microvilli of polarized epithelial cells, where it binds filamentous actin (f-actin) with its c-terminal domain, while the n-terminal domain is connected to the plasma membrane via specific binding to l-a-phosphatidylinositol-4,5-bisphosphate (pip 2 ). nanoporous anodic aluminum oxide (aao) films provide similar dimensions as microvilli and are thus a versatile template to investigate the interaction of ezrin with f-actin within spationally confined areas. owing to their optical transparency, functionalized aaos can be used to measure the binding process of ezrin to a pip 2 containing solid supported membrane by means of time resolved optical waveguide spectroscopy (ows). confocal laser scanning microscopy (clsm) will elucidate, whether f-actin binding to ezrin takes place within or atop the nanopores. furthermore, elasticity mapping of f-actin filaments by means of atomic force microscopy will allow determining binding forces and the lateral tension of the actin cytoskeleton. in vitro application of porphyrin photosensitisers on mcf7, hela and g361 tumour cell lines binder s., kolarova h., bajgar r., tomankova k., daskova a. deparment of medical biophysics, faculty of medicine of palacky university, olomouc, czech republic tumour treatment presents a challenge to all scientists and clinicians. contemporary methods like radiotherapy, chemotherapy or surgery have many undesirable side effects. photodynamic therapy (pdt) seems to be one of alternatives which can be helpful in malignant cell therapy. pdt is not only limited to cancer treatment but is also used as an alternative for cardiovascular, skin and eye disease treatment. pdt employs photosensitive agents which need to be activated by light which is not harmful to a patient. the activated photosensitive agent provokes a formation of reactive oxygen species leading to cell damage or death. the phototoxicity of the two porphyrin photosensitizer (tmpyp, zntpps 4 .12h 2 o) on the malignant cell lines (g361, hela, mcf7) irradiated with the 1 jcm -2 doses was evaluated by ros production assay, mtt assay and comet assay. our results indicate higher efficiency of tmpyp over zntpps 4 .12h 2 o. as for the photodynamic effectiveness of the used photosensitizers on chosen cell lines we found that hela cell line is the most sensitive to phototoxic damage induced by tmpyp. p-018 nmr analysis of the respiratory syncytial virus m2-1 protein structure and of its interaction with some of its targets c. sizun 1 the respiratory syncytial virus (rsv) is a major cause of acute respiratory tract infections (bronchiolitis, pneumonia) in human and a leading cause of viral death in infants and immunocompromised patients. rsv genome is formed of a single non-segmented negative strand rna which transcription and replication is ensured by a specific rna-dependent rna polymerase complex formed of the large (l) polymerase subunit and of several cofactors. this complex has no cellular counterpart and represents an ideal target for antiviral drugs. among the cofactors, m2-1 acts as an antitermination factor and increases the polymerase processivity. its central domains has been shown, in vitro, to bind the phosphoprotein p and genomic rna in a competitive manner. here we report the nmr structure of this central domain and its interaction with p and rna fragments. m2-1 shares structural similarity with vp30, a transcription factor of ebola virus. the binding surfaces for rna and p are distinct but overlapping. rna binds to a basic cluster located next to residues found to be critical for transcription both in vitro and in vivo by mutational analysis. we speculate that m2-1 might be recruited by p to the transcription complex, where interaction with rna takes place, stabilized by additional elements. force spectroscopy at the membrane-cytoskeleton interface: interactions between ezrin and filamentous actin julia a. braunger 1,2 , ingo mey 1 and claudia steinem 1 1 institute for organic and biomolecular chemistry, georg-august-university of gö ttingen, tammannstraße 2, 37077 gö ttingen, germany, 2 ggnb doctoral program: imprs ezrin, a member of the erm (ezrin/radixin/moesin) protein family, provides a regulated linkage between the plasma membrane and the actin cytoskeleton. it contributes to the organization of structurally and functionally distinct cortical domains participating in adhesion, motility and fundamental developmental processes. ezrin is negatively regulated by an intramolecular interaction of the terminal domains that masks the f-actin binding site. a known pathway for activation involves the interaction of ezrin with phosphatidylinositol 4,5bisphosphate (pip 2 ) in the membrane, followed by phosphorylation of the threonine 567 residue in the c-terminal domain. to date, it is unclear to what extent both regulatory inputs contribute to the activation. we developed an in vitro system that facilitates the specific analysis of the interaction forces between ezrin and f-actin by means of atomic force spectroscopy (afm). applying ezrin wild type and the pseudophosphorylated mutant protein ezrin t567d, respectively, permits to monitor the individual influence of phosphorylation on the f-actin-ezrin interaction. thus, a thorough characterization of the acting forces at the ezrin-actin interface will elucidate the activation mechanism of ezrin. delivery system even more efficient, we have constructed nano-carrier by coating of ldl by polyethylene glycol (peg) . the hydrophilicity of peg should reduce the interaction of ldl with other serum proteins and consequently decrease the redistribution of loaded drug from ldl to the (lipo)proteins. dynamic light scattering was used for determination of hydrodynamic radius of ldl-peg particles. cd spectroscopy measurements didn't reveal structural changes of apoliprotein b-100 (ligand for ldl receptors on cell surface), after conjugation of peg with ldl. interaction of ldl-peg complexes with hypericin (hyp) a natural photosensitizer was studied by fluorescence spectroscopy. we have demonstrated accumulation of higher number of hyp in ldl-peg than ldl particles. however, the kinetics of hyp redistribution from hyp/ldl-peg complex to free ldl have similar parameters as those for the kinetics of hyp transfer between non-modified ldl molecules. we suggest that hyp molecules are mostly localized in the vicinity of the surface of the ldl-peg particles and they are prone to redistribution to other serum proteins. grant support: lpp-0072-07, vega-0164-09. modification of the head-group of aminophospholipids by glycation and subsequent lipid oxidation affect membrane's structure causing cell death. 1 these processes are involved in the pathogenesis of aging 2 and diabetes. 3 non-enzymatic glycation forms in the first step a schiff base (sb), which rearranges to a more stable ketoamine, amadori product, which leads to the formation of a heteregenous group of compounds (ages). although several studies have been focused on identification of aminophospholipid glycation products, 4 less attention has been paid to kinetic mechanism of the reaction. for that reason, in the present work, we compare the kinetic reactivity of polar head-group of phosphatidylethanolamine (pe) and phosphatidylserine (ps), the two target phospholipids components of mammalian cell membranes. the reaction of pe and ps's head-group with glycating compounds (glucose and arabinose) was studied in physiological conditions by using nmr spectroscopy. the obtained formation rate constants for sb are lower than those determined for the sb of the peptide ac-phe-lys with the same carbohydrates. 5 it suggests that the phosphate group may delay the glycation process. moreover, the ps's head-group has a carboxilic group in the structure, which affects the stability of the sb. we developed ultrasensitive, elisa-like nanoimmuno assays suitable for proteomics/interactomics studies in low sample volumes. we exploit the approach of dna microarray technologies applied to proteomics [1] , in combination with atomic force microscopy (afm) to generate functional protein nanoarrays: semisynthetic dna-protein conjugates are immobilized by bioaffinity within a nanoarray of complementary ssdna oligomers produced by afm nanografting (ng). a nanoarray of different antibodies or synthetic molecular binders can be generated in a single operation, once the dna nanoarray is produced. moreover, ng allows adjusting the packing density of immobilized biomolecules to achieve optimum bio-recognition. afm-based immunoassays with these nanoarrays were shown to achieve detection limit of hundreds of femto molar, in few nanoliters volumes, with very high selectivity and specificity [2] . to detect the hybridization efficiency of our devices, we run a combined experimental-computational study that provides quantitative relations for recovering the surface probe density from the mechanical response (afmcompressibility measurements) of the sample. nucleoside analogues used as anticancerous drugs can be rapidly degraded within treated cells, constituting a major obstacle of their therapeutic efficiency. among the enzymes responsible for this degradation, the cytosolic 5'nucleotidase ii (cn-ii) catalyses the hydrolysis of some nucleoside monophosphates. in order to improve the efficacy of anticancerous drugs and to define the precise role of cn-ii, new original inhibitors have been developed against cn-ii. virtual screening of chemical libraries on the crystal structure has allowed us to identify very promising candidates that turned to be competitive inhibitors of cn-ii. one molecule was included in the anticancerous treatment of tumoral cell lines in order to evaluate the potential benefit and could induce in fine a sensitization of certain anticancerous drugs. we also explore other inhibitors targeting the allosteric sites of this enzyme using a strategy that takes into account the dynamics of cn-ii. the chemical structures of the newly identified allosteric inhibitors as well as the atomic interactions with enzyme residues will be presented. the final goal of this study is to find molecules that can freeze the enzyme in a conformation for which its dynamics is severely limited and therefore its function. native mass spectrometry to decipher interactions between biomolecules sarah cianferani laboratoire de spectromé trie de masse bio-organique, université de strasbourg, iphc, 25 rue becquerel 67087 strasbourg, france. cnrs, umr7178, 67037 strasbourg, france mass spectrometry is generally understood as ''molecular mass spectrometry'' with multiple applications in biology (protein identification using proteomic approaches, recombinant protein and monoclonal antibody characterization). an original and unexpected application of mass spectrometry emerged some twenty years ago: the detection and the characterization of intact biological noncovalent complexes. with recent instrumental improvements, this approach, called native ms, is now fully integrated in structural biology programs as a complementary technique to more classical biophysical approaches (nmr, crystallography, calorimetry, spr, fluorescence, etc.). native ms provides high content information for multiprotein complexes characterization, including the determination of the binding stoichiometries or oligomerization states, sitespecificities and relative affinities. recent developments of ion mobility / mass spectrometry instruments (im-ms) provide a new additional level for ms-based structural characterization of biomolecular assemblies allowing size and shape information to be obtained through collisional cross section measurements. these different aspects of native ms for structural characterization of biomolecular assemblies will be illustrated through several examples, ranging from multiprotein-complexes to protein/nucleic acid assemblies. complex coacervation is a process which may result by electrostatic interaction between charged polysaccharides. it depends essential on ph, ionic strength and biopolymers properties like ratio, concentration and charge density. in this case, the main work was to study the structural properties of a colloidal system of opposite charge -chitosan and gum-arabic by atomic force microscopy (afm). according to some of complexes show tendency to agglomerate. this depends on the molar ration of the macromolecules and their relative molecular weights. afm micrographs show, too, that some formation of irregular aggregates by both polymers were due to presence of noncharged polar monomers in chitosan molecule. at higher gum-arabic/chitosan ratios biopolymer concentrations, coacervates appear like a core-shell miccelar structure composed of hydrophobic core (charge neutralized segments) stabilized by the excess component (positive zeta potential) and non-charged segments of gum arabic. interaction of human serum albumin with rutin theoretical and experimental approaches ícaro p caruso 1 human serum albumin (hsa) is the principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. flavonoids are a large class of naturally occurring polyphenols widely distributed in plants. rutin (quercetin-3-rutinoside) is the glycoside between flavonoids quercetin and disaccharide rutinose. like other flavonoids, rutin displays anti-inflammatory and anti-oxidant properties. the interaction between hsa and rutin was investigated by fluorescence spectroscopy, ab initio and molecular modeling calculations. fluorescence titration was performed by keeping the hsa concentration (4 lm) constant and stoichiometrically varying the rutin concentration (1-4 lm) . the emission spectra were obtained in the range of 305 to 500nm, with the excitation wavelength at 295nm. the obtained fluorescence data were corrected for background fluorescence and for inner filter effects. the stern-volmer quenching constant values were 3.722 9 10 5 and 1.868 9 10 5 m -1 at 298 and 303 k, respectively. from the modified stern-volmer association constants 2.285 9 10 5 (at 298 k) and 2.081 9 10 5 m -1 (at 303 k) were calculated the thermodynamic parameters dh = 14.048 kj mol -1 , dg 298k = -30.557 kj mol -1 and dg 303 k = -30.834 kj mol -1 , and ds = 55.4 kj mol -1 k -1 . fluorescence quenching method was used also to study the binding equilibria thus determining the number of binding sites 1.085 and 1.028, and binding constant 1.094 9 10 6 m -1 and 0.255 9 10 6 m -1 at 298 and 303 k, respectively. the efficient quenching of the trp214 fluorescence by rutin indicates that the binding site for the flavonoid is situated within subdomain iia of hsa. the distance r = 2.397 nm between the donor (hsa) and the acceptor (rutin) was obtained according to fluorescence resonance energy transference (fret). wavelength shifts in synchronous fluorescence spectra showed the conformation of hsa molecules is changed in the presence of rutin. the structure of rutin utilized in molecular modeling calculation was obtained by gaussian 98 program. the optimization geometry of rutin was performed in its ground states by using ab initio dft/b3lyp functional with 6-31g(d,p) basis set used in calculations. the molecular electrostatic potential (mep) was calculated to provide the molecular charge distribution of rutin. the gap energy value between the homo and lumo of the rutin molecule was about 4.22 ev which indicates that rutin is classified as a reactive molecule. from molecular modeling calculation the interaction between hsa and rutin was investigated using the autodock program package. the three-dimensional coordinates of human serum albumin were obtained from the protein data bank (entry pdb code 1ao6) and of rutin were obtained from output optimization geometry of dft. the best energy ranked result shows that rutin is localized in the proximity of single tryptophan residue (trp214) of hsa that is in agreement with the fluorescence quenching data analysis. the effect of toxofilin on the structure of monomeric actin lívia czimbalek, veronika kollá r, roland kardos, gá bor hild university of pé cs, faculty of medicine, department of biophysics, pé cs, hungary actin is one of the main components of the intracellular cytoskeleton. it plays an essential role in the cell motility, intracellular transport processes and cytokinesis as well. toxoplasma gondii is an intracellular parasite, which can utilise the actin cytoskeleton of the host cells for their own purposes. one of the expressed proteins of t. gondii is the 27 kda-sized toxofilin. the long protein is a monomeric actinbinding protein involved in the host invasion. in our work we studied the effect of the actin-binding site of toxofilin 69-196 on the g-actin. we determined the affinity of toxofilin to the actin monomer. the flourescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of toxofilin. in the presence of toxofilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. the results of the completed experiments can help us to understand in more details what kind of cytoskeletal changes can be caused in the host cell during the invasion of the host cells by intracellular parasites. t7 bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. both tmpcp and bmpcp and their peptide conjugates proved to be efficient photosensitizers of virus inactivation. the binding of porphyrin to phage dna was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by dna bound porphyrin. mechanism of photoreaction (type i. versus type ii) and the correlation between dna binding, singlet oxygen production and virus inactivation capacity was also analyzed. dna binding reduced the virus inactivation due to the reduced absorbance and singlet oxygen production of bound photosensitizer, and altered mechanism of photoinactivation. as optical melting studies of t7 nucleoprotein revealed, photoreactions of porphyrin derivatives affected the structural integrity of dna and also of viral proteins, even if the porphyrin did not bind to np or was selectively bound to dna. anesthesia is a medical milestone (friedman & friedland, medicine's 10 greatest discoveries, 2000) and local anesthetics (la) are the most important compounds used to control pain in surgical procedures. however, systemic toxicity is still a limitation for la agents as well as low solubility, as for tetracaine (ttc). approaches to improve la effects include macrocyclic systems formation, such as in cyclodextrins (cd). we have studied complexes formed between ttc and b-cd or hydroxylpropyl (hp)-b-cd through nmr and other (uv-vis, fluorescence, dsc and x-ray diffraction) techniques. at ph 7.4 a 1:1 stoichiometry of complexation was detected for both complexes, with association constants of 777 m -1 and 2243 m -1 for ttc:b-cd and ttc:hp-b-cd, respectively. the nuclear overhauser nmr data disclosed trough the space proximities between hydrogens h h and h iat the aromatic ring of ttc -and hydrogens from the inner cavity of the cyclodextrins, allowing us to propose the topology of ttc:cd interaction. complex formation did not curb ttc association with model (liposomes) and biological membranes since the total analgesic effect (infraorbital nerve blockade in rats) induced by 15mm ttc increased 36% upon complexation. supported by (fapesp # 06/121-9, 03838-1) brazil. p-033 itc as a general thermodynamic and kinetic tool to study biomolecule interactions philippe dumas 1 , dominique burnouf 1 , eric ennifar 1 , sondes guedich 1 , guillaume bec 1 , guillaume hoffmann 1 isothermal titration calorimetry (itc) is a powerful technique for thermodynamic investigations that is little used to obtain kinetic information. we have shown that, in fact, the shape of the titration curves obtained after each ligand injection is strictly governed by the kinetics of interaction of the two partners. a simple analysis allowed us to explain several facts (e.g. the variation of time needed to return to equilibrium during a titration experiment). all simplifications were further released to obtain a very realistic simulation of an itc experiment. the method was first validated with the binding of the nevirapine inhibitor onto the hiv-1 reverse transcriptase by comparison with results obtained by biacore tm . importantly, for more complex systems, the new method yields results that cannot be obtained in another way. for example, with the e. coli transcription-regulator thiamine pyrophosphate riboswitch, we could resolve kinetically and thermodynamically the two important successive steps: (1) the binding of the tpp ligand and (2) the subsequent rna folding. our results show that initial tpp binding is controlled thermodynamically by tpp concentration, whereas the overall transcription regulation resulting from rna folding is kinetically controlled. gfps, due to their tendency to dimerize at high concentration. we have characterized for the first time the selfassociation properties of cfp (cyan fluorescent protein), the fluorescent protein mostly used as fret donor. we found that the fluorescence quenching observed at high expression level in the cell cytoplasm and the fluorescence depolarization measured at high concentration in vitro are insensitive to the a206k mutation, shown to dissociate other gfp dimers. both phenomena are satisfactorily accounted for by a model of non-specific homo-fret between cfp monomers due to molecular proximity. modeling the expected contributions to fluorescence depolarization of rotational diffusion, homo-fret within a hypothetical dimer and proximity homo-fret shows that cfp has a homo-affinity at least 30 times lower than gfp. this difference is due to an intrinsic mutation of cfp (n146i), originally introduced to increase its brightness and that by chance also disrupts the dimers. biomolecular recognition typically proceeds in an aqueous environment, where hydration shells are a constitutive part of the interacting species. the coupling of hydration shell structure to conformation is particularly pronounced for dna with its large surface to volume ratio. conformational substates of the phosphodiester backbone in b-dna contribute to dna flexibility and are strongly dependent on hydration. we have studied by rapid scan ftir spectroscopy the isothermal b i -b ii transition on its intrinsic time scale of seconds. correlation analysis of ir absorption changes induced by an incremental growth of the dna hydration shell identifies water populations w 1 (po 2 --bound) and w 2 (non-po 2 --bound) exhibiting weaker and stronger h-bonds, respectively, than those in bulk water. the b ii substate is stabilized by w 2 . the water h-bond imbalance of 3-4 kj mol -1 is equalized at little enthalpic cost upon formation of a contiguous water network (at 12-14 h 2 o molecules per dna phosphate) of reduced !(oh) band width. in this state, hydration water cooperatively stabilizes the b i conformer via the entropically favored replacement of w 2 -dna interactions by additional w 2 -water contacts, rather than binding to b i -specific hydration sites. such water rearrangements contribute to the recognition of dna by indolicidin, an antimicrobial 13-mer peptide from bovine neutrophils which, despite little intrinsic structure, preferentially binds to the b i conformer in a water-mediated induced fit. in combination with cd-spectral titrations, the data indicate that in the absence of a bulk aqueous phase, as in molecular crowded environments, water relocation within the dna hydration shell allows for entropic contributions similar to those assigned to water upon dna ligand recognition in solution. segmental-labeling expression of sh3 domains of cd2ap protein to study interaction with their ligand i.f. herranz-trillo 1 , j.l. ortega-roldan 1 , n.a.j. van transient and low affinity interactions within the cell can be enhanced by the combination of more than one domain. up to now most of the effort has been put on the study of the regulation in the affinity and specificity of the binding to isolated single domains but little is known about the effect of the presence of a second or third domain. multiple examples of proteins containing tandem domains exist in the genome like the cin85/cms family of adaptor proteins. in this family all three n-terminal sh3 domains are involved in a wide variety of different interactions, they share higher similarity among themselves than to any other sh3 domains, suggesting an overlapping specificities in binding. cd2 associated protein (cd2ap) is an adaptor protein and belongs to this family, its n-terminus consists of three sh3 domains and the interaction of each one of them with its target(-s) might be ultimately modulated by the presence of its next-door-neighbor. in this work we present the expression and purification of the tandem cd2ap-sh3a/ sh3b produced by segmental labeling techniques that allow us to express the domains with different isotopic label, improving the nmr signal and facilitating to study the interaction of the natural ligand in the presence of nextdoor-neighbor domain. there are plenty of molecules that exert their effects at the cell membrane. the evaluation of these interactions, frequently quantified by the nernst lipid/water partition constant (kp), helps to elucidate the molecular basis of these processes. we present here a recently derived and tested method to determine kp for single solute partitions using fpotential measurements. the concept was then extended to the interaction of supramolecular complexes with model membranes. a simultaneous double partition with an aqueous equilibrium is considered in this partition model. the results were validated by dynamic light scattering -dls, f-potential, fluorescence spectroscopy and laser confocal microscopy experiments. we evaluated the interaction of supramolecular complexes (peptides derived from dengue virus proteins with oligonucleotides) with luv to study our biophysical models. dengue virus (dv) infects over 50-100 million people every year and may cause viral hemorrhagic fever. no effective treatment is available and several aspects of its cellular infection mechanism remain unclear. the extension of the interactions of these complexes with biomembranes helps to elucidate some steps of dv life cycle. the aggregation of amphotericin b in the lipid membranes induced by k + and na + ions: langmuir monolayers study marta arczewska, mariusz gagoś department of biophysics, university of life sciences in lublin, poland the polyene antibiotic amphotericin b (amb) is currently the drug of choice in the treatment of fungal infections despite its undesirable side effects. according to the general conviction, the biological action of the drug is based on the formation of transmembrane channels which affect physiological ion transport, especially k + ions. this work reports the results of langmuir monolayers study of the effect of k + and na + ions on the molecular organizations of amb in the model lipid membrane. the two-component monolayers containing amb and phospholipid (dppc) have been investigated by recording surface pressure-area isotherms spread on aqueous buffers containing physiological concentration of k + and na + ions. the strength of the amb-dppc interactions and the stability of the mixed monolayers were examined on the basis of surface pressure measurements, the compressional modulus and the excess free energy of mixing. the obtained results proved a high affinity of amb towards lipids in the presence of k + than na + ions. the most stable mixed monolayers were formed with the 1:1 and 2:1 stoichiometry in the presence of k + and na + ions, respectively. this research was financed by ministry of education and science of poland within the research project n n401 015035. microcalorimetric study of antibiotic amphotericin b complexes with na + , k + and cu 2+ ions arkadiusz matwijczuk, grzegorz czernel, mariusz gagoś department of biophysics, university of life sciences in lublin, poland amphotericin b (amb) as a metabolite of streptomyces nodosus is one of the main polyene antibiotics applied in the treatment of deep-seated mycotic infections. we presented microcalorimetric (dsc) study of molecular organization of amphotericin b in lipid membranes induced by na + , k + and cu 2+ ions. the analysis of dsc curves indicates the influence of na + and k + ions on the main phase transition of pure dppc lipid. for the molar fractions of 3, 5, 10, 15 mol% amb in dppc we observed the thermal shift towards higher temperatures in respect to pure lipid, both in the presence of na + and k + ions. this result may be connected with the changes in dynamic properties of the model membrane system. in case of amb-cu 2+ complexes in aqueous solution at two ph values, 2.0 and 10.6, the dsc measurements reported endothermic heat effect. this phase transition was related to the dissociation process of amb-cu 2+ complexes. the formation of amb-cu 2+ complexes are accompanied by changes of the molecular organization of amb especially disaggregation. these all observed effects might be significant from a medical point of view. this research was financed by ministry of education and science of poland within the research project n n401 015035. membrane proteins and peptides are acting in an environment rich in other proteins or peptides. aim of our study was to understand how such molecular crowding and resulting intermolecular interactions can influence the behavior of membrane proteins, using various antimicrobial peptides and membrane proteins as examples. in the case of antimicrobial peptides we have previously described a change in their alignment in the membrane at a characteristic threshold concentration. to understand whether this change is due to unspecific crowding or specific peptidepeptide interactions, we tested if this re-alignment depends on the presence of additional peptides. in most cases we found a similar re-orientation behavior irrespective of the added peptide type, indicating unspecific crowding. when pairing pgla and magainin-2, however, we observed a distinctly different sequence of pgla re-orientation in the membrane, indicating a specific interaction between these two peptides, which correlates well with their known synergistic activity. a rather different effect of crowding was observed for the larger channel protein mscl, which was found to form clusters of functionally active proteins in the membrane. we propose that this clustering is caused by lipid-mediated protein-protein interactions. water, hydrophobic interaction, and protein stability j. raul grigera and c. gaston ferrara instituto de física de líquidos y sistemas bioló gicos (iflysib), conicet-unlp, la plata, argentina although there are several forces maintaining protein structure, it is well know that hydrophobic interaction is the dominant force of protein folding. then, we can infer that any factor that alters hydrophobic interaction will affect the protein stability. we have study by computer simulation a model system consisting in solution of lenard-jones particles in water (spc/e model) at different pressures and temperatures and analyzed the solubility i.e. the aggregation properties, of such a system. from the obtained data we are able to build up the phase surface determining the critical point. the computing results where compared with experimental data of binary mix of non polar substance in water and of protein denaturation, finding high coincidence on the critical point. since the behavior of our model system can only be due to hydrophobic effects, the coincidences with the denaturation of proteins allow us to conclude that the dominant factor that determine temperature and pressure denaturation of proteins is the hydrophobic interaction. the temperature and pressures at which the denaturation, as well the disaggregation of simple non-polar particles, starts agree with what we could expect based on the cross over line of the low to high density structure water transition. the functional reconstitution of a mitochondrial membrane protein into a lipid bilayer was studied using a quartz crystal microbalance. the 6xhis-tagged protein was immobilised via specific binding to a cu 2+ terminated sensor surface, with a change in frequency indicating approximately 75% coverage of the sensor surface by the protein. a lipid bilayer was reconstituted around the protein layer, with a final change in frequency that is consistent with the remaining area being filled by lipid. incubation with a specific ligand for the protein resulted in a significant change in frequency compared to the interaction with the surface or lipid alone. the change is greater than expected for the mass of the ligand, indicating a possible conformational change of the protein, such as the opening of a channel and increased water content of the layer. electrical impedance measurements on the same system have provided additional evidence of protein-lipid bilayer formation, and it is intended that this system will be studied with neutron reflectometry to characterise potential ligand induced channel formation. valuable functional and structural information about this membrane protein was obtained by using surface sensitive techniques to study the protein in a biomimetic lipid bilayer. visualizing and quantifying hiv-host interactions with fluorescence microscopy jelle hendrix 1, *, zeger debyser 2 , johan hofkens 3 and yves engelborghs 1 1 laboratory for biomolecular dynamics, university of leuven, belgium, 2 laboratory for molecular virology and gene therapy, university of leuven, belgium, 3 laboratory for photochemistry and spectroscopy (*present address), university of leuven, belgium protein-chromatin interactions are classically studied with in vitro assays that only provide a static picture of chromatin binding. fluorescence correlation spectroscopy (fcs) is a non-invasive technique that can be used for the same purpose. being applicable inside living cells it provides dynamic real-time information on chromatin interactions. transcriptional co-activator ledgf/p75 has well characterized protein and chromatin interacting regions. we studied ledgf/p75 in vitro and inside living cells with fcs and other techniques (luminescent proximity assay, spot/half-nucleus fluorescence recovery after photobleaching, continuous photobleaching). protein-protein interactions in living cells can be monitored with fluorescence cross-correlation spectroscopy (fccs) using fluorescent proteins as genetic labels. advantages over using fö rster resonance energy transfer (fret) are the independence from intermolecular distance and knowledge of absolute protein concentrations. we characterized fccs with fluorescent proteins in vitro and then studied the intracellular complex of ledgf/p75 and the hiv-1 integrase (in) enzyme both with fret and fccs. nucleus and its compartment nucleolus are a seat of enormous biosynthetic activity in human cancer cells. nucleolar proteins, e.g. b23 or c23, play an important role in regulation of cell division and proliferation. one of the strategies how to intermit malignant cell proliferation is affecting, e.g. by drug treatment, a net of intracellular protein interactions to bring the cell on a way of apoptosis. a cytostatic agent actinomycin d initiates apoptosis in human cancer cells, as well as in normal peripheral blood lymphocytes. at the same time, translocation of b23 and c23 into nucleoplasm is observed in the treated cells. therefore interaction between nucleolar and apoptotic proteins comes into a question. co-immunoprecipitation, fluorescence microscopy and yeast two hybrid analysis are used to answer it. in co-immunoprecipitation experiments, tumor suppressor p53 showed up to be a promising candidate for the interaction. fluorescence deposits mostly constituted by variants of transthyretin (ttr), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol [1] . nowadays, the only effective therapy for ttr amyloidosis is liver transplantation. new therapeutic strategies are being developed taking advantage of our current understanding of the molecular mechanisms of amyloid formation by ttr [2] . a significant effort has been devoted to the search and rational design of compounds that might decrease ttr tetramer dissociation, for example, through ligand binding at the thyroxine binding sites of ttr [3, 4] . here, we use isothermal titration calorimetry (itc) to characterize the thermodynamic binding signature of potential ttr tetramer stabilizers, previously predicted by computerassisted methods [3] . itc allows the measurement of the magnitude of the binding affinity, but also affords the characterization of the thermodynamic binding profile of a protein-ligand interaction. high affinity/specificity ttr ligands, enthalpically and entropically optimized, may provide effective leads for the development of new and more effective drug candidates against ttr amyloidosis. we have established a set of vectors to promote easy cloning of ecfp and eyfp fusions with any protein of interest. we exploit these fluorescent fusion proteins to study protein-protein interactions by fluorescence lifetime of ecfp. the decrease of ecfp lifetime reveals fret between ecfp and eyfp and hence the interaction between proteins in question. groel-groes chaperonin complex is required for the proper folding of eschericia coli proteins. bacteriophage t4 and its distant relative coliphage rb49 encode co-chaperon proteins (respectively gp31 and coco) that can replace groes in the chaperonin complex. gp31 is also required in the folding of the major capsid protein of the phage. prd1 is a large membrane-containing bacteriophage infecting gram-negative bacteria such as e. coli and salmonella enterica. it has 15 kb long linear dsdna genome and the capsid has an icosahedral symmetry. the groel-groes chaperonin complex is needed in the assembly of prd1. we have found evidence that prd1 protein p33 can work similar way as other viral co-chaperones and substitute groes in chaperonin complex. fluorescence lifetime studies between proteins groel and p33 reveals an interaction that backs up the theory. structural modification of model membranes by fibrillar lysozyme as reaveled by fluorescence study a.p. kastorna v.n. karazin kharkiv national university, 4 svobody sq., kharkiv, 61077, ukraine recent experimental findings suggest that protein aggregation, leading to the formation and depositions of amyloids play a central role in the neurodegenerative diseases, type ii diabetes, systemic amyloidosis, etc. in the present study we focused our efforts on investigation of the influence of fibrillar lysozyme on the structural state of model lipid membranes composed of phosphatidylcholine and its mixtures with cardiolipin (10 mol %) and cholesterol (30 mol %). to achieve this purpose, two fluorescent probes with different bilayer location, 1,6-diphenyl-1,3,5-hexatriene (dph) distributing in membrane hydrocarbon core and 6-lauroyl-2-dimethylaminonaphthalene (laurdan) locating at lipid-water interface, have been employed. the changes in membrane viscosity under the influence of amyloid lysozyme were characterized by fluorescence anisotropy of dph. this fluorescence parameter was not markedly affected by fibrillar protein in all types of model membranes. the changes in emission spectra of laurdan were analysed by the generalized polarization value (gp). it was found that adding of amyloid lysozyme resulted in the increment of gp value. our data suggest that lysozyme fibrils cause reduction of bilayer polarity and increase of lipid packing density. isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions. however, reliable determination of the dissociation constant (k d ) is typically limited to the range 100 lm [ k d [ 1 nm. nevertheless, interactions characterised by a higher or lower k d can be assessed indirectly, provided that a suitable competitive ligand is available whose k d falls within the directly accessible window. unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of highand low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-and low-affinity protein-ligand interactions. interaction of myoglobin with oxidized polystyrene surfaces studied using rotating particles probe m. kemper 1,2 , d. spridon 1 , l.j. van ijzendoorn 1 , m.w.j. prins 1,3 1 eindhoven university of technology, department of applied physics, eindhoven, the netherlands, 2 dutch polymer institute, eindhoven, the netherlands, 3 philips research, eindhoven, the netherlands the interaction of proteins with polymer surfaces is of profound importance for the sensitivity of biosensors. polymer surfaces are often treated in order to tune their chemical and physical properties, for example by oxidation processes. to get a better understanding of the association of proteins to treated polymer surfaces, we use the rotating particles probe (x.j.a. janssen et al., colloids and surfaces a, vol. 373, p. 88, 2011). in this novel technique, protein coated magnetic particles are in contact with a substrate and the binding is recorded for all individual particles using a rotating magnetic field. we investigate the interaction of myoglobin coated magnetic particles to spincoated polystyrene surfaces that have been oxidized with a uv/ozone treatment. the surfaces have been characterized by xps, afm and water contact angle measurements. we will demonstrate a clear influence of polystyrene oxidation on the binding fractions of the myoglobin coated particles. we interpret the results in terms of dlvo-theory: electrostatic as well as electrodynamic properties of the surfaces will be influenced by the oxidation. interact with monomeric and/or filamentous actins. twinfilin is a 37-40 kda protein composed of two adf-homologue domains connected by a short linker. in our work we studied the effects of the mouse twinfilin-1 (twf1) on the monomeric actin. we determined the affinity of twf1 to the atp-actin monomer with fluorescence anisotropy measurement (k d = 0.015 lm). the fluorescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of twf1. in the presence of twinfilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. it was confirmed with stopped-flow experiments that the kinetics of nucleotide-exchange of actin decreased in the presence of twf1. we determined the thermodynamic properties of twf1 and investigated the effect of twinfilin on the stability of actin monomer with differential scanning calorimetry. the twf1 stabilized the stucture of the g-actin. our results can help us to understand the regulation of actin cytoskeleton in more details. magnetic np have attracted attention due to their potential of contrast enhancement of magnetic resonance imaging and targeted drug delivery, e.g. tumor magnetic hyperthermia therapy. potential nephrotoxicity of single i.v. administration of fe 3 o 4 np was studied in female wistar rats i.v. administered either placebo (10% v/v rat serum in 0.9% nacl), suspension of tio 2 np (positive control, bimodal 84/213 nm distribution), or fe 3 o 4 np (bimodal 31/122 nm distribution) in doses of 0.1, 1.0 or 10.0 mg/kg. rats were sacrificed 24h, 7-, 14-and 28-days after np injection (n=9-10/each group). administration of np did not alter kidney size significantly; renal function of np administered rats as monitored by plasma creatinine and urea concentrations, creatinine clearance and protein excretion rate did not differ significantly in either interval from rats administered placebo. one week after administration significant rise in plasma ca, its urinary and fractional excretion was observed in rats administered 10 mg fe 3 o 4 /kg. plasma mg levels rose in this group 1 and 2 weeks after administration. no significant changes in the expression of tnf-a, tgf-b, and collagen iv genes in renal cortex were revealed. no obvious nephrotoxic effects were observed in rats after a single i.v. dose of fe 3 o 4 np. study was supported by 7fp ec eu: nanotest (development of methodology for alternative testing strategies for the assessment of the toxicological profile of nanoparticles used in medical diagnostics.), grant no.: 201335. biomimetic supramolecular assemblies for studying membrane interactions in vitro and in vivo s. kolusheva, r. jelinek ben-gurion university, beer-sheva, israel we designed a novel biomimetic sensor, composed of conjugated polydiacetylene (pda) matrix embedded within lipid vesicles. the system is capable of detecting various compounds occurring within lipid membranes through rapid colorimetric as well as fluorescent transitions. the colorimetric response of the sensor is correlated to the extent of compound-membrane binding and permeation and quantified binding sensitivity to lipid composition. we describe a new disease diagnostic approach, denoted ''reactomics'', based upon reactions between blood sera and an array of vesicles comprising different lipids and polydiacetylene (pda), a chromatic polymer. we show that reactions between sera and such a lipid/pda vesicle array produce chromatic patterns which depend both upon the sera composition as well as the specific lipid constituents within the vesicles. through attachment of chromatic polydiacetylene (pda) nanopatches onto the plasma membrane, real-time visualization of surface processes in living cells is possible. the ras protein is mutated in 30% of human tumors. ras acts as a switch, transmitting a growth signal in an active gtp-bound form and turning the signal off in an inactive gdp-bound form. the switch off is accomplished by gtp hydrolysis, which is catalyzed by ras and can be further accelerated by gtpase activating proteins (gaps). mutations which prevent hydrolysis cause severe diseases including cancer. we investigate the reaction of the ras gap protein-protein complex by time-resolved ftir spectroscopy. 1 detailed information on the mechanism and the thermodynamics of the reaction was revealed: 2 first, the catalytic arginine-finger of gap has to move into the gtp binding pocket, then cleavage of gtp is fast and h 2 po 4 hydrogen-bonded in an eclipsed conformation to the b-phosphate of gdp is formed. further, we performed for the first time atr-ftir spectroscopy of ras in its native environment, a lipid membrane. 3 in this setup we are able to do difference spectroscopy of the immobilized protein. interactions with other proteins can be determined in a similar way as in spr experiments but with the additional information from the infrared spectra. galectins are a family of animal lectins that specifically bind b-galactosides and have gained much attention due to their involvement in several biologic processes such as inflammation, cell adhesion and metastasis. in such processes, several issues are still not clear including the mechanisms of interaction with different carbohydrates. galectin-4 (gal-4) is a tandem-repeat type galectin that contains two carbohydrate recognition domains (crd-i and crd-ii) connected by a linking peptide. in this study, we performed spectroscopic studies of the carbohydrate-recognition domains from human gal-4. our goals are two-fold: (1) to monitor conformational changes in each domain upon its binding to specific ligands and then to correlate the observed changes with structural differences between the crds and (2) to investigate the interaction between the crds and lipid model membranes. to achieve such objectives we used a combined approach of spectroscopic techniques involving circular dichroism and electron spin resonance. overall the results obtained so far show that crd-i and crd-ii have distinct behaviors in terms of carbohydrate recognition and membrane binding. this may be due to specific differences in their structures and certainly suggests a non-equivalent role in protein function. hemoglobin influence on lipid bilayer structure as revealed by fluorescence probe study o.k. kutsenko, g.p. gorbenko, v.m. trusova v.n. karazin kharkov national university, kharkov, ukraine hemoglobin (hb) is a red blood cell protein responsible for the oxygen transport. its affinity for lipid bilayers represents interest for gaining insight into protein biological function as well as for some applied aspects such as development of blood substitutes or biosensors. hb influence on lipid bilayer structure was investigated using fluorescent probes pyrene and prodan. model membranes were prepared of phosphatidylcholine (pc) and its mixtures with phosphatidylglycerol (pg) and cholesterol (chol). hb penetration into membrane interior is followed by the increase of relative intensity of pyrene vibronic bands and decrease of prodan general polarization value suggesting an enhancement of bilayer polarity. this implies that hb incorporation into membrane interior decreases packing density of lipid molecules, promoting water penetration into membrane core. chol condensing effect on lipid bilayer prevents protein embedment into bilayer, thus decreasing membrane hydration changes as compared to pc bilayers. in the presence of anionic lipid pg hb-induced increase of bilayer polarity was found to be most pronounced, pointing to the modulatory role of membrane composition in hb bilayer-modifying propensity. we present optimized sialic acid-based mimics binding in the low nanomolar range. molecular interactions were determined with surface plasmon resonance (spr), characterizing the affinity and the kinetics of binding. furthermore, isothermal titration calorimetry (itc) was applied to dissect the standard free energy of binding (dg°) into the standard enthalpy of binding (dh°) and the standard entropy of binding (ds°). in order to pass the cell membranes, most of these medicines has to be administrated to patients as nucleoside pro-drugs and not directly as triphosphorylated forms. because of the poor phosphorylation of the nucleoside analogues used in therapy, it is important to understand and to optimize their metabolism. our aim is to understand how compounds of chirality l turn away 3-phosphoglycerate kinase (pgk) from its normal glycolytic function to be converted into the triphosphate forms. in order to elucidate pgk mechanism and substrate specificity, we have measured the kinetics of the different steps of the enzymatic pathways by rapid mixing techniques and studied the influence of the nature of the nucleotide substrate thereon. we first performed an extensive study with d-and l-adp (see poster by p. lallemand). we are now extending the studies to other nucleotide diphosphates (some of them used in therapies). changes in the nature of the nucleobase or deletion of hydroxyl group of the sugar affect the efficiency of phosphorylation by pgk, either by decreasing dramatically their affinity or by altering the phospho-transfer step itself. structural explanations are given based on docking data. probing drug/lipid interactions at the molecular level represents an important challenge in pharmaceutical research, drug discovery and membrane biophysics. previous studies showed that enrofloxacin metalloantibiotic has potential as an antimicrobial agent candidate, since it exhibits antimicrobial effect comparable to that of free enrofloxacin but a different translocation route. these differences in uptake mechanism can be paramount in counteracting bacterial resistance. in view of lipids role in bacterial drug uptake, the interaction of these compounds with different e. coli model membranes were studied by fluorescence spectroscopy. partition coefficients determined showed that lipid/antibiotic interactions were sensitive to liposomes composition and that the metalloantibiotic had a higher partition than free enrofloxacin. these results corroborate the different mechanism of entry proposed and can be rationalized on the basis that an electrostatic interaction between the metalloantibiotic positively charged species, present at physiological ph, and the lipids negatively charged head groups clearly promotes the lipid/antimicrobial association. oligomerization and fibril assembly of amyloid b peptide amyloid b peptide (ab) forms a large amount of extracellular deposits in the brain of alzheimer's disease (ad) patients and it is believed that this peptide is related to the pathogenesis of that disease. the most abundant monomeric form of physiological ab (*90%) is constituted by 40 amino acids and is benign, but by an unknown mechanism this endogenous material becomes aggregated and neurotoxic. increasing evidence suggests that membrane interaction plays an important role in ab neurotoxicity. in this work it will be studied the interactions of ab(1-40) with ctac (cationic), sds (anionic), pfoa (anionic with fluorine atoms) and og (nonionic) amphiphiles in monomeric and micellar forms. the results demonstrated that ab(1-40) forms fibrils with different morphologies in the presence of micelles. in addition, the presence of micelles accelerates the formation of fibrils and decreases the lifetime of oligomers. we present here the exploitation of the powerful approach of surface plasmon resonance imaging and mass spectrometry coupling for protein fishing in biological fluids such as human plasma at the same sensitivity. on one hand, multiplex format spri analysis allows direct visualization and thermodynamic analysis of molecular avidity, and is advantageously used for ligand-fishing of captured bio-molecules on multiple immobilized receptors on a spri-biochip surface. on the other hand, maldi mass spectrometry is a powerful tool for identification and characterization of molecules captured on specific surface. therefore, the combination of spri and ms into one concerted procedure, using a unique dedicated surface, is of a great interest for functional and structural analysis at low femtomole level of bound molecules. to reach these goals, particular surface engineering has been engaged to maintain a high level of antibody grafting and reduce non-specific adsorption. thus, various chemistries have been tested and validated towards biological fluids such plasma, keeping in mind the capacity of the in situ investigation by ms. finally, signal to noise ratio was magnified leading to the characterization of protein lag3, a potential marker of breast cancer, in human plasma. atenolol incorporation into pnipa nanoparticles investigated by isothermal titration calorimetry mihaela mic, ioan turcu, izabell craciunescu, rodica turcu, national institute for r&d of isotopic and molecular technologies, 400293 cluj-napoca, romania e-mail: mihaela.mic@itim-cj.ro poly(n-isopropylacrylamide) (pnipa) is a thermo-sensitive hydrogel undergoing a volume phase transition at about of 32°c close to the body temperature. this volume phase transition is envisaged as a key property for drug binding and release. the purpose of our research is the thermodynamic characterization of the binding of atenolol by pnipa polymeric nanoparticles. the thermodynamic parameters which characterize the binding process are obtained using the isothermal titration calorimetry (itc) as the main investigation technique. when polymeric nanoparticles bind drug molecules, heat is either generated or absorbed depending on the amount of bond molecules and also on the exothermic or endothermic character of the binding process. the heat measurement allows the determination of binding constants, reaction stoichiometry and the thermodynamic profile of the interaction. itc technique has been used to investigate the binding properties of nanoparticles which shrink from the swollen to the collapsed state. the capacity of such nanogels to bind atenolol molecules is directly related to relevant differences between the binding properties in the swollen and in the collapsed state respectively. aggregation study of x-(alkyldimethylamonium)alkylaldonamide bromides p. misiak 1 , b. ró _ zycka-roszak 1 , e. woźniak 1 , r. skrzela 1 , k.a. wilk 2 1 department physics and biophysics, wrocław university of environmental and life sciences, wrocław, poland, 2 department of chemistry, wrocław university of technology, wrocław, poland sugar-based surfactants are of considerable research interest because they have improved surface and performance properties, reduced environmental impact, and have potential pharmaceutical and biomedical applications. x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lenghs (n = 10, 12, 14, 16) belonging to cationic sugar-based surfactants were newly synthesised. the aggregation processes of c n gabs were studied by means of isothermal titration calorimetry (itc), electric conductance method and molecular modelling methods. the critical micelle concentrations (cmc), the degree of micelle ionization (b), the enthalpies (dh m ) and the entropies (ds m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (dg m 0 (hy)) were calculated. the obtained values were compared with those for dodecyldimethylethylammonium bromide and literature data for analogical glucocationic surfactants. the latest compounds differ from c n gab surfactants by substitution of sugar chain by gluco ring. molecular modelling methods were used to relate the molecular properties of the compounds with their experimentally studied properties in solution. this work was supported by grant n n305 361739. every year over 50 million people are infected with dengue virus (denv), transmitted by a mosquito (aedes aegypti). this enveloped virus, member of the flaviviridae family, has four distinct serotypes. it has a single stranded positive rna molecule with a single open reading frame that encodes a single poliprotein, which, after appropriate processing by viral and host proteases, gives rise to three structural proteins (c, prm and e) and seven non-structural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b and ns5) [1] . the surface of the immature virion is composed of e and prm heterodimers that are arranged as trimers protruding from the membrane [2] . the virus is thought to enter the host cell via a receptor-mediated endocytosis, although, if any, the specific dengue receptors have not been described. once inside the cell, the acidified environment inside the endocytic vesicle triggers an irreversible trimerization of the envelope (e) protein, inducing the release of the nucleocapsid (composed of viral rna and multiple copies of c protein) to the cytoplasm, thus starting the infection process, where the poliprotein is translated and processed, originating all viral proteins. considering the structural proteins c and e, these are essential for the viral infection, specifically, protein c is thought to be involved in the viral assembly and specific encapsidation of the genome and protein e (a class ii fusion protein) plays a major role in the fusion process. as recently described by some studies [3] , protein c is composed of four a helices connected by four short loops and has a highly hydrophobic region forming a concave groove that could interact with lipid membranes and a region with an increased concentration of positive charges, possibly interacting with the viral rna. as for protein e, it is composed of three b stranded domains. it is proposed that the fusion loop is located in domain ii of this protein and the putative receptor binding sites, considered essential for the viral entry, are supposedly located in domain iii. in this work, we describe the identification of the membrane active regions of both these proteins, considering both theoretical studies, hydrophobic moments, hydrophobicity and interfaciality values as well as experimental ones, namely fluorescence spectroscopy, where a fluorescent probe is encapsulated in model membrane systems, and differential scanning calorimetry [4] . we have found one region in protein c and four regions in protein e with membranotropic activity. this is the first work describing experimentally the putative membrane interacting zones of both these proteins. this work was funded by grant bfu2008-02617-bm from ministerio de ciencia y tecnologia, spain, granted to jose villalaín. investigation of membrane-membrane interaction mediated by coiled coil lipopeptides gesa pä hler, andreas janshoff georg-august-university, tammannstrasse 6, 37077 gö ttingen, germany e-mail: gpaehle@gwdg.de specific cellular membrane interaction and fusion are crucial points in vivo which are in eukaryotic cells mediated by snare proteins. the definite mechanism behind these processes is still poorly understood, but the coiled coil formation of a snare core complex consisting of four a-helices seems to generate a fusogenic driving force. this offers the possibility to design a straightforward experimental setup to mimic the complex protein-mediated membrane-membrane interaction by using mere protein fragments or peptides attached to artificial lipid bilayers which self-assemble to a coiled coil structure. in our approach, two artificial three heptad repeat coiled coil forming peptides were synthesized and attached to maleimide functionalized membranes via an in situ-coupling reaction. thus, secondary structure changes, kinetic characteristics and binding energetics were monitored during coiled coil formation with real time ellipsometry, ir and cd spectroscopy. the lipopeptide mediated membrane-membrane interaction itself is investigated by colloidal probe spectroscopy and tirfm. these techniques and the setup of our model system allow screening the energetic and structural properties of variable coiled coil forming peptides, i.e. linker-modified or biologically inspired sequences. enzymatic reactions in nanostructured surfaces: unzipping and cutting the double helix pietro parisse 1 , matteo castronovo 2 , bojana lucic 3 , alessandro vindigni 3 , giacinto scoles 2 and loredana casalis 1 1 sincrotrone trieste s.c.p.a., trieste, italy, 2 temple university, philadelphia, usa, 3 protein-dna interactions are vital for living organisms. from viruses to plants and humans, the interactions of these two different classes of biopolymers control processes as important and diverse as the expression of genes and the replication, rearrangement, and repair of dna itself. to understand these processes at the molecular level, and to follow changes in cellular pathways due to different kinds of perturbations and/or diseases it is necessary the identification and quantification of proteins and their complex network of interactions. we have exploited the high spatial resolution given by atomic force microscopy to generate dna arrays of variable density by means of nanografting. on such nanostructures, we investigate the mechanism of different enzymatic reactions (from restriction enzymes to helicases). registering with high precision the height variation due to the action of the enzyme onto the engineered dna sequences (in the case of restriction enzymes) or taking advantage of the different mechanical properties of single and double stranded dna (in the case of helicases, where for the first time kinetic data were obtained on recq1 human helicase), we were able to monitor either the activity and/or the action mechanisms of these two important classes of enzymes. in this study an attempt has been made to investigate the structure, dynamics and stability of cyclic peptide nanotubes (cpnts) formed by the self-assembly of cyclic peptides (cps) using classical molecular dynamics (md) simulation and semiempirical quantum chemistry calculation employing pm6. the structure and energetics of monomer and various oligomeric cpnts have been investigated by considering the (cyclo-[(d-ala-l-ala) 4 ]) peptide as the model for cp. various geometrical parameters extracted from the md simulation reveal that the terminal residues are loosely hydrogen bonded to the inner subunits regardless of degree of oligomerization. the hydrogen bonds present in the inner core regions are stronger than the terminal residues. as the degree of oligomerization increases, the stability of the tube increases due to the hydrogen bonding and stacking interactions between the subunits. the results show that the binding free energy increases with the extent of oligomerization and reaches saturation beyond cpnt5. in addition, hydrophobic and electrostatic interactions play crucial roles in the formation of cpnts. analysis of both structural and energetics of formation of cpnts unveils that the selfassembly of dimer, trimer and tetramer cpnts are the essential steps in the growth of cnpts. monolayers on a langmuir trough constitute a great biomimetic model to characterize protein-protein or protein-lipid interaction, where the physical state of the interfacial layer is completely controlled. we present here three studies performed on monolayers, with a wide panel of experimental (optical, spectroscopical, rheological) techniques. i) surface properties and conformation of nephila clavipes spider recombinant silk proteins (maspi and masp2) was studied at the air-water interface: we show that the mechanism of assembly of both proteins is different, although both proteins share the same sequence pattern and a close hydrophobicity. they both exhibit a certain propensity to form b-sheets that may be important for the efficiency of the natural spinning process. ii) the dystrophin molecular organization and its anchoring in a lipidic environment depend on the rod fragment used and on the lipid nature. moreover the interaction is guided by the lateral surface pressure. this lipid packing variation is essential to understand the role of the dystrophin during compression-extension cycle of the muscle membrane. iii) we evidence that non additive behavior of mixtures of food globular proteins leads to enhanced foaming properties or to self assembled objects. nucleolar-targeting peptides (nrtps) were designed by structural dissection of crotamine, a toxin from the venom of a south-american rattlesnake. at lm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work was to pursue with the study of nrtps molecular mechanism for translocation, as well as to determine the ability of nrtp to delivery large molecules into cells. for the translocation experiments, rhodamine b-labeled nrtps were used and tested with giant multilamellar vesicles. confocal microscopy results show that there is an efficient translocation across model membranes. high levels of intracellular peptide were also seen in different cell lines and pbmc, soon after incubation with nrtp. furthermore, a conjugate of nrtp (nrtp6c) bound to b-galactosidase was prepared by chemical synthesis and tested in hela cells. this conjugate maintains enzymatic activity and is stable at 4°c for several days. the work done so far with this new family of cell-penetrating peptides revealed strong interaction and translocation with lipid model systems. moreover, successfully cellular delivery of bgalactosidase was observed and quantified. interaction of zinc phthalocyanine with ionic and non ionic surfactants: uv-vis absorption and fluorescence spectroscopy for application in photodynamic therapy m. p. romero, s. r. w. louro physic department, pontifícia universidade cató lica de rio de janeiro puc-rio, brazil among the second-generation photosensitizer (ps) developed for the treatment of neoplastic diseases by photodynamic therapy (pdt), metallo-phthalocyanines (mpc) have been proposed as an alternative to the currently used ps in clinical application. unsubstituted mpc are not soluble in physiological solvents and their in vivo administration relies upon their incorporation into carriers or their chemical conversion into water-soluble dyes by the attachment of selected substituents. in this work, uv-vis absorption and fluorescence spectroscopy were used to study the ability of different micelles for dispersing zinc phthalocyanine (znpc). the following surfactants were tested: sds, ctab, hps, tween 80, tween 20, and pluronic f127. znpc has low solubility in virtually all solvents, but dmf and dmso are observed to dissolve znpc in concentrations of the order of 0.9 and 0.2 mmol/l, respectively. stock solutions of znpc in dmf and dmso were prepared. micelles of the different surfactants containing znpc were prepared by dissolving in aqueous medium (milli-q water or phosphate buffer ph 7.4) small amounts of the stock solutions previously mixed with each surfactant. the amounts of each surfactant were calculated to give an average ratio of one znpc molecule per micelle in the final solution. the absorption and fluorescence spectra of znpc in the micellar systems were obtained, and were observed to change in time. immediately after dissolution the spectra are characteristic of monomeric znpc, suggesting formation znpccontaining nanoemulsions with the mixture of znpc-organic solvent in the hydrophobic region of the micelle. since dmso and dmf are miscible with water, the solvent diffuses out of the micelle and znpc stays inside the micelle in a monomeric or aggregated form. the different surfactants lead to different time evolution of znpc aggregation. aggregation lifetimes vary from one hour, in the case of pluronic f127, to more than twelve hours, in the case of ctab and hps. it was observed that the ionic surfactants were more efficient than non ionic ones for monomeric delivery of znpc . work partially supported by cnpq, inami and faperj. nucleobase-containing peptides are an important class of molecules comprising both artificial (synthetic nucleopeptides) and natural (peptidyl nucleosides and willardiine-containing peptides) compounds characterized in many cases by interesting biological properties. 1, 2 in this work, we report a spectroscopic study on the properties of a chiral nucleobase-bearing peptide obtained by chemical synthesis starting from commercial sources. the findings of this research strongly encourage further efforts in the field of the use of nucleopeptides as supramolecular assembling systems and open the way to novel drug delivery approaches based on nucleobase recognition. conformational plasticity. their structure depends tremendously on their local environment and confinment, and may accommodate several unrelated conformations, that are a strong challenge for the traditional characterizations of structure, supramolecular assembly and biorecognition phenomena. atomic force microscopy (afm) has been successfully exploited for both highly controllable nanolithography of biomolecules and for biorecognition studies, such as oriented prion protein -antibody interaction (sanavio et al., acs nano (2010) 4(11): 6607, bano et al. nano lett (2009) 9(7): 2614-8). here, we report different strategies for selective, oriented confinement of alphasynuclein at the nanoscale for sensitive and accurate direct detection, via precise topographic measurements on ultraflat surfaces, of biomolecular interactions in confined assemblies. a new class of cell penetrating peptides (cpps) was generated by splicing the (1-9) and (38-42) segments of crotamine, a toxin from crotalus durissus terrificus venom [1] . as they localize preferably on the nucleolus, these novel cpps were named nucleolar-targeting peptides (nrtps). the extent of nrtp partition to zwitterionic (popc; popc:cholesterol 67:33) and anionic (popg; popc:popg 70:30) lipid vesicles was studied following the intrinsic tyr or trp fluorescence of the peptides. the partition curves into popc zwitterionic vesicles were characterized by downward slopes and higher partition coefficients (k p *10 4 -10 5 ). for pure popg, an upward curve and smaller partition coefficient point out for a different type of membrane-peptide interaction. popc:popg membranes present characteristics of both types of interaction. from red edge excitation shift and quenching experiments similar conclusions were attained. leakage assays ruled out lipid vesicle disruption by crotamine or nrtps. further studies on nrtp cellular translocation mechanism and large molecule delivery are currently in progress. dystrophin is essential to skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeleton and membrane. we characterized the behaviour of dys r11-15, a five spectrin-like repeats from the central domain of human dystrophin, in the presence of liposomes and monolayers as membrane models. interaction of dys r11-15 depends on the lipid nature, anionic or zwitterionic, with suvs, and on the lipid packing when comparing luvs to suvs. lateral pressure of lipid monolayers modifies the protein organization and leads dys r11-15 to form a regular network as revealed by afm. trypsin proteolysis assays show that the protein conformation is modified following its binding to monolayer and suvs. label free quantification by nano-lc/ms-ms allowed identifying the helical amino acid sequences in repeats 12 and 13 that are involved in the interaction with anionic suvs. results indicate that dys r11-15 constitutes a part of dystrophin that interacts with anionic as well as zwitterionic lipids and adapts its interaction and organization depending on lipid-packing and lipid nature. we provide here strong experimental evidence for a physiological role of the central domain of dystrophin on sarcolemma scaffolding through modulation of lipid-protein interactions. extracellular matrix proteins. overexpression of the mmps has been associated with a variety of diseases ranging from periodontal disease and arthritis to tumor invasion and metastasis. the majority of the more powerful synthetic inhibitors of mmps incorporate a hydroxamate group, but exhibit low selectivity and are toxic. in a recent modeling study, astaxanthin (ast), a carotenoid with potent antioxidant property, has been shown to be a potential inhibitor of mmp-13 function by occupying a binding site near the active center of the enzyme (bika 0 di et al. 2006). in our ongoing project, we investigate the binding of ast to the catalytic domain of mmps using biochemical and ultimately crystallization to validate the proposed action of ast. along these lines, the catalytic domain of mmp-13 (cdmmp-13) was expressed in e.coli bl21(de3) codon-plus and refolded using a novel effective refolding method. our results reveal that ast has a potent inhibitory effect on cdmmp-13 activity, however, determination of ic 50% or k i is difficult due to fast oxidation and structural instability of ast. ongoing work aims at optimizing the inhibition conditions and improving the refolding yield to allow analyzing structure and function of the ast-bound mmp-13 in more detail. hyaluronic acid (hyaluronan, ha) is a linear polysaccharide with a molar mass in the range of 10 5 to 10 7 da and is built from alternating units of glucuronic acid and n-acetylglucosamine. synthesized in the cellular plasma membrane, it is a network-forming and space-filling component in the extracellular matrix of animal tissues. here, we create hyaluronic acid films atop a porous alumina substrate, where they act as a barrier for macromolecular transport depending on their length and geometry. the geometry of the hyaluronic acid switches between a fully stretched and a mushroomlike state and is dependent on the concentration of hyaluronic acid. to bind hyaluronic acid selectively atop the nanoporous anodic aluminum oxide (aao), the aao is orthogonally functionalized by silane chemistry. by means of time resolved optical waveguide spectroscopy (ows) the transport of macromolecules, e.g. avidin, across the hyaluronic acid barriers can be recorded as a function of the pore diameter and hyaluronic acid concentration in a time resolved and label free manner. confocal laser scanning microscopy (clsm) provides an alternative method to investigate the orthogonal functionalization of the pores and to elucidate whether a molecule can cross the barrier at the pore entrance. we functionalized gold surfaces with a hydroxy-terminated self-assembled thiol monolayer exposing an adjustable fraction of biotin moieties. [1] by in-situ acetylation or fluorination, surface properties could be fine-tuned to different protein immobilization scenarios. using streptavidin as a linker protein, immobilization of human abcc3 [2] in liposomal and planar bilayer systems was possible. abcc3-containing proteoliposomes doped with a biotinylated anchor lipid were successfully tethered to our streptavidin-coated surfaces. biotinylation of the extracellular glycosylation of abcc3 allowed direct immobilization with inside-up orientation and subsequent assembly of a lipid bilayer. outside-up orientation was achieved by exploiting the c-terminal histidin tag of recombinant abcc3 for immobilization via ni 2+ and biotinnitrilotriacetate. all systems were thoroughly characterized by quartz crystal microbalance, atomic force microscopy and surface plasmon resonance techniques with respect to monitoring abcc3mediated substrate transport in real time. because of its role in the apoptotic pathway, conformational transitions of cytochrome c (cyt c) have gain on interest. in native cyt c, met 80 and his 18 residues serve as heme axial ligands. cyt c interaction with the membrane causes the disruption of the iron-met80 bond. this allows the binding of others endogenous ligands forming alternative low spin species 1,2 or induces peroxidase activity through the formation of a five coordinated high spin iron specie. acquisition of this peroxydase activity by cyt c has been shown to be a key stage before its release from the mitochondria 3 . in order to study these non native low spin species by checking the possible amino acids able to bind the human cyt c heme, different mutants have been designed and produced: h26q, h33n, and the double one h26q/h33n. sds in countries where seafood is an integrate part of the diet, fish are among the most common food allergen sources. the major fish allergen parvalbumins are abundant in the white muscle of many fish species. parvalbumin belongs to the family of ef-hand proteins and has a globular shape containing six helical parts. high pressure is known to unfold proteins. we performed high pressure ftir experiments, to explore the p-t phase diagram of cod parvalbumin, gad m 1, to test the possibility of its inactivation by high pressure treatment. the infrared spectrum of parvalbumin is characteristic for the helical conformation, in agreement with the crystal structure. a marked transition in the structure of the parvalbumin was observed with the central point of 0.5 gpa (at room temperature). the amide i position shifts to a wavenumber which is between the helical and the unfolded position. we assign this change to a native-molten globule transition. it was reversible as seen from the infrared spectra. it has been proven in the past that reflectometric interference spectroscopy (rifs) is a powerful measurement system for the detection of protein-protein interactions 1 . we present here the development of a reflectometric sensor which allows for the detection of membrane-protein interactions in the micromolar regime. in this study we employ two different instrumental assemblies. the first installation enables direct detection and quantification of the interaction of membrane proteins with solid supported lipid bilayers. in the second assembly the original instrument is combined with an upright fluorescence microscope. the advantages of this installation are the direct optical control of the experiment as well as a smaller sensing area. the set-up allows for the detection of interactions on lipid-patches of just several micrometers in diameter. the aim of this work is an experimental system that enables the measurement of transport processes through lipid membranes. we attempt to achieve this by covering a closed porous substrate with a lipid membrane. the first steps to reach this goal were done by spanning membranes over anodized aluminum oxide substrates. initiation of actin polimerization in cells requires nucleation factors. a pointed-end-binding protein of f-actin -the lei-omodin2-acts as a strong filament nucleator in muscle cells. the dynamical, structural and kinetic properties of a protein can provide important information to understand the intramolecular events underlying its function. we are interested in how does the leiomodin2 regulate the actin polimerization. our aim is to determine the dissociation constant of the actin-leiomodin2 complex, and study a possible side-binding effect of the leiomodin2. the cardiac leiomodin2 of rattus norvegicus is a 50 kda molecular weight protein, which contains a 17 kda n-terminal, a 18 kda leucin reach repeat (lrr) and a 15 kda c-terminal region. the n-term and the lrr regions are together tropomodulin homologues. we expressed the wild type the n-term+lrr the lrr+c-term and the c-term protein fragments by using a ptyb1 vector that contains an amplicillin resistance gene. the expression of the proteins was carried out with the twin-cn (ne bio-labs) kit, which is a chitin-intein self-cleavage and purification system. the nucleation activity of leiomodin and the polimerization speed of actin in the presence of tropomyosin and leiomodin were studied by using pyrene-actin polimerization assay. we measured the stoichiometric, conformational and kinetic properties of the leiomodin-actin complexes with co-sedimentation assay, fluorescence spectroscopic and rapid-kinetic methods. the results showed that the rate of actin polimerization depended on the leiomodin2 concentration. the nucleator activity of leiomo-din2 was ionic strength dependent. the data also confirmed that leiomodin2 is a side-binding and pointed-end binding protein of f-actin. the binding of leiomodin2 to the sides of the actin filaments was slower than to the pointed-end of the f-actin. the structure of f-actin was changed by the sidebound leiomodin2. these observations will contribute to the better understanding of the development and function of thin filaments in cardiac and other muscle tissues. leukemias are one of the most common malignancy worldwide. there is a substantial need for new chemotherapeutic drugs effective against these diseases. doxorubicin (dox), used for treatment of leukemias and solid tumors is poorly efficacious when administered systemically at conventional doses. therefore, in our study, to overcome these limitations, we used transferrin (trf) as a drug carrier. we compared the effect of dox and doxorubicin-transferrin conjugate (dox-trf) on human leukemic lymphoblasts (ccrf-cem). the in vitro growth-inhibition test, xtt assay, indicated that dox-trf was more cytotoxic for leukemia cells than dox alone. in our researches we also evaluated the alternations of mitochondrial transmembrane potential (dw m) , and production of reactive oxygen species (ros). we monitored the dw m using dye jc-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine) . the level of ros was studied using the fluorescent probe dcfh 2 -da (2', 7'dichlorodihydrofluorescein diacetate). the results demonstrate that dox-trf induced, decrease of mitochondrial membrane potential and significantly higher production of ros compared with dox treated cells. moreover, all these results seem to be correlated with dna fragmentation analyzed by dna ladder. the tested processes were partially inhibited by the antioxidant, n -acetylocysteine (nac). the changes induced by dox-trf conjugate and free drug were suggest the different mechanism of action of dox alone and conjugated with transferrin. time-resolved detection of protein-protein interaction masahide terazima kyoto university, kyoto, 606-8502, japan revealing molecular mechanism of a protein reaction has been a central issue in biophysics. for that purpose, a variety of time-resolved spectroscopic methods have been developed. however, most of them can monitor only dynamics associated with an optical transition and it has been very difficult to trace processes without optical transition. we used the pulsed laser induced transient grating (tg) method to study spectrally silent reactions of various proteins in time-domain. here we will show studies on pixd. pixd is a 17 kda short protein which consists of the bluf domain and additional short helices, and is involved in phototactic movement. the photochemical reaction studied by absorption spectroscopy revealed that this protein exhibits typical photochemistry of the bluf proteins. the red-shifted intermediate is generated within a 100 ps. the spectrum does not change after this initial reaction, and returns back to the dark state with a time constant of 12 s at room temperature. we studied the reaction of this protein by our method and found that the proteinprotein interaction is drastically changed during the reaction. the details and the biological meaning will be presented. human ileal bile acid-binding protein (i-babp) plays a key role in the enterohepatic circulation of bile salts. previously we have shown that the protein has two binding sites and triand dihydroxy bile salts bind with strong and moderate positive cooperativity, respectively. positive cooperativity is thought to be related to a slow conformational change in the protein. our current study is directed at the structural and dynamic aspects of molecular recognition in human i-babp using nmr spectroscopy and other biophysical techniques. as a first step in the investigation, 15 n relaxation nmr experiments have been employed to characterize the backbone motion in the apo and holo protein on a wide range of timescales. our results show a moderately decreased ps-ns flexibility in the ligated protein, with most significant ordering near the portal region. in addition, the measurements indicate a slow ls-ms fluctuation at four distinct segments in the apo protein, a motion not observed in the doubly-ligated form at room temperature. our studies support the hypothesis of an allosteric mechanism of binding cooperativity in human i-babp. to shed more light on the molecular details of the binding mechanism, a site-directed mutagenesis study is in progress. cationic porphyrin-peptide conjugates were recently shown to enhance the delivery of peptide moiety to the close vicinity of nucleic acids but their interaction with dna is not yet studied. we synthesized two cationic porphyrin-peptide conjugates: tetra-peptides were linked to the tri-cationic meso-tri(4-n-methylpyridyl)-mono-(4-carboxyphenyl)porphyrin and bi-cationic meso-5,10-bis(4-n-methylpyridyl)-15,20di-(4-carboxyphenyl)porphyrin. dna binding of porphyrins, and their peptide conjugates was investigated with comprehensive spectroscopic methods. evidences provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and cd signals reveal that peptide conjugates of di-and tricationic porphyrins bind to dna by two distinct binding modes which can be identified as intercalation and external binding. the peptide moiety does not oppose the interaction between the dna and cationic porphyrins. we compared the effect of complexation on structural stability of dna and nucleoprotein complex : hela nucleosomes and t7 phage. uv and cd melting studies revealed that the porphyrin binding increases the melting temperature of dna and destabilizes the dna protein interaction in the nucleosomes but not in the t7 phage. the wide nucleotide specificity of 3-phosphoglycerate kinase (pgk) allows its contribution to the effective phosphorylation (activation) of nucleotide-based pro-drugs against hiv. here the structural basis of the nucleotide-pgk interaction is characterised in comparison to other kinases, namely pyruvate kinase (pk) and creatine kinase (ck) by enzyme kinetic and structural modelling studies. the results evidenced favouring the purine vs. pyrimidine base containing nucleotides for pgk rather than for pk or ck. this is due to the exceptional ability of pgk in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate chain for catalysis. the unnatural l-configurations of the nucleotides (both purine and pyrimidine) are better accepted by pgk than either by pk or ck. further, for the l-forms the absence of the ribose oh-groups with pgk is better tolerated for the nucleotides with purine rather than pyrimidine base. on the other hand, positioning the phosphate chain of both purines and pyrimidines with l-configuration is even more important for pgk, as deduced from the kinetic studies with various nucleotide-site mutants. these characteristics of the kinase-nucleotide interactions can provide a guideline in drug-design. the role of the enzyme types atp-ases in the muscle contraction g. vincze-tiszay 1 , j. vincze 1 , e. balogh 2 1 hheif, budapest, hungary, 2 nové zá mky, slovakia the myofibrilla assuring muscle contraction gains energy to the slipping in mechanisms and the degree of efficiency of this process will decisively be determined by the velocity of recombination of the atp molecule. in this there play a particular part the na + -k + -atp-ase and mg ++ -atp-ase enzymes. chemical reactions taking place in the living organism are catalyzed by enzymes, so the recombination from adp to atp, too. this transport process can be modelled from the energetic point of view on the basis of the general transport theorem through the following formula: grad a x dx dt: from the point of view of muscle contraction it is of interest that, dependent from the type of the motions whether the length of time is very short, some seconds, or we can speak about a long lasting process. in the first case one can compare the decomposition of the atp with the avalanche effect while in the spot. its degree of efficiency is determined by the migration and linkage velocity of the ions. conclusion: the degree of efficiency of the muscle contraction is determined by the quantities of the two enzymes (na + -k + -atp-ase and mg ++ -atp-ase) as related to each other. [1] . experiments with deletion mutants have shown that the aminoterminal domain contains a beta sheet with an ordered array of acidic residues, which mediates the attachment to basic calcium phosphates [2, 3] . the inhibition is based on the formation of nanometer-sized, spherical mineral-fetuin-a colloids, denoted as calciprotein particles (cpps) [2, 4] . the initially formed cpps show hydrodynamic radii in the range of 50 nm and are only transiently stable. after a distinct lag time, they are subject to a morphological change towards larger prolate ellipsoids with hydrodynamic radii of 100-120 nm [5] . in this context, we studied the role of fetuin-a in the formation and ripening of cpps. on the one hand, dynamic light scattering (dls) was used to study the influence of temperature, fetuin-a concentration and mineral ion supersaturation on the kinetics of cpp ripening [6] . on the other hand, the protein fetuin-a was investigated by means of small angle x-ray scattering (saxs) and fluorescence correlation spectroscopy (fcs degradation of the mrna cap (mrna 5' end) by dcps (decapping scavenger) enzyme is an important process of the gene expression regulation, but little is known about its mechanism. the biological role of dcps and its potential therapeutic applications, e.g. as a novel therapeutic target for spinal muscular atrophy, make it an interesting object for biophysical investigations. the ability of dcps to act on various short capped mrnas will be presented. we have examined the substrate specificity and binding affinity of the enzyme in a quantitative manner, employing experimental physics' resources, such as atomic force microscopy (afm) and fluorescence spectroscopy for enzyme kinetics and timesynchronized-titration method (tst 2007) . in this study we extended the application of mqd-ihc to investigate potential biomarkers associated with prostate cancer (pca) invasiveness and lethal progression. objectives: to establish a mqd-ihc protocol using qd light-emitting nanoparticles 1) to detect the expression/activation of critical cell signaling proteins at the single cell level; 2) to image the plasticity and lethal progression of human pca with specific emphasis on emt and c-met signaling; and 3) to examine the utility of mqd-ihc in clinical pca specimens to determine its invasion ability and predict its metastatic capability. results: we analyzed the co-expression and activation of osteomimicry associated biomarkers: b2-microlgobulin (b2-m), phosphorylated cyclic amp responsive element binding protein (pcreb) and androgen receptor (ar) in 2,100 cells from 14 localized pca tissue areas (gleason 3 and 4) of 10 patients with known metastatic status. the overall median % triple positive for b2-m + /pcreb + /ar + cells was 51.5%. the median triple positive for the samples with metastatic potential was 61% compared with those without metastatic potential (median = 0%); p = 0.01 by a wilcoxon rank sum test. the results were confirmed in 11 pca bone metastatic specimens. we also investigated the c-met signaling in castration-resistant human pca model or crpc xenografts and the clinical pca specimens and found that the downstream signal components including pakt and mcl-1 were activated. conclusion: to validate our findings, additional clinical specimens with confirmed survival data will be analyzed and the cell-signaling-network-based mqd-ihc will be automated by vectra image analysis system in a high throughput manner with the hope to predict the lethal progression of pca prior to clinical manifestation of distant metastases. protein ligand binding is an important field of biopharmaceutical research. there are many techniques for quantitative determination of the ligand binding. the combination of isothermal titration calorimetry (itc) and thermal shift assay provides a robust estimate of the binding constant. many binding reactions are coupled to the absorption or release of protons by the protein or the ligand, conformational changes of the protein and other processes. to correlate the structural features of binding with the energetics of binding one needs to carry out a detailed thermodynamic study of the binding reaction and to determine dependencies such as ph, buffer and temperature. here we present a detailed thermodynamic description of radicicol binding to human heat shock protein hsp90 and determined proton linkage contributions to observed binding thermodynamics. we calculated the pk a of the group responsible for proton linkage, the protonation enthalpy of this group and intrinsic thermodynamic parameters for radicicol binding. the intrinsic enthalpy of radicicol binding to hsp90 is one of the largest enthalpies observed for any protein -ligand binding. the structural features responsible for such large binding enthalpy and very favorable intrinsic binding gibbs free energy are discussed. neuronal systems and modelling o-111 optogenetic electrophysiology walther akemann, amelie perron, hiroki mutoh, and thomas knö pfel laboratory for neuronal circuit dynamics, riken brain science institute, japan the combination of optical imaging methods with targeted expression of protein-based fluorescent probes enables the functional analysis of selected cell populations within intact neuronal circuitries. we previously demonstrated optogenetic monitoring of electrical activity in isolated cells, brain slices and living animals using voltage-sensitive fluorescent proteins (vsfps) generated by fusing fluorescent proteins with a membrane-integrated voltage sensor domain. however, several properties of these voltage reporters remained suboptimal, limiting the spatiotemporal resolution of vsfpbased voltage imaging. a major limitation of vsfps had been a reduced signal-to-noise ratio arising from intracellular aggregation and poor membrane targeting upon long-term expression in vivo. to address this limitation, we generated a series of enhanced genetically-encoded sensors for membrane voltage (named vsfp-butterflies) based on a novel molecular design that combines the advantageous features of vsfp2s and vsfp3s with molecular trafficking strategies. the new sensors exhibit faster response kinetics at subthreshold membrane potentials and enhanced localization to neuronal plasma membranes after long-term expression in vivo, enabling the optical recording of action potentials from individual neurons in single sweeps. vsfp-butterflies provide optical readouts of population activity such as sensoryevoked responses and neocortical slow-wave oscillations with signal amplitudes exceeding 1% dr/r 0 in anesthetized mice. vsfp-butterflies will empower optogenetic electrophysiology by enabling new type of experiments bridging cellular and systems neuroscience and illuminating the function of neural circuits across multiple scales. opsin molecules are a burgeoning new tool for temporally precise neuronal stimulation or inhibition. opsin properties are commonly characterized in cell culture or acute brain slice preparations using whole cell patch clamp techniques, where neuronal membrane voltage is fixed at the resting potential. however, in vivo, where neurons are firing action potentials, opsins are exposed to large fluctuations in membrane voltage and transmembrane ionic concentrations which can influence opsin function. in the case of implanted light delivery devices, stimulation light power varies as a function of brain tissue volume. we therefore investigated the stability of opsin properties across a variety of in vivo-like stimulation conditions. we find that off-kinetics of excitatory opsins vary significantly with holding membrane potential; channelrhodopsin (chr2) slowing with depolarisation and chief (chr1/chr2 hybrid) in contrast, accelerating. new chr2 point mutation variants demonstrate stability across all membrane potentials. we additionally explore responses to initial and subsequent light pulses and find that chief has the unique property of accelerating kinetics after the first light stimulation. inhibitory opsins vary in sensitivity to light in a manner which correlates with their off-kinetics. slower opsins, such as mac (leptosphaeria maculans), have higher sensitivity at low light power densities, saturating early relative to fast inhibitory opsins such as arch (archaeorhodopsin) and nphr (halorhodopsin). we discuss the relative merits of stability versus versatility of opsins under variable stimulation conditions. it has been previously shown that overexpression of ndm29 ncrna in a sknbe2-derived neuroblastoma (nb) cell line leads to cell differentiation, with a decrease of malignant potential. here we use the patch-recording technique to characterize the ionic channel apparatus of nb cells expressing ndm29 at its basal level (mock cells) or at 5.4 fold higher levels (s1 cells). the two cell lines shared very similar pools of functional k channels, but s1 cells displayed larger ttx-sensitive na currents and were able to generate action potentials, while mock cells were not. in addition, while mock cells barely express functional gaba receptors, in the majority of s1 rapid application of gaba elicited a current with a ec 50 =11.4 lm; this current was antagonized by bicuculline (10 lm) and potentiated by zaleplon (ec 50 = 35 nm). in mock cells, real time pcr evidenced a high level of gaba a a 3 subunit, while in s1 cells a significant expression of a 1 and a 4 was detected, whereas a 3 mrna was downregulated by 70%, confirming the development of functional gaba a receptors. in the same cell lines, the presence of specific markers and the secretion of specific cytokines confirmed that ndm29 expression leads to a differentiation process toward a neuron-like, rather than glial-like, phenotype. was planned therefore to reconstitute a model of brain tumors in rats by orthotopic implantation of xenogenic transformed human cells. iron is important element used for chemical reaction catalysis and physiological cell functions. the reason of iron deposition is still unknown. under conditions prevailing in human brain it is expected the formation of an amorphous or minute crystalline phase. we used light, scanning (sem) and transmission electron microscopy (tem), energy-dispersive microanalysis, electron diffraction and electron paramagnetic resonance (epr) for investigation of iron deposits in globus pallidus of human brain. sem revealed iron rich particles with na, si, p, s, cl, ca and cu around glial cells. tem revealed bumpy, solid particles of platy and sometimes rounded shape with the size of 2 lm to 6 lm. these ones were identified as hematite. epr measurements showed the presence of fe(iii) and cu(ii), but little amount of fe(ii) can not be excluded. we consider low-temperature process of hematite formation in human globus pallidus in aqueous environment influenced by organic and inorganic factors. chemical processes leading to nanoparticles formation can be associated with neurodegenerations such as alzheimer or parkinson disease. over the past 50 years our understanding of the basic biophysical mechanisms governing the spatio-temporal dynamics of neuronal membrane potentials and synaptic efficacies has significantly expanded and improved. much research has focussed on how ionic currents contribute to the generation and propagation of action potentials, how subthreshold signals propagate along dendritic trees, how the active properties of dendrites shape the integration of incoming signals in a neuron, and how pre-and postsynaptic activities â and potential heterosynaptic effects a determine the way synaptic efficacies change on the short-and long-term. yet, despite these advances, there have been no systematic efforts to relate the basic dynamical repertoire of neurons to the computational challenges neural circuits face, and in particular to explain systematically how the biophysical properties of neurons are adapted to process information efficiently under the constraints of noise and uncertainty in the nervous system. as an initial step in this direction, i will show how various biophysical properties of neurons, in particular short-term synaptic plasticity and dendritic non-linearities, can be seen as adaptations to resolve an important bottleneck in neuronal information processing: the loss of information entailed by the conversion of analogue membrane potentials to digital spike trains. the optogenetic toolbox has greatly expanded since the first demonstration of genetically-targeted optical manipulation of neural activity. in addition to the cation channel channelrhodopsin-2 (chr2), the panel of excitatory opsins now includes an array of chr2 variants with mutations in critical residues, in addition to other, related cation channels, and channel hybrids. the inhibitory opsin panel has similarly expanded beyond the first-described halorhodopsin (nphr), a chloride pump, to include trafficking-enhanced versions of nphr as well as the proton pumps mac and arch. while the expansion of available opsins offers researchers an increasingly powerful and diverse selection of tools, it has also made it increasingly difficult to select the optimal tool for a given experiment. one cannot extract a comparison of opsins from the current literature, since studies differ across multiple variables known to contribute to opsin performance (e.g. expression method, light power density, stimulation protocols, etc.). here, we provide the first empirical comparison of both excitatory and inhibitory opsins under standardized conditions. furthermore, we identify the set of parameters that describe the properties of an opsin in a way that is maximally relevant for biological application. o-120 subcellular compartment-specific distribution of voltage-gated ion channels zoltan nusser institute of experimental medicine, hungarian academy of sciences, budapest, hungary voltage-gated na + (nav) channels are essential for generating the output signal of nerve cells, the action potential (ap). in most nerve cells, aps are initiated in the axon initial segment (ais). in vitro electrophysiological and imaging studies have demonstrated that dendritic nav channels support active backpropagation of aps into the dendrites, but the subunit composition of these channels remained elusive. here, i will present evidence for the somato-dendritic location of nav channels in hippocampal pyramidal cells (pcs). using a highly sensitive electron microscopic immunogold localization technique, we revealed the presence of the nav1.6 subunit in pc proximal and distal dendrites, where their density is 40-fold lower than that found in aiss. a gradual decrease in nav1.6 density along the proximo-distal axis of the dendritic tree was also detected. we have also investigated the subcellular distribution of kv4.2 voltage-gated k + channel subunit and found a somato-dendritic localization. in contrast to that of nav1.6 channels, the density of kv4.2 first increases then decreases as a function of distance from the somata of pcs. such subcellular compartment-specific distribution of voltage-gated ion channels increases the computational power of nerve cells. keywords: memory, extra cellular matrix, random walk we expose first a biological model of memory based on one hand of the mechanical oscillations of axons during action potential and on the other hand on the changes in the extra cellular matrix composition when a mechanical strain is applied on it. due to these changes, the stiffness of the extra cellular matrix along the most excited neurons will increase close to these neurons due to the growth of astrocytes around them and to the elastoplastic behavior of collagen. this will create preferential paths linked to a memory effect. in a second part, we expose a physical model based on random walk of the action potential on the array composed of dendrites and axons. this last model shows that repetition of the same event leads to long time memory of this event and that paradoxical sleep leads to the linking of different events put into memory. myelinated nerve fibres were studied with fluorescent microscopy and laser interference microscopy. ca 2+ redistribution during prolonged stimulation, changes in morphology and rearrangement of cytoplasmic structures were compared in normal conditions and after membrane modification by lysolecithin and methyl-b-cyclodextrin. lysolecithin is a detergent known to provoke demyelination, and methyl-b-cyclodextrin extracts cholesterol from membranes. cholesterol extraction could lead to disruption of membrane caveolae-like microdomains or ''rafts'' and solubilisation of different proteins connected to them. our data suggest that methyl-b-cyclodextrin and lysolecithin lead to different changes in morphology and distribution of cytoplasmic structures. the effect was different for different regions of the nerve (node of ranvier, paranodal and internodal regions). the agents also altered the kinetics of ca 2+ response to stimulation in myelinated fibres. extracellular carbonic anhydrase contributes to the regulation of ca2+ homeostasis and salivation in submandibular salivary gland nataliya fedirko, olga kopach, nana, voitenko lviv national university, human and animal physiology, lviv, ukraine the maintenance of ph in the oral cavity is important for the oral heath since even a minor drop in ph can result in dental caries and damage to the teeth. submandibular salivary gland (smg) is main source of fluid and electrolytes enriched saliva therefore its core for oral ph homeostasis. smg secretion is activated by acetylcholine (ach) in [ca 2+ ] idependend manner and accompanied with oral ph acidic shifts. ph shifts could be due to changes in buffering capacity that is regulated by carbonic anhydrase (ca). despite the expression of different subtypes of ca in smg the role of ca in the regulation of smg function is unclear yet. we found that ca inhibition by benzolamide (bz) decreased of fluid secretion in vivo extracellular na 2+ concentration in situ. the latter confirm the ability of ca to modify both primarily and final saliva secretion. we also found correlation between the secretion and ca 2+ -homeostasis since bz-induced decrease of: in striated muscle ca 2+ release from the sarcoplasmic reticulum (sr) occurs when ryanodine receptors (ryr-s) open either spontaneously or upon the stimulation from dihydropyridine receptors that are located in the adjacent transverse-tubular membrane and change their conformation when the cell is depolarized. recent observations demonstrated that muscles from animal models of ptdinsp phosphatase deficiency suffer from altered ca 2+ homeostasis and excitation-contraction coupling, raising the possibility that ptdinsp-s could modulate voltage-activated sr ca 2+ release in mammalian muscle. the openings of a single or a cluster of ryr-s can be detected as ca 2+ release events on images recorded from fibres loaded with fluorescent ca 2+ indicators. to elucidate the effects of ptdinsp-s on ca 2+ release events, images were recorded from skeletal muscle fibers enzymatically isolated from the m. flexor digitorum breavis of mice utilizing a super-fast scanning technique. a wavelet-based detection method was used to automatically identify the events on the images. three different ptdinsp-s (ptdins3p, ptdins5p, and ptdins(3,5)p) were tested. all these ptdinsp compounds decreased the frequency of spontaneous ca 2+ release events. supported by the hungarian national science fund (otka 75604), té t. calcium sparks elicited by 1 mmol/l caffeine and by a depolarization to -60 mv were recorded at high time resolution on both x-y (30 frames/s) and line-scan images (65 lines/ ms) on intact skeletal muscle fibers of the frog. while a typical spark appeared in one frame only, 17.3 and 26.0% of spark positions overlapped on consecutive frames following caffeine treatment or depolarization, respectively. while both caffeine and depolarization increased the frequency of sparks, as estimated from x-y images, the morphology of sparks was different under the two conditions. both the amplitude (in df/f 0 ; 0.49 ± 0.025 vs. 0.29 ± 0.001; n = 22426 vs. 23714; mean ± sem, p \ 0.05) and the full width at half maximum (in lm; parallel with fiber axis: 2.33 ± 0.002 vs. 2.21 ± 0.005; perpendicular to fiber axis: 2.07 ± 0.003 vs. 1.88 ± 0.004) of sparks was significantly greater after caffeine treatment than on depolarized cells. these observations were confirmed on sparks identified in line-scan images. in addition, x-t images were used to analyze the time course of these events. calcium sparks had significantly slower rising phase under both conditions as compared to the control. on the other hand, while the rate of rise of signal mass was decreased after depolarization, it increased in the presence of caffeine. prolonged depolarisation of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. skeletal muscle excitation-coupled calcium entry relies on the interaction between the 1,4 dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. in this study we exploited tirf microscopy to monitor with high spatial resolution excitationcoupled calcium entry (ecce) in primary coltures of human skeletal muscle cells harbouring mutation in the ryr1 gene linked to malignant hyperthermia and central core disease. we found that excitation-coupled calcium entry is strongly enhanced in cells from patients with central core disease compared to individuals with malignant hyperthermia and controls. in addition, excitation-coupled calcium entry induces generation of reactive nitrogen species and causes the nuclear translocation of nfatc1. the activation of nfatc1 dependent genes is consistent with an increase of the il6 secretion from primary coltures human myotubes from ccd patients and with fibre type 1 predominance of skeletal muscle of ccd patients. membrane lipids, microdomains & signalling p-132 ftir and calorimetric investigation of the effects of trehalose and multivalent cations on lipid structure sawsan abu sharkh, jana oertel, and karim fahmy division of biophysics, institute of radiochemistry, helmholtz-zentrum dresden-rossendorf, germany e-mail: s.sharkh@hzdr.de the structure of membrane lipids is of fundamental importance for the integrity of cell and organelle membranes in living organisms. membrane lipids are typically hydrated and their headgroup charges counter-balanced by solvated ions. consequently, water loss can induce severe structural changes in lipid packing (lyotropic transitions) and can lead to the damage of lipid membranes even after rehydration. this can be one out of several factors that affect the viability of organisms undergoing desiccation. many organisms, however, are resistant to even extreme water loss. some of them synthesize trehalose which has been shown to be associated with survival of desiccation in phylogenetically diverse organisms (yeast, nematodes, brine shrimp, insect larvae, resurrection plants, and others). here we have studied hydration sensitive transitions in model lipids to determine the effect of trehalose and electrostatics on lipid order. hydration pulse-induced time-resolved fourier-transform infrared (ftir) difference spectroscopy was used to address hydration-dependent lipid structure as a function of trehalose. in combination with differential scanning calorimetry and studies of langmuir-blodget films we arrive at a structural and energetically consistent picture of how trehalose can affects lipidic phase behaviour and support a native lipid structure under water loss. experiments were performed on model lipids with different headgroups and native lipids from desiccation-tolerant organisms. controlled self-assembly and membrane organization of lipophilic nucleosides and nucleic acids: perspectives for applications martin loew 1 , paula pescador 3 , matthias schade 1 , julian appelfeller 1 , jü rgen liebscher 2 , oliver seitz 2 , daniel huster 3 , andreas herrmann 1 , and anna arbuzova 1 1 humboldt universitä t zu berlin, institute of biology/ biophysics, berlin, germany, 2 humboldt universitä t zu berlin, institute of chemistry, berlin, germany, 3 universitä t leipzig, department of medical physics and biophysics, leipzig, germany lipophilic conjugates of nucleosides and nucleic acids such as dna, rna, and peptide nucleic acid (pna) -combining assembly properties of amphiphiles and specific molecular recognition properties of nucleic acids -allow numerous applications in medicine and biotechnology. we recently observed self-assembly of microtubes, stable cylindrical structures with outer diameters of 300 nm and 2-3 lm and a length of 20-40 lm, from a cholesterol-modified nucleoside and phospholipids. morphology and properties of these microtubes and functionalization with lipophilic dna will be characterized. we also observed that lipophilic nucleic acids, pna and dna differing in their lipophilic moieties, partition into different lipid domains in model and biological membranes as visualized by hybridization with respective complementary fluorescently-labeled dna strands. upon heating, domains vanished and both lipophilic nucleic acid structures intermixed with each other. reformation of the lipid domains by cooling led again to separation of membrane-anchored nucleic acids. by linking specific functions to complementary strands, this approach offers a reversible tool for triggering interactions among the structures and for the arrangement of reactions and signaling cascades on biomimetic surfaces. conformational dependent trafficking of p-glycoprotein with rafts zsuzsanna gutayné tó th, orsolya bá rsony, katalin goda, gá bor szabó and zsolt bacsó university of debrecen, mhsc, department of biophysics and cell biology, debrecen, hungary p-glycoprotein (pgp), an abc-transporter playing a prominent role in multidrug resistance, demonstrate conformationdependent endocytosis on the surface of 3t3-mdr1 cells. these cell surface transporters have a uic2 conformationsensitive-antibody-recognizable subpopulation, which is about one-third of the rest persisting long on the cell surface and perform fast internalization via rafts. we have identified that the rapid internalization is followed by quick exocytosis, in which the other subpopulation is not or only slightly involved. the exocytosis presents a cholesterol depletion dependent intensification, in contrast to the internalization, which is inhibited by cyclodextrin treatment. this continuous recycling examined by total internal reflection (tirf) microscopy increases the amount of the raft associated subpopulation of pgps in the plasma membrane, and it might have a role in restoring the cholesterol content of the membrane after cholesterol depletion. our presentation will summarize related endocytotic, exocytotic and recycling processes and that how does our data fit into our current notions regarding to the cholesterol and sphingomyelin trafficking. membrane nanodomains based on phase-segregating lipid mixtures have been emerged as a key organizing principle of the plasma membrane. they have been shown to play important roles in signal transduction and membrane trafficking. we have developed lipid-like probes carrying multivalent nitrilotriacetic acid (tris-nta) head groups for selective targeting of histagged proteins into liquid ordered or liquid disordered phases. in giant unilamellar vesicles strong partitioning of tris-nta lipids into different lipid phases was observed. for a saturated tris-nta lipid, at least 10-fold preference for the liquid ordered phase was found. in contrast, an unsaturated nta lipid shows a comparable preference for the liquid disordered phase. partitioning into submicroscopic membrane domains formed in solid supported membranes was confirmed by superresolution imaging. single molecule tracking of his-tagged proteins tethered to solid supported membranes revealed clear differences in the diffusion behavior of the different nta-lipids. by using bsa as a carrier, multivalent nta lipids were efficiently incorporated into the plasma membrane of live cells. based on this approach, we established versatile methods for probing and manipulating the spatiotemporal dynamics of membrane nano domains in live cells. il-9 is a multifunctional cytokine with pleiotropic effects on t cells. the il-9r consists of the cytokine-specific a-subunit and the c c -chain shared with other cytokines, including il-2 and -15, important regulators of t cells. we have previously shown the preassembly of the heterotrimeric il-2 and il-15r, as well as their participation in common superclusters with mhc glycoproteins in lipid rafts of human t lymphoma cells. integrity of lipid rafts was shown to be important in il-2 signaling. we could hypothesize that other members of the c c cytokine receptor family, such as the il-9r complex, may also fulfill their tasks in a similar environment, maybe in the same superclusters. co-localization of il-9r with lipid rafts as well as with the il-2r/mhc superclusters was determined by clsm. molecular scale interactions of il-9ra with il-2r and mhc molecules were determined by microscopic and flow cytometric fret experiments. the role of lipid rafts in il-9r signaling was assessed by following the effect of cholesterol depletion on il-9 induced stat phosphorylation. our results suggest the possibility that preassembly of the receptor complexes in common membrane microdomains with mhc glycoproteins may be a general property of c c cytokines in t cells. to unravel the molecular processes leading to fas clustering in lipid rafts, a 21-mer peptide corresponding to the single transmembrane domain of the death receptor was reconstituted into model membranes that display liquid-disordered/ liquid-ordered phase coexistence, i.e. mimicking cells plasma membranes. using the intrinsic fluorescence of the peptide two tryptophans residues (trp 176 and trp 189 ), fas membrane lateral organization, conformation and dynamics was studied by steady-state and time-resolved fluorescence techniques. our results show that the fas has preferential localization to liquid disordered membrane regions, and that it undergoes a conformational change from a bilayer inserted state in liquid-disordered membranes to an interfacial state in liquidordered membranes. this is a result of the strong hydrophobic mismatch between the (hydrophobic) peptide length and the hydrophobic thickness of liquid-ordered membranes. in addition, we show that ceramide, a sphingolipid intimately involved in fas oligomerization and apoptosis triggering, does not affect fas membrane organization. overall, our results highlight ceramide role as an enhancer of fas oligomerization, and unravel the protective function of fas transmembrane domain against non-ligand induced fas apoptosis. organization and dynamics of membrane-bound bovine a-lactalbumin: a fluorescence approach arunima chaudhuri and amitabha chattopadhyay centre for cellular and molecular biology, hyderabad 500 007, india e-mail: amit@ccmb.res.in many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. interestingly, apo-bovine alactalbumin (bla), a soluble protein, specifically interacts with negatively charged membranes and the membranebound protein exhibits a molten globule conformation. we have used the wavelength-selective fluorescence approach to monitor the molten globule conformation of bla upon binding to negatively charged membranes as compared to zwitterionic membranes. tryptophans in bla exhibit differential red edge excitation shift (rees) upon binding to negatively charged and zwitterionic membranes, implying differential rates of solvent relaxation around the tryptophan residues. our results utilizing fluorescence anisotropy, lifetime and depth analysis by the parallax approach of the tryptophans further support the differential organization and dynamics of the membrane-bound bla forms. in addition, dipole potential measurements and dye leakage assays are being used in our ongoing experiments to explore the mechanism of bla binding to membranes. these results assume significance in the light of antimicrobial and tumoricidal functions of a-lactalbumin. role of long-range effective protein-protein forces in the formation and stability of membrane protein nano-domains nicolas destainvill laboratoire de physique thé orique, université paul sabatier toulouse 3 -cnrs, toulouse, france we discuss a realistic scenario, accounting for the existence of sub-micro-metric protein domains in plasma membranes. we propose that proteins embedded in bio-membranes can spontaneously self-organize into stable small clusters, due to the competition between short-range attractive and intermediate-range repulsive forces between proteins, specific to these systems. in addition, membrane domains are supposedly specialized, in order to perform a determined biological task, in the sense that they gather one or a few protein species out of the hundreds of different ones that a cell membrane may contain. by analyzing the balance between mixing entropy and protein affinities, we propose that protein sorting in distinct domains, leading to domain specialization, can be explained without appealing to preexisting lipidic micro-phase separations, as in the lipid raft scenario. we show that the proposed scenario is compatible with known physical interactions between membrane proteins, even if thousands of different species coexist. lipid rafts are cholesterol and sphingolipid-enriched functional microdomains present in biomembranes. rafts have been operationally defined as membrane fractions that are detergent insoluble at low temperature. here we have characterized drms from erythrocytes treated with the nonionic detergents brij58 and brij98, at 4°c and 37°c, and compared them to drms obtained with triton x-100 (tx100). we have also investigated the effect of cholesterol depletion in drms formation. brij drms were enriched in cholesterol as well as tx100 drms. hptlc analysis showed a very similar distribution of phosphatidylcholine-pc, phosphatidylethanolamine-pe and sphingomyelin-sm in brij drms to that found in ghost membranes. sm-enriched drms were obtained only with tx100 while pe content was decreased in tx100 drms, in comparison to brij drms. immunoblot essays revealed that rafts markers (flotillin-2 and stomatin) were present in all drms. contrary to tx100 drms, analysis of electron paramagnetic resonance spectra (with 5-doxyl stearate spin label) revealed that brij drms are not in the liquid-ordered state, evincing the differential extraction of membrane lipids promoted by these detergents. supported by fapesp/cnpq (brazil). several biological membrane mimics have been built to investigate the topology of molecules in membranes. among them ''bicelles'', i.e., mixtures of long-chain and short-chain saturated phospholipids hydrated up to 98%, became very popular because they orient spontaneously in magnetic fields. disk-shaped systems of 40-80 nm diameter and 4-5 nm thickness have been measured by electron microscopy and solid state nmr and can be oriented by magnetic fields with the disc-plane normal perpendicular to the field. we have been developing recently lipids that contain in one of their chains a biphenyl group (tbbpc) affording an orientation parallel to the magnetic field, in the absence of lanthanides. a large number of hydrophobic molecules including membrane proteins have been successfully embedded and static nmr afforded finding the orientation of protein helices in membranes; mas nmr provided the 3d structure of peptides in bicelles. biphenyl bicelles keep their macroscopic orientation for days outside the field, thus leading to combined nmr and x-rays experiments. tbbpc allows also construction of lm vesicles showing a remarkable oblate deformation in magnetic fields (anisotropy of 3-10) and opens the way to applications for structural biology or drug delivery under mri. imaging membrane heterogeneities and domains by super-resolution sted nanoscopy christian eggeling, veronika mueller, alf honigmann, stefan w. hell department of nanobiophotonics, max planck institute for biophysical chemistry, am fassberg 11, 37077 gö ttingen, germany cholesterol-assisted lipid and protein interactions such as the integration into lipid nanodomains are considered to play a functional part in a whole range of membrane-associated processes, but their direct and non-invasive observation in living cells is impeded by the resolution limit of [200nm of a conventional far-field optical microscope. we report the detection of the membrane heterogeneities in nanosized areas in the plasma membrane of living cells using the superior spatial resolution of stimulated emission depletion (sted) far-field nanoscopy. by combining a (tunable) resolution of down to 30 nm with tools such as fluorescence correlation spectroscopy (fcs), we obtain new details of molecular membrane dynamics. sphingolipids or other proteins are transiently (* 10 ms) trapped on the nanoscale in cholesterol-mediated molecular complexes, while others diffuse freely or show a kind of hopping diffusion. the results are compared to sted experiments on model membranes, which highlight potential influences of the fluorescent tag. the novel observations shed new light on the role of lipid-protein interactions and nanodomains for membrane bioactivity. ca2+ controlled all-or-none like recruitment of synaptotagmin-1 c2ab to membranes sune m. christensen, nicky ehrlich, dimitrios stamou bio-nanotechnology laboratory, department of neuroscience and pharmacology, nano-science center, lundbeck foundation center biomembranes in nanomedicine, university of copenhagen, 2100 copenhagen, denmark e-mail: ehrlich@nano.ku.dk & stamou@nano.ku.dk synaptotagmin-1 (syt) is the major ca2+ sensor that triggers the fast, synchronous fusion of synaptic vesicle with the presynaptic membrane upon ca2+-mediated membrane recruitment of the cytosolic c2ab domain. the ca2+-dependent recruitment of syts c2ab domain to membranes has so far been investigated by ensemble assays. here we revisited binding of wild type c2ab and different c2ab mutants of syt to lipid membranes using a recently developed single vesicle assay. the hallmark of the single vesicle approach is that it provides unique information on heterogeneous properties that would otherwise be hidden due to ensemble averaging. we found that c2ab does not bind to all vesicles in a homogenous manner, but in an all-or-none like fashion to a fraction of the vesicles. the fraction of vesicles with bound c2ab is regulated by the amount of negatively charged lipids in the membrane as well as by [ca2+] . this ca2+ controlled all-ornone like recruitment of syt to membranes provides a possible explanation for the strongly heterogeneous behavior of the in vitro model system for neuronal membrane fusion. furthermore, heterogeneity in release probability among synaptic vesicles is a critical property in determining the output of a neuronal signaling event. new insights in the transport mechanism of ciprofloxacin revealed by fluorescence spectroscopy mariana ferreira, sílvia c. lopes, paula gameiro requimte, faculdade de ciê ncias da universidade do porto, porto, portugal keywords: fluoroquinolones; liposomes; proteoliposomes; ompf; ciprofloxacin; fluorescence; anisotropy. fluoroquinolones are antibiotics that have a large spectrum of action against gram negative and some gram positive bacteria. the interaction between these species and liposomes has been cited as a reference in the understanding of their diffusion through phospholipid bilayer and can be quantified by the determination of partition coefficients between a hydrophobic phase (liposomes) and an aqueous solution. it is also known that some porins of the bacterial membranes are involved in transport mechanism of many fluoroquinolones. ompf, a well characterized membrane protein characteristic of the outer membrane of gram negative bacteria assumes the conformation of homo-trimer, whose monomers have two tryptophan residues (one located at the interface of monomers and the other at the interface lipid/protein). thus, we proceeded to study the interaction of ciprofloxacin, a second generation fluoroquinolone, with unilamellar liposomal vesicles and ompf proteoliposomes of pope/popg, pope/popg/cardiolipin and e. coli total. partition coefficients (kp's) and the association with ompf proteoliposomes were determined by steady state fluorescence spectroscopy under physiological conditions (t=37°c; ph 7.4). the membrane mimetic systems used were characterized by dls and fluorescence anisotropy. motivation is whether there exist differences in the patternforming capabilities of two adhesion molecules of different roles: cd44, mediating ,,dynamic'' adhesion in cell rolling and icam-1, mediating ,,static'' adhesion during the formation of immune-synapse. homo-and hetero-associations of cd44, icam-1 and the mhci is investigated on the nm-and lmdistance levels on ls174t colon carcinoma cells in two different conditions of lymphocyte homing: (1) with ifnc and tnfa, both lymphokines up-regulating the expression level of mhci and icam-1 and down-regulating that of cd44. (2) crosslinking of cd44 and icam-1 representing receptor engagement. the observations are explained by assuming the existence of a kinase cascade-level crosstalk between the cd44 and icam-1 molecules which manifests in characteristic complementary changes in the properties of cell surface receptor patterns. for the characterisation of cluster morphology new colocalization approaches were developed: (i) ,,number of first neighbours'' distribution curves, (ii) ,,acceptor photobleaching fret-fluorescence intensity fluctuation product'' correlation diagrams, and (iii) ,,random gradient-kernel smoothing assisted decay'' of pearson-correlations, and (iv) k-function formalism. analyzing janus kinases in living cells with single-color fcs using confocal and tir-illumination thomas weidemann 1 , hetvi gandhi 1 , remigiusz worch 1,3 , robert weinmeister 1 , christian bö kel 2 , and petra schwille 1 1 biophysics research group, technische universitä t dresden, germany, 2 crtd, center for regenerative therapies dresden, technische universitä t dresden, germany, 3 institute of physics, polish academy of science, warsaw, poland cytokine receptors of the hematopoietic superfamily transduce their signal through non-covalently bound janus kinases. there are only 4 such kinases in humans (jak1, 2, 3 and tyk2), which associate to 46 different cytokine receptor chains. here we study the dynamics of gfp-tagged jak1 and jak3 in epithelial cells with fluorescence correlation spectroscopy (fcs). jak1 and jak3 behave differently in various aspects: in the absence of receptors, jak1 still binds the membrane, whereas jak3 diffuses homogeneously in the cytoplasm. we used fcs under total internal reflection illumination (tir-fcs) and determined the membrane binding affinity of jak1 to be 60±36 nm. the association of jak3 with the common gamma chain (c c ) is very tight as shown by fluorescence recovery after photobleaching (frap). molecular brightness analysis of single-point fcs shows that jak1 diffuses as a monomer in the rather small cytoplasmic pool, whereas jak3 diffuses as dimers, which undergo a defined oligomerization. the degree of oligomerization decays at higher concentrations, indicating that some unknown, saturable scaffold is involved. characterizing the binding and mobility schemes of the janus kinases may be important to further elucidate their specific and redundant effects in signal transduction. plasma membrane (pm)-enriched fraction obtained through subcellular fractioning protocols are commonly used in studies investigating the ability of a compound to bind to a receptor. however, the presence of mitochondria membranes (mi) in the pm-enriched fraction may compromise several experimental results because mi may also contain the interest binding proteins. aiming to analyze the subcellular fractioning quality of a standard sucrose density based protocol, we investigated (a) the na + k + -atpase (pm marker) and succinate dehydrogenase -sd (mi marker) activities; (b) the immunocontent of the adenine nucleotide translocator (ant -mi membrane marker) in both pm-and mi-enriched fractions. since several binding protocols may require long incubation period, we verified the quality of both fractions after 24 hours of incubation in adequate buffer. our results show that pmand mi-enriched fractions exhibit contamination with mi or pm, respectively. we did not observe any effect of incubation on na + k + -atpase activity and ant content in both fractions. surprisingly, sd activity was preserved in the pm-but not in mi-enriched fraction after incubation. these data suggest the need of more careful use of pm-enriched fraction preparation in studies involving pm proteins characterization. human neutrophil peptide 1 (hnp1) is a human cationic defensin that present microbial activity against various bacteria (both gram-positive and negative), fungi and viruses. hnp1 is stored in the cytoplasmic azurophilic granules of neutrophils and epithelial cells. in order to elucidate the mode of action of this antimicrobial peptide (amp), studies based on its lipid selectivity were carried out. large unilamellar vesicles (luv) with different lipid compositions were used as biomembranes model systems (mammal, fungal and bacterial models). changes on the intrinsic fluorescence of the tryptophan residues present in hnp1 upon membrane binding/insertion were followed, showing that hnp1 have quite distinct preferences for mammalian and fungal membrane model systems. hnp1 showed low interaction with glucosylceramide rich membranes, but high sterol selectivity: it has a high partition for ergosterol-containing membranes (as fungal membranes) and low interaction with cholesterolcontaining membranes (as in mammalian cells). these results reveal that lipid selectivity is the first step after interaction with the membrane. further insights on the hnp1 membrane interaction process were given by fluorescence quenching measurements using acrylamide, 5-doxylstearic acid (5ns) or (16ns). nanoparticles (np) are currently used in many industrial or research applications (paints, cosmetics, drug delivery materials…). recent papers demonstrate clearly their activity with biological membranes (nanoscale holes, membrane thinning, disruption). different studies of the np-membrane interaction suggest that parameters are particularly important, such as the np size, their surface properties or their aggregation state. composition of biological membranes being particularly complex, supported lipid bilayers (slb) composed of a restricted number of lipids are usually used as simplified membrane model. moreover, these two-dimensional systems are convenient for surface analysis techniques, such as atomic force microscopy (afm), giving information on the morphology of the slb and its mechanical properties. in this work, we study the behaviour of slbs made of lipids representative of the membrane fluid phase (popc) or of the raft phase (sphingomyelin). these slbs are deposited on planar surfaces (mica or glass) previously recovered with silica beads (10 or 100 nm in diameter) in order to mimick the np-membrane interaction. we will present in this work our first results obtained by afm and fluorescence microscopy. it is well known that the eukaryotic nuclei are the sphere of lipids active metabolism. the investigations demonstrated the existence of numerous enzymes in nuclei which modulate the changes of nuclear lipids during different cellular processes. although the nuclear membrane is accepted as the main place of the lipids localization, nearly 10% of nuclear lipids are discovered in chromatin fraction. the ability of chromatin phospolipids to regulate dna replication and transcription was already demonstrated. the chromatin phospolipids seems to play an important role in cell proliferation and differentiation as well as in apoptosis. it seems also possible that chromatin phospholipids may be participated in realization of cisplatin antitumor effects. the 24-hour in vivo effect of cisplatin on rat liver chromatin phospholipids was investigated. the phospholipids of rat liver chromatin were fractionated by microtlc technique. the quantitative estimation of fractionated phospholipids was carried out by computer program fugifilm science lab. 2001 image gauge v 4.0. the alteration of total phospholipids content as well as the quantitative changes among the individual phospholipids fractions in rat liver chromatin after in vivo action of cisplatin was established. the total content of chromatin phospholipids was significantly decreased after the cisplatin action. four from five individual phospholipids fractions were markedly changed after the drug action. two cholin-content phospholipids, particularly phosphatidylcholine and sphingomyelin exhibit diversity in sensitivity to this drug: the increase of sphingomyelin content accompanied by quantitative decrease of phosphatidylcholine. the quantity of cardiolipin was markedly increased while the amount of phosphatidylinositol was decreased after the cisplatin treatment. the phosphatidylethanolamin content remained unchanged after the drug action. it seems that high sensitivity of chromatin phospholipids exhibited to cisplatin action may play an important role in antitumor effects of this drug. membrane lipids and drug resistance in staphylococci r. d. harvey institute of pharmaceutical science, king's college london, 150 stanford street, london se1 9nh, uk staphylococci express numerous resistance mechanisms against common antimicrobials, including peptide components of the innate immune system which have been trumpeted as being likely candidates to replace our increasingly ineffective antibiotics. the membrane phospholipid lysylphosphatidylglycerol (l-pg) appears to play a key role in staphylococcal drug resistance, since its absence in mutant bacteria renders them susceptible to a range of cationic antimicrobials. the current assumption about the role l-pg plays in drug resistance is that of facilitating charge neutralisation of the plasma membrane, leading to loss of affinity towards cationic moieties. we have investigated this phenomenon using a range of model membrane systems composed of both synthetic lipids and reconstituted natural lipid extracts, using such techniques as stopped-flow fluorescence, circular dichroism and neutron scattering. our conclusions indicate that the initial assumptions about the role of l-pg in drug resistance are over-simplistic and certainly do not tell the whole story of the physical and biological properties of this fascinating moderator or membrane behaviour. our findings show that l-pg does not inhibit antimicrobial drug action by charge dampening, hinting at a different protective mechanism. modulation of a-toxin binding by membrane composition m. schwiering, a. brack, c. beck, h. decker, n. hellmann institute for molecular biophysics, university of mainz, mainz, germany although the alpha-toxin from s. aureus was the first poreforming toxin identified, its mode of interaction with membranes is still not fully understood. the toxin forms heptameric pores on cellular and artificial membranes. the present hypothesis is that the initial binding to the membrane occurs with low affinity, and that an efficient oligomerisation, relying on clusters of binding sites, is the reason for the overall high affinity of the binding process. in order to separate the effects of increasing concentration of binding sites from this topological effect, we investigated the oligomer formation based on pyrene-fluorescence for a series of lipid compositions, where the fraction of toxin binding lipids (egg phospatidylcholine (epc) or egg sphingomyelin (esm)) was varied while their concentration remained constant. the results indicate that an increased local density of toxin binding sites occurring due to phase separation facilitates oligomer formation. furthermore, the change in local environment (number of neighboring cholesterol molecules) upon domain formation also enhances oligomer formation.we thank the dfg (sfb 490) for financial support, s. bhakdi and a. valeva for production of the toxin and helpful discussions. we explored quercetin effects on lipid bilayers containing cholesterol using a spectrofluorimetric approach. we used the fluorescent probe laurdan which is able to detect changes in membrane phase properties. when incorporated in lipid bilayers, laurdan emits from two different excited states, a non-relaxed one when the bilayer packing is tight and a relaxed state when the bilayer packing is loose. this behavior is seen in recorded spectra as a shift of maximum emission fluorescence from 440 nm at temperatures below lipids phase transition to 490 nm at temperatures above lipids phase transition values. emission spectra of laurdan were analyzed as a sum of two gaussian bands, centered on the two emission wavelengths allowing a good evaluation of the relative presence of each population. our results show that both laurdan emission states are present with different shares in a wide temperature range for dmpc liposomes with cholesterol. quercetin leads to a decrease in the phase transition temperature of liposomes, acting in the same time as a quencher on laurdan fluorescence. this paper is supported by the sectorial operational programme human resources development, financed from the caveolins are essential membrane proteins found in caveolae. the caveolin scaffolding domain of caveolin-1 includes a short sequence containing a crac motif (v 94 tkywfyr 101 ) at its c-terminal end. to investigate the role of this motif in the caveolin-membrane interaction at the atomic level, we performed a detailed structural and dynamics characterization of a cav-1(v94-l102) nonapeptide encompassing this motif and including the first residue of cav-1 hydrophobic domain (l102), in dodecylphosphocholine (dpc) micelles and in dmpc/dhpc bicelles, as membrane mimics. nmr data revealed that this peptide folded as an amphipathic helix located in the polar head group region. the two tyrosine sidechains, flanked by arginine and lysine residues, are situated on one face of this helix, whereas the phenylalanine and tryptophan side-chains are located on the opposite face (le lan c. et al., 2010, eur. biophys. j., 39, 307-325). to investigate the interactions between the crac motif and the lipids, we performed molecular dynamics simulations in two different environment: a dpc micelle and a popc bilayer. the results obtained are in good agreement with nmr data and the comparison between both systems provided insight into the orientation of the crac motif at the membrane interface and into its interactions with lipids. this work was partially supported by the strategic grant posdru/21/1. our study suggests that conformation and positioning of hydroxyl groups significantly affects thermotropic properties of sphingolipids and sterol interaction. the polymorphism of a new series of bolaamphiphile molecules based on n-(12-betainylamino-dodecane)-octyl b-d-glucofuranosiduronamide chloride is investigated. the length of the main bridging chain is varied in order to modify the hydrophilic/lipophilic balance. the other chemical modification was to introduce a diacetylenic unit in the middle of the bridging chain to study the influence of the pp stacking on the supramolecular organisation of these molecules. dry bolaamphiphiles self-organize in supramolecular structures such as lamellar crystalline structure, lamellar fluid structure and lamellar gel structure. the thermal dependence of these structures, as well as the phase transition is followed by smallangle and wide-angle x-ray scattering. once the thermal cycle is accomplished, the system remains in the kinetically stabilized undercooled high-temperature phase at temperature of 20°c. subsequently, the time dependence of the relaxation to the thermodynamically stable phase is followed and very slow relaxation on the order of hours or days is observed. the study of polymorphism and the stability of various phases of this new series of bolaamphiphiles is interesting for potential application in health, cosmetics or food industry, is undertaken in this work. alkylphospholipids have shown promising results in several clinical studies and among them perifosine (opp) is promising for breast cancer therapy. antitumor effect was much better in estrogen receptor negative (er-) than in estrogen receptor positive (er+) tumors in vivo. it is believed that apl do not target dna, but they insert in the plasma membrane and ultimately lead to cell death. liposomes made of opp and different amount of cholesterol (ch) showed diminished hemolytic activity as compared to micellar opp, but in most cases cytotoxic activity was lower. in order to find optimal liposomal composition and to understand better the difference in the response of er+ and er-cells the interaction opp liposomes with er+ and er-cells was studied. for liposomes with high amount of ch both cell types showed slow release of the liposome entrapped spin probe into the cytoplasm. liposmes with low amount of ch interact better with cells but the release is faster for er-as for er+ cells at 37°c. experiments with nitroxide-labeled opp (sl-opp) liposomes suggest that the exchange of sl-opp between liposomes and cellular membranes is fast. however, translocation of sl-opp across the plasma membrane is slow, but seems to be faster for opp resistent, er+ cells as for er-cells at 37°c. estimation of a membrane pore size based on the law of conservation of mass krystian kubica 1 , artur wrona 1 and olga hrydziuszko 2 1 institute of biomedical engineering and instrumentation, wroclaw university of technology, wroclaw, poland, 2 centre for systems biology, university of birmingham, birmingham, uk the size of biomembrane pores determines which solutes or active compounds may enter the cell. here, using a mathematical model of a lipid bilayer and the law of conservation of mass, we calculate the radius of a membrane pore created by rearranging the lipid molecules (the pore wall was formed out of the lipid heads taken from the membrane regions situated directly above and below the pore, prior its formation). assuming a constant number of lipid molecules per bilayer (with or without the pore) and based on the literature data (60% decrease in the area per chain for a fluid-to-gel transition and a matching change of one chain volume not exceeding 4%) we have shown that the pore radius can measure up to 4.7nm (for a 7nm thick lipid bilayer) without the lipid molecules undergoing a phase transition. a further assumption of area per chain being modified as a consequence of the lipids conformational changes has resulted in an increase of the calculated radius up to 7.1nm. finally, a comparison of the pore volume with the corresponding volume of the lipid bilayer has led to a conclusion that for the system under consideration the membrane pore can only be created with the lipids undergoing fluidto-gel conformational changes. the key signaling pathway involves tyrosine phosphorylation of signal transducers and activators of transcription (stat1 and stat2) by receptor-associated janus kinases. we aim to unveil of the very early events of signal activation including ligand-induced receptor assembly and the recruitment of the cytoplasmic effector proteins stat1 and stat2 in living cells. to this end, we have explored the spatiotemporal dynamics of stat recruitment at the membrane on a single molecule level. highly transient interaction of stats to membrane-proximal sites was detected by tirf-microscopy, allowing for localizing and tracking individual molecules beyond the diffraction limit. thus, we obtained a pattern of the spatio-temporal recruitment of stat molecules to the plasma membrane revealing distinct submicroscopic structures and hotspots of stat interaction with overlapping recruitment sites for stat1 and stat2. strikingly, these stat binding sites were independent on receptor localization and expression level. simultaneous superresolution imaging of the cytoskeleton revealed the organization of stat recruitment sites within the cortical actin skeleton. characterization of molecular dynamics on living cell membranes at the nanoscale level is fundamental to unravel the mechanisms of membrane organization andcompartmentalization. we have recently demonstr ated the feasibility of fluorescence correlation spectroscopy (fcs) based on the nanometric illumination of near-field scanning optical microscopy (nsom) probes on intact living cells [1] . nsom-fcs was applied to study the diffusion of fluorescent lipid analogs on living cho cells. the experiments allowed to reveal details of the diffusion hidden by larger illumination areas and associated with nanoscale membrane compartmentalization. the technique also offers the unique advantages of straightforward implementation of multiple color excitation, opening the possibility to study nanoscale molecular cross-correlation. furthermore, the nsom probe evanescent axial illumination allows to extend diffusion study to the membrane-proximal cytosolic region. as such, nsom-fcs represents a novel powerful tool to characterize the details of many biological processes in which molecular diffusion plays a relevant role. the growing interest in supported lipid bilayers (slbs) on conductive substrates, such as gold, is due to the possibility of designing lipid-based biosensor interfaces with electrochemical transduction. due to the hydrophobicity of gold it is still a challenge to deposit planar and continuous bilayers without previous surface modification. most studies on gold concern single-phase slbs without cholesterol or gangliosides, two vital components of biomembranes. in this work the experimental conditions suitable for the formation of complex slbs with phase-separation directly on gold are exploited. the mixtures dopc/dppc/cholesterol (4:4:2) with 0 or 10 mol % of ganglioside gm1, which should yield lipid raft-like domains according to reported phase diagrams, were studied. slb with lipid rafts were successfully formed onto bare au (111), although surface modification with 11-mercapto-undecanoic acid sam stabilized the slbs due to its charge and hydrophilicity. the different experimental conditions tested had an impact on nano/microdomains organization observed by atomic force microscopy in buffer solution. surface characterization through the combined use of ellipsometry, cyclic voltammetry and afm allowed to optimize the conditions for the formation of more planar and compact slbs. it is widely accepted that the conversion of the soluble, nontoxic amyloid b-protein (ab) monomer to aggregated toxic ab rich in b-sheet structures is central to the development of alzheimer's disease. however, the mechanism of the abnormal aggregation of ab in vivo is not well understood. we have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by ab, the toxicity and physicochemical properties of which are different from those of ab amyloids formed in solution [1, 2] . in this presentation, we report a detailed mechanism by which ab-(1-40) fibrillizes in raft-like lipid bilayers composed of gm1/cholesterol/sphingomyelin. at lower concentrations, ab formed an a helix-rich structure, which was cooperatively converted to a b sheet-rich structure above a threshold concentration. the structure was further changed to a seed-prone b structure at higher concentrations. the seed recruited ab in solution to form amyloid fibrils. [ hepatitis c virus (hcv) has a great impact on public health, affecting more than 170 million people worldwide since it is the cause of liver-related diseases such as chronic hepatitis, cirrhosis and hepatocarcinoma. hcv entry into the host cell is achieved by the fusion of viral and cellular membranes, replicates its genome in a membrane associated replication complex, and morphogenesis has been suggested to take place in the endoplasmic reticulum (er) or modified er membranes. the variability of the hcv proteins gives the virus the ability to escape the host immune surveillance system and notably hampers the development of an efficient vaccine. hcv has a single-stranded genome which encode a polyprotein, cleaved by a combination of cellular and viral proteases to produce the mature structural proteins (core, e1, e2, and p7) and non-structural ones (ns2, ns3, ns4a, ns4b, ns5a and ns5b), the latter associated with the membrane originated from the er in the emerging virus. the ns4b protein, a fundamental player in the hcv replicative process and the least characterized hcv protein, is a highly hydrophobic protein associated with er membranes. it has recently been shown that the c-terminal is palmitoylated and the n-terminal region has potent polymerization activity. the expression of ns4b induces the formation of the so called membranous web, which has been postulated to be the hcv rna replication complex. thus, a function of ns4b might be to induce a specific membrane alteration that serves as a scaffold for the formation of the hcv replication complex and therefore has critical role in the hcv cycle. due to the high hydrophobic nature of ns4b, a detailed structure determination of this protein is very difficult. the ns4b protein is an integral membrane protein with four or more transmembrane domains. the c-terminal region of ns4b is constituted by two a helices, h 1 (approximately from amino acid 1912 to 1924) and h 2 (approximately from amino acid 1940 to 1960), which have been studied as potential targets for inhibiting hcv replication. previous studies from our group, based on the study of the effect of ns4b peptide libraries on model membrane integrity, have allowed us to propose the location of different segments in this protein that would be implicated in lipid-protein interaction. additionally, the h 1 region could be an essential constituent in the interaction between protein and membrane. in this study we show that peptides derived from the c-terminal domain of ns4b protein of hcv are able to interact with high affinity to biomembranes, significantly destabilizing them and affecting their biophysical properties. there were also differences in the interaction of the peptide depending on the lipid composition of the membranes studied. we have also applied fluorescence spectroscopy, infrared spectroscopy and differential scanning calorimetry which have given as a detailed biophysical study of the interaction of the peptide with model biomembranes. this work was supported by grant bfu2008-02617-bmc (ministerio de ciencia y tecnología, spain) to j.v. a semi-quantitative theory describing the adhesion kinetics between soft objects as living cells or vesicles was developed. the nucleation-like mechanism has been described in the framework of a non-equilibrium fokker-planck approach accounting for the adhesion patch growth and dissolution (a. raudino, m. pannuzzo, j. chem. phys. 132, 045103 (2010)). a well known puzzling effect is the dramatic enhancement of the adhesion/fusion rate of lipid membranes by water-soluble polymers that do not specifically interact with the membrane surface. we extend the previous approach by molecular dynamics simulations in the framework of a coarse-grained picture of the system (lipid+polymer+ions embedded in an explicit water medium) in order to test and support our previous analytical results. simulations show that the osmotic pressure due to the polymer exclusion from the inter-membrane spacing is partially balanced by an electrostatic pressure. however, we also evidenced an interesting coupling between osmotic forces and electrostatic effects. indeed, when charged membranes are considered, polymers of low dielectric permittivity are partially excluded from the inter-membrane space because of the increased local salt concentration. the increased salt concentration means also a larger density of divalent ions which form a bridge at the contact region (stronger adhesion). the overall effect is a smaller membrane repulsion. this effect disappears when neutral membranes are considered. the model could explain the fusion kinetics between lipid vesicles, provided the short-range adhesion transition is the rate-limiting step to the whole fusion process. the role of ceramide acyl chain length and unsaturation on membrane structure sandra n. ceramide fatty acid composition selectively regulates distinct cell processes by a yet unknown mechanism. however, evidence suggests that biophysical processes are important in the activation of signalling pathways. indeed, ceramide strongly affects membrane order, induces gel/fluid phase separation and forms highly-ordered gel domains. the impact of ceramide n-acyl chain in the biophysical properties of a fluid membrane was studied in popc membranes mixed with distinct ceramides. our results show that: i) saturated ceramide has a stronger impact on the fluid membrane, increasing its order and promoting gel/fluid phase separation, while their unsaturated counterparts have a lower (c24:1 ceramide) or no (c18:1 ceramide) ability to form gel domains at physiological temperature, ii) differences between distinct saturated species are smaller and are related mainly to domain morphology, and iii) very long chain ceramide induces the formation of tubular structures probably associated with interdigitation. these results suggest that generation of different ceramide species in cell membranes has a distinct biophysical impact with acyl chain saturation dictating membrane lateral organization, and chain asymmetry governing interdigitation and membrane morphology. extra high content of cholesterol (chol) in fiber-cell membranes in the eye lens leads to chol saturation and formation of cholesterol bilayer domains (cbds). it is hypothesized that high enrichment in cholesterol helps to maintain lens transparency and protect against cataractogenesis. in model studies, the cbd is formed in a phospholipid bilayer when cholesterol content exceeding the cholesterol solubility threshold, thus, the cbd is surrounded by phospholipid bilayer saturated with cholesterol. in the present study, we carried out molecular dynamics (md) simulation of two bilayers: a palmitoyloleoylphosphatidylcholine (popc) bilayer (reference) and a 1:1 popc-chol bilayer, to investigate the smoothing effect of the saturating amount of cholesterol on the bilayer. to our knowledge, this effect has not been studied so far so this study is certainly providing new results. our results indicate that saturation with cholesterol significantly narrows the distribution of vertical positions of the center-of-mass of the popc molecules and the popc atoms in the bilayer and smoothes the bilayer surface. we hypothesize that this smoothing effect decreases light-scattering and helps to maintain lens transparency. the phospholipid content of staphylococcus aureus membranes displays a high degree of variability (1-3). the major phospholipids found in s. aureus are phosphatidylglycerol (pg), cardiolipin (cl) and lysylphosphatidylglycerol (lpg), (1) the concentrations of which are environment dependent and see fluctuations on exposure to high concentrations of positively charged moieties (4) . up regulation of lpg has a suspected role in neutralisation of the plasma membrane in response to cationic threats. studies have been conducted to probe biomimetic models of this theory however our focus is to look at atomic details of membrane extracts in the presence of magaininf5w. s. aureus 476 lipid extracts from cells grown at ph 5.5 and 7.0, studies by neutron diffraction with and without peptide at two contrasts. d-spacings were assessed by vogt area fitting and bragg's law. bilayer separation at low ph was *1-2 å less than ph 7.0. with peptide, bilayer separations of ph 5.5 and 7.0 extracts were reduced by *2 å and *4 å , respectively. reduced pg content of low ph extracts is suggested to reduce d-spacing, however presence of peptide further reduces this, possibly by an anion neutralisation effect. abnormal d-spacing on increased humidity may be due to the breakdown of lpg. activation of neutrophils releasing hocl and apoptosis of vein endothelial cells are the events documented to occur in the course of atherosclerosis. as lipid chlorohydrins, which are the key products of the reaction between hocl and unsaturated fatty acid residues, were found in atherosclerotic plaques, we decided to check their biological activity in the context of their ability to act as the mediators of hoclinduced oxidative stress and apoptosis in the culture of immortalized human umbilical vein endothelial cells (hu-vec-st). the concentration of reactive oxygen species was found to be elevated after 1 h cell incubation with phospahtidylcholine chlorohydrins. this effect was at least partially caused by the leakage of superoxide anion from mitochondria and followed by depletion of gsh and total thiols. the significant decrease of antioxidant capacity of cell extracts was also observed. the intracellular red-ox imbalance was accompanied by the increase of the ratio between phosphorylated and dephosphorylated forms of p38 map kinase. after longer incubation a significant number of apoptotic cells appeared. summing up, phosphatidylcholine chlorohydrins may be regarded as signaling molecules, able to initiate signalling pathways by induction of oxidative stress. giant unilamellar vesicles (guvs) are a valuable tool in the study of lateral distribution of biological membrane components. guv dimensions are comparable to typical cell plasma membranes and lipid phase separation can be observed through fluorescence microscopy. guv studies frequently require immobilization of the vesicles, and several methods are available for that effect. one of the most common methodologies for vesicle immobilization is the use of avidin/streptavidin coated surfaces and biotin labeled lipids at very low concentration in the vesicles. here, we analyze the effect of using this methodology on lipid domain distribution for different lipid compositions. we show that as a result of non-homogeneous distribution of biotin labeled lipids between liquid disordered, liquid ordered and gel phases, distribution of lipid domains inside guvs can be dramatically affected. monitoring membrane permeability: development of a sicm approach christoph saßen 1,2 and claudia steinem 1 1 institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße 2, 37077 gö ttingen, germany, 2 ggnb doctoral program: imprs, physics of biological and complex systems scanning ion conductance microscopy (sicm) utilises a nanopipette containing an electrode as a probe for surface investigations with resolutions of 1/3 of the inner pipette diameter. experiments are conducted under physiological conditions, in situ and without mechanical contact of probe and sample. hence, sicm serves as a well-suited technique for the investigation of soft objects such as cells or artificial lipid membranes. using pore-suspending membranes (psm) as a model system, interactions of melittin as an example for cell penetrating peptides (cpps) and lipid membranes are investigated by means of sicm. formation of a range of solvent free psm from lipid vesicles has been achieved as confirmed by means of fluorescence microscopy and sicm. application of melittin results in rupturing of the lipid bilayer. putative insights gained from this assay are critical concentrations of membrane permeabilising ccps and answers to mechanistic questions, e.g. whether ccps translocate only or form pores within the lipid bilayer. positioning of the z-ring in escherichia coli prior to cell division is regulated by intracellular pole-to-pole oscillation and membrane binding of min proteins, allowing assembly of ftsz filaments only at the center plane of the cell. in order to investigate the influence of membrane geometry on the dynamic behavior of membrane binding of min proteins, we combined concepts of synthetic biology and microfabrication technology. glass slides were patterned by a gold coating with microscopic windows of different geometries, and supported lipid bilayers (slb) were formed on these microstructures. on slbs, min-proteins organize into parallel waves. confinement of the artificial membranes determined the direction of propagation.min-waves could be guided along curved membrane stripes, in circles and even along slalom-geometries. in elongated membrane structures, the protein waves always propagate along the longest axis. coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and viscosity of the aqueous media above the bilayer. this indicates the existence of an inhomogeneous and dynamic protein gradient above the membrane. minimal systems for membrane associated cellular processes petra schwille bio technology center biotec, technical university of dresden, germany the strive for identifying minimal biological systems, particularly of subcellular structures or modules, has in the past years been very successful, and crucial in vitro experiments with reduced complexity can nowadays be performed, e.g., on reconstituted cytoskeleton and membrane systems. in this overview talk, i will first discuss the virtues of minimal membrane systems, such as guvs and supported membranes, in quantitatively understand protein-lipid interactions, in particular lipid domain formation and its relevance on protein function. membrane transformations, such as vesicle fusion and fission, but also vesicle splitting, can be reconstituted in these simple subsystems, due to the inherent physical properties of selfassembled lipids, and it is compelling question how simple a protein machinery may be that is still able to regulate these transformations. as an exciting example of the power of minimal systems, i show how the interplay between a membrane and only two antagonistic proteins from the bacterial cell division machinery can result in emergence of protein self-organization and pattern formation, and discuss the possibility of reconstituting a minimal divisome. quantitative microscopic analysis reveals cell confluence regulated divergence of pdgfr-initiated signaling pathways with logically streamlined cellular outputs á rpá d szö } or, lá szló ujalky-nagy, já nos szö ll} osi, gyö rgy vereb university of debrecen, department of biophysics and cell biology, debrecen, hungary platelet derived growth factor receptors (pdgfr) play an important role in proliferation and survival of tumor cells. pdgf-bb stimulation caused a redistribution of pdgf receptors towards gm1 rich domains, which was more prominent in confluent monolayers. pdgf-bb stimulation significantly increased relative receptor phosphorylation of the ras / mapk pathway specific tyr716 residues and the pi3-kinase / akt pathway specific tyr751 residues in nonconfluent cultures. tyr771 residues that serve as adaptors for ras-gap which inactivates the mapk pathway and tyr1021 residues feeding into the plc-gamma / camk-pkc pathway were the docking sites significantly hyperphosphorylation following ligand stimulation in confluent cells. we found that p-akt facilitated cell survival and pmapk dependent proliferation is more activated in dispersed cells, while phospholipase c-gamma mediated calcium release and pkc-dependent rhoa activation are the prominent output features pdgf stimulus achieves in confluent cultures. these observations suggest that the same stimulus is able to promote distinctly relevant signaling outputs, namely, cell division and survival in sparse cultures and inhibition of proliferation joined with promotion of migration in confluent monolayers that appear contact inhibited. a thermodynamic approach to phase coexistence in ternary cholesterol-phospholipid mixtures jean wolff, carlos m. marques and fabrice thalmann institut charles sadron, université de strasbourg, cnrs upr, 22, 23 rue du loess, strasbourg cedex, f-67037, france e-mail: thalmann@ics-cnrs.unistra.fr we present a simple and predictive model for describing the phase stability of ternary cholesterol-phospholipid mixtures. assuming that competition between the liquid and gel organizations of the phospholipids is the main driving force behind lipid segregation, we derive a phenomenological gibbs free-energy of mixing, based on the calorimetric properties of the lipids main transition. gibbs phase diagrams are numerically obtained that reproduces the most important experimental features of dppc-dopc-chol membranes, such as regions of triple coexistence and liquid orderedliquid disordered segregation. based on this approach, we present a scenario for the evolution of the phase diagram with temperature. results for other phospholipid species, such as popc or psm will also be presented. interleukin-2 and -15 receptors play a central role in the activation, survival and death of t lymphocytes. they form supramolecular clusters with mhc i and ii glycoproteins in t cells. in damaged or inflamed tissues the extracellular k+ concentration increases, which can depolarize the membrane. the common signaling beta and gamma chains of il-2/15r are phosphorylated upon cytokine binding and get a permanent dipole moment, thus their conformation, interactions, mobility and activity may be sensitive to the membrane potential. we induced depolarization on ft 7.10 t lymphoma cells by increasing the ec. k+ level or by blocking kv 1.3 voltage gated k+ channels with margatoxin. fcs measuremens showed that the lateral mobility of fab-labeled il-2/ 15r and mhc i and ii decreased upon depolarization, while that of gpi-linked cd48 did not change. fret efficiency measured between some elements of the il-receptor/mhc cluster increased, which may reflect an increase of cluster size. il-2-induced receptor activity, as monitored by measuring stat5-phosphorylation, increased upon depolarization, whereas il-15 induced phosphorylation did not change. our results may reveal a novel regulatory mechanism of receptor function by the membrane potential. cytokines play an important role in t cell activation and immunological memory, whereas mhcs are known for the role in antigen presentation. we applied rnai to silence the expression of mhc i in order to study its possible role in receptor assembly and function. fret data indicated that the association of il-2r and il-15r with mhc i as well as between il-2r and il-15r weakened. fcs indicated an increase of receptor mobility also suggesting the partial disassembly of the clusters. mhc i gene silencing lead to a remarkable increase of il-2/il-15 induced phosphorylation of stat5 transcription factors. in search for the molecular background of this inhibition of signaling by mhc i we checked il-2 binding and the formation of the receptor complex (il-2r alpha -il-2r beta association), but we did not find a difference as compared to the control. our results suggest that mhc i plays an organizing role in maintaining supramolecular receptor clusters and inhibits il-2r signaling, revealing a nonclassic new function of mhc i beyond its classical role in antigen presentation. interleukin-4 (il-4) is an important cytokine involved in adaptive immunity. il-4 binds with high affinity the singlepass transmembrane receptor il-4ra. the occupied complex, il-4/il-4ra then engages either il-2rc or il-13ra1, to form an activated type i or ii receptor, respectively. this formation of heterodimers is believed to trigger cross-activation of intracellular janus kinases. here we follow a fluorescently labeled ligand through various stages of receptor activation in hek293t: using fluorescence correlation spectroscopy (fcs), we see that the receptor chains diffuse as monomers within the plasma membrane. using dual-color fccs provides direct evidence for ligand induced co-diffusion of occupied il-4ra and il-13ra1. in contrast, type i complexes containing il-2rc could not be observed. however, ectopic expression of gfp-tagged il-2rc/jak3 induced stable fluorescent speckles in or close to the plasma membrane. we identified these structures as early sorting endosomes by colocalization of surface markers like eea1 and rab gtpases. the il-4ra chain is continuously trafficking into these compartments. these observations suggest that the formation of a type i il-4r heterodimer may require internalization and that early endosomes serve as a platform for il-4 signaling. among the membrane associated proteins, the ras family, which is lipid-anchored g protein, plays a key role in a large range of physiological processes and, more importantly, is deregulated in a large variety of cancer. in this context, plasma membrane heterogeneity appears as a central concept since it ultimately tunes the specification and regulation of ras-dependent signaling processes. therefore, to investigate the dynamic and complex membrane lateral organization in living cells, we have developed an original approach based on molecule diffusion measurements performed by fluorescence correlation spectroscopy at different spatial scales (spot variable fcs, svfcs) (1). we have shown in a variety of cell types that lipidbased nanodomains are instrumental for cell membrane compartmentalization. we have also observed that thesenanodomains are critically involved in the activation of signaling pathways and are essential for physiological responses (2-3). more recently, weextend the application of svfcs to characterizethe dynamics of k-rasproteinat the plasma membrane. as major result, we demonstrated that the rate of k-ras association/dissociation from the membrane is fast but vary as a functional of the activation state of the molecule as well as of specific intracellular protein interactions. we have so demonstrated that an helical lid sub-domain in the sbd is essential for monomeric as binding, but not for the anti-aggregation activity of the chaperone, suggesting that hsp70 is able to interact with pre-fibrillar oligomeric species formed during as aggregation and that, then, the mechanism of binding for these species is different from that of the monomeric protein. aggregation of the acylphosphatase from sulfolobus solfataricus (sso acp) into amyloid-like protofibrils is induced by the establishment of an intermolecular interaction between a 11-residue unfolded segment at the n-terminus and the globular unit of another molecule. we have used data from hydrogen/deuterium exchange experiments, intermolecular paramagnetic relaxation enhancements and isothermal titration calorimetry measurements on an aggregation-resistant sso acp variant lacking the 11-residue n-terminus to characterize the initial steps of the aggregation reaction. under solution conditions that favour aggregation of the wild-type protein, the truncated protein was found to interact with a peptide corresponding to the n-terminal residues of the full length protein. this interaction involves the fourth strand of the main b-sheet structure of the protein and the loop following this region and induces a slight decrease in protein flexibility. we suggest that the amyloidogenic state populated by sso acp prior to aggregation does not present local unfolding but is characterized by increased dynamics throughout the sequence that allow the protein to establish new interactions, leading to the aggregation reaction. amyloid-like aggregates alter the membrane mobility of gm1 gangliosides martino calamai and francesco pavone university of florence, lens -european laboratory for non-linear spectroscopy, sesto fiorentino, florence, italy neuronal dysfunction in neurodegenerative pathologies such as alzheimer's disease is currently attributed to the interaction of amyloid aggregates with the plasma membrane. amongst the variety of toxic mechanisms proposed, one involves the binding of amyloid species to gm1 gangliosides. gm1 takes part into the formation of membrane rafts, and exerts antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. in this study, we investigated the effects of amyloid-like aggregates formed by the highly amyloidogenic structural motif of the yeast prion sup35 (sup35nm) on the mobility of gm1 on the plasmamembrane of living cells. preformed sup35nm aggregates were incubated with cells and gm1 molecules were subsequently labeled with biotinylated ctx-b and streptavidin quantum dots (qds). single qds bound to gm1 were then tracked. the mobility of gm1 was found to decrease dramatically in the presence of sup35nm aggregates, switching from brownian to mainly confined motion. the considerable interference of amyloid-like aggregates with the lateral diffusion of gm1 might imply a consequent loss of function of gm1, thus contributing to explain the toxic mechanism ascribed to this particular interaction. insights into the early stages of fibrillogenesis of insulin using mass spectrometry harriet l. insulin is a vital hormone in metabolic processes as it regulates the glucose levels in the body. insulin is stored in the b cells of the pancreas as a hexamer, however its biologically active form is the monomer. the formation of fibrillar aggregates of insulin rarely occurs in the body; however localised amyloidosis at the site of injection for diabetes patients and aggregation of pharmaceutical insulin stocks present problems. in the current study oligomers formed early in the process of fibril assembly in vitro are observed by mass spectrometry (ms). ms is the only technique which allows early species to be characterised as it can identify different oligomeric orders by mass to charge ratio and show protein abundance and aggregation propensity. on mobility ms is used to examine rotationally averaged collision cross sections of oligomers in the aggregating solution. a wide array of oligomers is observed and the stability of specific species is remarked. the presence of multiple conformations for the highly charged oligomers is particularly noted and their assignment confirmed using fourier transform ion cyclotron resonance ms and collision induced dissociation. molecular modelling has been used to further explore the conformational space the oligomers inhabit. amyloid fibrils consisting of different proteins have been recognized as an accompanying feature of several neurodegenerative diseases. many proteins without known connection to any diseases have been found to form amyloid fibrils in vitro, leading to suggestion that the ability to form fibrils is the inherent property of polypeptide chain. the observed common character of protein amyloid formation enables to seek further clues of fibrillation mechanism by studying generalized sequenceless polypeptide models, e.g. polylysine. we have studied conformational transitions of polylysine, with different chain length at various ph, ionic strength and temperature by means of novel approachviscometric method. this polypeptide undergoes alfa-helix to beta-sheet transition and forms amyloid fibrils in special conditions. temperature induced a-helix to b-sheet transitions occurs at ph interval form 10 to 11.8 and with increasing chain length is slightly shifted to the lower ph. we have found narrow ph interval, in which the thermal transition is fully reversible, suggesting the high sensitivity of polypeptide conformation on subtle changes in charge on its side chains. this work was supported within the projects vega 0079,0038 and 0155, cex sas nanofluid and by esf project no. 26220120033. understanding the mechanisms of the conversion from the native state of a protein to the amyloidal state represents a fundamental step in improving the purification, storage and delivery of protein-based drugs and it is also of great relevance for developing strategies to prevent in vivo protein aggregation. amyloid fibrils have a structural arrangement of cross b-sheet but they can also experience different packing into three dimensional superstructures, i.e. polymorphism. it is well known that, among others, both the geometric confinement of the molecules and shear forces can affect the final morphology of the aggregates. importantly, due to the complexity and crowding of the cellular region, such parameters also play a crucial role in in vivo processes. we present an experimental approach to study in vitro amyloid aggregation in a controlled and uniform shear force field and within microscale environments. in particular we focus on the effect of these two parameters on the formation of spherical aggregates, known as spherulites. using micro channels of different cross-sections from 5 to1000 lm x 12 lm and flow rates in the range of hundreds of ll/min, the number and diameter of spherulites within the channels have been characterized using crossed polarizers optical microscopy. inhibition of insulin amyloid fibrillization by albumin magnetic fluid k. insulin amyloid aggregation causes serious problems for patient with insulin dependent diabetes undergoing long-term treatment by injection, in production and storage of this drug and in application of insulin pumps. recent studies indicate that protein amyloid aggregation causes the cell impairment and death; however, the prevention of amyloid aggregation is beneficial. we have investigated ability of albumin magnetic fluid (amf) to inhibit insulin amyloid aggregation by spectroscopic and microscopic techniques. albumin magnetic fluid consists of magnetic fe 3 o 4 nanoparticles sterically stabilized by sodium oleate and functionalized with bovine serum albumin (bsa) at various weight ratios bsa/fe 3 o 4 . we have found the positive correlation between inhibiting activity of afm and nanoparticle diameter and zeta potential. the ability of amf to inhibit formation of amyloid fibrils exhibits concentration dependence with ic 50 values comparable to insulin concentration. the observed features make amf of potential interest as agents effective in the solving of problems associated with insulin amyloid aggregation. (this work was supported within the projects vega 0079, 0155 and 0077, cex sas nanofluid, apvv-0171-10, sk-ro-0012-10 and esf project 26220220005). amyloid formation of peptides causes diseases like alzheimer's and parkinson's disease. however, the conditions for the onset of the neurotoxic beta-sheet formation are poorly understood. we focus on aggregation triggers and their interplay: interactions with hydrophobic-hydrophilic interfaces, orientation of peptides in 2d, metal ion complexation and lipid layers. the tailor-made model peptides exhibit defined secondary structure propensities and metal ion binding sites. the interactions of the peptide with the air-water interface and with metal ions are studied using surface sensitive methods connected to film balance measurements. x-ray diffraction, x-ray reflection, infrared reflection-absorption spectroscopy and total reflection x-ray fluorescence were applied to reveal the layer structure, peptide conformations and metal ion binding at the interface. we found that amyloid formation in 2d is dominated by the hydrophobic-hydrophilic interface and not comparable to the bulk behaviour. the interface can enhance or inhibit betasheet formation. the effect of metal ion complexation depends on the arrangement of the binding sites in the peptide and the preferred metal complexation geometry. the two triggers interface and metal ion complexation, can oppose each other. effect of apoe isoform and lipidation status on proteolytic clearance of the amyloid-beta peptide hubin, e. alzheimer's disease (ad) is the most common type of dementia in the elderly. the most important genetic risk factor identified for ad is the isoform, e2, e3 or e4, of apolipoprotein e (apoe), a lipid-carrying protein. one hallmark of ad is the accumulation of amyloid-beta peptide (ab) in the brain which is thought to result from an imbalance between the production of ab and its clearance. previous studies report an important role for apoe in ab degradation. we sought to determine the effect of apoe isoform and lipidation status on the degradation of soluble ab by proteinases such as insulin-degrading enzyme and neprilysin. in this study an in vitro ab clearance assay based on the competition between ab and a fluorogenic peptide substrate is developed to quantify ab degradation. to elucidate the proteolytic clearance mechanism, the fragments resulting from cleavage are identified by mass spectrometry and further analyzed to identify the interacting stretch of the ab sequence with the different apoe isoforms. the results suggest that apoe influences the rate of ab degradation. the aggregation of proteins into fibrillar nanostructures is a general form of behaviour encountered for a range of different polypeptide chains. the formation of these structures is associated with pathological processes in the context of alzheimer's and parkinson's diseases but is also involved in biologically beneficial roles which include functional coatings and catalytical scaffolds. this talk focuses on recent work directed at understanding the kinetics of this process through the development and application of experimental biophysical methods and their combination with kinetic theories for linear growth phenomena. lbs contains not only as, but also other proteins including 14-3-3 proteins. 14-3-3 proteins exist mainly as a dimer and its exact functions are remain unclear. however, recent work has shown that the association of 14-3-3(eta) with as in lbs. herein we show how 14-3-3(eta) can modulate as in vitro aggregation behavior, by rerouting it toward the formation of stable non-fibrillar aggregates. we also show that the resulting populations of fibrillar and pre-fibrillar aggregates exhibit a modified toxicity in vivo with respect to the unperturbed aggregates. interestingly, 14-3-3(eta) does not show any binding affinity for monomeric as, nor for the mature fibrillar aggregates. we provide evidence that it acts on the oligomeric species which form during the amyloidogenesis process of aggregation. since 14-3-3(eta) can influence the toxicity of amyloidogenesis without perturbing the functional as monomers, we are convinced that once fully understood, its mode of action could represent a promising model to mimic with synthetic drugs and peptides. what makes an amyloid toxic: morphology, structure or interaction with membranes? more than 40 human diseases are related to amyloids. in order to understand why some amyloids may become toxic to their host and some others are not, we first developed a genetical approach in the yeast saccharomyces cerevisiae. we have chosen the amyloid/prion protein het-s prion forming domain (218-289) from the fungi podospora anserina, which is not toxic in yeast. some toxic amyloids mutants were generated by random mutagenesis. in vitro the most toxic mutant called ''m8'' displays very peculiar nanofibers, which polymerized mainly in amyloid antiparallel b-sheets whereas the non-toxic wt exhibits a parallel polymerization. we further established the dynamic of assembling of the m8 toxic amyloid, in comparison to the wt non-toxic amyloid, and showed the presence of specific oligomeric intermediates also organized in antiparallel b-sheet structures. a more global structure/toxicity study on more than 40 mutants clearly identified an antiparallel b-sheet signature for all the toxic mutants. therefore size, intermediates and antiparallel structures may account for amyloid toxicity in yeast but we still wonder what their cellular targets are. recently, we established the first evidences that toxic mutants may specifically bind in vitro to lipids, particularly negatively charged. interconnected mechanisms in abeta (1-40) we present an experimental study on the fibril formation of ab(1-40) peptide at ph 7.4. the kinetics of this process is characterized by the occurrence of multiple transient species that give rise to final aggregates whose morphology and molecular structure are strongly affected by the growth conditions. to observe in details the aggregation pathway as a function of solution conditions, we have used different experimental techniques as light scattering, thioflavin t fluorescence, circular dichroism and two-photon fluorescence microscopy. this approach gives information on the time evolution of conformational changes at molecular level, on the aggregates/fibrils growth and on their morphologies. the selected experimental conditions allowed us to highlight the existence of at least three different aggregation mechanisms acting in competition. a first assembly stage, which implies conformational conversion of native peptides, leads to the formation of small ordered oligomers representing an activated conformation to proceed towards fibril growth. this process constitutes the rate limiting step for two distinct fibril nucleation mechanisms that probably implicate spatially heterogeneous mechanisms. the formation of amyloid fibrils of amylin 10-29 was studied by means of molecular dynamics (md) and energy partition on three peptide ß-sheet stack systems with the same amino acid composition: wild type amylin 10-29 (amyl 10-29), reverse amylin 10-29 (rev-amyl 10-29) and scrambled amylin version scr-amyl 10-29. the results show that for amylin10-29 peptides, amino acid composition determines the propensity of a peptide to form amyloid fibrils independent of their sequence. the sequence of amino acids defines the shape and the strength of amyloid protofibril, which conforms with the atom force microscope (afm) data [1] . md show that the 6x6revamyl-10-29 stack has looser selfassembly than the 6x6amyl-10-29 stack, which conforms with the results of fourier transform infrared spectroscopy (ftir) measurements for the peptides studied [1] . the results of md show that 6x6amyl-10-29 could have a turn, which consists with ftir data [1] . data on ab aggregation kinetics have been characterized by a large spread between experiments on identical samples that contain so many molecules that stochastic behaviour is difficult to explain unless caused by uncontrolled amounts of impurities or interfaces. we have therefore spent considerable effort to eliminate sources of inhomogeneity and reached a level of reproducibility between identical samples and between experiments on separate occasions that we can now collect data that can lead to mechanistic insights into the aggregation process per se, and into the mechanism of action of inhibitors. data on ab42 aggregation will be shown that give insight into the influence of physical parameters like peptide concentration, shear and ionic strength, as well as the effect of inhibitory proteins, model membranes and the effects of sequence variations. monte carlo simulations of amyloid formation from model peptides corroborate the finding from experiments and underscore that the very high level of predictability and reproducibility comes from multiple parallel processes. negatively-charged membranes were reported to catalyze ''amyloid-like'' fiber formation by non-amyloidogenic proteins [1] . our study aims to elucidate the factors that govern the formation of these amyloid-like fibers. lysozyme was selected as a model of non-amyloidogenic protein and was fluorescently-labeled with alexa fluor 488 (a488-lz). first, a488-lz partition towards phosphatidylserine-containing liposomes was characterized quantitatively using fluorescence correlation spectroscopy (fcs), in order to calculate the protein coverage of liposomes. secondly, the interaction between a488-lz and negatively-charged lipid membranes was studied using both steady-state and time-resolved fluorescence techniques. this interaction was found to switch from a peripheral binding to the anionic headgroups, at high lipid/ protein molar ratio (l/p), to a partial insertion of protein into the hydrophobic core of the membrane, at low l/p. finally, the lipidprotein supramolecular complexes formed at low l/p were characterized by fluorescence lifetime imaging microscopy (flim). the mean lifetime of a488-lz in these supramolecular structures is much lower compared to the values obtained for the free and bound a488-lz at high l/p. the fiber characterization will be complemented by fcs studies. [ the conversion of normal prp c to its pathological isoform prp sc is a key event in prion diseases and is proposed to occur at the cell surface or more probably in acidic late endosomes. a convergence of evidence strongly suggests that the early events leading to the structural conversion of the prp seem to be in relation with more or less stable soluble oligomers, which could mediate neurotoxicity. as commonly shared by other amyloidogenic proteins, membrane-bound monomers undergo a series of lipid-dependent conformational changes, leading to the formation of oligomers of varying toxicity rich in b-sheet structures (annular pores, amyloid fibrils). here, we have used a combination of biophysical techniques (dynamic and static light scattering, fluorescence techniques, and quartz crystral microbalance) to elucidate the interaction of native monomeric prp and that of purified b-rich oligomeric prp on model lipid membranes. under well established conditions, three b-sheet-rich oligomers were generated from the partial unfolding of the monomer in solution, which were found to form in parallel. from single mutation and/or truncation of the full length prp, the polymerization pathway is strongly affected, revealing the high conformational diversity of prp. in our previous work, we identify the minimal region of the prp protein leading to the same polymerization pattern of the full length prp. soluble 12-subunits and 36-subunits oligomers were obtained depending on the single mutation or truncation and purified. we compare their structural properties (ftir, cd) when associated with anionic lipid bilayers and study their propensities to permeabilize the membrane. fluorescence kinetics suggest different mechanisms of membrane perturbation for the monomer and the prp oligomers. deciphering this complex network of lipid interactions and conformational diversity of the prp protein will help for understanding of how amyloidogenic proteins induce neurotoxicity. the traditional view of the lipid bilayer described as a ''sea'' of lipids where proteins may float freely, is going to be inadequate to describe the increasingly large number of complex phenomena which are known to take place in biological membranes. membrane-assisted protein-protein interactions, formation of lipid clusters, protein-induced variation of the membrane shape, abnormal membrane permeabilities and conformational transitions of membrane-embedded proteins are only a few examples of the variegated ensemble of events whose tightly regulated cross-talk is essential for cell structure and function. experimental work on the above mentionated problems is very difficult and some time not accessible, especially when the studied systems have a fast dynamics. due to the large size of the systems usually involved in this multifaceted framework, a detailed molecular description of these phenomena is beyond the possibilities of conventional amyloid aggregation, a generic behavior of proteins, is related to incurable human pathologies -amyloid-related diseases, associated with formation of amyloid deposits in the body. all types of amyloid aggregates possess rich bsheet structural motif. the recent data confirm the toxic effect of aggregates on the cells, however, it was found that reduction of amyloid aggregates plays important role in prevention as well as therapy of amyloidosis. we have investigated effect of phytoalexin derivatives on amyloid fibrillization of two proteins, human insulin and chicken egg lysozyme, by tht and ans fluorescence assays. we have found that amyloid aggregation of both studied proteins was significantly inhibited by phytoalexin derivates cyclobrassinin and benzocamalexin. for most effective phytoalexins the estimated ic 50 values were at low micromolar concentration. the observed inhibiting activity was confirmed by transmission electron microscopy. our data suggest the potential therapeutic use of the most effective phytoalexins in the reduction of amyloid aggregation. (this work was supported within the projects vega 0079, 0155, cex sas nanofluid, apvv-0171-10, sk-ro-0012-10 and esf project 26220220005). the amyloid pore hypothesis suggests that interactions of oligomeric alpha-synuclein (as) with membranes play an important role in parkinson's disease. oligomers are thought to permeabilize membranes and interfere with ca 2+ pathways. permeabilization by as requires the presence of negatively charged phospholipids. whether as can bind and permeabilize membranes with physiologically relevant lipid compositions has not been extensively explored. here we report on the binding of as to giant unilamellar vesicles (guvs) with physiologically relevant lipid compositions. comparing different protocols of oligomer preparation, leakage assays on both large unilamellar vesicles (calcein release) and guvs (hpts efflux assay) show that as is not able to permeabilize these membranes. the presence of cholesterol has a stabilizing effect on these membrane systems. in agreement with these findings, we do not observe concentration dependent as toxicity using in vivo mts assays. however, in the calcein release assay, different as preparations show differences in kinetics and as concentrations that cause 100% leakage. these results motivate us to critically reassess the amyloid pore hypothesis, and suggest that membrane permeabilization may be attributable only to a very specific as species. alpha-synuclein oligomers impair membrane integrity-a mechanistic view martin t. stö ckl, mireille m. a. e. claessens, vinod subramaniam nanobiophysics, mesa+ institute for nanotechnology, university of twente, enschede, the netherlands one of the most prevalent neurodegenerative diseases is parkinson's disease (pd), which is accompanied with the loss of dopaminergic neurons. although the mechanisms leading to the death of these cells are still unclear, the protein alpha-synuclein (as) is one of the pivotal factors. previous studies indicate that especially oligomeric forms of as show a detrimental effect on membrane integrity. as an intact membrane is crucial to many cellular processes, the impairment of the membrane integrity is a likely pathway for neuronal death. we use different phospholipid bilayer model systems to investigate the mechanisms underlying this process. atomic force microscopy in combination with suspended asymmetric phospholipid bilayers, which closely mimic the plasma membrane, allows the identification of the binding sites, the measurement of penetration depths of the as oligomers into the phospholipid bilayer, and the detection of membrane thinning or creation of membrane defects. using an approach based on phospholipid vesicles we were able to observe for the first time that as oligomers cause an enhanced lipid-flip flop. suggesting that the loss of lipid asymmetry is a novel mechanism which may contribute to or trigger neuronal death in pd. amyloid protein-membrane interactions and structuretoxicity relationship study h.p. ta (toxic) has a much higher and more specific effect on negatively charged phospholipids (dops, dopi and dopg) than in the case of wt (non-toxic). therefore the insertion of protein into phospholipid monolayers, which occurred similarly for both wt and m8, is not a key factor for these effects (h.p. ta et al. langmuir, in press). we are now using unilamellar vesicles as a membrane model to investigate the amyloid protein (toxic and nontoxic) -phospholipid interactions. results confirmed the high specific and strong effects of m8 on negatively-charged membrane. in this project, we study the chemical, physical and biological properties of fibrillar networks. the formation and the network mechanics are investigated by combining droplet-based microfluidics with optical microscopy and small angle x-ray scattering (saxs). the chosen system, fibrin network formation, plays an important role in blood coagulation processes. crosslinking of fibrinogen induced by an enzymatic reaction with thrombin leads to 3d fibrin network formation. the fibrillar networks are formed within picoliter droplets of aqueous solutions in an continuous oil phase. droplets containing fibrinogen and thrombin can be produced of different sizes and stored for fibrin network formation. the formation of the fibrillar networks is imaged by fluorescence microscopy. to analyze the elastic properties of the networks, the droplets flow through a microchannel device of alternating width in order to squeeze and stretch the networks. additionally, saxs experiments will give structural information about the molecular dimensions of the networks. the amyloid beta peptide (ab), implicated in alzheimer's disease (ad), is released from the amyloid precursor protein (app) by secretase-induced cleavage. this process results in the release of a range of ab peptides varying in length. the brains of ad patients often contain longer ab peptides while the total concentration of ab is unaffected. longer peptides are more hydrophobic having far-reaching consequences for their toxicity and aggregation. as ab is necessary for normal neuronal function, research activities into ad therapeutic development currently explore the possibilities of modulating c-secretase activity to produce short ab peptides. whether such an approach effectively ameliorates the toxic effect of ab has not been explored yet. to answer this question, we studied the impact of heterogeneity in ab pools in an in vitro biophysical and in cellulo context using microelectrode array to assay the synaptic activity of primary neurons. we show that various lengths of the ab peptide and mixtures thereof aggregate with distinct kinetics and notoriously affect synaptotoxic and cytotoxic response. we also show that small amounts of less abundant peptides ab38 and ab43 induce aggregation and toxicity of ab40 while the behavior of ab42 is unaffected. one of the most important irreversible oxidative modifications of proteins is carbonylation, a process of introducing the carbonyl group in reaction with reactive oxygen species. importantly, carbonylation increases with the age of cells and is associated with the formation of intracellular protein aggregates and the pathogenesis of age-related disorders such as neurodegenerative diseases and cancer. however, it is still largely unclear how carbonylation affects protein structure, dynamics and aggregability on the atomic level. here, we use classical molecular dynamics simulations to study structure and dynamics of the carbonylated headpiece domain of villin, a key actin-organizing protein. we perform an exhaustive set of molecular dynamics simulations of native villin headpiece together with every single combination containing carbonylated versions of its seven lysine, arginine and proline residues, the quantitatively most important carbonylable amino acids. surprisingly, our results suggest that high levels of carbonylation, far above those associated with cell death in vivo, may be required to destabilize and unfold protein structure through the disruption of specific stabilizing elements, such as salt bridges or proline kinks, or tampering with the hydrophobic effect. on the other hand, by using thermodynamic integration and molecular hydrophobicity potential approaches, we quantitatively show that carbonylation of hydrophilic lysine and arginine residues is equivalent to introducing hydrophobic, chargeneutral mutations in their place, and, by comparison with experimental results, demonstrate that this by itself significantly increases intrinsic aggregation propensity of both structured, native proteins and their unfolded states. finally, our results provide a foundation for a novel experimental strategy to study the effects of carbonylation on protein structure, dynamics and aggregability using site-directed mutagenesis. septins are an evolutionarily conserved family of gtp-binding proteins involved in important cellular processes, such as cytokinesis and exocytosis, and have been implicated in neurological diseases, such as alzheimer's and parkinson's diseases. the focus of this study was two septins of schistosoma mansoni, (the causative agent of schistosomiasis in south america) named smsept5 and smsept10, which were produced in a recombinant system. our objective was to verify if these septins from a simpler organism display similar characteristics to human septins. analysis of protein structure by circular dichroism showed that both recombinant smseptins produced were folded. the gtpase activity assay showed that smsept5 was able to hydrolyze gtp, whereas smsept10 was not. aggregation studies for amyloid fibril detection by right angle light scattering and thioflavin t fluorescence assay were performed. both proteins showed a temperature dependent increase in light scattering and fluorescence emission of tht probe. this indicated that s. mansoni septins tend to aggregate into amyloid-like fibers in high temperatures, with thresholds of 30°c for smsept5 and 37°c for smsept10. these results are in accordance to that previously reported for human septins. in our work we investigated the response to standard chemotherapy of blood lymphocytes of patients suffering with melanoma. dna single and double strand breaks were determined using comet assay; intracellular levels of marker proteins were detected using immunocytochemistry. ultimately this set of parameters allows to characterize two mechanisms of dna repair (base excision repair, ber and mismatch repair, mmr) which together with apoptosis proneness underlie response of tumor cells to chemotherapy. cell death caused by o 6 mg adducts is promoted by mmr system by inducing unrepaired double strand breaks in dna. there was a linear correlation between the level of dsdna breaks in lymphocytes after 1-st cycle of chemotherapy and mmr efficiency in them. the level of double strand breaks in dna after 1-st cycle of chemotherapy is predictive of clinical outcome. otherwise damage at the level of ssdna (ap-sites and single strand breaks) and ber mechanism associated with it couldn't be a good prognostic factor of chemotherapy. high level of double strand breaks in dna in blood lymphocytes of melanoma patients 48 hours after 1-st cycle of chemotherapy appears to be a marker of a good prognosis. self-assembly and stability of g-quadruplex: counterions, pressure and temperature effects e. baldassarri jr., p. mariani, f. spinozzi, m. g. ortore saifet dept. & cnism, marche polytechnic university, ancona, italy the important role of g-quadruplex in biological systems is based on two main features: composition and stability of telomeres, and activity of telomerase. the g-quadruplex structures are formed by supramolecular organization of basic units called g-quartets that are planar rings constituted by four guanosines linked by hoogsten hydrogen bonds. gquadruplex requires the presence of monovalent cations playing a key role in stabilizing these structures, since they give rise to coordination bonds needed for the stacking of more tetrads. we performed x-ray diffraction experiments at different pressures (ranging from 1 to 2000 bar), and small angle x-ray scattering (saxs) changing the temperature (between 20-60°c retinoic acid receptor (rar) is a member of the nuclear receptor superfamily. this ligand-inducible transcription factor binds to dna as a heterodimer with the retinoid x receptor (rxr) in the nucleus. the nucleus is a dynamic compartment and live-cell imaging techniques make it possible to investigate transcription factor action in real-time. we studied the diffusion of egfp-rar by fluorescence correlation spectroscopy (fcs) in order to uncover the molecular interactions determining receptor mobility. in the absence of ligand we identified two distinct species with different mobilities. the fast component has a diffusion coefficient of d 1 = 1.8-6 lm 2 /s corresponding to small oligomeric forms, whereas the slow component with d 2 = 0.06-0.12 lm 2 /s corresponds to interactions of rar with the chromatin or other large structures. the rar ligand binding domain fragment also has a slow component probably as a result of indirect dna-binding via rxr, with lower affinity than the intact rar:rxr complex. importantly, rar-agonist treatment shifts the equilibrium towards the slow population of the wild type receptor, but without significantly changing the mobility of either the fast or the slow population. by using a series of mutant forms of the receptor with altered dna-or coregulator-binding capacity we found that the slow component is probably related to chromatin binding, and that coregulator exchange, specifically the binding of the coactivator complex, is the main determinant contributing to the redistribution of rar during ligand activation. formation of inactive nuclear with high level of dna compaction in sperm cells is accompanied by a substitution of linker histones h1 by a number of other proteins. among them sperm-specific histones (ssh), which are characterized by elongated arginine-rich polypeptide chain compared to the somatic h1. the secondary and tertiary structure of the ssh and their interactions with dna were studied using spectroscopic and thermodynamic approaches. the histones were isolated from sperm of marine invertebrates and rat thymus. all studied ssh demonstrate no considerable compaction of dna in solutions of low ionic strength. however, at physiological conditions, ssh h1 from s.intermedius and a.japonica compact dna more intensively than other ssh. the somatic h1 from rat thymus revealed a minimal ability to compact the dna. we suggest that the ssh h1 are able to interact with dna not only in the major groove but also in the minor groove of the double helix inducing considerable structural changes in dna and facilitating the formation of the supercompact sperm chromatin. the authors are grateful for the financial support from the russian foundation for basic research (grants § 10-04-00092 and 09-08-01119) and from administration of saint-petersburg. ionizing radiation causes modification and destruction of nitrogenous bases in dna molecule. there are also local breakages of hydrogen bonds both in the lesion sites mentioned above and in other sites of the macromolecule. to reveal the amount of some of these damages we applied cd and uv absorption spectroscopy. radiation-induced changes in dna structure influence its uv absorption spectrum in different ways: partial denaturation causes hyperchromic effect, while destruction of the bases results in hypochromic shift. at the same time both of them result in the same changes in dna cd spectra: the decrease in intensity. we attempted to segregate the described damages in dna structure and studied the influence of dna ionic surroundings on the radiation effect. it is shown that the radiation efficiency of base destruction and partial denaturation increases with decreasing concentration of nacl in irradiated solution. udu (ugly duckling) has been first identified from a zebrafish mutant and shown to play an essential role during erythroid development; however, its roles in other cellular processes remain largely unexplored. facs analysis showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased dna damage in udu mutants. we further showed that the extensive p53-dependent apoptosis found in udu mutants is a consequence of activation in the atm-chk2 pathway. udu appears not to be required for dna repair, because both wild-type and udu embryos similarly respond to and recover from uv treatment. yeast two-hybrid and coimmunoprecipitation data demonstrated that pah-l repeats and sant-l domain of udu interacts with mcm3 and mcm4. furthermore, udu was colocalized with brdu and heterochromatin during dna replication, suggesting a role in maintaining genome integrity. recently, we started to work on the second zebrafish homolog, udu2, and its mammalian counterpart, gon4l. preliminary data showed that udu2 and gon4l mrna injection can rescue zebrafish udu mutant phenotypes. furthermore, pah-l and sant-l domains of udu2 and gon4l can bind to mcm3 and mcm4 and they are localized in the nucleus. these data suggest that udu2 and gon4l are functionally equivalent to zebrafish udu. their molecular mechanism leading to udu phenotypes is currently under investigation. chromatin condensation: general polyelectrolyte association and histone-tail specific folding nikolay korolev, nikolay berezhnoy, abdollah allahverdi, renliang yang, chuan-fa liu, james p. tam, lars nordenskiö ld school of biological sciences, nanyang technological university, 60 nanyang drive, 637551, singapore the major component of chromatin, dna, is a densely charged polyanion. electrostatic interactions between dna and dnapackaging proteins contribute decisively to formation of its elementary unit, the nucleosome, and are also important in chromatin folding into higher-order structures. we investigate condensation of dna and chromatin and find that electrostatics and polyelectrolyte character of dna play dominant role in both dna and chromatin condensation. by comprehensive experimental studies and using novel oligocationic ligands, we suggest simple universal equation describing dna condensation as a function of oligocation, dna and monovalent salt concentrations and including the ligand-dna binding constant. we found that similar dependence was also observed in condensation of the nucleosome arrays. next, we studied how general electrostatic and specific structural alterations caused by lysine acetylations in the histone tails influence formation of 30-nm chromatin fibre and intermolecular nucleosome array association. for the first time, a structural model is suggested which explains critical dependence of chromatin fibre folding on acetylation of the single lysine at position 16 of the histone h4. exceptional importance of the h4 lys 16 acetylation in general and gene specific transcriptional activation has been known for many years but no structural basis for this effect has yet been proposed. detection of specific dna sequences is central to modern molecular diagnostic. ultrasensitive raman measurements of nucleic acids are possible through exploiting the effect of surface-enhanced raman scattering (sers). in this work, the sers spectra of genomic dnas from leaves of different apple trees grown in the field, have been analyzed [1] . a detailed comparative analysis of sers signatures of genomic dnas is given. sers wavenumbers (cm -1 ) are reported here for all types of vibrations of plant genomic dnas, including bands assigned to localized vibrations of the purine and pyrimidine residues, localized vibrations of the deoxyribosephosphate moiety, etc. proposed band assignments are given. a strong dependence of the sers spectra on dna concentration and on time have been observed. in biochemical fields, nucleic acids might be used to explore the interaction between dna and small molecules, which is important in connection with probing the accurate local structure of dna and with understanding the natural dnamediated biological mechanisms [2] . the ph-dependent structure of dna studied by fourier transform infrared spectroscopy the region of the infrared spectrum studied covered the wave number range from 4000 cm -1 to 700 cm -1 . ir spectra show that in ph 8.0-9.0 interval carbonyl (c=o) band at 1689 -1 cm (assigned to guanine) is reduced in intensity and slightly shifted to lower frequencies. at the ph 10.0 significantly decreases band intensity at 1712 cm -1 due to unbounded c2=o of thymine and shifts to lower frequencies, indicating at the transition of this group in bounded form, supposedly by means of excess polarized hydroxyl ions. together this, in basic region a new intense absorption band has been observed in 1500-1300cm -1 frequency interval, corresponding to o-h group in-plane bending vibration (1410-1310cm -1 ). as for acidic conditions, it was observed that under the extreme ph (*2.8) value carbonyl absorption region shifts to higher frequencies and absorption intensity significantly increased, indicated at releasing of c=o groups from h-bonding between base pairs. moreover, bands intensity at 1418cm -1 and 1022cm -1 corresponding to out-of-plan deformation of nh 2 groups increased due to rupture of connections between the dna strands. during the last decade it was found that in many cases specific structural organization of multi-molecular protein and dna-protein complexes determines their functioning in living cells. although these functioning structures are usually unique, it is often possible to identify their common structural elements. one of the interesting examples of such universal elements are hmgb domains: structurally conservative functional domains of non-histone proteins hmgb1/2 also identified in many nuclear proteins. using afm, thermodynamic approaches, circular dichroism and molecular absorption spectroscopy in far-uv and mid-ir regions we have studied structural organization of the complexes between dna and different proteins, including hmgb1, hmgb-domain recombinant proteins and linker histone h1. we have demonstrated, that interaction with dna leads to increasing both a-helicity of the proteins and thermal stability of dna. also, this interaction may result in formation of highly ordered supramolecular complexes facilitated by hmgb-domains. the c-terminal sequence of hmgb1/2 regulates affinity of the proteins to dna and can be ''inactivated'' by interaction with histone h1. based on the data obtained a model of the interaction of multy-domain hmgb-proteins with dna is suggested. darmstadt, germany, 4 lmu biozentrum; munich, germany *these authors contributed equally to this work chromatin in living cells displays considerable mobility on a local scale. this movement is consistent with a constrained diffusion model, in that individual loci execute multiple, random jumps. to investigate the connection between local chromatin diffusion (lcd) and the changes in nuclear organization, we established a stable hela cell line expressing gfp-pcna. this protein, a core component of the replication machinery, serves as a cell-cycle marker and allows us to visualize sites of ongoing dna synthesis within the nucleus. to monitor lcd, we labeled discrete genomic loci through incorporation of cy3-dutp. this experimental system, in conjunction with particle tracking analysis, has enabled us to quantitatively measure chromatin mobility throughout the cell cycle. our results demonstrate that lcd is significantly decreased in s-phase. to explore the connection between dna replication and reduced chromatin movement, we undertook a more detailed examination of lcd in s-phase nuclei, correlating chromatin mobility with sites of replication. our results demonstrate that labeled chromatin in close proximity to gfp-pcna foci exhibit significantly decreased mobility. we therefore conclude that presence of active replication forks constrains the movement of adjacent chromatin. single-molecule studies of dna replication antoine m. van oijen zernike institute for advanced materials, groningen university, nijenborgh 4. 9747 ag groningen, the netherlands e-mail: a.m.van.oijen@rug.nl advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. in a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important future direction will be the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. we are applying a single-molecule approach to study dna replication. i will present recent results of single-molecule studies of replication in bacterial and eukaryotic systems. by combining the stretching of individual dna molecules with the fluorescence observation of individual proteins, we visualize the dynamic interaction of replication factors with the fork. in the bacteriophage t7 replication system, we show that dna polymerases dynamically associate with and dissociate from the fork during replication. further, i will present new data from single-molecule replication studies in x. laevis oocyte extracts. we have developed a novel imaging scheme that permits single-molecule fluorescence experiments at concentrations of labeled protein that were hitherto inaccessible. using this method, we visualize, in real time, origin firing and fork movement. in force-extension diagrams of reference models of naked dna (freely jointed chain, wormlike chain) as well as extensionrotation diagrams of naked dna have been successfully recovered. of note, plectonemic structures are most efficiently simulated thanks to ode's collision detection code. new insights into nucleosome and chromatin fiber structure and dynamics will be presented. the study of the pkm.101 plasmid effect on the repair of dna j. vincze 1 , i, francia 2 , g. vincze-tiszay 1 1 hheif, budapest, hungary, 2 univ. debrecen, debrecen, hungary in our experiments was studied the effect of pkm.101 plasmid on repair of single strand breaks in dna induced by 60cogamma irradiation in e.coli k 12 ab 1157 (wild type) and its different rec mutant cells. the pkm.101 resistant-factor in case of the control decreases the sensitivity of radiation, which as we suppose, is reached by the help of dna conformation change. it can be supposed from the well known effect of radiation biology that by the effect of pkm.101, the ratio of dna radiation sensitive volumes by appearing its new conformation decreases. the pkm.101 r-factor in rec mutants in case of gamma irradiation shows effects in two ways. one is the ''chemical'' connection between the r-factor and dna, though the other relate to positive and negative ''induced'' radiation resistance from the local type effect of the connection of an r-factor and a rec mutant, and the two radiation resistant effects are added algebraically. as a result from the view of biology we have to categorize the radiation resistance and the connected repair processes as two different classes according to the change either in the chemical or in the induced radiation resistant effect. recent studies have indicated that two trimethylated peptides (k6, k7), derived from the parental hybrid peptide ca(1-7)m(2-9), strongly interact with a bacterial membrane model (mixture of zwitterionic and negatively charged lipids), but not with a membrane model of mammalian erythrocytes (zwitterionic lipids) [1] . a reduction of the cytotoxicity effect and an improvement of the therapeutic index have also been reported for the derivatives when compared with the parental ca(1-7)m(2-9) [2] . in this work, with the aim of providing insight on the interaction phenomena of the indicated peptides with zwitterionic and negatively charged membrane models, a systematic molecular dynamics study was carried out. full hydrated bilayers of dmpc:dmpg (3:1) and pope:popg(3:1) were studied in the presence of each peptide, and results analyzed in terms of peptide structure and membrane composition. lipid-water and lipid-lipid interactions at the membrane/water interface play important role in maintaining the bilayer structure, however, this region is not easily available for experimental studies. we performed molecular dynamics simulations of two bilayers composed of two different types of lipids: (1) dioleoylphosphatidylcholine (dopc); (2) galactolipid monogalactosyldiacylglycerol (mgdg). to investigate the properties of the membrane/water interface region, we performed analysis of lipid-lipid interactions: direct, via charge pairs (dopc) and hydrogen bonds (mgdg) as well as indirect, via water bridges. we also examined water-lipid interactions. existence of well-defined entities (lipids) linked by different types of interactions (hydrogen bonds, charge pairs, water bridges) makes the analysis of the membrane/ water interface region a suitable for a graph theoretical description. we applied a network analysis approach for comparative analysis of simulated systems. we note a marked difference between the organization as well as the dynamics of the interfacial region of the two bilayers. l-nucleoside analogues form an important class of antiviral and anticancer drug candidates. to be pharmacologically active, they need to be phosphorylated in multiple steps by cellular kinases. human phosphoglycerate kinase (hpgk) was shown to exhibit low specificity for nucleotide diphosphate analogues and its catalytic efficiency in phosphorylation was also affected. to elucidate the effect of ligand chirality on dynamics and catalytic efficiency, molecular dynamics simulations were performed on four different nucleotides (d-/l-adp and d-/l-cdp) in complex with hpgk and 1,3-bisphospho-d-glycerate (bpg). the simulation results confirm high affinity for the natural substrate (d-adp), while l-adp shows only moderate affinity for hpgk. the observed short residence time of both cdp enantiomers at the active site suggests very weak binding affinity which may result in poor catalytic efficiency shown for hpgk with d-/l-cdp. analysis of the simulations unravels important dynamic conditions for efficient phosphorylation replacing the single requirement of a tight binding. using the van der waals density functional based on the semilocal exchange functional pw86 together with a longrange component of the correlation energy [1] implemented in the siesta program code, we have calculated the band structure of the double stranded dna. the unit cell was built by taking together 10 gc (or at) homogenous base pairs and we have considered the translational symmetry as the periodic boundary condition. the results obtained are compared with the oligomer calculations taking up to seven base pairs. the band structure obtained with this van der waals density functional is also compared with results obtained with other exchange-correlation functionals as well as with band structure obtained by the hartree-fock crystal-orbital method taking into account the helical symmetry of the double stranded dna. the role of different parts of dna (base pairs, sugar-phosphate backbone, na ions) is also presented. transmembrane (tm) proteins comprise some 15% to 30% of the proteome but owing to technical difficulties, relatively few of these structures have been determined experimentally. computational modeling techniques can be used to provide the essential structural data needed to shed light on structurefunction relationships in tm proteins. low-resolution electrondensity maps, obtained from cryo-electron microscopy (cryo-em) or preliminary x-ray diffraction studies, can be used to restrict the search in conformational space. at the right resolution, the locations of tm helices can be roughly determined even when the amino acids are not visible. when these data are combined with physicochemical characteristics of amino acids (such as their hydrophobicity) and with evolutionary conservation analysis of the protein family, the location of the amino acids can be modeled. the modelstructure may provide molecular interpretations of the effects of mutations. moreover, it can be used to suggest molecular mechanisms and to design new mutations to examine them. the overall approach will be demonstrated using two human proteins: copper transporter 1 (ctr1), which is the main copper transporter in the human cell, and the 18 kda translocator protein (tspo) of the outer mitochondria. modelstructures of these proteins and their functional implications in health and disease will be discussed. calcium channels play a crucial role in many physiological functions and their selectivity mechanism is still an unresolved question and a subject of debate. a physical model of selective ''ion binding'' in the l-type calcium channel is constructed, and consequences of the model are compared with experimental data. this reduced model treats only ions and the carboxylate oxygens of the eeee locus explicitly and restricts interactions to hard-core repulsion and ion-ion and ion-dielectric electrostatic forces. according to the charge/space competition mechanism, the charge of structural ions attracts cations into the filter, while excluded volume effects are trying to keep them out. this is a competition between energy and entropy, where the balance of these terms minimizes free energy and determines selectivity. experimental conditions involving binary mixtures of alkali and/or alkaline earth metal ions are computed. the model pore rejects alkali metal ions in the presence of biological concentrations of ca2+ and predicts the blockade of alkali metal ion currents by micromolar ca2+. conductance patterns observed in varied mixtures containing na+ and li+, or ba2+ and ca2+, are predicted. ca2+ is substantially more potent in blocking na+ current than ba2+. in apparent contrast to experiments sing buffered ca2+ solutions, the predicted potency of ca2+ in blocking alkali metal ion currents depends on the species and concentration of the alkali metal ion, as is expected if these ions compete with ca2+ for the pore. these experiments depend on the problematic estimation of ca2+ activity in solutions buffered for ca2+ and ph in a varying background of ulk salt. equilibrium binding affinity (expressed as the occupancy of the selectivity filter by various ions) is computed by equilibrium grand canonical monte carlo (gcmc) simulations. the conductivity of the channel is estimated from the equilibrium concentration profiles using the integrated nernst-planck equation. our simulations show that the selectivity of l-type calcium channels can arise from an interplay of electrostatic and hard-core repulsion forces among ions and a few crucial channel atoms. the reduced system selects for the cation that delivers the largest charge in the smallest ion volume. we have also performed dynamic monte carlo (dmc) simulations for a model ca channel to simulate current directly and present our results for the dynamical selectivity (expressed as the flux carried by various ions). we show that the binding affinity of ca2+ versus na+ is always larger than the dynamical selectivity because ca2+ ions are tightly bound to the binding site of the selectivity filter of the channel and, at the same time, their mobility and drift velocity is smaller in this region. carotenoids are used in light-harvesting complexes with the twofold aim to extend the spectral range of the antenna and to avoid radiation damages. the effect of the polarity and conformation of the environment is supposed to be responsible for the tuning of the electronic, optical and vibrational properties of peridinin carotenoid both in solution and in protein matrix. we investigate the effect of vibrational properties of peridinin in different solvents by means of vibrational spectroscopies and qm/mm molecular dynamics simulations 1 . the shift of vibrational fingerprints in the 1500-2000 cm -1 frequency region, due to the solvent polarity and proticity, is studied in three cases: cyclohexane (apolar/aprotic), deuterated acetonitrile (polar/aprotic) and methanol (polar/ protic). the frequencies and vibrational modes of the carbonyl, the allene, and the polyene chain were identified using effective normal mode analysis 2 and compared with the present and previous experimental data 3 . on the basis of our calculations and experiments in different solvents, we propose a classification of the four peridinins of the high-salt pcp form. the controlled self-assembly of functional molecular species on well defined surfaces is a promising approach toward the design of nanoscale architectures. by using this methodology, regular low-dimensional systems such as supramolecular clusters, chains, or nanoporous arrays can be fabricated. small biological molecules such as amino acids represent an important class of building blocks that are of interest for molecular architectonic on surfaces because they inherently qualify for molecular recognition and selfassembly [1] . the interaction between amino acids and solid surfaces is decisive for the development of bioanalytical devices or biocompatible materials as well as for a fundamental understanding of protein-surface bonding. we investigate the adsorbtion mechanism of the cysteine on au(111) surfaces by means of the dft [2] . our main concern is to describe the molecule-metal bonding mechanism. therefore we present a complex study, including the full determination of the density of states for the free and adsorbed molecule, the determination of molecule-surface bonding energy. the method of crystal orbital overlap populations is used in order to determine the contribution of specific atomic orbitals to the molecule-metal bond. it is now widely accepted that myoglobin (mb) is not simply an o 2 storage/delivery system but, depending on oxygen concentration, it exerts other fundamental physiological roles. recent studies revealed a widespread expression and, in particular, an over-expression in response to hypoxia, in various non-muscle tissues, including tumor cells. in human five different mb isoforms are present. the two most expressed ([90%) differ only at the 54 th position, k54 (mb-i) and e54 (mb-ii) respectively. since high-altitude natives from tibet are characterized by a higher mb concentration and locomotion efficiency, together with the observation that the mb overexpression is totally attributable to mb-ii, the idea that the latter might be one of the responses to high-altitude evolutionary adaptation, i.e. hypoxic environment, started to emerge. however, this is not yet supported by any structure/function investigation. we performed hundred nanoseconds md simulations on human mbs to investigate the structure and dynamics of both protein and surface water. important differences have been protein kinases play key roles in cell signaling and constitute crucial therapeutic targets. in normal cell, upon substrate binding, tyrosine kinase receptor kit undergoes extensive structural rearrangement leading to receptor dimerization and activation. this process is initiated by the departure of the juxta membrane region (jmr) from the active site, allowing the activation loop (aloop) deployment. the deregulation of kit activity is associated with various forms of cancer provoked by abnormalities in signal transduction pathways. mutations v560g (jmr) and d816h/v (a-loop) have been reported as oncogenic and/or drug-resistant. to contribute further in the understanding of kit activation/ deactivation mechanisms, we applied a multi-approach protocol combining molecular dynamics (md), normal modes analysis (nma) and pocket detection. disturbing structural effects, both local (a-loop) and long-range (jmr), were evidenced for kit d816h/v in the inactive state. nma showed that jmr is able to depart its position more easily in the mutants than in the wild type. pockets analysis revealed that this detachment is sufficient to open an access to the atp binding site. our results provided a plausible conception of mutant dimerization and a way to explore putative allosteric binding sites. transmembrane association of leukocyte integrin heterodimer might be mediated by a polar interaction choon-peng chng 1 and suet-mien tan 2 1 biophysics group, a*star institute of high performance computing, and, 2 school of biological sciences, nanyang technological university, republic of singapore the lateral association of transmembrane (tm) helices is important to the folding of membrane proteins as well as a means for signaling across the cell membrane. for integrin, a hetero-dimeric protein important for cell adhesion and migration, the association of its a-and b-subunits' tm helices plays a key role in mediating bi-directional mechanical signaling across the membrane. we found evidence from experiment and simulation for a polar interaction (hydrogenbond) across leukocyte integrin alb2 tm that is absent in the better-studied platelet integrin aiibb3 [1] . our coarse-grained molecular dynamics simulations of tm helix-helix selfassembly showed more native-like packing achieved by alb2 within the simulation timescale as compared to its 'lossof-function' b2t686g mutant or aiibb3 [2] . association free energy profiles also showed a deeper minimum at a smaller helix-helix separation for alb2, suggestive of tighter packing. the likely conservation of this polar interaction across the b2 integrin family further reinforces its importance to the proper functioning of leukocyte integrins. active extrusion of drugs through efflux pumps constitutes one of the main mechanisms of multidrug resistance in cells. in recent years, large efforts have been devoted to the biochemical and structural characterization of rnd efflux pumps in gram-negative bacteria, in particular the acrb/a-tolc system of e.coli. specific attention has been addressed to the active part of the efflux system, constituted by the acrb unit. despite the presence of several data, crucial questions concerning its functioning are still open. the understanding of the structure-dynamics-function relationship of mexb, the analogous transporter in p. aeruginosa, encounters even more difficulties, because of the lack of structural data of the transporter in complex with substrates. to shade some light on the activity of mexb, we performed computational studies on mexb interacting with two compounds, meropenem and imipenem, the first known to be a good substrate, and the second a modest one. several techniques were used in the present work, ranging from flexible docking [1] to standard and targeted molecular dynamics (md) simulations. starting from the published crystal structure [2] we identified the most probable poses of the two compounds in both the original experimental and in the md-equilibrated structures. we used information from acrb binding pocket in order to find relevant binding sites of the two compounds in the analogous binding pocket of mexb. meropenem frequently lies with appropriate orientation in a pocket similar to the one identified for doxorubicin in acrb [3] , while the occurrence of imipenem poses in the same pocket is very scarce. additionally, when present in the pocket, imipenem is located in a position that renders very unlikely its extrusion toward the oprm docking domain during the simulation of the functional peristalsis. the analysis of the trajectories has provided a complete inventory of the transporter and antibiotic hot spots, which is key information in terms of screening and design of antibiotics and inhibitors. clathrins are three-legged proteins with the intriguing ability to self-assemble into a wide variety of polyhedral cages. the nucleation and growth of a clathrin lattice against the cytosolic face of a cell membrane enables the endocytosis of membrane proteins and various external molecules, by wrapping the membrane around the cargo to produce a coated transport vesicle within the cell. clathrins can also self-assemble, in slightly acidic solutions devoid of auxiliary proteins, into empty cages. our simulations of this process, using a highly coarsegrained model, indicate that the key to self-assembly is neither calthrin's characteristic puckered triskelion shape, nor the alignment of four legs along all cage edges, but an asymmetric distribution of interaction sites around the leg's circumference. based on the critical assembly concentration, the binding strength in these cages is estimated at 25 to 40 k b t per clathrin. the simulations also answer the long-standing conundrum of how flat patches of purely hexagonal clathrin lattices, which in cryo electron microscopy are frequently seen to decorate cell membranes, can produce highly curved cages containing twelve pentagonal faces interdispersed between hexagonal faces. we present experimental evidence supporting this pathway. in eukaryotic cells, the exchange of macromolecules between the cytoplasm and the nucleus is mediated by specialized transport factors. by binding to these transporters, cargo molecules, which are otherwise excluded from entering the nucleus, can traverse the nuclear pore complex efficiently. most of the proteins mediating nuclear import and export exhibit a characteristic _-solenoid fold, which provides them with an unusual intrinsic flexibility. crm1 is an essential nuclear export receptor, which recognizes a very broad range of export cargoes. crm1-dependent nuclear export is ran-gtpase-driven, and recognition of rangtp and cargo is highly cooperative. however, recent crystal structures show that the binding sites for export cargos and rangtp are located at distant parts of crm1 [1] [2] [3] . we have used a combined approach of all-atom molecular dynamics simulations and small-angle x-ray scattering to study rangtp and cargo binding to crm1. we have found that the allosteric effect in crm1-dependent nuclear export arises from a combination of subtle structural rearrangements and changes in the dynamic properties of crm1. light-induced phototactic responds in green algae chlamydomonas reinhardtii are mediated via microbial-type rhodopsins, termed channelrhodopsin-1 (chr1) and channelrhodopsin-2 (chr2)1, which carry the chromophore retinal covalently linked to lysin via a schiff base and were shown to be directly light-gated ion-channels2. the n-terminal putative seven-transmembrane region of chr2 was shown to be responsible for the generation of photocurrents and exhibits sequence similarity to the well understood proton pump bacteriorhodopsin (br) and the sensory rhodopsin anabaena sensory rhodopsin (asr)3. as for the majority of membrane proteins, there is no 3d-structural data for chr2 available yet. here we present homology models of chr2 using two high-resolution x-ray template structures of br (1qhj4) respectively asr (1xio5) in order to get structural and functional insights into chr2. with both homology models we performed molecular dynamics (md) simulations in a native membrane/solvent environment using gromacs 4.0.36. comparison of energetic and structural results revealed obvious advantages of the br-based homology model of chr2. here we show that the br-based homology model is a reliable model of chr2 exhibiting structural features already found experimen-tally7. our br-based homology model of chr2 allows predictions of putative crucial residues within chr2. so we proposed several mutations within the chr2 sequence which are already accomplished. electrophysiologic and spectroscopic studies of these mutations are underway in order to confirm the functional relevance of these residues and to contribute to an optimized usage of chr2 as a powerful tool in optogenetics. ( neuroglobin is a recently discovered globin protein predominantly expressed in brain. its biological function is still elusive. despite the fact that neuroglobin shares very little sequence homology to the well-known globins as mioglobin and hemoglobin, they all have a characteristic globin fold with heme molecule bound to the distal pocket. the structural investigations and co binding kinetics revealed existence of cavities and tunnels within the protein matrix, where small ligands can be stored even for hundreds of microseconds [1] . in human neuroglobin there is one internal disulfide bond possible which existence is proved to have significant effect on ligand affinity [2] . in this study effects of temperature, ph, distal histidine mutation and presence of disulfide bond on co rebinding to neuroglobin are investigated by flash photolysis experiments. in parallel, the molecular dynamics simulations are performed in corresponding conditions in order to investigate structural change of neuroglobin and especially changes in distribution of internal tunnels and cavities able to bind diatomic ligands in response to different physical conditions listed above. the thrombospondin family, being extracelluar proteins, is known to be implicated in various physiological processes such as wound healing, inflammation, angiogenesis and neoplasia. the signature domain of thrombospondins shows high sequence identities and thus allows us to transfer results obtained, studying this complex calcium reach part of the proteins, from one member of the family to the other. the domain is known to play a key role in hereditary diseases such as psach or med. in this part of thrombospondins lies a binding site to integrins, important for cell attachment. it is further known that the lectin like globe binds to cd-47, a feature known to be important in cancer research. as the theoretical unit we are trying to resolve these problems by numerical means and are constantly challenged by the size, where thrombospondin can be a huge trimeric protein as one strand can measure 430 kda, and the large variety of subdomains found in this proteins. we are thus facing a multiscale problem which can range from solving, by means of quantum mechanics a specific ion binding site, to large scale abstraction by continuum mechanics. in our talk we will show you our newest results that we obtained by simulating calcium rich c-terminal domain which is known to be conserved across the entire family, and give you an outlook into the future of our research. the process of swift heavy ions energy deposition while penetrating a solid or scattering on its surface can result in a strong and nonequilibrium excitation of matter. an extremely localized character of this excitation, meanwhile, can make possible both selective changes in chemistry of matter 1 and its surface nanomodifications 2 . since possible applications have been found in bio-and it-technologies (cancer curing and nanostructuring respectively) in the last decade, the heavy ion bombardment technique has attracted a lot of scientific interest 3, 4 . the processes of fast energy deposition into the solid and its further dissipation, however, are essentially perturbed with highly excited and nonequilibrium state of both lattice and electron systems. at such conditions therefore, the precision in treatment of processes of electron thermalization, fast electron heat conduction, and phase transformation of the overheated solid becomes crucial. having several physical models to handle the mentioned processes, it is nevertheless difficult to describe all of them within a scale of a single computational approach. our work is aimed on elaboration of the atomistic-continuum model 5 of heavy ion bombardment of solids. in particular, the model will be applied to study the formation of nanohillocks in the experiments on swift heavy ion xe + scattering on srtio 3 surface 2 . [3] g. aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). the presence of the human aquaporin 5 (hsaqp5) in cells proximal to airinteracting surfaces (eyes, lacrimal glands, salivary glands, lungs, stomach etc.) suggest its potentially important role in ''wetting'' these surfaces. the high-resolution x-ray structure of the hsaqp5 tetramer (pdb code 3d9s) exhibits two important features: (i) lack of the four fold symmetry, common in most of the aquaporins, and (ii) occlusion of the central pore by a phosphatidylserine lipid tail. in this study we investigate the importance of these two features on the transport properties of the human aqp5 by means of molecular dynamics simulations. we found that the asymmetry in the tetramer leads to a distribution of monomeric channel structures characterized by different free energy landscapes felt by the water molecules passing through the channel. furthermore, the structures' distribution is influenced both by the presence/absence of the lipid tail in the central pore, and by the lipid composition of the bilayer that solvates the hsaqp5 tetramer. elucidating the modular structure of the protein g c2 fragment and human igg fc domain binding site using computer simulations hiqmet kamberaj faculty of technical sciences, international balkan university, skopje, r. of macedonia protein-protein recognition plays an important role in most biological processes. although the structures of many protein-protein complexes have been solved in molecular detail, general rules describing affinity and selectivity of proteinprotein interactions break down when applied to a larger set of protein-protein complexes with extremely diverse nature of the interfaces. in this work, we will analyze the non-linear clustering of the residues at the interface between proteins. the boundaries between clusters are defined by clustering the mutual information of the protein-protein interface. we will show that the mutations in one module do not affect residues located in a neighboring module by studying the structural and energetic consequences of the mutation. to the contrary, within their module, we will show that the mutations cause complex energetic and structural consequences. in this study, this is shown on the interaction between protein g c2 fragment and human igg fc domain by combining molecular dynamics simulations and mutual information theory, and computational alanine scanning technique. the modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. the results test our understanding of the dominant contributions to the free energy of protein-protein interactions, can guide experiments aimed at the design of protein interaction inhibitors, and provide a stepping-stone to important applications such as interface redesign. membrane proteins can form large multimeric assemblies in native membranes that are implicated in a wide range of biological processes, from signal transduction to organelle structure. hydrophobic mismatch and membrane curvature are involved in long range forces largely contributing to such segregation. however, the existing assembly specificity is thought to be coded in the atomic details of protein surface and topology. these are best described in high resolution structures and atomistic molecular dynamics simulations. in order to explore more systematically such forces and energetics arising at intermediate time scales and resolution, we use coarse grained molecular dynamics simulations applied to 20 membrane systems spanning over 5 to 15us. as a first glimpse we study how proteins induce lipid perturbations using a previously developed conformational entropy estimator. we show that in the model membrane where hydrophobic mismatch is present, lipid perturbations extend up to *40a from the protein surface. however, significant variations in perturbation profiles are seen. parameters such as protein shape, surface topology, and amino acid physicochemical properties are studied to discover the parameters governing such perturbations. crossing energy barriers with self-guided langevin dynamics gerhard kö nig, xiongwu wu, bernard brooks national institutes of health, national heart, lung and blood institute, laboratory of computational biology, rockville, md, usa even with modern computer power, the applicability of molecular dynamics simulations is restricted by their ability to sample the conformational space of biomolecules. often high energy barriers cause normal molecular dynamics simulations to stay trapped in local energy minima, leading to biased results. to address this problem, self-guided langevin dynamics (sgld) were developed. it enhances conformational transitions by accelerating the slow systematic motions in the system. this is achieved by calculating the the local average of velocities and adding a guiding force along the direction of systematic motions. thus, the efficiency of sgld is governed by three factors: a.) the friction constant involved in the langevin dynamics b.) the local averaging time and c.) the guiding factor that determines the guiding force. however, the guiding force also causes deviations from the original ensemble that have to be corrected by reweighting the data, thus decreasing the efficiency. here, we explore the three-dimensional parameter space of sgld for several benchmark systems with particularly rough energy surfaces. based on our data, we supply guidelines for the optimal selection of sgld parameters, to allow the extension of our method to other biological problems of interest. propagation of d816v/h mutation effects across kit receptor e. laine, i. c. de beauchê ne, c. auclair and l. tchertanov lbpa, cnrs -ens de cachan, france receptor tyrosine kinases (rtks) regulate critical biological processes. constitutive activation of rtks provokes cancers, inflammatory diseases and neuronal disorders. biological data evidenced that oncogenic mutations of the rtk kit, located either in the juxtamembrane region (jmr) or in the activation loop (a-loop) -as is the case of d816v/h, displace the equilibrium of conformational states toward the active form. we present a multi-approach study that combines molecular dynamics (md), normal modes (nm) and pocket detection to characterize and compare the impact of d816v/h on the structure, dynamics and thermodynamics of kit. we have evidenced a local structural destabilization of a-loop induced by the mutation and a long-range effect on the structure and position of jmr. we have further correlated these observations with experimental data and decipher some details about the activation mechanisms of the mutants, involving leverage of the jmr negative regulation and release of an access to the catalytic site. through the identification of ''local dynamic domains'' and the recording of interactions within the protein, we propose a model of the mutational effects propagation, which highlights the importance of both structural distortion and local conformational fluctuations. investigation of biological active azolidinones and related heterocycles refer to one of the most successful scientific projects of dh lnmu. it is based on three strategic vectors: organic synthesis, pharmacological research, rational design of ''drug-like'' molecules (including in silico approaches). while applying the research strategy we succeeded in gaining a number of interesting results that make possible to extend the field, especially in the scope of ''drug-like'' molecules design, notably it has focused on the search of new anticancer agents. anticancer activity screening was carried out for more than 1000 compounds (us nci protocol (dtp) based on obtained directed library over 5000 new compounds, among them 167 compounds showed high activity level. for the purpose of optimization and rational design directions of highly active molecules with optimal ''drug-like'' characteristics and discovering of possible mechanism of action sar, compare analysis, molecular docking and qsa(p)r were carried out. obtained results allowed to form main directions of possible anticancer activity mechanisms, which probable are apoptosis-related. nowadays the investigation of cellular and molecular aspects of anticancer effects is in progress. regulation of (bio)chemical reactions by mechanical force has been proposed to be fundamental to cellular functions [1, 2, 3] . atomic force microscopy experiments have identified the effect of mechanical force on the reactivity of thiol/disulfide exchange, a biologically important reaction [4] . in order to understand the influence of the force at an atomistic level, we have performed hybrid quantum mechanicsmolecular mechanics (qm/mm) transition path sampling simulation of the reaction under external forces. the results of the simulations supported the experimental findings and demonstrated that the location of the transition state on the free energy surface was shifted due to force [5] . in contrast to our findings, however, a recent experimental study suggests only a weak coupling between the mechanical force and the reaction rate [6] . in this study, the reactants were covalently linked to a stilbene molecule. in this system a force can be applied by photo-isomerization from the relaxed trans to the strained cis configuration. a drawback of this system is that one cannot easily determine the forces that acting on the reaction coordinate. therefore, we have developed a force distribution analysis method for quantum mechanical molecular dynamics simulations. the results of the analysis show how isomerization of stilbene alters the forces acting on the reacting atoms. the force distribution is essential for understanding how chemistry is controlled by external forces. [ conformational space modelling (csm) is a promising method for membrane protein structure determination. it is based on the concept of the side-chain conformational space (sccs), which is formed by the allowed side-chain conformations of a given residue. each sccs can be calculated from a 3d structure or measured via epr-sdsl experiments. for structure determination a set of singly spinlabelled mutants is needed. the final structure is obtained by altering an initial (possibly random) 3d structure until the best fit between the calculated and measured sccs for the whole set is found. such optimization is computationally intensive; therefore csm includes several empirical approximations. one of them describes the effect of the lipid tails on the sccs. the implementation is not trivial as lipids diffuse in the plane of the membrane and the lipid tails behave differently at different membrane depths. to unravel this relationship adaptive biasing force md simulations were used. an alanine peptide helix was made in silico, spin-labelled at the middle and inserted perpendicularly into a dmpc membrane. the free energy of the spin-label orientation at various membrane depths was calculated. a 3d free energy surface describing the membrane ''depth'' effect was obtained. it is known that b-cyclodextrins (bcds) are able to modify the cholesterol content of cell or model membranes. however the molecular mechanism of this process is still not resolved. using molecular dynamics simulations, we have been able to study the bcd-mediated cholesterol extraction from cholesterol monolayers and lipid-cholesterol monolayer mixtures. we have investigated many conditions that would affect this process (e.g. lipid-cholesterol ratio, lipid chain unsaturation level) our results can be summarized as follow: i) dimerization of bcds, ii) binding of the dimers to the membrane surface assuming either a tilted (parallel to the membrane normal) or untilted (90°respect to the normal of the membrane) configuration, iii) the latter configuration is suitable to extract cholesterol at a reasonable computational time scale (100-200 ns), however, this process may be affected by unfavorable energy barriers (from 10 to 80 kj mol -1 ), iv) desorption of the complex brings cholesterol in solution, v) the bcd-cholesterol complex is able to transfer cholesterol to other membranes. with a clearer understanding of the basic molecular mechanism of the bcd mediated process of cholesterol extraction, we can begin to rationalize the design of more efficient bcds in numerous applications. the mechanism of the complex formation of biopolymers with ligands including the solvent molecules is an actual problem of modern biophysical and biological science. polypeptides form a secondary structure and mimic the motifs of the protein architecture. the study of complexation between polypeptides and solvent molecules leads to deeper understanding of the basic interaction of proteins with environment at atomic level. besides polypeptides are promising for the development of applications which encompass some of the following desirable features: anti-fouling, biocompatibility, biodegradability, specific biomolecular sensitivity. on this account polypeptides have a great significance for a variety modern applications ranging from the nanoscale medicine devises up to food technology and others. we compare the results of calculations of complexes between helical polypeptides (polyglutamic acid in neutral form and poly-c-benzyl-l-glutamate) and water molecules at dft pbe level and the results of ftir-spectroscopic study of the film of wetted polypeptides. vibrational spectroscopy is one of the most useful experimental tools to study non-covalent bonded complexes, and calculated spectra in comparison with experimental data are reliable test for reality of simulated complexes. platelet aggregation at the site of vascular injury is vital to prevent bleeding. excessive platelet function, however, may lead to thrombus formation after surgery. therefore, an accurate measure and control of platelet aggregation is important. in vitro platelet aggregometry monitors aggregate formation in platelet rich plasma triggered by agonists such as adp, epinephrine or collagen. the fraction of aggregated platelets is plotted versus time, and platelet function is assessed by analyzing the plot's morphology. we propose new measures of platelet function based on a compartmental kinetic model of platelet aggregation induced by adp. our model includes three compartments: single, aggregated and deaggregated platelets. it is simpler than earlier models and agrees with experimental data. the model parameters were determined by non-linear least squares fitting of published data. we associated the kinetic parameters with the activity of the adp receptors p2y1 and p2y12. to this end, we studied published data obtained in the presence and in the absence of specific antagonists of these receptors. comparison of kinetic parameters of healthy subjects with those of patients with myeloproliferative disorder (mpd) shows that the function of p2y12 is significantly reduced in mpd. coarse-grained modeling of drug-membrane interactions manuel n. melo, durba sengupta, siewert-jan marrink groningen biomolecular sciences and biotechnology institute, university of groningen, groningen, the netherlands the martini coarse-grained (cg) forcefield was used to simulate the actions of the antimicrobial peptide alamethicin and of the anti-tumoral drug doxorubicin. both drugs were shown to interact strongly with a fluid phospholipid bilayer, and aggregate there, in agreement with experimental results. because doxorubicin may establish intermolecular h-bonding, and thus lower its dipole moment, the cg representation of a doxorubicin dimer was adjusted. this less polar dimer was then tested for translocation and/or pore formation. contrary to results of atomistic simulations, alamethicin aggregates did not spontaneously open pores. they did so, however, when the size of the water beads was decreased. several small independent pores could then be observed. the magnitude of the permeability of these pores is analyzed and compared to experimental values. the occurrence of multiple small pores could indicate that the different conductance levels experimentally observed for alamethicin might simply result from the association of different numbers of these small pores. polarization refers to the asymmetric changes in cellular organization in response to external or internal signals. neuronal polarization begins with the growth of a single neurite shortly after cell division, followed by the growth of a second neurite at the opposite pole. this early bipolar shape is critical for brain function, as it defines axis of migration and consequently proper three dimensional organization and nerve circuitry. however, it is not known if a direct relationship exists between the formation of the second, opposite, neurite and the mechanisms involved in the formation of the first. we tackled this issue through mathematical modeling, based on membrane traffic (exocytosis-endocytosis), and lateral diffusion. with this approach, we demonstrated that a single pole of molecular asymmetry is sufficient to induce a second one at the opposite side, upon induction of growth from the first pole. our work gives mathematical proof that the occurrence of a single asymmetry in a round cell is sufficient to warrant morphological bipolarism. trypsin is one of the best characterized serine proteases. enzyme acylation process is required for substrate degradation. there is a lot of information about how this process undergoes. however, in order to obtain a more detailed description of the catalytic triad mechanism, a qm/mm approach was used. we used the hybrid qm/mm potential implemented in amber11 package. in the qm calculations a dft hamiltonian was used. we develop an approach based on the adaptively biased md in order to obtain the free energy surface of the conformational space defined by the reaction coordinates. this approach presents some characteristics of steered md and umbrella sampling procedures. our results offer information about the lowest energy trajectory, the barrier profile of the reaction, and the geometry of the transition state. this method also provides a further insight into the role of specific residues in the reaction. substituting asp102, member of the catalytic triad, for ala we were able to detect an increase of the barrier profile. this was due to the loss of the interaction of carbonyl group of asp102 with nd of his57, which make ne of this residue a worst proton acceptor. this results show our approach as a valuable method in the study of enzymatic mechanisms. the intracellular media comprise a great variety of macromolecular species that are not individually concentrated, but being preset in the same compartment they exclude each other's volume and produce crowding. crowding has a profound impact on protein structure and determines conformational transitions and macromolecular association. we investigated macromolecular association on a 50% w/w bovine serum albumin (bsa) solution by time-domain terahertz (thz) spectroscopy and molecular modeling. molecular crowding was simulated by including two bsa molecules in the same water box. we generated *300 such dimeric models, computed their thz spectra by normal modes analysis and compared them with the experimental data. the best bsa dimer model was selected based on the agreement with the experiment in the lowest frequencies domain of up to 1 thz. symmetry constraints improve accuracy of ion channels models. application to two-pore-domain channels adina l. ion channels are important drug targets. structural information required for structure-based drug design is often filled by homology models (hm). making accurate hm is challenging because few templates are available and these often have substantial structural differences. besides, in molecular dynamics (md) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate hm refinement using md. we evaluate the ability of symmetry-constrained md simulations to improve hm accuracy, using an approach conceptually similar to casp competition: build hm of channels with known structure and evaluate the efficiency of various symmetry-constrained md methods in improving hms accuracy (measured as deviation from x-ray structure). results indicate that unrestrained md does not improve accuracy, instantaneous symmetrization improves accuracy but not stability during subsequent md, while gradually imposing symmetry constraints improves both accuracy (by 5-50%) and stability. moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting hm accuracy. we further used this method to refine hm of trek channels. we also propose a gating mechanism for mechanosensitive channels that was further experimentally confirmed. nucleotide modifications and trna anticodon-mrna codon interactions on the ribosome olof allné r and lennart nilsson karolinska institutet, stockholm, sweden molecular dynamics simulations of the trna anticodon and mrna codon have been used to study the effect of the common trna modifications cmo 5 u34 and m 6 a37. in trna val these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. previous data suggest entropic effects are mainly responsible for the extended reading capabilities but detailed mechanisms have remained unknown. the aim of this paper is to elucidate the details of these mechanisms on an atomic level and quantify their effects. we have applied: extensive free energy perturbation coupled with umbrella sampling, entropic calculations of trna (free and bound to the ribosome) and thorough structural analysis of the ribosomal decoding center. human neuroserpin (hns) is a serine protease inhibitor (serpin) of a tissue-type plasminogen activator (tpa). the conformational flexibility and the metastable state of this proteins underlies to misfolding and to dysfunctional mutations causing a class of rare genetic diseases which share the same molecular basis. the conformational transition of the native form, triggered upon the cleavage at reactive center loop (rlc), releases a complex of the cleaved form bound to the inactivated target protease. without rlc cleavage a stable inactive latent form can be obtained by intra/inter molecular loop insertion leading to polymerization. this work concerns the study of the three above mentioned forms of hns by md simulations to investigate the relation between their conformational stability and. the starting native and cleaved configurations are based on the x-ray structure, while the latent form is here modelled. the results of the simulation reveal a striking conformational stability along with the intrinsic flexibility of selected regions of the fold.the analysis of the essential collective modes of the native hns shows that the initial opening of the b-sheet a coincides with several changes in the local pattern of salt bridges and of hydrogen bonds. regulation of ubiquitin-conjugating enzymes: a common mechanism based on a pattern of hydrophobic and acidic residues enzyme temperature adaptation generally involves a modulation of intramolecular interactions, affecting proteins dynamics, stability and activity [1] [2] . in this contribution, we discuss studies of different classes of extremophilic enzymes, focusing on cold-adapted variants, as well as their mesophilic-like mutants, performed by all-atom molecular dynamics simulations with particular attention to structural communication among residues within the threedimensional architecture [3] [4] . common adaptation strategies turned out to be based on improved local flexibility in the proximity of the functional sites, decrease in interconnected electrostatic interactions, and modulation of correlated motions and networks of communicating residues. specific differences related to the diverse protein folds can also be detected. bneurexins and neuroligins are cell adhesion molecules and play important role in synapse junction formation, maturation and signal transduction between neurons. mutations in genes coding these proteins occurs in patients with cognitive diseases like autism disorders, asperger syndrome and mental retardation [1] . it has been found that the complex bneurexin-neuroligin has also an important role in angiogenesis [2] . herein we will present molecular foundations of bneurexin-neuroligin interactions obtained from all-atom molecular dynamics simulations of bneurexin, neuroligin and their complex (3b3q) [3] . 50 ns md trajectories (charmm force field) were analyzed and roles of ca2+ and n-actetyl-d-glucosamine posttranslational modifications in intermolecular interactions were scrutinized. advances in hardware and software have enabled increasingly long atomistic molecular dynamics simulations of biomolecules, allowing the exploration of processes occurring on timescales of hundreds of microseconds to a few milliseconds. increasing the length of simulations beyond the microsecond time scale has exposed a number of limitations in the accuracy of commonly employed force fields. such limitations become more severe as the size of the systems investigated and the length of the simulations increase. here i will describe the force field problems that we have encountered in our studies, how we identified and addressed them, and what we have learned in the process about the biophysics of the systems we are investigating. while the quest for a ''perfect'' force field is not over (and may never be), our work has greatly improved the accuracy and range of applicability of simple physics-based force fields, to the point that reliable predictions can now be obtained from millisecond-timescale simulations of biomolecules. local anesthetics (la) are pain-relief drugs, widely used in medicine and dentistry. the relatively short duration of analgesia still restricts their clinical use for the treatment of chronic pain. nowadays, intensive research is focused on anesthetics entrapped into liposomes to enhance their activity and pharmacokinetic properties [1] . in this work, we investigated the encapsulation of prilocaine (plc), an aminoamide local anesthetic, into a small unilamellar liposome. on the line of our previous work [2] , we have carried out molecular dynamics (md) simulations using a coarse grain model up the microsecond time scale. in this way, we compare the effects of the concentration of la at fisiological ph. we were able to capture important features of the plc-vesicle interactions. the behavior of plcs at fisiological ph is essentially a combination of high and low ph: we found that all neutral plc molecules rapidly diffuse into the hydrophobic region of the vesicle adopting an asymmetric bimodal density distribution. protonated plc molecules (pplc) initially placed in water were instead only found on the external monolayer, with a high rate of exchange with the water phase and no access to the inner part of the liposome in a concentration dependent way. we focus on applications of molecular and mesoscale simulation methodologies to the cellular transport process of endocytosis, i.e., active transport mechanisms characterized by vesicle nucleation and budding of the cell membrane orchestrated by protein-interaction networks, and functionalized carrier adhesion to cell surfaces. we discuss theoretical and computational methodologies for quantitatively describing how cell-membrane topologies are actively mediated and manipulated by intracellular protein assemblies. we also discuss methods for computing absolute binding free energies for carrier adhesion. we present rigorous validation of our models by comparing to diverse range of experiments. the importance of delta-opioid receptors as target of a large number of drugs is well recognized, but the molecular details of interaction and action of the compounds are largely unknown. in an effort to shade some light on this important issue we performed an extensive computational study on the interaction of two compounds, clozapine and desmetilclozapine, with a delta-opioid receptor. according to experiments, the lacking of a single methyl group in desmetilclozapine with respect of clozapine makes the former more active than the latter, providing a system well suited for a comparative study. we investigated stable configurations of the two drugs inside the receptor by simulating their escape routes by metadynamics, an algorithm that allows the simulation of events that are otherwise out of range for standard molecular dynamics simulations. our results point out that the action of the compound is related to the spatial distribution of the affinity sites it visits during its permanency. desmetilclozapine has a larger distribution of residues, which is interacting with, than clozapine. however, large conformational changes of the receptor were not observed in the presence of both compounds. thus, a more dynamical picture of ligand-receptor affinity is proposed on the basis of the results obtained, involving the competition among different stable states as well as the interaction with the solvents. such information might be useful to provide hints and insights that can be exploited in more structure-and-dynamics-oriented drug design. the coupling between the mechanical properties of enzymes and their biological activity is a well-established feature that has been the object of numerous experimental and theoretical works. in particular, recent experiments show that enzymatic function can be modulated anisotropically by mechanical stress. we study such phenomena using a method or investigating local flexibility on the residue scale, which combines a reduced protein representation with brownian dynamics simulations. we performed calculations on the enzyme guanylate kinase in order to study its mechanical response when submitted to anisotropic deformations. the resulting modifications of the protein's rigidity profile can be related to the changes in substrate binding affinities that were observed experimentally. further analysis of the principal components of motion of the trajectories shows how the application of a mechanical constraint on the protein can disrupt its dynamics, thus leading to a decrease of the enzyme's catalytic rate. eventually, a systematic probe of the protein surface led to the prediction of potential hotspots where the application of an external constraint would produce a large functional response both from the mechanhiv-1 protease autocatalyses its own release from gag and gagpol precursor polyproteins into mature functional proteins. as it is functional in the dimeric form, whilst initially, only a single monomer is embedded within each gagpol chain, the question arises as to what cut's the cutter. two individual monomers in different gagpol chains are known to come together to form an embedded-dimer precursor protease. mature-like protease activity is concomitant with n-terminal intramolecular cleavage of this transient embedded-dimer precursor, but how this crucial maturation-initiating step is physically achieved, has remained unknown. here, we show via 400 all-atom explicit solvent molecular dynamics simulation runs of 400 ns each that the n-terminal of an immature-like protease, with the n-terminal initially unbound as in the gagpol polyprotein, can self-associate to the active site and therefore be cleaved under conditions of thermodynamic equilibrium, identifying possible binding pathways at atomic resolution, in agreement with previous indirect experimental evidence [1] . the binding pathway predominantly makes use of the open conformation of the beta-hairpin flaps characterised by us previously [2] , and the n-terminal binds across the entire active site in good agreement with crystal structures of a cleavage-site peptidebound protease. the n-terminus serves two roles, firstly in the maturation of the protease itself by self-associating to the protein and then as a stabilizing component of the dimer interface in a mature protease. targeting the prior mechanism could be the focus of a novel therapeutic strategy involving immature protease inhibition. [ knotted proteins are the object of an increasing number of experimental and theoretical studies, because of their ability to fold reversibly in the same topologically entangled conformation. the topological constraint significantly affects their folding landscape, thus providing new insight and challenges into the funnel folding theory [1] . recently developed experimental methods to trap and detect knots have suggested that denaturated ensembles of the knotted proteins may be knotted [2] . we present numerical simulations of the early stage of folding of the knotted proteins belonging to protein families mtase (methyltransferase) and sotcase (succinyl-ornithine transcarbamylase), and of their unknotted homologues [3] . our results show that native interactions are not sufficient to generate the knot in the denaturated configurations. however, when non-native interactions are included we observe formation of knots only in the chains whose native state is knotted. in addition, we find that the knots are formed through a universal mechanism. such a knot formation mechanism correctly predicts the fraction of the knotted proteins found in nature and can be used to make qualitative prediction on their folding times. shape and motility and also for numerous signaling processes. adhesion is based on non-covalent interactions between transmembrane proteins and the extracellular matrix. cells are able to create two-dimensional assemblies of integrins, so called focal adhesions, which they use to stick to the substrate and collect information about the environmental properties. the goal of this work is a deeper understanding of the formation and the stability of these adhesion clusters. bond cluster formation and disintegration is dynamically modeled with the aid of monte carlo simulations. in the model, a membrane is attached to a flat surface via a variable number of adhesion bonds. the spacial configuration of these adhesion points subjected to an inhomogeneous stress field maps into a distribution of local membrane/ surface distances. we introduce a model which explicitly accounts for the membrane elasticity and demonstrate that such models are able to explain the spontaneous formation of adhesion bond clusters. structure based models are successful at conjugating the essence of the energy landscape theory of protein folding [1] with an easy and efficient implementation. recently their realm expanded beyond single protein structure, been used profitably to widely study large conformational transitions [2] [3] . still, when dealing with conformational transition between two well-defined structures an unbiased and realistic description of the local backbone and sidechain interactions is necessary. the proposed model merges a precise description of these interactions with a structure-based long range potential that takes into account different conformers. we present the results of the activation of the catalytic domain of human csrc tyrosine kinase for which we reconstructed the transition free energy and the description of the activation loop flexibility. the excellent model performances in terms of speed and the satisfactory accuracy of the description of the system and its flexibility are promising for a more systematic study of the tyrosine kinase family activation mechanisms. [ we introduce a previously undescribed technique for modelling the kinetics of stochastic chemical systems. we apply richardson extrapolation, a sequence acceleration method for ordinary differential equations, to a fixed-step tau-leaping algorithm, to produce an extrapolated tau-leaping method which has weak order of accuracy two. we prove this mathematically for the case of linear propensity functions. we use four numerical examples, two linear and two nonlinear, to show the higher accuracy of our technique in practice. we illustrate this by using plots of absolute error for a fixed-step tau-leap and the extrapolated tau-leap. in all cases, the errors for our method are lower than for a fixedstep tau-leap; in most cases they are second order of accuracy. the major tripartite efflux pump acrab-tolc is responsible for the intrinsic and acquired multidrug resistance in escherichia coli. at heart of the extrusion machinery there is the homotrimeric transporter acrb, which is in charge of the selective binding of structurally and chemically different substrates and energy transduction. the effects of conformational changes, which have been proposed as the key features of the extrusion of drugs, are investigated at molecular level using different computational methods like targeted molecular dynamics. simulations, including almost half a million atoms, have been used to assess several hypotheses concerning the structure-dynamics-function relationship of the acrb protein. the results indicate that, upon induction of conformational changes, the substrate detaches from the binding pocket and approaches the gate to the central funnel. in addition, we provide evidence for the proposed peristaltic transport involving a zipper-like closure of the binding pocket, responsible for the displacement of the drug. using these atomistic simulations the role of specific amino acids during the transitions can be identified, providing an interpretation of sitedirected mutagenesis experiments. additionally, we discuss a possible role of water molecules in the extrusion process. virus inhibitory peptide (virip), a 20 amino acid peptide, binds to the fusion peptide (fp) of human immunodeficiency virus type 1 (hiv-1) gp41 and blocks viral entry. molecular dynamics (md) and molecular mechanics/poisson-boltzmann surface area (mm/pbsa) free energy calculations were executed to explore the binding interaction between several virip derivatives and gp41 fp. a promising correlation between antiviral activity and simulated binding free energy was established thanks to restriction of the flexibility of the peptides, inclusion of configurational entropy calculations and the use of multiple internal dielectric constants for the mm/pbsa calculations depending on the amino acids sequence. bases on these results, a virtual screening experiment was carried out to design enhanced virip analogues. a selection of peptides was tested for inhibitory activity and several improved virip derivatives were identified. these results demonstrate that computational modelling strategies using an improved mm/pbsa methodology can be used for the simulation of peptide complexes. as such, we were able to obtain enhanced hiv-1 entry inhibitor peptides via an mm/pbsa based virtual screening approach. an essential step during the hiv life cycle is the integration of the viral cdna into the human genome. hiv-1 integrase mediates integration in a tight complex with the cellular cofactor: ledgf/p75 [1] . disruption of the interaction interferes with hiv replication and therefore provides an interesting new drug target for antiretroviral therapy [2, 3] . here we present the structure based discovery and optimization of a series of small molecule inhibitors that bind to hiv-1 integrase and block the interaction with ledgf/p75. the work flow was set up according to a funnel principle in which a series of virtual screening tools were applied in such way to discard at each step molecules unlikely to be active against the desired target (including 2d filtering, pharmacophore modelling and molecular docking) the activity and selectivity of the selected molecules were confirmed in an alpha screen based assay, that measure protein-protein interaction in vitro, and furthermore by in vivo experiments. active compounds proceeded towards crystallographic soaking into the receptor protein crystals. these crystal structures not only validated the binding mode and activity of the hit compounds, but furthermore were used as a platform for structure based drug design which resulted in the rational discovery of new hit compounds and optimized lead compounds. in vitro and in vivo experiments validated the mechanism of action of these compounds and show that they are a novel class of antiretroviral compounds with in vivo inhibitory activity by targeting the interaction between ledgf/p75 and hiv-1 integrase. cross resistance profiling indicate that these compounds are active against current resistant viral strains. [4] currently the most potent inhibitors show an in vivo ic 50 of 55nm. these compounds are promising for future pharmaceutical optimizations to be used in the clinic as new antiretroviral agents. crystallography was used to validate the binding mode of the discovered inhibitors. insights in the interaction of the ligand-protein complex allowed for rational design of optimized inhibitors. ligand-induced structural changes are small, thermal fluctuations can play a dominant role in determining allosteric signalling. in thermodynamic terms, the entropy change for subsequent binding is influenced by global vibrational modes being either damped or activated by an initial binding event. one advantage of such a mechanism is the possibility for long range allosteric signalling. here, changes to slow internal motion can be harnessed to provide signalling across long distances. this paper considers homotropic allostery in homodimeric proteins, and presents results from a theoretical approach designed to understand the mechanisms responsible for both cooperativity and anticooperativity. theoretical results are presented for the binding of camp to the catabolite activator protein (cap) [1] , where it is shown that coupling strength within a dimer is of key importance in determining the nature of the allosteric response. results from theory are presented along side both atomistic simulations and simple coarse-grained models, designed to show how fluctuations can play a key role in allosteric signalling in homodimeric proteins. [ reversibly switchable fluorescent proteins (rsfps) can be switched between a fluorescent (on) and a nonfluorescent (off) state which is accompanied by a cis-trans isomerization of the chromophore on the molecular level 1,2. this unique property has already provided new aspects to various microscopy techniques, such as high resolution microscopy, fcs or monochromatic multicolor microscopy 3-5. despite of their established potential, rsfps still have a major drawback: the wavelength for fluorescence excitation is always one of the two switching wavelengths. the imaging process thus inevitably results in the switching of a small fraction of the rsfps, which might hinder or complicate some experiments. we developed a new reversibly switchable fluorescent protein which eliminates the problem of the coupling between switching and fluorescence excitation. this fluorescent protein follows an unusual and currently unknown mechanism of switching between a fluorescent and a nonfluorescent state. it is brightly fluorescent and exhibits an excellent signal to noise ratio. in parallel studies [2] , qd-based ligands (egf, mabs) were targeted to egfr in gliomas. cell-cultures, animal models and ex vivo biopsies of human high-grade as well as low-grade gliomas showed high probe specificity.. the aim is to define more precisely the tumor boundaries at the time of resection. we used the programmable array microscope designed for sensitive, high-speed optical sectioning, particularly of living cells. the pam is based on structured illumination and conjugate detection using a digital micromirror device (dmd) [3] located in a primary image plane. the unique feature is the rapid, (re)programmable adjustment of local excitation intensity, dynamic, on-the-fly optimization is thus achieved, e.g. multipoint frap [4] , light exposure minimization and object tracking [5] , or super-resolution strategies. the features and operation of the 3rd generation pam will be presented. contraction of muscle cells, motility of microorganisms, neuronal activity, and other fast cellular processes require microscopic imaging of a three-dimensional (3d) volume with a video-rate scanning. we present 3d video-rate investigations of structural dynamics in biological samples with the multicontrast third-and second-harmonic generation as well as fluorescence microscope. the multidepth scanning is achieved by two combined laser beams with staggered femtosecond pulses. each of the beams is equipped with a pair of deformable mirrors for dynamic wavefront manipulation enabling multidepth refocusing with simultaneous corrections for optical aberrations. combined, more than 250 frames per second lateral scanning with fast refocusing enables the 3d video-rate imaging of dynamically moving structures. in addition, combination of two laser beams is accomplished at two perpendicular polarizations enabling live imaging of sample anisotropy, which is important for structural studies particularly with the second harmonic generation microscopy. investigations of beating chick embryo hearts with the 3d video-rate scanning microscope revealed multidirectional cardiomyocyte contraction dynamics in myocardial tissue. intricate synchronization of contractions between different layers of myocytes in the tissue will be presented. the video-rate 3d microscopy opens new possibilities of imaging fast biological processes in living organisms. confocal fluorescence microscopy is an invaluable tool to study biological systems at cellular level also thanks to the synthesis of always new specific fluorescent probes, e.g. multiprobe labelling enables complex system characterization. however, only the recent employment of narrowband tunable filters overcomes the problems due to the use of the broadband ones. the possibility of acquiring the emission spectra in a spatially resolved way extends simple image intensity studies into characterization of complex probeenvironment relationship through the sensitivity of fluorescence spectra to the local molecular environment differences. consequently, fluorescence microspectroscopy (fms) is able to provide the spectral information in a well defined spatial region allowing the researcher to simultaneously obtain spatial and spectroscopic information. our instrument has been specially built to study live cells and their interaction with nanomaterials, drug carriers and modified cell environment. other main characteristics are reducing the bleaching effect and employing a white light source that does not limit the use to specific probes. graphical tools, such as colour coded images, have also been introduced to provide explicit and straightforward visual information. high speed fpga based multi-tau correlation for singlephoton avalanche diode arrays jan buchholz 1,2 , jan krieger we demonstrate the use of fret-imaging (forster resonance energy transfer) as an assay to directly monitor the dynamics of cross-bridge conformational changes in single fibres of skeletal muscle. we measured nm-distances of several fret pairs located at strategic positions to sense myosin head conformational changes: we focused our attention on the essential light chain, elc (specifically labelling a modified elc and exchanging it with the natural elc of the fibre) and we investigated its interaction with the sh1 helix, with the nucleotide binding pocket and with actin. we characterized fret in single rigor muscle fibres, determining distances in agreement with those from the crystallographic data. the results demonstrate the viability of the approach in sensing different fret efficiencies over a few nanometres, an essential requirement to follow the expected small fret variations in contracting muscle fibres. we are now performing dynamic experiments on rigor and active fibres by applying small stretch/release cycles to alter the interaction distances (estimated time resolution of nearly 20ms/frame). in this configuration, it will be possible to measure functional changes, shedding light on the myosin head dynamics during contraction. focal stimulation of cultured neurons is crucial since it mimics physiological molecules release. indeed, the nervous system finely tunes the activity of each synapse by regulating the secretion of molecules spatially and temporally. currently used techniques have some drawbacks such as a poor spatial resolution or a low flexibility. we propose a novel approach based on optical tweezers (ot) [1] to overcome these limitations. ot allow an ease manipulation with sub-micrometric precision of silica beads, which can be functionalized with any protein. for a proof-of-principle study we coated 1,5lm large beads with brain-derived neurotrophic factor (bdnf) or bovine serum album (bsa) as control. we showed that a single bead was able to activate the bdnf receptor trkb, inducing its phosphorylation. moreover bdnf beads but not control beads were able to induce c-fos translocation into the nucleus [2] , indicating that the whole pathway was activated. finally, we positioned vectors in proximity to the growth cones of cultured hippocampal neurons [3] . control beads didn't affect the normal development of these structures while bdnf beads significantly did. these findings support the use of the ot technology for long-term, localized stimulation of specific subcellular neuronal compartments. a key role of its photoactivity, due to the singlet oxygen production, which has a very short lifetime (ns-ls, depending of hyp environment). hyp sub-cellular localization depends on its concentration in the medium, incubation time and used delivery system. variations in activity of protein kinase c, (anti-apoptotic pkca and pro-apoptotic pkcd) in correlation with the activity of bcl-2 protein, cytochrome c release from mitochondria and decreasing of mitochondrial membrane potential after photodynamic action were monitored. the study was performed for two different delivery modes of hyp to u-87mg glioma cells-hyp alone (membrane diffusion) vs. hyp loaded in low density lipoprotein (ldl) (endocytosis). confocal fluorescence microscopy, flow-cytometry and specific fluorescence labeling were used as main experimental techniques. our results show that hyp photoaction strongly affects apoptotic response of the cells and that the dynamics of this action significantly depends on used delivery system. correlation analysis between the monitored parameters (see above) determined for both delivery system is presented and critically discussed. surface contamination by bacteria is a natural and spontaneous process occurring in any natural environment on biotic (mucosa, tissues…) and abiotic surfaces (medical equipments, food surfaces…). whatever the bacterial nature (gram-positive or -negative), the environmental fluid (air, water, blood…) and the receptor surface (implants, medical equipments, food surfaces…), the surface contamination initiated by the first adherent bacteria can evolve to a three dimensional structure named biofilm (cohesive bacteria assembly ensured by an autoproduced extracellular organic matrix). the mechanisms by which these biofilms offer protective environment to viral particles or hypertolerance to antimicrobial action are not yet elucidated. to reach a better understanding of biofilm reactivity, we reported for the first time successful applications of correlative time-resolved optical microscopy approach by time-lapse (tl), frap, fcs, flim, for real-time analysis of molecular mobility and reaction inside biofilms. by means of non-biological or biological (virus, biocides and antibiotics) reactive compounds, significant advances to understand the roles of the extracellular matrix and bacteria physiological properties were obtained, an important step to improve pathogenic biofilm inactivation. here we present a feasibility study to develop two-photon microscopy (2pm) into a standard diagnostic tool for noninvasive, skin cancer diagnosis. the goal is defining experimental parameters to maximize image quality of optical biopsies in human skin, while avoiding tissue damage. possible diagnostic indicators will be compared for healthy tissue, benign, and malignant melanocytic lesions. we report on preliminary results of a study on 2pm imaging of ''ex-vivo'' biopsy samples, were autofluorescence intensity and contrast between lesion and surrounding tissues were optimised varying excitation wavelength, detection band, and dispersion pre-compensation. moreover, we determined modulation functions for laser power and detector gain to compensate losses in deep tissue imaging. as the main process of photo-damage, thermo-mechanical modifications were quantified and damage threshold powers were determined. in order to image structural changes in ordered tissue like collagen fibres, second-harmonic generation signals were recorded and optimised. in-vivo two-photon imaging of the honeybee antennal we adapted a two-photon microscope for in-vivo imaging of the honeybee olfactory system, focusing on its primary centres, the antennal lobes. the setup allowed obtaining both 3d-tomographic measurements of the antennal lobe morphology and time-resolved in-vivo calcium imaging of its neuronal activity. the morphological data were used to precisely measure the glomerular volume in both sides of the brain, investigating the question of volumetric lateralization. functional calcium imaging allowed to record the characteristic glomerular response maps to external odour stimuli applied to the bees' antennae. compared to previous neural imaging experiments in the honeybee, this work enhanced spatial and temporal resolution, penetration depth, and it minimized photo-damage. final goal of this study is the extension of the existing functional atlases of the antennal lobe to 3d and into the temporal dimension by investigating the time-resolved activity pattern. the use of voltage sensitive fluorescent dyes (vsd) for noninvasive measurement of the action potential (ap) in blood perfused heart have been hindered by low interrogation depth, high absorption and auto-fluorescence of cardiac tissue. these limitations are diminished using new near infrared (nir) vsd (di-4-anbdqbs). here we validated toxicity and photo-toxicity of these dyes in guinea pig and human cardiac muscle slabs. application of nir vsd showed no effect on cardiac muscle contraction force or relaxation. optical action potentials closely tracked kinetics of microelectrode recorded aps in both field and electrode stimulated preparations. for phototoxicity assessment dye (50 lm) preloaded cardiac slabs were exposed to prolonged laser radiation of various power. microelectrode ap recordings show that exposure to prolonged laser radiation (10min.; 2mw/mm 2 ) of dye loaded tissue had no statistically significant effect on apd50 or conduction velocity, thus indicating no or weak photo-toxicity on the nir vsd. in contrast, exposure to 5 min. laser radiation of phototoxic dyes (mitotracker deep-red) preloaded tissue caused significant reduction in apd50 (by 13%) and conduction velocity (30%). thus, due to the low photo-toxicity, nir vsd are well suited for in vivo cardiac imaging. streptomycesetes are filamentous gram-positive soil bacteria well known for their complex morphological development and secondary metabolite production. during their life cycle spores germinate to form a network of hyphae, which later develops into aerial mycelium when cross-walls are generated and spores are formed. we have examined and compared the last stage of the differentiation process in a wild-type s. coelicolor (m145) and its dcabb mutant lacking a calmodulin-like calcium binding protein. the strains were grown on four kinds of media: smms, smms with 10 % saccharose, r5 and r5 with reduced calcium in order to study the effect of environment and osmotic stress on the sporulation of the two strains to assess the function of cabb protein. from the cultures pictures were taken at 48 hours and after 7 days using phase contrast, atomic force and confocal laser scanning microscope and the sizes of spores were measured. our results showed that the dcabb mutant made smaller spores, its differentiation and stress response were slower. we could conclude from it that the aberrant protein slows the metabolism, the signal transduction and has effects on sporulation, septation and air-mycelium formation. based on it we can tell that the cabb has a significant role in normal development. the mobility and reaction parameters of molecules inside living cells can be conveniently measured using fluorescent probes. typically fluorescence correlation spectroscopy (fcs) based on confocal microscopy is used for such measurements. this implies high time-resolution but only for a single spot at a time. in order to achieve a high time-resolution at multiple spots, we built a single plane illumination microscope (spim) equipped with high-speed image acquisition devices and a high-na detection optics. this allows us to do parallel fcs measurements in a thin plane (width * 2-3 lm) of the sample. our setup is equipped with a fast emccd camera (full frame time resolution 2000ls) and a 32932 pixel array of spads. the spad array has a full frame time resolution of 3-10ls, which is even fast enough to resolve the typical motion time-scale of small molecules (like egfp) inside living cells. the performance of the system is characterized by diffusion measurements of water-soluble fluorescent beads, as well as fcs measurements in living cells. our data acquisition system uses programmable hardware for some tasks and is fast enough to allow real-time correlation of 1024 pixels, as well as saving the complete dataset for later evaluation. electron cryo-microscopy (cryo-em) covers a larger size range than any other technique in structural biology, from atomic resolution structures of membrane proteins, to large noncrystalline single molecules, entire organelles or even cells. electron crystallography of two-dimensional (2d) crystals makes it possible to examine membrane proteins in the quasi-native environment of a lipid bilayer at high to moderately high resolution (1.8-8å ). recently, we have used electron crystallography to investigate functionally important conformational changes in membrane transport proteins such as the sodium/proton antiporters nhaa and nhap, or the structure of channelrhodopsin. ''single particle'' cryo-em is well suited to study the structure of large macromolecular assemblies in the 3.3 to 20å resolution range. a recent example is our 19å map of a mitochondrial respiratory chain supercomplex consisting of one copy of complex 1, two copies of complex iii, and one of complex iv. the fit of the x-ray structures to our map indicates short pathways for efficient electron shuttling between complex i and iii by ubiquinol, and between complex iii and iv by cytochrome c. electron cryo-tomography can visualize large protein complexes in their cellular context at 30-50å resolution, and thus bridges the gap between protein crystallography and light microscopy. cryo-et is particularly suitable for studying biological membranes and large membrane protein complexes in situ. we found that long rows of atp synthase dimers along ridges of inner membrane cristae are an ubiquitous feature of mitochondria from all 6 species we investigated (2 mammals, 3 fungi, 1 plant). the proton pumps of the respiratory chain appear to be confined to the flat membrane regions on either side of the ridges. this highly conserved pattern suggests a fundamental role of the mitochondrial cristae as proton traps for efficient atp synthesis. single-particle analysis: advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. chief among these fluorophores are the on/off photoswitchable and green-to-red photoconvertible fluorescent proteins. irisfp was recently reported as the first fluorescent protein to combine irreversible photoconversion from a green-emitting to a red-emitting state with reversible on/off photoswitching in both the green and red states. the introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging, but the properties of irisfp are far from being optimal from a spectroscopic point of view and its tetrameric organization complicates its use as a fusion tag. we have demonstrated how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins by developing and characterizing a new set of such enhanced optical highlighters derived from meo-sfp and dendra2. one of these, which we called nijifp, was identified as a promising new multi-photoactivatable fluorescent protein with optical properties that make it ideal for advanced fluorescence-based imaging applications. introducing to medicine and biology concept of optical markers in tremendous way has changed the recent status of these two important disciplines. this was mainly due to strong development in imaging techniques which recently allow us to investigate both static as well dynamic properties of living cells, their components and their interactions with external factors. recently used molecular markers including organic dyes, fluorescent proteins or chelates containing lanthanide ions have several significant limitations. one of the alternatives for molecular markers are inorganic quantum dots (ie. cdse, cds) which are recently commonly used in many academic works. however, even if they are much better from physicochemical point of view, from the application point of view at this moment they are rather useless mainly because of their high risk of toxicity. one of the solution combining advantages of both concepts is to make nontoxic inorganic nanocrystals doped by lanthanide ions. in this work, we will present optical results obtained for nayf 4 nanocrystals doped by different lanthanide ions. the aim of this work was to design and to synthesize these markers and to understand physical processes responsible for their emission and to optimize these processes to the physical limits. intravital microscopy has fostered a full blown of publication regarding the behavior of cells in different tissues and physiological conditions. however, a few papers describe how the motility parameters can be used to understand whether an interaction is occurring and, on balance, the distinction between interacting and non interacting cells is performed visually on the image time stack. here we describe a multi-parameter approach that allows to discern among different cell behaviors on an objective ground and we demonstrate its effectiveness valuating the mutual fate of natural killer (nk) and dendritic (dc) cells at the draining lymph-nodes in inflammatory and stationary conditions. the method is time saving and allows a wide scale characterization of the lymphocyte tracks and to build up statistics of the cell-cell interaction duration. this has allowed the development of a numerical model of the nk-dc interaction, based on a molecular-stochastic dynamic approach, whose output can be directly compared to the data. hemozoin is formed during malaria infection of red blood cells, the malaria parasite cleaves hemoglobin, leaving free heme which is then toxic to the parasite. the free heme is then bio-crystalized to form hemozoin which allows the parasite to remain viable. the hemozoin is released during the breakdown of the red blood cells, is small and can be difficult to resolve spatially. since it contains an abundance of heme protein, which has a strong absorbance at 532 nm, it can be readily detected and tracked by using resonant raman scattering spectroscopy. here we use slit-scanning confocal raman imaging to detect the hemozoin and resolve it against the background molecules. inside a red blood cell, hemoglobin is the strongest background signal since it also contains large amounts of heme. nevertheless, the discrimination is possible, and the time-resolved observation of hemozoin is an important tool to understand effects of malaria since the hemozoin can trigger the immune response and cause inflammation in tissue. muscle performance at the molecular level is determined by the elementary displacement (working stroke) produced by the motor protein myosin ii, and its dependence on load. we developed a laser trap assay (the optical leash) capable of applying controlled loads to a single myosin head before the working stroke is initiated and probing actin-myosin interaction on the microsecond time scale. we found that the working stroke size depends both on load and on the detachment pathway followed by myosin. in a first pathway, myosin detaches very rapidly from actin (\1ms) without producing any movement. in a second pathway, myosin steps and remains bound to actin for a time inversely proportional to atp concentration; the working stroke remains constant (*5 nm) as the load is increased, until it suddenly vanishes as the isometric force is reached (5.7±0.6pn). a third dissociation pathway becomes more populated as the force is increased, due to premature unbinding of myosin from actin, resulting in a working stroke that decreases with load. taken together, these results give a new insight into the molecular mechanism of load dependence of the myosin working stroke, which is a primary determinant of skeletal muscle performance and efficiency. previously we have deleted either or both of these terminal helices genetically. surprisingly, all mutants rotated in the correct direction, showing that the shaft portion is dispensable. here we inquire if the rest of the c rotor, the globular protrusion that accounts for *70 % of the c residues, is also dispensable. keeping the n-and c-terminal helices that constitute the shaft, we have replaced the middle *200 residues with a short helix-turn-helix motif borrowed from a different protein. the protrusion-less mutant thus made retained a high atpase activity and rotated fast in the correct direction. this may not be unexpected because, in crystal structures, most of the removed residues do not contact with the a 3 b 3 ring. combined with the previous results, however, the present results indicate that none of the c residues are needed for rotation. the rotary mechanism of a molecular engine, the vacuolar proton-atpase, working in a biomembrane csilla ferencz 1 , pá l petrovszki 1 , zoltá n kó ta 1 the rotary mechanism of vacuolar proton-atpase (v-atpase) couples atp hydrolysis and trans-membrane proton translocation. we tested the effect of oscillating electric (ac) field on v-atpase activity in yeast vacuoles. the ac technique has several advantages over direct observations: it can be applied on native membranes, there are no labels and attachments involved, and the target protein is in its natural environment. this was/is the first of its kind of experiment on v-atpase, and we got strikingly different results from previous studies on other proteins: both low and high frequency ac field reduces atpase activity in a wide frequency range. a sharp resonance is seen at 88.3 hz, where the atpase activity reaches or exceeds the control (no ac) level. we think that the resonance happens at that of the 60 degrees rotor steps, meaning that the rotation rate of the rotor is around 15 hz, under the given conditions. synchronisation of individual atpases by slow or matching, but not fast ac is likely via a hold-and-release mechanism. we can explain the above observations by assuming that the ac field interacts with the proton movements, and if we consider the estimated geometry of the hydrophilic proton channels and the proton binding cites on the rotor. [2] . the ttss constitutes a continuous protein transport channel of constant length through the bacterial envelope [3] . the needle of the type three secretion system is made of a single small protein (protomer). we analyzed the assembly and the structure of the ttss needle using different biophysical methods including fourier transform-infrared spectroscopy, nmr spectroscopy and x-ray crystallography. we show that the ttss needle protomer refolds spontaneously to extend the needle from the distal end. the protomer refolds from a-helix into b-strand conformation to form the ttss needle [4] . regulated secretion of virulence factors requires the presence of additional protein at the ttss needle tip. x-ray crystal structure analysis of the tip complex revealed major conformational changes in both the needle and the tip proteins during assembly of the s. typhimurium ttss. our structural analysis provides the first detailed insight into both the open state of the ttss needle tip and the conformational changes occurring at the pathogen-host interface [5] . [ the membrane-bound component f o of the atp synthase works as a rotary motor and plays the central role of driving the f 1 component to transform chemi-osmotic energy into atp synthesis. we have conducted molecular dynamics simulations of the membrane-bound f o sector with explicit lipid bilayer, in which the particular interest was to observe the onset of helix motion in the c ring upon the change of the protonation state of asp61 of the c 1 subunit, which is the essential element of the boyer's binding-change mechanism. to investigate the influence of transmembrane potential and ph gradient, i.e., the proton motive force, on the structure and dynamics of the a-c complex, different electric fields have been applied along the membrane normal. correlation map analysis indicated that the correlated motions of residues in the interface of the a-c complex were significantly reduced by external electric fields. the deuterium order parameter (s cd ) profile calculated by averaging all the lipids in the f o -bound bilayer was not very different from that of the pure bilayer system, which agrees with recent 2 h solid-state nmr experiments. however, by delineating the lipid properties according to their vicinity to f o , we found that the s cd profiles of different lipid shells are prominently different. lipids close to f o formed a more ordered structure. similarly, the lateral diffusion of lipids on the membrane surface also followed a shell-dependent behavior. the lipids in the proximity of f o exhibited very significantly reduced diffusional motion. the numerical value of s cd was anti-correlated with that of the diffusion coefficient, i.e., the more ordered lipid structures led to slower lipid diffusion. our findings will not only help for elucidating the dynamics of f o depending on the protonation states and electric fields, but may also shed some light to the interactions between the motor f o and its surrounding lipids under physiological condition, which could help to rationalize its extraordinary energy conversion efficiency. this work has been published 1 in march 2010, and it was selected as one of the two featured articles of that issue. the research project is directed toward the construction of a synthetic bio-inorganic machine that consists in a single actin filament that interacts with a linear array of myosin ii motors regularly disposed on a nano-structured device. the motor array is intended to simulate the unique properties of the ensemble of motor proteins in the half-sarcomere of the muscle by providing the condition for developing steady force and shortening by cyclic interactions with the actin filament. the mechanical outputs in the range 0.5-200 pn force and 1-10,000 nm shortening will be measured and controlled the bacterial flagellar motor is a membrane-embedded molecular machine that rotates filaments, providing a propulsive force for bacteria to swim. the molecular mechanism of torque (turning force) generation is being investigated through the study of the properties and three-dimensional structure of the motor's stator unit. we are taking both topdown and bottom-up approaches, combining data from the molecular genetics studies, cross-linking, x-ray protein crystallography and molecular dynamics simulations. we have recently determined the first crystal structure of the protein domain that anchors the proton-motive-force-generating mechanism of the flagellar motor to the cell wall, and formulated a model of how the stator attaches to peptidoglycan. the work presented at the meeting will inform the audience on our latest work that establishes the relationship between the structure, dynamics and function of a key component of the bacterial flagellar motor, the motility protein b (motb). this work will be put in the perspective of the mechanism of rotation, stator assembly, anchoring to peptidoglycan and interaction with the rotor, and discussed in the light of the elementary events composing the cycle of electrochemical-to-mechanical energy conversion that drives flagellar rotation. members of the conserved kinesin-5 family fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plusend directed motors. the mechanisms that regulate kinesin-5 function are still not well understood. we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin-5 cin8 using single-molecule motility assays and single-molecule fluorescence microscopy and found that cin8 motility is exceptional in the kinesin-5 family. in vitro, individual cin8 motors could be switched by ionic conditions from rapid (up to 50 lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert of 99 amino acids in loop 8 of cin8 induced bias towards minus-end motility and strongly affected the directional switching of cin8 both in vivo and in vitro. we further found that deletion of the functionally overlapping kinesin-5 kip1 and of the spindle-organizing protein ase1 affected cin8 velocity and processivity, but directionality was not affected. the entirely unexpected finding of switching of cin8 directionality in vivo and in vitro demonstrates that the ''gear box'' of kinesins is much more complex and versatile than thought. many biological motor molecules move within cells using stepsizes predictable from their structures. myosin-vi, however, has much larger and more broadly-distributed stepsizes than those predicted from its short lever arms. we explain the discrepancy by monitoring qdots and gold nano-particles attached to the myosin vi motor domains using high-sensitivity nano-imaging. the large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large (72 nm) and one small (44 nm). these results suggest there exist two tilt-angles during myosin-vi stepping, which correspond to the pre-and post-powerstrokes states and regulate the leading head. the large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with adp. switching between these two mechanisms in a strain sensitive, adpdependent manner allows myosin-vi to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring. http://www.fbs.osaka-u.ac.jp/labs/yanagida/, http:// www.qbic.riken.jp/. ferritin deposits iron in oxyhydroxide iron core surrounded by protein shell. the iron core structure may vary in different ferritins in both normal and pathological cases. to study iron core variations the mö ssbauer spectroscopy with a high velocity resolution was applied for comparative analysis of normal and leukemia chicken liver and spleen tissues, human liver ferritin and commercial pharmaceutical products imferon, maltoferò and ferrum lek as ferritin models. mö ssbauer spectra of these samples measured with a high velocity resolution at room temperature were fitted using two models: homogenous iron core (one quadrupole doublet) and heterogeneous iron core (several quadrupole doublets). the results of both fits demonstrated small variations of mö ssbauer hyperfine parameters related to structural variations of the iron cores. these small structural variations may be a result of different degree of crystallinity, the way of iron package, nonequivalent iron position, etc. obtained small differences for normal and leukemia tissues may be useful for distinguishing ferritins from normal and pathological cases. this work was supported in part by the russian foundation for basic research (grant # 09-02-00055-a). invasion of epithelial cells by salmonella enterica is mediated by bacterial ''effector'' proteins that are delivered into the host cell by a type iii secretion system (ttss). the collaborative action of these translocated effectors modulates a variety of cellular processes leading to bacterial uptake into mammalian cells. type iii effectors require the presence in the bacterial cytosol of specific tts chaperones. effectors are known to interact with their chaperone via a chaperone binding domain (cbd) situated at their n-terminus. this work focus on sopb, an effector with phosphoinositide phosphatase activity and particularly its interaction with the specific chaperone sige by biochemical, biophysical and structural approaches. we have co-expressed sopb with its specific chaperone sige and purified the complex, determined the limits of the cbd and purified the sopb cbd /sige complex. the structure of sige has been solved previously but no crystals could be obtained for structure determination of both complexes. we used saxs experiment combined with biophysical approach to analyse the interaction between sopb and its chaperone as well as the quaternary structure on the complex that will be described in this presentation. guanylate monophosphate kinase (gmpk) is a cytosolic enzyme involved in nucleotide metabolic pathways. one of the physiological roles of gmpks is the reversible phosphoryl group transfer from atp to gmp (its specific ligand), yielding adp and gdp. the gmpk from haemophilus influenzae is a small protein, with a number of 208 amino acids in the primary structure. in order to determine the secondary structure changes of this enzyme, as well as some physical characteristics of its complexes with gmp and atp ligands, circular dichroism (cd) and atr -ftir studies were performed. the enzyme and its ligands were dissolved in tris -hcl buffer, at ph 7.4 and 25°c. from the cd spectra the content of the secondary structure elements of gmpk and gmpk/gmp, gmpk/atp (with and without mg 2+ ) were determined. the major secondary structure elements of gmpk from haemophilus influenzae were a-helix (* 37 %) and b-sheet (* 36 %). atr -ftir experiments show that the amide i and amide ii bands of the gmpk are typical for a protein with great a-helix content. from the second derivative spectra, the content of the secondary structure elements were estimated. these data were in agreement with those obtained by cd. assembly of the mature human immunodeficiency virus type 1 capsid involves the oligomerization of the capsid protein, ca. the c-terminal domain of ca, ctd, participates both in the formation of ca hexamers, and in the joining of hexamers through homodimerization. intact ca and the isolated ctd are able to homodimerize in solution with similar affinity (dissociation constant in the order of 10 lm); ctd homodimerization involves mainly an a-helical region. in this work, we show that peptides derived from the dimerization helix (which keep residues energetically important for dimerization and with higher helical propensities than the wild-type sequence) are able to self-associate with affinities similar to that of the whole ctd. moreover, the peptides have a higher helicity than that of the wild-type sequence, although is not as high as the theoretically predicted. interesting enough, the peptides bind to ctd, but binding in all peptides, but one, does not occur at the dimerization interface of ctd (helix 9). rather, binding occurs at the last helical region of ctd, even for the wild-type peptide, as shown by hsqc-nmr. as a consequence, all peptides, but one, are unable to inhibit capsid assembly of the whole ca in vitro. the peptide whose binding occurs at the ctd dimerization helix has an val?arg mutation in position 191, which has been involved in dimer-dimer contacts. these findings suggest that event keeping the energetically important residues to attain ctd dimerization within a more largely populated helical structure is not enough to hamper dimerization of ctd. putp is an integral membrane protein located in the cytoplasmic membrane of escherichia coli, being responsible for the coupled transport of na + and proline. it belongs to the family of sodium solute symporters (sss). structural data for putp is not available, but secondary structure predictions together with biochemical and biophysical analyses suggest a 13 transmembrane motif. from a recent homology model based on the x-ray structure of the related na + /galactose symporter vsglt, previously published electron paramagnetic resonance (epr) studies, and recent crystallographic and epr studies on the cognate bacterial homolog of a neurotransmitter:na + symporter, leut, it has been proposed that helices viii and ix as well as the interconnecting ''loop 9'' region determine the accessibility of the periplasmic cavities which bind sodium and proline. we performed site-directed spin labeling of ''loop 9'' in combination with epr spectroscopy to investigate the structural features of this region and possible conformational changes induced by sodium and proline. analyses of spin label mobility and polarity as well as accessibility to paramagnetic quenchers allow us to refine this region in the present homology model. furthermore, our data suggest conformational changes in this region upon substrate binding including an overall motion of a helical segment. fatty acid-binding proteins (fabp) are a family of low molecular weight proteins that share structural homology and the ability to bind fatty acids. the common structural feature is a b-barrel of 10 antiparallel b-strands forming a large inner cavity that accommodates nonpolar ligands, capped by a portal region, comprising two short a-helices. b-fabp exhibits high affinity for the docosahexaenoic (dha) and oleic acid (oa). it is also postulated that b-fabp may interact with nuclear receptors from ppar family. in the present work, we used molecular biology and spectroscopic techniques to correlate structure, dynamics and function. site-directed mutagenesis was used to produce 5 mutants of b-fabp with a nitroxide spin probe (mtsl) selectively attached to residues located at the portal region. esr spectra of the labeled b-fabp mutants were sensitive to the location of the mutation and were able to monitor interactions in three cases: (1) shsp are ubiquitous proteins involved in cellular resistance to various stress (oxidative, heat, osmotic…). they are able to prevent aggregation of non-native proteins through the ability of forming large soluble complexes and preventing their nonspecific and insoluble aggregation. in consequence to this molecular chaperone function, they can regulate many processes (resistance to chemotherapy, modulation of the cellular adhesion and invasion, inflammatory response in skin), and the modulation of their expression has been found to be a molecular marker in cancers, spermatogenesis, or cartilage degeneration. furthermore, they are involved in several pathologies: myopathies, neuropathies, cancers, cataracts. among the 10 human members (hspb1-10), this study focused on hspb1 (hsp27 involved in some cancers), hspb4 (lens specific), hspb5 (lens, muscle, heart, lung) and hspb5-r120g (responsible for a desmin-related myopathy and a cataract). as shsp form large, soluble (but polydisperse in mammals) hetero-oligomers, molecular biology, biochemistry, biophysics and bioinformatics were successfully combined to compare the functional/dysfunctional assemblies in order to understand the critical parameters between shsp members depending upon their tissue and cellular localization. ionizing radiation is a type of radiation that contains enough energy to displace electrons and break chemical bonds. this can promote the removal of at least one electron from an atom or molecule, creating radical species, namely, reactive oxygen species (ros) [1, 2] . these are often associated with damages at cellular level, such as, dna mutations, cell cycle modifications and, in animal cells, cancer. to overcome this problem, organisms developed different protection/repair mechanisms that enable them to survive to these threats. dna glycosylases are enzymes that are part of base excision repair (ber) system, mainly responsible for dna repair. they can recognize a dna lesion and, in some cases, are able to remove the mutated base. here we propose to study one of those enzymes, endonuclease iii, which contains a [4fe-4s] cluster [3, 4] . samples were exposed to different doses of uv-c radiation and the effects were studied by electrophoretic and spectroscopic methods. [ na,k-atpase is an integral protein present in the plasma membrane of animal cells, and consists of two main subunits: the a and b. cholesterol is an essential constituent of the animal membrane cells. in order to study the interaction between na,k-atpase and cholesterol, we have used the dsc technique, and a proteoliposome system composed by the enzyme and dppc:dppe, with different percentage in mol of cholesterol. the heat capacity of purified na,k-atpase profile exhibits three transitions with 31, 189 and 60 kcal/mol at 55, 62 and 69°c. multiple components in the unfolding transition could be attributed either to different steps in the pathway or to independent unfolding of different domains. this denaturation of na,k-atpase is an irreversible process. for the proteoliposome, we also observed three peaks, with 180, 217 and 41 kcal/mol and 54, 64 and 72°c. this increase in dh indicates that the lipids stabilize the protein. when cholesterol was used (from 10 to 40 mol %), the first transition was shifted to a lower temperature value around 35°c. these results confirm that cholesterol has an influence on the packing and fluidity of lipid bilayer and changes in lipid microenvironment alter the thermostability as well as the activity of na,k-atpase. financial support: fapesp. we have undertaken to study the structure and function of peroxisomal multifunctional enzyme type 2 (mfe-2) from different organisms. mfe-2 is a key enzyme in long-and branched-chain fatty acids breakdown in peroxisomes. it contains two enzymes in the same polypeptide and also consists of differing amount of domains depending on the species. crystal structure and enzyme kinetics of drosophila melanogaster mfe-2 has revealed the domain assembly and raised a question about existence of a substrate channeling mechanism. small-angle x-ray scattering studies have further resolved the assembly of domains in the human mfe-2. mutations in the mfe-2-coding gene in humans may cause d-bifunctional protein deficiency -a metabolic disease characterized by accumulation of fatty acyl-coa intermediates due to inactive or residually active mfe-2 protein. we have also studied the structure, stability and dynamics of such mutant proteins both experimentally and in silico. the latest results on all these studies will be presented. ftsz is a protein that plays a key role in bacterial division, forming a protein ring directly related to the constriction of the membrane. this process has been observed to occur without the help of molecular motors. nonetheless, the details of the self-assembly and subsequent force generation of the septal ring are still obscure. afm observations allows the study of the behaviour of ftsz solutions on a substrate with unprecedented resolution, permitting the identification of individual protein filaments. the different resulting structures can be compared to monte carlo models for a 2d lattice accounting for the essential interactions between monomers. these include a strong longitudinal bond that allows a limited flexibility (i.e., curvature of the filaments) and a weaker lateral interaction. the work we present follows this approach, focussing on the latest experiments with ftsz mutants. by using these mutants, it is possible to choose the specific region of the monomer that will anchor to the substrate, thus generating new structures that provide an insight into monomer-monomer interactions. in this way, we explore the anisotropy of the lateral bond in ftsz, a factor that has not been taken into account before but may prove to be of importance in fstz behaviour in vivo. modeling protein structures and their complexes with limited experimental data dominik gront university of warsaw, pasteura 1, 02-093 warsaw, poland conventional methods for protein structure determination require collecting huge amounts of high-quality experimental data. in many cases the data (possibly fragmentary and/or ambiguous) on itself cannot discriminate between alternative conformations and a unique structure cannot be determined. small angle xray scattering is an example of such a ''weak'' experiment. the spectrum encodes only several independent degrees of freedom that provide a global description of a molecular geometry in a very synthetic way. in this contribution we utilized both local information obtained from nmr measurements and global description of a macromolecule as given by saxs profile combined with a knowledge-based bimolecular force field to determine tertiary and quaternary structure of model protein systems. saxs curve as well as various kinds of local nmr data such as isotropic chemical shifts and their tensors, j-couplings, rdc, backbone noe and redor from nmr in solid phase are parsed with the ''experimental'' module of bioshell toolkit and utilized by rosetta modeling suite to generate plausible conformations. obtained results show the new protocol is capable to deliver very accurate models. noenet-use of noe networks for nmr resonance assignment of proteins with known 3d structure dirk stratmann, carine van heijenoort and eric guittet structural genomics programs today yield an increasing number of protein structures, obtained by x-ray diffraction, whose functions remain to be elucidated. nmr plays here a crucial role through its ability to readily identify binding sites in their complexes or to map dynamic features on the structure. an important nmr limiting step is the often fastidious assignment of the nmr spectra. for proteins whose 3d structures are already known, the matching of experimental and back-calculated data allows a straightforward assignment of the nmr spectra. we developed noenet, a structure-based assignment approach. it is based on a complete search algorithm, robust against assignment errors, even for sparse input data. it allows functional studies, like modeling of protein-complexes or protein dynamics studies for proteins as large as 28 kd. almost any type of additional restraints can be used as filters to speed up the procedure or restrict the assignment ensemble. noenet being mainly based on nmr data (noes) orthogonal to those used in triple resonance experiments (jcouplings), its combination even with a low number of ambiguous j-coupling based sequential connectivities yields a high precision assignment ensemble. we observed, that t. thermophilus isopropylmalate dehydrogenase (ipmdh) has higher rigidity and lower enzyme activity at room temperature than its mesophilic counterpart (e. coli), while the enzymes have nearly identical flexibilities under their respective physiological working conditions, suggesting that evolutionary adaptation tends to maintain optimum activity by adjusting a ''corresponding state'' regarding conformational flexibility. in order to reveal the nature of the conformational flexibility change related to enzymatic activity, we designed a series of mutations involving non conserved prolines specific to thermophilic ipmdh. proline to glycine mutations substantially increased conformational flexibility and decreased conformational stability. the mutant enzyme variants did not show enhanced catalytic activity, but the non arrhenius temperature dependence of enzyme activity of the wild type was abolished. this phenomenon underlines the fact that the delicate balance between flexibility, stability and activity which is required for the environmental adaptation of enzymes can be easily disrupted with mutations even distant from the active site, providing further evidence that optimization of proper functional motions are also a selective force in the evolution of enzymes. the kinetoplastids trypanosoma brucei, t. cruzi and leishmania major are responsible for causing great morbidity and mortality in developing countries. the all a-helical dimeric dutpases from these organisms represent promising drug targets due to their essential nature and markedly different structural and biochemical properties compared to the trimeric human enzyme. to aid in the development of dutpase inhibitors we have been structurally characterizing the enzymes from these species. here we present the structure of the t. brucei enzyme in open and closed conformations, completing the view of the enzymes from the kinetoplastids. furthermore, we sought to probe the reaction mechanism for this family of enzymes as a mechanism has been proposed based on previous structural work but has not received any further verification. the proposed scheme is similar to that of the trimeric enzyme but differs in detail. using tryptophan fluorescence quenching in the presence of the transition state mimic alf 3 we have been able to identify which species is the likely transition state in the reaction. the crystal structure of t. brucei in complex with this transition state analogue confirms the nature of the nucleophilic attack clearly showing how it differs from trimeric enzymes. the structure of factor h-c3d complex explains regulation of immune complement alternative pathway circular dichroism (cd) spectroscopy is a widely used technique for studying the secondary structure (ss) of proteins. numerous algorithms have been developed for the estimation of the ss composition from the cd spectra. although, these methods give more or less accurate estimation for proteins rich in a-helical structure, they often fail to provide acceptable results on mixed or b-rich proteins. the problem arises from the diversity of b-structures, which is thought to be an intrinsic limitation of the technique. the worst predictions are provided for proteins of unusual b-structures and for amyloid fibrils. our aim was to develop a new algorithm for the more accurate estimation of ss contents for a broader range of protein folds with special interest to amyloid fibrils. using synchrotron radiation cd (srcd), we were able to collect high quality spectra of amyloid fibrils with good s/n ratios down to 175nm. the novel reference dataset with spectra that significantly differ from present reference sets, extends the information content for ss determination. our algorithm takes into account the diverse twist of the b-sheets that has a great influence on the spectral features. for the first time, we can reliably distinguish parallel and antiparallel b-structure using cd spectroscopy. monitoring the assembly of the membrane protein insertase alexej kedrov 1 , marko sustarsic 2 , arnold j.m. driessen 1 1 groningen biomolecular sciences and bioengineering institute, university of groningen, the netherlands, 2 university of oxford, uk molecular forces that govern membrane protein integration and folding remain a major question in current molecular biology and biophysics. each nascent polypeptide chain should acquire its unique three-dimensional folded state within a complex environment formed by the anisotropic lipid membrane and the membrane-water interface. secyeg translocase and members of a recently described yidc/oxa1/alb3 chaperone family are recognized as primary players in the membrane protein genesis. these proteins, so called insertases, serve as membraneembedded molecular pores where the newly synthesized protein is loaded prior its release into the bilayer. here we apply fluorescence correlation spectroscopy to monitor the assembly of the insertase:ribosome:nascent polypeptide chain complexes in solution and reconstituted into nanodiscs and model membranes. results provide insights on molecular mechanisms and dynamics of the insertase functioning. conformational changes during gtpase activity induced self-assembly of human guanylate binding protein 1 revealed by epr spectroscopy correct assembly and regulation of multi-component molecular machines is essential for many biological activities. the type iii secretion system (t3ss) is a complex molecular machine that is a key virulence determinant for important gram-negative pathogens including shigella, yersinia and salmonella species [1] [2] . the t3ss consists of multiple copies of *25 different proteins (totalling *7mda), spans both bacterial membranes and drives insertion of a contiguous pore into the host-cell membrane. virulence factors are secreted via this apparatus directly into the host cell. in all t3ss various levels of regulation occur with switching between secretion off and on states overlaid on control of which substrates are secreted. genes involved in a variety of these switches have been identified but the molecular mechanisms underlying these functions is poorly understood. we are studying the t3ss of shigella flexneri, the causative agent of dysentery and will present the structure of the so-called ''gatekeeper protein'' mxia. the diacylglycerolacyltransferase1 (dgat1) is an integral protein from the reticulum endoplasmic membrane that plays an essential role in triacylglyceride synthesis. in cattle, this enzyme is associated to the fat content regulation on milk and meat. in this study, synthetic peptides corresponding to both dgat1 binding sites (sit1 and sit2) were designed, purified and employed to investigate the enzyme interaction with substrates and membrane models. different binding specificities in the interaction with phospholipid vesicles and micelles were noted. sit1 showed to bind more strongly in nonpolar membrane models, while sit2 was electrostatically attracted to negative phospholipid surfaces. the binding of both peptides was followed by significant conformational changes (like unordered to helix transition) in circular dichroism spectra and a 20nm blue shift in fluorescence emission. the binding of sit1 and sit2 peptides to negative liposome gave dissociation constants (k d ) of 170 and 0.44lm, respectively, and a leakage action 24-fold higher to sit2. the difference in specificity is related to the features of the putative substrates (acyl-coas and diacylglycerol) and can be attributed to the distinct role of each dgat1 binding site during lipid synthesis. supported by fapesp. ftsz, the bacterial homologue of tubulin, assembles into polymers in the bacterial division ring. the interfaces between monomers include a gtp molecule, although the relationship between polymerization and gtpase activity is still controversial. a set of short ftsz polymers were modelled and the formation of active gtpase structures was monitored using molecular dynamics. only the interfaces nearest the polymer ends exhibited geometry adequate for gtp hydrolysis. conversion of a mid-polymer interface to a close-to-end interface resulted in its spontaneous rearrangement from an inactive to an active conformation. fluorescent proteins (fps) have become extremely valuable tools in the life sciences. due to the latest advances in the light microscopy, there is a steady need for fps with improved spectral properties. mirisfp is a monomeric fp that can be switched reversibly between a bright green fluorescent and a dark state by illumination with light of specific wavelengths [1] . structurally, this photo-switching is based on a cis-trans isomerization of the chromophore. upon illumination with violet light, mirisfp can be irreversibly photoconverted from the green-emitting to a red-emitting form. the red form can again be switched reversibly between a fluorescent and a dark state. to elucidate the mechanistic details of the photoinduced reactions, we have generated mirisgfp1. this variant can still undergo reversible photoswitching, but lacks the ability to photoconvert to the red state so that the photoinduced transitions of the green form can be studied without 'artifacts' due to green-to-red photoconversion. using uv/visible spectroscopy, we have characterized the on-and off-switching processes in great detail. several light-activated reaction pathways have been identified. they are highly intertwined so that the net effect achieved with light of a particular wavelength depends on the relative probabilities to photoinduce the various processes. phosducin (pd) is a g t bc-binding protein that is highly expressed in photoreceptors. pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. dephosphorylated pd binds g t protein bc-heterodimer with high affinity and inhibits its interaction with g t a or other effectors, whereas phosphorylated pd does not. therefore pd down-regulates the light response in photoreceptors. phosphorylation of pd at s54 and s73 leads to the binding of the 14-3-3 protein. the 14-3-3 proteins function as scaffolds modulating the activity of their binding partners and their role in pd regulation is still unclear. the 14-3-3 protein binding may serve to sequester phosphorylated pd from g t bc or to decrease the rate of pd dephosphorylation and degradation. we performed several biophysical studies of the 14-3-3:pd complex. analytical ultracentrifugation was used to determine the complex stoichiometry and dissociation constant. conformational changes of pd induced both by the phosphorylation itself and by 14-3-3 binding were studied using the time-resolved fluorescence spectroscopy techniques. mö ssbauer spectroscopy with a high velocity resolution was used for comparative study of various oxyhemoglobins for analysis of the heme iron electronic structure and protein structure-function relationship. samples of pig, rabbit and normal human oxyhemoglobins and oxyhemoglobins from patients with chronic myeloleukemia and multiple myeloma were measured using mö ssbauer spectrometric system with a high velocity resolution at 90 k. mö ssbauer spectra were fitted using two models: one quadrupole doublet (model of equivalent iron electronic structure in a-and b-subunits of hemoglobins) and superposition of two quadrupole doublets (model of non-equivalent iron electronic structure in a-and b-subunits of hemoglobins). in both models small variations of mö ssbauer hyperfine parameters (quadrupole splitting and isomer shift) were observed for normal human, rabbit and pig oxyhemoglobins and related to different heme iron stereochemistry and oxygen affinity. small variations of mö ssbauer hyperfine parameters for oxyhemoglobins from patients were related to possible variations in the heme iron stereochemistry and function. the different types of silk produced by orb-weaving spiders display various mechanical properties to fulfill diverse functions. for example, the dragline silk produced by the major ampulate glands exhibits high toughness that comes from a good trade-off between stiffness and extensibility. on the other hand, flagelliform silk of the capture spirals of the web is highly elastic due to the presence of proline and glycine residues. these properties are completely dictated by the structural organization of the fiber (crystallinity, degree of molecular orientation, secondary structure, microstructure), which in turn results from the protein primary structure and the mechanism of spinning. although the spinning process of dragline silk begins to be understood, the molecular events occurring in the secretory glands and leading to the formation of other silk fibers are unknown, mainly due to a lack of information regarding their initial and final structures. taking advantage of the efficiency of raman spectromicroscopy to investigate micrometer-sized biological samples, we have determined the conformation of proteins in the complete set of glands of the orb-weaving spider nephila clavipes as well as in the fibers that are spun from these glands. domain of rgs3 at 2.3å resolution was solved. the stoichiometry of 14-3-3 f /rgs3 protein complex was elucidated using the analytical ultracentrifugation. to map the interaction between 14-3-3f and rgs3 protein we performed a wide range of biophysical measurements: h/d exchange and cross link experiments coupled to mass spectrometry, fret (fö rster resonance energy transfer) time-resolved fluorescence experiments, time-resolved tryptophan fluorescence spectroscopy and saxs (small angle x-ray scattering) measurements. based on all these results we build 3d model of 14-3-3 f /rgs3 protein complex. our model revealed new details on architecture of complex formed by 14-3-3 proteins. to date all known structure of 14-3-3 proteins complexes suggests that the ligand is docked in the central channel of 14-3-3 protein. our results indicate that the rgs domain of rgs3 protein is located outside the central channel of 14-3-3f protein interacting with less-conserved residues of 14-3-3f. the receptor for advanced glycation end-products (rage) is a multiligand cell surface receptor involved in various human diseases. the major alternative splice product of rage comprises its extracellular region that occurs as a soluble protein (srage). although the structures of srage domains were available, their assembly into the functional full length protein remained unknown. here we employed synchrotron small-angle x-ray scattering to characterize the solution structure of human srage. the protein revealed concentration-dependent oligomerization behaviour, which was also mediated by the presence of ca 2+ ions. rigid body models of monomeric and dimeric srage were obtained from the scattering data recorded at different solvent conditions. the monomer displays a j-like shape while the dimer is formed through the association of the two n-terminal domains and has an elongated structure. the results provided insight into the assembly of i) the heterodimer srage:rage, which is responsible for blockage of the receptor signalling, and ii) rage homodimer, which is necessary for signal transduction, paving the way for the design of therapeutical strategies for a large number of different pathologies. clpb is a hexameric aaa? atpase that extracts unfolded polypeptides from aggregates by threading them through its central pore. the contribution of coiled-coil m domains is fundamental for the functional mechanism of this chaperone, and its location within the protein structure in previous structural models is contradictory. we present cryo-electron microscopy structural analysis of clpb from e. coli in several nucleotide states. the study reveals a novel architecture for clpb and shows that m domains form an internal scaffold located in the central chamber of clpb hexamers. this inner structure transmits local signals due to atp binding and hydrolysis by aaa? domains. surprisingly, coiled-coil m domains are seen to bend significantly around a hinge region that separates two structural motifs. our results present a new framework to understand clpb-mediated protein disaggregation. streptomyces clavuligerus isoenzymes involved in clavulanic acid biosynthesis: a structural approach clavulanic acid (ca) is a potent b-lactamase inhibitor produced by streptomyces clavuligerus. n 2 -(2-carboxyethyl) arginine synthase (ceas) and proclavaminate amidino hydrolase (pah) catalyze the initial steps in the biosynthesis of ca. recently ceas1and pah1genes (paralogous of ceas and pah) were related to the ca biosynthesis but their products have not been studied yet. here we present the initial structural analysis of ceas1 and pah1 using biophysical techniques. pah1 and ceas1 genes were isolated from the genomic dna of s. clavuligerus and overexpressed in e. coli. the recombinant proteins were purified by affinity chromatography and analyzed by size exclusion chromatography, non-denaturing page, dynamic light scattering, far-uv circular dichroism (cd) and fluorescence spectroscopy. our results showed that pah1 and ceas1 were obtained as hexamer and dimer respectively. both proteins showed an a/b folding, being stable up to 35°c. above this temperature protein unfolding was observed but the complete unfolding was not observed, even at 100°c. moreover ceas1 and pah1 showed to be stable over a wide ph range (ph 5.5 -9.5). we are currently working on improving ceas1 crystals which are a promising step towards the elucidation of the ceas1 structure. supported by fapesp. • synchrotron radiation circular dichroism • mass spectrometry following vuv photoionisation • fluorescence imaging with lifetime and spectral measurements here we will present the srcd experiment. high photon flux of 10 10 photons / sec, improved detector performances as well as user-orientated software developments have proven to be the garants for successful data collections,which considerably increased the information content obtained. the exploration into the charge transfer region of the peptide bonds is adding specifically new insights. low sample volumes of as little as 2ll per spectra as well as convenient sample chamber handling allow for economic and efficient data collections. typical spectra acquisition from 280 to 170nm, last for 9 min for three scans with a 1nm step size. prior to high resolution based techniques, srcd spectrawill answer questions about folding states of macromolecules including dna, rna and sugar macromolecules as well as their complexes with proteins and specially membrane proteins. sporulation in bacillus subtilis begins with an asymmetric cell division producing a smaller cell called the forespore, which initially lies side-by-side with the larger mother cell. in a phagocytosis-like event, the mother cell engulfs the forespore so that the latter is internalised as a cell-within-a-cell. engulfment involves the migration of the mother cell membrane around the forespore until the leading edges of this engulfing membrane meet and fuse. this releases the forespore, now surrounded by a double membrane, into the mother cell cytoplasm. membrane migration during engulfment is facilitated by the interacting proteins spoiiq and spoiiiah that are membrane-associated and expressed in the forespore and the mother cell respectively 1 . they interact in the intercellular space and function initially as a molecular zipper and later they participate in a more elaborate complex in which spoiiq and spoiiiah are integral components of an intercellular channel. this channel is a topic of much current interest, having initially been proposed as a conduit for the passage from the mother cell to the forespore of a specific, but putative, regulator of the rna polymerase sigma factor, r g 2 and later as a gap junction-like feeding tube 3 through which the mother cell supplies molecules for the biosynthetic needs of the forespore. here we present data on the structure and interactions of spoiiq and spoiiiah gleaned from biophysical methods and protein crystallography. these data lead to a plausible model for the intercellular channel. the glycine receptor (glyr) is a chloride permeable ligand gated ion channel and that can mediate synaptic inhibition. due to a possible involvement in the pathophysiology of temporal lobe epilepsy, the different properties of glyrs containing alpha3l and k subunit isoforms are currently investigated. previous characterizations of homomeric receptors consisting of these isoforms have shown a difference in their electrophysiological properties and their membrane distribution as observed by diffraction-limited fluorescence microscopy. we studied these isoforms, when separately expressed in transfected hek 293t cells, by using single molecule tracking (smt) in living cells and direct stochastic optical reconstruction microscopy (dstorm) on fixated cells. for both techniques the glyrs are stained using a primary antibody directly labeled with alexa fluor dyes. the dstorm experiments support the observation that alpha3l glyr are clustered, while the alpha3k glyrs are more uniformly spread. the analysis of the short range diffusion coefficients obtained by smt reveals the presence of heterogeneous motion for both isoforms. the k-isoform has a higher fraction of fast diffusion. in contrast, the l-isoform is more associated with slow diffusion and appears to undergo hindered diffusion. since nanoparticles are suitable for tumor therapy due to their passive targeting to cancer cells by enhanced permeability and retention effect [1] , it is important to understand mechanisms of their delivery into the living cancer cells. in this respect we have developed a modular spectral imaging system based on a white light spinning disk confocal fluorescence microscope and a narrow tunable emission filter. firstly, interaction of polymer nanoparticles and cells labeled with spectrally overlapping probes was examined. the use of fluorescence microspectroscopy (fms) allowed co-localization, which showed that the size of polymer nanoparticles strongly influences their transfer across the cell plasma membrane. next, the delivery of liposomes (composed of cancerostatic alkylphospholipid (opp) and cholesterol) labeled with environment-sensitive fluorescent probe was monitored. we were able to detect a very small shift in emission spectra of cholesterol-poor opp liposomes inside and outside the cells, which would not be possible without the use of fms. this shift implies that the delivery of these liposomes into cancer cells is based on fusion with the cell membrane [2] . [ high-resolution optical imaging techniques make now accessible the detection of nanofeatures in bio-and soft-matter by non-ionizing visible radiation. however, high-resolution imaging is critically dependent by the fluorescent probes used for reporting on the nano-environment. on account of our long-standing interest in the development of fluorescent probes, we set out to design and engineer new fluorescent systems for nanoscale imaging and sensing of biological specimens and soft-matter. these fluorophores report on fast subtle changes of their nanoscale environment at excited state and are meant to fulfill these requirements: a) optical responses (intensity, wavelength-shift, lifetime, anisotropy) predictably related to the environmental polarity, viscosity, macromolecular structure, b) high brightness allowing for single-molecule detection, c) easily conjugable to biomolecules or macromolecules of interest. notably, we aim at conjugating these properties with the capability of nanoscopy imaging based on stimulated emission depletion or stochastic reconstruction optical microscopy. in this lecture the main features and applications of the engineered probes will be reviewed and future developments in this exciting field will be discussed. foamy virus (fv) is an atypical retrovirus which shares similarities with hiv and hepatitis b viruses. despite numerous biochemical studies, its entry pathway remains unclear, namely membrane fusion or endocytosis. to tackle this issue, dual color fluorescent viruses were engineered with a gfp labeled capsid and a mcherry labeled envelope. using high resolution 3d imaging and 3d single virus tracing, we followed the entry of the fluorescent viruses in living cells with a precision of 30nm in the plane and 40 nm along the optical axis. to distinguish between the two possible pathways, we developed a novel colocalization analysis method for determining the moment along every single trace where the colors separate, i.e. the fusion event. the combination of this dynamical colocalization information with the instantaneous velocity of the particle and its position within the reconstructed 3d cell shape allows us to determine whether the separation of capsid and envelope happens at the cell membrane or from endosomes. we then compared two types of fv and demonstrated, consistently with previous ph-dependency studies, that the prototype fv can enter the cell by endocytosis and membrane fusion, whereas the simian fv was only observed to fuse after endocytosis. phosphatidylinositol 4,5-bisphosphate (pi(4,5)p 2 ) is a minor component of the plasma membrane known to a critical agent in the regulation of synaptic transmission. clustering of pi(4,5)p 2 in synaptic active zones is important for synaptic transmission. however, pi(4,5)p 2 does not spontaneously segregate in fluid lipid membranes and another mechanism must be responsible for the lateral segregation of this lipid in active zones. clustering of pi(4,5)p 2 is expected to be associated with lipid-protein interactions and possibly partition towards lipid rafts in the plasma membrane. here we analyze the influence of protein palmitoylation on the formation of pi(4,5)p 2 clusters and on synaptic protein-pi(4,5)p 2 interaction by means of fö rster resonance energy transfer measurements by fluorescence lifetime imaging (fret-flim) and fret confocal microscopy. . during sporulation an entire chromosome is transferred into the forespore. this process starts by the formation of an asymmetrically located division septum that leads to the formation of two unequally sized compartments: a large mothercell and a smaller forespore. the septum traps about 30% of the chromosome to be transferred into the forespore. the remaining (*3mbp) are then translocated from the mother cell into the forespore by an active mechanism involving the spo-iiie dna translocase. the mechanisms of translocation, particularly the control of the directionality, still remains unknown and various models have been proposed so far. since each model predicts very different distribution of spoiiie proteins at the sporulation septum, we used palm microscopy (photoactivated localization microscopy) to investigate proteins localization in live-sporulating bacteria. using this technique, we showed that spoiiie proteins are forming a single tight focus at the septum with a characteristic size of around 30nm. more surprising, the focus is usually localized in the mother cell compartment and the mean distance between the spoiiie focus and the septum is 35nm. our data suggest that during the translocation process, spoiiie proteins are only forming stable complexes on the mother cell side, allowing then for a control of the chromosome translocation from the mother cell to the forespore. morphogenetic gradients determine cell identity in a concentration-dependent manner and do so in a way that is both incredibly precise and remarkably robust. in order to understand how they achieve this feat, one needs to establish the sequence of molecular mechanisms starting with morphogen gradient formation and leading to the expression of downstream target genes. in fruit flies, the transcription factor bicoid (bcd) is a crucial morphogen that forms an exponential concentration gradient along the embryo ap axis and turns on cascades of target genes in distinct anterior domains. we measured bcd-egfp mobility in live d. melanogaster embryos using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. we found that bcd-egfp molecules had a diffusion coefficient on the order of * 7 lm 2 /s during nuclear cycles 12-14, both in the cytoplasm and in nuclei. this value is large enough to explain the stable establishment of the bcd gradient simply by diffusion. on the other hand, in the context of the extremely precise orchestration of the transcription of the hunchback bcd target gene, it is too slow to explain how a precise reading of bicoid concentration could be achieved at each interphase without the existence of a memorization process. single molecule studies of key processes during the initiation of innate and adaptive immune response the two pillars of the vertebrate immune system are the innate and adaptive immune response, which confer resistance to pathogens and play a role in numerous diseases. here we exploit single molecule fluorescence imaging on live cells to study the key molecular processes that underpin these responses. the first project looks at the changes in the organisation of toll-like receptor 4 (tlr4) on the cell surface of macrophages upon activation via lipopolysaccharide (lps), as it is currently not known whether a higher level of tlr4 organisation is required for the signalling process. macrophages natively express tlr4 at a low level which allows for oligomerisation to be analysed in live cells by dynamic single molecule colocalisation (dysco) using data obtained by totalinternal reflection fluorescence (tirf) microscopy. the experiments of the second project aim at determining the critical initial events in t-cell triggering by labelling key proteins like the tcr receptor and cd45 on the surface of live t-cells and following how their spatial distribution changes following the binding of the t-cell to a surface. this enables us to distinguish between the different models of t-cell triggering which are based on aggregation, segregation or a conformational change of the tcr. the study of cells using scanning force microscopy the motility of unicellular parasites in mammalians seems very interesting, yet very complex. in a world, were inertia cannot be used for propulsion, in a world at low reynolds numbers, most of our everyday strategies of self-propulsion do not work. one class of parasites that know their way around, the flagellate trypansome, manage not only to survive in the blood stream, which is a lot faster than its own propulsion velocity and where the trypanosome is constantly attacked by its host's immune response, but also to penetrate the bloodbrain-barrier, which actually should be to tight to enter. even though trypanosomes are known for more than 100 years, their motility behaviour is not completely elucidated yet. now, using high-speed darkfield-microscopy in combination with optical tweezers in microfluidic devices and analyzing the recorded data, new light has been shed on the motility of these parasites. astonishing results show that trypanosomes are very well adapted to their hosts environment, they even can abuse red blood cells for their self-propulsion and use the bloodstream itself to drag antibodies bound to their surface to their cell mouth, where the antibodies are endocytosed and digested. the first part of the presentation will discuss nanoparticle (quantum dot, qd) biosensors and nanoactuators that exploit novel and unusual fret phenomena in the induction/detection of protein aggregation [1] , reversible on-off qd photoswitching [2] , and ph sensing [3] . the second part of the presentation will feature the application of an integrated chemical biological fret approach for the in situ (in/on living cells) detection of conformational changes in the ectodomain of a receptor tyrosine kinase (the receptor for the growth factor egf) induced by ligand binding [4] . the measurements were conducted with a two-photon scanning microscope equipped with tcspc detection; novel methods for lifetime analysis ad interpretation were employed to confirm the concerted domain rearrangements predicted from x-ray crystallography. the study of protein-protein interactions in vivo is often hindered by the limited acquisition speed of typical instrumentation used, for instance, for lifetime imaging microscopy. anisotropy polarization is altered by the occurrence of foerster resonance energy transfer (fret) and anisotropy imaging was shown to be comparatively fast and simple to implement. here, we present the adaptation of a spinning disc confocal microscope for fluorescence anisotropy imaging that allowed to achieve in vivo imaging at high spatial and temporal resolution. we demonstrate the capabilities of this system and in-house developed analysis software by imaging living caenorhabditis elegans expressing constitutive dimeric and monomeric proteins that are tagged with gfp. measuring intracellular viscosity: from molecular rotors to photodynamic therapy of cancer marina k. kuimova department of chemistry, imperial college london, exhibition road, sw7 2az, uk viscosity is one of the main factors which influence diffusion in condensed media and is crucial for cell function. however, mapping viscosity on a single-cell scale is a challenge. we have imaged viscosity inside live cells using fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent [1] [2] [3] . this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to 1009 higher than that of water. we demonstrated that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt) [2] . we also demonstrated that the ability of a fluorophore to induce apoptosis in cells during pdt [4] , or to act as a benign molecular rotor, measuring viscosity, can be controlled by carefully selecting the excitation wavelength in viscous medium [5] . in the field of biophysics and nanomedicine, the cellular reaction and the kinetics of gene expression after transfection of live cells with plasmid dna or gene-silencing sirna is of great interest. in a previous study on the transfection kinetics of non-viral gene-transfer [1] we realised that the development of single-cell arrays would be a great step towards easy-to-analyse, high-throughput transfection studies. the regular arrangement of single cells would overcome the limitations in image-analysis that arise from whole populations of cells. in addition to that, the analysis of expression kinetics at the single-cell can help to identify the cell-to-cell variability within a cell population. in order to develop suitable single-cell arrays, we are currently adjusting the different parameters of such a microenvironment (e.g. size, shape, surface-functionalisation) in order to end up with a defined surrounding for single-cell transfection studies. in addition to that, we try to find the optimal uptake pathway for each of the different applications. the neurodegenerative disorder alzheimer's disease (ad) causes cognitive impairment such as loss of episodic memory with ultimately fatal consequences. accumulation and aggregation of two proteins in the brain -amyloid beta and tau -is a characteristic feature. these soluble proteins aggregate during the course of the disease and assemble into amyloid-like filaments. recently it was found that the toxicity of soluble amyloid beta oligomers must also be taken into account for the pathogenesis of cognitive failure in ad. if oligomers are the predominant toxic species it would be pertinent to determine how they disrupt and impair neuronal function. the prion protein (prp) receptor has been proposed to mediate amyloid beta binding to neuronal cells. we have characterised the interaction of the amyloid beta and the prp receptor expressed on hippocampal and neuroblastoma cells at the single-molecule level. we do not detect any colocalisation of either the 40 or 42 amino acids variants with the prp receptor. bacterial biofilms are of the utmost importance in the study of environmental bioremediation and the design of materials for medical applications. the understanding of the mechanisms that govern cell adhesion must be analysed from the physics point of view in order to obtain quantitative descriptors. the genus rhodococcus is widely spread in natural environments. the species are metabolically diverse and thus they can degrade a wide range of pollutants. due to their high hydrophobicity, these cells are very resistant to harsh conditions, are able to degrade hydrophobic substances (e.g. oil) and attach to high-contact angle surfaces. the hydrophobicity of several strains of rhodococcus is measured and mapped using chemical force microscopy (cfm) in the present study. cfm relies on the functionalisation of scanning force microscopy (sfm) tips using hydrophobic or hydrophilic groups. in cfm, the microscope is operated in the forcevolume mode, which combines adhesion data with topographic images. the careful control of the tip chemistry permits the study of interactions between the functional groups on the tip and the bacterial surface, thus allowing the assessment of hydrophobicity. in order to perform a cfm study, the cells need to be firmly anchored to a substrate under physiological conditions (i.e. under a nutrient media or a saline buffer). to this end, several adhesive surfaces have been tested in order to find the one that gives the best results. optical microscopy is arguably the most important technique for the study of living systems because it allows 3d imaging of cells and tissues under physiological conditions under minimally invasive conditions. conventional far-field microscopy is diffraction-limited; only structures larger than *200 nm can be resolved, which is insufficient for many applications. recently, techniques featuring image resolutions down to *20 nm have been introduced such as localization microscopy (palm, storm) and reversible saturable optical fluorescent transition microscopy (resolft, sted). these methods are well suited for live-cell imaging and narrow the resolution gap between light and electron microscopy significantly. we have used palm imaging to study the formation and disassembly of focal adhesions of live hela cells in a high resolution pulse-chase experiment using monomeric irisfp [1] . mirisfp is a photoactivatable fluorescent protein that combines irreversible photoconversion from a green-to a red-emitting form with reversible photoswitching between a fluorescent and a nonfluorescent state in both forms. in our experiments a subpopulation of mirisfp molecules is photoconverted to the red form by irradiating a specified region of the cell with a pulse of violet light. migration of tagged proteins out of the conversion region can be studied by subsequently localizing the proteins in other regions of the cell by palm imaging, now using the photoswitching capability of the red species. real-time image reconstruction developed in our lab [2] allowed instant control imaging parameters. live cell imaging of cancer cells is often used for in-vitro studies in connection with photodynamic diagnostic and therapy (pdd and pdt). especially in presence of a photosensitizer, this live cell imaging can only be performed over relatively short duration (at most 1 hour). this restriction comes from the light-induced cell damages (photodamages) that result from rapid fluorescence photobleaching of photosensitizer. while these studies reveal exciting results, it takes several hours to discover the detailed effects of the photosensitizer on cell damage. up to our knowledge, however, there is no general guideline for modification of excitation light dose to achieve that. in this paper, the relation between excitation light doses, photobleaching of photosensitizer (pvp-hyperycin) and cell vitality are investigated using human lung epithelial carcinoma cells (a549). the strategy of this paper is to reduce the excitation light dose by using a low-power pulsed blue led such that the structures are visible in time-lapse images. fluorescence signals and image quality are improved by labelling the cells with an additional non-toxic marker called carboxyfluorescein-diacetate-succinimidyl-ester (cfse). in total we collected 2700 time-lapse images (time intervals 2 min) of dual-marked a549 cells under three different light intensities (1.59, 6.34 and 14.27 mw/cm 2 ) and a variety of pulse lengths (0.127, 1.29, 13, 54.5 and 131 ms) over five hours. we have found that there is a nonlinear relationship between the amount of excitation light dose and cell vitality. cells are healthy, i.e. they commence and complete mitosis, when exposed to low light intensities and brief pulses of light. light intensities higher than 6.34 mw/cm 2 together with pulse durations longer than 13 ms often cause cell vesiculation, blebbing and apoptosis. in all other cases, however, we found no cell death. in the future, this striking nonlinearity will be studied in more detail. progressive advances in scanning ion conductance microscopy (sicm) [1] enabled us to convert ordinary scanning probe microscope (spm) in to versatile multifunctional technique. as an imaging tool, ion conductance microscopy is capable to deliver highest possible topographical resolution on living cell membranes among any other microscopy techniques [2] . also, it can visualize surfaces complexity of those makes them impossible to image by other spms [3] . ion conductance microscopy combined with a battery of powerful methods such as fluorescence resonance energy transfer (fret) [4] , patch-clamp, force mapping, localized drug delivery, nano-deposition and nano-sensing is unique among current imaging techniques. the rich combination of ion conductance imaging with other imaging techniques such as laser confocal and electrochemical [5] will facilitate the study of living cells and tissues at nanoscale. ) and coleoptiles of wheat (triticum aestivum l.) seedlings, which were growing in light and dark conditions, were used to determine fluorescence of whole cells. fluorescence emission spectrum was monitored by fluorescent microscopy using the spectrometer usb 4000. fluorescence intensity f490, f680, f710 and f740 was determined and data was statistically analyzed in annova. we observed that bgf, rf and frf intensity increased in the first leaves with the age of the seedlings. in the coleoptiles was observed great bgf intensity increase with the age of the seedlings. in the coleoptiles decreased rf intensity of the 144 and 196 hours old seedlings, and bgf intensity decreased of 196 hours old seedlings. it was found that emission spectrum and fluorescence intensity changes are induced by the lack of light and salt (nacl) stress. analysis of fluorescence spectrum can quickly and accurately indicate the outset of light and salt stress in plants. there are analogical changes in fluorescence emission spectrum of plant cells in senescence and stress conditions. it was assumed that environmental stress and senescence have common mechanisms in plants. this changes can be monitored by fluorescent microscopy. triple-colour super-resolution imaging in living cells markus staufenbiel, stephan wilmes, domenik lisse, friedrich roder, oliver beutel, christian richter and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastraße 11, 49076, germany, markus.staufenbiel@biologie.uniosnabrü ck.de super-resolution fluorescence imaging techniques based on single molecule localisation has opened tremendous insight into the sub-micrometre organisation of the cell. live cell imaging techniques such as fluorescence photoactivation localization microscopy (fpalm) are currently limited to dual-colour detection due to the restricted availability of red-fluorescent photoswitchable proteins. we employed photoswitching of the oxazine dye atto655 under reducing conditions for super-resolution imaging in the cytoplasm of living cells. for efficient and specific covalent labelling of target proteins, we have made use of the halotag system. atto655 was coupled to the halotag ligand (htl) and fast reaction of htl-atto655 with the halotag enyzme was confirmed in vitro by solid phase binding assays. efficient labelling of the membrane cytoskeleton using lifeact fused to the halotag was observed and super-resolution imaging was readily achieved. based on this approach, we managed to follow the nanoscale dynamics of the actin cytoskeleton as well as clathrincoated pits using clathrin light chain fused to the halotag. we combined this technique with fpalm for triplecolour super-resolution imaging of the spatial distribution of membrane receptors in context of the membrane skeleton. the erbb family of receptor tyrosine kinases consists of four transmembrane proteins that transduce signals across the membrane to control cell fate. growth factor binding results in homo-and hetero-interactions between these receptors at the membrane. erbb receptors are implicated in many cancers, making them a target for therapeutic drugs. to date, studies of erbb interactions have been limited to individual family members or specific pairs, giving an incomplete picture of the highly complex behaviour controlling positive and negative feedback loops and signalling outcomes. to investigate erbb receptor interactions, we have developed tirf-based single molecule fluorescence microscopes capable of simultaneously imaging three, and soon five, fluorescence probes in live cells. we have also developed a catalogue of extrinsic fluorescent probes for 1:1 labelling of both endogenous and transfected erbb family members in mammalian cells, plus a bayesian approach to the analysis of single molecule data. this allows us to track active and inactive erbb family members at the basal surface of a model breast cancer cell line that expresses physiological levels of all four receptors. we present here initial characterisation of the entire erbb family together in the cell membrane. the human genome contains more than 800 g proteincoupled receptors (gpcrs); overall, 3-4% of the mammalian genome encodes these molecules. processes controlled by gpcrs include neurotransmission, cellular metabolism, secretion, and immune responses. however it is the stoichiometry of these receptors that is the most controversial. the starting point for understanding gpcr function was the idea that these receptors are monomeric. on the other hand a lot of recent studies favour the concept that gpcr form dimers and are not capable of signalling as independent monomers. recent single molecule studies try to solve this dilemma by suggesting that gpcrs form transient dimers with a lifetime of *100 ms. however questions remain about the physiological relevance of the preparations necessary for these studies, since they have not been performed on endogenous receptors. here, we directly image individual endogenous receptors using an equimolar mixture of two colour fluorescent fab fragments. we can then determine the receptors stoichiometry by quantifying its dynamic single molecule colocalisation (dy-sco) recorded by total-internal reflection fluorescence (tirf) microscopy. we have recently investigated the domain dynamics of pgk (1) . structural analysis by small angle neutron scattering revealed that the structure of the holoprotein in solution is more compact as compared to the crystal structure, but would not allow the functionally important phosphoryl transfer between the substrates, if the protein would be static. brownian large scale domain fluctuations on a timescale of 50 ns was revealed by neutron spin echo spectroscopy. the observed dynamics shows that the protein has the flexibility to allow fluctuations and displacements that seem to enable function. [ many physiological and pathological processes involve insertion and translocation of soluble proteins into and across biological membranes. however, the molecular mechanisms of protein membrane insertion and translocation remain poorly understood. here, we describe the ph-dependent membrane insertion of the diphtheria toxin t domain in lipid bilayers by specular neutron reflectometry and solid-state nmr spectroscopy. we gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. at ph 6, the native tertiary structure of the t domain unfolds, allowing its binding to the membrane. the membrane-bound state is characterized by a localization of the c-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. in contrast, the amphiphilic n-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. at ph 4, repulsive interactions vanish; the n-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. the c-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. these helices do not adopt a transmembrane orientation. interestingly, the t domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. we propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane. the limited stability in vitro of mps motivates the search of new surfactants (1) (2) (3) (4) . fss with a polymeric hydrophilic head proved to be mild towards mps (1) .new fss were designed with chemically defined polar heads for structural applications. lac-derivative was efficient in keeping several mps water soluble and active but formed elongated rods (2) . the glu-family was synthesized, characterized in by sans and auc and for its biochemical interest. the formation of rods is related to the low volumetric ratio between the polar head and hydrophobic tail. the surfactant bearing two glucose moieties is the most promising one, leading to both homogeneous and stable complexes for both br and the b 6 f. it was also shown be of particular interest for the structural investigation of membrane proteins using sans (3). by combining elastic and quasi-elastic neutron scattering data, and by applying theory originally developed to investigate dynamics in glassy polymers, we have shown that in lyophilised apoferritin above t * 100 k the dynamic response observed in the pico-to nano-second time regime is driven by ch 3 dynamics alone, where the methyl species exhibit a distribution of activation energies. our results suggest that over the temporal and spatial range studied the main apoferritin peptide chain remains rigid. interestingly, similar results are reported for other smaller, more flexible lyophilised bio-materials. we believe this work elucidates fundamental aspects of the dynamic landscape in apoferritin which will aid development of complex molecular dynamic model simulations of super-molecules. a detailed appreciation of the relationships between dynamics and biological function will require analysis based on such models that realize the full complexity of macromolecular material. biological systems must often be stored for extended periods of time. this is done by lyophilisation in the presence of lyoprotectants, such as sugars, which results in stable products at ambient conditions. [1] in an effort to understand the mechanism of preservation and stabilization, the interactions between sugars and liposome vesicles, which serve as a simple membrane model, have been studied extensively. amongst the common sugars, trehalose has superior preservative effects [1] and accumulates to high concentrations in many anhydrobiotic organisms. despite many experimental and numerical studies three mechanisms are proposed: vitrification [2] , preferential exclusion [3] and water replacement [4] . to gain more insight into the stabilization mechanism we have recently investigated the effect of trehalose on the bending elasticity of fully hydrated unilamellar vesicles of 1,2-dipalmitoyl-phosphatidylcholine (dppc) in d 2 o at temperatures below and above the lipid melting transition (tm) using neutron spin-echo. the data was analyzed using the zilman-granek theory. at all temperatures measured, trehalose stiffens the bilayer suggesting strong interactions between trehalose and the lipid. trehalose appears to broaden the melting transition but does not change the tm. this agrees with observations using differential scanning calorimetry. influence of macromolecular crowding on protein stability sté phane longeville, clé mence le coeur laboratoire lé on brillouin, gif-sur-yvette, france cell interior is a complex environment filled with a variety of different objects with respect to shape and size. macromolecules are present at a total concentration up to several hundred grams per litre and the overall occupied volume fraction can reach ö & 0.3-0.4. under crowding environment protein-protein interaction play a fundamental role. the crowding environment can affect some physical, chemical, and biological properties of biological macromolecules [1, 2] . traditionally, protein folding is studied in vitro at very low concentration of proteins. under such conditions, small globular single chain proteins can unfold and refold quite rapidly depending mainly to the nature of the solvent. such processes have been very intensively studied, since folding of proteins into their native structure is the mechanism, which transforms polypeptide into its biologically active structure. protein misfolding is involved in a very high number of diseases [4] (e.g. alzheimer, parkinson, and kreuzfeld-jacob diseases, type ii diabetes, …). theoretically, the problem was studied by the introduction of the concept of excluded volume [5] . in recent papers [6, 7] , minton uses statistical thermodynamic models to address the question. he predicted that inert cosolutes stabilize the native state of proteins against unfolding state mainly by destabilizing the unfolded state and that the dimension of the unfolded state decreases with increasing the concentration of cosolute in a measurable way. small angle neutron scattering (sans) is a technique of choice for such study because, by using appropriate mixtures of light and heavy water, it is possible to match the scattering length density of the solvent to the one of the cosolute and thus to measure the conformation of a molecule at low concentration in a presence of a high concentration of another one. we will present a complete experimental study of the mechanism that leads to protein stabilization by macromolecular crowding [7, 8] . coupled dynamics of protein and hydration water studied by inelastic neutron scattering and molecular dynamics simulation hiroshi nakagawa 1,2 , mikio kataoka 1,3 1 japan atomic energy agency, tokai, japan, 2 juelich centre for neutron science, forschungszentrum juelich gmbh, 3 nara institute of science and technology, ikoma, japan proteins work in an aqueous environment at ambient temperature. it is widely accepted that the proteins are flexible and mobile. the flexibility and mobility, that is, protein dynamics are essential for protein functions. neutron incoherent scattering is one of the most powerful techniques to observe protein dynamics quantitatively. here i will talk about dynamics of protein and its hydration water. the structure of a soluble protein thermally fluctuates in the solvated environment of a living cell. understanding the effects of hydration water on protein dynamics is essential to determine the molecular basis of life. however, the precise relationship between hydration water and protein dynamics is still unknown because hydration water is ubiquitously configured on the protein surface. we found that hydration level dependence of the onset of the protein dynamical transition is correlated with the hydration water network. hydration water dynamics change above the threshold hydration level, and water dynamics control protein dynamics. these findings lead to the conclusion that the hydration water network formation is an essential property that activates the anharmonic motions of a protein, which are responsible for protein function. thermal motions and stability of hemoglobin of three endotherms (platypus -ornithorhynchus anatinus, domestic chicken -gallus gallus domesticus and human -homo sapiens) and an ectotherm (salt water crocodile -crocodylus porosus) were investigated using neutron scattering and circular dichroism. the results revealed a direct correlation between the dynamic parameters, melting -, and body temperatures. on one hand, a certain flexibility of the protein is mandatory for biological function and activity. on the other hand, intramolecular forces must be strong enough to stabilize the structure of the protein and to prevent unfolding. our study presents experimental evidence which support the hypothesis that the specific amino acid composition of hb has a significant influence on thermal fluctuations of the protein. the amino acid sequence of hb seems to have evolved to permit an optimal flexibility of the protein at body temperature. macromolecular resilience was found to increase with body and melting temperatures, thus regulating hb dynamics. where k is the trap spring constant, a is the subdiffusion exponent and e a is the mittag-leffler function. the parameters obtained by fitting this equation to the experimental msds are summarized in table 1 . at short lag times we have not found any difference between the two cell types, contrarily to the previous results obtained by afm 2 . for both cell lines the subdiffusion exponent, a was found close to , the value predicted by the theory of semiflexible polymers. but the crossover frequency x 3 , was found smaller for the cancerous cells for all datasets. it corresponds to passage to the confined regime at longer times. we attribute it to the bigger impact of molecular motors. we study the spatiotemporal evolution of fibrous protein networks present in the intracellular and extracellular matrices. here, we focus on the in vitro actin network dynamics and evolution. in order to study the hierarchical self-assembly of the network formation in a confined environment and provide external stimuli affecting the system minimally, we introduce a new microfluidic design. the microfluidic setup consists of a controlling channel to which microchambers of different shapes and sizes are connected through narrow channels. this design results in having mainly convective transport in the controlling channel and diffusive transport into the microchamber. rhodamine labeled actin monomers diffuse into the chamber. after polymerization is induced, they form a confined entangled network. cross-linking proteins can then be added to increase the network complexity. moreover, we can generate gradients of reactants across the microchambers and vasodilator-stimulated phosphoprotein (vasp) is a crucial regulator of actin dynamics. it is important in cellular processes such as axon guidance and migration, promoting assembly of filopodia and lamellipodia. vasp's multiple domain structure increases the range of interactions it has with actin monomers, filaments, and other proteins and it displays multiple binding modes both in vitro and in vivo, including barbed end elongation and filament bundling. however, it is not fully understood how vasp affects the structural and mechanical properties of actin networks. we characterize vasp-mediated bundling of actin networks in a simplified in vitro system using confocal microscopy and quantify mechanical properties with rheology measurements. we show that the network properties differ from other actin bundling proteins and reflect vasp's multiple domain structure, displaying a complex bundling phase space that depends upon solution conditions. we observe the formation of large bundle aggregates accompanied by a reduction in network elasticity at high protein ratios. in addition, we change vasp's actin binding mode and eliminate bundling by introducing free actin monomers. finally, we show preliminary results from a biomimetic system that extends the range of actin-vasp interaction. cell migration or proliferation? the go or grow hypothesis in cancer cell cultures tamá s garay, é va juhá sz, jó zsef tímá r, balá zs heged} us 2 nd department of pathology, semmelweis university, budapest, hungary background: cancer related death is constantly growing in the past decades. the mortality of solid tumors is mostly due to the metastatic potential of tumor cells which requires a fine adjustment between cell migration and cell proliferation. as the metabolic processes in the cell provide a limited amount of available energy (i.e. atp) the various biological processes like cell motility or dna synthesis compete for the atp available. the go or grow hypothesis postulates that tumor cells show either high migration or proliferation potential. in our study we investigate on a large series of tumor cell lines whether this assumption stands for malignant cells. materials and methods: twenty tumor cell lines derived from malignant mesothelioma (mesodermal origin) and malignant melanoma (neuroectodermal origin) were subjected to three-days-long time-lapse videomicroscopic recordings. cell motility and proliferation were characterized by the probability of cell division within 24 hours and the 24-hour migration distance of the cells. results: we found a wide range in both the cell migratory activity and the proliferation capacity in our series. the 24-hour migration distance ranged from 40 to 300 micron and from 10 to 130 micron in mesothelioma and melanoma cells, respectively. the lowest 24-hour cell division probability was found to be 0.21 in both the melanoma and mesothelioma series while the highest proliferation activity reached 1.1 and 1.4 in melanoma and mesothelioma, respectively. interestingly, in the melanoma cell lines we found a significant positive correlation (r=0,6909; p=0,0186) between cell proliferation and cell migration. in contrast our mesothelioma cell lines displayed no correlation between these two cellular processes. conclusions: in summary our findings demonstrate that the investigated tumor cells do not defer cell proliferation for cell migration. important to note the tumor cells derived from various organ systems may differ in terms of regulation of cell migration and cell proliferation. furthermore our observation is in line with the general observation of pathologists that the highly proliferative tumors often display significant invasion of the surrounding normal tissue. many cell types are sensitive to mechanical signals. one striking example is the modulation of cell proliferation, morphology, motility, and protein expression in response to substrate stiffness. changing the elastic moduli of substrates alters the formation of focal adhesions, the formation of actin filament bundles, and the stability of intermediate filaments. the range of stiffness over which different primary cell types respond can vary over a wide range and generally reflects the elastic modulus of the tissue from which these cells were isolated. mechanosensing also depends on the type of adhesion receptor by which the cell binds, and therefore on the molecular composition of the specific extracellular matrix. the viscoelastic properties of different extracellular matrices and cytoskeletal elements also influence the response of cells to mechanical signals, and the unusual non-linear elasticity of many biopolymer gels, characterized by strain-stiffening leads to novel mechanisms by which cells alter their stiffness by engagement of molecular motors that produce internal stresses. the molecular mechanisms by which cells detect substrate stiffness are largely uncharacterized, but simultaneous control of substrate stiffness and adhesive patterns suggests that stiffness sensing occurs on a length scale much larger than single molecular linkages and that the time needed for mechanosensing is on the order of a few seconds. to explore potential role of cytoskeletal component in cardiomyocyte for adaptation to extreme conditions was carried out the comparative study of expression of cytoskeletal sarcomeric protein titin in myocardium of ground squirrels during hibernation and gerbils after spaceflight. we have revealed a two-fold increase in content of long n2ba titin isoform as compared to short n2b titin isoform in different heart chambers of hibernating ground squirrels. the prevalence of the long titin isoform is known to determine the larger extensibility of heart muscle that promotes, according to frank-starling law, the increase in force of heart contractility for pumping higher viscous blood during torpor and adapting the myocardium to greater mechanical loads during awakening. moreover, titin mrna level showed seasonal downregulation in which all hibernating stages differed significantly from summer active level. it is possible that the decline of mrna and protein synthesis during hibernation may be regarded as the accommodation for minimization of energetic expenditures. we have not revealed differences in titin mrna levels between control gerbils and gerbils after spaceflight. but we have also observed the two-fold growth in the amount of n2ba titin isoform in left ventricle of gerbils after spaceflight that is likely to be directed to the restoration of the reduced heart contractility at zero-gravity. these results suggest that the increase of the content of the long n2ba titin isoform may serve as universal adaptive mechanism for regulating of heart function in response to the extreme conditions. nuclear migration is a general term for a non-random movement of the nucleus toward specific sites in the cell. this phenomenon has been described throughout the eukaryotes from yeast to mammals. the process is however still poorly understood in mammalian cells. by using microcontact printing we are able to regulate the geometry and spreading of cultured cells. adhesive micropatterns of fibronectin provide an attachment surface for the cells whereas the passivation of the surface by pll-peg prevents protein, thus cell adhesion. live cell imaging by time-lapse microscopy has shown that under these conditions cells gain a bipolar shape, and more interestingly, the nuclei of the cells showed auto-reversed motion. our research tries to understand the molecular cues and mechanisms behind the observed cellular and nuclear movement. we have already shown that the cytoskeleton plays an important role in this phenomena but the exact players and the detailed mechanism remain to be clarified. in order to identify the most important components and their relationship have drug treatments and sirna experiments have been applied. although our research focuses mainly on the motility of the nucleus, it may also help to get a better understanding of the general theme of cell migration. cell motility involves a number of strategies that cells use to move in their environments in order to seek nutrients, escape danger and fulfil morphogenetic roles. when these processes become uncontrolled, pathological behaviours, like cancer or metastasis of cancerous cells, can occur. here we present a new method for the contextual quantification of cellular motility, membrane fluidity and intracellular redox state, by using the ratiometric, redox-sensitive protein rxyfp and the ratiometric fluidity-sensitive probe laurdan. we provide evidence that dynamic redox and fluidity changes are correlated with signaling processes involved in cellular motility. these findings may pave the way to novel approaches for the pharmacological control of cell invasiveness and metastasis. manipulation of cellular mechanics anna pietuch, andreas janshoff georg-august university, tammanstraße 6, 37077 gö ttingen, germany, e-mail: anna.pietuch@chemie.unigoettingen.de rheological properties of cells determined by the underlying cytoskeleton (cortex) are key features in cellular processes like cell migration, cell division, and cell morphology. today it is possible to investigate local cellular elastic properties under almost physiological conditions using the afm. by performing force indentation curves on local areas on a cell surface the use of contact mechanic models provides the young's modulus, comprising information about the elastic properties of cells. the administration of cytoskeleton modifying substances into the cell is achieved by microinjection. we are also investigating morphological changes and rearrangements of the cytoskeleton in time resolved impedance measurements. electric cell-substrate impedance sensing is a label-free and minimal invasive technique which allows monitoring morphological changes of cells in real time. readout of the impedance is sensitive to changes in cell-substrate contacts as well as density of cell-cell contacts yielding important information about the integrity of the cell layer and changes in the properties of the cell membrane. we are studying the cellular response to modification of the cytoskeleton e.g. by introducing proteins which affect directly the organization of the actin structure like ezrin. mechanical characterization of actin gels by a magnetic colloids technique thomas pujol, olivia du roure, julien heuvingh pmmh, espci-cnrs-umpc-p7. paris, france the actin polymer is central in cell biology: it is a major component of cytoskeleton and it plays a fundamental role in motility, division, mechanotransduction…. its polymerization just beneath the cell membrane generates forces responsible for cell movement. actin filaments form a network whose architecture is defined by the nature of the binding proteins and depends on the location in the cell. for example, in the structure which leads cell migration, the lamellipodium, the gel is branched due to its interaction with arp2/3 protein complex. determining the mechanical properties of such actin network is a crucial interest to understand how forces are generated and transmitted in living cells. we grow a branched actin network from the surface of colloids using the arp2/3 machinery. the particles are super paramagnetic and they attract each other via dipole-dipole interaction to form chain. by increasing the magnetic field we apply an increasing force to the gel and we optically measure the resulting deformation. from those measurements we deduce a young modulus for a large amount of data. we are characterizing different networks by varying the concentration of the capping and branching proteins and we show how mechanics can be regulated by the different proteins. microtubules (mts) are central to the organization of the eukaryotic intracellular space and are involved in the control of cell morphology. in fission yeasts cells mts transport polarity factors to poles where growth is located, thus ensuring the establishment and maintenance of the characteristic spherocylindrical shape. for this purpose, mt polymerization dynamics is tightly regulated. using automated image analysis software, we investigated the spatial dependence of mt dynamics in interphase fission yeast cells. we evidenced that compressive forces generated by mts growing against the cell pole locally reduce mt growth velocities and enhance catastrophe frequencies. in addition, our systematic and quantitative analysis (in combination with genetic modifications) provides a tool to study the role of ?tips (plus-end tracking proteins) such as mal3 and tip1 in the spatial regulation of mt dynamics. we further use this system to decipher how the linear transport by mt interferes with the feedback circuitry that assures the correct spatial distribution of tea1, the main polarity factor in fission yeast cells. the dynamics of the cytoskeleton are largely driven by cytoskeletal motor proteins. complex cellular functions, such as mitotsis, need a high degree of control of these motors. the versatility and sophistication of biological nanomachines still challenges our understanding. kinesin-5 motors fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plus-end directed motors. here we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin-5 cin8 using single-molecule motility assays and single-molecule fluorescence microscopy. in vivo, the majority of cin8 motors moved slowly towards mt plus-ends, but we also observed occasional minus-end directed motility episodes. in vitro, individual cin8 motors could be switched by ionic conditions from rapid (up to 50 lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert in loop 8 of cin8 induced bias towards minus-end motility and strongly affected the directional switching of cin8 both in vivo and in vitro. the entirely unexpected in vivo and in vitro switching of cin8 directionality and speed demonstrate that kinesins are much more complex than thought. these results will force us to rethink molecular models of motor function and will move the regulation of motors into the limelight as pivotal for understanding cytoskeleton-based machineries. morphological and dynamical changes during tgf-b induced epithelial-to-mesenchymal transition david schneider 1 , marco tarantola 2 , and andreas janshoff 1 1 institute of physical chemistry, georg-august-university, gö ttingen, germany, 2 max planck institute for dynamics and self-organization, laboratory for fluid dynamics, pattern formation and nanobiocomplexity (lfpn), goettingen, germany the epithelial-to-mesenchymal transition (emt) is a program of cellular development associated with loss of cell-cell contacts, a decreased cell adhesion and substantial morphological changes. besides its importance for numerous developmental processes like embryogenesis, emt has also been held responsible for the development and progression of tumors and formation of metastases. the influence of the cytokine transforming growth factor 1 (tgf-b1) induced emt on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines nmumg, a549 and mda-mb231 was investigated by time-resolved impedance analysis and atomic force microscopy (afm) performing force-indentation measurements. the three cell lines display important differences in cellular morphology mirrored in changes of their elastic response (young modulus), as well as their dynamics upon tgf-b1 treatment. impedance based measurements of micromotility reveal a complex dynamic response to tgf-b1 exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. additionally, the investigation of cellular elasticity via afm depicts the different cytoskeletal alterations depending on the metastatic potential of the used cell type. physics of cellular mechanosensitivity studied with biomimetic actin-filled liposomes bjö rn stuhrmann, feng-ching tsai, guido mul, gijsje koenderink fom institute amolf, amsterdam, the netherlands biological cells actively probe the mechanical properties of their tissue environment by exerting contractile forces on it, and use this information to decide whether to grow, migrate, or proliferate. the physical basis for cell contractility is the actin cytoskeleton, which transmits motor generated stresses to mechanosensitive adhesions sites that anchor the cell to the tissue. the origins of mechanosensing are far from understood due to the complex interplay of mechanical effects and biochemical signaling that occurs far from equilibrium. we use a quantitative biophysical approach based on biomimetic constructs to elucidate physical principles that underlie active mechanosensing in biological cells. we have built realistic in vitro models of contractile cells by encapsulating cross-linked actin networks together with myosin motors in cell-sized membranous containers (liposomes). our method has several advantages over prior methods, including high liposome yield, compatibility with physiological buffers, and chemical control over protein/lipid coupling. i will show contour fluctuation spectra of constructs and first data on mechanical response obtained by laser tweezers microrheology. our work will yield novel insights into stress generation and stiffness sensing of cells. setting up a system to reconstitute cytoskeleton-based protein delivery and patterning in vitro nú ria taberner, liedewij laan, marileen dogterom fom institute amolf, amsterdam, the netherlands keywords: microtubules, fission yeast, cell polarity, protein patterning, plus end binding proteins. many different cell types, from mobile fibroblasts [1] to fission yeast cells [2] , display non-homogenous protein patterns on their cell cortex. in fission yeast the cell-end marker protein tea1 that among others is responsible for recruiting the actin dependent cell-growth machinery, is specifically located at the growing cell ends. tea1 travels at the tips of growing microtubules and is delivered to the cell ends [2] . we aim to in vitro reconstitute a minimal microtubule plus-end tracking system that leads to cortical protein patterning in functionalized microfabricated chambers. our model will allow us to perturb microtubule-based transport and diffusion independently and evaluate the resulting protein patterns. [ the tropomyosins (tm) are dimeric actin-binding proteins that form longitudinal polymers along the actin filament groove. there is a great variety of isoforms, but the division of labour between the individual tms and their significance is poorly understood. as in most cell types, also in the neurons several isoforms are present, whose spatio-temporal localisation is differentially regulated. the neuron-specific brain 3 isoform (tmbr-3) can be found in the axon of the mature cells. we aimed to clone, express and charaterise this protein in terms of its effects on the kinetic parameters of the actin filament. using a pet28a construct we purified native, tag-free protein, and examined if it influences the rate of actin polymerisation or the stability of the filaments in the presence of either gelsolin or latrunculin-a, two depolymerising agents. in cosedimentation experiments the affinity of tmbr-3 to actin was* 3lm, about six times that of skeletal muscle tropomyosin. the net rate of actin polymerisation was reduced by 17% in the presence of tmbr-3. the depolymerisation induced by gelsolin or latrunculin-a was inhibited in a concentration-dependent manner. tmbr-3 seems to stabilise actin filaments against disassembly without significant effect on the net polymerisation. cell mobility and metastatic spreading: a study on human neoplastic cells using optical tweezers f. tavano the primary causes of death in cancer patients are local invasion and metastasis but their mechanisms are not yet completely understood. metastatization is accompanied by alterations of the cytoskeleton and membrane structure leading to changes in their biomechanical properties [1] . in this study we analyzed by means of optical tweezers the mechanical properties of two different breast adenocarcinoma cell lines corresponding to different metastatic potential. ot were used to grab the plasma membrane by a 1,5 um silica bead and form a plasma membrane tether. we measured the force exerted by the cell membrane on the bead and drew the force-elongation curves. fitting data in the kelvin body model [2] we found out the values for the viscoelastic parameters influencing the pulling of the membrane tethers. the first cell line analyzed, mcf-7, associated to a low metastatic potential showed tether stiffness of 153 pn/um in average. the second cell line, mda-mb 231, poorly differentiated with a high metastatic potential had a tether stiffness of 36pn/um in average, that is a four times lower value. these results seems to confirm the hypotesis that metastasis prone cells are softer than less aggressive cancer cells, and support the use of ot for these measurements for its sub-pn force resolution and because cells are manipulated without damage. [ tubulin polymerization promoting protein (tppp/p25) is a brain-specific protein that primary targets the microtubule network modulating its dynamics and stability. tppp/p25 is a disordered protein with extended unstructured segments localized at the n-and c-terminals straddling a flexible region. tppp/p25 is primarily expressed in oligodendrocytes where its multifunctional features such as tubulin polymerization promoting and microtubule bundling activities are crucial for the development of the projections in the course of oligodendrocyte differentiation enabling the ensheathment of axons with a myelin sheath that is indispensable for the normal function of the central nervous system. microtubule network, a major constituent of the cytoskeleton, displays multiple physiological and pathological functions in eukaryotic cells. the distinct functions of the microtubular structures are attained by static and dynamic associations of macromolecules and ligands as well as by post-translational modifications. tppp/p25 is actively involved in the regulation of microtubule dynamics not exclusively by its bundling activity, but also by its tubulin acetylation-promoting activity. atypical histone deacetylases, such as nad-dependent sirt2 and histone deacetylase-6, function outside of the nucleus and control the acetylation level of cytosolic proteins, such as tubulin. acetylation-driven regulation of the microtubule network during cellular differentiation is an ambiguous issue. tppp/p25 has been recently identified as an interacting partner and inhibitor of these deacetylases and their interaction decreased the growth velocity of the microtubule plus ends and the motility of the cells. we have established cell models for the quantification of the acetylation degree of microtubule network in correlation with its dynamics and stability as well as in relation to aggresome formation, that mimics the pathological inclusion formation. the intracellular level of tppp/p25 is controlled at posttranscription level by microrna and at protein level by the proteosome machinery. under pathological circumstances this disordered protein displays additional moonlighting function that is independent of its association with microtubule system or deacetylases; it enters aberrant proteinprotein interaction with a-synuclein forming toxic aggregates within the neuronal and glial cells leading to the formation of inclusions characteristic for parkinson's disease and multiple system atrophy, respectively. the cell membrane separates the intracellular from the extracellular environment while intimately interacting with the cytoskeleton in numerous cellular functions, including cell division and motility. cell shape changes are for a large part mediated by the contractile actomyosin network forming the cortex underneath the cell membrane. to uncover molecular mechanisms of cell shape control based on actin-membrane interactions, we built a novel biomimetic model system: a cell-sized liposome encapsulating an actively contracting actin-myosin network. our fabrication method is inspired by a recent report of liposome preparation by swelling of lipid layers in agarose hydrogel films. 1 we extensively characterize important liposomal properties, finding diameters between 7 and 20 lm, unilamellarity, and excellent and uniform encapsulation efficiency. we further demonstrate chemical control of actin network anchoring to the membrane. the resulting liposomes allow quantitative tests of physical models of cell shape generation and mechanics. in the cohesive structure of the cytoskeleton functionally distinct actin arrays orchestrate fundamental cell functions in a spatiotemporally controlled manner. emerging evidences emphasize that protein isoforms are essential for the functional polymorphism of the actin cytoskeleton. the generation of diverse actin networks is catalyzed by different nucleation factors, like formins and arp2/3 complex. these actin arrays also exhibit qualitative and quantitative differences in the associated tropomyosin (tm) isoforms. how the molecular composition and the function of actin networks are coupled is not completely understood. we investigated the effects of different tm isoforms (skeletal muscle, cytoskeletal 5nm1 and br3) on the activity of mdia1 formin and arp2/3 complex using fluorescence spectroscopic approaches. the results show that the studied tm isoforms have different effects on the mdia1-, and arp2/3 complexmediated actin assembly. the activity of the arp2/3 complex is inhibited by sktm and tm5nm1, while tmbr3 does not have any effect. all three tm isoforms inhibited the activity of mdia1. these results contribute to the understanding of the mechanisms by which tropomyosin isoforms regulate the functional diversity of the actin cytoskeleton. chronic thromboembolic pulmonary hypertension (cteph) is a dual pulmonary vascular disorder, which combines major vascular remodelling with small-vessel arteriopathy. the presence of fibroblasts in the clot, occluding the pulmonary arteries, and its composition create a microenvironment with increased collagen level, which might affect the local endothelial function. in this study, human pulmonary artery endothelial cells (hpaec) were exposed to collagen type i to address the effect of the thrombotic microenvironment on the vessel wall forming cells. the hpaecs, cultured under standard conditions were treated with 1, 10 and 100lg/ml of collagen type i for 6h and 24h. the changes in the endothelial cell barrier function were investigated by performing permeability and migration test as well as ve-cadherin staining. collagen type i treatment led to a decrease in ve-cadherin signal in hpaec. the loosening of cell-cell contacts could be proven with a significant increase in permeability after 6h of collagen treatment with different concentrations. besides the loosening of the cell-cell contacts, the hpaec migration was also dose dependently retarded by collagen application over time. our data show that collagen-rich microenvironment leads to a disruption of the junctional proteins in hpaecs, indicating an environmental induced possible alteration in the function of endothelial cells in the clots of cteph patients. the implementation of miniaturisation and high throughput screening has quickened the pace of protein structure determination. however, for most proteins the process still requires milligram quantities of protein with purity [ 95 %. these amounts are required as a result of unavoidable losses during purification and for the extensive screening of crystallisation space. for integral membrane proteins (imps), one of the initial steps in the structure determination procedure is still a major bottleneck -the over-expression of the target protein in the milligram quantity range. with a view of developing guidelines for over-expression of human imps, a systematic approach using the three most common laboratory expression systems (e. coli, s. cerevisiae, sf9 insect cells) was implemented. initial expression levels were determined by either partial purification using ni 2? -nta (e. coli), green fluorescence protein (gfp) fluorescence using a c-terminal gfp tagged protein (s. cerevisiae) or flag tagged partial purification (sf9 cells). the results show that e. coli is suitable for the over-expression of human imps in the required quantity range however protein size and complexity is an important factor. the yeast system is fast and affordable but, for the group of human imps tested, the expression levels were borderline. finally for the insect cell system, the timelines are slower and it is in comparison costly to run, however, it can produce relatively large quantities of human imps. the cu?-atpase copb of enterococcus hirae is a bacterial p-type atpase involved in resistance to high levels of environmental copper by expelling excess copper. the membrane protein copb was purified from an over-expressing strain and solubilized in dodecyl-maltoside. by uv circular dichroism the secondary structure is predicted to contain 40-50 % a-helices and 10-15% b-sheets in agreement with estimates based on homology with the ca atpase serca1. we present cd-spectroscopic data on thermal unfolding of the protein to address the influence of the binding of the atp analogs atpcs and the fluorescent analog mant-atp on the protein stability. such analogs are used to mimic functional states of the atpase but undergo different interactions with the binding site that are not well characterized. we propose a competition-based assay for nucleotide binding using cdspectroscopy to deduce the occupancy of the nucleotidebinding site by non-fluorescent nucleotides. alternatively, the change of intrinsic fluorescence of mant-atp upon binding to the atpase is exploited in these assays. finally, we show how the simultaneous measurement of protein cd and nucleotide fluorescence in thermal denaturation experiments may help to determine the stability of several functional conformational states of copb. showing the steady-state distribution of electric potential, ionic concentrations are obtained efficiently. channel current, a summation of drift and diffusive currents, can be further computed from the flux of ionic concentrations. the influence of finite size effect will be also addressed. effect of cholesterol and cytoskeleton on k v 10.1 membrane distribution jimé nez-garduño am 1,2 , pardo la 2 , ortega a 1 , stü hmer w 2 1 unam, mexico-city, mexico, 2 mpi-em, gö ttingen, germany the potassium channel k v 10.1 is expressed nearly exclusively in the central nervous system. besides its function as an ion channel, k v 10.1 has also been associated with non-canonical signaling functions. various membrane proteins associated with cholesterol-sphingolipids enriched microdomains are involved in signaling pathways. in this work we studied the membrane distribution of k v 10.1 in highly purified brain-tissue plasma membranes as a function of cholesterol content versus cytoskeletal proteins. the results show that one fraction of kv10.1 associates to cholesterol-rich domains or detergent resistant membranes (drm) and another fraction to non-drm domains. the kv10.1 fraction inserted in drm is dependent on cholesterol as well as on cytoskeleton proteins. depletion of cholesterol leads to a doubling of k v 10.1 current density. we suggest that k v 10.1 coexists in two different populations: one where the transmembrane domain fits cholesterol enriched membranes and another able to fit into a less packed lipid bilayer. the importance of this distribution on signaling processes needs to be further investigated. we use the reduced model of an axis-symmetric water-filled channel whose protein wall has a single charged site. the channel length, radius and fixed charge are selected to match experimental data for gramicidin a. the ion current, occupancy and escape rate are simulated by the 1d self-consistent bd technique with account taken of the electrostatic ion-ion interaction. the bath with non-zero ion concentration on one side of the channel is modelled via the smoluchowski arrival rate. it is shown that: a) the occupancy saturates with michaelis-menten kinetics. b) the escape rate starts from the kramers value at small concentrations and then increases with concentration due to the electrostatic amplification of charge fluctuations. the resulting dynamics of the current can be described by modified reaction rate theory accounting for ionic escape over the fluctuating barrier [1] . many membrane-protein functions are amenable to biophysical and biochemical investigation only after the protein of interest has been reconstituted from a detergent-solubilised state into artificial lipid bilayers. unfortunately, functional reconstitution has remained one of the main bottlenecks in the handling of numerous membrane proteins. in particular, gauging the success of reconstitution experiments has thus far been limited to trial-and-error approaches. to address this problem, we have established high-sensitivity isothermal titration calorimetry (itc) as a powerful method for monitoring the reconstitution of membrane proteins into liposomes. itc has previously been employed for characterising liposome solubilisation and reconstitution in the absence of protein. here we show that itc is also excellently suited for tracking the complex process of membrane-protein reconstitution in a non-invasive and fully automated manner. the approach is exemplified for the prokaryotic potassium channel kcsa, which we first purified in detergent micelles and then reconstituted into stable proteoliposomes at very high protein densities. electrophysiological experiments performed in planar lipid membranes confirmed that kcsa regained its functional activity upon itc-guided reconstitution. gating currents of low-voltage-activated t-type calcium channels family má ria karmažínová , ľ ubica lacinová institute of molecular physiology and genetics, sav, bratislava, slovak republic t-type calcium channels are distinguished by relatively low voltage threshold for an activation and steep voltage dependence of activation and inactivation kinetics just above the activation threshold. kinetics and voltage dependence of macroscopic inward calcium current through ca v 3 channels was described in a detail. in contrast, very little information is available on gating current of these channels. therefore we compared gating currents measured from all three ca v 3.1, ca v 3.2 and ca v 3.3 channels. voltage dependencies of charge movement differ dramatically from those for macroscopic current. first, their slope factors are several-fold bigger that slope factors of macroscopic current activation. second, activation mid-point for ca v 3.3 channels on-gating is shifted to more positive membrane potentials by about 20 mv compare to ca v 3.1 and ca v 3.2 channels, whose activation mid-points are similar. the same is truth for off-gating voltage dependences. kinetics of both onand off-gating is remarkably faster for ca v 3.1 and ca v 3.2 channels compare to ca v 3.3 channels. further, more charge is moved per unit of macroscopic current amplitude in ca v 3.3 channels compare to ca v 3.1 and ca v 3.2 channels. supported by apvv-0212-10 and vega 2/0195/10. the local anaesthetic lidocaine (lid) is generally believed to reach its binding site in the intracellular vestibule of the voltage-gated sodium channel via the cell membrane. qx222 (qx) is a permanently charged, quaternary amine analogue of lid, that can access this binding site via a hydrophilic route across the channel protein. the mutation i1575e of the adult rat muscle-type sodium channel (rna v 1.4) opens such a hydrophilic pathway. when bound to the internal vestibule, lid stabilizes both fast and slow inactivated states. we wondered whether qx, once bound to the internal vestibule, exerts a similar modulatory action on inactivated states as lid. the construct i1575e was expressed in tsa201 cells and studied by means of the patch-clamp technique. when applied from the extracellular side 500 lm qx stabilized the slow but not the fast inactivated state in i1575e. when applied internally, qx entered the channel, but stabilization of inactivated states could not be observed. these results suggest that binding site for use-dependent block is in the inner vestibule of the channel, fast inactivation is modulated only by the hydrophobic form of lid, and the binding site for modulation of slow inactivation by qx is only accessible from the extracellular side of the channel. we observed that lipophilicity (quantified by the logarithm of the calculated water-octanol partition coefficient, logp) is important in determining both kr and ki, but had a greater effect on ki. distribution coefficients (logd) discriminated better between kr and ki than partition coefficients (logp). the ratio of positively charged/neutral forms (quantified by the acidic dissociation constant, pka) was a significant determinant of resting affinity: predominantly charged compounds tended to be more potent against resting channels, while neutral compounds tended to be more state-dependent. aromaticity was more important for inactivated state affinity. the acidification of intracellular compartments is critical for a wide range of cellular processes. a recent candidate for ph regulation within early endosomes and tgn is the highly conserved intracellular na ? /h ? exchanger 7 isoform (nhe7), whose mutation leads to neurological syndromes in human patients. however, due to its intracellular localization, nhe7 biochemical features are still poorly characterized and its biological function remains elusive. we have developed somatic cell genetic techniques that enable the selection of variant cells able to resist h ? killing through plasma membrane expression of h ? extruders. this enabled us to obtain stable cell lines with forced plasma membrane expression of nhe7. we used them to measure its functional and pharmacological parameters with high accuracy, using fast transport kinetics. to summarize, this exchanger displays unique features within the nhe family, especially with respect to its affinity for its substrates, lithium, sodium and protons and for its guanidine-derived inhibitors. taken together with our results on the subcellular localization of the native nhe7, these unique biochemical features provide new insights on the biological function and pathological implications of this intracellular na ? /h ? exchanger. analysis of the collective behaviour of ion channels j. miśkiewicz, z. trela, s. przestalski wrocław university of environmental and life sciences, physics and biophysics department, ul. norwida 25, 50-375 wrocław, poland a novel approach to the analysis of the ion current recordings is proposed. the main goal of the standard patch clamp technique is to measure single channel activity (however the whole cell configuration is also used in various researches). in the presented study the ion channels time series recordings were several (up to four) ion channels were present are analysed and the collective behaviour of ion channels is investigated. the time ion current time series are converted into dwelltime series and the channel activity is analysed. the hypothesis of collective ion channels behaviour is verified and the influence of organolead compounds (met3pbcl) on collective ion channel activity is measured. the analysis is performed on the sv cation channels of vacuolar membrane of beta vulgaris. the aim of our computed study was to examine the possible binding site of primaquine (pq) using a combined homology protein modeling, automated docking and experimental approach. the target models of wild-type and mutant-types of the voltage-dependent sodium channel in rat skeletal muscle (rna v 1.4) were based on previous work by tikhonov and zhorov. docking was carried out on the p-loop into the structure model of rna v 1.4 channel, in the open state configuration, to identify those amino acidic residues important for primaquine binding. the threedimensional models of the p-loop segment of wild types and mutant types (w402. w756c, w1239c and w1531a at the outer tryptophan-rich lip, as well as d400c, e755c, k1237c and a1529c of the deka motif) helped us to identify residues playing a key role in aminoquinoline binding. in good agreement with experimental results, a 1000-fold inhibition loss was observed, tryptophan 756 is crucial for the reversible blocking effects of pq. as a result, w756c abolished the blocking effect of primaquine in voltage-clamp assays. hydrogen bond formation accelerates channel opening of the bacterial mechanosensitive channel mscl yasuyuki sawada and masahiro sokabe department of physiology, nagoya university graduate school of medicine, nagoya, japan the bacterial mechanosensitive channel mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer (tm1, tm2) a-helices, and its 3d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. to address this question, we performed molecular dynamics (md) simulations for the opening of mscl embedded in the lipid bilayer. in the closed state of mscl, neighboring tm1 inner helices are crossed each other near the cytoplasmic side and leu19 and val23 in the constricted part form a stable hydrophobic environment called gate. upon membrane stretch, phe78 in tm2 outer helices was dragged by lipids, leading to an opening of mscl. thus phe78 was concluded to be the major tension sensor. during opening, tm1inner helices were also dragged and tilted, accompanied by the outward sliding of the crossings. this led to a slight expansion of the gate associated with an exposure of oxygen atoms of the backbone to the inner surface of the gate. this allows water penetration in the gate and formation of hydrogen bonds between water and the exposed oxygen, which in turn weakened the hydrophobic interaction at the crossings, causing a further opening of the gate and water permeation. mitochondrial bk ca , mitobk ca has been proposed to be cardioprotective and formed by proteins of *50 to *125 kda. thus, we investigated the molecular characteristics of this channel in isolated mitochondria from murine heart. labeling of adult mouse cardiomyocytes with plasmalemma bk ca antibodies, mitotracker, and wheat germ agglutinin yielded remarkable mitochondrial but not plasma membrane localization. nanoscale fluorescence microscopy (stimulated emission depletion) revealed 7 to 15 of *40-50 nm bk ca clusters per mitochondria. further, western blot analysis of purified mitochondria showed the presence of a full length *125 kda protein. systematic rt-pcr exon scanning of isolated cardiomyocyte mrnas were consistent with a full length *125 kda alpha-subunit protein and revealed the expression of three splice inserts. insertless-bk ca robustly localized to the plasma membrane of cho cells but when a c-terminal splice insert was present bk ca was readily targeted to the mitochondria (protein proximity index was *1.0 indicating 100% colocalization). hence, cardiac mitobk ca is composed by full-length bk ca protein but with splice inserts which may facilitate its targeting to mitochondria. supported by nih and aha. patch-clamp technique was used to examine effect of trimethyl-lead and -tin on the sv channel activity in the red beet (beta vulgaris l.) taproot vacuoles. it was found that the addition of both investigated compounds to the bath solution inhibit, in a concentration-dependent manner, sv currents. when single channel properties were analyzed, only little channel activity can be recorded in the presence of 100 lm of organometal. compounds investigated decreased significantly (by about one order of magnitude) the open probability of single channels. the recordings of single channel activity obtained in the presence and in the absence of organometal showed that compounds only slightly (by ca. 10%) decreased the unitary conductance of single channels. it was also found that organometal diminished significantly the number of sv channel openings, whereas it did not change the opening times of the channels. taken together, these results suggest that organometal binding site is located outside the channel's selectivity filter and that the inhibitory effect of both compounds investigated on sv channel activity probably results from organometal-induced disorder in compatibility between membrane lipids and membrane proteins. the research was financed by polish ministry of science and higher education by grant no. nn305 336434. electrophysiological investigation of the hvdac1 ion channel in pore-spanning membranes conrad weichbrodt, claudia steinem georg-august-universitä t gö ttingen, iobc, tammannstr. 2, d-37077 gö ttingen, germany, e-mail: cweichb@gwdg.de the human voltage-dependent anion channel 1 (hvdac1) plays an important role in cell life and apoptosis since it is the main porin of the outer mitochondrial membrane. as hvdac1 is believed to play a pivotal role in apoptosis-related diseases such as stroke, alzheimer, parkinson and cancer, the alterations of its electrophysiological properties under different conditions are of great value. to perform different investigations, refolded hvdac1 is reconstituted in artificial membranes which typically consist of dphpc with a cholesterol fraction of 10-30%. they are prepared via the mü ller-rudin-technique on a functionalized porous alumina substrate containing pores with a diameter of 60 nm. the quality of these so-called nano-black-lipid-membranes (nano-blms) is verified via electrochemical impedance spectroscopy (eis), hvdac1 is reconstituted and single channel recordings are made. membranes are also prepared by spreading proteoliposomes on hydrophobized porous silicon nitride with pores of 1 lm diameter. information about altered gating-characteristics and related conductivities is gained by application of holding potentials up to ±100 mv and evaluation of the resulting currents. the hvdac1 was a kind gift of prof. c. griesinger, mpibpc, gö ttingen. pharmacological inhibition of cardiac herg k ? channels is associated with increased risk of arrhythmias. many drugs bind directly to the channel, thereby blocking ion conduction. ala-scanning mutagenesis identified residues important for drug block. two aromatic residues y652 and f656 were found to be crucial for block of most compounds. surprisingly, some cavity blocking drugs are only weakly affected by mutation y652a. in this study we provide a structural interpretation for this observation. md simulations on the y652a mutant suggest side chain rearrangements of f656 located one helical turn below y652. loss of p-p stacking induces reorientation of f656 from a cavity facing to a cavity lining conformation, thereby substantially altering the shape of the binding site. docking studies reveal that due to their rigid shape and compactness y652 insensitive drugs can still favorably interact with the reoriented f656 aryl groups, while molecules with more extended geometries cannot. the ankyrin transient receptor potential channel trpa1 is a transmembrane protein that plays a key role in the transduction of noxious chemical and thermal stimuli in nociceptors. in addition to chemical activation, trpa1 can be activated by highly depolarizing voltages but the molecular basis of this regulation is unclear. the transmembrane part of the tetrameric trpa1 is structurally related to the voltagegated k ? channels in which the conserved charged residues within the fourth transmembrane region (s4) constitute part of a voltage sensor. compared to these channels, the voltage-dependence of trpa1 is very weak (apparent number of gating charges * 0.4 versus 12 in k ? channels) and its putative voltage-sensing domain most likely lies outside the s4 because trpa1 completely lacks positively charged residues in this region. in the present study we used homology modelling and molecular dynamics to create models of the transmembrane part and the proximal cytoplasmic c terminus of trpa1. in combination with electrophysiological data obtained from whole cell patchclamp measurements we were able to point out several positively charged residues which mutation strongly alter the voltage sensitivity of trpa1 channel. these may be candidates for as yet unrecognized voltage sensor. photosynthesis p-511 action of double stress on photosystem 2 aliyeva samira a., gasanov ralphreed a. institute of botany, azerbaijan national academy of sciences, baku, azerbaijan the simultaneous effect of photoinhibitory illumination and toxic action of heavy metals ions (cd 2? and co 2? ) on activity of ps2 in vitro measuring by millisecond delayed fluorescence (ms-df) of chlorophyll a was studied. during action on chloroplasts only of cd 2? (10 -3 m) the fast component of ms-df, which originates via radiative recombination of reaction center with the camn 4 -cluster or y z on donor side of ps2, is inhibited stronger than at action of only co 2? . the steadystate level at cd 2? treatment is remain stable, while at co 2? action it is increased. simultaneous action of cd 2? and photoinhibitory illumination (4000 lmol photons m -2 s -1 ) have shown that fast component of ms-df was inhibited faster with time than in case of action of co 2? and excess light. result indicates that damage sites of action cd 2? and co 2? are donor and acceptor side of ps2, accordingly. we assume that binding site of cd 2? is y z or camn 4 -cluster, one of the recombination partners with p 680 ? on the donor side of ps2. thereby, action of cd ions on donor side of ps2 leads mainly to development of mechanism of donor-side photoinhibition. field instrument for determination of the photosynthetic capacity of intact photosynthetic bacteria e. asztalos 1 , z. gingl 2 and p. maró ti 1 1 department of medical physics and informatics, university of szeged, hungary, 2 department of experimental physics, university of szeged, hungary a combined pump and probe fluorometer and spectrophotometer with high power laser diodes has been constructed to measure fast induction and relaxation of the bacteriochlorophyll fluorescence yield and light-induced absorption changes in intact cells of photosynthetic bacteria. the construction is the upgraded version of our previous set up 1 with better time resolution (5 ls). the compact design of the mechanics, optics, electronics and data processing makes the device easy to use as outdoor instrument or to integrate into larger measuring systems. the versatility and excellent performance of the apparatus will be demonstrated on different fields: 1) organization and redox state of the photosynthetic apparatus of the whole cells under different growth conditions deduced from fluorescence characteristics including the lag phase, the amplitude and the rise time of the variable fluorescence, 2) electron transfer in the reaction center, cytochrome bc 1 complex and in between obtained from relaxation of the fluorescence and 3) re-reduction kinetics of the oxidized primary donor of the reaction center and energetization and relaxation of the intracytoplasmic membrane tracked by absorption changes at 798 and 525 nm, respectively. previous work has established that the iron stress induced protein a (isia) synthesized by cyanobacteria under stress conditions, has at least two functions: light harvesting [1] and photoprotection [2] . under prolonged iron starvation isia becomes the main chlorophyll-binding protein in the cell and occurs without a photosystem association. these isia aggregates have a strong ability to dissipate light energy and there is evidence of carotenoid participation in the quenching mechanism via downhill energy transfer from chlorophyll to the s1 state of a carotenoid [3] . in the present work we have measured the temperature dependence of the fluorescence of carotenoid depleted mutants (echinenone and/or zeaxanthin) and isia monomers in order to investigate the role of carotenoid and aggregation in the quenching process. pigment analysis confirms the absence of the carotenoid mutated in its biosynthesis but shows that it is mainly replaced. the monomers are lacking two carotenoids, echinenone and one of the two ß-carotenes found previously in isia aggregates. temperature dependent fluorescence shows that quenching properties are affected in the monomers and the mutants lacking zeaxanthin. soon exhausted oil resources and global climate change have stimulated research aiming at production of alternative fuels, ideally driven by solar energy. production of solar fuels needs to involve the splitting of water into protons, energized electrons and dioxygen. in photosynthetic organisms, solar-energy conversion and catalysis of water splitting (or water oxidation) proceed in an impressive cofactor-protein complex denoted as photosystem ii (psii). the heart of biological water-oxidation is a protein-bound manganese-calcium complex working at technically unmatched efficiency. in an attempt to learn from nature, the natural paragon is intensely studied using advanced biophysical methods. structural studies by x-ray spectroscopy with synchrotron radiation play a prominent role in this endeavor. time-resolved methods provide insights in the formation of intermediate states of the reaction cycle. an overview is presented focusing on (i) the efficiency of solar energy usage in psii, (ii) the interrelation between electron transfer and proton relocations, and (iii) the mechanism of water oxidation. as an outlook, new results on water oxidation by biomimetic manganese and cobalt oxides, which may become a key element in future solar-fuel systems, are presented. the peridinin-chlorophyll-protein (pcp) is a light-harvesting complex (lhc) that works as antenna in the photosynthetic process of dinoflagellates. the protein contains both chlorophylls and carotenoids molecules, the latter being responsible to extend the spectral range of captured light to regions where chlorophylls are transparent. pcp crystal structures [1] reveal that each chlorophyll is surrounded by 3 or 4 molecules of the carotenoid peridinin, located in non-equivalent positions. the different protein environment of the sites might be responsible of a spectral shift of the pigments with the functional role to extend the absorption spectra of the complex and enhance its light harvesting capabilities. high resolution x-ray diffraction data on a reconstructed pcp, the refolded peridinin-chlorophyll a-protein (rfpcp) [2] , and on the less common high salt-pcp (hspcp) opened the way to the mechanistic understanding of peridinin spectral tuning, peridinin chlorophyll energy transfer and photoprotective mechanism [3] . the two pcp forms differ in various features: spectral properties, molar mass, amino acid sequence and, above all, pigment stoichiometry, the peridinin:chlorophyll ratio being 4:1 for the rfpcp and 3:1 for the hspcp [4] . in the present work we perform classical molecular dynamics simulations of the rfpcp and the hspcp in explicit water solution. we analyse the structure and dynamics of the proteins and of their pigments to characterize the different peridinin sites in both pcp forms in terms of quantities that can affect the chromophore spectra, such as distorsion, fluctuations and nature of the protein environment. the comparison between the data suggests correspondences between the pigments of the two forms. quantum and mixed quantum/ classical molecular dynamics simulations are also under progress to investigate the effect of the protein environment on the electronic and optical properties of the pcp pigments. peculiar applications, like in optoelectronics, biosensors, photovoltaics. among the existing carrier matrices conductive metal oxides (e.g. indium tin oxide, ito), carbon nanotubes, graphenes, silicon (si) are the most frequently used materials because of their unique characteristics such as good conductivity, good optical properties and excellent adhesion to substrates. in our work we combined purified photosynthetic reaction center protein (rc) and porous silicon (psi) investigating the morphology and optoelectronic properties of the bio-nanocomposite material. ftir spectroscopy, scanning electron microscopy and energydispersive x-ray spectroscopy indicated the binding of the protein to the psi. specular reflectance spectra showed a red shift in the characteristic interference peak of the psi microcavity which was saturated at higher concentration of the protein. the binding was more effective if the functionalization was done by the si-specific oligopeptide compared to the classical covalent binding via aminopropyl-triethoxysilane (aptes). excitation by single saturation flashes indicated that the rc still exhibited photochemical turnover after the binding. the role of reactive oxygen species (ros) in plant stress, both as damaging agent and as potential signal molecule is often assessed in experiments using photosensitized elicitor dyes. for these studies, it is essential to know how efficiently these chemicals generate ros, whether they are specific ros sources, as well as their cellular localization and additional side effects. the present study addresses these issues using a variety of dyes known and traditionally applied as singlet oxygen ( 1 o 2 ) sources. rose bengal (rb), methylene violet (mvi), methylene blue (mb), neutral red (nr) and indigo carmine (ic) were studied as putative ros sources in tobacco leaves. ros products of photosensitized dyes were measured in vitro, using spin trapping epr spectroscopy. dye concentrations and irradiation concentration leading to equal absorbed excitation quanta were determined spectrophotometrically. in vivo studies were carried out using tobacco leaves infiltrated with water solutions of the putative 1 o 2 sources. cellular localizations were identified on the basis of the dyes' fluorescence. rb, nr and mvi reached into mesophyll cells and were used to study the effects of these dyes on photosynthesis. photochemical yields and quenching processes were compared before and after photosensitization of the elicitor dyes inside the leaf samples. chlorophyll-chlorophyll charge transfer quenching is the main mechanism of non-photochemical quenching in higher plants alfred r. holzwarth max-planck-institut fü r bioanorganischechemie, stiftstraße 34-36, d-45470 mü lheim a.d.ruhr, germany non-photochemical quenching (npq) in plants protects against photochemical destruction of the photosynthetic apparatus under excess light conditions.while one location of the npq process has been shown to be centered on the major light harvesting complex ii (lhcii) (q1 type or qequenching), an additional quenching center responsible for qi type (identical to q2 center) quenchinghas been suggested to be located on the minor light-harvesting complexes upon accumulation ofzeaxanthin (zx), in particularon cp24 and cp29 we have performed femtosecond transient absorption and time-resolved fluorescence measurements of npq quenching in intact leaves of higher plants, on isolated light harvesting complexes in the minor (non-aggregated)light harvesting complex cp29 reconstituted with violaxanthin (vx) or zx, and in the isolated major lhc ii complex in the aggregated state. in all of these situations we find the formation of chl-chl charge transfer (ct) states to be the dominant quenching mechanism. the yield of formation of carotenoid cation states and/or carotenoid s 1 state is either extremely low or absent, thus excluding their involvement in npq quenching as a major quenching mechanism. single-molecule spectroscopy (sms) is a powerful technique that allows investigation of fluorescence properties from single fluorescing systems. this technique enabled us to investigate the dynamics of the fluorescence intensity and spectral profiles of single, isolated light harvesting complex (lhc) on timescales of milliseconds to seconds, during continuous laser illumination. we were able to observe how each complex can rapidly switch between different emission states [1, 2] and to characterise the intensity and the spectral dynamics of major and minor antenna complexes from plants, in two different environments, mimicking the light harvesting and the light dissipating state, respectively. the results will be discussed with respect to the current models for nonphotochemical quenching (npq) mechanisms [3, 4, 5] , a vital photoprotection mechanism during which the lhcs of plants switch between a state of very efficient light utilisation and one in which excess absorbed excitation energy is harmlessly dissipated as heat. phaeodactylum tricornutum is one of the most utilized model organisms in diatom photosynthesis research mainly due to availability of its genome sequence (1). it's photosynthetic antennae are the fucoxanthin chlorophyll a/c binding proteins (fcps) which share a high degree of homology with lhcs of higher plants and green algae (2) . for detailed investigation of the antenna system of p.tricornutum, a transgenic strain expressing recombinant his-tagged fcpa protein was created which simplified the purification of a specific stable trimeric fcp complex consisting of fcpa and fcpe proteins. excitation energy coupling between fucoxanthin and chlorophyll a was intact and the existence of a chlorophyll a/fucoxanthin excitonic dimer was demonstrated (3). we investigated in detail the existence of specific antenna system for psi and psii in p.tricornutum as in case of higher plants. our studies indicated that at least the main light harvesting proteins fcpa and fcpe are most probably shared as a common antenna by both psi and psii. harvesting complex ii (lhciib) from spinach or in native thylakoid membranes by picosecond time-resolved fluorescence. the domain size was estimated by monitoring the efficiency of added exogeneous singlet excitation quenchers -phenyl-p-benzoquinone (ppq) and dinitrobenzene (dnb). the fluorescence decay kinetics of the systems under study were registered without quenchers and with quenchers added in a range of concentrations. stern-volmer constants, k 0 sv and k sv -for aggregates (membranes) and detergentsolubilized complexes, respectively, were determined from the concentration dependence of the ratio of the mean fluorescence lifetimes without/with quencher (s 0 , s). the ratio k 0 k sv • s 0 /s 0 0 was suggested as a measure of the functional domain size. values in the range of 15-30 were found for lhcii macroaggregates and 12-24 for native thylakoid membranes, corresponding to domain sizes of 500-1000 chlorophylls. although substantial, the determined functional domain size is still orders of magnitude smaller than the number of physically connected pigment-protein complexes; thus our results imply that the physical size of an antenna system beyond these numbers has little or no effect on improving the light-harvesting efficiency. the interaction between photosynthetic reaction center proteins (rcs) purified from purple bacterium rhodobacter sphaeroides r-26 and functionalized and non-functionalized (single (swnt) and multiple (mwnt) walled) carbon nanotubes (cnt-s) has been investigated. both structural (afm, tem and sem microscopy) and functional (flash photolysis and conductivity) techniques showed that rcs can be bound effectively to different cnts. both physical sorption and binding through -nh 2 or -cooh groups gave similar results. however, it appeared that by physical sorption some sections of the cnts were covered by multiple layers of rcs. after the binding the rcs kept their photochemical activity for a long time (at least for three months, even in dried form) and there is a redox interaction between the cnt and rcs. the attachment of rc to cnts results in an accumulation of positive and negative charges followed by slow reorganization of the protein structure after excitation. in the absence of cnt the secondary quinone activity decays quickly as a function of time after drying the rc onto a glass surface. the special electronic properties of the swnt/protein complexes open the possibility for several applications, e.g. in microelectronics, analytics or energy conversion and storage. the decay of the high fluorescence state generated by actinic illumination of different durations was measured in whole cells of various strains and mutants of photosynthetic purple bacteria. although similar method is used in higher plants, its application in photosynthetic bacteria is novel and highly challanging. the available data are restricted and only the re-oxidation of the reduced primary quinone (q a -? q a ) is usually blamed for the decay kinetics. here, we will analyse the complexity of the kinetics over a very broad time range (from 5 ls to 5 s) and show that the dark relaxation of the bacteriochlorophyll fluorescence reflects the overlap of several processes attributed to the intra-and interproteinous electron transfer processes of the reaction center (rc) and cytochrome bc 1 complex of the bacterium. in the shorter (\ 1 ms) time scale, the dominating effect is the re-reduction of the oxidized primary donor (p ? ? p) that is followed by the re-oxidation of the acceptor complex of the rc by the cytochrome bc 1 complex. as the life times and amplitudes of the components depend on the physiological state of the photosynthetic apparatus, the relaxation of the fluorescence can be used to monitor the photosynthetic capacity of the photosynthetic bacteria in vivo. circular dichroism (cd) spectroscopy is an indispensable tool to probe molecular architecture. at the molecular level chirality results in intrinsic cd, pigment-pigment interactions in protein complexes give rise to excitonic cd, whereas ''psitype'' cd originates from large densely packed chiral aggregates. it has been well established that the anisotropic cd (acd), measured on samples with defined orientation, carries specific information on the architecture of molecules. however, acd can easily be distorted by linear dichroism of the sample or instrumental imperfections -which might be the reason why it is rarely studied in photosynthesis research. here we present acd spectra of isolated intact and washed, unstacked thylakoid membranes, photosystem ii membranes (bby), and tightly-stacked lamellar macroaggregates of the main light-harvesting complex ii (lhcii). we show that the acd spectra of face-and edge-aligned stacked thylakoid membranes and lhcii lamellae exhibit profound differences in their psi-type cd bands. marked differences are also seen in the excitonic cd of bby and washed thylakoid membranes. thus acd provides an additional dimension to the light induced conformation changes of quinone depleted photosynthetic reaction centers (rcs) purified from the carotenoid-less rhodobacter sphaeroides r-26 were investigated by transient absorption (ta) and grating (tg) methods. surprisingly, the decay of the ta signal measured at 860 nm was divided into a 15 ns and a 40 ls components. the latter coincides with the life time of the tg signal, which was assigned earlier [nagy et al. (2008) febs lett. 582, 3657-3662] to spectrally silent conformational changes. the nature of the 40 ls phase was investigated further. although, the probability of chlorophyll triplet formation under our measuring conditions was small, possible contribution of the triplet states was also studied. the presence of carotenoid in the wild type rcs eliminated the 40 ls component indicating the role of carotenoid in the energy transfer within the rcs. there was no significant effect of the molecular oxygen on the ta. this fact may be explained if the chlorophyll triplets inside the protein have reduced accessibility to molecular oxygen. a differential effect of osmotic potential and viscosity on conformation changes accompanying the primary charge separation was measured by the effect of ficoll, glucose and glycerol as compared the ta to the tg signals. variable chlorophyll fluorescence: in part a yield change due to light-induced conformational change gert schansker 1 , szilvia z. tó th 1 , lá szló ková cs 1 , alfred r. holzwarth 2 and gy} oz} o garab 1 1 institute of plant biology, biological research center, hungarian academy of sciences, szeged, hungary, 2 max-planck-institut fü r bioanorganische chemie, mü lheim an der ruhr, germany on a dark-to-light transition the chlorophyll fluorescence rises from a minimum intensity (f 0 ) to a maximum intensity (f m ). conventionally, this rise is interpreted to arise from the reduction of the primary quinone acceptor, q a , of photosystem ii -although this cannot explain all presently available observations. in untreated leaves, at room temperature, the fluorescence rise follows the reduction of the electron transport chain (etc). once induced, *30-40 % of the variable fluorescence intensity relaxes within 100 ms in darkness and can be re-induced within 3 ms as long as the etc remains reduced. analyzing the fluorescence relaxation kinetics, ?/-dcmu, *30% of the amplitude cannot be explained by q a -re-oxidation. special properties of this phase determined on dcmu-inhibited samples: at cryogenic temperatures (below -40°c), where the q a -/s 2 recombination is blocked, it still relaxes and it exhibits a strong temperature dependence with an apparent e a & 12 kj/mol, whereas the reduction of q a is nearly temperature insensitive. a fluorescence yield change, driven by light-induced conformational change in the reaction center complex, can explain all these observations. tuning function in bacterial light-harvesting complexes katia duquesne 1 , edward o'reilly 2 , cecile blanchard 1 , alexandra olaya-castro 2 and james n. sturgis 1 1 lism, cnrs and aix-marseille university, marseille, france, 2 department of physics, university college, london, uk purple photosynthetic bacteria are able to synthesize a variety of different light-harvesting complexes, sometimes referred to as lh2, lh3 and lh4. here we have investigated the structural origins of these different forms and the manner in which the sequence tunes the absorption spectrum of the light-harvesting system. we then consider the functional consequences of this tuning for the organization of the light harvesting system and on the ecology of the organisms. specifically by spectroscopic techniques, in particular circular dichroism and resonance raman spectroscopy, we have been able to obtain information on the organization of the bacteriochlorophyll binding sites in the unusual lh4 of roseobacter denitrificans. this provides a picture of how different peripheral light-harvesting complexes are able to modulate the absorption spectrum. the structure and organization of this complex is the put in the context of the the recently published variability of the light-harvesting complexes. in particular the observation of their ability to form mixed complexes containing different polypeptides. we examine quantitatively the possible reasons for maintaining such variability by considering the transport properties of membranes containing either pure or mixed complexes and show that mixed complexes can permit light-harvesting to continue during adaptation. we then consider the different constraints that may be behind this type of adaptation in different bacteria and the conditions under which different types of antenna system might be optimal finally we integrate this into the evolutionary context of adaptation to variable light intensity and the ecological niches where such organisms are found. interaction between photosynthetic reaction centers and ito t. szabo 1 , g. bencsik 2 , g. kozak 1 , cs. visy 2 , z. gingl 3 , k. hernadi 4 , k. nagy 5 , gy. varo 5 and l. nagy 1 1 departments of medical physics and informatics, 2 physical chemistry and materials science, 3 technical informatics and of, 4 applied and environmental chemistry, university of szeged, hungary, 5 institute of biophysics, has biological research center, szeged, hungary photosynthetic reaction center proteins (rc) purified from rhodobacter sphaeroides purple bacterium were deposited on the surface of indium-tin-oxide (ito), a transparent conductive oxide, and the photochemical/-physical properties of the composite was investigated. the kinetics of the light induced absorption change indicated that the rc was still active in the composite and there was an interaction between the protein cofactors and the ito. the electrochromic response of the bacteriopheophytine absorption at 771 nm showed an increased electric field perturbation around this chromophore on the surface of ito compared to the one measured in solution. this absorption change is associated with the chargecompensating relaxation events inside the protein. similar life time, but smaller magnitude of this absorption change was measured on the surface of borosilicate glass. the light induced change in the conductivity of the composite as a function of the concentration showed the typical sigmoid saturation characteristics unlike if the chlorophyll was layered on the ito which compound is photochemically inactive. in this later case the light induced change in the conductivity was oppositely proportional to the chlorophyll concentration due to the thermal dissipation of the excitation energy. the supramolecular organization of photosystem ii in vivo studied by circular dichroism spectroscopy tü nde tó th 1,2 , herbert van amerongen 2,3 , gy} oz} o garab 1 , lá szló ková cs 1 1 institute of plant biology, biological research center, hungarian academy of sciences, hungary, 2 wageningen university, laboratory of biophysics, wageningen, the netherlands, 3 microspectroscopy centre, wageningen university, wageningen, the netherlands the light reactions of photosynthesis in higher plants take place in granal chloroplast thylakoid membranes, which contain chirally organized macrodomains composed of photosystem ii (psii) supercomplexes associated with light harvesting antenna complexes (lhciis). the physiological relevance of this hierarchic organization, which often manifest itself in semicrystalline assemblies, has not been elucidated but the diversity of the supramolecular structures and their reorganizations under different conditions indicates its regulatory role. the present work focuses on the structural and functional roles of different components of lhcii-psii supercomplexes. we used various growth conditions, influencing the protein composition, and different arabidopsis mutants (kocp24, kocp26, kopsbw, kopsbx, dgd1), with altered organization of the membranes, and measured their circular dichroism (cd) spectra as well as their chlorophyll fluorescence kinetics to characterize the chiral macro-organization of the chromophores and the functional parameters of the membranes, respectively. we show that the formation of chiral macrodomains require the presence of supercomplexes. our data also reveal specific functions of some of the protein or lipid compounds in the light adaptation processes of plants. excitation energy transfer and non-photochemical quenching in photosynthesis rienk van grondelle department of physics, vu university, de boelelaan 1081, 1081hv, amsterdam, the netherlands the success of photosynthesis relies on two ultrafast processes: excitation energy transfer in the light-harvesting antenna followed by charge separation in the reaction center. lhcii, the peripheral light-harvesting complex of photosystem ii, plays a major role. at the same time, the same light-harvesting system can be 'switched' into a quenching state, which effectively protects the reaction center of photosystem ii from over-excitation and photodamage. in this talk i will demonstrate how lhcii collects and transfers excitation energy. using single molecule spectroscopy we have discovered how lhcii can switch between this light-harvesting state, a quenched state and a red-shifted state. we show that the switching properties between the light-harvesting state and the quenched state depend strongly on the environmental conditions, where the quenched state is favoured under 'npq-like' conditions. it is argued that this is the mechanism of non-photochemical quenching in plants. photobiology in the soil: arrested chlorophyll biosynthesis in pea epicotyl sections beá ta vitá nyi, katalin solymosi, annamá ria kó sa, bé la bö ddi eö tvö s university, institute of biology, department of plant anatomy, pá zmá ny p. s. 1/c, h-1117 budapest, hungary the key regulatory step of chlorophyll (chl) biosynthesis is the nadph:protochlorophyllide oxidoreductase (por) catalyzed reduction of protochlorophyllide (pchlide)which is light activated in angiosperms. this process is usually described on artificially dark-grown plants. in this work, we studied epicotyl segments developed under the soil surface, which were dissected from pea plants grown under natural light conditions. using 77 k fluorescence spectroscopy, pigment analyses, electron microscopy and fluorescence microscopy, we found that upper segments showed transitional developmental stages, i.e. chl appeared besides pchl(ide) and etio-chloroplasts were typical. in regions under 5 cm depth, however, the characteristics of the segments were similar to those of plants germinated artificially in complete darkness, i.e. only pchl(ide) and etioplasts were present. the results of this work prove that these latter symptoms may occur in shaded tissues of fully developed, photosynthetically active plants grown under natural conditions. in this overview talk it will be shown how atomistic computations can complement experimental measurements in our quest to understand biological electron and proton transfer reactions. at first the molecular simulation methods for calculation of important electron transfer parameter such as reorganization free energy, electronic coupling matrix elements and reduction potentials will be explained. then three applications will be discussed where such computations help interpret experimental data on a molecular level. the first example concerns electron tunneling between heme a and heme a3 in the proton pump cytochrome c oxidase. this reaction is very fast, occurring on the nano-second time scale, and it is unclear if this is due to an unusually low reorganization free energy or due to high electronic coupling. carrying out large-scale all-atom molecular dynamics simulation of oxidase embedded in a membrane, we do not find evidence for unusually small values of reorganization energy as proposed previously, implying that the nanosecond tunneling rate between heme a and a3 is supported by very efficient electronic coupling. the second example is on electron transport in a deca-heme 'wire'-like protein, used by certain anaerobic bacteria to transport electrons from the inside of the cell to extracellular substrates. the crystal structure of such a protein has been solved recently for the first time. however, it is unclear if and in which direction the wire structure supports electron transport. here we present results of heme reduction potential calculations that help us reveal the possible electron flow in this protein. in a third example we explain how quantum mechanical/molecular mechanical methods (qm/mm) recently helped us understand why the catalase from h. pylori is prone to undergo an undesired protein radical migration reaction during catalysis. proton pumping activity of purple and brown membranes regenerated with retinal analogues k. bryl 1 , k.yoshihara 2 1 university of warmia and mazury, department of physics and biophysics, olsztyn, poland, 2 suntory institute for bioorganic research, wakayamadai, osaka 618, japan the retinal protein bacteriorhodopsin (br) acts as a light-driven proton pump in the purple membrane (pm) of halobacterium salinarium (h.s.). the aim of these studies was to clarify whether the specific crystalline structure of protein and protein-substrate interactions are significant for h ? transfer into the aqueous bulk phase. two membrane systems were prepared: purple membranes (br arranged in a two-dimensional hexagonal lattice) and brown membranes (br not arranged in a crystalline lattice) were regenerated with 14-fuororetinals. light-induced proton release and reuptake as well as surface potential changes inherent in the regenerated systems reaction cycles were measured. signals of optical ph indicators residing in the aqueous bulk phase were compared with signals of ph indicator covalently linked to the extracellular surface of proteins and with surface potential changes detected by the potentiometric probe. the energies of activation of proton transfer have been calculated. experimental results and thermodynamic parameters (energies of activation) suggest the different mechanism of proton transfer into the aqueous bulk phase in these two systems. the implications for models of localized-delocalized energy coupling by proton gradients will be discussed. iron regulation is a vital process in organisms and in most of them it is accomplished through the metal solubilisation and storage by ferroxidase enzymes of the ferritin family, which have the ability of sequester, oxidize and mineralize ferrous ions using oxygen or hydrogen peroxide as substrate. dnabinding proteins from starved cells (dps) belong to this ferritin's family. dps belongs to the sub-type designated as miniferritins and, besides iron storage and release capability, is responsible for hydrogen peroxide resistance showing the ability to form stable complexes with dna. the preferable cosubstrate of this enzyme is h 2 o 2 although the reaction can occur in the presence of oxygen with lower rate [1, 2] . in this work, the electrochemical behaviour of the recombinant dps from pseudomonas nautica, was assessed as a function of metal content in anaerobic environment with h 2 o 2 as co-substrate. the obtained electrochemical results together with spectroscopic studies allowed inferring some new hypothesis on the dps iron uptake mechanism. for myoglobin electrostatically immobilized on au-deposited mixed self-assembled monolayers (sams) of the composition: -s-(ch 2 ) 11 -cooh/-s-(ch 2 ) 11 -oh. our approach allows for a soft switching of the haem group charge state and accurate probing of the accompanying reorganizational dynamics of conformational (quasi-diffusional) and quantum (e.g. protonrelated) modes. the electron transfer rate constants were determined with h 2 o or d 2 o as solvent, under the variable temperature (288-328 k) or pressure (5-150 mpa) conditions, revealing the overall reorganization free energy of 0.5±0.1 ev, activation volume of -3.1±0.1 cm 3 mol -1 and inverse solvent kinetic isotope effect of 0.7±0.1 (25°c). on the grounds of an extended charge-transfer theory, we propose specific protoncoupled et mechanism additionally coupled to the slow conformational dynamics of the protein matrix accompanied by translocation(s) of haem-adjacent water molecule(s). proton gradients across pore spanning membranes: towards on-chip energy conversion daniel frese, claudia steinem institute for organic and biomolecular chemistry, georg-august-university goettingen, germany in cell organelles, chemiosmotic potentials resulting from proton gradients across membranes are widely used to fix chemical energy in forms of atp. the high efficiency of this protein-mediated energy conversion raises interest for artificial proton gradient setups. to investigate proton transport across artificial membranes, we prepared pore spanning membranes (psms) on porous silicon substrates via painting technique. this allowed us to trap aqueous content of well-defined composition and volume inside the substrates microcavities. nigericin, a peptide that acts as an h ? -k ? -antiporter and bacteriorhodopsin, a transmembrane protein which is well known to be a light driven proton pump, were reconstituted into the pre-formed psms to achieve proton transport from one aqueous compartment to the other. changes of proton concentrations inside the pores were monitored by means of confocal laser scanning microscopy (clsm). therefore, pores were filled with pyranine, a ph-sensitive fluorescence dye and variations in intensity were measured to analyze proton translocation. we were able to show that both, nigericin and bacteriorhodopsin are capable of building up a proton gradient across psms and plan to co-reconstitute atp synthases for on-chip energy conversion by formation of atp. application of the gibbs free energy profiles to sequential biochemical reactions pé ter farkas, tamá s kiss and eugene hamori department of biological physics, eö tvö s ló rá nd tudomá ny egyetem, budapest, hungary, and department of biochemistry, tulane university, new orleans, la., usa the full understanding of the energetic details of complex metabolic reaction sequences requires a step-by-step analysis of the gibbs free energy (g) changes of the ''parasystem'' (i.e., a collection of atoms comprising all the molecules participating in a given reaction) as it gradually changes from its initialreactants state to its final-products state along the reaction pathway. knowing the respective equilibrium constants of each of the participating reaction steps and also the actual in vivo concentrations of the metabolites involved, a free-energy profile can be constructed that will reveal important information about the progress of the reaction as driven by thermodynamic forces. this approach will be illustrated on some biochemical reactions including the glycolytic/gluconeogenetic pathways. furthermore, the often misleading text-book representation of enzymatic catalysis will be reexamined and explained in thermodynamic terms using the free-energy profiles of both the non-catalyzed and the enzyme-catalyzed reactions. redox-active proteins can be diversely functionalized at metaldeposited self-assembled monolayers (sams) of a widely variable composition and thickness. the voltammetric methodology in combination with the advanced data processing procedures allow for comprehensive kinetic data within the congruent series of nano-devices and the subsequent calculation of the key physical parameters, such as the medium reorganization energy of et, the donor-acceptor electronic coupling, effective relaxation time (related to the protein's and environment's fluctuational dynamics), etc. in our studies the ''model'' redox protein, cytochrome c (cytc), was either freely diffusing to sam terminal groups (mode 1), or attached to sams through the electrostatic interaction (mode 2), or specific ''wiring'' (mode 3). another redox-active protein, azurin, was confined at terminal sam groups through the hydrophobic interaction (mode 4). diverse experimental strategies including the variation of sam thickness, solution viscosity temperature and hydrostatic pressure, allowed for a severe demonstration of the full adiabatic and nonadiabatic control (thinner and thicker sams, respectively), and the intermediary regime, in a nice agreement with the major theoretical predictions. proton transfers in a light-driven proton pump j.k. lanyi dept. physiology & biophysics, university of california, irvine, usa illumination of bacteriorhodopsin causes isomerization of alltrans retinal to 13-cis,15-anti and a cyclic reaction ensues, in which the protein and the chromophore undergo conformational changes with an overall ten millisecond turn-over time and a proton is transported from one membrane side to the other. with crystal structures of six trapped intermediate states and plausible structural models for the remaining two intermediates, structures are now available for the initial bacteriorhodopsin state and all intermediates. they reveal the molecular events that underlie the light-induced transport: protonation of the retinal schiff base by asp85, proton release to the extracellular membrane surface, a switch event that allows reprotonation of the schiff base from the cytoplasmic side, side-chain and main-chain motions initiated in the cytoplasmic region, formation of a single-file chain of hydrogen-bonded water molecules that conducts the proton of asp96 to the schiff base, and reprotonation of asp96 from the cytoplasmic surface. the observed changes can be summarized as a detailed atomic-level movie in which gradual relaxation of the distorted retinal causes a cascade of displacements of water and protein atoms results in vectorial proton transfers to and from the schiff base. electron transfer (et) processes are fundamental in photosynthesis, respiration and enzyme catalysis. the relative importance of superexchange and sequential mechanisms in biological et is still a matter of debate. the identification of any ''stepping stones'' necessary for electron hopping is a key point in the understanding of long range et. hence, the study of a single event in the sequence of reactions occurring in these phenomena is a fundamental but formidable task. muon spin relaxation (lsr) has been shown to be sensitive to charge transport on a molecular lengthscale. the muon is a very sensitive probe of electron transport, as any changes to the electronic density sampled by the muon can change its spin polarization, which can easily be measured. in this context, a very useful tool is the detection of the so-called avoided level crossing (alc) resonances [1] . the enhancement in the loss of polarization of the muon's spin on these resonances dramatically increases sensitivity. we show that a laser pump -lsr probe technique can measure et processes at particular, and most importantly known, sites within the amino acids chain, and therefore track the time evolution of the electron over the molecule. keywords: photosynthesis, reaction center, electron transfer, proton transfer, fourier transform infrared, l210dn, isotopic labeling, band assignment, histidine, mechanism in photosynthesis, the central step in transforming light energy into chemical energy is the coupling of light-induced electron transfer to proton uptake. in the photosynthetic reaction center (rc) of rhodobacter sphaeroides, fast formation of the charge separated state p ? q a is followed by a slower electron transfer from the primary quinone q a to the secondary quinone q b and the uptake of a proton from the cytoplasm to q b . previous fourier transform infrared (ftir) measurements on rc suggested an intermediate x in the q a -q b to q a q b transition. mutation of the amino acid aspl210 to asn (l210dn mutant) slows down proton uptake and oxidation of q a -. using time-resolved ftir spectroscopy we characterized this rc mutant and proposed specific ir bands that belong to the intermediate x. to study the role of the iron-histidine complex located between q a and q b , we performed fast-scan ftir experiments on the l210dn mutant marked with isotopically labelled histidine. we assigned ir bands of the intermediate x between 1120 cm -1 and 1080 cm -1 to histidine vibrations. these bands show the protonation of a histidine, most likely hisl190, during the q a -q b to q a q b transition. based on these results we propose a new mechanism of the coupling of electron and proton transfer in photosynthesis. complex i of respiratory chains is an energy transducing enzyme present in most bacteria and in all mitochondria. it is the least understood complex of the aerobic respiratory chain, even though the crystallographic a-helical structures of bacterial and mitochondrial complexes have been recently determined [1] [2] . this complex catalyses the oxidation of nadh and the reduction of quinone, coupled to cation translocation across the membrane. rhodothermus marinus complex i, our main model system, is a nadh:menaquinone oxidoreductase and has been extensively characterized. we have made an exhaustive study in order to identify all the subunits present in the complex [3] . the nature of the coupling charge of r. marinus complex i was investigated using inside-out membrane vesicles, which were active with respect to nadh oxidation and capable of creating and maintain an nadh-driven membrane potential (dw) positive inside. it was observed that this bacterial complex i is able of h ? and na ? transport, although to opposite directions. the coupling ion of the system was shown to be the h ? being transported to the periplasm, contributing in this way to the establishment of the electrochemical potential difference, while na ? is translocated to the cytoplasm [4] . the sodium ion extrusion from the membrane vesicles was due to the activity of complex i, since it was sensitive to its inhibitor rotenone, and it was still observed when the complex i segment of the respiratory chain was isolated by the simultaneous presence of cyanide and external quinones. additional studies have shown that although neither the catalytic reaction nor the establishment of the dph requires the presence of na ? , the presence of this ion increased the proton transport. combining all these results, a model for the coupling mechanism of complex i was proposed, suggesting the presence of two different energy coupling sites, one that works only as a proton pump (na ? independent), and the other functioning as a na ? /h ? antiporter (na ? dependent) [4] . this model was reinforced by further studies performed in the presence of the na ? /h ? antiporter inhibitor, 5-(n-ethyl-n-isopropyl)-amiloride (eipa) [5] . a deeper inside into the coupling mechanism of this enzyme was provided by studying the influence of sodium ions on energy transduction by complexes i from escherichia coli and paracoccus denitrificans. it was observed that the na ? / h ? antiporter activity is not exclusive of r. marinus complex i, since the e. coli enzyme is also capable of such a transport, but is not a general property given that the p. denitrificans enzyme does not performed sodium translocation [6] . due to the fact that r. marinus and e. coli enzymes reduce menaquinone while p. denitrificans complex i reduce ubiquinone, it is suggested that the na ? /h ? antiporter activity may be correlated with the type of quinone used as substrate. under anaerobic conditions some bacteria can use nitrate instead of oxygen in a process called denitrification. during denitrification, the reduction of no to n 2 o is catalyzed by a membrane-bound enzyme nitric oxide reductase (nor). this enzyme is an important step in the evolution of a respiratory system: nor belongs to the superfamily of o 2 -reducing heme-copper oxidases and is assumed to be the evolutionary ancestor of cytochrome c oxidase. the understanding of nor functioning was limited by the lack of structural information, but recently the first structures (cnor type from ps. aerug. and qnor type from g. stearoth.) were solved [1] [2] . we will present results of the first computational studies of nor (both cnor and qnor types) [2] [3] . the studies include: (i) large-scale all-atom md simulations of proteins in their natural environment (i.e. embedded in membrane and solvent), which were performed to describe water dynamics inside the protein and to identify potential proton transfer pathways, and (ii) free-energy calculations by the empirical valence bond (evb) method [4] for the explicit proton translocations along the pathways established by md. among important findings are new proton pathways, which were not predicted from the x-ray structure and could be identified only by means of computer simulations. simulations also reveal that, despite a high structural similarity between cnor and qnor, these enzymes utilize strikingly different proton uptake mechanisms. our results provide insights into the functional conversion between no and o 2 reductases, and into evolution proton transfer mechanisms and respiratory enzymes in general. the genome of the bacterium geobacter sulfurreducens (gs) encodes for 111 c-type cytochromes (1). genetic studies using cytochrome deficient gs strains and proteomic studies identified cytochromes that were produced under specific growth conditions (2) (3) (4) . a putative outer-membrane cytochrome, omcf, is crucial for fe 3? and u 6? reduction and also for microbial electricity production (4). omcf is a monoheme c-type cytochrome with sequence similarity to soluble cytochromes c 6 of photosynthetic algae and cyanobacteria (4). the structure of oxidized omcf was determined (5) being the first example of a cytochrome c 6 -like structure from a nonphotosynthetic organism. the structural features of omcf hinted a different function compared to cytochromes c 6 from photosynthetic organisms, being an excellent example of how structurally related proteins are specifically design by nature to perform different physiological functions. in order to elucidate omcf structural-functional mechanism, isotopic labeled protein ( 15 n and 13 c) was produced and its structure in the reduced form determined by nmr. single point-mutations at key residues were produced by site-directed mutagenesis and their impact on the structural and functional properties of omcf will be presented. in the early 90s, the search for the source of nitrogen monoxide (no) production in mammals led to the discovery of three major isoforms of no-synthases (nos): the neuronal nos (nnos), the inducible nos (inos), and the endothelial nos (enos) ( (1)). 20 years later, based on genomic analysis, numerous nos-like proteins have been identified in the genome other organism and in particular of several bacteria (bacillus anthrax, staphylococcus aureus… (2)). in spite of superimposable 3d structures and the ability to catalyse no production, all these enzymes are carrying different (if not opposite) physiological activities including cgmp signalling, cytotoxic activities, anti-oxidant defence, metabolism… moreover, noss become increasingly associated to oxidative-stress related pathologies ranging from neurodegenerative disorders, cardiovascular and inflammatory diseases, diabetes, cancers (3)…. this apparent paradox seems related to the belief that the strong similarity of sequence and structure of no-synthases must lead to a unique and identical functioning (no production) for all isoforms. this is blatant for bacterial nos-like proteins that are lacking the essential components required for no biosynthesis but remain considered as genuine no synthases. this approach might remain an obstacle to the understanding of nos actual biological role and could prevent the design of efficient nos-targeted therapeutic strategies. to elucidate this ''nos paradox'' our group has initiated a multidisciplinary approach that aims to relate the wide diversity of nos biological activities to variations in the catalytic mechanism of noss, to modifications of their regulation patterns and to adaptations to their physiological environment. in this context we have been investigating the mechanism of bacillus subtilis nos-like proteins with a special focus on the features that are specific to nos mechanism: i) electron and proton transfers and the role of the substrate and the pterin cofactor ii) oxygen activation and the role of the proximal ligand iii) the very molecular mechanism and the variations in the nature of reaction intermediates. for that matter we have been using a combination of radiolytical techniques (cryoreduction with 60 co y-irradiation, pulse-radiolysis with the elyse electron accelerator), stateof-the-art spectroscopies (epr, atr-ftir and resonance raman, and picoseconds uv-visible absorption spectrosocopies), organic synthesis (synthesized substrates and cofactors analogues) biochemistry and molecular biology (site-directed mutagenesis). we will present our results on the coupling of electron and proton transfers, on the tuning of the proximal ''push effect'' and we will discuss the conditions that favour for each nos isoform no production versus other reactive nitrogen and oxygen species. photosynthetic iron oxidation (pio) is an ancient form of photosynthesis with relevant consequences in the shaping of the planet. this form of metabolism may have been involved in the deposition of geological structures known as banded iron formations, which hold key information regarding the co-evolution of photosynthesis and earth. rhodopseudomonas palustris tie-1 and rhodobacter ferroxidans sw2 both use ferrous iron as an electron donor to support photosynthetic growth (i.e. photoferrotrophy). the sw2 foxeyz operon can stimulate light-dependent iron oxidation by other bacteria. it codes a two-heme cytochrome, a pyrroloquinoline quinone protein and an inner membrane transporter, respectively. in tie-1 the pioabc operon is required for photoferrotrophy. it codes for a ten-heme cytochrome, an outer membrane beta-barrel and a high potential iron-sulfur protein (hipip) respectively. here we present functional and structural characterization of proteins involved in pio. this molecular characterization is essential for understanding this mode of bioenergetic metabolism, and may one day aid the development of biotechnological applications like microbial fuel cells and bioremediation. alongside classical, cytochrome respiratory pathway, phycomyces blakesleeanus possess alternative, cyanideresistant respiration (crr) facilitated by alternative oxidase (aox). in order to study role of oxygen in regulation of crr, the effects of cyanide on respiration of 24h old mycelia in aerated (control), hypoxic and anoxic conditions were measured. mycelium was incubated in these conditions for 1.5h, 3h and 5h. after 1.5h, aox activity was increased only in specimens incubated in anoxic conditions (13.6%). after 3h, increase in aox activity was significant in both hypoxic and anoxic specimens (18.9% and 18.8%, respectively), with even greater increase after 5h, 20.7% for hypoxic and 23.3% for specimens in anoxic conditions. mycelia treated for 5h was then oxygenated for 10 minutes. this induced decrease in aox activity of 17% in anoxic and even 23.9% in hypoxic mycelia. aox is recognized as one of the mechanisms for maintaining low levels of reduced ubiquione which can function in conditions in which cytochrome chain is disabled, such as anoxia. this is in concordance with results obtained on p. blakesleeanus, where aox levels rise in hypoxic and anoxic conditions and decrease close to control level shortly after introduction of oxygen into the system. influence of escherichia coli f 0 f 1 -atpase on hydrogenase activity during glycerol fermentation k. trchounian 1,2 , g. sawers 2 , a. trchounian 1 1 department of biophysics, yerevan state university, 0025 yerevan, armenia, 2 institute for microbiology, martin-luther university halle-wittenberg, 06120 haale, germany e. coli encodes four hydrogenases (hyd); only three of these, hyd-1, hyd-2 and hyd-3 have been well characterized. hyd-2 has been shown recently to reversibly evolve hydrogen during glycerol fermentation at ph 7.5 [1] . proton reduction was inhibited by n,n'dicyclohexylcarbodiimide suggesting a link with the proton-translocating f 0 f 1 -atpase. indeed, at ph 7.5 in an e. coli mutant (dk8) lacking f 0 f 1 overall hyd-activity was reduced to approximately 50% of the wild type activity; hyd-2, but not hyd-1, was detected in an in-gel activity assay. f 0 f 1 is therefore suggested to be required for hyd-1 activity. at ph 6.5 in glycerol medium hyd-activity in dk8 was *10% of wild type activity and hyd-1 and hyd-2 exhibited only weak activity. this indicated a significant f 0 f 1 contribution towards hyd-activity as ph decreased. furthermore, at ph 5.5 hydactivity was negligible and only a very weak activity band corresponding to hyd-2 could be observed. these results suggest that f 0 f 1 -atpase is essential for hydrogenase activity during glycerol fermentation at ph 5.5. taken together, the results suggest an interdependence between hyd-1, hyd-2 and f 0 f 1 -atpase activity. [ ion channels and transporters control many facets of cancer cell biology 1 and blocking the activity of these impairs tumor cell growth in vitro and in vivo. this new paradigm has opened new opportunities for pharmaceutical research in oncology 1,2 . we have contributed to this field showing that k v 11.1 (herg1) channels are aberrantly expressed in several human cancers where they control different aspects of the neoplastic cell biology such as proliferation and apoptosis, invasiveness and angiogenesis, the latter through the regulation of vegf secretion (reviewed in 1 ). the herg1dependent effects were shown in vitro and, more recently, in vivo. in preclinical models of both leukemia 1 and colorectal cancer 3 , herg1 overexpression confers a higher malignancy to neoplastic cells. moreover, herg1 blockers have therapeutic potential, since preclinical tests showed that treatment with specific herg1 blockers overcame chemoresistance in acute leukemias 4 as well as reduced gi cancer growth, angiogenesis and metastatic spread 3 . the overall message emerging from our data is that the herg1 protein represents a novel biomarker and drug target in oncology. up to now, herg1 was considered an ''antitarget'' due to the cardiac side effects that many (but not all) herg1 blockers produce and that result in lengthening the electrocardiographic qt interval. we report here recent studies on known and newly developed herg1 blockers that exhibit no cardiotoxicity and are more specific for the herg1 channels expressed in cancer cells. we reported previously that the increase of the cholesterol content of the cell membrane (in vitro) modified the biophysical parameters of the gating of kvl.3 k ? ion channels in human t-lymphocytes. in our present study we aimed to determine the effect of hypercholesterolemia on the biophysical parameters of kv1.3gating and the proliferation of t cells. t-lymphocytes were isolated from the peripheral blood of patients with cholesterol level considered normal (\5.2 mmol/l,control group) and patients with hypercholesterinaemia (hc). whole-cell k? currents were measured in patchclamped t cells and the kinetic (activation and inactivation kinetics) and equilibrium parameters (voltage-dependence of steady-state activation) of kv1.3 gating were determined. lymphocyte proliferation was measured using cfse staining with and without anti-cd3 and anti-cd28 stimulation. our results indicate that the biophysical parameters of kv1.3 gating are similar in the control group and in the 'hc' samples. the cfse-based assay showed that hypercholesterolemic t cells had higher spontenaous activation rate compared to control group. however, t cells from high cholesterol level patients challenged by anti-cd3 and anti-cd28 exhibited lower proliferation rate than control cells. generalized epilepsy with febrile seizures plus (gefs?, omim 604233) is a childhood genetic epilepsy syndrome correlated to mutations in the ancillary b-subunit of neuronal voltage-gated sodium channels (nachs). b1-subunit is non-covalently associated with nach a-subunits, serving as modulator of channel activity, regulator of channel cell surface expression and as cell adhesion molecule. the first and best characterized gefs? mutation is the c121w. this mutation changed a conserved cysteine residue into a tryptophan, disrupting a putative disulphide bridge that should normally maintain an extracellular immunoglobulinlike fold. in this study, we investigated the presence of this putative disulphide bond using 2-d-diagonal-sds-page, where the proteins were separated in the first dimension in absence of a reduction agent and in presence of it in the second dimension. this method allows to visualize the protein above the diagonal experimentally confirming that the disulphide bond is intramolecular. duchenne muscular dystrophy (dmd) is associated with severe cardiac complications. recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. it is, however, unknown if ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiomyopathy. here, we studied na and ca channel impairments in dystrophic neonatal cardiomyocytes, derived from dmd mouse models, prior to cardiomyopathy development. dystrophin-deficiency reduced na current density. in addition, extra utrophin-deficiency altered na channel gating. moreover, also ca channel inactivation was reduced, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. to assess developmental changes, we also studied na channel impairments in dystrophic adult cardiomyocytes, and found a stronger na current reduction than in neonatal ones. the described na channel impairments slowed the action potential upstroke in adult cardiomyocytes, and only in dystrophic adult mice, the qrs interval of the ecg was prolonged. ion channel impairments precede pathology development in the dystrophic heart, and may be considered cardiomyopathy triggers. supported by austrian fwf (p19352). it has been over 15 years since sensory neuron-specific sodium channel na v 1.8 was identified. since then na v 1.8 has been shown to play a crucial role in pain pathways, and it became a prominent drug target for novel pain killers. in contrast to myelinated neurons, mechanisms that target voltagegated sodium channels to the unmyelinated c-fibre axons are largely unknown. we investigated the localisation of na v 1.8 in unmyelinated primary sensory neurons. na v 1.8 was found to be clustered in lipid rafts on unmyelinated axons. when the lipid rafts are disrupted, remarkable reduction in both the conduction velocity and the number of cells responsive to mechanical stimuli to the unmyelinated axons were seen. using a compartment culture system, we also found that disruption of rafts in the middle region of the sensory axons caused a significant reduction in the responsiveness of the neurons to chemical stimuli to nerve endings. this is due to the failure of action potential propagation through the axons. these data suggest that clustering of na v 1.8 in lipid rafts of unmyelinated fibres is a key factor for the functional properties of the channel, which may due to a change in the voltage threshold. disruption of the na v 1.8 cluster and modifying lipid rafts in primary sensory neurons may be a useful new approach to control the excitability of nociceptive neurons. ion currents in are crucially important for activation of t-lymphocytes. our aim was to investigate how the blockage of various ion channels in isolation or in combination affects mitogen-dependent activation and proliferation of t-cells. we activated human peripheral blood lymphocytes using monoclonal antibodies against the tcr-cd3 complex and cd28. we applied specific channel blockers inhibiting the major ion channels of the t-cell: either the kv1.3 (tea or anuroctoxin), ikca1 (tram-34), or the crac channel (2-apb) alone or in combination. five days after the stimulus we measured the change in cell size and cellular granulation with flow cytometry along with the proportion of dividing cells, using cfse (carboxyfluorescein succinimidyl ester) dilution assay. our measurements indicated that the ion channel blockers suppressed the proportion of dividing cells in a dosedependent manner. increasing the strength of the stimulation reduced the potency of the blockers to inhibit cell proliferation and eventually the blockers were ineffective in decreasing lymphocyte proliferation. we experienced the greatest amount of inhibition using the combination of the blockers, which indicates synergy in the regulation pathway of the various ion channels. recently, sodium dependent phosphate transporter napi2b was revealed as potential marker for breast, thyroid and ovarian cancer. in vivo, napi2b is involved in maintenance of phosphate homeostasis and mutations or aberrant expressions of its gene (slc34a2) are associated with several diseases including cancer. however, data about napi2b mrna expression in different types of cancer and correspondent normal tissues are controversial and restricted. we investigated slc34a2 gene expression level in normal ovarian tissues and different histomorphological types of ovarian tumors using real-time pcr analysis. it was found that slc34a2 gene was highly expressed in well-differentiated endometrioid and papillary serous tumors, but was not expressed in low-differentiated tumors, benign tumors and in most normal tissues. mrna expression of slc34a2 in serouse and endometrioid ovarian tumors accurately correlated with protein expression that was detected in these tumor samples by western blot analysis and immunohistochemistry in our previous investigation. upregulation of slc34a2 gene expression in well-differentiated tumors may reflect cell differentiation processes during ovarian cancerogenesis and could serve as potential marker for ovarian cancer diagnosis and prognosis. in the present contribution a procedure for molecular motion characterization based on the evaluation of the mean square displacement (msd), through the self-distribution function (sdf), is presented. it is shown how msd, which represents an important observable for the characterization of dynamical properties, can be decomposed into different partial contributions associated to system dynamical processes within a specific spatial scale. it is shown how the sdf procedure allows us to evaluate both total msd and partial msds through total and partial sdfs. as a result, total msd is the weighed sum of partial msds in which the weights are obtained by the fitting procedure of measured elastic incoherent neutron scattering (eins) intensity. we apply sdf procedure to data collected, by in13, in10 and in4 spectrometers (institute laue langevin), on aqueous mixtures of two homologous disaccharides (sucrose and trehalose) and on dry and hydrated (h 2 o and d 2 o) lysozyme with and without disaccharides. the nature of the dynamical transition is highlighted and it is shown that it occurs when the system relaxation time becomes shorter than the instrumental energy time. finally, the bioprotectants effect on protein dynamics and the amplitude of vibrations in lysozyme are presented. we evaluated q for genotoxicity in mcf-7 breast cancer cells in the presence or absence of doxorubicin (dox), docetaxel (dtx) and paclitaxel (ptx), anticancer drugs commonly used in chemotherapy of different solid tumors. damage to dna was determinated by comet assay. the cells, after treatment with investigated compounds, were washed up and cultured in fresh medium for 0, 24, 48, 72 and 96 hours. we have found that q by itself caused significant dna damage. moreover flavonol enhanced genotoxic effect of anticancer drugs. the highest amount of dna in the comet tail was observed 48 h after treatment with combination of q with dox. similar changes were found in cells incubated with combination of q with taxanes -ptx or dtx. however, damage to dna in this case was considerably lower than damage caused by combination of q with dtx. our results confirmed anticancer and genotoxic activity of quercetin, which makes it a promising candidate for a potential use as a modulator of cytotoxicity and anticancer activity of anthracycline and taxane chemotherapeutics. although the health effects of low-frequency and intensity electromagnetic fields (lfi-emfs) are controversial, increasing evidence suggests that lfi-emfs are capable for initiating various healing processes. many (bio)physical ideas were suggested to explain the influence of lfi-emfs in living systems but the main effect of lfi-emfs on cell functions remains vague. however, some effects of lfi-emfs may be explained by redox and membrane processes. during diseases, cells not only demonstrate altered biochemical processes but also produce altered non-linear bioelectromagnetic complex patterns. thus, it is reasonable to use non-linear bioelectric and bioelectromagnetic signals from cells of the body for potential therapeutic applications that may be more effective than the artificial lfi-emfs signals. our novel emost (electromagnetic-own-signal-treatment) method is based on the utilization of the non-linear, bioelectric and bioelectromagnetic signals of the patients without any electromagnetic wave modulation and inversion of recorded output signals of subjects. here, we report our some restorative results after emost application. we also suggest that the possible effects of the emost may be achieved via redox-related processes. background or leak potassium conductances are a major determinant of resting potential and input resistance, two key components of cell excitability. these currents are not passive but finely tuned and adapted to cell specific functions. k 2p channels producing these currents are tightly regulated by a variety of chemical and physical stimuli including temperature, membrane stretch, free fatty acids, ph, ca 2? , neurotransmitters and hormones as well as protein partners. these different stimuli converge on gating mechanisms that show remarkable conservation between intracellular k2p channels (twik1 channels) and k2p channels located at the plasma membrane (trek1/2 channels). living at the edge: volume 2 the conduction system of interfacial forces into the alveolar type ii cell in previous investigations, using new microscopic approaches, we found that the presence of an a li leads to a paradoxical situation: it is a potential threat that may cause cell injury, but also a important stimulus: at ii cells respond promptly, and show sustained ca 2?signals that activate exocytosis. exocytosed surfactant, in turn, clearly prolonged the time to irreversible cell damage, and may be an adaptive and evolutionary defense mechanism against the harmful nature of surface forces. recently we published that at ii cells are sensing the a li but how this stimulus is conducted and converted into the cell, is still obscure. currently we are searching for potential calcium sources and it seems that the cells signal ca 2? by extracellular ca 2? entry probably through mechanosensitive channels. specialdesigned gene chips allowed a whole genome profiling of a li exposed single cells. these cells react with rapid changes on the transcriptional level: cellular pathways that are involved include e.g. defense response and lipid metabolism, and we identified genes associated with several lung diseases and injuries. we summarize, interface forces are strong, they are acting on the cells and triggering cellular events that are closely related with classical concepts in mechanotransduction, it is very plausible that those forces play a crucial role in the lung surfactant homeostasis. calorimetric method and instrumentation were worked out and applied for investigation aqueous solutions of proteins. thermal effects were analyzed by own construction heat-fluxtype dsc cell designed for temperature range from the boiling point of water down to 120 k. the achieved sensitivity of heat flow rate (hf) of the instrument is better than 50%w tested using 1 ll of 150 mm aqueous solution of nacl. from the integral value of hf -the total enthalpy change dh total , the enthalpies of transitions were separated from the heat capacities. using the method, several types of proteins (bsa, erd10, ubq, a-, b-casein, and wt, a53t, a-synuclein mutants) were investigated in that temperature range. the results are shown in detail as an illustrative example. potential applications are outlined, which include (i) the distinction between the solvent accessible surfaces of globular and intrinsically disordered proteins, (ii) the distinction between protein mutants, and (iii) the identification of monomer and polymer protein states. this method provides a possibility to study the polymerization process (amyloid formation) and to investigate in-situ the reason and circumstances of that. morphometry due to self gravity in living organism is an integral part in understanding self gravitation bio. computational studies of biological system, especially in ab-initio embryo as self gravitating mass, floating over amniotic fluid, as if maintaining anti -gravitation (extrinsic) mechanism, would be an interesting one to explore biomechanics of intrinsic gravity. since the work would be of exploratory nature, both from the point of view of identifying appropriate stage of development of embryological morulla and available computational logics, it was contemplated to initiate functional study with a few reported ultrasound evidential works on small animals like mice, grasshopper including compact human embryo as mathematical structure that may allow the formal definition of concepts such as convergence, mapping and continuity. as many of the finite-dimensional function analysis in topological vector spaces are available, we initiated our simulation and studies on concentrating locally compact banach spaces. 3 institute for x-ray physics, university of gö ttingen, germany we report on hard x-ray phase contrast imaging of black lipid membranes (blms), which are freely suspended over a micro machined aperture in an aqueous solution. this new way of membrane structure analysis allows investigating bio molecular and organic substances in aqueous environments by parallel and divergent beam propagation imaging, using partially coherent multi-kev x-ray radiation. the width of the thinning film is significantly smaller than the detector pixel size, but can be resolved from quantitative analysis of the intensity fringes in the fresnel diffraction regime down to its native thickness of about 5nm. to our knowledge this is the first time that such small features of a very weak phase object have been visualized by direct x-ray imaging techniques. we have put forward a simplified but extendable model, which enables the theoretical description of image formation and characterization of membrane thickness and its decrease during the thinning process from a bulk to a bimolecular film. on the basis of recent experiments, future investigations will be performed to study the interactions of membranes, as they are for example known from synaptic fusion, with high spatial resolution. shown that the acid incorporates mainly in the exterior part of the erythrocyte membrane, inducing creation of echinocytes. this suggests that it interacts predominantly with the outer part of the lipid layer of erythrocytes and liposomes. it was also shown that the cga decreases the packing order of the hydrophobic part of the membranes, without changing the anisotropic fluorescence of the hydrophobic part. one of the unique features of single molecule absorption/ emission is their anisotropy due to the well-defined transition dipoles for both processes allowing the determination of the molecule's 3d orientation. therefore, several techniques have been proposed in order to determine the full 3d orientation of dipole emitters on a single molecule level. we recently demonstrated a technique that combines emission distribution and polarization detection [1, 2, 3] . as the method is an intensity distribution technique and based on single photon detection in principle, one can extend the 3d orientation determination to fluorescence correlation spectroscopy (fcs) as well as dynamical anisotropy measurements. this allows for the determination of the dynamics in 3d orientation of single molecules down to a nanosecond timescales. the 3d orientation is particularly interesting in non-isotropic environments. a lipid membrane is such a non-isotropic environment of enormous importance in biological systems. we therefore use giant unilamellar vesicle (guv) labeled with dyes like dio as a model system. due to the defined curvature of such vesicles all possible dipole orientations can be achieved. this allows us to show the capabilities of our method on different timescales and to quantify the error in determination of 3d orientation dynamics in lipid membranes. [ the aim of the studies was to determine the changes that occur in a biological membrane and the model lipid membrane as a result of interaction with strawberry leaf extract. numerous studies conducted all over the world have documented a beneficial effect of polyphenolic compounds on the human organism. however, the mechanism of the interaction on the molecular and cell level is not yet known. in the work presented, the effect of strawberry leaf extract on the erythrocyte and black lipid membranes has been investigated. the applied methods -spectroscopic, fluorimetric and electric -allowed to determine the hemolytic and antioxidant activity, and the packing order in the erythrocyte membrane as well as the electric capacity of blms. the results obtained indicate that the extract is efficient in protecting membrane lipids against oxidation, does not induce hemolysis, increases osmotic resistance and decreases packing order in the hydrophilic region of the erythrocyte membrane. moreover, it increases stability and life-time of flat lipid membranes, without altering their specific capacity. supported lipid bilayers are an abundant research platform for understanding the behavior of cell membranes as they allow for additional mechanical stability and enable characterization techniques not reachable otherwise. however, in computer simulations these systems have been studied only rarely up to now. we present systematic studies on different length scales of the changes that a support inflicts on a phospholipid bilayer using molecular modeling. we characterize the density and pressure profiles as well as the density imbalance induced by the support. it turns out that the changes in pressure profile are strong enough that protein function should be impacted leading to a previously neglected mechanism of transmembrane protein malfunction in supported bilayers. we determine the diffusion coefficients and characterize the influence of corrugation of the support. we also measure the free energy of transfer of phospholipids between leaflets using the coarsegrained martini model. it turns out that there is at equilibrium about a 2-3% higher density in the proximal leaflet. these results are in agreement with data obtained by very large scale modeling using a water free model where flip-flop can be observed directly. we are additionally characterizing the intermediate states which determine the barrier height and therefore the rate of translocation. we also study the influence of surface roughness and curvature on the behavior. simulations in atomistic detail are performed for selected systems in order to confirm the findings. [ the inverse bar (i-bar) domain is part of the superfamily of the membrane-deforming protein is bin-amphiphysin-rvs (bar) proteins which induce either positive or negative membrane curvature both in vitro and in cells. generation of membrane curvature by these membrane deforming proteins often works together with actin dynamics. i-bar shares its function between actin bundling and membrane binding but it is still obscured what molecular mechanisms are responsible for these functions. the aim of our project is to investigate the detailed membrane binding properties of the i-bar of irsp53 and its relations to the actin cytoskeleton. in vitro fret experiments and fluorescence quenching studies were carried out between the i-bar and liposomes made up from different lipid constructs. we have found that the i-bar has preference to bind to the negatively charged lipids however it can also bind to the uncharged lipids. the fluorescence quenching studies reflected that the accessibility of the i-bar surface was higher toward the negatively charged lipids than for the uncharged ones. tns fluorescence assay reflected that the i-bar domain binds to the surface of the micells rather than penetrating into its core. lipid bilayers present a well-known order-disorder chain transition at ambient temperatures. this transition may become anomalous if the lipid head-group presents ionic dissociation at low ionic strength, as detected by several experimental techniques: between the gel and the liquid phases an intermediate phase appears as a shoulder in the specific heat, a deep in turbulence or a maximum in conductivity. we propose a statistical model which allows ionic dissociation of the polar group on the membrane surface and thus introduces competition between the hydrophobic interaction of hydrocarbonic chains, which favours the gel phase, at low temperatures, and the electrostatic interaction of charged head-groups, which favours the fluid phase, at higher temperatures. the model presents an intermediate fluid phase with higher dissociation and charge ordering on the membrane surface, beyond a sharp gel-fluid transition. the model presents increasing temperature of the main transition by addition of salt, as well as the shrinking of the anomalous region as chain length increases. model thermodynamic behavior is compared to results for pgs, phospholipids with a glycerol head-group. the well-programmed membrane fusion systems, operating in a weakly acidic environment, have attracted attention in the fields of biochemistry, biophysics, and pharmacy because these acidic conditions are generally observed in endosomal membranes or tumor tissues. we have reported a selective liposomal membrane fusion system toward a sugar-like cyclic cis-diol structure on the target liposome. this system consists of a lapidated phenyl boronic acid derivative as membrane -bound fusogen and phosphatidylinositol as target. here we report the preparation of a boronic acid / ph-responsive polypeptide conjugate as a novel membrane fusion device and the development of a target selective liposomal membrane fusion system with endosomal ph-responsiveness. during the course of lipid-mixing, inner-leaflet lipid-mixing, and contents-mixing assays to characterize membrane fusion behavior, we clearly observed a liposomal membrane fusion phenomenon when the ph of the experimental system was changed from 7.4 (physiological) to 5.0 (endosomal). our highly effective methods, which include a target selective liposomal membrane fusion, can be useful in the area of nanomedicine such as hybridoma technology and liposomebased drug or gene delivery. complete and reversible chemical denaturation of an a-helical membrane protein jana broecker, sebastian fiedler, sandro keller molecular biophysics, university of kaiserslautern, erwin-schrö dinger-str. 13, 67663 kaiserslautern, germany the question of how an unordered polypeptide chain assumes its native, biologically active conformation is one of the greatest challenges in molecular biophysics and cell biology. this is particularly true for membrane proteins. chemical denaturants such as urea have been used successfully for in vitro un-and refolding studies of soluble proteins and b-barrel membrane proteins. in stark contrast with these two protein classes, in vitro unfolding of a-helical membrane proteins by urea is often irreversible, and alternative denaturation assays using the harsh detergent sodium dodecyl sulphate suffer from a lack of a common reference state. here we present the complete and reversible chemical denaturation of the bacterial a-helical membrane protein mistic out of different micellar environments by urea. we applied multidimensional spectroscopy and techniques typically used in b-barrel membrane protein unfolding. mistic unfolds reversibly following a two-state equilibrium that exhibits the same unfolded reference state. this allows for a direct comparison of the folding energetics in different membrane-mimetic systems and contributes to our understanding of how a-helical membrane proteins fold as compared with both b-barrel membrane proteins and water-soluble proteins. in recent years, buckwheat has been of great interest in the world markets of healthy food, due to its high energy value, the content of unsaturated fatty acids, mineral constituents and vitamins. its seeds contain flavonoids which are natural, efficient antioxidants. the aim of the present studies was to investigate the effect of buckwheat extracts on the properties of biological membrane, which is the main site of the interaction between the substances buckwheat contains and the organism. the research was conducted on red blood cells and their isolated membranes, using the spectrophotometric, microscopic and fluorimetric methods. from the results obtained it follows that the compounds contained in buckwheat extracts increase the osmotic resistance of erythrocytes, making them less sensitive to the medium's osmotic pressure, induce changes in cell shape, producing increased number of echinocytes, and decrease the packing order of the polar heads of membrane lipids. it can thus be inferred that the compounds contained in the extracts penetrate the hydrophobic region of the erythrocyte membrane and alter its properties. .due to its small size, symmetric structure, amphipathicity, proteolytic stability and testable mode of activity, the gs backbone is a convenient model system to examine the structure-activity relationship of individual amino acid substitutions. we have previously reported the structure analysis of two gs analogues in which either the val or leu residues on the hydrophobic face of the molecule were substituted by the aliphatic 19 f-labeled amino acid 4f-phg. using 19 f-ssnmr in oriented lipid bilayers, we observed a re-alignment of the peptide that is compatible with the formation of a putative pore [top.curr.chem. 273:139]. here, we present novel analogs of gs with different 19 f-prolines in the b-turn region, and with cf 3 -bpg in place of leu. based on these 19 f-ssnmr results and supported by cd, dsc and activity tests, we could demonstrate that all analogues are structurally intact and antimicrobially active. we observe, however, differences in the re-alignment propensity when comparing these gs analogues in dlpc and dmpc bilayers. these differences can be rationalized in terms of molecular shape being changed upon incorporation of unnatural amino acids at various sites of the molecule. the beta-propiolactone (bpl) is an inactivating reagent commonly used to produce viral vaccine preparations (whole virions or split-virions). although bpl has been reported to inactivate nucleic acids, its mechanism of action on proteins and the outcome on viral infection remains ill-defined. in this work, h3n2/victoria/210/2009 influenza virus strain has been submitted to various bpl inactivation conditions (from 2lm to 1 mm). cell infection ability was progressively reduced and entirely abolished at 1 mm bpl. to clarify the bpl effect, we focused on membrane fusion infection steps using kinetic fluorescence molecule leakage from liposome and lipid fret assays combined with cryo electron microscopy. membrane fusion measured at ph 5 on gm3 liposomes was reduced in a dose-dependent manner. interestingly the fusion activity was partially restored using the proton-ionophore monensin as confirmed by cryoem images. in addition, a decrease of molecule leakage irrespective to bpl concentration was measured suggesting that the hemagglutinin affinity for gm3 was slightly modified even at low bpl concentration. altogether these results strongly suggest that bpl treatment impairs m2 protein activity likely by preventing proton transport and bring new light on the mechanism of action of bpl. cellular membranes have a heterogeneous lipid composition, potentially forming nano-domains or membrane rafts, believed to be platforms of altered fluidity involved in protein sorting and trafficking 1 . an alternative mechanism, potentially leading to protein sorting, has recently been proposed, suggesting that the curvature of membranes can also actively regulate protein localization 2 . recently we showed that a variety of protein anchoring motifs are membrane-curvature sensors and thus up concentrate in regions of high membrane curvature 3 . furthermore the curvature sensing ability of the anchoring motifs persisted independently of their structural characteristics. this leads us to speculate that curvature sensing might be an inherent property of any curved membrane and as a consequence, the lipid composition of the bilayer could potentially regulate this recruitment by membrane curvature. thus there might be an intimate, yet unrecognized, link between the way raft-like membrane domains and membrane-curvature promotes the localization of membrane-anchored proteins. we examined how changing the lipid composition of liposomes influenced the recruitment by membrane curvature of a model amphiphilic protein-anchoring motif. employing our single liposome curvature assay, we tested lipid mixtures with different ratios of dopc, sphingomyelin and cholesterol, giving rise to liposome populations of different phase-states. we found an amplified recruitment by membrane curvature for all raft-like l o phase-state mixtures when compared to the l d phase-state counterparts. based on these findings we suggest a synergetic effect when combining a raft-like lipid phase-state and high membrane curvature, resulting in a highly potent mechanism for selective localization of membrane-anchored proteins. keywords: non-lamellar lipid structure, phase transition, minerval, 2-hydroxylated fatty acid. minerval (2-hydroxyoleic acid), a potent antitumoral drug, is known to modulate the lipid membrane structure by decreasing the lamellar-to-non-lamellar phase transition temperature (t h ). a series of 2-hydroxy fatty acid derivatives, varying in acyl chain length and degree of unsaturation, have been analyzed in terms of their ability to stabilize the inverted hexagonal (h ii ) phase in palmitoyl-oleoyl-phosphatidylethanolamine membranes. differential scanning calorimetry and 31 p-nuclear magnetic resonance showed that mono-and polyunsaturated, but not saturated, 2-hydroxylated fatty acid molecules were able to decrease the t h . lipid vesicles mimicking the lipid composition of a cell membrane were solubilized at 4°c in the presence of triton x-100. the results demonstrated that the amount of detergent-resistant membranes, which are related to liquid ordered (lo) structures, decreased in the presence of 2-hydroxylated fatty acids. the so-called lipid membrane therapy focuses on the reversion of cell disfunction through the modulation of the membrane structure, thus altering the activity of membrane-associated proteins. the ability to modify the biophysical properties of a lipid membrane makes the studied 2-hydroxylated fatty acid molecules be prospective candidates for the use in the lipid membrane therapy. ]. however, a significant downward divergence occurs above 4 mol % of probe content, which might indicate deviations to ideal mixing in fluid phase. results for b-py-c 10 -hpc, in mixtures of popc with 10 and 20 mol % pops were indistinguishable from those obtained with pure popc vesicles; however excimer formation in pure pops bilayers appears to be appreciably higher. we also compared the excimer formation findings with quenching of the same probes by low concentrations of doxyl quencher groups labeled acyl phospholipid chain at the same depth of the pyrenyl group. the results are also scrutinized by the same two-dimensional kinetic formalism and good correlation was also found. derek marsh max-planck-institut fü r biophysikalische chemie, 37070 gö ttingen, germany the amassing of comprehensive data on the lipid composition of biological membranes by lipidomics initiatives provides a potent challenge to the membrane biophysicist interested in lipid structure. this resolves itself essentially into two aspects. the first systematises the dependence of membrane biophysical parameters on lipid molecular structure. lipid volumes, membrane dimensions, chain-melting temperatures and enthalpies, nonlamellar phase formation and structure, critical micelle concentrations and thermodynamics of membrane formation, membrane-membrane interactions and lipid transfer are amongst the properties of central biophysical interest. the relevant structural parameters are lipid chain length, degree of unsaturation, chain branching and headgroup configuration. the second, more complex and less well developed, aspect concerns the lipidlipid interactions that determine the membrane properties of lipid mixtures. in part, these can be obtained from binary phase diagrams, and the more limited number of ternary phase diagrams -notably with cholesterol -that are available. extrapolation to higher order mixtures lies in the future. i shall attempt to summarise some of the progress in these directions. the immediate aim is a second edition of my handbook of lipid bilayers, which, in addition to a vastly expanded database, will include interpretative features and will be available in the early part of next year. lateral diffusion dynamics in phosphatidylcholine/cholesterol bilayers has been mostly accessed by means of epr, nmr and fcs spectroscopic techniques. reliable stady-state florescence quenching analysis of diffusion-controlled processes has been ampered by the lack of a self-consistent kinetic formalism for the two-dimensional (2d) counterpart of the classical stern-volmer analysis for three-dimensional (3d) solvents. we studied the excimer formation of phospholipid-labeled pyrenyl probes (proportion of 4 mol %) in mixed popc/cholestrol mlv liposomes by combined steady-state and timeresolved fluorescence the findings are in very good agreement with the theoretical predictions of the kinetic formalism specific for fluorescence quenching processes occurring in the small hydrophobic head group, the closely packed acyl chains, and the capability of interfacial hydrogen bonding have been suggested to govern the characteristic membrane behavior of long chain saturated ceramides: the self-segregation, and the formation of hexagonal phases and highly ordered gel phases. while it has been shown that structural alterations of the ceramide acyl chains induce position dependent effects on their behavior, we wanted to study the effect of interfacial properties, including hydrogen bonding, on ceramide membrane properties. the h-bond donor functions of 2nh and 3oh in the sphingosine backbone of palmitoylceramide were disrupted either separately or simultaneously by replacing the hydrogen with a methyl-group. when the lateral phase behavior of mixed bilayers containing cholesterol/sphingomyelin-rich domains was studied in the presence of the ceramide analogs, the 3o-methylated ceramide appeared to form a thermally stable, sterol-excluding gel phase with sphingomyelin, whereas the 2o-methylated ceramide failed in both thermal stabilization and sterol displacement. the doubly methylated analog was the poorest ceramide mimic. together with the possible steric effects induced by the methylations, the lack of 2nh h-bond donor function impaired ceramide membrane behavior to a greater degree than the lack of 3oh h-bond donor function. sugar-based surfactants are made from renewable resources using the ''green chemistry'' methods, are easily biodegradable and used in washing agents, cosmetics, and drug carriers. besides, there are attempts to use them as nonviral vectors in gene therapy. we studied the influence of new x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lengths (n = 10, 12, 14, 16) on the thermotropic phase behavior of dppc, and dppc/chol bilayers by means of differential scanning calorimetry. the surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). we analyzed the changes in the temperatures, enthalpies and shapes of the main phase transitions as a function of concentration. molecular modeling methods were also used. cytotoxicicity of the c n gabs was determined in the cell line l929 and a549. for cytotoxicity test, the cells were seeded in 96-well plates 1 ml of 2á10 6 cells/ml in the culture medium eagle'a or dulbecco with 2% calf serum, penicillin and streptomycin was deposited into each plates. the cells were treated with various doses of surfactants and incubated. the minimal concentration which was toxic to approximately 50% of cells was taken as tccd 50 . this work was supported by grant n n305 361739. membrane fusion is ubiquitous in life requiring remodeling of two phospholipid bilayers. as supported by many experimental results and theoretical analyses, merging of membranes seems to proceed via similar sequential intermediates. contacting membranes form a stalk between the proximal leaflets which expand radially into a hemifusion diaphragm (hd) and subsequently open to a fusion pore. direct experimental verification of the hd is difficult due to its transient nature. using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (guvs) containing fluorescent membrane protein anchors and fluorescent lipid analogues in the presence of divalent cations. time resolved imaging revealed that fusion was preceded by displacement of peptides and lipid analogues from the guv-guv contact region being of several lm in size. a detailed analysis showed that this structure is consistent with the formation of an hd. a quantitative model of the hemifusion equilibrium and kinetics of the growing hd was developed. bilayer tension could be shown to drive hd expansion and interleaflet tension was found to act as a counterforce, because the outer leaflets are compressed upon hd growth. the model and its predictions fit nicely with observations above. concentration effects of trehalose in the equivalent polarity of fluid popc bilayers c. nobre 2 , d. arrais 1 , j. martins 1,2 1 ibb -cbme, faro, portugal, 2 dcbb -fct, universidade do algarve, faro, portugal trehalose is an important disaccharide, formed by two units of glucose linked by a a-1,1 glicosidic bond. it is capable of replacing water molecules in the hydration shell of the phospholipid headgroups, in cases of extreme dehydration, by establishing hydrogen bonds with their -co and -po groups, preserving this way the membrane structure. the polarity gradient is a significant feature of lipid bilayers and is influenced by the amounts of water within this medium. it is therefore important to understand the effects of different concentrations of trehalose in simple model membranes. using the pyrene empirical polarity scale, we monitorized changes in the polarity values when varying trehalose concentration in the bounding aqueous phase. for lower concentrations (until 0.25 m), we observed a decrease in polarity, comparing with popc bilayers in pure water. for higher trehalose concentrations (above 0.5 m), the polarity values are indistinguishable from those popc in water. using the freeze and thaw technique we obtained the same results, except for the lower trehalose concentrations. general anesthetics are indispensible tools of daily surgery. yet, their molecular mode of action remains elusive. while one school favors specific (direct) interactions with proteins of the central nervous system, another school adheres to a nonspecific modulation of biophysical membrane properties. one of the strongest arguments against lipid theories is the absence of stereo-specific effects in model membranes, as opposed to their detection by electrophysiological measurements on ionchannels. we have combined x-ray scattering and molecular dynamics simulations on palmitoyl-oleoyl-phosphatidylcholine bilayers with fluorescence microscopy on live cells to study the effects of the stereoisomers of ketamine on membrane properties. we find significant effects of both enantiomers on the distribution of lateral pressures at clinically relevant concentrations, being more pronounced for s-(?)-ketamine. we further calculated the effect of the lateral pressure profile changes on the opening probability of an ion-channel using crystallographic information. the observed channel inhibition compares remarkably well with clinically observed effects of the enantiomers. we thus provide first evidence for a stereo-specific, but indirect effect of general anesthetics on ion-channels. dependence of gramicidin a channel lifetime on membrane structure obtained from x-ray scattering measurements horia i. petrache department of physics, indiana university purdue university indianapolis, in 46202, usa the activity of ion channels, in particular the lifetime of their conducting (open) state depends on the physical properties of lipid bilayers [1, 2] which in turn depend on lipid headgroup and acyl chain composition. in order to investigate this dependence, we have performed measurements of gramicidin a (ga) channel lifetimes in three different lipid series. in each series, the lipid headgroups were phosphatidylcholine (pc), phosphatidylethanolamine (pe), and phosphatidylserine (ps), while the acyl chains consisted of symmetric monounsaturated di(18:1), mixed (16:0)(18:1), and methylated di(16:0-4me). in order to minimize the effect of headgroup electrostatics, measurements where performed in 1m kcl salts. we show how ga lifetimes depend on headgroup and acyl chain composition and on structural parameters determined by x-ray scattering. for the lipids considered, ga lifetimes cover a range from 0.7 seconds in the dope lipid to 18 seconds in dphps. in this range, we find a gaussian dependence of ga lifetime on bilayer thickness, consistent with hydrophobic matching models. we discuss different aspects of channel-lipid interactions and to what extent measurements of ga lifetime in binary mixtures are consistent with measurements in pure lipid systems. [ the aim of the studies was to determine the effect of chlorogenic acid (cga), which is the main constituent of plant extracts, on properties of the model membranes. its effect was studied on temperature of the main phase transition of various lipids with and without presence of cholesterol, using the differential scanning calorimetry (dsc) method and the fluorimetric method. in particular, the degree of packing order of the hydrophilic phase of liposomes was determined using the laurdan and prodan probes, and fluorescence anisotropy of the hydrophobic phase with the probes dph and tma-dph. it had also been studied the effect of chlorogenic acid on the structure and capacity of black lipid membranes (blms), formed of egg lecithin and lipids extracted from erythrocytes. the results obtained indicate that cga lowers the main phase transition temperature slightly, without changing the fluorescence anisotropy in the hydrophobic part of the bilyer, and causes a decease in the packing order of the hydrophilic phase. by monitoring the capacity during blm formation we have found that the presence of chlorogenic acid accelerates the process of lipid self-organization into a bilayer, and increases stability and life of the blms. however, the was no effect of cga on specific capacity of the membranes, and thus on thickness of the liposome membrane hydrophobic layer. this work was sponsored by the ministry of science and education, scientific project no. n n312 422340 and n n304 173840. many examples have recently been found where biological processes in the lipid bilayer are affected by the changes in the physicochemical properties of the membrane, e.g. the local curvature, the membrane tension and certainly the membrane structure. it has been shown that the activity of polyene antibiotics is strongly correlated to the phase diagram in a membrane composed of a mixture of popc and ergosterol or cholesterol (j membrane biol, 237:41-49, (2010)). it is known that polyene action is quite sensitive to the type of sterol in the membrane, which enables its medical use, mainly as antifungals. it has been proposed that this selectivity of the drug to fungi is related to structure modulation by the sterols (see for example, biophys. j., 85, 2323, (2003)) and therefore the correlation found could be due to structural differences between popc/ergosterol and popc/ cholesterol along the corresponding phase diagrams. to investigate this, molecular dynamics simulations of the above mixtures along their phase diagrams were performed. it was found that there are indeed marked differences in structure along the phase diagrams, but for the sterol-sterol distribution function. an analysis of the behavior of this observable and the implications on polyene action is discussed. acyl transfer from lipids without enzyme catalysis: a new paradigm for membrane protein ageing? john sanderson, catherine pridmore, jackie mosely, paul yeo durham university, department of chemistry, durham, uk membrane proteins are recycled in cellulo with half-lives ranging from minutes to days. in other systems, such as enveloped viruses, proteins may equally remain membranebound for periods of days. it is therefore of interest to examine the behaviour of proteins in model membranes over extended periods in order to determine the long-term stability of the mixed systems, both in kinetic terms (attainment of equilibrium states) and chemical terms (reactivity). the reactivity of proteins towards membranes has been examined using the peptide melittin as a model for membrane proteins. acyl transfer from phospholipids to the peptide was found to occur over a period of several days, in the absence of any enzyme catalysis. transfer was detectable after 2 days and reached 50% conversion in 8 days. using tandem mass spectrometry approaches, the sites of melittin modification were localised. these sites included the side chain of lysine, opening the possibility that this residue may be modified in any membrane protein where this residue has an appropriate disposition. these observations challenge preconceptions concerning the membrane as an inert medium and highlight potential new mechanisms for membrane protein ageing. interaction of poly(l-arginine) with negatively charged bilayers studied by ft-ir spectroscopy christian schwieger, alfred blume martin-luther-university, halle-wittenberg, institute of chemistry, van-dankelman-platz 3, 06108 halle / saale, germany e-mail: christian.schwieger@chemie.uni-halle.de oligoarginine residues attached to macromolecules are known to facilitate the transport through lipid membranes. since the mechanism of this transport is still unclear, the effect is often called ''arginine magic''. we studied the interaction of poly(larginine) (pla) of different molecular weight with negatively charged lipid bilayers. we have shown by calorimetric and monolayer techniques that the interaction is due to a combination of electrostatic and hydrophobic forces. now we present an ft-ir spectroscopic study to reveal the effect of pla binding on membrane organisation and peptide conformation. we will show that pla binding reduces the lipid miscibility of negatively charged (pg or pa) and zwitterionic (pc) lipids within the bilayer. from the shift of the c=o stretching vibration we deduce that arginine side chains penetrate into the hydrophobic/ hydrophilic interface and replace hydration water molecules. the binding reduces the rotational freedom of the lipid molecules, as could be shown by an analysis of the ch 2 -streching vibrations. pla binds in a b-sheet conformation to pg or pa gel phase membranes whereas its structure in bulk is random coil. the shift of the guanidyl vibration frequencies shows that also hydrogen bonds contribute to the pla -lipid interactions. neutron scattering studies of model membrane as a function of hydration and temperature federica sebastiani 1,2 , alessandra filabozzi 1 and giovanna fragneto 2 1 dipartimento di fisica, università degli studi di roma ''tor vergata'', roma, italy, 2 institut laue-langevin, grenoble, france cell membranes carry out highly specialised functions in living materials. the composition of bacterial membranes is essential to understand the mechanism of action of antimicrobial peptides. in order to understand the role of the various components contributing to the overall behaviour, we have reproduced the membrane of bacillus subtilis and carried out neutron diffraction studies on d16 (small momentum-transfer diffractometer) and d17 (reflectometer used as a diffractometer), at ill. an ordered and homogeneous sample has been obtained by using the widely studied dmpc. the measured d-spacing of dmpc as a function of the relative humidity (rh) is related to the physical and chemical conditions affecting the sample. consequently the reliability of the humidity chamber, which has been previously upgraded, has been stated. moreover, the most suitable preparation technique has been set up. in order to investigate the component roles within bacillus subtilis membrane, three samples of phospholipids were prepared (with pope, popg and cardiolipin). neutron diffraction measurements, performed at controlled rh and temperature, suggested the presence of interesting phase transitions or coexistence of phases. the rupture of membrane vesicles near solid surfaces annamá ria taká ts-nyeste, imre deré nyi department of biological physics, eö tvö s university, h-1117 budapest pazmany p. stny. 1/a, hungary the behavior of lipid membranes near solid surfaces has a great significance both in medicine and in technology. in spite of the widespread use and study of such membrane phenomena, their theoretical analysis is rather scarce. our main goal here is to understand the process during which membrane vesicles first adhere to solid surfaces, then rupture (or go through a series of transient ruptures) due to the mechanical tension induced by the adhesion, and finally spread along the surface forming a supported lipid bilayer. in our theoretical description we simultaneously consider the dynamics of spontaneous pore opening and closing; volume loss via leakage through the pores; and the advancement of the adhesion front. all these processes are supposed to follow an overdamped dynamics and coupled to each other through membrane tension. our numerical simulations reveal that the rupture process consists of three well distinguishable phases: a fast initial volume loss; followed by a slow volume loss; ending with a final burst and surface spreading. the second phase can be skipped if either the first phase advances far enough or the third phase sets in early enough. the smaller the vesicle, the further the first phase can advance. the third phase can start earlier if either the surface is smooth enough, or the adhesion energy is large enough, or the line tension is small enough. when the second phase is not skipped the time needed for the rupture process can take very long with a large variance. in the realistic range of the material properties (line tension, bending rigidity) the process is qualitatively always the same, so the most decisive parameter remains the size of the vesicle: the smaller the vesicle the faster and easier it ruptures. (3)). we chose four plant-derived polyphenols (flavonoids and stilbenes) of documented biological activity to study their influence on lipid domain number, area, shape, and borderlength. we found that resveratrol elevated the number of domains per vesicle, decreased their area and markedly increased the total length of domain border without affecting domains' circular shape. surprisingly, no such effect was observed for piceatannol differing from resveratrol by one hydroxyl group only. neither genistein nor 8-prenylnaringenin changed the morphology of lipid domains significantly. the possible mechanism of resveratrol-induced effect on lipid domains' morphology could be its selective accumulation in the interfacial regions between liquid ordered and liquid disordered domains. putative cholesterol recognition amino acid consensus (crac) motif in hiv coreceptors cxcr4 and ccr5 mikhail a. zhukovsky, albrecht ott biological experimental physics department, saarland university, 66041 saarbruecken, germany we identified a cholesterol recognition amino acid consensus (crac) motif in transmembrane domain 5 (tmd5) of two g protein-coupled receptors (gpcrs), human chemokine receptors cxcr4 and ccr5, coreceptors of human immunodeficiency virus (hiv). we suggest that residues belonging to this crac motif are involved in cholesterol binding to cxcr4 and ccr5 that is responsible for cholesterol requirement for cxcr4 and ccr5 conformation and function and for the role that cell cholesterol plays in the cell entry of cxcr4-using and ccr5-using hiv strains. putative crac sequences involve residues v 214 /l 216 -y 219 -k 225 in cxcr4 and l 208 /v 209 /v 211 -y 214 -k 219 in ccr5. in cxcr4, crac motif is highly conserved across chordata species, whereas in ccr5, crac motif is less conserved. t curve describe quantitatively the interfacial landscape around the protein molecules and can be used for the distinction between the globular and idp states. the behavior of the t 1 and t 2 data showed that there are two reorientation types present for every protein solutions below 0°c, irrespective for the nature of the protein or the solvent composition. local field fluctuation and the bpp models were applied, which failed for the buffered protein solutions and for the idps dissolved in water. a main cause of the failure is the changing h in the analyzed temperature range. this case is valid for the solutions of idps and for buffered solutions of both protein types. another cause can be the active relaxation channels other than dipolar when ions of quadrupolar nuclei are present. ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. this trait is exhibited by rtx (repeat in toxin) motifs found in more than 250 virulence factors secreted by gram-negative pathogenic bacteria. we investigated several cyaa rtx polypeptides of different lengths ranging from 111 to 706 residues. we showed that the rtx proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium: they adopt premolten globule conformations and exhibit a strong timeaveraged apparent hydration, due in part to the internal electrostatic repulsions between negatively charged residues, as revealed by the high mean net charge. calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the rtx proteins. we propose that the intrinsically disordered character of the rtx proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. these results support the hypothesis that the folding reaction is achieved upon protein secretion and, in the case of proteins containing rtx motifs, could be finely regulated by the calcium gradient across bacterial cell wall. occupational exposure to heavy metals has been recognized to be a risk factor for parkinson's disease via metaltriggered deposition of alpha-synuclein (as) 1,2 . in the present work, al 3? induced conformational change and instant oligomerization of as have been studied using fret and fcs as main techniques. donor and acceptor were labeled in the c-terminal at positions a107c and a140c. the average lifetime of donor in the presence of acceptor increases with the increase of al 3? concentration, indicating as adopts a more extended conformation upon al 3? binding. the intrinsic tyr fluorescence rises sharply within the mixing dead time, reflecting an enhanced hydrophobicity of the tyr environment and a fast conformational change of as. al 3? also induces an immediate oligomerization of as as monitored by fcs. the diffusion coefficient of as changes from 85 ± 5 lm 2 /s as monomer state to 23 ± 5 lm 2 /s as oligomer state. the oligomerization is supposed to be induced by the ligand bridging of trivalent al ions. nearly 2% of human genes encode protein-kinases (pk), enzymes involved in cellular signaling and several other vital biochemical functions, which transfer phosphate groups from atp to specific target molecules, modifying their activity. [1] deregulated pk have been linked to numerous diseases including cancer and diabetes, making them attractive targets for drug design. [2] conformational transitions play a central role in regulating the phosphorylation activity. pk adopt an on state that is maximally active and one or more inactive states that shows minimal activity. [3] the similarity of the relatively rigid and largely conserved atp binding site makes the design of selective inhibitors binding to the active state very difficult. indeed some of the best cancer therapies available are based on inhibitors, as imatinib, that bind to inactive states peculiar to a small subset of pk (abl, c-kit and pdgfr in the case on imatinib). thus, understanding the atomic details of the active to inactive transitions in kinases has a great importance. here we study a particular active-toinactive transition of c-src, a fundamental proto-oncogene involved in cancer and metastasis, by using multi-microsecond long fully solvated molecular dynamics simulations, metadynamics and ptmetad calculations [4, 5] . the results, validated by mutagenesis, x-ray crystallography and binding kinetics, are suggestive of a functional role for the conformational transition. moreover, we were able to single out the most important residues affecting the conformational transition and to show that even a very conservative amino-acid substitution can have a dramatic effect on the conformational free energy landscape. the time-scales of protein folding events range over many orders of magnitude. in order to understand the complex folding mechanisms, peptides with well-defined secondary structure are often used as model systems as they may be regarded as smallest folding units of proteins. the formation of secondary structure elements occur on the nanosecond to low microsecond time scale. thus, stopped-flow techniques are too slow whereas pulsed laser techniques are capable to trigger folding processes in nanoseconds and to analyze faster folding events. we study ns-to-ls peptide dynamics by temperature-jump infrared spectroscopy. after initiation of a nanosecond temperature jump, the spectral response is monitored at single wavelengths in the amide i region reflecting the dynamics of the peptide backbone. relaxation rates are obtained. the helix-to-coil relaxation of polyglutamic acid is a multi-step process and requires more complex models than two-state kinetics. however, there are kinetic steps that are well described by single-exponential behavior and a two-state model. we demonstrate how equilibrium and time-resolved infrared spectroscopic data can be combined to deduce folding rates. unfolding and refolding studies using chemical denaturants have contributed tremendously to our understanding of the thermodynamics and kinetics of protein folding and stability. however, a major limitation of this approach lies in the large uncertainty inherent in the extrapolation of the free energy of unfolding in the absence of denaturant from free energy values measured at finite denaturant concentrations. here we show that this limitation can be overcome by combining multiple spectroscopic signals-including fluorescence, circular dichroism, and absorbance-recorded in a quasi-simultaneous and fully automated way at different wavelengths. we have optimised the number of wavelength values used, the integration time per data point, the increment in the denaturant concentration, and the weighting scheme applied for global data fitting. compared with the traditional approach based on the use of a single or a few wavelengths, we could thus improve the precision of the free energy value by an order of magnitude. we exemplify and validate this novel approach using representative, well-studied globular proteins and explain how it can be exploited to quantify subtle changes in membrane-protein stability which have thus far remained elusive. the rates of protein conformational changes are usually not only limited by external but also internal friction, however, the origin and significance of this latter phenomenon is poorly understood. it is often found experimentally that a linear fit to the reciprocal of the reaction rate as a function of the viscosity of the external medium has a non-zero 1 , the physical basis of pressure unfolding is still largely unknown. we report here a specific study of cavities contributions to the volume difference between unfolded and folded states (dv u ), using four single point mutants of staphylococcus nuclease (snase). each mutation is localised in a strategic position on the protein structure and was designed to change a large buried hydrophobic side chain into alanine, thus opening tunable cavities in the snase 3d structure. measuring hsqcs peaks intensities up to 2500 bar monitored the equilibrium high pressure unfolding and leads us to precise estimations of dv u for more than two-thirds of the 143 residues of each mutant. so-fast hmqc experiments 2 were also performed to measure folding and unfolding rates from 200 bar pressure jumps. high-pressure fluorescence experiments were done on six additional alanine mutants to complement the nmr study, allowing a more complete exploration of the local pressure sensitivity along the protein 3d structure. all these highly reliable measurements shed light on the real signification of the thermodynamic parameter dvu, and bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. determination of contributing factors to the volume change magnitude between unfolded and folded states (dv u ) is a longstanding question in the high-pressure field. we provide here new experimental and computational data using two wellcharacterized model proteins: notch ankyrin repeat domain (nank) and staphylococcal nuclease (snase). the repetitive nature of the nank protein was used to study influence of the protein size on dv u in a systematic way with a set of deletion mutants. high-pressure fluorescence data provided new evidences that neither peptide bonds hydration nor side chains differential hydration could be considered as major contributor to the measured dv u value. additional molecular dynamics (md) simulations rather suggested that the heterogeneous distribution of void volume in the folded states structures could explain the dv u variations among the nank deletion mutants. the specific issue of the void volume contribution to dv u values was studied using 10 cavity mutants in snase, allowing a large structural mapping of the alanine mutations on this globular protein. combination of x-ray crystallography, highpressure fluorescence, high-pressure nmr and md simulations provided a first clear determination of the void volume contribution to the dv u values. these results also bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. we expressed an ig domain (i27) and a 170-residue-long fragment of the pevk domain in order to investigate the effect of temperature and pressure on their conformation. ftir spectroscopy is a useful method for investigating the secondary structure of proteins. we analyzed the amide i band to obtain information on protein structure. fluorescence labeling was also used in some experiments. to generate high pressures, a diamond anvil cell was employed. the ftir and fluorescence spectra of the protein fragments were recorded across the pressure and temperature ranges of 0-1 gpa and 0-100°c, respectively. moderate changes were observed in the conformation of the pevk fragments in the explored range of the t-p plane, suggesting that the domain is a highly flexible, random-coil across the entire studied t-p range. by contrast, the i27 domain showed quite stable secondary structure. intrinsically disordered proteins participate in important regulatory functions in the cell, including regulation of transcription, translation, the cell cycle, and numerous signal transduction events. disordered proteins often undergo coupled folding and binding transitions upon interaction with their cellular targets. the lack of stable globular structure can confer numerous functional advantages, including, paradoxically, both binding promiscuity and high specificity in target interactions. nmr is unique in being able to provide detailed insights into the intrinsic conformational preferences and dynamics of unfolded and partly folded proteins, and into the mechanism of coupled folding and binding. the function of intrinsically disordered protein domains in transcriptional regulation and signaling will be described, with particular reference to the general transcriptional coactivators cbp and p300, the tumor suppressor p53, and the adenovirus e1a oncoprotein. the globular domains of cbp/p300 are targets for coupled folding and binding of disordered transactivation motifs of numerous transcription factors and viral oncogenes, which compete for binding to limiting amounts of cbp/p300. many intrinsically disordered proteins contain multipartite interaction motifs that perform an essential function in the integration of complex signaling networks. the role of multipartite binding motifs and post translational modifications in regulation of p53-mediated signaling pathways will be discussed. the early vascular network is one of the simplest functioning organs in the embryo. its formation involves only one cell type and it can be readily observed and manipulated in avian embryos or in vitro explants. the early vascular network of warm-blooded vertebrates self-organizes by the collective motility of cell streams, or multicellular ''sprouts''. the elongation of these future vascular network segments depends on a continuous supply of cells, moving along the sprout towards its tip. to understand the observed self-organization process, we investigate computational models containing interactions between adherent, polarized and self-propelled cells. by comparing the simulations with data from in vivo or simplistic in vitro experiments, we explore the role of active migration, leader cells, invasion of the ecm, and cell guidance by micromechanical properties of adjacent cell surfaces. boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron-10. the biophysics of bnct is very complicated; primarily due to the complexity of element composition of the brain. moreover; numerous components contributes to the over all radiation dose both to normal brain and to tumor. simple algebraic summation cannot be applied to these dose components, since each component should at first be weighed by its relative biological effectiveness (rbe) value. unfortunately, there is no worldwide agreement on these rbe values. thermal neutrons were formerly employed for bnct, but they failed to prove therapeutic efficacy. later on; epithermal neutrons were suggested proposing that they would be enough thermalized while transporting in the brain tissues. however; debate aroused regarding the optimum source neutrons energy for treating brain tumors located at different depths in brain. insufficient knowledge regarding the rbe values of different bnct dose components was a major obstacle. a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding condition on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating 65 different source neutron energies, between 0.01 ev and 13.2 mev. mcnp4b monte_carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about 30 kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. simulation of mutation induction by inhaled radon progenies in the bronchial epithelium balá zs g. madas, á rpá d farkas and imre balá shá zy hungarian academy of sciences kfki atomic energy research institute, konkoly-thege mikló s ú t 29-33., budapest, h-1121, hungary radon is considered as the second most important cause of lung cancer after smoking. to understand the mechanisms leading from radon exposure to cancer formation is of crucial importance. this study focuses on the description of mutation induction by radon progenies in the bronchial epithelium. computational fluid and particle dynamics approach was applied to determine the radio-aerosol deposition distribution in the central airways. a numerical replica of a small fragment of the bronchial epithelium was prepared based on experimental data. microdosimetric computations were performed to quantify the cellular radiation burdens at the very site of deposition accumulation. a mutagenesis model was applied supposing that radiation induces dna damages and enhances the cell turnover rate. the results show that both considered mutagenic effects of densely ionising radiation contribute significantly to mutation induction and mutation rate depends non-linearly on exposure rate. furthermore, simulations suggest that the local maintenance capacity of the bronchial epithelium can be exhausted by chronic exposure to radon progenies with activity concentration characteristic of some uranium mines. the present work demonstrates possible applications of numerical modelling in radon related carcinogenesis studies. the neural crest is a group of cells found in all vertebrate embryos. it forms in the neural folds at the border of the neural plate and gives rise to a huge variety of cells, tissues and organs. one of the astonishing characteristic of neural crest cells is that they are able to migrate very long distances in the embryo. the neural crest has been called the ''explorer of the embryo'' as it is one of the embryonic cell types that migrate most during development, eventually colonizing almost every tissue. in this talk i will discuss our recent finding about neural crest migration. we have shown that neural crest cells, classically described as mesenchymal cells, migrate in large clusters cytokinesis relies on tight regulation of the mechanical properties of the cell cortex, a thin acto-myosin network lying under the plasma membrane. although most studies of cytokinetic mechanics focus on force generation at the equatorial acto-myosin ring, a contractile cortex remains at the poles of dividing cells throughout cytokinesis. whether polar forces influence cytokinetic cell shape is poorly understood. combining cell biology and biophysics, we demonstrate that the polar cortex makes cytokinesis inherently unstable and that any imbalance in contractile forces between the poles compromises furrow positioning. we show that limited asymmetric polar contractions occur during normal cytokinesis, and that perturbing the polar cortex leads to cell shape oscillations and division failure. a theoretical model based on a competition between cortex turnover and contraction dynamics accurately accounts for the oscillations. we further propose that blebs, membrane protrusions that commonly form at the poles of dividing cells, stabilise the position of the cleavage furrow by acting as valves releasing cortical contractility. taken together, our findings show that the physical properties of the entire cell are integrated into a finetuned mechanical system ensuring successful cytokinesis. collective motion of individual cells marks the onset of the transition to multicellularity in many microorganisms. this transition is often mediated by intercellular communication signals between cells. here, we show, in contrast, that the transition from single cell to collective motion in an ensemble of gliding bacterial cells can be understood as a dynamical selfassembly process of self-propelled rods. experiments were carried out with a mutant of the bacterium myxococcus xanthus moving by means of the a-motility system only and without undergoing reversals. the collective motion phase is confined to a monolayer and is characterized by the organization of cells into larger moving clusters. a transition to collective motion is detected in experiments by image analysis, that reveals a qualitative change of the cluster-size distribution at a critical cell packing fraction around 17%. this transition is characterized by a scale-free power-law cluster size distribution with an exponent 0.88. we provide a theoretical model for cluster formation of self-propelled rods that reproduces the experimental findings for the cluster size distribution. our findings suggest that the interplay of selfpropulsion of bacteria and volume exclusion effects of the rodshaped cell bodies is sufficient to explain the onset of collective motion and the related changes in the cluster statistics. despite much speculation on the existence of structurally distinct oligomeric species associated with the conversion of certain monomeric proteins into amyloid fibrils, it has not previously been possible to observe them directly or to relate them to any key mechanistic steps involved in the interconversion process. we have developed a novel application of singlemolecule intermolecular fret to investigate in unprecedented detail the aggregation and disaggregation of alpha-synuclein, the protein whose pathogenic deposition as intracellular lewy bodies is a characteristic feature of parkinson's disease. our study reveals that a range of oligomers of different size and structure are formed, even at physiologically relevant concentrations. interestingly, the resistance to degradation of the aggregated state of alpha-synuclein, which is a wellwe focused on the structure-dynamics interplay and showed how the fractal-like properties of proteins lead to such anomalous dynamics. we used diffusion, a method sensitive to the structural features of the protein fold and them alone, in order to probe protein structure. conducting a large scale study of diffusion on over 500 pdb structures we found it to be anomalous, an indication of a fractal-like structure. taking advantage of known and newly derived relations between vibrational dynamics and diffusion, we demonstrated the equivalence of our findings to the existence of structurally originated anomalies in the vibrational dynamics of proteins. more specifically, the time dependent vibrational mean square displacement (msd) of an amino acid is predicted to be subdiffusive. the thermal variance in the instantaneous distance between amino acids is shown to grow as a power law of the equilibrium distance. the autocorrelation function in time of the instantaneous distance between amino acids is shown to decay anomalously. our analysis offers a practical tool that may aid in the identification of amino acid pairs involved in large conformational changes. more recently, we studied the effect of the hydrodynamic interaction between amino acids using a zimm-type model. we computed the time-dependent msd of an amino acid and the time-dependent autocorrelation function of the distance between two amino acids, and showed that these dynamic quantities evolve anomalously, similar to the rouse-type behavior, yet with modified dynamic exponents. we also studied the dynamic structure factor s(k,t) of proteins at large wavenumbers k, kr g [ [1, with r g the gyration radius, that are sensitive to the protein internal dynamics. we showed that the decay of s(k,t) is dominated by the spatially averaged msd of an amino acid. as a result, s(k,t) effectively decays as a stretched exponential. we compared our theory with recent neutron spin-echo studies of myoglobin and hemoglobin for the rouse and zimm models of hydrodynamic friction. in addition, i will mention two other projects currently underway: (i) a new elastic network model that accounts for the tensorial aspects of protein elasticity and is a combination of stretch-compress springs and bond-bending energies. (ii) the unfolding of a protein under the exertion of a large pulling force. allosteric regulation of enzymatic activity is crucial for controlling a multitude of fundamental cellular processes. yet the molecular level details underlying regulation often remain poorly understood. here we employed single molecule activity studies to dissect the mechanistic origin of enzymatic activity regulation. as a model system we employed a lipase and measured its activity as a function of accessibility to surface tethered liposomes (1), which are known regulators of its activity. our results surprisingly revealed that the lipase oscillates between 2 states of different activity. we accurately quantified for the first time both the interconversion rates between activity states and the inherent activity of these states. based on these we calculated the energetic landscape of the entire reaction pathway and identified that regulatory interactions redistributed the probability to reside on preexisting enzymatic activity states but did not alter the activity of these states. our findings provide the missing link between conformational and activity substates supporting and represent the first direct validation of the textbook hypothesis of conformational selection for regulation of enzymatic activity to identify the potential targets of cgmp in arabidopsis plants we adopted a proteomic approach to isolate possible cgmp-binding proteins. purification of soluble cgmp-binding proteins was performed using cgmp-agarose-based affinity chromatography procedure. next eluted proteins were analyzed by sds-page which revealed ten bands. we focused the subsequent analysis on low-molecular peptides of 15, 16 and18 kda which were bound cgmp more intensively. after 2d-ief-page of the proteins isolated by cgmp-agaroseaffinity chromatography eight most abundant protein spots in the low-molecular area were visualized. these spots of interest were excised from the gel and in gel digested by trypsin. then tryptic peptides were analyzed by maldi-tof mass spectrometry and identified as isoforms of nucleoside diphosphate kinase (ndpk) from arabidopsis. thus, our data suggest that ndpk is a potential target of cgmp signaling in arabidopsis. dual-color fluorescence-burst analysis (dcfba) was applied to measure the quaternary structure and high affinity binding of the bacterial motor protein seca to the protein-conducting channel secyeg reconstituted into lipid vesicles. dcfba is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. seca binds to secyeg as a dimer with a nucleotideand preprotein-dependent dissociation constant. one of the seca protomers binds secyeg in a salt-resistant manner, while binding of the second protomer is salt-sensitive. since protein translocation is salt-sensitive we conclude that the dimeric state of seca is required for protein translocation. a structural model for the dimeric assembly of seca while bound to secyeg is proposed based on the crystal structures of the thermatoga maritima seca-secyeg and the escherichia coli seca dimer. • dcfba is a flurorescence based single molecule technique that allows assessment of the stoichiometry of ligands bound to membrane receptors • dimeric seca binds asymmetrically to the protein-conducting membrane channel secyeg • monomeric seca binds secyeg but dimeric seca is required for protein translocation • protein translocation depends on receptor cycling of the dimeric seca if the dna charge is sufficiently neutralized by counter-ions, electrostatic interactions between helical charge patterns can cause attraction [1] . helix specific interactions also cause tilt, in one direction, between two dna fragments [1] . in braids and supercoils, this impetus to tilt breaks positive-negative supercoil symmetry. we show that these effects may cause spontaneous braiding of two molecules, lowering the dna pairing energy [2] . the pairing is more energetically favourable for homologues (same base pair text) than for nonhomologous pairs. this might explain pairing between only homologues observed in nacl solution [3] . also, we construct a simple model for a closed loop supercoil, including chiral electrostatic interactions. there are very interesting effects, for sufficient charge neutralization and groove localization of counter-ions. i.) positive super-coils are more energetically favourable than negative ones. ii.) a transition between loosely and tightly wound supercoils as one moves from negative to positive values of the supercoiling density. iii.) in positive super-coils the chiral interaction underwinds dna. [ von willebrand factor (vwf) is a large multimeric protein that is crucial for the force sensing cascade triggering primary hemostasis. it mediates binding of activated thrombocytes to injured epithelial tissue and serves as a transporter for coagulation factor viii. while it was shown that the hemostatic activity of vwf is affected by shear stress [1] , the exact impact that shear forces have on the inflammatory cascade remains unclear. it is assumed that hydrodynamic forces lead to partial unfolding of vwf, which in consequence exposes more binding sites. in order to observe shear-induced changes of the protein's functionality, we measure conformational changes of vwf under flow with fluorescence correlation spectroscopy (fcs). we aim to measure the degree of uncoiling of vwf multimers under various buffer conditions, e.g. in the presence of colloids, vesicles or platelets. as only large multimers show significant hemostatic activity we intend to monitor the molecular weight distribution of vwf. shifts in this distribution indicate various pathological conditions making our multimer analysis a fast diagnostical tool for vwf-related diseases. this will serve as a basis for studies of vwf binding to collagen, fviii, gpib, vesicles and membranecoated nanoparticles under shear flow. [ data on mechanical properties of medically important proteins located in neural junctions are very limited. contactins (cntn) and paranodin proteins, located in extracellular part of ranvier nodes, are important for proper brain wiring. here we study a new series of fniii modules from human cntn-1 and -4 using a single molecule afm force spectroscopy and advanced, all-atom steered molecular dynamics (smd) computer simulations. mutations in cntns are responsible for numerous brain disorders including autism or pathological development of odor maps. perhaps mechanical properties of individual fniii mutated protein modules are compromised, thus we address this problem. a comparison of our afm force spectra with those of reference proteins will be presented [1] [2] , and the molecular level interpretation fniii nanomechanics, based on our smd data will be given. we believe that these data should help to understand a role of cntn in regulation of sodium ion channels in both normal and autistic subjects. supported in part by polish ministry of education and science, grant no. n202 262038, the computational center task in gdansk and license for accelrys software. recent achievements in rational dna-motors engineering demonstrate the possibility to design nano-motors and nanorobots capable of performing externally controlled or programmed tasks. a major obstacle in developing such a complex molecular machine is the difficulty in characterizing the intermediates, the final products and their activity. typically, non in-situ gel and afm and in-situ bulk fluorescence methods are used. i will present two dna-motors recently developed and studied using in-situ single-molecule fluorescence resonance energy transfer (smfret), alternating laser excitation (alex) and total internal reflection fluorescence spectroscopy (tirf), and will demonstrate that these methods can improve the way we design, construct, measure and understand highly complex dna-based machines. a motor made of bipedal dna-walker, which walks on a dna track embedded on a dna-origami, capable of long walking distance and maintaining structural stability, will be presented. the motor is non-autonomous; it receives ss-dna fuel/anti-fuel commands from outside (as in shin & pierce, jacs, 2004). the motors assembly stages and singlemotor's walking steps are monitored using smfret. the second motor is based on published bipedal autonomous dna-motor (seeman, science 2009). it is characterized by coordinated activity between the different motor domains leading to processive, linear and synchronized movement along a directionally polar track. to prove that the motor indeed walks, the authors chemically froze the motor at each step and use a complicated radioactive gel assay. i will demonstrate that using single-molecule approach, we are able to directly and in-situ measure single-motor's movements in few simple experimental steps, and measure its structural dynamics and kinetics. translation by a single eukaryotic ribosome using single molecule total internal reflection fluorescence microscopy, we observed translation of a short messenger rna (mrna) strand by single eukaryotic ribosomes. the ribosome-mrnas complexes are fixed to a microscope coverslip through the mrna, and mrnas are located through fluorescently labelled oligonucleotides hybridized to it downstream start codon. because of the ribosome helicase activity, the double strand formed by the oligonucleotide and the mrna is opened while the ribosome translates this region of the mrna. thus, the loss of the fluorescence signal allows us to measure the distribution of translation speed of single ribosomes. careful attention was given to photobleaching for the data analysis. this experiment opens the door to the study of eukaryotic translation at the single molecule level. erythrocyte hyperaggregation, a cardiovascular risk factor, has been associated to high plasma concentrations of fibrinogen. using atomic force microscopy (afm)-based force spectroscopy measurements, we have recently identified the erythrocyte membrane receptor for fibrinogen, an integrin with a a3 or a3-like subunit [1] . after this, we extended the study to the influence of erythrocyte aging on fibrinogen binding [2] . force spectroscopy measurements showed that upon erythrocyte aging, there is a decrease of the binding to fibrinogen by decreasing the frequency of its occurrence (from 18.6% to 4.6%) but not its force. this observation is reinforced by zeta-potential and fluorescence spectroscopy measurements. knowing that younger erythrocytes bind more to fibrinogen, we could presume that this population is the main contributor to the cardiovascular diseases associated with increased fibrinogen blood content, which disturbs the blood flow. our data also show that sialic acid residues on the erythrocyte membrane contribute for the interaction with fibrinogen, possibly by facilitating the binding to its receptor. antimicrobial peptides are usually polycationic and amphiphilic with high affinity for bacterial membranes. in order to characterize their therapeutic potential it is crucial to disclose which properties of the peptide/lipids are important for target selectivity, and to examine the peptide structure and its association with lipid bilayers. in this work, first experiments have been carried out on a promising peptide called sb056, which might represent the basis for developing a novel class of antibiotics. with the goal of enhancing the activity of a new semi-synthetic sequence, two identical peptides (wkkirvrlsa) were assembled via a lysine-linker, carrying also an octanoyl-lipid anchor. a highly active compound was obtained, but its structure and mode-of-action remain unexplored. this dendrimeric peptide and its linear deca-peptide counterpart are being studied in parallel to highlight the relevant properties and differences between dendrimeric structure and the sequence. monolayer intercalation is investigated with microtensiometry, fluorescence spectroscopy is applied to study thermodynamics and kinetics of the binding process. circular dichroism, nmr and md simulations are employed with the aim of elucidating the 3d structure in the membrane-bound state. the capability of proteins to build structures via self-organization is fascinating biophysicists since decades. with the advent of single-molecule methods, namely fluorescence correlation spectroscopy (fcs) and fluorescence resonance energy transfer (fret), the process of complex formation is becoming accessible to direct observation. coronaviruses (cov) are enveloped positive-stranded rna viruses. for sars-cov, it was shown that coronaviruses encode a rna-dependent rna-polymerase (rdrp) build from non-structural protein 7 (nsp7) and non-structural protein 8 (nsp8). this hexadecameric nsp7-nsp8 complex is a hollow, cylinder-like structure assembled from eight copies of nsp8 and held together by eight nsp7 molecules [1, 2] . we are aiming at understanding the assembly process and conformational changes of the complex for the related feline coronavirus. first results implicate that nsp8 alone forms a dimer, where interchain fret is more efficient than intrachain fret. for the complex the results indicate that nsp7-nsp8 form a heterodimer which is different from sars-cov. our experiments highlight the potential of single-molecule fret for the study of protein complex formation. diffracted x-ray tracking (dxt) has been considered as a powerful technique for detecting subtle dynamic motion of the target protein at single molecular level. in dxt, the dynamics of a single protein can be monitored through trajectory of the laue spot from the nanocrystal which was labeled on the objective protein. in this study, dxt was applied to the group ii chaperonin, a protein machinery that captures an unfolded protein and refolds it to the correct conformation in an atp dependent manner. a mutant group ii chaperonin from thermococcus strain ks-1 with a cys residue at the tip of the helical protrusion was immobilized on the gold substrate surface and was labeled with a gold nanocrystal. we monitored diffracted spots from the nanocrystal as dynamic motion of the chaperonin, and found that the torsional motion of the chaperonin in the presence of atp condition was 10 times larger than that in the absence of atp condition. and uv-light triggered dxt study using caged atp revealed that the chaperonin twisted counterclockwisely (from the top view of chaperonin) when the chaperonin closed its chamber, and the angular velocity from open to closed state was 10 % faster than that from closed to open state. peptides or proteins may convert (under some conditions) from their soluble forms into highly ordered fibrillar aggregates. in vivo such transitions can lead to neurodegenerative disorders such as alzheimer's disease. alzheimer's disease is characterised by the extracellular deposition of abeta peptide in amyloid plaques, and the intracellular formation of neurofibrillary tangle (nft) deposits within neurons, the latter correlating well with disease severity. the major constituent of nft deposits are paired helical filaments (phf) composed of a microtubule-associated protein known as tau. studying the process by which tau forms these large aggregates may be an essential step in understanding the molecular basis of alzheimer's disease and other tauopathies. we have applied a two-colour single molecule fluorescence technique, and single molecule intermolecular fret measurements to study the soluble oligomers of tau which are formed during the aggregation and disaggregation of phf's. the neuronal protein alpha-synuclein is considered to play a critical role in the onset and progression of parkinson's disease. fibrillar aggregates of alpha-synuclein are the main constituents of the lewy bodies that are found in the brains of parkinson patients. however, there is growing evidence suggesting that oligomeric aggregates are significantly more toxic to cells than fibrillar aggregates. very little is known about the structure and composition of these oligomeric aggregates. we present results using single-molecule photobleaching approaches to determine the number of monomeric subunits constituting the oligomers. our results show that the oligomers have a narrow size distribution, consisting of *13-20 monomers per oligomer. fluorescence correlation spectroscopy data confirm the narrow size distribution and additionally indicate a very loose packing of the oligomers. in combination with bulk fluorescence spectroscopy results of tryptophan containing mutants of alpha-synuclein, we present a structural model for the alpha-synuclein oligomer. gold colloids are widely used for in vitro and in vivo imaging. compared to the traditional optical tags sers-coded nanoparticles show a narrow emission bandwidth with structured spectra typical of the molecule used, a wider excitation bandwidth, higher emission intensity, a better photo-stability, and a lower toxicity. this is why in cancer therapy, besides being considered good tools for the delivery of anti-tumor drugs, aunp can be also good optical tags for the analyses of both np localization by laser scanning microscopy and the process of drug release inside the cells by raman. in our work we used 10 nm diameter aunp loaded with rhodamine 6g, a molecule with a high raman and fluorescence efficiency, and with a chemical structure similar to doxorubicin, the antitumoral drug used in our system. the data showed that aunp are internalized by cells and sers can be performed. 10 nm and 60 nm diameter aunp loaded with doxorubicin were incubated at different time points with a549 cell line (human adenocarcinomic alveolar basal epithelial cells). only 60 nm aunp showed intense raman emission typical of the doxorubicin phonon transitions. in recent years biomedical applications of diamond nanoparticles have become of significant interest, which rises questions of their biocompatibility and mechanisms of interactions with cells. the aim of this study was to compare the effect of nonmodified diamond nanoparticles (dnps) and dnps modified by the fenton reaction on human endothelial cells. dnps (\10 nm particle size, sigma) were modified by the fenton reaction introducing surface -oh groups. immortalized human endothelial cells (huvec-st) were incubated with 2-100 lg/ml dnps in the optimem medium. diamond nanoparticles modified by the fenton reaction had smaller hydrodynamic diameter estimated by dynamic light scattering and the surface potential (zeta potential) measured using laser-doppler electrophoresis. they were more cytotoxic as evaluated by the mtt reduction assay. dnps augmented generation of reactive oxygen species in the cells, estimated by oxidation of 2',7'-dichlorofluorescin, the effect being higher for the fenton-modified dnps after 48-h incubation. cellular production of nitric oxide, estimated with daf-fm, was also affected by dnps; after 72h, fentonmodified oh, in contrast to non-modified diamond, decreased no production. diamond nanoparticles affected also the cellular level of glutathione and activities of main antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione s-transferase). we aim at investigating how photoreactions of proteins can be controlled by means of intense thz radiation tuned in resonance to specific vibrational modes, much in analogy to coherent control experiments conducted by fs nir laser pulses [1] . for this we will combine a time-resolved ir difference spectroscopic setup with uniquely intense, tunable narrow bandwidth thz radiation (3 -280 lm) at the ps beamline of the thz free electron laser felbe. these experiments will be performed on bacteriorhodopsin (br) which is the sole protein of the purple membrane of the archaebacterium halobacterium salinarum [2] . upon illumination, the chromophore retinal isomerizes around the c 13 -c 14 double bond [3] and br pumps a proton from the cytoplasmic to the extracellular side. this proton gradient is used by the bacterium to drive photosynthetic atp production under low oxygen tension [4] . in our experiment, the photoreaction is initiated by a visible laser pulse as in standard experiments, but then the sample will be irradiated by a thz pulse from the free electron laser tuned into resonance with low-energy vibrational modes which is supposed to influence the photoreaction [1] . such vibrational control will be monitored by time-resolved ftir spectroscopy using the step-scan technique [5] . liposomes are increasingly studied as nanoscale drug delivery systems and biomembrane models. however the exact structure dynamics and mechanical behavior of liposomes is little known. atomic force microscopy (afm) is a powerful tool to characterize nanoscale morphology and enables the mechanical manipulation of submicron-sized vesicles. a drawback of afm, however, is that liposomes may flatten and rupture on substrates to form patches or supported planar bilayers (spb). our aim was to obtain better understanding of factors affecting liposomes on substrates and find experimental conditions at which liposomes preserve their structural integrity. in the presence of divalent cations dppc liposomes formed spb on mica. vesicles sedimented subsequently preserved their integrity and showed stronger attachment to spb. in addition to cross-bridging lipid head groups, divalent cations influence the surface charge of liposomes, thereby modulating liposome-substrate and liposome-liposome interfacial interactions. preserved vesicles stabilized by divalent cations may provide a unique experimental system for studying membrane-protein interactions. the influence of e.g. ph and ionic strength on various chromatographic bead -biomass combinations. to analyze the force curves, possible elastic contributions, e.g. from deforming cellular membranes, have to be decoupled from the interaction forces. then, bead-biomass interactions will be modeled using (extended) dlvo theory and resulting data can also be compared to real-life eba processes. the project aims for a better understanding of the interaction forces in chromatography and might help to improve the process quality of eba. long-term non-invasive in vivo monitoring of the survival, migration, homing and fate of transplanted cells is of key importance for the success of cell therapy and regenerative medicine. tools for in vivo magnetic resonance (mr) imaging of labeled cells are therefore being developed. we have prepared superparamagnetic iron oxide nanoparticles by the coprecipitation of fe(ii) and fe(iii) salts and oxidation. to stabilize the particles and to facilitate their internalization by the cells, the nanoparticles were coated with several novel low-and highmolecular weight compounds including d-mannose, poly(llysin), poly(n,n-dimethylacrylamide) and dopamine-hyaluronate conjugate. the surface-modified magnetic nanoparticles were thoroughly characterized by a range of physico-chemical methods, which proved the presence of the coating on the particles. the particles were then investigated in stem cell experiments in terms of real time cell proliferation analysis, viability, labeling efficiency and differentiation. the iron oxide concentration of the labeled cells was assessed using mr relaxometry. the advantages/disadvantages of particular iron oxide coatings will be discussed and the optimal coating suggested. excellent contrast was achieved by labeling the cells with dopamine-hyaluronate-coated nanoparticles. support of the as cr (no. kan401220801) is acknowledged. in the last decades the interest towards the fabrication of innovative bio-sensors with improved sensitivity and reliability for medical-diagnostics applications has been constantly risen. among the different techniques, microfluidic systems are playing a major role. in order to detect extremely low concentrations of biomolecules (pm and fm), attention should be placed on the controlled, selective functionalization of micro-and nano-channels. in this work we propose a new approach to functionalize gold patches inside fluidic channels. we start from self-assembled monolayers (sams) of thiolated molecules on a gold electrode deposited inside the channel. then, by using an electrochemical approach [1, 2] we remove molecules from the sam at selected locations, by applying a negative voltage to the electrode. the newly exposed gold surface can be re-functionalized by using a thiolated biomolecule (i.e an antibody) capable to bind specific proteins flowing inside the channel. the cycle can be applied to other electrodes in the microfluidic system, creating a multiplexing device which, as we will show, can differentially measure ionic current flows in different channels. optically actuated micromanipulation and micro-probing of biological samples are increasingly important methods in the today's laboratory. microbeads as probes are the most commonly used tools in this field, although the only manipulative motion they allow is translation. we present polymerized 3d microstructures which can also be used for optical micromanipulation with more degree of freedom than microbeads. the two-photon polymerization (tpp), based on focused fs laser beam into appropriate photopolymers is a powerful method to build structures of arbitrary complexity with submicrometer resolution. the presented tools have the advantage of being capable of twisting and rotational manipulative motion, and also that the position of biological manipulation and optical trapping is spatially separated. different manipulative interfaces, the positioning stability and surface activation of the manipulators will be discussed. the potential application of multiplexed quantum dot labeling, mqdl, in clinical detection, prognosis and monitoring therapeutic response has attracted high interests from bioengineers, pathologists and cancer biologists. mqdl is superior comparing with conventional organic dye staining in its narrow emission bandwidths, wide signal dynamic ranges, high detection sensitivity, and low noise to signal ratios. however, the majority of the mqdl application has been limited to identification of specific cell type or cancer subtype and the improvement in labeling methodology. in this study, we focused on simultaneous detection and analysis of 5 proteins in the c-met activation pathway, i.e. rankl, vegf, nrpln-1, p-c-met and mcl-1, which are known to be associated with human prostate cancer progression and metastasis. two experimental systems were analyzed: 1) fixed xenograft tissues from an established ltl313 castration resistance human prostate cancer or crpc model; and 2) clinical prostate tissue specimens from localized cancer and bone metastasis. in the presentation we will report our experience in 1) the mqdl protocol optimization for the sequential reactions of individual primary antibody, the biotinylated secondary antibody and streptavidin-coated qd conjugate with nuclear dapi staining; and 2) the multiplexed image catching, image unmixing, and subsequent per cell base quantification. for future multi-specimen analyses and validation, we will introduce a high throughput vectra image analysis system. carbon nanotubes (cnts) 1 are already quite popular among many scientific and technological disciplines 2 . in recent years they have been targeted for biotechnological and medical applications. in this work we have investigated the nanostructural self assemblies of biological lipid molecules in presence of cnts. advantage of using highly aligned cnts 3 for this purpose being the possibility of studying the interactions of lipid molecules on the macromolecular surface as well as in the confinement of aligned cnts. we have observed various lyotropic nanostructures that are found for corresponding lipids in the bulk under dry and hydrated conditions. 4 nanostructural studies were mainly performed using small and wide angle x-ray scattering techniques. this work is crucial for designing the nano-micro-fluidic architectures and supported model membranes where both -functionalization of cnts and nanostructural assembling of lipids, could be employed simultaneously. alternative of the commonly used measuring tools in protein research and medical diagnostics. thazolidone derivatives are novel synthetic compounds possessing various biological activities. we selected three such compounds les-3120, 3166, 3372 which passed national cancer institute in vitro tests. annexin v/pi and dapi staining, dna electrophoresis in agarose gel, and western-blot analysis using specific antibodies against 30 cellular proteins involved in apoptosis were applied to study molecular mechanisms of tumor cell death induced by these compounds. it was found that molecular targets of thiazolidones in target cells strongly depend on structure of their side groups: les-3120 containing isatine fragment, activated caspase-8 involved in receptor-mediated apoptosis, while les-3166 possessing benzthiazol residue, induced mitochondrial apoptosis mediated by caspase-9, and les-3372 which has a unique chlorine atom in side chain, also led to mitochondrial apoptosis mediated by aif (apoptosis-inducing factor). to increase anticancer potential of these molecules, in silico study was performed and most active groups of les-3120 and les-3372 were combined into one molecule. in vitro studies showed that such hybrid molecule called les-3661 possessed 10 times higher anticancer potential (ic 50 =1 lm) comparing with initial compounds. we report on the synthesis and characterization of vaterite microcontainers for controlled drug release. moreover, we present experiments on possible release strategies of encapsulated substances via recrystallization, ph controlled, or by desorption methods. vaterite spherical particles were fabricated with controllable average sizes from 400±12nm till 10±1hm. we considered two ways of functionalization of the containers: encapsulation of the substances during the vaterite synthesis or their adsorption onto the prepared particles. as model experiments, vaterite containers, encapsulating rhodamine 6g, were imaged by two-photon microscopy, showing dye release into the aqueous medium due to recrystallization to calcite within 3 days. differently, in ethanol only small amounts of the encapsulated markers were diffusion released after one week. the release mechanisms can be further controlled by covering the microcontainers with additional polymer layers to increase diffusion and recrystallization time. a change of the ph from neutral to acid conditions leads to the destruction of the vaterite matrix followed by a quick release of the encapsulated materials. these flexible control mechanisms make this system an interesting candidate for pharmaceutical applications. magnetic nanoparticles (np) in combination with therapeutic molecules represent one of most promising methods for targeted drug delivery. one of major current limitations of magnetic drug targeting is to achieve efficient concentration of magnetic carrier-drug complexes at the targeted sites due to poor mobility of nanoparticles in tissue structures. interstitial delivery is hindered by microscopic extracellular matrix, which represents a major barrier for nanoparticles motilities. in order to achieve efficient magnetic drug targeting it is crucial to know particle mobility in a given in vivo environment as well as to apply magnetic field having appropriate field gradient which drags magnetic nps. we used gel magnetophoresis in order to measure motilities of different magnetic nps (co-ferrite, c-fe 2 o 3 ) in agarose gel. numerical modeling using fem method was used to determine appropriate settings of magnets, which generate sufficient magnetic field gradient. further, we used the numerical modeling to evaluate the magnetic force on the nps for different geometries. we obtained that one of crucial factors which determines final mobility in tissue is formation of larger aggregates of nanoparticles under physiological conditions and interaction of nanoparticles with surrounding matrix. defining the forces required to gate mechanosensitive channels in mammalian sensory neurons kate poole and gary lewin department of neuroscience, max delbrueck center for molecular medicine, robert-roessle str 10, 13125, berlin-buch, germany our sense of touch and mechanical pain is based on mechano-electrical transduction (met) at the terminal endings of subsets of dorsal root ganglion (drg) neurons innervating the skin. to quantify the stimulus strengths required to gate mechanosensitive channels in these subsets of neurons, we developed an approach using microstructured surfaces. the drg neurons are grown on laminin-coated pdms pillar arrays, mechanical stimuli are applied by deflecting individual pili and the deflection is monitored using light microscopy. as the pili behave as light-guides, the center of each pilus can be determined from a fit of the intensity values, allowing detection of movements of a few nanometers. the response to such stimuli is monitored using whole-cell patch-clamp. pili deflections of 10nm can gate the rapidly adapting-current in mechanoreceptor cells, while deflections above 150nm are required for gating of slowly adapting-currents in nociceptors. smaller stimuli are required to generate currents via pili deflection (10nm) vs neurite indentation (70-100nm), suggesting that gating occurs at the cell-substrate interface. we have also characterized the met currents present in n2a cells which we show are modulated by the substrate to which the cells are attached. enhanced stimulation of toll-like receptor 9 via immunostimulatory nanoparticles jan rother, anna pietuch, andreas janshoff georg-august university gö ttingen, institute of physical chemistry, tammanstr. 6, d-37077 gö ttingen, germany e-mail: jrother@gwdg.de among the toll-like receptor family (tlrs), the tlr9 has been subject of intensive research because of its predominant localization in the lysosomes of immune cells and its ligand rendering it a potential candidate for immunotherapy of autoimmune diseases and cancer. additionally, a use as an adjuvant in vaccination is aimed using synthetic cpg-oligodeoxynucleotides (cpg-odn's). albeit, immunostimulatory cpg-odns already showed promising results in animal experiments and clinical trials, several groups found that tlr9 is also expressed by tumor cells. first experiments show that activation of tlr9 displayed by cancerous cells leads to a decreased apoptosis rate and proliferation posing unpredictable threat to tumor patients exposed to cpg-odns. therefore, detailed knowledge about the impact of cpg-odns on cancer cells is inevitable for a save use in pharmaceutics. herein, we describe a sophisticated way to address tlr9 in cancer cells using cpg-odn functionalized ''superparamagnetic'' mno-and c-fe 2 o 3 -nanoparticles (nps) to stimulate tlr9 in a549 cells. analysis of impedimetric measurements revealed a cytotoxic effect of the mno-nps. cells treated with immunostimulatory fe 2 o 3 -nps showed an increased micromotility as well as a higher long-term correleation of the impedance signal. biomedical diagnostics like high-sensitivity, single-molecule study, easy sample preparation. furthermore, sers allows to conduct non-invasive studies of conformations of the molecules without destruction of living cells, i.e. in vivo [1] . this work presents a sers study of cytosolic hemoglobin (hb c ) using silver nanoparticles (agnps). the hb c was isolated from cytoplasm of red blood cells taken from rat erythrocytes and diluted. agnps were prepared by developing leopold and lendl method [2] . three types of colloids were prepared at various temperatures (25, 40 and 60°c). the resulted agnps were characterized by uv-vis-, ftirspectroscopy, dls and tem. reduction of ag ions leads to the formation of predominantly spherical agnps but also silver nanorods, faceted and aggregated agnps in small quantities with a surface plasmon resonance band in the range of 413 -445 nm. for agnps synthesized at 25°c, for example, a bimodal size distribution was observed (about 7 and 45 nm medium sizes, respectively). sers measurements were optimized for each type of agnps. it was demonstrated that agnps gave strong raman enhancement from hb c and types of sers spectra differ from each other. nano-zno is characterized by unique properties, low toxicity and high biocompatibility instead of a lot of others nanomaterials. for this fact nanoparticles of zno have great potential for applications in biosystems, for example biolabeling, biosensoring, delivery systems and others, which can be used in genetics, pathology, criminology, safety of food and many other industries. for these bioapplications are necessary surface modifications, which can made to the nanostructures to better suit their integration with biological systems, leading to such interesting properties as enhanced aqueous solubility, bio-recognition or applicability for biological systems. for synthesis of zno nanoparticles in aqueous solution we used 11-mercapto-undecanoic acid (mua) as stabilizing agent. the coating of nanoparticles with mua could allow their solubility in the water and the binding through carboxyl groups present in its structure. we defined the optimal ph for mua modificated nano-zno solubility and their ability interaction with positive charges. we studied the optical properties of pure and surface modificated nanoparticles and their conjugates with cytochrome c and also the effect of ph on the interaction between nano-mua and horse cytochrome c. the permeation of water soluble molecules across cell membranes is controlled by channel forming proteins and particularly the channel surface determines the selectivity. an adequate method to study properties of these channels is electrophysiology and in particular analyzing the ion current fluctuation in the presence of permeating solutes provides information on possible interactions with the channel surface. as the binding of antibiotic molecules in the channels of interest is significantly weaker than that of preferentially diffusing nutrients in substrate-specific pores, the resolution of conductance measurements has to be significantly increased to be able to resolve the events in all cases. due to the limited time resolution, fast permeation events are not visible. here we demonstrate that miniaturization of the lipid bilayer; varying the temperature or changing the solvent may enhance the resolution. although electrophysiology is considered as a single molecule technique, it does not provide atomic resolution. molecular details of solute permeation can be revealed by combining electrophysiology and all atom computer modeling. [ novel functionalized nanocomposites (nc) were designed and synthesized on the basis of polymeric surface-active oligoelectrolytes. the developed technology permits controlling: 1) quality and quantity of structural blocks of nc, and size unimodality; 2) branching at specific sites in polymer chain of nc; 3) providing nc with reactive chemical groups; 4) covalent conjugation of specific bio-targeting molecules. provided bioactive elements were: a) specific anticancer drugs, antibiotics, alkaloids; b) dna and sirna; c) immunoglobulins and lectins; d) lipids and amino acids; e) polyethylene glycol. fluorescent, luminescent, super-paramagnetic, or x-ray detectable compounds were also incorporated in nc to make them detectable and measurable. biocompatible nc possessing low toxicity towards mammalian cells in vitro and in vivo (mice) were created. they were effective in delivery of: 1) drugs (doxorubicine and antibiotics) for chemotherapy in vitro and in vivo; 2) dna for transfection of mammalian, yeast and bacterial cells; 3) protein antigens for animal immunization and specific lectins for targeting apoptotic cells. these and other approaches in application of developed nc and nanobiotechnologies are considered. this work was supported by stcu grants #1930, #4140, #4953. in the anti-cancer drug delivery domain, nanotechnologies are a promising tool, providing a good tissue distribution and a low toxicity. drug delivery vehicles relying on solid nanoparticles have been proposed, among which diamond nanoparticle (size\20 nm) is a very promising candidate [1] . we have investigated the delivery of sirna by nanodiamonds (nd) into cells in culture, in the context of the treatment of a rare child bone cancer (ewing sarcoma), by such a gene therapy. sirna was bound to nds after nds coating by cationic polymers, so that the interaction is strong enough to pass the cell membrane without loss of the drug and does not prevent its subsequent release. the cellular studies showed a specific inhibition of the gene expression at the mrna and protein level by the nd vectorized sirna. we also uses the fluorescence of color center created in the nanodiamonds [2] to monitor the release of fluorescently-labeled sirna in the intracellular medium. this technique brings a quantitative insight in the efficiency of sirna to stop cell proliferation. considering the success of the cell model we recently started the drug delivery in tumor xenografted on nude mice. silica nanoparticles are stable aqueous suspension of condensed siloxane nanocomposites, having an average diameter between 10 and 100 nm. particles containing organic functional groups on their surface are called organically modified silica nanoparticles (ormosil). due to the various chemical and physical properties of the surface groups, ormosil nanoparticles may have an enormous variety of biological applications, such as in vivo bioimaging, non-viral gene delivery or targeted drug delivery. our aim was to synthesize both void and fluorescent dye doped amino functionalized ormosil nanoparticles through the microemulsion method and use them for gene delivery. the obtained nanoparticles have been characterized by transmission electron microscopy and dynamic light scattering. furthermore, the nanoparticles have been investigated to exploit their transfection efficiency and the possible toxicity caused by surfactants used in the synthesis. the transfection efficiency was tested on various cell cultures. our further aim is the in vivo transfection of salivary glands using ormosil nanoparticles. our work has shown that the nanomedicine approach, with nanoparticles acting as a dna-delivery tool is a promising direction for targeted gene therapy. in vivo amperoetric cells for detection of fast diffusing, physiologically important small molecules lívia nagy, bernadett bareith, tü nde angyal, erika pinté r, gé za nagy university of pé cs, pé cs, hungary h 2 s is a naturally occurring gas that is toxic in high concentration. it exists also in different tissues of living animals sometimes in concentrations as high as 20 lm. it is generally accepted, that h 2 s has important roles in modulating different, physiologically important biochemical processes similarly to other, fast diffusing molecules like no, co and h 2 o 2 . for investigation of the physiological effects of these species their local concentration in the studied biological media is important to know. this means methods needed for measuring the instantaneous concentration with high spatial resolution in living tissues without major invasion. electrometric micro, and ultramicro sensors are often gain application in experimental life sciences for measurement of local ion concentration or following neurotransmitter species in vivo measurements. in our work efforts are being carried out to improve the applicability of selective electrometric sensors in life science experiments. as a result of these work an improved h 2 s measuring cell and improved electrode and method was developed for measurement of electroactive small molecules like no or h 2 o 2 . in the poster to be presented the structure, the working principles and the performances of the different sensors mentioned will be described. bacteriorhodopsin (br) is the only protein in the purple membrane of the halophilic organism halobacterium salinarium. it is a light-driven proton pump converting light into a transmembrane proton gradient through isomerization of the covently bound retinal chromophore. its stability, as well as its photoactivity in dried films, has made br an attractive material for biomolecular devices. such studies, however, have used br within the membrane, on relatively large surfaces. here, conducting-probe atomic force microscopy (c-afm) analysis was performed after isolating the protein from its native membrane environment while keeping its basic trimeric structure, and demonstrated that the molecular conductance of br can be reversibly photoswitched with predictable wavelength sensitivity. intimate and robust coupling to gold electrodes was achieved by using a strategically engineered cysteine mutant located on the intracellular side of the protein which, combined with a 75% delipidation, generated protein trimers homogenously orientated on the surface. c-afm proximal probe analysis showed a reproducible 3 fold drop of br mean resistance over *5 cycles of interspersed illuminations at the same gold-br-gold junction when k[495 nm, while no shift was observed with other wavelengths. capture of circulating tumor cells with a highly efficient nanostructured silicon substrates with integrated chaotic micromixers shutao wang 1 ). this core technology shows significantly improved sensitivity in detecting rare ctcs from whole blood, thus provides an alternative for monitoring cancer progression. by assembling a capture-agent-coated nanostructured substrate with a microfluidic chaotic mixer, this integrated microchip can be applied to isolate ctcs from whole blood with superb efficiency. ultimately, the application of this approach will open up opportunities for early detection of cancer metastasis and for isolation of rare populations of cells that cannot feasibly be done using existing technologies. this technology helped to find a needle in a haystack and will open up the opportunity for single cell genomic and epigenetic sequencing and gene expression profiling. results from further development of this technology will assist the physicians in follow-up patients and testing vigorously the concept of personalized oncology with individualized therapy. this novel technology has recently been reviewed and highlighted by nature medicine the growing crisis in organ transplantation and the aging population have driven a search for new and alternative therapies by using advanced bioengineering methods. the formation of organized and functional tissues is a very complex task: the cellular environment requires suitable physiological conditions that, presently, can be achieved and maintained by using properly-designed bioreactors reproducing all specific functions and bioactive factors that assure viability/regeneration of cells cultured in an appropriate scaffold. the creation of biomimetic environment requires the use of biomaterials such as membranes with specific physico-chemical, morphological and transport properties on the basis of the targeted tissue or organ. tailor-made membranes (organic, functionalized with specific biomolecules, in hollow-fiber configuration), designed and operated according to well-defined engineering criteria are able to sustain specific biotransformations, to provide adequate transport of oxygen, nutrients and catabolites throughout the cellular compartment, and to supply appropriate biomechanical stimuli of the developing tissue. in this talk the author will show the development of membrane engineered constructs focusing on liver and neuronal systems. the role of membrane surface and transport properties in providing instructive signals to the cells for the guiding of proliferation and differentiation will be discussed. membrane bioreactors, which through the fluid dynamics modulation may simulate the in vivo complex physiological environment ensuring an adequate mass transfer of nutrients and metabolites and the molecular and mechanical regulatory signals, will be presented. here we present a novel but simple system for cell-based assays enabling simultaneous testing of multiple samples on a same tissue without cross-contamination between neighbouring assays, as well as sequenced or repeated assays at the same tissue location. the principle of this method lies in the spatially-controlled diffusion of test compounds through a porous matrix to the target cells. a simple microfabrication technology was used to define areas where diffusion processes are allowed or inhibited. we performed proof-of-principle experiments on madin-darby canine kidney (mdck) epithelial cells using hoechst nuclear staining and calcein-am cell viability assay. fluorescent staining superimposed properly on membrane pattern with a dose-dependent response, indicating that both compounds specifically and selectively diffused to the target cells. mdck cells similarly treated with cytochalasin b showed their actin network rapidly altered, thus demonstrating the suitability of this system for drug screening applications. such a well-less cell-based screening system enabling multiple compounds testing on a same tissue and requiring very small volumes of test samples appears interesting for studying potential combined effects of different biochemicals applied separately or sequentially. it is generally believed that all-optical data processing is the most promising direction to achieve serious improvements both in capacity and speed of internet data traffic. one of the bottlenecks of the state-of-the-art photonic integration technology is to find the proper nonlinear optical (nlo) materials that are supposed to serve as cladding media in waveguidebased integrated optical circuits performing light-controlled active functions. recently, the unique chromoprotein bacteriorhodopsin (br) has been proposed to be used as an active, programmable nlo material in all-optical integrated circuits. in integrated optical applications of br, its light-induced refractive index change is utilized. in this paper we exploit the refractive index changes of a dried br film accompanying the ultrafast transitions to intermediates i and k, which allows even sub-ps switching, leading beyond tbit/s communication rate. in the experiments direct pulses of a femtosecond laser system at 800 nm were used along with synchronized ultrafast laser pulses at 530 nm. we believe that the results may be the basis for the future realization of a protein-based integrated optical device, and represent the first steps to a conceptual paradigm change in optical communication technologies. last years, such autoantibodies attract an increasing attention of researchers as potential cancer biomarkers. since the sera of cancer patients typically contain a unique set of antibodies that reflect the tumor-associated antigens expressed in a particular malignant tissue, diagnosing and predicting the outcome of disease such as breast cancer based on serum autoantibody profiling is an attractive concept. to create a representative panel of antigens for detecting of breast cancer autoantibody profile we selected 18 breast cancer associated antigens. these antigens were identified by screening of tumor cdna libraries with autologous sera using serex (serological investigation of recombinantly expressed clones) approach. all antigens were cloned, expressed, purified in bacteria and tested with sera of breast cancer patients and healthy donors in large-scale allogenic screening using elisa. the utility of selected tumor associated antigens for detecting of autoantibody profile in different types of breast cancer was evaluated. (controlled by architectural software) is carried out according to a design template, consistent with the geometry and composition of the desired organ module. structure formation occurs by the post-printing fusion of the discrete bio-ink units. when the bio-ink units contain more than one cell type, fusion is accompanied by sorting of the cells into the physiologically relevant pattern. thus structure formation takes place through self-assembly processes akin to those utilized in early embryonic morphogenesis. we demonstrate the technology by detailing the construction of vascular and nerve grafts. spherical and cylindrical bio-ink units have been employed to build fully biological linear and branching vascular tubular conduits and multiluminal nerve grafts. upon perfusion in a bioreactor the constructs achieved desirable biomechanical and biochemical properties that allowed implantation into animal models. our results show that the printing of conveniently prepared cellular units is feasible and may represent a promising tissue and organ engineering technology. femtosecond lasers have become important tools for noncontact microprocessing of biological specimens. due to the short pulse length and intensity-dependent nature of the multiphoton ionization process, fs-laser pulses affect only a small volume of a treated cell, providing a high degree of spatial localization. we employed fs-laser to address topical bioengineering and biomedical problems such as cell fusion and embryo biopsy respectively. a tightly focused laser beam (cr:f seed oscillator and a regenerative amplifier, 620nm, 100 fs, 10 hz) was used for a fusion of blastomeres of two-cell mouse embryos and for a polar body (pb) biopsy. in order to fuse blastomeres the contact border of cells was perforated by a single laser pulse. the fusion process usually completed within *60 min. in order to perform a noncontact laser based pb biopsy we initially drilled an opening in the zona pellucida with a set of laser pulses, and then extracted the pb out of zygote by means of optical tweezer (cw laser, 1064 nm). the energy of laser pulses was thoroughly optimized to prevent cell damage and increase the fusion and biopsy rates. the proposed techniques demonstrate high efficiency and selectivity and show a great potential for using fs lasers as a microsurgical tool. new insights into mechanisms of electric field mediated gene delivery maš a kanduš er and mojca pavlin university of ljubljana, faculty of electrical engineering, si-1000 ljubljana, slovenia gene electrotransfer is widely used for transfer of genetic material in biological cells by local application of electric pulses and is currently the most promising non-viral delivery method for gene therapy for a series of diseases as well as for dna vaccination. current description of the process defines several steps: electropermeabilization, dna-membrane interaction, translocation, trafficking to nucleus and into nucleus. but the mechanisms of electrotransfer are still not fully understood. we present results of the systematic in vitro analysis using pegfp of all steps involved in electrotransfection from electropermeabilization, analysis of different pulsing protocols, theoretical analysis of plasmid mobility to visualization of the processes of dna-membrane interaction. we demonstrate that in order to translate in vitro results to tissue level sub-optimal plasmid concentrations have to be used. furthermore, sofar the method of dna entry into cytoplasm was only speculated. our results suggest that it is crucial that first, membrane is electropermeabilized, then sufficient electrophoretic force is crucial for insertion of dna into destabilized lipid bilayer followed by dna translocation into cytoplasm via a slow process. efficiency of electrotransfer depends also on the stage of of cell culture -cells in dividing phase are easer to electrotransfect. gentamicin interaction with b16f10 cell membrane studied by dielectrophoresis dielectrophoresis (dep) is the translational motion of polarizable particles due to an electric field gradient. positive-dep and negative-dep correspond to particle movement forward or backward the region of high field intensity, respectively. our study reveals some of the cell membrane modifications induced by gentamicin (gt), as they are reflected in the crossover frequency f co of b16f10 murine cells incubated with gt for different concentrations and durations. f co is the ac frequency when cells turn from positive-dep to negative-dep. gentamicin is a positively charged aminoglycosidic antibiotic, with concentration-dependent killing action; it is widely used because of its low cost and reliable bactericidal activity. gt drawbacks consist in high toxicity for renal and hearing cells; the molecular mechanisms of this toxicity are still unclear. for low external medium conductivities (& 0.0012 s/m), f co of control and gt-cells was found to range from 3 to 10khz. f co shifts to higher frequencies with the increase of gt concentration and incubation time. cells dielectrophoretic behavior is discussed using the cell singleshell based model. extracellular matrix (ecm) is a major obstacle for succesful delivery of genes. chitosan is a versatile and biocompatible polysaccharide derived from chitin and is a promising gene carrier. chitosan-dna interactions, and hence dna polyplexation and release can be controlled through chitosan de-acetylation degree, molecular weight and functionalization of chitosan cationic groups. grafting of poly(ethylene glycol) peg to gene delivery vectors increases circulation time of gene delivery systems in blood vessels and reduces polyplexes charge. diffusion and unpacking of pegylated and non-pegylated chitosan-dna polyplexes through articial ecms based on collagen and collagen-hyaluronic acid (ha) gels were compared using fluorescence correlation microscopy, confocal microscopy and colocalization analysis. non-pegylated polyplexes were immobilized in the gels whereas pegylated polyplexes were diffusing. the smaller charge of pegylated polyplexes seems to decrease interactions between polyplexes and ecm components. furthermore, ha might also screen collagen fibers-pegylated polyplexes interactions. pegylated polyplexes also showed a higher degree of unpacking in gels, probably due to a looser compaction of dna by pegylated chitosan compared to non-pegylated chitosan. fabrication of vesicles, a close membrane made of an amphiphile bilayer, has great potentiality for encapsulation and controlled release in chemical, food or biomedical industries but also from a more fundamental point of view for the design of biomimetic objects. methods based on lipid film hydratation 1 , inverse emulsion techniques 2 and more recently microfluidic techniques such as double emulsion 3 or jetting 4 method are limited either by a low yield, a low reproducibility, a poor control on the size, or by the presence of remaining solvent or defects. we propose a fast and robust method 5 easy to implement: continuous droplet interface crossing encapsulation (cdice), that allows the production of defect-free vesicles at high-yield with a control in size and content. the vesicles have controlled bilayer composition with a polydispersity in size lower than 11%. we have shown that solutions as diverse as actin, cells, micrometric colloids, protein and high ionic strength solutions can easily be encapsulated using this process. by adjusting the parameters of our set-up, we are able to produce vesicles in the range 4-100 lm in diameter, stable for weeks. we believe this method open new perspectives for the design of biomimetic systems and even artificial tissues. under appropriate conditions. the extremely variable d3 domain of flagellin subunits, comprising residues 190-283, protrudes at the outer surface of flagellar filaments. the d3 domain has no significant role in the construction of the filament structure. thus, replacement of d3 may offer a promising approach for insertion of heterologous proteins or domains without disturbing the self-assembly of flagellin subunits. our work aims at the construction of flagellinbased fusion proteins which preserve the polymerization ability of flagellin and maintain the functional properties of the fusion partner as well. in this work a fusion construct of flagellin and the superfolder mutant of green fluorescent protein (gfp) was created. the obtained gfp variant was highly fluorescent and capable of forming filamentous assemblies. our results imply that other proteins (enzymes, binding domains etc.) can also be endowed by polymerization ability in a similar way. this approach opens up the way for construction of multifunctional filamentous nanostructures. generation 5 polyamidoamine (pamam) dendrimer has been shown to be highly efficient nonviral carriers in gene delivery. however, their toxicity limits their applications. in this study, to improve their characteristics as gene delivery carriers, g5 pamam dendrimer was modified with anti-tag72 nanobody through hetrobifunctional peg, then complexed with t-bid coding pdna, yielding pamam-peg-anti-tag72 nanobody/pdna nanoparticles (nps). nuclear magnetic resonance (nmr) spectroscopy, zeta sizing and gel retardation assay results provided evidence that the nanovector was successfully constructed. the transfection efficiency of vector/pdna complexes were evaluated in vitro. real time pcr results also demonstrated that anti-tag72 nanobody modified nps are more efficient in t-bid killer gene expressing in colon cancer cell line than the unmodified nps. in conclusion, pamam-peg-anti-tag72 nanobody showed great potential to be applied in designing tumour-targeting gene delivery system. 3 dept of chemistry, faculty of science, national university of singapore, singapore, 4 dept of biochemistry, yong loo lin school of medicine, national university of singapore, singapore, 5 division of bioengineering, faculty of engineering, national university of singapore, singapore macromolecular crowding (mmc) is a biophysical tool which has been used extensively to enhance chemical reactions and biological processes by means of the excluded volume effect (eve). the in vivo stem cell microenvironment contains macromolecules which are crucial for stem cell selfrenewal and cell fate determination. in order to mimic this physiological microenvironment, crowders are included in cell culture medium. we have observed that the ex vivo differentiation of human mesenchymal stem cells (hmscs) into the adipogenic lineage is significantly amplified when a crowder mixture comprising ficoll 70 and ficoll 400 is added to the culture medium. stem cell differentiation is modulated by soluble chemical substances as well as interactions between cells and the extracellular matrix (ecm), and both these external influences may be affected by mmc. measurements we have performed by fluorescence correlation spectroscopy (fcs) show that ficoll additives cause anomalous subdiffusion within a crowder concentration range of 0 to 300 mg/ml. the diffusion of fluorophorelabelled molecules in artificial lipid bilayers and membranes of living cells is not changed by crowders, suggesting that these crowders do not directly alter membrane properties and cell surface signalling. however, we have data to suggest that crowders increase actin polymerization reaction rates in vitro. we have also observed that crowders are taken up by stem cells and that they localize to specific compartments. based upon our observations, we hypothesize that crowders can influence stem cell differentiation by influencing molecular kinetics. lignocellulose-based composites are becoming extremely important and perspective sustainable and renewable natural materials. fibre modification enhancing their existing properties can be obtained to broaden the application areas. in response to shortcomings of traditional chemical and physical methods, enzymes and chemo-enzymatic methods have emerged as eco-friendly catalysts working under mild conditions and enable tailoring of the material surface properties by substrate specificity and regional selectivity. recently, binding of different functional molecules to lignin-rich fibres by using an oxidative enzyme (e.g. laccase) has been reported leading to their functionalisation through free radical reactions. by the application of electron paramagnetic resonance spectroscopy (epr) laccase action was inspected. consumption of substrates was investigated and their polymerization traced. stable radical intermediates were detected with epr when substrate molecules were in contact with active enzymes. secondly, oxidation of mediators like nitroxides was determined via epr spectroscopy of stable water-soluble nitroxide radicals. finally, the generation of short-lived radicals as well as their reduction was measured via epr spin trapping using dmpo as sensitive water soluble spin trap. mammalian ovary hormone stimulation (ohs) is known to be an inalienable stage of reproductive biotechnology as well as human infertility treatment. the basic aim of the ohs is to receive a stock of valuable oocytes and early embryos for subsequent utilization in the reproductive technology, experimental work et al. however, it is known that ohs itself affects the character of ovulation and oocyte quality, which in its turn affects the development of embryos and even has distant consequences. the wideness of cell parameters and appropriate methods for investigation of gamete/embryo quality are very important. the aim of this study is determination of specific electric conductivity of mouse oocytes and early embryos which have been received after ohs in comparison with the ones that have been received in natural animal sex cycle. using techniques of electroporation the dependence of specific electric conductivity of mouse oocytes, zygotes, 2-cell and 8-cell embryos on the external electric field intensity has been studied. it is shown that the whole pool of oocytes that were obtained in the result of ohs consists of two groups of oocytes that don't differ from each other morphologically, but differ by their electric parameters and resistance to electric breakdown. at the zygote stage, dividing of embryos into two groups is preserved, but is less expressed. at the stage of 2-cell and 8-cell dividing of embryos into two groups on their electric conductivity disappeared but certain scattering of the parameters due to individual embryo peculiarities is observed. the obtained data show that ohs may lead to latent changes of oocyte state that in their turn affect embryo quality. many microbes synthesize and accumulate granules of polyhydroxyalkanoates (pha, biodegradable storage materials alternative to traditional plastics), which help them survive under stresses. in particular, the plant-growth-promoting rhizobacterium azospirillum brasilense, that is under investigation worldwide owing to its agricultural and biotechnological significance, can produce poly-3hydroxybutyrate (phb) [1] . in our work, phb synthesis in a. brasilense cells was studied under various stresses using diffuse reflectance ftir spectroscopy. phb in cells was determined from the band intensity ratio of the polyester m(c=o) at *1740 cm -1 to that of cell proteins (amide ii band at *1550 cm -1 ), showing a. brasilense to be able to produce phb up to over 60% of cells' dry weight. stresses induced phb accumulation, enhancing ir absorption in phb specific regions. analysis of a few structure-sensitive phb vibration bands revealed changes in the degree of intracellular phb crystallinity (related to its enzymatic digestion rate) at different stages of bacterial growth, reflecting a novel trait of the bacterial adaptability to an enhancing stress, which is of great importance to agricultural biotechnology. the aim of this work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. the d3 domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. d3 is not essential for filament formation. the concept in this project is to replace the d3 domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. to test the feasibility of this approach, xylanase a (xyna) from b. subtilis was chosen as a model enzyme for insertion. with the help of genetic engineering, a fusion construct was created in which the d3 domain was replaced by xyna. the flic(xyna) chimera exhibited catalytic activity as well as polymerization ability. these results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created ( table 1) . the support of the hungarian national office for research and technology and the hungarian scientific research fund (otka) (grants ck77819, nk77978, nanoflag) is acknowledged. cluster phases of membrane proteins an alternative scenario for the formation of specialized protein nano-domains (cluster phases) in biomembranes dna fragmentation induced in human fibroblasts by accelerated 56 fe ions of differing energies swift heavy ion irradiation of srtio 3 under grazing incidence'' proc. natl. acad. sci. usa 105 forespore engulfment mediated by a ratchet-like mechanism a channel connecting the mother cell and forespore during bacterial endospore formation a feeding tube model for activation of a cell-specific transcription factor during sporulation in bacillus subtilis the scanning ion-conductance microscope imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy nanoscale live-cell imaging using hopping probe ion conductance microscopy beta2-adrenergic receptor redistribution in heart failure changes camp compartmentation simultaneous noncontact topography and electrochemical imaging by secm/sicm featuring ion current feedback regulation timasheff in protein-solvent interactions the roles of water in foods on motor and electrical oscillations in urinary tract: computer evaluation daniele martin 1,2 , viktor foltin 1,2 , erich gornik 2 , rumen stainov 3,4 , tanya zlateva 4 nü rnberg. icsd e.v. postfach (pob) 340316, d-80100 mü nchen method: parameters: motor patterns (guinea-pig) -frequency/f, amplitudes/a (% init. length, isot. & intracell. rec.) of spontaneous phasic/spc & tonic/stc contractions, also electrical spikes/s, bursts/b, burst plateaus/bp (neu et al. biophys.j. 125/6a/jan2008 stretch (3-80mn), k-/ca-influence induced specific changes in motor/electrical parameters. special computer programme reflects exactly biophysical parameters. conclusion: acc. to earlier/recent results mechano-sensitive ca ?? -activated k ? -channels participate in electrical oscillations of detrusor/ureteral myocytes. further experiments/evaluations incl effect of hydrophobic mismatch on the light-induced structural changes in bacterial reaction centers s. s. deshmukh, h. akhavein, and lá szló ká lmá n department of physics mechanism of proton transfer in nitric oxide reductase: computational study andrei pisliakov riken advanced science institute, wako-shi proteins 74, 266. acknowledgments this work was supported by project grant ptdc/qui/70182/ 2006 (cas) and doctoral grants sfrh th anniversary conference aicr this work was supported by the italian association for cancer research (airc), the istituto toscano tumori and the associazione noi per voi differential hydration, void volume: which factor provides the main contribution to dv u ? inserm umr 1054 fr; roumestand@cbs.cnrs.fr introduction: globalization needs new organizational models also for biophysics. reports on necessity of int. institutes for biophysics (iib) c/o int. universities (proposed by british nobel laureate b.russell) are given conception: proposals for ebsa-discussion: 1. enlargement of executive committee by a. honorary & presidents (permanent 1-3: moral support & 1-3 fixed term), b. interdisciplinary commission: scientists from biology, medicine, physics, etc. (feps/iups, iuphar, iupab, etc). 2. implication of interdisciplinary topics to esba/iupab congressprogrammes, 3. also for biophysical journals. 4. organization of common interdisciplinary sessions not only to biophysical, but also to other congresses. 5. co-operation between esba/iupab with int. interdisciplinary organisations (waas, icsd/ias, eur. academies) for creation of iib by network of national ones: successive common personnel, possibility for whole life work, etc. conclusion: realization of proposals 1.-5. could increase scientific/political authority of ebsa/iupab, leading to model for renewal of scientific organizations collective migration of neural crest cells: a balance between repulsion and attraction roberto mayor university college london goodilin 2,3 , olga v single-molecule cut-and-paste surface assembly (smcp) has been also used to build up a biotin scaffold that streptavidin utilizing specific molecular interactions, for example between dna-binding proteins and dna or antibodies and antigens, this technique is capable of providing a scaffold for the controlled self-assembly of functional complexes. furthermore, this allows for the introduction of smcp into protein science. we aim to employ dna-binding zinc-finger variants and gfp-binding nanobodies as shuttle-tags fused to the proteins of interest. thus a fully expressible system that can be used for the step-wise assembly of individual building blocks to form single-molecule cut-and-paste surface assembly optically monitoring the mechanical assembly of single molecules nanoparticle self-assembly on a dna-scaffold written by single-molecule cut-and-paste torsional motion analysis of group ii chaperonin using diffracted x-ray tracking nanomechanical manipulation of mason-pfizer monkey retroviral rna fragment with optical tweezers melinda simon 1 , zsolt má rtonfalvi 1 , pasquale bianco 1 , beá ta vé rtessy 2 , mikló s kellermayer 1 micro-viscosimeter generated and manipulated by light andrá s buzá s 1 , lá szló oroszi 1 , ló rá nd kelemen 1 , pá l ormos 1 temesvá ri krt proc. natl. acad. sci. usa 105 neural signal recordings with a novel multisite silicon probe gergely má rton 1 , anita pongrá cz 1 , lá szló grand 2,3 , é va vá zsonyi 1 pé ter pá zmá ny catholic university, faculty of information technology, h-1083, 50/a prá ter st multiscale pattern fabrication for life-science applications francesco valle 1 , beatrice chelli 1 , michele bianchi 1 , eva bystrenova 1 , marianna barbalinardo 3 , arian shehu 1 , tobias cramer 1 , mauro murgia 1 , giulia foschi 1 miroslava kuricova 1 , jana tulinska 1 , aurelia liskova 1 , eva neubauerova 1 , maria dusinska 2,1, ladislava wsolova acceleration neuronal precursors differentiation induced by substrate nanotopography gianluca grenci 1 , jelena ban 2 , elisabetta ruaro 4 , massimo tormen 1 , marco lazzarino 1,3 and vincent torre 2 light-induced structural changes are reported near the primary electron donor of bacterial reaction centers (brc) dispersed in detergent micelles and in liposomes from lipids with different fatty acid chain lengths. in this study we present evidence for the correlation between the light-induced increase of the local dielectric constant, determined by the analysis of the electrochromic absorption changes, and the lifetime of the charge-separated state at physiologically relevant temperatures. the increase of the local dielectric constant induced a significant decrease of the oxidation potential of the primary electron donor and a slow proton release, which appears to be the rate limiting step in the overall process. systematic selection of the head group charges of detergents and lipids, as well as the thickness of the fatty acid chains of the liposome forming lipids can increase the lifetime of the charge-separated state by up to 5 orders of magnitude. such extensions of the lifetime of the charge-separated state were reported earlier only at cryogenic temperatures and can provide new opportunities to utilize the brc in energy storage. ontogenesis of photosynthetic bacteria tracked by absorption and fluorescence kinetics m. kis, e. asztalos, p. maró ti department of medical physics and informatics, university of szeged, hungarythe development of photosynthetic membrane of rhodobacter sphaeroides was studied by absorption spectroscopy and fast induction of bacteriochlorophyll fluorescence in different phases of the growth, under various growing conditions (oxygen content, light intensity etc.) and in synchronous cell population. the results are: 1) the newly synthesized components of the membranes were imbedded immediately into the proteinous scaffold independently on the age of the cell (no ,,transient'' membranes were observed). 2) under aerobic conditions, the pigments were bleached and under anaerobic conditions the pigment systems showed greening. the relative variable fluorescence (f v /f max ) had small age-dependent (but not cellcycle-related) changes. the fluorescence induction kinetics was sensitive marker of the aerobiosis: the f v /f max ratio dropped from 0.7 to 0.4 and the photochemical rate constant from 5á10 4 s -1 to 3á10 4 s -1 with an apparent halftime of about 4-5 hours after change from anaerobic to aerobic atmosphere.3) the electrogenic signal (absorption change at 525 nm) reflected the energetization of the membrane which showed cell-cycle dependent changes. that included periodic production and arrangement of protein-lipid components of the membrane synchronized to the cell division. interfacial water in b-casein molecular surfaces: wide-line nmr, relaxation and dsc characterization t. verebé lyi 1 , m. bokor 1 , p. kamasa 1 , p. tompa 2 , k. tompa 1 1 research institute for solid state physics and optics, hungarian academy of sciences, pob. 49, 1525 budapest,hungary, 2 institute of enzymology, biological research center, hungarian academy of sciences, pob. 7, 1518 budapest, hungarywide-line proton nmr fid, echoes, spin-lattice and spin-spin relaxation times were measured at 82.55 mhz frequency in the -70°c to ?40°c temperature range, in lyophilized bcasein and aqueous and buffered solutions, and dsc method were also applied. the motivation for the selection of b-casein is the uncertainty of structural order/disorder. naturally, the nmr and thermal characteristics were also evaluated. the melting of hydration water could be detected well below 0°c and the quantity of mobile water molecules (hydration) was measured. the hydration vs. melting temperature curve has informed us on the bonding character between the protein surfaces and water molecules. the generally used local field fluctuation model and the bpp theory were applied in the interpretation, and the limits of the models were concluded. the torsional properties of dna play an important role in cellular processes such as transcription, replication, and repair. to access these properties, a number of single-molecule techniques such as magnetic tweezers have been developed to apply torque to dna and coil it. i will briefly refer to investigations of dna-protein interactions using these techniques, and describe what has been learnt. i will then focus on the development of novel magnetic techniques that go beyond standard magnetic tweezers, such as the magnetic torque tweezers 1 and the freely-orbiting magnetic tweezers 2 . these approaches allow one to quantify conjugate variables such as twist and torque. for example, the magnetic torque tweezers rely on high-resolution tracking of the position and rotation angle of magnetic particles in a low stiffness angular clamp. we demonstrate the experimental implementation of this technique and the resolution of the angular tracking. subsequently, we employ this technique to measure the torsional stiffness c of both dsdna molecules and reca heteroduplex filaments. lastly, i will describe novel applications of the optical torque wrench 3, 4 . the optical torque wrench is a laser trapping technique developed at cornell capable of applying and directly measuring torque on microscopic birefringent particles via spin momentum transfer. we have focused on the angular dynamics of the trapped birefringent particle 4 , demonstrating its excitability in the vicinity of a critical point. this links the optical torque wrench to non-linear dynamical systems such as neuronal and cardiovascular tissues, non-linear optics and chemical reactions, which all display an excitable binary ('all-or-none') response to input perturbations. based on this dynamical feature, we devise a conceptually novel sensing technique capable of detecting single perturbation events with high signal-to-noise ratio and continuously adjustable sensitivity.for the first time we report a comparative approach based on surface enhanced raman spectroscopy (sers) and raman spectroscopy to study different types of haemoglobin molecules in living erythrocytes. in erythrocytes there are two fractions of haemoglobin: cytosolic (hb c ) and membranebound (hb m ). the concentration of hb m is less then 0,5% and therefore it is impossible to study hb m with traditional optical techniques. modifications of cellular membrane can affect conformation of hb m . therefore, it can be used as a sensitive marker of pathologies. firstly, we investigated enhancement of sers signal of hb m depending on ag nanoparticles' size. we found that the intensity of sers spectra of hb m and enhancement factor increase with the decrease in ag nanoparticles' size. secondly, we investigated the dependence of haemoporphyrin conformation in both hb c and hb m ion ph values. we observed different sensitivity of hb c and hb m to the ph and found that conformational movements of haemoporphyrin (vibrations of pyrrol rings and side radicals) in hb m are sterically hampered comparably with hb c . our observation is an evidence of a benefit of application of surface enhanced raman spectroscopy to investigate properties of the hb m in erythrocytes and provide new information about conformational changes and functional properties of hb m . rna nanotechnology is an emerging field with high potential for nanomedicine applications. however, the prediction of rna three-dimensional nanostructure assembly is still a challenging task that requires a thorough understanding the rules that govern molecular folding on a rough energy landscape. in this work, we present a comprehensive analysis of the free energy landscape of the human mitochondrial trna lys , which possesses two different folded states in addition to the unfolded one. we have quantitatively analyzed the degree of rna tertiary structure stabilization, firstly, for different types of cations, 1 and, secondly, for several naturally-occurring nucleotide modifications in the structural core of the trna lys . 2,3 thus, notable variations in the rna binding specificity was observed for the divalent ions of mg 2? , ca 2? and mn 2? , that can be attributed to their sizes and coordination properties to specific ligands. furthermore, we observed that the presence of m 2 g10 modification together with the principal stabilizing m 1 a9 modification facilitates the rna folding into the biologically functional cloverleaf shape to a larger extent than the sum of individual contributions of these modifications. in order to elucidate the mechanism of the recognition, we used diffracted x-ray tracking method (dxt) that monitors real-time movements of individual proteins in solution at the single-molecule level. we found that peptides move distinctly from i-a k , and the rotational motions of peptides correlate with the type b t cell activation. in the case of diabetogenic i-a g7 , immediately after peptide exchange, all the peptides moves magnificently but the motion ceased in a week, then new ordered motion appears; the rotational motion of peptides correlate to t cell activation, which is analogous to the peptide in i-a k . the rotational motion of peptides may create transient conformation of peptide/mhc that recognized by a population of t cells. dxt measurement of peptide/mhc complex well correlated to other biological phenomenon too. our finding is the first observation that fluctuations at the level of brownian motion affect to the functions of proteins.mason-pfizer monkey virus (mpmv) is an excellent model for the analysis of retrovirus assembly and maturation. however, neither the structure of the viral rna, nor its modulation by capsid-protein binding are exactly known. to explore the structure of the mpmv genome, here we manipulated individual molecules of its packaging signal sequence with optical tweezers. the 207-base-long segment of mpmv rna corresponding to the packaging signal, extended on each side with 1200-base-long indifferent gene segments for use as molecular handles, was cloned into a pet22b vector. rna was synthesized in an in vitro transcription system. rna/ dna handles were obtained by hybridization in a pcr with complementary dna initiated with primers labeled with either digoxigenin or biotin. the complex was manipulated in repetitive stretch and relaxation cycles across a force range of 0-70 pn. during stretch, transition occurred which increased the rna chain length and likely corresponds to unfolding. the length gain associated with the unfolding steps distributed across three main peaks at *13, 20, 32 nm, corresponding to *35, 57, 85 bases, respectively. often reverse transitions were observed during mechanical relaxation, indicating that refolding against force proceeds in a quasi-equilibrium process. structural investigation of gpcr transmembrane signaling by use of nanobodies jan steyaert 1,2 1 structural biology brussels, vrije universiteit brussel, pleinlaan 2, 1050 brussel, belgium, 2 department of structural biology, vib, pleinlaan 2, 1050 brussel, belgiumin 1993, scientists at the vrije universiteit brussel discovered the occurence of bona fide antibodies devoid of light chains in camelidae. the small and rigid recombinant antigen binding fragments (15kd) of these heavy chain only antibodies -known as vhhs or nanobodies -proved to be unique research tools in structural biology. by rigidifying flexible regions and obscuring aggregative surfaces, nanobody complexes warrant conformationally uniform samples that are key to protein structure determination by x-ray crystallography:• nanobodies bind cryptic epitopes and lock proteins in unique native conformations • nbs increase the stability of soluble proteins and solubilized membrane proteins • nbs reduce the conformational complexity of soluble proteins and solubilized membrane proteins • nbs increase the polar surface enabling the growth of diffracting crystals • nbs allow to affinity-trap active protein i will focus my talk on the use of nbs for the structural investigation of gpcr transmembrane signaling to illustrate the power of the nanobody platform for generating diffracting quality crystals of the most challenging targets including gpcrs and their complexes with downstream signaling partners. dynamics of the type i interferon receptor assembly in the plasma membrane stephan wilmes, sara lö chte, oliver beutel, changjiang you, christian paolo richter and jacob piehler university of osnabrü ck, division of biophysics, barbarastrasse 11, 49076 osnabrü ck, germanytype i interferons (ifn) are key cytokines in the innate immune response and play a critical role in host defense. all ifns bind to a shared cell surface receptor comprised of two subunits, ifnar1 and ifnar2. detailed structure-function analysis of ifns has established that the ifn-receptor interaction dynamics plays a critical role for signalling specificities.here we have explored the dynamics of receptor diffusion and ifn assembly in living cells. by using highly specific orthogonal posttranslational labelling approaches combined with tirf-microscopy we probed the spatio-temporal dynamics of receptor diffusion and interaction in the plasma membrane of live cells on the single molecule level. for this purpose, we employed posttranslational labelling with photostable organic fluorophores. this allowed us to map diffusion and lateral distribution of ifnar1 and ifnar2 with very high spatial and temporal resolution by using single particle tracking (spt) and single molecule localization imaging. observed events of ''transient confinement'' and co-localization with the membrane-proximal actin-meshwork suggest partitioning of ifnar1/2 in specialized microcompartments. this will be investigated in terms influence on receptor assembly and recruitment of cytoplasmic effector proteins. cytoplasmic dynein moves through uncoordinated action of the aaa1 ring domains ahmet yildiz department of physics, and department of molecular and cell biology, university of california, berkeley, ca 94720 usa cytoplasmic dynein is a homodimeric aaa? motor that moves processively toward the microtubule minus end. the mechanism by which the two catalytic head domains interact and move relative to each other remains unresolved. by tracking the positions of both heads at nanometer resolution, we found that the heads remain widely separated and move independently along the microtubule, a mechanism different from that of kinesin and myosin. the direction and size of steps vary as a function of interhead separation. dynein processivity is maintained with only one active head, which drags its inactive partner head forward. these results challenge established views of motor processivity and show that dynein is a unique motor that moves without strictly coordinating the mechanochemical cycles of its two heads.o-715 self-controlled monofunctionalization of quantum dots and their applicaitons in studying protein-protein interaction in live cells changjiang you, stephan wilmes, sara loechte, oliver beutel, domenik lisse, christian paolo richter, and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastrasse 11, 49076 osnabrü ck, germanyindividual proteins labeled with semiconductor nanocrystals (quantum dots, qd) greatly facilitate studying protein-protein interactions with ultrahigh spatial and temporal resolution. multiplex single molecule tracking and imaging require monovalent quantum dots (mvqd) capable of orthogonally labeling proteins with high yield. for this purpose, we prepared monovalance qd-trisnta by a chemical conjugation method. our results indicated that monovalent qd-trisnta was obtained in high yield by restricting the coupling by means of electrostatic repulsion. monovalent functionalization of the qd-trisnta was confirmed by assays in vitro and in vivo. two-color qd tracking of interferon receptors ifnar1 and ifnar2 based on mvqd-trisnta were realized on live cell [1] .to broaden the multiplex toolbox of mvqds, we extended the electrostatic-repulsion induced self-control concept for mono-functionalizing quantum dots with different affinity moieties. as a first instance, we used negatively-charged biotin peptide to produce qd with biotin mono-functionalization. we confirmed our approach was a general method to rend qd monovalent by single molecule assays based on stepwise photobleaching. these mvqds facilitate obtaining spatiotemporal information of ifnars' organization in live cells. by orthogonal labeling u5a cells stably expressing ifnar2 at low level with biotin mvqd and mvqd-trisnta-ifn, we verified colocalization and colocomotion of individual ifn and ifnar2 at minute scale. combined with super-resolution imaging of ifnars' cytosolic effecter stat2, we observed the dynamic coming-and-going contact between the microcompartments of ifnar2 and stat2.a micron sized viscometer was fabricated using the couette type geometry that is capable of measuring the complex viscosity of fluids. the viscometer was produced by two photon polymerization of su8 photopolymer using a femtosecond laser system, a high na objective and a piezo translator stage. the viscometer was manipulated by holographic optical tweezers and operated in the 0.005-1 hz frequency range. video analysis algorithm was used to evaluate our measurements. we tested the viscometer with water-glycerol solutions. one of the main reasons for lack of reliability in protein analysis for disease diagnostics or monitoring is a lack of test sensitivity. this is because, for many tests, to be reliable, they need to be performed on a homogeneous, and therefore very small, sample. current in-vitro techniques fail in accurately identifying small differences in protein content, function and interactions starting from samples constituted of few or even single cells. a nanotechnology approach may overcome the current limits in low abundance protein detection. we aim at designing a microwell device for the trapping (in native environment) and the parallel characterization of rare cells (e.g. adult stem cells). such versatile device, based on soft and nanolithography, will promote cell adhesion and viability on differently functionalized bio-compatible materials, allowing for the morphological characterization of the cells, at a single cell level. in parallel, by facing our microwell device with a protein nanoarray, produced via atomic force microscopy nanolithography, we can run proteomic studies at a single/few cells level. moreover, we could foresee the possibility to deliver different stimuli to each cell, correlating the changes in chemistry/ morphology with the protein profile at a single cell level. using an electrophysiological assay the activity of nhaa was tested in a wide ph range from ph 5.0 to 9.5. forward and reverse transport directions were investigated at zero membrane potential using preparations with inside out and right side out oriented transporters with na ? or h ? gradients as the driving force. under symmetrical ph conditions with a na ? gradient for activation, both the wt and the ph-shifted g338s variant exhibit highly symmetrical transport activity with bell shaped ph dependencies, but the optimal ph was shifted 1.8 ph units to the acidic range in the variant. in both strains the ph dependence was associated with a systematic increase of the k m for na ? at acidic ph. under symmetrical na ? concentration with a ph gradient for nhaa activation an unexpected novel characteristic of the antiporter was revealed; rather than being down regulated it remained active even at ph as low as 5. these data allowed to advance a transport mechanism based on competing na ? and h ? binding to a common transport site and to develop a kinetic model quantitatively explaining the experimental results. in support of these results both alkaline ph and na ? induce the conformational change of nhaa associated with nhaa cation translocation as demonstrated here by trypsin digestion. furthermore, na ? translocation was found to be associated with the displacement of a negative charge. in conclusion, the electrophysiological assay allowed to reveal the mechanism of nhaa antiport and sheds new light on the concept of nhaa ph regulation. swimming motility is widespread among bacteria. however, in confined or structured habitats bacteria often come in contact with solid surfaces which has an effect on the swimming characteristics. we used microfabrication technology to quantitatively study the interaction of swimming cells with solid boundaries. we tracked bacteria near surfaces with various engineered topologies, including flat and curved shapes. we were able to study several surface related phenomena such as hydrodynamic trapping and correlated motion. we think that our results may help to understand how physical effects play a role in surface related biological processes involving bacteria such as biofilm formation. cell labeling efficiency of oppositely charged magnetic iron oxide nanoparticles-a comparative study raimo hartmann 1 , christoph schweiger 2 , feng zhang 1 , wolfgang. j. parak 1 , thomas kissel 2,# , pilar rivera_gil 1,# 1 biophotonics, institute of physics, philipps university of marburg, 2 pharmaceutical technology, institute of pharmacy, philipps university of marburg e-mail: kissel@staff.uni-marburg.de; pilar.riveragil@physik.uni-marburg.dethe interaction of nanomaterials with cells is a key factor when considering their translocation into clinical applications. especially an effective accumulation of nanoparticles inside certain tissues is beneficial for a great number of applications. predominantly size, shape and surface charge of nanoparticles influence their cellular internalization and distribution. to investigate this, two series of maghemite (c-fe 2 o 3 ) nanoparticles were synthesized either via aqueous coprecipitation or via thermal decomposition of organometallic precursor molecules. size and the spherical shape of both nanoparticle types were kept constant whereas the charge was changed by modifying the surface of the nanoparticles with polymers of opposite charge, in detail poly(ethylene imine) (pei) and a polymaleic anhydride derivative (pma). the positively and negatively charged c-fe 2 o 3 nanoparticles were characterized with respect to size, zeta potential, colloidal stability and magnetic properties. furthermore, the uptake rate and localization of both formulations into a549 carcinoma cells after fluorescent labeling of the carriers as well as the resulting alteration in mr-relaxation times were evaluated. membrane proteins are the target of more than 50% of all drugs and are encoded by about 30% of the human genome. electrophysiological techniques, like patch-clamp, unravelled many functional aspects of membrane proteins but usually suffer from poor structural sensitivity. we have developed surface enhanced infrared difference absorption spectroscopy (seidas) 1,2 to probe potential-induced structural changes of a protein on the level of a monolayer. a novel concept is introduced to incorporate membrane proteins into solid supported lipid bilayers in an orientated manner via the affinity of the his-tag to the ni-nta terminated gold surface 3 . full functionality of surface-tethered cytochrome c oxidase is demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c. general applicability of the methodological approach is shown by tethering photosystem ii to the gold surface 4 . in conjunction with hydrogenase, the basis is set towards a biomimetic system for h 2 -production. recently, we succeeded to record ir difference spectra of a monolayer of sensory rhodopsin ii under voltage-clamp conditions 5 . this approach opens an avenue towards mechanistic studies of voltage-gated ion channels with unprecedented structural and temporal sensitivity. initial vibrational studies on the novel light-gated channelrhodopsin-2 will be presented 6 . probing biomass-chromatographic bead interactions by afm force spectroscopy gesa helms, marcelo ferná ndez-lahore, rami reddy vennapusa, and jü rgen fritz school of engineering and science, jacobs university bremen, 28759 bremen, germany e-mail: g.helms@jacobs-university.dein expanded bed adsorption (eba), bioproducts are purified from an unclarified fermentation broth by their adsorption on chromatographic beads in a fluidized bed. the unspecific deposition of biomass onto the adsorbent matrix can severely affect the process performance, leading to a poor system hydrodynamics which then decreases the success of this unit operation. to quantify the bead-biomass interactions different chromatographic beads are attached to afm cantilevers, and force spectroscopy experiments are performed with these colloidal probes on model surfaces and cells in solution. the experiments are conducted under varying conditions to study uncovering physiological processes at the cellular level is essential in order to study complex brain mechanisms. using multisite signal recording techniques in the extracellular space, functional connectivity between different brain areas can be revealed. a novel microfabrication process flow, based on the combination of wet chemical etching methods was developed, which yields highly reproducible and mechanically robust silicon-based multielectrode devices. the fabricated shaft of the probe is 280 lm wide, 80 lm thick, has rounded edges and ends in a yacht-bow like, sharp tip. its unique shape provides decreased invasivity. the sensor contains 24 platinum recording sites at precisely defined locations. murine in vivo experiments showed that the probes could easily penetrate the meninges. high quality signals, providing local field potential, multi-and single unit activities, were recorded. the interfaces between the tissue and the platinum contacts were further improved by electrochemical etching and carbon nanotube coating of the metal sites. the integrated optical mach-zehnder interferometer is a highly sensitive device, considered a powerful lab-on-a-chip tool for specific detection of various chemical and biochemical reactions. despite its advantages, there is no commercially available biosensor based on this technique. the main reason is the inherent instability of the device due to slight changes of environmental parameters. in this paper we offer a solution to this problem that enables the optimal adjustment of the working point of the sensor prior to the measurement. the key feature is a control unit made of a thin film of the lightsensitive chromoprotein bacteriorhodopsin deposited on the reference arm of the interferometer. after showing the transfer characteristics of such a device, we demonstrate its applicability to sensing of specific protein-protein interactions. we expect our method to become a rapid and cost-efficientthe combination of unconventional fabrication technology and biomaterials allows both to realize state-of-the-art devices with highly controlled lateral features and performances and to study the main properties of the biomolecules themselves by operating at a scale level comparable with the one crucial for their activity. soft lithography and microfluidic devices offer a tool-box both to study biomolecules under highly confined environments [1] and to fabricate in an easy way topographic features with locally controlled mechanical and chemical surface properties, thus leading to a finer control of the interplay of mechanics and chemistry. i will present an application of this technology to the control of cell fate that is becoming a key issue in regenerative medicine in the perspective of generating novel artificial tissues. patterns of extracellular matrix (ecm) proteins have been fabricated, by a modified lithographically controlled wetting (lcw), on the highly antifouling surface of teflon-af to guide the adhesion, growth and differentiation of neural cells (shsy5y, 1321n1, ne-4c) achieving an extremely accurate guidance [2] . local surface topography is also known to influence the cell fate [3] , thus, integrating this parameter in the substrate fabrication could increase the complexity of the signals supplied to the cells. in this perspective we have developed a novel fabrication technique, named lithographically controlled etching (lce), allowing, in one step, to engrave and to functionalize the substrate surface over different lengthscales and with different functionalities. i will conclude showing how we have been developing ultrathin film organic field effect transistors (ofets) as label-free biological transducers and sensors of biological systems. ofets are low dimensional devices where ordered conjugated molecules act as charge transport material. unconventional patterning techniques and microfluidics have been adapted to proteins and nucleic acids to dose the molecules on the ofet channel with a high control of the concentration. in another set of experiments, we have also been addressing the signalling from neural cells and networks grown on pentacene ultra-thin film transistosr [3, 4] .advances in nanotechnology are beginning to exert a significant impact in medicine. increasing use of nanomaterials in treatment of diseases has raised concerns about their potential risks to human health. in our study, the effect of poly(lactic-co-glycolic acid) (plga) and titanium dioxide (tio 2 ) nanoparticles (nps) on function of b-and t-lymphocytes was investigated in vitro. human blood cultures were treated with plga and tio 2 nps in concentrations: 0.12; 3 and 75 lg/cm 2 for 72h. lymphocyte transformation assay was used to assess the effect of nps on lymphocyte function. lymphocytes were stimulated with mitogens: concanavalin a, phytohaemmagglutinin (t-cell response) and pokeweed mitogen (b-cell response). our findings indicate immunomodulatory effect of plga nps. proliferative response of t-and b-lymphocytes exposed in vitro to the highest dose of plga for 72h was suppressed significantly (p.01, p.05). on the other hand, we observed stimulative effect of exposure to middle dose of plga nps on b-lymphocyte proliferation (p.05). no alteration was found in lymphocyte proliferation treated in vitro with tio 2 nps for 72h. in conclusion, proliferation of lymphocytes in vitro might be one of the relevant tests for evaluation of nps immunotoxicity.embryonic stem (es) cell differentiation in specific cell lineage is still a major challenge in regenerative medicine. differentiation is usually achieved by using biochemical factors (bf) which concentration and sides effects are not completely understood. therefore, we produced patterns in polydimethylsiloxane (pdms) consisting of groove and pillar arrays of sub-micrometric lateral resolution as substrates for cell cultures. we analyzed the effect of different nanostructures on differentiation of es-derived neuronal precursors into neuronal lineage without adding biochemical factors. neuronal precursors adhere on pdms more effectively than on glass coverslips but the elastomeric material itself doesn't enhance neuronal differentiation. nano-pillars increase both precursors differentiation and survival with respect to grooves. we demonstrated that neuronal yield was enhanced by increasing pillars height from 35 to 400 nm. on higher pillar neuronal differentiation reaches *80% 96 hours after plating and the largest differentiation enhancement of pillars over flat pdms was observed during the first 6 hours of culture. we conclude that pdms nanopillars accelerate and increase neuronal differentiation. key: cord-279598-xzionafe authors: chang, chia-yu; cheng, ivan-chen; chang, yen-chen; tsai, pei-shiue; lai, seiu-yu; huang, yu-liang; jeng, chian-ren; pang, victor fei; chang, hui-wen title: identification of neutralizing monoclonal antibodies targeting novel conformational epitopes of the porcine epidemic diarrhoea virus spike protein date: 2019-02-21 journal: sci rep doi: 10.1038/s41598-019-39844-5 sha: doc_id: 279598 cord_uid: xzionafe since 2010, newly identified variants of porcine epidemic diarrhoea virus (pedv) have caused high mortality in neonatal piglets which has devastated the swine industry. the spike (s) glycoprotein of pedv contains multiple neutralizing epitopes and is a major target for pedv neutralization and vaccine development. to understand the antigenicity of the new pedv variant, we characterized the neutralizing epitopes of a new genotype 2b pedv isolate from taiwan, pedv pintung 52 (pedv-pt), by the generation of neutralizing monoclonal antibodies (nmabs). two nmabs, p4b-1, and e10e-1–10 that recognized the ectodomain of the full-length recombinant pedv s protein and exhibited neutralizing ability against the pedv-pt virus were selected. recombinant truncated s proteins were used to identify the target sequences for the nmabs and p4b-1 was shown to recognize the c-terminus of co-26k equivalent epitope (coe) at amino acids (a.a.) 575–639 of the pedv s. interestingly, e10e-1–10 could recognize a novel neutralizing epitope at a.a. 435–485 within the s1(a) domain of the pedv s protein, whose importance and function are yet to be determined. moreover, both nmabs could not bind to linearized s proteins, indicating that only conformational epitopes are recognized. this data could improve our understanding of the antigenic structures of the pedv s protein and facilitate future development of novel epitope-based vaccines. determination of neutralizing antibodies. after purification and quantification of mabs, the neutralizing ability of each mab against the g2b taiwan pedv-pt strain was assessed using the virus neutralizing assay. the starting concentration for each mab in the neutralizing assay was 20 μg/ml, with a two-fold serial dilution to obtain a final neutralizing concentration of 2.5 μg/ml. the mabs, p4b-1, and e10e-1-10, possessed potent neutralizing ability that completely blocked the cpe of the taiwan pedv-pt strain under 5 μg/ml. on the contrary, the mabs, r7h-2, and unk-1, showed no neutralization ability against pedv-pt even at 20 μg/ml. synthesized codon-optimized complete gene sequence of the pedv-pintung 52 s protein (pedv-pt; genbank accession no. kp276252) was used to amplify dna amplicons of varying lengths that encode different truncated forms of the s protein genes. using the primer pairs listed in supplementary table 1 , dna sequences of different lengths were amplified namely s 1-435 (1305 bp), s 1-485 (1455 bp), s 1-501 (1503 bp), s 1-509 (1527 bp), s 1-575 (1725 bp), s 1-639 (1917 bp) and the full-length s ectodomain (4086 bp) of the pedv-pt strain. these dna fragments were digested with restriction enzymes and ligated into the pcdna3.1-v5-his vector. seven plasmids containing the v5-tag, namely pcdna3.1-pedv s 1-435 -v5-his, pcdna3.1-pedv s 1-485 -v5-his, pcdna3.1-pedv s 1-501 -v5-his, pcdna3.1-pedv s 1-509 -v5-his, pcdna3.1-pedv s 1-575 -v5-his, pcdna3.1-pedv s 1-639 -v5-his, and pcdna3.1-pedv s-v5-his were constructed. we have shown the construction of the full-length spike ectodomain of the taiwan pedv-pt strain in a previous publication 19 . the expression levels of various truncated recombinant s proteins, s 1-435 , s 1-485 , s 1-501 , s 1-509 , s 1-575 , and s 1-639 of the pedv-pt strain, were successfully detected using icc staining with the anti-v5 tag antibody ( fig. 1) , as well as western blotting (fig. 2) . the v5 tag-positive hek293 cells for each construct occupied 70% to 90% of the total population. the positive ratio of each construct was adjusted between 60% and 70% by mixing with untransfected hek293 cells, which were used as internal negative control in the icc staining. the corresponding molecular weights of s 1-435 , s 1-485 , s 1-501 , s 1-509 , s 1-575 , s 1-639 , and the full-length spike protein were detected to be approximately 60, 75, 75, 80, 85, 90, and 250 kda, respectively (fig. 2) . mapping the epitopes of the pedv-pt by icc staining. as summarized in table 1 , the two nmabs, p4b-1 and e10e-1-10 and the non-neutralizing mab, r7h-2, all exhibited the ability to recognize full-length pedv spike-expressing hek293 cells but were unable to recognize pedv s 1-435 -expressing hek293 cells in icc staining. more precisely, p4b-1 recognized pedv s 1-639 expressing hek293 cells but did not recognize the hek293 cells expressing s 1-575 , s 1-509 , s 1-501 , s 1-485 , s 1-435 of pedv-pt (fig. 3) . in other words, p4b-1 was unable to bind to the amino acids upstream of a.a. 575 of the spike protein of pedv-pt. therefore, the targeting epitope of p4b-1 must be located between a.a. 575-639 on the pedv spike protein. surprisingly, e10e-1-10 exhibited good binding affinity for hek293 cells expressing pedv proteins s 1-639 , s 1-575 , s 1-509 , s 1-501 , and s 1-485 , but with reduced binding affinity for the pedv s 1-435 . this evidence obtained from the icc staining confirmed this nmab recognizing the novel epitope present within the 435-485 a.a. region of pedv-pt spike protein (fig. 3) . in contrast, the non-neutralizing mab, r7h-2, that recognized only the full-length pedv spike-expressing hek293 cells, was unable to bind to the pedv spike protein upstream of a.a. 639 (fig. 3) . these results suggest that the targeting epitope of r7h-2 is downstream of a.a. 639 of the pedv spike protein. elisa results showing reactivity of mabs to various truncated pedv s proteins. to further confirm the reactivity of each mab to the targeting epitopes, elisas were performed using plates coated with www.nature.com/scientificreports www.nature.com/scientificreports/ various truncated pedv spike proteins. the pedv nmabs (p4b-1 and e10e-1-10) and the non-pedv recognizing mab (unk-1) were all serially diluted to conduct the elisa. as shown in fig. 4 , p4b-1 had a binding affinity with appropriate dilution effects toward the purified full-length pedv spike protein, as well as pedv s 1-639 , but had its affinity towards purified pedv proteins, s 1-575 , s 1-509 , s 1-501 , s 1-485 and s 1-435 was weak. however, e10e-1-10, which targets a novel neutralizing epitope as shown with icc staining, was also capable of binding to the full-length pedv spike protein as well as truncated pedv proteins, s 1-639 , s 1-575 , s 1-509 , s 1-501 , and s 1-485 with appropriate dilution effects. similar to the results obtained by icc staining, e10e-1-10 showed no binding ability www.nature.com/scientificreports www.nature.com/scientificreports/ toward pedv s 1-435 by elisa and therefore, an nmab targeting a novel epitope of the new variant of pedv specifically at a.a. 435-485 in the s1 region was confirmed. the non-pedv recognizing mab, unk-1 showed no binding affinity towards the various truncations of the spike protein and hence, it was used as the external control for the elisa assay. binding affinity of nmabs to linearized epitopes. to further determine whether the binding of neutralizing epitopes require conformational integrity, immunodot blotting was performed by denaturing the various truncated pedv spike proteins. the conformational proteins as well as the denatured proteins of s 1-435 , s 1-485 , s 1-501 , s 1-509 , s 1-575 , s 1-639 , and the full-length spike of pedv-pt were dotted onto the membrane. as shown in fig. 5 (a-c), p4b-1 was able to detect the conformational s 1-639 and the full-length spike protein on the membrane but was unable to detect the proteins upstream of a.a. 575 of spike, consistent with the findings from the elisa and icc staining. the other nmab, e10e-1-10, could bind to s 1-485 , s 1-501 , s 1-509 , s 1-575 , s 1-639 and the full-length spike proteins in conformational structures. the r7h-2, a spike-recognizing but non-neutralizing mab, was only capable of binding to the conformational full-length spike protein. p4b-1, e10e-1-10, and r7h-2 were unable to stain the denatured target proteins on the membrane, indicating the loss of binding affinity after protein linearization. moreover, to ensure all the proteins were equally dotted on the membrane, the results of mab-stripping and re-probing with anti-v5 tag antibodies are shown in fig. 5 (d-f). a molecular model of recombinant pedv s ectodomain protein was established using swiss model and re-edited using ucsf chimerax molecular modelling system. as shown in fig. 6 , the monomer of pedv s protein was divided into s1 (a.a. 1-735, in light-blue) and s2 (a.a. 736-1378, in grey). the novel neutralizing epitope (a.a. 435-485 of the s protein), characterized by the e10e-1-10 nmab labelled in yellow in fig. 6(a) , was inferred to be at the c terminal region of the s1 a domain. the two mutations, v 441 i, and mab s 1-435 s 1-485 s 1-501 s 1-509 s 1-575 s 1-639 s www.nature.com/scientificreports www.nature.com/scientificreports/ s 477 a that were different from the cv777 strain were highlighted in red sphere; and the one mutation, e 459 q that was different from the taiwan historic pedv hc070225-s strain was highlighted with a green sphere. on the other hand, the region of the neutralizing epitope (a.a. 575-639 of the s protein; labelled in yellow) characterized by the p4b-1 nmab was deduced at the c-terminal region of the s1 b domain in fig. 6(b) . the a.a. mutation (g 596 s) different from the cv777 strain was highlighted with a purple sphere; and the a.a. mutations, d 608 e and l 615 f, different from the taiwan historic pedv strain hc070225-s were highlighted with pink spheres. the mutation, q 636 e that was different from both cv777 and taiwan historic pedv strain hc070225 was highlighted with an orange sphere. the six truncated as well as full-length spike protein of pedv-pt were coated into separate wells and probed with two-fold serially diluted mabs, p4b-1 and e10e-1-10. the non-pedv recognizing mab, unk-1, was added as the external control for elisa under the same dilution conditions. the two-fold serially diluted anti-v5 tag antibody was used as the standard to show the dilution effect of the binding affinity assay. the data is represented as the s/p ratio. the wells incubated with only secondary antibodies and the wells incubated with anti-v5 tag antibody at 1,250 ng/ml were used as negative and positive controls for s/p ratio respectively. the results of the elisa for the various truncations of the spike proteins are shown as follows: www.nature.com/scientificreports www.nature.com/scientificreports/ since 2010, outbreaks of infections caused by new pedv variants have been correlated to viral mutations in critical neutralizing epitopes of the spike protein 8, 20 . although several sequences of the new variant pedv spike genes have been decoded 6, 21 and multiple domain architectures of the pedv s protein have been predicted by three-dimensional structures of alphacoronaviruses 16 , the effects of genetic mutations on the antigenicity of pedv remains poorly understood. a thorough evaluation of the neutralizing epitopes and antigenicity of the new pedv variants is therefore needed. in the present study, we generated a panel of mabs using pedv-pt viral particles. by selecting mabs that exhibited high binding affinity to full-length spike ectodomain-expressing hek293 cells, we successfully identified two representative nmab against pedv-pt. the targeting epitopes identified by these two nmabs were located at a.a. 575-639 and a.a. 435-485 of the pedv spike protein, respectively, as shown by icc staining and elisa. further, none of these nmabs were able to bind to the linearized forms of the recombinant ectodomain of s protein of pedv-pt strain was modelled using swiss model and edited using ucsf chimerax. the s protein was present as monomer and the s1 and s2 regions of pedv were displayed in light-blue and grey, respectively. the conformational neutralizing epitope regions characterized by the mabs are given as follows: (a) e10e-1-10 mab labelled in yellow; two mutations (v 441 i and s 477 a) on the epitope different from the cv777 strain were highlighted in red sphere and one mutation (e 459 q) on the epitope different from the taiwan historic pedv strain (hc070225-s) was highlighted in green sphere. (b) p4b-1 mab was labelled in yellow; the g 596 s mutation on the epitope different from the cv777 strain was highlighted in a purple sphere; mutations, d 608 e and l 615 f, on the epitope different from the taiwan historic pedv strain (hc070225-s) were highlighted in pink sphere. the orange sphere represented the common mutation (q 636 e) noted on the epitope of pedv-pt s protein that was different from both of cv777 and taiwan historic pedv (hc070225-s) strains. (2019) 9:2529 | https://doi.org/10.1038/s41598-019-39844-5 www.nature.com/scientificreports www.nature.com/scientificreports/ the truncated spike proteins, indicating that they recognize the integral epitopes with appropriate conformation. taken together, we discovered a novel conformational neutralizing epitope in the s1 a domain at a.a. 435-485, whose function has not been clearly determined in coronaviruses. in addition, we also discovered a conformational neutralizing epitope close to the c-terminus of the coe domain, at a.a. 575-639. the neutralizing epitopes of the historic strains of pedv on the s protein that have been previously published include the co-26k equivalent epitope (coe epitope; a.a. 499-638 in the brl strain; a.a. 501-640 in the pedv-pt strain) 12 , 2c10 epitope (a.a. 1368-1374 in the cv777 strain; a.a. 1373-1379 in the pedv-pt strain) 13 , and s1d (a.a. 636-789 in the cv777 strain; a.a. 640-794 in the pedv-pt strain) 14 . in case of the new variants of pedv, the domain b (a.a. 510-640 in the pedv gdu and the pedv-pt strain) and domain 0 in the s1 region (a.a. 1-219 in the pedv gdu and the pedv-pt strain) are demonstrated to have neutralizing epitopes 17 . furthermore, okda et al. also discovered several linear epitopes at the n-terminus of s2 18 . previously, the identification of neutralizing epitopes of the historic pedvs led to the proposal that several single nucleotide polymorphisms (snps), such as three serine amino acid substitutions in the coe epitope and two serine amino acid substitutions in the s1d epitope, may be related to newly-occurring global outbreaks 6, 22 . in the present study, the nmab, p4b-1, was able to recognize the epitope within a.a. 575-639 on the s protein, which overlaps with the c-terminus of the coe epitope, echoing the findings of previous studies 12 . as the s1 b domain consists of the main coe neutralizing epitope of pedv and is the receptor binding domain for the majority of the coronaviruses, its importance has been emphasized in many studies 11, 23, 24 . li et al. have demonstrated that the binding affinity of neutralizing antibodies could be dramatically altered by exchanging one amino acid on the s protein of pedv 17 . hence, mutations in s1 b domain, especially in neutralizing epitopes of the pedv s protein, are speculated to represent the evolution of viral escape from antibodies, like the one evoked by cv777-based vaccine immunization of a historic pedv strain infection 4, 25 . interestingly, the other nmab, e10e-1-10, presented a high binding affinity toward a.a. 435-485 of the s protein that precludes the coe epitope and locates in the s1 a domain has also been identified in our study. to our knowledge, the function of the s1 a domain of the pedv s protein has not been well studied. the s1 a domain of human coronavirus hcov-nl63 is speculated to interact with heparan sulfate proteoglycans on the host cells to mediate the virus anchoring and infection 26 . the n-terminus of s1 a domain of bovine coronavirus has been shown to have two sugar binding loops which can recognize the carbohydrate receptor on the host cells 27 . moreover, heparan sulfate has also been demonstrated as an attachment factor for pedv 28 . however, as the actual topography of the pedv trimeric s protein remains to be solved and the function of s1 a domain is yet to be determined, the actual interaction of the s1 a domain targeting mab with these sugars or the heparan sulfate proteoglycans-binding domains on the pedv s glycoprotein remains to be further evaluated. visualization of the topology and interaction of pedv s glycoprotein with these neutralizing antibodies for guiding future immunogen and therapeutics design is an important future goal. epitopes for antibody recognition can generally be divided into two main classes: linear and conformational forms. linear epitopes are formed by a continuous sequence of amino acid in a protein, while conformational epitopes consist of amino acid sequences that are discontinuous in the protein but are brought together upon three-dimensional protein folding. it has been demonstrated that 90% of b cell-recognizing epitopes are conformational epitopes that result from the antigen internalizing process and special antigen-recognizing ability 29, 30 . many conformational epitope-targeting antibodies, including neutralizing antibodies, are particularly difficult to determine because the antibody-antigen complex is formed solely in the native structure of the protein. over the past several decades, with the use of phage-display peptide probing, the neutralizing epitope of cv777, named 2c10 has been identified 13 . using elisa or pepscan assays, several linear neutralizing epitopes have been discovered on the s2 glycoprotein subunit of new pedv variants 18 . additionally, two b cell epitopes, named ss2 and ss6, in the region of the s1d neutralizing epitope were evaluated using a combination of phage-display peptide probing and pepscan 14, 15 . however, these epitope mapping approaches focus mainly on linear epitopes 31 . using icc staining, elisa, and denatured immunodot blotting, all of the b cell-recognizing epitopes identified in the present study were found to be conformational epitopes. thus, these affinity-binding assays could serve as valuable platforms for studying the conformational epitopes of nmabs. in conclusion, the nmabs and various truncated s proteins constructed generated in the present study could contribute to a better understanding of the antigenicity and immunogenicity of highly virulent pedvs, especially in revealing the antigenic role of s1 a domain, and the pathogenesis of immune escape that leads to an outbreak of pedv. moreover, our study may provide important fundamental information for the future development of novel epitope-based vaccines. collection (atcc) no. crl-1586) as previously described 32 . after freezing and thawing, the harvested viral supernatant was centrifuged at 3000 rpm for 30 min to remove cell debris and filtered through a 0.22 µm ultrafiltration cup (corning, new york, usa). the viral supernatant was then added onto a 20% sucrose (sigma, missouri, usa) cushion, purified by ultracentrifugation at 75,000 × g for 2.5 h. the viral pellet was re-suspended in phosphate buffered saline (pbs) (gibco, gaithersburg, usa), and then applied to a 20-60% sucrose-tne (20 mm tris-hcl (ph 7) (sigma), 100 mm nacl, 2 mm edta (sigma)) gradient, and centrifuged at 75,000 × g for 2.5 h in an optima ™ l-100xp preparative ultracentrifuge using an avanti j-25 rotor (beckman coulter, sykesville, usa). purified virions were diluted in tne buffer, pelleted by centrifugation at 75,000 × g for 1.5 h to remove the sucrose and then, re-suspended in tne buffer. www.nature.com/scientificreports www.nature.com/scientificreports/ mab production. three balb/c mice were intramuscularly (im) immunized with 20 μg purified pedv viral particles mixed with 100 μl complete freund's adjuvant (sigma). after two weeks, two im booster injections were administered using 20 μg purified pedv viral particles with 100 μl incomplete freund's adjuvant (sigma) at intervals of 3 weeks. three days before sacrifice, mice were immunized with 20 μg purified pedv viral particles in pbs (gibco) via intrasplenic (is) injection. serum antibody titres at each immunization were monitored using a complete pedv viral particle elisa and the mouse with the highest titre was sacrificed for hybridoma preparations. hybridoma preparation. splenocytes were isolated from the mice immunized with purified pedv particles. after gentle washing with brief centrifugation, splenocytes were fused with sp2 myeloma cells at a cell ratio of approximately 10:1 using 50% polyethylene glycol (sigma). hybridomas were seeded onto 96-well culture plates in rpmi-1640 medium supplemented with 20% foetal bovine serum (gibco), 100 mg/ml streptomycin, and 100 iu/ml penicillin (sigma), and incubated overnight at 37 °c in a humidified incubator with 5% co 2 . after incubation, approximately 50% medium was removed from each well, and a selective hat rpmi-1640 medium (hat-rpmi) (sigma) was added to achieve a final concentration of 20% foetal bovine serum (gibco), 100 mg/ml streptomycin, 100 iu/ml penicillin, 100 mm hypoxanthine (sigma), 400 mm aminopterin (sigma), and 16 mm thymidine (sigma). wells containing growing hybridoma cells were screened for antibody production by icc staining using pedv-infected vero cells or hek293 cells (atcc crl-1573 ™ ) expressing the full-length pedv s protein. positive clones were isolated for limiting dilution and incubated in selective ht rpmi-1640 medium without aminopterin. after two limiting dilutions, the supernatant from each line was further tested for anti-pedv s-specific antibodies by icc staining using pedv-infected vero cells or hek293 cells expressing the full-length recombinant pedv s protein. mab purification and quantification. to purify mabs from cultured supernatants, pierce ™ protein l magnetic beads (thermo fisher scientific, waltham, usa), which selectively bind to mouse immunoglobulin were utilized following the manufacturer's instructions. the beads were mixed with 40 ml supernatants of each mab and incubated for 1 h after which the antibody-bound beads were collected using a magnetic stand. after washing thrice wash buffer (tris-buffered saline (tbs), 0.05% tween-20 detergent), the mabs were eluted with 60 μl elution buffer (0.1 m glycine, ph 2.0) for 10 min and then, alkalized pbs buffer (ph 8.5) was added for neutralization. the concentrations of each purified mab were determined by the pierce ™ bca protein assay kit (thermo fisher scientific). of the full-length pedv s protein of the taiwan pedv-pintung-52 (pedv-pt) strain (genbank: ky929405.1) was performed as previously described 33 . to prepare recombinant truncated pedv s proteins, regions of the s gene coding for amino acids 1-435 (s 1-435 ), 1-485 (s 1-485 ), 1-501 (s 1-501 ), 1-509 (s 1-509 ), 1-575 (s 1-575 ), 1-639 (s 1-639 ) were amplified using specific primers (supplementary table s1 online) and the resultant proteins were purified as described previously 19 . the amplicons encoding different truncated genes were subcloned into a bamhi-noti restriction site in the pcdna tm 3.1/v5-his topo ® vector (invitrogen, carlsbad, ca, usa), transfected into hek293 cells and selected by using 750 μg/ml geneticin (g418, gibco). after two weeks of selection, cells stably expressing the truncated pedv s proteins were subjected to icc staining and western blotting. purification and western blot detection of truncated spike proteins. large-scale purification of the truncated s proteins of pedv-pt was performed as previously described 19 . the protein expressing hek-293 cells were harvested, resuspended, and cultured in freestyle 293 expression medium (gibco) for one week. after supernatant collection and removal of cell debris through a 0.22 μm filter, the proteins were banded with hispur cobalt resin (thermo fisher scientific) following the manufacturer's protocol. the purified proteins were subsequently concentrated with a 30 kda vivaspin ® 20 (ge healthcare life sciences, ny, usa), complete ™ edta-free protease inhibitor cocktail (roche molecular biochemicals, quebec, canada) was added and the concentrations were measured using the pierce ™ bca protein assay kit. the purified proteins were denatured in a buffer containing nupage ® lds sample buffer and nupage ® reducing agent, and heated for 5 min at 95 °c. using bio-rad mini-protein ® electrophoresis system(bio-rad, hercules, ca, usa), the samples were separated using 10% sodium dodecyl sulfate (sds)-polyacrylamide gel electrophoresis (page) cast in a gradient t-pro ez gel solution (t-pro biotechnology, taiwan) according to the manufacturer's protocol. following this, proteins were wet-blotted onto a polyvinylidene difluoride (pvdf) membranes (bio-rad)blocked with 5% skim milk in tris-buffered saline with 0.05% tween 20 (tbs-t) buffer at room temperature (rt) for 1 h. membranes were incubated with a 1:5000 diluted anti-v5 tag antibody for 1 h at rt. after washing with tbs-t, horseradish peroxidase (hrp)-conjugated goat anti-mouse igg secondary antibody (1:10,000 dilution in blocking buffer; jackson immunoresearch laboratories, philadelphia, usa) was added and incubated at rt for 1 h, the signals were visualized using clarity ™ western ecl blotting substrates (bio-rad) and detected with a chemidoc ™ xrs+ imaging system (bio-rad). neutralizing assay for the pedv-pt strain. a neutralizing assay was conducted as previously described with some modifications 32 . next, 100 μl suspended vero cells (atcc no. crl-1586) were seeded onto 96-well culture plates at 3 × 10 5 cells/ml and incubated overnight at 37 °c in a humidified incubator with 5% co 2 until cells reached 90% confluence. purified mabs were two-fold serially diluted from 20 μg/ml to 2.5 μg/ml in post-inoculation medium (pi medium) containing dulbecco's modified eagle medium (gibco) supplemented with tryptose phosphate broth (0.3%) (sigma, missouri, usa), yeast extract (0.02%) (acumedia, ca, usa), and 10 μg/ml trypsin (gibco). fifty microliters of diluted culture supernatant from each hybridoma clone were mixed and incubated with an equal volume of 100 tcid 50 /ml pedv-pt passage 5 (pedv-pt-p5) at 37 °c in 5% co 2 porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis journal of veterinary diagnostic investigation: official publication of the american association of veterinary laboratory diagnosticians a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea phylogenetic analysis of the spike (s) gene of the new variants of 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virus spike protein are key targets of neutralizing antibodies the s2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes efficacy of heat-labile enterotoxin b subunit-adjuvanted parenteral porcine epidemic diarrhea virus trimeric spike subunit vaccine in piglets antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains new variant of porcine epidemic diarrhea virus evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains receptor recognition mechanisms of coronaviruses: a decade of structural studies structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies epidemiology and vaccine of porcine epidemic diarrhea virus in china: a mini-review glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy crystal structure of bovine coronavirus spike protein lectin domain porcine epidemic diarrhea virus uses cell-surface heparan sulfate as an attachment factor mapping epitope structure and activity: from one-dimensional prediction to four-dimensional description of antigenic specificity continuous and discontinuous protein antigenic determinants prediction of residues in discontinuous b-cell epitopes using protein 3d structures evaluation and comparison of the pathogenicity and host immune responses induced by a g2b taiwan porcine epidemic diarrhea virus (strain pintung 52) and its highly cell-culture passaged strain in conventional 5-week-old pigs display of porcine epidemic diarrhea virus spike protein on baculovirus to improve immunogenicity and protective efficacy ucsf chimera-a visualization system for exploratory research and analysis washing six times, 50 μl of abts ® peroxidase substrate system (kpl, seracare) was used to obtain a positive signal. the coloration procedure was stopped after 20 min by adding 50 μl stopping solution (kpl, seracare). the signals were detected at 405 nm wavelength by emax plus microplate reader (molecular devices, crawley, uk). the final data is shown as a sample to positive ratio (s/p ratio), representing the difference between the od values of the sample and the negative control divided by the difference between the od value of the positive and negative controls. the od value of the positive control was obtained from the result of 80,000x diluted anti-v5 tag antibody, and the od value of the negative control was obtained from the result of the secondary antibody only wells.linear epitope mapping by immunodot blotting and western blotting. to confirm the conformational significance of the neutralizing epitopes, we also denatured the six purified truncated spike and the full-length spike protein of pedv-pt by mixing them with nupage ® reducing agent (thermo fisher scientific) and heating for 5 min at 95 °c. the purified proteins and the denatured proteins were separately dotted on nitrocellulose membranes (merck millipore, ma, usa) at 50 ng. the reducing agent mixed with water was also dotted on the membrane as a negative control. following a 1-h block in 5% skim milk, the membranes were stained with nmabs (p4b-1 and e10e-1-10) and non-neutralizing mab (r7h-2) at 2.5 μg/ml diluted in tbs-t buffer at rt for 1 h. after washing three times, hrp-conjugated goat anti-mouse igg secondary antibody (1:10,000 dilution, jackson immunoresearch laboratories) was added and incubated at rt for 1 h. protein signals were visualized using the clarity ™ western ecl blotting substrates (bio-rad) and detected with a chemidoc ™ xrs+ imaging system (bio-rad) as previously described. to ensure the denatured proteins were correctly dotted on the membrane, after the first run of probing, the mabs were stripped with stripping buffer (thermo fisher scientific) and the membranes were separately re-probed by anti-v5 tag antibodies (invitrogen). a full-length homology model of the ectodomain of recombinant pedv s protein was generated by swiss-model and built with promod3 version 1.0.0 (https://swissmodel.expasy. org). the manipulated protein sequence of the pedv s ectodomain was constructed using the trimeric human coronavirus nl63 spike structure (pdb accession no. 5szs) as a template. the images were edited using ucsf chimerax molecular modelling system 34 . the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-39844-5.competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-252536-gfx4cq03 authors: bieniossek, christoph; imasaki, tsuyoshi; takagi, yuichiro; berger, imre title: multibac: expanding the research toolbox for multiprotein complexes date: 2011-12-07 journal: trends biochem sci doi: 10.1016/j.tibs.2011.10.005 sha: doc_id: 252536 cord_uid: gfx4cq03 protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. however, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. to overcome this bottleneck, powerful recombinant expression technologies are being developed. in this review, we describe the use of one of these technologies, multibac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. among other applications, multibac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms. protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. however, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. to overcome this bottleneck, powerful recombinant expression technologies are being developed. in this review, we describe the use of one of these technologies, multibac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. among other applications, multibac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms. multiprotein complexes catalyze cellular function our understanding of cellular function has remarkably expanded in recent years, brought about by technological advances in dna and protein analysis [1] . the sequencing of complete genomes, such as the human genome, has set the stage to address proteome-wide interaction studies, which have revealed that proteins do not typically exist as isolated entities [2] [3] [4] . rather, they are assembled in complex molecular machines consisting of numerous proteins and, often, other biomolecules (such as nucleic acids, sugars, lipids and small molecules), arranged into functional modules that catalyze essential cellular processes. this molecular organization has been recently termed 'protein sociology' [5] . understanding cellular processes requires detailed knowledge of the 3d structure of the molecules involved, and the parameters and architectural features that dictate their interaction. structural genomics efforts strive to analyze comprehensively single proteins and protein domain structures on a whole-genome scale, and have provided atomic structures of thousands of protein structures and folds. furthermore, the architectures of essential macromolecular complexes, such as ribosomes, nucleosomes and rna polymerases, have been revealed at near atomic resolution, providing a wealth of structural detail and crucial insight into the functions of these multicomponent machines [6] [7] [8] . integrated approaches combining structural, functional and computational data are emerging, and provide views of protein organization in space and time in entire organisms [5, 9] . notwithstanding, our molecular understanding of the very large number of protein complexes in the cell remains limited to a handful of examples for which detailed nearatomic structures are known. this is mostly because of the difficulty in obtaining samples in sufficient quality and quantity for molecular studies. many essential complexes remain intractable because they exist in very low amounts in their endogenous host, which hinders their purification review glossary cre recombinase: enzyme that recognizes, binds to, cuts and religates specific dna sequences (called loxp sequences). four copies of cre recombinase bind to two loxp dna sequences, and then cre recombinase exchanges one strand each from the two loxp sequences. if the two loxp sequences are present on two different dna molecules, then these will be conjoined as a result, resulting in one dna molecule with two loxp sites. homing endonucleases: restriction enzymes with particularly long (20-30 base pairs) recognition sequences. after cutting the dna, they often leave fournucleotide overhangs that can be compatible with overhangs generated by another restriction enzyme, bstxi; then, dnas processed with homing endonucleases can be efficiently ligated to dnas tailored by bstxi. both recognition sequences are destroyed in the process, and the ligation product cannot be cut by either enzyme. ligation independent cloning (lic): a method for inserting dna sequences (i.e. genes) into a plasmid dna molecule without using dna ligase. dnas to be conjoined are treated by exonucleases that trim back one strand of the dna double helix, exposing the complementary strand as a long sticky end. if two dna molecules have complementary sticky ends, they can be conjoined simply by mixing. lic methods have become popular in recent years because they do not require preselected dna sequences that serve as recognition sites for restriction enzymes. a version of lic that is entirely independent of any dna sequence is called slic (sequence and ligation independent cloning). loxp sequence: short imperfect inverted repeat of 34 base pairs which serves as a recognition site, cleavage site and religation site for cre recombinase. the loxp sequence is not symmetric, therefore, the conjoining of two loxp sequences by cre recombinase is directional. nuclear polyhedrosis virus (npv): npv attacks caterpillars, such as the larvae of the alfalfa looper or the silkworm. npvs are highly specific for their host and thus useful as pest control agents. polyprotein: long polypeptide which contains several individual proteins that are connected by linker amino acids which either are capable of self-cleavage, or are recognition and cleavage sites of highly specific proteases. tn7 transposon: originally discovered as a large (14 kb) dna segment from tn7 phage, which can insert into a specific location in the e. coli genome. the transposon encodes the tn7 transposase, a four-subunit protein complex that carries out this insertion reaction. three dna elements are minimally required for this reaction: the tn7l and tn7r dna sequences which flank the dna to be transposed, and a sequence called tn7 attachment site (atttn7) into which the transposed dna is inserted. this insertion mechanism can be efficiently exploited to conjoin recombinant dna by providing the tn7l and tn7r sequences on a plasmid at each end of a dna sequence of interest, and the atttn7 sequence on another dna which will serve as recipient. the reaction is started by addition of the transposase to these dnas. corresponding author: berger, i. (iberger@embl.fr). from native source material. recombinant overproduction can resolve this bottleneck, and numerous expression systems, mostly for heterologous protein expression in escherichia coli, have been developed and refined over the past few decades. more recently, e. coli expression systems have been designed for coexpression of several proteins by using polycistronic mrna transcripts, or two or more plasmids that coexist in the same cell [10, 11] (box 1). however, many eukaryotic protein complexes cannot be produced efficiently in e. coli. they may contain subunits that are too large for the e. coli transcription and translation machinery, or may require either specific chaperone systems for proper folding or protein modifications (such as phosphorylation or acetylation) that e. coli cannot provide. thus, the successful overproduction of these complexes, which is required to decipher their structure and function, depends on the availability of powerful eukaryotic expression technologies. in this review, we describe multibac, a box 1. coexpression toolbox for production of protein complexes expression systems for production of protein complexes in e. coli frequently make use of polycistronic expression cassettes with several genes of interest, spaced apart by ribosome binding sites (shine-dalgarno sequences), placed under the control of a single promoter, and typically followed by a sequence for efficient termination of mrna transcription [10, 11, 21, [60] [61] [62] (figure ia) . in eukaryotic hosts, an analogous design can involve internal ribosomal entry sites (iress), which are inserted between gene coding regions under control of a single promoter [24] [25] [26] [27] (figure ib ). iress exist in the 5 0untranslated regions of many viral and cellular mrnas [24] , and facilitate cap-independent translation by recruiting ribosomes for efficient protein production. ires sequences differ greatly and exhibit species specificity [26] . for example, ires elements from encephalomyocarditis virus (emcv) work well in mammalian cells, whereas iress from perina nuda virus (phv) and rhopalosiphum padi virus (rhpv) have been successfully used for protein complex production in insect cells [27] . polyproteins are long polypeptides containing individual proteins spaced by specific proteolytic cleavage sites. certain viruses, such as coronavirus, produce their entire proteome by proteolytic processing of polyproteins encoded by a few open reading frames. polyprotein approaches have proven to be particularly powerful for balancing the stoichiometry of coexpressed proteins [22, [28] [29] [30] (figure ic) . polyprotein constructions can involve self-cleaving peptides found in picornavirus (called p2a peptides) or variants thereof [28, 29] . alternatively, constructions can be used that mimic open reading frames in positive-sense single-stranded rna viruses and provide a highly specific protease gene together with genes of interest arranged in a single large open reading frame [22, 30] . individual proteins are then liberated from the encoded polyprotein by the protease, which cleaves the proteolytic sites placed between the protein subunits ( figure ic) . the elucidation of protein-protein interactions in complexes is of crucial importance for many applications including structural biology. a novel approach, coesprit, utilizes library-based construct screening for the identification and expression of soluble protein complexes in e. coli [31] . in coesprit, a subunit of a (putative) protein complex is provided as bait. the interacting partner is provided in the form of a random incremental library generated by exonuclease digestion of the full-length gene. prior input from bioinformatics analyses such as homology alignments or domain identification is not required for this approach. coexpression of the library with the bait protein allows identification of soluble complexes by immunofluorescence-assisted colony screening using labeled antibody markers against affinity tags present on the proteins screened. production of protein complex from high-expressor colonies thus identified can then be scaled up to milligram amounts for high-resolution studies by nmr or x-ray crystallography [31] . trends in biochemical sciences february 2012, vol. 37, no. 2 recent eukaryotic expression system that is specifically designed to tackle and overcome this crucial production bottleneck [12, 13] . we summarize the technology concepts underlying multibac and review its wide range of applications. the multibac system yeast, mammalian cells and insect cells have been successfully used for recombinant production of eukaryotic proteins [14] . in particular, the use of insect cell cultures infected by a recombinant baculovirus, carrying the eukaryotic gene of interest, was first demonstrated many years ago [15] . the exciting evolution of baculovirus from a pest control agent to a powerful recombinant protein production tool has recently been reviewed [16] , and baculovirus expression systems have become increasingly popular for many applications [17] [18] [19] . multibac is a baculovirus expression system particularly designed for the production of eukaryotic multiprotein complexes with many subunits (figure 1 ). it consists of an array of small synthetic dna plasmids, an engineered baculovirus genome derived from the autographa californica nuclear polyhedrosis virus (acnpv; see glossary) that is used to infect cells of the caterpillar spodoptera frugiperda, and a set of protocols detailing every step from gene insertion into the plasmids to production of protein complexes in cultured insect cells [19, 20] . the presence of many subunits in a protein complex requires the assembly of many encoding genes and their integration into the baculovirus in a multicomponent co-production experiment. this process is laborious and technically challenging using conventional methods that typically involve serial insertion of genes into increasingly large and difficult-to-handle dna plasmids. multibac applies a different concept for multigene assembly that relies on recombination of small, custom-designed, synthetic dna plasmid molecules (< 3 kb) that are called 'acceptors' and 'donors' (figure 1a ). acceptors and donors can be easily loaded with one or several genes each, and recombined in a single-step reaction into a multigene construct. acceptors contain an origin of replication that allows propagation in standard cloning strains of e. coli, whereas donors harbor a conditional origin of replication (derived from phage r6kg) that requires the presence of a specific protein (known as p) for replication. this protein is expressed from the pir gene inserted into the chromosome of specifically tailored e. coli strains that are used to propagate donor plasmids [13, 20] . donors and acceptors contain a resistance marker, a short imperfect inverted repeat (loxp), an expression cassette consisting of a baculoviral promoter (p10 or polh), a dna segment for inserting heterologous genes, and an efficient signal for eukaryotic polyadenylation (figure 1a ). the expression cassettes are flanked by a homing endonuclease site and a compatible bstxi site, which allow for iterative assembly of several expression cassettes on each plasmid [21] . importantly, donors can survive in a pir-negative background only if they are conjoined with an acceptor that provides a nonconditional origin of replication, and this is the central feature that enables flexible and efficient generation of multigene constructs. these are achieved in vitro multibac consists of an array of small (3 kb), synthetic dna plasmids called acceptors and donors. acceptors have a regular origin of replication (ori cole1 ), whereas donors have a conditional origin derived from r6kg phage (ori r6kg ) that requires special bacterial strains for their propagation. donors and acceptors contain expression cassettes controlled by late baculoviral promoters (polh or p10) as well as strong eukaryotic polyadenylation signals (from sv40 or hsvtk). all plasmids contain the loxp sequence (circles filled in red) for fusing donors to an acceptor by the cre recombinase. each plasmid has a different resistant marker: gentamycin resistance (gn r ) for acceptors, and either chloramphenicol (cm r ), kanamycin (kn r ) or spectinomycin (sp r ) resistance for donors. in addition, a 'multiplication' module is present to facilitate the assembly of several expression cassettes on a donor or acceptor based on specifically designed restriction sites (e.g. homing endonucleases and bstxi restriction endonuclease, shown as blue boxes flanking promoter and terminator, respectively) [18, 19, 21] . acceptors contain the dna sequences (tn7l and tn7r) required for transposition by the tn7 transposase. (b) the assembly of a composite multibac baculoviral genome for multigene expression. genes are inserted into donors and acceptors by using either restriction endonucleases and ligase, or sequence-independent and ligation-independent cloning methods (bottom). cre-mediated fusion produces a multigene construction that is inserted into the baculoviral genome by tn7 transposition in specifically tailored e. coli cells that contain the viral dna (also known as a bacmid). the dna located between the tn7r and tn7l sequences in the multigene fusion construct is inserted by the transposase enzyme into the tn7 attachment site (mini-atttn7). the multibac baculovirus was engineered for improved protein production and delayed host cell lysis by deleting specific genes [12] . the combination of sequence and ligation independent cloning (slic) for gene insertion and cre-loxp fusion for multigene construct generation is called tandem recombineering and can be performed in a parallelized mode on microtiter plates, optionally on a robot [21] . further heterologous genes can be inserted into an additional loxp site present on the bacmid. composite baculoviral dna is then purified from the bacteria and used to transfect insect cells. virus is expanded by infecting small-volume (50-100 ml) insect cell cultures and harvesting the budded virus particles released into the culture media. this virus is then used for protein complex expression in larger (typically one to several liters) insect cell cultures [13, 20] . trends in biochemical sciences february 2012, vol. 37, no. 2 by cre recombinase, which fuses one or several donors (each with one or several inserted genes) to a single acceptor in a one-step reaction by conjoining via the loxp sites; this results in plasmid dimers, trimers and tetramers. the resulting multigene expression constructs are characterized by the precise combinations of resistance markers present on the fusions, and this can be exploited for combinatorial assembly strategies based on multiple antibiotic selection [21] . the process of inserting genes into acceptors and donors, which can be optionally done by ligation independent methods followed by cre-loxp fusion, is termed 'tandem recombineering' [22] . gene insertions into the multibac genome take place in bacterial strains that contain the multibac viral genome as an artificial chromosome together with a plasmid encoding the tn7 transposon enzyme complex. multigene acceptordonor fusion constructs are transformed into these bacterial cells, and the tn7 transposon enzyme inserts the collection of expression cassettes present on the acceptor-donor fusion in a single-step reaction into the tn7 attachment site engineered into the baculoviral genome ( figure 1b) . productive transposition disrupts a laczaencoding gene, which enables blue/white screening of colonies. the multibac baculoviral genome has been engineered for improved protein complex production by removing genes encoding viral protease and apoptotic activities, thereby reducing proteolytic breakdown of the heterologous gene products and delaying lysis of the infected cells [12, 13] . as a second site of entry in addition to the tn7 attachment site, the multibac genome contains also a distal loxp site for adding further functionalities. for example, a gene encoding l-phosphatase was inserted into this site to remove phosphates from a coexpressed protein complex [23] . the composite multibac virus is then purified and used to transfect insect cells for protein production in petri dishes containing cell monolayers or, for larger volumes, shaker flasks containing suspension cultures [13, 19, 20] . multibac resolves several challenges encountered in protein complex production. these include constraints on handling many and often very large encoding dnas and the necessity to revise expression experiments rapidly and flexibly if a subunit or purification tag needs to be altered or exchanged, by replacing the respective donor or acceptor with a modified version in the tandem recombineering pipeline. in addition to multibac, a growing number of technologies are currently being used for expressing protein complexes (box 1). examples of these approaches are the use of internal ribosome entry sites (iress) for multigene expression, and the use of polyprotein constructions involving self-cleaving peptide sequences or proteolytic processing of large precursor polypeptides by specific proteases [22, [24] [25] [26] [27] [28] [29] [30] . particularly promising for structural biology, where often a large number of constructs need to be scrutinized in parallel, are new methods coupling gene libraries to coexpression (box 1) [31] . these approaches are compatible with the multibac technology concept; for example, the polyprotein approach has been successfully used to produce a human transcription factor core complex by multibac, with improved production efficiency and yield [22] . multibac expression of protein complexes for structural studies the multibac system, originally designed by x-ray crystallographers interested in studying multiprotein complexes [12] , has been well received and put to good use by the structural biology community (figure 2, box 2) . many proteins and their complexes have been produced by multi-bac, often for the first time, for structure elucidation, providing important insight into their biological function ( figure 2 ). in some cases, for example for producing intact full-length protein kinase cbii, an existing transfer plasmid was combined with the optimized multibac baculovirus genome for protein production [32] . the modular concept of gene assembly by tandem recombineering has turned out to be instrumental for accelerating the process of obtaining high-quality samples for structural biology of numerous complexes [33] [34] [35] [36] . two outstanding examples of the utility of multibac are the elegant production of the entire anaphase promoting complex, apc/ca large (1.1 mda) 13-subunit multiprotein assembly that regulates defined cell cycle transitions [34] and the recent crystal structure elucidation of the mediator head modulea transcription factor complex that is essential for the expression of class ii genes in eukaryotes [35] . the gene assembly encoding the complete apc/c was inserted into two multibac baculoviruses, encoding eight and five subunits, respectively. co-infection of insect cells with the two viruses allowed the purification of the entire 1.1-mda complex and its structural determination by electron microscopy (em), revealing the structural basis for subunit assembly (figure 2 ) [34] . the production of the mediator head module from yeast (seven subunits, 223 ti bs figure 2 . multibac expression for structural biology. the multibac system has been successfully used to produce many proteins and their complexes in highquality for structural analysis. the structures of lkb1-strada-mo25a (pdb 2wtk) [55] , a tumor suppressor kinase complex, the pkcbii kinase (pdb 3pfq) [32] , and the rad9-rad1-hus1 complex, a dna damage checkpoint complex (pdb 3g65) [56] , were determined by x-ray crystallography providing crucial insight into the function of these important proteins. the crystal structure of the n-terminal domain (ntd) of the chromatin remodeler mot1 bound to the tata-box binding protein (tbp) showed a molecular bottle-opener for tbp (pdb 3g65) [57, 58] . in a hybrid approach involving em and x-ray crystallography, a structure of a different chromatin remodeling enzyme, isw1 (lacking the atpase domain), bound to a nucleosomal template was elucidated (pdb 2y9z and emd-1877) [33] . strikingly, the entire 13 subunit yeast apc/c, an e3 ubiquitin ligase essential for the cell cycle, was assembled by multibac (box 3) as well as two multisubunit apc/c subcomplexes (tpr5 and sc8), leading to structures revealing many details of apc/c assembly by em (emd-1844, 1841 and 1845 ) [34] . molecular illustrations were prepared with pymol (http://www.pymol.org/). trends in biochemical sciences february 2012, vol. 37, no. 2 kda), a paradigmatic case study for the successful application of the multibac system, is detailed in box 2 [35] . clearly, multibac is only one of many tools that bring such studies to fruition. nonetheless, quality sample preparation is a crucial component of the structural determination pipeline. the studies already available exemplify the aptitude of the system, and hint at recurring challenges encountered when using multibac in multiprotein complex research (box 3). the list of illuminating structures of complexes produced by using multibac can be expected to grow rapidly in the future. complexes come in many forms: the coatomer proteins and their complexes in their native tissue can exist as several isoforms, which may complicate their biochemical and structural characterization when extracted and purified from endogenous source material by immunoprecipitation or classical biochemical approaches. an example is the coatomer, the central protein component that covers certain vesicles, known as coat protein i (copi)-coated vesicles, which are thought to play an important role in the early secretory pathway. coatomer consists of seven subunits and exists in four isoforms of different subunit composition. the four isoforms co-purify when extracted from animal tissue, which hampers their structural analysis and impedes attempts to understand the potentially different functions of the individual isoforms. for example, it remains unclear whether or not copi-coated vesicles are uniformly coated with individual isoforms, although these have been found to be unequally distributed in the golgi stack [37] . recently, all four coatomer isoforms have been successfully produced using multibac, setting the stage to individually address their structure and function [38] . this has been achieved by integrating the genes encoding the five subunits that are common to all isoforms into the tn7 attachment site of the multibac viral genome. the genes for the other two subunits, which are variable and define the four isoforms, were inserted by using a donor plasmid into the loxp site that is distal to the tn7 attachment site on the multibac viral genome. coatomer complex isoforms can then be purified successfully from insect cell cultures infected with the respective multibac virus [38] . silkworms, the larvae of the moth bombyx mori, have been used for thousands of years to produce silk for textile production, but they have gained a new role as protein production bioreactors after the successful expression of human a-interferon in these insects. a growing number of proteins, including interleukins and antibodies, have since been produced using this system [39, 40] . interestingly, it seems that silkworms can sometimes produce much higher yields of recombinant proteins than can be obtained from liquid cell cultures. for example, the activity of interleukin-3 produced in silkworms is 10 000-fold higher than that produced in cultures of immortalized african green monkey kidney (cos) cells, and 30-fold higher than that produced in insect cell suspension culture. notably, 1 mg of purified human macrophage colony-stimulating factor can be extracted from 10 silkworms [39] . mediator is a large multiprotein complex that is central to gene expression in eukaryotes and is organized into three modules termed head, middle or arm, and tail [63] . the size and complexity of mediator has long posed a major challenge for structural studies. mediator head (seven subunits, 223 kda) was first successfully produced recombinantly by infecting insect cells with single baculoviruses, each encoding one subunit, opening the door for structural studies [36] . however, this procedure required lengthy optimization to indentify suitable, highly producing recombinant baculoviruses by repeated rounds of laborious screeninga process of 8 weeks or longer for each baculovirus. the demanding procedures constrained logistics and severely compromised attempts at structure determination of mediator head by x-ray crystallography, which typically necessitates flexible, stream-lined and efficient production protocols for generating many variants of the protein specimen studied. the multibac system elegantly resolved this fundamental impediment [35] . all genes encoding the subunits of the mediator head module were inserted into a single multibac baculovirus by using tandem recombineering [36, 64] . combinatorial assembly of the genes lead to a series of multibac baculoviruses encoding either full mediator head (med17, med6, med18, med8, med20, med11 and med22) or subcomplexes including core head (med17, med6, med8, med11 and med22) and mini head (med17, med11 and med22). investigation of these complexes by em allowed initial assignment of subunit positions [64] . modification of med17 and med18 by eliminating flexible regions was crucial to obtain diffraction-quality head module crystals, and this was easily accomplished by simply exchanging the respective unmodified genes in the original multibac baculovirus expressing the full head [35] . the modular acceptor-donor construction principle of multibac is crucial to facilitate this alteration, alleviating a need to assemble the entire multigene construct every time again de novo. the recombinant head module has been purified and crystallized, and the architecture of this 223-kda complex has been determined by x-ray crystallography ( figure i) . the crystal structure of the mediator head reveals how this essential complex is built from its components to provide stability as well as flexibility for transcription regulation, resulting in a platform for other transcription factors [35] . notably, a portion of the head named 'neck domain' confers stability and integrity of the complex by forming a striking, novel multihelical bundle, engaging five of the seven subunits of mediator head. these impressive results have prompted the development of a version of the multibac system adjusted for protein complex production in a silkworm bioreactor [41] . in this system, the original baculovirus genome was replaced with that of a b. mori nuclear polyhedrosis virus (bmnpv), likewise provided as an artificial chromosome in bacterial cells. a tn7 attachment site was introduced into this bmnpv genome, and a plasmid expressing the tn7 transposon complex was co-transformed. thus, the silkworm multibac system retains the ability to accept multigene vector constructions prepared by tandem recombineering or by conventional restriction and ligation cloning. a distinct feature of the b. mori multibac system is the method of infecting the silkworms, which occurs via injection of virus solution with a needle. protein complexes are then purified from the hemolymph of infected silkworms after several days. recombinant human dna polymerase d has been produced using silkworm multibac [41] . although the enzyme is thought to contain four subunits, the extraction from animal tissue usually leads to loss of two subunits during purification. four milligrams of active enzyme containing all four subunits can be purified from 350 silkworms infected with a silkworm multibac virus carrying four genes, providing a simple, fast, reliable and economic platform to produce human dna polymerase d. therefore, the silkworm multibac system might be useful for the production of other protein complexes, especially if an economic option for protein production is required. extending the concept to mammalian cells the concept of multigene delivery of fused acceptor and donor plasmids, each carrying one or several genes, is not restricted to baculovirus expression systems. originally, tandem recombineering was developed for generating multiexpression plasmids for production of protein complexes in e. coli to alleviate imbalances in expression levels observed with multiplasmid co-transformation [21] . expression of protein complexes in mammalian cells can be achieved by transfection with multiple plasmids, in analogy to multiplasmid co-transformation in e. coli. however, this approach can lead to imbalanced production from the plasmids and to heterogeneous cell populations with differences in the ratio of plasmids incorporated. this impediment can be efficiently resolved by using a single plasmid that contains all the heterologous genes. this multigene plasmid can be conveniently generated by tandem recombineering of properly modified acceptors and donors, in which the baculoviral promoters are exchanged for mammalian promoters [22, 42] . the resulting multigene plasmid is introduced into mammalian cells by using a transfection reagent [42] . the efficacy of this approach was demonstrated in a study where an assortment of proteins, systematically tagged with fluorescent markers, was expressed transiently or stably in animal tissue cells [42] . this multibacderived system, called multilabel, can be used for simultaneously visualizing proteins and their actions in homogeneous cell populations by tracking the fluorescent signals. importantly, the ratio of the heterologous proteins is constant at the single-cell level. multilabel has been recently implemented to dissect the interactions of epidermal growth factor receptor (egfr), rab gtpases and phosphoinositol phosphates, which are components of membrane trafficking processes [42, 43] . gene therapy for obesity? gene therapy involves either the targeted delivery of one or several wild-type genes that replace or substitute in for disease-causing genes (which bear aberrant mutations) and produce the desired gene products. or the delivery of genes encoding therapeutic proteins that prevent, modulate or correct the disease [44] . baculoviruses have been intensively studied as potential delivery vectors for gene therapy because they transduce mammalian cells efficiently, do not seem to be toxic, and are nonhazardous (for the target cells and for those carrying out the experiment) as they do not replicate in mammalian cells [17, 45, 46] . further advantages are the ease of producing baculoviruses and the large tolerance for heterologous gene insertions into the viral genome without compromising virion integrity. however, there are formidable obstacles to overcome, such as the observed inactivation of baculoviruses by the human complement system and the lack of specificity with respect to cell types transduced. these problems have not box 3. multibac debugged: challenges and solutions a multitude of factors can compromise the production of multiprotein complexes. among the major impediments are: insufficient knowledge of the composition of the complex. recombinant production of such a complex by coexpression will invariably fail if a subunit crucial for complex integrity has not been properly identified in original preparations. although proteomics and genomics technologies increasingly contribute to improve and validate the data available (reviewed in [1] ), protein complex coexpression can already reveal important information about interacting partners even if the (presumed) complete complex cannot be obtained. post-translational modifications (ptms). ptms such as phosphorylation and acetylation can be essential for proper assembly of a complex or for its activity. thus, the modification activity needs to be either coexpressed or supplied during purification. an additional problem is that insect cells may decorate proteins with nonphysiological ptms (frequently phosphorylation), which compromise complex formation or activity. therefore, mutations that abolish the modification activity must be introduced in the host genome, or enzymes (such as l-phosphatase) that remove the modification have to be coexpressed or supplied [23] . alternatively, inhibitors of the particular modifying enzyme (if known) can be added to the growth media if compatible with cell growth and protein production. complex instability. coexpression of an entire complex from a single virus may not be the ultimate solution. for example, a complex may dissociate during purification in a salt gradient. it may be worth identifying exactly what components dissociate during purification; stable subcomplexes may then be produced separately to reconstitute the complex in vitro by choosing proper conditions. effort required to place all subunit genes on a single baculovirus. for very large complexes, this may be challenging, and applying coinfection with a small number (two or three) of multigene viruses instead may be a viable option. virus instability. this is a drawback of every baculovirus expression system, because baculoviruses deteriorate during amplification and passaging. gene deletions, notably affecting heterologous dna inserts, can potentially abolish complex production. to minimize such deleterious events, specific protocols have been delineated [13, 20] , and new approaches to overcome viral in stability by genome engineering are being developed [65] . satisfactorily been resolved, therefore, baculoviruses have not yet played a major role as gene therapy vectors. by contrast, recombinant adeno-associated virus (raav) does not have the drawbacks of baculovirus and is currently the best choice for efficient gene delivery for gene therapy [47] . however, clinical grade production of raavs, which are necessarily rendered replication incompetent, remains a major impediment for the field. a highly efficient scale-up protocol for raav production utilizes recombinant baculoviruses to produce gene-therapy-competent raav particles in insect cell cultures [48] [49] [50] [51] . this protocol involves three recombinant baculoviruses that carry genes encoding different components of raav and the transgene to be delivered (figure 3 ). co-transfection of insect cells with the three baculoviruses results in the assembly of intact raav particles, which can be further processed and purified to achieve clinical grade. a particularly noteworthy recent study has involved the production of raav particles for gene therapy of obesity in laboratory rodents (figure 3 ) [52] . the raav administered to laboratory rats fed on a high-fat diet contained leptin cdna as the transgene, introduced into the multibac baculoviral genome via cre-loxp fusion of a donor containing the leptin gene under control of a promoter that is active in mammalian cells (figure 3 ). in this review, we have presented approaches, notably the multibac technology, for producing important eukaryotic protein complexes that were hitherto not amenable to structural and functional analysis. molecules as complex as the apc/c have been successfully produced using multi-bac, enabling structure determination and architectural dissection. more recently, the technology concepts underlying multibac have been extended to mammalian gene delivery and even gene therapy, and we anticipate that many more applications will benefit from this approach. in addition, optimized multigene expression technologies that involve polyproteins and library approaches have become available, and the polyprotein technology has been integrated into multibac, increasing the prowess of the system. a beneficial next step could be to incorporate also library approaches such as coesprit [31] (box 1). this could conceivably accelerate the discovery of protein-protein interactions for complexes that rely on a eukaryotic expression host. protein-protein interactions are intensively studied in the pharmaceutical industry, with the aim to identify intermolecular surfaces that can be targeted for drug design [53] . it will be interesting to see whether this field of discovery will also benefit from the multibac technology in the future. considerable effort has been devoted to generating custom-designed insect and mammalian host cells, which provide specialized functionalities such as humanized glycosylation [54] . a logical extension of the multibac system will be to make use of these host cells, for example to produce antibodies with a close-to-human glycosylation pattern for therapeutic applications. the multibac system continues to catalyze progress in many fields of research. an entirely open question is where the limits of the multibac production technology may be. for instance, the transcriptional machinery producing eukaryotic mrnas contains, in addition to the genetic dna template, a stunning 100 proteins organized in multisubunit complexes. it may seem frivolous to set out with multibac to address this complexity structurally and functionally in a defined, fully recombinant setup. nonetheless, the first glimpses of the molecular architectures involved are emerging: the mediator head, produced by multibac, has been crystallized and the structure resolved. the stage is set. exciting times are ahead of us. the authors declare no competing financial interest. bac-vp chicken î²-actin promoter loxp donor ti bs figure 3 . application of the multibac system in gene therapy. raav particles are produced by co-infection with three different baculoviruses [48, 49] . bac-rep harbors two expression cassettes that contain genes for the major aav replication enzyme, rep78, and an n-terminal truncation of rep78, rep52d. raav-bac contains aav inverted terminal repeat (itr) elements that are required for rescue, replication and packaging of transgene sequences, together with rat leptin cdna under the control of a chicken b-actin promoter, which is inserted into the raav-bac baculoviral genome by cre-loxp-mediated fusion of a specifically tailored donor plasmid [52] . leptin is a hormone that acts in the brain to reduce food intake and stimulate energy expenditure. bac-vp produces the aav virion coat proteins. complete raav virions containing the leptin gene are produced in triply co-infected insect cells and purified [50, 52] , and then administered to dietinduced obese rats. an obese rat is shown compared to a normal rat for illustration (bottom). diet-induced obesity renders laboratory rats (and presumably other species) resistant to leptin treatment. therefore, it is close to impossible to curtail diet-induced weight gain. this could be overcome by circumventing leptin resistance or restoring leptin actions in obese animals. the surprising outcome of the study involving baculovirus-produced leptin raav as a gene therapy vector was that exercise, in this study wheel-running, was required to prevent completely weight gain when combined with the leptin gene therapy intervention, leading to the conclusion that work-out, in tandem with leptin gene delivery, may actually develop into a potential antiobesity treatment [52] . the baculovirus schematic drawing is adapted from an image kindly supplied by k. j. airenne (university of kuopio, finland). the raav particles shown are based on pdb entry 1lp3 [59] . 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procedures, database tracking and case studies structure of eukaryotic mediator complexes mediator head module structure and functional interactions stabilized baculovirus vector expressing a heterologous gene and gp64 from a single bicistronic transcript we thank christiane schaffitzel, darren hart, sergei zolotukhin, francisco asturias and all members of our laboratories for helpful discussions, and tim richmond and roger kornberg for support. cb is recipient of a swiss national science foundation (snsf) advanced researcher grant. ti is a fellow of the human frontier of science program (hsfp). yt is supported by the us national science foundation (grant mcb 0843026) and the american heart association (grant 073595n). ib acknowledges support from the snsf, the agence nationale de la recherche (anr), the centre national de la recherche scientifique (cnrs), the embl and the european commission (ec) through the joint eipod program, and the ec projects spine2-complexes and 3d-repertoire (framework program 6 (fp6)), as well as instruct, pcube, biostruct-x and complexinc (ec fp7). key: cord-271693-7tg21up3 authors: zheng, fan; zhang, she; churas, christopher; pratt, dexter; bahar, ivet; ideker, trey title: identifying persistent structures in multiscale ‘omics data date: 2020-10-03 journal: biorxiv doi: 10.1101/2020.06.16.151555 sha: doc_id: 271693 cord_uid: 7tg21up3 in any ‘omics study, the scale of analysis can dramatically affect the outcome. for instance, when clustering single-cell transcriptomes, is the analysis tuned to discover broad or specific cell types? likewise, protein communities revealed from protein networks can vary widely in sizes depending on the method. here we use the concept of “persistent homology”, drawn from mathematical topology, to identify robust structures in data at all scales simultaneously. application to mouse single-cell transcriptomes significantly expands the catalog of identified cell types, while analysis of sars-cov-2 protein interactions suggests hijacking of wnt. the method, hidef, is available via python and cytoscape. significant patterns in data often become apparent only when looking at the right scale. for example, single-cell rna sequencing data can be clustered coarsely to identify broad categories of cells (e.g. mesoderm, ectoderm), or analyzed more sharply to delineate highly specific subtypes (e.g. pancreas islet β-cells, thymus epithelium) [1] [2] [3] . likewise, protein-protein interaction networks can inform groups of proteins spanning a wide range of spatial dimensions, from protein dimers (e.g. leucine zippers) to larger complexes of dozens or hundreds of subunits (e.g. proteasome, nuclear pore) to entire organelles (e.g. centriole, mitochondria) [4] . many different approaches have been devised or applied to detect structures in biological data, including standard clustering, network community detection, and low-dimensional data projection [5] [6] [7] , some of which can be tuned for sensitivity to objects of a certain size or scale (so-called 'resolution parameters') [8, 9] . even tunable algorithms, however, face the dilemma that the particular scale(s) at which the significant biological structures arise are usually unknown in advance. guidelines for detecting robust patterns across scales come from the field of topological data analysis, which studies the geometric "shape" of data using tools from algebraic topology and pure mathematics [10] . a fundamental concept in this field is "persistent homology" [11] , the idea that the core structures intrinsic to a dataset are those that persist across different scales. recently, this concept has begun to be applied to analysis of 'omics data and particularly biological networks [12, 13] . here, we sought to integrate concepts from persistent homology with existing algorithms for network community detection, resulting in a fast and practical multiscale approach we call the hierarchical community decoding framework (hidef). hidef works in the three phases to analyze the structure of a biological dataset (methods). to begin, the dataset is formulated as a similarity network, depicting a set of biological entities (e.g. genes, proteins, cells, patients, or species) and pairwise connections among these entities (representing similarities in their data profiles). the goal of the first phase is to detect network communities, i.e. groups of densely connected biological entities. communities are identified continually as the spatial resolution is scanned, producing a comprehensive pool of candidates across all scales of analysis (fig. 1a) . in the second phase, candidate communities arising at different resolutions are pairwise aligned to identify those that have been redundantly identified and are thus persistent (fig. 1b) . in the third phase, persistent communities are analyzed to identify cases where a community is fully or partially contained within another (typically larger) community, resulting in a hierarchical assembly of nested and overlapping biological structures ( fig. 1c,d) . hidef is implemented as a python package and can be accessed interactively in the cytoscape network analysis and visualization environment [14] (availability of data and materials). we first explored the idea of measuring community persistence via analysis of synthetic datasets [15] in which communities were simulated and embedded in the similarity network at two different scales (supplementary fig. 1a; methods) . notably, the communities determined to be most persistent by hidef were found to accurately recapitulate the simulated communities at the two scales (supplementary fig. 1b-g) . in contrast, applying community detection algorithms at a fixed resolution had limited capability to capture both scales of simulated structures simultaneously (supplementary fig. 2; methods) . we next evaluated whether persistent community detection improves the characterization of cell types. we applied hidef to detect robust nested communities within cell-cell similarity networks based on the mrna expression profiles of 100,605 single cells gathered across the organs and tissues of mice (obtained from two datasets in the tabula muris project [16] ; methods). these cells had been annotated with a controlled vocabulary of cell types from the cell ontology (co) [17] , via analyses of cell-type-specific expression markers [16] . we used groups of cells sharing the same annotations to define a panel of 136 reference cell types and measured the degree to which each reference cell type could be recapitulated by a hidef community of cells (methods). we compared these results to toomanycells [18] and conos [19] , two recently developed methods that generate nested communities of single cells in divisive and agglomerative manners, respectively (methods). reference cell types tended to better match communities generated by hidef than those of other approaches, with 65% (89/136) having a highly overlapping community (jaccard index > 0.5) in the hidef hierarchy ( fig. 2a,b, supplementary fig. 3a,b) . this favorable performance was observed consistently when adjusting hidef parameters to formulate a simple hierarchy, containing only the strongest structures, or a more complex hierarchy including additional communities that are less persistent but still significant (fig. 2c, supplementary fig. 3c) . the top-level communities in the hidef hierarchy corresponded to broad cell lineages such as "t cell", "b cell", and "epidermal cell". finer-grained communities mapped to more specific known subtypes (fig. 2d) or, more frequently, putative new subtypes within a lineage. for example, "epidermal cell" was split into two distinct epidermal tissue locations, skin and tongue; further splits suggested the presence of still more specific uncharacterized cell types (fig. 2e) . hidef communities also captured known cell types that were not apparent from 2d visual embeddings (supplementary fig. 4a,b) , and also suggested new cell-type combinations. for example, astrocytes were joined with two communities of neuronal cells to create a distinct cell type not observed in the hierarchies of toomanycells [18] , conos [19] , or a two-dimensional data projection with umap [20] (fig. 2f, supplementary fig. 4c ). this community may correspond to the grouping of a presynaptic neuron, postsynaptic neuron, and a surrounding astrocyte within a so-called "tripartite synapse" [21] . next, we applied hidef to analyze protein-protein interaction networks, with the goal of characterizing protein complexes and higher-order protein assemblies spanning spatial scales. we benchmarked this task by the agreement between hidef communities and the gene ontology (go) [22] , a database that manually assigns proteins to cellular components, processes, or functions based on curation of literature (methods). application to protein-protein interaction networks from budding yeast and human found that hidef captured knowledge in go more significantly than previous pipelines proposed for this task, including the nexo approach to hierarchical community detection [23] and standard hierarchical clustering of pairwise protein distances calculated by three recent network embedding approaches [24] [25] [26] (fig. 3a, fig. 7) . we also applied hidef to analyze a collection of 27 human protein interaction networks [27, 28] . we found significant differences in the distributions of community sizes across these networks, loosely correlating with the different measurement approaches used to generate each network. for example, bioplex 2.0, a network characterizing biophysical protein-protein interactions by affinity-purification mass-spectrometry (ap-ms) [29] , was dominated by small communities of 10-50 proteins, whereas a network based on mrna coexpression [30] tended towards larger-scale communities of >50 proteins. in the middle of this spectrum, the string network, which integrated biophysical protein interactions and gene co-expression with a variety of other features [31] , contained both small and large communities (fig. 3c) . in agreement with the observation above, the hierarchy of bioplex had a relatively shallow shape in comparison to that of string (and other integrated networks including giant and pcnet [27, 32] ), in which communities across many scales formed a deep hierarchy (fig. 3d ,e; availability of data and materials). in contrast to clustering frameworks, hidef recognizes when a community is contained by multiple parent communities, which in the context of protein-protein networks suggests that the community participates in diverse pleiotropic biological functions. for example, a community corresponding to the mapk (erk) pathway participated in multiple larger communities, including ras and rsk pathways, sodium channels, and actin capping, consistent with the central roles of mapk signaling in these distinct biological processes [33] (supplementary fig. 8) . the hierarchies of protein communities identified from each of these networks have been made available as a resource in the ndex database [34] (availability of data and materials). to explore multiscale data analysis in the context of an urgent public health issue, we considered a recent application of ap-ms that characterized interactions between the 27 sars-cov-2 viral subunits and 332 human host proteins [35] . we used network propagation to select a subnetwork of the bioplex 3.0 human protein interactome [36] proximal to these 332 proteins (1948 proteins and 22,835 interactions) and applied hidef to identify its community structure (methods). among the 251 persistent communities identified (fig. 3f) , we noted one consisting of human transducin-like enhancer (tle) family proteins, tle1, tle3, and tle5, which interacted with sars-cov2 nsp13, a highly conserved rna synthesis protein in corona and other nidoviruses (fig. 3g) [37] . tle proteins are well-known inhibitors of the wnt signaling pathway [38] . inhibition of wnt, in turn, has been shown to reduce coronavirus replication [39] and recently proposed as a covid-19 treatment [40] . if interactions between nsp13 and tle proteins can be shown to facilitate activation of wnt, tles may be of potential interest as drug targets. community persistence provides a basic metric for distilling biological structure from data, which can be tuned to select only the strongest structures or to include weaker patterns that are less persistent but still significant. this concept applies to diverse biological subfields, as demonstrated here for single cell transcriptomics and protein interaction mapping. while these subfields currently employ very different analysis tools which largely evolve separately, it is perhaps high time to seek out core concepts and broader fundamentals around which to unify some of the ongoing development efforts. to that effect, the methods explored here have wide applicability to analyze the multiscale organization of many other biological systems, including those related to chromosome organization, the microbiome and the brain. consider an undirected network graph , representing a set of biological objects (vertices) and a set of similarity relations between these objects (edges). examples of interest include networks of cells, where edges represent pairwise cell-cell similarity in transcriptional profiles characterized by single-cell rna-seq, or networks of proteins, where edges represent pairwise protein-protein biophysical interactions. we seek to group these objects into communities (subsets of objects) that appear at different scales and identify approximate containment relationships among these communities, so as to obtain a hierarchical representation of the network structure. the workflow is implemented in three phases. phase i identifies communities in at each of a series of spatial resolutions . phase ii identifies which of these communities are persistent by way of a panresolution community graph ! , in which vertices represent communities, including those identified at each resolution, and each edge links pairs of similar communities arising at different resolutions. persistent communities correspond to large components in ! . phase iii constructs a final hierarchical structure that represents containment and partial containment relationships (directed edges) among the persistent communities (vertices). community detection methods generally seek to maximize a quantity known as the network modularity, as a function of community assignment of all objects [41] . a resolution parameter integrated into the modularity function can be used to tune the scale of the communities identified [9, 42, 43] , with larger/smaller scale communities having more/fewer vertices on average (fig. 1a) . of the several types of resolution parameter that have been proposed, we adopted that of the reichardt-bornholdt configuration model [42] , which defines the generalized modularity as: where ⃗ defines a mapping from objects in to community labels; " is the degree of vertex ; is the total number of edges in ; is the resolution parameter; ( , ) indicates that vertices and are assigned to the same community by ⃗ ; and is the adjacency matrix of . to determine two values satisfying the above formula are defined as -proximal. the sampling step, which was practically set to 0.1 to sufficiently capture the interesting structures in the data; it is conceptually similar to the nyquist sampling frequency in signal processing [44] . we used $"% = 0.001, which we found always resulted in the theoretical minimum number of communities, equal to the number of connected components in . we used $&' = 20 for single-cell data ( fig. 2 to identify persistent communities, we define the pairwise similarity between any two communities and as the jaccard similarity of their sets of objects, ( ) and ( ): we initialize a hierarchical structure represented by , a directed acyclic graph (dag) in which each vertex represents a persistent community. a root vertex is added to represent the community of all objects. the containment relationship between two vertices, and , is quantified by the containment index (ci): which measures the fraction of objects in shared with . an edge is added from to in if ( , ) is larger than a threshold ( is -contained by ). since ( , ) < for all , (a property established by the procedure for connecting similar communities in phase ii), setting ≥ 2 /(1 + ) guarantees to be acyclic. in practice we used a relaxed threshold = , which we found generally maintains the acyclic property but includes additional containment relations. in the (in our experience rare) event that cycles are generated in , i.e. ( , ) ≥ and ( , ) ≥ , we add a new community to , the union of and , and remove and from . finally, redundant relations are removed by obtaining a transitive reduction [45] of , which represents the hierarchy returned by hidef describing the organization of communities. the biological objects assigned to each community are expanded to include all objects assigned to its descendants. throughout this study, we used the parameters = 0.75, = 5, = 75. note that since is a threshold of minimum persistence, the results under a larger value of ′ can be produced by simply removing communities with persistence lower than ′ (figs. 2c, 3afig. 9 ). different combinations of parameters and typically do not significantly change the performance of hidef in the benchmark tests on protein-protein interaction networks (supplementary fig. 6 ), except that certain parameters (e.g. = 0.9) are less robust to network perturbation (i.e. randomly deleting edges from networks). we found that combining hidef with node embedding resolved this issue and further improved the performance and robustness (supplementary fig. 7 ; see sections below). simulated network data were generated using the lancichinetti-fortunato-radicchi (lfr) method [15] (supplementary figs. 1,2) . we used an available implementation (lfr benchmark graphs package 5 at http://www.santofortunato.net/resources) to generate benchmark networks with two levels of embedded communities, a coarse-grained (macro) level and a fine-grained (micro) level. within each level, a vertex was exclusively assigned to one community. two parameters, c and f, were used to define the fractions of edges violating the simulated community structures at the two levels. all other edges were restricted to occur between vertices assigned to the same community (supplementary fig. 1a) . we fixed other parameters of the lfr method to values explored by previous studies [9] . some community detection algorithms include iterations of local optimization and vertex aggregation, a process that, like hidef, also defines a hierarchy of communities, albeit as a tree rather than a dag. we demonstrated that without scanning multiple resolutions, this process alone was insufficient to detect the simulated communities at all scales (supplementary fig. 2) . we used louvain and infomap [46, 47] , which have stable implementations and have shown strong performance in previous community detection studies [48] . for louvain, we optimized the and other parameters to default. in general, these settings generated trees with two levels of communities. note that infomap sometimes determined that the input network was nonhierarchical, in which cases the coarse-and fine-grained communities were identical by definition. mouse single-cell rna-seq data ( fig. 2; supplementary fig. 3 identical analyses were applied to the facs and the droplet datasets respectively, yielding a hierarchy of 273 and 279 communities respectively (fig. 2d) . scanpy 1.4.5 [49] was used to create tsne or umap embeddings and associated two-dimensional visualizations [20] as baselines for comparison (fig. 2e,f; supplementary fig. 3a,b) . through previous analysis of the single-cell rna data, all cells in these datasets had been annotated with matching cell-type classes in the cell ontology (co) [17] . before comparing these annotations with the communities detected by hidef, we expanded the set of annotations of each cell according to the co structure, to ensure the set also included all of the ancestor cell types of the type that was annotated. for example, co has the relationship "[keratinocyte] (is_a) [epidermal_cell]", and thus all cells annotated as "keratinocyte" are also annotated as "epidermal cell". the co was obtained from http://www.obofoundry.org/ontology/cl.html and processed by the data driven ontology toolkit (ddot) [50] retaining "is_a" relationships only. we compared hidef to toomanycells [18] and conos [19] as baseline methods. the former is a divisive method which iteratively applies bipartite spectral clustering to the cell population until the modularity of the partition is below a threshold; the latter uses the walktrap algorithm to agglomeratively construct the cell-type hierarchy [51] . we chose to compare with these methods because their ability to identify multiscale communities was either the main advertised feature or had been shown to be a major strength. toomanycells (version 0.2.2.0) was run with the parameter "min-modularity" set to 0.025 as recommended in the original paper [18] , with other settings set to default. this process generated dendrograms (binary trees) with 463 communities. the walktrap algorithm was run from the conos package (version 1.2.1) with the parameter "step" set to 20 as recommended in the original paper [19] , yielding a dendogram. the greedymodularitycut method in the conos package was used to select n fusions in the original dendrogram, resulting in a reduced dendrogram with 2n+1 communities (including n internal and n+1 leaf nodes). here we used n = 125, generating a hierarchy with 251 communities (fig. 2c) . the communities in each hierarchy were ranked to analyze the relationships between celltype recovery and model complexity (fig. 2c, supplementary fig. 3c) . hidef communities were ranked by their persistence; conos and toomanycells communities were ranked according to the modularity scores those methods associate with each branch-point in their dendrograms. conos/walktrap uses a score based on the gain of modularity in merging two communities, whereas toomanycells uses the modularity of each binary partition. we obtained a total of 27 human protein interaction networks gathered previously by survey studies [27, 28] , along with one integrated network from budding yeast (s. cerevisiae) that had been used in a previous community detection pipeline, nexo [23] . this collection contained two versions of the string interaction database, with the second removing edges from text mining (labeled string-t versus string, respectively; fig. 3 ). benchmark experiments for the recovery of the gene ontology (go) were performed with string and the yeast network ( fig. 3a,b, supplementary fig. 4) . the reference go for yeast proteins was obtained from http://nexo.ucsd.edu/. a reference go for human proteins was downloaded from http://geneontology.org/ via an api provided by the ddot package [50] . hidef was directly applied to all of the above benchmark networks. the nexo communities were obtained from http://nexo.ucsd.edu/, with a robustness score assigned to each community. to benchmark communities created by hierarchical clustering, we first calculated three versions of pairwise protein distances (hc.1-3; fig. 3a,b; supplementary fig. 4) using mashup, dsd and deepnf [24] [25] [26] . mashup was used to embed each protein as a vector, with 500 and 800 dimensions for yeast and human, as recommended in the original paper. a pairwise distance was computed for each pair of proteins as the cosine distance between the two vectors. similarly, deepnf was used to embed each protein into a 500-dimensional vector by default. dsd generates pairwise distances by default. given these pairwise distances, upgma clustering was applied to generate binary hierarchical trees. following the procedure given in the nexo and mashup papers [23, 24] communities with <4 proteins were discarded. since all methods had slight differences in the resulting number of communities, communities from each method were sorted in decreasing order of score, enabling comparison of results across the same numbers of top-ranked communities. hidef communities were ranked by persistence. nexo communities were ranked by the robustness value assigned to each community in the original paper [23] . to rank each community c of hierarchical clustering (branch in the dendrogram), a one-way mann-whitney u-test was used to test for significant differences between two sets of protein pairwise distances: (set 1) all pairs consisting of a protein in c and a protein in the sibling community of c; (set 2) all pairs consisting of a protein in each of the two children communities of c. the communities were sorted by the one-sided p-value of significance that distances in set 1 are greater than those in set 2. we adopted a metric average f1-score [52] to evaluate the overall performance of multiscale structure identification, focusing on the recovery of reference communities. given a set of reference communities * and a set of computationally detected communities ⃗ , the score was defined as: where ( ) is the best match of " in ⃗ , defined as follows: and 1( " , 1 sss⃗ ) is the harmonic mean of precision( " , 1 sss⃗ ) and recall( " , 1 sss⃗ ). the calculations were conducted by the xmeasures package (https://github.com/exascaleinfolab/xmeasures) [53] . hidef was directly applied to the original networks in in most of our analyses of protein-protein interaction networks, and compared with the results of hierarchical clustering following the network embedding techniques [24, 26] . we sought to explore if we can combine the strength of network embedding and hidef to further improve the performance and robustness to parameter choices (supplementary fig. 7) . we borrowed the idea of shared-nearest neighbor (snn) graph that we had been using in the analyses of single-cell data. we made a customized script to use the 500-dimensional node embeddings of the string network as the input of the seurat findneighbors function [3] . the parameters of this function remained as the default. the output snn graph has 1.65 ´ 10 6 edges, which is on the same magnitude as the original network (2.23 ´ 10 6 edges). we then applied hidef to this snn graph with different combinations of parameters ( supplementary fig. 7) . 332 human proteins identified to interact with sars-cov-2 viral protein subunits were obtained from a recent study [35] . this list was expanded to include additional human proteins connected to two or more of the 332 virus-interacting human proteins in the new bioplex 3.0 network [36] . these operations resulted in a network of 1948 proteins and 22,835 interactions. hidef was applied to this network with the same parameter settings as for other protein-protein interaction networks (see previous methods sections), and enrichment analysis was performed via g:profiler [54] (fig. 3f,g) . not applicable. not applicable. these models include the hierarchy of murine cell types (fig. 2) , the hierarchies of yeast and human protein communities identified through protein network analysis, and the hierarchy of human protein complexes targeted by sars-cov2 (fig. 3) . t.i. is cofounder of data4cure, is on the scientific advisory board, and has an equity interest. t.i. . a yeast network [23] and the human string network [31] were used as the inputs of a and b, respectively. hc.1-3 represent upgma hierarchical clustering of pairwise distances generated by mashup, dsd, and deepnf [24] [25] [26] , respectively. c, distributions of community sizes (x-axis, number of proteins) for three human protein networks: bioplex 2.0 [29] , coexpr-geo [30] , and string [31] . supplementary figure 1 . exploring simulated networks. a, the lfr generative model [15] was used to simulate networks with 1000 vertices and average degree 10 (methods). the simulation included two layers of communities, "coarse" (10-20 communities, 50-100 vertices per community) and "fine" (25companion plots to panels (b-d). points represent identified communities, delineated by size (y axis) and persistence (x axis). blue/gray point colors indicate a match/non-match to a true community in the simulated network (jaccard similarity > 0.75). note that when noise is low (e), the highest persistence communities correctly recover simulated communities with near-perfect accuracy, e.g. for persistence threshold >20. hidef is compared with the louvain and infomap algorithms [46, 47] , with louvain and infomap fixed at their default single resolutions (methods). the three plots (a-c) compare the performance of the three algorithms in recovering simulated communities at different settings of the coarse/fine mixing parameters (see supplementary fig. 1 clustering following any of three protein pairwise distance functions (mashup, dsd, and deepnf) [24] [25] [26] . using the performance analysis depicted in fig. 3b , the area under curve (auc) was computed for different sets of hidef parameters (p, ). this auc was compared to that of the best baseline tool, hc.3 (i.e. hierarchical clustering of pairwise distances generated by deepnf [26] ) to generate an equal number of communities (methods). note the ratio hidef auc / hc.3 auc is usually higher than 1, indicating the favorable performance of hidef except for very high values of the t parameter. as per fig. 3b , the analysis was undertaken using the string network and the go cellular component branch. b, similar analysis with subsampling of network edges (in which a random 10% of network edges are removed prior to community detection at each resolution). higher persistence (y axis) than a given threshold (x axis). e-f, scatterplots of community size (y axis) versus persistence (x axis). the left column characterizes the single-cell transcriptomics data (fig. 2, supplementary fig. 3) . the right column (panel b, d, f) characterizes the yeast and human protein-protein interaction datasets ( fig. 3a-b) . the human cell atlas data-driven phenotypic dissection of aml reveals progenitor-like cells that correlate with prognosis integrating single-cell transcriptomic data across different conditions, technologies, and species molecules into cells: specifying spatial architecture data clustering: a review community detection in networks: a user guide visualizing data using t-sne analysis of the structure of complex networks at different resolution levels van dooren p: significant scales in community structure persistent homology-a survey a topological paradigm for hippocampal spatial map formation using persistent homology homological scaffolds of brain functional networks cytoscape: a software environment for integrated models of biomolecular interaction networks benchmark graphs for testing community detection algorithms organ collection and p, library preparation and s, computational data a, cell type a, writing g, supplemental text writing g, principal i: single-cell transcriptomics of 20 mouse organs creates a tabula muris the cell ontology 2016: enhanced content, modularization, and ontology interoperability toomanycells identifies and visualizes relationships of single-cell clades joint analysis of heterogeneous single-cell rna-seq dataset collections dimensionality reduction for visualizing single-cell data using umap tripartite synapses: astrocytes process and control synaptic information gene ontology: tool for the unification of biology. the gene ontology consortium a gene ontology inferred from molecular networks compact integration of multi-network topology for functional analysis of genes going the distance for protein function prediction: a new distance metric for protein interaction networks deepnf: deep network fusion for protein function prediction systematic evaluation of molecular networks for discovery of disease genes assessment of network module identification across complex diseases architecture of the human interactome defines protein communities and disease networks a next generation connectivity map: l1000 platform and the first 1,000,000 profiles string v11: protein-protein association networks with increased coverage, supporting functional discovery in genomewide experimental datasets understanding multicellular function and disease with human tissue-specific networks activation and function of the mapks and their substrates, the mapk-activated protein kinases ndex 2.0: a clearinghouse for research on cancer pathways a sars-cov-2 protein interaction map reveals targets for drug repurposing dual proteome-scale networks reveal cellspecific remodeling of the human interactome the nonstructural proteins directing coronavirus rna synthesis and processing molecular functions of the tle tetramerization domain in wnt target gene repression inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide broad spectrum antiviral agent niclosamide and its therapeutic potential finding and evaluating community structure in networks statistical mechanics of community detection introduction to digital signal processing the transitive reduction of a directed graph fast unfolding of communities in large networks maps of random walks on complex networks reveal community structure scanpy: large-scale single-cell gene expression data analysis ddot: a swiss army knife for investigating data-driven biological ontologies computing communities in large networks using random walks overlapping community detection at scale: a nonnegative matrix factorization approach accuracy evaluation of overlapping and multiresolution clustering algorithms on large datasets profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update) the reactome pathway knowledgebase we are grateful for the helpful discussions with drs. jianzhu ma, karen mei, and daniel carlin. reactome [55] . key: cord-274366-t138l6px authors: benetti, elisa; tita, rossella; spiga, ottavia; ciolfi, andrea; birolo, giovanni; bruselles, alessandro; doddato, gabriella; giliberti, annarita; marconi, caterina; musacchia, francesco; pippucci, tommaso; torella, annalaura; trezza, alfonso; valentino, floriana; baldassarri, margherita; brusco, alfredo; asselta, rosanna; bruttini, mirella; furini, simone; seri, marco; nigro, vincenzo; matullo, giuseppe; tartaglia, marco; mari, francesca; renieri, alessandra; pinto, anna maria title: ace2 gene variants may underlie interindividual variability and susceptibility to covid-19 in the italian population date: 2020-07-17 journal: eur j hum genet doi: 10.1038/s41431-020-0691-z sha: doc_id: 274366 cord_uid: t138l6px in december 2019, an initial cluster of interstitial bilateral pneumonia emerged in wuhan, china. a human-to-human transmission was assumed and a previously unrecognized entity, termed coronavirus disease-19 (covid-19) due to a novel coronavirus (sars-cov-2) was described. the infection has rapidly spread out all over the world and italy has been the first european country experiencing the endemic wave with unexpected clinical severity in comparison with asian countries. it has been shown that sars-cov-2 utilizes angiotensin converting enzyme 2 (ace2) as host receptor and host proteases for cell surface binding and internalization. thus, a predisposing genetic background can give reason for interindividual disease susceptibility and/or severity. taking advantage of the network of italian genomes (nig), here we mined whole-exome sequencing data of 6930 italian control individuals from five different centers looking for ace2 variants. a number of variants with a potential impact on protein stability were identified. among these, three more common missense changes, p.(asn720asp), p.(lys26arg), and p.(gly211arg) were predicted to interfere with protein structure and stabilization. rare variants likely interfering with the internalization process, namely p.(leu351val) and p.(pro389his), predicted to interfere with sars-cov-2 spike protein binding, were also observed. comparison of ace2 wes data between a cohort of 131 patients and 258 controls allowed identifying a statistically significant (p value < 0.029) higher allelic variability in controls compared with patients. these findings suggest that a predisposing genetic background may contribute to the observed interindividual clinical variability associated with covid-19, allowing an evidence-based risk assessment leading to personalized preventive measures and therapeutic options. in december 2019, a new infectious respiratory disease emerged in wuhan, hubei province, china [1 -3] . an initial cluster of infections likely due to animal-to-human transmission was rapidly followed by a human-to-human transmission [4] . the disease was recognized to be caused by a novel coronavirus (sars-cov-2) and termed coronavirus disease-19 . the infection spread within china and all over the world, and it has been declared as pandemic by the world health organization (who) on 2nd march 2020. the symptoms of covid-19 range from fever, dry cough, fatigue, congestion, sore throat, and diarrhea to severe interstitial bilateral pneumonia with a ground-glass image at the ct scan. while recent studies provide evidence of a high number of asymptomatic or paucisymptomatic patients who represent the main reservoir for the infection progression, the severe cases can rapidly evolve towards a respiratory distress syndrome which can be lethal [5] . although age and comorbidity have been described as the members main determinants of disease progression towards severe respiratory distress, the high variation in clinical severity among middle-age adults and children would likely suggest a strong role of the host genetic asset. a high sequence homology has been shown between sars-associated coronavirus (sars-cov) and sars-cov-2 [6] . recent studies modeled the spike protein to identify the receptor for sars-cov-2 and indicated that angiotensin converting enzyme 2 (ace2) is the receptor for this novel coronavirus [7, 8] . zhou et al. conducted virus infectivity studies and showed that ace2 is essential for sars-cov-2 to enter hela cells [9] . although the binding strength between sars-cov-2 and ace2 is weaker than that between sars-cov and ace2, it is considered as much high as threshold necessary for virus infection. the spike glycoprotein (s-protein), a trimeric glycoprotein in the virion surface (giving the name of crown -corona in latin-), mediates receptor recognition throughout its receptor binding domain (rbd) and membrane fusion [10, 11] . based on recent reports, sars-cov-2 protein binds to ace2 through leu455, phe486, gln493, ala501, and tyr505. it has been postulated that residues 31, 41, 82, 353, 355, and 357 of the ace2 receptor map to the surface of the protein interacting with sars-cov-2 spike protein [12] , as previously documented for sars-cov. following interaction, cleavage of the c-terminal segment of ace2 by proteases, such as transmembrane protease serine 2 (tmprss2), enhances the spike protein-driven viral entry [13, 14] . thus, it is possible, in principle, that genetic variability of the ace2 receptor is one of the elements modulating virion intake and thus disease severity. ace2 is located on chromosome x. although it is one of the genes escaping x inactivation several lines of evidence suggest that a different degree of x-chromosome inactivation (xci) is present in distinct tissues [15] . taking advantage of the network of italian genomes (nig), a consortium established to generate a public database (nig-db) containing aggregate variant frequencies data for the italian population (http://www.nig.cineca.it/), here we describe the genetic variation of ace2 in the italian population, one of the newly affected countries by the sars-cov-2 outbreak causing covid-19. three common c.2158a>g p.(asn720asp), c.77a>g p.(lys26arg), and c.631g>a p.(gly211arg) variants and 27 rare missense variants were identified, 9 of which had not previously been reported in public databases. we show that p.(asn720asp), which lies in a residue located close to the cleavage sequence of tmprss2, likely affects the cleavagedependent virion intake. along with the other two common variants, this substitution is represented in the italian and european populations but is extremely rare in the asian population. we also show that two rare variants, namely, c.1051c>g p.(leu351val) and c.1166c>a p.(pro389his) are predicted to cause conformational changes impacting rbd interaction. as the uncertainty regarding the transmissibility and severity of disease rise, we believe that a deeper characterization of the host genetics and functional characterization of variants may help not only in understanding the pathophysiology of the disease but also in envisaging risk assessment. the work has been realized in the context of the nig, with the contribution of centers: azienda ospedaliera universitaria senese, azienda ospedaliera universitaria policlinico sant'orsola-malpighi di bologna, città della salute e della scienza di torino, università della campania "luigi vanvitelli", ospedale pediatrico bambino gesù. the nig (http://www.nig.cineca.it/) aim is to create a shared database (nig-db) containing data from nucleic acids sequencing of italian subjects. this database allows defining an italian reference genome for the identification of genes responsible for genetic diseases or italian population susceptibility to complex disorders and for the detection of genetic variants responsible for interindividual differences in disease progression ad /or drug response among the italian population. individuals coming to our centers were offered to participate to the nig study and blood withdrawal was performed upon informed consent. individuals provided signed informed consents at each participating center for whole-exome sequencing analysis (wes), and clinical and molecular data storage and usage. all subjects were unrelated, healthy, and of italian ancestry. italian origin was ascertained asking for parents and grandparents origin. dna has been stored in the telethon network of genetic biobanks (project no. gtb12001), funded by telethon italy. the study was consistent with institutional guidelines and approved by the university hospital (azienda ospedaliera universitaria senese) ethical committee, siena, italy (prot n. 16929, dated march 16, 2020). written informed consent was obtained from all patients and controls. peripheral blood samples in edta-containing tubes and detailed clinical data were collected. all these data were inserted in a section of the established and certified biobank and registry of the medical genetics unit of the hospital dedicated to covid-19. the cohort of covid-19 patients consists of 131 individuals out of whom 34 females and 97 males belonging to the gen-covid multicenter study ([16] , late breaking abstract eshg 2020.2 virtual conference "wes profiling of covid-19"). the cohort of controls consists of 258 italian individuals (129 males and 129 females). all patients are of italian ethnicity. the median age is 64 years (range 31-98): median age for women 66 years and for males 63 years. the population was clustered into four qualitative severity groups depending on the respiratory impairment and the need for ventilation: high care intensity group (those requiring invasive ventilation), intermediate care intensity group (those requiring noninvasive ventilation i.e., cpap and bipap, and high-flows oxygen therapy), low care intensity group (those requiring conventional oxygen therapy) and very low care intensity group (those not requiring oxygen therapy). targeted enrichment and massively parallel sequencing were performed on genomic dna extracted from circulating leukocytes of 6930 individuals. genomic dna was extracted from peripheral blood samples using standard procedures. exome capture was carried out using sur-eselect human all exon v4/v5/v6/v7 ( broad institute, harvard, usa). alignment of raw reads against reference genome hg19, variant calling and annotation were attained using in-house pipelines [17] [18] [19] which take advantage of the gatk best practices workflow [20] and of annovar, vep [21, 22] . the genome aggregation database gnomad (https://gnomad.broadinstitute.org/) was used to assess allele frequency for each variant among different populations. the mean depth of coverage of each ace2 exon in all participants was 55×. variants with a depth of coverage lower that 20× were filtered out according to ashg guidelines for germline variants [23] . the the structure of native human angiotensin converting enzyme-related carboxypeptidase (ace2) was downloaded from protein data bank (https://www.rcsb.org/) (pdb id code 1r42) [24] . the duet program [25] was used to predict the possible effect of amino acids substitutions on the protein structure and function, based on the use of machine-learning algorithms exploiting the threedimensional structure to quantitatively predict the effects of residue substitutions on protein functionality. molecular dynamics (md) simulations of wild-type and variant ace2 proteins were carried out in gromacs 2019.3 [26] to calculate root mean square deviation (rmsd) to define structural stability. the graphs were plotted by the xmgrace software [27] . md simulations were performed using a high parallel computing infrastructure (hpcs) with 660 cpu within 21 different nodes, 190t of ram, 30t hard disk partition size, and six nvidia tesla gpu with cuda support. pymol2.3 was used as a molecular graphic interface. the protein structures were solvated in a triclinic box filled with tip3p water molecules and na + /cl − ions were added to neutralize the system. the whole systems were then minimized with a maximal force tolerance of 1000 kj mol −1 nm −1 using the steepest descendent algorithm. the optimized systems were gradually heated to 310 k in 1 ns in the nvt ensemble, followed by 10 ns equilibration in the npt ensemble at 1 atm and 310 k, using the v-rescale thermostat and berendsen barostat [28, 29] . subsequently, a further 100 ns md simulations were performed for data analysis. the extent of variability along the entire ace2 coding sequence and flanking intronic stretches was assessed using 6930 italian wes, out of which 4171 males and 2759 females which sum up to 9689 alleles. identified variants and predicted effects on protein stability are summarized in tables 1, 2, and table s1 , and represented in exomes. in addition to these variants, 28 rare missense variants were identified, out of which ten had not previously been reported in gnomad database and nine truncating variants that had not been reported in gnomad database (table 1 and supplementary table 1 ). out of these variants, two fall in the neck domain, which is essential for dimerization and one in the intracellular domain. many of them truncate the protein in different positions of the protease domain embedded in the extracellular domain, which contains the receptor binding site for sars-cov-2. only three truncating variants have been previously described for ace2 likely due to a low-tolerance for loss-of-function variants. in line with this evidence, all these variants were very rare and no homozygous females were detected for the identified variants. three missense changes c.1517t>c p. (fig. 3a) , the c.1166c>a p.(pro389his) and c.1051c>g p.(leu351val) variants show a difference in comparison with the native protein with a gradual increase in rmsd value, which stabilizes at an average of 0.5 nm (fig. 3a) . finally, the c.631g>a p. (gly211arg) shows a bigger difference with a higher increase in rmsd value, which stabilizes at an average of 0.6 nm (fig. 3a) . structural analysis between wt and mutant c.1517t>c p.(val506ala) md simulations showed that the c.1517t>c p.(val506ala) forms a hydrophobic center together with leu456, leu503, and phe516 with minimum differences in protein rearrangements when the residue is mutated in ala as reported in fig. 2 and supplementary video s1. the c.77a>g p.(lys26arg) is located at the n-terminus and the sidechain engages a hydrogen bond with asn90 thus determining a minimal destabilizing predicted effect as shown in table 2 during md simulations, we have also investigated the surrounding region of ace2 wt and previously selected variants by calculating change in solvent accessibility surface area (sasa). differences in average sasa value would suggest for the native protein a wider surface exposed to solvent and subsequently a different ability to interact with spike sars-cov-2 in comparison with the studied variants (fig. 3b) . in order to shed light on the role of ace2 variants on interindividual variability and susceptibility to covid-19 in italian population we performed wes analysis on a cohort of 131 patients and 258 controls who agreed in participating to the study (see "materials and methods"). data analysis of ace2 variants identified a different distribution of variants in controls compared with patients ( fig. 4) according to recent reports, ace2 is essential for sars-cov-2 to enter cells. recent single-cell rna studies have also shown that ace2 is expressed in human lung cells [30] . the majority of ace2-expressing cells are alveolar type 2 cells. other ace2-expressing cells include alveolar type 1 cells, airway epithelial cells, fibroblasts, endothelial cells, and macrophages although their ace2-expressing cell ratio is low and variable among individuals. the expression and distribution of the ace2 receptor can thus justify the route of infection and the main localization at the alveolar level. although the different density of ace2 receptors in the upper respiratory tract among individuals can partially give reason for the clinical variability, which (5 å) y180, l456, r460, p500, a501, s502, l503, f504, h505, n506, s507 y207, e208, v209, n210, g211, v212, y215, d216, y217, p565, t567 h373, h374, e375, m376, g377, h378, i379, a380, y381, f315, h401, v404, g405, m408 l262, p263, a264, h265, l266, l267, w271, w478, v487, v488, e489, p490, w165 y497, c498, d499, p500, a501, s502, g173, r177, l176, y180, w459, w473, m474 a242, y243, v244, r245, a246, k247, l248, duet program results that display predicted change in folding free energy upon ace2 missense variant (δδg in kcal/mol). in the first three columns are reported single missense variants with specific position on ace2 protein. the residues in the first column highlighted in gray are involved in n-glycosylation pattern nxt/s, therefore those missense variants determine the loss glycosylation of asparagine 53 and 90, respectively. in the fourth column is reported δδg analysis predict effects of missense variants on protein stability using an integrated computational approach. the column "interaction network around (5 å)" shows for each single missense variant the residues around 5 å. in this column, we highlight in green residues involved in spike sars-cov protein interaction, in yellow residues involved in zinc coordination and finally in magenta residues of asn involved in n-glycosylation. the last column defines the outcome of protein stability for each single missense variant. an increasing negative value for the δδg is correlated with a higher destabilizing effect, while a positive value is associated with a variant predicted as stabilizing. ranges from asymptomatic/paucisymptomatic patients to severely affected ones, it could not be the only reason for such variability. in addition, recent works did not observe significantly different viral loads in nasal swabs between symptomatic and asymptomatic patients [31] . italy has been the first european country that experienced the covid-19 outbreak with a rapid increase in the positive cases in a very short-time period and a morbidity and lethality (~10%) definitely higher in comparison with asian countries, such as china (4%) and south korea (1.2%) [32] . these considerations raise the possibility of a predisposing genetic background accounting for or contributing to the wide interindividual clinical variability, as well for the differential morbidity and lethality observed among different countries, population awareness, and constrictive measures apart. we integrated genomic wes data produced by five italian centers (siena, naples, turin, bologna, and rome) interconnected by the nig in the attempt to identify variation encompassing the ace2 gene, which could account for a difference in sars-cov-2 spike binding affinity, processing, or internalization. previous studies showed that the residues near lysine 31, and tyrosine 41, 82-84, and 353-357 in human ace2 are important for the binding of s-protein to coronavirus [12] . in line with previous reports [33] , we did not find polymorphism or rare variants in these residues in the italian population. however, we identified three variants namely c.2158a>g p.(asn720asp), c.1166c>a p.(pro389his), and c.1051c>g p.(leu351val), one of which polymorphic c.2158a>g p.(asn720asp), moderately expressed in the italian and european non-finnish populations and with a very low allele frequency or not occurring in the eastern asia population. these variants which surround residual essentials for the sars-cov-2 spike protein binding were predicted to likely affect the cleavage-dependent virion intake, such as the polymorphic c.2158a>g p.(asn720asp) (allele frequency 0.011) which lies four amino acids from the cleavage sequence of tmprss2 or to have a substantial impact on protein structure and spike protein interaction by md simulation (fig. 3a) . the relatively frequent c.631g>a p.(gly211arg) (allele frequency 0.0012, 12/6930 individuals) was predicted to confer a wide flexibility to the region because of the ability to engage different interactions with the nearby amino acid residues. along with these more common variants we also identified very rare variants such as the c.1166c>a p.(pro389his) and the c. finnish european population, that could give reason for a different affinity for the sars-cov-2 spike protein (figs. 2, 3a and supplementary video s4). interestingly all the studied variants affect residues highly conserved among species (supplementary fig. s1 ). given their rarity in other populations, we cannot exclude that these variants can fig. 3 structure superimposition snapshot between wild-type protein and variant proteins. a root mean square deviation (rmsd) trends for the backbone of ace2 wt (black line) and some selected variants (colored lines, see legend) during 100 ns of simulation. the molecular dynamics simulation shows a good stability for all systems with exception of g211r mutants. rmsd is a parameter used to define the stability of an element. wild type shows a steady course in the rmsd value, stabilizing at an average of 0.2 nm, while, the g211r variant shows a gradual increase in rmsd value, stabilizing at an average of 0.6 nm. b sasa graphical representation of ace2 wt (black line) and ace2 variants (colored lines, see legend). partially account for the clinical outcome observed in the italian population. wes data generated from a wide cohort of covid-19 italian patients revealed a statistically significant (p < 0,029) higher allelic heterogeneity for ace2 in controls compared with patients with a higher chance to find at least one ace2 variant in the cohort of controls compared with the cohort of patients. therefore, it is plausible to think that the effect of allelic variability on ace2 conformation would at least partially account for the interindividual clinical differences and likely modulate clinical severity. this finding reinforces the hypothesis that at least some of the identified variants or the cumulative effect of few of them confer a different susceptibility to virus cell entry and consequently to disease onset and progression. we cannot exclude that also silent variants such as the c.2247g>a (p. val749val) with no effect on the protein could play a role because of an unpredictable impact at a posttranscriptional level. notably, morbidity and lethality have been reported definitely higher in men compared with women (~70% vs. 30%, 20th march 2020 iss report). although several parameters have been brought to case to explain this difference, i.e., smoking, differences in ace2 localization and/or density in alveolar cells, hormonal asset, it is noteworthy that ace2 is located on chromosome x and that given the low allele frequency of the identified variants the rate of homozygous women is extremely low (see results section). the xci is incomplete in humans and some genes show a degree of xci escape which vary between individuals and tissues [34] . ace2 is one of the genes escaping x inactivation, but it belongs to a subgroup of x-chromosome genes showing a higher expression in men in several tissues thus mostly suggesting that ace2 gene xci is present although different in distinct tissues [15] . therefore, the impact of x inactivation on the alternate expression of the two alleles would guarantee, in the affected tissues, a heterogeneous population of ace2 molecules, some of which protective towards the infection until the point of a complete or almost complete protection in the case of a x inactivation skewed towards the less sars-cov-2-binding prone allele. this hypothesis would justify the high rate of asymptomatic or paucisymptomatic patients. however, the presented data does not allow to confirm a clear cause-effect relationship and, since most of the identified variants have very low frequencies, further functional studies are needed to validate these results. ace2 is definitely one of the main molecules whose genetic heterogeneity can modulate infection and disease progression; however, a deeper characterization of the host genetics and functional variants in other pathwayrelated genes may help in understanding the pathophysiology of the disease opening up the way to a stratified risk assessment and to tailored preventive measures and treatments. questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. conflict of interest the authors declare that they have no conflict of interest. ethical approval the nig study was approved by the ethical committee of each center, prot name nig prot n 10547_2016, approved ceavse on 18.07.2016 n 88/16. the covid-19 patients study was approved by the university hospital (azienda ospedaliera universitaria senese) ethical committee, siena, italy (prot n. 16929, dated march 16, 2020). publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. department of specialized and internal medicine, pneumology unit and utip, san donato hospital 33 department of health sciences, clinic of infectious diseases department of molecular medicine, university of padova, padova, italy; 47 department of infectious and tropical diseases, university of brescia and asst spedali civili hospital clinical features of patients infected with 2019 novel coronavirus in wuhan a novel coronavirus outbreak of global health concern a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2): facts and myths networkbased drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 pathological findings of covid-19 associated with acute respiratory distress syndrome viral dynamics in mild and severe cases of covid-19 a pneumonia outbreak associated with a 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the genome analysis toolkit: a mapreduce framework for analyzing next-generation dna sequencing data annovar: functional annotation of genetic variants from high-throughput sequencing data the ensembl variant effect predictor acmg clinical laboratory standards for nextgeneration sequencing ace2 x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis duet: a server for predicting effects of mutations on protein stability using an integrated computational approach gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. soft-warex version 5.1.19 canonical sampling through velocity rescaling molecular dynamics with coupling to an external bath single-cell rna expression profiling of ace2, the putative receptor of wuhan the early phase of the covid-19 outbreak in lombardy how deadly is covid-19? a rigorous analysis of excess mortality and agedependent fatality rates in italy comparative genetic analysis of the novel coronavirus (2019-ncov/sars-cov-2) receptor ace2 in different populations x-inactivation profile reveals extensive variability in x-linked gene expression in females 8 • francesco musacchia 9 • tommaso pippucci 10 • annalaura torella 11 • alfonso trezza 3 • floriana valentino 7 • margherita baldassarri 7 • alfredo brusco 5,12 • rosanna asselta 13,14 • mirella bruttini 2,7 • simone furini 1 • marco seri 8,10 • vincenzo nigro 9,11 • giuseppe matullo 5,12 • marco tartaglia 4 • francesca mari 2,7 • gen-covid multicenter study • alessandra renieri author contributions eb, rt, os, amp, and ar have made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, and have been involved in drafting the paper. ra, gb, abruselles, abrusco, gd, ag, fm, tp, atorella, atrezza, and fv has made substantial contributions to acquisition and analysis of the data. mbaldassarri, mbruttini, ac, sf, fm, gm, vn, ms, and mt have made substantial contributions to interpretation of data and clinical evaluation. all authors have been involved in drafting the paper; have given final approval of the version to be published and agree to be accountable for all aspects of the work in ensuring that key: cord-017887-pj6pal35 authors: ouyang, bo; dong, ying; chou, james j. title: structural and functional properties of viral membrane proteins date: 2018-06-29 journal: advances in membrane proteins doi: 10.1007/978-981-13-0532-0_6 sha: doc_id: 17887 cord_uid: pj6pal35 viruses have developed a large variety of transmembrane proteins to carry out their infectious cycles. some of these proteins are simply anchored to membrane via transmembrane helices. others, however, adopt more interesting structures to perform tasks such as mediating membrane fusion and forming ion-permeating channels. due to the dynamic or plastic nature shown by many of the viral membrane proteins, structural and mechanistic understanding of these proteins has lagged behind their counterparts in prokaryotes and eukaryotes. this chapter provides an overview of the use of nmr spectroscopy to unveil the transmembrane and membrane-proximal regions of viral membrane proteins, as well as their interactions with potential therapeutics. we will focus our discussion on two classes of transmembrane (tm) proteins encoded by viruses, viroporins (or viral channels) and membrane fusion proteins, as these proteins have been sought after as antiviral targets and they often exhibit peculiar structural features not seen in other membrane proteins. as implied by their name, the viroporin proteins can form channel-like structures in lipid bilayer that permeate ions or solutes. now over a dozen viroporins from various sources have been characterized, covering a wide range of ion substrates including h + , k + , na + , ca 2+ , and cl − , as well as larger substrates such as rna. the exact function of ion channel activity in many viruses is not yet known, though their roles have been implicated in entry, virus assembly and virus release. the function of membrane fusion proteins is much better defined, that is, to enable viral entry by mediating virus-host membrane fusion. the viral fusion proteins have large extramembrane domains for receptor recognition and for grabbing onto the host cell membrane, but the function of their tm and membrane-proximal regions have been elusive. functional mutagenesis studies have suggested that, at least in the cases of hiv-1 and influenza a viruses, the tm domains (tmds) of fusion proteins are not merely membrane anchors, but play important roles in membrane fusion and viral infectivity. apart from the channels and fusion proteins, some viruses have developed enzymatic domains anchored to the membrane, e.g., the polymerases of the hepatitis c virus and the neurominidase of the influenza viruses. in these cases, the tmds are believed to only serve as membrane anchors and thus will not be discussed in this chapter. viroporins serve highly diverse functions in viral infection cycles (illustrated in fig. 6.1 ). early studies of viroporins have focused on only a few systems including 2b of poliovirus, 6k of togavirus, and m2 from influenza a virus (am2). the 2b is a well-known viroporin that can modify membrane permeability and allow the passage of ions and small molecules during later stages of viral infection [1] [2] [3] . the 6k is a small, hydrophobic acylated protein that has been shown to form cation-selective channels when inserted into lipid bilayers [4] . the 6k is proposed to be involved in virus budding, but the exact role of 6k in the viral life cycle is poorly characterized [5] [6] [7] . the am2 proton channel is the most studied viroporins because (1) it is the drug target of the first anti-flu drug (amantadine or rimantadine) and (2) it is one of the smallest proton channels with proton selectivity motif not found in any of the proton channels from other organisms [8] [9] [10] . since the definition of viroporin has been put forward, more and more viroporins have been classified (table 6 .1). the influenza b virus encodes the bm2 proton channel that is a functional homolog of the am2 but cannot be blocked by the adamantane family of drugs [11] . the human immunodeficiency virus type 1 (hiv-1) vpu and the hepatitis c virus (hcv) p7 have been shown to form oligomeric channels to conduct cations [12] [13] [14] [15] [16] . the 2b protein from the enterovirus 71 (ev71), however, displayed altered channel activity, as it conducts anions (e.g., cl − ) instead of cations [17] . paramecium bursaria chlorella virus 1 (pbcv-1) has a viral k + channel named kcv that contains the canonical potassium selectivity filter [18] and was proposed to adopt similar structure as kcsa [19] . in addition to the ion channels, the vp4 from rhinovirus can form pores that (1) . virion attaches directly host cell receptors on the membrane (2) . and subsequently enters the cell by receptor-mediated endocytosis (3) . acidification of the endosomal vesicles triggers conformational changes in the virion, resulting in fusion between the viral and the endosomal membranes, allowing the release of the nucleocapsid into the cytoplasm. the viral rna is then released into the cytoplasm and presented to the endoplasmic reticulum (er) (4) . at the er, viral rna is translated into protein that is processed by viral and host proteases (5) . after the viral replication complex is synthesized, rna synthesis begins by the transcription of an antisense viral rna followed by the amplification of viral rna (6) . the newly synthesized rna is subsequently packaged by capsid protein, forming a nucleocapsid (7) . virus assembly occurs on the surface of the er when the nucleocapsid buds into the er lumen, then transported through the golgi, where acidification induces virus maturation (8) . mature viral particles are released in the neutral ph of the extracellular millieu (a) viral entry, which acidifies the virion interior immediately after endocytosis and facilitates rna release (b) dissipation of the proton gradient in the golgi and the trans-golgi network. the viroporins influenza a virus (iav) matrix protein 2 (m2) and hepatitis c virus (hcv) p7 equilibrate the proton concentration with the cytosol to reduce the acidification of vesicular acidic compartments (c) viroporins play an essential role in assembly, budding and release of the viral particles (d) alteration of cellular ca 2+ homeostasis. calcium uptake by the mitochondria can lead to dissipation of the inner-mitochondrial-membrane potential (δψm), permeabilization of the outer mitochondrial membrane and, finally, the release of cytochrome c. in the cytosol, cytochrome c promotes the subsequent formation of apoptosome that is involved in apoptosis allow single-stranded viral rna to cross the membrane [20, 21] , further expanding the list of substrates that viroporins transport. probably the best-defined roles of viroporins are that of the m2 proton channels of influenza a and b viruses, both of which involve equilibrating ph between compartments. one important role of the m2 is to equilibrate ph across the viral membrane during viral entry, which acidifies the virion interior immediately after endocytosis and facilitates rna release [22, 23] (fig. 6.1a ). another role of m2 is to equilibrate ph across the trans-golgi membrane of the infected cells during viral maturation, which preserves the pre-fusion form of the hemagglutinin fusion protein through de-acidification of the golgi lumen [24] (fig. 6.1b) . the roles of other cation-selective viroporins are less clear. one of the general roles proposed for viroporin is to depolarize either the er or plasma membrane to facilitate membrane curvature formation during virus budding [25, 26] (fig. 6.1c ). in the case of hcv, for example, the p7-mediated channel activity has been reported to facilitate virus release [27, 28] . another general role for viroporin is simply to cause cation leakage, which would induce cellular stress and programmed cell death [29] (fig. 6.1d ). in addition to ion permeation, several viroporins are known to participate in viral assembly through interaction with other proteins. for example, the influenza am2 and bm2 proteins both have significant cytoplasmic domains that are believed to recruit matrix proteins m1 during virus assembly [30] [31] [32] . the hcv p7 protein has also been reported to interact with other non-structural proteins such as ns2, and this interaction appears to be crucial for the production of infectious hcv particles [33, 34] . pore formation indicated but a defined oligomeric state either does not exist or is not characterized enveloped viruses infect host cells by the fusion of viral and cellular membrane. depending on the type of the virus, fusion can occur either with the host plasma membrane or with the endosomal membrane. the membrane fusion must occur without consuming energy; it is catalyzed by major conformational changes of specialized envelope proteins generally known as viral fusion proteins. despite their diversity, all known viral fusion proteins convert from a prefusion state (dimers or trimers, depending on the class), through a key intermediate state (extended conformation), to a postfusion state (a trimer of hairpins) that brings the fusion peptide, attached to the target membrane, and the tm domain, attached to the viral membrane, into close proximity thereby facilitating the fusion of viral and target membranes ( fig. 6 .2a) [35] . three distinct classes of viral fusion proteins (exemplified in fig. 6 .2b) have been defined based on structural criteria [36] . the class i includes fusion proteins from some of the best characterized human pathogens, such as influenza and hiv-1. these proteins are trimers of a single chain precursor that requires a proteolytic cleavage to generate two fragments. for example, the influenza a hemagglutinin (ha) contains the n-terminal fragment ha1 and c-terminal fragment ha2 [37] , and the hiv-1 envelope protein is cleaved into the gp120 and gp41 fragments [38] . the fusogenic fragment gp41 bears a hydrophobic fusion peptide capped by the outer receptor binding fragment gp120 [39] . receptor binding accompanies cap protein release, allowing major conformational changes in the gp41 that ultimately leads to fusion [40] . in the case of influenza, the cap ha1 is released by low ph of the endosome after endocytosis [41] . the class ii fusion proteins are found on flaviviruses, alphaviruses, and bunyaviruses (e in flaviviruses, e1 in alphaviruses, gn and gc in bunyaviruses) [42, 43] . although these fusion proteins are architecturally different from the class i fusion proteins, i.e., class ii fusion proteins are mostly made of β-sheets as opposed to the helical structures of the class i fusion proteins, they operate on the same physical principle. their fusion peptides are formed as loops at the tips of β-strands that serve as an anchor to insert into the host membrane after endocytosis. unlike class i proteins, which remain trimeric, their conformational change involves a change in oligomeric state from pre-fusion dimers to post-fusion trimers [44, 45] . the reduced ph of the endosome induces a cascade of subsequent refolding and trimerization events that achieve membrane fusion [46] . members of the class iii include g protein of vsv, gb of herpes simplex virus and epstein-barr virus (ebv), and gp64 of the insect-cell baculovirus [47] . the class iii fusion proteins combine some of the features of class i and ii fusion proteins [48] . each protomer is composed of five domains that contain a central trimeric coiled-coil, a hallmark of class i proteins in their postfusion states, three of the domains are predominantly made of β-sheets and their fusion peptide more closely resemble those of class ii proteins in that their fusion loops are found at the tips of extended β-strands [49] . due to the essential roles of viral fusion proteins during infection, they have long been pursued as targets for antiviral intervention. for example, the approved small molecule drug arbidol is used as a broad-spectrum inhibitor of influenza a and b virus, as well as hepatitis c virus [50, 51] . unlike many other broad-spectrum antivirals, arbidol has an established mechanism of action against the has in influenza a and b viruses that involves the inhibition of virus-mediated membrane fusion and thus viral entry [50] . in addition, enfuvirtide (t20, fuzeon) is an approved peptide drug that blocks the hiv-1 entry [52] ; it is derived from the c-terminal heptad repeat 2 region of the hiv-1 gp41 envelope glycoprotein, designated the c-peptide, that disrupts membrane fusion by competing with an intra-molecular interaction that is important for the refolding of gp41 during membrane fusion [53] . apart from being therapeutic targets, another important medical use of the fusion proteins is vaccine development. viral fusion proteins are usually presented on the virion surface with high copy numbers, and thus are popular antigens for eliciting broadspectrum neutralizing antibodies. for example, the native conformations of the the n terminus of ha1 and the c-terminus of the ha2 ectodomain are labeled. blue arrow: position of fusion peptides inserted near threefold axis in prefusion form. only ha2 is shown on the bottom. the n-terminus (blue arrow; note that the fusion peptide is not part of the structure shown) and c-terminus of the blue-colored subunit are indicated class ii: dengue virus type 2 e protein (1oan and 1ok8). the radial ("top") view shows just the "stem-less" ectodomain (1oan). colors: domain i: magenta; domain ii: cyan; domain iii: yellow; stem: cyan; colors for domains i, ii and iii are the same in the postfusion representation. a dashed cyan arrow on the postfusion trimer shows where the stem emerges from domain iii. red asterisks: fusion loops class iii: vsv g ectodomain (2j6j and 2cmz). the three chains are in gold, sea green, and magenta. dashed lines show the location of a disordered, c-terminal segment that connects the folded protein to the transmembrane anchor. only the magenta-subunit c-terminal segment is shown on the bottom. the curved gold arrow indicates that in the transition from the conformation on the top to the conformation on the bottom, the domain bearing the fusion loops flips over by about 180° to engage the host-cell membrane. red asterisks: fusion loops hiv-1 envelope glycoprotein have being pursued intensely as immunogens for b-cell based vaccine development [54, 55] . for most single-pass tm proteins, the role of their tmds beyond membrane anchoring is unresolved, partly because of the difficulty in structural studies: they are notorious for resisting crystallization and due to their small sizes, unfeasible for visualization by cryo-electron microscopy (em). there have been only a few examples of crystal structures of the small tmds, including the tmd of the am2 [56, 57] and the more recent crystal structure of the tm helix dimer of the glycophorin a protein [58] . as cryo-em is rapidly approaching atomic resolution, it is becoming increasingly used to examine the structures of the much larger viral fusion proteins containing the tmd. most notably, a recent cryo-em study of the hiv-1 envelope glycoprotein (env) including the tmd achieved a high resolution structure of the prefusion state of the hiv-1 env [59] . this study, however, showed that the tmd and the membrane-proximal regions of the env are disordered, possibly due to incompatibility between the membrane-associated regions of the env and the detergent used to solubilize the env. in this chapter we mainly focus on the use of nmr to study the tm and membrane-proximal regions of viral membrane proteins, including methods for structure determination and for investigating drug binding. the first challenge of nmr studies is preparing sufficient quantities of isotope labeled proteins and this is not trivial for small and hydrophobic proteins. a robust approach is to force highlevel expression of the hydrophobic peptides, which are toxic to cells, into inclusion bodies. in our experience, the most effective implementation of this approach is to express the peptide as a c-terminal fusion to the trple protein (which drives inclusion body formation) in the pmm-lr6 vector [60] [61] [62] (fig. 6 .3a). the trple has an n-terminal 9×his tag to permit nickel affinity purification. a methionine between the trple and the peptide is added for cleavage by cyanogen bromide so that the peptide can be released from the trple. purification of the peptide involves (1) purifying the trple-peptide fusion protein from the inclusion bodies by nickel affinity, (2) cleavage by cyanogen bromide of the fusion protein in formic acid, and (3) separation of the peptide from trple or fusion protein by reverse-phase hplc. while the above protocol is quite general for hydrophobic peptides, it requires that the peptides have no methionine in the middle of the peptide sequence. if the native methionines cannot be mutated, an alternative method is to use soluble fusion domains (e.g., the maltose binding protein) with specific protease cleavage site for releasing the peptide. the purified hydrophobic peptides can be reconstituted in any membrane mimetic media, including detergent micelles, bicelles, and lipid nanodiscs. while nanodiscs are the closest mimic of native membrane, we find that the lipid/detergent bicelle system is a good compromise between having the capacity to provide a near lipid bilayer environment and generating good nmr spectra. previous studies on the bicelle system with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (dmpc) as lipid and (1,2-dihexanoyl-sn-glycero-3-phosphocholine (dhpc) as detergent have shown that when the molar ratio of lipid to detergent (q) > 0.5, the assembly reaches the ideal bicelle condition in which the lipid and detergents are well segregated [63, 64] . at q = 0.5, for example, the estimated diameter of the lipid bilayer region of the bicelle disc is ~45 å according to the equation describing bicelle assembly [64] [65] [66] , and this size is sufficiently large to accommodate small tmds. remarkably, even bicelles of such size could generate nmr spectra of sufficiently high quality to enable full-scale structure determination, as was demonstrated for the structure fig. 6 .3a schematic of hydrophobic peptide sample preparation using hcv p7 as an example 1. trple plasmid with targeted peptide is transformed into and bl21(de3) competent cells for expression 2. the cells were grown in m9 media 3. cells were collected by centrifugation and lysed by sonication 4. after lysis, the inclusion bodies and membranes were collected by centrifugation and dissolved in 6 m guanadine buffer. insoluble aggregates were removed by centrifugation. the supernatant was then subjected to ni-nta purification. 5. the protein was eluted using 400 mm imidazole 6. cyanogen bromide cleavage followed by dialysis in water 7. the protein was further separated by reverse-phase hplc or ni-nta 8. the lyophilized peptide was then dissolved in detergent and refolded by dialysis against the nmr buffer 9. nmr experiments were performed at spectrometers determination of the trimeric tmds of the fas death receptor [67] and hiv-1 env [68] . here we provide an example of bicelle reconstitution from a previous study on the tmd of hiv-1 env [68] . the hydrophobic peptides (lyophilized) are first completely dissolved in strong organic solvent (e.g., hexafluoro-isopropanal) with calculated amount of dmpc. the solution is slowly dried to a thin film under nitrogen stream. the dried film is then dissolved in 8 m urea containing calculated amount of dhpc. the denaturant is removed by dialysis, during which the dmpc: dhpc ratio is monitored by 1d nmr, and the loss of dhpc during dialysis is compensated by further addition of dhpc. owing to the small size of most of the tmds of viral membrane proteins, structure determination by nmr is normally straightforward. probably the biggest challenge is measuring inter-protomer restraints in cases of oligomer complexes. in particular, structure determination of symmetric oligomers faces the challenge of measuring noe-based 1 h-1 h distances between structurally equivalent protomers having the same nmr peaks, which are needed as inter-protomer restraints. a proven way to solve this problem is using a mixed sample in which half of the protomers are ( 15 n, 2 h)-labeled and the other half 13 c-labelled, for measuring, exclusively, noes between the 15 n-attached 1 h of one protomer and 13 c-attached 1 h of the neighboring protomer [60, 69] (example from the tmd of the fas receptor shown in fig. 6 .3b). once the trimer solution is established, conventional 15 n-and 13 c-edited noe experiments can then be used to collect self-consistent backbone-sidechain and sidechain-sidechain noe restraints. in addition to structure determination, an important aspect of small tmds is interaction with membrane. the bicelle system allows accurate determination of the position of tmd in the bilayer region of the bicelles using a solvent paramagnetic relaxation enhancement (pre) analysis [63] . in this method, titration of the water-soluble and membrane-inaccessible paramagnetic agent, gd-dota (gadolinium (iii) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate), is used to generate the solvent pre. this approach is based on the notion that if the bicelle is sufficiently wide, the lateral solvent pre becomes negligible, thus allowing the use of measurable solvent pre to probe residue-specific depth immersion of the protein along the bicelle normal. at each titration point, a 2d 1 h-15 n correlation spectrum is recorded to measure residue-specific pre. for each of the residues, the pre titration curve is fitted to exponential decay to derive the residue-specific pre amplitude (pre amp ). the pre amp vs. (residue position) along the trimer symmetry axis (parallel to the bilayer normal) is then analyzed using the sigmoidal fitting method [63] to determine the tmd region that resides at the center of the bilayer as well as the thickness of the bilayer around the protein. for studying small molecule drug binding to viral membrane proteins, chemical shift perturbation (csp) induced by drug titration is a convenient probe for identifying the approximate binding site. but, cautions need to be taken due to complications associated with membrane-mimetic media. first, many small molecule inhibitors are hydrophobic and preferentially partition into detergent micelles or lipid/detergent bicelles, and thus the csp can be induced simply by alteration of the micelle/bicelle environment. second, in addition to csp caused by close ligand contact, csp could arise from changes in conformation and/or dynamics. hence, the more direct noe data is preferred wherever feasible. the simplest and the most sensitive approach for collecting protein-drug noe is using proteins that are 15 n-labeled and completely deuterated so that noe between the protein backbone amide protons and drug protons could be measured unambiguously (fig. 6.3c ). once the binding site has been unambiguously spotted, further contacts between the drug and protein sidechains can be measured using conventional 13 c-edited methyl or aromatic noesy. details of these experiments have been described in studies that identified the drug binding sites in the influenza am2 and hcv p7 channels [60, 70, 71] . structural studies of viroporins have been challenging because these small membrane proteins are typically dynamic and very hydrophobic. in the past 10 years, multiple biophysical techniques including solution nmr, solid-state nmr, x-ray crystallography, and em have been used to gradually fill the structural gaps. taking the influenza am2 for example, there is now structural information in crystal and solution states, in lipid bilayer, under different phs, and bound to different small molecules. these complementary structural data allow elucidation of the functional mechanism from different view angles. the am2 of influenza a and the bm2 of influenza b are 97-and 109-residues single-pass membrane proteins, respectively, that form homotetramers in membrane [72] [73] [74] [75] [76] . the sequences consist of three domains: an extracellular n-terminal domain, a transmembrane domain (tmd) and an intracellular c-terminal domain. these domains arrange into different structures. the only homologous sequence between the two proteins is the hxxxw sequence motif of the tmds that is essential for channel activity. the tm region of the am2 contains residues 24-46. the unstructured extracellular segment of am2 is relatively conserved and has been sought after as a vaccine epitope [77] [78] [79] [80] [81] . bm2 has a similar sized tmd (residues 4-33), but a much larger c-terminal cytoplasmic domain [32, 82] . the bm2 cytoplasmic region also assembles into oligomers which are important for recruiting the matrix proteins to the cell surface during the viral assembly [30, 31, 83, 84] . the first high-resolution structures of the am2 channel were reported concurrently by solution nmr spectroscopy and x-ray crystallography in 2008 [56, 62] . subsequent x-ray and solution nmr studies also determined structures under different conditions [57] and with different drug resistance mutations [85, 86] . moreover, structural characterization by solid-state nmr studies generated the channel structures in the lipid bilayer environment [87] [88] [89] . the above am2 structures solved under different conditions show significantly different conformations ( fig. 6.4a) , suggesting that the tetrameric assembly of the am2 is quite dynamic and is sensitive to the reconstitution environment. the structural plasticity observed in am2 is likely present in many other viral membrane proteins, which could explain why structural study of this type of proteins has been so difficult. despite the differences, the experimental structures converged to a common mode of channel assembly: a left-handed four-helix bundle forms the channel pore, and that tetramerization of the four tm helices is further stabilized by intermolecular contacts between c-terminal amphipathic helices flanking the tmd [62, 88] . this mode of assembly places the "h" and "w" of the hxxxw sequence motif inside the channel (fig. 6.4a) . four imidazole rings of his37, which are ph-gating features and are essential in transporting protons, are packed closely inside the pore (fig. 6.4b) . moreover, packing of the trp41 indoles creates a channel gate, which occludes the c-terminal end of the pore. the structure of the bm2 channel was solved using solution nmr methods [32] . although the overall assembly of the bm2 tmd is similar to that of the am2 tmd, i.e., both show left-handed helical packing, the two differ substantially in details ( fig. 6.4c) . unlike the am2, the bm2 channel shows strong coiled-coil characteristics with regular heptad repeats [8] . in the bm2 structure, the two heptad repeats show that positions a and d are occupied mostly by hydrophilic residues such as serines and his19; positions g and e are occupied by hydrophobic leucines and phenylalanines, respectively, to allow for peripheral hydrophobic interactions between the leucines and phenylalanines (fig. 6.4d ). this arrangement for coiled-coil assembly in membrane allows the tm segment to form a stable tetramer by itself and is the opposite to that of water-soluble coiled-coil tetramer, in which positions a and d are typically hydrophobic residues and positions g and e are polar residues [90] . the histidine (his19) and tryptophan (trp23) of the hxxxw motif are also pore-lining in the inverse coiled-coil assembly in membrane. the cytoplasmic domain of the bm2 also oligomerizes into a left-handed coiled-coil tetramer, but it is water-soluble and shows strong bipolar distribution in the surface charges. this charged domain supports the interaction with the m1 matrix protein [32] . the structural arrangement of the histidine and tryptophan inside the pore of the am2 and bm2 suggests that the two residues play an essential role in the channel selectivity and channel gating (fig. 6.4e ). functional mutagenesis [91] and nmr measurements [92] showed that proton transport across the membrane through the am2 channel involves cycles of histidine protonation and deprotonation, and that the histidines serve as proton shuttling devices. not all histidines can be protonated at the same time in the narrow channel, as protonation of one histidine would increase the energy barrier for the protonation of another histidine. indeed, multiple pka values (8.2, 6.3 and one below 5.0) have been detected in the am2 [91, 93] . therefore, our current understanding of the proton conduction mechanism is that the minimally required unit for ph-dependent proton conduction in the am2 and bm2 is the his-trp complex. it was proposed in ref. [93] that in the non-conductive state two pairs of histidines in the tetramer each share one proton, which explains structures of the influenza proton channels and mechanism of proton conduction. the many structures of the influenza m2 channel. the pdb codes 2rlf and 2kwx represent the solution nmr structures of the wildtype and the v27a mutant determined using residues 18-60. the 3c9j and 3lbw are crystal structures of the tm domain (residues 22-46) determined at ph 7.3 and ph 6.5, respectively. the structures 2l0j and 2kqt were obtained using solid-state nmr using protein constructs that encompass residues 22-62 and residues 22-46, respectively the high pk a ~8.2. lowering ph results in the protonation of the third histidine from the n-terminal side, which, in turn, disrupts the two histidine dimers and leads to the proton conductive state. it was also proposed that protonation of histidines leads to cation-π interactions between the histidine and tryptophan [94, 95] . the remaining question to be addressed is how does the third protonation affect the tryptophan conformation, allowing proton to be relayed to the c-terminal side of the tryptophan gate [91] . the viroporin p7 encoded by the hcv genome is a 63-residue protein that oligomerizes in membrane to form cation-selective channels [15, 16] , with higher selectivity for ca 2+ than k + /na + [96, 97] . the channel activity of p7 is important for the assembly and release of infectious viruses, although the molecular mechanism of this function remains unknown [27, 28] . as in the case of am2, structural characterization of p7 was confronted with challenges of coping with the hydrophobic and dynamic nature of the protein. earlier nmr studies of p7 under conditions that support the monomeric state of the protein showed that p7 has three tm helical segments: two in the n-terminal half of the sequence and one near the c-terminus [97, fig. 6 .4b isolated view of the pore-lining histidine (magenta) and tryptophan (cyan) sidechains in m2 and bm2 channels. images are from the high resolution crystal structure (3lbw) and nmr structure (2rlf) of m2 and nmr structure of bm2 (2kix) 98] . although the monomeric state should not conduct ions, it could be involved in interacting with the ns2 protein during virus assembly [33, 34] . the oligomeric form of p7 was first examined using single-particle em, which showed that p7 from hcv genotype 2a (jfh-1 strain) assembles into hexamers in 1,2-diheptanoylsnglycero-3-phosphocholine (dh 7 pc) micelles and the complex adopts a flowerlike shape that does not resemble any of the known ion channel structures in the database [99] . later, a more detailed structure of the p7 hexamer was determined by solution nmr using p7 from genotype 5a (euh1480 strain) reconstituted in dodecylphosphocholine (dpc) micelles [60] . consistent with the flower-shaped em images, the nmr structure shows a funnel-like architecture with six minimalist chains, each containing three helical segments: h1, h2, and h3. the h1 and h2 form the narrow and wide regions of the funnel-shaped cavity, respectively, and the h3 helices wrap the channel peripheral by interacting with h1 and h2 (fig. 6.5a) . the assembly strategy adopted by p7 differs significantly from those known channels from bacteria and eukaryotes. ion channels typically have two essential features: (1) pore elements that support selective ion dehydration; (2) a gate or constriction that prevents non-specific permeation, but can open in response to regulating factor such as ph, voltage, ligand, or the ion of selection itself [100, 101] . the channel interior of p7 has a number of strongly conserved residues that are likely candidates to serve the above functions. one suspect is asn9, which forms a ring of carboxamide near the narrow end of the channel (fig. 6.5b) . residue 9 is asparagine in all strains except being substituted with histidine in genotype 2 viruses. formation of a ring of carboxylates or carboxamides has been a recurring theme in prokaryotic and eukaryotic channels that have selectivity for divalent cations. for examples, the cora mg 2+ channel has a pentameric ring of asparagines [102] , and the calcium release-activated calcium (crac) channel orai has a hexameric ring of aspartic acids [103] . these channels all have strong selectivity for divalent cations, although they can also conduct monovalent cations such as na + and k + . in addition to the asn9 ring, residues near the hinge between h1 and h2 (ser12, asn16, trp21) are in an arrangement that may also bind in addition to what appears to be the cation selectivity ring near the narrow, n-terminal exit of the channel, the wider, c-terminal entrance of the channel is decorated with a conserved ring of arginines or lysines (fig. 6.5b) . placement of a positively charged ring at the entrance was anticipated because it may repel cations. but an earlier study reported that a designed tm barrel with internal argininehistidine dyads forms efficient cation selective channels [104] because the immobile arginines can recruit mobile anions, which in turn facilitate cations to diffuse through the pore. it is interesting to note that a highly basic region containing arg155, lys159 and lys163 in the pore was also found in the crac orai structure [103] . one possible role of these basic residues in cation selective channels is binding and obstructing anions while allowing cations to diffuse into the pore. this mechanism would be consistent with the observation that replacing arg35 with negatively charged aspartic acid largely abrogated conductance [60] . there are still many unanswered questions. does the nmr structure, solved in the absence of ca 2+ and inhibitors, represent the open or closed state of the channel (if the two states exist)? how strong is the ca 2+ selectivity of p7? or was p7 developed as a general, unspecific cation channel for the purpose of dissipating membrane potential? for example, another recently study reported p7 activity in dissipating proton gradients within cell membrane compartments [105] . it is unclear what ion flux mediated by p7 plays a dominant role in the hcv life cycle. from a structural perspective, the funnel-like architecture is formed with multiple helical segments connected by hinges and short loops and we believe this flexibility can afford the dynamic opening and closing of the tip of the channel. probably the most intriguing aspect of small molecule interaction with viroporin is the finding that the adamantane derivatives amantadine and rimantadine have inhibitory effect on multiple viroporins including the influenza am2 and hcv p7. amantadine (symadine) or rimantadine (flumadine) was the first licensed drug for treating influenza infections [106] . in fact, the compound also played critical roles in the early days of functional characterization of the am2 channel [107] [108] [109] . the bm2 channel is a functional and structural homolog of am2 but is not sensitive to the adamantane family of drugs [11] . remarkably, the hcv p7 channel structure is completely different but showed detectable, though not strong, sensitivity to rimantadine [15, 110] . the mechanism of how amantadine/rimantadine inhibit the am2 channel has been elusive for quite some time. previous confusion came mainly from the multiple binding sites that have been observed experimentally. in a crystallographic study of the am2 tmd (residues in the presence of amantadine, the drug density was found inside the channel near residue ser31, but at structural resolution of 3.5 å, it was difficult to confirm the position of amantadine binding [56] . at the same time, however, a solution nmr study of a longer version of am2 (residues showed that rimantadine binds to an external, lipid-facing pocket around residue asp44 between adjacent tm helices [62] . the two different binding sites obviously suggest very different mechanisms of inhibition. one is the drug directly blocking the channel passage, and the other is the drug binding to the external site allosterically favors the closed state of the channel. subsequent solid-state nmr measurements of the am2 in lipid bilayer showed that both sites exist, with higher affinity for the internal site [87] . the strongest evidence that the internal site is the primary site of drug action came from functional studies using an am2-bm2 chimera protein [111, 112] . in this study, the authors constructed a chimera of m2 variants from influenza a and b viruses that contains only internal site showed that the chimera channel is still amantadine sensitive, indicating that the internal site is the primary site of drug inhibition. later, the complex structure of the tmd of the chimera with rimantadine was determined by solution nmr, providing a detailed view of the drug binding inside the channel pore [70] . the drug binding site consists of eight methyl groups of the m2 tetramer (two from each subunit: val27 c γ1 h 3 and ala30 c β h 3 ) that form a deep internal hydrophobic pocket surrounding the adamantane cage of the drug (fig. 6.6a ). the structure also shows that the nitrogen of the rimantadine amino group may form a hydrogen bond with the backbone carbonyl oxygen of ala30 of one of the four subunits. the terminal methyl group of the rimantadine is in the middle of the pore, facing the open space in the channel around the gly34 position. the structure also shows that rimantadine binding is slightly tilted: its vertical axis is on average ~20° from the c4 symmetry axis of the channel. the tilt angle is consistent with the amantadine tilt in the m2 channel observed with solid-state nmr spectroscopy [87] . in addition to blocking the am2 channel, the amantadine and its derivatives have also been shown to pose some inhibitory effects on the p7 channel conductance [15, fig. 6.6 the amantadine and rimantadine binding sites in the m2 and p7 channels (a) the precise nmr structure of the am2-bm2 chimeric channel with rimantadine determined in dhpc micelles and at ph 7.5. left panel: detailed illustration of the methyl groups (in green) that interact with the adamantane cage. right panel: surface representation for showing the hydrophobic pocket that fits the drug snuggly. one of the four subunits is omitted for drug visibility (b) the amantadine or rimantadine binding site of the p7 channel determined by nmr in dpc micelles and at ph 6.5. left panel: the drug binds to six equivalent hydrophobic pockets of the p7 channel. right panel: a close view of amantadine docked into the binding pocket as determined using nmr noe restraints (c) comparison between adamantane binding sites of influenza m2 and hcv p7 channels top: the internal pocket that wraps around rimantadine in the am2-bm2 chimeric channel. the am2-bm2 chimeric channel is a well-behaved model system wth its n-terminal half is from influenza a m2 protein (sensitive to amantadine or rimantadine inhibition) and its c-terminal half is from influenza bm2 protein (insensitive to amantadine or rimantadine). on the right panel, one subunit of the tetrameric complex is removed to unveil the channel interior bottom: amantadine binds to the peripheral pockets between the h2 and h3 helices; and a representative pocket among six equivalent pockets in the p7 hexamer 113 ]. the physical binding sites of amantadine and rimantadine have been identified in p7 of genotype 5a using intermolecular noe experiments [60] . the noe data revealed that amantadine or rimantadine binds to six equivalent hydrophobic pockets (due to the six-fold symmetry of the p7 channel) between the pore-forming and peripheral helices (fig. 6.6b) . in each site, leu52, leu53, and leu56 from h3 and val25, val26, and phe20 from h2 appear to form a hydrophobic pocket that wraps around the adamantane cage of the drug. the amino group of amantadine or rimantadine is facing the largely hydrophilic channel lumen. an important property of the drug binding site is that it consists of elements from different helical segments and from different monomers. as rationalized above, permeation of cations through the p7 channel may depend on opening the narrow end of the funnel, which in turn depends on the reorientation of the helical segments. the binding of adamantane derivatives to the pocket may inhibit channel activity allosterically by causing the channel to close. indeed, an nmr relaxation dispersion study showed that residues at the h1-h2 hinge (phe19) and the narrow end of the cavity (val7, leu8) experienced substantial chemical exchanges (k ex~1 000 ± 79 s −1 and ~10% excited state). this data is consistent with movements of the h1 helices that cause the tip of the funnel to open and close. more importantly, addition of rimantadine slowed down motion at the tip of the channel, as relaxation dispersion curve for val7, which has significant chemical exchange in the apo state, is completely flat in the drug-bound state [114] . the dispersion curve of phe19 is also significantly flatter, and individual curve fit yielded k ex value of 67 ± 182 s −1 . clearly, rimantadine binding makes the channel less dynamic. therefore, the rimantadine may thus act as a "molecular wedge" that prevents the dynamic "breathing" of the channel required for ion conduction. comparing the amantadine or rimantadine binding mode of hcv p7 to that of influenza am2 shows two fundamentally different mechanisms of drug inhibition. in the case of am2, one drug binds to one channel. drug binding inhibits proton transport by directly blocking the channel passage; it also prevents channel from opening. in the case of p7, amantadine and rimantadine are clearly too small to block the channel. they instead bind to six equivalent sites outside of the channel cavity, which can afford up to six drugs per channel. if rimantadine binding to this site is relevant to inhibition, as crudely suggested by previous functional mutagenesis, drug binding to these sites inhibits cation conduction with an allosteric mechanism, possibly by stabilizing the closed state of the channel. although the mechanisms of drug inhibition may be completely different, the structural bases that govern drug-binding affinity for am2 and p7 are actually similar, and they involve hydrophobicity and size of the pocket, and position of the drug amino group (fig. 6.6c ). in the case of the am2 tetramer, the drug adamantane cage fits snuggly in a hydrophobic pocket formed by eight methyl groups from val27 and ala30 (two from each subunit), while the drug amino group forms polar contact with the backbone oxygen of ala30 and points to the polar region of the channel cavity. for the p7 channel, the adamantane cage is in contact with ten methyl groups and an aromatic group from the protein. these hydrophobic groups form a deep hydrophobic pocket that also matches closely the size of the adamantane cage. the amino group of amantadine or rimantadine points to the channel lumen; it is in position to form polar contacts with the electronegative groups such as backbone carbonyl of residues 15-17. hence, having a greasy pocket for the adamantane cage and a nearby electronegative group to interact with the amino group may be a general requirement for amantadine or rimantadine binding. previous functional mutagenesis studies have suggested that the tmds of viral fusion proteins are not only limited to the function of membrane anchoring, but also involved in other functions such as membrane fusion or assembly of the fusion protein on the viral membrane. for example, sequence analysis of the ha from mutant viruses and site-specific mutagenesis of the fusion peptide identified a group of mutations in the n-terminal half of the influenza ha tmd severely affected membrane fusion (the hemifusion to pore formation) [115] [116] [117] . in the case of hiv-1, multiple lines of evidences suggest that the tmd of gp41 is not merely a membrane anchor, but plays critical roles in membrane fusion and viral infectivity [118] [119] [120] [121] [122] . the amino acid sequence of gp41 tmd is also highly interesting. there is a gly rich motif in the tmd, which suggests some sort of oligomerization. even more peculiar is the presence of a conserved arginine in the middle of the predicted tm region. unlike the structural biology of viroporins, there is essentially no structural information of tmds of viral fusion proteins except for that of the tmd of hiv-1 env. hence, we focus on the discussion on the trimeric membrane anchor of the tmd of the hiv-1 envelope spike. hiv-1 envelope spike [env; trimeric (gp160) 3 , cleaved to (gp120/gp41) 3 ] is a type i membrane protein that fuses viral and host cell membranes to initiate viral infection [123] . the gp120 and gp41 are the receptor recognition and membrane fusion proteins, respectively. conformational changes in gp120 when triggered by binding to receptor (cd4) and co-receptor (e.g., ccr5 or cxcr4) lead to a cascade of refolding events in gp41 (similar to those illustrated in fig. 6.2a) , and ultimately to membrane fusion [39, [124] [125] [126] . the mature and functional env spikes, (gp120/gp41) 3 , are the sole antigens on the virion surface and thus important candidates for vaccine development [127, 128] . the native prefusion conformation of hiv-1 env is recognized by most broadly neutralizing antibodies (bnabs) [129] [130] [131] and it is generally believed to have the potential to induce such antibody responses. thus, the native conformation of env spikes on the surface of virions is extremely important to immunogen design in b-cell based vaccine development. a vast amount of structures of the ectodomain (ecd) of the gp120/gp41 complex have been determined [39, 124, 125, [132] [133] [134] [135] [136] [137] [138] [139] , but relatively little is known about the tm and membrane proximal regions of gp41 due to challenges of preserving the native-like folding of these regions in membrane mimetic media. it has been shown that truncations in the cytoplasmic tail (ct) of gp41 could alter the antigenic surface of the env ecd on the opposite side of the membrane [129] , suggesting that the ecd, the tmd, and the membrane proximal regions are conformationally coupled, and thus the conformational stability of the tmd is an important consideration for immunogen design in b-cell based hiv-1 vaccine development. recently, the nmr structure of the gp41 tmd has been solved in bicelles (made of dmpc lipid and dhpc detergent; q = 0.5) [68] using a gp41 fragment (residues 677-716) from a clade d hiv-1 isolate 92ug024.2, designated gp41 hiv1d (677-716). the tmd forms a well-structured trimer, almost helical all the way from the n-to the c-terminal end. it shows two peculiar features not seen with other known oligomeric tm helices (tmhs) (fig. 6.7a) . one unusual feature is that the tmd trimer appears to be stabilized by two separate packing modes (fig. 6.7c) . the n-terminal half encompassing the gxxxg motif forms a coiled-coil trimer, whereas the c-terminal half is held together by a network of polar contacts, which we named the hydrophilic core. for the n-terminal coiledcoil, the helical wheel representation of the trimer clearly indicates packing of hydrophobic residues such as ile and val at the "a" and "d" positions of the heptad motif, forming a hydrophobic core. the gxxxg is a well-known motif that drives tmh dimerization [58, 140, 141] . in the classic example of the glycophorin a tmd dimer structure, the two glycines of one tmh allow close packing with the gxxxg face of another tmh, resulting in a very strong tmh dimeric complex [58, 140] . there has been no previous report, however, of the gxxxg involvement in tmh trimerization. in the coiled-coil region of the hiv-1 env tmd, only g690 is involved in the trimer assembly, i.e., its small sidechain allows a close vdw contact with v689 of the adjacent tmh. the other glycine, g694, is on the periphery of the trimer facing outwards and its mutation to alanine or valine has essentially no effect on tmd trimerization, as well as env functions [68] . therefore, the key difference in the structural role of the gxxxg motif between the glycophorin a tmd dimer and hiv-1 env tmd trimer is that both the glycines are required for forming intermonomer contacts in the dimer, while only one glycine is important for the trimeric assembly. tm segments of many viral fusion proteins contain a gxxxg motif or "smallxxxsmall" motifs ("small" refers to residues with a small side chain, such as, glycine, alanine, serine or cysteine) [142] [143] [144] , suggesting that oligomerization of their tmds may be a common property. for example, recent biochemical evidence has shown that the tmds of hepatitis c virus envelope glycoproteins e1 and e2 form stable dimers or trimers that are also resistant to sds [143] . no highresolution structure of any tmd oligomer from other viral fusion proteins has been reported, due to technical challenges for structural studies of such constructs in the context of lipid bilayer. the nmr structure of the hiv-1 env tmd may provide some clues for how other viral fusion proteins oligomerize in the membrane. another peculiar feature is the presence of three copies of arginine (r696) near the middle of the tmhs (fig. 6.7b) , suggesting three unbalanced charges in the hydrophobic core of the membrane if the arg remains protonated. in the nmr structure, the tips of the long sidechains of these arginines are facing lipids and each of them is surrounded by three hydrophobic residues (l692, l695, and i697). it is also interesting to note that r696 occupies a "d" position in the coiled-coil, with its cβ facing inwards to the trimer interface. precise physical basis of how r696 is accommodated in the highly hydrophobic environment remains unclear, but the underlying mechanism must tolerate a lys residue, which is present in some viral isolates at this position. it is interesting to mention that the noe experiment for the r696 epsilon protons showed clear water noe even with a short noe mixing time of 60 ms, suggesting that the arg is somehow hydrated in the membrane. a positively-charged arg or lys in the tm segment of viral fusion protein is present in some related enveloped viruses, including simian immunodeficiency virus, caprine arthritis and encephalitis virus, equine infectious anemia virus, visna virus, and foamy virus, as well as in hepatitis c virus [145] [146] [147] [148] [149] [150] , but absent in many others. it is therefore not a prerequisite for viral membrane fusion in general [121] . functional mutagenesis indicated that the r696a mutant of hiv-1 env showed some defect in cell-cell fusion, but it could be fully compensated by high env expression [68] . moreover, this mutant has wildtype viral infectivity. in vitro infectivity and cell-cell fusion do not, however, mimic all the conditions under which the virus moves from one cell to another in an infected individual, nor are those assays particularly sensitive to physiologically relevant kinetic parameters. the structures of viroporins discussed above are all substantially different from any of the channel structures found in prokaryotes and eukaryotes, and are thus clean structural targets for developing antiviral compounds. the available structures also suggest that viroporins generally adopt minimalist architecture and can possess structural features compatible with selective ion transport. the viral channels, however, may not be as functionally robust or specifically regulated as some of their counterparts in prokayrotes and eukaryotes, because viruses often do not need intricate regulation of channel activities during their infection cycles. having minimalist structure also makes viroporins fragile and sensitive to the membrane environment. this is reflected by the conformational variations observed for the am2 channel in different reconstitution media. therefore, it remains important to examine these viroporins in more native environments, e.g., lipid bilayer and full-length proteins. as solid-state nmr continues to improve spectral resolution [87, 89] , obtaining detailed structures of viroporins in liposomes should in principle be feasible in the near future. alternatively, the ideal bicelle system can be explored with solution nmr to revisit some of the viroporin structures determined previously in detergent micelles. future establishment of the nmr systems for viroporins under more native conditions would certainly provide versatile and effective platforms for investigating inhibitor binding. apart from the progress in structure determination, major challenges lie ahead in the functional aspects of viroporin research. the precise functional roles of many viroporins are still unclear. moreover, due to the lack of robust single-channel recording setups suitable for viroporins, the ion conductance properties of most viroporins have not been fully characterized. therefore, better definition of the functional roles and channel properties of viroporins will certainly draw greater enthusiasm for developing therapeutics that target this interesting family of membrane channels. the example from hiv-1 env suggests that tmds of viral fusion proteins can adopt very interesting structures, and the distinct structural features certainly allude to their roles in viral fusion protein assembly and incorporation into the envelope, as well as in the process of membrane fusion. the nmr structural study of the hiv-1 gp41 tmd in bicelles with q = 0.5 [68] demonstrates that the structures of most of the viral fusion protein tmds can in principle be determined in essentially lipid bilayer environment using modern nmr techniques. moreover, the combined use of ideal bicelles (q ≥ 0.5) and solvent paramagnetic relaxation enhancement measurements can be used to determine the membrane partition of the tmds in the bilayer region of the bicelles [63] . we believe the next major challenge lies in understanding the roles of the fusion protein tmds in the fusion mechanism. in the cases of the flu ha and hiv gp41, for example, new experiments need to be designed to address questions such as, "does the tmd interact with the fusion domain in the hemifusion stage in which the two domains are presumably in close proximity?" and "does the tmd play a role in facilitating the conversion from the hemifusion to the pore formation?" understanding the structural properties and conformational stability of the tmd may also have far reaching implication to vaccine development and this has been recognized at least for hiv-1. as mentioned above, truncations in the ct of the env could drastically alter the sensitivity of the env ecd to the known trimer-specific bnabs [129] . the continuous structure from the n-to c-terminal ends of the env tmd, as observed by nmr, suggests that the env ecd can be structurally coupled via the tmd. indeed, it has also been shown that the env tmd can also modulate the antigenic structure of the ecd in a cell-cell fusion assay and a pseudovirusbased neutralization assay [68] . the results show direct correlation that more disrupted tmd trimer led to less inhibition or neutralization by the trimer-specific bnabs that target only the ecd. the results suggest that the stability of the tmd trimer is an important consideration when designing immunogens for hiv vaccines. the hiv env tmd is, however, not directly linked to the env ecd. another important segment known as the membrane-proximal external region (mper) is the direct link that connects the tmd to the ecd, and thus it could in principle also affect the antigenic properties of the env ecd. the mper sequence is extremely conserved with five absolutely conserved tryptophans, suggesting that the mper probably also has interesting structural features. the conformation of the mper attached to the tmd trimer in a lipid bilayer 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viruses viroporin-mediated membrane permeabilization. pore formation by nonstructural poliovirus 2b protein the hpv-16 e5 protein represses expression of stress pathway genes xbp-1 and cox-2 in genital keratinocytes hpv-16 e5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside gm1 at the plasma membrane of cervical cells the human papillomavirus type 16 (hpv-16) e5 protein sensitizes human keratinocytes to apoptosis induced by osmotic stress viral encoded potassium ion channel is a structural protein in the chlorovirus paramecium bursaria chlorella virus-1 (pbcv-1) virion key: cord-287266-sd5izamc authors: song, zhenhui; yang, yang; wang, li; wang, kai; ran, ling; xie, yilu; huang, leishi; yang, zhou; yuan, peng; yu, qiuhan title: eif4a2 interacts with the membrane protein of transmissible gastroenteritis coronavirus and plays a role in virus replication date: 2019-04-30 journal: research in veterinary science doi: 10.1016/j.rvsc.2018.12.005 sha: doc_id: 287266 cord_uid: sd5izamc abstract transmissible gastroenteritis coronavirus (tgev) is enteropathogenic coronavirus that causes diarrhea in pigs, and is associated with high morbidity and mortality in sucking piglets. the tgev membrane (m) protein is a decisive protein for the proliferation of viral proteins, and is associated with virus assembly and budding. to identify the cellular proteins that interact with the tgev m protein, yeast two-hybrid screening was employed, and seven cellular proteins were identified m-binding partners. using the gst pull-down approach and a co-ip assay, the m protein was found to interact with porcine intestinal cells via eukaryotic translation initiation factor 4-alpha (eif4a2), an essential component of the cellular translational machinery. additionally, confocal microscopy revealed that eif4a2 and m were colocalized in the cytoplasm. furthermore, the function of eif4a2 in intestinal cells during tgev infection was examined. a knockdown of eif4a2 by sirna markedly decreased m protein proliferation and tgev replication in target cells. thus demonstrating that eif4a2 plays a significant role in tgev replication. the present study provides mechanistic insight into the interaction between the tgev m protein and intestinal cells which contributes to the understanding of coronavirus replication and may be useful for the development of novel therapeutic strategies for tgev infection. porcine transmissible gastroenteritis (tge) is a highly contagious disease caused by transmissible gastroenteritis virus (tgev), particularly in the colder seasons of winter and early spring (piñeyro et al., 2018) . the clinical symptoms of tgev include acute diarrhea, vomiting, and dehydration. although all aged swines can be infected with this virus, suckling piglets are the most susceptible and have a mortality rate as high as 100% (mullan et al., 1994) . tgev is a positive single stranded rna virus with a full length of 28.5 kb genome, which have a typical cap structure at 5′ end and a poly (a) tail at the 3′ end, and encodes four structural proteins including spike [s] , membrane [m] , nucleocapsid [n] , and envelope [e] , as well as other five non structural proteins (lai and cavanagh, 1997; eleouet et al., 1995) . the m and e proteins might be thought to be involved in the formation of virus-like particles (vlps) (baudoux et al., 1998) . in addition, the gene of m protein is highly conserved and encoded a decisive protein for viral proliferation (zhenhui song et al., 2015) . as an important structural protein of tgev particles, the m protein is located in the lipid envelop and associates with the golgi complex in the cell, suggesting that the m protein plays a pivotal role in virus assembly and budding. additional evidence indicates that the m protein is an indispensable part for the replication of virus particles in host cells, which may also be used for virus-specific diagnostics and treatment (cologna and hogue, 1998; de haan et al., 1998; zou et al., 2013) . however, there have been few reports regarding the interaction between the tgev m protein and intestinal cells. the identification of the cellular ligands which interact with viral proteins is likely to provide a better understanding of the dynamics of viral replication, virus-mediated cellular modulation, and pathogenic mechanism. in the present study, using yeast two-hybrid screening, seven cellular proteins, including eukaryotic translation initiation factor 4-alpha (eif4a2), were identified to be m-ligands. the eukaryotic translation initiation factor, eif4a, is a number of rna helicase, which is required for the binding of mrna to 40s ribosomal subunits with function to unwind rna secondary structures in the 5′-utr of mrna. there are three closely related eif4a proteins in, eif4a1, eif4a2, and eif4a3, which not only participate in initiation and translationare but also https://doi.org/10.1016/j.rvsc.2018.12.005 received 21 may 2018; received in revised form 4 december 2018; accepted 13 december 2018 associated with multiple life processes such as embryogenesis (lu et al., 2014) . although both eif4a1 and eif4a2 display high protein homology and assemble into the eif4f complex required for micrornamediated gene regulation, evidence suggests that eif4a2, but not eif4a1, plays a key role in this process of formation of the eif4f complex (meijer, 2013) . eif4a3 is localized in the nucleus, where it plays a role as an mrna clamp and is related to nonsense-mediated mrna decay (chan et al., 2004) . moreover, previous reports have shown that eif4a2 interacts with vp1 of infectious bursal disease virus (ibd) and inhibits the rna polymerase activity of ibdv to reduce its replication in host cells (tacken et al., 2004; gao et al., 2017) . therefore, we aimed to elucidate whether eif4a2 interacts with the m protein of tgev and impacts virus replication. in this study, we demonstrate that eif4a2 in intestinal cells associates with the tgev m protein and further examined the specific role of eif4a2 on tgev replication by knocking down eif4a2 expression using small interfering rna (sirna). porcine intestinal epithelial cells (piecs) were developed at our institute. the cells were isolated from the jejunum of a newborn piglet, grown in dulbecco's modified eagle's medium (dmem)/f-12 medium (gibco) with 10% fetal bovine serum (fbs, gibco brl), and maintained in maintenance medium (dmem/f-12 supplemented with 2% fbs) in a 5% co 2 incubator. the tgev chongqing (cq) strain was isolated from sick piglets with symptoms of diarrhea by our lab (zhenhui et al., 2015) . c-myc antibodies, gst-tag antibodies, and his-tag antibodies were purchased from sangon biotech, china. horseradish peroxidase (hrp)conjugated goat anti-rabbit igg was purchased from proteintech (usa). fitc-conjugated goat anti-rat igg (h + l) was purchased from bbi life sciences (usa), and cy3-conjugated goat anti-rabbit igg (h + l) secondary antibody was purchased from boster, china. the beta tubulin rabbit polyclonal antibody (β-tubulin) and eif4a2 rabbit polyclonal antibody were purchased from proteintech (usa). the mab to the tgev m protein was kindly donated by yu bai from wenzhou college of sciences and agriculture. y2hgold and y187 were purchased from clontech, japan. the escherichia coli rosetta strain was provided by prof. ji xiang li. the plasmids applied in the yeast two-hybrid, pfastbac™-m was stored in the laboratory at −80°c, and both pgbkt7 and pgbkt7-53 were purchased from clontech, japan. pmd19-t simple was purchased from takara, japan. the tgev m protein was amplified from pfastbac™-m by pcr that added the ecor i and sal i restriction enzyme sites, and was then cloned into a pmd19-t simple vector. after verification by pcr, it underwent directional cloning into the bait vector, pgbkt7, of the yeast two-hybrid system and was identified by pcr. y2hgold yeast were transformed with either pgbkt7-m or pgbkt7 (control). the expression product of y2hgold (pgbkt7-m) was tested by western blot using the c-myc tag antibody (1:2000) as the primary antibody and hrp-conjugated goat anti-rabbit igg (1:5000) as the secondary antibody. the yeast cultures were diluted 1/10 and 1/100, plated onto sd/−trp plates, and incubated at 30°c for three to five days. the size and number of yeast colonies treated with pgbkt7-m were compared to that of the control to test the bait for toxicity. the yeast cultures were diluted at 1/10 and 1/100 and 100 μl and spread onto sd/−trp, sd/− trp/x-α-gal, and sd/−trp/x-α-gal/aba (aureobasidin a) to test whether the bait induced autoactivation. a yeast two-hybrid assay was performed according to the instructions of the matchmaker @ gold two-hybrid system (catalog no.630489; clontech). briefly, the bait strain y2hgold (pgbkt7-m) and y187 (piecs cdna) were mixed and incubated at 30°c for 24 h until a threelobed structure similar to a "mickey mouse" face was visible at 40× magnification under a microscope. the culture mixture was diluted at 1/10, 1/100, 1/1000, and 1/ 10000, and 100 μl was spread onto sd/−trp/−leu/−ade. all monocolonies that grew were plated out onto higher stringency. sd/−trp/−leu/−ade/-his. finally, all of the white colonies from the second screening that grew on sd/−trp/−leu/−ade/-his/x-α-gal/aba. the positive colonies were selected and inoculated into sd/ −trp/−leu/−ade/-his liquid medium. the positive prey plasmids were rescued and their cdna inserts were sequenced to identify the candidate proteins. eif4a2 appeared at a high frequency among the candidate proteins, which could be connected with virus proliferation and synthesis of viral proteins (jk ndzinu et al., 2018; gao et al., 2017) . therefore, the candidate protein, eif4a2, was selected as a protein of interest for further verification of its interaction with the tgev m protein. the gst-m fusion protein was expressed in e.coli rosetta under induction by 1 mm isopropyl-b-d-thiogalactopyranoside (iptg), then immobilized on glutathione-sepharose 4b serving as a bait protein at 4°c for 6 h, followed by the addition of purified his-eif4a2 protein at 4°c for 8 h. the isolated pull-down proteins were then analyzed by 12% sds-page analysis and western blot. gst protein expression was used as a control. the co-ip assay was carried out using ip kit kip-1(protientech, china). piecs infected with tgev (moi = 0.1) and control cells were harvested at 24 h and washed three times with cold-pbs. ip lysis buffer containing 1 mm protease inhibitor was added for 30 min on ice. after centrifugation at 12,000 ×g for 15 min, the lysate supernatant which contaning 1-2 mg of total protein was incubated with rabbit pab to eif4a2 (2 μg) overnight at 4°c. then, 50 μl resuspended protein a/g-agarose was added to this mixture and subjected to rotated incubation at 4°c for 4 h. after washing five times with washing buffer containing 1 mm protease inhibitor and centrifugation at 18 ×g for 30 s, 40 μl elution buffer was added to the elute immune complex. the isolated bound proteins were analyzed by western blot. the tgev mock-infected piecs lysate was used as a control. piecs were cultured in six-well plates, and the cell monolayer was table 1 primers for qrt-pcr. primer sequence grown to a confluence of 70%-80%. transfection with the sirnas (three sirnas targeting eif4a2 produced by ribo bio, china) was performed with lipofecatmine @ 3000 in accordance with the manufacturer's instructions. the three sirnas (20 nm) were complexed with lipofecatmine @ 3000 by incubating them together at room temperature for 5 min. after removing the cell culture supernatant, the complex was added and incubated for 24 h prior to analysis. piecs were transfected with the best sirna targeting (sirna2, 5′-ggagactatatgggagcaa-3′) against eif4a2 for 24 h, then infected with tgev (moi = 0.1). the virus suspensions were collected at 0 h, 12 h, 24 h, and 48 h, serially diluted from 10 −1 to 10 −7 , and added to st cells in 96-well culture plates. each dilution was added to eight wells. the tcid 50 was calculated using the reed and muench method. the mock-treated cells and cells treated with tgev at each time point served as controls. 2.9. immunofluorescence assay piecs were infected with tgevfor 48 h. the cells were washed three times with pbs, and fixed in paraformaldehyde (4%) for 15 min at room temperature and permeabilized with 0.3% triton x-100 for 10 min. after blotting with 5% bsa (bovine serum albumin, bbi), the cells were incubated with tgev m protein (1:100) mab and rabbit pab to eif4a2 (1:50) overnight at 4°c. the cells were then incubated with fitc-labeled goat anti-rat igg (1:100) and cy3-labeled goat anti-rabbit igg (1:100). subsequently, the cells were stained with 4,6-diamidino-2phenylindole (dapi) for 5 min and examined under a zeiss lsm 800 with airyscan. piecs were transfected with sirna2 against eif4a2 for 24 h and then inoculated with tgev for 48 h. total rna was extracted using the rnaiso plus (invitrogen, usa) reagent and subjected to reverse transcription with primerscript™ rt master mix (takara, japan) according to the manufacturer's instructions. quantitative pcr analysis was performed to amplify the membrane protein (m) gene and eif4a2 using the cdna as the template and the β-actin gene as the internal standard. data analysis was based on the measurement of the cycle threshold (ct). the relative level of m gene and eif4a2 expression was calculated using the 2-△△ct method. the primers are presented in table 1 . 2.11. western blotting piecs were transfected with sirna2 and infected with tgev for 48 h. piecs were washed three times with cold-pbs and lysed in radioimmunoprecipitation assay (ripa, 200 μl/well) buffer (beyotime, china) containing the protease inhibitor, pmsf (0.2 μl). the protein concentrations of the resulting lysates was determined using a pierce bca (beyotime, china). after centrifugation at 11,000 ×g for 12 min, proteins in the supernatant (40 μg protein) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) on 12% gradient gels, and transferred to polyvinylidene fluoride (pvdf) membranes (merck millipore, darmstadt, germany). the membranes were blocked for 1 h in tris-buffered saline (tbs) containing 5% non-fat dry milk at room temperature, and incubated with the primary antibodies (1:600) at 4°c overnight. after washing three times with tbst, the membranes were incubated with hrp-conjugated goat anti-rabbit igg (sangon biotech) at 37°c for 1 h and the proteins were visualized using 3,3′-diaminobenzidine and detected by enhanced chemiluminescence (ecl; thermo scientific) and autoradiography. all calculations were performed using graphpad prism 7.0. all data are presented as the mean ± sd or with the standard errors of the mean from three independent experiments. a one-way analysis of variance (anova) and t-test were employed to determine the statistical differences between multiple groups. p-values < .05 were considered statistically significant (*p-value < .05; **p-value < .01;***p-value < .001). the bait vector, pgbkt7, of the yeast two-hybrid system was constructed to screen the interactions between the tgev m protein and host proteins, and the recombinant plasmid was successfully transformed into y2hgold yeast cells (fig. 1a) . yeast harboring pgbkt7-m and those harboring pgbkt7 (control) were spread at two dilutions onto sd/trp plates, and the yeast size and number of colonies produced by pgbkt7-m were compared with that produced by the control pgbkt7. the results suggest that the bait vector displayed no toxicity toward the yeast (fig. 1b) . the yeast harboring pgbkt7-m were spread at two dilutions on three different selective media plates. no colony growth was observed on the sd/−trp/x-α-gal/aba, indicating that there was no self-activation phenomenon to the reporter genes (fig. 1c) . a western blot showed that pgbkt7-m produced an approximately 50 kda fusion protein (fig. 1d) . these results indicate that the constructed pgbkt7-m bait vector could be used for screening host proteins that interact with the tgev m protein using the yeast twohybrid system. tgev m protein-associated cellular proteins were identified using the yeast two-hybrid system. the zygotes typically displayed a threelobed structure similar to a "mickey mouse" face at. 40× magnification under the microscope ( fig. 2a) after the bait strain y2hgold (pgbkt7-m) and y187 (piecs cdna) were mixed and incubated. after three rounds of screening on different nutritionallydeficient medium plates, a total of 108 blue clones that grew on the sd/ −trp/−leu/−ade/-his/x-α-gal/aba were visible and identified by pcr ( fig. 2b and c) . finally, a database search with sequence analysis (table 2) . these proteins included the multifunctional protein, ade2, regulator of g protein signaling, small nuclear ribonucleoprotein smd3, p2x purinergic receptor 7, rna polymerase ii transcription subunit 1,eukaryotic initiation factor 4a2 (eif4a2), and centromere-related protein e. since it has been previously reported that the host translation initiation factor, eif4a2, is involved in the promotion of virus proliferation and viral protein synthesis (ndzinu et al., 2018; li et al., 2016) . eif4a2 was used as the candidate protein for further research and validation. next, the interaction between the m protein and candidate protein eif4a2 was verified using a gst pull-down assay and co-ip. the his-eif4a2 fusion protein solution was added to glutathione ™4b agarose beads adsorbed with the gst-m fusion protein at the same concentration. gst protein was used as a control to eliminate non-specific protein binding. as shown in fig. 3a , sds-page electrophoresis and the western blot results revealed that the eif4a2 protein was present in the gst-m protein immobilized beads but not in the other gels and the western blot indicated that the size of the his-eif4a2 fusion protein was approximately 50 kda (fig. 3b.) . a co-ip assay was performed to further verify the interaction between eif4a2 and tgev m in vitro.the western blot presented in fig. 3c shows that the m protein was precipitated by the antibody in the tgev-infected group but not in the mock-treated group. these results confirm the interaction between eif4a2 and the m protein both in vitro and in vivo. indirect immunofluorescence was used to validate where eif4a2 and m were localized in piecs following tgev infection. the results indicated that eif4a2 was distributed in both the cytoplasm and nucleus. the green fluorescence of m protein and the red fluorescence of eif4a2 were observed to be partially overlapping in the cytoplasm, indicating that eif4a2 was co-localized with the m protein within piecs during tgev infection (fig. 4) . to further study the role of eif4a2 in tgev replication, the eif4a2 protein of piecs was inhibited using sirna2. at 24 h post transfection, the cells were lysed with trizol and the level of eif4a2 mrna expression was detected using rt-qpcr. at 48 h post transfection, the cells were lysed for eif4a2 protein expression by western blot. as shown in fig. 5a , sirna2 knocked down protein expression more efficiently than the other interference fragment, and quantitative analysis further confirmed a significant inhibitory effect of sirna2 against eif4a2 (fig. 5b) . to investigate the role of eif4a2 on tgev replication, eif4a2 was knocked down in piecs with sirna2, which were subsequently infected with tgev at an moi of 0.1. the viral loads in the supernatants treated with sirna2 were significantly lower compared to those of the negative sirna-and mock-treated groups (p < 0.01 and p < 0.001, respectively) (fig. 6) . similarly, compared with the tgev group, the level of m protein and mrna expression decreased following the knockdown of eif4a2 in piecs and infected with tgev for 48 h (fig. 7a and b) . together, these results suggest that eif4a2 interacts with the tgev m protein and impairs tgev replication. the culture supernatants of the cells and the virus-associated cells infected with tgev were collected at different points. the viral titers are presented as the averages from three independent samples. **p < 0.01 and ***p < 0.001. tgev is a highly contagious disease in the coronavirus that can cause acute diarrhea in piglets at 1-2 weeks of age, and causing severe economic losses (duque and ospina, 2017) . thus, identification of host proteins that interact with tgev viral proteins is a vital for studying the mechanisms of viral pathogenesis. in this study, using a yeast two-hybrid system, we identified seven cellular proteins that can interact with the tgev m protein. these proteins are primarily related to gene transcription, protein folding, protein degradation and metabolism. of these, eif4a2 was identified as a novel m ligand. however, eif4a2 had not been identified in intestinal cells in previous study, likely due to the different cell types used to construct the cdna library. in vivo, tgev replicates in the intestinal epithelial cells of susceptible animals, inducing cell injury, villi atrophy, as well as reduced villous height and crypt depth (haelterman, 1972; schwegmann and herrler, 2006; xia et al., 2018) . therefore, it is important to identify host proteins in the natural target cells of tgev to study viral replication and pathogenesis. previous studies have found that viral nucleic acids and structural proteins are assembled into viral particles in the region between the endoplasmic reticulum (er)and golgi, and that the particles are subsequently transported into the extracellular by budding of vesicles (corse and machamer, 2000; kuo and masters, 2010) . our previous research demonstrated that infectious virions entered the intestinal cells by membrane fusion and mature viruses budded into vacuoles, which were gradually to the cell membrane before being released (song et al., 2016) . moreover, research into tgev has established that the m protein as a linker may contribute to initiate the viral particle assembly by interacting with genomic rna and nucleoproteins in pre-golgi compartments (risco et al., 1998) . therefore, this study provides evidence that host cellular proteins interacting with the tgev m protein will be serve to futher study of viral replication and pathogenesis. in the present study, eif4a2 was identified to interact with the tgev m protein using a gst pull-down assay and co-ip. eif4a2 is one of variants (eif4a1 and eif4a2) reported in mice and rabbits (nielsen and trachsel, 1988) and associates with eif4a1 to form a complex (marintchev et al., 2009) . a previous study showed that eif4a2 acts to limit ibdv replication in infected df1 cells, which is consistent with our findings that knocking down eif4a2 expression can inhibit the replication of tgev in intestinal cells. furthermore, we identified the mechanism by which eif4a2 reduced tgev replication in intestinal cells to be via the down-regulation of m expression. some studies have found that eif4a2 plays an significant role in the virus infection cycle. for example, eif4a2 reduces ibdv replication by inhibiting rna polymerase activity in host cells (li g et al., 2016) . similar findings have also been reported that human eif4a2 was found to interact with the rdrp ns5b of hepatitis c virus by using a yeast two-hybrid system, which may facilitates the genome rna synthesis of ns5b protein (kyono et al., 2002) . in the present study, our results suggest that eif4a2 may be required for tgev replication. therefore, eif4a2 may potentially serve as new therapeutic target for controlling tgev infection. elucidating the interactions between host cell and viral components is essential for understanding coronavirus pathogenesis. in this study, the tgev m protein was shown to interact with the eukaryotic translation initiation factor, eif4a2. moreover, the knockdown of eif4a2 expression with small interfering rna (sirna) led to the inhibition of m protein synthesis and decreased viral copy numbers. since such inhibition occurs during the later stages of the virus replication cycle, inhibition of m protein expression, which plays a key role in the morphological processes of coronaviruses, likely affects the assembly of viral particles, there by leading to decreased tgev titers released into the supernatant of piec. therefore, eif4a2 can play a dual role by either forming the eif4f complex to activate translation or binding to the ccr4-not complex to facilitate mirna-mediated transcription repression (mathys et al., 2014) . furthermore, one study showed that cellular mirnas can inhibit viral infection and that the virus mutates to escape suppression by cellular mirnas (zheng et al., 2013) . therefore, since the underlying mechanism by which eif4a2/tgev affects tgev/ eif4a2 replication is extremely intricate and may involve cellular mirnas, further research is required to confirm this mechanism. in summary, this study is the first to identify proteins involved in the porcine intestinal epithelial interaction with the tgev m protein using a yeast two-hybrid system. a total of seven cellular proteins were identified, which provided novel insight into the mechanism of coronavirus replication. the interaction between the cellular eif4a2 and the tgev m protein was confirmed in tgev-infected piecs. moreover, the knockdown of eif4a2 reduced tgev replication in piecs. therefore, our results may be useful in understanding the mechanisms involved in tgev replication and developing novel strategies for infection control. coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes eif4a3 is a novel component of the exon junction complex coronavirus nucleocapsid protein rna interactions infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles coronavirus particles assembly: primary structure requirements of the membrane protein understanding tgev-etec coinfection through the lens of proteomics: a tale of porcine diarrhea complete sequence (20 kilobases) of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus eukaryotic translational initiation factor 4ii reduces the replication of infectious bursal disease virus by inihiting vp1 polymerase activity on the pathogenesis of transmissible gastroenteritis of swine evolved variants of the membrane protein can partially replace the envelope protein in murine coronavirus assembly human eukaryotic initiation factor 4aii associates with hepatitis c virus ns5b protein in vitro the molecular biology of coronaviruses the diverse roles of the eif4a family: you are the company you keep topology and regulation of the human eif4a/4g/4h helicase complex in translation initiation structural and biochemical insights to the role of the ccr4-not complex and ddx6 atpase in microrna repression translational repression and eif4a2 activity are critical for microrna-mediated gene regulation simulation of the economic impact of transmissible gastroenteritis on commercial pig production in australia eif4a2 is a host factor required for efficient hiv-1 replication the mouse protein synthesis initiation factor 4a gene family includes two related functional genes which are differentially expressed first retrospective studies with etiological confirmation of porcine transmissible gastroenteritis virus infection in argentina two types of virus-related particles are found during transmissible gastroenteritis virus morphogenesis sialic acids as receptor determinants for coronaviruses the assembly of virus-like particles of porcine transmissible gastroenteritis virus in vitro by baculovirus expression system morphogenesis and proliferative rule of porcine transmissible gastroenteritis virus in porcine intestinal epithelial cells vp1, the rna-dependent rna polymerase and genome-linked protein of infectious bursal disease virus, interacts with the carboxy-terminal domain of translational eukaryotic initiation factor 4aii impact of tgev infection on the pig small intestine human microrna hsamir-296-5p suppresses enterovirus 71 replication by targeting the viral genome isolation and identification of a new strain of porcine transmissible gastroenteritis virus from chongqing, southwestern china transmissible gastroenteritis virus: identification of m protein-binding peptide ligands with antiviral and diagnostic potential this work was supported by the graduate scientific research in chongqing, china. (no. cys2015076). the authors declare that there are no conflicts of interest. key: cord-277424-9aimvogs authors: criscitiello, michael f.; kraev, igor; lange, sigrun title: deiminated proteins in extracellular vesicles and serum of llama (lama glama)—novel insights into camelid immunity date: 2019-11-13 journal: mol immunol doi: 10.1016/j.molimm.2019.10.017 sha: doc_id: 277424 cord_uid: 9aimvogs peptidylarginine deiminases (pads) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, causing functional and structural changes in target proteins. protein deimination causes generation of neo-epitopes, affects gene regulation and also allows for protein moonlighting. furthermore, pads have been found to be a phylogenetically conserved regulator for extracellular vesicle (evs) release. evs are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. in this study, post-translationally deiminated proteins in serum and serum-evs are described for the first time in camelids, using the llama (lama glama l. 1758) as a model animal. we report a poly-dispersed population of llama serum evs, positive for phylogenetically conserved ev-specific markers and characterised by tem. in serum, 103 deiminated proteins were overall identified, including key immune and metabolic mediators including complement components, immunoglobulin-based nanobodies, adiponectin and heat shock proteins. in serum, 60 deiminated proteins were identified that were not in evs, and 25 deiminated proteins were found to be unique to evs, with 43 shared deiminated protein hits between both serum and evs. deiminated histone h3, a marker of neutrophil extracellular trap formation, was also detected in llama serum. pad homologues were identified in llama serum by western blotting, via cross reaction with human pad antibodies, and detected at an expected 70 kda size. this is the first report of deiminated proteins in serum and evs of a camelid species, highlighting a hitherto unrecognized post-translational modification in key immune and metabolic proteins in camelids, which may be translatable to and inform a range of human metabolic and inflammatory pathologies. lamoids, or llamas, belong to a family of camelids which are economically important livestock and have developed complex features and immunological traits related to their habitat (wu et al., 2014; saadeldin et al., 2018a) . the llama (lama glama), bactrian camel (camelus bactrianus), dromedary (camelus dromedarius) and alpaca (vicugna pacos) differ in their habitat. the bactrian camel and dromedary are adapted to arid-desert-adapted environments, alpacas to plateaus, and the llama to higher altitudes. llamas are historically found in the andean highlands, specifically the altiplano of southeast perú and western bolivia, as well as in chile and argentina, which has the third highest population of llamas (wu et al., 2014) . the domesticated llama is closely related to two extant wild south american camelids, the vicuña (vicugna vicugna) and guanaco (lama guanicoe). previous genomic studies have revealed a range of specific adaptions in camelids relating to fat and water metabolism, osmoregulation, blood glucose level regulation, stress responses to heat, aridity, as well as to intense ultraviolet radiation and dust (wu et al., 2014) . furthermore, a particularly important feature in camelid immunity is the production of small, homodimeric heavy chain-only, antibodies (hcabs) which are of great value for the biomedical industry . peptidylarginine deiminases (pads) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner. this can cause functional and structural changes in target proteins (vossenaar, and intrinsically disordered proteins, while the position of the arginine is also important; arginines sitting next to aspartic acid residues are most prone to citrullination, arginines next to glutamic acid residues are rarely citrullinated and those flanked by proline are poorly citrullinated (nomura, 1992; tarcsa et al., 1996; györgy et al., 2006) . protein deimination can affect gene regulation, cause generation of neoepitopes (witalison et al., 2015; lange et al., 2017) and may also allow for protein moonlighting, an evolutionary acquired phenomenon facilitating proteins to exhibit several physiologically relevant functions within one polypeptide chain (henderson and martin, 2014; jeffrey, 2018; magnadóttir et al., 2018a) . pads have been identified throughout phylogeny from bacteria to mammals, with 5 tissue specific pad isozymes in mammals, 3 in chicken, 1 in bony fish and arginine deiminase homologues in bacteria (vossenaar et al., 2003; rebl et al., 2010; magnadóttir et al., 2018a, a; kosgodage et al., 2019) . while studies on pads in relation to human pathophysiology, including cancer, autoimmune and neurodegenerative diseases (wang and wang, 2013; witalison et al., 2015; lange et al., 2017; kosgodage et al., 2017 kosgodage et al., & 2018 and cns regeneration (lange et al., 2011 and 2014) exist, relatively little phylogenetic research has been carried out on pads in relation to normal physiology and evolutionary acquired adaptions of the immune system. recent comparative studies focussing on roles for pads in teleost fish have identified post-translational deimination in key proteins of innate, adaptive and mucosal immunity (magnadóttir et al., 2018a, b; magnadottir et al., 2019a; magnadóttir et al., 2019b) . a recent study in shark also revealed novel insights into this post-translational modification in relation to key immune factors, including shark immunoglobulins (criscitiello et al., 2019) . as the camelid family has developed unusual small immunoglobulins similar as to shark through convergent evolution, indicating common factors between shark and camelid immunity, we felt that an investigation of post-translationally deiminated proteins in camelids was warranted. as pads have been identified to be a key regulator of extracellular (ev)-release, a mechanism that has been found to be phylogenetically conserved from bacteria to mammals (kholia et al., 2015; kosgodage et al., 2017 kosgodage et al., , 2018 gavinho et al., 2019; kosgodage et al., 2019) , the characterisation of evs in camelids is of further interest. extracellular vesicles (evs) are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material (inal et al., 2013; colombo et al., 2014; lange et al., 2017; turchinovich et al., 2019; vagner et al., 2019) . evs in body fluids, including serum, can also be useful biomarkers to reflect health status (hessvik and llorente, 2018; ramirez et al., 2018) . previous work on evs has hitherto mainly been in the context of human pathologies, while recently comparative studies are growing (iliev et al., 2018; yang et al., 2015; magnadóttir et al., 2019b; criscitiello et al., 2019; gavinho et al., 2019; kosgodage et al., 2019) . few studies have been performed on evs in camelids but therapeutic effects of evs isolated from camel milk have been identified in halting cancer progression (badawy et al., 2018) . a recent study in shark identified for the first time deiminated small immunoglobulin proteins as part of ev cargo (criscitiello et al., 2019) . due to the link between camelid and shark immunity through convergent evolution including the unusual immunoglobulin structure of small heavy chain-only ig's, a comparative study on evs and deiminated ev cargo in a camelid species may provide further insights into shared immunological traits. camelids have an unusual ig repertoire and a large diversity of functional nanobodies has been identified in the llama (harmsen and de haard, 2007; deschaght et al., 2017) . this has made camelids an important source for small immunoglobulins that can be used for immunotherapy purposes, including for tumour targeting (van lith et al., 2016) , as well as for assessing cancer metastasis (ramos-gomes et al., 2018) . as these nanobodies can also penetrate the blood-brain barrier (širochmanová et al., 2018) they are of great value for a range of therapeutic treatment applications, including for brain cancers (iqbal et al., 2010) . furthermore, as the camelid family has acquired unique metabolic features, they are also of interest as a model species for informing metabolic diseases. in the current study we assessed post-translationally deiminated proteins in llama serum and serum-derived evs, and report for the first time ev-mediated export of deiminated key immune, metabolic and nuclear proteins in serum of a camelid species. llama (lama glama l. 1758) serum was shared from excess blood collected in routine health checks of a resident male llama at the texas a&m winnie carter wildlife center. blood collected from the jugular vein of this 21 year old llama was allowed to clot at room temperature for 2 h before serum was collected by centrifuging at 300 g for 10 min. serum was aliquoted and immediately frozen at −80°c until further use. evs were isolated by step-wise centrifugation according to established protocols using ultracentrifugation and the recommendations of misev2018 (the minimal information for studies of extracellular vesicles 2018; théry et al., 2018) . llama serum was diluted 1:5 in ultrafiltered (using a 0.22 μm filter) dulbecco's pbs (dpbs, 100 μl serum added to 400 μl dpbs) and then centrifuged at 4000 g for 30 min at 4°c for removal of cells and cell debris. the supernatant was collected and centrifuged at 100,000 g for 1 h at 4°c. the pellet was then resuspended in dpbs and washed again at 100,000 g for 1 h at 4°c. the resulting evenriched pellet was resuspended in 100 μl dpbs, diluted 1/100 in dpbs and analysed by nta, based on brownian motion of particles in suspension, using the nanosight ns300 system (malvern, uk). the na-nosight was used in conjunction with a syringe pump to ensure continuous flow of the sample, with approximately 40-60 particles per frame and videos were recorded for 5 x 90 s. the replicate histograms generated from the recordings were averaged. evs were isolated from serum as described above, the ev pellets were fixed with 2.5 % glutaraldehyde in 100 mm sodium cacodylate buffer (ph 7.0) for 1 h at 4°c, resuspended in 100 mm sodium cacodylate buffer (ph 7.0), placed on to a grid with a glow discharged carbon support film, stained with 2 % aqueous uranyl acetate (sigma-aldrich) and thereafter viewed in tem. imaging was performed using a jeol jem 1400 transmission electron microscope (jeol, japan) operated at 80 kv at a magnification of 80,000-100,000. digital images were recorded using an amt xr60 ccd camera (deben, uk). llama serum and ev isolates (an ev pellet derived from 100 μl serum, reconstituted in 100 μl dpbs after isolation and purification) were diluted 1:1 in 2x laemmli sample buffer, boiled for 5 min at 100°c and separated by sds-page on 4-20 % gradient tgx gels (biorad uk). approximately 5 μg protein was loaded per lane and transferred to nitrocellulose membranes using semi-dry western blotting. blocking of membranes was performed in 5 % bsa in tbs-t for 1 h at room temperature (rt) and incubation with primary antibodies, diluted in tbs-t, was carried out at 4°c overnight (f95 mabn328, merck, 1/1000; pad2 ab50257, abcam, 1/1000; pad3 ab50246, 1/1000; pad4 ab50247, 1/1000; cith3 ab5103, 1/1000; cd63 ab216130, 1/1000; flot-1 ab41927, 1/2000). the membranes were washed in tbs-t for 3 × 10 min at rt and thereafter incubated in the corresponding secondary antibody (anti-rabbit igg biorad or anti-mouse igm biorad, diluted 1/4000 in tbs-t) for 1 h at rt. membranes were washed for 6 × 10 min in tbs-t and visualisation performed using electrochemiluminescence (ecl) and the uvp biodoc-ittm system (thermo fisher scientific, uk). for isolation of total deiminated proteins from llama serum and serum derived evs, the catch and release immunoprecipitation kit (merck, uk) was used together with the f95 pan-deimination antibody (mabn328, merck), which has been developed against a deca-citrullinated peptide and specifically detects proteins modified by citrullination (nicholas and whitaker, 2002) . for f95 enrichment, 50 μl serum was used according to the manufacturer's instructions (merck). for evs, total protein was first extracted from ev-enriched pellets derived from 100 μl serum, using 100 μl radioimmunoprecipitation assay (ripa) buffer, containing protease inhibitor cocktail (p8340, sigma, uk), and shaken gently on ice for 2 h. thereafter proteins were isolated from the evs by centrifugation at 16,000 g for 30 min, collecting the supernatant containing the proteins. immunoprecipitation was carried out according to the manufacturer's instructions (merck), using a rotating platform overnight at 4°c. the f95 bound proteins were eluted using denaturing elution buffer, according to the manufacturer's instructions (merck). the f95 enriched eluates were then either analysed by western blotting or by lcems/ms (cambridge proteomics, cambridge, uk). for lcems/ms, the f95-enriched eluates were run 1 cm into a sds-page gel and the whole f95-enriched eluate was cut out as one band, whereafter it was processed for proteomic analysis (carried out by cambridge proteomics). peak files were submitted to in-house mascot (matrix science; cambridge proteomics). databases used for protein identification (in house, cambridge proteomics) were as follows: camelidae_family_20190613 (21429 sequences; 9086806 residues) and also specifically for llama: lama_glama_20190613 (234 sequences; 52757 residues). evs from llama serum were characterised, following step-wise ultracentrifugation, by size exclusion using nta, by morphological analysis using tem and by western blotting using ev-specific markers ( fig. 1) . a poly-dispersed population of evs in the size range of 30-576 nm, with main peaks at 38, 119, 167, 237, 323 and 403 nm was identified by nta analysis (fig. 1a) . western blotting confirmed that the llama serum evs were positive for the ev-specific markers cd63 and flotillin-1 (fig. 1b) . tem analysis confirmed a poly-dispersed evs population (fig. 1c) . a cross-reaction with human pad2, 3 and 4 isozyme-specific antibodies was observed in llama serum by western blotting, at an approximate 70-75 kda size range as expected for mammalian pads ( fig. 2a) . deiminated histone h3 was also detected in llama serum by western blotting at the expected approximate 20 kda size ( fig. 2a) . total deiminated proteins in llama serum-evs were detected by western blotting using the f95 pan-deimination antibody, revealing a range of proteins between 25-100 kda in size (fig. 2b ). deiminated proteins were also assessed by western blotting after f95 enrichment, using immunoprecipitation (ip) from llama serum and serum-derived evs (fig. 2c ). deiminated proteins from the f95 enriched eluates were further identified by lc-ms/ms analysis. in llama serum, 103 hits for camelid proteins were identified, as listed in table 1 and supplementary table 1 . overall, 43 of these deiminated protein hits were common to whole serum and serum-derived evs, while 60 hits were specific for whole serum (fig. 2d ). llama serum-derived evs showed positive for deiminated proteins by western blotting, using the pan-deimination f95 antibody (fig. 2b ). deiminated proteins were further identified by f95 enrichment and lc-ms/ms analysis, revealing 68 deiminated protein hits in total for evs, with 25 hits unique to evs (not identified from serum). peptide sequences of hits with camelid proteins and m/z values are listed in table 2 and supplementary table 2 . overlap with deiminated proteins identified in whole llama serum and evs is represented in the venn diagram in fig. 2d . for the first time deiminated proteins are described in a camelid, using the llama (lama glama) as a model species. post-translational deimination was identified in key immune, nuclear and metabolic proteins. a llama pad homologue was identified at an expected 70-75 kda size similar as for human pads by western blotting via cross reaction with anti-human pad2, pad3 and pad4 antibodies. pad2 is known to be the phylogenetically most conserved pad form (vossenaar et al., 2003; magnadóttir et al., 2018a) and has also been seen in shark (criscitiello et al., 2019) . deiminated histone h3, a marker of neutrophil extracellular trap formation (netosis), was also detected in llama serum by western blotting and is described for the first time in a camelid species but was recently described in shark (criscitiello et al., 2019) . netosis is driven by pads (li et al., 2010) , is conserved throughout phylogeny and is important in innate immune defences against a range of pathogens including bacteria, viruses and helminths (brinkmann et al., 2004; branzk et al., 2014; schönrich and raftery, 2016) . netosis has also been associated with clearance of apoptotic cells and during tissue remodelling in teleosts (magnadóttir et al., 2018a (magnadóttir et al., , 2019a . furthermore, netosis is linked to a range of autoimmune diseases, due to net activation via neo-epitopes (o'neil and kaplan, 2019), which can also lead to organ damage (lee et al., 2017) . netosis is also associated with cancer (gonzalez-aparicio and alfaro, 2019) and neurodegenerative diseases (pietronigro et al., 2017) . further deiminated proteins identified in llama serum and serumderived evs by f95 enrichment and lcems/ms analysis included key proteins of camelid innate and adaptive immunity, nuclear proteins, as well as proteins involved in metabolic function. using string (search tool for the retrieval of interacting genes/proteins) analysis (https:// string-db.org/) for protein-protein interaction, the ppi enrichment value for deiminated proteins in whole llama serum was found to be p < 1.0e-16 for 43 deiminated proteins out of 103 identified in serum, which indicates that these proteins have more interactions among themselves than what would be expected for a random set of proteins of similar size, drawn from the genome (string analysis, see fig. 3a ). for deiminated proteins in 29 out of the 68 deiminated ev cargo proteins, the ppi enrichment value was found to be p = 0.0193 (string analysis, see fig. 4a ). such enrichment indicates that the proteins are at least partially biologically connected, as a group. as the camelid proteins are not present in the string database, corresponding protein homologues for human were chosen to create the protein-protein interaction networks shown in figs. 3 and 4. out of the 103 camelid proteins identified as deiminated in serum, protein ids for 43 were present for homo sapiens to perform the assessment of protein-protein interactions and identification of main biological go pathways (response to stress, response to wounding, oxygen transport, small molecule metabolic process, vesicle mediated transport and regulated exocytosis; fig. 3b ). for llama evs, out of 68 proteins identified as deiminated, 29 corresponding protein homologues were found in the string database for homo sapiens and were analysed highlighting main biological go pathways (response to stress, cytoskeleton organisation and vesicle-mediated transport; fig. 4b ). deimination protein candidates identified here in llama serum and evs, which are involved in immune, nuclear and metabolic functions, are further discussed below, including where appropriate in a comparative context with relevant human diseases. proteins that have previously been identified as deiminated in other species are listed first. nanobodies are based on immunoglobulin single variable domains, derived from the variable domains of heavy chain-only antibodies, which occur naturally in camelids (deschaght et al., 2017) . in the llama the heavy chain-only antibodies are comprised of at least two subclasses . these types of homodimeric, light chain-less antibodies have evolved through convergent evolution (brooks et al., 2018) , in at least two groups (camelids and cartilaginous fish), and their variable binding domains (v h h s ) are of great value for therapeutic and diagnostic applications (muyldermans, 2013; criscitiello, 2014; steeland et al., 2016; könning et al., 2017; henry et al., 2019) . nanobodies can act against challenging targets such as small molecules and toxins (wesolowski et al., 2009; bever et al., 2016) , viruses (wei et al., 2011; hassiki et al., 2016; vanlandschoot et al., 2011; cohen, 2018) , enzymes (muyldermans, 2013) , ion channels (wei et al., 2011; danquah et al., 2016) and g protein-coupled receptors gpcrs (cromie et al., 2015) . llama nanobodies have been shown to tether to early endosomes and to mitochondria (traub, 2019) , be used for diagnostics (shriver-lake et al., 2018) , be used for design of cancer immunotherapeutics (hussack et al., 2018; bannas and koch-nolte, 2018; rossotti et al., 2019) and have been approved for passive immunotherapy (sheridan, 2019) . our current finding, that llama nanobodies can be post-translationally deiminated may shed some light on their observed structural variation which still remains to be fully explained as sequence alignment does not fully elucidate their diversity (mitchell and colwell, 2018) . as a structurally analogous immunoglobulin in shark, new antigen receptor (nar) (greenberg et al., 1995; barelle et al., 2009; flajnik and dooley, 2009; de silva et al., 2019) was recently also found to be deiminated (criscitiello et al., 2019) , our current finding may provide novel insights into function of these immune proteins and be useful for refinement in therapeutic nanobody development. llama single-chain antibodies were here found to be deimination candidates both in llama whole serum and in serum derived evs, highlighting also their ev-mediated export. ig proteins were identified here as being deiminated in llama serum and in serum-derived evs, scoring with ig components from other camelids. immunoglobulins (ig) are key molecules in adaptive immunity but post-translational deimination of ig's has hitherto received little attention. deimination of the igg fc region in patients with bronchiectasis and ra has been identified (hutchinson et al., 2017) . furthermore, deimination of ig's in teleost fish was recently described (magnadóttir et al., 2019a) , as well as in shark (criscitiello et al., 2019) . given the increased focus on understanding of ig diversity throughout phylogeny (smith et al., 2012; zhang et al., 2013; de los rios et al., 2015; zhang et al., 2016 zhang et al., , 2017 stanfield et al., 2018) and the unique features of camelid immunoglobulins (plasil et al., 2019) , our current finding of deimination of llama ig's highlights a novel concept that may further understanding of ig diversity throughout evolution. of additional interest is the finding that some deiminated ig proteins were also found to be exported in evs. complement components identified to be deiminated in llama serum included c3, c4 and c1q, while only c3 was identified to be deiminated in serum derived evs. complement component c3 plays a central role in all pathways of complement activation and can also be directly activated by self-and non-self surfaces via the alternative pathway without a recognition molecule (dodds and law, 1998; dodds, 2002) . in camelids, the alternative and classical pathway haemolytic activity of serum has been assessed in the dromedary camel with respect to age and gender (olaho-mukani et al., 1995a; and 1995b) . hitherto little is known about roles for post-translational deimination of complement components throughout phylogeny. in teleost fish, c3 has been identified in serum in deiminated form (magnadóttir et al., 2019a) and also found to be deiminated in mucosal and serum evs of teleost fish (magnadóttir et al., 2019b (magnadóttir et al., , 2019c , while post-translationally deiminated c3 was identified in shark total serum but not evs (criscitiello et al., 2019) . other complement components, including c4 and c1q, which belong to the classical pathway of complement activation were here identified as deiminated in llama whole serum and some of those llama serum evs are positive for the ev-specific markers cd63 and flotillin-1 (flot-1). c. transmission electron microscopy (tem) imaging of evs isolated from llama serum shows a polydispersed population; scale bar represents 100 nm. have also recently been reported to be deiminated in teleost fish (magnadóttir et al., 2019a) . the c1q subcomponent can bind to the fc region of immunoglobulins that are bound to antigen and activate the classical part of the complement pathway (reid et al., 2002; reid, 2018) . interestingly, an essential role for arginine in c1q has previously been suggested for c1q-igg interaction (kojouharova et al., 2004) . c1q also serves as a potent pattern recognition molecule which recognises self, non-self and altered self-signals (nayak et al., 2012; reid, 2018) and may therefore also bind to deiminated neo-epitopes (magnadóttir et al., 2019a ). the complement system has multifaceted roles. it forms part of the first line of immune defence against invading pathogens, acting in clearance of necrotic or apoptotic cells (dodds and law, 1998; sunyer and lambris, 1998; fishelson et al., 2001; carroll and sim, 2011) . complement also has roles in regeneration (del rio-tsonis et al., 1998; haynes et al., 2013) and tissue remodelling (lange et al., 2004a (lange et al., , 2004b lange et al., 2005 lange et al., , 2006 lange et al., , 2019 . furthermore, c1 is also implicated in multiple non-complement functions including binding of apoptotic cells, cleavage of nuclear antigens and cleavage of mhc class i (lu and kishore, 2017) . post-translational deimination of complement components may possibly influence their function including cleavage ability, binding, deposition and generation of the convertase. apolipoprotein a-i is primarily involved in lipid metabolism where conformational plasticity and flexibility are regarded as key structural features (arciello et al., 2016) . apo a-i is associated with regulation of llama pad homologues were identified at the expected size of approximately 70-75 kda using the anti-human pad2, pad3 and pad4 isozyme specific antibodies respectively. deiminated histone h3 (cith3), representative of neutrophil extracellular traps (nets), was verified in llama serum. b. total deiminated proteins were assessed by western blotting in llama serum evs, using the f95 pan-deimination specific antibody. c. immunoprecipitated (ip) deiminated proteins after f95-enrichment were assessed both in serum-evs and whole serum of llama by western blotting. d. the venn diagram represents deiminated proteins identified in total serum and serum-derived evs by f95 enrichment and lc-ms/ms analysis. overall, 43 proteins were identified in common with both samples, while 60 proteins were found deiminated in serum only and 25 proteins were identified as deiminated in evs only. (continued on next page) mitochondrial function and bioenergetics (white et al., 2017) . furthermore, apo a-i has been shown to have a regulatory role in the complement system by affecting membrane attach complex (mac) assembly and thus the final lytic pathway (hamilton et al., 1993; jenne et al., 1991; french et al., 1994) . given the diverse roles of apo a-i, the current finding of deiminated forms in serum may be of quite some relevance and has previously been identified in teleost fish (magnadóttir et al., 2019a) . apo a-i was here found to be deiminated in whole llama serum only. serum albumin is a major acidic plasma protein in vertebrates and serves as a transport molecule for fatty acids, bilirubin, steroids, amino acids and copper, as well as having roles in maintaining the colloid osmotic pressure of blood (peters, 1996) . in camelids, total albumin levels have been assessed, for example in relation to reproductive efficiency (el-malky et al., 2018) and as biomarkers of oxidative stress 1 28 centromere protein j ⱡ ions score is -10*log(p), where p is the probability that the observed match is a random event. individual ions scores > 20 indicated identity or extensive homology (p < 0.05). protein scores were derived from ions scores as a nonprobabilistic basis for ranking protein hits. cut-off was set at ions score 20. ⱡ ions score is -10*log(p), where p is the probability that the observed match is a random event. individual ions scores > 22 indicated identity or extensive homology (p < 0.05). protein scores were derived from ions scores as a nonprobabilistic basis for ranking protein hits. cut-off was set at ions score 20. (caption on next page) m.f. criscitiello, et al. molecular immunology 117 (2020) [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] (el-deeb and buczinski, 2015). while albumin has been identified as a glycoprotein in some species (metcalf et al., 2007) investigation of posttranslational deimination has been limited, but was recently identified in teleost fish (magnadóttir et al., 2019a) . serum albumin was identified as deiminated in both whole llama serum and evs. hemopexin is a scavenger protein of haemoglobin and a predominant heme binding protein, which contributes to heme homeostasis (smith and mcculloh, 2015; immenschuh et al., 2017) . hemopexin also associates with high density lipoproteins (hdl), influencing their inflammatory properties (mehta and reddy, 2015) . hemopexin is a plasma glycoprotein that has been previously identified as a deimination candidate in teleost fish (magnadóttir et al., 2019a) and shark (criscitiello et al., 2019) . here, hemopexin was found deiminated in whole llama serum only. inter-alpha-trypsin inhibitor (heavy chain h1) belongs to the serpin family of proteins, which have protease-inhibitory functions and are involved in diverse physiological and pathological processes including fertilisation, ovulation, coagulation, inflammation, as well as tumorigenesis, metastasis and dementia (zhuo and kimata, 2008; weidle et al., 2018) . inter-alpha-trypsin inhibitor is synthesised in the liver, circulates in the blood and has two chains, a light and heavy chain, whereof the heavy-chain (itih) includes a von willebrand domain and can interact with the extracellular matrix (bost et al., 1998) . itih is downregulated in tumours via methylation and itih2 is strongly reduced in invasive cancers (hamm et al., 2008) . while itih was recently identified as a deimination candidate in teleost fish (magnadóttir et al., 2019a) further studies on the regulation of itih2 via post-translational deimination have not been carried out. itih was here identified as deiminated in whole llama serum only. fibrinogen is a glycoprotein, synthesised in liver (tennent et al., fig. 3 . protein-protein interaction networks of deiminated protein hits identified in whole llama (lama glama) serum. reconstruction of protein-protein interactions based on known and predicted interactions using string analysis. due to annotations for camelids not being present in string, proteins are based on corresponding human protein identifiers. a. coloured nodes represent query proteins and first shell of interactors; white nodes are second shell of interactors. b. biological go processes are highlighted for the same protein network as follows: red = response to stress; blue = response to wounding; green = vesicle mediated transport; yellow = oxygen transport; purple = regulated exocytosis; dark green = small molecule metabolic process. coloured lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see colour key for connective lines in a). protein-protein interaction networks of deiminated protein hits identified in evs of llama (lama glama) serum. reconstruction of protein-protein interactions based on known and predicted interactions using string analysis. due to annotations for camelids not being present in string, proteins are based on corresponding human protein identifiers. a. coloured nodes represent query proteins and first shell of interactors. b. biological go processes are highlighted as follows: red = response to stress; blue = cytoskeleton organisation; green = vesicle mediated transport. coloured lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see colour key for connective lines in a). 2007) and forms part of the acute phase response as part of the coagulation cascade (tiscia and margaglione, 2018) . in camelids, fibrinogen is a biomarker for stress and infection (greunz et al., 2018; el-bahr and el-deeb, 2016; el-deeb and buczinski, 2015) . impaired mechanism of fibrinogen formation and fibrin polymerization are implicated with various pathologies including coagulopathies and ischemic stroke (weisel and litvinov, 2013) , while acquired fibrinogen disorders can be associated with cancer, liver disease or post-translational modifications (besser and macdonald, 2016) . fibrinogen is indeed a known deimination candidate and this post-translational modification contributes to its antigenicity in autoimmune diseases (hida et al., 2004; muller and radic, 2015; blachère et al., 2017) . fibrinogen was here identified as deiminated both in whole llama serum and evs. tubulin beta-chain participates in cytoskeletal rearrangement and its deimination has previously been linked to ev release (kholia et al., 2015) . deimination of tubulin may therefore be crucial for facilitating ev-mediated processes in homeostasis, immune responses and in relation to pathologies. here, tubulin was identified as deiminated in both whole llama serum and evs. histone h2b was identified as being deiminated in both llama serum and evs and in addition, histone 1 (h2ai isoform 3-like protein) and core histone macro-h2a.1 isoform 2 were identified as deiminated in evs only. histones undergo various posttranslational modifications that affect gene regulation and can also act in concert (latham and dent, 2007; bird, 2007) . in addition to acetylation, phosphorylation and ubiquitination, histones are known to undergo deimination, including h2b (sohn et al., 2015) and h2a (hagiwara et al., 2005) , as identified in this study in llama. other histones that are known to undergo deimination include h3 and h4 (chen et al., 2014; kosgodage et al., 2018) . heat shock protein 90 (hsp90) was here found to be deiminated both in llama whole serum and evs. hsp90 has been described in camelid (saeed et al., 2015) . hsp90 is a phylogenetically highly conserved chaperone protein involved in protein folding, stabilisation of proteins against heat stress, and aids in protein degradation (buchner, 1999; picard, 2002) . hsp90 also stabilizes a number of proteins required for tumour growth and is therefore important in anti-cancer drug investigations (goetz et al., 2003) . hsp90 is responsible for most of the atpase activity of the proteasome (imai et al., 2003) and has an atp binding region, which also is the main binding site of drugs (chiosis et al., 2006) . in camelids, hsp90 has been related to adaptive tolerance of camel somatic cells to acute and chronic heat shock (up to 20 h at 45 degrees celsius), which is lethal to many mammalian cells (saadeldin et al., 2018b) . hsp90 has previously been described to be post-translationally deiminated in rheumatoid arthritis, allowing deiminationinduced shifts in protein structure to generate cryptic epitopes capable of bypassing b cell tolerance (travers et al., 2016) . it is of some interest to find that hsp90 is also found deiminated in llama serum, further highlighting protein moonlighting functions of hsp90 in physiological and pathophysiological context via post-translational deimination. in addition, finding post-translational deimination of the same protein throughout phylogeny also supports translational value between species to further understanding of this post-translational modification in human pathologies. the following deimination protein candidates identified in the current study in llama serum and serum-evs have to our knowledge not been previously reported as deiminated: alpha 2-macroglobulin is closely related to other thioester containing proteins, such as complement proteins c3, c4 and c5 (sottrup-jensen et al., 1985; davies and sim, 1981) . alpha-2-m is phylogenetically conserved from arthropods to mammals and found at high levels in mammalian plasma. alpha-2-m forms part of the innate immune system and clears active proteases from tissue fluids (armstrong and quigley, 1999) . here, alpha-2-m was found deiminated in whole llama serum only and has not reported as deiminated before, to our knowledge. adiponectin is the most abundant secreted adipokine with pleiotrophic roles in physiological and pathophysiological processes (fiaschi, 2019) . it has received considerable interest in the field of metabolic and obesity research (frankenberg et al., 2017; spracklen et al., 2019) , as well as in diabetes (yamauchi et al., 2003) , due to its key function in regulating glucose (yamauchi et al., 2002) . adiponectin is furthermore linked to longevity (chen et al., 2019a (chen et al., , 2019b , regenerative functions (fiaschi et al., 2014) and has roles in myopathies, such as duchenne muscular dystrophy and collagen vi-related myopathies (gamberi et al., 2019) . adiponectin is also implicated in a range of cancers, often in relation to obesity (parida et al., 2019) . furthermore, adiponectin plays roles in reproduction, embryo pre-implantation and embryonic development (barbe et al., 2019) . due to the range of functions in relation to key pathophysiologies there is great interest in drug development for modulating adiponectin signalling (fiaschi, 2019) . given the unique metabolic adaptive features of camelids, the identification of post-translational deimination of this key metabolic protein may be of some interest. recent studies in rheumatoid arthritis made a correlation between inflammation, autoantibodies and adiponectin levels (hughes-austin et al., 2018; liu et al., 2019) , while adiponectin itself has not been previously identified to be deiminated to our knowledge. post-translational deimination may be a hitherto unrecognized mechanism for adiponectin, also in humans, to adapt moonlighting functions via changes in protein folding and therefore interaction with other proteins. adiponectin is a small 244 aa protein (np_001171271.1) in humans and contains 2 unfolded regions and 7 arginine sites, while camel adiponectin has 8 arginine sites. these could be subjected to pad-mediated deimination and therefore modulate adiponectin folding and function, depending on which arginine is deiminated. here, deiminated adiponectin was identified in whole llama serum only. dystonin is a plakin-family adhesion junction plaque protein and was here identified as deiminated in both llama serum and evs. dystonin, along with xvii collagen, form hemidesmosomes and both proteins are autoantigens believed to be responsible for the type ii hypersensitivy pathologic in the pruritic skin disease bullous pemphigoid (bağcı et al., 2017; basseri et al., 2018) . dystonin has also been linked to sjögren's syndrome and linked to a hypermethylation status (gonzalez et al., 2011) , but post-translational deimination of dystonin has not been previously described to our knowledge, although deimination is associated with a range of autoimmune diseases, including sjögren's syndrome (konsta et al., 2014; selmi et al., 2015) . xaa-pro dipeptidase, also known as prolidase, is an enzyme that in humans is encoded by the pepd gene. post-translational modifications of prolidase regulate its enzymatic abilities. deficiency in prolidase leads to a rare, severe autosomal recessive disorder (prolidase deficiency) that causes many chronic, debilitating health conditions in humans (viglio et al., 2006) . these phenotypic symptoms vary and may include skin ulcerations, mental retardation, splenomegaly, recurrent infections, photosensitivity, hyperkeratosis, and unusual facial appearance. furthermore, prolidase activity was found to be abnormal compared to healthy levels in various medical conditions including: bipolar disorder, breast cancer, endometrial cancer, keloid scar formation, erectile dysfunction, liver disease, lung cancer, hypertension, melanoma, and chronic pancreatitis (kitchener and grunden, 2012) . in some cancers with increased levels of prolidase activity, such as melanoma, the differential expression of prolidase and its substrate specificity for dipeptides with proline at the carboxyl end suggests the potential of prolidase in becoming a viable, selective endogenous enzyme target for proline prodrugs (mittal et al., 2005) . serum prolidase enzyme activity is also currently being explored as a biomarker for diseases including chronic hepatitis b and liver fibrosis (duygu et al., 2013; şen et al., 2014; stanfliet et al., 2015) . phosphorylation of prolidase has been shown to increase its activity while dephosphorylation leads to a decrease in enzyme activity. post-translational deimination of prolidase has not been described before to our knowledge and may add to understanding of how this enzyme is regulated. prolidase was here identified as deiminated in both llama serum and evs. dipeptidylpeptidase 4 (dpp4, also known as cd26) was here identified to be deiminated in both llama whole serum and evs. dpp4 controls glucose homeostasis and has complex roles in inflammation and homeostasis, including in liver cytokine expression, while its activity in plasma has been shown to correlate with body weight and fat mass (varin et al., 2019) . interestingly, in camel milk, ddp4 inhibitory peptides have been identified and suggested to play roles in the regulation of glycaemia in humans (nongonierma et al., 2018) . furthermore, roles for ddp4 in cancer have been found to relate to its posttranslational processing of chemokines, thereby limiting lymphocyte migration to sites of inflammation and tumours (barreira da silva et al., 2015) . as the success of antitumour immune responses depends on the infiltration of solid tumours by effector t cells, a process which is guided by chemokines, dpp4 inhibitors have been suggested as a strategy to enhance tumour immunotherapy (barreira da silva et al., 2015) . furthermore, serum ddp4 activity levels in primary hiv infection were found to be significantly decreased and to correlate with inflammation and hiv-induced intestinal damage (ploquin et al., 2018) . middle east respiratory syndrome coronavirus (mers-cov) has been found to utilize dipeptidyl peptidase 4 (dpp4) as an entry receptor, via glycosylated sites (peck et al., 2017) . therefore the identification of dpp4 as a deimination candidate may be of some relevance as such post-translational modification can affect dpp4 structure and function, allowing for moonlighting functions which may vary in pathological compared to pathophysiological milieus. e3 ubiquitin-protein ligase roquin, belongs to the roquins which are a family of highly conserved rna-binding proteins involved in ubiquination, are crucial for t-cell-dependent b-cell responses (athanasopoulos et al., 2016) and play important roles in modulating tcell activity (akef and muljo, 2018) . roquins repress constitutive decay elements containing mrnas and play a critical role in rna homeostasis and immunological self-tolerance essig et al., 2018; mino and takeuchi, 2018) . roquin plays multifaceted roles both in the generation of a homeostatic immune response, as well as during chronic inflammation and autoimmunity (schaefer and klein, 2016; lee et al., 2019) . while roquin causes post-translational ubiquination of target proteins, post-translational deimination of roquin itself may modulate is function and is described here for the first time. roquin was here identified as deiminated in whole llama serum only. serine-trna ligase, mitochondrial was here identified as a deimination candidate protein in llama evs only. it has been linked to hupra syndrome which is a rare mitochondrial disease characterized by hyperuricemia, pulmonary hypertension, renal failure in infancy and alkalosis (rivera et al., 2013) . it has furthermore been linked to progressive spastic paresis (linnankivi et al., 2016) . post-translational deimination of this mitochondrial protein has not been described before to our knowledge. nucleoredoxin (nrx) was here identified to be deiminated in evs only. it is an oxidoreductase of the thioredoxin family of proteins with numerous functions in the redox regulation of metabolic pathways, cellular morphology, and signal transduction (urbainsky et al., 2018) . nrx has been shown to inhibit wnt-beta-catenin signalling (funato et al., 2006) and is linked to ca 2+ -mediated mitochondrial reactive oxygen species metabolism (rharass et al., 2014) . nrx has furthermore been identified as an epigenetic cancer marker related to the oxidative status of human blood (schöttker et al., 2015) , but hitherto not been described as deiminated. dyslexia-associated protein was here identified to be deiminated in total serum of llama. it is associated with developmental dyslexia (levecque et al., 2009) and has been shown to have roles in cell-cell interactions and signalling and neuronal migration (velayos-baeza et al., 2008) , as well as in axon guidance (poon et al., 2011) . dyslexiaassociated protein has previously been found to follow the classical clathrin-mediated endocytosis pathway and its surface expression seems regulated by endocytosis, indicating that the internalization and recycling of the protein may be involved in fine-tuning its role in neuronal migration (levecque et al., 2009) . it has been descried to be highly glycosylated in different mammalian cell lines (velayos-baeza et al., 2008) , while deimination has not been described before. metabotropic glutamate receptor 3 was identified here as deiminated in llama evs only. it has, like other components of the glutamatergic system, a widespread distribution outside the central nervous system (cns) and has been linked to regulation of the brain-gut axis (julio-pieper et al., 2013) . it has also been linked to pain (acher and goudet, 2015) and psychotic disorders (matrisciano et al., 2016) . the group iii mglu receptors have been described in human stomach and colon, revealing a huge potential for these receptors in the treatment of peripheral disorders, including gastrointestinal dysfunction (julio-pieper et al., 2013) . as post-translational deimination has not been described before in this receptor, but may well affect is tertiary structure, our current finding may be of some relevance in relation to strategies for developing antagonistic probes (wenthur et al., 2019) . such pharmacological tools originally designed for mglu receptors in the cns may also be directed towards new disease targets in the periphery. ulcerative colitis and crohn's disease are potential targets, as irritable bowel diseases can be co-morbid with anxiety and depression (julio-pieper et al., 2013) . glutathione synthetase (gss) was here identified to be deiminated in both llama whole serum and evs. gss is the second enzyme in the glutathione (gsh) biosynthesis pathway involved in homeostasis and cellular maintenance and also acts as a potent antioxidant (njålsson and norgren, 2005) . in camelids, glutathione peroxidase, which also belongs to the gsh biosynthesis pathway and is a potent anti-oxidant, has been identified as a seminal plasma fertility biomarker (waheed et al., 2015) . furthermore, camel milk has been shown to boost glutathione and total anti-oxidant capacity in sera and exudates in animal studies (arab et al., 2017) . in a diabetic mouse model, the activities of gsh, alongside glucose insulin and ros levels, were restored after camel whey protein treatment (sayed et al., 2017) . in humans, gss deficiency is an autosomal recessive disorder with varyingly severe clinical manifestations that include metabolic acidosis, hemolytic anemia, hyperbilirubinemia, neurological disorders and sepsis (guney varal et al., 2019) . deimination of gss identified here has not been assessed before and may possibly affect the gsh biosynthesis pathway and such posttranslational regulation remains to be further investigated. rootletin, also known as ciliary rootlet coiled-coil protein (crocc), is a protein that is required for centrosome cohesion and is therefore important in mitosis (bahe et al., 2005; graser et al., 2007) . it was identified here as deiminated in whole llama serum only. rootletin has been shown to be phosphorylated and to have the ability to form centriole-associated fibers, suggesting a dynamic model for centrosome cohesion based on entangling filaments (bahe et al., 2005) . deletion of rootletin in mouse models causes photoreceptor degeneration and impaired mucociliary clearance, supporting its key function in rootlet structures (yang et al., 2005) . post-translational deimination of rootletin, as identified here, may possibly facilitate its dynamic functions. centromere protein j is is a highly conserved and ubiquitiously expressed centrosomal protein, involved in microtubule disassembly and plays a structural role in the maintenance of centrosome integrity, genome stability and normal spindle morphology during cell division (tang et al., 2009; mcintyre, 2012) . it was here identified as deiminated in whole llama serum only. knockout mouse models targeting the gene encoding this protein have phenotypes of impaired glucose tolerance, hypoalbuminemia and increased micronuclei, indicative of genomic instability (gerdin, 2010) . in humans, it is associated with primary autosomal recessive microcephaly (gul et al., 2006) and the microcephalic primordial dwarfism disorder seckel syndrome (al-dosari et al., 2010; mcintyre et al., 2012) . deimination has not bee described before in this protein to our knowledge. telomere-associated protein rif1 isoform 1 is involved in the repair of double-strand dna breaks in response to dna damage (silverman et al., 2004; escribano-díaz et al., 2013; drané et al., 2017) and protects teleomeres (fontana et al., 2018) . it was identified here as deiminated in evs only. rif1 has been associated with cancer via activation of human telomerase reverse transcriptase expression . it is also required for immunoglobulin class-switch recombination during the germinal centre reaction for humoral antibody immunity (di virgilio et al., 2013) . rif1 is involved in telomere homeostasis and was recently found to be post-translationally s-acylated, identifying a novel posttranslational modification regulating dna repair (fontana et al., 2019) . post-translational deimination has hitherto not been described. rabenosyn-5-like protein was here identified as a deimination protein candidate in both llama whole serum and evs. it acts in early endocytic membrane fusion and membrane trafficking of recycling endosomes (naslavsky et al., 2004; rai et al., 2017) . it plays a role in the lysosomal trafficking of ctsd/cathepsin d from the golgi to lysosomes and also promotes the recycling of transferrin directly from early endosomes to the plasma membrane (navaroli et al., 2012) . rabenosyn-5-like protein also binds phospholipid vesicles and plays roles in regulating protein sorting and recycling to the plasma membrane (nielsen et al., 2000; de renzis et al., 2002; naslavsky et al., 2004) . rabenosyn-5 was recently identified as an upregulated urinary biomarker associated with malignant upper gastrointestinal cancer (husi et al., 2019) and found to be increased in diabetic kidney disease (dumont et al., 2017) . rabenosyn-5 has been shown to be phosphorylated, regulating its recruitment to membranes (macé et al., 2005) . post-translational deimination has hitherto not been described.. spermatogenesis-associated protein 2 (spata2) was here identified as deiminated in whole llama serum only. it is expressed in testis and to a lesser extent in spleen, thymus, and prostate (graziotto et al., 1999) . spata2 has been identified as a bridging factor that regulates tnf-alpha-induced necroptosis and is instrumental for tnf-induced cell death (elliott et al., 2016; kupka et al., 2016; schlicher et al., 2016; wagner et al., 2016; schlicher et al., 2017) . spata2 ensures normal secretory function of sertoli cells (zhao et al., 2017) and has recently been identified as a novel predictor in ovarian cancer outcome (wieser et al., 2019) . while spata2 has been linked to ubiquination (schlicher et al., 2016) , post-translational deimination has not been invesigated. vitamin d-binding protein (vdbp) belongs to the albumin gene family, together with human serum albumin and alpha-fetoprotein. it is a multifaceted protein mainly produced in the liver, where its regulation is influenced by estrogen, glucocorticoids and inflammatory cytokines (bikle and schwartz, 2019) . it is secreted into the blood circulation and is able to bind the various forms of vitamin d including ergocalciferol (vitamin d2) and cholecalciferol (vitamin d3), the 25hydroxylated forms (calcifediol), and the active hormonal product 1,25dihydroxyvitamin d (calcitriol) (verboven et al., 2002; norman, 2008) . it transports vitamin d metabolites between skin, liver and kidney, and then on to various target tissues (norman, 2008) . vdbp is a macrophage activating factor (maf) and has been tested as an anti-cancer agent via activation of macrophages against cancer cells (yamamoto et al., 2008) . some association has also been made between polymorphisms of vdbp and the risk of coronary artery disease (tarighi et al., 2017) . post-translational modifications (which still remain to be identified) of vdbp have been associated with multiple sclerosis (ms) (perga et al., 2015) , while protein deimination is well known to be associated with ms (moscarello et al., 2013) . vdbp has previously been identified to be glycosylated (kilpatrick and phinney, 2017) but was here identified as a deimination candidate in whole llama serum only. post-translational deimination may contribute to various functions of vdbp in physiological as well as pathophysiological processes. pseudopodium-enriched atypical kinase 1 (peak1) was here identified as deiminated in evs only. it is a tyrosine kinase and scaffold protein that transmits integrin-mediated extracellular matrix (ecm) signals to facilitate cell movement and growth. while aberrant expression of peak1 has been linked to cancer progression, its normal physiological role in vertebrate biology is still relatively unknown. deletion of the peak1 gene in zebrafish, mice and human endothelial cells (ecs) induced severe defects in new blood vessel formation due to deficiencies in ec proliferation, survival and migration. peak1 seems therefore to play roles in modulation of cell adhesion and growth factor cues from the extracellular environment necessary for new vessel formation during vertebrate development and cancer (wang et al., 2018) . peak1 has not been described as deiminated before. desmoplakin was here identified to be deiminated in llama evs only. desmoplakin is a unique and critical component of desmosomal cell-cell junctions and involved in integrity of the cytoskeletal intermediate filament network (bendrick et al., 2019) . it has been shown to be required for epidermal integrity and morphogenesis in the xenopus laevis embryo (bharathan and dickinson, 2019) and novel roles in coordination of cell migration were recently established (bendrick et al., 2019) . mutations in desmoplakin have been linked to multiple allergies, severe dermatitis and metabolic wasting (sam) syndrome (liang et al., 2019) . it is also linked to carvajal syndrome, involving altered skin and hair abnormalities, and heart diseases (yermakovich et al., 2018) , including cardiomyopathies (reichl et al., 2018; chen et al., 2019a chen et al., , 2019b . desmosomal proteins have been shown to have both tumourpromoting and tumour-suppressive functions, depending of cancer types and can regulate cell proliferation, differentiation, migration, apoptosis, and impact treatment sensitivity in different types of cancers (zhou et al., 2017) . as the roles of desmosomal proteins in cancer and metastasis are not fully understood, the identification of deiminated desmoplakin in llama evs here, and not described before, may be of some interest and add to understanding of diverse functional ability via such post-translational modification. tsc22 domain family protein 3, also called glucocorticoid-induced leucine zipper protein (gilz), was here identified as a deimination candidate in llama evs only. it is a glucocorticoid-responsive molecule involved in immune regulation and glucocorticoid actions. its interactions with signal transduction pathways, many of which are operative in ra and other inflammatory diseases, suggest that it is a key endogenous regulator of the immune response including a key glucocorticoid-induced regulator of inflammation in rheumatoid arthritis (ra) (beaulieu and morand, 2011) . giltz is a small, 135-amino acid protein with anti-inflammatory properties and has been shown to inhibit nf-κb and mapk pathways (bereshchenko et al., 2019; ricci et al., 2019) . it has also been shown to be induced in response to hypoxia by a hif1α-dependent mechanism and in response to cholesterol starvation, leading to downstream shedding of procoagulant evs in ovarian cancer (koizume et al., 2019) . post-translational deimination of gilz has not been described before but may indeed affect its multifaceted functions, including in inflammation and cancer. in the current study we report deiminated proteins in llama serum and serum-derived evs. due to the fact that the llama genome is not yet fully annotated, the hits identified here may underestimate the amount of deiminated proteins present in llama serum and evs. therefore a wider protein-hit analysis was carried out including other members of the camelid family, using known sequences from camelus ferus, camelus dromedaries, lama guanicoe and vicugna pacos. deimination of key immune factors of innate and adaptive immunity and key metabolic proteins is identified here for the first time in a camelid species, highlighting putative protein moonlighting functions via post-translational deimination. it must be noted that in relation to previously observed increases of deiminated proteins with age (ding et al., 2017) , the llama used in this study would be considered relatively old at 21 years of age, as typical llama lifespans are 15-25 years, with some individuals surviving 30 years or more. our findings presented here furthermore complement expanding research in the comparative ev research field. previous studies on the camel urinary proteome revealed enriched proteins from evs and relevance to stress and immune responses as well as antimicrobial activities (alhaider et al., 2012) . research on evs is a relatively new field in comparative immunology and to our knowledge; this is the first description of evs in serum of a camelid species. previous studies on evs in camelids focussed on evs in camel milk, which were shown to have anti-cancerous properties (badawy et al., 2018) . as pads have been identified to play major roles in the regulation of ev release (kholia et al., 2015; kosgodage et al., 2017 and , including in host-pathogen interactions (gavinho et al., 2019) , such ev-mediated communication may be of great relevance also for addressing diverse zoonotic diseases identified in camels (el-alfy et al., 2019; zhu et al., 2019) . in continuation of the current pilot study, the assessment of changes in deiminated proteins in camelid serum, and their lateral transfer via evs, will be of great interest to assess animal health in response to infection and environmental stress. our findings will further current understanding of the roles for evs, pads and posttranslational deimination throughout phylogeny and in relation to adaption to a range of, including extreme, environments. furthermore, novel identification of post-translational deimination in key proteins of metabolism and immunity may reveal hitherto unrecognized moonlighting function of these proteins throughout phylogeny, in relation to physiological and pathological processes, as well as being translational to and informing inflammatory and metabolic diseases. pad-mediated contribution to protein moonlighting and in ev-mediated communication in response to physiological and pathophysiological changes remains therefore a field of further studies. this is the first report of deiminated proteins in serum and serum-evs of a camelid species, using the llama as a model animal. our findings highlight a hitherto unrecognized post-translational modification in key immune and metabolic proteins in camelids, which may be translatable to and inform a range of human metabolic and inflammatory pathologies. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this 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therapy in danio rerio novel desmoplakin mutations in familial carvajal syndrome shark igw c region diversification through rna processing and isotype switching new insights into the rna-binding and e3 ubiquitin ligase activities of roquins preferential combination between the light and heavy chain isotypes of fish immunoglobulins molecular characterization and expression analysis of three subclasses of igt in rainbow trout (oncorhynchus mykiss) deletion of spata2 by crispr/cas9n causes increased inhibin alpha expression and attenuated fertility in male mice the role of desmosomes in carcinogenesis structure and function of inter-alpha-trypsin inhibitor heavy chains a review of zoonotic pathogens of dromedary camels the authors would like to thank yagnesh umrania and michael deery at the cambridge centre for proteomics for the lc-ms/ms analysis, as well as alice blue-mclendon of texas a&m university's winnie carter wildlife center for sharing llama serum. this study was funded in part by a university of westminster start-up grant to sl and u.s. national science foundation grant ios-1656870 to mfc. thanks are due to the guy foundation for funding the purchase of equipment utilised in this work. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.molimm.2019.10.017. key: cord-048344-ps3mnpzq authors: zhu, xiaowei; gerstein, mark; snyder, michael title: procat: a data analysis approach for protein microarrays date: 2006-11-16 journal: genome biol doi: 10.1186/gb-2006-7-11-r110 sha: doc_id: 48344 cord_uid: ps3mnpzq protein microarrays provide a versatile method for the analysis of many protein biochemical activities. existing dna microarray analytical methods do not translate to protein microarrays due to differences between the technologies. here we report a new approach, procat, which corrects for background bias and spatial artifacts, identifies significant signals, filters nonspecific spots, and normalizes the resulting signal to protein abundance. procat provides a powerful and flexible new approach for analyzing many types of protein microarrays. dna microarray technologies have proven to be extremely valuable for probing biological processes by measuring mrna expression profiles. however, studies at the protein level have the potential to provide more direct information since most genes function through their protein products. traditional investigations focus on individual proteins in a system and then combine such individual analyses to provide a more global perspective. recently, technologies to analyze proteins in a high throughput and unbiased fashion have become feasible [1] . one particular powerful technology is protein microarrays, which contain a high density of proteins and allow a systematic probing of biochemical activities [2, 3] . there are two types of protein microarrays [3] . a 'functional protein microarray' contains a set of proteins individually produced and positioned in an addressable format on a microarray surface. functional protein microarrays are useful for identifying binding activities or targets of modification enzymes. the first version of a proteome microarray was reported in 2001 and contained 5,800 yeast proteins with amino-terminal glutathione s-transferase (gst) tags printed on the array [4] . a second version of yeast protein microarrays was generated recently and contained 5,600 proteins with carboxy-terminal 6his-ha-zz domain tags [5] . proteins from both collections were overexpressed, purified and spotted onto the protein microarrays. global proteome studies were performed on these chips to understand various biological mechanisms. for example, 87 yeast kinases were examined for their substrates using yeast protein microarrays and over 4,200 in vitro substrates representing 1,325 unique proteins were identified [6] . compared with the approximately 150 known in vivo kinase-substrate interactions, this global study served as an important first step for dissecting yeast signaling networks. in addition to searching for kinase substrates, proteome chips can be probed with labeled proteins, dna, lipids, antibodies and many other molecules to search for interacting proteins [4, 7, 8] . large amounts of data have been generated using protein microarrays, presenting significant challenges in developing robust methods to process the raw data and building reasonable biological hypotheses from the datasets. the second type of protein microarray, the 'analytical protein microarray' or 'antibody microarray', shares similarities with immunoassays and uses antibodies to detect specific probes. studies have shown that these antibody arrays can recognize specific targets and generate dose-dependent signal intensities, indicating that they can be used to quantify levels of various targets in a crude mixture [9, 10] . because of the crossreactivity of certain antibodies with a variety of proteins, only highly specific antibodies are suitable for this type of study. this remains a limiting factor in preparing antibody microarrays. both dna and protein microarrays are prone to systematic errors that are usually generated from different sources, such as surface defects and spatial artifacts. many studies have offered insight on noise subtraction in dna microarrays [11] [12] [13] [14] , but little investigation has been done for protein microarrays. functional protein microarrays differ in many respects from dna microarrays. first, the goals of these two microarrays are different. dna microarrays measure the relative dna levels in a pool of probes, whereas functional protein arrays often aim at discovering global interactions of a single probe molecule. second, a typical dna microarray experiment measures signal ratios between two color channels, one for a tested mrna sample and the other for a reference sample [15] . signals in the second channel may serve as intrinsic controls that can help to decrease the effects of various amounts of reagent on the arrays and any local array nonuniformity. furthermore, many current scaling methods are then based on the assumption that signal intensities should be balanced between the two color channels despite variation in slide location, intensity and other sources of systematic variation [16] [17] [18] . however, such controls are missing in onecolor-channel protein microarrays. third, several scaling approaches in dna microarrays are based on a set of 'housekeeping' genes that give constant signal intensities at different conditions [19, 20] . however, in protein microarrays, such a control group must be customized according to the type of activities that are assayed, and, therefore, a ubiquitous reference group does not exist. fourth, unlike dna microarrays, in which non-specific binding can often be addressed by signal comparison with mismatch probes [21] , cross-reactivities of protein microarrays can not be as directly corrected for. a separate slide is, therefore, often required to be probed in parallel as a negative control in protein microarray experiments. finally, several protein-specific artifacts serve as common noise sources in protein microarrays. in the kinase assay, for example, the signal from strongly phosphorylated spots can bleed into neighboring spots, leading to incorrect background measurement. these differences are particularly applicable to functional protein microarrays in comparison to antibody arrays, and, therefore, the normalization techniques used for dna microarrays are usually not directly applicable to functional protein microarrays. we have developed a new protein chip analysis tool (procat) to deal with various artifacts specific to functional protein microarrays. the work started from a careful survey and characterization of all potential sources of systematic errors in protein microarrays. specific approaches were then designed to deal with each type of noise. a correction approach is applied to reduce measurement errors in the background signals. in addition, spatial variations can be reduced efficiently through a novel two-parameter signal normalization approach and calling positive spots locally. after generating a list of positives, negative control slides are analyzed in the same approach and spots are subtracted from the list if they appear in the control slide. slide features with poor signal qualities are also removed. finally, signal intensities of the positives are normalized according to their protein amounts. all modules that account for the challenges in data processing specific to protein microarrays are built into procat and tested. procat contains a flexible modular design whose individual components can be adjusted according to the experimental designs and stringency level selected by the users. six sequential modules are currently implemented in procat before a final annotation report is assembled ( figure 1 ). these modules carry out: background correction; signal normalization; positive spot identification; spot cross-reactivity filter; signal qualities inspection; and protein amount normalization. the performance of many of the steps was tested using several types of experiments as described below. a fundamental issue in all microarray experiments is background correction, which aims at reducing noise in background quantification. signal intensities are generally quantified by subtracting the foreground intensities with the local background intensities, which are measured as the background signals immediately surrounding the spot of interest (termed here the 'adjacent background'; figure 2b ). however, in protein microarrays local background regions can be easily skewed by artifacts such as small speckles. in addition, strong positive signals from on-chip kinase assays tend to produce signal smears on both film and phosphoimagers that exceed the normal feature size (figure 2a) . in both cases, the measurement for that spot will be inaccurate. first, the background intensity will be arbitrarily high, which will diminish the real signal intensity for that spot. second, the intensities will be affected by the alignment of the grid and extent of the smear, and, therefore, the variance of the same protein at replicate experiments will be increased. two methods can reduce the artifacts in local background. the user can manually adjust the grid size to fit the circles to each individual spot. however, the aligning process requires considerable time and effort. the size of the smear may even prevent refitting the grid without adversely affecting neighboring spots. additionally, a larger spot size can diminish the signal of the spot because the signal density decreases with increasing spot size. the second method for background correction, which is applied in procat, replaces the background intensity of the central spot with the background from its local neighborhood. a three by three surrounding window is assigned to each protein spot, and the median background of the nine spots will be used as the 'neighborhood background' value for the central spot (see materials and methods for more details). no additional time is needed for further alignment, yet this method will significantly reduce artifacts that can produce erroneous measurements on spots background. in the analysis of the phosphorylome dataset [6] , we applied the neighborhood background correction and observed a high sensitivity in identifying positive targets. to further characterize the effects of neighborhood background correction, we performed a test kinase assay with 100 nm protein kinase a (pka) spotted at 96 locations on one slide (figure 2a) . each of the 48 blocks on the slide contains two pka pairs with random yeast proteins spotted elsewhere (approximately 12,000 spots). after incubating the slide with 33 p-γ-atp, all of the pka spots autophosphorylated and showed strong signals, and in many cases the signal went beyond the grid circle boundaries (figure 2b ). we then applied the neighborhood background correction to the pka spots. as expected, the median for pka signal intensities was enhanced by 53%. furthermore, the pka signals from different positions are more similar to each other; the variance within them is decreased by 41% (p value = 0.006; fig 2d) . therefore, the neighborhood method for accessing background provides more robust measurements than that of the adjacent background method. spatial artifacts arise from uneven signal distribution across the slide, in part due to uneven probing conditions and smear artifacts [13] . uneven probing can occur by several means, such as uneven mixing of the probe, exposure to the probe solution, or uneven washing and drying of the slides. twocolor-channel experiments of dna microarrays provide intrinsic controls that can be used to account for spatial artifacts. functional protein microarrays often use only one color channel and, therefore, are especially prone to spatial artifacts. spatial artifacts will cause inaccurate measurements of signal intensities and can hinder the identification of significant interactions. adding more controls can help remove spatial artifacts since the signal of each spot can then be normalized according to its local controls. due to the variable shape and size of spatial artifacts, ideally a large number of controls would be needed. however, space constraints of the protein chip and an inability to anticipate all the uses of the arrays usually prevent the necessary number of controls to fully account for spatial artifacts on the array. a scaling method that reduces signal variations among spots of the same proteins at different array locations decreases spatial artifacts. we developed a new normalization method to deal with the spatial artifacts specific to functional protein microarrays. by assuming that signal distribution in large windows is consistent across the slide, the foreground signal of each spot can be normalized according to signal intensities in its surrounding neighborhood. this assumption is usually valid in protein microarray experiments in which proteins are randomly printed on the array ( figure 3 ). two parameters, the median and the median absolute deviation (mad), are calculated to represent the signal distribution in the local window ( figure 4 ). to perform the normalization, the median and mad of all sliding windows are averaged. the average values are then used to correct the signal of the central spot to to test the performance of this two-parameter scaling approach for signal normalization within one slide, we designed a test microarray containing multiple positive controls printed at different positions on the slide. the test array was organized in the same format as the commercially available protein microarrays (invitrogen). each protein was printed in duplicate, and the array contained 24 blocks of 16 by 16 printed proteins (figure 5a ). two gst-fusion proteins, sla2p and myo4p, were purified separately and a 1:1, 1:5, and 1:25 dilution of each protein was prepared. sla2p and myo4p at each concentration were printed at eight random positions on the array. other spots were occupied with bovine serum albumin (bsa) as negative controls. in order to visualize the two fusion proteins, anti-gst antibody was used to probe the slide, and one probing with typical spatial artifacts is shown in figure 5 . the artifact-containing slide showed different signal levels between the edges and the middle portion of the array. this produced blocks that had a variable signal distribution that ranged from high to low from one edge of the slide to the opposite edge; the variability occurred across blocks and simple block normalization methods adopted in dna microarray normalization approaches [17] would not be suitable for dealing with this problem. we applied procat to normalize the slide with several different parameters ( figure 5) . five window sizes were tested, termed windows 1, 3, 5, 7, and 9. these numbers correspond to the window size as a function of the number of spots on one edge of a block. for example, a block of 20 by 20 spots analyzed using window 1 would have a window size of 0.1 that of the block edge, or in this case 2 spots above, below, and to either side of the central spot, whereas a window size of 9 would contain a 37 by 37 area roughly as large as 4 blocks. three observations were made from the analysis of different window sizes. first, as the window size increases, the computational time used for the normalization also increases. second, no obvious spatial artifacts were left after the normalization with any of the window sizes tested ( figure 5b ). third, a small window size diminishes any signal inequality that exists between positive signals and background noise. indeed, a small scaling window tends to introduce extreme changes to the original signals and, therefore, increases the discrepancy between the duplicate spots of the same protein. the variance of the signals for the same protein after normalization with different window size was calculated. in five out of the six cases (three dilutions of two proteins) the scaling window 9 can successfully reduce the signal variance in a range from 31% to 90% (figure 5c ). decrease of signal variation suggests that a large scaling window will help to reduce spatial artifacts. although larger window sizes are possible, 9 was used as the default number for procat because the analysis can be done in a reasonable time and minimal improvement has been achieved after window size 7 (additional data file 1). in addition to providing accurate measurements of spot intensities, procat has been developed to assign thresholds for identifying positive targets in one experiment. traditionally, a global cutoff can be calculated from all spots and applied to the whole slide. due to variable spatial artifacts, cutoffs were assigned locally in procat. for each spot on the array the signal distribution within a nine by nine window was calculated and a cutoff defined as a number of standard deviations away from the mean; the default for procat is two standard deviations. this cutoff corresponds to 5% significance level if the signal distribution within this local window is normal. when many spots with strong signals are included in the window, the cutoff will be arbitrarily high and thus decrease the sensitivity of detecting positive spots by the program. to avoid this loss in sensitivity, procat has a built in function to identify possible outliers, to remove those outlier spots that have extremely strong signals, and then to calculate a cutoff for identifying positive spots using the remaining spots. a receiver operating characteristic (roc) curve was used to compare the performance of local window cutoffs versus a global cutoff on the test slide [22] . area under roc curve (auc) is a performance indicator that ranges from 0 to 1, with 1 for the best performing method. using gst-sla2p and gst-myo4p as positive controls and bsa as negative controls, the sensitivity and specificity for both local and global cutoff methods was estimated. five window sizes were tested and compared with the global cutoff ( figure 6 ). prediction performance is increased significantly when using local windows with nine or more spots on one edge. thus, a nine by nine window is used as the default in procat since a larger a representative protein microarray with high-quality data figure 3 a representative protein microarray with high-quality data. the slide image was reconstructed from a protein microarray experiment with minimal noise in the data. density plots of signals in local 37 by 37 windows (window size 9) for all spots were computationally combined, and they showed high similarities. window size results in increased computing time with only minimal improvement in sensitivity. the auc value is much larger in local cutoffs (0.992) compared to global cutoffs (0.916) and the improvement is unlikely to be due to random chance (p value = 0.002) [20] . therefore, we can conclude that the local cutoff is significantly better in identifying positive spots than a global cutoff. scheme for the signal scaling method figure 4 scheme for the signal scaling method. the signal of one spot on the array is normalized according to the distribution in its local neighborhood. for each spot, a surrounding window is chosen and all spots in this window are defined as its neighborhood. the signal of a center spot will then be normalized by comparing the local median and mad with the average values. norm, normalized signals; origin, original signals. average distribution two layers of filters are implemented in procat. first, all positive spots from negative control experiments are removed. for example, in on-chip kinase assays, kinase dead alleles were probed on separate arrays using the same experimental conditions as used with wild-type kinases. spots that produce signals in the absence of active kinase were identified by procat and removed from the target lists of kinase probings. when probing tagged protein to detect protein-protein interaction, testing the epitope tag in the absence of the protein of interest is also an essential control. if proper negative control experiments are available, procat will analyze them in the same way as regular experiments to construct experimental positive spot lists void of proteins producing positive signals under control conditions. the second filter checks the quality of each positive spot. all proteins are spotted in duplicates on protein microarrays, hence should have very similar signal intensities. procat then uses the difference between duplicate signals as an indicator of the signal qualities. the difference between signals of two duplicate spots (s 1 , s 2 ) is calculated as (s 1 + s 2 )/(|s 1 | + |s 2 |) and then fitted to a normal distribution. proteins with exceptionally large differences in their duplicate spots are more likely to be biased by certain artifacts, and thus are removed from the positive list. the default threshold for the duplicate spot difference in procat is set at two standard deviations away from the mean. one of the goals for protein microarray experiments is to identify the affinity of a binding interaction (in a protein-protein interaction assay) or the extent of phosphorylation (in a kinase assay) so that one can compare the relative strength of the reaction for each positive protein. ideally, the spot intensity would directly correspond to the strength of interaction. however, a number of other factors contribute to the array signal intensities, including the systematic noise from various artifacts, as was already discussed, and the amount of protein printed on the chip. nonetheless, semi-quantitative estimates can be obtained. after background correction and signal normalization, the raw signals can be standardized by relative protein amounts before they can be used to estimate the interaction strength. although proteins on the microarray can have very different amounts, they do share the same epitopes for the purpose of large-scale protein purification [4, 5] . therefore, probing with anti-epitope antibodies will provide an estimate of the relative protein amounts in each spot on the array. after the protein amount is determined for one spot at row i and column j, procat divides the raw signal intensities s i,j by the protein amount signals a i,j and uses the quotient as an approximation of the strengths of interactions: this approximation generally works well across the slide except for the following two situations. less abundant proteins will be biased because the a i,j values estimated in antiepitope probings are more susceptible to background noise and slide artifacts. on the other hand, overpowering spots can also be biased if they have saturated signal intensities. a saturated s i,j value is an underestimate to the real signal. for these two reasons, only proteins with amounts more than a minimal cutoff and signal intensities lower than a saturation threshold will be normalized with protein amounts. proteins that do not conform to these two requirements will be recorded with unnormalized signals and flagged for further inspection. an additional caveat is that the relative protein amount assessed using antibodies includes both native and denatured protein at a given spot. therefore, the estimation of interaction strength will be an underestimate since the amount of functional protein may be an overestimate. procat was designed as a flexible tool to analyze functional protein microarray data. the program was scripted in perl (version 5.6.1) on top of a tomcat (version 5.0.30) web server [23] . each module discussed above was implemented independently and can be included or excluded depending on various experimental designs. to input a dataset, the user has to characterize the data in three aspects: experimental designs, data file formats and normalization parameters. experimental design contains parameters such as the number of test arrays and negative control arrays for one particular assay. data file format describes the layout in the uploaded dataset so that procat can recognize and extract the useful information from it. normalization parameters allow users to try different stringency levels. these three levels supply sufficient information to uniquely characterize an experiment while still allowing ample flexibility for the individual user to customize parameters to suit many different types of experimental designs. after inputting all three descriptions and uploading the dataset, procat takes five minutes on average to complete all analysis modules for each array. the time may vary depending on the selected analysis modules and the size of the protein microarrays. each task is assigned a unique id and results are organized into a database for future queries. processed data including analysis parameters, a list of positive spots with protein annotations, and normalized signal intensities will be available for the users to download from the server. functional protein microarrays serve as an efficient platform for screening protein biochemical functions. here we present procat as a systematic approach to process and analyze data specific to functional protein microarrays. calibrated by explicit test experiments, procat has proven to be able to handle many types of functional protein microarray studies with three unique features. procat includes novel scaling methods that provide robust and reproducible measurement for quantitative signals. this is crucial for protein microarrays as chip signal intensities often indicate strength of interactions. in addition, by calling positive candidates locally, procat demonstrated excellent performance in identifying positives in comparison to global thresholds. finally, each step has been integrated into a modular design to fit various experimental designs and stringency requirements. a major challenge in designing any automated data processing method is thinking of and anticipating all possible situations that may arise. procat uses a local three by three window to correct background containing signal smears or dust speckles. this method assumes the artifacts are sparse enough so that the majority of the nine spots in the local window still provide correct measurements of the background signals. since the median value of nine spots is used to correct the background, a few biased spots within the window will not severely affect the corrected background value. this assumption is usually valid since the percentage of spots that are either positive or whose signal is contaminated by artifacts in protein microarray experiments is generally quite low. in extreme cases where such spots are likely to be very close to each other, a larger window (five by five for example) can be used. large artifacts such as bright speckles and incubation bubbles may affect many spots in a particular region. since the shapes of these artifacts are variable, it is necessary to manually flag these spots initially and then remove them from future analysis. many commercially available software packages for microarray experiments have a built in flagging function, and procat will automatically discard flagged spots. a key aspect of procat is the two-parameter approach for reducing spatial nonuniformity. several factors can affect the performance of procat's normalization. first, procat normalizes the signal of a spot according to the signal distribution in its local neighborhood. it diminishes the signal intensity if the spot is located in a high signal neighborhood, while compensating the intensity if it is in a low signal neighborhood. this approach is based on the assumption that signal intensities across the slide share the same distribution, and it holds true if and only if the regional variations observed on the slide are due to technical artifacts and not from real biological differences. since proteins are printed in a random order on most of the current protein microarrays, it is unlikely a particular region of the slide will gain high intensities as a result of biologically relevant reasons. second, the size of the neighborhood window can also largely affect the performance of the normalization. small window sizes tend to add biases to signals and diminish all local variations, whereas large window sizes increase the computational burden and tend to preserve local variations. we found that the optimal window size of procat is 9 for protein-protein interactions; this figure corresponds to approximately four blocks on the chip and is used as the default. other window sizes can also be chosen to fit various shapes of spatial artifacts. procat can be applied to many experiments using protein microarrays, such as kinase assays, protein-protein interactions and protein-dna interactions. thus far, the twoparameter scaling approach has only been used in single chip normalization; however, a similar strategy can be extended to rescale multiple slides by assuming signals in neighborhood windows on different slides are similarly distributed. overall, procat provides a powerful and flexible new approach for optimal processing and analysis of functional protein microarrays. for the slide used for testing background correction, 100 nm pka (sigma, st. louis, mo, usa) was spotted at 96 different places as positive control. the slide was incubated with 200 μl of kinase buffer (100 mm tris ph 8.0, 100 mm nacl, 10 mm mgcl 2 , 20 mm glutathione, 20% glycerol) plus 0.5 mg/ ml bsa, 0.1% triton x-100, and 2 μl 33 p-γ-atp in a humidified chamber at 30°c for 1 hour. the slide was then washed twice with 10 mm tris ph 7.4, 0.5% sds and once with double distilled h 2 o before being spun dry and exposed to x-ray film (kodak, rochester, ny, usa). for the anti-gst probing, slides were printed with sla2p and myo4p as positive controls and 150 nm bsa as a negative control. the array surface was blocked using superblock (pierce, rockford, il, usa) at 4°c for 1 hour. rabbit polyclonal igg (santa cruz biotechnology, santa cruz, ca, usa) was incubated with the slides at 1,000-fold dilution. the array was then washed with pbst (sigma) and incubated with a 1:1,000 dilution of cy5-conjugated anti-rabbit igg antibody (jackson laboratories, bar harbor, me, usa). slides were then washed with pbst five times and scanned in an axon testing experiment for the signal scaling approach figure 5 (see previous page) testing experiment for the signal scaling approach. (a) the design of the test slide with positive spots shown as red spots and the five tested normalization window, indicated by red squares, for a given spot on the array, shown in blue. (b) comparison of signal intensity before and after normalization using window size 9 on the testing experiment. the two images were computationally reconstructed from the signal files, either without or with normalization. genepix scanner (molecular devices, sunnyvale, ca, usa). raw signals were extracted with genepix pro 6.0 software (molecular devices). for one spot, let i be the row and j the column on a protein microarray. thus, b i,j represents the adjacent background intensity and f i,j denotes the foreground intensity. the raw signal intensity s i,j is calculated as: in neighborhood background correction, we use neighborhood background to replace the adjacent background. a local three by three window around b i,j is chosen and the neighborhood background is defined as: in a protein slide with n rows and m columns, a local window w i,j around one spot (i, j) is defined as signals of a set of spots s i,j that satisfy: the size parameter k is dependent on window size factor f win and the block size f block : in which f block represents the number of spots on one edge of the block, and f win is chosen by users from five options: 1, 3, 5, 7 and 9. different windows can overlap with each other and go beyond the block edges. let s denote signal intensities of spots within the local window; procat uses two parameters to characterize the signal distribution of s: median (med) and median absolute deviation (mad): after calculating med i,j and mad i,j for all the spots on the array, they are averaged to obtain the two parameters and for the reference distribution. for one spot (i, j), procat normalizes its raw signal s i,j by comparing med i,j and mad i,j with the average values: for a given spot at row i and column j, its normalized signal is compared to surrounding spots in a nine by nine window w ij (4) . signals within this window are fit to a normal distribution. the mean μ i,j and standard deviation σ i,j will be calculated and the default threshold is set at two standard deviations above the signal mean. a spot (i, j) will be called positive only if its signal is above the threshold: when positive spots are likely to be close to each other, pro-cat uses box plots to examine and remove possible outliers from the surrounding window [24] . let q 1 be the lower quartile (25th percentile) and q 2 be the upper quartile (75th percentile); the difference between q 1 and q 2 is termed interquartile range δq. a spot (i', j') is then defined as an outlier if its signal: roc curve comparing the global cutoffs and local cutoffs in calling positive spots figure 6 roc curve comparing the global cutoffs and local cutoffs in calling positive spots. the test slide has six unique positive controls (sla2p and myo4p in three different titrations). the performance of identifying the positive controls is increased by using local cutoffs generated in relatively large surrounding windows. five window sizes were tested and the best performance was achieved using nine by nine or larger windows. to obtain a robust threshold, the corrected mean and standard deviation are generated using the non-outlier spots. the following additional data files are available with the online version of this paper. additional data file 1 is a figure illustrating the variance reduction in positive controls using different normalization window sizes. additional data file 2 is a table listing the raw signals generated in the autophosphorylation experiment for testing the background correction method. additional data file 3 is a table listing the raw signals generated in the anti-gst probing experiment calibrating the signal scaling approach. additional file 1 variance reduction in positive controls using different normaliza-tion window sizes variance reduction in positive controls using different normaliza-tion window sizes. click here for file additional file 2 raw signals generated in the autophosphorylation experiment for testing the background correction method raw signals generated in the autophosphorylation experiment for testing the background correction method. click here for file additional file 3 raw signals generated in the anti-gst probing experiment cali-brating the signal scaling approach raw signals generated in the anti-gst probing experiment cali-brating the signal scaling approach. click here for file proteomics technologies: probing the proteome printing proteins as microarrays for high-throughput function determination protein analysis on a proteomic scale global analysis of protein activities using proteome chips biochemical and genetic analysis of the yeast proteome with a movable orf collection global analysis of protein phosphorylation in yeast regulation of gene expression by a metabolic enzyme severe acute respiratory syndrome diagnostics using a coronavirus protein microarray immunophenotyping of leukemias using a cluster of differentiation antibody microarray profiling of cancer cells using protein microarrays: discovery of novel radiation-regulated proteins bioconductor: open software development for computational biology and bioinformatics analysis of variance for gene expression microarray data identification and correction of spurious spatial correlations in microarray data microarray data normalization and transformation quantitative monitoring of gene expression patterns with a complementary dna microarray expressyourself: a modular platform for processing and visualizing microarray data normalization for cdna microarray data: a robust composite method addressing single and multiple slide systematic variation statistical methods for identifying differentially expressed genes in replicated cdna microarray experiements profound effect of normalization on detection of differentially expressed genes in oligonucleotide microarray data analysis new normalization methods for cdna microarray data high density synthetic oligonucleotide arrays the meaning and use of the area under a receiver operating characteristic (roc) curve procat statistical algorithm for assuring similar efficiency in standards and samples for absolute quantification by real-time reverse transcription polymerase chain reaction we thank k nelson, l kung and d gelperin for help in synthesizing the testing protein microarrays, and j ptacek, j mok and t gianoulis for comments on the manuscripts. this research was supported by grants from nih. key: cord-281552-zfjy3m3i authors: alsaadi, entedar a. j.; neuman, benjamin w.; jones, ian m. title: identification of a membrane binding peptide in the envelope protein of mhv coronavirus date: 2020-09-22 journal: viruses doi: 10.3390/v12091054 sha: doc_id: 281552 cord_uid: zfjy3m3i coronaviruses (covs) are enveloped, positive sense, single strand rna viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. following replication, covs assemble on intracellular membranes including the endoplasmic reticulum golgi intermediate compartment (ergic) where the envelope protein (e) functions in virus assembly and release. in consequence, e potentially contains membrane-modifying peptides. to search for such peptides, the e coding sequence of mouse hepatitis virus (mhv) was inspected for its amino acid conservation, proximity to the membrane and/or predicted amphipathic helices. peptides identified in silico were synthesized and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (guvs) consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dppc), sphingomyelin and cholesterol. to confirm the presence of membrane binding peptides identified in the context of a full-length e protein, the wild type and a number of mutants in the putative membrane binding peptide were expressed in lenti-x-293t mammalian and insect cells, and the distribution of e antigen within the expressing cell was assessed. our data identify a role for the post-transmembrane region of mhv e in membrane binding. the coronavirus envelope protein (e) is a small hydrophobic protein ranging from 74-109 amino acids [1, 2] . it has an n-terminal domain, a long alpha helical transmembrane domain and a c-terminal hydrophilic domain, and is found incorporated into the virus particles of all coronavirus groups at low levels [3] [4] [5] [6] [7] . two membrane topologies have been suggested for the e protein: hairpin or transmembrane [5, 8] . e also interacts with the m protein, and mutants of m that are unable to bud from cells can be complemented by forms of e [9, 10] . the membrane curving properties of e are such that the coexpression of m and e is adequate for the efficient formation of virus-like particles (vlp) [11, 12] , and these can also incorporate the s protein if it is coexpressed [13] . for many coronaviruses, including mhv, e protein also functions as an ion channel, a viroporin [14, 15] , affecting the trafficking of virions in the secretory pathways and membrane permeability, both of which are essential for virus growth [3, 16, 17] . while e function is critical for virus assembly, its viroporin activity in mobilizing calcium ions and its interactions with host cell tight junction proteins have also been implicated in the pathologies of some coronavirus infections [16, 18] . an additional role for e in viral pathogenesis is an anti-apoptotic effect on host cells during virus replication [5] . the relative significance of the e encoded ion channel activity to virus morphogenesis, as opposed to its structural role in binding m, is uncertain. however, its role as a virulence factor has been demonstrated by many studies on sars-cov e protein which have shown altered pathogenesis in a mouse model, either by an effect of virus production and by effects on expressing cells [7, 19, 20] . stimulated by the pandemic of sars-cov-2 [21] , a recent protein interaction map of virus infected hek cells identified host proteins of the vesicular sorting pathway and bromodomain proteins as interacting with e, the latter of which may have therapeutic potential [22] . notwithstanding its multifunctional nature, the interaction of e with membranes is central to its biological role, and peptides with direct membrane binding properties are present within the coding region. the mapping of such peptides may help to interpret e mechanisms of action and offer the potential for inhibitor development. here, we test e-derived peptides for membrane binding activity in vitro and confirm those identified as positive in the context of the full length protein expressed in two different cell types. all chemical reagents were purchased from thermofisher scientific unless otherwise stated, and were used following the vendor's recommendations. lipids were obtained from avanti polar lipids. peptides used for the in vitro analysis (table 1) were derived from the e protein of mhv coronavirus (accession number aau06359), synthesized and supplied as a lyophilized product with a stated mean purity of ≥70% by cambridge research biochemicals, uk. peptide stock solutions were prepared in a buffer consisting of 0.1mm sucrose, 0.1 mm glucose, 10 mm dtt and 0.5% (v/v) dmso. a positive control peptide, the m2-influenza peptide, a well characterized amphipathic helix sufficient for budding into guvs and the formation of large luminal vesicles (luvs) [23] , was used to validate the guv assay. negative controls were provided by buffer only in order to match test samples in all but the test peptide. guvs were generated by electroformation using a vesicle prep pro (nanion technologies gmbh, munich, germany) with a mix of 5 mm 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dppc), 4 mm egg sphingomyelin and 0.5 mol% cholesterol, which were first dissolved in chloroform. to visualize the guvs, 0.5 mol% of naphtha [2-alpha] pyrene (tokyo chemical industry uk ltd, oxford, uk) was added. lipids were mixed in an amber vial to a final total lipid concentration of 9 mm before preparation by electroformation. briefly, 20µl of lipid stock was spread on the conductive side of an indium tin oxide slide (ito-slide), air dried and put into a vacuum desiccator for 1 h to remove all solvent. a 28-mm o-ring was coated on one side with silicon grease and placed around the dried lipid film, which was then hydrated with 750 µl of 0.1 mm sucrose solution in deionized water. a second ito slide was placed on top of the o-ring with the conductive sides facing, and a voltage of 3 v peak to peak at a frequency of 5 hz was applied to the ito slides over a period of 2 h at 50 • c [24] . guvs were imaged using an evos-fl digital fluorescence microscope (evos, usa). following electroformation, the chamber was disassembled and the sucrose solution removed to leave the guvs attached to one ito slide. diluted peptide was added immediately at the desired concentration and the field was imaged 0, 1, 2 and 5 min thereafter. imagej was used to measure the shape and the relative size of the guvs. experiments were performed in triplicate and the average and standard deviation were calculated. buffer only and m2-influenza peptide controls were treated in the same way. guv-peptide incubations were at room temperature. statistical significance was calculated using spss version 22, using linear mixed model (lmm) (p•0.05). results were expressed as mean ± sem. figures were generated using graphpad prism 7.0b software. lenti-x 293t cells were cultured and maintained in dulbecco's modified eagle medium (dmem) (sigma aldrich) supplemented with 10% fetal bovine serum (fbs) (ge healthcare) and 0.2% penicillin-streptomycin solution (gibco/invitrogen, paisley, uk) at 37 • c with 5% co 2 . spodoptera frugiperda (sf9) cells (invitrogen) were maintained in ex-cell 420 medium (sigma, gillingham, uk) supplemented with 2% fetal bovine serum and 1% penicillin/streptomycin solution at 27 • c with shaking. the coding sequence of mhv e (genbank accession: ay700211.1) was amplified by pcr from cdna kindly supplied by dr. volker thiel using the primers hsv-e-fw (5'gcgccatggcaca gccagaactcgccccggaagaccccgaggattttaatttattccttacagacacagtatggtat gtgggg-3') and hsv-e-rv (5'-gcgctcgaggatatcatccacctctaatag ggg-3') and the amplicon cloned into ptriex 1.1 (merck millipore, darmstadt, germany) between the ncoi and xhoi restriction sites. to provide markers for the detection of expression by western blot, the wt e coding region was cloned in frame with an upstream hsv and downstream his tag, both provided by the vector. following initial expression experiments, detection of the hsv tag was found to be poor. as a result, mutant e sequences were synthesized de novo (integrated dna technologies, dresden, germany) and cloned similarly, except they were in frame only with the downstream his tag. all constructs were confirmed by dna sequencing prior to use. one day before transfection, 1.25 × 10 5 lenti-x 293t cells (takara, saint-germain-en-laye, france) were seeded into 12 well plates containing a glass coverslip in each well and incubated for 24 h at 37 • c/5% co 2 . the following day, the cells were transfected using lipofectamine 3000 transfection reagent (invitrogen) with plasmids encoding wildtype or mutant mhv-e and incubated for 24 h at 37 • c/5% co 2 . a control vector ptriex1.1-gfp expressing green fluorescent protein (gfp) gene was used to visualize the efficiency of transfection, typically 20-30%. twenty four hours after transfection, the media was removed and the cells were washed twice with cold pbs for 5 min, then incubated in fix and permeabilization buffers for 1 h at room temperature (ebioscience, san diego, usa). fixed and permeabilized cells were incubated with anti-his-alexa fluor 488 conjugate ab (4e3d10hh2/e3, thermofisher scientific) for 1 h at room temperature diluted 1:100 in 1× permeabilization buffer, washed twice with tbs for 15 min each at room temperature in the dark, and then counterstained with dapi. the fixed cells were mounted with a drop of slowfade™ gold reagent before being imaged using an evos-fl digital fluorescence microscope. typical images were captured at 20× magnification and further manipulated, if required, using imagej software [25] . the flashbac™ gold (fbg) baculovirus expression system (oxford expression technologies, oxford, uk) was used to produce recombinant baculoviruses. small-scale protein expression was performed by infection in a 6-well plate seeded with 1 × 10 6 sf 9 cells per well using 200 µl of a high titre stock of the recombinant baculovirus, typically passage 3, and incubated for 3 days at 27 • c. after incubation, cells were harvested and used for western blot with an anti-his antibody. proteins were separated by sds-page using 4-12% precast tris-glycine sds polyacrylamide gels (invitrogen) for 30 min at 170v. after electrophoresis, gels were either stained by coomassie blue r250 or transferred to pvdf membranes for western blot analysis. following transfer to pvdf membranes (whatman, chalfont st. giles, uk) using a semidry western blotting apparatus, membranes were incubated in blocking buffer (5% skimmed milk powder in tbst (0.2% tween-20, 1× tbs)) for 1 h. membranes were incubated with the primary antibody at 1:10,000 in 1× tbst buffer for 1 h, followed by three washes for 5 min each in tbst buffer, and subsequently, with a secondary horseradish-peroxidase (hrp) antibody conjugate (dako, glostrup, denmark) diluted 1:10,000 in 1× tbst for 1 h followed by three washes for 5 min each with tbst buffer. the membrane was finally washed with tbs and the bound hrp activity was detected using chemiluminescence imagery on a syngene g: box. to allow reprobing of a pvdf membrane with a different antibody, the membrane was stripped using a stripping buffer (100 mm β-mercaptoethanol, 2% sds, 62.5 mm tris-hcl ph 6.7) for 30 min at 50 • c. the membrane was then washed three times with tbst buffer, 10 min each, and then reblocked and probed as before. a total of 2.5 × 10 6 sf9 cells were seeded in t25 flasks as monolayers, infected with recombinant baculoviruses at an moi = 3 and incubated for 72 h at 27 • c. the infected cells were harvested by loosening the monolayer into the media and collected by centrifugation at 3000× g at 4 • c for 20 min. cell pellets were resuspended in 500 µl of cold pbs and lysed by sonication for 10 m with on/off pulses at 20-s intervals (sonics, vibracell tm ). the cell lysates were briefly clarified (1500× g for 5 min) and then centrifuged at low speed, i.e., 10,000× g, at 4 • c for 30 min to collect large membrane debris. the pelleted material was kept as a low-speed pellet (lsp). the collected supernatants were then centrifuged at high speed, i.e., 100,000× g, for 90 min at 4 • c in a beckman tl-100 ultracentrifuge and the pellets were retained as high-speed pellets (hsp). low-and high-speed pellets were tested for the presence of mhv e by western blot using an anti-his antibody, as before. several viral proteins encode amphipathic helices that modulate membrane curvature, e.g., the m2 protein of the influenza virus and the nonstructural protein 4b of the hepatitis c virus [23, 26, 27] . the program amphipaseek [28] was used to assess local amphipathy and putative membrane-binding regions for a phylogenetically diverse set of e proteins representing 17 covs, and the output was visualized using jalview v 2.9.0b2 [29] . cov e proteins have related topologies but otherwise limited direct homology outside of the clearly predicted tm domain. however, amphipathicity occurred in a region immediately downstream of the tm domain, a region which contains the previously mapped cysteine region [30] at the end of the predicted transmembrane domain ( figure 1 ) and which is also palmitoylated intracellularly [8, 31] to facilitate contact with the lipid bilayer [32] . in addition, two conserved proline residues occur in this region, plausibly providing conformational flexibility that may be important for e-e interactions or between e and other proteins [8, 33] . spanning the first conserved proline, a 15 residue sequence, i.e., 50-64 in mhv a59 e, including a number of hydrophobic residues, was evidently relatively well conserved among most α and β covs, and was also shared with the more distantly related γ and δ covs, with the exception that insertions containing further multiple hydrophobic residues were also present ( figure 1 ). based on these criteria this peptide, the e post tm peptide (eptm), was considered to have potential for direct membrane interaction. ; green represents polar amino acids (n, q, s, t); pink represents cysteines (c); orange represents glycines (g); yellow represents prolines (p); cyan represents aromatic amino acids (h, y); white represents any unconserved/gap. the sars-cov-2 e protein is 95% identical to sars e, so was not included as a distinct entry. to address its potential as a membrane active peptide, the 15 residue wild type peptide was synthesised and reconstituted with giant unilamellar membranes (guvs), and the effect on size and morphology were measured. in addition, peptides etm, representing the e protein tm domain itself, and the m2 influenza peptide were tested (table 1) . guvs, composed of 5 mm dppc, 4 mm egg sm and 0.5 mol% cholesterol were reconstituted with the peptides at a concentration of 10 µm and the guvs were measured at 1, 2 and 5 min thereafter by fluorescence microscopy, as described elsewhere [34] . shape was measured as the ratio between the longest and shortest radii, while relative guv size was estimated by ramanujan's first approximation, taking an average of 40 guvs per experiment for three separate experiments. peptide eptm changed the size but not the shape of the guvs membrane, leading to membrane deformation that increased with time, while guvs treated with buffer only showed no deformation ( figure 2) . interestingly, peptide etm showed no effect on either the shape or size of the guvs. interaction of mhv-eptm peptide with the guv membranes made the guvs smaller in size and more rugged in appearance, which was possibly indicative of fragmentation of the lipid bilayers after peptide insertion. the lack of effect by the etm peptide suggested that this peptide alone is not membrane active and may need to be presented in the context of the complete e protein for biological activity. the assay was validated by use of the m2-influenza peptide which showed a statistically significant change in guv shape with the formation of intraluminal-vesicles (ilvs) as described [23] , although no significant change in size was apparent ( figure 2 ). to validate the eptm peptide sequence as membrane active in the context of the full length e protein, e was expressed in both mammalian and insect cells through the use of a dual promoter vector, ptriex1.1, which permits expression from the same vector in both systems. mhv e protein was expressed as the wild type protein and also as a series of alanine scanning mutants in which conserved and hydrophobic residues were exchanged for alanine ( table 2 ). in addition, the entire region was deleted within the e coding sequence. mammalian expression was done by transfection of lenti-x 293t cells where expression is driven by the vector resident cag promoter, and the expressed e protein was detected by immunofluorescence imaging with an anti his tag antibody 24 h post-transfection. the wt mhv e protein was found to be distributed evenly throughout the cytoplasm with a slight concentration near the nucleus, plausibly the golgi body. in all the alanine scanning mutants of e, expression levels were similar with little diminution of the overall signal, but in most cases, the pattern of staining was altered to a more granular punctate appearance (figure 3 ). this was particularly notable for mutation l52a, where almost all of the fluorescence signal was associated with a punctate distribution. this data suggests that the eptm sequence, identified as causing membrane association in an isolated peptide, also influences the behaviour of the complete protein in a physiologically relevant environment. del ---------to provide a more quantifiable measure of membrane association, a more productive protein expression system, i.e., expression in insect cells via the construction of recombinant baculoviruses, was investigated with the same panel of e variants. following the generation of recombinant viruses, insect cells were infected at high moi and their ability to express e protein assessed by western blot at 3 days postinfection when synthesis from the vector encoded p10 promoter was maximal. to facilitate detection, the wt e protein was expressed with both hsv and his tags, at the n-and c-termini respectively, a predicted molecular mass of~13 kda. of these two tags, however, only probing with the his antibody detected the expressed product consistently. as a result, all mutant e proteins were expressed with only the c-terminal his tag, a predicted molecular mass of~11 kda. all e proteins were expressed at the molecular weight expected, but the expression level varied with mutation ( figure 4a ). half of the mutants expressed at levels similar to the wild type, but mutations l50a, v51a, l52a and y57a were present at reduced levels, consistent with a potential role of these residues in protein folding and stability. to ensure the levels of infection were equivalent, the blot was stripped and reprobed with a monoclonal antibody to the major baculovirus glycoprotein, gp64, a marker for infection level, which showed near equivalent infection in all cases ( figure 4b ). to investigate whether the mutations introduced into mhv-e resulted in an effect on cellular localization in more detail, plausibly by altered membrane association, infection of insect cells with each recombinant virus was repeated on a larger scale and the infected cells harvested at 2 days postinfection. the cells were lysed by sonication in the absence of detergent, clarified and subjected to differential membrane fractionation to produce low-speed (lsp) and high-speed membrane pellet (hsp) fractions. to confirm the fractionation, lsp and hsp samples were probed with an antibody to calnexin, an er marker, which was largely found in the lsp fraction ( figure 5 ). the expression of the wt mhv e was found in both fractions, but was more strongly associated with the hsp fraction ( figure 5a ), consistent with its known primary localization in mhv-cov infected cells [35] . similarly, mutations l50a and v51a partitioned mainly into the hsp fraction, although expression levels overall were reduced. strikingly, the "all" mutant, in which all targeted residues were mutated to alanine, and the "del" mutant, in which the target peptide was deleted from mhv e, were found almost exclusively in the lsp fraction, despite high levels of expression. the remaining mutants carrying mutations l52a, p54a, y57a and y59a also associated preferentially with the lsp, although, as before, expression levels were lower in some cases, notably l52a and y57a. relative densitometry of the hsp and lsp bands revealed significant differences among the mutants with regard to their localisation to the different membrane fractions of e expressing insect cells ( figure 5b ) and confirmed a role for the amphipathic mhv cov e 50-64 peptide in membrane interaction. the cov e proteins consist from a short hydrophilic amino terminal region, a hydrophobic transmembrane region and a carboxy-terminal region that encompasses the majority of the protein [30] . the e proteins are multifunctional proteins involved in virus assembly and release, and have been located in the ergic and golgi of infected cells but do not traffic to the infected cell surface [35] . as studies of e have often involved different coronaviruses and different experimental systems, the unambiguous basis of this localisation has not been described. an amphipathic helix, eptm, detected in the post-tm region of e, was suggested by bioinformatics analysis and assessed for direct membrane interaction in vitro by binding to guvs. for comparison, the predicted e protein tm domain, etm, and an established membrane active peptide from the influenza m2 protein were also included. eptm, but not etm, caused a change in guv size and appearance, consistent with direct membrane binding. following expression of the complete e protein with mutations in the same identified peptide, altered membrane binding in two distinct cell types, mammalian and insect, was apparent. these data support the hypothesis that the membrane binding observed for the eptm peptide in vitro occurs also in the context of the complete e protein expressed in a physiologically relevant environment. cov e protein is a known integral membrane protein, and it has been proposed that the basis of membrane interaction is by one of a number of possible topologies, e.g., as a type iii membrane protein, as a membrane hairpin or as a glycosylated type ii membrane protein [5] . the expression of e in insect cells resulted in a single band detected by western blot that did not have the appearance typical of a glycosylated protein. moreover, in the latter topology, the eptm region is predicted to be free on the luminal side of the membrane and not to interact with it. the data here are inconsistent with this prediction and are more explicable by assuming that either the type iii membrane protein or the membrane hairpin topology is more likely. in both cases, it is postulated that the post-tm region folds back against the membrane, where it may be additionally anchored by cysteine palmitoylation, although the precise mechanism of interaction remains unknown. assuming that the predominant punctate staining in mammalian cells represents misfolded e protein that induced stress granules, then mutants l52a, y57a and y59a formed a group with a shared phenotype. the "all" mutant, which necessarily included these mutations, was also punctate in appearance. by contrast, l50a and v51a were more similar to the wt straining pattern. biochemical fractionation of membrane from insect cells following the construction and validation of recombinant baculoviruses broadly recapitulated this division. the relative level of e protein following differential centrifugation of the cell membranes showed that wt, l50a and v51a mutants were predominantly associated with the hsp fraction with a minority in the lsp fraction (31.4%, 12.7 % and 20.2% respectively), while the remaining mutants were predominantly associated with the lsp fraction. some mutants, notably l52a and y57a, were expressed at lower levels but were hardly present in the hsp faction. as the wt exhibited an hsp pattern, we interpret this to be authentic partitioning into membranes, whilst the lsp pattern is likely to be aggregated protein trapped in the er by virtue of its inability to associate with membranes correctly. despite its conservation, the role of the p54a mutant was equivocal, i.e., some way between the two extremes but tending towards non-wt-like mutants. these data suggest a possible model for eptm interaction with the membrane in which the c-terminal hydrophobic residues within the 15 residue region are the most relevant. interestingly, these fall broadly on one side of the helix prediction for eptm, while those broadly tolerated fall on the other ( figure 6 ). plausibly, this distinction relates to membrane binding verses the requirement for e to also bind other ligands. further work will be required to address the level of membrane curvature afforded by this interaction and the extent to which it relates to the various functions reported for cov e, such as reorientation of the plasma membrane within the golgi [33] , interaction with the m protein [3, 36, 37] and viral particle scission [38, 39] . in sars cov, e residues which reportedly bind to the membrane include valine-52, tyrosine-59 and lys-63 [40] . these residues are fully conserved in sars-cov-2 within an overall e similarity of 98%, and are also equivalent in the mhv e peptide identified here. membrane binding by these residues in sars e is thus supported by the observations made here for v52a and y59a, although lys 62 was not examined. e has been considered a therapeutic target for sars in relation to its viroporin function which is linked to inflammation [41] , and more recently, for sars-cov-2 as a result of its mapped interactions with bromodomain proteins, for which drugs already exist [21] . a more detailed mapping of membrane binding by e and how it functions once bound may facilitate future drug discovery programs targeting this small, variable but multifunctional protein. a highly unusual palindromic transmembrane helical hairpin formed by sars coronavirus e protein characterization of the coronavirus mouse hepatitis virus strain a59 small membrane protein e coronavirus structural proteins 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nonstructural protein 4b mediates membrane association prediction of amphipathic in-plane membrane anchors in monotopic proteins using a svm classifier jalview version 2-a multiple sequence alignment editor and analysis workbench model of a putative pore: the pentameric alpha-helical bundle of sars coronavirus e protein in lipid bilayers expression and purification of coronavirus envelope proteins using a modified beta-barrel construct conformations of peptides corresponding to fatty acylation sites in proteins. a circular dichroism study identification of a golgi complex-targeting signal in the cytoplasmic tail of the severe acute respiratory syndrome coronavirus envelope protein a fusion peptide in the spike protein of mers coronavirus coronavirus envelope (e) protein remains at the site of assembly the production of recombinant infectious di-particles of a murine coronavirus in the absence of helper virus the missing link in coronavirus assembly. retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-golgi compartments and physical interaction between the envelope and membrane proteins assembly of the coronavirus envelope: homotypic interactions between the m proteins analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly structure of a conserved golgi complex-targeting signal in coronavirus envelope proteins severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank fellow members of the virology laboratory for their help during the course of this study. the authors declare no conflict of interest. key: cord-266444-rw94yls8 authors: dominguez andres, ana; feng, yongmei; campos, alexandre rosa; yin, jun; yang, chih-cheng; james, brian; murad, rabi; kim, hyungsoo; deshpande, aniruddha j.; gordon, david e.; krogan, nevan; pippa, raffaella; ronai, ze’ev a. title: sars-cov-2 orf9c is a membrane-associated protein that suppresses antiviral responses in cells date: 2020-08-19 journal: biorxiv doi: 10.1101/2020.08.18.256776 sha: doc_id: 266444 cord_uid: rw94yls8 disrupted antiviral immune responses are associated with severe covid-19, the disease caused by sar-cov-2. here, we show that the 73-amino-acid protein encoded by orf9c of the viral genome contains a putative transmembrane domain, interacts with membrane proteins in multiple cellular compartments, and impairs antiviral processes in a lung epithelial cell line. proteomic, interactome, and transcriptomic analyses, combined with bioinformatic analysis, revealed that expression of only this highly unstable small viral protein impaired interferon signaling, antigen presentation, and complement signaling, while inducing il-6 signaling. furthermore, we showed that interfering with orf9c degradation by either proteasome inhibition or inhibition of the atpase vcp blunted the effects of orf9c. our study indicated that orf9c enables immune evasion and coordinates cellular changes essential for the sars-cov-2 life cycle. one-sentence summary sars-cov-2 orf9c is the first human coronavirus protein localized to membrane, suppressing antiviral response, resembling full viral infection. sars-cov-2 is an enveloped, positive-sense single strand 29.9 kb rna virus (1, 2) that causes severe respiratory disease in humans . this coronavirus was first identified in wuhan, china, at the end of 2019 (3) . due to its easy human-to-human transmission and the lack of effective antiviral therapy, covid-19 has caused a pandemic with more than 19 million cases and over 740,000 deaths worldwide (https://covid19.who.int). mechanistically, the host protein ace2 serves as the viral receptor and host cellular proteases, such as tmprss2, play key roles in sars-cov-2 entry into host cells (4) (5) (6) (7) . ace2 expression is high in alveolar epithelial cells (8) , making the lung a highly vulnerable target for the virus. sars-cov-2 infection causes a wide range of disease, from asymptomatic to mild disease to severe disease that can lead to death (9) . sars-cov-2 is most similar to the coronaviruses sars-cov and mers-cov (10, 11) . however, neither of those became a global pandemic. current therapies are primarily palliative and supportive (9) . more than 2000 clinical trials are currently in progress worldwide (12) (https://clinicaltrials.gov/ct2/who_table). without effective vaccines or treatments, there is an urgent need to understand the pathology of sars-cov-2 infection, the roles of each of the 29 proteins encoded within the viral genome in the life cycle, virulence, and pathogenicity of the virus, and identify strategies for intervention or treatment. various therapeutic and vaccine strategies target viral entry mechanisms, such as vaccines or antibodies targeting on the spike (s) protein (13) (14) (15) (16) ; others target viral replication or assembly processes, such as the antiviral drug remdesivir, which interferes with rna replication and has emerged as superior to placebo in shortening recovery time in adults (17). another strategy for treatment is interfere with viral immune evasion mechanisms and thus enable the body's natural 4 antiviral responses to be more effective at clearing the virus. indeed, investigation of mechanisms of immune evasion by sars-cov-2 is an active area of translational research with immune evasion properties discovered for nonstructural protein 1 (nsp1) (18) . the sars-cov-2 genome contains 15 open reading frames (orfs), which encode 29 viral proteins (19) (20) (21) . orf1a and orf1ab encode polyproteins that are cleaved into 16 nonstructural proteins (nsp1 -nsp16) that comprise the replicase-transcriptase complex. spike (s) is encoded by orf2, envelope (e) by orf4, membrane (m) by orf5, and nucleocapsid (n) by orf9. an additional 9 orfs encode "accessory" proteins: orf3a, orf3b, orf6, orf7a, orf7b, orf8, orf9b, orf9c, and orf10. various studies have investigated the functions of the virally encoded proteins by performing interactome analysis in cells expressing individual viral proteins (19) or by evaluating the proteomic or transcriptomic changes associated with either viral infection (22) (23) (24) (25) (26) (27) . others have used computational approaches to investigate protein-protein interactions between sars-cov-2 viral proteins and host proteins (28) . the interactome and proteome studies identified cellular processes affected by sars-cov-2 infection or specific viral proteins, notably innate immune signaling (19, 20, 23, (28) (29) (30) , ubiquitin ligase activities (19, 20, 23, (28) (29) (30) , p38 mitogenactivated protein kinase (mapk) signaling (19, 20, 23, (28) (29) (30) . the transcriptomic studies identified interferon signaling (24) , cell death (27) , interleukin 1 (il-1), il-6, and chemokine signaling (22) . given the intense interest in catalytically active cov-2 proteins (31) (32) (33) (34) , we examined the lessstudied group of orfs encoding accessory proteins, which are largely thought to maintain viral 5 structural organization in replication organelles and within the viral particle (35, 36) . here, we showed that expression of only orf9c is sufficient to alter cellular networks in a manner that resembles full sars-cov-2 virus infection. seven of the 29 cov-2 proteins are orfs that lack catalytic activity and, in some cases, lack a known function (19) . each of these were tagged with strep at the n terminus and expressed them individually in the lung cancer epithelial cell line a549 in the presence or absence of the proteasome inhibitor mg132 (fig. 1a ). the protein encoded by orf9c was particularly unstable, with a profound increase in abundance evident in mg132-treated compared to that in vehicle-treated a549 cells (fig 1a) . orf9c is present in previously characterized strains of sars-cov (37), a conservation suggesting a function in coronavirus pathogenesis. phylogenetic analysis and alignment of the protein sequences showed that mutations are present in orf9c among different coronavirus strains with bat sars-like coronavirus orf14 as the closest ortholog sharing 94% sequence identity and only 77% identity with orf14 of sars-cov (fig. 1b, fig. s1a ). tmhmm analysis (38) of sars-cov-2 orf9c predicted a transmembrane sequence in the c-terminal domain, a motif not present in sars-cov-1 (or other human coronaviruses) orf9c sequence ( fig. 1b, fig. s1b ). additionally, a single nucleotide mutation in sars-cov-2 orf9c altered a termination codon, enabling the reading frame to extend by 3 amino acids (fig. 1c ). we assessed potential functions of orf9c by performing transcriptome, interactome, and proteome analysis of a549 lung cancer cells transfected with orf9c tagged at the n terminus with 2 copies of the strep tag (19) . to map the orf9c interactome, we conducted liquid chromatography tandem mass spectrometry (lc-ms/ms) of 2xstrep-tagged orf9c compared with control 2xstrep-tagged gfp) immunoprecipitated from a549 cells 24 h after transfection. orf9c interactome analysis revealed that most interacting proteins were classified as membrane proteins (fig. 1d , table s1) according to gene ontology cellular component. as a protein with a transmembrane domain, this was not surprising. however, we were surprised to find that the orf9c interactome was distributed throughout the membrane-bound organelles (fig. 1d ), including >30 proteins in the protein biosynthesis and transport systems of the endoplasmic reticulum (er) and golgi, >15 proteins in the mitochondria, and >30 other membrane-related proteins. given the instability of orf9c, we were not surprised to identify a group of membraneassociated proteins that function with the proteasome. comparison between orf9c and orf10, both expressed using the strep-tagged vector, confirmed that enrichment of membranal proteins as part of the interactome was selectively seen for the orf9c (fig. s1c). we conducted both label free quantification (lfq) and tandem mass tag (tmt) mass spectrometry analysis to identify changes in the cellular proteome in a549 cells expressing orf9c. we compared the proteomic changes associated with orf9c expression in the presence 7 or absence of proteasomal inhibition with mg132, using dmso as the vehicle control for comparison in each set. principle component analysis (pca) revealed that orf9c contributed to major variance in all data sets ( fig. s2a, s2b ). pairwise comparisons between orf9c and control untransfected samples identified differentially expressed proteins in both the dmso and mg132 groups (fig 2a) . in both the dmso and mg132 datasets, most changes induced by orf9c were a reduction in protein abundance (downregulation) ( fig. 2a, table s1 ). downregulated proteins identified using both approaches consistently showed ~60% overlap, while no overlap were identified among the upregulated proteins. thus, to maximize the discovery of orf9c dysregulated proteins, results from both technologies were combined. including the differentially regulated proteins identified by both the tmt and lfq analysis revealed 14 proteins were upregulated in common by orf9c expression in the presence or absence of the proteasome inhibitor and 144 proteins were downregulated in common (fig. 2b ). using the downregulated proteins and upregulated proteins identified for either the dmso or mg132 condition separately, we performed ingenuity pathway analysis (ipa) to assess signaling pathways deregulated in orf9c-expressing cells. in both the dmso and mg132 condition, interferon (ifn) signaling exhibited the greatest difference, both in terms of the intensity of the downregulation and the number of proteins significantly associated with this pathway, in response to orf9c (fig. 2c, table s1 ). other pathways affected by orf9c and of particular importance to virulence were antigen presentation and innate immune response pathways, such as irf/cytosolic pattern recognition receptors. we further examined potential upstream regulators of these pathways using ingenuity pathway analysis for proteins that exhibited a change in abundance in the orf9c-expressing cells. this analysis revealed that several components of the ifn machinery [interferons (ifnl1, ifna), interferon responsive 8 transcription factors (irf7, irf1), and an interferon receptor (infar)] were reduced, consistent with the impaired ifn signaling, and an increase in mapk1 (also known as erk2) abundance (fig. 2c, table s1 ). to assess if there were notable differences in the intensity of the changes in protein abundance in response to proteasome inhibition, we calculated relative changes in protein abundance between control and orf9c-expressing cells from both the dmso and mg132 conditions for proteins associated with ifn signaling or the ubiquitin proteasome (ubp) system and antigen presentation (fig. 2d ). the intensity of the changes was similar in the presence or absence of mg132, suggesting even small amounts of the unstable orf9c are sufficient to induce cellular changes including those that contribute to immune evasion. consistent with the ipa-based analysis (fig. 2b) , ifn signaling components, including ifi35, multiple ifit proteins, irf9, isg15, mx1, psmb8, and stat proteins, were downregulated in orf9c-expressing cells in both the presence and absence of mg132 (fig. 2d, left) . indicative of a decrease in antigen presentation capacity, multiple proteins involved in this process were decreased, including proteins involved in antigen loading and display [hla proteins, β2m, and antigen transporters (tap1 and tap2)] and proteins involved in ubp [ubiquitin-conjugating enzymes ube2i and ube2l6), deubiquitination enzymes (usp18 and ups41), and proteasome components (psmb and psme proteins)] (fig 2d, right) . these changes in the proteome indicated that the expression of only orf9c, even in the absence of proteasomal inhibition to stabilize this protein, is sufficient to elicit effective inhibition of ifn, immune recognition, and ubp components at the protein level. such a response suggested that orf9c contributes to immune evasion of sars-cov-2. we assessed transcriptional changes elicited by orf9c expression in a549 cells using rna-seq analysis. pca showed that changes induced by orf9c in both dmso-and mg132-treated cells cluster in distinct experimental groups (fig. s2c) . in contrast to the proteomic results that revealed predominant downregulation of proteins following orf9c expression, rna-seq analysis showed a similar number of transcripts were increased or decreased in the presence or absence of mg132 (fig. 3a, table s2 ). additionally, the number of differentially regulated transcripts was higher than that for the differentially regulated proteins. using ipa, we identified the pathways significantly enriched in differentially regulated transcripts. the same set of pathways were identified in the dmso and mg132 conditions, and similar to the proteomic results, most related to immune signaling (fig. 3b , upper, table s2). however, many of the specific pathways were different from those identified at proteomic level. at the transcriptional level, we detected the greatest effects on the complement system and several pathways involved in inflammatory signaling. thus, some components of antigen presentation and immune signaling pathways showed comparable changes at the protein and mrna levels; other changes elicited by orf9c expression were unique to the transcriptional level, such as induction of il-6 signaling and p38 mapk signaling, or the protein level, such as impairment of ifn signaling. we analyzed the orf9c-regulated transcripts for those encoding upstream regulators of the pathways altered at the transcriptional level by orf9c. this analysis identified the classic immune modulators tumor necrosis factor (tnf), il-1b, ifng, transforming growth factor β (tgfb), and nf-kb signaling components (fig. 3b , lower, table s2). we evaluated transcripts associated with the complement system or il-6 signaling in detail. in both the presence and absence of proteasome inhibition, complement system transcripts were mostly downregulated by orf9c expression in a549 cells (fig. 3c, left) . for il-6 signaling, we found some differences between the mg132 and dmso conditions (fig. 3c, right) . for several transcripts the intensity of the upregulation was greater in the absence of proteasome inhibition (map3k14 and map2k3, il6 and il6r, socs1 and socs3); for others the presence of the proteasome inhibitor resulted in a greater reduction in transcript abundance (il1b, tnfaip6, cd14, il1r2). thus, these results suggested that orf9c had a dose-dependent effect on some transcripts. we combined the results of the proteomic and transcriptomic (fig. 4a ), which revealed a small set of commonly upregulated or downregulated genes by orf9c at both the transcription and protein levels (fig. 4b ). we performed ipa canonical pathway analysis and found commonly altered pathways at both the transcript and protein levels (fig. 4c , table s1, s2). the direction of regulation (increased or decreased activity) was consistent between the transcripts and proteins. however, the number of components significantly enriched in most of the pathways differed between the protein and transcript levels. we compared our findings at the transcriptome, proteome, and interactome levels with those reported by stukalov et al. (39) for proteins with altered ubiquitination (ubiquitinome) in response to sars-cov-2 infection of a549 cells. within the top 10 enriched ipa canonical pathways, we noticed enrichment across all 5 protein ubiquitination data sets, sirtuin signaling, phagosome maturation, tight junction signaling, and caveolar-mediate endocytosis (fig. 4d , table s3). seven of the 10 were common between the ubiquitinome data (39) and our proteomic analyses by lfq or tmt mass spectrometry. we also compared the pathway enrichment across our transcriptomic data and those from blanco-melo et al. (22) reporting transcriptomic changes upon sars-cov-2 infection in human primary epithelial cells or ace-expressing a549 cells and with those from stukalov et al. (39) reporting transcriptomic changes in ace2-expressing a549 cells 12 and 24 hours after infection in the presence or absence of the proteasome inhibitor, we observed substantial overlap (55 -65%) in the orf9c-downregulated proteins ( fig. 2a) and 54 -72% overlap in orf9cupregulated transcripts and 45 -64% in the downregulated transcripts (fig. 3a) . thus, despite orf9c increasing in mg132-treated a459 cells, the persistent changes in mg132-treated cells suggested that even a low amount of orf9c is sufficient to elicit its cellular effects. however, some transcripts (399) and proteins (97) were downregulated by orf9c in cells not treated with mg132 and were upregulated in cells treated with mg132 ( fig. 5a ). pathway analysis on the transcripts and proteins showed discordant regulation in dmso-or mg132-treated cells in response orf9c. the most pronounced among all three datasets were components of the ubp and the unfolded protein response (upr) (fig. 5b, table s4 ). changes in events or pathways associated with the cell cycle was not surprising given the critical role ubp plays in their regulation. we thus hypothesized that both the ubp and upr were involved in degradation of orf9c. finding that upr signaling was also reversed upon mg132 treatment ( fig 5b) is consistent with the role of upr signaling in degradation of orf9c. to directly assess the importance of ubp and upr components in orf9c instability, we performed an sirna-based screen targeting over 1100 genes that encode components of both machineries in a549 cells stably expressing strep-tagged orf9c (fig. s3 ). the top hits independently validated as blocking orf9c degradation were sirnas targeting vcp [also known as p97, an atpase involved in export of unfolded proteins from the er for er-associated degradation (erad) and in er to golgi transport] (40, 41) , the proteasomal subunit psmd2, and the proteasome maturation factor pomp (42) , which has been also implicated in erad (43) 13 and in ifn-induced reorganization of proteasomes into immunoproteasomes (44) (fig. 5c , table 1 ). to assess if interfering with erad affected the cellular effects induced by orf9c, we compared the transcript abundance for 6 genes (ifngr1, igs15, irf9, socs1, psmb8, tap1) that were downregulated by expression of orf9c in dmso-treated cells with their abundance in cells treated with the vcp inhibitor mns-873, the heat shock protein 90 (hsp90) inhibitor geldanamycin, or the proteasome inhibitor bortezomib. although the hsp90 inhibitor and the proteasome inhibitor increased transcript abundance for some of the gens tested, vcp inhibition was the most consistently effective at enabling expression of each of these transcripts in the orf9c-expressing cells (fig. 5d ). these observations suggested that orf9c ability to attenuate key cellular signaling involved in antiviral responses, including antigen presentation, immune, and ifn pathways, requires the activity of vcp. the key to our ability to control spread of the sars-cov-2 virus is to understand its mechanism of action and how the concerted action of its 29 encoded proteins subvert cellular regulatory networks. one can divide viral "success" into two key phases: infection, which is the ability to enter a given cell type, and multiplication that enables continuous infection through viral replication and packaging, which exploits host cell machineries (45) . a third aspect to viral success is evasion from immune clearance. disruption of either the infection or replication phases should effectively inhibit the sars-cov-2 life cycle. accordingly, many efforts focus on neutralizing interaction of the viral s protein with ace2 (4, 46) . other efforts strive to interfere 14 with the viral life cycle after it invades target cells, and many focus on catalytically active proteins encoded by the sars-cov-2 genome (47). here, we analyzed one small, unstable sars-cov-2 protein, orf9c in the context of an epithelial lung cancer cell line. limited overlap between published studies of the orf9c interactome can be attributed to their use of different cell system (hek293 compared with a549 lung cancer cells used here, as the use of different filtering criteria (19) . however, many of the cellular changes elicited by orf9c in our study also occur following infection with full replicative sars-cov-2 virus (22) . those phenotypes included changes in ifn and other cytokine signaling, immune recognition (including antigen presentation; dendritic cell, t cell, and acute immune responses; and pattern recognition), cell cycle, and the complement system, all of which were downregulated by orf9c. additionally, similar to cells infected with the virus or expressing orf9c, il-1, il-6, and p38 mapk signaling pathways were upregulated. the primary change identified in our analysis was deregulation of the ifn system, coupled with changes in cytokines associated with tnf and stat signaling and factors implicated in innate immunity. in addition to mediating an antiviral response, aberrant ifn signaling is also critical for numerous pathological indications linked to covid-19 (48). thus, we concluded that sars-cov-2 orf9c elicits pathologies not seen with previously characterized coronavirus prototypes, primarily through effective modulation of ifn signaling. our findings suggested that orf9c enables cells to escape from immune surveillance through by reducing hla abundance and antigen presentation, while also slowing cell replication, which could viral replication of infected cells. strikingly, orf9c is predicted have a transmembrane domain and we found that the orf9c interactome was mostly comprised of membrane-associated proteins in multiple organelles, 15 including er, golgi, mitochondria, cell surface membrane, and peroxisomes. indeed, many of the cellular changes that we observed following orf9c expression are associated with membrane proteins or pathways mediated by proteins that associate with the membranes of various cellular compartments. importantly, sars-cov-2 orf9c is the first human coronavirus orf9c protein that has acquired this putative transmembrane sequence. mutations have been acquired along the course of evolution of orf9c, although ~80% of the sars-cov-2 orf9c sequence is identical to the ortholog in other coronaviruses, although greater similarity was identified with the bat sars-cov-2 sequence. the membranal anchoring capability identified in sars-cov-2 orf9c is novel feature that may mediate the effect on ifn signaling, antigen presentation, and immune evasion phenotypes, characteristics that make sars-cov-2 much more virulent and pathogenic than other coronaviruses. notably, 0.7-1.4% of patients were found to possess a mutation that is expected to impair transmembrane domain of sars-cov-2 orf9c (19) ; awaiting future assessment of clinical outcome, our data would predict a better clinical outcome, distinguishing these patients from those harboring the transmembrane domain. correspondingly, the interactome for the less virulence and pathogenic sars-cov-1 orf9c (49) did not overlap with that for sars-cov-2 orf9c. a notable signature that we identified is the upregulation of histone and histone deacetylaserelated factors, which suggested that histone modification may underlie the transcriptional repression. the increased transcription of ap1 family members (fosb, fosl1, creb, and atf3), which participate in the cellular stress response, may reflect the response to stress imposed by orf9c, which, in turn, can limit immune-related signaling identified in our study. another remarkable signature of orf9c expression in a459 cells was the association with ubp components. together with the observations on cellular immune pathways, this association with 16 the ubp suggested that orf9c induces changes in ubp components that alter the stability of cellular proteins implicated in cytokine signaling, antigen presentation, innate immunity, and the cell cycle. additionally, we identified upr components as important for orf9c instability, suggesting that this protein is misfolded or at least recognized as a misfolded protein by the host cell. in this scenario, we propose that misfolded orf9c engages upr (through vcp) and the ubp, which clears this protein. by engaging the ubp, orf9c promotes enhanced proteasome activity as suggested by our proteome analysis. our analysis revealed that interfering with vcp activity blunted the transcriptional repressive effects of sars-cov-2 orf9c on impact immune system components, such as irf9, infgr1, isg15, socs1 and tap1. proteasome inhibition with bortezomib was also effective, although not as consistently effective as mns-873, the vcp inhibitor. these findings suggested that inhibition of vcp or the proteasome, which has inhibitors currently in clinical trials for cancer (50, 51), may be considered among therapeutic measures to fight sars-cov-2 virulence and pathologies. however, we cannot exclude the possibility that orf9c stability and degradation mechanisms differ based on cell type or the activity of other viral proteins in infected cells. another potential therapeutic opportunity involves targeting the membrane association of orf9c, because this is a unique feature of the protein in the sars-cov-2 coronavirus. thus, identifying small molecules that could interfere with orf9c localization to the membrane could limit orf9c function and impede the ability of the virus to evade the immune response and reduce viral replication. given that orf9c is expected to affect immune evasion, virulence and pathogenesis, additional studies should assess the consequences of orf9c inhibition in vivo, using primates and possibly mouse models where sars-cov-2 shown to impact ifn signaling and immune response (48). secondary antibodies were used at 1:5000. immunoprecipitation of streptavidin-tagged cov-2 orfs was performed as previously described (19) . briefly, frozen cell pellets were thawed on ice for 15-20 minutes and suspended in 1ml lysis buffer with 50 mm tris-hcl, ph 7.4 at 4°c, 150 mm nacl, 1 mm edta and supplemented with 0.5% nonidet p-40 substitute, complete mini edta-free protease and phosstop phosphatase inhibitor cocktails (roche). samples were centrifuged 10 minutes at 4°c at 13,000g. protein quantification was performed using pierce's bca quantification kit as per the manufacturer's indications. supernatants (1 mg protein) were incubated 2h at 4°c with magstrep "type3" beads (30 μl; iba lifesciences) that had been previously equilibrated twice with 1 ml wash buffer (ip buffer supplemented with 0.05% np40). beads were washed five times with 1 ml wash buffer and then five times with 1 ml ammonium bicarbonate 50mm. raw fastq files were processed using cutadapt v1.18 (52) figure 4d , figure 5a and 5b were selected based on bh correct p <0.05 without fold change cutoff. differentially expressed proteins in figure 4d , figure 5a and 5b were selected based on p <0.01 without fold change cutoff. protein sequences similar to sars-cov-2 orf9c were retrieved through ncbi blastp using the nr database (58). sequence alignment was performed using clustalo (59). phylogenetic tree was built using phyml algorithm by 100 times bootstrap, and visualized using seaview (60) and geneious version 2020.2.2 (san diego, ca). transmembrane domain prediction was performed using tmhmm web server v2.0 (38) . transmembrane domain was predicted based on tmhmm posterior probability more than 0.5. statistical test results of rna-seq, proteomics and interactome data provided in supplementary tables. analyses of omics data in this study were performed using r customized scripts. statistical analysis of proteomics data sets was performed using msstats (label-free data) and mstatstmt (tmt data) bioconductor package. differential expression of rna-seq was performed using deseq2 bioconductor package following negative binomial distribution and wald test. pathway enrichment and upstream regulator analyses were performed using ipa following fisher's exact test and z-score calculation considering directional changes in ipa database. ana dominguez andres 1^, yongmei feng 1^, alexandre rosa campos 1^, jun yin 1^, chih-cheng beads were resuspended in 8m urea, 50 mm ammonium bicarbonate, and cysteine disulfide bonds were reduced with 10 mm tris (2-carboxyethyl) phosphine (tcep) at 30°c for 60 min. high confidence interacting proteins were selected using the following filtering criteria: log2fc > 3.3 (10x) and a p-value <0.025 (to include the p-value of proteins detected in at least 2 orf9c pulldown replicates but not detected in the negative controls). we also considered the 'crapomescore' < 0.5, which is the fraction of single affinity purification experiments a given protein-interacting candidate receives in the crapome database (crapome.org). a score of 1 means the candidate is identified in all experiments in that database. cells were lysed in uab buffer (8m urea, 50 mm ammonium bicarbonate (abc) and benzonase 24u/100ml) with vigorous shaking (20 hz for 10 min at room temperature using a retsch mm301 instrument). lysates were centrifuged at 14,000xg for 10 minutes to remove cellular debris, and protein concentration in supernatants was determined using bicinchoninic acid (bca) protein assay (thermo scientific fragmented precursors were detected in the ion trap as rapid scan mode with automatic gain control target set to 1 x 10 4 and a maximum injection time set at 35 ms. the dynamic exclusion was set to 20 seconds with a 10 ppm mass tolerance around the precursor. for processing label-free lc-ms/ms data, all raw files were processed with maxquant (version 1.5.5.1) using the integrated andromeda search engine against a target/decoy version of the curated human uniprot proteome without isoforms (downloaded in january of 2020) and the gpm crap sequences (commonly known protein contaminants). first search peptide tolerance was set to 20 ppm, and main search peptide tolerance was set to 4.5 ppm. fragment mass tolerance was set to 20 ppm. trypsin was set as the enzyme in specific mode, and up to two missed cleavages was allowed. carbamidomethylation of cysteine was specified as fixed modification and protein n-terminal acetylation and oxidation of methionine were considered variable modifications. in addition, the phosphopeptide-enriched samples were also searched with phosphorylation of serine, threonine or tyrosine considered as variable modification. the target-decoy-based false discovery rate (fdr) filter for spectrum and protein identification was set to 1%. statistical analysis of label-free proteomics data was carried out using in-house r script (version 3.5.1, 64-bit), including r bioconductor packages. first, peptide feature intensities (maxquant evidence table) were log2-transformed and normalized (loess normalization) across samples to account for systematic errors. then all non-razor peptide sequences were removed from the list. protein-level quantification and statistical testing for differential abundance were performed using msstats bioconductor package. cells were imaged with an ic200 high-content screening system (vala sciences) using a 20x objective to visualize strep-orf9c proteins (alexa 488) and nuclei (dapi). four images were obtained from different fields in each well for 384-well plates. images were analyzed with acapella high-content imaging and analysis software for valid cell numbers per field and to determine average alexa 488 intensity per cell. a549 cells expressing sars-cov-2 strep-orf9c were treated with dmso or mg132 (10um) served as negative and positive imaging controls, respectively. plate-to-plate variability was normalized using a control-based method; associated control samples were aggregated, and the mean and variance across wells were determined. the alexa488 mean intensity for all wells with sirna knockdown was normalized using unique non-targeting sirnas included in each plate as reference data points. the top 36 scoring hits were obtained using a threshold of > 1.46-fold increase in average intensity from duplicates (p-value <0.05). ten of the 36 sirna pools were selected for confirmation in a secondary deconvolution screen. for that screen, quantification data were converted to a z-score, and the average z-score from data in triplicate plates was determined. genes were defined as confirmed screen hits if they had 3 or more individual positive sirna score (cut-off of >3 sd). table s1 . interactome and proteome data analysis, pathway enrichment and upstream regulators analyses table s2 . transcriptome data analysis, pathway enrichment and upstream regulators analyses table s3 . canonical pathway comparison of different omics technologies and public data sets table s4 . canonical pathway analysis of mg132 reversed genes and proteins a novel coronavirus outbreak of global health concern a new coronavirus associated with human respiratory disease in china a novel coronavirus from patients with pneumonia in china sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor cell entry mechanisms of sars-cov-2 structural and functional basis of sars-cov-2 entry by using human ace2 a pneumonia outbreak associated with a new coronavirus of probable bat origin single-cell rna expression profiling of ace2, the receptor of sars-cov-2 sars-cov-2: a comprehensive review from pathogenicity of the virus to clinical consequences genome composition and divergence of the novel coronavirus (2019-ncov) originating in china systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov characteristics of registered studies for coronavirus disease 2019 (covid-19): a systematic review an alphavirus-derived replicon rna vaccine induces sars-cov-2 neutralizing antibody and t cell responses in mice and nonhuman primates structure, function, and antigenicity of the sars-cov-2 spike glycoprotein a vaccine targeting the rbd of the s protein of sars-cov-2 induces protective immunity potently neutralizing and protective human antibodies against sars-cov-2 remdesivir for the treatment of covid-19 -preliminary report structural basis for translational shutdown and immune evasion by the nsp1 protein of sars-cov-2 a sars-cov-2 protein interaction map reveals targets for drug repurposing structural genomics of sars-cov-2 indicates evolutionary conserved functional regions of viral proteins the proteins of severe acute respiratory syndrome coronavirus-2 (sars cov-2 or n-cov19), the cause of covid-19 imbalanced host response to sars-cov-2 drives development of covid-19 proteomics of sars-cov-2-infected host cells reveals therapy targets bulk and single-cell gene expression profiling of sars-cov-2 infected human cell lines identifies molecular targets for therapeutic intervention. biorxiv transcriptional landscape of sars-cov-2 infection dismantles pathogenic pathways activated by the virus, proposes unique sex-specific differences and predicts tailored therapeutic strategies a dynamic immune response shapes covid-19 progression transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients interactome of sars-cov-2 / ncov19 modulated host proteins with computationally predicted ppis the global phosphorylation landscape of sars-cov-2 infection covid-19: viral-host interactome analyzed by network based-approach model to study pathogenesis of sars-cov-2 infection drug design targeting the main protease, the achilles' heel of coronaviruses crystal structure of sars-cov-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors structure of the rna-dependent rna polymerase from covid-19 virus structural and evolutionary analysis indicate that the sars-cov-2 mpro is a challenging target for small-molecule inhibitor design characterization of accessory genes in coronavirus genomes. biorxiv sars coronavirus accessory proteins insights into sars-cov-2 genome, structure, evolution, pathogenesis and therapies: structural genomics approach predicting transmembrane protein topology with a hidden markov model: application to complete genomes multi-level proteomics reveals host-perturbation strategies of sars-cov-2 and sars-cov. biorxiv ubiquilin and p97/vcp bind erasin, forming a complex involved in erad vcp/p97-mediated unfolding as a principle in protein homeostasis and signaling the proteasome maturation protein pomp facilitates major steps of 20s proteasome formation at the endoplasmic reticulum sirna silencing of proteasome maturation protein (pomp) activates the unfolded protein response and constitutes a model for klick genodermatosis ifn-gamma-induced immune adaptation of the proteasome system is an accelerated and transient response covid-19 infection: origin, transmission, and characteristics of human coronaviruses sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues competing interests: zar is a co-founder and serves as scientific advisor to pangea therapeutics. all other authors declare no competing interests.data and materials availability: all datasets will be deposited in publicly available data sets prior to publication; all reagents and study protocols are available by requests from the corresponding authors. key: cord-277811-j58qvyum authors: mehrani, hossein; ghanei, mostafa; aslani, jafar; tabatabaei, zahra title: plasma proteomic profile of sulfur mustard exposed lung diseases patients using 2-dimensional gel electrophoresis date: 2011-01-07 journal: clin proteomics doi: 10.1186/1559-0275-8-2 sha: doc_id: 277811 cord_uid: j58qvyum introduction: sulfur mustard "bis (2-chlroethyl) sulphide" (sm) is a chemical warfare agent that remains a threat to human health. the aim of this study was to identify protein expression signature or biomarkers that reflect chronic lung damages induced by sm exposure. methods: prior to analysis, plasma was fractionated using ethanol precipitation. using two dimensional sds-page; fractionated protein profiles of 20 healthy and 20 exposed patients with lung diseases were established. selected protein spots were successfully identified with maldi tof ms/ms. results: the results show that α1 haptoglobin isoforms were detected in plasma of the all lung disease patients but none of the healthy controls. amyloid a1 isoforms was also detected in plasma of the lung disease patients but none of the healthy controls. moreover, low molecular weight proteins were enriched in ethanol supernatant compared to ethanol precipitate. conclusion: our present results and previous studies suggest that ongoing tissue remodeling is involved in sm exposed lung damage patients. these finding might improve patient care and suitable therapies. sulfur mustard is a chemical warfare agent that remains a threat to human health.. more than lethality, sm causes debilitating effects that can leave an exposed individual incapacitated for days, months, or years. lung injury is a common health problem after inhalation, which leads to chronic bronchitis and interstitial lung diseases [1] . the clinical picture of the poisoning is well known from the thousands of victims during world war i and the recent iran-iraq conflict. in the latter, sulfur mustard was heavily used and at the present time about 30,000 victims still suffer from late effects of the agent, such as chronic obstructive lung disease, lung fibrosis, recurrent corneal ulcer disease, and chronic conjunctivitis [2] . late complications of mustard gas exposure and main clinical findings include; chronic bronchitis, bronchiectasis and bronchiolitis obliterans (bo) [3] [4] [5] . however, clinical manifestation in lung disorders due to sulfur mustard is different from other lung diseases, due to the fact that mustard lung is not responsive to corticosteroids. there is no common consensus about the pathophysiological basis of chronic pulmonary disease caused by this chemical warfare agent [6] . proteomics technologies can identify and quantify novel proteins in the plasma that can function as biomarkers of the presence or severity of disease states. in general, human plasma proteome profiling is challenging. albumin is present at about 40 mg/ ml and several other proteins are highly abundant including immunoglobulins (iggs), transferrin and fibrinogen which typically constitute greater than 90% of total protein mass [7] . these abundant proteins may hinder the detection of low-abundant proteins that can be of specific interest in the search for biomarkers of disease [8] . however, it is the low abundant proteins that are most likely to be biologically relevant as the markers of a disease state. for analysis of low-abundant proteins in plasma, many strategies have been developed for the selective removal of albumin and other highabundance proteins. albumin can be removed by immune affinity columns chromatography [9] , isoelectric trapping [10] , heparin chromatography [11] and peptide affinity chromatography [12] . however, it is well known that albumin and other high-abundance proteins may also act as carrier or transport proteins and thus are likely to bind many species of interest, such as peptide hormones, cytokines, and chemokines. there are wide-ranging interests in using the proteomics approach to define markers of lung disease. although respiratory tract lesions represent the major disability after sm exposure, only a few studies have investigated the long term pathophysiology of sm induced respiratory damages, in particular their proteomes. we have recently examined the proteomics pattern in bronchoalveolar lavage (bal) fluid of sm exposed patients and identified families of proteins whose expression is up or down regulated compared to healthy controls [13] . plasma proteins and peptides are from almost every tissue and cell, and their change in quantity and quality is specific not only to the tissue affected by disease, but also to the disease process itself. in addition, plasma is the most easily accessible, less invasive, and widely collected sample. we attempted to explore plasma proteomics patterns of these patients, using ethanol fractionation. tow-dimensional gel electrophoresis was applied and followed by maldi-tof ms to look for new markers in the plasma of exposed patients which may help in further understanding the nature of long term effects of mustard gas. these finding might improve patient care and finding suitable therapies. the plasma protein content of the patients and the controls are presented in table 1 . no significant differences were observed in plasma protein contents of patients and controls. the ethanol fractionation was used to enrich low molecular weight proteins. as shown in figure 1 , most of the low molecular weight proteins were enriched in the ethanol supernatant rather than the precipitate. we found that 50% (v/v) ethanol was more efficient in fractionating low molecular weight proteins. to avoid any protein losses from the sample, we used both supernatants and precipitates of these fractions for 2-de analysis. for the first dimension 24 cm ipg strips with ph values in the range of 4-7 were used. the proteins were resolved in homogeneous 15% acrylamide gels in the second dimension to obtain greater resolution for small proteins. about 300 spots in each colloidal cbb-stained gel can be visualized by imagemaster software. representative 2-de images of plasma profiles from a healthy control for 50% (v/v) ethanol precipitate and ethanol supernatant are shown in figure 2a and 2b respectively. comparing the proteomics patterns of these two gels shows that both immunoglobulin heavy and light chains are separated in ethanol precipitate ( figure 2a ) and albumin is distributed both in ethanol supernatant and precipitate (figure 2a and 2b) . moreover, most of the small molecular weight proteins in the range of 15-50 kda, have significantly (p < 0.05) higher spot volume and intensity in ethanol supernatant rather than precipitate ( table 2 and figures. 2a and 2a) . we analyzed the differences in the plasma protein patterns, comparing the gels of the diseased and healthy controls. the analysis of protein patterns of the plasma was focused on those protein spots which showed differences, comparing the patients and the controls. they were compared with image master 2-de software and indicated only protein results in all cases (100%) with the same condition. as shown in figures 2 b and 2c, twenty six protein spots were subjected to maldi tof ms analysis. all selected proteins and their isoforms were subsequently identified by pmf and ms/ms u s25% p25% s50% p50% s75% p75% figure 1 sds-page of plasma proteins fractionated with different concentrations (v/v) of ethanol. u represents plasma proteins before precipitation; s, ethanol supernatant; p, ethanol precipitate. samples were analysed as described in the method section and gels were stained using silver staining. analysis. table 3 lists the identities of the proteins and their isoforms which were analyzed in this experiment using maldi tof ms. figures 2b and 2c shows the location of these protein spots in the 2-de gels of a healthy controls and an exposed patients respectively. volume and intensity of those protein spots which were only present in all patients' plasma but none of the healthy controls are shown in table 4 . images from other healthy volunteers and patients were similar (data not shown). haptoglobin α1 chain isoforms (spots 21, 22 and 23) were only detected in the plasma of the severe lung diseases patients but were not detectable in healthy controls ( figure 2b and 2c). furthermore, serum amyloid a1 isoforms (spots 24 and 26) were only seen in the plasma of the patients but none of the healthy controls ( figure 2b and 2c). in this study we present plasma proteome analysis of sm exposed patients compared to the healthy controls. human plasma and serum represent important biological materials for disease diagnosis. however, the wide dynamic range in protein concentrations remains a major challenge in the development of diagnostic assays. human plasma albumin and the various forms of immunoglobulin represent the most abundant proteins in the plasma, constituting up to 80% of the total plasma proteins. the table 3 . classical depletion strategy for albumin involves using hydrophobic dye cibacron blue, a chlorotriazine dye which has high affinity for albumin [14] . as a group, the immunoglobulins represent the second most abundant proteins in the plasma or serum. it has been reported that albumin depletion may also remove some small low copy number proteins [15] . therefore, in these experiments we used ethanol fractionation and found that this simple and low cost experimental procedure can be used for removing immunoglobulins and part of the albumin. moreover, using 50% (v/v) fractionation, we showed that the ethanol supernatant contains all the protein spots that were found in the ethanol precipitate and recovers more small mw proteins. we have identified some proteins that could give a novel insight into the pathogenesis of mustard lung. one of the main findings was different isoforms of haptoglobin in the plasma of severe lung disease patients but not in healthy controls. haptoglobin is present in normal human plasma at a concentration range of 0.3-1.9 mg/ml, accounting for 0.4-2.6% of total plasma protein. human haptoglobin is an inflammation-inducible plasma protein. it consists of 2 different types of α chains and a single type of β chain connected by disulfide bridges (β-α-α-β) giving 3 major phenotypes (hp 1-1, hp 2-1, hp 2-2), the numbers 1 and 2 representing α1 (8.9 kda) and α2 (16 kda) chains, respectively. the β chain (40 kda) is heavier than α chain and is identical in all hp types [16] [17] [18] [19] . our results ( figures 2b and 2c) show that under our experimental conditions, the α2 chain (spots 11, 12, 13) is similarly expressed in the plasma of both experimental data are mean ± sem of the % volume and intensity of protein spots on the 2d gels multiplied by 100. significant difference between the samples were calculated using the student's t-test and marked by * if p < 0.05, ** if p < 0.01. a. spot numbers correspond to those in figures 2a and 2b groups, but α1 chain (spots 21, 22, 23) is only expressed in the patients plasma samples but not in the healthy controls. in our recent study of bal fluid proteomics patterns in sm exposed patients we also found that haptoglobin isoforms were significantly elevated in moderate and severe lung disease patients compared to mild and healthy controls [13] . acute-phase proteins are induced shortly after exposure to triggering events such as inflammation, infection, and trauma, and are thought to be part of a general defense-response in injured tissue. but, these patients have been exposed to sm more than 20 years ago. it seems that an ongoing damage is occurring in lung of these patients. a recent proteomic study has also shown that levels of haptoglobin are elevated in bal fluid in patients with mild asthma, and reported that haptoglobin may play a role in the differentiation of fibroblast progenitor cells, suggesting a novel role for haptoglobin in airway remodeling in patients with asthma [20] . conversely nishioka et al. showed that the serum concentration of haptoglobin was decreased during acute exacerbation in asthmatic children [21] . haptoglobin isoforms and fragments are also elevated in plasma of severe acute respiratory syndrome (sars) patients [22] . haptoglobin plays a crucial role in defense against hemoglobin-induced oxidative stress by a mechanism thought to involve its high-affinity binding with hemoglobin and preventing iron release from hemoglobin. however, it has yet to be shown that haptoglobin itself is an antioxidant molecule [23] . arredouani et al. [24] further described the role of haptoglobin affecting the immune system, showing that haptoglobin directly affects t-cells and suppress t-helper-cells through down-regulation of cytokine production. we found that amyloid a1 isoforms only detected in the plasma of severe lung diseases patients but not in healthy controls ( figure 2b and 2c) . similar results were reported for sars patients [22] . although serum amyloid a1 is not as commonly used in human medicine as c-reactive protein (crp), it is more sensitive to acute response than crp [25, 26] . serum amyloid a1 is an acute-phase protein that is induced, like crp, by inflammatory mediators, including il-6, il-1β, and tnf-α, that rises in acute exacerbation of copd [27] . serum amyloid a1 is secreted from the liver as the predominant apolipoprotein associated with plasma high density lipoprotein. we postulate that in our sm exposed patients ongoing tissue damage and repair (remodeling) is occurring which leads to an increase in acute phase reactant proteins such as haptoglobin and amyloid a1. in conclusion, this study complements our previous bal fluid proteome analysis of patients exposed to sm gas which resulted in identification of number of differentially expressed proteins. to our knowledge this is the first study of plasma fluid proteome in sm exposed subjects. in this and our previous study the patterns of differentially expressed proteins identified in sm exposed patients are somewhat different from other lung diseases. it seems that sm exposed lung has an aberrant tissue remodeling which results in abnormal tissue architecture. all chemicals used in these studies were analytical grade or equivalent and were obtained from sigma (st. louis, mo) unless otherwise noted. milli q water (18.2 mω) was used throughout. according to the american thoracic society (ats) classification and based on our spirometric and high resolution computed tomography (hrct) findings, the patients were classified as severe lung diseases condition. patients group included 20 male subjects. a group of 20 healthy age-matched male individuals was used as the control. this study was approved by the ethics committee of the research center of baqiyatallah university of medical sciences, and informed consent was obtained from all patients and healthy controls. all participants were free to leave the study at will. the participant patients were suffering from pulmonary disorders due to previous exposure to a single high dose of sm gas during the iran-iraq conflict in 1987. inclusion criteria were as follows: documented exposure to sm and documented diagnosis of chronic pulmonary disease due to mustard gas. exclusion criteria for the patients and the control subjects were history of a chronic disease (tuberculosis, diabetes, hypertension, heart disease, hepatic diseases, etc.), resection of one or more lobes of the lungs, pneumonia and/or acute bronchitis, cigarette smoking or substance abuse. none of the patients or control subjects had a history of allergy or asthma. all patients and controls were in a stable condition and none of the participants had been administrated corticosteroids during the two-month period immediately preceding the studies. fasting venous blood samples were collected in the morning (8-10 am). blood samples were drawn into tubes containing k2edta and then immediately centrifuged at 1300 × g and 4°c for 10 min according to hupo plasma proteomics project recommendation [28] . supernatant was removed to a new tube and one tablet of complete protease inhibitor cocktail (roche, mannheim, and germany) was added. then, sample were promptly frozen in aliquots and stored at -80°c until used. plasma samples were thawed at room temperature and centrifuged at 10,000 × g for 10 min at 4°c. then clear supernatants were transferred to new microfuge tubes for further processing. total protein in plasma fluid was determined by the bicinchoninic acid (bca) assay and employed bovine albumin as the standard (pierce, rockford, il). the concentrations of proteins in plasma and different fractions were determined using a standard curve generated by the absorbance at 562 nm. following procedures for ethanol fractionation were applied. all steps were performed at 4°c. plasma samples were diluted 1:1 (v/v) with milli q water in a new microfuge tube and equilibrated to 4°c by gentle mixing (continuously on a vortex mixer at a low setting) for 10 min. in new separate microfuge tubes containing 500 μl of diluted plasma samples, different volumes of cold ethanol were added and total volume was adjusted to 1000 μl with milli q water. samples were incubated for an additional hour with gentle mixing on the vortex mixer and were centrifuged at 10,000 × g for 15 min at 4°c. supernatants were removed to new tubes, pellets were briefly re-centrifuged and any residual supernatant was removed and added to the previous supernatant fraction. for electrophoresis, the supernatants were dialyzed against 50 mm tris-hcl (ph 7.4) overnight to remove ethanol. for one dimensional sds-page analysis the collected ethanol supernatants and pellets were reconstituted in 50 mm tris-hcl (ph 6.8), 2% (w/v) sds, 0.1% (w/v) bromophenol blue and 10% (v/v) glycerol. proteins were separated by mini 1-de (10% t resolving gels with 4% stacking gels; 10 μg protein/lane). gels were electrophoresed at 30 v for 30 min and then at 50 ma per gel for 45 min, and stained using vorum silver staining procedure as described recently [29] . for 2-de analysis dialyzed ethanol supernatants and pellets were reconstituted in buffer consisting of 7 m urea, 1 m thiourea, 20 mm tris, ph 7.5, 4% (w/v) 3-[(cholamidopropyl) dimethylamino]-1-propanesulfonate (chaps), and centrifuged at 10,000 × g at room temperature for 5 min. supernatants were taken for 2-de gel analysis. protein concentration in the recovered samples was determined using a modification of the method as described by bradford [30] . two dimensional gels were analysed using 400 μg proteins per 24-cm linear ipg strips ph 4-7 (bio rad) as described previously [13] . ipg strips were reduced and alkylated, and then proteins were separated in the second dimension on homogeneous 15% polyacrylamide gels in an ettan daltsix electrophoresis unit (ge healthcare, uppsala, sweden). ipg strips were placed on the top of polyacrylamide gels (1 × 200 × 260 mm) and sealed with a solution of 1% (w/v) agarose containing a trace of bromophenol blue. gels were run in a running buffer, containing 25 mm tris, 192 mm glycine, and 0.1% (w/v) sds at 2w/gel for 45 min, followed by 6w/gel at 20°c until the bromophenol blue had migrated to the bottom of the gel. gels were fixed overnight in acetic acid, methanol, water, 10:45:45 (v/v) and stained with colloidal coomassie brilliant blue (cbb) g-250 as described previously [13] . analysis of spot patterns was performed using imagemaster 2d platinum software (version 6.0; ge healthcare). cbb stained gels were scanned in transmission scan mode using bio-rad gs-800 calibrated densitometer at a resolution of 600 dots per square inch (dpi). the scanned gels were saved as tif images for subsequent analysis. protein spots were detected automatically. manual spot editing or deleting (of artifacts) was performed when necessary. the spots intensity, volume, or saliency was adjusted in the preview mode using image master software. furthermore spots on all gels were visually carefully inspected for inappropriate matching, staining artifacts, or bad spot detection and conserved for analysis. to measure the volume and intensity of protein spots on cbb-stained gels the volume and intensity of each spot was divided by the total volume or intensity of all spots of the same gel. since this method of normalization produces extremely small values, the result was multiplied by a scaling factor of 100, which produced spot percentage volumes or intensity. only those spots that were reproducibly and statistically significant (p < 0.05) in intensity or volume were used for analysis. for mass analysis the protein spots of interest were aseptically removed under a laminar flow hood. processing of gel plugs, trypsin digestion and maldi tof ms/ms analysis using a bruker autoflex iii maldi tof/tof instrument (alphalyse, denmark) and database searching was performed as described previously [13] . the ms and ms/ms spectra were combined and used for a database search using the mascot software (matrix science, version 2.2.03) for database searches with the selection of following criteria: database search program: nrdb1 (6655203 protein sequence), species of origin homo sapience, peptide ion mass tolerance 60 ppm,, ms/ms tolerance 0.2 da, peptide cut-off of 25, and digestion by trypsin allowing for no more than one missed cleavage. the accuracy of mass detection was mh+ of 0.01 assuming possibility of modification of cysteine by acrylamide and oxidation of methionine. proteins identification was based on a combination of the peptide mass fingerprint (pmf) with peptide masses, and several ms/ms spectra of selected peptides in each maldi ms spectrum. the protein and protein isoforms shown on the identification list is based purely on the human protein database accession that gives the highest mascot score. to the extent that the different sequence isoforms are represented in individual database accession numbers, these have been considered in the database search. other modifications and isoforms are not considered in this analysis. the positive protein identification was based on a probability-scoring algorithm (http://www.matrixscience. com) and the 95% confidence level for positive identification was a score = 80 all data are representative of at least three independent experiments with twenty individuals in each group and are expressed as means ± standard error of the mean (sem). comparisons between two groups were performed using unpaired student's t-test. the criterion for statistical significance was p < 0.05 for all comparisons. tracheobronchomalacia and air trapping after mustard gas exposure medical aspects of sulfur mustard poisoning the diversity of the effects of sulfur mustard gas inhalation on respiratory system 10 years after a single, heavy exposure: analysis of 197 cases bronchiolitis obliterans in a survivor of a chemical weapons attack an international collaborative pathologic study of surgical lung biopsies from mustard gas-exposed patients long term consequences from exposure to sulfur mustard: a review the human plasma proteome: history, character and diagnostic prospects efficient prefractionation of low-abundance proteins in human plasma and construction of a two-dimensional map sample preparation of human serum for the analysis of tumor markers. comparison of different approaches for albumin and gamma-globulin depletion depletion of the highly abundant protein albumin from human plasma using the gradiflow heparin chromatography to deplete high-abundance proteins for serum proteomics development of mammalian serum albumin affinity purification media by peptide phage display bronchoalveolar lavage fluid proteomic patterns of sulfur mustardexposed patients studies on the mechanism of binding of serum albumin to immobilized cibacron blue f3ga albumin depletion of human plasma also removes low abundance proteins including the cytokines differentially expressed serum haptoglobin alpha chain isoforms with potential application for diagnosis of head and neck cancer covalent structure of human haptoglobin: a serine protease homolog gene action in the human haptoglobins. 3. isolation of the α-chains as single gene products. isolation, molecular weight, and amino acid composition of α-and β-chains hplc analysis of discrete haptoglobin isoform n-linked oligosaccharides following 2d-page isolation specific haptoglobin expression in bronchoalveolar lavage during differentiation of circulating fibroblast progenitor cells in mild asthma alpha-1-antitrypsin and complement component c7 are involved in asthma exacerbation plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry antioxidant role of human haptoglobin haptoglobin directly affects t cells and suppresses t helper cell type 2 cytokine releases serum amyloid a is a biomarker of acute exacerbations of chronic obstructive pulmonary disease the association of sensitive systemic inflammation markers with bronchial asthma copd exacerbations. 2: etiology. thorax spirometric classification hupo plasma proteome project specimen collection and handling: towards the standardization of parameters for plasma proteome samples a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding plasma proteomic profile of sulfur mustard exposed lung diseases patients using 2-dimensional gel electrophoresis the authors are grateful to baqiyatallah university research branch and chemical injuries research center for a supporting grant. we also thank dr. ejvind mørtz in alphalyse, denmark for mass analysis. authors' contributions hm carried out study design, analysis of proteomics profile, data mining and drafted and managed the manuscript. mg participated in the design of study and patient handling. ja participated in the patient handling and funding management. zt participated in the experimental laboratory work on proteomic profiling. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-023208-w99gc5nx authors: nan title: poster presentation abstracts date: 2006-09-01 journal: j pept sci doi: 10.1002/psc.797 sha: doc_id: 23208 cord_uid: w99gc5nx nan background and aims: homodimerization of myd88 adapter protein is essential for nf-kb activation in the inflammatory pathway triggered by il-1 and tlr [1] . we designed a peptidomimetic of the myd88 tir domain consensus peptide arg-asp-val-leu-pro-gly-thr [2] , named st2825. here, we report its synthesis and biological activity. we also report the synthesis and biological activity of its enantiomer, st3511, and its diastereoisomers, st3489 and st3558. methods: the structure of the myd88 tir domain consensus peptide is subdivided into three distinct portions, the most important of which is a b-turn. in the peptidomimetic design we changed the b-turn with a tricyclic spirolactam [3] , already known [4] . we synthesized this building block, its enantiomer and two of 8 possible diastereoisomers by "in solution" synthesis. based on semiempirical calculation of heat of formation [5] , we could predict the right stereochemistry of the 4 products selectively obtained in the last cyclization step. results: these four compounds were tested for their biological activity by reporter gene assay (rga). some coimmunoprecipitation experiments were also carried out and we report their results. conclusions: the results show the activity of st2825 and its isomers on our target, with limited specificity towards their stereostructure. introduction of a methylene bridge between the cα(i+1) and the n(i+2) atoms in an open peptide (i) to mimic simultaneously the cαh(i+1) and hn(i+2) protons (β-lactam scaffold assisted design -β-lsad) has proven to be a practical tool for the preparation of monotopic β-turn peptidomimetics (ii, r2 = r3 = h), according to the principle of separation of constraint and recognition elements1. in this work we report a short, general, and stereocontrolled synthesis of multitopic β-lactam scaffolds of type vi. α-alkyl serinates iii are converted into the corresponding enantiopure nnosyl-aziridines iv which undergo "in situ" ring-opening with amino acids v. subsequent base-promoted cyclization affords the n-protected α-alkyl-α-amino-β-lactams vii. incorporation of the novel scaffolds into linear and cyclic peptides and their conformational features are also presented, most of them showing stabilized β-and γ-turn conformations. poly(amino acids) are emerging as promising therapeutic carriers finding widespread application in the field of drug delivery. in this context, polyproline polymers have been used to solubilize poorly water-soluble proteins, in affinity chromatography for the purification of platelet profilin, and more recently, in the design of dendrimers. poly(amino acids) are most conveniently synthesized by polymerization of the corresponding amino acid n-carboxyanhydride (nca). in spite of the interest of polyproline, the preparation of proline n-carboxyanhydride (pro-nca) renders poor synthetic yields. in this work a new method for the preparation of pro-nca in high yields and purities is described. amino acid n-carboxyanhydrides are obtained by the method described by fuchs. but, in the case of proline, the n-carbamoyl chloride does not cyclise spontaneously as it takes place with other amino acids, and the use of a non-nucleophilic base is required for the cyclisation. a tertiary amine, such as triethylamine, is commonly used but it renders a low conversion of the n-carbamoyl chloride to the expected pro-nca, together with the presence of the pro-pro diketopiperazine byproduct. in the present work, polymer-supported bases have been used instead of triethylamine. higher yields of pro-nca, and very low percentages of diketopiperazine have been obtained. in addition, no tertiary amine contamination was observed. polymer-supported bases could also be recycled and pro-nca yields were reproducible. in conclusion, we have developed an efficient method for pro-nca preparation with polymer-supported bases. the introduction of novel nonproteinaceous heterocyclic amino acids into peptides results in new compounds with interesting structural, physicochemical and biological properties. the transformation of amino acid side chains after the peptide assembly is a convenient method of generating such modified peptides. taking into account the biological activity and complexing abilities of nitrogen-containing heterocycles, we investigated the formation of imidazole, benzimidazole and quinoxaline moieties using condensation with various aldehydes and α-dicarbonyl compounds after classical peptide synthesis on solid support. the imidazole synthesis utilizes the n-terminal or side chain amino group of amino acids, whereas a derivative of phenylalanine, β-(4-amino-3-nitrophenyl)alanine, was developed for benzimidazole and quinoxaline synthesis. the modified peptides were purified by preparative hplc and characterized by esi-ms, uv and nmr. in conclusion, we developed a straightforward method of synthesis of peptides with specific ion affinity and spectral characteristic. the broad range of commercially available aromatic aldehydes and dicarbonyl compounds makes possible the synthesis of combinatorial libraries of modified amino acids and peptides. part of this work was supported by a grant no. 3t09a 110 28 from the ministry of education and science. nmda receptors belong to the ionotropic group of glutamate receptors. the activity of the receptor can be altered by compounds acting at binding sites. the (r,s)-(tetrazol-5-yl)glycine (tg) has been shown to be a highly potent nmda (n-methyl-d-aspartic acid) receptor agonist with exitotoxic effects [1] . the aim of our studies was to investigate the chelating ability of tg towards copper(ii) ions. copper is widely distributed throughout the body with a distinct concentration in the brain. copper enters cells as complex and seeks out targets requiring it to function. for these reasons it was interesting to evaluate stability and structure of tg -copper(ii) complexes. the equilibrium and structural properties of complex species were characterized by ph-metric and spectroscopic (uv-vis and epr) methods. in the system, polymeric species are dominant at acidic ph range having { nh2, coo-} coordination with possible ntetr bridging elements. monomeric complexes were found at physiological ph. the two tg molecules are bound to copper ion via four nitrogen donors. the formation of two {nh2, ntetr} donor sets results in very strong metal-ligand interactions and the complex species are very stable over a wide ph region. we have also performed an investigation on similar tetrazole compounds in order to compare the chelating ability of the tetrazole moiety . the targets of our studies were 1,5-diamino-1h-1,2,3,4-tetrazole [2] and tetrazole aspartic acid. references continuing work in that field, we synthesized oxytocins containing tetrazole analogues of amino acids. the 5-tetrazolyl group is widely used in medicinal chemistry as an isostere of the carboxyl group. compounds containing tetrazole ring appear to be metabolically more stable than their carboxylic analogues and have comparable acidity. we synthesized derivatives of aspartic, glutamic, and alpha-aminoadipic acids containing 1h-tetrazole ring in side chains. these derivatives were then used for syntheses of oxytocin analogues substituted in position 4. apart from above we also obtained two analogues with tetrazole analogue of glycine in position 9. the first one contains 1h-tetrazole ring, the second one has tetrazole ring substituted with methyl group in position 1. oxytocin analogues possessing amino acids with tetrazole ring in side chains were synthesized on amide resin using fmoc methodology. in the case of analogues with c-terminal tetrazole ring, fragments 1-7 were synthesized on resin and then coupled with suitable dipeptides in solution. all obtained peptides show no pressor and rather low uteronic activity. however, for some analogues the uterotonic activity when measured in the presence of magnesium ions was several times higher. in humans, two classes of defensins, α-defensin and β-defensin, have been identified on the basis of tissue specificities and structural features including their modes of disulfide pairing. in general, particular combinations with disulfide bonding in cysteine-containing peptides are critical for expressing their intrinsic biological activities. in the case of human α-and β-defensins, however, disulfide isomers without the native pairing were demonstrated to exhibit similar antimicrobial activity to that of the native defensins. therefore, to assess the biological activities of defensins as well as defensin-based therapeutics, extreme care is required in the chemical synthesis of these peptides to avoid ambiguity in quality. in the present study, we synthesized human α-defensin-1, -2 and -4, and human β-defensin-1, -2, -3 and -4 by employing boc chemistry, and determined the optimal conditions for folding the respective reduced peptides preferentially into a native conformation. among the factors affecting the oxidative folding in the presence of reduced and oxidized glutathione, the buffer concentration and reaction temperature were essential. all the synthetic human α-and β-defensins were confirmed to have the respective native disulfide pairing by sequential analyses and mass measurements with cystine segments obtained by enzymatic digestion. all the human α-and β-defensins could be efficiently oxidized to the α-and β-defensin-type disulfide structure, respectively, under several conditions determined in the present study. these synthetic peptides of high homogeneity were used to accurately assess the antimicrobial activity. native chemical ligation is based on the reaction of a peptide bearing a c-terminal thioester group with an n-terminal cysteinyl peptide, leading to the formation of an amide bond at the aa-cys junction. the key starting materials for native chemical ligation are unprotected c-terminal thioester peptides. thioester peptides are often prepared using boc/benzyl solidphase peptide chemistry. however, the widespread use of the fmoc/tert-butyl chemistry for peptide synthesis, over the boc/benzyl method, has stimulated the development of methods allowing the preparation of thioester peptides that are compatible with the basic treatments used to remove the fmoc alpha-amino protecting group. we report here a novel method for thioester peptide synthesis that is based on the use of the sulfonamide safety-catch linker. once the peptidyl chain is assembled by fmoc/tert-butyl chemistry, the thioester function is generated on the solid-phase through an intramolecular n,s-acyl shift. the procedure seems to be insensitive to the bulkiness of the amino acid directly attached to the sulfonamide linker. the thioesters were successfully used for native chemical ligations in solution or on the solid support. we optimized the recognition sequence of the substrate and the reaction conditions with respect to the yield. the sortase-mediated ligation was successfully applied to the synthesis of cellpenetrating peptide-pna conjugates which showed enhanced activity in antisense experiments compared to pna alone. this ligation strategy was also employed for the coupling of a chemically synthesized construct of the extracellular loops of the crf-receptor with the corresponding n-terminal receptor domain, which was expressed in e. coli. this 23 kda protein behaves like an artificial receptor, binding specifically natural ligands. linear gramicidins represent the most investigated family of antibiotic peptides forming ionic channels. gramicidins produced by bacillus brevis are hydrophobic peptides composed of 15 amino-acids with d and l configuration strictly alternate. the presence of d-amino acids in the sequence of gramicidin a (hco-val-gly-ala-dleu-ala-dval-val-dval-trp-dleu-trp-dleu-trp-dleu-trp-nhch2ch2oh) should possible make the peptide highly resistant to proteolysis [1] . striking features like ethanolamine group in c-terminus, the n-terminal n-formylated valine and the high hydrophobicity of the peptide sequence, make the solid-phase synthesis of gramicidin a very tricky. therefore, we followed a new synthetic strategy for peptide chain elongation assisted by microwave energy. in fact, microwave energy has been demonstrated to produce highpurity compounds with more rapid reaction times, enhancing coupling rates and efficiency in difficult syntheses [2] . however, microwave-assisted solid phase peptide synthesis (mw-spps) has not been yet extensively investigated. in this context, we synthesized gramicidin a by mw-spps in high yield and purity, enhancing reaction rate compared to the traditional spps. thermal disruption of peptide aggregation, induced by microwaves, is possible favorable for obtaining this particularly difficult sequence. gramicidin a was incorporated in synthetic lipid bilayers, self-assembled on mercury electrodes, characterized by hydrophilic spacers interposed between the metal and the lipid bilayer. we tested the behaviour of gramicidin a in biomimetic membranes using electrochemical impedance spectroscopy (eis), ac voltammetry and other electrochemical techniques [3] . [ csf114(glc) is an n-glucosylated peptide to be produced in large scale by peptlab because it is the active molecule of the first specific diagnostic/prognostic test for monitoring disease activity and guiding therapeutic treatments of multiple sclerosis patients [1] . in order to develop a synthetic protocol by an automated instrumentation, increasing yield, purity of the crude, and reaction time, a microwave-assisted solid phase peptide synthesis was validated comparing the use of the new generation of triazine-based coupling reagents (tbcrs) with a series of commonly used ones. activation of carboxylic acids by tbcrs is particularly effective because of formation of triazine "superactive esters". the usefulness of tbcrs as coupling reagents has been recently confirmed in the synthesis of z-, boc-, and fmoc-protected dipeptides, sterically hindered amino acids, in the synthesis of esters, in manual and automated spps of difficult peptide sequences, and head-to-tail constrained cyclopeptide analogues [2] . moreover, we also demonstrated tbcrs efficient in a microwave-assisted solution synthesis of the n-glucosylated building block fmoc-asn(glcoac4)-oh using a manual monomode microwave instrument [3] . this building block was used to obtain csf114(glc) comparing the efficacy of a monomode microwave automatic instrument with the traditional solid-phase peptide synthesizers such as the manual and automatic in batch systems, as well as the continuous-flow one. it is known that enzymatic peptide synthesis is more advantageous than chemical synthesis in many aspects; it is highly stereoselective, racemization-free and requires minimal side-chain protection. the method is, however, limited to the use of amino acid derivatives which meet the enzymatic specificity as a coupling component. this problem may be solved using enzymes which have wide specificity of substrate. but in this case, secondary hydrolysis of the resulting peptide may arise from the inherent nature of the protease. in this matter, ficin and ficin-like enzymes were used as cysteine protease to analyze the diminishment of specificity for the substrate. the cysteine protease-catalyzed peptide coupling reaction has been studied by using synthetic fourteen boc-amino acid phenyl and naphthyl esters as acyl donor. the reaction conditions were optimized for organic solvent, ph, and concentration of acceptor. the coupling reaction was carried out by incubating an acyl donor (1 mm) with an acyl acceptor (ala p-nitroanilide, 35 mm) and enzyme (0.1u) in a mixture of gta buffer (50 mm, ph 9.0) and dmso (3:2) at 37ْc. the progress of the coupling reaction was monitored by rp-hplc. the products were obtained in satisfactory yields. non-enzymatic glycosylation, also called glycation, is a common modification in living organisms formed by the reaction of carbohydrates with free amino groups of peptides and proteins. it is a slow chemical reaction yielding amadori products undergoing further oxidation and degradation reactions finally leading to advanced glycation end-products (age). amadori products are early markers for ageing, diabetes mellitus and alzheimer's disease. despite the clinical importance of these amadori products, universal protocols to synthesize amadori modified peptides are still missing. here we describe a solid phase strategy for the glycation of specific amino groups on partially protected resin bound peptides using a global post synthetic approach. the peptides were synthesized by standard fmoc/tbu-chemistry using carbodiimide activation. the lysine position to be modified was incorporated with a methyltrityl protected ε-amino group, which can be selectively cleaved after completion of the peptide synthesis with 1% tfa in dichloromethane. the partly deprotected peptide was glycated in methanol using a ten-fold molar excess of 2,3-4,5-di-o-isopropylidene-aldehydo-β-d-arabino-hexos-2-ulo-2,6-pyranose and nabh3cn for 18 h at 70°c. after cleavage the overall yields were in the range of 50 -70 % for the tested octapeptides. all byproducts were well separated by rp-hplc allowing a simple purification strategy even for medium-sized peptides. thus the general strategy presented here allows routine synthesis of amadori peptides at reasonable yields and purities using standard protocols established in most laboratories synthesizing peptides. 2-chlorotrityl chloride resins are recommended for the synthesis of c-terminal proline peptide acids to overcome diketopiperazine formation during chain assembly. however, we have found these (and similar) resins to be unsuitable for the synthesis of peptides greater than 20 residues. for example, the chemokine guinea pig eotaxin, (73 residues c-terminal proline) assembles poorly if not at all on a 2-chlorotrityl resin. we sought to circumvent these problems in the chemical synthesis of peptides and proteins, through the development of a resin-swap procedure. whereby the initial c-terminal protected tripeptide is assembled on a 2-chlorotrityl resin, liberated from the solid-support, then reattached to a resin that is suited for long chain peptide / protein synthesis. using this approach, the synthesis of guinea pig eotaxin is reported. the tripeptide fmoc-thr(but)-lys(boc)-pro-oh was assemble on 2-chlorotrityl resin, cleaved with 20% tfe in dcm and attached to wang resin using standard protocols. peptide assembly gave the gp eotaxin in 53% overall yield (as determined by uv monitoring). fmoc-on cleavage, purification and tag removal followed by folding gave the native chemokine in good yield. choice of resin is one of the most critical factors in ensuring a successful peptide synthesis, we have shown the superiority of wang resin over chlorotrityl resin in the synthesis of medium and long peptides and developed a method for the synthesis of c-terminal proline containing peptides which overcomes the problem of diketopiperazine formation. the technique is being applied to the synthesis of other c-terminal proline peptides e.g. human eotaxin and ip10. dimerization of cell receptors, involved in antigen presentation, is an essential step in several cellular signal transduction processes, therefore substances that are able to modulate such a process are of potential therapeutic value. dimeric peptide ligands could represent useful tools to cause dimerization of such receptors. a similar strategy applies dimerization of ligands, interacting with dimeric proteins or proteins with multiple binding sites, to design molecules with enhanced affinity. dimeric analogs of the immunosuppressory hla class ii fragments were synthesized using suitably modified, standard fmoc solid-phase protocols and mbha-resin. the dimerization was achieved by crosslinking n-terminal amino groups of the peptides with the commercially available mixture of poly(ethyleneglycol)biscarboxylic acid (average mw 600, length range 30-45å), activated by esterification with pentafluorophenol. the same procedure was applied to synthesize a series of dimeric analogs of c-terminal fragments of plexin-b, consisting of two undecapeptides, linked by the polyethyleneglycol spacers. other biand polyvalent linkers were also investigated. our results demonstrated that the amino-terminal dimerizations of the tested hla-fragments resulted in enhanced immunosuppressive activities, whereas interaction of pdz dimer with the plexin fragments led to about 20-fold increase in affinity, as compared to their monomeric counterparts. [background and aims] elucidation of alzheimer's disease (ad)-related aß1-42 dynamic events is a difficult issue due to uncontrolled polymerization. [methods] based on the "o-acyl isopeptide method" (chem. commun. 2004, 124; j. am. chem. soc. 2006, 128, 696), we have developed a novel photo-triggered "click peptide" of aß1-42 (1), e.g., "26-n-nvoc-26-aiaß42 (2)", in which a 6-nitroveratryloxycarbonyl (nvoc) group was introduced at ser26 in 26-o-acyl isoaß1-42 (26-aiaß42, 3). [results] i) the click peptide 2 did not exhibit the self-assembling nature under physiological conditions due to one single modified ester; ii) photo-irradiation of the click peptide 2 and subsequent o-n intramolecular acyl migration afforded the intact aß1-42 (1) with a quick and one-way conversion (so-called "click"); and iii) no additional fibril inhibitory auxiliaries were released during conversion to aß1-42 (1). [conclusions] this method provides a novel system useful for investigating the dynamic biological functions of aß1-42, such as the self-assembly and aggregation processes in ad. several insulin analogues have recently been introduced clinically for improved treatment of diabetes. industrial productions of such insulins are based on microbial expression systems, which are highly efficient, but generally limited to the 20 proteogenic amino acids. also, some sequences form inclusion bodies or fail to express. the total chemical synthesis of insulin in research scale was a landmark achievement in peptide science. however, the most commonly used method relies on recombination of a-and b-chains under "random" folding and pairing of the three disulfide bridges. this folding/oxidation step is difficult and low yielding. a general approach using a removable auxiliary which can direct correct formation of disulfide bridges is highly desirable. in the pancreas as well as in microbial expression systems, insulins are prepared and folded as single chain precursors, with a c-peptide connecting the a and bchains. the c-peptide helps direct the orientation of a and b-chains in obtaining the correct disulfide pairing and overall peptide folding. upon folding, the c-peptide is removed enzymatically. we report here a new method for total chemical synthesis of insulin by use of fmoc-based step-wise solid-phase synthesis of single-chain precursors followed by cpeptide directed folding and cleavage of c-peptide, thereby allowing total chemical synthesis of novel insulins with unnatural substitutions. 2-chloro-4-methoxy-1,3,5-triazines 1a-c anchored on cellulose, silica or wang resin were prepared by the treatment of 2,4-dichloro-6-methoxy-1,3,5-triazine with appropriate solid support in the presence of a base. immobilized, environmentally friendly triazine coupling reagents 3a-c were obtained by treatment of 1a-c with n-methylmorpholinium p-toluenesulfonates 2 in the presence of hcl acceptor. the loading of the solid carriers were calculated from n, s contents, determined by microanalysis. all prepared immobilized n-triazynylammonium toluenosulfonates 3a-c have been found stable at room temperatures. activation of carboxylic components afforded triazine activate esters 4a-c connected to the support. treatment of 4a-c with appropriate amino components gave amides or peptides. the final products, chromatographically homogenous amides and peptides, were isolated by filtration or extraction from the solid support. mutter's pseudoproline dipeptides and sheppard's hmb derivatives are powerful tools for enhancing synthetic efficiency in fmoc spps. they work by exploiting the natural propensity of n-alkyl amino acids to disrupt the formation of the secondary structures during peptide assembly. their use results in better and more predictable acylation and deprotection kinetics, enhanced reaction rates, and improved yields of crude products. however, these approaches have certain limitations: pseudoproline dipeptides can only be used for sequences containing serine or threonine, and the coupling of the amino acid following the hmb residue can be extremely difficult. to alleviate some of these shortcomings, we have prepared fmoc-ala-(dmb)gly-oh and fmoc-gly-(dmb)gly-oh. these dmb-dipeptides can be incorporated into peptides in place of ala-gly and gly-gly, resulting in peptides containing structure breaking (dmb)gly residues. by introducing the (dmb)gly residue as part of a dipeptide unit, the need to acylate the highly hindered secondary amino group of (dmb)gly is avoided. on treatment with tfa the dmb group is cleaved regenerating gly. to test the efficacy of our new derivatives in expediting the synthesis of hydrophobic peptides, we undertook the preparation of the challenging neurotoxic prion peptide 106-126 <b>1</b>; this peptide reportedly can not be made using fmoc spps methods. the dipeptides marked in bold were systematically substituted with the appropriate dmb peptides. the effects of the substitution were evaluated using conductivity monitoring and lc-ms analysis of the crude peptides. h-lys-thr-asn-met-lys-his-met-<b>ala-gly</b>-ala-ala-ala-<b>ala-gly</b>-ala-val-val-<b>gly-gly</b>-leu-gly-oh <b>1</b> th120 efficient dipeptide production form unprotected l-amino acids with the novel enzyme l-amino acid α-ligase. k. tabata 2 , h. ikeda 1 , m. yagasaki 1 , s. hashimoto 1 background and aims: application of α-dipeptides has been limited due to the lack of cost-effective manufacturing methods. the known methods require the protection of amino acid(s) to fix the order of the amino acids ( fig. 1) . furthermore, they usually accompany the formation of longer peptides. to establish the costeffective manufacturing method, a novel activity which synthesizes α-dipeptides from two unprotected l-amino acids was screened. methods and results: a gene was found in the genome of bacillus subtilis by in silico screening based on a putative reaction mechanism. the purified protein coded on the gene, i) catalyses α-dipeptide formation from unmodified l-amino acids with a specific order in an atp-dependent manner, ii) never forms tri-or longer peptides, and iii) takes a wide variety of l-amino acids but no d-amino acids. the enzyme was tentatively named l-amino acid α-ligase (lal). the whole cell reaction of a recombinant e. coli strain expressing lal and polyphosphate kinase (ppk) with two l-amino acids and polyphosphate (polyp) enable the efficient production of many dipeptides with a certain order of the constituent amino acids through the coupling reaction of lal and ppk (fig. 2) . conclusion: a novel enzyme, lal, enables to synthesize dipeptides cost-effectively directly from unmodified l-amino acids. t. ye 1 marine organisms continue to provide rich sources of structurally unique and pharmaceutically active compounds. due to the difficulties in the isolation of significant quantities of these natural products, synthetic chemistry serves an important role in their structural assignment and biological evaluation. antifungal agents have received considerable attention recently since the spread of hiv has left many people open to fungal infections, and there is a rapidly growing number of drug resistant strains of fungus emerging. ll-15g256gamma is a cyclodepsipeptide isolated from the marine fungus hypoxylon oceanicum and structurally assigned in 1998 by schlingmann. the structure of ll-15g256gamma was determined by a combination of chemical degradation, chiral chromatography and spectroscopic analysis. ll-15g256gamma uniquely combines a beta-ketotryptophan and a polyketide portion within a macrolactone ring. ll-15g256gamma has exhibited potent activity against fungal strains and as such, is an attractive compound to develop as a future therapeutic agent. to date, there have been no reported studies towards the synthesis of ll-15g256gamma. we have completed the total synthesis of ll-15g256gamma by employing the macrolactamization followed by a c-h oxidation as the key step. aspartimide (aminosuccinimide, asu) formation is the first step in the degradation of asp/asn containing peptides and proteins. the reaction is especially prevalent at asx-gly sites and results in a variety of rearranged and racemized products. the bases used in fmoc-tert-based spps promote the formation of asu and related products. we recently found that the dmb backbone protection efficiently prevents secondary structure formation at gg sites and is orthogonal with respect to standard fmoc spps. here we explore the use of dmb, tmb and nbzl groups (z) for the synthesis of "difficult"/asu-prone peptides, in three different schemes: a) fmoc-asx-(z)gly-oh dipeptide building blocks; b) fmoc-(z)gly-oh monomer building blocks, and c) two steps "submonomeric" approach for synthesis of substituted n-benzyl glycines on the resin. we tested the new methods on two model peptides vkd/ngyi and ha21-20hiv-tat48-57 (h-g1lfgaiagfi engwegmidg20grkkrrqrrr30-oh) fusion peptide. the yield and purity of the products reach and even exceed the level in control experiments obtained with hmb protection and the peptides were found free of asu/piperidides. the acid removal of the dmb protection is ~30% faster than that of hmb. the submonomeric route (strategy c) is especially simple, efficient, cost effective and it allows the use of different amines for halogen-displacement. the backbone protecting groups used were in many respects superior to the commercial reagents and applicable for synthesis of both peptide acids and peptide amides. the use of nbzl-nh2 for halogen displacement represents a new method for preparation of backbone-caged peptides. alkyl bonded silica gels historically have been the standard in reversed phase (rp) purification of biomolecules such as synthetic peptides, small proteins, and oligonucleotides. silica gels provided the resolving power needed for challenging separations and the mechanical stability required to be operated industrially under high pressure conditions. the chief disadvantage of silica gels is poor chemical stability under alkaline conditions, which limits their capability to withstand rigorous clean and sanitization -in-place (cip/sip) protocols. as a result, polymeric media have gained recent market attention because of their excellent chemical stability, which enables full compatibility with modern cip/sip protocols. however, first generation polymeric gels lacked both the resolving power and the mechanical stability to be compatible with industrial high pressure dynamic axial compression (dac) hardware. rohm and haas' advanced biosciences division recently introduced a new, monospheric, 10 micron, high performance polymeric rp material. unlike existing softer polymeric gels, this product has higher mechanical stability which enables it to be used effectively with industrial dac / hplc hardware. in addition, this material provides high resolving power for the most challenging industrial separations, because of its unique and selective pore structure, as well as its small monospheric particle size. finally, because of its excellent chemical stability, the media is not limited in the range of ph that can be used. the combination of mechanical stability for high throughput, chemical stability for long lifetime in use, and high resolution for high yield, together translate to an effective cost-in-use solution for industrial polishing processes. we have developed new types of peptide nucleic acids with improved water solubility by introducing ether linkages and pyrrolidine rings in the main chain; pyrrolidine-based oxy-pnas (popnas). in this work, cellular uptake and endosomal release of the trans-l-popna oligomers, one of stereoisomes of the popna, were investigated. the cellular uptake was achieved by combining the popna oligomer with an n-terminal 23-mer peptide of an influenza virus hemagglutinin protein (ha2) that is labeled with a rhodamine fluorophore at the n-terminal and covalently linked with a hepta-arginine unit at the c-terminal (rho-ha2-r7). the fluorescence images of the cho cells after incubation with fam-po(13) [fam-o-cag tta ggg tta g-gly-nh2] in the absence and presence of rho-ha2-r7 were observed with confocal laser-scanning microscopy. incubation with fam-po(13) alone, no internalization of the oligomer was observed. in the presence of rho-ha2-r7, however, fam-po(13) was successfully internalized into cho cells and, more importantly, the fluorescence spread over the whole cell. the fluorescence image indicates that the popna oligomer in combination with the ha2-r7 peptide was transferred into cytoplasm within 1 h. since both the red (rho) and green (fam) fluorescence spread over the cytoplasm, the popna oligomers that were taken up into endosomes together with the rho-ha2-r7 were released into cytoplasm as the disruption of the endosomes by the ha2 peptide. in summary, the popna oligomers were readily taken up into cytoplasm of cho cells, when combined with a ha2-r7 peptide. most of functional rnas have post-transcriptional modifications, some of which are quite important for their structure and function. thus, for studying such rnas, it is necessary to use purified raw rnas obtained from living organisms. isolation of native rna is necessary also in the case of analyzing the sequence and modifications of mature rna, which may be different from simple transcript of its gene. therefore, rna isolation method is required. many previous reports demonstrated isolation of rnas, especially trnas. most common and traditional purification methods are based on successive column chromatographies. it seems difficult to apply such method to every trna because effective combination of columns varies among individual trnas. to overcome the difficulty, a sequence-specific selection method using a solid-phase dna has been devised. in this method, a trna can be purified from rna mixture by a single step. however, this method needs high temperature treatment, which might assist hydrolysis of rna strand and might impair heat labile modifications. pna-rna hybrid has been known to be much more stable than dna-rna hybrid. thus pna-based rna purification method seems to be possible for wider variety of rnas in lower temperature, in comparison with dna-based method. in this study, we attempted to purify a single rna, such as a trna and a noncoding rna, from rna mixture by using immobilized pna. r. pipkorn 1 , w. waldeck 2 , h. spring 3 , j. jenne 4 , k. braun 5 background and aims: safe drug delivery technologies are pivotal for genetic interventions, but viral vectors baer the risk of inflammatory reaction. questions concerning the efficacy of delivery of the genetic substances, the desired topical gene activation and targeting must be answered. therefore we attempted to develop a membrane non-perturbing delivery system for transport of inactive functional genes into cells and tissues. genes can be subsequently activated at the target site. our concept bases on the use of peptide-nucleic-acids (pnas) resistant against proteases and nucleases, oligonucleotide derivatives, in which the phosphate-backbone has been replaced with ethylen-amin connected alpha-amino-ethylglycine-units. methods: peptides conjugates were composed and synthesized according to the solid phase synthesis and protecting group chemistry strategies. pna sequences were conjugated covalently, non cleavable, with a capronic acid spacer to the nls, pkkkrkv. pnas have gained broad attention in antisense/antigene experiments and as diagnostic tools. in principal, they can be synthesised with several activating reagents known from peptide synthesis. namely, hatu or pybop are often used. synthesis with hatu is more laborious, because preactivation is needed in order to avoid guadinylation of the n-terminus of the growing pna-chain. we wanted to use pybop, because preactivation should not be needed in this case, which is especially useful in automated synthesis. surprisingly, in the pybop-mediated syntheses of 18mer pnas we obtained products showing molecular masses approx. 67 da above the expected ones. detailed analysis revealed, that the modification occurred at the only guanine residue in the sequence. in order to further characterise the side reaction, a short pna fragment was synthesised using hatu and pybop activation, respectively, and cleaved from the resin with and without the n-terminal fmoc-group. while synthesis with hatu gave the desired products, pybop partly activates the aromatic carboxy group of the guanine residue, which is substituted by piperidine during subsequent fmoc cleavage. the modified sequences could be further characterised by ms/ms-fragmentation. our results show that care must be taken when synthesising pnas with pybop activation. on the other hand, this reaction possibly opens an opportunity to synthesise guanine derivatives. the opioid receptor system in the central nervous system (cns) controls a number of physiological processes including pain, reward, gastrointestinal and cardiovascular functions. as a consequence, most pain modulating compounds currently available cause a variety of side-effects. the endogenous ligands for the opioid receptors are a series of peptides that includes endomorphin-1. endomorphin-1 has been shown to elicit potent anti-nociception through the highly selective activation of µ-opioid receptors. it is this receptor that mediates supraspinal analgesia and thus, selectivity for this receptor results in analgesia without affecting other processes. therefore, endomorphin-1 is considered a promising lead compound for the development of a new, safer pain medication. we have synthesized a large number of lipid-and carbohydrate-modified endomorphin-1 analogues and screened these compounds for their binding and activation of µ-and δ-opioid receptors in sh-sy5y cells as well as caco-2 cell monolayer permeability and plasma stability. compounds conjugated with either a lipoamino acid or sugar moiety on the c-terminus lost binding affinity by several orders of magnitude, whilst n-terminal conjugations resulted in minimal loss of binding affinity. a number of analogues showed pm binding affinity and high apparent permeability, and of these compounds, one has been selected for assessment in nociceptive and neuropathic pain models. in addition to these pre-clinical studies, internalization and tolerance formation of these compounds has also been measured in an effort to synthesise a non-tolerant opioid agonist. endomorphin-1 analogues with a high degree of amphiphilicity cause increased receptor internalization and subsequently less tolerance formation. a. marcinkowska 1 , l. borovičkova 2 , j. slaninowá 2 , z. grzonka 1 carbohydrate moieties of glycopeptides and glycoproteins play different decisive roles in various biological phenomena. conformation and solubility of proteins are influenced by the oligosaccharide chains, which can also inhibit the proteolytic degradation. as a result, the synthesis of glycopeptides is an attractive field that contributes to understanding of mutual interactions between both moieties and for their biological interest. the synthesis of glycopeptides requires a combination of synthetic methods from both carbohydrate and peptide chemistry. moreover, this synthesis needs stereoselective formation of the glycosyl bond between a carbohydrate and a peptide (amino acid) part, and also an appropriate protecting group methodology that allows selective deblocking of only one functional group in these polyfunctional molecules. in the present work we modified the oxytocin and vasopressin structure with glycoamino acids. transformations of fmoc-protected serine and threonine derivatives into appropriate o-glycosylated precursors suitable for solid phase peptide synthesis were worked out. the -and -o-glycosides were synthesized from fmocserine and fmoc-threonine allyl esters and appropriate glycosyl bromide using hanessian's modification of the koenigs -knorr reaction. these n--fmoc-protected glycosides were used in synthesis of glycopeptides. eight analogues of oxytocin modified in position 4 were obtained. we have also prepared two types of lysin-vasopressin analogues modified with glycoamino acid, in which the glucuronic acid was attached to the ω-amino group of lysine in position 8 through the amide bond. glycosylated analogues of oxytocin and vasopressin display an increased stability towards enzymatic degradation, and retain some hormonal activities. supported by grants: ds/8350-5-0131-6 (zg) and z40550506 (js) according to many authors the formation of amadori products is a key stage in the glycation process. glycated proteins may show allergenic properties and potentially initiate autoimmunological processes. they may also serve as the markers of diabetes. to our best knowledge, all procedures concerning the synthesis of peptide-derived amadori products reported in literature are based on "in solution" approach which makes them tedious and time consuming. a modified method of the solid phase synthesis of peptide-derived amadori products based on direct alkylation of the deprotected ε-amino groups with 2,3:4,5-di-oisopropylidene-β-d-arabino-hexos-2-ulo-2,6-pyranose in the presence of sodium cyanoborohydride was proposed. isopropylidene groups, protecting the sugar moiety in the obtained conjugate, were removed with trifluoroacetic acid containing 5% water. studies on optimization of the reaction performed on the model peptide attached to a wang resin, fmoc-lys-leu-leu-phe-(resin), showed that the best yield of the product is attained with a two-fold excess of 2,3:4,5-di-oisopropylidene-β-d-arabino-hexos-2-ulo-2,6-pyranose and a five-fold excess of sodium cyanoborohydride. the identity of the product was confirmed by high resolution ms. the several side products were isolated and their structures will be discussed. our results prove that the synthesis of glycated peptides in the solid phase is feasible. the lack of homogeneous glycoproteins in sufficient quantities is an ongoing challenge in glycobiology. in order to solve this problem researchers have turned to a variety of approaches ranging from mutant eukaryotic strains to the highly demanding total synthesis of glycoproteins. [1] using rnase b as a model nglycoprotein [2] we have searched a path to assemble this enzyme employing a combination of chemical and recombinant methods. native chemical ligation [3] allows the coupling of protein segments of unrestricted size in a chemoselective manner. we have developed solid phase methods to produce the required thioester building blocks 1-25-sr (a) and glycopeptide thioester 26-39-sr (b) containing an n-glycan at asn34 on a dual linker pega resin. [4] the remaining segment 40-124 (c) was expressed in e. coli as a fusion protein and released by intein mediated protein cleavage. [5] sequential coupling of the three rnase segments requires the use of a protective group at the n-terminus of segment b compatible with the oligosaccharide part. dysfunctional mutations of antitrypsin can result in a loss of elastase inhibitory activity or allow self-aggregation to occur and cause emphysema and cirrhosis, respectively. insights of the mechanism of disease provide strategy to cope with the aberrant protein aggregation and may bring potential therapeutic agents. in the present work, we describe our effort to identify effective anti-protein polymerization ligands by the employment of combinatorial technology. antitrypsin from human plasma was purified by glutathione sepharose and mono q-sepharose column chromatography. both ala-scanning and peptide shortening were carried out systematically to explore the structural requirements necessary for binding. combinatorial chemistry was then employed to conduct the library screening experiments. assessment of peptide binding was achieved through an unique gel electrophoresis assay. the structural requirements and the minimal peptide length required for binding were revealed by our systematic approach. this information was critical for the design of combinatorial library and the discovery of antitrypsin binding peptides with much improved affinity and specificity. there is currently no effective cure for z antitrypsin related cirrhosis and emphysema. the synthesis and screening of combinatorial libraries offer avenues to increase throughput and ultimately lead to the discovery of inhibitory peptides to the polymerization of pathogenic antitrypsin. with the rapidly increasing number of biopharmaceuticals in the industrial pipeline the need for efficient and expedient purification procedures is growing ever greater. affinity chromatography is one of the most promising technologies in this regard, as it offers very high selectivity and can often replace lengthy and expensive traditional chromatographic procedures. the use of combinatorial split-and-mix libraries is a powerful tool for discovering new affinity ligands but the technique has been limited by the laborious spectroscopic and chemical analysis needed to identify the binding ligand. we have previously introduced a novel bead encoding technology based on a 3-dimensional image recognition of patterns made by fluorescent particles randomly distributed inside larger beads. [1] the beads are read prior to each chemical transformation by an instrument featuring three fluorescence microscopes at a rate of 5,000 beads per hour. we here present the development of small peptidomimetic affinity ligands for the human growth hormone (hgh) by the use of this technology. the library was sought enriched prior to synthesis by in silico screening of a virtual combinatorial library using a large number of diverse building blocks. binding ligands were identified by incubation with fluorescence tagged hgh. [ the cinnamic acids and their derivatives have been found to possess a variety of biological effects, including antiviral, antimicrobial, antitumor and antioxidant activity. for example, several hydroxycinnamic acid conjugates with amino acids, isolated from plant sources showed enhanced antioxidant activity. the synthesis of cinnamic acid amides and their opioid activity was also cited in the literature. however the synthesis and pharmacological properties of sinapoyl-peptide amides continues to be virtually unexplored. on the other hand, the synthesis and opioid activity of analogs of tyr-mif-1 has been well documented by us. herein we present a synthesis of a series of sinapoyl -peptide amides where sinapic acid were attached consecutively to both c-and n-end of the tyr-mif-1 peptide chain: sa-pro-xaa-gly-nh2; sa-tyr-pro-xaa-gly-nh2; pro-leu-gly-nh(ch2)nnh-sa sa=sinapic acid; xaa=leu, unusual aminoacid; n=2,3 to obtain the sinapoyl-peptide-amides, both fmoc-and boc-based spps approach were used. analgesic activity was determined by the randall-sellitto paw-pressure test. the antioxidant effects were examined by dpph test as well. studies to establish the importance of introducing the sinapoyl moiety in the tyr-mif-1 molecule for the antioxidant and opioid activities are underway. several proteins are involved in the transcription of dna to mrna, among which the basic leucine zipper (bzip) proteins. these transcription factors bind specific dna sequences by dimerization and inserting short alpha-helices into the dna major groove. because the dimerization domain is only required to obtain the correct geometrical positioning of the alpha-helices, we will replace it by a dipodal steroid scaffold with defined stereochemistry. due to orthogonal protecting groups, a unique feature of this scaffold is the possibility to design not only homodimers, but also heterodimers. therefore this strategy allows for the construction of both major/major groove and major/minor groove binding peptides, either mimicking naturally occurring proteins or designing peptides with new binding properties. native chemical ligation and staudinger ligation are both suitable for the construction of these peptide dimers. moreover, a combination of solution-and solid-phase chemistry allows for the generation of combinatorial libraries. the increasing number of antibiotic-resistant bacteria is a global health problem. therefore the development of new highly efficient drugs is one of the major tasks of this century. as an example of peptides, which inhibit the growth of e. coli, we demonstrate an easy and rapid method for finding peptides with optimized antimicrobial properties. as a first step we built a modular construct. this construct consists of a constant cationic and a variable module. the cationic module was choosen to achieve cellpenetrating properties. the variable module was expected to act as the virtual active part of the peptide. to increase the proteolytic stability of the peptide we synthesized them in cyclic form. in the first step we used the combinatorial approach to screen approximately 64.000.000 peptide sequences in the variable region in order to find highly active peptides against e. coli. to optimize the identified sequence, we substituted all amino acids of the sequence with other amino acids and building blocks. additionally, in order to increase stability we modified the bridging. in this way we were able to uncover peptides with high antimicrobial activity as well as proteolytic stability and reasonable solubility. a series of melanocortin active core tetrapeptide hfrw nonpeptide imitations has been prepared using a combination of solution and solid phase synthesis. most of them included residue of 3-(1-imidazolyl) propylamine or histamine as substitutes of histidine. phenylalanine residue, which is included in melanocortins was replaced by residues of derivatives of 4, 4'-disubstituted isopropylidenedicyclohexane, 4, 4'-disubstituted bicyclohexane, 1,4-disubstituted cyclohexane, 1,5-disubstituted cyclooctane, and 1,2-, 1,3-or 1,4disubstituted benzenes. instead of arginine, residues of oligomethylene diamines, 2-butyl-2-ethyl-1,5-pentanediamine, 4,4'-methylene-bis(cyclohexylamine), and 4,4'-diaminodiphenylmethane were introduced. 2-naphtyloxyacetyl-, (4-1h-indol-3-yl)-butyryl-, 2-phenyl-ethanesulfonyl-and naphthalene-2-sulfonyl-groups served as replacement of tryptophan residue. tested on binding assay on melanocortin receptors, active core imitations exhibited a micromolar affinity to them. isopropylidenedicyclohexane and bicyclohexane derivatives showed about 10 fold higher affinity compared with corresponding derivatives of cyclohexane, cyclooctane or disubstituted benzene. obestatin is a novel endogenous ghrelin-associate peptide, which is involved in the regulation of food intake and weight gain. it was shown to be anorexigenic, able to decrease food intake, gastric emptying and jejunal motility. although obestatin and ghrelin originate from a common prepropeptide of 117 residues, they are reported to exert opposing physiological roles, by binding distinct receptors belonging to the subgroup of type a gpcrs [1] . obestatin was found to be the natural ligand of the orphan gpr39 receptor, a gpcr, expressed in jejunum, duodenum, stomach, pituitary, ileum, liver and hypothalamus. as many other peptides involved in the obesity process, it is a new and interesting drug target for the discovery of new anti-obesity molecules. in particular, the first step for the design of new molecules with potential improved anti-obesity activity, is the elucidation of the obestatin conformational features. here, we present the synthesis and the conformational analysis by nmr and cd spectroscopies of obestatin and its related 13-mer c-terminal sub-fragment, in aqueous solution and in membrane mimicking environment. the data outline the obestatin c-terminal portion as the region characterized by significant conformational features potentially opened to interesting future developments. [ a total of 50 isolates of rhizobium were collected from root nodules of medicago sativa and melilotus officialis plants in different regions of isfahan province .all of isolates on ty medium formed white ,slimy colonies with smooth margins and their inoculation on to roots of young alfalfa plants produced spindly nodules . the nodules developed with some of the isolates were big and pinkish ,although the rest of isolates produced small and white nodules .the speed of nodulation for all the isolates was almost similar and the related nodules were appeared within two weeks . the production of brown pigments on aged colonies of some isolates on ty or ty supplemented with l_tyrosine and copper sulfate revealed that these isolates of s. meliloti are melanin-producing rhizobia.based on the motility and sensitivity to antibiotics tests ,all of the isolates formed a reasonably homogenous group .however a few of them were able to produce an anti-microbial compound which was found to inhibit a number of isolates of s. meliloti .the compound did not suppress the growth of other bacteria . partial purification and spectrophotometery of the compound suggested that it likely belong to the antimicrobial polypeptides .considering on their physiological and biochemical properties ,none of the isolates were selected as a superior and competitive strain ,although based on nodulation efficiency , melanin and antimicrobial compounds production capability the isolate s. meliloti sm2 and sa23 were nominated to investigate in details. cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. this conserved structural architecture, termed the cyclic cystine knot, is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. cyclotides have a variety of biological activities but their insecticidal activities suggest that their primary function is in plant defense. in this study we determined the cyclotide content of the sweet violet viola odorata, a member of the violaceae family. we identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. the new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. as many of the biological activities of cyclotides appear to be associated with membrane interactions, we used hemolytic activity as a marker of bioactivity for a selection of the new cyclotides. the new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. the results show that while biological activity varies with the sequence the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. the structure of one of the new cyclotides, cycloviolacin o14, was determined and shown to contain the cyclic cystine knot motif. this study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens. furthermore, the inherent stability of the framework makes it an excellent scaffold for protein engineering applications. warfarin is the most widely prescribed anticoagulant drug for the prevention and treatment of arterial and venous thromboembolic disorders.because of large interpatient variability in the dose-anticoagulant effect relationship and a narrow therapeutic index careful dosage adjustment based on inr is essential. warfarin is available as a racemic mixture of two enantiomers,(s)-and (r)-warfarin. in contrast to (r)-warfarin, which is metabolized by multiple cytochrome p450s(cyps), including cyp1a2 and cyp3a4,(s)-warfarin, is predominantly metabolized to 7-hydroxywarfarin by polymorphic cyp2c9. since the potency of (s)-warfarin is much higher than that of (r)-warfarin, about 3-to 5-fold,any change in the activity of cyp2c9 gene is likely to have a significant influence on the anticoagulant response. previous in vitro findings revealed that certain variants in the cyp2c9 gene are associated with large interindividual differences in the pharmacokinetic and pharmacodynamic outcomes of warfarin therapy. three major alleles have been found to date in humans:arg144/ile359, and cys144/ile359 and arg144/leu359, and arg144/leu359, which have been designated cyp2c9*1 (wild-type), cyp2c9*2, and cyp2c9*3, respectively. we have investigated this polymorphism in iranians that has not been described previously. genomic dna was isolated from whole blood. for detection of cyp2c9*2, and cyp2c9*3 variants, a protocol based on pcr technique and endonuclease digestion with kpni, ava ii was used. in this research work, we have studied a group of 56 patients, in which warfarin therapy was initiated. recently new 21-residue antimicrobial peptides -arenicins were isolated from coelomocytes of marine polychaeta arenicola marina and their sequences were determined [1] . there are two isoforms of arenicins which differ only with single amino acid. these peptides have no structure similarity to any previously identified antimicrobial peptides. we have synthesized and estimated the antibacterial properties of arenicin-1: rwcvyayvrvrgvlvryrrcw. the linear peptide was prepared by solid phase method using boc-technology without any problem. however the cyclization caused the appreciable difficulties. the following methods of oxidation were used: oxygen of air, k3fe(cn)6 and hydrogen peroxide in aqueous or organic media. the best results were obtained by using hydrogen peroxide in methanol, but and in this case the yield of the aim peptide did not exceed 5%. synthetic arenicin had the same hplc profile and maldi-tof spectra as a natural molecule. the peptide showed an antimicrobial activity against gram-positive bacteria: peptidergic hormones and neurotransmitters are known to be produced by the specific cleavage of their precursor proteins that per se have no biological functions. the neutrophil-activating peptides we recently identified, however, are the peptides cleaved from mitochondrial proteins by proteolysis. therefore, we named them "functional cryptic peptides" because they are hidden in protein sequences. some of these peptides activate gi type of g proteins directly, and neutrophils are suggested to be stimulated by the direct (i.e., not via gpcrs) activation of g proteins. these peptides had features, in common, in their distributions of charged and hydrophobic amino acid residues, but homologies in their primary structures were not apparent. in the present study, we predicted functional cryptic peptides that activate g proteins, based on the distribution of charged and hydrophobic residues. receptors for these peptides were also investigated by the direct cross-linking experiments between peptides and their targeted proteins. the finding of functional cryptic peptides is expected to lead to the identification of novel signaling mechanisms where such peptides are involved in the regulation of bio-functions. the fragment 81-88 of the precursor of human interleukin-1alpha (pil-1α) (gk-vlkkrr) appeared to have more than 80% homology with corticotropin fragment 10-18 (gkpvgkkrr). we have previously synthesized the octapeptide gkvlkkrr (referred to as leucocorticotropin, lct) and found its high affinity binding to corticotropin receptors on various immunocompetent cells in human and mouse. in this study we investigated the interaction of lct with rat adrenal cortex membranes and the effects of lct on the level of 11-oxycorticosteroids (cs) in rat adrenal glands and plasma in vivo. lct was labeled with tritium by the high-temperature solid-state catalytic isotope exchange reaction to specific activity of 22 ci/mmol. receptor binding studies revealed that tritium-labeled lct bound with high affinity and specificity to corticotropin receptor on rat adrenal cortex membranes (kd = 2.2 nm). lct at concentrations of 0.1 -1000 nм was found to have no influence on the adenylate cyclase activity in adrenal cortex membranes, while intranasal injection of lct to rats at doses of 10 -50 microg/kg was found to inhibit the secretion of cs from the adrenals to the bloodstream. thus, lct is an antagonist of corticotropin receptor. comarin derivatives such as warfarin are prescribed widely for treatment and prevention of thrombosis. warfarin is the widespread oral anticoagulant drug employed, but its required dose is highly variable both inter-individually and inter-ethnically. so it is desirable to develop strategies to predict the warfarin dose response in patients before initiation of anticoagulation. the vitamin k-dependent γ-carboxilation system, consists of the vitamin k-dependent γ-carboxylase, which requires the reduced hydroquinone form of vitamin k1 as a cofactor and the warfarin sensitive enzyme vitamin k1 2,3-epoxide reductase (vkor), which produces the cofactor. warfarin exerts its anticoagulant effect by inhibiting the vitamin k epoxid reductase enzyme complex (vkor) that recycles vitamin k1 2, 3-epoxide to vitamin k1 hydroquinone. a component of the vkor termed vkorc1, has now been identified as a therapeutic target site of warfarin. point mutations were identified within the gene encoding vkorc1 in individuals who required large doses of wafarin to maintain therapeutic anticoagulation. however the relationship between the primary structure of vkorc1 and the mechanism of action of warfarin is poorly understood. in previous works we have shown that naturally occurring functional protein fragments affect cell proliferation [1] . their mechanisms of action involve receptors of "classical" regulatory peptides, or are non-receptoric [1] . among protein kinases involved are pka, camkii, mapk [2] . in organism bioactive functional protein fragments could participate in maintenance of tissue homeostasis. in present work, homeostatic potential of functional protein fragments was studied in compare with classical regulatory peptides. the panel of test substances was formed, including signal transduction modulators (pka, pkc, ca2+-channel activators), classical regulatory peptides (bradykinin, somatostatin, met-enkephalin, endothelin, neurotensin) and fragments of functional proteins (β-actin fragments from (75-90) and (69-77) segments; valorphin (β-globin (33-39)); neokyotorphin (α-globin (137-141); short acidic peptides from multiple precursors). their activity was tested in cultures derived from similar sources but differing in transformation degree (e.g., mouse embryonic vs. tumor fibroblasts) and/or culturing conditions. the factors most affecting cell sensitivity to the test substances were (in order of the importance decrease): (1) cell type; (2) transformed vs. normal phenotype; (3) cell density; (4) serum supply. activity of fragments of functional proteins, showing general correlation with other test substances, was more influenced by the culturing conditions (i.e., cell population status). thus, fragments of functional proteins could be regarded as partners of "classical" homeostasis regulators, playing role of finer tuners of tissue proliferative status. the study was supported by ras presidium programme "molecular and cell biology" histone-like proteins in bacteria are small basic proteins that contribute to the control of gene expression, recombination and dna replication. they are also an important factor in compressing the bacterial dna in the nucleoid. among the hlps, hu protein is attracted to dna containing structural aberrations such as four way junctions or single stranded lesions. this protein plays an important role in binding as a dimer and bending dna. it also contributes to the beginning of the dna replication. in this study we showed that a 10-kda protein, probably hu exists in halobacillus karajiensis which is a novel gram positive moderate halophile bacteria that was recently isolated from surface saline soil of the karaj region, iran. since hu is purified and characterized in e.coli we used this bacteria as the control in this study. the 10-kda protein extraction was carried out by using pca 5% which is normally used for extracting histones from eukaryotic cells. the results of running the protein extracts on sds-page demonstrated a band around 10-kda which was seen in protein extracts of this protocol. these results supported the hypothesis of the existence of a 10-kda protein in halobacillus karajiensis. sufficient oxygen and nutrient delivery is a necessity for tumors. when oxygen supply decreases, tumors initiate growth of new blood vessels. low grade astrocytomas, a class of malignant brain tumors, grow along the existing vessels in a process called co-option. hypoxia is induced in the progression from grade iii to grade iv astrocytomas (glioblastoma multiforme, gbm) which in turn triggers the formation of a new and distinct tumor vasculature. the new vessels formed by tumor-triggered angiogenesis differ by molecular composition from their normal vascular counterparts. we are utilizing phage displayed peptide libraries to identify peptides that specifically home to either co-opted or angiogenic brain tumor vessels. furthermore, we aim at characterizing differentially expressed endothelial markers (receptor molecules) to get a better understanding of the molecular changes in the vasculature. several rounds of in vivo biopanning was performed in mouse models of astrocytomas to isolate a phage pool that has up to a hundred-fold homing to low grade tumor lesions. out of the selected pool we discovered peptides capable of homing and accumulating to the tumor islets and co-opted vasculature. the homing potential of our newly identified peptides has shown to be highly specific for clusters formed by the tumor cells and co-opted "early" vessels within these palisades. these homing peptides represent promising candidates to selectively target co-opted vessels and tumor lesions in the brain and act as lead compounds in identification of surface molecules (receptors) differentially expressed by co-opted tumor vessels. the αvβ3 integrin receptors play an important role in human tumor metastasis and growth. the inhibition of these receptors by antibodies or by cyclic peptides containing the arg-gly-asp( rgd) sequence may be used as selectively treatment to suppress the disease. our research group has previously described that the formal introduction of a single carbon atom to bridge the cα (i) and n(i+1) contiguous residues of a linear or cyclic peptide leads to α-amino-β-lactam peptidomimetics containing predictably placed β-turn and γ-turn motifs, respectively. the combination of these results with the well-known capacity of rgd tripeptide for inhibition of the biological answer in integrin led us to the design of the following cyclic peptide. the adhesion and cell-growth "in vitro" assays using human umbilical vein endothelial cells (huvec), as well as "in vivo" assays with xenograph mice revealed that the rgd peptidomimetic was active to micromolar concentrations, slightly better than the reference compound in this field: cilengitide®. whole saliva is composed of secretions from parotid, submandibular and sublingual glands, and smaller ones from saliva of minor salivary glands (e.g. palatal and labial). saliva contains a variety of proteins and polypeptides. one of them is statherin, a multifunctional 43-amino acid residue, phosphominiprotein. this peptide is present in human parotid and submandibular saliva . the aim of our study was to investigate the stability of statherin in extracts of the major salivary glands. submandibular, sublingual and parotid gland tissues were obtained at autopsy 12 h after death. samples of the gland tissues were homogenized, centrifugated (30,000 g, 30 min., 4 c) and the supernatants were frozen and stored at -70 c prior to analysis. synthetic statherin was added to the supernatants before analysis (45 microgram/ml). the samples were analysed for the presence of the peptide by the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (maldi-tof ms}technique and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds page). statherin has been found to be decomposed in extracts of parotid and submandibular glands and also in the extract of sublingual glands. the dramatic increase in research for new anthrax therapeutic approach was prompted by potential use of the causative agent of anthrax bacillus anthracis as a biological weapon. anthrax toxin consists of three proteins, the protective antigen (pa) and the two enzymes lethal factor (lf) and edema factor (ef) that are carried through the membrane of the target cell upon binding to specific site on the membrane receptor-bound pa. lethal factor and edema factor were found to cooperate to promote immune evasion of the bacterium. here we describe the production of peptide inhibitors of pa-lf binding, obtained by selecting pa-binding peptide by a competitive panning of a phage peptide library, using recombinant lf. we selected several 12 mer peptides, which were synthesized in tetra-branched multiple antigen peptide (map) form, inducing resistance to proteolytic degradation (1) and maintaining biological activity of phage peptides. lead tetra-branched peptides were systematically modified by progressive shortening and residue randomization, to obtain an increase of peptide affinity and inhibitory efficiency. affinity maturation of lead sequences enabled selection of a peptide which has an ic50 at least one log lower that any other lethal-toxin-inhibiting peptide described so far and is effective for in vivo neutralization of anthrax toxin activity (2) . the same peptide can also efficiently inhibit the binding of ef to pa and ef-induced camp increase in different cell lines. microtubules are dynamic polymers that have important roles in eukaryotic cellular processes such as signal transduction, cell polarity, vesicular transport and chromosomal movement. the dynamic behavior of microtubules has been studied both in vivo and in vitro. the effect of arsenic trioxide on microtubule polymerization has been studied under in vivo experimentation shown that it inhibits formation of mitotic bundles. we studied the mechanism of arsenic trioxide effect on polymerization of microtubule protein purified from sheep brain in vitro. microtubule polymerization has been conducted by adding 1mm gtp to purified tubuline in pem buffer at 37oc for 30 minutes and simultaneously followed by measuring turbidity (350 nm). the results shown that lag time of polymerization (nucleation step) is affected by increasing concentrations of arsenic trioxide from 0-5 micromolar. moreover the rate of elongation step was decreased exponentially by increasing arsenic trioxide concentration. electron micrographs also showed microtubules length decrement due to arsenic trioxide. the results have shown the inhibitory effect of arsenic trioxide on microtubule polymerization via its effect on nucleation step as well as elongation rate. background and aims: alzheimer's disease (ad) is the major cause of dementia among the elderly. the increase in life expectancy worldwide demands new therapies for ad urgently. self-association of the amyloid ß-protein (aß) into neurotoxic assemblies, a seminal event in the etiology of ad, is considered to follow interactions of the c-terminus of the 42-residue form of aß (aß42). we hypothesized that molecules with high affinity for the c-terminus of aß42 will disrupt aß42 oligomerization. a series of c-terminal fragments (ctfs) of aß42, aß(x-42) with x = 28-39, has been prepared to study their potential to inhibit aß42 oligomerization and neurotoxicity. methods: attenuation of aß42 assembly by ctfs was studied by quantitative analysis of oligomer size distributions using a photo-cross-linking assay followed by sds-page. biological activity of ctfs themselves and as inhibitors of aß42-induced neurotoxicity was assessed by mtt reduction assay using differentiated pc-12 cells. the structure of the ctfs was studied by circular dichroism (cd) spectroscopy and ion mobility spectrometry-mass spectrometry (ims-ms) coupled with molecular dynamics (md) simulations. results: ctfs were found to inhibit aß42 oligomerization in a length dependent manner with minimal or no toxicity of the ctfs themselves. certain ctfs were found to inhibit aß42-induced neurotoxicity. cd spectra indicate that increasing peptide length results in growing ß-sheet content. structures based on experimentally determined cross-sections support the existence of a previously proposed turn around residues gly37-gly38. the data suggest that aß42 ctfs can serve as lead compounds for development of peptidomimetic drugs for treatment and prevention of ad. background and aims: human kallikrein hk2 is a prostate specific serine protease, which expression level is elevated in aggressive human prostate cancer suggesting a possible role in a tumour growth and spreading. since hk2 protease is highly prostate specific, inhibition of its activity is a possible method to prevent tumour growth without interfering the function of the other proteases. we have identified hk2 specific linear peptide inhibitor by using phage display techniques. in order to design peptide for in vivo studies we tested the protease stability of the linear and the cyclic forms of the peptide. methods: the prerequisite of the binding was studied by using conventional ala-replacement method and the most optimal sequence was selected for further studies. the stability of the original linear form, acetylated form, peptide with cystein bridge and head-to-tail cyclic peptide was tested with modified trypsin (sequencing grade) and with human plasma. results: both linear versions and peptide with cystein bridge were unstable and were degraded during the first 30 minutes in both stability tests. head-to-tail form of the peptide was stable in both tests during the first 180 minutes. conclusions: since our peptide contains arginine there was a possibility that our peptide is sensitive to trypsin and other serum proteases. indeed both linear and one cyclic from degraded in our tests. only head-to-tail peptide was stable during the first 3 hours suggesting protease resistant folding. background and aims: a large number of anticancer agents has been developed in recent years. however, these agents have very little or no specificity which leads to systemic toxicity. among them paclitaxel is considered to be one of the most important drugs in cancer chemotherapy; however, this agent has also lack of selectivity to the tumor tissue. therefore, development of tumor-targeting prodrug is highly promising. methods: to activate cytotoxic agent specifically at the tumor tissue, we developed a new prodrug strategy based on o-n intramolecular acyl migration, which is a well-known reaction in peptide chemistry, and photodynamic therapy. results: we synthesized a prodrug which has a coumarin derivative conjugated to the amino acid moiety of isotaxel (o-acyl isoform of paclitaxel). the prodrug was selectively activated by visible light irradiation (430 nm) leading to cleavage of coumarin. finally, paclitaxel was released by subsequent o-n intramolecular acyl migration. conclusion: we synthesized and evaluated a novel type of paclitaxel prodrug. this prodrug showed promising kinetic data. therefore, we believe that photoactivation can be promising novel strategy for design of tumor-targeting prodrugs. the search of new immunosupressants, exhibiting the mechanism of action characteristic for cyclosporine a (csa) and fk-506 is an important challenge for medicinal chemistry. cyclolinopeptide a (cla) natural cyclic nonapeptide [cyclo(leu-ile-ile-leu-val-pro-pro-phe-phe)] possesses a strong immunosuppressive activity comparable with that of csa in low doses. the possibility of practical application of cla as a therapeutic agent is limited due to its high hydrophobicity. it has been suggested that the tetrapeptide sequence pro(6)-pro(7)-phe(8)-phe(9) is responsible for the interaction of the cla molecule with the proper cellular receptor. in order to evaluate the role of this tetrapeptide unit for biological activity of native peptide, we decided to modified this fragment. in this communication we present linear and cyclic cla analogues in which phenylalanine residues in position 8 and/or 9 have been replaced with amphiphilic; alphahydroxmethylphenylalnine 1 or homophenylalanine 2. the synthetic strategy and biological activity will be evaluated. resistance to currently used small molecule antibiotics develops at an alarming rate. while resistance to β-lactams in clinical isolates is primarily due to hydrolysis of the ring by β-lactamases, when bacteria develop resistance to fluoroquinolones or aminoglycosides, the sequences of the target biopolymers are altered. earlier we developed a family of antibacterial peptide derivatives that kill bacteria by inhibiting protein folding and are active in animal models of infection. in the current study we examined the synergy between antibiotics acting by different modes of action. inhibition of properly folded active resistance enzymes was completely efficacious to recover the activity of amoxicillin, a β-lactam antibiotic against strains that were originally resistant to this molecule. some activity of ciprofloxacin was also recovered by reducing the load of the induced self-defense dnak protein, but the synergy between the antibacterial peptide and the fluoroquinolone did not yield full bacterial killing. the mode of action of the synergy is indeed inhibition of protein folding because no such effect could be observed with kanamycin where resistance involves changes in the target protein sequence. as opposed to current β-lactamase inhibitors and combination therapies that work against only a limited number of strains, inhibition of all protein folding in bacteria is a universally applicable treatment option. elimination of resistance to β-lactams by proline-rich peptide derivatives may represent a viable avenue to give second life to these antibiotics for which large stockpiles are available for pharmaceutical companies in both patented and generic forms. the integrin αiibβ3 is the major integrin-adhesion receptor on platelets. in unstimulated platelets αiibβ3 is present in a resting conformation state. upon platelet activation by agonists, αiibβ3 receives intracellular signals (inside-out signaling) that allow its rapid conversion to a high-affinity state capable of binding soluble ligands, resulting in platelet aggregation. the intracellular signals include proteins that bind to the cytoplasmic tails of the two subunits α and β of the integrin, or integrin-associated membrane proteins. in vivo charge swapping mutation studies suggested that αiib and β3 tails have a direct site of interaction between αiib (r995) and β3 (d723). peptides derived from the cytoplasmic tail sequences can specifically induce or block αiibβ3 activation in platelets. the aim of this study is to develop peptide analogues based on the cytoplasmic tail sequences of both αiib and β3 subunits that could inhibit platelet thrombus formation by specifically disrupting the inside-out signaling pathway. peptide analogues of the αiib and β3 subunits spanning the sequences αiib-989-1008, αiib -997-1003, αiib -997-1008, αiib-1000-1008 and β3-743-750, β3-743-756, β3-749-756 were synthesized in their free state, palmitoylated and/or tagged with the tat fragment 48-60 and carboxyfluorescein-labeled, in order to investigate their membrane permeability, as well as their inhibition of the platelet aggregation. inflammatory pain begins when noxious stimuli (thermal, chemical or mechanical) excite sensorial neurons called nociceptors. the activation of nociceptors leads to the opening of some ionic channels and depolarization of the cell membrane. one of these channels is trpv1, which is directly implied in thermal hyperalgesia associated to inflammation. in previous work it has been found that peptoid h-arg-15-15c ( fig. 1) inhibits the activation of trpv1 by blocking the pore entrance. however, this compound showed toxicity in vivo. the aim of our work is the design and synthesis of new compounds, based on the structure of h-arg-15-15c, with better therapeutic properties. we synthesized some new non-competitive antagonists of trpv1 that exhibit notable anti-inflammatory and analgesic activity in vivo. th. skarlas, e. panou-pomonis, d. krikorian, m. sakarellos-daitsiotis, c. sakarellos erf is a transcriptional repressor with tumor suppressor activity regulated by the ras/erk signaling pathway. it has been shown that erf interacts with, and is phosphorylated by erks in vitro and in vivo. this phosphorylation determines its subcellular localization and biological function. erf exhibits a high degree of specificity and sensitivity for erks. the major objective of our study is to provide proof of principal for a specific anti-cancer approach targeting the ras-pathway, which is commonly activated in human tumors, via the stimulus of the downstream effector erf. this will be attained by modeling specific peptide inhibitors that block the erf phosphorylation and inactivation by the ras/erk signaling pathway. we present the design and synthesis of peptide inhibitors incorporating the fsf and fkf motifs, known to play a critical role in the erf/erk interaction, in their free forms or conjugated to a carrier. ubiquitinium is a well known mechanism in protein degredation of eukaryotic cells ,in which many obsolte and corrupted three dimentional structure protein ,become marked by covalent attachment of ubuquitin through a multi-step enzymatic pathway.ubiquitin is a small ,8.5 kda peptide of 76 amino acid residues that targets such substrtes for proteolysis in proteasome .recnt studies showed that an extra cellular ubiquitination process also taking place in the epididymes of humans and other animals marks protein on the surface of the defective sperm .it appears that structurally and functionally defective sperm become surface ubiquitinated by epididymal epithelial cells. a certain portion of ubiquitin -labeled sperm is phagocytosed and the remaining is ejaculated .hence ubiquitin on the sperm surface could be a good marker of semen quality control in men. the aim of present study is to purify ubiquitin from packed blood cells , to produce and purify antiubiquitin antibodies,to design an immunofluorescence assy for detection of defective sperm, to compare the percentage of ubiquitinated sperm in oligoasthenotertozoospermia and normozoospermia and finally to determine correlations between sperm parameters and sperm ubiquitination. p. vakalopoulou, ch. anastasopoulos, g. stavropoulos, s. yiannis c-terminal analogues of substance p (sp) have been studied for their ability to prevent tumor growth or the proliferation of several cancer cell lines. the incorporation of damino acids into the sequence of sp and n-methylation of peptide bonds have shown to protect sp from the action of plasma and tissue peptidases. aiming to design and prepare more potential antagonist of cancer cells proliferation and taking into account that all the metabolites of the c-terminal hexapeptide analogue [arg6, d-trp7,9, mephe8]sp6-11 (antagonist g) possess the n-me group and d-trp residue, we proceeded to the synthesis of peptoid-peptide hybrids. they are oligomeric peptido-mimetics containing the residue [-ν(bzl)-ch2-co-]=(nphe). the incorporation of n-substituted glycine in peptide chains has been proved to improve their stability against proteases and give biologically active peptides. thus, the tetrapeptoid-peptide hybrids h-arg1-d-trp2-nphe3-d-trp4-oh and h-d-trp1-nphe2-d-trp3-leu4-oh, corresponding to metabolites of antagonist g and also the hexapeptoid-peptide hybrids glp1-d-trp2-νphe3-d-trp4-leu5-glu(obzl)6-νη2 and glp1-d-trp2-νphe3-d-trp4-leu5-glu(obzl)6-oh have been synthesized. the latter have incorporated the amino acid residues glp at the n-terminal and glu(obzl) at the c-terminal of the analogue, which have shown to give to the analogues increased resistance and biological activity. all the products were purified (hplc), identified (esi-ms) and set about for study their biological properties and activity against cancer cells proliferation. a chemokine receptor cxcr4 has multiple critical functions in normal and pathologic physiology. cxcr4 is a gpcr that transduces signals of its endogenous ligand, cxcl12 (stromal cell-derived factor-1, sdf-1). the cxcl12-cxcr4 axis plays an important role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems and so on. this axis has recently been proven to be involved in several problematic diseases, including hiv infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (ra) and pulmonary fibrosis. thus, cxcr4 is a great therapeutic target to overcome the above diseases. fourteen-mer peptides, t140 and its analogs, were previously found to be specific cxcr4 antagonists that were identified as hiv-entry inhibitors, anti-cancer-metastatic agents, anti-chronic lymphocytic/acute lymphoblastic leukemia agents and anti-ra agents. cyclic pentapeptides, such as fc131 [cyclo(d-tyr-arg-arg-l-3-(2-naphthyl)alanine-gly)], were previously found as cxcr4 antagonist leads based on pharmacophores of t140. in this symposium, we would like to report the development of low molecular weight cxcr4 antagonists involving fc131 analogs and other compounds with different scaffolds including leaner-type structures. erythropoietin (epo) controls the proliferation and differentiation of red blood cells. it activates epor by inducing dimerization and reorientation of two receptor chains. peptides mimicking the action of epo, epo mimetic peptides (emp), have been discovered by phage display, interacting with the receptor on the active site and competing with the hormone [1] . another peptide, epor derived peptide (erp), was reported to activate the receptor through an alternative site distant from the hormone binding site, and to have synergic action with epo [2] . we report the design of new synthetic epo-r agonists by dimerization of active peptides. pegbased polyamide linkers of precise length were used to link the molecules, using oxime chemistry [3] . these peptides include emps that have been homo-dimerized through their n-or c-terminus. a hetero-dimer of one emp and one erp peptides was also created. biological characterization of the molecules is currently under investigation. [ the envelope spike (s) glycoprotein of the severe acute respiratory syndrome associated coronavirus (sars-cov) mediates the entry of the virus into target cells. recent studies point out to a cell entry mechanism of this virus similar to other enveloped viruses, such as hiv-1. as it happens with other viruses peptidic fusion inhibitors, sars-cov s protein hr2 derived peptides are potential therapeutic drugs against the virus. it is believed that hr2 peptides block the six-helix bundle formation, a key structure in the viral fusion, by interacting with the hr1 region. it is a matter of discussion if the hiv-1 gp41 hr2 derived peptide t20 (enfuvirtide) could be a possible sars-cov inhibitor given the similarities between the two viruses. we used fluorescence spectroscopy techniques to test the possibility of interaction between both t20 (hiv-1 gp41 hr2 derived peptide) and t-1249 with s protein hr1 and hr2 derived peptides. our biophysical data show a significant interaction between a sars-cov hr1 derived peptide and t20. however the interaction is only moderate (kb = (1.1±0.3) × 105 m-1). this finding shows that the reasoning behind the hypothesis that t20, already approved for clinical application in aids treatment, could inhibit the fusion of sars-cov with target cells is correct but the effect may not be strong enough for application. [1] were used to investigate the structure, dynamics and thermodynamics of the known complex between erythropoietin mimetic peptides (emp) and erythropoietin receptor (epor). with gromacs 3.2 bioinformatics software, we have obtained from the known emps about the key functional amino acids required for effective epo mimetic action. then we systematically altered the amino acids in those peptides, and simulated the complex to observe the differences between the altered peptides with the original ones. based on these results, we designed new emps of potential significance. in order to fast identify the mimetic action of these new peptides, we synthesized these peptides and labeled the epor binding peptide (ebp) with quantum dots [2] , to study the binding of these new emps to epor. our results illustrate a principle for fast identifying receptor-specific sites importance for receptor internalization, and for enhancing sensitivity to hormones and other agonists. blood vessel formation largely contributes to the pathogenesis of numerous diseases, including ischemia and cancer [1] [2] . in this regard therapeutic strategies aim to stimulate vascular growth in ischemic tissues and suppress their formation in pathologies like in tumour and diabetic retinopathy. placental growth factor (plgf), an homolog of vascular endothelial growth factor (vegf), (42% amino acid sequence identity), stimulates angiogenesis and collateral growth in ischemic heart and limb. whereas vegf exerts it biological function through the binding to both vegf receptor-1 (vegfr-1or flt1) and vegfr-2 (or kdr) plgf binds specifically to flt1. the complex plgf/flt1 constitutes a potential candidate for therapeutic modulation of angiogenesis and inflammation [3] . the binding between plgf and flt-1 has multipunctual features [4] and potential antagonist must have a sufficient molecular surface to spatially distant contact points. we have used an elisa-like screening assay to select antagonists of plgf/flt-1 complex from a large random library of tetrameric unnatural peptides (complexity: 3^30=27.000 molecules) identifying two active molecules with an about 10 m ic50. the relative stability of identified peptides were assessed in human serum and their inhibitory properties were tested in a capillary-like tube formation assay performed with human umbilical vein endothelial cells (huvec the αvβ3 integrin is a cell adhesion receptor involved in angiogenesis and tumor cell invasion. the tripeptide motif rgd is the αvβ3 minimal recognition sequence and many rgd-containing peptides have been investigated as radiopharmaceuticals for targeting angiogenesis and tumor metastatic phenotype. since rgd sequence binds also to other integrins, the aim of the present study was to develop and characterize a selective αvβ3 ligand suitable for imaging. a novel peptide containing the rgd loop covalently linked to an echistatin domain (crgdechi) was designed, synthesized and then tested for selective binding to αvβ3 integrin. a panel of peptides were used for comparison. adhesion assays showed that the novel peptide was able to inhibit adhesion of αvβ3 overexpressing cells but not αiibβ3 and αvβ5 overexpressing clones. in conclusion the novel peptide showed a high affinity and specificity for αvβ3 integrin. the design of new molecules, based on the lead compound presented here, is currently ongoing with the aim at developing novel anticancer drugs and/or new class of diagnostic noninvasive tracers as suitable tools for αvβ3 -targeted therapy and imaging. background and aims: short peptides like leu-pro-phe-phe-asp (lpffd) and leu-pro-tyr-phe-asp-amide (lpyfda) can influence the structure and aggregation of ß-amyloid peptides. soto's pentapeptide lpffd has been published as a ß-sheet breaker (bsb). it is necessary to gain more information about the nature of the interaction of aß and the pentapeptides mentioned above for the understanding of their action and for the possible development of future therapeutic agents. methods: in this study radioligand binding assay, diffusion ordered nmr spectroscopy, dynamic light scattering, circular dichroism and ft-infrared spectroscopy was used. results: it was shown by radioligand binding assay and diffusion ordered nmr spectroscopy that both pentapeptides bind to aggregated aß. dynamic light scattering, circular dichroism and ft-infrared spectroscopy revealed, that after the treatment of the aß with the pentapeptides aß fibrils are still present. conclusion: both peptide can bind to aß and can cause small conformational changes of aß, however, they cannot prevent completely the formation of aß fibrils in 50-100 micromolar concentration using 1:1 molar ratio of aß and the bsb peptide. peptide arrays are convenient tools for the analysis of antibodies, protein binding domains and to address other biological questions. here we present a new method to produce identical copies of arrays on microscope slides. the peptides are synthesized on modified cellulose-discs, using a variation of the spot-method introduced by ronald frank more than 10 years ago [1] . the new array format overcomes several limitations of the spot-method, e.g. the low throughput with only one copy of the library and the large sample volumes that are needed for membrane incubations. for the presented arrays modified cellulose discs with covalently bound peptides are dissolved after synthesis. the resulting solutions can be spotted onto glass slides by conventional spotting techniques. three dimensional layers of cellulosepeptide molecules are formed on the surface of the supports used for spotting. a virtually unlimited number of identical arrays can be printed and assays are performed with a sample volume of 100 µl or less. as application example we show mapping experiments of the streptavidin recognition site with a peptide library containing histidine-proline motives. because of the much higher peptide loading compared to conventional arrays, the formed 3-dimensional structure might be superior for protein-interaction studies with even low binding constants. [ the interaction between the cap binding protein, eukaryotic initiation factor (eif) 4e, and the scaffolding protein eif4g is critical for the formation of the heterotrimeric eif4f translation initiation (ti) complex (eif4e/eif4g/eif4a). elevated levels of eif4e and eif4g found in several human solid tumor cancers and the induction of malignant transformation in animal models by overexpression of eif4e and the reversal of this phenotype by treatment with anti-sense rna, suggest the importance of the eif4e/eif4g interaction in the excessive translation of oncogenic proteins. eif4g binds to eif4e through a conserved eif4e-binding motif yx4l (non-specified (x) and hydrophobic ( ) amino acids) that interacts with an hydrophobic hot spot on eif4e. we report here the identification of a putative eif4e anionic exosite that is distinct from the hot spot and contributes to the binding of eif4g-derived ligands. our strategy focuses on in situ eif4e-templated click reaction-mediated assembly of hybrids comprised from an anchoring minimal eif4g-derived peptide fragment, which binds to the hot spot, and a series of complementing positively charged fragments targeting the anionic exosite. we synthesized a training set of [1, 2, 3] triazole-containing hybrid peptides that are potent inhibitors of eif4e/eif4g interaction. moreover, we achieved in situ eif4e-templated assembly of these hybrids from the corresponding fragments via click reaction in the absence of cu(i) catalysis. as such, we demonstrate a proof-of-concept for a new paradigm in the development of inhibitors of protein-protein interaction merging click reaction with fragment-based and in situ target-templated approaches. goodpasture disease is an autoimmune pathology caused by the accumulation of reactive autoantibodies against the alpha-3 of collagen iv. goodpasture antigenbinding protein (gpbp) is a ser/thr protein kinase that phosphorylates the alpha-3 chain and might be important in human autoimmune pathogenesis [1] . we are carrying out in our laboratories the biophysical and functional characterization of gpbp protein. in the presence of some proteins and at specific experimental conditions, gpbp participates in structurally ordered intra-and inter-protein aggregation processes. structure prediction programs identify four different domains for gpbp: an n-terminal domain showing pleckstrin homology (ph domain); a central domain with high tendency to form coiled-coils; a domain with ww features; and a c-terminal start domain ('star-related lipid-transfer'). using the tango algorithm [2] , we have identified several aminoacid sequences in the gpbp start domain of vertebrates with high tendency to participate in protein aggregation. in this work we present the synthesis and structural characterization of a collection of peptides derived from the sequences described above. we recently developed a combinatorial library screening protocol to identify hpq-containing cyclopeptides that bind streptavidin more tightly than its linear analogues. the relative affinities in ic50 of these structurally constrained ligands and its linear counterparts were measured by a captured enzyme-linked immunosorbent assay. however, their intrinsic binding kinetics remained to be elucidated. in this work, surface plasmon resonance (spr) was employed to directly determine the kinetics and thermodynamics of the ligands binding to a streptavidin chip. solid-phase peptide synthesis was carried out using standard fmoc chemistry. spr experiments were carried out using biacore3000 optical biosensor. streptavidin was immobilized onto a cm5 sensor chip using the standard amine coupling procedure. the equilibrium dissociation constants and kinetic on/off rates of n-to-side chain and n-to-c cyclopeptides were deduced by scatchard analysis and computational simulation, respectively. it was found that both cyclopeptides exhibited similar binding kinetics and bound streptavidin far more avidly than its linear form (1000-fold). in addition, the reversed (qph) linear and cyclic peptidyl ligands were hardly recognized by streptavidin. not only the binding specificity was distinguished qualitatively, but also the entropic advantage acquired by the pre-organized conformation over its linear analogues was demonstrated quantitatively by spr in this study. the mutation of tumour suppressor genes in the progression of cancer is well characterized. for example, p53 is found to be mutated in approximately 50% of cancers and the loss of this proteins activity has been shown to lead to the deregulation of cell growth and apoptosis. the potential of peptide aptamers to inhibit protein/protein interactions in a highly specific manner makes them very attractive as research reagents or as target validation tools in anti-cancer drug discovery. more interestingly, these molecules have the potentially to inhibit the activity of proteins which are key regulators of cancer cell growth and therefore could act as synthetic tumour suppressor proteins. we used peptides based on known protein/protein interactions, as well as peptides isolated using display technologies, for the design of protein aptamers that were used to analyze pathways critical in controlling cancer cell growth. a range of scaffolds were used to present these peptides in an effort to optimize the peptides activities. data relating to the activity of these peptide aptamers in vitro as well as in cellular systems will be discussed. the cyclic undecapeptide urotensin ii (u-ii) is the endogenous agonist for the u-ii receptor (ut), a gq coupled gpcr. current views suggest that binding by agonist, but not antagonist, leads to induction of stabilization of an active receptor conformation. we have previously probed the interactions of urotensin ii with rat ut (rut) using a series of photolabile u-ii analogues containing p-benzoyl-l-phenylalanine (bpa). it was found that the c-jun n-terminal kinases (jnks) are important mitogen-activated protein kinases. these ser/thr protein kinases are activated by various growth factors, cytokines, and cellular stresses. jnks have been shown to play a key role in phosphorylation of proteins in signal transduction of different diseases including cancer, neurodegenerative, cardiovascular, and inflammatory diseases. therefore, these enzymes are considered as important therapeutic target proteins. the interactions of jnk with peptides are of special interest for development of novel specific atp-noncompetitive inhibitors. interactions of this kinase and its mutants with various substrates were demonstrated in vivo using yeast ras-recruitment system. bioinformatical tools have been developed to predict optimized binding peptides as well as to correlate sequence position and amino acid with binding effiency to extract binding determinants. biomolecular interaction analysis have been performed for selected peptide sequences using surface plasmon resonance (spr) technology. real time measurements of the binding of peptides to the different isoforms jnk2 and jnk3 resulted in the determination of affinities as well as kinetic constants for association and dissociation. experimental results and their bioinformatic analysis are discussed with respect to critical features of potential atpnoncompetitive inhibitors. the b-domain of is one of the five nearly homologous domains of staphylococcal protein a. this domain contains three alpha-helices which are assembled in an anti parallel three-helix bundle. the b-domain binds the fc region of mammalian immunoglobulins through the n-terminal fragment that contains two alpha-helices. the c-terminal helix does not interact with fc but it is necessary for the correct folding and immunoglobulin recognition of the b-domain. to search for new peptide analogues of the c-terminal helix that bind the n-terminal fragment, a ¨one-bead one-compound¨ library of 300 peptides was designed based on the sequence of the c-terminal helix. active peptides were obtained after incubation of the library with the n-terminal fragment and rabbit immunoglobulin g labelled with fluorescein. new peptides were found and their sequences identified by maldi tof-tof mass spectrometry. the synthesis of the two most active peptides was carried out and the binding with the n-terminal fragment was confirmed by cd spectroscopy. the nterminal fragment peptide showed an increase in helicity when the c-terminal wild type peptide or some analogues were present in solution. the complete domains with the c-terminal fragment mutations were synthesized and structurally characterized by cd and nmr spectroscopy. the wild type and the new mutants adopt predominantly an alpha-helical structure. the interaction between rabbit immunoglobulin g and the wild type b-domain and the new analogues was investigated using surface plasmon resonance. although compared with the wild type, the mutants exhibited different kinetics, they were able to bind the immunoglobulin with high affinity. a. jaśkiewicz, e. bulak, h. miecznikowska, k. rolka sfti-1, a strong trypsin inhibitor, was isolated in 1999 from seeds of sunflower. it is homodetic 14-amino-acid-residue peptide containing a disulfide bridge. because of its small size and strong trypsin inhibitory activity, this inhibitor became an interesting model for studying enzyme -inhibitor interactions. sfti-1 possesses one reactive site located at the lys5-thr6 peptide bond and therefore is able to interact with the enzyme in a 1:1 stoichiometry. in this report we describe chemical synthesis and kinetic studies of a series of sfti-1 analogues containing double sequences of the wild inhibitor. their structures contain combinations of disulfide bridges and/or head-to-tail cyclization. each of these analogues contains two trypsin-specific reactive sites. we expect that kinetic studies should answer the question whether such dimeric analogues are able to interact simultaneously with more then one trypsin molecule and how this fact affects their inhibitory potency. in addition, we alsopresenttwo analogues in which we substituted the disulfide bridge with a carbonyl one. since carbonyl bridge has not been previously introduced into molecules proteinase inhibitors, we decided to check its impact on the activity and proteolytic stability of such modified analogues. ubiquitination, the covalent attachment of one or multiple polymerized ubiquitins is a post-translational modification of proteins, which has manifold functions. it mainly determines the protein for degradation, but also activation, deactivation or substrate alteration. due to its ubiquitinous distribution in all eucarionts no high-affinity antibodies could be originated. highaffinity ligand peptides are of interest to study ubiqutination. based on bioinformatical considerations and investigations of ubiquitin-interacting proteins short peptide sequences were selected. by using a peptide array specific ubiquitin binding was monitored and quantified with label free detection based on reflectometric interference spectroscopy (rifs). the results from rifs were confirmed by detection of binding in solution with fluorescence correlation spectroscopy (fcs) using carboxyfluorescein and s0387-labelled peptide amides. binding constants were determined by isothermal calorimetry (itc) and rifs. finally 1h,15n-nmr chemical shift analyses of the peptides with the highest affinity were carried out, which allowed the localisation of the interaction site of ubiquitin with the peptide the results from all four methods correlated very good. they showed fast equilibria within 30 s and binding constants down to the low micromolar range. nmr results revealed hints for discrimination possibilities between lys48 and lys63 polymerized ubiquitins. ( 101f is a potent neutralizing mab that binds the human respiratory syncytial virus (hrsv) f protein and is a promising candidate for clinical development. the majority of neutralizing antibodies to hrsv f protein map to two regions of the protein designated site ii and site iv,v,vi. to further characterize the 101f epitope, we employed a trypsin digestion of a hrsv f protein-101f mab complex, followed by mass spectrometry analysis of the resulting recovered mab bound peptide. one peptide at m/z 3330 was captured by the 101f mab. sequence assignment was based upon mass and matched with the database from a virtual digest. this peptide was assigned as residues 420-445 [tkctasnknrgiiktfsngcdyvsnk] of the hrsv f protein which spans antigenic site iv,v,vi. to further delineate the epitope, the binding of 101f mab to a series of peptides corresponding to antigenic site iv,v,vi in the hrsv f protein was determined. based on the peptide elisa data, the 101f-binding region could be reduced to 422-436 sequence [ctasnknrgiiktfs] . as demonstrated by the substitution analysis, r429 and k433 significantly contribute to epitope binding, but another positively charged residue, k427 makes a minor contribution to the binding. both, the peptide elisa and proteolytic digestion of the mab-antigen complex approaches identified the same region of hrsv f protein as being critical for the binding of 101f. furthermore, these data confirm the results obtained using complementing genetic approaches using a panel of mutations in recombinantly expressed f protein and selection of antibody escape virus mutants (data not presented). the recently identified uracil-dna specific nuclease (ude) is the first representative of a new family of nucleases. the protein sequence has no detectable homology to other proteins except a group of sequences present in genomes of other pupating insects (vertessy et al, submitted). to analyze the physiological function of this protein, peptide conjugates were prepared to serve as synthetic antigens for the generation of antibodies against isoforms of dutpase, an enzyme inherently involved in preventing the synthesis of uracil-dna [1, 2] . we used poly[lys(seri-dl-alam)] (sak) as a synthetic branched polypeptide [3] or bovine serum albumin (bsa) as a natural macromolecular carrier. peptides were prepared by solid phase method utilizing syro2000 (mulltisyntech gmbh, germany) peptide synthesizer, using fmoc chemistry and dipci/hobt-mediated coupling on rink-amid mbha resin. a c-terminal cys(acm) was added to the native sequences for incorporation of sh group into the peptide. in case of sak choroacetylated polypeptide was conjugated with sh-peptide to form thioether linkage. the maleimidobenzoyil-n-hydroxyszukcinimid (mbs) derivative of bsa was used to introduce the peptide into the macromolecule. antibodies have been developed as diagnostic tools and therapeutics for many different diseases. however, the isolation and preparation of intact specific antibodies is often very tedious or even unfeasible. recent studies have shown that single paratope peptides might be well capable to mimic corresponding antigen ligands [1, 2] , suggesting that paratope peptides from a native antibody might have many advantages, e.g. for molecular vaccine design and targeting. we have developed a new method for identification of paratope-containing peptides by proteolytic affinity-proteome analysis in combination with high resolution fticr-mass spectrometry (fticr-ms) [3] . in the present study we used hen eggwhite lysozyme (hel) and a polyclonal rabbit anti-lysozyme antibody (helpab) as a model system. the direct determination of paratope peptides was obtained by selective binding of a dtt-cleavage mixture of the anti-lysozyme antibody to immobilised hel, followed by proteolytic digestion of the antibody-antigen complex (paratope excision, parexprot). two specific paratope peptides were identified by maldi-fticr-ms, and the corresponding peptide sequences were identified by database search within a 1-2 ppm threshold. additionally, the identified paratope peptides were synthesised and characterised by affinity mass spectrometry, which ascertained their full binding specificity to lysozyme. the propeptide blocks the active site of inactive zymogen of cathepsin d and is cleaved off during maturation. we have designed a set of peptidic fragments derived from the propeptide structure and evaluated their inhibitory potency against mature cathepsin d using kinetic activity assay. the mapping localized two segments in the propeptide involved in the inhibitory interaction with the enzyme core: n-terminus of propeptide plays a major role and the active site anchor plays a minor role according to their respective ki values. in addition, a fragment derived from the mature n-terminus of cathepsin d displayed inhibition, which supports its proposed regulatory role. the mechanism of interaction of both propeptide segments was characterized by the mode of inhibition and by spatial modeling of propeptide in cathepsin d zymogen. using fluorescence polarization measurements, kd in nanomolar range was determined for the n-terminal propeptide segment. the inhibitory potency of the active site anchor segment was modulated by ala38val mutation that was reported to be associated with cathepsin d pathology. . by comparing the resulting low-energy conformations using different sets of atoms, specific conformational features common only to the high/medium affinity compounds were identified. they included the spatial arrangement of the three most important pharmacophoric side chains tyr2, arg4, and nal5 as well as the orientation of the xaa3-arg4 amide bond, which together represent a "minimalistic" 3d pharmacophore model for binding of the cyclopentapeptide antagonists to cxcr4. this model rationalizes the data for the cyclopentapeptides as well as for the peptidomimetic cxcr4 antagonist krh-1636. automated docking of the pharmacophore model to the 3d structure of the tm region of cxcr4 revealed that the pharmacophoric groups of the cyclopentapeptide ligands were involved in favorable interactions with their counterparts in cxcr4. for instance, the hydroxyl group of tyr2 formed a hydrogen bond with lys38, the guanidino group of arg4 formed a salt bridge with glu288, and the backbone carbonyl of xaa3-arg4 formed a hydrogen bond with lys282. this finding gives additional support for the suggested 3d pharmacophore model, and also provides opportunities for rational design of cxcr4 mutants to map potential contacts with peptide ligands. with the successful completion of the human genome project, the next challenge is to assimilate enormous amount of genetic information generated and to assign functions to a large number of proteins encoded. although the dna chip technology to detect the abundance of mrnas has been established, it is known that the abundance of mrnas and proteins does not correlate. thus, protein detection methods for reproducible and quantitative investigation of protein networks are strongly required. we attempted to establish a novel protein detection system based on a fluorescent measurement that does not require labeling of target molecules and preparation of secondary antibodies. we focused on a steric hindrance caused by the interaction between a target protein and a specific capture agent. when a target protein interacts with a specific capture agent immobilized on solid surface, we assumed that a steric hindrance in the vicinity of a capture agent increases. in order to detect the differences in the steric hindrance, we utilized a fluorescent system with the staudinger reaction. this reaction is a chemical ligation between a phosphine and an azide group. these two functionalities are unreactive with protein surfaces under biological conditions. we incorporated an azide group into an immobilized capture agent and investigated the efficiency in the staudinger reaction between the azide and an external triphenyl phosphine derivative. it was found that a target protein bound to the capture agent immobilized onto the solid support interferes with the efficiency in the staudinger reaction. the major histocompatibility complex (mhc) has a crucial role to initiate the immune response via the binding of the peptide fragments (epitopes) of foreign antigens and their presentation to the t-cell receptors (tcr). the co-receptor molecule cd4 enhances the binding between tcr and mhc ii. small molecules that mimic surfaces of mhc-ii may lead to blockage of the autoimmune response and the development of drugs for immunotherapy. hla-dqa1*0501/dqb1*0201 (dq2) and hla-dqa1*0501/dqb1*0301 (dq7) are highly correlated to autoimmune diseases as sjogren syndrome (ss) and systemic lupus erythematosus (sle). the non polymorphic β regions of the modelled hla-dq7, which are exposed to the solvent and may disrupt the interaction of dq7 with cd4+ t lymphocytes were determinated using the getarea program. it was found that the regions 133-140 (arg-asn-asp-gln-glu-glu-thr-thr) and 59-66 (glu-tyr-trp-asn-ser-gln-lys-glu) display the highest solvent accessibility. peptide analogs of these regions were synthesized, by the fmoc/otbu solid phase strategy, purified by rp-hplc and characterized by mass spectrometry esi-ms. the dimeric analogs of the peptides, designed to mimic the superdimeric nature of the immunosuppressory fragments of hla class ii molecules were also synthesized and investigated. conformational studies were performed with cd spectroscopy and biological experiments are in progress. background and aims: aggregates of β-amyloid peptide (aβ) play central role in the etiopathology of alzheimer's disease (ad). short peptides like c. soto's pentapeptide lpffd and lpyfd-amide synthesized in our laboratory are neuroprotective agents against aβ assemblies both in vitro and in vivo. however, the mechanism of their neuroprotective effect has not yet been fully understood. methods: transmission electron microscopy (tem), cd, ft-ir, diffusion ordered nmr spectroscopy, dynamic light scattering, and radioligand binding assays were used. results: all the methods applied showed that the pentapeptides mentioned above do not break the fibrillar structure of aβ, that is these molecules are not real β-sheet breakers (bsb). the pentapeptides bind to aβ fibrils and cause small structural changes by intercalating into the aβ assemblies. fibrils of aβ survive one week treatment with the pentapeptides using them in 2 to 5-time molar excess. conclusion: all the results in our laboratory show that the short peptides have long-term interaction on aβ-assemblies. in the first step they bind tightly to the aβ surface and prevent further interaction of aβ fibrils with the neuronal membranes. after this step the short peptides can be built into the structure of aβ-assemblies with intercalation causing a less ordered β-conformation. proteolytic enzymes (neprilisin, ide) could cleave and hydrolyze aβ peptides after this structural change, therefore the short peptides are good drug candidates for the treatment of ad. cellular processes in normal and pathogenic cell states are regulated by external stimuli via complex networks of catalytic and non-catalytic protein-protein interactions. we have developed methodology for the synthetic variation of peptides and peptidomimetics using polymer reagents including linker reagents enabling polymer-supported cacylations. [1] in combination with the virtual screening of protein subsites, we have demonstrated the application of the novel synthetic methods to inhibitor optimization for various proteases including plasmepsin ii, hiv protease, and sars coronavirus main protease. [2, 3] moreover, multivalent peptide polymers have been developed for the intracellular targeting of proteins. [4] this methodology was now extended to the inhibition of peptide-protein interactions by small molecules. for this purpose, we have composed a library of 20,000 small molecules by algorithmic searching of a database of bioactive molecules with virtually designed substructures (fragments). high throughput assays were developed on the basis of fluorescence and fluorescence polarization detection. despite the scepticism regarding the inhibition of protein-protein interactions with small molecules, efficient hit molecules have been developed for several intracellular targets and were subjected to synthetic variation and cellular follow-up assays. the essential event in platelet adhesion to the blood vessel wall after injury or in thrombosis is the binding to sub-endothelial collagen of plasma von willebrand factor (vwf), a protein which interacts transiently with platelet glycoprotein (gp) ibalpha , slowing circulating platelets to facilitate their firm adhesion through other collagen receptors, e.g. integrin alpha2beta1 and gpvi. to locate thevwf-binding site in collagen iii; we synthesized 57 overlapping triple-helical peptides which comprise the whole native sequence of collagen iii . peptide #23 (gpogpsgprgqogvmgfogpkgnd (o is hydroxyproline)) alone bound vwf, with an affinity comparable to that of native collagen iii. immobilized peptide #23 supported platelet adhesion under static and flow conditions, processes blocked by an antibody which prevents the vwf a3 domain from binding full-length collagen. truncated and alaninesubstituted triple-helical peptides derived from #23 either strongly interacted with both vwf and platelets, or lacked both vwf and platelet binding. thus, we identified the sequence rgqogvmgf as the minimal vwf-binding sequence in collagen iii. the present work completes our understanding of the collagen-vwf interaction, providing information on crucial sequences in collagen that perfectly complements our existing knowledge of the collagen-binding site in vwf and may assist in targeting the collagen-vwf interaction for therapeutic purposes solid phase assay systems such as enzyme-linked immunosorbent assay (elisa), surface plasmon resonance (spr), and overlay gels are used to study processes of protein-protein and protein-peptide interactions. the common principle of all these methods is that they monitor the binding between soluble and surfaceimmobilized molecules. following the use of bovine serum albumin (bsa)-peptide conjugates or isolated synthetic peptides and the above-mentioned solid phase assay systems, we were able to demonstrate that positively charged peptides, which would be expected to repulse each other, can interact with each other. both the elisa and spr methods showed that the binding process reached saturation with kd values ranging between 1 and 14 nm. no interaction was observed between bsa conjugates bearing positively charged peptides and conjugates bearing negatively charged peptides or with pure bsa molecules, strengthening the view that interaction occurs only between positively charged peptides. however, interactions between the same peptides were not observed in solution when was monitored by nuclear magnetic resonance (nmr) or by native gel electrophoresis. thus, it appears that for positively charged molecules to interact one of the binding partners must be immobilized to a surface, a process that may lead to the exposure of otherwise masked groups or atoms. the relevance of our findings for the use of solid phase assay systems to study interactions between biomolecules will be discussed. the hematopoietic progenitor kinase 1 (hpk1), a mammalian hematopoiesis-specific ste20 kinase, contains a cluster of four proline-rich sequences called p1, p2, p3 and p4 located after the kinase domain. these pro-rich regions play an important role in the interactions of this kinase with different adapter proteins. previous studies showed that p1, which contains the canonical pxxpxr motif, and p2 and p4 with the canonical pxxpxk motif interact with the c-terminal sh3 domain of hematopoietic lineage cell-specific protein 1 (hs1) even if with different affinity. hs1 protein shares a high amino acid sequence and structural similarity to cortactin although their functions differ considerably. here we report the results of our investigation on the interaction between the c-terminal sh3 domain of cortactin and the four proline-rich motifs of hpk1. these interactions were analyzed by non-immobilized ligand interaction assay by circular dichroism (nilia-cd). upon peptide addition, the binding was monitored by the cd changes of the trp side-chains of the conjugate gst-sh3cort. the dissociation constants kd were determined analyzing the cd data at 294 nm using a nonlinear regression method. the results demonstrate that gst-sh3cort displays an affinity binding higher than that found with the corresponding hs1 domain and that the four hpk1 pro-rich regions are not equivalent. p2 appears to bind with the highest affinity (kd=0.5 µm), followed by p1 (kd=10 µm) and p4 (kd=33 µm), whilst p3 does not interact at all. the generation of a fibrin clot is mediated by the regulated activation of a series of serine proteases and their cofactors. factor viii in its activated form, fviiia, acts as a cofactor to the serine protease fixa, in the conversion of the zymogen fx to the active enzyme fxa. both fviii and fix are essential for normal coagulation, deficiencies of either are associated with the bleeding diatheses hemophilia a and b, respectively. the role of fviiia is to bind factor ixa, generating the phospholipid-dependent intrinsic factor xase complex. at least two interactive sites have been identified for the enzyme-cofactor interaction. the ser558-gln565 region within the a2 subunit has been shown to be crucial for viiia-ixa interaction. in an attempt to study this interaction, we synthesized a series of peptides of 558-565 loop of the a2 subunit. the syntheses of these peptides were carried out by using spps and fmoc/but methodology. the synthesized compounds were purified by rp-hplc and lyophilized to give fluffy solid, identified by ft-ir, nmr and es-ms spectra. these compounds were tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation reagents to citrated platelet rich plasma (prp). the aggregation was determined using a dual channel electronic aggregometer by recording the light transmission. and 120 ci/mmol, respectively. both tritiated aβ peptides were used in cat brain( in vivo) experiments and it was found that the peptide aggregates enter the neurons within 30 min (electronmicroscopic autoradiography). this transport is most probably an endocytotic pathway. aβ aggregates could interact also with cytoplasmic proteins such as 3-phosphoglyceraldehyde dehydrogenase etc. we suppose that aβ assemblies can interact both with membrane receptors (nmda, ampa, ach) and with cytoplasmic proteins triggering neuronal dysfunction and death. background and aims: ww domains are the shortest known protein domains and contain a stable three-stranded b-sheet, which presents the binding site for prolinerich ligands. this interaction is mediated by hydrophobic interactions between aromatic and hydrophobic residues of the domain, and the polyproline core of the ligand. as part of our ongoing efforts aimed at synthetically mimicking conformationally defined protein binding sites (1), we have designed and synthesized linear and cyclic peptides covering the binding site of the ww domain of human yes-associated protein (hyap-ww), whose structure in complex with a proline-rich ligand had been solved by nmr spectroscopy (2) . methods: peptides were synthesized by spps, purified by hplc, and characterized by 2d-nmr spectroscopy, as well as by molecular dynamics calculations. affinities of the peptides to a hyap-ww ligand were determined in direct and competitive binding assays. results and conclusions: a cyclic peptide covering the sequence stretch of hyap-ww that contains its primary contact residues for proline-rich ligands, was found to compete with the domain for binding to a hyap-ww ligand. long-ranging noes identified in the nmr spectra of this peptide indicate a conformation, in which sequentially distant residues are brought into spatial proximity, likely through formation of a beta-sheet. these result demonstrate the feasibility of functional, as well as structural, mimicry of conformationally defined protein binding sites through synthetic peptides. the rockefeller university, new york, ny, usa integrins constitute a family of transmembrane cell surface receptors. they are involved in cell-cell and cell-extracellular matrix interactions. thus, they participate in many physiological and pathophysiological processes and are of crucial importance for the living organism. integrins possess two non-covalently bound subunits, &alpha; and &beta;, that jointly participate in ligand binding. these dimeric proteins show very high specificity in recognition of natural ligands. for example, α4β1 integrin recognizes vcam-1 (vascular cell adhesion molecule 1) and fibronectin through binding amino acid motifs tqidspln and ldv, respectively. on the other hand, fibronectin is a classical ligand for α5β1 integrin with the recognition motif rgd. as shown, identification of the integrin ligands occurs through small recognition amino acid sequences (mostly tripeptides). thus, small cyclic peptides possessing a recognition motif in the appropriate three-dimensional conformation are able to interfere with the integrin-ligand interactions and act as inhibitors. the aim of this investigation is the characterisation of small cyclic peptides containing the rgd motif and the determination of the selectivity and specificity of these inhibitors. two new pentapeptides with 3-amino-cyclopropane-1,2-dicarboxylic acid monomethyl ester ((+/-)acc) were synthesized and tested. peptides were characterized in biological assays with living cells (k562 and wm115) and in surface plasmon resonance binding studies. experiments have shown that cyclic peptide cyclo-(arg-gly-asp-(+)acc-val) is a very potent inhibitor (ic50-value in nm range) of interaction between vitronectin and αvβ3 or αvβ5 integrins. when preparing biotin-labelled peptides as ligands for avidin-based assays, it is chemically most expedient to locate the biotin label on the n-terminal group of the peptide. this is done without any regard to how this may affect peptide-target interactions, biotin-avidin binding, and the solubility properties of the resultant peptide. in many instances, the products are poorly soluble, have little biological activity, and poor affinity for avidin. problems can also arise during the synthesis of such nterminally biotinylated peptides due to the poor solubility and reactivity of many of the reagents used for biotin introduction. to overcome these limitations, we have developed an extremely simple method for synthesising peptides c-terminally with biotin. peptides can now be easily prepared by standard solid phase techniques either n-or c-terminally labelled, and screened to determine the optimum presentation for the biotin. in cases studies using protein-protein interaction and kinase assays, peptides c-terminally labelled with biotin gave better sensitivity. y. yang 1 , j. eble 2 , n. sewald 1 many bacterial pathogens bind and enter eukaryotic cells to establish infection. invasin is an outer membrane protein required for efficient uptake of yersinia into m cells. invasin mediates its entry into eukaryotic cells by binding to members of β1 integrin family that lack insertion domains (i domains), such as α3β1,α4β1,α5β1,α6β1, and αvβ1. this type of peptide-protein interaction is an ideal subject for the rational design of inhibitors. the integrin binding motif consists of one loop region with a conservative asp911 residue and two synergistic regions. the aim of this project is to synthesize cyclic peptides based on the invasin binding epitope sdms. this sequence has to be positioned in a β-turn with asp in i+1 position for optimal activity of the peptide. also the arg883 and asp811 residue, which are about 27.29å and 31.54å respectively away from the asp911 residue of the sdms loop in invasin, should be investigated. peptides that mimic these recognition sites have been synthesized and tested as ligands for the integrin peptide-dna cross-linking is a very powerful tool for studying peptide-dna complexes. it transforms non-covalent complexes into covalent complexes, which renders characterization of the adduct by classical techniques (mass spectroscopy, nmr,…) much easier. the aim of our research is to develop a new method for peptide-dna cross-linking involving the incorporation of a furan moiety. the strategy is inspired by the naturally occurring process of oxidative furan ring opening by cytochrome p450. the resulting cis-butene-1,4-dial has been shown to react with amino-and sulfhydryl groups of macromolecules such as proteins and dna. in our research, dna binding peptides are modified with a furan moiety and then chemically oxidized into a reactive enal. this enal can react with dna to form a covalent peptide-dna complex. previous attempts to selectively oxidize furan modified minor groove binding peptides consisting of n-methylpyrrole building blocks failed. we are now applying the same strategy on major groove binding peptides consisting of natural amino acids. currently the oxidation conditions are being optimized so that the furan moiety undergoes selective oxidation. these optimized conditions will be applied to known dna binding peptides, in order to obtain a peptide-dna cross-link. we coupled octanoyl or palmitoyl group to the n-terminus of an analogue of sv40 nuclear localization signal (nls) peptide, sv126-133(ser128) to investigate the effect of fatty acid chain length on the conformation of the lipopeptide-antisense oligodeoxynucleotide (odn) complexes and to establish the optimal peptide/odn molar ratio (rm) for the effective delivery of odn into the cells. the odns used in this study were targeted towards either the green fluorescent protein (gfp) mrna and the junction sequence between ews and fli1 genes. the conformational change of odn at different rm values was followed by circular dichroism (cd), attenuated total reflection-fourier transform infrared (atr-ftir) spectroscopy and atomic force microscopy (afm). the sv40 peptide-mediated odn transfer into nih/3t3 cells was studied by epifluorescence microscopy. the interaction between the hiv-1 regulatory protein rev and rev responsive element (rre) of hiv-1 mrna has emerged in the last decade as an important target in antiviral therapy. the rev-rre interaction is essential for the replication of hiv. the rev protein binds to the rre site located in the env coding region of the full length viral mrna and facilities the export of the rna from the nucleus, while protecting it from the cell's splicing machinery. in the published nmr structure of the rre/rev-derived peptide complex, an -helical segment of rev binding domain recognises a specific region of rre. an approach is described to design a new class of -hairpin peptidomimetic ligands for hiv-1 rev protein, which inhibit its binding to the rre rna. a model -hairpin peptide served as a scaffold to pre-organise side chains into a geometry similar to that seen in a helical peptide. a library of peptidomimetics was prepared by grafting sequences related to the rna recognition element in rev onto a hairpin-inducing d-pro-l-pro template. the electrophoretic mobility shift assay (emsa) revealed that all of the designed peptidomimetics bind to rre and the best examples show affinities (kd) in a nanomolar range. these new ligands show a novel approach to designing rev peptidomimetics, represent interesting leads for the development of more potent hiv rre/rev inhibitors and permit more detailed studies of the mechanism of binding to rna. a. napiorkowska, a. sawula, m. olkowicz, p. mucha, p. rekowski tat (trans-activator of transcription) is the protein which controls the early phase of hiv-1 replication cycle. it is a potent viral trans-activator containing from 86 to 101 amino acid residues which binds to tar rna. the fundamental role of tat is promoting effective elongation of viral mrna (vmrna). binding of tat to tar is mediated by a 9-amino-acid, highly basic arg49-lys-lys-arg52-arg-gln-arg-arg-arg57 sequence of the arm (arginine rich motif); the key role in these interactions is played by arg52. the goal of our research was to investigate the interaction of 27-nucleotide tar rna with synthesized tat peptide analogues using capillary electrophoresis (ce), a powerful analytical technique of biochemical studies. changes in electrophoretic mobility of the tar peak are employed for monitoring tar-tat complex formation. ce experiments were performed using lpa-coated capillary and a physical gel containing buffer. native arm fragment tat(49-57)nh2, its analogues ac-tat(49-57)nh2 and ac-[lys52]tat(49-57)nh2 and analogues substituted in position 52 with alanine-, homoalanine and lysine-derived amino acids containing nucleobases (adenine, guanine, cytosine, uracil, thymine) and nucleosides (adenosine, guanosine, uridine and cytidine) in the side chain were studied. specific interactions and complex formation were observed for both the native arm peptide fragment and selected tat analogues. the research is aimed at improving our understanding of the molecular mechanism of peptide-nucleic acid interaction, as well as evaluating the usefulness of selected nucleobase-containing amino acids as point probes for investigating peptide-rna interactions. interactions between proteins and dna are important to all living organisms. the goal is to investigate the molecular recognition between dna and the transcription factor phob of e. coli on the single molecule level and to identify amino acids required for dna binding. phob is composed of a transactivation domain (amino acids 1-127) and a dna binding domain (amino acids 123-229) that binds to specific dna sequences (pho boxes) containing a tgtca sequence. [1] chemical synthesis of peptide epitopes present in the dna binding domain of phob and isolation of the whole dna binding domain of phob was performed. the protein was purified using intein mediated protein purification. an additional cysteine residue was ligated to the protein using intein mediated ligation reactions and will be used for immobilization and labeling. in single molecule force spectroscopy (afm) experiments it has been shown that both a peptide with a native phob-sequence and the recombinant protein bind to dna. competition experiments were performed to prove specific dna binding. [2] mutated peptides and proteins where strategic amino acids were replaced by alanine have also been examined to reveal the contributions of single residues to molecular recognition. the binding contribution of the proteins is determined by surface plasmon resonance, electrophoretic mobility shift assays and fluorescence correlation spectroscopy. we investigated the biophysical characteristics and the pore formation dynamics of naturally occurring and synthetic peptides forming membrane-spanning channels by using isolated rod outer segments (os) of reptilia and amphibia recorded in the whole-cell configuration. once blocked the two os endogenous conductances (the cgmp channels by light and the retinal exchanger by removing one of the transported ion species from both sides of the membrane, i.e. k+, na+ or ca2+), the os membrane resistance (rm) could be >5 gώ. therefore, any exogenous current can be studied down to the single channel level. macroscopic currents of amplitude of ~300 pa were recorded in symmetric k+ or na+ (>100 mm) and ca2+ (1 mm) from the commercially available alamethicin mixture, the synthetic alamethicin f50/5 (a major component of the natural mixture), and selected analogues applied at 1 µm concentration at -20 mv. once applied and removed the peptide, the current activates and deactivates with a time constant of about 160 ms. the synthetic analogues [glu(ome)7,18,19] and [glu(ome)18,19] produce a current of about 100 pa at 1 µm concentration, and they show a strong activation by hyperpolarization as alamethicin f50/5 itself. clear single channel events were observed when the concentration of all of the alamethicin peptides is reduced to <250 nm.<br> these results indicate that the three gln residues at positions 7, 18 and 19 of alamethicin f50/5 are not a key factor for pore formation and its conduction properties. in general, the pore assembly and disassembly are very fast and cooperative events. the translocation mechanism of penetratin (rqikiwfqnrrmkwkk) is not clear, but the involvement of cell membrane was supposed. recent studies with phospholipid model membranes have shown that penetratin interacts only with negatively charged liposomes. we aimed to analyse the effect of penetratin on liposomes composed of different phospholipids (dppc/dppg 2:8-8:2) by fluorescence spectroscopy. in the first set of experiments, liposomes labelled with fluorescent markers (dph, ans and tma-dph) were incubated with penetratin and the fluorescence polarisation was determined as a function of the temperature. in the range of 15-200 mol/mol phospholipid/penetratin ratio, no change in the transition temperature was observed indicating that penetratin has no influence on the membrane structure. next, we have analysed the interactions between phospholipids and penetratin through changes in the intrinsic fluorescence of the peptide due to the presence of two w residues in its sequence. comparing the emission spectra corresponding to penetratin in aqueous media or in presence of vesicles one can clearly appreciate a blue shift. this indicates that that tryptophan residues are mainly exposed to a hydrophobic environment. analysis of the main band shows low values of polarization suggesting a free motion of the peptide chain. on the contrary polarization measured for penetratin mixed with liposomes results in higher values. this indicates that hydrophobic residues, like trp, are inserted into the bilayer and their motion is restricted. these data suggest the presence of interation sensed strongly by trp properties. cyclopeptide antibiotic gramicidin s (gs) has antimicrobial activity against gram-positive and gram-negative bacteria and some fungi. but non-specific action of gs and its high lytic potential limits therapeutic application of gs. we attempted to elucidate in which way gs molecule could be modified to lose its haemolytic side effects. gs molecule interacts directly with membrane phospholipids due to electrostatic and hydrophobic interaction. naturally, changes in the state of a lipid bilayer cause changes in the gs molecule binding to a bilayer. we studied the effect of gs on human blood platelets and the effect of platelet membrane state on the gs-induced disaggregation of cells with the help of turbidimetric and microscopy techniques. we modified the membrane state by temperature, osmotic stress, ionizing irradiation, lipid oxidation. depending on concentration gs causes platelet shape change and activation. when added to preliminary aggregated (in response to physiological agonists -thrombin, epinephrine, adp) platelets, gs causes crumble of cells aggregates. the rate and extent of platelet disaggregation under the effect of gs non monotonously depends on temperature (range of 5-40°c) and irradiation dose (up to 200 gy). parameters of the gs interaction with membranes are determined by the mobility of membrane lipids. factors modifying the lipid bilayer change the degree and the speed of the gs interaction with platelet membranes. results obtained permit to use gs for testing the state of membrane lipids and on the other hand allow to suppose ways of gs molecule modifications to achieve its tolerance to blood cells. g. bai 1 , p. gomes 1 , r. seixas 1 , m. hicks 2 , m. prieto 3 , m. bastos 1 eukaryotic antibiotic peptides (eaps) have been widely studied for the past years as an alternative to conventional antibiotics due to emergence of multi-resistant microbial strains, and significant efforts targeting increasingly potent and specific antimicrobial peptides are being made. one interesting approach in peptide antibiotics is based on hybrid sequences derived from natural eaps, with ca ( resistance to conventional antibiotics has stimulated a search for alternative therapeutics for microbial infections, a possible source that has gained much interest in recent years are antimicrobial peptides. antimicrobial peptides target the cell membrane directly, which is a key feature as evolution has shown bacteria have had difficulty in altering their membrane composition and organization to mount a suitable defence against these peptides. a common theory is that peptides that bind strongly exhibit high biological activity, but our real-time quantitative binding studies via surface plasmon resonance (spr) have shown that this correlation does not always hold. as more information on the molecular details of membrane disruption is required, we have used atomic force microscopy (afm) to visualise peptide insertion and changes in membrane morphology by a range of antimicrobial peptides in situ. interaction studies were performed with a series of phospholipid mixtures that mimic either mammalian cells (high in phosphatidylcholine and cholesterol) or microbial cells (high in phosphatidylethanolamine and phosphatidylglycerol). the present study may assist in the design of new specific antimicrobial peptides with high antimicrobial activity and low host toxicity. proportions of popc and popg as models. very high molar ratio partition constants ((18.9+-1.3)x10^3 and (43.5+-8.7)x10^3) were obtained for the bacterial models (popg:popc 4:1 and 2:1, respectively), these being about one order of magnitude greater than the partition constants obtained for the less anionic mammalian model systems ((3.7+-0.4)x10^3 for the 100% popc system). at low lipid:peptide ratios there were significant deviations from the usual hyperbolic-like partition behavior of peptide vesicle titration curves, especially in the case of the most anionic systems. membrane saturation was shown to be related to such observations and mathematical models were derived to further characterize the peptide-lipid interaction under these conditions. the calculated peptide-to-lipid saturation proportions, together with the determined partition constants, suggest that the minimal inhibitory concentrations of omiganan pentahydrochloride could represent the conditions required for bacterial membrane saturation to occur. the hemolytic pore-forming toxin sticholysin ii (stii) produced by the sea anemone stichodactyla heliantus belongs to the actinoporin protein family. the n-terminal domain of these proteins is required for interaction with membranes. to investigate the role of stii´s n-terminal domain in membrane binding and in the molecular mechanism of hemolysis, peptides corresponding to residues 1 to 35, or shorter fragments from this region, were synthesized. in some peptides leu was replaced by trp. all peptides exhibited hemolytic activity, albeit to a lesser extent than the whole protein. moreover, peptides lacking the 1-14 hydrophobic stretch were less active. the longer peptides were also able to permeabilize phospholipid vesicles. conformational studies were performed in aqueous solution and in membranemimicking environments. cd spectra showed that, while the shorter, more hydrophilic peptides, displayed a random conformation, the longer peptides underwent aggregation with increasing concentration, ph, and ionic strength. in the presence of trifluoroethanol and upon binding to detergent micelles and phospholipid bilayers, the peptides showed a propensity to acquire -helical conformation, as expected for the sequence comprising residues 14 to 26. fluorescence spectra demonstrated that the first residues of stii´s n-terminus penetrate more deeply into the bilayer, whereas residues 14-26 are located more superficially. this is in agreement with the predicted amphipathic nature of the helix formed by these residues and corroborates the existing hypotheses for the role of the n-terminal domain in the process of membrane insertion and pore formation. among a great number of antibacterial peptides a group of trp-rich peptides is of special interest. taking into consideration, that in most of proteins tryptophan is not frequently occurring amino acid, the biological meaning of a high content of tryptophan in structure of these antimicrobial peptides is particularly interesting. in the present study we carried out the investigation of antimicrobial and hemolytic activities of selected trp-rich peptides and their action on microbial membrane: ilpwkwpwwpwrr-nh2 indolicidin (i) pitwpwkwwkgg-nh2 3b3 (ii) plswffprtwgkr-nh2 gsp-1a (iii) fpvtwrwwkwwkg-nh2 puroindoline (iv) vrrfpwwwpflrr-nh2 tritrypticin (v). sunflower trypsin inhibitor sfti-1 is the smallest and the most potent known peptidic trypsin inhibitor from the bowman-birk class of proteins [1] . this head-to-tailcyclized 14-amino-acid peptide contains one disulfide bridge and a lysine residue (lys5) present in the p1 position, which is responsible for inhibitor specificity.as was reported by us and other groups, sfti-1 analogues with one cycle only retain trypsin inhibitory activity. very recently we have shown [2] that introduction of nsubstituted glycine residues mimicking lys and phe (denoted as nlys and nphe) in the p1 position of monocyclic sfti-1 with disulfide bridge yielded potent trypsin and chymotrypsin inhibitors, respectively. in this novel class of proteinase inhibitors contains completely proteolytic resistant p1-p1' reactive site. in the present communication we report chemical synthesis and determination of trypsin and chymotrypsin inhibitory activity of a series of ten sfti-1 analogues modified in the p1 position by these peptoid monomers (nlys and nphe). each of the synthesized peptomeric (peptide-peptoid hybrid polymer) sfti-1 analogues contains one of the following cycles: head-to-tail, disulfide bridge formed by cys, by pen and by cys/pen residues. the impact of the different cycles introduced into sfti-1 structure on proteinase inhibitory activity will be discussed. s-protein contains a proteolytic processing site and two interacting heptad repeat regions denoted as hr-n and hr-c. following processing of s-protein mediated by host cellular protease/s, the c-terminal s2-fragment fuse with host cell membranes via its hr-n and hr-c domains that form coiled coil 6-helix bundle (trimeric of dimers)-crucial for its receptor-mediated viral fusion. our objective in this work is to study the proteolytic site using model peptides and also to examine the interaction of hr-n and hr-c domains using fluorescence microscopy and other techniques. thus we synthesized an intramolecularly quenched fluorogenic peptide containing the proposed cleavage site [abz-eqdrntr761 evfatyx, abz=2-amino benzoic acid and tyx=3-nitro tyrosine] and showed by kinetic measurements that this cleavage is mediated most efficiently by furin, followed pc5 and pc7. other potential substrates were also tested and compared. above cleavage can be blocked by specific-pc-inhibitors in a dose-dependent manner. in addition using fluorescent-labeled peptides derived from hr-n and hr-c domains, circular dichroism spectra and surface assisted laser desorption mass spectral interest has grown to develop specific and potent inhibitors of this enzyme. our objectives in this study are to generate soluble recombinant human (h)ski-1 enzyme, design potent inhibitors and study its 3dmodel structure. we have successfully expressed hski-1 enzyme lacking its transmembrane domain in hek-293 cells and purified the enzyme via chromatography. in addition we developed ski-1 inhibitors by using pseudo-and multi-branch peptide approaches. in first approach we inserted dipeptide isosteres amino oxy acetic acid (aoaa) or 8-amino 3, 6 dioxa octanoic acid (adoa) at scissile p1-p1' position ((r175 l) of hski-1. a typical example is 167gryssrrl(adoa)aip179. other dipeptide isosters were also incorporated at the cleavage site of either ski-1 prodomain or lassa virus glycoprotein. in second approach we prepared 2 and 4-branch peptides containing hski-1128-137 segment. these peptides inhibit ski-1 in competitive manners with varying degrees ranging from low m to high nm ic50. circular dichroism spectra indicated strong interactions of inhibitors with ski-1 consistent with observed inhibition profile. a 3d-model structure of catalytic domain of ski-1 indicated a broad catalytic pocket cysteine proteases are of great importance in biochemical processes and these enzymes are used in biotechnology, food industry and agriculture. in this connection synthesis of high selectivity and high specificity substrates for cysteine proteases is of importance. enzymatic synthesis of peptides is a good tool for obtaining different biologically active peptides. immobilized serine proteases, subtilisin carlsberg and α-chymotrypsin immobilized on poly(vinyl alcohol) cryogel (pvacryogel), proved to be a convenient biocatalyst for such kind of syntheses. the high specific chromogenic substrate for cysteine proteases assay glp-phe-ala-pna was obtained with high product yields (up to 88% in 24 h) using subtilisin and chymotrypsin immobilized on pva-cryogel. the reaction was carried out according to the following scheme: glp -the residue of pyroglutamic acid, pna -p-nitroanilide. the influence of initial concentrations of components, the reaction mixture composition, the biocatalyst content and time on product yield was studied. it was shown that the optimal conditions are: dimethylformamide-acetonitrile mixture 20:80 (v/v), initial concentrations -85 mm, and enzyme-to-substrate ratio -1:3900. this approach was used in order to synthesize analogous substrates, containing different fluorogenic and chromogenic groups as well as other amino acids in p1position. the obtained substrates were tested for the papain assay. peptidyl-a-ketoaldehydes 3 represent attractive lead compounds and intermediates in the development of potent protease inhibitors due to their structural similarity with peptide aldehydes, previously known to be excellent inhibitors of serine-and cysteine protease. recently, we demonstrated the application of polymer cyano methylene-and carboxylato methylene phosphoranes in the assembly of a-hydroxy-b-amino esters (norstatines), a,b-diketoesters, and a,b-unsaturated ketones. [1, 2, 3] we now present a further development of our reagent linker 2 approach employing peptidyl-a-ketoaldehydes 3 and diamino propanoles 4. carboxylato methylen phosphoranes 1 derived from bromo acetic esters which are readly acylated without racemization, play the key role in our synthetic concept. herein we show the oxidative cleavage to peptidyl-a-ketoaldehydes 3 using dimethyldioxirane (dmd) in acetone as oxidant, after saponification and decarboxylation on the solid support. diamino propanoles 4 were furnished via the reductive amination of resin-bound peptides. over the past few years nuclear magnetic resonance has emerged as a powerful means for lead molecular identification and optimization.on the other hand, the 19f nmr has been used succesfully in several structural studies, protein folding studies and for the identification of active compounds, using a very similar methodology that the one used in the present work. the methodology required the labeling of the substrate with a cf3 moiety. the enzymatic reaction is performed with the cf3 substrate and quenched, using an enzyme inhibitor. 19f nmr is then used to monitor the evolution of both substrate and product. only two peaks are observed, the starting substrate and the cleaved substrate. this nmr method has some advantages: fluorine nmr is very sensitive, 0,83 times that of the proton. there are no spectral interference from protonated solvents, buffers or detergents typically present in the enzymatic reactions.the 19f isotropic chemical shift is extremely sensitive to small structural perturbations resulting in different chemical shift for the signals of the substrate and product. isotopic labeling of the protein is not required. as a model, caspase-8, which play a critical role in the initiation of apoptosis process5 and hiv-1 protease were chosen. two different kind of libraries were screened: one based on natural products from plant and animal extracts used in tradicional chinese medicine and a second one corresponding of a synthetic library with two sublibraries of 160 and 144 compounds.with this methodology it has been possible to identify some compounds with very promising inhibitory properties. background and aims: human kallikrein 2 (hk2), a prostate specific serine protease, regulates the activity of several factors that may participate in proteolytic cascades promoting tumor growth and metastasis. thus, inhibition of its enzymatic activity is a potential way of preventing growth and metastasis of prostate cancer. moreover, specific ligands for hk2 have potential use for targeting and in vivo imaging of prostate cancer and for development of novel assays. methods: to find peptide ligands we panned several phage display peptide libraries against active recombinant hk2 captured by a monoclonal antibody exposing the active site of the enzyme. alanine scanning and amino acid deletion analyses were performed to elucidate the motifs required for hk2 inhibition. results: from libraries expressing 10 and 11 amino acid long linear peptides we isolated six different hk2-binding peptides. three of these peptides are specific inhibitors of the enzymatic activity of hk2. amino acid substitution and deletion studies indicated that motifs of 6 amino acids are necessary for the inhibitory activity. conclusions: we have developed specific hk2 inhibitors by phage display technology. these novel hk2 specific peptides are potentially useful for treatment and targeting of prostate cancer. peptidylarginine deiminase iv (padiv) catalyzes the citrullination of arg residues in various peptides and proteins, such as histone, resulting in the production of citrullinated proteins in granulocytes [1, 2] . the citrullination mechanism of histone subunits and its functional effects in cells are not well known yet in detail. recently, it has been reported that the protein deimination/citrullination by pad iv plays a role in rheumatoid arthritis [3] . this implicates that the citrullination of histone may be related to rheumatoid arthritis. in order to further study the citrullination mechanism of histone, we explored the citrullination sites of histone h2a and h3 by pad iv using a series of synthetic peptides. recently, hagiwara et. al. reported that pad iv only citrullinates the arg3 of histone h2a as well as the arg3 in histone h4 [4, 5] . in order to investigate the citrullination mechanism, the n-terminal peptides of histone h2a and h3 were chemically synthesized and examined the citullination by pad iv. the n-terminal acetylation effect of the n-terminal synthetic peptide was also estimated on the citrullination by padiv. the velocity of each arg residues in the n-terminal peptides were estimated in vitro. the results indicated that padiv recognizes the specific arg residues in the synthetic peptide, and that the n-terminal acetylation of the histone peptides dramatically affects on the substrate recognition of padiv. in addition, the cd spectra of the n-terminal peptides were measured to elucidate the structural specificity for the recognition of pad iv. background and aims. prolyl oligopeptidase (pop) is a serine peptidase that cleaves oligopeptides after prolyl residues. it has been associated with cognitive disorders. pop inhibitors have been shown to enhance cognition in monkeys (1) and to improve performance in verbal memory tests in humans (2) . in the present study, the p2 l-prolyl residue of pop inhibitors was replaced by two l-proline mimetics, the 5-t-butyl-l-prolyl group and the (r)-cyclopent-2-enecarbonyl group. the effect of the mimetics on in vitro potency, lipophilicity and binding kinetics were studied. methods. the l-proline mimetics were synthesized according to the published procedures (3, 4) with minor modifications. the ic50 and ki values and the binding kinetics were determined for porcine pop. the log p values were determined with the shake-flask method. results. the replacement of the p2 l-prolyl residue by the l-proline mimetics gave compounds which were equipotent with their parent structures. both l-proline mimetics increased lipophilicity but the effect of the 5-t-butyl-l-prolyl group was more pronounced. while the 5-t-butyl-l-prolyl group increased the dissociation half-life of the enzymeinhibitor complex, the (r)-cyclopent-2-enecarbonyl group decreased it. conclusions. both l-proline mimetics perfectly mimicked l-proline at the p2 position of pop inhibitors. these mimetics can be used to modify the lipophilicity and the binding kinetics of pop inhibitors. the proteasome is an essential multicatalytic protease of the ubiquitin proteasome pathway. as a prime executor of regulated proteolysis, the proteasome controls almost all aspects of cell metabolism from signal transduction to cell cycle and differentiation. pharmacological intervention into proteasome activity leads to cell apoptosis. this observation was applied to successfully treat multiple myeloma, since the cancer cells exhibit substantially higher sensitivity to competitive inhibition of proteasome than normal cells. however, the complete shutting down of the proteasome catalyzed proteolysis leads to serious side effects resulting from the disruption of proteolytic homeostasis even in noncancerous cells. here, we show an alternative approach to control the proteasome activity using peptide based noncompetitive regulators. the cathelicidins derived peptides rich in proline and arginine (pr) residues have been found to affect activity of all the proteasome complexes both in vivo and in vitro, likely by binding to the face of the enzyme. mechanism and structural constrains of the pr peptides dictating their influence on the proteasome remain elusive. our results indicate that there are three sequence related properties of the pr peptides controlling their effectiveness as proteasome regulators: length of the peptide, distribution of a set of positive charges at the peptide n-terminus, and positioning of proline residues. far uv cd spectroscopy demonstrates that these properties also correlate with the structure of pr peptides. in particular, it seems that structural propensity of the pr peptides to form beta-turns are required to bind to proteasome as regulatory competent molecules. our work is focused on the search of selective, low-molecular cathepsin b peptide inhibitors acylated with the (e)-3-(benzylsulphonyl)acroyl group (bsa). the double bond, embedded in the bsa moiety is activated by two electron-withdrawing groups and may be a good target for the michael-type addition of the catalytically active -sh group. three series of peptide derivatives possessing general structures: bsa-phe-asn(r)-oh, bsa-ile-x(oh)-n(ch3)2 and bsa-x-pro-oh were synthesized in solution and characterized by enzyme kinetic studies against papain, cathepsins b and k. it should be noted that all the investigated compounds were competitive and reversible inhibitors of the enzymes examined. using 2d 1h nmr (tocsy, cosy, roesy) and 13c nmr spectroscopy along with theoretical calculations (analyse program) we determined the conformational properties of two most potent and selective cathepsin b inhibitors. this work was supported by grant ds/8350-5-0131-6. background and aims: we have developed peptides inhibiting human kallikrein-2 (hk2) activity. as hk2 is overexpressed in prostate cancer tissue, these peptides are potentially useful for treatment and diagnosis of prostate cancer. two of the potential physiological substrates for hk2 are proform of prostate specific antigen (propsa) and insulin-like growth factor-binding protein-3 (igfbp-3). both of these might participate in the regulation of prostate cancer growth: igfbp-3 by inhibiting igf-dependent tumor growth and psa by degrading extracellular matrix. we aimed to study whether our hk2-inhibiting peptides inhibit also hk2 activity towards natural protein substrates, i.e. activation of propsa and degradation of igfbp-3. methods: the effect of the peptides on the activation of propsa by hk2 was studied by preincubating the peptides with hk2, followed by addition of psa and specific psa substrate. igfbp-3 degradation was studied by two specific immunoassays, one detecting only native igfbp-3, while the other one also detected proteolytically cleaved forms of the protein. results: hk2-inhibiting peptides were found to inhibit propsa activation and igfbp-3 degradation by hk2 in a dose dependent fashion. conclusions: we have developed new peptides inhibiting hk2 activity towards natural substrates, like propsa and igfbp-3. the peptides might be useful for treatment of prostate cancer and other diseases associated with increased hk2 activity. from the seeds of garden four-o'clock and spinach we isolated two serine proteinase inhibitors (mjti i -mirabilis jalapa trypsin inhibitor and soti i -spinacia oleracea trypsin inhibitor), which are probably representatives of a new family of inhibitors. the purification procedures of these inhibitors included affinity chromatography on immobilized methylchymotrypsin in a presence of 5 m nacl, ion exchange chromatography and/or preparative electrophoresis and finally rp-hplc on c18 column. their primary structures (fig. 1 ) differ from those of known trypsin inhibitors, but showed significant similarity to one another, as well as to the antimicrobial peptides isolated from the seeds of mirabilis jalapa (mj-amp1, mj-amp2), mesembryanthemum crystallinum (amp1) and phytolacca americana (amp-2 and pafs-s) and from hemolymph of acrocinus longimanus (alo-1, 2 and 3). the equilibrium association constants (ka) of mjti i and soti i with bovine -trypsin were found to be about 107-109 m-1. mjti i and soti i have been synthesized using solid-phase method. the synthesized inhibitors and inhibitors isolated from plants have similar properties. the disulfide bridge pattern in both inhibitors was established after digestion with thermolysine, followed by the maldi-tof: cys1-cys-4, cys2-cys5 and cys3-cys6. s. cosgrove, l. rogers, c. hewage, j.p. malthouse aspartyl proteases are required for the multiplication of the aids virus and for producing the amyloid protein which causes alzheimer's disease. hiv protease inhibitors have been highly effective in treating aids patients and it is hoped that potent inhibitors of the beta secretases will also prove effective in treating alzheimer's disease. therefore inhibitors of the aspartyl proteases have great therapeutic potential. we have shown that the peptide glyoxals are potent inhibitors of the thiol protease papain and of the serine proteases subtilisin and chymotrypsin. using 13c-nmr we have been able to show that glyoxal inhibitors react reversibly with an active site nucleophile in these enzymes to form a tetrahedral adduct which is tightly bound by the enzyme. in the present work we synthesise 13c-enriched peptide glyoxals, we assess their inhibitor potency, and use 13c-nmr to examine how the inhibitors interact with the aspartyl protease pepsin. z-ala-ala-[2-13c]phe-glyoxal was synthesised from [1-13c]phenylalanine which was converted to its methyl ester. this was then coupled with z-ala-ala to give z-ala-ala-[2-13c]phe-ome which was hydrolysed to the free acid. this was converted to the diazoketone and transformed into z-ala-ala-[2-13c]phe-glyoxal using dimethyldioxirane. nmr spectra at 11.75 t were recorded with a bruker avance drx 500 standard-bore spectrometer. we show that peptide glyoxal inhibitors can be potent inhibitors of pepsin and that pepsin only binds one of the four glyoxal forms (one non-hydrated, one fully hydrated and two partially hydrated forms). alzheimer`s disease (ad) is the most common cause of dementia in older people. a major factor in the pathogenesis of ad is the cerebral deposition of amyloid fibrils, consisting of amyloid β peptides (aβ), as senile plaque. the 40-to 42 amino acid long aβ is generated by the proteolysis of β-amyloid precursor protein (app) by β-and γ-secretases. since bace1, a unique member of the pepsin family of aspartyl proteases initiates the pathogenic processing of app by cleaving at the n-terminus it is a molecular target for therapeutic intervention in ad proteolytic activity was found to occur, to a variable degree, in digestive organs of all studied organisms over the entire ph range. the common feature was the existence of two activity peaks, in the acid (ph 2.5 -3.5) and alkaline (ph 7.5 -8.5) zones, as well as a similar protease set containing e and d cathepsins, a trypsin-like enzyme, elastase, and collagenolytic proteases. proteolytic activity in the hepatopancreas of crab and sea star was found to be an order higher than in other study objects. high protease activity in crab hepatopancreas is an evolutionary mechanism compensating for a poor differentiation of digestive system, low substrate specificity of enzymes, and cold environment. trypsin activity in digestive organs of invertebrates suggests that a trypsin-like enzyme is a genetically old one, an evolutionary origin of all serine proteases. a difference of kind between vertebrates and invertebrates is that the latter have cathepsine activity (absent in vertebrates) and no pepsin activity. it is of interest to develop enzyme inhibitors containing a light activated switch that can be used to control binding and inactivation of an enzyme. several inhibitors containing the azobenzene photoswitch group have previously been developed and have shown changes in activity of around two times on photoswitching. this study aimed to improve this switching by more extensive derivatisation of azobenzene to closer resemble the peptide substrates of proteases. a series of peptidomimetics containing the azobenzene photoswitch group were synthesized and assayed against the protease alpha-chymotrypsin. these compounds contained azobenzene, linked to a known chymotrypsin inhibitory group (either a trifluoromethylketone or boronate ester), and otherwise designed to be peptide-like. in some cases both ends of the azobenzene moiety were derivatized in order to increase the impact of photoswitching on the shape of the compound and thus its enzyme binding strength. assays showed that most compounds were reversible inhibitors of chymotrypsin, with low micromolar inhibition constants (ki or ic50). up to four times increase in enzyme inhibition on light activated switching of the azobenzene group conformation was obtained. a number of peptidyl derivatives structurally based upon the inhibitory sites of cystatins has been synthesized. these compounds are prone to proteolytic degradation, are rapidly excreted and poorly bioavailable. the majority of this problems might be overcome by use of peptidomimetics with structures resembling those of previously synthesized peptidyl derivatives. among the peptidomimetics are azapeptides, in which alpha-ch group of amino-acid residue is replaced by a nitrogen atom. the azapeptides have recently been demonstrated as potent and selective inhibitors of cathepsins b and k. it was shown that azapeptide inhibitors bind along the active site cleft of cathepsin b in a bent conformation. this bent structure is likely to result from the mobility of the bonds in the vicinity of the inserted azaamino acid residue as well as from the interaction with enzyme. in our present work we have studied the peptide of a sequence: z-arg-leu-arg-gly-ile-val-ome, which is characterized by one major and three minor conformations. the replacement of alpha-ch group in the gly residue of peptide chain of z-arg-leu-arg-gly-ile-val-ome by the nitrogen atom likely results in rigid conformation. our aim was a comparison of structure of the parent peptide z-arg-leu-arg-gly-ile-val-ome and a selective cathepsin b inhibitor z-arg-leu-arg-agly-ile-val-ome by using 1h-nmr. severe acute respiratory syndrome corona virus associated main protease (sars cov mpro protease), alternatively known as chymotrypsin-like protease (3clpro), is a mediator of virus infection cyclus and from there a therapeutic target. a peptide aldehyde library targeting the sars corona virus main protease (sars-cov mpro, alternatively known as 3clpro) was designed on the basis of three different reported binding modes and supported by virtual screening. a set of 25 peptide aldehydes were prepared by a newly developed methodology and investigated in an inhibition assay against sars-cov mpro. [1] protected amino acid aldehydes furnished by the racemization-free oxidation of amino alcohols with dess-martin periodinane were immobilized on threonyl resins as oxazolidines. following boc-protection of the ring nitrogen yielding the n-protected oxazolidine linker, peptide synthesis was performed efficiently on this resin releasing deprotected products under mild hydrolysis conditions. the library was tested in a new fluorimetric enzyme assay for sars cov mpro. via immobilization of the fluorophor, 2-(7-amino-4-methyl-3-coumarinyl)-acetic acid, the substrate actsavlq-amca was prepared, surprisingly displaying a higher affinity than the native substrate. several potent inhibitors were found with ic50 values in the low micromolar range. interestingly, the most potent inhibitors seem to bind sars-cov mpro in a non-canonical binding mode. currently, the initial screen is extended towards the discovery of small molecule inhibitors of sars corona virus main protease. literature: a method of bromelain cleavage of surface glycoprotein hemagglutinin (ha) from the influenza a virions was initially employed for ha ectodomains crystallographic study [1] . the remaining spikeless subviral particles were used by us earlier for ha2 c-terminal fragment extraction and mass spectrometric (ms) investigation [2] . now sds-page analysis of the subviral particle preparations revealed several additional bands in a range of 9-23 kda together with major viral proteins comparing to intact virions (figure, m1matrix protein, f1-f5-m1 protein fragments, np-nucleoprotein). maldi-tof ms analysis of the in-gel trypsin hydrolyzates has shown that the additional bands are fragments of м1 protein. this was confirmed by n-terminal sequencing of the protein fragments electroblotted from the bands. concentration of sh-reducing reagent in bromelain digestion reaction influenced on the m1 fragment bands intensity. we conclude that due to membrane destabilization during ha spikes removing, m1 protein localized under viral membrane inside intact virions becomes accessible to limited proteolysis by bromelain. [ dipeptidyl peptidases (dpp's) sequentially release dipeptides from polypeptides. among those enzymes, dppiv, fapα, dpp8, dpp9 and dppii cause the release of n-terminal dipeptides containing proline or alanine at the penultimate position. they are all members of clan sc, a group of serine proteases that contains proline-specific peptidases. dipeptidyl-peptidase iv (dppiv) is the best studied member of this group of enzymes and has become a validated target for the treatment of type 2 diabetes over the last years. the development of inhibitors for the related enzymes (i.a. dppii) has only started recently. this poster presents selected products synthesised to further elaborate the structure-activity relationship for dpp ii inhibitors with a 2,4-diaminobutyrylpiperidine basic structure. this class of compounds was described earlier by our group as the hitherto most potent and selective inhibitors of dpp ii. starting from n4-p-chlorobenzyl-substituted uamc00039, our lead compound, two types of modifications were proposed: • the synthesis of n4-(di)alkyl-and arylalkyl analogues; • the synthesis of 3-methyl analogues. in our previous study, we reported potent and small-sized bace1 inhibitors containing phenylnorstatine [(2r,3s)-3-amino-2-hydroxy-4-phenylbutyric acid; pns] at p1 position as a transition-state mimic. in developing more active compounds, we focused our attempts on the p1 position, where we replaced the pns by its thioderivative. herein, we present the synthesis of a novel phenylthionorstatine [(2r,3r)-3-amino-2-hydroxy-4-(phenylthio)butyric acid; ptns] as a p1 moiety with hydroxymethylcarbonyl (hmc) isostere, and then an application to the bace1 inhibitors design. we have synthesized ptns starting from readily available n-benzyloxycarbonyl-serine and after multistep reaction (including weinreb amide formation, thiophenyl group introduction, through cyanohydrin derivative the transformation into the 2-hydroxy ester and then acid). purification was done by column chromatography and rp-hplc. peptides were synthesized by the fmoc based solid phase method and characterized by maldi-tof ms. the peptide inhibitors were adopted to enzyme assay using a recombinant human bace1 and a fluorescence-quenching substrate. bace1 inhibitory activity was determined based on the decrease% of the cleaved substrate by the enzyme.we have synthesized ptns and then the (2r,3r)-enantiomer was applied to spps (solid phase peptide synthesis). we synthesized octa-and pentapeptide-type inhibitors of bace1 containing pns or ptns at the p1 position. these compounds were enzymatically tested and showed high bace1 inhibitory activity. a novel derivative of pns, ptns, was synthesized, and evaluated in comparison to corresponding pns. the inhibitors with ptns exhibited a slightly higher inhibitory activity against bace1 comparing to those with pns. this study suggests possibilities of the application of ptns to design other aspartyl proteases inhibitors. the αν β3 integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis, mainly by interacting with matrix proteins through recognition of an arg-gly-asp (rgd) motif. inhibition of the αν β3 integrins with a cyclic rgd peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. the aim of this study was to investigate the effects of replacement of amino acids by aza-β3-amino acid analogs in cyclic rgdpeptides as αν β3 -integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. the selectivity profile of these antiadhesive cyclopeptide is rationalized by a special presentation of the pharmacophoric groups. thr rgd motif resides in position i to i+2 of a regular γ-turn. we synthetized linear and cyclic aza-β3 rgd-peptide with the purpose to examine the effect on the conformation and the activity. are aza-β3 amino acids γ-turn mimetics? the preferred conformations were determined by nmr. prostaglandins are involved in a large number of biological activities mediated by their g-protein coupled receptors (gpcrs). the prostaglandins pgf2 alpha receptors are found specifically in uterine muscle, where they initiate parturition and labor. the pgf2 alpha receptor plays a key role in preterm labor, for which medical and social costs are estimated at $ 9 billion per year in the usa (the highest per patient cost of any disorder). peptide mimics have been developed in our laboratory (1, 2) , that serve as allosteric antagonists of the pgf2 alpha receptor. the importance of the turn geometry of the central residue in these peptide mimics has been investigated using enantiomeric indolizidin-2-one beta-turn mimics which can respectively induce type ii and ii' geometry. our presentation will discuss the synthesis and biology of these novel allosteric modulators of prostaglandin pgf2 alpha receptor activity. it was shown that luteinising hormone -releasing hormone (lhrh) receptors are overexpressed in the most of adenocarcinoma cells in contrast to their low content in normal tissues. these data create the basis for lhrh analogues application in therapy of breast, ovary, prostate, lung, intestine, liver and kidney cancers. both agonists and antagonists utility for the targeting of cytotoxic moiety to the tumor cells is well documented. however, the number of lhrh analogues possessed their own cytotoxic activity is still very limited. we nicotianamine (na) that was first isolated from the leaves of nicotiana tabacum l [1] , is known as a key biosynthetic precursor of phytosiderophores. various studies have proved that nicotianamine plays a significant role in plants as an iron, nickel, zinc ... transporter [2] . the aim of our study was to synthesize unnatural analogues of na via peptide intermediates, to investigate the mechanisms of metal transport and accumulation within the plant. we found that the strategy developed for na synthesis could not be applied when the azetidine ring was changed for pyrrolidine ring and we investigated a new route to synthesize such analogue. these synthetic pathways will be discussed. the primary physiological roles of arginine vasopressin (avp), [cycle1-6 (h-cys1-tyr2-phe3-gln4-asn5-cys6-pro7-arg8-gly9-nh2)], involve vasoconstriction of vascular smooth muscles, via v1a receptor, and antidiuretic action in kidney (blood osmolality regulation) via v2 receptor. binding of avp to the v1a receptor subtype also stimulates glycogenolysis in the liver and promotes platelet aggregation. in addition, activation of the v1b (also known as v3) receptor causes adrenocorticotropic hormone release from the anterior pituitary. v1b receptors are also present in the brain where avp functions as a neurotransmitter. in the recent years by the salivary glands of several bloodsucking animals like, teaks, leeches, vampire bats and so forth are isolated plenty of proteins and peptides with different molecular weight and well established anticoagulant activity. many of the strongest anticoagulants isolated by bloodsucking animals are found in the extract of salivary glands of different kinds of leeches. such leech is the haementeria officinalis, from which is isolated the most active inhibitor of factor xa -ats. in order to study the role of some amino acids in the process of interaction among peptides mimetics and the active site of serine proteinases, some fragment analogues of ats's active site by replacement of some amino acids with the other with similar structure or with unnatural amino acids were synthesized. in the present work the synthesis and the anticoagulant activity according to the aptt and ic50 of the newly synthesized peptides and structure-activity relationship will be discussed. rational design of peptides is a challenge which would benefit from a better knowledge of their rules of sequence-structure-function relationships. peptide structures can be approached by spectroscopy and nmr techniques but data from these approaches too frequently diverge. structures can also be calculated in silico from primary sequence information using three algorithms: pepstr, robetta and peplook. the most recent algorithm, peplook introduces indexes for evaluating structural polymorphism and stability. the method uses a de novo search of energy minima by an iterative boltzmann-stochastic procedure and using a combination of 64 phi/psi couples derived from the structural alphabet for protein structures proposed by etchebest et al. for peptides with converging experimental data, calculated structures from peplook and, to a lesser extent from pepstr are close to nmr models. the peplook index for polymorphism is low and the index for stability points out possible binding sites. for peptides with divergent experimental data, calculated and nmr structures can be similar or, can be different. these differences are apparently due to polymorphism and to different conditions of structure assays and calculations. the peplook index for polymorphism maps the fragments encoding disorder and the mean force potential score indicates which residues will be most available for interactions with partners. this should provide new means for the rational design of peptides. several diseases like cancer metastasis, rheumatoid arthritis and chronic lymphocytic b-cell leukemia are linked to the interaction of the cxcr4 chemokine receptor to its natural ligand, the 68 amino acid protein stromal cell-derived factor-1α (sdf-1α). [1] one strategy for the treatment of these diseases could be to block the interaction between cxcr4 and sdf-1α with small cxcr4 antagonists. furthermore, radiolabeling of suitable compounds with appropriate radioisotopes could provide agents for imaging of cxcr4 expression in vivo via pet. previous studies by fujii et al. on cxcr4 antagonists led to a high affinity cyclic pentapeptide with the sequence cyclo[gly-d-tyr-arg-arg-nal]. [2] to further improve this structure, different approaches have been chosen with respect to metabolic stability, bioavailability, conformational rigidity and chemical versatility for radiolabeling. first, an n-methyl scan of the backbone amides was performed to influence conformational freedom and to increase metabolic stability and bioavailability. n-methylation of arginine residues yielded peptides with moderate affinity (ic<sub>50</sub>-values: 23nm (n-me)arg<sup>3</sup> and 31nm (n-me)arg<sup>4</sup>, resp.) whereas n-methylation of other amino acids significantly decreased the affinity (ic<sub>50</sub>>100nm). by substitution of arg<sup>3</sup> by ornithine, the affinity was mostly retained. [3] the amino group of orn can be alkylated or acylated via radiolabeled groups containing short lived isotopes. moreover, the bioavailability should be improved as the high basicity of the two guanidino groups could be reduced. first ornithine-acylated derivatives showed ic<sub>50</sub> values between 11-35nm enabling for the first time <sup>18</sup>f-radiolabeling of small cxcr4 antagonists for pet imaging in vivo. binding of ligands to integrins plays a major role in cell adhesion, migration, and signal transduction of cells. these interactions are important not only for normal cell functions, but also in pathogenic processes. the v 3 integrin for example is involved in tumor cell adhesion and osteoporosis. the association of ligands is specific and requires minimal recognition sequences. therefore, suppression of integrin activity using competitive inhibitors bears great pharmacological potential. the tri-peptide sequence rgd is a prominent recognition sequence of integrin ligands. two new cyclic pentapeptides were synthesized containing the tripeptide sequence rgd as well as 3-amino-cyclopropane-1,2-dicarboxylic acid monomethyl ester (acc) and valine varying only with respect to the stereochemistry of acc. both the (+) (all r) and (-) (all s) isomers of acc were incorporated. acc is a cyclic -amino acid as well as a cyclopropyl analogue of aspartic acid. biological tests with cell lines expressing mainly v 3 and v 5 integrin show a higher inhibitory activity of cyclo-(-arg-gly-asp-(+)acc-val-). in order to derive a structure-activity relationship of these two isomers, solution structures in dmso-d6 were investigated by nmr spectroscopy. subsequently, structural information was obtained by applying distance restraints derived from the nmr spectra in distance geometry/simulated annealing and molecular dynamics calculations. due to the rigidity of the cyclopropyl unit in acc, the structure of the cyclopeptide is significantly influenced by the integrated propane ring, thus explaining the different biological properties. integrins are an important family of cell adhesion molecules. currently, 24 members are known. among other functions, integrin α 4 β 1 is involved in inflammatory processes, leukocyte migration and tumor angiogenesis. the structure of its natural ligand vcam-1, including the binding loop sequence tqidspln, has been determined by x-ray crystallography. therefore, it is possible to apply the concept of spatial screening: using small cyclic peptides with structure inducing building blocks, the binding motif is presented in different well-defined structural arrangements. for this study, a series of cyclic penta-and hexapeptides based on the tqidspln sequence has been synthesized. β-homoamino acids, i.e. β 3 -amino acids with proteinogenic side-chains, have been incorporated as structure inducers for spatial screening. although β 3 -amino acids are supposed to prefer the central position of ψγ-turns, less data exist than for e.g. d-amino acids. apart from the structural characterization of potential high affinity ligands for integrin α 4 β 1 , a major goal of this work is to provide a better understanding of the influence of β 3 -amino acids on the structure of cyclic peptides. the structures of the peptide library have been investigated by nmr spectroscopy, followed by dg/sa and md calculations. the results substantiate the γ-turn inducing capability of β-homoamino acids, but also prove the formation of different turn structures in certain cases. a comparison to the x-ray structure of vcam-1 shows that the structure of the binding sequence has been successfully approximated by some of the peptides. biological activity tests should lead to meaningful structure-affinity relationships. neuropeptide y (npy) is a 36-amino acid peptide amide and binds to the so-called y receptors. its most dominant element is the c-terminal alpha-helix spanning amino acid residues 12-36. residues 1-10 form a polyproline helix with highly conserved proline residues at positions 2, 5 and 8, followed by a loop structure. the importance of the polyproline helix strongly varies between different receptor subtypes. it obviously plays no role in ligand binding at the y2 receptor subtype, whereas the n-terminal segment is of importance for ligand binding at y1 and y5. in order to further study the importance of the polyproline helix we introduced a conformationally constrained pyridone dipeptide mimetic at different single positions by solid phase peptide synthesis using fmoc/tbu strategy. the resulting peptides have been investigated in cell lines that selectively express y1 and y5 receptor, respectively. different methods including radioactive competitive binding assay, cd and nmr have been applied to investigate conformation and interaction of receptor and ligand. loss of affinity at the y1 receptor is independent of the position and about 10-, 20-and 30-fold, respectively, when introduced once, twice and thrice. introduction of the building block in position 8/9 leads to the most reduced affinity at the y5 receptor subtype but, surprisingly, affinity can partially be regained by introduction of the dipeptide at two additional positions. the position of the dipeptide is of greater importance at y5. these novel peptides clearly indicate the importance of proline residues and the structure of the n-terminus for ligand binding. interactions of src homology 2 (sh2) domains with phosphotyrosine (py) containing ligands is critical for regulating cellular processes. the cytosolic protein tyrosine phosphatase shp-1 contains two sh2 domains. an intramolecular interaction of the n-terminal sh2 domain with the catalytic (ptp) domain renders the enzyme inactive in the native state. binding of a py-ligand to shp-1 n-sh2 leads to a conformational shift and the dissociation of the sh2-ptp complex [1] . in previous studies we investigated the topographical and conformational preferences of the n-sh2 domain of shp-1 using conformationally restricted linear and cyclic peptides derived from the natural interaction partner ros py2267 [2] . we identified peptides that showed an increased binding affinity for the n-sh2 domain and partially inhibited ros-mediated shp-1 activity. on the basis of these results we hypothesized that an imperfect fit of the py+1 and py+3 side chains might be responsible for the inhibitory effect. in order to confirm this hypothesis we synthesized a new series of peptides and evaluated their biological activity. to better understand the role of each individual sh2 domain in the activation process we also determined the binding affinity against the c-sh2 domain and the activation profile of different shp-1 mutants. pull-down assays of the interactions of the py-ligands with full length shp-1 confirmed the results obtained for the binding to the individual sh2 domains. proteins are targets for photo-destruction due to absorption of incident light by endogenous chromophores. mass spectroscopic data presented evidence that structural modification observed upon irradiation of goat alpha-lactalbumin at 290 nm results from tryptophan (trp) mediated cleavage of disulfide bonds [1] . the aim of the recent studies is to define structural elements that direct the destructive influence of near-uv light on the disulfide bridges of proteins. most of the proteins of the immunoglobulin superfamily contain a so called triad, consisting of two s atoms, forming a disulfide bridge, and a single trp in their close vicinity [2] . we have indications that this arrangement gives rise to a photolytic degradation similar to that described in our earlier studies for goat alpha-lactalbumin [3] . we therefore investigated the influence of uv light on the single chain variable fragment (scfv) of a monoclonal antibody (82d6a3) [4] which contains two triads. the results showed that after irradiation of the wild type scfv (i) new bands (degradation products) appeared in electrophoresis experiments and (ii) the affinity for its antigen, von willebrand factor decreased. by site-directed mutagenesis, we modified the critical trp-residues to perform a parallel study on these mutants. background and aims: it is known that thrombomodulin has important function which prevents thrombus. we found kmylcvckn (m, n >= 2) peptides derived from thrombomodulin had strong anti-thrombus activity in our recent studies. these peptides formed two structures, parallel and anti-parallel, as dimers, we examined the relation between structure and activity. methods: two peptides of kkkylc(acm)vckkk and kkkkylcvc(acm)kkkk were synthesized by fmoc chemistry. dimer peptides were made by removing acm with iodine, after dissolving in 0.1 m tris hcl buffer (ph 8.0) and oxidizing the mixture of these synthesized peptides spontaneously. then three peptides shown in figure were separated using rp-hplc. the peptide concentration in normal human pooled plasma was 10 micro moles / l when measuring aptt (activated partial thromboplastin time). results: the anti-parallel formed peptide, peptide b, was prolonged aptt approximately 2.7 times, although two parallel formed peptide, peptide a and c, were not significantly different from the aptt of normal plasma. conclusions: these peptides have structure-activity relationship, we observed that the anti-parallel formed peptide had strong anti-thrombus activity. insect kinins share a highly conserved c-terminal pentapeptide sequence phe-xaa-xbb-trp-gly-nh2, where xaa can be tyr, his, ser or asn and xbb can be ala but is generally ser or pro. they are potent diuretic peptides that stimulate the secretion of primary urine by malpighian tubules, organs involved in the regulation of salt and water balance (1). the insect kinins preferentially form a cis-pro, type vi β-turn. insect kinin analogs containing tetrazole (1) and 4-aminopyroglutamate (2), both cis-peptide bond, type vi β-turn motifs, demonstrate significant activity in the in a cricket diuretic assay. in this study, we compare the diuretic activity of insect kinin analogs incorporating the four stereochemical variants of the 4-aminoglutamate (apy) motif. three of the insect kinin analogs incorporating the stereochemical variants, ( the need for new effective and to mammalian cells non-toxic antifungal agents increases in parallel with the expanding number of immunocompromised patients at risk for invasive fungal infections. in our laboratory we have produced a serie of low-molecular peptide derivatives of the general structure: x-arg-leu-nh-ch(ipr)-ch2-nh-y (where x and y were acyl groups with aromatic carbocyclic system). we have found and earlier reported that some of these display high antimicrobial activities against several clinically important gram-positive pathogenic bacteria. in this study we have by solution methods synthesized a group of low-molecular compounds and investigated their antifungal activity. the study included both candida and aspergillus species. we have found that some of the compounds were highly fungicidal. we also made a conformational study in which the residues were separately replaced by selected hydrophobic amino acids and their equivalents. the conformational study showed that the desirable stable intramolecular structure could only be formed in the presence of some vital components. this work was supported by grant ds/8350-5-0131-6. increased resistance of bacterial pathogens to currently employed antibiotics has resulted in efforts to develop antimicrobial compounds with new mechanisms of action. previously, we have synthesized some high potent antimicrobial compounds based upon the n-terminal binding fragment of human cystatin c. some derivatives of the general structure: x-arg-leu-nh-ch(ipr)-ch2-nh-y (1) (where x and y were acyl groups with aromatic carbocyclic system) have displayed the broad antibacterial spectrum and high activity against several clinically important gram-positive pathogens, including multi-resistant staphylococci. herein, the synthesis and structure -antibacterial properties relationship for two series of analogues of 1 are presented. the x and y groups in 1 were replaced by selected substituents with various geometry and distance between aromatic moieties and carbonyl. we have established the general structural features which the discussed class of peptide derivatives should possess in order to displaying the particular antimicrobial activity. this work was supported by grant ds/8350-5-0131-6. we have synthesized beta-endorphine-like decapeptide immunorphin sltclvkgfy which corresponds to the 364-373 sequence of the heavy chain of human igg. immunorphin was found to be a selective agonist of non-opioid (naloxone-insensitive) beta-endorphin receptor. the purpose of this study was to prepare [3h]immunorphin and characterize by its using the non-opioid beta-endorphin receptor on mouse peritoneal macrophages and membranes isolated from various rat organs. by use of tritium-labeled immunorphin ([3h]sltclvkgfy) with specific activity of 24 ci/mmol, non-opioid beta-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. since dehydroamino acids are quite reactive and various thiol nucleophiles are known to add to their double bonds [1, 2] , we hoped that these compounds might act as alkylating inhibitors of cathepsin c (dipeptidyl-peptidase i). its main function is protein degradation in lysosymes, but it is also found to participate in the activation of neuraminidase and proenzymes of serine proteinases (leukocyte elastase, cathepsin g, granzyme a) [3, 4] . it is well known, that phosphonodipeptides structural analogues of synthetic substrates of cathepsin c are the model substances in designing the new inhibitors of this enzyme. for that reason we have undertook the synthesis, theoretical and structural investigations of phosphonic analogues of dehydropeptides. gly-∆zphe-abupo(ome)2 gly-∆zphe-alapo(oet)2 gly-∆zphe-leupo(ome)2 gly-∆zphe-valpo(oet)2 gly-∆zphe-glypo(ome)2 gly-∆zphe-nbupo(oet)2 the structure and conformational preferences in this group of peptides had been investigated by mean of nmr techniques. in order to find the interactions between compounds-enzyme (cathepsin c) and interpret the results of biological test, the molecular modelling methods had been used. the interaction of v 3 integrin receptor with its ligands is selectively implicated in various processes, like angiogenesis, bone-formation, tumor genesis and tissue-genetic migration of embryonic cells. several cyclic rgd pentapeptides are known as selective ligands for v 3 integrin receptor. the aim of this study was to prepare a new conjugate, composed of the cyclo[rgdfc] derivative and a branched chain polycationic polypeptide, poly[lys(dl-alam)] (ak). the cyclopeptide was prepared on 2-cl-trityl chloride resin by fmoc/tbu strategy. the "head-to-tail" cyclisation was achieved in a diluted solution of dmf in the presence of bop and hobt coupling reagents and diea base. coupling of the cyclopeptide to ak polymer was carried out by thioether linkage. adhesion properties of soluble cyclic rgd peptides and their plate-immobilized forms were studied. free cyclopeptides evoke aggregation of cultured primary neural and cloned neural stem cells, while their plate-immobilized forms fail to support cell adhesion. on the contrary, in case the newly synthesized ak-c(rgdfc) conjugate such induction of cell aggregation was not observed. whereas immobilizing this derivative to either glass, or plastic was found to support cell-attachment in case of various cell types. in addition, all cell lines investigated -including also the primary neural cells -attached to ak-c(rgdfc) coated surface and survived, grew or differentiated even in the absence of serum. our data suggest that cyclic rgd -polypeptide conjugates represent a new tool to investigate selective cell adhesion and may provide a novel scaffold-material for directed cell-seeding. in the ph-induced channel closure in combination with the pip2 interactions. however, their detailed regulations are still remaining unclear. therefore, in the present study, we investigate these crucial residues with electrophysiological recordings and rationally designed mutagenesis based upon our structural analysis of kir1.1 tetramer. lys-80 is located fairly close to the intracellular channel gate and protrudes its long side chain positive charge into the pore. this may interfere with the potassium flow by providing repulsion charge while ph is lowered, which pushes the channel towards its closed state. mutation to met-80 therefore reduces such ph-sensitivity. on the other hand, arg-188 is supposed to be responsible for the maintenance of channel opening in the presence of pip2. loss of positive charge at this site may lead to the enhanced ph-sensitivity due to an abolished or reduced pip2 interaction. more interestingly, the double mutant for both sites reveals a compensation scenario. in combination with the discussion for the role of previously known r-k-r triad, our data provide very clear structural explanation for the exact functional roles of these basic residues in the regulation of ph-sensitive channel gating. mouse obese cart peptides are neurotransmitters involved in feeding, stress and endocrine regulation. leptin, a long-term adiposity signal, upregulates expression of cart in the hypothalamus. recent findings of co-localization of cart and cholecystokinin (cck)-a receptor (responsible for satiety effect of cck) in brain and gastrointestinal tract suggest a neurochemical link between cart peptides and cck. in normal fasted mice, cart(61-102) peptide decreased food intake after intracerebroventricular (icv) administration in a dose-dependent manner. anorectic effect of cart peptide was enhanced by peripherally administered cck-8, while cck-a receptor antagonist, devazepide blocked the effect of cart peptide on food intake. we used two mouse obesity models in this study: monosodium glutamate (msg) and diet-induced obese (dio) c57bl mice. both dio and msg mice had substantially increased fat to body mass ratio compared to their controls and were hyperleptinemic. msg mice were hypophagic and neither cart peptide nor cck-8 and devazepide had any effect on food intake of these mice. dio mice fed high-fat diet showed slightly decreased sensitivity to central administration of cart peptide, effect of cck-8 on food intake was preserved. in conclusion, cart peptide and cck-8 showed a synergistic effect on feeding in control mice that pointed to their probably integrated action in the central nervous system. analogously, devazepide suppressed cart anorectic effect. in msg obese mice, effects of both cart peptide and cck-8 on food intake were diminished due to disrupted signaling in hypothalamus. in dio mice, additive effects of cart and cck-8 were partly preserved inspite of hyperleptinemia and increased adiposity. b. chini 1 , s. stoev 2 , l.l. cheng 2 , m. manning 2 , were subsequently shown not be selective for the rat v1b receptor [2] . peptides a-d served as excellent leads to the design of selective agonists for the rat vp v1b receptor [3] . replacement of the arg8 residue in a-d by lys, orn, dap and dab, led to the first potent and selective agonists for the rat v1b receptor [3] . we now report that three of these; d the aim of the de novo peptide synthesis and the incorporation of cofactors is the construction of artificial protein models. these model systems can be used for understanding the structure-function relationship of native proteins and might open a way for possible applications. protegrin-1 (pg-1) is an 18-amino acid peptide with an amidated c-terminus, which forms an antiparallel beta-sheet, constrained by two disulfide bridges. the native sequence of pg-1 is highly cationic, containing six positively charged arginine residues. it was found that the structural features such as amphiphilicity, charge and shape are important for the cytolitic activity of pg-1. in this study we investigate the sar (structure activity relationship) of two pg-1 analogues: rglcycrgrfcvcvg-nh2 (bm-1) and rglcyrprfvcvg-nh2 (bm-2). our antimicrobial activity studies of these peptides show that the bm-1 peptide is active against microbe species as well as the native pg-1, whereas the bm-2 is completely inactive. the bm-1 analoque is shorter than native pg-1 and contains only three arginine residues, therefore is much cheaper in the chemical synthesis, what could be an advantage of this antimicrobial peptide. the conformational studies of both analogues were performed by using 2d 1h-nmr technique (in dmso-d6) and molecular dynamics studies. the 3d solution structure of both analogues was established using interproton distances and torsion angles. for simulated annealing calculations the xplor program was used. our conformational studies show that the bm-1 forms a regular beta-hairpin structure, which is very similar to that of the native pg-1 peptide, whereas the bm-2 analogue is very flexible, what could be a reason of the antimicrobial inactivity. copper amine oxidases (ec 1.4.3.6) catalyze the oxidative deamination of primary amines to the corresponding aldehydes, ammonia and hydrogen peroxidase. these enzymes are ubiquitous, occurring in micro-organisms, plant and animals. activity of this enzyme increases under various stress conditions including thermal and water stresses. although lsao is not a thermostable enzyme, it is in maximum stability and activity above physiological temperatures. in this study we have investigated the kinetics of thermal denaturation of lentil seedling amine oxidase (lsao) by measuring its denaturation constant (kden) at various temperatures from 37 to 67 degrees centigrade in 100 mm phosphate buffer, ph 7.0. the results of thermal inactivation curves as well as measuring of a280 at various temperatures were used to calculate kden. moreover, activation energy (ea) for denaturation reaction was obtained from corresponding arrhenius plot. our results showed that unfolding process started to occur at 56 degree centigrade and ea of denaturation was changed at 65 degree centigrade proving a dominant conformational change of the enzyme at this temperature. the results of the kinetic study are coincident with previously reported equilibrium studies denoting the optimum and melting temperature of the enzyme are 56 and 65 degree centigrade, respectively. development and advancing of enzymatic processes used for production and modification of natural polysaccharides are now major biochemistry challenges. the paper investigates enzymatic systems in invertebrates, in particular, an enzymatic complex obtained from the hepatopancreas of red king crab paralithodes camtschaticus, and clarifies its effect on the mechanism of chitin and chitosan hydrolysis. chitinolytic activity was estimated with spectrophotometer using 4-(dimemylamine)-behzaldehyde method by the concentration of n-acetyl-d-glucosamine which is educed under chitinolysis. total glycolytic activity was defined by the sum of n-acetyl-d-glucosamine and d(+)-glucosamine in the reaction with potassium hexaferricyanide (iii). content of d(+)glucosamine in the hydrolysates of chitin and chitosan was estimated by highly effective reverse-phase liquid chromatography (helc) of aminosaccharides with ortho-phthalaldehyde. the paper studies the process of chitin and chitosan glycolysis and the effects of different factors (ph, temperature and time of incubation, enzyme/substrate ratio) on the total glycolytic activity of the enzymatic complex from crab hepatopancreas, which is compared with a previously studied proteolytic and exochitinase activities. a mechanism of enzymatic hydrolysis of chitin and chitosan is suggested. study results allowed the following conclusions concerning glycolytic and deacetylase activity of ep: 1) ep induces the formation of a monomer (n-acetyl-d-glucosamine) and oligomers (chitin and chitosan) with low deacetylation. thus, ep is characterised by a marked endochitinase (endochitosanase) activity; 2) n-acetylglucosamine deacetylase and, apparently, exochitosanase activity was not revealed; 3) it was found that chitinase and protease activities of ep are associated with different enzymes. [background] in opioids, the n-terminal amino acid 2',6'-dimethyl-l-tyrosine (dmt) enhances bioactivity by orders of magnitude. c-terminal modification of the dmt by a methyl group, h-dmt-nh-ch3, exhibited µ-opioid receptor affinity (kiµ = 7.5 nm) equivalent to that of morphine; however, antinociception was only 0.64-0.85% [1] . dmt plays an important role in the message domain to anchor opioid ligands into the active site of opioid receptors, specifically to trigger biological activity by the µ-opioid receptor. [methods] dimerization of dmt through diaminoalkanes [2] or 3,6-bis-(aminoalkyl)-2(1h)-pyrazinone produced potent opioidmimetics with high affinity for µ-opioid receptors (kiµ = 0.02-0.115 nm), agonism (gpi, ic50 = 1.3-1.9 nm), and antinociception in mice after systemic and oral administration, which verified passage through the epithelial membranes of the gastrointestinal tract and blood-brain barrier [3] . (1-aminocycloalkane-1-carboxylic acid) . in this case the ring consists of five atoms. knowing that acylation of the nterminus of several known b2 blockers with a variety of bulky groups has consistently improved their antagonistic potency in the rat blood pressure assay, the apc substituted analogues were also synthesized in n-acylated form (with 1-adamantaneacetic acid (aaa)). the activity of eight new analogues was assayed in isolated rat uterus using a modified holton method in munsick solution and in rat blood pressure tests. the results clearly demonstrated the importance of the position in the peptide chain into which the sterically restricted apc residue was inserted. apc at positions 7 led to preservation or reduction of antagonistic qualities, respectively. acc at position 8 enhanced antagonistic qualities in blood pressure test and led to preservation of activity in antiuterotonic test.. in most cases acylation of the n-terminus led to enhancement of antagonistic potencies. our findings offer new possibilities for designing new potent and selective b2 blockers. background: during the course of developing opioidmimetic analgesics, data revealed that the n-terminal residue 2',6'-dimethyl-l-tyrosine (dmt) plays an important role in anchoring opioid ligands in the active site of opioid receptors. as a single residue c-terminally extended with an aminomethyl group exhibited µ-opioid receptor affinity (kiµ = 7.5 nm) similar to morphine; however, antinociception was only 0.64-0.85% [1] . in order to develop potent µ-opioid agonists, the dimerization of tyr or dmt through diaminoalkanes [2] or 3,6-bis-(aminoalkyl)-2(1h)-pyrazinones [3] resulted in production of unique opioidmimetics with high receptor affinities and potent biological activities [3] . methods: the synthesis of opioids and opioidmimetics and the determination of their receptor binding characteristics were performed as described previously [1] [2] [3] . results and conclusion: newly synthesized 3-(tyr-nh-butyl)-6-(tyr-nh-propyl)-2(1h) pyrazinone and 3-(tyr-nh-propyl)-6-(tyr-nh-butyl)-2(1h) pyrazinones (i and ii) exhibited fairly high binding affinity towards µ-opioid receptor (kiµ = 7.6 and 27.4 nm, respectively). replacement of tyr with dmt in i and ii gave opioidmimetics iii and iv (kiµ = 0.021 and 0.051 nm, respectively); they exhibited 361-and 537-fold higher binding affinity than the tyr derivatives. while iii is a dual µ-/δ-opioid agonist, iv is only a µ-opioid agonist. these findings pave the way to design additional µ-opioid receptor agonists and antagonists for therapeutic application. divalent cations have been known for a long time to influence significantly binding to receptors and biological activity of the peptide oxytocin (ot). there is very low binding of 3h-ot to the receptors in the absence of these ions. it has been speculated where the divalent cations work. recently an article appeared showing formation of a complex divalent cation-ot and stressing the importance of n-terminal amino group for binding and activity [1] . however deamino analogues of ot are also very active and their binding is also influenced by divalent cations. we have studied ot, deaminooxytocin (dot) and an ot antagonist (antag) by means of electrospray ms and we have observed that all these compounds form molecular adducts with zn2+, mg2+, mn2+ and ca2+. in binding experiments using 125-i antag, the quantity of tracer bound to membranes of hek cells having stable expressed human ot receptor strongly depends on the character and concentration of divalent ions. displacement curves using unlabelled antag do not change in the absence or presence of 10 mm of tested divalent ions. on the other hand, displacement curves using unlabelled ot and dot are shifted to the left in the presence of mg2+ and mn2+, and to much lesser extent by zn2+ and ca2+. all this points to the idea that the divalent ions do work on the site of membrane receptors. biologically active peptides exhibit multiple conformations in solution. thus, the synthesis of conformationally restricted analogues is a valuable approach for determining structure -activity relationships. restrictions can be imposed e.g. through the formation of cyclic structures within the peptide framework by disulfide bridges, or by substitution of chosen amino acid residues that limit conformational freedom, thus forcing the peptide backbone and/or side chains to adopt specific orientations. in recent years, conformationally constrained analogues of bioactive peptides seem to be a feasible approach to providing useful informations concerning threedimensional structure of such compounds which, in turn, could rationalize our knowledge about structure-biological activity relationships and thus help to design peptides with desired pharmacological properties. steric restrictions can be introduced by the formation of cyclic structures within the peptide backbone or by incorporation of amino acids with limited conformational freedom, which in turn results in specific orientations of the peptide backbone and its side chains. another approach to reduce the flexibility of the analogue is substitution of chosen amino acids with various types of pseudopeptides prepared trough short-range cyclizations. the present work is a part of our studies aimed at clarifying the influence of sterical constraints in the n-terminal part of arginine vasopressin (avp) and its analogues on the pharmacological activity of the resulting peptides. we describe the synthesis of four new analogues of avp substituted at positions 2 and 3 or 3 and 4 with two diastereomers of 4-amino-pyroglutamic acid and four peptides in which we combined the above modification with the placement of 3mercaptopropionic acid (mpa) at position 1. all the peptides were tested for their in vitro uterotonic, pressor and antidiuretic activities in the rat. different strategies to modulate shp-1 activity protein tyrosine phosphatase shp-1 consists of two sh2 domains n-terminal to the catalytic (ptp) domain and a short c-terminal tail. the binding of a py-ligand to the n-sh2 domain is required for an efficient activation of shp-1 phosphatase activity. the specificity of the shp-1 sh2 domains is determined by the py-residue (position 0) and residues at positions -2, +1 and +3. combinatorial peptide library methods revealed different classes of consensus sequences for both sh2 domains [1, 2] . in addition, the importance of residues c-terminal to py+3 (+4 to +6), in particular for binding to the n-sh2 domain, has been demonstrated [3] . together with investigations of the determinants for optimal sh2 domain binding and stimulation/inhibition of shp-1 activity [4] , these informations were useful for the generation of different strategies for effectors of shp-1 activity. peptides cyclized between different positions of the general consensus py-2 to py+3 were synthesized and evaluated with respect to n-sh2 domain binding and stimulation of phosphatase activity. structure-activity studies have revealed that the specificity of an integrin towards its rgd-containing ligands can be evaluated through the distances between the cβ atoms and/or the distance between the charged centers of arginine and aspartic acid as well as, the pseudo-dihedral angle (pdo), composed by the r-cζ, r-cα, d-cα and d-cγ atoms, which defines the relative orientation of the arg and asp side chains. in a previous study [1] , the antiaggregatory activity of rgd peptide analogues, i.e. their ability to act as fibrinogen receptor αiibβ3 antagonists, was correlated with the above structural criteria. our results suggested that the fulfillment of the criterion -45ο < pdo < +45ο is a prerequisite for an analogue to exhibit activity. in the present study, we examine the above criteria to rgd-containing 15peptides, derived from the active sites of the ecm proteins fibrinogen, fibronectin and vitronectin, as well as, from the cryptic rgd site of von willebrand factor. the correlation of the structural data with the biological activity of compounds, are in good agreement with the previously mentioned -45ο < pdo < +45ο criterion. furthermore, our results show that the differences in activity of compounds, which display similar distances between the charged centers of arg and asp, can be better evaluated by the pdo structural criterion. acknwolegments : this work was supported by grants from eu and the hellenic ministry of education ( heraklitos). references : the gpiib/iiia receptor, which is a member of the integrin family, is the most abundant receptor in the surface of platelets and can interact with a variety of adhesive proteins including fibrinogen, fibronectin and von willebrand factor. fibrinogen binding on gpiib/iiia is an event essential for platelet aggregation and thrombus formation. mapping of the fibrinogen binding domains on gpiib subunit suggested the sequence 313-332 as a putative binding site [1] . this region was restricted to sequence gpiib 313-320 (ymesradr) using synthetic octapeptides overlapping by six residues [2] . the ymesradr octapeptide inhibits adp stimulated human platelets aggregation and binds to immobilized fibrinogen. in this study we present the conformational analysis of three synthetic analogues yaesradr (a2) ymesaadr (a5), and ymesraar (a7), using nmr spectroscopy and distance geometry calculations. common structural characteristic of peptides a2 and a7 is the interaction between the side chains of arg5 and glu3, however in a2 the guanidino group of arg5 seems to form salt bridges with both glu3 and asp7. peptide a5 is stabilized only by a week interaction between arg8 and glu3 side chains. the interactions between the residue side chains provoke different overall shape of the three molecules. the most populated structural family of a2 exhibits a π backbone shape, a5 a turn around -s4a5-, while a7 an almost extended shape. background and aims: endomorphin-2 (em-2: h-tyr-pro-phe-phe-nh2), endogenous opioid peptide isolated from bovine and human brain, has high affinity and selectivity for the mu receptor and produces potent and prolonged analgesia in mice [1] . in this presentation, the incorporation of ethylene-bridged phe-phe unit (eb[phe-phe]) or piperidine carboxylic aid (pic) in position 2 was carried out to obtain more potent agonist or antagonist with stability against dipeptidyl peptidase iv (dpp iv). methods: the synthesis of eb[phe-phe] unit was achieved according to the procedure of lammek b. et al. [2] protected peptides were synthesized by a solution method using boc-chemistry. the final products were identified by maldi-tof mass spectrometry and elemental analyses. the receptor binding affinity of peptides was assessed by radio-ligand receptor binding assay using mu and delta opioid receptors from rat brain membranes or cos-7 cell membranes expressing each opioid receptors. muc2 glycoprotein, produced by the epithelium of the colon, built up mainly of repeat units of 1ptttpitttttvtptptptgtqt23, can be underglycosylated in colon carcinoma. we have been studying the epitope structure of the muc2 repeat unit with the mucin peptide specific mab 996 monoclonal antibody. this antibody recognizes the 18ptgtq22 sequence as minimal, and 16ptptgtq22 as optimal epitope. our interest lies in the modification of this epitope with maintained or enhanced specificity, and we aim to clarify the effect of different epitope modifications on mab 996 antibody binding: a) amino acid changes in the flanking region, b) glycosylation in the epitope core and in the flank. for this we have prepared a) libraries of ax(1)ptgtqaa and atptgtqx(2)a peptides, and x(1)ptgtqx(2) heptapeptides based on the antibody binding properties of the libraries; and b) glycopeptides pt(galnac)ptgtq, ptpt(galnac)gtq and ptptgt(galnac)q. the peptides were prepared by solid phase synthesis; after purification, esi-ms and amino acid analysis characterisation their antibody binding properties were studied by competitive elisa. our results show that a) although all amino acids in positions x(1) and x(2) resulted in antibody binding; in position x(1) hydrophobic, in x(2) aromatic residues provided stronger binding than that of the native peptide; b) glycosylation on thr(17) did not influence the binding of mab 996, but on thr(19) the presence of n-acetyl-galactosamine, interestingly, slightly increased the antibody recognition. these findings could be useful in designing synthetic peptide vaccines for tumour therapy. histidines play essential role in binding of biological metal ions, either in small or macromolecular chelating molecules, e.g. in metalloenzymes. therefore the low molecular weight polyhistidine type ligands are of potential importance as model substances. continuing our investigations on a novel branched oligopeptide type ligand -(his)4(lys)2lys-nh2 -prepared by solid phase peptide synthesis, we investigated the metal ion binding properties with zinc(ii) and copper(ii). the eight primary metal-binding sites are the four imidazole and four ammine groups on the ligand. phpotentiometric titrations revealed, that up to ph 8 all these donor atoms loose their protons on increasing ph. the competition between the protons and the metal ions results the decrease of pka values to about 1-3 in the case of copper(ii) and to about 4-6 in case of zinc(ii) ion. this reflects the higher stability of the complexes formed with copper(ii) in spite of the weak axial coordination that seems to occur in zinc(ii) complexes. combined potentiometric, spectrophotometric, cd and nmr spectroscopic methods were utilized to investigate the speciation and the structure of the complexes formed in aqueous solution. the prepared cu(ii) complexes cleaved dna, but it is not known whether in oxidative or in hydrolytic manner. because of this ambiguity further studies with zn(ii) complexes will be undertaken. this work has received support through sapstclg97697 nato collaborative linkage grant and from the hungarian science foundation (otka t43232). lgr8. further studies have shown that in both male and female gonads, insl3 and lgr8 represent a paracrine system important for meiosis induction in the ovary and male germ cell survival in the testis. thus insl3 may have clinical applications in fertility management. we undertook to determine the key structural elements responsible for its unique actions. methods: alanine-scanned analogues of human insl3 and mimetics of the b-chain alone were prepared by solid phase peptide synthesis. each was subjected to cd spectroscopy for secondary structure analysis and assayed for in vitro lgr8 binding and activation activity. the tetrapeptide h-dmt-d-arg-phe-cbp was found to be a selective µ agonist [ic50 (gpi) = 78.8 ± nm] with 40-fold lower potency than the corresponding, highly potent tiq4-tetrapeptide, but with still 3-fold higher potency than leu-enkephalin. in conclusion, we developed selective, cbp-containing δ antagonists and µ agonists with significant potency. recently, we described the syntheses and biological activities of several opioid peptide analogues that contained the n-terminal sequence 1-4, common to dermorphin and deltorphin. some of them showed very high agonist potency both in the gpi assay and in the mvd assay [1, 2] . in this work, we designed new analogues in which the sequences were elongated at the c-terminal to obtained the full sequences of dermorphin (a) and deltorphin (b). the syntheses of compounds and their biological activity profiles will be discussed. background and aims: endomorphin-2 (em-2: tyr-pro-phe-phe-nh2) is very potent endogenous opioid peptide, which exhibits high affinity and selectivity for the mu-opioid receptor [1] . previously, we had reported that [ac3c2]-em-2 containing 1-aminocyclopropane-1-carboxylic acid (ac3c) exhibited higher affinities than em-2 for the mu-opioid receptor [2] . in order to clarify that the substitution of 1-aminocycloalkane-1-carboxylic acids (acnc: n indicates the number of carbon atoms in a ring) for pro in position 2 of em-2 is efficient to obtain higher affinity for the mu-receptor, we synthesized . therefore, the replacement of pro to ac3c and ac4c will be efficient to make these analogs adopt bioactive conformation and exhibit high affinity for mu-receptor. in the past few years, many attempts have been made to prepare a synthetic insulin. the biological activity of insulin is known to be closely related to the c-terminal octapeptide fragment of its b-chain.it was found that b23 gly and b24 phe were present in all insulins so far obtained from various animal species indicating the significance of these two residues.it would therefore seem desirable to study the effect of each of these two amino acid residues or both on biological activity of the octapeptide fragment of the b-chain. a heptapeptide arg-phe-tyr-thr-pro-lys-ala-och3, corresponding to (b22-b30) insulin des gly23-phe24, and an octapeptide arg-phe-phe-tyr-thr-pro-lys-ala-och3, des gly23 were synthesized using the solid phase method. the c-terminal ends of both peptide were converted to methyl ester by transesterification cleavage from the resin. the side chain protecting groups were removed by hf. manual counter current distribution method was used for purification of the free peptides. the way to solve the evaluation of tyrosine containing peptide was studied. the free methyl ester peptides were administered for insulin-like activity test by glucose metabolism in the rat fat cells technique in vitro. aim of this study is to develop peptides as useful tools for degradation of synthetic dyes, which are often pollutants. we focused our interest in peroxidases, a class of enzymes reported to efficiently degrade azo and anthraquinonic dyes. in particular, the fungus versatile peroxidase (vp) of pleurotus eryngii can perform this degradation. therefore, our goal is the synthesis of a peptide based on this peroxidase able to emulate its biological function. the linear and cyclic peptide sequences were derived by the theoretical model of vp [pdb: 1a20], which determined the amino acids fundamental for the desired function of the active sites. in particular, the residues instrumental for the coordination of the heme, the mn binding site, and the long range electron transfer pathway [1] , were pin-pointed. moreover, we calculated the radius of the heme cavity. the next step was the synthesis of these peptides in order to verify the coordination of the heme and optimize their sequences. the syntheses were carried out by solid-phase following the fmoc/tbu strategy. because of purification difficulties of the fully-protected peptide, we undertook an alternative synthetic pathway, based on a solid phase head-to-tail cyclisation strategy, following the fmoc/tbu/allyl three-dimensional protection scheme [2] . next steps will be to test the coordination properties of the synthetic peptides, with respect to the heme, and further computational studies based on the new model of pleurotus the calcium plays an important role in biochemical pathways. it binds to enzymes and proteins in a different process. aspartic (asp, d) and glutamic (glu, e) acid side chains are the main ligands of calcium, but the contribution of the backbone carbonyl groups in the binding is also important. generally the binding places in the proteins are an unstructured loop between two helixes (310-or alpha-helix). the common sequence is the so-called ef-hand motif, which contains 12 amino acids [1] . it is already known that some proteins also bind calcium with a non-ef-hand loop. for example alpha-lactalbumins have a ten amino acid long sequence for binding [2] . it is an asp rich sequence where 5 asps are closer to each other than in ef-hand motif (-k79fldddltdd88-) but only 3 asps side chains take part in calcium coordination. we constructed a series of cyclopeptides to mimic the loop structure of alpha-lactalbumin [3] . in this study we focus on determining the importance of conservative amino acids within the ca2+ binding loop of this protein, using microcalorimetry (itc). the itc measurements were performed in different organic solvents and at different temperature. the synthesis of fatty acids in adipose tissue. in this article, we present the solution structure of gip in water and tfe/water determined by nmr spectroscopy. the calculated structures are characterised by the presence of an -helical motif between residues ser11-gln29 and phe6-gln29 respectively. the helical conformation of gip is further supported by cd spectroscopic studies. six gip(1-42)ala1-7 analogues were synthesised by replacing individual n-terminal residues with alanine. alanine scan studies of these n-terminal residues showed that the gip(1-42)ala6 was the only analogue to show insulin secreting activity similar to that of the native gip. however, when compared with glucose its insulinotropic ability was reduced. for the first time, these nmr and modelling results contribute to the understanding of the structural requirements for the biological activity of gip. a knowledge of the solution structure of gip and of the role of its individual residues will be essential in the understanding of how they interact with the gip receptor. efrapeptins are pentadecapeptides produced as a mixture of six closely related analogues (efrapeptin c-g) by the fungus tolypocladium niveum and other members of this species. they consist predominantly of the nonproteinogenic amino acids -aminoisobutyric acid (aib), isovaline (iva), -alanine ( ala) and pipecolic acid (pip), have an acetylated n-terminus and bear an unusual cationic c-terminal headgroup derived from leucinol and 1,5-diazabicyclo[4.3.0]non-5-ene. efrapeptin c is a competitive inhibitor of the f1-atpase and active against the malaria pathogen plasmodium falciparum. an anti-proliferative effect was also reported. conformational analysis of efrapeptin c in trifluoroethanol and dimethylsulfoxide was conducted to obtain structure-affinity relationships. the absence of amide-and -protons resulted in an imperfect assignment and unsatisfying conformational study. specific deuteration of methyl groups in aib did not simplify the assignment. cd and ft/ir spectra hint to helical or beta-turn secondary structures as main structure elements. residual dipolar couplings (rdc) were measured in a stretched cross-linked poly(dimethylsiloxane) gel in dichloromethane. the impact of the rdc on the conformational analysis led to an improved high resolution structure from simulated annealing protocols and consolidated the formation of a helical structure of efrapeptin c in nonpolar solution which is comparable with the binding pocket of the f1-atpase. finally, the dynamics of the resulting structures was studied using the gromos96 force field in explicit solvent. serotonin selective reuptake inhibitors (ssris) are currently among the most frequently prescribed therapeutic agents of depression. their therapeutic use includes also obsessive-compulsive disorder, panic disorder, bulimia. the serotonin transporter (sert) is the target of serotonin selective reuptake inhibitors (ssris). altough the inhibition is the proximal event in antidepressant action, the clinical benefit of antidepressant medications requires weeks of continuous dosing, indicating that their mechanism of action involves events downstream from acute transporter blockade. long-term effects of ssri treatment may be due to changes in intrinsic properties of sert structure, function, or regulation. thus, understanding the mechanism of action of sert remains a primary goal in the search for developing novel treatments for diseases associated with serotonergic dysfunction. in the present study experimentally determined ligand selectivity of the buspirone analogues toward the serotonin transporter was theoretically investigated on the molecular level. the model of serotonin transporter based on the crystal structure of bacterial homologue from aquifex aeolicus (leutaa) was constructed using the traditional homology modelling approach. a series of docking experiments with ssri's were conducted, using interactive molecular graphics techniques combined with energy calculations and analysis of the transporter-ligand complexes. structural information about the serotonin transporter and its molecular interactions with ssri's is important for understanding the mechanism of action of these drugs and for development of drugs with improved potency and selectivity. the protein kinase c (prkc) is a member of a super-family of the eukaryotic receptor protein kinases. it forms dimers and is anchored in the membrane, with a cytoplasmic kinase domain and an external domain, presumably acting as a sensor. prkc enables formation of biofilms of bacillus subtilis which show a high degree of spatial organization. they colonize various surfaces and produce complex antibiotic resistant communities. prkc acts as a ser/thr kinase with features of the receptor kinase family of eukaryotic hanks kinases. our current study involved theoretical modeling of the protein kinaze prkc complexes with the modified atp. the ligands were selected from a set of molecular probes developed by k. shah and coworkers [1] . each modified atp molecule was docked to the active site of the kinase molecule using autodock genetic algorithm procedure. the optimized structures of the complexes were submitted to the molecular dynamics simulations in the amber force field. we obtained four optimized structures of prkcc complexes in water. the results suggest the great similarity of our complexes with human cyclin-dependent kinase 2 [1] complexes. background and aims. indolicidin is a 13-residue antimicrobial peptide, which was isolated from bovine neutrophils. this molecule possesses a wide spectrum of antibacterial, antifungal and antiviral activity, furthermore it has also haemolytic effect. data derived from structural investigations led to considerably diverse conclusions regarding the secondary structure of this peptide, therefore the aim of this study was to examine the effect of cis-trans isomerization on the conformational properties of this antimicrobial peptide. methods. the conformational analysis of indolicidin containing cis or trans xxx-pro peptide bonds was performed by simulated annealing calculations with the use of amber force field. results. for the conformers of indolicidin with cis or trans xxx-pro peptide bonds, the evolving secondary structural elements were examined and poly-proline ii helix and type vi beta-turn were identified. in the case of this peptide, various intramolecular interactions may play an important role in stabilizing the structure of conformers. therefore the presence of the h-bonds between backbone atoms, the aromatic-aromatic interactions between the side-chains of trp amino acids and the proline-aromatic interactions between the side-chains of trp and the pyrrolidine rings of pro amino acids was investigated. conclusions. the conformational comparison of the peptides possessing cis or trans xxx-pro peptide bonds resulted in different secondary structural elements for both isomers, which are the poly-proline ii helix and type vi beta-turn for the trans and cis isomers of indolicidin, respectively. the occurrences of various intramolecular interactions are in agreement with the observed secondary structures. we have shown the monte carlo conformational search using macromodel is useful for conformational study of oligopeptides prepared from alpha, alphadisubstituted alpha-amino acids. moreover, we have studied conformational analysis of oligopeptides containing chiral alpha, alpha-disubstituted alphaamino acids to predict the helical screw sense of helical structures. here we report computational study on conformation of oligopeptides containing cyclic alpha, alpha-disubstituted alpha-amino acids with side-chain chiral centers. background and aims. the homopolymeric amino acids (hpaas) are polypeptides consisting of the same amino acids. some of them play a relevant role in the formation of several neurodegenerative diseases. most probably the poly-(ala) and poly-(gln) are the best representatives of these peptides because of their important biological effects. our aim was to perform conformational analysis and structural investigation of these two hpaas. methods. to explore the conformational spaces of the peptides, simulated annealing (sa) and random search (rs) calculations were carried out using amber force field. two different forms of the hpaas were modelled: either with charged n-terminal amino group and c-terminal carboxyl group, or with the n-and c-termini blocked by acetyl and n-methyl amide groups, respectively. results. for the conformers obtained by sa and rs calculations, the occurrences of various secondary structural elements like different types of beta-turns, gammaand inverse gamma-turns, alpha-helix, 310-helix, poly-proline ii helix and beta-strand were investigated. in the cases of various helices and beta-strand, segments with different lengths characterized by these secondary structures were determined along the entire sequence of peptides. for the conformers of the hpaas, the intramolecular h-bonds formed between the backbone atoms as well as between the backbone and side-chain atoms were identified. the vasopressin and oxytocin receptors (v1ar, v2r and otr) are membrane-embedded proteins belonging to the large family a g protein-coupled receptors (gpcrs). they are involved in crucial physiological functions as the regulation of water metabolism, control of blood pressure and stimulation of labor and lactation, mediated via v2r, v1ar and otr, respectively. as such, they are involved in a number of pathological conditions and are important drug targets. understanding their inhibition and activation mechanisms may improve design of ligands capable of selective stimulation or blockade of the respective receptors presenting the therapeutic targets. to investigate the otr, v1ar and v2r interactions with agonists and antagonists thirty computer models of receptor-ligand complexes have been modeled via docking and molecular dynamics (md) and analyzed in details. the receptor models were built on rd crystal structure template or using the coordinates of mii-gtα(338-350), for non-active and activated models, respectively. the ligands (arginine vasopressin, oxytocin, desmopressin, atosiban ([mpa1,d-tyr(et)2,thr4,orn8]ot) and barusiban (mpa1,d-trp2,ile3,allo-ile4,asn5,abu6,mol7) were docked into the receptors. the complexes have been embedded into the hydrated popc bilayer and submitted to 1ns unconstrained md in the amber force field. the relaxed systems have been obtained and analyzed in details. the receptor residues responsible for agonists/antagonists binding have been identified and mechanism of binding involving the highly conserved residues has been proposed. a three-dimensional models of the neurohypophyseal hormone receptors were constructed using a multiple sequence alignment and either the crystal structure of bovine rhodopsin or the complex of activated rhodopsin with gta c-terminal peptide of transducin rd*-gt(338-350) prototype to obtain nonactive or activated receptor models, respectively. analogs were docked to v1ar, v2r and otr, both non-active and activated models. the low-energy receptor-ligand complexes, with properly docked analogs were submitted to the constrained simulated annealing (csa), in vacuo. the relaxed receptoranalog models were obtained. the residues responsible for analogs binding to v1ar, v2r and otr have been identified and presumable biological activity of these compounds was determined. n-methyldehydroamino acids belong to non-standard amino acids found in nature. n-methyl-(z)-dehydrophenylalanine was found in tentoxin, a selective weed killer, having been produced by several phytopathogenic fungi of the alternaria genus. n-methyl-(z/e)-dehydrobutyrine and n-methyldehydroalanine are components of nodularins and microcystins, families of hepatoxins produced by species of freshwater cyanobacteria, primarily nodularia spumingena and microcystis aeruginosa. the simplest n-methyl dehydropeptides, ac-delta(me)xaa-nhme (where xaa = ala, (z/e)-abu, (z/e)-phe, and val) and, for comparison, the saturated ac-l-(me)ala-nhme analogue were investigated using computational methods. cis-trans b3lyp/6-31+g**//hf/3-21g ramachandran potential energy surfaces were created. the conformers found were optimised at the b3lyp/6-31+g** level. the effect of the electrostatic solute/solvent (water) interaction on the solute energies was investigated within the scrf method using the polarisable continuum model (pcm) on the geometries of solutes in vacuo. it was found that for all the studied dehydropeptide molecules the lowest conformer (phi, psi = ~ -109°, 10°) has the cis n-methyl amide bond. this feature seems to be independent of the dehydroamino acid moieties, the c-beta substituent and the z/e configuration. the pi-electron conjugation as well as the n-h···n hydrogen bond play the dominant role in the stability of this conformer (see figure) . the preliminary nmr investigations into the conformational preferences of the studied molecules in solution confirm the theoretical results obtained. the strong tendency of the n-methyl amide bond to adopt the cis configuration seems to be the reason why n-methyldehydroamino acids are found in small natural cyclic peptides, where they ensure the conformational flexibility necessary for biological action. the purpose of this study was to determine the potentials of mean force (pmf) of the interactions between models of nonpolar amino acid side chains in water. the potentials of mean force (pmf's) dependent on orientation were determined for systems forming hydrophobic and diagonal complexes composed of side-chain models of alanine, valine, leucine, proline and iso-leucine, respectively, in water. for each hydrophobic pair in water a series of umbrellasampling molecular dynamics simulations with the amber force field and explicit solvent (tip3p water model) were carried out and the pmfs were calculated by using the weighted histogram analysis method (wham). in all cases a characteristic shape of pmf plots for hydrophobic association were found, which was manifested as the presence of contact minima and solvent separated minima. depths of contact minima for all systems studied were about 1 kcal/mol. in this work we compared the ability of two theoretical methods of ph-dependent conformational calculations to reproduce experimental potentiometric-titration curves of two models of peptides: ac-k5-nhme in 95% methanol (meoh)/5% water (h2o) mixture and ac-xx(a)7oo-nh2 (xao) (where x is diaminobutyric acid, a is alanine, and o is ornithine) in water, methanol (meoh) and dimethylsulfoxide (dmso), respectively. in theory, in all three solvents, the first pka of xao is strongly downshifted compared to the value for the reference compounds, the water and methanol curves have one, and the dmso curve has two jumps characteristic of remarkable differences in the dissociation constants of acidic groups. the predicted titration curves of ac-k5-nhme are in good agreement with the experimental ones; better agreement is achieved with the md-based method. the titration curves of xao in methanol and dmso, calculated using the md-based approach, trace the shape of the experimental curves, reproducing the ph jump, while those calculated with the edmc-based approach, and the titration curve in water calculated using the md-based approach, have smooth shapes characteristic of the titration of weak multifunctional acids with small differences between the dissociation constants. quantitative agreement between theoretically predicted and experimental titration curves is not achieved in all three solvents. the poorer agreement obtained for water than for the nonaqueous solvents suggests a significant role of specific solvation in water, which cannot be accounted for by the mean-field solvation models m337 a. papakyriakou 1 , g.f. vlachopoulos 2 , g.a. spyroulias 2 , e. manessi-zoupa 3 , p. cordopatis 2 angiotensin-i converting enzyme (ace) belongs to the m2 family of the ma clan of zinc metallopeptidases and can act either as a dipeptidyl carboxypeptidase, which catalyses the proteolytic cleavage of dipeptides from the carboxy terminus of a wide variety of peptides, or as an endopeptidase, which hydrolyses peptides bearing amidated c-termini. among the former category of ace peptide substrates, the most distinguished are those involved in blood pressure regulation, such as angiotensin i (angi) and bradykinin (bk). in the latter category falls the gonadotropin-releasing hormone (gnrh) in an attempt to analyze molecular interactions at atomic level we simulated the ace-substrate complexes, using the recently determined 3d crystal structure of ace testis isoform and a knowledge-based docking method in order to insert the peptide substrate (angi, bk and gnrh) of ace into its catalytic cleft. in order to introduce the effect of protein mobility and gain information about enzyme-substrate recognition and interaction we have sampled the conformational space of these complexes via molecular dynamics simulations with explicit solvent representation. we have also performed molecular dynamics calculations with tace-inhibitor complexes, such as lisinopril, as well as with tace mutated at specific sites, such as the ligands of the two buried chloride ions that have been shown to affect substrate activity. our results provide new insights into the role of specific domains of tace and their implication in the enzyme activity, which is not readily apparent from the available crystal structures. two main mechanisms for the propagation of action potential in myocytes are: 1) the free flow of local circuit current through gap junctions and 2) the effect of electrical field. here we study effect of each mechanism and their importance during action potential propagation. method: we simulated the cardiac myocyte by the orcad software, then used the model of sinoatrial node to stimulate the myocytes model and studied the propagation of action potential with and without gap junction. result: our results show that, although gap junction solely is not able to mimic physiological condition, but it is necessary for normal cardiac functioning. on the other hand, electric field is not sufficient for successful propagation of action potential and the existence of gap junction is necessary. anthrax is a disease of animals and humans, caused by the bacterium bacillus anthracis. anthrax toxin (at) consists of three proteins, one of which is the anthrax lethal factor (alf). alf is a gluzincin zn-dependent highly specific metalloprotease (~90.000 kda), which belongs to the m34 family of the ma clan of zinc metalloproteases. alf cleaves most isoforms of mitogen-activated protein kinase (mapk)-kinases (meks) close to their amino termini, leading to the inhibition of one or more signaling pathways. no data are available on the enzyme-substrate interaction at the molecular level. therefore, we performed classical molecular dynamics simulations on the alf-mkk/mek complexes in order to probe protein-substrate interactions. the simulations pinpointed specific hydrophobic as well as electrostatic alf-peptide substrate interactions and these data were exploited in the building of virtual combinatorial libraries of di-and tri-peptides using the twenty native aminoacids. by applying docking simulations to anthrax zn-metalloprotease around 1.000 peptide substrates were virtually screened according to their binding affinity. data suggest that complexes of alf with peptides substrates bearing arg, trp, lys and phe aminoacids, exhibit the highest binding affinity providing evidence for electrostatic interactions between negatively charged residues of alf's active site and positively charged side-chains of di/tri-peptides. new libraries of substrates were built incorporating non-protein residues, organic moieties and chelating groups. alf-substrate complexes with the best score (in terms of binding energy) are further analysed. in the present studies we designed and synthesised seven new bradykinin (bk) analogues and evaluated them in the in vivo rat uterotonic assay using a modified holton method in munsick solution on a strip of rat uterus and in blood pressure test. we used [arg0, hyp3, thi5, 8, d-phe7]bk, the b2 antagonist of vavrek and stewart as a model, when designing our analogues. in all cases, the n-terminus of our peptides is acylated with bulky substituent. we previously reported that acylation of the n-terminus of several known b2 antagonists with various kinds of bulky acyl groups has consistently improved their antagonistic potency in rat blood pressure assay. on the other hand, our earlier results seem to suggest that effects of acylation on the contractility of isolated rat uterus depend substantially on the chemical character and size of the acyl group, as we observed that this modification may either change the range of antagonism or even transform it into agonism. the peptides were synthesized by the solid-phase method using the fmoc-strategy the modifications proposed either preserved or increased the antagonistic potency in the rat blood pressure test. on the other hand, the seven substituents, differently influencend the interaction with the rat uterine receptors and except one led to decrease of antiuterotonic activity. in both cases acylation of the n-terminus led to enhancement of antagonistic potencies. our results may be of value in the design of new b2 agonists and antagonists. the formations of amyloid fibrils have been reported as for various amyloidosis. several structural models of fibrils are proposed for respective proteins so far. however, their common basic structures and universal features to induce amyloid fibril formations are not known in detail. previously, we examined intermolecular interactions among the several amino acid residues in barnase, which is known to form amyloid-like fibril. based on the experimental results using a series of mutant barnase, we discovered that the interactions between hydrophobic side-chains are the most essential driving force to form the fibrils and that both intermolecular and inter-sheet interactions in the fibril maintain highly ordered molecular packing. in the present paper, we describe a novel prediction method for core regions of various fibril-forming proteins and show the verification of the above possible structural principle. at first, we calculated the interaction's score between side-chains in the antiparallel orientation of beta-strands. next, the peptides with predicted sequences of fibril cores, a couple of high-scored regions with a designed turn moiety to induce a hairpin-like form, were chemically synthesized by spps. as a result, the formation of amyloid fibrils was confirmed for most of high-scored sequences. in addition, we also applied this method to prion protein, we could predict 4 possible beta-strands with hetero-paired orientation. some synthetic peptides involving these strands were proved to have fibril-forming ability. thus, we have developed the novel method to predict the core regions that induce amyloid fibrils. a principal factor analysis (pfa) is a very efficient way of identifying patterns in the data sets even if the patterns are hard to find (e.g. in the high dimensional data sets). this is the reason why the pfa method can be powerful tool for analyzing molecular dynamics (md) trajectories. it is possible to reduce dramatically the trajectory size without loosing significant structural information by applying the pfa procedure. we used this tool for interpretation of results from the molecular dynamics simulations of the model of the transcription factor nf-kb. nf-kb is a protein involved in the numerous biological processes such as regulation of immune response, inflammation, various autoimmune diseases and is used by many viruses, including human immunodeficiency virus (hiv), to activate transcription of their own genes. only the trajectory of the backbone atoms of the nf-kb were subjected to the further analysis. peptides contain many basic sites such as side chains of basic amino acid residues, oxygen and nitrogen atoms of amide groups, and terminal amino groups. these parts can interact with protons. this interaction can change conformational behaviour of peptides and, consequently, their biological functions. the interaction becomes even stronger in the gas phase. in that case, the stability of the peptide chain is influenced, which may have impact on peptide fragmentation during mass spectroscopy analysis of peptide structures. in this study, we will present the interaction of proton with carbonyl oxygens in the model of alanine tripeptide. quantum chemical calculations employing density functional theory using hybrid b3lyp functional and 6-31++g** basis set were used to describe this interaction and also to find possible pathways of proton transfer among interaction sites. two different mechanisms of proton transfer were found. the first mechanism is represented by an isomerization of the proton around the double bond of the carbonyl group. the second mechanism is based on the large conformational flexibility of the tripeptide model where all carbonyl oxygens cooperate. the later mechanism exhibits nearly half energy barrier of the rate-determining step compared to the first one. we focus our attention on situation, in which methyl groups attached to alpha atoms in tripepetide model influence the conformational behavior. results will be presented for all four possible stereochemical configurations. a. papakyriakou 1 , p. galanakis 2 , p. gazonis 2 , g.a. spyroulias 2 p53 protein is one of the most effective defensive weapons of human body against carcinogenesis, due to its tumor suppression properties. it has been noticed, in many types of cancer, that the functions of p53 are being downgraded or even suppressed and this fact is ought to the presence of mutated forms of p53 or to the complete absence of the protein. the suppression of p53 levels is being indirectly regulated by the protein itself, which activates the expression of a gene, the oncogene mdm2 (murine double minute 2), which expresses the mdm2 protein, known as human-mdm2 or just hdm2. hdmx protein is a homologue protein to hdm2 and is being implicated, through various biological processes, in the suppression of p53. however, recent experimental evidence suggests that hdm2 and hdmx proteins are not the only ubiquitin ligases that negatively regulate p53 through ubiquitin pathway. two recently discovered e3 ligases, cop1 and pirh2, are also proposed to promote p53 for degradation. all these proteins function as e3 ligases bearing a ring finger domain. these domains are characterized by their high content in cysteines and the binding of two zn(ii) ions while they catalyze the latter stage of protein signaling for proteolysis by the 26s proteasome, through the ubiquitin pathway. the structure variation and the stability of these ring fingers is studied through molecular dynamics simulations of 2-5 ns and structure variations are analyzed in a structure-function correlation basis. semax is a synthetic analogue of adrenocorticotropic hormone acth 4-10. it is a nootropic agent containing seven amino acids met-glu -his-phe-pro-gly-pro without hormonal (adrenocorticotrophic) activity. semax is neuroprotective via a mechanism involving the regulation nitric oxide (no) and lipid peroxidation. semax proved to be highly effective in abating the rise in no and restoring neurologic functioning [1] . it was found to improve intellect and memory in healthy human. it is effective in rehabilitation of people with memory and motor disorders, parkinson's and hantington's diseases, after cerebral stroke and head trauma [2] . to study conformation dynamics in connection with in vivo activity of semax the molecular dynamics method of standard protocol was applied [3] . semax and about twenty its analogs were studied. using cluster analysis method semax was found to be more labile among various synthesized analogous (met-gln -his-phe-pro-gly-pro; gly-glu-his-phe-pro-gly-pro; lys-glu-his-phe-pro-gly-pro; glu-his-phe-pro-gly-pro; his-phe-pro-gly-pro). because of collective degree of freedom it has one more stable configuration that is unreachable in analogs. singularities of semax and analogous were studied using 2-d, 3-d poincare maps, auto and crosscorrelation functions of special type in terms of topological structure of energy hypersurface. this work was supported by rfbr (pr. 04-04-49645), russian ministry of education and science, moscow government and crdf. rhodopsin (rd) is the only representative of g-protein coupled receptors (gpcrs) whose structure has been described with high resolution. thus, it has become the structural prototype for other gpcr. these receptors are involved in transduction of various signals into the cell and actions of many hormones and neurotransmitters. about 50% of all drugs act through gpcr. growing evidence that rd and related gpcrs form functional dimers/oligomers, followed by direct proof (using atomic force microscopy -afm) that in the retina rd associates into a paracristalline network of rows of dimers, need models of rd-transducin (g t -heterotrimeric protein) complex that would envision an optimal rd dimer/oligomer amenable to satisfy all well documented interactions with gt. current model includes tetramer built of two activated (metaii) and two inactive rd molecules, ligands stabilising metaii: gtα(ile338-phe350) and gtγ(asp60-cys71)farnezy, lipid bilayer built of 36 pc (phosphatidylocholin head groups) , 6 ps (phosphatidyloserine) and 30 pe (phosphatidyloetanolamine) (all three types of phospholipids contain the polyunsaturated docosahexaenoyl chain -dha) and water. experimental data concerning shape of oligomer, conformational changes in metaii, proper interactions and distances among residues have been looked upon. the poster shows results of the molecular dynamic carried in amber force field for ~6000ps in the periodic box. conformational changes which took place during simulation caused proper adaptation one another monomers in tetramer and ligands to activated receptors. the human cystatin c (hcc) is a one of known domain swapping proteins. during this process, one of the hcc β-hairpins (β2-l1-β3) changes its conformation forming long β-strand. this conformational transition destabilizes the monomer structure and leads to domain-swapped dimer. the causative force for changing the βhairpin conformation is assumed to be the alleviation of distortions of the l1-loop val57 amino acid residue's backbone. following the above assumption and our previous conformational studies of the hcc β-hairpin peptide we investigated the influence of the point mutations, v57d, v57p and v57n of the val57 residue, on the β-hairpin peptide structure. the conformational studies by means of cd spectroscopy and molecular dynamics studies were performed. the study revealed that the hcc peptide with the wild-type sequence has the strongest tendency from all studied peptides to form a β-hairpin structure. on the basis of these results we conclude that the presence of distortions in the val residue of l1-loop is unlikely to cause the 3d domain swapping of the human cystatin c. acknowledgments: this work was financially supported by the ministry of scientific research and information technology of poland under grant 1t09a10430. temporin a (ta) (flpligrvlsgil-nh2) and temporin l (tl) (fvqwfskflgril-nh2 ) are small, basic, hydrophobic, linear antimicrobial peptides amide found in the skin of the european red frog, rana temporaria. these peptides have variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin-sensitive and resistant staphylococcus aureus as well as vancomycin-resistant enterococcus faecium strains. to gain further insight into the mechanism of action of these small antimicrobial peptides, we have investigated their conformational behaviour in different environmental conditions. more specifically, we deeply investigated by solution nmr spectroscopy in water and water/dmso (8:2) solutions as isotropic solutions and 200 mm aqueous solution of dpc (dodecylphosphocholine) was used as membrane mimetic environment. understanding the basis of the interactions of temporins with membranes could be crucial for the design and synthesis of potent antimicrobial agents. cripto is the founding member of a family of soluble and cell bound growth factors known as egf-cfc [1] distinguished by the presence of an n-terminal signal peptide, two distinct cysteine-rich domains (crd) and a c-terminal hydrophobic region involved in cell surface attachment by a post-translational gpi modification. the characteristic crds, known as egf-like and cfc domains (from the first members cripto, frl1 and cryptic), both span about 40 residues with 3 disulfide bridges [2] each, which, presumably, beside a possible functional modularity, confer them also a structural independence. in this work we have focused our attention on the cfc domain of mouse cripto. the domain has been produced by ssps, along with variants bearing mutation on h104 and w107, that have been described as crucial for alk4 receptor recognition. the two variants have been purified and refolded, achieving the correct disulfide bridges, and then comparatively analyzed by cd spectroscopy under different ph conditions; thus obtaining experimental insights on the structural arrangements of this new class of protein domains. furthermore, the binding properties of wild type and mutants cfc domains to alk4 receptor have been determined by using an elisa-based assay. our results demonstrated that the cfc domain alone can directly bind alk4 in the absence of additional ligands and, furthermore, confirmed a role of h104/w107 in cripto/alk4 interaction. there is considerable interest in the pharmacology of the two cholecystokinin (cck) receptors ccka-r (or cck-1) and cckb-r (or cck-2) that mediate the biological action of the cck hormone. they are membrane receptors belonging to the superfamily of g-protein coupled receptors (gcpr) and are predominantly located in the gastrointestinal tract and in the central nervous system, respectively. a library of 14 cyclic peptide analogues derived from the octapeptide c-terminus sequence of the human cholecystokinin hormone [cck(26-33), or cck8] has been designed, synthesised and characterised. the 14 peptide analogues have been rationally designed to specifically interact with the cck type b receptor (cckb-r) on the basis of the structure [2] of the bimolecular complex between cck8 and the third extracellular loop of cckb-r [namely, cckb-r(352-379)]. the new ligands showed binding affinities generally lower than that of parent cck8. anyway, structure activity relationship data underline that preservation of the trp30-met31 motif is essential, and that the phe33 side chain and a carboxylic group close to the c-terminal end must both be present. the nmr conformational study in dpc micelles of the compound endowed with maximal binding affinity (cyclo-b11, ic50=11 m) shows that this compound presents the turn-like conformation, centred at the trp30-met31 segment, as planned by rational design, and that such conformation is stabilised both by the cyclic constrain and interaction with the micelle. cripto is the founding member of a family of extracellular growth factors called egf-cfc found in mouse, human, chicken, xenopous and zebrafish [1] . these proteins are characterized by the presence of an n-terminal signal peptide, a c-terminal hydrophobic region and two highly conserved cysteine-rich domains, the egf-like (epidermal growth factor) and the cfc (cripto/frl1/cryptic). cripto is strictly required in the early embryonic development and contributes to deregulated growth of cancer cells in adults, since it is highly over-expressed in many solid carcinomas. it has been proposed that each single domain of cripto could bind different protein partners, playing different functional roles [2] . on this grounds, investigation of the single domains 3d-structures can have also strong functional implications. we present here an extensive conformational analysis of the mouse cfc domain (96-134 sequence) and of the w107a mutant based on nmr data. sequences have been synthesized by spps and refolded reconstituting the correct disulfide bridges [3] . the molecular models have been built by computational methods using the nmr data collected under both acidic (ph 3) and nearly physiological (ph 6) conditions. both domains show a globally extended folding with three strands linked by the three disulfide bridges and two connecting loops, in which h104 and w107, key residues in receptor binding, are exposed to the solvent urantide, a selective antagonist. thus, we carried out a study aiming at the characterization of conformational arrangement and affinity properties of ut extracellular segments.we measured by surface plasmon resonance (spr) technology the binding affinities of the three ligands, u-ii, urp and urantide towards the three extracellular loops of ut. furthermore, the secondary structures of the synthetic receptor fragments in presence of dodecylphosphocholine micelles and interaction with ut ligands were analysed using nmr spectroscopy. spr data showed that the ec loop ii was able to recognize the ligands u-ii, urp and urantide with similar affinities while none of these two ligands were able to interact with the extracellular loop i. furthermore, the absence of binding of urantide, a peptide antagonist, suggested strongly that loop iii would be involved in the signal transduction process and implies that u-ii and urp, but not urantide, would bind to ut according to a common pattern. moreover, the results indicate that potent ut antagonists could be designed by producing highaffinity ligand targeting the extracellular loop ii. also, the spr and nmr studies revealed that the synthetic structural ut domains contained some of the conformational and chemical features essential for the binding of hu-ii, urp and urantide to hut. synthetic cysteine-rich replicates of naturally occurring peptides such as hormones, neurotransmitters, enzyme inhibitors, defensins and toxins often can be oxidatively folded in high yields to their native structure. the presence of identical cysteine patterns in the sequence were found to lead to identical disulfide connectivities and homologous spatial structures despite significant variability in the non-cysteine positions. therefore, it is generally accepted to attribute the disulfide connectivities based on the homology of their cysteine pattern. minicollagen-1 from the nematocysts of hydra is a trimeric protein containing n-and c-terminal cysteine-rich domains involved in the assembly of an intermolecular disulfide network. examination of three-dimensional structures of peptides corresponding to these folded domains by nmr spectroscopy revealed a remarkable exception from the general admitted rule [1] . despite an identical cysteine pattern, they form different disulfide bridges and exhibit distinctly different folds. additionally, comparative analysis of the oxidative folding revealed for the c-terminal domain a fast and highly cooperative formation of a single disulfide isomer, the n-terminal domain proceeding mainly via an intermediate that results from the fast quasi-stochastic disulfide formation according to the proximity rule. to our knowledge, this is the first case where two short peptides with identical cysteine pattern fold uniquely and with high yields into defined, but differing, structures. therefore, these cysteine-rich domains may well represent ideal targets for structure calculations to learn more about the elementary information encoded in such primordial molecules. the conformational change of the cellular prion protein, prpc, to its virulent "scrapie" form, prpsc, is believed to be responsible for prion infectivity. and several studies suggest that the prion disulfide bond is important for the stability, structure, and propagation of prion oligomers. to test this hypothesis, we selected two conserved peptides flanking the disulfide bond in the sheep prion protein, and measured the secondary structure of these peptides with circular dichroism, hydrogen/deuterium exchange, and molecular dynamics simulations. our preliminary data suggests that the two peptides do not adopt stable secondary structure, native or otherwise. thus, the folding intermediate of a prion protein seems unlikely to comprise local structure around the disulfide bond. the conformationally labile cα-tetrasubstituted α-amino acid residue bip possesses non isolable (r) and (s) atropoisomers. we have previously reported that in the linear dipeptides boc-bip-α-xaa*-ome with α-xaa* = ala, val, leu, phe, (αme)val and (αme)leu residues at the c-terminal position of bip, the onset of an equilibrium between diastereomeric conformers with unequal populations could be observed by cd and 1h nmr. the phenomenon of induced circular dichroism (icd) represents the basis for the "bip method", an easy and fast configurational assignment for chiral α-amino acids. in search for an extension of the bip method, we investigated the boc-bip-β-xaa*-ome dipeptide series with β-xaa* = β3-hala, β3-hval, β3-hleu, β3-hpro, β3-hphe, or the cyclic β2,3-amino acids (1s,2s)/(1r,2r)-achc and (1s,2s)/(1r,2r)-acpc. low-temperature (233 k) 1h nmr spectra in cd3od revealed the presence of two conformers. significant d.r. (diastereomeric ratio) values were observed for all combinations of bip with both β3-and cyclic β2,3-amino acids. cd analysis in meoh solution of the boc-bip-β-xaa*-ome dipeptides allowed us to conclude that the cd resulting from the induced axial chirality in the biphenyl core of the bip residue gives clear information on the β-xaa* configuration for both β3-and cyclic β2,3-amino acids (except the aromatic β3-hphe), with a p torsion of the biphenyl axial bond of bip being preferentially induced by (l)-β3-xaa* as well as cyclic (1s,2s)-β2,3-xaa* c-terminal residues. we have recently reported that the induced circular dichroism (icd) of the biphenyl core of boc-bip-xaa*-ome dipeptides based on the conformationally labile cαtetrasubstituted α-amino acid residue bip could allow an easy and fast configurational assignment for both α-and β-xaa* amino acid residues. in search for other biphenyl/xaa* architectures in which a transfer of central to axial chirality could result in a potentially useful icd, we considered n-substituted 6,7-dihydro-5hdibenz[c,e]azepine (daz) derivatives from α-and β-amino acids as interesting candidates. in the present communication, we report the syntheses, and the 1h nmr and cd analyses of a series of (daz)xaa*-ome amino esters derived from α-, β3-, and cyclic β2,3-xaa* residues, namely dβ-peptide molecules possess interesting conformational characteristics and biological properties. they may represent a new class of rigid foldamers potentially useful as templates or spacers. 3d-structures of β-peptides have been experimentally investigated using x-ray diffraction and various spectroscopic techniques, but they have never been doubly spin labelled and studied by epr. a terminally protected β-hexapeptide, based on trans-(3r,4s)-β-toac and trans-(1s,2s)-achc, synthesized using classical solution methods, was found by ft-ir absorption and cd techniques to adopt the 3-14-helical conformation. a set of four, terminally blocked, hexapeptide sequences, each characterized by four strongly helicogenic aib residues and all combinations of the two isomeric ile/allo-ile residues at positions 2 and 5 was synthesized by solution methods and fully characterized. a detailed solution (by ft-ir absorption, nmr, and cd) and solid (crystalline)-state (by cd and x-ray diffraction) conformational investigation allowed us to validate our assumption that all four peptides are folded in well developed 3-10-helical structures. however, the most relevant conformational conclusion extracted from this 3d-analysis is that the handedness of the 3-10-helical structures formed does not seem to be sensitive to the configurational change at the β-carbon atom of the constituent ile versus the diastereomeric allo-ile residues (in other words, the dominant control on this important structural parameter appears to be exerted by the chirality of the amino acid α-carbon atom). these results complement published findings on the diverging relative stabilities of the intermolecularly h-bonded β-sheet structures generated by ile versus allo-ile homo-oligopeptides. taken together, these data represent an experimental proof for the intuitive view that potentially different conformational properties are magnified in a strongly self-aggregated homo-peptide system (as compared to weakly self-aggregated, helical, host-guest peptides such as those investigated in this work). in a first approach to β-sandwich proteins the hydrophobic core between two symmetrical sheets each with four antiparallel β-strands was computationally designed by packing of amino acid side chain conformations (rotamers) in an initially given backbone structure. the proteins were synthesized by coupling four peptides with β-hairpin structure to a cyclic decapeptide template (tasp). an aggregation observed by equilibrium ultracentrifugation with the first designed proteins was decreased to a dimer by increasing the surface charge in two further variants of this protein from -1 to +3 and +5. replacement of l-pro by d-pro in the loops and the template proved to stabilize the β-structure. these results led us to an improved design of an asymmetric core with algorithms for selection of proteins with a minimal number of atom clashes and cavities in the core, and a maximum number of hydrogen bonds after energy minimization. this protein termed beta-mop (modular organized protein) was synthesized in amounts to allow a characterization by cd, ftir, tryptophan fluorescence during reversible unfolding, and by high resolution nmr. nmr measurements of diffusion indicate a dimeric structure. the β-structure is stable up to 80 °c (353 k) as determined by 1d 1h nmr showing sharp resonance lines. the 2d 1h,1h dqf-cosy spectrum at 750 mhz shows a typical βsheet distribution extending well into the characteristic regions >8.5 ppm (for amide protons) and >5.0 ppm (for hα signals). all data indicate a well folded protein with β-structure. a.s. galanis 1 , z. spyranti 1 , n. tsami 1 , g.a. spyroulias 1 , e. manessi-zoupa 2 , g. pairas 1 , i.p. gerothanassis 3 , p. cordopatis 1 angiotensin-i converting enzyme (ace) belongs to the m2 family of the ma clan of zinc metallopeptidases and can act either as a dipeptidyl carboxypeptidase, or as an endopeptidase. among the ace peptide substrates, the most distinguished are angiotensin i (angi) and bradykinin (bk) due to their role in blood pressure regulation. despite the fact that biological data strongly suggest that the two active sites exhibit different selectivity and activity towards physiological and exogeneous substrates none experimental evidence for the interaction of angi and bk with ace catalytic sites, is available so far. a dual approach for studying the structure and physicochemical determinants of ace-angi/bk interaction has been performed. the first involves the application of molecular dynamics simulations (presented elsewhere in this book) and the second is making use of the solid-phase synthesized 36-46 aa ace catalytic site maquettes (csm) bearing the native sequence and the application of the nmr spectroscopy, and presented herein. therefore, high-resolution multinuclear nmr spectroscopy was applied to analyze the conformational features of ace substrates angi and bk in dmso or aqueous mixtures. then titration experiments were conducted and ace csms were titrated by angi/bk peptides, while monitored by nmr. 2d 1h-1h tocsy and noesy experiments were used in order to map the interaction site of both substrates and csm through chemical shift perturbation and comparison of noe signal differentiation. competitive binding studies were also carried out through titration studies of csm-angi/bk and known ace inhibitors. a. carotenuto 1 , p. grieco 1 , l. auriemma 1 , e. novellino 1 , v.j. hruby 2 the melanocortine receptors are involved in many physiological functions, including pigmentation, sexual function, feeding behavior, and energy homeostasis, making them potential targets to treat obesity, sexual dysfunction, etc. understanding the conformational basis of the receptor-ligand interactions is crucial for the design of potent and selective ligands for these receptors. the conformational preferences of the cyclic melanocortin agonists and antagonists mtii, shu9119, [pro6]mtii, and pg911 (table 1) when two chromophores are chirally oriented and close enough to one another in space, their excited states couple and become non-degenerate. this phenomenon, termed exciton coupling, produces a typical bisignate cd curve. the intensity of the cd couplet is dependent on the molar extinction coefficient and the distance between the interacting chromophoric moieties, while the sign is governed by the angle between the effective electron transition moments. in particular, exciton coupling over a long distance can be observed only with strongly absorbing chromophores, e. g. porphyrin derivatives, characterized by their extremely intense and sharp soret band near 415 nm. in this work we examined by the exciton coupled cd method the combined distance and angular dependencies, generated by the seven conformationally restricted β-turn and 3-10-helical spacer peptides -l-ala-[l-(αme)val]n-(n = 1-7) on a system formed by two intramolecularly interacting 5-carbamido-5,10,15,20-tetraphenylporphyrin chromophores. these porphyrin derivatives are confirmed to be excellent reporter groups. we find that not only the centerto-center separation (from 19 to 34 å) between the two chromophores, but the orientation (roughly parallel or perpendicular) between the directions of their effective transition moments as well, are responsible for the onset or even for the modulation of the intensity of the exciton coupling phenomenon. in particular, the porphyrin…porphyrin interaction is still clearly detectable over the long distance of ca. 30 å when the two chromophores are about perpendicularly oriented. a. hetényi 1 , g.k. tóth 2 , c. somlai 2 , t.a. martinek 1 , f. fülöp 1 β-peptides are probably the most thoroughly investigated peptidomimetic oligomers. to extend the field of β-peptides towards the construction of possible new secondary structures, the replacement of the cα and cβ atoms of the β-amino acid with heteroatoms could be an attractive modification, for example cβ-atom of β-peptides by an nr moiety, leading to hydrazine peptides. in the literature, there are only a few studies [1] [2] [3] about hydrazine peptides, and hydrazine peptides with cyclic side-chain have not been studied yet. in order to determine the secondary structure preference of 1-amino-pyrrolidine-2s-carboxylic acid homo-oligomers (figure 1 ), their potential energy hypersurface were probed at the ab initio b3lyp/6-311g** level. the calculations predicted the 8-strand to be the most stable structure. the hydrazino-peptides in question were synthetized on solid support, and their structures were characterized by nmr and cd methods. the results were found to be in good accordance with the 8-strand structure. cathepsin c [ec 3.4.14.1](1), which belongs to family of cysteine proteases, catalyzes hydrolysis of n-terminal peptide, preferential glyphe. this enzyme may play a part in chronic airway diseases (2) . also increaser level of enzyme was found in case of cancer, rheumatism and muscle's distrophy (3) (4) (5) . for this reason we have undertook investigations of peptides containing two dehydroamino acid residues, which could act as alkylating inhibitors of this enzyme. to define structure and conformation of investigated peptides we were used different methods of nmr spectroscopy, including standard 1d experiments, protonproton correlations, proton-carbon correlations, and 2d noe experiments. to complete structural research computational chemistry methods had been used. in order to predict the biological activity of investigated peptides, the simulation of docking process of these peptides to enzyme active site had been made and after that correlated with results of enzymatic test.. the obtained results suggest, that investigated peptides containing two ∆phe residues (z and e isomers respectively) in solution have bent conformation, which is stabilized by intermolecular hydrogen bonds. these results are confirmed by the results of theoretical calculations. also simulation of docking process have showed two possible peptide's orientation in active site of cathepsin c and allowed the rational interpretation of biological test's results. turns are important elements of secondary structure in peptides and proteins. different types of turns are distinguished according to the number of residues involved. the most abundant is the β-turn, which involves four consecutive amino acids with the co at position i hydrogen-bonded to the i+3 nh. the γ-turn is centred at a single residue and is generally stabilized by a hydrogen bond between the i co and the i+2 nh. model dipeptides rco-l-pro-xaa-nhr' are the smallest systems able to adopt the β-turn conformation, which is favoured by the presence of proline at i+1. a peptide of this series, incorporating a cyclopropane amino acid (xaa), has been shown to accommodate two consecutive γ-turns in the solid state [1] , instead of the expected β-turn conformation. the double γ-turn encountered is unique among crystalline short linear peptides. in fact, the γ-turn is observed almost exclusively in low-polarity solvents, and only a few oligopeptides of cyclic structure exhibit a γ-turn in the crystal. this is the first time that the strong tendency of pro-xaa dipeptides to adopt a β-turn in the solid state has been switched to the γ-turn. theoretical calculations [2] also show the high preference of this cyclopropane amino acid for the γ-turn conformation. [ oxidative stress plays an important part in the development of cardiovascular disease (cvd). haptoglobin is a hemoglobin-binding protein that has a major role in providing protection against heme-driven oxidative stress. there are two common alleles for haptoglobin (1 and 2), and the three phenotypes, haptoglobin 1-1, haptoglobin 2-1, and haptoglobin 2-2, differ in their ability to function as antioxidants. we determined whether there was a relation between the haptoglobin phenotype and the development of coronary artery diseases. haptoglobin (hp) phenotypes were determined in iranian patients with coronary artery diseases. we performed haptoglobin (hp) genotyping by polymerase chain reaction (pcr) using allele-specific primer-pairs. in multivariate analyses controlling for conventional cvd risk factors, haptoglobin phenotype was a highly statistically significant, independent predictor of cvd. the odds ratio of having cvd in patients with the haptoglobin 2-2 phenotype was 5.0 times greater than in patients with the haptoglobin 1-1 phenotype. an intermediate risk of cvd was associated with the haptoglobin 2-1 phenotype. these results suggest that haptoglobin phenotype is an important risk factor in determining susceptibility to cardiovascular disease which may be mediated by the decreased antioxidant and antiinflammatory actions of the haptoglobin 2 allelic protein product. the special feature of proteins involved in alzheimer's or prion diseases is their ability to adopt at least two different stable conformations. the conformational transition that shifts the equilibrium from the functional to the pathological isoform can happen sporadically. it can also be triggered by mutations in the primary structure, changes of different environmental conditions, or the action of chaperones. elucidation of the molecular interactions that occur during the transformation from α-helix to β-sheet and the consecutive formation of amyloids on a molecular level is still a challenge. therefore, the development of small peptide models that can serve as tools for such studies is of paramount importance. we succeeded in generating model peptides that, without changes in their primary structure, predictably react on changes of diverse environmental parameters by adopting different defined secondary structures. these de novo designed peptides strictly follow the characteristic heptad repeat of the α-helical coiled coil structural motif. furthermore, domains that favour β-sheet formation and aggregation can be generated.1 alternatively, those peptides can be equipped with functionalities that allow either the binding of metal ions or the interaction with membranes. as proof of our concept we showed that the resulting secondary structure of such peptides will strongly depend on environmental parameters. thus, this system allows to systematically study the interplay between peptide / protein primary structure and environmental factors for peptide and protein folding on a molecular level. the pathogenesis of alzheimer's disease (ad) is strongly linked to neurotoxic assemblies of the amyloid β protein (aβ). aβ is a soluble component of human plasma which by an unknown mechanism becomes aggregated and neurotoxic. some genetic mutations within the aβ sequence cause very early onset of ad-like diseases, probably by facilitation of aβ assembly into neurotoxic species. recently, it was found that not amyloid fibrils, but smaller aβ assemblies initiate a pathogenic cascade resulting in ad. therefore, preventing the folding of nascent aβ monomers would have therapeutic benefit. to uncover details of structural changes accompanying the aggregation process, especially its initial stage, we have decided to study the aβ(11-28) fragment and its mutation-related variants. our recent studies on this aβ fragment using the cd method and the aggregation test have proved it a good model for structural studies. the obtained results confirmed that the aggregation process follows the scheme with an α-helical intermediate and pointed out differences in the behaviour of aβ variants. to further confirm the scheme of the structural changes accompanying aggregation we have applied ftir spectroscopy and analysed aggregation-induced changes of the amide i band which is directly related to peptide backbone conformations. the ftir spectra analysis indicate that water addition provoked conformational changes are strongly dependent on the aβ(11-28) variant and in some cases the formation of α-helical intermediate seems to be preceded by 310 helix formation. to verify this hypothesis the temperature dependent atr ftir spectra will be analysed. supported by ug bw grant. amino acid octarepeats present in the prion protein bind to cu2+ and are considered as a potential periplasmic copper ion transporters. this octarepeat is located in the unstructured region of the prion protein, which is supposedly not intricately involved in prion aggregation. our group is involved in exploring the function of octarepeats with a special emphasis on their possible role in amyloid fibril formation and aggregation.1 in this context, we have prepared truncated peptide constructs derived from the prion protein octarepeat phgggwgq and have reported their fibrillation activity. we will present aggregative behavior of a truncated bis-pentapeptide, containing gggwg segment, when tethered with a flexible linker diaminobutane. fibrillar architectures were observed by this bis conjugate after incubation in water which was probed by different microscopic and steady state fluorescence techniques. further investigations with ki revealed a homogeneous environment of the two tryptophan moieties in the conjugate. in the absence of other side-chains, it is likely that fibril formation involves hydrophobic interaction between tryptophan indole moieties and main chain backbone interactions. interestingly, a facilitator role for aromatic-glycine motifs for amyloid aggregation has been proposed based on bioinformatics search of the swiss-prot and trembl databases. collagens are known to fold into a highly ordered rode-shaped triple helix with stretches of lower and higher suprastructural stability and even disruptions to modulate recognition by other proteins that interact with the extracellular matrix [1] . to increase understanding of folding and stability of the collagen triple helix, we have adressed the design of photocontrolled collagenous peptides. our aim was to crosslink two side chains of the repetitive (xaa-yaa-gly) sequence motifs of collagen model compounds via an azobenzene chromophore in analogy to our previous studies on photomodulation of the conformational preferences of cyclic peptides and more recently of hairpin-peptide model systems [2] . molecular modeling experiments suggested appropriate sequence positions that could result in triple-helical peptides with conformational stabilities that can be modulated by cis/trans isomerization of the azobenzene moieties. as light switchable crosslinker azobenzene-4,4'-n-(4-iodo-2-butynenyl)carboxyamide was synthesized for reaction with two (4s)-mercaptoproline residues placed in suitable xaa and yaa positions, respectively. by this approach a fully folded triple helix was obtained upon thermal relaxation, and unfolding was induced by irradiation at 350 nm. the favorable optical properties of the azobenzene derivative together with the regular suprastructure resulted in a valuable model system that allows for ultrafast time-resolved studies of collagen folding and unfolding. amyloid formation is connected with alzheimer's disease, parkinson's disease, finnish familial amyloidosis. after protein misfolding short peptide sequences act as "hot spots" providing the driving force for protein aggregation in amyloid fibrils. we have identified one of these sequence stretches in the abl-sh3 domain of drosophila (dlsfmkge) whereas the human homologous region (dlsfkkge) is predicted to be less amyloidogenic. the possible reason for the difference of amyloid formation propensities of the two peptides was investigated by molecular dynamics (md) of β-sheet structures. the antiparallel alanine β-sheets consisting of two and ten strands were constructed, minimized, and mutated to the sequences dlsfmkge and dlsfkkge. all four systems: 1) dlsfmkge -two strands, 2) dlsfkkge -two strands, 3) dlsfmkge -ten strands, 4) dlsfkkge -ten strands, were surrounded by 10 å layer of water molecules over the solute and subjected to md, amber 8.0 force field, ntp protocol. the md runs were started at the temperature of 10 k and the temperature was elevated stepwise by 10 degrees till 300 k. the results show considerably higher hydrogen bond percentage for dlsfmkge than that one for dlsfkkge during the course of the simulation, thus suggesting that dlsfmkge is a potential fibril-maker, but dlsfkkge is not. two strand β-sheet systems were stable until 170 k. the ten strand β-sheets are more stable. angiotensin-i converting enzyme (ace) has a critical role in cardiovascular function, which consists of cleaving the carboxy terminal his-leu dipeptide from angiotensin-i producing a potent vasopressor octapeptide, angiotensin-ii. there are two isoforms of ace. the somatic isoform is present in all human cells except the testis cells, where the testicular isoform is produced. the major difference between these two types is that, the somatic form has two active sites, at the n-and c-end respectively while the testicular has only one, which is almost identical to the somatic c-terminal active site. here we report the structural study of a 108aa peptide (previously expressed in bacteria), which corresponds to an extended domain of the human somatic n-terminal active site of ace (ala361-gly468) by circular dichroism experiments, and the overexpression in bacteria, purification and structural study, using circular dichroism techniques, of a 108aa peptide which corresponds to an extended domain of the human somatic c-terminal active site of ace (ala958-gly1065). following the subclonning into an appropriate expression vector and the expression, the peptide was isolated from the inclusion bodies using chromatography techniques. the recombinant protein fragment had a molecular weight, measured by esi-ms, of 12102 kda which was in consistence with the theoretical calculation based on the dna sequence. the recombinant peptides acquired their theoretically calculated secondary structure only when 1,1,1-trifluoroethanol is present at a concentration of ~70%. in order to elucidate their structures, solutions of these peptides, labeled with 15n and/or 13c, will be studied by nmr spectroscopy. aggregation of peptides is believed to trigger various degenerative diseases but it also plays an important role for the preparation of peptide fibres and peptide-based biomaterials. it is therefore extremely important to understand the mechanisms involved in peptide aggregation and be able to control them. studies were performed on a library of amphiphilic peptides, designed around the sequence of a model antimicrobial peptide rich in leucine and lysine. the library also included peptide hybrids in which natural amino acids were replaced by non-proteinogenic omega-amino acids, such as 6-aminohexanoic acid and 9-aminononanoic acid. the aim was to estimate the aggregation and its correlation with the biological activity by using a fluorescence technique commonly employed to calculate the cmc (critical micelle concentration) of surfactants. peptides and peptide hybrids were synthesized on solid support using the fmoc polyamide protocol. they were purified by semi-preparative rp-hplc and characterized by esi-ms and analytical rp-hplc. the aggregation behaviour of the synthesized molecules was investigated in water by steady-state fluorescence measurements using pyrene as fluorescent probe. peptides were dissolved in water/pyrene or water/pyrene/0. fluorescence spectroscopy has become an extremely valuable technique for conformational studies of biopolymers, the development of peptide-based chemosensors, and biochemical research in general. in this connection, synthetic amino acids as fluorescent probes to be incorporated into a peptide chain may exhibit significant advantages over the related protein (trp and tyr) residues in terms of potentially different and ameliorated properties. we recently designed and prepared a new fluorescent amino acid, antaib, based on a planar anthracene core and belonging to the class of achiral, ciα↔ciα cyclized, cα-tetrasubstituted α-amino acids (strong β-turn and helix inducers in peptides). peptides based on antaib combined with (l)-ala residues were synthesized and subjected to a conformational analysis. more specifically, the protected derivatives boc-antaib-oet (oh) and fmoc-antaib-otbu (oh) were prepared in seven steps from 1,2, 4 conformational transitions in peptides and proteins emerge to play the major role in the genesis and evolution of prion related diseases and alzheimer's disease (ad).1 in this context, conditions influencing this transition and the following aggregation process are of paramount interest. peptides and proteins that are involved in aggregation processes contain potential metal binding sites. the concentration of metal ions in the brain tissue is naturally high and zn in the mm range has been found in ad amyloid plaques. thus, it is widely accepted that metal complexation is one of the key incidents that lead to conformational transitions and aggregation. we present here a coiled coil based model peptide system with an intrinsic amyloid forming tendency which can be used to study the impact of different metal ions on secondary structure and aggregation. metal complexing histidine residues were incorporated to create potential binding sites which, depending on their position and the nature of the metal ion, dictate folding and aggregation. the time dependent conformational transition was monitored by cd-spectroscopy. aggregates were characterized by cryo tem. high resolution fticr-ms experiments revealed information on the stoichiometry of the peptide-metal complexes. in the absence of metal ions the presented peptides formed amyloids in a time range of weeks. depending on the his positions and milieu conditions, the nature of the metal ion determines folding and aggregate morphology. furthermore, metal binding was shown to inhibit the amyloid formation. a challenge to our understanding of protein folding is the design of a protein from first principle, i.e. starting from geometric restraints and applying properties of amino acids expected to be essential for folding to a defined structure. we developed a program to calculate the backbone coordinates of antiparallel strands to match the surface of an elliptical cylinder. various parameters like the number and shearing of the strands and the ellipticity of the structure can be varied. the relative orientation of the β-strands and the geometrical features of the hydrogen-bonds were derived from statistical analyzes of natural β-sheet structures. iterative cycles of core-packing with amino acid rotamers, molecular dynamics simulation and energy minimization with the charmm forcefield are used to include backbone movements and to minimize the risk of trapping an energetically unfavorable structure. the quality of residue packing is assessed with the help of criteria which proved to be successful in our design of β-sandwiches. at the protein surface, a network of salt-bridges with an excess of positive charges has been designed to increase the stability and the solubility of the protein. the final sequence is synthesized by standard solid phase fmoc-chemistry. insights gained from the analysis of the synthesized structure with ftir and cd spectrometry should help us to refine the parameters for subsequent designs. with this strategy, we hope to contribute to a better understanding of protein folding. the immunoglobulin binding protein g (1igd) from streptococcus species consists of 61 amino acids residues, which form two antiparalell-packed beta-hairpins and an alpha-helix in the middle of the sequence packed to the beta-sheet. the second hairpin was found to be stable in isolation. this fragment is therefore likely to be the first folding initiation site of the protein which could provide an adequate nucleation center on which the rest of the polypeptide chain would find a favorable environment to fold. thus, among the two beta-hairpins, the 48-59 fragment of 1igd corresponding to the c-terminal beta-hairpin was synthesized. in our studies, we investigated different environmental and temperature conditions for formation of the 48-59 beta-hairpin structure. its structure was examined by means of cd spectroscopy in water, buffer solutions (ph = 3 to 9) and in aqueous solutions of trifluoroethanol. additionally, its structure was investigated in the solid state by ftir spectroscopy. the cd studies revealed that the 48-59 fragment of 1igd in water forms mainly a statistical-coil structure, whereas the ftir technique shows formation of a regular beta-sheet structure. nmr spectroscopy and calorimetric measurements were carried out at various temperatures. our studies show that the 48-59 fragment at low temperature exists in an equilibrium between two conformations -a regular beta-hairpin and a statistical-coil. although increasing temperature resulted in shifting the equilibrium in the direction of the statistical-coil structure, the overall beta-hairpin shape of the 48-59 fragment was maintained. prion diseases are characterized by the conversion of the physiological cellular form of the prion protein (prpc) into an insoluble, protease-resistant abnormal scrapie form (prpsc) with an highly beta-sheet content [1] . the analyses of intrinsic structural propensities of the prp c-terminal domain showed an high conformational flexibility for the αhelix 2 fragment which indicates that this region may be particularly important in the prpc→prpsc transition [2] . therefore conformation-based approaches focalized on helix 2 region appear to be the most promising for the study of prion protein misfolding. recent studies on tetracycline properties showed that this molecule binds and disrupts prp peptide aggregates and inactivates the pathogenic forms of prp [3, 4] . a fluorometric titration of the fluoresceinated peptide corresponding to prion protein helix-2 with tetracycline has been carried out to determine the value of the apparent dissociation constant of this interaction (estimated to be 189 ± 7 nm). remarkably, the fluoresceinated peptide exhibits in water a canonical α-helical cd spectrum, that is maintained even in presence of tetracycline. accordingly, docking calculations and molecular dynamics simulations suggest that tetracycline interacts preferentially with the c-terminal end (residues 183-195) of helix-2 with a significant involvement of the treonine rich region. in the last decades, a series of discoveries have shed light on the role played by the carbohydrate moiety in glycoproteins. it has been shown that covalently linked sugar moieties influence peptide/protein properties such as hydrophobicity, conformation, biostability and bioactivity. the design of carbohydrate-peptide analogs with increased, retained or modified biological activity requires an understanding of their conformational preferences both in solution and in the receptor-bound state. in our recent work we have created two classes of well-structured linear and cyclic carbohydrate-modified analogs of opioid peptides, leu-enkephalin and leuenkephalin amide. the first class represents a group of compounds in which the linear peptide is alkylated at the n-terminal position by 1-deoxy-d-fructose unit, while its cyclic analog possesses an ester bond between the c-6 hydroxy group of the sugar moiety and the c-terminal carboxy group of peptide, 1-deoxy-dfructofuranose acting as a bridge between the leu-enkephalin terminal parts. the rigid 5-membered imidazolidinone ring is characteristic for the second class of compounds. in these adducts an imidazolidinone moiety connects the acyclic sugar residue with the linear peptide chain. in the corresponding bicyclic imidazolidinone analogs 19-membered ring is formed through an ester bond between the primary hydroxyl group of the d-gluco-pentitolyl residue and the cterminus of peptide. this work reports the comparative cd and ftir spectroscopic properties of the prepared glycopeptides in comparison with data on the non-modified flexible parent peptides performed in different solvents in order to expose the structural and conformational differences caused by a keto-sugar, rigid 5membered imidazolidinone ring and/or cyclization. the α-helical coiled coil structural motif consists of two to five α-helices which are wrapped around each other with a slight superhelical twist. the simplicity and regularity of this motif have made it an attractive system to study the role of complementary interactions for protein folding. here we present a systematic study showing that intermolecular electrostatic interactions between positions e and g of the helices are in competition with the intramolecular interactions between positions e/b and g/c. those competitive interactions affect folding and stability of the motif which were monitored by temperature dependent cd-spectroscopy. incorporation of oppositely charged amino acids in positions e/b and g/c reduced considerably electrostatic repulsion between equally charged amino acids in positions e and g. in addition coiled coil stability can be increased by the alkyl part of the amino acid side chains in positions e and g. studies with natural and unnatural amino acids showed that the longer this alkyl part the better is the hydrophobic core protected from solvent. therefore the repulsion of equally charged amino acids in positions e and g can be overruled by involving them either into attractive intrahelical electrostatic interactions or into hydrophobic core formation. human cystatin c (hcc) is a 120 amino acid residues protein that reversibly inhibits papain-like cysteine proteases. this inhibitor belongs to the amyloidogenic proteins shown to oligomerize through 3d domain swapping mechanism. the crystal structure of hcc reveals the way the protein refolds to produce symmetric dimer while retaining the secondary structure of the monomer. the monomeric form of hcc consists of a core with a five-stranded antiparallel β-sheet wrapped around a central α-helix. the hcc dimerization is preceded by an opening movement of l1 loop from β2-l1-β3 hairpin and separation of the β1-helix-β2 fragment from the remaining part of the molecule. the amino acid sequence of β1-helix region suggests additional possible partial unfolding in the n-terminal part of helix. in order to investigate the structural stability of β1-helix region the peptide corresponding to the helix and its n-terminal truncated analogs was synthesized along with the peptide analogs of helix containing point mutations that could stabilize helical structure of the n-terminus. the peptides were synthesized by the solid-phase method using fmoc/tbu tactics. purified products were identified by maldi-tof. the secondary structure content was calculated from the cd spectra using selcon3. the random coil was the predominant structure of the peptide corresponding to the α-helical fragment of hcc and its n-truncated analogs. however, an increase of αhelix content was observed in some of the peptides corresponding to the helix containing point mutations. we expect that these mutations could stabilize the hcc monomer and suppress dimerization. the tumor suppressor protein p53 is a trarnscription factor that triggers cell-cycle arrest and apoptosis in response to genotoxic stress signals. the tetramereric structure of the p53, which is essential for its activity as a transcription factor, is formed as a dimer of dimers. while the primary dimer is constructed from inter molecular formation of a two-stranded anti-parallel b-sheet and a two anti-parallel a-helix bundle, the secondary dimer is stabilized through interactions between residues on the surface of the primary dimer. from various substitution experiments on p53, it has been shown that hydrophobicity of phe341 is critical for the tetramer formation of p53. also we have substituted three phenyl groups of p53 with cyclohexylalanine (cha) and showed that phe341cha is dramatically stabilized against temperature, chemical denaturant, and organic solvent by cd measurements. here, to clarify the mechanism of the stabilization of phe341cha, we analyzed its three dimensional structure using x-ray crystallography. we obtained two kinds of crystals, one is a hexagonal bipyramid crystal in the space group of p<i>6<sub>4</sub>22</i> with <i>a</i>=50.12 å, <i>b</i>=50.12 å, <i>c</i>=48.18 å diffracted to about 2.0 å, and another is tabular crystal in the space group c<i>2</i> with <i>a</i>=77.25 å, <i>b</i>=50.04å, <i>c</i>=55.10 å diffracted to about 2.1 å. in these crystals, the peptides formed tetramers which are very similar to those observed in the wild-type. the structure of the pocket where the side chain of cha341 is incorporated was defined to elucidate the hydrophobic interaction to determine the stability. helices shown in proteins, as a secondary structure, almost always form right-handed screw sense. this right-handedness is believed to result from the chiral center at the αposition of proteinogenic l-α-amino acids. among proteinogenic amino acids, l-isoleucine and l-threonine possess an additional chiral center at the side-chain β-carbon besides the α-carbon. however, no attention has be paid how the side-chain chiral centers affect the secondary structures of their peptides. recently, we have reported that sidechain chiral centers of chiral cyclic α,α-disubstituted amino acid (s,s)-ac(5)c(dom) affected the helical secondary structure of its peptides, and the helical-screw direction could be controlled without a chiral center at the α-carbon atom. herein, we synthesized a chiral bicyclic α,α-disubstituted amino acid (r,r)-ab(5,6)c and its homopeptides, and studied the relationship between the chiral centers and the helical-screw handedness of peptides. contrary to the left-handed helices of (s,s)-ac (5) four threefinger-toxins (tfs) have been purified from the pooled venom of golden krait (bungarus fasciatus, i.e. bf) from thailand and studied previously. these peptide toxins contain 60-65 residues and 4 or 5 pairs of disulfide bonds, and are rich in β-structure. we herein analyzed the tf-isoforms in bf venoms from kolkata (eastern india), hunan province (eastern china) and indonesia to study the geographic variations and structure-function relationships of the venom polypeptide family. a total of five or six tfs of low lethality were purified from each of the geographic venom samples, the n-terminal sequences and accurate masses of the peptides were determined. the cdnas encoding some of these tfs were also cloned and sequenced. full peptide sequences were deduced and match with those of the tfs purified from crude venom. intra-species variations of the venom tfs were found to be surprisingly high since sequence-identities between the majorities of orthologous toxins in different geographic samples are only 75-80 %. most of the bf proteins were not neurotoxic by electrophysiological assays using chick biventer-cervicis and mouse diaphragm neuromuscular tissues. the toxins appear to be associated with weak toxins or non-conventional snake venom tfs as analyzed by a phylogenetic tree. the reason behind their lack of neurotoxicity would be discussed. v. moussis, e. panou-pomonis, c. sakarellos, v. tsikaris peptides involved in neurodegenerative diseases can adopt at least two different stable secondary structures. amyloid-forming proteins can experience a conformational transition from the native, mostly α-helical structure, into a ß-sheet rich isoform. the latter conformation is probably present in intermediates for the formation of amyloids. the conformational change can be triggered by protein concentration or environmental changes. therefore, our aim was to generate a de novo designed peptide that contains structural elements for both, stable α-helical as well as ß-sheet formation. this model peptide can be used to elucidate the conformational changes dependent on concentration and ph.1 the design is based on the well studied α-helical coiled coil folding motif. the conformation and structure of the resulting aggregates were characterized by cd-spectroscopy and cryo transmission electron microscopy. as a result, three distinct secondary structures can be induced at will by adjustment of ph or peptide concentration. low concentrations at ph 4.0 yield globular particles of the unfolded peptide whereas, at the same ph but higher concentration, defined ß-sheet ribbons are formed. in contrast, at high concentrations and ph 7.4, the peptide prefers highly ordered α-helical fibers. in conclusion, we successfully generated a model peptide that, without changes in its primary structure, predictably reacts on environmental changes by adopting different defined secondary structures. thus, this system allows to systematically study now the consequences of the interplay between peptide primary structure and environmental factors for conformation on a molecular level. cells respond simultaneously to a multitude of different signals. inside a cell signals from activated receptors are integrated by networks of enzymatic reactions and molecular interactions, leading to a spectrum of cellular responses. in order to understand the relationship between a specific cellular stimulus and a cellular response, methods are required to detect in parallel the pattern of molecular interactions. a large number of molecular interactions is mediated by protein domains, binding to linear peptide motifs. lysates of activated and resting cells were incubated on peptide microarrays carrying peptides corresponding to such binding motifs of signalling domains. binding of proteins to a spot of the array was probed by immunofluorescence. jurkat t cells were stimulated either with the phosphatase inhibitor pervanadate or with antibodies directed against cell surface receptors. upon activation of t cells, numerous changes in the pattern of molecular interactions were detected for a total of 10 proteins and 30 peptides. these changes were caused either (i) by masking or unmasking of a binding domain which resulted in a reduced or increased binding of a protein to the microarray or (ii) by recruitment of a protein into a complex that in turn bound to the microarray. the changes were dependent on the nature of the stimulus. the human melanocortin receptor 1 (hmc1r) was constructed to contain a flag epitope and a hexahistidine tag at the amino-terminus as well as at the carboxyl terminus to facilitate purification. stably transfected hmc1r in human embryonic kidney (hek293) cell lines that expressed the receptor resulted in a kd value of 0.1 and 0.2 nm respectively in each case when the super potent agonist mtii was competed with [125i]ndp-α-msh. treatment of the tagged receptors in the hek293 cells with agonist resulted in down-regulation which indicates that these tagged receptors retain their biological functions. the hmc1r was solubilized from cell membranes with n-dodecyl-β-d-maltoside and purified at a nickel chelating resin and a newly constructed affinity column. the purified hmc1r was a glycoprotein that migrated on sds/page with a molecular mass of 58 kda. the results from matrix-assisted laser desorption ionization-time of flight (maldi-tof) mass spectrometry was used to identify and characterize peptides derived from the hmc1r following in-gel digestion with chymotrypsin. the phosphorylation sites were identified on the purified human melanocortin receptor 1 with agonists (peptide vs small molecule) treatment. the discovery of antibiotics in the 1930s has been one of the most revolutionary events in the history of medicine. however, during last decades, the increase of antibiotic resistance has significantly hampered the application of antibiotics. therefore, further scientific effort to find new antibiotics with novel mechanisms of action is of high importance. insects are the largest and the most diverse group of living animals on earth. they have potentially been confronting high variety of microorganisms. as a result, they have evolved powerful defense system, thus representing vast source of novel potential therapeutics. we chose larvae of fleshfly neobellieria bullata for identification and characterization of new promising molecules, peptides or proteins, which participate in immunity response against microbial infections. the hemolymph of the third-instar larvae of neobellieria bullata was used for isolation. the larvae were injected with bacterial suspension of escherichia coli or staphylococcus aureus to induce antimicrobial response. the hemolymph was separated into crude fractions, which were subdivided by rp-hplc. isolated fractions were characterized by uv-vis spectroscopy, amino-acid analysis, mass spectroscopy, 1-d and 2-d sds electrophoresis, capillary zone electrophoresis, ion-exchange hplc, tryptic digests and n-terminal sequencing. we found out significant antimicrobial activities against escherichia coli, staphylococus aureus or pseudomonas aeruginosa in several fractions. using real-time pcr, we followed and compared levels of mrna of different proteins and peptides in induced and non-induced larvae. despite the fact that many technological advances are currently involved in proteome analysis, like two-dimensional gel electrophoresis and mass spectrometry, there is still a great need for the development of novel engineered chemical probes for proteomics and interactomics. here, we describe our approach concerning the study of proteome and interactome of proteins involved in cell-matrix interactions. it relies on the use of a small synthetic inhibitor chemically modified to allow for its immobilisation to magnetic beads or affinity chromatography materials. proteins will be detected together with their native interaction partners because of nondenaturing conditions. this general procedure is applied for the enrichment of metalloproteinases, especially matrix metalloproteinases, which are potential target in tumour therapy. hydroxamic acids are known to be potent inhibitors of metalloproteinases. marimastat is a reversible inhibitor with a good potency and shows activity towards a wide range of metalloproteinases. the synthesis of new marimastat derivatives will be reported. the parent compound is modified with a linker to allow immobilisation on a solid surface. binding studies were performed using surface plasmon resonance. this approach is not only appropriate for the generation of metalloproteinase proteome subsets by affinity column or using magnetic beads, but also to enrich and isolate interaction partners of the target proteins. the present report summarizes the latest data devoted to theoretical and experimental investigation of the high temperature solid state catalytic isotope exchange reaction (hscie) that takes place in peptides and proteins by the action of deuterium and tritium [1] . the available ms-procedures, designed to estimate the amount of protein, are aimed at derivatization at different stages of sample preparation, and as the best result, it is only possible to achieve quality comparison of the objects involved. the hscie reaction allows the production of evenly deuterium labelled proteins and peptides, and their application makes it possible to create a qualitative mass spectrometry method for protein analysis. introduction of definite amounts of these deuterium-labeled proteins into biological objects, prior to isolation, separation and trypsinolysis, will generate quantitative information concerning the composition of the proteins under study. tritium labelled proteins produced at a temperature of 100-120oc carry the isotopic label in all the peptide fragments and completely retain their enzymatic activity. the proteins' reactivity is dependent on their three-dimensional structure. the hscie reaction has been shown to be used both in the production of tritium labelled proteins and in the investigation of spatial interactions in protein complexes. in addition, evenly deuterium and tritium labelled peptides can be used in studies of the kinetics and transformation paths of peptides in the organism's tissues. immunization of mice with type ii collagen (cii) from rat leads to development of collagen-induced arthritis (cia). susceptibility to cia is associated with the major histacompatibility class ii protein h-2aq that binds the glycopeptide epitope cii260-267 (1) and presents it to helper t cells. [1] to explore the interactions in the system and to stabilize 1 towards in vivo degradation, amide bond isosteres have been introduced in its backbone.glycopeptide 1 was virtually docked into the binding site of a comparative model of h-2aq. based on the hydrogen bonding network between the peptide backbone and h-2aq, the amide bond between ala261-gly262 was chosen for isosteric replacement. to vary the geometric and hydrogen bonding properties, mimetics of the dipeptide were synthesized with the amide bond replaced by ψ[ch2nh], ψ[coch2] and ψ[(e)-ch=ch], respectively. these were introduced in 1 using solid-phase synthesis to give glycopeptidomimetics that were biologically tested for their ability to bind to the h-2aq protein and for recognition by t cells. shown to be a potent neuroprotective factor in various pathophysiological models. despite its therapeutic potential in diverse neurodegenerative diseases, its short in vivo half-life limits its utility as a useful clinical agent. moreover, the development of a peptidomimetic that reproduces the pharmacological activity of pacap is unlikely since the pharmacophores are spreaded throughout the entire peptide chain. therefore, the development of pacap analogues with lower susceptibility to proteolysis represents a first step toward clinical applications. in the present study, derivatives of both pacap27 and pacap38 with particular chemical modifications were developed targeting specific peptidase sites of action. results indicate that the incorporation of an acetyl or a hexanoyl group at the n-terminus and modifications at the ser2 residue contributed to increase stability against dipeptidyl peptidase iv, the major enzyme involved in pacap degradation. moreover, after determination of pacap metabolites in human plasma, the amide bond between residues 21 and 22 was substituted by a ch2nh surrogate and this derivative showed increased plasma stability. all modified peptides were tested for their ability to induce pc12 differentiation. the effects of pacap analogs on pc12 cells are mediated through the pac1 receptor which is the major receptor involved in the neuroprotective effects of pacap. this study exposes interesting data concerning pacap metabolism in isolated human plasma and demonstrates the possibility of increasing the metabolic stability of pacap without significantly reducing its biological activity. species of the fungal genus trichoderma are commercially used as bioprotective agents against fungal plant diseases. more than 400 strains were collected from their natural habitats and evaluated for biocontrol properties. seven of the most active isolates exhibiting strong biological activity towards eutypa dieback and esca diesease of grapevine were classified as trichoderma brevicompactum, or shown to be closely related to that species. these strains were screened for production of peptaibiotics. the formation and synergistic action of hydrolytic enzymes and peptaibiotics were to play an important role in mycoparasitism. after background and aims: conotoxins are short, disulfide-rich neurotoxins that target various ion channels and receptors. these peptides have desirable pharmacological properties to become therapeutics for neurological disorders; several conotoxins have already reached clinical development stage. our long-term goal is to improve bioavailability, metabolic stability and pharmacokinetics of conotoxins using a variety of chemical modifications. methods: we designed and chemically synthesized conotoxin analogs containing two distinct types of backbone modifications: (1) peptide-peptoid chimeras (conopeptoids) of alpha-conotoxin imi and (2) peptide chimeras of mu-conotoxin kiiia containing non-peptidic "backbone spacers". results: conopeptoid-imi, containing ala9 replaced by n-methyl glycine potently blocked activity of nicotinic acetylcholine receptors. in mu-conotoxin kiiia, aminohexanoic acid or amino-3-oxapemtanoic acid were inserted to be a part of the peptide backbone. the two oxidized analogs containing "backbone prosthesis" differed in their hydrophibicity profile, but they both potently inhibited neuronal sodium channels. conclusion: our results suggest that backbone engineering may become an effective method of producing conotoxin analogs with modified bioavailability. to increase the stability and the therapeutic efficacy of peptide sequences from myelin oligodendrocyte protein (mog) that act as multiple sclerosis (ms) antigens, we grafted them onto a framework of a particularly stable class of peptides, the cyclotides. they are a recently discovered family of cyclic plant peptides with superb intrinsic stability. the limitations of linear peptides as drugs due to their instability and poor bio-availability can be overcome by using the cyclotide scaffold as a framework for novel drug design. peptide epitopes from mog protein were incorporated onto the framework of the model cyclotide kalata b1 by means of boc-spps approach. after successful backbone cyclisation and oxidation of the cysteine residues, the peptides were purified to high purity with rp-hplc. nmr chemical shift analysis was used to assess whether the grafted analogues have a stable scaffold, similar to that of kalata b1. a structure of a representative peptide was determined and it shows remarkable resemblance to the native scaffold of kalata b1. the activity of the bioengineered peptides has been tested in vivo. a group of mice injected with one of the peptides have shown a depression in the clinical score and have not fallen ill. this is an exciting result that shows the first active bioengineered cyclotide in an animal model of disease. the structural information from nmr studies will be used in conjunction with the results from the activity studies in a feedback loop to design second-generation lead molecules. a.d. de araujo, p.f. alewood conotoxins are small, disulfide-rich peptide neurotoxins produced in the venom of marine cone snails that enable these molluscs to capture their prey. these compounds exhibit a high degree of selectivity and potency for different ion channels and their respective subtypes, making them useful tools in the investigation of the nervous system. due to the key role of ion channels in many physiological processes, conotoxins are also excellent drug lead candidates in drug discovery, with some examples currently undergoing clinical trials and one recently approved drug (prialt®). like other peptide drugs, the use of conotoxins in drug development is limited by the low oral bioavailability of these compounds due to pre-systemic enzymatic degradation and poor penetration through the intestinal mucosa. peptide analogs that mimic the native structure and incorporate rationally designed non-natural modifications in the disulfide framework or peptide backbone may exhibit increased resistance to proteolytic degradation. in biological environments, disulfide linkages are susceptible to attack by enzymes and reducing agents (such as glutathione). therefore we carried out the synthesis of redox-stable conotoxin analogs in which intrinsic disulfide bonds were replaced by thioether linkages. in this work, wehave explored two solid-phase synthetic routes in the preparation of thioether conotoxin mimetics: the first route based on peptide assembly using a tetra-orthogonally protected lanthionine building block, and the second route based on intramolecular on-bead cyclisation between cysteine and betabromoalanine residues. thyrotropin releasing hormone (trh, l-pglu-l-his-l-pro-nh2), a tripeptide synthesized in the hypothalamus, operates in the anterior pituitary to control levels of tsh (thyroid-stimulating hormone) and prolactin. the thyrotropin-releasing hormone (trh) receptor (trh-r) belongs to the rhodopsin/β-adrenergic receptor subfamily of seven transmembrane (tm)-spanning, g protein-coupled receptors (gpcrs). the two g-protein-coupled receptors for trh, trh receptor type 1 (trh-r1) and trh receptor type 2 (trh-r2), have been cloned from mammals and are distributed differently in the brain and peripheral tissues. the trh receptor subtype-1 appears to mediate the hormonal and visceral effects, whereas trh receptor subtype-2 has been implicated in its central stimulatory actions. identification of critical features of the trh, separation of its multiple activities through design of selective analogues and affinity labels have been elusive and unfulfilled goals for more then 30 years. this presentation will highlight our studies on effect of the biological activity of trh with the introduction of alkyl groups of varying sizes at the n-1 and c-2 position of the centrally placed histidine residue of trh peptide. the requisite n--boc-dialkyl-l-histidines as building scaffolds have been synthesized in six steps from l-histidine methyl ester dihydrochloride and used for the synthesis of various trh analogues. ghrelin, the natural ligand for the growth hormone secretagogue receptor (ghsr-1a), has received a great deal of attention due to its ability to stimulate growth hormone secretion and to control food intake. during these last years, ghrelin analogues or mimetics gained interest for their implication in food intake regulation. in the course of our studies concerning ghrelin analogs, we developed new ligands of the ghsr-1a based on heterocyclic structures. interestingly, one heterocyclic family presented high affinity for the ghrelin receptor. a structure activity study was performed within this family and led to potent ghsr-1a agonists and antagonists. the binding affinities were determined by displacement of radiolabelled ghrelin and the agonist or antagonist character was measured by intracellular calcium mobilisation. the first in vivo results revealed that in vitro activities and in vivo responses were not correlated. particularly, binding to ghsr-1a and in vivo gh release and food intake control were not fully correlated. these results strongly suggest the presence of receptor subtypes that modulate ghrelin actions. some examples will be reported. further investigations are going on in the laboratory. background and aims: alzheimer's disease (ad) related beta-amyloid (aβ) peptides possess high propensity towards aggregation. nowadays one of the major directions of the drug-design against ad is the synthesis of putative amyloid aggregation inhibitor molecules (aai) which are able to hinder the formation of these toxic amyloid aggregates. in the present work we report the design, synthesis and investigation of putative aais derived form the peptide sequence aiigl identical to aβ(30-34). methods: aβ(1-42) peptide and putative aggregation inhibitors were synthesized manually using fmoc-chemistry and dcc/hobt activation. studies on both the aβ aggregation and the effect of the aais were performed with several instrumental techniques. the total amount of the aggregates was determined by thioflavine-t binding assay, while their size distribution was characterized with dynamic light scattering (dls). aggregated aβ forms were visualized with transmission electron microscopy (tem). the binding affinity of the aais to aβ fibrils was studied in saturation transfer nmr experiments, while in vitro viability assays were performed on cultured human sh-sy5y neuroblastoma cells to monitor the neuroprotective effect of the amyloid aggregation inhibitors. results and conclusions: 32 derivatives of aβ(30-34) were synthesized and tested concerning their neuroprotective effect against aβ-mediated toxicity. the most promising candidates were examined with physico-chemical methods to characterize their aggregation altering ability. the pentapeptide riigl-amide proved to be the most powerful neuroprotective compound, and it was also able to alter considerably the aggregation kinetics of aβ(1-42) . this molecule might serve as a lead compound in the drug-design against ad in the future. m. mattiuzzo, a. bandiera, m. benincasa, i. samborska, m. scocchi, r. gennaro antimicrobial peptides (amps) are ancient molecules of the innate immune defence system. most of them kill bacteria by lysing their membranes. the proline-rich group of amps represents an exception, as they act via a permeabilization-independent mechanism that is likely based on recognition of molecular targets/transporters. however, specific internal targets have not yet been identified for most of these amps. bac7 is a pro-rich amp isolated from bovine neutrophils that is capable to translocate across membranes to target hypothetical intracellular proteins. in this study, we used a molecular approach to identify putative targets for bac7 by selection of peptide-resistant mutants followed by identification of the genes responsible of this resistance. to this aim, an e. coli strain susceptible to the fully active fragment bac7(1-35) was subjected to chemical mutagenesis and a number of peptide-resistant mutants was obtained. a pool of genomic dna from these mutants was used to construct a plasmid library that was used to transform a susceptible strain. this approach allowed the identification of 14 different clones that provided a high level of resistance to bac7. sequence analysis revealed the presence of genes originating from five different regions of the e. coli chromosome. among them, one clone contained ptrb, a gene coding for a serine peptidase broadly distributed among gram -bacteria, which could be involved in resistance by degrading the peptide. other resistanceconferring clones, which encode for membrane proteins that may be involved in peptide translocation across the memmbrane, are currently under investigation. lycotoxins are peptides from the venom of the wolf spider that were predicted to have an amphipatic alpha-helical structure and confirmed to possess significant antimicrobial and pore-forming activities. [1] we became interested in these peptides as potential leishmanicidal agents against leishmania donovani promastigotes and leishmania pifanoi amastigotes. thus, lycotoxin i (lyi) and lycotoxin ii (lyii), [1] and shortened analogues lyi1-19, lyi1-15, lyii1-21 and lyii1-17, were synthesized as c-terminal carboxamides. short-and long-term parasite proliferation measurements showed all peptides except lyi1-5 to be active against both promastigotes and amastigotes at the micromolar range, and suggest peptide effects on parasites to be irreversible. lyii, that showed the lower activity, became more leishmanicidal upon residue trimming, whereas the most active lyi displayed the opposite behaviour. a set of complementary techniques showed that lycotoxin peptides are membrane-disruptive to promastigotes. electron microscopy showed that two populations of promastigotes, one with intact parasites and the other extensively damaged, are formed upon addition of the peptides at their ec50. all peptides were non-hemolytic for sheep erythrocytes below 20 micromolar. tissue transglutaminase (tg2) is an enzyme that plays a key role in the pathogenesis of the celiac disease. tg2 is the main autoantigen recognized by the antiendomysium antibodies and, furthermore, catalyzes the deamidation of strategic glutamine to glutamic acid within the sequence of immunodominant gliadin epitopes. recently, another unexpected role for surface tg2, in the innate immune response in the celiac disease, has been suggested. it follows that tg2 inhibitors might represent a potential attractive pharmacological alternative to the gluten-free diet that, nowadays, is the only possible therapy for celiac patients. starting from the sequence of the heptapeptide pqpqlpy, known to be a high affinity substrate of human tg2, we have synthesized new analogs replacing pro3 with different constrained amino acids (d-pro, pip, chg, ind, deg, inp, hyp, thz)) with the aim to develop specific inhibitors of tg2. actually, proline residues present in the gluten epitopes are important in determining the immunogenicity of the epitopes and the specificity of tg2. herein, we describe the preliminary conformational studies of the synthesized analogs by cd and nmr spectroscopy and molecular modeling methods. the structural features of the peptides have analyzed in different environment. given the role of the domain pqpqlpy in the gliadin proteins, structural analysis on its analogs are of considerable interest. the results of our studies might be useful to clarify the role of the proline residues in the interaction of the gluten epitopes with tg2 and, consequently, to gain new insight in the molecular mechanism of celiac disease. carnosine and related histidine-containing dipeptides are known to react with high efficiency with the products of lipid peroxidation, namely 4-hydroxy-trans-2,3nonenal (hne) and other alpha, beta-unsaturated aldehydes, preventing their reaction with nucleophilic residues in proteins and nucleic acids. histidyl hydrazide alone or conjugated with aminoacids, long chain fatty acids, cholesterol and alpha-tocopherol synthesized in our laboratories demonstrated higher aldehydesequestering efficiency than carnosine, and were also efficient in protecting sh-sy5y neuroblastoma cells and rat hippocampal neurons from hne-mediated death. the cytoprotective efficacy of these compounds suggests their potential as therapeutic agents for disorders that involve excessive membrane lipid peroxidation. lantibiotics are antibiotic natural products that are synthesised ribosomally and undergo extensive post-translational modification, resulting in multiple thioether bridges and dehydro amino acids in the mature peptide. one such lantibiotic is mersacidin, which is produced by bacillus sp. and exhibits extremely promising in vitro and in vivo efficacy versus a number of clinically relevant pathogens, most notably methicillin-resistant staphylococcus aureus (mrsa). mersacidin is believed to function by binding to the bacterial cell wall precursor lipid ii, thereby preventing its incorporation into the growing peptidoglycan network. in an attempt to better understand this mode of action and develop more active analogues, we have embarked upon its chemical derivatisation. some of these modifications resulted in an altered antibacterial spectrum, permitting some insight into tentative structure-activity relationships. the characterisation of these structurally complex compounds via a combination of multidimensional nmr and tandem mass spectrometry will also be presented. conjugation of peptides with different moieties, such as peg, lipids, carrier proteins and toll-like receptor ligands is an established approach to improve their pharmacokinetic profile for drug use and/or to enhance their immunogenicity as subunit vaccines. the development of suitable conjugation strategies for peptides of any complexity is therefore fundamental for their effective use in human therapeutic applications. here we describe our strategy to engineer a trimeric coiled coil to obtain a covalently linked, structurally stable construct endowed of extra functionality for further derivatization. we previously showed that covalent stabilization of the designed trimeric coiled coil izn17, by interchain disulfide bonds, yielded an extremely potent and broad inhibitor of viral infection, (ccizn17)3, [1] . this potent inhibitory activity makes (ccizn17)3 not only attractive as an antiviral compound, but also as an immunogen to elicit a neutralizing antibody response [2] . we have now developed an alternative synthetic strategy to obtain the covalently-linked izn17 trimer, which allows the presence in the molecule of a free thiol for subsequent chemoselective reactions. first, we showed that stable interchain thioether bonds can be effectively substituted for the disulfides. second, we devised an orthogonal cysteine protection scheme which allows formation of the thioether bonds, while leaving an extra free cysteine on demand. this thiol group can be used for conjugation of the trimeric coiled coil to adjuvant/carrier proteins or, as reported more specifically here, to a peg40kda moiety. human igg is a most abundant type of immunoglobulin in serum and most of antibody drugs applied for therapy of cancer and autoimmune diseases also belong to this group of human immunoglobulin. specifically binding peptide to human igg is very useful for detection and purification as an affinity ligand of human igg. although some previous reports described such peptides, we tried here to isolate high-specific and high affinity ligands by employing random peptide-displayed t7 phage library. our t7 random peptide phage library possesses a total diversity of 10 powered 10th consisting of different sequence length constrained by disulfide bond. by biopanning against human igg, we isolated several igg specific clones from our library. the peptides displayed on these phages shared some common sequences in the limited region surrounded by cys residues, which suggests they are essential for binding. these clones bound only to fc region of igg and did neither other types of human immunoglobulin nor igg of other animals. a synthetic peptide derived from a phage clone showed a sub-nanomolar of kd value in binding to human igg fc on spr analysis. these results indicate that the peptide motifs obtained here are a strong candidate of human igg-specific affinity ligand for detection and purification of igg. therefore, we are now going on constructing detection and purification system using modified and improved peptide motifs. synthesis of glp-1 analogues as potential agents for blood glucose control p. kanda, r.p. sharma, c.p. hodgkinson in this study, we synthesised a panel of glp-1 analogues stabilised against dpp-iv degradation through either selective amino acid substitutions for ala8, or introduction of amide bond surrogates into the peptide backbone between ala8 and glu9. each was made by standard fmoc or boc chemistry, purified by hplc, and characterized by electrospray mass spectroscopy. all derivatives except one bearing a hydrazine modification were stable to dpp-iv proteolysis for up to 48 hours at ph 7.5, 37o. each was tested for its ability to augment insulin release from a glucose-sensitive, murine insulinoma-derived tc-6 cell line in culture. it was found that each compound acted as a glp-1 agonist to varying degrees, with some exhibiting higher activity than native glp-1 toward promoting insulin release. the most active analogues have been chosen as candidates for stabilisation against renal clearance in efforts to develop new glp-1 analogues with therapeutic potential. the high propensity of the glucose regulatory hormone human islet amyloid polypeptide (iapp) to misfold and aggregate into cytotoxic beta-sheets and fibrils is strongly associated with beta-cell degeneration in type ii diabetes (t2d) and precludes its pharmacological use for the treatment of diabetes. iapp analogs that combine solubility, lack of cytotoxicity, and bioactivity with the ability to block iapp aggregation and cytotoxicity could thus be of high biomedical interest. here we apply a minimalistic conformational restriction strategy to redesign the extremely insoluble and amyloidogenic 37-residue iapp sequence into a soluble, nonamyloidogenic, noncytotoxic, and bioactive iapp mimic (yan et al., pnas (2006) ). the designed mimic has nearly the same sequence as iapp but is highly soluble, nonamyloidogenic, noncytotoxic and a full iapp receptor agonist. in addition, the mimic binds with high affinity iapp and blocks and reverses with nanomolar activity its cytotoxic self-assembly which makes it to the most potent known iapp cytotoxic self-assembly inhibitor. due to its bifunctional nature, the mimic might find therapeutic application for the treatment of diabetes both as an inhibitor of amyloid cytotoxicity and as a soluble iapp receptor agonist. our findings offer a proof-of-principle of a chemical design approach for generating a novel class of highly potent inhibitors of polypeptide cytotoxic selfassembly which are nonamyloidogenic mimics of the native amyloidogenic sequence as well. such reengineered biomolecules -the design of novel mimics is in progress-are of high biomedical significance for understanding the mechanism of protein aggregation diseases and for the development of prospective therapeutic treatments. peptides that are capable of targeting abnormal changes of living tissue can be very useful in early detection or diagnosis of, e.g., cancer. conjugating a functional agent, an effector unit, to such a peptide may provide the agent with improved pharmacodynamic properties.the specificity of a thiol group for reactive groups offer a unique way to attach effector units to cysteine-free linear or cyclic peptides. tumor targeting peptides were synthesized by fmoc-type solid phase methods. peptides cyclic by cystine were modified by lactam bridges. fluorescein, metal (e.g. lanthanide) chelates and cytotoxins were coupled to tumor targeting peptides via e.g. a peg-type spacer, or the conjugates were immobilized on plates for adhesion assays. the method was uncomplicated and gave stable conjugates with good solubility. the approach is useful in making stable peptide-effector conjugates and sets of them for e.g. detection assays such as delfia method and has prospective use in development of diagnosis and therapy. antibacterial proline-rich peptides -synthesis and antibacterial activity d. knappe, r. hoffmann the antibacterial proline-rich peptide family, originally isolated from insects, shows remarkable activity against diverse bacterial and fungal pathogens. while more and more bacterial pathogens become resistant to common drugs, this part of insect innate immunity provides a new promising approach to develop future peptide-drugs. proline-rich peptides possess a significant sequence homology and share a common mechanism of action. in addition oglycosylated threonine residues of drosocin and formaecin appear to be necessary for full antimicrobial activity, although the significance of the carbohydrate moiety in interaction with intracellular targets is still unknown. we synthesized analogues of different antibacterial peptides on solid phase by the fmoc-tbustrategy. the combination or insertion of sequence regions from different native antibacterial peptide sequences offers several advantageous effects, including further reduction of toxicity and broadening of the antimicrobial activity. furthermore, mimicking the o-glycosylation site and changing the carbohydrate moieties, may yield new synthetic approaches to increase both the activity and the selectivity of these oligopeptides. new immunotherapeutic approaches have been developed for treatment of neurodegenerative diseases of the alzheimer's dementia (ad) type. the identification of a ß-amyloid-plaque specific epitope, aß(4-10) (frhdsgy) [1] , recognised by therapeutically active antibodies from transgenic ad mice, provides the basis for development of new ad vaccines and for molecular ad diagnostics. in order to produce immunogenic conjugates, the aß4-10 epitope was attached via thioether linkage to synthetic carriers of well-defined structures, such as tetratuftsin derivative (ac-[tkpk(clac)g]4-nh2) and its elongated version by a helper t-cell epitope (ac-fflltriltipqsld-[tkpk(clac)g]4-nh2); sequential oligopeptide carrier (ac-[lys(clac)-aib-gly]4-oh) and multiple antigenic peptide (clac-lys(clac)-lys(clac-lys(clac))-arg-arg-ßala-nh2). the epitope conjugates containing a cysteine residue either at the c-or n-terminus, and the chloroacetylated carriers were prepared by spps according to boc/bzl strategy. conjugation reactions were performed in solution under slightly alkaline conditions, and monitored by hplc and high resolution-ms. structures and molecular homogeneities of all epitope peptides, carriers and conjugates were ascertained by hplc, maldi and esi-fticr-ms. conformational preferences of the synthesized compounds in water and in tfe were examined by cd spectroscopy. comparative binding studies of the conjugates with a mouse anti-amyloid protein beta-(1-17) monoclonal antibody were performed by indirect elisa. experimental data showed that the chemical nature of the carrier, the epitope topology and the presence of a pentaglycine spacer between the epitope peptide and the carrier, had significant effects on the antibody recognition and on the secondary structures of the conjugates. the new cla analogue 1, containing ethylene bridge between phe nitrogen atoms, was found to exhibit unexpected stimulatory effect in the model of the in vitro humoral immune response in mice. to disclose the structure-activity relationship the nmr solution conformational analysis was carried out. the solid-state and solution conformational analysis of native cla indicated the existence of this cyclic system as a complex mixture of conformations [1] . the nmr spectra recorded at 600 mhz in chloroform at 214 k showed the different conformational behaviour of both cyclopeptides: cla exists as one isomer [1] , peptide 1 is in an equilibrium among at least of three conformers. the picornaviruses are small nonenveloped rna viruses with a single positive strand rna. the virus replication cycle starts after the penetration of the virus in the cytoplasm of the host cell. there are several stages of the virus life cycle used for attack. one of the most useful strategies for attacking of the virus includes inhibition of important for the virus lifecycle enzymes. the key enzymes in the replication of the picornaviruses are 3c and 2a proteases. changes in the active center of these enzymes make them incapable to produce polyprotein in vitro, therefore the inhibition by low molecular weight molecules could stop the viral replication in vivo. 3c protease plays a major roll in the enzymatic proteolysis of the initial viral polyprotein. the target compounds were based on structural modifications in the known as crucial for the 3cp inhibition activity dipeptides phe-gln by incorporation of additional amino acid and pyrrole moiety. the synthesis was cared out as multiple-peptide synthesis in parallel using stepwise spps, fmoc-strategy. the obtained compounds were tested for antiviral activity by agar-diffusion plaque inhibition test against coxsackievirus b1 replication in fl cell and on this base some structure-activity interpretations were made. histone deacetylases (hdac) play important roles in various aspects of regulation such as proliferation, differentiation, and aging by counteracting with histone acetyl transferases. the hdacs categorized in class-i and ii have a metalloprotease-related mechanism in its catalytic activity. these enzymes could be inhibited by small molecules bearing various zinc ligands such as hydroxamic acid and mercaptan. based on the structure of chlamydocin, which has a cyclic tetrapeptide framework, cyclo(-l-aoe-aib-l-phe-d-pro-), where aoe is (2s,9s)-2-amino-8-oxo-9,10-epoxydecanoyl, we have developed potent hdac inhibitors as shown in the figure. in the present study, we examined the effects of the chlamydocin hydroxamic acid (1) and ss-hybrid (2) on the diabetes model mice, kk-ay. the peptide (1) exhibited satisfactory activity to reduce both the blood glucose and blood insulin levels comparable to or even superior to those by pioglitazone, a pparγ agonist. the ss-hybrid (2) , which is expected to be reduced inside of cells to generate the corresponding thiol-containing cyclic tetrapeptide, also showed a significant effect but less than (1). the effect was dose-dependent from 3 mg/kg to 30 mg/kg. the effects of hdac inhibitors were also confirmed by the observation of in vivo histone hyperacetylation induced in the lymphocyte cells. synthetic peptides have a number of advantages over current vaccines. however, exploitation of synthetic peptides as vaccines has been limited by the small size; copy number, inefficient delivery, poor peptide immunogenicity and mhc restriction. we have developed chemical methodologies that overcome these limitations by synthesising and polymerising vinyl-peptides. protective b-cell and t-cell peptide epitopes from the oral pathogen porphyromonas gingivalis were identified for three different mhc restricted mouse strains using pepscan techniques. fmoc chemistry for spps was used to synthesize these peptides and a vinyl moiety was incorporated at the n-terminus using acryloyl chloride. after rp-hplc purification free radical polymerisation using ammonium persulphate and temed was used to polymerise the vinyl-peptides in the presence of either acrylamide or other vinyl-monomers to produce single peptide or multi-peptide polymers. size exclusion chromatography indicated that the peptide polymers were >2 million da. the peptide polymers were used to immunise each mouse strain (balb/c, cba and c57bl6) and the t-cell response induced was evaluated using proliferation and cytokine (elispot) assays. the peptide polymers were found to be highly immunogenic, the single peptide polymers were found to only induce a response in their respective mouse strain, however, the multi-peptide polymer containing all of the t-cell epitopes was found to induce a response in all three mouse strains. in conclusion, our data shows that the polymerisation method overcomes all of the limitations in developing a peptide vaccine and most importantly that of mhc restriction. cytotoxic substances are auspicious weapons in tumour therapy. this compounds inhibit cell division and proliferation, hence, they affect all cells that are able to divide. however, all these compounds act intracellularly, i.e. at first they have to enter the tumour cell efficiently. this is a serious obstacle when using highly effective cytostatica and the cause of severe adverse effects by using higher doses. our aim is to overcome this problem by using synthetic hybride molecules composed of the cytostatic agent, in our case derivatives of arglabin, covalently linked to shuttle peptides. in order to identify the most effective possibility we tested two different strategies. by using the peptide hormone npy, whose specific y receptors are often overexpressed by tumour tissues, we intended to address the chemotherapeutic selectively to y receptor expressing tumour cells via receptor mediated endocytosis. on the other hand, the cytostatic agent was covalently bound to a cell penetrating peptide derived from human calcitonin (hct). recently, a c-terminal fragment of human calcitonin was found to internalize into excised nasal epithelium while the receptor activating n-terminal part is lacking. for the class of hct derived carrier peptides previous studies suggested a receptor independent "lipid raft" mediated endocytotic mechanism of uptake. here we present comparative data investigating both strategies, the highly selective receptor mediated delivery and the highly efficient receptor independent delivery. we investigated the peptide uptake by various cell lines and examined the release of the cytostatic agents inside the cells and its toxic effects. background and aims: synthetic peptides provide a straight-forward access to rationally designed inhibitors of molecular interactions based on structural information of proteins. poor membrane permeability may be overcome via cell-penetrating peptides, but low stability remains a major drawback. an increase of stability and bioactivity of peptides by coupling to polymers is intended. methods: for the development of peptide-functionalized cell-penetrating polymer conjugates, peptides were coupled chemoselectivly to hpma (n-(2hydroxypropyl)methacrylamide, average mw 28.5 kda), as an inert backbone polymer by native chemical ligation. hpma is water soluble and its capacity in drug delivery has been demonstrated. peptide-functionalized polymers and free peptides were incubated with proteases or cell lysates and proteolytic break-down was determined by fluorescence correlation spectroscopy (fcs) deriving information on the number and size of fluorescent particles based on temporal fluctuations of a fluorescence signal caused by diffusion of particles through a femtoliter-size confocal detection volume. the diffusion time depends on molecular size. therefore this technique is suited for the detection of proteolytic fragments. results: efficient chemoselective conjugation of unprotected fluorescein-labelled peptides was accomplished by means of native chemical ligation. our fcs investigations revealed that conjugation of peptides to hpma increased their biostability. this data also indicate that this effect is peptide-dependent. a proapoptotic peptide coupled to hpma and introduced into mammalian cells by electroporation retained its bioactivity. cofunctionalization of hpma with this peptide and nonaargine yielded an efficient cellular import. macrolides, a rather large group of natural or semi-synthetic antibiotics, are widely known translation inhibitors whose structure is based on 14-16-member lactones with carbohydrate substitutes attached. macrolides bind to ribosomal tunnel (rt) in a way that their lactone ring is located orthogonally to the long axis of the rt, covering most of its cleft. carbohydrate residues of the macrolides are spread along the walls of the rt. hence, the mechanism of protein synthesis inhibition by macrolides relies on the mechanical obstruction they provide to the passage of nascent polypeptide chain through the rt. the goal of this study was to design and obtain peptide derivatives of macrolides interesting both as antibacterial agents and potential probes for investigation of nascent peptide chain topography in the rt. tylosin ( in order to reach target sites inside the cns, neurotherapeutic candidates must overcome the blood-brain barrier (bbb). while several transport mechanisms occur at the bbb, this work has focused on the passive diffusion mechanism. the prediction of a peptide's ability to cross the bbb is not a simple task; hence there exists the need for the rational study of the relevant factors that affect the movement across this physiological barrier. here we present two new approaches based on in vitro non cellular assays for this type of studies. firstly, the evaluation of compound mixtures on parallel artificial membrane permeability assays (pampa). this approach increases the throughput of the study and structure-activity relationships can be easily establish. secondly, the transport and biological activity evaluation in a single assay. this second approach has been applied to the search of inhibitors for cns proteases involved in different neurological diseases (such as prolyl oligopeptidase for schizophrenia) able to cross the bbb. these two new approaches allow assaying compound's permeability in the early stages of a drug development project, and then designing novel analogues with improved bbb transport properties or using blood-brain-barrier shuttles for their delivery. two newly synthesized compounds are derivatives of l-valin and are positional isomers of nicotinic (m-6) and isonicotinic acid (p-6). these functional groups, as well as established good lipid solubility suggest that the main target for their biological action probably will be central nervous system. the presence of aminoacid l -valin, supposes their low toxicity, confirmed by our earlier experiments. the aim of the present work is to study their pharmacological activity as putative drugs. methods: male albino mice (18-20g, 10 in groups) were used. next neuropharmacological parameters were studied: analgesic effect (acetic acid test), neuromuscular coordination (rota-rod test), orientation ("hole board" test) and learning and memory (passive avoidance-step down test). results: significant analgesic effect of both compounds was established (more pronounced by dose 250 mg/kg i.p.). slight depressing effect on the orientation reactions in animals was registered, but the neuromuscular coordination and locomotor activity of treated animals were not changed. good dose-dependent effect on learning and memory was established and м-6 had stronger effect than р-6. the compounds modified the effects of some model substances with central nervous activity. hexobarbital sleeping time was significantly prolonged by p-6, but was antagonized by m-6. pentileneterazole threshold was increased significantly and suggests some anticonvulsant activity of both compounds. conclusion: as positional isomers m-6 and p-6 demonstrated some variations in their pharmacological activity probably due to the differences in their kinetics, metabolism and excretion. registered significant neuropharmacological activity, accompanied by low toxicity motivates the new synthesis and future experimental studies. the glyproline family of regulatory peptides includes pro-gly-pro, gly-pro, pro-gly and also the simplest peptides with hyp substituted for pro. a distinctive feature of these peptides is that they exhibit a broad spectrum of biological activities: the antiulcer activity, the inhibition of blood clotting and thrombosis, the reduction of degranulation activity of mast cells, and the normalization of stressogenic behavioral disorders. alzheimer's (ad) and parkinson's (pd) disease are progressive neurodegenerative disorders which are characterized by amyloid plaques. the main components of the plaques are β-amyloid peptides (aβ1-40 and aβ1-42) and α-synuclein. we have previously shown that small peptides structurally related to the sequence of aβ(1-42) protect against the neurotoxicity of aβ peptides. recent studies by other groups have shown that β-synuclein can counteract the aggregation of α-synuclein in the neurodegenerative process of pd, hereby might protect the central nervous system from the neurotoxic effects of alpha-synuclein. it was found that a tetrapeptide (kegv) protected against the neurotoxicity of aβ peptides in vivo. based on the previous findings, the following sequences of β-synuclein have been synthesized: kegv-nh2 and kegv-oh. after comparing the sequences of α-and β-synuclein, we found common sequences which are keqv, regv, kqgv, keqa. we have synthesized these tetrapeptides in amide forms at their c-termini. the peptides have been synthesized on mbha resin using a manual solid phase peptide synthesis equipment and boc-chemistry. the neuroprotective effects of peptides have been investigated in vitro in mtt test in differentiated neuroblastoma cell culture (sh-sy5y) and in electrophysiological test on rats using multibarrel electrodes in vivo. the neuroprotective peptides might stop neuronal death and can influence ad and pd progression. the present study was carried out to determine antinociceptive effect in vivo of plant peptide hormone phytosulfokine-alpha (h-tyr(4-so3h)-ile-tyr(4-so3h)-thr-gln-oh ) (i) and its selected analogues, such as h-phe(4-no2)-ile-tyr(4-so3h)-thr-gln-oh (ii), h-d-phe(4-so3h)-ile-tyr(4-so3h)-thr-gln-oh(iii), h-tyr-ile-tyr(4-so3h)-thr-gln-oh(iv), h-tyr(4-so3h)-ile-tyr-thr-gln-oh(v) and h-tyr-ile-tyr-thr-gln-oh (vi) in rats. peptides were injected into the lateral brain ventricle at the dose of 100 nmol. in the preliminary investigation we found the psk-as well analogues ii and iii induced a significant antinociceptive effect determined in the test of hot plate. the probable mechanism of this effect was discussed. a.b. bozhilova-pastirova 1 , b.a. landzhov 1 , p.v. yotovski 1 , e.b. dzambazova 2 , a.i. bocheva 2 the tyr-mif-1 family of peptides (tyr-mif-1`s) includes mif-1, tyr-mif-1, tyr-w-mif-1 and tyr-k-mif-1, which have been isolated from bovine hypothalamus and human brain cortex. all these peptides interact with opioid receptors and in addition bind to non-opiate sites specific for each of the peptides. data in the literature suggest that tyr-mif-1`s have antiopioid and opioid -like effects. we used wistar rats to study distribution and density of the tyrozine hydroxylase (th) imunoreactive fibres and nadph-d reactive neurons in the rat ventral and dorsal striatum. our results showed that neuropeptides mif-1 and tyr-mif-1 may affect them. opioid peptides have been recognized as modulators of reactive oxygen species (ros) in mouse macrophages and human neutrophils. data in the literature suggest that peptides of the tyr-mif-1 family -mif-1 and tyr-mif-1 have antiopioid and opioid -like effects. these neuropeptides are isolated from bovine hypothalamus and human brain cortex. so far no data about direct scavenger properties of tyr-mifs peptides were available. in this study we tested the hypothesis that they may scavenge ros in vitro. the antioxidant activity of these two substances was studied in the concentration range of 10-6 -10-4 mol/l. we investigated the luminol-dependent chemiluminescence to test their ability to scavenge the biologically relevant oxygen-derived species: hydroxyl radical, superoxide radical, hypochlorous acid in vitro. we found that tyr-mif-1 was a powerful scavenger in all tested systems. the effects were higher for hypochlorous anion and weaker for superoxide radical. mif-1 had no scavenge activity against the hydroxyl and superoxide radicals and showed a moderate scavenger effect on hypochlorous anion. we have compared different strategies to increase the immunogenicity of an antigenic hiv peptide as a vaccine candidate. our selected b-cell epitope comprises 15 amino acids (317-331) of the v3 region of hiv-1, jy1 isolate (subtype d), and is in tandem with a t-helper epitope corresponding to the 830-844 region of tetanus toxoid. several presentations, including oligomerization, map dendrimer, conjugation to dextran beads or to other macromolecular carriers, have been synthesized and evaluated. murine sera from the different presentations of the v3 epitope have been compared with regard to antibody titers and cross-reactivity with heterologous hiv subtypes. the map dendrimer version of the peptide, conjugated to recombinant hepatitis b surface antigen protein, was a better immunogen than the dendrimer alone, and showed higher immunogenicity than other multimeric presentations, or than the peptide alone conjugated to dextran beads. the map dendrimer version, either alone or conjugated to hbsag, enhanced cross-reactivity towards heterologous v3 sequences relative to monomeric peptide. group a streptococcus (gas) responsible for critical diseases (eg. acute rheumatic fever and rheumatic heart disease) are classified over 100 serotypes according to their surface virulence m proteins. development of vaccine to prevent infection with gas is hampered by the widespread diversity of circulating gas strains and m protein serotypes, and multivalent vaccine strategy would contribute to prevention against various gas infections and provide better protective immunity. we have studied the efficacy of incorporating four different epitopes derived from gas m protein into a single synthetic lipid core peptide (lcp) construct, in inducing broadly protective immune responses against gas following parenteral delivery to mice. peptide vaccine was synthesized on mbha resin by manual spps in situ neutralization/hbtu activation in boc-chemistry. immunisation with the mono-or multi-epitopic lcp vaccine led to high titers of antigen-specific systemic igg responses, and the production of broad protective immune responses as demonstrated by the ability of immune sera to opsonise multiple gas strains. systemic challenge of mice after primary vaccination showed that mice were significantly protected against gas infection in comparison with control mice demonstrating that vaccination stimulated long-lasting protective immunity. these data support to the usefulness of lcp system in the development of synthetic multiepitope vaccine to prevent gas infection. glycoprotein d represents a major immunogenic component of the virion envelope of herpes simplex virus and able to induce high titres of neutralizing antibodies. one of its optimal epitopes is the 9-22 region (9lkmadpnrfrgkdl22). several cyclic peptides possessing thioether bond and different ring size have been already prepared and some of them were conjugated with tetratuftsin derivative (ac-[tkpkg]4-nh2) by thioether bond formation using selectively removable cys protecting groups. antibody recognition results suggested that the size of the cycle has considerable influence on antibody recognition, however, the replacement of met in position 11 by nle is permitted. conjugation of cyclic peptide might increase the antibody recognition, but the binding depends on the structure and/or conjugation site of the cyclic peptide. conjugate with the best binding capacity (7.2 pmol/100ul) as well as the conjugate containing the linear (9-22c) epitope (0.7 pmol/100ul) were selected for immunization. in order to increase the production of antibodies a new group of conjugates was prepared. in these constructs promiscouos t cell epitope peptide derived from tetanus toxoid (ysyfpsv) was attached to both amino groups of lysine residue coupled to the n-terminus of the carrier (ac-ysyfpsv-k(ac-ysyfpsv)-[tkpk(clac)g]4-nh2). the cys containing linear and cyclic epitope peptides were conjugated to the carrier in solution (0.1m tris buffer, ph 8). this work was supported by grants of the spanish-hungarian intergovernmental program and cost chemistry action. background. primary hyperparathyroidism (pht) is characterized with increased parathyroid hormone (pth) secretion and in 70% of pht patients with hypertension. it was previously shown that pro-analogue of pth with a reversed sequence (which include strong alkali sequence -arg-lys-lys-) induced significant hypertensive response at dose 10-10m/kg b.w. one of the hypothesis attributed hypertension in pht patients to the presence of fragments of degraded pth possessing -arg-lys-lys-sequence. aim. to compare influence on mean arterial blood pressure (map) of analogue of 25-34 pth fragment (amide) and 25-34 fragment of pth, with -arg-lys-lys-sequence and also responsible for binding to pth receptor. methods. chosen peptides were synthesized manually by a solid phase peptide synthesis method. the purity of the products was tested by reversed-phase high performance liquid chromatography. the synthesized peptides showed the right molecular mass. the influence on map of synthesized peptides was tested in wistar rats. sequential increasing boluses of each peptide: 10-10, 10-9 and 10-8m/kg.b.w. in the same animal were given i.v. blood pressure was measured continuously in carotid artery. results. injection of synthesized analogue of 25-34 fragment of pth does not show influence on map vs. control group. synthesized 25-34 fragment of pth increased map in 92min. of experiment for 12mmhg ± 3mmhg vs. time of administration of first dose and for 17mmhg vs. control group. conclusion. it seems to be possible, that in case of alternate degradation of pth, accumulation of 25-34 fragment of pth may partially play role in the mechanism inducing hypertension in pht patients. biologically active domains of a high affinity receptor for ig е (fcεri) were determined, the fragments 111-114 and 111-117 of the receptor. the program of research of biological properties of synthesized tetrapeptide rnwd and heptapeptide rnwdvyk included the study of their binding with ige, which was contained in standard solutions and in patients' blood serum. the binding of peptides with ige was explored by the ifa method using ige antibodies labeled with horse-radish peroxidase (hrpo). peptides in the concentration of 100 mkg/ml were used for sorption on immunological plotting boards. higher correlation between the ige concentration and the optical density of the solution after introducing monoclonal antibodies labeled with hrpo and substrate chromogenic mixture (r=0.99) was found in the diagnostic system with sorption rnwdvyk-peptide than in the diagnostic system with sorption rnwd-peptide (r=0.94). similar investigations were conducted with the diagnostic systems with sorption rnwd and rnwdvyk peptides, but rwnd peptides conjugated with hrpo were used as antibodies against immunoglobulin e, instead of hrpo-labeled monoclonal antibodies. almost equal correlation was found between the concentration of ige in standard serum and serum of allergy patients with the known concentration of ige and the optical density of the solutions after introducing the rnwd peptide, conjugated with hrpo. after introducing allergy patients' blood serum in the holes on the plotting board, the heptapeptide bound 23.79% more ige than the tetrapeptide. our experiments demonstrated a high ige binding activity of synthetic rnwd-and rnwdvyk-peptides. in this study, the information spectrum method (ism) of the ha1 subunit of the h5 hemagglutinin protein of the influenza virus, h5n1, of different reference isolates was performed in order to identify possible antigenic determinants resistant to virus mutations. results of this analysis demonstrated that the primary structures of ha1 subunit of h5 hemagglutinins encode a common information corresponding to one characteristic frequency in their iss, which is probably important for the biological function of these proteins, including their possible recognition by the immune proteins targeting this molecule. besides, comparison of the iss of ha1 proteins of h1 "spanish flu" and h5n1 isolates demonstrated an informational similarity between them. based on these results, a segment of the n-terminus of ha1 h5n1 was identified to play a crucial role in the inhibitory and immunological properties of all possible h5n1 variants. the identified core segment, being highly conserved in all h5 strains, was selected as an antigenic determinant and coupled to the sequential oligopeptide carrier (socn), (lys-aib-gly)n, to the lys-nεh2 groups, in order to develop a diagnostic immunoassay and formulate a vaccine candidate for the highly pathogenic h5n1 influenza virus. background and aims: thrombin plays a key role in various disorders such as arterial thrombosis, atherosclerosis, restenosis, inflammation and myocardial infarction. insights into the way in which thrombin interacts with its many substrates and cofactors have been clarified by crystal structure and site-directed mutagenesis analyses, but until recently there has been little consideration of how its non-proteolytic functions are performed. we investigated cardiovascular effects of seven modified proline-and rgd-containing peptides designed from three surface-exposed sites of prothrombin, corresponding to residues 218-223, 332-347, and 445-454. methods: cardioprotective effects of synthetic peptides were tested on the two rat models of heart failure produced by coronary artery occlusion (10-or 45-min) and reperfusion (30-or 240-min). arterial blood pressures from left carotid artery, heart rate and ecg ii standard lead were measured throughout experiment. at the end of second experiment hearts were morphologically investigated by light microscopy and electron microscopy methods. results: on animal model with short-term ischemia investigated peptides did not protected from myocardium ischemia during occlusion, however, tp-l13, bk-mc and tp-h7 protected rat hearts from ventricular fibrillation, contributed more significant functional recovery during reperfusion and raising survival rate. on the model with prolong ischemia, acceptable cardioprotective effect revealed tp-h7 and bk-mc. these peptides significantly diminished necrotic zone of left ventricle, protected hearts from ischemia-reperfusion induced functional and morphological changes. conclusions: investigated proline-containing peptides revealed activity on cardiovascular system -decreasing of blood pressure, cardioprotective properties and improved recovery from ischemia. r. mansi, d. tesauro, c. pedone, e. benedetti, g. morelli the widespread use of compounds containing the gamma-emitting radionuclide 99mtc in nuclear medicine for the scintigraphic imaging, as well as the recent introduction of the beta-emitting radionuclides 188re and 186re in radiotherapy, have led to a rapid development of their chemistries, in order to produce novel radiopharmaceuticals. we have developed new peptide based radiopharmaceuticals based on a scaffold in which the radioactive metal ion is complexed by two different peptides that are able to bind two target receptors (see figure) . the 3+1 mixed-ligand approach has been used for the preparation of neutral oxotechnetium(v) and oxorhenium(v) peptide complexes. the complex preparation requires the simultaneous action of a dianionic tridentate ligand and a monoanionic monodentate thiolato on a suitable metal precursor. the dianionic tridentate ligand is based on the snn donor set able to stabilize the metal complex. the chelating agent (hsc(ch3)2conhch2ch(co-r)nh2) was coupled step by step to a bioactive peptide synthesized on solid phase. the second ligand, based on monodentate thiolato moiety, was coupled on n-terminus of the second peptide. labelling procedures and biological tests on tumour cells overexpressing receptors are described for 99mtc(o) complexed by cck8 and octreotide peptide derivatives. background: endostatin inhibits the proliferation of endothelial cells and induces their apoptosis. the measurement of serum endostatin can predict tumor vascularity. tumor angiogenesis is a strong prognostic factor in patients with hepatocellular carcinoma(hcc). significantly high levels of endostatin were noted in patients with trabecular-type tumors , and with hepatitis infection. methods : 20 patients with hcc, 16 patients with git malignancies, 8 patients with liver metastasis and 8 without metastasis, and 10 normal persons . all subjects were tested for alfa-feto protein (afp) , ca19.9 , carcinoemberyonic antigen (cea), and endostatin by elisa results : endostatin in normal control persons was 47.5 ± 14.22 ng/ml with a significant elevation (p< 0.001) between the hcc group and all the other tested groups .afp was 1.9 ± 0.98 ng/ml in normal persons with a significant elevation between hcc and all the other tested groups ( p< 0.01). ca19.9 was 8.14 ± 1.89 u/ml in normal persons with a significance elevation ( p< 0.01) relative to hcc., and a significance of ( p< 0.001 ) relative to git cancers with metastases. cea was tested to be 1.12 ± 0.71 µg/l in normal persons , and had a significance of ( p< 0.001) relative to git metastases. endostatin was elevated in 2 of 8 patients with git cancers not proved to have metastasis. conclusion : endostatin can be used to denote metaplasia and can also detect possibilities of metastasis or liver cell affection even before the frank development of metaplasia affibody® molecules are a novel class of affinity proteins which are generated by combinatorial engineering of the 58 aa three-helix bundle scaffold, originating from the b domain of staphylococcal protein a. we have used fmoc/tbu chemistry for total chemical synthesis of the affibody zher2:342, binding with picomolar affinity to the cell surface receptor her2. the synthetic protein was investigated for molecular imaging of her2-overexpressing tumours. in vivo detection of her2 in malignant tumours provides important diagnostic information which may influence patient management. to enable gamma camera imaging of the tumours, a panel of potential 99mtc-chelating sequences was designed and introduced into the affibody. the well-studied tc-chelating sequence mercaptoacetyltriglycyl (mag3) was compared to serine-containing sequences with increased hydrophilicity, such as mercaptoacetyltriserinyl (mas3). the total synthetic yield was 14-16 % and the her2-binding affinity of the affibody conjugates were all in the range 200-400 pm. binding specificity of tc-labelled affibody molecules was determined on her2-expressing skov-3 ovarian carcinoma cells. all variants showed receptor-specific binding. the tumour-targeting properties were studied in skov-3 tumour-bearing nude mice. all conjugates demonstrated high tumour uptake, quick blood clearance and low uptake in most other organs. the biodistribution results further showed that the more hydrophilic, serine-containing chelators resulted in a reduced hepatobiliary excretion, which significantly decrease the background in the abdomen area and provide for more sensitive detection. gamma camera images of mice with grafted tumours showed clear visualization of her2-expressing tumours using the 99mtclabelled mas3-affibody conjugate, suggesting a potential future application of this agent for diagnostic imaging. antimicrobial peptides are molecules with a unique mechanism of action. they are widespread in nature and play the role of an effective weapon of innate immune system against bacteria, fungi and viruses. the purpose of this study was to investigate the in vitro activity of natural antimicrobial peptides: citropin, piscidin, protegrin, temporin, uperin and the analogues of antimicrobial peptides: iseganan, pexiganan and omiganan. the peptides were synthesized using the solid-phase method and purified by high-performance liquid chromatography. the peptides were subjected to microbiological tests [mic (minimal inhibitory concentration) and mbc (minimal bactericidal concentration)] on reference strains of bacteria, according to the procedures outlined by the national committee for clinical laboratory standards (nccls). for comparison, conventional antibiotics (vancomycin, rifampycin, piperacillin, chloramphenicol) were included in this research. both the natural antimicrobial peptides and the analogues inhibited the growth of bacteria, but at higher concentrations than did conventional antibiotics. nevertheless, both natural origin of antimicrobial peptides and their low toxicity constitute a considerable advantage and this is an argument for considering the antimicrobial peptides as good candidates for medicines. the linear hexapeptide cypate-grdspk (compound 1; the cypate moiety is a near-infrared fluorescent label) whose rgd sequence was rearranged to grd showed high uptake in the αvβ3 integrin-positive tumor tissues in vitro and in vivo. despite low affinity of 1 to the integrin in the binding assays, the uptake was inhibited by equimolar amounts of the cyclic peptide c(rgdfv), which possesses high affinity to αvβ3 integrin. these observations led to hypothesis that cell internalization of compound 1 may be mediated mostly by only one of the integrin subunits, as the β3 one. indeed, blocking of αv integrin by the specific antibody did not inhibit the internalization of 1 in tumor cells, which was in the contrast with successful blocking the cell internalization by the anti-β3 integrin antibody. similar results were obtained in immunocytochemical assays employing the anti-αv and anti-β3 integrin antibodies. also, studies utilizing the β3-knockout and wild-type mouse cell lines demonstrated that deletion of the β3 subunit markedly decreased internalization of compound 1 in the β3knockout cells. the preferential interaction of compound 1 with the β3 subunit of integrins relative to the αv subunit was supported also by molecular modeling studies. summarizing, the bulk of our experimental and modeling data emphasizes interaction with the β3 integrin as the primary mechanism of the uptake of cypate-grdspk by tumors. since this compound showed the superior biodistribution profile in vivo, our results may provide a strategy to image and monitor the functional status of the β3 integrin in cells and live animals. background and aim: a growing tumor is accompanied by tumor intoxication development. intoxication independs on tumor size and intensity of its break-up. tumor intoxication is one of variant of endogenous intoxication. concentration of tyrosine-contained peptides (tcp) in blood plasma have been proposed as biochemical marker of endogenous intoxication at different organs cancers. our aim was to determine the tcp concentration in blood plasma patients with ovary tumor and its association with the severity of tumor. materials and methods: 178 patients with ovary tumor, mean age is 53 years, were studied. the control group consisted of 20 healthy women without tumor. patients were divided into 2 groups: people with non-malignant and people with malignant ovary tumor. tcp content in blood plasma was estimated by our technique. results: tcp concentration in the control group were 0,32±0,13 mmol. the tested marker was present in increased concentration in blood plasma of the patients with ovary tumor. the mean concentration tcp in patients with non-malignant tumor was 0,53±0,16 mmol. the content of this marker in blood plasma of patients from second group was increased 1,32±0,20 mmol compared with healthy control group. after treatment a significant decrease in tcp content was observed. conclusions: the result indicate that content tcp in blood plasma depends of the type of tumor. it could be suggested that determination of tcp concentration in blood plasma could be useful for improve the diagnostic of ovary tumor and monitoring of its progression. c. strongylis 1 , ch. papadopoulos 1 , k. naka 2 , l. michalis 2 , k. soteriadou 3 , v. tsikaris 1 troponin is a structural protein complex, which is responsible for the regulation of skeletal and cardiac muscle contraction. it consists of three components: troponin i (24kda), troponin c (18kda), and troponin t (37kda), each of which carries out different functions in the striated muscles. cardiac troponins are released into the bloodstream of patients after the onset of a cardiovascular damage. even minimal elevations over the normal values, of serum troponin t and i are being used to diagnose acute myocardial infarction and also to rule out the patients' condition. the development and commercialization of highly specific biological assays for the detection of cardiac troponins is based on the production of specific antibodies against the whole complex or individual subunits. however, the specificity and sensitivity of these assays vary due to problems mainly originated from the fact that cardiac troponins have a high homology with the skeletal isoforms. the aim of this work is to select and synthesize appropriate regions of the cardiac isoforms of troponin i, c and t, suitable for the production of more sensitive and specific cardiac troponin detecting reagents. in order to construct the immunogenic complexes, the selected sequences were conjugated to the tetrameric sequential oligopeptide carrier (soc4), either by the classic solid phase step-by-step methodology or by chemoselective ligation reactions. using the carrier conjugated troponin sequences, anti cardiac troponin complex specific antibodies in high titers were produced. the increasing problems with the reproductive systems of man and animals are recently linked to the presence of polluting chemicals with endocrine activity, the so called endocrine disrupting chemicals or edc's . the family of edc's is a heterogeneous one and consists of natural and synthetic hormones (like estradiol, ethynylestradiol and diethylstilbesterol), phyto-estrogens (like genistein and coumestrol) and industrial chemicals (like bisfenol-a, ftalates and various pesticides). because of the complexity of the environmental matrices and the low physiologically active concentrations of the edc's there is still a need for an efficient routine analysis protocol. we want to develop a solid-phase bound receptor that possesses a high selectivity for edc's and thus can be used in a simple solid-phase extraction protocol. this receptor must have the right functional groups that bind the edc's with a strong affinity and must be able to create a cavity in which the edc's can fit. by looking at nature's own estrogenic receptor for humans we have found the different amino acids responsible for the specific interactions . in order to create the cavity which mimics the behaviour of the hormone-binding domain of the human estrogenic receptor we have made a tripodal scaffold. this tripodal scaffold has three orthogonal protected amino groups that will allow the generation of three independent peptide chains. milk proteins are a source of opioid peptides. these peptides are liberated from milk proteins during enzymatic hydrolysis. some of these peptides are characterized with agonistic (β-casomorphins) and some with antagonistic (casoxins) properties. the aim of the investigations was to determine the presence of opioid peptides with antagonistic properties in milk products. the experimental material included cheeses, yogurts and kefirs. peptides were extracted with a methanol-chloroform mixture (2:1 v/v). the peptide extracts were purified by spe method on c18 or stratax columns and characterized by sds-page electrophoresis. the agonist opioid peptides (casoxins) were identified by hplc using standard agonist peptides. the opioid activity was measured by examining the effects of peptide extracts on the motor activity of isolated rabbit intestine. the results of sds-page electrophoresis indicated the presence of 5 to 9 fractions in peptide extract derived from cheeses and yogurts and 17 to 20 ones from kefirs. the presence of casoxin a (0.22-0.68 µg/mg of extract) was proved in all examined the milk products. lactoferroxin a (0.31-1.88 µg/mg of extract) was identified only in kefirs and yogurts. those products were also found to contain trace amounts of casoxin c. all peptide extracts showed the antagonistic activity in the relation to motor activity of isolated rabbit intestine. the highest antagonistic activity was reported of peptide extract from kefirs (3.62-17 .20%) and gouda cheese (15.68-16.36%), as compared to morphine. the physiological and nutritional function of these antagonist peptides requires elucidation. a. péter 1 , r. berkecz 1 , f. fülöp 2 the past decade has seen a growing interest in β-amino acids, which are important intermediates for the synthesis of compounds of pharmaceutical interest and can be used as building blocks for peptidomimetics. oligomers of β-amino acids (β-peptides) fold into compact helices in solution. recently, a novel class of β-peptide analogues adopting predictable and reproducible folding patterns (foldamers) was evaluated as a potential source of new drugs and catalysts. studies on synthetic β-amino acids can be facilitated by versatile and robust methods for determining the enantiomeric purity of starting materials and products. highperformance liquid chromatography (hplc) is one of the most useful techniques for the recognition and/or separation of stereoisomers including enantiomers. the aim of the present work was to evaluate hplc methods for the separation of enantiomers of eighteen 3-amino-3-aryl-substituted propanoic acids (β-amino acids). direct separations were carried out on different macrocyclic glycopeptide based stationary phases, such as ristocetin a containing chirobiotic r, teicoplanin containing chirobiotic t, teicoplanin aglycon containing chirobiotic tag, vancomycin containing chirobiotic v columns and on a chiral crown ether based column. the effects of different parameters on selectivity, such as the nature of the organic modifier, the mobile phase composition, the flow rate and the structure of the analytes are examined and discussed. the separation of the stereoisomers was optimized by variation of the chromatographic parameters. the efficiency of the different methods and the role of molecular structure in the enantioseparation were noted. the elution sequence of the enantiomers was determined in most cases. a rational approach to evaluating peptide purity a. swietlow 1 , r. lax 2 1 amgen, inc., pharmaceutics, thousand oaks, ca 2 polypeptide laboratories, inc., torrance, ca, usa recent years have seen an enormous increase in interest in peptide therapeutics. new peptide leads are often chosen by screening procedures using microgram to milligram quantities of peptides, frequently provided by specialized manufacturers utilizing automatic synthesizers to maximize output. the purity of the resulting compounds is often not very high. the use of spps synthetic procedures predetermines that most impurities are closely related and difficult to resolve by reversephase purification. these factors, combined with the use of generic analytical methods not specifically optimized for the peptide in question (e.g. the ubiquitous 0.1% tfa/water/acetonitrile system), lead to erroneous results that frequently severely overestimate the purity of the peptide. the use of poorly characterized materials in pharmaceutical development leads to significant risks of obtaining false negative or false positive results that may cause potential leads to be overlooked or misinterpretation of their pharmacological profiles. we describe a rapid, systematic and reliable hplc procedure for evaluation of peptide purity. utilizing the increased separation efficiency by increasing the column temperature and adjusting the gradient in two steps in reverse-phase buffers containing tfa, naclo4, or ion-exchange buffers containing kcl, we demonstrate that methods -suitable for preclinical research -can be developed rapidly. the proposed approach will be illustrated with examples of peptides ranging between 9 and 28 amino acids and a model peptide vypnga. it will be demonstrated that peptides showing an hplc purity close to 100% are often 10 -25% less pure. to approach a high-throughput cell assay format using peptides, we attempt to design and construct a peptide microarray for examination of cell activities of peptides including apoptotic cell death. peptides were immobilized onto solid surfaces via a novel multi-functional linker. the linker enable us to examine various types of peptide cell assays in an array format. we also designed and synthesized peptidyl capture agents on the basis of the cell-active sequences suitable for the peptide microarray. the utility of targeted nanoparticles as fluorescent probes for tissue imaging has recently been subject to widespread interest. one exciting prospect is the further development of nanoparticles conjugated to both targeting peptides and cytotoxic cargoes. these nanodevices could preferentially bind to specific cells and/or tissues to provide effective tools for drug delivery. hence, such multifunctional nanoparticles could provide both diagnostic and therapeutic functions by acting as fluorescent probes that offer targeted delivery of therapeutic agents. we have coated the surface of quantum dots (qdots) with cell-penetrating peptides (cpp) to target and label u251m cells for fluorescence imaging. qdots were initially coupled to polyethylene glycol linkers via carboxyl functionalities on their surface. a heterogeneous mixture of poly-arginine peptides of varying lengths (arg(6)-arg(10)) were covalently coupled via amide bonds to the polyethylene glycol linkers, conferring a cell penetrating capacity to the modified qdots. fluorescence imaging of u251m cells, after incubation with the conjugated qdots at concentrations of 20nm, gave clear signals indicating cell binding and internalisation of the modified qdots across the plasma membrane. we aim to further expand this work by employing racemic mixtures of cpp and cytotoxic agents to engineer conjugates that will facilitate both imaging and the therapeutic delivery of cytotoxic moieties. the ability of cell penetrating peptides (cpp) to deliver biologically active cargoes into different cell types has been successfully applied in several experimental systems. despite the progress and growing number of described cpps, reports about the internalization mechanisms and the intracellular routes of cpps still remain controversial. we have characterized the membrane interaction and cellular localization of proteins delivered into hela cells by cell penetrating peptide transportan (tp) and its shorter analogue tp10 on ultrastructural level. our previous results obtained by transmission electron microscopy showed that complexes of transportans with gold-labeled streptavidin translocated into cells inducing large invagination of plasmamembrane, suggesting the uptake by macropinocytosis. the complexes of transportan with gold-labeled neutravidin, in contrary, were taken into cells mostly via caveosomes and clathrin-coated vesicles. the cell-transduced transportanprotein complexes localized mainly in the vesicular structures of different size and morphology. most of the complexes-containing vesicles in the perinuclear area contained also lamp2 protein, marker of late endosomes and lysosomes. still, the transportan-protein complexes were not confined in the membrane-surrounded vesicles, but spread in the cytosol suggesting the escape of transportan-protein complexes from endosomes. our findings show the involvement of different endocytic pathways in the transportan-mediated uptake process of proteins. the concentration of a cpp and the properties of cargo protein seem to determine the pathway for the cellular uptake of a particular construct. rgd peptides (r = arginine; g = glycine; d = aspartic acid) have been found to promote cell adhesion upon interaction with alphav-beta3 receptors, which are strongly overexpressed during neoangiogenesis by solid tumor associated cells compared to healthy cells. in this study we designed new targeting motifs aimed to deliver various antitumoral drugs specifically to cells involved in tumor vascularization. we inserted this short rgd sequence in tetracyclopeptides closed with various means. we expect these new cyclotetrapeptides to be more specific for the targeted receptor. moreover, these new type of cyclic peptides were multimerized on different scaffold to further improve the receptor avidity. our purpose is first to scrutinize and to quantify the efficient cellular uptake of these molecules and second, to address the specific cell targeting of a fluorescent cargo by differents tools such as fluorescence activated cells sorting (facs) analysis or fluorescence microscopy. these new targeting units were evaluated on two different cell lines: human umbilical vein endothelial cells (huvec) with an over expression of the alphav-beta3 integrin receptor and a549 cells expressing a much lower level of this receptor. preliminary results about the selectivity and the efficacy of these new targeting units will be presented. we have recently developed new approaches for obtaining highly immunogenic peptide conjugates: synthetic polyelectrolytes (pe) were used for the conjugation with peptide molecules in which pe carry out the carrier and adjuvant roles simultaneously. in this study, 4 epitopes of antigenic parts of surface antigen of hepatitis b virus (2-16, 22-35, 95-109 and 115-129 of the s gene.) had been synthesized.the synthesis of peptides was performed by explorer pls ® automated microwave synthesis workstation (cem). peptide conjugates of synthetic anionic polyelectrolytes (copolymers of acrylic asid and n-vinylpyrrolidone) were synthesized by carbodiimide condensation following the modification procedures described early. composition and structure of bioconjugates were characterized by hplc (shimadzu), nanospr-3, zetasizer nano zs, steady state fluorescence spectrometer qm-4 and viscotek tda 302 size exclusion chromatography. it was obtained that a single immunization of mice with pe-peptide conjugates without classical adjuvant increased the primary and secondary peptide-specific immune response to hbsag. moreover, these conjugates possess own selectivity for recognizing the antibody in blood sera of hepatitis virus injected people. tissue engineering requires delivery of transplanted cells to organ sites needing repair/regeneration. we have demonstrated that several active laminin peptide-conjugated chitosan membranes enhanced the biological activity and promoted cell adhesion in a cell-type specific manner. the most active laminin peptide (ag73: rkrlqvqlsirt)-conjugated chitosan membrane could deliver keratinocytes to a wound bed. when human keratinocytes were seeded onto the ag73-chitosan membranes under serum-free condition, more than 70% of the cells attached within 2 hrs. the membranes carrying keratinocytes were stable enough for handling with forceps and were inverted onto the muscle fascia exposed on the trunk of nude mice. keratinocyte sheets were observed after 3 days and colonies appeared after 7 days on the fascia of host mice. these cells were multilayered on day-3 and expressed various keratinocyte markers, including cytokeratin-1, involculin, and laminin ?2-chain. these results suggest that the ag73-conjugated chitosan membrane is useful as a therapeutic formulation and is applicable as a cell delivery system, such as delivering keratinocytes to the wound bed. the peptide-chitosan approach may be a powerful cell transplantation tool for various tissues and organs. the fluorescent semi-conductive (cdse/zns) nanocristals possess very attractive optical properties that could be used for tracking individually biological receptors in vivo. our aim is to design functionalized water-soluble semi-conductive nanocristals (or quantum dots) that interact selectively with lipidic or biological membranes. to valid our approach, the interaction between the decorated qd and giant vesicles were observed by optical fluorescent and dark field microscopies. in view to solubilize and selectively bind fluorescent nanocristals to a lipid membrane, heterobifunctionalized peptidic ligands (liipe) that presented an adhesion domain for the nanocristal surface, an hydrophilic spacer and a terminal recognition function, were synthetized. the colloïdal stability of the water-soluble nanocristals (nc-lipe) was checked by dynamic light scattering, optical and electron microscopies the interaction of grafted nanocristals (nc-lipe) with positive or neutral giant vesicles was observed by optical fluorescence and dark field microscopies. as shown in figure, negatively charged nanocristals (nc-lipe) selectively adsorbed onto the surface of positively charged giant vesicles without altering the morphology of the vesicle. the nanocristals appeared as fluorescent patches growing on the surface of the vesicle until completely recovering. therefore these ligands (lipe) permitted to chemically functionalize the nanocristals by keeping their colloïdal stability and their fluorescence in water. furthermore it was possible to selectively label vesicle membrane. creatine analogues for treatment of obesity: us patent 5 bioactive peptides containing pairs of basic amino acids are rapidly metabolized as a result of cleavage by trypsin-like enzymes. to increase the metabolic stability of opioid peptides containing arg-arg and arg-lys pairs, the arg residues were replaced by homoarginine (har) kenes international leprince 1 , f. cavelier 2 , p. gandolfo 1 , m. diallo 1 , h. castel 1 , l. desrues 1 m304 new bradykinin analogues modified with 1-aminocyclopentane-1-carboxylic acid effect on rat blood pressure and rat uterus o. labudda-dawidowska 1 , d. sobolewski 1 , m śleszyńska 1 , i derdowska 1 synthesis and heparin binding sites identification f. baleux 1 , f. arenzana-seisdedos chemokines are small proteins involved in numerous biological processes (inflammation, immunity, morphogenesis, tissue repair, and tumour development) the general goal of our project is to elucidate the role that hs plays in vivo in the physiologic and pathologic activities of the chemokine cxcl12/stromal derived factor-1, and to characterise the molecular and structural determinants accounting for the interaction. three sdf isoforms, alpha (68 aa), beta (72 aa) and gamma (98 aa) have been identified. we previously identified the major heparin binding site on sdf alpha and demonstrated the importance of hs/sdf interaction in hiv entry cell inhibition (1,2). sdf gamma amino acids sequence corresponds to the sdf alpha sequence extended by a c-ter 30 amino acids sequence containing putative heparin binding sites. in order to determine sdf gamma heparin binding sites, wild type and mutants proteins were synthesised by stepwise solid-phase peptide synthesis using fmoc chemistry prusiner proc. natl. acad. sci here we describe xenome's drug development process for the chi family, conopeptide -mria[1] of the predatory marine snail conus marmoreus, leading to a suitable drug candidate (xen2174). xen2174 is highly selective for the norepinephrine transporter (net) compared to other transporters, such as dopamine and serotonin, and inhibits net via an allosteric mechanism. xen2174 is currently in a phase i/iia clinical trial for the treatment of severe pain. an intensive synthetic analogue and screening program around -mria, incorporating early stage animal data xen2174 isomers were synthesized via selective disulfide bond formation to identify the active connectivity. data from alanine-scans, single amino acid mutations and probing of backbone interactions combined with the full 3d nmr structure, led to the development of a pharmacophore for xen2174. this model is refined from further studies where structure-activity relationships were developed utilising binding and functional assay data for a range of peptides anti-cancer drug design anti-cancer drug design published structural data for hdac-like protein, a bacterial enzyme sharing high homology to the hdacs in its active site, confirmed that this protein contains a zinc in the active site. for the discovery of specific hdac inhibitors, a number of hydroxamic acis and related compounds have been designed based on the ligating function to the zinc atom. the mechanism also involves an appropriate nucleophile in the active site. chlamydocin is a cyclic tetrapeptide on the other hand, we have been focusing the cyclic tetrapeptide to develop the potent and specific inhibitors of hdacs. in the present report, we employed the chlamydocin scaffold and successfully introduced the series of thioether as the functional group to the cyclic tetrapeptide. it is well argued that the strong inhibition of hdac requires the best combination of zinc ligand, capping group, and appropriate spacer between them jerusalem 3 department of biological regulation 4 department of organic chemistry 5 department of biological chemistry, weizmann institute of science, rehovot, israel estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. the use of estrogen and selective estrogen receptor (er) modulators in treatment of osteoporosis is limited due to substantial risks for breast cancer. recently, we developed peptides having estrogen-like activity peptide emp-1 had no effect on bone growth in normal mice, and did not influence the ovx-induced bone-loss. we then developed a new µct methodology to evaluate uncalcified and calcified growth-plate parameters. in the ovx mice, peptide emp-1 reduced volume and thickness of the uncalcified growth-plate, a possible cause for the inhibition of bone longitudinal growth. based on a reported enhancement of er-in female mice during protein biosynthesis, after the release of the nascent polypeptide chain, ribosome recycling factor (rrf) disassembles the post-translational complex. rrf has been shown to be essential for bacterial growth. thus, we are attempting to design suitable compounds to inhibit the rrf function as candidates for new-type antibiotics. we have determined the structure of rrf with 185 amino acid residues by nmr and x-ray analyses and shown that rrf has two domains domain i with three stranded helical bundles and domain ii with &beta;/&alpha;/&beta furthermore, we recently determined the structures of the 70s ribosome-rrf complex by cryo-electron microscopy and the 50s ribosome-rrf domain i complex by x-ray analysis using the results of these experiments, we elucidated the interaction profiles between rrf and ribosome and found that the cationic center consisting of three arginine residues on the surface of the helical bundle, which we have shown to be essential for the activity of rrf, is bound to helix 69-71 of 23s ribosomal rna. we synthesized the rna and peptide fragments around this interacting site and characterized them by physico-chemical analyses. the results of cd and biacore experiments to investigate the details of the interactions between them showed that a 27 mer of rna fragment is bound to rrf <i>biochemistry</i> <b>42</b> causes an increase of pain by inhibiting the opioid response [1]. recent research has shown further that melancortin receptors, mainly subtype mc4r, produce an increase in response to pain stimuli [2]. based on this previous work, we are developing chimeric ligands which will be of benefit to therapeutic pain treatment with enhanced opioid efficacy by acting as agonists at opioid receptors and antagonists at both cck and melanocortin receptors cck (i) and melanocortin (ii) pharmacophores were overlapped by trp, and different profiles of opioid pharmacophores (iii) were linked to the n-terminal of the melanocortin pharmacophore (figure). the designed ligands showed moderate to high biological activity at all three receptors depending on their respective structures. design considerations and structure-activity relationships will be discussed in detail along with in-vivo assay results china synthetic exendin-4 is a 39-amino acid peptide that exhibits potent anti-diabetic and dose-dependent glucose-regulatory activity. exendin-4 is susceptible to degradation in plasma, so its activity is limited. our aim is to find sites in exendin-4 that are susceptible to cleavage and provide information for designing new exendin-4 analogues. in this study the stability of exendin-4 in human plasma was evaluated in vitro. exendin-4 was incubated in plasma at 37 ℃, extracted with sep-pak octadecyl columns and subsequently analyzed using high performance liquid chromatography (hplc). the results showed that exendin-4 was slowly broken down in plasma with a half-life of 9.57 h. the degradation products were identified by quadrupole time of flight mass spectrometry (q-tof-ms) with electrospray ionization pharmacology of exenatide(synthetic exendin-4): a potential therapeutic for improved glycemic control of type 2 diabetes one of the proposed solutions for the pharmacotherapy of obesity, a major health problem in the western world, is to regulate the biochemical pathways that control the metabolic balance in the body. the melanocortin pathway regulates energy balance by binding of the catabolic endogenic neuropeptide αmsh to its mc4 receptor and thus causes a decrease in food intake. we have synthesized a backbone cyclic peptide library, based on the minimal αmsh sequence phe6-d-phe-arg-trp9 [1], that activates the mc4 receptor. all the members of the library shared the same sequence analysis using colorimetric liposomes confirmed that bbc-1 penetrate the intestinal cells by the transcellular mechanism. moreover, bbc-1 have high metabolic stability to intestinal enzymes (100% recovery after 5 hr). ec50 analysis showed that bbc-1 selectively binds and activate the mc4 and mc5 receptors (ec50 3.97±0.63 and 7.27±0.40 respectively). oral administration of bbc-1 in mice showed reduces food intake melanocortin tetrapeptides modified at the n-terminus, his, phe,arg and trp positions 2 department of experimental and health sciences graduate school of pharmaceutical sciences 2 graduate school of agriculture 3 graduate school of medicine consisting of 54 amino acids, is a product of the metastasis suppressor gene kiss-1. this c-terminally amidated peptide was identified as the endogenous ligand of an orphan g-protein coupled receptor metastin-gpr54 signaling may regulate gonadotropin secretion and negatively regulate cancer metastasis. it is interesting that activation of gpr54 signaling negatively regulates the function of sdf-1-cxcr4 axis in cho and hela cell transfectants we conducted the structure-activity relationship (sar) study on kisspeptin-10 using the neuropeptide-derived rw-amide scaffold to identify five-residue peptide amides as novel gpr54 agonists equipotent to kisspeptin pro-), where aoe is (2s,9s)-2-amino-8-oxo-9,10-epoxydecanoyl. in continuation of our study to design and synthesize analogues bearing a zinc ligand to develop potent inhibitors of histone deacetylase (hdac) inhibitors, we shifted the aromatic ring of phenylalanine at the aminoisobutric acid (aib) position and also at the imino acid position. the aim is to explore the interaction of cyclic scaffold with the rim of hdac paralogs. we replaced the epoxyketone moiety of aoe with sulfhydryl group, which is protected as disulfide hybrid, as zinc ligand. benzene ring was introduced to aib structure to design amino-1-indane carboxylic acid and amino-2-indane carboxylic acid. aromatic ring-containing imino acids, such as d-tic were also employed to replace d-pro. the cyclic tetrapeptides were profiled by the inhibition of hdac1, hdac4, and hdac6 in adult goats, however, the infection remains unapparent and the virus may cause abortion, vulvovaginitis or balanoposthitis. the use of a vaccine could provide a powerful tool for the control of cphv-1 infection. synthetic peptide-based vaccines have advantages of being selective, chemically defined and safe. in order to further localize immunogenic epitopes, glycoproteins b, c and d of cphv-1 were analyzed with several prediction programs. peptide conjugates incorporating t and b cell determinants in multiple copies in branched architecture are better immunogens for the preparation of goat vaccines, we synthesized peptide conjugates bearing t cell epitope on the n-terminal of the core. b cell epitopes were conjugated via a thioether bond on the ne-amino group of four choroacetylated lysine residues of the core. elisas confirm that the b cell epitopes and the conjugates t celltetratuftsin induce epitope-specific and antibody responses two recorded electrodes implanted bilaterally. eeg recorded after the mirror focus was arises. trh applicated intranasal in ultra low doses (10-9 m and 10-12 m) or intravenously in high doses (25mg, 50mg, 100 mg). for eeg registration and analysis the computer system conan was used and new modification of fractal analyze of quantized eeg. results: the synchronization of epileptic activity between primary and mirror focuses observed on the third day after operation. intranasal application of trh induced reduction of spontaneous focal epileptic activity as in primary cobalt damage focus as in the mirror focus more than 1h. the inhibition of mirror focus was more expressed. quite the contrary intravenous trh administration provocated the epileptic discharges in both local focus. the intense synchronized generalized activity was record during 30-40 min deraos 1 , t.v. tselios 1 , i. mylonas 2 , g.n. deraos 1 it is known that cyclic peptides are more stable in enzymatic degradation and conformationally restricted compared to linear. the cyclization was achieved using o-benzotriazol-1-yl-n,n,n',n'-tetramethyluronium tetrafluoroborate (tbtu) and 1-hydroxy-7-azabenzotriazole, 2,4,6 collidine allowing fast reaction and high yield final product. the purification was achieved using reversed phase high performance liquid chromatography (rp-hplc) and the peptide purity was assessed by analytical hplc and mass spectrometry (esi-ms). the synthesized cyclic plp analogue was found to exhibit lower agonist eae activity compared to linear plp139-151 epitope in sjl/j mice. this implies that the conformation of cyclic analogue does not trigger autoimmune reaction in the central nervous system and therefore encephalomyelitis the netherlands 3 department of experimental and health sciences on the other hand, in both central and peripheral nervous system, cck acts as neurotransmitter. recently, cck is focused at modulation effects on feeding especially. in this study, we tried to establish a sensitive and specific enzyme immunoassay (eia) for detecting cck and to investigate the effect of some dopamine receptor antagonists using this eia, we measured plasma cck-like immunoreactive substance (is) levels in five healthy human subjects after single oral administration of some prokinetics. the minimum amount of cck detectable by our eia system was 2.0 pg/ml, and the ic50 of the calibration curve was 75 pg/ml. we revealed that domperidone and itopride caused significant decreases in plasma cck-is levels but metoclopramide and sulpiride did not. we established a sensitive and specific eia for cck. furthermore pro-pro-phe-phe-), isolated from linseed oil [1], possesses a strong immunosuppressive activity comparable at low doses with that of cyclosporin a [2]. it has been postulated that the tetrapeptide sequence pro-pro-phe-phe is important for biological activity of cla on the basis of this information we have synthesized a series of cla analogues in which the alpha-proline residue was replaced by beta2-iso-proline and beta3-homo-proline residues, respectively (fig.1). the synthesis of titled beta-amino acids has been achieved according to the literature procedure italy synthetic peptides are largely used as antigens in solid-phase immunoenzymatic assays (elisa) for recognition of antibodies (abs) in biomedical research and, most importantly, in the set up of diagnostic methods. it is well known that the method of peptide immobilization on the solid support is very important for a correct ab recognition atherosclerosis in patients infected with helicobacter pylori (hp) we synthesized appropriate oligopeptides immobilized on cellulose via n-or c-termini, using standard -alanine linkers as well as a new linker, developed for this particular studies glc)ghsvflapygwmvk) we found the strongest recognition when the peptide was linked to the cellulose support via the c-terminus. however, in the case of ureb f8 hp urease smallest epitope (sikedvqf), and epitope ub-33 (321-339 hp fragment: chhldksikedvqfadsri) the strongest reactions with sera of atherosclerosis suffering patients were obtained for n-terminally anchored peptides the synthetic dipeptide gamma-d-glutamyl-l-tryptophan (scv-07) has been shown to stimulate t-lymphocyte differentiation and specific immune responses, and enhance il-2 and inf-gamma production. due to this preferential activation of th1 cytokine production, scv-07 may show utility in treatment of infectious diseases cba mice at a dose of 2,500 µg/kg. the same animals were used for all three methods of administration with a dosing interval of 2 weeks. blood samples were taken from the right retro-orbital sinus. for determination of the scv-07 concentration in blood samples, an "eia-scv-07" competitive solid-phase immuno-enzyme assay was developed (loq 20 ng/ml) with a mean residence time of 10 minutes. 5 minutes postdose, indicating very rapid absorption. mean concentrations then declined and were measurable through 1.5 and 3 hours postdose (mrt 20 and 50 minutes, for i/p and p/o, respectively). the estimated bioavailability of scv-07 after i/p and p/o administration was almost equivalent gastrin-17 (g17) is a peptide which promotes gastric acid secretion, cell proliferation, and occasionally gastrointestinal cancer in the gastric antrum. g17 also promotes the growth of cancerous colonic epithelial cells, but the cck2/gastrin receptor, which mediates its activity, is largely not expressed on such cells. instead, our previous studies have shown that some other receptor mediates stimulation of proliferation of dld-1 and ht29 human colonic carcinoma cells by ncarboxymethyl gastrin (g17gly) at namomolar concentrations and inhibition at micromolar concentrations, indication separate binding sites. we have shown previously that g17(1-12)-oh stimulates cell proliferation of ht-29 cells -6)-nh2, in order to determine their selectivity for and activation of the putative proliferation-stimulatory receptor. the results revealed that g17(1-12)-oh is not selective for a single receptor, but binds both sites as do g17 and g17gly. g17(1-6)-nh2 promotes dose-dependent and non-biphasic proliferation of dld-1 cells and binds a single receptor with low affinity. m484 comparative study of ige-binding activity of synthetic peptides rnwd and rnwdvyk v th486 involvement of l-name in the antinociceptive effects of newly synthesized analogues of tyr-mif-1 peptide in rats tyr-mif-1 is able to interact with opioid receptors with a higher potency at m sites as well as to its specific non-opiate receptors in the brain. nitric oxide and tyr-mif-1`s are potent modulators of opioid activities. involvement of no in nociceptive effects is well documented in various physiological and pathological processes in the cns. l-name when administrated i.c.v. or systemically exhibit antinociceptive activity in rats as evaluated by the pp test. in the present study, we investigated the involvement of l-name in the antinociceptive action of newly synthesized analogues of tyr-mif-1 peptide: nα-(me)tyr-mif-1, d-tyr-(me)-mif-1, tyr(cl2)-mif-1 and tyr(br2)-mif-1. the experiments were carried out on male wistar rats (180-200 g). the changes in the mechanical nociceptive greece paclitaxel is one of the most important anticancer drugs used mainly in treatment of breast, lung, and ovarian cancer and is being investigated for use as a single agent for treatment of lung cancer, advanced head and neck cancers, and adenocarcinomas of the upper gastrointestinal tract. however, the development of resistance to paclitaxel, the side effects and low solubility of this drug remain major obstacles for its optimal use in the clinical practice. in this work, we present the synthesis of various analogues in which paclitaxel is covalently bound to peptides or as multiple copies to synthetic carriers. these peptide-paclitaxel derivatives possess greater solubility in water, could be suitable in producing anti-paclitaxel antibodies and inhibit the proliferation of human breast, prostate and cervical cancer cell lines. although, no major differences were found concerning the extent of the antiproliferative effect between various paclitaxel derivatives and paclitaxel, the analogue with four molecules of paclitaxel covalently bound to synthetic carrier [ac-(lys-aib-cys)4-nh2] when used at low concentrations inhibited cell proliferation more potently than paclitaxel th489 involvement of the histaminergic system in the nociceptin-induced pain-related behaviors in the mouse spinal cord the purpose of the present study was to determine whether histamine-containing neurons in the spinal cord are involved in nociceptin-induced behaviors in mice. the i.t. injection procedure was adapted from the method of hylden and wilcox. immediately following the i.t. injection, the time spent for nociceptive behaviors including scratching, biting and licking were measured. the i.t. administration of nociceptin resulted in nociceptive behavioral responses, which were eliminated by the i.t. co-administration of opioid receptor like-1 (orl-1) receptor antagonists. the nociceptive behaviors were significantly attenuated by the i.t. co-administration of the h1 receptor antagonists, but not the h2 receptor antagonists. i.t. co-administration of the h3 receptor antagonist significantly increased the behavioral responses, whereas the behavioral responses were completely attenuated by the i.t. co-administration of the h3 receptor agonist. an antiserum against histamine injected i.t. reduced the nociceptin-induced behavioral responses. the same result was observed by i.p. pretreatment with histidine decarboxylase inhibitor. in conclusion, i.t.-administered nociceptin elicits the orl-1 receptor-mediated nociceptive behavioral responses. the activation of the orl-1 receptor by nociceptin may induce the release of histamine in previous studies, we demonstrated that highly constraint cyclic (s,s)-cxaac-containing peptides inhibit platelet aggregation and fg binding [1,2]. cyclization reduces the allowed conformations, of both the backbone and the side chains, and possibly induces a favourable for the biological activity orientation of the charged side chains. conformational studies revealed that orientation of the charged side chains toward the same side of the molecule increase the anti-aggregatory activity of the inhibitor. in this work we present the synthesis and the inhibitory activity of new cyclic compounds. for the design of the studied compounds we combined the available information from the -cdc-containing inhibitors and the gpiib 313-320 (ymesradr) sequence which has been shown to inhibit the adp induced human platelet activation however, its pharmacological effects and physiological functions are still unclear. the present study was designed to characterize the nociceptive behaviours induced by intrathecal (i.t.) administration of hk-1 in mice. the i.t. administration was made in conscious mice according to the method described by hylden and wilcox. immediately after the i.t. administration, the accumulated time for nociceptive behaviours was measured for 10 min. the i.t. administration of hk-1 dose-dependently produced characteristic nociceptive behaviours consisting of scratching, biting and licking, which peaked at 0-5 min and almost disappeared by 15 min after injection. the subcutaneous pretreatment with morphine dose-dependently attenuated the hk-1-induced nociceptive behaviours. the nociceptive behaviours elicited by low-dose of hk-1 were significantly inhibited by i.t. co-administration with nk1 receptor antagonist, however, the nociceptive behaviours elicited by high-dose of hk-1 were not affected by i.t. coadministration with nk1 receptor antagonist. on the other hand, nmda receptor antagonists significantly suppressed both high-and low-doses of hk-1-induced nociceptive behaviours in a dose-dependent manner. these results suggest that the nociceptive behaviours induced by low-dose of hk-1 may be mediated through both nk1 and nmda receptors, whereas high-dose of hk-1 may induce the nociceptive behaviours through nmda receptor the bacterial lp are strong modulators of the innate immune system. until recently, it was generally assumed that triacylated lp, like the synthetic pam3cys-sk4, are recognized by tlr2/tlr1 heteromers, whereas diacylated lp, like fsl-1, induce signalling through tlr2/tlr6 heteromers. contrary to this model, we could show that depending on the peptide moiety, diacylated lp also signal in a tlr6-independent and tlr1-dependent manner. the aim of this study was to analyse more closely the structural basis of this heteromer usage. the synthesis of lp was carried out by fully automated solid phase peptide synthesis and fmoc/tbu chemistry on tcp or rink amide resin. information on the structural factors determining the tlr2/tlr1 versus tlr2/tlr6 heteromer usage was obtained by testing of ligands with cells obtained from tlr2 , tlr6 , and tlr1-deficient mice. when stimulating b-lymphocytes of wild-type mice we found that ester-bound long-chain fatty acids are necessary to induce considerable responses. for triacylated lp with long chain length ester-bound acyl residues (like pam3c-ssnask4) the response in tlr1-deficient cells was only slightly decreased, whereas for lp with short length ester-bound fatty acids (like pamoct2c-ssnask4) the response was completely abolished. in summary, a tri-acylation pattern is necessary but not sufficient to render an lp tlr1-dependent and a di naples 2 department of organic chemistry "ugo schiff sera from patients suffering from autoimmune disorders often contain multiple types of autoantibodies, some of which can be exclusive of a disease and thus used as biomarkers for diagnosis. identification of these autoantibodies, as disease biomarkers, should be achieved using native antigens in simple biological assays. however, post-translational modifications, such as glycosylation, may play a fundamental role for specific autoantibody recognition. in line with these observations, we previously described synthetic glycopeptides able to detect high autoantibody titers in sera of patients affected by multiple sclerosis, an inflammatory, demyelinating disease of the central nervous system. we also demonstrated that glycopeptides able to reveal high antibody titers in multiple sclerosis sera are characterized by a type i we describe here the result of a conformationally driven rational design exercise, which led to the preparation of new, optimized glycopeptides endowed with enhanced antigenic properties. most importantly, the same approach, based on structure alignment, was used to shed light on the native antigen(s), target of pathogenetic autoantibodies involved in demyelination processes vitro and in vivo evaluation for cholecystokinin-b receptor imaging istituto nazionale per lo studio e la cura dei tumori ome (f0)<br> (aib, α-aminoisobutyric acid) to investigate its binding properties to tb(iii) ions. according to our published spectroscopic results f0 populate a set of ordered conformations involving 310/α-helical segments and compact structures generated by the formation of a turn around the flexible gly5-gly6 central motif. cd experiments showed that the binding of tb(iii) to f0 gives rise to a structural transition of the peptide chain from a helical to a folded conformation. peptide binding is also responsible for the dramatic increase in the tb(iii) fluorescence intensity, suggesting that the tb(iii)/f0 complex may represent an interesting system for imaging applications or bioanalytical sensing the 16 kda protein of mycobacterium tuberculosis provokes specific immune response, therefore related epitope peptides and peptide-conjugates can be considered as potential diagnostics. in our previous study we have determined the functional human t-cell epitope within the 91-110 region. based on this we synthesised two groups of peptides: a) nand c-terminal alanine and beta-alanine elongated variants of the 91-104 epitope and b) 91-104 peptides with alanine substitution at different position according to the hla dr and tcr binding sites. peptides were prepared by solid phase synthesis using boc/bzl or fmoc/tbu strategy. the homogeneity and the primary structure of peptides were checked by analytical rp-hplc, amino acid analysis and esi-ms. the t-cell stimulatory activity of the compounds was investigated using in vitro assays (proliferation and ifn-gamma production) on the 91-110 epitope specific human t-cell clones and pbmc (peripherial blood mononuclear cells) from patients and healthy (ppd+, ppd-) subjects. the effective peptides were conjugated to branched polypeptides with polylysine backbone (sak, eak), tetratuftsin derivative (h-[thr-lys-pro-lys-gly]4-nh2) and lysine dendrimer (h-lys-lys(h-lys)-arg-arg-beta-ala-nh2) (map) carrier via thioether bond formation. the subtitution degree of the conjugates was determined by amino acid analysis. pbmc and human t-cell clones were stimulated with the free peptide alone or with peptide-conjugates containing an equimolar amount of peptide or with a mixture of free peptide and carrier italy we demonstrated, for the first time, that an aberrant post-translational modification (ptm, n-glucosylation) is possibly triggering autoantibody response in multiple sclerosis. this was possible because of a "reverse approach", which led to csf114(glc), a structure based designed glycopeptide, as the first multiple sclerosis antigenic probe accurately measuring high affinity autoantibodies (biomarkers of disease activity) in sera of a statistically significant patients' population universal peptide scaffold" to be modified with a series of glycosyl amino acids (different in sugars and linkages), in the aim of developing personalized diagnostic/prognostic tests. the csf114 beta-turn structure, exposing at the best the aberrant ptm specific for antibody-mediated forms of other autoimmune diseases, will lead to a family of antigenic probes to be used in diagnostic this information is encoded by the distribution of the electron-ion potential (eiip) of amino acids along the sequence and is represented by the frequency components in is. proteins with the same biological functions or interacting proteins (e.g. antibody/antigen) share the information corresponding to the common frequency components in their iss. investigation of the hiv-1 envelope glycoprotein gp120, as a model system for hypervariable proteins, revealed that this information is strongly conserved and is not significantly affected by natural mutations. the c-terminus of the second conserved region (c2) of gp120, encompassing ntm peptide is important for infectivity and neutralization of hiv-1, while human natural anti-vasoactive intestinal peptide (vip) antibodies reactive with gp120 play an important role in control of hiv disease progression. ntm/vip multiple copies were coupled to an artificial sequential oligopeptide carrier for developing an immunoassay (elisa) as a reproducible, reliable and sensitive tool for the detection of anti-ntm/vip derived antibodies these peptides have been utilized in an immunopeptidometric assay for specific measurement of active, noncomplexed psa. however, this assay has not been sensitive enough for the measurement of active psa in clinical samples. therefore, we aimed to develop an improved assay utilizing the same principle as previously, but using a more sensitive detection method based on proximity ligation assay. methods: in the assay, psa is first captured on a solid phase by a psa antibody czech republic rapidly increasing knowledge of new gonadotropin-releasing hormones (gnrh)of different species of the animal kingdom induces the need to prepare new synthetic derivatives and fragments of these peptides with higher potency and metabolic stability and suitable for the formulation of new immunogens. the species related differences in the sequence of the native mammalian gnrh pglu-his-trp-ser-tyr-gly-leu-arg-pro-glynh2 concern predominantly the positions 5, 7 and 8, particularly tyr in position 5 is replaced for his or leu, leu in position 7 by val or trp, and arg in position 8 is substituted by lys, ser, asn or gln. several gnrh derivatives with with the above substitution and gnrh fragments were prepared by solid phase peptide synthesis and purified by rp-hplc. purity of the synthetic peptides was checked by capillary zone electrophoresis (cze); peptides were analysed as cations in acidic backround electrolytes (ph 2.25 -2.quantitative analyses for determination of their effective electrophoretic mobilities and the estimation of their effective charges.supported by grant of ministry of agriculture of cr-nazv qf 3028 by rants of ga cr nos we use peptaibiomics for the structural determination of peptaibiotics from fungi grown on single agar plates thus avoiding time-consuming isolation and purification procedures. the method comprises fast and effective solid-phase extraction followed by on-line rp-hplc coupled to tandem esi-ms. here we present a survey of the peptaibiome of hypocrea species. in extracts of hypocrea semiorbis, h. vinosa, h. dichromospora, h. gelatinosa, h. nigricans, h. muroiana and h. lactea a multitude of short and long-chain peptides containing aib could be characterized. the formation of new and known peptaibiotics could be established by comparison with sequences stored in data bases japan process scale rp hplc purification of peptides and proteins is increasingly important in bio-pharmaceutical production. besides selectivity, other crucial factors are loadability, recovery loadability is believed to depend on the surface area of the packing material. consequently, smaller pores providing larger surface area should lead to increased loadability. this principle is misleading in the case of large molecules, because they cannot penetrate smaller pores. therefore the chromatographically accessible surface area has to be taken into account. recovery problems like irreversible adsorption or aggregation are frequently caused by hydrophobic surface properties of ods phases. the less hydrophobic c8 is a substituent to avoid considerably these problems. however c8 is less durable than ods under extreme acidic conditions. our new proprietary c8 modification technology combined with a perfect end-capping minimizes the presence of residual silanol groups and protects the silica surface sp-200-c8-bio demonstrates high mechanical stability by no obvious alteration of back pressure and particle size after 10 repeated packing cycles in dac columns. by overcoming the common weaknesses of the conventional c8 rp silica phases, daisogel sp-200-c8-bio opens new avenues for process scale separation of peptides and proteins. m514 determination of peptide: protein molecular ratio in conjugates by seldi-ms method synthetic peptides are widely used as antigens in various research and practical areas of biology and medicine. peptides with molecular masses < 5000 kda should be conjugated with carrier proteins in order to ensure their immunogenicity and protect from proteolysis. in these cases the comparison of peptide immunogenicities and immunotest system development should be performed having in mind exact peptide-to-protein ratios. 23 conjugates of peptide fragments of hepatitis c virus envelope protein e2 with ovalbumin, bovine serum albumin, and myoglobin were prepared using glutaraldehyde (ga), m-maleimidobenzoyl n-hydroxysuccinimide ester, dimethyl suberimidate (dms), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide as conjugating reagents. the rough evidence of the peptide-protein conjugate formation was obtained by page. the exact peptide:protein molar ratio was estimated in all 23 conjugates by seldi-ms. almost all conjugates had oligomeric structures due to the formation of intermolecular linkages between proteins. the peptide : protein molar ratio in conjugates varied from 1:1 to 13,6:1. conjugates obtained with the ga were more diversified in the number of peptide molecules linked to carrier proteins (peptide:protein ratios ranged from 3:1 to 13:1) than other conjugation reagents mazur-marzec 1 poland posttranslational modifications (ptms) like phosphorylation, acetylation, or methylation have been shown to play a significant role in directing the function of various proteins [1]. in eukaryotes, most of proteins have been shown to be posttranslationally regulated by a variety of different modifications. many effects of ptms include a change of enzymatic activity capillary electrophoresis (ce) has been used to study electrophoretic behavior of ptm-peptides gal-nh2, gal(1-15)-nh2 by capillary electrophoresis. using a phosphate buffer most of ptm-peptides were poorly separated at acidic or neutral ph. the best results were obtained using trifluoroethanol containing separation buffers. optimization of ce separation of maps of peptides containing ptms should allow to detect ptm-proteins and characterize their role in the living cell. comparison of modification events occurring in diseased and healthy cell may iran the purpose of this study was to use the application of multiplex reverse transcription polymerase chain reaction(rt-pcr) assay for detecting the two most common leukemia translocations t(1;19) and t(9;22) in childhood acute lymphoblastic leukemia in iranian children. 32 cases of leukemia patients were screened with the rt-pcr assay. this assay will identify the all type bcr-abl transcripts encoded by the t(9;22) and all described variants of the e2a-pbx1 transcript encoded by the t(1;19). rna was isolated from leukocyte cells of patient's samples. through the construction and optimization of specific primers for each translocation,we have been able to set up multiplex rt-pcr reactions.then pcr products was electrophoresed on agaros gel and were compared with size markers and expected fragtments key words: acute lymphoblastic leukemia -multiplex rt-pcr tu521 study on the syntheses and lc/esi-ms analyses of the glutathione conjugates of bile acids t. wakamiya 1 , m. sogabe 1 a carboxylic acid-containing drug, is metabolized to a glutathione (gsh) conjugate in vivo, and the conjugate is excreted in human urine [1]. although bile acids, compounds with carboxylic acid in molecules, are also expected to form gsh conjugates in liver, no evidence is so far obtained to confirm such metabolism, since there are no suitable standard samples for the research. in the present paper we report the syntheses of the gsh conjugates of main bile acids in human, i.e., cholic acid (ca), chenodeoxycholic acid (cdca), deoxycholic acid (dca), ursodeoxycholic acid (udca) and lithocholic acid (lca) as shown below, and the detailed analyses of these synthetic conjugates by means of linear ion trap lc/esi-ms. furthermore, the evidence for conversion of cholyl adenylate [2] and ca-coa thioester into ca-gsh conjugate will be presented these peptides do no exhibit such strong side effects as csa, but their practical application is hindered because of their poor solubility in water. the 49-57 fragment of tat protein and its analogs, including oligoarginine sequences, are known for their unusual ability to cross cell membranes, skin, and blood-brain barriers. moreover, these peptides are able to transport other substances into cells. this strategy was successfully applied in cases of csa, taxol, and other drugs to improve their bioavailability. now we have synthesized a series of analogues of cyclolinopeptide a, clx, and the immunosuppressive fragment of ubiquitin, covalently bound to the cell-penetrating fragment of tat and its analogues. the ability to cross the biological membranes and the immunosuppressive activities of these conjugates were tested. the conformation of the peptides was determined by circular dichroism methods: we used fluorescein-labelled cationic cell-penetrating peptides and analyzed the uptake efficiency (flow cytometry) as well as the intracellular distribution (confocal laser scanning microscopy). the bioactivity of a proapoptotic cargo-peptide, delivered into the cells either via electroporation or via cpps was quantified using a caspase-3 activity assay and cellular assays. to address the integrity of cpps during their trafficking, a fluorescent double-labelled antp peptide was designed and used as an intracellular fret-sensor. results: endocytosis-mediated uptake of the cpp-cargo conjugate led to a significant reduction of cargo bioactivity compared to its direct transfer via transient membrane permeation. this finding was related to the sequestration of peptides within endocytic vesicles but also, in the case of the tnf response, to the induction of receptor internalization during cell entry. moreover, during endolysosomal passage peptides undergo significant proteolytical degradation. conclusions: the endocytosis-dependent uptake mechanism of cholesterol pullulan (cp), in which maltose moieties are partially modified by cholesterol, is unique in forming self-assembled nanoparticles (20-30 nm) in water. combination of these characteristics is considered to be promising for development of effective non-viral vectors without toxicity. a conjugate of hiv-tat and cp was synthesized and its gene expression efficiency was evaluated. fully protected hiv-tat-(48-57)-cys(snm)-gly-nh-r was obtained by conversion of the corresponding cys(acm) peptide which was synthesized by the solid-phase method [snm: (n-methyl-n-phenylcarbamoyl)-sulfenyl] [1]. the sulfhydryl function was introduced to the hydroxyl groups of cp by acylation with trt-3-mercaptopropionic acid followed by acid treatment. resulting 3-mercaptopropionyl-cp was coupled with cys(snm) peptide to form disulfide bridge and the protecting groups of the peptide were removed to give the cp-tat conjugate. cp-tat and pcmv-luc complex was transfected into cos7 cells and luciferase activity was analyzed after 24 h. cp-tat elicited remarkable cytoplasmic luciferase activity and low toxicity this finding provides a possibility to use gnrh-iii as a targeting moiety for intracellular drug delivery. therefore we have prepared methotrexate and doxorubicine conjugates of gnrh-iii. the drug molecules were attached to the lys side chain in position 8 of gnrh-iii by thioether bond formation through the gflg spacer elongated with cys either at the c-or n-terminus. since we found earlier that the dimer derivative of gnrh-iii was more effective, new dimer derivatives with a combination of antitumour agents were also prepared. branched gnrh-iii derivative (pyrhwshdwk(clac-glfgc(acm))pg-nh2]) was synthesised by spps. the drug molecules were attached to this compound by thioether bond and finally disulfide bridge was formed between two peptide chains. the cytotoxicity of new derivatives was characterised by mtt test th531 oligopeptide antifungals are exceptionally active against multidrug-resistant yeast previous studies have demonstrated that longer sirnas that are processed by dicer can result in more potent knockdown than the corresponding standard 21-mer sirnas. dicer-substrate 25-27-mer sirnas were conjugated with different structural classes of peptides and their cell uptake properties evaluated. peptides were conjugated to the 5' end of the sirna sense or antisense strand via a thioether bond under denaturing conditions to prevent aggregation and precipitation. the ability of conjugates to translocate fluorescently-labeled sirna across the plasma membrane was evaluated by flow cytometry. the results indicate that some peptides can mediate higher efficiency uptake of sirna into cells compared with lipofectamine or cholesterol-conjugated sirna. the peptide-sirna formulation with 27-mer sirna conjugates showed higher knockdown of tnf-alpha mrna and protein levels in activated human monocytes in vitro compared to the conjugated 21-mer sirna species. the products resulting from in vitro digestion of peptide-conjugated rna duplexes with recombinant human dicer were identified using esi-ms and consisted predominantly of the desired 21-mer sirna several peptides containing the sequence arg-gly-asp (rgd) were studied and developed for their nanomolar affinity to the membrane receptor alfa v beta3 and alfa v beta 5 integrins, which are over-expressed by endothelial cells during proliferation and by tumor cells. to improve the pharmacological profile of some camptothecin derivatives (cpts), five conjugates were designed, where the cytotoxic drugs were covalently attached to the rgd peptide analogues for preferential uptake into tumor cells. the peptides to be used have been selected among a series of new pentacyclic peptides bearing at 5-position a trifunctional pseudoamino acid with a carboxy-terminal side-chain. peptide analogues showing the highest affinity to alfa v beta 3 and alfa v beta 5 integrins were coupled with cpts at different positions. the conjugates have been optimized for binding to the receptors, proteolytic stability and an overall improvement in tumor selectivity. the nature of the linkage between rgds and cpts has a major impact on stability and biological activity of the conjugates. the conjugates with amide bond, but not those with ester bond, are sufficiently stable and show in vitro antitumor activity against a498 and a2780 cell lines combination of amaranth protein with other plant proteins (cereals) enables to formulate the composite protein (near to milk or beef protein, but exclusively on vegetal basis). it is shown on graphs. the aim of the projects' proposals is a development and realization of the technology for fractionation of amaranths defatted flour product is a top protein obtained by removing starches and next polysaccharides decomposed on soluble monosaccharide by specific enzymes. there are shown the chromatograph measuring. we can see complete disintegration of starch and the unchanged proteins. the separate solution monosaccharide is usable for others fermentative processes or as a nutrient solution for yeasts there are description methods for isolation amaranth protein -extraction processes, enzymatic removal starch. the product is a isolate protein rich in essential amino acids. the waste monosaccharide solution was used to production yeasts biomass rich in proteins vitamins amaranth protein isolate have high nutritional value and can be used as food ingredients, for functional, probiotic formulation to begin with, we made epitope mapping with the highly sensitive spot array method (2) in order to study antigenic regions of parvovirus b19 vp1 and vp2 capsid proteins. epitope mapping identified highly reactive, immunodominant early epitope on parvovirus surface that centered to kyvtgin residues of vp1. in the subsequent phases we developed the kyvtgin epitope type specific (ets) igg serodiagnostics. a correlation between enhancing igg avidity to b19 capsid and a transient reactivity with the point-of-care kyvtgin peptide was clear. together the two assays enhanced the value of early diagnosis of b19 infections (3) acute phase-specific heptapeptide epitope for parvovirus b19 diagnosis synthetic peptide arrays on membrane supports-principles and applications human parvovirus b19 infection during pregnancy--value of modern molecular and serological diagnostics 1 laboratory of peptide & protein chemistry & biology 2 department of organic chemistry "ugo schiff" and cnr-iccom 3 department of chemistry 4 department of pharmaceutical sciences glc) able to recognize specific autoantibodies in multiple sclerosis (ms) patients' sera has been developed by the laboratory of peptide & protein chemistry and biology [1and bio-plex suspension array system, biorad. the biosensor technology and bio-plex suspension array system will offer advantages such as rapid analysis, and high sensitivity for a high throughput screening. immobilisation will be based on different strategies that are anchoring the synthetic antigen on different solid supports such as polystyrene well plates (elisa) optimisation of the different techniques was performed with anti-csf114(glc) autoantibodies isolated using affinity chromatography from ms patients' sera. the analytical parameters such as specificity, sensitivity, and matrix effect were evaluated. the different technologies have been used for a high throughput screening of ms sera which control specific cell adhesion.2 here we discuss the route for preparation of amphiphilic block copolymers composed of hydrophobic polylactide and hydrophilic polyethylene oxide (peo) blocks, carrying various cell-adhesion oligopeptide sequences at the end of peo block. fully protected peptide fragments were prepared by solid-phase peptide synthesis by using fmoc strategy and chlortrityl resin. the side-chain protected peptides were cleaved from resin by 25% hfip solution in dcm. the copolymers peptide-polytehyleneoxide were prepared by coupling of the activated peptide fragments with α-amino-ω-hydroxy-peo in dmf using pypop as an activation reagent. subsequently, the polylactide block was grafted to the ω-hydroxy end group of the peptide-peo copolymer via a controlled rop polymerisation of lactide 2r)-2-aminocyclopentanecarboxylic acid (acpc) and beta-methylphenylalanine (beta-mephe) were designed and synthesized to obtain more potent and selective mu-opioid receptor ligands with higher stability against proteolytic enzymes. we have prepared the peptides by spps methods using racemic amino acids. the diastereomeric peptides were separated by hplc. the configuration of the unnatural amino acids in the peptides was determined by chiral tlc using enantiomeric standards. radioligand binding assays and in vitro gpi and mvd assays indicated that several analogues showed high, subnanomol affinity and high selectivity for mu-opioid receptors having agonist or antagonist properties. the incorporation of alicyclic amino acids into the endomorphins resulted in enzyme resistant peptides. the most promishing analogues (dmt-pro-phe-phe-nh2 and tyr-(1s,2r)acpc-phe-phe-nh2) were labeled with tritium using precursor peptides containing dehydroproline or dehydro-(1s,2r)acpc amino acids and tritium gas and pd/baso4 catalyst. the novel peptides and their radiolabelled analogues with high specific radioactivity (1.4-2.8 tbq/mmmol) have become useful pharmacological and biochemical tools for the opioid research iran background and aims: injectable drug delivery based on polymer solution platforms has gained in resent years, particulary for protein-based therapies. the influence of polymer molecular weight (rg 502h, rg504h) on the morphology, erosion of matrices and also on their in-vitro drug release behavior over a period of 28 days was assessed for leuprolide acetate in this study. methods: each formulation was composed of 33% (w/w) polymer and 3% (w/w) leuprolide acetate dissolved in nmp. release studies were performed in a home-made diffusion cell at 37°c. the polymer erosion was studied using two different methods as follows. (a): l-lactic acid detection (b): ph change study. the morphology of the matrices was then analyzed by scanning electron microscope as is shown, the lower molecular weight polymer formulation shows higher porosity and pore diameter due to a rapid phase inversion phase i) can be divided into three more phases with different release rates. results showed that burst effect for rg 502h, 32%, was significantly (p<0.05) higher than rg 504h (13%) italy fabrication of photocurrent-generating systems based on bioinspired organic-inorganic hybrid materials is currently of great interest. more specifically, the photoelectronic properties of nanometric films formed by peptide self-assembled monolayers have been actively investigated. in this work interdigitated gold microelectrodes were modified by covalently linking a hexapeptide ester functionalized by a lipoic acid (lipo) at the n-terminus. the peptide chain [lipo-(aib)4-trp-aib-otbu] comprises five α-aminoisobutyric acid (aib) residues and one trp, a fluorescent amino acid with strong absorptions in the uv region. due to the very high percentage of conformationally constrained aib residues in the chain, the peptide adopts a rigid 3-10-helical structure. cyclic voltammetry measurements indicate that the peptide forms a homogeneously and densely packed monolayer on the gold surface, while current/voltage curves exhibit interesting rectifying properties of the peptide sam. photocurrent generation experiments, performed on the peptide-layered microelectrode, show peculiar modifications of the spectrum. at 240 nm a notably higher photocurrent/voltage response was observed for the peptide-modified electrode, suggesting that a photoinduced electron transfer process from trp to gold does take place with high efficiency this may lead to randomly orientated enzymes and subsequently limited activity. the aim of this work is to selectively activate enzymes at their c-terminal position in order to allow specific immobilisation. we chose akr1a1, an enzyme of the aldo/keto reductase superfamily, for the synthesis of an artificially monolabeled redoxprotein. akr1a1 is a monomeric enzyme and catalyzes the nadph dependent reduction of aliphatic/aromatic ketones and aldehydes. to produce monofunctionalized enzymes we applied the strategy of expressed protein ligation (epl). accordingly, we used the impact®-system and cloned the aldo/keto-reductase as fusion protein with an additional intein/chitin binding domain. through intein mediated splicing we could produce c-terminal thioester of the akr1a1. in the next step, the thioester was coupled to a biotin containing peptide by native chemical ligation. this specifically modified enzyme was immobilised on avidin coated surfaces. the attachment on the surface was tested by tryptic digestion, followed by maldi-tof-ms analysis since safe organic solvent waste disposal is an important environmental problem, we aimed to perform peptide synthesis in water. we have reported solid phase peptide synthesis in water using water-soluble n-protected amino acids, such as 2-[phenyl(methyl)sulfonio]ethoxycarbonyl and 2-(4-sulfophenylsulfonyl)-ethoxycarbonyl amino acids. following to study on water-soluble n-protected amino acids, we developed a new technology based on nanochemistry for solid phase peptide synthesis in water. the new technology is based on coupling reaction of suspended nanoparticle reactants in water. fmoc-amino acids are used widely in peptide synthesis, but most of them show poor water-solubility. we prepared well-dispersible fmoc-amino acid nanoparticles in water by pulverization using a planetary ball mill in the presence of poly(ethylene glycol) (peg). the size of fmoc-amino acid particles was 300-500 nm. to evaluate the utility of this technique, leu-enkephalin was prepared using the nanoparticulate fmoc-amino acids on a peg-grafted rink amide resin in water supramolecular structures formed from n-lipidated oligopeptides immobilized in the regular pattern on the cellulose surface are able to bind ligand molecule, thus acting like artificial receptors. due to the conformational flexibility of lipidated oligopeptide chains, the supramolecular structure is highly flexible, forming the cavities with the shape and prosperities adjusted most effectively to requirements of the guest molecule. structural requirements for a peptide providing the most efficient fit the guest molecules are not known, therefore an array of the artificial receptors have been synthesized and used in the studies. thus, even in the case, when the single receptor in an array does not necessarily have selectivity for a particular analyte, the combined fingerprint response can be extracted as a diagnostic pattern visually, or using chemometric tools in order to improve the sensitivity of the competitive binding and to study the mechanism of molecular recognition, experiments involving fluoresceine and fluoresceine marked acp fragment were performed. we found that λmax and intensivity of fluorescence depends on the structure of the peptide motif and lipidic fragment of receptor this mts was linked with dhhp-6 by disulfide bond, and the new molecular was named mts~dhhp-6. the peroxidase activity of mts~dhhp-6 (2.1x103 u•µmol-1) was tested and similar to that of mp-11 (4.2x103 u•µmol-1). mts~dhhp-6 coated with quantum dots (qds) [3] were observed to accumulate into neonatal rat cardiomyocytes (nrcms) of wistar rats and co-localized with mitotracker red in mt. these results suggest that mts~dhhp-6 is an excellent apx mimics and may have potential proceedings of the twenty-eighth european peptide symposium, kenes international, israel, 2005, 551. references: 1 elastin-based polypeptide, poly(val-pro-gly-val-gly), undergoes self-assembly called coacervation, in which microcoacervate droplets with approximately 1000 nm diameters are formed [1]. nanoparticles cross-linked by cobalt-60 γ-irradiation of these microcoacervate droplets are useful as drug release devices. to investigate the size optimization of nanoparticles, the stability of nanoparticles in the treatment of enzyme, and the drug release profiles from nanoparticles, the three copolymers; poly[10(val-pro-gly-val-gly), (val-ala-pro-gly-val-gly)], poly[4(val-pro-gly-val-gly) application of polyelectrolytes and theoretical models the synthetic heptapeptide rnwdvyk is a fragment of a high affinity receptor (fcεri) for immunoglobulin e (fragment 111-117). it is the active domain for binding with ige. the program of studies of biological properties of the heptapeptide included the investigation of its binding to ige contained in standard solutions and in patients' blood serum. the binding of rnwdvyk with ige was investigated by the ifa method using the ige antibodies labelled with horse-radish peroxidase (hrpo). we determined the optimum sorption concentration of the peptide in this experimental immunoenzyme system to be 100 mkg/ml. the ability of synthetic rnwdvyk peptides to bind with ige was studied as a function of ige concentration in standard serum (0.47 to 60 ng/ml ige). a high correlation was found between the ige concentration and the optical density of the solution after introducing monoclonal antibodies labeled with hrpo and the substrate chromogenic mixture (r=0.99). similar investigations were conducted using the allergy patients' blood serum. the serum with a known concentration of ige was added to immunological plotting boards with sorbed synthetic rnwdvyk peptides. a high correlation was also found between the concentration of ige in the patients' blood serum and the optical density of the solution after introducing monoclonal antibodies labelled with hrpo and substrate chromogenic mixture (r=0.94). our experiments showed the high ige binding activity of synthetic rnwdvyk peptides. we demonstrated the possibility of construction of diagnostic systems for the quantitative determination of ige and ige-antibodies. the fusion protein nucleocapsid-dutpase is present in virions of mason-pfizer monkey betaretrovirus and in virus-infected cells where it potentially contributes to rna/dna folding and reverse transcription (barabas, et al., 2003; bergman et al., 1994; berkowitz, et al., 1995) . in addition to trimeric dutpase core, the protein possesses flexible n-and c-termini consisting of the nucleocapsid segment, and a peptide motif conserved in dutpases. to analyze the function of the flexible cterminal peptide segment, reconstitution experiments were designed with truncated enzyme lacking the c-terminal 14mer oligopeptide and the synthetic oligopeptide prepared on rink-amid resin by solid-phase peptide synthesis, using fmoc strategy. the truncated enzyme proved to be practically inactive. addition of the synthetic 14mer (pyrgqgsfgssdiy) at 100fold molar excess resulted in partial complamentation of the catalytic activity (to 10% of original). a mixture of the truncated enzyme and the 14mer oligopeptide (this latter at 100fold excess) was put to crystallization trials. we conclude that the c-terminal 14mer is essential for catalytic activity. antifolate drugs are inhibitors directed to interfere with folate metabolic pathway. methotrexate (mtx) and pemetrexed (alimta®) are known folic acid analogues used in cancer treatment. different peptide conjugates of mtx have been prepared for intracellular delivery. (1) in octaarginine conjugates one of the carboxylic groups of mtx was attached to the n-terminal of the peptides. (2) however, as results showed, that both carboxylic groups are required to the biological effect of mtx. therefore we decided to synthesize peptide conjugates of folic acid analogues in which the carboxylic groups are untouched. octaarginine, penetratin and a cyclic peptide cgnkrtrgc, which can deliver a cargo molecule in the lymphoid system, were used as delivery peptides. we introduced squaric acid or aminoxy acetic acid as linker moiety between the peptides and cargo molecules. the conjugation was monitored by rp-hplc, the crude products were purified by rp-hplc and were identified by mass spectrometry. the biological activity of the conjugates was evaluated in vitro on sensitive and resistant human leukemia (hl-60) cell lines. besides its endocrine activity, trh (the tripeptide pglu-his-pro-nh<sub>2</sub>) has also been long recognized as a modulatory neuropeptide with broad range of physiological and pharmacological activities in the central nervous system (cns). although numerous centrally active and metabolically stable analogues and peptidomimetics have been synthesized using trh as a template, selectivity of their cns action has remained an issue to be addressed. we aimed at discovering novel analogues with enhanced cns-selectivity by incorporating pyridinium building blocks. the design also allowed for enhancing transport across the blood-brain-barrier and increasing residence time in the cns through prodrug strategy. solid-phase chemistry was used to prepare the analogues and novel methods previously not used to incorporate pyridinium moieties into resin-bound peptides such as the zincke reaction were also introduced. comprehensive evaluation included measurement of affinity to trh-receptor, acetylcholine-releasing, analeptic and antidepressant-like activity in animal models, as well as prediction of membrane affinity, determination of in vitro metabolic stability, and pharmacokinetics and brain uptake/retention studies that employed in vivo microdialysis sampling. a strong connection between acetylcholine-releasing potency and analeptic effect in animals was obtained for close analogues of trh, while pyridinium compounds designed from the structurally related pglu-glu-pro-nh<sub>2</sub> maintained the antidepressant-like effect of the parent peptide, while showing significant decrease in analeptic action.in conclusion, an increase in the selectivity of cns-activity profile was obtained by the incorporation of pyridinium moieties. we have also demonstrated the benefits of the prodrug approach on the pharmacokinetics, brain uptake and retention of the analogues upon systemic administration. the use of small radiolabelled compounds such as peptides is a very attractive tool for the diagnosis of several different pathologies, specially cancer, through the use of nuclear medicine techniques.1 among the various membrane receptors, the two cholecystokinin receptors ccka-r and cckb-r are very promising biological targets for radiolabelled compounds due to their overexpression in many tumours2. in order to develop radiolabelled peptide derivatives able to target these receptors, the binding mode of the c-terminal cholecystokinin octapeptide (cck8), toward the two cholecystokinin receptors ccka-r and cckb-r has been, recently, structurally characterized. 3 the structural data suggest that modifications on the n-terminal end of cck8 obtained by introducing chelating agents and their metal complexes should not affect the interaction of the derivatized cck8 peptides with both ccka-r and cckb-r. here we report the labelling procedures and the in vitro and in vivo characterization of new 99mtc cck8 derivatives. a stable 99mtc-nitrido complex is obtained by using the coordinative set formed by: 1) the n-terminal amino group and the sh cystein of the cck8 derivative cys-gly-cck8 peptide; and 2) a pnp aminodiphosphine used as coligand. several phosphines are used in order to define the best labelling procedures and to optimize the in vivo biodistribution properties of the 99mtc labelled peptides.references various combinations of pore size and chemistry of silica-based materials were investigated for high performance liquid chromatography (hplc) of peptide separation. incorrect pore size and ligands have been suggested to cause peak broadening, poor resolution and poor recovery. our study suggests that an appropriate combination of pore sizes and ligands is necessary to obtain the most efficient usage of reversed-phase hplc columns according to the molecular weights of peptides and proteins. we will also show the possibilities of an improved method development for the separation of complex peptide mixture by ph or additives.the development of new biopolymer materials as drug delivery systems is of enormous interest on biomedicine. dendrimers are polymers with particular properties; they are highly branched polymers with well-defined chemical composition, and show compact globular shape, monodisperse size and controllable surface functionalities. peptide dendrimers incorporates amino acids in their structures and have additional features such as biocompatibility and biodegradability.in previous studies we described the solid-phase synthesis of a new class of polyproline-based dendrimers. these biopolymers have the capacity to cross the mammalian cell membrane and moderate toxicity. these promising results open up the possibility to explore these dendrimers as delivery systems.to design more versatile polyproline dendrimers, we have developed a methodology that involves the combination of solid-phase and solution strategies. diverse multivalent peg-and proline-based cores were synthesized to attain dendrimers with distinct topologies. dendrimers were synthesized by iterative building block addition [(glypro5)2imdoh], around an inner core, using a peptide solution convergent approach. a variety of coupling methodologies and protecting groups for the n-terminal function were explored. the novel high throughput protein detection system using designed peptide arrays has been successfully indicated on their capabilities as the "protein-chip" [1] [2] [3] [4] . our concept has many advantages especially for high-quality industrial production and practical applications compared to arrays with antibodies or recombinant proteins. the deposited peptide solution can be dried without covalent immobilization, although, when the resulted arrays are exposed in protein-solution they showed planned conformation [5] . based on these basic results, several hundreds of α-helical, β-loop and β-sheet peptides, which involved a cysteinyl residue for covalent immobilization and tamra as a fluorescent label, were successfully synthesized, characterized and used as capture molecules. a novel material for chips made from amorphous carbon suitable for our concept has been developed, which has significant advantages over conventional glass or polymer plates, such as no selffluorescence, mechanically more stable, easy manufacturing in the aspects of precised and high throughput processing. thus, chip-plate with nano-l wells could also be easily manufactured. peptides were deposited on these chips surface covalently as well as non-covalently in 350 picol/spot (diameter: ca 200 µm). the resulted chips were used for protein detection. a part of this work was funded by the okinawa-bio-project and nedo-grant. key: cord-283035-tpqf458q authors: thanthrige-don, niroshan; abdul-careem, mohamed f.; shack, l. allen; burgess, shane c.; sharif, shayan title: analyses of the spleen proteome of chickens infected with marek's disease virus date: 2009-08-01 journal: virology doi: 10.1016/j.virol.2009.05.020 sha: doc_id: 283035 cord_uid: tpqf458q marek's disease virus (mdv), which causes a lymphoproliferative disease in chickens, is known to induce host responses leading to protection against disease in a manner dependent on genetic background of chickens and virulence of the virus. in the present study, changes in the spleen proteome at 7, 14 and 21 days post-infection in response to mdv infection were studied using two-dimensional polyacrylamide gel electrophoresis. differentially expressed proteins were identified using one-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (1d lc esi ms/ms). comparative analysis of multiple gels revealed that the majority of changes had occurred at early stages of the disease. in total, 61 protein spots representing 48 host proteins were detected as either quantitatively (false discovery rate (fdr) ≤ 0.05 and fold change ≥ 2) or qualitatively differentially expressed at least once during different sampling points. overall, the proteins identified in the present study are involved in a variety of cellular processes such as the antigen processing and presentation, ubiquitin–proteasome protein degradation (upp), formation of the cytoskeleton, cellular metabolism, signal transduction and regulation of translation. notably, early stages of the disease were characterized by changes in the upp, and antigen presentation. furthermore, changes indicative of active cell proliferation as well as apoptosis together with significant changes in cytoskeletal components that were observed throughout the experimental period suggested the complexity of the pathogenesis. the present findings provide a basis for further studies aimed at elucidation of the role of these proteins in mdv interactions with its host. marek's disease (md) in chickens is caused by gallid herpesvirus 2 (gahv-2) or marek's disease virus (mdv). hereafter, this virus is referred to as mdv. mdv is a highly prevalent alpha-herpesvirus and the disease it causes is characterized by transient neurological signs and immunosuppression at early stages that could subsequently be followed by lymphoma formation in various visceral organs in susceptible birds. upon infection via inhalation, in all infected birds, mdv is taken to various lymphoid organs, such as spleen, thymus and bursa of fabricius where early cytolytic infection in b cells that proceeds to latent infection of t cells occurs. while a lifelong latent phase could occur in genetically md-resistant birds and in those protected by vaccination, late reactivation of latent virus in susceptible birds could cause transformation of mainly cd4+ t cells, leading to lymphoma formation. meanwhile, active replication of mdv occurs in feather follicles of infected birds regardless of their genetic susceptibility rendering them a continuous source of infectious viruses (baigent and davison, 2004) . although md is currently controlled by vaccination, there have been periodical md outbreaks caused by new strains of mdv with increased virulence (witter, 1997) . the potential of mdv to evolve and overcome vaccinal immunity is considered as a major threat for a sustainable md control strategy. however, many aspects of mdv-host interactions are still being elucidated (baaten et al., 2004) . to date, several studies have been conducted to examine host gene expression in response to mdv infection on a relatively large scale using various genomic techniques, such as microarrays. changes in gene expression of chicken embryo fibroblasts (cef) infected with rb1b, a very virulent strain of mdv, were studied by morgan et al. (2001) using a microarray containing 1126 expressed sequence tags. these authors reported the differential expression of a number of host genes including those associated with inflammation, antigen presentation and cell growth. an in vivo study by our group using the same virus strain and a small-scale microarray has revealed significant changes in the expression of genes encoding cell surface molecules, transcription and signal transduction molecules as well as cytokines (sarson et al., 2006) . while studies of this nature, in the context of md in particular and various other viruses in general (reviewed in piersanti et al., 2004) would certainly enhance our understanding of hostpathogen interactions, further expansion of this knowledge with proteomic studies is still important (reviewed in burgess, 2004; zhang et al., 2005) . this is partly because of the possible inconsistency between the expression of genes at the transcript and protein levels (gygi et al., 1999) . furthermore, viruses can induce post-translational modifications in host proteins without affecting the mrna expression (liu et al., 2001) . fig. 1 . representative 2d gel images of mdv-infected and uninfected control spleen proteomes with their respective sampling points. arrows with accompanying spot numbers show successfully identified protein spots that were uniquely expressed (qualitative differences) in each group at corresponding time point. please refer to table 1 for identities of corresponding spot numbers and to fig. 2 for the map of quantitatively differentially expressed spots. in a recent in vitro proteomic study, ramaroson et al. (2008) inventoried 1460 and 1676 proteins expressed in mdv-infected and mock-infected cef, respectively. several other proteomic studies in the context of md have been conducted to model the proteome of mdvtransformed cd4+ t lymphocytes in vitro. notably, proteomic modeling of mdv-transformed cd30 hi cd4+ t lymphocytes has suggested that these cells exhibit a regulatory t cell phenotype (buza and burgess, 2007; shack et al., 2008) . further, such studies have been able to describe the fundamental differences between mdv-transformed t cells and their non-transformed healthy counterparts with regard to activated signalling pathways . with respect to viral protein expression, liu et al. (2006) identified a number of unique proteins expressed during the lytic phase of mdvinfected cef using a mass spectrometry-based proteomic approach. these studies highlight the potential of various proteomic tools in understanding the dynamics of host-pathogen interactions during various phases of md. the current study was intended to investigate the dynamics of host protein expression across the various phases of mdv life cycle in mdvinfected chickens. while we were able to detect more than 500 separate protein spots for each sample, here we report more than 60 significantly differentially expressed proteins identified using two-dimensional gel electrophoresis (2de) and mass spectrometry. putative importance of some of these proteins in the context of md is discussed. proteins from spleens of mdv-infected and uninfected control chickens at 7, 14 and 21 days post-infection (dpi) were extracted and analyzed by 2de in order to compare the protein expression profiles between each group. on average, 517 ± 84 distinct protein spots could be resolved by 2de using ph 3-10nl ipg strips loaded with fig. 2 . a representative gel image showing 2d gel electrophoresis map of the relative locations of spots that displayed significant quantitative differential expression (fdr ≤ 0.05 and fold change ≥ 2) at least once during different sampling times. this image represents the proteome of 7 dpi mdv-infected spleen. because of the absence of certain spots in this gel, arrows for spot 111, 968, 1576 and 1609 represent the relative positions only. please refer to table 1 for identities of corresponding spot numbers and to fig. 1 for maps of qualitatively differentially expressed spots. fig. 3 . comparison of total numbers of significantly differentially expressed protein spots in mdv-infected spleens at various sampling time points. in calculation of total number of spots in each category, newly induced protein spots were considered as upregulation and the absence of spots compared to uninfected controls was considered as down-regulation. 200 μg of total proteins. the molecular weights of spots ranged from 8 to 120 kda. differences in spot intensity were identified as either qualitative or quantitative changes (figs. 1 and 2). on average there were over 500 spots on each gel, however only 309, 375 and 341 spots from each group at consecutive sampling points were considered for statistical comparison. because, according to our selection criteria, only those spots which were present in at least 3 of 4 gels in both infected and control groups at a given sampling point were considered for quantitative comparisons. the highest number of qualitative differences in spots was detected at 7 dpi (n = 26). this number decreased in the subsequent sampling time point at 14 dpi (n = 19) and the lowest number was at 21 dpi (n = 7). although a similar pattern was seen in the total number of spot differences, the highest number of quantitatively significant differences (false discovery rate or fdr ≤ 0.05 and fold change ≥ 2) in spot expression was detected at 7 dpi (n = 33), which was followed by 21 dpi (n = 12) and 14 dpi (n = 2). among these spots, there were 16, 1 and 5 spots identified as significantly up-regulated at 7, 14 and 21 dpi, respectively. the rest were significantly down-regulated (fig. 3) . taken together, 61 protein spots were detected as either quantitatively or qualitatively differentially expressed at least once during different sampling points. in order to obtain the identities of the differentially expressed spots, the spots were manually excised from preparative gels prepared by loading 1 mg of total proteins and staining with coomassie blue. subsequently, trypsin-digested spots were identified by 1d lc esi ms/ ms. peptide identities with fdr b 0.01 were considered significant and, in total, the identity of proteins in 61 different spots representing 48 different proteins was determined (table 1) . moreover, several spots contained peptides generated from multiple proteins. while we have used the protein with the highest relative abundance under corresponding spot number throughout our discussion, the complete list of spots with multiple identities has been provided as supplementary table 1 . several proteins were differentially expressed at more than one time point, e.g. cathepsin d (cathd), natural killer cell enhancing factor isoform 4 (nkef), eukaryotic elongation factor 2 (eef2), aldolase b (aldob), membrane associated guanylate kinase (magi3), a predicted hypothetical protein (hp5) and beta actin (actb) at all three times. in total, 30 proteins were differentially expressed exclusively at 7 dpi. notably, several proteins involved in antigen presentation pathways, the ubiquitin-proteasome protein degradation system and a number of spots representing several cellular structural proteins were among those that were differentially fig. 4 . venn diagram summarizing the spots that were significantly differentially expressed in the spleen tissues of mdv-infected chickens according to their corresponding time of sampling. these identities include both quantitatively and qualitatively differentially expressed spots. the identities of spots which were commonly expressed were placed in overlapping areas accordingly. corresponding spot numbers are in parentheses. refer to table 1 for the respective protein names. expressed exclusively at 7 dpi. there was only one spot specific to each of 14 and 21 dpi. these spots were identified as glutathione-stransferase theta 1 (14 dpi) and proliferating cell nuclear antigen (21 dpi). the significant changes detected with the rest of the spots overlapped between each time point in varying numbers. interestingly, some of the spots representing a particular protein showed opposite directions of regulation even within the same group. for example, three spots with different molecular weights and pi were identified as cathd (table 1) . of these three spots, two spots with molecular weights of 13.3 and 28.4 kda were found to be up-regulated in infected birds at more than one time point. the summarized distribution of the identities of spots identified at each time point is presented in fig. 4 . forty-percent of the identified proteins were associated with the cytoplasm and the plasma membrane (terms go:0005737 and go:0005886 respectively). furthermore, there were 24% nuclear and nuclear envelope proteins (go:0005634 and go:0005635), 22% cytoskeleton-associated proteins (go:0005856) and 5% extracellular proteins (go:0005576). nine-percent of proteins did not have any go annotation with respect to their cellular compartment, hence, were categorized very broadly as being associated as a "cellular component" (go:0005575). for biological processes, the highest associations (18%) were with metabolic processes (go:0008152). another 16% associations were with nucleic acid metabolism (go:0006139), while 14% and 13% were associations with transport (go:0006810) and cell communication (go:0007154), respectively. among the remaining associations, 4% were associated with cell death (go:0008219) (fig. 5) . virus genome copy numbers in infected spleens were determined using quantitative real-time pcr (qpcr). using conventional pcr screening, mdv-meq gene could be amplified from dna of all infected spleens but not from any of the uninfected controls (data not shown). results of the subsequent qpcr analysis of infected samples are presented in fig. 6 . the average mdv-meq copy numbers were 3.82 × 10 6 ± 1.48 × 10 6 (n = 3), 2.66 × 10 7 ± 7.78 × 10 6 (n = 3) and 2.98 × 10 7 ± 1.22 × 10 7 (n = 4) per 100 ng of spleen dna at 7, 14 and 21 dpi, respectively (fig. 6 ). there was a statistically significant difference between genome copy numbers at 7 dpi compared to other time points. in the present study, we have profiled the global protein expression changes in the chicken spleen in response to mdv infection at various time points representing the different phases of mdv life cycle. furthermore, we have determined the identity of the spots that were differentially expressed between infected and uninfected birds using mass spectrometry. among the 48 proteins that were differentially expressed, there was considerable number of proteins that had multiple spots in 2d gels. in addition, there was some degree of disagreement between the expected and experimental molecular weights in case of some of the proteins. this could be due to several reasons. first, some proteins exist as different isoforms (therefore different pi) as well as different intermediate stages between translation and functionally mature form (i.e. pre-pro-and pro-forms of protein). this could change the molecular weight and/or the isoelectric point (for example, please see the discussion regarding different spots of cathd below). another reason is possible protein degradation between sample collection and processing. interestingly, no protein with multiple matches showed any specific migration pattern in our 2d gels (such as trains of spots). while current data are not enough to determine the exact reason for such discrepancies, it should be noted that several previous studies using 2d gels have also reported the presence of similar patterns of proteins with multiple spots (dupont et al., 2008; liu et al., 2008; saldanha et al., 2008; zheng et al., 2008) . analysis of viral load in spleen tissue revealed a significant increase over time. although viral genome load may not be a direct indicator for the degree of infection, there is a correlation between mdv genome load and the number of infected cells (bumstead et al., 1997) as well as with subsequent md incidence (islam et al., 2006) or protection conferred by vaccines against md (abdul-careem et al., 2007) . based on our previous observations, virus genome load of around 1 × 10 5 copies/100 ng of spleen dna is correlated with development of tumors in infected birds (abdul-careem et al., 2007) . therefore, it is conceivable that the virus copy numbers observed in the present study would be an indicative of a high level of infection. the proteins identified in the present study are involved in a variety of cellular processes, notably the antigen processing and presentation, ubiquitin-proteasome protein degradation, formation of the cytoskeleton, cellular metabolism, signal transduction and regulation of translation (table 1 ). the significance of these processes in chicken-mdv interaction is discussed below. although this was not determined in the present study, temporal changes in the cellular composition of the spleen may have, at least in part, influenced the whole spleen organ proteome. however, the advantage of our method is that the proteins that are differentially expressed could be used to point to cell subsets and/or mechanisms that have a potential involvement in mdv-host interactions and could be targeted for future studies. among the differentially expressed spots, several proteins were identified that are either directly or indirectly involved in the ubiquitin-proteasome (upp) and antigen presentation pathways (table 1 ). in our study, we identified three differentially regulated proteins, namely ubiquitin c-terminal hydrolase l3 (uchl3), proteasome (prosome, macropain) subunit beta type 7 (psmb7) and predicted protein similar to mouse proteasome 26s subunit atpase 5 (psmc5) (also known as msug1), which are involved in upp (table 1) . among them, chicken uchl3 (alias uch-6) has 86% amino acid similarity with its human counterpart (baek et al., 1999) , which is a deubiqutinating enzyme (dub) that functions as a negative regulator of ubiquitination of proteins as well as facilitates recycling of ubiquitin. increasing evidence suggests that dubs are important regulators of many cellular processes such as endocytosis, apoptosis and various signalling pathways (wing, 2003) . several virus-encoded proteins such as epstein-barr virus (ebv)-encoded epstein-barr nuclear antigen 1 and herpes simplex virus (hsv-1) regulatory protein icp0 have been shown to interact with dubs for viral survival in infected cells (reviewed in lindner, 2007) . although there is a high amino acid identity between human uch-l3 and chicken uch-6, they differ with respect to their substrate specificity and tissue distribution (baek et al., 1999) . therefore, the chicken ortholog of uch-l3 may not have a similar role in viral infections. more functional studies are needed to understand the significance of the down-regulation of uchl3 in mdv infection. down-regulation of psmb7 at 7 dpi probably indicates the modification of proteasome into immunoproteasome under the influence of interferon (ifn)-γ to enhance the generation of peptides for binding to major histocompatibility complex (mhc) class i molecules. in this process, three constitutive beta subunits of the 20s proteasome, namely delta, x and z (psmb7), are replaced by ifn-γ inducible low molecular mass peptide (lmp)-7, lmp-2 and multicatalytic endopeptidase complex-like (mecl) catalytic subunits, respectively (griffin et al., 1998) . although we could not detect upregulation of ifn-γ inducible subunits, given the reciprocal regulation between constitutive and ifn-γ inducible subunits (hisamatsu et al., 1996) , down-regulation of psmb7 may indicate the effect of induced ifn-γ. in line with that, mdv is known to induce ifn-γ in chickens as early as 3 dpi and remains up-regulated until at least 15 dpi (xing and schat, 2000) . moreover, although mdv is capable of down-regulating mhc-i expression in vitro, elevated ifn-γ has been shown to reverse the effect of mdv on mhc-i (levy et al., 2003) . our present observations do not provide evidence for differential regulation of mhc-i expression. however, given the possible enhancement of immunoproteasome activity, it is conceivable that there is an enhanced mhc-i-mediated antigen presentation. the 19s proteasome subunit sug1 up-regulates mhc-ii expression by interaction with class ii transactivator (ciita) gene and mhc-ii proximal promoter in human. mhc-ii expression is reduced in the absence of the expression of sug1 (bhat et al., 2008) . in agreement with this, the observed absence of predicted protein, msug1 in infected birds is associated with a significant decrease in the expression of the mhc class ii alpha chain (b-la) in infected spleens at 7 dpi. reduction of the mhc-ii expression is known to be a predominant way of evading the host immune response by a number of viruses including herpesviruses (hegde et al., 2003) . in agreement with our present observation of down-regulation of mhc-ii expression, previous work from our laboratory showed that mdv infection causes significant downregulation of invariant (ii) chain gene expression in the chicken spleen tissue (sarson et al., 2006) . in contrast to this observation, niikura et al. (2007) have shown an up-regulation of mhc-ii in bursa cells of chickens infected with md11, another very virulent strain of mdv. however, in contrast to our present infection model which used outbred spf chickens, they have used chickens from a cross between two inbred lines, 15i 5 and 7 1 , both of which are susceptible to md (bacon et al., 2000) . therefore, it is possible that the difference in genetic background of infected birds may have, at least in part, contributed to these contradicting observations. taken together, our observations suggest that there is enhanced antigen processing, presumably mediated by elevated ifn-γ for mhc class i pathway, while there is a down-regulation of mhc class iimediated antigen presentation at least in our experimental model at early stages of the disease. in the current study, altered spot profiles were observed for a number of cytoskeleton-associated proteins representing all three main categories of cytoskeleton proteins, namely microfilaments, intermediate filaments and microtubules. among the microfilament proteins, all detected actin spots, except for alpha-2 actin (acta2) and a predicted protein similar to coactosin-like 1 (cotl1) at 21 dpi, were either newly induced or significantly up-regulated across the experimental period. intermediate filament, lamin b2 (lmnb2) spots also were either up-regulated or newly induced in infected birds at all times. however, microtubule protein, tubulin β 2b (tubb2b) was significantly down-regulated in infected birds at 7 dpi (table 1) . changes in cytoskeleton proteins have been previously described in several other viral infections, such as ibdv (zheng et al., 2008) , severe acute respiratory syndrome (sars)-associated coronavirus (jiang et al., 2005) and human papillomavirus type 8 (akgül et al., 2009) . herpesviruses are known to interact with the actin filament system and its regulatory protein, rho gtpase, at various stages of infection (favoreel et al., 2007) . in addition to actins, here we have identified a rho gtpase regulatory protein, d4-gdi, as a protein that was differentially expressed in spleen of infected chickens (discussed in detail elsewhere in the discussion). this may indicate a putative role of the same system in the context of mdv infection. schumacher et al. (2005) showed that mdv-encoded us3 ortholog protein causes depolymerization of the actin stress fibers. however, its role in mdv replication and/or spreading is still unclear. stathmin 1 (stmn1), which also plays a role in the regulation of the microtubule system, has been shown to be up-regulated in ebv-infected b lymphocytes in human as early as 7 dpi (baik et al., 2007) . in the present study, we could not detect changes in stmn1 in spleen at 7 dpi. however, the expression of this protein was induced by 21 dpi. as a possible result of regulation by stmn1, tubb2b, which is a component of the microtubule filament system, also showed a similar pattern of expression in mdv-infected spleen tissues. however, if that change has any direct relationship with the stmn1 is not known. apart from its interaction with the microtubule system, stmn1 has been shown to interact with heat shock protein 70 (hsp70) proteins, particularly with heat shock cognate 70 (hsc70) (manceau et al., 1999) . in line with that, we have also identified significant changes in the expression of hsp70 proteins, including hsc70. another cellular structural protein, lmnb2, which is associated with the nuclear membrane, was up-regulated in infected birds throughout the experimental period as a probable result of the egress process of virus nucleocapsids from the infected nuclei. the nucleocapsids of herpesviruses acquire a temporary envelope from the inner nuclear membrane before egress by budding off from the infected cell nuclei (granzow et al., 2001) . in line with that, camozzi et al. (2008) have reported various biochemical and structural modulations, including mislocalization of lamin proteins in the nuclear envelope of cells infected with human cytomegalovirus. although camozzi et al. (2008) did not observe any quantitative changes in the expression of lamin proteins, qualitative changes in the expression of the same protein have been described in infections with several other viruses such as ibdv (zheng et al., 2008) , enterovirus 71 (leong and chow, 2006) and sars-associated coronavirus (jiang et al., 2005) . similar to the other members of the family, mdv particles egress from the nucleus of infected cells through budding (baigent and davison, 2004) . therefore, it is conceivable that the up-regulation of lmnb2 protein observed in the present experiment is, at least partly, the result of such interaction. proliferating cell nuclear antigen (pcna), which is one of the critical proteins in cell survival, was significantly up-regulated in infected spleens at 21 dpi. pcna is an essential component in dna synthesis process in the cell. therefore, it is likely that induction of pcna represents highly replicating cell populations in the spleen at 21 dpi. while it is possible that elevated pcna expression represents a transformed cell population, possible interactions between pcna and viral proteins might be also occurring. for example, hsv-1 encoded protein, icp34.5, interacts with pcna of infected cells, presumably preventing virus-induced translational arrest (brown et al., 1997; harland et al., 2003) . the possibility of a yet unidentified mdv protein interacting with pcna remains to be investigated. icp34.5 has also been shown to interact with phosphatase 1, preventing the inactivation of eukaryotic elongation factors (eef), a group of proteins which play an important role in protein biosyntheses, hence being important in various cell processes including proliferation (thompson and sarnow, 2000) . eef2 was among the proteins that were up-regulated at both 7 and 14 dpi. increased levels of eef2 have been shown in response to human immunodeficiency virus (hiv) protein, vpr, and have been shown to have the potential of preventing vpr-mediated apoptosis in cd4+ t cells (zelivianski et al., 2006) . d4-gdp-dissociation inhibitor (d4-gdi) (also known as ly-gdi) was differentially regulated in infected birds compared to uninfected controls. as mentioned above, ly-gdi represents a group of proteins i.e. gdi, which are involved in the regulation of another group of proteins, rho family gtpases. apart from its involvement in the organization of the cytoskeleton, rho-gtpases are also involved in cell signalling and proliferation. gdis are involved in the regulation of shifting of rho gtpase between the active gtp-bound form and the inactive gdp-bound form. our present observations showed a more than 5-fold increase of an 18.4 kda d4-gdi spot while there was a significant decrease of the presumably intact protein of 27 kda. while activation as well as apoptosis of different types of cells in response to viral infections has been well documented, our present observations may highlight some of the molecules involved in these processes in the context of mdv infection. cathepsin d (cathd), a lysosomal aspartic proteinase, was among the proteins that were differentially expressed in infected spleens at all three time points. there were two spots with molecular weight of approximately 13 and 28 kda which were up-regulated, while another one (39.2 kda) was down-regulated. cathd is synthesized as a single chain pre-pro-enzyme and after being cleaved into several successive intermediates, it forms the mature 34 + 14 kda form consisting of a heavy and a light chain in the lysosome (laurent-matha et al., 2006) . therefore, processing of the 39.2 kda intermediate into the mature enzyme appears to be induced by mdv infection. cathd has been shown to be an important mediator of apoptosis induced through the lysosomal pathway (guicciardi et al., 2004) . while cathd appears to be involved in the intrinsic pathway of the induction of apoptosis in activated t lymphocytes in human (bidere et al., 2003) , it may also play a significant role in the resolution of inflammation by inducing apoptosis in neutrophils (conus et al., 2008) . apart from its role in the induction of apoptosis, increasing evidence suggests that cathd plays a significant role in cancer progression and metastasis. in the context of md, several studies have shown the occurrence of cell death in mdvinfected organs such as the bursa of fabricius (st hill and sharma, 1999) , thymus (morimura et al., 1996) and in peripheral blood mononuclear cells (morimura et al., 1995) . while it is conceivable that up-regulated cathd may play a role in apoptosis during the early stages of md, it may also have a role in t cell transformation in the later stages of the disease; however, this needs to be further studied. transglutaminase 4 (tgm4) is among the proteins with the highest fold increase in spleens of infected chickens. generally, transglutaminases are involved in post-translational modification of proteins (beninati and piacentini, 2004) . tgm4 is highly expressed in the prostate gland of humans but little information is available about the function of this protein in chickens. tgm2, which is a ubiquitously expressed protein in human (also known as tissue tgm, ttgm), has been shown to play a significant role in stress response (ientile et al., 2007) notably in apoptotic cell death. while tgm2 is up-regulated in apoptotic cells, it acts as a molecular glue and appears to stabilize the dying cells to prevent release of intracellular molecules prior to clearance by phagocytosis (fesus and szondy, 2005) thereby preventing adverse effects, such as excessive inflammatory reactions. assuming chicken tgm4 has a similar role as that of human tgm2, above observations may explain the putative role of the former in the context of host-mdv interactions in the spleen to prevent collateral damage to the neighbouring cells. according to go classification based on the biological process, the highest association of identified proteins in the current study was with metabolic processes (19%) (fig. 5) . notably, several metabolic enzymes associated with glycolysis have been found differentially regulated. among them, a 38.76 kda spot representing aldolase b fructose-bisphosphate (aldob) was constantly up-regulated in infected spleens at all time points. interestingly, another spot with similar identity and molecular weight but slightly different pi was significantly down-regulated only at 7 dpi. while different pis for the same protein is possible with structural changes such as phosphorylation, functional relevance in this context needs to be elucidated. another two newly induced glycolytic enzymes, each at 7 and 21 dpi, were identified as triosephosphate isomerase 1 (tpi1) and phosphoglycerate mutase 1 (pgam1), respectively. viruses utilize host cell metabolic process for their replication process. in agreement with the present observations, significantly elevated levels of several serum enzymes, including aldolase b, in response to mdv infection in vivo has been previously described (ivanov et al., 1974) . further, mdvencoded protein pp38 has been shown to up-regulate cellular metabolic activities in vitro as determined by the enhanced activity of mitochondrial dehydrogenases (li et al., 2006) . similarly, human cytomegalovirus infection in fibroblasts has also shown overall upregulation of a number of glycolytic enzymes (munger et al., 2006) . in conclusion, findings of the present study highlight some of the mechanisms involved in the host response in the spleen to mdv infection during various time points representing different stages of mdv pathogenesis. although the functions of the proteins, which were identified here, were not studied, it is likely that all or some of them are involved in host-virus interactions. one of the limitations of the tools used in this study is the inefficiency of detecting low abundance proteins or those with low molecular weights, such as cytokines and chemokines. therefore, a more comprehensive study is needed to elaborate on our present observations and to further explore other proteins that may play a role in pathogenesis of the virus as well as host responses to this virus. all the chickens used in this experiment were one-day old specific pathogen free (spf) chickens obtained from the animal disease research institute, canadian food inspection agency (ottawa, ontario, canada). birds were kept in an isolation facility at the ontario veterinary college throughout this experiment. chickens were infected with the rb1b strain of very virulent marek's disease virus (passage 9) (schat et al., 1982) which was obtained from dr. k.a. schat (cornell university, ny, usa) . twenty-four, one-day old chicks were randomly divided into two groups and were housed in the isolation facility. one group of birds (n = 12) was given 750 plaque-forming units (pfu) of the rb1b strain of very virulent mdv intraperitoneally on day 5 of age. the rest (n = 12) were kept as uninfected controls. infected and uninfected control birds were kept in separate units with similar environmental conditions. on 7, 14 and 21 dpi, representing different stages of mdv pathogenesis, four chickens that were randomly selected from each group were euthanized using co 2 inhalation. at necropsy, a portion of spleen was collected from each bird and was snap-frozen in liquid nitrogen. subsequently, frozen tissues were kept at −80°c until further processing. another portion was preserved in rnalater (qiagen inc., missisauga, on, canada) . animal experiments were conducted in accordance with the guidelines provided by the canadian council on animal care. all experiments complied with institutional animal care guidelines and were approved by university of guelph animal care committee (protocol number 06r015). each frozen spleen tissue was briefly homogenized in a lysis buffer (ph 8.5) containing 30 mm tris-cl, 2 m thiourea, 7 m urea and 4% (w/v) chaps. the volume of lysis buffer used for each tissue sample was equal to 15 times of tissue mass. samples were further solubilized by sonication for 5 min on ice and insoluble tissue debris was removed by centrifugation under 18,000 ×g at 4°c. subsequently, supernatant was collected separately from each sample and protein concentrations were determined using the bio-rad protein assay as prescribed by the manufacturer. the spleen protein sample from each chicken was analyzed separately. there were a total of 24 chickens in two groups (infected and uninfected) i.e. 24 protein samples for analysis by 2d-page. each analytical 2d-page gel was prepared with 200 μg of proteins mixed with rehydration buffer (8 m urea, 2% chaps, 90 mm dtt, 5 μl/ml appropriate ipg buffer, 12 μl/ml destreak reagent (ge healthcare) and 0.005% bromophenol blue) to a total volume of 250 μl. the first dimension separation was performed in 13 cm, ph 3-10 non-linear immobiline drystrips (ge healthcare) using ettan ipgphor isoelectric focusing unit (ge healthcare). after rehydration at 30 v for 12 h, isoelectric focusing was performed at 500 v for 1 h, 1000 v for 1 h and 8000 v until a total of 57,000 volt hours was reached. each focused strip was incubated at room temperature, initially in 10 ml of equilibration buffer (50 mm tris-cl (ph8.8), 6 m urea, 30% (v/v) glycerol, 2% (w/v) sds and 0.005% bromophenol blue) containing 1% (w/v) dtt for 15 min and subsequently in a similar volume of equilibration buffer containing 2.5% (w/v) iodoacetamide for a similar time. for the second dimension separation, each ipg strip was placed on a 12.5% sds-polyacrylamide gel and four such gels were simultaneously run each time subjecting them to 25 ma/gel of current at 25°c in a ruby apparatus until the bromophenol blue dye front reach the opposite edge of the gel. each gel was subsequently fixed for 1 h in a solution containing 10% (v/v) methanol and 7% (v/v) acetic acid, stained with sypro ruby stain (bio-rad) overnight and destained in the fixing solution for 2 h. gel images were digitized using typhoon 9400 variable mode imager (ge healthcare) at 532 nm using a 610 nm filter. preparative gels were prepared in a similar manner, using 1 mg of protein from each sample and stained them with coomassie brilliant blue instead of sypro ruby. digitized gel images were used to estimate the expression of different proteins in each analytical gel using 2004 version of phoretix 2d software (nonlinear dynamics). the pixel volume of each detected spot was referred to as the spot volume and was used in the subsequent comparisons. background correction for pixel volumes of each spot was done using the mode of non-spot and normalized spot volumes were calculated as a fraction against the total volume of spots in each gel. the spots which were not present in the expected position or showed decreased intensity were considered "down-regulated", while the spots that appeared in only one group or showed enhanced intensity were considered "up-regulated". although this is an indicator of protein abundance, the possibility of post-translation modifications, hence changes in location of spots on the 2d-page gels, could not be ruled out in our study. there were eight 2d-page gels per time point: 4 derived from infected spleens and 4 from those of uninfected birds. only the spots that were present in all gels and those that were absent from a maximum of one analytical gel per group at a given time point were considered for the statistical comparison. statistical analysis was done using sas (version 9.1). normalized volumes of each corresponding spot from each group at similar time point were compared with student t-test. the resulting p values were used to calculate the fdr. spots that were having both p ≤ 0.01 and fold difference n2 in mean normalized volumes were considered as significantly differentially expressed. fdr for any selected spot was less than 5%. the spots that showed a significant difference, and those expressed only in a particular group at a given sampling point, were selected for identification by 1d lc esi ms/ms using a lcq deca xp plus mass spectrometer coupled with two thermo surveyor ms pump quaternary gradient pumps at the life science and biotechnology institute, mississippi state university as described below. each selected spot was excised manually from coomassie brilliant blue stained preparative gels and "in-gel" digested exactly as previously described (shevchenko et al., 1996) . proteins were reduced with 5 mm dtt at 65°c for 5 min and alkylated with 10 mm iodoacetamide at 30°c for 30 min. trypsin digestion was done using molecular biology grade porcine trypsin (2 μg; 37°c; 16 h; 50:1 ratio of protein:trypsin; promega corporation, madison, wi). liquid chromatography was done with a reverse phase (c18) lc column coupled directly in line with the mass spectrometer. peptides were loaded into a liquid chromatography gradient ion exchange system containing a thermo separations p4000 quaternary gradient pump (thermoelectron corporation; san jose, ca) coupled with a 0.18 × 100 mm biobasic c18 reverse phase liquid chromatography column of a proteome x workstation (thermoelectron). the reverse phase gradient used 0.1% formic acid in acetonitrile and increased the acetonitrile concentration in a linear gradient from 5% to 30% in 15 min and then 30% to 65% in 5 min followed by 95% for 5 min and 5% for 10 min. the mass spectrometer was configured to optimize the duty cycle length with the quality of data acquired by alternating between a single full ms scan followed by three tandem ms scans on the three most intense precursor masses (as determined by xcalibur software in real time) from the full scan. the collision energy was normalized to 35%. dynamic mass exclusion windows were set at 2 min and all of the spectra were measured with an overall mass/ charge (m/z) ratio range of 300-1700. identification of all spots was done as a single run in a randomized order. to prevent "carry-over" after the peptides generated from each spot were analyzed, and before those from the next spot were analyzed, the lc column was washed with 95% acn, a negative control sample was run to confirm no carryover and the lc column was washed again. resulting mass spectra were analyzed using bioworks 3.2 (thermoelectron) using a non-redundant proteome database containing protein from both chicken and mdv-rb1b (build 3.2) downloaded from the ncbi. we used the bioworks reverse database function to create the decoy database from this proteome database. the probability of the tandem mass spectrometry match occurred by chance was calculated using; (a) the decoy database searching exactly as described by elias and gygi (2007) and (b) the orthogonal p(pep) function in bioworks 3.2 (which calculates probability based on a theoretical y and b ion spectrum calculated based on theoretical amino acid dissociation). these two probabilities were the used to calculate the fdr as described by benjamini and hochberg (1995) and only peptides with fdr b 0.01 were considered as a significant and retained for protein identification. the probability of protein identity was then calculated from the peptide probabilities exactly as described (maccoss et al., 2002; nesvizhskii et al., 2003) . to calculate the percent protein coverage by identified peptides, the protein sequence was digested in silico using "peptidecutter" (http://us. expasy.org/tools/peptidecutter/ (gasteiger et al., 2005) ) and the total number of amino acids in peptides between 6 and 30 amino acids (the size range of 99% of the peptides detected by the mass spectrometer) was used as the denominator; the peptides identified were used as the nominator. for spots with multiple protein identities, the protein with the highest peptide coverage and highest protein score (i.e. σxcorr; a surrogate for the amount of precursor ion) (nanduri et al., 2005) was considered as the dominant protein and the most likely protein to contribute to the differential expression in the gel. subsequently, their biological relevance was discussed. spot identities were submitted to goretriever (http://www. agbase.msstate.edu/) to obtain the go annotations. if no annotation was returned, goanna was used to retrieve go annotations assigned depending on the sequence similarities. the resulting annotations were summarized based on the goa and whole proteome goslim set using goslimviewer (mccarthy et al., 2006) . dna extraction, conventional pcr confirmation of the presence of mdv-meq gene and subsequent determination of the absolute mdv genome copy number from infected spleen samples were essentially performed as previously described . quantitative real-time pcr reactions to determine viral genome load in samples were performed in duplicate. development of a real-time pcr assay using sybr green chemistry for monitoring marek's disease virus genome load in feather tips cytokine gene expression patterns associated with immunization against marek's disease in chickens proteomic analysis reveals the actin cytoskeleton as cellular target for the human papillomavirus type 8 study of host-pathogen interactions to identify sustainable vaccine strategies to marek's disease a review of the development of chicken lines to resolve genes determining resistance to diseases molecular cloning of chick uch-6 which shares high similarity with human uch-l3: its unusual substrate specificity and tissue distribution marek's disease virus. inbiology and life cycle identification of stathmin 1 expression induced by epstein-barr virus in human b lymphocytes the transglutaminase family: an overview: minireview article controlling the false discovery rate -a practical and powerful approach to multiple testing the 19s proteasorne atpase sug1 plays a critical role in regulating mhc class ii transcription cathepsin d triggers bax activation, resulting in selective apoptosis-inducing factor (aif) relocation in t lymphocytes entering the early commitment phase to apoptosis the herpes simplex virus virulence factor icp34.5 and the cellular protein myd116 complex with proliferating cell nuclear antigen through the 63-amino-acid domain conserved in icp34.5, myd116, and gadd34 quantification of marek's disease virus in chicken lymphocytes using the polymerase chain reaction with fluorescence detection proteomics in the chicken: tools for understanding immune responses to avian diseases modeling the proteome of a marek's disease transformed cell line: a natural animal model for cd30 overexpressing lymphomas different signaling pathways expressed by chicken naive cd4 + t cells, cd4 + lymphocytes activated with staphylococcal enterotoxin b, and those malignantly transformed by marek's disease virus remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pul50 and pul53 caspase-8 is activated by cathepsin d initiating neutrophil apoptosis during the resolution of inflammation application of saturation dye 2d-dige proteomics to characterize proteins modulated by oxidized low density lipoprotein treatment of human macrophages target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry actin and rho gtpases in herpesvirus biology transglutaminase 2 in the balance of cell death and survival the proteomics protocols handbook egress of alphaherpesviruses: comparative ultrastructural study immunoproteasome assembly: cooperative incorporation of interferon gamma (ifn-gamma)-inducible subunits lysosomes in cell death correlation between protein and mrna abundance in yeast the herpes simplex virus (hsv) protein icp34.5 is a virion component that forms a dna-binding complex with proliferating cell nuclear antigen and hsv replication proteins viral inhibition of mhc class ii antigen presentation newly identified pair of proteasomal subunits regulated reciprocally by interferon gamma tissue transglutaminase and the stress response relationship between marek's disease virus load in peripheral blood lymphocytes at various stages of infection and clinical marek's disease in broiler chickens changes in the activity of several serum enzymes in chicks with marek's disease quantitative analysis of severe acute respiratory syndrome (sars)-associated coronavirus-infected cells using proteomic approaches -implications for cellular responses to virus infection processing of human cathepsin d is independent of its catalytic function and autoactivation: involvement of cathepsins l and b transcriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection major histocompatibility complex class i is downregulated in marek's disease virus infected chicken embryo fibroblasts and corrected by chicken interferon expression of marek's disease virus phosphorylated polypeptide pp38 produces splice variants and enhances metabolic activity deubiquitination in virus infection a strategy to identify positional candidate genes conferring marek's disease resistance by integrating dna microarrays and genetic mapping a mass spectrometry-based proteomic approach to study marek's disease virus gene expression proteomics analysis of differential expression of cellular proteins in response to avian h9n2 virus infection in human cells probability based validation of protein identifications using a modified sequest algorithm stathmin interaction with hsc70 family proteins agbase: a unified resource for functional genomics analysis in agriculture induction of host gene expression following infection of chicken embryo fibroblasts with oncogenic marek's disease virus immunomodulation of peripheral t-cells in chickens infected with marek's disease virusinvolvement in immunosuppression apoptosis and cd8-down-regulation in the thymus of chickens infected with marek's disease virus -brief report dynamics of the cellular metabolome during human cytomegalovirus infection proteomic analysis using an unfinished bacterial genome: the effects of subminimum inhibitory concentrations of antibodies on mannheimia haemolytica virulence factor expression a statistical model for identifying proteins by tandem mass spectrometry marek's disease virus up-regulates major histocompatibility complex class ii cell surface expression in infected cells use of dna microarrays to monitor host response to virus and virus-derived gene therapy vectors changes in the gallus gallus proteome induced by marek's disease virus differential proteome expression associated with urokinase plasminogen activator receptor (upar) suppression in malignant epithelial cancer transcriptional analysis of host responses to marek's disease viral infection characterization of 2 highly oncogenic strains of marek's disease virus the protein encoded by the u s 3 orthologue of marek's disease virus is required for efficient de-envelopment of perinuclear virions and involved in actin stress fiber breakdown the neoplastically transformed (cd30 hi ) marek's disease lymphoma cell phenotype most closely resembles t-regulatory cells mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels response of embryonic chicken lymphocytes to in ovo exposure to lymphotropic viruses regulation of host cell translation by viruses and effects on cell function deubiquitinating enzymes-the importance of driving in reverse along the ubiquitin-proteasome pathway increased virulence of marek's disease virus field isolates expression of cytokine genes in marek's disease virusinfected chickens and chicken embryo fibroblast cultures suppressive effect of elongation factor 2 on apoptosis induced by hiv-1 viral protein r host-pathogen interactions: a proteomic view proteomics analysis of host cells infected with infectious bursal disease virus key: cord-261375-6fu3dzi9 authors: hoppe, sebastian; bier, frank f; von nickisch-rosenegk, markus title: microarray-based method for screening of immunogenic proteins from bacteria date: 2012-03-21 journal: j nanobiotechnology doi: 10.1186/1477-3155-10-12 sha: doc_id: 261375 cord_uid: 6fu3dzi9 background: detection of immunogenic proteins remains an important task for life sciences as it nourishes the understanding of pathogenicity, illuminates new potential vaccine candidates and broadens the spectrum of biomarkers applicable in diagnostic tools. traditionally, immunoscreenings of expression libraries via polyclonal sera on nitrocellulose membranes or screenings of whole proteome lysates in 2-d gel electrophoresis are performed. however, these methods feature some rather inconvenient disadvantages. screening of expression libraries to expose novel antigens from bacteria often lead to an abundance of false positive signals owing to the high cross reactivity of polyclonal antibodies towards the proteins of the expression host. a method is presented that overcomes many disadvantages of the old procedures. results: four proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. one protein with no known immunogenic behaviour before suggested potential immunogenicity. we incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. this enhances the specific binding of the proteins compared to nitrocellulose. thus, it helps to reduce the number of false positives significantly. it enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. we validated our method by employing several known genes from campylobacter jejuni nctc 11168. conclusions: the method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. it could provide a powerful and attractive alternative to existing methods and help to detect and identify vaccine candidates, biomarkers and potential virulence-associated factors with immunogenic behaviour furthering the knowledge of virulence and pathogenicity of studied bacteria. results: four proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. one protein with no known immunogenic behaviour before suggested potential immunogenicity. we incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. this enhances the specific binding of the proteins compared to nitrocellulose. thus, it helps to reduce the number of false positives significantly. it enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. we validated our method by employing several known genes from campylobacter jejuni nctc 11168. the method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. it could provide a powerful and attractive alternative to existing methods and help to detect and identify vaccine candidates, biomarkers and potential virulence-associated factors with immunogenic behaviour furthering the knowledge of virulence and pathogenicity of studied bacteria. campylobacter jejuni is one of the principal causing agents of bacterial gastroenteritis in industrialized countries [1] . from january to mid september 2011 52.940 campylobacter infections have occurred in germany alone [2] . although infections predominantly result in mild self-limiting gastroenteritis, in some cases severe post-infection ramifications have been reported with the guillain-barré syndrome the major contributor [3] . the main route of infection and transmission is believed to be the incorrect handling and incomplete cooking of poultry. in fact, studies mention 62% of poultry carcasses to be contaminated with campylobacter after slaughter [4] . on account of its widespread occurrence and clinical relevance testing for contamination of meat or the presence of campylobacter infections in patients is necessary. although several genomic typing methods exist [5, 6] these are often time-consuming and laborious. in comparison, a rapid point-of-care device would need a more direct approach like the presence of antigens which react swiftly with specific antibodies. for many pathogens several different methods based on the antigen-antibody-reaction are already commercially available, e.g. latex-agglutination-tests [7] . however, the knowledge of antigens for pathogens is often limited. traditionally, testing for immunogenic proteins has been carried out by screening of expression libraries using nitrocellulose membranes [8] . despite its simplicity, this method has a key shortcoming regarding bacterial proteins. the elucidation of novel antigens is extremely difficult as a result of the high cross-reactivity of polyclonal antibodies against the host bacterium of the library [9] . therefore, a high number of false positives occur, which hinder a fast and successful search for new antigens. with the emergence of fusion tags, protein purification has become more convenient. several tags have been established and frequently used in all kinds of applications. apart from other commonly used tags such as gst [10, 11] , mbp [12, 13] or 6xhis [14, 15] the halotag ® (promega) features several unique characteristics. the halo-tag ® and its ligand bind covalently, leading to a strong, irreversible bond [16] . further, it increases the amount of soluble protein expressed in contrast to other tags [17] , as it reduces the formation of inclusion bodies during recombinant protein expression. in this paper, we describe a method to covalently attach different halotag ® fusion proteins on halolink™ slides (see figure 1 ) and consequently perform an immunoscreening using polyclonal antibodies in a microarray format, which is a suitable method for high-throughput applications such as screening of entire expression libraries (see figure 2 ). this method reduces the pitfall of crossreactive signals normally encountered with screenings and leads to a more rapid detection of immunogenic proteins compared to conventional methods. furthermore we show the outstanding performance of this method by expressing and detecting several proteins from campylobacter jejuni previously described as immunogenic. as controls of the screening process, we included proteins from campylobacter which have not been described as immunogenic before. we successfully amplified all but one gene (cfra) from the genomic dna of camyplobacter jejuni. all eleven genes that were successfully amplified showed the correct length (see figure 3 ) and were subsequently cloned to krx single-step competent cells. the correct insert size was determined by colony pcr. plasmids from clones containing the correct-sized inserts were isolated and the mcs was sequenced using both a forward and a reverse primer (ht7 for and flexi r). during cloning an extra gtt, which encodes for a valine residue in the proteins' primary structures, was inserted immediately prior to each stop codon. as a universal stop codon taa was used, replacing other stop codons if present. this was mainly done to gain maximum flexibility during cloning as the flexi vector system enables the direct transfer to other vectors with different tags. a gtt is mandatory to later transfer the gene of interest to a vector encoding a c-terminal tag. for that reason, no 100% identity can be observed, when comparing the sequencing results with the original gene sequences from embl using a global alignment with free-end gaps. nine of the plasmids differed only in the additional gtt mentioned above. for pyrc the usual stop codon tga was replaced by taa causing minor discrepancy in the alignment. the longest gene under investigation, flaa demonstrated the least conformity to the respective sequence from embl. the length of each sequence reaction was approximately 1300 nucleotides. due to the numerous alterations in the sequence causing a shift in amino acid sequence, flaa was omitted from future investigation. the correct expression of the encoded fusion proteins was assessed by sds-page. analyzing the gel under fluorescent conditions reveals protein bands which have the halotag ® ligand attached. the pageruler plus prestained protein ladder possesses two fluorescent bands, at 25 and 70 kda respectively. halotag ® standard protein with a figure 1 detection of immunogenic proteins on microarrays. the cell lysates are spotted onto halolink™ slides (shaded area) with immobilized ligand (curved lines). the halotag ® (pink sector) is fused to the n-terminus of the protein of interest (purple sector) and attaches covalently to the ligand. thus, the protein of interest is covalently attached to the surface prior to screening, whereas other proteins from the cell lysate are unbound and washed away after the coupling reaction. subsequently, primary antibodies (blue) bind to the protein of interest. afterwards, secondary antibodies (black) conjugated with a fluorophore (yellow) bind to the respective primary antibody. the fluorescent dye is used for detection of positive spots identifying sites of potential immunogenic proteins in the process. size of 60 kda was also analyzed and helps as an additional size reference. the halotag ® features a size of 34 kda alone. for ease of use and brevity the expressed proteins are abbreviated and referred to as their corresponding gene names in plain text, e.g. pseb as the protein encoded by the gene pseb. full protein names for each gene as taken from the kegg database can be found in table 1 . most of the fusion proteins investigated fall into a range between 61 and 73 kda, namely halotag ® fused to argc (73 kda), pyrc (72 kda), pseb (71 kda), gapa (70 kda), cjaa (65 kda), peb1 (62 kda), hisj (62 kda) and flac (61 kda). outside of this size range, only halotag ® -flaa (93 kda) and the small halotag ® -pal (52 kda) are found. for each protein, bands with the correct size could be detected, see figure 4 . additionally, bands of smaller size are visible (34 kda) which might be due to untimely termination of translation, potentially comprising only the halotag ® , which features the corresponding size. the cell lysates containing the expressed fusion proteins were spotted to halolink™ slides (promega) by the qarray2 microarray spotter. for the spotting layout see figure 5 . the immunogenicity of the immobilized proteins was assessed using polyclonal antibodies raised against whole and partially lysed attenuated cells of campylobacter jejuni. secondary antibodies conjugated with a fluorophore were used to detect signals. for better comparability the raw data was processed and contrast values were calculated. figure 6 shows the resulting bar chart of the processed data of one slide. four proteins showed contrast values above the cutoff. the contrast values ± s.d. for each of these proteins were 0.63 ± 0.17 (cjaa), 0.47 ± 0.10 (hisj), 0.35 ± 0.05 (flac) and 0.68 ± 0.15 (peb1a) respectively. in comparison, the contrast values of the following proteins were significantly below the cutoff: 0.06 ± 0.20 (gapa), 0.08 ± 0.15 (pyrc) and 0.09 ± 0.04 (argc). the last two proteins -pal and pseb -led to contrast values of 0.17 ± 0.11 (pal) and 0.25 ± 0.05 (pseb), which albeit closer are still slightly below the cutoff of 0.25. subsequently clones are cultivated, protein expression is induced and finally cells are lysed. the whole lysates can directly be spotted to the halolink™ arrays rendering additional time-consuming and costly purification steps obsolete. as only fusion proteins will bind to the ligand, remaining proteins from the lysates are simply washed off after spotting minimizing background and crossreactivity during the subsequent screening steps. the microarray platform allows for several thousand spots per slide allowing for easy high-throughput screening applications. table 2 for expected gene lengths. as markers hyper ladder i (m) and ii (m2) were used both supplied by bioline. to validate the results from one slide technical replicates (n = 5) were analyzed. summing up the results of all slides, a box-whisker-plot was composed, see figure 7 . the replicates emphasized the results from the one slide above. cjaa, hisj, flac and peb1a were clearly above the respective cutoffs and led to positive signals in all the slides. on the contrary, gapa, argc and pyrc were significantly below the cutoff. the tendency of pal and pseb to fall within close proximity of the cutoff is observed throughout the replicates. further verification of the results was performed by using a standard western blot experiment to test for immunogenicity. figure 8 shows the results of the investigated proteins after purification with halolink™ magnetic beads was performed prior to sds-page and blotting. only two of the investigated immunogenic proteins (cjaa, and hisj) show strong visible bands in western blot matching the expected sizes. the three remaining immunogenic proteins, peb1a, flac and pal, the full names of each protein investigated are given according to the kegg database with their coding genes. the gene names were used as abbreviations for the proteins in this paper cannot be distinguished in western blot analysis as well as all the other proteins. additionally, a dotblot was performed ( figure 9 ) with the purified proteins, which showed clear positive signals for cjaa and hisj, a rather weak signal for peb1a and extremely low signals for flac and pal. in contrast, figure 10 shows a western blot performed directly with whole lysates after recombinant expression without further purification. at least four bands are visible in all the samples with the most prominent band at 70 kda. bands of lower intensity appear at approximately 55 kda, 28 kda and 18 kda. the first five lanes, corresponding to the known immunogenic proteins, cjaa, hisj, peb1a, flac and pal, show bands of higher intensity than the remaining five lanes. however, as the investigated fusion proteins fall either into the 70 kda or in case of ht-pal into the 55 kda range, a clear differentiation between positive bands and background caused by krx cross-reactive proteins is hardly possible. using our new method, we were able to express, immobilize and screen all of nine different proteins from campylobacter jejuni using halotag ® and krx cells. cjaa, hisj, flac, peb1a and pal act immunogenic as described elsewhere [18] [19] [20] [21] [22] [23] . gapa, pyrc, argc and pseb have not been reported as immunogenic before. immunoscreening on figure 6 contrast value for one microarray slide. the bars represent the median. the standard deviation (sd) is accounted for by the error bars. krx represents cell lysate without halotag ® protein and pbs was spotted as a buffer control. the cutoff is represented by a bold horizontal line. four proteins namely cjaa, hisj, flac and peb1a are above the cutoff. in comparison, gapa, pyrc and argc lie significantly below the cutoff value. two proteins -pal and pseb -are approximately at the cutoff. the contrasts for krx and pbs are at or below zero. microarrays showed that gapa, pyrc and argc were significantly below the cutoff and can be considered negative. further, different codon usage between these two organisms (campylobacter jejuni and escherichia coli) might be the cause for low expression (see additional file 1: codonusage 1). this could have been a problem in the expression of pal and possibly to an extent for flac explaining the low signals in microarray and more prominently in western blot analysis and dotblot. nevertheless, four proteins with known immunogenic ability have been successfully detected with strong signals clearly above the cutoff. so far, pseb has not been described as immunogenic, although it is involved in the glycosylation of the flagellar apparatus of campylobacter jejuni [24, 25] . although the signals are far weaker than for known antigens such as cjaa, hisj or peb1a, the results suggested immunogenic potential of pseb to be in the realm of possibility. the close proximity and involvement in the modification of flagellin, which is known to be immunogenic as well as highly exposed, suggests immunogenic behavior of pseb to be plausible. however, western blot and dot blot analyses have revealed no band or signal for pseb, which is in accordance to the microarray data. the contrast values were below the cutoff and no band was visible in western blot experiments. directly analyzing the whole lysates by western blot failed to discriminate between positive bands and background as the whole lysates of krx cells clearly show cross-reactive signals with the used polyclonal antibody. however, as these bands bear similar sizes as the investigated fusion proteins, a secure identification of true positives is difficult at best using standard western blot without previous purification methods. even then identification of immunogenicity is sometimes difficult at best as sensitivity might be low due to differences in expression rate and losses of protein content during purification. still, the signal intensities of the five immunogenic proteins were well represented by all three methods -microarray, western blot and dot blot, as those with highest contrast values, namely cjaa, hisj and peb1a, have also shown bands in western blot and signals in dot blot albeit lower for peb1a. in contrast, pal failed to generate a positive signal during microarray experiments and this result could be confirmed by western blot and dot blot. for flac no band is visible in western blot and the signal in dot blot is extremely weak, yet our microarray method has identified the protein as being immunogenic, which has been reported elsewhere. a possible explanation for this is the high sensitivity of microarrays. this is well in accordance to ekins "ambient analyte theory" [26] which has been the basic concept of microarrays for several decades. thus, the microarray method might well be superior for immunoassays than common blotting methods. we were able to clone several genes from campylobacter jejuni into krx cells, to express the respective proteins as fusion constructs with a halotag ® attached to their n-terminus and to immobilize these proteins on microarray surfaces. subsequently, we have succeeded in screening the proteins using polyclonal antibodies to detect their immunogenic abilities. the usage of pfn18a flexi vector in combination with krx cells offers various advantages. first, flexi vectors inhabit several intriguing features. they encode a bacterial rnase -barnase -which is only removed during successful cloning. if cloning fails, the barnase remains intact and leads to cell death and no visible colonies on the plates. the halotag ® -a derivative of a dehalogenase -is fused during expression to the n-terminus of our protein of interest. the expression of the fusion proteins is under control of t7 rna polymerase. additionally, an ampicillin cassette is present to allow for antibiotic selection. second, with commonly used bl21(de3) expression cells, induction is realized by isopropyl β-d-1-thiogalactopyranoside (iptg). however, the iptg-induced expression of t7 rna polymerase in bl21(de3) is not tightly regulated, i.e. the promoter is leaky, causing a basal expression even if cells are not induced. this is a figure 10 western blot of whole cell lysates. four bands are visible in the krx whole cell lysate with a more prominent band at 70 kda and three weaker bands at 55 kda, 28 kda and roughly 18 kda. these bands are most likely due to cross-reactive proteins within the e.coli lysates. all four proteins previously described as non-immunogenic (argc, gapa, pseb and pyrc) show band patterns highly similar to krx. this underlines their lack of immunogenic potential. the five proteins previously described as immunogenic elsewhere show stronger bands at roughly 70 kda for cjaa, hisj, peb1a, flac and 55 kda for pal respectively. these sizes correspond closely to the expected sizes of the halotag ® fusion proteins. however, the interference of the background bands from krxbeing in the same range as the target proteins under investigations -prevents a secure identification of immunogenic proteins. major problem especially if toxic substances are to be expressed [27] . in our method, we used krx cells to counter this problem. these cells are under regulation of l-rhamnose and induction can completely be turned off by addition of glucose to the medium during growth. in fact, for the proteins investigated, expression has failed in bl21(de3) for all but two proteins (data not shown), whereas krx cells were able to express all the proteins with satisfying yields as we showed by sds-page. third, the halotag ® offers covalent binding to its specific ligand, therefore leading to a strong attachment of the proteins to the microarray surface in comparison to other tags such as gst or 6 × his. expression, immobilization to modified surfaces and detection of antigens was difficult at best using 6 × his and gst (data not shown). combining these features with microarrays, we have been able to employ a method to rapidly screen for immunogenic proteins without the need for an additional purification step. instead whole lysates can directly be spotted to the microarray surface. still, cross-reactivity is prevented by the special use of the tag and its specific ligand on the microarray slide. further, the use of microarrays as a screening platform offers several advantages in itself. high throughput, statistical testing of the data and numerous replicates are easily achieved compared to the more traditional screening on nitrocellulose membranes. in fact, numerous replications with nitrocellulose membranes are difficult, the selection of positive clones errorprone as replicate and master filters have to be compared visually and selection occurs manually. moreover, nitrocellulose membranes bind entire protein lysates unable to discriminate between proteins of interest and proteins from the expression host. this high background content leads to numerous false positive signals when using polyclonal sera raised against whole cells to search for new antigens. although, these effects might differ for different bacteria, in case of campylobacter jejuni and escherichia coli this has been observed previously as their outer membrane proteins (omp) show high similarities [28] . on top of that, identification of positive clones is merely done visually, usually by chemiluminescence. with our method, these problems are minimized. it allows screening of numerous clones on microarrays with sufficient replicates to gain statistically significant data. it reduces the occurrence of false positive signals to a minimum, thus focusing on the real positive proteins and leading to a faster detection. it allows for statistical and mathematical analysis of the data compared to nitrocellulose membranes illuminating proteins with different strength in their immunogenic ability. further, the choice of our expression host -krx -and the halotag ® have brought other benefits, as well. the expression is tightly regulated, offers high yields and the covalent attachment of the proteins to the surface makes separate purification obsolete as it combines spotting and purification directly. the overall time frame from cultivation to screening is roughly 30 hours, whereas immunoscreening on nitrocellulose membranes [8] takes at least 3 days. the benefit of low turn-around time increases, when taking the cloning into consideration. normally, cloning to recombinant expressed proteins is first done in propagation cells such as jm109 or dh5α and afterwards a second cloning procedure to expression cells as bl21(de3) is performed. with krx single-step cells both propagation and expression can be performed sequentially avoiding time-consuming plasmid preparations and additional cloning steps. our results proved to be reproducible, which we showed using several technical replicates. the halotag ® has previously been used for an increasing spectrum of applications, such as the monitoring of single molecules during trans-translation processes [29] , reporter protein assays for magnetic resonance imaging (mri) analysis [30] and flow cytometry [31] . with our method, we have added another intriguing application to the mix. this method ought to become an attractive alternative to traditional methods for immunoscreening as it features several unique advantages. it provides a low turnaround time, significantly reduces background and false positive signals and enables high-throughput screenings of expression libraries or other sources of hundreds and thousands of different clones. with our results we were able to show the great potential of this method in future full proteome screenings as an attractive alternative to other broad screenings. its major advantages being the complete deletion of cross-reactivity to proteins of the expression host as these are simply washed off after spotting. thus an additional purification step, which is usually performed in microarray-based screening approaches of expression libraries or proteome analysis before immobilization to nitrocellulose slides [32] , becomes obsolete. therefore, this method might decrease time and material costs associated with extensive screenings of libraries. these advantages increase significantly with the number of different samples processed, making library screening a preferred application. for screening of whole expression libraries and the detection of novel antigens the use of different polyclonal sera and antibodies is necessary as these might detect different antigens. only if a protein leads to positive signals in all screenings it can be considered immunogenic. additionally, controls using established methods such as western blot, immunoprecipitation and elisa ought to be considered to validate the potential of a novel antigen. consequently, the method presented herein may be useful for broad screening of bacterial expression libraries to identify and detect novel immunogenic proteins. our approach gives the possibility to discover new proteins that could be used as biomarkers in diagnostic assays or for the production of vaccines. additionally, further knowledge about potentially new virulence-associated factors and antigens will grant deeper insight and understanding of virulence and pathogenicity of many enigmatic bacteria. therefore, we strongly believe this to emerge as an important tool in the search for antigens, virulence factors, biomarkers and vaccine candidates. campylobacter jejuni nctc 11168 was used for isolation of genomic dna and amplification of genes used in this study. campylobacter jejuni nctc 11168 was streaked on karmali agar (oxoid) and incubated under microaerophilic conditions at 42°c for at least 2 days. for genomic dna isolation a flask of 100 ml brainheart-infusion (bhi) broth was inoculated with a loop of bacterial culture and cultivated in an innova 40r (new brunswick) incubation shaker for 48 h at 42°c in an anaerobic jar. genomic dna of campylobacter jejuni nctc 11168 was isolated using a phenol-chloroform protocol. briefly, cells were harvested by centrifugation (1000 g, 10 minutes) in a benchtop centrifuge 5415 r (eppendorf) and the pellet resuspended in 1 ml of 1 × pbs (1.9 mm nah 2 po 4 , 8.1 mm na 2 hpo 4 , 150 mm nacl). after a second centrifugation the pellet was resuspended in 500 μl of lysis buffer (10 mm tris, ph 7.5, 25 mm edta, 75 mm nacl, 0.1% sds and 0.5 mg/ml proteinase k) and incubated for 1 h at 50°c. afterwards one volume of phenol-chloroform-isoamyl alcohol (25:24:1) was added and after centrifugation (> 13.000 g, 1 min) the dna-containing top phase was carefully removed and transferred to a clean tube. this step was repeated three times and the combined top phases washed twice with chloroform-isoamyl alcohol (24:1). after addition of 1/10 volume of 3 m sodium acetate and one volume of 100% isopropanol the solution was centrifuged for 30 min at 13.000 g. the supernatant was discarded, the pellet air-dried and resuspended in 100 μl tris-cl (ph 8.5). the concentration and purity of the isolated dna was measured using the nanodrop nd-1000 device (peqlab). pcr was carried out to selectively amplify several genes of interest from the genomic dna. the primers were designed using flexi vector primer design tool (promega) and possessed overhangs for subsequent cloning into flexi vectors (promega). the melting temperature of each primer was determined using a t m calculator (finnzymes). table 2 summarizes primer sequences and melting points. the amplification was performed in separate tubes containing a reaction mixture comprised of 2 μl dna (200 ng), 10 μl 5× phusion hf buffer, 1 μl dntps (10 mm each), 0.5 μl 50 mm mgcl 2 (final concentration 2 mm) 5 μl (10 μm) of each primer, 0.5 μl phusion high fidelity polymerase (2 u/μl, finnzymes) and pcr-grade sterile water to a final volume of 50 μl. amplification of the targets was performed in a gradient thermocycler (biorad): initial denaturation 98°c for 3 min, 30 cycles of 98°c for 10 s, annealing temperature (t m + 3°c of the lower primer) for 30 s and 72°c for 30 s and a final extension of 72°c for 3 min. the pcr products were purified using qiaquick pcr purification kit (qiagen) according to the manufacturer's instructions. the concentration and purity of dna were measured with the nanodrop device and the size of the amplicon determined by using an agarose gel electrophoresis. agarose gel electrophoresis was carried out in a per-fectblue gelsystem mini (peqlab) for 90 min at 80 v. the dna was visualized by ethidium bromide staining and detected on a transilluminator. hyper ladder i (bioline) served as a size reference. after the correct size was determined the purified pcr product was used in restriction digest according to the flexi vector system (promega). briefly, the dna and the acceptor vector, flexi pfn18a (promega) were cut by sgfi and pmei. for the reactions 500 ng of dna were mixed with 4 μl 5× flexi digest buffer, 4 μl of flexi enzyme blend (sgfi and pmei) and the total volume adjusted to 20 μl using nuclease-free water. respectively, 200 ng of acceptor vector were cut after combining it with 4 μl of 5× flexi digest buffer, 2 μl of flexi enzyme blend (sgfi and pmei) and 12 μl of nuclease-free water. the reactions were incubated for 30 min at 37°c. afterwards, the reactions containing the different amplified genes were purified by qiaquick pcr purification kit (qiagen), whereas the digest of vector was heated for 20 min to 65°c to inactivate both enzymes. directly after purification and inactivation 4 μl of restricted dna was added to 10 μl 2× ligase buffer, 5 μl of digested acceptor vector and 1 μl t4 dna ligase (hc, 20 u/μl). the ligation reaction proceeded for one hour at room temperature. the ligated constructs of vector and gene of interest were cloned into krx single-step competent cells (promega) by following the manufacturer's instructions. 200 μl of each transformation reaction were spread on lb agar plates containing ampicillin (100 μg/ml). the plates were incubated overnight at 37°c in an incubator. from each plate seven clones were selected and used as templates in colony pcr. the reaction mixture contained 11.2 μl nuclease-free water, 4 μl 5× phusion hf buffer, 0.4 μl dntps (10 mm each), 0.2 μl 50 mm mgcl 2 (final concentration 2 mm), 2 μl of each primer (10 μm) and 0.2 μl of phusion high fidelity polymerase. the colony pcr and gel electrophoresis conditions were identical to the ones described above. positive clones were used to inoculate 5 ml of lysogeny broth containing ampicillin (lb-amp) and cultivated under shaking conditions (270 rpm) at 37°c, until an optical density of od 600 = 0.6 was reached. the cell suspension was centrifuged (1000 g, 5 min). the supernatant was discarded and the pellet resuspended in 0.9 ml of fresh lb-amp. subsequently 100 μl sterile dmso (roth) was added. clones were stored in cryo tubes at -80°c. for protein expression, clones from cryo stocks were used to inoculate 5 ml of lb-amp and incubated for 8 h at 37°c in an incubation shaker. the temperature and shaking were decreased to 18°c respectively 180 rpm. protein expression was induced by addition of 25 μl of 20% sterile-filtrated l-rhamnose (promega). the incubation continued overnight. lysis of cells was achieved using easylyse bacterial protein extraction kit (epicentre) according to manufacterer's instructions. afterwards lysates were spotted to halolink™ slides (promega) using the qarray2 microarray spotter (molecular devices). humidity was set to 70% during spotting and the slides were left in the humidity chamber for 1 h after spotting was completed. the slides were washed after spotting with pbsi (1× pbs, 0.05% igepal ® ca-630) and a 3 well proplate™ module (grace biolabs) was attached to each slide. each chamber was filled differently, as follows: top chamber with rabbit-polyclonal antibody to campylobacter jejuni (acris, 5 μg/ml), middle chamber with rabbit-polyclonal to halotag ® (promega, 5 μg/ml) and bottom chamber with pbs only. incubation was performed for one hour at the gene name, accession from embl, primer names and sequences, their melting points, length of gene and immunogenic characteristics of the encoded protein are given room temperature under gentle rocking. the slides were washed with pbsi. secondary antibody (goat polyclonal to rabbit igg conjugated with chromeo™-547, abcam, 10 μg/ml) was applied to each chamber. the slides were incubated under gentle rocking for one hour at room temperature in the dark. finally, slides were washed with pbsi, the proplate™ module was removed and the slides were dried by a nitrogen stream. the scanning of each slide was performed on an axon genepix 4200a laser scanner (molecular devices) with the following settings: 532 nm laser, pmt 400, 40% power, lines to average 1, 10 μm resolution, standard green emission filter 575 nm. the raw data of the microarray experiment [geo: gse33295] was analyzed using originpro 8 g (origi-nlab). the local background of each spot was subtracted from each spot's intensity to gain relative fluorescence intensities (rfi). the signals from chamber one were corrected through substraction of rfis of chamber three to account for unspecific signals by secondary antibody immobilization alone. from the krx extract samples without halotag ® protein the median and the standard deviation (sd) of the background are calculated. for each sample the contrast is computed by: with rfi s the relative fluorescence intensity of each spot and the median of the background median(b). as samples were spotted in multiple replicates, mean contrast values and their respective standard deviations were calculated. the cutoff was calculated using the median and sd of the background: plasmids were isolated from 1 ml aliquots of uninduced cells by qiaprep spin miniprep kit (qiagen) according to the manufacturer's instructions. the purified plasmid dna was eluted in 50 μl tris-hcl (ph 8.5). concentration and purity were determined by nanodrop measurements. each plasmid was sent to sequence laboratories göttingen gmbh for sequencing using two different sequencing primers ht7 f (5' acatcggcccgggtctgaatc 3') and flx r (5' cttcctttcgggctttgttag 3'). the generated sequences were aligned with the corresponding gene sequences by global alignment with free-end gaps (modified needleman-wunsch) using geneious pro™ 5.4.4 (biomatters). the expression of the desired halotag ® fusion proteins was checked by sds-page. after lysis of cells, 2 μl of each protein extract was mixed with 1 μl of 10 μm halo-tag ® alexa 488 ligand. after addition of 7 μl 1× tbs (100 mm tris, 150 mm nacl, ph 7.6) the reaction was incubated at room temperature for 30 minutes. 2 μl of each reaction were removed, mixed with 8 μl of 5× loading buffer (fermentas) and 1 μl dtt and heated for 5 min at 70°c. the separation was performed on a mini-protean tgx gel (biorad, any kd, 15 wells) in a protean ii xi cell chamber (biorad) for 30 min at 200 v. as a size reference pageruler plus prestained protein ladder (fermentas) was used in combination with halotag ® standard protein (promega). fluorescence was measured in a fla-5100 (fujifilm) with excitation at 473 nm. for western blot analysis the samples were run on an sds-page, the gel blotted to a pvdf membrane using the iblot ® western blotting system (life technologies). the blotting time was set to 7 minutes. after blotting, membranes were rinsed with water and blocked for 1 h in 5% fat-free milk powder in pbs with gentle rocking. the membranes were then washed with pbs-tween (0.05%) twice for five minutes each. the primary antibody (4 μg/ml) was added in 1% fat-free milk powder in pbs and incubation was performed for 1.5 h under gentle rocking. afterwards, membranes were washed twice with pbs-tween (0.05%). the ap-conjugated secondary antibodies (4 μg/ml) were added in 1% fat-free milk powder in pbs and incubation proceeded for 1 h under gentle rocking. finally, membranes were washed twice with pbs-tween and western blue ® stabilized substrate for alkaline phosphatase (promega) was added. after 5 min the membrane was taken out, rinsed with water and dried. a picture of the membrane was taken using bio-docanalyze digital and its software (biometra). for dot blot analysis, 2 μl of the purified protein solution were added as small droplets to a nitrocellulose membrane. after drying, the membrane was blocked using 5% fat-free milk powder in pbs for 1 h. the membrane was washed twice with pbs-tween (0.05%) and primary antibody (4 μg/ ml) was added in 1% fat-free milk powder in pbs. incubation prolonged for 30 min with gentle rocking, afterwards the membrane was washed twice as above and ap-conjugated secondary antibody (4 μg/ml) was added and incubation proceeded for additional 30 min. after a final two washes, the western blue ® stabilized substrate for alkaline phosphatase (promega) was added and after 5 min the membrane was rinsed with water. the visualization was performed with the biodocanalyze digital software. campylobacter, from obscurity to celebrity epidemiologisches bulletin 040 chronic effects of campylobacter infection bundesinstitut für risikobewertung: stellungnahme nr. 010 typing of campylobacter jejuni and campylobacter coli isolated from live broilers and retail broiler meat by flaa-rflp, mlst, pfge and rep-pcr rapid pulsedfield gel electrophoresis protocol for subtyping of campylobacter jejuni evaluation of three commercial latex agglutination tests for identification of campylobacter spp molecular cloning: a laboratory manual. 3 edition. cold spring harbor: cold spring harbor press immunogenic cross-reaction among outer membrane proteins of gram-negative bacteria purification of annexin v and its use in the detection of apoptotic cells purification of proteins fused to glutathione stransferase van voorhis wc: expression of proteins in escherichia coli as fusions with maltosebinding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline bacterial expression, purification, and model membrane reconstitution of the transmembrane and cytoplasmic domains of the human app binding protein lr11/sorla for nmr studies spatially addressed combinatorial protein libraries for recombinant antibody discovery and optimization correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site halotag: a novel protein labeling technology for cell imaging and protein analysis protein-protein interactions: analysis of a false positive gst pulldown result the campylobacter jejuni/coli cjaa (cj0982c) gene encodes an nglycosylated lipoprotein localized in the inner membrane genetic diversity of the campylobacter genes coding immunodominant proteins immunological characterization of the campylobacter jejuni 72dz/92 cjad gene product and its fusion with b subunit of e. coli lt toxin immunogenicity and protective efficacy of recombinant campylobacter jejuni flagellum-secreted proteins in mice localization of immunogenic regions on the flagellin proteins of campylobacter jejuni 81116 immunogenicity and immunoprotection of recombinant peb1 in campylobacter-jejuniinfected mice characterization of cj1293, a new udp-glcnac c6 dehydratase from campylobacter jejuni functional characterization of dehydratase/aminotransferase pairs from helicobacter and campylobacter: enzymes distinguishing the pseudaminic acid and bacillosamine biosynthetic pathways multianalyte microspot immunoassay. the microanalytical "compact disc" of the future use of bacteriophage t7 lysozyme to improve an inducible t7 expression system conformational analysis of the campylobacter jejuni porin single molecule imaging of the trans-translation entry process via anchoring of the tagged ribosome reporter protein-targeted probes for magnetic resonance imaging the use of halotag-based technology in flow and laser scanning cytometry analysis of live and fixed cells severe acute respiratory syndrome diagnostics using a coronavirus protein microarray submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the recombinantly expressed target-proteins were purified from whole lysates by using halolink™ magnetic beads (promega) according to the manufacturer's instructions. subsequently, target protein was cleaved off using protev plus protease and the supernatant used for sds-page. additional file 1: excel sheet comparing the codon usage of campylobacter jejuni nctc 11168 and escherichia coli strain k12.authors' contributions sh carried out the design of the method, the testing of the method including all cloning steps, interpretation of sequence data, data analysis and drafted the manuscript. mvnr conceived and coordinated the study, participated in the analysis of the data and helped draft the manuscript. ffb participated in the critical review and drafting of the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-286301-7sjw5ci7 authors: sadasivan, jibin; singh, manmeet; sarma, jayasri das title: cytoplasmic tail of coronavirus spike protein has intracellular targeting signals date: 2017-04-18 journal: j biosci doi: 10.1007/s12038-017-9676-7 sha: doc_id: 286301 cord_uid: 7sjw5ci7 intracellular trafficking and localization studies of spike protein from sars and oc43 showed that sars spike protein is localized in the er or ergic compartment and oc43 spike protein is predominantly localized in the lysosome. differential localization can be explained by signal sequence. the sequence alignment using clustal w shows that the signal sequence present at the cytoplasmic tail plays an important role in spike protein localization. a unique gyqel motif is identified at the cytoplasmic terminal of oc43 spike protein which helps in localization in the lysosome, and a novel klhyt motif is identified in the cytoplasmic tail of sars spike protein which helps in er or ergic localization. this study sheds some light on the role of cytoplasmic tail of spike protein in cell-to-cell fusion, coronavirus host cell fusion and subsequent pathogenicity. coronaviruses are enveloped positive strand rna viruses which acquire their membrane envelope by budding into the lumen of the intermediate compartment between the endoplasmic reticulum and golgi complex (ergic) (krijnse-locker et al. 1994) . matured virus are thought to move through the vesicles via the secretory pathways and exit the cell when the vesicles fuse with the plasma membrane (holmes et al. 1984; tooze et al. 1987) . mature coronavirus virion normally consists of structural proteins, s (spike), m (matrix), and e (envelope) that surrounds a helical nucleocapsid. an additional protein, he (hemagglutinin esterase) is expressed in some beta-coronaviruses. m is a triple spanning membrane protein believed to play a key role in maintaining viral core structure; whereas e, a small enveloped glycoprotein, is critical for virion assembly. the s protein is a type i membrane glycoprotein which (vennema et al. 1990 ) constitutes the characteristic coronavirus spike. he is also a type i glycoprotein with a single membrane spanning anchor and a short cytoplasmic tail. detailed studies on virus-like particle formation (vlps) revealed that only m and e proteins are required for vlp formation. most glycoproteins of the enveloped viruses sort to the cellular membrane, where their budding occurs (garoff et al. 1998) . some viral glycoproteins sort to the specialized apical or basolateral plasma membrane of the polarized epithelial cells. for example, glycoprotein gp160 from type 1 human immunodeficiency virus (hiv-1) sorts to the basolateral domain (owens and compans 1989) , but a prototype glycoprotein, hemagglutinin from the influenza virus, sorts to the apical domain (mora et al. 2002) . to mediate budding of infectious viral particles through the intracellular membranes, viral glycoproteins must embed a signal for sorting to an intracellular compartment. for example, golgi targeting signal (teasdale and jackson 1996) in the membrane spanning domains of the m protein of coronaviruses (machamer and rose 1987; swift and machamer 1991) and the g1 protein of bunya viruses (matsuoka et al. 1991 ) specify the glycoprotein accumulation at golgi complex (andersson and pettersson 1998) . the dilysine signal (dekkmp) in adenovirus glycoprotein e19 carboxy terminal localizes it to the er (paabo et al. 1987; nilsson et al. 1989) . a similar dilysine signal partitions human foamy virus maturation to intracytoplasmic compartments (goepfert et al. 1999) . proteins containing the dilysine motif have been observed to bind directly to well-characterized coat proteins (copi) (cosson and letourneur 1994; gaynor et al. 1998; corse and machamer 2002) . the copi coated vesicles mediate retrograded transport of the dilysine signal-containing proteins from the proximal golgi compartments to the er. recent studies on coronavirus spike protein signals have revealed a dilysine-based er localization signal in the infectious bronchitis virus (ibv, a gamma-coronavirus) (figure 1). the ibv s protein also carries a tyrosine-based (yytf) endocytotic signal in its cytoplasmic tail (corse and machamer 2002; bonifacino and traub 2003) which is more crucial for the intracellular retention of the s protein than its canonical dilysine-based signal (winter et al. 2008) . a similar dibasic motif (kxhxx) in the cytoplasmic tail of s protein from alpha-coronavirus tgev and from the newly emerging pathogen sars, an outlier of beta-coronavirus, localizes the s protein in the ergic (teasdale and jackson 1996; lontok et al. 2004) . a tyrosine-based sorting motif (yepi) has also been shown to be an intracellular localization signal in the cytoplasmic tail of tgev (schwegmann-wessels et al. 2004 ). very few studies have convincingly demonstrated a localization signal in the cytoplasmic tail of beta-coronaviruses that would lead to their sequestration within the intracellular compartment. the intracellular transport and targeting of proteins from their site of synthesis to their correct destination is a process instrumental to maintenance of virion integrity. a major effort is underway to understand the interplay of factors that guide the envelope proteins to the intracellular budding compartments avoiding the bulk flow through the secretory pathways (bonifacino and traub 2003) . there is ample evidence that the localization signalling motifs in the virus mimic those used by the endogenous cellular proteins of the host cell subverting their cellular machinery (bonifacino and traub 2003) . we studied the intracellular trafficking and localization of the full-length s protein from two human coronaviruses (hcov): sars and oc43 (a beta-coronavirus). full-length sars s protein when expressed exogenously is observed to accumulate predominantly in intracellular compartments resembling er and ergic with occasional surface staining. hcov-oc43 s protein is localized in distinct puncta that could represent the endocytic structure. exogenously expressed hcov-sars s protein occasionally induces cell-to-cell fusion when transported to surface in transfected cells, but hcov-oc43 s protein fail to induce the same. the intracellular distribution of the s protein differs significantly in the two viruses. we identified a unique tyrosine-based motif (gyqel, an yxxø motif where ø = bulky hydrophobic side chains) at the cytoplasmic tail of hcov-oc43 (figure 1). yxxø is known to function as a signal for rapid internalization (mellman 1996; kirchhausen et al. 1997; marks et al. 1997) , lysosomal targeting and localization to basolateral surface in polarized epithelial cells (mellman 1996) . the position of this motif in the protein sequence figure 1 . alignment of the amino acid sequence in the cytoplasmic tail downstream of the membrane-spanning domain in the coronavirus s proteins. the yxxø internalization signals are coloured red, and the di-lysine-based motifs, grey. the di-basic motifs from hcov-229e and fipv are putative intracellular signals not experimentally verified (lontok et al. 2004) . localization signals experimentally identified from this study are marked in bold. the sequence alignment was done using the program clustal omega (mcwilliam et al. 2013) . specific virus, localization signal, cellular localization and the authors describing them are elaborated as follows: (sars; klhyt; er/ergic; this study, also lontok et al. 2004 on truncated protein); (oc43; gyqel; lysosome/internalization; this study); (tgev; kvhvh, yepi; er; (schwegmann-wessels et al. 2004) ; (ibv; kksv, yytf; er/internalization; (lontok et al. 2004). relative to the transmembrane domain determines its cellular location. this signal is not present in the cytoplasmic tail of other two beta-coronavirus, bcv (bovine coronavirus) and mhv-a59 (mouse hepatitis virus). our findings allow rationale for the observed differential localization of the s proteins in different coronaviruses from previous published studies. the rabbit polyclonal anti-sars spike antisera was a kind gift of jim wilson (university of pennsylvania, pa). the rabbit polyclonal anti-bcv s antibody that recognizes hcov-oc43-s protein was a kind gift from brenda hogue (arizona state university). rabbit anticalnexin was purchased from stress gene (san diego, ca), rabbit anti-β-cop from affinity bioreagents (golden, co), rabbit anti-mannii (chemicon), rabbit anti-eea1 and rabbit anti-lamp1 from santa cruz biotechnology, inc. (santa cruz, ca). fluorescent-tagged secondary antibodies were obtained from jackson immunoresearch (west grove, pa), triton x-100 from roche diagnostics and tissue culture reagents form invitrogen. unless otherwise specified, all other reagents were from sigma. the s gene of sars-cov strain urbani was amplified by reverse transcription-pcr from viral rna isolated from cell lysate of infected vero cells (provided by dr w bellini, cdc). reverse transcription was performed with superscript ii (invitrogen, carlsbad, ca) and random primers using 1 μg of total cellular rna, as described by the manufacturer. the cdna was amplified with a mix of tth dna polymerase (roche, indianapolis, in) and vent dna polymerase (new england biolabs, beverly, ma) with primers 5′-gttaacaactaagaattcatgt ttattttc-3 ′ and 5′ -aatctcataaa cctcg agtaaagttcgtttatgtg-3′ using a hot-start long-pcr consisting of one cycle of 94°c, 2 min and 80°c, 3 min, followed by 30 cycles of 94°c, 30 s; 55°c, 20 s; 72°c, 3 min, and 72°c, 7 min extension. the resulting pcr product was cloned into topo-ii ta vector (invitrogen) and its sequence was verified by automated sequencing using bigdye terminator v3.1 cycle sequencing kit (applied biosystems, foster city, ca). a wild-type urbani spike sequence was confirmed by sequence analysis using macvector (accelrys, san diego, ca). the s gene was then inserted between the ecori and xmai sites downstream of the cmv ie enhancer and the chicken β-actin promoter into the mammalian expression vector pcaggs-mcs (niwa et al. 1991) . oc43 spike in pcdna3 was used as template. vectors encoding either hcov-sars or hcov-oc43 s protein we engineered the yellow fluorescent protein (yfp)-tagged hcov-sars s protein (sars-s) and hcov-oc43 s protein (oc43-s) by constructing the full-length cdna of hcov-sars-s and hcov-oc43-s in commercially available pyfp-n1 vector (promega). full-length cdna of hcov-sars-s and hcov-oc43-s was amplified by pcr amplification in a robocycler (stratagene) using expand long template pcr system (roche diagnostics, indianapolis, in), starting with either hcov-sars cdna or hcov-oc43 cdna as a template. the resulting pcr products were restricted with xhoi and bamhi and ligated in pyfp-n1 vector to make in frame fusion protein. ligated product was transformed into bacterial stocks and dna was isolated and purified by using qiagen midiprep kit according to the manufacturer's instructions. positive clones of hcov-sars-s/pyfp-n1 and hcov-oc43-s/pyfp-n1 were screened by restriction digestion and sequenced to confirm the in frame fusion of s protein-yfp (s-yfp). for transient transfection in hela cells, the full-length cdna encoding hcov-sars-s-yfp (sars-s-y) and hcov-oc43-s-yfp (oc43-s-y) was subcloned into pcaggs vector. the full-length cdna of sars-s-y and oc43-s-y was amplified by pcr by using hcov-sars-s/pyfp-n1 and hcov-oc43-s/pyfp-n1 as a template respectively. pcr products were restricted with ecori and xmai and ligated into pcaggs mammalian expression vector (niwa et al. 1991) . ligated product was transformed and plasmid dna was isolated and purified as described previously. positive clones of sars-s-y/pcaggs and oc43-s-y/ pcaggs constructs were sequenced. the dibasic residues (k and h) at the c terminus (klhyt) of sars-s protein were mutated using quick change site directed mutagenesis kit (stratagene, la jolla, ca), generating the sars-s 2a -y/pcaggs mutant signal (lys→ala and his→ala). same kit was also used to mutate the critical tyrosine after the glycine residues, a lysosomal targeting signal of hcov-oc43-s (gyqti) cytoplasmic tail, to generate oc43-s gtoa -y/pcaggs mutant and hcov-oc43-s ytoa -y/pcaggs mutant. for transient transfection and immunofluorescence, hela cells were plated on 25 mm circular coverslips in 35 mm dishes 1 day prior to transfection and transfected with s protein from either wild-type hcov-sars, hcov-oc43, or tagged spike cdna constructs using fugene (roche diagnostics, indianapolis, in) at 1 μg/ml dna using fugene/dna ratios of 6:1 (l μg). for protein analysis, cells were plated on 100 mm tissue culture dishes and transfected with cdna constructs as described above. for immunofluorescence after 48 h of transfection, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (pbs) for 10 min at room temperature, then washed 3x with pbs and permeabilized with pbs+0.5% triton x-100 and then blocked with pbs + 0.5% triton x-100+ 2% heatinactivated goat serum (pbs/gs). the cells were incubated with primary antisera diluted into pbs/gs for 1 h, washed, and labelled for 1 h with secondary antisera (texas red goat antirabbit) diluted into pbs/gs. the cells were then washed with pbs, mounted into mowiol. cells were next visualized by fluorescence microscopy using an olympus ix-81 microscope system with a 60x uplanapo oil immersion objectives with the iris diaphragm partially closed to the limit the contribution of out of plane fluorescence and filter packs suitable for green (u-mwiba bp460-490 dm505 ba515-550) and red (u-nmg bp530-550 dm570 ba590-800+) fluorescence. images were acquired with a hammatzu orca-1 ccd camera and image pro images analysis software (media cybernetics, silver spring, md). for staining with anti-calnexin, cells were fixed with meoh/acetone (1:1) (v/v) instead of paraformaldehyde. to label surface sars-s-y and oc43-s-y fusion proteins, transfected cells were washed with ice-cold pbs and incubated with rabbit anti-sars-s or rabbit anti-bcv-s antisera for 10 min at 4°c. cells were then washed 2x with ice cold pbs and fixed by 4% paraformaldehyde for 10 min, followed by washing with 2x pbs and mounted into mowiol and visualized as described above. transfection ability of yfp-tagged spike protein constructs are much more efficient compared to wild-type spike construct, the reasons for which are unclear (data not shown). because of efficient detection of yfp fluorescence and the limitations of spike specific antibodies, we decided to use the yfp-tagged spike constructs instead of wild-type constructs throughout this study. the expression of the s protein via the cmv promoter was below the limits of detection, we subcloned s-y fusion protein and the full-length untagged s into pcaggs, a mammalian expression vector under the control of a chicken β-actin promoter that, in the past, has proved useful for the efficient expression of rna virus glycoprotein (niwa et al. 1991) . transient expression in hela cells by using pcaggs/ s-y resulted in detectable expression s-y under epifluorescence microscopy. it is not clear why pcaggs was more efficient at driving s expression compared to cmv promoter containing vectors. yfp-tagged full-length cdna of hcov-sars spike and hcov-oc43 spike in pcaggs constructs were generated as described in materials and methods. to detect the intracellular localization of these constructs, hcov-sars-s-y (sars-s-y) and hcov-oc43-s-y (oc43-s-y) were transiently transfected into hela cells and then examined by indirect immunofluorescence microscopy, utilizing the yfp autofluorescence properties of the constructs. full-length hcov-sars s (sars-s) protein was predominantly accumulated in intracellular compartment resembling er and ergic (figure 2c). in contrast full-length hcov-oc43 s (oc43-s) protein was localized in the puncta that could represent the endocytotic structures (figure 2d). we observed similar intracellular distribution of the s protein in both human kidney (293 cell line; figure 3a, b) and human lung epithelial cells (a549; figure 3c, d). our observation is consistent with the hypothesis by vennema et al (vennema et al. 1990 ) that exogenously expressed coronavirus s protein mostly remain intracellular. when yfp alone plasmid was transfected in hela cells, the fluorescence was detected all through the cells (figure 2a, b). to further confirm the intracellular localization of sars-s and oc43-s protein, and to rule out that the intracellular retention is not due to yfp, we transfected hela cells with wild-type full-length cdna construct of sars-s protein and oc43-s protein in pcaggs. transfected cells were immunolabelled with respective antisera and examined by immunofluorescence microscopy (figure 2e, f). wild-type s constructs of both hcov-sars and hcov-oc43 were predominantly localized in intracellular compartments as with yfp-tagged fusion s proteins. 3.2 confirmation of intracellular localization of yfptagged hcov-sars and hcov-oc43 s protein using respective anti-spike antisera to confirm that antisera is also detecting the intracellular d i s t r i b u t i o n o f y f p -t a g g e d s p i k e p r o t e i n , w e immunolabelled the yfp-tagged s protein transfected cells with respective antiserarabbit anti-hcov-sars s protein antisera for sars-s-y construct and rabbit anti-bcv antisera for oc43-s-y construct. bcv antisera are known to cross-react with hcov-oc43 (hogue et al. 1984) . as shown in figure 4a-f, spike antisera and yfp are significantly co-localizing, demonstrating that both hcov-sars (figure 4a-c) and hcov-oc43 (figure 4d-f) antisera also detects the similar localization of the s fusion protein in the intracellular compartments. localization of the s protein (figure 2) was further confirmed by surface staining and antibody uptake experiments. to distinguish the surface expression from the internally localized yfp auto fluorescence of sars-s-y and oc43-s-y, intact hela cells expressing sars-s-y and oc43-s-y were stained with respective rabbit antisera at 4°c for 10 min and then fixed and stained with fluorescent-tagged goat anti-rabbit igg. sars-s-y was absent from surface in most of the cells and localized at the intracellular compartments (figure 5a), and oc43-s protein was mainly localized in distinct puncta that could represent endocytic structures following internalization from the plasma membrane (figure 5b). to test whether sars-s protein after reaching the surface can be internalized and oc43-s puncta could represent endocytic structures following internalization from the plasma membrane, live cells expressing s protein from either sars or oc43 were incubated for 15 min at 37°c with either anti-sars antisera or anti-bcv antisera, and then fixed, permeabilized, and stained with fluorescence conjugated secondary antibodies. in some transfected cells where the sars-s protein expressed in higher levels, we observed antibody staining mainly restricted to the surface (figure 5c; colour merged). the perinuclear intracellular fluorescent s protein rarely labelled with the exogenously added antibody, indicating that the portion of the sars-s protein which reaches the plasma membrane may not efficiently endocytosed after reaching the plasma membrane. interestingly, oc43-s protein expressing cells did not show any surface antigen staining, and the intracellular puncta containing oc43-s-y were not labelled by exogenously added antibody (figure 5d; colour merged), indicating that oc43-s-y could not reach the plasma membrane. to determine the specific intracellular localization compartment of sars and oc43 spike protein, transiently transfected hela cells were double labelled with antibodies recognizing various resident proteins localized in different intracellular compartments (calnexin for er, β cop for ergic, mannii for golgi and mainly localized in the puncta that could represent the endocytotic structures (more description below). examination of the cytoplasmic tail sequence of the s protein from hcov-oc43 (figure 1) revealed a previously known motif, gyxxø, at the 9 residues from the carboxyl terminus. yxxø signals are known to be much more widely involved in protein sorting, and are required for the rapid internalization from the plasma membrane. these signals also involved in targeting of transmembrane proteins to lysosomes and lysosome-related organelles in addition to endocytosis. the presence of glycine, preceding the critical tyrosine, a characteristic of lysosomal targeting signals may inhibit the flexible or unhindered recognition of the yxxø residues in close proximity to the membrane. full-length oc43 spike protein was unable to demonstrate surface expression either by surface labeling or by antibody uptake experiment. to verify whether the motif signals for oc43-s-y transport to endocytic organelles, we used immunofluorescence co-localization with the early endosomal marker (eea1) (figure 7a-c) and late endosomal-and lysosomal-marker (lamp1) (figure 7d-f). oc43-s-y intracellular puncta showed significant colocalization with the lysosomal marker lamp-1 (figure 7df) but no significant overlap with early endosomal marker eea1 (figure 7a-c). this suggests that gyxxø motif may after 48 h of post-transfection, cell was immunolabelled using rabbit anti-hcov-sars s protein antisera for sars-s-y construct (b) and rabbit anti-bcv antisera for oc43-s-y construct (e) which were then stained with texas red goat anti-rabbit igg secondary antibodies. yfp is denoting the spike protein (a and c); merged images (c and f) (n>3). be directing the oc43-s-y in late endosomal or lysosomal compartments with or without recycling from the plasma membrane. examinations of cytoplasmic tail sequence of the s protein from sars (figure 1) show a unique motif klhyt, at the c termini. s protein from other alpha-coronavirus (human coronavirus 229e and feline infectious peritonitis virus) also contains the sequence at their c termini (figure 1). the klhyt fits the criteria for the dibasic kxhxx motif if the histidine residue is protonated. lontok et al., in their chimeric s protein studies used c terminal 11 amino acids of sars-s protein attached to the plasma membrane reporter protein vsv-g to show kxhxx motif is an intracellular localization signal for sars, and the intracellular distribution closely overlapped with ergic. they also demonstrated that mutagenesis of the lysine and histidine residues in the kxhxx signal results in the loss of intracellular localization (lontok et al. 2004 ). in our fulllength sars spike yfp fusion protein though the kxhxx motif is no more near the c-terminal; it still acts like the kxhxx motif in the c terminal tail of sars wild-type protein. to test that the internal kxhxx motif could function as a genuine intracellular localization signal, the lysine and histidine residues in sars-s-y pcagg was mutagenized to alanines to construct the sars-s 2a -y pcaggs. hela cells were transiently transfected with mutated sars-s 2a -y pcaggs and were examined by epifluorescence microscopy (figure 8a, b) . sars-s 2a -y pcaggs fluorescence was observed all through the cell and not localized to any particular organelle. our studies clearly demonstrated that the kxhxx motif is the major intracellular localization signal of the full-length sars-s protein and the c-terminal proximity is not essential. 3.7 mutation of critical tyrosine residues of oc43 lysosomal targeting signal causes hcov-oc43 spike to traffic to cell surface to resolve whether the yxxø motif in the cytoplasmic tail of hcov-oc43 is indeed essential for its intracellular distribution by rapid turnover, we mutated the critical tyrosine residues in the gyxxø motif to alanines generating the oc43-s ytoa -y construct. transiently transfected hela cells expressing oc43-s ytoa -y was examined by immunofluorescence microscopy ( figure 8c, d) . the majority of cells showed reduction of mutated s protein localization in the intracellular puncta and increased surface expression. to test whether the glycine residue preceding the critical tyrosine play any role in hcov-oc43 s protein trafficking, we mutated the glycine residue to alanine residue to generate a construct oc43-s gtoa -y. if yxxø in the gyxxø motif plays a major role in rapid internalization and g facilitates the lysosomal targeting from endocytosed spike protein after reaching the plasma membrane then the mutated construct are expected to continue to be internalized at the same rates but recycle to the plasma membrane, instead of going to the lysosomes. if gyxxø motif acts as a direct lysosomal targeting signal then the construct are expected to localize primarily in the perinuclear region (due to its large luminal domain) as well as in the surface (by default bulk flow) like other beta-coronavirus. when we transfected the cells with the g to a mutated construct interestingly we observed that s protein goes readily to the surface and also retains in the perinuclear region (figure 8e, f). the expression level was much higher in the same experimental condition in compare to the non-mutated wild-type oc43-s-y construct (data not shown). due to higher level of expression we observed a blazing signal in the nucleus. g to a construct also induces cell to cell fusion in transfected cells which we never observed before. most likely g residue plays a critical role in the gyxxø motif of hcov-oc43-s tail. we can conclude that the tyrosine residue may be essential for rapid internalization, and glycine residues is involved in facilitating the distribution of rapidly internalized s protein to lysosome or it playing a role in direct lysosomal sorting. the control of protein translocation during trafficking in coronaviruses have features common to other organisms. previous studies have suggested that the presence of dibasic residue pair and its specific location relative to the transmembrane domain and the carboxy terminal is critical for er localization activity. specific studies propose a minimum of 5 amino acid residues to be mandatory between the first charged residue of the cytoplasmic tail and the −3 lysine such that the signal sequence be exposed from the membrane lipids in order to function (teasdale and jackson 1996) . our alanine mutation studies on the kxhxx motif confirm the importance of the lysine and histidine in the full-length wild-type hcov-sars s protein; the mutant protein showed localization in plasma membrane instead of the usual er and ergic. the carboxy terminal proximity of the motif was not found mandatory. the c-terminal s-yfp-tagged fusion protein also showed er/ergic localization. yfp is a 120-residue protein, and when fused to the c-terminal end along with an artificial linker makes the dibasic motif xxx residues carboxy terminal distal. we have not systematically examined the maximum length of a cytoplasmic domain of s protein on which a lysinebased motif still functions. there is putative evidence of dilysine motif 546 amino acids from the predicted membrane-spanning region and x residues from the carboxy terminal end in the er localizing hmg coa reductase (luskey and stevens 1985) . other er localization signals like the rxr motif identified in certain oligomeric ion channels do not require proximity to either the n or c terminus. this suggest carboxy terminal proximity of the dibasic motif is not a general requirement for localization function, although, exposure from the membrane lipids may be necessary. the dibasic signals are present in the s protein from alpha and gamma and the sars-coronavirus; there was no such obvious dibasic motif in the cytoplasmic tail of beta-coronavirus. however, we reported that a tyrosine-based signal (gyqel), at the 23-27 residues of the carboxy terminal tail of hcov-oc43 s protein, is a motif of the class yxxø conferring sorting information onto transmembrane proteins. the y residue is essential for function in most cases. x can be any amino acid and are highly variable but tend to be hydrophilic. the ø position can accommodate several residues with bulky hydrophobic side chains, although the exact identity of this residue can specify the properties of the signal (canfield et al. 1991) . however, the x residues and residues flanking the motif also contribute to the strength and fidelity of the signals. the topological location of the yxxø motif on the context of a specific protein is also as important as the actual amino acid sequence. depending on the context of the yxxø motif in the cytoplasmic tail of the protein, yxxø motif can function as a rapid internalization from the surface (mellman 1996; kirchhausen et al. 1997; marks et al. 1997) , lysosomal targeting, localization to specialized endosomal-lysosomal organelles such as antigenprocessing compartments as well as basolateral surface in polarized epithelial cells. purely endocytic yxxø signals are most often situated at 10-40 residues from the transmembrane domains, but not at the carboxy termini of the proteins (bonifacino and traub 2003) . in contrast, lysosomal targeting yxxø signals are conspicuous for their presence at 6-9 residues from the transmembrane domain and are often located at the carboxy terminus of short cytoplasmic tail preceded by a glycine (bonifacino and traub 2003) . their effectiveness may be modified by spacing the gyxxø motif relative to the membrane bilayer (rohrer et al. 1996) . lysosomal-sorting yxxø motifs tend to have acidic residues at the x positions, and these may also contribute to the efficiency of lysosomal targeting (rous et al. 2002) . these signals can be found in all types of transmembrane protein, including type i (e.g. lamp-1 and lamp-2), type ii (e.g., the transferring and a sialoglycoprotein receptors) and multispanning integral membrane proteins (e.g., cd63 and cystinosin). the importance of the distance from the transmembrane domain has been emphasized by a study showing that changing the spacing of the gyqti signal from lamp-1 impairs targeting to lysosomes (rohrer et al. 1996) . the mutant proteins continue to be internalized at the same rates but recycle to the plasma membrane, a behavior typical of endocytic receptors (rohrer et al. 1996) . these observations indicate that the placement of yxxø signals at 6-9 residues from the transmembrane domain allows their recognition as lysosomal targeting signals at the tgn and/or endosomes. very recently it has been demonstrated that the gyxxø motifs in their cytosolic tail can take both direct and indirect (via plasma membrane) traffic routes from the trans-golgi network (tgn) to lysosomes. lamp-1 and lamp-2 which contain the gyxxø at the extreme carboxy terminal tail (after 6 residues from the transmembrane domain) were delivered from tgn to lysosomal compartment via an intracellular route. in contrast, lysosomal acid phosphatase, also a type i membrane protein with a cytosolic tail gyxxø motif located 7 residues from both transmembrane domain and the carboxy termini (tm-rmqaqppgyrhvadgedha) delivered mainly via the cell surface (braun et al. 1989) . the gyxxø motif in hcov-oc43 spike cytoplasmic tail could act as an endocytic signal or lysosomal targeting signal. it has been known that the position of actual amino acid sequence of yxxø signals within the cytoplasmic domain is important (bonifacino and traub 2003) . as gyxxø motif is not in close sequential proximity to the transmembrane domain, it predominantly acts as lysosomal targeting signal rather than rapid internalization or endocytic signal. it is also be possible that when yxxø motif acts as a rapid internalization signal, the presence of glycine (due to its special physico-chemical properties) preceding the critical tyrosine may facilitate the targeting of internalized protein to the lysosome. while hcov-sars is a deadly pathogen causing acute respiratory syndrome, hcov-oc43 causes only benign common cold. it has been reported that this virus hcov-oc43 has neuroinvasive properties (arbour et al. 2000) . the molecular determinants that may account for the dramatic difference in pathogenic potential of the two hcov are currently unknown. previous attempts to uncover the basis of pathogenicity have shown several directions. the primary target of such studies has been the s proteins which are most amenable for interaction with the host cells due to their convenient location on the mature virion surface. several evidences support a major role for the s protein in determining the tropism and pathogenesis (casais et al. 2003 , das sarma 2010 . the nucleotide sequencing revealed that alterations in virus virulence were more closely associated with differences in the s protein. the s protein is responsible for attachment to the cellular receptor both for virus cell fusion during viral entry and cell to cell fusion later during infection (reviewed in (belouzard et al. 2012) . it contains epitopes for viral neutralization and t-cell response. zinc metallopeptidase, angiotensin converting enzyme 2 (ace2) has been identified as the functional receptor for hcov-sars (li et al. 2003) . no functional receptor is yet known for hcov-oc43. the fate of the s protein during the virion assembly is therefore important in understanding the basis of the host-pathogen interaction and virus infectivity. the differential localization of the s protein in two closely related hcov with distinct pathogenic potential offers several insights into the pathologic determinants. the budding and maturation of the virion in the host cell determines the turnover of the coronavirus infection. unique localization signals in the corona viral envelope proteins permit them to follow different intracellular trafficking pathway to reach the same budding compartment at the same time for proper virion assembly. although the localization signals vary from protein to protein as well as virus to virus, in most of the coronaviruses, all three envelope proteins m, e and s possess targeting signals that maintain the localization near the budding site (vennema et al. 1990 ). there is evidence to suggest that the localization signal in s protein is only active in the unfolded structure in er, and its specific interactions in the folded form between the m protein leads to its incorporation into the virion, where the retention signals are no longer recognized causing excess s protein to go to the surface. when they reach the surface, they induce cell-cell fusion with uninfected cells forming multinucleated syncytia causing rapid spread of the infection. the syncytia formation is a late stage phenomenon of the infection due to the over production of the s protein, which cannot be retained intracellular. our studies on differential s protein localization in the intracellular compartment indicates the probable mode of infection to be dependent on signal-sequence-based protein trafficking. in our study exogenously expressed the fulllength hcov-sars had a steady state distribution in the er and ergic, and hcov-oc43 was mostly sorted to lysosomes. the predominant intracellular localization or retention of s protein in the early stage of infection have two implications in pathogenesis: (i) delayed cell-damaging effect which may contribute to maximum virus production, and (ii) absence from the cell surface may help to avoid the defense mechanism, e.g. complement activation. at the same time the surface expression could promote direct cell-to-cell spread of the infection. the low virulence of hcov-oc43 can be clearly attributed by its restricted surface transport of its s protein during infection. the sorting of the excess s protein of hcov-oc43 in the lysosomal compartment may hinder its cell-to-cell spread during infection. viral production is mainly dependent on the intracellular budding. cellto-cell fusion cannot help to spread the disease. targeting of a short peptide derived from the cytoplasmic tail of the g1 membrane glycoprotein of uukuniemi virus (bunyaviridae) to the golgi complex neuroinvasion by human respiratory coronaviruses mechanisms of coronavirus cell entry mediated by the viral spike protein signals for sorting of transmembrane proteins to endosomes and lysosomes lysosomal acid phosphatase is transported to lysosomes via the cell surface localization of the signal for rapid internalization of the bovine cation-independent mannose 6-phosphate/insulin-like growth factor-ii receptor to amino acids 24-29 of the cytoplasmic tail recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism the cytoplasmic tail of infectious bronchitis virus e protein directs golgi targeting coatomer interaction with dilysine endoplasmic reticulum retention motifs a mechanism of virus-induced demyelination copi in er/golgi and intra-golgi transport: do yeast copi mutants point the way? an endoplasmic reticulum retrieval signal partitions human foamy virus maturation to intracytoplasmic membranes antigenic relationships among proteins of bovine coronavirus, human respiratory coronavirus oc43, and mouse hepatitis coronavirus a59 coronavirus maturation characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus intracellular targeting signals contribute to localization of coronavirus spike proteins near the virus assembly site human 3-hydroxy-3-methylglutaryl coenzyme a reductase. conserved domains responsible for catalytic activity and sterol-regulated degradation a specific transmembrane domain of a coronavirus e1 glycoprotein is required for its retention in the golgi region protein sorting by tyrosine-based signals: adapting to the ys and wherefores bunyavirus protein transport and assembly analysis tool web services from the embl-ebi endocytosis and molecular sorting apical budding of a recombinant influenza a virus expressing a hemagglutinin protein with a basolateral localization signal short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum efficient selection for high-expression transfectants with a novel eukaryotic vector expression of the human immunodeficiency virus envelope glycoprotein is restricted to basolateral surfaces of polarized epithelial cells a short sequence in the cooh-terminus makes an adenovirus membrane glycoprotein a resident of the endoplasmic reticulum the targeting of lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane role of adaptor complex ap-3 in targeting wild-type and mutated cd63 to lysosomes a novel sorting signal for intracellular localization is present in the s protein of a porcine coronavirus but absent from severe acute respiratory syndromeassociated coronavirus a golgi retention signal in a membrane-spanning domain of coronavirus e1 protein signal-mediated sorting of membrane proteins between the endoplasmic reticulum and the golgi apparatus sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-golgi network of att20 cells biosynthesis and function of the coronavirus spike protein the spike protein of infectious bronchitis virus is retained intracellularly by a tyrosine motif ms received 02 saumitra das this work was supported by the national multiple sclerosis society grant rg3774a2-11 and research grant (bt/ pr14260/med/30/437/2010) from department of biotechnology, india, to jds. we thank iiser kolkata for providing dst inspire support to js, and university grant commission, india, for providing support to ms. key: cord-276966-wmelyonk authors: roe, kevin title: a proposed treatment for pathogenic enveloped viruses having high rates of mutation or replication date: 2020-07-08 journal: scand j immunol doi: 10.1111/sji.12928 sha: doc_id: 276966 cord_uid: wmelyonk several enveloped viruses, particularly some rna viruses, have high rates of mutation or replication, which can make them virulent pathogens in humans and other mammals. a proposed treatment could use synthesized proteins to mask pathogenic viral surface proteins to quickly induce an immune attack on specific enveloped viruses by using existing immune cells. one treatment could inject dual‐protein ligand masks into patients' blood streams to mask pathogenic surface proteins used to infect mammalian cells. the mammalian immune system already uses an analogous, more complex structure called a pentraxin to neutralize some pathogens by connecting their surface proteins to immune cells. and several types of antiviral peptides have already experimentally demonstrated effectiveness in blocking various viral pathogen infections. these treatments offer advantages, especially for currently untreatable viral pathogens. furthermore, using dual‐protein ligands and the antigenic memory of some subpopulations of nk cells would also allow the creation of defacto vaccines based on a host's nk cells, instead of vaccines utilizing cd4 and cd8 α:β t cells, which are limited by the requirement of mhc presentation of the target antigens to α:β t cells. targeted nk cell vaccines could attack host cells latently or actively infected by intracellular pathogens, even host cells having pathogen downregulated mhc antigen presentation. eight postulates concerning the effects of pathogen mutation, or change in phenotype from genetic recombination or rearrangement, and replication rates on pathogen versus host dominance are also listed, which should be applicable to viral and non‐viral pathogens. every human has barrier surfaces to suppress viral infections through the epithelium of the respiratory system and the gastrointestinal tract, the endothelium of blood vessels, placental barrier tropoblasts, and skin. 1 the 2018b.v2 report of the international committee on taxonomy of viruses (ictv) lists 1019 viral genera and 5560 viral species. 2 but only a small percentage are pathogenic to humans, and presently 155 viral genera are known to cause human viral diseases. 3 yet despite anti-viral medicines and vaccines, pathogenic viruses infect millions of children and adults yearly world-wide. [1] [2] [3] [4] even though some viral infections can be treated or at least avoided by vaccination, for many of the untreatable viral infections, a large fraction of the survivors frequently suffer mild to severe life-long impairments. for example, the eastern equine encephalitis (eee) virus, endemic to the western hemisphere, has no human vaccine or even a treatment, and has been called the most deadly mosquito-borne pathogen in north america, because it can kill 35% to 75% of infected humans depending on the medical care received, and an estimated 35% to 50% of the survivors suffer severe and permanent neurological brain damage. [5] [6] this makes the need for a eee virus vaccine obvious, but the only vaccine currently available is for horses, not humans. 6 another untreatable and lethal virus, transmissible by bodily secretions of humans and other mammals, and even considered fully capable of a world-wide pandemic spread after mutation, is the nipah virus, with a mortality rate ranging from 72% to 86% in the indian subcontinent. 7-10 peptide inhibitors against the nipah virus have been designed and modeled to target and inhibit interacting sites on the viral attachment g receptor, the f fusion protein trimer used to fuse the viral envelope with the host cell membrane, and the m matrix protein dimer used for initiating the budding of the virus; and the bonding stabilities of various antiviral peptide inhibitors were assessed with molecular dynamics (md) simulations. 11 but at this time, there is still no vaccine and not even a treatment for humans. this article is protected by copyright. all rights reserved the two preceding viruses illustrate the virulence of some viral pathogens after transmittal to humans, but some extremely virulent viruses can have both high rates of mutation and high rates of replication. some dna viruses, and particularly several rna viruses, have high mutation rates, which can be expressed as nucleotide substitutions per nucleotide per cell infection (s/n/c); and these mutation rates typically range from 10 -8 to 10 -6 s/n/c for dna viruses to 10 -6 to 10 -4 s/n/c for rna viruses, although nucleotide insertions and deletions can also make a smaller contribution to the overall mutation rate. 12 and independently from mutation rates, some viral pathogens can have a high replication rate among host cells, especially after transmission to secondary hosts of other species, such as viruses that originally evolved high replication rates while they infected animals such as bats and were thus selected by the fast immune responses of bats. 13 [13] [14] and it will obviously be quite challenging to treat virulent viruses that have both high mutation rates and high replication rates. however, there are several known antiviral peptides that can inhibit or block viral infections, and some of them could potentially treat infections by virulent enveloped rna viruses having high rates of mutation and replication. [15] [16] there are even databases of experimentally verified antiviral peptides, typically derived from micro-organisms. 16 these antiviral peptides are members of the larger group of antimicrobial peptides that contribute to the innate immune response of many species, and they are known to act either directly or by creating an immune response. 17 several antiviral peptides have been experimentally demonstrated to be effective against different viruses, even in serum solutions with micro-molar concentrations, and their actions typically include blocking one or more stages of a virus's infection cycle; such as host cell attachment, host cell entry, replication inside a host cell, transcription, translation, maturation, or release from a host cell. [18] [19] this paper is focused on antiviral applications of custom designed dual-protein ligand masks that can be synthesized by conventional recombinant dna biotechnology, and these ligand masks are accepted article theoretical and functional analog extensions of pentraxins, which are more complex immune structures used by the mammalian immune system to neutralize some pathogens. 20 in targeting specific viral pathogens, dual-protein ligand masks (for brevity, henceforth called dualprotein ligands) should be able to create a quick and powerful immune memory response with existing memory immune cells against some viral pathogens or virus infected cells, without some of the practical limitations of vaccines. which viral pathogens or virus infected cells are susceptible to memory t cell and memory b cells? enveloped viruses, and some non-enveloped viruses, typically have pathogenic surface proteins, such as glycoproteins, needed for viral infection of host cells. [8] [9] some of these surface proteins cannot mutate too much without losing their functionality for infection of a host cell, so critical sections of these essential surface proteins can be targeted to block viral pathogen infections. most t cell activations require that an antigen (i.e., a molecular pattern that a patient's immune system recognizes as foreign to the patient) be presented by another cell, such as a dendritic cell, on a specific surface protein known as a major histocompatibility complex (mhc), in humans this is also called a human-leukocyte-associated (hla) protein. 21 t cells predominantly are α:β t cells with the mhc requirement for antigen presentation to activate α:β t cells; whereas a different subset of t cells called γ:δ t cells have no need for any mhc presentation. 22 although activating γ:δ t cells is challenging, there are several distinct sub-populations of γ:δ t cells; while they are more abundant in mucosal tissues, sub-populations of γ:δ t cells are also found in several types of tissues and they are even found in blood. 22-25 furthermore, γ:δ t cells can be transformed into memory t cells like α:β t cells; and for γ:δ t cell sub-populations residing in various tissues, peptides of distinct proteins, such as an endothelial protein c receptor β sheet, in the presence of other co-stimulating peptides of proteins, such as icam-1 (known also as cd54) can activate their γ:δ t cell receptors. [23] [24] [25] activating the γ:δ t cell receptor and this article is protected by copyright. all rights reserved one or two co-stimulatory receptors, can activate γ:δ t cells, which can also result in antibody production by activated b cells. 26 so γ:δ t cells are the t cells referenced herein. dual-protein ligands could make specific viral pathogens targets for existing immune memory cells or innate immune cells. dual-protein ligands could induce an immune response by mimicking the key parts of antigens that activate existing immune memory cells or innate immune cells to attack tagged viral pathogens. there are significant benefits in using the immune memory system to neutralize viral pathogens. one benefit is that when memory immune cells are triggered by a dual-protein ligand antigen, the antibody response of the immune memory system will produce antibody numbers much larger than the antibody numbers released by a primary immune response, or released by long lived plasma cells resident in the bone marrow, or b cells in the respiratory and intestinal mucosal tissue. 27 other potential benefits of using dual-protein ligands include avoiding the need to use many different preventative anti-viral vaccinations, and sufficient mass bondings to pathogenic viral surface proteins could potentially minimize t cell death (lymphopenia) from too many inflammatory stimulating signaling proteins (a "cytokine storm") caused by some viral pathogens, such as the ebola virus. 28 in summary, surface proteins are used by viral pathogens to infect mammalian cells. however, dualprotein ligands could mask and block these surface proteins before the widespread viral pathogen infection of mammalian cells. a suggested treatment for humans uses a dual-protein ligand, that includes a first protein ligand that will mask (i.e., specifically bond to) a unique virus surface protein, or alternatively bond to a distinctive surface protein of a virus infected cell, and a second protein ligand that matches or mimics the section of an antigen that would activate memory immune cells, wherein the second protein ligand is connected to the first protein ligand. both the first protein and the second this article is protected by copyright. all rights reserved protein ligands can separately have their three dimensional conformations strengthened by covalent disulfide bonds (s-s bonds) formed between the sulfhydryl (-sh) groups of precisely placed cysteine amino acid residues. 29 both the bonding between the first protein ligand to a viral surface protein, and the bonding between the second protein ligand to an immune cell, could be bondings utilizing non-covalent forces, such as electrostatic forces, van der waals forces, hydrophobic forces, cation-pi interactions and hydrogen bonds. 30 some component amino acid residues can produce several bonding forces; the aromatic amino acid residue tyrosine is typical and can bond by hydrogen bonding, hydrophobic forces, and through it's side chain pi-electron system have cation-pi interactions with nearby cations. 30 in the brain or central nervous system (cns) especially, synthesis of the second protein ligand to mimic an antigen to cause an attack on virus infected cells by memory γ:δ t cells must also take into consideration the risk of excessive inflammation. there are several sub-populations of γ:δ t cells, that can be distinguished by their t cell receptor variable regions (vγ), and they can be separated into bigger classifications, such as γ:δ t1 cells and γ:δ t17 cells. 26 however, there are some classifications of γ:δ t cells, the pro-inflammatory interleukin-17 producing γ:δ t17 cells for example, that can stop certain infections, but they can also potentially create excessive inflammation and promote cancers and some auto-immune diseases. 31 other alternatives for treatments involving the cns and brain are second protein ligands that utilize the less inflammatory γ:δ t1 cells, which are relatively scarce in blood, but more common in tissues and organs, that produce interferon-γ, which activates macrophages, but this can also cause some inflammatory interleukin-6 release. 32 some potential ligands that could be mimicked by the second protein ligand to activate the γ:δ t cell receptors are listed later in this paper in the section that discusses the nk cell activating receptor nkg2d. another alternative is to synthesize a second protein that utilizes specific innate immune cells. one option is the microglia, the main resident macrophages for neurons in the cns and brain, and they and cns border-associated macrophages in general are among the innate immune cells that could be used. 33 microglia are essentially accepted article macrophages, so the second protein ligand could be synthesized to bond to one of the many distinct types of receptors expressed by macrophages -including the pattern recognition receptors (prr), such as the toll-like receptors, the fc antibody receptors, or the complement receptors such as cr3, for example. 34 the receptor structures of some macrophages and other immune cells, such as the nk cell nkg2d activating receptor, are known to some extent; but any targeted receptor structure will probably require considerable research to determine enough detailed structure to enable the design of a second protein ligand having a strong bond to the targeted immune cell receptor. the nk cell nkg2d activating receptor and the structure of its ligands are discussed in more detail later in this paper. there are several potential surface proteins on specific viral pathogens that can be targeted. for example, these surface proteins include the glycoproteins e1 and e2 on the surface of the hepatitis c virus of the hepacivirus genus. 35-36 another example is human immunodeficiency virus 1 (hiv-1), an enveloped virus of the lentivirus genus with a surface protein, a trimeric glycoprotein, to induce membrane fusion for viral entry into a human host cell, and this glycoprotein is known to be targeted by antibodies. 37 measles virus is an enveloped virus of the morbillivirus genus, with a morbillivirus surface protein complex with tetrameric attachment (h) and trimeric fusion (f) glycoproteins for viral entry and infection of human host cells. 38 eastern equine encephalitis virus is an enveloped virus of the alphavirusgenus with two trans-membrane envelope glycoproteins e1 and e2, where the surface protein e2 bonds to human cells for viral entry. [39] [40] in summary, there are several possible target viral surface proteins for bonding to dual-protein ligands for the immune system neutralization of viral pathogens. as previously discussed, the structures of some viral surface proteins are already known to some extent; but any targeted viral surface protein will probably require considerable research to determine enough detailed structure to enable the design of a first protein ligand having a strong bond to the targeted viral surface protein. this article is protected by copyright. all rights reserved one treatment option injects dual-protein ligands into the blood stream or localized regions to mask pathogenic surface proteins used by viruses to infect mammalian cells. the dosage of dual-protein ligands necessary to treat a viral pathogen infection will vary, depending on how long the dual-protein ligands will reside inside the patient before they are removed or inactivated, depending on the concentrations of the targeted virus and the immune cell utilized, and depending on how strongly each dual-protein ligand will bond with both the targeted viral surface protein and the immune cell utilized. the strength of bonding between ligands and proteins is quantified by an association constant (k a ), which is defined as the equilibrium molar concentration of a protein bound to a ligand, divided by the multiplication of the molar concentration of the unbound ligand and the molar concentration of the unbound protein, as seen in equation (1). 41 other papers use the dissociation constant (k d ), the reciprocal of the association constant k a , as seen in equation (2) as mentioned before, a different option is utilizing cells of the innate immune system (e.g., innate lymphoid cells, macrophages or other phagocytes). as a specific example, the first protein ligand would bond to either a surface protein of a viral pathogen, or bond to a surface protein of a cell infected by the viral pathogen, and the second protein ligand would be designed to strongly bond to and activate phagocytes to consume the viral pathogen or the virus infected cell, by designing the second protein ligand to bond to a surface protein of human macrophages or neutrophils, for instance the fcαri receptor (cd89). 42 this option would bypass some viral defenses against memory t cells or b cells, and would also quickly initiate an attack by innate immune system cells, in this instance by phagocytic cells. an injection of dual-protein ligands into the blood stream of patients will be easier than trying to introduce dual-protein ligands from inside the gastrointestinal tract, whether by using oral capsules or therapeutic bacteria. [43] [44] and injections into the blood stream, or injections into accepted article localized regions, can be used separately or in a combination. a combination of both treatment approaches is probably better for treating viral pathogen infections that also include the brain or cns. in such cases, a lumbar puncture through the patient's spine can safely inject the dual-protein ligands into the cerebrospinal fluid of the patient, and this is a standard procedure to deliver antibiotics directly into the brain and cns, while avoiding the blood brain barrier, a serious impediment to introducing most medicines into the brain by blood circulation. 45 these proposed treatments raise the question of whether there is any risk of creating dangerous immune system reactions by injections of dual-protein ligands. this risk is small, because as discussed in an earlier paper, by themselves most pure proteins and peptides rarely induce an immune response. 46 this is one motivation to combine a chemical adjuvant with many vaccinations to make their antigens immunogenic to induce a strong immune response, and influence the antibody titer and isotype and increase cell-mediated responses. 47 however, since memory t cells and memory b cells already are activated effector cells, dual-protein ligand injections should be able to activate these targeted memory cells without requiring any adjuvants combined with the injections. but including certain cytokines can be helpful; cytokine therapies using type i interferons and interleukin-2 have been approved for some cancer treatments; and specific cytokines help nk cell receptor activation, which will be discussed in more detail later. 48 it should be possible to synthesize dual-protein ligands in advance in the quantities needed to stop viral pathogens at relatively low cost. the modification of bacterial genomes, such as the genome of escherichia coli, using restriction enzymes can induce expression of specific and desirable peptides and proteins, and this is a very mature and long commercialized technology. 49 thus, recombinant dna techniques, applied to one of the commercially available strains of bacteria, could make dualprotein ligands. protein ligands having an equivalent number of amino acid residues, this implies that entire dualprotein ligands could be synthesized with several hundred amino acid residues, in some cases with as little as 600 amino acid residues, possibly including a short section between the first protein ligand to the second protein ligand to provide more flexibility. each protein ligand only needs to be long enough to provide a strong bond with its respective target, and each protein ligand may need conformal stabilization provided by disulfide bonds between precisely placed cysteine amino acid residues within each protein ligand. one last question concerns the therapeutic duration of the dual-protein ligands in the blood stream of a mammal. one significant factor determining the duration of a dual-protein ligand will be the presence and concentrations of pathogen and mammalian proteolytic enzymes, also known as proteases or peptidases, having nucleophilic active sites, that typically cleave peptide bonds by hydrolysis, such as at the carbonyl (c=o) of the peptide bond. 59 any peptide segment of the dualprotein ligand is a potential peptidase target, but the peptide segment in the linkage of the two protein ligands of a dual-protein ligand will be particularly accessible to peptidases. fortunately, many peptidases also have known inhibitors, and carefully chosen specific inhibitors for the most inconvenient peptidases could also be injected with the dual-protein ligands to prevent or slow peptidase attacks, preferably without causing major disruption to the normal physiological functions of mammalian peptidases. the most appropriate inhibitor for a peptidase can be found in an extensive database of peptidases, their substrates and their this article is protected by copyright. all rights reserved inhibitors, such as the merops database. 60 as of september 2017, there were 5267 peptidase identifiers and 868 inhibitor identifiers listed in the merops 12.0 database, along with the peptidase's substrate. 60 eight postulates concerning the effects of pathogen mutation, pathogen change in phenotype by genetic recombination and pathogen replication rates on pathogen versus host dominance are listed below and should be applicable not only to viral pathogens, but also applicable to bacterial, fungal, and protozoan pathogens. the term "pathogen strain" as used below is defined as a sub-species or sub-type of a pathogen species. the term "dominate" as used below is defined as survive, or at least maintain an infection, where a pathogen strain possibly, but not necessarily, could kill its host; whereas when a host "dominates" a pathogen strain, it may, or may not, be able to eliminate the pathogen strain. the term "mutation" as used below is defined as random changes in one or more genetic nucleotides, by substitution, deletion or insertion of nucleotides, etc. the term "beneficial mutation" as used below is defined as a mutation that helps the pathogen strain, against the host or against other pathogen strains. a "beneficial change in phenotype" as used below is also defined as helping the pathogen. the term "genotype" as used below is defined as the entire genome of a pathogen strain, and the term "phenotype" as used below is defined as the characteristics of a pathogen strain, expressed as the interaction of a pathogen strain's genotype with its age, its various conditions of activity or latency, or its environment. it should be noted that a pathogen's phenotype can also change as a result of genetic recombination or rearrangement (called "genetic recombination" for brevity henceforth), either randomly or by programming, recombining or rearranging groups of nucleotides within a single pathogen strain or from multiple pathogen strains; such as a change in viral phenotype after random genetic recombination during co-infection of a host cell by two different viral strains. 61 one specific example of a viral change in phenotype would be the antigenic shift in one or more antigenic determinants of an influenza virus resulting from the genetic recombination of various bird and mammalian influenza virus strains. 61 a beneficial mutation for a pathogen strain could be a higher rate of replication, an improvement in its infectivity or the transmission of the pathogen strain, an improvement in reducing environmental, resource or metabolic requirements for the survival, replication or spread of the pathogen strain, an improvement in evading or overcoming the immune defense of a host by changing one or more antigenic determinants, and so forth. and as previously discussed, a beneficial change in phenotype by genetic recombination of a pathogen strain can possibly result in an antigenic shift in one or more antigenic determinants that were essential to the host's immune system for recognition and defense against the pathogen strain. examples of how the effectiveness of host defenses could be improved include an improved blocking of the infectivity of the pathogen, or an improvement in targeting, accessing or in neutralizing the pathogen, such as provided by adaptive immune system immunoglobulin antibody isotype switching or somatic hypermutation and affinity selection of antibodies against the pathogen, producing more effective cytokines, activating other more effective innate or adaptive immune cells, and so forth. 27, 62 introduction of medicines or treatments are an alternative equivalent means to improve the effectiveness of the host defenses. a host can replicate effective defenses include increasing the number of effective antibodies, increasing the number of innate or adaptive immune cells, increasing the number of effective this article is protected by copyright. all rights reserved cytokines, increasing the number of activated immune cells, and so forth. introduction of medicines or treatments are an alternative equivalent means to replicate host defenses faster. recombination against a first host's immune defenses faster than its first host can improve the effectiveness of its immune defenses will dominate its first host, and after transmission to a second host it will be enabled to more virulently dominate a second host individual or species having a slower or weaker immune defense, which otherwise provides an equivalent host environment for the pathogen strain. host environment for the pathogen strain. can beneficially mutate or beneficially change its phenotype by genetic recombination against the host's immune defenses can eventually dominate the pathogen. replicate can eventually dominate the pathogen. this article is protected by copyright. all rights reserved these eight postulates summarize the effects of pathogen mutation, pathogen change in phenotype by genetic recombination, and pathogen replication rates on pathogen versus host dominance and their scope is limited to pathogen versus host dominance, but these postulates should be applicable to viral pathogens, and also applicable to bacterial, fungal, and protozoan pathogens. the host could be a mammal, but could possibly be non-mammalian, or even outside of the animal kingdom, since plants also struggle for dominance over several types of pathogens. targeted dual-protein ligands could mask viral surface proteins to quickly treat some untreatable virus infections by using already existing immune cells. one treatment uses injection of the dual-protein ligands into the blood of patients, and another treatment injects the dual-protein ligands into less accessible viral pathogen infections, such as the brain or central nervous system, to bond to a viral surface protein used to infect mammalian cells. these treatments could have advantages, especially for enveloped rna viruses having high rates of mutation or replication, although the initial development and implementation of these new treatment approaches will require substantial resources. furthermore, using dual-protein ligands and the antigenic memory of some subpopulations of nk cells would also allow the creation of defacto vaccines based on a host's nk cells, instead of vaccines based on α:β t cells, which are limited by the requirement of mhc presentation of the target antigens to α:β t cells. targeted nk cell vaccines could attack host cells latently or actively infected by intracellular pathogens, even host cells having less mhc antigen presentation capabilities, such as neurons. eight postulates concerning the effects of pathogen mutation, or change in phenotype from genetic recombination or rearrangement, and replication rates on pathogen versus host dominance have also been listed, which should be applicable to viral and non-viral pathogens. the author confirms that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. no ethical approval was required as this is an article with no original research data. data sharing is not applicable to this article as no new data were created or analyzed in this study. the author attests that he conceived the paper, wrote the paper, and approved the final version of the manuscript, and attests that he meets all of the icmje criteria for authorship. type iii interferons in antiviral defenses at barrier surfaces international committee on taxonomy of viruses (ictv) global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young childer in 2015: a systematic review and modelling study eastern equine encephalitis virus -old enemy heparan sulfate binding by natural eastern equine encephalitis viruses promotes neuro-virulence morbidity and mortality due to nipah or nipah-like virus encephalitis in who south-east asia region world health organization nipah virus infection. world health organization zoonotic potential of emerging paramyxoviruses: knowns and unknowns accepted article this article is protected by copyright. all rights reserved 11 viral mutation rates accelerated viral dynamics in bat cell lines, with implications for zoonotic emergence appendix -structure, genome organization, and infectious cycles viral escape from endosomes and host detection at a glance avpdb: a database of experimentally validated antiviral peptides targeting medically important viruses the gamma interferon(ifn-gamma) mimetic peptide ifngamma (95-133) prevents encephalomyocarditis virus infection both in tissue culture and in mice hipdb: a database of experimentally validated hiv inhibiting peptides avppred: collection and prediction of highly effective antiviral peptides pentraxins in the activation and regulation of innate immunity protective immunity against hepatitis c: many shades of gray human vδ2 versus non-vδ2 γδ t cells in antitumor immunity tissue adaptations of memory and tissue-resident gamma delta t cells cytomegalovirus and tumor stress surveillance by binding of a human γδ t cell antigen receptor to endothelial protein c receptor endothelial cell protein c receptor-dependent signaling six-of-the-best: unique contributions of γδ t cells to immunology ige repertoire and immunological memory: compartmental regulation and antibody function disabling of lymphocyte immune response by ebola virus recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins antigen recognition by b-cell and t-cell receptors t cell receptor signaling for γδ t cell development gamma-delta γδ t cells: friend or foe in cancer development harnessing microglia and macrophages for the treatment of glioblastoma innate immunity: the induced response to infection role of hepatitis c virus envelope glycoprotein e1 in virus entry and assembly identification of novel functions for hepatitis c virus envelope glycoprotein e1 in virus entry and assembly conformational states of a soluble, uncleaved hiv-1 envelope trimer measles virus fusion protein: structure, function, and inhibition cryo-em structures of eastern equine encephalitis virus reveal mechanisms of virus disassembly and antibody neutralization protective antibodies against eastern equine encephalitis virus bind to epitopes 2 in domains a and b of the e2 glycoprotein antigen-antibody interactions: principles and applications the humoral immune response stopping untreatable pathogen infections using peptide ligands to sabotage pathogenic cell surface proteins dual-peptide ligand masks: a proposed treatment approach to stop prion disease dementias penetration of drugs through the blood-cerebrospinal fluid/bloodbrain barrier for treatment of central nervous system infections accepted article this article is protected by copyright. all rights reserved 46. roe, k. a proposed treatment approach to treat lethal mutating cancers effects of adjuvants on the humoral immune response to congopain in mice and cattle cytokines in the treatment of cancer construction of biologically functional bacterial plasmids in vitro toxoplasma gondii infection drives conversion of nk cells into ilc1-like cells the "less-is-more" in therapeutic antibodies: afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity redefining memory: building the case for adaptive nk cells hiv-1 nef: taking control of protein trafficking two human ulbp/raet1 molecules with transmembrane regions are ligands for nkg2d γδ t cells in cancer: a small population of lymphocytes with big implications the global cysteine peptidase landscape in parasites the merops database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the panther database influenza virus evolution, host adaptation and pandemic formation innate immunity this article is protected by copyright. all rights reserved key: cord-290088-g9559ux3 authors: loh, hwei-san; green, brian j; yusibov, vidadi title: using transgenic plants and modified plant viruses for the development of treatments for human diseases date: 2017-08-08 journal: curr opin virol doi: 10.1016/j.coviro.2017.07.019 sha: doc_id: 290088 cord_uid: g9559ux3 production of proteins in plants for human health applications has become an attractive strategy attributed by their potentials for low-cost production, increased safety due to the lack of human or animal pathogens, scalability and ability to produce complex proteins. a major milestone for plant-based protein production for use in human health was achieved when protalix biotherapeutics produced taliglucerase alfa (elelyso(®)) in suspension cultures of a transgenic carrot cell line for the treatment of patients with gaucher's disease, was approved by the usa food and drug administration in 2012. in this review, we are highlighting various approaches for plant-based production of proteins and recent progress in the development of plant-made therapeutics and biologics for the prevention and treatment of human diseases. hwei-san loh 1,2 , brian j green 3 production of proteins in plants for human health applications has become an attractive strategy attributed by their potentials for low-cost production, increased safety due to the lack of human or animal pathogens, scalability and ability to produce complex proteins. a major milestone for plant-based protein production for use in human health was achieved when protalix biotherapeutics produced taliglucerase alfa (elelyso 1 ) in suspension cultures of a transgenic carrot cell line for the treatment of patients with gaucher's disease, was approved by the usa food and drug administration in 2012. in this review, we are highlighting various approaches for plant-based production of proteins and recent progress in the development of plant-made therapeutics and biologics for the prevention and treatment of human diseases. infectious diseases remain as one of the leading causes of mortality and morbidity in developing countries and are exacerbated by the lack of resources and infrastructure to prevent, treat and control diseases. therefore, emerging and re-emerging pathogens have frequently resulted in epidemics in these countries. over the past several decades, production of proteins in plants has been shown to be a promising approach for the manufacture of targets for human health applications. plants, when compared to other production systems, offer some advantages, including ease of scaling and lack of human and animal pathogens [1] [2] [3] (table 1) . this review focuses on several approaches that have been used to produce proteins in plants for prophylactic and therapeutic applications to combat human disease conditions. the various approaches for plant-based production of proteins are illustrated in figure 1 . stable nuclear and chloroplast transformations are the two approaches utilized to express heterologous recombinant proteins in plants. agrobacterium-mediated stable transformation has a long history in plant genetic manipulation, and is achieved by stable integration of t-dna into plant nuclear genome [4] . however, the approach is time consuming, with a lead time ranging from 12 to 18 months and typically has low levels of the target protein expressed [5] . stable introduction of target genes into chloroplast genome, that is, chloroplast transformation or transplastomics, however, allows for higher levels of target expression as compared to nuclear transformation, largely due to the lack of gene silencing and high gene copy number [6] , but it is technically difficult, lacks most post-translational modifications and has only been successful in a limited number of plant species. transient expression of target proteins in plants using modified plant viruses or viral vectors integrated into binary vectors delivered via agrobacterium [7, 8 ] is often considered a more robust approach when compared to stable transformation, due to its rapid production capabilities and relatively high protein expression [8 ] . the majority of plant viral vectors used to date are based on single-stranded rna viruses, such as tobacco mosaic virus, potato virus x and cowpea mosaic virus (cpmv), which encode for at least three proteins with functions in viral replication (replicase), encapsidation (coat protein) and movement from cell-to-cell (movement protein) [9]. the initial strategy involved production of recombinant proteins using plant viruses by exploiting their natural ability to infect (full virus) plants. however, this approach generally failed due to instability of viral genome modified by the introduction of large target genes [7] . this issue was largely resolved by using agrobacterium-mediated gene delivery or agroinfiltration. the target gene can either be directly cloned into an agrobacterium vector or through a modified plant viral vector which has been integrated into an agrobacterium binary plasmid, and delivered into the plant tissues by infiltration with the transformed agrobacterium [7, 8 ] . agroinfiltration allows for high levels of target protein expression with the table 1 general comparison of expression hosts for the production of heterologous proteins for medical and pharmaceutical applications potential for cost-effective production [5, 10] . the peak protein expression is typically observed in less than 7 days postinfiltration which is significantly faster when compared to the full virus strategy which requires more than 2 weeks in order to generate a systemic infection for expression. the promise of this platform has been evidenced in numerous successful clinical trials, which demonstrated safety and efficacy of plant-made protein therapeutics and biologics [11 ] . for example, in responding to the h1n1 influenza virus pandemic that occurred in 2009, medicago, a canadian company, reported producing the vaccine candidate, hemagglutinin in 19 days in nicotiana benthamiana [10] . as such, agroinfiltration provides a rapid response capability and is currently the preferred approach for the production of proteins in plants. modified plant viruses for treatment of human diseases loh, green and yusibov 83 detection of serum igg in immunized mice (sc) and fecal iga in immunized mice via oral administration. [33 ] hepatitis b virus (hbv) small surface antigen (s-hbsag) evlp vaccine against hbv lactuca sativa (lettuce)/transgenic (nuclear) detection of serum igg in immunized mice via oral administration. [34] hbv surface antigen (hbsag) suv against hbv solanum tuberosum (potato)/transgenic (nuclear) induction of serum antibodies and stable immunological memory in immunized mice fed with transgenic potato tubers. [35] human immunodeficiency virus (hiv) gp120 multi-epitopic envelope protein (c4(v3)6) lettuce/transgenic (nuclear) detection of cell-mediated and humoral immunities in immunized mice via oral administration. [36] hiv gp120 and gp41 multi-epitopic envelope proteins (multi-hiv) tobacco/transgenic (chloroplast) detection of antibody and cellular responses as well as specific ifn-g production in immunized mice via oral administration. [37] hiv-1 envelope proteins (gag/dgp41) evlps vaccine against hiv-1 purified ctb-ex4 increased level of insulin secretion from pancreatic cells. oral feeding of lyophilized ctb-ex4 lowered blood glucose level in mice. [55] keys for abbreviations: ade, antibody-dependent enhancement; c h , constant domains of immunoglobulin heavy chain; c l , constant domain of immunoglobulin light chain; ctb, cholera toxin b; cvlp, chimeric virus-like particle; diii, domain iii; dpp, dipeptidyl peptidase; e, envelope; evlp, enveloped virus-like particle; ex, exendin; f, coagulation factor; gaa, acid alpha glucosidase; glp, glucagon like peptide; gp, glycoprotein; ha, hemagglutinin; hi, hemagglutination-inhibition; hc, heavy chain; ig, immunoglobulin; ltb, heat-labile enterotoxin b subunit; im, intramuscular; in, intranasal; ip, intraperitoneal; iv, intravenous; mab, monoclonal antibody; n, nucleocapsid; pa, protective antigen; rtb, ricin toxin b; sag, surface antigen; sc, subcutaneous; scfv, single-chain variable fragment of immunoglobulin; suv, subunit vaccine; vlp, virus-like particle. numerous examples of plant-produced proteins targeting prophylactic and therapeutic applications (subsectioned as vaccines, antibodies and other biopharmaceuticals) in preclinical development are shown in table 2 . several lead candidates have gone through clinical trials (table 3) and have been comprehensively reviewed [12,13 ]. vaccines are highly effective tools for the prevention of infections. over the last three decades, plant-produced antigens targeting various pathogens have been shown to be effective in animal models ( table 2) . several of these candidates have progressed into early stage clinical development and were evaluated in phase 1-2 human clinical trials ( (table 3) . planet biotechnology (hayward, ca) produced the world's first plant-derived clinically tested secretory iga monoclonal antibody which recognizes the surface antigen i/ii of streptococcus mutans (carorx tm ) that predominantly causes dental caries. following the successful demonstration of safety and efficacy in a phase 2 clinical trial, carorx tm has been licensed in europe in a medical device category [17, 18] and applied as an oral topical solution to prevent tooth decay. in 1986, the recombinant human growth hormone was the first plant-based biopharmaceutical protein produced in plants [19] . then over two decades later, the fda in may 2012 approved elelyso 1 (human recombinant taliglucerase alfa or glucocerebrosidase), an enzyme produced in genetically engineered carrot cells for treating type 1 gaucher's disease (gd) by protalix biotherapeutics and its partner, pfizer [20] . gd is a lysosomal storage disorder caused by a hereditary deficiency of the enzyme, glucocerebrosidase (gcd). gd is currently treated by enzyme replacement therapy using this recombinant gcd that is administered intravenously every 2 weeks [21] . in addition to offering a versatile production platform for numerous plant-made proteins, plant viruses have been engineered to provide medical applications in other ways [22] . vlps offer advantages over recombinant protein vaccines as they tend to elicit a higher immune response [23] . virus nanoparticles have also been developed for the targeted delivery for disease treatment and diagnostic purposes. for example, cpmv represents an icosahedral nanoparticle with its capsid surface displaying 300 accessible lysine residues; each of these can be conjugated to various chemical moieties like fluorescent dyes/arrays, polyethylene glycol polymers and subcellular targeting molecules [24, 25] . the use of this technology includes 86 engineering for viral resistance the construction of cpmv nanoparticles displaying gastrin-releasing peptide receptors that are overexpressed in human prostate cancers [26] . another example, cowpea chlorotic mottle virus can stably assemble in vitro and package the rna derived from sindbis virus, a mammalian virus. these hybrid cowpea chlorotic mottle virusbased vlps were shown to protect against rna degradation by cellular nucleases and were able to deliver and release their rna contents within the cytoplasm of mammalian cells. moreover, these hybrid vlps with the fusion of subcellular targeting moieties could be directed toward distinct sites within the cell [27] and potentially applied as a medical targeted delivery tool. plant viruses have also been engineered to act as adjuvants to elicit an immune response that is more potent and effective. the rodshaped papaya mosaic virus nanoparticles have been engineered to express an influenza epitope on their surface, and mice and ferrets immunized with these recombinant nanoparticles exhibited an increase in robust humoral response to influenza virus infection [28] . there is growing evidence that plants are capable of making proteins with desired quality to address a range of human health-related issues. plant production platforms for protein therapeutics and biologics, in particular the transient agroinfiltration approach, have demonstrated the ability to be used for broad research and development, as well as commercial needs. it has been extensively discussed that the transient agroinfiltration approach is the ideal platform for fast and scalable production in response to new outbreaks of highly infectious diseases and has been demonstrated under various programs. the success of protalix biotherapeutics in gaining fda approval for the therapeutic enzyme, elelyso 1 for human use was a significant milestone for the plant molecular pharming field. more importantly, the primary benefits of plant-made protein therapeutics and biologics in terms of product safety and potential cost-effectiveness will further contribute to global public health in both developed and developing nations. yao j, weng yq, dickey a, wang yj: plants as factories for human pharmaceuticals: applications and challenges. int j mol sci 2015, 16:28549-28565. this paper illustrates the plant molecular farming or pharming concept in relation to human pharmaceutical applications. several types of plantbased production platforms are described. the challenges such as planttype glycosylations, downstream bioprocesses and biosafety concerns are also discussed. besides, some of the preclinical and clinical studies that have been enlisted in our review paper are elaborated here. the production of recombinant pharmaceutical proteins in plants production of vaccines and therapeutic antibodies for veterinary applications in transgenic plants: an overview comparative evaluation of recombinant protein production in different biofactories: the green perspective the integration of t-dna into plant genome magnifection -a new platform for expressing recombinant vaccines in plants engineering the chloroplast genome for hyperexpression of human therapeutic proteins and vaccine antigens a launch vector for the production of vaccine antigens in plants. influenza other respir viruses production of secretory iga antibodies in plants production of antibodies in plants: status after twenty years the expression of a nopaline synthase -human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue first plant-made biologic approved plant-based oral delivery of b-glucocerebrosidase as an enzyme replacement therapy for gaucher's disease plant virus expression vector development: new perspectives virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development plant viral capsids as nanobuilding blocks: construction of arrays on solid supports cowpea mosaic virus nanoparticles target surface vimentin on cancer cells intravital imaging of human prostate cancer using viral nanoparticles targeted to gastrinreleasing peptide receptors reconstituted plant viral capsids can release genes to mammalian cells improvement of the trivalent inactivated flu vaccine using papmv nanoparticles a plantproduced protective antigen vaccine confers protection in rabbits against a lethal aerosolized challenge with bacillus anthracis ames spores. hum vaccines immunother generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine novel vaccination approach for dengue infection based on recombinant immune complex universal platform expression of an immunogenic ebola immune complex in nicotiana benthamiana expression of an immunogenic ltb-based chimeric protein targeting zaire ebolavirus epitopes from gp1 in plant cells brief background about ebola virus is mentioned in this paper. development of plant-based vaccine against ebola virus is in fact scarcely reported till date. the authors have demonstrated the successful expression of the chimeric protein, ltb-ebov in plant cells and its immunogenicity in balb/c mice via subcutaneous and oral administrations freeze-drying of plant tissue containing hbv surface antigen for the oral vaccine against hepatitis b study of the immunogenicity of hepatitis b surface antigen synthesized in transgenic potato plants with increased biosafety immunogenic properties of a lettuce-derived c4 (v3)6 multiepitopic hiv protein a plantderived multi-hiv antigen induces broad immune responses in orally immunized mice immunological characterization of plant-based hiv-1 gag/dgp41 virus-like particles transgenic tobacco expressed hpv16-l1 and lt-b combined immunization induces strong mucosal and systemic immune responses in mice immunization with an hpv-16 l1-based chimeric virus-like particle containing hpv-16 e6 and e7 epitopes elicits long-lasting prophylactic and therapeutic efficacy in an hpv-16 tumor mice model a plant produced h1n1 trimeric hemagglutinin protects mice from a lethal influenza virus challenge immunogenicity of h1n1 influenza virus-like particles produced in nicotiana benthamiana. hum vaccines immunother the authors have developed the recombinant hemagglutinin from the a/ california/04/09 strain of h1n1 influenza a virus in the form of enveloped vlps (hac-vlps) in plants. hac-vlps resemble the influenza a virus by morphology expression of h3n2 nucleoprotein in maize seeds and immunogenicity in mice purification and immunogenicity of hemagglutinin from highly pathogenic avian influenza virus h5n1 expressed in nicotiana benthamiana expression of rabies glycoprotein and ricin toxin b chain (rgp-rtb) fusion protein in tomato hairy roots: a step towards oral vaccination for rabies antigen production in plant to tackle infectious diseases flare up: the case of sars a non-glycosylated, plant-produced human monoclonal antibody against anthrax protective antigen protects mice and non-human primates from b. anthracis spore challenge. hum vaccines therapeutic intervention of ebola virus infection in rhesus macaques with the mb-003 monoclonal antibody cocktail structural and functional characterization of an anti-west nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants generation and analysis of novel plantderived antibody based therapeutic molecules against west nile virus a plant produced antigen elicits potent immune responses against west nile virus in mice suppression of inhibitor formation against fviii in a murine model of hemophilia a by oral delivery of antigens bioencapsulated in plant cells low cost industrial production of coagulation factor ix bioencapsulated in lettuce cells for oral tolerance induction in hemophilia b oral delivery of acid alpha glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of pompe mice the authors have demonstrated the successful expression of acid alpha glucosidase (gaa) in fusion with a transmucosal carrier, cholera toxin b in chloroplasts. by using oral administration of gaa bioencapsulated in plant cells, an induction of oral tolerance and significant suppression of gaa-specific inhibitory antibody (which will jeopardize the enzyme replacement therapy) were evidenced in pompe mice oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-tc6 cells the authors would like to thank dr stephen streatfield (fhcmb) for editorial assistance. the authors declare no conflict of interest.papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest key: cord-298242-iuskpoug authors: yu, alvin; pak, alexander j.; he, peng; monje-galvan, viviana; casalino, lorenzo; gaieb, zied; dommer, abigail c.; amaro, rommie e.; voth, gregory a. title: a multiscale coarse-grained model of the sars-cov-2 virion date: 2020-10-02 journal: biorxiv doi: 10.1101/2020.10.02.323915 sha: doc_id: 298242 cord_uid: iuskpoug the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the causative agent of the covid-19 pandemic. computer simulations of complete viral particles can provide theoretical insights into large-scale viral processes including assembly, budding, egress, entry, and fusion. detailed atomistic simulations, however, are constrained to shorter timescales and require billion-atom simulations for these processes. here, we report the current status and on-going development of a largely “bottom-up” coarse-grained (cg) model of the sars-cov-2 virion. structural data from a combination of cryo-electron microscopy (cryo-em), x-ray crystallography, and computational predictions were used to build molecular models of structural sars-cov-2 proteins, which were then assembled into a complete virion model. we describe how cg molecular interactions can be derived from all-atom simulations, how viral behavior difficult to capture in atomistic simulations can be incorporated into the cg models, and how the cg models can be iteratively improved as new data becomes publicly available. our initial cg model and the detailed methods presented are intended to serve as a resource for researchers working on covid-19 who are interested in performing multiscale simulations of the sars-cov-2 virion. significance statement this study reports the construction of a molecular model for the sars-cov-2 virion and details our multiscale approach towards model refinement. the resulting model and methods can be applied to and enable the simulation of sars-cov-2 virions. the onset of the global covid-19 pandemic has brought intense investigation into the molecular components of sars-cov-2 encoded by the virus's 30 kilobase (kb) genome. structural biology efforts using cryo-electron microscopy (cryo-em) and x-ray crystallographic techniques are currently reporting new structures of viral proteins every week (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) , and computational structure prediction efforts are targeting unresolved sections of the genome using a variety of protein folding algorithms. computational and experimental studies are underway to find new molecular therapeutics that can inhibit viral activity or further elucidate the mechanisms of action of sars-cov-2 proteins (13) (14) (15) (16) . the computer simulation of large-scale sars-cov-2 processes, such as virion assembly, budding, entry, and fusion, will remain intrinsically challenging to investigate using all-atom (aa) molecular dynamics (md), owing to the computational cost of meaningfully simulating the hundred of millions to billions of atoms involved. a holistic model of the sars-cov-2 virion can provide insight into the mechanisms of large-scale viral processes and the collective behavior of macromolecules involved in viral replication and infectivity. sars-cov-2 virions contain four main structural proteins: the spike (s), membrane (m), nucleocapsid (n), and envelope (e) proteins (17) . s proteins are glycosylated trimers that mediate fusion and entry, in part by attaching enclosed fusion peptide sequences into the membranes of host cells (18) . m proteins appear as dimeric complexes embedded within the virion envelope, and are believed to anchor ribonucleoprotein complexes to the envelope (19, 20) . n proteins associate with and organize rna into ribonucleoprotein structures found in the interior of virions (21, 22) . lastly, e proteins are thought to form pentameric ion channels that are found at the lipid bilayers of virion membranes and contribute to viral budding (23) . in this paper, we construct a largely "bottom-up" coarse-grained (cg) model of the sars-cov-2 virion from the currently available structural and atomistic simulation data on sars-cov-2 proteins. in general, this model serves as a resource for researchers working on covid19 , and as a platform to incorporate computational and experimental data. this model also enables the multiscale study of sars-cov-2 processes for the development of treatment and prevention strategies against covid-19. atomistic trajectory and experimental structural data deposited in the nsf molecular sciences software institute (molssi) will be incorporated, as they become publicly available (24) . in this work, we detail several of our cg methods used to iteratively develop a cg model for the full sars-cov-2 virion, in which molecular interactions between cg particles are derived using a combination of phenomenological, experimental, and atomistic simulation approaches. building models from structural data we first constructed atomic models of the structural proteins of the sars-cov-2 virion, ( figure 1 ). aa models of the open and closed state of the s protein were built based on the cryo-em structures of the spike ectodomain (pdb id: 6vyb, 6vxx) (5), respectively, and atomic models of the n protein were constructed based on the x-ray crystallographic structure of the nucleocapsid ntd (pdb id: 6m3m) (25) . glycosylation sites were modeled using glycan reader & modeler in charmm-gui (26) and the site-specific glycoprofile derived from mass spectrometry and cryo-em analysis (27, 28) . homology models for the s protein stalk, including the hr2 and tm domains were assembled as α-helical trimeric bundles using modeller (29) on the basis of secondary structure assignments in jpred4 (30) . homology models for the sars-cov-2 n protein ctd were created from the x-ray crystallographic structure of the sars-cov n protein ctd (pdb id: 2cjr) (31) . missing amino acid backbones in loop regions were built in modeller, and side chain rotamers were built using scwrl4 (32) . for the m protein dimer and the pentameric e ion channel, we used atomic models derived in the recent critical assessment of structure prediction (casp) commons competition (33, 34) . aa protein models (see discussion below) were subsequently simulated and coarsegrained to generate the cg models ( figure 2 and see sections below). a previously developed cg model for lipids was used, consisting of three cg beads per lipid and distinct bead types for lipid head groups and hydrophobic tails (35) . a single component cg lipid bilayer was generated in a spherical configuration and equilibrated using cg molecular dynamics simulations under constant nvt conditions in lammps (36) . we note that in the future more complex cg lipid models (37) can be added. transmembrane segments of component membrane proteins were visually identified and assigned based on secondary structure motifs. individual lipids on the outer leaflet of the spherical bilayer were randomly selected and used as initial positions for embedding spike, membrane, and envelope proteins. for each initial position, the center-of-mass of the transmembrane domain was aligned with the center of the lipid bilayer, and the principal axis of the protein was aligned with the vector normal to the lipid bilayer. transmembrane regions were then substituted for the overlapping cg lipids to embed the proteins. the procedure was iterated until a spike, membrane, and envelope protein density on the virion surface was achieved that was approximately consistent with current available experimental estimates of ~25, 1000, and 20 per virion, respectively, from cryo-em and biochemical data (38) (39) (40) . the diameter of the membrane envelope is approximately 100 nm and 120 nm including the s proteins on the virion surface. as higher-resolution experimental data are released, the overall structure of this model can be refined. two glycosylated models of the open and closed spike were inserted into a symmetric 225 å × 225 å lipid bilayer mimicking the composition of the endoplasmic reticulum-golgi intermediate compartment (41, 42) . the lipid patch was built using charmm-gui. the complete proteinmembrane system was solvated with using the tip3p water model (43) , and neutralized with chloride and sodium ions to maintain a 150 mm concentration. each system contained ~1.7 million atoms. minimization and equilibration was performed using the charmm36 forcefield (44, 45 ) and namd 2.14 (46) . production runs were performed in the npt ensemble using a langevin thermostat at 310k and nosé-hoover langevin barostat at 1 atm. all production runs used a 2-fs timestep and the shake algorithm. multiple replicas of aa md simulations of the open (3x) and closed (3x) systems were performed on nsf frontera at the texas advanced computing center (tacc), achieving an aggregate sampling of 3.0 and 1.8 µs, respectively. the cg model of the glycosylated s protein (figure 2 a) was parameterized from the aa md simulations described below (47) . reference statistics used conformations sampled equally from both open and closed states with aa trajectories spanning the 3.0 and 1.8 µs, respectively. first, the protein was mapped to cg beads using essential dynamics coarse-graining (edcg) (48) . we used 60 and 50 cg beads for the s 1 and s 2 domains, respectively, and the 22 n-linked glycans were each mapped to a single bead. intra-protein interactions were represented as a heterons prior to a 4 µs production run on anton2. all simulations were run in the constant npt ensemble at 310k and 1 atm using the charmm36m forcefield. a cg model containing approximately 5 residues per cg bead was mapped from the reference statistics of the aa md simulations using the edcg ( figure 2c ), and henm approaches. a cg model for the e protein was developed by linearly mapping the amino acid sequence to particles at a resolution of 1 cg bead per 5 amino acids ( figure 2b ). several studies suggest that the c-terminal domain (ctd) of the n protein assembles into a helix that contains two rna binding grooves (21, 51) . based on these studies, we constructed atomic and cg models of the viral ribonucleoprotein complex (vrnp) by iterating between cg and aa simulations. we first constructed an atomic model of the n protein ctd helix with two rna binding grooves by stacking 3 copies of the ctd octamer structure (pdb: 2cjr), which is composed of 4 ctd dimers and homology modeled from the x-ray crystallographic structure of the sars-cov n protein ctd (31) . the ctd helix was simulated in the charmm36m force field for 400 ns. we then constructed the ctd helix model using edcg combined with henm followed by manually placing cg rna beads into the groove of the helix ( figure 2d ). the positions of the cg beads were used as restraints to build an atomic model of the vrnp complex. finally, the vrnp model was relaxed and simulated in the charmm36m force field for 400 ns. it is important to note that recent cryo-em studies have found granule-like densities within the virion for the vrnp complex (22) . structural detail into how ctd oligomers (including the previously proposed helical model) and rna fit into these densities will likely require higher-resolution images. several computational approaches have been developed to build or refine cg models using data from aa or fine-grained simulations. our approach to coarse-graining the sars-cov-2 virion is to couple several cg methods in a hierarchical fashion. cg sites or "beads" are mapped from atomic structures using edcg, a method designed to preserve the principal modes of motion sampled during atomic-level simulations (48) . in edcg, a given cg mapping operator, where, similarly, in the rem approach, the objective function for minimization is the kullback-liebler divergence, which provides a metric for the differences between the atomistic and cg probability in the canonical ensemble, and ܼ is the configurational partition function. furthermore, the relative entropy can be expressed as a difference between the potential energy and free energy of the atomistic and cg ensembles: minimization of the relative entropy is performed using iterative newton-raphson techniques. it is important to note, however, that the quality and fidelity of such cg models is determined by the molecular behavior sampled in the underlying aa simulations. macromolecular complexes, such as virions, undergo a wide range of behavior including physical and chemical transitions that will be difficult to capture through aa simulations alone, as well as experimental techniques. this is especially true for processes that involve large conformational changes that are not sampled effectively in aa simulations, whether due to the long timescales required, free energy barriers, or inherent limitations of the simulation force field. for instance, the s protein of sars-cov-2 exhibits two proteolytic cleavage sites (at the s1/s2 and s2' locations) as well as binding to the host cell receptor, angiotensin-converting enzyme 2 (ace-2). cleavage and binding events trigger dramatic conformational changes in the spike that result in the insertion of the fusion peptide into the host cell membrane. high-resolution structural studies of the s-ace-2 complex have made protein binding simulations amenable to enhanced sampling techniques at the aa level (4, 56) . the proteolytic cleavage of the spike and large-scale conformational shift towards fusion peptide insertion, however, are more difficult to sample in atomistic simulations. to address these issues, one can use cg molecular simulation techniques that allow cg particles to adaptively switch discrete "states" and interactions, such as "ultra-coarse-graining" (ucg) (57) (58) (59) experiments can probe longer timescales than are available from aa md simulations. in recent cryo-em images of sars-cov-2 particles, the s1 domain of the s protein was found to transiently open and close in order to bind the ace-2 receptor (3, 5) , which are subtle conformational changes that are difficult to sample in atomistic simulations. for these conformational changes -in the case that they cannot be treated as discrete state switchesplastic network models (60) or multi-configuration coarse graining (mccg) methods (61) can be used to construct a cg model that continuously transitions from one state to the next. for plastic network models, two known experimental configurations of the protein are used to build a multibasin enm that represents deviations away from each of the individual conformational minima. a phenomenological interaction hamiltonian is constructed that couples and mixes the enms between the two structural endpoints. in mccg, the primary difference is that the coupling terms in the hamiltonian are constructed from a two-state mixing approach, derived on the basis of a mapped potential of mean force which is explicitly computed from aa simulation data along collective variables that distinguish between the two (or more) conformational states at a cg level. an alternative (and sometimes necessary "top-down") route to deriving cg models is to construct a model hamiltonian and then analyze the model's resulting behavior in the context of the assumed interactions. typically, parameterization of such models is designed to fit or reproduce particular observables measured in experimental data and perhaps particular sets of aa simulation data. these can be performed based on variational optimization of some systemspecific functional that depends on the experimental observable. model hamiltonian approaches have the advantage that physical intuition is clearer, but are not systematic, since each new problem requires a different treatment for the set of interactions involved. furthermore, these approaches often require orthogonal experiments to validate the underlying model. such coarsegraining methods are, however, especially useful in cases where atomistic simulation and/or experimental data is difficult or infeasible to obtain on the system or if the bottom-up methods described above are not expected to yield converged results for the cg effective potential, owing to limited atomistic sampling. here we present results from the first cg simulations of the sars-cov-2 virion (figure 3 ). it should be noted that these are early results and we can thus expect additional simulations to become available from this model as more experimental data and aa simulations become available for the various virion components. in addition, the overall cg methodology and modeling of the virion will continue to evolve, and are works in progress. a cgmd simulation was performed on the complete cg virion model using lammps for (figure 4 a) . in general, the cg model captured the positions and peaks in the pair correlation functions; however, error in the fine structure of the peaks was also present, indicating that refinement involving the addition of more expressive basis cg potentials (e.g., splines) may be necessary. we performed principal component analysis (pca) on a subset of the cg particles to examine collective modes of motion of the virion ( figure 4b ). the cartesian coordinates of 1 particle for every 15 cg lipids, 1 for every m and e protein, and 1 for every 3 s particles were extracted from the trajectory data and used to compute the covariance matrix, the s1/s2 during cgmd. in general, there was a high degree of variance in the s protein, and these correlated modes of motion are likely informative of how the virion collectively utilizes spike proteins to explore and detect receptors. longer cg simulations with more expressive cg models will likely be required to uncover additional modes of motion in the virion, including modes that involve the structural m, n, and e proteins. this work provides an initial cg molecular model of the sars-cov-2 virion and details a bottomup cg approach capable of further refining the model as new atomistic and experimental data becomes available. currently, the lipid envelope is described using a particle-based the protein data bank announcing the worldwide protein data bank cryo-em structure of the 2019-ncov spike in the prefusion conformation structural basis for the recognition of sars-cov-2 by full-length human ace2 structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structural basis of receptor recognition by sars-cov-2 structure of replicating sars-cov-2 polymerase cryo-em structure of the sars-cov-2 3a ion channel in lipid nanodiscs structural model of the sars coronavirus e channel in lmpg micelles molecular basis 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amplitude conformational change in proteins explored with a plastic network model: adenylate kinase multiconfigurational coarse-grained molecular dynamics coarse-grained simulation reveals key features of hiv-1 capsid selfassembly 2020. trim5α self-assembly and compartmentalization of the hiv-1 viral capsid off-pathway assembly: a broad-spectrum mechanism of action for drugs that undermine controlled hiv-1 viral capsid formation discovery of a novel binding trench in hiv integrase structural basis for modulation of a g-protein-coupled receptor by allosteric drugs glutamate and glycine binding to the nmda receptor energetics of glutamate binding to an ionotropic glutamate receptor neurotransmitter funneling optimizes glutamate receptor kinetics molecular lock regulates binding of glycine to a primitive nmda receptor atomic-scale characterization of mature hiv-1 capsid stabilization by inositol hexakisphosphate (ip6) research. key: cord-274424-juj71nc5 authors: pulford, david j.; britton, paul title: intracellular processing of the porcine coronavirus transmissible gastroenteritis virus spike protein expressed by recombinant vaccinia virus date: 1991-06-30 journal: virology doi: 10.1016/0042-6822(91)90617-k sha: doc_id: 274424 cord_uid: juj71nc5 abstract the spike (s) protein from a virulent british field isolate of porcine transmissible gastroenteritis virus (tgev) fs772/70 was constructed from cdna and inserted into the vaccinia virus (vv) thymidine kinase gene locus under the control of the vv early/late gene p7.5k promoter. recombinant s protein was synthesized as an endo-β-n-acetylglucosamini-dase h (endo h)-sensitive glycoprotein with high mannose simple oligosaccharides (gp190) that underwent post-translational modification to an endo h-resistant glycoprotein with complex oligosaccharides (gp210). immunofluorescence analysis demonstrated that the majority of recombinant s protein was retained at the golgi but some s protein was expressed on the plasma membrane. monoclonal antibodies (mabs) raised against native s protein reacted with this recombinant s protein; also, mice infected with the recombinant vaccinia virus (rvv) expressing the s protein induced tgev neutralizing antibodies. a truncated s protein (sδ) was also expressed in rvv-infected cells by introducing a deletion into the s protein cdna that removed 292 amino acids from the c-terminus. the sδ protein (gpl70) was shown to be antigenically similar to tgev s protein by immunofluorescence and immunoprecipitation tests but was retained in the endoplasmic reticulum and not expressed on the cell surface. transmissible gastroenteritis virus (tgev) belongs to the family coronaviridae, a large group of enveloped viruses with a positive-stranded rna genome. the virus causes gastroenteritis in neonatal pigs, resulting in a high mortality and morbidity. tgevvirions are composed of three structural proteins; a basic phosphorylated nucleoprotein (n) m, 47,000 was shown to associate with the viral genomic rna to form the nucleocapsid and interact with a glycosylated membrane protein (m) observed as a series of polypeptides n/r, 28-31,000, and the peplomer or spike (s), a surface h/l, 200,000 glycoprotein (garwes and pocock, 1975) . the tgev s protein has been shown to elicit a neutralizing antibody response (laude et a/., 1986; jimenez et al., 1986; gatwes et a/., 1987) capable of conferring some protection to suckling pigs (garwes eta/., 1978/79) . by analogy to the s protein of mouse hepatitis virus (mhv), the tgev s protein may possess the cell receptor binding components (collins et a/., 1982) and virulence determinants of the virus (fleming et al., 1986) . the s protein of tgev and coronaviruses antigenically related to tgev such as feline infectious peritonitis ' present address: department of pathology and microbiology, school of medical sciences, university of bristol, university walk, bristol, 858 ltd, uk. *to whom all correspondence and reprint requests should be addressed. virus (fipv), canine coronavirus, and porcine respiratory coronavirus, are not cleaved (for a review see spaan et al., 1988) . however, the s proteins from mhv, infectious bronchitis virus, bovine coronavirus, human coronavirus (oc43 and 229e), and porcine hemagglutinating encephalomyelitis virus which are not antigenically related to tgev are proteolytically cleaved into two subunits (spaan et a/., 1988) . some coronavirus s proteins have been demonstrated to induce cell fusion (collins et a/., 1982; sturman et al., 1985; de groot eta/., 1989) , generating multinucleated syncytia. the complete amino acid sequences for the s proteins of the virulent fs772/70 strain ) and the avirulent purdue strain (jacobs et al., 1987; rasschaert and laude, 1987) have been published and compared with other coronaviruses. the fs772/70 strain s protein has a 1449-amino acid precursor polypeptide with 33 potential n-linked glycosylation sites and 97% sequence homology at the nucleotide and the amino acid level with the purdue strain. from the deduced amino acid sequence the coronavirus s protein was shown to contain characteristic features: a 16-amino acid cleavable secretory signal; a heptad repeat sequence that forms a-helical structures which may interact with other subunits to form a coiled-coil oligomeric structure ; a hydrophobic sequence near the c-terminus that is probably responsible for anchoring the s protein to the virion envelope. this membrane anchor region is imme-diately followed by a cysteine-rich domain, a feature common to all other coronaviruses, that may stabilize protein-lipid interactions. in this paper we report the construction of a fs772/ 70 cdna s gene and its expression by a recombinant vaccina virus (rvv) to study the antigenicity and cellular localization of the s protein. the role of the membrane anchor domain was investigated by the introduction of a c-terminal gene deletion downstream of the heptad repeat sequence. tgev, strain fs772/70, was cultivated in a porcine continuous cell line (llc-pkl) maintained with medium containing 10 pg ml-' trypsin (hofmann and wyler, 1988) . cv-1 and human 143 thymidine kinase negative (htk-) cells were grown in eagles mem medium (flow labs) containing 10% heat-inactivated fetal calf serum. transfections were performed on subconfluent monolayers of cv-1 cells previously infected with wild-type vaccinia virus (vv) (wr strain) using plasmid dna calcium phosphate precipitates (mackett et a/., 1985) . recombinant vaccinia viruses were cultivated in htk-cells in the presence of 25 pg ml-' 5-bromodeoxyuridine (budr; sigma) as described by mackett et al. (1985) . of the tgev s and sa genes from cdna cloning procedures were as described by maniatis et a/. (1982) . enzymes were used according to manufacturers' instructions (new england biolabs, bethesda research labs). plasmids ptg47, ptg25, and ptg30 were used to reconstruct a dna copy of the tgev s gene; the procedure is outlined in fig. 1 . essentially barnhi linkers (pcggatccg, no. 1021 biolabs) were added to the hoal site known to be 11 bp upstream of the s gene initiation codon ) and the 1.29kb hindill fragment was cloned into puc9 and excised as a 0.85-kb xbal-barnhi fragment. the barnhi-sty1 fragment derived from ptg47, the styl-kpnl fragment derived from ptg25, the kpnl-xbal fragment derived from ptg30, the 0.85-kb xbal-barnhi fragment, and barnhi dephosphorylated pbr322 were mixed, ligated in a five-way reaction mixture, and transformed into dhl escherichia co/i cells. as a result of the different cohesive ends an insert of 4.6 kb would correspond only to the complete s gene. an aprtcs transformant was found to contain a plasmid, ppbp1, with a 4.6-kbp insert in pbr322, corresponding to the tgev s barnhi gene cassette. the reconstructed gene was verified by sequencing the junction regions. the tgev cdna s gene was subcloned from ppbp1 and inserted into the barnhi cloning site of the vv plasmid insertion vector pgs20 (mackett et al., 1984) downstream of the w early/late p 7,5k promoter. the recombinant plasmid, pgsp-1, containing a correctly orientated s gene, was identified by restriction endonuclease digestion with sall. the s protein with a c-terminal deletion was generated by cloning a 3.7-kbp sali fragment from pgsp-1 into the sali site of pucl2. restriction endonuclease digestion of recombinant plasmids produced either a 0.25-or 3.45-kbp barnhi fragment and the latter was recloned back into barnhi-digested pgs20. the recombinant plasmid insertion vector pgspa-38 was found to contain a correctly orientated sa gene by sryl restriction endonuclease digestion of plasmid dna. a synthetic oligonucleotide primer 5'-gtgtgcggctac-tataacta-3', that binds to the complementary dna strand 43 bp downstream of the barnhi cloning site in pgs20, verified the position of the sa protein stop codon in pgspa-38 by sequencing ( fig. 2a) . the rvvs, vts-1 and vtsa-1, were generated with pgsp-1 and pgspa-38 using methods described by mackett et al. (1984) . thymidine kinase negative viruses were screened by dna dot blot and dna from vts-1 and vtsa-1 infected cells was analyzed by southern blot using a [32p]-labeled s gene cdna probe as described in . groups of three female balb/c mice were immunized by intraperitoneal (ip) inoculation with l-2 x 1 o5 pfu/ mouse of partially purified recombinant or wildtype vv as described by . after 4 weeks, one animal from each group was sacrificed to obtain convalescent serum, the remaining animals were hyperimmunized with homologous virus by ip inoculation and bled after a further 4 weeks. mice initially inoculated with either phosphate-buffered saline (pbs) or wr strain vv were inoculated after 4 weeks with a dose of vts-1 to establish if age or a previous w infection affected the stimulation of tgev neutralizing antibody. tgev neutralizing antibody was assessed by plaque reduction assay as described by garwes et a/. (1987) . viral proteins were routinely radiolabeled by incubating tgev-infected llc-pkl cells or rvv-infected htk-cells, at 8 hr p.i. or 17 hr p.i., respectively, in methionine-free medium for 1 hr and then in the presence of 50-l 00 &i ml-' l-[35s]methionine (amersham international; see figure legends for further details). after pulse labeling, cells were washed in pbs (10 mm potassium phosphate, 150 m/l/l naci, ph 7.2) and chased in medium supplemented with 2 mm l-methionine. cells were washed in pbs before treating with test lysis buffer (20 mm tris-hci, ph 7.6, 2 mm edta, 100 mm naci, 1% triton x-l 00, 0.1% aprotinin). nuclei and ceil debris were pelleted from infected cell lysates by centrifugation at 100,000 g using a beckman tla-100 ultracentrifuge. tissue culture medium was clarified by low speed centrifugation. immunoprecipitations were performed by mixing 1 vol of cell lysate with l/l 00th vol of porcine tgev hyperimmune antiserum at 4' for 1 hr and the resulting immune complexes were incubated overnight with formalinfixed staphy/ococcus aureus cells (sac; brl) previously washed three times in test buffer. immune complexes were washed three times with test buffer and resuspended in laemmli sample buffer, and proteins separated on 6% sds-polyacrylamide gels (laemmli et a/., 1970) . 14c-methylated proteins (amersham, code cfa.626) were routinely used as molecular weight markers. proteins were detected by fluorography after immersing gels in 0.8 m sodium salicylate for 30 min. pelleted immune complexes were washed as described above, resuspended in 40 ~i50 mll/l tris-hci, ph 6.8, containing 0.25% sds, and boiled for 2 min. the solubilized proteins were incubated in the presence or absence of 1 mu endo h (boehringer-mannheim) in 90 mmsodium citrate, ph 5.5, at 37" for 18 hr. the proteins were analyzed on 6% polyacrylamide gels and detected by fluorography. glass coverslips with subconfluent monolayer cultures of htk-or llc-pkl cells were infected with tgev and wild-type or rvvs. at 20 hr p.i. cells were either fixed with cold 80% acetone and air dried or washed in cold pbs and maintained at 4" for surface staining. cells were incubated for 1 hr with porcine tgev hyperimmune antiserum diluted 1 :loo in pbs and then extensively washed with pbs. cells were then incubated for 30 min with fluorescein isothiocyanate-conjugated rabbit anti-pig immunoglobulin g (nor-die immunology) diluted 1:40 in pbs. coverslips were washed, air dried, and mounted on glass slides with 80% glycerol. fluorescent cells were observed and photographed with a leitz wetzlar uv microscope. a 4.6-kbp barnhi s gene cassette was constructed from tgev fs772/70 cdna and used to generate ppbp1 (fig. 1) . the 4350-bp s gene was capable of encoding a precursor polypeptide of 1449 amino acids with a m, 159,811. after cleavage of the n-terminal signal peptide, shown to be absent in virion-associated purdue strain s protein (rasschaert and laude, 1987) the protein would consist of 1433 amino acid residues with a ai, 157,891. the s gene barnhi gene cassette was subcloned into the vv insetion vector pgs20 to give pgsp-1 as described under materials and methods. the sa gene, contained in pgspa-38, was 3474 bp long, capable of encoding a 1157-amino acid polypeptide, corresponding to a deletion of 292 residues from the complete s protein and also included eight amino acid residues derived from the pucl2 polylinker and pgs20 sequences. the sa gene terminated in a new tga stop codon contained within the vv tk sequences (fig. 2) . the predicted size of the precursor sa polypeptide was m, 126,748, which when modified by the removal of the 16-amino acid n-terminal signal peptide was reduced to m, 124,916. the c-terminal deletion also resulted in removal of eight potential nlinked glycosylation sites predicted for the s protein. the s gene initiation codon was 54 bp away from the vv early promoter rna start site for both pgsp-1 and pgspa-38 insertion vectors. the tgev s genes were inserted into the vv genome by homologous recombination into the tk locus, and southern blot analysis was used to confirm that the resulting rvvs vts-1 and vtsa-1 contained the 4.6-and 3.45-kbp tgev barnhi gene fragment, respectively. two plaque-purified rvv clones, vts-1 and vts-2, were used to immunize balb/c mice, and the level of tgev neutralizing antibodies was measured by a plaque reduction assay (table 1 ). both recombinant virus clones induced tgev neutralizing antibodies that were boosted with a second inoculation. mice previously inoculated with the wild-type vv and inoculated with vts-1 produced a 22-fold lower level of tgev neutralizing antibody compared to the convalescent serum of vts-1. a primary infection with wild-type vv could have stimulated the mouse immune system fig. 1. construction of the tgev s gene from the fs772/70 cdna. the complete tgev s gene was generated by a four-way ligation into pbr322. the thin lines represent tgev cdna and the thick lines vector dna sequence. the top line represents the three cdna clones used to generate specific cdna fragments. if more than one enzyme was used they are listed in a descending order next to the appropriate arrow. the 0.4-bp hpal-pstl fragment from ptg47 had barnhi linkers added and was then digested with barnhi and sty1 to generate a 0.2.kb fragment with a barnhi site upstream of the s gene initiation codon. the 1.29.kb hindill-hindill fragment from ptg30 was initially cloned into puc9 and removed as a xbal-barnhi fragment, with the barnhi site derived from the puc9 polylinker sequence, to provide a barnhi site within the tgev orf-3a gene 3'to the end of the s gene. the relevant fragments were then ligated together with pbr322 such that only the correct alignment of the cohesive ends would give a fragment of 4.6 kb in pbr322 corresponding to the tgev s gene bamhl cassette. into producing a rapid clearance of a subsequent rw infection and prevented the expression of large amounts of s protein. mice given a single inoculation with vts-1 4 weeks after the other mouse group had a fourfold reduction in the level of tgev neutralizing antibody. this suggested that age may have a significant effect on the induction of tgev neutralizing antibodies, but further animal studies are needed to make any firm conclusions. expression of the recombinant spike antigens tgev gene products synthesized by vts-1 and vtsa-1 were analyzed by pulse labeling rvv-infected htk-cells with l-[35s]methionine in the presence or absence of 10 pg ml-' tunicamycin for 6 hr. tgev s protein was immunoprecipitated from cell lysates or tissue culture fluids using porcine tgev hyperimmune antiserum. this resulted in s polypeptides of aj 190,000 (gpl90) and aj 210,000 (gp210) being immunoprecipitated from vts-1 -infected cell lysates in the absence of tunicamycin (fig. 3 , lane 2) while in the presence of tunicamycin, a aa, 160,000 (~160) precursor s polypeptide was synthesized (fig. 3, lane 3) . cells infected with vtsa-1 expressed a m, 170,000 glycosylated polypeptide (gpl70) only (fig. 3, lane 4) that was expressed as a aa, 130,000 (~130) precursor protein (fig. 3, lane 5) in the presence of tunicamycin. it was noted that the levels of s protein expressed were appreciably less in the presence of tunicamycin (fig. 3) . comparison of the nonspecifically precipitated proteins in the presence or absence of tunicamycin (fig. 3) indicated that the reduction in s protein synthesis was not due to inhibition of viral replication in the presence of tunicamycin. these observations suggested therefore that either glycosylation was required for efficient synthesis of the s protein or the majority of the anti-s antibodies were directed against the glycosylated form of the protein resulting in less efficient immunoprecipitation of the nonglycosylated form. although gp210 was immunoprecipitated from the vts-1 -infected cell culture medium in the absence of tunicamycin (fig. 4 , lane 3) no detectable extracellular sa protein (gpl70) was recovered from medium of vtsa-l-infected cells (fig. 4, lane 6) suggesting that the membrane anchor is required for export out of cells. the lack of detectable ~160 or ~130 in the culture medium of vts-l-and vtsa-l-infected cells, respectively (fig. 4 lanes 4 and 7) , could result from the levels being too low to be detected by the antibodies due to reduction in synthesis or the lack of glycosylation of the s protein in the presence of tunicamycin. the rate of s protein intracellular transport was compared by measuring the acquisition of endo h resisnote. tgev neutralizing antibody was titrated by a 50% plaque reduction assay and expressed as the reciprocal of the antiserum dilution. tance in tgev-, vts-i-, and vtsa-1 -infected cells. after a i-hr pulse, the tgev s protein consisted of a major gp190 component and a minor gp210 component (fig. 5a) . after a 1-hr chase, gpl90 and gp210 were present in approximately equal proportions but after a 2-hr chase, gp210 was the dominant s protein component and the majority was endo h resistant. tgev s protein steadily became endo h resistant with time such that after a 4-hr chase virtually all s protein had been modified in the golgi. s protein was also detected in the tissue culture medium of tgev-infected cells after a 1 -hr chase and accumulated steadily with time. digestion of gp210 from extracellular or intracellular sources with endo h reduced the size of the s protein to m, 205,000 . this observation has also been made for the fipv s protein from fipv-infected feline cells (vennema eta/., 1990) and suggested that coronavirus s proteins still have high mannose or hybrid oligosaccharide structures following golgi processing. the recombinant s protein was completely endo h sensitive after a 1-hr pulse and after chasing became partially endo h resistant with approximately half of the protein observed as unresolved components of high molecular weight (fig. 5b ). extracellular s protein was not detected in the culture medium after chasing for 4 hr because s protein transport from the rer through the golgi stack and out of the cell was significantly disrupted in the absence of coronaviral morphogenesis. infection of llc-pkl cells with vts-1 produced a discrete mr 2 10,000 endo h-resistant form of s protein (data not shown). this was in contrast to the partial endo h-resistant forms observed in vts-1 -infected htk-cells, implying that the tgev s protein may be post-translationally modified to a different extent depending on the origin of the cell line. the sa protein remained completely endo h sensitive, even after a 4-hr chase, demonstrating that the truncated s protein was glycosylated with high mannose oligosaccharides only and was not subject to golgi-specific modifications (fig. 5b ). the cellular location of the s and sa protein in rvvinfected cells was analyzed by indirect immunofluorescence microscopy (fig. 6) . acetone-fixed tgev-infected llc-pkl cells, probed with anti-s-specific serum, had a granular appearance with occasional bright accumulations in localized parts of the cell (data not shown). in vts-l-infected htk(fig. 6a ) and llc-pkl (fig. 6c) an intracellular compartment in all infected cells (represented with arrows), while vtsa-1 -infected cells had a uniform distribution of sa protein throughout the cytoplasm ( fig. 6b and 6d ). recombinant s protein was also observed on the cell surface of unfixed infected htk-cells (fig. 6e ), unlike sa protein (fig. 6f) , demonstrating that the full-length protein has all the intrinsic properties for cell surface transport and does not require the cooperative effect of other tgev proteins. the possibility that soluble s protein released from human cells may bind back to cell surface receptors and give the appearance of cell surface fluorescence can be disregarded as tgev s protein only binds porcine cell surface receptors. studies described in this paper with tgev recombinant s protein have shown that it is an n-linked glyco-protein with a m, 190-210,000. mice inoculated with two separate clones of vts demonstrated that the recombinant s protein was immunogenic and elicited tgev neutralizing antibodies. this was in contrast to previous studies with tgev n and m proteins expressed by rvvs that induced an immune response but no neutralizing antibodies . the tgev s protein neutralizing epitopes and major antigenic sites were retained by the sa protein but this protein was not transported to the plasma membrane or through the golgi stack and must therefore be accumulated in the endoplasmic reticulum. hu et al. (1984) constructed an incomplete tgev s protein gene that initiated 8 bp downstream of a hpal site but ended 80 bp upstream of a xbal site. this gene construct expressed an endo h sensitive m, 180,000 polypeptide that was not transported to the cell surface in rvv-infected cells (hu et al., 1985) . sequence analysis of the tgev purdue-l 15 (jacobs et al., 1987; rasschaet-t et al., 1987) and fs772/70 strains demonstrated that the xbal site was 3776 bp from the s protein initiation codon and within an orf of 4350 bp, implying that the s gene product expressed by hu et a/. (1985) was a truncated protein. coronavirus s proteins are known to be highly glycosylated with n-linked oligosaccharides and undergo modification with complex sugars at the medial compartment of the golgi stack during virus maturation (niemann et al., 1982) . simple high mannose and hybrid structures can be removed from glycoproteins by digestion with endo h but glycoproteins modified with complex sugar structures are resistant to cleavage with endo h (hubbard and ivatt, 1981; dunphy and rothman, 1985) . the rvv vts-1 produced two s protein species in human cells with different oligosaccharide composition, assigned gpl90 and gp2 10, while the sa protein was expressed in human cells as a single glycoprotein species designated gp170. the gp210 was partially resistant to endo h, suggesting that gpl90 was modified to gp210 by the addition of complex oligosaccharides at the golgi. the sa protein remained endo h sensitive even after extensive chasing, implying that it was not transported to the golgi stack. pulse-chase analysis of tgev-or vts-1 -infected cells demonstrated that s protein was initially synthesized as gpl90 but gradually accumulated as gp210 due to post-translational modifications. endo h digestion of recombinant s protein expressed in htk-produced a range (n/l, 160-210,000) of partially resistant polypeptides. both of these observations were made for the fipv (79-l 146) s protein expressed in a bovine papilloma virus transformed mouse cell line (de groot et a/., 1989) . however, the tgev s gene expressed by a rvv in porcine llc-pkl cells produced an endo h-resistant gp210 product with no partially resistant endo h intermediates. a similar observation was also made for the fipv s gene expressed by a rw in feline cells (vennema et al., 1990) , suggesting that expression of coronavirus s proteins in a cell line compatible with their origin may have profound effects upon the extent of post-translational processing of these proteins. the acquisition of endo h resistance was significantly retarded for recombinant s protein compared to s protein expressed during a tgev infection. retardation of coronavirus s protein intracellular transport has also been observed for the fipv and mhv s proteins expressed by rvvs (vennema et al., 1990) . in this study, retardation between the rer and the golgi in the absence of virus budding was interpreted as a transient accumulation of the s protein at or near the site of virus budding and suggested that the s protein may have a role in defining the site of virus budding, a func-tion previously ascribed to the m proteins of coronaviruses (booze et al., 1985) . lmmunofluorescence studies with vts-infected cells demonstrated that recombinant s protein accumulated in a polar perinuclear compartment of the cell, a similar observation was seen for the cellular localization of rvv expressed tgev m protein (pulford and britton, 1990 ) and at the cell surface. the presence of tgev gp210 in the culture medium of vts-infected cells suggested that gp210 may be released from the cell. proteins can be transported from the rer to the golgi by interacting with carrier proteins or following membrane incorporation, by the flow of membrane from the er to the cell surface. this membrane flow may be responsible for the cell surface presence of coronavirus s proteins. the sa protein was not processed in the golgi apparatus, nor found on the cell surface nor found to be exported into the extracellular medium, indicating that the c-terminal membrane anchor domain of tgev s protein may be required for all of these functions. puddington eta/. (1986) used a mutant vsv g protein to establish that the cytoplasmic tail of the g protein was essential for its transport through the golgi stack. in addition, bray eta/. (1989) used rws to demonstrate that a dengue virus envelope protein truncated at the c-terminus, unlike the wild-type protein, was not secreted into the extracellular medium. however, sveda et a/. (1982) found that c-terminal sequences of the influenza virus hemagglutinin (ha) were essential for cell surface expression and that mutants with a disrupted anchor domain were secreted, suggesting that influenza virus ha, unlike tgev, vsv, and dengue virus envelope proteins, underwent a different protein maturation pathway. evidence suggests that unless a membrane glycoprotein folds into a native or near-native conformation it will not be exported from the rer (lodish, 1988) . however, the general conformation and antigenicity of the sa protein appeared to be retained as it reacted with both polyclonal and monoclonal tgev s antisera in contrast to denatured s protein or s protein expressed in e. co/i cells (correa et a/., 1990; delmas et a/., 1990; pulford, unpublished observations) . the results presented in this paper suggest that the sa protein was retained in the rer possibly due to the removal of essential c-terminal transport signals. we would like to thank miss k. mawditt for synthesizing the oligonucleotide used in the sequencing, mrs. a. waite for carrying out the animal inoculations and dr d. pocock for helpful comments on the manuscript. this research was supported by the biotechnology action programme of the commission of the european communities contract n" [bap-0235.uk(hl)]. mice immunised with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein nsl are protected against fatal dengue virus encephalitis sequence of the s gene from a virulent british field isolate of transmissible gastroenteritis virus monoclonal antibodies to murine hepatitis virus-4 (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion localization of antigenic sites of the e2 glycoprotein of transmissible gastroenteritis coronavirus cdna cloning and sequence analysis of the gene encoding the peplomer protein of feline infectious peritonitis virus stably expressed fipv peplomer protein 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proteins: implications for virus assembly key: cord-297960-4x1j0iqg authors: bösl, korbinian; ianevski, aleksandr; than, thoa t.; andersen, petter i.; kuivanen, suvi; teppor, mona; zusinaite, eva; dumpis, uga; vitkauskiene, astra; cox, rebecca j.; kallio-kokko, hannimari; bergqvist, anders; tenson, tanel; merits, andres; oksenych, valentyn; bjørås, magnar; anthonsen, marit w.; shum, david; kaarbø, mari; vapalahti, olli; windisch, marc p.; superti-furga, giulio; snijder, berend; kainov, denis; kandasamy, richard k. title: common nodes of virus–host interaction revealed through an integrated network analysis date: 2019-10-04 journal: front immunol doi: 10.3389/fimmu.2019.02186 sha: doc_id: 297960 cord_uid: 4x1j0iqg viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. a collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. here, we performed an integrative meta-analysis to elucidate the 17 different host-virus interactomes. network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. we also identified the core cellular process subnetworks that are targeted by all the viruses. integration with functional rna interference (rnai) datasets showed that a large proportion of the targets are required for viral replication. furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis c virus and human metapneumovirus. altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape. viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. a collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. here, we performed an integrative meta-analysis to elucidate the 17 different host-virus interactomes. network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. we also identified the core cellular process subnetworks that are targeted by all the viruses. integration with functional rna interference (rnai) datasets showed that a large proportion of the targets are required for viral replication. furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis c virus and human metapneumovirus. altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape. viruses continue to be a major contributor to the global burden of disease through acute and chronic infections that cause substantial economic impact in addition to increased mortality and morbidity (1) . despite the tremendous improvement in the understanding of the antiviral immune response and the availability of therapeutics, existing and emerging viral diseases are an ever-growing problem, particularly in developing countries. development of antiviral resistance of hepatitis c virus (hcv), influenza a virus (iav), herpes simplex virus (hsv), human cytomegalovirus (hcmv), human immunodeficiency virus (hiv), and other viruses is a major concern (2-4). one of the main reasons for increasing resistances is the accumulation of mutations in the viral genome caused by multiple factors including the polymerase infidelity (5, 6) . therefore, the world health organization (who) and the united nations have urged for better control of viral diseases. this has led to turning the focus on the host for therapeutic intervention. targeting the host factors has been proven to be useful for restricting viral infections (7, 8) . the small molecule ccr5 inhibitor maraviroc and the anti-cd4 monoclonal antibody ibalizumab are examples of successful use of host-directed therapies for combating hiv in clinic (9) (10) (11) . viruses have evolved to evade the host antiviral response at various stages starting from viral sensing to antiviral proinflammatory responses (12) (13) (14) . multiple studies attempted to understand global principles of the viral evasion employed by various viruses, including dengue virus (denv), ebola virus (ebov), iav, and hiv (15) (16) (17) (18) (19) (20) . global systems-level approaches including functional rnai screens, interactome mapping technologies such as affinity-purification mass spectrometry (ap-ms), quantitative proteomics, and crispr/cas9-based screens have provided unparalleled details and insights into the dynamics of host proteome in immune cells (21) (22) (23) (24) , host-virus interactome (15-17, 25, 26) , and also identified important host dependency factors of various viruses (25, 27, 28) . meta-analyses of such high-dimensional datasets have been crucial for identifying novel host factors as drug targets such as ubr4 in iav infection (29) . moreover, some of these factors represent drug targets for multiple viruses (30) . we hypothesized that combining a meta-analysis of host-virus protein-protein interactions of multiple viruses and functional rnai screens would provide novel insights for developing broadspectrum antiviral strategies. for this, we assembled a host-virus protein-protein interactome of 5,781 host-virus interactions (hereafter referred to as "hvppi") covering 183 viral proteins from 17 different viruses and 2,381 host proteins. we performed extensive bioinformatics and network analysis and integrated this dataset with genome-wide or druggable-genome rnai screen data from published studies. this resulted in the assembly of critical nodes of viral evasion and identification of core cellular processes and druggable nodes that were verified by a drug re-purposing screen using broad-spectrum antivirals. host-virus protein-protein interactions were downloaded from published studies (15-17, 25, 31-34) we performed gene-set enrichment analysis using david bioinformatics resources [version 6.8, (38) ]. for all enrichment analysis, a p-value cutoff of ≤0.01 was used as significant. protein disorder analysis was performed using iupred2a software. we used the offline version with protein sequences downloaded from uniprot. statistical analysis of disordered region distribution was performed by kolmogorov-smirnov test in r statistical environment. annotation of human proteins was mapped from uniprot id to ensembl using ensdb.hsapiens.v86. the index of subcellular localization of interaction partners of single viral proteins was calculated for all viral proteins with ≥ five host targets. localization of host targets was mapped using compartments (39) , filtered for a minimum evidence score of 3 in the knowledge channel, excluding non-experimental based localization predictions. evidence for all protein was subsequently divided by the absolute number of host-targets per viral protein. multiple sequence alignment was performed using clustal x [version 2.0, (40)]. genome-wide rnai screen data for hcv (41) and hpv18 (42) , through genomernai database [(43)-gr00197], as well as druggable rnai screen data for hpv16 (44) , vacv (45) , and sv40 (46) were integrated in the existing network as z-scores. drug-gene interaction data was downloaded from dgidb and drugbank. the identifiers were mapped to uniprot ids and then compared with hvppi. for the hmpv nl/1/00-gfp screen, approximately 4×10 4 human retinal pigment epithelial (rpe) cells were seeded per well in 96-well plates. human non-malignant rpe cell line represents excellent model system for studying replication of many viruses including respiratory (30, 47, 48) . the cells were grown for 24 h in dmem-f12 medium supplemented with 10% fbs, 0.35% nahco 3 , and 100 µg/ml streptomicine and 100 iu/ml penicillin. the medium was replaced with virus growth medium (vgm) containing 0.2% bovine serum albumin (bsa), 2 mm l-glutamine, 0.35% nahco 3 , and 1 µg/ml l-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin in dmem-f12. hcv screen-associated cell culture conditions are described in kim et al. (49) . the compounds were added to the cells in 3fold dilutions at seven different concentrations starting from 50 µm. no compounds were added to the control wells. the cells were mock-or virus-infected at a multiplicity of infection (moi) of one. after 48 h of infection, the medium was removed from the cells. to monitor cell viability, celltiter-glo reagent was added (30 µl per well). this assay quantifies atp, an indicator of metabolically active living cells. the luminescence was measured with a plate reader. to determine compound efficacy, hmpv nl/1/00-mediated gfp expression was measured. the half-maximal cytotoxic concentration (cc 50 ) and the half-maximal effective concentration (ec 50 ) for each compound were calculated after non-linear regression analysis with a variable slope using graphpad prism software version 7.0a. the relative effectiveness of the drug was quantified as the selectivity index (si = cc 50 ec 50 ). cytotoxicity and antiviral activity of the compounds against gfp-expressing hcv in huh-7.5 cells was determined as previously described (49). to provide new and critical insights into viral evasion mechanisms we performed a comprehensive meta-analysis of the host-virus interaction landscape. we assembled the hostvirus protein-protein interaction data ("hvppi") from published studies ( figure 1a ) (15-17, 25, 31-34) . this dataset comprised of protein-protein interactions from two different types of experimental methods-affinity purification mass spectrometry (ap-ms) and yeast two-hybrid screens (y2h). altogether, this combined dataset includes 183 viral proteins, 2,381 host proteins, and 5,781 protein-protein interactions ( figure 1b and figure s1 ). many interactome networks including yeast and human are scale-free networks, where a large portion of the nodes (e.g., a protein in the network) have few interactions and only a few nodes have large number of interactions. the latter are often referred to as "hubs" which are crucial in keeping the network intact (34) . we performed network topology analysis to infer the properties of the host proteins targeted by the viral proteins in the context of the human protein interactome. we considered two important parameters-relative betweenness centrality (which reflects the amount of information that passes through this protein in the human interactome) and degree (number of binding partners in the human interactome) of the host proteins targeted by each virus. the targets of all the viruses showed higher betweenness centrality and degree as compared to an average protein in the human interactome (figures 1c,d) . this shows that viruses, by targeting "hubs" and proteins that serves as key communication nodes, have evolved the best way to disrupt the scale-free human interactome. this topological property thereby shows how viruses having small genomes achieve the maximal effect in rewiring the human interactome to benefit viral survival and replication. our analysis is in agreement with several previous studies, which have highlighted this property (15, 16, 31, 50, 51) . we propose that this could be a general principle for all viruses. proteins typically fold into stable three-dimensional structures that mediate specific functions. in addition, there are substructures in proteins termed "intrinsically disordered regions (idrs)" which lack stable structures under normal physiological conditions. idrs are required for multiple cellular functions even though they lack these defined structures (52) . many studies have highlighted the presence of such idrs in viral proteins (53) (54) (55) , such as e6 from human papilloma virus, that are crucial for hijacking the cellular machinery. we analyzed the host proteins from the hvppi for presence of idrs using the prediction software iupred (56) . it is estimated that the human proteome more than 100,000 short linear binding motifs in idrs (57, 58) . proteins with idrs are often signaling hubs and might form dynamic complexes with other proteins through specific motif based interactions (58) . we found a statistically significant enrichment (p-value < 6.246 × 10 −06 ) of idrs in the host proteins targeted by viruses (figure 2a and figure s2 ). we then identified the subnetwork in the hvppi which contained the top host targets with high disorderness score ( figure 2b) . the top five proteins with large idrs include cd44 antigen (cd44), serine/arginine repetitive matrix protein 2 (srrm2), myristoylated alaninerich c-kinase substrate (marcks), bag family molecular chaperone regulator 3 (bag3), and mitochondrial antiviralsignaling protein (mavs; figure 2c ). cd44 is a marker of exhausted cd8+ t cells (59) and replication of hcv in t cells was shown to decrease cell proliferation by inhibiting cd44 expression and signaling (60) . srrm2 is a serine/arginine-rich protein involved in rna splicing (61) . srrm2 is differentially phosphorylated in hiv-1 infected cells and absence of srrm2 lead to increased hiv-1 gene expression, since it regulates the splicing of hiv-1 (62) . in the hvppi, srrm2 is targeted by multiple viral proteins including the tat protein from hiv-1. tat protein has an important role in the stimulation of the transcription of the long terminal repeat (ltr) (63) . in addition, ns1 protein from influenza b virus has also been reported to interact with srrm2 (64) . proteins of the marcks family are involved in a range of cellular processes including cell adhesion and migration (65) . marcks is a negative regulator of lipopolysaccharide (lps)-induced toll-like receptor 4 (tlr4) signaling in mouse macrophages (66) . mavs is an adaptor protein in the rig-i signaling pathway involved in the sensing of rna. ablasser et al. (67) reported that double-stranded dna serves as a template for rna polymerase ii and is transcribed into a 5 ′ triphosphate containing double-stranded rna, which activates the rig-i signaling pathway. in the hvppi, mavs is targeted by several proteins from dsdna viruses such as ebv and hpv. altogether, our analysis shows that the idr-high part of the human proteome is an essential part of the viral evasion strategy and some of the selected targets highlighted here could show novel insights into the viral evasion mechanisms. however, the very flexible protein structure of disordered proteins also makes them also difficult to target with drugs. to assess the signaling pathways and cellular processes within the hvppi, we identified highly connected subnetworks within hvppi network. we constructed a host-host interaction network based on the host targets in the hvppi and identified a number of highly connected subnetworks/clusters (figure 3) . we then performed a gene-set enrichment analysis of significantly enriched biological processes. we found one or more enriched processes for each of this subnetwork including core cellular processes such as proteasome, spliceosome, protein translation, protein/rna transport, and cell cycle. next we listed the viruses that target one or more of these processes, and found that almost all the core pathways and processes are targeted by all the 17 viruses that are part of the hvppi (figure s3 ). this analysis highlights the core components of the cellular process subnetworks which are targeted as part of the viral evasion strategies and thus could be broad-spectrum antiviral hot-spots from a therapeutic point of view. given that the viral proteins were interacting with a large number of host proteins, we analyzed the sub-cellular location of the host proteins. we performed gene-set enrichment analysis of sub-cellular localization information provided by uniprot database. we binned the localization into 11 compartments and estimated the percent of host proteins in a given compartment as compared to the total number of host proteins targeted by a given figure 3 | clusters of hvppi involved in core cellular processes. network view of the "clusters" or highly-connected sub-networks and their associated cellular processes. each cluster is marked in a unique color. virus. we found that the viral targets were distributed across multiple subcellular compartments with cytoplasm being the most common ( figure s4a ). the hvppi includes two different strains of iav-pr8 (h1n1) and udorn (h3n2). the subcellular localization analysis showed that both strains were enriched for nuclear proteins. non-structural protein 1 (ns1) from both the strains had the highest number of nuclear targets but their targets were very different ( figure 4a) . ns1 of udorn was enriched for a large number of histones as compared to ns1 of pr8 that had large number of heterogeneous nuclear ribonucleoproteins (hnrnps), such as hnrnpu−a known restriction factor for many viruses. this corroborates with the observation that ns1 protein has short linear histone mimicry motifs that can suppress the host antiviral response (68) . in our analysis, we found that it is ns1 of udorn that has a histone mimicry motif "arsk" (figure s4b) . similarly, hpv11 and hpv18 e5 proteins interact more often with host proteins located in the endoplasmic reticulum (er). we found both common and specific subsets of er proteins targeted by the e5 protein ( figure 4b ). hpv18 e5 protein er targets were enriched for phospholipid biosynthesis as well as gpi anchor related proteins, such as phosphatidylinositol glycan anchor biosynthesis class s/t/u (pigs, pigt, and pigu), glycosylphosphatidylinositol anchor attachment 1 (gpaa1) and phosphatidylserine synthase 2 (ptdss2). hpv11 e5 protein er targets were enriched for er-associated ubiquitindependent protein catabolism involving host proteins such as er degradation enhancing alpha-mannosidase-like protein 3 (edem3) and er lipid raft associated 1 (erlin1). er targets common to hpv18 and hpv11 e5 protein were enriched for unfolded protein response, n-linked glycosylation and protein folding involving host proteins such as srp receptor alpha/beta subunit (srpra/srprb) and catalytic subunits of the oligosaccharyltransferase complex (stt3a and stt3b). two independent crispr/cas9 screening studies identified multiple er associated components including stt3a and stt3b as host factors for denv, zika virus (zikv) and japanese encephalitis virus (jev) (27, 28) . the non-canonical function of stt3a and stt3b is required for denv replication and that ns1 protein of denv interacts with these proteins (28) . our orthogonal approach can lead to the identification of critical host factors, and similar functions of er components, such as stt3a and stt3b, are used by hpv11 and hpv18 as well. thus, targeting the non-canonical function of stt3a and stt3b could be a broad antiviral strategy. overall, the enrichment analysis clearly shows that there is commonality and specificity in the subcellular targets of the viral proteins and that detailed interrogation of these targets can give vital clues into the viral evasion mechanisms. rnai screens have been a powerful high-throughput method to identify various cellular functions, including for identification of host restriction factors of viruses (69) . in order to explore the functional relevance of the host targets in the hvppi, we integrated it with five published rnai screens that performed genome-wide or druggable-genome-wide rnai screens for identifying host factors of hcv (41), hpv18 (43), hpv16 (44), sv40 (46) , and vacv (45) . we found that host targets from the hvppi were spread across the spectrum of genes with proviral as well as antiviral phenotype (figure s5) , thus showing that targeting of the host protein by the virus could lead into any direction that favors the virus. we then investigated the top 50 proviral genes that are also targeted by the viral proteins as seen in the hvppi. we identified 42 host proteins ( figure 5a ) that were significantly enriched for coatomer protein complex 1 (copi), protein translation/transport and proteasome ( figure 5b) . this further substantiates the findings from the earlier section on the core cellular processes targeted by the viruses. network analysis of these top hits showed high level on connectivity and crosstalk-for example between the translation and proteasome machinery (figure 5c ). vesicle carriers are involved in the transport of membranes and proteins. copi system is one of the three vesicular carrier systems that is involved in the early secretory pathway (70) (71) (72) . moreover, it has been pointed out that there is a strong similarity between vesicular transport and viral transport [viral entry to budding process, (73) ]; thus making copi system important for the viral life cycle. in addition to the findings of the present study, sirna-based silencing of copi lead to a decrease in entry and subsequent gene expression of iav, vsv, lcmv, and hpiv3 and disruption of the copi complex inhibited the production of infectious progeny virus (74, 75) . copi coatomer inactivation results in a direct decrease of vsv attachment and uptake, but not for membrane fusion or rnp release; however the direct mechanism remains unclear (76) . altogether, these top hits including the copi system could serve as targets for developing therapeutic antiviral intervention strategies for a broad group of viruses. our analyses pointed out that viral evasion mechanism observed in one virus could also be relevant for other viruses. to test this, we obtained known drug-gene interactions from dgidb (77). we selected 28 investigational/experimental/approved antivirals compounds (30) which had dgidb annotated targets that are part of the hvppi. we added 12 agents as controls (table s1 and figure s6) . we tested 40 broad-spectrumantivirals against gfp-expressing human metapneumovirus (hmpv) nl/1/00 strain (78) . seven different concentrations of the compounds were added to hmpv or mock-infected cells. hmpv-induced gfp expression and cell viability was measured and after 48 h to determine compound efficiency and toxicity. after the initial screening, we identified five compounds, which lowered gfp-expression without detectable cytotoxicity (with si > 3). we repeated the experiment with these compounds. the experiments revealed novel activity of azacytidine, lopinavir, nitazoxanide, itraconazole, and oritavancin against hmpv ( figure s7a and table 1, table s2 ). similarly, we examined toxicity and antiviral activity of broad-spectrum-antivirals against gfp-expressing hcv in huh-7.5 cells using previously described procedures (49) . our test identified azithromycin, cidofovir, oritavancin, dibucaine, gefitinib, minocycline, and pirlindole mesylate as novel anti-hcv agents with si > 3 ( figure s7b and table 1, table s2 ). in summary, our metaanalysis approach of the hvppi could provide novel and faster approaches for the re-purposing of existing drugs as antiviral agents. using integrative analysis of orthogonal datasets our study provides a comprehensive view of viral evasion mechanisms. in particular, our analysis of the hvppi network revealed that all the viruses have evolved to target proteins that are central and have strong control over the human interactome. host proteins targeted by viruses contain a high proportion of intrinsically disordered regions. we identified the core cellular processes and associated proteins that are targeted by all viruses. detailed comparative analysis of the subcellular localization of the host proteins showed commonality and specificity both between viral proteins from different strains of the same virus; and between viruses. integrating hvppi with functional rnai screens showed that 28% of the hvppi are host factors of one or more virus. hvppi data-based drug re-purposing screen identified novel activities for various broad-spectrum antivirals against hmpv and hcv. this unique dataset can be used for further detailed interrogation of the mechanisms behind viral evasion. this could serve as a starting point for identifying novel host targets and generating hypothesis in the context of viral evasion and development of pan-viral therapeutic intervention strategies. the methods described here also provide unique ways of dissecting the orthogonal datasets. various analyses from this study have highlighted the existence on pan-viral evasion points that could be utilized for the development of host-directed antiviral therapies. it is also intriguing to see that there is commonality and specificity at the level of sub cellular localization of the viral targets. our analyses have underlined some salient features in the context of iav, hpv, denv, and hcv. further detailed analysis in this context along with protein sequence features, such as short linear motifs [slims; (79) ] would provide novel insights as well as deeper understanding of how small sequence features are involved in the hijacking of the host machinery. integration of such data with known drug-gene interactions provides a clear estimate of the druggable proportion in the hvppi. our meta-analysis approach of the hvppi could provide novel avenues of re-purposing existing drugs for antiviral targeting strategies. our meta-analysis approach of the hvppi could provide novel avenues of re-purposing existing drugs for antiviral targeting strategies. to prove the concept, we tested 40 bsas against hmpv, hcv, sindbis virus (sinv), cytomegalovirus (cmv), and hepatitis b virus (hbv). importantly, 28 bsas have dgidb annotated targets that are part of the hvppi, whereas 12 were used as controls. these safein-man drugs have already been used as investigational agents or experimental drugs in different virus infections (table s2) . we demonstrated novel antiviral effects of azacytidine, itraconazole, lopinavir, nitazoxanide, and oritavancin against hmpv, as well as cidofovir, dibucaine, azithromycin, gefitinib, minocycline, oritavancin, and pirlindole against hcv. azithromycin, is an fda-approved antibiotic of the macrolide family. it is also an investigational agent against rsv and experimental agent against ebov, hrv-a, zikv, and rsv. cidofovir is an fda-approved anti-cmv drug. it is also investigational agent against adv, bkv, hpv, hsv-1, hsv-2, and experimental drug against b19v. dibucaine is an fdaapproved amide local anesthetic. in addition, it is experimental anti-hev-a, hev-b, hev-d, and ebov agent. gefitinib is an fda approved anticancer drug. it has also antiviral activity against bkv, cmv, and vacv. minocycline is a broad-spectrum antibiotic and experimental anti-denv, hiv-1, and wnv agent. oritavancin is a semisynthetic glycopeptide antibiotic used for the treatment of gram-positive bacterial skin infections. it also inhibits ebov, mers-cov, and sars-cov infections. pirlindole is an antidepressant, which is also experimental anti-hev-a, hev-b, and hev-d agent. azacitidine is a chemical analog of cytidine, which is used in the treatment of myelodysplastic syndrome. it is also an experimental anti-adv, the measurements were repeated three times (p < 0.05). fluav, rvfv, hiv-1, and hiv-2 agent. itraconazole is an antifungal medication. it is also used as experimental anti-hev-b, hrv-b, hrv-a, par-a3, and safv agent. nitazoxanide is a broad-spectrum antiparasitic drug, which is also investigational agent against fluav and hcv and experimental anti-chikv, rsv, hbv, hiv-1, vacv, rv, jev, mers-cov, nov, ruv, and zikv agent. lopinavir is an fda-approved antiretroviral of the protease inhibitor class. it is also investigational anti-mers-cov and experimental anti-zikv agent (table s2 ). in addition to inhibition of viral proteases (table s2) , lopinavir was reported to induce host rnasel production in infected and non-infected cells (80) . rnasel is endoribonuclease that is a part of interferon (ifn) antiviral response, which is the most critical node of virus-host interactions. although, the antiviral mechanisms of action of other compounds are still unknown, these agents could inhibit steps of viral infections, which precede reporter protein expression from viral rna. in summary, our results indicate that existing bsas could be re-purposed to other viral infections. to further expand a spectrum of their activities, these bsas could be tested against other viruses. re-purposing these and other safe-inman antiviral therapeutics could save resources and time needed for development of novel drugs to quickly address unmet medical needs, because safety profiles of these agents in humans are available. effective treatment with broad-spectrum-antivirals may shortly become available, pending the results of further pre-clinical studies and clinical trials. this, in turn, means that some broad-spectrum-antivirals could be 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viruses hijack cell regulation lopinavir up-regulates expression of the antiviral protein ribonuclease l in human papillomavirus-positive cervical carcinoma cells key: cord-290638-7ro72sv3 authors: lenstra, johannes a.; kusters, johannes g.; koch, guus; van der zeijst, bernard a.m. title: antigenicity of the peplomer protein of infectious bronchitis virus date: 1989-01-31 journal: molecular immunology doi: 10.1016/0161-5890(89)90014-x sha: doc_id: 290638 cord_uid: 7ro72sv3 abstract to study the antigenic structure of the peplomer protein of the avian coronavirus infectious bronchitis virus, fragments from the peplomer gene were generated by restriction-enzyme cleavage or by limited dnase digestion and inserted in the escherichia coli expression plasrnid pex (stanley and luzio, 1984). the antigenicity of the expression products was tested using a number of polyclonal antisera and monoclonal antibodies. the polyclonal antisera recognized different sets of epitopes in the 1162-residue sequence. the n-terminal region of one of the two subunits, s2, was recognized by all polyclonal sera and by two monoclonal antibodies. this clearly immunodominant region contains at least two adjacent or overlapping epitopes, one of which has been localized within 18 residues. the epitopes found as antigenic pex expression products do not coincide with the regions in the s1 subunit that have been found to contain hypervariable sequences. we suggest that these regions constitute conformation dependent neutralization epitopes that cannot be detected in the pex system. the relevance of our finclings for vaccine development is discussed. coronaviruses act as causative agents of a number of infectious diseases in domesticated animals (for a review about these positive stranded rna viruses, see siddell et al., 1983; sturman and holmes, 1983) . conventional vaccines do not exist or are rendered ineffective by antigenic variation. this latter mechanism is thought to be relevant for the epidemiology of infectious bronchitis virus (ibv), which causes a highly contagious respiratory disease in chickens. after recent outbreaks of the disease, new serotypes have been isolated that may have originated from the live attenuated strains used for vaccination (kusters et al., 1987) . obviously, recombinant-dna expression products or peptide vaccines would offer a valuable alternative for conventional vaccines. the antigenic properties of ibv or other coronaviruses most likely reside in the peplomer (cavanagh et al., 1984) , a club-shaped spike projecting from the viral membrane and consisting of a dimer or trimer of the e2 peplomer protein (cavanagh, 1983) . analysis of the amino acid sequences of the peplomer proteins from representatives of the three main coronavirus serotypes (de groot et a[., 1987) has led to a model, in which two §author to whom correspondence should be addressed. abbreviations: elisa, enzyme-linked immunosorbent assay; ibv, infectious bronchitis virus; mab, monoclonal antibody; pbs, phosphate-buffered saline. long cc-helices are located in the c-terminal half of the monomer subunit. in the oligomer, those helices intercalate and form a coiled-coil structure, which constitutes the stalk of the peplomer. the transmembrane segment at the c-terminal end serves as membrane anchor. the n-terminal half is thought to form the spherical outer part of the peplomer. in ibv, proteolytic cleavage results in an n-and a c-terminal subunit, designated sl and s2, respectively. sl is thought to be the most relevant part for the antigenic properties of ibv (cavanagh et al., 1986) . it is recognized by strongly neutralizing monoclonal antibodies (mockett et al., 1984; koch et al., unpublished) . furthermore, isolated sl is capable of eliciting neutralizing antisera (cavanagh et al., 1986) . by comparing the amino acid sequences of different strains, it was found that most amino acid substitutions, likely to reflect antigenic variation, have occurred in the sl subunit (niesters et ul., 1986; binns et al., 1986; kusters et al., unpubljshed) . in this study, we have localized epitopes by expressing parts of the peplomer sequence in a prokaryotic expression system, and measuring the antigenicity of the expression products (stanley and luzio, 1984) . most probably, this method detects epitopes that are essentially conformation independent. antigenic regions were almost exclusively found in the s2 subunit, suggesting that the antigenic variation in sl reflects nonformation dependent epitopes. one immunodominant region has been localized near the n-terminus of s2. this region was recognized by all polyclonal antisera tested as well as by two neutralizing monoclonal antibodies, and contains at least two different epitopes. the sequence of this region may be the starting point for the development of peptide vaccines. schuell). the hinfl ends of hl were made blunt by treatment with the klenow fragment of dna polymerase. depending on the desired reading frame, the fragments were ligated in compatible sites of pex i, pex2 or pex3 (stanley and luzio, 1984) . which had been treated with calf intestine phosphatase if linearized by a single digestion. the cdna clones p39 and p42 of the peplomer gene of ibv strain m41 in the vector puc9 have been described previously (niesters et al., 1986) . rabbit antisera against m41 were raised by intramuscular injection of purified virus (1 mg protein), lysed by triton x-100, in freund's complete adjuvant, followed by the same dose in freund's incomplete adjuvant after two and three weeks, respectively. mouse antiserum was raised by intraperitoneal injection of 100 pg virus protein, followed by a second dose after 1 month. chicken antisera were collected from convalescent (chickens 1 and 3) or hyperimmune (chickens 2 and 4) animals, infected with ibv strain m41 (chickens 1 and 2) or strain d207 (chickens 3 and 4). recombinant plasmids were introduced into e. coli strain pop 2136 via the caclz transformation procedure. recombinants expressing the ibv gene fragments were selected by immunoscreening with a rabbit antiserum or, when the expression product was not recognized antigenically, by hybridization followed by gel electrophoresis of the protein expression products. as hybridization probe, a mixture of hindiiiihinfi fragments of the insertion from plasmid p39 was used, which could be isolated free from the 2.7-kb puc fragment by gel electrophoresis. the preparation of the monoclonal antibodies 26.1 and 31.5 against ibv has been described previously (koch et al., 1986) . alkaline phosphatase-conjugated goat anti-(mouse igg, h + l) and goat anti-(rabbit igg, fc) were from promega (madison); anti-(chicken igg, h + l) conjugates were from nordic, tilburg, the netherlands (horseradish peroxidase) or from miles (alkaline phosphatase). as pex host strain, e. coli pop 2136 was used, which was constructed by dr raibaud (institut pasteur, paris). unless mentioned otherwise, all dna manipulations used were done essentially as described by maniatis et al. (1982) or davis et al. (1986) . genomic cdna clones p39 and p42 were cut with restriction enzymes as indicated in fig. 1 tris-hc1 (ph 7.5) 1.5 mm mncl, and 0.1 mg/ml bsa at 22 c for 6 min (first 5 /tl of the reaction mixture) or 10 min (remaining 5 ~1). as judged by gel electrophoresis and autoradiography after end-labelling with the klenow fragment of dna polymerase, fragments of 2&500 bp were generated. these fragments were made blunt-ended by treatment with t4 dna polymerase, followed by treatment with the klenow fragment. after sequential extractions with phenol/chloroform and ether, the fragments were ligated directly in pex2 dna, which had been cut with smai and dephosphorylated with calf intestine phosphatase. the reaction was carried out overnight this rapid cloning procedure obviates the use of linkers (haymerle ez al., 1986 ) but generates nevertheless a sufficient number of clones from a low amount of insert dna. immunoscreening of bacterial colonies transferred to nitrocellulose membranes (ba85 from schleicher and schuell) was performed as described (stanley, 1983) , using a gelatin/triton x-1oojpbs buffer and an e. coli lysate to quench aspecific reactions. the dilutions of the primary antisera were 1: 1000 for polyclonal sera and 1: 100 or 1:500 for ascites fluids containing monoclonal antibodies. binding of antisera to colonies was visualized using alkaline phosphatase-conjugated secondary antisera (diluted 1:5000) and nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates (blake et al., 1984) . to localize dnase fragments from pex clones that were positive after immunoscreening or to check the correct reading frames for the expression of restriction fragments, dna from pex/ibv recombinants was isolated by the alkaline-lysis method, cut with two restriction enzymes flanking the insertion and with hind111 to cleave the pex vector sequence. the resulting mixture of fragments was ligated in m13mp9 dna cut with the appropriate enzymes. subsequently, the nucleotide sequence of the insertion was determined by the dideoxynucleotide termination method and compared to the sequence of the ibv m41 peplomer gene. expression of the pex fusion gene, leading to the synthesis of a hybrid b-galactosidase protein, was induced in a 5 ml exponential culture (odmm ca. 0.25) by incubation at 42°c for 90 min. hybrid proteins were purified as triton x-100 pellet (stanley and luzio, 1984) . cells were spun down (10 min at 5ooog), resuspended in 100 ~1 15% (w/v) sucrose, 50mm tris-hci (ph 8.0), 50mm edta, and treated with lysozyme (1 mg/ml) for 10 min. after addition of 140 ~10.2% (v/v) triton x-100 in 10 mm tris-hcl (ph 8.0) 1 mm edta, the suspension was sonicated in a bath sonicator for 15 min. the lysozyme digestion as well as use of the sucrose buffer appeared essential for the purity of the final preparation. after spinning down the insoluble hybrid protein (10 min at 10,ooog) and removing the viscous supernatant as completely as possible, the pellet was resuspended in triton x-100 buffer. sonification, centrifugation and removal of the supernatant were repeated (l-2 times) until no viscosity remained. finally, the pellet was resuspended in 250~1 pbs. after adding concentrated sds-containing lysis buffer (laemmli and favre, 1973) , hybrid proteins were spotted via a manifold on nitrocellulose membranes or fractionated by sdspolyacrylamide gel electrophoresis (7.5%) followed by western blotting. hybrid proteins thus immobilized were analysed as described for the filters of the immunoscreening (see above). binding of chicken antisera was visualized using peroxidase-conjugated secondary antisera (diluted 1: 1000) and 3'-3' diaminobenzidine and h,oz as substrates. in the pex expression system, foreign gene fragments are inserted at the 3' end of a cro-iacz expression vector (stanley and luzio, 1984) . expression of this gene is under control of the lambda p, promotor and induced by inactivation of the temperature sensitive cl repressor at 42°c. the resulting expression products precipitate inside the cell and are thus protected against proteolysis. after solubilization, the product of the foreign gene fragment, linked to the c-terminal residue of the /?-galactosidase part, may be recognized by appropriate antisera. figure 1 shows the hinfl, pst i, sau3ai and sau3ai/psti fragments that have been excised from the genomic cdna plasmids p39 or p42 (niesters et al., 1984) and expressed in pex plasmids. the hinfl site in the region encoding the n-terminal signal sequence of the peplomer protein allowed the cloning of the 5' part of the coding sequence without the stop codons just upstream of (and in frame with) the atg start codon. as judged from an sds-polyacrylamide gel [ fig. 2(a) ], the hybrid proteins have sizes predicted by the lengths of the insertions. exceptions were ps4, which contains the stop codon of the peplomer gene, and the 144 bp fragment p4, which was inserted twice, both fragments expressed in tandem and in the same reading frame. analysis of the sequence showed that only in the case of hl and p4, hybrid proteins of (almost) the same size could be obtained after translation in a wrong reading frame; the proper reading frames of these clones have been checked by sequencing. fragment pl starts with a stretch of 12 dg residues (originating from ~42); apparently, this did not disturb the reading frame by ribosome slippage along the homopolymer track (stanley, 1983) . the ibv peplomer sequences contained in the hybrid proteins are listed in table 1 . only residue 1064 has not been overlapped by an expression product. further, a reactive region exactly on the junction of two fragments or in the gap between p4 and p5 can only be detected in an overlapping fragment if this fragment does not contain other reactive regions. in the theoretically worst case, the reactivity of 8% of the total peplomer sequence would not be detected, assuming an epitope size of 10 residues. in fig. 2(b) , a western blot of the ibv/pex hybrid proteins, incubated with a rabbit antiserum, is shown. only three regions were recognized strongly, one contained in p2, ~4, psl and ps2, the second in p3 and ~4, and the third in ps4. weaker signals were observed for the regions contained in p4, in p5 while not in ps3, and in the overlap of s5 and p6, respectively. have been carried out with another rabbit antiserum, with a mouse antiserum and with four antisera from chickens, infected with ibv strain m41 (chickens 1 and 2) or with strain d207 (chickens 3 and 4) . the binding patterns are summarized in fig. 3 . because of high background signals of the chicken antisera, only strong signals cquld be scored. apparently, each antiserum recognized a different set of epitopes. the number of the positions of the restriction-enzyme fragments of the peplomer gene are shown in fig. i . the fragments were ligated to linearized pex 1, -2 or -3 as indicated. the last column indicates which parts of the amino acid sequence of the peplomer protein are synthesized by the recombinants after induction of the expression. antigenic regions as defined by the restriction fragments varies from 1 (mouse) to 6 (rabbit 1). one region, corresponding to fragment ps2, was recognized by all homologous and heterologous antisera. (koch et al., unpublished results) . none of these sera bound any of the pex products, most likely because the epitopes of these antisera are conformation dependent. however, the lack of binding of other, non-neutralizing monoclonal antibodies recognizing conformation independent epitopes on the peplomer protein suggests that other factors than the conformation dependence may affect the reactivity of monoclonal antibodies with pex hybrid proteins. koch et al. (1986) described a number of monoclonal antisera with weak neutralizing activity that bound to the s2 subunit on western blots. one of these, mab 26.1, recognized all ibv strains tested and has a neutralization titre of about 10'. figure 2 (c) shows a western blot of hybrid proteins screened with this mab. the strong binding to fragments p2, s4, psl and ps2 localizes the epitope of mab 26.1 between residues 390 and 612, the same region that is recognized by all polyclonal sera. since subunit s2 starts at residue 538, the epitope must lie in the n-terminal region (residues 538 to 612) of s2. the same localization has been found for the epitopes of another s2-specific mab 31.5. to further localize the epitope of mab 26.1, a library of small random dnase fragments in pex has been constructed. after immunoscreening, 0.2% of the colonies were found to react positively with mab 26.1. six clones were selected for sequence analysis after subcloning in ml3 replicative-form dna. the results are summarized in fig. 4 . all clones contain nucleotides 1620-1673, restricting the localization of the epitope of mab 26.1 to residues 541-558. figure 5 shows the antigenicity of four expression products of the dnase fragments to mab 26. antisera and fragments are listed in table 2 . the antiserum from chicken 1 binds to the expression products of the same fragments as mab 26.1, indicating the recognition of the same or an adjoining epitope. the results with antiserum of chicken 2 were clouded by a particularly high background reaction, but the clear positive reactions with two of the fragments indicate a binding to the same region. interestingly, the negative reactions of fragments (15841682) and (1617-1673) demonstrate that the epitope(s) of mab 31.5 and of the scra of rabbit i, rabbit 2 and mouse are located partially or completely in the region 562-574. the positions of the dnase defined regions reactive with the polyclonal sera are graphically represented in fig. 3 by short horizontal lines. the delineations of the individual epitopes are indicated in fig. 4 by heavy lines, the dashed line indicating the possibility that the region 541-561 contributes to the epitopes recognized by the mab 31.5 and by the rabbit and mouse antisera. in indirect elisas on western blots with intact viral proteins, mabs 26.1 and 31.5 have different specificities (koch et al., 1986. unpublished results). mab 26.1 recognizes the $2 subunit of all strains tested, while mab 31.5 only binds to s2 of the h120/b222/m41 serotype. the same specificity was found with pex expression products: mab 26.1 binds to the hybrid proteins of strains m41, d207 and d1466; mab 31.5 only to m41 products. an accurate definition of the epitope of mab 26.1 and other epitopes in the same region, based on the sequences of several ibv strains, on pepscan peptide synthesis (geysen et al., 1984) and on competition experiments will be published elsewhere (kusters et al.; koch et al., in preparation) . in the pex system, the expression product of an inserted dna fragment constitutes the c-terminus of a hybrid /l-galactosidase protein, which precipitates in the cell. prior to the assay of its antigenicity, the hybrid protein is solubilized by sds and transferred to a nitrocellulose filter. presumably, epitopes found by cross-reaction with antisera are essentially conformation independent. the localization of epitopes within stretches of 10-20 residues in several coronaviruses (fig. 4 , unpublished results) demonstrates that the binding of antibody does not depend on particular flanking sequences, and that any nativelike folding of the epitope in the hybrid protein is confined to the same small region. classically, continuous epitopes are localized by testing the antigenicity of peptides (atassi, 1984) . prokaryotic expression of subgenomic fragments in pex or in i.gtll (nunberg et ul., 1984; mehra et al., 1986 ) offers a convenient alternative and is not limited to small proteins. we have combined the expression of large (13&1400 bp) restriction fragments to localize antigenic regions, and of small dnase fragments to delineate epitopes more accurately. another strategy is the subcloning of small kusters et al., luytjes et al., unpublished results) . this obviates the sequence analysis of dnase fragments, but depends on the presence of suitable restriction sites or on the availability of homologous sequences. another new and complementary approach to the localization of continuous epitopes is the synthesis of a large number of peptides coupled to a solid phase, the so-called pepscan (geysen et al., 1984) . this method has been used in conjunction with the pex expression system for the localization of a neutralization epitope of the peplomer protein of mouse hepatitis virus (luytjes et al., unpublished results). however, not all monoclonal antibodies that cross-react with pex expression products bind to pepscan peptides spanning the antigenic region (posthumus and meloen, unpublished results). for instance, mab 26.1 did not recognize any of the overlapping nonapeptides spanning the n-terminal region of the s2 subunit. whether this should be explained by the lengths of the peptides or by local conformations in the hybrid proteins will be the subject of further study. the reactivity patterns of the different polycionaf antisera (fig. 3 ) outline the variability of the humoral immune response against the ibv peplomer protein at the molecular level. there are also a number of other noteworthy features. firstly, all antisera recognize in the 1162-residue sequence a relatively small (l-6) number of regions, predominantly in the s2 subunit; the actual number of epitopes per antiserum depends on the degree of clustering of epitopes within the antigenic regions. so, if the whole surface of the proteins is potentially antigenic (berzofsky, 1985; geysen et al., 1987) , this is clearly not the case for the determinants detected as antigenic pex hybrid proteins. secondly, the n-terminal part of the s2 subunit is recognized by all polyclonal antisera tested and by a number of monocional antibodies. within this region, the monoclonal and polyclonal antibodies recognized different epitopes ( table 2 ). the mabs have neutraiization titres ranging from 10 to 1000 (koch et al., 1986 ) suggesting a role of this region in the neutralization of the infection. thirdly, the regions found to be antigenic do not coincide with the hypervariable regions in the sl subunit (niesters et al., 1986; binns et al., 1986) . these regions are supposed to be part of neutralization epitopes that are the targets of antigenie variation. in fact, although the sl subunit is thought to induce the protective immunity against ibv (cavanagh et al., 1986) we found it to be relatively, if not completely, devoid of antigenicity (fig. 3) . as the most obvious explanation, we propose that the neutralization epitopes in st are conformation dependent and cannot be mimicked by pex hybrid proteins. this would also explain the lack of reaction of the neutralizing mabs that were directed against epitopes in si. apparently, these epitopes are of the same category as the conformation dependent neutralization epitopes of the haemagglutinin of influenza virus (wiley et al., 1981) . in contrast, the epitopes localized in the n-terminal region of s2 may be comparable to the epitopes of foot-and-mouth disease virus, detectable by cross-reaction with peptides (geysen et al.. 1984; rossman et al., 1985) . respectively. the rabbit antisera were generously provided by ms n.m.c. bleumink-pluvm and dr h. g. m. niesters (veterinary faculty). the experimental contributions of antigenic structures of proteins intrinsic and extrinsic factors in protein antigenic structures comparison of the spike precursor sequences of coronavirus ibv strains m41 and 6/82 with that of ibv beaudette a rapid, sensitive method for detection of alkaline phosphatase-conjugated antiantibodies on western blots coronavirus ibv: structural characterization of the spike protein by virtue of their continuous epitopes often correspond to relatively inherent cross-reactivity with short peptides, these coronaviruses use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid chemistry of antibody binding to a protein efficient construction of cdna libraries in plasmid expression vectors using an adaptor strategy antigenic differentiation of avian bronchitis virus variant strains employing monoclonal antibodies molecular epidemiology of infectious branchitis virus in the netherlands mokectttar cloning, a laboratory manual. cold spring harbor laboratories efficient mapping of protein antigenic determinants monoclonal antibodies to the sl spike and membrane proteins of avian infectious bronchitis virus strain massachusetts m41 the pcplomer protein sequence of the m41 strain of coronavirus ibv and its comparison with beaudette strains epitopes on the peplomer protein of infectious bronchitis virus strain m4i as defined by monoclonal antibodies method to map antigenic determinants recognized by monoclonal antibodies: localization of a determinant of virus neutralization on the feline leukemia virus envelope protein ~~70 the biology of coronaviruses solubilization and immune-detection of p-galactosidase hybrid proteins carrying foreign antigenic determinants ~onst~ction of a new family of high efficiency bacterial expression vectors: identification of cdna clones coding for human liver proteins antigenic crossreactivity between proteins and peptides: new insights and applications correlation between segmental mobility and the location of antigenic determinants in proteins frankenberger identification of the antibodv-combining site of hone e mobile regions in the protein structure (westhof et regions are the prime candidates for the development of peptide vaccines (van regenmortel, 1987) . thus, for the n-terminal region of the ibv s2 subunit. structural considerations may outweigh the relatively weak neutralization, if compared with the epitopes in sl (mockett et al., 1984; koch et al., further, m41 sequences cross-react with chicken antisera against d207, belonging to a different serotype, (fig. 3, table 2 ) and vice versa (not shown). apparently, this region is not subject to antigenic variation. this would be another advantage of the epitopes found in 52.thirdly, the recognition of the n-terminal s2 sequence by several polyclonal antisera indicates that this region is strongly immunodominant.this would favour a response in all vaccinated animals.these considerations may become relevant as soon as the technology for designing effective peptide vaccines becomes available. key: cord-294575-kky8j9oy authors: li, jieqiong; sun, lin; xu, fang; xiao, jing; jiao, weiwei; qi, hui; shen, chen; shen, adong title: characterization of plasma proteins in children of different mycobacterium tuberculosis infection status using label-free quantitative proteomics date: 2017-09-23 journal: oncotarget doi: 10.18632/oncotarget.21179 sha: doc_id: 294575 cord_uid: kky8j9oy tuberculosis (tb), caused by mycobacterium tuberculosis (mtb), is an infectious disease found worldwide. children infected with mtb are more likely to progress to active tb (atb); however, the molecular mechanism behind this process has long been a mystery. we employed the label-free quantitative proteomic technology to identify and characterize differences in plasma proteins between atb and latent tb infection (ltbi) in children. to detect differences that are indicative of mtb infection, we first selected proteins whose expressions were markedly different between the atb and ltbi groups and the control groups (inflammatory disease control (idc) and healthy control (hc) groups). a total of 521 proteins differed (> 1.5-fold or < 0.6-fold) in the ltbi group, and 318 proteins in the atb group when compared with the control groups. of these, 49 overlapping proteins were differentially expressed between ltbi and atb. gene ontology (go) analysis revealed most proteins had a cellular and organelle distribution. the mtb infection status was mainly related to differences in binding, cellular and metabolic processes. xrcc4, pcf11, sema4a and atp11a were selected and further verified by qpcr and western blot. at the mrna level, the expression of xrcc4, pcf11and sema4a presented an increased trend in atb group compare with ltbi. at the protein level, the expression of all these proteins by western blot in atb/ltbi was consistent with the trends from proteomic detection. our results provide important data for future mechanism studies and biomarker selection for mtb infection in children. tuberculosis (tb), caused by mycobacterium tuberculosis (mtb), continues to be a serious infectious disease worldwide [1, 2] . it is estimated that most active tb (atb) cases originate from an initial latent tb infection (ltbi), a state without any clinical symptoms, radiological abnormality, and microbiological evidence. in contrast to adults, young children infected with mtb are more likely to progress to atb within the first year of primary infection; however, the molecular mechanisms behind this have long been a mystery [3] . mtb infection alters the expression of tbassociated proteins, which are released into the bloodstream through different pathways [4] . thus, analysis of tb-associated proteins in patients with different mtb infection status might reflect the mechanism of mtb infection and progression. plasma proteins, such as cytokines, extracellular secretory proteins, and other soluble factors involved in the immune system, are reported to be associated with the pathogenesis of infectious disease [5] . for instance, shuxian li et al. revealed that the proteins secreted by a549 cells were associated with the infection of mycoplasma pneumoniae [6] . dandan xu and her colleagues found that the level of serum proteins (s100a9, sod3, and mmp9) in atb reflected the immune response to the mtb infection [5] . together, the above information implies an important role for proteins in host-pathogen interactions. recently, advances in comprehensive analytical techniques, such as proteomics, make the analysis of all plasma proteins possible [7] . proteomic analysis using the label-free quantitative protocol is a novel approach for high throughput analysis, which provides for a rapid comparison of proteins. given that a specific mixture of plasma proteins has distinctive characteristics, this technique has been used to investigate the proteomic patterns in many diseases, including infectious diseases [8] [9] [10] [11] , vascular diseases [12] [13] [14] and cancer [15] . thus, analysis of the expression of tb-associated proteins in plasma could provide a better understanding of conversion from ltbi to atb in children. therefore, this study aimed to characterize the plasma proteins in children of different mtb infection status by label-free quantitative proteomics. we observed that 49 proteins were differentially-expressed in the ltbi and atb groups, and a subset was validated using quantitative real-time pcr (qpcr) and western blotting. our results will provide insight into the mechanism of mtb infection and progression, as well as potential biomarker selection for the diagnosis of atb in children. the demographic characteristics of participants showed no obviously differences between groups (table 1 ; age, p = 0.625; gender, p = 0.318). among the 19 children in the atb group, 12 had pulmonary tb, three had tubercular meningitis, one had bone tuberculosis and three had systemic tuberculosis. among the 17 children in the idc group, 10 had pneumonia (including both bacterial and viral infections), and seven children had upper respiratory tract infections. furthermore, the children in inflammatory disease control (idc) and healthy control (hc) groups had negative interferon gamma release assay (igras) and tuberculin skin test (tst) results, without a history of atb. all the subjects were vaccinated with bcg. as respiratory infections are often present in children who have atb or ltbi, both idc and hc groups were included in this proteomics analysis. based on the high throughput proteomics data, a total of 2170 proteins were identified. the plasma, pooled for each group was divided into 3 samples and each liquid chromatographymass/mass spectrometry (lc-ms/ms) analysis was repeated 3 times. to detect the proteins that are indicative of mtb infection, we first selected specific proteins whose expression was markedly different between the atb and control (idc and hc) groups, and between the ltbi groups and control (idc and hc) groups. as shown in figure 1 , a total of 318 proteins differed in the atb group, and 521 proteins in the ltbi group (> 1.5 fold or < 0.6 fold) when compared with the hc and idc groups. of these proteins associated with mtb infection, 49 proteins were differentially expressed between atb and ltbi. specifically, 41 proteins were up-regulated (> 1.5 fold) and 8 proteins were down-regulated (< 0.6 fold) in the atb group compared with the ltbi group (table 2) . to reveal the relationship between the 49 different proteins, a hierarchical clustering based on pearson correlation of variances was applied using r studio. figure 2a depicts hierarchical clustering of the 49 identified proteins, where an increasingred color indicates increasing protein expression levels. thus, the most prominent area of up-regulation in atb subjects was seen in the region where these protein peaks are shown in red. furthermore, important features were selected by a volcano plot ( figure 2b ). to further understand the functions of these identified proteins, we classified the 49 differential proteins between ltbi and atb by their gene ontology (go) categories, including cellular component (cc), molecular function (mf), and biological process (bp) using the web gene ontology annotation plotting (wego, figure 3 , supplementary table 1 and 2). according to the analysis of cc, the majority of the proteins had a cell and organelle distribution. according to the analysis of bp, the primary functions were cellular processes, metabolic processes and biological regulation; while others were related to cellular component organization, pigmentation, multicellular organismal processes and so on. according to the analysis of mf, these proteins could be classified into eight categories as follows: molecular transducer activity, translation regulator activity, enzyme regulator activity, transcription regulator activity, structural molecule activity, catalytic activity, binding, transporter activity. among them, a large proportion had binding as a molecular function. to identify the most important proteins involved in the conversion from ltbi to atb, proteins that were obviously up or down-regulated should be categorized into many different functional groups. kegg enrichment analysis was then implemented to test enrichment of the proteomics pathways between the ltbi and atb groups. the results indicated that mrna surveillance pathway (p = 0.018), non-homologous endjoining (p = 0.034) and spliceosome (p = 0.040), one carbon pool by folate (p = 0.049) were significantly associated with the conversion from ltbi to atb ( figure 4 and supplementary table 3 ). proteins identified as differentially expressed in atb and ltbi patients were validated using qpcr and western blotting. among the 49 different proteins, four (xrcc4, pcf11, sema4a and atp11a) were selected for further verification. the selection of the proteins for the verification stage was based on the following criteria: (1) high fold change; (2) being representative of the 49 different proteins; (3) p value of <0.05 in kegg analysis (xrcc4 and pcf11) or having biological functions associated with immunity and other infectious diseases (sema4a and atp11a). although these four proteins were mainly distributed in cellular parts, their functions most proteins different between the atb and ltbi groups were related to binding, cellular and metabolic process and these four selected proteins were associated with these processes. firstly, the four selected proteins all were also participate in the binding processes, including nucleic acid binding and protein binding. additionally, xcrr4 and pcf11 had an effect on metabolic process while sema4a and atp11a were related to the cellular process. thus, these four proteins, being representative of the 49 different proteins, reflected the different status of mtb infection. at the mrna level, the expression of xrcc4, pcf11and sema4a presented an increased trend in atb group compare with that in ltbi and this agreed with the proteomic results. the ratio of atp11a was apparently inconsistent with the proteomic data ( figure 5a ). the mrna result implies that regulation is occurring at the point of protein translation, not gene transcription. at the protein level, our results demonstrated that the plasma levels of xrcc4, pcf11, sema4a and atp11a in the atb group were significantly higher than the ltbi group (all p < 0.05; figure 5b ). representative western blots are shown in figure 5c . these ratios were consistent with the trends of proteomics results. total protein staining by coomassie blue employed as the loading control ( figure 5d ). it is generally known that the immune response induced by mtb infection influences the pathogenic mechanisms and protein expression [16] . specifically, tbassociated proteins appear in the circulation by a variety of mechanisms, including direct secretion of proteins, stimulated production of reactive proteins, or production of proteins during the infection of the mtb [17, 18] . accurate detection tb-associated proteins lead to a better understanding of the mechanism of mtb infection and progression. plasma proteins including cytokines, extracellular secretory proteins, growth factors, and other soluble factors, contribute to many physiological processes. recently, researchers have demonstrated that proteins are also significantly associated with different diseases. for instance, the secretion of adiponectin is associated with the development of many cardiovascular related diseases [19] . similarly, plasma proteins also have profound effects on a variety of infectious diseases. for example, during herpes simplex virus 1 (hsv-1) infection, increased ifninduced protein expression is reported to play an essential role in extracellular antiviral functions [20] . similarly, the secretion of proteins may increase the number of cd4 + t cells [21] . taken together, the above data show the importance of plasma proteins during pathogen infection. this study demonstrated a difference in plasma protein pattern in mtb infection in children. as respiratory infections are often present in children who have atb or ltbi, we included both idc and hc groups in this proteomic analysis. to detect differences that are indicative of mtb infection, we first selected specific proteins whose expression was markedly different in atb or ltbi groups compared with controls (the idc and hc groups). among these proteins, 49 were differentially expressed between ltbi and atb. these proteins were further performed by volcano plot. the traditional volcano plot is a combination of fold change and t-tests. note, for unpaired samples, the x-axis is log (fc). y-axis is -log10 (p-value) for both cases, and can be based on raw or fdr adjusted p values. however, our data alignment strategy is: disease control and normal control, respectively, compared with atb and ltbi, followed look at the difference between the significant difference between the part of atb and ltbi. in our data, the three biological repetitions and three technical repetitions combined after the comparison, so cannot achieve the traditional meaning of the volcano show. figure 2b just showed the different proteins between atb and ltbi. these plasma proteins were further subjected to a go enrichment analysis which clustered proteins based on three defined ontology terms: go-cc, go-bp and go-mf [22] . here, we observed that most of these proteins were present in the isolated cell or organelle fraction. similarly, released intracellular proteins were detected in mtb-infected plasma, suggesting the importance of these associated proteins at the lesions created by mtb infection. in addition, other identified proteins were associated with protein binding. these results demonstrate that the proteins affecting the mtb infection status were related to dna and protein binding, cellular and metabolic processes. among the 49 differentially expressed proteins, xrcc4, pcf11, sema4a and atp11a were selected for further validation. the above functional analysis demonstrated that most different proteins influencing the mtb infection status were mainly related to binding, cellular and metabolic processes. the four selected proteins were all associated with these processes. it is well known that binding is the first step of the mtb infection and these four selected proteins were all participate in the binding process, including nucleic acid binding and protein binding. furthermore, mtb infection also changes metabolic processes. xcrr4 and pcf11 influence metabolic processes and were significantly different between the atb and ltbi groups. according to the go analysis, sema4a and atp11a, which are related to cellular processes, also had an important role in mtb infection. the levels of mrna after mtb infection did not correlate with the proteins level. a similar result demonstrated that when the most induced proteins in human mesenchymal stromal cells (hmscs) were assayed by qpcr, the results do not match the results detected by ms [23] . this is a limitation of the current study. the selected proteins have previously been reported to be associated with immunity and other infectious diseases [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] . xrcc4 is an important component of non-homologous end-joining [24] . the expression of this gene is associated with many infectious diseases, including hsv-1 [25, 26] . in addition, pcf11serves as a target for regulated transcription and pre-mrna processing. recent studies showed that it is also involved in regulated transcription initiation, elongation, termination and alternative processing of pre-mrna [27] [28] [29] . sema4a is expressed by dendritic cells, macrophages, and activated t cells [30, 31] . it is involved in co-stimulation in helper t cell proliferation and cytokine production [32, 33] . higher levels of sema4a in atb group implied the higher t cell activation in mtb infection than in ltbi. atp11a, a member of p4-atpases, is a lipid flippase [34] . a previous study demonstrated that lipid flippases are essential to mediate the endotoxin-induced endocytosis of toll-like receptor 4 in human macrophages. macrophages play a primary role in tb transmission [35] . mtb usually replicates within macrophages and spreads to pulmonary lymph nodes. furthermore, lipid flippase is also important to dampen the inflammatory response, implying that atp11a had an important effect in mtb infection. our results indicate, for the first time, that the expression of these proteins were also correlated with the conversion from ltbi to atb in children. because of the difficulty of enrolling ltbi pediatric subjects, a limitation of this study is the relatively small sample size used for verification. thus, a larger sample size and further mechanism analysis should be the focus of future investigations. in conclusion, we identified 49 differentially expressed plasma proteins related to the differential status of mtb infection by the label-free quantitative method. after analyzing the protein functions and regulatory networks, xrcc4, pcf11, sema4a and atp11a were selected and further verified by qpcr and western blot. our results will provide important data for molecular mechanism studies and biomarker selection during mtb infection. respiratory infectious diseases overlapping with tb in children are very common. therefore, to detect the differences that are indicative of mtb infection, idc and hc were also included in this study. a total of 72 children (19 atb, 16 ltbi, 17 idc and 20 hc) were enrolled at beijing children's hospital between july 2010 to may 2013. the diagnosis of pediatric atb was according to the guidelines of the chinese medical association: (1) mtb culture result was positive, or (2) at least one symptom, sign, or radiographic evidence was consistent with atb, or (3) clinical and radiological improvement was seen following anti-tb chemotherapy [2] . to accurately reflect the mtb infection status, all the atb sera were collected before anti-tb chemotherapy. ltbi was defined as (1) positive by both igras and tst, (2) exposure history to a known atb case, (3) no clinical, radiological and microbiological evidence of active tb [36] . idc group subjects were enrolled according to their respiratory symptoms and negative igras and tst results (<5mm) to exclude atb and ltbi. hc group subjects were recruited from children admitted to beijing children's hospital for physical examination from june 2012 to may 2013. the idc and hc controls with negative igras and tst results, had not been previously infected with tb, hiv or other infectious diseases. each group was matched by age, sex, and ethnicity. at the qpcr verification stage, we utilized the peripheral blood mononuclear cell (pbmc) samples from 28 atb (18 boys and 10 girls; median age 5.0 years, range 0.4-12 years) and 18 age-and gender-matched ltbi subjects(12 boys and 6 girls; median age 5.6 years, range 0.25-15 years). at the western blot verification stage, the plasma of 5 atb (3 boys and 2 girls; median age 8.3 years, range 3-12 years) and 5 age-and gender-matched ltbi subjects (2 boys and 3 girls; median age 7.6 years, range 3-11 years) were collected. the diagnosis criteria of pediatric atb and ltbi outlined above. all the subjects had the positive igras and tst results. the study was approved by the ethics committee of beijing children's hospital. all the methods and experimental protocols in this research were performed according to the approved protocols and the ethics committees' existing guidelines. the parents of all children provided written, informed consent prior to their enrollment in the study. human plasma samples were pooled for each group and divided into 3 aliquots. albumin, igg, iga, antitrypsin, transferrin and haptoglobin were removed by the agilent multiple affinity removal column (multiple affinity removal column, 4.6 mm ã� 50 mm; agilent technologies, palo alto, ca, usa). after being resuspended with pbs (50 î¼l), the samples were centrifuged at 10,000g for 30 min in 4â°c and resuspended with 100 î¼l lysis buffer (7 m urea, 2 m thiourea). total proteins were extracted by ultrasonic sonication and precipitated with trichloroacetic acid (tca) for 30 min on ice before centrifuging at 40,000g for 30 min. next, the protein concentration was adjusted with 50 mm nh 4 hco 3 to a final concentration of 0.5mg/ml and then mixed with dtt (5î¼l, 1mol/l) and incubated at 37â°c for 60 min. after incubation with iaa (20î¼l, 1mol/l) for 60 min in the dark, the samples were digested with trypsin at 37â°c for 12 h [17] . peptide mixtures were used for protein identification by nano-liquid chromatography coupled with ms. the lc-ms/ms system consisted of an agilent 1100 quaternary hplc (easy-nlc1000) and a microtof-q ii mass spectrometer (bruker daltonics, usa) with the application of a distal 180â°c source temperature. a rp trapcolumn (thermo easy column sc200 150î¼mã�100mm) was used for desalting, and a c18 reverse-phase column (thermo easy column sc001 traps 150î¼m ã� 20mm) was used for separation. mobile phase a consisted of hplc-grade water containing 0.1% formic acid (fa), and phase b consisted of 84% hplc-grade acetonitrile (acn) containing 0.1% fa. the analytical separation was run at a flow rate of 400 nl/min using a linear gradient of phase b as follows: 0-45% for 100 min, 45-100% for 8 min and 100% for 12 min. to reduce the technical variation the analyses were repeated three times [37] . peptide spectral matches (psms) were validated using the percolator provided by the proteome discoverer software based on q-values at a 1% false discovery rate. normalization by a reference sample, also known as probabilistic quotient normalization, is a robust method to account for different dilution effects of biofluids. this method is based on the calculation of a most probable dilution factor (median) by looking at the distribution of the quotients of the amplitudes of a test spectrum by those of a reference spectrum [38] . for protein identification, the mascot 2.1 program (matrix science, boston, ma, usa) was used for fragmentation spectra research, which was searched against the human database. two missed cleavages were permitted, and an error of 6 ppm or 20 ppm was allowed for full ms or ms/ms spectra study, respectively. furthermore, we used the resulting mgf documents and data analysis 3.4 from bruker daltonics software to detect the fold-changes of the protein levels of each group. the identified proteins were analyzed by go categories using the web gene ontology annotation plotting (wego, http://wego.genomics.org.cn/cgi-bin/wego/index. pl). signaling pathway analysis was performed by kyoto encyclopedia of genes and genome (kegg) database (http://www.genome.jp/kegg/pathway.html) [39] . proteins used for validation were selected by molecular function and the biological process or pathway term in panther. total rna was obtained using mirneasy mini kits (qiagen, valencia, ca, usa) according to the manufacturer's instructions. subsequently, 500 ng of the rna was reverse transcribed to cdna using revertra ace qpcr rt kits(toyobo, osaka, japan), and quantitative real-time pcr was carried out with a 7900 ht sequence detection system (abi, foster city, ca, usa) using abi power sybr green pcr master mix. the raw data were normalized using the quantile algorithm in genespring 11.0 (agilent technologies, santa clara, ca, usa). the thermal cycling conditions were: 2 min at 95â°c for initial denaturation, followed by 40 cycles of 15 sec at 95â°c, 60 sec at 60â°c for amplification, and 15 sec at 95â°c, 15 sec at 60â°c and 15 sec at 95â°c for melting curve analysis [6] . target gene primers are listed in supplementary table 4 . western bloting was performed as described previously, with minor differences [6] . briefly, proteins extracted from plasma, were separated using 12% sds-page gels and transferred to pvdf membranes (millipore, billerica, ma, usa). for detection, after incubation with primary antibodies overnight at 4â°c, the membranes were washed three times with tris-buffered saline containing tween-20 (tbs-t), and incubated with horseradish peroxidase (hrp) -conjugated secondary antibodies (1:5000; santa cruz, dallas, tx, usa) for 2 h at room temperature. antibody binding was visualized using the enhanced chemi luminescence kit (ecl-kit, santa cruz) after being washed in pbs-t. the primary antibodies used in this experiment included rabbit polyclonal anti-xrcc4 antibody (abcam, cambridge, ma, usa, diluted 1:1000), rabbit polyclonal anti-pcf11 antibody (abcam, diluted 1:2000), rabbit polyclonal anti-sema4a antibody (abcam, diluted 1:1000), rabbit polyclonal anti-atp11a antibody (santa cruz, diluted 1:1000), and mouse monoclonal anti-î²actin antibody (upstate, lake placid, ny, usa; diluted 1:2000). ten î¼g protein was loaded per lane of a 1-mmthick mini polyacrylamide sds-gel. the proteins were stained with coomassie blue as an internal control for normalization [40] proteins were transferred to a nc membrane for 1h using bio-rad trans-blot. after being blocked with 3% bsa for 30min, the target proteins were incubated with primary antibody and associated secondary to each blot with at least four, 3-minute wash steps. www.impactjournals.com/oncotarget each experiment was repeated independently at least three times. data are expressed as the mean â± sd and differences between groups were evaluated with the student t-test or mann-whitney u test. values of p<0.05 were considered statistically significant. global, regional, and national incidence and mortality for hiv, tuberculosis, and malaria during 1990-2013: a systematic analysis for the global burden of disease study a 3'utr polymorphism of il-6r is associated with chinese pediatric tuberculosis paediatric tuberculosis the discovery and identification of a candidate proteomic biomarker of active tuberculosis serum protein s100a9, sod3, and mmp9 as new diagnostic biomarkers for pulmonary tuberculosis by itraq-coupled two-dimensional lc-ms/ms global secretome characterization of a549 human alveolar epithelial carcinoma cells during mycoplasma pneumoniae infection what can proteomics tell us about tuberculosis? identification of diagnostic markers for tuberculosis by proteomic fingerprinting of serum biomarker discovery in infectious diseases using seldi a novel and accurate diagnostic test for human african trypanosomiasis the use of proteomics in the discovery of serum biomarkers from patients with severe acute respiratory syndrome potential biomarkers of acute cerebral infarction detected by seldi-tof-ms the potential biomarkers for thromboembolism detected by seldi-tof-ms predicting left ventricular remodeling after a first myocardial infarction by plasma proteome analysis clinical proteomics: searching for better tumour markers with seldi-tof mass spectrometry the ins and outs of mycobacterium tuberculosis protein export diagnostic serum proteomic analysis in patients with active tuberculosis immunogenic membraneassociated proteins of mycobacterium tuberculosis revealed by proteomics therapy insight: adipocytokines in metabolic syndrome and related cardiovascular disease global secretome characterization of herpes simplex virus 1-infected human primary macrophages human cytomegalovirus latency alters the cellular secretome, inducing cluster of differentiation (cd)4+ t-cell migration and suppression of effector function the goa database in 2009-an integrated gene ontology annotation resource mechanisms of kidney repair by human mesenchymal stromal cells after ischemia: a comprehensive view using label-free ms(e) dynamic assembly of end-joining complexes requires interaction between ku70/80 and xrcc4 knockdown of dna ligase iv/xrcc4 by rna interference inhibits herpes simplex virus type i dna replication e1b 55k-independent dissociation of the dna ligase iv/xrcc4 complex by e4 34k during adenovirus infection the receptor tyrosine kinase ron represses hiv-1 transcription by targeting rna polymerase ii processivity a genetic screen for terminator function in yeast identifies a role for a new functional domain in termination factor nab3 the regulatory role of pcf11-similar-4 (pcfs4) in arabidopsis development by genome-wide physical interactions with target loci class iv semaphorin sema4a enhances t-cell activation and interacts with tim-2 semaphorin 4a exerts a proangiogenic effect by enhancing vascular endothelial growth factor-a expression in macrophages nonredundant roles of sema4a in the immune system: defective t cell priming and th1/th2 regulation in sema4a-deficient mice semaphorin4a is cytotoxic to oligodendrocytes and is elevated in microglia and multiple sclerosis atp11a is a novel predictive marker for metachronous metastasis of colorectal cancer phospholipid flippases attenuate lps-induced tlr4 signaling by mediating endocytic retrieval of toll-like receptor 4 a proteomics approach to the identification of plasma biomarkers for latent tuberculosis infection proteomic analysis of extracellular vesicles derived from mycobacterium tuberculosis probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures. application in 1h nmr metabonomics the kegg resource for deciphering the genome regulation of hp1-chromatin binding by histone h3 methylation and phosphorylation we are grateful to all our study participants. the authors have declared no conflicts of interest. key: cord-267475-6f4h3cck authors: kozak, marilyn title: pushing the limits of the scanning mechanism for initiation of translation date: 2002-10-16 journal: gene doi: 10.1016/s0378-1119(02)01056-9 sha: doc_id: 267475 cord_uid: 6f4h3cck selection of the translational initiation site in most eukaryotic mrnas appears to occur via a scanning mechanism which predicts that proximity to the 5′ end plays a dominant role in identifying the start codon. this ‘position effect’ is seen in cases where a mutation creates an aug codon upstream from the normal start site and translation shifts to the upstream site. the position effect is evident also in cases where a silent internal aug codon is activated upon being relocated closer to the 5′ end. two mechanisms for escaping the first-aug rule – reinitiation and context-dependent leaky scanning – enable downstream aug codons to be accessed in some mrnas. although these mechanisms are not new, many new examples of their use have emerged. via these escape pathways, the scanning mechanism operates even in extreme cases, such as a plant virus mrna in which translation initiates from three start sites over a distance of 900 nt. this depends on careful structural arrangements, however, which are rarely present in cellular mrnas. understanding the rules for initiation of translation enables understanding of human diseases in which the expression of a critical gene is reduced by mutations that add upstream aug codons or change the context around the aug(start) codon. the opposite problem occurs in the case of hereditary thrombocythemia: translational efficiency is increased by mutations that remove or restructure a small upstream open reading frame in thrombopoietin mrna, and the resulting overproduction of the cytokine causes the disease. this and other examples support the idea that 5′ leader sequences are sometimes structured deliberately in a way that constrains scanning in order to prevent harmful overproduction of potent regulatory proteins. the accumulated evidence reveals how the scanning mechanism dictates the pattern of transcription – forcing production of monocistronic mrnas – and the pattern of translation of eukaryotic cellular and viral genes. the scanning mechanism for initiation of translation postulates that the small (40s) ribosomal subunit enters at the 5 0 end of the mrna and migrates linearly, stopping when the first aug codon is reached. consistent with the postulated 5 0 end-dependent entry of ribosomes, translation in vivo is strongly augmented by the m7g cap (furuichi and shatkin, 2000; horikami et al., 1984; lo et al., 1998; neeleman et al., 2001) and ribosome binding in vitro is prevented by circularization of the mrna (kozak, 1979a; konarska et al., 1981) . perhaps because the scanning mechanism has been around for a while, the evidence for some basic points has been forgotten. one recent commentary even questions whether the 40s ribosomal subunit has anything to do with it (mathews, 2002) . the easiest answer is that the stopscanning step is clearly mediated by pairing of the initiation codon with the anticodon in met-trna i (cigan et al., 1988a) , and the 40s ribosomal subunit is the carrier of met-trna i ·eif2. but the 40s subunit was already implicated by experiments done earlier. the experiments that gave rise to the scanning model concerned unusual polysome-like complexes formed in the presence of edeine, an antibiotic which blocks recognition of the aug codon (kozak and shatkin, 1978) . analysis of the rapidly sedimenting complexes revealed 40s ribosomal subunits distributed throughout the body of the mrna. because control experiments showed that, even in the presence of edeine, ribosomes can enter only from the 5 0 end, the simplest explanation was that 40s subunits enter at the 5 0 end and then migrate into the interior of the mrna; in the absence of edeine, the migration would stop when an aug codon is reached. independent experiments confirmed that edeine is targeted to the ribosome (herrera et al., 1986) , and use of a fractionated translation system confirmed that the edeine-induced complexes are formed by 40s but not 60s ribosomal subunits (kozak and shatkin, 1978; kozak, 1979b) . subsequent experiments, with edeine omitted, showed that scanning can be interrupted by inserting a base-paired structure between the cap and the aug codon; the resulting abortive complexes sediment around 40s (kozak, 1989 (kozak, , 1998 paraskeva et al., 1999) . we are not yet sure which initiation factors are associated with the 40s ribosomal subunit during the scanning phase. the only factors whose role in scanning has been defined clearly are the gtp-binding protein eif2, which escorts met-trna i onto the 40s subunit, and eif5, which activates gtp hydrolysis by eif2 (asano et al., 2001; das et al., 2001) . by controlling the rate of gtp hydrolysis, eif5 controls the fidelity of initiation, i.e. the fidelity of the stop-scanning step . other protein factors have not yet been fitted in. (the voluminous literature on factors focuses on modifications -phosphorylation, cleavages -rather than on defining the initiation pathway. basic questions, such as when each factor enters and leaves, have not yet been answered.) one untested possibility is that the large initiation factor eif3, bound to the 40s ribosomal subunit, might form a clamp around the mrna that is opened and closed by cycles of atp hydrolysis. scanning appears to be dependent on atp hydrolysis (kozak, 1980) , thereby implicating eif4a, an rna-dependent atpase which might control the hypothetical clamp. some ideas about the function of other initiation factors are reviewed elsewhere (dever, 2002; mccarthy, 1998; pestova et al., 2001) . the strongest evidence that the scanning 40s ribosome/ factor complex advances linearly is the position effect on selection of the start codon: initiation at the first potential start codon has been demonstrated in rigorous experimental tests (cigan et al., 1988a; kozak, 1983 kozak, , 1995 and confirmed in many 'natural tests' wherein addition or removal of an aug codon produces the expected shift in the site of initiation (see below). the aforementioned blockade caused by inserting a base-paired structure between the 5 0 cap and the aug start codon is further evidence that 40s ribosomal subunits traverse the leader sequence linearly, rather than hopping (discontinuous scanning) or entering directly at the aug codon. although the scanning mechanism predicts that translation should initiate at the aug codon nearest the 5 0 end of the mrna, two ancillary mechanisms -reinitiation and context-dependent leaky scanning -enable additional initiation events at downstream aug codons in some mrnas. these well-defined mechanisms for escaping the first-aug rule are discussed below. an additional escape mechanism might involve direct entry of ribosomes at an internal site in the mrna. while there is evidence suggestive of direct internal initiation with picornavirus mrnas, the evidence for internal ribosome entry sites (ires) in cellular mrnas is problematic (kozak, 2001a) . the absence of shared structural features among candidate cellular ires elements makes it impossible to predict which mrnas, if any, might use such a mechanism. rather than attempting to summarize the extensive literature on internal initiation, i refer the reader to other detailed reviews on that subject (dever, 2002; hellen and sarnow, 2001; pestova et al., 2001) . the next section provides a terse summary of points that are easily explained by the scanning model. the bulk of the review then focuses on complicated examples and issues. 2. constraints imposed by the scanning mechanism explain many common aspects of gene expression in higher eukaryotes many plant and animal viruses produce dicistronic or polycistronic mrnas from which only the 5 0 cistron can be translated (table 1 ). all these viruses solve the problem of 'silent 3 0 cistrons' by producing -via splicing or discontinuous transcription or an internal promoteradditional forms of mrna in which the downstream cistron is repositioned closer to the 5 0 end. the reason for the complicated pattern of splicing seen with human immunodeficiency virus type 1 (hiv-1), for example, is simply to produce mrnas that allow downstream open reading frames (orfs) to be translated. the broad range of viruses represented in table 1 merits attention. the same problem and same solution -post-transcriptional processing of polycistronic mrnas -underlie the expression of many genes in caenorhabditis elegans (blumenthal et al., 2002; hough et al., 1999) . in mammalian cells, mrnas that contain two full-length nonoverlapping cistrons are extremely rare and, as with the aforementioned viruses, actual translation of the 3 0 cistron probably occurs from a second, monocistronic mrna (pardigol et al., 1998; westerman et al., 2001) or from a second mrna in which the two cistrons are fused into a single translation unit (gray and nicholls, 2000; hänzelmann et al., 2002) . a dicistronic transcript derived from the mouse snurf-snrpn locus barely supports translation of the second cistron, as discussed below in the section on reinitiation (section 4). recently discovered dicistronic transcripts produced from the mouse hyal locus support translation of only the 5 0 cistron (shuttleworth et al., 2002) . a few other reported dicistronic mrnas await testing. wold et al., 1995; ziff, 1985 parvovirus: adeno-associated capsid protein a capsid proteins b/c splicing muralidhar et al., 1994 hepatitis b virus core protein s proteins (envelope) promoter switch schaller and fischer, 1991 retrovirus: avian, murine gag (capsid) protein env protein splicing e pawson et al., 1977; van zaane et al., 1977 retrovirus: human foamy gag (capsid) protein pol precursor splicing jordan et al., 1996 lentivirus: hiv-1 tat rev and nef f splicing schwartz et al., 1992 alphavirus: semliki forest nonstructural proteins capsid protein internal promoter glanville et al., 1976; strauss and strauss, 1994 calicivirus: feline g nonstructural proteins capsid protein independent replication carter, 1990; neill et al., 1991 miller et al., 1985; shih and kaesberg, 1976 tobacco mosaic virus replicase coat and movement proteins internal promoters grdzelishvili et al., 2000; hunter et al., 1976 potato virus x 25 kda movement protein 12 and 8 kda movement proteins f ?? verchot et al., 1998 carmovirus: turnip crinkle g replicase (p28/p88) p8 and p9 movement proteins internal promoters li et al., 1998; wang and simon, 1997 fütterer et al., 1994 a the silent downstream cistron identified in the third column is expressed only upon being moved closer to the 5 0 end via production of a second, shorter mrna. translation of most genes derived from these viruses follows straightforward predictions of the scanning mechanism, although occasional deviations have been reported. in rare instances where a 3 0 cistron appears to be translated from a dicistronic mrna (grundhoff and ganem, 2001; kirshner et al., 1999; nador et al., 2001; stacey et al., 2000) , the virus in question employs a complicated pattern of splicing and therefore the existence of an undetected monocistronic mrna is not beyond the realm of reason. in some other cases only a small amount of the protein encoded by the 3 0 cistron was produced, and the published rna analyses were not sufficiently sensitive to rule out the presence of an additional subgenomic mrna (herbert et al., 1996). b in some cases the listed example is arbitrary, i.e. with retroviruses, coronaviruses, closteroviruses, etc., there are additional polycistronic mrnas wherein translation is restricted to the 5 0 cistron. c whereas dna viruses and retroviruses use conventional promoter-switching or splicing mechanisms to generate alternative forms of mrna that allow translation of the downstream cistron, more complicated mechanisms underlie the production of subgenomic mrnas by some rna viruses . d the presence of internal promoters that produce a shorter transcript for each downstream orf is suggestive, but testing of translation is still needed for the mrnas produced by cytomegalovirus and geminivirus. e whereas all retroviruses employ splicing to produce the subgenomic mrna from which envelope protein (env) is translated, some retroviruses also employ an internal promoter which is postulated to mediate expression of novel orfs, such as the superantigen of mouse mammary tumor virus (reuss and coffin, 1998) and orf-x of the virus that causes lung cancer in sheep (palmarini et al., 2002) . f see leaky scanning in table 3 and fig. 1 . g in place of the usual m7g cap, the 5 0 end of these viral rnas carries a covalently linked protein (vpg) or is unblocked. the need for a subgenomic mrna even in these cases emphasizes that translation is 5 0 end-dependent even when it is not cap-dependent. h the full-length genomic mrna supports translation of the 3 0 cistron in vitro but the 3 0 cistron is silent in vivo. the latter result is considered more reliable (meulewaeter et al., 1992) . notwithstanding the documented inability to translate the 3 0 cistron in natural dicistronic mrnas, synthetic dicistronic mrnas -constructed by inserting a putative ires element between two reporter genes -appear sometimes to allow translation of the downstream cistron. the interpretation that this occurs via direct internal initiation of translation has been questioned (kozak, 2001a) and defended (hellen and sarnow, 2001) in other reviews. the position effect, indicative of scanning, is seen when a mutation creates an aug codon upstream from the normal start codon and translation shifts to the upstream site (bergenhem et al., 1992; cai et al., 1992; gross et al., 1998; harington et al., 1994; liu et al., 1999; lock et al., 1991; mével-ninio et al., 1996; muralidhar et al., 1994; wada et al., 1995) . in the most stringent test of the rule, the first aug codon was shown to be the exclusive site of initiation even when the second aug was positioned just a few bases downstream from, and in the same optimal context as, the first (kozak, 1995) . the position effect is seen also when removal of the first start codon activates initiation from the next aug downstream. some genes require production of two versions of the encoded protein, wherein the shorter version, initiated from an internal aug codon, lacks the n-terminal domain of the longer isoform. the problem of how ribosomes can gain access to an internal start codon is solved by producing, via splicing or a downstream promoter, a second form of mrna from which the first aug start codon has been removed. table 2 lists some examples. the n-terminally truncated isoform thus produced may reside in a different cellular compartment, or may function as an antagonist to the full-length protein (as seen with various transcription factors listed in table 2 ), or may function in a surprising way. one such surprise was the discovery that a truncated form of tryptophanyl-trna-synthetase ('minitrprs') has angiostatic activity (wakasugi et al., 2002) . the entries in table 2 and some other examples (aichem and mutzel, 2001; beuret et al., 1999; falvey et al., 1995; nagpal et al., 1992) are what i call natural tests of the position rule. additional evidence comes from experimental manipulations wherein removal of the first aug was shown to activate initiation from a downstream site (cahana et al., 2001; chenik et al., 1995; tailor et al., 2001; thoma et al., 2001) . the scanning mechanism predicts that the 5 0 untranslated region (5 0 utr, an unfortunate misnomer) is actually traversed by ribosomes. this explains why translation of the major coding domain is reduced when adventitious out-offrame aug codons occur upstream. the upstream aug codons often create small orfs (uporfs) which are indeed translated, as shown by detecting the encoded peptide (hackett et al., 1986; raney et al., 2000; wang and wessler, 2001) or by fusing a reporter gene to the uporf (abastado et al., 1991; donzé et al., 1995; liu et al., 1999; steel et al., 1996; tanaka et al., 2001; xu et al., 2001) . the fusion test is the more reliable, as small peptides are usually degraded rapidly. even if upstream aug codons are arranged in a way that allows reinitiation, there is a penalty because reinitiation is usually inefficient. this topic will be discussed at length in section 4. the hypothesis that the 5 0 utr is traversed by ribosomes explains why a highly structured 5 0 leader sequence is so detrimental to translation. vertebrate mrnas characteristically have long, gc-rich -hence highly structured -leader sequences (kozak, 1991a; macleod et al., 1998) , and the resulting difficulty in translation has been discovered over and over in the course of cloning. even a short gc-rich 5 0 utr can inhibit profoundly, as illustrated in cases where a gene produces a mixture of mrnas with different leader sequences, and the worst-translated mrna was found to be the form with the shortest 5 0 utr (jiang and lucy, 2001; yang et al., 1998) . a stem-and-loop structure, stabilized in some cases by a repressor protein, is most inhibitory when its proximity to the 5 0 end blocks ribosome binding (goossen and hentze, 1992; kozak, 1989; wang and wessler, 2001) . if the structure is far enough from the 5 0 end to allow ribosome entry, the advancing 40s ribosome/factor complex apparently has some ability to disrupt base pairing, but this ability is notably less than that of 80s elongating ribosomes (kozak, 1986a (kozak, , 2001b lingelbach and dobberstein, 1988; paraskeva et al., 1999) and is curtailed in yeast (koloteva et al., 1997) . while there are mechanisms for reducing the inhibitory effects of upstream aug codons, as discussed below, no mechanism has yet been defined for modulating the inhibitory effects of secondary structure. some studies suggest that secondary structure might be less inhibitory to translation in vivo than in vitro (charron et al., 1998; curnow et al., 1995; hensold et al., 1997; hoover et al., 1997; morrish and rumsby, 2001; van der velden et al., 2002) . this could be due to production of an alternative transcript that simply eliminates the secondary structure -a reasonable possibility given that gc-rich domains often harbor promoter elements -or to modification of the translation machinery. interpretation of in vivo tests of translation could also be complicated by effects of secondary structure on mrna stability (stefanovic et al., 1999) . whether and how translation of gc-rich leader sequences might improve in exponentially growing cells (nielsen et al., 1995) remains an important open question. the scanning mechanism rationalizes the occurrence of initiation at upstream acg or cug codons in some mrnas. these alternative codons are usually too weak to actually substitute for the aug start codon (reviewed by kozak, 1999 ; for some exceptions see falvey et al., 1995; kiefer et al., 1994; riechmann et al., 1999; sadler et al., 1999) . it is not uncommon, however, for initiation to occur at an upstream non-aug codon in addition to the first aug (see leaky scanning in section 3). this is observed frequently with cellular genes that have highly structured, gc-rich leader sequences (kozak, 1991b) , perhaps because secondary structure slows scanning and thus allows more time for the mismatched codon to pair with met-trna i . with some viruses, the extra protein isoform initiated from an upstream non-aug codon serves an essential function (muralidhar et al., 1994; portis et al., 1994 portis et al., , 1996 . while the n-terminally-extended isoforms derived from some cellular genes also display distinct functions (arnaud table 2 partial list of vertebrate genes that produce a second, shorter version of the encoded protein via a second form of mrna in which an internal aug codon becomes a functional start site upon elimination of the upstream aug start sun et al., 1995 a production of long and short protein isoforms via this mechanism is seen also with genes from insects (mével-ninio et al., 1996) , plants (cunillera et al., 1997; wimmer et al., 1997) , yeast (beltzer et al., 1988; carlson et al., 1983; chatton et al., 1988; ellis et al., 1989; gammie et al., 1999; natsoulis et al., 1986; wolfe et al., 1996) and viruses (barbosa and wettstein, 1988; lambert et al., 1987; liu and roizman, 1991; liu and biegalke, 2002; weimer et al., 1987; welch et al., 1991; wu et al., 1993b; zheng et al., 1994). b in these cases, the long and short protein isoforms have different functional effects. other genes that resemble this pattern, producing long and short isoforms with contrasting functions, are not listed in the table because the aug start codon for the shorter protein is carried on an alternative exon present only in the shorter mrna (e.g. koski et al., 1999; molina et al., 1993) . that arrangement does not illustrate the main point of the table, which is that a silent internal aug codon in the longer mrna can be activated simply by truncating the transcript. c the long and short isoforms are targeted to different cellular compartments. d the long and short isoforms are expressed in different tissues. e the long and short forms of b1,4-galactosyltransferase appear to function identically. the main significance of the promoter switch, which eliminates the first aug start codon, is that the shorter 5 0 utr supports translation more efficiently (charron et al., 1998 (charron et al., ). et al., 1999 calkhoven et al., 2000; spotts et al., 1997) or patterns of localization (acland et al., 1990; lock et al., 1991; packham et al., 1997) , it would not be surprising if some other upstream-initiated proteins turn out to be inadvertent byproducts generated in the course of slowly traversing a gc-rich leader sequence. because the ability of the scanning mechanism to explain the big picture is generally accepted, the remainder of this review directs attention, not to examples that can be seen readily to support the model, but to mrnas that seem to be poorly designed for a scanning mode of initiation. the main point is that the scanning mechanism applies even in these difficult cases. understandably, such mrnas are translated inefficiently and this brings out a second important point: some critical regulatory genes require protein synthesis to be inefficient. an earlier review raised awareness that genes that encode potent regulatory proteins -cytokines, growth factors, kinases, transcription factors -often produce mrnas in which the 5 0 leader sequence is gc-rich or burdened by upstream aug codons (kozak, 1991a) . some examples described herein validate the prediction that these encumbered 5 0 sequences are nature's way of limiting the synthesis of potent proteins that would be harmful if overproduced. i also suggested in earlier reviews that, when a cdna sequence has so many upstream aug codons as to challenge the applicability of the scanning mechanism, it is wise to ask whether the cdna correctly reflects the structure of the mrna. that advice is not changed by what is written here. very often, cdna sequences that appear incompatible with scanning have been found to derive from incompletely spliced transcripts or to have been misinterpreted in other ways (kozak, 1996 (kozak, , 2000 . in other cases, although an encumbered cdna sequence is correct, it derives from a transcript that does not support translation (hake and hecht, 1993; foo et al., 1994; larsen et al., 2002; lee et al., 2000) . only after one is certain of the mrna structure should the mechanisms below be considered. in mammals, the optimal context for recognition of the aug start codon is gccrccaugg. within this motif, the purine (r) in position 23 is the most highly conserved (see section 6.1) and functionally the most important position. the importance of a or g (a is somewhat better than g) in position 2 3 was proved by mutagenesis experiments on a wide variety of genes (kozak, 1986b; and see entries marked 'tested' in table 3 ). the g in position þ 4 is also highly conserved and, especially in the absence of a in position 2 3, contributes strongly (kozak, 1997) . adherence to the rest of the gccrccaugg motif varies, without major consequences as long as positions 2 3 and þ 4 conform; the upstream gcc motif can be seen to contribute, however, in the absence of other elements (kozak, 1987b) . the aforementioned mutagenesis experiments define two extremes: (i) when the first aug codon occurs in a strong context -annaugn or gnnaugg -all or almost all ribosomes stop and initiate at that point; (ii) when the first aug resides in a very weak context, lacking both r in position 2 3 and g in position þ 4, some ribosomes initiate at that point but most continue scanning and initiate farther downstream. this leaky scanning enables the production of two separately initiated proteins from one mrna, as documented below. it is harder to predict what happens at start sites that fall between the extremes, i.e. mrnas in which the first potential start codon has the sequence ynnaugg, gnnaugy or gnnauga. leaky scanning is seen in some but not all such cases. a possible explanation suggested by studies with test transcripts (kozak, 1990a) is that initiation might be restricted to the first aug codon, despite a suboptimal context, when downstream secondary structure slows scanning and thus provides more time for codon/anticodon pairing. suppression of leaky scanning via this mechanism requires a critical distance (13 -15 nt, which corresponds to half the diameter of the ribosome) between the aug codon and the downstream structured element. table 3 lists some examples in which two proteins are produced from one mrna via leaky scanning. the postulated link between context and leaky scanning has been tested in many of these cases by showing that, upon improving the context at the upstream site, initiation from the second site is reduced or abolished. (whether the second aug codon resides in a strong or weak context is not relevant; the ribosome reads the mrna linearly and thus the decision to stop or to bypass the first aug is not influenced by whether there is a better initiation site downstream.) the large number of genes that employ leaky scanning precludes discussion of the biological significance of the proteins thereby produced, but it merits noting that, for many of the viruses in table 3 , replication requires production of both listed proteins. for some other viruses, the second protein is a virulence factor that weakens host defenses (bridgen et al., 2001; chen et al., 2001; weber et al., 2002) . the biological importance of these downstream-initiated proteins shows that leaky scanning is a deliberately employed tool; it does not simply reflect sloppiness on the part of the translational machinery. the long list of examples in table 3 conforms to expectations in that the first start codon resides in a suboptimal context. there are, however, rare instances of leaky scanning despite a good context (r 23 and g þ4 ) around the first aug. this can happen when the first aug codon is too close to the 5 0 end to be recognized efficiently (kaneda et al., 2000; kozak, 1991c; ruan et al., 1994; sedman et al., 1990; slusher et al., 1991; spiropoulou and in all mrnas here listed, the sequence flanking the first start codon deviates from the consensus sequence in position 23 and/or position þ 4, highlighted by underlining. when the postulated link between context and leaky scanning was tested (so marked in this column), mutations that improved the context at the first start site diminished access to the downstream start site. this test failed only with cucumber necrosis virus, where the short distance between the m7g cap and aug#1 allowed some leaky scanning even when the context was optimized. c in some cases the first and second aug codons are in the same reading frame, generating long and short versions of the encoded protein which may function differently. in cases where the first and second start codons are in different reading frames, indicated by italicizing the second product, the extent of overlap between the two orfs ranges from a few codons (peanut clump virus, southern bean mosaic virus) to 626 codons (turnip yellow mosaic virus). d access to the downstream initiation site via leaky scanning is augmented by a reinitiation shunt, as explained in the text (section 4.3) and diagrammed in fig. 1 for c/ebpb mrna. e mutations that eliminate aug#1 usually increase production of the second, downstream protein. in rare cases where the expected increase was not seen (e.g. von hippel-lindau, turnip yellow mosaic virus), it might be because translation of the second protein was restricted at the level of elongation. for a similar reason, improving the context around aug#1 occasionally fails to elevate production of the protein there initiated (fajardo and shatkin, 1990) . these entries nevertheless satisfy the main prediction of the leaky scanning mechanism, which is that improving the context around aug#1 prevents initiation from the second, downstream site (fajardo and shatkin, 1990; iliopoulos et al., 1998) . f whereas feline leukemia virus produces an n-terminally-extended, glycosylated form of gag (gp80 gag ) from the indicated weak aug codon, the corresponding upstream start site in murine leukemia virus is acccugg (portis et al., 1994) . when that site was experimentally ablated, however, revertants expressed the extended protein from a weak upstream aug codon (uuuaugg) created by a point mutation. those revertants were selected because the extra glycosylated form of gag contributes to viral spread (portis et al., 1996) . g in the mrnas from baboon reovirus, influenza a virus, and southern bean mosaic virus, the indicated proteins derive from the first (weak) and fourth aug codons. aug#2 and aug#3 initiate small orfs that terminate before aug#4. thus, a combination of leaky scanning and reinitiation probably mediates access to the downstream start site. nichol, 1993; werten et al., 1999) or when the facilitating effect of g in position þ 4 is canceled by u in position þ 5 (kozak, 1997; sloan et al., 1999; stallmeyer et al., 1999) . other occasional claims of leaky scanning despite a strong context at the first aug codon were simply mistaken (scherer et al., 1995) ; the shorter protein turned out to be translated from a second form of mrna (kogo and fujimoto, 2000) . at the opposite extreme, there are rare mammalian mrnas in which, despite a very unfavorable context flanking the first aug codon, translation appears to initiate exclusively at that site (arai et al., 1991; hickey and roth, 1993; leslie et al., 1992; mcneil et al., 1992; plowman et al., 1990; wu et al., 1993a) . leaky scanning might be suppressed in these and a few other cases because of downstream secondary structure, or because the wider context (c in positions 21, 22, 24, 2 5; g in position 2 6) compensates to some extent for the absence of r 23 and g þ4 , or for other unknown reasons. the same principle that allows initiation from the first and second aug codons when the first aug is in a suboptimal context (table 3 ) applies in cases where translation initiates at an upstream non-aug codon in addition to the first aug (acland et al., 1990; arnaud et al., 1999; carroll and derse, 1993; fajardo et al., 1993; florkiewicz and sommer, 1989; fütterer et al., 1996; fuxe et al., 2000; lock et al., 1991; muralidhar et al., 1994; packham et al., 1997; saris et al., 1991; spotts et al., 1997) . recognition of an upstream acg, cug or gug start codon requires a strong context (portis et al., 1994) , despite which scanning is usually leaky because the initiator codon itself is weak. production of long and short protein isoforms via leaky scanning is harder to regulate -e.g. to achieve tissue specific expression of one or the other form -than when a unique mrna encodes each isoform, as in table 2 . there are hints, however, that dual initiation via leaky scanning might be regulable (probst-kepper et al., 2001; spotts et al., 1997) . this could conceivably be accomplished via proteins that stabilize downstream secondary structure or, perhaps, via a combination of leaky scanning and regulated reinitiation, if the mrna also has small uporfs. among the examples in table 3 are many plant viruses, indicating that the basic context rules extend to plant systems. mutagenesis experiments confirm the functional importance of r in position 23 and g in position þ 4 in plant mrnas (jones et al., 1988; lukaszewicz et al., 2000) and surveys of plant cdna sequences confirm the conservation of those key positions (pesole et al., 2000; rogozin et al., 2001) . unlike mammalian mrnas, however, plant mrnas do not show a predominance of c in positions 2 1, 2 2, 2 4 and 2 5. the foregoing discussion pertains to mrnas from plants and vertebrate animals. there is some evidence for contextdependent leaky scanning in fungi (arst and sheerins, 1996) , but context effects on initiation have not yet been studied carefully in protozoa, insects, and various other systems. the observation that trans-splicing of mrnas in c. elegans sometimes brings a purine into position 2 3 is interesting (hough et al., 1999) but the significance awaits testing. with a number of yeast genes, there is a hint of leaky scanning when the usual a in position 2 3 is replaced by a pyrimidine (gaba et al., 2001; slusher et al., 1991; vilela et al., 1998; welch and jacobson, 1999; wolfe et al., 1994) . context effects were not evident, however, in other studies of translation in yeast (cigan et al., 1988b) . for whatever reason, leaky scanning is rare in yeast, apart from a few cases attributable to the first aug codon residing too close to the 5 0 end. mrnas that initiate translation from three sites provide a striking illustration of how far leaky scanning, alone or in combination with reinitiation, can be pushed. fig. 1 shows some examples. the predominant translation product obtained from cmyc mrna is a 65 kda 'long form 2' which initiates at the first aug codon (fig. 1a) . a small amount of a longer isoform (68 kda) derives from an upstream cug codon which is a weak start site (i.e. very leaky) because the codon is not aug. although the first aug codon has the required a in position 2 3, a small percentage of ribosomes bypass that site and initiate at the next aug, producing a third (50 kda) form of c-myc. this happens apparently because the context flanking the first aug codon is good but not perfect. thus, production of the 50 kda isoform was eliminated when the upstream site was changed from acgaugc to accaugg (spotts et al., 1997) . fig. 1b depicts another example in which ribosomes initiate from three in-frame aug codons. with the mrna that encodes c/ebpb, access to the far downstream site via leaky scanning is augmented by a reinitiation shunt, as explained in the legend to fig. 1 and discussed further in section 4. translation of c/ebpa mrna occurs by a mechanism similar to that depicted for c/ebpb except that the first start site in c/ebpa is a cug codon (calkhoven et al., 2000) , which generates a smaller amount of the longest protein (isoform a) than does the aug codon in c/ ebpb. with c-myc, c/ebpb and c/ebpa mrnas, leaky scanning is biologically important because the long and short versions of the protein have opposing effects as regulators of transcription. it is striking that leaky scanning can operate even when the second initiation site resides far downstream from the first. with synthetic transcripts designed to test the processivity of scanning, there was no reduction in initiation from the downstream site when the inter-aug distance was expanded stepwise from 11 to 251 nt (kozak, 1998) . in some remarkable viral mrnas, the second functional initiation site is more than 500 nt downstream from the fig. 1 . examples of 'maximally leaky' scanning wherein one mrna produces three independently initiated proteins. major (thick arrow) and minor (thin arrow) translation products are identified below their respective start codons. sequences that cause the initiation site to be weak, and thus promote leaky scanning, are highlighted in red. offset rectangles represent orfs in different reading frames. (a) with c-myc mrna, a leaky scanning mechanism was inferred from experiments in which optimizing the context around the first aug codon suppressed production of the 50 kda isoform, while changing the upstream cug codon to aug suppressed production of both the 65 and 50 kda isoforms (spotts et al., 1997) . access to the downstream start site might be more complicated than here depicted, as there is a small out-of-frame orf between the 65 and 50 kda start sites. (b) with c/ebpb mrna, a mutation that strengthens the first start codon (uucaugc ! accaugg) blocked production of all shorter isoforms, implicating a leaky scanning mechanism (calkhoven et al., 2000) . a small uporf (blue) superimposes another level of control, causing more ribosomes to bypass the start site for isoform b1 than would be expected from leaky scanning alone. presumably because the aug start codon for isoform b1 is positioned close to the termination site of the uporf, reinitiation at site b1 is inefficient and some ribosomes thus reach the far downstream start site for the 20 kda isoform (lip). as evidence for this reinitiation shunt, calkhoven et al. (2000) showed that eliminating the aug codon of the uporf abolished production of lip and that strengthening or weakening the context around the uporf start codon caused corresponding changes in the yield of lip. although the smallest form of c/ebpb can be generated in some situations by proteolysis (dearth et al., 2001) , the effects of the aforementioned mutations clearly implicate a translational mechanism. the lap/lip ratio shows tissue and stage specific variation (dearth et al., 2001; descombes and schibler, 1991) . (c) whereas leaky scanning allows initiation at multiple sites within a single orf in c/ebpb and c-myc mrnas, leaky scanning allows translation of three separate orfs in the pregenomic mrna of rice tungro bacilliform virus. these orfs (not drawn to scale) have overlapping start and stop codons of the form auga. translation via leaky scanning was inferred from the strong reduction (.13-fold) in translation of orf2 and orf3 when the start codon of orf1 was changed from auu to aug (fütterer et al., 1997) and from the inhibitory effect on expression of orf3 when an adventitious aug codon was inserted into orf2. the 5 0 leader sequence that precedes orf1 has ten small uporfs which are not depicted here because that peculiar leader sequence, postulated to be translated by ribosome hopping (fütterer et al., 1996) , is not required for the leaky scanning mechanism that underlies translation of orfs 1, 2 and 3. (d) the avian reovirus s1 mrna supports translation of one structural and two nonstructural proteins (bodelón et al., 2001) . the depicted mechanism postulates that orf1 has a dual function, encoding its own polypeptide (p10) and facilitating translation of orf3 by shunting some ribosomes past the strong aug start codon for orf2. the absence of extraneous aug codons in the 310 nt region between the end of orf1 and the start of orf3 is consistent with the idea that orf3 might be translated by reinitiation. some ribosomes would be expected to translate p17 (orf2) by leaky scanning, engendered by the poor context at the start of orf1. improving the context at the start of orf1 indeed increased production of p10 (shmulevitz et al., 2002) ; unfortunately, the yield of p17, which would be expected to decrease, was not monitored. the observation that strengthening the context at the start of orf1 had no effect on the yield of sc is not surprising because the reinitiation mechanism postulated to underlie translation of orf3 would probably be limited by other features, such as the relatively large size of orf1. first aug (herzog et al., 1995; sivakumaran and hacker, 1998) . the pregenomic mrna of rice tungro bacilliform virus (fig. 1c) provides the most dramatic illustration of these points. use of a weak (non-aug) codon to initiate orf1 and an unfavorable context at the start of orf2 (uacauga) enables the majority of ribosomes to reach and initiate at the start of orf3. the orf3 polyprotein is thought to be a precursor from which coat protein, protease and reverse transcriptase are derived by proteolysis. the remarkable absence of aug codons from the long (563 nt) coding domain of orf1 and the presence of but one weak aug codon within orf2 underscore how carefully this mrna is constructed to support translation via scanning. the careful construction includes minimizing the overlap between adjacent cistrons. without that precaution, elongational occlusion might work against utilization of a far downstream start codon, as documented in other cases (kozak, 1995) . the avian reovirus rna diagrammed in fig. 1d offers another example of initiation from three sites in one mrna. additional experiments are needed to validate the postulated mechanism. in contrast with the 'maximally leaky' mrnas in fig. 1 , the mrnas in fig. 2 are minimally leaky: only a small fraction of ribosomes bypass the first aug codon and initiate downstream. here the leaky scanning mechanism has been pushed to the limits in the sense that there is (lowlevel) initiation from a second site despite the presence of a strong context around the first aug codon. the explanation is that the context flanking the first aug start codon is good but not perfect. the resulting low-level leaky scanning enables the viruses depicted in fig. 2a ,b to produce two proteins -one abundant, the second in small amountsfrom a single mrna. experimental manipulations that support this interpretation are summarized in the legend to fig. 2. a few other viral genes that might fit this category have been described (chenik et al., 1995; jayakar and whitt, 2002) . the hepatitis b virus example is noteworthy because, via the rube goldberg mechanism diagrammed in fig. 2b , reverse transcriptase encoded by the p gene is initiated independently from a far downstream site, unlike most other reverse transcriptase genes which lack an independent start codon and therefore require frameshifting during translation of the preceding core gene. production of a second protein isoform via low-level leaky scanning is seen also with some cellular mrnas. an interesting example is the production in rats of an osteogenic growth peptide (ogp) initiated from codon 85 in the histone h4 gene (fig. 2c) . the leaky scanning explanation was tested by showing that production of ogp increased upon deleting the upstream h4 start codon, and that production of ogp was suppressed upon changing the h4 start codon from a good (aggaagaugu) to a perfect (gccaccaugg) context. a similar mechanism might operate with a few other cellular genes that produce a trace amount of a second protein isoform short and pfarr, 2002; it is not clear whether leaky scanning or a change in splicing underlies the translational switch described by land and rouault, 1998) . the fourth example in fig. 2 differs from the others in that a good-but-not-perfect context at the first start site serves, not to enable production of two proteins, but simply to modulate the yield of a 2a -r from the second aug. examination of a 2a -r genes from various organisms shows conservation of the overlapping orf, with the upstream aug codon always in a context that allows only low-level leaky scanning (lee et al., 1999) . conservation of the structure supports the interpretation that this is a device contrived to limit the production of a 2a -r protein. low-level leaky scanning caused by a not-quite-perfect context around the first aug codon might occur with other mrnas where it normally goes unnoticed because the downstream start site(s) are out-of-frame. antigenic peptides recognized by cytotoxic t-lymphocytes (ctls) might be produced in this way, as discussed in section 5.4. a small degree of leaky scanning that normally goes unnoticed could become significant if a mutation that shifts the normal start codon out of frame moves a downstream aug codon into the main reading frame. in some cases where low-level internal initiation was observed with such a mutated gene (e.g. maser et al., 2001) , the possibility that the downstream site is reached via a combination of leaky scanning and reinitiation -a mechanism such as that proposed for hepatitis b virus (fig. 2b ) -merits consideration. reinitiation occurs with mrnas, such as those depicted in fig. 3 , that have small orfs near the 5 0 end. our rudimentary understanding of what happens following translation of the first uporf may be summarized as follows. when the 80s ribosome reaches the termination site of the uporf, the 60s ribosomal subunit is thought to be released (this has not actually been shown) while the 40s subunit remains bound to the mrna, resumes scanning, and may initiate another round of translation at a downstream aug codon. for the downstream reinitiation event to occur, the 40s subunit must reacquire met-trna i and this appears to be an important point of control. reacquisition of met-trna i is promoted by lengthening the intercistronic domain (abastado et al., 1991; kozak, 1987c) , which provides more time for met-trna i to bind, or by increasing the concentration of eif2 (abastado et al., 1991; hinnebusch, 1997) . genetic experiments also implicate eif3 in the met-trna i rebinding step (garcia-barrio et al., 1995) . another potential point of control is at the termination site of the uporf, where certain features -perhaps nearby secondary structure (grant and hinnebusch, 1994; vilela et al., 1998) -might prevent the resumption of scanning or, in some other way, prevent reinitiation. this brief summary is based on studies carried out in yeast and mammals. some studies of reinitiation in plants suggest that the intercistronic sequence may have effects beyond simply providing time for ribosomes to reacquire met-trna (wang and wessler, 1998) . some results obtained in early experiments with mammalian vectors were interpreted as evidence that ribosomes can scan backwards and thus reinitiate at an aug codon positioned upstream from the termination site (peabody et al., 1986 ), but recent experiments contradict fig. 2 . examples of minimally leaky scanning in which a strong, but not quite perfect, context at aug#1 causes most ribosomes to initiate there while allowing a low level of initiation downstream. with the depicted viral mrnas (a,b), the predominant product of translation is the capsid protein initiated from aug#1. low-level leaky scanning generates a small but adequate amount of the indicated second protein. with bovine coronavirus, a mutation in position þ4 (u ! g, indicated in red) flanking aug#1 strongly reduced translation from the downstream site (senanayake and brian, 1997) , supporting the interpretation that the natural mrna is slightly leaky because the context flanking aug#1 is not a perfect match to the consensus sequence. with hepatitis b virus, ribosomes en route to the p start site (aug#5) apparently bypass the weak aug#2 by leaky scanning, while translation of the small orf initiated at aug#3 enables some ribosomes to miss the inhibitory aug#4 (inhibitory because it resides in a strong context and overlaps the p orf) and thus to reach aug#5. whereas the core protein start codon (aug#1) here depicted resides in a context which allows a low level of leaky scanning, a slightly longer mrna which encodes the pre-core protein has a stronger start codon (a in position 23) and polymerase cannot be translated from that form of mrna (fouillot and rossignol, 1996) . the publications on which the scheme shown here is based (fouillot et al., 1993; hwang and su, 1998 ) also discuss some alternative possibilities vis-à-vis translation of polymerase. (c) the first aug codon in rat histone h4 mrna initiates translation of the full-length protein. the second aug, 85 codons downstream and in the same reading frame, initiates production of a peptide which has growth-regulatory properties (bab et al., 1999) . (d) with rat a 2a r adenosine receptor mrna, an overlapping uporf that initiates at an aug codon in a strong context is used to minimize production of a 2a r protein. the overlapping arrangement precludes reinitiation but the not-quite-perfect context at the upstream start site allows low-level leaky scanning. this interpretation is supported by the observed ten-fold increase in translation of a 2a r in vivo when the start codon of the uporf was eliminated (lee et al., 1999) . via a second promoter, the rat a 2a -r gene produces some transcripts with additional uporfs, but no transcript has yet been found that lacks the inhibitory uporf discussed here. here and in fig. 3 , the major coding domain is shaded gray. small regulatory orfs (blue rectangles) are not drawn to scale. that view (kozak, 2001b) . indeed many studies have shown that the strongest inhibition is caused by an uporf that overlaps the start of the downstream cistron (babik et al., 1999; bates et al., 1991; byrne et al., 1995; cao and geballe, 1995; ghilardi et al., 1998; hansen et al., 2002; kos et al., 2002; lee et al., 1999; liu et al., 1999) , which would not be the case if ribosomes could move backwards to reinitiate. the size of the first orf is a major limitation on reinitiation in eukaryotes: reinitiation can occur following the translation of a 'minicistron' (a small first orf) but not following the translation of a full-length 5 0 cistron. the long list of mrnas that contain silent 3 0 cistrons (table 1) underscores the point. the only apparent exception occurs with cauliflower mosaic virus, where a protein encoded by the virus appears to promote reinitiation following the translation of a full-length first cistron (park et al., 2001) . the reason why reinitiation is usually restricted by the size of the first orf is not known, but a possible explanation is that certain initiation factors dissociate from the ribosome only gradually during the course of elongation. if the elongation phase is brief -i.e. if the first orf is a fig. 3 . small upstream orfs in eukaryotic mrnas function in various ways to modulate translation. only the 5 0 end of each mrna is depicted. (a) the presence of uporfs forces translation of the major orf to occur by a reinitiation mechanism, which is usually inefficient. the extent of inhibition depends on the number and arrangement of uporfs and whether the context flanking the upstream start codon(s) allows some escape via leaky scanning. (b) because reinitiation can occur only in the forward direction, an overlapping uporf strongly impairs translation of the major orf. (c) whereas type b mrnas have a single in-frame start codon which is bypassed due to the overlapping uporf, type c mrnas initiate from two in-frame start codons; the uporf serves to divert some ribosomes to the downstream start site. the depicted sequence is a simplified representation of glyrs mrna (mudge et al., 1998) . translation of bag-1 mrna can also be fitted to this pattern: the first start site is an in-frame cug codon which produces the 50 kda form of bag-1; the next start site (aug#1, out-of-frame) initiates a small uporf within which the first in-frame aug codon (aug#2) resides, and that aug is thereby skipped; the 36 kda form of bag-1 is produced from aug#3 which is accessed by reinitiation following translation of the small uporf (packham et al., 1997) . some other mrnas that use an uporf to dodge one aug codon in favor of another are described elsewhere (mittag et al., 1997; sarrazin et al., 2000) . note that the reinitiation shunt as here defined adheres to the linear scanning mechanism, unlike a shunt postulated to operate with cauliflower mosaic virus mrna (ryabova et al., 2000) . (d) the common feature of mrnas that use mechanism d is inhibition of translation in cis by a peptide encoded within the uporf. the amino acid sequence of the inhibitory peptide is different in each case (morris and geballe, 2000) . in the column at the far right, asterisks indicate examples in which the translational control mechanism is regulated, e.g. via a change in concentration of eif2 (gcn4) or arginine (cpa1) or polyamines (adometdc) or, more commonly, via an alternative promoter that generates a simpler form of mrna devoid of uporfs (c-mos, mdm2, il-12; see text for other examples, e.g. in alderete et al., 2001) . minicistron -the factors required for reinitiation would still be present when the 40s subunit resumes scanning. although the postulated factors have not been identified, there is evidence for the idea that the duration of the elongation phase matters: when a short uporf which normally permits reinitiation was reconfigured to contain a pseudoknot that is known to slow elongation, reinitiation failed (kozak, 2001b) . that result makes it difficult to specify a cutoff size, i.e. one cannot say 'an uporf this long will allow reinitiation' while a longer orf will not. the permissible size is likely to vary depending on features, such as secondary structure or codon usage, that affect the rate of elongation. as a rough guide, however, one may note that reinitiation often has been observed following translation of a ten to 12 codon uporf, and that reinitiation was substantially reduced, but not abolished, when a 13 codon uporf was lengthened to 33 codons (kozak, 2001b) . in a different study, reinitiation occurred following a 24 codon uporf but not when the orf was lengthened to 40 codons (luukkonen et al., 1995) . some naturally occurring uporfs that strongly inhibit translation, perhaps because their size precludes reinitiation, include a 36 codon uporf in mitochondrial uncoupling protein 2 mrna (pecqueur et al., 2001) , a 71 codon uporf in polyoma virus jc mrna (shishido-hara et al., 2000) , and a 53 codon uporf in plant s-adenosylmethionine decarboxylase (adometdc) mrna (hanfrey et al., in press) . that the size of the uporf might be what limits translation of adometdc is suggested from the five-fold increase in translation observed when the uporf was shortened from 53 to 25 codons, but that result could also be explained in other ways. (the suggested interpretation is not contradicted by the fact that an alternative uporf in adometdc mrna caused little inhibition even when lengthened to 66 codons; the alternative uporf initiates from an aug codon in a weak context which would allow it to be bypassed to some extent by leaky scanning. the 53 codon uporf, in contrast, has a strong start codon.) with the mouse snurf-snrpn transcript, where the first cistron is 71 codons long , a very low level of reinitiation might account for translation of the downstream snrpn cistron. a naturally occurring atg-to-agg mutation in the start codon of the upstream snurf cistron was found to elevate translation of snrpn . 15fold (tsai et al., 2002) , which implicates a scanning/ reinitiation mechanism and rules out direct internal initiation. from cdna sequencing data, it is clear that many vertebrate mrnas have small orfs upstream from the start of the major coding domain, but an accurate count of genes in this class is difficult. the tallies that have been attempted (e.g. pesole et al., 2001; suzuki et al., 2000) are invariably flawed by inclusion of misinterpreted cdna sequences, such as cdnas in which a putative 5 0 utr with 'upstream' aug codons turned out to be part of the coding domain or part of an intron that gets removed from the functional mrna (di fruscio et al., 1998; kozak, 1996 kozak, , 2000 kubu et al., 2000; nishitani et al., 2001; santamarina-fojo et al., 2000; wagner et al., 1998) . some transcripts with long, aug-burdened leader sequences are not associated with polysomes (hake and hecht, 1993; sanchez-góngora et al., 2000) or not able to support protein synthesis (foo et al., 1994; larsen et al., 2002; lee et al., 2000) , emphasizing that not all cdnas correspond to functional mrnas. a more fundamental complication vis-à-vis which genes to count is the propensity for a single gene to produce transcripts with different 5 0 leader sequences, only some of which have upstream aug codons (anant et al., 2002; aplan et al., 1991; eerola et al., 2001; huo and scarpulla, 1999; kawakubo and samuel, 2000; laurin et al., 2000; perälä et al., 1994; perrais et al., 2001; sanchez-góngora et al., 2000; suva et al., 1989; tanaka et al., 2001; tsuda et al., 2000; zimmermann et al., 2000) . the significance of a particular form of rna cannot always be deduced from its abundance, inasmuch as a minor transcript is sometimes the major functional mrna (andrea and walsh, 1995; babik et al., 1999; ghilardi et al., 1998; mitsuhashi and nikodem, 1989; nielsen et al., 1990) and incompletely processed transcripts are sometimes more abundant than the fullyspliced, translatable mrna (boularand et al., 1995; frost et al., 2000; xie et al., 1991; zachar et al., 1987) . translational regulation mediated by small uporfs is important, as discussed below, but equally important are non-translational mechanisms -use of alternative promoters or splice sites -that simplify the 5 0 utr by eliminating uporfs in certain tissues or at certain times when elevated synthesis of the protein is required (aizencang et al., 2000; anusaksathien et al., 2001; arrick et al., 1994; babik et al., 1999; brown et al., 1999; horiuchi et al., 1990; landers et al., 1997; lee et al., 2000; nonaka et al., 1989; phelps et al., 1998; ren and stiles, 1994; steel et al., 1996; teruya et al., 1990) . because vertebrate mrna leader sequences are often gc-rich (section 2.4), secondary structure near the 5 0 end might impair translation even more than the presence of upstream aug codons. thus, it is not surprising that eliminating upstream aug codons does not improve translation in every case (rao et al., 1988; wood et al., 1996) . in many cases, however, mutations targeted to the upstream aug codons confirmed their role in restricting translation from downstream (anant et al., 2002; babik et al., 1999; bates et al., 1991; brown et al., 1999; child et al., 1999a; gereben et al., 2002; ghilardi et al., 1998; griffin et al., 2001; harigai et al., 1996; kos et al., 2002; lee et al., 1999; marth et al., 1988; meijer et al., 2000; pecqueur et al., 2001; ren and stiles, 1994; steel et al., 1996; tanaka et al., 2001; tsai et al., 2002; wang and wessler, 1998; wang and rothnagel, 2001; wera et al., 1995; wu et al., 2002) . this occurs by a variety of mechanisms, as summarized in fig. 3 and discussed next. while the efficiency of reinitiation varies, there is almost always a penalty -demonstrable by showing an increase in translation when the uporf is deleted -and the penalty can be severe. thus, the simplest function of small uporfs is to limit production of the protein encoded in the full-length orf by making downstream translation dependent on an inefficient reinitiation mechanism (fig. 3a) . the best studied example is yeast gcn4, which initiates from the fifth aug codon in the mrna; the long leader sequence contains four small uporfs. in a series of classic experiments, hinnebusch (1997) was able to reconstruct gcn4 regulation using only the first and fourth uporfs, and i will explain what happens in that simplified case. uporf1 is always translated efficiently (it is the first aug in the mrna), after which ribosomes resume scanning and reinitiate, usually, at uporf4. uporf4 is unusual in that its translation precludes further reinitiation events: thus, when uporf4 is translated, gcn4 is not. that is the situation in yeast cultures that have adequate nutrients. starvation for amino acids, however, causes some ribosomes to bypass the inhibitory uporf4 and reinitiate instead farther downstream. this happens because starvation creates a pool of uncharged trnas which activate a protein kinase that phosphorylates, and thus partially inactivates, eif2. when eif2 levels fall, it takes longer for ribosomes to reacquire met-trna i and thus become competent to reinitiate. the slower acquisition of competence means that some ribosomes, scanning in the reinitiation mode, will bypass the nearby uporf4 and can thus reach the downstream gcn4 start site. three general lessons from the gcn4 story appear to carry over to mammals. (i) fig. 3a lists some examples of mammalian mrnas that are translated inefficiently due to small uporfs; many other examples were cited in section 4.2. (ii) experimental manipulations with c/ebpb mrna (fig. 1b ) support the interpretation that an aug codon which follows the uporf too closely is skipped (presumably because ribosomes have not yet reacquired met-trna i ), allowing reinitiation to occur farther downstream. the same mechanism might be invoked to explain how an internal start codon is accessed in minitrprs mrna (wakasugi et al., 2002) and baculovirus ie0 mrna (theilmann et al., 2001) , and how c-myb gets translated from a rearranged transcript generated by retrovirus insertion (jiang et al., 1997) . in each of these mrnas, the first aug codon that follows a small uporf must be bypassed to reach the functional start codon downstream. (iii) the third lesson from gcn4 pertains to regulation of reinitiation by manipulation of eif2 levels. although hints of this have been described with mammalian genes that encode c/ebp transcription factors (calkhoven et al., 2000) , macrophage receptor protein cd36 (griffin et al., 2001) and activating transcription factor 4 (harding et al., 2000) , the point requires much more careful study. the mrnas discussed in connection with fig. 3a have uporfs that terminate before the start of the major coding domain, thus allowing (inefficient) translation of the main orf by reinitiation. in fig. 3b , however, the uporf overlaps the start of the major coding domain. this precludes reinitiation and profoundly reduces the translational yield. limited access to the main orf in some of these mrnas might be achieved by leaky scanning, as was discussed for a 2a -r (fig. 2d) . mrnas derived from the human thrombopoietin (tpo) gene have structures similar to that depicted in fig. 3b and much can be learned from the tpo story, as outlined in fig. 4 . the normal gene produces a mixture of transcripts with different leader sequences, all of which translate tpo poorly because of an overlapping uporf (uporf7 in fig. 4 ). targeted mutagenesis (ghilardi et al., 1998) confirmed that upstream aug#7 is primarily responsible for blocking translation of tpo. this is because its near-optimal context (gccgccuccaugg) prevents leaky scanning and the overlapping arrangement precludes reinitiation. various mutations that restructure the 5 0 utr in ways that increase production of tpo cause hereditary thrombocythemia. translation of tpo normally initiates at aug#8 in exon 3, but a splice-site mutation that causes deletion of exon 3 causes initiation to shift to a previously silent inframe codon (aug#9) in exon 4; this is diagrammed in the center of fig. 4 . the resulting truncated form of tpo lacks only the first four amino acids and appears to function normally . the problem -the cause of the pathology -is that the mutation greatly increases translation of tpo by removing the inhibitory uporf7. in two other families affected with hereditary thrombocythemia, production of tpo is elevated by mutations that restructure uporf7. in one case, deletion of a g residue shifts uporf7 into the same reading frame as tpo, thereby causing overproduction of an elongated form of tpo initiated from aug#7 kondo et al., 1998) . in the other case, a g ! t mutation creates a terminator codon within uporf7 and this shortening of the orf, which now terminates 31 nt before aug#8, enables efficient reinitiation at aug#8 . these insightful studies of tpo expression make two important points: (i) the bulk of the transcripts produced by the wild type gene are virtually untranslatable; and (ii) it is necessary for this potent cytokine to be translated poorly; overproduction results in disease. with tpo as precedent, one suspects that in other cases where -despite the production of alternative leader sequences -it is hard to find even one form of mrna devoid of upstream aug codons (e.g. larsen et al., 2002; lee et al., 1999; pecci et al., 2001; peterson and morris, 2000; wang et al., 1999) , the goal is to ensure that translation is very, very inefficient. the wig-1 growth-regulatory gene might be another example: an overlapping uporf initiates from a strong aug codon, while the wig-1 start codon itself is weak, and these distinctive features are conserved between the human and mouse genes (hellborg et al., 2001) . whereas an overlapping uporf functions simply to down-modulate translation in the examples depicted in fig. 3b , with the mrnas in fig. 3c the overlapping uporf qualitatively affects the protein output. ribosomes that translate the uporf thereby miss the first in-frame aug codon but proceed to reinitiate at another start codon fig. 4 . a low-level reinitiation mechanism normally prevents overproduction of tpo. translational yields from various forms of tpo mrna in transfected cos cells (far right column) are expressed relative to a control transcript that has a short, unencumbered 5 0 utr. p1 and p2 are alternative promoters; a cluster of arrows indicates that p2 produces staggered start sites. the tpo coding domain (horizontal black bar) begins at an aug codon which is labeled #8 because, in the longest form of mrna (line 1), it is preceded by seven aug codons that initiate small uporfs. superscript letters indicate whether each upstream aug resides in a strong (s) or weak (w) context and horizontal blue lines depict the approximate length and arrangement of the uporfs. vertical lines demarcate the boundaries of exons; carets depict the introns in alternatively spliced transcripts. only the beginning of the tpo coding domain (exons 3 -7) is shown. the key point is that the normal set of transcripts supports translation poorly because uporf7 overlaps the tpo start site. various mutations (shown in red) that relieve this constraint elevate the translation of tpo, and this overproduction causes hereditary thrombocythemia. among the normal set of mrnas, the 'rare' transcript from promoter p1 (line 2) supports translation slightly better than the others, perhaps because the short distance between uporf2 and aug#7 enables some reinitiating ribosomes to bypass aug#7 and thus reach aug#8. because of the strong context at augs #1 and #2, uporfs 1 and 2 would be more effective than uporfs 5 and 6 in setting up this reinitiation shunt. the depicted scheme is based on experiments described by ghilardi et al. (1998) and wiestner et al. (1998) . additional mutations diagrammed near the bottom of the figure were described by , , and kondo et al. (1998). downstream. if the uporf itself has a suboptimal initiation site (u in position 2 3 in the depicted example), leaky scanning will allow some production of the long protein isoform from the first in-frame aug codon while the reinitiation shunt promotes production of the shorter protein isoform. the operation of a reinitiation shunt is most obvious when the uporf overlaps a potential start codon, as shown in fig. 3c , but the same principle applies in cases (discussed in section 4.3) where, although the uporf terminates prior to a potential downstream start codon, the intervening distance is too short to allow reinitiation. the fourth regulatory mechanism diagrammed in fig. 3 is used only rarely. mammalian adometdc mrna is the best studied example in which a small uporf encodes a peptide which functions in cis to inhibit downstream translation. the nascent peptide (magdis) produced during translation of the uporf is thought to interact with ribosomes in a way that prevents completion of the termination process and thus prevents reinitiation (law et al., 2001) . the stalled ribosome, held at the termination site of the uporf, would also block other ribosomes from reaching the downstream start site via leaky scanning. biologically, this mechanism is important because ado-metdc is a key enzyme in polyamine biosynthesis and, at least in vitro, elevated polyamine levels stabilize the ribosome complex stalled at the end of the uporf (law et al., 2001) . in other words, elevated polyamine levels down-regulate translation of adometdc. it is interesting to note parenthetically that antizyme, a protein that downregulates polyamine levels, is also translated via a polyamine-sensitive mechanism. elevated polyamine levels up-regulate production of antizyme by promoting a ribosomal frameshift needed to translate the full-length protein (ivanov et al., 2000) . the foregoing examples illustrate how reinitiation operates as part of the normal translation mechanism in cases where uporfs are constitutively present in mrnas. there are other cases in which a reinitiation mechanism kicks in only when a nonsense mutation is introduced in a way that truncates the coding domain. in effect, the normal aug initiator codon becomes the start of an uporf, following which reinitiation occurs at a normally silent internal aug codon (chang and gould, 1998; ledley et al., 1990; zoppi et al., 1993) . the n-terminally truncated protein thus produced sometimes retains enough function to mitigate the pathological effects of the nonsense mutation (chang and gould, 1998) . this potential rescue device often fails, however, because many mrnas are rapidly degraded when a nonsense codon is introduced (frischmeyer and dietz, 1999; he and jacobson, 2001) . the mrna decay pathway that targets these abnormal mrnas is activated in part by cis elements located in the coding domain (gudikote and wilkinson, 2002) , which might explain why normal uporf-containing mrnas (e.g. those discussed in figs. 3 and 4) are not rapidly degraded. initiation factor eif2 plays a key role in translational control (clemens, 2001; dever, 2002) and mutations that perturb regulation of eif2 have profound pathological consequences (delépine et al., 2000; han et al., 2001; harding et al., 2001; van der knaap et al., 2002) . human genetic disorders have been traced also to disruption of regulatory mechanisms mediated by mrna binding proteins cazzola and skoda, 2000; cazzola et al., 2002; kaytor and orr, 2001; mikulits et al., 1999) . here, however, i focus on pathologies resulting from increases or decreases in translation caused directly by changes in mrna structure. the preceding paragraph mentioned some cases in which an n-terminally truncated protein is produced, apparently by reinitiation, when a mutation introduces a premature nonsense codon. the effects of some other types of mutations can also be understood in light of the scanning mechanism, as outlined next and discussed elsewhere in more detail (kozak, 2002) . recent investigations have identified diseases that result from failure to produce one of the two protein isoforms derived from genes that encode certain transcription factors (table 4 ). because the second isoform often functions as a modulator, the transcriptional imbalance caused by these changes in translation can have serious consequences. hereditary diseases have been traced occasionally to point mutations that alter the context flanking the aug start codon. the list includes a-thalassaemia caused by an a ! c change in position 23 of the a-globin gene (morlé et al., 1985) , androgen insensitivity syndrome caused by a g ! a mutation in position þ4 of the androgen receptor gene (choong et al., 1996) , and ataxia with vitamin e deficiency caused by a c ! t mutation in position 21 of the a-tocopherol transfer protein gene (usuki and maruyama, 2000) . there is an interesting report of a somatic point mutation (g ! c in position 23) in the brca1 gene in a highly aggressive case of sporadic breast cancer (signori et al., 2001) . in mice, a screen for mutations that cause defects in eye development uncovered an a ! t change in position 23 of the pax6 gene (favor et al., 2001) . each of these mutations was shown to cause a decrease (generally two-to four-fold) in translation. not every mutation or polymorphism within the consensus motif can be explained simply, however. other considerations, such as codon usage, might prevent an increase in translation even when the context is improved (i.e. translation might be limited at the level of elongation rather than initiation), and some mutations near the aug codon might affect mrna processing or stability rather than translation per se. a clinically relevant polymorphism in position 2 1 of annexin v appears to have an effect on translation which is inconsistent with the context rules (gonzález-conejero et al., 2002) , but the effect was small and documented only by assaying translation in vitro, which is not always reliable (section 6.2). a natural polymorphism in position 2 5 of the glycoprotein iba gene displayed a small effect on translation in vitro that was consistent with the rules (c worked better than t; afshar-kharghan et al., 1999) but, in the same study, mutations that changed position þ 4 from c to g did not augment translation. testing mutations in position þ 4 is tricky, however, because the change in identity of the penultimate amino acid might affect protein stability in ways that obscure the effects on translation. the solution is to use an assay that directly monitors the initiation step of translation (kozak, 1997) . the scanning mechanism predicts that a mutation which weakens or destroys the normal start codon should activate initiation from the next aug downstream. in some hereditary diseases in which the aug start codon is ablated, a truncated protein is indeed produced in this way but it does not function well enough to prevent the disease (cahana et al., 2001; huang et al., 1999; o'neill et al., 2001) . in the case of a mutated vasopressin gene in which the g of the aug start codon is deleted, the shorter signal peptide initiated from the second aug codon is not recognized by signal peptidase (beuret et al., 1999) . the resulting uncleaved vasopressin-precursor protein folds incorrectly, causing subsequent processing steps to fail, and therefore vasopressin never gets released from the endoplasmic reticulum. the second aug is only four codons downstream from the first, but the processing defect caused by this slight shift in the site of initiation causes diabetes insipidus. the scanning mechanism predicts that, when an out-offrame aug codon is introduced into the 5 0 utr, the adventitious upstream start codon should supplant the normal start site. a number of pathologies result from this kind of translational block. sometimes the upstream aug codon is created by a rare mutation (cai et al., 1992; liu et al., 1999) . other times it derives from a common polymorphism (bergenhem et al., 1992; endler et al., 2001; kanaji et al., 1998; kraft et al., 1998; zysow et al., 1995) . the reduction in translation is more or less severe depending on the context of the upstream aug codon and whether reinitiation is possible. i have already explained how hereditary thrombocythemia is caused by mutations that elevate translation of tpo by restructuring or eliminating an inhibitory uporf (fig. 4) . translation of proto-oncogenes is also elevated in some cases by eliminating small uporfs from the mrna. the mdm2 oncogene is one example: whereas the normal mrna has a long 5 0 utr that includes two upstream aug codons, in tumor cells the use of a different promoter eliminates the upstream augs and thus increases translational efficiency 20-fold (brown et al., 1999; landers et al., 1997) . in the case of oncogene gli1, the upstream aug codons that restrict translation in normal cells reside in an intron which is eliminated by more efficient splicing in basal cell carcinomas (wang and rothnagel, 2001) . translation of many other human or rodent oncogenes is restricted in normal cells by an encumbered (aug-burdened or gc-rich) leader sequence (arrick et al., 1991; bates et al., 1991; child et al., 1999a; harigai et al., 1996; hoover et al., 1997; horvath et al., 1995; manzella and blackshear, 1990; sarrazin et al., 2000) ; in some of these cases, a shorter 5 0 utr that better supports translation is produced in transformed cells (arrick et al., 1994; marth et al., 1988) . for other oncogenes, although there are alternative leader sequences that might regulate expression in normal tissues (link et al., 1992; sasahara et al., 1998) , there is no evidence that switching leader sequences contributes to tumorigenesis. table 4 pathologies resulting from a change in mrna structure which selectively abolishes production of the long or short form of a transcription factor translational mechanism that normally generates two protein isoforms disease-associated change in mrna structure and translation references c/ebpa (human) two proteins from one mrna via leaky scanning þ reinitiation shunt in acute myeloid leukemia, mutations near amino terminus eliminate production of longer isoform. pabst et al., 2001 gata1 (human) two proteins from one mrna via leaky scanning in down syndrome-related leukemia, premature stop codon eliminates production of longer isoform. wechsler et al., 2002 lef1 (human) two proteins from two mrnas (via two promoters) in colon cancer, failure to activate downstream promoter prevents production of shorter (inhibitory) isoform. hovanes et al., 2001 rx/rax (mouse) two proteins from one mrna via leaky scanning a in eyeless mice, mutation of second aug, leaving only the weak upstream start codon, results in inadequate yield. tucker et al., 2001 a here the long and short isoforms appear to function identically; the significance of the second aug start codon pertains to boosting the overall protein yield. the eyeless mouse serves as a spontaneous model for human anophthalmia. whereas removal of small uporfs elevates the translation of the aforementioned mdm2 and gli1 oncogenes in tumor cells, addition of small uporfs shuts off the translation of some tumor suppressor genes. in the case of hyal1, retention of an intron which contains eight upstream aug codons renders the mrna untranslatable in squamous cell carcinomas (frost et al., 2000) . a striking example of translational inactivation of a tumor suppressor gene is seen in some individuals with a predisposition to melanoma. in certain families, a point mutation (g ! t) creates an upstream, out-of-frame aug codon in the cdkn2 gene . the small uporf initiated from this new aug codon overlaps the cdkn2 start codon, and the resulting inhibition of translation is profound. structural changes that attenuate the translation of viral mrnas can contribute to the development of persistent infections. the 5 0 leader sequence on bovine coronavirus mrnas, for example, was found to evolve -by acquiring a small uporf -during the course of establishing a persistent infection (hofmann et al., 1993) . shishido-hara et al. (2000) speculate that human polyomavirus jc might cause persistent rather than acute infection because all capsid-protein encoding transcripts produced by the jc virus have a small uporf. with the related simian virus 40, in contrast, the uporf is sometimes eliminated by splicing, generating transcripts that better support translation of the major capsid protein. attenuating effects caused by introducing an upstream aug codon have been described also with other viruses (petty et al., 1990; slobodskaya et al., 1996) . a more drastic restructuring of mrnas sometimes occurs during the establishment of persistent infections by the measles virus. instead of the normal monocistronic mrna for the fusion protein, the predominant transcript in some persistently infected cells was a dicistronic mrna from which the f cistron, located at the 3 0 end, could not be translated (hummel et al., 1994) . a similar problem encountered in studies with recombinant rhabdoviruses provides insight into the transcriptional defects that can generate untranslatable dicistronic mrnas (quiñones-kochs et al., 2001) . in the case of a human parvovirus, productive infection is restricted to a subset of erythroid cells in which splicing generates a monocistronic mrna for each of the major capsid proteins. in nonpermissive cells, a slight shift in the position of a splice site imposes an upstream orf which is postulated to restrict translation of the capsid proteins (brunstein et al., 2000) . translational twists sometimes generate antigens which, by stimulating the ctl response, are important in the host defense against tumor cells and viruses (shastri et al., 2002) . leaky scanning is a likely explanation in several cases where the major orf starts with an aug codon in a suboptimal context and the ctl antigen derives from initiation at the next (out-of-frame) aug (aarnoudse et al., 1999; bullock et al., 1997; probst-kepper et al., 2001; rimoldi et al., 2000) . in one notable case, translation shifts upstream to an in-frame aug codon created during insertion of a provirus, and the resulting novel n-terminal amino acid extension functions as a tumor rejection antigen (wada et al., 1995) . the scanning mechanism cannot explain the translation of ctl antigens for which the start codon resides far in the interior of the mrna (ronsin et al., 1999; wang et al., 1996) . in these cases the antigenic peptide might be produced from an undetected alternative form of mrna. sensitive new assay techniques employed with some genes indeed reveal an array of alternative transcripts from which novel tumor antigens can be translated (behrends et al., 2002) . in another study, a potent tumor rejection peptide, which maps to an internal aug codon in the full-length cdna, was expressed experimentally from a truncated cdna wherein the start codon for the antigenic peptide was made the first aug (rosenberg et al., 2002) . additional analyses are needed to determine whether, in the melanoma cells wherein this antigen is expressed naturally, a transcript similar to the experimentally truncated cdna is produced via a downstream promoter or splice site. 6. surveys and assays and problems therein 6.1. cdna surveys surveys of mrna/cdna sequences differ in other details, but every survey confirms the presence of a purine in position 2 3 in most ($ 90%) vertebrate mrnas (kozak, 1987a; pesole et al., 2000; rogozin et al., 2001; sakai et al., 2001) . the occasional survey that purports to challenge the context rules involves distortions, such as emphasizing the low percentage of cdnas that have the full consensus sequence while ignoring the high percentage of cdnas that have the critical purine in position 2 3 (peri and pandey, 2001) . a major uncertainty pertaining to all cdna surveys concerns the validity of the database. when i re-examined the entries in one study (suzuki et al., 2000) , i found numerous instances in which the aug start codon had been misidentified; the corrected start sites adhered more closely to the consensus motif (kozak, 2000) . some authors pre-emptively defend their conclusions on the grounds that the (unidentified) cdna sequences used for their analysis derive from refseq, which is a curated database (pruitt et al., 2000) . but the entries in refseq are not without errors, some of which -e.g. misidentified start codons, mistaken claims of upstream aug codons -can be traced by comparing curated genbank entries nm_005493, nm_005502, nm_000282 and nm_003605 with results published else-where (campeau et al., 2001; nishitani et al., 2001; nolte and müller, 2002; santamarina-fojo et al., 2000) . some cdna surveys use misleading terminology, e.g. referring to upstream aug codons as 'unused' (peri and pandey, 2001) . given that upstream aug codons are used, as proved by detecting the encoded peptide or by fusing the uporf to a reporter gene, it is not anomalous to find a good context around some upstream aug codons. all surveys tend to overestimate the incidence of upstream augs by scoring only the longest cdna isoform, ignoring the existence of alternative transcripts that have shorter, unencumbered 5 0 utrs (section 4.2). the significance of upstream aug codons also tends to be misstated: the presence of small uporfs in vertebrate mrnas which are thereby translated inefficiently (see the foregoing discussion of tpo, oncogenes, etc.) constitutes evidence for, rather than against, the scanning model. the first-aug rule, which i cite as evidence for the scanning mechanism, derives not from statistical analysis of cdna sequences but from the experimentally observed fact (section 2.2) that translation shifts predictably upstream or downstream when an aug codon is added or removed. in short, it makes more sense to use the scanning/context rules to evaluate cdna sequences (hatzigeorgiou, 2002) than to attempt the reverse. while conclusions about translation derived from experimental studies are arguably more meaningful than those derived from statistics, the interpretation of experimental results can be complicated. in vivo assays avoid the problem of reaction-conditions-dictating-the-outcome (see next paragraph), but there are other potential traps. the usually-valid assumption that polysomal association identifies actively translated mrnas is called into question by the recent discovery of mrnas trapped on large polysomes from which there is no polypeptide production (rüegsegger et al., 2001) . the major problem when translation is studied in vivo is uncertainty about the structure of the mrna. for example, a claim that ires-mediated translation is developmentally regulated (créancier et al., 2000 (créancier et al., , 2001 is premature, inasmuch as those studies monitored the amount but not the form of mrna. when mrna structure is examined, the developmentally regulated expression might be found to reflect activation of an internal promoter rather than activation of an ires. other studies wherein translation of an encumbered leader sequence appears to improve under certain conditions or in certain cell types require better analyses to rule out a possible change in structure of the 5 0 utr (bernstein et al., 1995; child et al., 1999b; li et al., 2001; zimmer et al., 1994) . some useful hints may be found in reports that describe the belated discovery of alternative forms of mrna that were missed the first time around cortner and farnham, 1990; déjardin et al., 2000; deng et al., 2002; frost et al., 2000; grundhoff and ganem, 2001; jordan et al., 1996; kastner et al., 1990b; kiss-lászló et al., 1995; laurin et al., 2000; peremyslov and dolja, 2002; zhang and liu, 2000; zheng et al., 1994) . in vitro translation assays pose a different set of problems. the commercial availability of in vitro translation kits is both a blessing -the systems are easy to useand a curse. the latter because insufficient attention is paid to reaction conditions that can affect the selection of aug start codons. when the magnesium concentration is too low, the first aug codon may be bypassed despite an adequate context; when the magnesium concentration is too high, initiation may occur at upstream non-aug codons that are not naturally used. one solution is to include control transcripts for which start-codon selection was determined in vivo, and to adjust the in vitro reaction conditions to give the same result (kozak, 1990b) . some suppliers of translation kits make it possible to adjust the magnesium concentration, but there is little awareness of the need to do so and the use of coupled transcription/translation systems makes it difficult. for whatever reason, in vitro translation results sometimes deviate significantly from what is seen in vivo vis-àvis access to internal aug codons (grove et al., 1991; land and rouault, 1998; meijer et al., 2000; meulewaeter et al., 1992; mitchelmore et al., 2002; saucedo et al., 1999) and the degree of inhibition caused by small uporfs (ghilardi et al., 1998; harigai et al., 1996; pecqueur et al., 1999 pecqueur et al., , 2001 tanaka et al., 2001; wang and wessler, 1998) . the fidelity of initiation in vitro is clearly impaired, possibly due to degradation of the mrna, in cases where extraneous, low molecule weight polypeptides are produced (herbert et al., 1996; liu and biegalke, 2002; lekven et al., 2001, fig. 3c; maser et al., 2001, fig. 4b; packham et al., 1997, fig. 5) . the possibility that the input mrna might undergo cleavage during incubation in vitro complicates attempts to study the expression of dicistronic mrnas, as discussed in the next section. this type of artifact is not ruled out by finding that only certain downstream orfs are translated (o'connor and brian, 2000) . extrapolating from what is seen when mrnas are deliberately cleaved in vivo (thoma et al., 2001) , activation of internal start codons in vitro would depend on where the accidental cleavage occurs and whether the endolytic cleavage product persists long enough for a ribosome to engage the newly created 5 0 end before exonucleases take over. this line of reasoning could explain the claim that an 'artificial ires', consisting of a multiple cloning region and a portion of the escherichia coli laci gene, supports internal initiation of translation in starved yeast cells (paz et al., 1999) : starvation is likely to promote mrna degradation, and the 'ires' might fortuitously stabilize certain intermediates in degradation. the discovery that ires elements are actually targeted by some ribonucleases (elgadi and smiley, 1999; nadal et al., 2002) should be remembered. recent studies that use a primer-extension inhibition (toeprinting) assay to monitor the binding of ribosomes to mrnas have the advantage of focusing directly on the initiation step, but care is needed to distinguish authentic initiation complexes from artifactual pauses in primer extension caused by base-paired structures or extraneous proteins bound to the mrna. the complicating effects of mrna secondary structure, which are prominent when avian reverse transcriptase is used for toeprinting, can be minimized by using a form of the enzyme derived from murine leukemia virus (kozak, 1998) . attempts to explain the origin of multiple isoforms of eif4g (bradley et al., 2002) illustrate how challenging it can be to interpret translation assays. in vitro experiments presented in support of the idea that translation can initiate from five aug codons, in a single form of eif4g mrna, might have been compromised by mrna breakage; this would explain the production of an array of extraneous smaller polypeptides (byrd et al., 2002, fig. 3c , lanes 1, 3 and 5). translation of some eif4g isoforms from broken mrnas could also explain the variability in yields noted throughout that study. when the endogenous eif4g gene is expressed in vivo, access to certain downstream aug codons might occur via alternative splicing or internal promoters; both mechanisms have been documented in studies of eif4g by other investigators (han and zhang, 2002 , and references therein). thus, even though one could rationalize the production of at least three isoforms of eif4g from one mrna via established translational mechanisms -an overlapping uporf could shunt some 40s ribosomal subunits past the first in-frame aug codon (position 275), and the unfavorable context at aug 395 might allow some ribosomes to reach aug 536 by leaky scanning -it would be premature to propose that solution. the in vitro experiments need to be repeated with careful attention to magnesium levels and with efforts to minimize mrna breakage. the latter might be accomplished by lowering the temperature to 25 8c and limiting the window for initiation to 5 or 10 min. (addition of edeine after the first 5 or 10 min, followed by another period of incubation, would allow polypeptides to be elongated without further initiation events.) the possible production of some eif4g isoforms by proteolysis also needs to be ruled out, as this protein is notoriously susceptible to cleavage. the study by byrd et al. (2002) included experiments carried out with dicistronic transcripts, predicated on the belief that eif4g mrna contains ires elements which allow direct internal initiation of translation. an eif4g/ egfp (enhanced green fluorescent protein) fusion gene positioned at the 3 0 end of a dicistronic transcript was translatable in vitro, but the aforementioned possibility of mrna breakage complicates the interpretation. indeed, the unexpected translation of egfp from the 3 0 position even without fusion to eif4g (byrd et al., 2002, fig. 7a , lane 3) is most easily explained by mrna cleavage. (the authors invoke reinitiation as the explanation, but reinitiation cannot occur following translation of a large 5 0 cistron.) fragmentation of the mrna could explain why translation of the 3 0 eif4g/egfp cistron persisted when translation of the 5 0 cistron was blocked by a hairpin structure (byrd et al., 2002, fig. 7b ). the hairpin test, widely used to test for internal initiation, is meaningless without evidence that the dicistronic input mrna remains intact. the in vivo tests of eif4g translation (byrd et al., 2002, fig. 8 ) also require careful rna analyses to document that the vector produces only the intended dicistronic mrna; the quality of the northern blot in that figure falls far short of what is required. to rule out the possibility that the 3 0 cistron might be translated from an unintended monocistronic mrna, a promoter-deletion control is needed -a control which shows that, upon deleting the promoter that precedes the 5 0 cistron, expression of the 3 0 cistron is also abolished. this test failed in studies with some other sequences, revealing that the candidate ires actually harbors a cryptic promoter (han and zhang, 2002; larsen et al., 2002) . the foregoing discussion of eif4g translation alludes to only some of the problems associated with dicistronic vectors; a more detailed critique may be found elsewhere (kozak, 2001a) . use of a certain popular vector which harbors an intron near the 5 0 end (jopling and willis, 2001) increases the likelihood of producing an unintended monocistronic mrna via splicing; the candidate ires need contribute only a cryptic 3 0 splice site. this indeed happens in some cases (grundhoff and ganem, 2001; pinkstaff et al., 2001) . claims of ires activity are problematic when supported by in vitro assays in which translation of the 3 0 orf is very, very weak (e.g. deffaud and darlix, 2000, fig. 2; lekven et al., 2001, fig. 3b, lane 3; maser et al., 2001, fig. 4e ). the simple idea that an ires can be identified based on the ability to support translation of a 3 0 cistron runs into trouble when, for example, the bglobin mrna leader sequence, intended to serve as a negative control, turns out somehow to allow translation of a downstream cistron ( van der velden et al., 2002) . in another study, merely lengthening the intercistronic domain enabled substantial translation of the 3 0 cistron (gallie et al., 2000) , perhaps by providing room for rnases to cleave and thus release a translatable 3 0 fragment. even with the paradigmatic ires elements derived from picornaviruses, the ability to support internal initiation was found to depend inexplicably on the choice and arrangement of 5 0 and 3 0 reporter genes (hennecke et al., 2001) . these odditiesalong with the notable inability to translate the 3 0 cistron in natural dicistronic mrnas (table 1) -are reason to worry about the validity of experiments that employ synthetic dicistronic constructs. the proffered rationale for a cap-independent internal initiation mechanism is that it would enable certain mrnas to be translated when eif4e levels decline, but recent experiments presented in support of that idea used the 5 0 utr from poliovirus rather than 5 0 utrs from cellular mrnas, such as eif4g, that are supposedly regulated via 'a dynamic interplay between cap-dependent and cap-independent processes'. even if the proffered rationale is valid, convincing evidence for direct internal initiation with particular mrnas is needed. the widely used dicistronic assay has flaws, as outlined above. an alternative assay which involves circularization of the mrna has been attempted with only one viral ires element (chen and sarnow, 1995) ; the results await independent verification and extension to other sequences. the scanning model provides a framework for understanding basic patterns of eukaryotic gene expression, such as the reliance on monocistronic mrnas, and for understanding how translation is perturbed by mutations that restructure the 5 0 utr. a growing number of human diseases have been traced to such mutations. the scanning mechanism has been shown to operate not only with simple mrnas that have a short 5 0 utr and initiate at the first aug codon, but also with mrnas that have complicated leader sequences and multiple start codons. one often hears the suggestion that an alternative, iresmediated mechanism of initiation is required when a long leader sequence is encumbered by secondary structure or upstream aug codons (dever, 2002; pestova et al., 2001) . that view is not well taken. scanning can occur over long distances, as evidenced by some bifunctional viral mrnas in which the second start site is more than 500 nt downstream from the first (e.g. peanut clump virus, southern bean mosaic virus, rice tungro bacilliform virus; table 3 and fig. 1c ). the structure-prone, gc-rich leader sequences on mammalian mrnas strongly reduce translational efficiency but do not preclude operation of the scanning mechanism (van der velden et al., 2002) . upstream aug codons also reduce translational efficiency and that is why they are there. to postulate the need for an alternative mechanism is to miss the point that an encumbered leader sequence ensures that translation via scanning will be inefficient, and thus ensures against harmful overproduction of cytokines (fig. 4) and other potent proteins. the high frequency of intron-containing cdna sequences (kozak, 1991a (kozak, , 1996 might reflect another type of regulation. inefficient or regulated removal of the first intron has been documented in some cases (boularand et al., 1995; frost et al., 2000; van der leij et al., 2002; wang and rothnagel, 2001; xie et al., 1991; zachar et al., 1987) and i suspect that additional examples might be found -miscategorized -among the aforementioned cdnas that are postulated to require an alternative mechanism of initiation. removal of the intron, or use of a cryptic promoter therein, would eliminate the upstream aug codons that are barriers to scanning. examples in which translation is prevented deliberately by splicing-out the exon that contains the aug start codon (lin et al., 1998) or by other regulated splicing events (rueter et al., 1999) underscore the point that not every transcript -hence not every cdna -corresponds to a functional mrna. before postulating the need for a new mechanism to explain how a funny looking cdna gets translated, one must be certain that it is translated. the mechanisms discussed herein for escaping the first-aug rule, within the constraints imposed by the scanning model, obviously cannot explain every report of initiation from an internal position. more information is needed to understand how n-terminally truncated versions of some proteins are produced apparently without truncation of the mrna (goss et al., 2002; maser et al., 2001; santagata et al., 2000; scharnhorst et al., 1999; vanhoutte et al., 2001 ). an ires element was postulated in some of those cases, based on the dicistronic test, but in one study there were no accompanying analyses of rna structure in vivo (goss et al., 2002) , and in another study the use of an in vitro translation system produced too little of the truncated protein to be convincing (maser et al., 2001) . speculation about how some other interesting genes are translated (klemke et al., 2001 ) also must be postponed pending a search for possible additional forms of mrna. although i listed the von hippel-lindau tumor suppressor gene as a possible example of leaky scanning (table 3) , definitive tests are needed to distinguish between that and other mechanisms for producing the short isoform (iliopoulos et al., 1998) . those of us with an interest in translation have a tendency to interpret every change in mrna structure as a means to control translation, but transcriptional requirements -the need to turn on a gene in various tissues via whatever promoter works in each tissue -underlie most switches in 5 0 leader sequences. in some cases the actual sequence of the 5 0 utr is dictated by the presence therein of transcriptional control elements (akiri et al., 1998; minami et al., 2001; solecki et al., 1997; yin and blanchard, 2000; yu et al., 2001; zimmermann et al., 2000) . regulation of transcription is the major reason for the gc-rich domains near the 5 0 end of many mammalian genes; the accompanying down-modulation of translation is an inevitable consequence -arguably a useful consequence because, given the long half-life of most mammalian mrnas, inefficient translation might be a necessity. it merits repeating that, although the m7g cap strongly promotes ribosome binding, the scanning mechanism is not dependent on the presence of the cap. the essence of the scanning model is 5 0 entry of ribosomes and positiondependent selection of the aug start codon. those key points hold with naturally uncapped mrnas produced by some viruses (footnote g in table 1 ) and with synthetic uncapped mrnas used to study translation in vitro (kozak, 1979a . the inclination to invoke internal initiation based on indirect criteria -absence of a cap, or the ability to be translated in extracts from poliovirus-infected cells -should be resisted. it is a mistake to think that, because archaeal mrnas lack a 5 0 cap, translation in that system cannot occur via scanning. the discovery in archaea of proteins similar to certain eukaryotic initiation factors (kyrpides and woese, 1998) is intriguing for other reasons but has no direct bearing on whether the start codon in archaeal mrnas might be recognized via a prokaryotic-or eukaryotic-type mechanism. that interesting question, which bears on the evolutionary origin of scanning, awaits answering. fundamental questions about the molecular workings of the scanning mechanism also await answering. what drives migration of the 40s subunit during the scanning phase? how does the 40s subunit hold on at a terminator codon, in order to reinitiate? what prevents reinitiation when the size of the first orf exceeds a certain length? we know nothing about how recognition of the start codon is aided by a purine in position 23 and g in position þ 4. there is no evidence for base pairing between the gccrcc motif and rrna (or for binding of rrna to any other sequence in eukaryotic mrnas). there is as yet no convincing evidence for recognition of gccrcc by a trans-acting protein factor. it would be easy, and meaningless, simply to find proteins that bind an rna fragment which contains the motif. credible experiments would require controls based on what we know about the consensus sequence: that the purine (a . g) in position 2 3 plays a dominant role, and the full effect requires that the gccrcc motif abut the aug codon (kozak, 1999, fig. 1 ). with so much effort being directed to searching for possible exceptions to the scanning mechanism, one can only wish that some enterprising soul would tackle these important questions. interleukin-2-induced, melanoma-specific t cells recognize camel, an unexpected translation product of lage-1 suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for gcn4 translational control subcellular fate of the int-2 oncoprotein is determined by choice of initiation codon kozak sequence polymorphism of the glycoprotein (gp) iba gene is a major determinant of the plasma membrane levels of the platelet gp ib-ix-v complex specific sequences in p120ctn determine subcellular distribution of its multiple isoforms involved in cellular adhesion of normal and malignant epithelial cells unconventional mrna processing in the expression of two calcineurin b isoforms in dictyostelium uroporphyrinogen iii synthase. an alternative promoter controls erythroid-specific expression in the murine gene regulation of vascular endothelial growth factor (vegf) expression is mediated by internal initiation of translation and alternative initiation of transcription abundant early expression of gpul4 from a human cytomegalovirus mutant lacking a repressive upstream open reading frame apobec-1 transcription in rat colon cancer: decreased apobec-1 protein production through alterations in polysome distribution and mrna translation associated with upstream augs identification of a brain-specific protein kinase cj pseudogene (cpkcj) transcript tissue-specific and ubiquitous promoters direct the expression of alternatively spliced transcripts from the calcitonin receptor gene structural characterization of sil, a gene frequently disrupted in t-cell acute lymphoblastic leukemia cloning and characterization of the gene encoding rabbit cardiac calsequestrin a new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-aug start codon and behaves as a survival factor inhibition of translation of transforming growth factor-b3 mrna by its 5 0 untranslated region enhanced translational efficiency of a novel transforming growth factor b3 mrna in human breast cancer cells translational initiation competence, 'leaky scanning' and translational reinitiation in area mrna of aspergillus nidulans multiple roles for the cterminal domain of eif5 in translation initiation complex assembly and gtpase activation biosynthesis of osteogenic growth peptide via alternative translational initiation at aug 85 of histone h4 mrna expression of murine il-12 is regulated by translational control of the p35 subunit the two proteins encoded by the cottontail rabbit papillomavirus e6 open reading frame differ with respect to localization and phosphorylation biosynthesis of human fibroblast growth factor-5 novel products of the hud, huc, nnp-1 and a-internexin genes identified by autologous antibody screening of a pediatric neuroblastoma library yeast leu4 encodes mitochondrial and non-mitochondrial forms of a-isopropylmalate synthase mutation creates an open reading frame within the 5 0 untranslated region of macaque erythrocyte carbonic anhydrase (ca) i mrna that suppresses ca i expression and supports the scanning model for translation the translational repression mediated by the platelet-derived growth factor 2/c-sis mrna leader is relieved during megakaryocytic differentiation positive and negative regulation of myogenic differentiation of c2c12 cells by isoforms of the multiple homeodomain zinc finger transcription factor atbf1 mechanism of endoplasmic reticulum retention of mutant vasopressin precursor caused by a signal peptide truncation associated with diabetes insipidus alternative splicing of the imprinted candidate tumor suppressor gene zac regulates its antiproliferative and dna binding activities a global analysis of caenorhabditis elegans operons the avian reovirus genome segment s1 is a functionally tricistronic gene that expresses one structural and two nonstructural proteins in infected cells the 2.2 kb e1b mrna of human ad12 and ad5 codes for two tumor antigens starting at different aug triplets the human tryptophan hydroxylase gene. an unusual splicing complexity in the 5 0 -untranslated region mass spectrometric analysis of the n terminus of translational initiation factor eif4g-1 reveals novel isoforms the procaspase-8 isoform, procaspase-8l, recruited to the bap31 complex at the endoplasmic reticulum bunyamwera bunyavirus nonstructural protein nss is a nonessential gene product that contributes to viral pathogenesis role of two upstream open reading frames in the translational control of oncogene mdm2 microarray identification of fmrp-associated brain mrnas and altered mrna translational profiles in fragile x syndrome identification of a novel rna splicing pattern as a basis of restricted cell tropism of erythrovirus b19 initiation codon scanthrough versus termination codon readthrough demonstrates strong potential for major histocompatibility complex class i-restricted cryptic epitope expression generation of multiple isoforms of eukaryotic translation initiation factor 4gi by use of alternate translation initiation codons translational control of mammalian serine hydroxymethyl-transferase expression targeted mutagenesis of lis1 disrupts cortical development and lis1 homodimerization two novel b-thalassemia mutations in the 5 0 and 3 0 noncoding regions of the b-globin gene translational control of c/ ebpa and c/ebpb isoform expression alternative translation initiation site usage results in two functionally distinct forms of the gata-1 transcription factor structure of the pcca gene and distribution of mutations causing propionic acidemia translational inhibition by a human cytomegalovirus upstream open reading frame despite inefficient utilization of its aug codon the secreted form of invertase in saccharomyces cerevisiae is synthesized from mrna encoding a signal sequence translation of equine infectious anemia virus bicistronic tat-rev mrna requires leaky ribosome scanning of the tat ctg initiation codon transcription of feline calicivirus rna translational pathophysiology: a novel molecular mechanism of human disease a novel deletion of the l-ferritin iron-responsive element responsible for severe hereditary hyperferritinaemia-cataract syndrome phenotype-genotype relationships in complementation group 3 of the peroxisome-biogenesis disorders the increased level of b1,4-galactosyltransferase required for lactose biosynthesis is achieved in part by translational control the yeast vas1 gene encodes both mitochondrial and cytoplasmic valyl-trna synthetases structure of the gm2a gene: identification of an exon 2 nonsense mutation and a naturally occurring transcript with an in-frame deletion of exon 2 initiation of protein synthesis by the eukaryotic translational apparatus on circular rnas a novel influenza a virus mitochondrial protein that induces cell death translation initiation at alternate in-frame aug codons in the rabies virus phosphoprotein mrna is mediated by a ribosomal leaky scanning mechanism translational control by an upstream open reading frame in the her-2/neu transcript cell type-dependent andindependent control of her-2/neu translation a novel missense mutation in the amino-terminal domain of the human androgen receptor gene in a family with partial androgen insensitivity syndrome causes reduced efficiency of protein translation two distinct protein isoforms are encoded by ntk, a csk-related tyrosine protein kinase gene alternative transcription and splicing of the human porphobilinogen deaminase gene result either in tissue-specific or in housekeeping expression trna met functions in directing the scanning ribosome to the start site of translation mutational analysis of the his4 translational initiator region in saccharomyces cerevisiae initiation factor eif2a phosphorylation in stress responses and apoptosis tissue-and development-specific alternative rna splicing regulates expression of multiple isoforms of erythroid membrane protein 4.1 identification of the serum-responsive transcription initiation site of the zinc finger gene krox-20 fibroblast growth factor 2 internal ribosome entry site (ires) activity ex vivo and in transgenic mice reveals a stringent tissue-specific regulation c-myc internal ribosome entry site activity is developmentally controlled and subjected to a strong translational repression in adult transgenic mice the arabidopsis thaliana fps1 gene generates a novel mrna that encodes a mitochondrial farnesyldiphosphate synthase isoform alternatively spliced human type 1 angiotensin ii receptor mrnas are translated at different efficiencies and encode two receptor isoforms eukaryotic translation initiation factor 5 functions as a gtpase-activating protein the s4 genome segment of baboon reovirus is bicistronic and encodes a novel fusion-associated small transmembrane protein expression and function of ccaat/enhancer binding protein b (c/ ebpb) lap and lip isoforms in mouse mammary gland, tumors and cultured mammary epithelial cells rlk/txk encodes two forms of a novel cysteine string tyrosine kinase activated by src family kinases characterization of an internal ribosomal entry segment in the 5 0 leader of murine leukemia virus env rna a novel subgenomic murine leukemia virus rna transcript results from alternative splicing eif2ak3, encoding translation initiation factor 2-a kinase 3, is mutated in patients with wolcott-rallison syndrome transcriptional regulation of the interleukin-6 gene of human herpesvirus 8 (kaposi's sarcomaassociated herpesvirus) era gene expression in human primary osteoblasts: evidence for the expression of two receptor proteins a liver-enriched transcriptional activator protein, lap, and a transcriptional inhibitory protein, lip, are translated from the same mrna gene-specific regulation by general translation factors genomic structure of unp, a murine gene encoding a ubiquitin-specific protease control of start codon choice on a plant viral rna encoding overlapping genes the first and third uorfs in rsv leader rna are efficiently translated: implications for translational regulation and viral rna packaging identification of eight novel 5 0 -exons in cerebral capillary malformation gene-1 (ccm1 ) encoding krit1 picornavirus internal ribosome entry site elements target rna cleavage events induced by the herpes simplex virus virion host-shutoff protein nucleotide sequence and expression of the small (s) rna segment of maguari bunyavirus amino-terminal extension generated from an upstream aug codon increases the efficiency of mitochondrial import of yeast n 2 ,n 2 -dimethylguanosine-specific trna methyltransferases gastric cancers overexpress darpp-32 and a novel isoform, t-darpp a common c ! t polymorphism at nt 46 in the promoter region of coagulation factor xii is associated with decreased factor xii activity translation of bicistronic viral mrna in transfected cells: regulation at the level of elongation a 31-amino acid n-terminal extension regulates c-crk binding to tyrosine-phosphorylated proteins the rat hepatic leukemia factor (hlf) gene encodes two transcriptional activators with distinct circadian rhythms, tissue distributions and target preferences molecular characterization of pax6 2neu through pax6 10neu : an extension of the pax6 allelic series and the identification of two possible hypomorph alleles in the mouse mus musculus human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non-aug codons identification of a new isoform of the human estrogen receptor-alpha (her-a) that is encoded by distinct transcripts and that is able to repress her-a activation function 1 a testisspecific promoter in the rat vasopressin gene translational stop codons in the precore sequence of hepatitis b virus pre-c rna allow translation reinitiation at downstream augs translation of the hepatitis b virus p gene by ribosomal scanning as an alternative to internal initiation upstream organization of and multiple transcripts from the human folylpoly-gglutamate synthetase gene nonsense-mediated mrna decay in health and disease hyal1 luca-1 , a candidate tumor suppressor gene on chromosome 3p21.3, is inactivated in head and neck squamous cell carcinomas by aberrant splicing of pre-mrna viral and cellular mrna capping: past and prospects splicing in a plant pararetrovirus position-dependent att initiation during plant pararetrovirus rice tungro bacilliform virus translation rice tungro bacilliform virus open reading frames ii and iii are translated from polycistronic pregenomic rna by leaky scanning translation of p15.5 ink4b , an n-terminally extended and fully active form of p15 ink4b , is initiated from an upstream gug codon physical evidence for distinct mechanisms of translational control by upstream open reading frames the role of 5 0 -leader length, secondary structure and pabp concentration on cap and poly(a) tail function during translation in xenopus oocytes the two forms of karyogamy transcription factor kar4p are regulated by differential initiation of transcription, translation, and protein turnover gcd10, a translational repressor of gcn4, is the rna-binding subunit of eukaryotic translation initiation factor-3 the mrna structure has potent regulatory effects on type 2 iodothyronine deiodinase expression a single-base deletion in the thrombopoietin (tpo) gene causes familial essential thrombocythemia through a mechanism of more efficient translation of tpo mrna thrombopoietin production is inhibited by a translational mechanism hereditary thrombocythaemia in a japanese family is caused by a novel point mutation in the thrombopoietin gene initiation of translation directed by 42s and 26s rnas from semliki forest virus in vitro a common polymorphism in the annexin v kozak sequence (21 c . t) increases translation efficiency and plasma levels of annexin v, and decreases the risk of myocardial infarction in young patients position is the critical determinant for function of iron-responsive elements as translational regulators attenuated apc alleles produce functional protein from internal translation initiation characterization of the cis-acting elements controlling subgenomic mrnas of citrus tristeza virus: production of positive-and negativestranded 3 0 -terminal and positive-stranded 5 0 -terminal rnas effect of sequence context at stop codons on efficiency of reinitiation in gcn4 translational control diverse splicing mechanisms fuse the evolutionarily conserved bicistronic mocs1a and mocs1b open reading frames an imprinted, mammalian bicistronic transcript encodes two independent proteins mapping of the tobacco mosaic virus movement protein and coat protein subgenomic rna promoters a link between diabetes and atherosclerosis: glucose regulates expression of cd36 at the level of translation the vitamin d receptor gene start codon polymorphism: a functional analysis of fok i variants cloning and expression of two human p70 s6 kinase polypeptides differing only at their amino termini mechanisms governing expression of the v-flip gene of kaposi's sarcoma-associated herpesvirus t-cell receptor sequences that elicit strong down-regulation of premature termination codon-bearing transcripts mapping and expression of southern bean mosaic virus genomic and subgenomic rnas synthesis in vitro of a seven amino acid peptide encoded in the leader rna of rous sarcoma virus utilization of an alternative transcription initiation site of somatic cytochrome c in the mouse produces a testisspecific cytochrome c mrna heme-regulated eif2a kinase (hri) is required for translational regulation and survival of erythroid precursors in iron deficiency regulation of gene expression by internal ribosome entry sites or cryptic promoters: the eif4g story human ubiquitin-activating enzyme, e1. indication of potential nuclear and cytoplasmic subpopulations using epitope-tagged cdna constructs abrogation of upstream open reading frame-mediated translational control of a plant s-adenosylmethionine decarboxylase results in polyamine disruption and growth perturbations mouse atf5: molecular cloning of two novel mrnas, genomic organization, and odorant sensory neuron localization functionality of alternative splice forms of the first enzymes involved in human molybdenum cofactor biosynthesis regulated translation initiation controls stress-induced gene expression in mammalian cells diabetes mellitus and exocrine pancreatic dysfunction in perk 2 /2 mice reveals a role for translational control in secretory cell survival characterization of two cis-regulatory regions in the murine b1,4-galactosyltransferase gene a cis-acting element in the bcl-2 gene controls expression through translational mechanisms subcellular relocalization of a long-chain fatty acid coa ligase by a suppressor mutation alleviates a respiration deficiency in saccharomyces cerevisiae translation initiation start prediction in human cdnas with high accuracy upflp, nmd2p, and upf3p regulate the decapping and exonucleolytic degradation of both nonsense-containing mrnas and wild-type mrnas human wig-1, a p53 target gene that encodes a growth inhibitory zinc finger protein internal ribosome entry sites in eukaryotic mrna molecules composition and arrangement of genes define the strength of ires-driven translation in bicistronic mrnas the conserved 5 0 -untranslated leader of spi-1 (pu.1) mrna is highly structured and potently inhibits translation in vitro but not in vivo detection of the orf3 polypeptide of feline calicivirus in infected cells and evidence for its expression from a single, functionally bicistronic subgenomic mrna an altered ribosomal protein in an edeine-resistant mutant of saccharomyces cerevisiae translation of the second gene of peanut clump virus rna 2 occurs by leaky scanning in vitro characterization of the gene encoding human platelet glycoprotein ix translational regulation of yeast gcn4. a window on factors that control initiator-trna binding to the ribosome translation initiation and assembly of peripherin in cultured cells a translationattenuating intraleader open reading frame is selected on coronavirus mrnas during persistent infection molecular basis for the dual mitochondrial and cytosolic localization of alanine:glyoxylate aminotransferase in amphibian liver cells pim-1 protein expression is regulated by its 5 0 -untranslated region and translation initiation factor eif-4e characterization of the infections of permissive and nonpermissive cells by host range mutants of vesicular stomatitis virus defective in rna methylation translational regulation of complement protein c2 expression by differential utilization of the 5 0 -untranslated region of mrna multiple elements in the 5 0 untranslated region down-regulate c-sis messenger rna translation caenorhabditis elegans mrnas that encode a protein similar to adars derive from an operon containing six genes b-cateninsensitive isoforms of lymphoid enhancer factor-1 are selectively expressed in colon cancer rh mod syndrome: a family study of the translation-initiator mutation in the rh50 glycoprotein gene gtp hydrolysis controls stringent selection of the aug start codon during translation initiation in saccharomyces cerevisiae genomic structure of the locus encoding protein 4.1. structural basis for complex combinatorial patterns of tissue-specific alternative rna splicing restriction of fusion protein mrna as a mechanism of measles virus persistence messenger rna for the coat protein of tobacco mosaic virus multiple 5 0 -untranslated exons in the nuclear respiratory factor 1 gene span 47 kb and contribute to transcript heterogeneity and translational efficiency translational regulation of hepatitis b virus polymerase gene by termination-reinitiation of an upstream minicistron in a length-dependent manner pvhl 19 is a biologically active product of the von hippel-lindau gene arising from internal translation initiation conservation of polyamine regulation by translational frameshifting from yeast to mammals identification of two additional translation products from the matrix (m) gene that contribute to vesicular stomatitis virus cytopathology decreased riz1 expression but not riz2 in hepatoma and suppression of hepatoma tumorigenicity by riz1 variants of the 5 0 -untranslated region of the bovine growth hormone receptor mrna: isolation, expression and effects on translational efficiency minimal truncation of the c-myb gene product in rapid-onset b-cell lymphoma both codon context and leader length contribute to efficient expression of two overlapping open reading frames of a cucumber necrosis virus bifunctional subgenomic mrna expression of bacterial chitinase protein in tobacco leaves using two photosynthetic gene promoters n-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells expression of human foamy virus reverse transcriptase involves a spliced pol mrna a common genetic polymorphism (46 c to t substitution) in the 5 0 -untranslated region of the coagulation factor xii gene is associated with low translation efficiency and decrease in plasma factor xii level production of three distinct mrnas of 150 kda oxygen-regulated protein (orp150) by alternative promoters: preferential induction of one species under stress conditions two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms a and b transient expression of human and chicken progesterone receptors does not support alternative translational initiation from a single mrna as the mechanism generating two receptor isoforms human rna-specific adenosine deaminase (adar1 ) gene specifies transcripts that initiate from a constitutively active alternative promoter rna targets of the fragile x protein molecular cloning of the human p120 ctn catenin gene (ctnnd1): expression of multiple alternatively spliced isoforms competition between nuclear localization and secretory signals determines the subcellular fate of a single cug-initiated form of fgf3 expression of the open reading frame 74 (g-protein-coupled receptor) gene of kaposi's sarcoma (ks)-associated herpesvirus: implications for ks pathogenesis splicing of cauliflower mosaic virus 35s rna is essential for viral infectivity two overlapping reading frames in a single exon encode interacting proteins -a novel way of gene usage murine phospholipid hydroperoxide glutathione peroxidase: cdna sequence, tissue expression, and mapping a positive-strand rna virus with three very different subgenomic rna promoters caveolin-1 isoforms are encoded by distinct mrnas the position dependence of translational regulation via rna-rna and rnaprotein interactions in the 5 0 -untranslated region of eukaryotic mrna is a function of the thermodynamic competence of 40s ribosomes in translational initiation binding of ribosomes to linear and circular forms of the 5 0 -terminal leader fragment of tobacco mosaic virus rna familial essential thrombocythemia associated with one-base deletion in the 5 0 -untranslated region of the thrombopoietin gene upstream open reading frames regulate the translation of multiple mrna variants of the estrogen receptor alpha independent promoters regulate the expression of two amino terminally distinct forms of latent transforming growth factor-b binding protein-1 (ltbp-1) in a cell typespecific manner inability of circular mrna to attach to eukaryotic ribosomes migration of 40s ribosomal subunits on messenger rna when initiation is perturbed by lowering magnesium or adding drugs role of atp in binding and migration of 40s ribosomal subunits analysis of ribosome binding sites from the s1 message of reovirus. initiation at the first and second aug codons translation of insulin-related polypeptides from messenger rnas with tandemly reiterated copies of the ribosome binding site influences of mrna secondary structure on initiation by eukaryotic ribosomes point mutations define a sequence flanking the aug initiator codon that modulates translation by eukaryotic ribosomes an analysis of 5 0 -noncoding sequences from 699 vertebrate messenger rnas at least six nucleotides preceding the aug initiator codon enhance translation in mammalian cells effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mrnas downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources an analysis of vertebrate mrna sequences: intimations of translational control structural features in eukaryotic mrnas that modulate the initiation of translation a short leader sequence impairs the fidelity of initiation by eukaryotic ribosomes adherence to the first-aug rule when a second aug codon follows closely upon the first interpreting cdna sequences: some insights from studies on translation recognition of aug and alternative initiator codons is augmented by g in position þ 4 but is not generally affected by the nucleotides in positions þ5 and þ6 primer-extension analysis of eukaryotic ribosome-mrna complexes initiation of translation in prokaryotes and eukaryotes do the 5 0 untranslated domains of human cdnas challenge the rules for initiation of translation (or is it vice versa new ways of initiating translation in eukaryotes? constraints on reinitiation of translation in mammals emerging links between initiation of translation and human diseases migration of 40s ribosomal subunits on messenger rna in the presence of edeine significant impact of the þ 93 c/t polymorphism in the apolipoprotein(a) gene on lp(a) concentrations in africans but not in caucasians: confounding effect of linkage disequilibrium identification of the translational initiation codon in human maged1 genomic organization and biosynthesis of secreted and cytoplasmic forms of gelsolin archaeal translation initiation revisited: the initiation factor 2 and eukaryotic initiation factor 2b ab-d subunit families the molecular biology of coronaviruses a transcriptional repressor encoded by bpv-1 shares a common carboxy-terminal domain with the e2 transactivator the genetics of bovine papillomavirus type 1 targeting of a human iron-sulfur cluster assembly enzyme, nifs, to different subcellular compartments is regulated through alternative aug utilization translational enhancement of mdm2 oncogene expression in human tumor cells containing a stabilized wild-type p53 protein nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus genomic organization of the mouse peroxisome proliferator activated receptor b/g gene. alternative promoter usage and splicing yield transcripts exhibiting differential translational efficiency the hormone-sensitive lipase gene is transcribed from at least five alternative first exons in mouse adipose tissue polyamine regulation of ribosome pausing at the upstream open reading frame of sadenosylmethionine decarboxylase mutation eliminating mitochondrial leader sequence of methylmalonyl-coa mutase causes mut o methylmalonic acidemia molecular cloning and characterization of the mouse peroxiredoxin v gene the 5 0 untranslated regions of the rat a 2a adenosine receptor gene function as negative translational regulators zebrafish wnt8 encodes two wnt8 proteins on a bicistronic transcript and is required for mesoderm and neurectoderm patterning the human galactose-1-phosphate uridyltransferase gene translational control of cell fate: availability of phosphorylation sites on translational repressor 4e-bp1 governs its proapoptotic potency cell-to-cell movement of turnip crinkle virus is controlled by two small open reading frames that function in trans a translationally regulated tousled kinase phosphorylates histone h3 and confers radioresistance when overexpressed a 30-kda alternative translation product of the ccaat/enhancer binding protein a message: transcriptional activator lacking antimitotic activity intron-exon structure of the met gene and cloning of an alternatively-spliced met isoform reveals frequent exon-skipping of a single large internal exon an extended rna/rna duplex structure within the coding region of mrna does not block translational elongation characterization of the 5 0 untranslated region of the human c-fgr gene and identification of the major myelomonocytic c-fgr promoter the promoter, transcriptional unit, and coding sequence of herpes simplex virus 1 family 35 proteins are contained within and in frame with the u l 26 open reading frame initiation of translation from a downstream in-frame aug codon on brca1 can generate the novel isoform protein dbrca1(17aa) the retinoblastoma interacting zinc finger gene riz produces a pr domain-lacking product through an internal promoter mutation of the cdkn2a 5 0 utr creates an aberrant initiation codon and predisposes to melanoma the human cytomegalovirus ul35 gene encodes two proteins with different functions rna polymerase i-promoted his4 expression yields uncapped, polyadenylated mrna that is unstable and inefficiently translated in saccharomyces cerevisiae two isoforms of murine hck, generated by utilization of alternative translational initiation codons, exhibit different patterns of subcellular localization the human aqp4 gene: definition of the locus encoding two water channel polypeptides in brain cloning and origin of the two forms of chicken vitamin d receptor in vivo evaluation of the context sequence of the translation initiation codon in plants alternative splicing and promoter usage generates an intracellular stromelysin 3 isoform directly translated as an active matrix metalloproteinase efficiency of reinitiation of translation on human immunodeficiency virus type 1 mrnas is determined by the length of the upstream open reading frame and by intercistronic distance expression of nfat-family proteins in normal human t cells an alternative promoter in the mouse major histocompatibility complex class ii i-ab gene: implications for the origin of cpg islands regulation of rat ornithine decarboxylase mrna translation by its 5 0 -untranslated region translational activation of the lck proto-oncogene an alternative mode of translation permits production of a variant nbs1 protein from the common nijmegen breakage syndrome allele lost in translation molecular biology of luteoviruses posttranscriptional control of gene expression in yeast. microbiol isolation, characterization, and transcription of the gene encoding mouse mast cell protease 7 translational control of the xenopus laevis connexin-41 5 0 -untranslated region by three upstream open reading frames human mxb protein, an interferon-a-inducible gtpase, contains a nuclear targeting signal and is localized in the heterochromatin region beneath the nuclear envelope subgenomic rnas mediate expression of cistrons located internally on the genomic rna of tobacco necrosis virus strain a the three dominant female-sterile mutations of the drosophila ovo gene are point mutations that create new translation-initiator aug codons posttranscriptional control via iron-responsive elements: the impact of aberrations in hereditary disease synthesis of subgenomic rnas by positivestrand rna viruses synthesis of brome mosaic virus subgenomic rna in vitro by internal initiation on (2)-sense genomic rna transforming growth factor-b 1 -mediated inhibition of the flk-1/kdr gene is mediated by a 5 0 -untranslated region palindromic gata site characterization of two novel nuclear btb/poz domain zinc finger isoforms. association with differentiation of hippocampal neurons, cerebellar granule cells, and macroglia regulation of expression of the alternative mrnas of the rat a-thyroid hormone receptor gene differential translational initiation of lbp mrna is caused by a 5 0 upstream open reading frame identification of murine p120 cas isoforms and heterogeneous expression of p120 cas isoforms in human tumor cell lines inducibility and negative autoregulation of crem: an alternative promoter directs the expression of icer, an early response repressor a-thalassaemia associated with the deletion of two nucleotides at position 22 and 23 preceding the aug codon upstream open reading frames as regulators of mrna translation the 5 0 utr of protein kinase c 1 confers translational regulation in vitro and in vivo complex organisation of the 5 0 -end of the human glycine trna synthetase gene mapping of the polycistronic rnas of tomato leaf curl geminivirus site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity specific cleavage of hepatitis c virus rna genome by human rnase p expression of kaposi's sarcoma-associated herpesvirus g protein-coupled receptor monocistronic and bicistronic transcripts in primary effusion lymphocytes rar-b4, a retinoic acid receptor isoform is generated from rar-b2 by alternative splicing and usage of a cug initiator codon the hts1 gene encodes both the cytoplasmic and mitochondrial histidine trna synthetases of s. cerevisiae translation of a nonpolyadenylated viral rna is enhanced by binding of viral coat protein or polyadenylation of the rna nucleotide sequence and expression of the capsid protein gene of feline calicivirus translational discrimination of mrnas coding for human insulin-like growth factor ii growthdependent translation of igf-ii mrna by a rapamycin-sensitive pathway full-sized ranbpm cdna encodes a protein possessing a long stretch of proline and glutamine within the nterminal region, comprising a large protein complex human o-glcnac transferase (ogt): genomic structure, analysis of splice variants, fine mapping in xq13 tissue-specific initiation of murine complement factor b mrna transcription downstream ribosomal entry for translation of coronavirus tgev gene 3b generation from a single gene of two mrnas that encode the mitochondrial and peroxisomal serine: pyruvate aminotransferase of rat liver abcd1 translation-initiator mutation demonstrates genotype-phenotype correlation for amn ccaat/enhancer-binding protein mrna is translated into multiple proteins with different transcription activation potentials dominant-negative mutations of cebpa, encoding ccaat/enhancer binding protein-a (c/ebpa), in acute myeloid leukemia mammalian cells express two differently localized bag-1 isoforms generated by alternative translation initiation spliced and prematurely polyadenylated jaagsiekte sheep retrovirus-specific rnas from infected or transfected cells ribosomal pausing and scanning arrest as mechanisms of translational regulation from cap-distal iron-responsive elements hcc-2, a human chemokine: gene structure, expression pattern, and biological activity a plant viral "reinitiation" factor interacts with the host translational machinery genetic manipulation of arterivirus alternative mrna leader-body junction sites reveals tight regulation of structural protein expression the size of rous sarcoma virus mrnas active in cell-free translation starved saccharomyces cerevisiae cells have the capacity to support internal initiation of translation effect of upstream reading frames on translation efficiency in simian virus 40 recombinants promoter choice influences alternative splicing and determines the balance of isoforms expressed from the mouse bcl-x gene functional organization of the human uncoupling protein-2 gene, and juxtaposition to the uncoupling protein-3 gene uncoupling protein 2, in vivo distribution, induction upon oxidative stress, and evidence for translational regulation the exon structure of the mouse a2(ix) collagen gene shows unexpected divergence from the chick gene identification of the subgenomic mrnas that encode 6-kda movement protein and hsp70 homolog of beet yellows virus a reassessment of the translation initiation codon in vertebrates characterization of human mucin gene muc4 promoter p76 mdm2 inhibits the ability of p90 mdm2 to destabilize p53 analysis of oligonucleotide aug start codon context in eukariotic mrnas structural and functional features of eukaryotic mrna untranslated regions molecular mechanisms of translation initiation in eukaryotes human myeloid zinc finger gene mzf produces multiple transcripts and encodes a scan box protein systematic movement of an rna plant virus determined by a point substitution in a 5 0 leader sequence coupled transcriptional and translational control of cyclin-dependent kinase inhibitor p18 ink4c expression during myogenesis hepatocyte-nuclear factor 3b gene transcripts generate protein isoforms with different transactivation properties on the glucagon gene internal initiation of translation of five dendritically localized neuronal mrnas the amphiregulin gene encodes a novel epidermal growth factor-related protein with tumor-inhibitory activity characterization of hypersensitive sites, protein-binding motifs, and regulatory elements in both promoters of the mouse porphobilinogen deaminase gene identification of a sequence in the unique 5 0 open reading frame of the gene encoding glycosylated gag which influences the incubation period of neurodegenerative disease induced by a murine retrovirus the glycosylated gag protein of mulv is a determinant of neuroinvasiveness: analysis of second site revertants of a mutant mulv virus lacking expression of this protein an alternative open reading frame of the human macrophage colony-stimulating factor gene is independently translated and codes for an antigenic peptide of 14 amino acids recognized by tumorinfiltrating cd8 t lymphocytes introducing refseq and locuslink: curated human genome resources at the ncbi rat phospholipid-hydroperoxide glutathione peroxidase. cdna cloning and identification of multiple transcription and translation start sites mechanisms of loss of foreign gene expression in recombinant vesicular stomatitis viruses in vitro translation of the upstream open reading frame in the mammalian mrna encoding s-adenosylmethionine decarboxylase the 5 0 untranslated sequence of the c-sis platelet-derived growth factor 2 transcript is a potent translational inhibitor a single gene encodes two isoforms of the p70 s6 kinase: activation upon mitogenic stimulation posttranscriptional mrna processing as a mechanism for regulation of human a 1 adenosine receptor expression expression of the smoothelin gene is mediated by alternative promoters mouse mammary tumor virus superantigen expression in b cells is regulated by a central enhancer within the pol gene non-aug initiation of agamous mrna translation in arabidopsis thaliana efficient simultaneous presentation of ny-eso-1/lage-1 primary and nonprimary open reading frame-derived ctl epitopes in melanoma presence of atg triplets in 5 0 untranslated regions of eukaryotic cdnas correlates with a 'weak' context of the start codon a non-aug-defined alternative open reading frame of the intestinal carboxyl esterase mrna generates an epitope recognized by renal cell carcinoma-reactive tumor-infiltrating lymphocytes in situ identification of bing-4 cancer antigen translated from an alternative open reading frame of a gene in the extended mhc class ii region using lymphocytes from a patient with a durable complete regression following immunotherapy cell-specific translational regulation of s-adenosylmethionine decarboxylase mrna. influence of the structure of the 5 0 transcript leader on regulation by the upstream open reading frame block of hac1 mrna translation by long-range base pairing is released by cytoplasmic splicing upon induction of the unfolded protein response regulation of alternative splicing by rna editing continuous and discontinuous ribosome scanning on the cauliflower mosaic virus 35s rna leader is controlled by short open reading frames a complex translational program generates multiple novel proteins from the latently expressed kaposin (k12) locus of kaposi's sarcoma-associated herpesvirus correlation between sequence conservation of the 5 0 untranslated region and codon usage bias in mus musculus genes cot kinase proto-oncogene expression in t cells n-terminal rag1 frameshift mutations in omenn's syndrome: internal methionine usage leads to partial v(d)j recombination activity and reveals a fundamental role in vivo for the n-terminal domains complete genomic sequence of the human abca1 gene: analysis of the human and mouse atp-binding cassette a promoter the pim-1 oncogene encodes two related protein-serine/threonine kinases by alternative initiation at aug and cug negative and translation termination-dependent positive control of fli-1 protein synthesis by conserved overlapping 5 0 upstream open reading frames in fli-1 mrna normal developing rat brain expresses a platelet-derived growth factor b chain (c-sis ) mrna truncated at the 5 0 end multiple murine double minute gene 2 (mdm2) proteins are induced by ultraviolet light transcriptional control of hepadnavirus gene expression internal translation initiation generates novel wt1 protein isoforms with distinct biological properties caveolin isoforms differ in their n-terminal protein sequence and subcellular distribution identification and functional analysis of the turnip yellow mosaic tymovirus subgenomic promoter a second major native von hippel-lindau gene product, initiated from an internal translation start site, functions as a tumor suppressor an amino-terminal domain of mxi1 mediates anti-myc oncogenic activity and interacts with a homolog of the yeast transcriptional repressor sin3 mechanism of translation of monocistronic and multicistronic human immunodeficiency virus type 1 mrnas mechanisms of synthesis of virion proteins from the functionally bigenic late mrnas of simian virus 40 translation initiation at a downstream aug occurs with increased efficiency when the upstream aug is located very close to the 5 0 cap cloning, expression, and nucleotide sequence of rat liver sterol carrier protein 2 cdnas bovine coronavirus i protein synthesis follows ribosomal scanning on the bicistronic n mrna ubiquitinactivating enzyme (e1) isoforms in lens epithelial cells: origin of translation, e2 specificity and cellular localization determined with novel site-specific antibodies cloning and characterization of liverspecific isoform of chk1 gene from rat producing nature's gene-chips: the generation of peptides for display by mhc class i molecules preferential ribosomal scanning is involved in the differential synthesis of the hepatitis b viral surface antigens from subgenomic transcripts translation of the rnas of brome mosaic virus: the monocistronic nature of rna1 and rna2 analysis of capsid formation of human polyomavirus jc (tokyo-1 strain) by a eukaryotic expression system: splicing of late rnas, translation and nuclear transport of major capsid protein vp1, and capsid assembly sequential partially overlapping gene arrangement in the tricistronic s1 genome segments of avian reovirus and nelson bay reovirus: implications for translation initiation translational regulation of the jund messenger rna characterization of the murine hyaluronidase gene region reveals complex organization and cotranscription of hyal1 with downstream genes, fus2 and hyal3 polyoma virus has three late mrnas: one for each virion protein a somatic mutation in the 5 0 utr of brca1 gene in sporadic breast cancer causes down-modulation of translation efficiency the 105-kda polyprotein of southern bean mosaic virus is translated by scanning ribosomes the two subunits of human molybdopterin synthase: evidence for a bicistronic messenger rna with overlapping reading frames poliovirus neurovirulence correlates with the presence of a cryptic aug upstream of the initiator codon mrna leader length and initiation codon context determine alternative aug selection for the yeast gene mod5 chop-dependent stress-inducible expression of a novel form of carbonic anhydrase vi the promoters for human and monkey poliovirus receptors characterization of two bifunctional arabidopsis thaliana genes coding for mitochondrial and cytosolic forms of valyl-trna synthetase and threonyl-trna synthetase by alternative use of two in-frame augs a small highly basic protein is encoded in overlapping frame within the p gene of vesicular stomatitis virus identification of downstream-initiated c-myc proteins which are dominant-negative inhibitors of transactivation by full-length c-myc proteins leaky scanning is the predominant mechanism for translation of human papillomavirus type 16 e7 oncoprotein from e6/ e7 bicistronic mrna human molybdopterin synthase gene: identification of a bicistronic transcript with overlapping reading frames elements in the murine c-mos messenger rna 5 0 -untranslated region repress translation of downstream coding sequences regulatory role of the conserved stem-loop structure at the 5 0 end of collagen a1(i) mrna the alphaviruses: gene expression, replication, and evolution characterization of human mapre genes and their proteins calspermin gene transcription is regulated by two cyclic amp response elements contained in an alternative promoter in the calmodulin kinase iv gene structure of the 5 0 flanking region of the gene encoding human parathyroid-hormonerelated protein (pthrp) statistical analysis of the 5 0 untranslated region of human mrna using "oligo-capped" cdna libraries evidence for the existence of a coat protein messenger rna associated with the top component of each of three tymoviruses truncated forms of the dual function human asct2 neutral amino acid transporter/retroviral receptor are translationally initiated at multiple alternative cug and gug codons the 5 0 -untranslated region of the mouse glial cell line-derived neurotrophic factor gene regulates expression at both the transcriptional and translational levels testis-specific transcription initiation sites of rat farnesyl pyrophosphate synthetase mrna the baculovirus transcriptional transactivator ieo produces multiple products by internal initiation of translation generation of stable mrna fragments and translation of ntruncated proteins induced by antisense oligonucleotides transcriptional regulation of the interferon-g-inducible tryptophanyl-trna synthetase includes alternative splicing evidence for translational regulation of the imprinted snurf-snrpn locus in mice cancerassociated alternative usage of multiple promoters of human galcer sulfotransferase gene the eyeless mouse mutation (ey1 ) removes an alternative start codon from the rx/rax homeobox gene expression patterns of the multiple transcripts from the folylpolyglutamate synthetase gene in human leukemias and normal differentiated tissues ataxia caused by mutations in the atocopherol transfer protein gene mutations in each of the five subunits of translation initiation factor eif2b can cause leukoencephalopathy with vanishing white matter structural and functional genomics of the cpt1b gene for muscle-type carnitine palmitoyltransferase i in mammals ribosomal scanning on the highly structured insulin-like growth factor ii-leader 1 opposing roles of elk-1 and its brain-specific isoform, short elk-1, in nerve growth factor-induced pc12 differentiation identification of rauscher murine leukemia virus-specific mrnas for the synthesis of gag-and env-gene products in vivo translation of the triple gene block of potato virus x requires two subgenomic mrnas the yeast transcription factor genes yap1 and yap2 are subject to differential control at the levels of both translation and mrna stability rejection antigen peptides on balb/c rlf1 leukemia recognized by cytotoxic t lymphocytes: derivation from the normally untranslated 5 0 region of the c-akt proto-oncogene activated by long terminal repeat genomic architecture and transcriptional activation of the mouse and human tumor susceptibility gene tsg101: common types of shorter transcripts are true alternative splice variants a human aminoacyl-trna synthetase as a regulator of angiogenesis analysis of the two subgenomic rna promoters for turnip crinkle virus in vivo and in vitro inefficient reinitiation is responsible for upstream open reading frame-mediated translational repression of the maize r gene role of mrna secondary structure in translational repression of the maize transcriptional activator lc utilization of an alternative open reading frame of a normal gene in generating a novel human cancer antigen post-transcriptional regulation of the gli1 oncogene by the expression of alternative 5 0 untranslated regions a novel, testis-specific mrna transcript encoding an nh 2 -terminal truncated nitric-oxide synthase rna diversity has profound effects on the translation of neuronal nitric oxide synthase bunyamwera bunyavirus nonstructural protein nss counteracts the induction of alpha/beta interferon genomic organization of human mxi1, a putative tumor suppressor gene acquired mutations in gata1 in the megakaryoblastic leukemia of down syndrome infectious tymv rna from cloned cdna: effects in vitro and in vivo of point substitutions in the initiation codons of two extensively overlapping orfs expression of the hepatitis b virus core gene in vitro and in vivo cytomegalovirus assembly protein nested gene family: four 3 0 -coterminal transcripts encode four in-frame overlapping proteins an internal open reading frame triggers nonsense-mediated decay of the yeast spt10 mrna deregulation of translational control of the 65-kda regulatory subunit (pr65a) of protein phosphatase 2a leads to multinucleated cells the short 5 0 untranslated region of the betaa3/a1-crystallin mrna is responsible for leaky ribosomal scanning the human achaete scute homolog 2 gene contains two promoters, generating overlapping transcripts and encoding two proteins with different nuclear localization an activating splice donor mutation in the thrombopoietin gene causes hereditary thrombocythaemia cloning and characterization of two novel thyroid hormone receptor b isoforms a 6700 mw membrane protein is encoded by region e3 of adenovirus type 2 the glyoxysomal and plastid molecular chaperones (70-kda heat shock protein) of watermelon cotyledons are encoded by a single gene analysis and mapping of a family of 3 0 -coterminal transcripts containing coding sequences of human cytomegalovirus open reading frames ul93 through ul99 evidence that aguauauga and ccaagauga initiate translation in the same mrna in region e3 of adenovirus e3 transcription unit of adenovirus interplay of heterogeneous transcriptional start sites and translational selection of augs dictate the production of mitochondrial and cytosolic/nuclear trna nucleotidyltransferase form the same gene in yeast mechanisms leading to and the consequences of altering the normal distribution of atp(ctp):trna nucleotidyltransferase in yeast the 5 0 -untranslated region of the n-methyl-d-aspartate receptor nr2a subunit controls efficiency of translation characterization of an upstream open reading frame in the 5 0 untranslated region of pr-39, a cathelicidin antimicrobial peptide identification and characterization of a new mammalian mitogen-activated protein kinase kinase, mkk2 alternative transcriptional initiation as a novel mechanism for regulating expression of a baculovirus trans activator expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mrna splicing inhibition of corticotropin releasing hormone type-1 receptor translation by an upstream aug triplet in the 5 0 untranslated region ccaat/ enhancer binding protein 1 is preferentially up-regulated during granulocyte differentiation and its functional versatility is determined by alternative use of promoters and differential splicing identification of the start sites for the 1.9-and 1.4-kb rat transforming growth factor-b1 transcripts and their effect on translational efficiency dna methylation represses the expression of the human erythropoietin gene by two different mechanisms an ercc1 splicing variant involving the 5 0 -utr of the mrna may have a transcriptional modulatory function molecular identification and characterization of a and b forms of the glucocorticoid receptor evidence that a regulatory gene autoregulates splicing of its transcript identification of a new form of aqp4 mrna that is developmentally expressed in mouse brain identification of a noncanonical signal for transcription of a novel subgenomic mrna of mouse hepatitis virus: implication for the mechanism of coronavirus rna transcription novel short transcripts of hepatitis b virus x gene derived from intragenic promoter splicing in adenovirus and other animal viruses tissue specific expression of the retinoic acid receptor-b2: regulation by short open reading frames in the 5 0 -noncoding region analysis of the cc chemokine receptor 3 gene reveals a complex 5 0 exon organization, a functional role for untranslated exon 1, and a broadly active promoter with eosinophil-selective elements complete testicular feminization caused by an amino-terminal truncation of the androgen receptor with downstream initiation c/t polymorphism in the 5 0 untranslated region of the apolipoprotein(a) gene introduces an upstream aug and reduces in vitro translation research in my laboratory is supported by grant gm33915 from the national institutes of health. key: cord-282859-uxltqopq authors: rosati, a; graziano, v; de laurenzi, v; pascale, m; turco, m c title: bag3: a multifaceted protein that regulates major cell pathways date: 2011-04-07 journal: cell death dis doi: 10.1038/cddis.2011.24 sha: doc_id: 282859 cord_uid: uxltqopq bcl2-associated athanogene 3 (bag3) protein is a member of bag family of co-chaperones that interacts with the atpase domain of the heat shock protein (hsp) 70 through bag domain (110–124 amino acids). bag3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. in addition to the bag domain, bag3 contains also a ww domain and a proline-rich (pxxp) repeat, that mediate binding to partners different from hsp70. these multifaceted interactions underlie bag3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. in normal cells, bag3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. a growing body of evidence indicate that bag3 is instead expressed in several tumor types. in different tumor contexts, bag3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. in some tumor types, down-modulation of bag3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. this review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression. bcl2-associated athanogene (bag) proteins are a family of co-chaperones that interact with the atpase domain of the heat shock protein (hsp) 70 through a specific structural domain known as bag domain (110-124 amino acids). 1, 2 members of the family are found throughout the evolution in yeast (saccharomyces cerevisiae, schizosaccharomyces pombe), invertebrates (caenorhabditis elegans, ciona intestinalis, drosophila), amphibians (xenopus laevis), mammals (humans, mice) and plants (oryza sativa, arabidopsis thaliana), 1,3-6 suggesting a fundamental biological role. bag3 was originally identified by yeast two-hybrid screening using the atpase domain of the hsp (heat shock cognate (hsc)/hsp) 70 as a bait. 1 in addition to the bag domain, bag3 contains a ww domain and a proline-rich repeat (pxxp; figure 1a ) that mediate binding to other partners. 1, 7, 8 here we will outline the roles so far identified of bag3 in the regulation of major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy. by modulating these pathways, bag3 appears to mediate cell adaptive responses to stressful stimuli, and its alterations result in altered homeostasis and reduced cytoprotection, explaining why its expression is often found deregulated in a vast series of tumors. two bag3 forms have been described so far: one is the full-lenght product of the bag3 gene having an apparent mass of 74 kda, the other one is a shorter bag3 protein found in association to neuronal synaptosomes 2, [9] [10] [11] (figure 1b) . the bag3 full-length protein is localized in the cytoplasm, mainly concentrated in the rough endoplasmic reticulum; on cell exposure to stressors, a slightly different molecular weight variant of this form can also be observed, and both co-exist in some cell types and run as a doublet in a standard western blot. the origin of this doublet is currently unknown, but it could derive from post-translational modifications as phosphorylations, indeed bag3 protein contains several serinerich motifs and 10 tyrosine residues. tyrosine phosphorylation of bag3 occurs on egf stimulation in human breast cancer cell lines. 12 this post-translational variant of bag3 protein appears also to be associated to bruton's tyrosine kinase (btk) protein in defew cells on oxidative stress induction. 13 recently, a shorter form of bag3 (40 kda) has been characterized by immunoprecipitation from neural synaptosomes homogenates and successive mass spectrometry, 11 again it is yet unknown whether this form derives from the alternative splicing or proteolytic processing. bag3 expression has also been observed in the nuclei of some cell types, including pancreatic carcinoma cells (unpublished results from our laboratory). interestingly, bag3 was shown to localize in nuclei of cells infected by some viruses; those findings suggest an involvement of bag3 in viral expression and/or replication, and indeed some reports indicate a requirement for bag3 for virus gene expression and/or viral progeny production. 12, [14] [15] [16] [17] [18] in keeping with those results, viral infection was shown to induce the expression of bag3, as a possible tool contributing to virus life cycle. 12, 19 in humans, bag3 gene expression is constitutive in myocytes, a few other normal cell types and several primary tumors or tumor cell lines (lymphoid or myeloid leukemias, lymphomas, myeloma, neuroblastoma, pancreas, thyroid, breast and prostate carcinomas, melanoma, osteosarcoma, kidney, colon and ovary cancers, glioblastoma). [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] it is instead induced in different normal cell types (leukocytes, epithelial and glial cells, retinal cells) by a variety of stressors, such as oxidants, high temperature, serum deprivation, heavy metals, hiv-1 infection, elf exposure, electrophile stress, hemodialysis treatment, pulsed ultrasound, retinal light damage, kanaic acid-induced seizure and transient forebrain ischemia. 2, 10, 14, 19, 35, [37] [38] [39] [40] [41] [42] [43] [44] the expression of stress-responsive genes is regulated by the heat shock transcription factors (hsfs), 45 including hsf1, that is required for tumor initiation and maintenance in a variety of cancer models. 46 bag3 gene promoter activity is regulated by hsf1, 31, 47, 48 again suggesting a role in tumor formation. other known transcription factors that are known to regulate bag3 expression are egr1 29 aibzip 26 and wt1. 49 the regulation by wt1 is of particular interest because of the role played by this transcription factor in human leukemias 50 (see the section bag3 in leukemias). furthermore, in some cell types bag3 expression seems to be controlled by its own product. 51 a number of drugs -proteasome inhibitors, tnf-related apoptosis-inducing ligand, fludarabine, cytosine arabinoside and etoposide -increase bag3 protein levels, this in turn contributes to cell resistance to therapy, and indeed bag3 silencing improves neoplastic cell apoptotic response to drugs. 21, 22, 24, 30, 37, 52, 53 in some cell types, bag3 expression appears to be developmentally regulated. in the developing central nervous system (cns) of a rat, there is a transient expression, detectable by immunohistochemistry, of bag3 in the cerebral cortex and hippocampus, whereas a considerable expression is maintained in the rostral migratory stream and the subventricular zone of the lateral ventricle; there is an abrupt increase of bag3-positive neurons in the cortex and hippocampus during the first postnatal week, which declines thereafter. two specific populations of bag3-positive neurons can be identified in the developing forebrain of a rat. 54 furthermore, recent results indicated that bag3 is expressed in neural progenitors and sustains proliferation, mainly in response to fgf2, in those cells. 55 in addition, an early transient expression of bag3 was observed in midline radial glia in the developing brainstem and spinal cord of a rat. 56 together, these pieces of evidence indicate a role for bag3 in the development of both the neuronal and glial components of central nervous system. in agreement with the hypothesis of bag3 involvement in cns development, an altered bag3 expression was observed in the cerebellum of hypothyroid juvenile mice, and was suggested to contribute to impaired development of the hypothyroid brain. moreover, in rat and human cardiomyocytes, bag3 protein appears to be expressed during differentiation from cardiomyoblasts and to sustain myogenin expression. 57 these findings indicate an involvement of bag3 protein in late heart development and are in keeping with the role of bag3 in the survival and myofibrillar integrity in cardiocytes and, in general, in muscle cells. indeed, mice with homozygous disruption of bag3 gene develop post birth an early fulminant myopathy 23 and, in humans, a heterozygous p.pro209leu mutation in bag3 protein was recently recognized to be responsible for a severe muscular dystrophy with cardiomyopathy and severe respiratory insufficiency. [57] [58] [59] in one of the three studied families carrying this mutation, an axonal neuropathy was also present. this observation is intriguing in view of the reported localization of a 40 kd form of bag3 in synaptosomes 11 and of the above discussed role of bag3 in cns. finally, a recent paper reports a role of bag3 in the development of the hematopoietic system, showing that mice with a targeted disruption of bag3 exhibit a loss of hematopoietic stem cells and defective b-cell development, because of the microenvironmental defect, that is, an alteration in the vascular stem cell niche. 60 the authors observed a defective growth of stromal progenitor cells in colony-forming unit fibroblasts, a defect in sinusoidal endothelium, and the loss of stromal cells expressing cxcl-12 or il-7 in the bone marrow. 60 the molecular mechanisms underlying those perturbations could, once identified, disclose novel prospects in the understanding of the hematopoietic process. last but not least, recent evidence indicate that bag3 is also implicated in regulating the general metabolic state of the organism, as bag3-deficient mice were reported to show significant hypoglycemia, a decrease in triglyceride and cholesterol levels and growth retardation, and died by 3 weeks after birth. 61 altogether these data show a fundamental role of bag3 in regulating essential physiological events. a number of studies in tumor cell lines of different origin have shown that bag3 silencing or hyperexpression results in, respectively, enhancing or inhibiting spontaneous or drug-induced apoptosis. 9, 24, 25, 30, 31, 34, 36, 37, 44, 49 furthermore, caspases trigger bag3 cleavage, thereby facilitating the apoptotic process. 52, 62 bag3 seems to influence cell survival by interacting with different molecular partner, thus activating multiple pathways. a first demonstrated mechanism of bag3 anti-apoptotic activity is mediated by its role, as a co-chaperone, in protein delivery to the proteasome. indeed, although another member of bag family, that is, bag1, positively cooperates with hsp70 and chip (c-terminus of the hsc70-interacting protein) to direct, through its ubiquitin-like domain (figure 1 ), client proteins to proteasome, 63 bag3 can interfere with this process by competing with bag1. 2, 4, 29, 52 indeed, in osteosarcoma and melanoma cells, bag3 protects ikk-g from proteasome delivery and this results in sustained nf-kb activation and cell survival. 37 a different mechanism has been observed in glioblastoma cells, in which bag3 retains bax protein in the cytosol, preventing its mitochondrial translocation. 36 both mechanisms rely on an interaction between bag3 and hsp70. 36, 37 we can speculate that through its binding to hsp70, bag3 might also positively or negatively modulate folding of other apoptosis-regulating proteins, and expect that future research will disclose a very complex regulative mechanism mediated by this protein. more over, as hsp70 can bind to au-rich elements in the 3 0 -untranslated regions, regulating expression of a number of proteins, including the well-known pro-apoptotic, bh3 only protein bim, 64 bag3 might be expected to regulate hsp70 ability to stabilize bim mrna and possibly other mrnas, involved in various cell functions. finally, we can envisage functions of bag3 that are independent of hsp70, as it could also bind some client proteins through its ww or pxxp domain, directly influencing their stability, localization or activity (figure 2 ). it is likely that the availability of the different partners underlies the different mechanisms through which bag3 exerts its anti-apoptotic activity in different cell types. it has been shown that bag3 silencing reduces adhesion and/or motility of epithelial (breast, prostate) tumor cells. 27, 32, 65, 66 in mda435 human breast cancer cells, bag3 overexpression resulted in a decrease in migration and adhesion to matrix molecules; the decrease was reversed on deletion of the bag3 proline-rich (pxxp) domain, indicating that an interaction of bag3 with a sh 3 domain-containing protein was involved. 65 expression array studies showed that indeed bag3 regulated, in a pxxp-dependent manner, the expression of ccn (cyr61, connective tissue growth, nov) 1, a matricellular signaling protein that promotes cell adhesion through integrins and heparan sulfate-containing proteoglycans. 32 in addition, bag3 seems to regulate cell adhesion through binding to guanine nucleotide exchange factor 2 (pdzgef2). this protein induces the activation of rap1, a regulator of cell-cell junction formation and remodeling, and increases integrin-mediated cell adhesion. the ppdy motif at the c-terminus of pdzgef2 was shown to bind to the ww domain of bag3, and pdzgef2 knockdown reduced bag3 ability to induce cell adhesion in cos7 cells. 67 similarly to what we described above for apoptosis, the ability of bag3 to regulate cell adhesion appears to rely on multiple interactions of this protein through different structural domains. it is important to note that modulation of cell adhesion has profound effects on cell vitality, as cell detachment from matrix induces the apoptotic process known as anoikis. 68 therefore, bag3 alterations could underlie effects on both cell detachment/motility and resistance to associated anoikis, thereby favouring metastatization. finally, we recently reported that bag3, through its interaction with the cytosolic chaperonin cct (chaperonin containing tcp-1), regulates actin folding. 66 this property of bag3 highlights its involvement in cytoskeleton organization, possibly influencing not only cell survival and migration, but also membrane trafficking and organellar dynamics. the role played by bag3 in the cytoskeleton remodeling and membrane trafficking suggests the possibility that it might be involved in autophagy. with this term, we refer to a set of nonspecific bulk degradation processes, in which cells deliver cytoplasmic substrates for lysosomal degradation. 69 there is a chaperone-mediated autophagy (cma), selective for cytosolic proteins that contain a pentapeptide motif: this motif is recognized by the chaperone hsc70, which transfers protein substrates to lysosomes. 69 as bag3 is a hsc/hsp 70 co-chaperone, it is plausible to imagine its involvement in cma, but we can also envisage a role in the other two types of autophagy, namely micro-and macroautophagy. in macroautophagy, cells form double-membrane vesicles, called autophagosomes, around a portion of cytoplasm; autophagosomes are trafficked along microtubules and fused with lysosomes, resulting in degradation of their contents. microautophagy is a process in which lysosomes directly engulf cytoplasm. 69 similar to hsps and bag3, autophagy is upregulated under stress. indeed, the autophagic process is important in promoting cell survival in several conditions, such as protein aggregate formation, nutrient and growth factor deprivation, er stress and pathogen infection. defective autophagy is associated with diverse diseases, including neurodegeneration, lysosomal storage diseases, muscular dystrophies, cancers and crohn's disease. 69 bag3 participates, along with hspb8, a member of the hspb family of molecular chaperones, in the degradation of misfolded and aggregated proteins via macroautophagy. indeed hspb8 forms a stable complex with bag3 in cells and the formation of this complex is essential for the hspb8mediated degradation of the polyglutamine protein htt43q (huntingtin exon 1 fragment with 43 cag repeats), a pathogenic form of huntingtin prone to aggregation. 70, 71 hspb8 and bag3 induce, in a hsp70-independent manner, the phosphorylation of the a-subunit of the translation initiator factor eif2; this in turn causes a translational shutdown and stimulates autophagy. the mechanism by which the bag3/ hspb8 complex induces eif2 phosphorylation is not completely understood. 70, 71 bag3 binding to hspb8 is mediated by two conserved ile-pro-val (ipv) motifs located between the w-and the pro-rich domains of the co-chaperone; deletion of these motifs suppresses hspb8 activity in htt43q degradation. 72 interestingly, the p.pro209leu mutation responsible for dystrophy is in one of the two ipv motifs. 58, 72 this suggests that the disease pathogenesis could involve defective autophagy. through the same protein region, bag3 can bind also to another chaperone hspb6. this is particularly intriguing in view of the cardioprotective property of hspb6/ hsp20 73 and its role in myocyte contractility. 74 finally, bag3 complexes with the ubiquitin-binding protein p62/sqstm1, that, by binding simultaneously the autophagosome protein lc3 and polyub-proteins, controls the sequestration of polyub-proteins into inclusion bodies for autophagic degradation. 75 therefore, bag3 appears to negatively regulate protein delivery to proteasome, by competing with bag1, but at the same time it stimulates autophagy. this suggests that, although bag1 constitutively assures, in combination with hsc/hsp70, proteostasis through the ubiquitin-proteasome system (ups), stressful stimuli, by inducing bag3 increase, determine an inhibition of ups (because of the bag3 competition with bag1) and a stimulation of autophagy. in agreement with this hypothesis, bag3/bag1 protein ratio increases, concomitantly with an increase in autophagy, in an in vitro model of cell aging (replicative senescence: i90 cells) and in rodent brain neurons during aging. 75 the authors attribute bag3 increase to oxidative stress due to the enhanced pro-oxidant milieu characteristic of aging. as this milieu enhances the occurrence of protein aggregation, bag3-stimulated autophagy is interpreted as an adaptive response of the protein quality control system. 75 bag3 is expressed at low levels in normal blood cells, whereas it is highly expressed both in primary leukemic blasts or established cells lines from leukemic patients. 21, 22 furthermore, its levels are markedly higher in drug-resistant patients compared with patients that resulted responsive to chemotherapy. one mechanism responsible for bag3 overexpression in leukemic cells relies on the effect of the wt1 transcription factor on the bag3 gene promoter. 49 wt1 is overexpressed in acute lymphoblastic and myeloblastic leukemia, and high levels of the protein are associated with a poor response to therapy. 50 we recently showed that the wt1 isoform þ kts was able to positively regulates bag3 expression by a transcriptional mechanism, via a direct binding on two wt1 consensus sites on the bag3 gene promoter. wt1 knockdown induces apoptosis, whereas its overexpression protects leukemic cells from apoptosis inducers; these effect are in part because of the modulation of bag3 protein levels. 49 the role of bag3 modulation is even more evident in primary human leukemia cells. indeed in leukemic cells from 24 patients affected by b-cell chronic lymphocytic leukemia (b-cll) 21 and from 11 children affected by acute lymphoblastic leukemia, 22 we observed that bag3 down-modulation by specific antisense oligodeoxynucleotides results in enhancing the percentage of apoptotic elements by 4100%. cell apoptosis was enhanced in cells either untreated or incubated with chemotherapeutic drugs. those were the first reported evidence of the apoptosisregulating activity of bag3 protein in primary tumors. 21, 22 we also showed that bag3 down-modulation increased apoptosis of primary normal human peripheral blood mononuclear cells as well as of leukemic cells incubated with agents able to induced oxidative stress. 39 furthermore, a physical interaction was observed between bag3 and the btk under oxidative stress conditions. all those evidences assign to bag3 a critical role in the leukemic cell survival and responsiveness to chemotherapy and should prompt investigators to further investigate its role in these deseases and the possibility of exploiting it as a molecular target. from the first evidence of bag3-mediated survival in b-clls, 21 a number of reports confirmed the anti-apoptotic activity of the protein in tumors of the hematopoietic 2, 13, 21, 22, 25, 28, 30, 39, 40, 49 and other 2, 20, 24, 25, 29, 31, 34, 36, 37, 44 compartments. two principal elements contribute to the role of bag3 in vast range of tumors: bag3 induction by hsf1, 47 a transcription factor activated in many cancer types; 46 and bag3 ability to sustain of nf-kb activity. 76 in addition, bag3, because of its multifaceted ability to complex with many proteins, can regulate factors that are particularly relevant in the context of specific tumor types, such as bax in glioblastoma cells. 36 overall, studies on bag3 expression and activity in normal and neoplastic cells, and of its regulation during the development, increasingly delineate a complex picture of bag3 involvement in physiological and pathological processes. the pleiotropic functions attributed to bag3 probably reflect the adapter nature of this multidomain (bag, ww and pxxp) protein and its ability to assemble scaffolding complexes, which participate in more than one signal transduction pathway. it is of particular interest that bag3 deficiency can result in the loss of hematopoietic stem cells due to a defect in stem cell niche. 61 therefore, bag3 is required for the stem cellsupporting activity of microenvironmental cells. this finding suggests that bag3 may also regulate the cancer stem cell-supporting activity of stromal cells in the hematopoietic and possibly in other compartments. again, nf-kb factors could represent key targets of bag3, not only for the expression of tumor cell genes that sustain cell survival, but also for the expression of stromal cells' soluble and cell surface molecules that mediate bidirectional interactions between tumor cells and their environment. 77, 78 an evolutionarily conserved family of hsp70/hsc70 molecular chaperone regulators apoptosis inhibition in cancer cells: a novel molecular pathway that involves bag3 protein molecular chaperone targeting and regulation by bag family proteins drosophila starvin encodes a tissue-specific bag-domain protein required for larval food uptake identification and functional characterization of the bag protein family in arabidopsis thaliana gene and protein expression of drosophila starvin during cold stress and recovery from chill coma cair-1/bag-3 abrogates heat shock protein-70 chaperone complex-mediated protein degradation: accumulation of poly-ubiquitinated hsp90 client proteins death versus survival: functional interaction between the apoptotic and stress-inducible heat shock protein pathways what's in the 'bag'?-a functional domain analysis of the bag-family proteins regulation by heavy metals and temperature of the human bag-3 gene, a modulator of hsp70 activity identification of a synaptosome-associated form of bag3 protein epstein-barr virus nuclear antigen (ebna) 3a induces the expression of and interacts with a subset of chaperones and co-chaperones identification of a btk-bag3 complex induced by oxidative stress evidence for bag3 modulation of hiv-1 gene transcription the co-chaperone bag3 regulates herpes simplex virus replication bag3, a host cochaperone, facilitates varicella-zoster virus replication quantitative proteomics analysis reveals bag3 as a potential target to suppress severe acute respiratory syndrome coronavirus replication co-chaperone bag3 and adenovirus penton base protein partnership bag3 protein regulates caspase-3 activation in hiv-1-infected human primary microglial cells the anti-apoptotic protein bag-3 is overexpressed in pancreatic cancer and induced by heat stress in pancreatic cancer cell lines bag3 protein controls b-chronic lymphocytic leukaemia cell apoptosis bag3 protein regulates cell survival in childhood acute lymphoblastic leukemia cells bag3 deficiency results in fulminant myopathy and early lethality the antiapoptotic protein bag3 is expressed in thyroid carcinomas and modulates apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand apoptosis inhibition in cancer cells: a novel molecular pathway that involves bag3 protein transcriptional profiling of genes that are regulated by the endoplasmic reticulum-bound transcription factor aibzip/creb3l4 in prostate cells bag3 regulates motility and adhesion of epithelial cancer cells altered gene expression in busulfan-resistant human myeloid leukemia activation of bag3 by egr-1 in response to fgf-2 in neuroblastoma cells bag3 gene silencing sensitizes leukemic cells to bortezomibinduced apoptosis hsf1-mediated bag3 expression attenuates apoptosis in 4-hydroxynonenal-treated colon cancer cells via stabilization of anti-apoptotic bcl-2 proteins genomic and phenotypic analysis reveals a key role for ccn1 (cyr61) in bag3-modulated adhesion and invasion bag3 protein delocalisation in prostate carcinoma inhibition of the jnk signalling pathway enhances proteasome inhibitor-induced apoptosis of kidney cancer cells by suppression of bag3 expression light damage induced changes in mouse retinal gene expression bag3 protein is overexpressed in human glioblastoma and is a potential target for its therapy ikkg protein is a target of bag3 regulatory activity in human tumor growth induction of bis, a bcl-2-binding protein, in reactive astrocytes of the rat hippocampus following kainic acid-induced seizure bag3 protein regulates stress-induced apoptosis in normal and neoplastic leukocytes identification of genes responsive to low intensity pulsed ultrasound in a human leukemia cell line molt-4 acute effects of haemodialysis on pro-/anti-apoptotic genes in peripheral blood leukocytes systems analysis of protein modification and cellular responses induced by electrophile stress exposure to 50 hz electromagnetic field raises the levels of the anti-apoptotic protein bag3 in melanoma cells down-modulation of bis sensitizes cell death in c6 glioma cells induced by oxygen-glucose deprivation chaperone regulation of the heat shock protein response heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis bag3 gene expression is regulated by heat shock factor 1 activation of heat shock factor 1 plays a role in pyrrolidine dithiocarbamate-mediated expression of the co-chaperone bag3 wt1 protein is a transcriptional activator of the antiapoptotic bag3 gene a tumor suppressor and oncogene: the wt1 story autoregulation of co-chaperone bag3 gene transcription caspase-dependent cleavage of bag3 in proteasome inhibitors-induced apoptosis in thyroid cancer cells transcriptional upregulation of bag3 upon proteasome inhibition developmental expression of bis protein in the cerebral cortex and hippocampus of rats bag3 expression is sustained by fgf2 in neural progenitor cells and impacts cell proliferation transient expression of bis protein in midline radial glia in developing rat brainstem and spinal cord bag3 protein is induced during cardiomyoblast differentiation and modulates myogenin expression mutation in bag3 causes severe dominant childhood muscular dystrophy caught in the middle: the role of bag3 in disease myofibrillar myopathies disruption of bis leads to the deterioration of the vascular niche for hematopoietic stem cells characterization of bag3 cleavage during apoptosis of pancreatic cancer cells the ubiquitin-related bag-1 provides a link between the molecular chaperones hsc70/hsp70 and the proteasome cytokines direct the regulation of bim mrna stability by heatshock cognate protein 70 cair-1/bag-3 modulates cell adhesion and migration by downregulating activity of focal adhesion proteins the co-chaperone bag3 interacts with the cytosolic chaperonin cct: new hints for actin folding bag3 directly associates with guanine nucleotide exchange factor of rap1, pdzgef2, and regulates cell adhesion cytoprotective roles for autophagy the stress-inducible hspb8-bag3 complex induces the eif2alpha kinase pathway: implications for protein quality control and viral factory degradation? hspb8 participates in protein quality control by a non-chaperone-like mechanism that requires eif2\{alpha\} phosphorylation identification of the key structural motifs involved in hspb8/hspb6-bag3 interaction hsp20 and its cardioprotection the small heat shock protein, hspb6, in muscle function and disease protein quality control during aging involves recruitment of the macroautophagy pathway by bag3 nf-kappab a good target for cancer therapy? hopes and pitfalls nf-kappab as a critical link between inflammation and cancer tumor-host interactions: a far-reaching relationship the authors declare no conflict of interest. key: cord-291086-goidlh08 authors: walker, peter j.; dietzgen, ralf g.; joubert, d. albert; blasdell, kim r. title: rhabdovirus accessory genes date: 2011-09-14 journal: virus res doi: 10.1016/j.virusres.2011.09.004 sha: doc_id: 291086 cord_uid: goidlh08 the rhabdoviridae is one of the most ecologically diverse families of rna viruses with members infecting a wide range of organisms including placental mammals, marsupials, birds, reptiles, fish, insects and plants. the availability of complete nucleotide sequences for an increasing number of rhabdoviruses has revealed that their ecological diversity is reflected in the diversity and complexity of their genomes. the five canonical rhabdovirus structural protein genes (n, p, m, g and l) that are shared by all rhabdoviruses are overprinted, overlapped and interspersed with a multitude of novel and diverse accessory genes. although not essential for replication in cell culture, several of these genes have been shown to have roles associated with pathogenesis and apoptosis in animals, and cell-to-cell movement in plants. others appear to be secreted or have the characteristics of membrane-anchored glycoproteins or viroporins. however, most encode proteins of unknown function that are unrelated to any other known proteins. understanding the roles of these accessory genes and the strategies by which rhabdoviruses use them to engage, divert and re-direct cellular processes will not only present opportunities to develop new anti-viral therapies but may also reveal aspects of cellar function that have broader significance in biology, agriculture and medicine. the rhabdoviridae is arguably the most diverse family of rna viruses. rhabdoviruses have been isolated from a wide range of organisms including placental mammals, marsupials, birds, reptiles, fish, insects and plants (kuzmin et al., 2009) . their ecology includes marine, freshwater and terrestrial environments. transmission can be via insect vectors in which they replicate, by direct horizontal transfer via plant sap (wounds or grafts), aerosol, animal bite or immersion in infected water, or by vertical transovarial passage. the rhabdoviridae is one of only three virus families that include members that infect plants, vertebrates and invertebrates. replication can occur in the cytoplasm or the nucleus of infected cells. infection can be either acute or persistent and rhabdoviruses can cause infections that may be unapparent or result in disease that can vary from being mild to severe or even fatal. over 200 rhabdoviruses have been identified to date and most of them are poorly described (dietzgen et al., 2012) . for many years, knowledge of rhabdovirus molecular biology has been based primarily on extensive studies of vesicular stomatitis virus (vsv) and rabies virus (rabv). these rhabdoviruses, share very similar and relatively simple genome organisations with only five structural protein genes (n, p, m, g and l) that are transcribed sequentially from the (-) rna genome as monocistronic mrnas. although some variations are now evident, such as the long non-coding region in the rabv g gene (designated pseudogene ) (tordo et al., 1986 ) and the small alternative open reading frames (orfs) in the vsv p gene (c and c ) , these viruses have generally been regarded as the rhabdovirus archetypes and as very suitable models for studying virus-host interactions. however, it is becoming evident that the ecological diversity displayed by rhabdoviruses is also apparent in the complexity of their genome organisation and in the increasing array of additional proteins which they encode. although the functions of many of these proteins are yet unknown, preservation of the five canonical structural protein genes (3 -n-p-m-g-l-5 in negative polarity) by all known rhabdoviruses suggests that the additional proteins may not be essential for virus replication in culture and are likely to have functions associated with enhancing replication efficiency, blocking host innate immune defences, modulating cellular signalling pathways, re-directing normal cellular functions, and allowing effective cell-to-cell transmission. this expectation has been supported by limited studies to date on some of these proteins. by analogy with other rna viruses with relatively complex genome organisations (e.g., coronaviruses, lentiviruses, paramyxoviruses), we have therefore adopted the term 'accessory genes' to describe the orfs encoding these novel and potentially informative proteins. rhabdoviruses are non-segmented (-) ssrna viruses that, together with paramyxoviruses, filoviruses and bornaviruses, are classified in the order mononegavirales. the family rhabdoviridae currently comprises six genera containing 46 species, five species unassigned to a genus and more than 150 viruses that have not been formally classified (dietzgen et al., 2012) . several likely species and two proposed new genera (sigmavirus and tibrovirus) are currently under review for classification, and a number of other viruses have been identified as rhabdoviruses by morphology and/or serology but have not been further characterised. the genus lyssavirus includes rabies virus and the rabies-related viruses mokola virus, lagos bat virus, duvenhage virus, european bat lyssavirus 1, european bat lyssavirus 2 and australian bat lyssavirus, irkut virus, khujand virus, aravan virus and west caucasian bat virus as currently designated species. another unclassified lyssavirus, shimoni bat virus, has also recently been identified (kuzmin et al., 2010) . lyssaviruses naturally infect mammals and almost all have been isolated from bats which appear to be the primary natural reservoir (delmas et al., 2008) . transmission is usually by transfer of saliva in a bite puncture wound. they are neurotropic rhabdoviruses and most have been associated with rabies encephalitis in humans or animals (rupprecht et al., 2002) . the genus vesiculovirus comprises an ecologically diverse but genetically similar group of viruses. viruses causing vesicular stomatitis in cattle, pigs and horses in the americas are referred to collectively as serotypes of vesicular stomatitis virus (vsv) and classified as the species vesicular stomatitis indiana virus, vesicular stomatitis new jersey virus and vesicular stomatitis alagoas virus. other recognised mammalian vesiculovirus species include cocal virus, piry virus, carajas virus and maraba virus that are endemic in the americas, isfahan virus from asia and chandipura virus which occurs in both asia and africa. most of these viruses have been isolated from phlebotomine sand flies (lutzomyia, phlebotomus and sergentomyia spp.) or black flies (simulium spp.) which are considered the primary vectors. however, biting midges (culicoides spp.), mosquitoes and other insects have also been implicated as potential vectors and transmission can also occur by direct contact between infected vertebrates (comer and tesh, 1991; letchworth et al., 1999; rodríguez, 2002; stallknecht et al., 2001) . vesiculoviruses can also infect humans and some have been associated with serious disease (rao et al., 2004) . the genus vesiculovirus also currently contains the recognised species spring viraemia of carp virus and several other important pathogens of fish, including pike fry rhabdovirus, trout rhabdovirus, sea trout rhabdovirus, and siniperca chuatsi rhabdovirus, ulcerative disease rhabdovirus, eel virus american and eel virus european x, also appear to be members of the genus (dietzgen et al., 2012; hoffmann et al., 2005) . transmission of fish vesiculoviruses can occur by immersion in infected water and does not appear to involve arthropod vectors (ahne et al., 2002) . the genus novirhabdovirus comprises a second group of fish rhabdoviruses that appear to have a very different evolutionary history from the fish vesiculoviruses. species include infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus which are important pathogens of salmonids, but can also infect a very wide range of other fish species (walker and winton, 2010) . the species hirame rhabdovirus and snakehead rhabdovirus are pathogens of marine fish in temperate and tropical regions of asia, respectively (johnson et al., 1999; kim et al., 2005) . as for the fish vesiculoviruses, transmission is directly through mucosal surfaces or the skin by immersion in infected water and does not appear to involve an arthropod vector. the genus ephemerovirus comprises arthropod-borne rhabdoviruses that have been isolated from mosquitoes and/or biting midges (culicoides spp.) and infect primarily ruminants (walker, 2005) . the type species bovine ephemeral fever virus and the newly recognised species kotonkan virus each cause a disabling febrile disease in cattle and water buffalo (kemp et al., 1973; st. george and standfast, 1988) . other ephemeroviruses, including recognised species berrimah virus and adelaide river virus, and likely species obodhiang virus, kimberley virus, malakal virus and puchong virus, have been isolated only from insects or from healthy cattle and their association with disease is not yet known (walker et al., in press) . plant-adapted rhabdoviruses are separated taxonomically into two genera, cytorhabdovirus and nucleorhabdovirus, based on their site of virus replication and morphogenesis (dietzgen et al., 2012) . cytorhabdoviruses replicate in the cytoplasm of infected cells and virions bud in association with endoplasmic reticulum membranes. the type species of the genus is lettuce necrotic yellows virus and it includes eight additional recognised species. nucleorhabdoviruses replicate in the nuclei of infected plant cells. morphogenesis occurs at the inner nuclear membrane and virions accumulate in perinuclear spaces. the type species is potato yellow dwarf virus and there are nine additional recognised species in this genus. viruses in both genera infect monocot and dicot plants and are transmitted by leafhoppers, planthoppers or aphids in which they also replicate (jackson et al., 2005) . two other genera are currently under consideration for inclusion in the rhabdoviridae. the proposed genus sigmavirus comprises rhabdoviruses infecting only flies (drosophila spp.) with which they have co-evolved. they are transmitted vertically and confer co 2 sensitivity on the host. there are five proposed species including drosophila melanogaster sigmavirus (commonly called sigma virus of drosophila) which is the most extensively studied of the sigmaviruses (longdon et al., 2010 (longdon et al., , 2011 . like the genus ephemerovirus, the proposed genus tibrovirus comprises antigenically related rhabdoviruses that primarily infect cattle and water buffalo, and are transmitted by culicoides species midges. however, tibroviruses and ephemeroviruses have different evolutionary histories as is reflected in their phylogenetic relationships and genome organisations. tibrogargan virus and coastal plains virus, each isolated in australia, are currently proposed as species in the new genus (gubala et al., 2011) . bivens arm virus, isolated from culicoides insignis in the usa, also infects cattle and water buffalo and may be a third species in the proposed genus (gibbs et al., 1989) . complete genome sequences are also available for several other unclassified animal rhabdoviruses that are considered in this review. short sequences in the l and n gene have been used to determine phylogenetic relationships between viruses in the established and proposed genera and many other unclassified animal rhabdoviruses (bourhy et al., 2005; kuzmin et al., 2006) . one monophyletic cluster identified by this approach includes a geographically and ecologically diverse group of viruses termed the hart park group because of the close serological relationship of one of the members, flanders virus, to hart park virus. both of these viruses have been isolated from mosquitoes and infect birds in north america (boyd, 1972) . the hart park phylogenetic group also includes wongabel virus which was isolated from biting midges (culicoides spp.) in australia and appears to infect birds, and ngaingan virus, also isolated from biting midges in australia but whose vertebrate hosts include marsupials (doherty et al., 1973; gubala et al., 2010) . not included in the hart park group but related to each other phylogenetically (but not serologically) are tupaia rhabdovirus isolated from hepatocarcinoma cells of a tree shrew (tupaia belangeri) imported to the usa from thailand (kurz et al., 1986) , and durham virus, which was isolated from the brain of a moribund american coot (fulica americana) in the usa (allison et al., 2011) . oak vale virus, isolated from mosquitoes in australia and infecting feral pigs, and mandarin fish (siniperca chuatsi) rhabdovirus (scrv) are other currently unclassified rhabdoviruses that are considered in this review (cybinski and muller, 1990; zhang et al., 2007) . rhabdoviruses for which complete genome sequences are available are listed in table 1 . the (-) ssrna rhabdovirus genome ranges in size from approximately 11 kb to almost 16 kb. the five structural protein genes are arranged in the same linear order (3 -n-p-m-g-l-5 ) and may be interspersed with one or more additional genes. at each end of the genome, 3 leader and 5 trailer sequences of approximately 50-100 nt contain the promoter sequences that initiate replication of the genome and anti-genome, respectively. the 3 leader and 5 trailer sequences of plant-adapted rhabdoviruses are considerably longer at 84-206 nt and 145-389 nt, respectively (jackson et al., 2005) . in transcription mode, the rna-dependent rna polymerase moves progressively along the genome to transcribe the short leader rna (l) and then to transcribe, cap and polyadenylate mrnas from each gene in decreasing molar abundances (banerjee and barik, 1992) . critical to this process are transcription initiation (ti) and transcription termination/polyadenylation (ttp) sequences flanking each gene (barr et al., 2002) . these sequences are relatively well conserved in each species and are similar in all species within a genus. the termination of transcription and polyadenylation of mrnas are critically dependent on a stretch of seven uridine residues that are usually preceded by a guanosine residue. following polyadenylation, transcription is re-initiated at the next available ti sequence which follows a non-transcribed intergenic region that may be from one to approximately 50 nt in length (banerjee and barik, 1992) . corruption of the [u] 7 stretch in tpp sequences of animal rhabdoviruses leads to read-through, resulting in polycistronic mrnas (barr et al., 1997) . in plant-adapted rhabdoviruses, the incorruptible component of the polyadenylation signal is uumuuuu(u) (jackson et al., 2005) . as discussed below, rhabdovirus genes may contain multiple long open reading frames (orfs), either overlapping or sequentially in the transcriptional units, and these must be expressed by internal initiation of translation. all evidence suggests that the fundamental processes of genome replication and transcription are common to all rhabdoviruses infecting vertebrates, insects and plants. the five major structural proteins of vsv and rabv are amongst the most intensely studied of all viral proteins. although there are reported variations in their ancillary functions, the primary functions and structural characteristics of these proteins are conserved across all rhabdoviruses and have been reviewed in detail on many previous occasions assenberg et al., 2010; coll, 1995; jackson et al., 2005; jayakar et al., 2004; redinbaugh and hogenhout, 2005; roche et al., 2008; rupprecht et al., 2002) . in contrast, the number and locations of accessory genes varies widely both within and between taxa (figs. 1 and 2). in most cases, levels of amino acid sequence identity between cognate accessory proteins are quite low and, with a few exceptions, their specific functions do not appear to be conserved between taxa. as discussed in detail below, some occur as alternative or overlapping orfs within the major structural protein genes; many occur in the regions between the major structural protein genes as new genes containing single or multiple orfs. over 35 rhabdovirus accessory genes have been identified to date and it is likely that many more will emerge as the number of complete genome sequences continues to grow. although the vesiculovirus genome is the smallest and arguably the least complex of all rhabdoviruses, containing only the 5 canonical structural protein genes, additional proteins are expressed from both the p and m genes (fig. 1) . the p genes of most vesiculoviruses encode a small highly basic protein (c) in an alternative orf. in vsv new jersey and vsv indiana, two forms of the c protein (c and c ) are expressed in infected cells by translation initiation at alternative start codons spiropoulou and nichol, 1993) . there is evidence that the c protein is associated with nucleocapsids and increases the activity and fidelity of viral rna polymerase activity in vitro . however, c and c are not essential for replication in cell culture and a vsv infectious clone lacking these proteins has been shown to have identical growth kb 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 16 oth er animal rhabdoviruses table 1 . characteristics to wild-type virus (kretzschmar et al., 1996) . the predicted sizes of the putative c proteins in viruses examined to date range from 41 amino acids (piry virus) to 93 amino acids (cocal virus). interestingly, although spring viraemia of carp virus has the potential to encode highly basic proteins of 83 and 57 amino acids in an alternative orf in the p gene (hoffmann et al., 2002; teng et al., 2007) , there appears to be no alternative orfs in the pike fry rhabdovirus p gene (chen et al., 2009) , suggesting its function may not be conserved across all fish vesiculoviruses. however, these data should be viewed cautiously as a single point mutation kb 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 table 1. during adaptation to cell culture would be sufficient and inhibit expression of these accessory proteins by codon modification. it has also been reported that a small (7 kda) protein is expressed from the vsv p gene from the same orf as the p protein (herman, 1986) . the presence of alternative orfs in the p gene of vesiculoviruses mirrors the situation in paramyxoviruses in which the complex expression of the c and v family of proteins in overlapping open reading frame or by rna editing is very well characterised (nagai and kato, 2004) . the paramyxovirus c and v proteins have various functions associated with infection and pathogenesis including regulation of viral rna synthesis, virion assembly and budding, inhibition of apoptosis, and in binding to components of the jak/stat signalling pathway to antagonize the cellular interferon response (fontana et al., 2008; irie et al., 2010; kurotani et al., 1998) . the vsv m protein is a multi-functional protein that, as well as being a structural component of virions, is involved during infection in inhibition of viral transcription, assembly and budding, inhibition of host gene expression and perturbation of the cell cytoskeleton (black and lyles, 1992; blondel et al., 1990; coulon et al., 1990) . the m protein also blocks the host anti-viral response and promotes apoptosis both indirectly through inhibition of host gene expression and directly by targeting the extrinsic pathway and promoting expression of pro-apoptotic genes (kopecky et al., 2001; pearce and lyles, 2009 ). the vsv m gene is also expressed in infected cells from two alternative initiation codons in the same frame, producing the m2 and m3 proteins (jayakar and whitt, 2002) . little is known of the function of these proteins but they appear to be involved in cell rounding and cytopathology. although alternative initiation codons are available in the m proteins of other vesiculoviruses, it is not known if they are utilised. the lyssavirus genome also conforms to the basic canonical arrangement of the genes encoding the five major structural proteins but lyssaviruses share the common characteristic of a very long 3 non-coding region in the g gene (fig. 1) . the region varies in length from approximately 400 to 700 nt in different lyssaviruses and, in one lineage of laboratory-adapted rabv strains, it is truncated by an additional ttp signal 70 nucleotides downstream of the g protein orf, leading to the suggestion that it may be the remnant of an ancestral gene or pseudogene () (conzelmann et al., 1990; morimoto et al., 1989; tordo et al., 1986) . however, although the long 3 non-coding region is preserved, the additional ttp is absent from wild-type rabies viruses isolated directly from infected animals and from other lyssaviruses (ravkov et al., 1995) . in west caucasian bat virus (wcbv), the region extends 697 nucleotides beyond the g protein orf and, uniquely, contains an orf of 180 nucleotides but there is no evidence of expression of the second orf in wcbv-infected cells (kuzmin et al., 2008) . universal preservation of the region in wild-type lyssaviruses suggests it has functional significance but a rabv infectious clone lacking the region displayed normal growth characteristics in fibroblasts and neuronal cells in culture and no difference in axonal spread or pathogenicity in mice (ceccaldi et al., 1998; schnell et al., 1994) . nevertheless, there is some evidence that, in conjunction with the g protein, the rabv region does contribute to neuroinvasiveness from peripheral sites of infection (faber et al., 2004) . proposed functions of the region include attenuation of l gene expression, stabilisation of the g mrna and interaction of the mrna with host cell proteins (ravkov et al., 1995) . like the vesiculoviruses, lyssaviruses also commonly encode small, highly basic proteins in alternative orfs in the p gene (nadin-davis et al., 2002) . the lyssavirus orfs usually occur in the same region of the p gene as vesiculovirus c orfs but they vary in size in different isolates and do not appear to be selectively retained by a locally constrained mutation frequency (nadin-davis et al., 2002) . it is not known if lyssavirus c proteins are expressed and the variability of their detection in different isolates suggests they may have little functional relevance. however, if their role is to facilitate infection in vivo, sequence data on cell culture-adapted isolates should be regarded cautiously as introduced point mutations in the hypervariable p gene may be selected to interrupt functional c orfs. in addition to the full-length p protein, rabv p gene also encodes four smaller products (p2-p5) in the same reading frame as a result of leaky scanning of the ribosome on the p mrna (chenik et al., 1995) . due to a nuclear localisation signal (nls) in the c-terminal domain and a nuclear export signal (nes) in the n-terminal domain, the various forms of the p protein accumulate differentially in the cell such that the p1 (p) and p2 forms are cytoplasmic whereas p3, p4 and p5 are transported to the nucleus (moseley et al., 2007; pasdeloup et al., 2005) . the p isoforms antagonise cellular interferon responses by several different mechanisms including inhibiting the phosphorylation of cytoplasmic interferon regulatory factor 3 (irf3), binding to nuclear and cytoplasmic stat1 and stat2, and interacting with promyelocytic leukaemia (pml) nuclear bodies (blondel et al., 2002; brzozka et al., 2005 brzozka et al., , 2006 chelbi-alix et al., 2006) . the p3 isoform has also been shown to mediate the sequestration of stat1 away from the nucleus by a microtubule-associated mechanism, preventing nuclear import and activation of the interferon response (moseley et al., 2009 ). the interferon-antagonistic activity of the p protein isoforms are important determinants of the pathogenicity of rabv in mice (ito et al., 2010; rieder et al., 2011) . in the region of the genome occupied by the lyssavirus non-coding region (i.e., between the g and l genes), the novirhabdoviruses contain an additional gene of approximately 500 nucleotides (kurath et al., , 1997 (fig. 1) . the non-virion (nv) orf is flanked by ti and ttp sequences, and encodes a neutral to mildly acidic protein of 110-122 amino acids (13.2-13.6 kda) schutze et al., 1996) . the nv proteins of infectious haematopoietic necrosis virus (ihnv) and viral hemorrhagic septicaemia virus (vhsv) are expressed at low levels in infected cells but do not occur in virions (basurco and benmansour, 1995; schutze et al., 1996) . the level of amino acid sequence conservation amongst nv proteins is quite low (e.g., vhsv and hirame rhabdovirus share only 16.5% identity) but the nv gene is present in all four novirhabdovirus species and there is evidence that they are functionally equivalent (kurath et al., 1997; thoulouze et al., 2004) . the construction of novirhabdovirus infectious clones lacking the nv gene has indicated that it is not essential for virus replication in cell culture but reports vary on the replication efficiency and pathogenicity of the recombinant viruses (biacchesi, 2011) . deletion of the nv gene from an ihnv infectious clone resulted in reduced replication efficiency in epc fish epithelial cells and reduced cumulative mortalities following intraperitoneal injection of rainbow trout (oncorhynchus mykiss) (biacchesi et al., 2000; thoulouze et al., 2004) and similar observations have been reported for an nv-knockout infectious clone of vhsv in yellow perch (perca flavescens) (ammayappan et al., 2010) . however, snakehead rhabdovirus (shrv) infectious clones either lacking the nv gene or in which the nv orf was truncated by point mutations were shown to replicate normally in epc cells, and retained pathogenicity when injected intraperitoneally into zebra fish (danio rerio) (alonso et al., 2004; johnson et al., 2000) . it has been suggested that this apparent difference in the biological response to inhibition of nv expression may reflect the natural host range of the viruses which infect cold water fish species (vhsv and ihnv) or warm water fish species (shrv) and have corresponding temperature ranges for efficient virus replication (biacchesi, 2011) . recent evidence suggests that the vhsv nv protein has an anti-apoptotic function early in infection in vitro (ammayappan and vakharia, 2011) . however, as the m protein of ihnv has pro-apoptotic activity (chiou et al., 2000) and rhabdoviruses generally are known to promote apoptosis, the biological role of the nv protein remains unresolved. novirhabdoviruses also encode small basic proteins in alternative reading frames in the p gene but their functional relevance is unknown. sigmaviruses also contain a single additional gene but it is located between the p and m genes (teninges and bras-herreng, 1987) (fig. 1) . the x gene encodes a polypeptide (pp3) of 298-321 amino acids (landes-devauchelle et al., 1995) . although amino acid sequence identity between the pp3 proteins of sigmaviruses infecting drosophila melanogaster and drosophila obscura is relatively low (∼11%), sequence similarity is relatively high and each is predicted with high probability to have an n-terminal signal peptide. each also has three predicted n-glycosylation sites near the n-terminus and so the mature x protein appears to be a secreted glycoprotein. although original reports suggested that pp3 of drosophila melanogaster sigmavirus may be distantly related to reverse transcriptase of retrotransposons, the relevant amino acid motifs do not appear to be conserved in the drosophila obscura sigmavirus pp3 protein (landes-devauchelle et al., 1995; longdon et al., 2010) . as sigmaviruses have co-evolved with their hosts and are transmitted only vertically in ecological time frames, further studies of the function of pp3 may reveal interesting aspects of virus-insect interactions and innate anti-viral immunity in insects (longdon et al., 2011; tsai et al., 2008) . the genomes of plant rhabdoviruses are 12-14.5 kb in size and have the same basic organisation as their animal-infecting counterparts, 3 -n-p-m-g-l-5 (fig. 2) . the unique feature of plantadapted rhabdoviruses is that they encode a viral movement protein (mp) between p and m genes which facilitates cell-tocell transport (jackson et al., 2005) . there are no characteristic genus-specific differences in genome size or genome organisation between cytorhabdoviruses and nucleorhabdoviruses. however, nucleorhabdoviruses replicate in the nuclei of infected plant cells and most viral proteins are directed to the nucleus, while cytorhabdoviruses replicate in the cytoplasm (as all other rhabdoviruses) and viral proteins are mainly localised to the cell periphery and endomembranes (jackson et al., 2005) . individual members of both genera may encode up to four additional accessory proteins of unknown function interspersed between n-p, p-m and g-l genes (fig. 2) . the number and location of these additional genes transcends the two genera. rice yellow stunt virus (rysv) and rice transitory yellowing virus (rtyv) are strains of the same virus and the nucleotide sequences of their genomes are 98.5% identical (hiraguri et al., 2010) . all other fully sequenced plant rhabdoviruses are only distantly related and have been classified taxonomically as members of distinct virus species. genes located between the p and m genes encode 30-35 kda proteins that appear to be involved in viral intercellular cell-to-cell movement across plasmodesmata, symplastic connections that traverse the plant cell wall of neighbouring cells (benitez-alfonso et al., 2010) . orfs at this position encode the sc4, 4b and y proteins of sonchus yellow net virus (synv), lettuce necrotic yellows virus (lnyv) and potato yellow dwarf virus (pydv), respectively (bandyopadhyay et al., 2010; dietzgen et al., 2006; scholthof et al., 1994) . other plant-adapted rhabdoviruses also contain accessory genes at this location. single accessory genes (designated p3) are located at this position in rice yellow stunt virus (rysv), rice transitory yellowing virus (rtyv), maize mosaic virus (mmv), maize iranian mosaic virus (mimv), taro vein chlorosis virus (tavcv), lettuce yellow mottle virus (lymov) and strawberry crinkle virus (scv) (heim et al., 2007; hiraguri et al., 2010; huang et al., 2005; massah et al., 2008; reed et al., 2005; revill et al., 2005; schoen et al., 2004) . however, maize fine streak virus (mfsv) contains two genes (p3 and p4) and in northern cereal mosaic virus (ncmv) there are four genes (p3, p4, p5 and p6) at this position in the genome (fig. 2) (tanno et al., 2000; tsai et al., 2005) . secondary structure predictions have shown that the synv sc4, lnyv 4b, lymov p3, mmv p3, mfsv p4, rysv p3 and pydv y proteins have some structural similarities in common with those of the '30k' superfamily of plant virus movement proteins (melcher, 2000) . blastp database searches and detailed secondary structure prediction of the lnyv 4b and lymov p3 proteins also revealed close similarities with the movement proteins of viruses in the family flexiviridae which induce movement-associated tubules in infected cells (dietzgen et al., 2006; heim et al., 2007) . lymov p3 has a 58% amino acid sequence identity with lnyv 4b (heim et al., 2007) . the membrane and cell periphery associations of synv sc4 also suggest a role in cell-to-cell movement. direct experimental evidence of cell-to-cell movement function exists only for rysv p3 which is the most characterised protein encoded in the p-m region (huang et al., 2005) . p3 has been shown to trans-complement cell-to-cell movement of a movement-defective heterologous plant virus and binds nonspecifically ssrna in vitro, a common property of mps of numerous plant viruses (benitez-alfonso et al., 2010) . moreover, pull-down assays have shown that p3 interacts with the n protein, providing support for involvement of the nucleocapsid core in cell-to-cell movement (huang et al., 2005) . when expressed as fusion proteins with green fluorescent protein (gfp), pydv y and synv sc4 have been shown to localize to the cell periphery (bandyopadhyay et al., 2010; goodin et al., 2007) whereas mfsv p4 localized to nuclei . the putative mps of synv and pydv self-interact in bimolecular fluorescence complementation (bifc) assays (bandyopadhyay et al., 2010; min et al., 2010) . pydv y and m proteins interacted in planta and the complex accumulated inside the nucleus, while synv sc4 interacted with a soluble truncated form of g protein and complexes were seen on the nuclear envelope and on punctuate loci along the cell periphery. sc4 has also been shown to interact with microtubule-associated host proteins and cytoplasm-tethered transcription activators have been implicated in the intercellular movement of synv (min et al., 2010) . the available information for these four nucleorhabdoviruses indicates potentially different cell-to-cell movement pathways. the functions of the other accessory genes, including the four unique ncmv orfs located between p and m genes, are unknown. three plant rhabdoviruses, ncmv, scv and rysv (and the synonymous rtyv) contain a short orf between the g and l genes (fig. 2) . the ncmv p9, scv p6, and rysv p6 genes are predicted to encode proteins of 52, 68, and 93 amino acids, respectively. they share no identifiable sequence similarities but limited similarity can be identified with other rhabdovirus proteins. the rysv p6 protein shares 24% sequence identity with the n-terminal 110 amino acids of the synv l protein and has limited sequence identity with the nv proteins of novirhabdoviruses (up to 25%) and non-coding regions preceding the l gene in hendra virus (38.3%) and rabv (36.6%). the scv p6 and ncmv p9 proteins are moderately basic (pi ∼ 8.9) but rysv p6 is highly acidic (pi 3.49). the large negative charge of this protein is reminiscent of the acidic n-terminal domain of the rysv l protein, although they lack sequence similarity. of the three small proteins, only rysv p6 has been characterised experimentally (huang et al., 2003) . p6 is phosphorylated in vitro on both serine and threonine residues. it has also been shown to be associated with virions and may have a structural role but it is present only in low amounts during infection. the function of proteins encoded in this genome position is yet unknown. however, distant similarities of rysv p6 with the l proteins of rysv and synv may suggest a close evolutionary relationship with l proteins and potentially an ancillary role in rhabdovirus replication. pydv is unique amongst the sequenced plant-adapted rhabdoviruses in that it has an orf x of unknown function located between the n and p genes. the 11 kda x protein has a high content of proline, aspartic and glutamic acids, with a predicted pi of 4.5. these properties are similar to those of mfsv p3 (10 kda, pi 5.4). however, while mfsv p3-gfp fusions accumulate in punctuate loci in the cytoplasm , pydv x is positive in yeast-based nuclear import assay, indicating its likely nuclear localization (bandyopadhyay et al., 2010) . like the animal rhabdoviruses, some cytorhabdoviruses and nucleorhabdoviruses present alternative orfs in the p gene, but it is unknown if these are expressed in infected plants or vector insects (callaghan and dietzgen, unpublished). ephemerovirus genomes are amongst the largest and most complex of known rhabdoviruses with multiple accessory genes located in a region that extends up to 4.2 kb between the g and l genes (fig. 1) . perhaps the most interesting of these is a second glycoprotein (g ns ) gene that immediately follows the g gene (walker et al., 1992) . the g ns gene occurs in all ephemeroviruses and, until recently, has been regarded as a defining characteristic of the genus. the g ns proteins are 60-90 kda type 1 transmembrane glycoproteins that are highly glycosylated with n-linked glycans (walker et al., 1992; wang and walker, 1993) . they share significant amino sequence identity and structural homology with the g proteins of ephemeroviruses and other rhabdoviruses, and appear to have arisen by recombination or gene duplication (see below). the bovine ephemeral fever virus (befv) g ns protein has been detected in infected cells but is not present in virions (walker et al., 1991) . recombinant g ns expressed from a vaccinia virus vector has been detected in association with amorphous structures at the cell surface but not with budding virus particles (hertig et al., 1996) . despite the structural similarity, the befv g ns protein shares no neutralising epitopes with the g protein (johal et al., 2008) and polyclonal rabbit antibody to the befv g ns protein does not neutralise virus produced in either mammalian or insect cell cultures (hertig et al., 1996) . furthermore, unlike the g protein, befv g ns does not induce spontaneous cell fusion at low ph (johal et al., 2008) , suggesting it functions neither in cell attachment nor fusion in vertebrates or invertebrates. in an attenuated vaccine strain of befv (joubert and walker, unpublished data) and in a highly cell-culture-adapted strain of adelaide river virus (arv), corruptions in the ttp sequence at the end of the g gene allow expression of the g ns protein only as a polycistronic mrna (wang and walker, 1993 ) but this has not been observed in other wild-type or cell culture adapted ephemeroviruses. the function of ephemerovirus g ns proteins is currently unknown. immediately downstream of the g ns gene lies a set of relatively small accessory genes bounded by ti and ttp sequences that vary in number in different ephemeroviruses (mcwilliam et al., 1997; wang et al., 1994) . the ␣ gene occurs in all ephemeroviruses and contains two consecutive orfs (␣1 and ␣2) that are expressed from a single transcript due to the universal absence of intervening ttp and ti sequences. the ␣1 orf encodes proteins of 10.5-14.5 kda, each with a predicted n-terminal ectodomain containing clusters of aromatic residues, a central transmembrane domain and a highly basic c-terminal endodomain (fig. 3) . although sequence identity between the ␣1 protein is relatively low, the structure is highly conserved and strongly suggests they function as viroporins (gonzalez and carrasco, 2003; mcwilliam et al., 1997) . the ␣2 orf encodes proteins that range in size from 10.7 kda to 14.2 kda in different ephemeroviruses and have no identifiable motifs that may suggest a particular function. they share identifiable amino acid sequence homology with significantly higher levels of identity (up to 59%) between the more closely related viruses. conservation of the ␣2 orf in all ephemeroviruses and the preservation of sequence identity suggest that it is of functional significance. in most ephemeroviruses the ␣2 orf initiation codon is in close proximity to the ␣1 termination codon suggesting expression may occur by translation re-initiation as has been described for several other rna viruses (powell, 2010) . in befv, orf ␣2 also contains a 51 amino-acid orf (␣3) in an alternative reading frame but it does not occur in other ephemeroviruses and its functional significance is unknown (mcwilliam et al., 1997) . all ephemeroviruses also contain the ␤ gene which is located immediately downstream of the ␣ gene and is expressed as a monocistronic transcript encoding a 16.9-18.4 kda neutral protein (mcwilliam et al., 1997; wang et al., 1994) . the ephemerovirus ␤ proteins share significant amino acid sequence identity and have been detected both in infected cells and in virions. however, in highly adapted laboratory strains of befv and kimberley virus (kimv) the ␤ proteins are severely truncated by point mutations and are not expressed during infection, indicating that they are not essential for growth in vitro and are not essential components of virions (walker, joubert and blasdell, unpublished data) . they have no identifiable homology with any other known proteins but, although their function remains unknown, they may well have a key role in pathogenesis and/or blocking the host immune response. although absent from the genomes of arv and obodhiang virus (obov), befv, kimv, kotonkan virus (kotv) and berrimah virus (brmv) each also contain a ␥ gene, immediately downstream of the ␤ gene (mcwilliam et al., 1997) . proteins encoded in the ␥ orf are mildly basic and range in size from 11.8 kda to 13.6 kda. they share the highest levels of amino acid sequence identity of any of the ephemerovirus accessory proteins but are not related to any other known proteins and contain no identifiable sequence motifs that suggest a function. the ␥ proteins have been detected in infected cells and in virions. in wild-type viruses, the ␥ genes are transcribed as monocistronic mrnas but in highly cell culture adapted strains, expression of the ␥ protein is severely attenuated by corruption of the ttp sequence at the end of the ␤ gene, allowing expression only as a bicistronic ␤-␥ transcript. selective suppression of ␥ gene expression in culture suggests that its function may be important during infection in vivo. the 15,870 nt kotv genome is the largest yet discovered in any rhabdovirus due to an additional accessory gene immediately downstream of the ␥ gene (walker and blasdell, unpublished data) . the kotv ␦ gene is expressed as a monocistronic mrna encoding a mildly acidic 12.4 kda protein. the ␦ protein has been detected in kotv-infected cells. it is shares significant amino acid sequence identity with the pleckstrin homology (ph) domain of coactivatorassociated arginine methyl transferase (carm1) which is involved in chromatin re-modelling and transcriptional activation of nf-b dependent gene expression (covic et al., 2005; troffer-charlier et al., 2007) . kotv ␦ may mimic this domain, inhibiting activation of the anti-viral innate immune response (joubert and walker, unpublished data) . like most other rhabdoviruses, several ephemeroviruses also encode small highly basic proteins in alternative orfs in the p gene but their functional significance is unclear. unlike the ephemeroviruses, the complex genomes of the hart park group of rhabdoviruses contain multiple accessory genes inserted at various locations (fig. 1) . wongabel virus (wonv) contains five additional orfs (u1-u5) interspersed at various locations across the 13,196 nt genome (gubala et al., 2008) . the u1, u2 and u3 orfs are located between the p and m genes and are each bounded by ti and ttp sequences. orf u1 and orf u2 encode acidic proteins of similar size and net charge (21.2 kda/pi 5.2 and 21.9 kda/pi 4.5, respectively). they have no remarkable characteristics but do share a significant level of amino acid sequence homology (∼16% identity; 60% similarity) and are more closely related to each other than to any other known protein. the wonv u3 gene follows the u2 gene and also encodes a relatively small acidic protein (16.5 kda/pi 4.6) that is unrelated to any other known proteins but shares sequence homology with the wonv u1 protein (∼22% identity; 57% similarity), including the common sequence [ksxydfvwpxxxlxxg] in the central region of each protein (fig. 4a) . preliminary data suggest that the u3 protein binds to the iap (inhibition of apoptosis) protein apollon and a component of the chromatin remodelling complex and may be involved in promoting apoptosis (joubert and walker, unpublished data). wonv orf u4 is located within the n gene as a second orf in the 142 nt region that follows the n protein termination codon. it encodes a putative small acidic protein (5.8 kda/pi 4.0) of 49 amino acids that could only be translated by internal initiation on a bicistronic (n-u4) mrna. wonv orf u5 overlaps the g protein orf and thus also must be translated by internal initiation on a bicistronic (g-u5) mrna. translation from the first initiation codon would result in a 14.9 kda protein with that is similar in size and structure to the ephemerovirus viroporin-like ␣1 proteins (fig. 3) . several low molecular weight proteins (range 20-25 kda) have been detected in infected cells using wonv hyperimmune antiserum but they are larger than the predicted u1-u5 proteins and may well be degradation products of larger viral proteins (gubala et al., 2008) . the flanders virus (flav) genome organisation appears to closely resemble that of wonv. the available sequence data (gen-bank ah012179) is dated and appears to be corrupted either by sequencing ambiguities or as a result of cell culture adaptation of the virus. however, through alignment with the wonv genome, it is reasonable to infer that flav also contains three accessory genes between the p and m genes and a 13.8 kda viroporin-like protein encoded in a second orf in the g gene (fig. 3) . the flav u1, u2 and u3 orfs each appear to encode proteins of 18-19 kda and, as for the corresponding wonv proteins, they share an unusual level of sequence similarity suggesting a common origin and/or functional association. this is most evident between flav u1 and u2 (also referred to as the 19k protein) that share ∼20% amino acid sequence identity and ∼58% similarity (fig. 4b ). there is no second consecutive orf in the flav n gene corresponding to wonv u4 but there is a small alternative orf within the n gene encoding a polypeptide of 66 amino acids (7.9 kda). in addition to the n, p, m, g and l proteins, three virus-induced proteins have been reported in flav infected cells and in virions (boyd and whitaker-dowling, table 1. 1988). however, only one of these proteins (19 kda) corresponds to the predicted size of any flav accessory gene product. the 15,764 nt ngaingan virus (ngav) genome is one of the largest and most complex of known rhabdoviruses (gubala et al., 2010) . as in wonv and flav, the ngav genome contains three orfs between the p and m genes (u1, u2 and u3) encoding neutral to mildly acidic proteins of 15.1-17.1 kda that share identifiable sequence similarity. however, unlike wonv and flav, orf u1 and orf u2 are encoded in overlapping reading frames within the same gene and orf3 is encoded in a separate gene bounded by ti and ttp sequences. downstream of the u3 gene, the ngav genome organisation is very complex and varies significantly from wonv and flav. between the m and g genes the ngav u4 gene encodes a 9.7 kda protein with a tyrosine-rich c-terminal domain but otherwise has no remarkable features. downstream of the g gene, however, is a region that resembles the ephemerovirus genomes including a second glycoprotein gene (g ns ), followed by three additional orfs. the g ns gene encodes a type 1 transmembrane glycoprotein that is most closely related to the ephemerovirus g ns proteins and shares sequence homology and structural characteristics with the ngav g protein and those of all animal rhabdoviruses (fig. 5) . the two accessory genes immediately downstream of the ngav g ns gene also resemble the ephemerovirus ␣ and ␤ genes in format, size and location. the first contains two overlapping orfs (u5 and u6) that are similar in size to the ephemerovirus ␣1 and ␣2 orfs. however, the 12.6 kda u5 protein does not have the characteristic structure of the viroporin-like ␣1 proteins and, unlike the featureless ephemerovirus ␣2 proteins, the u6 protein has the structure of a small (10-12 kda) membrane-anchored class 1 glycoprotein with a signal peptide domain, a transmembrane domain and an nterminal ectodomain containing a single n-glycosylation site. the ngav u7 gene contains a single orf encoding an 18.2 kda protein that is similar in size to ephemerovirus ␤ proteins but shares no identifiable sequence homology and has no remarkable features. lyssavirus g a minimum evolution phylogenetic tree constructed from a clustal x multiple sequence alignment of animal rhabdovirus g protein and gns protein sequences. the tree was constructed using the mega program. viruses assigned to established genera and proposed genera are shaded. abbreviations of virus names are as listed in table 1 . in addition to these independent accessory genes ngav encodes two small basic proteins (p 1 and p 2) in alternative reading frames within the p gene and a 10.8 kda protein in an alternative reading frame in the m gene. if all these potential orfs in translated, ngav could potentially express a total of 16 functional proteins. whole genome sequences are available for two viruses that have recently been proposed as species in a new genus tibrovirus. the 13.2-13.3 kb tibrogargan virus (tibv) and coastal plains virus (cpv) genomes share a similar organisation with 3-4 accessory protein genes (gubala et al., 2011) (fig. 1) . in each virus, orfs u1 and u2 are located between the m and g genes. each is bounded by ti and ttp sequences and encodes a protein of 17-20 kda. the u1 proteins are acidic and the u2 proteins are basic, and there is extensive amino acid sequence homology between the corresponding proteins. an n-terminal signal peptide is predicted with high probability in the cpv u1 protein, suggesting it may be secreted but it is not predicted in the tibv u1 protein despite the high level of overall sequence homology and so its significance is uncertain. features of the u2 proteins are unremarkable. the tibv and cpv genomes also each contain orf u3 encoded in an independent gene located between the g and l genes. the u3 proteins have the structural characteristics of viroporin-like proteins that occur in ephemeroviruses as the ␣1 proteins and in wonv and ngav as the u5 proteins. in each case, the viroporin orfs are located between the g and l genes, either directly following the g gene or following the g ns gene in those viruses in which it occurs. in tibv, u4 is an alternative orf that overlaps the g protein orf and could potentially encode a very small basic protein (6.6 kda/pi 9.6). however, u4 does not occur in cpv and its significance, if any, is unclear. as in many other rhabdoviruses, the cpv p gene also contains an alternative orf encoding a small basic proteins but none is evident in the tibv p gene. several other unclassified rhabdoviruses isolated from insects, mammals, birds and fish share a similar genome organisation (fig. 1) . tupaia rhabdovirus (tupv), durham virus (durv) and oak vale virus (oakv) each encode small hydrophobic (sh) proteins in orfs flanked by ti and ttp sequences in the region between the m and g genes (allison et al., 2011; springfeld et al., 2005) . however, although superficially similar, the putative proteins expressed from these genes have different characteristics. the tupv orf encodes a small protein with a predicted signal peptide and a central transmembrane domain that would generate an 8.6 kda non-glycosylated membrane-anchored protein with short ectoand endo-domains. in durv, the sh protein is predicted to have similar membrane topology with predicted signal peptidase cleavage generating a 6.9 kda non-glycosylated membrane-anchored protein. these proteins resemble the small hydrophobic protein encoded in the ngav u5 gene (see above). the oakv sh protein is predicted to have an n-terminal signal peptide but no membrane anchor to generate a 4.2 kda non-glycosylated secreted product (genbank gq294474). the mandarin fish (siniperca chuatsi) rhabdovirus (scrv) genome does not contain an independent sh gene but does encode a small hydrophobic protein as an alternative reading frame in the upstream m gene (tao et al., 2008) . the predicted structure is similar to that of the oakv sh protein with a high probability signal peptidase cleavage site generated a secreted product of only 2.9 kda. although it has been reported that the putative scrv sh protein could not be detected in infected fish cells, this may not be unexpected if it is indeed secreted. the x genes of sigmaviruses also encode a protein that is predicted to be secreted (see above) but it is very much larger than these small peptides. several of these unclassified rhabdoviruses also encode small basic proteins in alternative orfs in the p gene. although characterised by remarkable diversity and complexity, a global view of rhabdovirus genomes does reveal some patterns in the nature and location of accessory genes (fig. 6) . in various rhabdoviruses, accessory genes occur in each of the junctions between the structural protein genes and as alternative or overlapping orfs within all structural protein genes except the l gene. alternative or overlapping orfs occur least commonly in the n gene (and of course the l gene), possibly reflecting the structural and functional constraints on amino acid sequence variations, and in the g gene that requires sufficient genetic flexibility to evolve under the pressure of neutralising antibody. alternative orfs occur most commonly in the p gene in which the preservation of function is far less dependent on conservation of the amino acid sequence, allowing more scope for the evolution and stabilisation of new orfs in alternative frames (nadin-davis et al., 2002; spiropoulou and nichol, 1993) . the n-p, p-m, m-g, and g-l gene junctions have all been utilised as sites for accessory genes, although in rhabdoviruses examined to date, the p-m and g-l junctions have been utilised more commonly. the primary evolutionary constraint on the occupation of these sites may be the resulting attenuation of transcription in the downstream genes. for example, rare use of the n-p gene junction may be due to the importance of the balance of expression of the n and p proteins in modulating replication and transcription (banerjee and barik, 1992) . alternative orfs in the p gene occur very commonly and usually encode small basic proteins. although their function has not been examined in any detail, by analogy with paramyxoviruses, they may have multiple roles in replication, virion assembly and in modulating host responses to infection (fontana et al., 2008; irie et al., 2010; kurotani et al., 1998) . the p-m gene junction has been utilised as a site of accessory genes in many rhabdoviruses including genes encoding movement proteins in cytorhabdoviruses and nucleorhabdoviruses and the apparently secreted and glycosylated pp3 protein of sigmaviruses. the p-m junction is also the location of the set of three related 15-22 kda proteins in the hart park group viruses that may be involved in the promotion of apoptosis. the m-g gene junction is occupied less commonly but genes encoding small proteins that are either secreted or membrane-anchored do occur here, including the tibv u1 protein, and the small hydrophobic (sh) proteins of the unclassified viruses tupv, durv and oakv (allison et al., 2011; springfeld et al., 2005) . interestingly, in the mandarin fish rhabdovirus (scrv), and sh protein is encoded just upstream as an alternative orf in the m gene (tao et al., 2008) . the g-l gene junction is the most commonly occupied site of accessory genes in animal rhabdoviruses. genes encoding the non-structural glycoprotein (g ns ) invariably occur immediately downstream of the g gene and share a common genetic lineage (gubala et al., 2010; walker et al., 1992; wang and walker, 1993) . genes encoding viroporin-like proteins also usually occur in this region, immediately following g ns gene in ephemeroviruses (mcwilliam et al., 1997; wang et al., 1994) and following the g gene in flav and tibroviruses which lack g ns (gubala et al., 2011) . interestingly, the wonv viroporin-like protein is encoded immediately upstream of the g-l junction in an orf that overlaps the end of the g gene. the viroporin-like proteins all have a similar structure with a central hydrophobic domain and a highly basic c-terminal tail, and so may have evolved from a common ancestor (fig. 3) . many other accessory genes are located in the g-l intergenic region including the novirhabdovirus nv gene, and genes coding proteins of unknown function in ephemeroviruses, ngav and some plantadapted rhabdoviruses. the g-l intergenic region is also the site of the lyssavirus region (tordo et al., 1986) . sequence insertion at this site may be most favoured as it would primarily affect expression of the l protein which is required only in catalytic amounts. the ecological diversity of rhabdoviruses suggests they have an ancient evolutionary history, perhaps originating as insect viruses that have adapted to replication in plants or animals (hogenhout et al., 2003) . there is recent evidence that endogenous viral elements (eves) derived from rhabdoviruses occur commonly in insect genomes with which they have co-evolved over many millions of years (katzourakis and gifford, 2010) . rhabdovirus genomic diversity will also therefore have evolved over geological time frames, possibly involving rare events that may appear highly improbable in a contemporary ecological context. we have argued previously that the evolution of rhabdovirus accessory genes may have occurred by a copy-choice mechanism involving polymerasejumping (wang and walker, 1993) . according to this model, upstream relocation of the polymerase during replication would generate a sequence duplication in a process mirroring the downstream relocation that has been proposed for the generation of defective-interfering (di) genomes (lazzarini et al., 1981; perrault, 1981) . there seems no prima face reason to suggest that upstream relocation and downstream relocation should not occur with similar frequency but purifying selection would enrich for rapidly replicating di genomes and quickly remove larger genomes unless the duplication event has provided a specific selective advantage. although survival of such larger genomes may be exceedingly rare, preservation of the region in lyssaviruses does indicate that the selective advantage of maintaining long stretches of apparently non-functional sequence may sometimes be cryptic (kuzmin et al., 2008; ravkov et al., 1995) and sequence similarities in adjacent genes of some rhabdoviruses do suggest that duplication may have contributed to the evolution of accessory genes. such sequence similarities are evident in the g ns genes in ephemeroviruses and ngav (gubala et al., 2010; walker et al., 1992; wang and walker, 1993) , the related u1, u2 and u3 genes in the p-m region of hart park group viruses (see above), and the p6 gene in rice yellow stunt virus (huang et al., 2003) . other possible mechanisms for the acquisition of accessory genes include homologous genetic recombination and lateral gene transfer (lgt). life-long persistent infections occur commonly in insects and mixed infections with different viruses have been reported (chen et al., 2004; mourya et al., 2001; thavara et al., 2006) . although genetic recombination is believed to occur rarely in (-) ssrna viruses (holmes, 2009) , possible recombination events have been detected and may have contributed to their long term evolution (chare et al., 2003; han et al., 2008; lukashev, 2005; qin et al., 2008; sibold et al., 1999) . unlike gene duplication, in which the new gene must be preserved while evolving a new function, recombination could offer immediate selective advantage in larger more complex genomes by increasing tissue tropism, host range or replication efficiency or more effective blocking of host defensive mechanisms. homologous genetic recombination must therefore be considered an equally plausible explanation for the generation of genomic structural diversity in rhabdoviruses. lgt also offers a mechanism by which accessory genes could be obtained from the host or an unrelated virus to provide immediate selective advantage (holmes, 2009) . there is evidence that the coronavirus haemagglutinin-esterase glycoprotein was acquired from influenza c virus (luytjes et al., 1988) and that the envelope glycoprotein of the tick-borne orthomyxovirus thogoto may have been derived from a baculovirus (morse et al., 1992) . several other examples of possible lgt involving transfer of host genes to viruses have been reported but these events appear to be very rare and none has yet been reported to our knowledge in rhabdoviruses or other unsegmented (-) ssrna viruses (holmes, 2009) . it can also be argued that purifying selection should drive evolution towards smaller and perhaps more efficient genomes rather than large complex genomes. indeed, the generation of di particles, which occurs with high frequency during infection in vitro, would appear to provide the mechanism for rapid elimination of redundant sequences (lazzarini et al., 1981; perrault, 1981) and phylogenetic analyses do suggest that accessory genes may have been lost during the evolution of some lineages. as shown in fig. 5 , phylogenetic analysis of available rhabdovirus g protein sequences places ngav with wonv and flav to form the hart park group and analyses of the l and n protein sequences supports these relationships (bourhy et al., 2005; gubala et al., 2010) . however, the ngav g ns protein clusters with the ephemerovirus g ns proteins (fig. 5) , suggesting that it was obtained by a common ancestral virus and subsequently lost from wonv and flav. alternatively, ngav may have acquired g ns independently in a later event involving recombination with a virus in the ephemerovirus lineage. all arguments considered, perhaps the most congruent hypothesis is that the evolution of rhabdovirus genomic diversity is a dynamic process in which accessory genes have been gained and lost during the course of continual adaptation and purifying selection. despite the importance of rhabdoviruses as pathogens of humans, livestock, fish and plants, it is evident from this review that very little is known of the functions of the proteins encoded in their many accessory genes. in some respects, this is very surprising as five major rhabdovirus structural proteins have been the subject of intensive investigation and have provided some of the most important models for understanding fundamental processes during viral infection. on the basis of available evidence, and by analogy with other viruses that are rich in accessory functions (e.g., hiv, sars coronavirus), we can anticipate that many of the rhabdovirus accessory proteins may be important determinants of virulence and pathogenesis and function to increase the efficiency of viral replication, block host innate and/or adaptive immune responses, regulate apoptosis or induce cytopathology. understanding the strategies employed by rhabdoviruses to engage, divert and re-direct cellular processes will not only present opportunities to develop new anti-viral therapies but may also reveal aspects that have broader significance in biology, agriculture and medicine. one of the most attractive targets for future study is the role of rhabdovirus accessory genes during infection in insects and in transmission to mammals or plants. despite their importance as vectors of disease, and over 100 years of immunology, the antiviral defences of insects remain poorly understood (huszar and imler, 2008) . rnai has been identified as an important anti-viral mechanism and there is evidence of involvement of the toll and imd pathways that function primarily in innate immune responses to bacteria and parasites (kemp and imler, 2009 ). in drosophila melanogaster, the innate immune response to sigmavirus infection has been shown to involve upregulated peptidoglycan reporter and antimicrobial peptide gene expression (tsai et al., 2008) . however, innate anti-viral sensor and effector mechanisms in insects are largely unexplored and may be critical determinants of their susceptibility to infection and ability to act as efficient vectors. as most rhabdoviruses naturally infect insects, their accessory genes may well be involved in engaging defensive responses and so provide useful tools for exploring invertebrate anti-viral pathways. there has been a significant increase in the number 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rhabdovirus sigma, the hereditary co2 sensitivity agent of drosophila: nucleotide sequence of a cdna clone encoding the glycoprotein double infection of heteroserotypes of dengue viruses in field populations of aedes aegypti and aedes albopictus (diptera: culicidae) and serological features of dengue viruses found in patients in southern thailand essential role of the nv protein of novirhabdovirus for pathogenicity in rainbow trout walking along the rabies genome: is the large g-l intergenic region a remnant gene functional insights from structures of coactivator-associated arginine methyltransferase 1 domains drosophila melanogaster mounts a unique immune response to the rhabdovirus sigma virus complete genome sequence and in planta subcellular localization of maize fine streak virus proteins bovine ephemeral fever in australia and the world ephemeroviruses: arthropod-borne rhabdoviruses of ruminants, with large and complex genomes proteins of bovine ephemeral fever virus the genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes emerging viral diseases of fish and shrimp complex genome organization in the gns-l intergenic region of adelaide river rhabdovirus adelaide river rhabdovirus expresses consecutive glycoprotein genes as polycistronic mrnas: new evidence of gene duplication as an evolutionary process isolation and characterization of a rhabdovirus from co-infection of two viruses in mandarin fish key: cord-296928-wu14k7u9 authors: hofmann, tim; krah, simon; sellmann, carolin; zielonka, stefan; doerner, achim title: greatest hits—innovative technologies for high throughput identification of bispecific antibodies date: 2020-09-08 journal: int j mol sci doi: 10.3390/ijms21186551 sha: doc_id: 296928 cord_uid: wu14k7u9 recent years have shown a tremendous increase and diversification in antibody-based therapeutics with advances in production techniques and formats. the plethora of currently investigated bito multi-specific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions in oncology and immunology, spanning from enhanced selectivity to effector cell recruitment, all of which cannot be addressed by monospecific antibodies. despite continuously growing efforts and methodologies, the identification of an optimal bispecific antibody as the best possible combination of two parental monospecific binders, however, remains challenging, due to tedious cloning and production, often resulting in undesired extended development times and increased expenses. although automated high throughput screening approaches have matured for pharmaceutical small molecule development, it was only recently that protein bioconjugation technologies have been developed for the facile generation of bispecific antibodies in a ‘plug and play’ manner. in this review, we provide an overview of the most relevant methodologies for bispecific screening purposes—the duobody concept, paired light chain single cell production approaches, sortase a and transglutaminase, the spytag/spycatcher system, and inteins—and elaborate on the benefits as well as drawbacks of the different technologies. the human body is continuously exposed to potentially harmful and life-threatening opponents such as pathogens or malignant cells. in order to assert oneself, several layers of innate defense mechanisms such as pathogen recognition receptors enable the early detection of 'danger signals' and, consequently, the elimination of the respective invader [1, 2] . moreover, the human immune system can also respond in an adaptive fashion to a foreign antigen via antibody-mediated processes, for instance antibody-dependent cellular cytotoxicity (adcc) or complement-dependent cytotoxicity (cdc) [3, 4] . as such, antibodies are crucial players in host defense. inspired by the natural mechanisms of antibody-mediated pathogen elimination, monoclonal antibodies emerged as one of the most promising classes of therapeutic molecules, with about 80 entities now being approved either in the usa or europe [5, 6] . however, one of the main limitations of antibody-derived therapeutics relies in their monospecific nature. this restricts targeting to one antigen only. since most diseases are typically rather complex, involving multiple disease mediators or factors simultaneously [7, 8] , much effort was made within the last decades to combat the multifaceted nature of oncology and immunology diseases more efficiently. in this respect, the advent of bispecific antibodies opened up new avenues for disease treatment such as effector cell redirection or the simultaneous blocking of two different disease mediators, with encouraging results in (mostly early stage) clinical trials [9] [10] [11] . consequently, an unprecedented multitude of bi-and multi-specific formats employing flexible valences and overall architectures were engineered to facilitate a plethora of modes of action (moa), as elegantly described elsewhere [9, 12] . for the generation of bispecific antibodies, one of the main tasks relies in the identification of paratope pairs that fulfill the desired moa in the best way possible when reformatted as bispecific. nowadays, antibody selection campaigns such as immunization combined with b-cell cloning, phage display, or yeast surface display of naïve, synthetic, as well as immune libraries typically enable the identification of up to hundreds of different initial hit candidates [13] [14] [15] [16] [17] . hence, testing of every combination of hits for each binding arm in a bispecific antibody format would result in the expression of several thousands of different molecules, making the whole process rather cumbersome and tedious. in this regard, kitazawa and co-workers initially expressed and analyzed 40,000 bispecific antibody combinations in order to identify a molecule that mimics the cofactor function of coagulation factor viii sufficiently [18] . in addition, other factors such as the order of paratopes in a given architecture or steric hindrance might impact biological activities, e.g., crosslinking abilities for effector cell redirection, and thus need to be taken into consideration [19] [20] [21] . consequently, full coverage of this multidimensional screening space would require huge efforts with respect to molecular biology, i.e., cloning, antibody expression, and purification. in order to overcome this bottleneck, technologies such as controlled fab-arm exchange used for duobodies [22] , paired light chain single cell production approaches [23] , microbial transglutaminase [24] or sortase a [25] mediated bioconjugation, the spytag/spycatcher system [26, 27] , or split inteins [28] were adapted to enable broad bispecific antibody screening ( figure 1 ). these methodologies harbor the benefit that bispecific entities can be generated from a pre-existing set of antibody fragments in a mix-and-match manner, significantly reducing hands-on time as well as overall efforts for bispecific screening. in this review, we aim at giving an overview of the most relevant protein bioconjugation technologies for the creation of bifunctional antibodies. we further discuss benefits as well as limitations of each specific methodology in the context of bispecific screening. [7, 8] , much effort was made within the last decades to combat the multifaceted nature of oncology and immunology diseases more efficiently. in this respect, the advent of bispecific antibodies opened up new avenues for disease treatment such as effector cell redirection or the simultaneous blocking of two different disease mediators, with encouraging results in (mostly early stage) clinical trials [9] [10] [11] . consequently, an unprecedented multitude of bi-and multi-specific formats employing flexible valences and overall architectures were engineered to facilitate a plethora of modes of action (moa), as elegantly described elsewhere [9, 12] . for the generation of bispecific antibodies, one of the main tasks relies in the identification of paratope pairs that fulfill the desired moa in the best way possible when reformatted as bispecific. nowadays, antibody selection campaigns such as immunization combined with b-cell cloning, phage display, or yeast surface display of naïve, synthetic, as well as immune libraries typically enable the identification of up to hundreds of different initial hit candidates [13] [14] [15] [16] [17] . hence, testing of every combination of hits for each binding arm in a bispecific antibody format would result in the expression of several thousands of different molecules, making the whole process rather cumbersome and tedious. in this regard, kitazawa and co-workers initially expressed and analyzed 40,000 bispecific antibody combinations in order to identify a molecule that mimics the cofactor function of coagulation factor viii sufficiently [18] . in addition, other factors such as the order of paratopes in a given architecture or steric hindrance might impact biological activities, e.g., crosslinking abilities for effector cell redirection, and thus need to be taken into consideration [19] [20] [21] . consequently, full coverage of this multidimensional screening space would require huge efforts with respect to molecular biology, i.e., cloning, antibody expression, and purification. in order to overcome this bottleneck, technologies such as controlled fab-arm exchange used for duobodies [22] , paired light chain single cell production approaches [23] , microbial transglutaminase [24] or sortase a [25] mediated bioconjugation, the spytag/spycatcher system [26, 27] , or split inteins [28] were adapted to enable broad bispecific antibody screening ( figure 1 ). these methodologies harbor the benefit that bispecific entities can be generated from a pre-existing set of antibody fragments in a mix-and-match manner, significantly reducing hands-on time as well as overall efforts for bispecific screening. in this review, we aim at giving an overview of the most relevant protein bioconjugation technologies for the creation of bifunctional antibodies. we further discuss benefits as well as limitations of each specific methodology in the context of bispecific screening. is selected to generate a bispecific igg full-length format with correct chain pairing. for the sake of simplicity in this example, fab fragments are reconstituted with one-armed monovalent fragments. the depicted antibody fragments contain a heterodimerization technology indicated by striped ch 3 regions. the bispecific antibody reconstitution is performed in a combinatorial setup to increase the number of variants. (c) high throughput antibody screening of reconstituted bispecific antibodies is performed in plate format, depending on the mode of action. (d) biological functional screening of several t-cell engager combinations is conducted in 1 to 2 weeks, depending on the combinatorial sample size. a very elegant and generic approach for the generation of bispecific antibodies was adapted from processes occurring within the human immune repertoire itself. igg4 antibodies are dynamic molecules that physiologically form bispecifics by a process called fab-arm exchange (fae). herein, half molecules (consisting of a paired heavy and light chain) from different igg4s recombine and form a polyclonal mixture of bispecific antibodies in vivo. by losing their ability to crosslink antigens and generate immune complexes, it is believed that igg4 may dampen inflammatory reactions by interfering with immune complex formation of other antibody isotypes in chronic inflammatory diseases [29] . the natural mechanism of fae also seems to be attractive to generate bispecific antibodies in vitro, because it may overcome limitations of other bispecific antibody platforms like knobs-into-holes [30] , wherein additional methods like crossmab [31] or paratope scfv grafting need to be applied to circumvent light chain mispairing [7, 32] . in 2013, genmab published an "efficient generation of stable bispecific igg1 by controlled fab-arm exchange" [22] . this work was inspired by the observation that human/rhesus intraspecies igg4 enhance fae (due to igg4 specific ch3 residues) and that 2-mercaptoethylamine-hcl (2-mea), as a mild reducing agent, enables fae with antibody pairs harboring native igg1-hinge regions. in controlled fab-arm exchange ((c)fae), two igg1s with matched ch3 mutations are produced separately and are subsequently recombined under reducing conditions. ch3 mutations need to destabilize the ch3 homodimer interaction and promote heterodimer formation. for this, the igg4 specific r409 mutation was incorporated into one igg1 ch3 and combined with surrounding mutations on positions l368, k370, d399, f405 and y407 (all amino acids except for c and p) on the opposite chain. in a dual-binding elisa, matched point mutations for all five antibody positions were obtained, enabling bispecific binding by fae. in depth analyses of (c)fae were conducted using igg1-f405l and igg1-k409r mutants, the variants that are also applied in genmab's proprietary duobody technology. the general process of bispecific antibody generation applying duobodies involves three basic steps: (1) separate production of monospecific antibodies harboring respective mutations in mammalian cell cultures, (2) purification according to standard processes (e.g., protein-a), (3) (c)fae under tailored laboratory conditions followed by another purification step. this typically yields bispecific antibodies with more than 95% heterodimer content. the beauty of duobodies lies in the fact that the bispecifics fully retain igg1 structure and that fc-mediated mechanisms, such as fcrn mediated recycling and fcγ-receptor interactions, stay unaffected [22] . today, several bispecifics based on (c)fae are under clinical development. one of these molecules is the met × egfr bispecific antibody jnj-61186372, jointly developed by genmab and janssen, currently in phase i, for the treatment of advanced nsclc, that was screened from five anti-met × 8 anti-egfr parental antibodies in the duobody format [33] . similar to the bioconjugation methods described in later sections, (c)fae serves as an ideal platform for bispecific antibody screening. for the given example of 40,000 antibody variants individually produced and screened by kitazawa et al. [18] , only 200 + 200 = 400 parental monospecific would need to be produced using complementary duobody mutations, while 200 × 200 = 40,000 variants could be screened, drastically reducing lab work and timelines applied during classical screening efforts [7, 32] . associated with that, astrazeneca recently published a method for high throughput bispecific antibody screening, applying matched f405l and k40r mutations [34] . in addition, h435r and y436f (also known as rf-mutation) were incorporated into one of the ch 3 mutants (k409r). the rf-mutation completely abrogates the interaction of an antibody-fc with protein-a (however, it should be noted, that antibodies harboring a vh3 segment will bind to protein-a, regardless of the rf-mutation in the antibody-ch 3 ). in a proof of concept study, bispecific antibodies were generated from four parental antibodies (anti-her2; anti-egfr; anti-nip228; anti-igf1r). all six resulting bispecific antibodies could be produced with high heterodimer purity as demonstrated by liquid chromatography-mass spectrometry (lcms) and reverse phase liquid chromatography (rplc) and showed bispecific target engagement proven by bli. compared to the conventional duobody generation, this approach enables the formation of bispecifics directly from culture-supernatants without any pre-purification steps of monospecific antibodies, making it perfectly suited as a bispecific screening tool [34] . finally, bispecific antibodies can be generated by the duohexabody platform, developed by genmab, combining the before mentioned duobodies with a hexameric formation of antibody complexes. the duohexabody technology offers a broad applicability for bispecific antibody generation with target-mediated enhanced potencies for cdc-mediated effector functions as elegantly demonstrated by oostindie et al. [35] . when aiming at screening of heterodimeric fc-based bispecific antibodies not affected by light chain (lc) pairing issues, the combination of two binding moieties' panels can also be executed on the dna rather than the protein level. genentech developed a tethered-variable cl bispecific igg (tcbsigg) platform based on knobs-into-holes heterodimerization combined with linked light chains. this approach allows robust production of intact bispecific antibodies in a single cell line, concurrently ensuring cognate light chain pairing and preserving structural and functional properties of heterodimeric fc antibodies, hence rendering it applicable to high throughput screenings [36] . another elegant example is a combination of a common lc with a dekk heterodimerization fc portion single cell production of 545 bispecific antibodies for a successful unbiased her2/her3 combinatorial screen at merus [23] . in addition to methodologies for screening of classical igg and heterodimeric fc architectures, plentiful approved or clinically-evaluated non-fc formats represent a need for further high throughput screening technologies beyond fc heterodimerization strategies. enzyme-mediated bioconjugation, up to now mainly applied for adc generation, could be a solution. enzyme-mediated bioconjugation has evolved in the last decades to a robust and versatile tool for protein labeling and covalent attachment of two or more protein building blocks by site-specific ligation. it also obviates the need for harmful chemicals as employed for chemical ligation. finally, also enzymatic bioconjugation avoids cumbersome genetic engineering or subsequent expression of individual multivalent binders but offers options for one-pot reactions yielding bispecific antibodies amendable to high throughput or functional screening approaches. first clues for the presence of transglutaminases (tgases) date back to the work of clarke and colleagues in 1957 describing the transamidation activity by pig liver [37] . microbial transglutaminases (mtgases) were first isolated from streptomyces mobaraensis [38] and are now widely used in the food, textile, and cosmetic industry as "biological glue", e.g., for meat [39] . belonging to the class of protein-glutamine γ-glutamyltransferases, tgases catalyze the transfer of primary amines (e.g., the ε-amino group of lysines as acyl-acceptor) to γ-carboxamides (e.g., γ-glutamyl group of glutamine as acyl-donor) under ammonia release, yielding a stable isopeptide bond resistant to proteolytic degradation [39] . although mtgases accept a promiscuous acyl-acceptor substrate repertoire [40] , surface-exposed glutamines of glycosylated native human igg1 antibodies are not accepted by mtgase [41] . mtgase has been engineered by directed evolution and rational design for improved catalytic performance and alternative recognition sequences [42, 43] , and has been exploited in particular for the generation of antibody-drug conjugates [41, 43, 44] or labeling of antibody fragments [45, 46] . multimerization of a single domain antibody fragment targeting tnf could be achieved by using mtgase and compatible peptidyl linkers [24] -an approach that could be generically employed for high throughput screening of non-fc bispecific antibodies. the staphylococcus aureus transpeptidase sortase a (srta) was identified in 1999 to be essential for cell wall assembly of gram-positive bacteria, recognizing a conserved "lpxtg" motif within secreted cell surface proteins, cleaving between the threonine and the glycine residues, forming an thioester acyl-enzyme intermediate and catalyzing the covalent linkage of the carboxyl group of the threonine to a nucleophilic amino group of a n-terminal pentaglycine within the cell wall anchored peptidoglycan [47] . the reaction is reversible, hence, dependent on an excess of donor and acceptor substrate to favor high yields, with the acylation step being the rate-limiting factor [48] . removal of unwanted byproducts can limit the reversibility of the reaction and could provide the basis for an equimolar ratio of the substrates. sortase a engineering yielded variants with improved reaction rates and the reduction of by-products [25, [49] [50] [51] [52] with versatile applications also termed "sortagging" [53] [54] [55] such as protein circularization [56] and antibody drug conjugates [57] . srta has been employed for the generation of bi-and multi-specific antibodies of several formats, e.g., conjugations of fab [55] or scfv fragments [58] as well as c-terminally linked antibody heterodimers in combination with click chemistry [59] . for a high throughput screening approach, the group of plückthun recently established a one-pot srta-mediated coupling reaction for the alternative binding protein darpin (designed ankyrin repeat proteins) with compatibility to functional assays, due to minimal levels of monovalent side products. combinations of 21 darpins, resulting in 441 bispecific molecules targeting c-met and epcam, were analyzed for cell proliferation inhibition and yielded a novel bifunctional darpin with superior cellular activity [25] . this illustrates the applicability of enzymatic bioconjugation for pharmaceutical high throughput biotherapeutics' functional screening approaches. another bacterial transpeptidase served as the basis for the engineering of a ligase, for the conjugation of two proteins. this system, also known as the spytag/spycatcher system, provides a fully intrinsic bioconjugation capability without the need to add enzymes and is described below. the spytag/spycatcher system originates from covalently-stabilized pilin polymers found in the pilus of gram-positive bacteria which usually undergo intramolecular amide bond formation to impart mechanical and proteolytic stability to pili [60] , precisely streptococcus pyogenes, inspiring the name spy technology. when split and engineered, zakeri and co-workers generated first generation isopeptags binding to pilin-n or -c [61, 62] , mediation of irreversible peptide-peptide interactions via spyligase [63] , and finally, the spycatcher and spytag [64] . this approach makes use of the collagen adhesin domain (cnab2) of fibronectin binding protein (fbab) that possesses an internal isopeptide bond between n-and c-domains' amino acids lys31 and asp117. when engineered, the domain is split between lys and asp into two fragments resulting in an n-terminal fragment (spycatcher) of 138 aa and a c-terminal fragment (spytag) of 13 aa. putting both fragments again in close proximity, a spontaneous formation of a covalent peptide bond between lys and asp occurs, resulting in a double hydrogen bond between glu77 and asp117, facilitating the peptide bond formation by nucleophilic attack and forming a zwitterionic intermediate. the bioconjugation of spytag and spycatcher is redox insensitive, efficient in a broad range of ph (5 to 8) and temperatures (4 to 37 • c) [62] . the ligation reaction rate is fast and was further increased 12-fold by engineering an optimized spycatcher002 variant yielding complete bioconjugation after a few minutes [65] . the technology is a promising tool for diverse biotechnological applications as reviewed by sutherland et al. [26] , as discussed also above, ranging from protein ligation via peptide bond formation to protein stabilization by cyclization [65] [66] [67] . specifically, the spytag/spycatcher fragments can either be fused c-or n-terminally or to internal positions within the protein, unlike, for example, split inteins, which are restricted to c-or n-terminal fusions. within the years, a plethora of applications has been published, ranging from the generation of bispecific antibodies to robo1 [68] , a generic trivalent scfv platform [69] , and the generation of site-specific conjugated adcs with high efficiency [70] for therapeutic approaches. akiba et al. described an elegant technology for generating biparatopic antibodies through two-step targeting using a pair antibody-spytag or spycatcher-fusions targeting different epitopes of the same target that spontaneously react to form a covalent bond between fragments to generate a biparatopic antibody in situ [71] . the system is applied in several modular "plug-and-display" approaches for the generation of enhanced immunostimulants against cancer or infectious diseases such as influenza or mers-cov [72] [73] [74] [75] [76] . further applications include optimized protein purification procedures (in parts, in combination with inteins) [77, 78] , specific immobilization of targets for enhanced phage display antibody discovery campaigns [79] , site-specific fluorescence labeling of antibodies for in vivo optical imaging [80] , up to in vivo assembling of proteins or enzymes [81, 82] , and versatile further applications, in depth reviewed by hatlem et al. [27] . in addition to a two-component spy system, veggiani and co-workers identified an orthogonally acting snooptag/snoopcatcher pair by engineering streptococcus pneumoniae adhesin rrga in a similar fashion, which also forms a spontaneous isopeptide bond with >99% yield and no cross-reaction to spytag/spycatcher [83] . similar to spy approaches, three-part splitting of rrga resulted in the respectively called snoopligase, catalyzing the formation of an isopeptide bond between the two peptide tags, snooptagjr and dogtag [84] . many biotherapeutics currently in pre-clinical research and early development are complex molecules, mostly bispecific antibodies, and require both a specific spatial arrangement and flexibility of their binding moieties to not only specifically bind but also fully elicit their functionality of often several desired moas [85, 86] . therefore, in-format screening of bispecific antibodies is desirable and can significantly impact the identification of the best binders' combination [87] . spytag/spycatcher fusions leave a peptide imprint of 151 aa to the protein of interest after bioconjugation. a 32 aa shortened, truncated version of an n-terminal spycatcher fragment has been developed by li et al. [88] to decrease an immune response in mice and to shorten the peptide footprint. in case of screening for effector cell engagers, the remaining 13 kda spy domain could lead to altered flexibility and paratope distances, to impaired biological functionality, and finally, to potentially suboptimal choice of binders or epitopes. despite these potential limitations for screening of certain biotherapeutics classes, the spytag/spycatcher system has, only ten years after its initial publication, been applied to a tremendous extent and very versatile manner and will continue to drive basic science as well as pharmaceutical development. another option for in vitro bioconjugation and screening of bispecific antibodies are split inteins. found in every organism of life, n-and c-termini of split inteins are located in their natural origin on two different genes to elicit splicing in trans at the protein level with well-understood mechanisms [89] . the process is called protein trans splicing (pts), and it yields-similar to enzymatic bioconjugation or spy technology-a stable peptide bond and is very specific for each split intein [90] . pts is a single irreversible turnover reaction, not dependent on thermal or energetic co-factors such as temperature or atp. the most common and well investigated split inteins are ssp dnab from cyanobacterium synechocystis [91] or npu dnae originated from nostoc punctiforme with a reported splicing rate of t 1/2 = 1 min at 37 • c [92] [93] [94] . most investigated split inteins are dependent on a mild reducing environment by the addition of reducing agents like tris (2-carboxyethyl) phosphine (tcep) or dithiothreitol (dtt) to undergo pts. pts is dependent on intein and extein sequences. exteins flank both split intein parts holding specific recognition sequences to initiate the pts mechanism. the amino acid residue at position +1 consists either of a cysteine, serine, or threonine, depending on the split intein. pts takes place in four steps: (1) a nucleophilic attack by the first amino acid residue of either a cysteine or serine located in the n-terminal intein part on the carbonyl carbon of the preceding amino acid residue (−1 position) located in the flanking n-extein leads to an n-s or n-o acyl shift; (2) a linear (thio) ester intermediate being trans-esterificated by a nucleophilic attack of the first amino acid residue of the so-called c-extein (+1 position) forms a branched intermediate [95] ; (3) the n-terminal intein part is now cleaved from its fusion protein and transferred to the n-extein. as a result, a succinimide ring is generated through cyclization of the conserved asparagine residue of the c-terminal intein part after the nucleophilic attack of the previously formed intein extein junction; (4) finally, the cleavage reaction of the c-terminal intein part followed by a spontaneous s-n or o-n acyl shift ligates the esterified c-and n-exteins by a native stable peptide bond [96, 97] . the process of pts is similar to native chemical ligation (ncl), which is well described elsewhere [98] . similar to (c)fae, mild reducing conditions for the activation of certain inteins have been proven to minimize the potential risk of interchain disulfide bond breaks but retain native lc pairing and the desired functionalities of resulting bispecific antibodies. non-antibody cysteine residues within extein sequences are essential for certain inteins and could lead to disulfide scrambling or the generation of trisulfide bonds [99] . of note, some split inteins rely on serine or threonine for catalytical activity, hence do not require cysteines and are engineered to undergo pts triggered by a shift in ph, salinity conditions, light or temperature, making them more applicable for a variety of biological applications [92, 100] . recently, the engineered cysteine-free version of the split intein aes polb1 intein was developed by bhagawati and coworkers, with fast kinetics and not depending on a reducing environment, enabling the screening of antibodies exhibiting disulfide bonds sensitive to reduction [101] . in addition, and similar to spytag/spycatcher, most inteins require the incorporation of a one to six extein amino acid footprint that may cause altered flexibility and geometry. to overcome this limitation, serine-or threonine-based inteins [100] could be applied for seamless bioconjugation at respective, naturally occurring sequence positions or exteins could replace amino acids within the igg hinge region or linker sequences in further formats during screening. finally, the incorporation of split inteins into a nonnative host protein generally could alter splicing kinetics resulting in non-ligated products [102, 103] , hence this needs to be individually assessed for each bispecific antibody format. han et al. first described in vitro bioconjugation of a full-length bispecific antibody via intein fusions to precursor antibody fragments within the hinge region and successfully demonstrated in vivo activity of a reconstituted cd3xher2 t-cell engager mediated by npu dnae [28] . a similar approach yielded a cd3xprlr bispecific antibody for t-cell activation and cytokine release towards prlr expressing breast cancer cells [104] . in addition to fc-based bispecifics, also non-fc, circularly connected vhh fragments (cyclobody) have been developed via siclopps (split intein circular ligation of peptides and proteins) reaction between both c-and n-termini, forming a cyclic conformation after pts [105] . these cyclobodies are, as discussed before for other applications, protected from proteolysis due to their cyclic topology, yet they retain their dual specificity. an anti-egfrxcd16 cyclobody was successfully generated to show cytotoxicity against egfr-positive cancer cells, able to bind simultaneously egfr and cd16 on the cell surface [105] . applicability of the aforementioned split intein aes polb1 intein was demonstrated by successful bioconjugation of several therapeutically relevant formats like full-length igg, fc, and vhh fusions [101] . split inteins have been used to bioconjugate toxic components to antibodies [106] , avoiding toxicity issues during antibody production, as exemplified by an anti-her2 immunotoxin conjugated via split intein derivative (m86) of the ssp dnab intein [107] . next to several split inteins applied in bioconjugation of single binders in diverse formats, we recently described the application of split inteins for automated high throughput screenings in pharmaceutical research and development [108] . similar to other presented methodologies, split intein screening could enable the comparison of complex formats with feasible low production needs and faster development times. in addition to such two component systems, and akin to the spy/snoop combinations discussed earlier, orthogonally-acting split intein pairs could enable three to multi component screens [109] , e.g., for trispecific antibodies. the group of pinto et al. [110] presented a mutually orthogonal split intein library for in vivo applications and further split intein pairs to be used for the in vitro seamless assembly of large repetitive proteins with biotechnological or pharmaceutical relevance. the past years have seen the unparalleled development of monospecific antibodies with natural moas as well as a broad range of engineered bispecifics to multi-functional biologics for therapeutic intervention in cancer or immunological diseases. the concept of bispecific antibodies for effector cell recruitment dates back to the 1980s, with reports on production [111] and effective ctl retargeting [112] and the design of a universal t cell engager [113] . however, it was only until a decade ago that catumaxomab (revomab ® ) for targeting epcam-positive tumors by fresenius biotech as well as micromet and amgen with cd19-specific blinatumomab (blincyto ® ) were the first to bring bispecific t cell engaging antibodies to clinical approval [114] . this resulted in a tremendous increase and diversification of bispecific antibody approaches for cancer and immunotherapy in research and clinical development [5, 9, 12] . many investigational bispecific antibodies still aim at retargeting t cells to kill tumor cells [115, 116] of both solid and hematological malignancies, exemplified by a bite platform review for cancer immunotherapy [117] . a similar, flourishing strategy in cancer immunotherapy involves the recruitment and activation of nk cells, as applied in bispecific or trispecific killer cell engagers (bikes or trikes), recently reviewed for approaches towards solid [118] and hematological diseases [119] . other moas aim at intervening with two different disease mediators such as cell surface receptors, soluble ligands, and other proteins [116] . diverse architectures allow various numbers and spatial relationship between different binding sites, valences, several secondary immune functions, and pharmacokinetic half-life. this plethora provides great opportunity to tailor the design of bispecific antibodies to match the desired moas and the intended clinical application to address the unmet need in several cancer indications [116] . advances have been reported in production, often by site-specific bioconjugation and self-assembly technologies, and reviewed elsewhere [120] . the sheer size of the combinatorial screening space spanning between diverse panels of targeting moieties, formats, and moas demands innovative methodologies for automated high throughput functional screenings of bispecific and even more complex formats in pharmaceutical development. as the plentiful formats and moas of the biology-driven discovery of bispecific antibodies was recently elegantly reviewed by nie et al. [121] , we sought to cover methodologies technically enabling broad bispecific screenings. until today, the successful development of highly innovative bispecific antibodies was carried out by efficient automated screening with appropriate robotics, as exemplified by the work of kitazawa et al. [18] or geuijen and co-workers [23] . in addition to the technologies described herein, classical screening will continue to yield highly differentiated biotherapeutics such as bispecific antibodies with optimized moas. complementary to this, most screening methodologies described herein share the opportunity to greatly reduce production needs and development times for complex biotherapeutics by bioconjugation on the protein level or when produced in a small scale from one cell. classical drug candidate screening is often straight forward and allows for functional interrogation in the desired final format, whereas methodologies presented herein add a layer of complexity with inherent advantages and drawbacks, discussed in the following paragraph. the choice of an appropriate method will often be based on locally established automation, expertise, and the respective desired format and associated moa to be screened. for heterodimeric fc architectures, (c)fae, tethered variable-cl, or common lc bispecifics avoid light chain mispairing issues and represent viable options with minimal engineering but robust conversion, enabling high throughput screenings in early stages even in crude supernatant. similarly, pts can be performed both in vitro and in vivo, hence enabling bioconjugation without preceding purification within the production cell or in the supernatant [120, 122] . this represents an advantage over enzymatic ligation for early bispecifics hts. enzyme bioconjugation, as exemplified by mtgase and srta, can be applied for non-fc formats, leaving minimal peptide footprints, but requires enzyme addition and, potentially, purification. unlike duobodies, the spy technology and split inteins are not restricted to the full-length igg antibody format and are applicable to pharmaceutical development high throughput screening of multiple formats by fusing binding moieties to the termini of a fc portion, or to each other and also overcome light chain pairing issues during screening. although the spytag/spycatcher system has been harnessed for versatile applications in the past years, the significant imprint of one additional protein domain in the resulting bispecific product might hinder its application for the screening of formats strongly relying on the respective paratope distance to elicit the desired moa, e.g., effector cell recruitment. split inteins generate a comparatively low scar of 3 to 6 amino acids remaining that, e.g., in linker sequences, can be engineered to mimic the envisioned format, representing a viable option for close-to-format or, under certain conditions, in-format screening. both the combination of spy/snoop technology and orthogonal split inteins offer the option to screen even larger combinatorial spaces of tri-to multivalent biologics, such as biparatopic effector cell engagers, bispecific, adcs, or trispecific antibodies. in addition to antibody-derived bispecifics, the plethora of alternative scaffolds fused to relevant antibody portions might further enlarge the combinatorial space that could be covered by the discussed screening methodologies, as recently exemplified for pts-generated, lectin-based antibody fusions termed "lectibodies" [123] . limitations and advantages apply similarly to the above discussed two component approaches. table 1 gives an overview of the properties, advantages, and shortcomings of the respective methodologies. the future will tell whether a broader coverage of these large screening spaces by application of reviewed or similar technologies will enable the identification of best-in-class innovative biotherapeutics. although this review discussed mainly the screening of igg-based therapeutic bispecific antibodies, as well, isotype-switched iga, igd, ige, and igm antibodies play important roles in the healthy and diseased immune system, with distinct differences in the architecture, tissue distribution, receptor binding, and effector function properties. respective isotypes and their application for bispecific antibodies were recently comprehensively reviewed [124] and might bear great potential for future bispecific antibody therapies. in summary, the reviewed methodologies offer great tools for broad functional screening campaigns of diverse bi-to multi-specific antibodies and formats, with versatile moas in early drug discovery. these methods could greatly shorten development times and enhance the probability of identifying optimal combinations, ultimately leading to the generation of better biotherapeutics. table 1 . comparison between different bioconjugation technologies for posttranslational antibody modification. the technologies presented here are able to reconstitute antibody fragments in vitro on the protein level. tethered variable cl as well as common lc bsabs are generated on a molecular biology basis and not recombined on the protein level. pathogen recognition and innate immunity role of toll-like receptors in pathogen recognition boosting therapeutic potency of antibodies by taming fc domain functions the ligands for human igg and their effector functions. antibodies 2019, 8, 30 antibodies to watch in 2020 development of therapeutic antibodies for the treatment of diseases engineering igg-like bispecific antibodies-an overview simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin bispecific antibodies: a mechanistic review of the pipeline bispecific antibodies: design, therapy, perspectives. drug des a review of bispecific antibodies and antibody constructs in oncology and clinical challenges the making of bispecific antibodies therapeutic antibody engineering by high efficiency cell screening phage display-derived human antibodies in clinical development and therapy designing human antibodies by phage display yeast surface display for screening combinatorial polypeptide libraries single b cell cloning and production of rabbit monoclonal antibodies a bispecific antibody to factors ixa and x restores factor viii hemostatic activity in a hemophilia a model ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design trifabs-trivalent igg-shaped bispecific antibody derivatives: design, generation, characterization and application for targeted payload delivery the effect of variable domain orientation and arrangement on the antigen-binding activity of a recombinant human bispecific diabody efficient generation of stable bispecific igg1 by controlled fab-arm exchange unbiased combinatorial screening identifies a bispecific igg1 that potently inhibits her3 signaling via her2-guided ligand blockade transglutaminasecatalyzed covalent multimerization of camelidae anti-human tnf single domain antibodies improves neutralizing activity high-throughput generation of bispecific binding proteins by sortase a-mediated coupling for direct functional screening in cell culture post-translational assembly of protein parts into complex devices by using spytag/spycatcher protein ligase catching a spy: using the spycatcher-spytag and related systems for labeling and localizing bacterial proteins efficient generation of bispecific igg antibodies by split intein mediated protein trans-splicing system anti-inflammatory activity of human igg4 antibodies by dynamic fab arm exchange knobs-into-holes' engineering of antibody ch3 domains for heavy chain heterodimerization immunoglobulin domain crossover as a generic approach for the production of bispecific igg antibodies engineering bispecific antibodies with defined chain pairing how the bispecific antibody targeting egfr and cmet has superior preclinical activity and potentially better safety profile fab-arm exchange combined with selective protein a purification results in a platform for rapid preparation of monovalent bispecific antibodies directly from culture media duohexabody-cd37 ® , a novel biparatopic cd37 antibody with enhanced fc-mediated hexamerization as a potential therapy for b-cell malignancies tethered-variable cl bispecific igg: an antibody platform for rapid bispecific antibody screening the incorporation of amines into proteins purification and characteristics of a novel transglutaminase derived from microorganisms transglutaminases: nature's biological glues comparison of substrate specificities of transglutaminases using synthetic peptides as acyl donors site-specific and stoichiometric modification of antibodies by bacterial transglutaminase microbial transglutaminase for biotechnological and biomedical engineering location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates locked by design: a conformationally constrained transglutaminase tag enables efficient site-specific conjugation enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase site-specific cross-linking of functional proteins by transglutamination staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall kinetic mechanism of staphylococcus aureaus sortase srt a a general strategy for the evolution of bond-forming enzymes using yeast display ca2+-independent sortase-a exhibits high selective protein ligation activity in the cytoplasm of escherichia coli recent advances in sortase-catalyzed ligation methodology sortase-mediated ligations for the site-specific modification of proteins sortase-mediated protein ligation: a new method for protein engineering sortagging: a versatile method for protein labeling protein-protein fusion catalyzed by sortase a sortase-catalyzed transformations that improve the properties of cytokines engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by sortase a-mediated protein ligation preparation of bispecific antibody-protein adducts by site-specific chemoenzymatic conjugation bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity pili in gram-positive pathogens spontaneous intermolecular amide bond formation between side chains for irreversible peptide targeting peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin superglue from bacteria: unbreakable bridges for protein nanotechnology synthetic biology: a new tool for the trade evolving accelerated amidation by spytag/spycatcher to analyze membrane dynamics spytag/spycatcher cyclization enhances the thermostability of firefly luciferase enhanced stability of a rumen-derived xylanase using spytag/spycatcher cyclization use of spytag/spycatcher to construct bispecific antibodies that target two epitopes of a single antigen a novel synthetic trivalent single chain variable fragment (tri-scfv) construction platform based on the spytag/spycatcher protein ligase system enzyme-based labeling strategies for antibody-drug conjugates and antibody mimetics generation of biparatopic antibody through two-step targeting of fragment antibodies on antigen using spytag and spycatcher improving the malaria transmission-blocking activity of a plasmodium falciparum 48/45 based vaccine antigen by spytag/spycatcher mediated virus-like display surface display of classical swine fever virus e2 glycoprotein on gram-positive enhancer matrix (gem) particles via the spytag/spycatcher system particulate multivalent presentation of the receptor binding domain induces protective immune responses against mers-cov a novel rapid modularized hepatitis b core virus-like particle-based platform for personalized cancer vaccine preparation via fixed-point coupling a self-adjuvanted, modular, antigenic vlp for rapid response to influenza virus variability spy chemistry-enabled protein directional immobilization and protein purification spy & go purification of spytag-proteins using pseudo-spycatcher to access an oligomerization toolbox integrating spycatcher/spytag covalent fusion technology into phage display workflows for rapid antibody discovery site-specific fluorescent labeling of antibodies and diabodies using spytag/spycatcher system for in vivo optical imaging genetically encoded spy peptide fusion system to detect plasma membrane-localized proteins in vivo crispr/cas-directed programmable assembly of multi-enzyme complexes programmable polyproteams built using twin peptide superglues snoopligase peptide-peptide conjugation enables modular vaccine assembly epitope distance to the target cell membrane and antigen size determine the potency of t cell-mediated lysis by bite antibodies specific for a large melanoma surface antigen format and geometries matter: structure-based design defines the functionality of bispecific antibodies in-format' screening of a novel bispecific antibody format reveals significant potency improvements relative to unformatted molecules structural analysis and optimization of the covalent association between spycatcher and a peptide tag inteins: structure, function, and evolution protein trans-splicing as a protein ligation tool to study protein structure and function protein trans-splicing by a split intein encoded in a split dnae gene of synechocystis sp unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources highly efficient protein trans-splicing by a naturally split dnae intein from nostoc punctiforme the naturally split npu dnae intein exhibits an extraordinarily high rate in the protein trans-splicing reaction inteins-a focus on the biotechnological applications of splicing-promoting proteins protein splicing mechanisms and applications chemical science a functional interplay between intein and extein sequences in protein splicing compensates for the essential block b histidine native chemical ligation and extended methods: mechanisms, catalysis, scope, and limitations disulfide bond structures of igg molecules: structural variations, chemical modifications and possible impacts to stability and biological function salt-inducible protein splicing in cis and trans by inteins from extremely halophilic archaea as a novel protein-engineering tool mesophilic cysteine-less split intein for protein trans-splicing applications under oxidizing conditions recent advances in in vivo applications of intein-mediated protein splicing intermolecular disulfide bonds between unpaired cysteines retard the c-terminal trans-cleavage of npu dnae a novel bispecific antibody targeting cd3 and prolactin receptor (prlr) against prlr-expression breast cancer construction of a circularly connected vhh bispecific antibody (cyclobody) for the desirable positioning of antigen-binding sites site-specific modification of ed-b-targeting antibody using intein-fusion technology generation of potent anti-her1/2 immunotoxins by protein ligation using split inteins intein mediated high throughput screening for bispecific antibodies segmental isotopic labeling of a central domain in a multidomain protein by protein trans-splicing using only one robust dnae intein an expanded library of orthogonal split inteins enables modular multi-peptide assemblies bispecific monoclonal antibodies from hybrid hybridomas human ovarian carcinoma lysis by cytotoxic t cells targeted by bispecific monoclonal antibodies: analysis of the antibody components universal bispecific antibody for targeting tumor cells for destruction by cytotoxic t cells bispecific antibodies for cancer therapy: a review t cell-redirecting bispecific antibodies in cancer immunotherapy: recent advances alternative molecular formats and therapeutic applications for bispecific antibodies utilizing the bite (bispecific t-cell engager) platform for immunotherapy of cancer monoclonal antibody therapy of solid tumors: clinical limitations and novel strategies to enhance treatment efficacy bispecific antibodies in the treatment of hematologic malignancies site-specific bioconjugation and self-assembly technologies for multi-functional biologics: on the road to the clinic biology drives the discovery of bispecific antibodies as innovative therapeutics intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for nmr studies an off-the-shelf approach for the production of fc fusion proteins by protein trans-splicing towards generating a lectibody in vitro sagacity in antibody humanization for therapeutics, diagnostics and research purposes: considerations of antibody elements and their roles key: cord-286219-qcx5ehnh authors: calistri, arianna; munegato, denis; carli, ilaria; parolin, cristina; palù, giorgio title: the ubiquitin-conjugating system: multiple roles in viral replication and infection date: 2014-05-06 journal: cells doi: 10.3390/cells3020386 sha: doc_id: 286219 cord_uid: qcx5ehnh through the combined action of ubiquitinating and deubiquitinating enzymes, conjugation of ubiquitin to a target protein acts as a reversible post-translational modification functionally similar to phosphorylation. indeed, ubiquitination is more and more recognized as a central process for the fine regulation of many cellular pathways. due to their nature as obligate intracellular parasites, viruses rely on the most conserved host cell machineries for their own replication. thus, it is not surprising that members from almost every viral family are challenged by ubiquitin mediated mechanisms in different steps of their life cycle and have evolved in order to by-pass or exploit the cellular ubiquitin conjugating system to maximize their chance to establish a successful infection. in this review we will present several examples of the complex interplay that links viruses and the ubiquitin conjugation machinery, with a special focus on the mechanisms evolved by the human immunodeficiency virus to escape from cellular restriction factors and to exit from infected cells. ubiquitin (ub) is a highly conserved protein of 76 aminoacids that can be covalently linked to target proteins through a multistep process known as ubiquitination. protein ubiquitination represents open access one of the best characterized post-translational modifications that controls the fate/function of proteins [1] . protein ubiquitination involves a series of cellular enzymes in an enzymatic cascade, starting with the ub-activating enzyme e1, followed by the ubiquitin-conjugating enzyme e2 and by the ub ligase e3, which form an isopeptide bond between the carboxyl terminus of ub and the ε-amino group of a lysine residue on the target protein [2] . the e3 ligase usually determines the substrate specificity, although the e2-conjugating enzyme can also play a role in the substrate selection. accordingly, while there are few known e1 enzymes in mammals and roughly thirty five e2s in humans, hundreds of e3/ub ligases have been identified so far. e3 enzymes are currently classified into three main classes with different structural and functional characteristics: the hect domain family of ub ligases, the cullin-ring family of ub ligases, and the u-box containing ub ligases [3] [4] [5] . the final outcome of the first round of the ubiquitination cascade is the mono-ubiquitination of the target protein. after mono-ubiquitination, a specific lysine of the first ub can be used by the same set of proteins to mediate the consecutive attachment of additional ubs, resulting in the formation of poly-ub chains ( figure 1 ). in the most studied event, the ubiquitinated misfolded or damaged cytoplasmic and nuclear proteins are delivered to the proteasome for degradation as final event of the well-known ub-proteasome system (ups) [6] . on the other hand, ubiquitination of the cytoplasmic domains of transmembrane proteins results in their sorting to lysosomes via the multivesicular body (mvb) pathway [7] . ub-mediated degradation is important not only for the regulation of protein turn-over, but it also plays a role in dna damage repair, cell-cycle regulation, cellular growth, as well as in the immune system functions [8] . moreover, it has been demonstrated that ub can also function act independently from its proteolytic activity, by regulating protein function and protein/protein interaction [8, 9] . an important role in this context is played by specific ub hydrolases (deubiquitinating enzymes or dubs) that catalyze the removal of ub from the target proteins. similar to the function of kinases and phosphatases during the phosphorylation process, ub ligases and dubs can affect substrate function by transient ubiquitination. thus, protein ubiquitination represents a highly versatile and reversible event that may influence different features of a protein and not only its stability. part of this versatility is clearly linked to the fact that ub contains at least seven lysines (k) and additional residues that can be employed by the ub ligases to generate different types of ub chains on the target proteins, which, in turn, will interact with different downstream factors ( figure 1 ) [1] . for instance, it is well established that k-48-based linkages lead mainly to the proteasome-mediated degradation of the ubiquitinated protein, while k-63-based ub chains control primarily protein endocytosis, as well as trafficking and enzyme activity ( figure 1 ) [10, 11] . in addition to ub, a number of ub-like (ubl) proteins can also be conjugated to target substrates by specific e1, e2, and e3 enzyme-like proteins. among them are sumo (small ub modifier), isg15 (interferon-stimulated gene 15), nedd8 (neural precursor cell expressed, developmentally downregulated 8), fat10 (hla-f adjacent transcript 10), atg12 (autophagy-related protein 12) and lc3 (microtubule-associated protein 1 light chain 3) which display different functions and roles in the cellular physiology [12] . schematic representation of the ub molecule and of the enzymatic cascade leading to protein ubiquitination. the seven lysines (k) involved in the process, the ubiquitin-activating enzyme e1, the ubiquitin-conjugating enzyme e2 and the ubiquitin ligase enzyme e3 are highlighted, along with the main fate of the target proteins. as obligate intracellular parasites with limited genome size, viruses must co-opt the host cellular machineries in almost every step of their life cycle, from entry into the host cell, replication and transcription of their genome (either rna or dna), synthesis of proteins, assembly of new particles, till the egress from the infected cell. in addition, in order to establish an infection, viruses must overcome different host immune defenses. thus, there is a constant interaction between the virus and its host, with the virus trying to either counteract or exploit different complex cellular mechanisms and pathways to efficiently produce infectious progenies, or to establish a long-term persistence in the host, depending on the virus taken into consideration. under this respect, given the role played in many cellular fundamental processes, it is to be expected that ub and ubl proteins are involved in almost every aspect of the viral life cycle and pathogenesis [13] . in this review, we will present different examples of how viruses interact with ub/ubl-mediated cellular processes in order to optimize their chance of survival, with a special focus on the mechanisms evolved, in this context, by one of the most important human pathogens, the human immunodeficiency virus type 1 (hiv-1). the first evidence supporting the ability of viruses to utilize the ups to their own advantage came from the study of small dna tumor viruses and of their capability to interfere with the regulation of the cell cycle [14] . since then, it has become clear that members of almost all viral families subvert or exploit both the cellular ub-conjugating and -deconjugating machineries in different phases of their replication cycle [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] . under this respect, studies based on the treatment of infected cells with proteasome inhibitors have been instrumental, as such a treatment not only blocks the ups, but also depletes the cellular pool of free ub, affecting de facto all the cellular pathways involving this protein. proteasome inhibitors have been shown to interfere with the replication of major human pathogens such as herpesviruses [15, 16] , poxviruses [17, 18] , hepadnaviruses [19] , adenoviruses [20] , influenzaviruses [21] , retroviruses [22] [23] [24] , coronaviruses [25] , paramyxoviruses [26] , picornaviruses [27] and rotaviruses [28] . ub already plays a role in the first steps of viral replication. as an example, proteasome inhibitors have been reported to inhibit herpes simplex virus (hsv) entry at an early step, immediately after the penetration of the viral capsid into the target cell [15, 29] . the kaposi sarcoma associated herpesvirus (kshv) [30] , the influenza virus [31] and adenoviruses [32] are additional examples of viruses which rely upon the ups for their entry into target cells. for instance, it has been demonstrated that the ups is linked to the ability of the human pathogen kshv to penetrate into endothelial cells and to traffic to the nuclei. not only entry and early post-entry events, but also other steps of the viral life cycle can be affected by impairment of the proteasome activity. indeed, ub-mediated mechanisms regulate gene expression in epstein-barr virus (ebv) [33] , hiv-1 [34] , and human t lymphotropic virus (htlv) [35] . in all these cases, specific viral proteins which function as transcriptional transactivators are able to interact with the ub/ubl-conjugating machinery, leading to an increase in viral protein activity. ups is also involved in the ability of herpesviruses to establish lifelong infections in their hosts, a phenomenon known as latency, as reported for kshv [36, 37] and ebv [38, 39] . specifically, in the case of ebv, the viral latent membrane protein 2a (lamp2a) [38] and lmp1 [39] regulate viral lytic/latent replication through the interaction with specific ubligases and dubs. finally, ub plays a role in viral release from infected cells [40] , as it will be discussed in more details later on. innate immunity represents a first line of defense employed by the cells against microorganisms. microorganisms are recognized by specific molecules named pattern recognition receptors (prr) that bind to pathogen-associated molecular patterns (pamps). this binding leads to the activation of different signaling cascades, with the involvement of the pro-inflammatory transcription factors ap-1, nf-kb and/or one or more members of the interferon-regulatory factor (irf) family, with the final production of pro-inflammatory cytokines and interferon (ifn). not only regulation of innate immune signaling relies on post-translational modifications such as conjugation of ub/ubls to keys cellular proteins [41] , but the ub/ubl conjugating system itself is adopted by many viruses to counteract this host defense response [42] . first of all, viruses prevent the induction of nf-kb and/or ifn. to this end, the use of the cellular ub conjugating machinery represents one of the most exploited strategy. nf-kb is a complex of dimeric transcription factors, which in mammals comprises rela (p65), relb, c-rel, nf-kb1 (p50) and nf-kb2 (p52) [43] . in normal conditions, nf-kb dimers are bound to nf-kb inhibitory proteins (ikbs) and retained in the cytoplasm. upon activativation, a multiprotein complex constituted by the enzyme transforming growth factor beta activated kinase-1 (tak1), the nf-kb essential modifier (nemo), and the ikb kinase (ikk) is formed. this complex phosphorylates the nfkb inhibitor (ikb), leading to its ubiquitination and degradation by the proteasome. once released by ikb, nf-kb can translocate into the nucleus where, along with specific irfs and other co-factors, is able to stimulate ifn transcription. among other mechanisms, it has been described that viruses can directly influence nf-kb stability. for instance, the case of the murid herpesvirus-4 (muhv-4) latency associated protein orf73 leads to p65/rela degradation. [44] . viruses can also use ups to impair nf-kb translocation to the nucleus by blocking ikb degradation. for instance, the rotavirus nsp1 protein mediates the ubiquitination and degradation of the β-transducin repeat containing protein (β-trcp) that, otherwise, would bind to and degrade ikb [45] . viruses can also affect the stability of irfs and in particular of irf3. one example is given by the varicella zoster virus (vzv) orf61, a protein displaying a ring finger e3 ubiquitin ligase activity. orf61 specifically interacts with the phosphorylated/activated form of irf3 leading to its ubiquitination and proteasome mediated degradation [46] . in addition, viruses can act downstream the ifn induction, by inhibiting the signal cascade activated by ifn binding to its receptor [47] . as an example of ups-mediated viral interference with proteins belonging to this cascade, viruses in the rubulavirus genus of the paramyxoviridae family lead stat proteins to proteasome-mediated degradation, by assembling stat-specific ubiquitin ligase complexes from cellular components [48] . finally, viruses can directly target the antiviral ifn-induced proteins (isgs) [49] . some of these proteins, such as the protein kinase r (pkr), the 2',5'-oligoadenylate-synthetase (oas), the ribonuclease l(rnasel), and the mx gtp-ase, along with the respective viral counteracting mechanisms have been extensively studied [49] [50] [51] . recently, different reports have been focused on the isgs belonging to the tripartite motif (trim) containing family, especially after the discovery of the role played by trim5α in the establishment of a cross-species barrier against hiv-1 infection [52] . interestingly, trim5α and many other members of this family of isgs function by ubiquitinating, sumoylating or isgylating host/viral proteins with different outcomes on the antiviral response [53, 54] . not only, the isg15 ub-like protein is itself an isg, and one of the most potently induced upon viral infection. it has been demonstrated that isg15 displays a broad antiviral activity [55] , even though the mechanism/s accounting for this effect is/are still under investigation. what is known is that isgylation can specifically inhibit the functions of an ub ligase, nedd4, which plays a role in the replication cycle of different rna viruses, such as ebola virus and oncoretroviruses [56] . moreover, it has been reported that viruses have evolved the ability to remove isg15 from target proteins. for instance, the coronavirus papain-like protease (plp) protein acts as a de-ubiquitinating and de-isgylating enzyme [57] . another interesting isg is represented by a peculiar cellular protein, tetherin, that being one of the most recently characterized targets of the hiv-1 accessory protein vpu, will be described in more details later. this rapid overview on the involvement of ups in crucial steps of the viral life cycle suggests immediately that viruses are connected to ub in different ways, either by usurping the host's ub-conjugating system or by evolving their own one. first of all, viral proteins have been described that can modify the substrate specificity of cellular ub ligases. as a consequence, specific cellular proteins are targeted for degradation. such a strategy, is exploited by viruses in several of the examples described in the previous paragraphs. for instance, the muhv-4 orf73 represents one of the viral proteins that can subvert the substrate recognition of a cellular e3 enzyme. indeed, by binding to orf73, the elonginc/cullin5/socs ub-ligase complex is able to poly-ubiquitinate p65/rela with its subsequent proteasomal degradation [44] . the rotavirus nsp1 protein leads to β-trcp ubiquitination and degradation through the recruitment of the ubiquitin-ligase complex skp-1/cul1/f-box (scf) [45] . furthermore, small dna viruses with known oncogenic activity, such as the human papillomavirus (hpv), adenoviruses and polyomaviruses, take control of the cell cycle by usurping specific cellular ub ligase complexes to target crucial cell cycle regulators such as p53 and the protein of the retinoblastoma (prb) for degradation [58] . in this way, two of the best studied tumor suppressor cellular pathways are inactivated. indeed, different types of hpv (high-risk) are strongly linked to the onset of cervical cancers, as well as to other type of cancer such as those affecting vagina, vulva, penis, and the head and neck [59] , while many others, classified as low-risk, are only very rarely associated with cancer. although the high-risk and low-risk hpvs share several biological features, the two groups display significant structural/functional differences at the level of the two main viral encoded oncogenes: e6 and e7. indeed, high-and low-risk e6 and e7 proteins have a different ability in affecting p53/prb stability and in modulating the activity of additional cellular proteins with an effect on cell cycle control and cellular proliferation [59] . interestingly, the studies of viral interactions with the host cell cycle have been instrumental in the identification and characterization of p53 and prb [60] . in addition to p53 and prb, another protein complex involved in cell cycle control, the anaphase-promoting complex (apc) is emerging as a key target for viral proteins. apc is a cullin-ring e3 ub ligase that leads to the proteasome degradation of multiple cell cycle regulators and, as a consequence, to the correct progression of the cell cycle itself [61] . different viruses have been reported to affect the apc function. in particular, the human cytomegalovirus (hcmv), a known human pathogen, encodes a protein, pul21a, that is able to induce proteasome-dependent degradation of apc subunits during viral infection [62] . additional viruses, and among them important human pathogens with known oncogenic activity, such as the htlv-1, hpv, hepatitis b virus (hbv) have been reported to interfere with apc [61] . viral proteins themselves can be directly modified by ub or ub-like proteins and, as a consequence, they can be recognized by cellular pathways that can be then exploited by the virus to perform specific tasks. examples of these processes are found in the mechanisms evolved by several enveloped viruses to egress from infected cells, as it will be explained later. viruses can also alter the activity of cellular de-ubiquitinating enzymes, by encoding, for instance, proteins which are able to interact with cellular dubs. under this respect, the ebv ebna1 protein, which is involved in several crucial aspects of viral replication and pathogenesis such as maintenance of the viral genome, transcription and translation of the viral dna, viral persistence, and cellular transformation, interacts with usp7 or herpesvirus-associated ub-specific protease (hausp) [63] [64] [65] . this cellular dub is able to remove ub from p53 and from ebna1 itself, thus preventing their degradation [66] . during ebv infection, not only usp7 but several additional cellular dubs are known to increase their activity and one of the effects appears to be the stabilization of β-catenin, a key factor of the wnt signaling pathway. the disregulation of this signaling pathway has been implicated in tumor development [67] . since ebv is associated, as well, with different types of cancers, such as burkitt's and hodgkin's and the nasopharyngeal carcinoma, the study of ebv interference with the wnt pathway and the role played by the cellular dubs in this context are relevant. some viruses, especially the large dna viruses such as herpeviruses and poxviruses, encode their own ubiquitinating enzymes (table 1) . vzv orf61 mentioned above is an example of such viral encoded ub ligases. additional examples are found in the processes evolved by different viruses to overcome the host immune defenses. under this respect, kshv encodes two e3 ub ligases, k3 and k5, able to ubiquitinate the class i major histocompatibility complex (mhc), leading to its downregulation from the cell surface or from the er and thus interfering with cell antigen presentation [68] . k5 also affects other surface proteins important for the stimulation of t cells, such as icam-1 and b7-2 [69] . interestingly, several other members of the herpeviridae family have been described to down-regulate mhci and t-cell activation markers from the cell surface by employing different mechanisms [70] , some relying again on ub, as in the case of hcmv [71] . another interesting example is given by the hsv-1 icp0 protein, a multifunctional factor which displays, among other features, a ring domain e3 ub ligase activity [72] . thanks to this activity, icp0 is able to disassemble cellular protein aggregates, known as promyelocytic leukemia nuclear bodies (pml nbs), that are present in the nucleus of infected cells where they are involved in different processes such as the interferon (ifn) response to viral infection [73] . both herpesviruses and adenoviruses have been described to interfere with pml nbs [73] and ups is at the basis of some of the mechanisms employed by these viruses to this end. hsv-1 icp0, in particular, mediates the proteasomal degradation of one of the protein complexes that constitute these bodies [74] . moreover, viral dubs have been described (table 2 ). among them, the large tegument protein of herpesviruses belonging to all the three known families of these pathogens (α, β and γ) not only is an essential component of the viral particle, but displays a conserved and unique deubiquitinating activity [96] . taking into account the functions played by the large tegument proteins in the herpesviral replication cycle, a role for these viral dubs during both the entry and the egress of the virus from infected cells is very likely. for instance, the ebv dub, bplf1, is known to deubiquitinate and downregulate the viral ribonucleotide reductase [97] , the cellular processivity factor pcna [98] and the e3 ubiquitin ligase rad18 with a positive effect on the production of infectious particles [99] . it is interesting to note that the herpes simplex virus 1 ub-specific protease, ul36, has been recently reported to inhibit β-interferon production by deubiquitinating the tnf receptor associated factor 3 (traf3) [100] . this finding would indicate a role for viral dubs in overcoming the cellular immune response, as in the case of several viral ub ligases. the impact played by the ub/ubl system on viral replication and on the establishment of a successful infection is particularly clear when the life cycle and the pathogenetic mechanisms evolved by one of the most studied human pathogens, the hiv-1, is analyzed. hiv-1 is a lentivirus and, like the other members of the retroviridae family, is characterized by a non-icosahedral particle enwrapped in a lipidic envelope. its genome comprises two copies of single-stranded rna with a short dimerized region. hiv-1 is the etiological agent of the acquired immunodeficiency syndrome, aids, a condition that after 30 years from its discovery and the introduction of the highly active antiretroviral therapy (haart), still represents one of the major public health problem world-wide [112] . the hiv-1 life cycle is a typical retroviral replication cycle starting with viral entry into specific target cells, reverse transcription of the viral rna into double stranded proviral dna, integration of this proviral genome into the host chromosomal dna, transcription and translation of viral proteins, assembly of viral particles, followed by their budding from the cell surface with the acquisition of an envelope. all retroviral genomes consist of at least 3 genes, gag, pol and env. in addition to these three main genes, complex retroviruses such as hiv-1 encode accessory proteins that enhance their replication and infectivity ( figure 2 ). in particular, hiv-1 is characterized by six auxiliary genes (tat, rev, nef, vpr, vpu and vif), of which only two, tat and rev, are essential for viral replication in vivo and in vitro [113] . on the other hand, nef, vpu, vif, vpr genes encode factors that are known as accessory proteins, as they appear to be dispensable for viral replication in several in vitro experimental settings. however, the high degree of conservation of these proteins suggest crucial functions in vivo. the relevance of hiv-1 as human pathogen and the consequent development and availability of different tools and techniques to manipulate its genome, has allowed to deeply dissect several aspects of hiv biology. even though different questions still need to be answered, the reliance of hiv-1 on numerous cellular pathways and factors for nearly every step of its replication is well appreciated [114] [115] [116] . moreover, it is becoming clear that hiv-1 proteins, and especially the accessory proteins, have the ability to antagonize host molecules that represent first lines of defense against retroviral infections. these cellular proteins are known as intrinsic immunity factors or restriction factors [117] . recent studies have highlighted how the hiv-1 accessory proteins nef, vif, vpu, and vpr have evolved in order to enable the virus to evade the host immune system [118] . interestingly, a common mechanism of action of these proteins is the use of the ups to interfere with cellular proteins that would affect hiv-1 replication. in particular, vif, vpu, and vpr exploit and subvert the physiological activity of specific cullin-ring finger ub ligases (crls) to induce the polyubiquitination and proteasomal degradation of specific cellular targets [119, 120] (table 3) . crls are the largest family of ub ligases and are responsible for ubiquitination of almost 20% of cellular proteins degraded through the ups. the choice as target of members of this particular family of e3 enzymes operated by these hiv-1 proteins is not accidental. indeed, crls are multisubunit complexes composed of a cullin (at least seven cullins are known in vertebrates), an rbx/roc ring finger protein, a variable substrate-recognition subunit (srs), and in most cases, an adaptor that links the srs to the complex [4, 121, 122] . thus, crls are extremely versatile comprising hundreds of distinct complexes with the potential to recruit several protein. this feature has been exploited by vif, vpu and vpr to optimize the chances of hiv-1 to overcome different restriction factors evolved by the cells to inhibit viral replication and spreading. vif is a 23kda protein that is required for production of infectious virus in a cell type-specific manner [134] . experimental evidence indicated that cells non permissive to vif-defective hiv-1 express a host factor inhibiting viral replication [135] , next identified in the cytidine deaminase apobec3g [136] . apobec3g is clearly expressed in vif-defective hiv-1 nonpermissive cells where it acts as an intrinsic restriction factor. in the absence of vif, apobec3g is incorporated into budding virions, and, in the newly infected cells, it determines cytidine to uracil mutations in the single stranded dna of hiv-1, during the process of reverse transcription. this hypermutation results in viral replication impairment. when expressed, vif recruits a multi-subunit e3 ub ligase complex composed of a scaffold protein, cullin 5, ring-box protein, a socs box binding protein complex, elongins b/c, as well as the core binding factor beta (cbf-β) [137] . vif directly binds cullin 5 [137] . the vif-cbf-β-elonginb-elonginc-cullin5-rbx e3 complex is then able to polyubiquitinate apobec3g leading to its protesomal degradation [138] . as a consequence, apobec3g is not incorporated into viral particle and hiv-1 can replicate in de novo infected cells. it has to be mentioned that additional apobec molecules (i.e. apobec3de/3f) have been characterized for their ability to restrict vif-defective hiv-1. in vif expressing cells, all these restriction factors are substrates of the same e3 complex described in the case of abobec3g and are as well subjected to proteasomal degradation [139] . interestingly the crystal structure of the vif/crl complex has been recently resolved [123] . this finding will help to further clarify the molecular basis of vif function. vpu is an 81 amino acid dimeric integral membrane protein. one of the first characterized functions of this hiv-1 accessory protein was the ability of recruiting a crl (skp1-cullin1 e3 ligase complex) to the cytoplasmic tail of cd4, inducing its downregulation at the level of the er [124] . in particular, vpu acts as an adaptor protein, directly interacting with cd4 in the er and with β-trcp, a component of the skp1-cullin-f box (scf) ubiquitin ligase complex, leading to the cd4 polyubiquitination, dislocation from the er to the cytosol and proteasomal degradation [124] . in addition to this important function, which is likely required for proper trafficking and maturation of the viral envelope glycoproteins, vpu has been more recently characterized for yet another crucial role, connected with the ability of the virus to evade a specific ifn-1 induced antiviral factor: the b cell stromal factor 2 (bst-2) or tetherin. the latter name perfectly reflects the activity of bst-2 that, in the absence of vpu, physically retains (tethers) fully assembled hiv-1 particles to the surface of the infected cells [140] . indeed, bst-2 is a type ii transmembrane protein with an unusual topology consisting of an n-terminal cytoplasmic tail, a transmembrane domain, a coiled-coil extracellular domain, and a glycosylphosphatidylinositol anchor at the carboxy-terminus. it has been demonstrated that this peculiar conformation, rather than the primary sequence, confers to tetherin the ability to physically retain budding virions on the cellular membrane [141] . hiv-1 is not the only virus sensitive to bst-2 [142] and human cells are not the only one encoding for such a restriction factor [142] [143] [144] [145] . indeed, tetherin represents also one of the major cross-species barrier factor especially in the context of lentiviral infection [143, 144, [146] [147] [148] . there is still some uncertainty as to how vpu antagonism is accomplished, as different mechanisms have been reported. indeed, it has been described that vpu directly interacts with tetherin and can mediate its down-regulation from the cell surface with an increase in viral release [125] . while some studies have indicated a vpu mediated β-trcp-dependent proteasomal degradation of tetherin [125, 126] , there are also data supporting a role for the β-trcp-dependent endo-lysosomal pathway in bst-2 degradation [127, 149] . in agreement with these latter studies rab7a, a small gtpase essential for the maturation of late endosomes and lysosomal fusion, appears to be required for tetherin degradation [150] , as along with the endosomal sorting complex required for transport (escrt)-0 [151] . interestingly, β-trcp2-dependent ubiquitination and subsequent degradation of tetherin do not seem to require lysine residues in the cytoplasmic domain of tetherin [152] . it has to be mentioned that other studies have revealed a β-trcp2-independent mechanism of tetherin antagonism by vpu, with the delocalization and retention of tetherin in a perinuclear compartment in the absence of degradation [153, 154] . even though degradation does not appear to be the primary system by which vpu counteracts bst-2, sequestration and degradation might be parallel existing and not mutually exclusive mechanisms by which vpu optimizes the chances to counteract the antiviral effect of tetherin. in agreement with the concept of multiple mechanisms, other viral proteins are able to counteract tetherin function, both by inducing its ub-dependent endosomal degradation, as in the case of the khsv k5 protein [82] , or by sequestration in a perinuclear compartment, as in the case of the hiv-2 env [155] . vpr is a 96 amino acid protein whose function has been difficult to elucidate. one of the clearest activities of vpr is its ability to delay or arrest cells in the g2 phase of the cell cycle. in addition, in this case, the recruitment of a specific crl appears to be essential. indeed, vpr interacts with the cullin4a-ddb1-dcaf1 ub ligase complex [130, [156] [157] [158] [159] [160] [161] [162] , a binding that is required for the induction of the g2 arrest [163] . however, the exact target/s of vpr-cullin4a-ddb1-dcaf1 activity that are linked to the g2 cell-cycle arrest is/are still unknown [164] . in particular, the two substrates of vpr-mediated degradation, that have been better characterized so far, the uracil-dna glycosylase (ung) 2 and the single-strand selective monofunctional uracil-dna glycosylase (smug1) [131] do not seem to account for the above described vpr effect. recently, it has been demonstrated that also the endoribonuclease dicer is subjected to proteosomal degradation via vpr-recruited cullin4a-ddb1-dcaf1 ub ligase, with enhanced hiv-2 replication in monocyte-derived macrophages [132] . it is well known that dicer is involved in the generation of mirna, a major component of the rna silencing machinery interfering with viral replication. thus, vpr would also act as a suppressor of silencing (srs) and vpr-mediated degradation of dicer would represent another example of a cellular restriction mechanism to hiv-1 infection bypassed by a viral "usurped" crl complex. while the vpr accessory protein is encoded by all primate lentiviruses, including hiv-1 and hiv-2, its paralog vpx is expressed only by hiv-2 and by certain simian lentiviruses. myeloid cell types are known to be less permissive to hiv-1 infection with respect to cd4-positive t lymphocytes [133, 165] . this difference in susceptibility to hiv-1 infection has been linked to the expression in myeloid cell types of the vpx-interacting protein samhd1 [133] . samhd1 is a nucleotide triphosphohydrolase that can inhibit lentiviral reverse transcription by depleting the intracellular pool of available dntps [133, 166] . the hiv-2 and siv vpx proteins counteract this restriction by inducing samhd1 degradation following its ubiquitination. once again, as just described in the case of vpr, the crl involved in such a process is the vpx recruited cullin4a-ddb1-dcaf1 [118, 167] . it has been recently reported that hiv vpr and vpx exploit not only cullin4a but also cullin4b to mediate ubiquitination of target proteins. interestingly, in primary macrophages vpx appears to need both cullin4a and cullin4b to obtain maximal samhd1 degradation [168] . since hiv-1 does not have a vpx protein and hiv-1 vpr is not capable of interacting with samhd1, myeloid cell types display resistance to hiv-1 infection. thus, samhd1 represents an additional restriction factor that interferes with hiv-1 infection, at least in a specific cell type, and that can be overcome by the virally recruited cullin4a-ddb1-dcaf1 ub ligase. viruses have developed sophisticated mechanisms for exiting from infected cells. enveloped viruses leave the cells through a complex process, known as budding, that requires two main steps, (i) the cell membrane deformation around the assembling virions; and (ii) a fission, resulting in the detachment of the viral particles from the cellular surface [169, 170] . studies on retroviruses, and in particular on hiv-1, have been instrumental in dissecting the complex viral/cellular interplay which ensures a successful egress of enveloped viruses. indeed, the gag polyprotein is the only retroviral protein necessary at this level. this feature allowed the development of viral mutants and tools to identify the molecular mechanisms involved in budding. in 1991, göttlinger and co-workers, along with other groups, identified in the c-terminal p6 domain of hiv-1 gag a highly conserved motif (pt/sap) as crucial player in the detachment of budded virions from the cell surface [171, 172] . starting from these initial findings, short proline-rich sequences, named late assembly or l-domains, functionally equivalent to the hiv-1 pt/sap motif, have been identified in the gag of different retroviruses [173] . to date, in addition to the pt/sap motif, typical of most lentiviruses, two further l-domains have been well characterized: the ppxy-type l-domain present in the gag proteins of oncoretroviruses and the ypx n l-type motif, identified in the gag protein of the equine infectious anemia virus (eiav) [56] . besides retroviruses, l-domains have been also found in the structural proteins of most rna enveloped viruses such as rhabdoviruses, filoviruses, arenaviruses, and paramyxoviruses [56] , and in some dna enveloped viruses [174] [175] [176] [177] [178] . several data have indicated a connection between ub, l-domains and retroviral egress from infected cells. firstly, a functional l-domain leads to gag ubiquitination [23] and when directly fused to different retroviral gags, ub can functionally replace the l-domains [179, 180] . furthermore, ub can be found in retroviral mature particles [181, 182] and its depletion inhibits virus budding [22, 23] , while the recruitment of hect ub ligases, belonging to the nedd4-like family, is clearly involved in retroviral particle release [23, [183] [184] [185] [186] [187] . interestingly, the ppxy type of l-domain interacts with the members of the nedd4-like family of ubiquitin ligases by directly binding the ww domain characteristic of these cellular proteins [183] . finally, the l-domains act independently from their position in the viral protein, frequently occur in combination and can be exchanged between unrelated viruses without losing their ability to mediate budding [188] [189] [190] [191] [192] . overall, these features are suggestive of a role for the l-domains as docking sites for cellular factors belonging to a specific pathway involving ub and exploited by retroviruses to efficiently execute budding. the role played by ub in the endocytosis of transmembrane proteins suggested initially that the endocytic pathway could represent this cellular pathway [6] . furthermore, some transmembrane proteins, such as the epithelial na+ channel, were known to contain sequences perfectly overlapping retroviral l-domains which are involved in the process of endocytosis [193] [194] [195] . moreover, it was demonstrated that the ub residues involved in retroviral budding were indeed the residues involved in protein endocytosis [196] . however, the vesiculation process taking place during endocytosis is topologically opposite to the one occurring during viral budding. indeed, while the budding of a virus happens from the cytosol toward the extracellular space and the factors that catalyze membrane fission must work from within the bud neck, during endocytosis the vesicles bud into the cytoplasm and membrane fission is driven by dynamin from outside of the bud neck. thus, the cellular machinery involved in the formation of endocytic vesicles could not be the one exploited by viruses for their egress. this apparent discrepancy between experimental data started to find a solution thanks to the seminal discoveries of the carter's laboratory [197] and of the sundquist's group [198] , that identified in the cellular protein tumor suppressor gene 101 (tsg101) the binding partner of the hiv1-1 pt/sap motif, an interaction which is crucial for hiv budding. these initial findings, along with the work done beforehand and in parallel by cellular biologists, allowed to establish the connection between a cellular pathway, to which tsg101 belongs, and the hiv-1 egress from infected cells: the biogenesis pathway of an organelle of the endocytic pathway, the multivesicular bodies (mvb) [7] . since then, more than 10 years of research have clarified that retroviruses, and in general most enveloped rna and some dna enveloped viruses, exploit mvbs during the latest steps of their replication cycle [173] . these organelles give reason of the connection between viral budding, ubiquitin, l-domains and the endocytosis of transmembrane proteins. indeed, mvbs represent the organelles that eukaryotic cells have evolved to make the degradation of transmembrane proteins possible [7] . when such a protein needs to be removed from the plasma membrane, it is ubiquitinated and endocytosed on the surface of endosome. then, a budding of vesicles from the membrane into the lumen of the endosome takes place, which allows the delivery of the trans-membrane protein into the lumen of the endosomes. this vesiculation event leads to the biogenesis of the mvb, which will eventually fuse with a lysosome resulting in the degradation of its cargo ( figure 3 ) [199, 200] . it is clear that, if the transmembrane protein would not get access to the lumen of the endosome, thanks to the formation of the mvb, its degradation through the lysosome could not occur. thus, the budding of vesicles from the endosomal membrane into the interior of the organelle (the biogenesis of the mvb) is the crucial step in this process. this vesiculation process, which takes place from the cytosol towards an environment that is equivalent to the extracellular space (the endosomal lumen), is now an event topologically identical to the budding of viruses from the plasma membrane. as a consequence, the connection between viral budding and mvbs biogenesis is functionally and physiologically sustainable. this vesicle budding step requires the sequential recruitment from the cytosol to the endosomal membrane of a highly conserved set of proteins, the escrt machinery [201] and different members of such a machinery, as tsg101 and aip1/alix are recruited by the viral l-domains to allow viral budding [56, 173] . what is still apparently missing is the link between ub, mvb and viral budding. instead, several links do exist, with the most interesting one represented by the evidence that ub plays a central role in the regulation of the mvb biogenesis pathway. indeed, ubiquitination is necessary and sufficient to trigger the escrt-dependent endosomal sorting of membrane proteins and their degradation through the mvb/lysosomal pathway [202] . moreover, precise and finely controlled cycles of ubiquitination and deubiquitination of target proteins and of specific components of the escrt machinery are essential for the function of the entire pathway of mvb biogenesis, till the vesicle budding [203] [204] [205] . indeed, different upstream components of the escrt machinery with an essential role in viral budding, such as tsg101 and aip1/alix, are ubiquitinated and are able to bind ub [206] [207] [208] [209] . the sequential recruitment of the escrt complexes to the mvb membrane is described along with the additional factors involved in the cargo protein delivery into the organelle lumen. the extracellular environment and its equivalents are colored in light blue, while the cytoplasmic environment is colored in yellow. details on the escrt proteins and on the other mvb key factors can be found in several comprehensive reviews [7, 169, 173, 201, 202] . overall, the aforementioned crucial role played by ub in the control of the mvb biogenesis pathway by itself supports the strong evidence of an involvement of this cellular protein in viral budding. however, in addition or in parallel to this regulatory function, there is evidence that also direct ubiquitination of specific viral/cellular proteins might be important for the escrt-dependent viral budding. in particular, the importance of a direct gag ubiquitination is supported by several studies, as reviewed by votteler and sundquist [173] . the working model foresees that ubiquitination of gag would facilitate its interaction with the escrt machinery. in other words, ubiquitination of gag would mimic ubiquitination of transmembrane proteins along the endocytic/mvb pathway, thus mediating the engagement of the escrt components. under this respect, it has been shown that the binding of tsg101 to gag has an increased affinity when gag is linked to ub [208, 209] . furthermore, the bouamr's group, by fusing the dub domain of the hsv-1 ul36 to gag and to specific escrt proteins, has recently reported evidence supporting a crucial role for gag ubiquitination in hiv budding [210] . however, along with these data, other studies indicate a more complex scenario. for instance, it has been demonstrated that ubiquitination of gag proteins can increase also in the absence of a functional l-domain and when budding is inhibited [211] . moreover, the artificial targeting of ub ligases to gag leads to its ubiquitination, but not always enhances budding [187] . finally, zhadina and coworkers have nicely shown that ub-dependent viral budding can take place without viral protein ubiquitination [212] . thus, the question whether gag ubiquitination is the result of a bystander effect or has a functional relevance is still open. interestingly, it has been reported that the identity of the protein to which ub is bound might not be the key element for viral budding [192] . what appears to be crucial is, instead, the presence of ub at the site of budding, thus emphasizing its crucial role in the process. as very simple intracellular parasites, viruses rely on host cell factors and pathways to perform their life cycle and to establish a successful infection. the extensive use of ub and ubl-mediated pathways by a number of unrelated viruses in different steps of their life cycle emphasizes the central importance of these cellular machinery in the cell physiology and, as a consequence, for viral replication and spreading. in this review, we have tried to give an overview of the complexity of the interplay between viruses and ub/ubl-conjugating systems. while it appears evident that different aspects have been deeply analyzed and better understood, such as the role played by ups in the context of the mechanisms evolved by viruses to control the cell cycle and to counteract innate immunity responses, more work needs to be done in order to clarify the functional relevance of ub involvement in specific steps of viral replication, such as viral budding. moreover, different emerging findings require further clarification both from the cellular and from the viral point of view, such as the role and the substrates of the newly characterized viral and cellular ubl proteins. under this respect, the study of the viral connection with the ub/ubl-conjugating machinery still represents one of the better examples of how viruses can serve as potent tools to dissect complex cellular processes and pathways. ubiquitin linkages make a difference mechanisms underlying ubiquitination the hect family of e3 ubiquitin ligases: multiple players in cancer development cullin-ring ubiquitin ligases: global regulation and activation cycles u-box proteins as a new family of ubiquitin ligases ubiquitin enters the new millennium multivesicular body morphogenesis ubiquitin and ubiquitin-like proteins as multifunctional signals back to the future with ubiquitin ubiquitin in chains polyubiquitin chains: polymeric protein signals ubiquitin-like protein conjugation and the ubiquitin-proteasome system as drug targets viral avoidance and exploitation of the ubiquitin system the e6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53 cellular proteasome activity facilitates herpes simplex virus entry at a postpenetration step proteasome subunits relocalize during human cytomegalovirus infection, and proteasome activity is necessary for efficient viral gene transcription inhibition of the ubiquitin-proteasome system prevents vaccinia virus dna replication and expression of intermediate and late genes orthopoxviruses require a functional ubiquitin-proteasome system for productive replication bortezomib inhibits hepatitis b virus replication in transgenic mice tip60 degradation by adenovirus relieves transcriptional repression of viral transcriptional activator eia inhibition of the ubiquitin-proteasome system affects influenza a virus infection at a postfusion step proteasome inhibition interferes with gag polyprotein processing, release, and maturation of hiv-1 and hiv-2 a role for ubiquitin ligase recruitment in retrovirus release retroviruses have differing requirements for proteasome function in the budding process the ubiquitin-proteasome system facilitates the transfer of murine coronavirus from endosome to cytoplasm during virus entry decreased replication of human respiratory syncytial virus treated with the proteasome inhibitor mg-132 ubiquitination is required for effective replication of coxsackievirus b3 replication of the rotavirus genome requires an active ubiquitin-proteasome system a pre-immediate-early role for tegument icp0 in the proteasomedependent entry of herpes simplex virus the ubiquitin/proteasome system mediates entry and endosomal trafficking of kaposi's sarcoma-associated herpesvirus in endothelial cells epsin 1 is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus a capsid-encoded ppxy-motif facilitates adenovirus entry ebna1-mediated recruitment of a histone h2b deubiquitylating complex to the epstein-barr virus latent origin of dna replication a non-proteolytic role for ubiquitin in tat-mediated transactivation of the hiv-1 promoter ubiquitination of human t-cell leukemia virus type 1 tax modulates its activity kaposi's sarcoma-associated herpesvirus transactivator rta promotes degradation of the repressors to regulate viral lytic replication kaposi's sarcoma-associated herpesvirus rta promotes degradation of the hey1 repressor protein through the ubiquitin proteasome pathway the epstein-barr virus latent membrane protein 2a py motif recruits ww domain-containing ubiquitin-protein ligases the a20 deubiquitinase activity negatively regulates lmp1 activation of irf7 wrapping up the bad news: hiv assembly and release regulation of the innate immune system by ubiquitin and ubiquitin-like modifiers ubiquitylation in innate and adaptive immunity shared principles in nf-kappab signaling termination of nf-kappab activity through a gammaherpesvirus protein that assembles an ec5s ubiquitin-ligase rotavirus nsp1 inhibits nfkappab activation by inducing proteasome-dependent degradation of beta-trcp: a novel mechanism of ifn antagonism varicella-zoster virus immediate-early protein orf61 abrogates the irf3-mediated innate immune response through degradation of activated irf3 viral evasion of the interferon system paramyxoviruses sv5 and hpiv2 assemble stat protein ubiquitin ligase complexes from cellular components interferon-inducible antiviral effectors the tiers and dimensions of evasion of the type i interferon response by human cytomegalovirus interferon-stimulated genes: a complex web of host defenses innate immune interferon responses to human immunodeficiency virus-1 infection trim family proteins and their emerging roles in innate immunity the roles of the trim e3-ubiquitin ligase family in innate antiviral immunity ifn-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses role of multivesicular bodies and their components in the egress of enveloped rna viruses deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases e3 ubiquitin ligases and human papillomavirus-induced carcinogenesis review of the current knowledge on the epidemiology, pathogenesis, and prevention of human papillomavirus infection how the rb tumor suppressor structure and function was revealed by the study of adenovirus and sv40 control the host cell cycle: viral regulation of the anaphase-promoting complex proteasome-dependent disruption of the e3 ubiquitin ligase anaphase-promoting complex by hcmv protein pul21a protein interaction domains of the ubiquitin-specific protease, usp7/huasp protein profiling with epstein-barr nuclear antigen-1 reveals an interaction with the herpesvirus-associated ubiquitin-specific protease huasp/usp7 huasp/usp7 as an epstein-barr virus target contributions of epstein-barr nuclear antigen 1 (ebna1) to cell immortalization and survival wnt signaling in stem cells and cancer what has the study of the k3 and k5 viral ubiquitin e3 ligases taught us about ubiquitin-mediated receptor regulation? viruses a viral protein that selectively downregulates icam-1 and b7-2 and modulates t cell costimulation herpesviruses and immunity: the art of evasion human cytomegalovirus immunity and immune evasion herpes simplex virus type 1 immediate-early protein icp0 and is isolated ring finger domain act as ubiquitin e3 ligases in vitro pml and pml nuclear bodies: implications in antiviral defence the two functions of herpes simplex virus 1 icp0, inhibition of silencing by the corest/rest/hdac complex and degradation of pml, are executed in tandem a proteomic perspective of inbuilt viral protein regulation: pul46 tegument protein is targeted for degradation by icp0 during herpes simplex virus type 1 infection herpes simplex virus 1 e3 ubiquitin ligase icp0 protein inhibits tumor necrosis factor alpha-induced nf-κb activation by interacting with p65/rela and p50/nf-κb1 centromere architecture breakdown induced by the viral e3 ubiquitin ligase icp0 protein of herpes simplex virus type 1 icp0 dismantles microtubule networks in herpes simplex virus-infected cells a viral e3 ligase targets rnf8 and rnf168 to control histone ubiquitination and dna damage responses ubiquitination, ubiquitin-like modifiers, and deubiquitination in viral infection haploid genetic screens identify an essential role for plp2 in the downregulation of novel plasma membrane targets by viral e3 ubiquitin ligases the ring-ch ligase k5 antagonizes restriction of kshv and hiv-1 particle release by mediating ubiquitin-dependent endosomal degradation of tetherin kaposi's sarcoma-associated herpesvirus k3 and k5 proteins down regulate both dc-sign and dc-signr the march family e3 ubiquitin ligase k5 alters monocyte metabolism and proliferation through receptor tyrosine kinase modulation the ring finger domain of varicella-zoster virus orf61p has e3 ubiquitin ligase activity that is essential for efficient autoubiquitination and dispersion of sp100-containing nuclear bodies adenovirus ubiquitin-protein ligase stimulates viral late mrna nuclear export murine gammaherpesvirus 68 orf75c contains ubiquitin e3 ligase activity and requires pml sumoylation but not other known cellular pml regulators, ck2 and e6ap, to mediate pml degradation newly discovered viral e3 ligase pk3 induces endoplasmic reticulum-associated degradation of class i major histocompatibility proteins and their membrane-bound chaperones the poxvirus p28 virulence factor is an e3 ubiquitin ligase viral ubiquitin ligase wssv222 is required for efficient white spot syndrome virus replication in shrimp arterivirus and nairovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling plp2 of mouse hepatitis virus a59 (mhv-a59) targets tbk1 to negatively regulate cellular type i interferon signaling pathway plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase mechanism of inhibiting type i interferon induction by hepatitis b virus x protein structure of a herpesvirus-encoded cysteine protease reveals a unique class of deubiquitinating enzymes the epstein-barr virus (ebv) deubiquitinating enzyme bplf1 reduces ebv ribonucleotide reductase activity epstein-barr virus bplf1 deubiquitinates pcna and attenuates polymerase η recruitment to dna damage sites the rad6/18 ubiquitin complex interacts with the epstein-barr virus deubiquitinating enzyme, bplf1, and contributes to virus infectivity herpes simplex virus 1 ubiquitin-specific protease ul36 inhibits beta interferon production by deubiquitinating traf3 autocatalytic activity of the ubiquitinspecific protease domain of herpes simplex virus 1 vp1-2 cleavage specificity of the ul48 deubiquitinating protease activity of human cytomegalovirus and the growth of an active-site mutant virus in cultured cells mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pul36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirus-encoded deubiquitinase orf64 molecular basis for ubiquitin and isg15 cross-reactivity in viral ovarian tumor domains mers-cov papain-like protease has deisgylating and deubiquitinating activities a compact viral processing proteinase/ubiquitin hydrolase from the otu family a viral deubiquitylating enzyme targets viral rna-dependent rna polymerase and affects viral infectivity the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions deubiquitinating function of adenovirus proteinase past, present and future: 30 years of hiv research regulation of hiv-1 gene expression identification of host proteins required for hiv infection through a functional genomic screen host cell factors in hiv replication: meta-analysis of genome-wide studies host factors mediating hiv-1 replication host restriction factors in retroviral infection: promises in virus-host interaction hiv accessory proteins versus host restriction factors viral modulators of cullin ring ubiquitin ligases: culling the host defense hiv: ringside views selective protein degradation: a rheostat to modulate cell-cycle phase transitions the emerging family of cullin3-ring ubiquitin ligases (crl3s): cellular functions and disease implications structural basis for hijacking cbf-β and cul5 e3 ligase complex by hiv-1 vif role of hiv-1 vpu protein for virus spread and pathogenesis hiv-1 antagonism of cd317 is species specific and involves vpu-mediated proteasomal degradation of the restriction factor hiv-1 vpu neutralizes the antiviral factor tetherin/bst-2 by binding it and directing its beta-trcp2-dependent degradation vpu directs the degradation of the human immunodeficiency virus restriction factor bst-2/tetherin via a {beta}trcp-dependent mechanism inhibition of β-trcp-dependent ubiquitination of p53 by hiv-1 vpu promotes p53-mediated apoptosis in human t cells besnard-gué rin, c. involvement of the betatrcp in the ubiquitination and stability of the hiv-1 vpu protein hiv1 vpr arrests the cell cycle by recruiting dcaf1/vprbp, a receptor of the cul4-ddb1 ubiquitin ligase vpr expression abolishes the capacity of hiv-1 infected cells to repair uracilated dna the hiv-1 protein vpr targets the endoribonuclease dicer for proteasomal degradation to boost macrophage infection samhd1 host restriction factor: a link with innate immune sensing of retrovirus infection new insights into the role of vif in hiv-1 replication evidence for a newly discovered cellular anti-hiv-1 phenotype isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein hiv-1 vif n-terminal motif is required for recruitment of cul5 to suppress apobec3 hiv-1 vif, apobec, and intrinsic immunity interactions between hiv-1 vif and human elonginb-elonginc are important for cbf-β binding to vif tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu tetherin inhibits hiv-1 release by directly tethering virions to cells the antiviral activities of tetherin tetherin-driven adaptation of vpu and nef function and the evolution of pandemic and nonpandemic hiv-1 strains tetherin antagonism by primate lentiviral nef proteins feline tetherin is characterized by a short n-terminal region and is counteracted by the feline immunodeficiency virus envelope glycoprotein ancient origin of a deletion in human bst2/tetherin that confers protection against viral zoonoses reacquisition of nef-mediated tetherin antagonism in a single in vivo passage of hiv-1 through its original chimpanzee host vpu antagonizes bst-2-mediated restriction of hiv-1 release via beta-trcp and endo-lysosomal trafficking rab7a is required for efficient production of infectious hiv-1 berlioz-torrent, c. the escrt-0 component hrs is required for hiv-1 vpu-mediated bst-2/tetherin downregulation serine-threonine ubiquitination mediates downregulation of bst-2/tetherin and relief of restricted virion release by hiv-1 vpu antagonism of tetherin restriction of hiv-1 release by vpu involves binding and sequestration of the restriction factor in a perinuclear compartment β-trcp is dispensable for vpu's ability to overcome the cd317/tetherin-imposed restriction to hiv-1 release hiv-1 vpu and hiv-2 env counteract bst-2/tetherin by sequestration in a perinuclear compartment hiv-1 vpr-mediated g2 arrest involves the ddb1-cul4avprbp e3 ubiquitin ligase hiv-1 vpr activates the g2 checkpoint through manipulation of the ubiquitin proteasome system lentiviral vpr usurps cul4-ddb1[vprbp] e3 ubiquitin ligase to modulate cell cycle hiv-1 vpr function is mediated by interaction with the damage-specific dna-binding protein ddb1 ddb1 and cul4a are required for human immunodeficiency virus type 1 vpr-induced g2 arrest the hiv1 protein vpr acts to promote g2 cell cycle arrest by engaging a ddb1 and cullin4a-containing ubiquitin ligase complex using vprbp/dcaf1 as an adaptor assembly with the cul4a-ddb1dcaf1 ubiquitin ligase protects hiv-1 vpr from proteasomal degradation human immunodeficiency virus type 1 vif induces cell cycle delay via recruitment of the same e3 ubiquitin ligase complex that targets apobec3 proteins for degradation defining the interactions and role of dcaf1/vprbp in the ddb1-cullin4a e3 ubiquitin ligase complex engaged by hiv-1 vpr to induce a g2 cell cycle arrest molecular mechanisms of hiv immune evasion of the innate immune response in myeloid cells intracellular nucleotide levels and the control of retroviral infections structural basis of lentiviral subversion of a cellular protein degradation pathway cullin 4a and cullin4b are interchangeable for hiv vpr and vpx action through the crl4 ubiquitin ligase complex membrane budding and scission by the escrt machinery: it's all in the neck viral membrane scission effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release p6gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease virus budding and the escrt pathway herpes simplex virus type 1 cytoplasmic envelopment requires functional vps4 intracellular trafficking and maturation of herpes simplex virus type 1 gb and virus egress require functional biogenesis of multivesicular bodies human cytomegalovirus exploits escrt machinery in the process of virion maturation cellular vps4 is required for efficient entry and egress of budded virions of autographa californica multiple nucleopolyhedrovirus functional characterization of the putative hepatitis b virus core protein late domain using retrovirus chimeras ubiquitin is part of the retrovirus budding machinery functional replacement of a retroviral late domain by ubiquitin fusion ubiquitin in avian leukosis virus particles ubiquitin is covalently attached to the p6gag proteins of human immunodeficiency virus type 1 and simian immunodeficiency virus and to the p12gag protein of moloney murine leukemia virus hect ubiquitin ligases link viral and cellular ppxy motifs to the vacuolar protein-sorting pathway efficient and specific rescue of human immunodeficiency virus type 1 budding defects by a nedd4-like ubiquitin ligase sundquist, w.i. nedd4l overexpression rescues the release and infectivity of human immunodeficiency virus type 1 constructs lacking ptap and ypxl late domains role of the feline immunodeficiency virus l-domain in the presence or absence of gag processing: involvement of ubiquitin and nedd4-2s ligase in viral egress rescue of hiv-1 release by targeting widely divergent nedd4-type ubiquitin ligases and isolated catalytic hect domains to gag positionally independent and exchangeable late budding functions of the rous sarcoma virus and human immunodeficiency virus gag proteins infectivity of moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses functional replacement and positional dependence of homologous and heterologous l domains in equine infectious anemia virus replication aip1/alix is a binding partner for hiv-1 p6 and eiav p9 functioning in virus budding functional interchangeability of late domains, late domain cofactors and ubiquitin in viral budding ww domains of nedd4 bind to the proline-rich py motifs in the epithelial na+ channel deleted in liddle's syndrome regulation of the epithelial na+ channel by nedd4 and ubiquitination late assembly domain function can exhibit context dependence and involves ubiquitin residues implicated in endocytosis pr55(gag) tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding the role of ubiquitination in lysosomal trafficking of δ-opioid receptors ubiquitination in the first cytoplasmic loop of μ-opioid receptors reveals a hierarchical mechanism of lysosomal down-regulation dynamics of escrt proteins ubiquitin: same molecule, different degradation pathways how ubiquitin functions with escrts escrt ubiquitin-binding domains function cooperatively during mvb cargo sorting the circuitry of cargo flux in the escrt pathway tsg101-specific e3 ubiquitin ligase, regulates receptor endocytosis and retrovirus budding the yeast alix homolog bro1 functions as a ubiquitin receptor for protein sorting into multivesicular endosomes structure and functional interactions of the tsg101 uev domain structure of the tsg101 uev domain in complex with the ptap motif of the hiv-1 p6 protein ubiquitin conjugation to gag is essential for escrt-mediated hiv-1 budding analysis of human immunodeficiency virus type 1 gag ubiquitination ubiquitin-dependent virus particle budding without viral protein ubiquitination owing to space limitations and to the complexity of the topics we have predominantly cited recent publications and reviews, therefore we apologize to those whose original contributions have not been specifically referenced. the studies carried on in the authors' laboratory have been supported by the miur-prin-2005 (#2005069559_004 to cp), miur-prin-2007 (#20072j9rwm_004 and 2007m52htt_004 to cp and gp, respectively), aids grants from the istituto superiore di sanità (rome-aids projects #30g.24 and 40h98 to cp, #30g.55 and 40f.57 to gp), grants from the university of padova (ex-60% to cp, gp, ac and cpda061582 to ac) and from regione veneto to gp. dm is a phd student in biomedicine, at the department of molecular medicine, university of padova. the authors declare no conflict of interest. key: cord-305602-yzc4bosn authors: llano, manuel; peña-hernandez, mario a. title: chapter seven defining pharmacological targets by analysis of virus–host protein interactions date: 2018-12-31 journal: advances in protein chemistry and structural biology doi: 10.1016/bs.apcsb.2017.11.001 sha: doc_id: 305602 cord_uid: yzc4bosn abstract viruses are obligate parasites that depend on cellular factors for replication. pharmacological inhibition of essential viral proteins, mostly enzymes, is an effective therapeutic alternative in the absence of effective vaccines. however, this strategy commonly encounters drug resistance mechanisms that allow these pathogens to evade control. due to the dependency on host factors for viral replication, pharmacological disruption of the host-pathogen protein–protein interactions (ppis) is an important therapeutic alternative to block viral replication. in this review we discuss salient aspects of ppis implicated in viral replication and advances in the development of small molecules that inhibit viral replication through antagonism of these interactions. chromatographic resolution of human cell extracts subsequently analyzed by quantitative tandem mass spectrometry resulted in the identification of 13,993 physical interactions established by 3006 individual proteins. computational analysis of the physical interactions led to the mapping of 622 complexes, suggesting an average of 4 proteins per complex (havugimana et al., 2012) . these estimates are also supported by other analysis indicating that most of the mammalian complexes are formed by the association of three or four different proteins. importantly, some host proteins are found in more than one complex at a time (wong et al., 2008) . similarly, a study in human cells combining large-scale immunoprecipitation of 331 bait proteins followed by high-throughput mass spectrometry analysis led to the discovery of 6463 interactions between 2235 unique proteins, suggesting an average of 3 proteins per complex (ewing et al., 2007) . protein complexes are stable assemblies that carry out most of the biochemical activities in the cell. corum, a public database of mammalian protein complexes, compiles complexes reported in nonhighthroughput, physical interaction experiments. of the proteins annotated in corum, 78% belong to one protein complex. fig. 1 represents the frequency of annotated complexes in the corum database. interestingly, protein complexes implicated in subcellular localization processes are notoriously overrepresented in this database, whereas others implicated in energy production, protein synthesis, and tissue differentiation are underrepresented. in these collection of experimentally characterized protein complexes, approximately 16% of all predicted human open reading frames are organized in 1815 protein complexes (ruepp et al., 2010) . however, this number is expected to be only a minor fraction of the human complexome considering that in yeast 45% of the encoded proteins reside in complexes (ruepp et al., 2010) . hypothesis-driven experiments conducted at small scale have notably contributed to our understanding of protein-protein interactions (ppis) implicated in viral replication. in addition, high-throughput technologies analyzing ppis, gene loss of function, or transcriptomic profiles have been utilized to define cellular complexes implicated in different steps of the viral life cycle. large-scale physical analysis of ppi is conducted by yeast two-hybrid (y2h) screens (calderwood et al., 2007; de chassey et al., 2008; fossum et al., 2009; lee et al., 2011; rajagopala, casjens, & uetz, 2011; shapira et al., 2009; trigg et al., 2017; uetz et al., 2006) and tandem affinity purification followed by mass spectrometry (tap-ms) (germain et al., 2014; pichlmair et al., 2012; ramage et al., 2015; rozenblatt-rosen et al., 2012) . y2h screens can analyze only binary interactions, whereas tap-ms allows for the identification of more complex interactors. then, the physical interactions identified with these methods are subjected to organization in functional networks by computational approaches. despite of their rate of success in ppis identification, both methodologies interrogating physical interactions of proteins fail to detect multiple types of ppis. for example, interactions involving transmembrane proteins, or transient/ weak interactions escape detection. similar to the physical methods, computational systems biology approaches successfully predict exploitation of biological processes by viruses (kitano, 2002a (kitano, , 2002b navratil, de chassey, combe, & lotteau, 2011; zak, tam, & aderem, 2014) . these methods use gene expression and gene loss-of-function systemic analysis to identify potential host factor networks implicated in viral replication (hirsch, 2010) . the main problem that the high-throughput functional approaches discussed earlier has is the poor data overlap resulting from these analyses. this is even though these methods have a high degree of similarity or complementary. therefore, validation of the discovered ppis is required (rozenblatt-rosen et al., 2012; shapira et al., 2009) . this validation involves the demonstration of the interaction by coimmunoprecipitation analysis, particularly during infection. prioritization analysis of ppis can be implemented by combining the findings from the direct interaction approaches with comprehensive gene loss-of-function screenings. an example of the effectiveness of this multifactorial approach for the identification of ppis is a study aimed to identify host-influenza ppis (shapira et al., 2009) . in this study, 35% of the 1745 genes initially found to be involved in viral protein interactions were demonstrated to influence viral replication by subsequent rnai screening. this high rate of positive validation may have been the result of the selection of the final candidate genes by a combined analysis of the results obtained in y2h screens using viral and host proteins (direct interactors), in transcriptional profiling experiments to define genes regulated by viral infection, and in in silico predictions of genes implicated in the protein networks found in the first two experimental approaches. comparison of the cellular complexes exploited by different pathogens indicates an significant overlap, leading to the definition of cellular processes implicated in infection (brander & walker, 2000; davis et al., 2015; dyer, murali, & sobral, 2008; hirsch, 2010; hiscott, nguyen, arguello, nakhaei, & paz, 2006; pichlmair et al., 2012; rozenblatt-rosen et al., 2012; shapira et al., 2009) . therefore, shared cellular complexes could constitute broad-spectrum therapeutic targets potentially impairing more than one pathogen. an example of this strategy is discussed later when we consider the implication of protein translation in the mechanism of replication of multiple viruses. the overlap in the utilization of cellular complexes between different pathogens also brings support to the hypothesis that the innate immune system has evolved the ability of detecting patterns of pathogenicity. these patterns are generated by the activation of similar biological processes by different pathogens, indicating the existence of pathogen-induced processes (dyer et al., 2008; pichlmair et al., 2012; vance, isberg, & portnoy, 2009) . the viral manipulation of the activity of type i interferon (ifn)-stimulated genes (isg) illustrates this concept. protein kinase, rna-activated (pkr) is an isg that upon activation phosphorylates the α-subunit of the translation initiation factor eif-2, inhibiting protein synthesis. viral dsrna is the main pkr activator in virus-infected cells, whereas the cellular protein pact (protein activator of pkr), that heterodimerizes with pkr, is an important activator of the kinase in stressed, noninfected cells in the absence of dsrna (li et al., 2006; patel & sen, 1998) . hiv-1 evades the robust type i ifn response mounted by plasmacytoid dendritic cells in infected individuals by rewiring the pact/pkr complex. in infected cells, hiv-1 tar nucleates a complex with the viral protein tat and the cellular proteins pact and adar1 (adenosine deaminase acting on rna 1). in this complex, adar1 inhibits pact pkr activatory function by direct interaction with pact (clerzius et al., 2013) . another example of pathogeninduced host complex rewiring will be discussed later in relation to the role of hiv-1 accessory proteins in the targeting of the ubiquitin/proteasome system to degrade different restriction factors. importantly (chukwurah, handy, & patel, 2017) . therefore, the pact-adar1-pkr axis is a cellular pathway commonly manipulated by different viruses that could constitute a therapeutic target with broad antiviral activity or pathogen-induced processes detected by the innate immune system. the hijacking of similar cellular pathways by different viruses also allows for the identification of broader biomarkers of viral infection. generally viral pathogens target cell cycle regulation, nuclear transport, and immune response processes (dyer et al., 2008; shapira et al., 2009) . ssrna(à) viral proteins tend to interact with processes implicated in the protection of rna from degradation and rna processing, whereas dsrna viruses are more connected to protein degradation processes. dna viruses, however, target proteins connecting the cell cycle with other processes such as chromosomal and transcriptional homeostasis (pichlmair et al., 2012; shapira et al., 2009 ). rnai-based screens have also been useful in identifying host factors implicated in viral replication. interestingly, the number of host factors identified for the different viruses analyzed in these screens outnumber by a factor of 10 the proteins encoded by these viruses (hirsch, 2010) . this disproportion could illustrate the multifunctional character of viral proteins. in this case viral proteins are expected to establish multiple independent binary interactions with independent host proteins. alternatively, viral proteins could interact with host proteins organized in complexes. therefore, rnai-mediated downregulation of the subunits of these protein complexes will produce similar phenotypes. correspondingly, y2h screenings have found that viral proteins establish a disproportionate number of binary interactions with the human proteome. these numbers of interactions registered for viral proteins exceed the predicted number of interactions estimated from the analysis of the human interaction network (shapira et al., 2009) . for example, assessment of the interaction of each of the 10 viral proteins encoded by influenza with 12,000 human orf through y2h screening found 135 interactions with 87 human proteins (shapira et al., 2009) . these evidences support a model indicating that viral proteins evolve multiple direct interactions. furthermore, computational analysis of host factors implicated in these pairwise interactions indicated that the host proteins occupy central positions, hub proteins, within the cellular interactome (shapira et al., 2009) . this higher than expected connectivity suggests that the direct interactions of viral proteins with host factors allow the access of the virus to cellular complexes. computational analysis of the interaction of human proteins binding to different viral proteins encoded by 35 different viruses showed that approximately 97% ae 9.1% of the 1396 unique targeted human proteins interact at least with one other human protein (dyer et al., 2008) . these data indicate that the majority of the host factors implicated in viral replication are in complexes. these conclusions are further supported by tap-ms and functional genomic studies. tap-ms studies of the virus-host interactome indicate that viral proteins tend to interact with proteins that have multiple interacting partners and participate in more cellular pathways (protein hubs), and also with proteins that are central to many pathways and, therefore, occupy a more central position within these networks (protein bottlenecks) (dyer et al., 2008; pichlmair et al., 2012; shapira et al., 2009) . for example, in a tap-ms experiment were mapped 3787 complex associations between 54 viral proteins from different viruses and 1079 host proteins (rozenblatt-rosen et al., 2012) , highlighting the high degree of connectivity of the interacting proteins. as discussed above, some of the host factors predicted, by the combined transcriptional profiling and in silico analyses, to interact with host proteins implicated in direct binary contacts with influenza proteins (y2h interactors) were demonstrated to influence viral replication in functional screenings (shapira et al., 2009 ). these findings demonstrate that influenza, and potentially other viruses, hijacks cellular networks by combining physical and regulatory (transcriptional level) interactions with these pathways. this multilayer regulation offers further alternatives of blocking physical or regulatory interactions aiming to impair viral replication. interestingly some of the host factors found in the networks of protein interacting with viral proteins are induced at the transcriptional level by infection in a type i ifnindependent manner (shapira et al., 2009 ). this indicates that viruses modulate, at the transcriptional levels, the abundance of proteins that will engage in physical binary or multiprotein complex interactions. therefore, computational analysis of the pathways discovered by functional genomics could lead to the identification of protein networks implicated in physical interactions with viral proteins. protein complexes established by viruses seem to determine the disease associated to the infection. for example, analysis of the interactome of e6 proteins from hpv types differing in their oncogenic potential associates with different subset of host proteins (rozenblatt-rosen et al., 2012) . therefore, disruption of these complexes could also prevent viral pathogenesis. almost 78% of the human-pathogen ppis reported belong to hiv-1 (dyer et al., 2008; fahey et al., 2011; ruepp et al., 2010) . generally host factors interacting with viral proteins tend to preserve the host protein interactions mapped in noninfected cells (rozenblatt-rosen et al., 2012; yu et al., 2011) , indicating that the normal host protein complexes rather than new virus-induced protein complexes are implicated in viral replication. this opens the opportunity to utilize the current body of knowledge on the human interactome to define the host complex hijacked by viruses. however, examples of virus-specific complex variants also occur. among them, one of the best characterized is the complex between hiv-1 vif and the transcriptional and protein degradation cellular machineries. vif removes apobec3 family restriction factors by targeting the ubiquitin ligase complex cul5 to this restriction factor, and inducing its proteasomemediated degradation. other hiv-1 proteins, vpu and vpr, also exploit cullin-ring e3 ligase to degrade other restriction factors. vif directly interacts with the substrate, the transcription factor cbf-β, the n-terminus of cul5 (cullin-ring e3 ligase), and the heterodimeric substrate adaptor elob/eloc. in turn, the c-terminus of cul5 binds rbx2 and recruits an e2 ubiquitin conjugation enzyme that mediates the transfer of polyubiquitin to the substrate proteins (jager, kim, et al., 2011; zhang, du, evans, yu, & yu, 2011) . cbf-β is required in this complex for the assembly of the vif-cul5 e3-ubiquitin-ligase complex, but not for the binding of vif to the substrate . cbf-β normally heterodimerizes with the runx family of transcription factors preventing its degradation and vif competes with runx1 for binding to cbf-β (guo et al., 2014) . the surface area buried at the interface of the interaction of vif and cbf-β is large ($4800 a 2 ) suggesting that is undruggable (salter, morales, & smith, 2014) , as we will discuss later for other surfaces of ppis. however, alanine scanning indicated that aa 5-11 of vif are a hot spot (defined in a later section of this review) in the interaction with cbf-β. furthermore, residues phe68 and ile55 of cbf-β establish important interactions with trp5 in vif through hydrophobic interactions (desimmie, smith, matsuo, hu, & pathak, 2017) , highlighting further opportunities for disruption of this complex with small molecules. therefore, these structural characteristics of the vif-cbf-β surface of interaction do not exclude its amenability to small-molecule interference, as we will show later for similar ppis. interfaces of ppi were considered undruggable for sometime, mainly because these surfaces of interactions are bigger than classical druggable protein surfaces, such as allosteric and catalytic sites in enzymes. the total area buried by the interactors in the binding site in ppis is in average 1600 (ae400) a 2 , with some proteins extending to 2000-4660 a 2 . in contrast, surfaces of interaction between proteins and small molecules are approximately from 300 to 1000 a 2 (arkin, tang, & wells, 2014; lo conte, chothia, & janin, 1999) . in addition, the surface of protein-protein interaction, although very variable in characteristics, was generally considered large patches of segmented complementary surfaces lacking small grooves or pockets (hopkins & groom, 2002; jones & thornton, 1996 lo conte et al., 1999) . furthermore, attempts to find small molecules interfering with ppis showed a very high failure rate ($60%) (brown & superti-furga, 2003) , therefore reinforcing the concept that surface of ppis is not druggable. in correlation with this, estimates using very stringent concepts for drugs indicate that only approximately 10% of the human proteome could be targeted by oral, drug-like small molecule. these predictions also indicate that of the druggable gene products only between 5% and 50% were implicated in diseases (hopkins & groom, 2002) . our understanding of the rules driving the ppis importantly changed with the discovery that the different residues buried in the large surfaces of interactions contribute differentially to the binding strength of the interacting proteins (clackson & wells, 1995) . this characteristic reduced considerably the area required to be targeted by small molecules. interrogation of the surfaces of binding in ppis by mutagenesis of individual residues (alanine scanning) indicated that mutation of a few residues implicated in these binding surfaces was sufficient to disrupt the ppis. residues importantly contributing to the binding free energy were defined as hot spots (clackson & wells, 1995) . these residues tend to cluster in tightly packed regions in the center of the surfaces of interactions and are called hot regions (clackson & wells, 1995; keskin, ma, & nussinov, 2005) . hot spots can be amenable to drug targeting due to their small surface area and important contribution to the total binding energy of the complex. however, not always hot spots are vulnerable to drugs, as additional topological constraints for drug binding limit the number of druggable hot spots (zerbe, hall, vajda, whitty, & kozakov, 2012) . in addition, in silico analysis indicated that interfaces could contain more than one hot region, and in some ppis the establishment of the complex depends on the cooperative interaction of different hot spots within the region (keskin et al., 2005) . by 2011, more than 40 ppis have been reported to be targeted by small molecules (morelli, bourgeas, & roche, 2011) . twenty-seven proteinprotein complexes were included in the 2016 release of the small-molecule orthosteric modulators of ppis database 2p2idb. in this site are listed only those ppis in which there is structural information of the protein-protein and protein-inhibitor complexes (basse, betzi, morelli, & roche, 2016) . the majority of the most successful ppis inhibitors target hot-spot residues that cluster in small binding pockets (250-900 a 2 ), and the surface of interaction of the binding proteins has short primary sequences (arkin et al., 2014) . structural analysis of protein-inhibitor complexes indicated that in some cases small molecules interrupting ppis bind to cavities not present in the surface of interaction of the protein-protein complex or in the interactor proteins when they are in isolation. these findings indicate some degree of adaptability in the surfaces of interaction initially considered lacking of druggable groves or pockets. according to in silico simulations the opening of some of these binding pockets is transient (wells & mcclendon, 2007) . most of the small molecules interfering with ppis bind directly to the implicated surfaces of interactions (orthosteric modulators) by targeting hot spot residues or by molecular mimicry of elements of secondary structures (arkin et al., 2014; basse et al., 2016; fry, 2006; wells & mcclendon, 2007; yin & hamilton, 2005) . however, several successful examples of small molecules disrupting ppis act through their binding to allosteric sites (crump et al., 2004; mcmillan et al., 2000; oswald et al., 2016; roche et al., 2016; szilagyi, nussinov, & csermely, 2013) . a well-characterized model of small-molecule disruption of a host ppi, deposited in the 2p2idb database, is the mdm2/p53 interaction. mdm2 binds to p53, impairing its transcriptional activity and stability. the interaction surfaces are formed by a hydrophobic pocket (aa 25-109) in the n-terminus of mdm2 that is occupied by the hydrophobic side of an amphipathic α-helix in p53 (aa 19-26) . mutations at any of the four residues within the mdm2-binding pocket or of three residues within the p53 α-helix block this ppi (moll & petrenko, 2003) . small molecules interrupting this interaction bind to the p53-binding pocket in mdm2 (vassilev et al., 2004) . most of the data on small molecules targeting ppis in the 2p2idb database correspond to host-host ppis and only two are relevant to viruses. that is the case of the interaction between the hpv proteins e2 and e1, and of allosteric inhibitors of hiv-1 integrase activity (allinis) (engelman, kessl, & kvaratskhelia, 2013) . hpv e2 and e1 bind cooperatively to the viral origin of dna replication and are required for initiation of dna replication (berg & stenlund, 1997) . indandione derivatives that bind to the e2 transactivation domain block this interaction. one of these small molecules was shown to contact 7 of the 20 residues implicated in this interaction. allinis bind to the dimer interface of integrase to the same residues of the host protein ledgf/p75 (engelman et al., 2013) . ledgf/p75 is a chromatin-bound protein required for efficient hiv-1 cdna integration (llano et al., 2006; shun et al., 2007) , an essential step in the viral life cycle. ledgf/p75 binds to the dimer interface of the catalytic core domain of integrase. two residues in the integrase-binding domain of ledgf/p75 are essential in this interaction. asp366 in an interhelical loop of the host protein contacts, via hydrogen bonds, with residues glu170 and his171 in one integrase monomer, whereas ledgf/p75 residue ile365 interacts with a hydrophobic pocket (leu102, ala128, trp132) in the other integrase subunit (cherepanov, ambrosio, rahman, ellenberger, & engelman, 2005; cherepanov, sun, et al., 2005) . allinis bind to the viral integrase at the ledgf/p75-binding site, preventing the binding of ledgf/p75 and affecting integrase catalytic core domain dimerization. this disrupts integrase assembly with viral dna and allosterically inhibits its activity (engelman et al., 2013) . 2-(quinolin-3-yl) acetic acid derivatives (ledgins), and in particular compound 6, showed potent inhibitory activity of hiv-1 replication (christ et al., 2010) . this compound establishes hydrogen bonds with integrase residues glu170, his171, and thr173 that are hot spots in the ledgf/p75-binding site in integrase (christ et al., 2010) . as allinis were more potent against hiv-1 in cells lacking ledgf/p75, the contribution of ledgf/ p75-binding inhibition to their mechanism of action is not clear (wang et al., 2012) . another successful strategy in the design of small molecules interfering viral replication has been the targeting of multimeric viral rna polymerases. influenza virus and vaccinia virus encode rna polymerases formed by the essential interaction of several subunits. in influenza virus each of the three viral subunits of the polymerase, pb1, pb2, and pa, is required to assemble in a complex for the polymerase activity. therefore, small molecules that interrupt the binding of pb1 into a hydrophobic groove in the c-terminus of pa (he et al., 2008) block replication of influenza a and b viruses (muratore et al., 2012) . similarly, vaccinia virus depends for replication on the formation of a trimeric complex between the dna polymerase e9, the uracil dna glycosylase d4, and the viral protein a20 (stanitsa, arps, & traktman, 2006) . small molecules disrupting this complex by impairing the interaction d4-a20 inhibit replication of vaccinia virus and cowpox virus (schormann et al., 2011) . there are two types of physical interactions that can be targeted to interrupt the utilization of cellular complexes by the virus: the virus-host interface or the host-host interface of complexes hijacked by the virus. targeting protein interaction surfaces containing viral proteins is always associated with the selection of mutant viruses that escape control of the inhibitor (de clercq, 2007; strasfeld & chou, 2010) . this is the result of the highly mutagenic rate of viruses. however, targeting host complexes utilized by viruses at the host-host interface is expected to do not exhibit mechanisms of resistance, since host proteins are genetically more stable than their viral counterparts. therefore, disruption of the host complexes exploited by viruses rather than interference of the virus-host protein interface is an attractive alternative to avoid the selection of escape mutants resistant to the inhibitors. a major disadvantage of this strategy is the potential toxicity associated to the disruption of cellular complexes. potentially, this could be minimized if the interruption of the complex is transient. viral replication is a fast process that occurs in a few hours, and disruption of these complexes for a few hours could greatly affect the ability of the virus to replicate without affecting cellular viability. we will later discuss experimental findings supporting this view. in addition, the high degree of functional overlap in the human proteome, that is absent in viruses because of their relative smaller genomes, could also ameliorate the toxicity caused by the disruption of host-host ppis implicated in viral replication. the differences in functional redundancy suggest that the disruption of cellular complex will be more tolerable for the host than the pathogen. in support of this view, shrna-based studies have showed that many host factors required for viral replication do not carry essential cellular functions. for example, out of 54,509 transcripts permanently targeted with lentivirus-encoded shrnas, 17% were dispensable for the cell and deficient stable cell lines were developed but a fraction of them were required for efficient hiv-1 replication (yeung, houzet, yedavalli, & jeang, 2009 ). finally, targeting host complexes relevant for different viruses could allow the development of broad-spectrum antivirals. protein translation is essential for the virus and the cell. however, transient interference with protein synthesis at the initiation step has been demonstrated to be more detrimental for the pathogen than the host at a cellular level. both cellular mrna and mrna from different viruses carry a 7-methylguanosine cap (cap) on their 5 0 terminal nucleotide. therefore, cap-dependent cellular translation is exploited by many different viruses. critical to this translation mechanism is the eukaryotic initiation factor 4f complex (eif4f). this complex is required for the efficient recruitment of ribosomes to capped mrnas. the eif4f complex includes a capbinding protein (eif4e), an rna helicase (eif4a), and a large scaffolding subunit (eif4g). eif4e protein binds to cap-mrna and then recruits eif4g that binds eif4a. therefore, inhibitors of eif4e-eif4g interaction (i.e., 4e2rcat ) or the helicase activity (hippuristanol) drastically impair translation. 4e2rcat completely blocks the replication of the human alphacoronavirus 229e (cencic, desforges, et al., 2011) or murine norovirus 1 (royall et al., 2015) at doses that impair approximately 40% of the cellular protein synthesis (cencic, desforges, et al., 2011) or do not alter at all cell viability. at these doses 4e2rcat affects eif4e-eif4g interaction as determined by coimmunoprecipitation (royall et al., 2015) . these results indicate that coronaviruses are more dependable on eif4f for ribosome recruitment mrnas than the host. hippuristanol is a natural product that inhibits the activity of eif4a by binding to its c-terminal domain. this compound impairs eif4a rnabinding, atpase, and helicase activities. hippuristanol specifically impaired replication of poliovirus , caliciviruses (chaudhry et al., 2006) , human cytomegalovirus (lenarcic, ziehr, de leon, mitchell, & moorman, 2014) , and junin virus (linero, thomas, boccaccio, & scolaro, 2011) . despite the cell toxicity of hippuristanol at the doses and length of the treatment used in these reports, viral replication was affected specifically since this compound did not alter cellular viability. for example, 6-h treatment of raw 264.7 cells with hippuristanol reduced cell viability by 10%, but murine norovirus production was reduced by over 3000-fold (chaudhry et al., 2006) . as described earlier, eif4e interacts with eif4g and assembles into the eif4f complex together with eif4a on the capped mrna. eif4f complex formation leads to eif4e phosphorylation at ser209 by the eif4g-associated kinase mnk1. inhibition of mnk1 by cgp57380 specifically impairs protein synthesis in a variety of large dna viruses (walsh, mathews, & mohr, 2013) . for example, this compound reduced by 10 2 -fold the replication of hsv-1 in quiescent primary fibroblasts without affecting cell viability (walsh & mohr, 2004) . in addition, cgp57380 inhibits reactivation of kaposi's sarcoma-associated herpesvirus (walsh et al., 2013) . because mnk1 is not essential for cell growth or development, it is a potential therapeutic target (ueda, watanabe-fukunaga, fukuyama, nagata, & fukunaga, 2004) . there are other examples of small molecules inhibiting ppis in host complex that are not essential for the cell. some compounds targeting host-virus ppis implicated in the earlier steps of the hiv-1 life cycle are in this category. for example, the hiv-1 entry inhibitor maraviroc, that reached clinical use, acts through an allosteric mechanism. this drug binds to a hydrophobic pocket located in the transmembrane domain of ccr5, altering the conformation of the extracellular domain of ccr5 and preventing in this manner the binding of env . in addition, the small molecule pf74, that inhibits hiv-1 replication by triggering early uncoating (shi, zhou, shah, aiken, & whitby, 2011) , binds to a pocket encompassing the n-terminal domain-c-terminal domain interface of ca in the assembled capsid (bhattacharya et al., 2014) . cleavage and polyadenylation-specific factor 6 (cpsf6) also binds to the same pocket in the capsid during hiv trafficking to the nucleus. this ppi delays uncoating, allowing hiv-1 to evade the innate immune activation through cytoplasmic nucleic acid sensor signaling (rasaiyaah et al., 2013) . we have presented literature evidences indicating that viruses interact for replication with cellular complexes. within these complexes, viral proteins bind to cellular proteins that are central (bottleneck proteins) in the interacting networks and establish multiple interactions in the human interactome (hub proteins). these ppis offer different opportunities for the development of small molecules to block viral replication. virusvirus, virus-host, and host-host ppis are amenable for pharmacological disruption. different viruses utilize similar host protein complexes during infection, showing overlap in the cellular processes hijacked. targeting these shared 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quiescent cells hrp2 determines the efficiency and specificity of hiv-1 integration in ledgf/p75 knockout cells but does not contribute to the antiviral activity of a potent ledgf/p75-binding site integrase inhibitor reaching for high-hanging fruit in drug discovery at protein-protein interfaces an evolutionary and structural characterization of mammalian protein complex organization a genome-wide short hairpin rna screening of jurkat t-cells for human proteins contributing to productive hiv-1 replication strategies for targeting protein-protein interactions with synthetic agents nextgeneration sequencing to generate interactome datasets systems-level analysis of innate immunity relationship between hot spot residues and ligand binding hot spots in protein-protein interfaces t-cell differentiation factor cbf-beta regulates hiv-1 vif-mediated evasion of host restriction we thank zachary s. martinez and angelica lopez, university of texas at el paso, for reviewing the manuscript. key: cord-298802-a5h91axf authors: ravindran, madhu sudhan title: molecular chaperones: from proteostasis to pathogenesis date: 2018-06-22 journal: febs j doi: 10.1111/febs.14576 sha: doc_id: 298802 cord_uid: a5h91axf maintaining protein homeostasis (proteostasis) is essential for a functional proteome. a wide range of extrinsic and intrinsic factors perturb proteostasis, causing protein misfolding, misassembly, and aggregation. this compromises cellular integrity and leads to aging and disease, including neurodegeneration and cancer. at the cellular level, protein aggregation is counteracted by powerful mechanisms comprising of a cascade of enzymes and chaperones that operate in a coordinated multistep manner to sense, prevent, and/or dispose of aberrant proteins. although these processes are well understood for soluble proteins, there is a major gap in our understanding of how cells handle misfolded or aggregated membrane proteins. this article provides an overview of cellular proteostasis with emphasis on membrane protein substrates and suggests host–virus interaction as a tool to clarify outstanding questions in proteostasis. protein biogenesis is a highly complex and errorprone process. cells maintain protein homeostasis via evolutionarily conserved protective mechanism called protein quality control (pqc), involving extensive chaperones and degradative pathways. when pqc encounters misfolded protein it is either repaired or disposed via the ubiquitin proteasomal system [1] . when this quality control fails, proteins can clump to form aggregates, which then undergo autophagic degradation [2] . although vast amount of information regarding the cellular mechanism of soluble protein quality control is available, membrane proteins pqc process is poorly understood, especially in the context of how quality control factors coordinate to rectify the misfolded or aggregated membrane protein problem. viruses are outstanding tools to break new grounds in cell biology and disease mechanisms. in order to replicate and propagate, viruses are highly dependent on their host and they achieve this by hijacking host factors called 'cues'. cues are receptors, enzymes, or chemicals, which directly or indirectly promote different stages of virus infection. the viruses, on the other hand trick these cues by either tuning or reprogramming their cellular role [3] . detailed understanding of these cues have paved way for the development of crucial antiviral targets and also helped us understand the basic cellular processes [4] . below, i will discuss our current knowledge and outstanding issues on proteostasis by comparing aberrant soluble versus membrane protein substrates, and also provide examples of host-virus interaction as a new strategy to tackle these issues. aberrant soluble proteins: recognition, correction, and/or degradation nascent proteins are highly unstable and tend to misfold and/or entangle due to their chemical and physical properties [5] . pqc pathway deploys powerful molecular chaperones that recognize and triage misfolded clients (fig. 1, step 1). different chaperones possess distinct modes of substrate recognition that determine their substrate range and specificity [6]. among them the ubiquitous 70-kda heat shock protein (hsp70) family of chaperones is shown to be associated with plethora of misfolded and aggregated substrates, possibly selecting their targets for proteasomal or autophagy degradation. the hsp70's activity, in turn, is regulated by a number of cofactors and cochaperones, together functioning as a 'machine' [7] . for instance, j-proteins prime the hsp70's folding property by selecting and supplying the substrate to hsp70 and also stimulate the hsp70's atpase activity, whereas nucleotide exchange factors (nef) promote the exchange of adp with atp, to accelerate the cyclic reaction [8] . however, the identity of these machineries and its components can vary for different clients. besides recognizing and selecting the misfolded proteins, chaperones and associated factors also promote refolding, prevent aggregation, or triage these targets for degradation (fig. 1, step 2). for instance, the atp-dependent refolding by chaperone binding and release involving hsp70, a j-protein, and a nef is well defined for several soluble proteins [7] . among them the mostly widely understood are the model substrates processed in the endoplasmic reticulum (er) lumen. the cellular organelle er is the most crowed environment in the cell performing diverse cellular roles. any dysfunction in the er activity leads to accumulation of misfolded and/or unfolded proteins. cells maintain er proteostasis by deploying diverse array of er-resident chaperones and enzymes, which process their client by correcting or priming them to degradative pathways. for example, in the case of misfolded secretory protein carboxypeptidase mutant cpy* and nonglycosylated pro-a-factor, the er lumen hsp70 called binding immunoglobulin protein (bip) and its associated cochaperones efficiently process the misfolded proteins for er-associated degradation (erad) (fig. 1, step 3 & 4) [9]. similarly, erad of terminally misfolded a1-antitrypsin variant null hong kong and transthyretin mutant d18g are handled by bip and a nef, 170-kda glucoseregulated protein (grp170) [10] and processed by degradation pathways. in case of aging diseases, when the above pqc system fails to repair or destroy severely damaged proteins, they tend to aggregate (fig. 1, step 5) causing diseases, such as amyotrophic lateral sclerosis, parkinson's disease, huntington's disease, alzheimer's aberrant soluble or membrane proteins (brown) are recognized (1) by chaperones (green and magenta) and promote its refolding (2). when proteins misfold they are then extracted (3) into the cytosol and degraded (4) by proteasomal machinery. when proteins aggregate (5), it is then sequestered (6) into quality control compartments, and degraded (7) by autolysosomal pathway. disease, type ii diabetes, etc. in most instances, the aggregate also recruits bystanders such as intermediately folded, and correctly folded species causing cytotoxicity and cell death [11] . cells counteract these aggregates by sequestering them in special cytoplasmic quality control compartment (fig. 1, step 6) for refolding or autophagic degradation (fig. 1, step 7) [12]. partitioning of misfolded proteins into compartments is an organized process that appears to be conserved from yeast to mammalian cells. distinct compartments with specific characteristics have been observed, including 'aggresome' colocalizing with microtubule organizing center, 'perinuclear inclusion' that costain with er markers, and 'insoluble inclusion' colocalizing with autophagic markers [13]. these structures serve several purposes, such as in concentrating toxic species, thereby reducing substrate burden on quality control systems, and orchestrating efficient repair. for instance, in yeast, specific quality control compartments are reported to possess hsp104 disaggregase activity. although metazoans lack hsp104 homolog, several recent reports have demonstrated the existence of a mammalian hsp110-dependent disaggregase activity [14] . for example, hsp110 is shown to stabilize apolipoprotein from undergoing erad [15] ; hsp70 has been demonstrated to be transiently associated with polyq protein aggregates, raising the possibility that it may be involved in disaggregating polyq aggregates [16] . similarly, overexpression of several hsp40 family proteins along with hsp70 has been shown to prevent accumulation of polyq ataxia-1/3 in inclusions [17] . in the er lumen, bip prevents aggregation of a misfolded client by binding to its exposed hydrophobic patches until the client is delivered to the erad machinery [18] . despite these findings, the normal cellular function of this machinery is poorly characterized, especially in the context of protein quality control. aberrant membrane proteins: recognition, correction, and/or degradation all membrane proteins are synthesized in the er and they comprise one-third of the human proteome. synthesis of the membrane proteins is a highly complex and error-prone process, which includes insertion of membrane domain into the bilayer and organizing domains on either side of the membrane. unsurprisingly, due to its complex organization, error in membrane protein synthesis, assembly, and delivery is associated with several diseases such as cystic fibrosis, retinitis pigmentosa, nephrogenic diabetes insipidus, another key question is how the protein quality control deals with the aggregated membrane proteins. similar to soluble pqc compartments, increasing evidence indicates the existence of quality control structures for membrane proteins [24], but the formation and composition of these structures are poorly characterized. recent studies have implicated requirement of certain pqc factors for the formation of these structures, including chaperones (hsp70, dnajb, bag3), molecular motors, microtubules, and microtubule-associated factors (histone deacetylase; hdac6) [25, 26] . however, the basic formation mechanism of these structures is vague. moreover, the manners in which substrates are recognized and targeted to aggresomes leading to autophagy are not known. recent studies have supported the notion of er membrane chaperones playing pivotal role in recognition and fate of aberrant clients. for instance, membrane-localized j-protein b12 along with cytosolic hsp70 is reported as a potential factor for membrane client recognition [27, 28] . another hsp70 cochaperone, bag3, was also reported to be involved in targeting misfolded client to the quality control sites for further processing [26] . also, an unbiased rnai screening analysis toward aggresome substrate (synphilin-1) has identified ruvbl proteins as aggresome-forming proteins with disaggregase activity [29] . in addition, little is understood about the underlying mechanism of retrotranslocation of membrane clients during erad, with several groups suggesting direct interplay of membrane channels hrd1 and derlin-1 in client selection and retrotranslocation [30, 31] . viruses hijack host factors called 'cues' by either exploiting their cellular role or modify to facilitate specific function [3] . several viruses trick host pqc factors into performing novel functions to support infection, which in turn has helped us to learn about the function and molecular mechanism of these host factors. it is well established that viruses exploit host pqc factors for many aspects of their life cycle [4], including entry, replication, and assembly (table 1) . a detailed overview of viruses, which use different steps of proteostasis during infection (as shown in fig. 2) , is discussed below. in the case of nonenveloped viruses host entry and genome delivery is poorly characterized. polyomavirus family is the most studied nonenveloped virus whose host entry is well understood. during entry, the virus reaches the er from the cell surface and co-opts erad factors to reach cytosol. specifically, the pdi family of enzymes reduces and isomerizes the viral disulfide bonds that often expose hydrophobic epitopes [32, 33] . these changes partially disassemble the virus and the particle now mimics a giant misfolded protein aggregate, which now recruits hsp70 homolog bip and its luminal cochaperones [34, 35] . the restructured, hydrophobic virus is primed for membrane penetration, by exploiting molecular motor kinesin-1 to drive the reorganization of er membrane chaperone b14 to form the virus membrane penetration site, called 'focus' [36] . the focus-localized virus is then extracted from the membrane by a b14-tethered cytosolic disaggregation machinery (b14, hsc70, and hsp110), consequently reaching the cytosol [37] . in summary, studies on polyomavirus have unraveled the interplay of host pqc components in the er lumen, er membrane, and the cytosol. a similar mechanism has been proposed for the entry and disassembly of human papillomavirus (hpv). for instance, several studies have proposed hpv reaching er during host entry and utilizing erresident pdi family proteins [38] . in addition, hsp70 chaperone system has been demonstrated to disassemble hpv in vitro, a mechanism similar to disassembly of polyomavirus [39] . but a detailed mechanistic understanding of the host membrane penetration and virus disassembly in hpv infection is poorly understood. another well-characterized example of host quality control machinery being utilized by a virus to promote its disassembly is influenza a virus (iav), an enveloped dna virus. during host entry, the iav capsid released from late endosome mimics as misfolded protein aggregate by carrying unanchored ubiquitin chains that activates histone deacetylase 6 (hdac6) to recruit cytoskeleton motors that generate opposing physical forces to break apart the capsid and disassemble the virus [40] . another example of an enveloped virus taking advantage of host pqc factors is in the case of vaccinia virus (vacv), the prototypic poxvirus. vacv has evolved a complex multistep core disassembly and genome release process due to its shape and structure. after 'core activation', host proteasome activity is required for core degradation and genome release [41] . overall, the examples illustrated above demonstrate how viruses hijack host protein quality control machinery and tweak them to promote virus entry and disassembly. nonetheless, studies on these viruses have demonstrated the key components of the aggresome formation and disassembly machinery and also provided a broad understanding of host components and cellular processes. postentry into the host cell, viral genome is transcribed and translated to promote virus replication and assembly and for all viruses this step depends entirely on the host proteostasis machinery. numerous viruses exploit host pqc factors to build site of replication and promote assembly. several members of flaviviridae family are reported to indirectly utilize er membrane chaperones to build and sustain their replication site. for instance, during hepatitis c virus (hcv) replication, virus induces er membrane rearrangement to form a viral replication factory. although, several chaperones (hsp70, hsp90, and calnexin) are implicated to play a role in virus replication, the exact composition and mechanism of replication factory formation is poorly defined and proposed to be closely related to pqc [42] . similarly, recent study on dengue virus (denv) has illuminated the requirement of hsp70 chaperone network that are required at distinct steps of the viral cycle, including entry, rna replication, and virion biogenesis. more importantly, the role of hsp70 at each step is specified by nine distinct dnaj cofactors [43] . of these, dnajb11 relocalizes to virus-induced replication complex, while dnajb6 facilitates virion biogenesis. studies on recently emerged zika virus (zikv) has demonstrated widespread remodeling of intracellular membrane and formation of cytoplasmic vacuoles. several er and cytosolic chaperones are implicated in formation of these vacuoles, but a thorough understanding is needed to reveal the importance and formation of these vacuoles [44] . globally, studies on flaviviruses have provided vital information on the membrane remodeling and role of chaperones during er membrane-derived compartment formations. another instance of pqc factors that are subverted to promote virus infection is for herpes simplex virus (hsv)-1, an enveloped dna virus. it has been proposed that the virus-induced replication compartment is enriched in chaperones such as hsc70, hsp90, bag3, and proteosomes, which perhaps remodel viral replication and regulatory proteins to promote hsv-1 replication [45] . although the virus-induced replication compartments have traces of pqc compartments, they vary in their protein composition and especially how they are built. nevertheless, studies on hsv-1, similar to flaviviruses, have provided key information on formation, maintenance, and functioning of the pqc compartments. in the case of enveloped rna rotavirus, the final assembly of the viral particle takes place in the er [46] , where er-resident chaperones grp78, pdi, calnexin, and calreticulin are reported to promote morphogenesis of the viral particle. specifically, these chaperones promote accurate trimming of the glycan chains on vp7 and nsp4, the correct formation of vp7 disulfide bonds, and the incorporation of properly folded vp7 into assembled rotavirus [47] . overall, studies on rotavirus assembly and morphogenesis have provided vital information on the interplay of chaperones and protein homeostasis in the er. in conclusion, the aforementioned example of viruses utilizing pqc factors as cues during infection has provided a broad understanding of host proteostasis mechanism. long-term research should focus on studying the quality control compartment for membrane protein aggregates. the key outstanding question is to understand the mechanism of membrane substrate recognition by the chaperone system. specifically, pinpointing the identity of er luminal, membrane, and cytosolic factors for a specific misfolded membrane client is vital. it is also important to clarify how misfolded/aggregated membrane substrates are refolded and sequestered and if not, how they are disaggregated and targeted toward degradation pathways. studies on virus should guide our understanding of how aggregated membrane proteins are processed from the cell in order to maintain cellular proteostasis and understanding how proteostasis pathways are affected in the cells infected with viruses. some of the experimental approaches should focus on unbiased proteomic analysis for specific membrane protein substrates to identify target pqc components. these targets should be further validated with gain and/or loss of function studies. from the virus perspective, the identities of the host quality control factors that influence the formation of virus-induced structures and also understanding how host proteostasis is impacted by formation of these structures are vital. currently several therapeutic options are explored for protein misfolding-related diseases, specifically targeting prevention, refolding, and degradation pathways [48] . future research should be directed toward unlocking further secrets of cellular protein homeostasis in conjunction with virus infection and provide therapeutic targets to combat diseases caused by these toxic agents, and to illuminate novel cellular mechanisms. for instance, these insights should help us develop molecular and pharmacological chaperones to prevent formation of protein aggregates thereby delaying the onset of misfolded protein-associated diseases or even develop antiviral agents. an allosteric hsp70 inhibitor, jg40, has been shown to potently block infection of different flaviviruses (dengue, yellow fever, west nile and japanese encephalitis viruses) without exerting toxicity to the host cells [43] . thus, targeting host chaperone networks should provide a path for broad-spectrum antivirals. der1 promotes movement of misfolded proteins through the endoplasmic reticulum membrane erp29 triggers a conformational change in polyomavirus to stimulate membrane binding simian virus 40 depends on er protein folding and quality control factors for entry into host cells erdj5 reductase cooperates with protein disulfide isomerase to promote simian virus 40 endoplasmic reticulum membrane translocation bap31 and bip are essential for dislocation of sv40 from the endoplasmic reticulum to the cytosol exploiting the kinesin-1 molecular motor to generate a virus membrane penetration site a non-enveloped virus hijacks host disaggregation machinery to translocate across the endoplasmic reticulum membrane downregulation of protein disulfide isomerase inhibits infection by the mouse polyomavirus chaperone-mediated in vitro disassembly of polyomaand papillomaviruses influenza a virus uses the aggresome processing machinery for host cell entry rnai screening reveals proteasome-and cullin3-dependent stages in vaccinia virus infection hepatitis c virus rna replication and assembly: living on the fat of the land defining hsp70 subnetworks in dengue virus replication reveals key vulnerability in flavivirus infection zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells virus-induced chaperone-enriched (vice) domains function as nuclear protein quality control centers during hsv-1 infection how viruses use the endoplasmic reticulum for entry, replication, and assembly endoplasmic reticulum chaperones are involved in the morphogenesis of rotavirus infectious particles protein misfolding in neurodegenerative diseases: implications and strategies a pdi family network acts distinctly and coordinately with erp29 to facilitate polyomavirus infection role of cell-type-specific endoplasmic reticulum-associated degradation in polyomavirus trafficking early events during bk virus entry and disassembly opposing effects of bacitracin on human papillomavirus type 16 infection: enhancement of binding and entry and inhibition of endosomal penetration sirna screen of early poxvirus genes identifies the aaa+ atpase d5 as the virus genomeuncoating factor ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors distinct classes of proteasome-modulating agents cooperatively augment recombinant adeno-associated virus type 2 and type 5-mediated transduction from the apical surfaces of human airway epithelia bag3, a host cochaperone, facilitates varicella-zoster virus replication the orf2 protein of hepatitis e virus binds the 5' region of viral rna coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting shortlived erad regulators, for replication unconventional roles of nonlipidated lc3 in erad tuning and coronavirus infection mouse mammary tumor virus signal peptide uses a novel p97-dependent and derlin-independent retrotranslocation mechanism to escape proteasomal degradation acknowledgements i thank billy tsai (university of michigan) for careful reading of the manuscript. i also thank members of tsai lab for discussions and suggestions. the author declares no conflict of interests. key: cord-287410-boxxlopy authors: devi, arpita; chaitanya, nyshadham s. n. title: in silico designing of multi-epitope vaccine construct against human coronavirus infections date: 2020-08-10 journal: journal of biomolecular structure & dynamics doi: 10.1080/07391102.2020.1804460 sha: doc_id: 287410 cord_uid: boxxlopy single stranded rna viruses were known to cause variety of diseases since many years and are gaining much importance due to pandemic after the identification of a novel corona virus (severe acute respiratory syndrome-coronavirus (sars-cov-2)). seven coronaviruses (covs) are known to infect humans and they are oc43 cov, nl63 cov, hku1 cov, middle east respiratory syndrome, sars cov, and sars cov-2. virus replication weakens the immune system of host thereby altering t-cell count and much of interferon response. although no vaccine or therapeutic treatment has been approved till now for cov infection, trials of vaccine against sars cov-2 are in progress. one of the epitopes used for vaccine production is of the spike protein on the surface of virus. the work focuses on designing of multi-epitope vaccine construct for treatment of seven human cov infections using the epitopes present on the spike protein of human covs. to address this, immuno-informatics techniques have been employed to design multi-epitope vaccine construct. band t-cell epitopes of the spike proteins have been predicted and designed into a multi-epitope vaccine construct. the tertiary structure of the vaccine construct along with the adjuvant has been modelled and the physiochemical properties have been predicted. the multi-epitope vaccine construct has antigenic and non-allergenic property. after validation, refinement and disulphide engineering of the vaccine construct, molecular docking with toll-like receptors (tlrs) have been performed. molecular dynamics simulation in aqueous environment predicted that the vaccine-tlrs complexes were stable. the vaccine construct is predicted to be able to trigger primary immune response in silico. communicated by ramaswamy h. sarma coronavirus (cov) belong to large and enveloped subfamily viruses contain sense single strand rna that are categorized into four genera namely alpha, beta, gamma, and delta, among them alpha-and beta-covs known to infect humans (yi et al., 2020) . seven different types of corona virus that infect humans of alpha covs includes 229e and nl63; and beta-covs includes oc43, hku1, mers-cov, and severe acute respiratory syndrome (sars-cov) and sars-cov-2 (waleed, 2020) . s, m, n, structural proteins are encoded by genome of cov and are common for all types of virus whereas e proteins are encoded in human coronavirus 229e (hcov-229e) and hcov-nl63 genome, while he protein is encoded by hcov-oc43 and hcov-hku1 genome apart from structural proteins. the spike (s) protein mediates receptor-binding and fusion with the host cell membrane. the membrane (m) protein plays an important role in viral assembly. the nucleocapsid protein (n) regulates viral rna synthesis, and may interact with m protein during virus budding. the hemagglutinin-esterase (he) glycoprotein found only in the beta-covs and hemagglutinin moiety binds to neuraminic acid on the host cell surface thereby permitting initial adsorption of virus to the membrane. hcov-229e genome consists of 27,317 bases that infects humans and bats (lim et al., 2016) . along with hcov-oc43, it causes common cold (gaunt et al., 2010) . hcov-229e is associated with a range of respiratory symptoms, ranging from the common cold to pneumonia, hcov-229e encodes four structural proteins, spike (s), envelope i, membrane (m), and nucleocapsid (n). hcov-nl63, a member of coronaviridae is large-envelope virus of positive-sense single-stranded rna genome of $27,553 bases in length that contains a cap structure at its 5 0 end and a poly (a) tail at its 3 0 end. the genome consists of 5 0 and 3 0 noncoding regions (ncrs), and the genes encodes for four structural proteins namely spike (s), envelope i, membrane (m), and nucleocapsid (n) (kim et al., 2018) . the hcov-oc43 genome encompasses 30,738 nucleotides of which 5 0 two-thirds of the genome consists of two large replicase open reading frames (orfs) and 3 0 end includes several structural and accessory protein genes: an envelope-associated he glycoprotein gene, a spike (s) glycoprotein gene; an envelope i protein gene; a matrix (m) glycoprotein gene; and a nucleocapsid (n) phosphoprotein gene (vijgen et al., 2005) . the genome of cov-hku1 is 29,926 nucleotides; polyadenylated rna with gc content is 32%, which is lowest among all known coronaviruses. the genome organization is with the characteristic gene order 5 0 -replicase, spike (s), envelope i, and membrane (m), nucleocapsid (n)-3 0 . both 5 0 and 3 0 ends contain short untranslated regions (utrs). the 5 0 end of the genome consists of a putative 5 0 leader sequence (woo et al., 2005) . the first imported mers-cov strain was named chinagd01 and 30,114 nucleotide long, including the 3 0 and 5 0 utrs (lu, 2015) . the genome structure is a single-stranded rna (ssrna) encoding replicase polyproteins (orfs 1ab and 1a), structural proteins (e, n, and m), a surface (spike) glycoprotein (s), and sulphidral proteins (orfs 3, 4a, 4 b, and 5) (mackay & arden, 2015) . mers-cov has been assumed to be transmitted from bats and spread to humans through intermediate hosts (coleman & frieman, 2014) . sars genome contains 29,751 bases with 5 0 cap structure and 3 0 polyadenylation tract. this complex then associates with the m protein in the membranes of the er, and virus particles form as the nucleocapsid complex buds into the lumen of the er (marra et al., 2003) . the single-stranded rna genome of the sars cov-2 was 29,891 nucleotides in size, contains two flanking utrs and a single long orf encoding a polyprotein, it is arranged in order of 5 0 -replicase (orf1/ab)-structural proteins (spike (s)-envelope i-membrane (m)-nucleocapsid (n))-3 0 and lacks the he gene (chan et al., 2020) . no drugs or vaccines are available to treat any of the hcov infections. therapies to treat covid-19 caused by sars cov-2 include antiviral drugs (lu, 2020) , which includes remdesivir (warren et al., 2016) , baricitinb, interferon-a, lopinavir/ritonavir, and ribavirin (zumla et al., 2016) ; immunosuppressant, steroids, and antibodies from plasma of recovered patients (kreil & farcet, 2018) . according to who, hydroxychloroquine is a promising candidate to reduce the pathogenicity of sars-cov-2 until date. studies revealed that several viral structural proteins such as small envelope i, membrane (m), nucleocapsid (n), and spike (s) play important roles in sars infection (simmons et al., 2004) . the 180-kda spike protein plays a central role in the host cell attachment and entry processes and comprised of s1 and s2 subunits. the s1 subunit contains a signal peptide, n-terminal domain, and receptor-binding domain, while the s2 subunit contains a fusion peptide, heptad repeat (hr) 1 and 2, transmembrane domain i, and cytoplasmic domain (cp) (chan et al., 2020) . coronavirus s proteins contain short amino-terminal hydrophobic signal sequence motifs (von heijne, 1984) . although some coronavirus spike proteins cleaved between the s1 and s2 regions as part of activation process, unlike sars-cov spike not cleaved before, internalized inside the host (xiao et al., 2003) . the more variable amino-terminal region of the spike protein (s1) seems to contain the receptor-binding activity (wong et al., 2004) . the more conserved s2 region contains the transmembrane anchor, palmitic acid acylation site (thorp et al., 2006 ) that is important for membrane fusion (mcbride & machamer, 2010) , and the coiled-coil fusion motor domain (bosch et al., 2003) . high-resolution x-ray crystallography structures obtained for coronavirus spike protein reveals sars-cov in conjunction with angiotensin-i-converting enzyme 2 (ace2) (pdb id: 3d0g, 3d0h, 3d0i, 6acc, 6acd) a cellular receptor for sars-cov (li et al., 2003) and hcov-nl63 (hofmann et al., 2005) (pdb id: 3kbh). image analysis of cryoelectron micrographs of sars-cov (beniac et al., 2006) and other coronaviruses (neuman et al., 2006) confirms that spikes exist as a homotrimer in the native perfusion state on virion-and protein-mediated fusion to its receptor-binding triggers conformational changes including disulphide reshuffling (lavillette et al., 2006) . spike protein is incorporated into virion through interactions with the membrane protein m (godeke et al., 2000) . there are different kinds of receptors for different virus for effective binding to its host. 229e virus enters the host cell by binding to the aminopeptidase n receptor (fehr & perlman, 2015) . nl63 virus enters its host cell by the ace2 receptor (brielle et al., 2020) . oc43 virus enters its host cell by binding to the n-acetyl-9-o-acetylneuraminic acid receptor (li, 2016) . hku1 virus enters its host cell by binding to the n-acetyl-9-o-acetylneuraminic acid receptor (lim et al., 2016) . mers-cov virus enters host cell by binding to the dpp4 receptor (fehr & perlman, 2015) . sars cov virus infects the epithelial cells within the lungs and enters the host cell by binding to ace2 (ge et al., 2013) . sars cov-2 virus enters the human cells by binding to ace2 receptor (letko et al., 2020) . three hcovs, mers cov, sars cov, and sars cov-2 causes severe symptoms in humans. middle east respiratory syndrome (mers) first reported in saudi arabia in september 2012 and sars was identified in southern china in 2003. recently, sars cov-2 became a pandemic spreading to 213 countries and territories. many researchers are working towards development of vaccine for sars cov-2. thus, our work focuses on designing of multi-epitope vaccine against all the hcovs (known so far) to avoid any repeated outbreak in near future. s-protein of coronaviruses is the main agent for attachment and entry of the viruses into the human cell. the epitopes from the s-protein of seven hcovs used to design a multi-epitope vaccine construct. sequences of full-length proteins of hcov-oc43, ku1, 229e, nlc3, mers, sars, and novel sars were retrieved from uniprot (http://uniprot.org) and ncbi (http://ncbi.nlm.nih.gov) databases. sequence of oc43, ku1, 229e, nlc3, and sars coronavirus was deposited with uniprot id p36334, q19u45, p15423, q6q1s2, and p59594, respectively. sequence of mers cov and novel sars cov-2 was deposited in ncbi database with accession nos. akj80137 and qih45053, respectively. clustal omega was used for multiple sequence analysis and phylogenetic analysis (madeira et al., 2019) . full length sequences of hcov-oc43, ku1, 229e, nlc3, mers, sars, and novel sars were provided in fasta format to perform the analysis. clustal omega uses seeded guide trees and hidden markov model profile-profile technique to align two or more sequences. the phylogenetic analysis was performed with a neighbour-joining tree. percent identity matrix was generated after phylogenetic analysis. multiple sequence alignment was done to check the sequence similarity of the s-protein of the coronaviruses. 2.3. b-cell, ctl, and htl epitope prediction b-cell epitopes were predicted using the modelled tertiary structures of s-protein of hcovs in ellipro webserver (ponomarenko et al., 2008) . each predicted epitope is given a score known as protrusion index value by the server. maximum score is 1 and higher the score better is the predicted epitope. for each protein, a number of epitope has been predicted. for cytotoxic t-cell lymphocyte (ctl) epitope prediction netctl 1.2 webserver was used (larsen et al., 2007) . this server predicts the mhc-i-binding epitopes by using neural network algorithm. the server gives a score based on predicted mhc class i-binding affinity, proteosomal c terminal cleavage and tap transport efficiency. higher score indicates high specificity of the epitopes towards mhc-i. the epitope with score >3 was further analysed. helper t-cell lymphocyte (htl) epitopes were predicted in iedb webserver (wang et al., 2010) . the server predicted the mhc-ii-binding epitopes using iedb recommended consensus approach. prediction of mhc-iibinding epitopes was done using hla-drb1 â 01:01, hla-dpa1 â 01/dpb1 â 04:01, hla-dqa1 â 03:01/dqb1 â 03:02 alleles. the server gives a percentile rank to the predicted epitopes. lower rank indicates good binders. hence, the epitopes with percentile rank of <1 were further analysed for their ability to induce ifn-c production by using the ifnepitope server (http://crdd.osdd.net/raghava/ifnepitope/). the b-cell, ctl and ifn-c producing htl epitopes were further screened for antigenicity, allergenicity, and toxicity properties. the antigenicity of the epitopes and vaccine constructs was predicted using vaxigen v2.0 (doytchinova & flower, 2007) webserver. vaxigen v2.0 uses alignment-free approach for antigen prediction. the allergenicity of the epitopes and vaccine construct was predicted using allertop v2.0 (dimitrov et al., 2014) webserver. allertop uses amino acid e-descriptors, auto-and cross-covariance transformation, and several machine learning methods for classification of allergens. the toxicity of the epitopes was predicted using toxinpred webserver (gupta et al., 2013) . toxinpred can predict toxicity of peptides with <50 residues. hence, the epitopes with more than 50 residues were chopped into fragments of 50 residues and checked for toxicity. the high scoring b-cell and ctl epitopes and low scoring htl epitopes with non-antigenic, non-allergenic and nontoxic properties were linked by appropriate linkers to design the multi-epitope construct. also, 50s ribosomal protein l7/ l12 (ncbi accession no. p9whe and uniprot id p0a7k2) was used as an adjuvant and was attached to the n-terminal through eaaak linker (kar et al., 2020; naz et al., 2020; samad et al., 2020) . the adjuvant is believed to improve the antigenicity of the vaccine. the b-cell and htl epitopes were linked by gpgpg linkers and ctl epitopes were linked by aay linkers (kar et al., 2020; samad et al., 2020) . a 6xhis tag was incorporated at the c-terminal end to aid in protein purification and identification. secondary structure of the vaccine construct was predicted in raptorx webserver (wang et al., 2011) . the secondary structures are predicted in the form of helix, b-sheet and loop. tertiary structures were predicted in i-tasser webserver (roy et al., 2010) . i-tasser server stands for iterative threading assembly refinement server and is an integrated platform for automated protein structure and function prediction. i-tasser first generates fragments of three-dimensional (3d) atomic models from the primary sequence by multiple threading alignments. then the fragments are reassembled into full-length models by replica-exchange monte carlo simulations. the 3 d model obtained for the vaccine construct was refined in the 3drefine server (http://sysbio.rnet.missouri.edu/3drefine/). the server combines iterative optimization of hydrogen bonding network and atomic level energy minimization using composite physics and knowledge-based force fields for tertiary structure refinement (bhattacharya et al., 2016) . disulphide engineering was done to rationally incorporate of disulphide bonds in the modelled structure of the vaccine construct. disulphide engineering was done using disulphide by design 2 v2.12 webserver (craig & dombkowski, 2013) . the server rapidly assessed for proximity and geometry of the amino acid residues consistent with disulphide formation. tertiary structure model validation detects potential errors in predicted 3 d models. the structure validation was performed in saves v5.0 server (https://servicesn.mbi.ucla.edu/ saves/). a ramachandran plot was obtained in this server to visualize energetically allowed and disallowed dihedral angles psi (w) and phi (/) of an amino acid. the server also gave errat score which is used to analyse non-bonded atom-atom interactions compared to reliable high-resolution crystallography structures. verify 3d score is also obtained from this server. this score determines the compatibility of an atomic model (3d) with its primary sequence (1d). the overall model quality was assessed in prosa webserver (https://prosa.services.came.sbg.ac.at/prosa.php). protparam webserver (https://web.expasy.org/protparam/) was used to predict various physiochemical properties of the constructs such as amino acid composition, theoretical isoelectric point (pi), instability index, in vitro and in vivo halflife, aliphatic index, and molecular weight. grand average of hydropathicity (gravy) was calculated in gravy calculator webserver (http://www.gravy-calculator.de/). toll-like receptors (tlrs) are immune receptors whose activation leads to intracellular signalling pathway. tlr2 and tlr8 induce the production of nf-jb. tlr3 induces the activation of irf3 and nf-jb. tlr4 activation leads to an intracellular signalling pathway nf-jb and inflammatory cytokine production. tlr5 induces the activation of activation leads to an intracellular signalling pathway nf-jb and il8. all the tlrs are located at the cell membrane thus; they are the first molecules to come in contact with pathogens. adjuvant, 50s ribosomal protein attached to the n-terminal of the vaccine construct has the ability to activate tlr4. however, other tlrs may also come in contact with the adjuvant. thus, molecular docking of the multi-epitope vaccine construct with tlr2 (pdb id: 6nig), tlr3 (pdb id: 2a0z), tlr4 (pdb id: 4g8a), tlr5 (pdb id: 3j0a), and tlr8 (pdb id: 4r0a) receptor was performed using the cluspro 2.0 (kozakov et al., 2017) and hdock (yan et al., 2020) webservers in order to evaluate the interaction between ligand and receptor and consequently the activation of an immune response. before docking, the x-ray crystallized structures of tlrs were prepared by removing solvent molecules and other ligands followed by addition of hydrogen atoms and energy minimization in swiss pdbviewer deep view software package. binding pockets of tlrs were searched using castp 3.0 webserver (http://sts.bioe.uic.edu/castp/calculation.html). the vaccine construct and vaccine construct-tlr complexes were subjected to 10 ns molecular dynamics simulation using gromacs 4.6.5 software package (van der spoel et al., 2005) . for the simulation, charmm forcefield and spc/e water model was used. the simulation system was neutralized by adding the suitable number of na þ /clions. the solvated system was energy minimized in 1000 steps using steepest descent method iterations. after setting the temperature at 300 k and pressure at 1 bar, production simulation was run for 30 ns to evaluate dynamics of vaccine construct and vaccine construct-tlr complexes. the rmsd, rmsf, and radius of gyration profiles of the vaccine construct and vaccine construct-tlr complexes were generated. codon biasness occurs between prokaryotic and eukaryotic genes. codon optimization therefore becomes necessary for protein expression in a prokaryotic host. codon optimization of the designed multi-epitope vaccine construct was done in java codon adaptation tool (jcat) server (http://www. prodoric.de/jcat) for gene expression in the e. coli (strain k12) host. rho-independent transcription termination, prokaryote ribosome-binding site, and restriction enzymes cleavage sites were avoided during codon optimization. codon adaptation index (cai) and percentage gc content was also received as output along with the optimized nucleotide sequence. the optimized nucleotide sequence of the final vaccine construct was cloned into the e. coli pet-28a (þ) vector using snapgene tool and ndei and bglii restriction sites were introduced to the n and c-terminals of the sequence, respectively. to predict the probable immune response of the designed multi-epitope vaccine construct in human immune system, in silico immune simulations were conducted using the c-immsim server (http://150.146.2.1/c-immsim/index.php) (rapin et al., 2010) . c-immsim is a novel in silico approach for the study of the mammalian immune system the tool is a combination of a mesoscopic scale simulator of the immune system with machine learning techniques for molecular-level predictions of major histocompatibility complex (mhc)-peptide-binding interactions, linear b-cell epitope discovery, and protein-protein potential estimation. all simulation parameters were kept as default and a three dose of injection (1000 antigens) at 4 weeks apart was given. the response after the injections was analysed. s-proteins of seven hcovs causing mild and severe disorders in human have been studied. primary structure comparison of the s-proteins revealed a sequence similarity towards the c-terminal of the proteins (supplementary figure 1) . based on similarity, it was found that s-protein of sars cov-2 and sars cov share the highest identity (77.46%) among all the hcovs. s-protein of oc43 cov and ku1 cov share 66.21% identity and 229e cov, and nl63 cov share 64.07% identity. s-protein of mers cov shares highest identity with oc43 cov (32.87%). the percent identity matrix of the s-proteins of hcovs is shown in table 1 . tertiary structure of full-length s-proteins of hcovs shows a similar y-shaped structure. arm a is the receptor-binding domain. arm c contains the transmembrane domain and the cytoplasmic domain. from figure 1 , it is clearly seen that the arms a and b of s-protein of 229e cov and nl63 cov is in closed conformation. the arms a and b of s-proteins of all other hcovs are in open conformation. another difference in the tertiary structure is seen in the c arm, which is the c-terminal tail of s-proteins. the c arm of s-protein of 229e cov and nl63 cov is in an elongated form, whereas the c arm is in a loop like structure in the other hcovs. b-cell epitopes of different lengths were predicted for each s-protein of hcovs (supplementary tables 1-7) . ctl epitopes of 9 residues along with their mhc-binding affinity, c-terminal cleavage affinity and transport affinity were predicted (supplementary table 8 ). htl epitopes of 15 residue length interacting with hla-drb1 â 01:01 and hla-dqa1 â 01:01/ dqb1 â 06:01 alleles were predicted (supplementary tables 9-13). the epitopes were subjected to allergenicity, antigenicity and toxicity prediction. the epitopes with best scores and non-allergenic, antigenic, and non-toxic properties were selected for the vaccine construct. eighteen epitopes from the s-protein of hcovs were linked by linkers to form the final vaccine construct ( table 2) . the b-cell and htl epitopes were linked by gpgpg linkers whereas the ctl epitopes were linked by aay linkers. the tlr4 agonist, 50s ribosomal l7/l12 protein was added to the amino terminus of the vaccine construct using an eaaak linker and 6xhis tag was added at the c-terminal to help in protein purification and identification. the final vaccine construct designed consisted of 1189 amino acid residues (figure 2) . the antigenicity of the vaccine construct including the adjuvant sequence and his-tag was predicted by the vaxijen 2.0 server to be 0.6452 with a bacteria model at a threshold of 0.4. the main vaccine sequence without adjuvant and histag gave scores of 0.6635 with bacteria model at a threshold of 0.4 on vaxijen 2.0 server. thus, the results indicate that the designed vaccine is antigenic in nature. the presence of adjuvant and his-tag doesn't change the antigenic property of the construct. the vaccine construct with and without the adjuvant and his-tag is predicted to be non-allergenic by allertop v.2 and allergenfp servers. the calculated molecular weight of the vaccine construct is 128.381 kda. theoretical pi value of the construct is 5.57, indicating the protein is slightly acidic in nature. the estimated half-life of the vaccine construct is 30 h mammalian reticulocytes in vitro, and >20 h in yeast in vivo and >10 h in e. coli in vivo. the instability index (ii) is computed to be 29.71, which classify the protein as stable. the aliphatic index is calculated to be 29.71 and grand average of hydropathicity (gravy) score is predicted to be à0.072, indicating that protein is hydrophilic in nature. the secondary structure of the multi-epitope vaccine construct with adjuvant and his-tag is predicted to be composed of 17% a-helix, 17% b-sheet, and 64% coils. of the total residues 42% are predicted to be exposed and 29% residues are predicted to be buried. tertiary structure prediction by i-tasser server predicted five tertiary structure models of the peptide aptamer. ten threading templates were used for the prediction, of which 6jx7a, 1rqua, 6nzka, and 5ijoj were the best templates. all the templates showed good alignment as per their z-score values, ranging from 1.40 to 18.24. the calculated c-score of the five predicted models ranges from à1.49 to à2.69. the c-score range is generally between à5 and 2 and higher value indicates higher confidence (lee et al., 2010) . thus, the model with the highest c-score was selected for further studies. this model had a tm-score of 0.51 ± 0.15 with an estimated rmsd of 13.6 ± 4.0 å. the tmscore is a measure of structural similarity between two structures (shaik, 2019) . a tm-score of >0.5 indicates a model of correct topology and a tm score <0.17 means random similarity (shaik, 2019) . these cut-off values are independent of the length of protein. the tertiary structure of the vaccine construct was refined on 3drefine server and it yielded five models. model 1 was found to be the best model based on model quality scores for all refined models (figure 3(a) ). the model quality scores included high accuracy global distance test (gdt-ha) score of 0.9899, global distance test total score of 0.9899, rmsd score of 0.263 å, molprobity score of 3.835, rwplus score of à201,345.267, and 3drefine score of 11,651.6. this model was chosen as the final vaccine construct model for further analysis. disulphide engineering was performed with 10 selected pair of residues (pro563-asn566, gly44-ala135, gly75-gly80, pro786-leu829, glu29-phe31, glu113-ala116, gln143-cys 239, phe1109-try1110, leu553-asn556, and ala1053-phe1072). the selection was done on the basis of energy, v 3 value, and b-factor. the residue pairs with energy of 1 kcal/mol to 2.2 kcal/mol (mean value is 1.0 kcal/mol, and the 90th percentile is 2.2 kcal/mol), v 3 value of à85 to à90 and þ95 to þ100 (mean value is à87 and þ97 ) and b-factor of 29-33 (mean b-factor for residues involved in stabilizing disulphide bonds is 31.6) (craig & dombkowski, 2013) . after refinement and disulphide engineering of the modelled structure, validation was done. the overall model quality was à2 as calculated by prosa. the ramachandran plot generated showed that 74.2% of the residues are in favourable region, 15.2% residues are in the allowed region, and 10.6% residues are in outline region. the quality and potential errors in the tertiary structure were verified by errat. the chosen model after refinement had an overall quality factor of 64.91% with errat. verify3d also passed the modelled structure as >80% of the residues have averaged 3 d-1d score !0.2. verify3d determines the compatibility of an atomic model (3d) with its own amino acid sequence (1d). binding pocket prediction of the studied tlrs revealed one pocket. however, the solvent accessible surface area and volume of the binding pocket of all the tlrs are not similar (table 3) . molecular docking was performed in hdock server and cluspro 2.0 server. hdock server predicted 100 complexes in each case from which the lowest scoring complex was studied. cluspro 2.0 predicted 10 complexes from which the lowest energy complex was studied. from figure 4 , it can be seen that the vaccine construct has almost similar binding pattern to tlr3, tlr4, and tlr5. the adjuvant containing arm of the vaccine could interact with the predicted binding pocket of the tlrs. based on binding energy, tlr5-vaccine construct complex is predicted to be the best complex in both the servers (table 3) . thus, although the adjuvant used is a known tlr4 agonist, the vaccine construct may have affinity towards tlr3 and tlr5 as well. molecular dynamics simulation of the vaccine construct revealed its stability throughout the simulation. the average rmsd of the vaccine construct alone was 0.08 å. upon binding to tlrs, the average rmsd of the vaccine construct increased. the vaccine construct-tlr complexes were found to be stable with no major fluctuation ( figure 5(a) ). the vaccine construct has shown a fluctuation at 12 ns but was stable after that. one major fluctuation in the vaccine construct-tlr4 complex was seen at 3 ns. the complex was found to be stable after that. the average rmsd of this complex was 0.5 å which is higher than all other complexes. the rmsf of vaccine construct showed that the n-terminal residues and the residues between 40 and 65 positions have highest movement ( figure 5(b) ). the n-terminal of the vaccine construct (residue 1-121) consists of the adjuvant and is involved in interaction with tlrs. however, when the vaccine construct is bound to the tlrs, the rmsf of the highest fluctuating residues of the construct has decreased. this can be due to interaction with tlrs. radius of gyration of vaccine construct after 30 ns simulation was calculated to be 45.74 nm. however, the radius of gyration of vaccine-construct-tlr complexes ranged between 29.47 and 35.67 nm. the radius of gyration profile against time (in ns) reveals that the vaccine construct alone and vaccine construct-tlr complexes were in a compact state till the end of molecular dynamics run (figure 5(c) ). codon optimization of the vaccine construct in e. coli (strain k12) was done for expression of protein. the optimized codon sequence was 3266 nucleotides long. the cai of the optimized nucleotide sequence was 1.0 which means that the nucleotide sequence contains the most frequently occurring codons (rapin et al., 2010) . the average gc content of the optimized sequence is 52.20%. higher the gc content, better is the expression of a protein in prokaryotes (zhou et al., 2014; du, 2018) . the optimized nucleotide sequence was inserted into the pet28a (þ) vector using snapgene software ( figure 6 ). immune simulation by c-immsim server showed the immune responses of three injections. the antigen is calculated to be present for about 5 days after each dose. after first dose, very low level of igm is seen to be present. but after second and third dose, the level of igm and igg subsequently increased. after third dose, the total level of igm and igg antibodies is found to be higher than the level of antigen. similarly, level of memory b-cell is seen to be increasing after each dose. the level of memory t-cells also increased after second dose. however, after third dose, there was no further increase in the level of memory t-cells. the level of ifn-c was found to be increasing at the same level after each dose. the level of ifn-c dropped to basal level at the end of fourth week and subsequent injection of the antigen again triggered the expression of ifn-c. another cytokine il-2 was found to reach its maximum expression after the second dose. the level of igg, igm, and cytokines dropped gradually after 4 weeks but the level of memory b-cells and memory t-cells were found to be constant. thus, the vaccine construct is predicted to trigger mostly igg, igm, b-cell, t-cell, and cytokines (ifn-c and il2) (figure 7 ). three hcov s are known to cause severe respiratory symptoms. mers cov outbreak was seen in 2003, sars cov other hcovs, 229e, nlc3, oc43, and ku1 causes mild respiratory symptoms but 229e and ku1 is also known to cause pneumonia. until the outbreak of sars cov-2, there is no approved treatment for any of the hcov infection. after the outbreak of sars cov-2, researchers are trying to develop vaccine against sars cov-2. b-cell, ctl, and htl epitopes are used as antigenic determinants for efficient vaccine production had much focused on spike (s) protein, which is gaining much attention in research. here in this paper we had focused on antigenic determinants on the sprotein of hcovs and their use as effective vaccine candidates through in silico approach. sequence comparison of sproteins of human corona viruses shows similarity towards the c-terminal end. however, tertiary structure shows a similar y-shaped conformation of the s-proteins. to get insights and details about various antigenic determinants of s-proteins of virus different lengths of b-cell epitopes were predicted using ellipro server. ctl epitopes were predicted using netctl 1.2 server and htl epitopes were predicted using iedb server. the htl epitopes were checked for their ability to produce ifn-c using ifn epitope server. htl epitopes predicted for hcovs 229e and ku1 didn't have the ability to produce ifn-c. all the predicted epitopes were further checked for allergenic, immunogenic, and toxic properties. the high-scoring epitopes (as per the servers) with antigenic, non-allergenic, and non-toxic properties were selected for vaccine construct designing. multi-epitope vaccine construct was designed by linking the selected b-cell and htl epitopes with gpgpg linkage. the ctl epitopes were linked by aay linkers. 50s ribosomal protein, which is a tlr4 agonist is used as an adjuvant and linked to the n-terminus with eaaak linkage .also, for ease of protein purification, 6â his tag is added at c-terminus, thus yielding a final construct of 1189 amino acids. immunogenic properties such as antigenic and allergic predictions performed using vaxijen v2.0 and allertop v.2 server indicates designed vaccine construct is antigenic and non-allergic in nature. the molecular weight of the designed multi-epitope vaccine construct is found to be 128.381 kda with pi of 5.57 and a half-life of 30 h in mammalian reticulocytes. the vaccine construct is also predicted to be stable and hydrophilic in nature. secondary structure prediction of multi-epitope vaccine construct as found to have 17% a-helix, 17% b-sheet, and 64% coils. of the total residues, 42% are predicted to be exposed and 29% residues are predicted to be buried. the tertiary structure of the vaccine construct was refined on 3drefine server. the refined model gdt-ha score of 0.9899, global distance test-total score of 0.9899, rmsd score of 0.263 ð, molprobity score of 3.835, rwplus score of à201,345.267, and 3drefine score of 11,651.6. disulphide engineering was performed with ten selected pair of residues (pro563-asn566, gly44-ala135, gly75-gly80, pro786-leu829, glu29-phe31, glu113-ala116, gln143-cys 239, phe1109-try1110, leu553-asn556, and ala1053-phe1072). the selection was done on the basis of energy, ꭓ3 value, and b-factor. after refinement and disulphide engineering of the modelled structure, validation was done. the overall model quality was à2 as calculated by prosa. the ramachandran plot generated showed that 74.2% of the residues are in favourable region, 15.2% residues are in the allowed region and 10.6% residues are in outline region. the model had an overall quality factor of 64.91% with errat. verify3d also passed the modelled structure as more than 80% of the residues have averaged 3d-1d score !0.2. molecular docking was performed in hdock server and cluspro 2.0 server. the vaccine construct has almost similar binding pattern to tlr3, tlr4, and tlr5. the adjuvant containing arm of the vaccine could interact with the predicted binding pocket of the tlrs. based on binding energy, tlr5vaccine construct complex is predicted to be the best complex in both the servers. the stability of the tlr-vaccine construct complexes were checked by performing a 30 ns molecular dynamics simulation in gromacs 4.6.5 software package. charmm force field and spc/e water model was chosen for molecular dynamics simulation. the average rmsd of the vaccine construct alone was 0.08 å. upon binding to tlrs, the average rmsd of the vaccine construct increased. the vaccine construct-tlr complexes were found to be stable. tlr4-vaccine construct complex showed fluctuation at 3 ns. after 3 ns, there was no major fluctuation in the rmsd profile of tlr4-vaccine construct complex. rmsf profile of the vaccine construct showed the presence of two highly fluctuating regions. the regions are the n-terminal region and the region between 40 and 65 positions. the nterminal of the vaccine construct (residues 1-121) consists of the adjuvant and is involved in interaction with tlrs. interaction of the vaccine construct with tlrs have decreased the fluctuations of the two regions. radius of gyration of vaccine construct after 30 ns simulation was calculated to be 45.74 nm. however, the radius of gyration of vaccine-construct-tlr complexes ranged between 29.47 and 35.67 nm. this change in radius of gyration reveals the compactness of the vaccine construct-tlr complexes. the vaccine construct must be cloned into a suitable vector for expression and purification. pet28a(þ) is a suitable vector for protein expression. prokaryotic vectors often have codon biasness while expressing eukaryotic proteins. thus, codon optimization was done to avoid codon biasness. the cai of the optimized nucleotide sequence was 1.0 and average gc content of the optimized sequence is 52.20%. these two parameters show that the vaccine construct will be highly expressed in the vector. in silico prediction of primary immune response after vaccine injection was also done in c-immsim server. three injections of 1000 antigen molecules without lps were given 4 weeks apart and immune response was recorded for 100 days. the antigen was seen to be present for about 5 days after each dose. after first dose, very low level of igm was generated. but after second and third dose, the level of igm and igg subsequently increased. after third dose, the total level of igm and igg antibodies is found to be higher than the level of antigen. similarly, level of memory b-cell increased after each dose. the level of memory t-cells also increased after second dose. however, after third dose, there was no further increase in the level of memory t-cells. the level of ifn-c was found to be increasing at the same level after each dose. the level of ifn-c dropped to basal level at the end of fourth week and subsequent injection of the antigen again triggered the expression of ifn-c. another cytokine il-2 was found to reach its maximum expression after the second dose. the level of their cytokines such as il4, il12, il10, il6, il18, ifn-a, tgf-b, and ifn-b did not undergo much change even after three doses of antigen injection. the level of igg, igm, and cytokines dropped gradually after 4 weeks but the level of memory b-cells and memory t-cells were found to be constant. thus, the vaccine construct is predicted to trigger mostly igg, igm, b-cell, t-cell, and cytokines (ifn-c and il2). complete eradication of a disease or infection is not possible without a specific treatment. there is always a possibility that previous epidemics can re-emerge and cause severe damage to coming generations. the outbreak of mers and sars cov caused severe damage to some countries but till date no vaccine or treatment has been approved. the present outbreak of sars cov-2 has triggered the importance of vaccine. thus, in this work, we have employed immuneinformatics tools to design multi-epitope vaccine construct against the s-protein of seven hcovs. the s-protein is the medium by which the viral genome transfers to human cells. thus, the vaccine construct designed is expected to trigger host immune system to generate antibodies against the sprotein epitope and restrict the entry of viral genome to host cells. this multi-epitope vaccine may help in boosting the host immune system against seven hcovs. architecture of the sars coronavirus prefusion spike 3drefine: an interactive web server for efficient protein structure refinement the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex the sars-cov-2 exerts a distinctive strategy for interacting with the ace2 human receptor genomic characterization of the 2019 novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan. emerging microbes and infections coronaviruses: important emerging human pathogens disulfide by design 2.0: a webbased tool for disulfide engineering in proteins allertop v.2-a server for in silico prediction of allergens vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines the gc content as a main factor shaping the amino acid usage during bacterial evolution process coronaviruses: an overview of their replication and pathogenesis epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein in silico approach for predicting toxicity of peptides and proteins human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry a candidate multi-epitope vaccine against sars-cov-2 complete genome sequence of human coronavirus nl63 cn0601/14, first isolated in south korea the cluspro web server for protein-protein docking immunoglobulins and virus antibody titers: of past needs, current requirements, and future options large-scale validation of methods for cytotoxic tlymphocyte epitope prediction significant redox insensitivity of the functions of the sars-cov spike glycoprotein: comparison with hiv envelope relative codon adaptation index, a sensitive measure of codon usage bias functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses structure, function, and evolution of coronavirus spike proteins angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus human coronaviruses: a review of virus-host interactions drug treatment options for the 2019-new coronavirus (2019-ncov) complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd the embl-ebi search and sequence analysis tools apis in 2019 the genome sequence of the sars-associated coronavirus palmitoylation of sars-covs protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with m protein designing multi-epitope vaccines to combat emerging coronavirus disease 2019 (covid-19) by employing immuno-informatics approach supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy ellipro: a new structure-based tool for the prediction of antibody epitopes computational immunology meets bioinformatics: the use of prediction tools for molecular binding in the simulation of the immune system i-tasser: a unified platform for automated protein structure and function prediction designing a multi-epitope vaccine against sars-cov-2: an immunoinformatics approach essentials of bioinformatics, volume i: understanding bioinformatics: genes to proteins characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry palmitoylations on murine coronavirus spike proteins are essential for virion assembly and infectivity gromacs: fast, flexible, and free complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event analysis of the distribution of charged residues in the n-terminal region of signal sequences: implications for protein export in prokaryotic and eukaryotic cells understanding the mosaic of covid-19: a review of the ongoing crisis peptide binding predictions for hla dr, dp and dq molecules protein 8-class secondary structure prediction using conditional neural fields therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia the sars-cov s glycoprotein: expression and functional characterization the hdock server for integrated protein-protein docking covid-19: what has been learned and to be learned about the novel coronavirus disease analysis of the relationship between genomic gc content and patterns of base usage, codon usage and amino acid usage in prokaryotes: similar gc content adopts similar compositional frequencies regardless of the phylogenetic lineages coronaviruses-drug discovery and therapeutic options a.d. acknowledges csir-srf for providing financial assistance. n.s.n.c. acknowledges icmr-srf for proving financial assistance. the authors acknowledge the anonymous reviewers for helping to improve the manuscript by providing critical suggestions. no potential conflict of interest was reported by the authors. key: cord-272268-8vrcwwll authors: kedersha, nancy; anderson, paul title: chapter 4 regulation of translation by stress granules and processing bodies date: 2009-10-27 journal: prog mol biol transl sci doi: 10.1016/s1877-1173(09)90004-7 sha: doc_id: 272268 cord_uid: 8vrcwwll stress necessitates rapid reprogramming of translation in order to facilitate an adaptive response and promote survival. cytoplasmic stress granules (sgs) and processing bodies (pbs) are dynamic structures that form in response to stress-induced translational arrest. pbs are linked to mrna silencing and decay, while sgs are more closely linked to translation and the sorting of specific mrnas for different fates. while they share some components and can interact physically, sgs and pbs are regulated independently, house separate functions, and contain unique markers. sg formation is associated with numerous disease states, and the expanding list of sg-associated proteins integrates sg formation with other processes such as transcription, splicing, and survival. growing evidence suggests that sg assembly is initiated by translational arrest, and mediates cross talk with many other signaling pathways. nuclear dna is packaged into chromatin, organized into active and inactive domains, and its availability is regulated by the many factors that control its access to polymerases, splicing factors, and the nuclear periphery. in the cytoplasm, rna is similarly structured, organized, and regulated by interactions with membranes, the cytoskeleton, different organelles, and by a host of aggregation-prone rna-binding proteins. localized gradients of specific mrna transcripts regulate the organization and the timing of gene expression that occurs during development. within individual somatic and germ cells, discrete mrna granules are transiently assembled as mrna transcripts move into and out of polysomes. the recruitment, retention, and removal of specific transcripts into and out of rna granules is orchestrated by numerous mrna binding proteins, which regulate the fate of specific transcripts. consequently, biochemical analysis of protein translation in cell lysates cannot completely recapitulate the translational regulatory pathways that occur in live cells. critical components of the ''cell biology'' of protein translation are mrnp granules known as processing bodies (pbs) and stress granules (sgs). these transient cytoplasmic ''structures'' are actively assembled from untranslated mrna by a host of rna-binding proteins, which determine whether specific transcripts will be reinitiated, degraded, or stored. the mrna within pbs and sgs is in a dynamic equilibrium with polysomes, and this equilibrium is shifted by changes in environmental conditions. specific rna-binding proteins promote the translation of some transcripts and inhibit the translation of others; silenced transcripts are assembled into sgs or pbs, whereas preferential translation occurs outside these bodies. in stressed cells, the selective translational repression of ''housekeeping'' genes conserves anabolic energy for the repair of stress-induced molecular damage. at the same time, enhanced translation of repair enzymes directly repairs this damage while promoting an adaptive response to the altered conditions. thus, shifting specific transcripts between polysomes and rna granules tailors translation to changes in environmental conditions. sgs and pbs are dynamic structures that are continuously assembled/ disassembled from the flux of mrnps that comprise them. drugs that arrest translational elongation and thereby stabilize polysomes promote the disassembly of sgs and pbs, whereas drugs that enhance polysome disassembly promote (but are not sufficient to cause) sg/pb assembly. numerous photobleaching studies indicate that the mrnps that comprise sgs and pbs are in very rapid flux (seconds), although sgs and pbs persist for minutes to hours. a useful analogy holds that mrnps are to sg/pbs as water is to a river; rapidly moving water creates a large stable river, but the water is always in flux. similarly, moving streams of mrnps create sgs and pbs. when examining the functional properties of sg/pbs, it is useful to consider that the functional properties of rivers (e.g., erosion, navigation, and hydroelectric power) are different from those of water alone. similarly, the discrete domains (sgs and pbs) created from relocalized mrnps alter the trafficking of other proteins and have important secondary effects (alternative splicing, transcription, nucleocytoplasmic transport) on overall cell metabolism. in 1999, it was noted that stress-induced translational arrest causes untranslated mrnps to assemble into large cytoplasmic ''sgs,'' whose formation is triggered by, and dependent upon, the phosphorylation of eif2a. 1 these granules contain a large proportion of cytoplasmic poly(a) mrna and pabp, suggesting that most of the cytoplasmic polya(þ) mrna is assembled into the granules upon stress. remarkably, the normally nuclear rna-binding protein tia-1 (t-cell internal antigen 1) rapidly colocalizes in these cytoplasmic granules, and a truncation mutant of tia-1 blocks their formation. as similar rnacontaining ''heat-shock granules'' had been described in plant cells, 2 it seemed likely that mammalian sgs were related to plant ''heat-shock granules.'' only recently 3 was it shown that rna-containing sgs and plant heat-shock granules are in fact distinct: plant heat-shock granules do not contain mrna as originally reported, although plant cells do indeed form bona fide sgs and pbs in addition to heat-shock granules. thus in hindsight, and despite the frequent use of the phrase ''it has long been known'' to describe rna-containing sgs, in actual fact these sgs were first described only a decade ago. sgs were then demonstrated to be dynamic sorting centers rather than stable repositories of mrna, as their apparent identity with plant heat-shock granules had predicted. 4 pharmacological data revealed that sg assembly is blocked by agents that arrest translation elongation (e.g., emetine and cycloheximide), but not by agents that promote translation termination (e.g., puromycin), indicating that polysome disassembly is required for sg assembly. importantly, preassembled sgs are forcibly disassembled by emetine even in the continued presence of phospho-eif2a suggesting that they do not represent static storage depots of mrnps but instead contain mrnps in equilibrium with polysomes. direct measurements using frap (fluorescence recovery after photobleaching) confirmed that both tia-1 and pabp rapidly shuttle into and out of sgs, with tia-1 moving more rapidly than pabp. the half-lives revealed by frap were on the order of 2-10 s, in marked contrast with the much slower kinetics of sg assembly (15-30 min) . the first movies of sg assembly and disassembly revealed that sgs form synchronously throughout the cell and progressively fuse to become larger and fewer. as cells recover from stress, the large ''mature'' sgs disassemble synchronously within individual cells, in a time frame of 4-6 min. these data established that sgs are dynamic microdomains into which pabp and tia-1 rapidly shuttle, and which are in equilibrium with polysomes. pabp is a translational enhancer, and tia-1 is a translational silencer, 5 hence we proposed that sgs constitute dynamic mrna triage domains, in which mrna processing, sorting, and remodeling events regulate the expression of specific transcripts. 6, 7 a more detailed in situ analysis of sgs 8 revealed that they contain small but not large ribosomal subunits, and the initiation factors eif3, eif4e, and eif4g. however, eif5 and eif2 are not prominent components of sgs, despite the fact that phospho-eif2a drives sg assembly. contemporary models of translation initiation suggested that eif5 normally links eif2/gtp/trna met i to eif3 and the small ribosomal subunit to form the 43s complex, which is thought to form prior to the recruitment of eif4f, pabp, and the mrna to form the 48s complex. as sgs contain most components of the 48s complex but lack eif5 and eif2, sgs appear to contain aggregates of aberrant 48s complexes, lacking eif5/eif2/gtp/trna met i but containing instead the tia proteins. in sucrose gradients, these tia-1-containing complexes migrate with small mrnps near the top of the gradient rather than with polysomes. interestingly, phospho-eif2a is detected in late sgs, but not newly formed ones, supporting a model in which lack of ternary complexes drives sg formation via the formation of aberrant 48s complexes. another report 9 partly confirmed these results, but differed in finding eif2a and eif2b in arsenite-induced sgs: whether this reflects differences between antibodies or cell lines remains unresolved. this study showed for the first time that thapsigargin, a specific activator of perk, induces sgs that are prevented by a kinase-dead form of perk. together, these studies established that sg formation is a result of stalled initiation and confirmed a central role for phospho-eif2a in their assembly. in 2003, it was shown that another rna-binding protein, ras-gap binding protein 3 (g3bp), is a sg component whose overexpression nucleates sg assembly, and that this ability is linked to the phosphorylation of a specific g3bp serine residue. 10 g3bp is a multifunctional protein linked to numerous pathways, 11 and its ability to nucleate dynamic (e.g., cycloheximide-reversible) sgs has subsequently been widely exploited in the field to study sg composition. although originally described as an endonuclease, no such activity has been demonstrated in vivo, so the functional consequences of g3bp-nucleated sgs are unknown. however, the au-rich mrna decay factor tristetraprolin (ttp) similarly nucleates sgs which are prevented by its phosphorylation and interactions with 14-3-3 proteins, 12 providing the first example of a protein that moves to sgs under some conditions but not others. a flood of proteins too numerous to cite individually were subsequently identified as sg-associated (summarized in table i ). some of these are considered below. importantly, a natural product isolated from new zealand sponge, pateamine a, induces sgs compositionally similar to those induced by arsenite: these sgs contain eif3, eif4e, eif4g, and tia-1 but are assembled in the absence of eif2a phosphorylation. pull-down studies established that the molecular target of pateamine a is eif4a, 13 the dead-box helicase necessary for cap-dependent translation requiring 48s scanning. toe-printing studies and polysome profiles on in vitro translated material revealed that pateamine a inactivates scanning, apparently by stabilizing interactions between eif4a and eif4b. this study added eif4a and eif4b to the growing list of initiation factors found in sgs. a similar compound named hippuristanol, found in the coral isis hippuris, also blocks eif4a scanning activity by preventing its binding to mrna. 14 remarkably, both pateamine a and hippuristanol induce sgs in mutant eif2a-s51a mefs, which contain only a non-phosphorylatable form of eif2a, 15, 16 and the sgs induced by these drugs contain eif2 and eif5. these drugs have been widely used in subsequent studies to determine whether a given protein is recruited to sgs or not, and as such have been invaluable tools. however, these drugs circumvent the physiological stress response that activates one or some of the multiple kinases that target eif2a (and perhaps other proteins: pkr targets rna helicase a; ref. 17) as well as other kinase cascades. moreover, eif4a is present in multiple isoforms and has other functions (e.g., nmd), and these drugs have other targets in addition to eif4a (e.g., pateamine a binds unrip; ref. 13) . the discovery that yeast 18 and human cells 19 contain dynamically organized sites of mrna decay enzymes and proteins later recognized as part of the microrna silencing machinery (reviewed in refs. 20-23) occurred somewhat later than the first sg studies. pbs exhibit similar dynamics and share some proteins with sgs, but are associated with mrna decay rather than translation. while regulated mrna decay is perhaps the ultimate form of translational control, this subject is beyond the scope of this chapter. sgs are absent from the cytoplasm of normally growing cells, but they are rapidly assembled in cells exposed to sudden changes in environmental conditions. in most cases, sg assembly results from stress-induced phosphorylation of eif2a, a component of the eif2/gtp/trna met i ternary complex that directs trna i met to the 40s ribosomal subunit. phospho-eif2a is a competitive inhibitor of eif2b, a gdp-gtp exchange factor that charges the eif2/gtp/trna met i ternary complex. thus, phosphorylation of eif2a effectively depletes active ternary complex resulting in translational repression. this translation control pathway is initiated by a family of eif2a kinases that are activated by different types of stress granules and processing bodies environmental stress. protein kinase r (pkr) senses heat, uv irradiation, and viral infection. 24, 25 perk/pek (pkr-like er kinase) senses endoplasmic reticular stress accompanying excess production of secretory proteins; gcn2 (general control non-derepressible 2) senses nutrient stress (e.g., amino acid starvation); hri (heme-regulated inhibitor) senses heme levels in erythroid progenitor cells to balance the synthesis of globin and heme, as well as arsenite-induced oxidative stress. lack of ternary complex triggers sg assembly by stalling initiation while allowing elongation to proceed normally, resulting in polysome disassembly, necessary but not sufficient for both sg and pb assembly. notably, pb formation is independent of phospho-eif2a, although it still requires free mrnps. the specificity of stress-induced translational repression correlates with sg assembly. whereas mrnas encoding ''housekeeping'' transcripts are concentrated at sgs, mrnas encoding molecular chaperones that refold stressdenatured proteins (hsp70) or alter the conformation of signaling proteins (hsp90) are selectively excluded from sgs. this has been proposed to account, in part, for the ability of certain transcripts to be selectively translated during stress. although the mechanism by which hsp70 transcripts are excluded from sgs is not known, it is likely that mrnas transcribed during stress acquire or lack distinct protein ''marks'' that determine their functional fate in the cytoplasm. hsp70 transcripts are unusual in several ways: they lack introns, and thereby lack proteins deposited at splicing junctions, one of which (mln51) is selectively recruited to sgs 26 -whether this allows a selective recruitment of spliced mrnas into sgs remains to be determined. in addition, hsp70 mrna possesses a long structured 5 0 -untranslated region (5 0 -utr) that allows translation initiation to occur via a shunting mechanism, 27 which would preclude reliance on eif4a, whose inactivation causes sg assembly. 15, 16 as certain viral 5 0 -utrs exempt transcripts from sg recruitment and translational silencing, 28 the hsp70 5 0 -utr may function similarly. a number of viral mrnas elude or suppress sg assembly by various mechanisms. stress-activated translation of some transcripts is a consequence of upstream open reading frames (orfs) found in the 5 0 -utr. under normal conditions, translation is initiated at the start codon of these orfs, preventing ribosomes from initiating translation at the protein-encoding orf. when stress-induced phosphorylation of eif2a depletes the levels of ternary complex, initiation at the upstream orfs becomes inefficient, allowing some initiation complexes to scan to the cryptic productive initiation codon. this mechanism allows stress-activated translation of atf4, a key transcription factor that regulates the integrated stress response. thus, eif2a-induced sg formation correlates with enhanced translation of this class of transcripts. most environmental stimuli which induce sgs (arsenite, clotrimazole, heat shock, osmotic shock, thapsigargin, uv) increase levels of phospho-eif2a, usually by activating a kinase and/or inactivating an eif2a phosphatase. these stimuli do not induce sgs in mutant mefs (s51a) expressing a nonphosphorylatable form of eif2a, 29,29a,30 which have been invaluable in discriminating between phospho-eif2a-dependent and phospho-eif2a-independent mechanisms of sg induction. eif2a-independent sgs can be induced through inactivation of eif4a helicase activity (pateamine a, hippuristanol, the lipid anti-inflammatory mediator 15-pgj2) which disrupts mrna scanning (table ii) . sgs can be nucleated by ectopic expression of a wide range of sg-associated proteins (e.g., caprin1, g3bp1, fmrp/fxr1, tia-1/tiar; see table i ). these proteins induce the formation of compositionally typical sgs (containing mrna, pabp, and initiation factors) and exhibit dynamic behavior: they are disassembled in response to cycloheximide or emetine, but not puromycin. to date, all tested nucleator proteins (g3bp, tia-1/tiar, ttp, fastk, fmrp/fxr1) fail to nucleate sgs in the s51a mutant mefs, so this type of sg induction is phospho-eif2a dependent. cotransfection of a pkr inhibitor usually prevents sg nucleation in wild-type cells, implicating pkr as the responsible kinase 28 . most sg nucleators bind mrna directly, and possess self-aggregation domains. in many cases, overexpression of the rna-binding domains alone has no effect on sg dynamics, whereas overexpression of the aggregation domains can dominantly inhibit sg assembly. deletion or knockdown of individual sg-nucleating proteins can have no effect, limit sg size, or delay sg assembly, or prolong sgs during recovery (table i) . much of our understanding of pbs comes from pioneering studies performed in saccharomyces cerevisiae, 21 but the situation in metazoans is complicated by (1) additional decapping proteins such as hedls/ge-1, which are lacking in yeast 23 ; (2) the fact that metazoan pbs also house the microrna machinery and associated noncoding rnas which are absent in budding yeast 20 ; and (3) metazoan pbs contain many proteins (4et, fast, apobe-c3ag) also lacking in yeast. moreover, yeast display a range of different dynamic granules that appear related, but may not be identical, to those in metazoans. glucose starvation triggers the formation of yeast ''egp'' bodies (containing eif4e, eif4g, and pabp) that exhibit dynamic behavior but lack eif3 and 40s subunits. 31, 32 these bodies contain other mrna-binding proteins related to mammalian sg-associated proteins, and were referred to as ''yeast sgs,'' 33 despite their lack of eif3/40s and nonreliance on phospho-eif2a for their formation. recently, eif3a/40s-containing ''yeast sgs'' have been described in s. cereviseae 34 exposed to robust heat shock, that require energy for their assembly but not phospho-eif2a, and appear to contain the pb marker dcp2. further studies and consistent definitions will be required to classify the yeast sgs/egpbs/pbs before we can understand their function(s) and relevance to metazoan systems, especially given the historical confusion between plant heat-shock granules and metazoan sgs. unlike sgs, mammalian pbs are found in normally growing cells, in a cellcycle-dependent manner. whereas sgs are highly variable in size and shape, pbs are uniform, spherical structures that may exhibit directed movements within the cytoplasm. the signature components of pbs are deadenylases (ccr4), decapping enzymes (dcp1 and dcp2), decapping enhancers (hedls/ge-1), and exonucleases (xrn1) that are required for 5 0 -3 0 mrna decay. pbs increase in size and number in cells lacking one or more components of the mrna decay machinery, 19 suggesting that mrna accumulates in pbs while awaiting decay. the number of pbs found in the cytoplasm of normally growing cells varies among different cell lines: hela and cos exhibit higher basal levels of pbs than u2os cells, most of which lack ''resting'' pbs entirely. pb number and size is increased several fold in response to some types of environmental stress (e.g., oxidative stress), however, in mammalian cells some stresses (heat shock, clotrimazole, glucose deprivation, etc.) induce sgs without inducing pbs, 35 providing additional evidence that other independent signaling pathways regulate sg and pb assembly, beyond their dependence (sgs) or independence (pbs) on phospho-eif2a. complicating the picture is the fact that mammalian pbs are usually, but not invariably, coincident with ''gw bodies,'' defined by the scaffolding proteins gw182, and housing components of the microrna machinery, notably argonaute-2 (ago-2). ago2 is recruited to sgs (here defined as containing eif3), whereas gw182 appears largely restricted to pbs (defined as containing dcp1a or hedls, which have not been reported to enter sgs). whether the decapping machinery and the microrna machinery share a common unidentified molecular link, or whether they are linked to some common cellular membrane or structure is not yet clear. two nuclear structures linked to mrnp maturation and processing, cajal bodies and gems, are coincident in many but not all cell types, so there is precedent for this duality. 36 this spatial confluence is ascribed to coupled mrna processing or remodeling steps that tether these structures together. factors regulating pb/gwb fusion have not been identified, although there are reports of pb heterogeneity. interactions between sgs and pbs are more clearly documented. 35, 37 some conditions (arsenite, overexpression of ttp or cpeb) similarly cause sgs and pbs to juxtapose and fuse, whereas under other conditions they are largely independent. these regulated interactions between sgs and pbs suggest that mrnp components move from one type of granule to the other. this could indicate the degradation of untranslated transcripts passing through sgs into pbs, or the rescue of decay-bound mrnps from pbs. an evolving model of sg assembly is shown in fig. 1 . it posits a series of reversible aggregation-driven stages, which allow for the rapid assembly and disassembly of sgs, as well as the inclusion or export of specific mrnps to pbs, such as those containing ttp. sg initiation requires sudden polysome disassembly caused by stalled or abortive initiation. metabolic labeling experiments indicate that translation must be severely affected (>90%) before sgs form in response to arsenite 38 (fig. 1a) . primary nucleation occurs when the stalled 48s mrnps, suddenly stripped of ribosomes, create localized regions in which the free mrna concentration suddenly spikes, favoring the binding of all locally available rna-binding proteins, regardless of sequence. many of these proteins (table i) normally shuttle between the nucleus and the cytoplasm, but are trapped by their affinity for the suddenly high concentrations of free mrna in the cytoplasm. this binding in turn localizes the rna-binding proteins, many of which (sg nucleators) self-aggregate. small mrnp aggregates rapidly form in an energy-independent manner, evenly dispersed throughout the cytoplasm (primary aggregation). within the small sg aggregates, competition and exchange occur, as high-affinity sequence-specific interactions exchange with low-affinity, sequence-independent ones. as most mrna transcripts bind multiple rna-binding proteins, crosslinking between mrnps (secondary aggregation) occurs just as readily as selfaggregation. in particular, the c-terminal domain of pabp is notoriously insoluble, and as pabp is present on most transcripts, it facilitates this stage. moreover, knockdown of pabp severely impairs sg assembly, as does overexpression of a c-terminal truncation of pabp (kedersha, tisdale, and anderson, unpublished data). small ribosomal subunits likely contribute to the aggregation, as o-glcnac modification of ribosomal subunits appears necessary to promote sg assembly downstream of polysome disassembly, but has less effect on p-body assembly. 39 in the second phase of sg assembly, small aggregates fuse to form larger aggregates (growth/fusion). this stage is facilitated by microtubules and motors, 40 model depicting the graded stages of sg assembly. stalled initiation allows elongating ribosomes to leave polysomes, resulting in stalled 48s mrnps which recruit available cytoplasmic rna-binding proteins (blue region), many of which would normally shuttle into the nucleus (pink area). locally high concentrations of mrnps promote aggregation into small complexes, which progressively fuse into larger aggregates, assisted by microtubule-dependent motors. sorting ensues as the higher affinity interactions prevail, and as other signaling molecules are recruited. subsets of mrnps may be removed from the sgs pending phosphorylation and 14-3-3 binding and shunted to pbs for decay (e.g., ttp). other mrnps may be exported for other fates. proteins and more proximal, core sg proteins that are highly concentrated in small foci. within the larger sgs as well as in the smaller ones, mrnp sorting and remodeling occur, as the most stable mrnps form, containing partners of the highest mutual affinity. the protein component of each mrnp could then determine the fate of its transcript. some mrnas might be reinitiated (left branch, yellow), others detached from the eif3/40s complex and transferred to the decapping and decay machinery within pbs (e.g., those bound to ttp, bottom, dark blue), while others packaged into more long-lived storage granules (right branch, light blue). sg disassembly occurs when translation returns to normal (or polysomes are artificially stabilized via drugs). note that the ''new normal'' following stress likely contains a different spectrum of transcripts, and that lack of proteins required to achieve this new normal state (zbp1, staufen) will prolong the time it takes for sgs to disperse. this model accounts for the following observations: (1) sg assembly is diminished, but not completely abolished, by knockdown of any individual sg-nucleating protein. (2) the abundance of a particular nucleator and its mrna targets is proportional to the effects of its loss on sg assembly rate or size. (3) proteins recruited to sgs by piggyback interactions are blocked from sg recruitment if their particular target protein is blocked or missing. as many of these are signaling proteins (ccar1, traf2, src3, rsk2), this could have important functional effects on cell survival or growth. (4) individual sg nucleators can exit sgs along with their associated mrnas, without disassembly of the sg. for example, ttp leaves sgs but not pbs upon phosphorylation and 14-3-3-binding, 12 whereas phosphorylated hmex3b leaves pbs but not sgs when bound to 14-3-3. 43 a large number of sg-associated proteins (dbpa, fastk, fmrp/fxr1, hnrnpa1, hur, mbnl1, mln51, sam68, tia-1/tiar) are nuclear shuttling proteins which regulate alternative splicing, a regulated process usually studied by overexpression/depletion experiments. although direct data are lacking, rerouting of these shuttling proteins to sgs should result in the altered splicing of genes transcribed during periods when sgs are present. this has been proposed to occur with hnrnpa1, a sg-associated multifunctional protein that regulates splicing (as well as microrna processing). its recruitment to sgs is predicated on its nuclear export, prior to its cytoplasmic phosphorylation which allows its targeting to sgs in an rna-binding-dependent manner. core components of sgs are stalled 48s preinitiation complexes. one approach to determine whether sg assembly is pro-or antisurvival is to eliminate a specific eif2a kinase and determine whether its loss sensitizes or promotes resistance to its activating stress. for example, hri kinase mediates arsenite-induced sg assembly, 30, 39 and hri knockout mefs and u2os cells in which hri is depleted by sirna fail to respond to phospho-eif2a or assemble sgs in response to arsenite, but exhibit unimpaired sg assembly in response to other stresses. both hri knockout mefs and mice are resistant to arsenite, 30 linking sg assembly to apoptosis. by sequestering specific proteins that regulate cell survival, sgs may influence different signaling pathways that determine whether a stressed cell repairs its damage and lives, or dies by apoptosis. deletion studies have shown that some sg-associated proteins regulate ''relative viability'' as assessed by atp levels following stress (hnrnpa1, 44 ; mln51, 26 ), whereas other proteins regulate survival/apoptosis in a localization-specific manner (rack1, 45 ; rsk2, 46 ). for example, rack1 is a scaffold protein required for the activation of the mapkkk. when rack1 is sequestered at sgs, this stress kinase cascade is inactive and stress-induced survival is enhanced. similarly, the recruitment of rsk2 to sgs and to the nucleus requires its binding to tia-1, which affects both its ability to induce cyclin d1 and promote survival following arsenite stress. 46 an important factor in modeling sg/pb dynamics must account for the contribution of nucleocytoplasmic shuttling proteins in sg assembly. as shown in table i , most sg-associated proteins other than initiation factors are nuclear shuttling proteins, including many (g3bp, fxr1/fmrp, tia-1/tiar) whose overexpression nucleates sgs. knockdown of any one of these proteins delays sg formation or reduces the size of sgs, but does not abolish sg formation altogether. this suggests that these shuttling proteins contribute to the size of sgs, but act downstream of polysome disassembly. the relocalization of these normally nuclear proteins into cytoplasmic sgs must prevent their nuclear function(s) such as alternative splicing, transcription, mrna processing, etc., thus allowing a mechanism whereby sg assembly can alter nuclear events. one nucleolar protein (sgnp) has been shown to accumulate in sgs, suggesting a link between sgs and ribosome assembly. after acting as a nuclear transcriptional coactivator of nfkb-induced inflammatory cytokine genes, src-3 (steroid receptor co-activator-3) moves to the cytoplasm where it binds tia-1 and promotes the translational silencing of tnfa mrna. this enables it to turn off expression of the same genes that it turns on early in the inflammatory process. 47 it requires binding to tia-1 for its localization into sgs, and provides a clear molecular link between nuclear transcription and cytoplasmic translational silencing. whether it also interacts with the tia proteins to regulate cytokine splicing remains to be determined. in addition to src-3, the transcriptional coactivator ccar1 (cell division cycle and apoptosis regulator 1) is recruited to sgs via interactions with g3bp/caprin1; the functional implications of ccar1 at sgs remain to be determined. 48 sg assembly is linked to membrane-associated signaling events as well as nuclear ones. traf2, a cytoplasmic signaling molecule that mediates tnfa signaling, is recruited to sgs upon heat shock, corresponding to its shift into an insoluble form due to its binding to eif4g. 49 its sequestration in sgs prevents its association with the tnf receptor, suggesting a mechanism whereby feverinduced sgs could mute the immune response. plakophilin-3 is recruited to sgs as part of a complex containing g3bp, 49 linking sg formation with cell adhesion. sg assembly is associated with a number of posttranslational modifications, among them phosphorylation, arginine methylation (cirp), 49 o-glcnac modification, 39 ubiquitinylation (tudor-3, hdac6), 41, 50, 51 and acetylation. it is also linked to the molecular chaperones hsp70 (which prevent aggregation of the prion-like domain of tia-1) and hsp90, required for ago-2 targeting to both sgs and pbs. 52 phosphorylation-driven binding to the chaperone 14-3-3 inhibits ttp interactions with sgs but not pbs, but is required for hmex3b/ago complexes to dock at pbs, yet has no effect on their targeting to sgs. polysome disassembly is not sufficient to induce sgs or pbs: whereas puromycin promotes rapid (30 min) polysome disassembly, sg assembly is not observed for several hours. 28 however, brief puromycin exposure lowers the threshold at which other sg/pb promoting drugs induce pb assembly, 4 implicating the activation of pathways downstream of termination in the sg assembly process. a single species of mrna can be assembled into a sg or a pb, even within the same cell. 35 while the factors that influence sorting into distinct types of granules are not well understood, there is evidence that the mode of polysome disassembly affects the type of rna granule that is assembled, leading to the following model. when polysome disassembly is initiated by deadenylation, depletion of pabp disrupts the link between the 5 0 -and 3 0ends of the transcript and allows the recruitment of decapping enzymes. these linearized transcripts recruit the decay machinery, and are thus assembled into pbs. when polysome disassembly is mediated by stalled initiation on a circularized mrna, ribosome run-off leaves a circular mrnp that is assembled into sgs, thus accounting for the fact that eif4e is present in both sgs and pbs, whereas only sgs contain eif3, eif4g, eif4a, and pabp, while pbs contain instead the eif4e binding protein 4-et. new information on sg/pb dynamics comes from an sirna-based screen for genes that are required for sg/pb assembly in response to arsenite. 39 strikingly, five different subunits of eif3 (eif3c, d, e, g, and i) are required for sg but not pb assembly, while eif3b is required for both sg and pb assembly. notably, eif3e mediates the interaction of eif3 with eif4g and stabilizes the circular form of the mrna; 53 its deletion would be predicted to destabilize circular mrnps that might be preferentially routed to sgs rather than pbs. not detected in the screen was eif3j, a protein required for ribosome recycling 54 suggesting that impairing recycling might prevent sg assembly. moreover, erf1 (eukaryotic translation release factor 1) was found to be required for pb, but not sg formation, also suggesting that the mode of polysome disassembly may influence whether sgs or pbs result. the importance of these rna granules in the regulation of protein expression is underscored by their involvement in several aspects of disease pathogenesis. our understanding of their roles in cellular physiology may allow us to exploit these regulatory pathways for the development of new classes of drugs for the treatment of disease. studies of the translation of viral rnas have provided general insights into mechanisms of protein translation. the finding that many viruses interact, functionally or physically, with sgs and pbs highlights their relevance to mrna translation and decay. the different ways that viruses interact with rna granules is summarized below. poliovirus is a plus-strand rna virus that encodes proteinases that cleave essential host proteins to allow preferential transcription and translation of viral rna. poliovirus infection results in the transient assembly of sgs. the disassembly of sgs correlates with the cleavage of g3bp by viral proteinase 3c. expression of a noncleavable g3bp mutant prevents the disassembly of sgs and inhibits virus production, suggesting that sgs function in host defense against virus infection. 55, 56 semliki forest virus is another plus-strand rna virus that shuts down host protein synthesis prior to initiating the synthesis of virus proteins. this is accomplished, in part, by the activation of a stress response program that phosphorylates eif2a to repress translation initiation. in mefs expressing a nonphosphorylatable eif2a mutant, host protein shutoff is markedly impaired. phosphorylation of eif2a results in the assembly of sgs during the early stages of virus infection, but sgs are disassembled as viral protein synthesis is initiated, and thereafter sgs cannot be induced by arsenite. infection of tia-1 à/à mefs that exhibit impaired sg assembly results in a significant delay in host protein synthesis shutoff. thus, tia-1 and sgs appear to facilitate host protein synthesis shutoff, and their disassembly is required for efficient translation of viral proteins. 57 reovirus is a double-stranded rna virus that also activates a stress response program in infected cells. this involves the activation of pkr, phosphorylation of eif2a, and transcription of atf4. individual strains of reovirus differ in the extent to which host protein synthesis is turned off following infection. the phospho-eif2a induced assembly of sgs parallels the extent of host protein shut off in different reovirus strains. thus, reovirus appears to utilize sgs to promote the preferential translation of virus proteins. 58, 59 west nile virus is a plus-strand rna virus that also induces host protein shut down in infected cells. tia-1 and tiar, related proteins that nucleate sg assembly, bind to a 3 0 -terminal stem loop in the minus strand viral rna. during virus infection, tia-1 and tiar are concentrated at perinuclear sites of viral replication, suggesting that these proteins play a role in virus replication. consistent with this possibility, virus replication is severely impaired in tiar à/à mefs. the sequestration of tia-1 and tiar at sites of virus replication has been proposed to contribute to the impaired assembly of sgs and pbs observed in virally infected cells. 59 sendai virus is a minus strand rna virus that induces the assembly of sgs during virus infection. sg assembly is regulated by a short rna transcribed from the 3 0 -end of plus-strand viral rnas. this rna binds and sequesters tiar to impair sg assembly. sequestration of tiar also inhibits virus-induced apoptosis, a phenomenon that may promote cell survival to allow optimal virus production. 60 rotavirus is a double-stranded rna virus that also promotes the phosphorylation of eif2a in infected cells. unlike sindbis virus, reovirus and west nile virus-infected cells, phospho-eif2a does not induce sg assembly in rotavirusinfected cells. moreover, the growth of rotavirus is not impaired in fibroblasts expressing a nonphosphorylatable eif2a mutant. it is possible that rotavirus encodes a factor that prevents phospho-eif2a-mediated translational repression and/or the aggregation of untranslated mrnps. mouse hepatitis coronavirus is a plus-strand rna virus that induces the phosphorylation of eif2a and shuts down host protein synthesis in infected cells. virus infection also induces the assembly of sgs and pbs suggesting that rna granules play a role in reprogramming mrna translation/decay during viral infection. 61 brome mosaic virus (bmv) is a plus-strand rna virus that replicates in yeast. the translation and replication of bmv rnas is dependent upon the pb components pat1p, dhh1p, and lsm1p-7p. moreover, bmv rnas are concentrated in pbs suggesting that pbs facilitate viral replication. 62 retroviruses and retrotransposons share the ability to reverse transcribe dna copies for insertion into the genome. apobec3g and apobec3f are antiviral proteins that deaminate cytidines in retroviral or retrotransposonencoded rnas. although both of these proteins are concentrated at sgs and pbs, the functional significance of this localization remains to be determined. 63 similarly, tsn (tudor staphylococcal nuclease) is a risc-associated nuclease 64 that specifically cleaves inosine-modified dsrna, and associates with sgs. 65 line-1 transposon, still active in the human genome, encodes line1p, a protein required for tranposition that nucleates sgs 66 and coaggregates the line-1 mrna, 67 suggesting that sg formation may be a manifestation of the host antiviral response. mutations in the fragile mental retardation protein (fmrp) are associated with an x-linked form of mental retardation. fmrp is an rna-binding protein that is expressed in dendrites and at synapses. it has been proposed to function as a translational repressor that dampens the expression of synaptic proteins to impair neuronal function. 68 in normal cells, fmrp is found in association with polysomes suggesting a possible role in protein translation. in cells subjected to oxidative stress, or in hippocampal neurons perturbed by electrode insertion, fmrp accompanies untranslated mrnas to sgs. 69 in cells lacking fmrp or expressing an fmrp mutant associated with fxs, sg assembly is markedly impaired 70 suggesting that fmrp actively contributes to sg assembly. impaired sg assembly may prevent the reprogramming of protein translation that is required to protect cells from the adverse effects of environmental stress. it is possible that mental retardation in fxs resulting from neuronal cell death is caused by impaired sg assembly. sg assembly has been shown to regulate several aspects of immune cell function and inflammation. sgs are assembled following t cell receptormediated activation of cd4þ t cells. in these cells, il-4 and il-13 mrnas are transcribed and held in a translationally repressed state. t cell receptormediated restimulation releases these transcripts from translational repression allowing cytokine secretion. lentiviral expression of a dominant-negative mutant of the sg nucleator tia-1 releases the activation-induced translational silencing of these transcripts, implicating sgs in the regulation of t cell activation. 71 roquin is an e3 ligase that regulates the expression of icos, a costimulatory molecule that promotes t cell activation. 72 mutant mice expressing mutant roquin overexpress icos and develop a severe autoinflammatory disease. roquin appears to promote the mirna-dependent repression of icos expression. as roquin resides at sgs and pbs, these rna granules may be involved in this autoimmune syndrome. sg and pb partitioning may also regulate the expression of inflammatory proteins in activated macrophages. ttp, an mrna destabilizing protein that represses the expression of inflammatory mediators (e.g., tnfa, il-1b, il-6), plays a major role in regulating interactions between sgs and pbs. 35 in activated macrophages, ttp is phosphorylated and complexed with 14-3-3 proteins, modifications that prevent it from delivering its associated mrnas to the degradation machinery. 73 because phosphorylation of ttp causes sg: pb conjugates to dissociate, inhibition of mrna decay may be a consequence of altered sg:pb interactions. reduced blood flow caused by transient occlusion of the carotid artery or by reduced cardiac output confers ischemic injury to susceptible neurons. resumption of blood flow after ischemia can potentiate this process by a number of different mechanisms. in postischemic neurons, protein translation is turned off to conserve anabolic energy for the repair of stress-induced damage. in neurons that are particularly susceptible to ischemic injury (e.g., hippocampal cornu ammonis 1; ca1), translational arrest is prolonged and translational reprogramming that allows the preferential translation of repair proteins (e.g., heat-shock proteins) is impaired. in neurons that are relatively resistant to ischemia, sgs are transiently assembled. in ca1 neurons, sgs persist for prolonged periods. the persistence of sgs correlates with the prolonged translational arrest observed in these cells. 74 in addition to sgs, a related rna granule characterized by the presence of hur and the absence of tia-1 is observed in ischemic ca1 neurons. besides exhibiting prolonged translational arrest, these cells fail to synthesize protective heat-shock proteins in response to ischemic insults. 75 thus, alterations in the assembly and function of different types of rna granules may impair translational reprogramming in the susceptible ca1 neuron leading to increased vulnerability to ischemic injury. sg and pb formation is a regulated consequence of translational arrest, but there appears to be many levels to the story. monitoring sg formation may be of diagnostic use in assessing viral infection or hypoxia, as it affords us a window on the translational status of individual cells within tissues. however, while sgs and pbs are primarily sites of remodeling, packaging, and sorting of mrna, their assembly is linked to other cellular processes and pathways via a growing number of specific proteins. sgs secondarily recruit proteins involved in splicing, transcription, and signaling as part of an adaptive process. defining the specific molecules that link sg formation to other pathways, and selectively targeting them may be of therapeutic value. the impact of pb/gwb formation on other pathways is presently less clear, but the ongoing river of data will doubtlessly flush out new ideas as these exciting young fields mature. rna-binding proteins tia-1 and tiar link the phosphorylation of eif-2a to the assembly of mammalian stress granules cytoplasmic heat shock granules are formed from precursor particles and are associated with a specific set of mrnas plant stress granules and mrna processing bodies are distinct from heat stress granules mammalian stress granules: highly dynamic sites of mrna triage during stress induced translational arrest tia-1 is a translational silencer that selectively regulates the expression of tnf-alpha visibly stressed: the role of eif2, tia-1, and stress granules in protein translation stressful initiations evidence that ternary complex (eif2-gtp-trna(i)(met))-deficient preinitiation complexes are core constituents of mammalian stress granules mammalian stress granules represent sites of accumulation of stalled translation initiation complexes the rasgapassociated endoribonuclease g3bp assembles stress granules rasputin, more promiscuous than ever: a review of g3bp mk2-induced tristetraprolin: 14-3-3 complexes prevent stress granule association and are-mrna decay inhibition of eukaryotic translation initiation by the marine natural product pateamine a rnamediated sequestration of the rna helicase eif4a by pateamine a inhibits translation initiation eukaryotic initiation factor 2alpha-independent pathway of stress granule induction by the natural product pateamine a inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation an antiviral response directed by pkr phosphorylation of the rna helicase a decapping and decay of messenger rna occur in cytoplasmic processing bodies cytoplasmic foci are sites of mrna decay in human cells p bodies: at the crossroads of post-transcriptional pathways p bodies and the control of mrna translation and degradation the highways and byways of mrna decay the control of mrna decapping and p-body formation perk is essential for translational regulation and cell survival during the unfolded protein response signal integration via pkr the exonjunction-complex-component metastatic lymph node 51 functions in stress-granule assembly distinctive properties of the 5'-untranslated region of human hsp70 mrna mammalian stress granules and processing bodies translational control is required for the unfolded protein response and in vivo glucose homeostasis metazoan stress granule assembly is mediated by p-eif2 {alpha}-dependent and -independent mechanisms heme-regulated inhibitor (hri) kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 (eif2) inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure stress-dependent relocalization of translationally primed mrnps to cytoplasmic granules that are kinetically and spatially distinct from p-bodies accumulation of polyadenylated mrna, pab1p, eif4e, and eif4g with p-bodies in saccharomyces cerevisiae p bodies promote stress granule assembly in saccharomyces cerevisiae robust heat shock induces eif2{alpha}-phosphorylation-independent assembly of stress granules containing eif3 and 40s ribosomal subunits in budding yeast, saccharomyces cerevisiae stress granules and processing bodies are dynamically linked sites of mrnp remodeling subnuclear organelles: new insights into form and function the translational regulator cpeb1 provides a link between dcp1 bodies and stress granules dynamic shuttling of tia-1 accompanies the recruitment of mrna to mammalian stress granules a functional rnai screen links o-glcnac modification of ribosomal proteins to stress granule and processing body assembly dynein motor contributes to stress granule dynamics in primary neurons the deacetylase hdac6 is a novel critical component of stress granules involved in the stress response disruption of microtubules inhibits cytoplasmic ribonucleoprotein stress granule formation interaction with 14-3-3 adaptors regulates the sorting of hmex-3b rna-binding protein to distinct classes of rna granules hnrnp a1 relocalization to the stress granules reflects a role in the stress response formation of stress granules inhibits apoptosis by suppressing stress-responsive mapk pathways codependent functions of rsk2 and the apoptosis-promoting factor tia-1 in stress granule assembly and cell survival an essential function of the src-3 coactivator in suppression of cytokine mrna translation and inflammatory response microtubuledependent association of akap350a and ccar1 with rna stress granules sequestration of traf2 into stress granules interrupts tumor necrosis factor signaling under stress conditions tdrd3 is a novel stress granule-associated protein interacting with the fragile-x syndrome protein fmrp tdrd3, a novel tudor domain-containing protein, localizes to cytoplasmic stress granules hsp90 regulates the function of argonaute 2 and its recruitment to stress granules and p-bodies translation initiation factor eif4g-1 binds to eif3 through the eif3e subunit recycling of eukaryotic posttermination ribosomal complexes inhibition of cytoplasmic mrna stress granule formation by a viral proteinase how viruses avoid stress importance of eif2alpha phosphorylation and stress granule assembly in alphavirus translation regulation reovirus induces and benefits from an integrated cellular stress response interaction of tia-1/tiar with west nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly sendai virus trailer rna binds tiar, a cellular protein involved in virus-induced apoptosis mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies interactions between brome mosaic virus rnas and cytoplasmic processing bodies antiviral protein apobec3g localizes to ribonucleoprotein complexes found in p bodies and stress granules a micrococcal nuclease homologue in rnai effector complexes inosine-containing dsrna binds a stress-granule-like complex and downregulates gene expression in trans line-1 orf1 protein localizes in stress granules with other rna-binding proteins, including components of rnai risc retrotransposons revisited: the restraint and rehabilitation of parasites fragile x syndrome: loss of local mrna regulation alters synaptic development and function fragile x mental retardation protein shifts between polyribosomes and stress granules after neuronal injury by arsenite stress or in vivo hippocampal electrode insertion cells lacking the fragile x mental retardation protein (fmrp) have normal risc activity but exhibit altered stress granule assembly activation of the integrated stress response during t helper cell differentiation a ring-type ubiquitin ligase family member required to repress follicular helper t cells and autoimmunity mk2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and are-mrna decay prolonged translation arrest in reperfused hippocampal cornu ammonis 1 is mediated by stress granules persistent redistribution of poly-adenylated mrnas correlates with translation arrest and cell death following global brain ischemia and reperfusion argonaute 2/risc resides in sites of mammalian mrna decay known as cytoplasmic bodies quantitative analysis of argonaute protein reveals microrna-dependent localization to stress granules the anti-hiv-1 editing enzyme apobec3g binds hiv-1 rna and messenger rnas that shuttle between polysomes and stress granules human retroviral host restriction factors apobec3g and apobec3f localize to mrna processing bodies ataxin-2 interacts with the dead/h-box rna helicase ddx6 and interferes with p-bodies and stress granules post-translational arginylation of calreticulin: a new isospecies of calreticulin component of stress granules distinct structural features of caprin-1 mediate its interaction with g3bp-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2alpha, entry to cytoplasmic stress granules, and selective interaction with a subset of mrnas identification of fuse-binding proteins as interacting partners of tia proteins the cold-inducible rnabinding protein migrates from the nucleus to cytoplasmic stress granules by a methylationdependent mechanism and acts as a translational repressor the eif4g-homolog p97 can activate translation independent of caspase cleavage mbnl1 associates with yb-1 in cytoplasmic stress granules the dead-box rna helicase ddx3 associates with export messenger ribonucleoproteins as well as tip-associated protein and participates in translational control recruitment of the rna helicase rhau to stress granules via a unique rna-binding domain a functional link between disrupted-in-schizophrenia 1 and the eukaryotic translation initiation factor 3 the enhancer of decapping proteins, edc1p and edc2p, bind rna and stimulate the activity of the decapping enzyme multiple processing body factors and the are binding protein ttp activate mrna decapping a role for the eif4e-binding protein 4e-t in p-body formation and mrna decay regulation of stress granule dynamics by grb7 and fak signalling pathway trapping of messenger rna by fragile x mental retardation protein into cytoplasmic granules induces translation repression the multifunctional fus, ews and taf15 proto-oncoproteins show cell type-specific expression patterns and involvement in cell spreading and stress response functional dissection of the human tnrc6 (gw182-related) family of proteins ge-1 is a central component of the mammalian cytoplasmic mrna processing body nuclear rna export factor 7 is localized in processing bodies and neuronal rna granules through interactions with shuttling hnrnps hnrnp k interacts with rna binding motif protein 42 and functions in the maintenance of cellular atp level during stress conditions human hnrnp q re-localizes to cytoplasmic granules upon pma, thapsigargin, arsenite and heat-shock treatments stress granule assembly is mediated by prion-like aggregation of tia-1 hur binding to cytoplasmic mrna is perturbed by heat shock importin 8 is a gene silencing factor that targets argonaute proteins to distinct mrnas intracellular localization of human inositol 1,3,4,5,6-pentakisphosphate 2-kinase localization of the developmental timing regulator lin28 to mrnp complexes, p-bodies and stress granules the human lsm1-7 proteins colocalize with the mrna-degrading enzymes dcp1/2 and xrnl in distinct cytoplasmic foci identification of novel argonaute-associated proteins neural rna-binding protein musashi1 inhibits translation initiation by competing with eif4g for pabp inhibition of g(1) to s phase progression by a novel zinc finger protein p58(tfl) at p-bodies identification of patl1, a human homolog to yeast p body component pat1 selective localization of pcbp2 to cytoplasmic processing bodies identification of the junctional plaque protein plakophilin 3 in cytoplasmic particles containing rna-binding proteins and the recruitment of plakophilins 1 and 3 to stress granules polysome-bound endonuclease pmr1 is targeted to stress granules via stress-specific binding to tia-1 dendritic localization of the translational repressor pumilio 2 and its contribution to dendritic stress granules rna-associated protein 55 (rap55) localizes to mrna processing bodies and stress granules rpp 20 interacts with smn and is re-distributed into smn granules in response to stress the rna polymerase ii subunit rpb4p mediates decay of a specific class of mrnas sam68 functions in nuclear export and translation of hiv-1 rna sgnp: an essential stress granule/nucleolar protein potentially involved in 5.8s rrna processing/transport mammalian smaug is a translational repressor that forms cytoplasmic foci similar to stress granules staufen recruitment into stress granules does not affect early mrna transport in oligodendrocytes mammalian staufen 1 is recruited to stress granules and impairs their assembly survival motor neuron protein facilitates assembly of stress granules probing the mrna processing body using protein macroarrays and "autoantigenomics zbp1 regulates mrna stability during cellular stress a pkr-like eukaryotic initiation factor 2alpha kinase from zebrafish contains z-dna binding domains instead of dsrna binding domains key: cord-279418-3r1ijafm authors: nevers, quentin; albertini, aurélie a.; lagaudrière-gesbert, cécile; gaudin, yves title: negri bodies and other virus membrane-less replication compartments() date: 2020-08-21 journal: biochim biophys acta mol cell res doi: 10.1016/j.bbamcr.2020.118831 sha: doc_id: 279418 cord_uid: 3r1ijafm viruses reshape the organization of the cell interior to achieve different steps of their cellular cycle. particularly, viral replication and assembly often take place in viral factories where specific viral and cellular proteins as well as nucleic acids concentrate. viral factories can be either membrane-delimited or devoid of any cellular membranes. in the latter case, they are referred as membrane-less replication compartments. the most emblematic ones are the negri bodies, which are inclusion bodies that constitute the hallmark of rabies virus infection. interestingly, negri bodies and several other viral replication compartments have been shown to arise from a liquid-liquid phase separation process and, thus, constitute a new class of liquid organelles. this is a paradigm shift in the field of virus replication. here, we review the different aspects of membrane-less virus replication compartments with a focus on the mononegavirales order and discuss their interactions with the host cell machineries and the cytoskeleton. we particularly examine the interplay between viral factories and the cellular innate immune response, of which several components also form membrane-less condensates in infected cells. during their replication cycle, many viruses induce the formation of specialized intracellular compartments in the host cell. these structures often referred as viral inclusions, viral factories or viroplasms, concentrate viral proteins, nucleic acids and specific cellular factors. in many cases, those viro-induced compartments harbor essential steps of the viral cycle and constitute a platform facilitating viral replication and assembly but also protecting the viral genome from cellular defense mechanisms. such viral factories have been now identified for a variety of non-related viruses [1] [2] [3] . viral factories are very heterogeneous. they can be associated with membranes from diverse organelles (mainly endoplasmic reticulum -er-, late endosomes, lysosomes, and mitochondria) that they rearrange. this is the case for positive-strand rna viruses leading to the formation of double-membrane vesicles (coronaviridae, arteriviridae, picornaviridae and flaviviridae) or spherules derived from diverse cellular membranes (togaviridae, nodaviridae and flaviviridae) (fig. 1 ). however, for several negative-strand rna, double stranded rna and dna viruses, viral factories are devoid of membranes. these compartments can be either cytosolic or nuclear and, in several instances, have been demonstrated to have properties like those of liquid organelles (fig. 1 ). membrane-less liquid organelles contribute to the compartmentalization of the eukaryotic cell interior [4] [5] [6] [7] . they are referred as droplet organelles, proteinaceous membrane-delimited organelles. finally, they are highly enriched in some proteins that are much more concentrated in those structures than in the cytosol, as a result of liquid-liquid phase separation (llps) [7] . with the rapid identification of cellular membraneless compartments and proteins that undergo llps in vitro, a major challenge in the field is to demonstrate unambiguously that a specific structure is indeed a phase-separated liquid body in the cellular context. currently, common criteria for defining such a structure are that it is spherical, fuses, reversibly deforms when encountering a physical barrier, and recovers from photobleaching [14] . here, we will review membrane-less viral compartments with a focus on those that have liquid organelles properties. we will discuss their composition and their interactions with the host cell machineries, particularly those of the innate immune system, of which several components also form membrane-less condensates in infected cells, and of the cytoskeleton. mononegavirales (mnv) constitute a viral order which contains several viruses causing important human diseases (including rabies virus -rabv-, ebola virus -ebov-, measles virus -mev-, mumps virus -muv-, human respiratory syncytial virus -rsv-). their genome consists of a negative sense, single-stranded rna molecule ranging from 10 to 15 kb, starting and ending with a non-coding leader and trailer sequence, respectively. the viral rna is tightly associated with the nucleoprotein (n or np) to form the helical ribonucleoprotein (rnp) (fig. 2) . the rnp recruits the viral rna-dependent rna polymerase (rdrp) complex, composed of the protein l (the polymerase which bears polyribonucleotidyltransferase and methyltransferase activities) and its non-enzymatic j o u r n a l p r e -p r o o f cofactors (p for rhabdoviruses, paramyxoviruses, and pneumoviruses, vp35 for filoviruses), to form the nucleocapsid. in the nucleocapsid, p acts as a tether between l and the rnp. the nucleocapsid is enwrapped by a lipid bilayer that is derived from a host cell membrane during the budding process. the matrix protein (m, vp40 for filoviruses) is located beneath the viral membrane and bridges the rnp and the lipid bilayer, which contains one or two transmembrane glycoproteins that are involved in viral entry. the cell cycle of mnv is entirely cytoplasmic (with the notable exception of that of the bornaviridae family members). the negative sense rnp, once released in the cytoplasm, constitutes the template for viral gene expression and replication by the rdrp complex. in this complex, p is bridging l and the template-associated nucleoproteins. transcription begins at the 3' end of the genomic rna and results in the synthesis of a positive, uncapped and short leader rna and capped and polyadenylated messenger rnas (mrnas). viral mrnas are then translated by the host cell translation machinery providing a source of n protein necessary to encapsidate the nascent rna. this results in the switch of the activity of the rdrp complex from transcription to replication to produce rnps containing full-length antigenomic rna (positive sense), which in turn serve as templates for the synthesis of genomic rna (negative sense). during the replication stage, when not bound to the viral genomic or antigenomic rna, n is kept soluble by binding the amino-terminal region of p thus forming the so-called n 0 p complex (fig. 2b, 2d ) [15] [16] [17] . [34] are spherical (at least during the initial stages of infection), suggesting that they could be liquid organelles formed by phase separation. this liquid nature was confirmed by live-cell imaging for rabv [30] , vsv [31] and mev [25]. first, it was shown that when two spherical inclusions contact one another, they readily fuse and round up into a single larger spherical one [25, 30, 31] . they also reversibly deform when encountering a physical barrier and disappear when exposed to an osmotic shock [30] . beyond the mnv order, ibs are also observed during segmented negative strand rna virus infections. indeed, it has been shown that cells infected by influenza a virus contain inclusions located in the vicinity of the er exit site. these inclusions, which have been proposed to be involved in the control of the assembly process, have liquid properties [35] . similarly, the spherical aspect of the granules which concentrate the three bunyavirus genome segments [36] suggest the possibility that they also have liquid properties. co-expression of n and p proteins of paramyxoviruses and rabv after cell transfection also leads to the formation of spherical inclusions [25, 30, 37, 38] . in the case of vsv, the presence of l protein is also required for such inclusions to be formed [31] whereas in the case of ebov, np alone is sufficient for ib generation [39] . for both rabv and mev, the n-p inclusions formed in this minimal system have the same liquid characteristics as the viral factories [25, 30] . in the other cases, the spherical aspect of the inclusions is a strong argument in favor of their liquid nature, but additional experiments are necessary to definitively conclude on this point. rabv and mev minimal systems were used to identify p and n domains that are essential for inclusion formation. the phosphoprotein of paramyxoviruses and rhabdoviruses share a common modular organization [40] [41] [42] (fig. 3) . the n-terminal part of the protein is involved in the formation of the n 0 p complex [43] [44] [45] (fig. 2b, 2d , 3c) and, for rhabdoviruses, in the interaction with the l protein [46, 47] (fig. 3b, 3d ). the c-terminal domain (pctd for rabv and vsv, xd for mev) binds n associated with rna [48] [49] [50] ( fig. 3) . the xd domain of paramyxoviruses can also bind l [51] . two central intrinsically j o u r n a l p r e -p r o o f disordered domains (idd1 and idd2 for rabv, p tail and p loop for mev) are flanking an oligomerization domain. rhabdovirus p is dimeric in solution [52, 53] (fig. 3b , 3c) whereas paramyxovirus p form tetramers [54] (fig. 3e ). despite the absence of sequence similarity and the differences in the domain structures (fig. 3) , for both rabv and mev p, it has been shown that the oligomerization domain, the second intrinsically disordered domain (idd2 / more recently, it has been shown that mev n (produced in association with the 50 first amino-terminal residues of p and thus in the form n 0 p 50 ) and p expressed in e. coli form liquid-like membrane-less organelles upon mixing in vitro under physiological salt and protein concentrations [55] . as in the cell [25], the oligomerization domain, p loop and xd are required for llps. in this system, it was also shown that the interaction between the disordered c-terminal part of n (n tail residues 400-525) and xd of p is essential for droplet formation. indeed, a mutation of n (s491l) abrogating the n tail :pxd interaction and known to significantly decrease viral transcription in vivo resulted in suppression of phase separation. finally, when rna was added, it concentrated in the n-p droplets and was encapsidated by n protomers to form nucleocapsid-like particles. the rate of encapsidation within droplets was enhanced compared to the dilute phase, which suggests a function of llps in mev replication [55] . in the minimal systems in which n is associated with cellular rnas and forms n-rna rings and short rnp-like structures [43, 56] ), rna-binding proteins (n in this case) and proteins containing idds (p and sometimes n in this case). for several cellular proteins that phaseseparate, cation-pi interactions between arginine and aromatic residues have been shown to be key in the process [57, 58] . it is not known whether similar residues mediate the weak interactions that drive llps for mnv condensates. a comparison between rabv, mev and ebov nucleoprotein and phosphoprotein sequences with a focus on their idds do not reveal conserved specificities besides a slight enrichment in positive residues in rabv idd2 and mev p loop (both required to observe llps in the minimal systems) (table 1) . furthermore, alignment of the sequences of lyssaviruses phosphoproteins reveal a poor conservation of idds compared to the rest of the protein sequence in the viral genus [30]. the first viral factories which were characterized were those of large dna viruses such as the poxviridae, the iridoviridae and the asfarviridae [59] [60] [61] [62] . those cytoplasmic factories are devoid of membrane and located near the microtubule organizing center. they have several characteristics reminiscent of those of the cellular aggresomes which concentrate misfolded proteins in the cell [63] . they recruit mitochondria in their vicinity, contain molecular chaperones such as heat-shock proteins (hsp) and are surrounded by a vimentin cage [64] . however, no data are supporting the possible liquid nature of those cytoplasmic structures. other dna viruses belonging to herpesviridae, adenoviridae, parvoviridae, polyomaviridae and papillomaviridae families induce the formation of membrane-less assemblies inside the nucleus termed viral replication compartments (or centers), hereafter referred as vrcs [65] , which concentrate viral proteins and nucleic acids, incoming viral genomes and host proteins. herpesviruses and adenoviruses vrcs also coalesce as infection progresses and can fuse together in a liquid-like manner [66] [67] [68] [69] . however, unlike many other cellular liquid organelles [70] [71] [72] , hsv-1 vrcs are not disrupted by treatment with 1,6hexanediol and a model of their formation, not based on llps, has been proposed [69] . however, sensitivity to hexanediol is insufficient to unequivocally demonstrate that a structure is formed via llps [14] and the identification of the physicochemical principles double strand rna (dsrna) viruses are also known to induce the formation of membrane-less cytosolic electron-dense inclusions. this is particularly documented for the reoviridae family of viruses which contain 10 (for reoviruses) or 11 (for rotaviruses) j o u r n a l p r e -p r o o f segments of genomic dsrna. those organelles are referred as viroplasms. they constitute the site of viral genome transcription and replication, as well as the site of packaging of the newly synthesized pregenomic rna segments into the viral cores [75] [76] [77] [78] . viroplasms appear to be spherical and can fuse together [79, 80] which indicates that they have liquid properties. for rotaviruses, viroplasms are nucleated by two essential non-structural proteins, nsp2 and nsp5, and the inner virion capsid protein vp2, with nsp5 being crucial for both the recruitment of viroplasmic proteins and the architectural assembly of the viroplasms. in non-infected cells, co-expression of nsp5 either with nsp2 or vp2 leads to the formation of viroplasm-like structures [81, 82] . interestingly, nsp5 shares several characteristics with the phosphoproteins of mnv as it is phosphorylated, forms oligomers and contains intrinsically disordered segments [83] . similarly, for reoviruses, the expression of the non-structural protein µns alone leads to the formation of large inclusions that are similar to the viroplasms [84] . however, unlike nsp5, µns is not predicted to contain long intrinsically disordered segments. although the viral factories of positive stand rna viruses are associated with membranes, several of those viruses also form spherical intracytoplasmic inclusions that might have liquid properties. for example, depending on their genus, coronavirus nucleocapsid proteins (n) can form inclusion either in the cytoplasm [85] or in the nucleus in association with the nucleolus [86, 87] . in keeping with this idea, it has been shown in vitro that the sars-cov-2 n protein contains disordered regions and phase separates with rna [88] [89] [90] [91] . the phase separation is regulated by n phosphorylation [91] . interestingly, in vitro, n also partitions into liquid phases formed by several human rna-binding proteins [89] , which is reminiscent of coronavirus n ability to associate with the nucleolus. these finally, ebov np recruits several proteins into the ibs, which include the nuclear rna export factor nfx1 to drive viral protein synthesis [100] and the histone-lysinemethyltransferase smyd3 [101] as well as the cad protein [102] to facilitate mrna transcription and replication. mass spectrometry analyses of pull-down complexes of tagged nsp2 and nsp5 from human rotavirus incubated with extracts from uninfected rotavirus-permissive ma104 cells j o u r n a l p r e -p r o o f revealed the presence of several host heterogeneous nuclear rnps (hnrnps) and au-rich element-binding proteins (are-bps) [103] . finally, the composition of hsv-1 vrc has been investigated by immunoprecipitation of icp8 that appears as one of their most important components. this has allowed the identification of more than 50 viral and cellular proteins, mainly involved in dna replication, dna repair, chromatin remodelling, transcription, and rna processing [104] . in conclusion, although a significant number of proteins have been found associated with viral ibs, the precise role of those associations with, or sequestrations in, ibs or vrcs as well as the underlying molecular mechanisms remain to be determined and will certainly constitute a new field of research in the coming years. the cytoskeleton plays an important role in the morphogenesis and evolution all along tether cytoplasmic liquid organelles such as p-bodies and drive their fission in an active process [106] . it is not excluded that rabv has hijacked a cellular machinery specifically involved in this process or that the same basic physicochemical principles are at work in both cases. the subtle interplay between viral ibs and innate immunity is particularly exemplified by rabv for which p is not only one of the major component of the nbs but also the major viral counteractant of the innate immune response [108] . first, p has a critical role in suppression of ifn production by blocking the phosphorylation of the transcription factor interferon regulatory factor 3 (irf-3) [109] . second, the interaction of p with stat1 leads to the inhibition of ifn signaling by different processes including inhibition of stat1-dna binding [110] and stat1 sequestration away from the nucleus [111] . similarly, ebov vp35, which is concentrated in the ibs largely counteracts the innate immunity. it binds double-stranded rna and inhibits ifn production induced by rig-i signaling [112, 113] , prevents pkr activation [114] and impairs the function of ifn regulatory factor-activating kinases ikk and tbk-1 [115] . in the case of paramyxoviruses, the proteins that inhibit ifn production and counteract ifn response, although expressed from the p gene, are distinct from p [116] . they are either expressed from an alternate reading frame or from an alternate transcript due to the presence of an editing site. c protein of mev corresponds to the former case and has a completely different amino-acid sequence from p whereas v protein corresponds to the latter case and has the same amino-terminal part as p but a distinct c-terminal domain. consequently, v is j o u r n a l p r e -p r o o f missing the p domain required to associate with ibs and v protein has primarily a diffuse nuclear distribution [117] . therefore, mnv evolved different strategies to counteract innate immunity. some viruses may sequester the proteins involved in those pathways inside the viral ibs whereas others keep them away from the viral factory. several cytosolic sensors of the innate immunity are also associated with liquid organelles. the best characterized of those organelles are the sgs which are formed when the cell is under a cytoplasmic stress. they are storage sites containing translationally silenced mrnps that can be released to resume translation after the stress subsides. sgs can be induced by viral infections [9, 118] and are thought to have antiviral activities as they contain rig-i and mda-5 [119] [120] [121] . interestingly, during rabv infection, sgs come into close contact with nbs [9] but do not fuse with them [30] . this indicates that nbs and sgs are made of non-miscible liquid phases. however, they exchange some material as the mrnas (but not the genomes and antigenomes) are transported from nbs, where they are synthesized, into sgs [9] . for vsv, it has been shown that sgs associated proteins such as poly(rc) binding protein 2 (pcbp2), t-cell-restricted intracellular antigen 1 (tia1), and tia1-related protein (tiar) are associated with the viral factories. however, the bona fide sg markers, such as eukaryotic initiation factor 3 (eif3) or eif4a were not present in the factory [122] . in accordance with these results, it is worth noting that tia-1 exerts an antiviral effect on both rabv [9] and vsv [122] . sgs [123] . however, as infection proceeds, normal sg puncta are disrupted, and sgassociated proteins localized to the periphery of viral factories [124] . sgs alteration is due to j o u r n a l p r e -p r o o f mrv ns protein association with ras-gap sh3-binding protein 1 (g3bp1) [124] , a double-stranded nucleic acid helicase which is a master regulator of sg formation [125] . by comparing mrv replication on wild-type and g3bp1 -/-mefs, it was shown that g3bp1 inhibits viral growth [124] . it has been suggested that g3bp1 relocalization to the viral factory periphery induced by ns induces sg disruption in order to facilitate mrv replication in the host translational shutoff environment [124] . on the other hand, for rotaviruses, which also belong to the reoviridae family, it appears that most of the main sg components are present in the viroplasms but that there is a selective exclusion of g3bp1. this sequestration promotes progeny virus production [126] . taken together, all those examples indicate that there is a general, although remarkably diverse, interplay between sgs and viral factories. more recently, it has been shown that the cyclic gmp-amp synthase (cgas, which leads to the production of the secondary messenger cyclic gmp-amp), a major sensor of cytosolic dna from invading viruses which triggers innate immune responses, induced the formation of liquid-like droplets by binding dna in which cgas was activated [127] . in a cellular context, the liquid phase separation and the ifn response to intracellular dna are both dependent on the presence of g3bp1, which binds cgas. finally, an rna-dependent association with pkr promoted the formation of those g3bp1-dependent, membraneless cytoplasmic structures necessary for the dna-sensing function of cgas in human cells [128] . thus, the nucleic acid sensing pathways involved in viral infection detection require the formation of specialized subcellular structures having liquid properties. several isg products also form membrane-less condensates. this is the case of human mxa, a cytoplasmic 70-kda dynamin-family large gtpase which is induced in cells exposed j o u r n a l p r e -p r o o f to type i and iii ifns and has a broad spectrum of antiviral activities [129] . even in uninfected cells, mxa forms spherical or irregular bodies of variable size, filaments, and sometimes a reticulated network that reversibly disassemble/reassemble within minutes of sequential decrease/increase, respectively, in tonicity of extracellular medium [71] . promyelocytic leukemia protein (pml), which is the organizer of the pml nuclear bodies, is also induced by ifn. pml nuclear bodies also contain two other resident proteins, the isg product speckled protein of 100 kda (sp100) and death-associated dead protein (daxx), as well as several other proteins transiently recruited in pml bodies in response to different stimuli [130] . pml bodies as other nuclear bodies exhibit liquid-like properties [131, 132] . the initiation of vrcs of dna viruses occurs at sites near pml-nuclear bodies. human papillomavirus (hpv) infection requires the presence of pml protein suggesting that pml bodies are essential to establish infection [133, 134] . however, sp100 and daxx, which act as transcriptional repressors, restrict viral gene expression in adv [135] , hpv [136] , hsv-1 [137] , and hcmv [138] infections. therefore, it is not surprising that most of the dna viruses that replicate in the nucleus have evolved strategies to disrupt the pml bodies and to degrade or inhibit their components [138] [139] [140] [141] . as a well-characterized example, incoming hsv-1 genomes transiently co-localize with pml nuclear bodies [142] , which surround and encapsulate incoming viral genomes before being disrupted by newly synthesized icp0 viral proteins [137, 139, 143] . the antiviral action of pml nuclear bodies is also observed on rna viruses including dengue virus [144] , influenza virus [145] and rhabdoviruses [146, 147] . here again, viruses have evolved mechanisms that counteract pml j o u r n a l p r e -p r o o f function. as an example, rabv p interacts with pml and retains it in the cytoplasm to alter pml nuclear bodies [148] and consequently is thought to counteract the antiviral effect of isoform pml iv against rabv [146] . the discovery that several viro-induced membrane-less compartments have liquid properties and are formed by llps is a paradigm shift in the field (table 2) . indeed, these compartments provide a functional microenvironment, of which the physicochemical properties remain to be characterized, for the optimal working of the viral replication machinery. understanding the molecular basis of the weak interactions that keep the cohesion of those compartments might lead to the development of drugs that destabilize the viral factories or that concentrate inside to target the viral enzymes more efficiently. an exciting field of research is the identification of the proteome of those compartments. until now, only a few specific cellular factors, which directly interact with viral proteins such as the nucleoproteins and phosphoproteins of mnv, have been shown to concentrate in these structures. the proteome of the viral factories is probably much larger and some proteins might also preferentially partition in the organelle liquid phase due to their physicochemical properties without strongly interacting with any viral protein. as the liquid nature of mnv viral factories precludes their purification, original techniques will be required to map the complete proteome. a promising approach is proximity labeling which has already been used to map the proteome of sgs and processing bodies [125, 149, 150] . interactions between viral factories and several cellular components such as the cytoskeleton and the cellular membrane compartments and their role at distinct stages of the cycle also remain to be finely characterized. here again, understanding those processes may contribute to the development of novel antiviral strategies. however, it is very likely that the most significant impact of the discovery of the liquid nature of viral factories will be in the area of innate immunity. indeed, the liquid nature of viral factories as well as the increasing number of innate immunity actors that have been demonstrated to form biomolecular condensates, invite us to revisit the interactions between the viral infection and the cellular defenses. this interplay is certainly much more subtle than currently thought. therefore, we look forward to an integrated vision of the interactions between viruses and innate immunity that takes into account all the novel data of this booming field. [17] d. kolakofsky, paramyxovirus rna synthesis, mrna editing, and genome hexamer phase: a review, virology, 498 (2016) 94-98. [18] negri, a, contributo allo studio dell'eziologia della rabbia, boll soc med-chir pavia, (1903) 1459-1460. [151] . a number of asp residues and number of glu residues. frequency of negatively charged residues. b number of lys residues and number of arg residues. frequency of positively charged residues. c net charge per residue = (number of arg residues + number of lys residuesnumber of asp residuesnumber of glu residues) / total number of residues in the sequence d number of phe residues, number of tyr residues and number of trp residues. frequency of aromatic residues. 2 for hsv-1, a model of vrc formation that is not based on liquid-liquid phase separation has been proposed [69] . ntd stands for n-terminal domain ctd and ctd for c-terminal domain. left part: space-filling model of vsv n-rna complex (x-ray structure of a 10 n subunit ring (in shades of purple) associated with 90 rna bases (in red) (2gic.pdb) [158] . each vsv n subunit interacts with 9 rna bases. in this conformation, the rna molecule is clamped at the interface of the ntd and the ctd. each n subunit is shown in a different color indicating that the ntd arm from the n th subunit reaches over to the (n-1) th sub-unit and its ctd arm reaches over to the (n+1) th sub-unit. this arrangement leads to the interaction of both (n+1) th virus factories: associations of cell organelles for viral replication and morphogenesis virus factories, double membrane vesicles and viroplasm generated in animal cells virus factories: biogenesis and structural design biomolecular condensates: organizers of cellular biochemistry germline p granules are liquid droplets that localize by controlled dissolution/condensation droplet organelles? intrinsically disordered proteins in overcrowded milieu: membrane-less organelles, phase separation, and intrinsic disorder atpase-modulated stress granules contain a diverse proteome and substructure rabies virus infection induces the formation of stress granules closely connected to the viral factories cajal bodies, nucleoli, and speckles in the xenopus oocyte nucleus have a low-density, sponge-like structure active liquid-like behavior of nucleoli determines their size and shape in xenopus laevis oocytes coexisting liquid phases underlie nucleolar subcompartments higher-order oligomerization promotes localization of spop to liquid nuclear speckles considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates transcription and replication of nonsegmented negative-strand rna viruses negri bodies are viral factories with properties of liquid organelles phase transitions drive the formation of vesicular stomatitis virus replication compartments evidence that the paramyxovirus simian virus 5 can establish quiescent infections by remaining inactive in cytoplasmic inclusion bodies live-cell imaging of marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances analysis of borna disease virus trafficking in live infected cells by using a virus encoding a tetracysteine-tagged p protein influenza a virus ribonucleoproteins form liquid organelles at endoplasmic reticulum exit sites single-molecule fish reveals non-selective packaging of rift valley fever virus genome segments human metapneumovirus nucleoprotein and phosphoprotein interact and provide the minimal requirements for inclusion body formation in vivo interaction of rabies virus phosphoprotein (p) and nucleoprotein (n): existence of two n-binding sites on p protein ebola virus inclusion body formation and rna synthesis are controlled by a novel domain of np interacting with vp35 modular organization of rabies virus phosphoprotein structural disorder within paramyxovirus nucleoproteins and phosphoproteins structure, dynamics and phase separation of measles virus rna replication machinery, current opinion in virology isolation and characterisation of the rabies virus n°-p complex produced in insect cells structure of the vesicular stomatitis virus n0-p complex crystal structure j o u r n a l p r e -p r o o f journal pre-proof of the measles virus nucleoprotein core in complex with an n-terminal region of phosphoprotein structure of the vesicular stomatitis virus l protein in complex with its phosphoprotein cofactor structure of a rabies virus polymerase complex from electron cryo-microscopy solution structure of the c-terminal x domain of the measles virus phosphoprotein and interaction with the intrinsically disordered cterminal domain of the nucleoprotein structure of the vesicular stomatitis virus nucleocapsid in complex with the nucleocapsid-binding domain of the small polymerase cofactor structure and function of the c-terminal domain of the polymerase cofactor of rabies virus structure of a paramyxovirus polymerase complex reveals a unique methyltransferase-ctd conformation structure of the dimerization domain of the rabies virus phosphoprotein crystal structure of the oligomerization domain of the phosphoprotein of vesicular stomatitis virus tetrameric coiled coil domain of sendai virus phosphoprotein measles virus nucleo-and phosphoproteins form liquid-like phase-separated compartments that promote nucleocapsid assembly crystal structure of the rabies virus nucleoprotein-rna complex membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters fus phase separation is modulated by a molecular chaperone and methylation of arginine cation-π interactions poor viral relations no longer endoplasmic reticulum-golgi intermediate compartment membranes and vimentin filaments participate in vaccinia virus assembly replication of african swine fever virus dna in infected cells cytoplasmic organization of poxvirus dna replication aggresomes and pericentriolar sites of virus assembly: cellular defense or viral design? aggresomes resemble sites specialized for virus assembly replication compartments of dna viruses in the nucleus: location, location, location coalescing replication compartments provide the opportunity for recombination between coinfecting herpesviruses herpesviral replication compartments move and coalesce at nuclear speckles to enhance export of viral late mrna evidence for dna-mediated nuclear compartmentalization distinct from phase separation phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization human antiviral protein mxa forms novel metastable membraneless cytoplasmic condensates exhibiting rapid reversible tonicity-driven phase transitions toxic pr poly-dipeptides encoded by the c9orf72 repeat expansion target lc domain polymers phase separation of epstein-barr virus ebna2 and its coactivator ebnalp controls gene expression cytochemical, fluorescent-antibody and electron microscopic studies on the growth of reovirus (echo 10) in tissue culture rotavirus replication: plus-sense templates for double-stranded rna synthesis are made in viroplasms rotavirus genome replication and morphogenesis: role of the viroplasm function, architecture, and biogenesis of reovirus replication neoorganelles rotavirus viroplasm fusion and perinuclear localization are dynamic processes requiring stabilized microtubules the dynamics of both filamentous and globular mammalian reovirus viral factories rely on the microtubule network two non-structural rotavirus proteins, nsp2 and nsp5, form viroplasm-like structures in vivo rotavirus nsp5 orchestrates recruitment of viroplasmic proteins structural organisation of the rotavirus nonstructural protein nsp5 mammalian reovirus nonstructural protein μns forms large inclusions and colocalizes with reovirus microtubule-associated protein μ2 in transfected cells the nucleocapsid protein of human coronavirus nl63 the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division sars-cov-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by rna and partitions into phases of human ribonucleoproteins specific viral rna drives the sars cov-2 nucleocapsid to phase separate phosphorylation modulates liquid-liquid phase separation of the sars-cov-2 n protein hsp70 protein positively regulates rabies virus infection focal adhesion kinase is involved in rabies virus infection through its interaction with viral phosphoprotein p cellular chaperonin cctγ contributes to rabies virus replication during infection the chaperonin cctα is required for efficient transcription and replication of rabies virus: role of cctα in rabv replication immunohistochemical localization of endothelial and inducible nitric oxide synthase within neurons of cattle with rabies upon infection, cellular wd repeat-containing protein 5 (wdr5) localizes to cytoplasmic inclusion bodies and enhances measles virus replication actin-modulating protein cofilin is involved in the formation of measles virus ribonucleoprotein complex at the perinuclear region p38 and ogt sequestration into viral inclusion bodies in cells infected with human respiratory syncytial virus suppresses mk2 activities and stress granule assembly the ebola virus nucleoprotein recruits the nuclear rna export factor nxf1 into inclusion bodies to facilitate viral protein expression host factor smyd3 is recruited by ebola virus nucleoprotein to facilitate viral mrna transcription the cellular protein cad is recruited into ebola virus inclusion bodies by the nucleoprotein np to facilitate genome replication and transcription cytoplasmic relocalization and colocalization with viroplasms of host cell proteins, and their role in rotavirus infection proteomics of herpes simplex virus replication compartments: association of cellular dna replication, repair, recombination, and chromatin remodeling proteinswith icp8 further studies on the replication of rabies and rabies-like viruses in organized cultures of mammalian neural tissues endoplasmic reticulum contact sites regulate the dynamics of membraneless organelles interplay between innate immunity and negative-strand rna viruses: towards a rational model rabies viral mechanisms to escape the ifn system: the viral protein p interferes with irf-3, stat1, and pml nuclear bodies identification of the rabies virus alpha/beta interferon antagonist: phosphoprotein p interferes with phosphorylation of interferon regulatory factor 3 the nucleocytoplasmic rabies virus p protein counteracts interferon signaling by inhibiting both nuclear accumulation and dna binding of stat1 rabies virus p protein interacts with stat1 and inhibits interferon signal transduction pathways ebola virus vp35 protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling structural basis for marburg virus vp35-mediated immune evasion mechanisms ebola virus vp35 antagonizes pkr activity through its c-terminal interferon inhibitory domain ebola virus protein vp35 impairs the function of interferon regulatory factor-activating kinases ikkɛ and tbk-1 paramyxovirus evasion of innate immunity: diverse strategies for common targets inducible expression of the p, v, and np genes of the paramyxovirus simian virus 5 in cell lines and an examination of np-p and np-v interactions formation of antiviral cytoplasmic granules during orthopoxvirus infection dhx36 enhances rig-i signaling by facilitating pkr-mediated antiviral stress granule formation critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity mda5 localizes to stress granules, but this localization is not required for the induction of type i interferon induction of stress granule-like structures in vesicular stomatitis virus-infected cells mammalian orthoreovirus particles induce and are recruited into stress granules at early times postinfection mammalian orthoreovirus factories modulate stress granule protein localization by interaction with g3bp1 g3bp1 is a tunable switch that triggers phase separation to assemble stress granules rotavirus induces formation of remodeled stress granules and p bodies and their sequestration in viroplasms to promote progeny virus production dna-induced liquid phase condensation of cgas activates innate immune signaling pkr-dependent cytosolic cgas foci are necessary for intracellular dna sensing mx gtpases: dynamin-like antiviral machines of innate immunity role of promyelocytic leukemia protein in host antiviral defense establishment of papillomavirus infection is enhanced by promyelocytic leukemia protein (pml) expression the role of promyelocytic leukemia nuclear bodies during hpv infection control of human adenovirus type 5 gene expression by cellular daxx/atrx chromatin-associated complexes sp100 provides intrinsic immunity against human papillomavirus infection differential role of sp100 isoforms in interferon-mediated repression of herpes simplex virus type 1 immediate-early protein expression human cytomegalovirus infection causes degradation of sp100 proteins that suppress viral gene expression a viral ubiquitin ligase has substrate preferential sumo targeted ubiquitin ligase activity that counteracts intrinsic antiviral defence the adenoviral oncogene e1a-13s interacts with a specific isoform of the tumor suppressor pml to enhance viral transcription emerging role of pml nuclear bodies in innate immune signaling distinct temporal roles for the promyelocytic leukaemia (pml) protein in the sequential regulation of intracellular host immunity to hsv-1 infection replication of icp0-null mutant herpes simplex virus type 1 is restricted by both pml and sp100 cellular promyelocytic leukemia protein is an important dengue virus restriction factor differential suppressive effect of promyelocytic leukemia protein on the replication of different subtypes/strains of influenza a virus resistance to rabies virus infection conferred by the pmliv isoform implication of pmliv in both intrinsic and innate immunity rabies virus p and small p products interact directly with pml and reorganize pml nuclear bodies context-dependent and disease-specific diversity in protein interactions within stress granules high-density proximity mapping reveals the subcellular organization of mrna-associated granules and bodies analyzing protein disorder with iupred2a inclusion body fusion of human parainfluenza virus type 3 regulated by acetylated α-tubulin enhances viral replication rsv hijacks cellular protein phosphatase 1 to regulate m2-1 phosphorylation and viral transcription human metapneumovirus induces formation of inclusion bodies for efficient genome replication and transcription the spatio-temporal distribution dynamics of ebola virus proteins and rna in infected cells dna virus replication compartments linkage of transcription and translation within cytoplasmic poxvirus dna factories provides a mechanism to coordinate viral and usurp host functions structure of the vesicular stomatitis virus nucleoprotein-rna complex near-atomic cryo-em structure of the helical measles virus nucleocapsid solution structure of the c-terminal nucleoprotein-rna binding domain of the vesicular stomatitis virus phosphoprotein structure of the tetramerization domain of measles virus phosphoprotein structural basis for the attachment of a paramyxoviral polymerase to its template this work was supported by the cnrs, the university paris-saclay, la fondation bettencourt and by the agence nationale de la recherche (anr-19-ce15-0024-01). qn is a postdoctoral fellow supported by a grant on the anr contract. -ssrna j o u r n a l p r e -p r o o f key: cord-295381-0dqu3p3y authors: kamal, adeela; boehm, marcus f; burrows, francis j title: therapeutic and diagnostic implications of hsp90 activation date: 2004-06-01 journal: trends in molecular medicine doi: 10.1016/j.molmed.2004.04.006 sha: doc_id: 295381 cord_uid: 0dqu3p3y abstract the molecular chaperone heat-shock protein 90 (hsp90) is involved in the stabilization and conformational maturation of many signaling proteins that are deregulated in cancers. hsp90 inhibition results in the proteasomal degradation of these client proteins and leads to potent antitumor activity. the hsp90 inhibitor 17-allylaminogeldanamycin (17-aag) is presently in clinical trials. recent work has identified the role of hsp90 in multiple signal transduction pathways and revealed that the molecular mechanism of tumor selectivity by hsp90 inhibitors is the result of an activated, high-affinity conformation of hsp90 in tumors. this review discusses these recent advances in the understanding of tumor hsp90 for the treatment and diagnosis of cancer. in addition, the role of hsp90 in non-oncological diseases will also be discussed. the molecular chaperone heat-shock protein 90 (hsp90) is involved in the stabilization and conformational maturation of many signaling proteins that are deregulated in cancers. hsp90 inhibition results in the proteasomal degradation of these client proteins and leads to potent antitumor activity. the hsp90 inhibitor 17-allylaminogeldanamycin (17-aag) is presently in clinical trials. recent work has identified the role of hsp90 in multiple signal transduction pathways and revealed that the molecular mechanism of tumor selectivity by hsp90 inhibitors is the result of an activated, highaffinity conformation of hsp90 in tumors. this review discusses these recent advances in the understanding of tumor hsp90 for the treatment and diagnosis of cancer. in addition, the role of hsp90 in non-oncological diseases will also be discussed. hsp90 is a ubiquitous molecular chaperone protein that is involved in the folding, activation and assembly of many proteins, including key mediators of signal transduction, cell-cycle control and transcriptional regulation [1, 2] . hsp90 client proteins include transmembrane tyrosine kinases [her-2/neu, epidermal growth factor receptor (egfr), met and insulin-like growth factor-1 receptor (igf-1r)], metastable signaling proteins (akt, raf-1 and ikk), mutated signaling proteins (p53, kit, flt3 and v-src), chimeric signaling proteins (npm -alk, bcr-abl), steroid receptors (androgen, estrogen and progesterone receptors) and cell-cycle regulators (cdk4, cdk6) [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] . hsp90 is regulated by co-chaperone proteins, which participate in an ordered series of dynamic multi-protein complexes that are linked to the conformationally coupled atpase cycle of hsp90 [2, 14, 15] . client proteins bind to hsp90 in an intermediate complex, containing the cochaperone proteins hsp70, hsp40, hip and hop. upon atp binding and hydrolysis, hsp90 forms a mature complex, containing p23, p50cdc37 and immunophilins (ip), that catalyzes the conformational maturation of the hsp90 client proteins (figure 1 ). the naturally occurring ansamycin antibiotic geldanamycin binds to a conserved binding pocket in the n-terminal atp-binding domain of hsp90 [16] [17] [18] , inhibiting atp binding and atp-dependent hsp90 chaperone activity [19] [20] [21] and directing the proteasomal degradation of hsp90 client proteins [22, 23] . geldanamycin displays potent anti-tumor activity in vitro but is too hepatotoxic for clinical use [24] . the less toxic geldanamycin derivative 17-allylaminogeldanamycin (17-aag) also binds to hsp90 [3] , exerts a potent antitumor activity in preclinical models [12, 25] and is currently in clinical trials [26] . why hsp90 modulators preferentially targeted tumor cells, even though hsp90 is abundantly expressed in both normal and tumor cells, was unknown. furthermore, it was unclear why 17-aag binds to recombinantly isolated hsp90 protein with micromolar affinity but commonly kills tumor cell lines at nanomolar concentrations [3, 27] . recent data have revealed that the therapeutic selectivity of hsp90 inhibitors results from the presence of a high-affinity activated form of hsp90 in tumors, which is in a multi-chaperone complex with high atpase activity, whereas the hsp90 in normal tissues is in an inactive, uncomplexed form with low affinity [28] . in this review, we will discuss the role of hsp90 in multiple signal transduction pathways and the diagnostic and therapeutic implications of the molecular mechanism of tumor selectivity of hsp90 inhibitors. the structural implications of hsp90 activation by the formation of multi-chaperone complexes will also be discussed, as will the potential use of hsp90-inhibitor drugs in nononcological diseases. hsp90 inhibition affects multiple signaling pathways hsp90 influences the activity and stability of many of the client proteins that function as key regulators in cellular growth, differentiation and apoptotic pathways. more than 100 known hsp90 clients regulate multiple signal transduction pathways that are persistently activated in human cancers. inactivation of the ras-raf-1-mek -erk and phosphatidyl inositol-3 kinase -akt pathways by hsp90 inhibitors causes the downregulation of cyclin d1 and the functional inactivation of cdk4, both of which are important for the g1-s cell cycle transition ( figure 2 ). growth factor receptors that that are sensitive to hsp90mediated degradation include the receptors for her-2, igf-1, egf and platelet-derived growth factor (pdgf). hsp90 inhibitors inactivate multiple kinases, such as raf-1, akt and cdk4 [5, 6, 29] . furthermore, inactivation of akt by hsp90 inhibitors leads to reduced bad (bcl-xl/bcl-1associated death promoter) phosphorylation, which decreases the activity of the anti-apoptotic proteins bcl2 and bcl-x, thereby inducing apoptosis by cytochrome c release [30] . hsp90 inhibitors also inactivate steroid receptors, such as the androgen receptor and estrogen receptor, which, when bound to their cognate ligands, translocate to the nucleus and act as transcription factors [2] . therefore, hsp90 inhibitors simultaneously destabilize many oncoproteins in multiple signaling pathways, suggesting that the inhibition of hsp90 could be particularly beneficial in attacking late-stage cancer cells, which can easily circumvent the blockade of a single target or pathway. because of the wide array of hsp90 client proteins, hsp90 inhibitors can be used to target diverse cancers in which an hsp90 client protein is necessary for cancer proliferation, survival or progression. in particular, her-2 is one of the client proteins that is most sensitive to hsp90 inhibitors [4] , and promising preclinical data in her-2-overexpressing breast-and prostate-cancer models suggest that 17-aag could be used to treat such tumors [5, 12] . the discovery that the bcr-abl fusion protein, which is the sole causative factor in chronic myelogenous leukemia (cml), is an hsp90 client [31] led to studies showing that both geldanamycin and 17-aag induced apoptosis in cml-derived cell lines [32] . furthermore, cml cells that have become resistant to the bcr-abl inhibitor gleevec remain sensitive to 17-aag, suggesting this drug could be beneficial to gleevec-resistant patients [33] . in addition, there are data indicating that other leukemias and lymphomas that are dependent on chimeric or mutated proteins, such as npm -alk [9] , flt3(itd) [10] or c-kit [11] , can be targeted by hsp90-inhibitor drugs. therefore, hsp90 inhibitors could potentially affect a broad range of cancers, and various ongoing clinical trials using 17-aag aim to determine whether this is true [1, 26] . a high-affinity conformation of tumor hsp90 confers drug selectivity why do hsp90 inhibitor drugs selectively kill cancer cells, even though hsp90 is widely expressed in normal tissues? a recent study reported that the hsp90 in tumor cells is maintained in an activated conformation by the formation of multi-chaperone complexes that have increased atpase activity and 100-fold greater binding affinity for 17-aag compared with the uncomplexed, latent form of hsp90 that is present in normal cells [28] . interestingly, hsp90 from tumor cells that are overexpressing the hsp90 clientprotein her-2 exhibited the highest binding affinity for 17-aag, which is in accordance with a recent report that her-2 overexpression in tumor cells confers increased sensitivity to geldanamycin [34] . the increased binding affinity of tumor hsp90 for 17-aag also explains the remarkable ability of hsp90-binding drugs to accumulate selectively in tumors compared with normal tissues in vivo [28, 35] . furthermore, these results suggest that hsp90 usage, as indicated by binding-affinity, is a true predictor of the hsp90 dependence of tumor cells. it has been reported that hsp90 levels are elevated in many cancer tissues [36] [37] [38] but it is probable that it is the activity of hsp90 that is more closely linked to cancer progression, at least during the early stages. as malignant progression proceeds, the complete usage of tumor hsp90 within the cell might provide a selection pressure leading to the further upregulation of hsp90 that is observed in many advanced tumors [37 -40] . a model for hsp90-dependent malignant progression has been proposed in which, as tumor cells gradually accumulate mutant and overexpressed signaling proteins, hsp90 becomes engaged in the active chaperoning and stabilization of oncoproteins and adopts a high-affinity form that is induced by bound co-chaperone proteins ( figure 3 ) [28] . by contrast, the hsp90 in normal cells appears to be largely unused and remains in an uncomplexed form that has low-affinity binding for hsp90 inhibitors. this model would also predict, based on current knowledge, that proteotoxic stresses in normal cells would convert inactive hsp90 into complexed hsp90 with higher binding affinity. transcription of hsp90 is regulated by the heat-shock transcription factor hsf-1, which is thought to be regulated through its sequestration in an inactive state by cytosolic hsp90 [41] . under conditions of proteotoxic stress (e.g. heat), hsp90 is recruited to refold partially denatured proteins, liberating hsf-1 from sequestering complexes and enabling it to trimerize and translocate to the nucleus, where it transactivates hsp90 and other heatshock genes [42] . assuming that the stress-induced chaperoning activity of hsp90 is atp-dependent, this transition should be accompanied by a sharp increase in hsp90 usage and a commensurate increase in the affinity of the cellular hsp90 pool for inhibitors. similarly, it is interesting to speculate whether the de novo-induced hsp90 will be of high or low affinity for hsp90-binding drugs. because 17-aag and the other active agents that bind the n-terminal atp site of hsp90 also cause the dissociation of hsf-1 complexes and a robust heat-shock response [41] , it is possible that the time window for the cytotoxic activity of hsp90 inhibitors could be limited by the development of a drug-induced heat-shock response [43] . if the induced hsp90 is uncomplexed and has low affinity, it will compete poorly with the pre-existing highaffinity hsp90 complexes (containing client proteins) for binding to 17-aag. alternatively, repopulation of the cytosol with active high-affinity hsp90 complexes might be expected to dampen ongoing antitumor responses. the fact that several co-chaperone genes are targets for hsf-1, and are coordinately induced during the heat-shock response [42] , indicates that the latter possibility is more likely. the crystal structures of the n-terminal domain of hsp90 that is bound to either geldanamycin, radicicol, atp or adp ( figure 4 ) have been solved [17, 18, 44, 45] . key amino acids within the n-terminal binding pocket of hsp90 form crucial interactions with these hsp90-inhibitor compounds [44] . it is important to note, however, that all of the structural information is based on free, uncomplexed hsp90. because the active form of hsp90 comprises a multi-chaperone protein complex [28] , which imparts structural changes on the atp-binding site (as evidenced by the dramatic increase in binding activity for hsp90 inhibitors), the precise binding configuration of hsp90 inhibitors to the activated form of hsp90 remains to be determined. based upon the crystal structure of atp that is bound to hsp90, analogues such as pu3 (figure 4 ), which exhibit a c-shaped or compact conformation, have been synthesized [27, 46] and the crystal structure of pu3 with recombinant hsp90 has been solved [47] . these small-molecule hsp90-inhibitor compounds also exhibit many of the biological properties of a classical hsp90 inhibitor [46] , but it remains to be determined whether these compounds also bind to complexed hsp90 with a higher affinity, as do the geldanamycin derivatives. the difference in the binding activities of complexed versus free hsp90 might be explained by the conformational differences of geldanamycin in the liganded and unliganded forms. similar to atp, geldanamycin and its derivatives adopt a compact, c-shaped conformation when bound to hsp90 that is unlike the extended planar structure of unbound geldanamycin [17, 18] . this flexibility in conformation probably enables geldanamycin to adopt the precise conformation that is necessary for binding to hsp90 complexed with co-chaperones, resulting in exceptional selectivity for complexed hsp90 compared with free hsp90. an intriguing possibility that has been suggested is that either the multi-chaperone-bound hsp90, or other components of the multi-chaperone figure 3 . model for tumor selectivity of hsp90 inhibitors and hsp90-dependent malignant progression. hsp90 in normal cells exists in an uncomplexed form that has lowaffinity for hsp90 inhibitor drugs, which accumulate poorly in normal tissues, and normal cells exhibit poor drug sensitivity. by contrast, the hsp90 in cancer cells is involved in the active chaperoning of overexpressed oncoproteins and exists in a complexed form with co-chaperone proteins (p23 and hop are not in the same complex). complexed hsp90 in cancer cells exhibits high-affinity binding to hsp90 inhibitor drugs, which accumulate in tumor tissues, and tumor cells exhibit good drug sensitivity. this model predicts that the accumulation of mutant proteins in advanced cancer would further increase hsp90 usage and make tumor cells more hsp90 dependent. furthermore, this model suggests that the high-affinity change of hsp90 can be driven by the overexpression of oncoproteins, as well as by stressful conditions in normal cells (e.g. heat). complex, are responsible for catalyzing the conformational change in geldanamycin which enables it to bind with high-affinity to hsp90 [48] . alternatively, the hsp90 that is complexed with co-chaperone proteins might cause a conformational change in the nucleotide binding pocket of hsp90, such that geldanamycin derivatives bind with a higher affinity. recently, the crystal structure of another geldanamycin derivative, 17-allylamino-17-demethoxygeldanamycin (17-dmag), with human hsp90 has been solved, and molecular modeling studies suggested that 17-dmag, when constrained to a cis-amide bond in the ground state, would increase binding affinity for hsp90 [49] . these latter changes might occur more easily when hsp90 is complexed with co-chaperone proteins and thus modulate the higher binding affinity of complexed hsp90. the co-chaperone proteins hsp70, hsp40, p23 and hop were found to modulate hsp90 activation by increasing 17-aag binding affinity by 50-fold and hsp90 atpase activity by 32-fold [28] . this set of co-chaperones had been previously used for in vitro reconstitution experiments, where they were needed for hsp90-chaperoning activity of the progesterone receptor [15] . previous data showed that hop strongly inhibits and p23 weakly inhibits hsp90 atpase, but those experiments were performed using yeast hsp90 [50] . by contrast, human hsp90 is a much weaker atpase than is yeast hsp90, and hop has little effect on the basal atpase rate of hsp90 but inhibits the client-protein-stimulated atpase rate, whereas p23 exhibits a small suppression [51] . these results suggest that the formation of hsp90 multi-chaperone complexes is a dynamic process that regulates hsp90 activity. it will be interesting to determine the effects of two other cochaperone proteins on the affinity of hsp90 for inhibitors such as 17-aag: cdc37, which is important for chaperoning kinases [52] , and aha1, which increases hsp90 atpase activity 12-fold [53] . unlike most co-chaperones, which bind to the c-terminus of hsp90, the crystal structure of cdc37 with hsp90 revealed that cdc37 binds to the n-terminus of hsp90 and prevents the transactivating interaction of the n-terminal domains of hsp90, thus inhibiting hsp90 atpase activity [52] . the central region of hsp90 is involved in the direct interaction with aha1, and it has been suggested that aha1 increases hsp90 atpase activity by stabilizing interactions between the central and n-terminal domains of hsp90 [54] . therefore, future studies will be aimed at better understanding the dynamic role of co-chaperone proteins in modulating hsp90 activation by increasing the binding affinity for hsp90 inhibitors. diagnostic applications of hsp90 assays the profound differences in hsp90 activity between normal and malignant cells were revealed using three experimental approaches: a competitive-binding assay using hsp90 extracted from cell lysates, co-immunoprecipitation of hsp90-binding co-chaperone proteins and hsp90 atpase activity [28] . the magnitude of the divergence (100-fold, in terms of hsp90 binding affinity and percentage of free versus complexed hsp90) and the close correlation between binding affinity and cell-killing potency (r 2 â¼ 0.92) suggest that these analyses could have significant diagnostic value. the lysate-binding assay can be performed with a quantity of malignant tissue that could be obtained from a routine blood sample (for leukemias) or a fine-needle aspirate (other tumors), raising hopes that the current method, or a streamlined version of it, could be used to guide patient selection for the clinical development of hsp90 inhibitors. this type of assay could have advantages over existing molecular diagnostics such as hercepteste [55] because, in addition to measuring the levels of hsp90 in the sample, it indicates the extent to which the tumor is using (and is dependent upon) hsp90. clearly, a simpler methodology, such as an immunoassay, to determine hsp90 usage in clinical samples would be preferable. the hsp90 antibody ac88 recognizes uncomplexed hsp90 [28, 41] and might be a useful tool to measure decreased hsp90 usage, but a better reagent would be an antibody that specifically recognizes the high-affinity conformation of hsp90. such an antibody might recognize an epitope that is produced by the close juxtaposition of hsp90 and a co-chaperone protein, or an activated conformation-specific epitope on hsp90 itself. a conformation-specific antibody, 9g10, has been reported for grp94 [56] , the endoplasmic reticulum homolog of hsp90, and it remains to be seen if a conformation-specific hsp90 antibody can be developed. hsp90 inhibitors might have therapeutic potential in other conditions where diseased cells are dependent on increased hsp90 activity. because cellular hsp90 is recruited in cells that are undergoing viral infection or mediating autoimmune responses, hsp90 inhibition might be useful for treating these diseases. furthermore, hsp90 inhibitors can also upregulate the heat-shock proteins that provide protective functions in central nervous system (cns) disorders and cardiovascular diseases [57 -59] . it is possible that increased hsp90 usage in these diseases could provide a selective therapeutic target for the highaffinity binding of hsp90 inhibitors within the diseased tissue. in most cases, it is unlikely that the hsp90-related cellular changes in diseased cells will be as profound as those in highly malignant cells, but it will be interesting to determine whether hsp90 activity might represent a useful independent diagnostic and/or prognostic marker in these non-oncological diseases. viral infections are stressful for the infected cell, and this is reflected by an increased expression of heat-shock proteins, including hsp90. the virus essentially 'steals' the host hsp90 to facilitate its own assembly and replication, and studies have demonstrated that geldanamycin can block the viral life-cycle of hepatitis b virus (hbv), hepatitis c virus (hcv) and herpes simplex virus type 1 (hsv-1) [60 -62] . hsp90 is an essential host factor for hbv replication and interacts with the viral reverse transcriptase [60] , and it is also necessary for the activity of ns2 -3 protease of hcv [61] . furthermore, a recent study showed that hsv-1 replication in vitro was significantly inhibited by geldanamcyin with an ic 50 of 93 nm, whereas geldanamycin inhibited the cellular growth of uninfected cells with an ic 50 of 350 mm, suggesting good therapeutic selectivity [62] . the same study also demonstrated that geldanamycin exhibited broad-spectrum activity against a range of viruses with an ic 50 of 0.5 -4 mm: hiv-1 and sars coronavirus exhibited the highest sensitivity [62] . the mechanism of hiv-1 inhibition might be explained by the ability of geldanamycin to block tat activation of hiv-1, through its effects on the host cofactor cdk9 [63] . therefore, these studies suggest that hsp90 inhibitors could be used in multiple viral diseases, and it remains to be determined whether the hsp90 in virally infected cells exhibits high-affinity binding to hsp90 inhibitors and could provide therapeutic selectivity between the virally infected and uninfected cells. autoimmune diseases are the result of inappropriate and persistent lymphocyte activation in an aberrant response to self determinants. destructive symptoms of autoimmunity might arise by the action of t cells or from b-cellderived antibody and complement fixation. t and b lymphocytes use similar signal transduction pathways in their mitogenic and activation programs as do tumor cells, suggesting that hsp90 modulators might be potent inhibitors of lymphocyte function. yorgin et al. showed the connection between the inhibition of t-cell activation and the killing of activated t cells by geldanamycin with the degradation of the non-receptor tyrosine kinase p56 lck [64] . geldanamycin also depletes raf-1 (an inhibitorsensitive hsp90 client) from rat splenocytes and consequently blocks rat t-cell proliferation in response to repeated exposures to antigen [65] . furthermore, hsp90 inhibitors also block the raf-erk-mediated proliferation of murine b cells that are stimulated through the b-cell antigen receptor [66] . taken together, these studies suggest that antibody-mediated autoimmune disease could be doubly affected by hsp90 inhibition of both the t-cell and b-cell compartments. ischemia is a stress phenomenon that accompanies most cerebrovascular and cardiovascular disease states. heatshock proteins are upregulated during the stress response and provide neuroprotection and cardioprotection against ischemic damage. geldanamycin, via the hsf-1 pathway, also causes the upregulation of heat-shock proteins, including hsp27, hsp70 and hsp90. these have a protective role in stress-induced ischemic damage, exerting potent anti-apoptotic activity by inhibiting the assembly of the caspase 9-apaf-1-cytochrome c apoptosome [67] . studies have demonstrated that geldanamycin is an effective post-treatment neuroprotective agent in a cell-culture model of oxidative toxicity [58] and also protects the brain from focal ischemia in an in vivo rat model [59] , providing strong evidence that the hsp90 inhibitors could be used as a neuroprotective agents. geldanamycin has also been shown to induce a heat-shock response in myogenic cells and to confer protection against ischemic stress [57] , suggesting that hsp90 inhibitors offer a pharmacological method for increasing the level of heat-shock proteins in cardiac tissue, thus protecting the heart against ischemic injury. in addition to ischemic injury, there is evidence that hsp90 inhibitors could be utilized in protein-aggregation diseases of the cns, such as huntington's disease (hd), because nanomolar concentrations of geldanamycin activated a heat-shock response and inhibited hd protein aggregation in a cell-culture model for hd [68] . furthermore, a-synuclein-mutant flies fed on the drug were completely protected from neuronal loss in a drosophila model of parkinson's disease [69] . collectively, these studies suggest that hsp90 inhibitors could be used to modulate the heat-shock response and provide protective function in damaged cells in the brain and heart. inhibiting a protein that regulates multiple signal transduction pathways in cancer cells is an attractive approach for cancer therapy. because hsp90 is involved in the conformational maturation of various oncogenic signaling proteins, it is emerging as a prime target for anticancer drugs. the dependence of cancer cells on the activated form of hsp90 suggests that the multi-chaperone hsp90 complex could be a unique cancer target, and provides a mechanism for why 17-aag and other hsp90 inhibitors selectively destroy cancer cells but not normal cells. hsp90 might serve an analogous role in the somatic evolution of tumors as in darwinian evolution [70] , rescuing potentially misfolded mutant proteins to prevent the mutation becoming lethal to the cell and thus enabling these oncoproteins to support tumor-cell proliferation, survival and malignancy. further work will be required to determine how closely hsp90 usage mirrors disease progression in cancer and other conditions, but increased hsp90 usage in cancer cells might be useful as a diagnostic tool to stratify patients in clinical trials of hsp90 inhibitors or as an independent staging criterion in a range of diseases. heat shock protein 90 as a molecular target for cancer therapeutics regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery the benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin binds to hsp90 and shares important biologic activities with geldanamycin sensitivity of mature erbb2 to geldanamycin is conferred by its kinase domain and is mediated by the chaperone protein hsp90 ansamycin antibiotics inhibit akt activation and cyclin d expression in breast cancer cells that overexpress her2 disruption of the raf-1 -hsp90 molecular complex results in destabilization of raf-1 and loss of raf-1-ras association the heat shock protein 90 antagonist geldanamycin alters chaperone association with p210bcr-abl and v-src proteins before their degradation by the proteasome geldanamycin selectively destabilizes and conformationally alters mutated p53 nucleophosmin-anaplastic lymphoma kinase (npm-alk), a novel hsp90 -client tyrosine kinase: down-regulation of npm-alk expression and tyrosine phosphorylation in alk ã¾ cd30 ã¾ lymphoma cells by the hsp90 antagonist 17-allylamino,17-demethoxygeldanamycin flt3 expressing leukemias are selectively sensitive to inhibitors of the molecular chaperone heat shock protein 90 through destabilization of signal transduction-associated kinases 17-allylamino-17-demethoxygeldanamycin (17-aag) is effective in down-regulating mutated, constitutively activated kit protein in human mast cells 17-allylamino-17-demethoxygeldanamycin induces the degradation of androgen receptor and her-2/neu and inhibits the growth of prostate cancer xenografts stable and specific binding of heat shock protein 90 by geldanamycin disrupts glucocorticoid receptor function in intact cells reconstitution of progesterone receptor with heat shock proteins the assembly of progesterone receptor-hsp90 complexes using purified proteins inhibition of heat shock protein hsp90 -pp60v -src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation identification and structural characterization of the atp/adp-binding site in the hsp90 molecular chaperone crystal structure of an hsp90 -geldanamycin complex: targeting of a protein chaperone by an antitumor agent in vivo function of hsp90 is dependent on atp binding and atp hydrolysis atp binding and hydrolysis are essential to the function of the hsp90 molecular chaperone in vivo the importance of atp binding and hydrolysis by hsp90 in formation and function of protein heterocomplexes depletion of the erbb-2 gene product p185 by benzoquinoid ansamycins polyubiquitination and proteasomal degradation of the p185c -erbb-2 receptor protein-tyrosine kinase induced by geldanamycin preclinical pharmacologic evaluation of geldanamycin as an antitumor agent inhibition of heat shock protein 90 function down-regulates akt kinase and sensitizes tumors to taxol clinical development of 17-allylamino, 17-demethoxygeldanamycin a small molecule designed to bind to the adenine nucleotide pocket of hsp90 causes her2 degradation and the growth arrest and differentiation of breast cancer cells a high-affinity conformation of hsp90 confers tumor selectivity on hsp90 inhibitors mammalian p50cdc37 is a protein kinasetargeting subunit of hsp90 that binds and stabilizes cdk4 cellular survival: a play in three akts the hsp90 inhibitor geldanamycin selectively sensitizes bcr-abl-expressing leukemia cells to cytotoxic chemotherapy geldanamycin and its analogue 17-allylamino-17-demethoxygeldanamycin lowers bcr-abl levels and induces apoptosis and differentiation of bcr-abl-positive human leukemic blasts bcr-abl point mutants isolated from patients with imatinib mesylate-resistant chronic myeloid leukemia remain sensitive to inhibitors of the bcr -abl chaperone heat shock protein 90 erbb2 overexpression in an ovarian cancer cell line confers sensitivity to the hsp90 inhibitor geldanamycin physiologically-based pharmacokinetics and molecular pharmacodynamics of 17-(allylamino)-17-demethoxygeldanamycin and its active metabolite in tumor-bearing mice unusual expression and localization of heatshock proteins in human tumor cells differential expression of heat shock proteins in pancreatic carcinoma expression and roles of heat shock proteins in human breast cancer selective over-expression of mrna coding for 90 kda stress-protein in human ovarian cancer heat shock protein-90, il-6 and il-10 in bladder cancer repression of heat shock transcription factor hsf1 activation by hsp90 (hsp90 complex) that forms a stress-sensitive complex with hsf1 regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators induction of a heat shock factor 1-dependent stress response alters the cytotoxic activity of hsp90-binding agents structural basis for inhibition of the hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin atpases as drug targets: learning from their structure development of a purine-scaffold novel class of hsp90 binders that inhibit the proliferation of cancer cells and induce the degradation of her2 tyrosine kinase adenine derived inhibitors of the molecular chaperone hsp90 -sar explained through multiple x-ray structures cancer: the rules of attraction crystal structure and molecular modeling of 17-dmag in complex with human hsp90 regulation of hsp90 atpase activity by tetratricopeptide repeat (tpr)-domain co-chaperones stimulation of the weak atpase activity of human hsp90 by a client protein the mechanism of hsp90 regulation by the protein kinase-specific cochaperone p50(cdc37) activation of the atpase activity of hsp90 by the stress-regulated cochaperone aha1 structural and functional analysis of the middle segment of hsp90: implications for atp hydrolysis and client protein and cochaperone interactions quantitative immunohistochemical evaluation of her2/neu expression with hercepteste in breast carcinoma by image analysis radicicol-sensitive peptide binding to the n-terminal portion of grp94 induction of heat shock proteins by tyrosine kinase inhibitors in rat cardiomyocytes and myogenic cells confers protection against simulated ischemia geldanamycin provides posttreatment protection against glutamate-induced oxidative toxicity in a mouse hippocampal cell line geldanamycin induces heat shock proteins in brain and protects against focal cerebral ischemia hsp90 is required for the activity of a hepatitis b virus reverse transcriptase host cell factor requirement for hepatitis c virus enzyme maturation geldanamycin, a ligand of heat shock protein 90, inhibits the replication of herpes simplex virus type 1 in vitro requirement for a kinase-specific chaperone pathway in the production of a cdk9/cyclin t1 heterodimer responsible for p-tefb-mediated tat stimulation of hiv-1 transcription effects of geldanamycin, a heat-shock protein 90-binding agent, on t cell function and t cell nonreceptor protein tyrosine kinases immunosuppressive effects of the heat shock protein 90-binding antibiotic geldanamycin requirement for a hsp90 chaperonedependent mek1/2-erk pathway for b cell antigen receptor-induced cyclin d2 expression in mature b lymphocytes stress management -heat shock protein-70 and the regulation of apoptosis geldanamycin activates a heat shock response and inhibits huntingtin aggregation in a cell culture model of huntington's disease pharmacological prevention of parkinson disease in drosophila hsp90 as a capacitor for morphological evolution we thank our colleagues at conforma therapeutics corporation for useful comments and suggestions. do you want to reproduce material from a trends journal?this publication and the individual contributions within it are protected by the copyright of elsevier. except as outlined in the terms and conditions (see p. ii), no part of any trends journal can be reproduced, either in print or electronic form, without written permission from elsevier. please address any permission requests to:rights and permissions, elsevier ltd, po box 800, oxford, uk ox5 1dx. e-mail: permissions@elsevier.com review key: cord-034823-ogwjzfgf authors: guo, hao-bo; ma, yue; tuskan, gerald a.; qin, hong; yang, xiaohan; guo, hong title: a suggestion of converting protein intrinsic disorder to structural entropy using shannon’s information theory date: 2019-06-14 journal: entropy (basel) doi: 10.3390/e21060591 sha: doc_id: 34823 cord_uid: ogwjzfgf we propose a framework to convert the protein intrinsic disorder content to structural entropy (h) using shannon’s information theory (it). the structural capacity (c), which is the sum of h and structural information (i), is equal to the amino acid sequence length of the protein. the structural entropy of the residues expands a continuous spectrum, ranging from 0 (fully ordered) to 1 (fully disordered), consistent with shannon’s it, which scores the fully-determined state 0 and the fully-uncertain state 1. the intrinsically disordered proteins (idps) in a living cell may participate in maintaining the high-energy-low-entropy state. in addition, under this framework, the biological functions performed by proteins and associated with the order or disorder of their 3d structures could be explained in terms of information-gains or entropy-losses, or the reverse processes. protein structures have played a central role in molecular biology ever since the "lock-and-key" model for enzymatic catalysis [1], along with the sequence-structure-function dogma [2] and the protein folding landscape theory [3] . however, many proteins from different organisms have been found to lack stable, well-folded tertiary structures in their native states. these proteins are termed the intrinsically disordered proteins (idps) or intrinsically disordered protein regions (idprs) [4] . the term "intrinsic" here means that the quantity is encoded in the protein primary amino acid sequence, on the same ground as the idp denominator [5] . the abundance of idps and idprs, and the vital roles they play in cellular organisms and viruses, has been appreciated in a plethora of publications during the past two decades [6, 7] . despite the extensive discussions of idps in literature, "paradoxes" seem to present in idp structures and functions, including reduced structural content versus enhanced structural heterogeneity, simplified folding landscapes versus complexified functionalities, etc. [8] the intrinsic disorder content of each residue in a protein can be predicted from its primary amino acid sequence. it is now well understood that the idps lack the hydrophobic cores necessary for successful folding [9] and that the charged residues (r, k, h, e, and d) are disorder-promoting [10] . an early study showed the association between the ratios of the numbers of charged over hydrophobic residues and the disorder content for a relatively small set of proteins [11] ; however, this oversimplified approach was not predictive for larger protein sets. as representative present algorithms, the predictor of natural disordered regions (pondr) family predictors use artificial neural networks to integrate attributors, including the amino acid composition, sequence complexity, hydropathy, and net charge-and they yield reasonably accurate intrinsic disorder predictions [12] . developing accurate intrinsic disorder predictors is an endless path [13] , considering the astronomical size of the protein world-e.g., 20 100 different proteins of 100 amino acids-and the crowded and dynamic cellular environments surrounding the proteins. quality assessment has been performed among different protein disorder predictors [14, 15] , which adopt the same standard for the intrinsic disorder score, representing the probability of being disordered-i.e., a fully ordered residue scores 0 (0%) and a fully disordered residue scores 1 (100%), respectively. the need for a unified guideline to report a protein intrinsic disorder has also been demanded [16] . in intrinsic disorder interpretations, ambiguities exist for both amino acids and proteins. for example, if an amino acid has an id score of 0.5, is it an ordered or disordered residue? similarly, for a protein with 50% disordered residues, is it an ordered or disordered protein? in the present work, we suggest a conversion to the intrinsic disorder contents of the residues in the proteins into entropy contents, similar to the process developed by shannon over 70 years ago [17] . in information theory (it), the shannon entropy uses a scoring system very similar to the id predictors: a fully determined state scores 0 and a fully uncertain state scores 1, respectively. it is clear that the intrinsic disorder contents consist of the information that the protein carries. however, a closer examination must be taken to determine if the intrinsic disorder content could be treated as the disorder entropy, or structural entropy. below we will show that the protein intrinsic disorder (d) cannot be regarded as the structural entropy (h) directly. in addition, a means for converting d to h was proposed that provides a conceptual framework that quantitatively defines the structural information carried by a protein in vitro. hereafter, we will use structural entropy-or simply entropy-in accordance with shannon's insight that the information and entropy serve as measures of each other: information is negative entropy, or negentropy (equation (2) and see below). note that the term "structural" defines a meaning for the information; however, the semantic aspect of information was originally discarded by shannon [17] and later by some molecular biologists [18] . still, other biologists argued that meanings or purposes are crucial to biological information [19, 20] . discussions on syntactics versus semantics in it can be found in other publications [21] , but are beyond the scope of this paper. a fundamental difference between d and h is that the structural entropy, h, is an additive quantity, whereas the id score, d, is not (see the appendix in the supplementary material (sm)). that is, adding up structural entropies of all residues leads to the structural entropy of the entire protein, whereas we cannot infer the id score of a protein by adding up the id scores of all its residues. we suggest that the (intrinsic) structural entropy either of a single residue in the protein or of the entire protein can be directly calculated from a disorder predictor. our suggestion indicates that it is feasible to quantitatively compare structural entropies of two proteins, regardless of their respective disorder percentages, which are rough and qualitative measures of the protein disorder. our suggestion may also help to understand protein structures and functions in the cells quantitatively and provide an easy answer to the paradoxes mentioned above: reduction in information is equivalent to an increase in entropy or complexity. under this framework, the processes in a living cell might be quantitatively viewed as the procedures for maintaining the low entropy levels. shannon's equation [17] implies: where h(x) is defined as the entropy-or uncertainty-of x, which is any source of information with the number of states n and p i is the probability of the i-th state of x. k is an arbitrary scaling factor, which means that the base of the logarithm function could vary depending on the unit of the entropy. for example (set k = 1), a base of two corresponds to the unit of the binary digit (bit), and the natural base (ln) leads to the unit of the natural unit (nat). the maximum value of h(x) h max (x), determines the upper limit or the capacity of x, c(x), which shannon considered the channel capacity for communications [17] . the second half of equation (1), though not explicitly stated by shannon, indicates that, for a given capacity c(x), the entropy h(x) could serve as a measure of the information i(x) that one has received-that is, as a consequence, the information change ∆i was termed negentropy (negative entropy) [22] , which is what maxwell's demon receives to generate order out of disorder [23] . to connect the intrinsic disorder contents obtained by regular disorder predictors, the structural entropy of a protein x with a length of l is defined as: where d i is the intrinsic disorder content of the i-th residue. in the fully disordered state (d i = 1 for all residues), the protein exhibits the maximal entropy, expressed as the structural capacity of: which carries zero structural information i(x). when all residues are fully ordered (d i = 0 for all residues), h(x) = 0 since lim x→0 x log x = 0, and the i(x) reaches the maximum of l. normally, 0 ≤ d i ≤ 1 and 0 ≤ h(x) [or i(x)] ≤ l. the criteria for using the logarithmic measure suggested by shannon [17] then holds. in equation (3), each residue is in a two-state system, with the probabilities x i in one state and (1 -x i ) in the other state, and the total number of states is n = 2 l . more details and potential alternatives to this approach have been summarized in the appendix in supplementary. equation (4) indicates that the structural capacity (c) of a protein is its amino acid (aa) sequence length (l). in addition, it had been reported that c (protein length l was used in the original report, but since it is equivalent to c, only c will be used hereafter to prevent unexpected confusion) has an exponential distribution or a gamma distribution [24] . we used three models, the exponential, gamma, and power law distributions, to fit the structural capacities arranged in hierarchical ranks from the smallest to the largest (table s1 ). the exponential model provides the best overall fit to the structural capacities in all proteomes studied in the present work. the gamma model, in some cases, produces a better fit than the exponential model, but it fails to recapture the small-c regions for the two animal proteomes (human and fruit fly). the power law model fits the small-c but fails at the large-c regions. therefore, the exponential model is the best choice here to describe the protein structural capacity distributions, exemplified in figure 1a ,d. fittings of all proteomes studied here are summarized in table s1 in the sm. an exponential fit of c implies a linear fit of logc (with cs ranked hierarchically), which may therefore be a better measure of the protein-capacity distributions in proteomes [24] . interestingly, with a bin size of ∆log 2 c = 1, gaussian distributions can be observed in logc of all proteomes exemplified in figure 1b ,d. the underlying mechanisms of this gaussian distribution have not been discussed in the literature; however, the distribution density can also be understood as the potential of mean force, or free energy, using the relationship: where r is the gas constant, t is the temperature, and ρ i is the density of proteins at the i-th histogram of protein capacities. a preferential free-energy well (insets in figure 1b ,d) may present in the proteome, confining the protein structural capacity distributions. the locations and depths of this well vary among organisms: for the human proteome, the well depth is 3.16 kcal/mol centered at c = 592, whereas for jcvi-syn3.0 the well depth is 1.70 kcal/mol centered at c = 401. where di is the intrinsic disorder content of the i-th residue. in the fully disordered state (di = 1 for all residues), the protein exhibits the maximal entropy, expressed as the structural capacity of: which carries zero structural information i(x). when all residues are fully ordered (di = 0 for all residues), h(x) = 0 since lim → log = 0, and the i(x) reaches the maximum of l. normally, 0 ≤ di ≤ 1 and 0 ≤ h(x) [or i(x)] ≤ l. the criteria for using the logarithmic measure suggested by shannon [17] then holds. in equation (3), each residue is in a two-state system, with the probabilities xi in one state and (1 -xi) in the other state, and the total number of states is n = 2 l . more details and potential alternatives to this approach have been summarized in the appendix. in the left figures, the brown and green dots are the longest and shortest proteins of the proteomes, respectively. the red-dashed lines are the exponential fittings. the insets in b and d convert the probability distributions of proteins to the potential of mean forces (in kcal/mol) at 300 k using equation (5) . distributions of c using different fitting models are summarized in figure s4 and table s1 of the supplementary material (sm). equation (4) indicates that the structural capacity (c) of a protein is its amino acid (aa) sequence length (l). in addition, it had been reported that c (protein length l was used in the original report, figure 1 . distribution of the structural capacity c (left) and gaussian distributions of log 2 c (right) in proteomes of (a,b). homo sapiens (blue) and (c,d) jcvi-syn3.0 (red). in the left figures, the brown and green dots are the longest and shortest proteins of the proteomes, respectively. the red-dashed lines are the exponential fittings. the insets in b and d convert the probability distributions of proteins to the potential of mean forces (in kcal/mol) at 300 k using equation (5) . distributions of c using different fitting models are summarized in figure s4 and table s1 of the supplementary material (sm). the extremely dynamic environment in the cell renders difficulties in detection or prediction of the changes of the structural entropies and structural information. however, the sum of both quantities equals the structural capacity that is the same as the aa sequence length, and the information gain always equals the entropy loss and vice versa. hence, it is useful to know the relationships between the intrinsic structural entropies and the information that can be derived (equation (3)) from the residual disorder contents, which, in turn, can be predicted based on the protein amino acid sequences. here, we define the entropy-to-information ratio, r, as r = h:i. r ranges from 0 (for a fully ordered protein with h = 0 and i = c) to ∞ (for a fully disordered protein with h = c and i = 0). when r > 1, the protein is entropy rich (h > i), and when r < 1, the protein is information rich (h < i). we noticed no apparent correlation between c and r for all proteomes-e.g., the pearson correlation coefficients between c and r is -0.03 for both h. sapiens and arabidopsis thaliana, and a similar trend can be found in other proteomes studied in the present work. this lack of correlation permits the construction of a protein space with two attributes c and r: the former represents the sum and the latter represents the ratio of the structural entropy (h) and information (i), respectively. this space is termed the protein cr-space hereafter. in the cr-space, c i sets the upper limit and r i gives the intrinsic ratio between the two quantities of the total h i and i i of the i-th protein p i . based on the gaussian distribution in both log 2 c and log 2 r, the protein distributions in the cr-space are represented in figure 2 , for proteins expressed in representative prokaryotes (bacteria and archaea, figure 2a [25] and protein data bank (pdb) [26] (figure 2d ). similar trend can be found in other proteomes studied in the present work. this lack of correlation permits the construction of a protein space with two attributes c and r: the former represents the sum and the latter represents the ratio of the structural entropy (h) and information (i), respectively. this space is termed the protein cr-space hereafter. in the cr-space, ci sets the upper limit and ri gives the intrinsic ratio between the two quantities of the total hi and ii of the i-th protein pi. based on the gaussian distribution in both log2c and log2r, the protein distributions in the cr-space are represented in figure 2 , for proteins expressed in representative prokaryotes (bacteria and archaea, figure 2a ), eukaryotes ( figure 2b ), viruses ( figure 2c ), and datasets collected from the disprot database [25] and protein data bank (pdb) [26] ( figure 2d ). distributions of proteins in the cr-space from proteomes of (a) prokaryotes, (b) eukaryotes (c) viruses, and (d) protein sets from disprot and pdb [25, 26] . each protein i is represented by a black dot. the horizontal axis is c i = h i + i i and the vertical axis is the ratio between h i and i i such that r i = h i /i i . the horizontal dashed line (red) corresponds to r i = 1; proteins above this line are entropy-rich with h i > i i , and those under this line are information-rich with h i < i i . the vertical dashed line serves as a reference, using the median capacity of the human proteome at c i = 417. the total h-to-i ratios (σh:σi) are given in the maps. note that the boundaries of the horizontal (r) and vertical (c) axes vary across different proteomes and/or protein sets. table 1 summarized the protein distributions in the cr-space of all proteomes and datasets, and more discussions will be given as follows. all data are represented using (c, r), where c is the structural capacity (vertical axis in figures 2 and 4) and r is the h-to-i ratio (horizontal axis in figures 2 and 4) , respectively. the data for proteins with extreme c's or r's are given. for those with multiple entries, a range for c or r and the total number of proteins is listed. the infinite (∞) values in r come from the totally disordered proteins with i = 0 and h = c, respectively. n represents the total number of different proteins in the proteome or dataset; for eukaryotic proteomes, only the primary protein at each gene locus is counted. the last column gives the total h divided by total i of all proteins in the proteome or dataset. no average r's are given because there are proteins with r of ∞ in all eukaryotes, datasets, and the archaea lokiarchaeum and nanoarchaeum equitans. details of the names of organisms and datasets can be found in the methods section. figure 2a ,b show the protein distributions of prokaryotic and eukaryotic proteomes, respectively. the total number of proteins vary from the smallest synthesized bacterium jcvi-syn3.0 that has only 438 proteins [27] to the monocot plant oryza sativa (rice) that has 48,782 proteins [28, 29] . a significant difference between prokaryotes and eukaryotes is the overall r (σh:σi) of the proteomes: values for prokaryotes range from 1.29 (nanoarchaeum equitans) to 1.57 (ignicoccus hospitalis) and values for eukaryotes range from 2.10 (amborella trichopoda) to 2.53 (h. sapiens). table 1 also shows the extreme c and r of all proteomes. the most distinct difference between prokaryotes and eukaryotes is the number of totally disordered proteins that have r max = ∞, which is induced by i = 0 (h = c) of these proteins. in the prokaryotes, only one such protein can be found in lokiarchaeum [30] , which exhibits strong potential to represent the ancestral host archaeon that accidentally swallowed the bacterium (rickettsiale being a potential relative [31] ), which fortunately turned into a symbiont and eventually evolved to the mitochondria of eukaryotic cells. all eukaryotic proteomes, however, contain fully disordered proteins, from two in saccharomyces cerevisiae to 37 in h. sapiens. it should be mentioned that there are 25 seleno-proteins in the human proteome, which has an unusual type of residue of selenocysteine (one letter code u, or three letter code sec), though the disorder contents of these proteins cannot be predicted by the current version of pondr predictors (see the sm for a list of the human seleno-proteins). these proteins were not included in the final analysis of the current study. figure 2c shows the protein distributions of two giant dna viruses (giruses) and two rna viruses. the r's of viruses vary significantly from 0.96 (human coronavirus) to 2.09 (pandoravirus). although the rna viruses generally contain less than 10 protein-encoding genes, the protein intrinsic disorder-i.e., structural entropy-may be reflective of immune evasion and virus-host interactions, especially for the ebola virus, in which all proteins are entropy-rich and have r > 1 [32] . on the other hand, the giruses have proteomes similar to those of some cellular organisms in both total protein numbers and their distributions in the cr-space. for example, σh:σi of mimivirus is 1.56, close to that of the prokaryotes, and σh:σi of pandoravirus is 2.09, close to that of some of the eukaryotes, such as the basal angiosperm a. trichopoda. moreover, pandoravirus has a fully disordered protein with a capacity of 29. the present work focuses on the intrinsic disorder and omits the environmental cues, which will certainly have great impact on determinations of the structural information and entropies. in contrast, the proteins collected in the datasets (disprot [25] and pdb [26] ) have been validated (or somewhat biased) experimentally. the protein distributions of the selected datasets are shown in figure 2d . not surprisingly, the proteins from the disprot database exhibit the highest σh:σi of 2.99. however, several proteins (8 out of 803, or 1%) have r < 1. this difference may be caused either by the protein disorder prediction methods used here or by inherent limits of the validation protocol of the database. the eight information-rich proteins from the disprot database, as well as their sequences, have been listed in the sm. apparently, the human titin [33] protein (c = 34,350, r = 2.52) is collected in disprot. however, a closer look finds that that protein in disprot points to the nuclear magnetic resonance (nmr) structure (pdb entry 1bpv) of the type-1 module of the titin protein (c = 112, r = 2.30). nevertheless, the titin protein does have an extremely long idpr based on our calculation ( figure s1 in the sm). we further analyzed the protein sequence sets from the pdb. after removing redundant entries, each sequence in the following datasets represents a unique sequence: nmr set for sequences of structures collected by solution nmr (8,476 entries), x-ray 1.5 set for sequences from x-ray crystallographic structures with resolution higher than 1.5 å (5,842 entries), and x-ray 3.0 set for sequences from x-ray structures with resolution lower than 3.0 å (10,636 entries). the nmr method largely limited the size of proteins with the majority of cs smaller than 200. however, the structures detected by nmr generally have higher flexibility, leading to larger r's (σh:σi = 2.65). the x-ray 1.5 and x-ray 3.0 sets have σh:σi of 1.78 and 1.73, respectively, but no significant difference was observed between these two datasets. it seems that the structural entropies of protein structures deposited in the pdb are method sensitive but are not determined by the resolution of the crystallography. it is worth noting that many structures deposited in the pdb (especially those solved by x-ray crystallography) have missing residues. however, here we are interested in their sequences that contain the missing residues. we also noticed that there are fully disordered (i.e., r = ∞) proteins in all datasets from pdb (table 1) . it may not be surprising to solve the highly disordered proteins using nmr, especially for relatively small proteins. however, even the x-ray 1.5 set contains fully disordered proteins. we listed the fully disordered proteins with c > 20 in both x-ray 1.5 and x-ray 3.0 sets in the sm (tables s2 and s3 ). first, these proteins are relatively short with the longest one (1jcd, x-ray 1.5 set) having 52 residues. it may be possible that the pondr predictor did not perform well for these sequences, particularly the collagen sequences (tables s2 and s3 ). on the other hand, all listed proteins either are subunits of big protein complexes or form oligomers (mostly homo-trimers), suggesting coupled folding and binding may help these idps attain the folded structures [34] . the mutual folding induced by the binding (mfib) database has collected 205 idps from pdb (both x-ray and nmr structures), with their folding induced by binding [35] . none of the proteins (tables s2 and s3 ) have been collected in the mfib database, but several entries listed in table s3 have also been collected in the intrinsically disordered proteins with extensive annotations and literature (ideal) [36] database. our findings may help expand the collection of mfib, ideal, and similar databases. we randomly generated 500 proteins with random capacities in the range [50, 800] . interestingly, this random set has a σh:σi ratio of 1.020 ( figure s5 in the sm), indicating that the total entropy and information are roughly 1:1. this ratio is smaller than those from cellular proteomes shown in figure 2 and is close to the viral proteome of human coronavirus ( figure 2c ), suggesting that when total structural entropy is found to be relatively larger than total structural information in cellular organisms, that relationship is not random. based on the analysis of cellular organisms and viruses, it seems that the σh:σi ratio may set a boundary at approximately 1.6 to 2.0 for separating prokaryotes and eukaryotes. in addition, if there were such a boundary, the two giant dna viruses (giruses) studied here would be located at different branches, with mimivirus close to or within the prokaryotic branch and pandoravirus close to or within the eukaryotic branch, respectively. previously, by using the protein length l (structural capacity c) and the disorder content d, distributions of proteins in the space with attributes l and d have been used to reconstruct a phylogenetic tree that indicates intriguing evolutionary dynamics associated with the variations of l and d in the proteomes [37] . a similar approach is used here to study protein distributions in the hi-space. the cr-space is split into 5 × 4 blocks ( figure 3a and table 2 ). the gene densities [38] at different blocks are then calculated, and the distance between two organisms a and b is defined using the euclidean equation: where d ab is the distance between organisms a and b and x ij (x = a or b) is the gene density at the ij-th (1 ≤ i ≤ 5, 1 ≤ j ≤ 4) block. the calculated distance matrix is converted to a phylogenetic tree, as shown in figure 3b . the rna viruses are excluded in this tree because both have fewer than 10 proteins and therefore are not statistically significant. and d in the proteomes [37] . a similar approach is used here to study protein distributions in the hispace. the cr-space is split into 5 × 4 blocks ( figure 3a and table 2 ). the gene densities [38] at different blocks are then calculated, and the distance between two organisms a and b is defined using the euclidean equation: as expected, the phylogenetic tree clearly separates eukaryotes from prokaryotes. this tree also correctly separates both animals (h. sapiens and drosophila melanogaster) and plants (a. thaliana and o. sativa), whereas the basal angiosperm (a. trichopoda) and moss (physcomitrella patens) are at the basal locations of the eukaryotic branch. mimivirus is located at the prokaryotic branch and pandoravirus is located at the eukaryotic branch, sitting with the moss p. patens. this trend is also expected from the σh:σi ratios. however, similar to our previous study [37] , for all prokaryotes, the phylogeny failed to separate bacteria from archaea without introducing other attributes. further investigations, such as considering the domain components in the proteins [39] , will be required to understand the evolution of the protein entropy. the phylogenetic tree presented in figure 3b (tree of proteomes (top)) is by no means for capturing the evolution of species that are usually derived based on multi-sequence alignments (msas) of genes. however, this top might be visualized as the evolution of structural entropies. it seems that higher complexity favors higher entropic content, especially the entropy-to-information ratios, in the proteins. two categories of proteins-the transcription factors (tfs) and kinases-are further examined from three model organisms, escherichia coli, a. thaliana, and h. sapiens. both categories are abundant in the organism and play regulatory roles in the cells. whilst regulations by the tfs often directly affect the expressions or translations of the genes or transcripts through recognitions and/or interactions with the nucleic acids, the kinases perform post-translational regulations by catalyzing the phosphorylation reactions to the target aas of proteins (often ser, thr, or tyr) through the adenosine triphosphate (atp) cofactors. figure 4 shows that the eukaryotes (the plant a. thaliana and animal h. sapiens) possess higher σh:σi ratios than the prokaryote (e. coli), which is consistent with the trend in figure 2 . moreover, the regulatory mechanisms may influence the h:i ratios (r's) of the proteins such that the recognition and interactions with the dna or rna targets for the tfs may involve larger amounts of information gains or entropy losses compared to the enzymatic catalysis by the kinases via a lock-and-key-like model. therefore, we may propose that more intrinsic structural entropies are required for the regulations by tfs than the those required by the kinases, or possibly other enzymes catalyzing different reactions in the cells. these differences are more significant in the eukaryotes (h. sapiens and a. thaliana) than in the prokaryotes (e. coli). moreover, the a. thaliana tfs have a larger σh:σi ratio compared to those of h. sapiens, indicating that the plant tfs may possess overall higher structural entropies than do the animal tfs. from three model organisms, escherichia coli, a. thaliana, and h. sapiens. both categories are abundant in the organism and play regulatory roles in the cells. whilst regulations by the tfs often directly affect the expressions or translations of the genes or transcripts through recognitions and/or interactions with the nucleic acids, the kinases perform post-translational regulations by catalyzing the phosphorylation reactions to the target aas of proteins (often ser, thr, or tyr) through the adenosine triphosphate (atp) cofactors. figure 2 , each protein is represented by a black dot in the maps, the horizontal axis is for the structural capacity ci, and the vertical axis is for the h:i ratios ri. σh:σi is given for each group of proteins. figure 4 shows that the eukaryotes (the plant a. thaliana and animal h. sapiens) possess higher σh:σi ratios than the prokaryote (e. coli), which is consistent with the trend in figure 2 . moreover, the regulatory mechanisms may influence the h:i ratios (r's) of the proteins such that the recognition and interactions with the dna or rna targets for the tfs may involve larger amounts of information gains or entropy losses compared to the enzymatic catalysis by the kinases via a lock-and-key-like model. therefore, we may propose that more intrinsic structural entropies are required for the regulations by tfs than the those required by the kinases, or possibly other enzymes catalyzing different reactions in the cells. these differences are more significant in the eukaryotes (h. sapiens and a. thaliana) than in the prokaryotes (e. coli). moreover, the a. thaliana tfs have a larger σh:σi ratio compared to those of h. sapiens, indicating that the plant tfs may possess overall higher structural entropies than do the animal tfs. figure 2 , each protein is represented by a black dot in the maps, the horizontal axis is for the structural capacity c i, and the vertical axis is for the h:i ratios r i . σh:σi is given for each group of proteins. many algorithms have been developed to predict the probabilities of being disordered for amino acids in proteins, which are presented as the disorder contents of the residues; however, it is not straightforward to quantify how "disordered" a protein is using these approaches. here, we suggest converting the disorder content to structural entropy, which is continuous and additive and therefore makes it feasible to quantitatively compare two proteins in terms of the degrees of structural entropy and/or information they possess. as erwin schrödinger noted, "life feeds on negative entropy" [40] . the nature of the negative entropy (negentropy) is, actually, the information. our results suggest that proteins and other biomolecules also feed on information. the capacity of every protein is fixed (equation (4)), and, therefore, the higher the information content it receives, the lower the entropy it possesses. a recent paper further indicates that the idps in the cell are either globally or partially structured. [41] in other words, the structural entropy carried by the idps is fully or partially registered by the structural information from the crowded and dynamic intracellular environment of the living cell. it might also be expected that the structural entropy/information proposed here has implications in biological processes where structural information-gain/entropy-loss (or the reverse) occur, such as in gene transcriptions, translations, protein folding pathways, protein-protein or protein-ligand interactions, and enzymatic reactions, to name a few. a recent work [42] reported the general transcriptional coactivator cbp that binds to two different proteins, the negative feedback regulator cited2 (target t1) and the hypoxia-inducible factor hif-1α (target t2). the binding affinities of cbp to both substrates are very close. however, even at a modest concentration, the substrate t1 rapidly substitutes t2 in binding to cbp. in addition, it was shown that t1 exhibits higher disorder contents-and therefore higher structural entropy-than t2. under our approach, the higher specificity of t1 can be understood as the higher information-gain of cbp upon binding to t1 than that of binding to t2. this approach might be extended to dna, rna, and other biomolecules in cases where their entropies and information could be estimated. it should be emphasized here that to quantify the information or entropy, the meaning of the information might be needed. this may especially be the case for the biological information. for instance, the meaning of the information discussed in the present work is the structure of proteins-or, more precisely, the order-to-disorder continuum in the protein world. proteomes of organisms from all three domains of life have been surveyed in the present work. three bacteria include the alphaproteobacterium rickettsiales bacterium ac37b [43] , which may be a close precursor of eukaryotic mitochondria, the cyanobacterium synechococcus elongatus pcc 7942 [44] , which may represent the precursor of plant plastids, and, recently synthesized, the smallest known free-living organism jcvi-syn3.0 [27] . three archaea include the smallest known free-living archaeon i. hospitalis; its parasite, the smallest known archaeon n. equitans [45] ; and lokiarchaeum (sp. gc14_75) [30] . the latter was reported to possess eukaryotic genes, such that its close relative, may have served as the host to accommodate an obligate parasitic bacterium, and the archaeon and bacterium accidently merged to produce the first eukaryotic cell some 2 billion years ago. the eukaryotic organisms include three plant species: the basal angiosperm amborella trichopoda [46] , the monocot plant o. sativa [29] (rice), and the eudicot and model plant a. thaliana [47] ; two animal species: h. sapiens [48, 49] (human) and d. melanogaster [50] (fruit fly); the moss p. patens [51] ; and the yeast s. cerevisiae [52] . several viral proteomes were also studied, including two rna viruses with less than 10 genes from the ebola virus and human coronavirus (which causes the common cold), plus two giant dna viruses (giruses), the mimivirus [53] , and pandoravirus (or p. salinus) [54] . in addition, protein sequences from the database of experimentally confirmed disordered proteins, disprot (v7.0) [25] , and those from the protein data bank [26] (pdb, up to december 16, 2016) are also analyzed. the redundant sequences from the pdb have been removed, and each entry represents a unique protein sequence in the analysis: for each of the protein sets, if there are multiple identical copies of a sequence, such as those from identical chains from a homo-dimer or other oligomers, only one unique sequence is retained for further analysis. the pondr-vsl2 algorithm [55] was applied to predict the id content of all residues in a protein. the obtained disorder content was then converted to probabilities that associated with the structural entropy, using equation 3. a neighbor-joining method, from the t-rex web server [56] , was used to convert the distance matrix to phylogenetic trees. more details on converting the disorder contents to structural entropy, and the fitting of the structural capacities using different models, can be found in the supplementary materials (sm). github repository: https://github.com/haoboguo/protein-structural-entropy. code written in r (random.seq.r) was used to generate 500 random sequences with random capacities in the range [50, 800] . a shell-script was written (entropy.csh) to perform protein structural entropy/information/capacity evaluations, using equation (3) . the pondr-vsl2 predictor was downloaded from http://www.dabi. temple.edu/disprot/predictor.php. java is required to run this code to perform disorder predictions. see descriptions in above repository. supplementary materials: the following are available online at http://www.mdpi.com/1099-4300/21/6/591/s1, figure s1 : profile of structural entropy of the residues in the giant human titin protein (c = 34350). appendix, on the derivation of the equations that convert the disorder contents to the probabilities of states (with figures s2 and s3 ), figure s4 : the exponential, gamma, and power law fittings to the structural capacities of the human and jcvi-syn3.0 proteomes, table s1 : summaries of the exponential, gamma, and power law fittings of the protein structural capacities of the proteomes studied in this paper list of 25 selenoproteins in human (h. sapiens) proteome, whose disorder contents cannot be predicted by the pondr list of eight information-rich proteins from the disprot database (v7.0) and their sequences, table s2 . x-ray structures from pdb with resolutions < 1.5 å, r = ∞ (fully disordered) and c > 20 in sequences, table s3 . x-ray structures from pdb with resolutions > 3.0 å, r = ∞ (fully disordered) and c > 20 in sequences, figure s5 . distribution of proteins in the cr-space from 500 randomly built protein sequences with capacity randomly chosen in the range [50, 800] . σh:σi ratio is 1.020 from this random set. the vertical dashed line represents the median capacity of 417 from h. sapiens proteome, and the horizontal dashed line is at r = 1. the key-lock theory and the induced fit theory principles that govern the folding of protein chains the energy landscapes and motions of proteins understanding protein non-folding what's in a name? why these proteins are intrinsically disordered introducing protein intrinsic disorder a decade and a half of protein intrinsic disorder: biology still waits for physics paradoxes and wonders of intrinsic disorder: complexity of simplicity the protein-folding problem, 50 years on top-idp-scale: a new amino acid scale measuring propensity for intrinsic disorder the conformation properties of proteins in solution predicting intrinsic disorder in proteins: an overview improved disorder prediction by combination of orthogonal approaches quality assessment for the putative intrinsic disorder in proteins quality and bias of protein disorder predictors on the need to develop guidelines for characterizing and reporting intrinsic disorder in proteins a mathematical theory of communication origin of life on earth and shannon's theory of communication the meaning of biological information what is information? phil formal theories of information. from shannon to semantic information theory and general concepts of information the negentropy principle of information maxwell's demon cannot operate: information and entropy. i protein-length distributions for the three domains of life disprot 7.0: a major update of the database of disordered proteins the protein data bank design and synthesis of a minimal bacterial genome international rice genome sequencing project. the map-based sequence of the rice genome a draft sequence of the rice genome complex archaea that bridge the gap between prokaryotes and eukaryotes evolution. pathogen to powerhouse intrinsically disordered side of the zika virus proteome titins-giant proteins in charge of muscle ultrastructure and elasticity mechanism of coupled folding and binding of an intrinsically disordered protein mfib: a repository of protein complexes with mutual folding induced by binding ideal in 2014 illustrates interaction networks composed of intrinsically disordered proteins and their binding partners classification of complete proteomes of different organisms and protein sets based on their protein distributions in terms of some key attributes of proteins only the prime protein is selected at each gene locus but other splicing isoforms are not counted at the locus; hence gene density instead of protein density is used here grammar of protein domain architectures the physical aspect of the living cell cell-wide analysis of protein thermal unfolding reveals determinants of thermostability hypersensitive termination of the hypoxic response by a disordered protein switch an integrated phylogenomic approach toward pinpointing the origin of mitochondria the essential gene set of a photosynthetic organism a genomic analysis of the archaeal system ignicoccus hospitalis-nanoarchaeum equitans the amborella genome and the evolution of flowering plants analysis of the genome sequence of the flowering plant arabidopsis thaliana the sequence of the human genome a high-resolution radiation hybrid map of the human genome draft sequence the genome sequence of drosophila melanogaster the physcomitrella genome reveals evolutionary insights into the conquest of land by plants saccharomyces genome database: the genomics resource of budding yeast the 1.2-megabase genome sequence of mimivirus amoeba viruses with genomes up to 2.5 mb reaching that of parasitic eukaryotes length-dependent prediction of protein intrinsic disorder a web server for inferring, validating and visualizing phylogenetic trees and networks this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: this work is supported by the department of energy (doe), office of science, genomic science program under award number de-sc0008834. oak ridge national laboratory is managed by ut-battelle, llc for the us doe, under contract number de-ac05-00or22725. hbg and hq acknowledge the support of nsf career award #1453078 (transferred to #1720215), bd spoke #1761839, and internal funding of university of tennessee at chattanooga. we thank three anonymous reviewers for providing valuable suggestions to our manuscript. we acknowledge bailey kirby for a thorough proofreading of the manuscript. the authors declare no conflict of interest. key: cord-280360-rh37d5wc authors: gibson, david s.; blelock, sarah; curry, jim; finnegan, sorcha; healy, adrienne; scaife, caitriona; mcallister, catherine; pennington, stephen; dunn, michael; rooney, madeleine title: comparative analysis of synovial fluid and plasma proteomes in juvenile arthritis – proteomic patterns of joint inflammation in early stage disease date: 2009-05-02 journal: j proteomics doi: 10.1016/j.jprot.2009.01.022 sha: doc_id: 280360 cord_uid: rh37d5wc synovial fluid is a potential source of novel biomarkers for many arthritic disorders involving joint inflammation, including juvenile idiopathic arthritis. we first compared the distinctive protein ‘fingerprints’ of local inflammation in synovial fluid with systemic profiles within matched plasma samples. the synovial fluid proteome at the time of joint inflammation was then evaluated across clinical subgroups to identify early disease associated proteins. we measured the synovial fluid and plasma proteomes using the two-dimensional fluorescence difference gel electrophoresis approach. image analysis software was used to highlight the expression levels of joint and subgroup associated proteins across the study cohort (n = 32). a defined subset of 30 proteins had statistically significant differences (p < 0.05) between sample types such that synovial fluid could be differentiated from plasma. furthermore distinctive synovial proteome expression patterns segregate patient subgroups. protein expression patterns localized in the chronically inflamed joint therefore have the potential to identify patients more likely to suffer disease which will spread from a single joint to multiple joints. the proteins identified could act as criteria to prevent disease extension by more aggressive therapeutic intervention directed at an earlier stage than is currently possible. comparative analysis of synovial fluid and plasma proteomes in juvenile arthritis -proteomic patterns of joint inflammation in early stage disease 1 around one in every thousand children in the uk suffers from juvenile idiopathic arthritis (jia) [1] . the worldwide reported incidence varies on a geographical basis, from 0.7 per 1000 in usa to 4.0 per 1000 in australia [2] . jia is a heterogeneous group of inflammatory disorders primarily affecting the musculoskeletal system. the most common symptoms of jia are persistent joint swelling, pain and stiffness. jia normally affects the load bearing joints of the knee and foot, with movement limited by pain [3] . adverse outcomes can present to varying degrees regardless of disease subtype, but persistently inflamed joints are a major risk factor [4] . the main outcomes include limitation of joint function, joint structural damage due to erosions and in severe cases disability or death. jia subtypes vary considerably in the pattern and severity of joint inflammation, disease progression and outcome. typically, the course of all subtypes of jia is one of exacerbations and remissions. however, some may have one or two "flare ups" and never have symptoms again, while others experience repeated "flare ups" or disease that is persistently active into adulthood. jia targets the lining of the joint, the synovial membrane, causing inflammation or synovitis [5] . when synovitis persists joint damage may occur. tissue destruction results from a dysregulation at the molecular level between synthesis and breakdown of the matrix components of joint cartilage [6] . in oligoarticular disease, children are managed with nsaids and intra-articular steroid injections to relieve pain and inflammation, slow down or prevent the joint destruction and restore joint function. to effectively manage and minimize the effects of recurrent synovitis however, an early and accurate diagnosis is essential [7] . successful clinical management can be induced in most (54%) jia patients with polyarticular disease, using combined anti-inflammatory treatment (nsaid and methotrexate mtx) [8] . even so, nearly half the patients will have relapses after treatment is discontinued. the rate of relapses may be influenced by residual synovial inflammation at the time when immunosuppressive treatment is withdrawn. once the disease is in clinical remission, it is important to know when it is possible to withdraw immunosuppressive drugs, without inducing a flare because of their potentially serious side effects [9] . for a clinician there are three main concerns: maintaining disease remission, prevention and pre-empting disease spread and ultimately averting permanent joint damage and disability. if there was a predictive test that would allow disease progression to be forecast and therefore modify treatment accordingly, significant benefit could be provided to patients. although the main clinical manifestation of arthritis is destructive joint inflammation, many other organs may be targeted by the inflammatory process. a holistic view inclusive of local and systemic aspects of the disease is more likely to achieve satisfactory disease resolution. we postulate that biomarkers may exist, which can make a distinction between local and systemic protein expression patterns across the disease subtypes. to date, there are no means of identifying those apparently clinically inactive patients at special risk of relapse. the current definition of disease activity in jia is exclusively based on clinical or routine laboratory measures. consequently, a large proportion of patients may be diagnosed at a relatively advanced stage of the disease, restricting the clinician's ability to modify disease outcome and reducing the efficacy of therapeutic intervention [10] . traditional acute phase response markers such as c-reactive protein (crp) and erythrocyte sedimentation rate (esr) show no significant difference between responders and non-responders to steroids [11] . even in early disease histopathological study of jia synovial membranes indicate that patients diagnosed with jia, exhibit increased vascularity, b and t cell infiltrates and hyperplasia [12] . all these features are evidence of an advanced stage in the inflammatory process. since samples within the current study are from newly diagnosed patients (<6 months), we have the potential opportunity to characterize early stage disease. work on the proteome of arthritic biological fluids and tissue is in its infancy, with little information available on juvenile disease. it was therefore essential that we undertook preliminary work to demonstrate the power and sensitivity of proteomics to study differentially expressed proteins in fluids from jia. our pilot study uncovered fragments of extracellular matrix and t-cell receptor proteins, products of the degradative proteolytic environs of the recurrently inflamed knee [13] . these peptides were observed as indicators of two processes which are likely to be partly interlinked-hypertrophic cartilage breakdown and t-cell infiltration [14] . the predictors and mechanisms of arthritic disease have focused on areas such as t cells, cytokines, fibroblasts, proteases or osteoclasts. what is clear though, is that no single agent or process is wholly responsible, but rather the interaction of these processes resulting in a characteristic protein expression pattern with a particular inflammatory nature. proteomics is therefore an ideal systems biology approach to examine the multifaceted processes at work in arthritis [15] . in addition multiplex diagnostics are more likely to reflect the true complexity of the arthritis disease process. fluorescence difference gel electrophoresis (dige) was applied in this study as the spectrally distinct cy dye fluorophores permit multiplexing of corresponding synovial fluid and plasma from an individual patient. the inclusion of an internal pooled standard (composed of all experiment samples), on each gel allows proteome comparisons to be made between patients and biological variation between body fluids to be reliably quantified. it is reasonable to propose that the most efficient biomarkers for diagnosing or classifying inflammation will be localized within the affected joints. on the other hand it is equally important to assess the systemic molecular foundations of the disease, which may not be clinically apparent. from a practicality viewpoint it is also preferable to be able to quantify biomarkers in plasma as it is more easily accessed with less trauma to children in particular, who may suffer repeated bouts of joint inflammation. the plasma proteome is not only composed of haemostatic proteins confined to the vascular system but also transient secretions of proteins from tissues as a part of normal developmental/aging processes or disease. the proteomic profile of plasma therefore encodes the physiological state of the tissues it is circulated around. however many authors caution that the most meaningful diagnostic plasma proteins are also likely to be found at the lowest abundance, since they are released from tissue into the blood with temporal and spatial restrictions [16] . moving further upstream of plasma to a biofluid proximal to the disease site is therefore likely to be more fruitful in the initial phases of biomarker candidate discovery. this study is concerned with revealing protein markers to allow us to not only track the process of inflammation in detail, but more importantly to dissecting out synovial protein expression patterns which may identify more reliable predictors of jia outcome. we initially performed a study of the proteins expressed within synovial fluid and plasma in early jia in order to discover novel biomarkers which distinguish between local and systemic components of joint inflammation in arthritis. a subsequent comparison of the synovial proteome across patient subgroups provided evidence that such analysis could be of potential use in patient stratification or even prognosis. thirty two patients with juvenile idiopathic arthritis (jia) according to ilar criteria entered this study. at the time of initial sampling there were 18 children with oligo-articular arthritis and 11 with rf-ve poly-articular arthritis. after a year, 8 oligo-articular had extended disease, with more affected joints than originally assessed, and were therefore reclassified as extended oligo-articular patients. this patient cohort was split into two study groups for proteome comparisons of: (a) synovial fluid versus plasma (n = 10), and (b) jia subgroups (n =22). see table 1 for patient demographics of each study group. all patients were examined by a consultant rheumatologist, (mr), who confirmed their diagnosis. for the purposes of this study, only initial synovial fluids from children with a disease duration of less than six months were studied. intra-articular steroid (triamcinolone acetonide) injections of the knee were performed with ultrasound guidance, under general anesthetic, with the dose determined by the size of the joint. (triamcinolone hexacetonide was not available during the time of this study). clinical details recorded included subtype of jia, age, sex, disease duration, local and general disease activity and where appropriate -time to local recurrence, esr and crp. local inflammation was defined as both joint swelling and pain on physical examination. all synovial fluids (sf) were aspirated using an aseptic technique and plasma obtained at the same procedure. the sf and plasma samples were immediately centrifuged at 5000 g for 15 min at 4°c to remove any particulate or cellular material, aliquoted and stored at − 80°c until analyzed. medical ethics committee approval was obtained for this study at green park healthcare trust and patient assent and parent informed consent given. proteins within patient fluids were extracted and stabilized for 1 h at 4°c by addition of m-per ® protein extraction reagent was added to make up the volume to 450 ml prior to ief. each sample was applied to a 24 cm immobiline drystrip ph 4-7 linear immobilized ph gradient (ipg) strip and re-hydrated overnight. previous runs with ph 3-10 carrier ampholytes and ipg strips revealed most of the proteins focused within the ph 4-7 range [15] . the first dimension separation of proteins by isoelectric focusing (ief) was performed for a total of 75,000 vh (2 ma/5 w limit per strip) including a final 8000 v step for 1 h to obtain high quality resolution. values are the mean ± standard deviation or the number of subjects. subgroups descriptors: oligo = oligoarticular 4 joints or less involved at diagnosis; poly = polyarticular, 5 joints or more involved at diagnosis; extended oligo = patients who become polyarticular 6 months after diagnosis. crp = c-reactive protein; esr = erythrocyte sedimentation rate. all study patients were rheumatoid factor negative. after ief, the strips were equilibrated in equilibration for two 15 minute steps with gentle rocking in 6 m urea, 50 mm tris ph8.8, 30% (v/v) glycerol, 2% (w/v) sds. in the first equilibration step 1% (w/v) dithiotreitol was added as a reducing agent and in the second 2.5% (w/v) iodoacetamide included to reduce protein streaking by alkylation. postequilibration, ipg strips were laid into single well 12% page gels (26 × 20 cm; 1 mm thick) and sealed in with 1% agarose (w/ v) in running buffer (25 mm tris, 192 mm glycine, 0.1% (w/v) sds and bromophenol blue). the second dimension separation was run at 0.75 w/gel, with temperature control for 19 h, until the bromophenol blue dye front had reached the end of the gel. two preparative pick gels (to isolate proteins for identification) were loaded with 500 mg of unlabelled protein consisting of an equal amount of each sample. these preparative gels were stained with silver and colloidal coomassie blue for maldi-tof and nesi-ms/ms, respectively. prelabeled proteins were visualized using a typhoon™ 9410 imager (ge healthcare, bucks, uk). the cy5 images were scanned using a 633 nm laser and a 670 nm band pass (bp) filter; cy3 images were scanned using a 532 nm laser and a 580 nm bp filter; cy2 images were scanned using a 488 nm laser and an emission filter of 520 nm. all gels were scanned at 100 nm pixel resolution. the photomultiplier tube was set so pixel intensity remained between 35,000 and 65,000 for any given cy dye. gel analysis was performed with progenesis samespots version 2.0 build 2644.18003 (nonlinear dynamics ltd. newcastle upon tyne, uk.), software comprising gel warping, dige normalization and comparison modules. briefly a single reference gel was assigned to the gel image with the most spots detected and all remaining gel images were aligned to this reference. the same spot outlines were overlayed onto all images ensuring no data was omitted at this early stage. landmark spots were used to confirm spot matching across all gels and manual verification of characteristic three-dimensional 'peaks' was used to screen out any dust artifacts or incorrectly identified spots. the protein spots in each image were automatically linked between the three cy dye images per gel. the cy2 spot intensities (volume) were used to normalize the cy3 and cy5 spot volumes. the normalized volume (nv) for each spot on each gel was calculated by the software from the ratio of the volume of the cy3 (or cy5) labeled sample to that of the cy2 labelled internal standard. the progenesis software performs log transformation of the spot volume ratios to generate normally distributed data. log normalized volume (lnv) was used in quantifying differential expression. spots were matched across replicate sample sets and normalized spot volumes were used to create master gels for experimental comparison. for example in study group a ten gel images were combined to create one sf and one plasma 'master' gel. subsequent qualitative subtraction of plasma from matched synovial fluid 'master' gels revealed a population of proteins uniquely over-expressed in the joint. quantitative differential spot analysis was performed on 'master' gels from both of the experimental groups. pairwise comparisons were made to establish synovial fluid-to-plasma, inter-subgroup, inter-patient and cy3-to-cy5 dye variations. within the samespots review module each comparison was filtered to find the spots (a) with a p value ≤0.05 for the paired t-test, (b) having a greater than two-fold change in expression between the groups, and (c) being correctly matched in at least six of the patients. fold change was calculated as the ratio of the average lnv between study groups. quantitative data sets were exported to excel spread sheet and analysed using epclust (http://www.bioinf.ebc.ee/ep/ep/ epclust/), a generic data clustering, visualization, and analysis tool for genomic or proteomic expression data. hierarchical analysis allows samples with inter-individual protein expression patterns that are highly similar to be reordered in an agglomerative fashion, using the unweighted pair-group average (upgma) clustering procedure. in this method, the distance between two clusters is calculated as the average distance between all pairs of objects in the two different clusters. euclidean distance was the similarity measure used to group or separate the expression data. the grouping of protein expression levels is presented in the form of heatmap accompanied by dendrograms with trees and branches depicting the extent of similarity between the different groups in the samples. protein spots were excised from silver-stained 2d gels and digested by use of the protocol described by [17] . briefly, the gel spots were washed with 50 mmol/l nh 4 hco 3 /acetonitrile (1:1), followed by dehydration with acetonitrile. the proteins were reduced in 10 mmol/l dithiothreitol/50 mmol/l nh 4 hco 3 for 1 h at 56°c and alkylated in 55 mmol/l iodoacetamide/ 50 mmol/l nh 4 hco 3 for 2 h at room temperature. the gel pieces were washed several times in 50 mmol/l nh 4 hco 3 , followed by dehydration with acetonitrile. the proteins were digested overnight with trypsin (promega, modified trypsin) at 37°c, and the resulting peptide mixtures were analyzed by maldi-tof mass fingerprint and/or maldi-tof/tof peptide sequencing at alphalyse a/s (odense, denmark). the instrument used was a maldi-tof/tof autoflex iii from bruker daltonics (bremen, germany). mass spectra were acquired in the 500-3000 m/z scan range. the mass accuracy was calibrated to within 50 ppm using calibration standards (standard peptide calibration mix from bruker daltonics) before each run. the peaklist was generated with bruker daltronics flexanalysis 3.0 software using the sophisticated numerical annotation procedure (snap) algorithm, a signal to noise ratio of >3, a quality factor of 20 and median baseline subtraction. the peptide masses obtained were used to query the non-redundant sequence database (nrdb-ncbi 2007.07.06) for protein identification using the mascot database search program version 2.10.03. a list of excluded contaminant ions and species restriction was not used. the nrdb database contains 4,655,816 entries and is maintained and updated by the european molecular biology laboratory. database search parameters considered: (i) that the trypsin enzyme cleaves on the c-terminal side of kr unless next residue is p, (ii) no fixed modifications, (iii) carbamidomethyl cysteine and oxidation methionine variable modifications, (iv) up to 1 missed cleavage permitted with no fixed modifications (v) peptide tolerance set at 60 ppm for the precursor ions and (vi) 0.7 da mass tolerance for the fragment ions. the acceptance criteria for pmf based identifications was a minimum mascot score of 50, using a 95% confidence interval threshold (p < 0.05). the peptide ions identified in this study by maldi-tof and further cid ms/ms analysis independently matched to single protein entries in the database. protein identifications were independently verified at queen's university belfast by nanoelectrospray-ionization (nesi-ms/ms) from a similar coommassie colloidal blue stained gel. digested peptides were extracted in 5% trifluoroacetic acid, 50% acetonitrile in ultrapure water with sonication for 6 min. after a gel pellet was formed by brief centrifugation, the supernatant was desalted using a c18 ziptip (pierce biotechnology inc., rockford, usa) and dehydrated in sc 210a speed vac vacuum evaporator (savant instruments inc., hicksville, usa). desalted peptide digests were subjected to nesi-ms/ms with a lcq deca ion trap-mass spectrometer (thermofinnigan, san jose, usa), operated with dynamic exclusion via lcq tune software (version 1.1). ms/ms data was recorded over a 400-2000 m/z scan range, with positive polarity and 35 kv collision energy. ms/ms data was collected by xcalibur (version 1.1) and integrated using the interactive chemical information system (icis) peak detection algorithm software provided by finnigan as default. ms/ms ion search was performed by mascot search engine (version 2.10.03) (matrix science inc., boston, usa), using the database ncbinr (2007.05.12) containing 4,914,404 sequences. the following are the standard search parameters: (i) trypsin cleavage, (ii) no fixed modifications, (iii) no constraints on protein mass or mass value, (iv) peptide and ms/ms tolerances set at 1.2 and 0.6 da, respectively and (v) peptide charge was set at 2+ and up to 1 missed cleavage permitted. mascot used the mowse scoring algorithm [18] . a threshold score of >49 indicates that the identity is significant if it would be expected to occur at random with a frequency of less than 5%. the simultaneous analysis of individual paired plasma and synovial fluids from ten patients ( table 1 , study group a) was used to initially isolate joint-specific protein expression profiles, without introducing bias from inter-individual differences. pilot 2-de gels revealed that the majority of proteins from the sampled fluids migrated to within the ph 4-7 range (results not shown). a characteristic pattern of protein migration is apparent in the 2-de gel scans of both synovial fluid and plasma samples (see fig. 1 ). a number of high abundance proteins form fig. 1 -dige reveals~900 spots per gel within the ph 4-7 range for synovial fluid. spot filtering on 'master gels' reveals 143 protein spots which predominate in synovial fluid or plasma. attention was focussed on a series of 26 synovial fluid proteins (circled in red) and 26 plasma proteins (circled in blue) as these spots remained unmatched or were expressed at a two-fold or higher level than could be detected in the corresponding fluid. interestingly, the synovial fluid 'specific' proteins were all under~60 kda, whereas the plasma 'specific' had a wider molecular weight range from~10 to 200 kda. all 30 proteins spots were cut from replicate preparative gels and maldi-tof and nesi-ms/ms was used to ascertain their identity. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) a series of typical charge trains. this spread of proteins is consistent with previous work by other laboratories and our own [13, 19] . interesting distinctions are revealed between the local (synovial fluid, sf) and systemic (plasma) molecular signatures of juvenile arthritis subgroups. on initial visual inspection it is apparent that as one of the predominant sets of high molecular weight proteins present in blood, fibrinogen is principally retained within blood. approximately 900 spots per synovial fluid gel image and 800 spots per plasma gel image were automatically detected and matched across patients by samespots software analysis. the upper and lower limits of detection for the cy dye stained body fluids spanned 5 orders of magnitude. protein spots were uniquely numbered by synchronization to those in a reference gel. intensity differences in spots matched between synovial and plasma master gels were quantified and normalized to the internal standard allowing real comparisons of individual protein spots from both sample types across the patient cohort. a master gel was constructed for each sample type with the gel analysis software and spot filtering performed to highlight spots which predominate in synovial fluid and those which predominate in plasma. spots which predominate in the sf/pl could be presumed to originate in either fluid and were described as sf/pl 'specific'. a number of proteins with consistent synovial and plasma 'specific' expression patterns are highlighted and quantified to demonstrate the ability to reliably differentiate molecular fingerprints of local and systemic disease across patient groups by this gel based approach. a spot filtering rationale to narrow our focus on the most prevalent and most consistent patterns was applied across the ten patients expression data set. images of areas from master gels (see fig. 2 ) demonstrate the inter-sample variation in the expression of 'synovial specific' proteins (spots 23, 29, 36, 25) in synovial fluids and absence in plasma samples. a series of 26 synovial fluid proteins and 26 plasma proteins were selected on the basis that they were expressed at a two-fold or higher levels than could be detected within the corresponding fluid. the inter-individual and inter-sample type variation in these 52 proteins is represented in heatmap form in fig. 4 . even though many of the protein constituents of synovial fluid are derived from plasma, some appear to be enriched in synovial fluid borne out by the higher synovial fluid to plasma ratios (listed in table 2 ). though an approximate 200 fold dynamic range is apparent between the highest and lowest sf/ pl ratios listed, we found the top and bottom detection range was over 5 orders of magnitude. interestingly, the synovial fluid 'specific' proteins were all under~55 kda, whereas the plasma 'specific' had a wider molecular weight range from~10 to 200 kda (as indicated in fig. 1 ). the statistical significance of a protein differing in abundance between the two sample types in one or more individuals is indicated by the p values listed in table 2 . we found that there is a degree of proteome variability across individual patient body fluid samples. to date, no results have been published on the variability of the synovial fluid proteome analysed by the dige method. statistical analysis of inter-patient variance revealed that 18 synovial 'specific' and 12 plasma 'specific' spots within the previously described spot trypsin digests were initially identified using matrix assisted laser desorption ionisation (maldi-tof) and confirmed with nano-electrospray mass spectra (nesi-ms/ms), correlated to compiled peptide data (matrixscience). the number of matching peptide and collision induced dissociation (cid) ions used to verify protein identity are listed alongside sequence coverage and mascot score. mascot used the mowse scoring algorithm [17] . this indicates that the identity is significant if it would be expected to occur at random with a frequency of less than 5%. a positive control spot picked from the 2-de gel molecular weight markers was correctly identified as myoglobin. peptide ion sequence and peak lists can be found in table 3 . subgroup of 52 proteins were consistently expressed in all patients studied. representative images of areas from individual gels (see fig. 3 ) demonstrate the inter-individual variation in the expression of a representative 'synovial specific' protein (spot 49) in synovial fluids and its absence in corresponding plasma samples. we calculated the expression variance of a selected group of 30 proteins (standard deviation / mean, expressed in percent) and observed that standard deviations can be high across the synovial samples in particular (e.g. proteins 11, 14, 53). a disparity is evident between synovial fluid and plasma in the degree of variation for given protein (e.g. proteins 56, 57). generally speaking, for a given protein, detected levels in synovial fluid vary on a wider scale (~30-70% on average) than those in plasma (~30-50% on average). exceptions to this rule are evident, where the standard deviation for certain proteins (6,16, 37 and 49) across the 10 patients is closely matched for both sample types. cluster analysis and protein identification of a defined synovial fluid proteome (study group a) the normalised expression data from the pool of 52 proteins, previously selected due their two fold abundance in one fluid fig. 3 -pairwise comparisons were made to establish patient-to-patient, synovial fluid-to-plasma and cy3-to-cy5 dye variations. statistical analysis of inter-patient variance revealed that 19 synovial 'specific' and 12 plasma 'specific' spots within this subgroup of proteins were consistently expressed in all patients studied (p < 0.05). the expression levels of properdin (spot 49) are visible in a section of gel images from individual patient (1-10) synovial fluid (a) and plasma (b) samples. the log normalized peak volume of properdin is plotted for each patient sample shown (c), such that the first ten points represent synovial fluid levels and the latter ten points represent plasma levels. type, were subjected to hierarchical cluster analysis to visualize their expression characteristics across each patient fluid (see fig. 4 ). the clustering algorithm applied distinguishes plasma and synovial fluid, forming two clear blocks of plasma and synovial fluid abundant proteins in the top and bottom halves, respectively. groups of co-regulated proteins can be rapidly identified by their absence or presence pattern as demonstrated by clusters labeled c1 and c2. certain groups of patients seem to form smaller clusters, whereby a fluid 'specific' expression pattern is apparent, synovial fluid clusters are apparent in regions labeled c3 and c4. the agglomerative nature of clustering reorders the data such that the degree of similarity between patients is strongest for proteins listed at the top and weakest at the bottom. this is represented by the constructed dendrogram, whereby the shortest arms signify the expression levels are closest. it is interesting therefore to note that the selected plasma proteome produces the most consistent expression patterns. 30 proteins selected on their ability to distinguish one fluid from another, were cut from replicate silver stained preparative gels and mass spectrometry was used to ascertain their identity. protein name, mass spectrometry data, fold differences between fluids and variation across patients are compiled for these proteins in table 2 . detailed information on each identification including peptide ion sequence identified is included in table 3 . albumin isoforms were common to both fluids, but even so, some unique expression traits were evident in this class of ubiquitous blood proteins. certain isoforms were detected exclusively in plasma (gi23307793 and gi28592) or synovial fluid (gi119626069). other albumin species detected in both plasma and synovial fluid could be distinguished by their molecular weights (ascertained from 2-de gel images), such that isoforms (gi62113341, gi11493459, gi27692693) of the same albumin species above 55 kda were predominant in plasma and those below 55 kda were more abundant in synovial fluid. for example the albumin gi11493459 (pro2619) was identified from the three separate gel cores of spots 16, 23 and 29, positioned at approximate molecular weights of 60, 48 and 47 kda respectively ( fig. 1 . the two lower molecular weight forms are more abundant in synovial fluid, with 4.7 and 3.7 fold more, respectively. conversely, transthyretin and transferrin chain fragments were identified at 29.4 and 2.2 fold higher levels in plasma, respectively. several apolipoprotein species were amongst those proteins commonly over expressed in synovial fluid. a disproportionate synovial abundance of chain c from apoliprotein a-1 compared to chain a from the same molecule is evident from the sf/pl ratios. apolipoproteins a-ii and c-iii precursor were also detected at significantly higher levels in synovial fluid. two isoforms of alpha-1 antitrypsin (gi28637 and gi1942629) were detected with molecular weight estimates ranging from 45-54 kda (spots 42, 73 and 99) according to gel markers. furthermore, inter alpha trypsin inhibitor and properdin were identified among those proteins enriched within synovial fluid. a number of proteins remained unidentified, probably attributable to a low protein yield from gel cores. a 'master' gel was constructed for each patient subgroup with the image analysis software and spot filtering performed to highlight spots which predominate in a particular subgroup. attention was focussed on a series of 40 synovial fluid proteins (circled and labelled in fig. 5a ) as these spots were expressed at a two-fold or higher level in pairwise comparisons of the other patient subgroups with statistical significance by anova (p<0.05). the group of selected synovial fluid proteins fell within~10 to 100 kda molecular weight range. certain proteins predominate in two patient subgroups e.g. proteins 37 and 892 abundant in both the polyarticular and extended oligoarticular patient subgroups, whereas others apparently predominate in a single subgroup e.g. proteins 5 and 907 in polyarticular or protein 865 in oligoarticular patients (highlighted in fig. 5b ). multivariate analysis was subsequently used to visualize inter-patient and inter-subgroup expression patterns of the 40 selected protein spots. the highlighted proteome was further explored by hierarchical cluster analysis of the 40 selected proteins and visualization in heat map form (fig. 6) . pearson ranked correlation revealed three distinctive clusters. cluster 1 contains proteins which are consistently over expressed in both extended oligoarticular and polyarticular patients, whereas proteins in cluster 2 predominate only in the polyarticular subgroup. intriguingly, proteins in cluster 3 are consistently overexpressed in the oligoarticular and polyarticular subgroups relative to those patients with disease extension. it seems conceivable that used in combination these three distinct clusters within the synovial fluid proteome could be used to differentiate disease subgroups. the unpaired t-test revealed proteins with significant fluctuations between combinations of any two patient subgroups. the p values are summarized in table 4 . having established the significance of protein expression patterns it was pertinent to assign identities using mass spectrometry. 3.6. the mass spectrometry derived identification and inter-subgroup variation of the 40 selected proteins are listed in table 4 . detailed information on each identification including peptide ion sequence identified is included in table 5 . in order to highlight proteins which may discriminate disease extension, emphasis was placed on statistically significant correlations to extended oligoarticular patients. as the most ubiquitous protein, six albumin isoforms (a, g, h, k, p and t) were identified as 11 distinct protein spots from picking gels and were distributed throughout the 3 identified protein clusters, predominating in cluster 1. within cluster 3, albumin isoforms t and k were over expressed 4.8 (p = 0.049), 2.9 (p =0.018) and 3.0 (p = 0.038) fold in extended oligoarticular patients when compared to the oligoarticular subgroup, whereas albumin isoforms a and g are conversely underexpressed 2.7 (p = 0.021) and 6.9 (p =0.021) fold, respectively, in patients with disease extension. intriguingly, albumin complexed with the non-steroidal anti-inflammatory drug (nsaid) s-naproxen (spot numbers 41 and 904) was detected at significantly raised levels in extended oligoarticular contrasted with oligoarticular patients with similar 2.4 (p = 0.006) and 2.3 fold (p = 0.045) differences recorded for isoforms isolated from two distinct protein spots. apolipoproteins ai and aii were significantly overexpressed 2.9 (p = 0.008) and 2.6 (p = 0.046) fold, respectively, in polyarticular patients when contrasted against oligoarticular and extended oligoarticular subgroups. in clusters 1 and 3, transferrin was identified as two independent protein spots (42,33) representing non-glycosylated and n-lobe forms of chain a. levels of the nonglycosylated transferrin were raised 2.0 (p = 0.014) fold in patients who showed disease extension, whereas the transferrin n-lobe displayed the inverse with 2.6 (p = 0.045) fold less, relative to oligoarticular patients. complement component c3c and hemopexin display analogous overexpression in polyarticular patients when balanced alongside oligoarticular and extended oligoarticular subgroups. interestingly, from the point of view of jia pathology vitamin d binding protein was detected in polyarticular patients at significantly overexpressed levels, 2.2 (p = 0.019) fold, when compared to oligoarticular patients in cluster 1. in addition, several spots (18, 542 and 699) were identified as immunoglobulin (ig) fragments within cluster 3 at significantly reduced levels in the patients with disease extension to oligoarticular patients. ig heavy chain variable region, igk1 and ig light chains were under expressed 5.4 (p= 0.030), 3.0 (p=0.023) and 2.9 (p=0.020) fold in the extended oligoarticular subgroup relative to the oligoarticular subgroup. ig heavy chain variable region was concomitantly overexpressed 3.3 (p=0.032) fold in the polyarticular patients when compared to the extended subgroup. a number of protein spots remained unidentified, attributable to low protein yield from the gel core extraction process. proteins and peptides actively or passively secreted and shed from the synovial membrane accumulate in synovial fluid. potential biomarkers could therefore be enriched within this fluid relative to plasma. in the first study (group a) we have shown that local and systemic disease can be differentiated by the clustering pattern of 52 proteins with consistent synovial or plasma 'specific' expression patterns. this discriminating group of proteins is expressed with at least twofold differences between the two sample types, but how relevant are these findings to the pathology of inflammatory arthritis in children. fragments or precursors of anti-inflammatory proteins, endogenous protease inhibitors, albumin and complement factors have been identified among these. a number of inhibitory acute phase proteins were associated with significant differences in local and systemic expression patterns. the levels of transthyretin, transferrin were raised in plasma whereas apolipoproteins (a-i, a-ii and c-iii) were conversely higher in synovial fluid. in rheumatoid arthritis patients, apolipoprotein a-i plasma levels are lower than normal controls [20, 21] , whereas in agreement with our findings the levels are increased in synovial fluid [22, 23] . apo a-i immunohistological staining is confined to perivascular areas within the synovium of ra patients [24] . it has been suggested that apo a-i could inhibit the production of proinflammatory cytokine production [25] and may limit disease recurrence by inhibition of interactions between t lymphocytes and monocytes [26] . investigation of the plasma from patients in the progressive stages of severe acute respiratory syndrome (sars) revealed that apolipoprotein a-i, transthyretin and transferring were at significantly lower levels than in normal controls and convalescent patients [27] . these proteins belong to the negative acute phase proteins (apps) and previous studies suggest that plasma concentrations decrease in response to inflammation because of increased rates of transcapillary escape degradation [28] . these proteins have also been identified at increased levels the in cerebrospinal fluid of alzheimer disease patients where they are thought to function in plaque clearance [29] . it has been suggested that transient infiltration of negative app plasma proteins may partially explain the relapse-remission cycles characteristic of jia and other forms of inflammatory arthritis [24] . raised levels of properdin, a component of the alternative complement activation pathway, were detected in synovial fluid. properdin activates compliment in the absence of immune complexes by stabilizing the c3 or c5 convertase complexes [30] . it is associated with the engulfing of foreign particles and invading cells by phagocytes and with tissue inflammation. as macrophages and t cells are able to secrete properdin into the synovial fluid, therefore these infiltrated cells are a likely source of the raised levels observed [31, 32] . the increase in blood proteins may be a function of their metabolism within the joint, such that certain isoforms or fragments of a given protein may be detected at higher than anticipated levels. a concentration gradient may also contribute to raised amounts of select proteins due to the fluid pressure differential that exists between blood supplied to the joint and the joint fluid itself [33] . a certain amount of 'leakiness' between the two fluids could be anticipated due to a breakdown in the integrity of the synovial capillary network, commonly observed in a range of rheumatic disorders as joint inflammation progresses [34] . the signal from a particular biomarker is likely to diminish with distance from the disease and in particular with dilution into the plasma. the interpatient differences observed may be indicative of the dilution of a protein into the larger volume of plasma thus generating a narrower concentration interval and as previously eluded to, the heterogenous degrees of joint inflammation which may result in a differential rates of protein turnover across the study patients. studies of capillary ultrafiltration and trans-synovial flow rates imply that macromolecular selectivity is a feature of the fenestrated membrane that separates synovial fluid and plasma. synovial fluid formation is driven by a net imbalance in the pressures between capillary plasma and intra-articular fluid. continuous drainage from and replenishment into the intra-articular space ensures the synovial fluid proteome remains in flux [33] . although capillary ultrafiltrate is the base solution for synovial fluid, secretions are made by the local synovial lining cells. size selectivity has been proposed on the theory that the hyaluronan lining the synovial space is able to filter proteins such as albumin by steric exclusion [35] . the rate of trans-synovial fluid loss and molecular sieving effects are determined by the chain length of hyaluronan concentrated at the synovial membrane surface [36] . our observation of albumin isoforms at variable stages of degradation across synovial fluid and plasma may reflect molecular sieving between the synovial endothelium and the joint fluid or a differential rate of degradation between the two fluids. our study also showed a number of variable mwt / pi isoforms of the same albumin species with variable ratios between synovial fluid and plasma. this also suggests that same protein was subjected to variable post translational modifications, which may direct proportions of a single gene product to select locations relevant to disease pathology [37] . inter alpha trypsin inhibitor and alpha 1 anti-trypsin were identified at higher synovial levels, compared to plasma which may represent the inherent anti-proteolytic character of normal synovial fluid. it is possible that these proteins are part of a homeostatic negative feedback system which protects the joint during inflammation.. inter alpha trypsin inhibitor and alpha 1 anti-trypsin were also detected alongside apolipoprotein, transferrin and transthyretin species in a recent 1de-ms study of normal and osteoarthritic synovial fluids [38] . support for our findings has been provided in a recent study of 500 peptide fragments from oa synovial fluid, which identified similar acute phase components apolipoprotein f precursor, inter-α inhibitor h4 and serum amyloid a [39] . untargeted analysis of peptides was performed on synovial fluid from osteoarthritis patients in the liquid phase by reversed-phase nanolc-ms. and it was suggested that peptide degradation products could reveal clinically relevant information on the activity of peptidases and proteases within the inflamed joint. in the second study (group b) jia patient subgroups, classified according to ilar criteria, can be segregated in early disease based on the clustering patterns of 40 synovial fluid proteins. by extension of this finding, it may be possible to differentiate jia patients who suffer disease extension within the first year of diagnosis from the other disease subgroups tested. the discriminatory group of proteins is expressed with at least twofold differences between jia patient subgroups, but how relevant are these findings to the pathology of inflammatory arthritis in children. several acute-phase proteins, albumins, glycoproteins and immunoglobulins comprise this discriminatory subproteome. substantial evidence suggests these molecules may govern a wide range of inflammatory pathways with relevance to the pathology of jia. a number of inhibitory acute phase proteins were associated with significant differences in jia subgroup expression patterns. when compared to the synovial fluid proteome levels of the oligoarticular patient subgroup, apolipoprotein a-i was reduced in patients with extended oligoarticular disease, whereas apolipoprotein a-ii was higher in the latter. both apolipoprotein species were significantly higher in the polyarticular patients. in rheumatoid arthritis patients, apolipoprotein a-i levels are increased in synovial fluid [22, 23] . it has been suggested that apo a-i could inhibit the production of proinflammatory cytokine production [25] and may limit disease recurrence by inhibition of interactions between t lymphocytes and monocytes [26] . in line with apolipoprotein a-i levels, extended oligoarticular patients display a general trend towards reduced transferrin, haptoglobin and complement c3c relative to the other two patient subgroups. in support of the assertion that these relatively abundant host-response proteins could be involved in the spread of inflammation to previously unaffected joints, raised sera levels of these proteins have been recorded in breast cancer patients with metastatic relapse [40] . furthermore, leucine rich alpha-2-glycoprotein identified in this study has been observed at elevated levels along with other acute phase protein in patients with severe acute respiratory syndrome [41] . vitamin d binding proteins (vdbp) have been identified in synovial fluid at reduced levels to those found in plasma, reflecting its limited dialysis into the joint space [42] . our synovial fluid analysis revealed significantly raised levels of vdbp in extended oligoarticular and polyarticular patient subgroups. the samples used in this study were only taken at the time of initial knee inflammation, therefore are most relevant to the disease pathology in early disease. the data provided indicates that synovial fluid proteome profiles could be used to classify patients based on existing clinical definitions. by virtue of the definition of the extended oligoarticular patient subgroup (become polyarticular after 6 months of initial diagnosis) we consider that both prediction of disease evolution and subgroup discrimination are possible by the proposed protein biomarker panel. the ability to predict disease course would be a powerful tool in the limitation of adverse outcome such as disease extension, as it may flag such patients for more effective/aggressive treatment strategies at an earlier stage in the disease process than is currently possible. the authors consider the collection of samples at the earliest stage in the disease course an important feature of the current study and vital in the discovery of biomarkers which not only 'describe' the initial disease processes, but may also act as sentinels to predict subsequent disease outcome. by cataloguing the protein profiles and identities which best describe the joint status as disease progresses, it may also be possible to monitor therapeutic response over time. furthermore joint proteome records could help predict disease spread and joint damage. considering the heterogeneity of patient outcome in jia, therapeutic suppression of synovial joint inflammation, while neglecting the systemic components of chronic disease, may not represent the best possible way to manage nonresponsive disease. longer lasting remission could be possible by taking systemic sentinels into account. the dige technique coupled with maldi-tof mass spectrometry limits biomarker detection to within an approximate dynamic range of 5 orders of magnitude. alternate strategies will be used in future investigations to enrich less abundant proteins in body fluid samples (affinity depletion). synovial fluid as a partial eluent of plasma is likely to mimic its protein concentration range over 10 orders of magnitude [15] . to reduce the dynamic range of the synovial fluid and enrich for lower abundance proteins, serum albumin and immunoglobulins could be removed by affinity depletion columns in future investigations [43] . intact and partially degraded proteins or peptide fragments are more likely to leach into the circulating plasma [44] . low molecular weight components could be enriched by size exclusion or reverse phase chromatography [45] . protein expression patterns localized in the chronically inflamed joint may have the potential to identify patients more likely to suffer disease which spread to multiple joints. the differences noted are currently being validated with a larger independent patient cohort, where alternate methods of expression and functional analysis will be used to further corroborate the proteomic findings. the proteins identified could act as criteria to prevent disease extension to allow earlier, more aggressive therapeutic intervention. pediatric rheumatology in the united kingdom: data from the british pediatric rheumatology group national diagnostic register worldwide prevalence of juvenile arthritis: why does it vary so much? measuring disability in early juvenile rheumatoid arthritis: evaluation of a norwegian version of the childhood health assessment questionnaire prognostic factors in juvenile rheumatoid arthritis: a case-control study revealing early predictors and outcome after 14.9 years patterns of joint involvement at onset differentiate oligoarticular juvenile psoriatic arthritis from pauciarticular juvenile rheumatoid arthritis regulation of human neutrophil-mediated cartilage proteoglycan degradation by phosphatidylinositol-3-kinase cd4+cd25bright regulatory t cells actively regulate inflammation in the joints of patients with the remitting form of juvenile idiopathic arthritis induction of invasive and degradative phenotype in normal synovial fibroblasts exposed to synovial fluid from patients with juvenile rheumatoid arthritis: role of mononuclear cell population methotrexate treatment in juvenile idiopathic arthritis: when is the right time to stop the knee joint in early juvenile idiopathic arthritis intra-articular glucocorticoids in early juvenile chronic arthritis juvenile idiopathic arthritis: joint destruction vs joint repair. clinical and histological findings in early untreated disease proteomic analysis of recurrent joint inflammation in juvenile idiopathic arthritis expression of myeloid-related proteins 8 and 14 in systemic-onset juvenile rheumatoid arthritis the human synovial fluid proteome: a key factor in the pathology of joint disease protein biomarker discovery and validation: the long and uncertain path to clinical utility in-gel digestion for mass spectrometric characterization of proteins and proteomes rapid identification of proteins by peptide-mass fingerprinting mass spectrometric proteome analyses of synovial fluids and plasmas from patients suffering from rheumatoid arthritis and comparison to reactive arthritis or osteoarthritis lipid profiles in untreated patients with rheumatoid arthritis analysis of changes in acute-phase plasma proteins in an acute inflammatory response and in rheumatoid arthritis using two-dimensional gel electrophoresis apolipoproteins a-i and b and cholesterol in synovial fluid of patients with rheumatoid arthritis plasma apolipoprotein(a) co-deposits with fibrin in inflammatory arthritic joints apolipoprotein a-i infiltration in rheumatoid arthritis synovial tissue: a control mechanism of cytokine production? apolipoprotein a-i inhibits the production of interleukin-1beta and tumor necrosis factor-alpha by blocking contact-mediated activation of monocytes by t lymphocytes the role of human t-lymphocyte-monocyte contact in inflammation and tissue destruction inflammation inhibitors were remarkably up-regulated in plasma of severe acute respiratory syndrome patients at progressive phase mass spectrometric protein structure characterization reveals cause of migration differences of haptoglobin alpha chains in two-dimensional gel electrophoresis comparative proteomic analysis of intra-and interindividual variation in human cerebrospinal fluid molecular biology and chemistry of the alternative pathway of complement expression of properdin in human monocytes properdin, a positive regulator of complement activation, is released from secondary granules of stimulated peripheral blood neutrophils a method for estimating macromolecular reflection by human synovium, using measurements of intra-articular half lives quantitative assessment of the rheumatoid synovial microvascular bed by gadolinium-dtpa enhanced magnetic resonance imaging fluid movement across synovium in healthy joints: role of synovial fluid macromolecules size selectivity of hyaluronan molecular sieving by extracellular matrix in rabbit synovial joints physiological and pathological changes in the redox state of human serum albumin critically influence its binding properties high abundance synovial fluid proteome: distinct profiles in health and osteoarthritis profiling of endogenous peptides in human synovial fluid by nanolc-ms: method validation and peptide identification postoperative serum proteomic profiles may predict metastatic relapse in high-risk primary breast cancer patients receiving adjuvant chemotherapy plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry vitamin d metabolites in synovial fluid use of mass spectrometry to identify protein biomarkers of disease severity in the synovial fluid and serum of patients with rheumatoid arthritis recent advances in blood-related proteomics rheumatoid arthritis, a complex multifactorial disease: on the way toward individualized medicine we thank janne skaarup crawford (alphalyse a/s) for careful compilation of the supplemental ms data. this work has been supported by the research and development office northern ireland grant rrg 8.42 (to d.g. and m.r.) key: cord-303494-tofch4j7 authors: bai, juan; chen, xinhui; jiang, kangfu; zeshan, basit; jiang, ping title: identification of vp1 peptides diagnostic of encephalomyocarditis virus from swine date: 2014-12-30 journal: virol j doi: 10.1186/s12985-014-0226-8 sha: doc_id: 303494 cord_uid: tofch4j7 background: encephalomyocarditis virus (emcv) can cause myocarditis, respiratory failure, reproductive failure, and sudden death in pre-weaned piglets, which has been isolated in china. emcv vp1 protein was one of the most important structural proteins and played an important role in the protective immunity. in this study, 10 monoclonal antibodies (mcabs) against emcv vp1 were screened and identified. results: epitope mapping results indicated that mcabs (6e11, 7a7, 7c9) specifically recognized the linear epitopes v(2)enaek(7), mcabs (1d1, 2a2, 5a1, 5a11, 5g1) recognized the epitope f(19)vaqpvy(25), and mcabs 1g8 and 3a9 recognized p(42)igaftvk(49). protein sequence alignment of vp1 with 16 emcv isolates indicated that the epitope f(19)vaqpvy(25) was conserved in all the reference strains. the epitopes p(42)igaftvk(49) and v(2)enaek(7) only had 1 or 2 variable amino acid among the reference strains. the 3d model analysis results showed that these epitopes presented as spheres were shown within the context of the complete particle. conclusions: in this study, ten mcabs against emcv vp1 were developed and three b-cells epitopes (2-7aa, 19-25aa and 42-49aa) were defined in vp1. all the results herein will promote the future investigations into the function of vp1 of emcv and development of diagnostic methods of emcv. encephalomyocarditis virus (emcv) is characterized by not only myocarditis and encephalitis, but also neurological diseases, reproductive disorders and diabetes in many mammalian species [1] [2] [3] [4] [5] [6] [7] [8] . rodents are considered to be natural hosts of emcv and are thought to be the primary reservoir and disseminators of the virus. out of all domestic animals, pigs are the most susceptible to emcv infection, which can cause severe economic losses on pig production due to high mortality in piglets as a result of respiratory failure [2, 9, 10] and in sows as a result of myocarditis and reproductive failure [11] [12] [13] . emcv is a member of the cardiovirus genus of picornaviridae [14] and has a single-stranded positive-sense rna of approximately 7.8 kb [15] . the orf encodes for a polyprotein that comprises both non-structural and structural elements divided into three primary precursor molecules, namely p1, p2 and p3, encoding for 11 distinct proteins. the structural proteins vp4, vp2, vp3 and vp1 make up the viral capsid and are encoded in the p1 region towards the 5′-end of the genome. the viral capsid proteins, in their capacity to interact with cellular receptors, are crucial for this entry step and may be considered to be factors that can modulate emcv virulence [1] . the three major capsid proteins, vp1, vp2 and vp3 that constitute the external virion shell of picornaviruses, are considered to play a pivotal role in viral infection and host recognition [16] . among them, vp1 is one of the most antigenic and can stimulate the organism to produce neutralization antibody [17, 18] . the detailed analysis of epitopes is important for the understanding of immunological events and for the development of epitope-based marker vaccines and diagnostic tools for various diseases [19] [20] [21] . in this paper, vp1 protein of emcv nj08 strain was expressed by the escherichia coli system and ten monoclonal antibodies (mcabs) against the recombinant emcv vp1 were screened and identified. three linear epitopes (2-7aa, 19 -25aa and 42-49aa) were identified in vp1 protein of emcv. after screening of the fusion cells by indirect elisa, the positive hybridoma cells secreting the antibodies against vp1 protein were selected and sub-cloned thrice by limiting dilution, and ten mcabs were generated and named as 1d1, 1 f3, 1g8, 1d1, 2a2, 5a1, 5a11, 5g1, 6e11, 7a7 and 7c9. the results of western blot showed that these mcabs were all directed against the purified emcv and rvp1 protein expressed in e. coli ( figure 1 ). meanwhile, ifa results also showed that these mcabs could react with the emcv in bhk21 cells (figure 1 ). the titres of antibodies in the cells cultures were 1:1600-3200. twenty-six overlapping vp1 protein gene fragments were prepared by pcr and cloned into a gst fusion protein expression vector. after validation by restriction analysis and nucleotide sequencing, recombinant proteins encoded by each of these constructs were expressed in e.coli. the resulting recombinant proteins were identified using western blot with gst-tagged monoclonal antibody. the results showed that all the proteins were reactive with gst-tagged mcab ( figure 2 ). in order to map the minimal sequences of the epitopes recognized by mcabs, the series of truncated recombinant proteins were used in western blot was to identify the reactivity of each of 10 anti-vp1 mcabs. the results showed that mcabs (6e11, 7a7 and 7c9) could reacted with fragments (f11, f9 and f3) containing the six amino acids v(2)enaek(7), but not reacted with the fragments with the deletion of any one of the six amino acids (f10, f7 and f8). it indicated that amino acids v(2) enaek(7) in vp1 were essential for this epitope ( figure 3a ). in the same way, mcabs (1d1, 2a2, 5a1, 5a11 and 5g1) reacted against the epitope comprising f (19)vaqpvy (25) , because they could react with the fragments f15, f13, f18 and f19, but not f12, f16 and f17 ( figure 3b ). mcabs (1g8 and 3a9) could reacted with fragments (f25, f26, f21 and f22) containing the eight amino acids p(42)igaftvk(49), but not reacted with the fragments with the deletion of any one of the eight amino acids (f24, f23 and f20) ( figure 3c ). meanwhile, those purified truncated fragment proteins were used as the antigens in elisa to detect the levels of those mcabs. the results showed that the od 450 values of mcabs (6e11, 7a7 and 7c9) in f1, f3, f9 and f11 groups were significantly higher than those in f6, f7, f8 and f10 (p < 0.01) ( figure 4a ). the od 450 values of mcabs (1d1, 2a2, 5a1, 5a11 and 5g1) in f1, f13, f14, f15, f18 and f19 groups were markedly higher than those in f12, f16 and f17 groups (p < 0.05) ( figure 4b ). the levels of mcabs (1g8 and 3a9) in the groups of f21, f22, f25 and f26 were notablely higher than those in f20, f23 and f24 (p < 0.01) ( figure 4c ). the synthetic peptides (p2-7aa, p19-25aa and p42-49aa), purified rvp1 and gst-1-72aa (f3) proteins were used as elisa antigens. the results showed that od 450 values of 6e11, 7a7 and 7c9 reacted with p2-7aa was highest in those mcabs. and the levels of mcabs (1d1, 2a2, 5a1, 5a11 and 5g1) reacted with p19-25aa were obviously higher than those of other mcabs (p < 0.05). mcabs 1g8 and 3a9 reacted with p42-49aa were significantly higher than other mcabs (p < 0.05). it indicated that the synthetic peptides p2-7aa, p19-25aa and p42-49aa have immune reactivity with the pepide specific mcabs, respectively ( figure 5 ). however, the od 450 values of anti-emcv mice serum were relatively lower than those of mcabs reacted with p2-7aa, p19-25aa and p42-49aa. it suggested that these synthetic peptides reacted weakly with emcv-specific serum by peptide-based elisa ( figure 5 ). alignment of the amino acid sequences of the different emcv isolates revealed that the minimal epitope f(19) vaqpvy(25) was highly conserved among all the reference emcv strains, the epitope v(2)enaek(7) was relatively conserved among all emcv strains, except for a k7 → r7 change in gxlc, d variant, emc-b and emc-d strains. in addition, the epitope p(42)igaftvk (49) was conserved among the strains isolated from china. whereas there were variable amino acids in different site of this region among the reference strains isolated from other countries ( figure 6 ). there existed a very close structural analog to nj08 in 2 structures published in 1990 and 1994 with pdb accession numbers 2mev and 1mec respectively both for the mengo virus. the vp1 protein of nj08 had very few amino acid changes and no deletions or additions in the sequence compared to the mengo virus vp1 sequence as shown in figure 7 . therefore any of the 2 structures could be used a 3d model to map the epitopes within the context of the complete viral particle. as show in figure 8 , the 3 epitopes presented as spheres were shown within the context of the complete particle with colors of red for vp1, green for vp3, yellow for vp2 and blue for vp4. the epitopes v(2)enaek(7) and f (19) vaqpvy (25) shown as cyan and magenta spheres were partially buried in the viron. p(42)igaftvk(49) shown as orange spheres was located circa the 5-fold icosahedral axis of symmetry and was on the surface. b cell epitopes are linear or conformational resulting from unique protein folding. they could be defined as regions on the surface of the native antigen that could bind to b-cell receptors or specific antibodies [21, [23] [24] [25] . in this study, ten monoclonal antibodies (mcabs) against the vp1 of emcv were prepared by inoculated with the purified killed emcv, and then three minimal epitopes [v (2)enaek(7), f(19)vaqpvy (25) , and p(42)igaftvk (49)] were identified in n terminus of the emcv vp1, using these mcabs and a series of recombinant truncated vp1. vp1 is one of the most important structures proteins of emcv, which can stimulate the organism to produce neutralization antibody [18] . immunization of mice with recombinant vp1 protein (plasmids or recombinant adenoviruses expressing small hairpin rnas targeted to vp1 protein genes of emcv), which could provide protective efficacy against emcv [26, 27] . dna vaccines encoding an emcv-d vp1 antigen confer protective immunity against heterologous challenge with emcv-k, indicating that vp1 is a potential vaccine candidate for controlling emcv infection [28] . thus it is need to explore the epitopes related to the protection or diagnosis of this disease, even thought the residue at position 49, 62 and 100 are related to neutralization epitopes. in this study, the ten anti-vp1 mcabs all did not have the ability to neutralize the emcv in cells cultures (data not shown here). however, they all could react with the emcv antigen in western blot and ifa and could be used for diagnosis of this virus. sequence analysis of both variants revealed one amino acid exchange within the capsid protein vp1, demonstrated that the diabetogenic and non-diabetogenic emcv variants differing in only one single amino acid [29] [30] [31] [32] . studies of theiler's murine encephalomyelitis virus (tmev) demonstrated that the amino acids on the loops exposed to the surface of the virion were important disease determinants [33] . the residue 49 was located in the loop connecting the βb and βc strands of vp1, which was exposed at the surface of the capsid and was known to be part of a neutralization epitope [34] . in this study, the results of alignment of the amino acid sequences of different emcv isolates revealed that the epitope f(19)vaqpvy (25) was highly conserved among all the reference emcv strains. there were only 1 or 2 amino acid residue different in the epitopes p(42)igaftvk(49) and v(2) enaek(7) between those strains. according to the model of mengo virus, the 3d model of vp1 of emcv was also analyzed. in the picture, p(42)igaftvk(49) shown as orange spheres was located circa the 5-fold icosahedral axis of symmetry and was on the surface. meanwhile, v(2) enaek(7) and f(19)vaqpvy (25) were at the n-terminus and located *under* the vp3 protein and sandwiched with the vp4 protein inside the particle. the obtain of the mc-abs against these two epitopes in this study might be explanted by the "breathing" of the n-terminal tail between the vp3 and vp2 proteins. these two epitopes could be used for diagnosis of this virus because of the results of alignment that these two regions were highly conserved among all the reference emcv strains. here, ten mcabs against emcv vp1 were developed and three b-cells epitopes (2-7aa, 19-25aa and 42-49aa) were defined in vp1. the results may contribute to understand the antigenic structure of vp1 protein deeply and facilitate the development of diagnostic method of emcv. emcv isolate nj08 (genbank accession no. hm641897) used in this study was isolated and kept in our lab (submitted 10-jul-2010). bhk-21 cells were kept in dulbecco's modified eagle medium supplemented with 10% fetal bovine serum. the sp2/0 cells were grown in rpmi 1640 medium supplemented with 20% fetal bovine serum. female balb/c mice, 4-6 weeks of age, were purchased from the laboratory animal centre of nantong university. hat and ht media supplement (50×) were purchased from sigma. horseradish peroxidase (hrp)-tagged goat anti-mouse igg was purchased from wuhan boster biological technology co. rapid elisa mouse mcab isotyping kit was purchased from pierce. the vp1 gene was amplified by pcr using the specific primers kept in our lab. pcr products were digested by bamh i and ecor i, and cloned into prokaryotic expression vector pet-28a(+) subsequently. the recombinant plasmids were verified by analysis and nucleotide sequencing, and then transformed into e.coli bl21 cells for fusion proteins expression. the transformants were cultured at 37°c in luria-bertani broth containing 0.05 mg/ml kanamycin until the optical density at 600 nm (od 600 ) reached 0.8. the cells were incubated for another 4 h in the presence of 1.0 mm isopropyl-β-d-thiogalactoside and harvested by centrifugation. the e. coli/pet-28a-vp1 transformants were lysed by sonication, and then centrifuged at 3000 g for 15 min at 4°c. the proteins were purified by probondtm purification system, according to the manufacturer's instructions (invitrogen). the purified recombinant vp1 (rvp1) protein was detected by sds-page and western blot, and the concentration was determined by nanodrop 2000 spectrophotometer (thermo). emcv was sedimented and purified from the culture medium with peg-6000 by ultracentrifugation. the purified emcv were analyzed by sds-page and western blot with anti-emcv sera. the mcabs were developed and obtained as follows. briefly, inactivated emcv was combined with equal volumes of complete/incomplete freund's adjuvant (sigma-aldrich) at final concentration of 0.2 mg/ml. each female balb/c mice (6-week-old) was immunized with 0.1 mg emulsion at 3 weeks intervals for 9 weeks. three days after the last injection, the spleens were surgically removed from the mice and fused with mouse myeloma cells (sp2/0) using 50% (v/v) peg. the fused hybridoma clones were screened by the indirect elisa for mcabs that have strong reactivity with the rvp1 but not his protein or bound emcv but not bhk-21 cells' protein. selected clones were subcloned by limiting dilution. ascites fluids were induced in pristine-primed balb/c mice. finally, the mcabs were identified by western blot and indirect immunofluorescence assay (ifa). for screening the mcabs, elisa plates were coated with the purified rvp1 protein and e.coli/pet-28a lysate (as the negative control) at the concentration of 0.002 mg/ ml in buffer bicarbonate (ph9.6) at 4°c overnight or coated with the purified emcv and bhk-21 cells' protein at the concentration of 0.01 mg/ml as the same way above. after washing with pbs containing 0.05% tween-80 (pbst) three times, the plates were blocked figure 8 the locations of the three epitopes. the picture was about the complete particle of mengo virus. vp1 is shown in red ribbon, vp3 is shown in green ribbon, vp2 is shown in yellow ribbon, and vp4 is shown in blue ribbon. the 3 epitopes presented as spheres, v(2)enaek (7) is colored cyan, f(19)vaqpvy(25) is colored magenta, and p(42)igaftvk(49) is orange. the figure was generated using ucsf chimera [22] . (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article). with 5% non-fat milk in pbst at 37°c for 2 h. then wells were incubated with the supernatant of hybridoma at 37°c for 1 h. the sera isolated from immunized mice and the supernatant of sp2/0 cells were used as positive and negative controls, respectively. after three washes with pbst, wells were incubated with a 1:10,000 diluted goat antimouse igg-hrp at 37°c for 1 h. after washing, the plates were incubated with substrate solution tetramethyl benzidine (tmb) at 37°c for 10 min, and the reaction was stopped with 2 m h 2 so 4 in each well (50 ml/well).the od was read at 450 nm in an automatic elisa plate reader. the criteria were judged as follows: the od 450 of the positive control should be more than 1.0, the od 450 of the negative controls should be lower than 0.2. the results were expressed as the ratio of od 450 produced by the samples compared to the negative control. samples, giving a ratio value higher than 2.1, were considered to be positive. for mapping the epitopes precisely, a series of truncated fragments of protein were expressed in e.coli. the epitopes recognized by mcabs were mapped by truncated vp1 fragments based on indirect elisa as described above. in order to identify the minimal antigenic epitopes recognized by mcabs, the vp1 gene was divided into 26 truncated fragments (figure 9 ). a series of primers (table 1 ) were synthesized to amply the different truncated fragments of vp1 protein. pcr products were separately cloned into the xho i and bamh i sites of pgex-6p-1 expression vector. the recombinant proteins were expressed in e.coli bl21 and identified by sds-page and western blot with the gst-tagged monoclonal antibody (boshide, wuhan, china), and then were used to determine the epitopes recognized by mcabs using indirect elisa and western blot methods. to identify the proteins expressed in e. coli (vp1 and truncated vp1 proteins), the proteins were separated by sds-page (5-12%) and transferred to nitrocellulose membrane (pall corporation). the mcabs (dilution of 1:10-1:50) bound to the proteins on the membrane was detected by goat anti-mouse igg-hrp (boshide, wuhan, china) at 37°c for 1 h. detection was performed using chemiluminescenceluminol reagents (supersignal west pico trial kit, pierce). bhk21 cells infected with emcv nj08 strain in 96-well culture plate were rinsed with pbs and fixed with cold ethanol for 45 min at 4°c. the cells were washed, and three linear epitopes (p2-7aa, p19-25aa and 42-49aa) identified in this study were synthesized using fmoc solidphase chemistry (invitrogen) with >98% purity. they were coated in reacti-bind™ amine-binding, maleic anhydride 96-well plates (pierce, u.s.a.) at the concentration of 10ug/ml for 5 h at room temperature. meanwhile, purified gst-1-72aa (f3) and rvp1 protein were used as positive antigens control. mice anti-emcv serum (at dilution of 1:100) were added as the primary antibody and incubated at 37°c for 1 h. the spf mice serum was used as negative antibody control. the following detection steps were the same as described above. nucleotide sequences of emcv strains from different countries were retrieved from genbank. the amino acid sequences of identified b-cell epitopes were aligned using dnastar megaligen software. the representative emcv strains were listed in table 2 . molecular graphics and analyses were performed with the ucsf chimera package. chimera is developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco (supported by nigms p41-gm103311). the differences in the level of antibodies between different groups were determined by one-way repeated measurement anova and least significance difference (lsd). differences were considered statistically significant when p < 0.05. the encephalomyocarditis virus pathogenesis of encephalomyocarditis experimental infection in young piglets: a potential animal model to study viral myocarditis an outbreak of encephalomyocarditis-virus infection in free-ranging african elephants in the kruger national park an encephalomyocarditis virus epizootic in a baboon colony fatal inflammatory heart disease in a bonobo (pan paniscus) screening for antibodies against zoonotic agents among employees of the zoological garden of vienna a wild-type porcine encephalomyocarditis virus containing a short poly(c) tract is pathogenic to mice, pigs, and cynomolgus macaques encephalomyocarditis virus infections in an australian zoo persistence of encephalomyocarditis virus (emcv) infection in piglets genetic variability of encephalomyocarditis virus (emcv) isolates outbreaks in quebec pig farms of respiratory and reproductive problems associated with encephalomyocarditis virus reproductive failure in sows following experimental-infection with a belgian emcv isolate serological survey on prevalence of antibodies to avian paramyxovirus serotype 2 in china retrospective genetic characterisation of encephalomyocarditis viruses from african elephant and swine recovers two distinct lineages in south africa the nucleotide and deduced amino acid sequences of the encephalomyocarditis viral polyprotein coding region host and virus determinants of picornavirus pathogenesis and tropism characterization of mengo-virus neutralization epitopes.2. infection of mice with an attenuated virus protective immunity against heterologous challenge with encephalomyocarditis virus by vp1 dna vaccination: effect of coinjection with a granulocyte-macrophage colony stimulating factor gene identification of immunodominant b-and t-cell combined epitopes in outer membrane lipoproteins lipl32 and lipl21 of leptospira interrogans identification of a conserved linear b-cell epitope at the n-terminus of the e2 glycoprotein of classical swine fever virus by phage-displayed random peptide library identification of b-cell epitopes in the nsp1 protein of porcine reproductive and respiratory syndrome virus ucsf chimera-a visualization system for exploratory research and analysis identification of continuous b-cell epitopes on the protein moiety of the 58-kilodalton cell wall mannoprotein of candida albicans belonging to a family of immunodominant fungal antigens identification of a linear b-cell epitope on avian reovirus protein sigmac selection of sars-coronavirus-specific b cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice inhibition of encephalomyocarditis virus replication by shrna targeting 1d and 3ab genes in vitro and in vivo protective efficacy of adenovirus-mediated small interfering rnas against encephalomyocarditis virus challenge in mice enhancement of vp1-specific immune responses and protection against emcv-k challenge by co-delivery of il-12 dna with vp1 dna vaccine molecular analysis of the capsid coding region of a virulent encephalomyocarditis virus isolate after serial cell passages and assessment of its virulence gain or loss of diabetogenicity resulting from a single point mutation in recombinant encephalomyocarditis virus analysis of sequence and pathogenic properties of two variants of encephalomyocarditis virus differing in a single amino acid in vp1 alteration of encephalomyocarditis virus pathogenicity due to a mutation at position 100 of vp1 mutations that affect the tropism of da and gdvii strains of theiler's virus in vitro influence sialic acid binding and pathogenicity efficient infection of buffalo rat liver-resistant cells by encephalomyocarditis virus requires binding to cell surface sialic acids this work was supported by grants from the jiangsu natural science foundation to young researcher (bk20130691), the china agricultural research system foundation (cars-36), the special fund for agroscientific research in the public interest (201003060-4), the national natural science foundation (31230071), and the priority academic program development of jiangsu higher education institutions (papd). the authors declare that they have no competing interests. key: cord-016594-lj0us1dq authors: flower, darren r.; davies, matthew n.; doytchinova, irini a. title: identification of candidate vaccine antigens in silico date: 2012-09-28 journal: immunomic discovery of adjuvants and candidate subunit vaccines doi: 10.1007/978-1-4614-5070-2_3 sha: doc_id: 16594 cord_uid: lj0us1dq the identification of immunogenic whole-protein antigens is fundamental to the successful discovery of candidate subunit vaccines and their rapid, effective, and efficient transformation into clinically useful, commercially successful vaccine formulations. in the wider context of the experimental discovery of vaccine antigens, with particular reference to reverse vaccinology, this chapter adumbrates the principal computational approaches currently deployed in the hunt for novel antigens: genome-level prediction of antigens, antigen identification through the use of protein sequence alignment-based approaches, antigen detection through the use of subcellular location prediction, and the use of alignment-independent approaches to antigen discovery. reference is also made to the recent emergence of various expert systems for protein antigen identification. of smallpox were reported annually from across the globe, leading to about 2 million deaths a year. yet, today, the disease has been completely eradicated. in the last 30 years, there have been no known cases. poliomyelitis or polio is the other largescale disease which has come closest to eradication. its success too has been formidable: in 1991, the pan american health organization effectively eradicated polio from the western hemisphere, since when the global polio eradication programme has significantly decreased the overall incidence of poliomyelitis through the rest of the world. in 1988, there were approximately 350,000 cases spread through 125 countries; in the past years, global figures amounted to less than 2,000 annually. yet, in spite of such remarkable success, death from vaccine-preventable diseases remains unacceptably high [2] . there are over 70 common infectious diseases responsible for one in four deaths globally. rotavirus and pneumococcus are pathogens causing diarrhoea and pneumonia, the leading causes of infant deaths in underdeveloped countries. in the next decade, effective, widespread vaccination programs against such pathogenic microbes could save the lives of 7.6 million children under 5 years of age. hepatitis b causes 600,000 deaths in adults and children aged over 5. seasonal, non-pandemic influenza kills upwards of half a million globally each year. for those aged under 5 in particular, a series of diseases causes an extraordinary and largely preventable death toll. for example, tetanus accounts every year for 198,000 deaths, pertussis is responsible for over 290,000 deaths, hib gives rise to in excess of 386,000 deaths, diphtheria accounts for 4,000 deaths, and yellow fever over 15,000 deaths. arguably, the most regrettable, the most lamentable situation is that of measles. measles accounts for the unneeded deaths of 540,000 under-fives and over 70,000 adults and older children. despite this, the situation is by no means bleak. by the close of 2008, approximately 42 million had been vaccinated against hib and 192 million children against hepatitis b. during its first decade, vaccinations against polio, hep b, hib, measles, pertussis, and yellow fever funded by gavi had prevented the unnecessary loss of over 5 million lives. there are approximately 50 vaccines licensed for use in humans, around half of these are widely prescribed. yet, most of these vaccines target the prevention of common childhood infections, with the remainder addressing tropical diseases encountered by travellers to the tropics; only a relatively minor proportion combat endemic disease in under-developed countries. balancing the persisting need against the proven success and anticipated potential, vaccines remain an area of remarkable opportunity for medical advance, leading directly to unprecedented levels of saved and improved lives. from a commercial perspective, the vaccine arena has long been neglected, in part because of the quite astonishing success limned above; today, and in comparative terms at least, activity within vaccine discovery is feverish [3, 4] . during the last 15 years, tens of vaccines and vaccine candidates have moved successfully through clinical trials, and vaccines in late development number in the hundreds. in stark contrast to antibiotics, vaccine resistance is negligible and nugatory. despite the egregious and outrageous success enjoyed by vaccines, many major issues persist. the world health organisation long ago identified tuberculosis (tb), hiv, and malaria as the three most significant life-threatening infectious diseases globally. no vaccine has been licensed for malaria or hiv, and there seems little realistic hope for such vaccines appearing in the immediate future. bacille calmette guérin (bcg), the key anti-tb vaccine, is of limited efficacy [5] . levels of morbidity and mortality generated by diseases already targeted by vaccines remain high. influenza is the key example, with a global annual estimated death toll in the region of half a million. in the twenty-first century, the world continues to be threatened by infectious and contagious diseases of many kinds: visceral leishmaniasis, marburg's disease, west nile, dengue, as well as sars potentially pandemic h5n1 influenza, and over 190 human and emerging zoonotic infections, as well as the persisting threat from hiv, tb, and malaria mentioned above. all this is further compounded by the additional risk arising from antibiotic-resistant bacteria and bioterrorism, not to mention major quasi-incidental issues, such climate change, an accelerating growth in the world's population, increased travel, and the overcrowding seen within the burgeoning populations concentrated into major cities [6] . for reasons we shall touch on below, the discovery of vaccines is both more urgent and more difficult than it has ever been. in an era where conventional drug discovery has been seen to fail-or at least as seen by cupiditous investors, for whom the current model of pharmaceutical drug discovery is broken-vaccines are one of a number of biologically derived therapies upon which the future economic health of the pharmaceutical industry is thought to rest. the medical need, as stated above, is clear. set against this is the unfortunate realisation that vaccines exist for most easily targeted diseases, those mediated by neutralising antibodies, and so outstanding vaccine-targets are those of more intractable diseases mediated primarily by cellular immunity. to address those properly requires what all discoveries required: hard work and investment; but they also need new ideas, new thinking, and new vaccine discovery technology. amongst, these are computational techniques, the most promising of which are those targeting the discovery of novel vaccine antigens: the candidate subunit vaccines of tomorrow see fig. 3 .1. vaccines are agents-either molecular (epitope-or antigen-based vaccines) or supramolecular (attenuated or inactivated whole pathogen vaccines)-which are able to create protective immunity against specific pathogenic infectious microorganisms and any diseases to which they might give rise. protective immunity can be characterised as an enhanced but highly specific response to consequent re-infection-or infection by an evolutionarily closely related micro-organismsmade by the adaptive immune system. such increased or enhanced immunity is facilitated by the quantitative and qualitative augmentation of immune memory, which is able to militate against the pernicious effects of infectious disease. vaccines synergise with the herd immunity they help engender, leading to reduced transmission rates as well as prophylaxis against infection. the term "vaccine" derives from vacca (latin for cow). the words vaccine and vaccination were coined specifically for anti-smallpox immunization by the discoverer of the technique, edward jenner (1749-1823). these terms were later extended by louis pasteur (1822-1895) to include a far more extensive orbit or remit, including the entire notion of immunisation against any disease [2, 3, 6] . several fundamentally distinct varieties of vaccine exist. these include inter alia inactivated or attenuated whole pathogen-based vaccines; subunit vaccines are based on one or more protein antigens, vaccines based upon one or more individual epitopes, carbohydrate-based vaccines, and combinations thereof. hitherto, the best-used and, thus, the most successful types of vaccine were built from attenuated-"weakened" or non-infective or otherwise inactivated-pathogenic whole organisms, be they bacterial or viral in nature. well-known examples include the following: the bcg vaccine which acts prophylactically against tuberculosis and albert sabin's anti-poliomyelitis vaccine based on attenuated poliovirus. the vast majority of subunit vaccines are immunogenic protein molecules, and are typically discovered using a somewhat haphazard search process. concerns over the safety of whole-organism vaccines long ago prompted the development of other kinds of vaccine strategy, including those based upon antigens as the innate or immanent active biological constituent of either single or composite vaccines. the vaccine which targets hepatitis b is a good exemplar of a so-called subunit vaccine as it is based on a protein antigen: the viral envelope hepatitis b surface antigen. other types of as-yet-unproven vaccines include those based on epitopes and others based on antigen-presenting cells; many have entered clinical trials, but none have fulfilled their medical or commercial potential. whole antigen discovery. when looking at a reverse vaccinology process, the discovery of candidate subunit vaccines begins with a microbial genome, perhaps newly sequence, progresses through an extensive computational stage, ultimately to deliver a shortlist of antigens which can be validated through subsequent laboratory examination. the computational stage can be empirical in nature; this is typified by the statistical approach embodied in vaxijen [115] . or this stage can be bioinformatic; this involves predicting subcellular location and expression levels and the like. or, this stage can take the form of a complex mathematical model which uses immunoinformatic models combined with mathematical methods, such as metabolic control theory [153] , to predict cell-surface epitope populations it is often difficult to capture the proper scientific meaning and use of recondite terms, often borrowed from common usage or archaic language. so, let us be more specific. an immunogen-a molecular moiety exhibiting the property of immunogenicity-is any material or substance capable of eliciting a specific immune response. an antigen, on the other hand, is a molecular moiety exhibiting the property of antigenicity. it is a substance or material recognised by a primed immune system. such a persisting state of immune readiness may be mediated by humoral immunity (principally via the action of soluble antibodies) or by cellular immunity (as mediated by t-cells, antigen presenting cells (apcs), or other phagocytic cells), or a combination of both, in what is often referred to as a "recall" response. immunogenicity is vital: it is the signature characteristic or property that prompts a certain molecular moiety to evoke a significant immune response. here, we shall strictly limit use of "immunogen" and "antigen" to a sole meaning. here, an "antigen" or an "immunogen" will mean a protein that is capable of educing some kind of discernible response from the host immune system. specifically, and for practical reasons, we will almost exclusively be referring to proteins derived from a pathogenic micro-organism. at present, the prophylaxis engendered by all current effective vaccines-all except bcg-is primarily mediated by the humoral immune system, via soluble antibodies. however, the disease mechanisms of most serious diseases for which vaccines are not available are usually mediated by cellular immunity. thus, for untreated disease, we seek to identify immunogenicity generated principally by cellular responses or by a combination of cellular and humoral responses, rather than by humoral immunity alone. to some extent, subunit vaccines can be thought to represent something of a compromise between vaccines based on attenuated or otherwise inactivated wholeorganisms and the many more recent and more innovative vaccine strategies typified by epitope or poly-epitope vaccines. vaccines based around whole pathogens have long engendered safety concerns [7] [8] [9] . from the lubeck disaster and the cutter incident [10] [11] [12] to the recent mmr debacle, issues over safety, real or imagined, have always dogged the development of vaccines [1, 9] . indeed, during the eighteenth century the pre-vaccination practice of variolation against smallpox prefigured much of the current debate over the perceived danger of vaccines [13] . while the case for vaccines is unanswerable, we should not be complacent. any live vaccine, however extensively attenuated, can revert to a pathogenic, diseaseinducing form. this is currently an on-going issue for polio vaccination [14] . other issues, particularly the chemical or biological contamination of vaccines during manufacture, remain enduring and persistent problems. undesired immunogenicity, the type leading to severe and pathological immune responses, rather than enduring immune memory, is a concern for both whole-organism and subunitbased vaccines, as well as putative biologics [15] . immunologists and vaccinologists have thus long sought alternatives to the use of whole organisms as vaccines. subunit vaccines and conjugate vaccines are one such. vaccines based on epitopes, singly or in combination, are another. the diversity of innovations in vaccine design holds much potential for success, but, thus far at least, has proved spectacularly unsuccessful in a clinical context. logically, a vaccine that relies solely on, at most, a few well-chosen epitopes, should be effective, efficacious, and, above-all, safe. epitopes, as peptides, may be cytotoxic and might possibly prompt some kind of inopportune immune response but cannot be infective or revert to infectivity. in many ways, epitopes are closer in size and share many properties with synthetic small molecules; possibly dealing with their pharmacokinetics as such may be better than thinking of them as biologic drugs. in practice, of course, epitope-based vaccines, like subunit vaccines, suffer from poor immunogenicity, necessitating the use of a complex combination of adjuvants and complicated delivery systems. for diverse reasons, including immunogenicity, stimulating protective immune responses against intracellular pathogens remains problematic when using nonreplicating vaccines. why should this be? first, the immune response is very complex, involving both the innate and adaptive immunity, and significant interaction between them. in all probability, and particularly when viewed in the context of the whole population, many epitopes and danger signals are involved; likewise, the many different immune actors, be they acting at the cellular or molecular levels, interact with each other and are subject to complex mechanisms of genetic, epigenetic, and system-level control and regulation. it may be that only the large and complex organism-sized vaccines can induce the range of immune responses necessary across the population to induce protection, since they comprise a potential host of immunogenic molecular moieties, not just a single immunodominant epitope see fig. 3 in that which follows, we shall seek to explore the availability and accessibility of informatic techniques and informatic tools used to identify candidate subunit vaccines of microbial origin. yet, we shall start by adding context with an examination of experimental approaches to antigen discovery: so-called reverse vaccinology. reverse vaccinology already relies on informatics, but, in a sense at least, what we would like to do using informatics is to reproduce as much as is possible the steps inherent in successful reverse vaccinology in silico rather than in vitro. reverse vaccinology, and the necessary computational support, is a much more prevalent means of identifying subunit vaccines [16] . see fig. 3 .1. even today, many experimentalists retain a deep and atavistic distrust of all computation. experimentalists seldom trust the reliability and dependability of computational methodology, choosing to trust instead in what they believe to be infallible, if actually rather elusive, empirical reliability of observations, experiments, and the whole paraphernalia of laboratory experimentation. yet, things are in the process of changing, and this change is likely to accelerate as we move forward into a future that looks more parsimonious and uncertain by the day. vaccines have come a long way from the days when they were prepared directly from the fluids of smallpox pustules or extracts of infected spinal cords. yet vaccine discovery and development remains firmly empirical. many modern vaccines still comprise entire inactivated pathogens. while vaccines targeting papillomavirus, tetanus, hepatitis b, and diphtheria are subunit vaccines, few are recombinant proteins devoid of contaminants. some would argue that the only molecular vaccines are glycoconjugates: oligosaccharides conjugated to immunogenic carrier proteins. conventional empirical, experimental, laboratory-based microbiological ways to identify putative candidate antigens require cultivation of target pathogenic micro-organisms, followed by teasing out their component proteins, analysis in a series of in-vitro and in-vivo assays, animal models and with the ultimate objective of isolating one or two proteins displaying protective immunity. unfortunately, in reality, the process is more complex, and more confusing, and much more confounding as this brief synopsis might suggest. cultivating pathogens outside the environment offered by their host organism can be difficult, even impossible. not every protein is readily expressed in adequate quantities in vitro, and many proteins are only expressed in an intermittent basis during the time course of infection. thus, a considerable number of potential, putative, and possible vaccine candidate antigens could be missed by conventional experimental approaches. reverse vaccinology [16] [17] [18] [19] has the potential to analyse genomes for potential antigens, initially scanning "open reading frames" (orfs), then selecting proteins because they are open to surveillance by the host immune system. this usually involves some complex combination of informatic-based prediction methodologies. recombinant expression of the resulting set of identified molecules can overcome their reduced natural abundance, which has often prevented us recognising their true potential. by enlarging the repertoire of native antigens, this technology can help to foster the development of a new cohort of vaccines. reverse vaccinology was originally established and has been established by studying neisseria meningitidis, which is responsible for meningococcal meningitis and sepsis. vaccines are currently available for all serotypes, except that serogroup b. n. meningitidis orfs were found initially [20, 21] ; 570 proteins were then identified, 350 expressed in vitro and 85 found to be surface exposed. seven proteins elicited immunity over many strains. the culmination of this work was a "universal" vaccine for serogroup b based on five antigens [22] . this protovaccine, when used with alum as adjuvant, induced murine bactericidal antibodies versus 78 % of 85 meningococcal strains drawn from the world population of n. meningitidis. strain coverage increases to over 90 % when used with cpg or mf59 as adjuvant. another key illustration is porphyromonas gingivalis, an anaerobic gramnegative bacterium found in the chronic adult inflammatory gum disease periodontitis. initially, 370 orfs were identified [23] ; of these, 120 protein sequences were open to immune surveillance and 40 were positive for several sera. two antigens were found to be protective in mice. yet another fascinating instance is provided by streptococcus pneumoniae, a prime cause of meningitis, pneumonia, and sepsis [24, 25] . in this study, 130 potential orfs were initially identified, with 108 of these proteins being readily expressed. finally, six proteins were seen to induce protection against the pathogen. more recently, other and more advanced experimental techniques, such as microarrays, are beginning to come on-stream, opening up a gallimaufry of possible technologies to the new but maturing field of reverse vaccinology. the following gives but a taste of what is to come. using ribosome display to undertake in-vitro protein selection, weichart et al. [26] identified within the methicillin-resistant col strain of the virulent human pathogen staphylococcus aureus 75 genes, the majority of which were secreted or surface-localized proteins; of these, 25 % had cell envelope function, 24 % were transporter proteins, and 9 % were virulence factors or toxins. using an ingenious combination of advanced proteomics techniques and in-vitro assays, giefing et al. [27] identified 18 novel vaccine candidates which prevented infections in children and in the elderly caused by a variety of pneumococcus serotypes; four demonstrating major protection versus sepsis in animals. two leads-stkp (a serine/threonine protein kinase) and pcsb (a structural protein with a role in cell wall separation of group b streptococcus)-showed clear cross-protection as potential candidate vaccines against four separate pneumococcal serotypes. using a whole proteome microarray, and in order to identify protein antigens, eyles et al. [28] probed serum from balb/c mice previously immunized with a vaccine comprising: killed francisella tularensis and two immunomodulatory adjuvants. eleven out of the top twelve immunogenic antigens were known already as immunoreactive, although 31 further proteins were discovered using this experimental approach. in further work from this consortium, titball and co-workers [29] constructed a protein microarray of 1,205 burkholderia pseudomallei proteins, treated it with 88 patient samples, identifying 170 antigens. this smaller set was treated with a further 747 distinct sera from 10 groups of patients, identifying 49 putative candidate antigens. this survey, brief though it is, helps to highlight the potential power of reverse vaccinology for vaccine discovery. however, since the number of antigens is high, given all the potential difficulties in characterising and expressing them, it is important to note that both computational and experimental techniques and methodologies will doubtlessly omit important and interesting proteins from further analysis, though not necessarily for the same or similar reasons. thus, with the burgeoning discipline of reverse vaccinology, both computational and experimental techniques are in need of constant development and improvement. compared to its role to drug discovery, genomics, and a host of other bioscience sub-disciplines, bioinformatics support for the preclinical discovery and development of vaccine is in its infancy; yet, as interest in vaccine discovery increases, the situation changes. there are two key types of bioinformatics support for vaccine design, discovery, and development. at the technical level, the first of these cannot be properly or meaningfully distinguished from general support for target discovery. it includes the annotation of pathogen genomes, more conventional host genome annotation, and the statistical analysis of immunological microarray experiments. the second form of support concentrates on immunoinformatics, that is, the informatics analysis of immunological problems, principally epitope prediction. b-cell epitope prediction remains defiantly basic or is largely dependent on a sometimes unavailable knowledge of three-dimensional protein structure. both structure[30] and data-driven [31] prediction of antibody-mediated epitopes evince poor results. however, methods developed to predict t-cell epitopes now possess considerable algorithmic sophistication. moreover, they continue to develop and evolve, as well as extend their scope and remit to address new and ever larger and more challenging epitope prediction problems. presently, accurate and reliable t-cell epitope prediction is restricted to predicting the binding of peptides to the major histocompatibility complex (mhc). class i peptide-mhc prediction can be reasonably accurate, or is for properly characterised, wellunderstood alleles [32] . yet a number of key studies have demonstrated that class ii mhc binding prediction is almost universally inaccurate, and is thus erratic and unreliable [33] [34] [35] . a similar situation persists for structure-driven prediction of mhc epitopes [36, 37] . irrespective of poor predictive performance, several other problems exist for epitope prediction. for t cell prediction in particular, a prime concern is with the availability or rather lack of availability of relevant data. it is now known that immunogenic t cell epitopes, thought previously to be peptides no more than 10 amino acids in length, can be 16 or more residues long. longmer epitopes now greatly expand the number of possible peptides open to inspection by t cells [38] [39] [40] [41] . the inadequate results generated by b cell epitope prediction algorithms may indicate that a fundamental reinterpretation of extant b cell epitope data is necessary before improved methods become feasible. these factors, when taken together, are consistent with the notion that methods relying only on the possession of certain epitopes will not be fully effective when tasked with antigen or immunogen identification. this is supported by information indicating a lack of correspondence between selected antigens and experimentally verified protective proteins. there are many means of identifying antigenic proteins. most focus on the properties of protein sequence and structure, but arguably one of the most insightful is instead to examine properties, both local and global, of the underlying nucleic acid. one notable way is to look for evidence of the horizontal or lateral transfer of so-called pathogenicity islands or pais. horizontal transfer, such as transformation, conjugation, or transduction, is distinct from the vertical transfer of genetic material from an ancestor within its lineage. it typically involves an organism incorporating genetic material from an evolutionarily distant organism without being its offspring. pais are a specific type of genomic island; that is, part of a genome acquired through direct transfer between microbes. a genomic island can occur in distantly related species and may be mono-or multi-functional; there are many sub-classes classified by function. other examples include antibiotic resistance islands, metal resistance, and secretion system islands. the gene products of pais are crucial to the propagation of disease pathogenesis, much as the pais themselves are key to the evolution of pathogenesis. pathogen-associated type iii and type iv secretion systems are, for example, often found together in the same pai. detecting such large (>10 kb) and discrete clusters of genes clusters, habitually possessing a characteristically atypical g/c content, at least when compared with the remainder of the genome, leads, in turn, to the individual identification within clusters of virulence-associated protein antigens. prokaryotic pais are frequently associated with trna-encoding genes, many are flanked by repeat structures, and many contain fragments of mobile genetic elements such as plasmids and phages. pais can be identified by combining analysis of nucleotide composition and phylogeny, amongst others. composition-based approaches rely on the natural variation between genome sequences from different species. regions of the genome with abnormal composition, as demonstrated by nucleotide or codon bias, may be potentially transferred horizontally. such methods are prone to inaccuracies; these result from inherent genomic sequence variation, such as is seen in highly expressed genes, and the observation that over time the sequences of genomic islands alter to mirror the composition of host genomes. evolution-based approaches seek regions that may have been transferred horizontally by comparing related species. put at its simplest: a putative genomic island present in one species, but absent from several related species, is consistent with horizontal transfer. of course, the island may have been present in the last common ancestor shared by the species compared and subsequently been lost from the other species. a less likely explanation would be that the island arose by mutation and selection in this species and no other. to decide, a body of extra evidence would need to be explored, such as the size of the pai, the mechanistic ease of deletion, the consistent presence of the island in more distantly related species, the relative pathogenicity of island-less species, and the divergence of the genome relative to that of other related species. many methods, which seek to quantify and leverage these somewhat vague notions, are now available [42] [43] [44] . such analysis at the nucleic acid level shares many features in common with approaches used to identify cpg islands in eukaryotic genomes [45] [46] [47] [48] . recently, langille et al. tested six sequence-composition genomic island prediction methods and found that islandpath-dimob and sigi-hmm had the greatest overall accuracy [49] . island path was designed to help identify prokaryotic pais, through the visualisation of common pai characteristics such as mobile element-associated genes or atypical sequence composition [50] . sigi-hmm is a very accurate sequence composition-based genomic island predictor, which combines a hidden markov model (hmm) and codon usage measurement to identify genomic islands [51] . in another work, yoon et al. coupled heuristic sequence searching methods, which aimed simultaneously to identify pais and individual virulence genes, with composition and codon-usage bias [52] . exploiting a machine learning approach, vernikos and parkhill sampled the structural features of genomic islands using a hypothesis-free, bottom-up search, with the objective of explicitly quantifying the contribution made by each feature to the overall structure of different genomic islands [53] . arvey et al. sought to identify large chromosomal regions with atypical features using a general divergence measureable to quantify the compositional difference between genomic segments [54] . islandpick is a comparative genomic island predictor, rather than a composition-based approach, that can identify very probable genomic islands and very probable non-genomic islands within investigated genomes but does require that several phylogentically related genomes are available [49] . observing pais as having a g + c composition closer to their host genome, wang et al. used so-called genomic barcodes to identify pais. these barcodes are based on the fact that the frequencies of 2-mers to 7-mers, and their reverse complement, are very stable across a whole genome when using a window size of over 1,000 bps and that this constituted a characteristic signature for genomes [55] . the ready detection of pais, as a tool in computational reverse vaccinology, has been greatly aided by the deployment of several web-based resources. a key example of a server that successfully integrates several accurate genomic island predictors is islandviewer [56] , which combines the methods: islandpick [49] , islandpath [50] , and sigi-hmm [51] and is available at the url: http://www. pathogenomics.sfu.ca/islandviewer/query.php. the gui facilitates the visualisation of genomic islands and downloading of data at the gene and chromosome levels in a variety of formats. another important, web-accessible resource is paidb or the pai database. this is a wide-ranging database of pais, containing 112 distinct pais and 889 genbank accessions present in 497 strains of pathogenic bacteria [57] . paidb may be accessed via the url: http://www.gem.re.kr/paidb. thus, alternative techniques and methodologies are required in order to select and to rank proteins likely to be protective antigens and thus candidate vaccines. below, we shall explore three key approaches: subcellular location prediction, alignment-dependent sequence similarity searching, and alignment-independent empirical statistical approaches. in this section, we consider, perhaps, the clearest and cleanest way to identify potential new antigens in any microbial genome to alignment-dependent sequence similarity searching. there are two complimentary but distinct ways of identifying the immunogenicity of a protein from its sequence. one is to look for significant similarity to proteins of known immunogenicity. this idea seems so straightforward as to be almost facile. the other approach is somewhat less obvious conceptually but almost as straightforward logistically and involves seeking to identify antigens as proteins without discernible sequence similarity to any host protein. let us turn to the first of these two alternatives. let us begin by stating or rather reiterating the obvious. if we know the sequence of an existing antigen or antigens, we can use sequence searching to find similar sequences in the target genome [58, 59] . any candidate antigens selected by this process can then be selected for further verification and validation. the same old, familiar caveats apply here: are chosen thresholds appropriate? are high-scoring matches an artefact or are they real and meaningful? the litany of such conditions is all too familiar to anyone well versed in sequence similarity searching. clearly, when a sequence search is run, using blast or fasta3, for example, an enormously long list of nearly identical proteins might ensue, or one that does not get any hits at all, or almost any intervening result might be obtained. as reflective practitioners, we must judge which result can be classified as useful and which cannot, and in so doing, identify sets of suitable thresholds, above which we expect usefulness and below which we might anticipate little or no utility. thresholds are contingent upon the sequence family studied, as well as being dependent solely on the problem investigated. thus heuristically identified cut-offs are desirable, but much thinking and empirical investigation are required to select appropriate values. of course, the process adumbrated above presupposes that sufficient antigenic protein sequences are known. compilation of this data is the role of the database. recently, extensive literature mining, coupled with factory-scale experimentation, has created many functional immunology databases, although databases, such as syfpeithi [60, 61] , focussing on cellular immunology-primarily mhc processing, presentation, and t cell recognition-have existed for 15-20 years. arguably, the best extant database is the hiv molecular immunology database [62] , although clearly the depth of the database is at the expense of generality and breadth. other recent databases include mhcbn [63, 64] and epimhc [65] , amongst many others. two databases, warrant particular attention: antijen [66] , formerly known as jenpep [67, 68] ; and iedb [69] . implemented as a relational postgresql database, antijen integrates a wideranging set of data items, much of which is not stored by other databases. in addition to the kind of cellular immunological information familiar from syfpeithi, such as mhc binding and t cell data, antijen additionally archives b cell epitopes and also includes a significant stockpile of quantitative data: kinetic, thermodynamic, as well as functional, including measurements of immunological peptide-protein and protein-protein interactions. the iedb database is considerably more extensive than other equivalent database systems, benefiting from the input of 13 dedicated epitope sequencing projects. iedb has come to eclipse other work in this area. although both antijen and iedb are full of epitope-focussed information of many flavours, they remain incomplete concerning immunogenic antigens. fortuitously, specific antigen-orientated-rather than epitope-focusseddatabases are starting to be available. arguably, the most obvious and most unambiguous example of an antigen is virulence factor (vf): proteins, such as toxins, able to induce disease directly by attacking a host. analysis of known pathogens has allowed recurring vf systems of 40+ distinct proteins. often, sets of vfs exist as discrete, distinct genome-encoded pais, as well as being more widely spread through the genome. clearly, antigens do not need to be vfs in order to be immunogenic and thus candidates for subunit vaccines. instead, they need only be accessible to the immune system. they do not need to directly or indirectly mediate infection. thus, other databases are needed which capture, collate, and archive the burgeoning plethora of antigen-orientated data. recently, we have helped developed a very different database: antigendb [70] . it contains over 500 antigens collated from the primary scientific literature, as well as other sources. another related database system has been christened violin (vaccine investigation and online information network) [71] , which allows straightforward curation and the analysis and comparison of research data across diverse pathogens in the context of human medicine, animal models, laboratory model systems, and natural hosts. as we outline above, in addition to identifying sequence similarity to known antigens, another idea gaining ground is that the immunogenicity of an antigen is solely determined by the absence of similarity to host proteins. some think this is the prime determinant of potential protein immunogenicity [72, 73] . such ideas are supported by the belief that immune systems are actively educated to lack reactivity to self-proteins [74] , a process-often termed "immune tolerance"-which is generated via epitope-specific mechanisms [75, 76] . what we really want is a meaningful measure of the "foreignness" of a protein correlating with its immunogenicity. usually, "evolutionary distance" substitutes for "foreignness." clearly, such an evolutionary distance must be specified in terms of biomacromolecular structures or sequences. but, is this practically useful for selecting candidate vaccines? another way to formulate this idea is to say that the probability that a protein is immunogenic is exclusively a product of its dissimilarity, at the whole-sequence or sequence-fragment level, to each and every protein contained within the host proteome. most search software is well matched to this problem. in terms of fragment length, the typical length of an epitope might seem logical, since the epitope is the molecular moiety typically recognised during the initial phase of an immune response. yet, even at the epitope level-say a peptide of 8-16 amino acid residues-even a single conservative mutation or mismatch in an otherwise identical match might prove significant. single sequence alterations may totally abrogate or significantly enhance neutralising antibodies binding or recognition by the machinery of cellular immunology. we have attempted to benchmark sequence similarity and correlate it with immunogenicity in order to explore the potential of this idea in a quantitative fashion. to that end, we examined the differences between sets of antigens and non-antigen using sequence similarity scores. we looked specifically at sets of 100 known non-antigenic and 100 antigenic protein sequences from six sources: bacteria, viruses, fungi, and parasites, as well as allergens and tumours [77] [78] [79] , comparing pathogen sequence to those from humans and mice using blast [80] . most non-antigenic and antigenic sequences were non-redundant; implying a lack of homologues between pathogens and host proteomes, although certain parasite antigens, such as catalases and heat shock proteins, had a much greater level of similarity. we were not able to determine a suitable and appropriate threshold based on the hypothesis of non-redundancy to the host's proteome, suggesting that this is not a viable solution to vaccine antigen identification. however, rather than looking at nucleic acid sequences, or at protein sequences using an alignment-based approach, a new set of techniques, based upon alignmentfree techniques, has been and is being developed; as this approach begins to show significant potential, we shall examine it next. proteins accessible to immune system surveillance are assumed to lie external to the microbial organism or be attached to its surface rather than being sequestered and sequestrated within the cell. for bacteria, this means being located on-or in-the outer membrane surface or being secreted. thus, being able to accurately predict the physical location of a putative antigen can provide considerable insight into the likelihood that a particular protein will prove to be an immunogenic and possibly protective. there are two basic kinds of prediction method for identifying subcellular location: manual rule construction and the application of data-driven machine learning methods. data used to discriminate between compartments include sequence-derived features of the protein, such as hydrophobic regions; the amino acid composition of the whole protein; the presence of certain specific motifs; or a combination thereof. accuracy differs significantly between different methods and different compartments, mostly resulting from the deficiency and inconsistency of data used to derive models. gross overall sequence similarity is unable to predict protein sub-cellular location reliably or accurately. even nearly identical protein sequences may be found in distinct locations, while there are many proteins which exist simultaneously at several distinct locations within the cell, often having equally distinct functions at these different sites [81] . eukaryotes and prokaryotes have quite distinct subcellular compartments. the number of such compartments used in prediction studies varies. a common schema reduces prokaryotic to three compartments (cytoplasmic, periplasmic, and extracellular) and eukaryotic cells to four compartments (nuclear, cytoplasmic, mitochondrial, and extracellular). other structural classifications evince in excess ten eukaryotic compartments. ten compartments maybe a conservative estimate, such is the complex richness of sub-cellular structure. any prediction method must account for permanent, transient, and multiple locations, and, in addition, multi-protein complexes and membrane-bound organelles as possible sites. numerous signal sequences exist. several methods predict lipoproteins. the prediction of proteins translocated via the tat-dependent pathway is important but has yet to be addressed properly. however, amongst binary, single-outcome approaches, signalp is probably the most accurate and reliable method available. it uses neural networks to predict the presence and probable cleavage sites of type ii or n-terminal spase-i-cleaved secretion signal peptides [82] [83] [84] . this signal is common to both prokaryotic and eukaryotic organisms. signalp has recently been enhanced with a hmm intended to discriminate cleaved from uncleaved signal anchors. a limitation of signalp is its proclivity to over-predict: it cannot properly discriminate reliably between a number of very similar yet functionally different signal sequences, regularly predicting lipoproteins and integral membrane proteins as type ii signals. many methods have been devised capable of dividing a genome or virtualproteome between the various subcellular locations of a eukaryotic or prokaryotic cell. psort is a good example; it is a multicategory prediction procedure, comprising many different programmes [85] [86] [87] [88] . psort i predicts 17 subcellular compartments, while psort ii predicts ten different locations. ipsort deals with several compartments: chloroplast, mitochondrial, and proteins secreted from the cell, while psort-b focuses solely on predicting bacterial sub-cellular locations. another effective programme is hensbc [89] . hensbc can assign gene products to one of four different types (nuclear, mitochondrial, cytoplasmic, or extracellular) with an accuracy of about eight out of ten for gram-negative bacteria. another programme, subloc [90] , predicts prokaryotic subcellular location divided between three compartments. another programme is gpos-ploc [91] , which integrates several basic classifiers. other methods include phobius [92] , lipop 1.0 [93] , and tatp 1.0 [94] . a comparison of several such programmes, using 272 mycobacterial proteins as a gold standard [95] , showed subcellular localisation prediction and possessed high predictive specificity. we have developed a set of methods which predict bacterial subcellular location. using a set of methods for lipoprotein, tat secretion, and membrane protein prediction [96] [97] [98] [99] [100] [101] [102] , three different bayesian network architectures were implemented as software pipelines able to predict specific subcellular locations, and two serial implementations using a hierarchical decision structure, and a parallel implementation with a confidence-level-based decision engine [103] . the soluble-rooted serial pipeline performed better than the membrane-rooted predictor. the parallel pipeline outperformed the serial pipeline but was significantly less efficient. genomic test sets proved more ambiguous: the serial implementation identified 22 more of the 74 proteins of known location yet more accurate predictions are made overall by the parallel implementation. the implications of this work are clear. the complexity of subcellular structures must be integrated fully into sub-cellular location prediction. in extant studies, many important cellular organelles are not considered; different routes by which proteins can reach the same compartment are ignored; and proteins existing simultaneously at several locations are likewise discounted. clearly, combining high specificity predictors for each compartment appropriately must be the way forward [103] . many difficulties, problems, and quandaries persist; the most keenly felt is the lack of high-quality, verified, and validated datasets which unambiguously established the location of well-characterised proteins. this dearth is particularly serious for certain types of secreted protein, such as type iii secretion. in a similar manner, considerably more work is required to accurately predict the locations for proteins of viral origin; while certain studies are encouraging [104, 105] , the complexity of viral interaction with host organisms continues to confound attempts at analysis. predicting antigens in silico typically utilise bioinformatics tools. such tools can identify signal peptides or membrane proteins or lipoproteins successfully, yet the majority of algorithms tend to depend on motifs characteristic of antigens or, more generally, sequence alignment as the principal arbiter of definitive and meaningful sequence relationships. this is potentially a problem of some magnitude, particularly given the wide range of evolutionary rates and mechanisms amongst microbial proteins. certain protein families do not, however, show obvious or significant sequence similarity, despite having common biological properties, functions, and three-dimensional structures [106, 107] . thus alignment-based approaches may not always produce useful and unequivocal results, since they assume a direct sequence relationship that can be identified by simple sequence search techniques. immunogenicity, as a signature characteristic, may be encrypted within the structure and/or sequence instead. this may be encoded so cryptically or so subtlety as to completely confound or at least mislead conventional sequence alignment protocols. discovery of utterly novel and previously unknown antigens will be totally stymied by the absence of similarity to known antigenic proteins. alignment-dependent methods tend to dominate bioinformatics and, by extension, immunoinformatics. several authors have chosen to look at alternative strategies, implementing so-called alignment-independent or alignment-free techniques. the first authors to do so were mayer et al., who reported that protective antigens had a different amino acid composition compared to control groups of nonantigens [108] . such a result is unsurprising since it has long been known that the structure and sequence composition of proteins adapted to the different redox environments of different sub-cellular compartments [109] . mayer's analysis was formulated primarily in terms of univariate comparisons of antigens versus controls for different properties. subsequently, we explored bivariate comparison in terms of easily comprehensible scatter-plots. see fig. 3.3 for representative examples. what their results ably demonstrate is the potential for the discrimination of antigens and non-antigens by the appropriate selection of orthogonal descriptors. the challenge, of course, is to identify a robust choice of descriptors which are capable of extrapolating as well interpolating when used predictively. progressing beyond this type of analysis, and synergising with our other work on alignment-independent representation [110] [111] [112] [113] [114] , we have initiated the development of new methods to differentiate antigens-and thus potential vaccine candidates-and non-antigens, using more sophisticated alignment-free approach to sequence representation [115, 116] . rather than focus on epitope versus nonepitope, our approach utilises data on protective antigens derived from diverse pathogens to create statistical models capable of predicting whole-protein antigenicity. our alignment-independent method for antigen identification uses the auto cross covariance (acc) transformation originally devised by wold et al. [117, 118] to transform protein sequences into uniform vectors. the acc transform has found much application in peptide prediction and protein classification [119] [120] [121] [122] [123] [124] [125] [126] . in our method, amino acid residues are represented by the well-known and well-used z descriptors [127] [128] [129] , which characterise the hydrophobicity, molecular size, and polarity of residues. our method also accounts for the absence of complete independence between distinct sequence positions. we initially applied our approach to groups of known viral, bacterial, and tumour antigens, developing models capable of identifying antigen. extra models were subsequently added for fungal and parasite antigens. for bacterial, viral, and tumour antigens, models had prediction accuracies in the 70-89 % range [115, 116, 130] . for the parasite and fungal antigens, models had good predictive ability with 78-97 % accuracy. these models were incorporated into a server for protective antigen prediction called vaxijen [115] (url: http://www.darrenflower.info/ vaxijen). vaxijen is an imperfect but encouraging start; future research will yield significantly more insight as well-characterised protective antigens increase significantly in number [70] . as we have said, a number of bioinformatics problems are unique to the discipline of immunology: the greatest of these is the accurate quantitative prediction of immunogenicity. this chapter has in its totality been suffused and pervaded by the idea of immunogenicity and the challenge of predicting this property in silico. such an endeavour is confounding, yet exciting, and, as a key instrument in developing better, safer, more effective vaccines, is also of undisputed practical utility. successful immunogenicity prediction is at its simplest made manifest through the identification of b cell or t cell epitopes. epitope recognition, when seen as a chemical event, may be understood in terms of the relationships between apparent biological function or activity and basic physicochemical properties. delineating structure-activity or property-activity relationships of this kind is a key concern of immunoinformatics. at the other end of the spectrum, immunogenicity can be viewed is a cohesive, integrated, system property: a property of the entire and complete immune system and not a series of individual and isolated molecular recognition events. thus, the task of predicting systems-level immunogenicity is in all likelihood manifold more demanding than predicting peptide-binding say. the clinical manifestation of vaccine immunogenicity arises from the complex amalgam of many contributing extrinsic and intrinsic factors, which includes pathogen-side and host-side properties, as well as those just coming directly from proteins themselves. see fig. 3.2 . protein-side properties include the aggregation state of candidate vaccines and the possession of pamps. pathogen-side properties are clearly properties intrinsic to the pathogen, including expression levels of the antigen, the time-course of this expression, as well as its subcellular location. socalled host-side properties are innate recognition properties of host immunity, and most obviously include t cell epitopes or b cell epitopes. a bona fide candidate antigen should be available for immune surveillance and thus highly expressed, constitutively or transiently, as well as having several epitopes. a protein without immunogenicity would logically lack all or some of these characteristics. as a prediction problem, this is, to say the least, not uncomplicated; clearly consisting of a great variety of difficult-to-compute stages. in terms of mechanism, many of these stages are poorly understood. yet, each can be addressed using standard computational and statistical tools. they can all be predicted, however, presupposing, of course, the presence of relevant data in sufficient quantity. one of the strongest messages to emerge from this review is that immunogenicity is a strongly multi-factorial property: some protein antigens are immunogenic for one reason, or set of reasons, and other immunogenic proteins will be so for another possibly tangential reason or set of reasons. each such causal manifold is itself complex and potentially confusing. thus, the prediction of immunogenicity is a problem in multi-factorial prediction, and the search for new antigens is a search through a multi-factorial landscape of contingent causes and discombobulating decoys. some of the evidence will be highly precise and quantitative. the kind provided by predictive immunoinformatics, for example. this typically yields exact values for, say, the binding affinity of a peptide to a protein component of the immune system, or an unequivocal yes or no answer to the question: is this peptide sequence an epitope? however, for each such exact prediction, we have some notional associated probability concerning how reliable we regard this result. different methods evince a range of accuracy, which, in practice, equate to probabilities of reliability: we naturally have more confidence and assume a greater reliability for a highly accurate prediction versus one of average predictability, though it can still give wrong predictions and generally inaccurate predictors may work well for a specific subset of the data. other types of forms of evidence will have a distinctly more anecdotal flavour. take, for example, the case of bacterial exotoxins. together with endotoxins, such as lps, and so-called superantigens, exotoxins form the principal varieties of toxin secreted by pathogenic bacteria. exotoxins have evolved to be the most toxic substances known to science: in terms of the median lethal dose, botulinum toxin-the active ingredient of botox and causative agent of botulism, amongst others-is about ten times as lethal as radioactive isotope polonium-210 and a million times more deadly than mainline poisons, such as arsenic or potassium cyanide. virtually, all such potent bacterial exotoxins comprise two functionally distinct subunits, either separate proteins or distinct domains, usually denoted a and b. the a subunit is habitually an enzyme, such as a protease, which modifies specific protein targets, thus disrupting key cellular processes with host cells. the b subunit is a protein which binds to host cell surface lipids or proteins, enabling the toxin to be internalised efficiently. the high specificity of this dual action lends exotoxins much of their remarkable lethality. exotoxins are also extremely immunogenic, inducing the immune systems to produce high-affinity neutralising antibodies against them, and thus make excellent targets for vaccinology. a toxoid-a toxin which has been treated or inactivated, often by formaldehyde-is in essence a form of subunit vaccine and, as such, requires adjuvant to induce adequate immune responses. vaccines targeting tetanus and diphtheria, which usually need boosting every decade, are based on toxoids, albeit typically combined with pertussis toxin acting as an adjuvant. poisoning by exotoxins, on the other hand, requires treatment with antitoxin comprising preformed antibodies. however, and say that we were offered a newly sequenced pathogen genome, is such a classification for ab toxins helpful when trying to identify a potential exotoxins? the answer is neither yes nor is it no, but lies somewhere between these extremes. assuming we had extant knowledge or a reliable method predicting the presence of structural and functionally distinct domains, this very simple ruleof-thumb would become a useful tool for eliminating large numbers of possible toxin molecules. it would not directly identify an antigen but would enormously reduce the workload inherent in their discovery. as well as needing more and more reliable predictors, we also need a way of combining the information we gather from any set of reliable predictors to which we have access. thus, when analysing a pathogen genome, what we seem to need, at least in order to identify immunogenic proteins, is both a set of reliable and robust tools and a cohesive expert system within which to embed them. such systems, albeit still at a relatively crude and faltering level, do exist. because there is an implicit hierarchy of one prediction being based on others, there is a need to balance and judge different pieces of probabilistic evidence. an effective expert system should be capable of such a feat. to a first approximation, an expert system is a computer programme that undertakes tasks that might otherwise be prosecuted by a human expert ostensively by simulating the apparent judgement and behaviour of an individual or organization with expertise and experience within a particular discipline. an expert system might make financial forecasts, or play chess; it might diagnose human illnesses or schedule the routes of delivery vehicles. to create an expert system, one first needs to analyse human experts and how they make decisions, before translating this into rules that a computer can follow. such a system leverages both a knowledge base of accumulated expertise and a set of rules for applying such distilled knowledge to particular situations in order to solve problems. sophisticated expert systems can be updated with new knowledge and rules and can also learn from the success of its prediction, again mirroring the behaviour of properly performing experts. at the heart then of an expert system is the need to combine evidence in order to reach decisions. combining evidence, and reaching a decision based on that combined evidence, is no easier in the laboratory, be that virtual or actual, than it is in the court room. the problem of combining evidence is encountered across the disciplines, and various solutions have arisen in these different areas. within bioinformatic prediction, a particular variety of evidence combination, so-called meta-prediction, is a now a well-established strategy [131, 132] . this approach seeks to amalgamate the output of various predictors, typically internet servers, in an intelligent way so that the combined result is more accurate than any of those coming from a single predictor. indeed, combining results from multiple prediction tools does often increase overall accuracy. a consensus strategy was first proposed by mallios [133] , who combined syfpeithi [60, 61, 134] , propred [135, 136] , and the iterative stepwise discriminant analysis meta-algorithm [137] [138] [139] . multipred [140] integrates hmms and artificial neural networks (ann). six mhc class ii predictors were combined by dai and co-workers [141] [142] [143] basing its overall prediction on the probability distributions of the different scores. trost et al. have used a heuristic method to address class i peptide-mhc binding [144] . wang et al. [145] applied a consensus method to calculate the median rank of the top three predictive methods for each mhc class ii protein initially evaluated so as to rank all possible 8-, 9-, and 10-mers from one protein. this rank was used to identify the top 1 % of peptides from each protein. in probabilistic reasoning, or reasoning with uncertainty, there are many ways to represent espoused beliefs-or, in our domain, predictions-that effectively encode the uncertainty of propositions. these include fuzzy logic and the evidential method, among many others. for quantitative data, information fusion, in its various guises [146] , is one robust route to effective combination. another requires us to enter the world of bayesian statistics, or, at least, a special thread within it. bayes theory, and the ever-expanding strand of statistics devolving from it, is concerned primarily with updating or revising belief in the light of new evidence, while so-called dempster-shafer theory [147] is concerned not with the conditional probabilities of bayesian statistics but with the direct combination of evidence. it extends the bayesian theory of subjective probability, by replacing bayesian probabilities with belief functions that describe degrees of belief for one question in terms of probabilities for another and then combines these using dempster's rule for merging degrees of belief when based on independent lines of evidence. such belief functions may or may not have the mathematical properties of probabilities but are seemingly able to combine the rigor of probability theory with the flexibility of rule-based approaches. several expert systems of different flavours and hues have now become available within the vaccinology arena. sundaresh et al. developed a specialist software package for the analysis of microarray experiments that could easily be classified as an expert system and used it in the area of reverse vaccinology. this package, which was written in the open-source statistical package r, was used to help analyse a variety of complex microarray experiments on the bacteria f. tularensis, a category a bio-defense pathogen [148] . this programme implements a two-stage process for diagnostic analysis: selection of antigens based on significant immune responses coupled with differential expression analysis, followed by classification of measured antigen responses using a combination of k-means clustering, support vector machines, and k-nearest neighbours. we have already discussed vaxijen [115, 116, 130] , and the related server epijen [149] , which combines various methods for identifying epitopes within extant proteins. these two servers can also be classified as vaccine-related expert systems. nerve is another expert system, which has been developed to help automate aspects of reverse vaccinology [150] . using nerve, the prioritisation of potential candidate antigens consists of several stages: prediction of subcellular localisation; is the antigen an adhesion?; identification of membrane-crossing domains; and comparison to pathogen and human proteomes. candidates are filtered then ranked and putative antigens graded by provenance and its predicted immunogenicity. the web-based expert system, dynavacs [151] , was developed to facilitate the efficient design of dna vaccines and is available in the url: http://miracle. igib.res.in/dynavac. it takes a structured approach for vaccine design, leveraging various key design parameters, including the choice of appropriate expression vectors, safeguarding efficient expression through codon optimization, ensuring high levels of translation by adding specific sequence signals, and engineering of cpg motifs as adjuvant mechanisms exacerbating immune responses. it also allows restriction enzyme mapping, the design of primers, and lists vectors in use for known dna vaccines. vaxign is another expert system developed to help facilitate vaccine design [152] . vaxign undertakes dynamic vaccine target prediction from sequence. methodologically, it combines protein subcellular location prediction with prediction of transmembrane helices and adhesins, analysis of the conservation to human and/or mouse proteins with sequence exclusion from the genomes of nonpathogenic strains, and prediction of peptide binding to class i and class ii mhc. as a test, vaxign has been used to predict vaccine candidates against uropathogenic escherichia coli. however, nerve and its various and varied siblings are tasked with such a confounding and difficult undertaking that they are obliged to fall somewhat short of what is required. an obvious first step in tackling the greater problem is to address first subcellular location prediction. then, we can look at antigen presentation, modelling for each component step, before building these into a fully functional model. we can also develop empirical approaches-such as vaxijen [115, 116, 130] . we must also factor in antibody-mediated issues, properly address pamps, post translational danger signals, expression levels, the role of aggregation, and the capacity of molecular adjuvants to enhance the innate immunogenicity to usable levels. see fig. 3. 2. the value of vaccines is not yet unchallenged. however, most reasonable people would, in all probability, agree that they are a good thing, albeit with a few minor provisos. the idea underlying all vaccines is a strong and robust one: it is in the reification-that is, the realisation, manifestation, and instantiation-of this abstract concept that the trouble lies, if indeed trouble there is. existing vaccines are by no means perfect; again, most sensible and well-informed people would no doubt acknowledge this also. one might argue that their intrinsic complexity, and the highly empirical nature of their discovery over decades, and the fraught nature of their manufacture, has much to answer in this regard. why should this be? in part, it is due to the extreme complexity of immune response to an administered vaccine, which is largely specific to each individual or at least is different in different sub-groups within the totality of the vaccinated population. the immune responses is comprised, at least for whole-pathogen vaccines, of the adaptive immune response to multiple b cell and t cell epitopes as well as the responses made by the innate immune responses to diverse molecular structures, principally pamps. when one considers also the degree to which such a repertoire of responses is augmented and modified by the action of additives, be they designed to increase the durability and stability of vaccines or be they adjuvants, which are intended to raise the level of immune reactions. add in stochastic and coincidental phenomena, such as reversion to pathogenicity, and we can see immediately that navigating our way through the vaccine minefield is no easy task. all such problems engendered by this intrinsic complexity are themselves compounded by our comparatively weak understanding of immunological mechanisms, since, if we understood the mechanism of responses well enough, we could and would have designed our vaccines to circumvent these issues. part of the answer to this cacophony of conflicting and confounding quandaries is the newly emergent discipline of vaccinomics. a proper understanding of the relationships between gene variants and vaccine-specific immune responses may help us to design the next generation of personalised vaccines. vaccinomics addresses this issue directly. it seeks to identify genetic factors mediating or moderating vaccine-induced immune responses, which are known to be extremely variable within population. much data indicate that host genetic polymorphisms are key determinants of innate and adaptive response to vaccination. hla genes, non-hla genes, and genes of the innate immunity all contribute, and do so in many ways, to the variation observed between individuals for immune responses to microbial vaccines. vaccinomics offers many techniques that can help illuminate these diverse phenomena. principal amongst these are population-based gene/snp association studies between allele or snp variation and specific responses, supplemented by the application of next-generation sequencing technology and microarray approaches. yet, and for all this nay-saying and gainsaying, vaccines and vaccination have demonstrated their worth time after time; yet, to justify the continuing faith we invest in them, new and better ways of making safer and more focussed vaccines must be found. most current vaccines work via antibody-mediated mechanisms; and most target viruses and the diseases they cause. unfortunately, the stock of such disease targets is dwindling. low-hanging fruit has long since been cut down. only fruit that is well out of reach remains. vaccines based on apcs and peptides are new but unproven strategies; most modern vaccine development relies instead on effective searches for vaccine antigens. one of the clearest points to emerge from such work is that there are many competing concepts, thoughts, and ideas that may confound or help efficient identification of immune reactive proteins. certain such ideas we have outlined. some are indisputably persuasive, even compelling, yet many strategies-and the technical approaches upon which they are based-have singly failed to deliver on their promise. long ago, and based on his lifetime's experience of all things immunological, professor peter cl beverley sketched out a paradigm for protein-focussed vaccine development, which we have formalised further, and which schema is summarised in fig. 3.4 . some of his factors overlap with the factors from fig. 3 .2. he identified many of the factors that potentially contribute to the immunogenicity of proteins, be they of pathogen origin or another source entirely, and also other features which might make proteins particularly suitable for becoming candidate vaccines. of these, some are as-yet beyond prediction, such as the attractiveness for apcs or the inability to down-regulate immune responses. the status of proteins as evasins is currently only possibly addressable through sequence similarity-based approaches and likewise for the attractiveness for uptake by apcs is again, though possible there exist motifs, structural or sequence, which could be identified. currently, the dearth of relevant data precludes prediction of such properties; and, while it is possible to predict some of these properties with some assurance of success, and others are predictable but only incidentally, overall, we are still some way from realising the dream embodied in fig. 3 failure occurs for simple reasons: we deal with simplified abstractions and cannot hope to capture all that which is required for prediction by looking superficially at a single factor. protein immunogenicity comes instead from the dynamic combination of innumerable contributing factors. this is by no means a facile or easily solved informatics conundrum. a vaccine candidate should have epitopes that the host recognises, be available for immune surveillance, and be highly expressed. factors mediating protein immunogenicity are many; possession of b or t cell epitopes, post-translational danger signals, sub-cellular location, protein expression levels, and aggregation state amongst them. predicting such diverse, complex, confounding properties is-and remains-a challenge. vaccine antigens, once discovered, should, ultimately, and with appropriate manipulation, together with an apt, apposite, and appropriate delivery system and the right choice of adjuvant, become first a candidate for clinical trials, before, hopefully, progressing to regulatory approval. we require an integrative, systemsbiology approach to solve this problem. no single approach can be applied universally and with success; what we crave is the full integration of numerous equally wakefield's article linking mmr vaccine and 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in numbers: achieving greater accuracy in mhc-i binding prediction by combining the results from multiple prediction tools a systematic assessment of mhc class ii peptide binding predictions and evaluation of a consensus approach combination of fingerprint-based similarity coefficients using data fusion connectionist-based dempster-shafer evidential reasoning for data fusion from protein microarrays to diagnostic antigen discovery: a study of the pathogen francisella tularensis epijen: a server for multistep t cell epitope prediction nerve: new enhanced reverse vaccinology environment dynavacs: an integrative tool for optimized dna vaccine design vaxign: the first web-based vaccine design program for reverse vaccinology and applications for vaccine development enzymes, metabolites and fluxes key: cord-301128-woe6knpv authors: joyeux, marc title: requirements for dna-bridging proteins to act as topological barriers of the bacterial genome date: 2020-08-12 journal: biophys j doi: 10.1016/j.bpj.2020.08.004 sha: doc_id: 301128 cord_uid: woe6knpv abstract bacterial genomes have been shown to be partitioned into several kilobases long chromosomal domains that are topologically independent from each other, meaning that change of dna superhelicity in one domain does not propagate to neighbors. both in vivo and in vitro experiments have been performed to question the nature of the topological barriers at play, leading to several predictions on possible molecular actors. here, we address the question of topological barriers using polymer models of supercoiled dna chains that are constrained such as to mimic the action of predicted molecular actors. more specifically, we determine under which conditions dna-bridging proteins may act as topological barriers. to this end, we developed a coarse-grained bead-and-spring model and investigated its properties through brownian dynamics simulations. as a result, we find that dna-bridging proteins must exert rather strong constraints on their binding sites: they must block the diffusion of the excess of twist through the two binding sites on the dna molecule and, simultaneously, prevent the rotation of one dna segment relative to the other one. importantly, not all dna-bridging proteins satisfy this second condition. for example, single bridges formed by proteins that bind dna non-specifically, like h-ns dimers, are expected to fail with this respect. our findings might also explain, in the case of specific dna-bridging proteins like laci, why multiple bridges are required to create stable independent topological domains. strikingly, when the relative rotation of the dna segments is not prevented, relaxation results in complex intrication of the two domains. moreover, while the value of the torsional stress in each domain may vary, their differential is preserved. our work also predicts that nucleoid associated proteins known to wrap dna must form higher protein-dna complexes to efficiently work as topological barriers. the genetic information of most bacteria is encoded in a circular dna molecule comprising up to several millions of base pairs (bp). such closed molecules are topologically constrained, because they lack free ends capable of rotating and releasing the torsional stress (1). in vivo alterations of the torsional state of circular dna is mediated by the recruitment of enzymes called topoisomerases, which can either increase or decrease the twist of the double helix by opening transiently one or two strands (1). the net result of the action of all types of topoisomerases is that the genomic dna of most bacteria is significantly undertwisted (negatively supercoiled) and winds about itself to transfer part of the torsional stress to the bending degrees of freedom, thereby forming plectonemes (1). if the dna were not subject to any additional constraint beyond closure, then one single nick of one strand or one single break of the two strands would suffice to release the torsional stress of the full molecule. it has however long be known that at least several tens of nicks are required to achieve this goal in escherichia coli (2, 3) . this indicates that there exist barriers which block the diffusion of the torsional stress along the dna molecule and organize the chromosome of e. coli into many independent topological domains, whose torsional state is not affected by the relaxation of other domains. it is currently estimated that the genomic dna of bacteria like e. coli is composed of several hundreds of different topological domains with variable size (average size ≈10 kbp) and position (4) . more generally, the partitioning of the bacterial chromosome into ≈10 kbp independent topological domains is believed to hold in most bacteria (5) and to be related, in part, to the insulation of fundamental co-expression units that are neighbors along the genome (6) . the work reported here deals with the topological barriers which make this partitioning possible, that is, the barriers which block the diffusion of torsional stress along the dna molecule and are responsible for its division into independent topological domains. although this question has now been addressed for nearly two decades (7) (8) (9) , the mechanisms underlying the formation of topological barriers remain mostly elusive. most plausible models for the formation of topological barriers are (i) actively transcribing rnap, which generate both positive and negative supercoils (10) and may consequently block the displacement and dissipation of plectonemes (11, 12) , and (ii) formation of dna loops by certain nucleoid proteins, which may serve as topological barriers (13-16). in the present paper, we focus on this latter mechanism and establish under which conditions dna-bridging proteins may serve as topological barriers. several dna-bridging proteins have been studied in some detail, including transcription regulators like the laci repressor (17) , h-ns (18) and h-ns-like proteins (19) , lsr2 (20) , fis (21) and lrp (22) . in their functional form, all of these proteins have at least two independent dna-binding domains, so that they can interact with two dna duplexes simultaneously and form a bridge between two sites that are widely separated from the genomic point of view. most of these proteins also bind non-specifically but with high affinity to the dna molecule. however, among all these dna-bridging proteins, laci is to date the only one that has been proved to work as a topological barrier (15, 23, 24) . the guiding line of the present paper is consequently the experimental demonstration in (15) that the binding of a dna-binding protein to its recognition sites in two different locations on a supercoiled dna molecule can confine free supercoils to a defined region and divide the dna molecule into two distinct topological domains (15) . in order to gain more detailed information on this mechanism, we determined the minimal physical properties that must be added to a standard model of circular dna to reproduce the results described in (15) . the starting model for this study was similar to those we proposed recently to investigate facilitated diffusion (25) (26) (27) , the interactions of dna and h-ns nucleoid proteins (28) (29) (30) , the formation of the bacterial nucleoid (31) (32) (33) (34) (35) , and the interplay of dna demixing and supercoiling in compacting the nucleoid (36) . the results presented in this article reveal that the formation of dna loops is by no means sufficient to create topologically independent domains and that dna-bridging proteins must exert rather strong constraints on their binding sites in order to act as topological barriers: they must block the diffusion of the excess of twist through both binding sites and must additionally block the rotation of one dna segment relative to the other one. the coarse-grained bead-and-spring model developed for the present study is described in detail in model and simulations in the supporting material. in brief, bacterial dna is modeled, as in (30, 36) , as circular chains of n beads with radius 0 . as in (31, 36) , the torsional energy term was borrowed from (37) and requires the introduction of a body-fixed frame ( , , ) k k k u f v , where k u denotes the unit vector pointing from bead k to bead 1 k + . the torsional rigidity opposing rotation of ( , , ) ) was adjusted so that at equilibrium the writhe contribution accounts for approximately 70% of the linking number difference (38) , as is illustrated in fig. s1 . the values of the bending and torsional rigidities are close together, in agreement with experimental results (38) . it may be worth emphasizing that introduction of the body-fixed frame ( , , ) k k k u f v is crucial for modeling correctly the torsion of double-stranded dna with a "single-stranded" bead-and-spring model. this procedure is quite realistic and has already proved very useful for studying, for example, the interplay of dna demixing and supercoiling in nucleoid compaction (36) , the buckling transition in double-stranded dna and rna (39) , the influence of nucleoid-associated proteins on dna supercoiling (40) as a starting point, we recall the experimental results in (15) , where the torsional relaxation properties of 4100 bp plasmids with initial superhelical density 0.06 of special interest to us were the experiments performed with plasmids containing tandem copies of the binding site of the sequencespecific dna-binding protein laci at two different locations, such that the plasmid was divided into two stable loops of respective length 2900 bp and 1200 bp upon binding of laci. the 1200 bp region moreover contained the recognition site for one nicking enzyme (nt.bbvc1) and the 2900 bp region the recognition site for a second nicking enzyme (nb.btsi). in the absence of laci, addition of nt.bbvc1 alone or nb.btsi alone was sufficient to release the full torsional stress of the plasmid. in contrast, in the presence of laci, addition of nt.bbvc1 alone removed only the 7 negative supercoils of the 1200 bp region, while addition of nb.btsi alone removed only the 17 negative supercoils of the 2900 bp region (15) . simultaneous addition of both enzymes was required to relax the full plasmid (15) . this experiment demonstrates very clearly that pairs of laci bridges block the diffusion of torsional stress and divide the plasmids into two independent topological domains. we report below on our efforts to determine the minimal set of mandatory properties that allow molecular bridges to act as topological barriers and block the diffusion of torsional stress in out-of-equilibrium supercoiled chains. preparation of torsionally out-of-equilibrium circular chains. , which were chosen so that the distance between their centers is smaller than 10 nm. this step is illustrated in fig. 1 (a), which shows an equilibrated chain with where 12 r w quantifies the winding of one moiety of the chain around the other one, that is, loosely speaking, their intrication. the out-of-equilibrium chains prepared as described above the main purpose of this work is to understand how dna-bridging proteins like laci (15, 23, 24) can maintain such an unbalance when the whole chain is relaxed. since dnabridging proteins form dna loops by dynamically cross-linking widely separated dna sites, the question amounts here to determine the constraints that must be imposed to beads α and β j o u r n a l p r e -p r o o f to divide the circular dna chain into two topologically independent loops. note that dnabridging proteins are not introduced explicitly in the model: rather, constraints on beads α and β are meant to model the mechanisms that allow the proteins to act as topological barriers. twist equilibrates much more rapidly than writhe. in order to understand in more detail how the diffusion of torsional stress proceeds, the out-of-equilibrium circular chains prepared as described above were allowed to relax without constraint and all relevant quantities were monitored. from a practical point of view, four different initial out-of-equilibrium conformations were prepared for each value of 0 k l ∆ and 100 different relaxation trajectories were integrated for 1 ms for each initial conformation. , and r j w ( 1 j = , 2) was then averaged over these 100 trajectories. in the absence of any constraint on beads α and β, all conformations obtained at the end of the 1 ms integration time satisfy where w based on recent experimental results (12) (13) (14) (15) (16) , one may wonder whether dna loops are ipso facto independent topological domains, or whether some additional, more subtle properties of dna-bridging proteins are required to achieve this goal. we emphasize that the word "loop" is used here according to its biological meaning and not the geometrical one. more precisely, it does not refer to a closed curve, but rather to a conformation of the dna chain, where two dna sites that are widely separated from the genomic point of view are maintained close to each other by some other macromolecule, so that the segment of dna located between these two sites looks like a loop when it is observed from a long distance or projected on a plane. however, the chain is not closed and torsional stress cannot diffuse directly from one extremity of the loop to the other one through the bridging macromolecule. in order to ascertain this point, we launched a series of simulations with the crudest model for dna-bridging proteins, namely a simple bond between beads α and β. the , where pot e is the energy of the naked circular dna chain described in eqs. (s1)-(s5) and bp e an additional term, which models the action of dna-bridging proteins. for the simplest model, we used where d αβ is the distance between the centers of beads α and β and 0 4 d αβ = nm. for the sake of simplicity, we used the same value of the stretching rigidity as for the dna chain, that is topological barriers is consequently related to some property, which is subtler than the mere connection between two genetically widely separated dna sites. still, it is worth noting that the αβ bond has a significant effect on the time evolution of 12 r w , as can be checked in fig. s5 . the reason is the following. in order to compute the partial writhes r j w (see the supporting material), one has to introduce two "virtual" closed chains 1 c and 2 c . 1 c is composed of beads α to 1 β − , plus the "unphysical" segment between beads β and α, while 2 c is composed of beads β to 1 α − , plus the same "unphysical" segment between beads α and β. when beads α and β are bonded, they remain close to each other for 0 t > , as indicated by the positions of the two arrows in fig. 3(a) , so that the unphysical segment between beads α and β remains small and 1 c is a good representation of the initially torsionally relaxed dna loop (and 1 r w is a measure of its writhe), 2 c is a good representation of the initially torsionally stressed dna loop (and 2 r w is a measure of its writhe), and 12 r w is a measure of the intrication of the two loops. in this case, 12 r w remains close to zero at all times, as can be checked in fig. s5(b) . this indicates that, for bridged dna, equilibration of the torsional stress is not accompanied by any significant intrication of the two circular moieties. in contrast, for the system without the bp e term, beads α and β do not remain close to each other for 0 t > , as indicated by the positions of the two arrows in fig. 1(c) , so that the unphysical segment between beads α and β becomes arbitrarily large and 1 c , 2 c , the r j w and 12 r w lose their physical meaning. in this case 12 r w varies quite widely with changing dna conformations, as is illustrated in fig. s5 the reason why forming a loop is not sufficient to create independent topological domains is of course that the bond between beads α and β described in the previous subsection is not able to block the rapid diffusion of the excess of twist through beads α and β and the subsequent equilibration of the writhe. in order to form independent topological domains, the diffusion of the excess of twist must absolutely be blocked. stated in other words, rotation of the internal basis ( , , ) k k k u f v around k u must be forbidden at , from for , we note that the k δ remain small, and the corrections in eq. (5) where α ξ denotes the angle formed by beads β, α and 1 α + , and β ξ the angle formed by beads α, β and 1 β + . note that the bending rigidity for these two angles was assumed to be five time larger than the bending rigidity of the dna chain, in order for α ξ and β ξ to deviate only moderately from / 2 π . in this second model, the action of dna-bridging proteins is consequently modeled by the potential energy terms in eq. fig. 4(b) . it is stressed that this behavior has no precise physical meaning but arises instead from the mathematical definition of the total writhe r w in eq. (s11) and its somewhat arbitrary partitioning into the sum of the two partial quantities r j w and 12 r w in eq. (s17). in particular, r w does not display any sharp jump along single trajectories. as can be checked in fig. 4 fig. 4(b) ). according to eq. (1), this limit is equivalent to 12 r r w w = . ) superimpose at short times, which indicates that the relaxation speed is proportional to the total amount of torsional stress. however, for longer times and/or larger values of the linking number difference, a slower regime sets in, which is probably due to the resistance opposed by plectonemes against the reorganization of the chain and the intrication of the two moieties. finally, we note that after full relaxation the writhe contribution accounts for approximately 90% of the linking number difference, which is significantly larger than the 70% contribution in the absence of domains (see fig. s1 ). in conclusion, this second model of dna-bridging proteins indicates that blocking the diffusion of the excess of twist at the two dna loci bound by the proteins is sufficient to maintain a constant difference between the linking number difference, excess of twist, and writhe of the two moieties, but that it is not sufficient to insure true topological independence. indeed, the dna chain evolves towards very compact and intricated final conformations, where the topology of each moiety is different from the initial one. the relaxation limit 12 r r w w = (or equivalently 1 2 r r 0 w w + = ) appears non trivial and intriguing to us. we suspect that the explanation involves subtle considerations that should deserve further work. the question that remains to be answered is consequently: what additional constraint must the dna-bridging protein exert on its binding sites in order to preserve not only the difference in torsional stress between the two moieties of the dna chain, but rather the precise initial torsional stress in each moiety ? the feeling that emerges upon examination of conformations like the one shown in fig. 3(b) , is that the winding of the initially relaxed moiety around the initially stressed one probably requires the relative rotation of the two dna segments around the αβ bond, and that no winding would be possible if this rotation were blocked. in order to check this conjecture, a third bending term was added to bp e , namely 0 2 2 2 0 2 bp , , 16) . in this context, we surmise that the slow relaxation observed in (15) for single dna-bridges may correspond to the slow intrication obtained with our second model for dna-bridging proteins, while the more stable topological separation observed in (15) for bridges in tandem corresponds to true barriers obtained with the third model for dna-bridging proteins. we finally note that the intrication properties discussed here are also expected to contribute to the formation of topological domains that are, in effect, larger than the domain delineated by the binding sites of laci as recently observed in (16) . dna-bridging proteins form dna loops by dynamically cross-linking dna sites that are widely separated from the genomic point of view (17) (18) (19) (20) (21) (22) . they usually bind dna nonspecifically but with high affinity. dna-bridging proteins have now been investigated for more than two decades, but it is only very recently that it has been demonstrated that a dnabridging protein like the laci repressor is able to separate circular plasmids into two topologically independent loops (15, 23, 24) . to the best of our knowledge, this is at present the only demonstrated case of a dna-bridging protein acting as a topological barrier. in the present work, we aimed at going one step further and determining the conditions that dnabridging proteins must fulfill to act as topological barriers. we showed that these proteins this work also brings some light on another mechanism that has been proposed to explain the formation of independent topological domains, namely two proteins wrapping the dna in two different locations (ref. (15) , fig. 6 ). there is experimental evidence that certain wrapping proteins, like galr (15), the λ o protein (15) , and fis (62) another model which is frequently proposed for the formation of topological barriers, namely actively transcribing rnap (10) (11) (12) , is more difficult to discuss in terms of the results described in this paper, because several time scales need be considered. indeed, it is likely that the two parts of the dna molecule located respectively upstream and downstream from the transcribing rnap will tend to increase their intricacy, in order to reduce the torsional stress induced by the blocking of the diffusion of twist at the rnap. however, the rnap is itself moving forward along the dna, so that the ability of transcribing rnap to act as topological barriers probably depends on the relative speeds of the two mechanisms. more work is needed to clarify this point. similarly, the case of dna-bending nucleoid proteins, like hu (63), certainly deserves further investigations. finally, we note with interest that the intricate structure obtained with dna-bridging proteins that block the diffusion of twist along the dna but do not prevent the relative rotation of the dna segments are highly compact. it turns out that the mechanism leading to the formation of the bacterial nucleoid is a longstanding but still lively debated question (31, 32, (64) (65) (66) (67) . the point that has puzzled scientists for decades is that the volume of the fig. 1(b) under the constraint that beads α and β are bonded (eq. (3)). (b) representative snapshot obtained upon relaxation of the out-of equilibrium conformation shown in fig. 1(b) for the second model of dna-bridging proteins (eqs. (5) and (6)). (c) representative snapshot obtained upon relaxation of the out-of equilibrium conformation shown in fig. 1(b) for the third model of dna-bridging proteins (eqs. (5) and (7)). the imagined the research project and performed preliminary simulations. m.j. developed the models, ran the simulations and analyzed the results. m.j. and i.j. wrote the manuscript université lumière lyon 2) for her hospitality during the covid-19 epidemic confinement dna topology on the structure of the folded chromosome of escherichia coli chromosomes in living escherichia coli cells are segregated into domains of supercoiling topological domain structure of the escherichia coli chromosome universal and idiosyncratic characteristic lengths in bacterial genomes conserved units of co-expression in bacterial genomes: an evolutionary insight into transcriptional regulation the structure and function of the bacterial chromosome topological insulators inhibit diffusion of transcription-induced positive supercoils in the chromosome of escherichia coli supercoiling of the dna template during transcription transcription-induced barriers to supercoil diffusion in the salmonella 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cord_uid: ucguzgdm we have investigated the localization of kex1p, a type i transmembrane carboxypeptidase involved in precursor processing within the yeast secretory pathway. indirect immunofluorescence demonstrated the presence of kex1p in a punctate organelle resembling the yeast golgi apparatus as identified by kex2p and sec7p (franzusoff, a., k. redding, j. crosby, r. s. fuller, and r. schekman. 1991. j. cell biol. 112:2737). glycosylation studies of kex1p were consistent with a golgi location, as kex1p was progressively n-glycosylated in an mnn1dependent manner. to address the basis of kex1p targeting to the golgi apparatus, we examined the cellular location of a series of carboxyterminal truncations of the protein. the results indicate that a cytoplasmically exposed carboxy-terminal domain is required for retention of this membrane protein within the golgi apparatus. deletions of the retention region or overproduction of wild-type kex1p led to mislocalization of kex1p to the vacuolar membrane. this unexpected finding is discussed in terms of models involving either the vacuole as a default destination for membrane proteins, or by endocytosis to the vacuole following their default localization to the plasma membrane. s ~.crea'eo biologically active proteins and peptides are classically produced as precursors which undergo both endo-and exoproteolytic processing to release the mature species while traversing the secretory pathway. the kex/gene product (kexlp) is a carboxypeptidase, specific for basic amino acid residues, and is responsible for processing proteins secreted by saccharomyces cerevisiae (dmochowska et al., 1987; cooper and bussey, 1989) . in conjunction with the proteases kex2p and dipeptidyl aminopeptidase a (dpap a) ~ (stel3p), kexlp proteolytically matures proteins such as c~-factor and k1 killer toxin from their precursors (for reviews see bussey, 1988; fuller et al., 1988) . proteins that enter the secretory pathway are thought to be transported to the cell surface by default via a "bulk flow" mechanism unless they contain additional targeting information (pfeffer and rothman, 1987; rothman, 1987; wieland et al., 1987; karrenbauer et al., 1990) . such targeting information is found in soluble proteins resident in the er which maintain their localization by containing a retention signal at their carboxy termini (munro and pelham, 1987; pelham et al., 1988) . deletion of the retention signal results in secretion of the soluble er resident proteins to the cell surface. soluble proteins destined for the mammalian lysosome re-a. cooper's present address is the institute of molecular biology, university of oregon, eugene, or 97403. 1. abbreviation used in this paper: dpap, dipeptidyl aminopeptidase. 3158 ceive a mannose-6-phosphate signal which targets them to the lysosome in a receptor-mediated manner. the absence of the mannose-6-phosphate signal from these soluble proteins results in their secretion (for review see kornfield and mellman, 1989) . secretion also occurs when the targeting signals for yeast-soluble proteins destined for the lysosomelike vacuole are altered (vails et al., 1987; johnson et al., 1987; klionsky et al., 1988) . in addition to soluble proteins, the cell surface appears to be the default destination for mammalian er, golgi, and lysosomal membrane proteins as removal of their respective retention/targeting signals results in their delivery to the plasma membrane (machamer and rose, 1987; jackson et al., 1990; williams and fukuda, 1990) . as yet, no targeting or retention signals have been definitively assigned to yeast golgi or vacuolar membrane proteins, nor is it known where these proteins are delivered upon perturbation of their targeting signals. kexlp is predicted to be a type i transmembrane protein with a large amino-terminal protease domain in the lumen of the secretory pathway, a single membrane-spanning domain, and a smaller carboxy-terminai domain positioned cytoplasmicauy. the observation that kex/cells intracellularly retain kexlp activity prompted an analysis to determine in which secretory compartment kexlp resided, and how it achieved such retention. kexlp was found to be localized to the yeast golgi apparatus, with retention mediated via the cytoplasmic domain of the protein. removal of this domain or overproduction of wild-type kexlp resulted in delivery of the protein to the vacuole rather than to the cell surface. this surprising result raises the possibility that the vacuole is the default destination for yeast golgi membrane proteins. escherichia coli strains and associated dna manipulations were as described previously (cooper and bussey, 1989) . saccharomyces cerevisiae strains c13abys86 ($86), sc25k, and sc25k-13 (sc25k + kex/-a/) have been described previously (cooper and bussey, 1989) . other strains used were $6 (a strain sensitive to k1 killer toxin), tci06cr (mata leu2 his1 trpl ura3 ), seys016a (secl-1 leu2 ura3 gal20), xcy42-30d (mata ade2-10l ade x [a putative second ade mutation that results in white colonies] urn3 trpl lys2 leu2-3,h2 amnnl::leu2), and lb2134-3b (mata mnng), mnn mutants were crossed with either sc25 or sc25-jh (isogenic to sc25k except mata). the diploids were sporulated, asci dissected, and spores containing the relevant mutations selected. the resulting strains were hab595 (mnn/ura3), hab596 (ura3), and hab597 (mnn9 ura3). growth media, yeast transformation, and gane disruption have all been described previously (cooper and bussey, 1989) . restriction endonucleases, t4 dna polymerase, t4 dna ligase, and klenow fragment were purchased from either bethesda research laboratories, (gaithersburg, md) or new england biolabs (beverly, ma), and were used as recommended by the suppliers. unless otherwise stated, reagents used in these experiments were obtained from sigma chem. co., st. louis, mo. disruption of the kex/locus with the ura3-based construct pl6 resulted in the allele kexl-a2. an alternative disruption using the leu2-containing plasmid p17 produced the allele kex/-a3, p17 was constructed by digesting pl6 (dmochowska et ai., 1987) with ecorv to remove the ura3 gene and a portion of the kex/gene, and then replacing it with an hpai fragment carrying the leu2 gene. p17 was cleaved with hindiii and used to transform the strains $86, sc25, . transformants were initially screened for a kex-phenotype on the basis of the abolition of a k1 toxin killing zone for the transformants of sc25k and tc106a, or on the basis of a smaller cr zone for $86 transformants. disruption of the kex/locus was confirmed by southern blot analysis (not shown). mutagenesis of the carboxy-terminal region of kexi was performed on the same single-stranded dna template and in the same manner as described (cooper and bussey, 1989) . all of the mutations were confirmed by dna sequencing (sequenase, united states biochem. corp., cleveland, oh). fig. 3 shows the extent of each of the mutations listed below. plasmid pkx1-18ams contains kex./with a precise deletion of the membrane-spanning domain (ams) inserted into the multicopy plasmid pvt103-l (cooper and bussey, 1989) . the aberrant stop mutation (as) was identified while sequencing potential ams clones. the as clone contained a mutation at both bp1823 and bp1824, introducing a premature stop codon at codon 608. the hpa mutation was to introduce an arginine residue immediately after the membranespanning domain followed by a termination codon using the mutagenic oligonucleotide 5' ggagtttatgcgtatcgttaacgatagaagagtga 3'. to aid in identifying mutant clones an hpai site was introduced by the mntagenesis. all derivatives of the hpa mutagenesis were subsequently sequenced and found to have undergone a 2-bp deletion (a1908-1909 bp) upstream from the inserted termination codon resulting in the mutation shown in fig. 3 . the bcl mutation introduced a bcli cleavage site (5' gtttat-gcgtatgattgatcagtgaggagaaaag 31 immediately after the aspartate residue (codon no. 637) which borders the carboxy-terminal portion of the membrane-spanning domain. the hinc mutation introduced a termination codon 20 amino acid residues carboxy-terminal to the membrane-spanning domain using the mutagenic oligonucleotide 5' ccaaat-aatagttaacatgacagt 3'. to aid in identifying mutant clones an hincli site was introduced by the mutagenesis. introduction of the various kex/mutations into pvti03-l produced the following plasmids: pkxi-18as, pkxl-18hpa, pkxi-18bel, and pkxl-18hinc. a 3.1-kb hindlh fragment containing the kex/gene was inserted into ycps0, which had been digested with hindlii, to give the plasmid pkxi-20wt. the 1.4-kb xhoi-hindlh fragment of pkxi-15 containing the various kex/carboxy-terminal truncation mutations was isolated and ligated with the 1.7-kb hindlii-xhoi fragment containing the amino-terminal portion of kex/to reassemble the gene, and then inserted into hindlh-digested ycp50 to create pkx1-20as, pkx1-20ams, pkxi-20hpa, and pkx1-20hint. pad81 (dmochowska et al., 1987) , which contained the kex/ gene, was digested with xhoi, the site was filled in with klenow enzyme, and into this blunt-ended fragment was ligated the nhei nonsense codon linker 5' ctagctagctag 3' (new england biolabs). the resulting plasmid contained stop codons in all three frames in kex/at the xhoi site (+1760 bp). a 3.1-kb hindiil fragment containing the mutation at the xhoi site was then inserted into hindlh-digested ycp50 to create pkx1-20xho. ycp50-ct2 contains the adh/promoter and multiple cloning site from the plasmid pvt-100-l (veruet et al., 1987) cloned into the plnsmid ycps0. the termini of the 3.0-kb bglli-hindiii fragment of kex/were filled with klenow enzyme and inserted into ycp50-ct2 which had been digested with pvull. the resulting plasmid, pkxi-24, results in an ~10-fold overproduction of kexlp. the various truncation mutations of kex/were placed downstream from the gal/promotor in plasmid pmb258 to create the following plasmids: pkx1-25wt, pkx1-25hpa, pkx1-25bcl, and pkx1-25hinc. antisera production and procedures for sodium carbonate extractions, radiolabeling ceils, and associated immunoprecipitations have been described previously (cooper and bussey, 1989) . $86-16 transformants were grown to mid-log, and 6 • l0 s cells harvested, washed in i"i20, and resuspended in 3 ml of zsm (1.2 m sorbitol, 10 mm cac12, 10 mm tris-hc1, ph 7.5). 300/~g of zymolyase 60,000 (icn biomedicals, inc., costa mesa, ca) and/~-mercaptcethanol (final concentration 0.4%) were added to the cells before incubation at 30oc for 75 min. the resulting spheroplasts were gently pelleted, washed twice in 1.2 m sorbitol, 10 mm tris-hc1 (ph z5), and resuspended in 25 ml of ynb-sorb (yeast nitrogen base, 2% glucose, 1.2 m sorbitol), and incubated at 30oc for 3 h. spheroplasts were then concentrated to 5 ml, and bsa (200 mg ml -l) and pmsf (boehringer mannheim corp., indianapolis, in; final concentration i ram) were added before the addition of 500 mci of tram label (icn biomedicals, inc.). after 10 rain, half of the cells were harvested and the remainder chased for 30 rain as described above. after labeling, the spheroplasts were placed on ice, and sodium azide (fisher scientific co., pittsburgh, pa) was added to 1 ram. spheroplasts were then gently pelleted and both the supernatant and pellet fractions retained. the supernatant was microfuged for 2 rain and the resulting supernatant was concentrated with centricon 30 (amicon, beverly, ma) to a final volume of 200 ~tl before heating to 100~ in the presence of sds (1%). the spheroplasts werv washed once in ice cold 1.2 m sorbitol, i0 mm tris-hc1 (ph 7.5), pelleted, resuspended in bsb + 1% sds (cooper and bussey, 1989) , and boiled. the detergent-solubilized samples were then treated as above and immum~recipitated with kexlp antiserum. immunofluorescence studies fl-galactosidase-kexlp fusion protein (cooper and bussey, 1989 ) was isolated by preparative sds-page; electmeluted into 0.i m ni-~co3, 0.1% sds; lyophilised; resuspended in coupling buffer (0.5 m nacl, 0.1 m nahco3, ph 9.0); and then dialyzed against coupling buffer + 0.25% sds + 80 mg ml -~ pmsf. the extract was then coupled to 2 g of activated cnbr-sepharose 413 (pharmacia lkb biotechnology inc., piscataway, nj) overnight at room temperature. the coupling efficiency was judged to be > 80 % as determined by comparing unbound protein with the stming material. the sepharose was incubated with blocking buffer (1 m ethanolamine, 0.1 m nahco3, ph 8.0, 0.5 m naci) for 4 h at room temperature. the resulting column (bed volume, 2 ml) was then treated with successive washes of coupling buffer and acetate buffer (0.1 m sodium acetate, ph 4.0, 0.5 m nac1), and finally with pbs. a similar column (bed volume, 2 ml) was made with an extract made from the granules of e. coli cells producing/~-galactosidase. both the/~-galactosidase and ~-galactosidase-kexlp columns were washed successively with buffer a (50 mm hepes, ph 7.5, 150 mm naci, 1 mm edta), buffer b (buffer a + 1 m guanidine-hc1), and buffer c (50 mm hepes, ph z5, 15 mm nac1, 4.5 m mgci2). the two columns were then arranged so that the eluate from the #-galactosidase column flowed into the /~-galactosidase-kexlp column. all buffers used from this point on contained in addition: 1 mg/ml -i bsa, 0.2% nan3, 1 mm pmse columns were equilibrated with buffer a; kexlp antiserum was loaded onto the linked columns and was circulated for 3 h at 4~ the columns were disconnected and the o-galactosidase-kexlp column was washed first with buffer b and then with buffer a. antibodies were then eluted from this column with buffer c; fractions were collected and immediately dialyzed against buffer a at 40c. eluted fractions were tested for the presence of antibodies with a dotblot western approach and the positive fractions tested for their ability to immnnoprecipitate [35s]methionine-labeled kexlp from yeast. these fractions were concentrated by dialysis against buffer a containing 15% glycerol, and stored at -70~ immunofluorescence studies were performed following the procedure of redding et al. (1991) and used either affinity-purified anti-kexlp antibodies or a mouse monoclonal (13d11) which is directed against the 60-kd subunit of the yeast vacuolar membrane h+-atpase (kane et al., 1992) . the cells were observed using an axiophot microscope (carl zeiss, inc., thornwood, ny) (equipped for epifluorescence at excitation wavelengths appropriate for the described fluors) with a 100x objective and photographed with t-max 400 film (eastman kodak co., rochester, ny). mitochondria and nuclei were identified by 4',6-diamidino-2-phenyl-indole staining while vacuoles were identified by differential interference contrast (nomarski) optics. upon translocation into the yeast er, proteins that are destined for asn-linked glycosylation receive a core oligosaccharide which can be elaborated in the golgi apparatus by the addition of further mannose residues to produce the "outer chain: the outer chain consists of a backbone of u(1--~6) linked mannose residues to which are attached mannose sidechains in ot(l~2) linkages which then terminate in c~(1---3) linkages ( fig. 1 ; ballou, 1982) . the extension of the core is heterogenous in nature with different proteins receiving varying degrees of elaboration. kexlp is a glycoprotein which receives asn-linked glycosylation in a multistep process (cooper and bussey, 1989) . the initial event occurs in the er where core oligosaccharides are attached to the protein. the observed difference in molecular mass of ~ 3-4 kd between kexlp labeled for 10 min and that of kexlp produced in tunicamycintreated cells suggests that two of the three predicted lumenal asn acceptor residues are glycosylated. the ash-linked core oligosaccharides undergo a modification in a post-er compartment that increases the mass of kexlp by 1-2 kd, demonstrating that kexlp does not receive an extensive outer chain (cooper and bussey, 1989) . such a modification was reduced in a secl mutant which blocks intra-golgi transport, and was unaffected in a secl mutant that prevents secretory vesicle fusion with the plasma membrane (novick et al., 1981; cooper and bussey, 1989) . the yeast golgi apparatus has been functionally subdivided into several compartments on the basis of asn-linked oligosaccharide addition (franzusoff and schekman, 1989; graham and emr, 1991) . glycoproteins receive mannose outer chain modifications while traversing these golgi compartments. identification of the enzymes responsible for the (ballou, 1982; kukuruzinska et al., 1987) . a likely candidate for the mnn9-independent modification was the golgi-localized ~(1 ~3) mannosyltransferase which has been shown to be responsible for t~(l~3) mannose addition to both the core and outer chains ( fig, 1 ; nakajima and ballou, 1975; franzusoff and schekman, 1989; graham and emr, 1991) . this enzyme activity has been found to be deficient in an mnnl strain, and kexlp synthesized in a strain disrupted at the mnnl locus received only a small modification of its core oligosaccharide (fig. 2, lanes 3 and 4) . the ot(l~3) mannosyltransferase is therefore responsible for producing the majority of the post-er glycosylation modification to kexlp. the modification of kexlp by the golgi ot(l~3) mannosyltransferase suggested that kexlp reached the golgi compartment containing the mannosyltransferase, yet little kexlpdependent activity was detected at the cell surface (see below) and indicated that kexlp was retained within the secretory pathway. an analysis of mutant forms of kexlp was undertaken to examine which domain(s) of kexlp was responsible for its retention. the hydrophobic domain near the carboxy terminus of kexlp was previously shown to be responsible for conferring membrane association, as deletion of this 17-amino acid residue region resulted in a soluble protein, kexlp-ams (cooper and bussey, 1989) . cells expressing this protein showed a significant increase in kexlp-dependent activity in the cell medium (see below). that kexlp-ams was secreted rather than retained suggests that one of several domains may be responsible for retention of kexlp: (1) the membranespanning domain which was absent in kexlp-ams, (2) the carboxy-terminal domain which was no longer exposed in the cytoplasm in kexlp-ams, and (3) a domain aminos86-16/pkxl-18hpa was radiolabeled for a 10-min pulse (p) and harvested or chased for an additional 90 min (c). tunicamycin treatment (tcm) and high ph sodium carbonate extraction were performed as described previously (cooper and bussey, 1989) . the forms of kexlp were immunoprecipitated and analyzed by sds-page and fluorography. terminal to the membrane-spanning domain which is perturbed in kexlp-ams when the cytoplasmic domain was juxtaposed adjacent to it. a number of carboxy-terminal truncations of kexlp were produced to test the above possibilities. the construction of these mutations via site-specific mutagenesis or nonsense linker insertion is described in materials and methods and the predicted proteins are shown in fig. 3 . immunoprecipitations of the mutant kexlp proteins were performed to confirm that they were correctly synthesized by the cell (fig. 4) . all of the truncated proteins were smaller than kexlp by the predicted amount and entered the secretory pathway as shown by the addition of ash-linked glycosylation (data not shown). a concern was that the mutants designed to remain membrane associated may not attain a stable type i orientation and remain in the er. the most severely truncated mutant in this class, kexlp-hpa, lacks the entire endogenous cytoplasmic domain; it was further analyzed and shown to receive asn-linked glycosylation which was further modified within the golgi apparatus (fig. 4) . previous sodium carbonate extractions of whole cell extracts demonstrated that kexlp fractionated with the membrane containing pellet fraction whereas the soluble protein, kexlp-ams, fractionated with the supernatant (cooper and bussey, 1989) . carbonate treatment of whole cell extracts showed that kexlp-hpa fractionated with the membrane pellet (fig. 4 , lanes 5 and 6), consistent with it being an integral membrane protein. in addition, we have shown that cpy, a soluble kexlplike protein, is fully soluble under these extraction conditions (data not shown). kexlp activity was assayed in whole cells and their growth media to detect any potential mislocalization of the truncated forms of kexlp to the cell surface. to compare mutants, it was important that the kex/gene copy number be constant. the kexlp truncation constructs were, therefore, inserted into the vector ycp50, a centromeric-based plasmid which is normally maintained at a single copy per cell (rose et al., the indicated strains and transformants were inoculated in either yepd (a) or yep + galaetose (b) to a high cell density and grown to stationary phase. the intact cells were washed twice in water and a portion were lysed in 50 mm succinic acid (ph 6.0) with glass beads. the growth media, whole cells, and lysed cells were then assayed for kexlp activity as described (cooper and bussey, 1989) . a unit is defined as 1 pmol of product produced per minute at 30"c. (error -i-5%.) 1987). construction of these plasmids resulted in the deletion of an upstream portion of the k/ds this deletion resulted in reduced production of the encoded proteins relative to wild-type levels. these constructs (the pkx1-20 series of plasmids) were transformed into the yeast strain $86-17 (kex/-a3), a derivative ofs86 in which the kex/gene had been disrupted. the transformants were grown in liquid selective media; and the media, whole cells, and solubilized lysed cells were assayed for kexlp activity (data not shown). $86-17 produced no activity and the plasmid-borne wildtype kex/gene partially restored activity (10% of the level produced by the genomic allele of kex/) consistent with the promoter truncation. in comparing the partitioning of kexlp activity from various truncation mutants with that of the wild-type, two separate groups became apparent. the kexlp-hpa and kexlp-hinc truncated proteins (those that remained membrane associated; fig. 3 ) showed the same partitioning pattern as that of wild-type, whereas the proteins kexlp-xho, -as, and -ams (soluble proteins lacking the membrane-spanning domain) formed a different pattern with a 10-fold increase in activity at the cell surface relative to that of wild type. the total activity of each protein was approximately constant within a group. kexlp-hpa and kexlp-hinc had a total activity similar to that of wild type while the soluble forms of kexlp had ~50% of wild-type activity. although the decrease in activity may have been a direct consequence of the mutations, it was also possible that the reduced activity was due to secretion and subsequent degradation of the truncated soluble proteins. if such degradation of the soluble forms of kexlp-xho, -as, and -ams was occurring external to the plasma membrane, then the addition of bsa to the growth medium may lessen the extent of degradation. the addition of relatively high levels of bsa did not, however, alter the levels of total activity for kexlp-xho, -as, and -ams. a different approach to reduce potential degradation of truncated forms of kexlp was taken where transformants were grown in selective conditions (minimal media) and then transferred to nonselective yepd media for several generations before assay. yepd, a medium consisting primarily of yeast extract and peptone, should provide a substrate "buffer" against proteolytic degradation. although transformants were grown temporarily under nonselective conditions, plasmid loss was never >2% (data not shown). activity partitioning data suggested that kexlp-xho, -as, and -ams were secreted from the cell while the other constructs (kexlp, kexlp-hpa, and kexlp-hinc) remained intracellular (table i) . all the mutant forms of kexlp had approximately the same total activity as the plasmid-borne wild type. the percentage of kexlp extracellular activity for the membrane-associated forms of kexlp was comparable to the percentage of cells that stained positively with the vital dye methylene blue, suggesting that such extracellular activity resulted from cell lysis. kex/was placed downstream from the gal/ promoter which, upon induction by growth on galactose, resulted in a 70-fold increase over endogenous kexlp activity. however, such overproduction did not increase the percentage of extracellular kexlp activity relative to wild-type levels (table i) . to demonstrate the secretion of the soluble forms of kexlp, $86-16 (kex/-a2) was transformed with pkx1-8 (kexlp) or pkxi-18ams (kexlp-ams), and cells were then spheroplasted. the spheroplasts were radiolabeled (10 min), chased (30 min), and solubilized with sds, and kexlp was immunoprecipitated. media, containing proteins exported beyond the plasma membrane, were concentrated and kexlp was immunoprecipitated. wild-type kexlp remained associated with the spheroplasts, whereas kexlp-ams was shown to be secreted from the cell; the kexlp-ams signal associated with the cell fraction diminished with time while the signal from the medium showed the reverse trend (fig. 5) . thus, the results of the pulse-chase analysis correlated with the activity data and indicated that kexlp-ams, a soluble form of kexlp, was secreted from the cell. a stringent test of the effect of the truncations of kexlp was to determine the ability of the mutant proteins to carry out the golgi-based proteolytic processing of k1 killer toxin in vivo (for review see bussey, 1988) . sc25k is a strain harboring the dsrna virus that encodes k1 killer toxin, and produces a killing zone on plates seeded with a strain sensitive to the killer toxin (bussey et al., 1983) . the pkx1-20 series of plasmids (centromeric based) were transformed into sc25k-13 (kex/-a/), and the transformants analyzed for their ability to process k1 killer toxin and produce a killing zone (fig. 6 a) . sc25k-13 containing the vector plasmid produced no killing zone, whereas the introduction of a plasmidbased copy ofkexi (pkx1-20wt) enabled the strain to produce active k1 killer toxin as shown by the appearance of a zone (fig. 6 b) . this zone was smaller than that of sc25k (kexi) because of the kex/promoter truncation in pkx1-20wt (discussed above). the truncated forms of kexlp, both soluble and membrane associated, produced killer zones significantly smaller than that produced by pkx1-20wt (fig. 6 a) . this functional complementation assay was also conducted in a different k1 killer strain (tc106ot-17) with similar results. the reduced processing of k1 killer toxin by the soluble forms of kexlp can be explained because of their secretion and consequent reduced concentration of kexlp within the processing compartment. the membrane-associated truncated forms of kexlp remain intracellular and, therefore, secretion cannot account for their reduced processing ability. the above results indicated that kexlp-hpa was membrane associated, had received glycosyl modifications in the golgi apparatus, gave wild-type levels of total activity, and was retained intracellularly; yet processed the k1 killer toxin precursor to a lesser extent than kexlp. kexlp-bcl and kexlp-hinc showed similar phenotypes to that of kexlp-hpa. a likely explanation for such observations was that these membrane-associated mutant forms of kexlp were not retained within the correct golgi compartment, but instead were mislocalized within the secretory pathway. indirect immunofluorescent detection of kexlp and kexlp-hpa was undertaken to determine if the location of the two proteins differed. the expression of the proteins was such that a kexlp signal could only be detected upon overproduction of kexlp (fig. 7) . kexlp was found to be localized to a number of small punctate structures (on average, ,x,3-5 per cell) which were not associated with mitochondria, nuclei, or vacuoles, but were characteristic of proteins localized to (hpa) , or pkx1-20-hinc (hinc). transformants were grown to stationary phase in minimal media, harvested by centrifugation, washed in h20, pelleted, and than resusponded in h20 to a concentration of 5 x 108 cells ml -~. 10/~1 was then placed onto a minimal plate (ph 4.7), seeded with $6 (a strain sensitive to k1 killer toxin). plates were then incubated at 18"c for 24 h and zones of toxin killing examined. approximate relative toxin activities, determined by comparison with a toxin dilution series were wt 100%, xho 30%, as 40%, ams 15%, hpa 55%, and hinc 55%. (b) sc25k-13 (kex/), sc25k (kex1), and sc25k-13/pkxi-20wt (wt) were grown and spotted onto a plate seeded with a strain sensitive to k1 killer toxin and treated as described above. the yeast golgi apparatus (redding et al., 1991; cleves et al., 1991; franzusoff et al., 1991; roberts et al., 1992) . comparison of the kexlp-hpa localization with that of the vacuole (as determined by nomarski optics) indicated that almost all of kexlp-hpa was in the vacuole (data not shown). it is likely that kexlp-hpa was transported to the vacuole, where, in a protease-deficient strain such as $86-16, the protein was not degraded and remained active. to confirm this possibility, colocalization studies were undertaken between the 60-kd subunit of the s. cerevisiae vacuolar membrane h § and either kexlp or kexlp-hpa. the vacuole (as determined by nomarski optics) correlated to the ring structure in which the 60-kd atpase subunit was localized by indirect immunofluorescence. the level of kexlp detected varied among cells due to the variable plasmid copy number (2 #m based). observation of cells expressing both low and high levels of kexlp-hpa (as determined by the intensity of the fluorescence) indicated that >90% of the protein was located in the vacuole. a small percentage of cells (<5 %) showed both er and vacuolar staining. the protein was associated with the vacuolar membrane demonstrating that it remained membrane associated (fig. 8) . kexlp was predominantly restricted to punctate structures indicative of a yeast golgi location (fig. 7) . however, 10-15 % of the stained cells expressed high levels of kexlp (as determined by the intensity of the fluorescence) and showed kexlp in both golgi-like structures, and associated with the vacuolar membrane as defined by the 60-kd atpase subunit (data not shown). no kexlp or kexlp-hpa signal was detected at the plasma membrane. further indirect immunofluorescence analysis also localized the other membrane-associated forms, kexlp-bcl and kexlp-hinc, to the vacuolar membrane (data not shown). an analysis of the oligosaccharide modification of kexlp in-dicated secretory compartments to which the protein had been exposed, and the likely subcellular location of kexlp. the modification of the asn-linkod oligosaccharide cores of kexlp occurred within the golgi apparatus and likely involves several sequential steps. initially, in a pre-sec7 compartment, the core oligosaccharides are partially modified as judged by an increase in apparent molecular mass (cooper and bussey, 1989) . subsequently, in a post-sec7 compartment, the core is further modified in a mnnl-dependent manner. invertase produced in sec7 cells at the restrictive temperature does not receive c~(1--'3) linked mannose residues to the core or outer chain (franzusoff and schekman, 1989) , most likely because of the failure of secretory proteins to reach the compartment containing the cx(1--,3) mannosyltransferase. it is consistent that sec7 and mnnl mutations result in a similar reduction in the glycosyl elaboration of kexlp, as they would respectively either prevent kexlp from reaching, or reduce the activity of, the c~(1~3) mannosyltransferase. the golgi-based modification of kexlp can thus be explained as a result of two processes. the first occurs before the sec7 block and most likely involves the addition of an ot(l~6) mannose residue followed by the attachment of an oz(1--'2) linked mannose residue (indicated by * in fig. 1 ). the second step involves the mnn1dependent addition of up to three a ( l ' 3 ) linked mannose residues (indicated by a circle in fig. 1) to the oligosaccharide (ballou et al., 1990) . graham and emr (1991) provided evidence for at least three functional compartments in the yeast golgi apparatus that contain, from cis to trans, the following activities: (1) ct(l~6) mannosyltransferase, (2) cx(l~3) mannosyltransferase, and (3) kex2p endoprotease. kexlp must reach the golgi compartment housing the mnnl-dependent o~(1---3) mannosyltransferase as it is modified by this enzyme. in addition, to process killer toxin, kexlp must reach the proposed third golgi compartment containing kex2p as the toxin precursor substrate for kexlp is created by a kex2p-mediated endoproteolytic cleavage. no post-kex2p compartment before secretory vesicles has been observed (graham and emr, 1991) and, therefore, it is likely that kexlp resides with kex2p in the same late golgi compartment (redding et al., 1991) . it is interesting to note that both kexlp and kex2p figure 9 . model for the retention of kexlp within the secretory pathway. a model for the retention of kexlp is presented. kexlp is retained in a late golgi compartment because of its cytoplasmically exposed domain interacting with a receptor (recpt). kexlp-as, being soluble, is secreted by default to the cell surface. kexlp-hpa is membrane associated and residues in the vacuole because of several possible reasons: (1) kexlp-hpa is partially misfolded and is targeted to the vacuole via an unknown system; (2) the membranespanning domain of kexlp contains a cryptic targeting signal for the vacuole; (3) kexlp-hpa is secreted to the plasma membrane where it is endocytosed and delivered to the vacuole; and (4) membrane-associated proteins that leave the golgi apparatus are targeted to the vacuole by default. kexlp is delivered to the vacuole when overproduced to a high level, presumably due to saturation of a golgi retention receptor. the horizontal bar protruding from the kexlp-wt box represents the membrane-spanning and cytoplasmic domain, while for kexlp-hpa the bar represents the membrane-sparming domain. (wilcox and fuller, 1991) undergo a slow addition ofa(l~3) mannose units to their respective glycosyl groups. further evidence that kexlp is an enzyme that resides in the yeast golgi apparatus comes from indirect immunofluorescence results where a punctate pattern of staining is detected that is characteristic for a number of proteins localized to the golgi apparatus in s. cerevisiae: kex2p (redding et al., 1991) , dpap a (roberts et al., 1992) , secl4p (cleves et al., 1991) , and sec7p (franzusoff et al., 1991) . of the mutations that result in a soluble form of kexlp (kexlp-xho, -as, and -ams), kexlp-as is the most informative with regard to determining which domain(s) of kexlp is involved in its localization. kexlp-as is secreted, yet contains all but 11 residues of the 596-residue lumenal domain (fig. 3) . the 11 residues absent in kexlp-as are present in kexlp-ams which is also secreted. the results suggest that either the membrane-spanning or cytoplasmic domain, or both, are responsible for the retention of kexlp within the golgi apparatus. kexlp-hpa contains both the lumenal and transmembrane domains of kexlp yet is mislocalized to the vacuolar membrane. similar results were found for kexlp-bcl and kexlp-hinc which lack all or part of the cytoplasmic domain and strongly implicate this domain of kemp as being necessary for correct localization within the golgi apparatus. previous work has demonstrated the role that cytoplasmic domains of transmembrane proteins play in the retention and targeting of proteins within the secretory pathway. a consensus retention motif has been identified at the termini of the cytoplasmically exposed domain of mammalian er resident type i transmembrane proteins (nilsson et al., 1989; jackson et al., 1990) . the cytoplasmic domains of the transferrin receptor, cation-independent mannose-6-phosphate receptor, and the lysosomal acid phosphatase are involved in the correct targeting or endocytosis of these proteins (rothenberger et al., 1987; lobel et al., 1989; peters et al., 1990) . the three proteins involved in processing a-factor, kemp, kex2p, and dpap a, are all transmembrane proteins with cytoplasmic domains which are thought to be involved in retention of the respective proteins in similar if not identical compartments of the yeast golgi apparatus (fuller et al., 1989; roberts et al., 1992) . attempts to identify a consensus yeast golgi retention motif shared between these proteins have, however, showed no obvious amino acid sequence homology between the cytoplasmic domains of these three proteins. different mechanisms have been proposed for the retention of proteins within golgi compartments. interactions involving the cytoplasmic domain of kexlp may result in the aggregation of the protein in the appropriate compartment, and thereby prevent it from entering transport vesicles that are exiting the golgi compartment (pfeffer and rothman, 1987) . this retention via aggregation model is unlikely as it does not explain why aggregation would not occur earlier in the secretory pathway or why overproduction of a kexlp results in mislocalization to the vacuole rather than greater aggregation in the golgi compartment. a more likely explanation for the retention involves a receptor interacting with the cytoplasmically exposed domain of kexlp ( fig. 9 ). high level expression of kexlp would saturate the receptor with the result that excess kexlp is diverted to the vacuole. removal of the cytoplasmic domain would abolish such a receptor-ligand interaction, also resulting in mislocalization to the vacuole. retention of soluble er proteins has been shown to involve a receptor-mediated retrieval of proteins from a pre-cisgolgi compartment (semenz~ et al., 1990; lewis et al., 1990) . a cytoplasmicauy based retrieval system analogous to the er system can be envisaged in which kexlp is retrieved from a trans compartment, possibly the plasma membrane, and returned to the correct golgi compartment. to determine if kexlp is retrieved from the plasma membrane an immunofluorescence experiment was performed by inducing expression of kexlp in a secl strain, secl is a conditional mutation that, at the nonpermissive temperature, prevents the fusion of secretory vesicles with the plasma membrane (novick et al., 1981) . the delivery of kexlp to its golgi location was not blocked by secl, and if kex/expres-the jouma! of cell biology, volume 119, 1992 sion was constitutive before imposition of the secl block, the staining pattern of kexlp was not altered even after 3 h at the restrictive temperature (data not shown). therefore, if a receptor retrieval system exists, it is unlikely that kexlp is retrieved from the plasma membrane as outlined in one of several proposals presented by payne and schekman (1989) . if a retrieval system is responsible for the retention of kexlp, then the salvage compartment may instead be located between the golgi compartment and the vacuole where failure to bind the receptor would result in delivery to the vacuole. alternatively, the receptor may remain anchored within the golgi apparatus and not recycle. the clathrin heavy chain has been implicated in the retention of kex2p (payne and schekman, 1989) , raising the possibility that the receptor-based system that recognizes the cytoplasmic domain of kexlp, and potentially kex2p and dpap a, might be one of the clathrin-associated adaptin proteins known to occur in yeast (kirchhausen, 1990) . truncated forms of kexlp which lacked the cytoplasmic domain yet still remained membrane associated were not retained in the correct golgi compartment, and even at subwildtype expression levels v~ere diverted to the vacuole. similar mislocalization results have been found with dpap a, where overexpression of the protein results in mislocalization to the vacuole, as do mutations within its cytoplasmically exposed domain (roberts et al., 1992) . differing results were obtained with kex2p, where a mutation that deleted the cytoplasmic domain and part of the membrane-spanning domain resulted in a significant proportion of the kex2p activity being mislocalized to the cell surface (fuller et al., 1989) . the kex2p activity study did not, however, address whether the truncated protein produced was membrane associated; if not, then the resulting soluble protein would be expected to be secreted to the cell surface. in addition, the strain used for such kex2p activity studies contained wild-type activity levels of vacuolar hydrolases (pep4; jones, 1984) and, therefore, any kex2p potentially mislocalized to the vacuole might be degraded and hence go undetected. mislocalization of membrane proteins to the lysosome does not appear to occur in mammalian systems where mutations that interfere with the localization of er, golgi, and lysosomal integral membrane proteins result in their delivery to the plasma membrane (paabo et al., 1987; machamer and rose, 1987; peters et al., 1990) . one exception to this observation in mammalian systems is the coronavirus e1 protein, a type hi membrane protein normally resident in the golgi apparatus. mutations that affect the retention of this protein resulted in delivery to the lysosome rather than to the cell surface (armstrong et al., 1990) . a number of models could explain the observation that the membrane-associated mutant forms of kexlp are deh'vered to the vacuole while the soluble truncated forms of kexl are exported to the cell surface (fig. 9) . (1) the first model suggests that membrane-associated truncated forms of kexlp are misfolded, and as such may be targeted, via an unknown mechanism, to the vacuole for degradation. this model seems unlikely as all of the truncated forms of kexlp, both soluble and membrane associated, have similar total pro-tease activity to that of the wild-type protein, suggesting that at a minimum the catalytic domain of the mutants has folded to a conformation similar to that of wild type. in addition, these forms of kexlp all exit the er and reach the golgi apparatus where they are both glycosylated and process killer toxin. the misfolding targeting model would be unusual in that it must explain the mislocalization of the membraneassociated mutant forms of kexlp yet allow the soluble forms to be secreted. also, such a garbage pathway for delivery of misfolded mutant forms of kexlp cannot explain the vacuolar localization of highly expressed but presumably correctly folded kexlp. (2) the second model proposes that the membrane-spanning domain of kexlp contains a latent or cryptic targeting signal for the vacuole. studies are currently under way to address this possibility. it should be noted that although kexlp is homologous to the vacuolar protein cpy, the homology does not extend to include the vacuolar targeting signal found in procpy. (3) the third model involves kexlp-hpa (-bcl, -hinc) being secreted to the plasma membrane by default, where it is then endocytosed and delivered to the vacuole. such a "transient appearance" of kexlp-hpa (-bcl, -hint) at the cell surface would not be detected by the activity assay used or by indirect immunofluorescence. soluble kexlp-as, having reached the cell surface, would be released into the medium and, hence, would not be endocytosed to the vacuole. (4) the final model proposes that the vacuole is the direct default destination for membraneassociated proteins that enter the secretory pathway, such that membrane proteins lacking positive targeting/retention signals would be delivered to the vacuole. work with dpap b, a type ii vacuolar membrane protein homologous to dpap a, has shown that no domain of the protein contains positive targeting information for the vacuole (roberts et al., 1992) . the targeting signal for a second vacuolar membrane protein, alkaline phosphatase, has not been identified although the lumenal domain is not required for the correct localization of this protein (klionsky and emr, 1990) . the authors concluded that the vacuolar-sorting determinant must therefore reside in the cytoplasmic and/or membrane-spanning domain of alkaline phosphatase, but in light of the data presented here and elsewhere (roberts et al., 1992) , these results could be reinterpreted to suggest that the membrane association of alkaline phosphatase mediates its vacuolar delivery. given the results presented here, we favor models 3 and 4 which are variants of each other in that both result in the delivery of kexlp to the vacuole by default (no positive targeting signal involved) but differ in the delivery route. model 4, involving a direct golgi-to-vacuole route, may be the most likely as delivery of the vacuolar membrane protein dpap b does not involve transport to the plasma membrane and subsequent endocytosis (roberts et al., 1992) . in addition, kexlp-bcl contains no cytoplasmic tail to participate in classical endocytosis. an important implication of such default delivery is that yeast integral membrane proteins destined for the plasma membrane must have positive targeting information to either remain at the plasma membrane (proposal 3), or to avoid being diverted to the vacuole (proposal 4). it is pertinent that a mutation in the u-factor receptor (ste3p), an integral plasma membrane protein, results in delivery of this protein to the vacuole, independent of the plasma membrane (ho-recka, j., and g. sprague, personal communication), raising the possibility that a plasma membrane targeting signal has been destroyed. further work is proceeding to determine if the delivery route of kexlp to the vacuole is via the plasma membrane, and whether plasma membrane proteins have positive targeting/retention information. lysosomal sorting mutants of coronavirus e1 protein, a golgi membrane protein yeast cell wall and cell surface revision of the oligosaccharide structures of the yeast carboxypeptidase y proteases and the processing of precursors to secreted proreins in yeast secretion of saccharomyces cerevisiae killer toxin: processing of the glycosylated precursors mutations in the cdpcholine pathway for phospholipid biosynthesis bypass the requiremem for an essential phospholipid transfer protein characterization of the yeast kex1 gene product: a carboxypeptidase involved in processing secreted precursor proteins yeast kex/gene encodes a putative protease with a carboxypeptidase b-like function involved in killer toxin and alpha factor precursor processing functional compartments of the yeast golgi apparatus are defined by the sec7 mutation localization of components involved in protein transport and processing through the yeast golgi apparatus enzymes required for yeast prohormone processing intracellular targeting and the structural conservation of a prohormone-proeassing endoprotease compartmental organization of golgispecific protein modification and vacuolar protein sorting events defined in a yeast secl8 (nsf) mutant identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease the synthesis and function of proteases in saccharomyces cerevisiae: genetic approaches assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar h+-atpase rate of bulk flow from the golgi to the plasma membrane identification of a putative yeast homolog of the mammalian ~ chains of the clathrin-associated complexes a new class of lysosomal/vacuolar protein sorting signals intraceilular sorting and processing of a yeast vacuolar hydrolase: proteinase a propeptide contains vacuolar targeting information the biogenesis of lysosomes protein glycosylation in yeast the erd2 gene determines the specificity of the luminal er protein retention system mutations in the cytoplasmic domain of the 275 kd marmose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis a specific transmembmne domain of a coronavirus e1 glycoprotein is required for its retention in the golgi regiou a cooh-terminal signal prevents secretion of luminal er proteins yeast manno-protein biosynthesis: solubilization and selective assay of four mannosyltransferases short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum order of events in the yeast secretory pathway a short sequence in the cooh terminus makes an adenovirus membrane glycoprotein a resident of the endoplasmic reticulum clathrin: a role in the intracellular retention of a golgi membrane protein. science (wash, dc) sorting of soluble er proteins in yeast targeting of a lysosomal membrane prorein: a tyrosine-coutaining endocytosis signal in the cytoplasmic tail of lysosomal acid phosphatase is necessary and sufficient for targeting to lysosomes biosynthetic protein transport and sorting by the endoplasmic reticulum and golgi immunolocalizatiou of kex2 protease identifies a putative late gotgi compartment in the yeast saccharo-m2,~es cerevisiae membrane protein sorting in the yeast secretory pathway: evidence that the vacuole may be the default compartment a saccharomyces cerevisiae genomic plasmid bank based on a centromerecontaining shuttle vector endccytosis of the transferrin receptor requires the cytoplasmic domain but not its phosphorylation site protein sorting by selective retention in the endoplasmic retieulum and golgi stack erd2, a yeast gene required for the receptor-mediated retrieval of luminal er proteins from the secretory pathway protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase y resides in the propeptide a family of yeast expression vectors containing the phage fl intergenic region the rate of bulk flow from the endoplasmic reticulum to the celt surface posttranslational processing of the prohormone-cleaving kex2 protease in the saccharomyces cerevisiae secretory pathway accumulation of membrane glycoproteins in lysosomes requires a tyrosine residue at a particular position in the cytoplasmic tail we thank marc lussier, kathryn hill, and charlie boone for reading the manuscript; anne-marie sdicu for assistance in manuscript preparation; marc lussier for immunofluorescence quantitation data; joe horecka, steve nothwehr, chris roberts, george sprague, and tom stevens for communication of results prior to publication; chris roberts and tom stevens for providing the monoclonal antibody against the 60-kd subunit of the vacuolar h +-atpase; kevin redding for his immunottuorescence protocol; vivian mckay for the mnnl disrupted strain; and robert la.marcbe and guy l'i-ieureux for photographic work.this work was supported by natural sciences and engineering research council strategic and operating grams. at the time of this work a. cooper was a canadian commonwealth scholar.received for publication 7 november 1991 and in revised form 8 september t992. key: cord-034191-qqb2knmo authors: alayi, tchilabalo d.; tawalbeh, shefa m.; ogundele, michael; smith, holly r.; samsel, alison m.; barbieri, marissa l.; hathout, yetrib title: tandem mass tag-based serum proteome profiling for biomarker discovery in young duchenne muscular dystrophy boys date: 2020-10-06 journal: acs omega doi: 10.1021/acsomega.0c03206 sha: doc_id: 34191 cord_uid: qqb2knmo [image: see text] blood-accessible molecular biomarkers are becoming highly attractive tools to assess disease progression and response to therapies in duchenne muscular dystrophy (dmd) especially in very young patients for whom other outcome measures remain subjective and challenging. in this study, we have standardized a highly specific and reproducible multiplexing mass spectrometry method using the tandem mass tag (tmt) strategy in combination with depletion of abundant proteins from serum and high-ph reversed-phase peptide fractionation. differential proteome profiling of 4 year-old dmd boys (n = 9) and age-matched healthy controls (n = 9) identified 38 elevated and 50 decreased serum proteins (adjusted p < 0.05, fdr <0.05) in the dmd group relative to the healthy control group. as expected, we confirmed previously reported biomarkers but also identified novel biomarkers. these included novel muscle injury-associated biomarkers such as telethonin, smoothelin-like protein 1, cofilin-1, and plectin, additional muscle-specific enzymes such as utp–glucose-1-phosphate uridylyltransferase, aspartate aminotransferase, pyruvate kinase pkm, lactotransferrin, tissue alpha-l-fucosidase, pantetheinase, and ficolin-1, and some pro-inflammatory and cell adhesion-associated biomarkers such as leukosialin, macrophage receptor marco, vitronectin, galectin-3-binding protein, and prosaas. the workflow including serum depletion, sample processing, and mass spectrometry analysis was found to be reproducible and stable over time with cv < 20%. furthermore, the method was found to be superior in terms of specificity compared to other multiplexing affinity-based methods. these findings demonstrate the specificity and reliability of tmt-based mass spectrometry methods in detection and identification of serum biomarkers in presymptomatic young dmd patients. duchenne muscular dystrophy (dmd) remains a serious and fatal muscle disease with a worldwide incidence of 1:5000 male births. 1 dmd is due to mutations in the x-linked dystrophin gene, leading to the loss of expression of dystrophin, an essential skeletal muscle protein that maintains the integrity and function of muscle fibers. 2 there is no cure for dmd to date except the use of corticosteroids, which are known to delay the loss of ambulation by 2 to 3 years without changing the disease course. 2, 3 despite a number of completed and ongoing clinical trials, only two drugs received conditional approval from european medicines agency (ema) and us food and drug administration (fda) in the past 15 years. these include ataluren, a stop codon read-through, and eteplirsen, an exon skipping antisense oligonucleotide that restores the reading frame during dystrophin mrna translation. 4, 5 the slow development of new therapies in dmd has been hindered by the fact that dmd is a rare disease and that most clinical trials enroll ambulatory patients within a specific age range (4−10 years old), resulting in statistically unpowered studies to accurately assess the outcomes. furthermore, current clinical tests to assess disease progression and response to therapies such as 6 min walk test, 10 m run/walk velocity, and other physical tests are subjective and might require longer clinical trials to determine the efficacy or failure of an investigational drug. 6−8 major advances in biomarker discovery have been achieved in the past 7 years in dmd. 9−12 owing to the multiplexing capabilities of affinity-based methods such as antibody multiplexing bead technology and somascan aptamer technology, a large number of biomarkers were discovered in dmd patients and confirmed across different laboratories and different cohorts. 13−16 although these multiplexing affinitybased assays are superior to mass spectrometry in terms of sensitivity, dynamic range, and throughput, they are inherently not quantitative and lack accuracy in determining true fold change in biomarker levels. 17 furthermore, these multiplexing affinity methods only detect and measure targeted sets of biomarkers and are not suitable for de novo discovery of novel biomarkers or discrimination between biomarkers that exist as multiple isoforms. thus, alternative methods that are more versatile, specific, reproducible, and accurate and that can easily be implemented by other laboratories are needed. in this study, we sought to optimize and standardize a serum processing workflow in combination with tandem mass tag (tmt) multiplexing strategy to systematically survey the serum proteome of young untreated dmd boys and agematched healthy controls and identify biomarkers associated in the early stages of the disease. in this study, we focused on very young dmd boys for two reasons. first is to avoid confounding variables due to glucocorticoid use 14 and second is to define blood-circulating biomarkers that might be associated with early stages of the disease. these biomarkers might eventually be used as tools to assess disease progression and response to therapies in this younger dmd population and facilitate their enrollment into clinical trials from which they are often excluded because of lack of outcome measures for younger patients. our method successfully confirmed some of the previously reported biomarkers but also identified novel biomarkers in young untreated dmd patients. sample preparation and quality control check. to optimize our ms-based tandem mass tag (tmt) multiplex quantitation, we evaluated each step in the sample preparation and ms analysis through the workflow depicted in (figure 1 ). this workflow was evaluated in triplicate using commercial serum. we first checked for the depletion kit efficiency using three different aliquots of serum samples in triplicates, 4, 8, and 12 μl containing 300, 600, and 900 μg of total proteins, respectively. the results show a linear response between total protein used for depletion and total protein recovered after the depletion. averages of 17.9 ± 0.2 μg (1.1% cv), 49.9 ± 1.4 μg (2.9% cv), and 81.4 ± 5.9 μg (7.3% cv) of protein were collected from the 300, 600, and 900 μg aliquots, respectively ( figure 2a ). together, these findings suggest that the depletion columns have enough capacity to efficiently deplete serum-abundant proteins from serum aliquots in the 300 to 900 μg total protein range in a reproducible manner with an overall cv < 10%. moving forward, we decided to use serum aliquots containing 900 μg of total proteins as a starting material. depletion efficiency and reproducibility were further checked using 1d sds-page. nondepleted serum proteins exhibited very few intense protein bands, among which human serum albumin (hsa) was the most intense band on the 1d sds-page ( figure 2b ). the technical triplicate analysis of 3 μg of depleted serum revealed a large number of new protein bands (e.g., bands a, b, c, d, and e) on 1d sds-page and a considerable reduction in major protein bands such as hsa. optical density analysis of randomly selected new protein bands detected in the depleted serum sample by 1d sds-page (bands a, b, c, d, and e) showed reproducibility in depletion with cvs < 10% ( figure 2c ). furthermore, we checked the efficiency and reproducibility of the depletion kit using western blot analysis of two proteins ( figure 2d ), one of which is among the depleted proteins (haptoglobin, hp) and another one of which is not among the depleted proteins (gelsolin, gsn). the depletion process resulted in an enrichment of gsn protein by approximately 10-fold (cvs < 16%) and in a depletion of hp protein by approximately 2fold (cvs <14%), in comparison to nondepleted serum as expected ( figure 2e ). we then checked the reproducibility and stability of the entire workflow using 900 μg of total serum proteins as a starting material. aliquots of 10−14 μl of each sample were processed for top 12 abundant protein depletion kit and 50 μg of depleted sample was processed for tmt 6-plex tagging, as described in the method. the obtained peptides were labeled with tmt reagent, which covalently modifies the primary amines of the n termini and lysine side chains of peptides. 19 equal volumes of tagged samples were taken and mixed together and 18 fractions were collected from the high-ph reversed-phase spin column and analyzed by lc−ms/ms, as described above (figure 1 ). figure 1 . chart depicting the overall workflow from sample preparation to mass spectrometry analysis to data processing. serum samples (900 μg per aliquot) from 4 year-old dmd patients (n = 9) and age-matched healthy controls (n = 9) were processed for 12 most abundant proteins depletion. resulting samples were in-solution-digested and randomized for tmt tagging, as shown in the figure. one of the control samples (dark blue) was used as a reference to normalize the data. each sample mixture was further fractionated using a high-ph reversed-phase column and each fraction was analyzed by lc−ms/ms. data were processed as described in the method. the stability and reproducibility of the overall workflow ( figure 1 ) including serum depletion step, tandem mass tag (tmt) multiplexing step, and high-ph reversed-phase peptide fractionation were evaluated by lc−ms/ms in triplicate analysis using commercial serum. after removing contaminant proteins and missing data, this standardized tmt ms method enabled the identification and quantification of 618 serum proteins across the triplicate experiment with cv < 20% for 601 proteins (97% of protein quantified), cv < 15% for 588 proteins (95% of protein quantified), and cv < 5% for 396 proteins ( figure 2f ). the complete list of 618 quantified proteins by lc−ms/ms and associated normalized protein abundance ratios, means of normalized abundance ratios, standard deviation, and cvs of three technical replicate analysis of commercial serum are shown in table s2 . serum protein biomarker screening in young dmd patients. serum samples from 4 year-old glucocorticoid naive dmd patients (n = 9) and age-matched healthy controls (n = 9) were processed for proteome profiling using our standardized multiplexing tmt-based mass spectrometry method described above. a total of four experiment sets of tmt msbased assays were run in parallel including two 6-plex figure 2 . quality control of the immunoaffinity depletion of the 12 highly abundant proteins from serum samples. aliquots of serum samples with different amounts of total proteins (300, 600, and 900 μg) were used in triplicate to examine the variability and quality of the immunoaffinity depletion process. (a) linear response between the different starting amounts of total serum proteins and recovered total proteins in depleted samples. approximately 17.9 μg (cv 1.1%), 49.9 μg (2.9%), and 81.4 μg (cv 7.3%) of proteins were recovered from serum samples containing 300, 600, and 900 μg of total proteins, respectively. (b) sds-page showing side-by-side nondepleted serum (3 μg) and depleted serum samples in triplicate (3 μg each). the nondepleted serum proteins exhibited very few intense protein bands, among which human serum albumin (hsa) was the most intense band on the 1d sds-page, while the depleted serum samples revealed large and reproducible number on new protein bands (ex. bands a, b, c, d, and e). (c) detailed optical density analysis of randomly selected new protein bands observed on the depleted serum 1d sds-page (bands a, b, c, d, and e) allowed the comparison of the technical triplicate depletion of serum and estimated the cv < 9.1%. (d) western blot analysis of depleted proteins (haptoglobin, hp) and nondepleted proteins (gelsolin, gsn). (e) quantitative analysis of western blot shows an increase in gsn by approximately 10-fold (cvs < 16%) in comparison to nondepleted serum and a decrease in hp by approximately 2-fold (cvs < 14%), confirming the effectiveness and the reproducibility of the depletion kits. (f) quality control of in-solution digestion and tmt tagging efficiency. a total of 618 serum proteins were identified and quantified. the coefficient of variation between these three technical replicates is depicted in the pie chart with the majority of quantified proteins (97%) showing cv < 20%, with 64% of proteins showing cv < 5% and 95% of proteins showing cv < 15%. experiments comparing three controls and three dmd samples each, one 5-plex experiment comparing two dmd samples and three controls, and one 4-plex experiment comparing one dmd sample and three controls. to control for intra-and inter-experiment variation, the same amount of digest of tmt 126-labeled control serum sample was used as an internal standard in all four experiments ( figure 1 ). using our optimized and standardized workflow, 686 proteins were quantified across all 18 samples with only 13% of missing values in all samples (table s3 ). further statistical analysis identified 38 significantly elevated proteins and 50 significantly decreased proteins (student's t-test, adjusted for multiple testing, p < 0.05) in serum samples of 4 year-old dmd boys compared to 4 year-old healthy controls ( figure 3a ,b and table 1 ). twenty-nine proteins out of the 88 potential biomarker candidates had missing values in some samples and these are shown in figure s1 . fold change in protein levels in serum of young dmd patients compared to healthy controls. serum proteome profiling was performed on 4 yearold dmd patients (n = 9) and age-matched healthy controls (n = 9) using the tmt method. significance was defined between the two groups by independent two-sample t-test (two-sided) corrected with adjusted p value and by permutation-based fdr of 0.05. significantly elevated and significantly decreased proteins in dmd patients relative to controls are presented in green and red, respectively. (b) hierarchical clustering of serum proteins whose levels were significantly altered between the 4 year-old dmd (n = 9) group and age-matched healthy control (n = 9) group. intensities of proteins are normalized abundance ratios, which were log2-transformed. as expected, a large number of identified biomarkers (50%) that were found to be elevated in sera of these young dmd boys relative to the healthy controls were of muscle origin based on gene ontology molecular function annotations ( figure s2 ) and information collected using mass spectrometry data analysis tools. 20 most of these muscle-associated proteins were previously reported by other research groups and us 9,11,14−16 except telethonin (tcap), plectin (plec), smoothelin-like protein 1 (smtnl1), myosin-7 (myh7), pdz and lim domain protein 5 (pdlim5), tropomyosin alpha-1 and beta chain (tpm1, tpm2), troponin c skeletal muscle (tnnc1, tnnc2), troponin i slow skeletal muscle (tnni1), myomesin-2 (myom2), and fructose-bisphosphate aldolase c (aldoc), which are novel to this study. the fold change difference between dmd patients and healthy controls for this class of biomarkers ranged from 2.45 to 8.66 ( figure 3a ,b) and likely underlie the well-known sarcolemma instability in dmd and release of muscle-specific proteins. 12, 13, 21 the second class of elevated serum proteins found in young untreated dmd patients relative to controls included the well-known heme carrier protein myoglobin (mg) and muscle-specific enzymes such as creatine kinase band m-type (ckb, ckm), fructose-bisphosphate aldolase a (aldoa), and carbonic anhydrase 3 (ca3). additionally, novel enzymes in this class such as fructose-bisphosphate aldolase c (aldoc), 72 kda type iv collagenase (mmp2), utp−glucose-1-phosphate uridylyltransferase (ugp2), aspartate aminotransferase, cytoplasmic got1, pyruvate kinase pkm (pkm), atp-dependent 6-phosphofructokinase muscle type (pfkm), proteasome subunit beta type-1 (psmb1), and glutathione s-transferase mu 2 (gstm2) (table 1, figure 3b , and figure s1 ) were also found. 9, 11, 22 an example of a panel of these newly identified dmd biomarkers using mass spectrometry is shown in figure 3c ,d. serum proteins that were found to be decreased in young untreated dmd patients relative to healthy controls can be classified into three major groups. one group consisted of proteases such as beta-ala-his dipeptidase (cndp1), previously reported by others and us, 14−16 and others newly identified proteases such as lactotransferrin (ltf), tissue alpha-l-fucosidase (fuca1), pantetheinase (vnn1), plasminogen (plg), carboxypeptidase n catalytic chain (cpn1), glutamyl aminopeptidase (enpep), exostosin-1 (ext1), ficolin-1 (fcn1), and transketolase (tkt) ( figure 3a ,b and figure s2 ). the second group of decreased biomarkers consisted of actin-binding proteins such gelsolin (gsn), thymosin beta-4 (tmsb4x), coactosin-like protein (cotl1), and cofilin-1 (cfl1). circulating gsn was previously reported by others and our group to be decreased in dmd patients relative to controls, 14−16 while the decrease in the blood levels of the remaining actin-binding proteins is novel to this study. finally, the last group of serum protein biomarkers that were found significantly decreased in young dmd patients relative to age-matched controls are also novel to this study, which include proteins involved in cell signaling such as leukosialin (spn), macrophage receptor marco (marco), vitronectin (vtn), galectin-3-binding protein (lgals3bp), and prosaas (pcsk1n). the list of prominently decreased proteins in young dmd boys relative to healthy controls with a minimum 1.5-fold change is shown in table 1 , and a box plot example of these decreased biomarkers is shown in figure 3e ,f. the complete list of proteins identified and quantified in 4 year-old dmd (n = 9) versus age-matched controls (n = 9) and associated statistical analysis are shown in table s3 . biomarker correlation map reveals biomarker groups associated with different pathobiochemical pathways of muscle pathogenesis in dmd. serum or plasma protein levels can provide an indication on irregularities in the muscle integrity and health in dmd. for example, creatine kinase, lactate dehydrogenase, aldolase, troponin, and carbonic anhydrase caiii are commonly used serum markers associated with muscle injury. 12, 13, 21 to examine if newly identified protein biomarkers correlate with well-known muscle injury proteins or other group of proteins, we performed pairwise correlation analysis of biomarkers, which resulted in a data matrix of 59 proteins for 18 participants (9 dmd patients compared to 9 age-matched healthy control candidates). correlation of biomarkers to each other together with hierarchical clustering based on normalized intensity ( figure 3b ) of each protein led to a map of 3481 pearson correlation coefficients in total for both dmd patients and healthy controls. interestingly, one of these groups (orthogonal rectangles indicated with black arrows, figure 4a ) containing muscle-centric proteins such ttn, myom3, tnnc1, myh7, aldoa, and ckb is positively correlated in dmd patients (red rectangle indicated with black arrows, figure 4a , pearson correlation coefficient range between 0.47 and 0.82) and negatively correlated in healthy controls (blue rectangle indicated with black arrows, figure 4a , pearson correlation coefficient range between −0.86 and −0.53). furthermore, we observed an opposite correlation of the remaining muscle-derived proteins and skeletal muscle enzymes such as tnnc2, eno3, ckm, and tpm2 with the other group of proteins. meanwhile, this group of proteins was positively correlated with the other groups of proteins in the normal control (red rectangle indicated with green arrow, figure 4a , pearson correlation coefficient range between 0.32 and 0.89). these proteins turned out to be negatively correlated or have complete loss of correlation in dmd with other proteins such as plasma membrane antigen (kctd3) known for its implication in protein−protein homo-oligomer formation, e3 ubiquitin-protein ligase (rnf34) involved in apoptotic processes, and lactotransferrin (lft) known for its each protein is listed with its fold change (dmd/healthy controls) and two-sided p value corrected with adjusted p value and by permutationbased fdr of 0.05. b isoform. c adjusted. http://pubs.acs.org/journal/acsodf article antimicrobial activity (blue rectangle indicated with green arrow, figure 4a , pearson correlation coefficient range between −0.691 and 0.1). in drosophila, knockdown of rnf3 (drnf34) has been reported to promote mitochondrial biogenesis, improve exercise capacity in muscle, and extends climbing time to exhaustion in moderately aged flies. 20 the reduction of rnf34 in serum of dmd could be due to a compensatory mechanism in humans where the reduction of rnf34 levels in serum in dmd correlated to the muscle protein increase in the serum due to muscle damage. more importantly, we found a group of mostly significantly decreased extracellular matrix proteins in dmd (e.g., igfals, plg, c4bpa, c4bpb, cpn1, gc, vtn, and c2) to positively cluster with each other in dmd ( figure 4b , pearson http://pubs.acs.org/journal/acsodf article correlation coefficient range between 0.32 and 0.93) while exhibiting variable correlation in healthy controls. these proteins are involved in cell adhesion and tissue remodeling (e.g., igfals, vtn, and plg) as well as complement cascade activation and inflammation (c4bpa, c4bpb, c2, and cpn1). this finding suggests a regression or deficit in immunity in dmd, as reported previously. 23 we also found a group of proteins for which levels decrease in dmd and whose positive correlation in normal controls is disturbed in dmd patients ( figure 4c ). these proteins are mostly located in the extracellular matrix and plasma membrane. proteins such as agt, cpn2, basp1, spp1 and cndp1, fcn1, lgals3bp, and defa1 are involved in vasoconstriction, extracellular matrix assembly, complement activation, cell defense response, organ tissue development (e.g., diaphragm), biomineral cell differentiation (e.g., osteoblast), and positive regulation of bone resorption. these proteins are positively correlated to each other in the normal control (pearson correlation coefficient range between 0.38 and 0.89), while in dmd, their levels were significantly decreased, and the correlation was disturbed ( figure 4c ). this latest finding suggests that organ growth and bone development in natural defense systems are significantly impaired in dmd. this result corroborates with clinical observations from other groups. 23−25 confirmation of a subset of newly identified dmd biomarkers using elisa. to verify the reliability of our new multiplex workflow method approach, we performed elisa assays to confirm a set of biomarker candidates using the same cohort of dmd patients and healthy controls as in ms analysis. the confirmation analysis was performed using elisa assay for cfl1, fcn1, and plec. elisa analysis was performed in duplicate for both the standard curve and the samples with cv < 15% in all analysis. the elisa data agree with mass spectrometry data and show that cfl1 and fcn1 were both significantly decreased in dmd ( figure 4d ,e) by approximately 1.9-fold (p = 0.009) and 3-fold (p = 0.017), respectively. contrary to mass spectrometry data, plec was found to be slightly decreased by 1.2-fold (p = 0.008) using elisa in dmd patients compared to healthy subjects ( figure 4f ). plectin is a large actin-binding protein (highly expressed in muscle and heart) that links intermediate filaments (if) to dystrophin−glycoprotein complexes (dgc) and integrin complexes and anchors intermediate filaments to desmosomes. 26, 27 the elisa result of plectin analysis could be explained with the lack of specificity or interference observed with the immunoassay. 17, 28 indeed, the antibody could recognize a common sequence of a protein present in different proteoforms without being able to distinguish between them. 29 human plectin protein (approximately 500 kda) has several isoforms (nine isoforms in universal protein resource (uniprot)) with 96% sequence homology produced by alternative splicing. 26 antibody specificity for a specific isoform is needed for data interpretation but is not often possible. an example of such interferences has been reported for antibodies against the glycated hemoglobin form hba1c that also crossreacted with the glycosylated form of hemoglobin. 30 mass spectrometry was able to quantify two to six unique peptides of isoform-1 of plectin-1 (plec) and aqqqaeaer and qveeaer peptide sequences across all samples used for statistical analysis. development of blood-accessible biomarkers in dmd had become attractive in the past few years owing to the growing number of clinical trials and the need of reliable and sensitive outcome measures to assess disease progression and response to therapies. 31 current outcome measures such as timed functional tests, although important, are often subjective, can be used for ambulatory patients only, and might require longer clinical trials to observe meaningful changes. 4−6,8 serum biomarkers are rather objective and can be used as monitoring tools in clinical trials if associated with the disease outcomes and drug effect. in the past few years, affinity-based serum proteome profiling methods such as somascan aptamer-based technology and antibody bead array have contributed to the discovery of a large number of serum protein biomarkers in dmd. 8, 14, 16 although they are highly multiplexing and powerful, these techniques are inherently less quantitative and suffers from the epitope effect and specificity for certain target proteins that exist as multiple isoforms. 17, 32 furthermore, potential biomarker candidates for which there are no aptamers or antibodies available can be overlooked. in this study, we optimized and standardized a tmt multiplex assay workflow featuring high specificity and reproducibility. the workflow consisted of depletion of the 12 most abundant serum proteins, followed by in-solution digestion, tmt tagging, high-ph fractionation, and lc−ms/ms analysis. the overall workflow tested with three different starting amounts of total serum proteins and in triplicate for each experiment was found to be reproducible and stable over time. more than 600 proteins on average were identified and quantified across all samples with cvs < 16% for the serum protein depletion process and cv < 15% for 95% of protein quantification using the tmt-based mass spectrometry approach. our standardized workflow was implemented to compare the serum proteome profiles of untreated 4 year-old dmd patients (n = 9) and age-matched healthy controls (n = 9). we reliably identified 38 elevated and 50 decreased proteins in serum samples of the dmd group relative to the control group (p < 0.05, adjusted for multiple testing). as expected, the majority of elevated proteins were of muscle origin and reflect sarcolemma instability even in young preasymptomatic patients. although 60% of these muscle injury-associated biomarkers were previously reported by others and our group, 8,9,11,14−16 additional muscle-associated proteins were identified and are novel to this study. these included telethonin (tcap), plectin (plec), smoothelin-like protein 1 (smtnl1), pdz and lim domain protein 5 (pdlim5), and tropomyosin alpha-1 and beta chain (tpm1, tpm2), which to the best of our knowledge, were not described before. importantly, the fold change of these muscle injury proteins in dmd patients relative to controls was somewhat lower than the fold changes reported in earlier studies using affinity-based assays such as antibody bead arrays or somascan aptamer assay. for instance, circulating ck-m and ca3 were found to be 65-fold and 75-fold higher in young dmd patients relative to age-matched controls using the somascan aptamer 14 method, while these same proteins were found to be only 6.3-fold and 4.4-fold higher in dmd patients relative to controls using the tmt mass spectrometry method. the 4.4fold value in ca3 levels in dmd patients relative to controls determined by the tmt method was closer to the 10-fold acs omega http://pubs.acs.org/journal/acsodf article value obtained for this same protein using an absolute quantitative assay. 33 the discrepancy in fold change observed for ck-m between tmt mass spectrometry data and previously reported somascan data could be due to the fact that different techniques measure different things. in dmd, circulating creatine kinase exists as ck-m and ck-b and as homodimers and heterodimers. somascan might lack specificity and might measure both ck-m and ck-b and the different dimers. indeed, we have superimposed somascan data of ck-m and ck-b using the same set of samples and the r 2 was almost equal to 1 (data not shown), indicating that somascan was not able to distinguish between circulating ck-m and circulating ck-b. mass spectrometry, on the other hand, enabled measurement of ck-m and ck-b separately. further experiments using highly reliable absolute quantification methods are needed to resolve this discrepancy seen in the ck fold changes between dmd patients and age-matched healthy controls using these different techniques. other elevated biomarker candidates identified in this study included some glycolytic enzymes such as atp-dependent pfkm, ugp2, gstm2, and psmb1. these enzymes are also abundant in skeletal muscle and their release into the blood circulation could reflect sarcolemma instability. in this study, we have also identified 50 proteins whose circulating levels were significantly decreased in young dmd boys compared to controls. several of these decreased biomarker candidates in dmd patients relative to controls, namely, cnpd1, agt, spp1, gsn, omd, and plg, were previously reported to be decreased in young dmd patients relative to controls using an independent somascan technique. 14 additionally, several proteins that were found to be decreased in dmd patients relative to controls using tmt technology were reported to be unchanged in their levels between dmd patients and controls using the somascan technique. 14 these included carbonic anhydrase 2 (ca2), tkt, fcn1, and galectin-3-binding protein (lgals3bp). this discrepancy between the tmt mass spectrometry assay and somascan assay could be due to the epitope effect that might hinder the binding of the aptamer in the somascan assay in case the target is in complex with another protein and thus provide different quantitative results than mass spectrometry where all proteins are denatured and dissociated before analysis. the newly identified biomarkers that decreased in untreated young dmd patients relative to controls included tmsb4x, cotl1, cfl1, tkt, blvrb, among others. the serum biomarker proteins that were found to be decreased in young dmd patients relative to controls have mostly enzymatic activity, for instance, already described dmd biomarkers such as cndp1 9 but also new biomarkers such as ltf, fuca1, vnn1, plg, cpn1, enpep, ext1, and tkt. cpn1 is an enzyme that protects the body from potent vasoactive and inflammatory peptides. cpn1 cleaves cterminal arginine and lysine residues from complement anaphylatoxins (inactivation of c3a and c4a with significant reduction of c5a, kinins, and ckm) found in the bloodstream. 34, 35 we hypothesize that the decrease in circulating cpn1 is to reduce the complement activation cascade, a retro control mechanism used to reduce inflammation and attack of organ following fibrosis, muscle damage, and protein leakage in the bloodstream. 34−36 a small proportion of decreased serum biomarkers are of muscle origin, for example, known dmd biomarkers such as gsn but also new biomarkers such as tmsb4x, cotl1, and cfl1. these groups of proteins all interact with and bind actin. as reported for circulating gsn, these proteins might be involved in scavenging toxic circulating actin. 37 their decline in the circulation could be attributed to the fact that the formed complex with actin is cleared by the liver. 38 the last group of decreased biomarkers includes those involved in g protein-coupled receptor binding or transmembrane signaling receptor activity and scavenger receptor activity. this latest protein group includes new biomarkers such as spn, marco, lgals3bp, pcsk1n, and vtn. vtn is a glycoprotein largely found in serum or plasma, which is produced mainly in the liver. it has different roles in complement system regulation. 39 reduced level of vtn in serum has been shown to be related to liver disease. 40 indeed, in dmd and becker muscular dystrophy (bmd), several severe cases of defects in hepatocytes and nonalcoholic fatty liver disease were reported in pediatric patients. 41, 42 recently, cases of a reduction of the plasma vtn were reported in patients with myasthenia gravis (mg), a neuromuscular disease with similar symptoms as dmd, mainly muscle weakness and rapid fatigue. 43 another protein found in this group, prosaas (pcsk1n), is a neuroendocrine with multiple functions including fetal neuropeptide pro. 44 prosaas is known to inhibit proprotein convertase 1 (pc1/ 3), a major protein produced in neuroendocrine cells, which regulates the proteolytic cleavage of neuroendocrine peptide precursors. 45, 46 pcsk1n is enzymatically cleaved into different neurosignaling peptides including pen, len, and saas with roles as neurotransmitters. the alteration in pck1n levels can possibly affect pc1/3 and lead to endocrine system dysregulation reported in dmd 47, 48 or in other neuromuscular diseases such as fibromyalgia (fm). 49 as prosaas-derived peptides, two peptides were identified and quantified, one of which was specific to prosaas (aeaqeaedqqar) and not to pen, len, or saas fragment of neurotransmitters. this suggests that the change in the level of prosaas is driven by this peptide and not by the neurotransmitter peptides, which can bias prosaas protein quantitation. taken together, these findings add deeper insight into the understanding of the dmd disease mechanism and pathology in young patients. we correlated all serum biomarkers filtered based on 100% quantitative data completeness. this led to a data matrix of 59 proteins for 18 participants. the biomarkers' correlation to each other together with hierarchical clustering based on normalized intensity of each protein generated a map of 3481 pearson correlation coefficients in total for both dmd patients and healthy controls. this unbiased analysis led remarkably to the clustering of proteins into two main groups. our findings revealed impairments in the natural defense system, translated with decreases in the levels of a group of proteins in sera of dmd patients relative to healthy controls. these proteins are involved in cell adhesion and tissue remodeling (e.g., igfals, vtn, and plg) as well as proteins involved in complement cascade activation, regulation, and inflammation (c4bpa, c4bpb, c2, and cpn1). this finding suggests a regression or deficit in immunity in dmd, as previously reported. 23 also, we identified a group of proteins mostly located in the extracellular matrix and plasma membrane, such as agt, cpn2, basp1, spp1 and cndp1, fcn1, lgals3bp, and defa1. these proteins are involved in vasoconstriction, extracellular matrix assembly, complement activation, cell defense response, organ tissue development (e.g., diaphragm), biomineral cell differentiation (e.g., osteoblast), and positive regulation of bone acs omega http://pubs.acs.org/journal/acsodf article resorption. this finding indicates that organ growth and bone development are significantly disturbed in dmd. this result corroborates with clinical observation from other groups. 23−26 we recognize that sample size is one of the limitations of our current study. this is a recurrent question, especially when dealing with a rare disease such as dmd. we had some challenges enrolling and collecting more samples from 4 yearold boys during the period of the study. however, in this pilot study, although it is preliminary and uses a small sample size, we have confirmed previously reported biomarkers. we have also identified novel biomarkers that were significantly different between young dmd boys and age-matched healthy controls with a good p value adjusted for multiple testing. we have not noticed any large variations in the level of candidate biomarkers between patients. nevertheless, follow-up studies using a larger sample size are needed to validate these novel biomarker candidates and define their physiological significance and relation with early-stage dmd pathogenesis. the second limitation of our study is the identification and quantification of a lower number of serum proteins after the depletion step compared to previous studies. 50, 51 this is mainly due to differences in sample preparation between this study and previous studies. indeed, previous studies used intensive sample fractionation (>80 fractions per sample) and online chromatographic separation during the fractionation step, requiring more resources and machine time than our current workflow where only 18 fractions were collected from the spin column and analyzed by lc−ms/ms. our simplified method will be easy to implement by others for future validation studies. the third limitation of our study is the lack of a specific and validated elisa assay to confirm newly identified candidate biomarkers. this is also a recurrent question when it comes to validating proteomics data. biomarker discovery and validation are an ongoing effort and more specific and sensitive elisa assays or alternative orthogonal methods are needed to achieve this goal. we implemented and standardized a serum proteome profiling workflow, which implements serum-abundant protein depletion and the sensitivity gained from high-ph reversed-phase peptide fractionation and ms-based tandem mass tag (tmt) multiplex quantitation approach. we evaluated our workflow on 4 year-old untreated dmd cases and age-matched healthy controls to define candidate biomarkers associated with the early stage of the disease. a large number of these biomarkers confirmed the previous studies performed by other groups and us. however, several new biomarkers were identified in this study. these findings support the efficiency, reliability, sensitivity, and capability of our serum biomarker study workflow in biomarker discovery. also, we brought new insight into the assessment of the disease pathogenesis in young dmd patients with a new panel of biomarker signatures such as proteases and actin-binding proteins. further examination of serum protein biomarkers using our workflow in presymptomatic very young dmd patients less than 2 years old might provide further insight into muscle pathogenesis and provide tools to assess disease severity and response to intervention in dmd infants. collection. in this study, we used serum samples from a subset of young dmd patients (average age, 4.38 ± 0.24 years old; n = 9) and from age-matched healthy controls (average age, 4.47 ± 0.32; n = 9). dmd patients were enrolled through the cooperative international neuromuscular research group−duchenne natural history study (cinrg-dnhs). 18 the study protocol was approved by institutional review boards at all participating institutions and informed written consent was obtained from the parents of the participants or their legal guardians. serum samples from healthy control pediatric donors were obtained from a third party company (bioivt, hicksville, ny, usa). detailed patient demographics and characteristics are listed in table s1 . serum samples were prepared following a rigorous standardized operating protocol and stored in small workable aliquots at −80°c in polypropylene cryogenic vials (thermo scientific nalgene) to avoid repetitive freeze-thaw cycles. the commercial serum (catalogue #h4522) was purchased from sigma-aldrich. top 12 serum-abundant protein depletion and quality control. protein concentration was determined using the pierce bca protein assay kit (thermo fisher scientific, ll, usa) according to the manufacturer's instructions and measurements were performed using the spectramax i3x microplate reader (molecular devices, san jose, ca). serum aliquots containing 300, 600, or 900 μg of total proteins (corresponding to 5−14 μl of serum) were processed for depletion of the top 12 serum-abundant proteins using pierce top 12 abundant protein depletion spin columns (thermo fisher scientific, ll, usa). briefly, the depletion spin columns were equilibrated at room temperature (30 min), the column's screw caps were removed, and an adequate amount of total protein serum was directly added to the resin slurry into each column. columns were recapped and inverted several times until the resin was completely suspended in the solution and then incubated with gentle end-over-end mixing for 90 min at room temperature using a tube revolver (thermo fisher scientific, ll, usa) set at 19 rpm. after the incubation, the bottom closure of the columns was twisted off and the cap was loosened. the column was then placed in 2 ml protein lobind tubes (eppendorf ag, hamburg, germany) and centrifuged at 1000g for 5 min to collect the remaining serum depleted sample (430−470 μl). sample processing quality control was checked by gel electrophoresis using precast nupage 4−12% bis-tris protein gels (thermo fisher scientific, ll, usa). aliquots containing 3 μg of total proteins of depleted serum samples were mixed with 5 μl of nupage lds sample buffer and 2 μl of 500 mm pierce dithiothreitol (dtt) (thermo fisher scientific, ll, usa) and heated at 70°c for 5 min. samples were uploaded into the wells and gel electrophoresis was run for 1 h using mops sds running buffer (thermo fisher scientific, ll, usa) at a constant voltage of 200 v in an xcell surelock electrophoresis cell (thermo fisher scientific, ll, usa). the gel was stained with 15 ml of bio-safe coomassie (bio-rad laboratory, ca, usa) and then destained overnight with distilled water. gel images were taken using an azure c300 imager (azure biosystems, ca, usa) and analyzed using open-access software (gelquant.net). in-solution digestion of depleted serum samples and tmt derivatization. all the reagents used in this experiment were from tmtsixplex isobaric mass tagging kit (thermo fisher scientific, ll, usa) unless indicated. aliquots of 140−250 μl containing 50 μg of total proteins from each sample were vacuum-dried to 30 μl and denatured with 20 μl of 1% sodium dodecyl sulfate (sds) heated at 50°c for 20 min. disulfide bonds were reduced by adding 5 μl of 200 mm tris(2-carboxyethyl) phosphine (tcep) followed by incubation at 55°c for 1 h. free thiols were then alkylated by adding 5 μl of 375 mm iodoacetamide (iaa) followed by incubation at 40°c for 1 h in the dark. proteins were then precipitated by adding 370 μl of pure chilled acetone (−20°c), followed by centrifugation at 15000 rpm for 30 min using an eppendorf centrifuge 5424 (eppendorf ag, hamburg, germany). the precipitate from each sample was kept at −20°c overnight (16 h). the mixture was then centrifuged again the next day and the supernatant was gently removed from the protein pellet. then, 100 μl of 50 mm triethyl ammonium bicarbonate (teab) was added to the protein pellet and vortexed for 1 min. for protein digestion, a total of 20 μl of 0.05 μg/μl pierce trypsin protease, ms grade (thermo fisher scientific, ll, usa), was added to each sample in two steps for a total enzyme/protein ratio of 1:50 (w/w). for the first 4 h of proteolysis, only 10 μl of the trypsin was added to each sample and the remaining 10 μl was added to each sample for overnight protein digestion. during the entire digestion process, samples were incubated at 37°c using a thermomixer c (eppendorf ag, hamburg, germany) set 450 rpm. the peptide derivatization with tmt label reagents (thermo fisher scientific, ll, usa) was performed according to the manufacturer's procedure with slight modification. vials of tmt reagent (containing 0.8 mg of each tag reagent) were reconstituted in 100 μl of anhydrous acetonitrile and 50 μl of resulting solutions was added to the corresponding digested samples (50 μg of protein digested) and incubated at 27°c for 2 h with shaking at 450 rpm. to quench each reaction, 8 μl of 5% hydroxylamine was added to each sample and incubated for 1 h at 27°c with shaking. to multiplex the sample before the lc−ms/ms analysis, 42 μl of each tmt-labeled sample was combined in either 4-plex, 5-plex, or 6-plex in new eppendorf tubes, dried, and stored at −80°c until high-ph reversedphase fractionation. for the 4 year-old serum study, combined samples shared in common the same digested control sample labeled with tmt 126 tag, which was selected for the normalization of data. high-ph reversed-phase peptide fractionation. vacuum-dried samples were reconstituted in 300 μl of 0.1% trifluoroacetic acid (tfa) and fractionation was performed using pierce high-ph reversed-phase peptide fractionation kit (thermo fisher scientific, il, usa) according to the manufacturer's procedure with slight modification. briefly, columns were conditioned with successive cleaning steps using pure acn (300 μl twice) and washing steps with 0.1% tfa (300 μl twice) with centrifugation set at 1000g for a 4 min duration. each sample (300 μl) was loaded onto the columns and centrifuged at 500g for 8 min. the operation was repeated once again and the flow-through (ft) was collected. the columns were then washed with water (200 μl twice) by centrifugation at 500g for 6 min, and the wash (w) was then collected. peptide elution was performed with 300 μl of different elution buffer mixtures made with acn and 0.1% triethlyamine (tea) at 1000g centrifugation force for 8 min each. in total, 16 consecutive fractions were collected with elution buffers gradually increasing by 5% acn increments up to 80%. the ft, w, and 16 collected factions were dried using a speedvac vacuum concentrator and stored at −20°c until nanolc−ms/ms analysis. liquid chromatography−tandem mass spectrometry analysis. collected peptide fractions were reconstituted in 20 μl of water containing 0.1% formic acid (fa) (v/v) and 10 μl of samples was injected. peptide separation was carried out using an ultimate 3000 rslcnano system (dionex/thermo fisher scientific). for each analysis, the sample was loaded into an acclaim pepmap trap column (2 cm × 75 μm inner diameter, c18, 3 μm, 100 a; dionex, ca, usa) at 3.5 μl/min with aqueous solution containing 0.1% fa and 2% acn (v/v). after 8 min, the trap column was set online with an easy-spray acclaim pepmap rslc analytical column (50 cm × 75 μm inner diameter, c18, 2 μm, 100 a; dionex, ca, usa). peptides were eluted by applying a mixture of solvents a and b. solvent a consisted of hplc-grade water with 0.1% fa (v/ v), and solvent b consisted of hplc-grade acetonitrile (80% acn) with 0.1% fa (v/v). separations were performed using a linear gradient of 5 to 50% solvent b at 300 nl/min over 100 min followed by a 4 min linear increase of acn percentage up to a washing step (9 min at 99% solvent b) and a 5 min linear decrease of acn percentage up to an equilibration step (20 min at 2% solvent b). the total analysis run time was 145 min. lc−ms/ms data-dependent acquisition was performed using a q-exactive hf mass spectrometer (thermo scientific, bremen, germany) in positive mode. for ionization, an easy-spray es233 (thermo scientific, bremen, germany) was used with a voltage set at 2 kv, and the capillary temperature set at 350°c. full ms scans were acquired in the orbitrap mass analyzer over an m/z 375−1800 range with a resolution set at 60,000 for m/z 200. the target automatic gain control value of 3 × 10 6 was used with a maximum allowed injection time (maximum it) of 80 ms. for ms/ms, an isolation window of 1.2 m/z was utilized. the 20 most intense peaks with a charge state between 2 and 5 were selected for fragmentation using high-energy collision-induced dissociation with a stepped normalized collision energy of 27−32. the tandem mass spectra were acquired with fixed first mass of m/z 110 in the orbitrap mass analyzer with a resolution set at 30,000 for m/z 200 and an automatic gain control of 5 × 10 5 . the ion intensity selection threshold was 9.1 × 10 3 , and the maximum injection time was 110 ms. the dynamic exclusion time was 45 s for the total run time of 145 min. mass spectrometry data processing, protein identification, and quantitation analysis. all data files were collected and processed with a specific workflow designed in proteome discoverer 2.2 (thermo fisher scientific). searches were performed against homo sapiens (taxid = 9606) protein sequence database downloaded from www.uniprot.org on 15 november 2017 (42,182 entries) using sequest ht (thermo fisher scientific) with precursor and fragment mass tolerance respectively set at ±10 ppm and ±0.05 da and the following dynamic modifications: carbamidomethyl on cysteine, acetyl on protein n terminus, oxidation on methionine, tmt 6-plex modification on lysine, and n terminus of peptides. the targetdecoy database search allowed us to control and estimate the false positive discovery rate at 1% for the peptide and protein as well. quantitation of 4-plex (126, 127, 128, and 129), 5-plex (126, 127, 128, 129, and 130) , and 6-plex (126, 127, 128, 129, acs omega http://pubs.acs.org/journal/acsodf article 130, and 131) were set up in proteome discoverer by the activation or deactivation of unused channels from original tmt 6-plex quantification method. the reporter ion quantifier tolerance was set at 0.05 da with the integration method as the most confident centroid. the peptides used for the quantitation are only unique peptides and the spectra with missing channels were rejected. the reporter quantitation was corrected with isotopic impurity of reporter values for minimum signal-to-noise ratio (s/n) set at 1.5. the protein abundance was calculated as the sum of abundance of the corresponding protein in each fraction. all data were normalized to the same control sample labeled with tmt 126, which was used across all multiplexing analyses. the mass spectrometry proteomics data are made available via proteomexchange under the project named dmd biomarkers in 4 year-old boys, dmd patient vs control study, with identifier pxd013174. enzyme-linked immunosorbent assay (elisa). a subset of identified biomarker candidates including cofilin-1 (cfl1), ficolin-1 (fcn1), and plectin (plec) were examined using elisa assay with kits corresponding respectively to catalogue numbers okeh01091, okdd00565, and okbb002505 (aviva systems biology, san diego, ca, usa) on the same serum samples analyzed by mass spectrometry. serum samples from dmd patients and agematched healthy controls were diluted at 1:300 for cfl1, 1:250 for fcn1, and 1:400 for plec and the analyses were performed according to the manufacturer's instructions using the spectramax i3x microplate reader (molecular devices, san jose, ca). western blot. aliquots containing 20 μg of total proteins from each sample including molecular weight markers, ibright prestained protein ladder ranging from 11 to 250 kda, were loaded in different wells of precast criterion xt 12% bis-tris (1 mm, 18-well) gels. gels were run at 200 v for 1 h and transferred onto the nitrocellulose membrane (thermo fisher) at 0.07 a for 17 h. membranes were then washed with pbst and cut at the 55 kda ladder marker for separate antibody incubations for haptoglobin and gelsolin. the membranes were then washed in bio-rad blotting-grade blocker and separately incubated in anti-haptoglobin (ab131236) and anti-gelsolin (ab75832) at 1:10000 and 1:25000 dilutions, respectively. membranes were washed again and incubated in anti-rabbit igg-hrp (na934v) antibodies at 1:3000 dilutions. membranes were revealed using an azure c300 imager (azure biosystems, ca, usa) and image-analyzed using open-access software (gelquant.net) with fluorescence settings at 10 s of absorbance. the supporting information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c03206. supporting table descriptions; hierarchical clustering of other biomarkers found with missing data; gene ontology molecular function analysis (pdf) dmd patient and age-matched healthy control demographics; list of proteins identified and quantified in triplicate experiment using commercial serum; list of proteins identified and quantified in serum samples of young dmd patients relative to healthy controls; go molecular function ontology (mfo) analysis of the 38 elevated and 50 decreased proteins in serum samples of dmd patients relative to healthy controls (xlsx) ■ author information corresponding author united states; email: yhathout@binghamton.edu authors tchilabalo d. alayi − department of pharmaceutical science, school of pharmacy and pharmaceutical sciences united states holly r. smith − department of pharmaceutical science, school of pharmacy and pharmaceutical sciences and department of biochemistry children's national health system children's hospital of richmond at vcu the cinrg-dnhs was funded by the u.s. department of education/nidrr (#h133b031118 and #h133b090001) evidence-based path to newborn screening for duchenne muscular dystrophy the pathogenesis and therapy of muscular dystrophies corticosteroid treatments in males with duchenne muscular dystrophy: treatment duration and time to loss of ambulation advances in the treatment of duchenne muscular dystrophy: new and emerging pharmacotherapies restoring dystrophin expression in duchenne muscular dystrophy: current status of therapeutic approaches the 6-minute walk test and other endpoints in duchenne muscular dystrophy: longitudinal natural history observations over 48 weeks from a multicenter study revised north star ambulatory assessment for young boys with duchenne muscular dystrophy discovery of serum protein biomarkers in the mdx mouse model and cross-species comparison to duchenne muscular dystrophy patients proteomics profiling of urine reveals specific titin fragments as biomarkers of duchenne muscular dystrophy muscle-derived proteins as serum biomarkers for monitoring disease progression in three forms of muscular dystrophy affinity proteomics within rare diseases: a bio-nmd study for blood biomarkers of muscular dystrophies aartsma-rus, a. tracking disease progression non-invasively in duchenne and becker muscular dystrophies longitudinal serum biomarker screening identifies malate dehydrogenase 2 as candidate prognostic biomarker for duchenne muscular dystrophy the fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry cinrg investigators. the cooperative international neuromuscular research group duchenne natural history study-a longitudinal investigation in the era of glucocorticoid therapy: design of protocol and the methods used iii isobaric labeling-based relative quantification in shotgun proteomics proteomics software suite for in-depth mass spectrometry data analysis using grid computing biochemical markers of muscular damage mass spectrometry-based identification of muscle-associated and muscle-derived proteomic biomarkers of dystrophinopathies role of complements and immunoglobulins in duchenne muscular dystrophy short stature in duchenne muscular dystrophy: a study of 34 patients patterns of growth in ambulatory males with duchenne muscular dystrophy plectin isoforms as organizers of intermediate filament cytoarchitecture plectin 1 links intermediate filaments to costameric sarcolemma through -synemin, -dystrobrevin and actin advantageous uses of mass spectrometry for the quantification of proteins a review of variant hemoglobins interfering with hemoglobin a1c measurement aptamers they trust: the caveats of the somascan biomarker discovery platform from somalogic. circulation carbonic anhydrase iii in serum in muscular dystrophy and other neurological disorders: relationship with creatine kinase a pleiotropic regulator of inflammation structure and function of human plasma carboxypeptidase n, the anaphylatoxin inactivator targeted disruption of the gene encoding the murine small subunit of carboxypeptidase n (cpn1) causes susceptibility to c5a anaphylatoxin-mediated shock plasma gelsolin: indicator of inflammation and its potential as a diagnostic tool and therapeutic target role of plasma gelsolin and the vitamin d-binding protein in clearing actin from the circulation the complement factsbook quantitation of vitronectin in serum: evaluation of its usefulness in routine clinical practice the role of liver in muscular dystrophy duchenne and becker muscular dystrophy presenting as nonalcoholic fatty liver disease plasma vitronectin is reduced in patients with myasthenia gravis: diagnostic and pathophysiological potential coexpression of proprotein convertase spc3 and the neuroendocrine precursor prosaas 1 a novel function for prosaas as an amyloid anti-aggregant in alzheimer's disease prohormone convertase 1/3 is essential for processing of the glucose-dependent insulinotropic polypeptide precursor endocrine aspects of duchenne muscular dystrophy muscular dystrophy tracking and research network md star. bone health and endocrine care of boys with duchenne muscular dystrophy: data from the md starnet systematic analysis of the cerebrospinal fluid proteome of fibromyalgia patients quantitative workflow for sensitive biomarker discovery in plasma yields novel candidates for early myocardial injury the pride database and related tools and resources in 2019: improving support for quantification data key: cord-292688-w4zvfkyl authors: tooze, sharon a title: biogenesis of secretory granules in the trans-golgi network of neuroendocrine and endocrine cells date: 1998-08-14 journal: biochim biophys acta mol cell res doi: 10.1016/s0167-4889(98)00059-7 sha: doc_id: 292688 cord_uid: w4zvfkyl secretory granule formation requires selection of soluble and membrane proteins into nascent secretory granules, and exclusion of proteins not required for the function of secretory granules. both selection and exclusion presumably can occur in the compartment where assembly of the secretory granule begins, the trans most cisternae of the golgi complex. current research focused on the initial stages of secretory granule formation includes a search for the ‘signals’ which may mediate active sorting of components into secretory granules, and the role of aggregation of regulated secretory proteins in sorting. in addition, the temporal sequence of the sorting events in the golgi, and post-golgi compartments has gained much attention, as summarized by the alternative but not mutually exclusive ‘sorting for entry’ vs. ‘sorting by retention’ models. ‘sorting for entry’ which encompasses the most popular models requires selection of cargo and membrane and exclusion of non-secretory granule proteins in the tgn prior to secretory granule formation. ‘sorting by retention’ stipulates that protein selection or exclusion may occur after secretory granule formation: secretory granule specific components are retained during maturation of the granule while non-secretory granule molecules are removed in vesicles which bud from maturing secretory granules. finally, some progress has been made in the identification of cytosolic components involved in the budding of nascent secretory granules from the tgn. this review will focus on the recent data concerning the events in secretory granule formation which occur, in the trans-golgi network. dense core secretory granules are found in all exocrine, endocrine, neuroendocrine, and neuronal cells. vesicles which are similar to secretory granules are also found in cells of the haemopoetic and immune systems. formation of these vesicles is initiated in the trans-golgi network (tgn) and results in the secretory proteins being packaged in a concentrated form in the secretory granules. unstimulated exocytosis of these granules occurs at low levels, but ampli¢ed secretion from the cell of proteins within secretory granules is regulated exclusively via external stimuli which triggers the fusion of the secretory granule membrane with the plasma membrane. secretory granules in endocrine and neuroendocrine cells have a very similar composition. the limiting membrane contains proteins required for secretory granule acidi¢cation, transport, targeting and fusion, while the dense core content is composed of a set of soluble proteins, some of which are substrates, some of which are enzymes, others of which have an as yet unidenti¢ed function. it is known that there is variation in the content of the secretory granule, depending on the type of tissue the cell is derived from, however, very little is known about the membrane composition and the variation which might be present in the molecules involved in secretory granule biogenesis, targeting and fusion. research into the formation of the secretory granules from the tgn is mainly concerned with how the soluble regulated secretory proteins are targeted into the secretory granule, how soluble non-secretory granule proteins are excluded, if at all, what factors determine the membrane composition, and how secretory granule budding occurs from the tgn. this research is complicated by the fact that secretory granule formation could occur in parallel with formation of vesicles directed to endosomes or the plasma membrane. this review will explore the recent progress in these areas and attempt to compare the current models of secretory granule biogenesis from the tgn in endocrine cells and neuroendocrine cells. regulated secretory proteins are packaged into nascent secretory granules (which are referred to as immature secretory granules (isgs)) in a process that is thought to be initiated by selective aggregation of the secretory proteins in the tgn, followed by interaction of the aggregate with the tgn membrane which may involve receptor molecules. it is also possible that selection may be achieved by interaction of individual regulated secretory proteins with a putative membrane receptor prior to aggregation. proteins which are not present in regulated secretory protein aggregates, or do not interact with the recep-tor for the regulated secretory proteins, are thought to exit the tgn in constitutive secretory vesicles (csvs). an alternative hypothesis is that there is very little constitutive secretion originating from the tgn of regulated cells, and that most molecules in the biosynthetic pathway exit into isgs. in the isg, the non-secretory granule proteins would presumably be sorted from the regulated secretory proteins via interaction with a receptor and removed from the isg in constitutive-like vesicles. aggregation of the regulated secretory proteins would still occur in the isg and would function to allow retention in the isg by preventing any interaction with receptors for non-secretory granule proteins. in both models, one could make predictions about the stoichiometry of the receptor relative to the ligand, depending on whether it is a single secretory protein interacting with a single receptor, or an aggregate of regulated secretory proteins interacting with a single receptor. in both models, interaction with a receptor would allow removal of non-secretory granule proteins from the forming or maturing secretory granule. an important point of clari¢cation is that there may well be multiple classes of receptors for both regulated secretory proteins and constitutive secretory proteins in the tgn and the isg. in addition, interaction of any ligand with its receptor is most probably transient and disrupted, for example, by changes in the ph of the maturing isg. one mechanism for sorting soluble secretory proteins into secretory granules is via a selective aggregation of a subset of soluble proteins to the exclusion of other soluble, non-aggregating secretory proteins. for e¤cient aggregation, the local concentration of the secretory proteins should be high in the sorting compartment. soluble regulated secretory proteins are for the most part very abundant in cells with a regulated secretory pathway. in particular, a family of proteins known as the granins [1] are highly expressed in most endocrine and neuroendocrine cells. the members of this family of proteins include chromogranin a (cga), chromogranin b (cgb), secretogranin ii (sgii), secretogranin iii, and secretogranin iv (also known as 7b2). based on the primary sequence of the family members, it is thought they have similar structural features which may enable them to e¤ciently aggregate in the tgn [2] . although the exact function of the granins is not known, they might be acting as`helper proteins' [3] . if sorting only occurs via aggregation and does not rely on a speci¢c and stoichiometric receptor-ligand interaction, then e¤cient sorting of prohormones which either have a less than optimal ability to condense, or are present at very low concentration, might be enhanced by`helper proteins' that aid aggregate formation. recently, natori and huttner [4] have demonstrated that increasing the amount of cgb in att20 cells (a cell line derived from the pituitary corticotroph) indeed promoted e¤cient sorting of the 23 kda pomc (pro-opiomelanocortin) fragment into secretory granules, resulting in increased storage of the 23 kda pomc fragment and acth. aggregation of sgii has been shown in vitro as well as in permeabilized tgn membranes, under conditions (low ph, high ca 2 ) believed to be present in the lumenal environment of the tgn [5, 6] . other soluble secretory proteins, other members of the granin family and other proteins found in secretory granules, have also been shown to aggregate under these low ph, high ca 2 conditions (see e.g. [7, 8] ). indeed, morphological evidence demonstrating the presence of aggregates of regulated secretory proteins in the tgn [9, 10] , but not in earlier compartments under normal conditions supports both the location of the initiation of aggregation and its role in sorting. experiments performed several years ago with weak bases, such as chloroquine and ammonium chloride argued that perturbation of the ph in the tgn caused missorting of regulated secretory proteins into the constitutive pathway (see for example [11] ). although the data did not gain complete support [12] these experiments were the ¢rst to suggest that ph may have a role in the sorting of soluble proteins. more recent results obtained with streptolysin o-permeabilized pc12 cells support the idea in that ph may be crucial for sorting of regulated secretory proteins in the tgn [13] . concerning the exact ph and ca 2 requirements in vivo, values for the ph in the tgn of cells lacking a regulated secretory pathway have been proposed in two recent studies (6.17 þ 0.02 and 6.45 þ 0.03) [14, 15] supporting the notion that the tgn is slightly more acidic than previous golgi compartments, but there are still no data available on the free ca 2 concentration in the lumen of the tgn. interpretation of all these results to determine the precise role of ph and ca 2 in the tgn requires accurate measurements for ph and ca 2 in the lumen of the tgn in cells with a regulated secretory pathway. some controversy exists concerning the role of the tgn-localized aggregation event in sorting which may relate to di¡erences between the cellular systems studied. l-cells of the endocrine pancreas secrete large quantities of insulin [16] . insulin forms insoluble, close packed crystalline arrays in the presence of zinc, whereas proinsulin has distinct physical properties which prevent its condensation into insoluble crystals. processing of proinsulin to insulin, which requires two processing enzymes, prohormone converting enzymes (pcs) 1 and 2, acting at two distinct sites (for review see [17] ), is crucial for the condensation of insulin. however, the intracellular location where processing of proinsulin and other prohormones begins is unresolved. there are reports which demonstrate that processing of prohormones by pc enzymes can begin in the tgn [4, 18, 19] whereas other studies indicate that processing is initiated in the isg [20^22] . proinsulin conversion has been demonstrated to occur only in secretory granules ([23^25] and references included therein). therefore, if sorting of regulated secretory proteins into dense cores relies on exclusion of soluble non-granule material through aggregation, or condensation, insulin can only be sorted in a post-tgn compartment. this result also implies that proinsulin cannot be sorted by aggregation in the tgn or any post-tgn compartment. these results have provided the basis for the`sorting by retention' hypothesis [26] (fig. 1 ). this hypothesis suggests that regulated secretory proteins are sorted largely by retention in a post-golgi isg. non-secretory granule proteins, or processing remnants of regulated secretory proteins, such as the c-peptide (which is derived from proinsulin and not found in mature insulin), can be in part removed by constitutive-like vesicle budding from the isg, although the e¤ciency of this removal is likely to be related to whether a selection process (i.e. receptor-mediated) process is employed. thus, the e¤ciency for removal from isgs might be expected to vary for di¡erent proteins (see below). an interesting example which reinforces the importance of the precise location of prohormone processing on sorting comes from experiments done on aplysia californica [27] and lymnaea stagnalis [28] . these two model systems have been used to demonstrate that sorting of proelh (egg-laying hormone) can be in£uenced by the location of cleavage, which in turn presumably a¡ects the hormones ability to aggregate or condense. in aplysia, the proelh is cleaved in the golgi and the two n-terminal and c-terminal intermediates are sorted into two distinct populations of large dense core vesicles (ldcvs) [27] . lymnaea presents a more complicated scenario in that there are two di¡erent types of neurones which produce elh: in one type (type i neurones) processing occurs in the tgn and results in the appearance of two morphologically distinguishable condensed protein cores, containing either the n-terminal or c-terminal region of elh, which are sorted to di¡erent populations of secretory vesicles, leg (large electrondense granule) and ldcvs [28] . in the second type of neurone (type ii), the elh is not cleaved in the tgn and is sorted into the ldcvs as a prohormone, after which it undergoes cleavage to mature elh. this raises an interesting question concerning how proteins exit from the golgi in type i and type ii neurones. it would be of great interest to compare the sorting of true`constitutive' proteins in type i and type ii neurones to see if in type i neurones constitutive secretory proteins are secreted constitutively, implying that csvs are formed directly from the golgi, and likewise if in type ii neurones if these proteins are secreted in a constitutive-like fashion, implying secretion originates from isgs. interestingly, the ldcvs from the two types of neurones di¡er in size, the ldcv from the type i neurone having a size of approximately 150 nm whereas the ldcvs in the type ii neurone was 90 nm [28] suggesting that the composition of the content has an in£uence 6 fig. 1 . depicted in a very simple fashion is a scheme of the sorting mechanisms in the secretory pathway of neuroendocrine and endocrine cells proposed by arvan and colleagues [26] to explain the di¡erent models for formation of regulated secretory vesicles from the tgn, i.e. sorting for entry vs. sorting by retention. also included are other vesicle classes, such as csvs and constitutive-like secretory vesicles, as well as a pathway for lysosomal enzymes (blue circle) and mpr (black cup-shaped object). the low amount of missorted proteins, such as furin [62] , and mpr found in the isg in the top panel have not been illustrated for simplicity. the regulated secretory proteins are shown as yellow circles, and the constitutive secretory proteins are shown as red circles. for simplicity, all coats and coat proteins have been omitted, as well as any putative interactions of the regulated secretory proteins with the membrane. on the ¢nal size. in support of this idea, a size difference has also been seen in the secretory granules in endocrine cells of the pituitary (for review see [29] ). while the data to support`sorting by retention' [30] is compelling, the data to support the`sorting for entry' hypothesis, where the bulk of sorting has been demonstrated to occur in the tgn prior to isg formation, is equally compelling (see above). to reconcile these two hypotheses, it has been suggested [22, 31] that whether sorting is predominantly tgnbased (sorting for entry) or isg-based (sorting by retention) might depend both on the propensity of the major prohormone species to aggregate, or not, and the relative rates of prohormone synthesis and vesicle formation in the tgn. the paradigm for sorting of soluble proteins into vesicles is receptor-mediated endocytosis at the plasma membrane: a receptor binds a ligand and sequesters the ligand for transport to a di¡erent compartment in a coat-dependent fashion. this model has been extended to a variety of intracellular transport events including sorting of regulated secretory proteins in the tgn [32] . regardless of whether a single molecule or a molecular aggregate is sorted, this paradigm has remained attractive enough for investigators to continue to pursue the putative`sorting receptor'. the putative sorting receptor could then interact directly or indirectly with the cytosolic machinery involved in the vesicle formation. the search for a sorting receptor has been approached from both the ligand side, i.e. identi¢cation of sorting signals in the ligand, and the receptor side, i.e. identi-¢cation of membrane proteins which bind regulated secretory proteins. recent experiments have focused on an n-terminal disulphide loop structure found in pomc, provasopressin, pro-oxytocin, prodynorphin, proenkephalin, cga and cgb [1, 33] . the n-terminal 26 amino acids of pomc have been shown to contain a signal which sorts a reporter molecule into secretory granules in att20 cells [34] . it has been further demonstrated that a disulphide bond, within this sequence of 26 amino acids, forms an amphipathic loop structure which may play a role in sorting pomc to isgs [33] . this data is supported by experiments in which the analogous disulphide bond of cgb was disrupted by incubation of pc12 cells with dtt, leading to constitutive secretion of cgb [35] . in pursuit of a receptor which would recognize the sorting motif researchers have used assays based on the binding of solubilized secretory granule membrane components to immobilized regulated secretory proteins. as some granule`matrix' proteins such as cgb exist as membrane-bound isoforms [36] , it has been suggested that the membrane bound forms of such proteins would act as nucleating receptors to recruit soluble regulated secretory proteins [3] . indeed, recent experiments have demonstrated that the membrane associated form of carboxypeptidase e, a granule speci¢c carboxypeptidase [37] , can function as a sorting receptor [38] . these observations are the subject of some debate [31] ; in particular, in light of a recent report by halban [39] where the absence of carboxypeptidase e in a mouse mutant cpe fatafat [40] does not result in abnormal insulin processing. however, the principle behind the observations follows a common theme: soluble regulated secretory proteins appear to interact in a homo-or heterophillic manner with membrane associated forms of regulated secretory proteins. at this stage, however, it could also be that other unrelated bone ¢de membrane proteins act as receptors for the aggregate, such as the ip3 receptor [41, 42] identi¢ed using the same approach with immobilized, soluble cga. a variety of exogenous soluble proteins, such as renin, normally expressed in the kidney [43] to trypsinogen, normally expressed in pancreatic exocrine cells [44] , have been expressed by transfection in endocrine and neuroendocrine cell lines. these exogenous molecules have been found to be secreted in a regulated fashion from the transfected cells lines, suggesting that these proteins have been stored in secretory granules. from these data it was hypothesized that a putative sorting signal, a common sequence or structural feature, appears to be conserved across a variety of systems. this signal is not, however, dominant as fusion of proinsulin with the trans-membrane and cytoplasmic domain of cd5 resulted in targeting of proinsulin to the constitutive pathway and appearance on the cell surface [45] . these experiments demonstrate that a diverse set of soluble molecules can be targeted to secretory granules and cast doubts on the existence of a singlè sorting receptor' because it is di¤cult to reconcile the accurate sorting of such a variety of molecules with a single receptor. gerdes and colleagues [110] have argued that e¤cient sorting of these molecules was obtained because the exogenous molecules were sorted by co-aggregation with the endogenous regulated secretory proteins. therefore identi¢cation of a sorting sequence could only be achieved in essentially a null background of endogenous regulated secretory proteins. they have achieved this experimentally using a vaccinia virus expression system which shuts o¡ host protein synthesis, and have shown that exogenous cgb when expressed in vaccinia virus infected pc12 cells can be e¤ciently sorted to secretory granules but that cgb without the n-terminal disulphide loop (vcys-cgb) was not correctly sorted. furthermore, in the same set of experiments the authors demonstrated that vcys-cgb was correctly and e¤ciently sorted after conventional transfection protocols providing additional support for their hypothesis. using this powerful expression system the authors should be able to begin to identify the requirements for sorting of soluble proteins to the secretory granule. lastly, supplementary to the postulated role of receptors in sorting regulated secretory proteins or aggregates, sorting receptors may also serve the function of selecting an appropriate membrane for the nascent secretory granule. indeed, there must be a recognition mechanism between soluble granule proteins and granule membrane proteins or components to ensure that the membrane which ¢nally envelops the dense core contains all the proteins necessary for secretory granule maturation, storage and exocytosis. again, it is possible that this selection only occurs later in the pathway, in the isg, and that the proteins not required are removed from the isg instead of being left behind in the tgn. at present we have very little knowledge about the membrane composition of the nascent isg which would allow one to speculate on how and where the ¢nal selection is achieved. several years ago it was ¢rst shown that att20 cells, which have a regulated secretory pathway, have a distinct pathway by which they transport proteins to the plasma membrane, the constitutive secretory pathway [46] . more recently, the vesicles involved in constitutive secretion of proteins from pc12 cells, which also have a regulated secretory pathway, have been identi¢ed [47] . it was shown in pc12 cells, using two sulphated proteins (a hspg and sgii), as markers for constitutive and regulated pathways, respectively, that formation of csvs occurs concomitantly with formation of isgs from the tgn. these budding reactions have very similar characteristics to those described below for secretory granules and those identi¢ed using other systems which reconstitute vesicle formation from cells without a regulated pathway (see for example [48] ). formation of csvs appears to be regulated by heterotrimeric gtp-binding proteins [49, 50] , and be dependent upon arf1 [51, 52] . although the ¢delity of sorting into the constitutive and regulated pathways appears to vary in di¡erent pc12 cells clones (compare [47] and [53] ) the principle of sorting di¡erent populations of proteins in the tgn into distinct vesicle populations remains intact in neuroendocrine cells. in contrast, no one has to date identi¢ed a constitutive secretory pathway in endocrine l-cells using a bone ¢de constitutive secretory protein. the c-peptide, derived from processing of proinsulin, has been shown to be secreted constitutively, however, this might be through a constitutive-like pathway originating from the isg [54] . it may be that indeed that no constitutive secretory proteins in csvs are secreted directly from the tgn in l-cells, however, using immunogold labelling techniques csvs have been identi¢ed in l-cells using antibodies to the viral-spike glycoprotein ha of in£uenza virus [55] . at the same time, experiments were done which demonstrated that corona virus virions, which are released constitutively, were found segregated from dense-cores in att20 cells, a neuroendocrine cell line [56] . in retrospect, the ideal experiment would have been to study the transport of coronavirus particles, which have the advantage of being morphologically identi¢able marker for constitutive secretion, in l-cells. however, in the absence of experiments to prove whether secretory proteins are also conveyed by this pathway, the question remains an open one. transport of lysosomal enzymes from the tgn to endosomes occurs via clathrin-coated vesicles (ccvs), and is mediated by binding of the clathrincoat proteins to the mannose-6-phosphate receptor (mpr, see review by ho£ack in this issue). it has been assumed that this pathway exists in all cells. clathrin coats assemble on the tgn via the ap-1 adaptor complex (robinson, this issue) through an interaction with the cytoplasmic domain of receptors, such as mpr, and other proteins such as furin which tra¤c to and from the plasma membrane [57^59]. while the distribution of lysosomal enzymes has not been studied in pc12 cells, recent data using both gh 4 c 1 cells [60] and a l-cell line [26] have demonstrated that some lysosomal enzymes are present in isgs. the appearance of lysosomal enzymes in the regulated secretory pathway provides additional support for the`sorting by retention' hypothesis. these authors [26, 60] were able to demonstrate that lysosomal enzymes can be released from the cells by agents used to stimulate regulated secretion. some of the lysosomal enzymes (cathepsin b) were absent from the msg, while others (cathepsin l) were detected in msgs [30] . these results suggest that in the tgn newly synthesized lysosomal enzymes are incorporated into the nascent secretory granule. this could be due to a missorting of the receptors for lysosomal enzymes, mpr, into secretory granules. however, newly synthesized lysosomal enzymes that have diminished or abolished a¤nity for mprs continue to enter isgs in equal or even greater abundance [26, 30] . as an alternative explanation, there may be a decrease in the e¤ciency of sorting of lysosomal enzymes by the mprs in the tgn of cells with a regulated secretory pathway which allows them to escape from the tgn in other exit pathways, such as the regulated or constitutive secretory pathway, and be secreted or recaptured at a post-golgi location such as the endosome (see review [61] ). a variety of experiments, including those cited above, have con¢rmed the transient appearance of lysosomal enzymes in the regulated pathway, suggesting that lysosomal enzymes are being removed from the maturing isgs. it is assumed that this is occurs via the mpr and ccvs. indeed, this may explain the transient presence of clathrin coats on the isgs. further support for this idea comes from experiments which show that the ap-1 is used on the isg to assemble the clathrin coat [62] , and the mpr has been found in isgs but not msgs in isolated secretory granule fractions derived from pc12 cells (a.s. dittië, submitted). a second protein which is involved in recruiting ap-1 and therefore clathrin onto isgs is furin, an endopeptidase present in tgn membranes. furin, like mpr, is found in isgs but not msgs [63] . these results suggest that furin, like mpr, is removed from the isg via ccvs. the destination of the ccvs, containing either furin, mpr, or both, could be any one of several possibilities, for example, the tgn, early endosomes, or plasma membrane. there is no data at present regarding the content, composition and destination of ccv budding from the isg, and indeed the ccvs themselves have not been identi¢ed. a related question concerns the constitutive-like secretion of proteins which could be considered true markers of the constitutive pathway, and those which are unwanted products from the processing of prohormones, such as c-peptide. it has been proposed that ccvs might be a vesicular carrier for these population of molecules [64] . this raises a very intriguing set of questions: are there multiple classes of ccvs forming from the isg all with a di¡erent cargo?, would these ccvs all have a di¡erent destination? or is there a single species of ccvs forming with a mixed cargo? would this ccv deliver its cargo to one destination, for example, the endosome, from where further sorting would occur? while the mechanism for selection and transport via ccvs is clear when one assumes the cargo associates with a transmembrane receptor, it is more complex for other cargo proteins such as the c-peptide, or those proteins which follow a bulk-£ow pathway [65] which has not been thought to be signal-mediated. how does this signal-less type of cargo enter the ccvs allowing it to be removed? if it is simply a case of exclusion, as formulated by arvan et al. in their`sorting by retention' hypothesis, one would predict it could not be 100% e¤cient and some c-peptide would be found in the medium after stimulation of msg. this has been demonstrated for c-peptide (see [64] and the references therein). alternatively, it may be that multiple vesicular carriers bud from the isg with di¡erent cargo. depending on the cargo, distinct mechanisms could be used for selection to increase the e¤ciency of removal of nonsecretory granule proteins from the maturing isgs. while the mechanisms involved in the formation of both constitutive and regulated secretory vesicles from the tgn is largely unknown there have been recent reports which suggest that both these budding reactions maybe unique in that they function independently of known coat proteins. morphological data based on high voltage electron microscopy suggests that the formation of constitutive secretory vesicles from the tgn involves a`lace-like' coat [66] . the components of this coat have yet to be identi¢ed but may be associated with the p62 complex identi¢ed by howell and colleagues [67] and include p200 [68] . conventional electron microscopy and immunogold labelling has revealed that patches of clathrin are present on the tgn in the vicinity of forming isgs and are also found on isgs [10, 69] . however, there is no biochemical data available to date which supports the role of clathrin in secretory granule formation. recent results concerning the formation of secretory granules have been obtained primarily from two in vitro systems which reconstitute budding from tgn membranes; one using a post-nuclear supernatant derived from pc12 cells [70] and the second semi-intact gh 4 c 1 cells [18] . while in the former assay it is possible to identify two classes of vesicles budding from the tgn, in the latter this has not been possible. however, because there is no data which so far suggests the requirements for formation of an isg are di¡erent from that for a csv, they will be considered as congruent assays and the data ob-tained from both, and other assays monitoring constitutive secretory vesicle formation will be compared. the ¢rst observations made using the in vitro budding assays described above, both of which are atp and cytosol dependent, concerned the requirement for gtp. the formation of secretory vesicles from the tgn requires gtp and is inhibited by non-hydrolysable gtp analogs [18, 47] . in the pc12 cell assay, the same e¡ect was observed with [alf 4 ] à and several other lines of evidence demonstrated that heterotrimeric gtp-binding proteins are involved in the regulation of post-golgi vesicle formation (see [71] for review). the precise function of gtp is not yet clear as there maybe several di¡erent gtp-binding proteins involved in vesicle formation from the golgi, including arf (see below). an intriguing related observation concerns the role of an as yet unidenti¢ed cytoplasmic phosphoprotein as a modulator of the heterotrimeric g protein(s) that regulate vesicle formation from the tgn [50] . a similar requirement has been seen in the semi-intact gh 4 c 1 in vitro assay [72] in experiments using inhibitors of tyrosine kinases and phosphatases. this observation is supported by several other reports from cell-free budding assays which indicate that phosphorylation regulates vesicle formation [48, 73, 74] . a role for tyrosine-phosphorylation in secretory granule biogenesis and/or function has also been suggested by two recent reports that have iden-ti¢ed two highly homologous proteins in the membrane of secretory granules which appear to be nonfunctional protein-tyrosine phosphatases, called phogrin and the ica 512 autoantigen [75, 76] . most of the well characterized transport steps in the cell (for example from the endoplasmic reticulum to the cis-golgi, intra-golgi, plasma membrane to endosome) have been shown to be mediated by vesicles which form by coat-mediated budding reactions from donor compartments. in contrast no known coat, or coat protein, has been demonstrated by morphological or biochemical techniques to be involved in secretory granule formation. as mentioned above, if a coat were required, the most likely coat protein candidate would be clathrin. formation of clathrin-coats on the tgn requires ap-1 (see above) and the monomeric gtpase adp-ribosylation factor, or arf [77, 78] and these components might therefore be thought to function in the formation of secretory granules. however, to date there is no evidence which implicates clathrin in the formation of either constitutive secretory vesicles or secretory granules from the tgn. on the other hand, reports from the two cell-free assays described above have demonstrated that arf is required for the budding of secretory granules and post-tgn vesicles. myristoylated arf1 peptide stimulated both the formation of secretory granules and constitutive secretory vesicles from isolated tgn membranes [51] , while in a semi-intact cell system [52] arf1 peptide was su¤cient for stimulation of budding of gh-and prl-containing vesicles. importantly, although arf is known to be involved in the recruitment of cop i coats to membranes (see article by emr in this issue), barr and huttner [51] could demonstrate that depletion of cop i from cytosol did not inhibit post-golgi vesicle formation. several reports have recently demonstrated that arf may function by activating phospholipase d ([79^81] and see [82] for review). interestingly, addition of phospholipase d (pld) has been shown to stimulate budding from the tgn in the semi-intact assay using gh 4 c1 cells [83] . the stimulatory e¡ect of pld might be attributed to its ability to catalyse the hydrolysis of pc to pa and alter the lipid composition of the membrane. this local increase in charged lipids has been proposed to function to stabilize ap-2 association with endosomal membranes [84] . alternatively, the production of pa may have several consequences including the production of pip 2 (phosphatidylinositol 4,5-bisphosphate) in the membrane [85] . pip 2 has been implicated in a variety of membrane tra¤c events [86] and has been shown to regulate the nucleotide bound state of arf [87, 88] and to stimulate pld activity [89] leading to a feedback cycle of interactions between arf, pld, pa and pip 2 . it has been proposed [83] that the e¡ect of arf and pld on budding might alter-natively be to recruit ap-1 to regions of the membrane which are high in pa. however, from more recent experiments we now know that pld does not play a role in ap-1 recruitment [84] to tgn membranes, it would thus seem most likely that arf and pld are functioning to promote budding from the tgn via a signalling cascade. consistent with this scenario [73] shields and colleagues had previously shown that pkc can stimulate budding, and suggest that pa could function as a second messenger to trigger intracellular signalling events such as activation of pkc. further exciting results have been obtained with regard to cytoplasmic proteins required for vesicle formation from the tgn. a phosphatidylinositol transfer protein (pitp) has been implicated in the formation of both regulated and constitutive secretory vesicles [90] . the mammalian pitp activity can be substituted for by sec14p, a yeast protein from saccharomyces cerevisiae (which is, however, not homologous to the mammalian pitp) that is required for normal golgi function and shares with pitp the ability to exchange phosphatidylinositol for phosphatidylcholine ( [91] ; see also contribution in this issue by bankaitis). additional evidence recently published related to the function of sec14p, obtained from the study of sac1p, a sec14-bypass suppressor [92] would suggest that pitp can also function to cause a local production of diacylglycerol which may be involved in coat recruitment in much the same way the pa has been postulated to be involved in arf and ap-1 recruitment (see review [93] ). ptdins is also the substrate for the yeast kinase vps34 which has pi 3-kinase activity [94] . the synthesis of pdtins(3)p by vps34 has been implicated in the transport from the golgi to the vacuole in yeast and it has been suggested that vps34 activity is required for the formation of a speci¢c vesicle type from the golgi [95] . a wortmannin-sensitive pi 3kinase activity is also required for the sorting of lysosomal enzymes from the golgi to the endosome in mammalian cells (see [96] for review). in addition, a recent report has shown that a pi 3-kinase activity is required for formation of constitutive secretory vesicles from isolated rat liver golgi membranes [97] . interestingly, this pi 3-kinase regulatory subunit associates with the cytoplasmic domain of a tgn-resident protein tgn38 in a complex with a small molecular weight gtpase, previously identi¢ed as rab6 [67] . the authors hypothesis is that the activity of the pi 3-kinase is stimulated by the activation of the gtpase [97] . so far there has been no report implicating a pi 3-kinase in the budding of regulated secretory granules. in fact, incubation of pc12 cells with wortmannin had no e¡ect on either constitutive or regulated secretory vesicle formation, transport or exocytosis of secretory proteins (s. urbë and s.a. tooze, unpublished observations). it maybe that the activation of the pi 3-kinase is dependent upon the presence of particular receptors in the forming bud, for example, the presence of tgn38. data from experiments performed to examine the composition of the nascent isg demonstrates that there is no tgn38 present in isgs [63] . with regard to the role of ptdins-metabolites in vesicle-formation, it is of interest that a 105 kda protein, oculocerebrorenal syndrome of lowe (ocrl) [98] , belonging to the inositol (ins) 5-phosphatase family has recently been localized to the golgi complex [99] . ocrl dephosphorylates ins-(1,3,4,5)p 4 , ins(3,4,5)p 3 and most importantly pip 2 in vitro and seems to act preferentially as a lipidphosphatase in vivo, and could therefore regulate the levels of pip 2 in the golgi-membrane [100] . another inositol-5-phosphatase, synaptojanin, has been implicated in the recycling of synaptic vesicles, a process that involves the formation of clathrincoated vesicles [101] . synaptojanin interacts through amphiphysin [102] with the small gtpase dynamin which appears to play an important role in the budding of clathrin-coated vesicles from the plasmamembrane (for reviews see [103^105]). although the function of dynamin has predominantly, but not exclusively [106] , been linked to the function of clathrin, it has been speculated that there might be a dynamin for every budding event in the cell [107] . a protein with immunoreactivity to antibodies raised against conserved regions of the dynamin-family has been localized to the golgi complex in ¢broblasts and melanocytes [108] . as gtp-hydrolysis is required for dynamin function it is possible that the inhibitory e¡ect of gtpqs on the budding of secre-tory vesicles from the tgn re£ects not only an involvement of heterotrimeric g proteins and small gtpase, but also of dynamin. research on secretory granule biogenesis has revolved to a large extent around the granule content selection mechanisms. the only coat-proteins identi-¢ed so far on the secretory granule, which are present only on isgs are clathrin and adaptor-complex ap-1, and they function to mediate vesicle budding from the isg rather than formation of the isg from the tgn [109] . it has been suggested that budding of nascent secretory granules does not require a coat protein, but that the aggregated core of the granule is able to`drive' vesicle formation in a way analogous to the budding of a viral particle. this hypothesis predicts that there have to be speci¢c interactions between the granule core and the granule membrane that mirror the binding of viral nucleocapsid proteins to the membrane-bound envelope proteins. in this way the secretory granule core could mould its own membrane, driven only by a thermodynamically favourable binding of granule membrane proteins with the aggregating content. this hypothesis becomes less attractive if the nascent isg contains largely uncondensed, unaggregated secretory proteins which, along with the membrane proteins, are not sorted prior to budding (see the sorting by retention model above). work on this sorting mechanism is in progress and new results will no doubt be forthcoming to clarify the inconsistencies present. to address these questions, it would be most interesting to have an in vitro system for formation of isgs using the cells, or cell lines (i.e. l-cells), from which the sorting by retention hypothesis has been developed. the development of such a system would allow a direct test for common structural and regulatory molecules involved in formation of secretory granules in neuroendocrine and endocrine cells. network (erb-fmrxct 960023) for stimulating discussions on which a large part of this review is founded. in particular, i thank my network colleagues, hans-hermann gerdes and philippe halban for copies of manuscripts before publication. finally, i wish to thank peter arvan for taking the time to read this review and for his valuable comments. the granin (chromogranin/secretogranin) family what the granins tell us about the formation of secretory granules in neuroendocrine cells regulated secretion. helper proteins for neuroendocrine secretion chromogranin b (secretogranin i) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor the primary structure of human secretogranin ii, a widespread tyrosine-sulfated secretory granule protein that exhibits low ph-and calcium-induced aggregation milieu-induced, selective aggregation of regulated secretory proteins in the trans-golgi network calcium-and phdependent aggregation and membrane association of the precursor of the prohormone convertase pc2 secretory granule content proteins and the luminal domains of granule membrane proteins aggregate in vitro at mildly acidic ph conversion of proinsulin to insulin occurs coordinately with acidi¢cation of maturing secretory vesicles clathrin-coated vesicular transport of secretory proteins during the formation of acth-containing secretory granules in att20 cells chloroquine diverts acth from a regulated to a constitutive secretory pathway in att-20 cells the role of a low ph intracellular compartment in the processing, storage, and secretion of atch and endorphin transport via the regulated secretory pathway in semi-intact pc12 cells: role of intra-cisternal calcium and ph in the transport and sorting of secretogranin ii direct measurement of trans-golgi ph in living cells and regulation by second messengers the insulin factory: a tour of the plant surroundings and a visit to the assembly line sorting and processing of secretory proteins prohormone processing in the trans-golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells proteolytic processing of pro-acth/endorphin begins in the golgi complex of pituitary corticotropes and att-20 cells proteolytic maturation of insulin is a post-golgi event which occurs in acidifying clathrin-coated secretory vesicles an antibody speci¢c for an endoproteolytic cleavage site provides evidence that pro-opiomelanocortin is packaged into secretory granules in att20 cells before its cleavage ph-dependent processing of secretogranin ii by the endopeptidase pc2 in isolated immature secretory granules formation of the insulin-containing secretory granule core occurs within immature beta-granules ph-independent and -dependent cleavage of proinsulin in the same secretory vesicle distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic l-cells sorting within the regulated secretory pathway occurs in the trans-golgi network cell type-speci¢c sorting of neuropeptides: a mechanism to modulate peptide composition of large dense-core vesicles constitutive and regulated secretion of proteins di¡er-ential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic l-cells protein secretion : puzzling receptors pathways of protein secretion in eukaryotes identi¢cation of the sorting signal motif within pro-opiomelanocortin for the regulated secretory pathway the disul¢de bond in chromogranin b, which is essential for its sorting to secretory granules, is not required for its aggregation in the trans-golgi network chromogranin b (secretogranin i), a secretory protein of the regulated pathway, is also present in a tightly membrane-associated form in pc12 cells carboxypeptidase e is a regulated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in cpe fat mice proinsulin targeting to the regulated pathway is not impaired in carboxypeptidase e-de¢cient cpe fat /cpe fat mice hyperinsulinaemia in obese fat/fat mice associated with a carboxypeptidase e mutation which reduces enzyme activity ph-dependent interaction of an intraluminal loop of inositol 1,4,5-triphosphate receptor with chromogranin a ph-dependent interaction of chromogranin a with integral membrane proteins of secretory vesicle including 260-kda protein reactive to inositol 1,4,5-triphosphate receptor antibody a targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin the exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer a chimeric proinsulin-cd5 protein expressed in att20 cells is directed to the cell surface via the constitutive pathway two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells requirement for gtp hydrolysis in the formation of secretory vesicles the in vitro generation of post-golgi vesicles carrying viral envelope glycoproteins requires an arflike gtp-binding protein and a protein kinase c associated with the golgi apparatus multiple trimeric g-proteins on the trans-golgi network exert stimulatory and inhibitory e¡ects on secretory vesicle formation an elevation of cytosolic protein phosphorylation modulates trimeric g-protein regulation of secretory vesicle formation from the trans-golgi network a role for adp-ribosylation factor1, but not cop i, in secretory vesicle biogenesis from the trans-golgi network adp-ribosylation factor-1 stimulates formation of nascent secretory vesicles from the trans-golgi network of endocrine cells intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells protein discharge from immature secretory granules displays both regulated and constitutive characteristics the trans-most cisternae of the golgi complex: a compartment for sorting of secretory and plasma membrane proteins sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-golgi network of att20 cells intracellular tra¤cking of furin is modulated by the phosphorylation state of a casein kinase ii site in its cytoplasmic tail two independent targeting signals in the cytoplasmic domain determine trans-golgi network localization and endosomal tra¤cking of the proprotein convertase furin an acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-golgi network localization and internalization from the cell surface cysteine proteinases in gh4c1 cells, a rat pituitary tumor cell line, are secreted by the constitutive and regulated secretory pathways mannose 6-phosphate receptors in sorting and transport of lysosomal enzymes the ap-1 adaptor complex binds to immature secretory granules from pc12 cells, and is regulated by adp-ribosylation factor interaction of furin in immature secretory granules from neuroendocrine cells with the ap-1 adaptor complex is modulated by casein kinase ii phosphorylation protein targeting via the`constitutive-like' secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment biosynthetic protein transport and sorting by endoplasmic reticulum hvem tomography of the trans-golgi network: structural insights and identi¢cation of a lace-like vesicle coat a cytosolic complex of p62 and rab6 associates with tgn38/41 and is involved in budding of exocytic vesicles from the trans-golgi network distinct coated vesicles labeled for p200 bud from trans-golgi network membranes perrelet, clathrin-immunoreactive sites in the golgi apparatus are concentrated at the trans pole in polypeptide hormone-secreting cells cell-free protein sorting to the regulated and constitutive secretory pathways trimeric g proteins and vesicle formation formation of nascent secretory vesicles from the trans-golgi network of endocrine cells is inhibited by tyrosine kinase and phosphatase inhibitors regulated formation of golgi secretory vesicles containing alzheimer beta-amyloid precursor protein metabolism of alzheimer beta-amyloid precursor protein: regulation by protein kinase a in intact cells and in a cell-free system ica 512, an autoantigen of type i diabetes, is an intrinsic membrane protein of neurosecretory granules molecular cloning of phogrin, a protein-tyrosine phosphatase homologue localized to insulin secretory granule membranes the binding of ap-1 clathrin adaptor particles to golgi membranes requires adp-ribosylation factor, a small gtp-binding protein mannose 6-phosphate receptors and adp-ribosylation factors cooperate for high a¤nity interaction of the ap-1 golgi assembly proteins with membranes adp-ribosylation factor, a small gtp-dependent regulatory protein, stimulates phospholipase d activity phospholipase d: a downstream e¡ector of arf in granulocytes human adp-ribosylation factor-activated phosphatidylcholine-speci¢c phospholipase d de¢nes a new and highly conserved gene family arf proteins: the membrane tra¤c police? phospholipase d stimulates release of nascent secretory vesicles from the trans-golgi network the role of adpribosylation factor and phospholipase d in adaptor recruitment type i phosphatidylinositol 4-phosphate 5-kinase isoforms are speci¢cally stimulated by phosphatidic acid phosphoinositides as regulators in membrane tra¤c e¡ects of acid phospholipids on nucleotide exchange properties of adp-ribosylation factor 1. evidence for speci¢c interaction with phosphatidylinositol 4,5-bisphosphate gtp hydrolysis by adp-ribosylation factor is dependent on both an adp-ribosylation factor gtpase-activating protein and acid phospholipids cantley, novel function of phosphatidylinositol 4,5-bisphosphate as a cofactor for brain membrane phospholipase d a role for phosphatidylinositol transfer protein in secretory vesicle formation an essential role for a phospholipid transfer protein in yeast golgi function essential role for diacylglycerol in protein transport from the yeast golgi complex greasing the golgi budding machine vps34p required for yeast vacuolar protein sorting is a multiple speci¢city kinase that exhibits both protein kinase and phosphatidylinositol-speci¢c pi 3-kinase activities receptor-mediated protein sorting to the vacuole in yeast: roles for a protein kinase, a lipid kinase and gtp-binding proteins phosphoinositide 3-kinases and membrane tra¤c phospatidylinositol 3-kinase is required for the formation of constitutive transport vesicles from the tgn the lowe's oculocerebrorenal syndrome gene encodes a protein highly homologous to inositol polyphosphate-5-phosphatase the oculocerebrorenal syndrome gene product is a 105-kd protein localized to the golgi complex the protein de¢cient in lowe syndrome is a phosphatidylinositol-4,5-bisphosphate 5-phosphatase a role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals the role of clathrin, adaptors and dynamin in endocytosis dynamin and receptor-mediated endocytosis molecular mechanisms in synaptic vesicle endocytosis and recycling rapid endocytosis coupled to exocytosis in adrenal chro-ma¤n cells involves ca2+, gtp, and dynamin but not clathrin ringing necks with dynamin association of a dynamin-like protein with the golgi apparatus in mammalian cells the ap-1 adaptor complex binds to immature secretory granules from pc12 cells, and is regulated by adp-ribosylation factor essential role of the disul¢de-bonded loop of chromogranin b for sorting to secretory granules is revealed by expression of a deletion mutant in the absence of endogenous granin synthesis key: cord-292958-k5d5fo3i authors: sekhon, simranjeet singh; nguyen, phat-loc; ahn, ji-young; lee, kyeong-ah; lee, lyon; kim, sang yong; yoon, hobaek; park, jihoo; ko, jung ho; kim, yang-hoon title: porcine epidemic diarrhea (ped) infection, diagnosis and vaccination: a mini review date: 2017-01-04 journal: toxicol environ health sci doi: 10.1007/s13530-016-0287-8 sha: doc_id: 292958 cord_uid: k5d5fo3i porcine epidemic diarrhea virus (pedv) is a main etiology causing severe enteric disease in piglets with clinical signs of anorexia, vomiting, diarrhea and dehydration resulting in loss of condition and death within a few days. historically, ped is one of major causes of loss in swine and remains prevalent in some parts of the world. even with increase in the available tests for ped diagnosis, which include histological diagnosis; virological diagnosis and serological diagnosis, there is no vaccine or specific treatment for this disease yet. in this mini review, the overview and current situation of ped is described with updated techniques, in an effort to comprehensively discuss and understand the disease characteristics. porcine epidemic diarrhea (ped) is a non-zoonotic viral disease of swine caused by a coronavirus and characterized by dramatic watery diarrhea and weight loss in swine with high mortality to neonatal piglets 1,2 . pigs of all age can be infected by ped virus from neonates to sows or boars but the fatality rate of ped in swine depends on age 3 . the morbidity and mortality of ped in 10-day-old piglets are less than piglets under 5 days old that may reach 100% in death because of severe diarrhea and dehydration 4 . the older pigs are able to recover within 7 to 10 days but can re-infect within five months 4 . the fecal-oral route is perhaps only transmission of ped and no other vector or reservoir in disease spreading 5 . however, the disease is highly contagious and rapidly infect throughout the year to both industrial and small pig farms. swine (sus scrofa) are the only natural hosts for ped virus infection. kamau et al. (2010) investigated the ability of using specific pathogen free mice as a potential vector in ped virus transmission. nevertheless, molecular tests and serological data showed no antibodies detection so mice and rat were not susceptible vectors of the ped transmission 6 . there are 2 forms of ped disease: the ped type i, which is only infected in weaning pigs, and the ped type ii affects pigs of all age 7 . the main etiologic agent of the disease is porcine epidemic diarrhea virus (pedv) which was firstly reported in europe in 1971 and was identified as coronaviruslike strain cv777 from pigs with watery diarrhea in belgium and united kingdom in 1978 [8] [9] [10] . after that the disease widely spread throughout many swine-raising countries in western europe: hungary (1981); germany (1981); to asian countries: japan (1982); south korea (2000) ; taiwan (2013) and north america (2013) 11 . during the 1970s and 1990s, a few severe ped outbreaks have been reported in europe 12 . nevertheless, pedv infection nowadays has become epidemic in asia pig industries, consist of china, japan, south korea, vietnam, thailand, the philippines and taiwan 13 . in china, a large-scale diarrhea outbreak was reported in the end of 2010 with the confirmation of pedv in pig population that over 1,000,000 piglets died with a mortality rate of 80%-100% and resulted enormous economic losses 14, 15 . in south korea, the pedv was firstly described in 1992 and re-emerged as a severe outbreak during 2013 with considerable variants that were different from previous korean isolates or vaccine strains 16, 17 . a massive ped outbreak suddenly occurred in the north american pig farm in april 2013 and rapidly spread across the country also to countries sharing the same border: canada (2014) and mexico (2013); causing high rates of mortality and huge economic losses 2, 3 . although the ped disease has similarly clinical signs to transmissible gastro-enteritis (tge) including anorexia; vomiting; diarrhea and dehydration, the causative agent ped virus is antigenically distinguishable from tge virus (tgev) and haemagglutinating encephalomyelitis virus (hev) 4 . the ped virus belongs to the member of genus alphacoronavirus in the family coronaviridae which constitutes the order nidoviracles and causes acute diarrhea in human and animals, especially fatal to newborn individuals 18, 19 . under the electron microscopy (em), the characteristic appearance of pedv contains an opaque and pleomorphic body about 90 to 190 nm diameter in range, with an electron-dense core, a large fringe and bulb-shaped projections of approximately 20 nm but the detailed internal structure of ped virus remains unknown 1, 7 . the ped virus adapts culture to be stable at wide range of ph from 5.0 to 9.0 at 4℃ and still keeps infectivity between ph 6.5 and 7.5 at 37℃ 1,20 . however, this virus is sensitive to high temperature when heating to or over 60℃ for 30 minutes and to chemical reagents such as ether and chloroform 1 . besides, most disinfectants are effective to against pedv, consisting of cresol, formalin (1%), anhydrous sodium carbonate (4%), sodium hydroxide (2%), iodophors (1%) in phosphoric acid, ionic and non-ionic detergents. more than 10 years after the first identification of pedv, the effort of growing pedv in cell culture was successfully proved in the study of hofmann m. and wyler r. (1988) 21, 22 . the vero cell (derived from african green monkey kidney) culture with the presence of trypsin was susceptible for ped virus propagation. in another research, shibata et al. (1999) described additional methods to isolate pedv using pig bladder cell-and kidney cell-derived cultures, also in the presence of trypsin in the medium because trypsin allowed the in vitro propagation of several enteric viruses and facilitated coronavirus growth as an important ingredient in the cell culture media 23 . the pedv is an enveloped virus with a single-stranded positive-sense rna genome with approximately 28,000 bases in size with a 5′ cap and a 3′ polyadenylated tail (poly a) 9, 24 . the pedv genome comprises at least 7 open reading frames (orfs) that encode three non-structural proteins (orf1a, orf1b and orf3) and four structural proteins (orf2, orf4-6) 1,8,25 (figure 1 ). the viral genome organization followed a conserved gene order: an untranslated region (utr) at 5′-end; the large orf1a and 1b that cover 70% at the 5′-end of genome for polymerase genes; a set of four genes encoding the structural proteins: 150-220 kda glycosylated spike protein (s), 7 kda envelope protein (e), 27-36 kda membrane protein (m) and 58 kda nucleocapsid protein (n); and a 3′utr 1, 8, 26 . table 1 based on all sequences of coronaviruses, the orf1a (nt 297-12650) and orf1b (nt 12605-20641) shared slightly overlapping sequences at a possible ribosomal frameshift (rfs) site 7, 8 . the orf1a translated for a replicase polyprotein 1a (pp1a) which could be extended into pp1ab at c-terminal of rfs site and encoded for several functional motifs such as three protease: papain-like proteinase (plp), x domain (x), poliovirus 3c-like proteinase (3c1) and one growth factor-like motif (gfl), whereas there were a metal ion binding domain (mb), a rna-dependent rna polymerase domain (rdrp) and a helicase motif (hel) that could be encoded within orf1b by analogy to amino acid sequences of other coronaviruses 25 . the post-translational cleavage of pp1a and pp1ab resulted in a group of 16 non-structural proteins (nsp1-16) by internal proteases. the orf3 is a functional accessory gene which located between s and e structural protein. it is conserved among canine coronavirus type i and encodes a glycoprotein gp3 that is concerned to virulence of pedv in the natural host and to be functional as an ion channel but not generally related to viral replication in cultured cells 1,27,28 . song et al. (2002) analyzed orf3 in restriction fragment length polymorphism (rflp) to differentiate the korean pedv, kpedv-9 field strains from wild-type strains for application in vaccine against pedv infection 28 . this study suggested that the loss of orf3 production or the changes in sequence of orf3 resulted in unexpected consequence of the adaption of pedv in cell culture. the e proteins, one of four structural components in pedv, are recognized as a small, hydrophobic transmembrane protein of coronaviruses with 76-109 amino acids (aa) in length that are rather divergent among different coronavirus strains 29, 30 . it was found that they play a remarkable role on the morphogenesis and viral assembly where they could prompt the curvature of membrane as well as assist in membrane scission 31, 32 . besides, the e proteins without modifications are also integrated into the endoplasmic reticulum (er) membrane where they are independent of inducing er stress 33 . another investigation of e protein expression in escherichia coli and mammalian cells demonstrate the crucial function in altering membrane permeability 34 and apoptosis promotion 35, 36 . additionally the e proteins are observed the interaction with host-cell proteins and in vitro ion channel activity which is more selective for monovalent cations and is blocked by hexamethylene amiloride in other coronaviruses 37 . the co-expression of e protein and a major membrane m glycoprotein, via their cytoplasmic domains localizing to pre-golgi membranes, allows the generation of coronavirus-like pseudoparticles which were identified same size with tgev virions 33, 38 . in recent studies of other coronaviruses, the absence of e proteins leads to two actions: non-infectious virions and the inhibition of full virus growth 39 . the membrane m proteins, the most abundant component of coronavirus envelope, have potential role in virus assembly. it is a glycoprotein type iii with molecular weight in 27-36 kda, consisting of triple-spanning transmembrane segments which flanked by one short n-terminal ectodomain on the outside of virus and one long c-terminal domain in the cytoplasm 40, 41 . when expressed in the absence of other viral proteins, m protein tends to accumulate in the golgi apparatus as detergent-insoluble, heterogeneous complexes in polymeric structures 42, 43 . by employing coimmunoprecipitation and immunofluorescence colocalization analysis in mouse hepatitis virus (mhv), m protein complexes appeared to immediately interact with the spike (s) proteins after their synthesis, forming heteromultimeric complexes in the pre-golgi membranes 42, 44, 45 . additional studies recently provided proof for the existence of m-m interactions which were observed that assembly-incompetent m could be rescued into viruslike particles (vlps) by assembly-competent one 42 . it is indicated that the self-association of coronaviruses envelope glycoproteins plays an essential role in arranging of the viral membrane proteins and is thought to drive virion envelope assembly but do not determine the viral budding site at the pre-golgi membrane. another function of pedv m protein is useful in developing epitope-based vaccines. zhang et al. (2012) identified epitopes on the ped virus m protein, highly conserved 4d4-defined epitope, to study the antigenic properties of this protein 40 . this epitope could be a candidate for development of protective antibodies for ped because it was recognized by positive serum of different pedv strains, comparing to tgev-positive serum. among pedv structural proteins, the s protein is a major type i membrane glycoprotein on virion surface, with average 1,300aa in length and 150-220 kda in weight 46 . this trimeric spike protein forms the characteristic peplomers (surface antigen) and consists of a signal peptide (1-18aa), four neutralizing epitopes (499-638, 748-755, 764-711, and 1,368-1,374aa), a transmembrane domain (1,334-1,356aa), and a short cytoplasmic tail 2 . the s protein can also have potential n-glycosylation sites and can be cleaved by exogenous or host cell protease into s1 (1-789aa) and s2 (790-1,383aa) domains 47 . the s1 domain is composed of a binding domain for host cell receptors whilst the s2 domain, involved in virus-cell attachment and in both virus-cell and cell-cell fusion, could be divided into three domains: a large ectodomain, a single transmembrane (tm) region and a cytoplasmic tail (ct) region 48 . the large ectodomain of s2 subunit was an identified role in membrane fusion activity and composed of a protease cleavage site, a putative fusion peptide and two heptad repeat (hr) region [48] [49] [50] . a few studies reported that the tm region with aromatic domain and the ct region carrying the cysteine-rich domain were concerned to regulate the fusogenic activities 51, 52 . further, another important role of ct region of the pedv spike protein, which was determined by a signal: a dibasic (kxxhxx-cooh), was accountable to retrieve this protein in the endoplasmic reticulum-golgi intermediate compartment (ergic), whereas the s protein with a mutation (h → r) or lack of this motif resulted in enhanced cell surfaces expression of s protein. nevertheless the role of a potential tyrosine-based (yxxf motif) signal of ct region remains unknown [53] [54] [55] . the interaction between s proteins and m or e proteins (as mentioned before), which is essential for virus assembly into those intracellular vesicles, involved in the localization of s protein in the ergic. some studies showed that the s protein were not essential for budding of coronavirus particle but needed for infectivity. by culturing coronaviruses mhv a59 in the presence of antibiotic tunicamycin, rottier et al. (1982) found that the spikeless and noninfectious virions were produced 56 . additionally, luytjes et al. (1996) conducted biochemical analysis and electron microscopy to survey the characteristic of temperature-sensitive mutants of coronavirus mhv a59 and found that the temperature-sensitive mutant s protein at 39.5℃ (non-permissive temperature) were fail to incorporate into virion particles and to mature to golgi membrane 57 . however, the synthesis of the m protein and the nucleocapsid n protein of mutant viruses was not sensitive to temperature. it is presumed that the s protein is the most antigenic of the pedv proteins because of containing virusneutralizing epitopes 58 . chang et al. (2002) targeted the s protein as a primary antigen to identify alternative epitope region for designing an effective vaccine against coronaviruses 59 . although the ped virus and tge virus are serological distinct, this study based on the co-26k, a collagenase-digested fragment, of the tgev s protein to find the critical virus-neutralizing epitope located in the s protein of pedv. by analyzing the neutralizing activity of the polyclonal antisera and comparing the sequences of coe (co-26k equivalent) gene of spike protein among pedv strains (pedv cv777 strain, korean isolate and pedv brl/87), this study suggested that the coe region of pedv spike protein contained an important virus-neutralizing epitope with no cross-neutralization (based on very low homology in the same region comparing with feline infectious peritonitis virus, fipv and tgev). the pedv n protein is a phosphoprotein which is associated with rna genome and indicates a basic structure for the helical nucleocapsid 1 . the encoded polypeptides range of pedv n protein is from 377 to 455 amino acids and has the similar physical properties with the other members of the family coronaviridae 60 . nevertheless one unique sequence with a 40-residuelong insertion in the central position of pedv n protein molecule could reflect recombinant event or stuttering of the viral polymerase. this sequence is particularly rich in arginine (arg), serine (ser) and asparagine (asp) residues, and presents in ped virus n protein with no counterpart in the remainder of coronaviruses. the n protein can be an alternative target to accurately diagnosis pedv in early infection. furthermore, xu et al. (2013) generated a specific cell line iec (porcine intestinal epithelial cell) expressed pedv n protein as well as investigated the function and localization of n protein 60, 61 . the study suggested that pedv n protein affected on cycle progression, interleukin-8 (il-8) expression, cell growth and survival. particularly the pedv n protein was identified in the er membrane, induced cell cycle prolongation at the s-phage, which is associated with a low level of cyclin a transcription and increased in cyclin a degradation, and was responsible for er stress, which included the initiation of an inflammatory react via activation of nf-kb, as well as up-regulation interleukin-8 expression 60,61 . this study was the first report to demonstrate novel functions of pedv n protein that was useful in investigating the molecular mechanisms and pedv pathogenesis. based on the partial sequence analysis of s, m and orf3 genes, the genetic and phylogenetic relation of pedv isolates were determined 26, 28, 46 . by phylogenetic analysis of s glycoprotein genes including epitope region, the diversity of koreans pedv strains were reported and divided into groups and subgroups. there were three groups of korean pedv isolates: the g1 group were highly homologous to reference pedv strains (cv777, js-2004-2, p-5v, sm98-1, kped-9, parent and attenuated dr13) but did not have specific nucleotides and amino acids comparing with other groups g2 and g3 korean pedv isolates, including highly homologous spk1 and chinju99 29 . otherwise the subgroup g1-1 korean pedv isolates had unique specific nucleotide sequences and had the dna sequence identities 95.3%-97.9%, 93.6-96.6%, 93.5-96.6%, and 88.7-90.7% with the subgroup g1-2, g1-3 and the group g2, g3 korean pedv isolates respectively. the study with analysis of s glycoprotein genes demonstrated the korean pedv strains were genetically diverse both among themselves and to other foreign reference pedv strains. the ped diagnosis cannot be determined only on the epidemiological investigation, the basic of clinical signs and the histopathological lesions. due to the dependence of ped clinical signs on the age of swine, the presence of secondary infection and the immunological status of the pigs, as well as the similarities in characteristics of ped syndromes from other causative agents of diarrhea such as tgev, rotavirus, bacteria (e. coli, salmonella sp., clostridium spp., etc.) or by parasites, the laboratory examinations are required to identify and confirm the pedv infection. many techniques are available for the detection of pedv from fecal materials, small infected intestines sample, including reverse transcript polymerase chain reaction (rt-pcr), direct immunofluorescence tests (if), indirect fluorescence antibody tests (ifa), immunohistochemistry techniques (ihc), in situ hydridization, electron microscopy and enzyme-linked immunosorbent assays (elisa) 2 . the popular diagnostic methods used to detect ped viral antigen, are immunofluorescence test while the most commonly applicable tests to detect pedv antibodies in serum and antigen in feces are enzyme-linked immunosorbent assays. of all available techniques to detect the causative agents of ped, electron microscopy (em) is the least sensitive technique. sueyoshi et al. (1995) evaluated the pedv-infected lesions using transmission em (tem) technique, nevertheless the result showed a mix of coronaviruses within the cytoplasm and microvilli of epithelial cells in small intestine 63 . after that they carried out a streptavidin-biotin (sab) technique to detect the specific viral antigen of ped in the cytoplasm of enterocytes, with light microscopy. this method examined formalin-fixed materials with a serial of washing step and no cryostat that demonstrated adequately detailed diagnosis methods for outbreak of pedv occurred in japan. in case the spikes of virus were lost or not clearly visible, the tem results were inconclusive to confirm that the causative agents were pedv or viruses with the similar size and morphology, especially tgev. for the reason that the em technique is not sensitive and specific, it could not be applied to differentiate ped virus from the remainder of coronaviruses, especially tge virus. shibata et al. (1999) employed virus neutralization (vn) test and immunofluorescence assay (ifa) to detect antibodies against pedv as well as the immunohistochemical examination for the detection of pedv antigens in the enterocytes of infected pigs by avidin-biotin (ab) technique 12 . the paper determined the isolation of pedv using porcine cell cultures (as mentioned before), whereas established the sb1 and sb2 cells derived from the epithelial cells of cesarean-derived colostrum-deprived (cdcd) pig bladder and the sk cells prepared from the cdcd pig kidney for different passage state, instead of using popular methods with vero cells. furthermore, this study discovered the effect of different pig age on the disease, in particular, the severe clinical signs were only found in two-day and one-week old piglets. so this research concluded that there was an age-dependent resistance to ped virus in swine. until now, the vero cell lines are the most popular and effective to isolate ped virus. pan et al. (2012) used the vero cell cultures to isolate chgd-01 pedv strain as well as employed direct immunofluorescence assay and em technique to investigate its specific cytopathic effects in the outbreak of diarrhea in guangdong (south china) swine 18 . the study also reported the susceptibility of vero cells to different pedv strains because the characteristic cpe caused by pedv isolate in this research was not confirmed until seven passages of vero cells. based on phylogenetic analysis of the whole genome, the chgd-01 isolates were placed in a cluster with two other chinese strains, sharing almost 98% of nucleotide sequence identities. however, this recent chinese strain might have originated from the korean pedv strains knu 0802, based on amino acid sequence analysis of viral spike protein gene. the rt-pcr could be applied for a rapid detection of pedv infection without showing any cross reaction with tgev or porcine rotavirus. kweon et al. (1996) designed three primer sequences from the membrane protein (m) gene of pedv for rt-pcr test 64 . the result of this study proved that a positive dna band of kpedv-9 m gene could be early detected from fecal specimens at 24 h post-infection in experimentally inoculated pigs. the research from ishikawa et al. (1997) was in the close agreement with the conclusion of rt-pcr as a practical, rapid and specific method for detecting pedv in swine 65 . the study developed a rt-pcr detection system by using primers to amplify an 854 bp fragment of m protein gene of pedv. the rt-pcr system in this study could effectively detect pedv rna from viral mixtures or small intestinal/ fecal samples in very low number of virus within short time. the virus isolation and the sab technique were employed to confirm pedv positive results by rt-pcr, based on virus and its antigen detection. moreover, a serial dilution of pedv jme2 strain for rt-pcr was conducted to evaluate the ability of this assay to amplify viral rna from small intestinal samples and fecal specimens as well as to eliminate the inhibitors of rt-pcr in specimens. previous studies have showed difference in the sensitivity of rt-pcr assay. this could be explained by the difference in rna extract processes, rt-pcr conditions or the presence of inhibitors in the intestinal and fecal samples that could affect the sensitivity of rt-pcr. there were many factors in the intestinal and fecal specimen that could inhibit the activity of thermostable polymerase in enzymatic reaction of pcr amplification, especially the presence of bilirubin and bile salts 66, 67 . to enhance the specificity and sensitivity of rt-pcr, the extraction process of viral rna and pcr conditions need to be optimized and chemical reagents that prevented the inhibition factors in raw materials, were advised to use. in the reverse transcription step, the rt-pcr also revealed high potential in cross contamination during mass screening, however. kim et al. (2007) improved the molecular detection methods of pedv by generating a multiplex real-time rt-pcr employing 2 sets of primers and different probes labeling with reporter dyes in a single reaction tube 68 . the primers and probes were designed and synthesized base on conserved sequence of the nucleocapsid n genes from a number of strains of tgev and pedv, and were labeled with specific dyes for each virus: 5′-reporter dye fam and 3′-quencher bhq1 for tgev, 5′-reporter dye cy-5 and 3′-quencher bhq2 for pedv. the selection of primers and probes which based on the highly conversed regions of nuclecapsid gene of the cv777 strain of pedv and the purdue 46-mad strain of pedv as well as using the same concentrations for both of them could optimize this assay. the multiplex real-time rt-pcr was able to differentially detect and qualify the pedv and tgev rapidly from both experimentally and naturally infected pigs, with no cross-reaction between diarrhea-causing viruses. this assay also saved the time-consuming comparing to complete rt-pcr-based dot blot hybridization and minimized the rate of contamination by reducing the number of experimental steps. hence, the rt-pcrbased technique was considered as a useful and practical method for rapid, sensitive and specific detection of pedv infection in fecal samples from live pigs, with out killing. the study of carvajal et al. (1995) was one of the first researches that focused on detection of pedv antigen and antibodies 69 . the study generated simultaneously 2 separate blocking enzyme-linked immunosorbent assays (elisas) and an indirect fluorescence test (ift) in order to detect both pedv antigen in fecal samples and pedv antibodies in serum, using monoclonal antibodies (mab) as capture and reporter agents. the results indicated that the antibodies in both natural and experimental pedv infections were detected by elisa in shorter time than by ift (3-5 days sooner). hence the mab-based elisa was considered higher sensitive method allowing the diagnosis of pedv infection in diarrhea endemic and on farm. however, there was unknown reason between elisa-negative but ift-positive results that remained in this study. in another research, the competitive blocking elisa (cb-elisa) was generated to identify pedv in culture medium and fecal samples, evolving non-conjugated monoclonal antibodies to membrane m protein of pedv. this study also showed the low correlation of sensitivity between the em technique and the cb-elisa that only 3/15 fecal samples was positive with coronavirus by em examination while the negative results of cb-elisa was 14% (9/65 fecal samples). hou et al. (2007) based on recombinant 48 kda nucleocapsid (n) protein of pedv as a useful antigen to develop an elisa (rnelisa) for detecting antibodies to pedv 18 . the rt-pcr was conducted to amplify nucleocapsid n protein gene from the pedv korean strain, using forward and reverse primer containing restriction enzyme sites (bamhi and saci). after subcloning into the prokaryotic expression vector pqe-30, the recombinant plasmid pqe30-pn was transformed into host cell (escherichia coli) to produce the pedv soluble nucleocapsid proteins with an n-terminal his6-tag of 442 amino acids. the recombinant n proteins were purified by affinity chromatography in ni-nitrilotriacetic acid (ni-nta) agarose and were analyzed by sds-page and western blotting with anti-his-tag monoclonal antibody before applying to rnel-isa for pedv detection. besides, the limitation of rnelisa was determined by a cut-off value that was calculated as absorbance values among 80 serum samples from field, based on the mean value plus two sds (standard deviation). in the additional confirmation from rt-pcr and the serum neutralization (sn) tests, the recombinant n protein igg elisa obtained 98% sensitivity (among 103 clinical pedv-infected sam-ples) and 98.7% specificity (among 80 pedv-free samples) compared to rt-pcr in methodologies as well as this recombinant protein-based serological test only reached 8.5% false-positive results by antibody detection in 18 of 213 sn negative results. the study indicated that the nucleocapsid n protein might be a sensitive antigen for the serological diagnosis of pedv and the rnelisa had high sensitivity and specificity for a rapid and simple method for the large-scale detection of pedv. the correlation between the das-elisa and the rt-pcr was also demonstrated by examining 506 specimens of pig herd from different farms in the po valley (italy), according to the study of sozzi et al. (2009) 70 . the double antibody sandwich (das) elisa was generated for detection of pedv in both swine intestinal and fecal samples, using monoclonal antibodies. the six mabs specific for pedv, which were generated and characterized from the screening of forty hybridomas by indirect if and indirect elisa using non-infected and pedv-infected cells, were purified and modified with horseradish peroxidase (hrpo) conjugation before applying to das-elisa. the six selected mabs were surveyed the intensity and specificity in different combination, i.e. 1f12 as conjugated mab and mab 4c3 as antigen catching antibody, for the best catcher and tracer in das-elisa. the comparison of das-elisa and rt-pcr indicated the complicated correlation between intestinal and fecal samples. for the fecal examining, the high kappa statistic value suggested an almost perfect agreement in two methods that only 2 of 215 samples were elisa-negative but rt-pcr-positive identification. this could be explained that the clinical samples were collected in the recovery period of disease with the low viral titres and the forms of specific antibody in faeces were immunocomplexes. however the intestinal examination by two methods gave a disagreement because there were 7 samples of rt-pcr negative but elisa positive. the disagreement might be due to the non-specific binding of antibodies in elisa reaction and the available inhibitors of pcr assay. these finding agreed with previous studies that serological test-elisas were rapid and sensitive for the screening of a large number of specimens so the pedv-elisas were suitable and effective in controlling ped disease during epizootic outbreaks. in a research by guscetti et al. (1998) , four methods were compared for diagnosis of wild-type pedv: an immunohistochemical detection using formalin-fixed tissues, a direct immunofluorescence assay using cryo-stat sections, an elisa and a pcr method 71 . this study showed that the elisa results confirmed a sensitive and reliable detection in fecal material for pedv but the presence of false positive reactions might affect the results when using for large number of specimen samples or samples from different infected animals in clinical practice. the pcr methods were ineffective in this study because wrong negative results were indicated by the other methods but the virus purification step before rna extraction could enhance this method. according to conclusion of this research, the ihc and if methods, which were used to detect viral antigen from gut tissues, demonstrated very high sensitivity and reliability, allowing detection for more than 75% enterocytes were positive for viral antigen by ihc in the mid-jejunum particularly. in contrast, the rt-pcr used in the recent study was indicated to be a simple, rapid, specific and sensitive method for the identification of pedv from fecal samples. kim and chae (2012) used and compared reverse-transcription polymerase chain reaction (rt-pcr), immunohistochemistry (ih) technique, and in situ hybridization for detection of pedv in fifteen experimental pedv-infected pigs and 94 samples of diarrheic piglets 72 . for the intestinal and fecal samples from experimental pedv-inoculated pigs, the rt-pcr could detect viral antigen and nucleic acid in all samples (15/15) while the ih and in situ hybridization could not identify the causative agent in 1/15 and 2/15 samples, respectively. for pedv detection of diarrheic pigs in fecal samples, 63/94 samples were positive and 15/94 samples were negative for causative pedv by all 3 methods, indicating a high agreement and correlation (83%) among these methods. the high rate of agreement demonstrated that the 3 methods could be applied independently to accumulate an accurate diagnosis for pedv infection. this study also indicated how to choose the suitable technique to diagnose pedv depends on the available types of sample. the rt-pcr test was recommended for virus detection if only fecal samples were provided whilst the immunohistochemistry (figure 2 ) and in situ hybridization were suggested for more sensitive detection of pedv in formalin-fixed intestinal samples 72 . due to the special features such as rapid detection, high sensitivity and specificity, and cost-effectiveness, the rt-pcr is considered as an important and successful test for detection of pedv both laboratory and field virus strains. vaccination is one of the traditional and required methods to prevent and control ped virus infection in swine. nowadays, the ped epidemic outbreaks, which neonatal pigs are involved, tend to be more serious and dangerous in asia countries than in europe and others with the sufficient economic loss. therefore, several trials of pedv vaccine were mainly researched and developed in asia countries such as attenuated vaccine ppedv-9 strain in korea, p-5v and 96-p4c6 strain in japan, cv777-attenuated or inactivated vaccines in china [73] [74] [75] . in sow, neonatal pigs are normally protected until they are 13-day-old by a transfer of igg maternal antibodies from colostrum and milk of immune mothers 1 . beside the 60% of igg accounting in colostrum immunoglobulin content, there are igm and especially iga which is more resistant and has high virus neutralizing activity than igg 76 . however, these antibodies from passive immunity are not able to prevent the intestinal infection of pedv. in south korea, researchers investigated attenuation of kpedv-9 isolate via serial passages in vero cell cultures (up to 93 passages) and its immunoprophylactic effects in pregnant sows 74 . the piglets intramuscularly or orally inoculated with attenuated virus of high passage level did not reveal any severe symptoms of diarrhea or death comparing to the animal with wild pedv-infection, so an alternative vaccine might be developed from attenuated virus deriving from serial passage of pedv. this study also indicated that the vaccinated pregnant sows by intramuscular inoculation had the low mortality rate of piglets. another researcher group generated a vero cell attenuated pedv dr13 which was differed from wild-type pedvs by a restriction fragment length polymorphism (rflp) pattern 77 . the study compared the ability of the cell attenuated dr13 virus after inoculation via oral (o) and intramuscular (im) routes in late-term pregnant sows to protect neonatal pigs against ped, employing the sn test and elisa. the experiment of the immunoprophylactic effect in pregnant pigs showed the similar agreement with the research by kweon et al. (1999) in decreasing mortality of piglets but cell-adapted dr13 vaccination in this study through oral route 74 . previously, pedv dr13 was also isolated in vero cells through serial passage at level 100 and was analyzed for differentiation from other korean field strain via rt-pcr rflp with hindiii and xhoii enzymes. moreover, the investigation of cell-adapted dr13 indicated the reduced virulence after high serial passage as well as its immune response in 14-day-old piglets and pregnant sows. the attenuation and safety of high serial passage dr13 after oral vaccination were also discussed in this study. hou et al. (2007) targeted the nucleocapsid n protein of pedv to develop a recombinant vaccine trial by employing a surface antigen display system on lactobacilli 78 . this system used the poly-γ-glutamate a (pgsa) protein, which is a synthetase complex of bacillus subtilis, as the transmembrane anchor to express heterologous antigens in the surface of lactobacillus casei525. the live and dead l. casei anchoring nucleocapsid protein of pedv were vaccinated to two animal models: intranasal and oral inoculation of mouse, oral inoculation in pregnant sows that induced the systemic and local immune responses in both. the rneli-sa were carried out to indicate the high levels of serum igg and mucosal iga after inoculation. moreover, the igg levels of piglets were highly increased after receiving colostrum secreted from vaccinated sows with recombinant l. casei. the surface antigen expression system on lab (lactic acid bacteria) was potential for vaccine applications to against ped but limited in the display size of foreign antigens. the pedv infection cause acute and severe enteritis with clinical symptoms: diarrhea and vomit, followed by extensive dehydration which is the main reason of death in neonatal pigs. this viral infection had a significant impact on the economy of the european and asia pig industries for the last three decades. the disappearance and re-emergence of epidemic ped de monstrated that the current vaccine application, the detection methods, the biosecurity system are old-fashioned and not effective in control and treatment of pedv infection. comprehensive knowledge of the pathogenic characteristics of epidemic and endemic pedv strains is required to generate effective tests for pedv detection in all affected countries and to develop suitable vaccines to different pedv strain for all regions. over the geographic limitation, a number of ped discoveries about the epidemiology, the virology, the pathogenesis, the immunology, the vaccinology as well as several detection methods such as histological examination, virological and serological diagnosis have been gained and upgraded 1, 2, 20 . the potential diagnosis of pedv should not only be rapid and robust but also cost-effective and able to detect a large number of samples in field. new researches and experiments are re quired to investigate cross-protection between field viruses and vaccine strains. more significant measures (such as physicochemical, genetic and antigenic analyses) need to be established to control and prevent the pedv infection. lastly the combined applications of early detection, vaccination, biosecurity programs and management play a major role in prevent and control ped. this review will provide basic and applied understanding of pathogenic pedv as well as the extensive discussion about the recent ped circumstance with modern and popular methods to against pedv. porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines 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characteristics of quebec isolates associated with acute and chronic outbreaks of porcine reproductive and respiratory syndrome this study was performed by the support of the "coo key: cord-290904-ngvhk0qy authors: zheng, zhiqiang; monteil, vanessa marthe; maurer-stroh, sebastian; yew, chow wenn; leong, carol; mohd-ismail, nur khairiah; cheyyatraivendran arularasu, suganya; chow, vincent tak kwong; lin, raymond tzer pin; mirazimi, ali; hong, wanjin; tan, yee-joo title: monoclonal antibodies for the s2 subunit of spike of sars-cov-1 cross-react with the newly-emerged sars-cov-2 date: 2020-07-16 journal: euro surveill doi: 10.2807/1560-7917.es.2020.25.28.2000291 sha: doc_id: 290904 cord_uid: ngvhk0qy background: a novel coronavirus, sars-cov-2, which emerged at the end of 2019 and causes covid-19, has resulted in worldwide human infections. while genetically distinct, sars-cov-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (sars) in 2002–2003, utilises the same host cell receptor as sars-cov-2 for entry: angiotensin-converting enzyme 2 (ace2). parts of the sars-cov-1 spike glycoprotein (s protein), which interacts with ace2, appear conserved in sars-cov-2. aim: the cross-reactivity with sars-cov-2 of monoclonal antibodies (mabs) previously generated against the s protein of sars-cov-1 was assessed. methods: the sars-cov-2 s protein sequence was aligned to those of sars-cov-1, middle east respiratory syndrome (mers) and common-cold coronaviruses. abilities of mabs generated against sars-cov-1 s protein to bind sars-cov-2 or its s protein were tested with sars-cov-2 infected cells as well as cells expressing either the full length protein or a fragment of its s2 subunit. quantitative elisa was also performed to compare binding of mabs to recombinant s protein. results: an immunogenic domain in the s2 subunit of sars-cov-1 s protein is highly conserved in sars-cov-2 but not in mers and human common-cold coronaviruses. four murine mabs raised against this immunogenic fragment could recognise sars-cov-2 s protein expressed in mammalian cell lines. in particular, mab 1a9 was demonstrated to detect s protein in sars-cov-2-infected cells and is suitable for use in a sandwich elisa format. conclusion: the cross-reactive mabs may serve as useful tools for sars-cov-2 research and for the development of diagnostic assays for covid-19. the severe acute respiratory syndrome coronavirus (sars-cov-1), a virus considered to have a zoonotic origin, is the aetiological agent for the infectious disease, sars, which first emerged in 2002-2003 [1, 2] . in december of 2019, another novel coronavirus (sars-cov-2), which causes coronavirus disease , appeared to have crossed species barriers to infect humans and was effectively transmitted from person to person, leading to an outbreak in wuhan, china [3] [4] [5] . this virus subsequently spread worldwide, leading the world health organization (who) to declare a pandemic on 11 march 2020 [6] . to date, sars-cov-2 continues to pose a high global health and economy burden, and as at 3 may 2020, covid-19 had affected 215 countries with over 3.35 million confirmed cases. to tackle the problems caused by sars-cov-2, improving its detection and knowledge of its infection mechanism is important. in this respect, the viral surface spike glycoprotein (s protein) has been demonstrated to play key role in host cell selectivity and binding. the s protein is functionally divided into two subunits, with the s1 subunit containing the receptor binding domain (rbd), which allows attachment to host cells, and the s2 subunit mediating fusion between viral and host membranes (reviewed by li, f.) [7] . phylogenetic analysis revealed that like sars-cov-1 and bat-derived sars-like coronaviruses (sl-covs), sars-cov-2 belongs to lineage b of the betacoronavirus genus [8, 9] . a study of 56 complete and partial sars-cov-2 genomes isolated from covid-19 patients showed very high sequence conservation of more than 99%, indicating a recent introduction of the virus into the human population [10] . although the animal source of sars-cov-2 is not clear, sars-cov-1 is believed to have originated from sl-covs residing in bats [11] [12] [13] [14] . for the majority of sl-covs, the s1 subunit has low sequence identity to that of sars-cov-1, which suggests species-dependent receptor binding [14, 15] . on the other hand, the high amino acid sequence identity of more than 90% in the s2 subunit suggests that the fusion mechanism during virus infection is well-conserved [14, 15] . while sars-cov-2 shares higher whole-genome sequence identity with bat-sl-covzc45 and bat-sl-cov-zxc21 (88-89%) than with sars-cov-1 (79-82%), the rbd of sars-cov-2 is more similar to sars-cov-1 rbd [8, 9] . in line with this, several research groups have demonstrated that sars-cov-2 utilises the same host receptor, angiotensin-converting enzyme 2 (ace2), as sars-cov-1 for viral entry [3, [16] [17] [18] . due to its role in virus entry, the s protein has been the target for the generation of monoclonal antibodies (mab). in our previous work, we used five different fragments of sars-cov-1 s protein to immunise rabbits. a fragment corresponding to residues 1029 to 1192 in the s2 subunit of sars-cov-1 was found to stimulate neutralising antibodies against sars-cov-1 [19] . this fragment was subsequently used to generate a panel of murine mabs with their respective binding domains characterised and described in lip et al. [20] . one of them, mab 1a9, which binds to the s protein through a recently identified epitope within the s2 subunit at amino acids 1111-1130, has the ability to bind and cross-neutralise pseudotyped viruses expressing the s protein of human sars-cov-1, civet sars-cov and bat sl-cov strains [21] . in this study, we aim to verify if the sequence of the immunogen used to generate mab 1a9, as well as three other mabs, is conserved in different coronaviruses and if these mabs bind to the s protein of sars-cov-2 expressed in mammalian cell lines. importantly, mab 1a9 is investigated for its ability to detect the s protein in sars-cov-2 infected cells and purified s protein in a sandwich elisa format when paired with another mab binding to the s1 subunit of sars-cov-2. the name of the viruses, for which sequences are being compared figure on the left side of the alignment, together with the respective sequences' genbank accession numbers. colour schemes represent the following categories of amino acids: blue -hydrophobic, cyan -aromatic, green -polar, magenta -negative charge, orange -glycines, pink -cysteines, red -positive charge, yellow -prolines, white -unconserved. and grown in dmem supplemented with 10% fbs, 100 units/ml penicillin, 100 µg/ml streptomycin and 500 µg/ml geneticin (thermo fisher scientific). cells were maintained at 37 °c with 5% co 2 . the hybridoma for mab 1a9 was previously generated [20] . all mabs were purified from cell culture supernatants using hitrap protein g hp affinity columns (ge healthcare, chicago, il, united states) and stored at −80 °c. the purity of the mab was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoretic (sds-page) analysis. the concentration of the purified mab was determined using the coomassie plus protein assay reagent (thermo fisher scientific). sars-cov-2 s-protein-expressing plasmids were codon-optimised and generated by gene synthesis (bio basic asia pacific, singapore) according to genbank accession number: qhd43416.1. one plasmid is for expressing untagged full-length s protein while the other is for expressing a myc-tagged s-protein fragment consisting of residues 1048-1206 (sars-cov-2 numbering). the pxj40-myc expression vector was used as an empty vector control and pxj40-myc-hbcag plasmid expressing myc-tagged hepatitis b virus core antigen (hbcag) was used as a negative control. 293ft cells were seeded onto 6-cm dishes 24 hours before transient transfection using x-tremegene hp dna transfection reagent (roche, basel, switzerland) according to the manufacturer's protocol. at 24 hours post-transfection, cells were harvested, spun down by centrifugation and washed with cold phosphate buffered saline (pbs) twice. cells were then resuspended in 2× laemmli sample buffer, boiled and sonicated. clarified supernatant containing the protein of interest was obtained by spinning down the cell lysate at 13,000 rpm at 4 °c to remove the cell debris and further analysed by western blot (wb) analysis. equal amounts of total cell lysates were loaded per lane and resolved using electrophoresis on sds-page gels and transferred onto nitrocellulose membrane (bio-rad, hercules, ca, united states). the membrane was blocked in 5% skimmed milk in tris-buffered saline with 0.05% tween 20 (tbst) for 1 hour at room temperature (rt) and incubated with primary antibodies at 4 °c overnight. after the membrane was washed with tbst, it was incubated with a horseradish peroxidase (hrp)-conjugated secondary antibody (thermo fisher scientific) at rt for 1 hour. the membrane was then washed with tbst again and bound antibodies visualised with enhanced chemiluminescence substrate (thermo fisher scientific) using chemidoc mp imaging system (bio-rad). mers: middle east respiratory syndrome; sars-cov-1: severe acute respiratory coronavirus; sars-cov-2: severe acute respiratory coronavirus; sb: shown below. high to low pairwise amino-acid identity are coloured coded respectively by contrasting green to red backgrounds. the sequence identity is not affected by the order in which paired sequences are compared so only one-way comparisons are shown to avoid redundancies; the abbreviation 'sb' is used when the pairwise amino-acid identity in question is already shown in a further cell of the table. for immunofluorescence (if) analysis, cos-7 cells on glass coverslips were transfected as above and fixed at 24 hours post-transfection in 4% paraformaldehyde for 10 min at rt followed by permeabilisation with 0.2% triton x-100 ( b. the abilities of 2b2, 1a9, 4b12 and 1g10 monoclonal antibodies to bind to sars-cov-2 s protein was determined by elisa. individual wells were coated with 20 ng of sars-cov-2 s protein and incubated with serially diluted mabs as indicated. a representative plot from three independent experiment is show for each antibody and error bars correspond to standard deviations of each mab experiment carried out in triplicates. c. each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (myc, 2b2, 1a9, 4b12, or 1g10). immunofluorescence analysis was performed on empty vector-transfected cos-7 cells (top photos) and cells expressing full-length sars-cov-2 s protein (bottom photos). the indicated primary antibodies were used followed by alexa fluor 488-conjugated secondary antibody (green). cell nuclei were counterstained with dapi (blue). scale bar = 50 µm. number: 40589-b08v1) was diluted with coating buffer (0.1 m nahco 3 , 34 mm na 2 co 3 ) and a total of 20 ng of protein was loaded into individual wells of a 96 well plate (nunc, roskilde, denmark) and allowed to coat overnight at 4 °c. plates were then washed four times with 0.05% tween 20 in pbs (pbst) and blocked with 5% bovine serum albumin (bsa)/pbst for 30 min before murine antibodies serially diluted with blocking buffer were added to desired wells for 1 hour. plate were washed four times with pbst before incubation for 1 hour with hrp-conjugated goat anti-mouse igg (thermo fisher scientific) secondary antibodies diluted in blocking buffer, and washed four times with pbst. visualisation of bound secondary antibodies was done by the addition of 3,3',5,5'-tetramethylbenzidine (tmb) substrate (thermo fisher scientific) for 5 min in the absence of light and the reaction was stopped with 2 m sulphuric acid. optical density at 450 nm (od450nm) was determined by a tecan (männedorf, switzerland) infinite m1000 reader and normalised od450nm was obtained by subtracting background absorbances determined in bsa coated wells. the human mab cr3022 was expressed in a similar manner as previously described [22] . the variable heavy (vh; genbank accession number: dq168569) and variable light (vl; genbank accession number: dq168570) genes of cr3022 were generated by gene synthesis (bio basic asia pacific) and cloned into pfusess-chig-higg1 and pfuse2ss-clig-hk cloning vectors (invivogen, san diego, ca, united states) respectively. transfection of suspension freestyle 293 cells (thermo fisher scientific) and purification of antibodies by fast protein liquid chromatography is as described in our previous study [23] . mab 1a9 was diluted with coating buffer (0.1 m nahco 3 , 34 mm na 2 co 3 ) and 0.1 µg of antibody was coated onto individual wells of a maxisorp flat-bottom plate (nunc) overnight at 4 °c. the plate was washed three times with pbst before blocking was done using 5% bsa/ pbst at 37 °c for 60 min. dilutions of his-tagged full length sars-cov-2 s protein (sino biological inc., catalogue number: 40589-b08v1) and his-tagged h7n7-ha (sino biological inc., catalogue number: 11082-v08b) were added to desired wells and incubated at 37 °c for 90 min followed by three washes with pbst. 100 µl of cr3022 antibody was added at a concentration of 1 µg/ml and incubated at 37 °c for 60 min followed by three pbst washes before hrp-conjugated goat antihuman igg (thermo fisher scientific) was added for 60 min at 37 °c. finally, after three pbst washes, tmb (sigma-aldrich) was added for 5 min and the reaction was stopped by 2 m sulphuric acid. the od450nm was determined by a tecan infinite m1000 reader. statistical analyses were performed using an unpaired, one-tailed student's t-test with welch's correction for unequal variances. p values < 0.05 were considered statistically significant. all works with live virus were performed in the biosafety level (bsl)3 facility at the public health agency of sweden. vero-e6 cells were infected with sars-cov-2 (sars-cov-2-iso/01/human/2020/swe; genbank accession number: mt093571) at a multiplicity of infection (moi) of one in dmem 2% fbs (thermo fisher scientific). at 24 hour post-infection, cells were fixed with chilled methanol/acetone and the cells were kept at −20 °c overnight. cells were then stained using mab 1a9 at 5 µg/ml at 37 °c for 30 min in if buffer (bsa 0.2%, triton ×100 0.1% in pbs, ph 7.4). the cells were washed three times with pbs and incubated, subsequently with alexa fluor 488-conjugated goat antimouse igg antibody (thermo fisher scientific) in if buffer containing dapi for an additional 30 min. cells were washed three times with pbs before visualisation and image acquisition with fluorescent microscopy. s protein reference sequences for sars-cov-1, sars-cov-2, batratg13, middle east respiratory syndrome (mers) and human common-cold coronaviruses 229e, nl63, oc43 and hku1 were downloaded from the national center for biotechnology information (ncbi). a multiple sequence alignment was created with multiple alignment using fast fourier transform (mafft) using the slow but accurate l-ins-i parameter settings [24] and the alignment curated, cut to the target region 1029-1192 (sars-cov-1 numbering) and visualised with jalview [25] . we used molecular evolutionary genetics analysis (mega) x [26] to calculate the number of amino-acid differences for all sequence pairs in the alignment of the mab target region and the full s protein normalised by the length of the aligned sequence of the respective reference protein to obtain per cent amino acid identities. to determine sars-cov-2 sequence diversity in the s protein within the current pandemic, 230 human and environmental viral sequences were downloaded from gisaid's epicov database on 1 march 2020. we gratefully acknowledge the authors, originating and submitting laboratories of the sequences on which this part of the research is based. the list is detailed in supplementary table 1 . the nt sequences were searched with basic local alignment search tool (blast)x against the reference s protein. 174 hits covered the full length of the s protein and amino-acid mutations were counted and tabulated using a custom perl script (supplementary table 2 ). ethical approval was not required for this study. sequence alignment of the s2 fragment corresponding to residues 1029 to 1192 shows that this fragment, which encompasses the heptad repeat (hr)2 but not hr1, is highly conserved in sars-cov-1 and sars-cov-2 ( figure 1 ). when compared with additional reference sequences from bat ratg13 (closest bat precursor), mers and human common cold coronaviruses 229e, nl63, oc43 and hku1 (figure 1) , it becomes apparent that the amino-acid identity between sars-cov-2 and sars-cov-1 is much higher in this region (93% , table) than over the full protein length (78%, table) and the similarity drops sharply (< 40% in this region) when considering mers and the other coronaviruses infecting humans regularly. we also studied the sequence diversity across 174 sars-cov-2 s proteins derived from nt sequences shared via the gisaid platform [27] . only four aminoacid mutations were found within the putative antibody-binding region compared with 30 mutations over the full length protein (supplementary table 2 ). two of these four amino-acid mutations are from a sequence flagged in gisaid's epicov database as lower quality due to many undetermined bases. four mabs with distinct binding profiles to sars-cov-1, as previously mapped by internal deletion mutagenesis study, were selected for testing to determine if they cross-react with sars-cov-2. a fragment containing residues 1048 to 1206 of sars-cov-2 s protein was expressed in 293ft cells via transient transfection and wb analysis was performed using the four mabs, namely 2b2, 1a9, 4b12 and 1g10. as shown in figure 2a , all four mabs detected this fragment of sars-cov-2, which is consistent with the sequence alignment shown in figure 1 . due to the easy detachment of 293ft cells, cos-7 cells were used for if assay instead. if analysis performed on transiently transfected cos-7 cells showed binding of the four mabs to this s protein fragment of sars-cov-2 ( figure 2b ). these interactions are also specific for the sars-cov-2 s protein (1048-1206) fragment as all four mabs did not show binding to the negative control hbcag. next, the full-length s protein of sars-cov-2 was overexpressed in 293ft and cos-7 cells and detected with each of the mabs using wb and if analyses. as shown in figure 3 , all four mabs bound to the full-length s protein of sars-cov-2 ( figure 3a ). the binding of these mabs to recombinant purified s protein was also determined using indirect elisa where different concentrations of antibodies were used for binding. binding to s protein was observed for all four mabs with 1a9 showing the strongest binding ( figure 3b ). similarly, all four mabs bound to the full-length s protein of sars-cov-2 when tested via if ( figure 3c ). collectively our data demonstrates the ability of all four mabs to bind full-length s protein in both its native and denatured forms. utility of monoclonal antibody 1a9 for detection of s protein in a sandwich elisa format and in sars-cov-2 infected cells based on indirect elisa data, mab 1a9 has the strongest binding to s protein when compared with the other three mabs. hence, a sandwich elisa was performed to determine if it can be paired with the human mab cr3022 which is known to bind to the s1 subunit of sars-cov-2. as shown in figure 4a , recombinant s protein was detected at 15.6 ng/ml and above when 1a9 was used as a capture antibody and cr3022 was used as a detector antibody. since the s protein was his-tagged, a his-tagged haemagglutinin (ha) protein of influenza a virus was used to check for specificity of binding. the absorbance readings in the presence of s protein were significantly higher than that in the presence of ha for protein concentrations of 15.6 ng/ml and above. next, 1a9 was tested on sars-cov-2-infected vero-e6 cells. as shown in figure 4b , mab 1a9 stained a considerable number of sars-cov-2-infected cells at 24 hours post-infection showing that it is sensitive enough to detect the expression of s protein during infection. numerous mabs against the s protein of sars-cov-1 have been generated for research and diagnostic assay development. some of these may be able to cross-react with the s protein of sars-cov-2 and serve as tools to aid research on this newly emerged virus. in this current study, an immunogenic domain in the s2 subunit of sars-cov-1 was found to be highly conserved in multiple strains of sars-cov-2 ( figure 1 and table) . consistently, wb and if analyses showed that four different mabs generated using this sars-cov-1 domain were cross-reactive against the s protein of sars-cov-2 (figures 2 and 3) . recent cross-reactivity studies have evaluated sars-cov-1 neutralising antibodies that bind to the rbdcontaining s1 subunit. although both sars-cov-1 and sars-cov-2 use ace2 as a receptor for viral entry [3, 16] , several sars-cov-1 rbd-directed mabs did not cross-react with sars-cov-2 rbd [28, 29] . interestingly, cr3022, which was isolated from a sars convalescent patient [22] , showed cross-reactivity to sars-cov-2 rbd and recognises an epitope that does not overlap with the ace2 binding site [28] . among the four mabs tested in this study, indirect elisa showed that 1a9 binds strongest to the s protein of sars-cov-2 ( figure 3b ). to determine if 1a9 is useful for detection of s protein in a sandwich elisa, it was paired with cr3022 since 1a9 binds to s2 subunit while cr3022 binds to s1 subunit. as would be expected, these two antibodies can be paired to detect s protein from 15.6 ng/ml ( figure 4a ). in addition, mab 1a9 stained a considerable number of sars-cov-2-infected cells at 24 hours post-infection showing that it is sensitive enough to detect the expression of s protein during infection ( figure 4b ). thus, mabs 1a9 will be useful for studying the kinetics of sars-cov-2 replication in vitro and development of diagnostic assays for covid-19. it is noteworthy that cytotoxic t-lymphocyte (ctl) epitopes also reside at residues 884-891 and 1116-1123 within the s2 subunit of sars-cov-1 [30] . interestingly, the latter ctl epitope overlaps with the epitope recognised by mab 1a9 [21] . hence, the s2 subunit may serve as an important antigen for inducing both humoral as well as cell-mediated immunity against sars-cov-1 and sars-cov-2. to our knowledge, this is the first study showing that mabs targeting the s2 domain of sars-cov-1 can cross-react with sars-cov-2 and this observation is consistent with the high sequence conservation in the s2 subunit. the ability of these antibodies, particularly 1a9, to detect sars-cov-2 s protein in indirect and sandwich elisas demonstrate their utility for detection of sars-cov-2 infections in a public health setting. whether or not the current sensitivity of these antibodies are sufficient for robust detection of sars-cov-2 infections in a clinical setting and how they compare to existing pcr-based detection remains to be determined. successful development of these antibodies into a point of care diagnostic kit will provide a complementary approach to existing detection methods. besides the mabs characterised here, several other mabs have been reported to bind to epitopes in the s2 subunit of sars-cov-1 [31] [32] [33] . thus, it will be important to determine if these mabs can also cross-react with sars-cov-2. maurer-stroh, s -designed experiments, performed experiments, analysed and organised data, wrote the manuscript. chow vtk -analysed and organised data. lin rtp -analysed and organised data. mirazimi a-designed experiments, performed experiments, analysed and organised data, wrote the manuscript. hong wj-analysed and organised data. tan yj-designed experiments, performed experiments, analysed and organised data, wrote the manuscript. identification of a novel coronavirus in patients with severe acute respiratory syndrome sars working group. a novel coronavirus associated with severe acute respiratory syndrome a pneumonia outbreak associated with a new coronavirus of probable bat origin an emerging coronavirus causing pneumonia outbreak in wuhan, china: calling for developing therapeutic and prophylactic strategies early 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escape mutants contribution of fc-dependent cell-mediated activity of a vestigial esterase-targeting antibody against h5n6 virus infection parallelization of mafft for large-scale multiple sequence alignments jalview version 2--a multiple sequence alignment editor and analysis workbench molecular evolutionary genetics analysis across computing platforms disease and diplomacy: gisaid's innovative contribution to global health potent binding of 2019 novel coronavirus spike protein by a sars coronavirusspecific human monoclonal antibody cryo-em structure of the 2019-ncov spike in the prefusion conformation characterization of cytotoxic t-lymphocyte epitopes and immune responses to sars coronavirus spike dna vaccine www.eurosurveillance.org expressing the rgd-integrin-binding motif fully human monoclonal antibody directed to proteolytic cleavage site in severe acute respiratory syndrome (sars) coronavirus s protein neutralizes the virus in a rhesus macaque sars model b-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the s2 domain of the surface spike glycoprotein a human sars-cov neutralizing antibody against epitope on s2 protein license, supplementary material and copyright the work performed in nus/nuhs was supported by nuhs research office under project number nuhsro/2020/033/ ro5+5/coronavirus/loa (wbs r-571-000-071-733). the work performed in imcb and bii was also supported by a*star through intramural funding and an a*cruse gap funding (accl/19-gap064-r20h-f). wjh and yjt declare that they are involved in the licensing of mabs 1a9, 1g10, 4b12 and 2b2 to commercial companies as research or diagnostic reagents. the other authors have declared that no competing interests exist. key: cord-279629-t1xjy12y authors: nazneen akhand, mst rubaiat; azim, kazi faizul; hoque, syeda farjana; moli, mahmuda akther; joy, bijit das; akter, hafsa; afif, ibrahim khalil; ahmed, nadim; hasan, mahmudul title: genome based evolutionary study of sars-cov-2 towards the prediction of epitope based chimeric vaccine date: 2020-04-15 journal: biorxiv doi: 10.1101/2020.04.15.036285 sha: doc_id: 279629 cord_uid: t1xjy12y sars-cov-2 is known to infect the neurological, respiratory, enteric, and hepatic systems of human and has already become an unprecedented threat to global healthcare system. covid-19, the most serious public condition caused by sars-cov-2 leads the world to an uncertainty alongside thousands of regular death scenes. unavailability of specific therapeutics or approved vaccine has made the recovery of covi-19 more troublesome and challenging. the present in silico study aimed to predict a novel chimeric vaccines by simultaneously targeting four major structural proteins via the establishment of ancestral relationship among different strains of coronaviruses. conserved regions from the homologous protein sets of spike glycoprotein (s), membrane protein (m), envelope protein and nucleocapsid protein (n) were identified through multiple sequence alignment. the phylogeny analyses of whole genome stated that four proteins (s, e, m and n) reflected the close ancestral relation of sars-cov-2 to sars-cov-1 and bat coronavirus. numerous immunogenic epitopes (both t cell and b cell) were generated from the common fragments which were further ranked on the basis of antigenicity, transmembrane topology, conservancy level, toxicity and allergenicity pattern and population coverage analysis. top putative epitopes were combined with appropriate adjuvants and linkers to construct a novel multiepitope subunit vaccine against covid-19. the designed constructs were characterized based on physicochemical properties, allergenicity, antigenicity and solubility which revealed the superiority of construct v3 in terms safety and efficacy. essential molecular dynamics and normal mode analysis confirmed minimal deformability of the refined model at molecular level. in addition, disulfide engineering was investigated to accelerate the stability of the protein. molecular docking study ensured high binding affinity between construct v3 and hla cells, as well as with different host receptors. microbial expression and translational efficacy of the constructs were checked using pet28a(+) vector of e. coli strain k12. the development of preventive measures to combat covid-19 infections might be aided the present study. however, the in vivo and in vitro validation might be ensured with wet lab trials using model animals for the implementation of the presented data. novel coronavirus named sars-cov-2/ 2019-ncov was identified at the end of 2019 in wuhan, a city in the hubei province of china, causing severe pneumonia that leads to huge death cases . gradually this virus emerged as a new threat to the whole world and affecting almost all parts of the world. to date, the pathogen has affected 198 countries, and thus becoming a global public health emergency. global public health concern with pandemic notion of covid-19 was declared on january 30th, 2020 by the world health organization (who, 2020) . agin, an adverse situation has also been announced on 13 march 2020 for increasing the infections of covid-19 (kunz and minder, 2020) . till april 10, 2020, total virus affected people around the world exceeded 1,633,272 and more than 97,601 committed death, while 366,610 people fully recovered from the infection (who, 2020) . the alarming situation is that the number of confirmed cases worldwide has exceeded one million by this time. it took more than three months to reach the first 10000 confirmed cases, while required only 12 days to detect the next 100000 cases. the situation is getting worse in european region. total death cases in italy, spain, usa, france, united kingdom was 14681,11744,7847,6507 and 4313 respectively (till april 4, 2020) and this number is exacerbating day by day (who, 2020) . some common clinical manifestations of covid-19 is fever, sputum production, shortness of breath, cough, fatigue, sore throat and headache which leads to severe cases of pneumonia. a few patients also have gastrointestinal symptoms with diarrhea and vomiting (guan et al., 2019) . though several early studies showed that the mortality rate for sars-cov-2 is not as high (2-3%), the latest global death rate for covid-19 is 3.4% which indicates the increasing trends . the investigation of chinese center for diseases control and prevention (2020) revealed that the prevalence of covid-19 is more apparent in the people ages 50 years rather than the lower age groups (jeong-ho et al., 2020) . high fever and lymphocytopenia were found more common in covid-19, though the frequency of the patient without fever condition is also higher than in the earlier outbreaks caused by sars-cov (1%) and mers-cov (2%) chen et al., 2020) . sars-cov-2 is a betacoronavirus that has a positive sense, 26-32 kb in length, single stranded rna molecule as its genetic material and belongs to the family coronaviridae, order nidovirales (hui et al., 2019) . it shares genome similarity with sars-cov (79.5%) and bat coronavirus (96%) zhu et al., 2020) . however, there are still obscured hypothesis regarding the vector or carrier of sars-cov-2, though its detection was primarily linked to wuhan's huanan seafood wholesale market (lu et al., 2020; who, 2020) . though the species of sars-cov-1 and bat coronavirus shares sufficient sequence similarities with the covid-19, the known way mechanism of infection to the host, and the death rate is quite different in case of the novel coronavirus. in addition, there is an evolutionary distance between sars-cov-1 and bat coronavirus as well as the covid-19 (hu et al., 2018; wu, 2020b) . because of high sequence variability of the pathogen, many of the efforts that have been undertaken to develop vaccine against sars-cov2, remain unsuccessful . therefore, there is an urgent need to develop vaccines for treatment of sars-cov-2 based on the understanding of actual evolutionary ancestral relationship. while some natural metabolites and traditional medication may come up with comfort and take the edge off few symptoms of covid-19, there is no proof that existing treatment procedures can effectively combat against the diseased condition (who, 2020). however, inactivated or live-attenuated forms of pathogenic organisms are usually recommended for the initiation of antigen-specific responses that alleviate or reduce the possibility of host experience with secondary infections (thompson & staats, 2011) . moreover, all of the proteins are not usually targeted for protective immunity, whereas only a few numbers of proteins are necessary depending on the microbes (tesh et al., 200, li et al., 2014) . depending on sufficient antigen expression from experimental assays, traditional vaccine could take 15 years to develop, while sometimes can lead to undesirable consequences (purcell et al., 2007 , petrovsky & aguilar, 2004 . reverse vaccinology approach, on the other hand, is an effective way to develop vaccine against covid-19. in this method, computation analysis towards genomic architecture of pathogenic candidate could predict the antigens of pathogens without the prerequisite to culture the pathogens in lab condition. although, few pathogens that challenge to develop effective vaccines so far may become possible through such approach (rappuoli, 2000) which initiates a huge move in the development of vaccine against the deadly pathogens. the strategy included the comprehensive utilization of bioinformatics algorithm or tools to develop epitope based vaccine molecules, though further validation and experimental procedures are also needed (moxon et al., 2019) . in addition, peptide based subunit vaccines are biologically safer due to the absence of continuous in vitro culture during the production period, and also implies an appropriate activation of immune responses (purcell et al., 2007; dudek et al., 2010) . such immunoinformatic approaches have already been employed by the researchers to design vaccines against a number of deadly pathogens including ebola virus (khan et al., 2015) , hiv (pandey et al., 2018) , areanaviruses , marburgvirus , norwalk virus (azim et al., 2019b) , nipah virus (saha et al., 2017) , influenza virus (hasan et al., 2019b) and so on. at present, a suitable peptide vaccine against sars-cov-2 is urgently necessary that could efficiently generate enough immune response to destroy the virus. hence, the study was designed to develop a chimeric recombinant vaccine against covid-19 by targeting four major structural proteins of the pathogen, while revealing the evolutionary history of different species of coronavirus based on whole genome and protein domain-based phylogeny. complete genomes of the covid-19 and other coronaviruses were retrieved from the ncbi (https://www.ncbi.nlm.nih.gov/), using the keyword 'coronavirus' and the search option 'nucleotide'. a total 61 complete genomes were retrieved, with unique identity (supplementary file 1). protein sequence of the spike, envelope, membrane and nucleocapsid were also retrieved from the corresponding genome sequences found in ncbi (supplementary file 1). tthe complete genome sequences of coronaviruses and the proteins of envelope, envelope, membrane and nucleocapsid were employed to construct different phylogenetic trees. multiple sequence alignment (msa) of the complete genome and protein sequences were performed using mafft v7.310 (katoh & standley, 2013) tool. for the whole genome alignment, we used mafft auto algorithm, while for the protein sequences alignment, mafft g-ins-i algorithm was used using default parameters. next, alignment was visualized using the jalview-2.11 (waterhouse et al., 2009) . alignment position with more than 50% gaps was pruned from coronavirus genome using phyutility 2.2.6 program (smith & dunn, 2008) . again, more than 20% gaps from the spike protein alignment was removed. partitionfinder-2.1.1 (lanfear et al., 2017) indicated the best fit substitution model of the completed genome sequences and the protein sequences. the phylogeny of the whole genome sequences of coronavirus was constructed using both the maximum likelihood method and bayesian method. raxml version 8.2.11 (stamatakis, 2014 ) with the substitution model gtrgammai was used using 1000 rapid bootstrap replicates. mrbayes version 3.2.6 (ronquist et al., 2012) with invgamma model was used for the corona virus genomes. phylogenetic analyses of four different protein sequences were performed by using raxml-8.2.11 tool. for spike and nucleocapsid proteins, we found protgammaiwag and protgammaiwag as the best fil model, respectively. again, protgammawag was the best fit model of evolution for both the membrane and envelope proteins. for the retrieval of the domain sequences of the stated protein sequences, interpro database (https://www.ebi.ac.uk/interpro/) was utilized. finally, the interactive tree of life (itol; embl, heidelberg, germany) was used for the visualization of the phylogenetic trees. all the trees were rooted in the midpoint. in the present study, reverse vaccinology technique was utilized to model a novel multiepitope subunit vaccine against 2019-ncov. the scheme in figure 1 represents the complete methodology that has been adopted to develop the final vaccine construct. among 496 proteins (available in the ncbi database) from different strains of novel corona virus, four structural proteins, i.e. spike glycoprotein, membrane glycoprotein, envelope protein and nucleocapsid protein, were prioritized for further investigation (supplementary file 2). after sequence retrieval from ncbi, the sequences were subjected to blastp analysis to find out the homologous protein sequences. multiple sequence alignment was done by using clustal omega to identify the conserved regions (sievers and higgins, 2014) . the topology of each conserved regions were predicted by tmhmm server v.2.0 (http://www.cbs.dtu.dk/services/tmhmm/), while the antigenicity of the conserved regions was determined by vaxijen v2.0 (doytchinova and flower, 2007a) . only the common fragments were used for t-cell epitopes enumeration via t-cell epitope prediction server of iedb (http://tools.iedb.org/main/tcell/) (vita et al., 2014) . again, tmhmm server was utilized for the prediction of transmembrane topology of predicted mhc-i and mhc-ii binding peptides followed by antigenicity scoring via vaxijen v2.0 server (krogh et al., 2001; doytchinova and flower, 2007b) . the epitopes which have antigenic potency were picked and used for preceding analysis. the level of conservancy scrutinizes the ability of epitope candidates to impart capacious spectrum immunity. homologous sequence sets of the chosen antigenic proteins were retrieved form the ncbi database by utilizing blastp tool. later, conservancy analysis tool (http://tools.iedb.org/conservancy/) in iedb was used to demonstrate the conservancy level of the predicted epitopes among different viral starins. the toxicity of non-allergenic epitopes was enumerated by using toxinpred server (gupta et al., 2013) . among different ethnic societies and geographic spaces, the hla distribution varies around the world. population coverage study was conducted by using iedb population coverage calculation server (vita et al., 2014) . to check the allergenicity of the proposed epitopes, four distinct servers i.e. allergenfp , allertop (dimitrov et al., 2013) , allermatch (fiers et al., and allergen online (http://www.allergenonline.org/) servers were utilized. three different algorithms i.e. bepipred linear epitope prediction 2.0 (jespersen et al., 2017) , emini surface accessibility prediction (emini et al., 1985) and kolaskar and tongaonkar antigenicity scale (kolaskar and tongaonkar, 1990) from iedb predicted the potential b-cell epitopes within conserved fragments of the chosen viral proteins. top ctl, htl and b cell epitopes were compiled to design the final vaccine constructs in the study. each vaccine constructs commenced with an adjuvant followed by top ctl epitopes, htl epitopes and bcl epitopes respectively. for construction of novel corona vaccine, the chosen adjuvants i.e. l7/l12 ribosomal protein, beta defensin (a 45 mer peptide) and haba protein (m. tuberculosis, accession number: agv15514.1) were used (rana and akhter, 2016) . several linkers such as eaaak, gggs, gpgpg and kk in association with padre sequence were incorporated to construct fruitful vaccine sequences against covid-19. the constructed vaccines were then analyzed whether they are non-allergenic by utilizing the following tool named algpred (azim et al., 2019) . the most potential vaccine among the three constructs was then determined by assessing the antigenicity and solubility of the vaccines via vaxijen v2.0 (doytchinova and flower, 2007b) and proso ii server (smialowski et al., 2006) , respectively. protparam tool (https://web.expasy.org/protparam/), provided by expasy server (hasan et al., 2019c ) was used to functionally characterize (gasteiger et al., 2003) the vaccine constructs. the studied functional properties were isoelectric ph, molecular weight, aliphatic index, instability index, hydropathicity, estimated half-life, gravy values and other physicochemical characteristics. alpha helix, beta sheet and coil structures of the vaccine constructs were analyzed through gor4 secondary structure prediction method using prabi (https://npsaprabi.ibcp.fr/). in addition, espript 3.0 (robert & gouet, 2014) was also used to predict the secondary structure of the stated protein sequences. vaccine 3d model was generated on the basis of percentage similarity between target protein and available template structures from pdb by using i-tasser (peng and xu, 2011) . the modeled structures were further refined via fg-md refinement server. structure validation was performed by ramachandran plot assessment in rampage (hasan et al., 2019b) . by utilizing dbd2 server, probable disulfide bonds were designed for the anticipated vaccine constructs (craig and dombkowski, 2013) . the value of energy was considered < 2.5, while the chi3 value for the residue screening was chosen between -87 to +97 for the operation (hasan et al., 2019b) . the b-cell epitopes of putative vaccine molecules were predicted via ellipro server (http://tools.iedb.org/ellipro/) with minimum score 0.5 and maximum distance of 7 ã� (ponomarenko et al., 2004) . moreover, ifn-inducing epitopes within the vaccine were predicted using ifnepitope with motif and svm hybrid detection strategy (hajighahramani et al., 2017). normal mode analysis (nma) was performed to predict the stability and large scale mobility of the vaccine protein. the imod server determined the stability of construct v3 by comparing the essential dynamics to the normal modes of protein (aalten et al, 1997; wuthrich et al., 1980) . it is a recommended alternative to costly atomistic simulation (tama and brooks, 2006; cui and bahar, 2007) and shows much quicker and efficient assessments than the typical molecular dynamics (md) simulations tools (prabhakar et al., 2016; awan et al., 2017) . the main-chain deformability was also predicted by measuring the efficacy of target molecule to deform at each of its residues. the motion stiffness was represented via eigenvalue, while the covariance matrix and elastic network model was also analyzed. patchdock server was prioritized for docking between different hla alleles and the putative vaccine molecules. in addition, the superior construct was also docked with different human immune receptors such as, ace 3, apn, dpp4 and tlr-8.the 3d structure of these receptors were retrieved from rcsb protein data bank. detection of highest binding affinity between the putative vaccine molecules and the receptor was experimented based on the lowest interaction energy of the docked structure. jcat tool was utilized for codon adaptation in order to fasten the expression of vaccine construct v3 in e. coli strain k12. for this, some restriction enzymes (i.e. bgli and bglii), rho independent transcription termination and prokaryote ribosome-binding site were put away from the work (grote et al., 2005) . after that, the mrna sequence of constructed v3 vaccine was ligated within bgli (401) and bglii (2187) restriction site at the c-terminal and n-terminal sites respectively. snapgene tool was utilized for in silico restriction cloning (solanki and tiwari, 2018 ). in the phylogenetic analysis, we introduced different coronavirus from three different genera: (forni et al., 2017; zhou et al., 2020; zumla et al., 2016) . among these, the first five species belong to the beta coronavirus genera, while the last two belongs to the alpha genera. apart from the human coronaviruses, we introduced other coronaviruses which choose different species of bats, whale, turkey, rat, mink, ferret, swine, camel, rabbit, cow and others as host (supplementary table domain analysis of spike protein of coronaviruses reveals that they contain mainly one signature domains namely, coronavirus s2 glycoprotein (ipr002552), which is present in all the candidates. all other betacoronavirus contains spike receptor binding protein (ipr018548), coronavirus spike glycoprotein hapted receptor 2 domain (ipr027400) and spike receptor binding domain superfamily (ipr036326). sars-cov-1 contains an extra domain, namely spike glycoprotein n-terminal domain (ipr032500), which is also present in some the sub-genera (embecovirus) of betacoronavirus, but not in covid-19. one important finding in our study is that the covid-19 candidates do not contain the domain spike glycoprotein (ipr042578), which is present in the sars-cov-1 (figure 3) . the secondary structure prediction study shows a large numbers of cysteine residues which contribute to the formation of disulfide bonds within the spike protein. most of them fall within the s1 spike protein, which is 654 amino acid long in sars-cov-1, while 672 amino acids long in covid-19. the rgd motif which is conserved within the covid-19 is present in the vicinity of the s1 protein. it exists as kgd that clearly demonstrates the mutation over the short time period. again, the receptor binding domain and receptor binding motif analyses disclose variations within several region between the covid19 and sars-cov-1 (supplementary file 2). the domain-based phylogenetic analysis reflects two main divisions, where the all the novel betacoronavirus i.e., covid19 form clade with the sars-cov-1; while other betacoronavirus fall in another clade which further divide to give rise different sub-genera. this clearly shows that the covid-19 exerts specific ancestral connection to the sars-cov-1 in terms of spike glycoproteins. interestingly, our study also revealed close relatedness of both the sars-cov-1 and covid-19 to the bat betacoronavirus that belongs to the hibecovirus sub-genus. however, in our study, the bat coronaviruses of nobecovirus subgenus did not fall into the same clade of novel coronaviruses. the phylogenetic study and msa also revealed that, the functional portion of the spike glycoprotein domain and spike glycoprotein n-terminal domain might be lost from the covid-19 during the course of evolution. the envelope proteins of both betacoronavirus and alphacoronavirus contain only one protein domain (ipr003873) namely, nonstructural protein ns3 or small envelope protein e (ns3/e). this domain is well conserved in coronavirus and also found in murine hepatitis virus. on the other hands, the gamma coronavirus shows the exception, which possess (ipr005296) ibv3c protein domain, which thought to be expressed from the orf3c gene of infectious bronchitis virus (jia & naqi, 1997 (figure 4) . in spite to the previous findings, where it was found that the envelope proteins of the mers virus and sars-cov-1 exerted close proximity in terms of secondary structure and functions (surya et al., 2015) . unlike to earlier finding, we got that gamma corona virus candidate in our study shows close connection with both sars-cov-1 and covid-19 in terms of envelope proteins. membrane the length of nucleocapsid proteins of betacoronavirus genus ranges from 410 to 450 amino acids. three signature domains are mainly present in the nucleocapsid proteins, which are: coronavirus nucleocapsid protein (ipr001218), nucleocapsid proteins c-terminal (ipr037179) and nucleocapsid proteins n-terminal (ipr037195). however, in our experiment, we didn't find these domains in hcov-hku1 ( figure 6) showed that among the immunogenic conserved sequences from the corresponding proteins except spike glycoprotein met the criteria of desired exomembrane characteristics (table 1) . a plethora of immunogenic epitopes were generated from the conserved sequences that were able to bind with most noteworthy number of hla cells (supplementary table 1, supplementary table 2, supplementary table 3 and supplementary table 4 ). top epitopes with exomembrane characteristics were ranked for each individual protein after investigating their antigenicity score and transmembrane topology (table 2) . epitopes from each protein showed high level of conservancy up to 100% (table 2 ). toxinpred server predicted the relative toxicity of each epitope which indicated that the top epitopes were non-toxin in nature (supplementary table 5 ). population coverage of four structures proteins were also done for the predicted ctl and htl epitopes. from the screening, results showed that population of the various geographic regions could be covered by the predicted t-cell epitopes ( figure 7) . finally, the allergenic epitopes were excluded from the list based on the evaluation of four allergenicity prediction server (supplementary table 5 ). top b-cell epitopes were predicted for spike glycoprotein, membrane glycoprotein, envelope protein and nucleocapsid protein using 3 distinct algorithms (i.e. bepipred linear epitope prediction, emini surface accessibility, kolaskar & tongaonkar antigenicity prediction) from iedb. epitopes were also allowed to analyze their vaxijen scoring and allergenicity (table 3) . three putative vaccine molecules (i.e. v1, v2 and v3) were constructed, each comprising a protein adjuvant, eight t-cell epitopes, twelve b-cell epitopes and respective linkers (supplementary table 6 ). padre sequence was included to extend the efficacy and potency of the constructed vaccine. the putative vaccine constructs, v1, v2 and v3 were 397, 481 and 510 residues long respectively. however, allergenicity score of v3 (-0.89886723) revealed that it was superior among the three constructs in terms safety and efficacy. v3 also had a solubility score (0.60) ( figure 8e ) and antigenicity (0.58) over threshold value (table 4) . protparam tool was employed to analyze the physicochemical properties of v3. figure 1) . tertiary structure of the putative vaccine construct v3 was generated using i-tasser server ( figure 8a and 8b). the server used 10 best templates with highest significant (measured via zscore) from the lomets threading program to model the 3d structure. after refinement, ramachandran plot analysis revealed that 92.7% and 5.7% residues were in the favored and allowed regions respectively, while only 8 residues (1.6%) occupied in the outlier region ( figure 8c ). the overall quality factor determined by errat server was 91.56% ( figure 8d ) ellipro server predicted a total 6 conformational b-cell epitopes from the 3d structure of the construct v3. epitopes no. 1 were considered as the broadest conformational b cell epitopes with 25 amino acid residues (figure 9 and supplementary table 8 ). stability of the vaccine construct v3 was investigated through mobility analysis ( figure 10a and 10b), b-factor, eigenvalue & deformability analysis, covariance map and recommended elastic network model. results revealed that the placements of hinges in the chain was insignificant ( figure 10c ) and the b-factor column gave an averaged rms ( figure 10d ). the estimated higher eigenvalue 6.341333e -06 ( figure 10e ) indicated low chance of deformation of vaccine protein v3. the correlation matrix and elasticity of the construct have been shown in figure 10g and figure 10h , respectively. the structural interaction between hla alleles and the designed vaccines were investigated by molecular docking approach. the server detected the complexed structure by focusing on complementarity score, ace (atomic contact energy) and estimated interface area of the compound ( table 5 ). the molecular affinity between the putative vaccine molecules v3 and several immune receptors were also experimented. the result showed that construct v3 interacted with each receptor with significantly lower binding energy (figure 11 ). the codon adaptation index (cai) and gc content for the predicted codons of the putative vaccine constructs v1 were demonstrated as 1.0 and 51.56% respectively. an insert of 1542 bp was found which lacked the restriction sites for bgli and bglii, thus providing comfort zone for cloning. the codons were inserted into pet28a(+) vector alongside two restriction sites (bgli and bglii) and a clone of 5125 base pair was generated ( figure 12 ). in december 2019, a new coronavirus prevalence flourished in wuhan, china, causing clutter among the medical community, as well as to the rest of the world . the new species has been renamed as 2019-ncov or, sars-cov-2, already causing considerable number infections and deaths in china, italy, spain, iran, usa and to a growing degree throughout the world. the major outbreak and spread of sars-cov-2 in 2020 forced the scientific community to make considerable investment and research activity for developing a vaccine against the pathogen. however, owing to high infectivity and pathogenicity, the culture of sars-cov-2 needs biosafety level 3 conditions, which may obstructed the rapid development of any vaccine or therapeutics. it had been found that about 35 companies and academic institutions are engaged in such works (spinney et al., 2020 , ziady et al., 2020 . among the potential sars-cov-2 vaccines in the pipeline, four have nucleic acid based designs, four involve non-replicating viruses or protein constructs, two contain live attenuated virus and one involves a viral vector (pang et al., 2020) , while only one, called mrna-1273 (developed by niaid collaboration with moderna, inc.), has confirmed to start phase-1 trial (nih, 2020) . however, in this study we emphasized on a different approaches by prioritizing the advantages of different genome and proteome database using the immunoinformatic approach. computational vaccine predictions were adopted by the researchers to design vaccines against both mers-cov (sudhakar et al., 2013; fernando et al., 2013) and sars-cov-1 (yang et al., 2003; oany et al., 2014) , targeting the outer membrane or functional proteins (sharmin and islam, 2014) . several in silico strategies have also been employed to predict potential t cell and b cell epitopes against sars-cov-2, either emphasizing on spike glycoprotein or envelope proteins (behbahani, 2020; rasheed et al., 2020) . none of the studies, however, focused on other structural proteins. moreover, random genetic changes and mutations in the protein sequences (yin, 2020) may obstruct the development of effective vaccines and therapeutics against human coronavirus in the future. hence, the present study was employed to identify the similarity and divergence among the close relatives of the target pathogen and develop a novel chimeric recombinant vaccine considering all major structural proteins i.e. spike glycoprotein, membrane glycoprotein, envelope protein and nucleocapsid protein simultaneously. the topology of the phylogenetic trees of the whole genome and the stated four proteins sequences from different species of coronaviruses reveal that sars-cov-1 and bat coronaviruses are the closest homologs of the novel coronaviruses. our results infer a significant level of similarities within the covid-19 and sars-cov-1 which was also aligned with the previous findings (jaimes et al., 2020; wu, 2020a) . the sequence similarities between the sars-cov, bat coronaviruses and the covid-19 from the reported studies (hu et al., 2018; wu, 2020b) suggests that those are distantly related, in spite those are capable of infecting the humans and therefore possess the adaptive convergent evolution. interestingly, the covid-19 envelope proteins form clade with the turkey coronavirus which belongs to gamma coronavirus genus. so, in terms of envelope proteins, the envelope gene of turkey coronavirus might contribute to the convergence process, which need further analysis. in addition, from the domain-based phylogeny of nucleocapsid proteins, it can be deduced that this protein might have originated in bats and was transmitted to camels and then later on choose human as the potential host. overall, the covid-19 might go through complex adaptation strategies in order to be transmitted into the human via different animals. the homologous protein sets for four structural proteins of coronavirus were sorted to identify conserved regions through blastp analysis and msa. only the conserved sequences were utilized to identify potential b-cell and t-cell epitopes for each individual protein (table 1) . thus, our constructs are expected to stimulate a broad-spectrum immunity in host upon administration. cytotoxic cd8+t lymphocytes (ctl) play a crucial role to control the spread of pathogens by recognizing and killing diseased cells or by means of antiviral cytokine secretion (garcia et al., 1999) . thus, t cell epitope-based vaccination is a unique process to confer defensive response against pathogenic candidates (shrestha, 2004) . approximately 800 mhc-i peptides (ctl epitopes) and 600 mhc-ii peptides (htl epitopes) were predicted via iedb server, from which we screened the top ones through analyzing the antigenicity score, transmembrane topology, conservancy level and other important physiochemical parameters employing a number of bioinformatics tools ( table 2 ). the top 10 epitopes from each protein was further assessed by investigating the toxicity profile and allergenicity pattern. different servers rely on different parameters to predict the allergenic nature of small peptides. therefore, we used 4 distinct servers for such assessment and the epitopes predicted as non-allergen at least via 3 servers were retained for further analysis (supplementary table 5 ). vaccine initiates the generation of effective antibodies that are usually produced by b cells and plays effector functions by targeting specifically to a foreign particles (cooper & nemerow, 1984) . the potential b cell epitopes were generated by three different algorithms (bepipred linear epitope prediction 2.0, kolaskar and tongaonkar antigenicity prediction and emini surface accessibility prediction) from iedb database (table 3) . suitable linkers and adjuvants were used to combine top finalized epitopes from each protein that led to develop a multi epitope vaccine molecules (supplementary table 6 ). as padre sequence was usually recommended to lessen the polymorphism of hla molecules in the population (ghaffari-nazari et al., 2015) , it was also considered to construct the final vaccine molecule. here, adjuvants would enhance the immunogenicity of the vaccine constructs and appropriate separation of epitopes in the host environment would be ensured by the linker (yang et al., 2015) . allergenicity, physiochemical properties, antigenicity and three-dimensional structure of vaccine constructs were characterized, and it had been concluded that v3 was superior to v1 and v2 vaccine constr. the final construct also occupied by several interferon-î± producing epitopes (supplementary table 8 ). the vaccine protein (v3) was subjected to disulfide engineering to enhance its stability. analysis of the normal modes in internal coordinates by imods was employed to investigate the collective motion of vaccine molecules (lopez-blanco et al., 2014) . negligible chance of deformability at molecular level was analyzed for the putative vaccine construct v3, thereby strengthening our prediction. moreover, molecular docking was investigated to analyze the molecular affinity of the vaccine with different hla molecules i.e. drb1*0101, drb5*0101, drb3*0202, drb1*0401, drb3*0101 and drb1*0301 (table 5) . it had been reported that a specific receptor-binding domain of cov spike protein usually recognizes its host receptor ace2 (angiotensin-converting enzyme 2) (li. et al., 2003; li, 2015) . previous studies also identified dipeptidyl peptidase 4 (dpp4) as a functional receptor for human coronavirus (raj et al., 2013) . therefore, we performed another docking study prioritizing these immune receptors to strengthen our prediction ( figure 11 ). results showed that the designed construct bound with the selected receptors with minimum binding energy which was biologically significant. finally, in-silico restriction cloning was adopted to check the suitability of construct v3 for entry into pet28a (+) vector and expression in e. coli strain k12 (figure 12 ). traditional ways to vaccine development are time consuming and laborious. moreover, the result may not be always as expected or fruitful (stratton et al., 2003; hasan et al., 2019) . in silico prediction and prescreening methods, on the contrary, offer some advantages while saving time and cost for production. therefore, the present study may aid in the development of preventive strategies and novel vaccines to combat infections caused by 2019-ncov. however, further wet lab trials involving model organism needs to be experimented 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coronavirus neutralization and protective immunity in mice genotyping coronavirus sars-cov-2: methods and implications atomic-level protein structure refinement using 857 fragment-guided molecular dynamics conformation sampling a pneumonia outbreak associated with a new coronavirus of probable bat origin network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 a novel coronavirus from patients with pneumonia in china biotech company moderna says its coronavirus vaccine is ready for first tests coronaviruses-drug discovery and therapeutic options authors would like to acknowledge the department of biochemistry and chemistry, department of microbial biotechnology and department of pharmaceuticals and industrial biotechnology of sylhet agricultural university for the technical support of the project. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. authors declare that they have no conflict of interests. supplementary table 1 : predicted ctl and htl epitopes of spike glycoprotein. key: cord-298251-u36lb44w authors: donaldson, julie g.; jackson, catherine l. title: arf family g proteins and their regulators: roles in membrane transport, development and disease date: 2011-05-18 journal: nature reviews molecular cell biology doi: 10.1038/nrm3117 sha: doc_id: 298251 cord_uid: u36lb44w members of the adp-ribosylation factor (arf) family of guanine-nucleotide-binding (g) proteins, including the arf-like (arl) proteins and sar1, regulate membrane traffic and organelle structure by recruiting cargo-sorting coat proteins, modulating membrane lipid composition, and interacting with regulators of other g proteins. new roles of arf and arl proteins are emerging, including novel functions at the golgi complex and in cilia formation. their function is under tight spatial control, which is mediated by guanine nucleotide exchange factors (gefs) and gtpase-activating proteins (gaps) that catalyse gtp exchange and hydrolysis, respectively. important advances are being gained in our understanding of the functional networks that are formed not only by the gefs and gaps themselves but also by the inactive forms of the arf proteins. guanine nucleotide exchange factors (gefs) . proteins that promote the release of gdp from guanin e-nucleotide-binding (g) proteins, which allows gtp to bind. these proteins often stabilize the nucleotide-free form and then are released upon gtp binding. gtpase-activating proteins (gaps) . proteins that promote gtp hydrolysis on gtp-bound guanine-nucleotide-binding (g) proteins. for adp-ribosylation factor (arf) proteins, gaps are critical, as arfs have negligible intrinsic gtpase activity. the catalytic regions of gaps often include an arg-finger motif that inserts into the gtp-binding pocket to stimulate hydrolysis of gtp. signalling is more complex and that gefs and gaps can initiate their own physiological responses. we see evidence of arf proteins acting in pairs or in series in the endoplasmic reticulum (er)-golgi system and at the plasma membrane. in this review, we emphasize how arf proteins function as a network in which the arf regulators participate. these regulators also integrate arf activities with other g protein signalling networks, as well as initiating their own distinct signalling pathways. we highlight new arf and arl activities, discuss a lipid modification, occurring co-or post-translationally, in which a myristoyl moiety is attached to a gly residue at the second position from the amino terminus, after cleavage of the n-terminal met residue. gdp dissociation inhibitor (gdi) . a protein that binds specifically to the gdp-bound form of a gtp-binding protein, preventing guanine nucleotide exchange. how gefs and gaps can act as scaffolds, both as effectors and in initiating signalling responses, and describe how they participate in development and disease. the reader is referred to two excellent prior reviews: one deals comprehensively with arf1 and arf6 function 1 and the other more broadly with arf, arl, gef and gap proteins 2 . arfs localize to membranes throughout the cell, including the plasma membrane and the membranes of the secretory, endosomal and lysosomal pathways. a distinguishing feature of arf family g proteins is the presence of an n-terminal amphipathic helix that is critical for membrane binding (fig. 1a,b ). in addition, all arf proteins are modified by myristoylation at the n terminus and this co-translational modification is required for membrane recruitment and biological activity. the myristoyl group and associated n-terminal amphipathic helix are inserted into the membrane upon gtp binding 3 . thus, in addition to changes in the effector-binding regions upon exchange of gdp for gtp, arf proteins undergo a second change in conformation that brings them into very close contact with the membrane 4 (fig. 1) . this distinguishes them from other small g proteins of the ras superfamily, including the ras, rho and rab families, which have a long carboxy-terminal linker to which their lipid membrane anchor is attached 2 . arf effectors are thus constrained to a position close to the membrane surface, in contrast to those of rab and rho, which can be located some distance from the membrane 2 . some arl proteins are myristoylated but most seem to lack this modification. in arl8b, loss of hydrophobic residues in the amphipathic helix abolishes lysosomal localization 5 . interestingly, arfrp1 (arl3 in saccharomyces cerevisiae), arl8a and arl8b are acetylated rather than myristoylated at their n terminus 2 . in sar1, the n-terminal amphipathic helix binds directly to membrane s and induces membrane curvature 6 . unlike for rab and rho g proteins, no gdp dissociation inhibitor (gdi) proteins have been identified for arfs or arls. arf1 and arf3 appear to be released from membranes on gtp hydrolysis in cells. arf6, however, remains bound to membranes in its gdp-bound conformation, and there is evidence that arf4 and arf5 remain bound to er-golgi intermediate compartment (ergic) membranes in their gdp-bound form 7, 8 . this raises the possibility that arf proteins that are bound to membranes in their gdp-bound form might interact with membrane-localized partners and mediate signalling. evidence for this idea is emerging for arf6 (see below), and suggests that distinct signalling pathways might be coordinated through the nucleotide state of these constitutively membrane-bound arf family proteins. sar1 and some of the arls, such as arl1, arl4 and arl8, are cytosolic when gdp-bound, similarly to arf1 (refs 2, 9) , and it remains to be determined whether this is true for other arl proteins. in humans, there are 15 arf gefs, which are divided into six subfamilies, as well as the sar1 gef sec12 (table 2) . no specific arl gefs have yet been identified, although the arf1 gef syt1 (suppressor of ypt3 1) in yeast apparently also has activity towards arl1 (ref. 10 ). the 31 identified mammalian arf gaps fall into nine major subgroups based on their domain structure (table 2) . two arl gaps have been identified (see below). gefs and gaps are recruited to very specific sites within cells to not only catalyse gtp exchange and hydrolysis, respectively, but also to assemble protein complexes at these sites independently of their catalytic activity (fig. 1c) . in this way, versatile signalling networks can be assembled that can respond dynamically to extracellula r and intracellular signals. following activation on membranes, gtp-bound arfs recruit coat proteins, lipid-modifying enzymes, tethers and other effector molecules that influence membrane trafficking and organelle structure 1,2 (table 1) . for example, arf1 recruits the cytosolic coatomer complex i (copi) to golgi membranes, allowing sorting of cargo proteins into copi-coated vesicles 11 . arf proteins at the trans-golgi network (tgn) also recruit heterotetrameric clathrin adaptor protein 1 (ap1), ap3 and ap4, as well as the three monomeric golgi-localized, γ-ear-containing, adp-ribosylation factor-binding figure 1 | the domain structure and regulation of arf and arls. a | a schematic of representative adp-ribosylation factor (arf), sar1 and arf-like (arl) proteins, indicating the conserved amino-terminal amphipathic helix and the protein-specific lipid modifications at the n terminus. these include myristoylation (myr) and acetylation (acet), both of which ensure tight membrane association. the effector regions of the guaninenucleotide-binding (g) protein, switch 1 (sw1) and sw2, and the interswitch region between them, are depicted. these regions change conformation upon exchange of gdp for gtp and are involved in interactions with effectors. b | arf•gdp reversibly associates with the membrane surface, and the myristoylated n-terminal helix ensures tight membrane association of arf•gtp. the switch and interswitch regions are also shown, and these undergo a conformational change upon gtp binding to enter the hydrophobic pocket that the n-terminal amphipathic helix occupies in the gdp-bound form. c | arf family g proteins undergo a cycle of gtp binding and hydrolysis, mediated by guanine nucleotide exchange factors (gefs) and gtpase-activating proteins (gaps), respectively. the gtp-bound form is thought to carry out g protein functions through interaction with 'classical effectors', including vesicle coat proteins and enzymes that can modify membrane lipid composition; however, increased attention has focused on networks of effectors that are targeted by proteins that interact with gefs and gaps themselves and unique effectors that associate specifically with the gdp-bound form of arf proteins. a slender extension on the cell surface. a non-motile, primary cilium is present on nearly all epithelial cells in the body and serves as a sensory organ that is important for regulating cell differentiation and division. proteins (ggas), gga1, gga2 and gga3 12 . these various coat proteins specifically bind cargo proteins and incorporate them into newly forming vesicles for sorting and transport to their correct destinations. arfs can also recruit and activate enzymes that alter membrane lipid composition. phospholipase d (pld), which hydrolyses phosphatidylcholine to generate phosphatidic acid, is activated by all arf proteins and also by arl1 (ref. 13 ). all arf proteins can both recruit and activate ptdins-4-phosphate 5-kinase (ptdins4p5k), an enzyme that phosphorylates ptdins4p at the 5-position to generate ptdins-4,5-bisphosphate (ptdins(4,5) p 2 ) 1 . for example, arf6 at the cell periphery directly affects the activity of ptdins4p5k at the plasma membrane, and thus regulates ptdins(4,5)p 2 levels there 1 . at the golgi, arf1 recruits and stimulates the activity of ptdins 4-kinase (ptdins4k), forming ptdins4p, which is an important membrane lipid for golgi function 14 . arf1 also binds to ptdins4p-specific pleckstrin homology (ph) domains contained in a family of oxysterolbinding proteins that are believed to function in lipid homeo stasis at the golgi 14 . new functions for golgi-associated arfs. the five arf proteins in humans, arf1, arf3, arf4, arf5, and arf6, are ubiquitously expressed. studies to date have focused mainly on arf1 at the golgi and arf6 at the plasma membrane, but arf3, arf4 and arf5 are also present on golgi membranes (fig. 2a) . surprisingly, depletion experiments using rna interference (rnai) show that no single arf, including arf1, is required for golgi function; instead, arfs function in pairs at particular steps in golgi transport 15 . for example, arf1 and arf4 act redundantly during transport in the early secretory pathway 15 . consistent with this observation, arf4 localizes to the ergic and cis-golgi 8 and, together with arf1 at the cis-golgi, it organizes traffickin g between these compartments 16 . arf1 and arf3 are identical except for seven amino acid differences in their n-terminal and c-terminal regions, and previously they were thought to function and localize identically. however, a golgitargeting sequence contained within the α3 helix of arf1 and arf3 targets a chimaera of arf6 and arf1 to the early golgi 17 . furthermore, arf3 localizes specifically to the tgn (fig. 2a) , and this localization depends on four arf3-specific amino acids contained in the n-terminal amphipathic helix, which are conserved among arf3 homologues 18 . arf3, but not arf1, becomes cytosolic at 20 o c, the temperature at which exit from the tgn is blocked 18 . thus, arf3 might have an additional crucial role during exit from the golgi. several important functions for class ii arfs at the tgn have now been defined (fig. 2a) . in an elegant series of studies, arf4 was found to specifically recognize the vxpx cytosolic targeting motif in retinal rhodopsin to facilitate its transport into the rod outer segment, which is a specialized cilium 19 (fig. 2b) . this ciliary targeting complex includes, in addition to arf4, rab11, fip3 (a shared arf and rab11 effector) and asap1 (arf gap containing sh3, ankyrin repeat and ph domains 1) 20 . exactly how this complex facilitates the packaging of rhodopsin into post-golgi carriers has yet to be determined but, interestingly, rhodopsin itself initiates complex formation by recruiting arf4. the rhodopsin-binding site on arf4 is in the α3 helix 19 , the same region that in arf1 binds the snare protein membrin (also known as gosr2) to mediate targeting to the early golgi 17 ; thus, this region might generally allow arf protein binding to membrane receptors. arf4 and arf5 can also directly bind to caps (calcium-dependent activator protein for secretion), which regulates exocytosis of dense core vesicles from nerve terminals 21 . it is the gdp-bound form of the arf that binds to the ph domains of caps proteins, and knockdown of caps, arf4 or arf5 causes retention of chromagrannin (a marker for dense core vesicles) in the golgi, suggesting that arf4 and arf5, together with caps, regulate the release of dense core vesicles from the golgi (fig. 2a) . how these roles of arf4 and arf5 at the tgn in specialized cells can be reconciled plants have numerous adp-ribosylation factors (arfs) that are homologous to human arf1 (ref. 125) and were originally thought to lack class iii arf6-like proteins. however, in arabidopsis thaliana, arfb (also called arfb1a) localizes to the plasma membrane and lacks the golgi-targeting motif (mxxe) that is found in other arf1 homologues in plants 126 and in mammals 17 . nevertheless, only the gbf and big subfamilies of arf guanine nucleotide exchange factors (gefs) seem to be present in plants, and these function in both endocytic and golgi trafficking pathways 127 . a. thaliana gnom (also known as emb30) is a homologue of mammalian gbf1 but acts at endosomes and the plasma membrane during the polar transport of the plant hormone auxin during development 127, 128 . another gbf-like protein in a. thaliana, gnom-like 1 (gnl1), functions at the golgi similarly to mammalian gbf1, but is also involved in endosomal trafficking 129 . big5 (also known as ben1 and atmin7) was identified in a screen for a. thaliana mutants defective for internalization of the pin auxin transporter from the plasma membrane. this arf gef is most closely related to big1 and big2 in mammalian cells, localizes predominantly to the trans-golgi network (tgn) and early endosomes, and is involved in early endosomal trafficking 130 . interestingly, big5 is targeted for degradation by a plant bacterial pathogen, pseudomonas syringae, to protect the latter from host defence systems at the cell wall 131 . a. thaliana arf gaps include four members of a family of mammalian acap homologues that are known as vascular network 3 (van3)-like after the first member to be characterized 125 . van3 (also known as scarface and agd3) regulates formation of plant vascular networks 132, 133 . in addition to its roles on endosomes, van3 cooperates with gnom during clathrin-mediated endocytosis of the pin auxin transporter 128 . another arf gap in a. thaliana, arf gap domain 5 (agd5; also known as nevershed), is a homologue of yeast arf gap effector 2 (age2) and mammalian smap family arf gaps that localizes to the tgn 134 . agd5 is required for floral organ cell separation 125 and regulates membrane trafficking though tgn-early endosomal compartments to trigger organ abscission 135 . interestingly, the protozoan parasite trypanosoma brucei expresses a single arf protein that has characteristics of both arf1 and arf6. t. brucei arf1 is a basic protein with a calculated isoelectric point (pi) value of 9.1, which is similar to that of human arf6, but t. brucei arf1 contains the golgi-targeting motif mxxe 136 that is found in human arf1 and arf3 (ref. 17 ). depletion of t. brucei arf1 by small interfering rna causes a major decrease in endocytosis and the formation of intracellular flagella, but the golgi remains intact 136 . trypanosomes also express an arf-like 2 (arl2) homologue, which is involved in microtubule biogenesis and cytokinesis 137 , and an arl1 homologue, which is important for golgi structure and exocytosis of glycosyl phosphatidylinositol (gpi)-anchored proteins 138 . arf and arl proteins in trypanosomes are myristoylated, a modification that is required for their activity. trypanosomes cause african sleeping sickness, a disease with no successful therapy. a selective inhibitor of trypanosomal n-myristoyl transferase has been shown to be effective in blocking trypanosome viability in a mouse model of this disease 139 . with findings of arf4 localization to, and arf4 and arf5 functioning at, the early golgi in other cells 8, 15, 16 is not known. recent discoveries show that arf1 regulates lipid transfer proteins within the golgi and promotes the formation of lipid droplets at the ergic (fig. 2a) . at the golgi, arf1 recruits the lipid transfer proteins ceramide transfer (cert) and fapp2 (ref. 14) through interaction with their ph domains, which can also bind ptdins4p. cert mediates the non-vesicular transport of ceramide from the er to the golgi and fapp2 mediates the transfer of glucosylceramide from the cytosolic side of the early golgi to the trans-golgi 22 . exactly how the directionality of this transfer occurs, and the role that arf1 has, is not yet clear. the finding that arf1 associates with gbf1 and copi during lipid droplet formation was un expected. these proteins were identified in an rnai screen of lipid droplet formation in d. melanogaster 23 and also appeared in proteomic analyses of lipid droplets along with other trafficking proteins, which led to the idea that lipid droplets interface with multiple membrane trafficking pathways 24 . in particular, the delivery of two proteins, adipose triglyceride lipase (atgl) and adipose differentiation-related protein (adrp; also known as adipophilin), to the surface of lipid droplets requires arf1, gbf1 and copi, and possibly the copii machinery, in mammalian cells 25 ; similar results were obtained in d. melanogaster s2 cells 26 . another arf family membe r, arfrp1, is highly expressed in adipocytes, and mice that lack arfrp1 in adipose tissue show severe defects in lipid storage and enhanced lipolysis 27 . finally, in some cell types arf1 at the plasma membrane affects endocytosis of proteins anchored to the membrane by a glyco syl ptdins (gpi) linkage 28 . this may also require the arf gef gbf1 (ref. 29) and could be related to the other lipid-regulating functions of arf1. a great deal of work on arf6 function has been summarized in a pre vious review 1 , so here we focus on more recent advances. in mammals, arf6 is not required for early embryonic development, but arf6-knockout mice die at and arf4 localize to the early cis-golgi and arf3 specifically localizes to the trans-golgi network (tgn). in addition to the recruitment of coat proteins (coatomer complex i (copi), gga (golgi-localized, γ-ear-containing, adp-ribosylation factor-binding protein) and adaptor protein 1 (ap1)) to the golgi, arf1 binds to ceramide transfer (cert) and fapp2 to mediate the transport of ceramide and glucosylceramide lipids from the cis-golgi to the trans-golgi. at the er-golgi intermediate compartment (ergic), arf1 and its guanine nucleotide exchange factor (gef) gbf1 act with copii to regulate the formation of lipid droplets and for the replication of several viruses. caps (calcium-dependent activator protein for secretion), which is involved in regulated secretion, is recruited to the tgn by arf4 and arf5. at the er, sar1, activated by sec12, recruits copii to allow vesicle transport to the golgi. b | in retinal cells, arf4 binds specifically to rhodopsin in the tgn membrane and, together with fip3, asap (arf gap containing sh3, ankyrin repeat and ph domains) and rab11, it facilitates the transport of rhodopsin in transport vesicles from the inner segment to the outer segment, which is a specialized cilium. arf-like 3 (arl3) has been found to be localized to the connecting cilium, and retinitis pigmentosa 2 (rp2; also known as xrp2), an arl3 gap, localizes to the tgn, the basal body and the membrane adjacent to the connecting cilium. c | in primary cilia, arl6 recruits the bbsome coat complex that facilitates the transport of membrane proteins into the cilium. arl13 is localized to the cilium and has been implicated in intraflagellar transport. adrp, adipose differentiation-related protein (also known as adipophilin); atgl, adipose triglyceride lipase; ptdins4k, phosphatidylinositol 4-kinase. new end take-off a switch in cellular growth of fission yeast, from monopolar extension to bipolar extension. mid-gestatio n or shortly after birth and exhibit impaired liver development 30 . this phenotype suggests that the critical physiological roles of arf6 take place after birth and is consistent with reported effects of arf6 on cell adhesion, cell migration, wound healing and metastasis. arf6 is present at the plasma membrane and influences both the cortical actin cytoskeleton and endosomal membrane recycling 1 (fig. 3) . at the plasma membrane, arf6 changes the membrane lipid composition through activation of ptdins4p5k and pld, resulting in the generation of ptdins(4,5)p 2 and phosphatidic acid. these lipids are important for sorting proteins within the membrane, for the formation of clathrin-coated pits during endocytosis, and for the recruitment and activation of rho family g proteins, such as rac, to alter actin poly merization. there is some evidence that arf6 can interact with ap2 (ref. 31) and clathrin during g proteincoupled receptor (gpcr) cell signalling 32 . a recent study has found that arf6 enters cells in clathrin-coated vesicles to facilitate the rapid recycling of the transferrin receptor back to the plasma membrane through interaction with the microtubule motor adaptor protein jnkinteracting protein 4 (jip4) after clathrin uncoating 33 . in some cells, arf6 associates with endosomal membranes derived from clathrin-independent forms of endo cytosis and mediates recycling of this membrane back to the plasma membrane 34 . recycling endosomes return membrane proteins that are important for cell adhesion and migration back to the plasma membrane 34, 35 . arf6 regulation of such endosomal membrane trafficking is required for the polarized delivery of cdc42, rac and the par6 complex to the leading edge of migratin g cells 36 , which can alter adhesion to the extracellular matrix through focal adhesions and actin-based protrusions. hence, regardless of the mode of endocytosis, arf6 is important for membrane recycling. the crucial functions of arf6 in membrane lipid modification, establishment of cell polarity and promotion of endocytic recycling are conserved in yeast and d. melanogaster 1 . arf3, the yeast arf6 homologue, contributes to ptdins(4,5)p 2 levels at the plasma membrane 37 and also affects polarization events, such as bud site selection 38 in s. cerevisiae and new end take-off growth in schizosaccharomyces pombe 39 . arfb, the arf6 homologue in the filamentous fungi aspergillus nidulans, localizes to both the plasma membrane and endomembranes, and regulates endocytosis and polarity establishment during hyphal growth 40 . in d. melanogaster, figure 3 | the localization and function of arf and arl proteins in endosomal-lysosomal trafficking. at the plasma membrane, adp-ribosylation factor 6 (arf6) activates phosphatidylinositol-4-phosphate 5-kinase (ptdins4p5k) to generate ptdins-4,5-bisphosphate (ptdins(4,5)p 2 ) and, together with arf-like 4 (arl4), recruits cytohesin (also known as arno) guanine nucleotide exchange factors (gefs) that can lead to further activation of arf6 or arf1. cytohesins associate with the ipcef (interactor protein for cytohesin exchange factors)-dock180 complex, which activates rac, but another rac gef, kalirin, can be recruited to membranes by arf6•gdp. arf6 at the plasma membrane can regulate the membrane lipid composition, alterations in cortical actin to drive protrusions (for example, during cell migration), and endocytosis of ligand-activated guanine-nucleotide-binding (g) protein-coupled receptors (gpcr) via clathrin-dependent endocytosis. arf6 and the microtubule motor adaptor protein jnk-interacting protein 4 (jip4) promote rapid recycling of endosomal membrane back to the cell surface, and arf6, together with the exocyst complex, also affects slow recycling from sorting endosomes. arf1 has been implicated in clathrin-independent endocytosis of glycosyl ptdins (gpi)-anchored proteins in some cells. arf6 and the arf6 gefs cytohesin and brag2 have been implicated in both assembly and disassembly of adherens junctions. two arf gtpase-activating proteins (gaps), asap1 (arf gap containing sh3, ankyrin repeat and ph domains 1) and git1, localize to focal adhesions that mediate adhesion to the extracellular matrix (ecm), and git1 interacts with pix, a gef for cdc42. arl8 is required for fusion of multivesicular late endosomes with lysosomes and is involved in transport along microtubules. hgf, hepatocyte growth factor. cellular adhesions that connect epithelial cells to form a polarized epithelium. made up of homotypic cadherin interactions and associated intracellular proteins. (roundabout homologue 4). acts as a receptor for slit2 protein and regulates vascular integrity. an adhesive, ring-like, actin-rich structure that is formed on the ventral surface of cells. a complex of proteins that facilitates membrane traffic into the cilium. mutant forms of several bbs components have been identified as causative agents for various ciliopathies. deletion of the arf6 homologue blocks the rapid endocytic recycling required for cytokinesis in spermatocytes, resulting in male sterility, but no other phenotypes were reported 41 . interestingly, in mammalian cells arf6 interacts with jip4 to control a motor switch mechanism regulating endosomal trafficking in cytokinesis 42 . the crystal structure of arf6 in complex with jip4 shows that residues adjacent to the switch regions are structural determinants for the specific binding of jip4 to arf6 (ref. 43 ). arf6 has been implicated in both the assembly and disassembly of adherens junctions in polarized epithelial cells 1 (fig. 3) . during adherens junction formation, par3 recruits a scaffolding protein, frmd4a, that binds to cytohesin gefs, which leads to activation of arf6 (ref. 44 ). treatment of fully polarized epithelial cells with hepatocyte growth factor leads to activation of arf6, most likely through the arf gef brag2 (ref. 45 ), and activation of rac, which causes disassembly of adherens junctions by stimulating endocytosis of epithelial cadherin (e-cadherin) 1 . hence, depending on the signalling complex assembled, either formation or dis assembly of adherens junctions can be achieved through activation of arf6. there is also some evidence that the arf6 gef efa6 affects tight-junction assembly 46 . arf6 activation has also been reported at the onset of tubulogenesis (a developmental progression from polarized epithelia to tubular structures), and perturbation of the arf6 gtp-gdp cycle inhibits tubule formation 47 . importance of turning off arf6. arf proteins carry out their actions through a regulated cycle of gtp binding and hydrolysis. this allows arfs to engage and disengage with their effectors with spatial and temporal specificity, and in some cases may allow arf•gdp to bind other classes of effector. arf6•gdp binds several tbc (tre2-bub2-cdc16) domain-containing proteins, which often have rab gap activity 48 . arf6•gdp binds both tbc1 domain family member 24 (tbc1d24; a protein mutated in familial infantile myoclonic epilepsy 49 ) and the tre17 oncogene 50 . tre17 binding to arf6 increases its activation 50 ; although tre17 does not itself have gef activity towards arf6, it may facilitate interaction of arf6 with another gef. arf6•gdp also binds to the kalirin family of rho gefs, through their spectrin-like repeat domain 51 , and recruits kalirin to the membrane, where it subsequently activates rac and rhog to regulate actin dynamics 51 (fig. 3) . hence, arf6•gdp and arf6•gtp both interact with regulatory proteins of other small g proteins, allowing alternative signalling pathways to be activated depending on which nucleotide is bound (fig. 1c) . this raises the intriguing possibility that other gdp-bound arf or arl proteins might also bind unique effector proteins. turning off arf6 is important for its biological function. in some cells, expression of the constitutively active mutant of arf6, q67l, leads to the accumu lation of early endosomes containing plasma membrane proteins that enter cells independently of clathrin; failure to in activate arf6 blocks further trafficking of this membrane towards recycling or to other destinations 52 . immediately upon platelet activation, arf6•gtp levels fall, and this inactivation precedes, and is required for, the subsequent activation of rac 53 . arf6 is important for the disassembly of adherens junctions 1 and, more recently, active arf6 was shown to disrupt the formation of epithelial cysts 54 . the slit2 -robo4 signalling pathway is important for maintaining barrier function in the vascular network, and robo4 interacts with paxillin to recruit arf gap proteins, such as git1, to inactivate arf6 (ref. 55); this arf6 inactivation suppresses protrusive activity of the endothelial cells and neovascularization. git2 and arf6 inactivation are also important for maintaining the podosome, an actin-rich sealing zone in osteoclasts 56 . finally, non-canonical ubiquitylation of arf6, catalysed by fbx8 (an f -box and sec7 domaincontaining protein) seems to be another, unusual, way to turn off arf6 (ref. 57 ). fbx8 is diminished or lacking in several cance r cell lines, which is consistent with roles for arf6 in cance r cell metastasis 58 . similarly to arf1, arl1 and arl2 arose early in evolution and share common effectors in plants, yeast and mammals. arl1 recruits grip-domain golgins to the tgn 2 . it also mediates tgnlocalization of arf-interacting proteins (arfaptins), which contain bin-amphiphysin-rvs (bar) domains that induce the formation of tubules and vesicles at the tgn 59 . whereas arl1 functions in vesicle trafficking similarly to arfs, arl2 has a highly conserved function in regulating microtubule-based processes 2 . arl3 is closely related to arl2, but is found only in cells with cilia, where it regulates microtubule-based processes at the cilial basal body 2,60 (fig. 2b) . elmod2 has been reported to be a gap for arl2, but also has activity against arf1 and arf6, which is surprising given that it has no homology to arf gaps 61 ; the physiological relevance of this activity remains to be determined. retinitis pigmentosa 2 (rp2; also known as xrp2) acts as a gap for arl3 during intraflagellar transport and ciliogenesis. arl3, arl6 and arl13 affect intraflagellar transport and ciliogenesis (fig. 2b,c) . cilia are vital for cell signalling and differentiation, and their impaired formation is responsible for many genetic disorders 62 . bardet-biedl syndrome is a complex genetic disease that can be caused by mutation in any one of 14 genes associated with ciliogenesis. transport of membrane proteins into the cilium is driven by a complex of proteins, called the bbsome. bbsome subunits have 'coat-like' attributes and simila r structural folds to those found in copi and adaptor protein complexes, suggesting that the bbsome can sort specific cargo for transport (fig. 2c) . arl6 is a bbs subunit (bbs3) and is required in its gtp-bound form to recruit the bbsome onto the plasma membrane to drive cargo sorting into cilia 63 . structural and biochemical analyses have shown that one of the mutations in arl6 that causes bardet-biedl syndrome, t31r, leads to a non-functional arl6 that cannot bind gtp 64 . this supports the idea that arl6 recruits the bbsome complex to membranes for formation of bbsome-coated vesicles. arl13 is mutated in patients with joubert syndrome, which is a rare, complex cerebral disorder that is characterized by developmental delays and cognitive dis ability. it is also involved in intraflagellar transport (fig. 2c) and, in c. elegans, arl-13 associates with the doublet segment of the cilium and its loss results in shortened cilia 65, 66 . retinitis pigmentosa is a retinal degeneration disease, and mutations in the rp2 gene are responsible for a large fraction of the most severe x-linked form. rp2 was identified as a gap for arl3, and mutations associated with retinitis pigmentosa compromise arl3 gap activity 67 . arl3 localizes to the photoreceptor segment connecting to the cilium (fig. 2b) , and arl3 -/mice have abnormal kidney and photoreceptor development, indicating the importance of this protein in primary cilia 68 . rp2 localizes to the basal body and centriole at the base of the photoreceptor cilium, but also to the adjacent golgi and apical plasma membrane 69 . furthermore, rp2 promotes vesicle trafficking from the golgi to the base of the cilium in mammalian cells 69 , presumably acting together with arf4, asap1 and fip3. intriguingly, d. melanogaster arl3 (also called dead end) regulates actin polymerization and vesicular trafficking to the plasma membrane, which are important for tracheal morphogenesis 70 . hence, arl3 appears to link microtubule-based processe s and vesicular trafficking during development. arl8 might also coordinate microtubule and vesicular trafficking. arl8 localizes to late endosomes and lysosomes (fig. 3) in both humans and worms, and mediates transport of endocytic proteins between these two compartments 71 . arl8 also facilitates the axonal transport of presynaptic cargo proteins in vesicles, preventing their premature aggregation 9 . exactly how these two functions of arl8 are related is not clear but they might both involve transport along microtubules 2 . a great deal of progress has been made in identifying arf gefs, and an unexpectedly broad range of roles has been revealed for these regulators, including both the coordination of membrane trafficking with lipid homeostasis and signalling at the plasma membrane (table 2) . because gefs ensure the precise temporal and spatial activation of arfs, their own localization mechanisms are crucial for understanding their cellular roles. these mechanisms are turning out to be quite complex, even for the simplest of the arf gefs, the members of the cytohesin (also known as arno) family. membrane trafficking is crucial to numerous developmental and physiological processes, and the specific functions of different arf gefs in these pathways and their links to disease are now being revealed. there is particular interest in understanding how arf gefs are recruited to membranes to regulate arf activation. big1 and big2 localize to the tgn and endosomes, where they have both distinct and overlapping functions 72, 73 . by contrast, gbf1 localizes predominantly to the cis-golgi 74 (fig. 2a) , where it controls transport of membrane proteins through the secretory pathway 75 . the activity of phosphodiesterase 3a is important for recruitment of big1 and big2 to the trans-golgi 76 . however, rab1 (ref. 77) and ptdins4p generated by ptdins4kiiiα 78 are involved in recruitment of gbf1 to membranes. other close connections between golgi arfs and ptdins4p have emerged recently. in yeast there is an interesting synergy observed between the arf1 gef gea2 and ptdins4p produced by pik1 (the yeast homologue of ptdins4kiiiβ). both are simultaneously required to activate the aminophospholipid translocase (flippase) drs2 at the tgn during formation of ap1-clathrin vesicles 79 . ptdins4ks are essential for viral replication, and notably produce the ptdins4p-enriched membrane environment that recruits the enteroviral rna polymerases 80 . gbf1 is required for the replication of numerous viruses, including enteroviruses, hepatitis c virus and corona viruses [81] [82] [83] [84] . in enteroviral systems, gbf1 and ptdins4kiiiβ are recruited coordinately to membranes by the viral 3a protein to promote formation of functional viral replication complexes 80 near er exit sites (fig. 2a) . yel1 is an efa6-like gef for the arf6 orthologue arf3 in yeast, and localizes to the plasma membrane of the emerging bud 85 . similarly to its mammalian orthologues, the ph domain of yel1 is required for membrane targeting but, interestingly, multiple regions of the protein are important for precise spatial localization of this gef 85 . brag2, an arf6 gef, also has a ph domain that is critical for membrane targeting and in breast cancer cells is specifically recruited to the egf receptor upon egf stimulation, through direct interaction of its ph domain with the egf receptor 86 . this interaction requires phosphorylation on specific tyr residues and thus the recruitment of brag2 couples receptor activation to arf6 activation 86 . brag2 is overexpressed in many breast cancer cell lines and depletion of brag2 by small interfering rna blocks cell invasion in vitro and in animal tumour models 86 . these observations add to others that have implicated arf6 and its activation in a number of models of cancer cell invasion and metastasis 1, 58 . autoinhibition of cytohesin gefs. at the cell periphery, the cytohesin gefs function in plasma membraneendosomal membrane trafficking pathways, and in signal transduction pathways that are important for cell proliferation, immune response and growth control 87, 88 . members of this gef family can catalyse exchange on both arf1 and arf6 in vitro and in cells, although in vitro they are more efficient gefs for arf1 (ref. 87 ). recent insights have been gained into how cytohesin activation is spatially regulated, and how its autoinhibition is relieved (fig. 4) . in addition to phosphoinositide binding at the membrane, the ph domains of cytohesin family members interact with the gtp-bound forms of arf6 (ref. 89) and arl4 (refs 90, 91) , leading to cytohesin recruitment and further activation of arf6 or arf1 at the membrane. a crystal structure of the sec7 domain in tandem with the ph domain of cytohesin 3 (also known as grp1) revealed that it adopts an autoinhibited conformation. the c-terminal helix that phagocytosis a cellular endocytic process for engulfing large particles, such as bacteria, and bringing them inside the cell. follows the ph domain and the linker between the sec7 and ph domains block the catalytic site 92 . interaction of the ph domain with arf6•gtp and phosphoinositides (either ptdins(4,5)p 2 or ptdins-3,4,5-trisphosphate (ptdins(3,4,5)p 3 )), as well as the interaction of the polybasic c terminus of cytohesin with acidic phospholipids, all contribute to relieving this autoinhibition 92 (fig. 4) . reconstitution of the cytohesin-exchange assay on liposomes, in the presence of both activating arf6•gtp and substrate arf1, revealed that mutations in the ph domain of cytohesin that abolished interaction with arf6•gtp were completely inactive 93 . together, these studies demonstrate how precise spatial regulation of cytohesin activation is achieved. a specific phosphoinositide (ptdins(4,5)p 2 and/or ptdins(3,4,5)p 3 ), additional acidic phospholipids and an active arf localized in the plasma membrane must all coincide to relieve autoinhibition, thus restricting the membrane domain at which these gefs can become active. we do not know whether arf6, arf1 or both are the primary substrates for the cytohesins. however, arf6•gtp is more efficient in relieving autoinhibition of cytohesins than arf1•gtp, both in vitro and in cells 89, 92 . the activation of cytohesins by a gtp-bound arf family member raises the question of whether they can engage in a positive feedback loop, whereby the substrate itself can stimulate further exchange. indeed, such a loop has been demonstrated in vitro for arf1 (ref. 93 ). there is also evidence that cytohesins might mediate a cascade of activation from arf6 to arf1. cells expressing constitutively active arf6q67l have increased levels of arf1•gtp 89 . arf1 affects several processes at the plasma membrane, including recruitment of proteins to focal adhesions and during phagocytosis. in the forming phagocytic cup, arf6•gtp is recruited earlier than arf1•gtp, at a stage that requires rapid insertion of new membrane 94 . hence, the arf6-cytohesin-arf1 cascade might ensure a high level of activated arf protein here. arf6 is less abundant than arf1 in cells, and as both arf1 and arf6 can recruit effectors such as ptdins4p5k and pld, processes requiring acute activation of such effectors may rely on the more abundant arf1 to provide an adequate supply. in support of this idea, both arf1 and arf6, through cytohesins, contribute to activation of ptdins4p5k and pld in the insulin signalling pathway 95 . in addition to arf6-cytohesin-arf1 or possible arl4-cytohesin-arf6 cascades, there is a conserved arl cascade, in which yeast arl3•gtp recruits arl1 to tgn membranes 2 . in this case, it is not known whether an arl gef is involved. hence, arf family cascades could be common and could explain the golgi arfs that act in pairs. use of the specific cytohesin inhibitor secinh3 has revealed roles for this family of gefs in the insulin and erbb receptor tyr kinase signalling pathways [96] [97] [98] . cytohesins are positive activators of insulin signalling in both d. melanogaster and mammalian cells, and they are important for cell growth and for insulin sensitivity in human liver cells 97, 98 . they regulate insulin signalling by binding cnk1, a scaffolding molecule that is important for ras, phosphoinositide 3-kinase (pi3k) and akt signalling 95 . cnk1 recruits cytohesins in an insulin-dependent manner to the plasma membrane, where they generate a ptdins(4,5)p 2enriched micro domain that is essential for pi3k-akt activation. other scaffolding proteins interact with the coiled-coil domain of cytohesin; these proteins include golgi reassembly-stacking protein (grasp) and ipcef (interactor protein for cytohesin exchange factor s), which mediate the interaction of dock180 with cytohesin 99 . interestingly, assembly of this scaffolding complex promotes rac activation and cell migration, indicating that these scaffolds assemble a signalling complex that determines a specific downstream output upon arf activation 99 . cytohesins also affect integrin signalling in the immune system, and cytohesin 1 can activate β2 integrins in dendritic cells 100 , possibly through a scaffoldin g role of cytohesins. levels of arf6 and the efa6 and cytohesin family gefs markedly increase in the mammalian brain after birth, suggesting important roles in postnatal nervous system development 101 . experiments in isolated hippocampal neurons indicate that arf6, efa6 and the cytohesins might affect neurite and dendritic spine development 102, 103 . in humans, mutations in the arf1 gef big2 are linked to autosomal recessive periventricular heterotopia (arph), a disease in which the cerebral cortex is severely underdeveloped owing to failure of neurons in the lateral ventricular proliferative zone to migrate to the cortex 104 . this impaired migration arises from a figure 4 | the recruitment of an arf gef to the membrane is coupled to relief of autoinhibition. an active gtp-bound adp-ribosylation factor (arf) family member (either arf-like 4 (arl4) or arf6), phosphoinositides (phosphatidylinositol-4,5-bisphosphate (ptdins(4,5)p 2 ) or ptdins-3,4,5-trisphosphate (ptdins(3,4,5)p 3 )), and additional acidic phospholipids such as phosphatidylserine, are all required for membrane recruitment of the cytohesin (also known as arno) guanine nucleotide exchange factor (gef), to convert it from its cytosolic inactive form to its fully active membrane-bound form. before recruitment, the sec7 catalytic gef domain, the pleckstrin homology (ph) domain and the carboxy-terminal α-helix of cytohesin are in an autoinhibited conformation (left), with the c-terminal α-helix (charged residues within this are shown as '+') and linker situated between the catalytic sec7 domain and the ph domain, which blocks the arf-binding site. upon binding of the ph domain to the gtp-bound gef at the membrane, the catalytic site is released from autoinhibition (right). this can in turn drive further activation of arf proteins, such as arf1, at the membrane, and may form the basis of an arf protein activation cascade. long-term depression (ltd) . a reduction in the efficacy or strength of neuronal synapses that is linked to learning and memory formation. defect in vesicular trafficking that alters the adhesive properties of these neurons 105 . disease alleles include an early frameshift mutation that deletes most of the big2 protein 104 . members of the brag (or iqsec) family of arf gefs are extremely abundant in neuronal postsynapti c densities, and can serve as gefs for arf6 (ref. 87 ). brag1 (also known as iqsec2) and brag2 are vital for neuronal development. brag1 is mutated in x-linked nonsyndromic intellectual disability (also referred to as mental retardation). three point mutations isolated from patients map to the sec7 domain and result in proteins that cannot activate arf6 normally 106 . brag2 has been linked to alterations in synaptic content during long-term depression (ltd). signalling through ampa (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors facilitates ltd, and downregulation of activated ampa receptors is normally regulated by ampa receptor-mediated recruitment of brag2, which in turn activates arf6 and endocytosis 107 . thus, brag gefs and arf6 are vital for neuronal developmen t and learning. cytohesin gefs may affect signalling through epidermal growth factor (egf) or erbb receptor tyr kinase receptors independently of their gef activity. egf receptors (egfrs) undergo ligand-induced dimerization and subsequent transphosphorylation, mediated by conformational changes in their cytoplasmic portion. cytohesins bind directly to these cytoplasmic domains and promote conformational changes that increase phosphorylation 96 . furthermore, treatment of an egf receptor-dependent lung cancer cell line with the cytohesin inhibitor secinh3 reduced proliferation 96 . surprisingly, this function of the cytohesins does not require their gef activity. similarly, in c. elegans, the gef efa-6 regulates microtubule dynamics at the cell cortex independently of its substrate arf6 (ref. 108 ). furthermore, essential functions of gbf1 in poliovirus replication are independent of arf1 activation 109 . the extent to which other arf gefs may have broader roles beyond arf activation warrants further investigation. there are also suggestions that some multidomain arf gap proteins have functions that are independent of their gap activity. all arf gaps contain the conserved zinc-finger arf gap catalytic domain in addition to other domains responsible for membrane recruitment, regulation of gap activity and other scaffolding functions (table 2) . arfgap1, the first arf gap to be cloned 110 , is golgilocalized and, together with arfgap2 and arfgap3, mediates most arf-bound gtp hydrolysis at the golgi. the complex, multidomain structure of the other arf gap families has stimulated much research. here, we highlight a few examples of how these multidomain arf gaps, by recognizing the gtp-bound form of their substrate arf, act as downstream effectors in addition to signal terminators. information about other arf gaps can be found in an excellent review article 111 . the asap proteins are the prototypical multidomain gaps that interact with many signalling molecules, including src and focal adhesion kinase 111 (table 2) . asap1 resides in focal adhesions but, in response to src activation, it facilitates formation of podosomes 112 , which are discrete actin-based structures that are formed at the cell substratum to degrade matrix. the crystal structure of arf6 in complex with the catalytic domain of asap3 revealed that a catalytic arg-finger of asap3 is responsible for gtp hydrolysis 113 , similarly to many other gaps, a finding that is consistent with an earlier structure of the gap domain of asap2 (ref. 114 ). there is also some evidence that calcium might bind to the complex and regulate gap activity 113 , although this needs to be confirmed with full-length asap3 and arf6 and in cells. the asaps all have n-terminal bar domains that can induce membrane curvature and tubule formation in transfected cells and in cell-free systems. the bar domain in asap1 negatively regulates its gap activity towards arf1 (ref. 115) , and binding of the rab11 effector fip3 to the bar domain of asap1 stimulates its gap activity 116 . as mentioned earlier, asap1 also promotes ciliary targeting together with arf4 and fip3 (ref. 20) (fig. 2b) . asap1 is upregulated in breast, pancreatic and colorectal cancer 58 . cbl-interacting protein 85 (cin85; also known as sh3kbp1) binds to asap1, recruiting the e3 ubiquitin ligase cbl, to trigger the mono ubiquitylation of asap1; this modification is important for invasion of breast cancer cells 117 but the role for ubiquitylation of asap in cell invasion is not known. one caveat to observations made when asap is expressed in cells is that a study designed to systematically look at arf gap function and arf specificity failed to detect an effect of asap1 expression on either arf1•gtp or arf6•gtp levels in cells 118 . this raises the possibility that the gap activity of asap1 might not always be critical for some of asap1's specific functions. the arf gap git1, originally identified as a gpcr kinase-interacting protein, can coordinate signalling by acting as a scaffold. git1 and its substrate arf6 affect ligand-stimulated endocytosis of several gpcrs through either clathrin-dependent or clathrin-independent endocytic pathways 119 . among the proteins interacting with git1 are the cdc42 and rac gef pix, focal adhesion kinase and paxillin. git1, similarly to asap1, is sometimes observed in focal adhesions and its influence on the activation of cdc42 and rac suggests that arf inactivation and rac activation are coordinated (fig. 3) . d. melanogaster git is required for muscle morphogenesis 120 and the git1-knockout mouse is defective in fear learning 121 and dendritic spine formation 122 . rac3 interacts with git1, disrupting git1 binding to paxillin; this in turn stimulates git1 gap activity, presumably towards arf6 (ref. 123 ), and inhibits cell spreading and neuritogenesis. in endothelial cells, robo4 interacts with paxillin, which recruits git1 to inactivate arf6, and this leads to vascular stability, blocking cellular protrusions and neovascular leak 55 . thus, these examples provide insights into how modular arf gaps promote spatially and temporally restricted assembly of signalling complexes, and allow a precise physiological output in response to a signal. intracellular pathogens can use a fascinating gapblocking mechanism to rewire the host cell's signalling network for their own purposes. enterohaemorrhagic escherichia coli produce the espg protein, which binds to gtp-bound arf1 and arf6, blocking their access to gaps and disrupting the function of both early golgi and recycling endosomes 124 . moreover, espg simultaneously binds to p21-activated kinase (pak), an effector of a distinct g protein family member, cdc42, and promotes pak localization at golgi membranes rather than at the plasma membrane. this raises the possibility that espg assembles its own signalling complex on intra cellular membranes to subvert membrane trafficking and polarit y processes in host cells. conclusions & perspectives arf activity is regulated in a spatiotemporal manner by the gefs and gaps, underlining the importance of precise localization of these regulators. in the case of cytohesins, such specificity can be achieved through a coincidence-detection mechanism, requiring both an activating arf or arl protein and a specific lipid composition. this example also reveals the existence of arf family activation cascades and how relief of autoinhibition can be coupled to precise spatial cues. it will be interesting to see how widespread these mechanisms are among arf family members. arf cascades, similarly to those demonstrated for rab g proteins, could transform one membrane domain into another during highly dynamic membrane trafficking. these transformations involve coordinated changes in the lipid and protein composition of each membrane domain, a specialty of many arf family members, which recruit both lipidmodifying enzymes and protein effectors such as coats and tethers. the signature feature of arf family proteins, their n-terminal membrane-binding amphipathic helix, ensures that they are closely associated with the lipid bilayer in their gtp-bound form. future studies on how arf family proteins function will therefore require in vitro reconstitution on model membranes. there appears to be a particularly important link between arf1 function and ptdins4p, a lipid that has a central role in the function of the golgi, which parallels the coordination of membrane trafficking and ptdins(4,5) p 2 signalling by arf6 at the plasma membrane. the gaps and gefs for the arf family proteins are multidomain proteins that can assemble signalling complexes and so place the arfs and arls into larger networks. these networks include cytoskeleton regulators, and it appears that some arl proteins (arl2, for example) have evolved exclusively to regulate the cytoskeleton. the role of arf6 in networks linking membrane trafficking to the actin cytoskeleton also involves interaction of arf6 with gefs and gaps of the rac and rho small g proteins, actin cytoskeleton regulators. another emerging concept is that some arf family members remain membrane-bound in their gdp-bound form so that they can interact with signalling complexes and promote alternative signalling pathways. ultimately, these arf family signalling networks will need to be studied through systems level analysis. so far, no gefs and only two gaps that are specific for an arl have been identified. several arl proteins affect ciliogenesis and, in some cases, ciliopathies; other arls function in neurons and have been associated with neurodegenerative disorders. hence, increased understanding of arls and their regulators should inform both fundamental questions in cell biology and disease mechanisms. finally, the use of model organisms to complement studies in mammalian cells has already provided valuable insights into the physiological roles of arf family proteins. this approach holds great promise for uncovering the unknown functions of most arls, as well as defining the full range of activities of all arf and arl proteins. arf proteins: roles in membrane traffic and beyond the small g proteins of the arf family and their regulators n-terminal hydrophobic residues of the g-protein adp-ribosylation factor-1 insert into membrane phospholipids upon gdp to gtp exchange toward a structural understanding of arf family:effector specificity an n-terminally acetylated arf-like gtpase is localised to lysosomes and affects their motility sar1p n-terminal helix initiates membrane curvature and completes the fission of a copii vesicle differential membrane association properties and regulation of class i and class ii arfs characterization of class i and ii adpribosylation factors (arfs) in live cells: gdp-bound class ii arfs associate with the er-golgi intermediate compartment independently of gbf1 an arf-like small g protein, arl-8, promotes the axonal transport of presynaptic cargoes by suppressing vesicle aggregation syt1p promotes activation of arl1p at the late golgi to recruit imh1p the copi system: molecular mechanisms and function coat proteins: shaping membrane transport phospholipid-and gtp-dependent activation of cholera toxin and phospholipase d by human adp-ribosylation factor-like protein 1 (harl1) protein-lipid interactions in membrane trafficking at the golgi complex isoform-selective effects of the depletion of adpribosylation factors 1-5 on membrane traffic adp ribosylation factors 1 and 4 and group via phospholipase a regulate morphology and intraorganellar traffic in the endoplasmic reticulum-golgi intermediate compartment targeting of arf-1 to the early golgi by membrin, an er-golgi snare arf3 is activated uniquely at the trans-golgi network by brefeldin a-inhibited guanine nucleotide exchange factors rhodopsin c terminus, the site of mutations causing retinal disease, regulates trafficking by binding to adp-ribosylation factor 4 (arf4) ciliary targeting motif vxpx directs assembly of a trafficking module through arf4 shows that the ciliary targeting motif in rhodopsin, vxpx, binds to arf4 and regulates its association with the tgn, where a ciliary targeting complex selects and packages cargo for delivery to the cilium interaction of calcium-dependent activator protein for secretion 1 (caps1) with the class ii adp-ribosylation factor small gtpases is required for dense-core vesicle trafficking in the trans-golgi network glycosphingolipid synthesis requires fapp2 transfer of glucosylceramide functional genomic screen reveals genes involved in lipid-droplet formation and utilization dynamic activity of lipid droplets: protein phosphorylation and gtp-mediated protein translocation demonstrates a novel pathway -requiring arf1, gbf1 and copi, as well as copii -by which the lipid droplet-associated proteins atgl and adrp are delivered copi complex is a regulator of lipid homeostasis the arf-like gtpase arfrp1 is essential for lipid droplet growth and is involved in the regulation of lipolysis arf1 is directly involved in dynamin-independent endocytosis analysis of endocytic pathways in drosophila cells reveals a conserved role for gbf1 in internalization via geecs crucial role of the small gtpase arf6 in hepatic cord formation during liver development the small g-protein arf6gtp recruits the ap-2 adaptor complex to membranes arf6 regulates angiotensin ii type 1 receptor endocytosis by controlling the recruitment of ap-2 and clathrin decoupling of activation and effector binding underlies arf6 priming of fast endocytic recycling pathways and mechanisms of endocytic recycling arf6 and microtubules in adhesion-dependent trafficking of lipid rafts cdc42 localization and cell polarity depend on membrane traffic yeast arf3p modulates plasma membrane ptdins(4,5)p 2 levels to facilitate endocytosis role for arf3p in development of polarity, but not endocytosis, in saccharomyces cerevisiae adp-ribosylation factor arf6p may function as a molecular switch of new end take off in fission yeast aspergillus nidulans arfb plays a role in endocytosis and polarized growth spermatocyte cytokinesis requires rapid membrane addition mediated by arf6 on central spindle recycling endosomes arf6 interacts with jip4 to control a motor switch mechanism regulating endosome traffic in cytokinesis the crystal structure of arf6 in complex with its effector jip4, which acts as a microtubule motor adaptor frmd4a regulates epithelial polarity by connecting arf6 activation with the par complex gep100/brag2: activator of adpribosylation factor 6 for regulation of cell adhesion and actin cytoskeleton via e-cadherin and α-catenin efa6, exchange factor for arf6, regulates the actin cytoskeleton and associated tight junction in response to e-cadherin engagement arf6-dependent activation of erk and rac1 modulates epithelial tubule development analysis of gtpase-activating proteins: rab1 and rab43 are key rabs required to maintain a functional golgi complex in human cells tbc1d24, an arf6-interacting protein, is mutated in familial infantile myoclonic epilepsy the tbc (tre-2/bub2/cdc16) domain protein tre17 regulates plasma membraneendosomal trafficking through activation of arf6 arf6 recruits the rac gef kalirin to the plasma membrane facilitating rac activation discovery of new cargo proteins that enter cells through clathrin-independent endocytosis arf6 plays an early role in platelet activation by collagen and convulxin unregulated arf6 activation in epithelial cysts generates hyperactive signaling endosomes and disrupts morphogenesis slit2-robo4 signalling promotes vascular stability by blocking arf6 activity src-dependent repression of arf6 is required to maintain podosome-rich sealing zones in bone-digesting osteoclasts fbx8 makes arf6 refractory to function via ubiquitination the egfr-gep100-arf6-amap1 signaling pathway specific to breast cancer invasion and metastasis arfaptins are localized to the trans-golgi by interaction with arl1, but not arfs arl2 and arl3 regulate different microtubule-dependent processes elmod2 is an arl2 gtpase-activating protein that also acts on arfs trafficking to the ciliary membrane: how to get across the periciliary diffusion barrier? shows that arl6, the bbs3 subunit, recruits the coat-like bbsome onto the plasma membrane to sort cargo for transport into cilia bardet-biedl syndrome-associated small gtpase arl6 (bbs3) functions at or near the ciliary gate and modulates wnt signaling the small gtpases arl-13 and arl-3 coordinate intraflagellar transport and ciliogenesis joubert syndrome arl13b functions at ciliary membranes and stabilizes protein transport in caenorhabditis elegans the retinitis pigmentosa 2 gene product is a gtpaseactivating protein for arf-like 3 adp-ribosylation factor-like 3 is involved in kidney and photoreceptor development the retinitis pigmentosa protein rp2 links pericentriolar vesicle transport between the golgi and the primary cilium the drosophila dead end arf-like3 gtpase controls vesicle trafficking during tracheal fusion cell morphogenesis the arf-like gtpase arl8 mediates delivery of endocytosed macromolecules to lysosomes in caenorhabditis elegans redundant roles of big2 and big1, guanine-nucleotide exchange factors for adpribosylation factors in membrane traffic between the trans-golgi network and endosomes specific functions of big1 and big2 in endomembrane organization localization of large adp-ribosylation factor-guanine nucleotide exchange factors to different golgi compartments: evidence for distinct functions in protein traffic dissecting the role of the arf guanine nucleotide exchange factor gbf1 in golgi biogenesis and protein trafficking interaction of phosphodiesterase 3a with brefeldin a-inhibited guanine nucleotideexchange proteins big1 and big2 and effect on arf1 activity rab1b interacts with gbf1 and modulates both arf1 dynamics and copi association the phosphatidylinositol 4-kinase pi4kiiiα is required for the recruitment of gbf1 to golgi membranes regulation of a golgi flippase by phosphoinositides and an arfgef shows that specific enteroviral proteins promote recruitment of ptdins4kiiiβ to viral replication membranes to produce ptdins4p, which the enteroviral rna polymerase binds to directly and requires for its membrane recruitment a critical role of a cellular membrane traffic protein in poliovirus rna replication mouse hepatitis coronavirus rna replication depends on gbf1-mediated arf1 activation gbf1, a guanine nucleotide exchange factor for arf, is crucial for coxsackievirus b3 rna replication identification of gbf1 as a cellular factor required for hepatitis c virus rna replication identification of a guanine nucleotide exchange factor for arf3, the yeast orthologue of mammalian arf6 shows that brag2 is recruited by its ph domain to the activated egfr, coupling egf stimulation to arf6 activation. depletion of brag2, which is overexpressed in many breast cancers, inhibits cell invasion and metastasis in animal models regulation of arf activation: the sec7 family of guanine nucleotide exchange factors guanine nucleotide exchange factors of the cytohesin family and their roles in signal transduction active arf6 recruits arno/cytohesin gefs to the pm by binding their ph domains the arl4 family of small g proteins can recruit the cytohesin arf6 exchange factors to the plasma membrane arl4d recruits cytohesin-2/arno to modulate actin remodeling structural basis and mechanism of autoregulation in 3-phosphoinositide-dependent grp1 family arf gtpase exchange factors presents the crystal structure of the cytohesin catalytic sec7 domain and the ph domain in tandem, revealing an autoinhibited conformation that can be relieved by binding of arf6 and phosphoinositides to the ph domain kinetic studies of the arf activator arno on model membranes in the presence of arf effectors suggest control by a positive feedback loop a phosphatidylinositol-3-kinase-dependent signal transition regulates arf1 and arf6 during fcγ receptor-mediated phagocytosis the cnk1 scaffold binds cytohesins and promotes insulin pathway signaling shows that cytohesin arf gefs facilitate conformational changes in the cytoplasmic portion of egf or erbb receptor tyr kinase receptors that promote ligand-induced signalling, and that inhibition of these gefs reduces proliferation of lung cancer cells the cytohesin steppke is essential for insulin signalling in drosophila inhibition of cytohesins by secinh3 leads to hepatic insulin resistance grasp and ipcef promote arf-to-rac signaling and cell migration by coordinating the association of arno/cytohesin 2 with dock180 cytohesin-1 controls the activation of rhoa and modulates integrin-dependent adhesion and migration of dendritic cells distinct spatiotemporal expression of efa6d, a guanine nucleotide exchange factor for arf6, among the efa6 family in mouse brain regulation of dendritic development by the arf exchange factor arno arf6 and efa6a regulate the development and maintenance of dendritic spines mutations in arfgef2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex disruption of neural progenitors along the ventricular and subventricular zones in periventricular heterotopia mutations in the guanine nucleotide exchange factor gene iqsec2 cause nonsyndromic intellectual disability ampa receptor signaling through brag2 and arf6 critical for long-term synaptic depression ltd, mediated by down-regulation of neuronal receptors by clathrin-mediated endocytosis, is shown in this study to require activation of arf6 by brag2 caenorhabditis elegans efa-6 limits microtubule growth at the cell cortex poliovirus replication requires the n-terminus but not the catalytic sec7 domain of arfgef gbf1 the arf1 gtpase-activating protein: zinc finger motif and golgi complex localization arf gaps and their interacting proteins src-dependent phosphorylation of asap1 regulates podosomes the structure of an arf-arfgap complex reveals a ca 2+ regulatory mechanism crystal structure of the arf-gap domain and ankyrin repeats of pyk2-associated protein β autoinhibition of arf gtpase-activating protein activity by the bar domain in asap1 arf gtpase-activating protein asap1 interacts with rab11 effector fip3 and regulates pericentrosomal localization of transferrin receptor-positive recycling endosome cin85, a cbl-interacting protein, is a component of amap1-mediated breast cancer invasion machinery substrate specificities and activities of azap family arf gaps in vivo regulation of receptor trafficking by grks and arrestins the drosophila homologue of arf-gap git1, dgit, is required for proper muscle morphogenesis and guidance during embryogenesis impaired fear response in mice lacking git1 impaired spine formation and learning in gpcr kinase 2 interacting protein-1 (git1) knockout mice rac3 inhibits adhesion and differentiation of neuronal cells by modifying git1 downstream signaling the assembly of a gtpase-kinase signalling complex by a bacterial catalytic scaffold analysis of the small gtpase gene superfamily of arabidopsis correct targeting of plant arf gtpases relies on distinct protein domains role of the gnom gene in arabidopsis apical-basal patterning -from mutant phenotype to cellular mechanism of protein action adp-ribosylation factor machinery mediates endocytosis in plant cells an arf-gef acting at the golgi and in selective endocytosis in polarized plant cells fluorescence imaging-based screen identifies arf gef component of early endosomal trafficking a bacterial virulence protein suppresses host innate immunity to cause plant disease van3 arf-gap-mediated vesicle transport is involved in leaf vascular network formation scarface encodes an arf-gap that is required for normal auxin efflux and vein patterning in arabidopsis agd5 is a gtpase-activating protein at the trans-golgi network in a screen for mutations affecting floral organ shedding in a. thaliana, this study identified mutations in an arf gap protein named nevershed, which localizes to the tgn and endosomes and is required for trafficking of cargo molecules involved in cell separation trypanosoma brucei arf1 plays a central role in endocytosis and golgi-lysosome trafficking the small gtpase arl2 is required for cytokinesis in trypanosoma brucei functional analysis of tbarl1, an n-myristoylated golgi protein essential for viability in bloodstream trypanosomes n-myristoyltransferase inhibitors as new leads to treat sleeping sickness consensus nomenclature for the human arfgap domain-containing proteins we apologize to authors whose work we could not cite owing to space limitations. we thank c. eyster, l. maldonado-baez, j. ménétrey and c. le clainche for critical reading of the manuscript. work in our laboratories is supported by the division of intramural research in the national heart, lung, and blood institute, us national institutes of health (j.g.d.) and grants from the agence nationale de la recherche and the centre national de la recherche scientifique, france (c.l.j.). the authors declare no competing financial interests. the authors would like to note that catherine l. jackson's address was incomplete as it appeared in the original version of this article. this has been corrected in the online version. key: cord-285647-9tegcrc3 authors: estrada, ernesto title: fractional diffusion on the human proteome as an alternative to the multi-organ damage of sars-cov-2 date: 2020-08-17 journal: chaos doi: 10.1063/5.0015626 sha: doc_id: 285647 cord_uid: 9tegcrc3 the coronavirus 2019 (covid-19) respiratory disease is caused by the novel coronavirus sars-cov-2 (severe acute respiratory syndrome coronavirus 2), which uses the enzyme ace2 to enter human cells. this disease is characterized by important damage at a multi-organ level, partially due to the abundant expression of ace2 in practically all human tissues. however, not every organ in which ace2 is abundant is affected by sars-cov-2, which suggests the existence of other multi-organ routes for transmitting the perturbations produced by the virus. we consider here diffusive processes through the protein–protein interaction (ppi) network of proteins targeted by sars-cov-2 as an alternative route. we found a subdiffusive regime that allows the propagation of virus perturbations through the ppi network at a significant rate. by following the main subdiffusive routes across the ppi network, we identify proteins mainly expressed in the heart, cerebral cortex, thymus, testis, lymph node, kidney, among others of the organs reported to be affected by covid-19. scitation.org/journal/cha hypothesized as a potential cause of the major complications of the covid-19. 11, 12 however, it has been found that ace2 has abundant expression on endothelia and smooth muscle cells of virtually all organs. 13 therefore, it should be expected that after sars-cov-2 is present in circulation, it can be spread across all organs. in contrast, both sars-cov and sars-cov-2 are found specifically in some organs but not in others, as shown by in situ hybridization studies for sars-cov. this was already remarked by hamming et al. 13 by stressing that it "is remarkable that so few organs become viruspositive, despite the presence of ace2 on the endothelia of all organs and sars-cov in blood plasma of infected individuals." recently, gordon et al. 14 identified human proteins that interact physically with those of the sars-cov-2 forming a high confidence sars-cov-2-human protein-protein interaction (ppi) system. using this information, gysi et al. 15 discovered that 208 of the human proteins targeted by sars-cov-2 forms a connected component inside the human ppi network. that is, these 208 are not randomly distributed across the human proteome, but they are closely interconnected by short routes that allow moving from one to another in just a few steps. these interdependencies of protein-protein interactions are known to enable that perturbations on one interaction propagate across the network and affect other interactions. [16] [17] [18] [19] in fact, it has been signified that diseases are a consequence of such perturbation propagation. [20] [21] [22] it has been stressed that the protein-protein interaction process requires diffusion in their initial stages. 23 the diffusive processes occur when proteins, possibly guided by electrostatic interactions, need to encounter each other many times before forming an intermediate. 24 not surprisingly, diffusive processes have guided several biologically oriented searches in ppi networks. 25, 26 therefore, we assume here that perturbations produced by sars-cov-2 proteins on the human ppi network are propagated by means of diffusive processes. however, due to the crowded nature of the intra-cell space and the presence in it of spatial barriers, subdiffusive processes more than normal diffusion are expected for these protein-protein encounters. [27] [28] [29] this creates another difficulty, as remarked by batada et al., 23 which is that such (sub)diffusive processes along are not sufficient for carrying out cellular processes at a significant rate in cells. here, we propose the use of a time-fractional diffusion model on the ppi network of proteins targeted by sars-cov-2. the goal is to model the propagation of the perturbations produced by the interactions of human proteins with those of sars-cov-2 through the whole ppi. the subdiffusive process emerging from the application of this model to the sars-cov-2-human ppis has a very small rate of convergence to the steady state. however, this process produces a dramatic increment of the probability that certain proteins are perturbed at very short times. this kind of shock wave effect of the transmission of perturbations occurs at much earlier times in the subdiffusive regime than at the normal diffusion one. therefore, we propose here a switch and restart process in which a subdiffusive process starts at a given protein of the ppi, perturbs a few others, which then become the starting point of a new subdiffusive process and so on. using this approach, we then analyze how the initial interaction of the sars-cov-2 spike protein with a human protein propagates across the whole network. we discover some potential routes of propagation of these perturbations from proteins mainly expressed in the lungs to proteins mainly expressed in other different tissues, such as the heart, cerebral cortex, thymus, lymph node, testis, prostate, liver, small intestine, duodenum, kidney, among others. a. settling a model the problem we intend to model here is of a large complexity as it deals with the propagation of perturbations across a network of interacting proteins, each of which is located in a crowded intracellular space. therefore, we necessarily have to impose restrictions and make assumptions to settle our modeling framework. as we have mentioned in sec. i, protein encounters should necessarily occur in subdiffusive ways due to the crowded environment in which they are embedded, as well as the existence of immobile obstacles such as membranes. by a subdiffusive process, we understand that the mean square displacement of a protein scales as where 0 < κ < 1 is the anomalous diffusion exponent. as observed by sposini et al., 30 these anomalous diffusive processes can emerge from (i) continuous time random walk (ctrw) processes or by (ii) viscoelastic diffusion processes. in the first case, the "anomaly" is created by power-law waiting times in between motion events. this kind of processes is mainly accounted for by the generalized langevin equation with the power-law friction kernel as well as by fractional brownian motion (fbm). while the first processes are characterized by the stretched gaussian displacement probability density, weak ergodicity, and aging, the second ones are ergodic processes characterized by the gaussian density probability distribution. therefore, our first task is to discern which of these two kinds of approaches is appropriate for the current scenario. we start by mentioning that weiss et al. 31 have analyzed data from fluorescence correlation spectroscopy (fcs) for studying subdiffusive biological processes. they have, for instance, reported that membrane proteins move subdiffusively in the endoplasmic reticulum and golgi apparatus in vivo. subdiffusion of cytoplasmatic macromolecules was also reported by weiss et al. 32 using fcs. then, guigas and weiss 27 simulated the way in which the subdiffusive motion of these particles should occur in a crowded intracellular fluid. they did so by assigning diffusive steps from a weirstrass-mandelbrot function yielding a fbm. they stated that ctrw was excluded due to its markovian nature. in another work, szymanski and weiss 33 used fcs and simulations to analyze the subdiffusive motion of a protein in a simulated crowded medium. first, they reported that crowded-induced subdiffusion is consistent with the predictions from fbm or obstructed (percolation-like) diffusion. second, they reported that ctrw does not explain the experimental results obtained by fcs and should not be appropriated for such processes. the time resolution of fcs is in the microsecond range, i.e., 10 −6 s. 34 however, an important question on biological subdiffusion may require higher time resolution to be solved. this is the question of how diffusive processes on short times, while the macromolecule has not felt yet the crowding of the environment, is related to the long-time diffusion. this particular problem was explored experimentally by gupta et al. 35 by using state-of-the-art neutron chaos article scitation.org/journal/cha spin-echo (nse) and small-angle neutron scattering (sans), which has a resolution in the nanosecond range, i.e., 10 −9 s. their experimental setting was defined by the use of two globular proteins in a crowded environment formed by poly(ethylene oxide) (peo), which mimics a macromolecular environment. in their experiments, nse was used to tackle the fast diffusion process, which corresponds to a dynamics inside a trap built by the environment mesh. sans captures the slow dynamics, which corresponds to the long-time diffusion at macroscopic length scales. from our current perspective, the most important result of this work is that the authors found that in a higher concentration of polymeric solutions, like in the intracellular space, the diffusion is fractional in nature. they showed this by using the fractional fokker-planck equation with a periodic potential. according to gupta et al., 35 this fractional nature of the crossover from fast dynamics to slow macroscopic dynamics is due to the heterogeneity of the polymer mesh in the bulk sample, which may well resemble the intra-cellular environment. as proved by barkai et al. , 36 the fractional fokker-planck equation can be derived from the ctrw, which clearly indicates that the results obtained by gupta et al. point out to the classification of the subdiffusive dynamics into the class (i). we should remark that independently of these results by gupta et al., 35 shorten and sneyd 37 have successfully used the fractional diffusion equation to mimic the protein diffusion in an obstructed media like within skeletal muscle. we notice in passing that the (fractional) diffusion equation can be obtained from the (fractional) fokker-planck equation in the absence of an external force. in closing, because here we are interested in modeling the diffusion of proteins in several human cells, which are highly crowded, and in which we should recover the same crossover between initial fast and later slow dynamics, we will consider a modeling tool of the class (i). in particular, we will focus our modeling on the use of a time-fractional diffusion equation using caputo derivatives. another justification for the use of this model here is that interacting proteins can be in different kinds of cells. thus, we consider that the perturbation of one protein is not necessarily followed by the perturbation of one of its interactors, but a time may mediate between the two processes. this is exactly the kind of processes that the time-fractional diffusion captures. in this work, we always consider g = (v, e) to be an undirected finite network with vertices v representing proteins and edges e representing the interaction between pairs of proteins. let us consider 0 < α ≤ 1 and a function u : [0, ∞) → r, then we denote by d α t u the fractional caputo derivative of u of the order α, which is given by 38 where * denotes the classical convolution product on (0, ∞) and g γ (t) t γ −1 (γ ) , for γ > 0,where (·) is the euler gamma function. observe that the previous fractional derivative has sense whenever the function is derivable and the convolution is defined (for example, if u is locally integrable). the notation g γ is very useful in the fractional calculus theory, mainly by the property g γ * g δ = g γ +δ for all γ , δ > 0. here, we propose to consider the time-fractional diffusion (tfd) equation on the network as with the initial condition x (0) = x 0 , where x i (t) is the probability that the protein i is perturbed at the time t; c is the diffusion coefficient of the network, which we will set hereafter to unity; and l is the graph laplacian, i.e., l = k − a, where k is a diagonal matrix of node degrees and a is the adjacency matrix. this model was previously studied in distributed coordination algorithms for the consensus of multi-agent systems. [39] [40] [41] the use of fractional calculus in the context of physical anomalous diffusion has been reviewed by metzler and klafter. 42 a different approach has been developed by riascos and mateos. 43, 44 it is based on the use of fractional powers of the graph laplacian (see ref. 45 and references therein). the approach has been recently formalized by benzi et al. 46 this method cannot be used in the current framework because it generates only superdiffusive behaviors (see benzi et al. 46 ) and not subdiffusive regimes. another disadvantage of this approach is that it can only be used to positive (semi)definite graph operators, such as the laplacian, but not to adjacency operators such as the one used in tight-binding quantum mechanical or epidemiological approaches (see sec. vi). theorem 1. the solution of the fractional-time diffusion model on the network is where e α,β (γ l) is the mittag-leffler function of the laplacian matrix of a graph. proof. we use the spectral decomposition of the network laplacian l = uλu −1 , where u = ψ 1 · · · ψ n and λ = diag (µ r ). then, we can write let us define y (t) = u −1 x (t) such that d α t x (t) = −uλy (t), and we have as is a diagonal matrix, we can write which has the solution we can replace which finally gives the result in the matrix-vector when written for all the nodes, we can write l = uλu −1 , where u = ψ 1 · · · ψ n and λ = diag (µ r ). then, which can be expanded as where ψ j and φ j are the jth column of u and of u −1 , respectively. because µ 1 = 0 and 0 < µ 2 ≤ · · · ≤ µ n for a connected graph, we have lim where ψ t 1 φ 1 = 1. let us take ψ 1 = 1, such that we have this result indicates that in an undirected and connected network, the diffusive process controlled by the tfd equation always reaches a steady state, which consists of the average of the values of the initial condition. in the case of directed networks (ppi are not directed by nature) or in disconnected networks (a situation that can be found in ppis), the steady state is reached in each (strongly) connected component of the graph. also, because the network is connected, µ 2 makes the largest contribution to e α,1 (−t α l) among all the nontrivial eigenvalues of l. therefore, it dictates the rate of convergence of the diffusion process. we remark that in practice, the steady state lim t→∞ x v (t) − x w (t) = 0, ∀v, w ∈ v is very difficult to achieve. therefore, we use a threshold ε, e.g., ε = 10 −3 , such that lim t→∞ x v (t) − x w (t) = ε is achieved in a relatively small simulation time. due to its importance in this work, we remark the structural meaning of the mittag-leffler function of the laplacian matrix appearing in the solution of the tfd equation. that is, e α,1 (−t α l) is a matrix function, which is defined as where (·) is the euler gamma function as before. we remark that for α = 1, we recover the diffusion equation on the network: dx (t) /dt = −lx (t) and its solution e 1,1 (−t α l) = exp (−tcl) is the well-known heat kernel of the graph. we define here a generalization of the diffusion distance studied by coifman and lafon. 47 then, we define the following quantity: we have the following result. proof. the matrix function f (τ l) can be written as f (τ l) = uf (τ λ) u −1 . let ϕ u = ψ 1,u , ψ 2,u , . . . , ψ n,u t . then, therefore, because f (τ l) is positively defined, we can write where consequently, d vw is a square euclidean distance between v and w. in this sense, the vector we have that d vw generalizes the diffusion distance studied by coifman and lafon, which is the particular case when α = 1. let µ j be the jth eigenvalue and ψ ju the uth entry of the jth eigenvector of the laplacian matrix. then, we can write the time-fractional diffusion distance as it is evident that when α = 1, d vw is exactly the diffusion distance previously studied by coifman and lafon. 47 the fractionaltime diffusion distance between every pair of nodes in a network can be represented in a matrix form as follows: where s = f(τ l) 11 , f(τ l) 22 , . . . , f(τ l) nn is a vector whose entries are the main diagonal terms of the mittag-leffler matrix function, 1 is an all-ones vector, and • indicates an entrywise operation. using this matrix, we can build the diffusion distance-weighted adjacency matrix of the network, the shortest diffusion path between two nodes is then the shortest weighted path in w (τ ). lemma 3. the shortest (topological) path distance between two nodes in a graph is a particular case of the time-fractional shortest diffusion path length for τ → 0. proof. let us consider each of the terms forming the definition of the time-fractional diffusion distance and apply the limit of the very small −t α . that is, chaos article scitation.org/journal/cha and in a similar way, therefore, lim , which immediately implies that the time-fractional shortest diffusion path is identical to the shortest (topological) one in the limit of very small τ = −t α . the proteins of sars-cov-2 and their interactions with human proteins were determined experimentally by gordon et al. 14 gysi et al. 15 constructed an interaction network of all 239 human proteins targeted by sars-cov-2. in this network, the nodes represent human proteins targeted by sars-cov-2 and two nodes are connected if the corresponding proteins have been determined to interact with each other. obviously, this network of proteins targeted by sars-cov-2 is a subgraph of the protein-protein interaction (ppi) network of humans. one of the surprising findings of gysi et al. 15 is the fact that this subgraph is not formed by proteins randomly distributed across the human ppi, but they form a main cluster of 208 proteins and a few small isolated components. hereafter, we will always consider this connected component of human proteins targeted by sars-cov-2. this network is formed by 193 proteins, which are significantly expressed in the lungs. gysi et al. 15 reported a protein as being significantly expressed in the lungs if its gtex median value is larger than 5. gtex 48 is a database containing the median gene expression from rna-seq in different tissues. the other 15 proteins are mainly expressed in other tissues. however, in reporting here, the tissues that were proteins are mainly expressed; we use the information reported in the human protein atlas 49 where we use information not only from gtex but also from hpa (see details at the human protein atlas webpage) and fantom5 50 datasets. the ppi network of human proteins targeted by sars-cov-2 is very sparse, having 360 edges, i.e., its edge density is 0.0167, 30% of nodes have a degree (number of connections per protein) equal to one, and the maximum degree of a protein is 14. the second smallest eigenvalue of the laplacian matrix of this network is very small; i.e., µ 2 = 0.0647. therefore, the rate of convergence to the steady state of the diffusion processes taking place on this ppi is very slow. we start by analyzing the effects of the fractional coefficient α on these diffusive dynamics. we use the normal diffusion α = 1 as the reference system. to analyze the effects of changing α over the diffusive dynamics on the ppi network, we consider the solution of the tfd equation for processes starting at a protein with a large degree, i.e., prkaca, degree 14, and a protein with a low degree, i.e., mrps5, degree 3. that is, the initial condition vector consists of a vector having one at the entry corresponding to either prkaca or mrps5 and zeroes elsewhere. in fig. 1 , we display the changes of the probability with the shortest path distance from the protein where the process starts. this distance corresponds to the number of steps that the perturbation needs to traverse to visit other proteins. for α = 1.0, the shapes of the curves in fig. 1 are the characteristic ones for the gaussian decay of the probability with distance. however, for α < 1, we observe that such decay differs from that typical shape showing a faster initial decay followed by a slower one. in order to observe this effect in a better way, we zoomed the region of distances from 2 to 4 [see figs. 1(b) and 1(d)]. as can be seen for distances below 3, the curve for α = 1.0 is on top of those for α < 1, indicating a slower decay of the probability. after this distance, there is an inversion, and the normal diffusion occurs at a much faster rate than the other two for the longer distances. this is a characteristic signature of subdiffusive processes, which starts at much faster rates than a normal diffusive process and then continue at much slower rates. therefore, here, we observe that the subdiffusive dynamics are much faster at earlier times of the process, which is when the perturbation occurs to close nearest neighbors to the initial point of perturbation. to further investigate these characteristic effects of the subdiffusive dynamics, we study the time evolution of a perturbation occurring at a given protein and its propagation across the whole ppi network. in fig. 2 , we illustrate these results for α = 1.0 (a), α = 0.75 (b), and α = 0.5 (c). as can be seen in the main plots of this figure, the rate of convergence of the processes to the steady state is much faster in the normal diffusion (a) than in the subdiffusive one (b) and (c). however, at very earlier times (see insets in fig. 2 ), there is a shock wave increase of the perturbation at a set of nodes. such kind of shock waves has been previously analyzed in other contexts as a way of propagating effects across ppi networks. 17 we have explored briefly about the possible causes of this increase in the concentration for a given subset of proteins. accordingly, it seems that the main reason for this is the connectivity provided by the network of interactions and not a given distribution of the degrees. for instance, we have observed such "shock waves" in networks with normal-like distributions as well as with power-law ones. however, it is possible that the extension and intensity of such effects depend on the degree distribution as well as on other topological factors. the remarkable finding here is, however, the fact that such a shock wave occurs at much earlier times in the subdiffusive regimes than at the normal diffusion. that is, while for α = 1.0, these perturbations occur at t≈0.1-0.3; for α = 0.75, they occur at t≈0.0-0.2; and for α = 0.5, they occur at t≈0.0-0.1. seeing this phenomenon in the light of what we have observed in the previous paragraph is not strange due to the observation that such processes go at a much faster rate at earlier times, and at short distances, than the normal diffusion. in fact, this is a consequence of the existence of a positive scalar t for which e α,1 (−γ t α ) decreases faster than exp (−γ t) for t ∈ (0, t) for γ ∈ r + and α ∈ r + (see theorem 4.1 in ref. 39 ). hereafter, we will consider the value of α = 0.75 for our experiments due to the fact that it reveals a subdiffusive regime, but the shock waves observed before are not occurring in an almost instantaneous way like when α = 0.5 , which would be difficult from a biological perspective. the previous results put us at a crossroads. first, the subdiffusive processes that are expected due to the crowded nature of the intra-cellular space are very slow for carrying out cellular processes at a significant rate in cells. however, the perturbation shocks occurring at earlier times of these processes are significantly faster than in normal diffusion. to sort out these difficulties, we propose a switching back and restart subdiffusive process occurring in the ppi network. that is, a subdiffusive process starts at a given protein, which is directly perturbed by a protein of sars-cov-2. it produces a shock wave increase of the perturbation in close neighbors of that proteins. then, a second subdiffusive process starts at these newly perturbed proteins, which will perturb their nearest neighbors. the process is repeated until the whole ppi network is perturbed. this kind of "switch and restart processes" has been proposed for engineering consensus protocols in multiagent systems 51 as a way to accelerate the algorithms using subdiffusive regimes. the so-called spike protein (s-protein) of the sars-cov-2 interacts with only two proteins in the human hosts, namely, zdhhc5 and golga7. the first protein, zdhhc5, is not in the main connected component of the ppi network of sars-cov-2 targets. therefore, we will consider here how a perturbation produced by the interaction of the virus s-protein with golga7 is propagated through the whole ppi network of sars-cov-2 targets. golga7 has degree one in this network, and its diffusion is mainly to close neighbors, namely, to proteins separated by two to three edges. when starting the diffusion process at the protein golga7, the main increase in the probability of perturbing another protein is reached for the protein golga3, which increases its probability up to 0.15 at t = 0.2, followed by prkar2a, with a small increase in its probability, 0.0081. then, the process switch and restarts at golga3, which mainly triggers the probability of the protein prkar2a-a major hub of the network. once we start the process at prkar2a, practically, the whole network is perturbed with probabilities larger than 0.1 for 19 proteins apart from golga3. these proteins are in decreasing order of their probability of being perturbed: akap8, prkar2b, cep350, mib1, cdk5rap2, cep135, akap9, cep250, pcnt, cep43, pde4dip, prkaca, tub6cp3, tub6cp2, cep68, clip4, cntrl, plekha5, and ninl. notice that the number of proteins perturbed is significantly larger than the degree of the activator, indicating that not only nearest neighbors are activated. an important criterion for revealing the important role of the protein prkar2a as a main propagator in the network of proteins targeted by sars-cov-2 is its average diffusion path length. this is the average number of steps that a diffusive process starting at this protein needs to perturb all the proteins in the network. we have calculated this number to be 3.6250, which is only slightly larger than the average (topological) path length, which is 3.5673. that is, in less than four steps, the whole network of proteins is activated by a diffusive process starting at prkar2a. also remarkable that the average shortest diffusive path length is almost identical to the shortest (topological) one. this means that this protein mainly uses shortest (topological) paths in perturbing other proteins in the ppi. in other words, it is highly efficient in conducting such perturbations. we will analyze this characteristics of the ppi of human proteins targeted by sars-cov-2 in a further section of this work. at this time, almost any protein in the ppi network is already perturbed. therefore, we can switch and restart the subdiffusion from practically any protein at the ppi network. we then investigate which are the proteins with the higher capacity of activating other proteins that are involved in human diseases. here, we use the database disgenet, 52 which is one of the largest publicly available collections of genes and variants associated with human diseases. we identified 38 proteins targeted by sars-cov-2 for which there is a "definitive" or "strong" evidence of being involved in a human disease or syndrome (see table s1 in the supplementary material). these proteins participate in 70 different human diseases or syndromes as given in tables s2 and s3 of the supplementary material. we performed an analysis in which a diffusive process starts at any protein of the network, and we calculated the average probability that all the proteins involved in human diseases are then perturbed. for instance, for a subdiffusive process starting at the protein arf6, we summed up the probabilities that the 38 proteins involved in diseases are perturbed at an early time of the process t = 0.2. then, we obtain a global perturbation probability of 0.874. by repeating this process for every protein as an initiator, we obtained the top disease activators. we have found that none of the 20 top activators is involved itself in any of the human diseases or syndromes considered here. they are, however, proteins that are important not because of their direct involvement in diseases or syndromes but because they propagate perturbations in a very effective way to those directly involved in such diseases/syndromes. among the top activators, we have found arf6, ecsit, retreg3, stom, hdac2, exosc5, thtpa, among others shown in fig. 3 , where we illustrate the ppi network of the proteins targeted by sars-cov-2 remarking the top 20 disease activators. we now consider how a perturbation produced by sars-cov-2 on a protein mainly expressed in the lungs can be propagated to proteins mainly located in other tissues (see table s4 in the supplementary material) by a subdiffusive process. that is, we start the subdiffusive process by perturbing a given protein, which is mainly expressed in the lungs. then, we observe the evolution of the perturbation at every one of the proteins mainly expressed in other tissues. we repeat this process for all the 193 proteins mainly expressed in the lungs. in every case, we record those proteins outside the lungs, which are perturbed at very early times of the subdiffusive process. for instance, in fig. 4 , we illustrate one example in which the initiator is the protein golga2, which triggers a shock wave on proteins rbm41, tl5, and pkp2, which are expressed mainly outside the lungs. we consider such perturbations only if they occur at t < 1. not every one of the proteins expressed outside the lungs is triggered by such shock waves at a very early time of the diffusion. for instance, proteins mark1 and slc27a2 are perturbed in very slow processes and do not produce the characteristic high peaks in the probability at very short times. on the other hand, there are proteins expressed outside the lungs that are triggered by more than one protein from the lungs. the case of golga2 is an example of a protein triggered by three proteins in the lungs. in table i , we list some of the proteins expressed mainly in tissues outside the lungs, which are heavily perturbed by proteins in the lungs. the table i. multi-organ propagation of perturbations. proteins mainly expressed outside the lungs are significantly perturbed during diffusive processes that have started at other proteins expressed in the lungs. act. is the number of lung proteins activators, p tot is the sum of the probabilities of finding the diffusive particle at this protein, and t mean is the average time of activation (see the text for explanations). the tissues of main expression are selected among the ones with the highest consensus normalized expression (nx) levels by combining the data from the three transcriptomics datasets (hpa, gtex, and fantom5) using the internal normalization pipeline. 49 boldface denotes the highest value in each of the columns. act. table s5 of the supplementary material. we give three indicators of the importance of the perturbation of these proteins. they are act., which is the number of proteins in the lungs that activate each of them; p tot , which is the sum of the probabilities of finding the diffusive particle at this protein for diffusive processes that have started in their activators; and t mean , which is the average time required by activators to perturb the corresponding protein. for instance, pkp2 is perturbed by 21 proteins in the lungs, which indicates that this protein, mainly expressed in the heart muscle, has a large chance of being perturbed by diffusive processes starting in proteins mainly located at the lungs. protein prim2 is activated by 5 proteins in the lungs, but if all these proteins were acting at the same time, the probability that prim2 is perturbed will be very high, p tot ≈ 0.536. finally, protein tle5 is perturbed by 13 proteins in the lungs, which needs as an average t mean ≈ 0.24 to perturb tle5. these proteins do not form a connected component among them in the network. the average shortest diffusion path between them is 5.286 with a maximum shortest subdiffusion path of 10. as an average, they are almost equidistant from the rest of the proteins in the network as among themselves. that is, the average shortest subdiffusion path between these proteins expressed outside the lungs and the rest of the proteins in the network is 5.106. therefore, these proteins can be reached from other proteins outside the lungs in no more than six steps in subdiffusive processes like the ones considered here. finally, we study here how the diffusive process determines the paths that the perturbation follows when diffusing from a protein to another not directly connected to it. the most efficient way of propagating a perturbation between the nodes of a network is through the shortest (topological) paths that connect them. the problem for a (sub)diffusive perturbation propagating between the nodes of a network is that it does not have complete information about the topology of the network as to know its shortest (topological) paths. the network formed by the proteins targeted by sars-cov-2 is very sparse, and this indeed facilitates that the perturbations occurs by following the shortest (topological) paths most of the time. think, for instance, in a tree, which has the lowest possible edge density among all connected networks. in this case, the perturbation will always use the shortest (topological) paths connecting pairs of nodes. however, in the case of the ppi network studied here, a normal diffusive process, i.e., α = 1, not always uses the shortest (topological) paths. in this case, there are 1294 pairs of proteins for which the diffusive particle uses a shortest diffusive path, which is one edge longer than the corresponding shortest (topological) path. this represents 6.11% of all total pairs of proteins that are interconnected by a path in the ppi network of proteins targeted by sars-cov-2. however, when we have a subdiffusive process, i.e., α = 0.75, this number is reduced to 437, which represents only 2.06% of all pairs of proteins. therefore, the subdiffusion process studied here through the ppi network of proteins targeted by sars-cov-2 has an efficiency of 97.9% relative to a process that always uses the shortest (topological) paths in hopping between proteins. in fig. 5 , we illustrate the frequency with which proteins not in the shortest (topological) paths are perturbed as a consequence that they are in the shortest subdiffusive paths between other proteins. for instance, the following is a shortest diffusive path between the two end points: rhoa-prkaca-prkar2a-cep43-rab7a-atp6ap1. the corresponding shortest (topological) path is rhoa-mark2-ap2m1-rab7a-atp6ap1, which is one edge smaller. the proteins prkaca, prkar2a, and cep43 are those in the diffusive path that are not in the topological one. repeating this selection process for all the diffusive paths that differs from the topological ones, we obtained the results illustrated in fig. 5 . as can be seen, there are 36 proteins visited by the shortest diffusive paths, which are not visited by the corresponding topological ones. the chaos article scitation.org/journal/cha average degree of these proteins is 7.28, and there is only a small positive trend between the degree of the proteins and the frequency with which they appear in these paths; e.g., the pearson correlation coefficient is 0.46. we have presented a methodology that allows the study of diffusive processes in (ppi) networks varying from normal to subdiffusive regimes. here, we have studied the particular case in which the time-fractional diffusion equation produces a subdiffusive regime, with the use of α = 3/4 in the network of human proteins targeted by sars-cov-2. a characteristic feature of this ppi network is that the second smallest eigenvalue is very small; i.e., µ 2 = 0.0647. as this eigenvalue determines the rate of convergence to the steady state, the subdiffusive process converges very slowly to that state. what it has been surprising is that even in these conditions of very small convergence to the steady state, there is a very early increase of the probability in those proteins closely connected to the initiator of the diffusive process. that is, in a subdiffusive process on a network, the time at which a perturbation is transmitted from the initiator to any of its nearest neighbors occurs at an earlier time than for the normal diffusion. this is a consequence of the fact that e α,1 (−γ t α ) decreases very fast at small values of t α , which implies that the perturbation occurring at a protein i at t = 0 is transmitted almost instantaneously to the proteins closely connected to i. this effect may be responsible for the explanation about why subdiffusive processes, which are so globally slow, can carry out cellular processes at a significant rate in cells. we have considered here a mechanism consisting in switching and restarting several times during the global cellular process. for instance, a subdiffusive process starting at the protein i perturbs its nearest neighbors at very early times, among which we can find the protein j. then, a new subdiffusive process can be restarted again at the node j and so on. one of the important findings of using the current model for the study of the pin of proteins affected by sars-cov-2 is the identification of those proteins that are expressed outside the lungs that can be more efficiently perturbed by those expressed in the lungs (see table i ). for instance, the protein with the largest number of activators, pkp2, appears mainly in the heart muscle. it has been observed that the elevation of cardiac biomarkers is a prominent feature of covid-19, which in general is associated with a worse prognosis. 53 myocardial damage and heart failure are responsible for 40% of death in the wuhan cohort (see references in ref. 53) . although the exact mechanism involving the heart injury is not known, the hypothesis of direct myocardial infection by sars-cov-2 is a possibility, which acts along or in combination with the increased cardiac stress due to respiratory failure and hypoxemia, and/or with the indirect injury from the systemic inflammatory response. [53] [54] [55] [56] as can be seen in table i , the testis is the tissue where several of the proteins targeted by sars-cov-2 are mainly expressed, e.g., cep43, tle5, prim2, mipol1, reep6, hook1, cenpf, trim59, and mark1. currently, there is no conclusive evidence about the testis damage by sars-cov-2. 57-60 however, the previous sars-cov that appeared in 2003 and which shares 82% of proteins with the current one produced testis damage and spermatogenesis, and it was concluded that orchitis was a complication of that previous sars disease. 57 we also detect a few proteins mainly expressed in different brain tissues, such as cep135, prim2, trim59, and mark1. the implication of sars-cov-2 and cerebrovascular diseases has been reported, including neurological manifestations as well as cerebrovascular disease, such as ischemic stroke, cerebral venous thrombosis, and cerebral hemorrhage. [61] [62] [63] kidney damage in sars-cov-2 patients has been reported, 64-66 which includes signs of kidney dysfunctions, proteinuria, hematuria, increased levels of blood urea nitrogen, and increased levels of serum creatinine. as much as 25% of an acute kidney injury has been reported in the clinical setting of sars-cov-2 patients. one of the potential mechanisms for kidney damage is the organ crosstalk, 64 as can be the mechanism of diffusion from proteins in the lungs to proteins in the urinary tract and kidney proposed here. a very interesting observation from table i is the existence of several proteins expressed mainly in the thymus and t-cells, such as tle5, retreg3, rbm41, cenpf, and trim59. it has been reported that many of the patients affected by sars-cov-2 in wuhan displayed a significant decrease of t-cells. 67 thymus is an organ that displays a progressive decline with age with reduction of the order of 3%-5% a year until approximately 30-40 years of age and of about 1% per year after that age. consequently, it was proposed that the role of thymus should be taken into account in order to explain why covid-19 appears to be so mild in children. 67 the protein tle5 is also expressed significantly in the lymph nodes. it was found by feng et al. 68 that sars-cov-2 induces lymph follicle depletion, splenic nodule atrophy, histiocyte hyperplasia, and lymphocyte reductions. the proteins hook1 and mipol1 are significantly expressed in the pituitary gland. there has been some evidence and concerns that covid-19 may also damage the hypothalamo-pituitary-adrenal axis that has been expressed by pal, 69 which may be connected with the participation of the previously mentioned proteins. another surprising finding of the current work is the elevated number of subdiffusive shortest paths that coincide with the shortest (topological) paths connecting pairs of proteins in the ppi of human proteins targeted by sars-cov-2. this means that the efficiency of the diffusive paths connecting pairs of nodes in this ppi is almost 98% in relation to a hypothetical process that uses the shortest (topological) paths in propagating perturbations between pairs of proteins. the 437 shortest diffusive paths reported here contain one more edge than the corresponding shortest (topological) paths. the proteins appearing in these paths would never be visited in the paths connecting two other proteins if only the shortest (topological) paths were used. what is interesting to note that 6 out of the 15 proteins that are mainly expressed outside the lungs are among the ones "crossed" by these paths. they are tle5 (thymus, lymph node, testis), pkp2 (heart muscle), cep135 (skeletal muscle, heart muscle, cerebral cortex, cerebellum), cep43 (testis), rbm41 (pancreas, t-cells, testis, retina), and retreg3 (prostate, thymus). this means that the perturbation of these proteins occurs not only through the diffusion from other proteins in the lungs directly to them, but also through some "accidental" diffusive paths between pairs of proteins that are both located in the lungs. all in all, the use of time-fractional diffusive models to study the propagation of perturbations on ppi networks seems a very promising approach. the model is not only biologically sounded but it also allows us to discover interesting hidden patterns of the chaos article scitation.org/journal/cha interactions between proteins and the propagation of perturbations among them. in the case of the pin of human proteins targeted by sars-cov-2, our current finding may help to understand potential molecular mechanisms for the multi-organs and systemic failures occurring in many patients. after this work was completed, qiu et al. 75 uploaded the manuscript entitled "postmortem tissue proteomics reveals the pathogenesis of multiorgan injuries of covid-19." the authors profiled the host responses to covid-19 by means of quantitative proteomics in postmortem samples of tissues in lungs, kidney, liver, intestine, brain, and heart. they reported differentially expressed proteins (deps) for these organs as well as virus-host ppis between 23 virus proteins and 110 interacting deps differentially regulated in postmortem lung tissues. according to their results, most deps (70.5%) appears in the lungs, followed by kidney (16.5%). additionally, qiu et al. 75 identified biological processes that were up-or down-regulated in the six postmortem tissue types. they found that most up-regulated processes in the lungs correspond to processes related to the response to inflammation and to immune response. however, pathways related to cell morphology, such as the establishment of endothelial barriers, were down-regulated in the lungs, which was interpreted as a confirmation that the lungs are the main focus of virus-host fights. other fundamental processes in the six organs analyzed postmortem were significantly down-regulated, which include processes related to organ movement, respiration, and metabolism. from the 59 proteins that we reported here as the ones with the largest effect on perturbing those 38 proteins identified in human diseases (see table s3 in the supplementary material), 18 were found to be down-regulated in the lungs by qiu et al. 75 if we make the corresponding adjustment, by considering that qiu et al. 75 considered 110 instead of 209 proteins in the ppi, the previous number represents 58.1% of proteins predicted here and experimentally found as down-regulated in the lungs. from the rest of the proteins, which were not found as having the largest effect on perturbing proteins identified in human disease, only 29.1% were reported by qiu et al. 75 to be down-regulated in the postmortem analysis of patients' lungs. among the proteins reported in table s3 of the supplementary material and by qiu et al., we have arf6, rtn4, rab7a, 6ng5, reep5, vps11 , rhoa, rab5c, among others. finally, among the proteins mainly expressed outside the lungs that are predicted in this work to be significantly perturbed, we have five that were found by qiu et al. 75 to be up-regulated in the different organs analyzed by them. from the proteins included in table i , qiu et al. 75 reported the following ones up-regulated: pkp2 (heart), reep6 (liver), hook1 (several organs), atp5me (heart), and slc27a2 (liver and kidney). they also reported cep43 (reported as fgfr1op) as downregulated in the brain. we should remark that we have considered here many more organs than the six ones studied by qiu et al. 75 there are no doubts that in considering a diffusive propagation of perturbations among proteins in a ppi, we have made a fig. 6. pi metaplex. in the metaplex, every node of the ppi corresponds to a protein and its crowded intracellular space. there is an internal dynamics in the nodes and an external between the nodes. few simplifications and assumptions. every protein is embedded in an intracellular crowded environment, which drives its diffusive mechanism. nowadays, it is well-established that this environment is conducive to molecular subdiffusive processes. as remarked by guigas and weiss, 27 far from obstructing cellular processes, subdiffusion increases the probability of finding a nearby target by a given protein, and therefore, it facilitates protein-protein interactions. the current approach can be improved using two recently developed theoretical frameworks: (i) metaplexes and (ii) d-path laplacian operators on graphs. a ppi metaplex, 70 a 4-tuple ϒ = (v, e, i, ω), where (v, e) is a graph, ω = { j } k j=1 is a set of locally compact metric spaces j with borel measures µ j , and i : v → ω is illustrated in fig. 6 . then, we define a dynamical ϒ = (v, e, i, ω = { k }) on the metaplex as a tuple (h, t ). here, h = h v : l 2 ( i (v), µ i (v)) → l 2 ( i (v), µ i (v))} v∈v is a family of operators such that the initial value problem ∂ t u v = h v (u v ), u v | t=0 = u 0 , is well-posed, and t = {t vw } (v,w)∈e is a family of bounded operators t vw : l 2 ( i(v) , µ i(v) ) → l 2 ( i(w) , µ i(w) ). this means that inside a node of the metaplex, we consider one protein and its crowded intracellular space. inside the nodes, we can have a dynamics like a time-fractional diffusion equation, the fractional fokker-planck equation, or any other in a continuous space. the inter-nodes dynamics is then dominated by a graph-theoretic diffusive model like the one presented here. the second possible improvement to the current model can be made by introducing the possibility of long-range interactions in the inter-nodes dynamics in the ppi metaplex. that is, instead of considering the time-fractional diffusion equation, which only accounts for subdiffusive processes in the graph, we can use the following generalization, which incorporates the d-path laplacian operators, 71 where l d is a generalization of the graph laplacian operator to account for long-range hops between nodes in a graph, d is the shortest path distance between two nodes, and s > 0 is a parameter. this equation has never been used before except that for the case α = 1 where superdiffusive behavior was proved in 1-and chaos article scitation.org/journal/cha 2-dimensional cases. 72, 73 other approaches have also been recently used for similar purposes in the literature. 74 we then hope that the combination of metaplexes and timeand space-fractional diffusive models do capture more of the details of protein-protein interactions in crowded cellular environments. see the supplementary material for a list of proteins targeted by sars-cov-2, which are found in the database disgenet as displaying "definitive" or "strong" evidence of participating in human diseases. the disease id is the code of the disease in disgenet. a list of proteins with the largest effect on perturbing those 38 proteins are identified in human diseases. p tot is the sum of the probabilities that the given protein activates those identified as having "definitive" or "strong" evidence of being involved in a human disease. there is an rna expression overview for proteins targeted by sars-cov-2 and mainly expressed outside the lungs. we select the top rna expressions in the four databases reported in the human protein atlas. there is a list of proteins mainly expressed outside the lungs and their major activators, which are proteins mainly expressed in the lungs. the author thanks dr. deisy morselli gysi for sharing data and information. the author is indebted to two anonymous referees whose clever comments helped in improving this work substantially. the data that support the findings of this study are available within the article and its supplementary material and also from the corresponding author upon reasonable request. scitation.org/journal/cha the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 a pneumonia outbreak associated with a new coronavirus of probable bat origin a new 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systems on diffusion path laplacian matrices: introduction and application to the analysis of consensus in networks path laplacian operators and superdiffusive processes on graphs. i. one-dimensional case path laplacian operators and superdiffusive processes on graphs. ii. two-dimensional lattice hopping in the crowd to unveil network topology key: cord-287729-pl88otue authors: gray, stewart m. title: plant virus proteins involved in natural vector transmission date: 1996-07-31 journal: trends in microbiology doi: 10.1016/0966-842x(96)10040-8 sha: doc_id: 287729 cord_uid: pl88otue abstract plant viruses transmitted by invertebrate vectors either reversibly bind to vector mouthparts or are internalized by the vector and later secreted. viral proteins mediate the binding of plant viruses to vector mouthparts and the transport of virus across vector-cell membranes. both mechanisms probably involve conformational changes of virus proteins during their association with the vector. the terminology used to describe plant virus transmission is biased towards aphid vectors, and does not accurately reflect the transmission processes of other vector taxa". in this review, i divide plant viruses into two broad cazgories with different transmission processes: circulative and noncirculative. circulative viruses are usually defined as moving from the abmentary canal of an insect vector into its hemocoel (its open circulatory system) and back out +hrougb the salivary secretory system. however, in this review, i expand the definition of circulative transmissioal to include any plant virus that must be actively transported across vector membranes and survive inside the vector to be transmitted. noncirculative viruses associate with the cuticular lining of the insect mouthpam or foregut and are released as the insect expels digestive secretions into the plant when it begins to feed. these viruses are not actively transported across vector-cell membranes, nor are they carried internally. the cxrernal cuticular lining of insects (and nematodes) exrecds well ir?to the mouthparts and foregut, but is shed when the animal molts. comoviruses, bromoviruses and sobemoviruses could also be included. as these viruses are tramported across gut membranes into the hemolymph of the insect"; however, this mipht not be an e-xntial requirement for transmis\inn". crrcf4larif 'c rransrrrcssion in i#se?cr rvcrors luteoviruses and pemv are the best studied of the circulative nonpropagative viruses, ard thcv share many fundamental katures and mechanisms related to their transmisgon by aphids'l'. afiet uptake into the alimentary canal, virus particles attach to and are transported across the hindgut, and occasionally the midgut, epitbelium into the hcmc~ ccl via a receptor-mediated & cytotic pathway's2'. the hindgut can act as a barrier to ttrteovirus movement into the hemocoel, but it does nor appear to inhibit the uptake of most luteovirus isolates=*. virus parricles are carried by the hemolymph from the abdomen to the head, where they can associatewiththea ccessory salivary glared (mc). here, virus is actively transported across two distinct barriers to transmi~ion, the asc, basal lamina and the -4% plasmalemma, and then released into the salivary canal. virus is then injected into a plant as the aphid feeds" (figs l-3) . the .4sg b-1 lamina is a fibrous network consisting primarily of collagen, which provides support snd may also act as a filtee. lutcovirus isolates differentially bind the asg basal lamina and selectively but move across the basal lamina matrix. luteoviruses do not interact with rhe basal lamina of other rissues, including the principal salivary gland""'-. the transport of luteoviruses across the asg plasma!emma, which is also selective for specific isolates, appears to occur by receptor-mediated endocytosis". the hindgut, asg basal lamina and asg plasmalemma have various effects on the transmission efficiency of different cornhinations of aphid species and virus isolate'-.".'-, suggesting that the moiecular mechanisms involved in virus movement across these three harriers are likely to involve different viral proteins or protein domains. the capsid of luteoviruses and pemv contains two proteins, a predominant coat prc,tc#u and r srcondary protein that is present in small amounts and isrranslated via .cadrhrough of the coat protein stop codon-."x-r'. heterologous encapsidation is common between barley yellow dwarf luteoviruses when multiple isolates infect the same plant"-". if complete or partial exchange of capsid proteins occurs, the vector-specific transmission phenotype of one or both isolates is altered".". the readthrough protein, although not required ior particle assembly or plant infection' '-.4. is required for aphid transmission-*-". although luteovirus particles lacking the readthrough protein are acquired by aphids and can cross the hindgut harrier to accumulate in he hemocoel, no transmission to plants occurs"*". these observations suggest that rhe readthrough protein is required for viruses to move across transmiscicm 5arriers in the asg., and that the coat protein is probably therefore responsible for virus movement across the hindgut. the circulative transmrssion of geminiviruses through their whitefly or leafhopper vectors has not been studied in detail, but is thought to be similar to that of luteoviruses in aphids. unlike the iuteovituses, only the coat protein is required for transmission, and the coar prorein appears to regulate the specificity of transmission by both whirefiies and leafhoppers'"*'-. the whiteflytransmitted gemirliviruses have a highly conserved amino acid sequence in their coat proteins, and are all transmitted efficiently by one whitefly species, bemisia tahci. in the amino terminus uc the coat protein is essential for aphid transmission. proteolytic treatment of virions, which removes the amino terminus of the coar-protein subunits, prevents the particles from being transmitted by aphids, although they remain infectious when mechanically inoculated into plantsas. the hc is presumably acquired along with virus particles as the aphid feeds on plant sap, hut the role of this protein in transmission is unknown. one hypothesis is that it mediates the binding of virus to sites within the aphid food canal, either directly, by iinking the virus to the aphid (fig. 4a ), or indirectly, by modifying viral attachment compounds in the aphid to allow virus binding (fig. 48) . virus-like particles have been observed embedded in a matrix material assocaated with the stylers and foregut of aphids fed on a mlxrurc of gus and hc. no bound particles were oherved when aphids were fed on virus alone*. in \ it-u+ mutants ccmraining deletions or substitutions making either the coat protein or the hc incompetent for aphid transmission, theassocianon of virus-like particles with the cuticle did not occur (t.p. pironc. pers. commun.). transmission -ompete; 7 hl is required for virus association and retention in the aphid mouthpatts and must be present before the virus or simultaneously with it'; this suggests that hc is involved in me&ating binding between the aphid cuticle and the virus. an alternative hypothesis is that i-k acts indirectly to modify the coat protein and allows a direct interaction between the virus partick md theaphid (fig. 4c) . when a recombinant protein containing the aminoterminal region: of; ;atyvirus coat protein was fed to aphids before feeding them virus lnd hc, transmission was abolished'-. salomon and bemardi interpret these data tosuggest that the recombinant protein saturated vircs-binding sites in the aphid and prevented subsequenr virus binding. they hypothesize that the aminoterminal region of the coat protein, rather than the hc. attacha to sites on the aphid. they further suggest that the am&-terminal region of the coat-protein monomers assembled into virus particles is not normally available for interaction with the aphids, but that hc mediates a conformational change in the amino termiof thecoat protein that allows binding of the virus ;o the aphid (fig. 4c) .mission is regulated mainly by the codt protein'". site-specific mutagenesis of cucumber mosaic virus (cumv) has identified two regions of the coat protein thar are involved in efficient transmission, and has pinpointed the amino acids needed<'. the same regions of the coat protein, but not the same amino b acids, have been implicated in a second poorly transmissible cumv strain. the rm isu&don effiiency of cumv has been attributed to properties of the coat protein, but not to identifiable linear amino acid sequences. these observations biggest that one or a few amino acid changes in the coat protein could alter the vectortransmission phenotype by pre\ enring direct interaction between the virus and the vector. alternatively, rhe amino c acid changes couid influence the threedimensional structure of the coat protein or capsid. and indirectly affect the ability of the virus to interact with its vector. irr leo, but cannot mediate transmission. if supplied along with functional hc and virus, the gst-hc fusion protein inhibits transmission'". schmidt eta/. suggest that the gst-hc protein outcompetes the native hc and saturates virus-binding sites. as the amino-terminal portion of the hc in the fusion is attached zo gst, it cannot interact with binding sites in the aphid, and so transmission is prevented. noncrrculuti~~e rims fransmissiorr by nentamdes the noncirculative nematode-transmitted nepoviruses and tobraviruses also associate with the cuticular lining of the vector food canal. acquired virus particles bind to specific regions of the stylet sheath, pharynx or esophagus, and a carbohydrate-containing material of unknown origin is associated with bound virus particles. the involvement of nonstructural virus proteins has not been established for the nepoviruses, but recent evidence suggests that a nonstructural protein might be required for transmission of to'uraviruses in addition to the coat proteitp. virus release is thought to be mediated by a ph change resulting from salivary secretions flowing through the foocl canal when the nematode begins to feed on a planta(l. like the amino-terminal domain of rhe potyvirus capsid protein, the carboxy-terminal domain of the tobravirus coat protein can be cleaved from the virus particle by some proteases without adversely affecting the virus". it is not known whether or not the lnge&ion the role of hc in mediating binding between the aphid cuticle and the virus. biologically active hc, isolated from infected plants or a baculovirus expression system, binds coat protein in &ro48*49. two of the carboxy-terminal 31 amino acids of the hc are required for hc to bind coat protein, and loss of binding abolishes aphid transmission. hc expressed in e.scbtichia ccli as an amino-terminat glutathione-s-transferase-hc (gst-hc) fusion protein can bind coat protein reviews ------modified particles can hc transmitted by nematodes. interestirgty, the aggregation state of coat-protcln monomers does rcrpond to chnngrs in ph. and the carboxyl terminus of the tobacco rattle tobravirus coat protein contains a segment that does not appear to he part of the struct!tral framework of the virus particlecl. could this be a conformationally active rcg)l,n of the coat protein that is exposed only in the nematode foregut and that acts as a cleavage site for virui release? in noncirculative transmission, the virus must associate with the cuticular lining of the food canal of the vector. binding require5 \ iral protein sequences and perhaps specific vector substances. the binding rnl!rt be reversible. and release can involve specific proieolyric cleavage events or can be passive. the ends <bithe coat-protein subunits of the virion are likely pomts of interaction with sites in the vector or with the viral helper component. although there is no dcfi. iti1.e proof. many of these observations suggest that cor.fortnational changes in the capsid-protein sub *lits facilitate transmission. the conforr,lational change might ho -ediated by a helper comp~ment. the environment wtthln the food canal or binding of the virus tc) the vector, all resulting in the exposure >f cleavage sites on the coat proteins. exposure of these sites to digestive uxre;ions during feeding would create a mechanism by which bou.~d \irt:s could bc released from rhr \'cilor and inirs---cl into a plant host (fig. 4) . recently. rapid progress has been m; de in dis=+ng the relatively simple genomes of p!ant vuuses and identitying the proteins that are involved in their rransmission by k-actors. changes in the linear sequence of transmission-associated proteins can alter the tratzsmission phenotype of a virus. but the three-dimensional structure of protein subunits or viruscapsid is also likely to be involved in virus-vector interactions. conformational changes in 3 virus protein could alter in response to environmental changes, and this could prevetrt virus transmission. however, most plant-infecting viruses do not replicate in their vectors and do not appear it) undergo major morphological changes within the \~'ctar. the environment within titc dumentary system or hemolymph of a vector 1s probably significantly different from that within a plant cell or in gtro. possibly. viruses undergo more-subtle conformational changes within the veczor that are not readily detectable. it is well known that changes in ph or ionic strength can alter the conformarion of plant viruses".". hence, the biochemical environment within a vector might alter the virion struc~~~re such that different protein domains are accessible for interaction with a vector. 1 discuss this hypothesis for noncirculative viruses, but it is also likely to apply to circulative viruses. the readthrough protein of luteoviruses is required for efficient transmission of vtrus through the aphid asg. the readthrough protein is thought to protrude from the particle surface, or at least to be part of the surface topography znjv.u. however, antibodies that specifically label the proteins in vitro do not iabel whole ----------. cd.). pp. l-14 ph~opofl?o&~ x4, i 155-1 i $6 iyys) v~i/~~~~ 112. i83-!ljl 5) \'rrobqy 2 i ;. (r blanc. 5. el d ( i 9931 vrro/r,i vtro/q,, l95,692-6y9 .<i i+. ill.. rt ~1. (lyy4) \'rrd~!c~gj~lli viroh~ rin press) 52 .\la<farlanr i19901 vrro/r~fl 177 51. jnd hen?. le. 119h6) r n. t\ppl. rx)/. ioy in eqclopedw u/ vnulw iwebster, rl. 199-ioh and granoff vtrrhgy 195. 16-32 fh hre -i htw: ~2li~. \. el~l. l 199.5) \'iro!~fl key: cord-007755-o2r8ktie authors: kokoszka, malgorzata e.; kay, brian k. title: mapping protein–protein interactions with phage-displayed combinatorial peptide libraries and alanine scanning date: 2014-10-20 journal: peptide libraries doi: 10.1007/978-1-4939-2020-4_12 sha: doc_id: 7755 cord_uid: o2r8ktie one avenue for inferring the function of a protein is to learn what proteins it may bind to in the cell. among the various methodologies, one way for doing so is to affinity select peptide ligands from a phage-displayed combinatorial peptide library and then to examine if the proteins that carry such peptide sequences interact with the target protein in the cell. with the protocols described in this chapter, a laboratory with skills in microbiology, molecular biology, and protein biochemistry can readily identify peptides in the library that bind selectively, and with micromolar affinity, to a given target protein on the time scale of 2 months. to illustrate this approach, we use a library of bacteriophage m13 particles, which display 12-mer combinatorial peptides, to affinity select different peptide ligands for two different targets, the sh3 domain of the human lyn protein tyrosine kinase and a segment of the yeast serine/threonine protein kinase cbk1. the binding properties of the selected peptide ligands are then dissected by sequence alignment, kunkel mutagenesis, and alanine scanning. finally, the peptide ligands can be used to predict cellular interacting proteins and serve as the starting point for drug discovery. very often in research projects, there is interest in mapping the protein-protein interactions of a protein of interest as a way of understanding its function in the cell or virus. while a variety of techniques exist for this purpose, such as yeast two-hybrid screening, mass spectrometry, and protein complementation assays, another approach is the use of phage-displayed combinatorial peptide libraries. in such an approach, one takes a purifi ed, recombinant protein and isolates peptide ligands to it through affi nity selection (fig. 1 ) . interestingly, the phage-displayed peptides bind at "hot spots" for molecular interaction and very often share the same primary structure as short regions within cellular interacting proteins. several examples where this approach has proven useful include such targets as protein interacting domains [ 1 , 2 ] . to demonstrate the utility of this approach, we describe its application to two targets, human protein tyrosine kinase, lyn, and the yeast serine/threonine-protein kinase cbk1. lyn was fi rst discovered as a viral oncogene [ 3 ] and later appreciated to be a proto-oncogene in humans. it is a non-receptor protein tyrosine fig. 1 a general workfl ow diagram for isolating and characterizing the peptide ligands to protein domains or fragments using phage display methods. in principle, coding regions of any protein domain of interest can be converted into recombinant dna and expressed as a fusion protein to a partner such as glutathione s-transferase (gst). with a soluble protein in hand, one can perform affi nity selections with phage-displayed combinatorial peptide libraries to identify its peptide ligands. the binding properties of those ligands can be then further characterized through affi nity and specifi city measurements. to assess the importance of each residue in the peptide ligand, a mutagenic analysis known as alanine scanning can be performed on the recombinant dna. with that knowledge, potential interacting partners of the protein of interest can be predicted [ 14 ] . finally, improving affi nity and specifi city of selected ligands through directed evolution may lead to the development of antagonists, which could be used as inhibitors of specifi c cellular interaction for proof-of-principle experiments of drug development kinase, which is a member of the src family of proteins. it has a modular architecture: a src homology 3 (sh3) domain, a src homology 2 (sh2) domain, several linker regions, and a catalytic domain that phosphorylate tyrosines in proteins [ 4 ] . the sh3 domain plays a role in mediating protein-protein interactions and has been described to bind proline-rich motifs in proteins. the cbk1 belongs to a large family of ndr/lats protein kinases, which is conserved across eukaryotes and includes members such as myotonic dystrophy kinase [ 5 ] . cbk1 plays a role in controlling cell separation after cytokinesis, cell integrity, and polarized growth in saccharomyces cerevisiae [ 6 ] . to date, only a few substrates of cbk1 have been reported. one of them, ace2, a transcription factor that is activated by cbk1 via three phosphorylation sites [ 7 ] , is responsible for transcriptional control of enzymes required for septum degradation after cytokinesis [ 6 ] . the other, ssd1, is an rnabinding protein that suppresses translation of certain mrnas and which loses activity when phosphorylated by cbk1 [ 8 ] . we chose these two proteins as targets in parallel affi nity selection experiments to illustrate that the same phage-displayed combinatorial peptide library can yield very different peptide ligands to different targets. the lyn sh3 domain has previously been used in affi nity selection experiments, and so it served as a positive control. the cbk1 protein had not been used previously in affi nity selection of combinatorial peptide libraries. we also show that the kunkel mutagenesis in combination with alanine scanning and elisa are very simple and expedient methods for evaluating the contribution of certain amino acids in the peptide ligands for binding to their targets. prepare using deionized water (dih 2 o). autoclave or fi lter sterilize and store at room temperature, unless indicated otherwise. single-stranded dna samples were isolated with the qiaprep spin m13 purifi cation kit (qiagen). 6. covalently closed, circular double-stranded m13 dna, synthesized via kunkel mutagenesis [ 9 , 10 ] , was purifi ed with qiaquick pcr purifi cation kit (qiagen). 7. concentrations of dna samples were determined using nanodrop nd-1000 uv spectrophotometer (thermo fisher scientifi c, inc.) and measuring the optical absorbance at 260 nm. 8. the following steps: phosphorylation of oligonucleotides, annealing to the template, and synthesis of covalently closed circular double-stranded dna (cccdna) were performed using dna engine dyad ® thermal cycler (bio-rad). in described methods, affi nity selections with phage-displayed combinatorial peptide libraries (figs. 2 and 3 ) were employed to identify peptide ligands that bind to human lyn sh3 domain and yeast cbk1 protein kinase. in general, once peptide ligands have been identifi ed for a protein target, an interacting motif can be deduced via sequence alignment (logo, fig. 4 ) of the primary structures of the displayed binding peptides. however, to assess the functionality of this motif, amino acid replacements of the critical residues are generally necessary. while this is possible through the chemical synthesis of variant peptides, it is more expedient to use mutagenesis techniques, such as alanine scanning (fig. 5 , [ 11 ] ). for the purpose of this selection protocol, the target domain was overexpressed in escherichia coli in the form of a glutathione s-transferase (gst) fusion protein. recombinant protein can be prepared via commercially available pgex vectors (ge healthcare). figure 2 presents a schematic diagram of the selection protocol utilizing glutathione-conjugated magnetic beads. to maintain optimum sterility and eliminate carryover, only fi ltered tips should be used throughout the selection procedure. 1. add 20 μl of glutathione magnetic bead slurry (5 μl of settled beads) into 2 ml eppendorf tube, and wash twice with 600 μl of ice-cold wash buffer #1 (pbs) on magnetic separator. remove the supernatant. 4. remove the supernatant. resuspend in 200 μl of blocking buffer #1. add 50 μg of gst protein to remove potential gst-binding phage clones and 20 library equivalents (e.g., if the library size is 10 10 clones and the titer 10 13 pfu/ml use 20 μl). bring the volume to 600 μl with ice-cold wash buffer #1. incubate at 4 °c for 1 h on the rotating shaker. 5. remove the supernatant. wash three times with 800 μl of icecold wash buffer #2 (pbst). remove supernatant, add 800 μl of ice-cold wash buffer #1, and transfer into a new 2 ml eppendorf tube to eliminate any plastic-bound phage. 6. repeat the washing step two more times with wash buffer #1. remove the supernatant. 7. elute the phage with 200 μl of elution buffer. incubate for 10 min at room temperature, and gently mix the content every couple minutes. 8. place the tube on magnetic separator, and transfer the eluted phage supernatant into a new 1.5 ml eppendorf tube containing 12 μl of neutralization buffer, and mix well. fig. 3c ) revealed a known y/fxfp docking motif to cbk1 [ 17 ] . also, it should be noted that sequence 3 (fig. 3c ) contains the fkfp motif, which is present in ssd1, a known cbk1 substrate [ 8 , 17 ] . our fi ndings clearly indicate that potential binding partners of certain targets can be deduced via selections with phage-displayed peptide libraries. as only three sequences were used for alignment, any additional amino acid preference analysis could be biased. ( c ) further characterization of the isolated y/fxfp motif showed preference to positively charged residues, r and k, at the "x" position. other allowed residues at that position included v, m, t, q, when at least one positively charged residue was observed outside the motif, or c, when a second cysteine was present following the motif. all sequences incorporated in the logo (32 isolates, data not shown) were isolated by screening phage-displayed focused library. the library was synthesized via kunkel mutagenesis (as described in subheading 3. 13. if blue-white screening can be performed, add 45 μl of 100 mm iptg and 80 μl of 2 % x-gal. 14. immediately before plating, add 4 ml of 2 × yt top agar (0.8 %), gently swirl, and pour over prewarmed 2 × yt agar plates. incubate overnight at 37 °c. 7. add anti-m13 hrp-conjugated antibody diluted 1:5,000 in pbst (same per well volume as the target protein), and incubate 30 min to 1 h at room temperature. 8. wash the plates with 200 μl of pbst three more times. 9. add chromogenic substrate solution (subheading 2.1 , item 21 ) (same per well volume as the target protein), and incubate for 10-30 min. 10. quantify the results by measuring the optical absorbance at 405 nm, using a microtiter plate spectrophotometer. all binding phage clones isolated via elisa are amplifi ed and their binding regions subsequently identifi ed by dna sequencing ( see note 7 ). if the selection generates enough unique isolates, the binding motif of the target can be predicted by sequence alignment known as logo plot (fig. 4 ) . with that knowledge, one can attempt to better defi ne the known interactions and to predict new substrates of the target. to further assess the importance of each residue in the motif, mutagenic analysis known as alanine scanning can be performed (fig. 5 , [ 11 ] ). in this method, each residue is replaced, one by one, by alanine (or by glycine if originally occupied by alanine). one way to generate all the variants is to incorporate them into m13 phage genome via kunkel mutagenesis (described in subheading 3.3.1 ) [ 10 , 12 , 13 ] . once the phage-displayed mutant pool is generated, the effect of the substitutions on their binding to the target is evaluated by phage elisa (described in subheading 3.2 ). for the purpose of this protocol, a modifi ed m13 phage vector containing an amber stop codon has to be fi rst obtained [ 9 ] . in the vector constructed by scholle et al., an amber stop codon, tag, has been placed at the n-terminus of the coding region of gene iii, following the signal sequence of protein iii (piii). that modifi cation eliminates the need for generation of uracil-containing single-stranded dna (u-ssdna) template, using e. coli strain cj236 ( dut − ung − ). a mutagenic oligonucleotide, containing both complementary and exogenous dna fragments, is then annealed to the ssdna template, with the exogenous fragment looping over the tag-containing vector region. once the double-stranded dna (dsdna) is synthesized from the ssdna template, it is electroporated into non-suppressor e. coli strain (e.g., ss320). if the tag triplet-containing region has not been replaced by the exogenous fragment, the phage will not be propagated as the minor coat protein, piii, cannot be translated. the wild-type phage can be propagated via the suppressor e. coli strain such as tg1 cells. the amber stop codon is then translated into glutamine. the use of tag-containing template facilitates nearly 100 % recombination effi ciency [ 9 ] . 2. if low quantities of target are available, a small amount can be used to coat the beads (e.g., 10 μg of protein and 10 μl of slurry, respectively). also, decreasing the amount of used target can facilitate isolation of higher affi nity clones. 3. for convenience, target-bound beads can be prepared for the entire selection procedure and stored at 4 °c in the blocking buffer #1 (pbs containing 3 % bsa (w/v)) for up to a week. 4. as long as no contamination is introduced, phage particles can be stored in a culture medium at 4 °c for many years. 5. since the phage titer, recovered after fi nal round of selection, depends on many controllable and uncontrollable factors such as stringency of the selections (i.e., number of washes, amount of target used) or type and structure of the target, from our experience, we recommend to cover a wide dilution range from at least 10 −4 -10 −8 . 6. if fi ltered tips are used for phage transfer, it is convenient to briefl y rinse and remove the tips with multichannel pipette. 7. in order to identify dna sequences of potential "binders," the phage is fi rst amplifi ed by infecting 200 μl of tg1 cells (from a mid-log phase culture) with 10 μl of phage supernatant (or single plaque) and incubated overnight in a shaking incubator (240 rpm), in 5 ml fi nal volume. the pelleted cells are then used for isolation of dsdna that consists of replicative form of phage dna. subsequently, the samples are analyzed via sequencing. remaining phage supernatant can be stored at 4 °c ( see note 4 ). 8 . in order to isolate high quantity of ssdna template, large amount of phage particles has to be fi rst collected. amplifi cation of phage by infecting cells with just a single plaque may result in low quantity of template. thus, we suggest a two-step preparation process, where the peg-precipitated concentrated virions are used to infect the fresh mid-log cells. 9. to remove any polyethylene glycol (peg) residues from the surface of the tube prior to phage reconstitution, it is recommended to fi rst briefl y rinse the side of the tube with pbs, avoiding the phage pellet. usually 1-3 ml of room temperature pbs is used to resuspend the pelleted virions. one of the major advantages of affi nity selection of phage-displayed combinatorial peptide libraries is the potential for rapid discovery of peptide ligands to a target protein. it is relatively straightforward to use homemade or commercially purchased libraries to isolate peptide ligands to a given target and then deduce a binding motif. a peptide with a consensus motif can then be used to study the biology of the target (i.e., predict cellular interacting partner, solve the three-dimensional structure of the peptide and target in a complex, and to inhibit the target in cells). however, when only one or a few peptide ligands are isolated to a target, it is diffi cult to know a priori what residues in the phage-displayed peptide ligand contribute to binding. while one can explore the binding parameters of the peptide sequence through chemical synthesis of peptides with systematic amino acid replacements across the primary structure, we present an alternative, faster, and easier approach involving kunkel mutagenesis, alanine scanning, and elisa. we fi nd that by coupling these three techniques, one can readily determine at fi rst pass many important aspects of the binding interaction. exploring protein -protein interactions using peptide libraries displayed on phage mapping intracellular protein networks the yesrelated cellular gene lyn encodes a possible tyrosine kinase similar to p56lck src family kinases: regulation of their activities, levels and identifi cation of new pathways cbk1p, a protein similar to the human myotonic dystrophy kinase, is essential for normal morphogenesis in saccharomyces cerevisiae the saccharomyces cerevisiae mob2p-cbk1p kinase complex promotes polarized growth and acts with the mitotic exit network to facilitate daughter cell-specifi c localization of ace2p transcription factor the ndr/lats family kinase cbk1 directly controls transcriptional asymmetry cbk1 regulation of the rna-binding protein ssd1 integrates cell fate with translational control effi cient construction of a large collection of phage-displayed combinatorial peptide libraries effi cient site-directed mutagenesis using uracilcontaining dna highresolution epitope mapping of hgh-receptor interactions by alanine-scanning mutagenesis improvements to the kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries rapid and effi cient sitespecifi c mutagenesis without phenotypic selection convergent evolution with combinatorial peptides can we infer peptide recognition specifi city mediated by sh3 domains? distinct ligand preferences of src homology 3 domains from src, yes, abl, cortactin, p53bp2, plcy, crk, and grb2 proteome-wide discovery of evolutionary conserved sequences in disordered regions this work was funded by the chicago biomedical consortium, with support from the searle funds at the chicago community trust. we would like to thank mr. kyle schneider, dr. brian yeh, and dr. eric weiss (nu) for providing the gst-cbk1 dna constructs and purifi ed gst-cbk1 fusion protein. we are grateful to dr. michael kierny and dr. renhua huang (uic) for their helpful comments on this manuscript. key: cord-298759-j965t808 authors: jiang, nan; ding, xuanwei; lu, yuan title: development of a robust escherichia coli-based cell-free protein synthesis application platform date: 2020-10-17 journal: biochem eng j doi: 10.1016/j.bej.2020.107830 sha: doc_id: 298759 cord_uid: j965t808 since the cell-free protein synthesis system is not limited by the cell growth, all the substrates are used to produce the protein of interest, and the reaction environment can be flexibly controlled. all the advantages allow it to synthesize toxic proteins, membrane proteins, and unnatural proteins that are difficult to make in vivo. however, one typical reason why the cell-free system has not been widely accepted as a practical alternative, is its expression efficiency problem. the escherichia coli-based system was chosen in this study, and the model protein degfp was expressed to explore a more efficient cell-free system. the results showed that mg2+ with a concentration of 15 mm in the cell-free system with bl21 star (de3) as the extract could better synthesize protein. the smaller the vectors, the lighter the burden, the higher the protein synthesis. simulating the crowding effect in the cell does not improve the protein expression efficiency of the optimized cell-free protein synthesis system. based on the optimized system, the cell-free fundamental research platform, primary screening platform, and portable biomolecular synthesis platform were established. this study provides a robust cell-free protein synthesis toolbox with easy extract preparation and high protein yield. it also enables more researchers to reap the benefits from the cell-free biosynthesis platform. the aim of synthetic biology is to advance biological functionality through engineering, and rapid progress has been made [1] [2] [3] [4] . its ability to engineer biological functionalities holds excellent prospects for applications in bioenergy production [5] , drug synthesis [6] , and biosensor development [7] . however, the development of synthetic biology faces enormous challenges [8] : hard to standardize, unwieldy complexity, incompatibility, and variability. to overcome these problems, cell-free protein synthesis (cfps) has received new attention as an emerging synthetic biology technology, because it is an open system, not limited by cell growth or cell membrane, and it allows the use of various reaction formats. cfps systems have many advantages such as controllable transcription, translation, and post-translational modification, high synthesis rate and product yield, convenient high-throughput screening format, easy incorporation of unnatural amino acids, high tolerance for toxic substrates or products, and accelerated design-build-test-learn cycle [9] . therefore, cell-free synthetic biotechnology has been used to make difficult-to-synthesize proteins, unnatural proteins, and complex proteins for various biological applications. despite its many advantages, the cfps system has not been widely accepted as a practical alternative. the problem of low productivity is considered to be one serious obstacle to cfps, so the optimization of the cfps system is very important [10] . the extract is one of the most j o u r n a l p r e -p r o o f important components in the cfps system. its preparation and selection affect the cfps efficiency [11] [12] [13] [14] . rational design of dna template is one of the keys for efficient cfps [15] . in most cases, a supercoiled plasmid dna containing the gene of interest is used in cell-free systems because of its good stability [16] . the substrate concentration in cfps is another key factor affecting the protein expression. the experimental conditions in cfps must be precise to achieve optimal expression. molecule density is another key plaguing cfps systems [17] . in recent years, the crowding effects of macromolecules have been transferred to cfps, which is an important function in living cells but ignored in cell-free systems. molecular crowding is a natural state of cells, which could lead to a fundamental impact on cellular properties and then may affect the efficiency of cfps [18] . in addition, metal cations are an indispensable part of biological machinery, involving many essential tasks ranging from biomolecular structure stabilization to enzyme catalysis. the change of metal cation type and concentration could affect the protein synthesis process, thus affecting the cfps efficiency [19] [20] [21] . cfps has a broad application prospect in basic scientific research and industrial production. with the advantages of cfps, it can be applied to the synthesis of difficult-to-express proteins, the embedding of unnatural amino acids, and the rapid screening of proteins. cfps systems have successfully expressed high levels of biopharmaceutical protein and membrane proteins, such as virus-like particles and g protein-coupled receptors [22] [23] . also, an orthogonal translation system can be used to incorporate unnatural amino acids into natural proteins to synthesize unnatural proteins with new functions [24] . in addition, through residue-specific and site-specific incorporation, cfps is becoming a platform for the production of enzymes with altered rapid screening functions [25] . it is believed that cfps can promote the development of basic science, biomedicine, and high-throughput screening platforms. in this paper, the issues that directly affect the cfps synthesis were concerned. the selection of extracts, the size of the plasmid, the molecular crowding effect and the metal ion j o u r n a l p r e -p r o o f effect, which are important component parameters, were explored to improve the protein expression efficiency. the final optimization results were used in three various applications. based on the optimized cfps systems, the cell-free fundamental scientific research platform, primary screening platform, and portable biomolecular synthesis platform were established. the target protein is the model protein degfp. first, inserted the coding sequence of degfp into the plasmid pet23a (tiandz, beijing, china) between ndeⅰ and salⅰ. a pcr cassette was generated using the primers w006f and w006r with the pet23a plasmid template and w003f and w003r with homemade pet21b-degfp plasmid template by a c1000 thermal cycler (bio-rad, hercules, ca, usa). two linear fragments were assembled by gibson. the plasmid pet23a-degfp was constructed after adding the coding sequence for a 6xhis-tag to the degfp gene by a pcr cassette (w005/w009) and gibson assembly. next, deleted the coding sequence of rop (w001f/w001r) and f1 ori (w002f/w002r) on the basis of pet23a (supplementary table s1 ). after pcr cassettes and gibson assembly, the small plasmid pet23a△rf-degfp was constructed (supplementary table s2 ). t7 rna polymerase was prepared from escherichia coli bl21(de3)/par1219 (sigma-aldrich merck, shanghai, china) as described previously [26] . cells were cultured in 1-l shake flasks with lb growth media (1% tryptone, 0.5% yeast extract, 1% nacl). cells were fermented at 37°c with 200 rpm agitation in a rotary incubator shaker for an hour, and then iptg induction was performed. another two hours later, cells were harvested at an od600 of 2.0 and centrifuged at 8000 rpm at 4°c for 20 min by a m41041-f000 centrifuge (kubota, tokyo, japan). cells were washed by suspending in 5 ml ice-cold s30 (10mm tris-acetate, j o u r n a l p r e -p r o o f 14mm magnesium acetate, and 60mm potassium acetate) per gram of cell for twice and centrifuged at 8,000 rpm at 4°c for 20 min. the pellets were resuspended in 4 ml ice-cold lysis buffer (50mm nacl, 10mm edta, 10mm k2hpo4, 1mm dtt, 10mm βmercaptoethanol, 1×protease inhibitor, 5% glycerin, ph 8.0) per gram of cell in preparation for cell lysis, ultrasonicated for 40 min (work 2s and intermittent 6s) and then centrifuged at 80,000 rpm at 4°c for 20 min. finally, the supernatant was dialyzed (6-8 kda) twice in dialysis buffer (50mm nacl, 1mm edta, 40mm k2hpo4, 1mm dtt, 20% sucrose, ph7.7) at 4°c overnight. the suspension was centrifuged at 10,000× g for 30 min at 4°c, and the pellets were discarded. the crude t7 rna polymerase was flash-frozen in liquid nitrogen and stored at -80°c until use. cell growth for extract preparation was performed using bl21 star (de3) cells in 1-l shake flasks with inner concave baffles. cells were cultured at 37°c with 240 rpm agitation in a rotary incubator shaker. the fermentation was performed in 2ⅹyt growth media (1% yeast extract, 1.6% tryptone, 0.5% nacl). cells were harvested at early logarithmic growth phase at an od600 of 3.2 to 4.0, about 3.5 h after induction by a m41041-f000 centrifuge (kubota, tokyo, japan) at 8000 rpm at 4°c for 20 min. cells were then washed by suspending in 5-ml ice-cold buffer a (1 mm dithiothreitol, 10 mm tris base, 14 mm magnesium acetate, and 60 mm potassium glutamate) per gram of cell and centrifuged at 10,000 rpm at 4°c for 10 min, and subsequently resuspended in 1-ml ice-cold buffer a per gram of cell in preparation for cell lysis. cell suspensions were lysed with two passes through a jn-3000plus low temperature ultra-high-pressure continuous flow homogenizer (jnbio, guangzhou, guangdong, china) with sample cooling for 1 min in an ice-water bath after the first pass. the lysate was centrifuged at 10,000 × g for 30 min at 4°c, and the pellet was discarded. the supernatant was carried forward for a run-off reaction by incubating at 37°c with 120 rpm j o u r n a l p r e -p r o o f agitation for 80 min. the suspensions were centrifuged at 10,000 × g for 30 min at 4°c, and the pellet was discarded. finally, dialyzed (6-8 kda) twice in buffer b (10 mm tris base, 14 mm magnesium acetate, and 60 mm potassium glutamate) at 4°c overnight. the suspensions were centrifuged at 10,000 × g for 30 min at 4°c, and the pellet was discarded. the extract was flash-frozen in liquid nitrogen and stored at -80°c until use. the cfps systems included the necessary components as shown in table 1 . combined transcription-translation reactions were carried out in 1.5 ml eppendorf tubes at 30°c for 12 h, unless otherwise noted. after the fermentation was performed in tb growth media (1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17mm kh2po4, 72mm k2hpo4), the plasmids were prepared using a qiagen plasmid maxi kit. the protein yield was determined by diluting the reaction volume to 200 µl with milliq water (millipore, billerica, ma, usa) in flat-bottomed 96-well black microtiter plates. measured the fluorescence with an infinite 200 pro microplate reader (tecan austria gmbh, 5082 grödig, austria). the excitation/emission wavelengths for degfp was 485 and 510 nm, and mcherry were 587 and 615 nm, respectively. 14 c-leucine-labeled protein synthesis was detected. added 0.534 μl 14 c -leucine into 20 μl cfps reaction system and then incubated reactions for 12 hours at 30°c. after the reaction, 3 μl reaction mixture was spotted onto both the r (total radiation), t (total protein) and s (soluble protein) filter paper strips corresponding to the correct experiment condition and replicate. the soluble fraction of the product protein was isolated by centrifuging the reaction the activity of tkadh was spectrophotometrically determined at 40 °c by monitoring the substrate-dependent change in the absorbance of nad(p)h at 340 nm (ε340 = 6.22 mm -1 cm -1 ). oxidation of alcohols was performed in a reaction mixture composing of 50 mm glycine-naoh buffer (ph 9.0), 100 mm phenylethanol, and 1.0 mm nad(p) + . the reaction started by adding an appropriate amount of the enzyme into the reaction mixture in a total volume of 1 ml. one unit of adh activity was defined as the amount of enzyme that oxidizes 1 μmol nad(p)h per minute. based on e. coli which was the most widely used host background for protein expression, five commercially available types were tested in the initial screening experiment ( table 2) . the microbiological contamination is encountered during the preparation of cell extracts, which are usually addressed by the addition of antibiotics. in order to explore whether the addition of antibiotics affected the protein expression of the extract, we also tested three different drug-resistant strains ( table 2 ). they were tested with or without antibiotics using different dna templates, which expressed the model protein (degfp). the result was obvious that the cell culture process without antibiotics were more suitable for cell extracts in cfps ( table 2 ). the reason for this may be that the toxicity of the antibiotics themselves inhibits cell growth and affects dna replication, which in turn affects the activity of the extract and thus hinders protein synthesis. interestingly, the results showed that the addition of antibiotics to bacterial seed fluids were needed to be grown in fresh media for 3 hours after overnight growth, and then the cell extract preparation was started ( supplementary fig. s1 ). in this section, a total of five strains were screened, bl21 star (de3) which had the highest protein expression was selected as the extract, and the culture medium was further optimized. these in most of the protocols for cell-free synthesis, protein synthesis is directed by plasmid templates, since the efficiency of the plasmid templates in cell-free synthesis is higher than those of the reactions utilizing linear templates. this is mainly due to the rapid decay of the linear templates by the exonucleases present in the cell-free extract [28] . according to the lowest cost principle, one of the points of concern is improving template utilization efficiency. the choice of vector affects the efficiency of cfps [29] . the pet system with the t7 promoter-driven system is the most widely used system yet developed for the cloning and however, no matter how the plasmid concentration changed, the protein expression level of pet23a△rf-degfp was always higher than that of pet23a-degfp (fig. 1c) . therefore, pet23a△rf-degfp was selected as the dna template. deleting the coding sequences unrelated to target protein transcription and translation made the plasmid size smaller, which meant the burden of plasmid became smaller, and the proportion of the target protein encoded gene was j o u r n a l p r e -p r o o f larger than before. the results indicated that plasmid size could be an important parameter for protein expression in the cfps system. this was an interesting discovery, but the specific reason was not yet known, and further investigation was needed. the biological significance of metal ions is that all organisms in the kingdom of life need metal ions as essential micronutrients. metal ions participate in various biological reactions and are estimated to be required in one third of all proteins [30] . protein-bound metal cations such as copper (cu 2+ , cu + ), iron (fe 3+ , fe 2+ ), magnesium (mg 2+ ), manganese (mn 2+ ), calcium (ca 2+ ), and zinc (zn 2+ ) are key elements for regulating gene expression and catalyzing enzyme activities [30] . among them, the electronic structure of the two redox states of iron, fe 2+ and fe 3+ , renders them the most versatile cofactors in biological redox reactions [31] . mg 2+ can play an essential role in folding, molecular recognition, and catalysis by rna [32] . in particular, divalent magnesium cations play an indispensable role in protein synthesis. in the cfps system, mg 2+ not only participates in transcription and translation but also acts as activators of enzymes such as rna polymerase and aminoacyl-trna synthase. however, excessive mg 2+ leads to premature termination of protein synthesis. consequently, the final concentration of mg 2+ has received widespread attention so that it is one of the most influential factors in the cell-free reaction mixture [33] . however, when it is necessary to synthesize metalloproteins in the cfps system or enzymes that require metals as cofactors, it is not enough to focus only on the concentration of magnesium ions. therefore, in this section, we explored the effect of different showed that the addition of the two metal ions promoted the protein expression, but the increase was very limited (fig. 2) . when ca 2+ and co 2+ were added to the system, the relative fluorescence value was lower than the control. in summary, mg 2+ was the optimal metal ion for this system, and the maximum yield was achieved when its concentration was 15mm. notably, the effects of mgcl and mgch16o8n2 on cfps were tested here. although the results showed a similar trend, there were differences in the protein expression level between these two. it indicates that the same metal ions were tested, but the paired anions had different effects on the protein expression. however, because the free mg 2+ has a complicated function of interactions with many cell-free reaction components, it is often beneficial to optimize mg 2+ for each lot of cell extract and cell-free reagents to obtain maximal protein synthesis. in addition, j o u r n a l p r e -p r o o f when the target protein to be synthesized is a metalloprotein or a specific metal ion is required as a cofactor, the corresponding metal ion can be added to the system. therefore, researchers need to optimize the metal ions and the corresponding concentration for the best cell-free protein synthesis. the interior of biological cells is a crowded environment. the biochemical interaction network that controls cell functions behaves very differently than the dilution environment of a typical test tube [34] . the biochemical reaction network involves the transcription, translation, and folding of proteins. one of the key distinguishing features between artificial cellular systems and living cells is the molecular density around cellular components [17] . molecular crowding is a natural state of cells, which could cause volume exclusion effects that reduce diffusion rates and enhance the binding rates of macromolecules, leading to a fundamental impact on cellular properties [18] . in this part, different kinds of crowding agents were selected to explore the influence of agents on the efficiency of protein synthesis in the cfps system. here, the effects of fifteen crowding agents (peg200, peg400, peg600, peg1000, peg4000, peg8000, dextran5, dextran500, trehalose, ethylene glycol, glycerol, carnitine, lactose, trimethylglycine and raffinose) at different concentrations were evaluated on the efficiency of protein synthesis in the cfps system. first, the effects of different crowding agents on protein synthesis at concentrations of 0 to 5% (w/v) were tested. the results showed that the relative fluorescence value decreased after adding crowding agents. among them, the addition of dextran5, dextran500, carnitine and raffinose had little effect on the protein expression. peg200, peg400, peg600, peg1000, peg4000, peg8000, ethyleneglycol, trimethylglycine, trehalose, glycerol and lactose had a great influence on the protein expression. among them, the addition of trehalose, glycerol and lactose inhibited the protein expression rapidly (fig.3) . the concentration range of the test was then reduced to 0 to 0.5% (w/v). the results showed that after the concentration range was reduced, the results were similar to before. it indicated that the crowding agents actually did not greatly improve the cfps efficiency. it might be due to the increased viscosity of some crowding agents, which increased the burden of the cfps system and therefore reduced the efficiency of protein expression. from the above, after optimizing the system (fig. 4) , important components of the cfps system have changed (table 1) . by comparing five e. coli extracts, the bl21 star (de3) extract was more suitable for the expression of the model proteins (degfp) due to its genetic features. since the smaller the plasmid size, the less the burden on cfps, the smaller pet23a△rf was selected as the carrier of degfp gene. in addition, the protein expression efficiency of cfps by metal ions was explored, and the protein expression level was highest when the metal ion was mg 2 + and the concentration was 15 mm. the crowding effect was explored and found that using additives to simulate the crowded environment would not improve the protein expression efficiency of the cfps system. after optimization, the fluorescence value of degfp increased from several thousand to one million. after the protein concentration determination, 0.535 mg/ml degfp were obtained in the optimized cfps system. the optimized cell-free system with better protein synthesis was obtained, which would be used in the following applications. the cell-free system is of significant benefit for expressing protein complexes consisting of hetero subunits, as it allows co-translation of multiple mrnas and forms the active protein complex. for example, active yeast trna methytransferase could be successfully expressed by co-translating mrnas for protein subunits trm8 and trm82 [35] . efforts to express the individual protein subunits and then reconstitute the complex, did not yield an active trm8trm82 heterodimer [35] . also, cfps can also be used in the research of basic education, avoiding the limitations of expensive special equipment for growing, storing and transporting cells and biosafety considerations [36] . here, the expression of the model proteins (degfp and mcherry) with genetic sequence similarity were demonstrated in the same cfps system. the addition of pet23a△rf-degfp and pet23a△rf-mcherry followed the proportion from 0:10 to 10:0. all the other reaction components were shown in table 1 . it showed that the transcription and translation were performed normally. however, the expression of two different fluorescent proteins did not affect each other, which were reflected in the relative fluorescence and visible colors. as their proportions changed, not only the fluorescence values of the two fluorescent proteins changed but also visible colors changed from green to red (fig. 5a) . thus, the expression of the target protein was methodical, even if multiple transcripts and translations were performed simultaneously. these results provided a reliable basis for fundamental research in the field of biochemistry. in addition, the reaction system only needed to simply mix several substances together, and the results could be observed through the change of fluorescence color and intensity. therefore, our system also provides a platform for basic education, avoiding the inherent limitations, so that untrained operators can experience synthetic biology research in such an easy-to-operate way. speeding up the design-build-test (dbt) cycles is a fundamental challenge that biochemical engineering faces. right now, in vivo approaches take, on average, 3~4 months to complete this cycle. in cell-free systems, the design cycle is not limited by how fast cells j o u r n a l p r e -p r o o f reproduce. rather than, it can be faster, potentially approaching the limit of synthesizing the components (dna, rna, and proteins). cell-free systems may therefore speed up the design cycle for engineering by more than 10-fold relative to in vivo approaches. in addition, cfps could be used as their own biomanufacturing platforms, or as feedback in the design of in vivo platforms [37] . at the same time, the active products can be monitored at any time. in order to explore whether cfps has the potential to become a preliminary screening platform, a novel thermostable alcohol dehydrogenase (adh) was selected and expressed in vitro. the enzyme has the activity toward aromatic secondary alcohols from the hyperthermophilic archaeon thermococcus kodakarensis kod1 (tkadh) and exhibits extreme thermostability as well as high resistance to organic solvents [38] . using the cell-free reaction mixture (fig. 5b) , the transcription and translation of tkadh were performed. the results indicated that the enzyme could be expressed, and enzyme activity could be quickly detected in the protein synthesis processes. the level of enzyme activity changed with the concentration of dna (fig. 5c) . the higher the concentration of dna, the higher the enzyme activity. in addition, the level of enzyme activity could be quickly confirmed by the color depth ( fig. 5b ). in summary, the use of cfps could not only express functional enzymes but also quickly and visually screen high-activity enzymes. the results provided a reliable basis for the cell-free preliminary screening platform. using living cells, molecular foundries for the biosynthesis of drugs, therapeutic proteins and other commodities required specialized equipment and refrigeration for their production and distribution. the traditional cell-free technology also has to store the main components, such as cell extracts and energy systems, below the freezing point in a bulky aqueous solution [39] . however, the delivery of these technologies to the field and low-resource areas face j o u r n a l p r e -p r o o f challenges. therefore, the portable freeze-dried cell-free (fd-cf) system has been developed [40, 41] . the fd-cf system provides the means for on-site, on-demand manufacturing of therapeutics, biomolecular biosensors, biocatalysts, and high-throughput protein synthesis [42, 43] . the flexible system is based on reaction pellets composed of freeze-dried cell-free transcription and translation machinery, which can be easily hydrated and utilized for biosynthesis through the addition of dna encoding the desired output (fig. 5d ). the preservation time, water loss rate, and activity changes of fd-cf components at different temperatures were investigated. the reaction components (table 1 ) prepared without the plasmid and water were needed to be stored at -80°c for 1 h. and then, they were freezedried into pellets by an fdu-1100 freeze dryer (eyela, tokyo, japan) and stored at -80°c. next, took samples every day and placed them at 4 °c or room temperature. finally, proteins could be synthesized by simply adding water and template dna at room temperature. the results showed that the water loss rate of the system was between 90% and 92% ( figure 5e ). using pet23a△rf-degfp as the dna template, the preservation conditions of the reaction mixture freeze-dried powders were tested in 25 days. it could preserve 20-30% activity for one week at room temperature and 5% activity after 25 days. more than 50% of the activity could be preserved after 2 weeks at 4 °c and up to 30% after 25 days (fig. 5f ). the results showed that our system could be stored for about one week at room temperature, 25 days or more at in order to better prove that our fd-cf system can become a portable therapeutic protein expression platform, the s1 region and the receptor-binding domain (rbd) of the sars-cov-2 surface spike (s) protein were expressed with the system. s protein is a structural protein of sars-cov-2, which plays the most important role in the virus attachment, fusion and entry. it is the target of antibodies, entry inhibitors and vaccine development [44, 45] . the s1 subunit of the s protein contains the rbd region, responsible for recognizing and binding to cell receptors. the studies on the synthesis of s1 protein or rbd protein could help the development of sars-cov-2 therapeutics [46] . therefore, we selected the s1 subunit and the rbd region in the s protein as the target protein to be expressed. the optimized freeze-dried cfps system was used to express both of them. as shown in the results, s1 protein and rbd could be successfully expressed (fig. s6) . the protein yield of s1 protein and rbd protein could reach about 21.7 μg/ml and 108.3 μg/ml, respectively. the results showed that our fd-cf system could quickly synthesize therapeutic proteins with only the addition of expression templates and water, which might solve significant practical limitations in the production and distribution of therapeutics and molecular tools, both to the developed and developing world. cfps has overcome the limitations of traditional cell-based synthesis and has become an emerging platform for in vitro protein synthesis due to its advantages of not limited by cell growth and allowing various formats. to improve the efficiency of cfps, the cell-free system was optimized in this study. by comparing five e. coli extracts, the bl21 star (de3) extract was more suitable for the expression of the model proteins (degfp) due to its genetic features. since the smaller the plasmid size, the less the burden on cfps, the smaller pet23a△rf was selected as the expression vector. besides, the effects of metal ions on the cfps were explored. when the metal ion was mg 2+ , and the concentration was 15mm, the protein expression j o u r n a l p r e -p r o o f synthetic biology: new engineering rules for an emerging discipline foundations for engineering biology the second wave of synthetic biology: from modules to systems reconstruction 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applications of cell-free biotechnology through enhanced shelf life and productivity of e. coli extracts the growing impact of lyophilized cellfree protein expression systems structural basis of receptor recognition by sars-cov-2 characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association figure 1. the cell-free protein expression by the plasmid with different sizes. (a) the schematics of two plasmids with different sizes supplementary material related to this article can be found, in the online version, at doi:https://doi.org/xxxx. key: cord-300625-fvirvpyl authors: srinivasan, suhas; cui, hongzhu; gao, ziyang; liu, ming; lu, senbao; mkandawire, winnie; narykov, oleksandr; sun, mo; korkin, dmitry title: structural genomics of sars-cov-2 indicates evolutionary conserved functional regions of viral proteins date: 2020-03-25 journal: viruses doi: 10.3390/v12040360 sha: doc_id: 300625 cord_uid: fvirvpyl during its first two and a half months, the recently emerged 2019 novel coronavirus, sars-cov-2, has already infected over one-hundred thousand people worldwide and has taken more than four thousand lives. however, the swiftly spreading virus also caused an unprecedentedly rapid response from the research community facing the unknown health challenge of potentially enormous proportions. unfortunately, the experimental research to understand the molecular mechanisms behind the viral infection and to design a vaccine or antivirals is costly and takes months to develop. to expedite the advancement of our knowledge, we leveraged data about the related coronaviruses that is readily available in public databases and integrated these data into a single computational pipeline. as a result, we provide comprehensive structural genomics and interactomics roadmaps of sars-cov-2 and use this information to infer the possible functional differences and similarities with the related sars coronavirus. all data are made publicly available to the research community. that of the 2003 severe acute respiratory syndrome coronavirus (sars-cov) and 2012 middle east respiratory syndrome coronavirus (mers-cov) outbreaks combined [1] [2] [3] . in spite of the instantaneous reaction by the scientific community and extensive worldwide efforts to address this health crisis, vaccines may be months and even years away [4, 5] . for instance, a phase i trial for a vaccine that treats sars was announced in december 2004, two years after the disease outbreak [6] . additionally, a vaccine against mers, another infectious outbreak of the related coronavirus that emerged in 2012, was patented in 2019, with phase i trials introduced in the same year [7, 8] . nevertheless, in the past two decades, a massive amount of work has been done to understand the molecular basis of the coronavirus evolution and infection, develop effective treatment in forms of both vaccines and antiviral drugs, and propose efficient measures for viral detection and prevention [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . structures of many individual proteins of sars-cov, mers-cov, and related coronaviruses, as well as their biological interactions with other viral and host proteins have been explored along with the experimental testing of the anti-viral properties of small-molecule inhibitors [16, [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] . however, experimental investigation of the same scale for sars-cov-2 may take the research community years to obtain. can it be facilitated? the answer lies in the use of the modern bioinformatics methods that can drastically streamline knowledge discovery by quickly providing important insights about possible molecular mechanisms behind infection, pinpointing likely protein targets for the anti-viral treatments, and predicting the efficacy of the existing antivirals developed for other coronaviruses. by leveraging previously known information on genome sequences as well as protein structures and functions, bioinformaticians have been successfully assisting virologists by structurally characterizing proteins of novel viruses, determining the evolutionary trajectories, identifying interactions with host proteins, and providing other important biological insights. in particular, a plethora of results has been achieved through comparative, or homology, modeling principles [32, 33] . in addition to the global structural genomics, an initiative that focuses on determining the 3d structures of individual proteins on a genome scale [34] , as well as to the specific efforts aimed at rapid structural characterization of proteins in emerging viruses [35] [36] [37] [38] , multiple works have used comparative modeling to predict the structures of protein-protein interaction complexes [39] [40] [41] , facilitate structure-based drug discovery [33, 42, 43] , infer protein functions [44] , determine the macromolecular interaction network [45] [46] [47] [48] , and provide molecular insights into the viral evolution [49] [50] [51] . here, using an integrated bioinformatics approach, we provide the first comprehensive structural genomics and interactomics analysis of the sars-cov-2. the structural information on the individual sars-cov-2 proteins and their interactions with each other and with human proteins allows us to accurately determine the putative functional sites. these functional sites, combined with an evolutionary sequence analysis of sars-cov-2 and the closely related human sars-cov and bat coronavirus proteomes, provide us with a structure-based perspective of the evolutionary diversity of sars-cov-2, and allow estimation of how similar the function of sars-cov-2 virus is when compared with sars-cov. consequently, we can forecast how likely the antibodies and candidate ligands that are efficient in inhibiting the sars-cov functions will be efficient in doing the same for sars-cov-2. the goal of this work is to identify the evolutionary differences between sars-cov-2 and the closest coronavirus species, human sars-cov and bat sars-like coronavirus, and to predict their possible functional implications. to do so, we structurally characterized individual proteins as well as intra-viral and human-virus protein complexes, extracted the information on their interaction interfaces and ligand binding, and superposed the evolutionary difference and conservation information with the binding information. specifically, our integrative computational pipeline included the following five steps. first, for each of the candidate sars-cov-2 proteins a set of sequentially similar coronavirus proteins was determined and aligned. second, structural models of sars-cov-2 proteins were obtained using template-based, or homology, modeling. third, the protein-protein interaction complex structures of sars-cov-2 proteins interacting with each other and/or with the human proteins were determined using a multi-chain comparative modeling protocol. fourth, the protein-binding sites were extracted from the obtained models of protein-protein interaction complexes, and protein-ligand binding sites were extracted from the evolutionarily close coronavirus protein-ligand templates and mapped to the relevant structural models of sars-cov-2 through structural alignment. fifth, the information on the evolutionary differences and conservations between the protein sequences of sars-cov-2 and the related coronaviruses were extracted from the above protein sequence alignments and mapped onto the structural models of the sars-cov-2 proteins to determine if the proteinand ligand-binding sites were functionally conserved. lastly, a joint human-virus and virus-virus interactome was constructed through homology and further analyzed. all the obtained models and sequence alignments were made publicly available to the research community. available sequences for protein candidates ws, worf3a, we, wm, worf6, worf7a, worf7b, worf8, wn, and worf10 were extracted from the ncbi virus repository [52] (collected on 29 january 2019) and then used in the sequence analysis and structural modeling (see supplementary materials containing sequence alignment files of each protein and the functional mappings of protein and ligand binding sites). the uniprot blast-based search was performed for each of the proteins using default parameters. from the results of each search, the final selection was done based on the pairwise sequence identity (>60%) as well as the evolutionary relationship (coronaviridae family). each of sars-cov-2 proteins was then aligned with the corresponding coronavirus proteins using a multiple sequence alignment method clustal omega (embl-ebi, cambridge, uk) [53] . the structure of each protein was determined using a single-template comparative modeling protocols with the modeller software (ucsf, ca, usa) [54] . first, the template for each protein sequence was identified using a psi-blast search in the protein data bank (pdb) (research collaboratory for structural bioinformatics) [55] . in general, a structural template with the highest sequence identity was selected out of those that covered at least 50 residues of the target sequence with at least 30% sequence identity. the polyprotein worf1ab was first split into 16 putative proteins based on its alignment with the human sars-cov polyprotein, with each protein then independently searched against pdb. in total, structural templates for 17 proteins were chosen ( table 1 , figures 1-3) . in some cases, several independent templates, each covering an individual protein domain of a target sars-cov-2 protein, were selected. the obtained template was used in the comparative modeling protocol, generating five models. each model was assessed using the dope statistical potential [56] ; the best-scoring model was selected as a final prediction. to model a protein-protein interaction complex, a multi-chain modeling protocol was used [39] . specifically, we aligned the corresponding pairs of homologous proteins and combined them into a single alignment where the individual chains were separated by "/" symbol. the alignment was used as an input together with the multi-chain structural template of a homologous complex. in the case of a viral-human interaction (these were the only virus-host structural templates found), the human protein remained the same in the alignment. in total, 18 structural templates of protein complexes involving homologs of 14 sars-cov-2 proteins were retrieved ( figure 3 ). similar to the single-protein protocol, five candidate models were generated for each complex conformation, and each model was assessed using the dope statistical potential, following by selection of the best-scoring model as a final prediction. we next extracted protein-and ligand-binding sites and mapped them onto the models of sars-cov-2 proteins. for protein binding sites, the obtained modeled structures of protein complexes that involved sars-cov-2 proteins were considered. for each sars-cov-2 protein in a protein-protein interaction complex, we identified all binding residues that constituted its protein-binding site. given an interaction between two proteins, a residue on one protein was defined as a protein binding site residue if there was at least one pair of atoms, one from this residue and another from a residue in the second protein, with van der waals (vdw) surfaces not farther than 1.0 å from each other. using this definition, the binding sites were identified with ucsf chimera (ucsf, ca, usa) [57] . the ligand binding sites were identified and mapped using a different protocol-we relied on the default definition of protein-ligand binding site residues from pdb 3d ligand view, since this standard was widely accepted. once all ligand binding site residues were identified for a protein from a related coronavirus, the residues were mapped onto the surface of the corresponding sars-cov-2 protein, guided by a structural alignment between the two proteins, which put residues from both proteins into a one-to-one correspondence. next, we leveraged the obtained protein homology information to predict and map all possible intra-viral and virus-host protein interactions at the systems level. since the sars-cov-2 genome exhibited substantial similarity to the 2002 sars-cov genome [58] and proteome [59] , we hypothesized that many of the interactions observed in the sars-cov proteome would be preserved in the sars-cov-2 proteome too, unless the corresponding binding sites were affected. the information in the interactome would help one understand the global mechanistic processes of the viral molecular machinery during the viral infection, survival within the host, and replication. with this knowledge, one could discern the protein interactions that were crucial for transmission and replication. these interactions could then be potential candidates for inhibitory drugs [60] . furthermore, using the network biology approach, one could identify hubs and bottlenecks, new to sars-cov-2, that could again be targeted by the antiviral drugs. for this purpose, we created a comprehensive integrated sars-cov interactome that consisted of both intra-viral and virus-host interactions. the sars-cov intra-viral interactome was created using the published data where the sars-cov orfeome was cloned, and a genome-wide analysis of viral protein interactions was performed through yeast-two-hybrid (y2h) matrix screens [61] . the y2h matrix screen was summarized by combining interactions in one direction, both directions and self-interactions. we also included intra-viral interactions gathered from the literature review [61] . the aggregated intra-viral interaction network consisted of 31 proteins and 86 unique interactions. we then constructed the sars-cov-host interactome through the published y2h interaction data [62] . we also included virus-host interactions mined from a literature survey of 5000 abstracts [62] . the curated virus-host interaction network consisted of 118 proteins, including 93 host proteins, and 114 unique virus-host interactions. next, to create an integrated network, the two individual networks were imported in cytoscape (cytoscape consortium) [63] and merged to form a unified interactome, representing both intra-viral and virus-host interactions. finally, we included our predictions from structural modeling of sars-cov-2 intra-viral and virus-host interactions, surveyed recent literature on predicted sars-cov-2 interactions and annotated them accordingly in the unified interactome. the unified interactome consisted of 125 proteins (94 host proteins) and 200 unique interactions. in addition, the networks were pruned for duplicate edges, and a network topology analysis was performed to compute a set of summary statistics (degree distribution, clustering coefficient, and other important characteristics) that characterized the three networks. finally, the 200 putative interactions were annotated based on the extent to which the protein binding sites of the sars-cov-2 proteins were altered, compared to their sars-cov homologs. based on the evolutionary conservation analysis of the putative protein binding sites extracted from the modeled complexes, some interactions were annotated as potentially disrupted. the recently sequenced genomes of sars-cov-2 strains combined with the comparative analysis of the sars-cov genome organization and transcription allowed us to construct a tentative list of gene products [64] . it was suggested that sars-cov-2 had 16 predicted non-structural proteins (referred to as wnsp1-wnsp16 here) constituting a polyprotein (worf1ab), followed by (at least) 13 downstream open reading frames (orfs): surface glycoprotein (or spike), orf3a, orf3b, envelope, membrane, orf6, orf7a, orf7b, orf8, nucleocapsid, orf9a, orf9b, and orf10, which we refer in this work to as ws, worf3a, worf3b, we, wm, worf6, worf7a, worf7b, worf8, wn, worf9a, worf9b, and worf10, respectively. the three viral species whose proteins shared the highest similarity were consistently the same: human sars coronavirus (sars-cov), bat coronavirus (btcov), as well as another bat betacoronavirus (btrf-betacov). searching against uniprot database (uniprot consortium) [65] resulted in matches for the polyprotein (worf1ab), all four structural proteins (ws, we, wm, and wn), and six orfs (worf3a, worf6, worf7a, worf7b, worf8, and worf10), also referred to as the accessory proteins. the closest protein matches from uniprot shared sequence identity with the related sars-cov-2 proteins as high as 91% (with worf1ab and wn) and as low as 57% (with worf8) (supplementary table s1 ). the majority of differences were single-residue substitutions spread across the protein sequence (see multiple sequence alignment files for all proteins in supplementary materials). perhaps the most profound differences lie in the sequences of the multi-domain protein wnsp3 and surface protein ws: our analysis revealed that, compared to related coronavirus proteins, the two proteins had large sequence inserts (multiple sequence alignments for both proteins can be found in supplementary materials, files worf1ab_matches.aln and ws_matches.aln). in particular, wnsp3 had a novel large (25-41 res., region 983-1023 of worf1ab, depending on the alignment method) insert between its two putative functional domains, which are homologous to the n-terminal domain and adenosine diphosphate ribose 1" phosphatase (adrp, recently renamed to macrodomain-1 or mac-1) of sars-cov [66, 67] (supplementary materials, file worf1ab_matches.aln). interestingly, the closest matching peptide was found in c-jun-amino-terminal kinase-interacting protein 4 (seq. identity is 46%) of labrus bergylta, a species of marine ray finned fish. being significantly more diverse than the other three structural proteins, ws was found to have 4 inserts (4-6 res.) that seemed unique to sars-cov-2 and two additional inserts shared with human sars-cov proteins (supplementary materials, file ws_matches.aln). the unusually low conservation of orf6, orf8, and surface proteins between sars-cov-2 and human sars-cov, bat coronavirus btcov, as well as another bat betacoronavirus, btrf-betacov, prompted us to perform an expanded search for orf8 homologs using the blatp tool of ncbi blast against a large non-redundant protein sequence repository (nr) [68] . our search resulted in three new homologs of orf8 not reported in uniprot that were originated from three different isolates of bat sars-like coronavirus: bat-sl-covzc45 (genbank id: mg772933, collected in 2017), bat-sl-covzxc21 (genbank id: mg772934, collected in 2015), and ratg13 (genbank id: mn996532, collected in 2013). the protein sequences from these isolates shared a striking similarity with worf8, unseen in other strains before: the sequence identities between each of these three homologs and worf8 ranged between 94% and 95%. further analysis showed that the proteomes of these isolates shared even higher sequence identity with the other proteins of sars-cov-2: from 88.4% to 100% for 2015 and 2017 isolates, and even higher, 97.4%-100% for the 2013 isolate (supplementary table s1 ). in spite of the significant similarity of the three isolates to sars-cov-2, important differences were observed. first, similar to other viruses, the 2017 and 2015 isolates did not have the four sequence inserts that were found in ws. second, neither of the two isolates had the large insert between the two domains of wnsp3 from worf1ab described above. on the contrary, the 2013 isolate had both, the four sequence inserts in its surface protein, matching those in ws, and the large insert in nsp3, although the sequence of the large insert is different from that one in wnsp3. next, as a result of a comprehensive comparative modeling effort, we were able to structurally characterize 17 individual proteins, including 13 non-structural proteins of worf1ab (wnsp1, wnsp3, wnsp4, wnsp5, wnsp7, wnsp8, wnsp9, wnsp10, wnsp12, wnsp14, wnsp15, wnsp16), three structural proteins (we, wn, and ws), as well as one orf (worf7a). for two proteins, wnsp3 and wn, multiple individual domains were modeled (figure 1, figure 2 ). the templates for the majority of the models were the homologous protein structures from other coronaviruses, with a high target-to-template sequence similarity (seq. ids: 75-96%, except for orf8). n-terminal and c-terminal domains of wn corresponded to n-terminal rna-binding domain and c-terminal dimerization domain of sars-cov, respectively. the modeled domains 1-6 of wnsp3 corresponded to (1) (figure 1) . a previously identified transmembrane domain of sars nsp3 was mapped to the sequence of wnsp3, but could not be modeled due to the lack of a template structure. the structural analysis of the modeled proteins combined with the sequence conservation analysis revealed several findings. first, we found that the mutated residues tended to locate on the protein's surface, supporting previous observations in other families of rna viruses that the core residues of viral proteins were more conserved than the surface residues [49, 69, 70] . furthermore, in a substantial number of proteins, distributions of mutated positions exhibited spatial patterns, with groups of mutations found to form clusters on the protein surfaces ( figure 2 ). these obtained models were then used as the reference structures to map and analyze the protein-binding and ligand-binding sites. next, using comparative modeling, we structurally characterized protein interaction complexes for both intra-viral interactions (homo-and hetero-oligomers) and host-viral interactions, where the host proteins were exclusively human. in total, we obtained structural models for 16 homo-oligomeric complexes, three hetero-oligomeric complexes, and eight human-virus interaction complexes including multiple conformations (figures 1 and 3) . the intra-viral hetero-oligomeric complexes included exclusively the interactions between the non-structural proteins (wnsp7, wnsp8, wnsp10, wnsp12, wnsp14, and wnsp16). the modeled host-viral interaction complexes included three types of interactions: non-structural protein wnsp3 (papain-like protease, plpro, domain) interacting with human ubiquitin-aldehyde, surface protein ws (in its trimeric form) interacting with human receptor angiotensin-converting enzyme 2 (ace2) in different conformations, as well as the same protein ws interacting with several neutralizing antibodies. based on the obtained models, the protein interaction binding sites were extracted and analyzed with respect to their evolutionary conservation. the analysis of the evolutionary conservation of protein binding sites revealed several patterns. first, we found that all protein binding sites of non-structural proteins involving in the intra-viral heteromeric complexes, wnsp7-wnsp8-wnsp12, wnsp10-wnsp14, and wnsp10-wnsp16, were either fully conserved or allowed at most one mutation on the periphery of the binding region, in spite of the fact that each protein had multiple mutations on its surface ( figure 4a , and file worf1ab_nsp7_nsp8_nsp10_nsp12_nsp14_nsp16_pbs_mapped.pdf in supplementary materials). furthermore, we observed the same behavior when analyzing the interaction between the papain-like protease plpro domain of wnsp3 and human ubiquitin-aldehyde ( figure 4b , and file worf1ab_wnsp3_domain5_human_pbs_mapped.pdf in supplementary materials): the only two mutated residues were located on the border of the binding region and thus were unlikely to disrupt the protein-protein interaction. protein ws presented a striking contrast to the majority of sars-cov-2 proteins due to its heavily mutated surface ( figure 5 , supplementary materials: ws_matches.aln and ws_human_pbs_mapped.pdf in supplementary materials) . first, the four novel sequence inserts and two inserts shared with the closest strains were expected to affect the protein's function. interestingly, three of the novel inserts were located in the first ntd domain, while the fourth one was located immediately before the s2 cleavage site and inside the homo-trimerization interaction interface. while the rbd domain of ws was not affected by those inserts, it was the most heavily mutated region of ws with potentially altering functional effects on the interactions with the human ace2 receptor and monoclonal antibodies mab 396 [71] and mab 80r [72] ( figure 4b ). the ntd domain had been considered a target of another antibody, ab g2, previously shown to work in mers-cov [73] . however, based on the structural superposition of the ntd domain, it seemed likely that the potential interaction would be disrupted by the novel sequence inserts to ws. finally, the analysis of seven ligand binding sites (lbs) for multiple candidate inhibitors previously identified, with their interactions structurally resolved, for four proteins in sars-cov and mers-cov, showed that many of the lbs were intact in the corresponding sars-cov-2 proteins, such as wn, wnsp3, wnsp5, and wnsp16 ( figure 4 and supplementary materials: wn_lbs_mapped.pdf, wnsp3-domain5_lbs_mapped.pdf, wnsp5-nonsars_lbs_mapped.pdf, wnsp5-sars_lbs1_mapped.pdf, wnsp5-sars_lbs2_mapped.pdf, wnsp14_lbs_mapped.pdf, and wnsp16_lbs_mapped.pdf). for wnsp14, the lbs for several inhibitors were mutated, while a co-localized binding site for another ligand was intact. after constructing the individual networks of intra-viral interactions and virus-host interactions, they were merged to form a unified network of sars-cov interactions, with the hypothesis that most interactions would be conserved in sars-cov-2 ( figure 6 ). the network analysis of all three networks showed that unifying the intra-viral and virus-host networks reduced the number of disconnected components (islands) in the sars-cov-host interactome ( table 2 ). the clustering coefficient and average node degree were both higher in the unified interactome compared to the virus-host interactome. table 2 . network parameters of the sars-cov intra-viral, virus-host and unified networks. the table shows topological statistics for the three networks. among the many computed statistics, the shown parameters include the number of nodes and edges in the networks, the average degree, number of components (independent networks), diameter (maximum shortest path), and clustering coefficient. the network topology of the unified interactome indicated the presence of several viral hubs for the structural, non-structural, and accessory proteins ( figure 6 ). the viral proteins exclusively formed all the hubs, with a few of specific interest. orf9b was one of the largest hubs in the network thought to play a secondary role only in intra-viral interactions and not necessary for replication [61] . however, a recent study showed that orf9b hindered immunity by targeting the mitochondria and limited the host cell responses [74] . nsp8 was the other major hub, which interacted with other replicase proteins, including two other hubs, nsp7 and nsp12, which together played a crucial role in replication [75] . another crucial non-structural protein was nsp1, also present in the sars-cov-2, which was known to be an inhibitor of the innate immune response. the host interaction partners of nsp1 modulated the calcineurin/nfat pathway that played an important role in immune cell activation [62] . overexpression of nsp1 was associated with immunopathogenecity and long-term cytokine dysregulation as observed in severe sars cases. additionally, inhibition of cyclophilins/immunopilins (host interaction partners) by cyclosporine a (cspa) blocked the replication of covs of all genera. there was an important interaction between the sars-cov spike protein (s) and the host angiotensin-converting enzyme 2 (ace2), as it was associated with cross-species and human-to-human transmissions. the same interaction was also inferred from structural modeling between sars-cov-2 ws and ace2. surprisingly, it was recently established that the interaction was preserved [76] , in spite of a number of mutations in the receptor binding site of ws. similar to sars-cov [77] , ws protein in sars-cov-2 was expected to interact with type ii transmembrane protease (tmprss2) [78] and was likely to be involved in the inhibition of antibody-mediated neutralization. thus, ws remained an important target for vaccines and drugs previously evaluated against sars and mers, while a neutralizing antibody targeting the ws protein could provide passive immunity [79] . in addition, there were seven interactions from sars-cov determined by structural characterization of the protein complexes that were predicted to be either conserved or potentially disrupted in sars-cov-2 (green edges in figure 6 ). an important target for vaccines was the surface (s) protein which was evaluated in sars-cov and mers-cov, with the idea that a neutralizing antibody targeting ws protein could provide passive immunity for sars-cov-2 [79] . we also structurally modeled interactions between the sars-cov-2 ws protein and three human monoclonal antibodies that were previously studied in sars-cov for immunotherapy and mapped the information about the evolutionary conserved and diverse surface residues. we found that two interactions of ws, with mab 396 [71] and mab 80r [72] , were likely to be disrupted due to the heavily mutated binding sites while another interaction, with ab g2 [73] , was likely to be disrupted due to a novel insert into the ws sequence. together, these findings may provide structure-informed guidelines in the search for potential antibody treatment. this work provides an initial large-scale structural genomics and interactomics effort towards understanding the structure, function, and evolution of the sars-cov-2 virus. the goal of this computational work is two-fold. first, by making the structural road map and the related findings fully available to the research community, we aim to facilitate the process of structure-guided research where accurate structural models of proteins and their interaction complexes already exist. second, by providing a comparative analysis between the new virus and its closest relatives from the perspective of protein-and ligand-binding, we hope to help experimental scientists in their deciphering of the molecular mechanisms implicated in infection by the new coronavirus as well as in vaccine development and antiviral drug discovery. through integrating the information on structure, function, and evolution in a comparative study of sars-cov-2 and the closely related coronaviruses, one can make several preliminary conclusions. first, the extended peptide sequence newly introduced to wnsp3 between two structurally and possibly functionally independent domains of this protein, might act as a long inter-domain linker, thus extending the conformational flexibility of this multi-domain protein. second, the presence of the four novel inserts and one highly variable region of the surface protein ws and the analysis of this large-scale sequence change with respect to intra-viral and viral-host interactions lead us to conclude that these inserts might have structural impact on the homo-trimeric form of the protein as well as impact the functions carried out by the ntd domain. third, the structurally modellable repertoire of the sars-cov-2 proteome also points to the interesting targets for structural biology and hybrid methods. for instance, the whole structures of the multi-domain proteins wn and wnsp3 could be resolved by integrating comparative models of the individual domains with the lower-resolution techniques that cover the entire protein, such as cryogenic electron microscopy (cryoem). the structure of wnsp3 is especially interesting because of the presence of the novel peptide introduced between the two structural domains of the protein. the evolutionary analysis of the protein-and ligand-binding sites mapped on the surfaces of sars-cov-2 may provide new insights into the virus functioning and its future treatment. the 100% or near 100% evolutionary conservation of the protein binding sites on the surfaces of non-structural proteins wnsp7, wnsp8, wnsp10, wnsp12, wnsp14, and wnsp16 that correspond to the 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treat the novel coronavirus originating in wuhan, china patterns of amino acid conservation in human and animal immunodeficiency viruses this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest.availability: all data generated in this work is freely available to the research community via korkin lab web-server (http://korkinlab.org/wuhan). key: cord-279586-likfvwwj authors: jin, jian; okagu, ogadimma; yagoub, abu elgasim ahmed; udenigwe, chibuike c. title: effects of sonication on the in vitro digestibility and structural properties of buckwheat protein isolates date: 2020-09-17 journal: ultrason sonochem doi: 10.1016/j.ultsonch.2020.105348 sha: doc_id: 279586 cord_uid: likfvwwj the present work investigated the effects of sonication at different amplitudes and durations on the in vitro digestibility of buckwheat protein isolates (bpis). the conformation, particle size and microstructures of the bpis were also studied to explicate the possible mechanisms of the sonication-induced changes. the results showed that sonication conditions of 20 khz, pulsed on-time 10 s, off-time 5s, amplitude of 60% and duration of 10 min (sa6t10) improved the digestibility of bpis from 41.4% (control) to 58.2%. the tertiary structure analysis showed that sonication exposed the hydrophobic core buried inside the protein molecules and broke the intramolecular crosslinks, based on the increase in the surface hydrophobicity and intrinsic fluorescence and the decrease in the disulphide content. the secondary structure analysis showed that sa6t10 decreased the content of β-turn and β-sheet by 40.9% and 22.4%, respectively, and increased the content of anti-parallel β-sheet, random coil, and α-helix by 40.9%, 30.6%, and 25.5%, respectively. the particle size of the control bpis (427.7±76.7 nm) increased to 2130.8±356.2 nm in the sa6t10 sonicated sample with a corresponding decrease in the polydispersity index from 0.97±0.04 to 0.51±0.13. moreover, scanning electron microscopy indicated that sonication broke the macroparticles into smaller fragments and changed the surface state of the proteins. taken together, sonication has proven to be a promising approach for improving the digestibility of buckwheat proteins, which can be explored as a source of plant-based alternative protein for food applications. buckwheat is mostly grown in some european countries and china. it can be categorized into two types, common buckwheat (fagopyrum esculentum moench) and tartary buckwheat (fagopyrum tataricum l. gaertn), based on differences in the composition and biological features. the total protein content of buckwheat ranges from 12-15% on a wet weight basis [1] . buckwheat protein is rich in lysine, which is the first limiting amino acid in cereal proteins, such as corn and wheat proteins [2] . the indispensable amino acid composition of buckwheat protein is comparable to the fao/who suggested requirements for children and adults. presently, studies on buckwheat proteins have focused on preparation of nutraceuticals with different beneficial effects on human health, such as cholesterol-lowering [3] [4] [5] , antioxidative [6, 7] , antihypertensive [8, 9] , antimicrobial and antitumor activities [10] [11] [12] . however, buckwheat proteins are underutilized in terms of human nutrition mostly because of their content of anti-nutritional factors, such as protease inhibitors, tannins, phytic acid and saponins, which decrease protein digestibility in the digestive tract [13] . furthermore, the molecular structure of proteins also affects their digestibility [14] ; for instance, corn protein contains numerous hydrophobic amino acid residues and α-helical domains that make it less digestible [15] . according to an fao report, over 690 million people globally were affected by hunger in 2019 and 132 million people may be added to the total number of undernourished in the world in 2020 because of covid-19 pandemic [16] . apart from lack of food, "hunger" encompasses food insecurity, reduced nutrient bioaccessibility and related health issues. meanwhile, the requirement for protein supply has increased due to the increasing world population and consumer preference for high-protein foods [17] . recently, the awareness of the food industry and consumers about plant-based proteins has increased because of their lower carbon footprint compared to animal proteins. however, one of the drawbacks of plant-based proteins is their lower digestibility and bioavailability, which can be improved by suitable processing. therefore, there is a need to discover alternative proteins and to develop new methods for improving the digestibility and nutritional quality of the proteins. power ultrasound, also known as high-intensity ultrasound, with intensity ranging from 10 to 1000 w·cm -2 and frequency of 10-100 khz, has extensively been used in the food industry. many studies reported that ultrasonic pretreatment of proteins before enzymolysis hydrolyzed the protein into smaller fragments and improved the reaction rate significantly [18] [19] [20] . this occurred because acoustic cavitation generated drastic physical forces (micro jets, shear forces, shock waves, turbulence, etc.) and highly reactive free radicals [21, 22] , which can attack the sidechains and backbone of protein molecules, leading to changes in their secondary and microstructures [23, 24] . these changes result in the exposure of hydrolysis sites buried inside a protein, making them accessible to proteases. acoustic frequency, power intensity, temperature of the medium, and duration of treatment have been extensively studied as the major parameters of high-intensity ultrasound [25] . however, the effect of the acoustic amplitude, square of which is directly proportional to the amount of energy applied to the sonication system [21] , on the protein structure and digestibility has not been investigated. theoretically, when a higher acoustic amplitude is used at fixed frequency of an ultrasonic system, the cavitation bubbles can grow and collapse more easily within a few acoustic cycles, and these collapsed bubbles can generate numbers of nuclei bubbles that finally intensify the cavitation effect. therefore, this study was aimed at investigating the effects of sonication duration and acoustic amplitude on the in vitro digestibility of buckwheat protein isolates (bpis). in addition, the effects of sonication on the tertiary structures (surface hydrophobicity, intrinsic fluorescence, sulfhydryl and disulfide bond contents), secondary structure, particle size, zeta-potential and microstructure of bpis were studied to elucidate the structural mechanism underlying the effect of ultrasound on the digestibility of the proteins. buckwheat flour was obtained from bulk barn (ottawa, ontario, canada where m is the mass of the medium (g), c p is the heat capacity of the medium (j·g -1 ·°c -1 ), and dt/dt is the slope of the temperature vs. time plot. the dissipated acoustic power was expressed as watts per unit area of the emitting surface (w·cm -2 ), and the values are presented in table 1 . the in vitro digestion of the control and sonicated bpis was conducted according to the cost consensus method [28] of cacl 2 (0.3 m) were added to the gastric digested mixture and the ph was adjusted to 7.0 using 1 m naoh. the mixture was incubated at 37°c for another 2 h with shaking at 120 rpm. the in vitro digestion was terminated by boiling the mixtures for 10 min, followed by centrifugation at 7 500 g for 15 min after cooling to room temperature. the in vitro digestibility (ivd) of the proteins was calculated using the following equation: where c is the concentration of peptides released (mg·ml -1 ) after deduction of a blank (buffer and digestive enzymes), which was determined using the lowry method [29] , v is the volume of digestion (ml), n is the dilution factor, and m is the mass of the protein (mg). the surface hydrophobicity (h 0 ) of the control and sonicated bpis was determined using ans as the fluorescence probe according to a previously reported method [30] . the total sulfhydryl (tsh) content of bpis was determined using the same protocol as rsh determination, with the exception that the buffer contained 8 m urea and 5 g·l -1 sodium dodecyl sulfate. the rsh and tsh contents were calculated according to the following equation: where d is the dilution factor (1.01), c is the diluted protein concentration determined by the lowry method (mg·ml -1 ), δabs 412 is the difference in absorbance measured at 412 nm with and without dtnb: the ss bond content of the control and sonicated bpis was determined according to the method developed by beveridge et al. [32] with some modification. bpis (20 mg the ss content was calculated using the following equation: (3) where is the content of tsh and sh derived from the reduction of ss, which was ' sh c determined using eq. (3), and is the total free thiol content which was calculated tsh c previously. the attenuated total reflectance fourier transform infrared spectra (atr/ft-ir) of the control and sonicated bpis were scanned at the wavenumber ranging from 400 to 4 000 cm -1 using a nicolet 6700 ftir (thermo fisher scientific, waltham, ma, usa) equipped with a 'itx smart orbit diamond' atr accessory. the surface of the crystal was cleaned with 99% ipa before the background was collected. after the background acquisition was completed, the sample was loaded onto the surface of the atr crystal and the pressing tip was pressed onto the sample to ensure good contact with the crystal surface. the spectra were collected using an omnic software (thermo fisher scientific cm -1 , β-turn: 1659 -1673 cm -1 ) as previously reported [24, [33] [34] [35] [36] . the content of secondary structure was reported as relative peak area to the total area of amide i. the control and sonicated bpis (2 mg) were dispersed in 2 ml of milli-q water. then, the particle size and zeta-potential of the suspensions were determined with a nano zeta-sizer (malvern instruments ltd, uk) after equilibrating for 120 s at 25 °c. the morphologies of the control and sonicated bpis were observed with a jsm-7500f field emission scanning electron microscope (jeol, japan) at an acceleration voltage of 2.0 kv, after coating the sample with a layer of gold with a thickness of 6.8 nm using an em ace 200 (leica, germany). all the data were reported as mean ± standard deviation (sd) of triplicate pretreatment experiments. statistical differences between the groups were analyzed by the one-way analysis of variance. significant differences between means were determined by tukey's test at p < 0.05. all analyses were performed with the statistical software spss 18.0 (ibm corporation, ny, usa) and the graphs were plotted with origin pro 8.5 (origin lab corporation, ma, usa). the results of the in vitro digestibility (ivd) of bpis are shown in table 2 acoustic amplitude is another parameter that influences the cavitation. generally, the intensity is proportional to the square of the amplitude of pressure waves [21] . as shown in similarly, the increase in ultrasound power was reported to decrease the degree of hydrolysis of oat protein isolates [37] . this observation may be because the smaller amplitude generated with lower intensity was dispersed in the protein solution and thus was not strong enough to disrupt the protein structure. however, the high intensity may have induced the formation of aggregates resulting in more compact protein particles that were less enzyme-digestible. bpis surface hydrophobicity, which correlates with the number of polar and non-polar groups on the surface of proteins, can reflect the change in molecular conformation of proteins. as shown in fig. 1(a) , higher amplitude and longer duration of sonication increased the surface hydrophobicity of bpis significantly (p < 0.05). this is possibly because acoustic amplitude is directly proportional to the amount of energy applied to the system. this increase in h 0 resulted from unfolding of the bpis and exposure of hydrophobic amino acid residues buried in the interior of the protein molecules, due to the disruptive effects of the micro jets, shear forces, shock waves, and turbulence generated by ultrasound waves. the unfolding of a protein may expose its cleavage sites, thus facilitating interactions with the enzymes and increasing digestibility. similar findings have been reported on studying sonicated plant proteins [23, [38] [39] [40] and animal proteins [27, 41] , resulting in improved enzymatic hydrolysis of proteins. some hydrophobic amino acid residues such as tryptophan, tyrosine and phenylalanine can emit fluorescence when they are exposed outside or located at the surface of proteins. this indicator can be used to characterize changes in tertiary structures of proteins because these residues form the hydrophobic core of proteins where they interact by hydrophobic bonding. as shown in fig. 1(b) , sonication increased the fluorescence intensity of the bpis. at 60% amplitude, the fluorescence intensity decreased due to the increase in the ultrasound treatment duration, except for sa6t10. this result is because the exposed hydrophobic groups aggregated under the turbulence effect of sound waves. on the other hand, the fluorescence intensity of sonicated bpis increased with increase in ultrasound amplitude, which might be ascribed to protein unfolding resulting from the higher intensity. however, extremely high intensity may have led to refolding or aggregation of the proteins, as observed in the decreased fluorescence intensity of sa10t10. taken together, the increase in fluorescence intensity of bpis may be because sonication destroyed the hydrophobic interactions, hydrogen bonds and van der waals forces between the protein molecules [23] . reactive sulfhydryl (rsh) groups, which are mostly located on the surface of protein molecules, can be involved in the formation of noncovalent bonds, such as hydrogen bonds. total sulfhydryl (tsh) is the sh group that exists in the free form rather than as ss bond, including tsh and sh groups buried inside the protein molecules. depending on the acoustic amplitude applied (fig. 2) , some sonication intensities increased both rsh and tsh contents significantly (p < 0.05). compared to the control, sonication at sa6t10 increased content of rsh from 13.86 to 22.05 μmol/g (by 59%), while sonication at sa4t10 increased the content of tsh from 69 to 80.67 μmol/g (by 16.9%). however, sonication at sa10t10 decreased the rsh content (p < 0.05) with no significant differences in tsh (p > 0.05). under the denaturation conditions, the content of tsh increased as a result of unfolding of the protein molecules. the increases in the content of rsh and tsh observed with the treatment may be ascribed to that sonication exposed the sh buried inside protein molecules and the ultrasound broke the disulfide bonds because of the physical and chemical effects of cavitation. the disulfide bond (ss) is one of the major forces that stabilize the structures of proteins. as shown in fig. 2 [42] . generally, the increase in sh content and the decrease in ss content is one of the mechanisms by which sonication improves the digestibility or enzymolysis of proteins [23, 40] . peaks located in the amide i region of atr/ft-ir are widely used to determine the secondary structure of proteins. after deconvolution, extraction of the overlapping components (see supplementary information, fig. s1 ) and assignments of attribution, the secondary structure contents of bpis were determined. the β-sheet, random coil, α-helix, and β-turn contents of the native buckwheat protein isolate ( table 3) were close to the values reported by choi and ma [33] for globulin from common buckwheat, i.e., 34.5% β-sheet, 14.4% random coil, 16.0% α-helix and 20.0% β-turn. notably, the increase in the in vitro digestibility of bpis by sa6t10, sa6t15, sa6t20 and sa6t30 ( table 2 ) might be because these treatments decreased the content of β-turn, which has a compact structure, compared to the control. moreover, these treatments, except sa6t15, increased the content of the anti-parallel β-sheet and, except sa6t10, increased the content of the β-sheet. however, only sa6t10 decreased the content of the β-sheet and increased the content of the random coil substantially; this treatment also had the most pronounced effect on ivd. jin et al. [23] attributed the improvement in enzymolysis of corn gluten meal to the increase in the β-sheet, but the study did not structurally differentiate the anti-parallel β-sheet from the β-sheet structure. in the present study, the total content of anti-parallel β-sheet and β-sheet of bpis treated by sa6t10 (45.6%) was close to that of the control (46.6%). interestingly, all the secondary structure contents of the sa6t15-treated sample decreased and this might be attributed to the increase in the relative peak area of side-chain vibration (1608 cm -1 ) ( table s1 ). the random-coil, found in the control, was not detected in the sa8t10-treated sample, but the contents of other secondary structures increased compared to the control. this is likely because sonication made the protein molecules arrange more regularly or because of the shift of bands. the increase in content of the ordered structure might have occurred during the renaturation of the proteins. in general, sonication can increase the digestibility of proteins because of changes in the content of secondary structures resulting from cavitation-induced physical forces and free radicals [21] . 3.5. effects of sonication on particle size, polydispersity index and zeta potential of bpis the particle sizes of the control and sonicated bpis, which were presented as z-average values, and polydispersity index (pdi) are shown in table 2 . the particle size of bpis was increased significantly by sonication, with the maximum 5-fold increase observed when the protein was sonicated at 100% amplitude for 10 min (sa10t10). some studies reported that ultrasound reduced the particle size of some animal and plant proteins [27, 43] . o'sullivan et al. [43] found that sonication of rice protein isolates by using a frequency of 20 khz and power intensity of ~34 w·cm -2 for 2 min slightly increased the particle size by 2.3%. jiang et al. [38] also reported that sonication increased the particle size of black bean protein isolates owing to protein aggregation. the large increase in particle size of bpis might be due to the disruption of the protein microstructure by the ultrasound, leading to protein particle swelling after dispersion in water or induction of protein self-assembly due to hydrophobic interaction between the unfolded regions. pdi represents the disperse homogeneity of particles, with higher values indicating more heterogeneous particles in the system. as shown in table 2 , the pdi of the control bpis indicated that the dispersed protein particles had a wide range of molecular weight distribution. sonication mostly decreased the pdi, suggesting that the dispersion of proteins occurred in a narrower molecular weight distribution, possibly ascribed to the formation of aggregates or to swelling of protein particles. similarly, stefanović et al. [44] reported that an increase in the sonication duration from 5 min to 20 min, decreased the pdi and increased the particle size of egg white proteins; the reduced pdi was attributed to the random aggregation induced by excessive protein denaturation. jiang et al. [38] indicated that the particle size of sonicated black bean protein isolates became larger because of the formation of soluble and unstable aggregates, and that the particle size distribution broadened due to the breakdown of the unstable aggregates. the surface of proteins shows a net charge resulting from the amino acid groups exposed to the exterior. typically, higher zeta potential means stronger electrostatic repulsion between protein molecules, and vice versa; thus, it plays a role in stabilizing the protein dispersion. in table 2 , the control sample had the highest zeta potential magnitude, which was decreased significantly (p < 0.05) by sonication, except for sa4t10. moreover, longer sonication duration and higher amplitude resulted in the most pronounced decrease in zeta potential magnitude because of higher energy input into the system. the zeta potential values indicated that the sonication-induced structural changes ( table 3) and protein aggregation, based on the increased particle size ( table 2) , may have reduced the number of negatively charged residues on the protein surface. in contrast, jiang et al. [38] reported that sonication increased the zeta potential of black bean protein isolates, resulting in the strengthening of the inter-particle electrostatic repulsion, thus disrupting protein aggregation and improving the dispersion stability of the proteins. the microstructures of the control and sonicated bpis are shown in fig. 3 . the thin lamellar structure is the typical feature of bpis; however, some blocks were present in fig. 3 (a)-(c). fig. 3 (a) showed that the structure of the control bpi was compact. the sa4t10-treated sample revealed a loose structure having a larger size (fig. 3 (b) ), which might be attributed to the destruction of the bonds between the protein subunits by the ultrasound waves, resulting in the extension of the distance between particles. in fig. 3 (c), the sa6t10 sample showed both blocks (marked with a circle) and multi-layer structure (marked with an arrow), which possibly resulted from the aggregation of the proteins. high intensity sonication can break the protein particles into smaller fragments, as observed in fig. 3 (c)-(g) . the quantity and size of the fragments correlated positively with the input energy of ultrasound waves to the protein solutions. the formation of fragments might be a result of the destruction of the crosslinks between the polypeptide chains of proteins, including the ss bonds, hydrogen bond and van der waals interactions, due to effects of shear forces, micro streaming, shock waves and free radicals generated by the ultrasound radiation [21, 23] . nonetheless, the fragmentation did not result in the reduction of particle size of the proteins ( table 2) . therefore, we postulated that the proteins might absorb and hold water in their inner structures when dispersed in the aqueous solution, thus increasing the particle size. surface morphology, including roughness, has substantial influence on the bio-adsorption [45] and mass transfer. as previously mentioned, the surface of the control bpi was tight ( fig. 4 (a) ), which may have protected the proteins from adsorption of water, thus the protein particles had smaller sizes when dispersed in water ( table 2) . sonicated samples (fig. 4(b)-(g) ), especially sa8t10 ( fig. 4(f) ), presented a rough surface of proteins with many micropores, which can allow water to enter into the proteins, thus increasing the degree of swelling and leading to larger particle sizes. furthermore, the adsorption between the proteins and enzymes can be enhanced because of the larger contact areas of the sonicated bpis. longer sonication duration led to rougher protein surfaces because of the extension of cavitation. on the other hand, the surface of sa10t10 sample ( fig. 4(g) ) appeared smoother than that of sa8t10 ( fig. 4(f) ), which might be because the surface protruding edges were excised by the high intensity ultrasound. a previous report had demonstrated the flattened surface of glutelin extracted from the sonicated corn gluten meal [24] . in general, the surface of the sa8t10-treated sample was substantially different from those of the other treated samples, which might be related to the lack of the random coil in the sa8t10 sample ( table 3) . our previous research on sonication of corn gluten meal indicated that sonication decreased the height and surface roughness of the proteins [24] , suggesting differences that may originate from the nature of the materials. treatment of buckwheat protein by sonication improved the in vitro digestibility of the proteins significantly. specifically, sonication under 20 khz, pulsed on-time 10 s, off-time 5s, amplitude of 60% and duration of 10 min (sa6t10) improved the digestibility significantly by 41% compared to the control. the structural analysis showed that sonication changed the tertiary structure by increasing the surface hydrophobicity, intrinsic fluorescence intensity, and total sulfhydryl contents. the secondary structure analysis showed that sa6t10 decreased the content of the β-turn and increased the content of the anti-parallel β-sheet, and the random coil. furthermore, sonication changed the particle size, dispersion characteristics, zeta-potential, and microstructures of the proteins. in conclusion, sonication can be further explored as an effective method to improving the digestibility of buckwheat proteins towards their utilization in food product development. sa6t5, sonication at 60% amplitude for 5 min; sa6t10, sonication at 60% amplitude for 10 min; sa6t15, sonication at 60% amplitude for 15 min; sa6t20, sonication at 60% amplitude for 20 min; sa6t30, sonication at 60% amplitude for 30 min; sa4t10, sonication at 40% amplitude for 10 min; sa8t10, sonication at 80% amplitude for 10 min; sa10t10, sonication at 100% amplitude for 10 min. different superscript letters on the bars represent significant differences (n = 6, p < 0.05). sa6t5, sonication at 60% amplitude for 5 min; sa6t10, sonication at 60% amplitude for 10 min; sa6t15, sonication at 60% amplitude for 15 min; sa6t20, sonication at 60% amplitude for 20 min; sa6t30, sonication at 60% amplitude for 30 min; sa4t10, sonication at 40% amplitude for 10 min; sa8t10, sonication at 80% amplitude for 10 min; sa10t10, sonication at 100% amplitude for 10 min. different superscript letters on the bars represent significant differences (n = 9, p < 0.05). ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: properties of protein concentrates and hydrolysates from amaranthus and buckwheat a buckwheat protein product suppresses gallstone formation and plasma cholesterol more strongly than soy protein isolate in hamsters preparation of tartary buckwheat protein product and its improving effect on cholesterol metabolism in rats and mice 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sh-and ss-groups in some food proteins using ellman's reagent conformational study of globulin from common buckwheat (fagopyrum esculentum moench) by fourier transform infrared spectroscopy and differential scanning calorimetry conformational study of globulin from rice (oryza sativa) seeds by fourier-transform infrared spectroscopy atr-ft/ir study on the interactions between gliadins and dextrin and their effects on protein secondary structure examination of the secondary structure of proteins by deconvolved ftir spectra ultrasonic treatment effect on enzymolysis kinetics and activities of ace-inhibitory peptides from oat-isolated protein effects of ultrasound on the structure and physical properties of black bean protein isolates effects and mechanism of ultrasound pretreatment on rapeseed protein enzymolysis effects of ultrasound and ultrasound assisted alkaline pretreatments on the enzymolysis and structural characteristics of rice protein structural and functional changes in ultrasonicated bovine serum albumin solutions situ monitoring of the effect of ultrasound on the sulfhydryl groups and disulfide bonds of wheat gluten the effect of ultrasound treatment on the structural, physical and emulsifying properties of animal and vegetable proteins effect of the controlled high-intensity ultrasound on improving functionality and structural changes of egg white proteins, food and bioprocess technology sensitivity of surface roughness parameters to changes in the density of scanning points in multi-scale afm studies. application to a biomaterial surface sonication at 60% amplitude for 5 min; sa6t10, sonication at 60% amplitude for 10 min sa6t15, sonication at 60% amplitude for 15 min; sa6t20, sonication at 60% amplitude for 20 min; sa6t30, sonication at 60% amplitude for 30 min; sa4t10, sonication at 40% amplitude for 10 min sa8t10, sonication at 80% amplitude for 10 min; sa10t10, sonication at 100% amplitude for 10 min the authors declare that there are no conflicts of interest.  buckwheat protein isolates (bpis) were treated by varied sonication time and amplitude.  sonication increased the in vitro digestibility of bpis significantly compared to control.  sonication changed the molecular conformations (tertiary and secondary) of bpis.  sonication broke bpis into smaller fragments and induced surface modifications. key: cord-285180-32bxx94u authors: lee, sunhee; lee, changhee title: functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus date: 2015-10-02 journal: virus res doi: 10.1016/j.virusres.2015.06.013 sha: doc_id: 285180 cord_uid: 32bxx94u porcine deltacoronavirus (pdcov) is a newly discovered enterotropic swine coronavirus that causes enteritis and diarrhea in piglets. like other coronaviruses, pdcov commonly contains 4 major structural proteins: spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. among these, the n protein is known to be the most abundant and multifunctional viral component. therefore, as the first step toward understanding the biology of pdcov, the present study investigated functional characteristics and expression dynamics of host proteins in a stable porcine cell line constitutively expressing the pdcov n protein. similar to n proteins of other coronaviruses, the pdcov n protein was found to interact with itself to form non-covalently linked oligomers and was mainly localized to the nucleolus. we then assessed alterations in production levels of proteins in the n-expressing pk (pk-pdcov-n) cells at different time points by means of proteomic analysis. according to the results of high-resolution two-dimensional gel electrophoresis, a total of 43 protein spots were initially found to be differentially expressed in pk-pdcov-n cells in comparison with control pk cells. of these spots, 10 protein spots showed a statistically significant alteration, including 8 up-regulated and 2 down-regulated protein spots and were picked for subsequent protein identification by peptide mass fingerprinting following matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. the affected cellular proteins that we identified in this study were classified into the functional groups involved in various cellular processes such as cell division, metabolism, the stress response, protein biosynthesis and transport, cytoskeleton networks and cell communication. notably, two members of the heat shock protein 70 family were found to be up-regulated in pk-pdcov-n cells. these proteomic data will provide insights into the specific cellular response to the n protein during pdcov infection. porcine deltacoronavirus (pdcov) is an emerging viral pathogen that was first reported in 2012 in hong kong, china (woo et al., 2012) . in february 2014, the detection of pdcov was first announced in ohio, united states, in conjunction with outbreaks of diarrhea without other etiologic agents. since its emergence, this novel coronavirus has been detected in 17 us states, and almost 80% of the tested samples corresponded to cases of coinfection of pdcov with other enteric viral pathogens such as a rotavirus or porcine epidemic diarrhea virus (li et al., 2014; utr. orf1a/b encompasses two-thirds of the genome encoding 2 overlapping viral replicase polyproteins, 1a and 1ab, which are then proteolytically processed into mature nonstructural proteins. as in other coronaviruses, production of polyproteins 1a and 1ab requires a −1 ribosomal frameshift during translation of the genomic rna. the last third of the genome encodes the 4 structural proteins, spike (s), envelope (e), membrane (m), and nucleocapsid (n), as well as two accessory genes, nonstructural gene 6 (ns6) and ns7 gene, between m and n, and within n, respectively (lai et al., 2007; lee and lee, 2014; li et al., 2014; marthaler et al., 2014a; woo et al., 2012) . among the structural proteins of coronaviruses, the n protein is abundantly produced in infected cells and has multiple functions in viral replication and pathogenesis (mcbride et al., 2014) . as the sole structural component of the viral capsid, the n protein of coronaviruses interacts with the nucleic acid and itself for selfassociation to protect the viral genome from extracellular agents, serving as the critical basis for ribonucleoprotein (rnp) complexes during virus assembly (mcbride et al., 2014) . the entire life cycle of coronaviruses takes place in the cytoplasm of infected cells, and accordingly, the n protein is distributed mainly in the cytoplasmic compartments. in addition to their cytoplasmic localization, coronaviral n proteins are commonly localized to the nucleolus, suggesting their non-structural functions in ensuring successful virus infection mcbride et al., 2014) . although a variety of studies have confirmed that n possesses multifunctional significance in coronavirology (mcbride et al., 2014) , detailed characteristics of this protein and its role in the replication of pdcov remain unknown. in the present study, alterations in cellular gene expression that are caused by the n protein were evaluated as a first step toward understanding the biological role of the n protein in pdcov replication. to accomplish this task, stable porcine-origin cell lines constitutively expressing the pdcov n protein were generated and characterized in this study. changes in expression patterns of various cellular proteins in the n protein-expressing porcine cells in comparison with control cells were examined by proteomic analysis at different time points. our proteomic data are expected to provide novel information for better knowledge of the properties and functions of the n protein during pdcov infection. hek-293t cells (crl-1573) were purchased from the american type culture collection (atcc, manassas, va) and cultured in dulbecco's modified eagle medium (dmem) with high glucose (invitrogen, carlsbad, ca) with 10% fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solutions (100×; invitrogen). pk-15 cells were grown in rpmi 1640 medium (invitrogen) supplemented with 10% fbs and antibiotic-antimycotic solutions (100×). the cells were maintained at 37 • c in an atmosphere of humidified air containing 5% co 2 . pam-pcd163-n cells that stably express the n protein of porcine reproductive and respiratory syndrome virus (prrsv; sagong and lee, 2010) were cultured in rpmi 1640 medium (invitrogen) supplemented with 10% fbs, antibiotic-antimycotic solutions (100×), 10 mm hepes (invitrogen), 1 mm sodium pyruvate (invitrogen), and nonessential amino acids (100×; invitrogen) in the presence of 50 g/ml zeocin (invitrogen) and 200 g/ml g418 (invitrogen). the anti-prrsv nucleocapsid (n) monoclonal antibody (mab) and anti-myc mab were purchased from median diagnostics (chuncheon, south korea) and invitrogen, respectively. antibodies to the 6× histidine tag, glucose-regulated protein 78 (grp78), heat shock cognate 70-kda protein (hsc70), ␤-actin, ␣-tubulin, and sp1 were purchased from santa cruz biotechnology (santa cruz, ca). dna manipulation and cloning were performed according to standard procedures (sambrook and russell, 2001) . the escherichia coli strain dh5␣ (rbc bioscience, taiwan) was used as the host for general cloning. the full-length n gene was amplified from the pdcov knu14-04 strain (lee and lee, 2014) with the following primer pair: knu14-04-n-fwd (5 -gccggtcgacatggctgcaccagtag-3 ) and knu14-04-n-rev (5 -cggctctagacgctgctgattcctgc-3 ), where underlines indicate the sali and xbai restriction enzyme sites, respectively. the pcr amplicon was initially inserted into the pbudce4.1 vector (invitrogen) that contains a myc epitope and 6 repetitive histidine codons, and the resulting plasmid pbud-pdcov-n was verified by nucleotide sequencing. because the pdcov genome contains one accessory ns7 gene within the n gene in a different reading frame, the presence of ns7 could affect our functional studies of the pdcov n protein. to prevent any such side effects, the translation initiation codon and the fifth codon of the ns7 orf were modified to disrupt its expression by pbud-pdcov-n. to accomplish this, overlapping pcr was conducted to simultaneously change the atg start codon and the fifth codon of the ns7 gene to tta and taa at genomic nucleotide positions 24,096-24,098 and 24,108-24,110, respectively, using pbud-pdcov-n as a template with the following primers for the t24097c/t24109a mutation: ns7-ko-fwd (5 -ggcaacggagttccgctaaactccgccatc-3 ) and ns7-ko-rev (5 -gatggcggagtttagcggaactccgttgcc-3 ), where lowercase letters indicate the mutated nucleotides. both mutations were translationally silent with respect to the orf encoding the n protein, and the resulting plasmid pbud-pdcov-n w/ons7 was verified by nucleotide sequencing. a fragment of pdcov n w/ons7 cdna that was prepared from pbud-pdcov-n was then subcloned into the pfb-neo retroviral vector (stratagene, la jolla, ca) using the sali and ecori restriction sites to construct the pdcov n gene expression plasmid pfb-neo-pdcov-n-myc/his that produces recombinant n protein. the retrovirus gene transfer system (stratagene) was used to generate cell lines constitutively expressing the recombinant pdcov n gene or an empty vector only as described elsewhere nam and lee, 2010; oh and lee, 2012) . antibioticresistant continuous cell clones were analyzed by rt-pcr to determine the presence of the full-length n gene, and the positive clones (pk-pdcov-n and pk-neo) were amplified for subsequent experiments. pk-pdcov-n cells were grown on microscope coverslips placed in 6-well tissue culture plates. at 48 h post-seeding, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature (rt) and permeabilized with 0.2% triton x-100 in pbs at rt for 10 min. the cells were blocked using 1% bovine serum albumin (bsa) in pbs for 30 min at rt and then incubated with an anti-his tag or anti-myc antibody for 2 h. after washing 5 times in pbs, the cells were incubated for 1 h at rt with a goat anti-mouse secondary antibody conjugated with alexa fluor 488 (molecular probes, carlsbad, ca). the cells were finally counterstained with 4 ,6-diamidino-2phenylindole (dapi; sigma, st. louis, mo), and the cell staining was visualized by the fluorescence leica dm il led microscope (leica, wetzlar, germany) or a confocal laser scanning microscope (carl zeiss, gattingen, germany). the n expression in pk-pdcov-n cells was analyzed by flow cytometry. the cells were trypsinized at 48 h post-seeding and centrifuged at 250 × g (hanil centrifuge fleta 5) for 5 min. the cell pellet was washed with cold washing buffer (1% bsa and 0.1% sodium azide in pbs), and 10 6 cells were resuspended in 1% formaldehyde solution in cold wash buffer for fixation at 4 • c in the dark for 30 min followed by centrifugation and incubation of the pellet in 0.2% triton x-100 in pbs at 37 • c for 15 min for permeabilization. after centrifugation, the cell pellet was resuspended in a solution of the primary anti-his tag antibody or normal mouse igg1 (santa cruz biotechnology) and the mixture was incubated at 4 • c for 30 min. the cells were washed and allowed to react with an alexa fluor 488-conjugated anti-mouse igg secondary antibody at 4 • c for 30 min in the dark. the stained cells were washed again and analyzed on the bd facsaria iii flow cytometer (bd biosciences, belford, ma). the growth properties of cells expressing pdcov n protein and control cells were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay (sigma) detecting cell viability as described previously (kim and lee, 2013) . all mtt assays were performed in triplicate. whole cell lysates were prepared from pk-pdcov-n cells grown at 5 × 10 5 cells/well in 6-well tissue culture plates at indicated time points using lysis buffer as described previously . for cell fractionation, pk-pdcov-n cells were fractionated using the nuclear/cytosol fractionation kit (biovision, mountain view, ca) according to the manufacturer's manuals. the protein concentrations of the cell lysates were determined using the bca protein assay (pierce, rockford, il). the cell lysates were mixed with 4× nupage sample buffer (invitrogen) and boiled at 70 • c for 10 min. equal amounts of total protein were separated in a nupage 4-12% gradient bis-tris gel (invitrogen) under non-reducing or reducing conditions, and electrotransferred onto an immobilon-p membrane (millipore, billerica, ma). the membranes were blocked with 3% powdered skim milk (bd biosciences) in tbs (10 mm tris-hcl [ph 8.0], 150 mm nacl) with 0.05% tween-20 (tbst) at 4 • c for 2 h and reacted with the primary antibody against the 6× his-tag, grp78, hsc70, or ␤-actin at 4 • c overnight. the blots were then incubated with a horseradish peroxidase (hrp)-labeled goat anti-mouse igg or goat anti-rabbit igg antibody (santa cruz biotechnology) at the dilution of 1:2000 for 2 h at 4 • c. finally, the proteins were visualized by enhanced chemiluminescence (ecl) reagents (amersham biosciences, piscataway, nj) according to the instructions of the manufacturer. to analyze the expression kinetics of cellular proteins in pk-pdcov-n cells, the band density of each protein was quantified relative to ␤-actin using densitometry with the wright cell imaging facility (wcif) version of the imagej software package (http://www. uhnresearch.ca/facilities/wcif/imagej/). a chemical cross-linking assay was performed as described previously . briefly, pk-pdcov-n cells and pam-pcd163-n cells were grown in a 6-well tissue culture plate for 48 h. for cross-linking studies, the cells were washed twice with cold pbs and then incubated at rt for 30 min with a 2 mm solution of a membrane permeable and thiol-cleavable cross-linker, 3,3dithiobis(succinimidyl propionate) (dsp; pierce), dissolved in 10% dimethyl sulfoxide (v/v in pbs). the reaction was quenched with 50 mm tris-hcl [ph 7.5] and incubated for an additional 15 min. the cells were lysed with lysis buffer, and the resultant cell lysates were then subjected to a western blot assay as described above. pellets of cultured cells were washed twice with ice-cold pbs, placed in sample lysis solution consisting of 7 m urea, 2 m thiourea, 4% 3-[(3-cholamidopropyl) dimethyammonio]-1propanesulfonate (chaps), 1% dithiothreitol (dtt), 2% pharmalyte (ph 3.5-10, amersham biosciences), and 1 mm benzamidine, and were then sonicated for 10 s using a sonoplus device (bandelin electronic, germany). the samples were subsequently incubated for 1 h at 4 • c. after centrifugation at 15,000 × g for 1 h at 4 • c, insoluble cellular debris were discarded, and the supernatant was collected and stored at −80 • c until use. the protein concentrations were measured by the bradford assay as described previously (bradford, 1976) . protein samples were analyzed by 2de using commercial ipg dry strips (ph 4-10, 24 cm; genomine inc., pohang, south korea) for the first-dimensional separation (isoelectric focusing; ief) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) for the second dimension. the ipg dry strips were loaded independently with 200 g of each protein sample and rehydrated overnight in rehydration buffer (7 m urea, 2 m thiourea, 2% chaps, 1% dtt, and 1% pharmalyte). ief was performed at 20 • c using a multiphor ii electrophoresis unit and an eps 3500 xl power supply (amersham biosciences) with the following parameters: gradient increase from 150 to 3500 v for 3 h and 3500 v for a total of 96,000 v h. following ief separation, the ipg strips were incubated for 10 min at rt with gentle shaking in equilibration buffer (50 mm tris-cl ph 6.8, containing 6 m urea, 2% sds, and 30% glycerol) containing 1% dtt and were subsequently incubated in the equilibration buffer containing 2.5% iodoacetamide under the same conditions. each equilibrated strip was then inserted into 10-16% polyacrylamide gels (20 cm × 24 cm) and sds-page was run using a hoefer dalt 2d system (amersham biosciences) at 20 • c for 1700 v h according to the manufacturer's instructions. the 2d gels were stained by the silver staining method as described previously with some modifications (oakley et al., 1980) . to compensate for the variability of 2de, three independent experiments were conducted for further statistical analysis. the stained gels were imaged using a high-resolution 2d gel ccd image analyzer, dyversity (syngene, frederick, md), and quantitative analysis of the digitized gel images was carried out using the pdquest software (version 7.0, bio-rad, hercules, ca) according to the protocols provided by the manufacturer. data representing 3 independent 2de experiments for each sample were statistically analyzed using student's t-test, and p-values of less than 0.05 were considered to be statistically significant. the quantitative data from each protein spot were normalized based on the total valid spot intensity for each gel. only protein spots that showed significant differential expression (p < 0.05) with a ±2-fold consistent change in the expression level in comparison with the control sample were selected for mass spectrometry (ms) analysis. each selected protein spot was excised manually from the silverstained gels and was enzymatically digested in-gel as previously described (shevchenko et al., 1996) and using modified porcine trypsin (sequencing grade; promega, madison, wi). the gel pieces were washed with 50% acetonitrile (acn), air-dried thoroughly at rt, and then rehydrated for 8-10 h at 37 • c with modified sequencing grade porcine trypsin (8-10 ng/l). the proteolytic reaction was terminated by addition of 5 l of 0.5% trifluoroacetic acid (tfa). the tryptic peptides were recovered by combining the aqueous phase from several extractions of gel pieces with 50% aqueous acn. after concentration, the peptide mixture was desalted using c 18 ziptips (millipore), and the peptides were eluted in 1-5 l of 50% acn. an aliquot of this solution was mixed with an equal volume of a saturated solution of ␣-cyano-4-hydroxycinnamic acid (chca; sigma) in 50% acn/0.1% tfa, and 1 l of this mixture was immediately spotted onto the target plate. maldi-tof analysis was performed on a microflex lrf 20 instrument (bruker daltonics, billerica, ma) as described by fernandez et al. (1998) . the spectra were collected from 300 shots per spectrum over the m/z range 600-3000 and calibrated by two point internal calibration using trypsin auto-digestion peaks (m/z 842.5099, 2211.1046). the peak list was generated using flex analysis 3.0. we used the following threshold for peak-picking: 500 for minimum resolution of monoisotopic mass, 5 for s/n. the search program mascot from matrix science (http://www.matrixscience.com/) was used for protein identification by pmf. the following parameters were used for the database search: trypsin as the cleaving enzyme, a maximum of one missed cleavage, iodoacetamide (cys) as a complete modification, oxidation (met) as a partial modification, monoisotopic masses, and a mass tolerance of ±0.1 da. the pmf acceptance criterion was probability scoring. total rna was extracted from the lysates of pk-pdcov-n cells at 24 and 48 h post-seeding by the trizol reagent (invitrogen) and was treated with dnase i (takara, otsu, japan) according to the manufacturer's protocols. the concentrations of the extracted rna were measured using a nanovue spectrophotometer (ge healthcare, piscataway, nj). quantitative real-time rt-pcr was performed using a thermal cycler dice real time system (takara) with genespecific primer sets as described previously (sagong and lee, 2011) . the primer sequences are available upon request. the rna levels of cellular genes were normalized to porcine ␤-actin mrna, and relative quantities (rq) of mrna accumulation were calculated using the 2 − ct method (livak and schmittgen, 2001) . to detect alterations of cellular mrna levels in the presence of the pdcov n protein, the relative fold change of each cellular gene was calculated in accordance with the comparison of pk-pdcov-n and control pk-neo cells. we used the student's t-test for all statistical analyses, and differences with p-values <0.05 were considered statistically significant. a set of seven novel mammalian and avian coronaviruses was recently discovered, including one porcine coronavirus, pdcov (woo et al., 2012) . pdcov is the fifth coronavirus infecting pigs in addition to transmissible gastroenteritis virus, porcine respiratory virus, porcine hemagglutinating encephalomyelitis virus, and porcine epidemic diarrhea virus (pedv). to date, interactions between this novel virus and the host have not been studied yet. furthermore, alterations of cellular protein expression in response to pdcov or each viral protein upon infection currently remain undetermined. in the current study, the pdcov n protein was chosen in the current study for the proteomic analysis to evaluate specific host cellular responses, which is not only the most abundant viral component but also performs several biological functions in completing viral replication. for this purpose, sublines of pk-15 cells were established to stably express recombinant pdcov n under the control of a retroviral longterminal-repeat (ltr) promoter. ten generated cell clones were initially collected and subjected to rt-pcr and western blotting to confirm the n gene expression at mrna and protein levels, respectively (data not shown). according to the results of the western blot analysis, one pk-pdcov-n cell clone that constitutively expressed the highest levels of n was selected for subsequent studies. to characterize pk-pdcov-n cells, we examined intracellular expression levels of n by ifa, facs analysis and western blotting. as shown in fig. 1a , the specific cell staining was clearly evident when pk-pdcov-n cells were reacted with the anti-his tag antibody, confirming the constant high expression level of the n protein. furthermore, the majority of the cells consistently exhibited specific fluorescent signals, indicating a homogenous population of cells in terms of n expression (fig. 1b) . time-course western blot analysis revealed that the pk-pdcov-n cells stably express and accumulate robust levels of a ∼45 kda recombinant n protein, larger than its predicted molecular weight of approximately 38 kda possibly due to post-translational modifications and the presence of c-terminal myc and histidine tags (fig. 1c ). in addition, the overall growth kinetics of pdcov n gene-expressing pk cells was found to be similar to that of the parental pk-neo cells, indicating that the pdcov n expression has no effect on cell proliferation (fig. 1d) . self-association of the n protein has been observed in many viruses, including coronaviruses, and is essential for assembly of the viral core constructing the basic architecture of viruses. sequence analysis of the pdcov n protein indicated that it is composed of 342 amino acids residues and contains no cysteine residues. as expected, no band corresponding to a disulfide-linked n dimer was detected by sds-page under non-reducing conditions ( fig. 2a, lane 4) , indicating that the pdcov n protein does not undergo cysteine-linked homodimerization. as a positive control, the prrsv n protein in the n-expressing stable pam cells was clearly demonstrated to form 35-kda n-n dimers under the same conditions (sagong and lee, 2010; fig. 2a, lane 2) . the ability of n to form non-covalent dimers was further investigated in a chemical crosslinking experiment. as shown in fig. 2b , the prrsv n protein in pam-pcd163-n cells formed a number of higher-order oligomers (lane 1) as reported previously . when the n protein in pk-pdcov cells was subjected to cross-linking, numerous multimeric forms of the n protein were identified (lane 2), indicating that the n protein of pdcov exists in the form of noncovalently linked oligomers that are used for assembly the viral capsid. we next determined whether the recombinant n protein expressed in pk-pdcov-n cells is subject to nucleolar localization that is known to be a common feature of coronaviral n proteins. the staining pattern in pk-pdcov cells was found to be predominantly cytoplasmic and nucleolic; this pattern persisted for up to 60 h after seeding (fig. 3a) . nearly all cells expressing pdcov n showed distinct fluorescent signals in the nucleolus at 48 h post-seeding, and thereafter, the pdcov n protein was localized mainly to the cytoplasm at 72 h post-seeding. the nucleolar localization of pdcov n was confirmed by transient transfection of bhk-21 or st cells with the plasmid pbud-pdcov-n (fig. 3b) . the cell fractionation assay also revealed the presence of the n protein in both the cytoplasmic and nucleic fractions (fig. 3c) . these observations demonstrated that, like n proteins of other coronaviruses, pdcov n protein is mostly distributed in the nucleolus along with the cytoplasm and has a conserved subcellular localization property. cellular proteins in the parental pk-neo cells and pk-pdcov-n cells were extracted and subjected to 2de analysis to compare the host protein expression profiles. to reduce the variability of gel electrophoresis, three independent 2de analyses of cellular extracts from control or n gene-expressing pk cells were performed, and spot intensity data from triplicate gels were selected for statistical analysis. on average, 1090 ± 63 protein spots were resolved by 2de within a ph gradient 4-10 and were visualized using silver staining; the molecular weights of the spots ranged from 10 to 200 kda. these spots were used for the comparative analysis. fig. 4a shows representative images of 2de gels from control pk cells (upper panel) and n gene-expressing pk cells (middle and lower panels). a total of 43 protein spots were initially found to be differentially expressed in pk-pdcov-n cells when compared with control pk-neo cells. on the basis of the statistical comparison, only those spots that consistently showed alteration in expression levels between pk-pdcov-n cells and the control cells were chosen for further protein identification. to identify the differentially expressed cellular protein spots in pk-pdcov-n cells at different time points, 10 protein spots with a statistically significant alteration, including 8 up-regulated and 2 down-regulated protein spots (fig. 4b) , were selected and manually excised from the stained gels. subsequently, the trypsin-digested spots were subjected to maldi-tof analysis. with combined pmf and database searching, identity of all 10 proteins was successfully determined. information on all these proteins in pk-pdcov-n cells, with their protein scores and sequence coverage, is summarized in table 1 . to better understand the implications of the cellular responses to the pdcov n protein, we further (c) nuclear and cytoplasmic fractionation of pk cells expressing pdcov n. each nuclear and cytosolic fraction was prepared from pk-pdcov-n cells at indicated time points post-seeding and subjected to western blot analysis with the antibody specific for his-tag (top panel), sp1 as a nuclear protein marker (second panel), or ␣-tubulin as a cytosolic protein marker (third panel). all blots were also reacted with ␤-actin antibody to verify equal protein loading (bottom panel). categorized the identified proteins by biological processes according to the gene ontology database as described previously (zhang et al., 2009) . these proteins showing altered expression were associated with various cellular functions including intracellular transport, metabolic processes, gene regulation, the stress response, protein synthesis, cytoskeleton networks, and cell division. since changes in cellular protein expression may be attributed to alterations in the corresponding mrna levels, transcriptional changes for all the identified proteins were tested by real-time rt-pcr to confirm the results of the proteomic analysis (fig. 5) . we were able to detect mrnas corresponding to nearly all proteins identified by proteomics, except for histone h4. although the altered expression levels of the remaining 9 proteins were consistent with the real-time rt-pcr results, we observed only modest increase or reduction in mrna levels. these results could be explained the case the correlation between mrna and protein abundance could be insufficient to predict protein expression levels from quantitative mrna data (gygi et al., 1999) , suggesting that the differences in protein levels might be due to post-translational modifications or protein stability rather than mrna levels. the quantity of each spot was normalized based on the total valid spot intensity for each gel and the relative fold change of each spot was then calculated between control pk-neo and pk-pdcov-n cells. data representing three independent 2de experiments for each sample were statistically analyzed and error bars represent standard deviations. *p = 0.001-0.05. two members of the hsp70 family, glucose-regulated protein 78 (grp78) and heat shock cognate 70-kda protein (hsc70), were found to be up-regulated in the pdcov n-expressing cells by the proteomic analysis. to further verify the dynamic alterations in fig. 5 . transcriptional alteration of identified cellular genes in pk-pdcov-n cells. the mrna level of each gene was assessed by quantitative real-time rt-pcr and normalized to that of porcine ␤-actin. relative quantities (rq) of mrna accumulation were evaluated by 2 − ct method and the relative fold change of each gene was then calculated between control pk-neo and pk-pdcov-n cells. results are expressed as the mean values from three independent experiments in duplicate and error bars represent standard deviations. *p = 0.001-0.05; † p < 0.001. expression of these stress-response proteins under the influence of pdcov n, we performed time-course western blot analysis. total cellular lysates were prepared from pk-pdcov-n cells at different time points, and the expression kinetics of the grp78 and hsc70 was compared between control and n protein-containing extracts. higher expression of both proteins was first evident in pk-pdcov-n cells as early as at 12 h post-seeding in comparison with control pk-neo cells, and the production of both proteins was persistently high during the later time points, suggesting that their enhanced expression is dependent on the n protein (fig. 6) . these results were consistent with the proteomic analysis of 2de gels and realtime rt-pcr, demonstrating that the synthesis of grp78 and hsc70 is indeed up-regulated in response to expression of the n protein of pdcov. since viruses are obligate intracellular parasites, they must utilize the cellular machinery and biosynthetic components of the host cell for their own replication. after viral infection, the infected cells launch a number of host antiviral defensive responses, which are turned on to eliminate the invading viruses. on the other hand, viruses employ their own evasion strategies to complete their replication and to successfully spread to neighboring cells. this series of interactions between the virus and host results in compensatory modifications in cellular gene production. accordingly, numerous studies have been extensively conducted by means of various molecular and genomic methods to understand how the altered host gene expression affects viral infection and the associated pathogenic processes. the proteomic analysis coupling high-resolution 2de and maldi-tof/ms is a tool that can generate ample data on cellular protein profiles modified by viral replication. with the proteomic techniques, it is now possible to identify relative changes in protein abundance for evaluation of host cellular fig. 6 . differential expression of the hsp70 family in pk-pdcov-n cells. cell lysates were prepared from pdcov n gene-expressing pk cells at indicated time points and immunoblotted to determine the expression profile of each protein with antibodies specific for grp78 and hsc70 (top to second panels). the blot was also reacted with anti-his tag (third panel) and anti-␤-actin (bottom panel) antibodies to confirm the status of n expression and equal protein loading, respectively. each cellular protein expression was quantitatively analyzed by densitometry in terms of the relative density value to the ␤-actin gene and pk-pdcov-n sample results were compared to pk-control results. values are representative of the mean from three independent experiments and error bars denote standard deviations. *p = 0.001-0.05; † p < 0.001. responses to viral infection or viral protein expression and to gain specific insights into the cellular mechanisms involved in viral pathogenesis (alfonso et al., 2004; brasier et al., 2004; neuman et al., 2008; oh and lee, 2012; ringrose et al., 2008; sagong and lee, 2010; zhang et al., 2008) . in the present work, a proteomic approach was applied for profiling the global protein expression changes in host cells in response to the n protein of pdcov, which is a major constituent of the virion and infected cells. therefore, we initially established a porcine cell line stably expressing the pdcov n protein and then characterized the n protein produced in those cells. due to the absence of cysteine residues, a disulfide-linked homodimeric n protein of pdcov was not detected in pk-pdcov-n cells. rather, the pdcov n protein was shown to be self-assembled via non-covalent oligomerization as a structural component for formation of the viral capsid. interestingly, the n protein of pdcov was found to be predominantly present in both the cytoplasm and nucleolus of pk-pdcov-n cells. the localization of nidovirus n proteins to the nucleolus may be necessary for control of rna synthesis or ribosome biogenesis via association with ribosomal subunits and interaction with nucleolar proteins (chen et al., 2002; wurm et al., 2001; yoo et al., 2003; ) . similarly, the pdcov n protein may participate in such a viral strategy to favor viral replication and pathogenesis. in addition, the nucleolar localization sequence detector program (http://www.compbio.dundee.ac. uk/www-nod/) predicted that pdcov n harbors a putative nucleolar localization signal (nols) consisting of a stretch of basic amino acids. therefore, further research is needed to identify a functional nols to confirm the intracellular localization of the pdcov n protein. these biological characteristics of n gene-expressing cells have yet to be confirmed using an authentic n protein in pdcovinfected cells, but according to our results, pdcov n appears to act as a multifunctional protein playing structural and non-structural roles contributing to completion of viral replication. our proteomic data revealed that 10 differentially expressed cellular proteins are identifiable in pdcov n gene-expressing porcine cells. the proteins that we identified in this study are involved in diverse cellular processes: metabolism, the stress response, protein biosynthesis and transport, cytoskeleton networks and cell communication, and cell division. the significance of the functional roles of the selected host proteins affected by the interaction of the pdcov n protein with the host cell is discussed below. the most interesting finding in the present study is that two cellular chaperone proteins belonging to the hsp70 family are up-regulated concomitantly by the n protein of pdcov. the first up-regulated protein is grp78 in cells expressing the pdcov n protein, which is associated with endoplasmic reticulum (er) stress. in eukaryotic cells, the er is the major site for synthesis and folding of transmembrane and secreted proteins, and accordingly, animal viruses also use the er as a site of synthesis and processing of their own proteins. the amount of protein entering the er can differ under physiological and environmental conditions. if protein synthesis exceeds the folding capacity of the er, unfolded proteins accumulate there, resulting in er stress. to maintain the er homeostasis, cells have developed a signaling pathway known as the unfolded protein response (upr) that transmits signals across the er membrane to the cytosol and the nucleus and ultimately reduces protein translation and enhances the er folding capacity by up-regulating chaperone proteins (ron and walter, 2007) . coronavirus infection of cultured cells is known to cause er stress and to induce the upr, which then crosstalks with various cellular signaling pathways, including mitogen-activated protein kinase cascades, autophagy, apoptosis, and innate immune responses, indicating the involvement of upr activation in virus-host interactions and viral pathogenesis . more interestingly, global proteomic and microarray analyses have shown that the expression of chaperon proteins, such as grp78 and grp94, is up-regulated in cells infected with a human coronavirus or in cells expressing the s2 subunit (jiang et al., 2005; yeung et al., 2008) . furthermore, the n protein of pedv, another porcine coronavirus, overexpression was shown to trigger er stress (xu et al., 2013) . thus, it appears that pdcov infection may induce er stress and the upr, leading to the up-regulation of grp78 to counteract the er stress; hence, the n protein may be responsible for this stress response pathway. the second identified hsp70 family protein is hsc70, also known as heat shock 70-kda protein 8 (hspa8). hsc70 is a molecular chaperone with multiple functions in protein folding and trafficking in all eukaryotic cells and in protecting cells from apoptosis or a wide array of stressors such as heat and infection (morano, 2007; powers et al., 2008; takayama et al., 1999) . accumulating evidence has shown that hsc70 plays important roles in certain processes involved in viral infection by modulating cell entry, virion disassembly and assembly, and the cellular antiviral response and apoptosis (chuang et al., 2015; gutiérrez et al., 2010; ivanovic et al., 2007; liu et al., 2013; radhakrishnan et al., 2010; yan et al., 2010) . apoptosis is considered an innate defense mechanism that limits propagation of a virus by eliminating infected cells (everett and mcfadden, 1999) . therefore, many viruses have evolved to employ various strategies that inhibit apoptosis in order to prevent premature cell death, thereby securing sufficient time for progeny production. thus, a conceivable explanation is that pdcov may take the advantage of suppressing apoptosis by up-regulating hsc70 in the early phase of the infection, and the n protein seems to be involved in this mechanism. in addition, hsc70 mediates the nuclear export of the influenza virus ribonucleoprotein complex via interaction with viral proteins m1 and ns2 (watanabe et al., 2006 (watanabe et al., , 2014 . in the present study, we found that pdcov n can be localized to the nucleolus in nexpressing cells. on the other hand, n proteins must be trafficked from the nucleolus to the cytoplasm to accomplish nucleocapsid assembly as well as viral rna synthesis and virus-host interactions. according to our results and existing data, hsc70 may facilitate the export of pdcov n from the nucleolus to complete the viral life cycle. like many other viruses, coronaviruses turn off host protein translation, while continuing to the synthesis of their own gene products to finish viral replication. one of the mechanisms behind this translational suppression is through interaction of the n protein with elongation factor 1␣ (ef1␣), a major translation factor in mammalian cells . in the present study, the amount of another translational factor, ef2, was affected by the host response to the pdcov n protein. although we do not know whether the increased expression of ef2 is related to its binding to pdcov n, such differential expression of a translation factor may be associated with regulation of both host and viral protein synthesis. we also found that expression of the cytoskeletal protein ezrin is significantly enhanced by the n protein. ezrin is a member of the ezrin-moesin-radixin (emr) family of host cytoskeletal proteins that organize the cortical cytoskeleton by mediating interactions between actin and the plasma membrane proteins and function as signal transducers in numerous signaling pathways (neisch and fehon, 2011) . the emr also modulate rna virus infection by regulating stable and dynamic microtubule formation (bukong et al., 2013; haedicke et al., 2008; naghavi et al., 2007) . given the biological features of the emr proteins, the up-regulation of ezrin in our study suggests that the pdcov n protein may manipulate the host cytoskeletal network and cell signaling, possibly to facilitate the processes of viral infection and replication. in contrast, the expression of translocon-associated protein subunit delta (trapd) is suppressed in cells expressing the pdcov n protein, according to our results. trapd is a part of the trap complex that is involved in translocating proteins across the er membrane and in regulating the retention of er resident proteins (fons et al., 2003) . coronaviruses acquire their lipid envelop via budding of the nucleocapsid through the er-golgi intermediate compartment (mcbride et al., 2014) . the down-regulation of trapd may lead to the release of cellular proteins from the er during pdcov replication, which in turn, promotes translocation of envelope-associated viral structural proteins to the er membrane -the site of budding -to facilitate virus assembly. eukaryotic cells reprogram their metabolism to adapt to stress-induced damage in response to environmental stressors. among our differentially expressed proteins, some are either directly or indirectly involved in metabolism. one hypothesis that can explain this result is that production of the n protein affects cellular biosynthesis by modulating the expression of metabolism-related proteins, which in turn remodels the intracellular environment for optimal pdcov replication. in conclusion, to our knowledge, this is the first report of a proteomic analysis of cellular responses to the pdcov n protein. although definite functions of the proteins that we identified here were not determined, it is likely that alterations in their expression are involved in virus-host interactions. to resolve these questions as well as performing various pdcov research, obtaining a korean pdcov isolate that can grow in cell culture is necessary; we are currently working on this task. in future studies, we are planning to assess cellular responses to pdcov by proteomic analysis to validate our present data; the proteins that are differentially expressed in the cells overexpressing pdcov n or in the cells infected with pdcov can be analyzed in detail in order to identify their precise function in the replication of pdcov. on the other hand, one limitation of the proteomic methodology used in this study is the inability to reliably detect low-molecular-weight or low-abundance proteins such as cytokines, which are involved in the host immune response. therefore, more comprehensive works are also needed to expand our present findings and to explore other proteins that may play a role in host responses to this protein. nevertheless, the data presented here are expected to advance the knowledge about the molecular mechanisms associated with pdcov-host interactions and viral pathogenesis. identification of cellular proteins modified in response to african swine fever virus infection by proteomics a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding nuclear heat shock response and novel nuclear domain 10 reorganization in respiratory syncytial virus-infected a549 cells identified by high-resolution two-dimensional gel electrophoresis human ezrin-moesin-radixin proteins modulate hepatitis c virus infection interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell heat shock cognate protein 70 isoform d is required for clathrin-dependent endocytosis of japanese encephalitis virus in c6/36 cells virus taxonomy: ninth report of the international committee on taxonomy 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chaperone hsc70 in reovirus outer capsid disassembly quantitative analysis of severe acute respiratory syndrome (sars)-associated coronavirus-infected cells using proteomic approaches: implications for cellular responses to virus infection pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs ribavirin efficiently suppresses porcine nidovirus replication coronaviridae the nuclear localization signal of the prrs virus nucleocapsid protein viral replication in vitro and antibody response in vivo complete genome characterization of korean porcine deltacoronavirus strain kor/knu14-04/2014 the small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties generation of a porcine alveolar macrophage cell line for the growth of porcine reproductive and respiratory syndrome virus full-length genome sequence of porcine deltacoronavirus strain usa/ia/2014/8734 analysis of relative gene expression data using real-time quantitative pcr and the 2(−delta delta c(t)) method heat shock cognate 71 (hsc71) regulates cellular antiviral response by impairing formation of visa aggregates complete genome sequence of strain sdcv/usa/illinois121/2014, a porcine deltacoronavirus from the united states rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus the coronavirus nucleocapsid is a multifunctional protein new tricks for an old dog: the evolving world of hsp70 moesin regulates stable microtubule formation and limits retroviral infection in cultured cells contribution of the porcine aminopeptidase n (cd13) receptor density to porcine epidemic diarrhea virus infection proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3 a simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels proteomic characterization of a novel structural protein orf5a of porcine reproductive and respiratory syndrome virus dual targeting of hsc70 and hsp72 inhibits hsp90 function and induces tumor-specific apoptosis protein analysis of purified respiratory syncytial virus particles reveals an important role for heat shock protein 90 in virus particle assembly proteomic studies reveal coordinated changes in t-cell expression patterns upon infection with human immunodeficiency virus type 1 signal integration in the endoplasmic reticulum unfolded protein response differential cellular expression in continuous porcine alveolar macrophages regulated by the porcine reproductive and respiratory syndrome virus nucleocapsid protein porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-␤ production by inhibiting irf3 activation in immortalized porcine alveolar macrophages molecular cloning: a laboratory manual mass spectrometric sequencing of proteins silver-stained polyacrylamide gels an evolutionarily conserved family of hsp70/hsc70 molecular chaperone regulators detection and genetic characterization of deltacoronavirus in pigs identification of hsc70 as an influenza virus matrix protein (m1) binding factor involved in the virus life cycle nuclear export of the influenza virus ribonucleoprotein complex: interaction of hsc70 with viral proteins m1 and ns2 discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus homo-oligomerization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein and the role of disulfide linkages localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division porcine epidemic diarrhea n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin-8 expression heat shock cognate protein 70 gene is required for prevention of apoptosis induced by wssv infection glucose-regulated protein 78 as a novel effector of brca1 for inhibiting stress-induced apoptosis colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rna-associated protein fibrillarin proteomic profiling of hepatitis b virus-related hepatocellular carcinoma in china: a seldi-tof-ms study changes in the cellular proteins of pulmonary alveolar macrophage infected with porcine reproductive and respiratory syndrome virus by proteomics analysis the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha key: cord-299270-fwbz3t25 authors: lemieux, m. joanne; overduin, michael title: structure and function of proteins in membranes and nanodiscs date: 2020-08-22 journal: biochim biophys acta biomembr doi: 10.1016/j.bbamem.2020.183445 sha: doc_id: 299270 cord_uid: fwbz3t25 abstract the field of membrane structural biology represents a fast-moving field with exciting developments including native nanodiscs that allow preparation of complexes of post-translationally modified proteins bound to biological lipids. this has led to conceptual advances including biological membrane:protein assemblies or “memteins” as the fundamental functional units of biological membranes. tools including cryo-electron microscopy and x-ray crystallography are maturing such that it is becoming increasingly feasible to solve structures of large, multicomponent complexes, while complementary methods including nuclear magnetic resonance spectroscopy yield unique insights into interactions and dynamics. challenges remain, including elucidating exactly how lipids and ligands are recognized at atomic resolution and transduce signals across asymmetric bilayers. in this special volume some of the latest thinking and methods are gathered through the analysis of a range of transmembrane targets. ongoing work on areas including polymer design, protein labelling and microfluidic technologies will ensure continued progress on improving resolution and throughput, providing deeper understanding of this most important group of targets. with a short sma(2:1) copolymer with a 2:1 ratio of s:ma subunits shows optimum copolymer concentrations of 0.5-1% under temperatures in the 25-37 °c range with incubation times of 1-2 hours. lower temperatures warrant longer incubations, while excessive polymer concentrations should be avoided to minimize aggregates. optimal ionic strength (300-450 mm nacl), divalent cations (<2 mm) and ph (8) (9) are evident for kcsa solubilization but can be protein and membrane dependent as they are subject to respective charge distributions. comparisons with the performance of conventional detergents suggests that the development of neutral or basic copolymers related to sma could offer advantages, providing avenues for solubilization and analysis of a broader array of biological membrane:protein assemblies (memteins). the utility of sma lipid particle (smalp) technology for analysis of memteins by mass spectrometry (ms) and x-ray diffraction methods remains limited. this problem stems from the requirements of the respective gas phase or crystalline samples and the heterogeneity of currently used sma copolymers. in order to address this, muench and colleagues have developed a method to transfer membrane proteins from smalps into amphipols or detergents for downstream analysis [3] . the homotrimeric e. coli multidrug transporter acrb was used as a test case. the vulnerability of sma to precipitation in the presence of divalent cations was exploited by using stepwise addition of mgcl 2 from 0.5 to 2 mm to cause the solubilizing copolymer to precipitate in the presence of a rescue solution composed of amphipol a8-35 or 1% n-dodecyl-β-d-maltoside (ddm) detergent. the resulting amphipol j o u r n a l p r e -p r o o f journal pre-proof and detergent complexes are more homogeneous, tolerate higher divalent cations levels, and yield observable signals by native mass spectrometry, including of phosphatidylglycerol in the amphipol-stabilized protein trimer. new methods are being applied to analyze the process of forming nanodiscs from membranes in greater detail. in particular, a microfluidic diffusional sizing (msd) system which was recently developed to detect protein hydrodynamic radii between 0.3 to 20 nm has been adapted to probe nanodisc formation [4] . this microfluidics technology requires small sample volumes (<10 μl) which are injected into a microfluidic chip. the complexes diffuse across from one side of the chamber to the other during a 15 minute time course and are then sensed via primary amines of, for example, phosphatidylethanolamine. several stages can be reliably seen from comparison of mds and dynamic light scattering (dls) data collected with increasing ratios of sma(3:1) to lipid. first the polymer inserts into bilayers until it reaches saturation. this is followed by solubilization into particles with 30 -10 nm diameters. as excess polymer is applied these become progressively smaller until reaching a limiting diameter of 6 nm. the lipid-specific association of an aggregation peptide derived from the tau protein is also demonstrated, indicating utility for detecting how fibers are nucleated on membranes. two longstanding challenges are the characterization of endogenous heteromultimeric membrane proteins and the development of conformation-specific antibodies for memteins. a study by mouro-chanteloup and coworkers of red blood cell ghosts addresses these confounding issues [5] . the individual components of erythrocyte membranes including rh proteins are well known, but their multimeric lipid complexes are dissociated in detergent-based preparations and hence no longer recognizable or extractable using conformation-specific antibodies. in contrast using sma(3:1) copolymer for solubilization preserves the native states of rh complexes, and membrane-associated cytoskeletal proteins can be gently removed by washing in very low ionic strength buffer. the biological relevant states of detergent-free rh complexes can then be isolated using gel filtration and monoclonal antibodies. the native states are preserved based on detection by conformation-dependent antibodies, paving the way for antibody screening and structure-function analysis. the extraction and purification of low abundance drug targets from membranes in stable, functional forms is a key goal for the pharmaceutical industry. the g-protein coupled receptor (gpcr) superfamily is critical, being targeted by many therapeutic agents. they respond to a diversity of signals and mediate cellular responses through interactions with heterotrimeric g proteins, gpcr kinases and arrestins. the complexity and conservation of the interactions of the phosphorylated and palmitoylated vasopressin receptor type 2, its peptide agonist vasopressin and arrestin is modeled by fanelli and coworkers, revealing coupled motions between key gpcr sites [6] . conformational changes that are induced by ligands including full agonists, partial agonist, antagonists and inverse agonists are j o u r n a l p r e -p r o o f journal pre-proof membrane-dependent. the adenosine 2a (a2a) receptor is a well-characterized gpcr from a structural perspective, although the roles of dynamics and lipids remain unclear. after expression in pichia pastoris, the wild-type a2a receptor and a pair of mutants with trp to tyr substitutions were solubilized into nanodiscs using sma(2:1) copolymer by wheatley and colleagues [7] . a fluorescent reporter (iaedans) was also attached to an introduced cys residue by the sixth helix, providing an additional probe. binding of specific ligands induces concentration-dependent changes in the fluorescence signals of trp residues, which exhibit greater steric hindrance based on fluorescence anisotropy measurements. the association with phospholipids from the plasma membrane is apparent by ms/ms analysis of the smalps. watts et al. show that the dopamine d1 receptor can be purified from human embryonic kidney (hek) cells with a yield of 255 g/l using sma(3:1) copolymer [8] . as with the a2a receptor, the purification of this gpcr was carried out at 4 °c to preserve the native state. binding studies of the protein in smalps provides estimated native-like affinities for a neurotensin peptide ligand and a specific antagonist. the transport of materials including drug molecules out of cells is carried out by the atp binding cassette (abc) family of membrane transporters. the structures and functions of these proteins are reviewed by kanelis and coworkers, with an emphasis on the intrinsically disordered regions that mediate key interactions in a phosphorylation-dependent fashion [9] . a set of abcg2 protein constructs were designed with n-terminal gfp or snap and his 6 tags and solubilized from hek cells using sma(2:1) copolymer, as were cd28 and cd86 antibiotics include lipid-specific peptides that self-associate into pores, which permeabilize bacterial membranes. however, the intact pores are difficult to resolve due to their dissociation in detergents. palmer's lab studies daptomycin, a cyclic lipopeptide consisting of 13 amino acids and a decanoic acid group that is a critical line of defense against infections caused by gram-positive bacteria. it works by binding phosphatidylglycerol-containing membranes in a calcium-dependent manner to form octameric pores. the design of alternating sma copolymers with methylamine sidechains allows octamers and tetramers to be obtained by varying on the amount of added polymer, illuminating the assembly of membrane complexes [13] . this paves the way for structure-determination of the native pore state and design of improved antibiotics to combat multi-drug resistant bacteria. aquaglyceroporin proteins allow the selective permeation of molecules across membranes. this family includes the e. coli glycerol facilitator, which mediates passive diffusion of glycerol across the inner membrane of the bacterium. although prone to aggregation, careful optimization of its expression and solubilization in various media showed that this helical bundle protein is most monodispersed in lauryl maltose neopentyl glycol [14] . this detergent j o u r n a l p r e -p r o o f journal pre-proof stabilizes the physiological tetramer, although a non-native octamer persists due to interactions between the disordered termini. the dynamics of lipids in nanodiscs formed by sma(3:1) and diisobutylene/maleic acid copolymer (dibma) copolymers, which differ in their hydrophobic groups, are compared by steinhoff and coworkers [15] . the application of electron paramagnetic resonance (epr) spectroscopy and a set of phosphatidylcholine lipids with nitroxide groups located at the 5 th , 12 th or 16 th carbon atom positions reveals that the lipids are more constrained in the more stable smalp discs. this can be explained by coarse-grain molecular dynamics simulations, which indicate that in the case of dibma a single belt of polymer contains lipids within the nanodisc with dynamics more closely resembling their mobility in a liposome. in the overduin lab, new amphipathic copolymers are being developed to broaden the utility of native nanodiscs. a series of alkylamine derivatives of sma with alternating sidechains were designed to reduce polymer sequence heterogeneity and improve resolution [16] . in the major facilitator superfamily, various solute carrier (slc) families exist. one of the challenges is to functionally characterize these transporters. in particular, a different transporter function may be present for the same transporter when it resides in different cell types. the cordat lab determined that the slc26a7 protein is a chloride/bicarbonate exchanger in kidney cells, which is in contrast to its role as a chloride channel in oocytes [17] . furthermore, they show that the abundance of this protein at the cell surface is dependent on the osmolarity of the cell culture medium, and ph of the cells. this is important considering the slc26a7 is expressed in outer medullary collecting duct cells near acid secreting cells that can render the local environment hyperosmotic compared to plasma. in human cells, both concentrative and equilibrative nucleoside transporters (cnt and ent) are found. with multiple transporters in this family having overlapping function it is important to characterize each individually. radioactive hplc was used by the young lab to determine the metabolism of nucleosides in oocytes, for which little previous study has been conducted [18] . this study revealed little metabolism occurred in oocytes and helped develop this novel means to examine nucleoside levels. next, in mice, the contribution of cnt and ents, which have overlapping specificities, were examined using radioactive j o u r n a l p r e -p r o o f hplc. nucleotide metabolites were assessed from plasma of mice with cnt or ent knockouts, illuminating the specificities of these transporters in vivo. the seca protein works to facilitate the transport of proteins destined for secretion through the secyeg translocon. despite structural information that reveals helical nucleotide binding domains, how seca interacts with diverse secretory proteins remain a question in the field. to address this, the bondar group conducted a sequence analysis of bacterial seca proteins (425), which was guided by sequence alignment, structural analysis and phylogeny [19] . they identified that seca proteins have varied length as a result of insertions and deletions, with clustering revealing three main groups. the first 220 residues housing the nucleotide binding region 1 (nbd1) is highly conserved in the family, with preferred residues at the n-terminus, which include a conserved phe at position 10, followed by hydrophobic sequences, and an arg/lys, are important for lipid anchoring. in addition they highlight the tyr residue located at the end of a helix finger structural motif, and other charged residues that may play a role in recognition of the positively charged pre-sequence. they further suggest diversity in sequence length contributes to increased conformational dynamics that may influence target interactions. the development of new tools to produce and analyze of integral membrane proteins is covered in a timely review by danmaliki and hwang [20] . these targets remain challenging to characterize at atomic resolution by any method. their analysis by nuclear magnetic j o u r n a l p r e -p r o o f journal pre-proof resonance (nmr) spectroscopy is complicated by the requirement for many milligrams of pure protein containing 2 h, 13 c and 15 n isotope labels to ensure high sensitivity and resolution, as well as the complex dynamics within the bilayer that broaden signals from transmembrane regions. advanced isotope labelling protocols allow chemical groups such as methyl and methylene groups and 19 f nuclei on specific residue types to be resolved, yielding structural information from such moieties. successes include an array of  barrel structures, which have been solved in various detergent micelles, as well as -helical structures that are inherently more challenging as they exhibit greater chemical shift degeneracy and fewer observable distances between secondary structure elements. hence nmr is well-positioned to provide unique insights into dynamics and interactions, as well as structures of proteins that are small or difficult to crystallize. single pass transmembrane proteins pose challenges for structural biology, being particularly dynamic, prone to aggregation and dependent on lipid microenvironments. consequently, very few have been characterized at high resolution. ramamoorthy and colleagues solubilized the full length form of the microsomal protein cytb5, which includes a heme-containing soluble domain, transmembrane helix and flexible linker, into nanodiscs using an 18-residue amphipathic helical peptide and various synthetic lipids [21] . through expression in e. coli the four tryptophan residues could be labelled with fluorine-19 for detection by 19 f-nmr spectroscopy. due to the rapid tumbling of the ~8 nm diameter nanodiscs, the 19 multi-domain proteins that reversibly bind membrane surfaces provide technical challenges for unravelling biological pathways. the phafin2 protein plays a critical role in inducing autophagy, but its membrane recognition mechanism is unclear. this modular protein contains a pleckstrin homology (ph) domain and a (fab1, yotb, vac1, and eea1) fyve domain, both of which bind phosphoinositol 3-phosphate (pi3p), a key signaling lipid that is found in endocytic membranes. the capelluto group showed that its fyve domain is indispensable for constitutive and specific pi3p recognition [16] . in contrast, its ph domain binds acidic lipids such as phosphatidylserine or pi3p when they are present in bilayers (but not as soluble lipids). the ph domain is autoinhibited by interactions with a conserved acidic c-terminal motif. thus the multivalent and tightly regulated binding of proteins to membrane surfaces can deciphered via the individual interactions. in the future soluble and homogeneous nanodiscs may offer opportunities to reveal the native binding mechanisms via concerted recognition of multiple lipids and protein elements that are heavily phosphorylated. together, these studies illustrate the challenges and potential of the growing field of membrane structural biology. membranes have traditionally been the least well understood components of the cellular ecosystem, and new tools have been sorely needed. the coming years promise further gains as the interfaces between proteins and lipids become better understood, and are certain to yield many more secrets into memtein formation and function. our ability to probe such mechanisms in native states and recombinant forms will in turn lead to improved understanding and exploitation of membrane targets for applications including synthetic biology and drug discovery. isolation of intramembrane proteases in membrane-like environments factors influencing the solubilization of membrane proteins from escherichia coli membranes by styrene-maleic acid copolymers styrene maleic-acid lipid particles (smalps) into detergent or amphipols: an exchange protocol for membrane protein characterisation microfluidic diffusional sizing probes lipid nanodiscs formation detergent-free isolation of native red blood cell membrane complexes dynamics and structural communication in the ternary complex of fully phosphorylated v2 vasopressin receptor, vasopressin, and beta-arrestin 1 ligand-induced conformational changes in a smalp-encapsulated gpcr detergent-free extraction of a functional low-expressing gpcr from a human cell line intrinsically disordered regions regulate the activities of atp binding cassette transporters application of fluorescence correlation spectroscopy to study substrate binding in styrene maleic acid lipid copolymer encapsulated abcg2 expression and detergent free purification and reconstitution of the plant plasma membrane na(+)/h(+) antiporter sos1 solution structure of the cytoplasmic domain of nhap2 a k(+)/h(+) antiporter from vibrio cholera characterization of multimeric daptomycin bound to lipid nanodiscs formed by calcium-tolerant styrene-maleic acid co-polymer solution structure and oligomeric state of the e. coliglycerol facilitator lipid dynamics in nanoparticles formed by maleic acid-containing copolymers: epr spectroscopy and molecular dynamics simulations the effect of hydrophobic alkyl sidechains on size and solution behaviors of nanodiscs formed by alternating styrene maleamic copolymer slc26a7 protein is a chloride/bicarbonate exchanger and its abundance is osmolarity-and ph-dependent in renal epithelial cells hplc reveals novel features of nucleoside and nucleobase homeostasis, nucleoside metabolism and nucleoside transport diversity and sequence motifs of the bacterial seca protein motor solution nmr spectroscopy of membrane proteins expression, purification, and functional reconstitution of (19)f-labeled cytochrome b5 in peptide nanodiscs for nmr studies where he serves as director of nanuc, canada's national nmr centre. his research focuses on membrane structural biology and the discovery of ligands of proteins involved in cell adhesion, signaling and endocytosis. he studies desmosomal protein interactions, phosphoinositide recognition by signaling proteins, and ligand binding by novel drug targets including calcium/calmodulin dependent ser/thr kinases and gtpase regulators. he co-developed the moda program to identify membrane binding domains and the smalp system for detergent-free purification of native membrane proteins into stable as director of the membrane protein disease research group she leads a multidisciplinary research program focused on membrane protease structure and function. she is internationally recognized as a leader in membrane protein crystallography having solved two distinct membrane protein crystal structures key: cord-004879-pgyzluwp authors: nan title: programmed cell death date: 1994 journal: experientia doi: 10.1007/bf02033112 sha: doc_id: 4879 cord_uid: pgyzluwp nan it is widely held that all developmental cell death is of a single type (apoptosis) and that neuronal death is primarily for adjusting the number of neurons in a population to the size of their target field through competition between equals for target-derived factors. we shall draw on our research and on that of others to criticize these views and replace them by the following. at least three types of neuronal death occur, only one of which resembles apoptosis; a neuron can choose between several self-destruct mechanisms depending on the cause of its death. the purpose of the death is to regulate connectivity, not neuron number. competitors for trophic factors are unequal, and many losers have made axonal targeting errors. a neuron's survival and differentiation depend on multiple anterograde and retrograde signals. activity affects retrograde signals and some but not all anterograde ones. the pattern of activity is more important than the overall amount. in rodents, the period of naturally occuring cell death of motoneurons is followed by a period of supersensitivity to axonal injury. thus, in newborn rodents lesion of the facial nerve leads to a rapid degeneration of the injured motoneurons. we have tested whether overexpression, in rive, of the bcl-2 proto-oncogene was capable of preventing death of axotomized motoneurons. to address this question we used transgenic mice whose motoneurons overexpress the bcl-2 protein. one of the two facial nerves of newborn mice was transected on the 2nd-3rd post-natal day. seven days after the lesion, the morphology of the facial nuclei was analyzed. in control mice, and when compared to the intact nucleus, 70 to 80 % of axotomized motoneurons had disappeared. in contrast, in the transgenic animals, the number of motoneurons on the lesioned side remained unchanged when compared to the eontralateral nucleus. furthermore, their axons remained visible up to the distal lesion site. these experiments show that, in rive, motoneurons overexpressing the bcl-2 protein survive after axotomy, and suggest that, in rive, bcl-2 protect neurons from experimentally induced cell death and could be a target for treatment of motoneurons degenerative diseases. messmer s., mattenberger l., sager y., blatter-garin m-c., pometta d., kate a., james r.w. drpt de mrdeeine, drpt. de pharmacologie, div. de neurophysiologie clinique, facult6 de mrdecine, gen~ve. clusterin is a widely expressed glycoprotein, highly conserved across species. numerous functions have been postulated for this protein. the most important are roles in lipid transport, as elusterin is associated with apolipoprotein ai in hdl, complement regulation and tissue remodelling, in particular during cell death and differentiation. using cultures of rat spinal cord neurones (90% neurons and 5-10% non-neuronal cells), we have studied the expression of clusterin and ape e in glutamate-induced neuronal cell death to examine potential roles in lipid management. up-regulation of the two proteins was observed. clusterin and ape e appear in the conditioned medium respectively 15h and 7.5h after incubation with glutamate. control studies, in the presence of a noncompetitive nmda receptor agonist showed the secretion of clusterin and ape e to be diminished by >60%. no up-regulation of either protein was observed in complementary studies with exclusively non-neuronal cell cultures. the cellular origin of the 2 secreted proteins is presently under investigation. programmed cell death and tissue remodelling are consequences of hormonally induced restructuring of the rat ventral prostate after castration and the rat mammary gland after weaning. we used the "differential display"-method (liang and pardee, 1992, science 257:967) to detect and isolate edna fragments whose corresponding rnas are regulated either coincidentally, or in an organ specific fashion during mammary gland involution and postcastrational prostate regression. partial sequencing of 12 clones revealed high, but not absolute homology of 5 fragments with sequences, previously characterized in different biological contexts. these five encode functions which could be anticipated to be important for cell growth and/or programmed cell death, we are presently investigating the functions of several of these transcripts in cell culture and in rive. antisense oligos are being employed in vivo to determine whether these genes contribute to the phenotype of programmed cell death. b epitopes derived from the envelope gp52 glycoprotein (ep3) or from the viral superantigen of mmtv have been incorporated into inert or live vaccines. the inert vaccine consists of purified chimeric proteins which contain the b epitopes alone or fused to multimeric promiscuous t helper epitopes from tetanus toxin. mice were immunized subcutaneously with these chimeric proteins. the live vaccine consists of an avirulent strain of salmonella typhimurium which expresses the mmtv epitopes in the form of chimeric proteins fused to the nucleocapsid protein of hepatitis b virus. this vaccine is given to mice in one oral dose. the level, duration and isotype of the immune response generated by each vaccine have been measured and compared. the level of protection has been investigated by systemically challenging immunized mice with the relzovims. a reduced binding of oxytocin (ot) occurs with aging in some, but not all, areas of the rat brain (arsenijevic et al., experientia 1993, 49, a75) . the candate putamen showed the most impressive loss of ot receptors. two other regions, the hypothalamic ventromedial nucleus (vmh) and the islands of caueja (icj) had also an important deficit of ot binding sites. on the other hand, these two regions were known to be sensitive to sex steroids. in the present work, we treated from 20 month old rats during one month with testosterone propionate (2 #g/kg s.c., once every 3 days) dissolved in oil. three rats of the same age injected with oil only served as controls. we labelled ot receptors throughout the brain of old rats using a 125i-labelled ligand specific for ot receptors. analysis of autoradiograms by an image analyzer revealed that the testosterone treatment increased ot binding sites in the vmh, in the icj, and, to a lesser extent, in the bed nucleus of the stria terminalis, a region also sensitive to sex steroids, by contrast, in the caudate putamen, the disappearance of ot receptors was not compensated. in conclusion, the decrease of ot receptors occurring in vmh and icj with aging can be reversed by administration of gonadal steroids. in contrast, the loss of ot receptors in the striatum appears to depend on another mecanism. vasopressin (avp) receptors are expressed transiently in the facial nucleus during development (tribollet et el., 1991, dev. brain res., 58, 13-24) . avp may therefore play a role in the maturation of neuromuscular connexions in the neonate rat, and possibly in the restanration of these connexions after nerve lesion in the adult. in order to investigate the latter proposition, we have sectionned the facial nerve in adult rats and used quantitative autoradiography to look at avp binding sites in the facial nucleus at various postoperative times. we observed a massive and transient increase of avp binding sites on the operated side. the number of facial avp binding sites reaches a maximum about one week after nerve section, remains stable during 2-3 weeks, then begin to decrease towards control level. the induction of avp receptors is markedly delayed if the proximal stump of the nerve is ligated. to assess whether other motor nuclei would also react to axotomy by up-regulating the expression of avp receptors, we have sectionned the hypoglossal nerve and the sciatic nerve. in both cases, the binding of avp receptor ligand increases massively in the respective motor nuclei, with a time-course similar to that found in the facial nucleus. altogether, our data suggest that central avp could be involved in the process of nerve regeneration. cytotoxic t-cell mediated apoptosis schaerer,e, karapetian,o.,adrian,m. and tschopp,j. inst.de biochimie, univ.de lausanne, 1066 epalinges. an apoptotic cell death mechanism is used by cytolytic t cells (ctl) to lyse appropriate target cells. ctl harbor cytoplasmic storage compartments, containing the lytic protein perforin and serineproteases (granzymes), whose content is released upon target cell interaction. we show that these granules are multivesieular bodies and that degranulation releases these intragranular vesicles (igv) having granzymes, t-cell receptor and yet undefined proteins associated. isolated igvs and perforin induce dna breakdown in target cells within 20 minutes. microscopic analysis demonstrates that igv specifically interact with target cell via the t-cell receptor and that their contents is taken up by the target cell. already 15 min. after interaction, 3 distinct igv proteins are found in the nucleus of the target cell.one of the molecules has been identified to be granzyme a, previously reported to be involved in apoptosis. we propose that lymphocytes transfer apoptosisinducing proteins to the nucleus of the target cells using vesicles as vehicles for delivery. cytotoxic t cells kill their targets by a mechanism involving membranolysis and dna degradation (apoptosis). recently, two sets of proteins have been proposed as dna breakdown-inducing molecules in t cells: granzyme a, b and tia-i. in this study, we cloned and further characterized the tia-i mouse homologue. aa sequence comparison with the human tia-1 showed an overall identity of 93%. devoid of a signal peptide, tia is yet localized to cytotoxic granules, probably targeted via a gly-tyr-motif. as tia-i, its mouse homolcgue contains three rnabinding domains. expression of tia during development shows a very strong signal in the brain and weaker signals in thymus, heart and other organs. during embryonic development several structures that contribute to organogenesis form transiently and are later eliminated by apoptosis. this pattern of tia expression could indicate its involvement in apoptosis. prostate involution occurs after castration in rats and is associated with the death by apoptosis of a large fraction of the epithelial cells. we have isolated several genes from a prostate involution bacteriophage lambda library using differential screening methods. among these clones, one d~monstrated an especially strong signal when used as a probe against northern blots of prostate mlhna obtained before, and at different times after castration. this gene is down-regulated after castration by 40-fold within 5 days. intramuscular injection of a testosterone depot resulted in complete restoration of expression within 24 hours. upon sequencing it became apparent that this clone has a high degree of homology to a known ndah dehydrogenase encoded in mitochondrial dna. the clone failed to hybridize to any transcripts from rat organs other than prostate. we are now in the process of isolating the htm~n hc~olog to this gene for use as a biomarker in study of benign hyperplasia and developing carcinoma. this gene is a possible indicator for testosterone-independent cell populations or of cells lacking ftl~ctional testosterone receptor. during the first three postnatal weeks the rat lung undergoes the last two developmental stages, the phase of alveolarization and the phase of microvascular maturation. the latter involves a decrease of the connective tissue mass in the alveolar septa and a merging of the two capillary layers to a single one. speculating that programmed cell death may play a role during this remodeling, we searched for the presence of apoptotie cells in rat lungs between days 10 and 24. lung paraffin sections were treated with y-terminal transferase, digoxigenin-dutp, and anti-digoxigeninfluorescein-f(ab)-fragments, and the number of fluorescent nuclei was compared between sections at different days. while the number of apoptotie ceils was low until the end of the second week and at day 24, we observed an about eight fold increase of fluorescent nuclei towards the end of the third week. we conclude that programmed cell death is involved in the structural maturation of the lung. brunner, a., wallrapp, ch., pollack, i, twardzik, t. and schneuwly, s. lehrstuhl genetik, biozentrum universit~t w~rzburg, mutants in the giant lens (g/l) gene show a strong disturbance in ommatidial development. in the absence of any gene product, additional phetoreceptors, cone cells and pigment cells develop. opposite effects can be seen in flies in which the gene product of the giant lens gene can be ectopically expressed by heat shock. a second very typical phenotype is the disturbance of photoreceptor axon guidance. molecular analysis of gil shows that it encodes a secreted protein of 444aa containing three evolutionary conserved cystein-motives very similar to egf-like repeats. we propose that gil functions as a secreted signal, most likely a lateral inhibitor for the development of specific cell fates and that gil, either directly or indirectly, is involved in targeting photoreceptor axons into the brain. the decrease in cellularity during scar establishment is mediated through apoptosis desmouliere, a., redard, m., darby, i., and g. gabbiani department of pathology, cmu, 1 rue michel server, 1211 gen~ve 4 dudng the healing of an open wound, granulation tissue formation is characterized by replication and accumulation of fibroblastic cells, many of which acquire morphological and biochemical features of smooth muscle cells and have been named myofibroblasts (sch0rch et el., histology for pathologists, t992). as the wound evolves into a scar, there is an important decrease in ceuuladty, including disappearance of myofibroblasts. the question adses as to which process is responsible for myofibroblast disappearance. during a previous investigation on the expression of (z-smooth muscle actin in myofibroblasts, we have obsewed that in late phases of wound healing, many of myofibroblasts show signs of apoptosis end suggested that this type of cell death is responsible for the disappearance of myofibroblasts (darby et al., lab. invest. 63:21, 1990) . we have tested this hypothesis by means of electron microscopy and morphometry and by in situ end-labeling of fragmented dna (wijsman et al., j. histochem. cytochem. 41:7, t 993) . our results show that the number of apoptotic cells increases as the wound closes and suggest that this may be the mechanism for the disappearance of myofibroblasts as well as for the evolution of granulation tissue into a scar. (supported by the swiss national science foundation, grant n~ s01-16 r. jaggl, a. marti and b. jehn. universit~t bern, akef, tiefenaustr. 120, 3004 bern at weaning the mammary gland undergoes a reductive remodelling process (involution) which is associated with the cessation of milk protein gene expression and apoptosis of milk-produclng epithelial cells. this process can be reversed by returning the pups to the mother within 1 day. elevated nuclear protein kinase a (pka) activity was observed from one day post-lactation, paralleled by increased c-los, junb, ]und and to a lesser extent c-]un mrna levels. ap-1 dna binding activity was transiently induced and the ap-1 complex was shown to consist principally of cfos/jund. oct-1 dna binding activity and oct-1 protein were gradually lost from the gland over the first four days of involution, whereas oct-1 m_rna levels remained unchanged. comparing nuclear extracts from normal mammary glands with nuclear extracts from glands which had been cleared of all epithelial cells three weeks after birth revealed that pka activation, ap-1 induction and oct-1 inactivation are all dependent on the presence of the epithelial compartment. the increased fos/jtm expression and the inactivation of oct-1 may be consequences of the increased pka activity. when involution is reversed, both, pica activity and ap-1 dna binding activity (and fos andjun mrna levels) are reduced to basal levels. our data suggests a role for pka and ap-1 on progranlmed cell death of manlnmry epithelial ceils. bcl-2~ does not require membrane attachment for its survival activity c. borner*, i. martinout, c. mattmann*, m. irmler*, e. sch&rrer*, j.-c. martinou-j-, and j. tschopp*. * institute of biochemistry, university of lausanne, 1066 epalinges, 1 institute of molecular biology, glaxo inc.,1228 plan los ouates. 8cl-2(z is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. how it exerts this activity is unknown but it is believed that membrane attachment is required. to identify critical regions in bcl-2o~ for subcellular localization and survival activity, we created by site-directed mutagenesis, various mutations in regions which are most conserved between the different bcl-2 species. we show here that membrane attachment is not required for the survival activity of bcl-2o< a truncation mutant of bcl-2(z lacking the last 33 amino acids (t3) including the hydrophobic domain is soluble, yet fully active in blocking apoptosis of sympathetic neurons induced by ngf deprivation or l929 fibroblasts induced by tnfc~ treatment. we further provide evidence for a putative functional region in bcl-2 which lies in the conserved domains 4 and 5 upstream of the hydrophobic cooh terminal tail. the breakdown of nuclear dna is considered to be a hallmark of apoptosis. we previously identified the perinuclear membrane localized dnase i as the endonuclease involved in the formation of oligonucleosomal-sized fragments (dna ladder). it is not clear how the nuclease is activated and has access to the dna. we show that in thymocytes induced to undergo apoptosis, lamin breakdown preceded dna laddering. by transfeeting hela cells with a constitutively active cdc2 mutant, nuclear envelope breakdown and typical apoptotic features (ehromatin condensation) were observed. moreover, co-transfection with cdc2 mutant and dnase i led to dna degradation. we propose that apoptosis can be induced by wrongly timed and hence abortive mitosis leading to uncontrolled nuclear membrane disintegration. s02-01 s02-04 platelet-derived growth factor (pdgf) is thought to play an active role in fibrosing diseases. bronchiolitis obliterans-organizing pneumonia (boop) is a condition characterized by intraluminal proliferation of connective tissue inside distal air spaces. to evaluate pdgf expression in boop we performed immunohistoehemistry on lung biopsies from 20 patients and 10 controls free of fibrosis. sedal sections were stained with an antibody against either pdgf or the monoeyte/macrophage marker cd68, in both groups the pdgf ~9 cells were essentially tissue macrophages. using point counting to measure volume fraction (vv) , pdgf-pesitive cells represented 4.65+1.63% (mean+sd) of the volume occupied by lung tissue in the boop cases, and 2,12+0.65% in the controls (!0<0,001). similarily, 10.73+4.69% of the lung tissue was occupied by cd68 e~ macrophages in the boop cases, compared to 5.37:~3.73% in the controls (p<o.005). a positive linear correlation was found between the v v of pdgf ~ cells and the v v of cd68@ cells on the 30 cases (r=g.50 p<0.0o5). we conclude that boop is characterized by a recruitment of tissue macrophages and that a significant proportion of them express pdgf. as pogf can also induce the macrophage chemo-attractant mcp-1, we speculate that maerophages are involved in a positive feed back regulation, and may play a pivotal role in the pathogenesis of boop. barrier lesions in hydrostatic pulmonary edema d.wu, h.bachofen, u.gyger and e.r. weibel department of anatomy, university of bern, ch 3012, bern pulmonary edema induced in isolated perfused rabbit lungs by moderate elevation of perfusion pressure (14 tort ) results in the formation of characteristic bleb-like extensions of the thin cytoplasmic leaflet of alveolar epithelium ( bachofen et al. am rev resp dis. vo1.147. pp989-996,1993) . in this study, we show by means of electron microscopy and morphometry that the frequency of the blebs correlate with the distribution of alveolar edema fluid. we find that 5% of the epithelial surface is involved in the formation of blebs. three-dimensional reconstruction showed that blebs had near-spherical shape and that their opening toward the interstitium is irregular. these finding demonstrate a great plasticity of epithelial cell extensions when subjected to moderate pressure stress. at high pressure the cell linings demonstrate clear breaks. two major roles have been defined for no: cellcell communication mediated by the stimulation of cgmp synthesis, and cytotoxicity by direct interaction of the free radical no with cellular target. to investigate these effects in human epithelia, which constitute a major source of no in man, we introduced the murine inos sequence into sv40-t immortalized human bronchial epithelial cells (beas-2b). inos activity was higher than 100 pmol citrulline/min/ mg prot in the first passages of inos transfected cells, but it decreased by subculturing. no inos activity was detected in cells transfected with control vector. in inos transfected cells, no stimulated a cgmp synthesis and c-los expression which could be inhibited by addition of lnma. thus, in human epithelial cells no is able to significantly alter signal transduction pathway. partial pneumoneetomy, i.e. the resection of lung tissue, is known to initiate compensatory growth in the remaining lobes. under appropriate conditions this results in a complete restoration of lung parenchymal structure and function. unfortunately the molecular mechanisms controlling the onset of this compensatory growth are poorly understood. the aim of our study was to find and eharacterise genes which are up or down regulated during this time. for isolating such genes, we chose the differential display approach (p.liang and b.pardee 1992, science 257:p 967). therein a 3'end fragment of a subset of reverse transcribed mrnas is amplified by the polymerase chain reaction. the fragments can be separated on a dna sequencing gel. genes differentially expressed in pneumonectomised and sham-treated rats show different patterns on these gels. the fragments of such genes can be isolated from the gel and cloned into vectors in order to provide tools to characterise their gene. a set of such genes, some up-regulated and others downregulated following pneumonectomy, have been isolated, partially sequenced and characterised. time periods. our new plasma marker consisting of highly concentrated gold nanospheres was infused via a femoral vein catheter into the right atrium of anaesthetised and ventilated rabbits. five or 10 seconds after start of infusion, the blood flow was stopped and the lung was fixed by instillation. silver enhanced paraffin sections revealed labeled and unlabeled blood vessels. electron-microscopic sections showed various plasma gold particle concentration. morphometric analysis of electron micrographs showed an increase of the portion of marked capillaries from shorter to longer circulation times. based on our results, we conclude the existance of inhomogeneous plasma flow velocities within the pulmonary microvascular bed. (supported by snsf grant 31-30946.91) in schizosaccharomyces pombe pairing of meiotic chromosomes is not accompanied by the formation of a tripartite synaptonemal complex (sc), a structure which connects the homologs in most other eukaryotes. meiotic nuclei revealed novel structures ("linear elements") that might be related to the axial elements ($c precursors) of other organisms and function in meiotic chromosome pairing, recombination, and segregation (baler et al. (1993) jcb 121:241). to get further insight into the behavlour of meiotic chromosomes, we performed fluorescence in situ hybridizations on spread nuclei with probes from different chromosomal regions. interestingly, all centromeres and telomeres become clustered during meiotic prophase. the centl:omeres are paired already before meiosis, whereas the telomeres initiate pairing specifically during meiotic prophase, followed by pairing of interstitial regions. painting of whole chromosomes revealed that the homelogs occupy together a specific territory in the nucleus. an expression vector containing the vai gene read by rna polymerase ill, injected into xenopus zygotes, yields high levels of rna in virtually all embryonic cells. anti sense fina is produced from a segment of the xhoxla gene inserted in opposite orientation into the vai gene. immunological staining of whole mount embryos shows that target mrna level and xhoxla protein expression of xhoxla protein is diminished in about half of the antisense treated individuals. accordingly, nearly half of the embryos develop typical malformations in regions where the xitoxla gene is normally expressed. somites el the anterior trunk are heavily disorganized and mesodermal denvalives surrounding the intestinal tract are reduced or missing. antisense inhibition, through production of rna in situ from expression vectors, thus represents a useful tool to study the functional role of zygotic genes in xenopus development. cloning and characterization of the mouse homolog to s.cerevisiae sep1 encoding a dna strand exchange and homologous pairing protein vladimir bashkirov and wolf-dietrich heyer. institute of general microbiology, university of bern, baltzer-strasse 4, 3012 bern. homologous pairing and dna strand exchange are the central steps postulated in the current models of genetic recombination. to date no mammalian gene encoding a homologous pairing protein has been cloned. to get insight into the mechanism of recombination in higher eukaryotes we were interested to clone such genes. protein sequence comparison of the s.cerevisiae homologous pairing protein, sepi, and its homolog from s.pombe, p140 ex~ revealed several conserved stretches of amino acids in their nh2-terminal regions. using fully degenerate primers and mouse testes edna as a template for the polymerase-chain-reaction a product with the expected length has been amplified. this 360 bp dna was shown to encode a putative polypeptide with high degree of homology to both proteins from the two yeasts. the mouse pcr-product was used as a hybridization probe for screening a 1 gtlo mouse testis cdna library. after several rounds of successive screening a total of about 4 kb of mouse edna (msep1) was cloned and sequenced. the putative translation product revealed high homology throughout the entire sequence to its s.pombe and s.cerevisiae counterparts, 38 % and 41% of amino acid identity, respectively. the msep1 sequence was shown to be unique in the genome and the chromosomal gone is likely to contain many introns. we want to establish a functional full length edna clone of msepi and characterize the gone and protein product. we have earlier developed a cell free system to study recombinational repair of dna double strand breaks and deletions. the method allowed us to purify and characterize a high molecular weight multiprotein complex (rc-1), which catalyzes dna recombination. a dna polymerase and dna ligase as well as other enzymatic activities copurify with rc-1.we have commenced an investigation of homologous dna recombination in nuclear extracts which were prepared fzom normal and mutated mouse thymocytes. the scid mutation in mice causes an aberrant v(d)j joining reaction, an elevated sensitivity to ionizing radiation and a defect in recombinational repair. moreover, the normal mouse thymocytes were sorted into the cd4/cd4 double negative (dn), double positive (dp), and single positive (sp) subclasses and their nuclear extracts, and extracts from rag-2 -/-mice, were tested. only the scid mice and the cd4/cd8 sp thymocyte extracts were inactive in the recombinational repair reaction. this indicates a development stage specific activity and a scid specific defect of recombinational repair in thymocytes. restoration of the recombination activity was observed in thymocyte extracts derived from scid, which express a wansgene for the t-cell receptor i~ chain. a protein could be purified from normal thymus cells to near homogeneity which specifically rescues the recombination activity in scid extracts. this protein, srsp ~cid recombination stimulatory ~otein), migrates as a single band of app. 7 2 kda in sds polyacrylamide gel electrophoresis. the ade6-m26 mutation in the ade6 gene of the fission yeast schizosaecharomyces pombe gives rise to a hot spot for meiotic homologous recombination. to address whether transcription plays a role in m26 activity, the effect of the deletion of the ade6 promoter in combination with either the m26 and m375 mutations on recombination frequency was studied at the tetrad level. deletion in eis of the promoter abolishes m26 hot spot activity relative to the m375 control deletion strain. it is possible that deletion of the promoter has eliminated a sequence necessary for m26 hot spot activity, rather than lack of transcription being responsible for the loss of m26 activity. the role of transcription in m26 hot spot activity is currently being examined. we are analysing meiosis in a temperature sensitive top2 mutant. complementary temperature-shift experiments showed that topoisomerase ii is required for both meiotic divisions; early meiotic events are not impaired. in a meiotic time course, dapi staining revealed that the cells are blocked before the first meiotic division. interestingly, in meiotic spreads we found that the spindle pole body (spb) cycle is not affected by the block: the final arrest point showed 1 nucleus with 4 completely separated spbs. we want to analyse the number of spbs and spindle formation with immunofluorescence. we are planning to analyse the course of meiosis in a top2 rec7 double mutant (rec7 is recombination deficient) to see if topoisomerase ii activity is necessary for separation of recombined chromosomes. analysis of meiosis in a top2 rec8 double mutant (rec8 is recombination deficient and does not form linear elements) could give us a clue about the function of the linear elements. genetic recombination is able to induce cancer and to mediate its further progression. in cells from cancer predisposed individuals, mitotic recombination can lead to loss of heterozygous tumor suppressor genes as is e.g. observed in the hereditary form of retinoblastoma. one mechanism for gene loss is inter-or intrachromosomal recombination involving short duplicated sequences. our aim is to identify xenobiotics that are able to induce mitotic recombination and to elucidate the involved molecular mechanisms. for that purpose we have constructed transgenic d. melanogaster (1). rn.). cell lines. cells were stably transfeeted with an extrachromosomal dna element providing a putative recombination substrate+ "l%e constructed element contained two ta'uncated fragments of the neomycin resistance gene (nee r) interrupted by a hygremycin resistance marker (hygr). the hyg r insert was flanked on both sides by identical 352 bp fragments of nee r, different d. m. promoters shown to be constitutively active in various d. m. tissues were inserted 5' to both e. coil genes. restoration of a functional nee r gone may occur by deletion of the hyg r after recombination between the homologous regions. stably transfected schneider line 2 cells were obtained and preliminary results concerning chemical induction of genetic rearrangements will be shown. fleck, 0., sch~r, p. and kohli, j., institute of general microbiology, baltzerstr. 4, 3012 bern to detect mismatch-binding proteins of s. pombe we performed band shift assays with radiolabeled oligonucleotides containing a defined mismatch. we found two distinct mismatch-binding activities. one shows high affinity to most of the mismatches, but is absent for c/c. this protein might belong to the general mismatch-repair system, which is thought to be related to the bacterial muts dependent pathway. the second activity preferentially binds to those mismatches containing a cytosine and therefore represents a novel type of mismatch-binding protein. baur m., sch~r p. and kohli j., institute of general microbiology, baltzerstrasse 4, ch-3012 bern homologs of the salmonella typhimurium (mutl, s and 59 and streptococcus pneumoniae (hexa, b) genes involved in mismatch repair have been identified in several distantly related organisms. so far no mismatch repair gone is known in schizosaccharomyces pombe. in order to get more insight into the ~chanisals of mismatch repair in s. pombe we started the cloning of a mutl homolog. degenerated oligonucleotide primers based on conserved regions of mutl homologs were used in the polymerase chain reaction (fcr) to amplify a corresponding dna fragment from s. pombe. a dna fragment of about 220 bp was amplified whose deduced amino acid sequence shared a high degree of homology with paiql of saccharomyces cerevisiae and mutl, hexb. with the help of this dna fragment the gone was isolated from a genomic dna library by colony hybridization. sequence analysis of this mlhl gene (mlh = mut~ homolog) shows an orf of the expected size with three putative introns. while the deduced amino acid sequence of the 5' region shares strong homology with the procaryotic genes mutl, hexb and the eucaryotic gene pmsi, the amino acid sequenoe of the 3' region shows homology only to pmsi and hexb. the central region of the protein seems to be conserved only weakly. parisi s., and kohli j., institute of general microbiology, university of bern, ch-3012 bern rec7 and rec8 are supposed to be genes with major functions in meiotic recombination since mutations in these genes reduce the frequency of meiotic intragenic recembination ~1000 fold. these genes have been isolated and sequenced but no enzymological activity could be assigned to the protein. a first step toward enzymological and biochemical characterization of rec7 and rec8 is to make beth protein products available in sufficient quantities and in reasonable pure form. for this purpose both genes will be cloned in a s. /x~nbe over-expression system using the strong and inducible nmtl promoter.in parallel both genes will also be expressed in e. coli as gst fusion proteins in order to produce polyclonal antibodies in rats and rabbits. these antibodies can then be used for protein purification in s. pombe as well as for immunofluorescence experiments. induction of expression in e. coli produces a band of the expected size of 56kda for rec7 and of 73kda for rec8. to remove the gst carrier, the rec7 and rec8 fusion proteins can successfully be cleaved by thrombin or factor xa respectively. production of antibodies is in progress. d6bbeling, u., nobi, r,, berchtold, m.w. and kuenzle, c.c. institut fdr veterinarbiochemie, universit6t zurich, wintertharerstrasse 190, normally, only pre b-and pre t-cells can rearrange their immunoglobulin genes by v(d)j recombination, but when other cell types are transfected with the rag-i and rag-2 genes they also become able to perform v(d)j recombination. by transiently transfeeting nih 3t3 fibroblasts with both intact rag genes we found da recombination rate of 0.06%. no recombination was detected, when we transfected the intact rag-1 gene with a mutant rag-2 gene, which lacks 89 c-terminal amino acids, or the intact rag-2 gene with a rag-i gene containing a deletion in the topoisomerase iz homology region. these experiments show that these two regions of the rag genes are essential for v(d)j recombination. when the l4 fibroblast cell line, which has been rendered recombination competent by stable transfection with the rag genes was overtransfected with both wild type rag genes, we found in contrary to an expected increase of v(d)j recombination a reduction by a factor of 2-2.5. this decrease was not observed with the mutant rag genes.this result indicates, that there exists an optimal intracellular rag concentration for efficient v(d)j recombination. m. fischer, a. sailer, h. blieler, t. riilicke*, a. aguzzi +, p. autenried and c. weissmann; 1nstitut fiir molekularbiologie l universitfit ziirich, h6nggerberg, 8093 ziirich, *biologisches zentrallabor and + lnstitut fiir neuropathologie , universitdtsspital ziirich, 8091 zfirich, switzerland the infectious agent of transmissible spongiform encephalopathies such as creutzfeldt-jakob disease in man or scruple in sheep is thought to be prpsc, a posttranslationally modified form of the normal host protein prpc we have shown that mice devoid of prpc (prn-polo) are completely resistant to scrapie. here we report that also heterozygous prn-p o/+ mice with reduced levels of prp c show enhanced resistance to scrapie, as manifested by a long delay in the onset and slow progression of clinical disease. conversely, prn-po/o animals overexpressing prp transgeues exhibit shortened incubation times and accelerated progression of the disease. propagation of the scruple agent is completely abolished in prnpolo animals. high titers of infectivity, about 108 logld50 units/g of brain, are detected in prn-po/+ mice 33 weeks after inoculation (the earliest time point measured), 24 weeks or more before the animals die of scrapie. in contrast, wildtype animals survive no more than 16 weeks after reaching similar titers of infectivity. a practical implication of our findings is that moderate inhibition of prpc synthesis could mitigate disease progression in spongiform encephalopathies. the ruvb protein has been shown, biochemically and genetically, to be involved in the process of homologous recombination in e. coli. one of its functions is to promote branch migration within holliday junctions formed during the process of recombination. to investigate the mechanism by which ruvb promotes branch migration we have used conventional electronmicroscopy, cryo-electron microscopy, scanning transmission electronmicroscopy, and image analysis to study the interaction of ruvb protein with dna. we observed that ruvb forms multimeric rings on dsdna which encircle the dna. we also observed that such rings tend to localize at the holliday junctions. we propose that ruvb rings can actively move along dna using the energy of atp hydrolysis.this movement continues upon encountering a holliday junction, thus forcing branch migration along the dna. gschwind m. and huber g., pharma division, preclinical research, in some families with early onset aizheimer's disease mutations on the 8-app gene have been identified which cosegregate with the disease. so far all these pathogenic mutations have been identified on exons 16 and 17 in humans framing the sequence of the 8-amyloid itself.a genomic clone was isolated from mouse strain c57 black/6 containing the coding regions of the last three exons (16) (17) (18) according to the human sequence. sequencing of all three exons including the large 3' non coding region showed a 100% homology to the previously described balb/c mouse cdna. exon-intron boundaries were determined at the same positions as in human and rabbit genome and the flanking regions in the introns are also very similar, not only is the homology between the human and mouse f$-app very high (97.6%) but also the genomic organisation of their genes is nearly identical. therefore homologous recombination can be applied to introduce molecular 8-app changes and to study their functional consequences on the protein level. we systematically investigated the ability of reca protein to push the dna strand exchange reaction through heterologous regions. we observed that reca can push the strand exchange through substantial heteroiogy (up to 150 bp) when the heterologous insert was placed on the so called proximal end of the linear duplex. in the case of the medial beterology the strand exchange reaction could overcome heterologies of up to about 50 bp. heterology at the distal end was most difficult for reca to resolve, and already 22 bp long insert was completely blocking the progress of the strand exchange. these results, together with earlier electron microscopy studies, allow to propose a molecular mechanism by which reca can exchange strands between partially heterohigous dna molecules. institut for veterin~r-virologie, universit~t bern, ch-3012 bern bovine viral diarrhae virus is a positive-stranded rna virus of the pestivirus group. two biotypes, cytopathic and non-cytopathic in cell culture, can be distinguished. all cytopathic strains characterized so far carry insertions in their genomes. they probably result from a strand-switching activity of the viral rna polymerase during viral replication. to study the switching capacity of the viral enzyme, rna isolated from cells infected with two different strains of bovine viral diarrhea virus were analysed for homologous recombination in an rt-pcr assay. performing the assay we found that fragmented rna present during reverse transcription, the reverse transcriptase enzyme itself and viral rna present in the pcr reaction result in the production of artificial recombination during the assay. it was not possible to detect a homologous recombination activity of the viral polymerase above the background recombination frequency of the rt-pcr assay. our results strongly question the use of the rt-pcr assay for the study of recombination of rna viruses. transpositional rearrangements mediated by insertion sequence (is) elements represent an important source of genetic variation within populations 1. we analyze dna rearrangements at the level of the complete genome by rflp, using 7 is elements as probes for hybridisation to southern blots samples taken at different time points from 12 independent cuttures of e coli b grown by serial transfer for 10'000 generations 2 and stored at -70~ are used to study structure and evolution of genetic diversity within populations. the seven is elements contribute differently to genetic variation and lead to a succession of mutants affecting the genetic structure of the population. we now search for correlations between rflp patterns and the increase of relative fitness over time observed previously 2 naas t., 81o~ m, fitch w.m and arber w, (1993) the complete dna sequence of the locusta migratoria mitochondrial genome has been derived from four cloned mtdna fragments. the sequence is 15.2 kb in length, contains 13 peptide coding sequences, and genes for 22 trnas and two rrnas. the organisation of the genome shows a strong resemblance to drosophila, the gene order and orientation being the same except for the positions two trnas. this contrasts with the only other non-dipteran insect mtdna to have been sequenced, apls mellifera, which shows differences in the positions of ii trnas. this finding is discussed in relation to the at content of the different genomes. the sequences of the three mtdna genomes are also compared directly and the results related to existing knowledge concerning the evolution of insects. udem, new jersey medical school, newark, nj 07103-2714, usa nonsegmented negative strand rna viruses are so far not amenable to reverse genetics. the genomes must associate with nucleocapsid (n), phosphoprotein and polymerase proteins to form a functional rnp. as a step towards reverse genetics of measles virus (mv), plasmid vectors were constructed allowing synthesis of rnas corresponding precisely to a mv genome in which all coding and intercistronic regions are replaced by the chloram-phenicol acetyl transferase (cat) coding region, transfection of these negative polarity transcripts into mv infected hela or 293 cell lines gave rise to cat activity which could be serially transferred and massively amplified together with progeny helper virus in cell cultures. both expected mv/cat chimeric mrna and replication product were identified in the progeny. preliminary experiments indicate that only rnas in which the total number of nucleotides is a multiple of six replicate and transcribe efficiently, consistent with the data on natural and modified copyback di rna of sendal virus (calain and roux, j. virol. 67 4822 (1993) ). precise end to end encapsidation of rna, each n protein contacting six nucleotides, might be required. the embryonic tissue specificity of the human cytomegalovirus immediate-early (hcmv-ie) promoter/enhancer (-522 to +13) was examined by l~-galactosidase staining of transgenic mouse embryos carrying hcmv-ie regulated lacz genes. embryos of three transgemc lines were analyzed between 8.5 -13.5 dpc. for all lines, hcmv-ie transcription was primarily confined to subsets of neural and vascular cells. however, extents of transgene expression along the embryonic primary axis were slightly and greatly restricted in two of the lines relative to the third line at all stages analyzed. these differences most likely represent integration site-dependent chromatin constraints that affect levels of hcmv-ie-directed lxanscripfion. together, the three lines can be taken to define a graded potential for hcmv-ie transcription along the anteriorposterior axis of the embryo with greatest permissivity in mid-axial structures which include the dorsal neural tube at the level of the hindbraln and upper cervical spinal cord, the inner ear and vestibular acoustic ganglia, branchial arteries, aortic sac, and lung buds. the cell typespecificity and axially graded permissivity for hcmv-ie transcription in the mouse embryo provide a basis for understanding the pathology and relative frequencies of different types of birth defects caused by congenital hcmv infection in humans. stauffer, y., 0fford, e. and beard, p., swiss institute for experimental cancer research (isrec), ch-i066 epalinges, switzerland. hpvi8 is associated with genital carcinoma. like other human papillomaviruses, hpvi8 cannot be easily propagated in cell culture, because the viral growth cycle is dependent on the differentiation of its host cell, the keratinocyte. moreover, very little viral capsid protein is found in vivo in infected tissues. the hpvi8 l1 and l2 genes code for the major and minor capsid proteins, respectively. to obtain the viral capsid proteins we made constructs with the l1 and l2 genes for expression in e. coli (the pqe system) and in recombinant vaccinie virus (a modification of the phgs-i system) infected cells. the l1 and l2 proteins expressed in e. cell contained a histidine tag encoded by the vector. this allowed us to purify the proteins on a sickelaffinity column in order to raise antibodies. these will permit us to detect the presence of capsid proteins expressed from recombinant vaccinia viruses. we expect the l1 and l2 proteins expressed together from the vaccinia-based vector to selfassemble to form virus-like particles. to papain-like proteinases. the role of these putative catalytic residues in the activity of the proteinase was studied by site-directed mutagenesis. a minimal proteinase has also been constructed by n-and c-terminal deletion to determine the boundaries of the proteolytic domain, sequence analysis of the c-terminal cleavage product should indicate the site of cleavage. cell entry, uncoat~ng and nuclear transport of adenovirus z . u. f. greber and a. helenius. yale university school of medicine, new haven, ct, u.s.a. to enter cells, viruses must achieve three major goals: transfer their genome across the plasma membrane into the cytosol, target the genome to the replication site and decondense it for replication. adenovirus, an icosahedral nonenveloped particle with a double stranded genomic dna and ii to 15 structural proteins~ enters into human kb cells by a sequential disassembly program during receptor-mediated endocytosi~, acid-dependent penetration into the cytosol, and targeting to the nucleus. during internalization, the fibers, primarily responsible for virus binding to the cell surface, are detached. in early endosomes, the capsid-stabilizing proteins ilia and viii are released. fenton base, a vertexassociated protein implicated in penetration across the endosomal membrane, is removed in endosomes, if penetration is blocked with lysosomotroplc reagents. in the absence of inhibitors, a large fraction of penton base stays associated with the cytoplasmic capsid. the viral dna is disconnected from the shell after proteolysis of the internal protein vi~ a capsid and dna binding component. removal of protein ix, a capsid binding factor, further destabilizes the coat, and the remaining 'stripped-down' virus particles associate with nuclear pore complexes to release their dna-genomes into the nucleoplasm. this stepwise dissociation program is thought to provide the high efficiencies of the entry process needed to successfully deliver the viral dna across multiple barriers to the cell nucleus, the site of replication. ch. schmidt and w. arber; biozentrum der universit~t basel, abt. mikrobiologie, klingelbergstr. 70, ch-4056 basel bacteriophage p1, carried as a single copy plasrnid in e. col~, is widely known for its horizontal gene transfer ability (transduction) in enterobscteria. as a temperate phage, it can lysogenize its host and thereby provides it with accessory functions (restriction of foreign dna by ecop1, functions expressed by transposons carried on the p1 genome or integrative suppression). these properties reflect both symbiotic and evolutionary functions of pi. p1 regulates expression from its genes by two different mechanisms: trans(:ription of early regulatory genes is repressed by the phage encoded repressors c1 and bof, whereas transcription of its morphogenetic genes from p1 specific late promoters is induced by the early expressed gpl0. by site-directed mutagenesis the late promoter ps, composed of the -22 inverted repeat aagttactt and the -10 box, was functionally characterized. p5 and its derivatives compare well with several other late promoters of pt with regard to their sequence dependent strengths: the strong promoters ps, pdar and lpr2b all share the perfect inverted repeat around position -22, whereas the weaker promoters lpr91, lpr82a, lpr82b and lpr83 contain mismatches in their inverted repeats or the inverted repeat is positioned mere upstream relative to the transcription initiation site. homologies. however, the phages formed two immunity groups. the results may suggest a common genetic trait providing a selective advantage to lysogenic aa. by using a dna substrate with defined gap size we found that human immunodeficiency virus type 1 reverse transcriptase (hiv~rt) was able to perform strand displacement dna synthesis. this activity was not affected first by calf thymus proliferating cell nuclear antigen and replication factor c and second by escherichia coli single-stranded dna binding protein~ which together allow dna polymerase ~ to perform strand displacement dna syrlthesis (podust , v. and h~bscher, u. (1993) nucl. acids res. 21, . 3'-azido-2',3'-dideoxy-thymidine tripbosphate inhibited displacement completely indicating that dna synthesis is required for this reaction. the hiv-rt p66 polypeptide alone could perform limited strand displacement dna synthesis, whereas the hiv-rt p51 polydeptide was completely inactive, likely due to its inability to replicate extensively on a mi3 dna template, on the other hand the hiv-rt psl polypeptide e~hanced the strand displacement activity of the hiv-rt d68 subunit at a molar ratio of 4:1 mainly by chasing short products into longer ones. furthermore kinetic experiments after complementation of hiv=rt p66 with kiv-rt psl indicated that hiv-rt psl can restore rate and extent of strand displacement activity by hiv-rt p66 compared to the hiv-rt heterodimer d66/d51, suggesting a function of the 51 kda polypeptide, the mouse mammary tumor virus proviral dna contains an open reading frame in the 3' long terminal repeat which can code for a 36 kda polypeptide with a putative transmembrane sequence and five n-linked glycosylation sites. this gene is known to code for a superantigen which deletes a specific subset el co4 + t-lymphocytes in vivo, the superantigen encoded by the exogenous mouse mammary tumor virus of the gr strain acts specifically on v[314 bearing t-cells. we produced recombinant vaccinia viruses to express either the complete or a truncated orf protein after infection of primate cells in culture. the complete err gene in mammalian cells leads to the production of a 47 kda protein which is specifically detected with an anti-orf-peptide antiserum. the 47 kda protein can be labeled with d-[2-3h]mannose and its synthesis is inhibited by tunicamycin, an n-linked glycosylation inhibitor, indicating that it is a glycoprotein. the truncated orf protein beginning at the second atg of the open reading frame is also modified, but the c-terminal half of orf, starting at the fifth atg (orf5), has the expected size of the non modified polypeptide. pulse-chase experiments indicate that the 47 kda orf g}ycoprotein has a short half-life of about 1.5-2 hours in this system whereas the orf5 truncated protein is even less stable; its half-life is about 20 minutes. the cellular localization of the orf protein and the role it could play in vivo are currently under investigation. conclusion was based on a restriction fragment length polymorphism (rflp) with probes of is-elements. the mutants displayed different growth behavior. the evolutionary stability of 32 of these mutants was further tested by competing a mixture of them for ten days. polymorphism was maintained during this competition but two fitter mutants took over 60% of the population. implications of these results will be discussed in terms of generation and selection of genetic variants for the adaptation of microbial population. mouse mammary tumor virus (mmtv) is produced in the mammary gland of infected females and transmitted by the milk to suckling new-boms. the passage of mmtv from the gut to the mammary gland is still poorly understood, the nature of the tissue tropism of mmtv has not been elucidated, a single receptor present on mammary epithelial and lymphoid cells might serve for the entry of the virus. a cell-type specific entry mechanism could in part be responsible for this phenomenon. alternatively different surlace molecules might be involved in its uptake. we have studied the cell susceptibility to mmtv using a sensitive method of detection of newly acquired exogenous proviral dna sequences, prior to viral integration and transcription. thereby we confirm that mmtv infects mouse, rat and monkey cell lines of different tissue origin in vitro demonstrating the lack of a strict host range specificity in tissue culture as previously described. we have also observed the infection by mmtv of various human cell lines. furthermore various infection susceptibilities are observed for cell lines of a same species. r. cattaneo, p. spielhofer, k. kaelin, m.a. billeter mo/eku/arbio/ogie /, universit~t zorich, hsnggerberg, 8093 zorich subacute sclerosing panencephalitis (sspe), a rare, lethal disease is caused by clonally selected defective measles viruses (dmvs) characteristically mutated. this emerged from sequencing of five mv genes from sspe cases and by functional tests of the envelope glycoproteins hemagglutinin (h) and fusion protein if). these expressed h and f variants migrate poorly to the cell surface but maintain some cell fusion activity whereas the envelope associated matrix protein (m) appears dispensable. thus, the spread of dmv genomes through the brain seems to occur by membrane fusion at local cellular contacts, essentially hidden from the massive immune responses. in more than half of sspe cases, the propagated dmvs contain m genes in which up to 50% of u residues are replaced by cs. such "biased hypermutations" are likely due to the simultaneous conversion of many a residues to inosine in double stranded rna regions accidentally formed, mediated by an ubiquitously occurring adenosine deaminase. similar events at less spectacular degrees have now been observed in functional genes of several negative strand rna viruses. thus, this enzyme might be an important evolutionary driving force for mononegavirates. pull, i., wieland, s., nieder~st, b., keist, r., walter, e., and blum, h.e. department of medicine, university hospital z'tirich infection by hbv-positive organ donors remains a serious problem in transplant surgery. here we present a case of a 58-year-old woman who received a heart transplant from a hepatitis b surface antigen (hbsag) positive donor. the patient was negative for all hbv markers prior to transplantation. two months after transplantation she became hbsag positive and died from subacute liver failure about seven months later. cloning and sequencing of hbv dna revealed several hbv mutants in the serum of the recipient, each with a 108 bp in-frame deletion in the pre-s 1 region. transfection studies in huh-7 cells with the predominant pre-s 1 deletion mutant showed a higher replication competence compared to wild-type hbv. sequence analysis of pcr products from serum of the donor showed mainly wild-type hbv dna in the pre-s region without the pre-s 1 deletion. these data demonstrate that mutant viruses accumulate in immunesuppressed patients. specific mutants may play a critical role in the severe or fatal clinical course of hbv-infection in transplanted patients. in non-neuronal cells herpes simplex virus (hsv) establishes a productive lyric infection starting with transcription of its immediate early (ie) genes by corecruitment of a viral protein (vpi6) and a cellular factor (oct-l) onto characteristic oetamer-like sequence motifs ftaatgarat) in the promotors of the ie genes. in neurons, however, hsv establishes latent infections whereby transcription of the ie genes is prevented and the lyric program aborted. our efforts are focused towards determining which neuronal factors are involved in the regulation of hsv ie gene transcription and whether they may be involved in hsv latency or reactivation in neurons. in electrophoretic mobility shift assays (emsa) using mouse brain nuclear extracts we found that neuronal oct factors (noct-2, noet-3 and noct-4) bind to taatgarat sequences from the viral icp0 and icp4 promoters. an additional brain protein also binds to these sequences but not to a consensus octamer sequence (atgcaaat) as do neuronal oct factors. emsa assays with mutated taatgarat probes show that the taat sequence is crucial for this binding and we have tentatively named this brain protein taat-1. we are currently testing the noct factors in in vivo and in vitro transcription assays to study their involvement in hsv ie promoter regulation. biochemical and functional analysis of a vaccinia virus recombinant containing two copies of the p37k gene. schmutz, c., and wittek, r. institut de biologie animate, univei-sit~ de lausanne, ch-1015 lausanne. the most abundant protein of the vaccinia virus envelope is the palmitated 37k protein. this protein is required for envelopment and subsequent release of virus from infected cells. the amount of extracellular enveloped virus (eev) produced varies considerably depending on the virus strain and host cell line but remains in any case low (<30% of the virus production). if p37k is a limiting factor for envelopment, its overexpression might be a taeans to increase the production of eev. for this purpose a vaccinia virus recombinant containing a second copy of the p37k gene was enginee,'ed. western blot analysis clearly showed increased levels of this protein. titration assays revealed slightly lower yields of both intracellular naked virus (inv) and extracellular enveloped virus (eeu in ceus infected with recombinant virus. unexpectedly, with recombinant virus, the ratio of eev versus inv was significantly lower when compared to wild-type virus infection, despite the higher level of p37k expression. we are currently testing whether this is a consequence of a lower production, or a reduced infectivity of eev particles in cells infected with recombinant virus. hanspeter stalder, christian hertig and ernst peterhans institut f~r veterin~r-virologie, universit-st bern, ch-3012 bern bovine viral diarrhoea virus (bvdv) causes acute transient infection in animals exposed to virus postnataly and persistent infection in animals infected in utero in the early phase of gestation. the latter is associated with specific immunotolerance to the infecting viral strain. animals infected in utero may be born normal and remain healthy despite the persisting infection with a noncytopathic biotype of bvdv. mutation to the cytopathic biotype, or superinfection with an antigenically similar cytopathic biotype, results in lethal mucosal disease. we have pcr amplified and sequenced parts of the region coding for the surface glycoprotein e2 and p80, a nonstructural protein associated with enzymatic activities. over a time of one year, virus obtained from an immunotolerant persistently infected animal remained identical in its nucleotide sequence in the analyzed regions. contrasting with this remarkable evolutionary stasis over some 600-800 virus generations is the heterogeneity of viral isolates obtained from different persistently infected animals. we propose that the muitilineage distribution of bvdv is the result of antigenic drift in the population of acutely infected animals, combined with evolutionary stasis in immunotolerant animals. in 20 to 30% of naturally infected goats, the sevedty of disease in infected animals correlates with the antibody titers to the viral envelope protein (enw) and especially to the gp 38 transmembrane podion (tm). in order to analyze linear b epitopes of env, we produced a random library of caev env polypeptides fused to ~-galactosidase and expressed in e. coli. the complete env gene obtained from an infectious clone of caev-co was subcloned in a puc18 plasmid and dnase digested, random fragments of 200 bp were inseded in ~,gtl 1 dna and expressed in e. coil y1090. as the first step of b epitope mapping and in order to identify immunodominant group epitopes we used high titer sera from naturally infected swiss goats to screen the library. six immunoreactive clones were obtained and subsequently sequenced. all six mapped to the tm region of env. a fine mapping of these immunoreactive regions was performed using pepscan technology and elisa with synthetic peptides. four epitopes could be identified including the eysteine loop corresponding to an immunodominant epitope in other lentiviruses. these peptides were used in vitro to test the reactivity of sera from naturally and experimentally caev-infected goats and in rive to modulate the immune response of goats to tm. the minute virus of mice (mvm) has a 5kb linear single stranded dna genome. at both termini, short selfcomplementary sequences are folded into stable hairpin structures. the 5' (right) hairpin is a single stem structure with only three unpaired bases within the stem. these unpaired form a bubble structure. we examined the requirement in mvm lytic infection for the bubble structure in the 5' terminal hairpin of mvmp. mvmp rf dna containing the entire 3' extremity and extending to the hha i site downstream of the axis of symmetry of the 5' extremity was cloned into pgem-szf(+). this construct contains more than half of the 5' palindrome, however it lacks the sequence information required to form a bubble. murine fibroblast a9 cells were transfected with this dna and the 5' terminal sequence of resulting virus was analysed. a bubble was seen to be generated, however the sequence of nucleotides contributing to the bubble was of n0n-viral, plasmid origin. the nucleotides in question were removed from the original plasmid construct and the experiment repeated. virus was obtained as before. sequence analysis of the 5' end indicated that both flip and flop forms were present. the corresponding 5' hairpin deduced from these two forms contains no unpaired bases at the position where the bubble is normally located in wild type mvmp. the immediate early lie) gene promoters of herpes simplex virus type-1 share a similar sequence which in some cases overlaps with a binding site for octamer binding proteins. this sequence confers responsiveness to a viral transaetivator, vp16. on these promoters, vp16 combines with multiple cellular proteins to form an activating complex. we set up a reconstituted in vitro system consisting of hela nuclear extract and recombinant oct-1/pou domain protein (without the activation domain of oct-1 ) and vp16 where we showed that the activation of ie genes is not mediated by oct-i itself. rather, oct-1 serves to recruit vp16 that has no intrinsic dna binding capacity. vp16, however, possesses a stong acidic c terminal activation domain of -80 amino acids which is indispensable for the in vivo function of the protein. as this and other acidic activation domains possess only a relatively weak activating capability when we test them in in vitro transcription assays we are interested in investigating the role of this acidic activation domain in vitro i.e. under condition of cell free extracts and on naked template dna. we are using recombinant oct-1/pou domain protein and recombinant vp16 that has been truncated at the c terminus. additionally, we are also testing different truncations of vp16 in vitro that have been produced in cos cells. furthermore, we are also trying to see whether these different truncations already affect the assembly of the actvating multiprotein complex. we have studied the kinetics of synthesis of transcripts initiated from the vaecinia virus 7.5 kd gene early promoter. unexpectedly, after a first burst of rna synthesis early in infection, the promoter showed a second peak of activity in the late phase.reactivation of transcription was neither dependent on the presence of the sequences upstream of the early promoter, nor on the location of the promoter in the genome. reactivation seems to be dependent on virus assembly since it is prevented by rifampicin, that specifically inhibits an early step of viral morpho-g~nesis. analysis of several other early genes showed that reactivation is not unique to the 7.5 kd early promoter. analyzing, by in situ hybridization using digoxigenin-labelled riboprobes, the expression of the viral genome relative to that of an array of cytokines including tnf-(z; ifn-% il-i~, il-2, il-8 and gm-csf. initial experiments suggest that viral genome expression is restricted in the inflammatory infiltrates whereas certain cytokines, particularly tnf-oq are strongly expressed in the synovial membranes. overall, the results point to a prominent role of macrophage activation in the maintenance of chronic inflammation and possibly also in the development of arthritic lesions. the core protein of the hepatitis b virus (hbv) interacts with the viral rna pregenome and the reverse-transcriptase.jdna-polymerase to form a core particle which allows for replication of the viral genome. deletion analysis of the core protein revealed, that the arg-rich carboxyterminal domain is required for rna encapsidation and productive viral dna synthesis. we demonstrate, that the duck hepatitis b virus (dhbv) core protein can be derided into two domains comprising amino acids 1-67 (n-ter) and 67-262 (c-ter), respectively. the co-expression of these two core subdomains leads to the formation of replication competent core particles. furthermore, we demonstrate that the carboxy-terminal domain of dhbv core (c-ter) can be functionally replaced by the corresponding domain of the human hbv. the complete hbv core protein, however, does not form replication competent core particles with the dhbv pregenome and rt. these results imply that the carboxy-terminal part of the hbv core proteins define an exchangeable, functionally conserved domain. influenza ha mediates the adhesion and penetration of host cell membranes. acylation of three conserved cysteine residues in the membrane spanning region of several ha subtypes has been shown. the biological significance of this hydrophobic modification remains to be elucidated. a possible role for the membrane fusing activity has been controversially discussed in the past. removal of covalently bound fatty acid from protein by incubation with hydroxylamine is a commonly applied method. as we shall show for a number of different envelope viruses with acylated glycoproteins, this treatment leads to quite different consequences in a functional seiase depending on the virus used. as an alternative approach to study the biological significance of palmitoylation we expressed wild-type influenza ha and an hamutant lacking the fatty acid binding sites using the baculovirns system. both wild type and fa--mutant ha, after expression in insect cells bind to red blood cells, induce hemolysis, and fuse efficiently with fluoreseently labeled erythrocyte ghosts. hydroxylumine treatment of both recombinant wt-and mutant-ha leads to a loss of hemolysis and of fnsion activity. at least in the ease of fa--i-ia its inhibitory action must be related to some other effect than fatty acid cleavage. these results indicate that hydroxylamine treatment may not always be suitable for the generation of fatty acid free glycoproteius or virus particles for functional studies. gesemann and j.g. nicholls, biocenter univ. basel, 4056 basel, switzerland depolarization of leech neurons leads to growth cone collapse and neurite retraction. this response to depolarization is influenced by the substrata on which the cells are growing and is mediated by the influx of calcium (s. grumbacher-reinert and nicholls, 1992, j. exp. biol. 167:1-14; neely, 1993 , i. neurosci. 13:1292 -1301 . the morphologieal changes induced by depolarization of leech neurons growing on leech extracellular matrix extract is accompanied by a loss of microfilaments in the growth cones. leech neurons growing on a different substrate, the plant lectin coneanavalin a (coma), did not retract their neudtes or lose rnierofilaments, but continued to grow after depolarization. we attribute this to the reduced calcium influx during depolarization of neurons on cona (ross et al., 1988, pnas 85:4075-4078) . increasing the intraeellular calcium concentration by incubating neurons with the calcium-ionophore a23187, led to cessation of motility and disruption of microfilaments but not microtubutes in growth cones on coma. to investigate the mechanism by which calcium affects the organization of microfilaments we stained neurons with antibodies against gelsolin, a protein that severs microfilaments when it is activated with calcium. we have observed that these antibodies colocalize with microfilaments in leech growth cones, we are now analyzing the nature of this antigen that cross-reacts with the anti-gelsolin antibodies with the goal of establishing its potential role in the calcium-induced disruption of microfilaments in leech neuronal growth cones. this work was supported by a grant from the swiss nationalfond no. 3127814.89 to j.g. nicholls. activation of metabotropic glutamate receptors (mglurs) was studied in voltage-clamped ca3 cells in rat hippocampal slice cultures using the whole-cell pateh-clamp configuration. in saline containing 16 mm k +, 1s,3r-acpd (50 ~tm), a mglur agonist induced an inward current associated with an increase in membrane conductance. the reversal potential, assessed with a voltage ramp protocol from -80 to -60 mv and calculated by linear regression, was -15.7 â�¢ 18.7 mv, and was shifted to -53.4 â�¢ 6.4 mv when the concentration of external monovalent cations was halved. these results indicate that the conductance underlying this current is selective for cations (mainly k + and na+). the steady-state i/v curve for the 1s,3r-acpd-induced inward current showed a slight inward rectification. in most of the cells, this inward current was followed (or overlapped) by an outward current concomitant with a decrease in a k + conductance (ere v = -51.2 ~ 5.6 mv in [k+]o = 16 mm and shifted to -74.0 :t: 2.8 mv in [i4+]o = 8 ram). this k + current was voltage-independent in the membrane potential range of-120 to -20 mv. similar results were obtained with methacholine, a cholinergic muscarinic agonist. the non-specific cationic current, seen only in the presence of elevated extracellular k + concentration, could play an important role during intense neuronal activity. the c-myc protein has been implicated in the regulation of cell growth and differentiation, c-myc specifically interacts with max which in turn forms heterodimers with mad and mxil and homodimera with itself. we present evidence that this network o! proteins is regulated both at the level of relative protein concentration as well as by post-translational mechanisms. in the hematopoietic cell lines u-937, ml-1 and hl-6o e-myc mrna and protein are rapidly downregulated after induction o| differentiation whereas max expression is not significantly altered. in contrast the expression of mad is induced with properties similar to immediate early genes, taxi1 is induced to a lesser degree and with slower kinetics, in an effort to study the regulation of o-myc we have mapped all the major in vivo phosphorylation sites in both c-mye and max. two sites in the n-terminal transactivation domain are important for the regulation of the transforming potential el c-myc both in established cell lines as well as in primary rat embryo tibroblasts. however, no difference in the transactivating potential of corresponding mutants was observed. phosphorylatien of max at sites close to the basic region increases both on-and off-rates of either myc-max hetero-as well as max homodimers to dna. brain research institute, university of z~rich, ch-8029 z~rich, switzerland gaba b and adenosine receptors act via pertussis toxinsensitive g-proteins of the gi/g o class to mediate longlasting inhibition in the cns. this class of g-protein is negatively coupled to the enzyme adenylyl eyclase. our aim was to determine whether the inhibition of adenylyl nyclase mediated by these receptors has functional consequences for electrical signaling in hippocampal neurons. the calciumdependent k + current, i~, underlying adaptation of cell firing, was used as an electrophysiological bioassay for adenylyl cyclase activity, as it is very sensitive to intracellular levels of camp. accordingly, the ~-adrenergic agoniat iaoproterenol (5-500 nm), that activates g,-linked receptors, reduced the amplitude of i~ by 51.5 â�¢ 3.6% in voltage-clamped ca3 pyramidal cells in ttx (n=20). reduction of imm by isoproterenol was curtailed to 27.3 â�¢ 2.9%, (p < 0.001) in the presence of baclofen (i0 ~m), acting at gaba b receptors, or adenosine (50 nm). thus, the inhibition of adenylyl cyclase activity due to activation of gaba a and adenosine receptors can modulate responses to other neuretransmitters by an interaction occurring at the level of intracellular signal transduction. furthermore, stimulation of these receptors will tend to enhance i~, suppressing repetitive cell firing. this represents a further mechanism which will result in prolonged inhibition of hippocampal neurons. a study of the possible modulation by nitric oxide (no) of endogenous dopamine (da) release was performed in bovine retina in vitro. isolated half retinas were incubated in kkg at 37 ~ for 3 successive 30-rain incubations, and da was measured in the incubation medium by hplc with ec detection. when retinas were incubated in the presence of the no-generating compound hydroxylamine (300 ttm), a decrease in either basal or k +-(50 ram) stimulated da release was observed during the 2 nd 30-rain period. tested under similar conditions, dibutyryl cgmp (1 mm) was ineffective. the no synthase inhibitor l-ng-nitro-arginine methyl ester (l-name, 100 gm) increased basal da release during the 1 st and 2 nd incubation, whereas it did not affects the k+-stimulated da release. in addition, its effect were potentiated in calcium-free medium. finally, we have also shown in cell-free experiments that retinal no synthase activity was totally calcium-dependent and blocked by l-name. taken together, these results suggest that endogenous no regulates da release via a cgmp independent mechanism. division of endocrinology, university hospital, ch-1211 geneva 14 while the stimulation of aldosterone biosynthesis by angiotensin ii (angii) in bovine glomerulosa cells clearly depends upon a sustained influx of ca 2+, the pathway for ca 2 ⧠entry is not yet accurately defined. at least two mechanisms seem to be involved: 1) the opening of voltage-operated ca z ⧠channels, and 2) the stimulation of a "capacitative" ca 2+ entry regulated by intraocllular ca 2 ⧠stores; however the relative contribution of these pathways to the stimulation of steroidogenesis needs to be determined. two pharmacological tools were used: nieardipine, a blocker of to and l-type ca 2+ channels, and thapsigargin, which releases ca ~+ and therefore induces a capacitative ca ~+ influx. nicardipine (1 lam) completely blocked the cytosolic ca 2+ response to k +, which exclusively activates voltage-operated ca 2. channels, but only partially (22 %) the response to angli. in the presence of nicardipine, angll was unable to stimulate a ca 2+ influx aiter depletion of ca 2 ⧠stores with thapsigargin, demonstrating that angli principally stimulates a capacitative ca 2+ entry pathway. in addition, nicardipine completely blocked the steroidogenic response to k +, but only 30% of the response to angll. thapsigargin-induced aldosterone formation, as the response to angll, was markedly potentiated by low concentrations of k +. in conclusion, this study shows that in bovine glomerulosa cells, angli activates a sustained ca 2+ influx, principally through a capacitativc pathway, leading to aldostcrone formation. this finding could partially explain the poor efficiency of dihydropyridine ca 2 ⧠antagonists for preventing aldosterone secretion. harald holder and john m. lucocq*. institute of anatomy, university of berne, ch 3012 berne *depanment of anatomy and physiology, university of dundee, uk dundee dd1 4hn. subjection of hela cells to 45~ for 30 minutes leads to a complex activation pattern of map-kinases as revealed with renaturadon gels. exponentially growing hela cells show activallon of a myelin basic protein (mbp) phospborylating kinase migrating with an apparent molecular mass of around 60kda upon heat shock induction. in contrast, in cells arrested in go by serum starvation for 24h, essentially three different proteins phosphorylafing mbp are activated subsequent to heat shock treatment. these kinases migrate with apparent molecular masses of around 32kda, 44kda and 55kda. western blot analysis with antibodies recognizing the human boraologues of erkl and erk2 revealed that considerable pools of erkl and erk2 show retarded elec/rophnretic migration behavioar indicating that they are phosphorylated and probably activated in heat subjected and growth-arrested hela cells. down-regulation of protein kinase c (pkc) by a prolonged treatment with 12-o-tetradecanoyl-phothol-13-acetate (tpa) did neither change the major activation pattern observed upon separation of cell lysates on renaturation gels nor that one observed on western blots decorated with anti-erkl or anti-erk2 antibodies. these results suggest that heat shock induced activation of at least erkl, erk2 and the 55kda mbp-kinase is not mediated via activation of pkc. this work was supported by swiss nsf grant no. 31-27636.89. effects of dominant negative glucocorticoid receptor mutants in cell cultures and transgenic animals stefano brenz verca, stefan schneider, sandro rusconi, institut fiir molekularbiologie h der universitat zf~rich, winterthurerstr. 190, switzerland the glueocorticoid receptor (gr) is involved in a large number of developmental and biochemical processes. recently, we obtained a trans-dominant negative gr mutant (tdn) -called gr[aia] -by a frame shift of the cag-repeat of the rat gr (see poster by hug et al.) . the gr[ala]-mutant shows normal llgand-binding and dna-binding but cannot stimulate transcription (lanz et al., steroids; in press) . modulatable versions of the tdn-mutant have also been created by substituting the hbd of the grmutant with the hbd of sex-hormone receptors. these constructs have only been tested so far in transient cotransfection assays. therefore, we are currently trying to express them permanently in cell lines (rat hepatoma cell line fto-2b) in order to verify whether they can affect the transcription level of a known endogenous gr-dependent target-gene, like the tyrosine aminotransferase (tat) gene or a permanently transformed mtv-iacz reporter. so far no rapid test-system has been described, that allows the quantitative analysis of such effects in transient expression assays. we are about to set up such a rapid and generally applicable transient test-system. in the future we plan to express dominant negative gr mutants in a tissuespecific manner in transgenic mice. it will be particularly interesting to investigate their effects on the immune system or on liver functions. these studies investigated growth cone responses m brief local encounters with surface molecules implicated in neuronal pathfinding using chick drg neurons. we tested two interrelated hypotheses: 1) discrete points of information (guideposts) override sustained surface information and dominate growth cone behavior and 2) guidance molecules provide such information by activating second messengers instead of simply increase adhesivity. the potential guidance molecule lamialn was covalentiy coupled to polystyrene beads in order to mimic guidepost cells. encounters with laminin model guideposts significantly altered the behavior and morphology of growth cones advancing on fibronestin or polylysine. growth cones steered towards theses beads in a saltatory medea once a long lasting contact with a single filopodium had been established. subsequent to a pause at the bead, growth cones advanced with a significantly increased growth rate for the next llmin. the average increase in rate stimulated by bead contact was 5 times the basal rate on polylysine and 2.5 times that on fibronectin. positioning of multiple model guideposts along a path and within filopodial reach maintained this bead stimulated rate of outgrowth and directed growth cone advance. the laminin induced pattern of events was totally blocked in the presence of either h7 or sphingosine, two pkc inhibitors, or neomycin, a plc inhibitor. neither of these inhibitors affected neurite outgrowth on the substrata alone. our results demonstrate the necessity of filopodial contacts, the importance of spatially restricted stimuli and the activation of transducing pathways as key mechanisms in neuronal pathfinding. mechanisms of glucocorticoid receptor desactlvation by homopolymeric alanines martin hug, manuela h&fferer and sandro rusconi, institut fiir molekularbiologie 11 der universitat uz zart'ch, winterthurerstrasse 190, 8057 ziirich, switzerland the rat glucocorticoid receptor (gr) edna contains in its y-portion a (cag-repeat that encodes a siring of 22 gin residues interrupted by 1 arg. a mutant with the frame-shifted repeat which codes now for poly-ala residues (called gr[aia]) is completely trans-inactive, is more cytoplasmitally located in absence of ligund and exhibits dominant-negative properties in presence of ligand (lanz et al., steroids, in press) . we generated gr recombinants with increasing numbers of cag-triplets in three possible reading frames (oligo-gln, -ser or -ala). the gr-oligo[ala]~-~.23 mutants showed an activity comparable to wild type gr, whereas the groligo[ala]>_25 were inactive. the severity of the effect of oligo a.la su'ings seems to be dependent on the position of the repeat along the primary sequence (e.g. progressive decrease of the effects by moving toward more earboxy-position). we have also examined the effects of the oligo[ala]21, 23, 25, 27, 31 on gal4 chimeric factors and found that the inhibition by ala repeats is probably factor-specific. our results made us speculate that possible differences in post-translational modifications may be at the root of the negative effects by the ala repeats. we believe that these effects are due to transient association of the nascent gr[aia] mutant proteins with intraceuular membranes. these hypotheses are currently tested. the activation of steroidogenesls in calcium-clamped bovine glomerulosa cells and its modulation by angiotensin ii and camp, python cp, laban op, rossier mf, vallotton mb and capponi am division of endocrinology, university hospital, geneva, switzedand. the ca2+-messenger system plays a crucial role in the regulation of steroid secretion in adrenal zona glomerulosa cells, as it is known to mediate the action of angiotensin ii and potassium ion. in the present study, we used intact isolated glomerulosa cells in which the cytosolic free ca 2+ concentration ([ca2+]c) was clamped at various levels with the ca2+-ionophore, ionomycin, in order to locate the sites of action of ca 2+, to determine their sensitivity towards ca 2+ and the modulation by other physiological factors. by measuring simultaneously steroid synthesis and [ca2+]c, we show that ca2+-clamping (50-860 nm) induced a concentration-dependent increase in the production of both pregnenolone (506___70 % of the basal level) and aldosterone (255+27 % of the basal level, ec50 = 273 nm). by contrast, ca 2+ did not influence the conversion of 1 t-deoxycorticosterone into aldosterone at concentrations up to 600 rim, although at higher concentrations we observed a modest but significant inhibition. in addition, ca2+-clamping did not affect the formation of pregnenolone from a freely diffusible analogue of cholesterol, 25hydroxycholesterol, indicating that ca 2+ acts at a step upstream of cholesterol side-chain cleavage. both angiotensin ii and a membrane-permeant surrogate of camp, dibutyryl cyclic amp, potentiated the steroidogenic response to increases in [ca2+]c . in summary, these studies reveal an exquisite sensitivity of the glomerulosa cell steroidogenic pathway toward very small physiological changes in [ca2+]c. radtke, rainer heuchel, oleg georgiev, gerlinde stark#, michel aguet# & walter schaffner institut fiir molekularbiologie 11, universilat ziirlch, 8057 z'6rich # lnstitut rir molehdarbiologie i, universitiit ziirich, 8093 ztirich we have previously described and cloned a factor (mtf-1) that binds specifically to metal-responsive dna sequence elements in the enhancer/promoter region of metallothionein genes. mtf-1 is a protein of 72.5 kda that contains six zinc fingers and multiple domains for transcriptional activation. here we report the disruption of both alleles of the mtf-1 gene in mouse embryonic stem ceils by homologous recombination. the resulting null mutant cell line fails to produce detectable mounts of mtf-1. moreover, due to the loss of mtf-1 the endogenous metalinthionein-i gene is silent, showing that mtf-i is required for both basal and metal-induced transcription. accordingly, reporter genes containing either synthetic or natural metal-reslxmsive promoters were not transcribed after transfection into the null mutant ceils. cotramsfection of the mtf-i expression vector rescued both basal and heavy metal-induced transcription. these results demonstrate that mtf-1 is essential for normal metallothionein gene regulation. rat pheochromocytoma cells (pc12) were differentiated with ngf for 3-4 days, a time sufficient for the formation of growth cones and elongation of neurites. addition of the phosphatase inhibitor calyculin a (cl-a) (0.2.50.0 nm) led to a concentrationdependent collapse of growth cones and retraction of the neurites within 15 minutes. after the retraction, the cell bodies showed the appearance of a grape-like domain opposite to the cell nucleus. they recovered to normal shape within about 30-60 minutes if the inhibitor was washed out. the neurite retraction was further analysed using both low-light level fluorescence video-microscopy and fura-2 single cell microfluorimetry. the onset of retraction started in the central part of the growth cone prior to a complete collapse of filopodia. the basal intracellular free calcium concentration ([ca2+] i) remained constant during neurite retraction. as a measure for the viability of cl-a-treated cells, agonist-isduced responses of [ca2+] i were analysed. ca2+ entry across voltage-activated ca 2-~ channels was inhibited by 50% after 30 minute treatment with cl-a, while the atp-induced ca2+ entry via ca2*-permeable cation channels was not significantly reduced. however, the onset of the atp-induced rise in [ca2+]i started at the grape-like domain. the speed of the ca 2+ wave across the cell body was slower than in control cells. the addition of 0.5 gm okadaie acid, a different type of phosphatase inhibitor, caused no cell shape change or neurite retraction within 24 hours. taken the specificity of the two phesphatase inhibiters into account, the observed effects seem to be induced by inhibition of a ppl-class phosphatase. myosin in nematocytes of hydra michel nakano & robert p. stidwill dept. zoology, university of ztirich, switzerland the functions of nonmuscle myosins during cell locomotion are not clear. several models have been presented reaching from very active participation of the molecules in different parts of cells to only minor or no roles at all. there is increasing evidence that the functions of myosin ii (capable of forming bipolar filaments) are quite distinct from the ones of myosin i and generally that the different members of the growing family of myosin-like proteins may not all have identieal functions. we have attempted to demonstrate the occurrence and determine pattern of myosins in tissue of hydra and especially in migrating nematoeytes mainly for two reasons. first, nematocytes are fairly rapidly moving cells which can be studied in vitro and, second, the question is of interest from a evolutionary point of view. we have utilized several antibodies against vertebrate and invertebrate myosins for immunofluoreseenee (confocal laser scanning microscopy) and western blotting studies. in addition to these results findings on the screening of a ~.zap c-dna library are presented. the jaki protein tyrosine kinase is a member of a family of intracellular kinases characterized by having two kinase catalytic domains: a bona fide tyrosine kinase domain and an amino terminally positioned kinase-related domain. recently it has been shown that jaki and the other members of this family (jak2, tyk2) are intimately involved in cytokine and growth factor signalling. the presence of two putative nuclear localizing sequences in the amino terminus of the protein prompted us to examine the subcellular localization of this protein. immunohistochemical and biochemical analysis revealed that a part of jaki was localized to the nucleolus. metabolic labelling showed that (a) the jaki protein partitioned rapidly into the nucleus and (b) the nuclear jaki became smaller with time. the size difference was not due to either co-translational glycosylation or phospherylation. these results suggest that part of the jaki protein is rapidly translocated to the nucleoli where it becomes processed. revealed that this clone is expressed predominantly in spleen, mammary gland and thymus as two transcripts, a major species of approximately 5kb and a minor species of approximately 4kb. nucleotide sequence analysis revealed a 84% homology to the porcine syk gene. the syk gene is expressed as a major 3.0kb transcript and has been reported to be lymphoid specific. due to the discrepancy of transcript size and number, we suggest that our cdna clone represents a novel member of this family of ptks. we are currently isolating a full length sequence from a mouse spleen cdna library. in addition, we want to characterize which cell(s) in the mammary gland are responsible for the observed expression. expression and function of g-protein isoforms were studied in differenflaring (6c8) and nondifferentiaflng ((33) subclones of red-1 rauscher murine erythroleukemia cells. erythroid differentiaflon induced by the combined action of erythropoietin (epo) and dmso was associated with a selective loss (>70%) of immunoreactive gai3. simultaneously, a iruncated form of g~2 accumulated in the cytosol, while the membrane concentrations of gai2, gets and gaq/11 remained unchanged. nondifferenfiating (33 cells contained sigrtificanfly higher levels of most get subunit isoforms that remained unchanged upon epo/dmso treamaent. changes in adenylyl cyclase activity and in intracellular ca ion levels ([ca]i) resulting from activation of g-protein-coupled receptors were monitored for differentiation-dependent effects on transmembrane signaling. adp-mediated changes of [ca]i in native and differenflaflng 6c8 cells were consistent with p2t receptor coupling to gai3. thrombin receptors seemed to couple preferentially to gai in g3 cells and to gaq in 6c8 cells. our results document substantial alterations in g-protein-mediated signaling during erythroid differentiation. to investigate the involvement of protein tyrosine kinases (ptks) in the growth control of the mammary gland, we have used pcr based cloning to identify ptks expressed during normal mammary gland development. this approach led to the isolation of three members of the eph-related family of receptor ptks: myk-;, a novel member expressed predominantly in lung, heart and mammary gland; m-eck, the murine homologue of the human eck gene and mek5, the murine homologue of the chicken cek5. all three ptks were expressed in a distinct and narrow window of mammary gland development: puberty and estrus cycle. no expression was found either in late pregnancy or in the end differentiated state of lactation. 0vet-expression was found in the undifferentiated and invasive tumors of transgenic mice expressing the h-ras oncogene. in contrast, no elevated expression of all three genes was detected in the well differentiated, non-invasive mammary tumors characteristic of c-myc expressing transgenic mice. these results suggest that members of this family of ptks are involved in the control of proliferation of the mammary gland but are incompatible with its differentiation. np-tcii is a lymphocyte specific transcription factor (lattion a.-l. et el., mol. cell. biol., 1992, 12:5217-5227 a nucleotide receptor that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases c and d, respectively. here we report that atp and utp potently stimulate mesangial cell proliferation. both nucleotides stimulate phosphorylation and activation of map kinase and the up-stream map kinase kinase. activation of map kinase by both nucleotides is dose-dependently attenuated by the p2 receptor antagonist suramin. stimulation of protein kinase c (pkc) by phorbol ester increased map kinase phosphorylation and activation, and downregulation of pkc partially inhibited atp-and utp-induced map-kinase activation. inhibitors of pkc which display a selectivity for the ca2+-dependent isoenzymes (c~, [3, 7) , as compared to the ca2+-independent isoenzymes (5, e, ~, rl) such as staurosporine and the specific pkc inhibitor cgp 41251 did not inhibit nucleotide-stimulated map kinase phosphorylation, thus implicating the involvement of a ca2+-independent pkc isoform. in conclusion, these data suggest, that atp and utp trigger the activation of the map kinase signalling cascade in mesangial cells and this may be responsible for the potent mitogenic acitivity of both nucleotides. subcellular ion-gradients in ca signaling p. lipp & e. niggli, department of physiology, univexsity of bern recent experimental evidence has indicated an important role for submicroscopic concentration gradients during ca-signaling in various cell types. in heart muscle cells influx of ca via l-type ca channels triggers ca release from the sarcoplasmic reticulum (sr) and links electrical excitation to mechanical contraction (ec-coupling). we used a ratiometric confocal method (fluo-3 and fura-red) to follow the intraceuular ca-concentration while ha-and ca-currents (inn, ica) were elicited in the whole cell mode of the patch-clamp technique. both, icand in, were able to trigger ca release from the sr. kinetic differences between ini and ic -induced ca-transients, li-substitution experiments and the existence of a residual inn-induced ca transient in the absence of sr function suggested that these ca signals were mediated by the ha-ca exchange running in the ca influx mode. control experiments showed that ins-induced ca transients did not result from spurious activation l-type ca channels. the significant increase of the na concentration required to activate the ha-ca exchange can only occur provided cytosolic diffusion of na is restricted in the vicinity of the exchangers. this limited diffusion results in significant ha-gradients in the subsarcolemmal space. in conclusion, i~iinduced ca influx may represent a safety factor for ec-coupling while ic, ensures sustained ca-release and is the source for refilling the sr with ca. supported by snf. the regenerative ca-induced ca release (cicr) mechanism is an important amplifier in cardiac signal transducilon. the cicr contributes to the ca transient responsible for mechanical activity, but also generates ca waves propagating within the cytosol. we investigated subcellular ca signals in neonatal rat and adult guinea-pig ventricular myocytes using ratiometric confocal microscopy with a mixture of two fluorescent ca indicators. individual resting myocytes exhibited spontaneous ca release events from the sarcoplasmic reticulurn (sr) that were attributable to three distinct patterns: i) local ca release events without propagation; ll) planar ca waves propagating across the cell; iii) spiral ca waves spinning around a nucleus or shifdng along a subcellular region without entering it. transitions between these patterns were frequently observed, suggesting that a particular release pattern is not the property of an individual cell but reflects the functional state of the sr. the existence of focal release and refractory zones within a single cell indicates that the sr is not uniform on the subcellular level. the sr seems to he a network of elements with distinct functional properties. a suhcellular variability in the positive feedback of the cicr mechanism can account for the observed features of ca signaling. the degree of positive feedback exhibited by a single sr element may depend on its ca content. to characterise the intracellular signalling and regulatory properties of the cloned parathyroid hormone (pth) receptor we have stably transfected two renal epithelial cell lines with cdna's encoding the pth receptor (provided by dr g. segre). the proximal tubular, porcine (llc pk1 [clone 4]), and the distal tubular, madine darby canine (mdck) cell lines were transfected since the culture of either cell line on permeant filter supports leads to the development of polarised epithelial cell layers that mimic the epithelial organisation in vivo. transfection of the cloned pth receptor into each of these cell lines allowed us to examine, independently, the functional properties of the receptors expressed at the apical and/or the basolateral cell surface. while the basolateral application of pth produces a large increase in camp production in both cell lines, the relative efficacy of an apical application of pth differs between the cell types. the transfected llc pk1 cells exhibit a greater response to apical pth compared to the transfected mdck cells. pth-mediated regulation of intrinsic phosphate transport was also examined in polarised, transfected mdck cells. neither apical or basolateral application of pth affects intrinsic apical or basolateral ha-dependent or phosphate transport activities. similar experiments are being performed with the transfected llc pk1 cells. a. and preiss, a. biozentrum der universit&t basel, ch 4056 basel hairless, a recessive larval lethal mutation, causes dominant defects in sensory organs of adult drosophila flies.manifold genetic interactions indicate that hairless antagonizes neurogenic gene function suggesting an involvment also in neurogenesis. in order to gain insight into its function we have cloned the hairless gene which encodes an extremely basic, very serine rich protein of ii0 kd calculated molecular weight. no significant homologies to proteins of known function could be identified. therefore, we are concentrating by various means on a structure -function analysis of the hairless protein. firstly, we are testing a series of hairless deletion constructs for rescue capacity and generation of anti-hairless phenotypes after overexpression compared with the wild type gene. secondly, we are analysing hairless protein distribution throughout development, also at the subcellular level. heat induced hairless protein runs at ~ 150 kd, possibly due to phosphorylation which might be intrinsic to hairless function, a hypothesis we are currently scrutinizing. thy-1 and cd26 surface glycoproteins are both capable of delivering proliferative signals to t cells upon cross-linking with appropriate antibodies. the tyrosine phosphatase cd45 is a key regulator of t cell proliferation as it controls the activity of src family kinases p56/~k and p59fy n. associations of thy-1 with cd45, and of cd26 with cd45 have been reported after chemical cross-linking of intact cells and it is likely that such associations constitute part of the structural basis for the t-cell stimulating capacity of thy-1 and cd26 accessory molecules. we report that thy-1, cd26 and cd45 can be specifically crosslinked at the cell-surface and isolated as multimolecular complexes containing a 78 kda protein that is recognized by an anti-bip (grp78) antibody. in vitro kinase assays show the selective association of p56 ick and p59fy n with thy-1 and cd45 contained in multimolecular complexes. the multimolecular structure we describe could representa transducing unit incorporating surface receptors and regulators of tyrosine phosphorylation that are stabilized through interaction with the bip protein in the plasma membrane. structure-function-analysis of hairless, a gene involved in drosophila nervous system development maier, d., functional coupling of ppars and trs ijpenberg, a., wahli, w., desvergne, b., institut de biologie animale, 1015 lausanne we are interested in a possible functional coupling of thyroid hormone (tr) and peroxisome proliferator-activated receptors (pfar). to this end, we performed cotransfection experiments in nih 3t3 cells using cat reporter constructs containing the promoter of either the t3 responsive malic enzyme (me) gene, or the ppar regulated acyl coa oxidase (aco) gene. the me reporter construct showed very moderate response to the activated mouse ppar (mppar), whereas combination of try1 and unactivated mfpar potentialized the t3 induction. a putative ppar response element was identified within this promoter and will be further investigated. the aco reporter constructs were not responsive to trs. in constrast, a tr and t3 dependent inhibition of the fpar mediated activation was observed. the mechanism of functional coupling of these receptors, which may possibly involve the 9-cisretinoic acid receptor rxr, will be further investigated by molecular analysis. unliganded steroid receptors form a complex with hsp90 via their hormone binding domain (hbd). the receptor activation involves a hormone-induced release of hsp90. we genetically addressed the role of hsp90 in budding yeast by analyzing three kinds of hsp82 (the essential yeast homologue of hsp90) mutants. these mutations affect either the quantity of the protein (low versus normal levels), its integrity (deletion analysis) or its nature (hsp82 homologues from other species). hsp82 mutant are examined for their ability to complement a hsp82 deletion mutant. moreover, they are tested for functional interaction with a hbd. interestingly, a viable deletion of a charged region, suspected to be involved in hsp82-hbd interaction, only slightly interferes with hormonal activation of both estrogen or glucocorticoid receptors. furthermore, a short deletion in the last third of hsp82 leads to the production of a dominant negative mutant which prevents ceil growth at elevated temperature. the ability of various hsp82 homologues to palliate or not the absence of the yeast protein allows us to define, using chimeras, the regions of the protein which are necessary and sufficient to support cell life. zhang,h.,reynaud, s. and spohr,g. d~partement de biologie cellulaire, sciences iii, 30, quai ernest-ansermet, ch-1211 gen&ve 4 id cdnas expressed during early xenopus development have been isolated and sequenced. the encoded protein is homologous to the proteins described in higher vertebrates. northern blot analysis reveals that transcription starts soon after mid blastula and decreases to some extent after tailbud-stage. lower levels of transcription are detected also in adult frog tissues such as liver and heart. microinjection into oocytes of in vitro-synthesized myodor/and id-mrna, along with a cat reporter gene whose expression is under the controll of an a3-actin promoter supplemented with four e-boxes shows that myo d function is repressed by id. to investigate the importance of negative regulatory sequences we have isolated and characterised the id promoter. as a strategy for identifying cis-regulatory elements, chimeric genes consisting of different 5' elements of the promoter were attached to the cat coding sequences and microinjected into embryos. we are studying the tissue distribution of the rat peroxisome proliferators activated receptor (ppar~), a member of the nuclear hormone receptors superfamily, during development and in adults. high levels of ppar~ mrnas are detected in adult liver, kidney and heart. muscle, stomach, and intestine show moderate amounts of the ppar~, whereas brain, testis and lung present a very low expression. during postnatal development, we observe a continuous increase of the ppar~ expression in the kidney, in contrast to a steady level in the liver. anjard, c., groux, b ., gamboni, s. and reymond, c.d., institut d'histologie, unil, rue du bugnon 9, 1005 lausanne. the camp dependent protein kinase (capk) from dictyostelium is composed of a single catalytic (c) and a single regulatory (r) subunit. the c subunit we characterised, pkac is about twice the size of its mammalian counterpart. we showed that it possesses all properties of a catalytic subunit. it associates with the r subunit in absence of camp, it copurifies with capk activity and an increased activity is observed in cells over-expressing pkac. furthermore, we dissected its role during development and cell differentiation. overexpressing pkac under cell type specific promoters allowed to show the requirement for its activity during spore differentiation. pkac seems to play a more complex role in prestalk cells. we are now dissecting the functional regions of the pkac protein, making use of the known tertiary structure of the c subunits of mammalian enzymes. dna binding properties and stimulation of gene transcription of baculovirus expressed xppar~. hihi, a.k, mermod, n., medin, j., ozato, k. and wahli, w., inst. de biol. animale, uni lausanne, ch-1015 lausanne. lab. of mol. growth reg., nih, bethesda, md, 20892 usa. the peroxisome proliferators activated receptors (ppars) are members of the steroid/thyroid nuclear receptor superfamily. so far, they have been found in amphibians, rodents and humans. in xenopus laevis, 3 subtypes (xppara,~.y} have been isolated. in order to study the mode of activity of the ~ form, the xppar~ gene product was overexpressed using a baculovirus expression system. the nuclear protein obtained has the correct molecular weight of 46 kd. gel shift assays showed that nuclear extracts containing the recombinant protein are able to specifically bind the 'ppar response element' which is a direct repeat of the core aggtca motif. this dna binding activity is potentiated by another nuclear receptor, the mouse rxr~ and is inhibited by phosphatase, suggesting a role for phosphorylation in ppar binding to dna. a functional approach, using an in vitro transcription system, was chosen to define ppar and rxr action on transcription stimulation, as well as the role of their respective activators. arachidonic acid (0.5 -20 ram) induces various patterns of ca2+i signal in bronchial smooth muscle cells, as revealed by single cell dynamic video imaging of fura-2 loaded cells. most frequently, ca2+i oscillations were observed, although other patterns (a single ca2+i transient; a single peak followed by a sustained elevated level, or a sustained ca2*i elevation solely) were occasionally seen. the amplitude of ca2*i oscillations was related to the concentration of arachidonic acid. the frequency histogramm was noticeably shifted to higher frequencies by nordihydroguaiaretic acid (ndga, 0.2 rtm, a lipoxygenase inhibitor), whereas it was markedly shifted to lower frequencies by indomethacin (50 pm, a cyclooxygenase inhibitor) and by 5, 8, 11, 14-eicosatetraynoic acid (etya, 3 ~tm, a lipoxygenase and cyclooxygenase inhibitor). these observations suggest that: (1) arachidonic acid per se elicits ca2+ i oscillations in these ceils; (2) cyelooxygenase activity products modulate the frequency of these oscillations. promoter analyses of a growth factor regulated gene t. triib, m. kalousek, r. kessler, and r. klemenz dept. of pathology, university hospital, 8091 ziirich stimulation of quiescent cells to enter the cell cycle results in altered gene expression. some of the first genes to be induced (immediate early (i.e.) genes) encode transcription factors which are thought to pass on the mitotic signal to the delayed early (d.e.) genes. we have analysed the promoter elements required for growth factor mediated induction of the d.e. mouse gene t1 which encodes a secreted glycoprotein of the immunoglobulin superfamily. a 448 bp region located between 3.6 and 4.0 kb upstream of the transcription start site is sufficient for growth factor mediated inducibility. it contains an ap-1 binding site which is absolutely required for t1 gene expression. it is surrounded by 3 e-boxes. at least 2 of them are essential for efficient gene induction. thus the i.e. proteins encoded by the fos and jun gene family which form the ap-1 complex can activate the d.e. gene t1 in collaboration with helixloop-helix transcription factors binding to the e-boxes. the increase of radiolabelled inositol phosphates ([3h]ips) production in agonist-stimulated human airway smooth muscle cells (asmc) was determined by hplc. in order to observe the role of calcium, pkc and pla2 on plc activity during agonist stimulation, the effects of pharmacological agents (able to alter calcium, pkc and pla2) were tested on ip production elicited by a 5 sec stimulation with histamine. the inhibitor of pkc staurosporine increased, while pma (an activator) decreased the histamine-stimulated production of [3hi 1,4,5-ip3 and of its degradation products. an inhibition was also observed with the two pla2 inhibitors, quinacrine and p-bromophenacyl bromide. in calciumfree buffer, no difference in the ip increase was shown, but an inhibition was observed when thapsigargin, an inhibitor of the ca 2+, mg 2+-atpasc of the sarcoplasmic reticulum, was added in the same conditions. the calcium ionophore ionomycin increased the lp production in presence of external calcium, whereas it completely blocked the stimulation in calcium-free buffer. the results suggest that plc activity, in human asmc, is modulated by a positive feedback of calcium and by a negative feedback of pkc and pla2. regulation of hormone-stimulated calcium signals m. boolman, afrc, laboratory of molecular signalling, dept. zoology, cambridge, uk stimulation of cells with hormones that activate inositol 1,4,5-trisphosphate (insp~) formation, leads to an increase in the intracellular ca 2. concentration ([ca2+]~). these hormone-stimulated [ca2 ⧠signals have a complex temporal and spatial regulation, with the [ca2*]~ increase often taking the form of a series of repetitive spikes or oscillations, arising from a steady basal level. the spatial counterpart of a [ca2+]~ spike is a wave, where [ca2 ⧠is first elevated in a localised region of a cell, and then spreads throughout the cell in a regenerative manner. the [ca~*]~ spike frequency is modulated by several environmental factors including hormone concentration, extracellular calcium and thiol reagents. current evidence suggests that a cell can interpret these complex [ca2+]~ changes to regulate a diverse range of cellular activities. although many of the biochemical steps between hormonereceptor activation and the [ca2 ⧠response are known, the precise mechanism generating these complex [ca2+]~ signals is unclear. in order to understand this mechanism more clearly, we have investigated the regulation of insp~-mediated ca ~ ⧠release from intracellular stores in intact cells. our current data suggests that under certain conditions, the insp3-sensitive intracellular stores behave as functionally discrete units, and that during a ca 2 ⧠spike these stores become functionally coupled by the diffusion of ca z+ from one store to the next, triggering a regenerative ca 2+ release. phenylarsenoxide as a tool for identifying proteins involved in the secretory process wiedemann. c., schaefer, t.,*gitler, c., burger, m. m. friedrich-miescher institut, p.o.box 2543, ch-4002 basel "department of membrane research and biophysics, weizmann institute of science, rehovot 76110, israel phenylarsenoxide (pao) at 20btm blocks exocytosis in chromaffm cells of the bovine adrenal medulla. this inhibition can be reversed by small dithiois, such as dithiothrcitol or 2,3~limercaptopropanol. because trivalent arsenicals interact specifically with dithiol4containing proteins, we conclude that dithiol-proteins do play an important role in regulated secretion in chromalym cells. to identify dithiol-proteins putatively involved in the secretory process, we compare pao-binding proteins from chromaffm and pci2 cells and from fibroblasts as well as from sulx:ellular fractions by 2-dimensional gelelectrophoresis. the proteins are identified by radioactive pao bound to the dithiol in a complex that witltslands sds-page. affinity chromatography on pao-scpharose with various protein fractions is used to purify the dithiol-proteins of interest. enzymatic activity, e.g. kinase or phosphatase activity, can be measured in the eluates of such a column to give further hints at the possible function of dithiol-proteins. rac-pks (~elated to pka_ and pkcc protein kinnses) represent a serine/threonine kinase family whose catalytic domain is closely related to those of protein kinases a and c. they contain a ph (pleckstrin homology) domain n-terminal to the catalytic domain. in addition, rac-pk~ was shown to be a proto-oncogene. in order to investigate the role of rac-pk in cellular signallimg we undertook genetic and biochemical analysis of the drosophila homolog (drac-pk). the drac-pk gene encodes multiple classes of transcripts. antisera raised against the drac-pk recombinant protein specifically recognize 3 distinct molecular weight forms (68, 85 and 120 kda). all forms are expressed throughout development and are both maternally and zygotically regulated. the drac-pk gene is localized at 89b4-i0 region of the third chromosome, betwee~ the pannie~ and the stubble genes. universit6 de lausanne, rue du bugnon 9, 1005 lausanne when spinal meninges homogenates were incubated with reduced glutathione (gsh), a cofactor of prostaglandin (pg) biosynthesis, [14c] arachidonate (aa) was bioconverted into a major and a minor product which comigrated on tlc with pge2 and pgd 2 respectively. in absence of gsh: 1) pgd2 could not be detected; 2) the level of pge2 was dramatically reduced but surprisingly counterbalanced by accumulation of an unusual [i4c] aa metabolite which exhibits particular traits of migration on tlc and of retention time on hplc. this aa metabolite: 1) does not correspond to a degradation product of pge2; 2) crossreacts with pge 2 antibody; 3) fits the conditions required for a 15 hydroperoxy pge2 (chemical degradation into pge2 by hydroperoxyde reducing reagents such as snc12 or gsh-hemin). it is therefore inferred that depletion of gsh favours accumulation of hydroperoxyprostaglandins which would participate to oxidative injury. the ecdysteroid receptor (ecr) and ultraspiracle (usp) from both, chironomus tentans and drosoplu'la melanogaster, were expressed in e. coil as fusion proteins with glutathione-s-trsusferase. the identity of the expressed recombinant proteins was confirmed by western blot analysis. the drosophila ecr binds to its cognate hormone response element as a heterodimer with usp as a partner. we now want to compare the capabilities of the two receptor partners from both insect species in regard to dna binding and heterodimerization by means of gel mobility shift assays. the use of naturally occurring andartificial hormone response elements will allow to define a hormone response element consensus sequence for ecr-usp heterodimers and, if existing, for ecr and usp homodimers, respectively. for other members of this receptor family it was shown that the dna binding domain and adjacent sequences alone sufficiently defines response element specificity for both homo-and heterodimers. the dna binding domains of ecr and usp from the two species show a high similarity (92% and 85% identity, respectively). the corresponding sequences were selected for overexpression in e. coli. in combination with immtmoprecipimtion of receptor-dna complexes and pcr amplification steps, the expressed proteins will be used for the detection of naturally occurring hormone response elements within genomic dna. university of fribourg, ch-1700 fribourg. various agonist-induced cell responses in neutrophils and fibroblasts, such as chemotaxis and cytoskeletal rearrangements, have been shown to parallel the synthesis of ptdins(3,4,5)p 3. but the importance of this rise is not clear. we show here that wortmannin blocks pdgf-induced production of ptdins(3,4,5)p 3 in human foreskin fibroblasts with an ic50 of about 5 nm. a similar inhibition was observed in in vitro assays (ics0=lnm) with pi 3-kinase ii~nunoprecipitated by antibodies directed against the 85 kd subunit (p85). on the other hand, wortmannin did not affect pdgf-mediated phosphorylation of p85, indicating the correct interaction of p85 with the pdgf-~ receptor. the pl10/p85 complex of the pi 3-kinase remained intact in the presence of ~m concentrations of wortmannln. these results are consistent with a direct, specific inhibition of pi 3kinase by wortmannin at concentrations relevant for its previously reported effects on cellular responses. when stimulated with pdgf, human foreskin fibroblasts form circular membrane ruffles rich in filamentous actin. the fact that wortmannin inhibits these pdgf-mediated aotin rearrangements suggests the need of pi 3-kinase activity as a signal for this cell response. caicineurin is a calcium/calmodulin-regulated protein phosphatase which is comprised of a catalytic subunit a and regulatory subunit b. although calcineurin was discovered in brain, the calmodulin stimulated protein phosphatase which has caicineurin like structure has been described in other tissues. the protein structure show that the calcineurin b is very conserved in different tissues and species, using anti-calcineurin b monocicnal antiserum, the tissue extracts from rat have been explored. immunoreactivity was obverved in all tissues, although its intensity was different. the highest intensity was found in brain. spleen, heart and thymus had an intermediate level intensity. the results were supported by the quantitative measurement of calcineurin. the caicineurin concentration was 10 to 50 fold higher in brain than that in other tissues. furthermore, the distribution of the calcineurin activity in rat tissue was studied using dephosphorylaticn assay of calctneurin. the highest caicineurin activity was found in brain too, but was only 5 to 15 fold more than that in other tissues. the specific activity of calcineurin was different in tissues. these results indicate the caicineudn activity might be regulated in tissue specific fashion. burst and cytoskeleton arcaro a. and wymann m.p., institute of biochemistry, university of fribourg, ch-1700 fribourg chemoattractants like n-formyl-met-leu-phe (fmlp) stimulate in neutrophils a rapid and transient increase in the levels of ptdins(3,4,5)p3 (pip3) which is due to the activation of ptdlns(3)-kinase. pip 3 has been proposed to be a second messenger molecule, but its cellular targets are presently unknown.here we report that pretreatment of human neutrophils with wortmannin inhibits the fmlp-etimulated production of pip 3 in a dose-dependent manner (ic50 = 5 nm). furthermore, ptdins(3)-kinase activity immunoprecipitated from resting cells is totally abolished by i0 nm wortmannin (ic50 = 1 nm). these results show a potent and direct effect of wortmannin on ptdins(3)-kinase. wortmannin also inhibits the respiratory burst of neutrophils at nanomolar concentrations, which suggests that pip3 is involved in the signalling pathway controlling activation of the nadphoxidase.when pretreated with wortmannin and stimulated with fmlp, neutrophils display oscillatory changes in factin content that correlate with oscillations in cell ~hape. this, and the inhibition of ptdins(3)-kinase, suggest a modulatory role for pip 3 in cytoskeletal rearrangements.we are currently investigating the effects of pip 3 on the functions of gelsolinl a protein that is proposed to mediate actin polymerization and can be regulated by polyphosphoinositides and ca 2+. matthias gstaiger, walter schaffner and christopher hovens institut f'tir molekularbiologie ill der universitat z'tirich, ch 8057 ziirich the octamer motif atgcaaat plays a central role in mediating the activity of a number of both lymphoid specific and ubiquitous promoters and in the immunoglubulin heavy chain intronic enhancer. the activity of thils motif in lymphoid cells is presumably mediated by the lymphoid cellspecific factor oct-2a. when ectopically expressed in non-lymphoid ceils, oct-2a is not sufficient to mediate enhancer activity of a multimerized 50 bp enhancer core element derived from the immunoglobulin heavy chain intronie enhancer, a segment, which is however highly active in b-cells. this and other findings support the supposition that additional b-cell specific factor(s) can account for the specificity and extent of the octdependent transcription observed in lymphoid cells. to further our understanding of the molecular mechanisms involved in mediating lymphoid-specific expression, we have employed the yeast two hybrid system to identify human proteins capable of physically associating with human oet-2a. to this end, a oct-2a expressing yeast strain has been constructed which bears two integrated reporter genes, his3 and lacz under the control of the octamer motif. transformation of this strain with a cdna library has allowed us to isolate clones interacting with oct-2a. we are currently analysing these candidates to determine the veracity of these interactions in higher enkaryotic cell background. in general, untransformed nuclear hormone receptors are predominantly confined to either the nucleus or the cytoplasm of a cell. receptors of the latter class are translocated to the nucleus upon interaction with ligand. our studies of the use of the ecdysteroid receptor complex from insects, consisting of a heterodimer formed between ecr and usp, as a tool for target gene activation in vertebrate cells have revealed a novel mechanism of nuclear translocation of ecr that does not depend on hormone action. after transient transfection of respective expression plasmids into hela or cv-1 cells, the subeellular distribution of the individual receptor types was analyzed by indirect immanofluorescence staining. while ecr alone was detected in both cellular compartments, nucleus and cytoplasm, usp was predominantly found in the nucleus of either host cell. cooxpression of both ecr and usp resulted in an efficient transloeation of ecr into the nucleus. usp appears to trap ecr in the nucleus, presumably by the formation of a heterodimeric complex. thus, heterodimer formation of ecr and usp not only appears to be required for high affinity binding to cognate hormone response dements (and subsequently for transactivation of an adjacent target gene) and for binding to ecdysteroid but in addition for an enhanced translocafion of ecr into the nucleus. activation of rodent macrophages with cytokines or bacteria is accompanied by the induction of a ca2+-independent nitric oxide (no) synthase which plays a key role in antimicrobial and antitumoral activity. firm evidence for expression of a similar enzyme in other mammals or in man has been lacking. we now show that bovine bone marrow-derived macrophages produce nitrite in an l-arginine-dependent manner upon stimulation with heat-killed grampositive or gram-negative bacteria. homologous interferon-7 and tumor necrosis factor-~ induced little no 2-production, but primed macrophages for enhanced n0 2-production induced by salmonella dublin or lps. this is one of the first demonstrations of no 2-production by nonrodent macrophages and encourages a search for an involvement of reactive nitrogen species in the killing of parasites or tumor cells by macrophages from mammals other than rodents. lectins are used in many analytical methods to study the carbohydrate structure of glycoproteins. we report here a technique which combines the high resolution of two-dimensional gel electrophoresis (2-dpage) to separate plasma proteins, the specificity of lectins to bind carbohydrates and the sensitivity of chemiluminescence. this method is compared with that of a commonly used colorimetrio reaction. since the reference plasma protein map obtained by 2-d page is available (i), the technique described here allows a quick and specific identification of plasma glycoproteins. therefore any ma j or glycosylation modification which may happen in some diseases can be detected. (i) electrophoresis 1992; 13:707-714. this work was supported in part by fcar (quebec, canada). in our laboratory we use genetic and biochemical approaches to study translation initiation, in particular the initiation factor eif-4a in yeast. this factor from higher eukaryofic cells is known as an rna dependent atpase and functions as a rna helicase together with another initiation factor, elf-4b. it belongs to a family of putative rna helicases, the d-e-a-d proteins. to find new genes involved in translation initiation, suppressors of a temperature sensitive eif-4a mutant were isolated. one of the suppressors (stm1) is a single copy gene which encodes a protein resembling the human translation initiation factor eif-4b. disruption of the gene is not lethal to the cell, but affects growth. polysome analysis of strains with a deleted stm1 gene have shown that this new gene is also involved in translation initiation. it carries six repeated sequences of 25 amino acids of unknown function and has -like the human eif-4b -a rna binding domain. the second suppressor (stm2) is also a novel gene. it encodes a large protein which shows no homology to other proteins present in the data libraries. it contains high portion of charged amino acids and is rich in glutamines and serines. its function in translation initiation is currently under investigation. tobacco translationini~ationfactore~4aisencoded by a large anddiverse genefamily karl brander, therese mandel, isabelle lutziger, george owttrim and cris kuhlemeier, institute of plant physiology, university of berne, altenbergrain 21, ch-3013 berne using heterologous probes we isolated cdnas coding for translation initiation factor eif-4a from tobacco, eif-4a is encoded by a large multigene family of which at least nine members are expressed in leaves and most if not all other organs of the tobacco plant. while screening genomic libraries we found several additional genes. two of them code for genes highly homologous with eif-4a throughout the coding region, but with changes in the gkt, ptrela and dead-box motifs. a third gene is expressed exclusively in the developing male gametophyte. this eif-4a-related gene may have a role in the well-documented regulation of translation in pollen. in order to study the function of these genes we have begun altering their expression in transgenic tobacco plants. seauence reauirements for a non-aug protein initiaf~jon non-aug initiation codons are still infrequent and were initially observed in animal viruses, but a number of cellular examples have now also been reported. several years ago we reported the use of an acg in the p/c mrna of sendal virus (sen) to initiate the non-structurar c' protein. we recently described a c' protein in the human parainfluenza virus type 1 (hpiv1), which is initiated from a gug. in both cases, the c' protein is an n-terminally ebngated form of the c protein which is initiated at the second aug, in the +1 reading frame relative to the first aug. this aug is used to initiate the p protein, an essential component of the viral rna polymerase. unlike the acg in sen, the gug in hplvt is clearly not a weak start codon since c' is the most abundant protein expressed from this mrna both in rive and in vitro. it appears that not any upstream gug in an optimal context will function as a start codon. for instance, the p gene of hpiv3 cannot express a c' protein (the orf is closed by stop codons) but it does contain a gug in an optimal context upstream of the p protein aug, which could encode a p' protein. nevertheless, we have been unable to demonstrate that this gug functions as a start cedon. with the use of chimeric mrnas between hpivl and 3, we have shown that positions +5 and +6 (the first g of the gug triplet being +1) are the major determinants of the efficiency of gug as an initiaton codon for protein synthesis. we are currently investigating the possibility that these nucleotides may increase the initiation rate of weak non-augs such as the acg of sen. biological activity of the heterodimeric subunit srp9/14 of the signal recognition particle is retained in 2 fusion proteins fabrice bovia & katharina strub, d6partement de biologie cellulaire, universit6 de gen~ve, ch-1211 gen~ve 4 the targeting of secretory proteins to the membrane of the endoplasmie reticulum is mediated by a cytoplasmic ribonucleoparticle, the signal recognition particle. it is composed of 6 proteins and f rna molecule (srp rna). the 2 smallest proteins srp9 and srp14 are required for the translational control function of srp. it effects a specific arrest in the biosynthesis of er-targeted proteins. these 2 polypeptides bind to the srp rna exclusively as a heterodimer (srp9/14). we have constructed single-chain variants of this dimer using a short peptide linker of 17 aa to connect the c-terminus of the 14 kd subunit to the n-terminus of the 9 kd subunit (14-9) and vice versa (9-14). we found that both proteins bind srp rna with a similar efficiency as the wild type protein. glutaraldehyde cross-linking experiments and sedimentation analysis indicate that the 2 fusion proteins fold into a similar structure as srp9/14 and bind srp rna as a monomer. furthermore, they can functionally replace srp9/14 in the elongation arrest and translocation assay. since the 2 fusion proteins are circular permutations of each other, we can conclude from this results that n-and c-termini of both proteins have no essential role in folding, rna-binding and in mediating their biological activity. furthermore, the possibility to express an oligomeric protein as a single polypeptide facilitates the analysis of its functions as well as its structure. a24 s06-08 studer, r. and hunziker, p. e., 8iochemisches institut der universit~t z0rich, ch-8057 z0rich metallothioneins (mts) are small cys-rich proteins which are inducible by a variety of agents such as bivalent transition-metal ions, tumor promoters, cytokines and hormones. the biological role of mts is still unclear. initially postulated to serve in the sequestration of the toxic heavy metals such as cadmium and mercury, they are now believed to play a major role in the cellular metabolism of essential metals such as zinc and copper. to explore this function further we have now established a method to quantify basal mt production in several human cell lines. thus, cellular isomt contents were monitored by h.pj.c, and the rate of synthesis was followed by the incorporation of ~ss-cysteine. the results show that maximum mt synthesis and accumulation occurs when the cells enter the exponential growth phase. with increasing celldensity the mt content decreases progressively until confluence is reached. in correlation with preliminary measurements our results suggest a close relationship between the mt content, the cell growth rate and the intracellular abundance of zinc. vincent bernard, adrian etter, heinz tobler and fritz mtiller. institute of zoology, university of fribourg, 1700 fribourg (switzerland). the nematode ascaris lumbricoides undergoes chromatin diminution which causes a loss of dna in all somatic ceils. we have identified a ribosomal protein (rp) gene (coding for alep1 = ascaris lumbrieoides eliminated protein 1) which is located within the germqine specific material. alep1 shows strong homologies with the ribosomal protein s19 (rps19). the transcription of its gene takes place only in the germ-line. 2d-gel electrophoresis on germ-line and somatic purified ribosome fractions shows that the alep1 protein (rps19) is present only in the germ-line ribosome. does the alep1 germ-line specific protein have a homologue in the somatic ribosomes? we have cloned a homologue of alep1 from ascaris somatic tissue named rps19', we are currently studying the expression pattern of this rps 19'. these results indicate the existence of a developmentally regulated ribosome heterogeneity between the germ-line and somatic ceils. what is the function of rps 19? since ascaris is not suitable for genetic analyses, we isolated rps19 from the nematode caenorhabditis elegans. the c. elegans rps19 cdna shows only 60% homology at the nucleic acid level. rps19 is located on the c. elegans chromosome iv. the identification of a rps19 mutant in c. elegans will indicate its function. favre, d, l, pavlovic, j2 and michel, m.r. t 1 institute of medical microbiology, university of beme, ch-3010 berne. 2 institute for immunology and virology, ch-8006 ziirich we have successfully purified the recombinant c (recc) protein from spodoptera frngiperda (sf9) insect cells which were infected with acnpv-c (a). the recc protein retained the biological activities of native c protein obtained from wild type sfv (b). our results suggest that the c protein is responsible for the cellular shut-off of host protein synthesis by inhibiting the initiation of translation. we are currently investigating the molecular mechanisms involved in this shut-off. tif3 is a new eukaryotic initiation factor which shows 26% identity with the sequence of mammalian translation initiation factor eif-4b. the tif3 gene is not essential for growth; however, its disruption results in a slow growth and coldsensitive phenotype. in vitro translation of total yeast rna in an extract from a tif3 gene-disrupted strain is reduced as compared to a wild-type extract. the translational defect is more pronounced at lower temperatures and can be corrected by the addition of purified yeast tif3,or mammalian eif-4b. in vivo translation of ~galactosidase reporter mrnas with varying degree of rna secondary structure in the 5' leader region in a tif3 gene-disrupted strain show preferential inhibition of translation of mrna with more stable secondary structure. chloroplast protein synthesis requires the import of elongation factors ef-tu (tuf-gene) and ef-g (fusgene). it seems that the expression of both genes is light regulated. we recently cloned and sequenced a chloroplast specific nuclear fus gene in soybean (biochim.biophys acta 1174 : 191-194, 1993 . further analysis revealed that a second very similar (sequence, anatomy) nuclear fus-2 gene exists which, however, shows strong sequence diversion in the 3'trailer region. several cdna clones were partially sequenced but sofar only transcripts from the fusi gene were retrieved. we currently study the promoter regions of either gene to test possible differential expression of fus genes. comparative mapping and sequencing studies of the two genes in the 3"-region indicate that a major dna insertion occurred distal to the coding part of both fus genes what may also influence gene expression. the rna-binding domains in the 9kd and 14kd subunits of the signal recognition part~clehavea putative~-h~licalstructure. nazarena buiand katharina strub dpt de biologie cellulalre, science ill, universit4gen~ve the signal recognition particle (sr2), a cytoplasmic ribonucleoprotein is important for targeting nascent secretory proteins to the endoplasmic reticulum. srp9and srpl4are constituents of srpandare, together with the alu sequences of srp rna, required for the elongation arrest activity of the particle. srp9 and srpi4 form a heterodimer in the absence of the rna and bind the rnaexclusively as a heterodimer. the primary sequence of srp9 and srpi4 revealed that both proteins are highly basic and lack structural similarities to other characterized rna-binding proteins. we have therefore been interested in characterizing the molecular nature of rna-protein interactions. the analysis of n-and c-terminal deletion mutants of the proteins have shown that both proteins contribute to the formation of an rnabinding pocket, the rna-binding and dimerization domains in both proteins, are found in regions that have a predicted ~~helical structure. in sp~pi4, this u-helical region is located betwen aa 72and i00 at the c-terminus; it contains the rna binding and dimerization domains adjacent to each other. our results support the model that the hydrophobic face of the putative u-helix is implicatedinprotein-protein interactions and the positively charged face in rna-protein interaction. in sp~pg, the two rna-binding domains found n-and c~ terminally, also have a predicted amphipatic s-helical structure. it has been proposed that the decline in protein synthesis observed in aging organisms may result from a decrease in the protein synthesis elongation factor ef-la. john shepherd's finding that transgenic drosophila melanogaster carrying an additional copy of the ef-la gene have an extended lifespan further indicated that the ef-la gene may play an important role in determining longevity (shepherd et al., 1989) . in order to test this hypothesis, we have quantitated ef-la mrna, ef-la protein, as well as catalytic ef-la activity in john's flies. young and aging ef-hx transgenic (ef) and control lines ((2) were examined. because the the additional ef-lc~ copy is under the control of the hsp70 promoter, flies were aged either at 25~ or at 29~ the results show that transgenic ef flies do not express more ef-lct than the control flies, neither at the rna nor at the protein level. the question arises, whether the lransgene can be induced at all. this was tested by doing rnase protection assays. transgenlc message can be detected only in ef flies heat shocked at 37~ but not in ef flies grown at 29~ nor in control flies at 37~ from this experiment we conclude that the transgene is indeed inducible, but is not expressed in detectable amounts at 29~ we conclude that the difference in the lifespan does not result from the overexpression of the ef-la transgene. phenobarbital (pb) induces the expression of liver enzymes involved in the metabolism of drugs and other foreign compounds (xenobiotics). these enzymes include several cytochromes p450, nadph:p450 reductase, aldeyde dehydrogenase, epoxide hydrolase, udp-glucoronyltransferases and glutathione-s-transferases. many other proteins not yet recognized may however be induced by pb. our first approach therefore is aimed at identifying gene products which respond to pb treatment. as model systems we are using either chicken embryos (induction in ovo) or primary cultures of chicken embryo hepatocytes (induction in culture) (althaus et al., jbc 254 pp 2148 , 1979 . to differentially display mrna of induced vs non-induced cells we are using a rt-pcr technique with sets of random primers. electrophoretic separation of amplification products allows to identify mrna species which are induced (or decreased) following treatment. data obtained from these experiments and sequence information revealed from extracted pcr products indicate that pb treatment results in the transcriptional avtivation (or repression) of a variety of so far unknown genes. antithrombin -binding heparan sulfates from ovarian granulosa cells hosseini g., de agostini, a., clinique de st6rilit6, hcug, gen~ve. at the time of ovulation, several serine proteases of the plasminogen activator and coagulation cascades are activated in the ovary. these proteolytic activities are tightly controlled in time and space, and the heparin-activated serpins plaminogen-aetivator-inhibitor-1, protease nexin-1 and antithrombin (at) are present in the follicular environment. we have observed that granulosa cells produce a subset of heparan sulfate proteoglycans that bind and activate at. these at-binding heparan sulfates (ahspgs) endow the vascular endothelium with antithrombotic properties. we are now examining the role of ahspgs in the extravascular compartment formed by the ovarian follicle. using 125i-at binding assays, we have detected ahspgs on granulosa cell surfaces and culture media, in amounts comparable to those found for endothelial ceils. after 48h of stimulation by the gonadotropin fsh (1-100 ng/ml) granulosa cells increased the amounts of ahspgs they released in culture media by 2-6-fold, while keeping their cell surfaceassociated ahspgs constant. analysis of 35s-labelled hspgs revealed that heparan sulfates constitute about 40 % of the surface-associated and 10% of the soluble granulosa cell glycosamineglyeans. finally, using 125i-at autoradiography on rat ovary cryosections, we have localized ahspgs on the granulosa cell layers of large antral follicles, while ahspgs could not be detected in smaller follicles. these data suggest that granulosa cell ahspgs could be critically located to modulate proteolytic activities in the changing environment of the growing follicle. division, cibabasel, switzerland. the development of chelators which can mobilise excess storage iron (fe) and permit its excretion is necessary in the treatment of fe overloaded diseases. although oral administration of such a pharmaceutical is highly desirable, no orally active fe chelator is so far in regular clinical use. searches for orally active and safe fe chelators require appropriate animal models of fe overload in order to evaluate the potential of these drugs. an animal model is particularly useful for testing the capacity of new fe chelating ce~oounds in enhancing the mobilisation of clinically relevant storage iron. iron loading in animals was achieved by dietary supplementation with carbonyl fe, fe fumarate and 3,5,5-trimethylhexanoyl ferrocene (tmh-ferrocene). liver fe concentrations were increased 6-21 times above normal levels. tmh-ferrocene was the most efficient compound to induce stable liver fe overload. the orally given con'pounds induced a predominantly hepatocellular iron distribution and was found distributed among reticuloendothelial cells only at a later stage. this pattern of liver fe storage is similar to that observed in the early stages of primazy ha6~ochromatosis and late stage of transfusional hae~ochromatosis. eeeently, we and ethers have cloned and characterized novel transcription factors called peroxisome proliferator-actirated receptors (ppak). they belong to the superfamily of nuclear hormone receptors as the steroid, thyroid and retinoid hormone receptors. ppars are activated, beside xenobiotic peroxisome proliferators, by natural fatty acids and induce, together with the receptor for 9-cis retinoic acld (rxr), the transcription of genes involved in the ~-and ~-oxidation of fatty acids. ppar and rxr bind as heterodimers to the ppar response element (ppre), consisting of a direct repeat of aggtca-iike sequences with one intervening nucleotide. now, we show by gel retardation analysis and transient transfection assays that ppar/rxr heteredimers also bind to and transactivate gene transcription through estrogen response elements (ere). the ere is a palindrome with aggtca half-sites separated by three nucleotides. the selectivity and specificity of the transactivation through an ere was demonstrated by the testing of various related palindromic and inverted palindromic repeats of aggtca sequences, which were all inactive. the possible activation of estrogen responsive genes by fatty acids via ppars in vivo is discussed especially with regard to a debated role of fatty acids in breast cancer. the purpose of this study was to evaluate whether intermittent doxorubicin (dox) treatment in vitro selects for multidrug resistance in rpmi 8226 myeloma cells and, if so, to determine the underlying mechanism(s). cells were exposed to constant doses of dox for 4 days alternating with growth in dox-free medium for 17 days. after > 9 months of treatment, the cells developed an atypical mdr phenotype with 4-to 6-fold resistance to topo ii poisons. cross-resistance to vincristine and taxotere was < 2-fold, sensitivity to cisplatin and melphalan remained normal. efflux of mdr1 drugs was incresed with no effect of verapamit; subcellular dox distribution was normal. expression of topo ii a was found to be reduced by around 50 and 60-70% at the rna and protein level, respectively. minimum mdrt rna overexpression was detected when using rt-pcr; p-glycoprotein was not detectable. mrp rna expression was increased by 60-90% relative to the wild type cells without detectable p190. this data suggests that atypical mdr might play a role in acquired dox resistance in human myeloma. mucosal surfaces cover a vast area in the human body. they satisfy its need to communicate with its environment, e.g. in terms of nutrient uptake and sexual reproduction. however, at the same time they offer a vulnerable gate for invasion by pathogenic microorganisms. for immunoprotection of these regions, large amounts of iga antibodies are produced in the bloodstream and transported across the epithelial barrier to the lumen by means of polymeric ig receptor. there the receptor is cleaved, and its extracellular portion remains associated as secretory component (sc) to the secretory iga complex. in the context of a project aiming to mass production of secretory iga as a passive vaccine, we evaluated a series of eukaryotic expression systems for production of sc. the cdna encoding polymeric ig receptor was engineered in a way that only its extracellular domains corresponding to sc were expressed in yeast, in insect cells infected with recombinant baculovirus, and in mammalian cells infected with recombinant vaccinia virus. furthermore, a c-terminal poly-histidine tag was introduced for simple purification of the products. we will show optimization of expression levels for each of the systems as well as a quantitative and qualitative comparison of the products. recombinant vaccinia viruses (rvv) that express gone products from other organisms has become a popular and widely used laboratory expression system. our current interest is directed toward the production of secretory component (sc), a subunit of secretory iga antibody complex involved in mucosal immunity. toward this goal, we constructed rvv governing sc transcription under the control of two viral-based and the t7 bacteriophage promoters. expression of the protein was assessed in several mammalian cell lines, such as human tk-, human hela (grown in suspension or as a monolayer) and monkey cv-i. optimization of infection parameters and culture conditions were also performed. institut de biologic animale de runiversit~ de lausanne protection of mucosal surfaces is mediated by secretory iga ant/bodies (alga) constituted of dimeric iga and secretory component (sc) . toward the aim of reconstituting slga complexes in vitro and using them for passive oral immunization, sc has been produced in yeast, insect and mammalian expression systems. the gone encoding sc has been engineered so that a) the c-terminus of the protein carries a 6xhis tag and b) the newly synthesized polypeptide is released in the culture medium. culture conditions have been established to permit recovery of sc in serum-free medium. purification procedures based on nickel chelate and classical chromatographies have been optimized to yield sc under native conditions. finally, in vitro reassociation with purified dimeric igas obtained from hybddomas has been used to assess biological activity of recombinant sc. using a magnet-assisted subtraction-hybridisation technique (mast), 17 cdna clones were isolated that are expressed in nqrm~l l~nq %issue but n__d_i expressed in the corresoondina nsclc tissue, ii of the 17 edna clones are highly homologous to edna sequences for the following proteins: pulmonary surfactant proteins sp-a and sp-b; receptor for advanced glycosylated endproducts of proteins (rage); calmodulin-like protein; natural killer gone 5 (nkg5); matrix gla protein (mgp); glutamine synthetase; ubiquitin; vascular smooth muscle alpha-actin; cytoskeletal beta-actin; vimentin. the remaining 6 edna clones may represent parts of still unknown genes. the mast edna clones were derived from a patient who developed squamous adenocarcinoma. northern blot analysis of normal/tumour rna from three other nsclc patients showed that the lack of gone expression was independent of nsclc type and stage. enzymatic methylation of cytosines in mammalian dna is believed to play a fundamental role in basic gene regulation. methylation in the vertebrate genome is restricted to cpg dinucleotides at the c-5 position of cytosine. in vitro studies have shown that methylation can drastically influence the dna structure. cpg islands remain unmethylated in normal cells, whereas several immortalised, transformed cell lines contain partially methylated cpg islands. transcription of genes can be inhibited by methylation of cpg's. this modification contributes e.g. to the stable repression of genes on the inactivated x chromosome of females. the collagen vi genes show typical cpg islands in their promoter region and they were shown to be down regulated at the transcdptional level in src or myc transformed cells. given these facts we dee~ded to investigate the effects of methylation in the collagen vi gwene. e could demonstrate that the promoter activity of (x2(vi) chicken collagen gone was strongly diminished by methylation in vitro. furthermore dna methylation completely prevented binding of a novel transcription factor to the promoter, whereas binding of sp1 was not affected. to show evidence of methylation in vivo, we are currently iocalising the exact positions of methylated cpg's in normal and transformed cells by genomic sequencing. alterations of putative tumour suppressor genes in human non-small cell lung carcinoma (nsclc). r. shipman and c.u. ludwig, molecular oncology, lab 405 (zlf), basel, ch-4031 chromosomal subregion llp13 displayed non-random allelic loss in 61 nsclcs. llp13 contains the genetic loci for catalase (cat), the wilms' tumour gene (wti) and the follicle-stimulating hormone ~-chain (fsh~). 76% of the nsclcs were deleted at cat suggesting that loss of a gene(s) near cat may be involved in the progression of nsclc. yac clones containing the cat locus are being prepared for use in a direct selection procedure for cdnas encoded at this region. although 38% of the nsclcs were deleted at wti, no mutations were detected in the remaining wti allele. wti expression was subsequently demonstrated in fetal lung, fetal hematopoietic tissue, normal adult lung and nsclc. allelic deletion at 17p13, immuno-histochemical and sequence analysis of the p53 gene were also examined in our nsclcs. these analyses support the contention that aberrant expression of the p53 gene is a pivotal event in the progression of nsclc. $08-11 recently, a number of advances have been made which enable the experimentalist to study the effects of low levels of ionizing radiation. the results suggest a threshold to radiation damage exists and that above a critical level, recovery mechanisms in the cell are induced and the biological response falls. thus low levels of radiation cause greater than expected levels of biological response. this has been demonstrated in studies of mutations, cell survival and cancer induction. because the low dose-rates and the low doses per fraction result in the total doses being small, the magnitude of the biological effects induced is insufficient to warrant alarm. identification of genes associated with the revertion of the malignant phenotype of a rhabdomyosarcoma cell line. michele genini, sabina solinas toldo*, ruedi fries* and beat w. schafer, dept. of pediatrics, university of ziirich, steinwiesstr. 75, 8032 ziirich, and *institut for nutztierwissenschaften, eth ztifich, 8092 ztirich. the human rhabdomyosarcoma cell line rd is tumorigenic and differentiates very poorly despite the expression of the myogenic factors myf3 and myf4. therefore it can be regarded as natural mutant. to identify genes which are involved in suppression of tumorigenicity or enhance differentiation we applied two different approaches. since the development of human embryonal rhabdomyosarcoma has been associated with abnormalities of chromosome 11 band p15, in the first approach we transferred a normal human chromosome 11 into rd ceils via microcell fusion. the microcell hybrids did not show a higher degree of differentiation. suppression of tumorigenicity was observed in one clone but is probably independent of chromosome 11, as it was shown by chromosome 11 specific painting. ' in the second approach we applied a subtractive hybridization procedure between primary myoblasts and rd cells to find genes specific for the normal phenotype. these genes will be tested in vivo for induction of differentiation and/or tumorsuppression. deficient synthesis of the gpi anchor is the molecular basis of idiopathic paroxysmal nocturnal hemoglobinuria ("pnh") and a similar phenotype can accompany aplastie anemia cpnh-iike"). the population expressing cd48, a gpi-anehored membrane glycoprotein, was quantified by flow cytometry in samples from patient p.g. (pnh) and patient m.m. (pnh-like) and amounted to 90% (p.g.) and 78% (m.m.), respectively. t lymphocytes from both patients were isolated, expanded and cloned. from both patients, clones expressing the cd48 marker (cd48+) and cd48 deficient clones (cd48-) were obtained. takeda et al. (cell 73, 1993, 703-11) showed that in some (cd59-) bcell clones, a deletion of the p1g-a gene product is responsible for the expression of the pnh-phenotype. pcr analysis of t cell mrna from extracts of both cd48+ and cd48-clones of patient m.m. (pnh-like) using pig-a specific primers revealed a band pattern from which can he concluded that no deletion is present in the pig-a gene product. this result suggests that cd48-t lymphocytes of the pnh-like patient express a normal pig-a gene product; a disorder in regulation of pig-a expression or an unrelated biosynthetic defect may explain the cd48phenotype in these t cells. further work is aimed at defining the differences in biosynthetic defects of pnh and pnh-like cd48-t cell clones. supported by grant 31-30757-91 of the snsf to egb. w@ have recently employed an experimental approach to turn the function of "nuclear messengers" such as c-fos (and c-jun) on and off at will in polarized me/nmary epithelial cells. to this end we used constructs encoding the c-fos (and c-jun) genes fused to the hormone-binding domain of the human estrogen receptor, designated c-foser (and c-juner), we could show that short-term activation (30 mins.) of c-foser by estradiole (e2) led to the disruption of epithelial cell polarity within 24 hours, as characterized by the expression of apical and basolateral marker proteins. during the next 3-5 days, however, the cells fully regained their polarized phenotype. conversely, long-term activation (24-48 hours) caused the irreversible loss of epithelial cell polarity, leading ultimately to an epithelial-fibroblastoid cell transition. these cells underwent dramatic changes in gene expression including the complete loss or reduction of epithelial markers such as e-cadherin/uvomorulin, zo-i and cytokeratins, and the de novo expression of mesenchymal proteins like vimentin, type i collagen and fibronectin. expression of the v-ha-ras oncogene (acting upstream of c-fos) did not markedly influence epithelial cell organization when the cells were grown on plastic. however, when cultured within type i collagen gels in the presence of serum, or when injected into the mammary glands of nude mice, rapid cell growth and a clear epithelial-fibreblastoid cell transition was observed. growth properties determine the organ colonization ability of a metastatic murine melanoma cell line. the ability of metastatic tumor cells to specifically colonize distant organ sites strongly depends on the interaction of the metastatic cells with the local organ environment. we have selected a b16 routine melanoma cell fine (b16-ls9) which has an increased ability to colonize the liver, and compared its behavior with either its parental (b16-f1) or inng-specific (b16-f10) cell line. we have found that adhesion in vitro and organ lodgement in vivo were not different for ls9 and f10. in contrast, b16-ls9 grew at slower rates than the other lines both in vitro and in vivo after subcutaneous or intra-foot-pad injection. analysis of tyrosine-phosphorylated proteins indicated a higher phosphorylation activity in b16-ls9 cells, which might suggest an altered regulation of some kinase(s) in this cell line. intercellular contact with hepatocytes in vitro, or the liver in vivo could restore an efficient growth of b16-ls9, enabling thus these cells to colonize this organ much better than others. hepatocytes or liver plasma membrane (p.m.) extracts conserved the growth stimulatory activity towards b16-ls9 ceils. chromatographic fractionations of these extracts showed that a major growth sfimulatory protein in this fiver p.m. fraction turns out to be ttansferrin, which in the liver appears to exist in at least two different mature forms. protein import into mitochondria requires atp in two locations: outside the organelle, and in the matrix space. depletion of matrix atp arrests translocation of precursors across the inner membrane, but does not prevent precursors from crossing the outer membrane. as a result, proteins that reach the inner membrane or intermembrane space without passing through the matrix are often sorted normally in atp-depleted mitochondria. external atp is used to maintain precursors in a translocationcompetent state. however, some precursors can fold and still remain translocation-competent, and import of these precursors is independent of external atp. with the folded cytochrome b2 precursor, it is matrix atp rather than external atp that drives translocation across the outer membrane. thus matrix atp generates a pulling force that can drive both the translocation and the unfolding of precursor proteins. mitochondrial hsp70 probably functions as the atp-dependent import motor. arsenijcvic d, dulloo ag & girardicr l, dpt of physiology, cmu. geneva, ch. the relationship between body weight, daily energy expenditure (assessed by indirect calorimetry), and immune status (determined by lymphocyte proliferation tests) was investigated in swiss webster mice maintaining a stable body weight several weeks after infection with toxoplasma gondii(me49). following an initial period of weight loss (infection-induced cachexia), the surviving mice could be differentiated into two main groups: (i) those showing a partial regain in body weight (the infected gainers) and eventually stabilizing al a mean body weight 25% below a non-infected control group, and (if) those showing no weight regain (the infected non-gainers) and eventually stabilizing at 45% below control levels. immune function was found to be markedly suppressed in the infected non-gainers, whereas it was normal (and similar to control levels) in the group of infecled gainers. daily energy expenditure (and food intake), after normalisation for differences in body weight, was found to be the same in both infected gainers and non-gainers, but lower by 15% when compared to controls (p<0.01); thereby indicating that the infected groups were able to increase their overall efficiency of food utilization in response to the low food inlake. in conclusion, this study conducted during a weight stable phase of chronic infection shows a clear-cut association between the inability to regain body weight and impaired immune function. in contrast, no relationship was found between metabolic rate and body weight nor between metabolic tale and immune function, but the chronically infected mice exhibited the same phenomenon of enhanced metabolic efficiency characteristic of chronic underfeeding per se. identification and functional analysis of chaperonin 10, the groes homolog of yeast mitochondria. biozentrum, university of basel, klingelbergstrasse 70, ch-4056 basel, switzerland. phone ++41-61-2672171, fax ++41-61-2672148. the mitochondrial chaperonin system consists of chaperonin 60 (cpn60; also termed hsp60), which is homologous to e. cull gruel, and chaperonin 10 (cpnlo), which is homologous to e. coil groes. we have used a functional assay to identify cpnl0 of yeast mitochondria. when dimeric rubisco is denatured and allowed to bind to yeast cpn60, subsequent refolding of the enzyme is strictly dependent upon yeast cpnl0. in the presence of mgatp, yeast cpn60 and yeast epnl0 form a stable complex that can be isolated by gel filtration. the atpase activity of hsp60 is not inhibited by complex formation with cpnl0. a different result is obtained with the e, coil system, where groes is able to completely inhibit the atpase activity of gruel (1). to test the in vivo role of cpnl0, we have cloned, sequenced and disrupted the corresponding nuclear gene cpnio. this gene encodes a protein of 11,372 da that is imported into the mitochondrial matrix without detectable cleavage. haploid cells lacking a functional copy of cpnio fail to grow at temperatures between 23 ~ c and 37 ~ c (2). (1) rospert, s. et al. (1993) proc. natl. acad. sci. 90, in press. (2) rospert, s. et al. (1993) febs lett., in press. characterization of yeast mitochondrial heat shock protein 70 , l. bolliger, b. s. glick and g. schatz, biocenter, university of basel, 4056 basel. mitochondrial hsp70 (mhsp70) is thought to use the energy of atp hydrolysis to pull precursor proteins across the mitochondrial membranes. to facilitate the purification of yeast mhsp70, we used the cloned gene (a gift of dr. n. morishima) to modify mhsp70 with a six-histidine tag at its c-terminus. this construct supports growth of yeast cells lacking wild-type mhsp70. we developed a purification procedure that allows us to recover this tagged protein with a purity of about 95%. experiments are in progress to characterize the nucleotidedependent interaction of purified mhsp70 with unfolded proteins. in addition, we are looking for partner proteins that may modulate the action of mhspt0. we have copurified a protein of about 20 kd together with mhsp70. the 20 kd protein could be released from mhsp70 by atp or adp. when tryptic fragments of this protein were subjected to microsequencing, one fragment showed strong homology to the grpe protein of e.coli. the transmembrane electrical potential of mitochondria (awmit) can be assessed in single hepatocytes by using epifluorescence microscopy. for this end the rhodamine 123 fluorescence of a mitochondria-rich (fr) and a mitochondria-poor region (fp) are measured. the ratio of these signals depends on the awmit according to a modified nernst equation (reber b .f.x., somogyi r. & stucki j.w. (1990) , bba, 1018, 190-193) . to calibrate this method the cell membrane was permeabifized for monovalent cations with nystatin and gramicidin. then the cytosolie k + was replaced by na + from a k+-free bath solution. subsequent addition of valinomycin made the mitochondrial membrane permeable for k + ions. by the addition of different k + concentrations to the bath, the awmit could be clamped to defined values. the relation between the ratio fr/fp and the awmit could be well fitted to our modified nernst equation. however, the calibration curve flattens out at potentials higher than about 110 inv. therefore the detection limit of changes of a~mit within the physiological range is about 10-20mv. maximal stimulation of the na+/k + atpase by addition of nystatin to the na + containing bath medium caused a drop of awmit of about 20inv. subsequent addition of ouabain resulted in an almost complete reversal of this drop. this shows that changes in atp consumption can be assessed via changes of audmit. schneiter ph., di vetta v., j6quier e. and tappy l. institut de physiologie de l'universitg bugnon 7,1005 lausanne. the metabolic effects of an exercise performed in the fasting or fed state were studied in 8 subjects over a 8 hour period in a respiratory chamber. a mixed meal containing 13c labeled carbohydrates was ingested at 9:30 am and an exercise session (walking at 5 km/h 45 rain, slope 10 %) was performed at 8:15 am (fasting) or 11 am (postprandial). net carbohydrate (net cho ox) and lipid oxidation (indirect calorimetry) and oxidation of exogenous carbohydrates (exo cho ox, breath 13co2) were measured. glycogen breakdown was calculated as net cho ox -cho exo ox, and glycogen synthesis as cho in -cho exo ox. energy expenditure over 8 hours was similar but net cho ox was 16 % lower, lipid ox 61% higher and exo cho ox 39 % lower when exercise was performed in fasting vs fed state; moreover, glycogen synthesis was increased by 40 % (p<0.0001), while glycogen breakdown was increased by 12 % (p=0.06). in conclusion, a 45 min exercise in the fasting vs fed state a) increases utilization of endogenous lipids, b) enhances muscle glycogen turnover over a 8 hour period. hormonal regulation of e-abl tyroslne kinase by fusion to a steroid binding domain t. mattioni, o. 8chini hooft van huijsduijnen, p.k. jackson and d. picard d4partement de bielogie cellulaire, universit4 de geneve, ch-1211 geneve 4 the activities of a wide variety of proteins can be subjected to hormonal control by fusion to the hormone binding domain of steroid receptors. we show that this strategy can also be applied to a tyrosine kinase. in particular, transforming derivatives of c-abl become hormone-inducible oncogenes by fusion to a steroid binding domain. it is noteworthy that the hormone-inducible transformation of nih3t3 cells is completely reversible upon removal of the specific ugand. reversion experiments reveal another facet of abl: its ability to inhibit cell proliferation. 48 hours after hormone depletion, cells are not only morphologically reverted, but the cell cycle appears to be blocked in g1. a cytostatic effect of abl overexpression has been reported by several groups but, until today, it could only be examined by comparing different abl derivatives or the same derivatives in different cellular contexts. in contrast, our hormone-regulable system has the advantage that the two distinct functions of abl (transformation versus growth inhibition) can be studied using the same derivative expresed in the same cell line. we are using the hormone inducible system to investigate the mechanisms regulating the transtorming and the cytostatic effects of abl in a variety of cell lines. jiewu yang and herbert zuber institute for molecularbiology and biophysic, eth, 8093 z'tirich enzymes from thermophilic organisms show higher thermostability and temperature optima than those of mesophilic organisms. it has been shown that these properties are based on a specific structure of the protein. the primary structure of thermophilic (b. stearothermophilus) and mesophilic(b, megaterium) triosephosphate isomerase(tim) have been determined by cloning and sequencing of the tim genes. comparison of the primary structures of the thermophilic and rnesophilic enzyme showed specific amino acid substitutions, particularly in the c~-helices of the tim barrel, which could play a critical role with respect to thermostability. we have consmacted mutants by exchanging (x-helices between tim of b. stearothermophilus and b. megaterium. helix h1, h2 and h7 of tim from b. megaterium, corresponding to amino acid residues 13-36, 44-55. and 220-227 respectively, have been exchanged. the properties (thermostability and temperature optimum) of the hybrid-mutants were compared with the wild type enzymes. in a comparative investigation of structural and functional parameters related to the oxidative substrate metabolism in skeletal muscle cells of dogs and goats, a close relationship was found between intracellular lipid stores and mitochondria. as revealed in serial sections with subsequent em analysis, each lipid droplet was in direct contact to one or several mitochondria. quantitatively, 23% of the lipid droplet surface in goats and 40% in dogs were in direct contact to outer mitochondrial membranes or -in other terms -1% of outer mitochondrial membranes in goats and 2% in dogs were in direct contact to lipid deposits. these findings were in good agreement with the physiological data showing a 2 times higher oxidation rate of fatty acids drawn from intracellular stores for dogs than for goats. human muscle adapts to altered load with changes in the expression of enzymes involved in energy metabolism. endurance exercise in humans is known to increase the oxidative capacity of skeletal muscles (hoppeler, int. j. sports med. (1986), 7, 187) . at the ultrastructurai level, a higher mitochondriai content contributes to the higher oxidative capacity of skeletal muscles. little is known about the molecular mechanisms that lead to such adaptations in humans. the expression of mitochondriai components involves complex regulation of both mitochondriai and nuclear encoded genes. we have developed a pcr approach to quantify dna and rna from human skeletal muscle cryostat sections and addressed the question, whether the structural adaptations in endurance trained athletes are accompanied by concomitant changes in respective rna steady state levels. preliminary data indicate a higher concentration of coxiv and coxl (1.8 and 1.6 times, respectively) in trained subjects. no differences were observed for mtdna, despite a two fold higher mitochonddal content. human skeletal muscle contains three main fiber types which are based on the myosin heavy chain composition of the individual fiber, the isoforms of other contractile proteins within a given fiber type can vary leading to a continuum of different phenotypes. we are studying the fiber type specific expression of the mrnas of myosin alkali light clmins (mlc) using in situ hybridization, especially the rarer slow form mlc lsa. differences were found between shoulder muscles and leg muscles: m. deltoideus yielded stronger signals for mlc lsa and mlc lsb in the least glycolytic type i fibers (ia), weak but detectable signals for mlc lsb and mlc lf/f mrna in more glycolytic type i fibers (ib) and strong signals for mlc lf/3f in type ii fibers. in m. vast. lat. mlc lsb was strongly expressed in type ia as well as ib fibers, mlc lsa mrna was only found in some ia fibers. mlc isa has been shown to be expressed at a particular time point during muscle development and regeneration. in a biopsy of a becker dystrophy patient, we found very strong signals in some small fibers displaying the morphology of regeneralmg fibers. thus this probe could be useful to dete~t regenerative processes in pathological muscle samples. the aim of this study was to validate the complementarity of two new non-invasive methods for the assessment of autonomic regulations. reactions to a moderale exercise (75 w on a cycloergometer) were compared in cardiac transplant recipients (ctr) who modulate their heart rate (hr) by means of circulating calecholarnines, their heart being donervated, and in normal subjects where such exercise level doesn't stimulate the sympathetic system. the methods used were: 1) the spectral analysis of hr variability where the high frequency component (hf, above 0.15 t4z, related to the respiratory frequency) is a marker of the vagal activity, the low frequency component (lf; at about 0.1 hz) depends on both sympathetic and parasympathetic activities and the lf/rf ratio indicates changes in the sympatbo-vagal balance; and 2) a pure sympathetic index obtained from the amplitude of the t-wave of the ecg (twa) which is decreased by adrenergic stimulation of the myocardium. our results indicate that in both groups hr was increased by the exercise. in ctr, a net decrease in twa (-39_+ 10%, n=6) was measured. contrasting with this, in normal subjects, no change in twa (+2_+12%, n=6) but a decrease in beth hf (-67_+13%, n=7) and lf (-85_+5%, n=7) without change in lf/hf ratio (-19+_.26%, n=7) were observed. these results indicate that ctr subjects increase their hr by an adrenergic stimulation, whereas the normal subjects regulate their hr for such moderate exercise only by redudng their vagal tone. there was a significant relationship (r=0.79, p<0.0005) between ps and the ree. the slope of the regression line indicated a net cost of ps of 3.6 kcal/g. we conclude that obesity in children is associated with an absolute increase in whole-body protein turnover consistent with the increased ffm and ree. the "glucose paradox" in the anoxic and reoxygenated embryonic myocardium raddatz, e., tran, l., rochat, a.-c., kucera, p. and de ribaupierre, y. institut de physiologie de l'universitd, ch-1005 lausanne. the hearts of 4-day-old chick embryos explanted in vitro react rapidly, reversibly and reproducibly to anoxia and reoxygenation. this work deals with the effects of exogenous glucose on the mechanical activity of the hearts submitted to strictly controlled normoxia-anoxia-normoxia transitions. the anoxic periods lasted from 10 to 60 seconds. instantaneous heart rate, amplitude of contraction, velocities of contraction and relaxation of the ventricle wall were determined optically. in presence of 8 and 20 mmol/1 of glucose 1) the arrest of cardiac activity under anoxia was delayed by 60 and 110 %, respectively, 2) the duration of the postanoxic cardioplegia was reduced and 3) recovery was faster. paradoxally, these concentrations of glucose induced arrhythmias both during anoxia and reoxygenation. the incidences of such arrhythmias increased with the duration of anoxia. the absence of glucose or blockade of glycolysis suppressed arrhythmias. image analysis showed that the observed myocardial dysfunctions originated in the atrium and were sometimes associated with disturbances of propagation. the microinjeetion of okadaie acid into incompetent oocytas leads to the release of the prophase block with entry into m-phase. these g2/m-like alterations include chromatin condensation, germinal vesicle breakdown (gvbd), microtubules reorganization and formation of an abnormal (often multipolar) spindle of meiosis i, with chromosomes not aligned on the metaphase plate. the polar body is never extruded. moreover okadaic acid microiujection induces the emergence of phosphorylated cytolasmic foci as detected by the monoclonal antibody mpm-2, known to react with mitotic phosphoproteins associated with centrosomes. the kinetics of gvbd following mieroinjection is extremdy retarded (24-48hrs) when compared to spontaneous maturation (30ran-lhr) and depends on the oocyte diameter. interestingly, when inc~ompetent oocytes are cultured during 48hrs and then microinjeeted with okadaic acid meiosis resumes within a short delay (3-rhrs). mpm-2 epitopes are present on ecntrosomes only after okadaic acid injection. following gvbd one of the step depending on protein synthesis, the expression of tissue type plasminogen activator, is triggered. indeed oa-injected incompetent oocytes produce small amounts of this activator. altogether these results argue for an effect of okadaic acid favoring entry into mphase including microtubule organization, centrosomes phosphorylatiou and the recruitment of one of the masked mrnas (tpa). st~rillt~, ncb~, c~-121l g~. ~t4rs-inse~, monf~llier, france. meiotic reinitiation of the mouse oocyte is caracterized by a slow entry into metaphas~ i, b6~jinnin9 with germinal ve~ir break@own (gvbd) and ending with spindle formation. it is accompanied by a cascade of protein kinases and phosphatases increasir~g protein phosphorylation. the activation of histone hi kinase (maturation promoting factor, mpf) and of the 1342 hapk have been compared during spontaneous or okedalc acid (oa)-induced rhetoric reinitiation. in spontaneously maturing oocytes, hist6ne hi kinase activity increases before gvbd (2x) and is a~sociated with the disappearance of the upper (tyrosine phosphorylated) migrating form of p34 cdc2, following gvbd, histone hi klnase activity culminates (8x) in metaphase i. activation by phesphorylation of p42 mapk occurs after gvfld. in contrast, no increase of histone hi kinase is detec~cable in oocytes induced to reinitiate meiosis by a transient exposure %o oa, neither before gvbo nor during the following 7 hours of culture. the upper migrating form of p34 cdc2 is present for 8 houra. the activation of p42 mapk b~lins before gvbd. furthermore, when oa is applied on coclrces microinjected with p13 sucl, neither iocrease of histone hi kinase activity nor p34 cdc2 dephosphorylation are observed although gvbd is induced; p42 mapk is activated. altogether these results suggest that gvbd may or may not be associated with a detectable activation of histene hi kinase, depending on the experimental conditions. activation of ps4 cdc2 and p42 mapk are separable events. we propose that, in the mouse c~yte, oa might be able to aetivaf8 an alternative pathw~ leading to gvbd that is cdc2-independent and that involves p42 mapk activation ensuing mpf-independent phosphorylations. s 10-04 urich, k., 4002 basel, switzerland transformation of cells by polyomavirus is mediated by middle-t antigen. this viral protein is able to form complexes with src family tyrosine kinases (e.g. c-src), phosphatidylinositol-3-kinase (pi 3-k) and phosphatase 2a (pp2a). c-src associated with middle-t is constitutively dephosphorylated at tyrosione 527, a site negatively regulating the activity of this enzyme. this results in high tyrosine kinase activity in interphase and mitotic polyoma-transformed cells. middle-t is transiently hyperphosphorylated during mitosis as reflected by an increase in the apparent lvl, on sds acrylamide gels. two putative phosphorylation sites for a cyclin-dependent kinase present in middle-t, threonine 160 and threonine 291, are also phosphorylated by purified p34 ~a~ in vitro. we constructed mutant middle-t genes where individual phosphorylation sites were converted to alanine residues. mutants lacking threonine 160 were still able to associate with all cellular enzymes described above yet defective for gell transformation. interestingly, the defect of this mutation could be compensated by additional changes in the middle-t protein sequence introduced further downstream in a domain suspected to play a role in the targeting of this protein to intracellular membranes. $10-05 schmidt, s., fa~khauser, c., and viesturs, s. isrec, 1066 epalin~es, switzerland little is understood of the mechanisms which regulate cytokinesis, or how it is integrated with mitosis. we have cloned a number of genes from the fission yeast s. po~be which are required for the initiation of septation and are characterising them. defects in the cdc7 and cdcll genes result in continued nuclear division without cytokinesis, while cdcl6 mutants undergo multiple rounds of septum formation without cell cleavage. our analysis suggests that phosphorylation will play an important role in the regulation of cytokinesis and its integration with mitosis. a functional cdcl6 product is required for maintenance of p34 r activity in mitosis and the primary sequence of the cdc7 protein indicates that it encodes a protein kinase. data concerning the role of the cdc7, cdcll and cdcl6 genes will be presented. rfc was originally identified in human cell extracts as one of the cellular factors essential for the replication of sv40 origin-containing dna in vitro. it is a five subunit protein with one large subunit of 100-i40 kda and four small subunits of 36-40 kda. rfc binds specifically to primertemplate structures at the 3'-end of the primer. together with proliferating cell nuclear antigen (pcna) it serves as a primer recognition factor for dna polymerase ~ and ~. to show the invobement of rfc in cellular dna replication we are currently cloning s. cerevisiae rfc. the amino acid sequences of a number of tryptic peptides have been obtained. this information was used to amplify parts of the s. cerevisiae genes by per and to screen a genomic library with these probes. three genes were cloned so far. the large subunit, rfc1, codes for a 95 kda polypeptide that is 36% identical to the large subunit of human rfc (lirfc). the sequences for two of the small subunits were also obtained. rfc2, coding for a 40 kda polypeptide, is 50% identical to the hrfc 37 kda subunit. rfc3, coding for a 38 kda polypeptide, is 51% identical to the hrfc 36 kda subunit. there is also considerable similarity between the different subtmits. rfcj, rfc2 and rfc3, as well as the hrfc subunits, have consensus sequences for a atp/gtp binding site. all three genes are essential in s. cerevisiae. p53 is a major tumor suppressor gene which spefically interferes with the onset of e phase. we have therefore cxa~ined the possibility that p53 modulates the normal functions of enzymes and proteins directly involved in dna replication. we have shown that human wtp53 protein binds physically to calf thymus single stranded dna binding protein a, rp-a, we have also found that p53 blocks dna helicases in a typical strand displacement assay. this may reflect the fact that p53 is able to reanneal complementary dna single strands. since both wt and some mutant forms of p53 share this function, they are unlikely to be related to the p53 tumor suppressor activity. to our surprise, among the helicases which were tested we determined that the p53 inhibition could be released by rp-a in the case of cellular but not in the case of viral dna helicases. additionally, we found that p53 can partially inhibit dna polymerase ~ and s holoenzymes on singly primed mi3 dna. this inhibition, however, was only evident when e. coli ssb was substituted for rp-a. our data thus show that p53 exerts an inhibitory effect on several enzymes involved in dna replication and dna repair. the p53-rp-a interaction may function to protect replication enzymes from inhibition by p53 under certain physiological conditions. catalytic subunit showed that pol ~ is the most conserved replicative pol (cullmarm g., hindges r., berehtold m.w. and htibscher u., gene, in press). we synthesized pcr primers and cloned three fragments spanning the whole catalytic subunit. the resulting pcr products, called n-term, middle and c-term have a size of 1600, 1570 and 1280 bp, respectively. the primer for the n-term and middle fragments contained a histidin tag in order to purify the recombinant proteins by using a ni-nta column. these fragments were cloned into the expression vector pgemex174. because of the natural termination codon in the c-term fragment, the histidin tag was placed directly in the vector in front of the fusion protein, generating a new vector, prhh1s. all three fragments were expressed in e.coli. the fusion proteins could be purified in a single step and were used to raise polyclonal antibodies. finally, the entire pol 8 sequence was joined together and could be expressed. data on the characterisation of the antibodies and the proteins will be shown. hsp 70 from calf thymus can influence dna polymerase under heat shock conditions. we have generated antibodies against dnak protein, the hap70 homolog from escherichia coil, and used them for detecting of hsp70 protein during its purification from calf thymus tissue. the apparently purified protein has a molecular weight of 70kda, and has been recognized by antibodies against dnak and eucaryotic hsp72/73, it possesses a very week atpase activity, which can be stimulated by different poly-and oligopeptide substrates. results will be presented showing the influence of hsp70 on dna polymerase r from calf thymus under heat shock conditions. identification of a vertebrate cdc2 mutant which is unable to complete the g1/s transition. nicole sr and viestars simanis. chemin boveresses 155, 1066 epalinges,.isrec. s. pombe strains have been constructed which should permit the generic and molecular analysis of the events which occur in late gi when cells become committed to the mitotic cell division cycle and the initiation of dna synthesis. a number of mutants of the chicken cdc2 gene were eonsuueted during the course of analysis of phosphorylation sites some of which lead to the interesting phenotype of cold sensitivity when expressed in a cdc2 null background, when the only source of p34 cde2 is the chicken homologue of the gene, expressed from a muldcopy plasmid. in order to create a genetically tractable strain, the wild-type chicken cdc2 gone and one mutant into the s. pombe genome to create a strain in which cell cycle progression is dependent upon the chicken cdc2 gene. analysis of this strain indicates gnat the cells block predominantly before replication of dna. in order to determine whether the cells were arrested before or after the execution of the start control their ability to conjugate at the arrest point was assessed. consistent with a block before the traverse of start and contmim~ent to s-phase, cells were able to conjugate with high efficiency. further support for the view that this mutant is defective only for the g1-s transition is provided by the observation that it is able to complement a cdc2a2l mutation in trans. this mutant is defective only for (32 function and not traverse of start. tl~e double mutant is viable at the restrictive temperature of either of tile parents alld is no longer cold sensitive. further work will concentrate on cloning genes involved in the traverse of gi into s phase in fission yeast. (masson and kreis, 1993, j. cell biol. 123:357) where it is localized on a subset of microtubulea. when caco-2 cells are grown to confluency and become polarized, e-map-115 expression levels increase concomitant with a higher microtubule stability. transfection of fibroblastic cells (veto), which do not contain any detectable e-map-115, with cdna encoding this protein, results in stabilization of microtubules to nocodazole treatment. these data suggest that e-map-115 may be involved in the stabilization and reorganization of microtubules in polarizing epithelial cells. since e-map-115 is a stabilizing protein, its interaction with microtubulen should presumably be modified at the onset of mitosis to allow disassembly of the interphase microtubule network and formation of the mitotic spindle. indeed, immunolabeling for e-map-t 15 varies during mitosis. in early prophase, no e-map-115 can be detected on the microtubules of the forming mitotic spindle. the protein is observed on the spindle microtubules of metaphase cells and staining becomes bright on the new interphase microtubules of telophase cells. analysis of the protein in cells blocked in mitosis by low concentrations of nocodazole indicates that e-map-115 is hyperphosphorylated. this hyperphosphorylated form of e-map-115 is no longer able to co-sediment with microtubules in in vitro microtubule-binding assays. phosphopeptide map and phosphoamino acid analysis show the existence of novel phosphorylation sites in e-map-115 during mitosis compared to the protein during interphase, we are currently characterizing the kinases involved in the modulation of e-map-115 activity. little is known about the specific functions of calcium-binding proteins in epithelial cells ~md in particular in tumoral cells of epithelial origin. calretinin (cr) and parvalbumin (pv) are supposed to be involved in cell proliferation. we observed the endogenous expression of cr in several cell lines from human colon adenoearcinomas, and we obtained stably transfected cells for pv in a human ovarian adenocarcinoma cell line. we studied the cell cycle in correlation with the endogenous or ectopic expression of cr and pv, respeetiveiy. g1 and mitotic celts are strongly labelled with the cr-antiserum 7696, while pv immunoreactivity is dominant in interphasic, but not in mitotic cells. in cr expressing cells, the cell cycle seems to be normal and the rate of proliferation to be proportional to the percentage of positive cells. the cell cycle is inhibited at mitosis after the addition of cr antisense oligo nucleotides to the culture medium. the cell cycle is blocked in gi/s and g2 in the cells eetopically expressing pv. the results support the hypothesis that these two calcium.binding proteins, cr and pv, could intervene at different moments and probably with different mechanisms on the mechanism of the cell cycle in the tumoral cells studied. production of polyclonal antibodies against calretinin cr-22k gander, j.-ch., gotzos, v., cello, m.r. and schwaller, 8. institute of histology and gen. embryol., university; 1700-fribourg a mrna for an alternatively spliced form of calretinin, ealretinin-22k (cr-22k) was detected in widr cells (a cell line from a human adenocarcinoma). we therefore decided to produce antibodies specific for this protein. it is directed against the 14 amino-acids localized at the c-terminus of the truncated protein which are unique for this protein and not present in full-length calretinin. two different approaches have been chosen to obtain a specific antiserum. we have produced a chimeric protein containing the cterminal region of cr-22k and the (h)6(nanp)19 polypeptide as carrier. the fusion protein was expressed in e. coil and purified on a nickel chelate column. as a second method, a synthetic peptide (corresponding to the last 15 amino acids of cr-22k) was crosslinked with the carrier protein klh (keyhole limpet hemocyanin). the antigens were used to immunize two rabbits. after the first boost, the 2 sera were tested. we have observed that both antisera recognize besides the protein used for immunization also cr-22k (expressed in e. coil) in a western blot specifically. no other protein bands of either e.coli extracts or from eytosolic extracts of widr cells crossreact with the 2 antibodies. both antisera detected the protein cr-22k in immunohistochemieal stainings of widr cells confirming the presence of cr-22k in widr cells. the tctp (p23) is a growth related tumour protein which is expressed under translational control. homologous acidic proteins have been found in higher plants and in saccharomyces cerevisiae (ykl312) . the striking conservation of this polypeptide between these very divergent organisms suggests some important but still, unknown cellular function. to address this question, a null mutation was constructed in yeast, by deleting almost all the ykl312 structural gone. the deleted strain shows no detectable phenotype. therefore, we conclude that, in yeast, this gene is dispensable. the protein ykl 312has been overproduced in e. coil and injected to rabbits to raise antibodies. we are now characterizing the ykl312 expression at rna and protein level. ellenrieder,c., forrer,p., kosovsky,j., rischle, m., nueller, r. and jaussi, r., institut f~r medizinische radiobiologie, paul scherrer institut & universitgt zh, ch-5232 villigen radiation therapy of tumors could be improved by influencing cellular radiation sensitivity. yeast strains with defective cell cycle checkpoint genes (e.g. radl, rad3, radg) show increased radiation sensitivity. cross-hybridization of a probe from the tad9 gene on human mrna northern blots yielded a single prominent signal. experiments to clone the corresponding 4 kb cdna are underway. we have cloned a hamster cdna encoding the homologue of human cdk2. probably, this cdna encodes the p40 protein from hamster whose phosphorylation responds to radiation checkpoint signals and who does not bind to p13 sucl beads. treatment of cells with cdk2 antisense oligonucleotides is hoped to modulate the radiation sensitivity. proliferation of smooth muscle cells (smcs) is a major event in vascular development and atheromatous plaque formation. in order to characterize smc replicative potential, newborn rats have been injected with 3h-thymidine (3h-tdr) during their first week of life when most of smcs were proliferating; then, 3h-tdr labelling was evaluated in adult rats. depending on the number of mitosis that smcs had accomplished after the first week of life, four different smc subpopulations could be defined indicating that rat aortic smcs are heterogeneous in their replicative activity. 5-bromo-2'-deoxyuridine (brdu) incorporation after balloon induced endothelial denudation of rat aorta in adult rats showed that smcs entering into the cell cycle were mainly devoid of 3h-tdr labelling, this category could derive from two distinct smc subpopulations: smcs which were arrested in their proliferation before or just after birth or smcs which had actively replicated during development. thus, one (or two) subpopulation(s) of rat aortic smcs, characterized by a particular replicative activity during development, is (are) selectively activated after balloon induced endothelial denudation. the situation in vivo of dermal fibroblasts embedded within the connective tissue can be emulated in vitro by growing the cells in collagen gels. we have investigated how fibroblasts cultivated within a matrix react upon wounding of this dermal equivalent. human skin-derived fibroblasts (kd-cells) growing within attached collagen matrices were monitored over a period of 8 days. fluorescently labeled cytoskeletal elements (tubulin, vimentin, acfin) and fibronectin served as phenotypica/ markers. the 3d-arrangement of fibroblast microtubules, intermediate filaments and microfilaments, as well as of fibronectin was visualized by clsm and optical sectioning. cells at the wound margin, adjacent to the wound, and in non-wounded controls, up to a depth of about 100gm were analyzed. upon wounding, i.e. upon excision of a 1 mm 0 "punch biopsy" from the center of the collagen matrix, fibroblasts in the wound region oriented towards the matrix defect. layer-like structures of increased cell density evolved over time at the wound margin. the tissue defect was covered by fibroblasts, reminescent of the situation in vivo. global yeast genome expression analyzed by 2-d page after tctp homolog gene (ykl312) disruption. sanchez a translationally controlled tumor protein(tctp) was identified and microsequenced from a human liver sample. previous publications described the presence of a gene related to tctp in plants, mouse erythroleukemia and ascetic tumor, human mammary carcinoma and more recently in the yeast. tctp has no known function but its high degree of homology from plants to human underlines its probable crucial role in cell function. in order to study multifactorial chan~es in protein expression as a function of the ykl312 gene disruption m the yeast, we used high resolution two-dimensional gel electrophoresis combined with an efficient computer system (melanie). two groups of gels (ykl312 +(n=10) and ykl312 -(n=l 0)) were analyzed, compared and classified using correspondence analysis. there was no significant difference between the two populations of gels according to either qualitative and quantitative spots expression except for two spots which completely disappeared. the first one corresponded to ykl312 gsne product. the second one corresponded to a 28 kda peptide with an isoelectric point of 4.6. this ykl312 associated peptide still needs to be characterized and linked to the yeast genome. demyelinating and neurodegenerative diseases are associated with altered cellular responses including processes mediated by receptor protein tyrosine kinases. using a pcr cdna cloning approach we have identified a novel member of the eck/elk/eph genefamily in myelinating serum-free brain cell cultures. alternatively spliced cdnas encoding complete open reading frames for putative transmembrane proteins have been isolated from both rat and human brain. a recombinant protein derived from the rat cdna was demonstrated to have protein tyrosine kinase activity. an affinity purified antiserum to this protein was used to identify a glycoprotein of 120 kd molecular weight in brain cultures, and developping and adult brain tissue. the protein expression is most prominent during embryonal development and during the first three weeks of postnatal life. by in-situ mrna hybridisation the transcripts were found predominantly in restricted neuronal populations. in order to investigate systematically the effects of geometrically defhied abrupt tissue expansion on the characteristics of impulse propagation, we constructed expanding cellular structures in vitro using patterned monolayer cultures of neonatal rat heart cells (photolithographically patterned substrates.) the preparations, which consisted of narrow cell strands (40-80 tim wide) inserting into large reetangular cell areas (side lengths >1500 ~m), were stained with the voltage-sensitive dye di-g-anepps and were imaged onto a 12 x 12 photodiode matrix array (maximal spatial resolution 151.tin x 151am in the object plane). while impulse propagation was uniform in linear cell strands (average conduction velocity 0.35 m/s), a transient decrease in conduction velocity hi the transitional region was invariably observed in the suddenly expanded structures. this decrease was accompanied by marked distortions of the local action potential shapes due to bidirectional electrotonic interactions across the transition: upstream from the expansion, the repolarization phase was interrupted, a few ms after its inception, by a small secondary depolarization ("spike and dome" configuration), while, downstream, the action potential upstrokes were preceded by prominent prepotentials. in those cases where conduction failed at the tlansition, action potential durations upstream were dramatically abbreviated. these findings showed that phenomena similar to those recorded in intact purkinje-fiber-ventricular preparations can be observed in cell ensembles in vitro having comparable geometlins but consisting of cells with uniform active membrane properties. supported by swiss nsf grant 823a-028424 (sr) and usphs grant ns 16824 03ms). it is a noncollagenous glycoprotein with a molecular mass of 148 kda consisting of three identical subunits. cmp is selectively extracted with edta-containing buffer what indicates a divalent cation dependent anchorage of the protein in cartilage matrix. electron microscopy revealed the presence of three compact globular subdomains and sequence analysis indicated the presence of a coiled-coil c~-helical assembly domain formed at the c-terminal end of each subunit. the trimeric structure was retained after complete reduction under native conditions which reveals that the subunit structure of cmp is not only stabilized by disulfide bridges but also by the coiled-coil assembly domain. tissue distribution studies in mouse revealed that cmp is selectively expressed in some but not all cartilages. adult rat cardiomyoeytes (arc) in culture degenerate the myofibrillar apparatus and after attachment to the substrate they grow and reassemble new rnyofibrils. the appearance of new, well organized rnyofibrils can be observerd in several distinct regions. they appear close to the membrane proximal to the culture substrate and in the perinuclear region close to the substrate. previous studies have shown that embryonic cultured cells assemble new myofibrils which insert in adherens junctions at sites of cell-cell contacts and in sub-sarcolemmal adhesions plaques (saps) at sites of cell-substrate contacts. this saps are thought to function as the nucleation sites for myofibril formation. we have investigated the formation of this saps in cultured adult rat cardiomyocytes. using confocal light microscopy we can show that the interaction between rnyofibrils and membrans occurs close to the membrane proxirnal to the substrate and that this saps are flanking both nascent myofibrils and sfls, the latter structures being believed to serve as scaffold for myofibrillogenesis, additionally we have used electron microscopy to investigate the formation of this structures at higher resolution. $11-05 muscle satellite cells (sc) are quiescent myoblasts, ready to be activated and to regenerate new muscle fibers in case of muscle damage. in vitro, single human muscle sc, cultivated as clones, give rise in proliferating conditions to two subpopttiations of cells, cells expressing both ~-striated muscle actin and desmin (c~-sr+dsm+), and cells expressing desmin alone (dsm+). in culture conditions promoting differentiation, a clone .typically gives rise to a fusing progeny, yielding c~-sr+dsm + myotubes, and to non fusing myoblasts (nfmb). the latter are dsm +, a phenotype similar to quiescent sc found in situ. in order to determine whether nfmb are the in vitro equivalent of in situ quiescent sc, this first generation of nfmb were selectively collected and subcultured. in proliferating conditions, nfmb were able to resume proliferation and to give rise to a progeny with both c~-sr+dsm + and dsm + cells. when cultured in differentiating conditions, nfmb formed ct-sr+dsm + myntubes, and a new generation of dsm + nfmb. when nfmb of the second generation were again selectively collected and subculture& they resumed proliferation and ~re again able to yield both myetubes and a third generation of nfmb. nfmb of the first generation were also cultivated as clones, and 5 to 25% of them were able to resume extensive proliferation, yielding in their progeny both c~-sr+dsm + and dsm + cells, which differentiated into myotubes as well as nfmb. these results strongly suggest that sc have the stem cell property, of selfrenewal, yielding in their progeny both cells ready to differentiate into muscle and ceils similar to themselves. extra cellular matrix (ec~ regulates the expression of b-casein in cultured mouse mammary epithelial cells. we have developed a functional ~ epithelial cell strain, which expresses high level of milk proteins, forms alveolar-like structures when plated onto a reconstituted basement membrane and secretes casein unidirectior~lly into a it~aen. we have further shown that fflv~dependent regulation of b-casein occurs mainly at the transcriptional level and that 5' sequences play an inloortant role in this regulation. we have located a 160bp transcriptional enhancer (bcei) within the 5' flanking region of the b-casein gene. using functional assays, we show that bcei contains responsive elements for ~ and prolactin-dependent regulation. bcei placed upstream of a truncated inactive b-casein promoter reconstitutes a promoter even more potent than the intact prcmoter, which contains bcei in its natural context more than 1.5kb upstream. this small fusion promoter reconstitutes the nonaal regulation pattern. we show that bcei mediates f/24 dependent regulation even when linked to a het~rologous viral promoter. purified satellite cells (sc) were obtained from human skeletal muscle biopsies following cell sorting by flow eytometry. a chemiluminescent assay for acetylnholine (ach) revealed the presence of 1,3 nmoles/weu of 100.000 sc. in elonal cultures of proliferating sc, this intracellular level of ach was 0.l nmoles/well. when cells were cultured in the presence of an esterase inhibitor (10 ixm phospholine), the ach amount was enhanced 2 fold. conversely, cultivating the cells in presence of a potent inhibitor of choline acetyl transferase (2 gm bromoach), gave a 50% decrease of ach content finally, the release of ach was detected in the supernatant of cultured sc, following a 15 min incubation, and the measured level of ach was 3 pmoles/well/min. the presence of an ach-like compound in myogenic cells in both freshly isolated and in proliferating sc suggests that sc may contain ach in situ. in additiou, the fact that ach was present in the extracellular medium suggests that ach can be released spontaneously by the cultured sc. type xii collagen is an extracellular matrix protein associated with collagen fibrils in vivo. as observed for several other extracellular matrix proteins, the synthesis of type xii collagen by chick fibroblasts cultured in 0.1% fcs is stimulated by tgf-i~i. two splice variants of type xii collagen are known, with subunits of either 220 kda or 350 kda. by using antibodies specific for the large (350 kda) form of type xii collagen, we could show a more restricted distribution of the large variant compared to the smaller form in the embryo. while only the large variant carries chondroitin sulfate, both variants are identical in their collagenous domain. it is thought that via this domain, type xii collagen binds to collagen type i fibrils. we demonstrate here that type xii collagen can bind to collagen i fibrils also in vitro. by neutralizing acid soluble collagen type i, we could coprecipitate type xii collagen together with newly formed collagen type i fibrils. this interaction occurs in physiological saline but is highly salt dependent. in further studies we investigated wether 35s-methionine labeled type xii collagen treated with chondroitinase abc, ct-chymotrypsin, or collagenase can still be precipitated together with type i fibrils. in addition, we found that type xii collagen also affects the rate of type i collagen fibril formation. h.u. keller department of pathology, university of bern, ch-3010 bern locomoting blebbing cells colchicine (10-sm) can induce locomotion associated with blebbing in walker carcinosarcoma cells. blebs expand at a rate (about 2~m/sec) which is much faster than known rates of actin elongation. this suggest that the membrane is directly pushed forward by forces other than actin elongation, possibly by hydrostatic pressure. this interpretation is suggested by the observation that there is significant f-actin staining all along the cell membrane but not with the cytoplasmatic content the blebs. blebbing is suppressed by 0.5 m sorbitol. the finding that cytochalasin d suppresses blebbing shows that actin polymerization is nevertheless indirectly instrumental in bleb formation, possibly by generating a high intracellular pressure. a tentative model explaining locomotion of blebbing cells is presented. osteopetrosis encompasses a family of diseases with different causes and the common phenotype of impaired bone resorption. the osteopetrotic mouse mutant oo/oo is deficient in csf-1, the growth factor for the cells of the mononuclear phagocytic system. the phenotype of oo/ou mice is characterized by a low number of peritoneal macrophages and peripheral monocytes, and a virtual absence of osleoclasts, leading to the osteopetrotic phenotype. injections of csf-1 into op/oo mice reversed the osteopetrotic phenotype, proving the requirement for csf-1 during the process of osteoclastogenesis. by in situ hybridization, expression of csf-1 was demonstrated during bone development. osteoclast precursors and mature osteoclasts were identified as putative target cells for the growth factor, since these cells express csf-1 receptors, which is encoded by the proto-oncogene c-fm~. subsequently, specific binding of csf-1 to osteoclasts was shown. expression of c-fms parallels the expression of csf-1 in bone both in time and place, suggesting a local action of this growth factor in osteoclast formation, but also in the modulation of osteoclastic activity. different transcripts, raised by alternative splicing of a commmon nuclear rna precursor, have been found to encode a secreted and a membrane associated forms of csf-i. the secreted form can be modified by attachment of a glycosaminoglycan side chain, which serves as an anchor to integrate the peptide into the extracellular matrix. investigation of the role the different csf-1 forms play during recruitment of osteoclasts and regulation of their activity will provide further insights into the mechanisms governing these processes. comparative sequence alignments of known voltage-gated k + channels revealed two conserved cysteine residues in the putative transmembrane segments $2 and $6. if the cysteines were connected by a disttifide bridge, it would put a structural consllaint on possible channel models by placing $2 next to $6. we used site-directed mutagenesis to study systematically the potential roles of these invariant cysteines. fourteen of 17 substitutions in $2 (c232), and 7 of 19 substitutions in $6 (c393) maintained channel function in xenopus oocytes microinjected with mutant crna. therefore, the conserved cysteines are not essential for k + channel expression in xenopus oocytes. however, electrophysiological characterization of the cysteine replacement mutants revealed distinct roles for the two cysteines. inactivation, deactivation, and ion permeation did not considerably change in $2 mutants. in $6, in contrast, cysteine replacement by leucine, asparagine, glycine and valine accelerated inactivation and deactivation kinetics substantially, whereas serine and threonine showed opposite effects. furthermore, the voltage dependence of deactivation was differently affected. in summary, it appears that the side-chain at position 393 in $6 of kv2.1 is involved in channel gating by participating in the transitions from the open to the closed and inactivated state of the channel. (supported by grants from the nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) the segment between $5 and $6 of voltage-gatedk + channels, called h5 or p region, has been implicated to form part of the ion conduction pathway. little is known about the conforroation of this region although various models have been proposed including a j3 barrel-like structure formed by four adjacent antiparanel j3 sheets. to gain insight into the secondary structure of this region, we used cysteine substitution mutagenesis and sulfhydryl-specific, membrane-impermeant reagents to probe the accessibility of amino acid side chains in and around the p region of kv2.1 (drki). twemy-eighi positions from k356 to t383 were each mutated to a cysteine. after expression of mutant k + channels in xenopus oocytes, cysteine side chain accessibilities were probed by superfusion with ch3so2sch2ch2nme3 + (mtset), mtset can form a mixed disulfide with an accessible cysteine, and this may lead to current reduction if the covalently modified side chain is in or close to the ion conduction pathway. thus far, we have identified four mutations that showed k + current reduction after superfusion with 3 mm mtset. the mutants p361c, i379c, y380c, and k382c showed 87%, 99%, 39%, and 21% reduction in current amplitude, respectively. in coutrast, mutants $363c, a367c, t368c behaved like wild type kv2.1 with less than 5% current reduction. our results suggest that the side chains of p361,1379, y380, and k382 are directly accessible from the extracellular environment. taken together, these residues may, therefore, face the lumen of the ion channel pore. furthermore, the degrees of inhibition are in agreement with a model in which the ion conduction pathway narrows from residue k382 to y380 to i379. (supported by grants from nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) motejlek k., h,,iuselmann r., and liig:her b.; pharmakologisches institut der universitiit ziirich, winterthmtr. 190, ch-8057 zlirich comparison of 5' flanking sequences of three gabaa-receptor subunit genes (atl, ~, 8) revealed a novel conserved purine sequence dement with the consensus sequence gagaggggagaoga gagag(gg/aa)g. this element is present once each in the (zl and 72 subunit gene promoters and in seven tandem copies in the 8 gene promoter. a novel dna binding activity (bsf1) was identified that binds to various versions of this purine element. bsf1 was found ordy in brain but not in any other tissues tested. furthermore, the factor is distinct of beta, another brain-specific dna binding .protein with a purine-rich dna recognition sequence. the expression of bsf1 during differentiation of eerebeuar granule cells in vitro correlates with the expression of gabaa-receptor subunit genes. this correlation suggests a role for bsf1 as a transcriptional activator of neuronal genes. therefore, bsf1 may be important for cell ty~specific regulation of gabaa-receptor gone expression. we also found that bsf1 binds to wheat germ agglutinin, suggesting that this protein is glycosilated. taking advantage of this property, we are in the course of purifying bsf1, the question, whether bsf1 is indeed a transcription factor is being investigated in vivo and in vitro. to produce a simple method of estimating the free magnesium concentration ([mgzq) in solution, we have manufactured dip cast mg 2+ macroelectrodes using the neutral mg carrier eth 7025.we have used the elecmides to measure the dissociation constant (k~) for mgatp in a background solutions mimicking the intracellniar milieu. for the mcasmement of the big atp dissociation constant, the background solution contained 2 mmol/l n%atp. this solution was titrated with mgci 2 and the changes in [mg ~] above 0.05 retool/1 monitored with the maeroelectrode. during the titration the ph was maintained constant. fitting such titration curves with the standard hyperbolic binding equation, after correction for zero drift, gave the following mean+sd values for the ka (pmol/1 at 37~ ph 6.4, 103.3-+-25,3 (n=9); ph 7, 59.7:k-27.4 (n=6) and ph 7.4, 30.g~15.4 (n=7). elevation of the [naq to 50 m~l/1 and reducing the [kq to 1(30 mmol/l was without effect at ph 6.4 (n=6). reducing the temperature to 25~ increased the k a at 7.4 to 49.0:k21.6 (n= 5). mg =+ macroelcctrodes provide an easy way of measuring [mg z ⧠in solution. [mg ~] can also be measured in a background solution containing i mn~i/l ca, although the electrode response is reduced. interference by protein to date prevents their use in plasma. similarily to the block by zn 2+ and ca 2+, protons only partially block the channel. the ability for all mutated channels to. conduct na + current, and the partial block induced hy zn 2+, ca 2+ or h + indicate that y401 is not located deep in the pore but rather in the extracellular mouth of the channel. progesterone modulates the activity of glycine receptor expressed in colliculli neurons of neonatal rats. maury, k. & bertrand, d. dpt of physiology, cmu, 1211 geneva 11. steroids are potent narcotics and have been shown to act on ligand-gated channels activated by gaba or nicotine. however, little is known about their possible actions on other receptor types. for instance, while it was demonstrated that progesterone inhibits glycine receptors expressed by spinal cord neurons isolated from chick embryos, nothing has yet been reported for brain receptors. in this study, we have determined the properties of glycine channels expressed by brain neurons of neonatal rats using the whole-cell voltage-clamp technique. in inferior colliculi neurons, glycine elicited little-desensitizing inward currents which reversed around -40 mv. these currents were blocked by strychnine in the nanomolar range. surprisingly, however, we found that progesterone, in the micromolar range, potentiated the glycine evoked currents. these results, contrasting those obtained in chick spinal cord neurons, suggest that progesterone enhances or antagonizes glycine currents depending on the subtype of the receptor. the mammalian auditory organ (organ of corti) relies on two groups of sensory cell for its normal hearing: inner hair cell & outer hair cell. ihcs are mainly innervated by afferent fibers while the ohcs essentially receive efferent fibers. the ohcs possess unique electromofile properties which are thought to enhance the motion the basilar membrane and to refine the mapping of the sound frequency. we found, using whole ceil recordings, that 5-10% of the fleshly dissociated ohcs displays fast inward currents. the magnitude of peak currents which can be as large as 2na vary from cell to cell. further experiments have revealed that this current is sensitive to ttx and strongly reduced when extracellular sodium is replaced by choline. its kinetic is relatively slow compared to that of sodium current of typelspiral ganglion cell, and has a more negative inactivation. other voltage activated currents and ligand-gated currents are under inverstigation. these results will provide further insights in the hearing transduction mechanism. the formation and the properties of homotypic and heterotypic gap junctions were studied using two types of insect cells, c6/36 and sf9. two single cells were pushed against each other and the subsequent de novo formation of gap junction channels was assessed by means of the dual voltage-clamp method (bukauskas and weingart: pfl~g. arch. 423:152, 1993 it was symmetrical for homotypic junctions. the vj-sensitivity was less pronounced in sf9 cells. hence, the relationship for heterotypic junctions was asymmetrical. all pairs examined revealed a s-shaped relationship between gj(ss) and v, which was virtually superimposable (gj(ss) declined upon depolarization). each channel contains a vm-gate and two ~-gates. the vj-gates are operated independently. they close when their intracellular aspect is made positive. shaped curve compatible with a two-state boltzmann process. half maximal inactivation was reached at -77 mv and +66 mv, implying an asymmetrical gating behavior. in case of an asymmetrical protocol (~ and vewas stepped simultaneous, but in opposite direction), the q-dependent asymmetry disappeared (half maximal inactivation at -59 mv and +60 mv). the asymmetry in gj-gating, attributable to the pulse protocol, was also reflected in the time constants of ij inactivation (r177 cerebellar granule cell cultures were used to study the regulation of the nmda receptor expression. we have shown previously that chronic membrane depolarisation (25 him k +) or treatment with nmda {i00 ~m) promotes the functional expression of nmda receptors, as assessed by nmda-evoked 45ca2+ influx. we have now developed a rnase protection procedure for the quantitative determination of the mrna levels of the nmda receptor subunits known so far (nr-i,2a,2b,2c,2d). the growth conditions mentioned above failed to alter the mrna levels of the different subunits. at the protein level, nmda receptors were evaluated quantitatively by the labeling pattern obtained by the new photoaffinity ligand 125i-cgp55802a. the intensity of the phctolabeled bands, thought to represent nr-i and nr-2a subunits, was increased by treatment with high k + or nmda compared with control cultures. this result suggest an increase in receptor protein by high k + or nmda treatment. thus, posttranscriptional mechanisms seem to play an essential role in the modulation of the nmda receptor activity. the gene encoding the pore-forming subunit of the rat epithelial na + channel (~renac) was cloned recently and shown to belong to a novel gene family coding for cation channels (nature 361, p467-470 (1993) . transport of na + ions through these channels is the rate-limiting step of na + absorption and thereby controls osmotic balance of body fluids and secretions. in order to study the implication of ~renac in regulating sodium balance, blood volume and blood pressure and therefore its involvement in hypertension, we expressed ~renac under the control of the human cmv promoter in transgenic mice. several lines of mice showing expression in different tissues were obtained. progress in the analysis of these mice will be discussed. activation of metabotropic glutamate receptors increases the excitability of neurones in the lateral septum of the rat. to elucidate the mechanism(s) of this action, we used whole-cell patch-clamp recordings from coronal brain slices of young animals. in voltageclamped cells, the selective agonlst (is,3r)-acpd, at 1-50 /~m, had the following effects, a) it evoked a sustained inward current, which was resistant to ttx and to cadmi~am and which persisted in neurones loaded with bapta, a calcium chelator. this current displayed inward rectification and was reduced, or suppressed, when extracellular sodium was partially replaced with n-methyl-d-glucamine. b) it elicited a ttx-insensitive, voltage-dependent inward current, which activated at -50/-40 mv and which reversed at aound 0 mv. this current was suppressed in a low-calcium/high-magnesium perfusion solution and was undetectable in the presence of intracelhilar bapta. we suggest that acpd exerts a dual action on lateral septal neurones. it causes a steady depolarization by generating a sodium-dependent current and it triggers transient plateau potentials by inducing a calcium-dependent cationic current. interaction between these currents results in a powerful, self-reinforcing neuronal excitation. we have investigated the effects of protein kinase c (pkc) activators (4[~-pma, oag) and of phosphatase inhibitors (okadalc acid, calyculin a) on voltage-gated ca 2+ and k + channels in ngf-differentiated pc12 ceils. whole-cell ba 2+ and k + currents were recorded with the patch-clamp technique. by using specific ca 2+ channel blockers (co-conotoxin (cgtx), isradipine) we found 3 types of ba 2+ currents (iba): a) a ~cgtx-sensitive iba; b) an isradipine-sensitive iba; and c) a t0-cgtx plus isradipineresistant iba. 4[~-pma and oag specifically down-modulated the isradipine-sensitive iba. the inhibition of iba was prevented by staurosporine and pkc (19-31) (2 pk inhibitors). the delayed rectifier k + current was unaffected by pkc activators. applied externally, okadaic acid and calyculin a inhibited the total iba by affecting several components of the ba 2+ currents. however, the 0~-cgtx plus isradipine-resistant iba was unaffected by okadaic acid. in conclusion, our results suggest a differential modulation of voltage-gated ca 2+ and k + channels by the pkc signalling pathway in ngf-differentiated pc 12 cells. agrin, a protein isolated from basal lamina extracts of the electric organ of the marine ray, is thought to mediate the motor neuron-induced aggregation of acetylcholine receptors (achrs) in muscle fibers at their neuromuscular junction. recent cloning in the marine ray, rat and chick has revealed that agrin can be alternatively spliced at two sites in its c-terminal half. site a encodes either 0 or 4 amino adds and site b encodes either 0, 8,11 or 19 (8+11) amino acids. studies of agrin mrna expression indicate that early in synaptogenesis, chick motor neurons contain high levels of a4b19. in contrast, non-neuronal cells and muscle calls contain a4b0 and aob0 mrna. in the adult ray, electric lobe motor neurons that innervate the electric organ contain a4b8, whereas agrin mrna in the electric organ again lacks both sites (mcmahan et al. (1992) , curr. up, cell biol. 4:869-874). to investigate the functional properties of agrin isoforms in more detail, we have now compared their specific activity to aggregate achrs on cultured chick myotubes. heterologous expression of the c-terminal half in cos-7 and 293 cells revealed that chick agrin isoforms a4b8 and a4b19 were highly active, while a4bll was up to 40 fold lower in activity. no activity could be detected for the a4b0 and aob0 isoforms. in agreement with these findings the a4b8 isoform of the marine ray also showed high achr-clustering activity. therefore, we conclude that motor neurons throughout development synthesize highly active agrin isoforms while the postsynaptic target ceils do not play a primary inductive role in the formation of synapses. we have used whole-cell patch-clamp recordings and hypothalamic slices in order to characterize the effect of n-methyl-d-aspartate (nmda) on suprachiasmatic neurones. in ceils clamped at or near their resting membrane potential, nmda (50-100 #m) generated an inward current of 10-60 pa, which was insensitive to ttx and which reversed at about 0 inv. the nmda current-voltage (i-v) relation contained a region of negative slope conductance. the nmda current was reduced or suppressed by d(-)-2-amino-5-phosphonopentanoic acid (d-aps) or by mk-801. it was potentiated by reducing the extracellular magnesium concentration from 1 to 0.01 ram, or by adding glycine (10 /~m) to the perfusion solution. in a majority of neurones, lowering the extracellular calcium concentration from 2 to 0.01 mm caused a 1.5to 4-fold enhancement of the nmda current. i-v relations indicate that in the low-calcium solution, the region of negative slope conductance was attenuated. this effect was due to extraceuular calcium, since it persisted in neurones loaded with the calcium chelator bapta. we conclude that nmda channels present in suprachiasmatic neurones may be modulated by extraeellular calcium. institut, and abt. pharmakologie*, biozentrum, basel. during innervation of skeletal muscle, the myonuclei underlying the synapse begin to express acetylcholine receptor (achr} ~-subunit gene and they remain activated after the nerve is removed. our experiments show that this is due to a factor in the synaptic portion of the muscle fibre basal lamina (bl). one candidate for this factor is agrin, which regulates ache clustering in the synaptic membrane: i) unlike in untreated muscle, the level of s at synapses was strongly reduced in cultured rat muscle fibres after proteolysis of synaptic bl. 2) conversely, when rat myotubes were cultured on bl isolated from adult muscle, they preferentially expressed achr accumulations and ~-mrna at sites where they contacted the synaptic portions of the isolated bl. 3) when various substrates were impregnated locally with agrin a4big, an isoform expressed by motor neurones in fetal spinal cord, it locally induced in the myotubes the expression of s-mrna. art increase of functional nicotinic acetylcholine receptors precedes the fusion of cultured human muscle satellite cells. r. m. kranse, c.-r. bader and l. berrtheim. department of physiology and division of clinical neurophysiohigy, university medical center, 1211 geneva 4, switzerland nicotinic acetylcholine receptors (nactlr) are expressed on embryonic myoblast and acb may play a role in mechanism of fusion. our study focuses on the appearance of functional nachr in freshly isolated and cultured satellite cells (sc) from normal human muscle biopsies. sc cart be conditioned to either proliferate or fuse to form myotubes depending upon culture media. presence of ach-activated current was investigated using the whole-cell and single.channel patch-clamp technique. in freshly isolated sc (whose properties should be close to the quiescent in vivo sc/no nachr were observed (n=16). in the proliferating state, 54% (n=35) of the satellite ceils (which are also called myoblasts) displayed a small ach-activated current (10 pa/pf) and, as expected, after fusion of salellite cells into myotabes, 100% of the ceils displayed an ach-activated current (n=6; 26 pa/pf). to examine, whether the increased expression of nachr precedes sc fusion, sc were cultured in a medium which promotes fusion, but were prevented from fusing by keeping them at a low density. in this culture condition, 93% of the sc (n=15) displayed an ach-activated current and, in addition, these cells expressed a 4 times higher ach-current density. (40 pa/pf) than the proliferating sc. we also observed that, in high density cultures, a small population of sc do not fuse even atter 3 months in the medium promoting fusion. these non-fusing myoblasts (nfmb) had no nachr. our results suggest that the appearance of nachr may be related to the process of sc fusion as i) quiescent sc in vivo do not express nachll ii) nachr expression increases in conditions promoting fusion and iii) no nachr were observed in nfmb. the latter cells may be equivalent of quiescent sc in vivo. $12-20 we have investigated the sensitivity of neuronal nicotinic acetylcholine receptors (nachrs) of known subunit composition to various cholinergic antagonists. neuronal nachrs were expressed in xenopus oocytes after injection of pairwise combinations of (x2 or c~3 with either 1~2 or 1~4 crnas. the two-electrodes voltage-clamp technique was used to measure currents induced by rapid application of low concentrations of acetylcholine (ach) together with increasing concentrations of antagonists. the response of ct31~4 neuronal nachrs to ach was halfmaximally inhibited by following antagonist concentrations (ic50): hexamethonium (0.3 i.tm), mecamylamine (0.2 p.m), pentolinium (0.2 i.tm) and trimetaphan (1.2 ~tm). with (x3132 nachrs the ic50s of the same antagonists were about 10 times higher. finally, (+)-tubocurarine was a conventional competitive inhibitor of ach in ~3132 and t~2~2 nachrs. in contrast, low concentrations of (+)-tubocurarine increased the response to low ach concentrations in a3(34 and t~2~4 nachrs. these results further underline the importance of [l subunits in determining the functional properties of neuronal nachrs. supported by the hochschnlstiftung and nf grant 31.31018.91 to abc. previously, the role of the transglial tubular system was shown for the squid giant axon: in na + free (tris) solutions the series resistance increased in correlation with a decrease of the tubular opening density (tod). in the crayfish giant axon, which show similar tubules, we correlate the change in tod, measured in freeze-fracture preparations, witl] the conduction velocity. the control to'd was estimated to be 16 per #ms. after 30 and 75 rain tris-inkubation the tod decreased to 10,4 and 1,5, respectively. this reaction was reversible when these axons were reincubated in na~-c41ntaining solution. after 120 rain reincubatiou the tod was 11.8 per tam'z. to determine the influence of decreased tod on the impulse conduction velocity, the nerve was incubated for 30 min in tris solution followed by reincubation. impulse propagation then recovered in two phases. during an initial fast phase with a short time constant of 1-2 rain na + diffused back into the periaxonai space and allowed impulses to occur with a low conduction velocity. in the second phase with a long time constant of 7 min, conduction velocity recovered slowly to normal values. this time course was comparable to the recovery of tod, as estimated from recent freeze-fracture preparations after short reincubation. these results indicate that the normal tod is a necessary condition for the normal excitability of the axon and determines its conduction velocity. most spinal cord neurons respond to the inhibitory action of gaba and glycine, suggesting co-expression of gaba a-and glycine receptors 4n individual ceils. while glycine-receptors are exclusively found in post-synaptic densities, the cellular localization of gaba areceptors has not yet been characterized. in this study, the distribution of gabaa-receptors in the spinal cord was analyzed with an antiserum recognizing the (xl-subunit. double-and tripleimmunofluorescence staining were employed to identify synaptic receptors apposed to gabaergic terminals (immunoreactive for glutamic acid decarboxylase) and to assess their co-localization with glycine-receptors (visualized with an antibody to the 93 kda protein gephyrin). staining for the gabaa-receptor ctl-subunit decorated the soma and dendrites of numerous neurons in laminae iii-viii and x, revealing their morphology in clear detail. by contrast, laminae 1i and ix contained little immunoreactivity for these gabaa-receptors. most gabaa-receptor-positive cells also exhibited a prominent glycine-receptor immunoreactivity. both types of receptors had very similar distribution patterns and were frequently co-localized in sites apposed to gabaergic boutons. these results indicate that gaba aand glycine-receptors may co-exist within single post-synaptic densities, suggesting a possible synergism between gaba and glycine neurotransmission in spinal cord neurons. prenatal benzodiazepine (bdz) exposure changes both behavioral and neuiochemical parameters of developing iats. these changes are not a consequence of remaining bdz in brain tissue, but rather indicate alterations in bdz receptol binding and function. since the bdz binding site is located on the gaba~ receptor complex, it is possible that prenatal bdz treatment influences the expression of gaba~ receptor subunits. to evaluate effects of pienatal bdz exposure on mrnas expression specific for several gaba~. receptor subunits (~i, ~5, ~ & y2), we treated plegnant rats with diazepam (l.25mg/kg/d; s.c.) from gestational day 14 to 20. we then analyzed specific mrna levels in offspring at four diffeient developmental stages (gd20, pns, pni5 & adult) by in situ hybridization. pleliminary data flom optical density measurements indicate a deciease in expression of mrna in particular for ~-subunit of gab~ receptors in different brain reglons of plenatally diazepam-treated zats. processing of sensory information within the auditory system has been well characterized using histological and eleetrophysiologlcal techniques. however, relatively little is known about the neurotransmitters involved. the present study was performed to elucidate the possible role of excitatory (eaa] and inhibitory (i.e., gaba) amino acids in neurotransmission within the primary auditory cortex (all the main afferent to the ai, the ipsilatera} medial geniculate body (mgb), was electrically stimulated and evoked responses were recorded intracellularly in the ai region in halothane anesthetized cats. a seven-barrelled iontophoresis pipette glued alongside the recording electrode allowed localized application of eaaergic and gabaergic compounds onto the recorded neuron. in most ai neurons, mgb stimulation evoked a short latency, short duration epsp followed by a long-lasting ipsp. pattern, duration, and amplitude of synaptic potentia{s were highly variable and strongly dependent on stimulation intensity, reduction of gabaa receptor-mediated inhibition by iontophoretic application of either bicuculline or sr95531 markedly enhanced mgb stimulation-evoked epsps. only the late component of this enhanced epsp was reversibly blocked by the competitive nmda receptor antagonistl ap7 at currents which selectively blocked neuronal excitation induced by iontophoretic application of nmda, our results demonstrate that in the eat 1) nmda receptors in ai are activated following mgb stimulation and that 2) a dominant gaba~ receptor mediated inhibition usually masks this nmda receptor activation under our experimental conditions, a highly purified and specific cell wall degrading endo-l,5-u-l-arabinanase was isolated from an a. niger pectinase preparation by low and medium (fplc) pressure column chromatographic separation methods. the isolated enzyme was most active on linear 1,5-~-l-arabinans (-90 i.u./mg), whereas branched arabinans from sugar beets were degraded to a lesser extent (-14 i.u./mg). the enzyme was shown to be electrophoretically pure after silver staining (sds-page) and its identity was confirmed through specific binding to an antiserum directed against endo-l,5-~-l-arabinanase. the major physico-chemical characteristics of the enzyme were the following: mr 42'000, iep ~ 3.0, ph optimum 4.8, temperature optimum 55~ ph stability 3.5-8.0, temperature stability s 45oc, km = 0.205 mg/ml, vmax = 17.7.10 -3 ~unol/min. zn and hg showed potent inhibitory effects. 3.2) is a specific enzyme of the glyoxylate cycle, which plays a key role in the initiation of gluconeogenesis in germination oilseeds, this pathway is localized in glyoxysomes, single membrane organelles which are converted into peroxisomes when lipid reserves are depleted. the glyoxylate cycle enzymes have been the subjects of numerous conflicting reports typically addressing the problems of their synthesis (on free or bound ribosomes), targetting and suborganelar localization (membrane bound or matrix proteins). recent biochemical studies have shown that ms from germinating soybean cotyledons is an interconvertible enzyme (membrane bound, aggregated or soluble fomls) which is significantly affected by its ionic environment (henry et al., 1992, plant science 82:21-27) . microscopy studies have now been carried out using immunofluorescence staining or immunogold-silver staining for light microscopy, and immunogold labelling for electron microscopy. all results consistently characterize ms as a glyoxysomal mawix enzyme. chorismate mutase (ec 5.4.99.5) catalyzes the first step in that branch of the shikimate pathway which leads to the aromatic amino acids phenylalanine and tyrosine. we have isolated a cdna for this enzyme from the higher plant arabidopsis thaliana by complementing a yeast strain (aro7) with a cdna library from a. this is the first chorismate mutase cdna isolated from a plant. the a. thaliana chorismate mutase expressed in yeast revealed allosteric control by the three aromatic amino acids as previously described for plastidic chorismate mutase isozymes. an attachment of rubisco to chloroplast membranes under cu++-stress has been described for wheat (mehta et al., j. biol. chem. 267, 2810 -2816 . the displacement to the membranes has been discussed as a possible step in the degradation of this predominant stromal protein, e.g. during leaf senescence. in our recent experiments it became evident that such interactions are not restricted to rubisco. several other stroreal, and even a peroxisomal enzyme (glycolate oxidase), were also detected in the membrane fraction after 6 to 30 hours of oxidative stress induced in wheat seedlings by a treatment with 10 mm cuso 4. the velocity and the degree of membrane attachment varied for different enzymes. based on these results it was interesting to check the solubility of rubiseo and its previously described 45 kd fragment which accumulates during the incubation of bean leaf discs under oxygen deficiency (hildbrand and feller, experientia 47, a67) . neither rubisco nor the breakdown product were found to accumulate in the membrane fraction. thus, at present, the steps involved in rubiseo catabolism cannot be generalized. different mechanisms depending on the metabolic situation and on environmental conditions should be considered. overexpression of the four parsley phenylalanine ammonia-lyase (pal) isoenzymes in e. coli. characterization of the enzymes and kinetic analysis of the inhibition by 2-aminoindan-2-phosphonic acid. christoph appert, j/irg schmid, jerzy zorn* and nikolaus amrhein institute of plant sciences, eth-z/jrich, 8092 zijrich. *technical university wroclaw, 50-370 wroclaw, poland. all four phenylalanine ammonia-lyase (ec 4.3.1.5) isoenzymes of parsley (petroselinum crispum nym.) were expressed as glutathione s-transferase fusion proteins in e. coil after affinity purification, the glutathione stransferase moieties were cleaved off with factor xa and the phenylalanine ammonia-lyases were characterized. the proteins form enzymatically active tetramers, even as fusion proteins. the four isoenzymes have comparable km values for l-phenylalanine (15gm to 24.5/.tm), similar temperature (58~ and ph optima (8.5). the aminooxy-and phosphonic-analogues of l-phenylalanine are competitive inhibitors of the enzymes. 2-aminoindan-2-phosphonic acid (j. zo{a & n. amrhein, liebigs ann. chem. 1992,625) was found to be a potent slow-binding inhibitor of these and other phenylalanine ammonnia-lyases, both in vivo and in vitro. chlorophyll a fluorescence has been used to compare the drought resistance of two varieties of solanum tuherosum (avr/i)c-12st-19 and sibtema). fast fluorescence rise kinetics were measured during the first second of illumination with a time resolution of l0 ms and 1z bits in fluorescence intensity. the rise kinetics shows the typical steps called fo -j -i -p. with this values different indexes have been defind with the goal to compare the behaviour of these two varieties of potato. salicylic acid (sa) was proposed as a putative signalling molecule for the induction of systemic acquired resistance (sar) in infected plants. we studied its biosynthesis as follows. cotyledons of cucumber plants were injected with a solution containing pseudomonas lachr~ans and fed with radioactive sa precursors by injection of 14c-benzoic acid (14c-ba) or 14cphenylalanine (14c-phe). alternatively, leaf 1 of cucumber plants were inoculated with tobacco necrosis virus (tnv) and leaf disks were then vacuum-infiltrated using 14c-ba or 14c-phe. free and bound 14c-phenolies were quantified by hplc. incorporation of 14c into sa was found in the two plant-pathogen systems. i~c-ba gave mostly 14c-sa and an unknown polar compound. 14c-phe gave also some 14c-sa, in addition to 14c-labelled lignin precursors like ferulic and p-coumaric acids. the biosynthetic pathway of sa from phe through cinnamic acid and ba will be discussed. after infection with a necrotizing pathogen, systemic acquired resistance (sar) is often observed in plants. in cucumber, salicylic acid (sa) increases in the lower infected leaf as well as in the upper uninfected leaves. sa was also found in the phloem sap of infected cucumber plants and was proposed as a signalling molecule for the induction of sar. we intend to clarify whether sa is translocated from the site of infection to the upper leaf, or wether it is made in the phloem tissue upon the action of a primary systemic signal. one cotyledon of cucumber plants was first infected with pseudomonas lachrymans and then fed with 14c-sa, 14c-benzoic acid (ba) or 14c-phenylalanine. after different times, cotyledons and first leaves were collected and the radioactive ~henolics were analyzed by hplc. in some cases, 4c-sa was found in the first leaf. in addition, two unknown radioactive compounds were detected in leaf 1 after treatment with 14c-sa and 14c-ba respectively. we will discuss signalling processes for the induction of sar in cucumber. one modern approach of creating virus resistant grapevine plants is the introduction of resistance genes into existing grapevine varieties by genetic engineering. the goal of this work is to use the coat protein (cp) mediated strategy to induce resistance to nepovirus in vitis spp. as a first step, several chimeric nepovirus cp genes, were constructed by addition of various promoter regions upstream of the gflv and armv cp regions. their ability of conferring resistance to nepovirus infection in transgenic nicotiana benthamiana and n. tabacum plants will be presented. the best constructions will be used to transform several grapevine varieties in order to create nepovirus resistant grapevine plants. $13-12 the bctalains axe a class of natural pigments found only in plants of the order caryophyllales and in some fungi. the first step in betalain biosynthesis is the conversion of tyrosine to dopa. subsequently , dopa is transformed to betalamic acid, the bctalain chromophorr through the action of dopa-4,5-dioxygenase. we have purified and characterised a copper-containing enzyme of -38kda from amanita muscaria pileus that catalyses the hydroxylation of tyrosine to dopa. considering that the enzymatic activity was restricted to the coloured parts of the mushroom, we postulate that it is involved in betalain biosynthesis. the enzyme featured a broad substrate specificity, and also oxidised the diphenols to their corresponding ortho-quinones, a reaction typical for tyrosinases. the implications of our findings on betalain biosynthesis axe discussed. chlorophyll a fluorescence was measured under steady state conditions of pea and tomato leaves adapted to low light intensity (30 wm-2s -i) at different temperatures (havaux and strasser, z. naturforsch. 45c, 1133 -1141 , 1990 ). when leaves were exposed for a short period (i0 min) to heat (25 to 45~ in darkness, the level of variable fluorescence decreased. when the heat stress was imposed in presence of low light, the variable fluorescence was much less affected and virtually no effect of heat treatment was observed until 37~ this protecting mechanism by low light was absent in algae and mostly absent in submerged water plants but fully present in free floating plants on the water surface. we conclude that a mechanism has been developed for higher land plants during evolution which protects the plants against heat damage on warmer days. caryophyllales, e.g. portulaca grandiflora, and in a few fungal species, e.g. amanita muscaria. we analysed the similarity of a key enzyme of the pathway, dopa-4,5-dioxygenase, in plants and fungi both at the protein and the dna level. antibodies against purified dopa-4,5-dioxygenase from the fungus a. muscaria crossreacted with protein from p. grandiflora petals and two cdna clones were obtained from this plant. their similarity with fungal sequences and with other dioxygenases is discussed. kinetics of prollne hydroxylauonj intrucellnlur transport and c-terminal processing of the tobacco vacuolar cbltlnase. ernst frcydl, thomas boiler and jean-marc neuhaus botenisehes instimt, abt. pflanzenphysiologie, hehoistxasse 1, ch-4056 basel the vacuolar chitinase a of tobacco (nicotlana tabacum) is syndietlzed as a preproprotein with ma n-teaminal signal peptide which causes its synthesis in the er mad a c-termlnal extension, which has been idcmified as the vacuolar targeting peptide (vtp) (1) and whleh is cleaved off from the maybe chitinase. it has recently been shown that mature chltin:~e a contains several hydi'oxyprolines within the short peptide spacer that links the n-terminal cysteine-rieh chitln-bindlng domain to the catalytic domain (2). we received specific antibodies against mature chitinasr (a kind gift from dr. f.meins. f/vii) and raised antihodiea against a synthetic vtp. we performed pulse-chase experiments and cell [ractlonation with 1) stably transformed tobacco plants expressing chitinase a or mutants lacking either the chitin-binding domain and spacer (chitah) or the vacuolar targeting poptid 9 (chitavtp) and 2) transient expression of the same conswacts in nicotiana plumbaginifolia protoplasts. in both systems, proline hydroxylatino in chltinase a was detectable after 30 vain. and complete after 90-120 rain. the ehltinase intermediate forms were detected in the mlcrosomal and soluble fractions for up to 90-120 rain. the mature chitinase continuously accumulated only in the soluble fraction. in both systems chitah showed the same kinetics of intraceilular transport and processing. whereas the transport of cbitavtp seemed more rapid.these results indicate that in vivo cbitinase a is synthetized as a proprotein that is than modified by proline hydroxylation. this second intermedinte form is then transported to the oolgi apparatus and sorted to the vacuole. c-terminal processing occurs late in this pathway or in the vacuole. infection of bean roots with the soil-borne phytopathogenic fungus fusarium solani f.sp. phaseoli leads to a rapid increase of chitinase activity (~five-fold). part of this increase in enzyme activity is due to transcriptional activation of the basic class i chitinase isoenzymes. semi-native polyacrylamide gels stained for chitinase activity using the substrate glycol-chitin revealed the appearence of three additional chitinase isoenzymes that are absent from uninfected control roots. conventional protein purification techniques showed that fusarium solani infected bean roots contain two basic and two acidic chitinase isoenzyrnes. their physiochemical properties and possible biological function will be discussed. proteasomes are multicatalytic protease complexes that function as a major nonlysosomal proteolytic system. they exhibit multiple endopeptidase activities that promote the intracellular turnover of abnormal polypeptides and short-lived regulatory proteins. moss proteasome has been purified from protonemata by successive chromatography on macroprep q, bio-gel a-1.5, bio-gel a-5 and ha. the molecular mass was estimated to be 1,300 kd by gel filtration. gel electrophoresis of proteasome under nondenaturing condition gave a single band and two-dimensional gel electrophoresis identified the moss proteasome to be composed of 14 different subunits with molecular weight in the range 22-35 kd. electron microscopy in aqueous solution showed that it is a cylindrical particle composed of four stacked rings with a diameter of 9 nm and a height of 14 rim. end-on projection established a 7-fold symmetry of the outer disks. substrate specificity of proteasome indicates that it contains endopeptidase activity against substrates bearing hydrophobic, basic, acidic and glycine residues immediately preceding the cleavage site. antibodies raised against moss proteasome reacted with proteasomes of other eukaryotes, including human. $14-01 hunger r.e., hess m.w., laissue j,a,, mueller c. the nod (non obese diabetic) mouse, a widely used animal model for insulin dependent diabetes mellitus, shows in addition to mononuclear cell infiltration of the islets of langerhans, massive cellular infiltrates of the submandibular and lacrimal glands with considerable tissue damage. several lines of evidence suggest that both insulitis and sialadenitis represent autoimmune disorders, to investigate a possible role of tumor necrosis faetor-a (tnf-c0 and activated cytotoxic cells (nk cells, t cells) in the inflammatory process, tissue sections of nod mice were hybridized with radiolabeled rna probes specific for the detection of mrna for tnf-r and the serine proteases granzyme a and b (gra and gr8) and perforin which are expressed in activated cytotoxic cells. tnf-e expressing cells were mainly located in infiltrates and are absent in non affected glands, whereas gra, grb and perforin expressing cells were distributed over the whole section with highest densities in the zones adjacent to parenchyma. the finding that activated cytotoxic cells are present in early stage of disease development indicates that cell mediated cytotoxicity may play a crucial role in initial tissue destruction. the role of tnf-a in autoimmune diseases is not known yet. the appearance, however, of cells expressing this gene in the infiltrate indicates a possible role of this cytokine in the progression of the inflammatory reaction, e.g. by increasing the lymphocyte traffic to this organ. we further demonstrated that the induction of inos was at the transcriptional level. in order to understand the underlying mechanisms we characterized the promoter region of the rat nos gene. a 9enomic rat library was screened using a cdna-fragment coding for the if-end of the inos cdna (nucleotides 1-817). a 1,8 kb hinc-ii fragment containing the 5"-flanking region of the inos gene was characterized by dna-sequencing. the transcriptional start site was determined by primer extension. sequencing analysis revealed a multitude of possible cis-acting elements homologues to consensus sequences for the binding of different transcription factors including ifn~' response element (ire), nuclear factor-kb (nf-~b), tumor necrosis factor response element (tnf-re), 7f-activated sites (gas) and one x-box. nf-kb, a transcription factor involved in signaling and immediate early gene activation during inflammatory processes was induced by treatment of mesangial cells with 2 nm of il-113. this was shown by the appearence in nuclear extracts of the dna binding activity of nf-~b using electrophoretic mobility shift assays (emsas) with a 32p-labeled ~r dna probe. pyrrolidinedithiocarbamate (pdtq), an inhibitor of nf-kb, suppressed the expression of inos mrna in mesangial cells without affecting the dibutyryl-camp-triggered increases in nos mrna levels. this data suggest that the il-i ~ signalling pathway responsible for nos expression requires nf-k8 activation and is definetly different from the signalling cascade directed by camp. recent findings indicate that the multifunctional cytokine il-6, which plays a key role in irm-nune and inflammatory responses, also exerts specific effects in the central nervous system (cns). for example, il-6 promotes neuronal survival, induces differentiation and modulates neurotrophin production. using the very sensitive technique of reverse transcription combined with polymerase chain reaction the developmental profile of il-6 and its receptor mrnas was analyzed in various rat brain regions. our results indicate that both genes are expressed in a region-specific manner and are developmentally regulated. these findings support the hypothesis that il-6 is involved in differentiation and maintenance of neuronal subpopulation in the cns. interleuldn 2 (il-2) expression is strictly dependent on a signal transmitted by the t cell receptor upon antigen stimulation. this signal can be mimicked in vitro with phorbol esters and calcium ionophores. we have found that stimulation with ca-ionophore alone induces expression of il-2 mrna, but does not induce secretion of il-2. in polysome gradient fractionation of cells stimulated with phorbol esters and ionophore, the fractions containing il-2 rrtrna are found at the bottom of the gradient, bound to polysomes. however, in ionophorestimulated cells the fractions containing il-2 mrna are clearly shifted to the top of the gradient, and therefore are not lzanslated. this effect is specific for il-2, as other mrna's like 1~2-microglobulin are not regulated. this data suggests that il-2 gene is translationally regulated. in addition, in vitro experiments have shown that the lack of translation of il-2 mrna in ionophore-stimulated cells is due, most likely, to the presence of a translational regulator that specifically inhibits translation of il-2 mrna. assuming the presence of a translational regulator that specifically represses il-2 mrna translation, we can predict that, if the represser is in excess, even upon a second stimulus that is by itself able to induce secretion of il-2, there will be no translation of il-2 mrna. the resuhs of such an experiment confirmed our prediction. polysome gradients showed that after ionophore stimulation, il-2 mrna gets "stacked" with one ribosome, and no further ribosome loading takes place. if malaria is highly endemic, every febrile child is a presumptive malaria case, and there is no differential diagnosis based on clinical, immunological or parasitological grounds. we evaluated the diagnostic usefulness of interleukin-2 receptor (il-2r), tumor necrosis factor-receptors 55 (tnf-r55) and 75 (tnf-r75) . sera came from tanzanian pediatric fever patients and controls, all enrolled in the kilombero malaria project. we found that all 3 receptors marked pediatric fever episodes, whereas stability of observed levels was highest for tnf-r75 and lowest for tnf-r55. tnf-r55 levels reflected severity of the fever attack. regardless of the presence of a febrile illness, tnf-r75 levels were strongly associated with parasite density. immunological markers can be applied in rapid pre-and post-intervention morbidity assessments, validation of health interviews and monitoring of convalescence. we have recently shown that human eosinophils produce mrna for interlenkin (il)-8 when stimulated with calcium ionophom (eur. j. immunol. 23 (1993), 956). we now present evidence that human eosinophils contain preformed il-8 protein although they do not express mrna for il-8 as measured by rt-pcr. il-8 protein expression was determined using specific anti-human il-8 monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il-8 was released into supernatant by 99% pure eosinophils after priming with gm-csf or il-5 and subsequent 25-min stimulation with paf, rantes, ionomycin or pma, however not with il-1 or il-2, as measured by elisa. this observation implies that activation of protein kinase c and/or inwacellular calcium mobilization am necessary events for il-8 release in human eosinophils. the determined amounts of preformed il-8 protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinophil derived il-8 in asthma. since the eosinophil is the predominant cell in the asthmatic airways, we determined 1l-8 concentrations in bronchoalveolar lavage (bal) fluids from normal individuals and asthmatic patients. indeed, il-8 concentrations in bals from both groups were similar to those seen in the supematants released by activated eosinophils. the role for 1i-,-8 in asthma remains to be determined. to study the effect of canthaxanthin in vitro, the hydrophobic compound was introduced in vivo into chick high density lipoproteins (hdl) by canthaxanthin feeding. the effects of hdl and hdl associated canthaxanthin on two types of cultures have been tested: flat sedimented cell cultures of embryonic chick neuronal retina, retinal figment epithelium (rpe), brain and meninges, and in reaggregate cell cultures of the neuronal retina. at high canthaxanthin concentrations the formation of colored, birefringent entities were induced in the neuronal retina in vitro. in line with human data, parameters of cellular function and cell differentiation remained unaffected by canthaxanthin at these concentrations. by contrast, fidl was found to induce various cell type dependent effects. proliferation of meninges cells was decreased at low hdl concentrations as concluded from.light microscopic examination and indicated by protein content, and lysosomal and mitochondrial activity of the cultures (ic50:15-30 mg hdl apoprotein/l medium). these parameters, however, were increased in brain cell cultures, while they remained unaffected in cell cultures of neuronal retina and rpe. concentrations above 0.3 g. hdl apoprotein/l caused a reduction of glial cell differentiation. $14-08 tnf-c~ had close-dependent antiproliferative activity on hormone-dependent human breast cancer cells that represent an early stage of the disease, whereas hormone-independent cells representing a late stage were not affected. however, in the presence of 1000 u/ml interferon ?(inf), high concentrations of tnf-c((lnm) were able to inhibit the growth of hormone-independent cells. using specific monoclonal antibodies in ftow cytometry, no significant changes of either the p75 or the p55 tnf receptors were observed upon inf treatment of hormone-independent cells. this indicates that the increased responsiveness of these cells for tnf-c~ is not due to a change in available tnf receptors. the affinity of the receptors for tnf-r are one order of magnitude different for the two cell types. inf treatment shifted the ecs0 of hormone-independent cells towards the ecs0 of hormone-dependent cells. thus, the basis for the increased tnf-sensitivity of hormone-independent cells in the presence of inf might be the interconversion of tnf receptors from low-to high-affinity. the adhesion of tumor cells to endothelium is the first step in the processus of metastasis, and is frequently dependent of cytokinemediated activation of endothelial cells. cytokines are also involved in nitric oxide (no) production by various cells. these experiments were designed to evaluate no production during tumor cell adhesion to endothelium. rat brain-derived endothelial and rat colon carcinoma cell lines were used during these experiments. no was evaluated with the griess reagent. activation of endothelial cells with cytokines (tnf-d, and ifn-~ ), only slightly increased (10-20 %) the adhesion of tumors cells to endothelium. activation of endothelial cells with tnf-ol and ifn-~, induced a 4-8 fold increase of no production. however, addition of tumor cells to the endothelial monolayer, either directly or in a semi-permeable membrane, decreased the cytokine-induced no production. these results indicate that no production is modulated during the proeessus of adhesion of tumor cells to endothelium. is induced in rat mesangial cells by inflammatory cytokines such as interleukin 1 ]3 (il-1 ~) and requires bh 4 as a cotactor. 2,4-diamino-6-hydroxypyrimidine (dahp), a selective inhibitor of gtp cyclohydrolase i, the rate-limiting enzyme for bh 4 synthesis, potently suppressed il-i~induced nos activity, measured as nitrite production. inhibition of no synthesis by dahp was reversed by sepiapterin, which provides bh 4 via a salvage pathway. sepiapterin dose-dependently augmented il-1 i],-stimulated no synthesis, indicating that availability of bh 4 limits the production of no in cytokine-stimulated mesangial cells. n-acetylserotonin, an inhibitor of the bh 4 synthetic enzyme sepiapterin reductase, completely abolished il-lj~-induced no formation whereas methotrexate, which inhibits the pterin salvage pathway, displayed only a moderate inhibitory effect, thus suggesting that mesangial cells predominantly synthesize bh 4 tom gtp. in conclusion, these data demonstrate that bn 4 synthesis is an absolute requirement for cytokine induction of nos in mesangial cells. inhibition of bh 4 synthesis may provide new therapeutic approaches to the treatment of pathological conditions mediated by no. -1 [3) can induce a macrophage-type of nitric oxide synthase (inos). northern-blot analyses of inos mrna-levels and nuclear run-on experiments of control and il-1 ~ stimulated mesangial ceils revealed that the induction of inos was at the transcripuonal level. here we demonstrate that platelet-derived growth factor bb (pdgf-bb) can suppress the il-113 dependent inducuon of the inos gene. nortllern-blot analyses of cellular rna isolated from mesangial ceils that were coincubated with il-1 [3 (2nm) and pdgf-bb (10 ng/ml, 100 ng/ml and 300 ng/ml) revealed a dosedependent suppression of inos mrna-levels. using run-on experiments with nuclei from mesangial ceils after stimulation with il-i~ (2nm) and pdgf-bb (100 ng/ml) we demonstrate that the inhibition occurs at the transcriptional level. basic fibroblast growth factor (bfgf), on the other hand, potentiates the il-1 dependent stimulation of inos. coincubation of mesangial cells with il-113 (2nm) and bfgf (3ng/m[, 10ng/m[, 30ng/m[, 100ng/m0 dose-dependently superinduces inos mrna levels as shown by northern-blot analyses of total cellular rna form control and stimulated cells. run-on experiments with nuclei from mesangial cells stimulated with il-113 (2nm) and bfge (100ng/ml) revealed an enhanced transcriptional activity of the inos gene. thus, pdgf-bb and bfgf differentaity modulate the il-i~ dependent expression of the inos gene and are therefore an excellent model system to study the crosstalk between signal transductjon pathways. we report that ifnyr-/-mice have an increased resistance to lipopolysaccharide-induced toxicity (lps). lps-induced lymphopenia, thrombocytopenia and weight loss seen in wild type mice were attenuated in ifntr-/-mice. ifntr-/mice survived in the d-galactosamine-lps model when conditions were 100% lethal for wild type mice, correlating with serum tnf levels of up to 10 fold higher in wild type mice. bone marrow and splenic macrophages from ifnyr-/-mice had a 3 to 5 fold decreased lpsbinding capacity, with serum from these mice lowering macrophage lpsbinding by a further 50%. thus, depressed tnf synthesis, diminished expression of lps receptors and low plasma lps-binding capacity in the mutant mice likely combine to manifest in the resistant phenotype of ifny r-/mice to endotoxin. in a complementary approach we have screened the 5' portion (-7.8 kb/+4.1 kb) of the gene for tissue specific and/or inducible dnasei hypersensitive sites. in chromatin from fibroblasts or kidney epithelial cells this segment of the il2ra gene does not contain any dnasei hypersensitive sites. in contrast, in resting t-and b-lymphocytes, as well as in activated t cells, two sites (dhi, -0.05 kb; dh3, -5.8 kb) were found. a third site (dh2, -1.35 kb) is detectable in t cells that have been activated with concanavalin a and il2. this site maps to the same position as cisacting regulatory sequences that are required for the response of the il2ra gene to signals from the il2 receptor. interleukin-6 (il-6) production in cultured human dermal fibroblasts can be stimulated by interleukin-i (il-i} added to the culture medium. absence of fetal calf serum and of growth factors (insulin, egf, t3) reduced the stimulation by more than 50 %. egf and t3 and even more choleratoxin increased the il-6 production in absence of fetal calf serum. the effect was dose dependent. 2% human ab serum substituted for i0 % fetal calf serum. pretreatment of the cultures with serum or growth factors prior to stimulation with il 1 had a stimulatory effect (serum, t3, egf) or an inhibitory effect (hydrocortisone, choleratoxin). our results suggest that the il-i signal transduction to il-6 is modulated by serum components and growth factors as well as through c-amp produced by choleratoxin. interleukin-6 (il-6) is produced by a variety of cells and plays a central role in host defense mechanisms. its function include induction of il-2 and il-2 receptor expression, proliferation and differentiation in t-cells. in cocultures of human dermal fibroblasts and allogenic human t-cells a cell number dependent 10 to 100 fold increase of the il-6 production could be demonstrated after 24h and 48h. conditioned medium from tcells and from fibroblasts failed to induce il-6 secretion in fibroblast and t-cell cultures, respectively. no up-regulation of il-6 production was found in cocultures of fibroblasts with tcells killed by ethanol and in t-cell culture incubated with recombinant hll-lc~. after separation of the cells il-6 production in fibroblast and t-cells returned to precoculture levels. our results indicate that cell-cell contact more than soluble factors must be involved in the up-regulation of il-6 synthesis in cocultures of t-cells and fibroblast. using cocultures of autologes human t-cells and fibroblasts, we are currently investigating whether the cell contact essential for the il-6 production depend on immunolcical interactions of t-cells and fibroblasts. rapid growth of tumor often results in oxygen and nutritional deprivation leading to extensive necrosis, which is one of the characteristics of glinblastomas (gbl). neoplastic cells surrounding necrotic areas often present a peculiar arrangement of cells called "pseudo-palisading" -as a hallmark of this entity, together with abundant neovascularization. a special biological milieu is probably formed in these palisading areas by certain unknown cytokines and/or growth factors induced by the necrotic process. plate et al. (1992) reported the expression of vascular endothelial growth factor (vegf) in the palisading cells. we previously demonstrated that gbl cells produce il-8, which is known to be an angiogenic factor. the aim of this study is first to assess if il-8 is expressed in the palisading ceils, and second to determine if hypoxia can trigger il-8 gene expression. in rive observation using in siru hybridization and immunohistuchemistry showed that il-8 is highly expressed specifically in the palisading cells. we also demonstrated the mrna expression of il-8 receptor in the endothelial cells adjacent to necrotic area. to simulate the in vivo condition, two in-vitro models are currently developed to determine if il-8 is induced by hypoxic insult on cells. first, gbl cell lines are cultured in the absence of oxygen and il-8 expression is assessed by northern blot analysis. preliminary results have demonstrated the induction of il-8 by hypoxia. secondly gbl cells are grown in spheroids until development of central necrosis. the il-8 expression will be assessed by in situ hybridization combined with immunohistochemistry to determine if the ceils surrounding necrosis express il-8. it has been proposed by taniguchi and his colleagues that activation of the interferon (ifn)-i3 gene by virus or double-stranded rna involves induction and modification of irf-1. overexpression of irf-1 can induce expression of the ifn-b gene in certain cells. a role of irf-1 in the activation of ifn-a genes has also been proposed. furthermore, irf-1 has been claimed to play the role of a tumor suppressor gene. we have generated mice in which both irf-1 alleles were disrupted and found them to develop normally. no spontaneous tumors have been observed so far. injection of poly(i)-poly(c) resulted in the same levels oflfn in blood of wildtype and null mice, and equal ifn-ct mrna levels in spleen. induction of wild type and irf ~ embryo fibroblasts (mefs) with virus resulted in the same levels of ifn-a and ifn-i] mrna respectively. in the case of polyo)-poly(c) induction, the levels of both 1fn mrnas were higher in wild type than in mutant cells; this difference was abolished if the cells were first primed with type i ifn. we conclude that irf-1 does not play an essential role in the induction oflfn genes. mitsuhiro tada, annie-claire diserens, isabelle desbaillets, marie-france hamon, nicolas de tribolet, erwin van meir; chuv, lausanne there is increasing evidence that a variety of cytokines produced by glioblastoma (gbl) cells modulate the tumor growth by affecting the host's immune response and by stimulating neovascularization. cytokines such as il-i, il-6, il-8, mcp-i, il-10 and tgf-132, affect each other's production and receptor system and seem to form a cytokine network. among them, il-1 may play a pivotal role, since il-i can induce or increase the production of other cytokines (il-6,il-8,mcp-i,tgf-132) and modulate their receptor expression. to test this hypothesis, we investigated co-expression of cytokines and their receptors in 15 gbl cell lines and 15 gbl tissues using rt-pcr, northern blot analysis, immunnhistnchemistry and elisa quantification. a majority of the gbl cell lines and gbl tissues expressed il-i~, il-113, type i and type ii receptors, indicating the presence of an il-1 autocrine loop. il-1 stimulated growth of some of the cell lines. a positive correlation of il-6 and il-8 expressions with the presence of an il-i autocrine loop in the cell lines was found. immunohistochemical studies showed the in rive co-expression of the il-i family and the secondary cytokines (il-6, il-8) in gbls. antisense oligonucleotide to il-lc~ and/or il-113 partly suppressed this secondary cytokine expression. these results demonstrate that the il-1 autocrine loop plays a central role in the formation of the cytokine network in gbls. double-stranded rna-dependent protein kinase (pkr) is induced by type i interferon (ifn) and is implicated in the establishment of the antiviral state. upon activation, pkr phosphorylates the eukaryotic initiation factor eif-2, thereby throttling protein synthesis. it was suggested that pkr may be essential for the induction of ifn-13 gene expression by both virus and double-stranded rna and for the activation of at least some ifninducible genes. recently it was proposed that pkr is a tumor suppressor gene because 3t3 ceils overexpressing inactive human pkr gave rise to tumors in nude mice (koromilas et al., 1992; meurs et al., 1993) . we have produced homozygous pkr knockout (pkr ~176 mice and found that they develop normally and have no striking phenotype. induction of ifn-c~ and ifn-13 gene transcription by virus was unimpaired in fibroblasts derived from pkr ~ mice, as was the induction of several ifn-stimulated genes. so far, no spontaneous tumor formation was observed. pkr o/o embryonal stem ceils showed no increased malignancy in nude mice as compared to their wild type counterparts. we conclude that pkr does not play an essential role in viral induction of the ifn genes and that its tumor suppressing effect, if any, is redundant. tgf-13 plays an important role as a negative regulator of hepatocyte proliferation in liver regeneration. exposure of rodents to nongenotoxic carcinogens like cyproterone acetate (cpa), thioacetamide (ta) and phenobarbital (pb) causes abnormal hepatocyte proliferation and liver tumors after long term treatment. this suggests that these chemicals have either a direct mitotic activity or impair the negative regulatory system for growth. therefore the inhibitory activity of tgf-6 on dna-synthesis induced whith cpa was investigated in cultured rat hepatocytes. after a 48h exposure to cpa, a dose dependent increase in dna synthesis (3h-tdr incorporation) was observed. the maximum effect was attained with 12 pm cpa (up to 4.3 fold) which was stronger than with 10 ng/ml egf (up to 2.8 fold). tgf-81 and tgf-132 inhibited this response dose dependently (idso = 0.1 ng/ml) and reduced it to control levels at 1 ng/ml. these findings show that the tgf-6 mediated pathway for negative growth control in hepatocytes is still responding after cpa treatment. mouse mxl protein is an interferon (ifn) induced gtpase with intrinsic antiviral activity against influenza a viruses. it accumulates in the cell nucleus and inhibits the transcription of influenza virus genes, recently, we have shown that mxl exerts its inhibitory activity by interfering with the function of the viral polymerase subunit p82. pb2 is responsible for recruiting 5" ends of cellular mrnas as primers and for the elongation of nascent viral transcripts. to elucidate which pb2 dependent step of viral mrna synthesis is the target of mxl action, we examined the activity of recombinant mxl protein in an in vitro transcription system of influenza virus. first, we expressed wildtype and mutant mxl proteins, carrying a hlstidinetag at their n-terminus, in e. coil and purified them under nondenaturing conditions by ni-chelate affinity chromatography. mxl efficiently inhibited viral transcription in vitro, while mutant mxl proteins, lacking antiviral activity in vivo ,were inactive. moreover, the use of capped synthetic rna primers revealed, that mxl almost completely abrogated the elongation of viral transcripts, whereas the the cap-binding and endonuclease activity of pb2 was not affected. a50 $14-28 neutralization rates of tnf-a from eight animal species by a monoclonal antibody against human tnf: implication for receptor action? we have recently developed a sensitive bioassay for measuring porcine tnf-a based on homologous pk(15) cells. this bioassay is 100-to 1000-fold more sensitive than the widely used l929 bioassay, also, human tnf-a and human tnf-8 were detected with a slightly higher sensitivity in the new test compared to the l929 bioassay, we now show that also canine, ovine, bovine, equine and caprine tnf-a can be detected with the pk(15) bioassay. we tested a murine monoclonal anti-human tnf-a antibody for its capacity to neutralize tnf-a from eight different animal species. the action of this antibody revealed that neutralization of tnf bioactivity is a complex phenomenon, indicating species-specific differences in the interaction with tnf receptors. to study this differential neutralization, we are cloning the porcine tnf receptors for expression in a heterologous cell system. administration.of high dose r-tnf(~ in combination with ifn 7 in isolation and perfusion of the limbs in melanoma patients has shown to be very promising with a response rate greater that 80%. this observation prompted us to investigate the possible expression of the tnf receptors in melanoma cells using the monoclonal antibodies utr-1 specific for the tnf type a (55 kda) receptor and htr-9 specific for the tnf type b (75 kda) receptor. flow cytometric analysis of cultured melanoma cells showed the presence at low level of the tnf type a and to a slightly higher level of the type b receptor. similar results were obtained in vivo by immunohistochemistry on fresh tumor raateriai, treatmem of melanoma cells in culture with the-amp induced up to a 2 fold increase in the number of type a tnf receptors with only a minimal change in the number of type b receptors. this increased expression of tnf receptors is conflrmed by direct binding experiments using 125i-labeled tnftx. the number of cpm's bound on dbc amp treated cells was about 0.5-3 fold higher than on untreated control calls, likewise incubation of melanoma cell lines with ifny increased the specific binding of subsequently added 125i-labeled tnfct. from flow cytometric analysis using the two anti-tnf receptor antibodies it became evident that flint was able to increase the expression of both type a and b receptors depending on the cell line used. characterization of a murine tumor necrosis factor (x-lacz reporter construct tumor necrosis factor ot (tnfcq is a prominent mediator of a variety of different pathologies such as endotoxic shock syndrom and rheumatoid arthritis. it also plays a crucial role in host defence against bacterial and viral infection. up to now, only few data about signal pathways leading to tnfo~ induction are available. an easy to handle tnfc~ -lacz reporter assay will represent a useful tool to study signal transduction on the one hand and provide data about compounds with potential tnfo~ inducible activity on the other hand. in order to develop a routine macrophage cell line capable to easily report tnfc~ induction, different tnfe-lacz reporter constructs were assembled. data about functionality of the different constructs in routine macrophages will be presented in the context of tnfot expression. cloning of a novel protein binding to lymphokine, fos and myc mrna with unexpected enzyme activity j. nakagawa, h-p. waldner, s. meyer-monard, and ch. moroni inst. medizinische mikrobiolegie, univ. basel an au-rich sequence in the 3' untranslated region of lymphokines, c-los, and c-myc proto-oncogene mrna is responsible for their rapid decay. we have purified a 32 kd protein by an auuua affinity column from human brain. partial amino acid sequence was obtained and the corresponding edna has been cloned. unexpectedly the edna sequence exhibited significant homology to enoyl coa hydratase and bacterially expressed recombinant protein indeed showed the activity. while rat hydratase did not show rna binding activity, the recombinant protein bound to cfos, c-myc, auuua cluster of il-3 mrna, and adeno virus iv, but not to mutated ad-iv, nor irrelvant transcript. the binding was competed out by poly u. interestingly the protein seems to be processed from a larger precursor. we suspect that the protein plays a role in mrna turnover and perhaps in oncogenesis. institute for medical microbiology, university of basel in t cells, the immunsuppressive agent cea is known to inhibit ca 2+ dependent induction of lymphokine mrna transcription. in contrast, the immunsuppressive drug rapamycin inhibits the lymphokine induced proliferation. we have analysed the effect of these drugs on a v-h-ras induced autocrine mast cell tumor line which expresses il-3 constitutively. csa and rapa inhibited autocrine growth by different mechanisms,, because added il-3 could antagonize inhibition by csa, but not by rapa. whereas csa acted by downregulation of il-3 mrna expression, rapa blocked il-3 induced proliferation. cea also inhibited il-3 superinduction by ca-ionophore, whereas rapa did not. nuclear run on assay indicated that the mechanism is posttranscriptional. therefore, we have introduced il-3 transgenes with and without the au-rich region in the 3' utr of the mrna into a tumor line. in contrast to the wild type, the expression of the mutant construct was insensitive to csa. since the mutant lacks sequences known to regulate rapid mrna decay, we suggest that csa affects the regulation of il-3 mrna stability. some non-hematopoietic cell lines produce both scf and its receptor, the c-kit protein (c-kit). testing the hypothesis that scf may be involved in secondary growth of nb bone marrow metastasis, we found and previously communicated (beck d. et al, proc aacr, 34, 52, 1993) a low or absent expression of c-kit in nb cells. the mrna and surface expressions of scf gene in nb cell lines grown in chemically-defined medium were evaluated by northern blot analysis and flow cytometry. the scf antigenic concentrations in culture supernatants were determined by elisa and growth-inhibition experiments performed by pre-incubating nb cells with anti-scf antibodies prior to 3h-thymldine uptake assay. the scf mrna was expressed in 4 of 5 tested lines but no membrane-bound antigen could be detected on those cells. detectable levels of scf in the supernatants were found in 5/7 lines (range : 39-96 pg/ml). blocking experiments resulted in a decrease of dna synthesis in i out of 7 lines only. from these and previous results, we conclude that scf is synthesized and released by many nb cells in vitro but the functional ~ole of it remains undetermined since scf does not appear to be usually involved in autocrine or paracrine growth stimulation of nb cells (supported by swiss fnrs grant 3200-031005). expression of the v-h-ras oncogene in the il-3 dependent pb-3c mast cells leads to the generation of two classes of il-3 autocrine tumors in vivo. autocrine il-3 expression in class-1 tumors results from increased il-3 mrna stability due to the loss of negative trans-acting posttranscriptional control. this defect can be corrected by somatic cell fusion to the nontumorigenic parental pb-3c resulting in downregulation of oncogenic il-3 expression and concomitant tumor suppression. expression of the v-h-ras oncogene remains unaffected in class-1 hybrids. in contrast, class-2 tumors are characterized by transcriptional activation of the il-3 gene due to the insertion of an endogenous retroviral element (intracisternal a-particle) which cannot be overcome by ceil fusion (see hirsch et al, 1993, j.exp.medicine 178, 403-411) . although v-h-ras is required for generation of either class of tumors, expression of anti-sense ras constructs inhibited only the proliferation of class-i tumor cells. experiments are underway to characterize the target and the nature of the class-1 alteration. nitric oxide (no) promotes vasorelaxatlon of renal vessels in vitro. the purpose of the present study was to assess the role of no in regulating renal hemodynamics in vivo. renal blood flow (rbf) and glomerular filtration rate (gfr) were studied in anesthetized mechanicallyventilated newborn rabbits both before and after the administration of a no synthesis inhibitor, ng-nitro-larginine methyl ester (l-name), at a dose of 50 pg/kg followed by 10 jzg/kg x rain. such a dose of l-name did not modify mean arterial pressure or pulse rate. by contrast, the renal vascular resistance increased by 31 + 9% (p < 0.005) while rbf decreased by 20-+6% (p < 0.02). gfr and urine flow rate remained constant. the overall results suggest that in normal conditions no may play a role in decreasing the renal vascular resistance of the immature kidney, without altering systemic blood pressure. interleukin-4 (il-4) is a t-cell derived lymphokine which, in addition to its effects on the immune system, is also able to suppress the growth of certain tumor cells. il-4 reduced the growth rate of four human colon tumor cell lines up to 70% in a dose-dependent manner. three other cell lines showed no significant alteration of 3h-thymidine incorporation or colony formation in the presence of 100 u/ml il-4. responder and nonresponder tumors were immunopositive for il-4 receptor (il-4r) expression in flowcytometric analysis, using monoclonal antibodies raised against recombinant il-4r and bound biotinyated il-4. responsive tumors expressed more receptors. 11-4 binds with high affinity to a 130 kda transmembrane receptor (il-4r), through which the extracellular signal has been shown to be transduced. coimmunoprecipitation and chemical crosslinking studies have enabled us to demonstrate il-4r target proteins. tyrosine phosphorylation of various proteins between 70 and 170 kda appears to be initiated during il-4mediated signaling events in colon tumor cells. the cloning of human il-4r target proteins will contribute to the understanding of the il-4 signal transduction pathway at the initial stage. the renal effects of endothelin-1 were investigated in 16 anesthetized and meehanleally-ventilated newborn rabbits. each animal acted as its own control. in 8 newborn rabbits, a bolus injection of 5 nmol.kg "1 of endothelin-1 caused an initial fall in mean blood pressure (mbp) followed by a gradual but significant increase in mbp that lasted for 45 rain. the dramatic increase in renal vascular resistance (+ 28 -+ 4%) induced by endothelin led to a fall in glomerular filtration rate (-12 -+ 4%) and renal blood flow (-16 _+ 3%). in spite of the reduction of gfr and rbf, urine flow and sodium excretion rates increased significantly (+ 20 _+ 5% and + 49 _+ 9%, respectively). in 8 additional newborn rabbits, a bolus injection of lnmol.kgaof endothelin-1 -a dose that usually induces marked renal and systemic vasoconstriction in adult models -did not affect systemic or renal hemodynamics. in conclusion, endothelin induces renal and systemic vasoconstriction, and affects water and sodium homeostasis during the neonatal period. however, these effects occur under higher doses than those used in adult animals, possibly reflecting receptor immaturity and/or interference of high levels of counteracting hormones. ontogeny of the endocrine pancreas in xenopus laevis c, maake 1 , w. hanke 2 and m. reine~ke 1 , 1 institute of anatomy, university of z0rich, switzerland and department of zoology ii, university of kaflsruhe, f.r.g. the pancreatic islets of anurans show insulin (ins), glucagon (gluc), somatostatin (som) and pancreatic potypeptide (pp) containing cells. since only limited information exists on the ontogeny and on the presence of insulin-like growth factor 1 (igf-1), we studied the development of the gastro-entero-pancreatic (gep) system in xenopus laevis using immunohistochemical techniques. singular ins-immunoreactive (-ir) cells were observed in the pancreas as early as on stage 41. at stage 43, the first pp-ir cells were found in the gep system followed by som-ir and gluc-ir cells. the first pancreatic islets consisting of ins-ir and gluc-ir cells occurred around stage 50. between stage 54 and 60, i.e. after the onset of metamorphosis, the endocrine cells decreased in number and the islets partly disintegrated. starting with stage 61, the islets reformed and the amount of endocrine cells increased reaching the adult status at stage 63. around stage 52, igf-l-immunoreactions appeared. igf-1immunoreactivity was found in pp-ir and gluc-ir cells but not in ins-ir and som-ir cells. it is assumed that islet-derived igf-1 may p~ay an important role during metamorphosis in xenopus. t. cathomen and r. cattaneo, institut for molekularbiologie i, universit~t zorich, hfnggerberg, ch-8093 zorich the signals used for synthesis of the measles virus fusion (f) protein are poorly understood: a long (>570 bases) untranslated region is followed by three in frame augs at codons 1,4 and 15. f is a type i transmembrane protein with a postulated signal sequence of 31 amino acids. we confirmed that the 46 aminoterminal residues contain a signal peptide by transfering them to another protein. with the other envelope protein hemagglutinin, f protein induces virus-cell and cell-cell fusion. mutagenesis of the three augs indicates that proteins initiated at codon 1, 4 or 15 are all functional, but show slightly different fusion efficiencies. forms translated from aug 1 or 4 appear larger in electrophoresis than forms initiated at aug 15, suggesting that two different signal peptide cleavage sites are used. we are currently investigating whether both f protein forms are incorporated in viral particles. the production of protein isoforms with staggered amino-termini is common in cytoplasmic and type ii transmembrane proteins, but signal sequences usually preclude a similar organization of type i transmembrane proteins. beguin p., beggah a.t., rossier b.c., jaisser f. and geering k. institut de pharmacologie et de toxicologie de l'universit6, ch-1005 lausanne recent experimental evidence suggests that na,k-atpase might be a candidate for regulatory phosphorylation by protein kinases a and c (pka and pkc). to verify this hypothesis, we have mutated several serine and threonine residues in consensus phosphorylation sequences of the ~subunit of bufo marinus na, k-atpase. the mutants were expressed in x~nopus ooeytes and phosphorylation was studied in oocyte homogenates upon stimulation of oocyte, pka and pkc. our results indicate that a unique phosphorylation site for pka is located at serine 943 in the c-terminus of the ~-subunit but its phosphorylation can only be revealed in the presence of detergent. on the other hand, mutations of several serine and/or threonine residues located in consensus sequences for pkc phosphorylation did not or only partially abollsh phosphorylation by pkc. in particular, mutations close to the catalytic phosphorylation site of the ~-subunit influenced phosphorylation by pkc, suggesting that either this region contains a real phosphorylation site or is part of a conformational domain necessary for phosphorylation by pkc. the heat-stable antigen (hsa or mouse cd24) gene is differentially regulated but has a housekeeping promoter. roland h. wenger*, georges kshler and peter j. nielsen. max planck institut f(jr immunbiologie, st0beweg 51, d-79108 freiburg i. bsg. "present address: physiologisches institut der universit&t zi.)rich, winterthurerstrasse 190, ch-8057 z0rich, expression of the gpi-anchored murine glycoprotein heat-stable antigen (hsa) shows tissue-specific as well as developmental regulation. during the maturation of several hematopoietic lineages, hsa expression is generally high in immature precursor ceils and low or absent in terminally differentiated cells. we present evidence suggesting that this regulation of the hsa gene (cd24a) occurs at the transcriptional level. in addition, sequence and methylation analysis of the cd24a promoter revealed characteristics of both "housekeeping" and tissue-specific promoters, including a methylation-free, hpall tiny fragment (htf) island, multiple putative sp1 and ap-2 consensus binding sites and a tata box. functional analysis of a 0.6 kb dna fragment containing these elements fused to the cat reporter gone in transient transfection experiments showed activity in both hsa expressing and non-expressing cell lines with a strength similar to that of the hsv tk promoter. large fragments from the flanking region of the cd24a promoter did not influence the ubiquitous nature of this promoter, finally, the cd24a, cd24b and cd24c genes were mapped to mouse chromosomes 10, 8 and 14 respectively. we have cloned, sequenced and characterized the gone for the m__itochondriat nadh-cytochrome b5 reductase (mcri) of saccharomyces cerevisiae. surprisingly, this gone encodes two mitochondrial isoforms of the flavoenzyme: a 34 kda integral protein exposed on the outer surface of outer mitochondrial membrane (mcrlp34), and a 32 kda soluble protein of the intermembrane space (mcrlp32). the smaller intermembrane space isoform is generated from the larger form by the action of the inner membrane protease i (impi) on the outer surface of the inner membrane. this is the first demonstration that a single gone encodes proteins which are located in different compartments of the same organelle. with special interest in any with a mitochondrial localization. degenerate primers were designed on the basis of high homology between members of the abc family, and pcr was performed on yeast genomic dna. ten dna fragments bearing significant homology to the abc family were amplified, nine of which were previously unidentified. disruptions of five of the corresponding genes were performed. disruption of one gene led to markedly impaired growth on rich medium and cessation of growth on minimal medium. the gene was cloned and found to encode a protein of 694 amino acids, a "half-transporter" which likely forms a dimer. ibi~tlnofluorescence on yeast expressing the gene cmyc-tagged at the c-terminus reveals co-localization of the protein with porin and with mitochondrial dna, visualized by dapi staining. nycodenz-purified mitochondria are enriched for the protein by immunoblotting. additionally,the c-myc epitope is protease-protected in mitoplasts, indicating an inner membrane sub-localization and a probable orientation with the c-terminus in the matrix. the gene, termed atmi for abc transporter of mitochondria, thus encodes the first member of the large abc transporter superfamily to be localized to this organelle. present work is aimed at revealing the substrate of this transporter. cycle in cardiac myocyte. the molecular study of this protein has been difficult even after its over-expression was made possible by the cloning of the corresponding cdna. the chief problem is the lack of convenient domains which could be used in the purification of the molecule. we have previously reported the efficient expression of the cardiac na+/ca 2+ exchanger in mammalian cell lines using the t7 rna polymerase-hybrid vaccinia virus system. we have now constructed the recombinant virus for the exchanger with a 6xhis-tag at the c-terminal. this tag has enabled the purification of the protein through a ni-nta agarose column. the expressed tagged protein induced na-dependent ca uptake which was not different from that in intact cells, i.e., cells in which the exchanger did not have the tag, the most important aspects of the purification produced and those of the reconstitution of the expressed protein will be described. $15-08 the neural cell adhesion molecule l1 is involved in the formation and specification of cell contacts in the central and peripheral nervous system during development, and thus may also play a role in synaptic plasticity of the adult central nervous system, using extracellular recordings of evoked field potentials in the rat hippocampal slice, we found that locally applied polyclonal anti-l1 and fusion proteins containing the ig-domains i-vi of l1 specifically block the stabilization of ltp in the ca1 region. baseline synaptic transmission was not affected by the presence of the anti-l1 antibodies. the anti-ll-induced effect was dependent on the antibody concentration and anti-l1 antibodies had no effect on previously established ltp. ltp was also reduced by an antiserum against the recombinantly expressed ig-domains i-vi of l1, whereas controt antibodies against liver cell membranes, which bind to neuronal membranes in the hippocampus, exhibited no effect on ltp. the contribution of l1 to stabilization of ltp within the ca1 synapses suggests that members of the ig-supertamily of cell adhesion molecules are involved in the structural modifications which are associated with synaptic plasticity in the mammalian central nervous system. an increasing number of surface proteins are described as being attached to the membrane by a glycosyl-phosphatidylinositol (gp1) anchor instead of the conventional transembranous hydropbobic domain. they are initially synthesized with a coohterminal polypeptide extension which is cleaved in the e.r. and replaced by the preformed glycolipid. this processing event is directed by a signal, located in the c-terminal region of the protein, which requires a group of three small amino acids positioned 10-12 residues upstream of an hydrophobic c terminal sequence. we previously transformed the transmembfanous glycoprotein d of herpes simplex type i into a gpi-anchored molecule by deleting its cytoplasmic domain and thus creating a molecule, gdww63, which meets the requirements for anchoring via a gpi structure. we now demonstrate, using gpi-deficient cell lines and biochemical studies, that a hybrid protein consisting of the thy-i ectoplasmic domain and of the c-terminal region of gdww63 can be alternative[y expressed in a gpi-anchored or in a transmembranous form. service de p6diatrie, chuv, ch-1oll lausanne proteolipid protein (plp) is a major myelin protein in the central nervous system (cns). a number of x-linked dysmyelinating disorders have been identified as mutations in the pip gene. we have identified a novel plp mutation in paralytic tremor (it) rabbit, characterized by body tremor, spastic limb paresis and hypomyelination in the cns. reduced levels of plp protein and its mrnas were observed in the cns as well as in the pns. plp sequence analysis revealed a single nucleotide change in exon 2 which results in the substitution of a histidine by a glutamine at position 36. histidine 36 is localized on the boundary between the first transmembrane region and the intracellular part of the protein. therefore, its position can be crucial for the efficient plp interaction with other proteins and lipids, and correct incorporation of the plp molecule into the membrane. histidine 36 is also part of a short fragment of ten amino acids which is conserved in all species studied. the same amino acid is altered in another hypomyelinated mutant, shaking pup, stressing its functional importance. furthermore, the pt mutation affects the plp "premyelin" functions, including oligodendrocyte maturation. equilibrium constants for octamer dissociation as well as dissociation rates in vitro increased in correlation to the number of charged residues eliminated, e.g. mutant 4-7, in which all four charged residues in the n-terminal heptapeptide are substituted with uncharged amino acids, showed a 100-fold higher equilibrium constant for octamer dissociation and a 145-fold increased dissociation rate compared to wild type. this strongly suggests that the charged amino acids in the n-termlnal heptapeptide of mib-ck, and therefore an ionic interaction, play an important role in forming the oetameric molecule. gangliosides and neutral glycosphingolipids (gsls) cell surface molecules are involved in a variety of biological processes and are assumed to play an important role in the mechanisms of hiv entry into lymphoid and epithelial cells. the total lipid-bound sialic acid (lbsa) content and the composition of gangliosides and gsls were investigated on pbmn cells from hiv+ve and normal donors. lbsa level was quantified by a fluorimetric assay, while gangliosides and gsls were assayed with high performance thin layer chromatography (hf'tlc) after multistep lipid extraction of 100 0013 x g membrane fractions. in normal pbmn cells the lbsa content accounted for 2.6/~g/108 cells or 20 nmol/mg protein whilst in pbmn cells from hiv+ donors the lbsa level decreased to 1.6 ,ug/108 cells or 13 nmol/mg protein. the qaantitation of neutral gsls and gangliosides was carried out by scanning spots revealed on hptlc plates and the integrated areas computed with a dedicated software. our findings showed that the most relevant ganglioside present in normal pbmnc was gm3 (65-80 % of total gangliosides) followed by small amounts of od3 (5 -15 %) and other acidic glycosphingolipids. neutral gsls included gl1, gl2, gl3 and gl4-hexosylceramide. hiv+ pbmn cells were characterized by a decrease of neutral glycosphingolipids as well as gm3 content which represented only 55 % of the ganglioside fraction, indicating an alteration in the glycolipid composition in the plasma membrane of infected cells. the neuropeptide y (npy) is one of the most abundant peptides of the mammalian brain. upon stimulation of cell surface receptors, npy generates diverse physiologic responses. npy acts on the cardiovascular system. it is a vasoconstrictor by itself and in addition, it potentiates the action of vasoactive substances such as angiotensin ii or norepinephrine. npy also inhibits glucose induced insulin secretion and is a potent inducer of appetite. to facilitate the development of npy antagonists we are investigating the interaction of npy with its cell surface receptor.we first constructed a series of mutants in which negatively charged residues present in putative extracellular domains of the y1 receptor were systematically replaced by alanines. the mutant cdnas were transiently expressed in hela ceils using a vaccinia virus derived expression system and the abi}ity of the encoded proteins to bind npy was evaluated. the capacity of the mutant proteins to be recruited to the cell surface was assessed by confocal microscopy. we identified initially 4 spacially clustered extracellular acidic residues of the human y1 npy receptor essential for npy binding. subsequently, we mutated 36 additional residues. based on these data a detailed computer model of the interactions between npy and its receptor was built. functional activity to energy metabolism. l. pellerin and p.j. magistretti. institut de physiologie, universit6 de lausanne, switzerland. astrocytes play a central role in brain energy metabolism. for example glucose, the exclusive brain energy substrate, is avidly taken up by astrocytes (pnas 90:4042, 1993) . application of glutamate (glu), the main excitatory neurotransmitter, stimulates in a concentration-dependent manner 3h-2-deoxyglucose (2-19(3) uptake by astrocytes in primary culture with an ec50 of ~ 100 ~m. the effect is not receptor-mediated since it can not be reproduced by glutamatergic agonists such as (nmda, kainate, quisqualate, t-acpd) nor prevented by antagonists (apv, cnqx, ap3). instead, it involves glutamate uptake since the effect is blocked by dl-threo-j3hydroxyaspartate, a potent inhibitor of the glu transporter. replacement of na + in the medium by choline also prevents the effect, suggesting a na+ driven process. finally ouabain, a selective inhibitor of the na+/k + atpase, completely prevented the effect of glu. these observations suggest that when glutamatergic synapses are active, glutamate, taken up by astrocytes, stimulates 2-dg uptake by astrocytes whose end-feet are known to surround capillaries, i.e. the source of glucose. they also provide a simple explanation for the mechanism linking functional activity to energy metabolism. cloning and molecular characterization of the mouse astrocyte glycogen synthase. g. pellegri, c. rossier, s. van berchem, p.j. magistre~i and j.-l. martin. institut de physiologie, universit6 de lausanne, switzerland. in the brain glycogen is predominantly localized in astrocytes, although its presence has been detected in certain large neurons, in ependymal and choroid plexus cells. glycogen is synthesized by the enzyme glycogen synthase (glys) from udp-glucose. out of the different glycogen synthase isozymes, only the muscle and the liver forms of glys have been cloned. we have isolated and characterized from a mouse astrocyte ~.zapii cdna library, a cdna clone encoding the mouse cerebral cortical astrocyte glys. the 3.5 kb clone isolated contains an open reading frame of 2214 bp encoding a protein of 738 amino acids. the coding region of the mouse astrocyte glys cdna shares 87% and 86% identity with the nudeotide sequences of the human muscle and the rabbit muscle giys cdna respectively. the cellular distribution of the glys mrna studied by northern blot shows the presence of a single transcript of 4 kb in cultures of astrocytes and, to a lesser degree, of neurons. the distribution of glycogen phosphorylase, which was also examined by northern blot shows the presence of a 4.2 kb transcript both in cultured astrocytes and neurons with however higher levels expressed in astrocytes. the functional molecular size of the vacuolar h+-pyrophosphatase (h +-ppase ec 3.6.1.1) from maize (zea mays l.) roots was estimated by radiation inactivation, both for substrate hydrolysis and for proton transport. light microsomal vesicles enriched in tonoplast membranes were prepared from young (2 days) maize roots using sucrose step gradients. these vesicles were still competent for ppi-dependent proton transport after freeze-drying and rehydration of the membranes. frozen native or freeze-dried samples were irradiated with 6~ for various periods of time. after thawing the samples, the activities of glucose-6phosphate dehydrogenase (added as an internal standard) and h+-ppase (hydrolysis of pyrophosphate (ppi) and ppi-dependent proton transport) were tested. by applying target theory, the functional size for hydrolysis was found to be around mr 155 000 when the native or freeze-dried samples were irradiated. no ppi-dependent proton transport activity was measurable after irradiation of the native samples. on the contrary, the freeze-dried membranes were still pumping protons after irradiation and a target size of around mr 250 000 was measured for the transport activity. these experiments suggest that the native h+-ppase from maize roots may exist as a dimer (at least) of the mr 80 800 catalytic subunit. $15-20 the na,k-pump moves 3 na + out and 2 k + ions into the cells. the domains of the protein involved in ion translocation have not been determined. we have studied the role of the n-terminal region in the na + translocation by measuring the kinetics of charge movements under na/na exchange conditions in wild type (wt) and two n-terminally truncated forms of the bufo ~ subunit with deletions of the 31 (t31) and 40 (t40) first amino acids expressed in xenopus oocytes. we have developped a new technique to perform fast (< i ms) voltage clamp on whole oocyte. the membrane on one side of the oocyte is permeabilized by digitonin allowing to measure current flowing across the other half. we observed presteady state ouabain-sensitive currents similar to that reported by other techniques (j.gen. physiol. 101:117,1993) . the estimated forward charge translocation rate was lower in both n-truncated mutants (wt 430 â�¢ 14, t32 215 z 8, t41 124 z 3 s-l). the midpoint potential of the charge translocation (best fit to boltzman equation) was -79 , -45, and -28 mv in the wt, t31 and t40 groups, respectively. the highly charged nterminus of the q subunit has a significant role in the forward translocation of na + ions. deletion mutants of human small intestinal lactase-phlorizin hydrolase (lph) were constructed to determine the role of protein subdomains in the intracellular transport of lph. the mutant lph1646mact (236 aa deletions), which contains the membrane anchor (ma) and the cytoplasmic tail (ct), was transported to the cell surface in a fashion similar to wild type lph. by contrast, lph1646 lacking the transmembrane domain persisted as a mannose-rich polypeptidc. this suggests that the membrane anchor and/or cytoplasmic tail are implicated in the er-egress of lph. another mutant, lph1559mact (323 aa deletions), was not efficiently transported to the golgi apparatus. epitope mapping with monoclonai antibodies as well as protease-sens{tivity assays provided an evidence that the tertiary structure of lph1646mact is very similar to that lph1559mact. in view of the variations in the proportions of the complex glycosylated molecules of the two mutants we conclude that the domain between lph1646mact and lphi559mact may play an essential role along the secretory pathway of lph. containing tyrosine. hussein y. naim, and michael g. roth; dept, of biocherni~try, ut-southwestem medical center, at dallas, dallas,texas-usa we have investigated an artificial internalization signal created by site-directed mutagenesis of the short cytoplasmic sequence of the influenza virus hemagglutinin (ha). mutation of cysteine 543 to tyrosine converted ha from a protein essentially excluded from coated pits to one that internalized at rate similar to some cellular endocytic receptors. to determine whether or not the ha-y543 signal is a degenerate form of the internalization signal found in proteins such as the transferrin receptor and the mannose-6-phosphate/igfii receptor, we have mutated amino acid positions in the ha-y543 shown to be important for internalization of the two receptors. we have changed amino acids on either side of y543 to those that either fit, or break the pattern of aromatic and hydrophobic residues proposed as consensus sequence. our results indicate that the ha-y543 mutant contains a sub-optimum sequence for a tyrosine-based internalization signal similar to those found in the receptors for transferrin, ldl, and mannose-6-phosphate/igfil. however, amino acids with side chains having different chemical properties functioned well in positions that are important for the internalization signal, indicating that the consensus sequence for this signal is more variable than previously proposed. n-linked glycesylation is an essential protein modification occuring in all eukaryotic cells. core-oligosaccharides are transferred by the enzyme n-oligosaccharyl-transferase frc~ the donor glc man gicnac -dolichol to selected asparagine 9 2 . resldues of t~e nascent polypeptlde chazn. the donor is build up in a stepwise fashion at the er-membraneby the products of the alg-genes (asparagine-linked-~lycosylafion) mutants defective in this pathway are available, but often lack a phenotype suitable for isolation of the corresponding genes. we found that double mutants of alg5 or alg8 with a wbpl mutation (defective oligosaecharyl-transferase) are severely growth retarded at 30oc. the double mutant phenotype allowed the isolation of both alg5 and alg8 genes which have been refractory to cloning so far. both genes encode potential trarsmenbrane proteins and are able to complement the biochemical defects of the corresponding alg5 or alg8 mutants. the na,k-atpase produces the driving force for the regulated transport of na across epithelial ceils. it is composed of an c~-subunit which carries the catalytic and ouabain binding sites and a b-subunit. to test the role of the usubunit in transport regulation, we transfected a6 cells with the b. marinus al-subunit (txl-tbm). this subunit confers a 300-fold higher resistance to ouabain (k i = 50/~m) compared to the endogenous pump. taking advantage of this difference, stable transfectants were selected with ouabain. expression of the cd-tbm subunit was visualized by western blotting. approximately half of the positive cell lines showed a high electrical resistance similar to untransfected cells, when cultured on filters. na-transport activity, measured as isc, was lower even in the absence of ouabain, but the relative increase in response to aldosterone was maintained. functional pumps containing the exogenous or endogenous al-subunit were quantified by equilibrium ouabain binding. interestingly, cells cultured on plastic dishes had a large fraction of low affinity binding sites (txi-tbm), while most sites were of the high affinity type when the cells were grown on filters. we conclude that the ouabain-resistant na,k-atpase cd-subunit of b. marinus can be expressed in a6 cells and used as a selective marker. however, the results suggest that the expression of functional ouabain-resistant pumps containing the al-tbm subunit could be influenced by the degree of cellular differentiation. in infected cells fully functional na/pi-cotransport was found in a time dependent manner; up to a 50-fold stimulation of na/p;-cotransport compared to uninfected cells was observed aft6r 3-4 days. transport of phosphate by infected cells was highly dependent on the presence of sodium, exhibited a kmtd~ of around 0.16 mm and was dependent on extraceuular i~h'. in agreement with expressed transport of phosphate, infected cells produced an immunoreactive polypeptide of 66 kda that represents a non-glycosylated form of the 80 to 90 kda mature na/p;-cotransporter. we conclud$ that the protein napi-2/rat when expressed in sf9 cells exhibits na/p-eotransport with similar kinetic characteristics as observdd in prox ma tubular apical na/pcotransport. therefore the napi-2 protein as expressed h sf9 cells can be used for further structural and functional studies. talin interacts in vitro with actin, vinculin, integrins and bilayers of acidic phospholipids, and nucleates actin filament growth at the bilayer surface. it is therefore believed to be a key protein mediating aetin-membrane linkage. we have analyzed functional properties of proteolytic fragments of purified platelet talin. using limited proteolysis two fragments of 47 and 190 kd were generated. preliminary results, obtained using viscometry and f-aetin-nucleating assays, suggest that the 190 kd fragment contains the actin binding site. we also assessed the lipid-binding capacity of the fragments. when a mixture of the two fragments was centrifuged in the presence of large multilamellar liposomes containing phosphatidylserine, only the 47 kd fragment, but not the 190 kd domain, co-sedimented with the liposomes, suggesting the presence of a specific lipid-binding site in the 47 kd domain. interestingly, this fragment contains the n-terminus of talin which shows homologies to the membrane-binding regions of protein 4.1. we are now analyzing membrane-binding properties of talin domains in more detail using hydrophobic photolabelling. recent studies suggest a role of the neural cell adhesion molecules l1 and ncam in mechanisms of memory formation (doyle e, et al, j. neurochem. 59:1570 -1573 , 1992 scholey a.b. et al, neuroscience 55:499-509, 1993; lijthi a. et al. unpublished data and see usgeb 94) . in the present study we analyzed the effect of chronic intraventricular infusion of polyclonal antibodies against l1 (anti-u) or ncam (anti-ncam) on the performance of male wistar rats during the acquisition and retention of a spatial learning task. briefly, rats had to remember the position of a submerged escape platform in a water tank (morris water-maze). when the escape platform was removed after acquisition training (= retention test), the performance of animals chronically injected with anti-l1 showed an impaired search pattern when compared with that of salineqnjected animals (p>0.05, mann-whitney). whereas control animals spent up to 45% of their time searching for the platform in the correct (= training) quadrant, the time anti-l1 animals swam in the correct quadrant was closer to chance level (31%). although monitoring penetration of antkncam into the hippocampus was almost as efficient as that of anti-l1, the performance of the anti-ncam-group was highly variable and did not differ from the performance of controls. these results agree with recent findings on the involvement of neural cell adhesion molecules in other forms of learning. we have isolated cdnas from mouse and from xenopus encoding the ~-subunit of the gastric h,k-atpase, which is expressed in the parietal cells of the stomach mucosa. the gastric h,k-atpase transports h + into the stomach lumen with the counter transport of k +, all at the expense of atp hydrolysis. when xenopus oocytes are injected with crna encoding ~h,k from either species, as well as a gastric ~h,k subunit, the two subunit proteins are synthesized, assemble, and form functional pumps located in the plasma membrane as demonstrated by 86rb+ uptake. the sensitivity to the gastric h,k-atpase inhibitor sch28080 was determined, with the ki for beth species found to be in the low ~m range. spodoptera frugiperda ceils are often used to overexpress eucaryotle proteins using baculovirus vectors. the atomic force microscope (afm) is a device which allows the observer to obtain high resolution images of biological samples immersed in a fluid medium; this instrument has therefore the potential to visualize overexpressed proteins on the surface of living cells. however, the interpretation of the images is sometimes difficult and requires a good knowledge of the specimen topography, i.e. the "normal" plasma membrane of the cell used for the overexpressisn. on our poster we present afm images of the plasma membrane of both fixed and living spodoptera frugiperda ceils. in additon, we discuss the possibility of achieving high resolution in the imaging of dynamic changes which affect /iving systems. the deduced protein sequence consists of 295 amino acids with about 55% amino acid sequence identicy when compared to mammalian ~h,k-subunits. data from other species and from the structurally similar na,k-atpase has shown that the ~-subunit is necessary for the formation of a functional pump, which consists of an ~/~ heterodimer. following co-injection of xenopus oocytes with crna for both the ~-and ~subunits, we have observed by rb + uptake h,k-atpase activity in the plasma membrane. we are presently studying the role of the ~-subunit in the maturation and function of the h,k-atpase. the atomic force microscope (afm) is a new type of microscope which allows visualisation of inorganic and organic samples with a very high resolution. a sharp tip fixed at the end of a cantilever is used as a topographic sensor. by scanning the sample with this device, a computer records the minute deformations of the cantilever and computes the topography of the sample. the instrument allows also the measurement of the interaction between the tip and the sample as a function of the distance. the knowledge of these interactions allows us to compute several mechanical properties like indentation, elasticity and stiffness. on this poster we report the results of these measurements on cells (sfg, c131), bacteria (e. coli, b. subtilis) and proteins (actin). in addition we discuss the use of the afm as a sensor for probing the mechanical properties of biological systems at a nanometric scale. neonatal thymectomy of balb/c mice induces aig. we have cloned the ~-and ~subunits of the balb/c h,k-atpase and expressed the proton pump in crna injected oocytes. both and ~ proteins are made, form functional pumps, and are immunoprecipated using auto-immune sera. the ~-subunit can also be in~unoprecipated independent of the ~-subunit when oocytes are injected with ~ crna alone. balb/c mice will be immunized with oocyte membranes containing either the ~/~ or ~ antigens in order to study the role of each h,k-atpase subunit in triggering aig. the prion protein is expressed in both astrocytes and oligodendrocytes of the neonatal brain and is down-regulated in adulthood. m. moser, colello, r, , pott, u, and b. oesch institut for hirnforschung der universit~t zorich, august forel str. 1, 8029 zorich the infectious agent causing transmissible spongiform encephalopathies consists largely or entirely of prp sc, a modified isoform o| normal prp (prpc). prpc is most abundant in the brain. the development of the disease strictly depends on the presence of prpc. and incubation times are shortened by overexpression of prpc. the normal function of prpe is not known and its cellular iocalisation in the cns is poorly characterised, except for its evident presence in neurons. here we describe the expression of prp c mrna in both oligodendrocytes and astrocytes during neonatal development of hamster and rat. in adult animals, expression is down-regulated in gila but not in neurons. our findings provide a link to the previously described accumulation of prpsc in astrocytes and the apparently faster course of prion disease in neonatal hamster. our results argue for a major involvement of non-neuronal cell types in prion diseases. such structures we raised mab against membrane protein fractions. a ~lab against a vitronectin receptor containing fraction was found, which if applied in vitro to bi6fi mouse melanoma cells, influenced growth, adhesion and morphology. ~tnen bi6fi cells prior to tail vein injection were treated, 2 different outcomes were observed. short treatment (lh) resulted in 85% inhibition of lung colonization whereas long-ter~ treatment ( 2weeks) resulted in 10-15 times more lung colonies. a candidate molecule likely involved in these observed effects could be identified as a 150 kda cell surface protein. sequence information from peptides revealed in several cases 100 % homology to mouse centrosomin a, i.e.a 32 kda cytosolic protein involved in the organization of the spindle apparatus. these data suggest that we may have found a protein which may participate in a link between the extra-cellular space and the microtubular system. we are currently trying to clone this 150 kda protein from a bi6fi c-dna library. wahlberg, j.m., and spiess, m. dept.biochemistry,biozentrum, university of basel. signals for correct basolateral sorting of subunit hi of the human asialoglycoprotein receptor are located in the cytoplasmic tail, involving a tyrosine at position 5. removal of this residue results in nonpolarized transport of the protein to the surface in mdckii-celis. a deletion mutant of hi, lacking 30 of the normal 40 residues of the cytoplasmic tail, including the tyrosine, has been found not to be transported to the cell surface, but to be accumulated intracellularly. the mutant protein is complex glycosylated and sialylated, indicative of passage into or through the trans-golgi. immunofluorescence analyses show defined juxtanuclear, tubular structures, which do not colocalize with markers for er, early endosomes or lysosomes, but which show similar staining pattern as markers for late endosomes and tgn. to define the determinants responsible for the intracellular accumulation a series of deletions and fusions have been constructed. the results suggest that the length of the cytoplasmic domain is one important determinant for missorting of hi. salerr~ n., medilansld, j., pellegrinelli, n. and eder-colli, l. departement of pharmacology, cmu, 1211 geneva 4 in chollnergic neurons the enzyme choline acetyltransferase (chat) exists as cytosolic(80-90%) and membrane-bound activity (10-20%}. are these two activities encoded by a single mrna? is membranebound enzyme preferentially associated with a given cellular membrane? in order to investigate these questions we isolated a fulllength cdna (4.2 kb) encoding drosophila chat and used it to transfect xenopus oocytes, rat fibroblasts and human neuroblastoma cell lines. triton x-114 fractionation of transfected cells showed that hydrophilic and amphiphilic chat activity were produced. the sp act. of hydrophflic enzyme reached 2, 0.8 and 0.25 ~mol ach/h/rng protein respectively in oocytes, fibroblasts and neuroblastoma whereas the sp. act. of amphiphilic enzyme was 1.8, 0.4 and 0.125 hinol ach/h/mg protein, respectively. amphiphilic chat was less sensitive to inhibition by the product acetylcholine and to heat denaturation than was hydrophilic enzyme. similar results were observed for the native drosophila chat. amphiphillc enzyme sedimentation was affected by the type of detergent present in linear density gradients of sucrose. transfected oocytes were subjected to subfractionation. besides a large amount of soluble chat activity, a significant proportion of enzyme was found associated with a fraction enriched in endoplasmic reticulum (er). preliminary results using transfected mammalian cells indicate that apart cytosolic chat, plasma mernbranes+golgi enriched fractions as well as er-enriched fraction contain chat activity. we have characterized a drosophila melanogaster gene encoding a very hydrophobic protein. the amino acid sequence shows considerable homology (33-42% identity) to neurotransmitter transporters. the gene is, however, not expressed in neural tissue, but in the male germline. transcription and translation occurs in early spermatocytes. antibody staining data suggest the following pathway of the protein during spermatogenesis: the protein is synthesized in early spermatocytes and is transported into the nucleus during prophase of rt i. at meiosis it associates with the mitochondria and it stays associated while they fuse to the nebenkern. during individualization it is stripped off in the cystic bulge. inspection of the putative protein sequence shows that it possesses alle the necessary targeting signals to allow its transfer from the cytoplasm through the nucleus into the mitochondria: three nuclear targeting signals, a mitochondrial import signal and, moreover, two pest-sequences for rapid protein degradation are present. we shall present a, currently tested, working hypothesis. in hg-treated toad skins, water permeability is high or low depending on whether the major anion of the inner bathing medium is so~ or ci. we report here the results of experiments designed to determine if, in skins bathed with so4-ringer, net water flow (j~,) could be diminished by agents added to the outer medium. toad skins were mounted inbetween glass chambers and exposed to a standard osmotic gradient of 200 mosm. jw was continuously monitored by automatic, volumetric techniques. exposure to lmm hgclz or chzcihg (external side) led to the typical anion-induced j,, changes. re-exposure to hg caused a mild (10%), although significant (n=8, p<0.001), fall in jw. since cuso4 is a sh-reagent like hg, we looked at the effects of cuso4 (lmm). there was a 68% reduction of j, (n=7, p<0.001), an effect totally reversible by simple washing of the skins. likewise, addition of dithiothreitol (dtt, 5mm) in the presence of cu, caused a 90% reversal of the cu effect ; there was no change in jw if dtt was given before cu, or if both agents were added simultaneously. finally, in hg-treated, glutaraldehyde-fixed skins, lmm and 5ram cuso4 caused 51% and 92% jw decrements, respectively, that were totally reversible. in conclusion, cu causes a dtt-sensitive inhibition of jr, in hgtreated skins. further work is needed to establish if the cu effect is exerted on sh-groups of apical aquapories of toad skin. $15-44 the amino acid sequences of the pc-645 e-subunits and other cryptomonad biliprotein ~-subunits pose the most intriguing question about the phylogenetic origin of these unique chromopeptides. cryptomonad pc-645 ~-subunits seem not to be closely related to the known phycobiliproteins, linker polypeptides (lpps), bpe or any other light-harvesting chl polypeptides from the thylakoid. for most of them the number of identical amino acid residues was 15%-20%. sequence alignment of c-pe associated lpps with t-subunits from cyanobacteria and red algae and the cryptophytan ~-subunits however show that the identity between c-pe associated lpps and cyanobacterial x-subunits is still significant, whereas it reaches the limit of significance between cyanobacteril and eucaryotic x-subunits. nevertheless it has to be assumed that x-subunit derive from cyanobacterial c-pe associated lpps as well as cryptomonad ~-subunits may derive from t-subunits. whereas the the phycobiliprotein ~-and 8subunits remained structurally conservative during evolution (e.g. -70% identity between cyanobacterial and red algal pe-subunits) the lpps and t-subunits show drastical changes in their chromophore binding domains. $15-45 functional and evolutionary relationships of the components of the primary events in photosynthesis have been traced using amino acid sequence comparison. basically, the question was posed regarding the interrelationship of the apoproteins of different photosynthetic organisms which gather light and/or separate charges across membranes. do their protein-chemical structure and organisation decipher us certain clues and parts of the path by which they have been selected and derived from a possible primordial reaction centre polypeptide? within that context the light-harvesting-and energy transfer systems of purple bacteria offered as ideal model components. a data set of more than 70 apoprotein sequences has pointed to some keystructural elements which have been partially verified as such by limited proteolysis and genetic engineering. a careful inspection of the determined bacterial antenna-structures revealed some remarkable similarities to eukaryotic antenna and reaction centre apoproteins indicating possible basic structural motifs for complexing pigment molecules, like chlorophylls or bacteriochlorophylls in very distant representatives of phototrophs. a number of glycoproteins of the nervous and/or immune system that are involved in cellular recognition and/or adhesion share the common lz/hnk-1 carbohydrate structure. in a large proportion of patients with peripheral demyelinating neuropathy associated with paraproteinemic gammopathy (ppn), monoclonal igm antibodies (m-igm) react with the lz/hnk-1 determinant of mag and p0. we plan to test the hypothesis that anti-mag m-igm autoantibodies may alter the expression of hnk-1 bearing myelin proteins such as mag, p0, mog, ncam or pmp22. by using monoclonal antibodies to myelin proteins on tissue sections, preliminary results indicate that mbp protein content seems to be strongly downregulated, whereas galc and $100 protein do not appear to be affected by the loss of myelin in ppn nerves. gfap protein expression appear to be upregulated in demyelinating biopsy specimens from ppn patients with anti-mag m-igm gammopathy. in conclusion, the expression of some l2/hnk-1 negative proteins might be disregulated by the presence of circulating anti-mag m-igm in the serum of ppn patients. we have isolated a human liver na+-taurocholate cotransporting polypeptide (ntcp) by screening a human liver edna library with a rat edna probe derived from ntcp (pnas 88, 10629, 1991) . the cdna codes for a protein of 349 amino acids which shows 77% amino acid homology with the rat ntcp. expression of ntcp in x. laevis oocytes resulted in na+-dependent bile acid uptake which exhibited saturability with an apparent km for taurocholate of 6 ~m. ntcp mediated taurocholate uptake into oocytes was inhibited by all major bile acid derivatives, bumetanide and bromosulphophthalein. southern blot analysis of genomic dna from a panel of human/hamster somatic cell hybrids mapped the human ntcp-gene to chromosome 14. in conclusion, these studies supplement the previously suggested selective mammalian distribution of the ntcp gene family and provide the possibility for direct molecular characterization of human bile acid transporter genes. diverse cell surface molecules of the nervous system play an important role in specifying ceil interactions during development. with the aid of a polyclonal antibody against l1, a 1.skb edna clone closely related to l1 (ch-l1) was isolated from a ~. gtl 1 library derived from poly(a)* l~qa of day 8 mouse brain. using this edna as probe the remaining cdi'ia 5' sequences were cloned out of a plasmid library constructed from postnatal day 6-14 mouse brain poly (a)+ p, na. two independent full length clones of 4.2 and 4.4 kb encompassing the entire coding region of chl] were isolated. these represent putative alternatively spliced mp, nas. the deduced primary structure of chl1 reveals that, like the cell adhesion molecules (cams) mouse l1, chicken ng-cam, and nr-cam (bravo), chl1 is composed of six ig-like domains, a transmembrane domain, and a short cytoplasmatic region. in contrast to the other l1 family members, only four and a half instead of five fibronectin type hi-like repeals are found in chl1. the fibronectin type [h-like repeats were expressed in e. col[ and the protein fragment was used for immunization. as observed for li, ng-cam, and nr-cam, chl1 is susceptible to proteolytic cleavage. bands of 185kd, 165kd, 125kd, and 50kd could be detected bywestern blot analysis. by in situ hybridization, a predominant expression by neurons was found in the adult mouse brain. western blot analysis showed expression of chl1 protein in brain and heart, but not lung, kidney and intestine. in liver, a 50 kd immunoreactive band was found. the relationship of chl1 to molecules known to be involved in cell adhesion and neurite outgrowth, combined with its pattern of expression, suggests a role for this molecule in cell interactions dndng neural development. work from this laboratory revealed that in hg-treated, glutaraldehydefixed toad bladders, the water permeability coefficient (pf) is high in sod-ringer and low in ci-ringer ; in all these studies the osmolality of the serosal medium was 220mosm. as an attempt to determine the mechanism(s) underlying such pf changes, experiments were carried out with bladders whose serosal surface was exposed to [so-, hypo-or hyperosmotic media ; the outer surface was always exposed to a hyposmotic solution (22 costa). net water flows were measured continuously with automatic, volumetric techniques. after exposure to 1 mm chzcihg (10', outer medium) followed by fixation with 0.25% glutaraldehyde (5', serosal medium), the bladders were thoroughly washed. in sod-ringer, pf (/~m/sec) was as follows (n=6) we wanted to determine the molecular weight (mw) of ntcp and its topology in the plasma membrane (pm) of rat liver. a rabbit antiserum generated against the c-terminus of ntcp was used to determine its mw on western blots and its subcellular distribution in liver and in hepatocytes by immunofluorescene (if). site directed mutagenesis and deletion experiments were used to determine the n-linked glycosylation sites. ntcp encodes a 50 kd protein in rat liver pm vesicles. the basolateral localization of ntcp in liver was confirmed by if. ntop could only be immunostained in permeabilized hepatocytes suggesting an intracellular localization of the cterminus, cnda constructs showed the ntcp is glycosylated at positions 5 and ii. the data show a selective basolateral expression of ntcp in rat liver and they suggest that the two ends of ntcp are located on opposite sides of the pm. zymogen granule membranes (zgm) are known to contain proteins with nucleoside phosphatase activity, but their exact nature and function are unknown. the activities require ca ++ and mg ++ and are sensitive to atpase inhibitors but not to inhibitors of na/k-atpase activity. the aim of this study was to isolate individual proteins of pig pancreatic zgm, to attribute enzymatic activity to individual proteins and to characterize them with various substrates (gtp, atp, amp etc.), inhibitors (amp-cp, amp-cpp, gtpgs etc.) and lectins (maa, dsa, gna etc.). we have subjected highly purified zgm solubilized in chaps to ief on immobiline dry strips | (pharmacia) with s ph range 3--10.5 (ist dim.). these strips were then electrophoresed in a polyacrylamide gradient gel containing 0.2% chaps and tris/hci at ph 9.5 (2nd dim.). nucleoside phosphatase activity was detected histochemically in the 2d gel by incubation with substrate in the presence of lead nitrate. focussed strips were also subjected to sds-page for comparison. several proteins showed strong activity to gtp, atp and itp. the role of these phosphatases are discussed in the context of the role of gtp-binding proteins and the de-/phosphorylating events on the zgm in intracellular targeting and exocytosis. the disease-specific isoform of the priori protein (prp sc ) is an essential part of the infectious particle causing scrapie or bse. prp sc differs from prp of normal animals (prp c ) by its relative protease resistance. the physical nature of this difference is still unknown. here we analyzed the protease-resistance of prp sc quantitatively. prp sc was rendered completely protease-sensitive at alkaline ph or in guanidinium thiocyanate (>1.5 m). denaturation in 2m guanidinium thiocxanate (gdnscn) completely abolished protease resistance of prp bc within 15 min while denaturation in 6 m urea showed a much slower time course. denaturation cuwes were used to calculate the gibbs free energy (as d ) in dependence of gdnscn or urea at different concentrations. the linear relationship between agd and the denaturant concentration is suggestive of a two state model involving the conformational change of a single protein domain. this is also reflected in the small number of side chains (11,6) additionally exposed to the solvent upon conversion of prp sc to its proteasesensitive isoform. our results suggest that minor rearrangements of the structure of prp sc are sufficient to abolish its protease resistance. of physiology, university of zurich, winterthursrstr. 190, ch-8057 zt~rich in contrast to mammalian kidneys where inorganic phosphate (pi) is reabsorbed unidirectionally, flounder renal epithelial cells both re.absorb and secrete pi. in order to investigate on a molecular level the cellular pi handling we have cloned a re,absorptive nalp i transport system from flounder renal tubules. based on homology with the cloned na/p i cotransporter from rat we have isolated a edna clone (flounder napi-ii, 2424 bp) encoding for a protein of 637 amino acids. in the eight transmembrane spanning domains the protein is 78% identical with the corresponding system from rat kidney, in the hydrophilic regions only 30%. expression of in vitro transcribed rna in xenopus oocytes specifically stimulated na-dependent pi transport. binding properties of na and pi as well as the ph-dependency were similar to the ones found in mammalian systems (rat, human, ok cells). by northern analysis three transcripts, 1.9, 2.7, and 4.2 kb in length, could be detected. rna from flounder renal tubules contained all of the transcripts, intestinal rna only the two lower bands, and non-epithelial tissue from kidney expressed only the 1.9 kb transcript. mammalian systems share only the 2.7 kb transcript but not the related species. the close functional relationship of flounder napi-ii with the known najp i transport systems and the pronounced differences in primary structure provide the basis for structure function analysis of na/p i cotransport. the amyloid 13/a4 protein precursor (app) is a transmembrane protein expressed in different isoforms throughout the organism. our work has consisted in a detailed study of the structure and biological function of app. specifically, we have shown that the secreted form (sapp) of app-695 is involved in the growth regulation offibroblast% and also stimulates neurite extension in b 103 neurnblastoma cell line. functional mapping studies have shown that the domain responsible for both the growth-regulating and the neurotrophic activities of sapp-695 is the rerms site, arg-328 to ser-332. the presence ofsapp binding sites on the surface of b 103 ceils (through the active rerms site) indicate that the neurotrophic activity of sapp is mediated by a receptor, and the subsequent activation of a signal transduction mechanism. in addition, we have shown that the rerms site is also involved in the neuronal survival properties of sapp-695. finally, we have initiated an in vivo study of app biological function, and shown that a peptide representing the neurotrophic domain ofsapp-695 can strengthen memory retention in rat through stabilization of synaptic structures in the frontal cortex. this work was supported by grants from the swiss national science foundation (823a-028366), and nih (ag 05131). we previously showed that in smooth muscle cells, the (~i-ar is rapidly phosphorymted following exposure to agonist. to understand this biochemical event, we investigated the phosphorylation of the c(1b adrenergic receptor stably expressed in rat1 fibroblasts (3.3 pmol/mg prot.). the =(1b-ar was solubilized from 32p-labeled rat cells and immunoprecipitated with antibodies raised against the n-terminus part of the receptor. treatment of ceils with 100 ~.m epinephrine showed a rapid and significant increase in receptor phosphorylation above basal. treatment of cells with a specific pkc inhibitor (ro-318220) did not affect agonist.promoted phosphorylation while it completely abolished phorbol esterinduced receptor phosphorylation. this strongly suggests that pkc is not involved in agonist-induced c{1b-ar phosphorylation. the elb-ar contains several putative phosphorylation sites in both its third intracellular loop and c-terminus tail. to investigate the role of these domains, a trunoated receptor (lacking its last 147 amino-acids) was constructed and stably expressed in rat cells (1.3 pmol/mg prot). this receptor mutant displayed similar ligand-binding properties and abirity to activate phospholipase c as compared to the wild-type receptor. phosphorylation experiments revealed that this mutant is not at all phosphorylated, neither by agonist nor by phorbol esters. agonistinduced decrease in cell surface receptors, measured by binding of the antagonist 3h-prazosin at 4~ was however similar for both the wild-type and mutant receptor. this suggests that loss of phosphorylation sites does not affect receptor internalization. human intestinal lactase-phlorizin hydrolase (lph) is synthesized as a precursor molecule (pro-lph), which undergoes proteolytic processing during maturation. lph expressed in cos-1 cells was enzymatically active, the electrophoretic properties of lph-species synthesized by transfected cos-1 cells correspond to those in organ cultured human intestinal explants, in caco-2 ceils and in transfected mock ceils. they included the pro-lph and a proteolytically processed form, which had similar electrophoretic properties to the mature enzyme. the appearance of the putative mature form was inhibited by brefeldin a, ammonium chloride and chloroquine. in addition, only complex glycosylated pro-lph (pro-lphc) was inserted into the cell surface membrane. these data indicate that in cos-1 cells pro-lph is inserted into the cell membrane, is internalyzed and enters lysosomes where proteolytic events lead to the appearance of a mature-like enzyme. $15-59 assembly and transport of paba peptide hydrolase alpha and beta subunits in cos-1 cells eldedng, j.a., gr0nberg, j. and sterchi, e., institut for biochemie und molekularbiologie, universit&t bern. paba peptide hydrolase (pph) (ec 3.4.24.18), a metalloendopeptidase from epithelial cells of human small intestinal mucosa, consists of two subunits, ~ and 13, which form homodimers and oligomers, cdna cloning has revealed homologies with developmentally important proteins from different species and has led to the definition of a new family of metalloendopeptidases, the astacin family (j. biol. chem. 266, 21381, 1991) . here, we report on the expression of the cdnas of pphcz and pphj3 in cos-1 cells. when expressed on its own, pph(z is synthesized in a core glycosylated form only, suggesting that it is transport-incompetent and not able to exit the er. immune-fluorescence studies have confirmed that pph~ is not expressed on the surface of cos-1 cells. pphl3, on the other hand, matures and is transported to the cell surface. when the two subunits are coexpressed, ppho~ can also be detected on the cell surface, suggesting that a heterooligomeric assembly is necessary for the surface expression of pphtz. truncated forms of both pphc~ and pphi~ could be detected in the medium of transfected cos-1 cells. in our previous work, we demonstrated by immunocytochemical reaction that sensory neurons expressed nuclear triindothyronin receptors (nt3r) in embryonic as well as adult rats. in contrast, in sciatic nerve, schwann cells exhibited only transient nt3r immunoreactivity from e17 to postnatal day 10. in the present work, we attempt to demonstrate by radioautography that the detected nt3r are effectively the binding sites of [125i] labeled triiodothyronin. cryostat sections of dorsal root ganglia (drg) or sciatic nerve were incubated with 0.1 nm of l, 3,5,3'-[t25i]triiodothyronin 2200 ci/m mol (nen), while the controls were incubated with a large excess (llxm) of unlabeled trfiodothyronin (t3). in aduit rat drg, radioautographs revealed that silver grains were exclusively restricted to neuronal cell bodies, while the schwann cells ensheathing the axons were free of significant label. in newborn rat sciatic nerve, silver grains accumulated over schwann cells; in contrast in adult rat sciatic nerve, schwann ceils were free of label. in control drg or sciatic nerve sections incubated in large exceas of unlabeled "1"3, all the radioautographs exhibited only scattered silver grains. in conclusion, our results show that sensory neurons and schwann cells are able to express functional nt3r which bind thyroid hormones (snf 31-33671-92). characterization of the maturepreeursor glycophospholipid for glycosylphophatidylinositoi anchors of saccharomyces cerevisiae. many attempts to isolate the mature yeast precursor gpi glycophospholipid ready to be attached to newly translated proteins for gpi-anehoring have failed although such precursors were previously identified in mammalian cells and protozoa. here we show that metabolically labeled glycolipids of the expected structure can be observed if the incorporation, their accumulation and their extraction are optimized. two very polar, (3h)-mannose-labeled glyeolipids named cpi and cp2 were identified and purified. they were structurally characterized using phnspholipase treatments, partial deacylation with methanolie ammonia, hydrofluoric acid dephosphorylation, nitrous acid deamination, acetolysis, exoglycosidase treatments and combinations thereof to produce labeled fragments which could be analyzed by paper chromatography or thin layer chromatography. cp1 and cp2 both contain the identical core oligosaccharide mangl,2(x->po4-6)manal,2mamxl,6(y->)mana-glcn-lansitol, x and y being hydrofluoric acid-sensitive substituents (most likely ethanolamine and phospheethanolamine, respectively). the inositol is acylated although no acyllaositol is present on protein-bound yeast gpi-anchors. cp1 and cp2 can also be observed after lableling with (3h)myo-inositol, the lipid moieties of cp1 and cp2 can be completely removed by mild alcaline hydrolysis altough the protein-bound gpi-anchors made by the pmi210 cells under identical labeling conditions are completely mild base resistant. this finding reinforces the notion that the ceramide moiety typically found on the majority of yeast gpi-proteins are added only after addition of the gpi precursor glycolipid to proteins. the distributions of the mannose-6-phosphat e/igfll-recept or and a2,6slalyltransferase in hepg2 cells: no evidence for co-localization by confocal laser scanning microscopy. the organization of the trans golgi apparatus (ga) with respect to (recycling) man-6-p/igfii receptor (mpr) and (resident) c~2,6sialyltransferase (st) has been investigated by double immunofluorescence laser scanning confocal microscopy in hepg2 cells. they were chosen because biochemical evidence suggested sialylation of mpr upon recycling (duncan & kornfeld, j. cell biol. 1988, 106, 617-28) implicating colocalization of both proteins. in the steady state st was confined to the ga whereas total (endogenous) mpr was localized to coarse vesicles without evidence for colocalization by confoeal microscopy. endocytosis of surface-labeled mpr was monitored in presence and absence of brefeldin a (bfa): without bfa, early endosomes (2' of warming up) showed no, late endosomes (20') little if any colocalization with st. in presence of bfa, the st-compartment disappeared whereas late endosomes persisted with some tubular emanations. under similar conditions, in "absence of bfa, endogenous mpr concentrated in the ga region suggesting colocalization with st. in presence of bfa the mpr compartment formed a redculum whereas the st compamnem disappeared. in conclusion, within the limits of the techniques used, exogenous mpr was not detectable in the st compartment whereas endogenons mpr concentrated in the ga region but obvious co-localization was exceptional. supported by grant 31-30757.91 of the snsf to egb. cultures of dissociated striatal neurons prepared from fetal rats were grown in the presence of neurotrophin-4/5 (nt-4/5) as well as the other known neurotrophins, ngf, bdnf and nt-3. we found that acute administration of nt-4/5 to seven days old cultures stimulates the hydrolysis of phosphatidyllnositol, an event involved in neurotrophin signal transduction. growth of stdatal cultures in presence of nt-4/5 resulted in increased cell survival as indicated by elevations in total cell number, protein content and number of viable cells. nt-4/5 exposure increased gaba uptake and staining intensity in gaba immunocytochemistry in these cultures indicating atrophic action on gabaergic neurons. to further identify responsive cell populations we analyzed for calretinin, a calcium binding protein known to colocalize with gaba in a number of neuronal cells. nt-4/5 strongly increased the number of calretinin positive cells in cultures prepared from rats of embryonic day 15, as we]l as calretinin levels determined by western blot analysis. when cultures were prepared from embryonic day 18 rats, nt-4/5 very strongly increased the morphological differentiation of calretinin positive cells. while all effects produced by nt-4/5 were mimicked by bdnf with similar potency, nt-3 was found to be only marginauy effective, our findings identify nt-4/5 as a potent neurotrophie factor for striatal gabaergic neurons. fusion of cells and organelles is a general patho-biological phenomenon: muscle cells, transport vesicles, langhans cells and virus induced fusion from within (ffwi, during replication) and without (ffwo, by the particles). in vivo and after virus isolation only little fusion can be seen, only after cultivation fusing hsv can be detected; a selective pressure must be released. 6 syn loci are known on the genome; the syn 3 locus is located inside of the cell in the carboxy terminal part of glycoprotein b, all other fusion domains are outside the ceil. fusion inducing mutations are in aa 816, 853, 854, 856, 857 and 872 and were correlated to fusion from within and without as well as to selective cyclosporin a effects. a model is presented for the 3 syn locus. -by recombination the fusion capacity could be transfected to fusion negative viruses. -hsv penetrates the cellmembrane by fusion by 2 ligand-recepter interactions. ffwo+-strains penetrate at 0~ cell/cell fusion was analysed and found to need also 2 receptor-ligand interactions if induced by syn 3 mutations. timing analysis of fusion events and by certain inhibitors allow further conclusions. quenching of lhcii isolated from dark-adapted (lhcii-d) or preilluminated (lhcii-l) rye leaves was similar in both preparations, but reversibility of this process within two hours of darkness was slower in lhcii-d. no differences in fluorescence quenching and full reversibility was observed for both lhcii preparations in reverse micellar solution. xanthophyll/chl concentrations in lhcii were consi-derably decreased under this conditions. excitation difference spectra (before and after illumination) of lhcii in asolectin liposomes showed typical changes of energy transfer between carotenoids and chl. a model is proposed according the xanthophyll cycle controls reversibility of light-driven excitation quenching as well as the xa~a~q~_x~tentof ~,,~nchi~g i/~ lhc~z. myelinogenesis is a complex, developmentally regulated process involving coordinate expression of myellnafion-related proteins. the myelin forming cells are the oligodendrocytes in the central nervous system (cns) and the schwann cells in the peripheral nervous system (pns). we differentially screened a rat postnatal day 16 (p16) spinal cord edna library with probes derived from normal (plus probe) and from myelin-free (minus probe) p16 spinal cord mrnas. we succeded in the isolation of several novel edna clones whose corresponding mrnas were expressed either selectively by oligodendroeytes or by oligodendroeytes and sehwann cells. here we describe one of the edna clones (tentatively denominated ns 2) whose mrna is specifically expressed by oligodandroeytes and schwann cells. the 3.5 kd transcript is expressed at highest level during myellnation and at much lower levels in the adult as it is known for other myelin-specific mrnas. sequence analysis of the full length edna sequence showed a 50% identity to the rat liver udpglucuronosyltransferase family, involved in the detoxificafion system. the 541 amino acid open reading frame encodes a 62 kd protein with a putative signal peptide and two transmembrane domains, whether this ns 2 protein is involved in a detoxification reaction or in glycosilation of proteins and lipids with glueuronie acid is under investigation. neuroblastoma (n8) are sympathetic tumors of childhood characterized by mycn amplification in advanced stages. cd44 standard (cd44s) and splice variants (cd44v) represent a group of surface glycoproteins whose expression has been recently linked to metastasis. we analysed the expression of cd44s and cd44v on n8 tumors and cell lines by immunohistology and northern blot. results showed high levels of cd44s on 33/44 nb, 6/10 n8 cell lines and 4/4 ganglioneuromas (mature, benign sympathetic tumors). lack of cd44s staining was observed on stage iv, mycn amplified tumors only. in cell lines, expression was not detected on cells with a neuronal phenotype while high levels of cd44s were expressed by cells with a glial differentiation, independently of mycn amplification. up-regulation of cd44s was obtained with agents that induce a glial differentiation, while neuronal differentiation by retinoic acid was not accompanied by a change in cd44s expression. no gd44v were detected on tumors or cell lines. we conclude that cd44s expression is down-regulated in a subset of nb ceils and tumors and that mycn product and additional mechanisms responsible for control of differentiation are involved in this regulation. the glycoprotein gc of herpesviruses is known to be non-essential for virus replication. non-essential herpesvirus proteins are suggested to perform "luxury functions" which may influence the outcome of an infection. we are aiming to analyse the function(s) of ibc specified by bovine herpesvirus 1 (bhv1), bovine herpesvirus 5 hv5) and caprine herpesvirus 1 (caphv1). these viruses are closely related but differ in their pathogenic potential. in a first step we have cloned and sequenced the individual genes. the nucleotide and the deduced amino acid sequence homologies of two 8hv1 reference strains was found to be >98%. the sequence homologies between bhv1 and bhv5 were >75% and those between bhv1 and caphvl were >65%. comparisons on the amino acid sequence level revealed that the differences were most prominent in the n-terminal parts, whereby the potential signal sequence region might be concerned. the putative glycosylation sites, however, were not affected, and the transmembrane sequences were similar in size and location. in order to characterize the significance of these differences, we are constructing gc-deletion mutants and recombinant viruses carrying a foreign gc gene. the in vitro and in vivo behaviour of these viruses will be compared to the wildtype virus. $15-67 o. moullet and j.-l. dreyer, instimt de biochimie, unlversit6 de fribeurg, ch-1700 fribourg camp has been recently shown to promote a cell survival response by retarding apoptosis (berridge, m.v& el. (1993) exp. hematol. 21,269-276) . on the other hand quinones are widely distributed substances of often potential toxicological significance. we have tested the effects of quinones on adenylate cyclase. the enzyme is rapidly inhibited by pbenzoquinone (ic50=40-45 ktm) or dicnlorophenol-indopbennl (/6'50=200 ilm), but 2-substituted quinones are inactive. the lc50's are decreased 3-10 times after membrane soinbilization but are not affected by gtp, gdp or analogues nor by cholera and pertussis toxins. the inhibition is not mediated by a g-protein or by the activation of a defined receptor. quinone inhibition stoichiometrically competes with forskolin activation of adenylate cyclase, equimolar concentrations of beth quinone and forskolin restoring the enzyme activity to its basal value. the cytotoxicity of benzoquinone, tested in vivo on hep-g2, a human bepatocellular carcinoma cell line, could be prevented with forskolin. in plasma membranes quinones tightly bind to only one major and two minor proteins that were purified by page electrophoresis under native conditions. together these observations indicate that quinone action can be attributed to targetting to a limited number of proteins at tile plasma membrane in a highly selective way. by affecting adenylate eyclase, a modulation of the camp pool induces apoptosis as a result of the cytotoxicity via. t.congolense is an african,unicellular hemoflagellate, pathogenic for domestic animals such as cattle.within the host bloodstream,parasites adhere to the wall of the microvasculature,eausing severe organ damage.we have set up and characterized an in vitro assay in order to study the metabolic requirements,reeeptor-ligand interactions and the ultrastructure of this adhesion phenomenon.our findings suggest that adhesion is an active process requiring metabolic energy on part of the parasite, but is independent of the target cells.we also show that t.congolense possess a leetin-like domain localized at specific sites along the surface of the anterior end of the flagellum, which interacts with specific carbohydrate receptors, probably sialic acid and/or n-aeetylglueosamine residues,on the endothelial cell surface. this suggests that adhesion is likely to be mediated by a trypanosomal surface component distinct from the variable surface glycoproteins(vsgs). we also suggest that the cytoskeletal protein aetin is involved in this process. local immune response based upon intestinal parasitespecific t and b cells to giardia lamblia may be impomrant in parasite clearance. experimental infections qf mice (cr:nih:s) with g. lamblia (clone gem-h7 which e~presses a major 72kd antigen on its surface recognized b~ mab g10/4) have demonstrated that the parasite undergoes surface antigenic variation in rive. experimental i~fection of immu/%odeficient mice (nu/nu mice and sci~d mice) and oontrol nu/+ littermates provided insights into associated immunological mechanisms: dominant surfade epitope-specific slga plays a major role in modulatin6] surface antigenic variation, and thymus-dependent t-lyn~phecytes in the induction of selfcure. athymio nu/nu mioe (zu. icr-strain) were reconstituted with immune peyer:~ patch lymphocytes obtained from self-healed littermat~ nu/+ mice. intestinal b-cells from these nu/+ mice @s well as from reconstituted athymic nude mice synthesized in vitro parasite-specific iga. this iga showed a predominant immunoblet reaction with the major surface antigen (a mr 72,000 polypep-tide) characterizing the giardia clone in question. nu/nu mice reconstituted wit~ immune cells acquired the potential to decrease significantly their intestinal parasite mass. thus the hypothesis on the causative role of intestinal iga in tl~ control of intestinal g. lamblia infection has found further experimental support. stephan jentsch, heinz tobler and fritz m011er, institute of zoology, university of fribourg, p4rolles, "1700 fribourg the process of chromatin diminution in the parasitic nematode ascaris lumbricoides leads to the formation of somatic cells that contain less dna than the germ-line cells. during this process, which occurs between the third and the fifth embryonic cleavage divisions in five different blastomeres, the termini of the chromosomes are cut off and eventually degrade in the cytoplasm. to analyze this process at the molecular level we constructed a xzap somatic telomere library. the analysis of three cloned telomeres and their corresponding germ-line genomic regions revealed that chromosomal breakage takes always place within short specific chromosomal regions (cbrs) and is followed by the addition of the telomeric sequences (ttaggc)n to the breakage sites. the different cbrs do not crosshybridize to each other. a comparative nucleotide sequence analysis is now being performed to screen for the existence of conserved sequence motifs. in a parallel approach we analyze the chromatin structure of the cbrs and their flanking regions in developmental stages before and during the elimination process, putative recognition or regulation sequences important for the elimination process might be recognized as dnasel hypersensitive sites. once such sequences will be identified, it should be interesting to establish their role in the elimination process and to look for specific binding factors. chromatin diminution takes place in all presomatie ceils of the early embryo of ascaris lumbricoides and is followed by the addition of many repeats of the telomeric sequence ttaggc. chromosomal fragmentation is developmentally regulated and occurs within specific chromosomal regions (cbrs). one of these cbrs (cbr1) was analyzed in detail. agene located close to the cbr1 encodes a putative gtp-binding protein, whose promoter region is located within a distance of only 2 kb from the telomeric ttaggc repeats of the corresponding somatic chromosome. a homologous gene was isolated from c. elegans. most interestingly, the c. elegans gtp-binding protein gene is also located at a telomeric position, namely at the end of chromosome v. in ascaris, transcripts of this gene are present in all developmental stages, though they seem to be stronger expressed in oocytes and early embryos than in later developmental stages and somatic tissues. a high degree of sequence conservation of these genes between the two nematode species and man (a corresponding partial cdna clone was identified in a human heart cdna library) suggests an important biological function. currently, we are using genetic methods to determine the possible function of this gene in c. elegans and to test whether its telomeric localization in both nematode species has any functional importance. it encodes a nuclear protein which is associated with she chromatin together with other probeins of the poiyoomb group. the mechanism of gene regulation caused by pelycomb seems to be similar to that of the modifier genes of the position variegation effect which also encode structural proteins of the heterochromatin. one of these proteins is hpi which shares with polycomb a region of similarity at the amino terminus called the ~omatin ~rganisation m~difier domain (chromo domain). the particular function of the chromo domain is not yet understood, but it seems to be important for the protein-protein interactions in chromatin associated complexes. furthermore, chromobox cdntai~ing genes were found in several species, suggesting that they are widespread in the animal and plant kingdoms. here we show chat ~. clemens contains at least one chromobox containing gene (cdna: cm08h7). length and szrusture of the putative ~. elegs~ chromo domain protein are similar to those of the chromo domain containing prozeins of other species. the expression pattern of cm08h7 is s:udied with antibodies raised against the putative chromo domain protein and by injecting a 5-ga! fusion construct inns the germ line of ~. eleman$. chromatin diminution in the parasitic nematode ascaris lumbricoides is a complex molecular mechanism, involving cleavage of the chromosomes at specific breakage regions, degradation of the eliminated chromatin and new telomere formation in all presomatic cells during the early embryonic development. the aim of the present project is to reproduce this dna elimination process in vitro, step by step or all in one, by using cell-free extracts of a. lumbricoides eggs. therefore, 4-8 cell extracts were established and their quality was assessed by the ability to transcribe 5s rrna genes (pol iii) and sl rna genes (pol li). faithful extracts were then tested for cleavage activity on dna substrates derived from different chromosomal breakage regions. finally, preliminary results revealed a possible non processiv rnase sensitive telomerase activity in the in vitro extracts, capable of adding specific nucleotide sequences to an oligonucleotide primer containing four repeats of the telomeric sequence traggc. as de novo synthesis of several kilobases of somatic telomere is likely to require a strong telomerase activity, we assume that this a. lumbricoides in vitro system is a good tool to isolate the telomerase protein and its corresponding gene or other implicated factors, s16-07 university of berne, ch 3010 berne a common feature of all mycobacteria -whether pathogenic or not -is their richness and variety of lipids. among numerous lipids widely distributed, pathogenic mycobacteria exhibit a group of sulfated surface lipids thought to be determinants of virulence. therefore, we studied sulfolipid-bioslrnthesis in pathogenic and apathogenic (opportunistic) mycobacteria species by monitoring the kinetics of [35s]sulfate incorporation into cellular lipids, pathogenic mycobaeterla (two virulent m. tuberculosis strains) showed a marked sulfolipid-synthesis with highest rates during exponential growth, while apathogenic ones (an avirulent m. tuberculosis strain and an opportunistic species) manifested negligible incorporation of the label. the partial characterization of the [35s]-sulfated lipids of pathogenic mycobacteria by thin layer chromatographic separation, combined with autoradiographic detection, revealed the presence of several radiolabeled lipid components of different polarities and with altering expressionpatterns during growth. these results strengthen the hypothesis, that sulfolipids are involved in the pathogenesis of mycobacterial disease. maiada (1,2) . some features of human pf maiada are observed in mudne malaria, although the pattern of sequestration changes due to the different plasmodial species involved, in mice, we showed by immunohistocbemistry that the expression of vcam-1 and icam-1 is upregulated in brain, lung and heart of p/asmodium bergheiinfected baib/c and also in lps-pdmed 8cid mice. in $gid mice injected with labeled human erythrocytes (e), a higher rate of sequestration to brain was observed with pfe than with uninfected e. lps-pdming increased the retention of pfe in the brain and lungs significantly (t test), but decreased it in the spleen. we conclude that pfe express surface structures which allow them to adhere to the vasculature of the brain more efficiently than uninfected e. lps increases the number of adhesive molecules on the host endothelium. interestingly, sequestration in the 8cid mouse resembles that of pfe in man. f. roth, f. riniker, k. schopfer and t. burkart inst. med. microbiology, univ. of berne, ch 3010 berne it has recently been shown that bacterial lipids cancarry antigenic determinants. the study of antibody specificity to such lipid epitopes in vitro turns out to be difficult since hydrophobic antigens have to react with watersoluble molecules. we have developed a solide phase lipid antigen immunoassay (laia) that allows to quantify the antibody reactivities with an array of lipid antigens derived from mycobacterial cells. defined amounts of extracted lipids were immobilized as equidistant lines on a nitrocellulose sheet. small strips of nitrocellulose, each carrying the same sets of lipids were incubated with different human sera. bound antibodies were then reacted with [125-i]-iodine labelled antihuman antibodies and visualized by autoradiography. the reaction was quantified by densitometry. assay conditions were optimized for antigen and antibody concentrations and appropriate incubation times. in addition, the effects of different ingredients (salts, proteins, detergent) on antigen immobilization and epitope accessability were studied. the presented laia-system is rapid, sensitive and reproducible. it allows to compare the specificities of different bacterial lipid antigens and to quantify their reactivity with serumantibodies. the atomic force microscope is a very convenient tool for studying hard and flat samples with atomic resolution. biological objects, usually soft and rough, are sometimes difficult to image using this technique. in contrast bacteria, which are too small to be observed with high resolution using the optical microscope, are hard enough to be observed with an afm at a relative good level of magnification. this kind of microorganisms can be prepared for afm imaging using very rapid and simple techniques. this method could be a convenient tool to observe the effect of bioactive substances such antibiotics. we show in this poster an exemple with the action of penicillin on b. subtilis. within the family trypanosomatidae, leishmania is a protozoan pathogen producing a broad spectrum of human disease. its life cycle is divided in two stages. promastigote is the flagellated form which colonizes the gut of the sandfly vector, and amastigote is the non-flagellated form that is specialized for growth and survival in the vertebrate host macrophage. to understand gene regulation during the transition between the promasttgote and the amastigote stage, we were interested in the characterization of stageregulated genes. we isolated a gene, sw3, from leishmania major whose mrna is more abundant in amastigote compared to promastigote. structural analysis of this gene showed that an additional polypeptide could be encoded by the opposite strand of sw3. we confn'med the presence of an open reading frame on the opposite strand by in vitro translation of the sw3 antisense rna. in vivo, an amastigote sw3 antisense transcript and the corresponding polypeptide were also detected suggesting that the sw3 is transcribed in both directions and that stage specific transcripts and polypeptides could be produced. analysis of other gene segments tend to indicate that an intensive usage of the leishmania genome to produce various polypeptides could be a general phenomenon in trypanosomatidae. the four variants and / or posttranslational modifications of histone h1 of procyclic trypanosoma brucei brucei (protozoa, kinetoplastida) were purified by hplc reversed phase chromatograpy and characterized by their amino acid composition and partial amino acid sequence. differences in sequence of up to 45% as compared to histone h1 of higher eukaryotes exist. alkaline phospatase digestion and analysis of h1 by means of hpce was used to demonstrate the presence of phosphorylated modifications. the unique biochemical properties of h1 of t. b. brucei contribute to the structural differences seen in the chromatin of the parasite as compared to that of the higher eukaryote host. larvae of the parasitic wasp chelonus inanitus (braconidae, hymenoptera) develop inside embryonic and larval stages of the host spodoptera littoralis (noctuidae, lepidoptera). parasitism induces in the host a precocious onset of metamorphosis and developmental arrest in the precocious prepupa. the wasp injects, along with the egg, pdv and venom into the host egg. injection experiments with pdv and venom revealed that pdv play a crucial role in altering host development and in suppression of the host's immune system. the genome of the pdv of c. inanitus consists of at least i0 segments of double-stranded circular dna ranging in size from 7-30 kb. we analyzed the expression of viral genes in the course of parasitization with cdna made from mrna at various developmental stages of s.littoralis. we also investigated the localization of the expressed viral genes on the various segments. in addition, these bacteria were isolated and cultured from brain tissue. to identify the infectious agent we used, among other techniques, an atomic force microscope (afm). when the. isolation fluids derived from the cortex of three alzheimer's cases were examined with afm and scanning electron microscopes, we observed bacteria whose size and morphology fit to the order of spirochaetales. these images are presented and discussed on our poster. ~ottstein bruno, institute of parasitology, ch-berne. %lveolar echinococcosis ae is due to infection with the metacestode (larval stage) of echinococcus multilocularls. an immunodominant antigen em2 of e. multilocularis was characterized by mab-gii, respective studies revealed, that (a) antigen expression metacestode stage-sdeci-fic and (b} that expression was restricted to the laminated layer. the em2-antigen i s a lectin-binding carbohydrate linked to a lipid residue. the "em2positive" laminated layer proved to be a crucial factor in protecting the parasite from host immune-effector mechanisms: only e. multilocularis larval structures exp ressing em2 were able to induce secondary alveolar chinococcosis in rodents. subsequent experimental studies in resistant (c57bl/10) versus susceptible (c57bl/ 6j and akr) mouse strains showed that resistance was as-sociated with synthesis of anti-em2 igg 3 and igg i. sus-ceptibility of c57bl/6j-mice was associated anti-em2 s and no igg 3 . the parasite-specific in vitro lym-~hoproliferative i~une response remained stationary ~ith time after infection in c57bl/10 and decreased in r and aki< mice. day 30 p.i. cd4 ⧠-lymphoblast cells dominated in all meuse strains; day 90 p.i. an in-creased nu/0ber of cd4-/cd8 + and cd4+/cd8 + cells were observed. only susceptible mice exhibited a significantly ~eereased response of lymph node cells to cona-stimula-~ien with time p.i. in conclusion, subtypes of parasitespecific cellular and humoral host immune responses seem leishmania species are protozoan parasites causing a spectrum of clinical manifestations in man ranging from cutaneous lesions to morbidity and death. the insect stage (promastigote) and intracellular stage (amastigote) differ in terms of morphology, physiology, antigenicity and gone expression. it is clear that the amastigote stage is uniquely adapted for survival within the inimical environment of the macrophage phagolysosome and that mechanisms regulating these adaptations must be called into play during the early stages of infection when the parasite undergoes transformation. in an effort to elucidate these regulatory mechanisms, we have constructed a "subtracted" cdna library which is specifically enriched for parasite transcripts expressed during the first 7 hours of infection ("switch" genes). several clones have been characterized by sequence, northern, and southern analysis. three clones, sw3, sw44, and sw20, are expressed at a several-fold greater level in the amastigote stage and potentially encode proteins which interact with nucleic acids. sw3 predicted protein is homologous with histone h1 proteins and sw44 and sw20 potentially encode ribosomal proteins. analyses of transcripts from this library are yielding clues not only to mechanisms underlying the regulation of parasite transformation, but also insights into post-transcriptional and translational control of gene expression in leishmania. $16-17 microbiology, university of geneva, medical school, c.m.u., 9 avenue de champel, 1211 geneva 4. a viral rna representing the sequence of a defective interfering rna, naturally selected for efficient replication, i.e. only containing the replication promoter, was cloned under the control of the t7 rna polymerase promoter. the expressed 17 transcript was then replicated in rive by supplying in trans the replication functions, equally expressed from plasmids. this system, completely accessible to genetic manipulations, allowed us to define a basic rule directing sendal virus replication, the "rule of six" (calain and roux, j.virol. 67 : 4822-4830, ig93). a second defective rna, representing a shorter version of the viral rna, in that it contains not only the replication but also the transcription promoters, was then cloned and replicated in the same system, although with lower efficiency. the replication of this "transcribing" viral rna was found to obey the "rule of six" as well. replicationitranscdpfion promoters were then achieved to define the cissequence requirements needed for efficient replication or for replication /transcdption. results obtained with these chimerical viral rnas will be presented and their contdbufion to the comprehension of the paramyxovirus replication and transcription processes bovine t-cells that become infected with the intracellular parasite theileria parva undergo lymphoblastoid transformation and acquire the ability to proliferate continuously. they cease to require specific antigenic stimulation, become independent of exogenous growth factors and cause tumors in nude mice. the il2 and il2r genes are constitutively expressed and the transcriptional activator nfwd3 which regulates these two genes is continuously activated. these processes are all strictly dependent on the presence of the parasite in the host cell cytoplasm and cease upon the specific killing of the parasite by the theilericidal drug bw720c. we have shown that the presence of the parasite results in the activation of the protein tyrosine kinases fyn and ick, which can participate in early t cell receptor signalling, suggesting that the parasite may use this signal transducti0n pathway to induce continuous proliferation. cysteine proteinases of leishmania as targets for chemotherapy. jacques bouvier*t and judy sakanari*. *department of pathology, ucsf school of medicine, san francisco, ca, and tanimal health, ciba-geigy, ch1566 st. aubin, switzerland. the overall goal of the study is to apply the structure-based approach to develop lead compounds and design novel enzyme inhibitors as potential therapeutic agents against leishmaniases. in this regard, we have identified and characterized the cysteine proteinases of leishmania major, and showed that they consist of excellent targets for drug design. we have purified a membrane-boand cysteine pmteinase not previously described in other trypanosomatids or protozoans and obtained amino acid sequence information from the purified enzyme. in addition, we have also characterized and purified the soluble cysteine proteinases of the parasite. consequently, we have isolated two genes encoding the soluble and membrane proteinases. the soluble cysteine proteinase gene occurs in multiple copies of 2.9 kb with flanking regions of 1.2 and 2.2 kb and is localized on one chromosome of approximately 440 kb. the membranebound enzyme gene is 3.1 kb and occurs as a single copy on a chromosome of approximately 1100 kb. a three dimensional computer model of both genes wiu be generated and enable us to scan the vast chemical database for new lead componds. we akeady have identified several cysteine proteinase inhibitors that are effective compounds in killing both promastigote and amstigote stages of the parasite in the micromolar range in vitro without affecting the host cells. dollenn~ier, g., cassinotti, p. and weitz, m., institute for clinical microbiology and irananology, 9000 st .gallen/swit zerland hav is a ~r of the picornaviridae. its rna gencrne rf~y be divided into 3 coding regions (pi, p2 and p3), qhe p3 region contains a protease 3cp ro that generates mature proteins by cis-cleavage of the initial polyprotein precursor. catalytic activity of 3cp r~ has been d6~aonstrated in a vacciniavirus/t7 ~qa-polymerase hybrid expression system. (balouter aided ali~ts of hav 3cp r~ sequence with that of other picornaviruses imply that amino acid residues his-44 and/or h) the complete hav open reading frame, hav specific expression products were identified by radioj_rmmnoprecipitation and lyy sds-page. the analysis with an antibody specific for putative nonstructural protein 2b revealed a 27,5 kda peptide. this is in contrast to its predicted size (12 kda). however, analysis with anti-(putative)2a antiserum confirmed an upstream location of the 2b n-terminus. the new 2bpeptide was also identified in lyeates of hav infected mrc-5 cells. hence, recombinantly expressed hav polyprotein underwent authentic processing and the predicted p2 organization has to be modified. we are interested in the development of animal models for one of the two major pathological changes found in the brains of patients with aizheirnar~s disease, the formation of fleuroitbrillary tangles, a main component of which is abnormally phosphorylaled tau-protaln. (tau itself is a protoin associated with mlcrotubuli.) two fundamental questions may be answered with our models: first of nit we woald like to reproduce the formation of tangles, and secondly we hope to answer the question, whether the formation of these pathological structures is sufficient for the bevalopment of senile dementia ot the alzheimer type (sdat). at present there are only a tow putative candidate genes known which may be in-voned in the hyperphosphor~lation ot tau in v/vo, one of which is the map-kinase erk2. we have cloned this candidate kinase from rat txaln rna via an rt-pcr approach and expressed the cona under,the co.rat of tour different promoters in transgenis mice. these promoters were tested for maximal expression. a similar approach was chosen to express the human taut0 isofonn in the brain ot transganic mice. we have stated to analyze different tau an~ erk2 expressing lines, northern blot analysis has revealed high levels of expression for all transgenes (up to fnetoid {n comparison to endocjenous levels). the neuronal spectficry of the transgenio erk2expression was c~nfirmsd by in situ hybridization analysis. ir~ addition several tauexpi'esalog tines were analyzed in wealernblots to determine the relative amount of htau= protein in the brain. sections of the brain were stained with tau-sp~c~c ardib~iies to identify the neurons expressing tau. from intemrosses of tau-with erk2-expressing lines, we have already identified double-positive mice which we currer~y analyze by immunohistocbemistry and in western nots to examine the phosphorylaiton status of tau. we are well aware of the possibility that only animals trans~enic for both tau and erk 2 might beveiop tang{es, whereas single transgenics mlsht not. pairs of individual neurons were recorded with sharp electrodes or in whole-cell configuratio~ in hippocampal slice cultures of rat. synaptic connections were studied between pre-and postsynaptie cells in ca3 and ca1 hippneampal fields by eliciting single aetinn potential in the presynaptic cell. monosynapfic excitatory connections were highly probable between ca3 and ca1 pyramidal ceils (p=0.76, n=82) with almost no feed-forward inhibition (p=0.01). by contrast, disynaptic ipsps, blocked by cnqx or bieuculline were observed with a very high probability (9--0.6) between ca3 pyramidal cells. monosynaptic excitation was found ha 56% of cases between ca3 neurons (n=43) but only in 27% of cases (n=ll) between two ca1 cells. monosynaptie ipsps with very short lateneies (<lms) and blocked by bicuculline, but not by cnqx, were recorded within area ca3 (5/6) and within cai (2/3), but not between ca3 and cat fields (2/2). probability of transmitlzr release, studied in recordings where failures could be unambiguously distinguished from spontaneous or small psps, was found to be close to 100% at both excitatory and inhibitory synapses. specific inhibitors of presynaptie release at excitatory and hahibitory terminals (i.e. adenosine and opioid peptides, respectively) gradually reduced the anaplitude of the psps indicating that the release at excitatory and inhibitory unitary synapses is multiquantal. the cns of a neonatal opossum, monodelphis domestic& shows profuse growth of fibers across a lesion. as in other mammals, such repair is not seen in adults. experiments have been made to define the age of the animal and stage of development at which repair ceases to occur. the entire cns was removed from animals aged 3-6 days or 11-14 days after birth. the spinal cord was crushed and growth of fibers assessed 5 days later by electrical recording of impulses conducted through the crush. repair after lesions to spinal cords of younger animals occurred in 38% of the trials (45 animals, aged 3-6 days). in nervous systems from older animals (11-14 days after birth) the success rate was low (only 4 out of 38 preparations). similarly, the carbocyanine dye dil labeled numerous fibers in spinal cords of younger animals (12 out of 21), whereas only one preparation out of 15 showed scant outgrowth into lesions in the older animals. our results suggest that there is a critical period for neuronal repair; at about 12 days nerve fibers lose the ability to grow across lesions. it is an advantage that at this later stage the nervous system is still small enough to survive in vitro. features correlated with these changes are oligodendrocyte differentiation and myelin formation. with immunofluorescent staining as well as electron microscopy myelination becomes apparent at 8 days after birth. we are now using time lapse-videomicroscopy to analyze molecular mechanisms that could play a part in promoting or preventing repair. supported by grants to j.g.n from the swiss nationalfonds (31-3626292) and from the internat. res. inst. for paraplegia. i. impulse conduction in axons has integrative functions. the dorsal root ganglion (drq) of the embryonic rat put into culture forms a monolayer and is thus accessible to eonventionalmieroelectrode technique, which allows testing the above hypothesis. we have chosen a dual approach by correlating ext~rimental el~ctrophysiological data from the chlture with results obtained from a compartrhental computer model. ii. the safety factor for conduction was found to be low. thus failures of ap invasion of the drg cell soma could occur at sites of impedance mismatch when a second stimulus felt into the relative refractory period of the first or when the axon was repetitively stimulated. the electrotonic resiuues (e.rs) of the failed aps had discrete amplitude levels, suggesting that the failures were always caused at the same site along the axon. these sites of low safety factor were found to be the branch point in the unipglar drg cell and the entrance of the stem piece into the soma in both cell types, the bipolar as well as the unipolar. a systematic comparison of 15oth cell types showed that the ap conduction through the latter is more reliable.'the length of this stem piece is inversely' correlated to the delay caused at the branch point, as tile electrical load of the soma is more efficiently masked by a long stem piece. the dendrites of motoneurons (and subsequently many other neurons) have been assumed to be electrically passive since the work of coombs, ecc]es and fatt in the 1950's. however, direct evidence has been impossible to come by until recently with the advent of appropriate dyes and equipment for measuring very small and very fast optical signals. we examined the propagation of action potentials, both spontaneous and evoked, in the dendritic trees of spinal cord neurons using a 12 â�¢ 1 2 array of photodiodes and a ccd camera as weft as conventional microelectrode techniques. organotypic slice cultures of embryonic rat spinal cord were stained with a voltage-sensitive dye (di-8-anepps) or a calcium dye (fluo-3) and recordings were made from motoneurons identified by their location and morphology. the results indicate that there is in fact an increase in calcium in the dendrites that accompanies action potentials whether synaptically evoked or evoked by current injection at the soma. spikes in the dendrites up to 1 60 i~m from the soma are much larger than could be explained by models assuming passive dendritic trees. work is currently underway to test the possibility that sodium conductances might contribute to the propagation of action potentials in the dendrites. cat auditory cortex contains multiple cochlear representations in both hemispheres that are interconnected through the corpus callosum (cc). the distribution of auditory callosal axons was studied on the mediosagittal level after injections of biocytin at electrophysiologically identified locations. single injections were done in ai (2 cats), aaf, aii and paf; multiple injections in ai & aaf in one cat. an additional cat received 1 injection in sii. the mediosagittal image of cc was subdivided into 30 segments along a rostro-caudal axis. labelled axons crossing the midline were counted in coronal or horizontal sections. axons from auditory fields were found in the posterior 2/3 and those from the somatosensory cortex in the anterior third of cc. axons from a discrete injection (ca imm in diameter) spread over as much as i/5 of cc. the rostrocaudal distribution of axons at the midline reflected roughly the rostrocaudal position of the field of origin, but apparently not the tonotopic order of a given field. the major efferent projections of substantia nigra pars reticulata (snr) convey information to the thalamus, to the tegmentum, to the superior colliculus, and to the spinal cord. such a range of targets suggests a highly organized activity within snr and this study investigates the temporal organization of spike trains. in the portion of snr between +2.7 and +4.5 mm interaural levels, we recorded 40 single units along 6 electrode tracks in 4 young female wistar rats. most units (n=32) fired in a non-regular poisson process, although only a small number (7/32) was characterized by an average burst size with more than 3 spikes. crosscorrelograrns were computed for 25 pairs of units simultaneously recorded from the same electrode and for 26 pairs simultaneously recorded from distinct electrodes. most crosscorrelograms (27/51) showed a symmetrical peak centered on time zero, which indicates a tendency to synchronous firing. when units were recorded from the same electrode tip we observed that 1/8 to 1/40 of their spikes were synchronous. about half of the significant interactions were characterized by a relatively long duration (>250 ms) and are likely to correspond to a different mechanism of synchronization. these results support the hypothesis that snr cells receive common afferences of two different types and their fine temporally organized activity yields coordinated activity between the target structures. the neuronal microtubule-associated protein map2 binds to microtubules via a domain containing 3 or 4 repeats of an 18 amino acid sequence. we have previously shown that when cultured nonneuronal cells are transfected with map2 their microtubules become stiffer and are then capable of supporting process outgrowth when their cortical actin cytoskeleton is depolymerised. we hypothesise that this stiffening effect of map2 is caused by the 18 amino acid sequences binding to neighbouring tubulin subunits in the walls of the microtubules thus reducing their freedom of movement relative to one another. to investigate this further we have created map2 mutants in which 1 or 2 repeats of the tubulin-binding motif were deleted. these were tested by transfection and expression in cultured cells. the results confirm that the number of repeats affects the stability, stiffness and the cytoplasmic arrangement of cellular microtubules. we were previously able to demonstrate the presence ot kinin-like immunoreactive structures in the mouse brain. in order to characterize them, we have now performed a donble-immunofluorescence study using both polyclonai auti-srudykinin (bk) and mouse monocloual anti-neeron-specificenolase (nse) antibodies. the latter is considered to be a specific neuronal marker. after fixation with bonin-hollande solution, the brains were removed, embedded in paraffin and serially sectioned at 10 am. the sections were incubated with the antibodies, which were visualized by an indirect immunofluorescence technique using fitc for nse and by an avidin-biotin technique using texas red for bk. double-immunofluorescent cells were found in the thalamus ventralis, in the nucleus cechleuris ventralis and in the nucleus mesencephaficns nervi trigemini (v). bk-positive cells in the nucleus principalis v were nse-negative ih spite of their typical neuronal aspect. the other bk-positive structures (pia, ependyma, hnic~es) also were alwa~ nse-negatlve. most of the nsepbsifive bk-negative cells were seen in the cortex. the neuronal nature of many kinin-like immunoreacfive structures in the mouse brain is thus ascertained. in contrast to the amphibian optic nerve (on), on of adult mammals is unable to regenerate after injury. astrocytes disappear from and macrophages accumulate at the lesioned site (blaugrund et al., j. neurocytol, 118, 105, 1992) , we have followed the distribution of astrocytes and microglial cells in xenopus tadpole ons over 10 d after mechanical crush. astrocytes were stained for cytokeratin and vimentin, and microglial cells for griffonia isolectin b4. soon after crush, immunoreactivity for intermediate filament proteins was strongly increased. astroeytic processes extended into the lesion from both proximal and distal stump. at ,5 d, astrocytes had crossed the injured region. in the distal stump, some of them contained vacuoles indicating phagocytic activity. while in the normal on microglial cells were ramified and scarce, 2.5 d after crush they were roundish and vacuolized, and scarce within the lesion but numerous distal to it. at 10 d, their number was decreased and most of them had reacquired ramifications. conclusively, in the tadpole on, astrocytes are virtually never absent from the lesion site but rather extend rapidly into it. astrocytes may thus provide a guiding support for outgrowing axons, microglial cells that are frequent in the distal stump, do not accumulate at the lesion. the behaviour of the two cell types differs profoundly from that in mammals and is likely to be one of the factors that may contribute to successful neuronal regeneration of the amphibian on. ci4mence, j. f. 1, ranieri, j. p.2, aebischer, p.2, and sigrist, h. 1, 1institute of biochemistry, university of berne, ch-3012 berne; 2division autonome de recherche chirurgicale, chuv, ch-1011 lausanne. small peptidic sequences of laminin (yigsr, ikvav) are known to promote attachment and/or neurite outgrowth of various neuronal cell lines (pc12, nb2, ng108-15). new and established heterobifunctional photo crosslinkers were used to immobilize these peptides in a topically defined fashion onto biocompatible surfaces such as hydroxylated fluorinated ethylene propylene (fep-oh) and pely(vinymlcohol) (pva) . n-[m[(3-trifluoromethyl) diazirine-3-yl]phenyl]-4-maleimido-butyramide (mad) was thermochemically linked to the synthetic peptide cdpgyigsr via its n-terminal cysteine, leaving the bioactive part (yigsr) free for cell receptor interaction. mad-cdpgyigsr was radioactively labeled by reductive methylation and purified by reversed phase hplc. patterned (20 and 300 #m) peptide immobilization was attained by photocoupling onto pva and fep-oh and visualised by autoradiography. light-structured biomaterial surfaces with molecularly oriented nerve growth promoting peptides were investigated for spatially controlled neuronal cell attachment and differentiation. it is striking to observe that these inhibitory or stimulatory effects were corroborated by binduced decrease or increase in 2-deoxyglucose uptake, respectively. this gives b a putative role in the limbic regulation. the sheet-like processes of oligodendrocytes wrap themselves spirally around central axons, thus formtng the myelin sheath. a single oligodendrocyte may donate internodal segments of myelin to each of 20-30 or more adjacent axons. it is not known, if one oligodendrocyte picks out axons randomly or if it prefers axons with a certain diameter or with a certain chemical specificity. we have studied this last possibility by combining intracellular injection of a fiuorochrome, and immunohistological detection of calciumbinding proteins (cabp's), markers for the axons ef certain subpopulafions of nerve ceils. lucifer yellow was injected into oligodendrocytes of the optic nerve, of living rat brain slices. the oligodendrocytes were identified by their electrophysiologieal properties. slices containing the iniected cells were immunostained /or parvalbumin or calretinin using second antibodies labelled with texas red. using co.focal laser-scanning microscopy combined with a three*dimensional reconstruction programm, the relationship between oligodendrocyte processes and axons positive for one of the cabp's was determined. for ultrastructural confirmation, single, lucifer-yellow injected, oligodendrocytes were photoconverted, and the cabp's-positive axor~ were revealed by gold-labelled antibodies. the few oligodendrocytes injected show the classic morphology, that is of a small cell body and few processes running parallel to the course of the axons. the proximity between glial processes and positive axons can be easily appreciated in confocal laser-scanning images. a preferential assodation of oligodendrocyte processes with parvatbumin-positive axons has not as yet been found. thus, the confirmation of the hypothesis that oligodendrocytes choose their axonal targets according to chemical cues awaits further work including the injection of a lar~er number of these cells. to initiate a molecular genetic analysis of brain development in insects, we are characterizing the mechanisms of embryonic brain development in the fly drosophila. early in embryogenesis, a small number of molecularly diverse brain neuroblasts generates the neurons of the brain. subsequently, pioneering brain neurons initiate axonal outgrowth along glialbound brain compartments. these pioneering neurons establish the primary brain tracts. the pioneering brain axons express the cell-cell adhesion molecule fasciclin i dynamically during outgrowth and initial fasciculation; a subset of the pioneering axons expresses fasciclin ii. the axonal framework of the embryonic brain tracts is used for outgrowth and fasciculation by subsequently differentiating axons and, thus, prefigures the tracts of the mature brain. a comparison of early brain development in drosophila and in vertebrates reveals common mechanisms for brain development. supported by the swiss nsf. institute ot histology, university of fribourg, p~rolles, ch-1700 fribourg calretinin (cr), an ef-hand ca2+-binding protein, is expressed in cajai-retzlus cells during development of the rat cerebral cortex. these cells are located in the marginal zone and are the ear{iest cortical neurons to be generated. they have been consldered as unusual on account of their morphology and their fate during corticogenesis. the functional significance of cajai-retzius cells and their destiny in adulthood is still controversial. in order to approach these questions we have applied organotypic culturing methods. slices of neocortex from brains of 0-6 days-old rat pups were cultured for 10-21 days applying the interphase technique (stoppini etal, j. neurosci. methods 37, 1991) or the roller-tube technique (g&hwiler, j. neurosci. methods 4, i981) . the fixed cultures were immunolabe0ed with an antibody against cr. the obtained results showed that cr positive structures develop in vitro like in vivo. however, in organotyp[c cultures their distribution corresponded to a more mature stage than the situation on the day of the explantations. many cr-positive cells were seen in layer l they were horizontally oriented or had a pyriform shape. based on their morphology these ceils were identified as cajai-retzius cells. orpositive cells were numerous in layer i1-l[i and showed mostly a bipolar morphology. some muripo[ar cells could also be observed. a fine net of crpositive fibers and puncta was found in layers [ and iv. the demonstration that cajai-retzius celts are detectable in these cultures will allow us to follow the fate of these cells dynamically and [nte~ene in their function by applying exogenous factors. cervical primary afferent input to vestibule-spinal neurones projecting to the dorsal horn: a double labelling study in the rat. wheat germ agglutinin-horseradish peroxidase (wga-hrp) was injected by pressure into cervical spinal ganglia c2 or c3. in the same experiments fluorogold (fg) was iontophorefically injected into the ipsilateral dorsal horn of different cervical spinal cord segments. anterogradely labelled fibers (wga-hrp) were present mainly in the external cuneate nucleus, but also to a lesser extent in the caudal part of the medial and descending vestibular nuclei (mvn, dvn) . the fg injections, restricted to the cervical dorsal horn, revealed retrogradely labelled cells in the central part of the caudal mvn. a double exposure (fluorescent and polarisadon optics) under the microscope showed primary afferent fibers surrounding like baskets retrogradely labelled fg cells. the projection from cervical ganglia cells to the mvn represents a pathway for direct afferent information from neck receptors (in particular muscles) to vestibular nuclei and supplies the mvn with afferent information which could enable the vestibulospinal nenrones, projecting to the dorsal horn, to influence the local information processing. in the sciatic nerve, all myelinated and non-myelinated axons were snap-ir. in peripheral tissues, all nerve endings were positive. the preterminal schwann cells of motor axons were also snap-25-ir. after sciatic nerve section, many myelinating sehwann cells also expressed snap-25-ir. in conclusion, snap-25 is expressed by neurons in the pns. it seems to be also expressed by preterminal schwann cells and by myelinating schwann cells during regeneration. $17-19 repair of completely transected spinal cord of neonatal opossum in culture h.a. vischer, dept. pharmacology, 8iocenter, uni. of basel, switzerland woodward et al. (j. exp. biol. 1993 , 1993 have shown that fibers grow rapidly and profusely across a lesion made by crushing the spinal cord of the neonatal opossum monodelphis domestica. in such experiments careful controls must be made to establish that fibers were in fact broken by the lesion. tests have now been made to determine whether similar repair can occur after a more drastic lesion involving complete transection of the cord. after dissection of the entire gns from animals aged 3-8 days, the cord was cut into two with scissors at the c5 -c7 level. the two-ends were glued together with matrigel on a sylgard mold, which encompassed and held the cns. the rostral end was labeled with the fluorescent carbocyanine dye dil in order to visualize growing fibers. in 33 preparations fibers grew over the cut into the separated part of the spinal cord. such growth could extend to 500 ixm. fibers grew straight, branched and multidirectionally, or in fascicles. in another set of experiments cords were combined from different animals of the same or different ages. again, fibers could grow across the cut but they did so less frequently. these results set the stage for investigating fiber growth after rotating the two ends at a defined angle and for analyzing factors that influence the direction of growth. map la is a microtubule associated protein of 360 kd. in rat brain two monoclonal antibodies, a and bw6, recognized map la in neurons and their axonal and dendritic processes. in mouse cerebellum, monoclonal a recognized purkinje ceils and granule cells uniformly, as already described for rat brain. in contrast, monoclonal bw6 stained selectively some dendrites of purkinje cells, forming bands in the molecular layer. we compared this striation with that obtained with a monoclonal antibody, anti-zebrin ii, which recognized aldolase c. in the mouse vermis, zebrin stained in a striated pattern, but complementary to bw6. these results demonstrate that map la is present in forms which are differentially distributed in the mouse cerebellum. these observations may be explained either by differences in metabolic states of neurons, or by differences in their regional functions, or by differences in the regional stability of microtubules. this work was supported by grant no 31-33447.92 from the swiss national science foundation. serum free aggregating brain cell cultures prepared from 16 day old fetal rat telencephalon are used as a model to study brain development. in these cultures it is possible to distinguish ontogenetic events such as cell proliferation, differentiation and myelination, in the present study we examined synaptogenesis by analyzing the expression of synaptic proteins such as synaptophysin, snap-25, synapsin i, and brain spectrin. different developmental stages were analyzed by western blotting. immunoreactivity for synaptic proteins was detectable already at day 12 in culture, suggesting that the first synapses are already present at this early stage. the expression of synaptic proteins strongly increased between day 20 and day 30 in culture, reflecting a period of intense synaptogenesis. treatment of the culture with an elevated concentration of kci (30mm) increased the expression of synaptic proteins, suggesting a stimulation of synaptogenesis. these findings demonstrate the utility of this approach to study brain synaptogenesis, and possibly synaptic plasticity. drenhaus, u.i gunten, a.v., rager, g. ; institute of anatomy, university of ch-1700 freiburg the retina of tupaia comprises three types of ganglion cells (rgcs) analogous to x-, y-, and w-cells found in other mammals. these cells differ in size and in their distribution pattern: large rgcs are frequent in temporal, small rgcs in nasal retina. as the fiber diameter correlates with the size of its respective rgc, it can serve as a parameter for the topographic representation of rgcs in the nerve. to study this we analyzed the optic nerves of three tupaia. using the electron microscope we recorded the density, diameter, and the position of fibers in the nerves. we found, that fibers with the largest diameter are located dorso-temporally and close to the center of the nerve. they are surrounded by zones of smaller fibers. the diameter decreases gradually towards the periphery and is smallest in the ventro-nasal region of the nerve. since the fiber diameter distribution corresponds to that of the size of rgc-types in the retina, we assume that the topographic relationships in the nerve and the retina are similar. (supported by s 17-23 stoppini luc, s. duport, l. parisl, c. oropes~, departement of pharmacology, cmu, 1211 geneva 4 the micro environment of the central nervous system is important for neuronal function. in this context, the blood-brain (bbb) provides and maintains the extracellular medium that is compatible with normal neuronal and synaptic activity. due to the difficulties inherent using whole animal as an experimental model to study permeability and metabolic processes at the cellular level, major efforts have been engaged in recent years, in order to bring up suitable /n vitro models. we are presently developping an in vitro bbb by interfacing a coculture of endothelial cells monolayem and nervous organotypic cultures. the main idea of this study is based on the premise that the complex intercellular interactions between the different cell types pertaining to the nervous tissue and the neighbeurlng endothelial cell is of fundamental importance to promote a realistic bbb system. we expect that erganotypic culture of nervous tissue, combined to endothelial cell monolayers, will initiate and maintain a bbb phenomena/n vilro. we will test more specifically the hypothesis that neuronal activities [spontaneous or elicited) will influence gila] cell response which will in turn modify endothelial properties. preliminary results clearely show a good nervous tissue and endothelial cell survival. tight junction llke structures could be identified using freezefracture or conventional transmission electron microscopic thechniques. {work supported by chemodyne gent~ve ). the process of myelination is a prerequisite for the proper function of the brain since it enables rapid saltatory conduction of axons. in the central nervous system (cns) myelination is performed by oligodendrocytes. a differential screening approach was used to isolate new rat cdna clones that are expressed in this glial cell type. here we report the further characterization of two of these novel cdnas which appear to be expressed specifically in oligodendrocytes. the two characterized cdna clones (tentatively called cns1 and cop-25.1) share an approximately 420 bp region of sequence identity which suggests that they are dedved from the same gene by alternative splicing, within this area a putative open reading frame is located. we demonstrated by northern blot analysis and in situ hybridization that the two mrnas of the novel gene are expressed exclusively in oligodendrocytes. however, the mrnas show a different specific spatial localization within the cell. we compared mrna expression of myelin basic protein (mbp) and proteolipid protein (plp), with that of cns1 and cop-25.1 in brain and optic nerve during development. it appeared that the mrnas of the novel gene were delayed significantly as compared to mbp and plp mrnas. streit. phyaiologisches institut, btihlplatz 5, 3012 bern in organotypic slice cultures of the embryonic spinal cord, rhythmic spontaneous activity arises when the inhibitory synaptie transmission is blocked. the rhythmic activity consists of bursts of regular oscillations of activity at 4 -5 hz. this study investigates the origin of the regular oscillations. when stimulated electrically at 5hz, the synaptic transmission in the cultures showed strong depression by about 50% on average. the synaptie depression was highly variable among individual connections and tended do be less pronounced in frequently used connections. when random depression ratios with an average of 50% at 5hz were implemented in a computer model consisting of 100 randomly connected excitatory cells, regular oscillations of activity did not arise, even in the presence of synchronous pacemaker cells. however, when individual depression ratios were adapted according to the rate of activity of individual connections, regular oscillations at 4 -5 hz arose in the presence of few unsynchronized pacemakers. this finding suggests that the regular oscillations in the cultures originate in a network formed by the plasticity of synaptic depression. maier a., schlumpf m., beer h.f., sehubiger p.a. and lichtensteiger w., inst. of pharmacology, univ. of zurich, the ontogeny of expression of dopamine d 1 receptor mrna in the rat brain and binding to the receptor was studied at 12 time points from gestational day (gd) 13 to postnatal day (pn) 60 by quantitative receptor autoradiography and in situ hybridization.long evans rat pups and fetuses of time pregnant rats were frozen and sectioned coronally and sagitally at i0 um. d 1 recepto~o~inding , determined with the selective antagonist ~ji-sch 23982 was first noted on gd 18 in the developing striatum, basal ganglia and the olfactory tubercle, reaching adult levels at around pn 14. the expression of d i receptor ~na was studied by using a mixture of 3-~js-labelled oligonu-specific cleotides. on gd 13 messages were noted in the developing stiatum, olfactory tubercle and retina. this study shows a good regional correlation between the development of receptor binding and mrna, however, mrna precedes detectable binding to the receptor by several days. earlier work had shown that q~ replicase forms complexes with q~, plus strand rna via interactions at two internal binding sites, the s-and the m-site, while binding of the 3'-end is mediated by host factor. in contrast, the 3'-end of the minus strand appeared to be directly accessible by replicase. we have prepared a series of plus and minus strand variant rnas containing either internal deletions or terminal deletions extending from the 5'-end. the template activities of the variants were determined by single-round replication assays. for the plus strand we find that while deletion of the s-site remains without effect (as expected from previous results), deletion of the m-site reduces template activity to 25 % or less compared to wild-type rna; the residual activity shows a decreased dependence on the presence of host factor. the results agree with an important role of the m-site interaction both with replicase and with host factor for the formation of productive initiation complexes. the template activity of the minus strand was unexpectedly found to be strongly dependent on the presence of a segment between nt 76 and 210 from the 5'-end. this shows that recognition of the minus strand by rep(icase does not only involve interactions near the 3'-end but requires a previously unknown structural feature near the 5'-end of this template. $18-02 karsten graning and angela kr&mer d~partement de biologie cellulaire, universite de gen~ve, 30, quai emest-ansermet, 1211 gen~ve 4 splicing of nuclear pre-mrnas takes place within multicomponent complexes (spliceosomes) that are assembled by interactions between the pre-mrna, snrnps and protein splicing factors. we have purified two splicing factors that function in the formation of the pre-splicing complex. sf3a consist of three subunits of 60, 66 and 120 kda and corresponds to three proteins present in the functional 17s but not in the 12s u2 snrnp. it binds to the 12s u2 snrnp only in the presence of sf3b, suggesting a two step assembly pathway of 17s u2snrnp: binding of sf3b to the 12s u2 snrnp generates a 15s intermediate particle that is converted to the active 17s u2 snrnp by the subsequent association of sf3a. comparison of proteins enriched in the sf3b fraction with polypeptides found in the purified 15s u2 snrnp suggests that sf3b comprises at least five of the other 17s u2 snrnp specific proteins and together with sf3a promotes the u2 snrnp/pre-mrna interaction. by edna cloning, two of the subunits of sf3a have been identified as homologs of well characterized yeast proteins that function at the same stage of spliceosome assembly in yeast. splicing factor sf1, a heat-stable 75-kd protein, functions early during spliceosome assembly. tryptic peptides of sf1 were sequenced and cdna libraries were screened with degenerate oligonucleotides. the information obtained by comparing the sequences of three overlapping cdnas suggests the existence of at least three mrnas that could be derived from a common pre-mrna by alternative splicing. in agreement with this assumption mrnas of the expected sizes that are differentially expressed depending on cell line or tissue are detected by northern blotting. in theory, three polypeptides could be generated that differ by the presence or absence of 7 internal amino acids and in their ctermini. the deduced amino acid sequence is rich in prolines and contains several possible phosphorylation sites and a putative leucine zipper. proline-rich domains, which are also present in splicing factors psf and sap62, as well as leucine zippers have been found in transcription factors and could mediate protein/protein contacts, suggesting that sf1 could function during splicing by interaction with other spliceesomal proteins. pre-mrna splicing is catalyzed by a multicomponent complex (the spliceosome) that consists of small nuclear ribonucleoprotein particles (snrnps) and non-snrnp protein factors. the spliceosome is assembled in a stepwise fashion on the pre-mrna. chromatographic fractionation of hela cell nuclear extracts and subsequent reconstitution of splicing in vitro has allowed the separation and isolation of snrnps and several protein factors. sf4 triggers the transition between splicing complex b, which contains all spliceosomal snrnps but is not competent for catalysis, and complex c, the active spliceosome. this transition involves a conformational change that leads to altered base pairing interactions between snrnas and pre-mrna. the purification of sf4 has been impeded by its low abundance; however, a correlation between sf4 activity and a polypeptide of 50 kd could be established in the most purified fractions. we are currently investigating whether this protein represents sf4. interestingly an atp-dependent rna-helicase cofractionates with sf4 through several purification steps. whether or not this activity is relevant for the splicing process is under investigation. selection of iron-responsive element rnas with high affinity for iron regulatory factor henderson, b.r., menotti, e., bonnard, c. , and kith_n, l.c., isrec, ch-i066 epalinges iron regulatory factor (irf) post-transcriptionally regulates iron homeostasis via binding to mrna iron-responsive elements (ires). the ire loop sequence (5'-cagugn-3') and "bulge" cytosine are phylogenetically conserved. we prepared a pool of 16,384 ire molecules randomized at these 7 nucleotide positions, and employed in vitro selection to identify optimal sequences which bind human irf. we define two major classes of high affinity rna ligand, the optimal loop sequences of each are 5'-cagugn-3' (wild-type) and 5'-uaguan-3'. this novel finding predicts base-pairing within the loop between positions i and 5. all nucleotide substitutions in the "bulge" or at loop positions 2,3 and 4 decreased binding by 36% to 99%. in addition, binding specificity of rat irf differed with that of the related rat ire-binding protein, irf b. in vitro analysis of ire-like stem-loops identified by computer search has not yet revealed any new ire-containing genes. a73 $18-08 3' processing of histone pre-mrnas: a phylogenetic comparison reto kohler, petra duda and daniel schiimperli. zoologisehes institut der universtit/it bern, abteihing fiir entwicldungsbiologie, baltzerstr. 4, ch-3012 bern, switzerland. we are using a phylogenetic approach to study the biochemical reaction by which animal histone pm-mrnas are processed at their 3' ends. in mammals, the efficiency and specificity of this reaction is known to depend absolutely on the u7 snrna which interacts with conserved spacer sequences downstream of the proc~sing site. a highly conserved hairpin element which precedes the cleavage site serves as a binding site for an additional processing factor, but its importanea for efficient processing varies greatly between different historic genes and between extracts of different mammalian cell lines. to determine whether the relative importance of the hairpin and spacer dements have been conserved during evolution, we analysed the processing of histone genes from three different animal phyla in vitro, i.e. vertebrates (mouse), arthropods (drosophila melanogaster) and nematodes (c. elegans). in the mouse system, processing was strongly dependent on the presence of a mouse spacer element. in nuclear extracts of drosophila ke cells, processing occurred exclusively when a drosophila spacer element was present in the rna. processing efficiency was not reduced by foreign (mouse, c. elegans) hairpins. in whole cell extracts of ascaris lumbricoides embryos, an inverted situation was observed. 3' processing products were only produced by rnas that included an upstream segment derived from a c. elegans histone gene, irrespective of the spacer sequences present. these surprising results correlate with certain unique features in the c. elegans upstream element. we are currently in the process of cloning ascaris lumbricoides histone genes, which will allow us to verify our results in a homologous system. in xenopus laevls ooeytes, wild type mouse u7 rna gets assembled into functional u7 snrnps, both when transcr~ed from an injected gene or when injected as/n vitro synthesized rna. if the sm binding site of u7 rna is converted into the canonical sm sequence (u7 sm opt), the rna assembles into a particle which accumulates more efficiently in the nucleus but wbach is nonfunctional. this u7 sm off particle inhibits the function of preformed endogenous u7 snrnps, most likely by nonproductive binding to histone pre-mrnas, histone pre-mrna processing can be demonstrated in c/s if u7 rna is placed 3' of hlstone pre-mrna and injected into oocytes. only u7 sm wt sequences are active in this c/s processing. three proteins can be photoaffinity cross]inked to u7 sm wt rna, the common snrnp proteins g and b/b' and a u7-specific protein of 40 kd (50 kd in mouse). these crosstinks map to closely spaced positions within the sm binding site with the u7-specific crosslink being located most 3'. u7 sm opt rna does not become crosslinked to the u7specific protein and is also more tightly associated with sm proteins. we now investigate the in vitro assembly of u7 snrnps using these characterized u7 rnas and a preparation of highly enriched snrnp proteins. u7 sm wt and u7 sm opt rnas associate with common sm proteins in this system, but the interaction with the u7-speciflc protein could not be demonstrated. the double-stranded (ds)rna modifying enzyme, which can convert adenine to inosine, was first identified in xenopus laevis, but has since then been detected in different species and in mammalian cells. the enzyme is a specific adenine deaminase which uses dsrna as substrate and recently, it has been postulated to be responsible for a specific mrna editing reaction. we have purified this enzyme to homogeneity, approximately 400,000 fold from calf thymus whole cell extracts. the protein was purified primarily by ion exchange chromatography over six columns, with the final step being chromatography on a dsrna affinity column. the purified protein has a molecular weight of 115 kd and is the only protein present when enzymatically active fractions are visualized on an sds-polyacrylamide gel stained with silver. the dsrna modifying enzyme is a very low abundant protein. we are in the process of further characterizing the purified enzyme and of cloning cdnas coding for it. detailed mutational analysis of histone rna 3' end formation carmen spycher, andr6 furger, adrian streit, daniel albrecht, d. schiimperli, zoologlsehes iustitut der universtit/it bern, abteilung thr entwicklungsbiologie. baltzerstr. 4, ch-3012 bern, switzerland histone rna 3' ends are formed by cndonucleolytic cleavage resulting in poly(a)" mrnas with a highly conserved 3'-terminal hairpin loop structure. for the mouse h4-12 gene major and minor processing sites are located 3 and 5 nucleotides downstream of the hairpin, respectively. for the cleavage reaction, the u7 snrnp interacts with the spacer element of histone pre-mrna by base-pairing through the 5' end of its rna moiety, u7 rna. we have observed that this base-pairing potential extends further than previously recognised in either direction. we have made site-directed rnutagenesis in fltis potential hybrid region and around the processing site. rna processing and complex formation with the u7 surnp were analysed by incubation in nuclear extract from mouse k 21 cells. our results indicate that base-pairing with u7 rna both in the classical spacer element and downstream of it are very important for histone rna processing. in contrast, sequences upstream of the spacer element do not seem to be involved in base-pairing with u7 rna, but mutations in this region may affect processing by other mechanisms. systematic mutations of the highly conserved four nucleofides immediately following the hairpin show no qualitative or quantitative effects in the processing reaction. alteration of nucleotides 5 to 7 around the minor processing site, however, result in a dramatic decrease in processing efficiency and alter the specificity of the cleavage site. in further experiments we will introduce specific nuclcetidc modifications at the two processing sites, which should allow us to characterize potential cleavage factors by chemical crosslinking. evolution callaerts p., glardon s., j.,oosli f., quiring r. and gehring w. cell biology, biozentmm, basel. the pax genes encode a family of dna binding factors which share the paired-box and play an important role in the control of development. they can be subdivided into four different classes which differ in the presence or absence of other conserved sequence motifs coding for a paired type homeobox or an octapeptide. the pax-6 gene, which contains a paired-box and a homeobox, has been cloned from humans, mice, rats and zebra fish. recently, we have cloned the pax-6 homologue of drosophila melanogaster. the five genes share a very high degree of sequence identity both in the paired-domain and the homeodomain. similar to its mammalian counterparts, the drosophila gene is expressed in the eye primordia and the nervous system. in addition, mutations in pax-6 affect eye development: aniridia in humans, small eye in mouse and rat, and probably eyeless in drosophila. this would imply that the pax-6 homologues share a conserved function in eye morphogeuesis in both vertebrates and invertebrates. furthermore, this suggests that pax-6 may have been present in a common ancestor. therefore, we are now trying to isolate pax-6 homologues from other, more primitive organisms by means of the polymerase chain reaction, and to determine whether they are implicated in eye development. putative candidates have been identified from a number of species and are being characterized. their sequence similarity and some evolutionary implications will be discussed. keegan liam and gehrin~ walter j., biozentrum, university of basel, klingelbergstr. 70, ch-4056 basel, switzerland using an enhancer map screen we have identified two genes that may be direct targets of antennapedia involved in forming the adult leg. spalt major is repressed in the leg imaginal disc and tea, shirt is activated by antennapedia. we wish to determine whether the control of these genes by antennapedia is direct, spalt major is expressed in a ring in the antennal disc and is repressed by antennapedia in the leg disc. the antennal ring is repressed by ectopie antennapedia expression that transforms the antenna to a leg. we are defining an enhancer at spalt major that is required for antennal ring expression and repression by antennapedia. we are using various approaches to show that antennapedia binds to the antennal ring enhancer at spalt major. these include polytene chromosome banding studies using antibodies to antennapedia as well as in vitro dna-binding and genetic experiments. in search for novel regulators potentially involved in myogenesis, we identified a homeobox of the paired type in human muscle. subsequent cdna cloning revealed that we had cloned the full length coding region of human pax7. our human sequence extends the known mouse edna both on the 5' and the 3' end. as a first step in order to test for a functional role of pax7 in myogenesis, we analyzed its expression pattern during chicken development. transcripts are present in the neural tube and in the dermamyotome of the developing somites. therefore, the pattern of expression of pax7 is conserved between mouse and chicken. next, we assessed the expression of pax7 in different mouse and human cell lines. we found pax7 transcripts specifically in myogenic cells and not in any other cell types. interestingly, pax7 is already present at the myoblast stage. moreover, 10ti/2 fibroblasts converted to myoblasts by either transfection of myod or treatment with 5-azacytidine expressed pax7, whereas the parental cells did not. while myod was able to activate pax7 in 10t1/2 cells, expression of pax7 was not sufficient to induce the myogenic phenotype. nevertheless, our expression data are consistent with a role of pax7 during the mesodermal commitment to the myogenic lineage. the segmental organization of the drosophila head is achieved by a flow of positional information from maternal gene products to the zygotic gap genes. recent genetic analysis has identified three new gap genes involved in head development. one of these genes is the empty spiracles (ems) gene. mutations in eras cause severe defects in the head and the filzk~rper at the posterior end are missing. the eros gene encodes a putative transcription factor with a homeodomain. the eros rna is expressed in two phases of embryonic development. first expression is seen at the blastoderm stage in a single anterior band, correlating with its function as an anterior gap gene. later during embryogenesis eros is expressed in the posterior spiracles as well as lateral regions of each segment where the tracheal pits form and lateral neuroblasts originate. using g-gal fusions we could identify at least five different regulatory elements in the eros promotor region responsible for tissue specific expression of the gene. a 300 bp element responsible for the early expression which is dependent on the maternal gene bicoid, the key gene of the anterior system, and the terminal gene tailless was identified and studied in detail. this element contains two bicoid and two tailless binding sites. the effects of muations in these binding sites on eros expression will be discussed.this analysis should allow us to elucidate the molecular mechanisms by which the morphogen bicoid regulates subordinate target genes like ems in the drosophila embryo. the pax-6 gene of the mouse encodes a transcription factor with both a paired-and a homeodomain. pax-6 is affected by several allelic mutations in the small eye (sey) gene. sey mutations cause a reduction of the eyes in heterozygotes, and homozygotes lack both eyes and nasal openings and die as newborns. the corresponding mutation aniridia in humans affects eye and cranlofacial development in a similar manner. we have isolated a pax-6 homolog from drosophila (dpax-6), which shares more than 90% sequence identity in the paired-and the homeodomain with the mammalian genes. also outside of these domains we find considerable sequence conservation arguing for a true pax-6 orthologue. the drosophila gene spans ~16 kb and is expressed in the brain and the ventral nervous system of the embryo, and in the eye imagjnal discs and the brain of the larva. the gene was mapped to the eyeless (ey) locus. mutations at the ey locus cause either the partial or complete absence of the compound eyes or embryonic lethality. in one ey allele we have identified a transposable element in the first intron of dpax-6, which affects dpax-6 transcription in the eye discs, suggesting that dpax-6 is encoded by the ey gene. this implies that pax-6 (sey) in the mouse is homologous to ey in drosophila. thus, despite of the different modes of eye morphogenesis, the same gene seems to control eye development in insects and vertebrates. the drosophila homolog of the vertebrate serum response factor (srf) was isolated by low stringency-hybridization. nucleotide sequence analysis revealed that the drosophila srf homolog (dsrf) codes for a protein which displays 93% of sequence identity with human srf in the mads domain, the region required for dna binding, dimerization and interaction with accessory factors. the dsrf gene is expressed during several phases of embryonic development. both the rna and the protein are maternally provided to the egg and slowly decrease in their levels during gastrulation. after germ band retraction, high levels of zygotic expression were observed in a distinct subset of peripheral tracheal cells distributed throughout the embryo. many of these cells are at the tip of tracheal branches and are in direct contact with the target tissues these branches tracheate. the dsrf gene was mapped to position 60c on the second chromosome, and overlapping deficiencies which remove the gene were identified. analysis of tracheal development in embryos carrying these deletions revealed a degeneration of most of the major branches of the tracheal system. although the initial migration of tracheal cells was not affected in those deficiency embryos, many tracheal cells appeared not to maintain their formerly correct position and continued to migrate. thus, the dsrf gene might play a role in the proper formation and maintenance of the major branches of the trachea. s 19-08 our experiments would be expected to provide insight into such problems as the evolution and function of transcription factors with multiple dna binding domains. ideally, we would be able to mimic "gene splitting" which occurred during evolution. the poan gene plays an important role during drosophila neurogenesis. it is expressed in specific subsets of sensory mother ceils (smcs) and neuroblasts. the smcs that express poxn differentiate into polyinnervated external sense (p-es) organs. in the absence ofpoxn, smcs differentiate into mono-innervated rather than poly-innervated external sense organs. as the poxn gene contains a paired-box, it probably encodes a transcription factor. since the function ofpoxn in the central nervous system is not known and no po~n mutants are available, an ems mutagenesis screen was initiated to isolate such mutants. based on the assumption thatpoxn null alleles are lethal, we screened for lethals uncovered by the deficiency df(2r)wmg, which includes poxn, but survive over the deficiencies df(2r)xte-18 and df(2r)kl-32 flankingpoxn. in a further test, such lethals are screened for the absence of embryonic p-es organs. ifpoxn mutants are not lethal, they will escape this screen. therefore, we are inducing local hopping of p-elements that have inserted in the vicinity of poxn. the offspring of flies that display an altered eye phenotype are screened for p-element insertion into poxn. we hope that these approaches will generate mutants that will be crucial for future studies ofpoxn function in neurogenesis. chromatin structures and transcription of rdna in yeast saccharomyces eerevisiae reinhard dammaun, renzo lucchini, thee keller and jest m. sogo; institute of cell biology, eth-honggerberg, ch-8093 ziirich the chromatin structure of yeast ribosomal dna was analyzed in rive by cross-linking intact cells with psoralen. we found that in exponentially growing cultures the regions coding for the 35s rrna precursor fall into two distinct classes. one class was highly accessible to psoralen and associated with nascent rnas, characteristic for transcriptionally active rrna genes devoid of nucleosomes, whereas the other class showed a cross-linking pattern indistinguishable from that of bulk chromatin and was interpreted to represent the inactive rrna gene copies. by cross-linking the same strain growing in complex or minimal medium, we have shown that yeast ceils can modulate the proportion of active (non-nueleosomal) and inactive (nucleosomal) rrna gene copies in response to variations in environmental conditions which suggests that yeast can regulate rrna synthesis by varying the number of active gene copies, in contrast to the vertebrate ceils studied so far. whereas intergenie spacers flanking inactive rrna gene copies are packaged in a regular uucleosomal array, spacers flanking active genes show an unusual cross-linking pattern suggesting a complex interaction of regulatory factors and histories with dna. $20-04 r. felleisen & b. gottstein; institute of parasitology, university bern, bern, switzerland most patients suffering from alveolar echinococcosis (ae) respond to infection with a marked igg synthesis directed against e.multilocularis metacestode antigen ii/3, which represents a novel member of the family of cytovillin related proteins. although the respective gene basically is also present and expressed in e.granulosus, most of the cystic echinococcosis (ce) patients do not recognize the antigen. this phenomenon was tackled by generating cdna derived from full length ii/3 genes from both echinococcus species, performed by rt-pcr. sequence analysis revealed a very high degree of conservation of the primary sequence (98.6% homology), cdna fragments were expressed as recombinant proteins and were comparatively assessed in elisa respective to antibody-binding characteristics. sera from patients suffering from ce were showing no significant differences in reactivity with the antigens derived from both species. therefore, parameters others than minor differences in the primary sequence seem to be responsible for the lack of antigen recognition with respect to ce. $20-02 patrick rigoni, sandro rusconi, institut f molekularbiologie h der universitat zarich, winterthurerstr. 190, 8057 z~rich, switzerland. trinuclcotide repeats are often found in the coding sequence of transcriptional regulators in both mammals and yeast, however, their function is yet unclear and data collected in our laboratory on the gheocorticoid receptor even suggest that they may not have any, and that their presence is probably the result of a selfish replicative behavior. nonetheless, we believe that these motifs can be used as tags to identify flexible protein domains typical of modular proteins such as transcription factors. another kind or repeat (coding for a poly-glutamine/alanine) has been discovered in the yeast regulators gall1 and ssn6. from northern blots, we know that such caggcn repeats are present also in higher eukaryotes and we want to isolate them by constructing an enriched edna library using a 5' race protocol. so far, no mammalian protein bearing the motif glrdala has been characterized. among the positive clones we hope to find the homologous or paralogous of gall1 and ssn6 that have escaped so far conventional cloning techniques. meanwhile, we have been testing the effect of coexpressed yeast gall 1 on different reporter-activator combinations in mammalian cells. preliminary results show that galll but not the mutant form galllp can inhibit the activation properties of some (not all) gal4 chimeras. we are also producing antibodies directed against oligo-ala and oligo-gln and oligo-gln/ala motifs in order to better characterize natural proteins containing these repeats. we are interested in the early development of c. elegans, particularly in the contribution of genes whose homologues in vertebrates and d. melanogaster mediate positional infolzzations during development. therefore, we are working on ceh-13 which belongs to a cluster of at least 4 homeobox containing genes. this cluster is considered to be equivalent to the homeotie cluster of drosophila and to the hox clusters in vertebrates. by generating ~-galactosidase transgenie lines, we have observed that ceh-13 is expressed very early during embryogenesis, embryonic expression appears to be restricted to the posterior half of the embryo, which is rather surprising, since the ceh-13 hemologues in drosophila and vertebrates are anterior-specific. during larval stages, ceh-13 is expressed in neuronal cells. these results are now in the process of being confirmed by immunolocalization of the ceh-13 protein product with a polyclonal antiserum. in order to clarify the function of ceh-13 in the c. elegans development, we have isolated a ceh-13 mutant from a tcl insertion library, in collaboration with r. h. a. plasterk from amsterdam. from this worm, which looks wt, we are now trying to obtain a deletion derivative. previous studies have shown that bovine herpesvirus 1 (shv-1) infected cell pratein (bicp) 0 acts -depending on the promoter -as a strong transactivator or as a repressor in transient expression assays. the 81cp0 polypeptide contains a cysteine-rich zinc finger domain (c3hc4) which is conserved in a number of viral and cellular regulatory proteins including the 8icp0 homologs icr3 of herpes simplex virus type 1 and protein 61 of varicella zoster virus. this type of zinc finger (the so-called ring tinge0 was shown to bind zinc ions but functional requirement for zinc has not yet been demonstrated, a gap which we aimed to fill with the present study. transient expression assays were performed in oocytes which had been microinjected with thionein to chelate and deplete the intracellular pool of zinc. 81cp0-induced cat activity of a promoter-cat construct was 72-fold higher than the basal cat activity of the reporter plasmid, in the presence of thionein, bicp0-induced transaotivatlon was reduced 54old. with a set of control experiments we excluded that thionein might affect transcription and protein synthesis in general, to our knowledge this is the first demonstration of zinc-dependence for a member of the ring finger family. we have identified by pcr and edna cloning five pou genes expressed during early embryogenesis of zebrafislx four of these genes show extended homology to the brn-1 class of pou genes. preliminary evidence suggests that the brn-1like pou genes overlap with most of their expression domains.the gene studied in most detail so far (zp-50) is first expressed on the ventral side of the future fore-and midbrain. slightly later, expression is also found in rhombomeres 3 and 5 in the hindbraln. these rbombomeres were identified by the specific expression of the krox-20 marker using a novel in situ hybridization protocol which allows the simultaneous detection of two different transcripts using different color substrates. in the 24 hours old embryo a complex expression pattern is found involving a variety of brain structures and the spinal cord. the distribution of the transcript suggests that zp-50 is mostly expressed in glia cells. another pou gene we have identified (gp-9) defines a novel class of pou proteins. as a maternal mp, na it is initially ubiquitously present. after gastrnlation its transcript is found most notably in the developing hindbrain in rhombomeres 2 and 4 for which so far no good molecular markers were known. we are investigating the roles the identified pou genes may have in zebrafish hindbrain segmentation. we are currently attempting to manipulate gene expression in developing embryos by injection of rna or by the production of transgenic fish. we are also screening the zebrafish genome for new developmental marker genes. progress about these experiments will be presented. $20-07 willimann, t. and trueb, b. to gain insight into the regulatory mechanisms of collagen vi synthesis we have characterized the cis-acting elements of the chicken ctl(vl) collagen promoter. footprinting experiments with nuclear extracts from chicken embryos revealed three distinct elements, designated a, b and c, that were protected from dnase i digestion. the nuclear proteins that interact with the three sites were identified by gel retardation assays in combination with the use of various oligonucleotide competitiors as well as specific antibodies raised against well-characterized transcription factors. site a was found to be a target for transcriptional activator apf, whereas sites b and c were shown to be recognized each by two distinct nuclear proteins which belong to the spl multigene family. to address the question whether the three sites alone are able to direct transcription, a minipromoter construct was created in which the sequences of sites a, b and c were placed in front of a reporter gene. after transfection into chicken fibroblasts, this construct exhibited a high relative promoter activity when compared to a large genomic fragment containing the basic ctl(vi) collagen promoter. thus, the three sites are sufficient to induce transcription of this gene. octamer transcription factors (oct or otf) are a subset of the pou family of transcription factors which regulate expression of cellular and viral genes by binding to the octamer sequence atgcaaat. neurons and astroglial cells harbour, in addition to the ubiquitous oct-i factor, brain specific factors termed n-oct 2, 3, 4 and 5. in the present study we determined the chromosomal localization of the gene encoding the n-oct 3 transcription factor and characterized the structure of isolated n-oct 3 genomie clones. the chromosomal mapping was performed by analysis of somatic cell hybrid panels and radioactive and fluorescence in situ hybridization of human metaphase chromosomes. it is interesting that the 5' end of the n-oct 3 coding sequence contains repetitive cag and ggc residues. several disorders have been discovered to be related to expansion of trinucleotide repeats. we are presently investigating if the n-oct 3 cag or ggc clusters are hot spots for such mutation mechanisms and if so, which diseases could be associated with it. two dna regions upstream of the distal glucocorticoid receptor binding site interact with nuclear proteins that are tissue-specific (region a) or ubiquitous (region c). the characteristics of the dna-protein interactions have been studied in vitro. these sequences, in different combinations with the natural mmtv promoter, were tested in transfection assays of fibrcblast or mammary cell lines. we show that they are able to modulate the level of glucocorticoid-stimulated transcription. the proximal region of the mmtv promoter has binding sites for the transcription factors ctf/nf-i and oct-1. p~asmids with a deletion or mutations in the oct-1 site were tested in stable transfections, that are most representative of the state of proviral dna with respect to both number of integrated dna templates and chromatin organization. we show that the oct-1 site is important for the basal level of promoter activity. the data further indicate that the functional outcome may depend beth on the relative ratio of factors to dna templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting. besides the fact that telomeres represent specialised structures important for chromosome stability, the particular significance of cloned c. elegans telomeres is that they will contribute to the completion of the physical mapping of chromosomes and that they provide one set of elements for the construction of artificial c. elegans chromosomes. the cloning of c. elegans telomeres, however, is impeded by the fact that tandem repeats of the sequence ttaggc are not only located at the ends of the c. elegans chromosomes, but also at many internal chromosomal sites. therefore, we have developed a special protocol for the construction of a c. elegans endlibrary in 2~ zap. the resulting telomeric library was screened for recombinants containing ttaggc repeats and yielded about 70 positive clones. so far, 30 were analysed by partial sequencing and twelve of them satisfy all criteria required for telomeric clones. their ttaggc tandem repeats are located at the expected position, namely just adjacent to the blunt end cloning site in the polylinker. furthermore, the g-rich strand of the repeats is oriented 5' to 3' towards the end of the fragment, thus corresponding exactly to the defined orientation of eukaryotic telomeric sequences. finally, hybridisation data with one of these putative telomeric clones show that a subtelomeric fragment hybridises to bal31-sensitive fragments on a southern blot with c. elegans dna, indicating that this clone represents a c. elegans telomere. $20-09 to investigate whether chromatin structure or transcription can interfere with replication, derivatives of the yeast trp1ars1 minichromosome were constructed that contain either the ded1 or pet56 promoter firing against the origin of replication ars1. while the pet56 constructs transformed yeast at high frequencies and were maintained as high copy number minichromosomes, the ded1 constructs transformed at low frequency and the constructs were integrated in the genome suggesting that ars function was impaired. insertion of the h4-ars, a second origin of replication, rescued a ded1 construct as a minichromosome (yrpdh3).the chromatin structure mapped at low and high resolution of the ars1 region in yrpdh3 was indistinguishable to that of the pet56construct yrpft61. ananlysis of rna showed that transcription was going through ars1 in yrpdh3 and in yrpft61, but the levels of transcripts in yrpdh3 were much higher. we conclude that transcription through are1 interferes with replication and prevents extrachromosomal maintenance. dna sequence of a repeated dna segment on circular and linear plasmids of borrelia burgdorferi w.r. z%ckert and j. meyer, abt. pzmom, zahn&rztliches institut der universit~t basel a plasmid-associated repeated dna segment of b. burgdorferi strain b31 was cloned. at least two copies of this segment appear to be present on the 29 kb circular plasmid, whereas one copy is carried on the 50 kb linear plasntid of this strain. dna sequence analysis revealed a region containing a novel 906 bp putative open reading frame (orfl) on the 50 kb linear plasmid, orfl displayed extensive sequence homology (95%) to putative open reading frames present on the clones obtained from the 29 kb circular plasmid. heterogeneity is mainly caused by 3rd-base-wobbllng. flanking sequences share 85-95% homology, including the 5' end of an additional putative open reading frame irmaediately downstream of orfl. whether orfl and/or the related sequences are being transcribed and yield, in the case of orfl, an approximately 36.5 kd, lysine-rich polypeptide, is yet unknown. the repeated sequence seems to be specific for b. burgdorferi sensu lato and therefore may be useful in nucleic acidbased diagnostics of lyme disease. $20-16 similar to type ib oculocutaneous albinism in humans, overall production of pigment is greatly reduced in dark eyed albino and only obvious in eyes. we have studied the molecular basis of the c 44h mutation and show that expression of the tyrosinese gene is not affected. after sequencing tyrosinase cdna isolated from c44h/c44h homozygotes we uncovered a single base alteration from wild type leading to a serine to isoleucine exchange. the importance of this mutation was demonstrated by generating transgenic mice containing a mutated tyrosinase minigene. this showed that the single base change is sufficient to severely depress pigment production in transgenic mice. we therefore conclude that the point mutation is responsible and sufficient to generate the dark eyed albino phenotype. members of the pou-family of transcription factors are involved in developmental and differentiation processes. using a pcr-based cloning strategy with degenerated oligonucleotides we identified several pougenes from the lactating and involuting mouse mammary gland. three of these cdna clones which contained as yet unknown sequences were chosen for further investigations: clone 5.80 which has a high homology to pit-l, clone 5.54 which is related to the brn-3 gene and clone 5.48 which belongs to the class iii of pou-proteins. the expression levels were analyzed in different mouse organs and in several developmental stages of the mammary gland, including the postlactational process of involution. because of the low abundance of the specific transcripts we used a semiquantitative, reverse transcriptase-mediated pcr assay to investigate the mrna levels of the three novel pou-family members. distinct and specific expression patterns for all three members were obtained in the different investigated organs. interestingly, the expression of the pit-1 related cdna clone 5,80 was elevated in all developmental stages of the lactogenic-hormone dependent mouse mammary gland and in sceletal muscle, whereas its expression was low in other organs. repetitive sequences in dna can allow ectopic (outof-register) homologous recombination, which is often undesirable and can even lead to disease in humans. to prevent this, long homologies should often be interrupted during evolution. accelerated mutation of repetitive sequences by so-called ripping may be one of the mechanisms used to accomplish this in fungi. we are investigating if introns in vertebrate genes have an undiscovered role as interrupters of homology within and/or between genes, in addition to their established role in exon shuffling. by inserting homology-poor introns in an otherwise homology-rich region the genome could prevent ectopic homologous recombination. such a mechanism could be especially useful where there is an advantage in encoding highly repetitive protein sequences. it appears that within the collagen gene family there is indeed a correlation between repetitiveness and intron density. the influence of prenatal diazepam exposume (i,25 mg/kg/d, s.c.) from gestational day 14 to 20 on the development of the ~-opioid binding sites in striaturn, nucleus accumbens and midbrain of pn 14, pn 28 and 8 week old male and female rats was studied. 8 week old prenatally diazepam exposed male rats showed a significant decrease of k-opioid binding sites in the nucleus accumbens. a sex-dependent difference in k-opioid binding site densities could also be detected between pn 28 prenatally diazepam exposed male and female rats. we now investigate by in situ hybridisation whether changes in the level of mrna encoding fo~ prodynorphin, the precttrsor for various endogenous ~-opioid agonists, could be responsible for the decrease of ~-opioid binding sites in the nucleus accumbens of 8 week old prenatally diazepam exposed male ~ats. $21-04 distribution and morphology of nitric oxide-positive neurons in the marmoset cerebral cortex during pre-and postnatal development j.p. hornung* and j. schultz** *institute of anatomy, university of lausanne, 1005 lausanne, and **university of fdbourg, 1700 fribourg nitric oxide, synthesized by the enzyme nitdc oxide synthase (nos), is a neuroactive substance and a limited number of neurons were shown to express this enzyme either by histochemistry or by immunocytochemistry. both techniques were performed on brains of marmosets with ages ranging from 6 weeks before birth to adult. the morphology and distribution of nos-positive neurons were analyzed in all lobes of the cerebral cortex. the same pattern was revealed by histochemistry or immunocytochemistry. three populations of nos-positive neurons were revealed. first, a transient population of neurons in the molecular layer of the embryonic cortex, many having the morphology of the retzjus-cajal cells. a second population was made of large multipolar neurons in the deep layers of the cortex and the underlying white matter, which persisted from embryonic to adult life. a third, permanent, population was made of small, weakly reactive neurons in the supragranular layers appearing postnatally, this study demonstrates that no can exert its actions in the cerebral cortex through several pathways at different developmental stages. trimethyltin (tmt), a well-known neurotoxicant, was used to study the sensitivity of the different brain cell types (i.e. neurons, astrocytes, oligodendrocytes and microglial cells) to a neurotoxic insult. aggregating brain cell cultures of fetal rat telencephalon were treated during 10 days with tmt at different concentrations, ranging from 10 -10 m to 10 -6 m. microglia were found to be the most sensitive cell type, since already at 10-10m of tmt an increased number and clustering of griffonia simplicifolia-positive ceils could be observed. at 10 -8 m of tmt astrocytes showed increased staining for glial fibrillary acidic protein, characteristic of gliosis. neurons and oligodendrocytes appeared to be the least sensitive cells, since a decrease in the activity of cell type-specific enzymes was observed only at 10 -6 m of tmt. these results show that microglial cells and astrocytes respond to a toxic insult before any neuronal changes could be detected, and that the microglial reaction may be the first target of tmt. this reaction could then trigger the astrocytic response. vif is widely distributed in brain with suggested functions related to development. vip expression was studied during rat brain development using hybridization histochemistry with a 48met probe. vip mrna was found in thalamic nuclei on el7, later recognized as the ventrolateral and reticular nuclei after further maturation during prenatal period, vip mrna was found in hypothalamus, especially suprachiasmatic nucleus on el9 and its expression matured over the next days. few cortical neurons contained vip mrna on e21, they continuously increased in number and signal intensity over the perinatal period. vip gradually matured in the first three postnatal weeks and adult-like patterns were found on p 22, when cerebral cortex, ventrolateral and reticular thalamic nuclei, hypothalamus, esp. suprachiasmatic nucleus, were the regions with most prominent vip expression. these results demonstrate the relatively late appearance of vip gene expression in the rat forebrain, as compared with peptides like srif and cck, not suggesting a major role in earlv brain maturation. in primary cultures of mouse cerebral cortical astrocytes, a rapid glycogenolysis followed by a massive glycogen resynthesis ( six-to ten-fold over basal levels after 9 hr) are induced by vasoactive intestinal peptide (vip) or noradrenaline (na). both actions of these neurotransmitters are mediated by camp. since the induction of glycogen resynthesis triggered by vip or na is abolished by inhibition of transcription and translation, we applied the 2-d gel electrophoresis technique to search for astrocytic proteins induced by vip or na. the comparison of 35s-labeled proteins from primary astrocyte cultures treated or not with vip 1 ~tm revealed two newly synthesized proteins: a 36 kda protein (pi=5) and a 23/24 kda protein (pi=6). only the latter is visible on a silver stained 2-d gel, which is a prerequisite to consider its purification and microsequencing. induction of the 23/24 kda protein by vip was time-dependent with a maximum at -12 hr. preliminary results indicate a similar induction by na and isoproterenol. in humans, the central analgesic effect of tramadol (t) is only weakly reversed by the ~t opioid antagonist naloxone (nx) (br j clin pharmacol 1993; 35:73p). t analgesia may thus result from an action on monoaminergic pathways as suggested by in vitro and animal data. we therefore investigated the effect of ct2-adrenoeeptor antagonism by yohimbine (y) on t analgesia. according to a randomised double-blind crossover and placebo (p) controlled design, healthy volunteers (n=10) received t (100mg orally), followed (+ 3 h) by y (0.1mg/kg intravenously), and y + nx (0.8mg intravenously). analgesia was assessed over 8 h by subjective pain threshold (numerical scale -ns-) and objective pain threshold (rill nociceptive reflex -riii-) monitoring. peak analgesic effect was observed at ca. 3.25 h (riii +8.8 +semi.6 ma and ns +8.4 +1.8) vs p (rih -0.3 +1.2 and ns +2.3 +1.6, p<0.05) and lasted ca. 6 h. y reversed t analgesic effects during ca. 1 h (r/ii -2.9 +1.8, p<0.05 and ns +2.8 +1.5, p<0.05), whereas y + nx abolished t effects thoughout the study period (rill -1.9 4-1.1 and ns +0.6 +1.3, p<0.05), y alone tended to lower pain thresholds (rill -4.1 +1.5 and ns -1.9 +1.4, p>0.05 anova). yohimbine, an r antagonist, reverses tramadol effects, thus pointing to the major role of monoaminergic modulation in tramadol anfinociception. a monoclonal antibody (in-l)was raised against neurite growth inhibitory proteins present in rat cns myelin (caroniand schwab, neuron 1: 85, 1988) . in-1 neutralizes the inhibitory ettect of cns tissue in vitro and in vivo, thus permitting regeneration of certain lesioned cns fiber systems. we have used this antibody to immunostain cryostat sections of the nervous system of the rat. we found that in-1 stains white matter and myehnated fiber tracts in the cns. the staining pattern corresponds to that of the known myelin specific antigens mbp (myelin basic protein), mag (myelin associated glycoprotein) and mog (myelin oligodendrocyte glycoprotein). the staining of in-1 in the cns is found in regions of the adult nervous system which have been shown to be inhibitory for axonal growth. interestingly, the regions which show a high abundance of in-1 antigens are low in staining for gap-43, a marker for growth and plasticity in the developing and adult brain. in contrast, gap-43 is strongly expressed in unmyelinated fiber tracts or nuclei. prevention of oligodendrocyte development in rat spinal cord resulted in suppression of in-1 expression and elevated gap-43 levels. this suggests that inhibitory myelin proteins may negatively influence plasticity in the rat cns. to date, the existence of anp and npy in the adrenal medulla has been shown only for a few mammalian and anuran species. thus the present immunohistochemical investigation attempts to localize anp-like and npy-like peptides n the adrena gland of representative mammals, birds, reptiles, amphibia and bony fish by the use of antisera specific for mammalian anp and npy. furthermore, the catecholamine-containing cells were characterized by antisera against enzymes of catecholamine synthesis. anp-and npy-immunoreactivies were detected in adrenal chromaffin cells of all vertebrates studied while no immunoreactivities were observed in the adrenal cortex or in its homolog, the interrenal. the representatives of the different vertebrate classes exhibited marked differences in the proportions of anp-immunoreactive and npy-immunoreactive cells, in the presence of anp-and npy-immunoreactivities in noradrenalineand/or adrenaline-containing cells and in the amount of of cells containing both anp-and npy-immunoreactivities. our results give evidence for a long phylogenetie history of adrenal anp-and npy-like peptides. thus, they stress the physiological impact of these peptides as adrenal paracrine and/or endocrine hormones. human neuronal growth inhibitory factor (gie) is involved in the regulation of neuronal growth and is deficient in the brains of alzheimer's disease victims. this brain-specific metalloprotein consists of 68 amino acids out of which 20 are cys and has been found to contain copper and zinc. proteins with similar primary structure were isolated from bovine and equine brain. the amino acid sequence of these proteins shows about 70 % homology to those of mammalian metallothioneins (mt), with two conserved inserts of 1 thr and a glutamic acid rich hexapeptide in the n-and c-terminal regions, respectively. in contrast to mts, which usually contain only zn(i1), the presence of cu(i) and zn(ii) (6-7 moles total) in all gif isolations suggests the functional importance of both metals ions. to spectroscopically probe the native zn-binding sites, the zn(ii) ions were replaced by cd(ii). both bovine and equine cd/cu-gif derivatives exhibit similar absorption and cd spectra with features characteristic of cd-thiolate clusters. the release of 3h-arachidonic acid evoked by glutamate was characterized in cerebral cortical neurons in primary culture grown under conditions that prevent glial cell proliferation. in these cultures, 99% of the total ceils are immunocytochemicauy characterized as being positive for specific neuronal markers such as neuron-specific enolase and neurofilament. specific markers for astrocytes, oligodendrocytes or microglia were not detected. glutamate induces a concentration-dependent release of 3h-arachidonic acid with a ec50 of 3 pm. the pharmacological profile of this glutamate response shows the involvement of two receptor types: the nmda and the ampa ionotropic receptors. the glutamate-evoked release of 3h-arachidonic acid is phsensitive. when the incubation buffer is alkalinized from 7.25 to 7.65, both basal and glutamate evoked release of 3h-arachidonic acid are enhanced. the glutamate response is also enhanced as intracellular ph is alkalinized by pharmacological treatments: such as inhibition of c1-/hco3-antiport by dids. these results further stress the role of intracellular ph in glutamate-mediated neurotransmission. $21-09 multiceli culture system for the study of drug kinetics wechsler d., *schittny j.c. and honegger u. e., depts. of pharmacology and *anatomy, university of bern, ch-3010 bern a cell culture system was developed for the investigation of cellular drug kinetics in cultures with up to 4 cell types. it was used for the study of uptake, competitive uptake, release and redistribution of drugs. individual cell types (human skin fibroblasts, rat astrocytoma cells (c6) and rat hybfidoma ceils (oligodendrocyte x astrocytoma, roc)) were separately grown to confluency on glass circle sectors. in the same petri dish 4 sectors in various combinations were exposed to media containing radiolabelled chlorpromazine (cpz). single and repetitive uptake and release of cpz were measured in each cell type after individual exposure or exposure in any combination of cell types: in 2 hour competitive uptake studies fibreblasts reached 1.7 and 2.6 times the concentrations of c6-and roc-cells, :respectively. in redistribution experiments the exchange of cpz from preloaded to unloaded cells was tested. the exchange rate was dependent on cell type and loading period. it decreased with increasing time of exposure suggesting the formation of more stable cpz stores. we have previously demonstrated that certain neurotransmitters such as vip, noradrenaline (na) and adenosine promote glycogenolysis in mouse cerebral cortical astrocyte cultures (brain res. 563: 227-233, 1991) . the question that we then addressed was the nature of the metabolic substrate released by astrocytes following glycogenolysis. we therefore determined the presence of various metabolic substrated released from astrocytes under static conditions and in the superfusate of a laminar flow system developed in our laboratory. neither glucose nor any intermediate of the tficarboxylic acid cycle could account for the decrease in glycogen stores; astrocytes however were shown to release great quantities of pyruvate and lactate at a rate of 50 nmol/mg protein/minute. noradrenaline but not vip promotes a significant increase (42%) in lactate release. the effect of noradrenaline is mimicked by isoproterenol and inhibited by propranolol, suggesting a i~adrenergic mediation. when astrocytes are incubated in an isotonic solution containing no energy suhstrate, the glycogen stores are rapidly depleted and lactate, but not glucose, is released in the medium. in conclusion, lactate appears to play a major role in cerebral energy metabolism homeostasis, as substrate released by astrocytes to prey!de energy fuel for neurones and other neighboring cells. it is known so far that calcium-binding proteins (cabps: calbindin, pan, albumin and ca/retinin) are expressed in some cells of the pineal gland of laboratory mammals. the exact phenotype of these cells is unknown. nevertheless, according to recent studies, it seems that some of them are ganglion cells. therefore we studied the pineal gland of different species (gerbils, rats, goats, cows and humans) using calretiain (cr) antibody which is considered as marker for neurons (andressen & al, cell & tissue rss, 27t:181, 1993) , the cr-expressing cells were found in all oar species: in gerbils, rats, cows and humans they were scattered throughout the parenchyrna, whereas in goats they lined mostly the pericapillary spaces. according to our findings, it seems that most of the reactive ceils are rather neuron-like elements since morphological criteria for true neurons (axosomatic synapses, nissl bodies, bundles of neurofilaments) are missing. however one cannot exclude that among these cells some are intrapineal ganglion cells, because of ranvier's nodes found along the processes of some crpositive cells. thus, it is possible that these cells are involved in the modulatory control mechanism of the pineal neurohumoral activity and/er by accumulation of intracellular calcium in the formation of pincal calcifications. besides the excitatory amino acid transmitter glutamate, its sulfur containing analogue homocystcic acid (hca) could play a role in excitatory processes in the central nervous system. hca is present in and released from nervous tissue and is a potent neuronal excitant, predominantly activating n-methyl-d-aspartate receptors (do et al., 1992) . these properties are suggestive of a classic synaptic neurotrausmitter role. on the other hand, hca seems to be localized not in neurones but in glial cells (grandes et al., 1991; tschopp et al. 1992) . the efflux of hca is temporally delayed following activation of specific pathways (klancnik et at., 1992) ; these latter observations are not in accordance with the classic concepts of synaptically-mediated neurotransmission. a laminar flow superfnsian system of monolayer cultures was developed and the released materials from mouse cortical astrocytes, previously preincubated with [35s]-methioulne, were investigated. during application of either noradrenaline or the b-adrenergie agonlst isoproterenol, the extracellalar level of [35s]-methianine was decreased and that of labelled hca was increased. this effect was not observed with the -adrenergic agonist methoxamine, and could be blocked by the ll-adrenergic antagonist propanolol these results, combined with the delayed hca release, the glial localisatian of hca and its neuroactivity, strongly suggest a potential role of hca as a gliotransmitter, modulated by noradrenaline inputs. vasoactive intestinal peptide (vip) induces long lasting metabolic effects in the mouse cns. we have investigated a shorter neuromodulatory action of vip on the synaptic responses in coronal slices of adult mouse cingulate cortex by means of intracelhilar current clamp recordings. electrical stimulation of the underlying white matter evoked a multiphasic synaptic potential consisting of early epsp~ early ipsp, late epsp and late ipsp mediated by non-nmda, nmda, gaba a and gaba b receptors, respectively. vip (0.1 ~d~l) superfused for 5 rain. had no effect, whereas concentrations over 0.2 ixm selectively and reversibly depressed the nmda-medlated responses in 12/16 cells. in the presence of cnqx (10 [tm) and biencuuine (10 ~tm), the isolated nmda-mediated late epsp was still attenuated by 0.3 ~m vip in 8/10 cells (mean:-42% al~er 30 rain.). in 3 other cells vip induced a spreading depression (sd) and a strong, reversals inhibition of the late epsp (mean:-64 % after 10 min.).these results indicate that in the absence of gabaergic inhibition vip can induce two types of effects in the mouse cingulate cortex, a prolonged decrease of the nmdamediated syaaptic response and a striking sd. interaction between vip and glutamate on c-fos mrna expression. |.-l. martin, d. gasser and p.] . magistretti. institut de physiologie, universit4 de lausanne, lausanne, switzerland. the regulation of c-los mrna expression by vip and glutamate was examined in primary cultures of neurons originating from the mouse cerebral cortex. in primary cultures of cortical neurons, vip increases camp levels and stimulates c-los mrna expression. interestingly vip stimulation is completely blocked by mk-801, an nmda receptor antagonist but not by cnqx or ap3, which antagonize ampa/kainate and metabotropic receptors respectively, c-los expression stimulated by glutamate shares the same pharmacology. these results suggest an involvement of endogenously released glutamate in the stimulation of c-fos mrna expression evoked by vip. furthermore, when added together, vip and glutamate interact synergistically to increase cfos mrna levels. consistent with the pharmacology of vip and glutamate, the synergistic interaction between vii' and glutamate on c-fos mrna expression is also inhibited by mk-801. interestingly, this synergism is completely inhibited by h-89, a protein kinase a inhibitor, whereas staurosporin and kn-62 which block protein kinases c and ca++/calmodulin dependent respectively exhibit smaller inhibitory effects. presynaptic proteins involved in neurotransmitter release (i.e. synapsin, b-50) and some postsynaptie gaba a receptor subunits possess phosphorylation sites for camp-dependent protein kinase (pka) and/or protein kinase c (pkc). the functional consequences of phosphorylation by these kinases is not known. we have studied the action of specific activators of pkc and pka, phorbol ester (pdbu) and forskolin, respectively, on gabaergic synaptic transmission in area ca3 of hippocampal slice cultures. pdbu significantly enhanced the amplitude of evoked monosynaptic ipscs (in cnqx and ap5), and both pdbu and forskolin increased the frequency of spontaneous miniature ipscs (m[pscs) in the presence of cnqx, ap5 and ttx. this effect by pdbu was antagonized by staurosporin, a protein kinase inhibitor. pdbu and forskolin did not change the amplitude or decay of mipscs, suggesting that only presynaptic kinases are involved. furthermore, pdbu enhanced mlpsc frequency by a comparable amount in the presence of the ca 2+ channel blocker cd 2+, indicating that pdbu did not enhance gaba release from presynaptic endings by facilitating ca 2+ influx into axon terminals. valiunas, v., sc~ilinsky fluri, g. and weingart, r. arachidonic acid (aa) reversibly impairs the gap junction conductance (g~) in cardiac cells. now, we examined whether aaacts directly or via its catabolites. experiments were performed on pairs of neonatal rat myocytes using a dual voltage-clamp method. we used blockers which interfere with enzymes of various catabolic pathways. pretreatment with i0 ~mpoca (blocks carnitine acyltransferase i) did not prevent the decline in g9 by i0 ~m aa; i.e. intermediates of p-oxidation are not involved. similarly, preexposure to i0 ~m indomethacin (blocks the cyclooxygenase pathway) did not prevent aa from uncoupling; this rules out an involvement of prostaglandins and thromboxanes. exposure to 10 ~mndga (blocks the 5-1ipoxygenase pathway) per se produced a decrease in gj. likewise, exposure to i0 ~m etya (blocks the 15-1ipoxygenase pathway) also impaired gj. these effects cannot result from accumulation of endogenous aa because preexposure to 50 ~m 4bpb (blocks phospholipase a 2) did not prevent ndga-or etya-induced uncoupling. exposure to 75 ~m skf 525-a (blocks the epoxygenase pathway) also produced a decrease in gj. hence, ndga, etya and skf 525-a, compounds with different biochemical actions and of different chemical structure, act on g~ either directly or via an unknown mechanism. $21-19 potential changes associated with electrical activity in nonmyelinated nerve fibres a. robert and p. jirounek d~partement de pharmacologie, cmu, 1211 gen~ve 4 simuheneous measurements of the extracellular concentration of k + ([k+]e), and of the concomitant changes in the membrane potential (era) were measured in the rabbit vagus nerve during and after a period of activity. during a 15 hz stimulation there was a large increase in the [k+]e , accompagned by a change in era, which roughly followed the depolarization expected from the increase in [k+] e . at the end of the stimulation, although the [k+]e was still elevated, the nerve developed a posttetanic hyperpolarization (pth). the pth was generated by the electrogenicity of the na+-k + pump, but its amplitude and time-course were strongly affected by two depolarizing currents: (i) an inward ba2+-sensitive current of k + and (ii) an outward current of ci-. we hypothesize that during activity and during the early phase of recovery there was a ba2+-sensitive uptake of potassium by the schwann cells. the ci" current, by short-circuiting the na+-k ⧠pump, contributes to the control of the e m, and by this way to the driving force for the inward k ⧠current. overdose of ifosfamide has ooo/red in a canc~c patient (25g i.v. iy0/24 hours with 20 g as ~utectarfe). urir~ collece{on was obtaj/3ed in 24 hour ~ fcr t~o days. c~ was measured ~ a gas liquid d~romatogr~ method of the u5~ ~ (s~@pl. v, pag.2627-262g) ~sir~ a ~e~s ar~ ni~, sensitive ths~mi(~3ic d~. ~ u~s p~mt ~ ~ ~ ~ ~ ~ 1500 m~/24h and 207 r~/24h at day one ard day res~ec~vely. 9~ _h=~=mmtly ~ has also bsm foa~ in mti~s ~ mgh e~e ~ ~ (~/o~e). w~n ~ ~s am/nist~ to rats (i~ m~/~ ~c 200 m~f~ intraperit~neally), no ~ omild be detected in urine within 24 ht~r~ after drug administl-4(cicn. from these ~ data we conclude that c~ is extensi~_ly metabolized in rats and ~ mscbard_sm of ~ el ~mlnaticn in pati~rfc t=cj_ne m~cjlr[c reflect a flmicticrsl der~,~t of c~ m~t~0olisn in man to ifo~=~de ~tion. there are numerous reports of improved memory functions in rats following a dietary supplementation with choline. in some cases, this improvement can be related to enhanced cholinergic transmission (mack et al., behav neurosci., 103, 1234 -1241 , 1989 ). but it is not clear whether there is a general improvement in memory or whether specific aspects of learning and memory are affected. this work was aimed at analysing the effects of perinatal cholinergic supplementation on the development of spatial abilities and upon adult performance. choline supplementation (3.5 g./l. in 0.02 m saccharine solution in tap water) was maintained during two weeks before birth. additional supplementation was maintained from the 5th to the 1oth week postnatal. spatial learning capacities were studied at the ages of 26, 65 or 80 days in a cimular swimming pool (morris place navigation task) and at the age of of 7 months on a homing board. adult and aged rats were tested on a radial maze. selective aspects of the performance were markedly improved. this treatment had no specific effect upon spatial memory per se, but, instead, affected learning abilities by promoting efficient behavioural strategies hypothetically based on active representation and anticipation processes. the effects of the treatment remained evident up to 6 months following the supplementation. barcelona, e-08193 bellaterra females of both lines (n=b/grp) were injected sc daily between days 15-20 of pregnancy with vehicle (v), i(di) or 3(d3) mg/kg diazepam, or not(c). in these studies(a,b) their 5-7 mo old offspring were subjected to a 50-run session in a shuttlebox(a:12 males, 6 females of each line/condition) or a 6min session in a hexagonal tunnel maze with strategically-placed barriers and a lit central arena(b_:6-7 males of each line/condition). a: the freezing behavior of ld3 rats was reduced ~s lc, this behavior reverting more to escapes in males & avoidances in females, although v had an effect in the latter also. no within-line differences in inter-trial responses were seen, but pre-session activity was increased in female lvs and ldis, returning to lc levels for ld3s. no differences existed among the h groups. b: whereas h maze activity exceeded that of l, no wtthin-line differences appeared. hd3s entered the lit arena less than hcs or hvs, indicating an increased timidity after pnd, and a trend in the same direction existed for ldi/ld3 vs lv. two groups of abstinent female smokers, performing either a fixed rate or a subject paced version of a rapid information processing task were compared. both groups participated in two test sessions (in which the task was performed twice) to compare two different types of cigarettes which were smoked before and during the second task trial: a) the subject's habitual cigarette with a nicotine yield of at least 0.7 mg/cigarette and b) a nearly nicotine free test cigarette with a tar yield comparable to that of the habitual cigarette. reaction times to correct detections (series of three odd or even digits in a pseudorandom sequence of single digits) were longer with the subject paced version and not affected by the type of cigarette but shorter with the fixed rate version and even more decreased with the smoking of the habitual cigarette. on the other hand, the pre-to posttreatment increase in the subject paced processing rate was greater with the habitual than the test cigarette. it was concluded that the two task versions assess different cognitive functions. whereas the fixed rate version primarily assesses vigilance performance and seems to be more suitable to measure changes in reaction time, the subject paced version is more sensitive to changes in speed capabilities in rapid information processing. heart rate, physical activity, and cigarette consumption were continuously recorded under field conditions, whereas subjective craving was assessed six times per day and saliva cotinine was measured once daily. abstinence, habitual cigarettes (with a nicotine yield of at least 0.7 mg/cigarette), and nearly nicotine free test cigarettes but with a tar yield comparable to that of the habitual cigarettes were compare~) in a sample of twelve female smokers for two days each. whereas physical activity was similar for the three conditions, heart rate was highest with the habitual cigarettes, lowest on abstinence days and in between with the test cigarettes. cigarette consumption was similar for both types of cigarettes and subjective craving was higher on abstinence than on smoking days but no differentiation between the two cigarette types was obtained. saliva cotinine values were highest on the days with the habitual cigarettes but lower and similar with the test cigarettes and on abstinence days. it was concluded that heart rate and saliva cotinine depended only on the amount of nicotine absorbed, whereas subjective craving was reduced by smoking independently of the actual nicotine yield of the cigarette. (ptps) is the rate limiting enzyme in the synthesis of bh, in man, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. the human and rat liver cdnas encoding the 16 kda subunit of ptps were expressed and the recombinant enzymes purified to homogeneity. the apparent k~ for the substrate, pi and heat stability of the re-combinant enzymes were similar to the native enzymes. the rat enzyme was crystallized and the x-ray structure showed a hexameric native form arranged as a sandwich of two trimers. three histidine, a glutamic acid, and -also shown by site-directed mutagenesis -a cysteine residue characterize the active center of the enzyme thought to be mg2+-dependent. according to the proposed ligands we replaced mg 2* by fe 2 ⧠or mn ~+ and observed an enhanced activity up to 100%. m. neerman-arbez, v. cirnlli and p. halban. laboratoires/eantet. centre m4dical universitaire, 1211 gen~ve 4. pci(3) and pc2, members of the mammalian family of pro-protein convertases homologous to the yeast kex 2, are both expressed in pancreatic islets of langerhans and are thought to be responsible for the conversion of proinsulin to insulin and c-peptide in beta cells. however, the insulin secreting beta ceils are not the only ceils present in these complex microorgans, prompting us to evaluate the expression of pc1 and pc2 in islet beta and non-beta cells. rat islet cells were sorted by autofluorescence activated flow cytometry to separate beta cells from non-beta ceils and conversion cndoprotease levels were analysed by western blotting. in beta cells, pc1 levels were higher than pc2 (pc1/pc2=2.6+0.2) whereas the opposite was found for non-beta cells (pc1/fc2=0.05â�¢ confirming that pc1 is important for proinsulin conversion while suggesting a role for pc2 in the conversion of proglucagon, prosomatostatin and propancreatic polypeptide. transformed murine cell lines (insulin-producing beta-tc and glucagonproducing alpha-tc) were found to faithfully reflect the primary rat cells in terms of their pc1/pc2 ratios. finally, post-translational modification of the convertases themselves was found to differ between cell-types, in particular, a precursor 75 kd form of pc2 accumulated in beta cells whereas only the fully processed 67 kd form was detected in the non-beta cells. the kringle (2+3) domains (k2+3) were successfully expressed in e. coil a n-terminal hexahistidine tag was fused to insure the isolation of k2+3 by affinity chromatography on a ni-2+-ntaagarose-column. to be able to remove the hexahistidine tag a factor xa cleavage site was introduced between the 6 histidine residues and the n-terminus of the kringle structures. the correct arrangement of the disulfid bonds was determined by amino acid-and sequence analysis. the lysine binding site was proven by affinity chromatography on iysine-bio-gel, as previously shown for k2. the dissociation constant of k2+3 was measured by intrinsic fluorescence titration with 6-aminohexanoic acid. a replication protein a copurifying dna helicsse from calf thymus anthl georgakl, narendra tuteja, blrglt sturzenegger and ulrich hobscher department of veterinary biochemistry university of z0rich-lrchel winterthurerstrasse f 90 ch-8057 z0rich, switzerland a dna helicase from calf thymus copurified with replication protein a through several steps of purification including deae-sephacel, hydroxyapatite and single stranded dna cellulose. it is finally separated from replication protein a on fplc mono q where the dna helicase elutes after replication protein a. we named this new calf thymus enzyme dna helicase f. characterization of the helicase f by affinity labeling with [a32p]atp indicated that the enzyme has a catalytic subunit of 72 kda. gel filtration experiments suggested that dna helicase f can exist both in a monomeric and an oligomedc form. the enzyme unwinds in the 5' ->3' direction in relation to the dna strand it binds. all eight deoxyribonucleoside-and ribonucleosidetriphosphates could serve as an energy source. testing a variety of dnndna substrates indicated that the dna heitcase f preferentially unwinds very short substrates and is slightly stimulated by a single stranded 3'-tail replication protein a allowed the dna helicase f to unwind longer dna substrates up to 400 bases suggesting that copurification of replication protein a with the dna helicase might be of functional relevance. 2,4,5,2',4',5'-hexachlorobiphenyl (6-cb) shows limited elimination and extensive storage in adipose tissue in vivo (lipophilie unmetabolizable compound) while chlorpromazine (cpz) is known for its lysosomal storage. uptake and release of 6-cb and cpz were studied in 3t3-preadipocytes (p) and 3t3-adipocytes (a). 3t3-cells were cultivated in dulbecco's minimal essential medium supplement with 10% fetal calf serum and grown to eonfluency. differentiation was stimulated by dexamethason and bezafibrate exposure for 48h. radiolabelled 6-cb (20~tm) or cpz (5~tm) was added to the medium. following a single dose 70-80% of cpz were taken up within an hour into p and a. in contrast, 6-cb reached an intracellular plateau of 30-50% of the dose after lh in p whereas dependent on the triglyceride content a accumulated up to 95% of the dose. 6-cb uptake into a was temperature dependent and not saturable with repetitive daily doses up to 1 week. 6-cb uptake into p was fully reversible while the release from a was limited to 10% of the accumulated drug. in contrast, the release of cpz was higher from p than from a as a consequence of the different lysosomal activities of the respective cells. the use of 3t3 cells allowed to show a remarkable difference in cellular handling of neutral and basic lipophilic drugs and may be helpful to further elucidate basic mechanisms involved. chronic granulomatous disease: an attempt to reconstitute the function of the x-linked form of the disease. chronic granulomatous disease (cgd) is a group of congenital immunodeficiencies that predispose patients to recurrent severe bacterial and fungal infections. the cause of the disease is lack or impairment of 02-production in phagocytic cells due to mutations in any of the four components of the superoxide producing system. approximately 70% of all cgd patients suffer from an x-linked form of the disease where the phagocyte oxidase gp91phox is affected. this project concentrates on the reconstitution of the deficient gene in phagocytic patient cells. expression of recombinant gp91 was assayed in vitro and in vivo. attempts to "tag" gp91 with a foreign epitope were unfortunately so far unsuccessful. the target cells for gene reconstitution are non-dividing monocytes. thus, we have chosen an adenovirus over other viral systems because of: a) its ability to infect nondividing cells, b) its genetic stability and, c) lack of human adenovirus related malignancies or side-effects. we constructed two recombinant adenoviruses: one recombinant expresses a lacz gene (control), another the gp9 lphox gene. we are trying the expression of recombinants adeno/lacz and adeno/gp91 in normal and patient-derived cells (for example ebvtransformed normal and patient b-cell lines), as well as the reconstitution of nadph oxidase function in peripheral blood monocytes of cgd patients. kinetics of molecular chaperone action schmid, d., gehring, h., *baici, a. and christen, p., biochemisches institut der universit&t z~rich, ch-8057 zorich; *rheumaklinik, universit&tsspital, ch-8091 z%rich molecular chaperones of the hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. target peptides labelled with an environmentally sensitive fluorophore allowed to follow their binding to the molecular chaperone dnak of escherichia coli in real-time. the two-step process is characterized by relaxation times z~ = 27 s and z2 = 200 s at 2 ~m dnak and 0.i ~m ligand at 25~ in the presence of atp, the formation of the complex is greatly accelerated and follows a single-exponential process with z : 0.4 s. the rate of dissociation of the complex was even more increased than that of association resulting in a decreased net affinity for ligands in the presence of atp. the binding-release cycle of dnak thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the atpase activity of the chaperone (ko~ ~ = 0.13 min i at 30"c). supported by the olga mayenfisch stiftung, z%rich, and the hartmann m~ller-stiftung, z~rich. comparison of the activities of native bovine seminal ribonuclease and that secreted from e. coil schein, ch* (siat, technopark, pfingstweidstr. 30, ch8005 ziirich) and haugg, m (lab. org. chemistry, e.tm., . seminal ribonuclease (bs-rnase) differs from the pancreatic rnase a in that it forms a cysteine-linked dimer and has a relatively higher specific activity in the cleavage of double-stranded (ds-) rna substrates. we expressed bs-rn in a secretion system similar to that described previously (biochem. j. 283:377-344, 1992) using the signal sequence of routine pancreatic ribonuelease. about i mg bs-rn was isolated per liter culture supernatant. human and bovine recombinant interfcron-~ (ifn-r) inhibit the degradation of ss-and ds-rna by bovine pancreatic rnase while they activate the activity of bs-rn on the same substrates (febs letters, 270:229-332, 1990) . recombinant bovine or routine pancreatic rnase (produced in our secretion system) are inhibited by ifn-y, while the recombinant bs-rn or a cysteine-linked dimer of rnase a is also activated by ifn-y. ifn-y activates bs-rn even in the presence of inhibitory concentrations (0.1-1 ram) of mononueleotides. thus the mode of activation cannot be attributed to alleviation of product inhibition. kinetic studies using 300 bp ds-rna as substrate suggest that ifn-~ interacts directly with the bs-rn to increase the rate of highmolecular weight producffsubstrate release, as the apparent ks increases proportionately to the increase in kcat. significantly smaller movements were observed in the simulation with the liganded enzyme. the structural differences between the protein in the crystal and the ensuing calculated structures might arise from the absence of lattice forces in solution. evolutionary relationships among pyridoxal-5'-p-dependent amino acid decarboxylases sandmeier, e., hale, t.i. and christen, p. biochemisches institut der universit~t zgrich, 8057 z~rich. the pyridoxal-5'-p (plp)-dependent amino acid decarboxylases (de) can be subdivided into four apparently unrelated groups. group i is represented by glycine dc, group ii comprises glutamate, histidine, tyrosine and aromatic-l-amino acid dc, group iii procaryotic ornithine and lysine dc as well as the procaryotic biodegradative type of arginine dc, group iv eucaryotic ornithine and arginine dc as well as the procaryotic biosynthetic type of arginine dc and diaminopimelate dc. (n-l) profile analysis, a more stringent application of profile analysis [gribskov et al. (1990) methods enzymol. 183, 146-159] established the homology among the enzymes of each group. a search with the profile of group ii indicated a distant relationship with aminotransferases and thus with the ~ family of plp-dependent enzymes. no evidence was obtained that groups i, iii and iv are related with other plp-dependent enzymes or any other protein in the database. apparently, the amino acid dc are of multiple evolutionary origin, in some cases even if they have the same substrate specificity. a. lo russo, a.c. passaquin, u.t. r~eg~, ecole de pharmacie, %hiv. lausanne, c~-1015 lallsaraqe drug-induced local vasoccmstricticn appears to be respmnsible for the hypertensive side effect of the imn/nosti~pressant cyclosporin a (csa). we have therefore ex~rnir~ the [ca 2+] i increase caused by the vasoccr~trictor hormcme [ar~]vascpressin (avp) in vascular ameoth ~uscle cells (vsmc) treated with csa. [ca2+] i levels were m~nitored either with a fluoresc~qce analyser using fura 2 or by 45ca 2+ efflux. pretreatment of v~mc with csa increased the avp induced rise in [ca2+]i. a leftward and upwsxd shift of the ccncentraticm-respmnse curve of the avp-induced rise in 45ca2+ efflux was also observed after csa treatxr~%t. ilzg/cticn of csa dote~tiaticn w-as detectable after 3 rain, took 1 to 2 h to become fully established and was reversed after washout. csa potentiaticm was cc~centraticn-deperdent with a threshold at 10 -7 m. %]]is potentiating effect of csa may be the underlyin~ cause for csa-ind/ced hypert~3sicn. 92) barja, f. 1 and turian, g. i, ilab. gen. microbiology, 2dept. biochemistry, 3station exp. zoology, university of geneva, 30 quai ernest-ansermet, 1211 geneva 4 the actin has been found from the simplest forms of cell to the highly evolved ones. in higher organisms it is transcribed by multigene families but in yeast and several filamentous fungi there appears to be only one actin gene. it was therefore of interest to study the expression of actin gene in n. crassa in which three actin isoforms have been characterized (barja et al., 1991, fems left. 77:19-24) . after isolating genemic dna from wild type n. crassa a pcr was performed with primers corresponding to actin consensus sequences and an amplified fragment of the gene (verified by sequencing) was obtained. this fragment was 'used as a probe in a southern to assess the number of aetin gene(s). our results suggest that n. crassa has alsc only one actin gene. the blocking effect of the n-terminal decapeptide of msmooth muscle actin (sma) (ac.eeedstalvc) on the monoclonal antibody anti-asm-1 was compared with that of synthetic peptides modified by changing the n-terminal acetyl group or by substituting a single amino acid in position 1 to 5. using immunofluorescence or immunoblotting techniques, anti-c~sm-1 activity was abolished after incubation with the native peptide and peptides modified in position 1 (only when e~a) or 5. on immunoblots running bsa crosslinked peptides on denatured gels, only the natural peptide and peptides with substitution in position 1 or 5 were detected by the antibody. our results indicate that ac.(e)eed is the epifope for anti-c~sm-l. binding of anti-~sm-1 to sma increased actin polymedzation by decreasing the critical concentration. as control this action was not exerted on skeletal actin. this is the first example of the role of a precise n-terminal sequence in the polymerization of a single actin isoform. double immunofluorescence for msma and total actin on cultured aortic sm cells microinjected with the native peptide showed a selective disappearance of msma staining suggesting that this peptide traps a protein involved in msma polymerization. work is in progress to search for the putative actin-binding protein(s) physiologically interacting with the n-terminal sequence of o~-sma. ( within mitochondria, the mechanism of selective protein degradation is poorly understood. while the bulk of mitochondrial proteins are long-lived, some are degraded rapidly. the selective degradation of mitochondrial proteins is likely to be mediated by proteases similar to those found in bacteria; this is based on the hypothesis that mitochondria have evolved from bacterial endosymbionts, in conjunction with the finding that mammalian mitochondria contain an atp-dependent proteolytic activity similar to that in bacteria. one atp-dependent protease from bacteria, lon, is of particular interest because it is involved in regulated protein turnover. degenerate oligonucleotide primers corresponding to two regions of the bacterial lon gene were used to pcr amplify a 1 kb fragment from yeast dna that encoded an open reading frame with striking similarity to the bacterial lon protease. by screening a yeast cdna library, we isolated a 3.6 kb clone potentially encoding a protein of 1133 amino acids. haploids bearing a disruptedlon gene were unable to respire or to grow on ethanol/glycerol. fractionation of rnjtochondrial matrix from wild-type cells revealed a peak of aip-dependent proteolytic activity which was absent in the disruptant. furthermore, we have identified matrix proteins that are degraded with a half-time of ~60 min in intact wild-type ceils but are stable in the ion disruptants. electron microscopy of lon disruptants revealed the presence of many electron dense bodies in the mitochondrial matrix which are not found in lon + cells. hydrolysis of high molekular weight kin1nogen by plasma kallikrein benner, s. and duffer, h., laboratory for organic chemistry, eth h6nggerberg, ch-8093 z/inch high molecular weight kininogen (hmwk) is cleaved by the serineprotease plasma kailikrein to generate the hypotensive peptide bradykinin in human blood. we established a measuring system for the time course of the hydrolysis of hmwk in its native and deglycosylated form and in the presence or absence of c~-inhibitor. analysis of the bradykinin release and the yielded protein fragments lead to the three following results: 1. the hydrolysis did not follow plane michaelis-menten kinetics due to a hmwk-fragment operating as a competitive inhibitor. 2. addition of the ci-inhibitor stopped the hmwk-hydrolysis immediately, which is in contrast to the physiological role of plasma kallikrein in blood. 3. so far we have no evidence that the high content of o-glycosylated side-chains of the hmwk-light chain has an effect on the kinin-liberation as proposed in literature. ( tetrahydrobiopterin (bh4) is the essential cofactor for the hepatic phenylalaoine hydroxylase, but also for tyrosine and tryptophan hydroxylases that are responsible for the production of nettrofransmitter precttrsors. furthermore, bh4 is required for the deavage of giyceryl ethers aztd is i~vo[ved in the biosynthesis of nitric oxide by the nitric oxide synthase. a vazia~t type of hyperphenylalani~emia is caused by a deficiency of bh4. the most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (ptps) activity. the human cdna for ptps was isolated, and the recombinant protein was found to be active when expressed in e. coil, thus allowing us to perform hmctional t e,3ting of mutant proteins. primary skin fibroblasts derived from ~everal ptps deficient patieilts were investigated for the molecular nature of this autosomal recessive disorder. all fibroblasts had f1"ps mrna expressed, and subsequent cdna sequence analysis revealed different mutatkons in the coding region of ptps (codon replacemenls, deletions, or nonseme codons) responsible for a lowered or no enzyme activity. in order to potentially correct ptps activity (and restore bi-i 4 biosynthesis) we are establishing a recombhtant rel~oviral-mediated gene transfer system to efficiently introduce the wild-type human ptps-cdna in these fibroblasts. the thylakoid membrane (tm) contains i0 molecular species of phosphatidylglycerol (pg). the relative amount of these species depends on the plant species and growth conditions. using phospholipase a2 (pla2) at 0~ to digest preferentially pg in the outer monolayer of spinach tm, we have shown previously that this monolayer was enriched in pg (ca 70~). the purpose of this investigation was to determine the transmembrane distribution of each pg molecular species. to this aim, we have studied the hydrolysis kinetics of each species in the presence of pla 2 in spinach and squash tm containing different relative proportion of molecular species. the hydrolysis rate and extent of the molecular species depended on the unsaturation degree of the fatty acid at the sn-i position as well as on the presence or absence of 16:1(3t) at the sn-2 position. for instance, the hydrolysis rate of pg ig:3/16:l(3t) was greater than that of pg 16:0/16:0. these results will be discussed in terms of the thylakoid transmembrane distribution of each pg molecular species. (supported by the snsf 3100-33693.92). from previous electrophysiological evidence, we concluded that metal ions (hgz+,cd2+,ag +) at low concentrations (i0-6m) increase rapidly (10-60 s) cation (na + , k + ) conductance at the apical membrane of epithelial cells. we investigated here the effects of these metals on [ca2+]i in mdck-i cultured cells, for a potential correlation between functional membrane changes and [ca2+]i . metals (10-4-10 -6 m) were added at the apical side of the epithelium, and [ca2+]i was measured with fura-2. hg 2+ induced a rapid (peak 5-10 s) and marked increase in [ca2+]i, whereas cd 2+ entailed a slower (peak 2-5 min) and marked response. the time-course of effects for both metals was similar to the electrophysiological changes. in contrast, the pattern of effects of ag + on [ca2+]i differed markedly from that on apical membrane conductance. thus, the effects of metals on [ca2+]i are conspicuous but not tightly associated with changes in membrane conductance. c. bulliard and l-l. dreyer, institut de biochimie, universit6 de fribourg, ch-1700 fribourg trans-plasma membrane oxydoreductases (pmo) play a significant role in energy transduction and post-receptor signal transmission, but the enzymes have not been characterized so far. we have achieved the purification of pmo from rat brain synaptic vesicles and from synaptic plasma membranes. the membrane enzymes were extracted by 1% lubrol xl tm, precipitated with ammonium sulfate (between 40 and 70% saturation) and purified by means of hydrophobic chromatography on butyl-agarose and gel f'fltration on superose-12, followed by page under native conditions. specific enzymatic staining of native page gels yields two homogenous diaphoraselike activities, pmo-i and pmo-ii . sds-page of the enzymes showed that pmo-i is made of two subunits of 52 kda and 40 kda whereas pmo-ii consists of a single sub-unit of 52 kda. the 52 ki)a subunits from pmo-i and pmo-[i are probably identical from their respective properties. partial n-terminal sequencing (13 aa) of the 40 kda subunit from pmo-i showed 84% homology with rat brain fructose-l,6-bisphophate aldolase. these data indicate that pmo is closely associated with the glycolytic enzyme that co-purifies in all steps under suitable conditions. the biochemical functions of pmo's remain to be established. ferredoxin:thioredoxin reductase (ftr) is the key enzyme in the ferredoxin/thioredoxin system, the light-dependent regulatory system in oxygenic photosynthesis. it is a nucleus encoded ironsulfur protein, composed of two dissimilar subunits, one of which is the catalytic subunit containing a redox-active disulfide bridge functional in the reduction of thioredoxins and a 4fe-4s cluster. using pcr we have isolated and characterized a cdna clone of 738 bp from spinach and a clone of 911 bp from corn coding for the catalytic subunits including the transit peptides. the first 31 (spinach) resp. 38 (corn) amino acids after the start exhibit typical features of chloroplast transit peptides. following the transit peptides are 113 (spinach) or 114 (corn) ami[~o acids representing the catalytic subunits. the primary structures are highly conserved between these two species with 91% similarity and 81% homology. seven of the eight cys residues found in the spinach subunit and important for the catalytic activity are at conserved positions. (snf 31-28811.90 and 31-37725.93) $23-20 r. zurbriggen and j.-l. dreyer, institut de biochimie. univarsit6 de fribourg, ch-1700 fribourg most mammalian cells display transplasma membrane oxidoreductase activity (pmo). the enzymes use intracellniar reduced pyridine nucleoticle to reduce an unspecific extracellular electron accepter as substrate. in order to set up a methodology for purifying, characterizing and quantifying pmo's, the plasma membrane from a neuroblastoma cell line, nb41a3, has been biotinylated. subsequently the biotinylated proteins were purified by immanoprecipitation with avidin, antiavidin-antibodies and insoluble protein a. the protein recovery of an immunopurified membrane preparation was <0.15% of the protein content in the cell extract. specific pmo activity was increased 15 to 20 fold compared to the activity in whole cells. this approach demonstrates the presence o1' pmo within the cell plasma membrane. this activity accounts for about one third of the total cellular diaphorase activity and cannot be attributed to an increased perraeabilization of the plasma membrane induced upon biotinylation nor to intracelluiar activity from lysed cells. pmo also promotes cell growth at low substrate concentrations (0.1-ll.tm). native gel electrophoresis of iarinobiotinylated and affinity purified plasma membrane extracts displays two diaphorase-positive bands, indicating that a homogeneous cell population may express several pmo activities at the plasma membrane. fluvastatin (fs) is a new hmg-coa reductase inhibitor. its affinity for 3 major human drug metabolizing p450 monooxygenases (cyp2c9, cyp2d6 and cyp3a4) was determined in liver microsomes. fs showed low affinity (ki > 50 ~tm) for cyp2d6 and cyp3a4 whereas cyp2c9 was selectively and competitively inhibited (ki = 0.1 ~tm). the potential for in vivo fs interactions with cyp2c9 substrates was therefore investigated in 14 volunteers using dicinfenac (df) as a probe (25 mg orally) on days 0, 1 and 8. df and 4'-ho-df kinetics were determined by hplc/uv. df cmax increased over time (0.28 [sd 0.12], 0.38 [0.20] and 0.45 [0.4] mg/l on day 0, 1 and 8, resp.). oral clearance was reduced on days 1 and 8 (14 % and 15 %, resp.). a time dependent decrease in urinary metabolic ratio (4'-ho-df/df) was noted (1.07 [0.34], 0.90 [0,23] and 0.70 [0.18] on day 0, 1 and 8, resp. (p < 0.0001)) only over the first 4 hours. fluvastatin therefore exhibits a two-phase interaction in vivo: an early transient and a late cumulative inhibition. the transient phase matches inhibition profiles predicted by quantitative models integrating in rive pharmacokinetics and in vitro inhibition data (q-dips). simple competitive inhibition by fs cannot explain the late phase. other mechanisms (i.e. fs or metabolite cumulation, mi complexation) are hypothesized. in some situations fs is expected to cause interactions with cyp2c9 substrates, which are partially predicted by quantitative modeling of in vitro data (snsf project n o 32-36600.92). 1211 gen~ve, switzerland. previously, a parvalbumin mutant, pvf~02w, was constructed with a unique reporter trp in the middle of the hydrophobie center. in the present study three new parvalbumin mutants, derived from pvft~w and containing alterations essential for ca2+-binding in either one, pv.câ�¢ and pv_m, or both, pv-co~, of the two ca2+-binding sites, were analyzed in order to study the metal binding properties of individual ca~+-binding domains and their contributions to the structure and stability of the protein. both, pv_ct, and pv.~, bind i ca ~ ⧠with affinity constants, kc,, of 1.1,107 and 3.2-106 m "~ in the absence of mg 2+. mg 2+ shifts the ca~+-binding isotherms of pv.co to higher free [ca z+] according to the competition equation with km~ = 2 9 103 m t. pv.m~ is much less effected by mg z* (kmg = 80 m "t) and can be considered as possessing a ca2+-specific site. nevertheless, in the absence of ca 2 ⧠both, pv.co and pv~, bind i mole of mg 2+ with much higher affinity than predicted from the competition studies. pv_cd;~ binds neither ca 2. nor mg 2+. trp-fluorimetry and uv-difference spectroscopy revealed that the ca~*-loaded conformations of pv_co, pv.ef and pvr~o~w are similar, with the trp-102 deeply buried in the hydrophobie core. however, striking differences between the mutants are found in the metal-free and mg~+-loaded forms. the conformation of pv~t~;~ is not affected by ca 2~ or mg z*. our results indicate that destroying the ca2+-binding of the ef loop, but not of the cd loop, strongly destabilizes the protein in the absence of ca z ⧠. biochemlsches institut der unlversitfit zfirich, switzerland, ch-8057. the sea urchin, strongylocentrotus purpuratus, metallothionein gene, mta, was constructed and expressed under the control of a heat shock promoter in a protease-deficient strain of e, coll. the nascent recombinant protein was stabilised by the addition of exogenous cd. the cd-containing protein was precipitated with ethanol and subsequently purified to homogeneity by gel permeation and ionexchange chromatography. the purified protein was identified as the desired gene product by tryptic peptide map analysis, sequence analysis and mass spectroscopy. typical yields of protein were 2mg per litre of culture. the presence of 7 cd ions per molecule was confirmed by the sh/cd ratio and by the existence of 7 h3cd nmr resonances. the apparent association constants of sea urchin mt with cd, which has a charge of -8, were found to be approximately 15 times weaker than for rabbit mt, at ph 7. (mt) are small metalloproteins displaying as their main feature clusters of d i~ metal ions bound to the thiolate ligands of the abundantly occurring cysteine residues (cys). from a set of over 85 sequences a general multialignment of 62 class i mt has been obtained on the basis of the needleman & wunsch algorithm. all cys are conserved in the man%mals and over 83% of them in other vertebrates and invertebrates. on the basis of similarity/identity scores we can isolate groups of sequences. evolutionary relationships between species and groups have been calculated with the maximum parsimony method of felsenstein and with the distance matrix method of fitch and margoliash. the divergences observed among the mammalian mts indicate a partition into four distinct subforms which all may have arisen before the separation of the species in evolution and which in part may differ in function. $23-28 bourque, a., singh, a., dykeman, a., macmahon*, a., and foster*, w. atlantic veterinary college, charlottetown, canada, and *reproductive toxicology section, environmental health centre, tunney's pasture, ottawa, canada. hexachlorobenzene (hcb) is a known environmental pollutant that is produced as an industrial by-product of certain chemicals. when administered in high dosage, hcb induces alterations in the rhesus monkey ovary. a purpose of the study was to determine a no observable adverse effect level (noael) for the compound. twenty, cynomolgus monkeys, 6-13 years old were placed in five groups. hcb was given orally in concentrations of 0.01, 0.1, 1.0, and 10 mg/kg b.w. in gelatin capsules, daily, for 13 weeks. one group of animals fed glucose only, served as the control. ovary specimens fixed in 2% buffered glutaraldehyde were prepared by conventional methods for transmission electron microscopy. ultrastructural alterations were revealed in the developing ova and follicular cells in all the ovaries from hcb-treated animals in a dose-related manner. clinical chemistry was unaffected by hcb. a noael for this reproductive toxicant is yet to be determined. a.b. was a recipient of the nserc undergraduate student award. an acidic haem-and zinc-containing protein has been isolated from bovine brain. native and sds-polyacrylamide-gel-electrophoresis reveal a monomeric protein with an apparent molecular weight of 47,2 kda. the absence of co-staining with tetramethylbenzidine implies that the haem group is non-covalently bound. the electronic absorption spectrum, with a soret-band absorption maximum at 412 nm and c~-and i~-bands at 540 and 575 nm, respectively, is similar to that of fe(ll)-myoglobin, consistent with the presence of haemiron in the ferrous oxidation state. in air the protein was easily oxidized to the fe(lll) form with a subsequent shift of the soretband maximum to 405 nm. atomic absorption measurements indicate approximately 1 mole of zinc and 1 mole of iron per mole of protein. the amino acid composition is similar to that of indoleamine-2,3-dioxygenase, although no such enzymic activity has been detected. in this study, we looked for this substance and its metabolites in young and old bovine lenses, because of their possible role in cataract formation. the substances were detected by hplc analysis. the kynurenine aminotransferase (kat) activity was determinated by the method of tobes (methods enzymol 1987~ 142:217) . the fluorescent substance formed from 3-hk was characterized by thin layer chromatography followed by reaction with rtinhydrin, uv and fluorescence spectrometry, and atom bombardment for molecular mass determination. 3-hk at concentrations of 0.08-+0.01, 0.2_+0.04, and 1_+0.4 btg/g of tissue was detected in iris/ciliary body, retina, and calf lens respectively. this substance is deaminated by kat. the activity of this enzyme was 2.6_+ 0.3, 3.7_+0.2, and 9.6+_2 ~tmol/g of tissue/hour in retina, iris/ciliary body, and lens respectively of old bovine eyes, but it was not found in calf eyes. the deamination of 3-hk resulted in the formation of a fluorescent substance which was identified as oxo-xanthurenic acid (oxa) with a molecular mass of 205 daltons. the accumulation of oxa acid possibly interacting with lens proteins could induce cataract formation. snf 32-36058.92, emdo, and hartmann-mtiller. the goal of covalent immobilization of oligonucleotide capture probes to solid supports has been accomplished by light-dependent, photolinker polymer mediated immobilization strategies. synthetic oligonucleotides were immobilized by facile layer coating procedures. the procedure does not require functionalization of the oligonucleotide probe or the matedal surface. oligonucleotides were linked to polystyrene with a biopolymer which was multiply substituted by photoactivatable diazirines. capture probe photoimmobilization (350 nm irradiation) was strictly light and diazirine dependent. using radiolabeled oligonucleotides as capture probes and polystyrene as material surface, the light dependent coupling efficiency was 40%. nonspecific oligonucleotide binding was below 2 %. photojmmobilized oligonucleotides retained the ability to form stable complexes with complementary dna strands, and target oligonueleotides of varying length were captured as well as dna fragments produced by pcr techniques. indoleamine 2,3-dioxygenase (ido), a superoxide radical scavenger, is present in transparent human lenses and produces 3-hydroxykynurenine, a uv-b filter. the synthesis of 3-hydroxykynurenine by ido and its degradation by kynurenine aminotransferase (kat) were measured in transparent lenses and cataracts. post-mortem eyes of elderly persons between 72 and 84 years of age with transparent lenses were obtained from the eye-bank of the department. fresh cataracts were obtained from cataract surgery patients. ido activity was measured by the method of yamazaki et al (biochem j 1985; 230:635) adapted for hplc. kat activity was measured by the method of tobes (methods enzymol 1987; 142:217) . ido activity found in post-mortem transparent lenses (cortex and nucleus) was 0.7_+0.3 nmol/mg protein/hour. in cataracts a low 1do activity of 0.3_+0.2 nmol/mg protein/hour was found in the cortex but not in the nucleus. kat activity, found in the eight cataracts examined, was 1.6_+0.9 nmol/mg protein/hour. inhibition of ido activity and induction of kat activity seems to be involved in human cataract formation snf 32-36058.92, emdo, and hartmarm-m011er photolinker polymer mediated covalent immobilization of antibodies and f(ab') fragments has been achieved by newly established lightdependent coupling procedures. anti alpha-l-fetoprotein monoclonal antibodies and f(ab') fragments derived from anti prostate specific antigen monoclonal antibodies were covalently linked to microplates. diazirine derivatized bovine serum albumin served as multifunctional light activatable linking agent. both immunoreagents, monoclonal antibodies and f(ab') fragments remained immunologically active after 350 nm irradiation (irradiance 0.7 mw cm -2 for 20 minutes). covalency of antibody binding was inferred from i) the photoreagent dependence, ii) the light dependence of the immobilization process, and iii) the reversibility of immunocomplexation after acid treatment. $23-33 two ~ of extracelluiar elecirical resistance in v~kwricular myocardil~ fleischhauer j.c., kl~ber a.g., dept. of physiol. bern in myocardial tissue, the electrical resistive properties of the extracellular space are important for local current flow during e~citation and therefore influence impulse conduction and the magnitude of the extracellular electrical field. to assess the r~tlti~t nature of the resistance of the extracellular space, electrical cable analysis was applied on an arterially perf~-sed rabbit papillary mascle. ~he resistivity of the intravascular space was changed (70 to 220~i~n) by variation of hematocrit frcrn 0 to 60% in the perfusate. in order to change the voltm~ and hence the electrical resistance of the interstitial space, colloidosmotic pressure in the perfusate was varied by changing dextran {m r 70000) concentration (i0 to 80g/l). altering the interstitial space volume had a marked influence on the extracellular resistance (ro) , conduction velocity and the magnitude of the extracellular field. variations of the resistive properties of the intravascular space had no significant influence on ro, conduction velocity and the magnitude of the extraeellular electrical field. our results suggest that the microvascular tree is insulated from the interstitial space and local electrical current flow during excitation in heart muscle is confined to the narrow interstitial clefts. s04-25, s 15-69 s05-18, s05-19 s 11-05, s11-06 bchini hooft van huijsduijnen s08-12 s05-30 s15-45 s08-13 s08-05, s08-06 s01-08 s15-67, $23-19 s13-03 nook, ek s01-15, s10-15 s05-02, s05-05 s 15-03, s15-29, s15-30 s19-03, s19-04, $19-05 s16-02, $16-15 s12-22 s15-48 s05-24 s03-01, s03-02, s03-03 s03-01 lrmler s05-28, s09-06 s03-07, s03-10, s03-11 s18-05 lipp, p. s05-26 so 1-02 s15-15, s15-16, $21-03, $21-11, $21-12, $21-14 $23-29,$23-30 s01-17 s 15-29, s15-30 s12-12, s12-24 molar s15-19 s08-05, s08-06 e s09-06, s09-07 s15-03, s15-29, s15-30 e. s03-05 s05-09, s05-10, s05-51 s05-52 s10-16 s08-14 s04-23, s04-29, s05-13, s05-53, $20-15 s10-16, s10-19 s09-01, s09-02 s15-68, s 17-24 stalder h. s03-19 s10-21 s09-09, s13-10, s13-13 s10-21 teheesen s01-10, s01-11, s01-18 s15-16 van dillewyn s05-32, s05-37, s05-38 s08-05, s08-06 s05-18, s05-19 huber d., ojha m. and turian g. laboratory of general microbiology, university of geneva, sciences iii, 30 quai ernest-ansermet, 1211 geneva 4, switzerland. ca2+-dependent protease in allomyces is predominantly localized in the apical region of the exponentially growing hyphae (huber and ojha, submitted) . many cytoskeletal proteins like actin and tubulin are also mainly localized in this growing region (heath, l~91). it is known that the ca2+-dependent protease proteolyzes a number of cytoskele~ ton proteins in order to regulate the plasticity of the cell (mellgren, 1987) . we have studied the proteolysis of some cytoskeletal proteins by this protease purified from the aquatic fungus allomyces erbuscula. actin, tubulin and desmin, were found to be rapidly proteolyzed in vitro at a high substrate to enzyme ratio. immunofluorescence studies of proteolyais made in situ in exponentially growing hyphae showed modification of apieally localized cytoskeletal proteins. this colocalization of the ca2+-dependent protease and cytoskeletal proteins in the same apical region is inferred in relation to hyphal elongation. jbiological databases grow exponentially. in order to provide access, as much data las needed and as little volume as possible shall be returned upon a query. the icurrentiy available dna and protein sequence databases are updated in a sophisticated network of procedures and expose a considerable amount of redundancy. research is performed on optimizing the creation of non-redundant data sets. the resulting data are disttibuted in switzerland, or made accessible to researchers who cannot utilize local resources. transparent access to the swiss resources, as well as paneuropean services, is provided with a job scheduling system which was developed in basel, the hierarchical access system for sequence libraries in europe ("hassle"), and various other computer programs which allow comprehensive browsing such as "gopher" and "www". training courses to use the resource effectively are running throughout the year. key: cord-308641-vqiipbo8 authors: jankauskaitė, justina; jiménez-garcía, brian; dapkūnas, justas; fernández-recio, juan; moal, iain h title: skempi 2.0: an updated benchmark of changes in protein–protein binding energy, kinetics and thermodynamics upon mutation date: 2019-02-01 journal: bioinformatics doi: 10.1093/bioinformatics/bty635 sha: doc_id: 308641 cord_uid: vqiipbo8 motivation: understanding the relationship between the sequence, structure, binding energy, binding kinetics and binding thermodynamics of protein–protein interactions is crucial to understanding cellular signaling, the assembly and regulation of molecular complexes, the mechanisms through which mutations lead to disease, and protein engineering. results: we present skempi 2.0, a major update to our database of binding free energy changes upon mutation for structurally resolved protein–protein interactions. this version now contains manually curated binding data for 7085 mutations, an increase of 133%, including changes in kinetics for 1844 mutations, enthalpy and entropy changes for 443 mutations, and 440 mutations, which abolish detectable binding. availability and implementation: the database is available as supplementary data and at https://life.bsc.es/pid/skempi2/. supplementary information: supplementary data are available at bioinformatics online. protein-protein interactions are central to almost all biological processes, from cellular signal transduction and the assembly of mesoscopic structures such as myofilaments, to viral adhesion and the immune response. consequently the effects of changes in protein sequence on the structure, thermodynamics and kinetics of proteinprotein interactions has wide implications for constraining the permissible substitutions that accrue over the course of evolution, and for understanding the molecular etiology of disease. methods which measure, predict or optimize these changes have applications in designing de novo interactions (fleishman et al., 2011) , enhancing the specificity and affinity of biological therapeutics (e.g. arkadash et al., 2017) , designing combinatorial protein libraries (e.g. guntas et al., 2010) , uncovering the effects of pathological mutations (e.g. tidow et al., 2006) , locating druggable binding sites (e.g. stevers et al., 2017) and binding hotspots for drug design (guo et al., 2014) , altering binding kinetics (cohen-khait and schreiber, 2016; rosenfeld et al., 2017) , protein-protein docking (e.g. epa et al., 2013) , and characterizing transition states (e.g. wu et al., 2002) , binding pathways (plattner et al., 2017) , and sequence-affinity landscapes (aizner et al., 2014) . skempi is a manually curated database of mutations in structurally characterized protein-protein interactions and the effect of those mutations on binding affinity and other parameters (moal and fernandez-recio, 2012) . the first release has been used as a basis for many further studies, including the development of energy functions (moal and fernandez-recio, 2013; moal et al., 2015b) which were subsequently implemented in the ccharppi web server for characterizing protein-protein interactions (moal et al., 2015a) , as well as being used for ranking docked poses (barradas-bautista et al., 2017; moal et al., 2013; moal et al., 2017; pfeiffenberger et al., 2017) . skempi has also been used to study human disease (das et al., 2014; peng and alexov, 2016; petukh et al., 2015a) , assessing the role of dynamics on binding (sumbul et al., 2015) , exploring the conservation of binding regions (hu et al., 2016) , evaluating experimental affinity measurement methods (geng et al., 2016) , as well serving as a data source for models which predict dissociation rate changes upon mutation (agius et al., 2013) , pathological mutations (gossage et al., 2014) , hotspot residues (e.g. hwang et al., 2014; liu et al., 2015; melo et al., 2016; simoes et al., 2017) and changes in binding energy (e.g. barlow et al., 2018; berliner et al., 2014; dehouck et al., 2013; dourado and flores, 2014; lai et al., 2017; li et al., 2016; moretti et al., 2013; pallara et al., 2013; pantazes et al., 2015; petukh et al., 2015b; pires et al., 2014; xiong et al., 2017; yan et al., 2017; zhao et al., 2014) . here we present a major update to the benchmark in terms of the number of mutations in the database and the number of different systems included (table 1) . we now also include details of the experimental method for all entries, based on the categories of geng et al., 2016 , as well as mutations which abolished detectable binding or for which only an upper or lower affinity limit could be ascertained for the wild-type or mutant. just over two fifths of the data come from the previous version of skempi (moal and fernandez-recio, 2012) , comprised mostly of data found in literature sources which came to the authors' attention, in some cases during the data collection for the structural affinity benchmark (kastritis et al., 2011) and following references therein. some entries in skempi 1.1 were found by checking the references in the asedb (thorn and bogan, 2001) and pint (kumar and gromiha, 2006) databases, although not all the data passed the checks required for inclusion (see section 2.2). similarly, most of the new data in skempi 2.0 was found by searching the literature, partly in tandem with the literature search for the more recent structural affinity benchmark (vreven et al., 2015) . during data collection three other relevant databases were published: abbind (sirin et al., 2016) , proximate (jemimah et al., 2017) and dbmpikt (liu et al., 2017) . their references were checked if they were not already included. data from these sources comprise 4%, 3% and 6% of skempi 2.0 respectively. as with asedb and pint, none of the data were directly copied into skempi. moreover, the cited papers were read and data entered using the same checks and procedures as other entries. each entry was found in the literature and manually vetted. to ensure quality, a number of stringent checks were applied. firstly, we ensured that the structure and the paper reporting the affinities refer to the same protein in the same species, and that structural and affinity data matched in terms of cofactors, ancillary chains and posttranslational modifications. for instance, we distinguish between rasgtp and rasgppnhp, as the nucleotide modulates the affinity of ras with its effectors. where the full-length protein was not used, we checked to ensure that the fragment in the crystal structure matched that for which affinities are reported. once the checks are passed, the data is collected, including the pdb file, the chains of the interacting subunits, the mutation, the wild-type and mutant affinities (k d , m), the reference, the names of the proteins, the temperature at which the experiment is performed (t, k), the experimental method used (an extension of the category scheme of geng et al., 2016) , notes on the entry and, when available, the association rate (k on ; m à1 s à1 ), dissociation rate (k off ; s à1 ), enthalpy (dh; kcal:mol à1 ) and entropy (ds; cal:mol à1 :k à1 ). for cases where multiple pdb entries are available, the higher resolution structure is chosen. where affinities or kinetic or thermodynamic parameters are reported in different units, these are converted to the units specified above. in some cases, when not reported directly, k d , k on ; k off ; dh and ds were calculated using the relationships dg to ensure consistency, the residue numbering in skempi is the same as that reported in the pdb file. thus, the numbering is often shifted or altered compared to that in the cited paper such that, for instance, if a crystal structure of an antibody is reported in the kabat numbering scheme but the mutation data is not, then the mutation data is converted before entry into skempi. for all entries it is the case that the affinity reported in the "wild-type" column corresponds to that of the pdb file, and the affinity in the "mutant" column is that after applying the specified mutation to the protein in the pdb file. thus, where there are cases in which the pdb reports a mutant form and the entry corresponds to the reverse mutation back to the wild-type, the affinity of the former appears in the "wild-type" column and the latter in the "mutant" column. such cases are noted in the database. in addition to checking new entries, we reappraised the papers cited in skempi 1.1 to collect data that were not collected previously, specifically to find mutants which abolish binding and to classify the experimental method used when not already included in the subset of skempi covered in geng et al., 2016 . it is worth noting that often an author's decision to report an interaction of affinity below the detection threshold as either non-binding, or as less affine than the weakest affinity presented in the paper, is arbitrary. thus, those wishing to use the non-binding data as an inequality on the affinity may do so. we also corrected entries for five wild-type and four mutant affinities identified by geng et al., 2016. in addition to the above data, skempi 2.0 also provides data on the location of the mutated residues, the homology between interactions in the dataset, and processed pdb files, which can be easily parsed. residue location: each mutated residue is classified according to the scheme proposed by levy (2010) ; residues at the interface are classified as support (mostly buried when unbound and entirely buried upon binding), core (mostly solvent exposed when unbound but buried upon binding) and rim (partly buried upon binding), while residues away from the binding site are classified as interior or surface. solvent exposed surface area was calculated using ccp4 (winn et al., 2011) . processed pdb files: the pdb files for the interactions, as downloaded from the protein data bank (berman et al., 2000) , often contain multiple copies of the interacting proteins in the unit cell or other chains irrelevant to the interaction. in one instance, the binding of dimeric myostatin to follistatin-like 3, the myostatin dimer must be created by tessellating the unit cell. further, some pdb files contain features that are not readily parsed by some software, such as residue insertion codes or negative residue numbers. to help users we provide "cleaned" pdb files which contain only the chains of interest, renumbered from one, as well as waters and other molecules with a non-hydrogen atom within 5 å of a non-hydrogen atom of any of the chains of interest. consequently, each mutation is reported with both pdb numbering and renumbered. defining homologous interactions: each entry also specifies which other entries are mutations to homologous interactions. two interactions are deemed homologous if they have a shared binding partner or homologous binding partner and at least 70% of the corresponding interface residues are common to both interactions. we determine the homology between proteins using the gap4 program (huang and brutlag, 2007) , and define homologous proteins as those with a similarity score greater than 50 and at least 30% sequence identity. interface residues are defined as those with a nonhydrogen atom within 10 å of a non-hydrogen atom on the binding partner. interactions falling within manually assigned clusters of homologous interactions are designated as pmhc/tcr, antibody/ antigen or protease/inhibitor. while the names of these clusters have been chosen to reflect the predominant function of their constituent interactions, they reflect the homologies within the dataset and are not functional assignments. thus, for instance, some nanobodies are classified as antibodies as they bind to the same site as cetuximab, 14.3.d is classified as tcr, even though it is only the b chain, and its binding partner, enterotoxin c3, is classified as a pmhc. in total 7085 entries were collected, summarized in table 1 and figure 1a . these data were derived from the literature and consequently, while encompassing a broad range of residues, proteins, interactions and systems, are biased toward the interests and capabilities of the research community. these biases are evident in the composition of the database according to parameters shown in figure 1 . the ddg values span a large range, but mostly fall within -3 to 7 kcal:mol à1 (fig. 1b) , for both biophysical and technical reasons (see section 3.3). almost three quarters of the data correspond to single point mutations, and more than half of those are mutations to alanine (fig. 1c) . charge swap mutations and mutations between aromatic residues are also over-represented. most single point mutations are located at the binding site, and most of those are at the core of the interface. similarly, most double mutations are both in the binding site, and most of those are both in the core. by far the most popular methods for measuring binding affinity were surface plasmon resonance and spectroscopic methods such as fluorescence. large biases toward specific interactions and classes of interaction are also present, such as early studies into protease inhibition and immunological interactions such as antibody-antigen complexes, the recognition of peptides presented on cell surfaces by t-cell receptors, cytokine signaling and the complement system. indeed, almost half of the data corresponds to protease-inhibitor, antibody-antigen and pmhc/tcr interactions alone. while many interactions within these classes share common binding sites or homologous binding sites (fig. 2) , there are also connections between these groups, for instance via inhibitory antibodies which bind to a protease active site, or due to common binding regions of proteins in the immunoglobulin superfamily, such as antibodies, tcrs, mhcs and b-2 microglobulin. in addition, present in the data are smaller clusters of shared and homologous interactions, such as the ras-effector cluster. these relationships are noted in the database and may be useful for avoiding overfitting when developing models or for validation and estimating generalization error, as described previously (moal and fernandez-recio, 2012) . the entries also vary in the degree of structural order. while most correspond to interactions between folded domains, the database contains entries in which structuring occurs upon binding, such as protein-peptide interactions and, in the extreme case, the actr/ ncbd interaction in which both binding partners become ordered upon binding (jemth et al., 2014) . indeed, the requirements of having a structure in order to be included in the dataset, ipso facto biases the data, and means that there is no representation of "fuzzy" complexes in which a diffuse structural ensemble in the bound state prevents the formation of a resolvable crystal. another source of variation is the origins of the interacting proteins. while entries range from viral and bacterial to the higher eukaryotes, biases are evident in the over-representation of model organisms including humans. with the exception of the pmhc-tcr, antibody-antigen and protease-inhibitor classes, most of the interactions are endogenous. nevertheless, the set also includes exogenous interactions ranging from those between proteins from different individuals within the same species, namely the sex fusion proteins juno and izumo1 from human sperm and egg respectively (aydin et al., 2016) , to host-pathogen interactions such as adenovirus and coronavirus interactions with human receptors during viral entry (howitt et al., 2003; seiradake et al., 2006) , to the inhibition of acetylcholinesterase by the snake venom neurotoxin fasciculin (aizner et al., 2014) . for the pmhc-tcr interactions, there are a variety of presented antigens, including exogenous viral peptides and gluten, as well as endogenous autoimmune and cancer peptides. the antibody interactions include pathogen antibodies, as well as antibodies raised and optimized to target extracellular therapeutic targets. the protease interactions are mostly exogenous, arising from their inherent cross-reactivity due to the convergent evolution of their canonical inhibitory loop. the investigations from which skempi data is derived are diverse, spanning many biological processes and reported in 295 publications including systematic scans, alanine and homolog scanning, design studies including computational design and designs derived from phage display, double mutant cycle studies, antibody engineering, biologic drug design and the evaluation of pathological mutations. the largest contribution comes from the group of the late michael laskowski jr., a systematic study of all possible mutations at selected sites in the turkey ovomucoid third domain and its inhibitory interactions with four proteases (lu et al., 2001) , as well as studies of interactions of the same domain in other bird species and the design of ultra-high affinity broad-spectrum inhibitors. substantial data also come from investigations into the inhibitory interactions of class a b-lactamases from the groups of gideon schreiber (e.g. reichmann et al., 2007) and timothy palzkill (e.g. brown et al., 2013) , as well as cytokine receptor interactions, in particular studies of type i interferons also from the schreiber group (e.g. roisman et al., 2001) and that of k. christopher garcia (thomas et al., 2011) , but also the study of the gm-csf/gmra interaction from the group of michael w. parker (broughton et al., 2016) . other prominent sources of data are studies into hormone receptor interactions, in particular the human growth hormone receptor from the group of jim wells (e.g. cunningham and wells, 1991) and the prolactin receptor from the group of michael e. hodsdon (kulkarni et al., 2010) , as well as studies into antigen recognition including the combined computational and experimental design study to enhance affinity of the aqc2 antibody to integrin a-1 from the group of herman van vlijmen (clark et al., 2006) , the dissection of the interactions of broadly neutralizing antibodies targeting hiv gp120 (clark et al., 2017) from the group of richard a. friesner, and various investigations from the group of roy a. mariuzza (e.g. dall'acqua et al., 1998) . also notable is the alanine scanning of the urokinase-type plasminogen activator and its receptor from the group of michael ploug (gardsvoll et al., 2006) , studies of ras effector interactions from the group of christian herrmann (e.g. kiel et al., 2004) , investigations of pmhc/tcr interactions from the group of brian baker (e.g. piepenbrink et al., 2013) , and investigations into the cognate and non-cognate recognition of escherichia coli colicin dnase bacteriotoxins by their immunity proteins from the group of colin kleanthous (e.g. li et al., 1997) . range: the changes in binding free energy upon mutation range from -12.4 to 12.4 kcal:mol à1 , as in skempi 1.1, with d log 10 k on ranging from -3.6 to 2.4, d log 10 k off ranging from -6.0 to 6.8, ddh ranging from -18.3 to 26.5 kcal:mol à1 , and dds ranging from -61 to 80 cal:mol à1 :k à1 . around 60 mutants are very destabilizing, reducing binding energy by 8 kcal:mol à1 or more. these are all in enzyme/inhibitor complexes such as the inhibition of acetylcholinesterase by the snake venom fasciculin, or the inhibition of enzymes which would be detrimental should they unbind and become active in the wrong location, such as nucleases (barnase/barstar, colicin e9 dnase/im9, rnase a/angiogenin) and proteases (such as trypsin/ bpti). these interactions tend to be around picomolar affinity and are at the upper limit of what can be detected, due to the time required to reach equilibrium and the low concentrations required by the mass action law to probe informative regions of the binding curve. these very destabilizing mutations reduce affinity into the micromolar range, near the lower limit of what can be quantified using standard methods. as a consequence of both mutant and wildtype affinities being near detection thresholds, errors in these entries are typically large. further, while some mutations may cause changes in affinity larger than seven orders of magnitude, the absence of affinities for such mutations in the benchmark can be explained by the fact that such mutations would involve affinities beyond the upper or lower limit. indeed, there are new entries in which single or double substitutions reduce binding from tens of picomolar to having no detectable binding. for many of the highly destabilizing mutations a crystal structure for the mutant has also been solved, and the 30 most stabilizing mutations in the database (ddg ¡ -5 kcal:mol à1 ) consist of the reverse mutation applied to these structures. these are mostly single or double mutants, but include mutations to up to 27 residues of the non-cognate colicin e2/ im9 complex, which move it toward the cognate e9/im9 in sequence space (li et al., 1997) . errors: standard errors in k d are typically reported in the order of 50%, around 0.25 kcal:mol à1 . these estimates are derived by repeat measurements using the same equipment, environment and protocol, and thus do not include errors arising from systematic bias. such biases can, however, be estimated from pairs of entries in which the same mutation is evaluated by different groups or using different techniques. for 84% of 1741 such pairs, both entries give a ddg value within 1 kcal:mol à1 of each other. for 704 pairs for which k on is available for both, 80% have d log 10 ðk on þ within 0.5 of each other. for 702 pairs for which k off is available, 83% have d log 10 ðk off þ within 0.5. for the 62 pair with both ddh and dds values, 61% have ddh within 3.0 kcal:mol à1 of each other and 58% have dds within 10 cal:mol à1 :k à1 of each other. within skempi, some entries can be combined to construct mutant cycles, which quantify the interactions between residues, the dependence of these interactions on other residues, and other higher order effects. the most common instances are double mutant cycles, where affinities are available for the wild type, a, b and ab mutations, of which there are 610 examples. of these, 53 involve at least one mutant for which binding was not observed, or only an inequality is available, and 235 involve mutations reported in the same reference, and thus the affinities are likely to have been measured using the same technique and conditions. a further 218 double mutant cycles can be constructed in the background of a third mutation (i.e. c, ac, bc and abc mutations are available), of which 209 are not composed of non-binding mutations or mutations with inequalities, and 131 involve affinities coming from the same reference. of the 766 double mutant cycles containing neither inequalities nor non-binding mutants, a number of parameters can be calculated, including ddg ab!ab ; ddg ab!ab and ddg ab!ab , the binding free energy change of both single and the double mutation respectively, as well as ddg ab!ab and ddg ab!ab , the energy of a single mutation within the context of the other mutation, and ddg int ¼ ddg ab!ab -ddg ab!ab ¼ ddg ab!ab à ddg ab!ab , the interaction energy of the two mutations (horovitz and fersht, 1990) . from these, it can be deduced that 345 are additive (ddg int < 0:5kcal:mol à1 ). of the non-additive cycles, 293 exhibit tighter binding in the double mutant than the sum of the single mutants (positive epistasis), of which six result in even tighter binding than individual effects of two single mutations that strengthen the interaction (synergistic positive), while 273 correspond to double mutants which reduce binding by less than the sum of two single mutants which reduce binding (antagonistic positive). similarly, 128 cycles have double mutants exhibiting weaker binding than the sum of the two single mutants (negative epistasis), of which 58 contain two destabilizing single mutations (synergistic negative) and 26 contain two stabilizing single mutations (antagonistic negative). the range of ddg int values rarely fall outside of the -5 to 3 kcal:mol à1 range. of the 421 non-additive cycles, 151 show noticeable sign epistasis, in which the sign of the effect of either the a or b mutation flips depending on the presence or absence of the background mutation (i.e. for the a mutation, jddg ab!ab j > 0:2 kcal:mol à1 and jddg ab!ab j > 0:2 kcal:mol à1 and jddg ab!ab à ddg ab!ab j > 0:4 kcal:mol à1 ). of these, 38 correspond to mutations, which destabilize the complex in the presence of the background mutation, but stabilize in its absence (destabilizing sign epistasis), while 113 correspond to mutations, which stabilize the complex in the mutant background but otherwise destabilize the complex (stabilizing sign epistasis). only eight cycles exhibit the more extreme reciprocal sign epistasis, which in six cases are where both single mutations are stabilizing ( < à0:2 kcal:mol à1 ), but the double mutant is destabilizing (>0.2 kcal:mol à1 ), and the remaining two correspond to two destabilizing mutations (>0.2 kcal:mol à1 ) for which the double mutation is stabilizing ( < à0:2 kcal:mol à1 ). the types of substitutions that can give rise to extreme effects such as stabilizing reciprocal sign epistasis can be illustrated with the mlc-iibglc interaction in e. coli (nam et al., 2008) , in which the removal of the f136 side-chain of mlc creates a large cavity at the binding interface, the addition of a phenylalanine at the a451 position of iibglc creates a large clash, however the double mutation creates an interaction that is even more stable than the wild-type by creating an anchor residue across the binding interface in which the cavity in mlc is filled by the new side-chain of iib. higher order interaction terms can be garnered from higher cycles, such as triple mutant cubes, constructed from the energies of the wild-type, three single mutants, three corresponding double mutants and the triple mutant (horovitz and fersht, 1990) . in skempi, 45 triple mutant cubes can be made, with 10 coming from the same reference. for these, third order interaction energies fall within the -1 to 1 kcal:mol à1 range. for fourth order interactions, constructed from energies of the wild-type, four single mutants, six double mutants, four triple mutants and the quadruple mutant, 14 examples exist within skempi. however, care should be taken in ascribing meaning to fourth order residue coupling energies due to the accumulations of errors, which in these cases are exacerbated by the affinities having been reported in different publications. no fifth or higher order interactions are present. the database is accessible online at https://life.bsc.es/pid/skempi2/, where the raw csv (comma-separated values) file containing all the data can be downloaded. the data can also be browsed online, ordered and searched by any field, such as the experimental method or the location of the mutation, or searching for a specific protein by its name or pdb code, and structures may be visualized. other pages on the web site offer a summary of the data, an faq and help page, and a page for user contributions, which will be evaluated to appear in future releases. characterizing changes in the rate of protein-protein dissociation upon interface mutation using hotspot energy and organization mapping of the binding landscape for a picomolar protein-protein complex through computation and experiment development of high affinity and high specificity inhibitors of matrix metalloproteinase 14 through computational design and directed evolution molecular architecture of the human sperm izumo1 and egg juno fertilization complex flex ddg: rosetta ensemble-based estimation of changes in protein-protein binding affinity upon mutation a systematic analysis of scoring functions in rigid-body protein docking: the delicate balance between the predictive rate improvement and the risk of overtraining combining structural modeling with ensemble machine learning to accurately predict protein fold stability and binding affinity effects upon mutation the protein data bank conformational changes in the gm-csf receptor suggest a molecular mechanism for affinity conversion and receptor signaling identification of the b-lactamase inhibitor protein-ii (blip-ii) interface residues essential for binding affinity and specificity for class a b-lactamases free energy perturbation calculation of relative binding free energy between broadly neutralizing antibodies and the gp120 glycoprotein of hiv-1 affinity enhancement of an in vivo matured therapeutic antibody using structure-based computational design low-stringency selection of tem1 for blip shows interface plasticity and selection for faster binders rational design of receptor-specific variants of human growth hormone a mutational analysis of binding interactions in an antigen-antibody protein-protein complex elucidating common structural features of human pathogenic variations using large-scale atomic-resolution protein networks beatmusic: prediction of changes in protein-protein binding affinity on mutations a multiscale approach to predicting affinity changes in protein-protein interfaces structural model for the interaction of a designed ankyrin repeat protein with the human epidermal growth factor receptor 2 computational design of proteins targeting the conserved stem region of influenza hemagglutinin characterization of the functional epitope on the urokinase receptor. complete alanine scanning mutagenesis supplemented by chemical cross-linking exploring the interplay between experimental methods and the performance of predictors of binding affinity change upon mutations in protein complexes an integrated computational approach can classify vhl missense mutations according to risk of clear cell renal carcinoma engineering a protein-protein interface using a computationally designed library hot spot-based design of small-molecule inhibitors for protein-protein interactions strategy for analysing the co-operativity of intramolecular interactions in peptides and proteins structural basis for variation in adenovirus affinity for the cellular coxsackievirus and adenovirus receptor conservation of hot regions in protein-protein interaction in evolution dynamic use of multiple parameter sets in sequence alignment binding interface prediction by combining protein-protein docking results proximate: a database of mutant protein-protein complex thermodynamics and kinetics a frustrated binding interface for intrinsically disordered proteins a structure-based benchmark for protein-protein binding affinity a detailed thermodynamic analysis of ras/effector complex interfaces two independent histidines, one in human prolactin and one in its receptor, are critical for ph-dependent receptor recognition and activation pint: protein-protein interactions thermodynamic database enhancing structure prediction and design of soluble and membrane proteins with explicit solvent-protein interactions a simple definition of structural regions in proteins and its use in analyzing interface evolution mutabind estimates and interprets the effects of sequence variants on protein-protein interactions protein-protein interaction specificity of im9 for the endonuclease toxin colicin e9 defined by homologue-scanning mutagenesis co-occurring atomic contacts for the characterization of protein binding hot spots dbmpikt: a web resource for the kinetic and thermodynamic database of mutant protein interactions predicting the reactivity of proteins from their sequence alone: kazal family of protein inhibitors of serine proteinases a machine learning approach for hot-spot detection at protein-protein interfaces skempi: a structural kinetic and energetic database of mutant protein interactions and its use in empirical models intermolecular contact potentials for protein-protein interactions extracted from binding free energy changes upon mutation the scoring of poses in protein-protein docking: current capabilities and future directions ccharppi web server: computational characterization of protein-protein interactions from structure inferring the microscopic surface energy of protein-protein interfaces from mutation data irappa: information retrieval based integration of biophysical models for protein assembly selection community-wide evaluation of methods for predicting the effect of mutations on protein-protein interactions analyses of mlc-iibglc interaction and a plausible molecular mechanism of mlc inactivation by membrane sequestration expanding the frontiers of protein-protein modeling: from docking and scoring to binding affinity predictions and other challenges the iterative protein redesign and optimization (ipro) suite of programs investigating the linkage between disease-causing amino acid variants and their effect on protein stability and binding on human disease-causing amino acid variants: statistical study of sequence and structural patterns predicting binding free energy change caused by point mutations with knowledge-modified mm/pbsa method a machine learning approach for ranking clusters of docked protein-protein complexes by pairwise cluster comparison the basis for limited specificity and mhc restriction in a t cell receptor interface mcsm: predicting the effects of mutations in proteins using graph-based signatures complete protein-protein association kinetics in atomic detail revealed by molecular dynamics simulations and markov modelling binding hot spots in the tem1-blip interface in light of its modular architecture structure of the interferon-receptor complex determined by distance constraints from double-mutant cycles and flexible docking improved antibody-based ricin neutralization by affinity maturation is correlated with slower off-rate values structural and mutational analysis of human ad37 and canine adenovirus 2 fiber heads in complex with the d1 domain of coxsackie and adenovirus receptor new parameters for higher accuracy in the computation of binding free energy differences upon alanine scanning mutagenesis on protein-protein interfaces ab-bind: antibody binding mutational database for computational affinity predictions structural interface between lrrk2 and 14-3-3 protein allosteric dynamic control of binding structural linkage between ligand discrimination and receptor activation by type i interferons asedb: a database of alanine mutations and their effects on the free energy of binding in protein interactions effects of oncogenic mutations and dna response elements on the binding of p53 to p53-binding protein 2 (53bp2) updates to the integrated protein-protein interaction benchmarks: docking benchmark version 5 and affinity benchmark version 2 overview of the ccp4 suite and current developments two-step binding mechanism for t-cell receptor recognition of peptide mhc bindprofx: assessing mutation-induced binding affinity change by protein interface profiles with pseudo-counts interaction entropy for computational alanine scanning determining effects of non-synonymous snps on protein-protein interactions using supervised and semi-supervised learning key: cord-294712-kvvxmvqo authors: pelosse, martin; crocker, hannah; gorda, barbara; lemaire, paul; rauch, jens; berger, imre title: multibac: from protein complex structures to synthetic viral nanosystems date: 2017-10-30 journal: bmc biol doi: 10.1186/s12915-017-0447-6 sha: doc_id: 294712 cord_uid: kvvxmvqo the multibac baculovirus/insect cell expression vector system was conceived as a user-friendly, modular tool-kit for producing multiprotein complexes for structural biology applications. multibac has allowed the structure and function of many molecular machines to be elucidated, including previously inaccessible high-value drug targets. more recently, multibac developments have shifted to customized baculoviral genomes that are tailored for a range of applications, including synthesizing artificial proteins by genetic code expansion. we review some of these developments, including the ongoing rewiring of the multibac system for mammalian applications, notably crispr/cas9-mediated gene editing. multiprotein complexes are vital cornerstones of most, if not all, biological processes in cells. to understand the essential mechanisms of these molecular machines, a detailed understanding of their architecture and interactions is required. several complexes, such as ribosomes, can be purified from native source material in sufficient quality and quantity for structural analysis. other complexes have low endogenous abundance, impeding their extraction. heterogeneity can further complicate protein complex purification and structure elucidation. in higher eukaryotes in particular, many complexes can contain distinct subunit isoforms in variable combinations in different tissues. moreover, the subunit composition can be altered according to cell state. for these complexes, recombinant production and purification by using heterologous host cell systems is typically required. we encountered this challenge over a decade ago, when working with tim richmond in his laboratory at eth zürich on multiprotein complexes that control * correspondence: imre.berger@bristol.ac.uk 1 the school of biochemistry and bristol synthetic biology centre brissynbio, university of bristol, tankard's close, bristol bs8 1td, uk full list of author information is available at the end of the article transcription initiation in higher eukaryotes, including humans (reviewed in [1] ). transcription is central to all biological function, and more than a hundred proteins contribute to its regulation. in humans, many of these are organized in multiprotein complexes with ten or more subunits. post-translational modifications that may require authentic recapitulation in heterologous expression experiments are likewise prevalent. moreover, subunits can be very large, with individual molecular weights exceeding 200 kda. after substantial trial and error, we realized that heterologous expression in escherichia coli, which dominated recombinant expression then and still is a preferred choice for many today, was unsuitable for the task at hand. we then turned our attention to eukaryotic methods, specifically an expression vector system relying on a recombinant baculovirus, to infect insect cell cultures for producing the complexes in which we had interest. baculovirus was originally trialed as a pesticide, to control crop damage caused by spodoptera frugiperda (the fall armyworm), with limited success initially, although more positive results have recently been reported [2] [3] [4] [5] . subsequently, pioneering work, in particular by max summers and colleagues, revealed that baculovirus is useful for very high-level expression of heterologous genes in insect cell laboratory culture (reviewed in [5, 6] ). among the many advantageous features of the baculovirus/insect cell system, the large dna cargo capacity was particularly notable. baculovirus, in contrast to other viruses that have a crystalline shell, has a flexible envelope that can grow with increasing size of the genome packaged within the baculovirion without adversely affecting baculovirus function. this particular feature, and the-at least conceptually-relative ease of manipulating baculovirus in the laboratory by non-expert users, was exciting and led us on to the development of multibac: a baculovirus/insect cell system specifically engineered for expressing functional multiprotein complexes in the quality and quantity required for high-resolution structural and mechanistic studies [7] [8] [9] [10] [11] . multibac went on to be broadly adopted and used for research in both industry and academic institutions [12] . the multibac system, and its application to protein complex production, has been described in considerable detail previously [8] [9] [10] [11] 13] . in summary, multibac consists of an engineered baculoviral genome, which is based on the a. californica multiple nuclear polyhedrosis virus (acmnpv). this genome was previously adapted for propagation and manipulation in bacterial cells in the form of a bacterial artificial chromosome (bac) [14, 15] . starting from this bac, we progressively removed functionalities that we identified experimentally to be detrimental to protein complex production. for instance, proteolytic and apoptotic viral factors were eliminated [10] . baculovirus infection of insect cells is a lytic process as the virus remodels the host cell machinery for producing baculovirions at high levels, resulting ultimately in cell death and lysis. in fact, the highest level of production of the recombinant protein of interest coincides with the onset of widespread cell lysis, which can compromise the protein produced. the introduced genome alterations resulted in a virus that exhibited delayed lysis of the insect cells, allowing the production of recombinant protein complexes at very high levels while the cells seemingly remained intact [11] . heterologous genes of interest are inserted into the multibac baculoviral genome by means of transfer plasmids, which are transformed into so-called dh10multi-bac cells harboring the bac (fig. 1) . we prepared an array of small synthetic plasmid dna precursor modules to generate multigene transfer plasmids that were easily inserted into the multibac genome by using tandem recombineering (tr), which can optionally be done in high-throughput [13, [16] [17] [18] [19] . tr is a method we conceived to facilitate dna assembly by iteratively exploiting sequence and ligation independent cloning (slic) to insert genes of interest into individual plasmid dna precursors. these are then concatenated by cre-loxpmediated plasmid fusion and then inserted into the mul-tibac baculoviral genome by transposition, catalyzed by the tn7 transposase. the transposase is provided in the dh10multibac cells from a separate helper plasmid coexisting with the bac. although we mostly use slic to manipulate our heterologous genes of interest, our plasmid modules also contain multiple cloning sites for the multibac baculovirus expression vector system. multibac consists of a baculoviral genome that we engineered for optimal multigene delivery and protein complex expression (left). the multibac genome is propagated as a bacterial artificial chromosome (bac) in escherichia coli cells that supply the tn7 transposition function from a co-existing helper plasmid. several methods have been successfully applied to assemble expression cassettes containing genes of interest and gene regulatory elements. we introduced tandem recombineering (tr), a method that relies on iterative cycles of sequence and ligation independent cloning (slic) coupled to cre-mediated fusion of plasmid dna precursor elements [9, 19] . other alternatives include the uracil-specific excision reagent (user) and bigbac methods [20, 21] , in addition to conventional restriction/ ligation-based cloning techniques. we engineered a second entry option into the viral backbone, which can accept additional functionalities by site specific recombination into the viral loxp site. composite multibac baculoviral dna containing all dna elements of interest is extracted. transfection of insect cell cultures in small scale yields live multibac virions. these can be used for a wide range of applications (right). the viral loxp site is shown as a circle filled in red. orf open reading frame, sbdd structure-based drug design, hcs high-content screening, vlp virus-like particle, cryo-em structure determination by electron cryo-microscopy, x-ray x-ray crystallography, kan kanamycin resistance marker, amp ampicillin, lacz laczα gene enabling blue/white selection, mini-atttn7 attachment site for tn7 transposition. the schematic drawing of the baculovirion was kindly provided by kari airenne conventional restriction/ligation cloning. moreover, others have provided valuable alternatives (such as uracil-specific excision reagent (user) cloning and bigbac) to assemble large transfer plasmids that contain many genes for integration into the multibac baculoviral genome [20, 21] (fig. 1) . the user approach, for instance, is a ligationindependent cloning method utilizing the incorporation of uracil in the primers to delimit sticky overhangs generated by exonuclease treatment; user facilitates assembly of multigene expression constructs for producing the apc/c multiprotein complex [20] . the bigbac method provides a selection of thoroughly validated primer dnas for multigene expression cassette assembly relying on the gibson method, and facilitated reconstitution of the kinetochore complex [21] . note that tr, user and bigbac (and other approaches such as conventional restriction digestion/ ligation) are not mutually exclusive methods to assemble dna into multi-expression cassettes for insertion into multibac baculoviral genomes but can be used in combination according to the requirements of the project. multibac was first adopted by the structural biology community and has enabled structure elucidation of many multiprotein complexes, illuminating their cellular functions. here, we show a selection of recent structures obtained with multibac-produced specimens [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] (fig. 2) . noteworthy in this context are the highresolution structures of influenza polymerase-the longelusive complex that the influenza virus uses to replicate and transcribe its genome [32] [33] [34] [35] . influenza polymerase consists of three subunits, pa, pb1 and pb2, with many functions, including cap-snatching and endonuclease activities. since its discovery decades ago, influenza polymerase has been studied intensively, but atomic resolution structures of this coveted drug target have remained elusive owing to lack of high quality samples. influenza polymerase production by using the multibac system allowed the polymerase to be crystallized. the crystal structures of polymerases from three major influenza types, a, b and c, were determined, revealing functional aspects of these protein machines in unprecedented detail (fig. 3) . interestingly, efficient production of influenza a and b polymerases prenecessitated the implementation of a polyprotein approach mimicking the strategy certain viruses, such as coronavirus, exploit to realize their proteome. in our polyproteins, the genes encoding the subunits of the complex studied are combined in a single large open multiprotein complex structural biology. a selection of recent near-atomic structures of multibac-produced specimens, determined by x-ray crystallography or electron cryo-microscopy [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] . in the case of mtor complex 1, heterologous raptor was produced by using multibac and crystallized, and the crystal coordinates fitted into a cryo-em map of endogenously purified holo-complex in a hybrid approach [31] . accession numbers from top left to bottom right are emdb 8166 (α-mannosidase), pdb 5k5s (ca 2+ sensing receptor), pdb 5u1t (separase-securin complex), pdb 5fib (sphingomyelinase), pdb 5l3x (nelf-ac), pdb 5i6i (acetyl-coa carboxylase), pdb 5udl (ifit1), pdb 5me3 (scc2), emdb 4029 (pds5b-cohesin complex) and pdb 5flc (mtor complex 1) reading frame (orf). this orf is integrated into the multibac genome, giving rise to a large protein that is subsequently processed by a highly specific protease (in our case tev nia) that is likewise encoded by the orf. the protease cleaves specific recognition sites present between the subunits, thus releasing them in a precise stoichiometric ratio, resulting in the functional complex [8, 36, 37] . when multibac was initially conceptualized, the dna sequence loxp was engineered into the viral backbone. this provided the means to integrate further genes of interest by cre-loxp fusion into this second locus, distal to the tn7 attachment site. our incentive was to provide the option for distributing the genes encoding a multiprotein complex, in case it became inconvenient to integrate many expression cassettes into a single site on the viral backbone. this turned out to be less of a concern than we had anticipated, and therefore this viral loxp site was initially used only to integrate a fluorescent marker, yellow fluorescent protein (yfp). this allowed real-time virus performance to be tracked and the optimal timepoint for harvesting the cell cultures to be determined [13] . in addition, the viral loxp site turned out to be useful for introducing specific functionalities, giving rise to customized genomes with tailored functions (fig. 4) , which we termed synthetic viral nanosystems (svns). with the grabherr group in vienna, we created sweet-bac, a svn designed to tailor the glycosylation pattern of secreted proteins expressed in insect cells [38] [39] [40] [41] . by exploiting the viral loxp site, mammalian glycosylases were integrated into the bac, which converted the insectcell-specific high-mannose type glycosylation into the complex sugars associated with human proteins. conversely, by introducing the deglycosylases pngase or endoh, respectively, we created svns that entirely removed sugars from the recombinant proteins. this is often preferred for structure elucidation by x-ray crystallography, in which a highly homogenous sample is required, ideally lacking variable extensions, such as sugars, which could impede crystallization. a further posttranslational modification that can lead to heterogeneity is phosphorylation, which may or may not be authentic in insect cell expression. in our study of isw, a chromatin remodeling enzyme complex, we found that the svn that expresses λ-phosphatase from the viral loxp site could remove the phosphate groups quantitatively from the recombinant complex, resulting in a highly homogenous sample that could be dissected mechanistically [42, 43] . following the same logic, further svns were developed for improved manufacturing of selected classes of protein biologics of pharmacological interest. kinases are cornerstones of signal transduction in cells, and many disease states, notably cancer, are caused by malfunction of kinases and their interactions. smallmolecule drugs acting as kinase inhibitors are prolifically used in cancer chemotherapy, and the discovery of new and better kinase inhibitors is a major driving force in oncology research and development. a vital prerequisite to sustain these efforts is the supply of high-quality recombinant kinases for screening and structure-based design approaches. kinases are often inherently fragile proteins that require helper molecules, such as chaperones, for proper folding during expression. to enhance recombinant production of properly folded and soluble kinases, we designed svns that contain up to seven chaperones in the viral loxp site (fig. 4) . to balance the quantities of individual chaperones produced, we again applied our polyprotein strategy. the resulting "kinase factory" produced previously unobtainable kinases at high levels, in soluble and functional form. another class of protein biologics of acute interest are virus-like particles (vlps), which mimic live viruses but are safe and non-infectious as they lack genetic content. vlps therefore represent promising candidates for vaccination to combat infectious diseases [44] [45] [46] . the influenza m1 capsid is a powerful driver of vlp formation in insect cells. we therefore integrated a gene that expressed m1 from influenza strain h1n1 into the viral loxp site, together with a gene encoding the fluorescent protein mcherry, which we used to monitor virus performance (fig. 4) . we used this svn for high-throughput production and validation of an array of influenza vlps with a large number of immune-modulating mutations in their hemagglutinin (ha) gene [47] . an unusual application of multibac technology involved the generation of recombinant adenovirus associated vector (raav) for gene therapy [48] [49] [50] [51] . here, multibac was used to provide all components (rep78, rep72, aav virion coat proteins and the transgenes) required to co-produce gene-therapy-competent raavs in sf9 insect cell cultures. the components spontaneously assemble to form intact raavs. the system was used to produce raavs encoding leptin, for gene therapy of obesity in laboratory rodents. although the gene therapy successfully resulted in weight-loss, it was nonetheless noted in the report that best results were achieved when the gene therapy regimen was accompanied with exercise using a tread-mill [48] , somewhat dampening optimism that exercise-free weight loss may be achievable. originally, this approach involved co-transfection of three individual multibac viruses to obtain intact raavs, constraining logistics. more recent refinement of the system, including integrating the encoding genes directly into the genome of the host sf9 cells, resulting in a streamlined manufacturing process representing a viable alternative for raav production [48] [49] [50] [51] . genetic code expansion (gce) incorporates artificial amino acids into polypeptide chains to create synthetic proteins with novel functions; it has many applications, ranging from discovery science to molecular medicine [52] [53] [54] . in gce, an orthogonal trna/cognate synthetase pair is used to suppress a rare stop codon introduced at a specific site in a gene of interest. the trna acts as a stop codon suppressor, effectively repurposing the rare stop codon of choice into a functional sense codon during protein synthesis, which leads to the site-specific incorporation of an artificial amino acid supplemented in the culture medium. a wide range of functionalities from protein labeling to photo-control can be introduced by using gce in biotechnology and biomedical research [55] [56] [57] . until recently, this method has been mostly confined to small individual proteins, expressed in e. coli or mammalian cells, representing a limited repertoire of cellular activity. together with the lemke and braese groups, we extended the scope of this method by combining gce with the multibac system. this gave rise to multibactag (fig. 4) , a protein engineering platform that enables gce in complex multiprotein machines expressed in insect cells [58] . to customize the multibac baculovirus for gce, the pyrolysyl trna/trna synthetase (pylrs/trna pyl ) pair from methanosarcina mazei was chosen. the gene encoding the synthetase and a cassette for producing trna pyl was inserted into the viral loxp site. the design of the trna production cassette proved to be surprisingly complicated. the available insect rna polymerase iiidependent promoters for rna production (bombyx mori u6 and drosophila melanogaster u6) did not function in the s. frugiperda sf21 cells that we typically use for multibac-based protein production. overcoming this bottleneck required sequencing the sf21 genome to identify a sf21 u6 promoter which supported efficiently the production of m. mazei trna pyl , leading to effective rare stop codon suppression and incorporation of artificial amino acids derived from pyrolysine into the proteins expressed with the multibactag svn. current multibactag applications include artificial amino acid cross-linking to map interactions in protein complexes, fluorescence labeling of specific targets to measure structure and dynamics in proteins and glycolengineering proteins compatible with human tissue studies. we anticipate that the platform will also be adopted for custom-design proteins for therapeutic biotechnology and pharmaceutical applications. as an example, we used multibactag to engineer trastuzumab (also known as herceptin), an antibody that associates with cancer cells, to recognize breast cancer cells in human tissue [58, 59] . multigene delivery and subsequent cellular expression is a key technology for a wide range of applications in biology, not only in structural research but also cellular reprogramming and functional pharmaceutical screening. the construction of multigene circuits in mammalian cells is a core concept in synthetic biology and requires efficient delivery of complex heterologous dna. for certain cell types, including the widely used hek293 and hela cells, this can be achieved by plasmid-based transfection. however, many cell lines, including primary cells, are recalcitrant to plasmid transfection and therefore require a different approach. baculovirus in its original form is highly selective for infecting insect cells, which is among the reasons why baculovirus/insect cell expression can be carried out at standard laboratory biological safety levels. at very high concentrations, however, baculovirus can also penetrate mammalian cells. this process is called transduction rather than infection as the baculovirus, in contrast to mammalian viral pathogens, will not replicate in mammalian cells. if the baculoviral genome that has been transduced into the mammalian host cell contains an expression cassette with a mammalian cell active promoter, expression of the transgene occurs [60, 61] . this "bacmam" gene delivery approach is attractive owing to the very large dna cargo capacity of baculovirus. in principle, a single baculovirus can deliver many genes of interest at the same time, a result that is difficult to achieve by plasmid-based co-transfection. we customized the multibac baculovirus by integrating the gene encoding vesicular stomatitis virus g protein (vsv-g), a protein which was shown to substantially increase the transduction efficacy [62] . the resulting multi-bacmam baculovirion displays vsv-g glycoproteins on the virion surface, and was well suited for transducing a wide range of established mammalian cells with unprecedented efficacy [63] . primary cells are a central focus of current biological research efforts, and multigene delivery in primary cells is highly desirable; however, suitable tools have been markedly lacking. we demonstrated that multibac-mam potently transduced primary cells (fig. 5) . moreover, multibacmam could be used to reprogram fibroblasts into neurons, with virtually identical efficiency as a lentiviral approach. of note, multibacmam transduction is transient in nature and does not permanently alter the genome, in contrast to the genomic integration of lentivirus. the very large dna cargo capacity of the baculovirion, and its aptitude to transduce mammalian cells, raises the attractive possibility to use this tool, in conjunction with powerful gene editing technologies, to rectify genetic aberrations, possibly in whole organisms. the gene encoding cas9 and the modalities to produce guide rnas and a gene encoding egfp as a model dna cargo were integrated into the multibacmam system. we showed that this multibacmam genome engineering tool could be used for crispr/cas9-mediated insertion of egfp into the hmg locus of a range of cell types, including neurons [63] . thus, multibacmam can now be developed into a bona fide genome-engineering tool, characterized by an unsurpassed dna cargo capacity that is orders of magnitude larger than the commonly used gene therapy viral vectors (lentivirus, aav and adenovirus) that currently dominate the field. the original acmnpv genome from which the multibac is derived contains all the information required for the virus to sustain its life cycle in nature, starting from uptake by the host (the fall armyworm), the infection of midgut cells, followed by massive amplification and release of occluded viral particles when the worm lyses. many of these functions are dispensable, or even detrimental, in laboratory cell culture. the advent of powerful dna synthesis and assembly technologies has now made it feasible to "rewire" entire genomes. genome improvement by eliminating undesired functionalities is a major ambition in current synthetic biology. the baculoviral genome is around 140 kb, which is the size of an average bac, and within the size that is capable of complete de novo synthesis, as recently shown [64] . a disadvantage of currently available recombinant baculoviral systems, including multibac, is genome instability during virus amplification. the baculovirus progressively eliminates dna from its genome when serially passaged over several generations, often most severely affecting the heterologous dna to be expressed [65] [66] [67] . in extreme cases, only a few full-size baculoviral genomes are present after several rounds of amplification in cell culture, and small circular dnas containing tiny fractions of the baculoviral genome dominate. these survive by scavenging on the packaging proteins produced from the full-size specimens, overtaking the viral population. for academic research requiring typically only a few milligrams of protein sample, this can be controlled in a relatively straightforward manner by avoiding virus overamplification [9, 10, 68] . however, this is much more difficult and often impossible to achieve at scales relevant for pharmaceutical manufacturing. here, large volumes of virus and therefore many cycles of amplification are required to infect fermenterscale production runs. an improved baculoviral genome resolving this handicap would thus be a major advance. we carried out a comparative study of all available baculoviral genomes in sequence data bases and combined this with data mining to delineate a putative minimal baculoviral genome, synbac, that would be capable of sustaining its propagation and high-level heterologous protein production in cell culture [69] . based on our data, the baculoviral genome was divided into segments to be individually streamlined through the elimination of dna elements that we identified as dispensable for our purposes (fig. 6) . the maximally condensed synthetic dna sections can then be subsequently grafted back into the multibac baculoviral genome to replace the wild-type sequence, and tested for functionality. synthetic sections that pass rigorous validation are then iteratively combined until the minimal, fully rewired synbac baculoviral genome is accomplished. we showed that 30 kb of the multibac baculoviral genome could be altered in this way. the firstgeneration virus generated (synbac1.0) was not only fully functional in terms of high-level protein production, but already showed a major improvement in genome stability during serial passaging of the virus (fig. 6 ). this validated the approach and addressed one of the major current shortcomings of baculoviral expression, genome instability. the multibac system was shown in its first configuration in 2004 [11] , which had been developed to address the challenge of efficient multiprotein complex production for structural biology applications. subsequently, we received requests from laboratories in both academia and industry exemplifying the need for such a tool-kit, a trend that has not since subsided. the numerous important structures elucidated using multibac-produced samples evidenced how useful the system is. we, and others, invested considerable effort over the years to streamline multibac, rendering it more user-friendly and accessible, with improvements such as alternative methods for inserting genes of interest. more recently, the scope of the system has been expanded by customizing the genome for specific applications, as well as altering the tropism of the virus to enable efficient multigene transfer in a wide range of mammalian cell types and organisms. in our view, genomic intervention mediated by nucleases (e.g. cas9, talens and zinc-fingers), is a particularly promising application. combining gene editing with the very large (>100 kb) heterologous dna cargo capacity of baculovirus could be instrumental for future gene therapy applications. we anticipate that next-generation genomic intervention strategies will critically depend on providing complex multicomponent functionalities, including targeting activities and host immune-system modulators in a single multifunctional dna delivery tool. synbac, the synthetic baculovirus, could be a tool of choice, providing currently unmatched dna cargo capacity in an optimized genome. minimizing and optimizing the baculoviral genome to create synbac. a the dna elements in the wild-type acmnpv baculoviral genome were classified according to perceived importance for cell culture applications, derived from exhaustive comparative genome analysis and data mining [69] , and distributed into fragments (i to vii) for rewiring and condensing, with the aim to eliminate surplus dna and improve virus performance in the laboratory and in manufacturing. b experiments with synbac1.0, a multibac-derivative comprising a condensed fragment i, showed powerful expression of a test protein (marked by arrowhead), indistinguishable from multibac based on coomassie-stained sds-page. numbers indicate molecular weight marker sizes (in kda). t total cell extract, s soluble protein. c serial passaging of synbac1.0 expressing a large polygenic test open reading frame (orf) containing a red fluorescent protein marker (dsred) evidenced a remarkable increase of synbac1.0 genome stability compared with multibac. more than 70% of infected cells still produce dsred in fluorescent micrographs, indicating the presence of intact genomes. by contrast, identically overamplified multibac virus frequently lost dsred, resulting in a much lower proportion (<20%) of infected cells producing dsred zooming in on transcription preinitiation baculovirus insecticide production in insect larvae insect pathogens as biological control agents: back to the future baculovirus insecticides in latin america: historical overview, current status and future perspectives milestones leading to the genetic engineering of baculoviruses as expression vector systems and viral pesticides thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology baculovirus expression: old dog, new tricks multibac: expanding the research toolbox for multiprotein complexes multibac: multigene baculovirus-based eukaryotic protein complex production protein complex expression by using multigene baculoviral vectors baculovirus expression system for heterologous multiprotein complexes the multibac baculovirus/insect cell expression vector system for producing complex protein biologics new baculovirus expression tools for recombinant protein complex production efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in escherichia coli baculovirus systems for the expression of human gene products automated unrestricted multigene recombineering for multiprotein complex production getting a grip on complexes robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells tandem recombineering by slic cloning and cre-loxp fusion to generate multigene expression constructs for protein complex research recombinant expression and reconstitution of multiprotein complexes by the user cloning method in the insect cellbaculovirus expression system bigbac enables rapid gene assembly for the expression of large multisubunit protein complexes higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the cvt vesicle structural mechanism of ligand activation in human calcium-sensing receptor structure of human ifit1 with capped rna reveals adaptable mrna binding and mechanisms for sensing n1 and n2 ribose 2′-o methylations molecular mechanism for the regulation of yeast separase by securin crystal structure of mammalian acid sphingomyelinase architecture and rna binding of the human negative elongation factor structure of the cohesin loader scc2 topology and structure of an engineered human cohesin complex bound to pds5b the dynamic organization of fungal acetyl-coa carboxylase structure of influenza a polymerase bound to the viral rna promoter structural insight into cap-snatching and rna synthesis by influenza polymerase structural basis of an essential interaction between influenza polymerase and pol ii ctd crystal structure of the rna-dependent rna polymerase from influenza c virus multiprotein complex production in insect cells by using polyproteins polyproteins in structural biology applying multibac technology towards flexible modification of insect cell glycosylation multibac turns sweet sweetbac: a new approach for the production of mammalianised glycoproteins in insect cells multiprotein expression strategy for structural biology of eukaryotic complexes structure and mechanism of the chromatin remodelling factor isw1a intrastructural help: improving the hiv-1 envelope antibody response induced by virus-like particle vaccines exploiting virus-like particles as innovative vaccines against emerging viral infections virus like particle-based vaccines against emerging infectious disease viruses production of influenza virus-like particle (vlp) array by using vlp-factorytm, a multibac baculoviral genome customized for enveloped vlp expression synergy between leptin therapy and a seemingly negligible amount of voluntary wheel running prevents progression of dietary obesity in leptinresistant rats a simplified purification protocol for recombinant adeno-associated virus vectors onebac: platform for scalable and high-titer production of adenoassociated virus serotype 1-12 vectors for gene therapy onebac 2.0: sf9 cell lines for production of aav1, aav2, and aav8 vectors with minimal encapsidation of foreign dna the use of unnatural amino acids to study and engineer protein function the expanded genetic alphabet genetic code expansion enabled site-specific dual-color protein labeling: superresolution microscopy and beyond the exploding genetic code expanding and reprogramming the genetic code of cells and animals adding new chemistries to the genetic code genetic code expansion for multiprotein complex engineering clinical pharmacology of trastuzumab baculovirus-mediated gene transfer into mammalian cells bacmam technology and its application to drug discovery efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus g glycoprotein highly efficient baculovirus-mediated multigene delivery in primary cells construction and rescue of a functional synthetic baculovirus autographa californica baculoviruses with large genomic deletions are rapidly generated in infected insect cells pivotal role of the non-hr origin of dna replication in the genesis of defective interfering baculoviruses spontaneous excision of bac vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells the multibac protein complex production platform at the embl gene gymnastics: synthetic biology for baculovirus expression vector system engineering we thank all members of the berger laboratory, in particular deepak balaji govinda raj, for helpful discussions. kapil gupta is acknowledged for help with structure representations. kari airenne (university of eastern finland) kindly provided the schematic drawing of the bacuvirion. we thank daniel fitzgerald (geneva biotech, switzerland) and ismail moarefi (crelux gmbh, germany) for useful comments on the manuscript, and tim richmond (eth zurich, switzerland) for encouragement and support at the early stages of the project. this work was supported by the science foundation ireland (grant number 14/ia/3295, to jr) and the european commission framework programme 7 projects synsignal (contract number 613879, to jr and ib) and complexinc (contract number 279039, to ib). the authors acknowledge support by brissynbio, a bbsrc/epsrc funded centre for synthetic biology at the university of bristol (bb/l01386x/1).authors' contributions mp and ib wrote the manuscript together with input from hc, bg, pl and jr. all authors have read and agreed the content. the authors declare competing financial interest. ib is inventor on international patents (us 61/762.607, ep2403940, ep1723246, ep1945773) covering parts of the multibac technology here described. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-023225-5quigar4 authors: nan title: posters date: 2012-08-21 journal: j pept sci doi: 10.1002/psc.2449 sha: doc_id: 23225 cord_uid: 5quigar4 no abstract is available for this article. laboratory of molecular biology and immunology, department of pharmacy, university of patras, patras, greece antimicrobial peptides (amps) are an important component of innate immune system of most living organisms. they have recently gained much attention as new anti-infective drugs with new modes of actions and few or no side effects. their antimicrobial spectrum covers gram-positive and -negative bacteria as well as fungi and certain viruses 1 . fish have proven to be a rich source of antimicrobial peptides. three chrysophsin peptides (chrysophsin-1, -2, -3) have been identified in the gills of the red sea bream, chrysophrys major, which are all bactericidal to pathogenic bacteria at low concentrations 2 . they are cationic α-helical peptides, rich in histidine residues and all end in an unusual rrrh motif. however, in addition to its high antimicrobial potency, chrysophsins have considerable hemolytic activity. the development of new analogues which would preserve high antimicrobial potency, but would lack the undesired hemolytic activity, could be a useful tool with possible commercial and clinical applications. in the present study, we synthesized a series of analogues of chrysophsin-1 with different ratios of lys and leu residues, utilizing the fmoc/but solid phase methodology 3 . the synthesized analogues were purified and isolated by rp-hplc. the antimicrobial properties of the above peptide analogues are currently testing in 3 gram positive (s. aureus, s. epidermidis, e. faecium) and 2 gram negative (e. coli, p. aeruginosa) bacteria. the goal is to identify the minimum bacteriostatic and bactericidal concentrations of the analogues, under conditions that simulate the best possible that of the human organism. hemolytic or cytotoxic activity of the peptides will also be determined. 1 the rise of antibiotic resistance demands the development of new antimicrobial agents. these should exhibit a novel mechanism of action so as to overcome the resistance and be invulnerable to 'not yet acquired resistance mechanisms'. such criteria are difficult to meet. however, cationic host defence peptides (hdps) have emerged as promising candidates. hdps target and disrupt bacterial membranes. in order to evade such a threat a bacterium would need to make substantial changes to its membrane composition disfavouring the development of resistance (1) . however, exact role and mechanism of hpds in the regulation and monitoring of microbial invasions remain to be established. herein we will present new potential mechanisms of antimicrobial regulation by helical hdps using de novo (2) and native systems (3) . biophysical and microbiology aspects of the experimental designs will be discussed. the low number of the newly discovered antibiotics, emergence of multiple-drug resistance, and the alarming death rate due to the infection disease led to development the alternative means to combat the infections. the researchers accumulate information about antimicrobial drugs that could be result of the innate immunity mechanisms. armed only with the innate immunity, the insect has developed into the most widespread class in living kingdom. they produce several antimicrobial peptides with complementary and rapid mode of action. so far there are hundreds of antimicrobial peptides isolated from insect and lot of them are waiting to be discovered. the fleshly neobellieria bullata was chosen for isolation of these active compounds. its larvae in the third instar were squeezed to collect the haemolymph, which was gradually centrifuged and precipitated by acidified methanol. supernatant was subsequently separated by chromatographic methods (spe column, rp-hplc) to obtain fractions of short peptides. identification and characterization of these fractions were performed by tricine electrophoresis, mass spectrometry maldi-tof analysis and n-terminal sequencing. several fractions showed antimicrobial activity against institute of chemical kinetics and combustion, 630090 novosibirsk, russian federation in this work, we extracted 3d-structural information on newly synthesized, medium-length, double spin-labeled peptaibiotics using peldor spectroscopy. we investigated the magnetic dipole-dipole interactions between spin labels and the orientation selectivity effects. in particular, the medium-length peptaibiotics tylopeptin b 1,2 and heptaibin 3 , double spin-labeled with the nitroxyl probe toac (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid), were studied by means of x-band peldor spectroscopy. this study was conducted on tylopeptin labeled at positions 3 and 13 (t313) and heptaibin labeled at positions 2 and 14 (h214) in frozen glassy methanol solutions at 77 κ. peldor data analysis was carried out using the theory developed for short interspin distances. the distance distribution functions between spin labels for τ313 (maximum at 1.78 nm, halfwidth of 0.08 nm) and η214 (maximum at 2.30 nm, half-width of 0.05 nm) were determined. the intramolecular distances observed between the labels allowed us to assign an essentially α-helical conformation to τ313 and a largely prevailing 310-helical structure to η214 under the aforementioned experimental conditions. are amidated at the c-terminus, as a result of a posttranslational enzymatic reaction. temporins are particularly active against gram-positive bacteria and are not toxic to eukaryotic cells. in this study we designed a series of analogues of tb with the aim to improve the peptide antimicrobial activity against both gram negative and gram positive strains and then to structurally elucidate the mechanism of interaction of active peptides with lps. the peptides have been synthesized substituting one or two amino acids with an alanine and lengthening the sequence with positively charged amino acids. among the 16 designed peptides, one of the analogues, tb_kkg6a, showed highly increased activity against gram negative bacteria and also a slightly increased activity against gram positive bacteria with a total lack of hemolytic activity. to develop ll-37-derived short amps with prokaryotic selectivity and lipolysaccharide (lps)neutralizing activity, a series of amino acid-substituted analogs based on ig-19 (residues 13-31 of ll-37) were synthesized. analog a4 showed the highest prokaryotic selectivity, but much lower lps-neutralizing activity compared to ll-37. the analogs, a5, a6, a7 and a8 with higher hydrophobicity displayed lps-neutralizing activity comparable to that of ll-37, but much lesser prokaryotic selectivity. these results indicated that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of lps-neutralizing activity and prokaryotic selectivity. to increase lps-neutralizing activity of the analog a4, we synthesized trp-substituted analogs (a4-w1 and a4-w2), in which phe 5 or phe 15 of a4 is replaced by trp. despite their same prokaryotic selectivity, a4-w2 displayed much higher lps-neutralizing activity compared to a4-w1. this result suggested that the effective site for trp-substitution when designing novel amps with higher lps-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. furthermore, d-enantiomeric peptides (a4-w1-e and a4-w2-e) of a4-w1 and a4-w2 possessed not only more improved prokaryotic selectivity and retained lpsneutralizing activity compared to a4-w2 but also protease stability. taken together, a4-w1-e and a4-w2-e can serve as promising templates for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection. department of zoology, faculty of science, charles university, prague, czech republic antimicrobial peptides (amps) are among the most promising lead compounds for developing medicines in the fight against resistant pathogenic bacteria. we have already shown that the venom of wild bee is a rich source of pharmacologically interesting antimicrobial peptides [1] [2] [3] [4] . from the venom of solitary bee macropis fulvipes, we isolated and characterized the novel antimicrobial peptide named macropin (mac-1). by edman degradation and mass spectrometry, its primary sequence was established as gfgmalkllkkvl-nh2. mac-1 possesses potent antimicrobial activity against both gram-positive andnegative bacteria and moderate hemolytic activity against human red blood cells. cd spectra confirmed that mac-1 can form an amphipathic α-helical secondary structure in the presence of membrane-mimicking substances as sodium dodecyl sulfate or organic solvents like trifluoroethanol. we prepared a series of mac-1 analogs to study the effect of incorporating d-amino acid residues into the sequence in various positions on antimicrobial and hemolytic activity, α-helicity and serum stability. the substitution of l-amino acid residues at n-terminal part of sequence by d-amino acid residues led to the improving hemolytic activity with maintaining or increasing antimicrobial activity. these modifications increased peptide stability in human serum. effect of the incorporation of d-amino acid residues into the mac-1 sequence on its α-helical structure will be discussed. the neutralization of endotoxins (lipopolysaccharide, lps) by suitable compounds has been shown to be a key step in the treatment of infectious diseases, in particular in the case of gram-negative bacteria. the active endotoxic center of lps is lipid a, its lipophilic part. an effective antimicrobial peptide against gram-negative bacteria is magainin 2, which was originally found in the skin of an african frog. here, we studied the interaction of hexa-acyl bisphosphoryl lipid a 1 prepared from erwinia carotovora lps with magainin 2 with some minor substitutions in the amino acid pattern. by using fourier-transform infrared spectroscopy, the gel to liquid crystalline phase transition of the acyl chains of lipid a, the conformation of their phosphate groups due to peptide binding, and the profile of the secondary structure of the peptides was investigated. the zeta potential of lipid a aggregates in the presence of the peptides was determined by measuring the electrophoretic mobility. small-angle x-ray scattering was performed for the elucidation of the aggregate structures in the absence and presence of the peptides, and isothermal titration calorimetry was applied for evaluating the thermodynamics of binding between peptides and lipid a. the data show that asp-or glusubstituted peptides improved the binding activity to lipid a correlated with characteristic changes in the physical parameters, which were stronger expressed for the aspsubstituted peptide. the new hydrogen bond connection between glu and asp by carboxylic acids apparently leads to a more pronounced -structure of the peptide. the conformation change of the peptide enhanced the activity of incorporation into the lipid a aggregates, along with changes in biochemical and biophysical parameters. royal college of surgeons, dublin, ireland cationic antimicrobial peptides (caps) have been reported to exhibit anticancer activity 1 . one such peptide, p18, has been shown to inhibit the growth of several cancer cell lines, with inhibiting concentration (ic50) in the range of 10 to 20 μm 2 . however the concentration at which p18 and other caps act is too high to be clinically relevant. the enhancement of their activity can be achieved through the modification of their amino acid composition or the addition of other molecules. conjugation of naturally produced hydroxylated fatty acids to p18 showed a 10-fold improvement in its anticancer activity on a variety of human-derived cell lines. in addition to the enhancement of activity we wished to understand the mechanism of action of the peptide and conjugates. we investigated the uptake of conjugated and unconjugated peptides into hela (cervical) and miapaca (pancreatic) human cancer cells and the localisation of the peptide in the cell once taken up. we investigated the effect of altering the carbon number of the hydroxylated fatty acids ranging from hydroxyhexanoic acid (r6) to hydroxydodecanoic acid (r12) conjugated to p18 peptide and tested on hela and miapaca cell lines. circular dichroism studies were performed to investigate the effect on α-helical content due to amino acid composition alteration and hydroxyalkanoic acid conjugation. the effect of the position of the hydroxyl moiety on enhancement of activity was also investigated. in the current study p18 and its derivatives also lacked haemolytic activity with concentrations up to 66 fold higher than ic50 values needed to observe any haemolysis. when current antibiotics become less efficient, there is a promise that some antibiotics can be replaced by other nature's substances, e.g. peptides. halictines are novel antimicrobial peptides isolated from the venom of the eusocial bee halictus sexcinctus. we obtained four analogues of the native peptide hal 1 from iocb av cr. they already characterized structural properties of these peptides and their antimicrobial activity against selected bacteria 1 . the analogues were prepared by point mutations of native peptide, which could increase antimicrobial activity and decrease undesirable hemolytic activity. our aim was to characterized membrane permeation activity of halictines through the use of a basic model of biological cells -large unilamellar phospholipid vesicles luvs. we prepared two basic types of leakage assays based on luvs with free dyes entrapped inside and one assay with laurdan content. we used classical steady state fluorescence spectroscopy 2 and advanced fluorescence methods 3 for study of dyes escape from luvs and we also used laurdan generalized polarization technique gp 4 for better understanding peptide insertion into membrane. in this way we received complementary information and we can conclude that the most active peptides are the native hal 1 and analogue hal 1/6. however hal 1/6 requires presence of negatively charged phospholipids in membrane which may explain its higher selectivity against bacteria. furthermore, fcs results have shown that the leakage happens via pore formation. results from gp revealed that peptide insertion in the membrane do not lead directly to formation of pores. against a wide range of microorganisms, mainly by perturbing the permeability of bacterial membranes through the formation of pores. however, amps effects on membrane properties probably extend beyond poreformation. we performed a systematic spectroscopic analysis of the effects on membrane structure and dynamics of two very different amps: the cationic pmap-23, which creates pores according to the "carpet" model 1 , and alamethicin, which forms "barrel-stave" channels 2 . by using fluorescence anisotropy measurements on liposomes comprising probes localized at different depths in the bilayer, we measured peptide effects on membrane fluidity and order. laurdan spectral shifts provided indications about water penetration in the bilayer. in the case of pmap-23, it was possible to focus specifically on the lipids surrounding the peptide by following the membrane-probe fluorescence due to fret from the peptide trp residues. finally, peptide-induced perturbation of lateral mobility and domain formation were determined by several methods. all experiments were compared with liposome-leakage measurements: while for pmap-23 all membrane-perturbing effects are correlated with the vesicle leakage process, alamethicin does not significantly influence membrane dynamics at the concentrations in which it forms pores. surprisingly, in all cases the most significant peptide-induced effect is a reduction in membrane fluidity. we have reinvestigated 20-residue peptaibols named metanicins from an ascomycetous fungus originally described as metarhizium anisopliae strain cbs 597.80 (cbs = centraalbureau voor schimmelcultures, utrecht, the netherlands). however, due to unusually shaped conidia and based on rna-sequencing of its internal transcribed spacer (its) region, the identification of cbs 597.80 as metarhizium has been withdrawn and this particular strain is currently under taxonomic reinvestigation 2 . sequencing of four isolated peptides by fab-ms, esi-ms and edman degradation of partial hydrolysates revealed structural relationship to 20-residue peptaibol antibiotics paracelsins from trichoderma reesei (=hypocrea jecorina). sequences determined are: ac-u-a-u-a-u-a(u)-q-u-v-u-g-l-u-p-v-u-u(j)-q-q-fol (exchange positions in parenthesis; ac, acetyl; u, aib, α-aminoisobutyric acid; j, d-isovaline; fol, l-upmc univ paris 06 laboratoire des biomolécules; cnrs umr 7203; ens lbm; address: laboratoire des biomolécules, ens dpt de chimie, 24, rue lhomond f-75005, paris, france current data suggest that the cellular uptake of cellpenetrating peptides (cpps) occur by two processes: direct translocation across the plasma membrane and endocytosis 1 . the large diversity of cpp sequences described in the literature (derived either from fragments of proteins, structurally constrained synthetic peptides, peptide libraries 2 or dendrimers) has hampered the identification of general rules for their efficacy of internalisation. we have used a reductionist approach, restricting the cpp functional groups (amide and guanidinium) and tailoring cpp amphiphilic properties. two families of cpps have been designed: 1) primary amphiphilic cpps corresponding to tetra-arginines functionalised with fatty acid chains of different lengths and 2) secondary amphiphilic cpps containing arginine and alanine or tryptophan residues 3 . these cpps were linked by a disulfide bridge to a peptide inhibitor of protein kinase c (pkci). the efficiencies of internalisation of the conjugates were quantified by a method based on maldi-tof mass spectrometry previously developed in our group 4 . the mechanism of internalisation was studied by comparing the amounts of cell-surface bound and internalized pkci cargo on cho-k1 cells and glycosaminoglycan-deficient cho cells at 4 o c and 37 o c. conjugates were found to enter by both direct translocation and glycosaminoglycandependent endocytosis. in addition, the primary amphipathic cpps were found to be more efficient than the secondary amphipathic ones. furthermore, structural or mechanistic novelty does not guarantee immunity from resistance, with strains resistant to linezolid identified prior to fda approval. therefore, modifying existing antibiotics to overcome resistance mechanisms presents an opportunity to rationally develop effective new drugs more rapidly than screening for new structures. vancomycin is a glycopeptide commonly used as a front line treatment for infections caused by methicillinresistant staphylococcus aureus (mrsa). the emergence of vancomycin-resistant enterococci (vre), vancomycinintermediate s. aureus (visa) and vancomycin-resistant s. aureus (vrsa) has prompted the development of semisynthetic glycopeptides 3 . we have generated a variety of glycopeptide derivatives that show superior antibacterial activity against mrsa and vre compared to vancomycin and second generation lipoglycopeptides. this was undertaken by employing a combination of solid phase and solution phase chemistry to attach a membraneassociative element that selectively binds to bacterial membranes in preference to eukaryotic membranes, thus increasing the local concentration at the lipid ii d-ala-d-ala peptidoglycan cell wall precursor target site. three novel antimicrobial peptides, named panurgines (png), were isolated from the venom of wild bee panurgus calcaratus. one of them is dodecapeptide with sequence lnwgailkhiik-nh2 (png-1). the next two peptides are almost identical. these are cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges ldvkkiicvackixpnpackkicpk-oh (x=k png-k and x=r png-r). all peptides exhibited antimicrobial activity against gram-positive bacteria and gram-negative bacteria, antifungal activity and low haemolytic activity against human erythrocytes. we prepared 11 analogues of α-helical amphipathic png-1 with the aim to improve its biological properties and a linear analogue of png-r to elucidate the importance of disulfide bridges for its activity. in the second part of the study, we followed the effect of panurgines on the degree of membrane disruption by observing the leakage of fluorescence dye (calcein) entrapped in artificial phospholipids vesicles [1] . specifically, we investigated membrane interactions of pngs with the vesicles made from negatively charged 1:2 dopc/dppg and 1:2 dopc/dopg vesicles as a general model of bacteria membrane and 15:80:5 dopc/dopg/cl as a possible model for a membrane of bacillus subtilis. the membrane interaction of pngs was also investigated on uncharged dopc vesicles as potential model membrane for erythrocytes. pngs exhibited weak dye-leakage activity for neutral vesicles, while they effectively induced dye leakage in the presence of negatively charged vesicles. these results indicate that pngs have stronger potency to disrupt bacteria-mimicking anionic membranes than those which mimic eukaryotic cell membrane. department of biochemistry and toxicology, university "lucian blaga", 550012 sibiu, romania a common tool to bias the conformation of linear peptides is the insertion of side-chain modified amino acids or sidechain/main-chain conformationally restricted building blocks. an alternative approach is a simple backbone modification. in this connection, backbone amide replacements with (almost) isosteric surrogates were extensively used. these modifications may impart resistance to enzymatic degradation and better bioavailability to the peptides, but also influence the secondary structure. a thioamide (ψ[cs-nh]) is perhaps the closest structural mimic of an amide. however, it possesses different and attractive features: (i) its nh group forms stronger hydrogen bonds, being more acidic than that of the amide. (ii) its c-n bond undergoes cis/trans isomerization by irradiation at 260 nm (π→π* transition). (iii) it may act as a "minimalist" fluorescence quencher. for all these reasons, we started a programme aimed at exploring how the endothioamide bond affects peptide folding and bioactivity. in this communication, we describe the synthesis and conformational results of the three analogs of the membrane-active peptaibiotic trichogin ga iv listed below: n-octanoyl-aib-gly-ψ[cs-nh]-leu-aib-gly-gly-leu-aib-gly-ile-leu-ome (2/3) n-octanoyl-aib-gly-leu-aib-gly-ψ[cs-nh]-gly-leu-aib-gly-ile-leu-ome (5/6) n-octanoyl-aib-gly-leu-aib-gly-gly-leu-aib-gly-ψ[cs-nh]-ile-leu-ome (9/10) the syntheses of the three peptides were accomplished in solution according to a fragment condensation approach. appropriate thioamide-containing tri-or tetrapeptides were prepared by treating the corresponding all-amide precursors with the lawesson reagent. ft-ir absorption, 2d-nmr and cd conformational investigations on the three analogs were conducted in comparison with the naturally occurring peptaibiotic. all three analogs maintain the capability to interact with the dope/dopg model phospholipid membranes and exhibit a comparable bioactivity against s. aureus. peptide-peptide interaction of lactococcin g class iib two peptide bacteriocin h. etayash, w.soliman and k. kaur* faculty of pharmacy and pharmaceutical sciences, university of alberta, edmonton, alberta, t6g 2e1 lactococcin g, a class iib two-peptide bacteriocin, consists of two complementary peptides lcng-α and lcng-β that act as one functional unit with optimal antimicrobial activity achieved by the presence of both peptides in approximately equal amounts. in this study we have investigated the mechanism of pairing of the two complementary peptides as well as explored any specific interaction that could take place between the peptides. molecular dynamics (md) simulation was employed to study the interactions at the atomistic level. four different md simulations with the peptides in a lipid bilayer system were conducted. md results from these simulations confirmed and pointed out that (i) the two putative gxxxg motif, g7xxxg11 in lcng-α and g18xxxg22 in lcng-β, were attracted and came closer to each other, showing the role of these motifs in attracting the two peptides to each other. closer views, however, showed no clear interactions between these two motifs. most likely, nonspecific interactions play a role in bringing the two peptides together; (ii) variations and loss in the secondary structure in both the peptide fragments were confirmed among the four simulations. on the contrary, stability of helical regions was identified between residues w3-g9 and d26-q29 in lcng-α and v9-e14 in lcng-β; and (iii) role of tryptophan at the n-terminal regions in positioning and setting the peptide orientations were confirmed which matched the previous reported results. faculty of pharmaceutical sciences, unesp -univ. estadual paulista, araraquara, sa͂ o paulo, brazil antibiotic resistant bacterial strains represent a global health problem. antimicrobial peptides (amps) are promising novel antibiotics because they have displayed little or no resistance effects. it is well known that the charge, amphipathicity, hydrophobicity and helicity of the peptide are fundamental for the biological activity. in addition, covalent dimerization appears as a new parameter to be studied. in this way, several bioactive sequences were dimerized obtaining pharmacotechnics advantages like enhanced antimicrobial activity, solubility and proteases resistant. however, the effect of this modification is unclear since dimeric versions of some amps are toxic 1 . to evaluate the effects of dimerization on the structure and biological activity of the amp aurein 1.2, the monomeric version (au) and the c-and n-terminal dimers, (au)2k and e(au)2, respectively, were synthesized. circular dichroism results indicated that dimeric versions showed more defined structures in aqueous solution. e(au)2 showed "coiled coil" structure while (au)2k an αhelix structure. in contrast, au displayed typical spectra for disordered structures. in tfe and lpc, all the peptides acquired a high amount of α-helix structure. the antimicrobial activity against bacteria and yeast decreased with dimerization. however, dimeric peptides promoted the aggregation of c. albicans. hemolytic and vesicle permeabilization assays showed that au has a concentration dependence activity, an effect that can be assigned to a "carpet" like mechanism peptide, whereas this effect was less pronounced for dimeric versions, suggesting that dimerization may change the mechanism of action. in conclusion, our studies showed that the effects of amp dimerization are complex and still unclear. 1 , the first antimicrobial peptide generated in vivo and isolated from the gut contents of the cattle tick boophilus microplus 2 . we have shown that these peptides are equally lethal to candida albicans mdm8 and practically not active on human erythrocytes 1 . to examine the properties and mode of action of hb40-61a, we synthesized it and its fluorescently labeled analogue (fam-hb40-61a) by the solid-phase method at 60 o c, purified them by rp-hplc and characterized their purified forms by lc-esims. at low salt concentration, both peptides were found to inhibit the growth of candida albicans atcc 90028, candida parapsilosis atcc 22019 and candida krusei atcc 6258, but hb40-61a was two-fold more active (mics of 12.5; 12.5 and 50.0 μm, respectively). at those concentrations, both peptides also kill the fungi. assays with human erythrocytes showed that, likewise hb40-61a, fam-hb40-61a present activity lower than 25% at 100 μm. apparently, hb40-61a targets the membrane cell because confocal microscopy analysis revealed that, at the half of mic value, fam-hb40-61 accumulates on the fungal cell membrane. in contrast, fluorescence activated cell sorting (facs) analysis revealed that, at the mic, more than 90% of the fam-hb40-61a penetrates into the cell. membrane permeability assay using hb40-61a, c. albicans atcc 90028 and the kit live/dead funga light confirmed progressive membrane damage associated with an increase in peptide concentrations. the use dibac4 (5) and facs analysis showed that hb40-61a alters the plasma membrane potential, leading to cell death. supported by fapesp, cnpq and capes. lasso peptides form a growing class of 16 to 21 residue ribosomally-synthesized and post-translationally modified peptides produced by bacteria. they share a rigid and compact interlocked structure consisting of a macrolactam ring at the n-terminus and a c-terminal tail that is looped back and threaded through the ring, forming a typical [1] rotaxane 1, 2 . the macrolactam is formed by condensation of an asp8 or glu9 side-chain with the free amino group of a gly1 or cys1. the lasso fold is stabilized either by steric hindrance assumed by bulky amino acid side-chains and/or by disulfide bonds between cysteines from the tail and the ring. given this structure, lasso peptides display a high stability against proteolytic and chemical degradation. they are biologically active on various enzymatic targets, which confer them in some cases an interesting antimicrobial activity. given its characteristics, the lasso scaffold thus represents a promising tool for biotechnological application in the development of bioactive peptides. until now, nine peptides had been structurally characterized as lasso peptides. based on a genomics-based approach, we identified a novel lasso peptide from streptomyces sviceus that we termed sviceucin. it was produced in high yield by heterologous expression in s. coelicolor and submitted to structural analysis by mass spectrometry and nmr. sviceucin is 20residue long and stabilized by two disulphide bonds. their connectivities were identified mainly from the typical noes between the beta-protons of the cysteines. the lasso structure of sviceucin was obtained by nmr-based molecular modelling. sviceucin was shown to exhibit antibacterial activity directed against gram positive bacteria, while gram-negative bacteria and fungi showed resistant. the penicillium chrysogerum antifungal protein (paf) is a cysteine-rich, cationic protein that inhibits the growth of a variety of filamentous fungi without toxic effect on mammalian cells 1 . although paf is used to be produced in p. chrysogerum or a similar microorganism, preparation of analogues of the protein for structural and functional investigations requires an efficient chemical method. the unsuccessful continuous synthesis of the 55-mer small protein prompted us to use native chemical ligation 2 . the syntheses of the fragments were performed by solid-phase method applying tboc chemistry. using the acid-labile tboc protecting group, the thioester end of the n-terminal fragment remains intact during the course of the synthesis. the first attempt was the synthesis of peptides with pmethylbenzyl groups on the side chains of all of the six cysteine residues. under no circumstances oxidative folding provided the natural disulphide bridge pattern. the failed attempts led us to orthogonal protection of the sulphydryl groups. different sets of protecting groups were tried and evaluated. our experiments showed that basic treatment triggered rearrangement of the previously formed disulphide pattern. thus, base-labile protecting groups (such as 9-fluorenylmethyl, fm) have to be avoided in the synthesis of paf. the alarming increase and spread of antibiotic resistance among bacterial pathogens has stimulated the development of new antibacterial agents with innovative mode of action. antimicrobial peptides with broad spectrum activity are widely distributed in nature and play an important role in innate immunity in several species, including humans. tigerinins are a unique family of 11-to 12-residue antimicrobial peptides found in skin secretion of the indian frog rana tigerina 1, 2 . characterized by a disulfide-bridged loop composed of nine amino acids, tigerinins do not show primary structural homology to any known antimicrobial peptides from amphibians. tigerinins could provide novel lead compounds for the design of effective antimicrobial peptides with a new mode of action. the peptide murdp1 has been identified after the screening of phage display libraries against pseudomonas aeruginosa cell wall biosynthesis murd amide ligase enzyme 3 . murdp1 is a low micromolar range inhibitor of murd enzyme and showed good antimicrobial activity. composed of nine amino acids, it is also characterized by a nine residues disulfide-bridged loop containing two prolines. this great similarity with tigerinins, led us to investigate if murd enzyme could be a potential target for these peptides. in silico analyses using modelling, molecular dynamics and docking with p. aeruginosa murd showed that murdp1 and tigerinin-1 and -2 make similar interactions in the binding site. these results suggest that murd may be an intracellular target for tigerinin-1 and tigerinin-2. synthesis, murd enzymatic inhibition assay, antibacterial activity evaluation and structure-activity relationships of murdp1 and tigerinins analogs will be presented. h. etayash, l. norman, t. thundat*, k. kaur* faculty of pharmacy and pharmaceutical sciences, department of chemical and materials engineering, university of alberta, edmonton, alberta t6g 2e1, canada listeria monocytogenes is a gram positive bacterium that accounts for about 28% of the deaths resulting from food borne illnesses in north america. moreover, l. monocytogenes is considered one of the most difficult bacteria to detect in contaminated food products. while standard microbiological and biochemical assays currently used are accurate and sensitive, they are time consuming and often require specialized instruments operated by a trained user making on-site testing difficult. to this end, we propose the development of an antimicrobial peptide (amp) or peptide fragment sensor for the on-site detection of l. monocytogenes. leucocin a, which is a naturally occurring amp consisting of a 37 amino acid sequence, is known to exhibit specific activity against l. monocytogenes at pico to nanomolar concentrations. for this reason, we have synthesized a shorter peptide fragment of leucocin a consisting of 24 amino acids using solid phase peptide synthesis. the peptide was purified by reversed phase hplc and maldi-tof mass spectrometry indicates the desired biological entity was achieved. by including an n-terminal cysteine group, the tailored amp was readily immobilized at a gold interface. the resulting thickness and molecular orientation, determined by ellipsometry and grazing angle infrared spectroscopy, respectively, indicate that the helical peptides were adsorbed on the interface with a preferred orientation parallel to the surface. the bacterial specificity of the anchored leucocin a fragment was tested against three gram positive bacteria and results reveal that the adsorbed amp exhibits a limit of detection of approximately one bacterium/μl which is a clinically useful detection range. faculty of science, university of south bohemia, české budějovice, czech republic during the last few years we have identified three novel defensins from arthropods. two of them, lucifensin 1 and lucifensin ii were purified from various tissues of lucilia sericata and l. cuprina larvae, respectively. larvae of these flies are routinely used in the hospitals around the world for the treatment of non-healing infected wounds in the procedure known as maggot therapy. these 40 amino acid residues and three disulfide bridges peptides differ from each other only in one amino acid residue in position 11 (val-ile). linear precursor of lucifensin was prepared by fmoc-spps chemistry which was then subjected to the oxidative folding yielding a peptide with a pattern of disulfide bridges identical to that of native lucifensin and other insect defensins. this was examined by the identification of the fragments resulting from the thermolysin digestion of lucifensin by means of mass spectrometry. however, this cyclization reaction proceeded via an intermediate having incorrect pairing of disulfide bridges. from the hemolymph of blood sucking tick dermacentor marginatus (d.m.) we purified defensin containing 38 amino acids and three disulfide bridges. its sequence determined by edman degradation and mass spectrometry was identical to that previously determined by molecular biology methods 2 . sequence of d.m.defensin shows no homology to insect defensins. by spps prepared linear precursor of d.m.-defensin was subjected to oxidative folding under the open air. the linear peptide was straightforwardly folded into cyclic one which was identical to the native peptide. in antimicrobial assay using a set of different bacteria all three studied defensins show activity preferentially against gram-positive bacteria including staphylococcus aureus but are inactive against gram-negative ones. the importance of disulfide bridges on tertiary structure of defensins and their antimicrobial activity will be presented. recently, the chemical structure and conformation of pseudodesmin a has been determined through x-ray diffraction and nmr spectroscopic analysis 1 . in this way pseudodesmin a was identified as a new member of the viscosin group of antimicrobial peptides (amps). in addition, it was demonstrated that individual molecules self-assembly in apolar environment into a supramolecular pore-like structure, providing structural support for its biological activity 2, 3 . to further explore the structure-function relationship, a viable synthesis strategy for pseudodesmin a analogues was developed, based on side-chain attachment of the first amino acid to the solid support, followed by stepwise fmoc solid-phase synthesis of the linear peptide precursor and on-resin head-to-tail cyclization. nmr study confirmed the molecular structure and thus the development of an efficient and successful synthesis of this type of amp's. these results and the synthesis route will be presented. trichogin ga iv, isolated from the fungus trichoderma longibrachiatum 1 , is the prototype of lipopeptaibols, a subclass of short-length peptaibiotics exhibiting membranemodifying properties. its primary structure is as follows: n-oct-aib 1 -gly-leu-aib-gly-gly-leu-aib-gly-ile 10 -lol, where n-oct is n-octanoyl, aib is α-aminoisobutyric acid, and lol is the 1,2-amino alcohol leucinol. this peptaibol is predominantly folded in a mixed 310-/αhelical conformation with a clear, albeit modest, amphiphilic character 2 . in this work, we synthesized by solution and solid-phase methodologies a set of trichogin ga iv analogs in which the four gly residues, lying on the poorly hydrophilic face of the helical structure, are substituted by one (or more) strongly hydrophilic lys residues. moreover, we synthesized another set of analogs where one (or more) aib residues are replaced by leu. the conformational preferences of these analogs were assessed by x-ray diffraction, cd, and 2d-nmr techniques 3 . we tested the role played by the substitutions on the peptide bioactivity, e.g. protease resistance, cytotoxicity, and hemolysis. cytotoxicity was tested using three in vitro cell-based assays: (i) human red-blood cells lysis; (ii) cell mortality in total human blood leukocytes and in separate subpopulations; (iii) cell mortality in three tumor-derived stable cell lines (hela, a541, and a431). our data show that some of our trichogin analogs are active against tumor cells, leaving the leukocytes unaffected. a convenient post-screening ring opening approach for the decoding of one-bead one-compound cyclic peptide libraries a. girard, e. biron* faculty of pharmacy, université laval and chuq research center, quebec, canada combinatorial chemistry has been widely used as an effective method for the generation and screening of synthetic peptide libraries. amongst the different combinatorial methodologies to discover new bioactive peptide-based compounds, we were particularly interested in the one-bead one-compound (oboc) approach 1 . this powerful approach fully exploit the great molecular diversity accessible with peptides and has been used to identify a great number of ligands and modulators for a wide variety of biological targets. however, its use with cyclic peptides is limited by difficulties in sequencing hit compounds by edman degradation or tandem mass spectroscopy due to the lack of free n-terminal amine and complicated fragmentation patterns, respectively. this problem has been overcome by pei and coworkers by using a bead segregation strategy in which the outer layer exposes the cyclic peptides and the inner layer the linear counterpart for sequencing 2 . more recently, lim et al. reported an elegant method to prepare and sequence oboc cyclic peptoid libraries without encoding by using a ring opening approach with triazine-based cyclic derivatives 3 . unfortunately this method is incompatible with amino acids bearing some functionalized side chains. based on this strategy, we have developed an efficient method to prepare oboc cyclic peptide libraries that does not require encoding by using a simultaneous ring opening/cleavage approach. the procedure is compatible with commonly used amino acids and allows rapid and efficient sequencing of selected hits after on-bead screening. the synthesis of an oboc cyclic peptide library, ring opening methodology and sequencing by mass spectrometry will be presented. cyclotides are a very abundant class of plant peptides that display immense sequence variability around a conserved cystine knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability 1 . their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals 2 . however, laborious isolation and purification prior de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development 3 . here we extend the knowledge about their sequence diversity by analyzing the cyclotide content of a violet species native to western asia and the caucasus region 4 . using an experimental approach, which we named 'sequence fragment assembly' by maldi-tof/tof-based peptidomics, we were able to characterize novel cyclotides from viola ignobilis. amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analyzing mixtures containing multiple peptides. using in-source decay and high energy collision induced dissociation of digested cyclotides allowed to distinguish isobaric residues ile and leu. overall this work underlines the immense structural diversity and plasticity of the unique cyclotide framework. the presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides. glycation is a nonenzymatic reaction occurring between reducing sugars and reactive amino groups of biomolecules. the reaction leads to a formation of a heterogeneous mixture of compounds which are classified as early, intermediate or advanced glycation end products (age). these compounds, especially advanced glycation end products, are involved in many pathological processes, mainly diabetic complications, and could be markers of certain diseases. detection of early products of glycation (amadori products) is a relatively easy task and can be performed by various methods including e.g. ms/ms techniques, isotopic labeling and affinity chromatography on immobilized boronic acid 1,2 . however, the diverse structures of ages make detection of these compounds more challenging. the aim of the study was testing a new method of ages identification based on isotopic 13 c labeling. a model protein (hen egg lysozyme) was modified with an equimolar mixture of [ 12 c 6 ]glc and [ 13 c 6 ]glc. then the glycated protein was subjected to reduction of the disulfide bridges followed by enzymatic hydrolysis. the obtained digest was analyzed by lc-ms methods. the glycation products were identified on the basis of characteristic isotopic patterns resulting from the use of isotopically labeled glucose. this method allowed for identification of 41 early maillard reaction products and 8 different structures of the glycation end products. isotopic labeling technique combined with lc-ms is a new and very sensitive method for identification of the advanced glycation end products even if their structures are unknown. this method could be also used as an alternative method of detection of amadori products. in the course of a project aimed to assess the significance of antibiotics for the producing organism(s) in the natural habitat, we screened a specimen of the fungicolous fungus hypocrea phellinicola growing on its natural host phellinus ferruginosus 1 . using a peptaibiomics approach 2,3 , we detected 19-and 20-residue peptide sequences by (u)hplc/hr-esi-qqtof-ms. structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of an agar plate culture of h. phellinicola cbs 119283 (ex-type) grown under laboratory conditions. notably, h. phellinicola could be identified as a potent producer of 18-, 19-, (culture) and 20-residue (specimen) peptaibiotics of the suzukacillin-type4. minor components of the 19-residue peptaibols, herein named suzukacillins c, are assumed to carry a c-terminal residue tentatively assigned as tyrosinol (tyrol). in addition, the previously isolated suzukacillin b 4 was sequenced and shown to be a microheterogeneous mixture of 11-residue peptaibols. in order to further investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened specimens of the fungicolous fungus hypocrea pulvinata growing on its natural hosts piptoporus betulinus and fomitopsis pinicola 1 . using a peptaibiomics approach2, we detected 17-, 18-, 19-(major sequences), and 20-residue peptide sequences in the five specimens analyzed by (u)hplc/hr-esi-qqtof-ms. structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of pure agar cultures obtained by single-ascospore isolation from the specimens 1 . major, 19-residue peptaibols were assigned as deletion sequences of the trichosporins b3 lacking the ala/aib residue in position 3 . our results corroborate that: i) peptaibiotics are, indeed, biosynthesized in the natural habitat, thus, ii) their membrane-perturbing formation of ion channels may support the parasitic life style of a fungicolous fungus. based on methodology that we have developed in our lab 2 , we identified specific and selective substrates for these serine proteases. we used a 10,000 membered pnaencoded peptide library to screen 10,000 possible peptide substrates in a single experiment. the library was incubated with the protease of interest and then hybridized on a custom designed dna microarray. microarray scanning and data analysis allowed the measurement of the changes in fam/tamra ratios resulting from the protease activity and the determination of the protease specificity. to verify the predicted activity and specificity, fret peptides were synthesized, incubated with the enzymes and the hydrolysis reaction was followed by monitoring fluorescence emission. specificity constants kcat/km were calculated and the cleavage sites of the peptides were identified. dubs were, moreover, found to be associated with several diseases and as such are emerging as potential therapeutic targets 2 . several directions have been pursued in the search for lead anti-dub compounds. however, none of these strategies have delivered inhibitors reaching advanced clinical stages due to several challenges in the discovery process, such as the absence of a highly sensitive and practically available high-throughput screening assay 3 . in this study, we report on the design and preparation of a fret-based assay for dubs based on the application of our recent chemical method for the synthesis of ub bioconjugates 4 . in the assay, the ubiquitinated peptide was specifically labeled with a pair of fret labels and used to screen a library comprising 1000 compounds against uch-l3. such analysis identified a novel and potent inhibitor able to inhibit this dub in time-dependent manner with kinact = 0.065 μm and ki = 0.8 μm. our assay, which was also found suitable for the uch-l1 enzyme, should assist in the ongoing efforts targeting the various components of the ubiquitin system and studying the role of dubs in health and disease. 3 . more recent work based on rna interference experiments on a mouse model suggested that isoform-specific inhibitors against nmt1 might be effective anti-cancer agents as a knockdown of nmt1 inhibits the tumour growth, whereas knockdown of nmt2 has no effect 4 . if residual nmt2 activity can compensate for loss of nmt function in healthy cells, potential toxicity may also be minimised. we developed a method to identify peptide or protein substrates of nmt1 and/or nmt2. peptides/ proteins are exposed to nmt1 and/or nmt2 and an alkyne-tagged analogue of myristoyl coa. subsequent azide-alkyne "click" cycloaddition allows visualisation of the myristoylated substrates in fluorescence or chemiluminescence, using a fluorescent or a biotin moiety on the capture reagent. this labelling technology was applied to peptide libraries prepared on microarrays to investigate nmt1/2 isozyme substrate specificity using recombinant nmt1 and nmt2. peptides made of the first 8 or 15 amino acids at the nterminus of known myristoylated proteins were functionalised with a biotin moiety at the c-terminus and immobilised on an avidin-functionalised glass plate before being screened for activity. selective peptide substrates will be developed as isozyme-specific inhibitors and applied in cancer cell lines. using chemical proteomics and the labelling technology, a selective nmt1 or nmt2 inhibitor could also be used to identify protein substrates of one isozyme. for this purpose computer programs are created which can generate fragments of one compared structure and to reveal homology by their scanning along the amino acid sequence of another. our analysis was performed by comparing the primary structures of all possible protein fragments with the amino acid sequences of all presently known natural regulatory oligopeptides. the oligopeptides were extracted from the erop-moscow database1 which at the time of analysis contained data on the structures and functions of more than 10,000 natural oligopeptide regulators. the structure-function analysis was performed using a specialized software package. the input data were the complete amino acid sequences of the proteins used as a source of fragments with a specified length. then the initial sequence was fragmented in a stepwise manner. for example, in the case of dipeptide fragments, this procedure produced fragments with the following numbers of amino acids from the n-terminus -1-2, 2-3, and so on until the fragment that started at the second residue from the c-terminus. the cases when the amino acid sequence of a fragment coincided with part of the primary structure of a natural oligopeptide were recorded in the total protein chemical synthesis requires a case by case design and optimization which is governed by factors such as the solubility of the individual peptide segments, their primary sequence and in particular the presence of "difficult" amino acid residues at ligation junctions such as proline or the location of cysteines. usually, a subset of chemical tools are selected among a vast array of methodologies to match the specificities of the target protein. in this context, methods enabling the assembly of three peptide segments in the n-to-c and c-to-n direction play a central role and must be considered as complementary as they can be selected for building subdomains of the target protein. to date, most of the proteins were assembled in the c-to-n direction. only few methods are available for the n-to-c sequential assembly of proteins, whose design is highly challenging. we have recently reported that sea ligation, that is the reaction of a bis(2-sulfanylethyl)amido group (called sea) with a cysteinyl peptide, allows the formation of a native peptide bond in water and at neutral ph 1 . in this communication we will show that native chemical ligation and the unique chemical properties of sea group 2,3 can be combined in order to design a highly efficient one-pot three segments protein assembly procedure, working in the n-to-c direction 4 amylin is one of the most amyloidogenic peptides, its fibrils are responsible for causing type ii diabetes. amyloid formation mechanism is investigated both to find amyloid inhibitors as potential medical drugs, and to use amyloids as potential self-assembling biomaterials [1] . amyloid formation of amylin 10-29, its reverse and designed analogue beta-sheets and beta-sheet stacks was studied by molecular dynamics (md), amber 9.0, f99 force field. md revealed that for amylin 10-29 and its reverse analogue both the parallel and antiparallel beta-sheet and beta-sheet stack structures are stable suggesting that this could explain the high tendency of amylin to form amyloid fibrils. parallel amylin 10-29 beta-sheet stacks are kept together by two hydrophobic cores, while for the antiparallel system the dominating is the backbone hydrogen bonding between neighbor strands. also the bent form of the amylin 10-29 beta-sheet is stable. this is in concordance with transmission electron microscopy (tem) experiments stating that all three peptides, amylin 10-29, its reverse and designed analogues, exhibited significant fibrillar polymorphism [2] university of gdansk, poland molecular dynamics (md) of two peptides dlsfmkge (mk) and dlsfkkge (kk) not related to any known disease was run to investigate the mechanism of the amyloid formation. the parallel and antiparallel [1] betasheets of mk and kk peptides were simulated by molecular dynamics (md), amber 9.0, f99 force field, ntp protocol. it was found that antiparallel beta-sheets both of mk-and kk-peptides show much higher stability than the corresponding parallel beta-sheets. this md result was supported by atr-ftir spectroscopy [2] . the betasheet stacks built from six ten stranded antiparallel beta-sheets of mk-and kk-peptides: 10x6xmk and 10x6xkk, were subjected to md. it was found that the mk-system, 10x6xmk, is strongly kept together due to hydrophobic core built from two metionines, two phenylalanines and two leucines, but the kk-system, 10x6xkk, which differs only by one mutation m5k dissolves already at 20 ns of md run, because the separate beta-sheets don't hold togather in the betasheet stack due to lost hydrophobic core. the hydrophobic core of the mk-system consists of hydrophobic units centered on the two phenylalaninetwo metionine hydrophobic interactions, and two leucines from the both sides stabilize the unit. this mechanism could be used in amyloid based biomaterials. urokinase plasminogen activator (upa) is a serine protease involved in the metastasis of several tumor types. upa is therefore an interesting target in cancer therapy. upain-2 is a new analogue of a highly specific peptidic inhibitor (upain-1) of upa. the peptide contains twelve amino acids and is cyclized through the cysteines at its termini (s 1 -s 12cyclo-ac-cswrglenhaac-nh2). upain-2 inhibits upa with a ki of approximately 40 μm. 1 one method to improve binding affinity is multivalent exposure of the inhibitor, where the local concentration at the binding site is increased. fusion of upain-1 to the trimeric tetranectin showed improved binding affinity compared to the single peptide. 2 here, we report efforts towards novel chemically linked upain-2 peptides to allow multivalent display. the ki value of an upain-2 dimer, linked by a short peg chain through the n-termini, was almost halved compared to that of the single peptide (23 μm). this motivated us to explore the role of the site (n-or cterminal) and the size of the linking segment on the binding affinity. additionally, the influence of the number of upain-2 peptides in the molecule (two vs. four) was investigated by synthesizing a carboprotein that displayed four upain-2 peptides. we present two novel nmr spectroscopic approaches to study reversible self-assemblies in solution. both methods were applied on the self-assembling pseudodesmin a, a pseudomonas produced cyclic lipodepsipeptide that has the capacity to form pores in cellular membranes. 1, 2 the first method is based on the dependence of the 13 c α relaxation rate constants on the anisotropy of the assembly. 3 when the monomer conformation is known and the multiple ch bonds in the monomer sufficiently sample all orientations, the rotational diffusion coefficients can be assessed, revealing assembly shape information. in addition, the orientation of the monomer within the assembly is obtained. the second method is based on fitting translational diffusion coefficient data as a function of concentration in a model-free way, i.e. without assuming an oligomer shape beforehand. here, it is assumed that the diffusion coefficient's dependence on the oligomer size behaves as a power law, which dramatically simplifies the expression for the average diffusion coefficient (measured by pfg-nmr) as a function of concentration. the fitted value of the exponent of the power law fully embeds all shape information of the assembly, and may be related to the socalled fractal dimension of the oligomer. moreover, this approach reveals mechanistic information concerning the assembly formation. both methods thus allow structural information of the assembly to be obtained, even when there is little or no prior knowledge available on the mechanism of the selfassembly. nucleotides and α-amino acids are crucial building blocks for living organisms. these chiral molecules are the biosynthetically precursors of two of the most important classes of biopolymers, dna and proteins, respectively. the 3d-structures of biomolecules are currently studied using a variety of techniques, while helical handedness is routinely detected by means of light pulses of opposite circular polarization. the difference in the uv absorption of these two circularly polarized pulses is called electronic circular dichroism (ecd). in nature, biomolecules explore a wide range of conformations with intrinsically strong ecd signals in the 200-300 nm region, but these signals are essentially absent in the visible. nanomaterials such as metallic nanoparticles (depending on their sizes) display absorptions in the visible region but are achiral. as a result, when biomolecules are co-assembled with nanomaterials their chirality is transferred to create a plasmon-induced ecd signal in the visible region. in this work, we present our results which underscore the occurrence of moderately strong ecd bands in the range 300-550 nm resulting from a series of appropriately thiolfunctionalized peptide oligomers (based on alternating l-ala and aib residues) covalently anchored to 2-2.5 nm sized gold nanoparticles. we related the (positive or negative) signs of the ecd plasmonic signal with the oligopeptide length, that in turn is strictly associated with their secondary structure. this latter property was simultaneously monitored via ecd in the 200-250 nm range. we believe that in our systems a peptide-tometallic surface chirality transfer would take place. light can be controlled with high temporal and spatial precision. if a specific molecule is made light-sensitive, then a precise spatiotemporal control of some of its properties can be achieved. azobenzene is the most widely used photochromic group due to its propensity to pass reversibly from the cis to the trans state under irradiation with light of the appropriate wavelength. the cisand transazobenzene isomers exhibit different spatial arrangement of the aromatic moieties that give rise to significantly distinct physical and chemical properties. the design of novel azobenzene-based molecules with precisely placed photochromic groups able to induce photomodulation of macroscopic properties is currently attracting much interest. in this work, we explored the behaviour of the conjugate formed by linking each of the four hydroxyl groups of pentaerythritol to the carboxylic function of bis[p-(phenylazo)benzyl]glycine. this c α -tetrasubstituted α-amino acid bears two side-chain azobenzene groups. the resulting system exhibits tetragonal symmetry, with a total of eight azobenzene moieties, and can be viewed as a central core surrounded by a shell of azobenzene groups at the periphery. up to eleven (out of the possible fifteen) discrete states produced by sequential trans-to-cis isomerization of the individual azobenzene units have been observed depending on the time of exposure to uv-light. this process is fully reversible (cis-to-trans) under vis-light irradiation for several cycles. in addition, this compound has been shown to exhibit photomodulated physical properties, such as polarity and hydrodynamic volume. moreover, it shows a high propensity to self-assemble in aqueous solution, giving rise to supramolecular vesicles. light-scattering and electron microscopy experiments confirmed that a conformational reorganization of the vesicles can be triggered under exposure to uv or vis light. the total chemical synthesis of native or modified proteins is gaining increase importance in the study of protein function, but also in the development of protein therapeutics. it is usually achieved by assembling in water unprotected peptide blocks using so-called native peptide ligation methods. recently, our group has developed a novel native peptide ligation method based on a peptide featuring a bis(2sulfanylethyl)amido (sea) 1 group on its c-terminus in reaction with a cysteinyl peptide in water at ph 7. we will discuss in this communication the scope and limitations of sea native peptide ligation. for this, model sea peptides featuring all the possible proteinogenic amino acids were synthesized. their rate of sea native peptide ligation with a model cys peptide were determined in the absence of presence of guanidinium hydrochloride or other additives frequently used in ncl. we will present also experiments intended to clarify the mechanism of sea ligation such as the effect of ph on the rate of ligation, or the ability of the transient thioester sea form produced by in situ n,s-acyl shift to participate in thiol-thioester exchange 2,3 . overall, the data show that sea ligation is an interesting method for native peptide ligation at various x-cys junctions, and thus an interesting alternative to ncl. plga copolymers were used as the support for inducing controlled biomarkers releasing system. visualization of the penetration in the hippocampus of mice with confocal microscope was carried out by testing both peptide-free and peptide-bearing nanoparticles, previously labeled with the phthalocyanine fluorescent probe. the encapsulation degree of the larger (12-24) segment was less effective than the others thus stressing the importance of the peptide length to this internalization process. the results showed that all peptide-containing nanoparticles were able to cross the blood-brain-barrier thus indicating improved bioavailability and uptake for peptide delivery into the brain. in regard to the radiolabeling approach, the 99m tc radioisotope was used to label the peptide sequences at his residues, as previously described 3 . stable metal-peptide complexes were obtained in 10 -5 -10 -6 m peptide concentration range. noteworthy, higher metal labeling yield was achieved with peptide segments bearing his residues at peptide c-terminal position, thus pointing to a positiondependent effect for the 99m tc coupling reaction. in conclusion, the findings indicate potentials for the proposed encapsulation and radiolabeling strategies applicable for in vitro and in vivo diagnostic assays with these peptides for the study of amyloid plaques. we have used bifunctional short peptides (ac-cg n c-nh 2 , n=2, 4, 6) to selectively link gold nanorods in an end-to-end manner. additionally, we have manipulated the gap distance between the rods by changing the length of the peptide linker. the presence of the peptide in the gaps was shown by incorporating a propargylglycine residue in the sequence, which was detected with surface-enhanced raman spectroscopy (sers). the acetylene moiety will allow further chemical modification of the linker in the gaps, opening a wealth of interesting molecular systems to be placed and studies inside self-assembled nanogaps. 1 in this work, the fragmentation pathways of alitame, neotame and andvantame in comparison to those of aspartame and aspartame-d3, were studied by negative ion electrospray ionization (esi) high resolution mass spectrometry (thermo orbitrap mass analyzer). accurate mass spectra of the dipeptides allowed proposing specific fragment ions. neotame and advantame, which are the n-(3,3dimethylbutyl) and n-[3-(3-hydroxy-4-methoxyphenyl)propyl] derivatives of aspartame, presented similar fragmentation to that of aspartame. for neotame and advantame, the "diketopiperazine'' pathway seemed to be the major one, while a pathway resulting to the formation of a pyrrolidine-2,5-dione derivative, through the involvement of the side chain carboxyl group of aspartate, was also observed. for alitame, the "pyrrolidine-2,5-dione" pathway was recorded. similarities in the fragmentation using either orbitrap or triple-quadrupole mass spectrometry have been observed. elucidation of the fragmentation is very useful for the trace-level determination of the artificial dipeptide sweeteners in complex matrices. generation of silver nanoparticles in the presence of oligoproline derivatives p. feinäugle, h. wennemers* eth zürich, switzerland in the last years, the generation of silver nanoparticles (agnps) attracts, due to its unusual physical and chemical properties, more and more attention. agnps offer great opportunities for applications in molecular electronics, catalysis, imaging and for antimicrobial coatings. 1 the characteristics depend on their shape and size. 1 many efforts have been made to optimise the generation process by, for example, varying the reducing agents, which usually are used for the synthesis or using manifold additives which should guide the nucleation and also stabilize the resulting particles. nevertheless, the generation of agnps in defined sizes and shapes still remains a challenge. we address this goal by utilizing functionalized oligoprolines that form a conformationally well-defined and rigid helical secondary structure (ppii) 2 as additives. recently, we showed that by decorating this template with aldehydes which allow for in situ reduction of the silver, they act as scaffolds in the generation process and allow the formation of defined nanoparticles. 3 we will report the results of the generation of agnps with various oligoprolines as additives, which differ in the attached functional groups as well as in the length of the peptides. laser desorption/ionization mass spectrometry (ldi-ms) using specific inert surfaces to promote ion formation has been widely investigated the last decade [1] . in addition to porous silicon through the original dios technique, different materials were tested as potent ldi-promoting agents. we explored a variety of inert silicon-based uvabsorbing materials that were presenting different physico-chemical properties for the analysis of peptides [2] [3] [4] . both material architecture (amorphous powders, structured particles, structured surfaces) and material hydrophilic/hydrophobic character tuned by specific chemical derivatization (oxidation, silanization) were probed as crucial parameters for achieving efficient and robust detection of an home-made array of model peptides covering a wide structural and mass diversity. through this set of experiments, we were able to compare the performances of all investigated silicon-based supports, especially taking into account peptide detection sensitivity (down to femtomolar concentrations) and reproducibility/repeatability (intra-spot/inter-spot signal variations) as well as the method robustness using conventional maldi-tof/tof instrument. having illustrated the capability to achieve both peptide detection and sequencing on these ionizing surfaces in the same run, high-throughput identification of protein tryptic digests by a rapid ms profiling and subsequent ms/ms analyses was achieved. comparison of the ms and ms/ms data with those obtained with sample conditioned in organic matrix [5, 6] showed a great behavior for low mass responses demonstrating the capability of ldi on nanostructured silicon supports to be a complementary method to maldi in proteomic workflow. the dipeptides aspartame, alitame, neotame and advantame are low caloric artificial sweeteners. advantame 1 , which is the n-[3-(3-hydroxy-4-methoxyphenyl)propyl] derivative of aspartame, is the most recent among them. an application for its approval has been applied in usa, australia and new zealand. such sweeteners are used in food products and beverages and they can help in managing body weight and disorders like obesity and diabetes. 2 in this work, the simultaneous determination of aspartame, alitame, neotame and advantame by negative and positive electrospray ionization (esi), under hydrophilic interaction chromatography (hilic), is presented. advantame, neotame and intermediates were synthesized in our laboratories for the present application. the key-step for the synthesis of advantame and neotame was the reductive amination of h-asp(obu t )-phe-ome with 3-(3-hydroxy-4methoxyphenyl)propanal and 3,3-dimethylbutanal, respectively. the chromatographic behavior of the artificial sweetener dipeptides was studied on two hilic columns: kinetex hilic (a fused core silica column) and zic-hilic column (a sulfoalkylbetaine column). the separation of dipeptides was achieved on kinetex hilic using 5 mm ammonium formate buffer ph 3.5 / methanol / acetonitrile (15/10/75), with a flow rate of 100 μl/min at 50 o c column oven temperature. at this ph, silica is neutral and the dipeptides are in positively charged form. the retention mechanism of all analytes seems to be partition to the water layer as well as hydrogen bonding. 3 département de pharmacologie, université de sherbrooke, sherbrooke, qc, canada plasma and in vivo stability are essential requirements for the successful development of potential drug candidates or diagnostic imaging probes. rapid degradation of compounds in plasma may result in insufficient concentration to produce the desired pharmacological activity or to be used as a diagnostic agent. there are several strategies to improve plasma half life of peptides including pegylation, modification of nand cterminal fragments of peptide, replacement of labile amino acids, and cyclization 1 . we have previously reported on probes which specifically detect matrix metalloproteinase-2 (mmp-2) activity with magnetic resonance and optical imaging 2,3 . mmps are zincdependent endopeptidases degrading the extracellular matrix (ecm) and involved in cancer progression. the main goal of this work was to find more stable probes without sacrificing enzyme specificity. we have selected specific mmp-2 substrates 4 and their stability was evaluated in three different conditions: in plasma, in plasma with a mmp inhibitor and in a mmp-2 solution. the samples were analyzed by hplc to detect the degradation pattern of our compounds and by lc-ms to determine the molecular mass of peptide fragments. based on these studies, the most stable peptide was selected and incorporated in a solubility switchable probe with radiolabelled (68)ga-dota. its in vivo stability was estimated up to 30 minutes, making it a suitable candidate for further investigations. cancer of thyroid gland is the most common malignancy of the endocrine system. the treatment improvement could be achieved by early diagnosis. the aim of the study was to identify cancer specific markers using the libraries of artificial receptors immobilized on the cellulose. an array of supramolecular structures formed from n-lipidated peptides attached to cellulose via aminophenylamino-1,3,5-triazine was prone to formation of monolayer of "holes" and "pockets" in dynamic equilibrium. this selforganized structures were found capable of binding small guest molecules very efficiently recognizing the shape, size, and polarity of ligands, thus resembling arti cial receptors 1 . recognition and binding properties of guest molecules by artificial receptors depends mainly on the character of peptidic pockets and structure of the fatty acid. proper construction of the binding pocket allows selective binding components of mixtures of compounds from a living organism 2 . the preliminary data indicates that it is possible to construct an array of artificial receptors with diversified structures of peptidic pockets which are able to distinguish between components of homogenates from tumor and normal tissue. century. most of opioid alkaloids and their derivatives have μ-opioid affinity, while endogenous enkephalins are rather δ-than μ-selective. morphine is still the drug of choice for treating severe pain caused by cancer or surgical operation, but its side effects are the reason for the searching and development of new, selective mor agonists. the aim of our study is to choose within recently published crystallographic structures templates for homology modeling of the human μ-opioid receptor. we generated several models using different templates and all of them were evaluated by docking procedure (gold 5.1) ligands used in this investigation were synthesized and evaluated for their biological activity in our previous studies. they are enekphalin analogues with substitutions in second position. the best model of the human mu-opioid receptor was chosen according to data obtained from docking and in vitro biological activity of analogues and endogenous enkephalins. acknowledgments: this work was supported by nfsr of bulgaria project dvu 01/197 and cost action cm0801 project do 02-135/31.07.2009. pneumoniae, h. pylori, proteus sp. are considered as important factor contributory to development of rheumatoid arthritis (ra). the aim of this study was to investigate the level and specificity of antibodies binding to the synthetic peptides corresponding to the bacterial ureases "flap" region sequences in the rheumatoid arthritis patient's sera. for these investigations, peptides with amino acid sequences derived from "flap" regions of different ureases were synthesized using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate 1 (dmt/nmm/bf4) as coupling reagent. peptides were immobilized on a cellulose membrane. the level of antibody binding as well as specificity of them was analyzed by quantitative dot blot method using sera 40 sera from rheumatoid arthritis patients (rap) and sera from 38 volunteer blood donors (vbd). the results of studies suggest that "flap" region may be involved in arising antibodies participating in autoimmunological processes but not to fight infection. this effect indicates that the peptides analyzed by us could be useful for investigation of ra pathogenesis. this suggestion was confirmed by the antibodies absorption experiment which indicates that specificity of antibodies present in rap serum is slightly lower in comparison with vbd serum. it has been found that antibodies present in rap serum recognize not only a specific peptide but also peptides containing fragments with different amino acid sequences. it means that immune system of rap is unstable and may produce a wide spectrum of antibodies recognizing not only a specific epitope but also a set of similar structures. autoantigen-specific t-cells also play a crucial role in the initiation and perpetuation of dsg3/dsg1-specific t-cell responses. t-cells recognize epitopes from dsg3 protein and produce different cytokines, e.g. interferon-γ (ifnγ). functional t-cell epitopes of dsg3 protein have outstanding importance in immunopathological research, development and the design of novel diagnostic tools. our previous studies have shown that certain t-cell epitope peptides are able to stimulate the peripheral blood monomorphonuclear cells (pbmc) of pv patients more effectively than those of healthy donors. our aim was to select a set of t-cell epitope peptides as potential synthetic antigens which are reliably able to distinguish between donors based on the in vitro t-cell stimulating activity. we have prepared synthetic dsg3 oligopeptides by fmoc/tbu solid phase methodology. after cleaving from the resin with tfa the peptides were purified by rp-hplc, and they were characterized by rp-hplc, mass spectrometry and amino acid analysis. pbmc of pv patients and healthy donors were isolated; and the cultures were stimulated by dsg3 peptides in a concentration of 0.025 mm for 20 hours, and the rate of ifnγ production was determined from the supernatants in sandwich elisa. synthetic dsg3 oligopeptides induced different in vitro ifnγ production rate on pbmc obtained from pv patients and healthy controls determined by elisa. our approach identified a synthetic antigen set as a promising biomarker for pemphigus vulgaris. [1] . in particular, cap2b (pelyafprvamide) has been shown to elicit antidiuretic activity in the green stink bug acrosternum hilare [2] , an important pest of cotton and soybean in the southern united states. analogs of cap2b containing either an (e)-alkene, cispro or a transpro isosteric component [3] were synthesized and evaluated in an in vitro stink bug diuretic assay, which involved measurement of fluid secretions of malpighian tubules isolated from a. hilare [2] . at a concentration of 1 μm, the conformationally constrained transpro analog demonstrated significant antidiuretic activity, whereas the cispro analog failed to elicit any activity. the results provide strong evidence for adoption of a trans orientation for the pro in cap2b neuropeptides during interaction with the receptor associated with the antidiuretic process in the stink bug. the work further identifies a scaffold with which to design biostable mimetic cap2b analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting cap2bregulated diuretic systems. the enkephalins are pentapeptides (tyr-gly-gly-phe-met/leu) with a proven antinociceptive action. it is believed that the interaction between them and the lipids composing the membranes is important for converting the peptides into a "bioactive" conformation 1,2 . using langmuir's monolayer technique the interaction of a synthetic methionine-enkephalin (met-enk) and its amidated derivative (met-enk-nh2) with mixed lipid monolayers composed of palmitoleoylphosphatidylcholine (popc), sphingomyelin and cholesterol was studied. the surface pressure-area (π-a) isotherms with regard to πmin, π max and the hysteresis curve shape of the pure lipid monolayers and after the addition of the respective enkephalins were detected. in addition, by using brewster angle microscopy (bam), the surface morphology of the mixed lipids-enkephalins monolayers were determined. our results suggest that there is a strong penetration effect of the enkephalins studied into the mixed monolayers. moreover, our results demonstrate the potential of lipid monolayers formed in langmuir's through in combination with bam to be successfully used as an elegant and simple membrane models to study lipid-peptide interactions at the air/water interface. acknowledgments: this work was supported by bulgarian ministry of education, youth and science, projects n do02-107/08, drg 02/5 and my-fs-13/07. dept pharmacolgy, temple univ, philadelpha, pa 19140, usa bioinformatic algorithms has predicted the existence of several potential hormone-like peptides transcribed from the ecrg4 gene 1 . previous publications indicated a highly level of gene expression of ecrg4 products has been found in the pancreas 1 , choroids plexus, epithelial cells, leukocytes, and macrophages 2 . however, the presence in the hypothalamus and the major form of derived peptides in each tissue haves not been clearly identified. knowingledge of the precise peptide generatesd within a given tissue is essential to understanding its functions. we have generated the peptide specific antibodies to against ecrg4-derived pprepro-augurin(71-147) and developed a specific ria kit for the quantification of the such peptide in question. a method for the purification of endogenous ecrg4-derived peptides from bovine hypothalamus also has been established. using ria to monitor the immunoreactive fractions and maldi-tof to identify the endogenous peptides, we foundhave detected the presence of ecrg4 derived molecular of bovine preproaugurin(71-147) from the homogenates of bovine hypothalamus. immunohistochemistrycal staining by antibody aalso confirmed the presence of thee peptide in some of the hypothalamic cells. of hypothalamus. the amount of prepro-augurin(71-147) in the hypothalamus although not soas high as pancreas, but is one third of the augurin level of the pituitary. conclusions: the native peptide derived from augurin preproteinecrg4 has been discovered.identified. we have confirmed the property of purified peptide,s prepro-augurin(71-147), along with the synthetic peptide standards. the present study provides the necessary procedures such as the elution from (1) c18 column, (2) p6 sizing column, and (3) a further purification conditions for hplc in order to enhance the immunoreactivity from tissue fractions and yield enough amount for identification. this dsip-related peptide (kn-dsip or knd) differs from dsip by only 2 amino acid residues in positions 2 and 5. we do not consider the homology between dsip and knd as accidental, bearing in mind functional significance of histone demethylases of the jmjc-group. methylation-demethylation of histones is known as an important mechanism of posttranslational modification playing a prominent role in epigenetic regulation of chromatin structure and gene transcription. dsip is also known as an effective "normalizer" and protector from homeostatic disorders induced by stress related disturbances. we suggest that histone demethylase of the jmjc-group containing dsip-related region can be considered as a possible protein precursor of endogenous peptides with dsip-like activity. in order to test our hypothesis we synthesized knd and studied its biological effects. in a preliminary assay cited below [1] knd showed similar and probably more pronounced effects than dsip as an agent that stimulates endurance and stress-resistance of animals in the forced swimming test. also knd provided a more active detoxifying action after administration of a semi-lethal dose of the cytostatic agent. in the present work we assessed neuroprotective and antioxidative potency of both peptides in vivo and confirmed the higher efficiency of knd. this study is supported by the moscow government. is a tridecapeptide (pglu 1 -leu 2 -tyr 3 -glu 4 -asn 5 -lys 6 -pro 7 -arg 8 -arg 9 -pro 10 -tyr 11 -ile 12 -leu 13 ) highly expressed in the central nervous system. this peptide elicits an analgesic response following peripheral or central administration. importantly, nt exerts a more potent analgesia than morphine at an equimolar dose, without having the associated side effects of opioid drugs. 1 structure-activity studies have identified the c-terminal fragment nt(8-13) as the biologically active minimal sequence. however, nor the full or truncated peptides cross the blood-brain barrier (bbb), thus hampering its clinical development. the substitution of pro 10 by an unnatural amino acid silaproline 2 (sip) increased bioavailability and plasma stability. structural properties conferred by the pro 10 were also retained as determined by nmr and ir. 3 aiming at delineating the mode of action of cl, three new cl derivatives bearing suitable labeling moieties, i.e the fluorescent molecule fitc, the streptavidin-counterpart biotinyl-group and the 99m tc-radiometal chelating unit dimethylgly-ser-cys, were designed, synthesized, purified, and characterized to be applied in in vitro and in vivo evaluation studies. the structure of the cl derivatives in aqueous solutions was studied with nmr, in parallel and in comparison with the parent molecule cl, in order to examine whether the presence of the labeling moieties has induced changes to the structure of the biologically active part of cl. cell survival assays with cl and the cl derivative bearing the fitc moiety were conducted in the pc12 cell line in order to explore their rescue effect. in parallel, the cl derivative bearing the dimethylgly-ser-cys moiety was successfully radiolabeled with 99m tc and its stability was assessed over time in its synthesis reaction mixture and in plasma. this 99m tc-radiolabeled derivative was subsequently administered to swiss albino mice in order to determine the biodistribution of cl in the living organism and its route of excretion, a study that has not been carried out so far for any peptide of the humanin family. furthermore, the potential interaction of cl with β-amyloid peptide, the hallmark of ad pathogenesis, was explored with circular dischroism. the results of this multifaceted approach to the biological action of cl will be presented. institute of biochemistry and biotechnology, martin-luter university, halle-wittenberg, germany kinins, such as the nonapeptide bradykinin, are important mediators of various physiological and pathophysiological responses including inflammatory disease, asthma, rhinitis, cell division, pain, vascular permeability, allergic reactions, pathogenesis of septic and endotoxic shock. there are two types of receptors for kinins, known as b1 and b 2 . b 2 receptors are constitutively expressed in wide variety of cells and required entire bk sequence for recognition, while b1 receptors have normally very limited expression and respond to [desarg 9 ]bk. b 1 receptors gene is turned on following either tissue damage or inflammation. accumulated evidence indicates that most of the clinically relevant effects of bk are functions of b2 receptors this being the reason why research on their antagonists is a topic of great interest. in our previous study we described the synthesis and some pharmacological properties of four new analogues of bradykinin (bk), designed by substitution of position 7 or 8 of the known [d-arg 0 ,hyp 3 ,thi 5,8 ,d-phe 7 ]bk antagonist with l-pipecolic acid (l-pip) (both analogues were also prepared in n-acylated form with 1-adamantaneacetic acid (aaa)). our results showed that presence of l-pip in position 7 slightly increased antagonistic potency in the blood pressure test, but it turned the analogue into an agonist in the rat uterus test. replacement of thi by l-pip in position 8 also enhanced antagonism in the rat pressure test but preserved the antagonism in the rat uterus test. in the present study we continue our previous investigations to find structural requirements which in the case of bk analogues result in high b2 antagonistic activity. several new bradykinin analogues modified in their cterminus with d-pipecolic acid were synthesized using spps method. the biological properties of the analogues were assessed by their ability to inhibit vasodepressor response of exogenous bk in conscious rats and by their ability to inhibit the contractions of isolated rat uterus evoked by bk. acknowledgements this work was supported by the university of gdansk (ds/8453-4-0169-2). peptides with beta-turn structure in peptide/mhc complexes a. stavrakoudis department of economics, university of ioannina, greece major histocombatibility complex (mhc) molecules interact with small peptides and form complexes. in most of the cases, peptide's structure in these complexes is found in extended conformation. however, notable exceptions exist where the peptide forms a beta-turn structure. this happens mainly in the central part of the peptide in class i complexes [1] , or at the c-terminal of class ii complexes [2] . several peptide/mhc complexes were, derived with xray studies, were extensively subjected to molecular dynamics simulations [3] in order to investigate the stability of this turn-like structural feature and to explore the factors that possibly contribute to this stability. it was found that both intra-peptide and peptide/mhc interactions might be responsible for peptide's conformation. the peptides were found to undergo several structural transitions indicating conformational plasticity and not a completely rigid structure inside the mhc groove. the results might be of special importance in designing defective peptide vaccines and beta-turn pharmaceuticals. the heptapeptide met-enkephalin-arg6-phe7 (merf) with the sequence of yggfmrf is a potent endogenous opioid located at the c-terminus of proenkephalin-a (penk), the common polypeptide precursor of met-and leuenkephalin. our systematic bioinformatic survey revealed considerable sequence polymorphism at the heptapeptide region of different penk prepropeptides among 56 vertebrate animals. four orthologous heptapeptides with single or double amino acid replacements were identi ed among 15 animals, such as yggfmgy (zebra sh), yggfmry (newt), yggfmkf (hedgehog tenrek) and yggfmri (mudpuppy). each novel hepta-peptide, together with the mammalian consensus merf and metenkephalin, were chemically synthesized and subjected to functionality studies, using radioligand binding competition and g-protein activation assays in rat brain membranes [1] . equilibrium binding af nities changed from good to modest as measured by receptor type selective [ 3 h]opioid radioligands. the relative af nities of the heptapeptides reveal slight mu-receptor (mop) preference over the delta-receptors (dop). [ 35 s]gtpγs assay, which measures the agonist-mediated g-protein activation, has demonstrated that all the novel heptapeptides were also potent in stimulating the regulatory g-proteins. all peptides were effective in promoting the agonist induced internalization of the green uorescence protein-tagged human mu-opioid receptor (hmop-egfp) stably expressed in hek293 cells. thus, the c-terminally processed penk heptapeptide orthologs exhibited satisfactory bioactivities, moreover they represent further members of the so-called "natural combinatorial neuropeptide library" emerged by evolution. corticotropin releasing factor (crf) exerts most of its physiological and pathophysiological actions by interacting with its type 1 receptor (crf1) and activating different intracellular signalling pathways. the crf1 is a plasmamembrane protein, which belongs to the family b of g-protein coupled receptors (gpcrs) and like the other gpcrs consists of an amino-terminal extracellular region, a carboxyl-terminal intracellular tail and seven, mostly hydrophobic, membrane-spanning segments (tm1-tm7), connected by alternating intracellular (il) and extracellular loops (el). binding of crf and its related peptides, such as sauvagine, to the extracellular regions of crf1 is associated with receptor activation and subsequent activation of different g-proteins and regulation of diverse signalling pathways. using a mutagenesis approach in combination with a radioligand binding study we found that trp259 and phe260 in the second extracellular loop of crf1 interacted with the amino-terminal portion of crf and sauvagine. interestingly only the interaction of sauvagine with trp259 and phe260 is important for crf1-mediated stimulation of camp accumulation. in marked contrast the interaction between crf and the residues trp259 and phe260 was unimportant for the activation of adenylate cyclase. thus it is possible for trp259 and phe260 of crf1 to regulate distinct signalling pathways, or different sets of them, after their interaction with different peptides. we are now performing experiments to fully elucidate the signalling pathways that are regulated by the interaction of crf and sauvagine with trp259 and phe260. these studies will advance the development of crf1-selective selective signalling-specific peptides that would be extremely useful for the elucidation of the role of crf1 in many physiological and pathophysiological situations, and possibly for the treatment of several crf1-related diseases. thymus humoral factor gamma-2 (thf-γ2), an octapeptide, purified from crude thf, retains essentially all the biological properties of thf [1] [2] . it regulates clonal expansion, differentiation and maturation of t-cell precursors, stimulates the production of lymphokine, maitains the normalization of impaired ratios between helper(cd4+) and suppressor / cytotoxic (cd8+) subsets and augments il-2 production in spleen cells. thf-γ2 has a calculated molecular weight of 918 and has the following amino acid sequence: leu-glu-asp-gly-pro-lys-phe-leu. its poor stability towards protein enzyme limits its extensive application. with the inte ntion to promote its bioavailability, bioactivity and develop ideal immunoregulatory drug candidat, four series of derivatives of thf were designed and synthesized: 1. n-and cterminal acylation. 2.restitution the flexible segment gly-pro by unnatural amino acids 6-aminohexanoic acid (aca) in order to shorten the synthetic steps and simultaneity improve the bioavailability and biostability of peptide; 3. reserve protected group of some amino acid residus as spot mutation. 4. mannich-based cyclization was carried out on resin [3] , phe was replaced by tyr serving as the active hydrogen component, a proline was introduced at the n terminal as the amine component and formaldehyde was used as the only component in solution. the bioactivity of synthesized products were detected. the leukocytopenia model in mice was induced by cyclophosphamide intraperitioneal injection. white blood cell count, thymus index and spleen index were detected to evaluate the immune function of compounds in mice. the results show that those compounds play a significant role in improving immune function in mice. the activity of compound lhl21 and lhl22 are also better than authentic compound tp-5 and tα1. marine organisms have been recognized as a promising source for the development of new pharmaceuticals. in the course of screening for antitumor substances from marine organisms, we found cyclic peptides containing many nonribosomal amino acids such as hydroxyasparagine, hydroxyleucine, or other supporting a hydrophobic side chain that were shown to be a key element for their biological activity. the laxaphycine b, a cyclic lipopeptide isolated from marine cyanobacteria anabaena torulosa harvested in french polynesia 1 constitutes an example of this peptide class. this compound has attracted our attention because of its micromolar cytotoxic activities on different cancer cell lines as well as its antiangiogenic properties which seems to be due to an interaction with the vegf receptor-1 2-3 . the synthesis of the non-natural amino acids 4-5 and of laxaphycine b analogues will be presented along with their preliminary biological activities. immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases including dermatology. cyclolinopeptide a (cla) c(pro 1 -pro 2 -phe 3 -phe 4 -leu 5 -ile 6 -ile 7 -leu 8 -val 9 -), a cyclic, hydrophobic nonapeptide isolated from linseed, possesses strong immunosuppressive and antimalarial activity. 1 it has been suggested that both the pro-pro cis-amide bond 2 and an 'edge-to-face' interaction between the two aromatic rings of adjacent phe residues 3 in tetrapeptide unit are important for biological activity. this edge-to-face interaction can be influenced when phenyl rings are replaced by naphtyl substituent. in this communicate new analogues of cla modified by 2naphtylalanine (2-nal) in positions 3 or 4 or both 3 and 4 (1-3 linear analogues, 4-6 cyclic analogues) will be presented. the synthetic strategy and biological activity as well as conformational analysis will be evaluated. the onset of type ii diabetes mellitus (t2dm) coincides with the deposition of fibrillar material in the islet of langerhans in the pancreas that is a clinical hallmark of more than 95% of patients suffering this disease. 1 the main component of the pancreatic amyloid deposits is a 37-residues polypeptide hormone called islet amyloid polypeptide (iapp) or amylin. 2 in this work we have examined, by means of cd spectroscopy and tht-fluorescence, the conformational polymorphism of both full-length 1-37 hiapp, and the related fragment hiapp17-29, and compared the results with the respective rat counterparts. moreover, the cytotoxic activity was determined toward different pancreatic β-cells lines in the attempt to correlate iapp's fibrillogenic properties with the ability to mediate cell death. together the results suggest that β-sheet conformational transition, that generally preludes to fibril formation, is not a prerequisite for eliciting toxicity toward β-cells cultures. interestingly, confocal microscopy indicated that both hiapp1-37 and hiapp17-29 can enter the cell and might exert their toxic action at intracellular level. acknowledgments: this work was supported by miur, firb-merit project rbne08hwlz. due to its physiological functions, 26s proteasome is considered the target molecule in overcoming several diseases [1] . its core particle 20s has three types of active sites: chymotrypsin-, trypsin-and caspase-like. many natural and synthetic compounds were tested for their ability to inhibit proteasome. a recent report describing the inhibition of 20s by the serine proteases inhibitor -bovine pancreatic trypsin inhibitor -was considered by us with great attention [2] . our scientific interest is focused on peptide inhibitors and their interaction with serine proteases. sunflower trypsin inhibitor (sfti-1) is the smallest and the most potent peptide inhibitor in the bowman-birk family. owing to its size and the rigid structure (disulfide bridge and "head to tail" cyclisation) sfti-1 is willingly chosen as the lead structure in the search for new inhibitors [3] . its sequence is shown below (lys 5the p1 residue responsible for specificity): & 1 gly-arg-cys(& 2 )-thr-lys 5 -ser-ile 7 -pro 8 -pro-ile-cys(& 2 )-phe-pro-asp& 1 since native sfti-1 is not able to inhibit 20s [2] , we have designed its monocyclic analogues (with disulfide bridge only) with lys or arg in position 5 (p 1) and at least one basic amino acid (lys or arg) in positions 7 (p 2') and/or 8 (p3'). all analogues inhibit chymotrypsin -(ic50 at the range of 1÷3 μm) and caspase-like (ic 50 at the range of 0.7÷7μm) activities in vitro, whereas their activity towards trypsin-like specificity is much weaker. in several rat tissues, our view on ras has changed. metabolism of the ang-(1-12) may represent alternative pathway of ang ii formation, importantly, independent on renin and ace activity 1,2 . ahmad 2 et al. have described metabolism of ang-(1-12) by human atrial tissues and showed that ang ii is formed mainly by chymase. this renin-inependent ang ii production could explain the "resistance" regarding use of ace inhibitors in patients with hypertension or diabetic nephropathy. noteworthy, the role of ang-(1-12) in circulation is still unclear and there are no information about possible pharmacological modulation of its metabolism. in our study, we compared the ex vivo metabolism of angiotensinogen (fragment 1-14) in hypertensive (shr) and normotensive (wky) rats in organ bath of aorta and heart using lc-ms method 3 . surprisingly, we identified ang-(1-12) formed via reninindependent pathway to be a main product of angiotensinogen metabolism in rat aortic tissue and heart. in this setting, ang-(1-12) appeared to be not only prevalent metabolite of angiotensinogen, but also served as a substrate for generation of ang i and ang ii. as compared to wky rats, formation of ang ii, from ang-(1-14), was much higher in shr aortas but not in the heart. the functional consequences of these findings require further investigation. this study was supported by the grant n n401 293939 polish ministry of science and higher education. the lysosomal cysteine protease cathepsin c (cat c), also known as dipeptidyl peptidase i (dppi), activates a number of granule-associated serine proteases with proinflammatory and immune functions by removal of their inhibitory n-terminal dipeptides. activity of this protease is associated with several pathologies in human body [1] . in this work the characterization of cat c specificity using combinatorial chemistry methods will be described. the main goal of this work was to determine of substrate specificity of the prime region of this enzyme. the chemical synthesis and deconvolution of two libraries will be described. the hemostatic mechanism has the crucial role to prevent loss of blood from injured blood-vessels. this loss is prevented by the integrity of the vessel walls, by platelets aggregation or by blood coagulation, which in normal conditions is limited onto the local trauma of the vessel wall. in generally, the blood coagulation mechanism is important for maintaining vascular integrity and thus for the precaution of an organism from bleeding, which may also occur by blood coagulation caused by thrombin production. the diversion rate of this production leads to an expansion of thrombin to the general blood circulation. thus, when thrombin generation is not controlled by the mechanisms of inhibition, a widespread undesirable intravascular thrombosis is occurred. the whole process of platelets adhesion requires the presence of clotting factor viii (fviii), a necessary for the blood coagulation cascade glycoprotein, which takes part in the intrinsic pathway and acts as a coenzyme for the activation of factor ix, a serine protease depended on the thrombin production. the target of the present research is the synthesis of biologically active cyclic, head to tail, peptides, analogs of the sequence 558-565 of a2 subunit of fviii, which are potentially capable to block fviiia-fixa complex, reducing the thrombin production and thus the blood coagulation. the synthesized peptides are investigated for their inhibitory activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (aptt) and the reduction of the % value of the fviiia, which they generate in samples containing recombinant fviiia, in vitro. the blood coagulation is part of an important host defence mechanism, which under pathological conditions results in inappropriate intravascular coagulation when thrombin is produced. clotting sequence is the result of a cascade of two biochemical pathways, intrinsic pathway, so called because all components are present in blood, and extrinsic pathway, in which tissue factor is required in addition to circulating components. the activated form of factor viii (fviiia) is a key component of the fluid phase of the blood coagulation and plays an important role formatting a trimolecular complex with factor ixa, ca 2+ and negatively charged phospholipids of the cells membrane. this complex is called tenase and participates in activation of prothrombin, which acts on fibrinogen to generate fibrin monomer, polymerized rapidly to form fibrin clot. the fviii is comprised of a heavy (a1-a2-b) and a light (a3-c1-c2) peptide chain, both cleaved by proteases at three sites, resulting in alteration of its covalent structure and conformation. its deficiency is known as haemophilia a. our research effort is focused on the synthesis, identification and biological evaluation of peptide analogs, expected to inhibit selectively the increasing of thrombin production. their sequence is based on the regions in which the fviii interacts with fix, specifically on the sequence 558-565 of the a2 subunit. the synthesized peptides are examined for their activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (aptt) and the reduction of the % value of the fviiia, which they generate in samples containing recombinant fviiia, in vitro. inhibitor with the following structure: ac-llllrvkr-nh2, which has potent effects on the proliferation of prostate cancer cells. the potency and stability of this compound was subsequently enhanced by substitution of arg residue in position p1 with its conformationally restricted mimetic -4-amidinobenzylamine (amba). nevertheless, the specificity toward pace4 was significantly reduced by this modification. thus, in order to improve its selectivity without sacrificing inhibitory potency we decided to use positional scanning approach. in this study we present synthesis of two series of peptide libraries, which were designed by substitution of leu in the p5, p6 position of our control peptide (ac-llllrvkr-amba) with each of nineteen amino acid residues in order to verifying its influence on activity and selectivity of the resulting analogues. all peptides were synthesized by a combination of solid phase peptide synthesis and solution synthesis and tested for their inhibitory potency against furin and pace4. the p2-p8 fragments were synthesized by fmoc/tbu spps strategy on hydrazinobenzoyl or acid labile 2chlorotrityl chloride resin. then coupling of the 4-amidinobenzylamine · 2 hcl was performed. the best modifications were combined to give as several multipoint substituted inhibitors. we believed that our work, will provide new important information about structure-activity relationship of these class of analogs in order to obtain potent and highly specific pace4 inhibitor. institute for research in biomedicine, parc cientific de barcelona, barcelona -spain a bacterial toxin-antitoxin (ta) system is composed of two genes organized in an operon encoding a toxin and an antitoxin that regulate the growth and death bacterial cell under various stress conditions. the operon parde encode a ta system formed by pare toxin and its antitoxin pard. pare is a 12 kda protein that inhibits dna gyrase activity and thereby blocks dna replication. however the pare-gyrase interactions and the gyrase activity inhibition mechanism have not been explored. as an approach for understanding of this mechanism and to elucidate the pare region responsible for protein-protein interactions we have designed and synthesized a series of linear analogues of pare and investigated the ability of peptides to inhibit dna topoisomerases activity. so, based on structural data inferred from pare three-dimensional model 1 , 12 peptides were synthesized by solid-phase method. four peptides (parelc3, parelc8, parelc10 and parelc12), showed complete inhibition of dna gyrase supercoiling activity, by gel electrophoresis assay 2 , with an ic100 of 20 to 50 μmol.l -1 . in addition, intrinsic fluorescence and fluorescence anisotropy assays showed that inhibition process must occur by interaction with the gyra subunit. differently of wild type pare, the peptide analogues were able to inhibit the dna relaxation of topoisomerase iv with lower ic100 values. interesting was that only parelc12 displayed inhibition of the relaxation activity of human topoisomerase ii. our results suggest a new class of molecules with simultaneous inhibitory activity in dna gyrase and topoisomerase iv. furthermore, we have obtained the first example of a synthetic peptide from a bacterial toxin with inhibitory activity on human topoisomerase ii. institute of experimental endocrinology, slovak academy of sciences, bratislava, slovakia the renin-angiotensin system (ras) has long been recognized as an important regulator of systemic blood pressure and electrolyte homeostasis. our understanding of ras has experienced remarkable change over the past two decades. besides, angiotensin ii, the new biologically active peptides [e.g. ang-(1-7), ang-(1-12), ang iv, ang-(2-10)] and pathways [e.g. angiotensin converting enzyme 2 -ace2] have been described 1 ; some of them, like ang-(1-7) may oppose many actions of ang ii. importantly, despite all components of classical ras are found in adipose tissue 2 , the data about fat formation of various angiotensins remain scarce. in our study, we compared the ex vivo metabolism of angiotensinogen, ang-(1-12) and ang i in hypertensive (shr) and normotensive (wky) rats in organ bath of retroperitoneal and periaortic fat tissue using lc-esi-ms method. additionally, qpcr measurements of mrna expression of main enzymes involved in ang i metabolism were performed. both in the periaortic and epidydymal fat, the formation of ang-(1-7) was higher than production of ang ii. fat tissue formation of two main ang i conversion products, ang ii and ang-(1-7), differed significantly between shr and wky rats. compared to wky rats, the formation of ang-(1-7) in periaortic fat tissue was decreased in shr. in opposite, in epidydymal fat tissue formation of ang-(1-7) and ang ii was higher in shr. interestingly, there were no differences in aorta formation of ang ii and ang-(1-7) between shr and wky rats. our results suggest that in hypertension visceral fat production of angiotensin peptides is increased, while generation of "beneficial" ang-(1-7) in periaortic fat is decreased. however, the functional importance of such finding require further investigation. department of chemistry and biochemistry, university of washington, usa phospholipases a2 (pla2) are a superfamily of enzymes involved in various inflammatory diseases. 1 in particular, human secreted giia spla2 is an attractive target for the development of novel medicines. 1 we have shown that 2oxoamides based on γ-or δ-amino acids are potent inhibitors of cytosolic giva pla2. very recently, we have demonstrated that a long chain 2-oxoamide based on (s)leucine displays inhibition of human and mouse giia spla2s (ic50 300 nm and 180 nm, respectively). 2 a combined experimental/computational study was undertaken to further understand the role of the α-amino acid of 2-oxoamides for the inhibitor-enzyme binding. the crystal structure of giia spla2s reveals a highly conserved ca 2+ -binding loop and a catalytic dyad consisting of his47/asp91. 2-oxoamides based on hydrophobic αamino acids showed better binding score prediction compared to polar α-amino acid derivatives. a number of new 2-oxoamides based on α-amino acids were synthesised and tested for their inhibitory activity against giia, gv and gx pla2. the 2-oxoamide based on (s)-valine displayed potent inhibition of giia spla2 (ic 50 218 nm) in accordance with the predicted docking score. docking results reveal that (s)-valine-based inhibitor forms key interactions with the active site of the enzyme. the carboxylic group participates in a hydrogen bonding with gly31 and lys62, and 2-carbonyl group with gly29. furthermore, both carbonyl groups are in the proximity with ca 2+ . the side chain of (s)-valine adopts a suitable orientation to interact with tyr51 and lys62. the long aliphatic 2-oxoacyl chain is accommodated in the hydrophobic region of the active site and creates proximal contacts with leu2, ile9, his6 and phe5. the search for novel classes of pharmaceutical molecules with enhanced therapeutic power has been the subject of numerous research groups all over the world. moreover, systems of immobilization and controlled release which are adapted to these new classes of molecules, has proven to be an area of extreme importance to provide the same therapeutic efficacy. using solid-phase chemistry a series of ccdb toxin analogous peptides were synthesized and were synthesized and tested against the capacity of inhibition of bacterial enzymes dna gyrase and topoisomerase iv (topo iv). subsequently those peptides were detained in drug delivery systems (dds) to be tested against the inhibition of growth of different bacterial species. in this data we could observed that the analogue ccdbsg2 could inhibit only dna gyrase and not the topoisomerase iv. in the other hand the analogue ccdbsg1 presents a hard inhibition potential against topo iv specially because of their structural difference. is possible conclude that topoisomerase iv presents the tertiary structure very similar to dna gyrase, but those mechanisms of action must be clearly distinct 1 . in the in vitro studies, as expected, results revealed that the drug delivery systems are the key to the power efficiency of peptide analogues against the bacterial growth inhibition which cannot be observed when the peptides are free in solution. some of the different lipid compositions of the dds are demonstrating to be more efficient in the membrane cell transverse and this data previously assumes that it is possible to apply different types of dds to promote the peptide molecules transport across the cellular membranes according to several specific therapies. with this studies we have obtained more knowledge about the interaction system of enzyme-toxin and hopes which helps in future studies to development a new antimicrobial molecules class. it is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. we propose to target an ancestral form of the proteasome, the hslvu protease, which is present in the parasite's single mitochondrion, essential for the growth of these organisms and has no analogue in the human host. originally discovered in eubacteria, this complex is constituted by two central hexameric hslv protease rings sandwiched between two hexameric hslu atp-ase rings. as hslv shares a similar enzymatic mechanism with the host proteasome, we propose to inhibit the assembly of the complex in order to be selective. according to studies on bacterial hslvu, 1,2 the c-terminal segment of hslu is essential in hslv activation and in complex assembly, therefore representing a privileged target. we produced recombinant hslv, which is inactive alone, and showed that a synthetic c-terminal hslu peptide was able to induce the digestion by hslv of a fluorogenic substrate that we developed. with this enzymatic test in hands, we started the characterization of the interaction of the c-terminal portion of hslu with hslv. we will present the results obtained with various series of analogues of the original c-terminal hslu peptide, including truncated forms, ala scan, constrained analogues and multivalent constructions. helped by molecular modelling studies, the aim is to establish structural requirements, which could lead to high affinity and stable ligands able to inhibit the interaction between the hslu and hslv rings, obligatory for the degradation of proteins by the hslvu complex. finally, we checked that hslv was inhibited by classical active-site directed proteasome inhibitors like bortezomib. f. babos a,d , e. szarka b , gy. nagy c , z. majer d , g. saŕmay b , a. magyar a , f. hudecz a,d citrullinated filaggrin peptide (ccp) were detected in ra sera and anti-ccp positivity is widely used for diagnostic purposes. identification of new epitopes of filaggrin 1 would be useful in the diagnosis of anti-ccp3 seronegative patients. in order to achieve optimal immune recognition of biotinylated epitope peptides it is important to analyse the effect of the labelling moiety on antibody binding. for these studies 5-as well as 19-mer peptides with nor c-terminal biotin were synthesised manually by spps, using fmoc/ t bu strategy. biotinylation was performed by using biotin, biotinyl-6-aminohexanoic acid or 4,7,10-trioxa-1,13-tridecanediamino succinic acid linker 2 modified biotin. labelled peptides were used in an indirect elisa, on neutravidin pre-coated plates and the binding was detected by anti igg-hrp. to examine the role of the presence/position of biotin in the secondary structure of the peptides, electronic circular dichroism (cd) method was used. we found that the ccp3+ serum samples specifically recognized the c-terminally biotinylated 5-mer filaggrin peptides, while showed no binding with the n-terminally biotinylated compounds. in case of the 19-mer epitope peptides there was no difference between the recognition of nand c-terminal biotinylated analogues. data presented suggest that the position of the biotin in case of the short filaggrin epitope peptides markedly influence the serum antibody binding. upon activation process, they are released from the granules and then involved in immunoresponse of the organism. when out of the cell those enzymes remain in free form or become associate with the cell membrane. the physiological role of this proteases is manifestated in several processes such cytokine and chemokine processing, platelet activation, and degradation of extracellular matrix's proteins [1] . in this work results of the specificity of two members of nsps pr3 and hne evaluated using the combinatorial chemistry methods will be presented . both enzymes share primary specificity and to obtain the selective substrate that will be recognized only by one enzyme, the prime sites should be investigated. the general formula of the designed library is as follows: where in positions x1', x2' and x3', the set of 19 proteinogenic amino acids (except cys) was introduced. abz is 2-amino benzoic acid served as donor of fluorescence and 3-nitro-l-tyrosine as acceptor. eukaryotic proteasome is a highly organized protease complex comprising a catalytic 20s core particle (cp) and two 19s regulatory particles (rp), which together form the 26s structure. the main function of this large intracellular protease is to degraded ubiquitine labeled proteins. the catalytic particle of the proteasome displays three distinct enzymatic activities: trypsin-like, chymotrypsin-like and glutamyl-like. the increase activity of the proteasome is associated with several disease including cancer [1] . the main aim of this work is to synthesized the cell permeable fret displaying peptides that will selective cleaved by single proteasome activity. additionally each peptide when independently cleaved by the proteasome subunit, should emit the fluorescence energy in a different spectral region. our intention was designing substrates which would allow to monitor simultaneously (in a single experiment) and independently of three proteasome activities in this report, we will describe the chemical synthesis of several peptides modified at on cand n-termini by synthetic fluorescent amino acids the general formula of these peptides is as follows: where x is a non proteinogenic amino acid that serve as a donor of fluorescence, y amino acid that is a acceptor of fluorescence. the obtained fluorescent peptides were examined for their ability to cross the cell membrane. also kinetic parameters (k cat, km, kcat/km) with proteasome will be presented. approximately 100 dubs are encoded in the human genome and are involved in a variety of regulatory processes, such as cell-cycle progression, tissue development, and differentiation. recently, several groups have introduced various methods for linking ubiquitin to different substrates via nonhydrolyzable isopeptide bonds, which resist the action of dubs. using these methods, one could explore the function and the mechanism of dubs and apply them in activity based profiling. here we present a new and convenient strategy for preparing nonhydrolyzable ubiquitinated peptides and proteins by nmethylating the isopeptide bond. using this method we prepared several nonhydrolyzable ubiquitinated peptides with different lengths derived from ubiquitinated h2b and examined their affinity to different dubs. f.i. nollmann, c. dauth, d. reimer, h.b. bode* goethe universität, frankfurt, germany bacteria of the genus xenorhabdus and photorhabdus are gram negative gamma proteobacteria that live in symbiosis with nematodes of the genus steinernema. undergoing their partly entomopathogenic life cycle these bacteria not only produce antibiotics 1,2 and insecticides 3 but also several different small molecular compounds and peptides. 4 for the most part the biological benefits of these secondary metabolites have not fully been understood yet. 5 with the help of inverse feeding experiments, hr-ms and nmr as well as molecular engineering we were able to characterize and/or isolate some of these peptides. since they are mostly produced in trace amounts, we synthesized them in order to make them accessible to continuative testing. given that not only linear but also highly methylated or cyclic peptides are produced, the synthesis was quite challenging. nevertheless, we were able to establish in our laboratory a general synthesis route for cyclic peptides 6 and depsipeptides 7 , as well as highly methylated hydrophobic linear sequences. testing several of these peptides has revealed activity against insect cells and against the causative organisms of neglected tropical diseases. cyclotides are a large class of plant peptides defined by a head-to-tail cyclized backbone and three conserved disulfide bonds in a knotted arrangement. these unique structural features confer them with remarkable stability and due to a range of bioactivities they are extensively investigated as templates in drug discovery 1 . based on the use of oldenlandia affinis in traditional african medicine for its uterotonic principle we investigated crude plant extracts and semi-pure cyclotide fractions for the ability to induce uterine contractions using a collagen-gel contractility model2. pharmacological analysis of the effects led to the identification of the oxytocin receptor, a representative of the g-protein coupled receptor (gpcr) family, as a molecular target for cyclotides. mass spectrometry-based sequence analysis of 'active' fractions revealed cyclotides with high similarity to the human oxytocin (h-ot) peptide that exhibited weak binding to the human oxytocin receptor. we further analyzed synthetic cyclotide-derived small ot-like peptides and grafted the h-ot sequence into the stable cyclotide frame. these peptides showed increased binding and activation as compared to native cyclotides. these findings may open new avenues for the discovery of gpcr ligands from natural peptide sources. gpcrs are promising drug targets and ~5 0% of currently used drugs act via binding to these receptors. natural combinatorial peptide libraries are likely to play an important role in identifying novel gpcr ligands 3 . particularly plant cyclotides cover a large chemical space based on their high sequence diversity. together with their range of bioactivities and unique stable structure suggests that cyclotides are of current and future interest for drug discovery and development. acknowledgements: this work is funded by the austrian science fund fwf (p22889). drosha and dicer are two key endonucleases for biogenesis of micrornas (mirnas) that regulate target mrna. drosha converts pri-mirna to ~7 0 nucleotide (nt) pre-mirna in nucleus and dicer converts pre-mirna to linear ~2 2 nt single-stranded mirnas in cytosol. even though dicer is potentially important to control availability of mature trans-acting rnas in cytosol, the enzyme itself does not seem to be the suitable target controlling mirna processing due to the lack of its substrate specificity. nature, however, might be intelligent enough to differentiate a variety of pre-mirna, so that a certain specific pre-mirna is converted to mature mirna in case it needs. therefore, other component(s) in the enzyme complex could be involved in recognition of auxiliary proteins from out sources to give extra specificity. we have synthesized trp-containing amphiphilic peptides against several pre-mirna. peptide 2b showed a picomolar binding affinity and a large specificity against pre-let7a-1. in vitro mirna processing, dicer activity was also selectively enhanced in the presence of this peptide. on treatment with this peptide on hct116 colon cancer and p19ec cell lines, let7a-1 mirna was more processed than reference mirnas. the toxicity of furan is known to rely on its selective oxidation in the liver by cyt p540 enzymes transforming it into the very reactive butenedial, which quickly reacts with proximate nucleophiles. 1 this principle was used in our laboratory to develop a high yielding dna interstrand crosslinking methodology. 2 in view of the demonstrated site-selectivity, the method further holds promise for sitespecific crosslinking dna to its binding proteins, which is highly relevant in the study of transient protein-dna interactions. furthermore irreversible dna binding can be achieved through such a covalent linkage, which is potentially useful for new generation therapeutics. 3 the reactive furan moiety can in principle be incorporated either in the dna or in the protein. in the former case, a furan modified nucleotide was built into an oligonucleotide positioning the furan moiety at the periphery of the dna, to avoid interstrand crosslinking. for the latter approach, we initially chose to synthetically access a furan modified dna binding protein mimic. next to a previously described non-covalent gcn4 mimicking dimer, 4 we have also investigated a new type of steroid-based dipodal dna binders. 5 synthesis of the latter constructs has proven challenging in view of the immobilization of two peptide chains with helix forming tendency at close distance on the template. results, showing the power of microwave assistance will be discussed. in an alternative approach, a full length protein was modified with furan by amber suppression based on the structural similarity between a furan modified amino acid and pyrrolysine. 6 pharmaceutical institute, university of bonn, an der immenburg 4, 53121 bonn, germany human matriptase-2 is a 80 kda protein with trypsin like specificity. this protein exhibits a domain organization similar to family of membrane-bound serine proteinases known as type ii transmembrane serine proteinases. among many ascribed function in human body, this enzyme is a potent negative regulator of hepcidin, the peptide involved in iron homeostasis [1] . matriptase-2 has a similar fold as other tmsp members, however their detailed specificity still remain unclear. the aim of this study was to determine the substrate specificity of this physiological important enzyme using combinatorial chemistry approach. in order to characterize the matriptase-2 specificity, the tetrapeptide library with c-terminal amide of aminocoumarin (acc-nh2) that serve as a fluorophore, was synthesized. its general formula is given below: x4-x3-x2-x1-acc-nh2, where in position x4-x2 the set of proteinogenic amino acid residues are present, whereas in position x1 lys or arg was introduced. deconvolution of such library was performed using iterative approach in solution. the results obtained indicate that matriptase-2 display diverse p4-p2 specificity as compare to matriptase-1. the most efficient hydrolyzed amino acid residue in position p4 appear to be ile, that is followed by arg in p3 and ser in p 2 . the arg in position p1 is 30% faster hydrolyzed then lys. for selected substrates, the kinetic parameters (kcat, k m ) were determined. amyotrophic lateral sclerosis (als) is a chronic progressive disease. it is characterized by degeneration of upper or lower motor neurons, but its pathogenesis is still unknown and no effective treatment currently exists. it is known that antibodies to gangliosides have been found in some als patients, and these antibodies are also well known to be present in the patients affected by a variety of autoimmune diseases including multiple sclerosis. up to now anti-gangliosides antibodies are detected in clinical immunology laboratories using isolated non consistent antigen mixtures. therefore, we are interested in developing reliable and univocally characterized synthetic antigens for efficient antibody detection. csf114(glc) is a family of structure-based designed glycopeptides that we previously developed as multiple sclerosis (ms) synthetic probes. these n-glucosylated peptides are able to detect specific autoantibodies in the sera of an antibody-mediated form of ms. 1 autoantibody recognition was favored because of the exposition of the sugar amino acid on the tip of type 1' β turn structures. aim of this study is the introduction, in the type 1' β turn peptide structure, of the sugar moiety specific for anti-gangliosides antibody recognition by synthesizing specific building blocks. these building blocks are amino acids carrying glycans mimicking the biological activity of complex oligosaccharides. we selected sialic acids (in particular the n-acetylneuraminic acid -neu5ac) 2 because they are involved in a significant number of biological events. neuraminic acid and its derivates are widely distributed in animal tissues and in bacteria, especially in glycoproteins and gangliosides. therefore, we synthesized fmoc-l-asn(neu5ac)-oh and fmoc-l-ser(neu5ac)-oh. these building blocks will be introduced in the type 1' β turn structure for the detection of anti-gangliosides antibodies in als. as a distinct pattern of ms could involve an antibodymediated demyelination, identification of autoantibodies as specific biomarkers is a relevant target. even if interesting data focused on the diagnostic and prognostic role of the detection of antibodies to myelin oligodendrocyte glycoprotein (mog) in adults' serum, its value remains dubious due to many other contrasting results. our research group identified csf114(glc), an nglucosylated peptide, able to detect disease-specific autoantibodies in the sera of a statistically significant number of ms patients. 1, 2 since this synthetic antigen may be considered as a mimic of aberrant post-translational modification (i.e. n-glucosylation) of myelin protein(s) triggering autoimmunity in ms, our goal is to obtain the extracellular domain of mog properly glucosylated thanks to a simplified native chemical ligation approach. 3 for this purpose, the n-glucosylation will be introduced in a synthetic peptide fragment following the building-block approach by spps. the other protein fragment bearing an n-terminal cysteine will be expressed in e. coli after introduction of a selective point mutation into mog. finally, our aim is to test the semi-synthetic protein by sp-elisa to study the ability to detect autoantibodies in ms patients' sera and to find a potential cross-reactivity with csf114(glc). this peptide is an endogenous ligand of the opioid receptorlike 1 (orl1), previously referred to as "orphan" receptor, structurally and functionally related to the classical opioid receptors. also the hexapeptide ac-ryyrwk-nh2 is shown to be a selective ligand for the nop receptor with marked analgesic effect. with a view to developing ligands for the nop receptor with more potent analgesic activity, new series of the ac-rfmwmk-nh2 and ac-ryyrwk-nh 2 , modified at position 4 and 5 respectively with newly synthesized β 2tryptophan analogues were synthesized 1 . the aim of the present study was to examine the effects of naloxone (nal) and jtc-801 (nop receptor antagonist) in the analgesic activity of newly synthesized hexapeptide analogues. all peptides (10 μg/kg), nal (1 mg/kg) and jtc-801 (0,5 mg/kg) were injected intraperitoneally (i.p.) in male wistar rats. antinociceptive effects were evaluated by two nociceptive tests -paw-pressure (pp) and hot-plate (hp) and statistically accessed by anova. the results will be discussed compared to the referent compound in both tests used and mechano-and thermo-receptors are involved. [1] . socs1 and socs3 have many similarities as well as some intriguing differences. both can block signalling by direct inhibition of jak enzymatic activity yet apparently require different anchoring points within the receptor complex. while the primary socs1 interaction is with a critical py residue within the jak catalytic loop [2] it interacts also with py residues in the ifnαr1 and ifn r1 subunits in a jak1-independent manner; the socs3-sh2 domain also interact with y1007 in jak2, albeit with slightly lower affinity, but subsequent studies demonstrated a high affinity interaction with py residues located within receptor subunits [3] . mutagenesis studies identified small regions at the n-termini of the socs1 and socs3-sh2 domains, and at the c-terminus of the socs3-sh2 domain, which were critical for phosphotyrosine binding. in order to gain insights in molecular discriminants for the interaction of both socs1 and socs3 toward jak2 and tyk2 we designed and synthesized peptides encompassing regions involved in proteins recognition. we set up a spr assay to evaluate the affinities of complexes formation. then through an alascanning approach we have designed new peptide sequences containing un-natural amino acids that are able to better recognize wild sequences and whole proteins. cellular experiments on stat1 activation signaling suggest their potential application as modulators of disorders involving socss overexpression. targeting proapoptotic death receptors (drs) to trigger apoptosis in cancer cells is a promising anticancer therapeutic approach. trail (tnf-related apoptosis inducing ligand) is a transmembrane homotrimeric protein belonging to the tnf family that triggers selective tumour cell apoptosis upon binding to its cognate receptors dr4 and dr5. several strategies are being developed to exploit the unique cancer selectivity of the trail-dr pathway in therapy, including the use of recombinant trail targeting dr4 or dr5. [1] recently, a disulfide-bridged macrocyclic 16-mer peptide (derived from phage display) that binds selectively to dr5 has been identified. [2] oligomeric versions of this macrocyclic peptide display increased binding avidity to the receptor and exhibit the capacity to activate the trail apoptotic pathway both in vitro and in vivo. [3] however, disulfide bonds are susceptible to reduction and scrambling in vivo potentially resulting in the loss of the desired biological activity. among alternative linkages with increased redox stabilities, lanthionine thioethers, in which one of the sulfur atoms of the disulfide bond is removed have previously been introduced into biologically active peptides with some success. [4] disulfide bridges can undergo a -elimination in alkaline conditions, followed by a michael addition to give a thioether bridge. optimization of this reaction led to the desulfurized analogue of the dr5-binding peptide. the native dr5-binding peptide and its desulfurized analog have been compared for their structural (nmr conformational analysis) and biological properties (affinity to dr5 and signaling pathways). the apelin/apj complex has been detected in many tissues and is emerging as a promising target for a number of pathophysiological conditions. in the central nervous system, apelin/apj was detected in brain regions involved in spinal and supraspinal control of pain, such as the amygdala, hypothalamus, dorsal raphe nucleus and spinal cord. we propose the hypothesis that apelinergic agonists represent a potential new approach to pain modulation and that the synthesis of stable analogues would lead to compounds with antinociceptive properties. there is currently little information on the structure/activity relationship (sar) of the apelin hormone. in an effort to better delineate sar, we synthesized analogs of apelin-13 modified at selected positions with unnatural amino acids, with a particular emphasis on the c-terminal portion. analogs were then tested in binding and functional assays by evaluating gi/o mediated reduction in camp levels and by assessing β-arrestin2 recruitment to the receptor. the plasma stability of new analogs was also assessed. several were found to possess increased binding and higher stability compared to the parent peptide. there is compelling evidence that the neuropeptide 26rfa and its cognate receptor gpr103, are involved in the control of food intake and bone mineralization. 1 among the gpcrs whose structures have been solved, gpr103 exhibits the highest sequence homology with the beta2adrenergic receptor. the aim of this work was to experimentally characterize predicted ligand-receptor interactions by site-directed mutagenesis of gpr103 and design of point-substituted 26rfa analogs. starting from the x-ray structure of the beta2-adrenergic receptor, a 3d molecular model of gpr103 has been built. the bioactive c-terminal octapeptide 26rfa(19-26), kggfsfrf-nh 2 , 2 was subsequently docked in this gpr103 model and the ligandreceptor complex was submitted to energy minimization. in the most stable complex, the phe-arg-phe-nh2 part was oriented inside the receptor cavity whereas the n-terminal lysine remained outside. a strong intermolecular interaction was predicted between the arg 25 residue of 26rfa and the gln 125 residue located in the third transmembrane helix of gpr103. in order to study this interaction, we have investigated the ability of 26rfa and arg-modified 26rfa analogs to activate the wild-type (wt) and the q125amutant receptors transiently expressed in cho cells. the platelet receptor αiibβ3 plays a critical role in the process of platelet aggregation and thrombus formation. upon platelet activation its conformation changes leading to an increased affinity for fibrinogen. the αiibβ3 activation is regulated by "outside-in" and "inside-out" signaling. among the protein-protein interactions, which contribute to «inside-out» signaling, the most important is that of talin with the β3 cytoplasmic tail. it has been recently suggested that talin-mediated αiibβ3 activation relies on the cooperative interaction of the membrane proximal (mp) and the membrane distal (md) β 3 regions with talin f3 domain and that the -n 744 ply 747 -motif of β3, which can be phosphorylated at y 747 , plays a critical role in this process. 1 to evaluate the interaction of talin with the β3 tail of integrin we designed and synthesized three peptides corresponding to the md and mp parts of β3 in their carboxyfluoresceinlabeled form (md: cf-r 736 akwdtannplyke 749 -nh 2 , cf-n 743 nplykea 750 -nh 2 and mp: cf-k 716 llitihdrke 726 -nh2). emission and anisotropy fluorescence spectroscopy was used to quantitatively assess the affinity of these peptides for talin. furthermore, to challenge the role of the y 747 phosphorylation in talin-α iib β 3 interaction we also studied the binding of talin to the modified analogues of md, cf-r 736 akwdtannpl(ptyr)ke 749 -nh 2 and cf-n 743 npl(ptyr)kea 750 -nh 2 . our experiments revealed that the md and mp parts of β3 bind tightly to talin and that y 747 phosphorylation has an inhibitory effect on this binding. functionalized oligoprolines as multivalent scaffolds in tumor targeting p. wilhelm, h. wennemers* eth zurich, zurich, switzerland oligoprolines are known to be structurally well-defined molecular scaffolds. in aqueous media, even short chain lengths of six proline residues adopt a polyproline ii helix (ppii). this secondary structure is a highly symmetric helix where every third residue is on top of each other in a distance of about 9.5 å. [1] the incorporation of azidoproline (azp) allows facile and versatile functionalization either via copper-catalysed azide-alkyne cycloaddition (cuaac) or an acylation that followed a staudinger reduction. [2] based on the structural integrity of the oligoproline scaffold, targeting vectors can be conjugated via coppercatalysed azide-alkyne cycloaddition in defined distances. recent studies on radiolabeled oligoproline-bombesin conjugates, to target the gastrin-releasing peptide receptor (grp-r), showed in vitro and in vivo superior internalization in prostate cancer cells compared to the established monovalent ligands. [3] a facile route to synthesize alkynylated ligands has been developed successfully. we are currently expanding this concept to the integrinligand c(rgdyk) as well as to [tyr 3 ]-octreotide, which binds to somatostatin-receptors. the monovalent analogue of the latter, dota-[tyr 3 ]-octreotide (dotatoc), is well established for diagnosis [4] and therapy [5] of somatostatinpositive tumors such as neuroendocrine tumors. the cu i -catalyzed azide-alkyne addition 1 (cuaaa), the useful variant of "click chemistry," has emerged as a powerful technique for specific addition. that chemistry is also commonly used for conjugation, and cyclization of peptides. it is known that cyclization can increase the metabolic stability of peptides, as well as enhance potency or selectivity. another useful application of the cuaaa, which we are reporting, is the n-terminal crosslink of two synergic peptides to gain their potency. cuaaa reaction is performed on solid phase (merrifield resin) where one of the peptide components with azido group on the linker (6azido-hexanoic acid) is "clicked" with second peptide component in solution, made by fmoc strategy in partially protected form containing at n-terminal side alkyne group (fmoc-l-propargylglicine). cuaaa coupling is performed in dmf/t-buoh/h2o with presence of cui and sodium ascorbate when reacting mixture was degassed. linked peptides are cleaved finally from resin and purified. as an application example we picked two endothelin active peptide analogues: bq123 derivative 2 (a highly potent and selective eta antagonist) and irl-1620 derivative 3 angiogenesis is a key step in the transition of tumors from a dormant state to a malignant state. the vascular endothelial growth factor (vegf) is a major contributor to tumor angiogenesis. its pro-angiogenic activity is mainly mediated through binding to two tyrosine kinase receptors located predominantly on the surface of endothelial cells: vegfr-1 and vegfr-2. vegf binding to these receptors triggers the activation of different signal transduction pathways responsible for the proliferation, survival and migration of endothelial cells 1 . vegf/vegfr system constitutes a target to stop tumour growth. an attractive approach is the development of peptides, or small-molecules, with a high affinity for the extracellular domain of the receptors to prevent vegf binding. based on the x-ray structure of vegf and the d2 domain of vegfr-1 2 , cyclic peptides had been developed in our group 3 . such peptides, mimicking simultaneously the 3-4 loop and helix ·1 of vegf, can bind to d2 domain of vegfr-1 and inhibit receptors phosphorylation and thus map kinase pathway 4 . we describe here our strategies to optimize peptidic antagonists of vegfr-1. chemical modifications are made in order to better mimic peptide conformations and to increase their receptor binding affinities. we introduce a hydrophobic functional group at the c-terminal of the original cyclic peptide 4 , some of such modified peptides reveal improved vegfr-1 binding affinity. otherwise, as the helix ·1 presents most of the important residues in vegfr1 binding according to alanine-scan in the literature5, we try to stabilize the helical conformation by insertion of aib residues or by peptide cyclisation. the peptides affinities are evaluated by an elisa test developed previously 3 . institute of chemistry and biochemistry, freie universität berlin, thielallee 63, d-14 195 berlin, germany new polypeptide was isolated from the azemiops feae viper venom by combination of gel filtration and reversephase hplc and called azemiopsin. its amino-acid sequence (dnwwpkpphqgprpprprpkp) was determined by means of edman degradation and mass spectrometry. it consists of 21 residues and does not contain cysteine residues. according to circular dichroism measurements, this peptide adopts a β-structure. peptide synthesis was used to verify the accuracy of the determined sequence and to prepare sufficient peptide amount for biological activity studies. azemiopsin efficiently competed with α-bungarotoxin for binding to torpedo nicotinic acetylcholine receptor (nachr) (ic50 0.18±0.03 m) and with lower efficiency to human α7 nachr (ic50 22±2 μm). ala scanning showed that amino-acid residues at positions 3-6, 8-11 and 13-14 are essential for binding to torpedo nachr. in biological activity azemiopsin resembles waglerin, a specific blocker of muscle-type nachr from tropidechis wagleri venom. however the sequences of these peptides are markedly different, and azemiopsin is the first natural toxin to block nachrs that does not possess disulfide bridges. laboratory of peptide science, nagahama institute of bio-science and technology, nagahama, shiga 526-0829, japan while neutrophils infiltrate into damaged sites immediately after tissue injury, endogenous factors which induce their acute transmigration and activation have not been thoroughly elucidated. for the candidates, we recently identified two novel neutrophil-activating cryptides, mitocryptide-1 (mct-1) and -2 (mct-2), which were hidden in mitochondrial cytochrome c oxidase and cytochrome b, respectively [1] [2] [3] . in addition, the presence of many neutrophil-activating peptides other than mct-1 and -2 was observed during their purification. these findings suggest that neutrophils are regulated by many unidentified peptides. here, we purified a novel neutrophil-activating octadecapeptide whose primary structure was identical to mitochondrial cytochrome c (68-85) from porcine hearts. we named this functional peptide as mitocryptide-cyc (mct-cyc). the structure-activity relationships of cytochrome c on β-hexosaminidase release from neutrophilic differentiated hl-60 cells demonstrated that cytochrome c (70-85) was the most potent cryptide among cytochrome c-derived peptides. since cytochrome c is known to be involved in the apoptotic process, our present results suggest that cryptides produced from cytochrome c play an important role in scavenging toxic debris from apoptotic cells by neutrophils. anthracis spores are very resistant and can remain dormant in soil for decades. therefore, an effective detection system for b. anthracis is urgently needed. recently, it was found that one of the components of the b. anthracis exosporuim is a collagen like protein whose carbohydrate portion is composed of the tetrasaccharide with the highly specific monosaccharide upstream terminal, named anthrose. 1 since anthrose was not found on other bacterial spores, including those closely related to b. anthracis, this monosaccharide is an attractive target for the development of new b. anthracis detection and identification methods. peptide cyclization represents particularly interesting approach for the design of artificial receptors for anthrose, because cyclic peptides provide the possibility of having a spherical lipophilic binding site of appropriate size and shape for a particular carbohydrate substrate. 2 the presence of hydrogen donor/acceptor groups within a three-dimensional structure permits carbohydrate substrates to be encapsulated, thereby allowing their binding in water. in order to determine whether the cyclic peptide receptor can selectively detect the anthrose, we have successfully prepared cyclic peptide combinatorial library (total 6859 peptides) by the process of divide, couple and recombine ("tea-bag" technology) using standard fmoc solid-phase peptide synthesis. 3 prepared combinatorial library is screened for anthrose binding in fluorescence-based assay, and individual cyclic peptides with enhanced affinity toward anthrose are identified by the positional scanning deconvolution process. 3 cyclization of linear sequences is a well-known approach used to restrict the flexibility of peptides. cyclization often increases selectivity of peptides towards one specific receptor type, increases metabolic stability and generally increases lipophilicity, which often improves the bloodbrain barrier permeability of peptides. in our previous study [1] we have reported on the synthesis of a cyclic endomorphin-2 (em-2) analog, tyr-c(d-lys-phe-phe-asp)-nh2, which elicited analgesia after peripheral administration. encouraged by the fact that this analog was able to cross the blood-brain barrier we designed and aliskiren is the first orally active, direct renin inhibitor to be approved for the treatment of hypertension. its structure and conformational analysis were explored using molecular dynamics (md) simulations. for the first time, md calculations have also been performed for aliskiren at the receptor site, in order to reveal its molecular basis of action. it is suggested that aliskiren binds in an extended conformation and is involved in several stabilizing hydrogen bonding interactions with binding cavity (asp32/255, gly34) and other binding-cavity (arg74, ser76, tyr14) residues. of paramount importance is the finding of a loop consisting of residues around ser76 that determines the entrapping of aliskiren into the active site of renin. the details of this mechanism will be the subject of a subsequent study. additionally molecular mechanics poisson-boltzmann surface area (mm-pbsa) free energy calculations for the aliskiren-renin complex provided insight into the binding mode of aliskiren by identifying van der waals and nonpolar contribution to solvation as the main components of favorable binding interactions. adamantyltripeptides and phospholipids in liposomal bilayers 1 . now, we were primarily interested to study incorporation profile of mannosylated adamantyltripeptides. we have demonstrated that the adamantyl moiety, due to its liphophilic properties, penetrates into the lipid core of the bilayer while the hydrophilic part with the mannosyl moiety is exposed on the liposome surface. after concanavalin a (con a), a lectin, which specifically binds α-d-mannosyl residues, was added to the liposome preparation, increase in liposome size and appearance of aggregates has been observed. the enlargement of liposomes was ascribed to the specific binding of the con a to the mannose present on the surface of the prepared vesicles. the afm analysis revealed that the adamantyltripeptide molecules grouped into small domains that raise above the bilayer surface. the molecule size and molecular geometry, as well as the hydrophilic and hydrophobic surfaces in the structure of mannosylated adamantyltripeptides, are responsible for arrangement of molecules in the lipid bilayer. this approach might be a useful model for investigation of specific protein interactions with membrane receptors. also, the adamantyl moiety may be considered as a potential membrane anchor for different carbohydrate or other molecules of interest, which could be bound on it and thus exposed on liposome surfaces and as such used in targeted drug delivery. the assay is carried out in a 96 well format p122 and images are captured throughout the course of the assay, thus we can not only determine a ligand's propensity to induce internalization, but also its efficacy and internalization rate. addition of test compound, followed by the standard agonist at a later interval, enables differentiation between agonist or antagonist activities. in the positional scanning format [1] , while the possibility of agonists and antagonists working against each other within a mixture exists, the effects are minimized in screening the whole library as there are as many arrangements of the sub-libraries as there are defined positions. therefore while an agonist and antagonist might be present in a particular mixture in one sub-library they will be in different mixtures in all other sub-libraries. we have used this assay format to simultaneously screen for novel agonists and antagonists against the orexin 2 receptor. assay development and library screening will be presented. [1] . since the pro residue in position 2 of em2 is very important in the proper conformational alignment of the two aromatic residues tyr 1 and phe 3 in em2 molecule at the receptor site, it is possible that structural modification around the pro 2 residue yields compounds with unique biological properties and improved metabolic stability. in the present study, we synthesized seven em2 analogues containing isopro or constrained residues with oxopyrrolidine or oxopiperadine ring, instead of pro residue in position 2. all peptide analogues were synthesized solid phase method. incorporation of oxopyrrolidine and oxopiperadine rings were carried out on a solid support by the methods of gellerman, et al. [2] and mohamed, et al. [3] , respectively. opioid receptor binding activity for μ and δ-receptors using the development of resistance to mainstay cancer therapies has become a major limitation for the treatment of many cancers. there is an urgent need to develop new antineoplasic agents with innovative anticancer approaches. to overcome resistance to cancer therapies, our attention has turned to proteins that regulate multiple signalling pathways essential for tumour survival. among the few known nodal proteins upregulated in cancer cells and involved in many hallmarks of cancer, we are interested in survivin. an essential regulator of cell proliferation and apoptosis, survivin is sharply overexpressed in cancer cells and plays a major role in resistance. 1 being a small protein, its bioactivity is relies mainly on protein-protein interactions (ppi) with different partners. a critical point for its multiple functions in cancer is its association with hsp90, which is required for its stability. a nonapeptide from survivin called shepherdin has been shown to modulate the interaction of survivin with hsp90 by binding to hsp90 and to induce death of tumour cells. 2 unfortunately, shepherdin is not cell permeable, has low proteolytic stability and shows poor bioavailability, limiting its use as anticancer therapeutic agent. to improve pharmacological properties of shepherdin, cyclic and peptidomimetic analogs of shepherdin have been synthesized followed by structure-activity relationship studies. in hsp90 binding studies, some cyclic hexa-and heptapeptidic analogs showed increased affinity compared to shepherdin. the synthesis of cyclic and peptidomimetic analogs and the results from the binding assays and the conformational analyses will be presented. the hexapeptides with formula ac-ryyr/kw/ir/k-nh2 have been identified as shortest peptide sequence with high nop receptor affinity, selectivity and marked analgesic effect. it was found that the following peptides act as partial or full agonists or antagonists of nop receptor in different in vivo and in vitro systems. these hexapeptides were used as chemical templates in sar studies 1,2 . the aim of the present study was the synthesis and the biological screening of new analogs of ac-rfmwmk-nh2 and ac-ryyrwk-nh 2 , modified at position 4 and 5 respectively with newly synthesized β 2 -tryptophan analogues 3 . these non natural amino acids were prepared using reaction of asymmetric friedel-crafts alkylation of various indoles with a chiral nitroacrylate to provide optically active β-tryptophan derivatives. the four newly synthesized ligands for the nociceptin/orphanin fq (n/ofq) receptor (nop) have been prepared by solidphase peptide synthesis-fmoc-strategy. these compounds will be tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of wistar rats. bacterial infections are a common problem associated with dermal wounds. these infections can prolong or impair wound healing. hydrogel materials that display inherent activity against bacteria can be used to directly treat accessible wounds to prevent or kill existing infection. in this work, we describe the design and utilization of injectable gels prepared from self-assembling β-hairpin peptides having a high content of arginine. these gels were found to be extremely effective at killing both gram-positive and gramnegative bacteria, including multi-drug resistant p. aeruginosa. importantly, no added antibacterial agents are necessary since the nanostructure of the gel, itself, is the active agent. using self-assembling peptides for material construction allows facile structure-activity relationships to be determined since changes in peptide sequence at the monomer level are directly transposed to the bulk material's antibacterial properties. structure-activity relationships studies show that arginine content largely influences the hydrogel's antibacterial activity, and influences their bulk rheological properties. these studies culminated in an optimized gel, composed of the peptide pep6r. pep6r gels prepared at 1.5 wt % or higher concentration, demonstrate high potency against bacteria, but are cytocompatible towards mammalian mesenchymal stem cells. the general mechanism by which pep6r exerts its action was explored and it is suggested that involves membrane disruption that occurs when cells come in contact with the gel's surface. atomic force microscopy (afm) was used to study the effect of the gel on the cell envelope morphology of e. coli. rheological studies indicate that the gel is moderately stiff and displays shear-thin recovery behavior, allowing its delivery via simple syringe. 1 they are intimately involved in the molecular process leading to the delicate nano-patterned silica shells of diatoms. deciphering the mechanisms of silica-biogenesis in diatoms will inspire the development of novel routes for the biomimetic synthesis of silicon-based materials under mild conditions and expand the scope of biotechnological applications, e.g. for immobilization of enzymes in silica matrices. we synthesized silaffin peptides derived from c. fusiformis that carry posttranslational modifications such as phosphorylation or polyamines linked to lysine side chains. a distinct alteration of silica precipitation activity depending on the particular modifications of the silaffins emerged. 2 these modified silaffin peptides were covalently linked to recombinant proteins by expressed protein ligation leading to stable protein-silaffin conjugates. 3 using egfp as model protein, we could show that egfp-silaffin conjugates can induce biomineralization of silica and ensure an efficient and homogeneous immobilization of egfp into silica particles, superior to simple co-biomineralization approaches. moreover, a significant stabilization of immobilized egfp against denaturing agents was observed. we established a method for controlled immobilization of biomolecules based on covalent attachment of silaffin peptides with well-defined silica precipitation properties. currently this method is applied to the immobilization of biotechnological relevant enzymes in order to test their activity and the stabilization effect. herein, we present the covalent functionalization of multiwalled cnts (mwcnts) with organocatalysts based on proline or proline derivatives carrying either a dipeptide or a sulfonamide moiety. two different approaches were followed, namely, covalent grafting of the organocatalysts either at the tips or at the sidewalls of the cnts. for the former approach, mwcnts were oxidized in order to introduce carboxylic units at their tips and make them easily dispersed in aqueous solutions. then, oxidized mwcnts readily reacted with proline-based derivatives carrying a free amino unit yielding the corresponding hybrid materials. for the latter approach, the functionalization methodology based on in-situ generated aryl diazonium salts was followed. in this context, mwcnts were modified with aryl units carrying free amino terminal groups, which were subsequently conjugated with proline-based derivatives carrying a free carboxylic unit. all newly formed hybrid materials were fully characterized with complementary spectroscopic (atr-ir, raman), thermal (tga) and microscopy (tem) techniques. the catalytic evaluation of the activity of the cnt-based organocatalysts in aldol reactions is in progress. financial support from gsrt/εσπα 2007-2013 συνεργασια through 09συν-42-691-νανοκαταλυση project is acknowledged. novel organogels based on self assembly of rationally designed pseudopeptides c. pappas, n. sayyad, a.g. tzakos, i. plakatouras section of organic chemistry and biochemistry, department of chemistry, university of ioannina, ioannina, gr-45110, greece self-assembly is becoming a rather intriguing way to build an array of nano-and micro-structured materials. low molecular weight organogelators can self-assemble into various architectural types in organic solvents through weak intermolecular interactions. such organogelators have potential applications in the generation of novel materials for nanobiotechnology1. herein, we report the synthesis of rationally designed pseudopeptides and the conditions to form organogels. the obtained gels are responsive to temperature, and the sol-gel process is thermoreversible. the architecture of the constructed organogels was characterized via tem and spectroscopic techniques. diffusion ordered nmr spectroscopy (dosy) was further utilized to determine differences in the molecular shape of the different pseudopeptides. applications of the resulted compounds in nanotechnology will be reported. since 2000, organocatalysis has met such a great rate of expansion that is nowadays considered the third major branch of modern asymmetric catalysis along with the transition metal catalysis and biocatalysis. after the seminal work of list, lerner and barbas on the enantioselective aldol reaction between acetone and 4-nitrobenzaldehyde catalyzed by proline, it became clear that amino acids and peptides could serve as an abundant pool full of potential to develop novel organocatalytic motives. following our recent report that the combination of a prolinamide with a thiourea group having as a spacer a chiral diphenylethylenediamine leads to an efficient organocatalyst for the aldol reaction, 1 we recently considered the possibility to couple the prolinamide unit with an urea moiety. one of our main interests was the substitution of the diphenylethylenediamine spacer by a gem diamine derived from an α-amino acid. the gem diamine is easily synthesized via a curtius rearrengement of the corresponding acyl azide. after synthesis and evaluation of a number of potential catalysts, the prolinamide derivative bearing a gem diamine derived from (s)-phenylalanine and an aryl urea moiety proved to provide the best results in the reaction between cyclic ketones and aldehydes. utilizing 10 mol% of our organocatalyst, the aldol products were obtained in high to quantitative yields (up to 98%), high to excellent diastereoselectivities(up to >98:2) and high to excellent enantioselectivities (up to 99% ee). peptide self-assembled monolayers are of current interest to study physicochemical properties of modified metal (e.g. au) surfaces. rigid peptide scaffolds could enhance the interaction between gold surfaces and labels by reducing and precisely monitoring the distance between the supported monolayers and gold. the c α -tetrasubstituted αamino acid 4-amino-1,2-dithiolane-4-carboxylic acid (adt) 1 , which contains a cyclic disulfide system, is interesting in this respect because it may allow the parallel binding of the peptide helical chain to the metal surface. adt occurs in nature 2 and has been utilized in medicinal chemistry 3 and in a model compound of [fefe] hydrogenase. 4 we synthesised a series of constrained helical peptides, based on the ala-ala or the ala-aib sequence, containing one or two adt residues. these peptides were functionalised with spectroscopic or opto-electronic labels. among the large number of reactions involving the formation of carbon-carbon bond, the addition of ketones to nitroolefins is a powerful tool for the synthesis of γ-nitrocarbonyl compounds, useful intermediates for pharmaceutical industry. our recently reported primary amine-thioureas based on tert-butyl esters of natural amino acids exhibit excellent performance for the michael reaction of ketones with nitroolefins providing the products quantitatively and almost stereospecifically (>99% ee). 1, 2 using this methodology, enantiopure baclofen and phenibut (analogs of gaba) have been synthesized. 2 polymersupported organocatalysts constitute a great challenge for the michael reaction. in the current study, we report the immobilization of amine-thiourea catalysts containing (1s,2s)or (1r,2r)-diphenylethylenediamine and tert-butyl aspartate, on various polymer supports, either directly or through spacer units. the solid-supported catalysts evaluated in the reaction between acetone and βnitrostyrene and highlighted the importance of the choice of the polymer as well as the presence of the spacer or not. the direct attachment of the primary amine-thioureaaspartate to a crosslinked polystyrene-divinyl benzene resin containing a uniform distribution of aminomethyl groups provides a supported catalyst that affords the product of the reaction between acetone and β-nitrostyrene quantitatively and in high enantioselectivity (91% ee a. theodorou, g.n. papadopoulos, c.g. kokotos* laboratory of organic chemistry, department of chemistry, university of athens, athens, greece after the pioneering report that proline can catalyze efficiently the intermolecular aldol reaction between acetone and a variety of aromatic aldehydes, 1 it became evident that amino acids and peptides 2 can afford a plethora of different structural scaffolds for novel catalysts. along the first decade of its life, organocatalysis has grown to such an extend that now it is considered the third major branch of asymmetric catalysis. recently, researchers have paid special attention to other amino acids rather than proline. some primary amino acids have already been applied to a number of transformations with success. 3 usually improved catalytic properties are observed when derivatives of primary amino acids are utilised. we have undertaken a study on the application of simple and cheap primary amino acids and amino acid derivatives, either commercially available or easily obtained, as organocatalysts for the asymmetric α-amination of aldehydes. in the present work, we report that the use of simple derivatives of primary amino acids like phenylalanine and aspartic acid can efficiently catalyze this transformation leading to products in high to quantitative yields and enantioselectivities up to 94% ee. the majority of the organocatalysts developed up to now for asymmetric organic transformations employ more than one functionalities in the catalytic mechanism that act through either covalent or non-covalent interactions. for example, proline employs the pyrrolidine nitrogen and the carboxylic acid group, while chiral thioureas combine the thiourea functionality with a tertiary or a primary amino group. we have recently shown that an amide of proline with a diamine carrying a thiourea group is a very good catalyst for the enantioselective aldol reaction. 1 trying to improve the activity, we have found that a tripeptide-like thiourea having as building blocks (s)-proline, (1s,2s)diphenylethylenediamine and (s)-di-tert-butyl aspartate provides the products of the reaction between ketones and aromatic aldehydes in high to quantitative yields and high stereoselectivities (up to 99:1 dr and 99% ee). a number of structural modifications of the catalyst were undertaken in order to understand the role of the hydrogen bond donors of the catalyst, i.e. the prolinamide hydrogen and the two hydrogen atoms of the thiourea group. we have come to the conclusion that the importance of the hydrogen bond donors of the catalyst follows the order: thiourea hydrogen originated from aspartate › amide hydrogen › thiourea hydrogen originated from diphenylethylenediamine. g eldrug s.a., patras 26504, greece a convenient and facile synthesis and in vitro biological evaluation of n-substituted 5-butylimidazole derivatives as potent angiotensin ii (ang ii) receptor type 1 (at1) antagonists have been reported in the present study. a series of imidazole based compounds bearing the biphenyl moiety at the n-1 position, a halogen atom at the c-4 and polar substituents such as hydroxymethyl at the c-2 position were synthesized. 1,2 these compounds were evaluated for binding to human at1 receptor and for ang ii antagonism in vitro on isolated rat uterus. in particular, 5butyl-1-[[2΄-(2h-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole derivatives complexed with the at1 receptor and showed high binding affinity. these analogues were also found to be active in the rat uterotonic test. importantly, their binding affinities and potencies were comparable to those of losartan. these results indicate that the hydroxymethyl at the c-2 position of the imidazole ring is favorable for high affinity binding and antihypertensive activity and in line with the activities of the losartan counterparts. experimental findings are in good agreement with docking studies, which were undertaken in order to investigate ligand/at1 receptor interactions. z-leu-glu-his-asp-aluc, suc-leu-leu-val-tyr-aluc) are good substrates for bioluminescence assays, for example in the detection of caspase activity during apoptosis 1 . these substrates generally offer significant advantages, such as increased sensitivity, ease of use, and high throughput screening capacity. luciferase-based assays are typically 10-to 100-fold more sensitive than the comparable fluorescent assays (rhodamine 110, 7-amino-4-methylcoumarin (amc) and 7-amino-4trifluoromethylcoumarin (afc)). the synthesis of different type peptide-amino-luciferin conjugates and their precursors have been published 2 and some of them are commercially available. however, because of their high price the in vivo application of these conjugates is limited. to solve this problem we successfully worked out a new, easier and more convenient and economical method for the preparing these derivatives starting from 2-chloro-benzothiazole. moreover this products have excellent purity (>99%) and adequate yield (82-93%). major health problems arising from bacterial resistance towards existing antibiotics make discovery of antibacterial drugs with new mechanisms of action pertinent. although proof of concept for a novel antimicrobial approach using peptide nucleic acid (pna) antisense targeting of essential bacterial genes was obtained a decade ago, this technology is still limited by the lack of carriers that facilitate effective bacterial delivery and confer optimal pharmacokinetic properties to the prospective drugs. [1, 2] in the past two decades, parallel efforts of exploiting naturally occurring antimicrobial peptides (amps) as drugs have been made. the cationic amp subclass appears to be directly involved in the innate immune response towards microbial infections. [3] so far only few cell-penetrating peptides, with activity on mammalian cells, and other membrane-active peptides, have been investigated as potential vehicles for bacterial delivery. for instance, cationic amps with an internal target appear not to have been investigated for bacterial delivery of antibiotics. the aim of this project is to develop highly potent genetic antibiotics by exploiting naturally occurring antimicrobial peptides as potential delivery vehicles for antisense peptide nucleic acid oligomers. the amps are chosen from amps reported to act via intracellular targets, and thus must possess an inherent ability to permeate bacterial cell membranes without direct killing of the bacteria. faculty of chemistry university of gdansk, gdansk, poland azt (3'-azido-2'3'-dideoksythymidine), a modified nucleoside used in antiretroviral therapy and peptide plant hormone -systemin were used as substrates of 1,3-dipolar cycloaddition (click chemistry). systemin is 18-aa peptide defense hormone released in response to plant (tomato, tobacco) damage or pathogen attack. we examinated whether systemin's fast movement through plant tissues could be used for cargo (azt) transport. the huisgen cycloaddition also known as 1,3-dipolar cycloaddition is a chemical reaction belonging to the larger class of cycloadditions. reaction between organic azide and alkyne appended substrates allows the synthesis of the desired conjugate in high purity and yields irrespective of the sequences and functional groups on either of the two substrates [1, 2] . conjugate of azt-systemin has been synthesized by click chemistry, using systemin modified at n-terminus with propiolic group and azt. the conjugation was catalyzed by cu(i). the reaction was fast, efficient and regioselective. its progress was easily monitored by capillary electrophoresis (ce). ce was also applied for characterization of systemin and azt-systemin stability and movement throughout tomato leaf and stem. despite the fact that systemin moves rapidly through tomato tissues, our calorimetric (itc) studies showed that the peptide does not interact with liposomes-cell membrane model. universitätsklinik für nuklearmedizin, inselspital, bern, switzerland regulatory peptides (e.g., somatostatin, bombesin) have been shown to be suitable vectors for the specific delivery of radioactivity to tumors for diagnostic and therapeutic applications in nuclear oncology. 1 a potential drawback of such vectors is their inherent instability in vivo. thus, new strategies are needed for the stabilisation of radiopeptides in order to improve their bioavailability and, consequently, increase their accumulation in the targeted tissue. it has been suggested that 1,2,3-triazoles, readily obtained by cuaac, are suitable amide bond surrogates which are resistant to proteases. 2 in the present study, we report the synthesis and pharmacological evaluation of radiolabelled, triazole-containing analogues of the gastrin releasing peptide receptor (grpr) targeting peptide bombesin (bbn). to study the effect of backbone modifications in the minimal grp-binding sequence, we synthesized a series of analogues of [nle 14 ]bbn (7) (8) (9) (10) (11) (12) (13) (14) , in which each amide bond is individually replaced by a 1,4-disubstituted 1,2,3triazole. after radiolabelling of the peptidomimetics, their binding affinity and internalization kinetics were determined using pc-3 cells. metabolic stability was evaluated in blood serum. a number of the novel tumor-targeting peptide analogues presented exhibit both a retained high affinity (nm) towards the grpr and an improved serum stability. first preclinical data on the in vivo evaluation of the most promising candidate will be presented. to the best of our knowledge, this is the first report of the systematic replacement of amide bonds with 1,2,3triazoles within the binding sequence of linear, high affinity peptides. the methodology can be applied to a variety of peptide vectors and thus, holds great potential for the development of novel, stabilized peptide-based radiopharmaceuticals. dna is the molecular target for many of the drugs that are used in cancer therapeutics, and is viewed as a nonspecific target of cytotoxic agents. although this is true for chemotherapeutics, other agents that were discovered more recently have shown enhanced specificity. 1 the development of new site-specific dna binders, which are associated with the recognition of the dna major groove, are based on the design of transcription factor mimics that bind the dna as a dimer 2 , and prevent specific genes from being transcribed. these could ultimately result in interesting biomedical applications as designed genome interfering agents or diagnostics. 3 in order to approach this biological constructs, we choose the bzip leucine zipper transcription factor as a model to mimic. as the entire structure cannot be synthesized without expensive, complicated and time-consuming biotechnological methods, the substitution of the dimerization domain by a less complex scaffold is the first step in the design. thus, we consider a steroid based scaffold as a candidate. the specific choice of the steroid scaffold as substituent is inspired by its known ability to enhance proteolytic stability of attached peptides, by its conformational properties ensuring correct positioning of the two appended chains and by its potential to increase bioavailability. this transcription factor binds specific dna sequences by dimerization and inserting short α-helices into the dna major groove. in order to attach the peptides to the scaffold, different strategies were studied. firstly, applying the well-known click chemistry, functionalizing the scaffold with an alkyne moiety, the peptide with an azide and viceversa. secondly, via the unknown resin to resin transfer reaction (rrtr), which has not been applied on peptide chemistry so far. this unprecedent methodology consists on the reaction of a peptide, which is attached on a safety-catch resin, with a second resin bearing a nucleophilic amino terminus resulting in amide bond formation. during the process, the peptide on solid support undergoes cleavage. an hexapeptide was synthesized on a preloaded safetycatch resin. deoxycholic acid derived scaffold with orthogonally protected amines was attached to tentagel resin that acts as acceptor resin. rrtr experiments were performed at both c12 and c3 positions of the deoxycholic acid derivative. in addition, this convergent strategy can be applied to other different peptide conjugated systems. we recently described a new kind of cyclized peptide in which the cyclization is performed between the side-chains of two diaminoacyl residues via a diversely substituted guanidine bridge. 1 we showed that the degree of bridge substitution could impact on the orientation of the bridge inside the cycle and therefore the peptide conformation. we prepared two series (22 and 15 atoms cycle size) of cyclic enkephalin analogues to assess the potential effect of this kind of bridge on the biological activity. the compounds were synthesized on the solid support via the formation of a thiourea bridge and with the variable substituent being introduced at the last step before cleavage. it is noteworthy that the synthesis afforded at least two stable and separable conformers for each analogue of the shortest cycle series. generally, one major and one minor species were recovered. but in the case of di-substituted compounds with a cyclic moiety (pyrrolidine or piperidine substituents), three significant species were obtained. analogues were submitted to various biological assays (binding to μ and δ opioid receptors and functional assays). we observed a significant variation in affinity and selectivity for the receptors as a function of the degree of bridge substitution. a structural analysis by 2d nmr has been undertaken and correlated the variation in activity with a variation in conformation. the origin of the multiple conformers observed for the analogue with a pyrrolidine susbtituent was also investigated. this kind of cyclization could represent a useful tool to easily modulate the conformation and biological activity of a unique peptide sequence. the t-cell response is triggered by the formation of the trimolecular complex between the major histocompatibility complex (mhc), the immunodominant myelin protein epitopes and the t cell receptor (tcr). herein, we report the design and synthesis of non-peptide analogues with the ability to mimic the immunodominant epitope 83-99 of mbp 1,2 . the mimetics were designed to block the formation of the trimolecular complex and therefore the t-cell activation 3, 4 . more specifically, indole analogues were synthesized with substitution at positions 2 and 4 or 6. these molecules contain a carboxyl or an ethyl ester group in position 2 and a benzylamino or phenylamino group in position 4 or 6. the synthesis of the indole ring was achieved by fischer reaction followed by catalytic hydrogenation, reductive amination or arylation and ester hydrolysis. the synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and 1h-nmr. laboratory of peptide science, nagahama institute of bio-science and technology, nagahama, japan amyloid β peptide (aβ), the main component of senile plaques in the brain of alzheimer's disease (ad) patients, is formed by proteolysis of amyloid precursor protein (app). as β-secretase (bace1: β-site app cleaving enzyme 1) triggers aβ formation by cleavage at the aβ domain nterminus, it is a molecular target for ad therapeutic intervention. 1 previously, we reported potent pentapeptidic 2 and non-peptidic 3 bace1 inhibitors containing a substrate transition-state mimic. although these inhibitors exhibited potent inhibitory activities, their molecular-sizes appeared a little too big (mw>600) for developing practical drugs. in this study, we designed a series of small molecular peptides, with bace1 inhibitory activity, lacking the p1-p1' region on the basis of the conformational structure bound in bace1. 4 design and synthesis of new 3'-peptidyl-trna analogues, in particular "hydrolysable" analogues, which represent covalent conjugates of peptide-nucleic acid (pna) with "stop-peptides," were carried out. such compounds are of interest as tools to study the ribosome functioning and as inhibitors of protein biosynthesis. (2 aminoethyl)glycine pna models 3'-end trna sequence cca in designed structures. computer simulations showed the formation of watson-crick pairing of the pna cytosine residues with 23s rrna nucleotides g2251 and g2252 involved in interactions with peptidyl-trna during its specific binding in p site of the ribosomal peptidyl transferase center (ptc). short "stop-peptides" were planned for conjugation with pna. these peptides form stable complexes with the ribosomal tunnel (rt) that leads to ribosome stalling and translational arrest. structures of "hydrolysable" 3'peptidyl-trna analogues that could form peptide bond with amino acid residue of aminoacyl-trna in a-site of ptc included 2'-deoxyriboadenosine instead of the pna adenine containing residue. such conjugates would permit to identify the chemical nature of specific sites localized in rt and responsible for interactions with amino acid residues of the nascent polypeptide chain. pna and "stop-peptide" as well as pna-"stop-peptide" conjugates were prepared by solid phase synthesis on sasrin polymer using fmoc/bhoc(boc) strategy. synthesis of "hydrolysable" conjugates included modification of the 3'hydroxyl of 5'-protected 2'-deoxyadenosine by n-blocked "stop-peptide", deprotection of the 5'-hydroxyl, its conjugation with n-protected pna and removal of protecting groups from the resulted conjugate. the binding of the new 3'-peptidyl-trna analogues with ribosome will be tested by chemical probing and in the cell free translation system. this study was supported by the russian foundation for basic researches (grant 10-04-01187-a). a close structural similarity of endomorphin-2 and another atypical opioid peptide, morphiceptin, which both have a phe residue in the third position, encouraged us to study antinociceptive activity of these two peptides and their analogues. in order to improve the affinity and chemical stability of these opioid peptides, we have designed, synthesized, and analyzed 9 novel analogues. the first modification included endomorphin-2 and morphiceptin analogues, where halogenated phenylalanines in position 3 or 4 were incorporated as surrogates of the native phenylalanine. another important modifying element is non-protein amino acid canavanine (cav) and its analogue (sarg). it is well documented that cav and sarg exhibit strong analgesic activity. two new morphiceptin analogues were synthesized by introducing cav and sarg in position 3. we further characterized their antinociceptive activities by the paw pressure (pp) test. the experiments were carried out on male wistar rats (180-200 g), treated with i.p. doses of 1 mg/kg. e eldrug s.a., patras 26504, greece the renin angiotensin system (ras) has been a prime target for the therapy of cardiovascular diseases. angiotensin ii type 1 (at1) receptor mediates vast majority of biologically detrimental actions. non-peptide at1 receptor blockers are presently the most specific means to block the ras enzymatic cascade. the dupont group was the first to develop losartan (dup 753), an orally effective angiotensin ii receptor blocker, which is metabolized in vivo to the more potent antagonist exp 3174. herein, we report on the preparation of e-urocanic1 acid based analogs, focusing our attention on the introduction and structural modifications of the substituents on the imidazole ring as well as the modifications on the acrylic side chain. in particular, we have designed and synthesized a series of urocanic acid analogs bearing the biphenylmethyl tetrazole moiety at the n-1 of the imidazole ring.2 additionally, the rigid acrylic chain was lengthened by esterification resulting in the ethyl ester and on the other hand the latter was readily converted to the corresponding acrylic alcohol or aldehyde which may proved to be effective structural elements for enhancing biological activity. finally, a lipophilic alkyl chain such as the n-butyl group was introduced at the 2-position of the ring which may possibly enhance the antihypertensive activity. docking studies and biological evaluation of the synthesized analogs are being undertaken. university of athens, department of chemistry, laboratory of organic chemistry, 15701, panepistimiopolis zografou, athens, greece the backbone modification of bioactive peptides with replacement of a scissile peptide bond in enzymatic hydrolysis is a well-established strategy for developing protease inhibitors. 1 in particular, for zinc metalloproteases, which contain a zinc atom in their active site, several successful modifications have been reported over the past years. 2 phosphinic pseudopeptides are among the best candidates when addressing the challenge to potent and selectively inhibit zinc proteases. a thorough search in the literature3 revealed the absence of any reference regarding thiophosphinic pseudopeptides. we thought that this class of compounds would add a valuable tool in the field of zincbinding groups. in the present study, we describe in detail the first synthesis of a new class of phosphorous compounds, thiophosphinyl dipeptide isosters (tdis). we prepared several fully protected thiophosphinate pseudodipeptides of the general formula pg-phe-ψ[p(s)(ox)ch2]-gly-pg' starting from the corresponding phosphinate pseudodipeptide using lawesson's reagent. selective deprotection of these compounds was also studied and the results are disclosed. these compounds can be used as building blocks for the synthesis of longer thiophosphinic pseudopeptides after suitable deprotection and elongation as well as transition transition state-mimicking inhibitors for several zinc metalloproteases. in the last decade, trypsin inhibitor sfti-1 isolated from sunflower seeds [1] has become one of the most studied peptidic inhibitors of serine proteases. owing to its small size and a strong trypsin inhibitory activity (ka = 1.1×10 10 m -1 ), sfti-1 is considered to be a very attractive template for designing proteinase inhibitors with the potential use as pharmacological agents [2] . it could also serve as an affinity probe for the isolation of trypsin like (sfti-1) or chymotrypsin like ([phe5]sfti-1) proteinases. following this idea, we decided to synthesize a set of cell-permeable monocyclic sfti-1 analogues with a fluorophore moiety attached at their n-termini. the presence of the fluorophore in the molecule enabled us to show that the analogues can cross the cell membrane. the cell penetration assay was performed using multiple cell lines (hela cells and human fibroblasts cell line (46br.1n) was obtained from european collection of cell cultures (ecacc)). for all the obtained peptidomimetics, we determined the association constants with cognate proteinases. selected peptides were also used as a probes for the detection of inhibitor -proteinase complex, which was achieved by the means of gel filtration chromatography equipped with fluorescence detector and acrylamide native gel electrophoresis. the functional reconstruction of folded protein surfaces with peptide-based mimics is an enormous scientific challenge. the majority of proteins show activity through a small area of their folded surface: "the binding site". however, linear peptides are too flexible and seldomly adopt the correct 3d-structure of the binding site spontaneously. therefore, they show limited or no activity at all 1 . crucial for activity is to control the secondary (αhelix, β-sheet and/or β-turn) and tertiary structure (relative orientation of subdomain structures). we present the development of a new type of watersoluble scaffolds that have the potential to control both secondary and tertiary structure of discontinuous (i.e. double-loop) protein mimics. the new scaffolds contain a first pair of reactive functionalities to constrain the linear peptide conformation via a 'clips' reaction2, stabilizing the secondary structure. next to this, a second functionality allows for ligation of two dissimilar constrained peptides to form a discontinuous binding site mimic via oxime-ligation or click-reaction. these ligations offer the ability to position different peptide loops in 3d, thus mimicking the tertiary structure of the native protein. most unique to our approach is the fact that all chemical conversions are performed in aqueous media, using side-chain unprotected peptides 3 . growth hormone-releasing peptide 6 (ghrp-6) is a synthetic hexapeptide (his-d-trp-ala-trp-d-phe-lys-nh2), which interacts with two kinds of receptors: growth hormone secretagogue receptor 1a (ghs-r1a) and cluster of differentiation 36 (cd36). the latter is a membrane glycoprotein member of the class b scavenger family, and decreases the internalization of oxidized lipids into macrophages, as well as causes inhibitory effects on angiogenesis associated with binding to thrombospondin. to increase activity and selectivity for the cd36 receptor, different analogues of ghrp-6 were synthesized. in particular, substitution of trp 4 in ghrp-6 by aza-amino acids has given selective analogs, due likely to induction of a β-turn secondary structure. for aza-peptide synthesis, a submonomer solid-phase approach has proven effective to introduce side chains onto the semicarbazide residue. studying influences of benzylidene, benzhydrylidene and fluorenylidene residues during the alkylation of the semicarbazide, superior conversion was observed with fluorenone derivative, and mild alkylation conditions employing et4noh as base have improved yields and minimized racemisation. our presentation will focus on the improved submonomer synthesis method for optimization of selective and potent cd-36 ligands with antiatherosclorotic and anti-angiogenic effects. for instance, the integrin αvβ3, vitronectin receptor, is expressed in a number of cell types and has been shown to mediate adhesion of osteoclasts to bone matrix, vascular smooth muscle cell migration, and angiogenesis. integrin αvβ3 also play a significant role in tumor growth, invasion and metastasis, and is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic (rgd) tripeptide sequence. rgd has been shown to be potent antagonist of the integrin αvβ3, and has excellent anti-angiogenic properties including its suppression of tumor growth in animal models. in this context, drug design based on the rgd structure may provide new treatments for diseases such as thrombosis, osteoporosis, and cancer. we designed and synthesized series of short rgdmimetics containing the sequence xaa-gd, where xaa is arg-mimetic. as promising candidates we have chosen canavanine (cav) and canaline (can) instead of the basic residue arg. in order to improve antitumor activity of the parent molecule, c-terminal modifications were also applied. their cellular uptake was determined on human breast (mcf7) cancer cell lines. furthermore, the in vitro cytostatic effect was evaluated by mtt assay on human liver hepatocellular carcinoma (hepg2) and human breast (mcf7) cancer cell lines after 24, 48 and 72 hours of treatment. in the case with the human tumor cell lines (hepg2, mcf7) and c-modified analogues, statistically reliable results were achieved for the most of concentrations used. acknowledgements: this work was supported by bulgarian ministry of education and science, project my-fs-13/07. microwave assisted solid phase synthesis of urea and urea/amide based foldamers k. pulka, c. douat-casassus, g. guichard* european institute of chemistry and biology, university of bordeaux 1 -cnrs umr 5248, pessac, france foldamers 1 are fully arti cial molecules that structurally and functionally mimic variety of biopolymers. among them, aliphatic n,n'-linked oligoureas with proteinaceous side chains can adopt extremely robust helical folds stabilized by intramolecular three-centred h-bonds. 2 owing to their resistance to enzymatic degradation, diversity of side chains and structural predictability urea-based foldamers represent unique scaffolds to elaborate functional mimetics of α-polypeptides. of note, heterogenous oligo(urea/γamides) backbones obtained by substituting nh groups by ch2 display very similar folding propensities. in our laboratory we are investigating the solid phase synthesis of urea and urea/γ-amide oligomers. urea bonds are incorporated into the growing chain by reaction of active succinimidyl carbamates. previously we have applied two different strategies involving fmoc-or bocchemistry, but both methodologies suffer some limitations. 3 therefore a new strategy (compatible with the use of tfa sensitive linkers and side chain protecting groups) featuring azide as a masked amine group has been developed. the synthesis of 15 new azido protected succinimidyl carbamate building blocks is reported. they were obtained in 6 steps from α-amino acids (12-45% overall yield). the staudinger reduction with pme3 was successfully applied to restore the amine group after urea formation on solid support. in addition, microwave irradiation has been found to dramatically accelerate the synthesis. overall, this azide-strategy combined with microwave irradiation was found to be very effective for the solid phase synthesis of oligoureas and related hybrids, surpassing previously developed approach utilizing fmoc chemistry. these antibiotics should have a mechanism different from currently used antibiotics to circumvent existing resistance mechanisms 1 . previous results have shown that "genetic" antibiotics operating by gene silencing in bacteria via rna interference may be successful new candidates. efficient silencing requires efficient crossing of cell membrane. this step can be alleviated using cell penetrating peptides (cpp) as carrier of drug candidates, such as peptide nucleic acids (pnas) which inherently have poor internalization properties 2 . the aim of this study is to elucidate mechanisms of uptake in bacteria using pna-cpp conjugates, which previously have shown promising antibacterial effects 2 . the fate of the pna and cpp parts of the conjugates, once inside the cell, is investigated regarding localization and possible degradation within the cell. furthermore, a method for toxicity testing of pna-cpps is being developed using histamine release in rbl-2h3 cells as a quantitative measure of allergenicity of pna-cpps. the prospect of this information is to define boundaries within which cpps can be found, thereby rationally designing novel efficient antibacterial biomolecular drug delivery systems. oxytocin and its fragments have the potential to influence behavioral and cognitive functions, including their disturbances in some brain disorders. therefore, there is an interest to synthesize new peptide-steroids chimeras for potential therapeutic use. oxytocin analogue was synthesized in solution by coupling azido-phenylalanyl residue or p-azidopegylated handle to the n-terminal end of oxytocin molecule. its c-terminal fragment pro-leu-gly-nh2 (mif-1) was elongated at proline residue by the same type of azido handles as well. both peptides were marked for fluorescent detection of their possible binding on brain slices. peptide chimeras with the suitable steroids were prepared via azide click to the triple bond on the modified steroid counterpart like (17α)-17-hydroxypregn-4-en-20-yn-3-one, 24-norchol-5-en-22-yn-3β-ol. steroidyl-peptides were then used in the trials using rat-brain slices. the sites of the peptide-steroids chimeras bound to the brain tissue were identified with the aid of fluorescent microscopy. the suitable chimeras will be tested for their penetration through blood brain barrier for the pharmacological effects. indicating that the orientation of the n-butyl group is of primary importance. docking studies revealed that the highly active analog affords an additional hydrophobic binding feature compared to losartan which fits to an extra hydrophobic cavity. these results may contribute to the discovery of new biologically active molecules by a convenient and cost effective synthetic strategy. the context of pain research, the co-administration of opioid agonists and nk1 antagonists previously led to an enhanced antinociceptive potency, 1 and recently largent-milnes and co-workers have shown that a hybrid opioid-nk1 octapeptide was able to attenuate tolerance development, related to sustained opioid treatment. 2 our group has prepared a compact opioid agonist-nk1 antagonist peptidomimetic chimera dmt-d-arg-aba-gly-nme-3',5'-bn(cf3)2 1 that served as a lead structure. 3 we report a solid phase method for the synthesis of the 4amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (aba) structure, which is used as a central unit in the investigated dual ligands. this method allowed the rapid assembly of new bifunctional ligands containing the aba structure. variations of the d-arg 2 , gly 4 and n-benzyl substituents were made. the introduction of d-cit 2 , a gly 4 → β-ala4 substitution and the removal of the trifluoromethyl substituents in 1 caused considerable shifts in receptor binding. the obtained structure-activity relationships will be presented. hence, a promising approach for the treatment of dmd is the use of drugs to force ptc readthrough. (+)-negamycin 1 is a dipeptidic antibiotic containing a hydrazide structure. although (+)-1 was not clinically developed due to some toxicity, it was recently reported that (+)-1 restore dystrophin expression in the muscles of mdx mice, an animal model of dmd. 1 therefore, (+)-1 is a promising therapeutic candidate for diseases caused by nonsense mutations. based on our own efficient total synthetic method of (+)-1, 2 structure-activity relationship (sar) study was perfromed to discover derivatives with a potent readthrough-promoting activity. we found a derivative, (5r)-5-hydroxy-6-aminohexanoyl-glycine exhibited not antimicrobial activity but a similar readthrough activity to (+)-1, suggesting that the ptc readthrough mechanism can be distinguished from the antimicrobial mechanism. 3 moreover, we synthesized 5-epi-negamycin and found that this analog exhibited a similar activity to (+)-1 in in vitro readthrough assay. this result hence prompted us to synthesize a 5-dehydro-derivative, e.g., 5-dehydro-3-epinegamycin 2, which is a natural product with little antimicrobial activity. surprisingly, we found that 2 showed a higher in vitro readthrough-promoting activity than (+)-1. this result suggests that mother nature independently evolved readthrough-promoting products like suppressor trna, in distinction from aminoglycosides, which show both antimicrobial and readthrough-promoting activities. agricultural university of athens, athens, greece high interest has been paid to synthetic structural motifs that promote specific conformations because of their importance for the development of new therapeutic peptidomimetics. 1 in addition, such motifs may show catalytic activity for asymmetric organic transformations. during the last two decades, various synthetic structural motifs that promote reverse turns have been studied. following our interest on chiral prolinamide-thioureas that present interesting organocatalytic activity, 2 we have undertaken a combined experimental/computational study to understand the structural features that may stabilize a reverse turn in short-length peptidomimetics containing a thiourea functionality. compounds with the sequence r-pro-diphenylethylenediamine-thiourea-asp(obut)-obut (r: boc or fmoc, or boc-ala), were synthesized and studied by nmr spectroscopy (tocsy, 1h-13 c hsqc, noesy, roesy spectra) for the sequential assignment and the exploration of the dipolar connectivities. sampling of the conformational space was driven by the noe intensities while molecular dynamics simulations were further applied to the consistent with the experimental data conformers in order to monitor the stability of the formed hydrogen bonding interactions in the course of time. energy refined produced conformers were subsequently modified by applying all combinations of d-and l-amino acids at each site in a stepwise manner. the modelled structures were studied in silico aiming to explore the combinations of heterochiral residues which would promote a folded structure and would favour the potential of β and γ turn motif. the most promising combinations were chosen for synthesis and subsequent nmr characterization. 3 in this research project we will deal with chemical strategies to produce suitable surface modifications in order to induce multidirectional cellular migration along gold surfaces. to achieve this objective we want to use and characterize self-assembled monolayers (sams) of thiolated dna chains (dna-sh) adsorbed on gold surfaces through the hybridization with complementary modified single-stranded pnas. pna is a structural dna mimic obtained by polymerization of n-(2aminoethyl) glycine monomers that replace the ribose-phosphate backbone characteristic of natural nucleic acids. it is an achiral, uncharged, and relatively rigid biopolymer of high biological and chemical stability, 4 and it can bind complementary dna strands with higher affinity than the corresponding dna sequences.for all these reasons we have chosen pna as a key molecule to promote and assist the movement of cells. by producing a chemical gradient of dna-sh along a gold surface in the presence of a chemotactic molecule it will be possible to obtain and control a directed cellular migration. the norwegian structural biology centre and the centre for theoretical and computational chemistry, department of chemistry, university of tromso, 9037 troms , norway renin is a highly selective aspartic protease which catalyzes the hydrolysis angiotensinogen, a protein secreted from the liver, to the decapeptide angiotensin-i. 1 angiotensin-i is further processed by the relatively nonspecific angiotensin converting enzyme (ace) to give the octapeptide angiotensin-ii, a potent vasoconstrictor and the dominant peptide produced by the reninangiotensin system. renin catalyses the rate determining step in the formation of angiotensin-ii, and has for several decades been an established therapeutic target for drug development in relation to hypertension. 1 in the search for renin inhibitors, substituted piperidine derivatives have been identified as promising, 2-4 and piperidines have proven to be efficient scaffolds for the development of novel non-peptide aspartic protease inhibitors, particularly towards renin. [5] [6] [7] we herein describe a series of 4-triazolyl substituted piperidine derivatives that have been synthesized from n-boc protected trans-4-ethynyl-3-hydroxy piperidine and tested as novel renin inhibitors. piperidine derivatives containing a 1-substituted 1,2,3-triazol-5-yl substituent were found to be most active and molecular docking experiments provides a rank order that is in very good agreement with experimental data. the cxcr4/sdf-1 axis is involved in many biological processes such as hematopoiesis, immune cell migration, as well as in cancer metastasis. cxcr4 also mediates the infection of t-cells with x4-tropic hiv functioning as a coreceptor for the viral envelope protein gp120. cxcr4, as a pharmaceutical target, is of utmost importance but the lack of synthetic agonists has seriously slowed down drug development. it has been recently described by our research group 1 , that grafting the sdf-1 n-terminus onto a side-chain of the inverse agonist t140 2 . generated high affinity synthetic agonists as well as partial agonists for the chemokine receptor cxcr4. to remain stable towards proteases and act as useful pharmaceutical tools, the pk-adme properties need to be improved with a gradual transition to peptidomimetic structures. medicinal chemistry witnessed major advances with the discovery of small synthetic molecules that mimic the natural peptidic substrates. these small molecules do not undergo proteolytic degradation, an advantage they hold over natural counterparts. in order to improve stability against proteases, part of the sdf-1 chain was replaced with variable lengths of polyethylene glycol and unnatural amino acids at differents positions. here, we have produced a series of compounds, most of which showing nanomolar affinities for cxcr4 and some are displaying partial agonistic properties. tlrs are the innate immunity receptors that recognize the epitopes found on surfaces of various cells and therefore they initiate and sustain the atherogenic inflammatory response [1, 2] . we assume that the use of small stat1 mrna−binding pna−inhibitors to manipulate the activity and expression of stat1 could prove an attractive therapeutic strategy in treatment of atherosclerosis. to that end we synthesized a specific stat1 mrna−binding pna inhibitor as well as a non-specific pna to compare their inhibition of gene expression. in our work we developed effective method of synthesis of pna−peptides conjugates by means of "click chemistry". determination of optimal conditions for conjugation (connection of pna with the peptide) will allow for the design of compounds useful in gene therapy. the specificity of pna hybridization to complementary dna fragment was verified by capillary electrophoresis (ce). as an artificially synthesized somatostatin analogue, tyr 3octreotate (toca) can specifically bind to somatostatin receptor (sstr), which are usually over-expressed on many tumor cells. carbohydration of n-terminus of toca has resulted in improved pharmacokinetics and tumor targeting (1) . 18 f is an ideal nuclide for positron emission tomography (pet) imaging; there may be significant uses of 18 f labeled glucitol-toca and its analogues as tumor probes for the diagnosis of sstr-positive tumors. in order to explore a novel pet probe for diagnosis of sstrpositive tumors, we designed a synthetic route to synthesize n-gluc-lys(nota)-toca, which uses 1,4,7-triazacyclononane-1,4,7-triacetic acid (nota) as the chelating reagent. n-gluc-lys([al 18 f]nota)-toca is radiosynthesized quickly and efficiently using the chelation reaction of al 18 f complex and n-gluc-lys(nota)-toca. the aim of this study is to develop an efficient method for the synthesis of monomers of triazolic nucleic acid (tna), a new class of artificial nucleic acids. but-2-yne-1,4-diol and nucleobases derivatives will be substrates of the monomers synthesis. tna oligomers could be used as specific inhibitor of tar rna hiv-1, the regulatory rna structure crucial for hiv replication. "click chemistry" based on 1,3-dipolar cycloaddition will be used to conjugate an alkyne and azide derivatives of monomers subunits. a ru (ii) complex will be used as a catalyst of internal alkyne (but-2-yn-based) cycloaddition. the reaction gives exclusively of 1,4,5-trisubstituted derivative of triazole ring 1 . the monomers will be characterized using rp hplc, capillary electrophoresis (ce) and 1 h and 13 c nmr. the resulting monomers containing fmoc-protected amino group and a free carboxyl group will be used for the classical spps method to synthesize tna oligomers. tna sequences will be designed against tar's bulge and an external loop. 1 through the recognition that the repertoire of polypeptide conformations can be greatly expanded by the creation of structures incorporating β-amino acids. 2 moreover, the numerous advantages of hybrid (mixed α-and β-) backbone peptidomimetics with respect to homogeneous ones were quite recently outlined. 3 we describe here various β-amino acid-based β-hphe-β-hphe dipeptide derivatives, also conformationally constrained, and their application to the synthesis and biological evaluation of hybrid analogues of the opioid endogenous peptide endomorphin-2 (em-2). the opioid system mediates a wide variety of pharmacological and physiological processes, including pain perception and modulation. the amidated tetrapeptide em-2 has been shown to be μ-opioid receptor (mor) agonist exhibiting a very high μ-receptor affinity and selectivity, and it is an important model in the search towards new analgesics. 4 structural investigation of em-2 reveals the high conformational freedom of the phe side chains and also the inherent flexibility of the peptide backbone, indicating many probable bioactive conformations, ranging from βturns to extended conformations. with the aim of better clarify the relevant role of the proper spatial orientation of the aromatic rings and in particular of the benzyl side chains at position 3 and 4, 1 h nmr studies, molecular modelling, and molecular docking to a homology mor model of our hybrid analogues are currently under way. the lantibiotics represent a class of antimicrobial peptides, in which the unusual amino acids dehydroalanine and dehydrobutyrine and the intramolecular thioether bridges (lanthionines) are important structural features for bioactivity.the lipid ii -nisin complex is responsible for pore-formation since the c-terminal part of nisin is inserted into the bacterial cell membrane which ultimately results in cell leakage and collapse of vital ion gradients. in order to increase the metabolic stability of nisin, the oxidationsensitive thioether bridges can be replaced by metabolically stable dicarba moieties, as successfully demonstrated by the synthesis of nisin ab(c) analogs containing alkane/alkene bridges [1] . to obtain more insight into the importance of the cross-bridged de-ring structure (i→i+3, i+2→i+5 connectivity) on nisin's bioactivity, we synthesized a series of all four diastereomers of the crossed alkene-bridged de-ring mimic, using ring-closing metathesis. all four diastereoisomers were obtained by hplc and structurally characterized by nmr spectroscopy. an orthogonal protection scheme was used, to enable the independent n-or c-terminal modification of the bicyclic hexapeptides with azide/alkyne functionalities. via cu(i)-catalyzed cycloaddition chemistries, alkyne-functionalized natural abc-fragments of nisin, which were obtained by tryptic digestion of full length nisin followed by hplc purification, have been conjugated to synthetic de-ring mimics to obtain novel nisin derivatives and their affinity toward lipid ii and pore-forming capacity have been studied. herein, we report on the details of the synthesis and characterization of the geometric isomers of the synthetic de-ring mimics, and their use as synthons in cu(i)-catalyzed click chemistry to obtain newly designed nisin hybrids as potential novel peptide antibiotics. università di ferrara, dipartimento di biochimica e biologia molecolare, ferrara, italy mirnas play an important role in regulation of gene expression, being involved in numerous processes such as cell proliferation, cell differentiation, apoptosis and also in the progress of diseases as cancer and cardiovascular disorders. mirnas associated to diseases recently become targets for the development of new drugs based on antisense oligonucleotides or analogues complementary to the chosen mirna, in order inhibit the binding of the mirna to its mrna target. therapeutic silencing of mirna has been also observed in several animal disease model. 1 in this work we propose a new approach to interfere in the mirna function, based on peptide nucleic acid (pna) oligomers designed to be complementary to selected regions of the mirna precursor (pre-mirna). as the pre-mirna bases belonging to the stem are not perfectly complementary, we hypothesized that the mismatched duplex of the pre-mirna could be opened by pnas inhibiting of its maturation into mirna. two pna sequences, targeting respectively the "sense region" and the " 5' end region" of the pre-mir210 were designed. pnas were conjugated to different carrier peptides, hiv-tat, r8, k4 and two nuclear localization signal (nls and binls), in order to increase their cellular uptake. to verify the ability of the designed pnas to give strand invasion on the pre-mirna, we conjugated also pnas to the thiazole orange, a probe which lights-up upon hybridization 2 the development of privileged molecular scaffolds efficiently mimicking reverse turn motifs has attracted remarkable interest when structural constraints are exploited to increase both binding and selectivity of model peptides. one of the successful approaches to restrict peptide conformation is the disubstitution in the α position of an α-amino acid, leading to a conformational constraint and a stereochemically stable quaternary carbon center. in particular, spirocyclic scaffolds are able to provide, upon the attachment of appropriate functional groups, useful high-affinity ligands, relevant to the field of drug discovery. 1 at present, we are interested to spirocyclic tryptophan (trp) analogues, in order to develop new reverse turn nucleating moieties able to be inserted into pharmacologically relevant peptidomimetic compounds. among peptides sharing a tryptophan-containing β-turn motif of which the trp residue is critical for binding, we looked at the hormone peptide somatostatin, 2 acting in various organ systems as a neuromodulator and a neurotransmitter, as well as a potent inhibitor of various secretory processes and cell proliferation. 3 somatostatin and its analogue octreotide (sandostatin® drug, clinically used for the treatment of endocrine tumors and acromegaly) are thought to interact with the sst1-5 receptors mainly by inserting a β-turn substructure, carrying a lysine (lys) and a trp side chain into a pocket of the g protein-coupled somatostatin receptor. we report here the preparation and structural characterization of a new 1,2,3,4-tetrahydro-β-carboline (thbc)-based spirocyclic lactam as type-ii β-turn model compound and the application of its core structure to the synthesis of a somatostatin mimetic, whose biological evaluation is under way. the analogues of sfti-1 modified in the p 1 position by, βand γ-amino acids and n-substituted β-alanines r. lukajtis, a m. filipowicz, a a. legowska, a d. debowski, a a. lesner, a k. rolka a a faculty of chemistry, university of gdansk, 80-952 gdansk, poland serine proteinases play very important roles in many physiological processes in humans, such as: food digestion, fertilization of the ovum, blood clotting and dissolution of blood clots, immune response. however, their uncontrolled activity can evoke serious pathological conditions. therefore, serine proteinase inhibitors are considered to be a promising class of therapeutic agents. trypsin inhibitor sfti-1, on which we focused our attention in the last decade, is an attractive template for the design of such compounds. its primary structure is shown below: & 1 gly-arg-cys(& 2 )-thr-lys 5 -ser 6 -ile-pro-pro-ile-cys(& 2 )-phe-pro-asp& 1 the inherent feature of natural peptides and proteins is their low stability towards proteases, which seriously reduces their bioavailability. there is a growing need for the development of artificial biopolymers with diverse side chains, capable of mimicing peptide function. β-and γpeptides are an interesting class of peptidomimetics with significant chemical and biological properties. the present communication describes the chemical synthesis and inhibitory activity of a series of trypsin inhibitor sfti-1 monocyclic analogues (with disulfide bridge only) modified in p1 position by βand γamino acids and n-substituted β-alanine (β-peptoid units). the following mimetics of proteinogenic lys or phe were used: β 3 hlys, β 3 hphe, γ 4 hhlys, γ 4 hhphe, βhnlys, βhnphe. all compounds were synthesized manually on solid support. β-peptoid monomers were introduced into the peptide structure by two steps method [1] . newly obtained sfti-1 analogues modified in p1 position by β-derivatives of lys and phe were able to inhibit bovine β-trypsin and bovine αchymotrypsin, respectively, whereas the remaining ones (except for [βhnphe5]sfti-1) appeared to be inactive. the notion that early soluble aß intermediates are endowed with cytotoxic effects suggests that a major effort should be directed toward the inhibition of amyloid aggregation at very early stages. inhibiting aß self-oligomerization could, therefore, provide a useful approach to treating and controlling the pathogenic pathways underlying alzheimer's disease (ad). likely, agents that target the basic molecular recognition process preceding the formation of early intermediates are the most valuable candidates. we have conjugated a trehalose moiety to the known ß-sheet breakers pentapeptides lpffd. 1 trehalose has received a special interest because it has been found to be effective in the treatment of neurodegenerative diseases associated with peptide or protein aggregation. 2 the glycosidic moiety was covalently linked to different regions of the peptides' primary sequence, including the n-terminus or c-terminus or the aminoacid side chain. this new class of peptides showed an increased resistance to proteases. 1 in this work, the inherent ability of these peptides to recognize and bind the monomeric form of recently reported a d-amino acid-containing hiv protease inhibitor with a sulfonyl group showed an activity enhancement against drug resistant viruses. x-ray crystallographic study of the derivative revealed existence of four bridging water molecules. we suggest that the additional indirect interactions through water molecules induced the inhibitor's flexibility in binding conformation, keeping the affinity with the mutated proteases. oxalyamide, so-called oxamide, has two carbonyl oxygen atoms as hydrogen bonding acceptor similar to sulfonyl group, which is promising to interact with water molecules. to increase the numbers of bridging water molecules, we built-in two oxamide structures to both terminals of pseudo-symmetric compounds with hydroxymethylcarbonyl-hydrazide isostere. the derivatives were tested for inhibitory activity using wildtype hiv protease and a highly mutated protease with lopinavir resistance. we found that the loss of potency against the mutated protease was relatively small in the oxamide derivatives. the molecular dynamic simulations suggested the ability of bridging water formation of the two oxamide groups. optimization of the pna-synthesis using different bases for fmoc-deprotection s. rawer 1 , k. braun 2 , r. pipkorn 2 1 life technologies, darmstadt, germany 2 dkfz, heidelberg,germany pna (peptide nucleic acids) are considered as highly sensitive and specific tools for antisense strategies especially conjugated with cell penetrating peptides. individual designed shuttle systems can be applied in cancer diagnostics and possible therapy (1) . it is, however, undisputed that proper pnas' syntheses prove to be a challenge for coupling and fmoc-deprotection. due to the structure-formation the success of the synthesis depends strongly from parameters, like activator's quality and deproctection kinetics correlating to the length of the pna polymer spps product. using the example of the spps pna synthesis' results of the coding sequence of c-myc human exon ii, different bases, acting as fmoc-deprotection reagents, are compared and analyzed aiming at optimizing the pna synthesis strategy (2) peptidoglycans are central structural components of the cell wall of bacteria. several plant receptors are known to recognize peptidoglycan fragments. it is believed that these receptors form part of the defense mechanism against bacterial infections in several plant species. peptidoglycans consist of long chains of alternating β(1-4)linked glcnac and murnac moieties that are crosslinked by short, non-ribosomal peptides. these peptides consist of several d-amino acids and the symmetrical (r,s)diaminopimelic acid (meso-dap). in particular, the latter complicates the synthesis of peptidoglycan fragments due to the requirement for individually addressing the two pairs of functional groups. some chemical syntheses of peptidoglycan fragments have been reported [1] [2] [3] [4] , hhich involved multi-step formation of an orthogonally protected dap moiety, and elaborate oligosaccharide synthesis. here we present a new and simple approach to peptidoglycan synthesis which is based on the use of commercially available building blocks for the dap and oligosaccharide components. this allows easy access to a range of peptidoglycan fragments for structure-activity studies. the introduction of solid-phase peptide synthesis (spps) and the subsequent refinement of resins, linkers, coupling reagents and amino acid protecting groups allowed access to a wide range of peptides. therapeutic peptides, in particular, have benefitted from the maturation of spps, as complex peptides can be synthesized more efficiently in comparison to conventional solution phase synthesis. however, peptides containing multiple disulfide bonds often still remain difficult to make due to a lack of orthogonal cysteine protecting groups that can be used in routine spps. the cysteine protecting group s-tertbutyl mercapto (s-tbu) is commercial and orthogonal to other cysteine protecting groups. removal of the protecting group is facilitated by reducing agents (e.g. thiols or phosphines) and is stable to tfa and piperidine, hence compatible with fmoc/o-tbu peptide synthesis. however, the protecting group cannot be used in routine spps due to long deprotection times (4-24h) . in certain cases it has been shown to be impossible to remove due to proximity of bulky protecting groups and sensitivity to certain sequences. additionally, reports of desulfurization of s-tbu protected cysteine to dehydroalanine, by the use of prolonged exposure to reducing agents, show the limitations of this protecting group. the concept of cysteine protecting groups labile to reducing agents is promising due to orthogonality to other cysteine protecting groups and the limitations of s-tbu initiated an investigation into novel reductive cysteine protecting groups. herein, we introduce s-tmp as a novel cysteine protecting group that is very labile to reducing agents. the increased lability, in comparison to s-tbu, allows utilization of reducing agent labile protecting groups in routine peptide synthesis of disulfide containing peptides. as modern automated spps protocols allow the assembly of larger and increasingly complex peptides, a precise control of the coupling reactions is a crucial prerequisite in peptide synthesis. monitoring the progress of synthesis allows the detection of undesirable products caused by side reactions, incomplete couplings or deprotections. 1 although different methods have been developed for monitoring spps, we observed that the use of colorimetric test or continuous-flow uv absorbance of the reaction column effluent was not informative enough to identify difficult steps in the synthesis. in this study we demonstrate the usefulness of the combination of a mw-assisted mini-cleavage protocol and the uplc-esi-ms analysis for monitoring the quality of the reaction steps. as a proof of concept, based on this strategy, we monitored the synthesis of pthrp(1-34)nh 2 (synthesised by fmoc/tbu rt-spps, liberty™, cem), characterised by a cluster of arginine residues in the 19-21 region. 2 by the use of mw irradiation during the mini-cleavage protocol, we optimized time for mini-cleavages particularly in case of multi-arginine containing peptides, protected by pbf group. the results obtained by uplc-esi-ms showed that the complete removal of the pbf groups from the arginine sidechain residues required 1h at rt. on the other hand, the mw-assisted mini-cleavage monitoring let us to obtain final results just in 15 min, confirming that the use of microwave irradiation in mini-cleavages is an efficient strategy to monitor also difficult peptide couplings, such as multiarginine peptides. identification of some deletion sequences was helpful to recognise critical couplings in order to adopt more efficient coupling strategies and therefore to optimise the final yield and purity of the crude peptide. development of green sustainable chemistry is currently regarded as a challenge in science and technology to reduce the use of organic solvents and utilize less toxic solvents instead. water and aqueous-based solvent systems represent an increasingly significant choice for replacing traditional solvents in synthetic chemistry. until recently, peptide synthesis in aqueous solution has remained largely unexplored. this is because the most common building blocks are sparingly soluble in water and are considered inappropriate for in-water peptide synthesis. we have developed a method for solid-phase peptide synthesis in water, which utilizes water-insoluble fmoc-amino acids that are converted to water-dispersible nanoparticles. in this way, the solubility problem is overcome. our technology, which uses suspended nanoparticle reactants for the coupling reaction, offers many advantages in terms of reaction efficiency over inwater synthesis using water-soluble or non-disperse reactants. however, there are two main problems with this nanoparticle approach; (i) slow reaction rates compared to general peptide synthesis in ordinary organic solvents (ii) poor yields for the synthesis of long peptides because the protected peptide chains on the resin have a tendency to aggregate in water. mw assisted spps is particularly attractive because of the widespread availability of the new technology, including automated peptide synthesizers. a trial of mw assisted inwater solid-phase synthesis using non-disperse boc-amino acids has been reported by albericio previously. currently, fmoc-amino acids are routinely used as building blocks for solid-phase peptide synthesis. with this in mind, we have developed a mw irradiation procedure aimed at reducing reaction time and increasing reaction yield for in-water solidphase synthesis using water-dispersible fmoc-amino acid nanoparticles. and we demonstrated in-water solid-phase synthesis of difficult sequence peptide with mw irradiation. m. lebl, z. flegelova spyder institute praha, czech republic cotton was shown as a convenient solid phase support earlier 1-3 , but did not find wide acceptance by the peptide community. we decided to try its application as (i) a support of choice for the synthesis driven by combination of capillary forces and gravity, (ii) support for synthesis utilizing in situ neutralization boc based protocol, (iii) support for combinatorial synthesis based on easy labeling and physical separability of cotton substrate, and (iv) support for multisupport synthesis. -we have built a simple synthesizer in which the cotton carrier (functionalized thread) is placed inside the capillary tubing and the appropriate reagents are introduced by connecting the inlet with appropriate reagents. the speed of "pumping" the reagents is driven by the difference between the elevation of the inlet and outlet of the capillary tubing. -we have shown that boc solid phase synthesis utilizing in situ neutralization is compatible with cotton substrate and provides high quality products. combining with the fact that cotton by itself 4 acts as the self-association breaking agent, makes cotton a suitable carrier for synthesis of "difficult" sequences. -labeling of individual solid support particles can be easily based on the length of the cotton thread pieces, number and positions of knots, or their attachment to a secondary carrier. in addition, it is possible to synthesize peptides differing by the partial structure (alternative linkers, terminal modifications, etc.) in a mixture of classical resin with labeled cotton carriers, which are easily separable at the end of the synthesis. . use of microwave irradiation provides peptides in a fraction of time compared to conventional methods, and the peptides are also often generated in higher yield and purity. while microwave technology is particularly suited for the synthesis of "difficult" to synthesize peptides, this tool can routinely be used for the synthesis of a wide variety of peptides without the need for extensive method optimization. the focus of this study is to demonstrate how a peptide can be synthesized on a small scale (for example 25 μmol) up through larger scales (>1 mmol) with ease. as a biologically relevant model peptide the last 13 residues of the human platelet factor 4 protein (hpf4 57-10) was selected due to its significant antimicrobial activity. 1 however, the problem of developing a robust fmoc thioester method is that the deprotection of the fmoc group with base at each cycle is not compatible with an active ester at the c-terminus. many ingenious approaches have been developed to generate the required thioester peptide. 2, 3, 4 the most popular has been to use an nacylsulfonamide as a base and acid stable (safety-catch) linker for peptide synthesis. alkylation of the sulfonamide after peptide assembly makes the linker labile to cleavage with nucleophiles. 5 whilst popular, it has been plagued by notoriously low yields which originate from the incomplete acylation of the resin-bound sulfonamide with the c-terminal residue, incomplete alkylation of the sulfonamide and the incomplete thiolysis of the resin-bound protected peptide. in this poster we describe the development of a novel dual linker strategy 6 , involving anchoring of the sulfonamide linker to a standard acid-labile resin. this variation overcomes many of the current limitations of the sulfamylbutyryl linker approach and provides a simpler and scalable method for peptide ligation via fmoc spps. m. ziovas, d. tataraki, p. manousou, n. parveri, f. satoglou, d. gatos and k. barlos department of chemistry, university of patras, 26500 patras, greece solid phase peptide synthesis is traditionally performed by the attachment of the c-terminal amino acid through its α-carboxyl function on a suitable solid support and elongating peptide chain towards the amino terminal of the peptide by adding sequentially the amino acid residues in the gradually growing peptide chain. several thousands of publications and patents describe this methodology and its application for the production of peptide pharmaceuticals. in contrary to the attachment of the c-terminal carboxyl function, attachment of amino acids and peptides through an amino acid side-chain on suitable resins and their application in spps is described in a small number of publications and patents. most of these publications describe the attachment of the amino acids through a side-chain carboxyl group of asp and glu. in the present work peptides were synthesized very efficiently in high yield and purity by anchoring of side-chain hydroxyl, amino or thiol groups of amino acids, amino acid amides, amino alcohols or small peptides on resins of the trityl or benzhydryl type. several peptides of pharmaceutical interest, such as exenatide, octreotide, pramlintide, calcitonin, bivalirudin, insulin b-chain and others were produced as examples of this technology, either by the step-by-step procedure or by fragment condensation in solution and on solid phase. step given that some of these diseases are caused by a mutation and/or malfunction of an essential protein, a better understanding of the structure and function of such proteins will allow us to prevent, slow down or even cure these diseases. to increase our knowledge, the synthesis of the target protein, a fragment involved in its activity or interacting peptides that modulate the protein activity is often required. in some cases the preparation of a protein analogue that improves its efficacy is envisage. however, conventional solid-phase peptide synthesis methods have some limitations when attempting to achieve these complex sequences of considerable length. using novel technologies, such as a microwave-assisted solid phase synthesis, commonly found in many peptide synthesis labs, here we performed the step-wise solidphase synthesis of a protein holding more than 100 residues (d-vegf). this synthetic achievement indicates the suitability of this approach for synthesis of long proteins or their analogues. the detailed synthesis of the enatiomeric version of vegf and the selenomethionine substituted analogues of huprp (106-140),1 proteins involved in angiogenesis and prion protein amyloidoses respectively, are described as study cases, where the use of microwaves allow us to obtain them in a fast and efficient manner. therefore, the development of novel peptide analogues with enhanced in vivo stability could potentially provide therapeutic alternatives. the pharmacological evaluation of a bioactive peptide [des-gly 10 ,tyr 5 (ome),d-leu 6 ,aze-nhet 9 ]gnrh, analogue 1, is presented herein. in vitro (kidney mouse membranes) and in vivo (clinically relevant pharmacokinetic mouse model) bioassays were coupled to liquid chromatographytandem mass spectrometry. analogue 1, an agonist of the gnrh receptor with a binding affinity in the nanomolar range, caused testosterone release in mice that was acutely dose-dependent, an effect blocked by cetrorelix. repeated dosing studies in mice demonstrated that analogue 1 was well tolerated and had potency similar to that of leuprolide, based on plasma and testis testosterone reduction and histopathological findings. analogue 1 also shared with leuprolide similar significant antiproliferative activity on androgen-dependent prostate cancer (lncap) cells. on the basis of pharmacokinetic advantages, we expect that analogue 1 or analogues based on this new design will be therapeutically advantageous for the treatment of cancer and endocrine disorders. cortexin is a polypeptide drug isolated from cattle and porcine brain cortex. cortexin is effective in monotherapy and in combination with traditional methods of treatment. cortexin produces tissue-specific, regulatory, and reparative effects on the brain cortex and contains active low-molecular-weight neuropeptides (<10 kda) penetrating through the blood-brain barrier. cortagen is a tetrapeptide h-ala-glu-asp-pro-oh (geropharm) produces nootropic and neuroprotective effects. oleylcortagen is a lipophylic analog of cortagen c 17 h 32 o-ala-glu-asp-pro-oh was created for increased proteolytic stability and increased penetrating through the blood-brain barrier. the main aim of our investigation is the analysis of psychopharmacological profile of 3 peptide preparations in comparison with piracetam. have been shown oleylcortagen (1 mg/kg) and piracetam (200 mg/kg) possess activating effect on motor and research components of behavior in «open field» test. two of peptides (oleylcortagen, cortexin) decreased period of immobilisation and demonstrated antidepressant effects on rat behavior in the porsolt's test, on other hand cortagen demonstrated depressant action. therefore, the significant psychoactivating properties are typical for oleyl-cortagen, cortexin. the mechanism of the action of these peptides can be explained from the viewpoint of the regulatory cascade. they produce a direct information impact on cell structures of the brain, and then promote release of the regulatory peptides, which in turn, induce the release of the next group of peptides. neurology, queen square, london wc1n 3bg, uk one of the hypotheses of alzheimer's disease neuropathology involves beta-amyloid (βa) binding with proteins on neuronal cell surface which leads to cell lysis and amyloid plaque formation. according to the latest data α7-type of the nicotinic acetylcholine receptor (achr) and the prion protein can be the target for betaamyloid toxicity [1, 2] . aggregated βa causes many pathological changes in cultures of mixed neurons and astrocytes such as sporadic cytoplasmic intracellular ca 2+ -signal, activation of reactive oxygen species (ros) production and cell death. in the present work we demonstrated the ability of affinity purified antibodies to synthetic fragment 173-193 of α7-subunit of the achr (achrabs) or to peptide 95-123 of the prion protein (prpabs) to protect cells from βa induced cell death. we also showed that both antibodies did not block βa induced ca 2+ -signal in astrocytes. however, preincubation of cortical co-culture of neurons and astrocytes with achrabs or prpabs significantly reduced the rate of caspase 3 activation and the rate of βainduced ros production via modulating nadph-oxidase. more detailed research of involvement of α7-type achr revealed that α-bungarotoxin was also very effective in the inhibition of caspase 3 activation and superoxide production. the observed positive effect of antibodies to α7-type achr or to the prion protein gives an additional explanation regarding the involvement of these proteins in ad pathology and provides new approach into an anti-ad vaccine design. capturing and macrophage-aided clearance of amyloid beta by surface modified proteineous particles m. richman, s. rahimipour department of chemistry, bar-ilan university, ramat-gan 52900, israel imbalanced homeostasis and oligomerization of amyloidβ (aβ) peptide in the brain are hallmarks of alzheimer's disease (ad). microglia and macrophages play a critical role in ad progression by clearing aβ from the brain or inducing inflammation. recent evidence suggests that the phagocytic pathway of aβ may be defective in ad microglia/macrophages that contributes to the build-up concentration of aβ in the brain. 1,2 therefore, efforts have been directed toward developing treatments that trigger these cells to clear aβ through alternative mechanisms. we have recently demonstrated that protein microspheres modified at their surface with multiple copies of an aβrecognition motif can strongly bind aβ, inhibit its aggregation and directly reduce its toxicity by sequestering it from the medium. 3 here, we describe how the aβ-bound microspheres can stimulate microglial cells and be phagocytosed through a mechanism that is distinct from that of aβ. the phagocytosis was mostly effective with microspheres having diameter size of about 0.7-1 mm and introduction of polyethylene glycol to the surface of the microspheres changed the kinetics of the phagocytosis. moreover, while aggregated aβ induced a significant inflammatory response that was manifested by the release of tnf-α from the microglial cells, the aβ-bound microspheres dramatically reduced the amount of the released cytokine. our data suggest that surface-modified microspheres could be utilized to detoxify other pathogenic or misfolded proteins that their accumulation may lead to genesis of other diseases. vasoactive intestinal peptide (vip) and its derivatives have been thought to be promising drug candidates for airway inflammatory diseases. however, the therapeutic potential of vips is highly limited because of rapid metabolic degradation and systemic side effects following systemic administration. previously, to overcome these drawbacks, our group developed a novel vip derivative, [arg 15, 20, 21 , leu 17 ]-vip-grr (ik312532), with improved metabolic stability (1), and respirable powder (rp) formulation of ik312532 (ik312532-rp) for pulmonary administration (2) . these attempts successfully led to enhanced pharmacological effects of ik312532 in the airway system and reduced systemic exposure; however, further chemical modification of ik312532 with a focus on metabolic stability might provide better clinical outcome. the present study aimed to design a pegylated vip derivative with improved metabolic stability and to develop its respirable powder (rp) formulation for inhalation therapy. ik312532 was chemically conjugated with peg (5 kda, p5k), the physicochemical and biochemical properties of which were characterized by cd spectral analysis, binding assay, and metabolic stability. the rp formulation of pegylated ik312532 (ik312532/p5k) was prepared with a jet mill, and in vitro inhalation performance and in vivo pharmacological effects in antigen-sensitized rats were also evaluated. the cd spectral analysis demonstrated that peg conjugation had no impact on the solution structure of ik312532. although receptor-binding activity of the ik312532/p5k (ic50: 82 nm) was estimated at ca. 30-fold less than that of ik312532 (ic50: 2.8 nm), metabolic stability for the ik312532/p5k was highly improved. according to the laser diffraction and cascade impactor analyses, ik312532/p5k-rp had fine in vitro inhalation performance. insufflation of ik312532/p5k-rp (150 μg of ik312532/p5k) in antigen-sensitized rats resulted in marked attenuation of inflammatory events, as evidenced by significant decrease of inflammatory biomarkers and granulocyte recruitment in pulmonary tissue at 24 h after antigen challenge. from these findings, pegylation of vip derivative, as well as its strategic application to the rp formulation, might be a viable approach to improve its therapeutic potential for treatment of airway inflammatory diseases. the previous studies have shown that trkb (tropomyocin receptor kinase) acts as an oncogenic agent and its binding to bdnf (brain derived neurotrophic factor) activates signaling angiogenesis of tumor proliferation [1] . for finding the most stable and potentially effective peptides against the trkb, we applied the following protocol. at the first step of this protocol we designed a peptide library by using sequence tolerance method in rosetta 3.3 package, then peptide energy optimization performed by backrub protocol for finding the most stable peptides. the five best peptides in energy optimization selected based on backrup scores by using r package [2] . 3d-structure prediction of the selected peptides was performed by using molecular dynamic in hyperchem 7 software. docking of peptides with trkb receptor was carried out in haddock software. we used cyclotraxin, a selective trkb inhibitor as positive control for this protocol. cyclotraxin and the peptides were compared by anova or t-test. the peptides are going to be tested against the trkb in an in vitro model. dirucotide (mbp 82-98 ) is a synthetic peptide analog of , that consists of 17 amino acids and tested in a phase trial were failed to reach his tolerance level on previous phase ii in rpms patients. one of the major disadvantages of peptide therapy is the activation of proteolytic enzymes, leading to peptide degradation. to address this problem cyclic peptide analogues have been synthesized. thus we synthesise a linear and cyclic analogues of dirucotide. the two analogues were synthesized by changing the amino acid residue at position 91 from to the synthesis of the linear peptide, as well as of the cyclic one, was carried out by the fmoc/tbu methodology, utilizing the 2-chlorotrityl chloride resin (cltr-cl). the purification was achieved using semi-preparative rp-hplc and the identification was assessed by analytical rp-hplc and by mass spectrometry (esi-ms). the linear and cyclic peptide analogues will be used in human t-cell cultures to test their immunogenicity in patients versus healthy controls. 1 in the first approach a reporter moiety was introduced to diagnose and treat cxcr4 related diseases. therefore, an anchor point to attach additional molecules to the ligand was elucidated. using sar studies to optimize the linker from the ligand to the detectable moiety the excellent receptor affinity could be retained. in a final step the reporter moiety was introduced to give a ligand for diagnosis via pet imaging and for possible endoradiotherapeutic applications. 2 originating from the dimeric motif present in many active cxcr4 ligands several dimers were prepared using a monomeric ligand identified in the prior study. comparison of monomers and dimers yielded a possible subsite binding mode explaining why the dimers exhibit enhanced affinity using a model derived from the origins of the monomer. 3 several peptidomimetic modifications were introduced to the ligand to reduce the peptidic structure. in a conformationally guided approach introduction of a peptoid motif could enforce a single active conformer that was enhanced through subsequent modifications. this yielded a compound 100 times better than the original cxcr4 antagonist which is the most affine cxcr4 ligand reported so far. our previous studies have demonstrated that pace4 represents a potential therapeutic target for the treatment of prostate cancer 1 . moreover, we have developed potent and selective pace4 inhibitor, (20-fold specificity over furin), known as multi-leu or ml peptide, which has a significant inhibitory effect on the proliferation of prostate cancer cell lines. peptide-based drug candidates can be limited by their poor metabolic stability and low bioavailability. thus, we performed structure-activity relationship studies to improve the pharmacokinetic properties of our ml inhibitor. we have designed and synthesized new ml peptide analogs having various chemical modifications. first, based on our previous results, we combined the most effective modifications of position p8 (d-amino acids) and p1 the arginine mimetic 4amidinobenzylamine (amba) to improve overall properties of our leading compound. second, the n-terminus of the resulting analogs was modified with a fatty acid, in order to enhance their cell permeability properties. third, we modified the inhibitors with a peg moiety to increase their stability and bioavailability. we tested the inhibitory activity, stability in plasma, cellular uptake, and cytotoxicity of each inhibitor. the results of this study demonstrate that the presence of the n-terminal extension (c8 or peg8) does not affect activity of our inhibitors. on the other hand, we show that introduction of the peg moiety does not increase cytotoxicity of ml analogs. it is interesting to note that the pegylated analog ac-peg8-d-leu-lllrvkr-amba has better cell-permeability activity than its counterpart without peg unit. this combination of pharmacological properties makes our new ml analogs promising candidates for the development of potential anti-prostate cancer agents. [1] peripheral coadministration of rf9 with opioid analgesics led to confirm the involvement of npff receptors as a part of the antiopioid system. indeed, rf9 was able to reverse the opioid induced hyperalgesia in rat (randall selitto test). then, a complete structure-activity relationships analysis was performed with rf9, assessing the involvement of each moiety for affinity and selectivity towards both npff receptors. a first exploration of the n-terminus part of rf9 (>80 synthesized derivatives) led to replace the hydrophobic adamantane moiety by a hindered aromatic group, providing a subnanomolar npff1 ligand with more than two log-units selectivity against npff2. then, the removal of the cterminal amide function led to reduce the dipeptide arg-phe-nh2 into an arginine derivative. in spite of an initial loss of affinity, optimization of the phenethyl moiety at the cterminus part of arginine led to non-selective nanomolar ligands for both npff1 & 2 receptors. next, we applied efficient methodologies in order to synthesize non-natural analogs of arginine, leading to various compounds exhibiting selectivity for either npff1 or npff2 receptors. in particular, compound rf313 was identified as a selective npff1 antagonist (npff1, ki = 64 nm; npff2: ki > 13 μm). lacking of analgesia properties, oral administration of rf313 (1 mg/kg per os) to rats was able to fully reverse fentanylinduced hyperalgesia. rf313 is the first orally available npff1 antagonist capable of reversing opioid induced hyperalgesia at low dose. moreover, this result allows identifying for the first time npff1 receptor as a key-partner of the anti-opioid system. administration of multiple antiplatelet agents has become the mainstay in the treatment of acute coronary syndromes in everyday clinical practice. we have previously reported significant antiplatelet effects of novel synthetic peptides' single administration on experimental carotid artery thrombosis in rabbits 1 . in the present study we sought to investigate the peptides' effects when administered in marginally effective doses (significantly lower than those utilized in the past), in animals that had previously received low doses of aspirin. the peptides when co-administered with aspirin preserved the carotid artery's blood flow, in contrast to the total artery occlusion observed in animals receiving aspirin and placebo. blood flow at 90 min after electrical stimulation was reduced to 56.7±7.9% and 33.2±0.3% in the ymesradr and psrcdcr-nh2 groups respectively (p<0.001 vs aspirin and control). thrombus weight was significantly reduced in animals receiving ymesradr and psrcdcr-nh2 versus aspirin and control (3.9±0.3 mg and 3.1±0.4 mg, vs 7.8±2.2 mg and 5.7±0.8 mg respectively, p<0.05). platelet aggregation was significantly inhibited in the ymesradr and psrcdcr-nh2 groups by 36.0±14.1% and 45.0±12.0% for adp (p<0.05 vs aspirin and control), and 35.5±6.3% and 54.2±5.6% for aa (p<0.05 vs aspirin and control), respectively. blood loss did not significantly differ among the various groups. administration of novel synthetic peptides, even at marginally effective doses, in animals previously treated with low doses of aspirin results in enhanced antiplatelet effects in an experimental model of arterial thrombosis. the study of peptide metabolism is particularly important when examining anticancer peptides; it can provide pivotal clues for the evaluation and improvement of stability of a peptide drug leading to enhanced pharmacokinetics and efficacy. gnrh analogues are used for the treatment of prostate cancer. as with other peptides a drawback is their short half-life due to their metabolic degradation. in order to examine the stability of these analogues we have developed several in vitro peptide stability and metabolism assays using specific tissues, isolated membranes, cancer cells that are analyzed subsequently using lc-ms/ms based approaches. such in vitro studies are followed up with pharmacokinetic studies in mice in order to establish the correlation between in vitro and in vivo approaches. the kidney is the main metabolic organ for peptide metabolism and for that reason we employed a peptide stability and metabolism assay previously described by our group using isolated mouse kidney membranes for the evaluation and comparison of different gnrh analogues. we tested gnrh analogues in other tissues such as mouse plasma, which is the "distributing" tissue for these drugs and mouse brain homogenate, a tissue known for its abundance in peptidases and relevance for centrally mediated effects. stability studies were also performed in cancer cells. in all cases lc-ms/ms based assays were developed for measurement of peptide drug and the resulting metabolites. for triptorelin, and a series of gnrh analogues, degradation and metabolite formation was studied by our mouse kidney membranes assay. studies of coadministering the peptide of interest with inhibitors that are presumed to block the metabolism in mice are ongoing. the vulnerability of gnrh analogues was verified after incubation with plasma and brain homogenate and by metabolite identification we obtained clues about the key cleavage sites. the described in vitro/in vivo protocols provide valuable information that could lead to the synthesis of more stable anticancer peptides with improved anticancer efficacy. in this work, we explored the use of a high-throughput synthesis and screening approach with peptide arrays to identify and structurally optimize shortened hiv-1 fusion inhibitors. the peptide array technology involves miniaturized synthesis of immobilized peptides, followed by affinity testing with a five-helix bundle, as a mimetic of the fusogenic gp41 protein. this exercise resulted in the identification of a class of truncated peptides which demonstrates a surprisingly high antiviral potency, despite the absence of the pocket-and the lipid-binding domain. the propensity of these peptides to adopt the bioactive α-helical conformation in solution as determined by circular dichroism, could be the key factor for this unexpected potency. these peptides are promising leads for the treatment and prevention of hiv. the pathological role of platelets in cardiovascular disease (cvd) is well established. platelets secrete adp to recruit additional platelets to a thrombotic site. we have previously identified novel cell-permeable peptide modulators of platelet function by using a bioinformatic screen based on patterns of evolutionary conservation in transmembrane proteins 1 . in this study we further explored peptides derived from cadherin cell adhesion molecules. we explored a range of 14 overlapping peptides derived from different cadherins varying from 6-15 amino acids long. peptides are synthesized and analyzed in a high-throughput platelet adp secretion assay. peptides (0.05-50μm) were assessed in the presence and absence of thrombin receptor activating peptide (trap;4μm). we identified cadh-5 and 6 proteins, but not cadh 1 or 2 in human platelets using western blotting and mass spectrometric analysis. peptides derived from paralogous juxta-membrane, cytoplasmic regions of these cadherins are potent modulators of platelet secretion. by systematically deleting amino acids from c or n-terminus of active peptides, we established that the minimal functional sequence for biological activity was a short six-residue motif, which corresponds to the known catenin-binding region of cadherin tails (kepllp) 2 . peptides alone have no biological activity. however, they potentiate the response induced by the platelet agonist trap at low doses. thus we have identified a cadherin-derived peptide that can modulate platelet secretion events. this highlights a previously unknown role of cadherins in the regulation of platelet function. agents that interfere with cadherin signaling in platelets might have a therapeutic role in cvd. ageing of the brain leads to impairments in cognitive and motor skills, and is the main risk factor for several common neurological disorders such as alzheimer's disease (ad) and parkinson's disease (pd). altered protein handling (proteolysis, repair system, chaperones) forms a basis for a large number of protein conformational disorders. extra-and intracellular, as well as intranuclear accumulation of abnormal proteins, in the form of protein inclusions and aggregates, and dysfunction of the quality control mechanisms are common in all these disorders. alterations in protein homeostasis occur with age, causing molecular changes such as protein misfolding and aggregation. many biologically active proteins lack stable secondary and tertiary structure, these are called intrinsically disordered proteins (idps), some of them (e.g. β-amyloid, α-synuclein) are coupled to neurodegenerative disorders. idps exist as assemblies of rapidly fluctuating structures undergoing coupled folding and aggregation process. protein aggregation is characterized by polymorphism, where a mixture of soluble oligomers, amyloid fibrils and amorphous aggregates is the final product. soluble oligomers are inevitable formed during the self-association process and might initiate the neurodegenerative cascades of ad, pd and similar diseases. the emerging consensus that protein misfolding (leading to idps) is the cause of several neurodegenerative disorders now offers the opportunity to develop a general therapy. soluble oligomers with id regions are potential drug targets. recently short peptide fragments and small peptidomimetic molecules have been found also in our laboratory; these molecules bind to the id regions of β-amyloid and are putative drug candidates. precise control of bleeding is ensured by anticoagulant mechanisms, which under normal conditions prevail over coagulants mechanisms. disrupting the balance between procoagulant and anticoagulant systems due to congenital or acquired defects leading to thrombotic disorders. anticoagulants are substances that inhibit blood clot formation. their action consists in inhibiting the synthesis of prothrombin, the substances forming thrombus as well as some coagulation factors. many peptides and proteins with different molecular weight, such as antistasin (ats), ghilantens, hirudin, etc. showing high anticoagulant activity are isolated from salivary glands of ticks and leeches. they inhibit the action of serine proteinases from blood coagulation cascade. this creates an opportunity for targeted synthesis of low molecular weight analogues of some of these proteins, which can be used in the prevention and treatment of thrombotic disorders. ats is competitive, slow-binding inhibitor of factor xa. it is well known that blood coagulation could be blocked at different stages of the coagulation cascade through inhibition of various enzymes. therefore, it is interesting to determine the place of action of newly synthesized antithrombotic agents in the blood coagulation cascade. this can be done by determining the inhibitory constants of newly synthesized peptides on different enzymes of this cascade. herein we report on our kinetic investigation of newly synthesized peptide amides, analogues of isoform 2 of ats 1 . ki, km, vmax and type of inhibition on the factor xa, thrombin and plasmin were determinate. some interesting differences between type of inhibition of ats, free acids and amide analogues of ats were revealed. to evaluate the relative anti-platelet aggregation activities of each peptide, the lebetins were chemically synthesized and fully characterized. here we described the synthesis, the solution structure of lebetin g1-g38 from the venom of vepera lebetina by 1 h bidimensional nmr and the relation structure-activity. this peptide has been demonstrated to be associated with a potent anti-platelet aggregating activity. the g1-g38 three dimensional structure consists in a compact β-bulged hairpain core from which emerges one loop and the cterminus and the n-terminus. we report on an approach whereby ligands are designed to bind and stabilize the 13-26 region of aβ in an α-helical conformation. these ligands reduce aβ toxicity to cells in culture and to hippocampal slice preparations. in addition, when these inhibitors are administered to drosophila melanogaster expressing human aβ (1-42) in the central nervous system, a prolonged lifespan, increased locomotor activity, and reduced neurodegeneration is observed 1 . stabilization of the central aβ α-helix appears to counteract polymerization into toxic assemblies and indicates that this approach holds promise for the development of orally available compounds against alzheimer's disease. encouraged by the above results we are currently developing a second generation of designed ligands. this involves synthesis of different new peptoids and unnatural amino acids. additional support for the concept comes from recent molecular dynamics simulations that also uncover details of the mechanism of unfolding of the aβ central helix 2 as well as retardation of the folding in presence of ligands designed to interact with the native helical conformation 3 . synthesis and methodology for new ligands, which includes synthesis of novel amino acids, will also be presented. triostin a is a well-known natural product with antibiotic, antiviral, and antitumor activities. it inhibits rna synthesis by bifunctional intercalation into dna base pairs through its two quinoxaline units showing cpg selectivity. triostin a must adopt an altered conformation upon dna bisintercalation that is substantially different than its preferred native x-ray or solution conformation. this fact suggests that the destabilizing conformational change in the cyclic octadepsipeptide counteracts much of the gains derived from a second intercalation. nonetheless, the wide range of pharmacological activities exhibited by this compound prompted interests in identifying novel and additionally potent lead compounds with improved pharmacokinetic properties for clinical applications. herein, a library of twelve simplified triostin a analogues has been synthesized by solid-phase peptide synthesis. the introduction of the key quinoxaline units was carried out in solution. the analogues' conformation corresponds to the staple form that bisintercalator cyclic (depsi)peptides adopt when binding to dna and, in addition, some of the synthesized compounds showed improved solubility. our library was evaluated for its antiproliferative activity against four human cancer cell lines and one analogue showed greater cytotoxicity than triostin a and even comparable activity to doxorubicin, a very commonly used drug in cancer chemotherapy nowadays. surprisingly, little is known about its mechanism of degradation in solution or the degradation products. a recent study identified monomeric polysulfides and dimeric degradation products, postulated to derive from β-elimination followed by deamidation and dimerization. 1 we recently reported that degradation of oxytocin and its analogues in aqueous solution at ph 7.4 produced monomeric polysulfides with up to 6 sulfur atoms as well as dimeric products. 2 unexpectedly, incubation of ot or of various analogues modified in position 1 resulted in identical dimeric degradants. we concluded that β-elimination via breakage of the c-s bond of cys1 must be a key step of the process, and that the resulting δala 1 residue would have to undergo further modifications to yield the same dimeric products independently of the substitution of the n-terminal nitrogen. here we further clarify the degradation mechanism and propose a structure for the dimers. we postulate that hydrolysis of the δala 1 residue yields an n-pyruvoyl linear peptide, which loses one sulfur atom and subsequently forms dimers, which we found are linked by one disulfide bridge and one non-reducible bond. the putative linear n-pyruvoyl oxytocin intermediate was synthesized and found to degrade to the same dimers as the ones in the incubations of ot. a [u-13 c]cys 1 ot analogue gave degradation products with 13 c-nmr spectra consistent with a non-stereospecific aldol-type condensation. detailed experimental procedures, structures of the degradants and the postulated mechanism of ot degradation in near neutral solutions will be presented. inserm u765 paris, france αiibβ3 is the main platelet integrin and is responsible for platelet aggregation. a lipid-modified peptide corresponding to αiib intracellular sequence 1000-1008 (palmitoyl-k-l 1000 eeddeege 1008 , pal-k-1000-1008), is platelet permeable and inhibits human platelet aggregation induced by thrombin 1 . ymesradr, a peptide corresponding to the extra-cellular sequence 313-320 of αiib, is a platelet activation and aggregation inhibitor 2 . the aim of the present study was to investigate the cooperativeness of the intracellular and extra-cellular peptides on platelet aggregation and their effect on the phosphorylation of fak and erk. pal-k-1000-1008 together with the extracellular ymesradr peptide, at concentrations lower than their ic50 values, showed cooperative inhibition of platelet aggregation. the peptide combination inhibited also fibrinogen and pac-1 binding to activated platelets. fak phosphorylation is a postaggregation event related to outside-in activation of the receptor. the combination of peptides inhibited fak phosphorylation. erk phosphorylation is independent from platelet aggregation, and is enhanced by rgd-peptide inhibitors. the combined peptides inhibited erk2 phosphorylation. ovarian cancer (oc) is considered a rare disease and represents the fifth most common cause of death from cancer in women. the standard first-line treatment consists of a combination of paclitaxel and carboplatin (ddp) or carboplatin alone. in the case of progressive disease or drug resistance to platinum-based agents, either alone or in combination, investigational compounds should be used (1) . the mechanisms behind acquired resistance to ddp and its derivatives are not clear yet, although it is evident that the process is multifactorial, including enhanced dna repair processes. some peptides designed from the interface subunit of the human thymidylate synthase (ts) have been identified recently (2), as effective anticancer agents against sensitive and resistant oc cells. one of them was also able to recover the cellular sensitivity towards cisplatin in resistant oc cells in the μm range. to improve its potency and selectivity structural studies have been performed in combination with cellular assays aimed at understanding the mechanism of action. a label-free quantitative proteomic approach has been undertaken to study the effects of the peptide on the proteins involved in the modulated metabolic pathways, in particular those involved in the folate metabolism. structure-activity relationships (sar) have been performed to improve the lead peptide pharmacodynamics. all the compounds have been assayed and a protein profile set was studied to mark and validate their behavior as inhibitor of oc cell growth. hepatitis c is a liver disease provoked by a virus known as hcv. the disease is insidious. hcv causes anorexia, nausea, vomiting, fever, fatigue and jaundice. in about 40% of sufferers the disease is short, but others become chronic. in the chronic form in about 20% of cases the final result is cirrhosis of the liver and in the remaining 20% it leads to liver cancer. hcv is a very serious problem today. about 3% of people infected with hcv worldwide, i.e. about 4 million are residents of europe. 170 million people carry the disease as a chronic illness with the potential to develop into cancer in their liver. all these people represent a "reservoir" for storage and distribution of hcv. ribavirin, the nucleoside analog 1-β-d-ribofuranosyl-1,2,4triazole-3-carboxamide, known by the trade name virazole (also known as rebetron in combination with interferon-α), exhibits antiviral activity against a variety of rna viruses (paramyxoviruses, flaviviruses, etc.) as well as some dna viruses. in humans ribavirin is currently used in combination with interferon-α to treat hcv infections. this lack of strict specificity and a broad spectrum of activity are due to its multifunctional mechanism of action against viruses. these characteristics have made ribavirin a drug of substantial research interest. unfortunately, ribavirin shows a significant toxicity, causing bleeding in accumulation [1] . herein, we report a total synthesis of modified in position 5' of ribose residue, ribavirin in order to be further linked to cell penetrating peptides (cpps). in addition the synthesis of some cpps as well as bonding between two parts of final hybrid molecules will be reported. in our case the design of new hybrid molecules is done in order to: (a) vectorize ribavirin into liver cells; (b) transport ribavirin molecule trough cell membrane and (c) decrease toxicity of ribavirin. to obtain oligomeric aβ peptide, our laboratory uses a precursor depsipeptide of aβ. this precursor, termed as "iso-aβ" has an ester bond between the side chain of ser 26 and gly 25 . at physiological ph this ester bond becomes an amide bond via an o→n acyl shift. binding partners by which the oligomeric aβ mediates its toxic effect has not been yet investigated in the proteome or subproteome level. we used protein array technology to study the interaction of oligomeric aβ with 8163 recombinant human proteins, immobilized on a protein chip. aβ binding proteins were identified with the aid of a monoclonal aβ antibody. altogether 324 proteins were found to interact with our aβ-oligomers. these proteins were grouped according to their function. one of the major groups contained 24 proteins participating in translation. these proteins were found in ribosomes. to prove our proteomic results ribosomes from rat hippocampus were isolated. elisa experiment revealed that aβ binds to ribosomes in a dose-dependent manner. using the sequence of the 324 aβ-binding proteins a homology search was performed to find oligopeptides, that possibly bind to aβ. based on these sequences a peptide chip containing hexapeptides was prepared. aβ interacting peptides were identified with a monoclonal antibody. several peptides were synthesized and tested on mtt assay. two out of four compounds inhibited the toxicity of aβ on rat hippocampal slices. summarizing our results aβ binding proteins and peptides were identified. knowledge about aβ binding proteins can help to understand the pathogenesis of ad, such us the possible involvement of ribosomes. oligopeptides can be lead compounds of future drug development. huge proteolytic complex named proteasome catalyzes protein degradation in every eukaryotic cell. it consists of 31 subunits forming four stacked rings and one or two regulatory caps. two inner rings of the proteolytic part contain three catalytic β-subunits that possess different substrate specificity. higher vertebrates can express γinterferon-inducible immuno-β-subunits. proteasome plays an essential role in continual turnover of intracellular proteins and in antigen processing. autoimmune diseases such as multiple sclerosis and its murine model eae are believed to rise from breakdown of tolerance of the immune system. it assumed that immunoproteasome could play an important role in autoimmune diseases. several classes of chemicals proved to be inhibitors of proteasome and the most active are boronate peptide derivatives. these inhibitors totally inactivate proteasome and result in full stop of intracellular protein turnover and cell death via apoptosis. another class of inhibitors, epoxy ketones, was shown to be more selective for immunoproteasome and could be used not for full stop of proteasome function, but for fine tuning of altered proteasome functioning. we examined properties of several inhibitors of four different classes, namely peptide boronate bortezomib, peptide aldehyde mg132, lactam lactacystin, and peptide epoxyketones epoxomicin, mg132ek, uk101 and pr-957. for inhibition experiments we used proteasome isolated from eukaryotic cell lines cho, nso and hek, treated and non-treated with γ-interferon, as a model cells contatinig constitutive and immunoproteasome. the upregulation of proteasome immunosubunits was revealed in cho and nso cells treated with γ-interferon. the ic50 values for all studied inhibitors were obtained, and ki in some cases were calculated. the epoxyketones were shown to selectively inhibit in submicromolar concentrations the proteasome sample which contain high amount of immunosubunits. in order to find an effective antimalarial, this study refers to some angiotensin ii (aii) analogues which were considered the important physicochemical characteristics described by silva et al. 1 to verify the biological activity against plasmodium gallinaceum and to understand the hydrophobic cluster influence, explained by tzakos et al. 2 these analogues were synthesized and characterized as described by silva 1 , as well as the biological assays and comprises, to verify: the hydrophobic cluster activity -a) drvyhipf; b) drvypr; c) ryhipf and d) fphiyvrd; the importance of these residues in aii molecule -e) rypf; the importance of aromatic residues -f) yhpf and the action of these hydrophobic residues, when interacting with the parasite membrane -g) vipf. it was observed that in a (94% of bioactivity), the phenol group of tyr is close to imidazole group of his that could promote a hydrogen bond formation. besides that, could occur van der waals interactions between ile and phe residues due its proximity and non-polar characteristic. these interactions could not be effective in native aii (88%) 3 , because ile residue promote a steric influence on the organization of his and tyr residues 4 that not exist in b (57%). in c (74%) and e (72%) analogues, the influence of the arg residue could promote a cation-π interaction with tyr residue 5 and the cluster may have suffered slight destabilization and its antiplasmodial activity was compromised subtly. in d (12%), the electrostatic change, obtained with the total inversion can have disordered its interaction with parasite membrane, since it is not related to membrane receptors, because d-aii presented 91% of biological activity. moreover, hydrophobic and aromatic residues importance was confirmed through the results obtained 93% and 89% of activity, with g and f, respectively. we conclude that hydrophobic cluster modifications and interactions of amino acid side-chain influences in the biological activity. closed joint-stock company "vertex", st-petersburg, russia creatine (cr), a small molecule synthesized in the kidney, liver and pancreas plays important role in atp synthesis, replenishing its store even in the absence of oxygen. cr is able to protect brain cells against ischemic damage; however it has poor ability to penetrate the blood-brain barrier without specific carrier protein. thus, synthesis of stable hydrophobic derivatives capable of crossing the bbb by alternative pathway is of great importance for the treatment of different neurological diseases including stroke, traumatic brain injury and hereditary crt deficiency. here we describe the synthesis and biological activity of new hybrid compounds -creatinyl amino acids. originally the title compounds were synthesized by guanidinylation of sarcosyl peptides. however, for large scale synthesis better results can be obtained using direct cr conjugation with amino acid or peptide derivatives by isobutyl chloroformate method. addition of equivalent amount of ptoluenesulfonic acid as lipophilic counterion ensures efficient cr dissolution in dmf along with its simultaneous protection towards intramolecular cyclization. it excludes the application of expensive guanidinylating reagents and permits to simplify the synthetic procedure. purification of final product and its conversion into appropriate salt form can be achieved by iec followed by crystallization from organic solvents. synthesized creatinyl amino acids and peptides exhibited significant biological activity in different assays including platelet aggregation test, ischemic stroke and nano2-induced hypoxia model. one of the most effective compounds -creatinyl-glycine ethyl ester increases life span of experimental animals more than two times in hypoxia model and has neuroprotective action in brain stroke model when applied both before and after ischemia. these data evidenced that creatinyl amino acids can represent promising candidates for the development of new drugs useful in stroke treatment. the efficient recognition and destruction of tumor cells via specific cellular markers is a major goal in cancer therapy. various growth factor receptors such as egfr, hgfr, vgfr and their downstream signaling networks have been proven to be effective molecular targets, as they are frequently involved in cancer proliferation and metastasis. downregulation of these receptors and/or blocking their signaling pathways have clear anti-tumoral effects. 1 drugs based on monoclonal antibodies (mab) targeting such cell surface receptors have attracted a lot of attention as a new generation of therapeutics. however, their production is costly and identifying new, variable routes to modified molecules with similar properties is currently a major focus. 2, 3 here we present an approach to chemically synthesize a molecule that combines the mode of action of antibodies with the advantages of smaller, chemically accessible molecules. these "synthetic antibody" (sab) molecules contain a chemoattractant that activates the innate immune response and resembles the fc domain of a typical antibody. specificity is imparted by two binder peptides that assume the function of the variable antibody domains and bind to a cell surface target. the fc and fab domains of the sab molecules are connected via polyethylene glycol linkers. sab molecules are prepared by solid phase synthesis, a flexible technique that allows fast production, full control of their properties and targeting two different cell surface receptors (bispecific tumor targeting). they are currently tested in vitro and in vivo for their effect on the innate immune system, general toxicity and selective binding to cancer cells. the key enzyme in the processing of polyproteins translated by viral rna genome of sars-cov is a 33kda protease called 3c-like protease (3cl protease). sars 3cl protease is a cysteine protease containing a cys-his catalytic dyad, and cleaves precursor poly proteins at as many as 11 conserved site involved a conserved gln at the p1 position and a small amino acid (ser, ala, or gly) at the p'1 position. due to its functional importance in the viral life cycle, sars 3cl protease is considered to be an attractive target for drug design against sars. recently, we found tetrapeptide aldehyde, ac-thr-val-cha-his-h, showed high inhibitory activity with ic50 value of 98 nm toward 3cl-r188i mutant protease 1,2 . to compare the inhibitory activity of small compounds with those containing active functional groups, we synthesized serine-derivatives within the essential functional groups and evaluated its inhibitory activity. the synthetic scheme was started from fmoc-ser(tbu)-oh, following modification of c-terminal carboxyl group with p2, n-terminal amine with p4 and side chain alcohol with p1 functionalities. 5 steps overall reaction led to obtain 44 novel serine derivatives for the small molecular inhibitors of sars 3cl protease. the assay with 3cl r188i mutant protease was examined to evaluate the inhibitory activity of the synthetic serine derivatives. then, molecular docking study of complex of 3cl protease with the ligand was carried out. docking simulation experiment with r188i (pdb id: 3aw0) and the inhibitor, which has the best activity in the serine derivatives, indicated that p1 fitting s1' pocket. at the result of assay, p1, p2 and p4 positions of the inhibitor should be modified by benzoyl group, cyclohexyl group and cinnamoyl group, respectively. their bioactivities are underpinned by their distinctive structure with exceptional stability, thus making cyclotides exciting, not only for agricultural and pharmaceutical purposes, but also as a template in drug design. in all of the reported activities, cell membranes seem to be the primary target for cyclotide activity. to unravel the importance of lipid membranes on the reported activities of cyclotides, a set of cyclotides belonging to möbius and bracelet subfamilies were compared in their mode of action. the lipid selectivity and membrane affinity were compared with their efficiency against different target cells (e.g. red blood cells, bacteria, hiv particles). we have found that the bioactivity of cyclotides is dependent on the lipid composition of the target cell membrane and independent of a protein chiral receptor. in particular, all the native cyclotides tested target the cell membrane through specific binding to phospholipids containing phosphatidylethanolamine (pe)headgroups, but the membrane binding affinity is further modulated thorough non-specific peptide-lipid hydrophobic interactions, which are dependent on the specific cyclotide. in addition, the bioefficiency of cyclotides broadly correlate with their ability to target and disrupt the cell membrane. overall, we have shown that even with a common specificity for membranes containing pe-phospholipids, a fine selection was found across the family. in particular, each cyclotide inserts and disturbs the membrane in a distinct way, which explains the diversity of this family but also their distinct activities. the observation that all the tested cyclotides have a preference for a specific lipid makes this family truly intriguing and brings insights to optimize the use of the cyclotide template in drug design. malaria is a disease that affects around 500 million people causing 0.5-1 million of deaths annually. based on our previous studies, angiotensin ii (aii) presented antiplasmodial activity against plasmodium gallinaceum, but due to pressure activity, it cannot be used as an antimalarial drug. in an attempt to increase antiplasmodial activity and reduce hypertensive activity, we synthesized by solid phase method, cyclic analogues of aii with i-(i+2) and i-(i+3) lactam bridge scaffold 1 using asp and lys residues. the bridge was more effective when inserted next to n-terminal extremity 1 , probably this insertion, on another portion of the peptide, provides a change in the conformation of the molecule and its hydrophobic cluster formed by tyr, ile and his 2 , which may have influence in the peptide-membrane interaction. thus, we have focused in the n-terminal extremity, testing new analogues, using glu/asp/orn/lys residues as bridgeheads components in i-(i+4) lactam bridge scaffolds, which showed that antiplasmodial activity is increased using glu residue and that larger lactam rings are better to biologically active. therefore, new restrict peptides by i-(i+2) and i-(i+3) lactam bridge were designed, using glu residue as bridgehead element, but the same effect was not verified, getting a maximum of 65% of bioactivity. on the other hand, we promoted an increase in the hydrophobic character of the molecule, replacing the asp residue of aii sequence by fmoc-glu and asp(ofm), in order to improve the interaction of these compounds in the sporozoite membrane. the replacement by fmoc-glu provided a decrease of activity, while that asp(ofm) kept the aii activity, because there are changes of charge in the peptide, which may have modified the conformation in physiological medium. this kind of approach may offer the basis for development of new drugs and chemotherapy against malaria. animal venoms are complex chemical cocktails, comprising a wide range of biologically active reticulated peptides that target with high selectivity and efficacy a variety of membrane receptors such as ion channels or g-protein coupled receptors. venoms can therefore be seen as large natural libraries of biologically active molecules that are continuously selected and highly refined by the evolution process. the vision associated with the venomics project is to investigate in depth the enormous structural and pharmacological diversity of venom peptides through the development, integration and implementation of a novel research paradigm combining cutting-edge "omics" technologies in a high-throughput workflow. this new paradigm enclosed in venomics aims at replicating in vitro the diversity of venoms to generate original peptide banks to be used in drug discovery programs. herein, we show the different strategies we adopted for efficient solid phase synthesis and folding with an easy purification of peptides rich in cysteine and containing posttranslational modifications (ptm). angiogenesis depends on the adhesive interactions of vascular cells. the adhesion receptor integrin av b3 was identified as a marker of angiogenic vascular tissue. the αν β3 integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis, mainly by interacting with matrix proteins through recognition of an arg-gly-asp (rgd) motif. inhibition of the αν β3 integrins with a cyclic rgd peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. the aim of this study was to investigate the effects of replacement of a-amino acids by aza-β 3 -amino acid analogs in cyclic rgd-peptides as αν β3 -integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. the selectivity profile of these antiadhesive cyclopeptide is rationalized by a special presentation of the pharmacophoric groups. we synthesized cyclic rgd peptidomimetics that include aza-β 3 -amino acid residues. modifications were added to the rgd skeleton in order to optimize the peptide activity. then, we investigated the pharmacokinetics activity of these pseudopeptides in hek (human embryonic kidney 293) and endothelails cells huvec (human umbilical vein endothelial cells) cell by analyzing cell viability and protein involved in the angiogneisis processes. since tenascin c is a factor expressed highly in the tumorassociated matrix, targeting it would be a desirable first step for targeting the tumor-specific microenvironment in fact, a high level of tenascin c expression has been reported in most solid tumors, including lung cancer, colon cancer and glioblastoma. therefore, the targeted binding of tenascin c in tumor stroma would inhibit tumor metastasis by modulating cancer cell growth and migration. we isolated a peptide that bound to tenascin c by phage display peptide library selection, and the selected peptide specifically recognized tenascin c protein in xenograft mouse tissue. we also observed exclusive staining of tenascin c by the selected peptide in tumor patient tissues. moreover, the peptide reduced tenascin c-induced cell rounding and migration. we propose that the tenascin c targeting peptide may be useful as a specific anti-cancer diagnostic and therapeutic tool for most human solid tumors. radiolabeled pansomatostatins are expected to enhance hsst1-5 tumor-uptake and to broaden clinical indications as compared to currently established sst 2-prefering radioligands. previous experience has revealed [111in-dota 0 ,dtrp 8 ]ss-14 ([ 111 in]at2s) as a true pansomatostatin analog, exhibiting however poor in vivo stability. in order to enhance metabolic stability, we introduced a second disulfide bridge to the at2s motif by formation of extra 6/12-amino acid (aa) or 8/12-aa ring generating at5s and at6s, respectively. the orthogonally protected sequences were assembled on the solid support, deprotected and cleaved from the resin with tfa. the first cys 6 -cys 11 (at5s) or cys 5 -cys 12 (at6s) cyclization was performed in dmso, while the second was completed with iodine oxidation after in situ deprotection of cys 3 (acm) and cys 14 (acm). during hsst 1-5+-autoradiography, at5s showed unexpected total loss of sst 1-5 affinity, whereas at6s showed high affinity (ic 50 in nm) to all hsst1-5 (hsst1= 11.5±3.3; hsst2= 6.3±0.6; hsst3= 9.7±3.6; hsst4= 5.4±0.8; and hsst5= 25.7±7.0). consistent with this finding, only at6s stimulated sst2 internalization during immunofluorescence-based internalization assays, showing agonistic properties for sst2. furthermore, [111in]at6s internalized rapidly and specifically in sst2+ ar4-2j and hek293-hsst3+-cells. hplc analysis of 5 min ex-vivo mouse blood samples revealed that >98% [111in]at6s remained intact. after injection in scid mice bearing ar4-2j and hek293-hsst3+ tumors [111in]at6s specifically localized in the rsst2a+ (1.9±0.2%id/g vs. 0.8±0.05%id/g + 100 nmol tate at 4 h postinjection (pi)) and in hsst3+ implants (3.7±0.4%id/g vs. 0.3±0.05%id/g + 80 nmol ke108 at 4 h pi). this study has shown that introduction of an extra disulphide bridge in at2s confers high metabolic stability. however, in a 6/12member ring combination it leads to total loss of affinity. the reasons for this effect are currently investigated by nmr conformational studies. transporter compounds are useful tools to solubilise and increase the delivery of therapeutic molecules in the human body. one system to improve the cellular uptake of such therapeutic molecules are cell-penetrating peptides (cpps). these short peptide chains are either polycationic (containing several arg and lys) or show a more amphiphatic structure. 1 it is known that the multivalency effect -the presentation of several copies of a cpp motif on a single molecule -can increase the cellular uptake. 2 peptide dendrimers represent a group of tree-like, multivalent macromolecules, which are synthesized for different chemical and biological applications in our group. 3 we now combine linear cpps with peptide dendrimers to get a well defined branched molecule made up of only natural amino acids. in our systematic study of peptide dendrimers decorated with different cpps we found that the potency of the single cpp as a transporter for small molecules can be increased and that these peptides show usually low cytotoxicity. additionally we designed new dendritic cell penetrating peptides with similar activities like linear cpps. all compounds are covalently linked to fluorescein for visualization with flow cytometry and confocal analysis. the results show that the peptides can transport efficiently a hydrophobic cargo into the cells. chemical stability of esters of acyclovir with amino acid and cholic acids k. chuchkov, r. chayov, i. g. stankova* south-west university "neofit rilski", blagoevgrad, bulgaria amino acid esters of antiviral drugs are a very good solution for improving oral bioavailability of the actual medicine. one of the most effective and tolerant prodrugs is valine ester of acyclovir -valaciclovir. taken orally exhibits three to four times higher bioavailability of acyclovir. the chemical stability of amino acids (4-fphenylalanine) (r,s) and bile acids (deoxycholic acid and chenodeoxycholic acid) esters of acyclovir was studied in experimental conditions simulating some relevant biological medias (ph 1.0 and 7.4, 37°c).the chemical stability experiments revealed that the examined amino acid ester of acyclovir were relatively unstable in acidic ph, but bile acid ester is stable in the same ph. the examined amino acid and bile acid esters of acyclovir in neutral ph are relatively stable. in ph 7,4 all of tested compounds are more stable than valacyclovir (t1/2 = 13 h) -the first effective prodrug of acyclovir. in acidic ph acyclovirdeoxycholat and acyclovirchenodeoxycholat are more stable than valacyclovir. acyclovirchenodeoxycholat is the most promising anti-ebv prodrug candidate with high activity and satisfying chemical stability. cell-penetrating peptides (cpp) have become efficient tools for the cellular internalization of bioactive molecules due to their ability to cross the plasma membrane of diverse cells and cell lines. [1] we recently reported that the cpp sc18, which consists of the residues 106-121 of the c-terminal region of the cationic antimicrobial peptide cathelicidin (cap18), is an effective carrier peptide for small organic molecules like fluorophors and toxic peptide sequences into various cell lines [2] . however, in general linear peptides are more susceptible to proteolytic degradation than their cyclic analogs [3] . therefore, we investigated the cyclization of cpp derived from sc18 by means of cui-mediated azidealkyne cycloaddition (cuaac) [4] . furthermore, we examined their conformation and proteolytic stability as well as their internalization efficiency and toxicity against various cell lines, in comparison to their linear equivalent and to other cpp. looking for the proper prodrug: a peptidomimetic approach to identify and inactivate bacterial mono-adp-ribosyltransferase toxins m. beich-frandsen, r. jørgensen division of microbiology and diagnostics, statens serum institute, copenhagen, denmark mono-adp-ribosylation is an endogenous posttranslational modification in eukaryotic cells, simultaneously utilized as virulence strategy by deadly secreted bacterial toxins. many bacterial toxins have been found to act as mono-adp-ribosylating enzymes, targeting anything from g-proteins to the actin skeleton. the diphtheria toxin from c. diphtheriae and exotoxin a from p. aeruginosa, both target the diphthamide-group of a unique modified histidine in eef2, inhibiting protein synthesis by ribosome mimicry 1,2 . we aim to inactivate these nad+-utilizing toxin enzymes by nad-conjugated peptidomimetics, in a target-specific prodrug-approach. the adp-ribosylation reaction follows a random third-order s n 1 mechanism. in the proposed model for the transition state of the reaction, the cleavage of the n c1-nn1 bond of nad + releases strain and generates a oxacarbenium ion intermediate with a positively charged nicotinamide (n)ribose, subject to a nucleophilic attack from the substrate 3,4,5 . adp-ribosylating toxins are commonly characterized by a artt-motif involved in substrate recognition 2 . studies suggests conformational rearrangement of the residues surrounding the substrate binding site to be required for optimal geometry 5 of the initial glycosidic nc1-nn1 bond cleavage within nad + . subtype specific nad-conjugated peptides, designed based on previous structural analysis of the adpribosylation reaction, act as substrate for the enzymatic adp-ribosyl-transfer, and hereby attach covalently to and inactivate the nad + -utilizing toxin. relying on previous structural studies, and established ligand-binding and kinetic data, an initial peptide library, designed by bioinformatics and evaluated for specificity of common targets in-silico identifies initial leads. lead-scaffolds are implemented in rational peptide-design, based on high-resolution structural-and biophysical studies of multiple peptide-enzyme complexes, to identify possible prodrug-strategies for enzyme inactivation. nanoparticles play a crucial role in medicine for their potential application as in vivo carriers of active principles [1] . liposome display unique pharmacokinetic properties slowly releasing drugs loaded in the inner aqueous cavity. in the last years we have developed supramolecular aggregates labeled by bioactive peptides able to recognize overexpressed receptors on tumour cells membrane delivering doxorubicin chemiotherapeutic drug [2] . neurotensin(nt), a 13 amino acid peptide, has dual functions of neurotransmitter or neuromodulator. the cterminus short fragment 8-13 preserve the activity but the half life of wild type form in vivo is very short. nt receptor type 1 (nts1) is overexpressed in severe malignancies such as small cell lung cancer and colon, pancreatic, and prostate carcinomas. we have designed new amphiphilic molecules containing in the hydrophobic moiety two aliphatic chains and in the hydrophilic moiety a the bioactive portion able to aggregates with phospholipid molecules achieving liposome. we have synthesized neurotensin wild type sequence, the truncated form and the tetra-branched neurotensin(nt1-13) or a truncated form(nt8-13) tetrabranched peptides(nt4) adopting an opportune synthetic strategy on solid phase. all liposome were formulated adding the neurotensin amphiphilic monomer in ratio 5:95 with dopc in order to evaluate the capability to recognize selectively receptors overexpress on cell membrane surface. the liposomes size was determined by dynamic light scattering measurements, values for the hydrodynamic radius(rh). the selective internalization and cytotoxicity of fully doxorubicin loaded liposomes as compared to pure dopc liposomes, was tested in ht29 human colon adenocarcinoma and te671 human rhabdomyosarcoma cells. recently, small interfering rna (sirna), one kind of rna interference (rnai) technology represent the most common and, to date, the most effective method to inhibit target gene expression in human cells. it is also a common recognition that non-toxic delivery of sirna is an urgent problem for the therapeutic application of sirna. for the efficient gene silencing in vivo, prolonged circulation of sirna with take efficient and non-toxic cellular uptake and resistance against enzymatic degradation are indispensably required. 1 ) telomerase activity has been regarded as a critical step in cellular immortalization and carcinogenesis and because of this, regulation of telomerase represents an attractive target for anti-tumor specific therapeutics. in this paper, we present the efficient and non-toxic cellular uptake of sirna using novel amphiphilic peptides and the application to silencing of htert in human cancer cell lines. in the present study, we investigated the intracellular delivery of sirna using some amphiphilic peptides and the silencing effect of sirna targeting htert mrna in 3 human cancer cell lines, jurkat, hela and k562. the complex of sirna and a specific amphiphilic peptide or its hybrid with an intracellular transport signal peptide could be effectively taken up into cells. the complex also showed a high silencing effect against htert mrna. moreover, the combination of sirna-nes conjugates and the amphiphilic peptides improved silencing effects up to 95.2 %. the amphiphilic peptides and their hybrids showed almost no cyto-toxicity and protected sirna against intracellular nuclease digestion in 10% fbs (half life time was over 48h). tumor targeting with the decapeptide gonadotropinreleasing hormone (gnrh) or its analogues is based on the discovery that gnrh receptors are overexpressed in many tumor cells, compared with their expression in normal tissues. using these peptides as carriers/targeting moieties in a conjugate with therapeutic agents can increase the selectivity and the stability of the conjugates, or eliminate the toxic side effects of the drug. gnrh-iii (<ehwshdwkpg-nh 2) is especially favoured as a targeting moiety because of its antiproliferative effect and weak endocrine activity in mammals. the aim of our work was to synthesize novel short-chain fatty acid (scfa) acylated daunorubicin-gnrh-iii bioconjugates with enhanced enzymatic stability, cellular uptake, in vitro and in vivo antitumor activity. ser in position 4 of gnrh-iii was replaced by lys and its ε-amino group was acylated with various fatty acids. gnrh-iii( 4 lys(x), 8 in conclusion, glu 6-10 /dglu 6-10 substitution in gi-derived radiopeptides enhanced stability and reduced renal values by ~2 .5 fold. on the other hand, removal of the (glu)5sequence in mg11 suppressed renal uptake but undermined stability. both modifications, however, compromised tumor targeting, demonstrating that more efficacious structural changes in the gi motif are warranted to improve the in vivo profile of resulting radioligands. synthesis and characterization of a 99mtc-labeled rhodamine-conjugated angiotensin peptide as a potential cardiac function imaging agent s.m. okarvi king faisal specialist hospital & research centre, cyclotron and radiopharmaceuticals department, riyadh 11211, saudi arabia angiotensin ii (ang ii) is an 8-amino acid peptide that has been known to play a vital role in cardiovascular system. the actions of ang ii have been implicated in many cardiovascular conditions, such as coronary heart disease and heart failure. in an effort to develop a cardiac imaging agent with enhanced targeting capacity, we attached ang ii to rhodamine (rh), a lipophilic cation that specifically accumulates in the myocardium. the rh conjugated ang ii was evaluated for its potential as a heart imaging agent. rh-lys-gly-cys-asp-arg-val-tyr-ile-his-pro-phe-conh2 was prepared by solid-phase peptide synthesis according to fmoc/hbtu methodology. nhs-rh was attached to the ang ii peptide via the free amino group of lys residue by manual synthesis. rh-ang ii conjugate was labeled with tc-99m by the stannous-tartrate exchange method. in vitro stability was determined in human plasma and in excess of cysteine. in vivo biodistribution and biokinetics were performed in balb/c mice and sprague dawley rats. the rh-ang ii conjugate radiolabeled efficiently with tc-99m (>75% labeling efficiency) as determined by hplc analysis. tc-99m-rh-ang ii exhibited good chemical stability against cysteine transchelation and sufficient metabolic stability in human plasma. in mice, the bioconjugate displayed efficient clearance from the blood and excreted mainly through the renal route with some excretion by the hepatobiliary pathway. the uptake in the heart was 1.8±0.5% id/g as early as 30 min post-injection; whereas, the uptake in the lungs, liver, stomach and kidneys varied between 1-10% id/g. in rats, the bioconjugate displayed relatively better pharmacokinetic characteristics, with low uptake in the major organs (<4% id/g). the uptake in the heart (1.7±0.4% id/g) was found to be higher than the uptake in the blood and muscle, resulting in good heart-to-blood and heart-to-muscle uptake ratios. this initial study towards the development of an effective cardiac imaging agent advocates that the use of hybrid conjugates appears to hold a great promise as a new and attractive approach for rapid and efficient imaging of heart. in humans two isoforms of gnrh are exist, gnrh-i (<ehwsyglrpg-nh 2, where <e is pyroglutamic acid) and gnrh-ii (<ehwshgwypg-nh2). in contrast to gnrh-i, exact function of gnrh-ii is not well identified. however, it was indicated that gnrh-ii derivatives have more potent antiproliferative activity on human endometrial and ovarian cancer cells in vitro than gnrh-i analogs. some kinds of tumor cells (e.g. breast, prostate, colon) produce gnrh-i and gnrh-ii, and express their receptors. however, the presence of functionally active gnrh-ii receptors on cancer cells is still not clear 1-2 . on the other hand, gnrh-ii analogs, both agonists and antagonists, induce apoptosis in many different cell types. in contrast to gnrh-i agonists, gnrh-ii agonist [d-lys 6 ]gnrh-ii does not activate nfκb, therefore does not protect the cancer cells from apoptosis [3] [4] [5] . in the regulation of apoptosis, not only the pro-and antiapoptotic proteins play a role 6 , but other molecules also attend (e.g. ck2, casein kinase ii enzyme) 7 . our aim was to synthesize new proapoptotic peptide or enzyme inhibitor containing [d-lys 6 ]gnrh-ii based derivatives and investigate their in vitro cytotoxic, cytostatic and apoptotic effects. the sythesis of [d-lys 6 ]gnrh-ii, which is one of the most potent gnrh-ii agonist, was carried out on solid phase using fmoc/tbu strategy. different proapoptotic peptides or enzyme inhibitors were attached directly to the gnrh-ii derivative. in vitro cytostatic and cytotoxic effects of gnrh-ii conjugates were investigated by mtt-assay using different human breast (mcf-7 and t47-d) and colon carcinoma (ht-29) cell cultures. early apoptotic effect of gnrh-ii conjugates was studied by flow cytometry on mcf-7, t-47d and ht-29 cell lines. according to our results, these conjugates might be potential candidates for cancer treatment, because the combination of a targeting moiety (gnrh derivative) with proapoptotic peptides or enzyme inhibitors and drug molecules might have synergistic activity resulting in highly potent multifunctional therapeutic agents. department of pharmaceutical and pharmacological sciences, university of padua, padua, italy melanoma is the most deadly skin cancer with an increasing incidence. its therapy continues to be a challenge since, regardless of the treatment used, longterm survival is quite uncommon. melanoma cells are characterized by high levels of glutathione s-transferase p1-1 (gstp1-1) isozyme. this enzyme is also highly expressed in solid tumours and drugresistant cells, where a role for this protein in the regulation of cell proliferation has been described. 1 in the past, lyttle et al. synthesised glutathione (gsh)linked anticancer prodrugs activated by physiological concentration of gsts. 2 structure and mechanistic studies showed that in the gst-gsh complex, the gst tyr residue placed near the s atom of the cys in gsh, is in the phenoxide form to facilitate the deprotonation of the sulfhydryl group in gsh, making it able to react with electrophilic species. 3 by taking advantage of this active-site geometry, we synthesised two tyrosinase activated melanoma prodrugs (4-methoxyphenol and n-acetyl-4-s-cysteaminylphenol) linked to gsh. in these compounds an ethoxycarbonyl group was placed between the gsh sulphur, oxidate to sulfone, and the tyrosinase-activated prodrugs. in this way the tyr phenoxide (tyr7 in gstp and tyr6 in gstm isozyme) would be able to abstract one of the acidic methylene protons linked to the sulfone moiety. such a deprotonation should trigger a beta-elimination, resulting in the release of melanoma prodrugs. the stability of the obtained putative prodrugs has been evaluated in different ph buffers and in rat liver and kidney cytosolic fractions. synthetic peptides represent first choice biomolecules, with respect to proteins and mab, for the monitoring of population variability and receptor functionality associated to a tumor pathology due to their favorable pharmacokinetic profile. it has been demonstrated that the surface molecule integrin alfa-v beta-3 could be an ideal target for the interaction with melanoma directed radiolabeled peptides. this integrin is indeed overexpressed both in tumor cells and in tumor vessels endothelium, while it is not expressed in the majority of normal tissues. cyclic peptides with the sequence arg-gly-asp (rgd), are known to bind the integrin alpha-v beta-3 with high affinity and selectivity and could conveniently target a radiotherapeutic to melanoma cells. 1 we previously demonstrated that the cyclic rgdfk peptide interact with high affinity to a375 human melanoma cells. 2 the aim of this work was to conjugate the 99mtc to the peptide by using the [ 99m tc(n)(pnp)] 2+ system (pnp=aminodiphosphines) to obtain a radiolabeled peptide useful in melanoma imaging. 3, 4 spectrum. a nh(i)→βch(i-1) cross peak more intense than the nh(i)→βch(i) cross peak is observed when the peptide adopts the 2.05-helixl conformation. by contrast, an opposite trend of intensities of the same noe cross peaks indicates the occurrence of a 310-helical conformation. by using this method, we were also able to determine that polar solvents, such as mecn or meoh, may induce a 310helical structure in deg homo-peptides which otherwise adopt the 2.05-helix conformation in cdcl3. this finding allows us to rapidly switch these peptide series from "short" (310-helix) to "long" (2.05-helix) by changing the solvent only, thus making them extremely attractive tools. school of biological sciences, university of auckland, auckland 1142, new zealand naturally occurring anti-freeze proteins (afps) enable organisms like polar fish to survive the freezing temperatures of their natural habitat. 1 as well as being cryoprotective, afps exhibit properties of ice recrystallization inhibition (ri). 2 the ability of afps to influence the size, morphology and aggregation of ice crystals can be used in food technology, where the growth of ice crystals in frozen foods is of primary concern. 3 chemical synthesis of afps enables access to pure material for structural and functional studies. our new research programme in the 'food and health' area, prompted us to develop tailor made anti-freeze peptides for potential applications in the frozen food industry. 'multiple salt-bridge analogues' of the naturally occurring winter flounder afp were synthesized and evaluated for their antifreeze activity in comparison to the natural sequence. details on the molecular modelling, structural characterization and anti-freeze activity analysis on the synthetic afps will be presented. several disorders are now believed to be conformational diseases caused by misfolding in the structure of specific proteins leading to the gradual aggregation and deposition of these abnormal proteins. attempts to develop effective therapies for these proteopathies (such as alzheimer's, parkinson's and huntington's disease) have been directed toward reducing the production of the proteins, inhibiting their aggregation, or promoting their removal. conformation-dependent antibodies that specifically target misfolded proteins are proving to be useful tools to study the pathogenesis and as possible treatments of these diseases. we have previously shown that active immunization with tetrapalmitoylated aβ1-15 peptide embedded in liposomes (aci-24) was capable of eliciting a conformation-specific immune response in mice. 1 the antigenic peptide was shown to mimic the aggregated β-sheet conformation of the pathological a β42 peptide on the surface of the liposomes by cd, ssnmr and tht assay. we have also reported the modulation of the conformation of aβ 1-15 derived sequences by means of different lipidation patterns, lipid chain lengths and liposome surface charge. here we present the characterization of this panel of liposomal peptide constructs by atr-ir and correlate the results with previous studies by cd. in summary, atr-ir secondary structure analysis of a battery of amyloid-derived lipopeptides allowed to study the key parameters responsible for the adopted conformation on the lipid bilayer. the presented strategy to control the conformation of liposomal peptide immunogens could be applied to the rational design of vaccines against a range of protein misfolding diseases, such as alzheimer's disease. cyclic lipodepsipeptides (clps) are secondary metabolites produced by various bacteria, most prominently bacillus and pseudomonas. these often display potential antimicrobial properties, frequently ascribed to the ability to disrupt cellular membranes or form ion-pores. clps are a structurally diverse group of molecules always consisting of an oligopeptide chain cyclized by a lacton bond and a fatty acid moiety. they are divided into several groups based on sequence similarity. one such group is the viscosin group, which contains clps produced by pseudomonas that all feature nine amino acids and a conserved sequence pattern of hydrophobic and hydrophilic residues. 1 recently, we have extensively described the self-assembly properties of one viscosin group member, pseudodesmin a. 2, 3 we have shown that in non-polar organic solvents this clp forms anisotropic supramolecular structures of unrestricted size, reminiscent of the ability to form ionpores in the non-polar cellular membrane environment. here, we report on how the most prominent structural variations within the viscosin group affect the conformation and self-assembly properties. since the well-defined monomer conformation of pseudodesmin a is key to this self-assembly, the most striking variation within the viscosin group is the variability of the leu5 stereochemistry. for the first time, a conformational study will be reported for clps featuring an l-leu5 residue, such as viscosin and viscosinamide. the results of this study contribute to a deeper understanding of the structure-function relationship of clps in general. effective design of peptide mimicking protein helical segments is a great challenge. an intriguing approach to improve the helical content in a peptide sequence is achieved through the "peptide stapling" strategy using a hydrocarbon cross-linker (staple) 1 . in this study we have investigated the effect of the introduction and location of staples on secondary structure of sequences derived from a reported peptide, mimicking cullin3 binding region to kctd11 2,3 , to gain an insight into the designing of new molecular entities able to modulate the kctd11 oncosuppressor activity. indeed, kctd11 plays a crucial role in the ubiquitination of hdac1 by acting, in complex with cullin3, as an e3 ubiquitin ligase, thereby affecting hedgehog activity and medulloblastoma growth. in the designed peptides the stapling has been achieved by ring-closing olefin metathesis between two s-2-(4'pentenyl) alanine residues incorporated at i and i+4 positions and into different sites within the peptide chains. the stapling position has been chosen on the basis of cullin3-kctd11 complex model. circular dichroism studies have highlighted that the stapled peptides exhibit remarkable and different changes in secondary structure contents in comparison with the linear precursors and the unmodified sequence. a nmr conformational analysis will allow the determination of the conformational preferences of the stapled peptides at atomic resolution with also relevant implications for the kctd-cullin3 recognition. based on our findings from protease-mediated ligation of cleavage-sensitive peptides and proteins using ionic liquids (ils) as additives 1 and from chemical acylation reactions of peptides in ils, 2 we hypothesized direct and unique interactions between biomolecules and ils as the molecular reason for il-mediated structural effects. to verify this, we performed systematic structural investigations of suc-ala-xaa-pro-phe-pna and ala-xaa-pro-phe model peptides dissolved in aqueous imidazolium-based ils using high resolution magic angle spinning (hr-mas) nmr spectroscopy. highly resolved nmr spectra are obtained that allow for complete 1 h resonance assignments of the peptides as solutes and the ils as solvents. the changes in the proton chemical shifts δδ( 1 η) of a peptide on dissolution in ils arise from direct interactions between both, including il-induced structural/conformational changes of the peptide molecules. 3, 4 the strength, position, and type of these interactions are influenced by the nature of the amino acid residues in the peptide sequence as well as the nature and type of the il anions. downfield shifts indicate strong interactions between the ils and the peptide backbone, whereas upfield shifts point to hydrophobic interactions and stacking effects with the il imidazolium ring. the pattern of δδ( 1 η) along the peptide chain reveals the sites and the type of interactions. the extent of δδ( 1 η) implies a significant effect on the structure of the peptides. different δδ( 1 η) were obtained for cis-and trans-configured peptide isomers and point to a conformer-specific il/peptide interaction. calcitonin is a 32-amino acid polypeptide hormone, which takes part in calcium metabolism in bones. it belongs to a protein family whose members may form amyloid fibrils. these fibrils (or pre-fibrillar aggregates) are related to serious diseases known as amyloidoses, whereas amyloid-like fibrils may have natural functional roles in many organisms. the amyloid form of human calcitonin is related to medullary thyroid carcinoma. using our online consensus prediction tool for 'amyloidogenic propensity' of protein molecules "amylpred", a novel hexapeptide of human calcitonin was predicted as a novel, possible amyloidogenic peptide. we investigated experimentally its ability to form amyloid fibrils, utilizing tem, x-ray fibre diffraction, atr ft-ir spectroscopy and polarized light microscopy. analysis of the results shows that this peptide self-assembles into amyloid-like fibrils and that amyloid fibrillogenesis is mediated via nuclei of liquid crystalline nature, known as spherulites. we thank the university of athens for the financial support. school of pharmacy, university of norwich nr4 7tj ; 3 inserm u982, université de rouen, 76821 mont saint aignan 26rfa, a rfamide neuropeptide, is the ligand of gpr103 and the system 26rfa/gpr103 is involved in feeding behaviour [1] . a structural study by nmr showed that 26rfa, in a membrane mimetic medium, possesses a helix p4-r17 -linker k18-g21 -turn f22-r25 motif. sar studies on truncated fragments of 26rfa indicate that, in addition to the turn motif, additional residues are required to maintain a correct biological activity (26rfa ec50=10,4nm; 26rfa10-26 ec50=37,5nm; 26rfa13-26 ec50=95,3nm; 26rfa16-26 ec50=237nm) [2] . our aim is to synthesize new gpr103 ligands using an iterative process combining rational conception, peptide and modified amino acid syntheses, biological and structural analyses. structural and biological results will allow us to find new modifications and conceive more potent analogues. using nmr, we demonstrated that 26rfa11-26 encompasses a nascent helix and a turn while 26rfa13-26 possesses only the c-terminal turn structuration. the destabilization of the helical structure on fragments with a shorter n-terminal region could explain the decrease of the biological activity of 26rfa fragments. we decided to focus on the importance of a stabilized helix on the biological activity. we chose the hydrogen bond surrogate method which stabilizes a helix by replacing the main hydrogen bond with a covalent link without modifying the side-chains [3] . syntheses, nmr characterization and molecular modelling of analogues will be discussed. acknowledgments: this work is supported by erdf funding through the is:ce-chem project and interreg iv a france-(channel)-programme. firstly, it induces a constraint by restricting the ı o-dihedral angle to values around -60°. secondly, the xaa-pro peptide bond is subject to cis-trans isomerization characterized by an increased cis population and an activation energy that is low when compared to the other amino acids. besides, the five-membered ring of the pro residue can adopt two main distinct conformations (up-puckered or downpuckered) that are almost equally abundant in peptides and proteins. a variety of mimics and analogs have been designed in order to control the conformation of the peptide backbone and/or to alter the cis/trans ratios and the rotational barriers for cis-trans isomerization. in this presentation, nmr studies 2 and theoretical calculations 3 have been performed on model peptides ac-ser(ψpro)-nhme, (s,s)-ac-ser(ψh, cf3 pro)-nhme, and (r,s)-ac-ser (ψ cf3 ,hpro)-nhme. 4 their thermodynamic and kinetic features have been analyzed in chloroform, dmso, and water, allowing a precise description of their conformational properties. we found that trifluoromethyl c δ -substitutions of oxazolidine-based pseudoprolines can strongly influence the cis-trans rotational barriers with only moderate effects on the cis/trans population ratio. in chcl3, the configuration of the cf3-c δ entirely controls the ψ-dihedral angle, allowing the stabilization of γ-turn like or ppi/ppii-like backbone conformations. moreover, in water and dmso, this c δ -configuration can be used to efficiently constrain the ring puckering without affecting the cis/trans population ratio. theoretical calculations have ascertained the electronic and geometric properties induced by the trifluoromethyl substituent and provided a rational understanding of the nmr observations. in contrast to formation of the native state, aggregation of misfolded protein molecules into so-called amyloid fibrils is a strongly irreversible, kinetically-controlled process which may lead to structural variants of fibrils composed of chemically-identical protein chains. this poorly understood aspect of polymorphism of amyloid fibrils has important implications in vivo in the course of amyloidassociated neurological disorders -e.g. in the form of prion strains in creutzfeldt-jakob disease. in this paper, we examine cross-seeding behavior of a model amyloidogenic polypeptide -bovine insulin (bov-ins) and recombinant lys b31 -arg b32 insulin (kr-ins). fibrils of bov-ins and kr-ins exhibit characteristic "fingerprint" features in the amide i' infrared region. these spectral traits are transferred to daughter generations of fibrils upon cross-seeding of native bov-ins with kr-ins fibrils and vice versa. this study highlights an interesting aspect of a non-anfinsenian behavior of aggregating insulin: the memory effect. our results indicate that fine infrared features in the amide i region of fibrils from closely related proteins may not reflect different amino acid composition but rather the fact that a particular amino acid sequence favors an amyloidogenic pathway that determines a certain and spectrally-distinct stacking mode of β-strands. an encounter with an alien seed on the amyloidgenic pathway would hijack the yetnative monomers and force them to follow the assembly pattern (and resulting ir characteristics) of the preformed fibril. phosphorylation and oglcnacylation are dynamic intracellular protein post-translational modifications that often are alternatively observed on the same protein serine and threonine residues. phosphorylation and oglcnacylation commonly occur on residues in natively disordered regions of proteins, and often have opposing functional effects. in the microtubule-associated protein tau, hyperphosphorylation is associated with protein misfolding and aggregation as the neurofibrillary tangles of alzheimer's disease, whereas oglcnacylation is stabilizing. a series of peptides derived from the prolinerich domain (residues 174-239) of tau was synthesized, with the free ser/thr hydroxyls, with phosphorylated ser/thr, with oglcnacylated ser/thr, and with the dietylphosphorylated ser/thr. phosphorylation and oglcnacylation were found by cd and nmr to have opposing structural effects, with phosphorylation favoring polyproline helix (ppii) formation, with oglcnacylation opposing ppii, and with the free hydroxyls intermediate. phosphorylation of serine and threonine residues in prolinerich sequences was found to induce ppii in other proline-rich sequences, with significant conformational restriction at phosphoserine and phosphothreonine residues observed by nmr. notably, serine and threonine residues are common in proline-rich domains of proteins, and ppii is a common conformation observed for phosphorylated ligands in protein-protein interactions. the ser/thr diethylphosphate was found to be a practical mimic of oglcnacylation, as an easily synthesized neutral amino acid with similar sterics to oglcnac and strongly opposing ppii. dept. of biology, university of padova, padova 35121, italy we recently established that designed hairpin peptides interfere with the amyloidogenesis of both human pancreatic amylin and α-synuclein ( α-syn), two 'unstructured' polypeptides associated with human diseases. 1 in the case of α-syn, the most potent 'amyloid inhibitors' act by diverting α-syn to non-amyloid aggregates. the binding sites for these agents and nonhairpin peptides of similar sequence have been determined by 2d-nmr-monitored titration studies using universally 15 n-labeled α-syn. in the case of the hairpins that produce non-amyloid aggregates from α-syn, initial binding occurs in the non-amyloidogenic c-terminal segment of α-syn and the hairpin peptide is incorporated in the resulting aggregates. nmr studies also serve to define binding interactions and conformational changes that may be essential early steps in aggregation and pre-amyloid state processes. mt-ii is a long acting, non-selective super-agonist of melanocortin gpcr receptors, 1 characterized by lactam bridge between residues i and i+5 stabilizing a type-ii β turn structure. 2 the recently introduced cui-catalyzed azide-alkyne 1,3-dipolar cycloaddition, presents a promising opportunity to develop a new paradigm for an orthogonal bio-organic and intramolecular side chain-toside chain cyclization. we introduced this [1,2,3]triazolyl-mediated cyclization in a pthrp model peptide, and demonstrated that i-to-i+4 side chain-to-side chain cyclization offers a powerful approach for generating stable helix mimetic structures. 3, 4, 5 in the current study we explored the potential of the i-to-i+5 side chain-to-side chain 1,4-disubstituted [1, 2, 3] triazolyl-bridge to stabilize -turn conformations. we designed a series of [1,2,3]-triazolyl containing cyclo-heptapeptides, derived from the model peptide lactam mt-ii, differing in the size of the bridge, the location and orientation of the [1,2,3]-triazolyl moiety. conformational and biological studies, performed on this series demonstrate that [1, 2, 3] triazolyl-mediated side chain-to-side chain cyclization results in the conformational stabilization of type-i β turn generating heterodetic cyclopeptides with enhanced in vitro potency and improved receptor subtype selectivity. this study confirms the versatile nature of the [1, 2, 3] triazolyl-mediated side chainto-side chain cyclization. moreover the chemical accessibility, functional compatibility, and metabolic stability suggest that the [1, 2, 3] international associated laboratory samuel de champlain, 5 regional platform for cell imaging of haute-normandie (primacen), institute for medical research and innovation (irib), university of rouen, 76821 mont-saint-aignan, france scorpion toxins are polypeptidic molecules capable of selectivity binding to ion channels with the highest affinities and represent natural peptide blockers that are good tools to characterize new or particular types of ion channels. astroglial cells appear to express all ion channels species even voltage-gated ion channels previously suspected to be present only in excitable cells; but these channels are poorly studied and their function are not still completely elucidated.thus, the purpose of the present study was to investigate for the first time, the global effect and the potential protective effect of one scorpion toxin acting on kv channels, the pi4,on h2o2-induced oxidative stress and cell death in rat astrocytes. pi4 is a 38-residue toxin crosslinked by 4 disulfide bridges, isolated from the venom of pandinus imperator1 and is one of the most potent and selective inhibitor of shaker b and kv1.2 channels2. in our study, pi4 was obtained by solid phase peptide chemical synthesis(spps) and named spi4. the identity and homogeneity of spi4 were also verified by hplc, mass spectrometry analysis(maldi-tof) and an enzyme-based cleavage to determine the assignment of spi4 half-cystine pairings.the synthetic spi4 was found to be indistinguishable from the natural toxin. incubation of cultured astrocytes with graded concentrations of h2o2 for 1h provoked a dose-dependent reduction of the number of living cells as evaluated by flow cytometric analysis and lactate dehydrogenase assay. interestingly, pre-treatment of astrocytes with very low concentrations of spi4(10-8 to10-12 m),dose-dependently prevented cell death induced by h2o2, and the effect of spi4 was accompanied by a marked attenuation of ros accumulation. also, spi4 stimulated sod and catalase antioxidant activities in a concentrationdependent manner,and blocked h2o2-evoked inhibition of sod and catalase activities. in addition, at low concentration (10-8 to10-14 m) the toxin exhibits no toxic effect on astrocytes.taken together these data indicate that the spi4 acts as a protective agent on astrocytes from oxidative assault and then might have therapeutic value in the potential treatment of a number of neurodegenerative diseases. [4] . in order to select small active peptides, the library was kept at the minimum size and was built using only 12 selected d-amino acids to avoid structural redundancies. to further increase the chemical diversity [5] a set of different carboxylic acids were coupled to the n-terminus. the iterative screening led to the identification of an n-terminally modified tetrapeptide with a very high radical-scavenging activity. by analyzing several peptide variants, we found that antioxidant properties were strongly sequence-and structure dependent, because scrambled or proline-scan variantswhose secondary structures are deeply altered -are much less effective. data on peptide structure-activity relationship will be presented. an investigation of the in vitro effects produced in cellular models of oxidative stress are also in progress. structural analysis of peptide-analogues of zp proteins with amyloidogenic properties: insights into mammalian zona pellucida formation. n.n. louros, v.a. iconomidou, s.j. hamodrakas* department of cell biology and biophysics, faculty of biology, university of athens, athens 15701, greece a filamentous structure surrounding mammalian oocytes, called zona pellucida, is responsible for species-specific sperm binding. this extracellular porous structure is composed of 3-4 glycoproteins, termed zp1-4, which assemble into filamentous polymers that are cross-linked by disulfide bonds. all zp proteins present a unit with high sequence homology at their c-terminal end, designated as the zp domain. this structural module is found in a family of extracellular proteins with various functions and from a wide variety of organisms (zp domain proteins). it is a binary structure of approximately 260 amino acids, divided into two domains, the n-terminal region named zp-n and the c-terminal region known as the zp-c domain. the zp-c block is associated with protein function, whereas the zp-n domain is responsible for protein polymerization. structural analysis revealed a possible polymerization model based on backbone interactions between specific parts of the zp-n units. peptide-analogues of the aforementioned segments were synthesized, since the ''amylpred'' application, an 'amyloidogenic determinant' prediction algorithm developed in our lab, predicted them as being potentially 'aggregation prone'. our experimental data clearly indicate that these peptides do actually fold and self-assemble into amyloid-like fibrils. therefore, all the derived data suggest that mammalian zona pellucida is a novel functional amyloid, similar to its biological equivalent, silkmoth chorion that has been established to be a natural, protective amyloid. we thank the university of athens for the financial support. biostrucutres and bioimmaging institute of national research council, naples, italy the definition of the molecular basis of human diseases is one of the most important goals of structural biology. nucleophosmin (nmp1) is an ubiquitously expressed protein is a nucleolar chaperon and has a key role in several biological processes from ribosome biogenesis, to cell-cycle regulation and is a frequently target of oncogenic mutations in acute myeloid leukaemia (aml) [1] . these mutations occur predominantly at the c-terminal domain of npm1 compromising its stability and causing the aberrant translocation of npm1 to the cytosol. this domain is structurally characterized by the presence of three alpha helices, and the folding of cter-npm1 proceeds via an extended nucleus and the denatured state retains significant malleable structure at the interface between the second and third helices [2] . here, we have further investigated the structural determinants in the folding process of the c-term npm1 domain and on the basis of available structural and composition data, several peptides covering the three helices were designed and synthesized. their conformational properties were investigated by cd and nmr spectroscopy: these studies demonstrated that once removed from the protein architecture helix1 and helix2 substantially miss fully ordered conformations while helix3 retain a high helical content also in mutated variants. our observations may help structure-based drug-design inserm u829 university of evry-val d'essonne, bâtiment maupertuis, 91025 evry, france sl21 (stop-like protein with mass of 21 kda) is a neuronal protein with a calmodulin-binding and microtubulestabilizing activity. microtubule binding is inhibited by calmodulin 1,2 . the lack of the stops gives rise to defects in synaptic plasticity, as observed in schizophrenia 3 . to study the sl21 protein's structure and its interaction with calmodulin, we have used a 44 residue peptide derived from sl21, containing the microtubule binding motif mn. the structure of the peptide has been analyzed using circular dichroism (cd) spectropolarimetry. the cd spectra showed that the peptide's secondary structure consists in a predominantly β-sheets, while the tertiary structure consists in rather folded 3d-structure. the temperature dependence of cd indicated that the secondary structure of the peptide remained very stable between 5 °c and 95 °c, while the tertiary structure changed at about 35 °c. the interactions between the sl21 peptide and calmodulin have been studied using the isothermal titration calorimetry (itc). the results showed that calmodulin binds the peptide with the affinity of 104 m-1, in a ca 2+ -independent manner, the reaction being driven by entropic contributions. our results might contribute to understanding the microtubule stabilizing activity of the sl21 protein. peptides 4, 5 . based on the structure in polar environment a transition from 310-helical to an α-helical conformation at the water/lipid interface in bilayer membranes is hypothesized 3 the efficient method development for the analysis of proteins, peptides, and synthetic oligonucleotides with a novel hybrid reversed phase column f. becker *1 , n. shoji *2 , a. matsui *2 , c. yokoyama *2 , t. sato *2 , t. takai *2 , and n. kuriyama *2 *1 ymc europe gmbh, *2 ymc co., ltd. the recent development of bio-pharmaceutical industry has been remarkable and an effective analytical method with higher sensitivity, superior selectivity, and increased speed has been required in the characterization of peptides, proteins and oligonucleotides by highperformance liquid chromatography (hplc). the method development of reversed phase hplc requires optimization of several conditions, such as bonded-phase, column efficiency, solvent type, ph and temperature. especially ph and buffer type is a most important parameter to control retention of biomolecules which have multiple ionic functional groups. furthermore, temperature often becomes a key tool to achieve better peak shapes and resolution for larger molecular-weight compounds. although silica based reversed phase columns have been widely used for separation of biomolecules, they have low stability under alkaline conditions and a limited usable ph range. to improve the chemical stability at an expanded ph and temperature range , we have developed a new type of organic/inorganic hybrid reversed phase column, ymc-triart c18 and ymc-triart c8. the novel technologies of manufacturing particles and surface modification provide outstanding chemical stability and excellent peak shape for many types of compounds under a variety of mobile phase conditions. in this poster, we will show characteristics of this new hybrid column, and some example cases of efficient method development in separation of the biomolecules such as peptides, proteins and synthetic oligonucleotides. the fully-extended peptide conformation (2.05-helix) is the longest possible for a single α-amino acid as it is characterized by an axial translation per residue of about 3.85 å. therefore, it is extremely attractive for its application as a molecular bridge. however, it is known that the stability of this type of helical structure (relative to the competing, much shorter 3 10-helix) appears to be dramatically governed by the choice of the c-terminal functionality (only esters seem to be appropriate). in this work, we examined the series pyrac-(deg)n-o-(pno2)bzl [where pyrac is the fluorophore 1-pyrenylacetyl, deg is cα,α-diethylglycine, n=1-5, and o-(pno2)bzl is para-nitrobenzoxy] with the deg homo-peptides as fully-extended bridges in a static fluorescence study. to perform a very stringent comparison with our ft-ir absorption and nmr results, the solvent used was chcl3. the trend observed in the quenching efficiencies for the shortest members of the series, the (deg)1-3 peptides, steadily decreases from 0.97 (n=1) to 0.89 (n=2), and 0.64 (n=3). this finding is compatible with the conclusion that these three peptides are all essentially fully-extended in chcl3 solution. however, the quenching efficiencies for the longest deg homo-peptides. 0.68 for n=4 and 0.65 for n=5, are not further reduced. the inevitable conclusion is that the tetra-and pentapeptides populate at least two different conformations (most probably, the 2.0 5-helix and the 310helix), in which the probe dyads are located at different distances. notably, these results fit well with those extracted from our ft-ir absorption and nmr studies in the same solvent of low polarity. we conclude that a combination of terminally aromatic groups, as in the deg homo-peptide series investigated in this work, is not eligible for the fabrication of a stable fully-extended conformation. the surfactant protein c (sp-c) is a 35-residue highly hydrophobic peptide with two covalently linked fatty acyl chains that adopts a mainly helix structure while associated with the membrane. however, it misfolds into a β-rich structure under certain environmental conditions, which suggests that sp-c is in a metastable state 1 . the slow α→β transitions are likely to be caused by the membranedissociation, deacylation1 and exposure to the neutral ph 2 of the alveolar subphase, leading to the formation of amyloid aggregates associated with pulmonary alveolar proteinosis. we have been studying the ph-dependent conformational behaviour of both the acylated and deacylated isoforms of sp-c in a membrane mimetics chloroform/methanol/water mixture using a constant-ph molecular dynamics method [3] [4] [5] . our simulations identify the increase of ph as a promoter of peptide-peptide interactions, which may contribute to the α→β transitions observed at neutral and alkaline ph conditions in the experimental results 2 . we also observe that the loss of structure is more prone to begin at the c-terminal region and that the β motifs start to form between the n and cterminal disordered regions of the sp-c. in addition, the presence of the acyl groups results in a slightly higher structural stabilization of the sp-c helix and in a decrease of the n-terminal plasticity at acidic and neutral ph. since a comprehensive conformational analysis and the elucidation of the mode of association between sp-c and the membrane are essential to understand the interactions involved in structure stabilization and in some specific functions of the peptide, we are now studying both sp-c isoforms in a membrane environment. these studies will not only be the most relevant for the in vivo situation but will also answer to some questions left open by the previous work. were rather small. vp plays a very important role in controlling resorption of water by the distal tubules of the kidney and in regulating the osmotic pressure of blood in mammals. vt, while exhibiting both oxytocin and vasopressin activities, has not changed over the last 300 million years until appearance of the mammals. currently, it occurs naturally in the non-mammal vertebrates [1] . here we report on studies of arginine-vasopressin (cyfqncprg-nh2, avp) and arginine-vasotocin ([ile3]avp, avt) in a mixed dodecylphosphocholine (dpc) and sodium dodecylsulfate (sds) micelles. dpc micelles are considered good models of eukaryotic cell membrane [2] , while the sds micelle improves peptide/micelle solubility [3] . for these studies we use 2d nuclear magnetic resonance (nmr) supported with molecular modelling methods. conformation-activity considerations relationships and the role of the peptide-micelle interactions on the structure of the analyzed peptides will be described and discussed. vibrational and chiroptical spectroscopic investigation on diamide peptide models k. knapp, a m. hollósi, a e. vass, a zs. majer a a eötvös lor nd university, institute of chemistry, laboratory for chiroptical structure analysis, budapest, hungary understanding of peptide folding has great importance in many fields of chemistry. since amino acid residues are the simplest building blocks of peptides, the investigation of their conformational properties could help us in understanding the structures of peptides or in designing peptides of specific 3d structure. describing the conformational building blocks of even the simplest peptide models (e.g., amino acid diamides) can serve as a good starting point to decipher higher-level structural properties of their polymers. among the simplest compounds which include the essential structural elements of polypeptide backbone are the n-methylamides of n'acetylamino acids. these compounds have been the subject of extensive spectroscopic analyses (ftir, vcd, ecd, nmr) [1, 2] . the conformational landscape of these model compounds were analyzed by solution phase vcd and ecd spectroscopy and density functional theory (dft) computations. this work reports systematic vcd and ecd spectroscopic studies in different solvents on synthesized diamide models (ac-α/βhomo-xxx-nhch3, xxx = ala, pro, phe). vcd and ecd data, combined with quantum chemical calculations performed at the b3lyp/6-31++g** and 6-311++g** level of theory was particularly useful for identifying the preferred dipartimento delle scienze biologiche, università di napoli "federico ii", napoli, italy c dipartimento di scienze ambientali, seconda università di napoli, caserta, italy β-hairpin is an important secondary structural element used by many proteins for biomolecular recognition, and thus is an attractive tool for mimetic design 1 . aβ -hairpin motif consists of two antiparallel strands linked by a turn region. in this work we aimed to stabilize a β-hairpin conformation by an interstrand covalent bridge, made through a click chemistry reaction, which yields 1,4-disubstituted 1,2,3, triazole, starting from alkyne and azide reactive groups. in particular, we explored the conformational stability of a series of β-hairpin peptides containing a 1,4-disubstituted 1,2,3 triazole bridge with variable length. we employed the well structurally characterized trpzip2 peptide as a convenient b-hairpin model system. 2 as alkyne amino acids were introduced propargylglycine (pra), homopropargylglycine (hpg) or bishomopropargylglycine (bpg), while the following azide amino acids were inserted: lβ-azidoalanine (dap (n3)), l-γ-azidohomoalanine (dab (n3)), and l-δ-azidoornitine (orn (n3)). all peptides were synthesized and purified. successively, the linear unprotected peptides were cyclized in solution by the cu(i)-catalyzed azide alkyne cycloaddition (cuaac) reaction and purified prior to the spectroscopic characterization. linear and cyclic peptides were analyzed by a combination of cd and nmr techniques. 3 have been investigated. 1 their behaviour has been compared with that of proline in homochiral and heterochiral dipeptide sequences including l-or dphenylalanine. nmr and ir studies as well as x-ray diffraction analysis provide evidence that the additional β-phenyl substituent does not disrupt the tendency of proline to occupy the i+1 position of a β-turn. moreover, the β-turn conformation is stabilised, with reference to l-pro, when l-cis(βph)pro or l-trans(βph)pro are combined with d-phe, that is, in the heterochiral sequences. the pyrrolidine conformation is significantly affected by the presence of the β-phenyl group. the puckering modes that alleviate most the steric hindrance introduced by the bulky phenyl substituent are preferred. the l-cis(βph)pro residue shows a marked propensity for the cγ-endo/cβexo half-chair arrangement whereas the trans derivative exhibits a higher flexibility, with different pyrrolidine shapes being accessible. interactions between the aromatic ring of (βph)pro and that of the contiguous l-or d-phe residue are observed in all the dipeptides crystallised. in solution, they seem to occur only for the heterochiral sequences. this intramolecular aromatic-aromatic interaction may be responsible for the higher β-turn ratio observed for such sequences and also seems to affect the conformational preferences of the pyrrolidine ring. in fmoc chemistry the trt and acm groups are widely accepted as a protecting group for cys. upon incorporation of these cys derivatives onto the growing peptide chain, however, considerable base-catalyzed racemization of cys is known to always occur at the activating and coupling steps with phosphonium or uronium reagents such as pybop or hbtu, respectively. in addition to this, racemization of the c-terminal cys esterified to resins also occurs during the repetitive n α -fmoc deprotection using piperidine. in order to find an s-protected cys derivative applicable for fmoc chemistry that can efficiently suppress racemization of cys both when its incorporation is conducted by phosphonium or uronium reagents and when it is linked to a resin via ester linkage, we introduced the 4methoxybenzyloxymethyl (mbom) group into the thiol function on cys. the mbom group was found to substantially supress racemization of cys (0.4%) during its incorporation mediated by phosphonium or uronium reagents in comparison with the trt and acm groups (8.0% and 4.8%, respectively). even after a 6-h treatment of the peptide resin, in which its c-terminal cys was linked to a trt-type resin, with 20% piperidine in dmf, the mbom group caused a significant reduction in the level of racemization with the c-terminal cys (6.4%) while trt and acm groups led to a considerable extent (30.2% and 27.9%, respectively). next, we demonstrated the usefulness of the mbom group on cys by applying it to ncl chemistry involving the coupling of a peptide thioester and a cys-peptide. in the synthesis of the latter peptide, however, a diastereoisomer with racemization of the nterminal cys tends to be difficult to separate from the intact peptide as its chain length increases. thus, this measure using fmoc-cys(mbom) can facilitate the avoidance of ambiguities in the quality of the synthesized peptides. labeled peptides are important tools in chemical biology to obtain information on their biological function in, for example, cellular communication processes as well as to study the molecular basis of diseases. the functionalization of bioactive peptides with biophysical reporter groups like strongly absorbing chromophores, is most often performed via post-synthetic chemoselective biocojugation reactions, involving amino, thiol, or azide specific reactions. an alternative approach for the installation intense chromophoric organic molecules, is the coupling of nonchromophoric precursor molecules to obtain chromophoric properties in the final product. recently, we have shown that the [2+2] cycloaddition-cycloreversion reaction between peptide-functionalized electron-rich alkynes and electrondeficient cyanovinyl derivatives results in the formation of a new class of π-conjugated peptidic donor-acceptor chromophores. 1, 2 the chromophoric properties of the final product can be tuned by varying the p-electron conjugation pattern. herein, we describe the synthesis of the versatile building blocks, n-a-(4-ethynylphenyl)-n-a-(methyl)-glycine and n-a-(4-(1,2,2-tricyanovinyl)benzoyl)-glycine, their subsequent application in spps to functionalize peptides with d-p-a chromophore precursor molecules, and their uvvis absorption characteristics. as a typical example, alkyne-functionalized aβ peptides, val-ala-ile and lys-leu-val-phe-phe-ala-glu, have been reacted with a series of cyanoolefins to obtain far-red intense imaging chromophores for amyloid-rich peptide aggregates. a novel method for regioselective disulfide formation in mini-proteins by kinetic oxidation control t. lindner, u. haberkorn, w. mier department of nuclear medicine, university hospital heidelberg, im neuenheimer feld 400, heidelberg, germany rigid frameworks are essential for the development of peptide-based alternatives to immunoglobulins. [1] disulfideconstriction covalently stabilizes tertiary structures, which allows further miniaturization of protein-derived drugs by chemical synthesis. while the amino acid assembly steps in solid phase peptide synthesis are highly efficient, regioselective formation of multiple disulfide bonds is still a challenging and time demanding task. even after applying laboriously optimized folding conditions and extensive purification, a successful synthesis of the correctly folded isomer is not guaranteed. [2] to circumvent these problems, a procedure that takes advantage of the different reactivity of free vs. acmprotected thiols during iodine oxidation was developed. this protocol to sequentially form two disulfide bridges in a one-pot reaction was applied to different peptides, such as (a) min23, a mini-protein scaffold for phage-display assisted drug design; [2] (b) α-conotoxin imi, a neurotoxic peptide isolated from the venom of marine cone snails; [3] and (c) endothelin-1, a peptide involved in blood pressure regulation. [4] the presented technique tolerates precarious residues within the to-be-folded-peptide, such as fluorescent dyes or metal chelators as used for bioactivityassays. compared to the individually optimized literature procedures, this novel approach offers significant improvements: overall yields >30% are obtained in first attempts and stepwise formation of the disulfide bridges is performed within a few hours instead of days. in recent thirty years, c-terminal modified peptides have been proved to have greater potential as apis (active pharmaceutical ingredients) due to their increased chemical and enzymatic stability and improved pharmacodynamic properties 2-4 . a prominent example, octreotide 5-6 , an octapeptidoalcohol, has witnessed as a potent anti-cancer agent targeted for gastro-entero carcinomas. in view of synthetic methodology, peptidoalcohol can not be directly prepared by standard spps protocol becouse of the c-terminal structure released from resin are not alcohol but always peptidoacid or peptidoamide. to overcome this problem, a novel protocol of shortened n-1 coupling cycles on merrifield resin and then the ammonolysis of peptedyl resin by an aminoalcohol as the c-terminal residue getting peptido-alcohol as targetting product has been devoloped in our lab. because of the cleavage treatment of peptidyl merrifield resin is not under acidic condition, such as hf or tfmsa, but ammonolysis, some side-chain producting groups(spg) related to boc chemistry like bzl, clz, tos…; must be avoided in sequence assembly. therefore a hybrid orthogonal protection (hop) of boc/fmoc protocol was adopted for the sake of producing naked peptidyl (without any spg) resin before ammonolysis. fifteen peptidoalcohols with different terminal alcohols were conveniently prepared, most of them released form resin with very good yields. due to its cyclic structure, proline is the coded amino acid with a more restricted conformational flexibility. the incorporation of additional groups into the pyrrolidine ring is a useful means to produce new amino acids that combine the conformational properties of proline with sidechain functionality. this is the case of β-phenylproline, (βph)pro, that can be regarded as a proline-phenylalanine hybrid in which the orientation of the aromatic substituent is dictated by the conformation of the five-membered ring and the cis or trans configuration of the phenyl group relative to the carbonyl moiety. accordingly, cis(βph)pro and trans(βph)pro combine the conformational properties of proline with an aromatic side-chain functionality that is rigidly oriented with respect to the peptide backbone, and this may be useful in the design of biologically active peptides and other applications relying on specificallyoriented side-chain moieties. we have developed synthetic procedures for the preparation of the cis(βph)pro and trans(βph)pro stereoisomers in enantiomerically pure form. the methodology is based on the preparation of racemic precursors of each amino acid and their subsequent hplc resolution on chiral columns. multigram quantities of the target amino acids have been isolated in optically pure form and suitably protected for use in peptide synthesis. the importance of peptide cyclization for studying peptide conformation, creating new structures, or for developing peptide therapeutics is well established. in particular, sidechain lactam bridges linking two amino acid residues that are several residues apart in the linear sequence or headto-tail backbone peptide cyclization enable rigidification of the structure and improvement of in vivo stability. native chemical ligation (ncl) is now an established method for producing backbone-cyclized peptides or proteins. the application of ncl to the synthesis of sidechain cyclized peptides is less frequent. head-to-side-chain cyclization by ligating a c-terminal thioester with a cys residue located on a lysine side-chain was used by few authors. the alternative tail-to-side-chain cyclization mode is rare, probably due to the difficulty of installing a thioester group on amino acid side-chains such as aspartic or glutamic acids the reaction of a bis(2-sulfanylethyl)amido (sea on ) group with an n-terminal cysteine residue in water and at neutral ph results in the formation of a native peptide bond. [1] oxidation of sea on results in a cyclic disulfide called sea off having a 1,2,5-dithiazepan-5-carbonyl structure. [2] sea off is a self-protected form of sea on . we show here that bis(2-sulfanylethyl)amido side-chain acid(dab), ornithine and lysine were selected as building block; 1a and n,n'-cbz-1-amidinopyrazole (1b) were selected as guanidinylating reagents for specific situation. for synthesis of n-terminus local cyclo-guanidine peptide, designated peptides were assembled on acid labile solid support such as rink amide resin by fmoc strategy. then either fmoc-dab(boc)-oh, fmoc-orn(boc)-oh or fmoc-lys(boc)-oh was incorporated respectively at n-terminus. fmoc was removed followed by guanidinylating by 1b and then peptide was cleaved by acid. by neutralizing with nmm in acetonitrile solution, side chain amino group and a-guanidine would form 6, 7 or 8 membered local cycloguanidine. the remaining cbz could be removed by hydrogenation. for synthesis of backbone side chain cyclic peptide, bis-fmoc-daa was introduced in the peptide previously on resin followed by removal of fmoc. selective guanidinylate side chain aminogroup by 1a followed by peptide assembling with an insertion of orthogonal protected daa at 1-4 aa apart from first daa. for synthesis of a-nh2sidechain cyclic peptide, first daa should be introduced with orthogonal protected form. 1b was used to guanidinylate a-nh2. after cleavage and neutralization of those two kinds of intermediates, guanidine-bridged marco-cyclic peptide was formed. the resin is also a multipurpose tool for the synthesis of carboxylic acids, esters and thioesters. when the synthesis is completed, the fully protected peptide hydrazide resin is oxidized with either n-bromosuccinimide (nbs) or copper(ii) acetate in pyridine. the resulting acyl diazene resin is then cleaved by peptide displacement at the c-terminus with amine. the fully deprotected peptide amide is finally obtained by treatment with trifluoroacetic acid (tfa). in our approach, we used a 4-fmoc-hydrazinobenzoyl am novagel resin to synthesize a peptide-substituted amide in the c-terminus. first, the oxidative cleavage was carried out with nbs in pyridine and a nucleophile [a protected 4 (aminomethyl) benzimidamide (amba)]. however, the yield of the reaction was very poor. in the next step, we applied copper(ii) acetate in the presence of pyridine and amba. following optimization, the efficiency of the process was significantly improved. herein we discuss the conditions needed to obtain a reasonably high efficiency of the oxidative cleavage in the synthesis of our c-terminal modified peptides using the aryl hydrazine resin linker. blood vessels on tumor tissues, similarly to integrin receptors. this observation suggests cd13 as a selective target for targeted delivery of drugs and nanoparticles to tumor neovasculature using ngr peptides as homing motif. in our work, new cyclic-ngr peptides containing a thioether linkage were prepared. the influence of their structure on the speed of succinimide ring formation and deamidation was evaluated and compared with the previously published data on cyclic-ngr derivatives containing amide bond or disulfide bridge in the cycle (c[kngre]-nh2 and c[cngrc]-nh 2 ). to avoid the deamidation under the conditions used for cyclization, the synthetic routes were optimized. the influence of the ph, ionic strength and temperature of the solution on their chemical stability was investigated. the structure of the cyclic peptides was investigated by circular dichroismand nmr-spectroscopy. receptor binding ability and the influence of the cyclic peptides on the cell adhesion and motility were also evaluated. this work was supported by grants from the hungarian national science fund (otka nk 77485 and k 81596) and the national innovation office (bio_surf, om-00146/2008). clickable peptides and their attachment to oligonucleotides m. wenska, m. alvira, r. strömberg department of biosciences and nutrition, karolinska institutet, novum, se-141 83 huddinge methodology for the ready conversion of peptides into "clickable" azido-peptides with the possibility of selecting either n-terminus or c-terminus connection is presented. 1 synthesis of peptide-oligonucleotide conjugates (poc's) include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals. a general procedure, based on a new activated alkyne linker, for the preparation of poc's has been developed. 2 with this linker, conjugation is effective at room temperature in mm concentration and submicromolar amounts. this is made possible since the use of a readily attachable activated triple bond linker speeds up the cu(i) catalyzed 1,3-dipolar cycloaddition ("click" reaction). the main scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of i) an h-phosphonate based aminolinker ii) the triple bond donor p-(npropynoylamino)toluic acid (pata) and iii) azido-functionalized peptides. the method gives excellent conversion of oligonucleotide to the poc on solid support, and only involves a single purification step after complete assembly. the procedure which makes use of a low concentration of copper ions leads to a product with very little copper left (similar or less than in drinking water). the synthesis is flexible and can be carried out in non-specialist laboratories without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. comparison of alternative deprotection reagents to piperidine for the synthesis of a poly-alanine peptide on the tribute® peptide synthesizer m.a. onaiyekan,* j.p. cain, c.a. chantell, m. menakuru protein technologies, inc. tucson, az, usa in peptide synthesis, piperidine is a common agent for fmoc removal. however, piperidine is a controlled substance which requires special handling and cannot be used in some countries. therefore, it would be useful to identify alternative deprotection reagents to piperidine for fmoc removal. it is well known that poly-alanine sequences have a high propensity to aggregate after the fifth residue. in this application, (a)10k-oh was synthesized using the tribute®'s intellisynth uvmonitoring and feedback system to compare the efficiency of fmoc removal by piperidine vs. three alternative bases (pyrrolidine, cyclohexylamine, and tertbutylamine) in the last 5 cycles of the synthesis. it was found that pyrrolidine produced a higher purity product with fewer deprotection repeats and shorter deprotection times per cycle than piperidine, proving it to be a highly efficient, viable alternative to piperidine for fmoc removal. the endogenous tripeptide gpe also nammed "glypromate" is made up by the three n-terminal residues (glycine-proline-glutamate) of the insulin-like growth factor 1 (igf1). this tripeptide is a partial glutamate antagonist and showed good results in different neuroprotective in vitro and in vivo experiments. 1,2 gpe also binds to glial cells regulating neurotransmitter levels in the brain. 3, 4 however, gpe suffers from poor lipophilicity and a short half-life in vivo. that's why there is a need for more lipophilic and protease resistant analogues of gpe. in this poster we present the synthesis of trifluoromethylated analogues of gpe based on the 2 or 5-cf3-pseudoproline residues. introduction of fluorine atoms on bioactive compounds is known to deeply modify their physico and biochemical properties increasing lipophilicity and resistance to protease. 5 thus, developing a trifluoromethylated analogues, we intend to increase the bioavailability of gpe, keeping the benefit of its neuroprotective properties. our research team is strongly involved in the synthesis of trifluoromethylated alpha-amino acids. recently we published the synthesis of 2-trifluoromethyl-1,3oxazolidines derived from fluoral and (l)-serine and we demonstrated that these five membered ring 5-cf3pseudoprolines are hydrolytically stable and can be considered as proline analogues. 6 that's the reasons why we are interested to replace the proline residue of gpe by those trifluoromethylated compounds. the development of original coupling conditions and the detailed synthesis of two pseudoprolines analogues of gpe will be presented in this poster. modifications. in combination with automated spps, unprecedented access to large peptides and small proteins for biological research has been achieved. we demonstrate the application of this methodology to the synthesis of a variety of peptides on the prelude® peptide synthesizer. exploring the space of fluorine-labeled α-amino acids for solid state 19 f-nmr structure analysis of peptides: rational design, synthesis and applications p. solid state 19 f-nmr is a powerful method to study membrane-active peptides, as it can reveal their conformation, orientation and dynamics when embedded in biomembranes. 1 for this purpose the native peptide has to be selectively labeled with a suitable 19 f-containing amino acid at several different positions. the resulting battery of singly 19 f-labeled analogues is then analyzed by solid state 19 f-nmr. the main limitation to this approach currently lies in the poor arsenal of available 19 f-labels. we have therefore rationally designed and synthesized several specific amino acids bearing a cf3-reporter group, which fulfil all strict criteria to a "proper" 19 f-label. 2, 3 to allow a geometry-based structure calculation, the cf3group has to be rigidly attached to the peptide backbone. we thus rigidified the side chain using either a [1.1.1]bicyclopentane moiety, a cyclobutane ring, or the intrinsic proline framework. this way, suitable cf3-labeled analogues were created as substitutes for bulky hydrophobic amino acids (leu/ile/val/met), for aromatic residues (phe), for polar side chains (ser/thr), and for proline (pro). by now we have applied the developed 19 f-labels for a comprehensive structure analysis of more than ten different membrane-active peptides (gramicidin s, pgla, mag 2, kigaki, sap, temporin a, bp100, etc). recently, several new activators have been introduced into the market, and they were evaluated along with some older activators for their ability to synthesize a range of peptides with shorter and longer reaction times on the symphony® peptide synthesizer. it was found that hdmc, pyclock, comu, hctu, and hatu worked well at shorter reaction times (2x1 min), but pyoxim and tffh only worked well at longer reaction times. the performance of pybop at shorter reaction times was poor only for more difficult sequences. these results are important for selecting an appropriate activator for fast spps applications. the plant cyclotides form the largest family of cyclic peptides 1 . they contain a signature motif referred to as the cyclic cystine knot, which is derived from the cyclic backbone and three inter-knotted disulfide bonds. intriguingly, cyclotides can be boiled, treated with chemicals or enzymes without disrupting their overall fold. thus, they are sometimes labeled as ultra-stable proteins. in addition, cyclotides are tolerant to mutations, and as a scaffold they can successfully accommodate foreign bioactive epitopes of variable sequences 2 . cyclotides share many of these properties with another disulfide containing cyclic plant peptide, the sunflower trypsin inhibitor 1 (sfti-1) 3 . emerging evidence indicates that cyclotides and sfti-1 are valuable not only as peptide stabilizing scaffolds; in combination with their cell penetrating properties, these disulfide rich cyclic peptides have significance as intracellular drug carriers. although both peptides are genetically encoded, studies to ascertain the exact mechanisms of their biosynthesis are currently on going. thus, the synthesis of cyclotides and sfti-1 are currently restricted to chemical means. we have recently adapted a fmoc-spps method for cyclic peptide synthesis, via n-acylurea intermediates with the assistance of microwave irradiation. this method is a safe and convenient alternative to boc-spps and has the ability to be automated conveniently. using this method, parent scaffolds as well as several cyclotide and sfti-1 analogues with potential antimicrobial and matrix metalloprotease activities were synthesized. with the rising interest in the cyclization concept as a tool to impart stability on unstable peptides, the cyclic peptide synthesis method adapted herein is anticipated to have numerous applications. fixed configuration. the nonnatural oligomers have an extended conformational space and are supposed to adopt non-canonical secondary structures 2 . in addition, the backbone modification makes these molecules more stable towards proteolytic degradation. the majority of proteins in nature are post-translationally modified, and the most abundant modification is the protein glycolysation, which introduces wide structural variety to proteins. glycoproteins have an important role in the biological recognition process, such as immunodifferentiation, cell adhesion, cell differenciation and regulation cell growth 3 . new aza-β 3 -amino acids bearing either an azide instead of amine on lys and orn chain or an alkyne group will be described and used in solid phase synthesis to finally performed a click chemistry to cyclize pseudopeptides or to introduced a glycosylated function 4 . true for a series of peptides that display strong corticotropin releasing factor (crf) antagonistic activity. seminal studies by rivier et al. have shown that the incorporation of a lactam bridge in the crf-sequence resulted in an enormous increase in activity and potency, due to stabilization of the bioactive a-helical conformation of the peptide; and the newly designed peptide was called astressin. 1 based on the astressin sequence, we started a truncation and deletion study to arrive at astressin analogs with a reduced size but still remain active as crf antagonists. this study resulted in the smallest active crf antagonist, astressin(30-41). 2 this sequence was further optimized by the introduction of novel covalent constraints, other than the well-known lactam bridge. as a first approach, the alkene/alkane bridge, which can be introduced via ring-closing metathesis via alkenesubstituted amino acid side chains, and as a second approach, the triazole bridge ('click' macrocyclization), via either a cu(i)-or a ru(ii)-catalyzed cycloaddition reaction between azide-and alkyne-derivatized amino acid residues were explored. herein, we will present the details of the synthesis of the alkene-, azide-, and alkyne-functionalized amino acids, their use in spps, and the optimized approaches for macrocyclization. furthermore, the peptides have been characterized by hplc, nmr, lcms, and studied by circular dichroism spectroscopy to obtain insight into the helical propensity of the peptides in relation to the cyclic constraint. the synthesis of a nitronyl nitroxide, c α -tetrasubstituted αamino acid (a class of sterically restricted amino acids that promote the formation of peptide β-turns and helical structures) was achieved by derivatisation of racemic 2amino-5-cyano-indan-2 carboxylic acid [aic(cn)]. racemic boc-aic(nn)-oh was prepared by bis(alkylation) of ethyl isocyanoacetate under phase transfer conditions with 3,4-(bis)bromomethyl benzonitrile as alkylating agent, followed by acidic hydrolysis, n α -boc protection, and saponification of the ester function. resolution was achieved through formation of the diastereomeric amides of (s)-phenylglycinol with chromatographic separation and mild acidic hydrolysis. reduction of the nitrile group to an aldehyde was carried out with raney nickel in the presence of sodium hypophosphite. condensation with 2,3-diamino-2,3-dimethylbutane gave the corresponding tetramethylimidazolidine, which was oxidised with 3chloroperbenzoic acid to the desired nitronyl nitroxide. the uv-vis absorption and epr spectra of the amino acid were recorded and its magnetic properties were examined. in order to develop the synthesis of this peptide using the fmoc solid-phase peptide synthetic methodology, orthogonally protected β-hydroxyaspartic acid was needed. more precisely we wish to dispose of (2r,3r)-n -fmoc-3-tbdm-silyloxy-aspartic acid α -allyl ester instead of the recently reported dmab ester 2-3 indeed, in preliminary assay using this protective group we experienced difficulties during the final cyclisation step 4 . the synthesis was developed starting from inexpensive l(+) dimethyltartrate and extended to the others stereoisomers of the β-hydroxyaspartic acid. structure. for that, we chose to replace proline by silaproline to afford polysilaproline. this study shows the comparison of two polyamino acids: polyproline and polysilaproline polymers. homopolypeptides were synthesized by polymerization of corresponding amino acid n-carboxyanhydride 3 . multicomponent reactions (mcrs) represent a chemical process involving at least three reactants for the formation of several covalent bonds in one operation 1 . by definition mcrs are chemo-and regioselective, convergent stepefficient procedures and take place with high atom economy. the copper(i)-catalyzed 1,3-dipolar cycloaddition of organic azides and terminal alkynes (cuaac) reported by meldal 2 and sharpless3 has been involved in various fields of chemistry and biochemistry research. however only few reports describe the implementation of cuaac and mcrs. 4, 5 recently our research focused on a novel threecomponent reaction based on a cu(ii)-triggered aminolysis of peptide hydrazide resin and an azide-alkyne cycloaddition sequence. 6 copper(ii)-induced oxidative aminolysis of hydrazides generates cu(i), catalyst of the azide-alkyne cycloaddition. this feature was exploited to design a solid phase detaching three-component reaction. the mcr process requires a peptide hydrazide resin, an amino azide linker and an alkyne, resulting in the formation of peptide modified at the c-terminus through an amino 1,2,3-triazole linker. this method can potentially be applied to the synthesis of a large variety of peptide derivatives starting from fmoc-spps assembled peptidyl resins. furthermore, it is not practical to compare hplc spectra from different resin samples (e.g., before and after reaction) directly. a comparison by analyzing the same (mg) amount of resin would involve tedious sample preparation that is extremely error-prone and would be impractical because factors resulting from the increase or decrease of the molecular weight of the resin-bound compounds may have a significant influence on the results. the use of internal reference compounds allows rapid assessment of reactions performed on solid supports. the internal reference compound is bound to the resin together with the substrate and cleaved with the products after completion of the reaction. commercially available compounds can be used for this purpose, or likewise, the reference compound can be generated from the substrate by partial capping of a functionality. the peak integration of the reference compound in the hplc-uv spectra can be correlated directly to those of the rest of the compounds present in the reaction mixture and therefore a quantitative interpretation of the spectra with respect to conversion and yield is possible. here we demonstrate the proof of principle as well as the accuracy of this method. modifier proteins such as ubiquitin are conjugated to protein substrates in cells and thereby mediate various biological processes. 1 of high interest, is the ubiquitin fold modifer 1 (ufm1, 83 residues) which has structural similarity to ubiquitin but has no sequence similarity. unlike ubiquitin, ufm1 has not been extensively studied and little is known about its biological role. to understand ufm1's biological functions, access to pure, homogeneous natural and modified ufm1 protein is essential. chemoselective ligation techniques are suitable for providing such proteins. recently, a variation of the α-ketoacid-hydroxylamine (kaha) ligation 2 was developed, which utilizes the chemoselective reaction between a c-terminal peptide αketoacid and a n-terminal 5-oxaproline. 3 this modified form of the kaha ligation furnishes a native peptide bond and a homoserine residue. this ligation is useful for the synthesis of proteins from two unprotected protein segments in aqueous buffers. for the synthesis of larger proteins, a sequential ligation strategy is necessary. using ufm1 as the model system we have developed a sequential ligation procedure using kaha ligation with 5-oxaproline. applying the new sequential ligation strategy we have prepared ufm1 by total chemical synthesis. we have also prepared a cterminal thioester surrogate of ufm1 protein, which is suitable for conjugation to proteins of interest. the syntheses required the development of a bifunctional peptide segment bearing an α-ketoacid and an orthogonally protected 5-oxaproline. the preparation of the protein segments, their intermediates, the deprotection, and sequential kaha ligations towards the syntheses of ufm1 protein and c-terminal modified thioester ufm1 protein will be discussed. affinity and biological activity, we have designed and synthesized new analogues by multiple n-methylation of hut-ii(4-11) backbone amide bonds. all the peptides were performed by a novel synthetic approach, in which the introduction of n-methyl groups occur during regular solidphase peptide synthesis. on these new ligands we evaluated the binding affinity and biological activity at the ut receptor and performed preliminary nmr conformational studies. since that time a number of different machines have been used to automate peptide synthesis. modern machines are following two general setups; the so called "single approach" and the "parallel approach". in the single approach, the machine is developed to synthesize one or few peptides simultaneously. the user is able to optimize the synthesis conditions on each single peptide and each single coupling step. the maximum product quality regarding purity and yield is the major task of this approach. in the parallel approach, the machine is developed to synthesize a huge number of different peptides in the same single setup and time-frame. the user always has to find a synthesis protocol appropriate for the needs of each peptide to reach the maximum quality, knowing that there will be always a number of failed peptides. as a result you will find both types of peptide synthesizers in laboratories all over the world: the single machine, for the complicated peptides, and the parallel machine, allowing generation of multiple peptides with standardized protocols for each. the tetras is the first instrument combining the advantages of both machine types and allows the user to synthesize up to 106 different peptides in parallel. each peptide can have its own individual synthesis protocols, separate of all others. the user can combine different synthesis scales, peptide lengths, and activator reagents in one run. finished peptides can be removed and new peptides can be started while the tetras is still running. the tetras allows the user to establish an uninterrupted production shop using one instrument only. siemion, i. z.; peptide res the peptides: analysis, synthesis and biology monitoring peptide folding in membrane-active peptides: a time-resolved spectroscopic study e. gatto a cordopatis p. 31st european peptide symposium sar studies of triazolyl-containing cyclopeptides: a defined -turn structure increases potency and selectivity to melanocortin receptor subtypes c. testa, a,b proc. natl. acad. sci proc. natl. acad. sci usa multicomponent reactions microglobulin: a "difficult" protein s. abel, m. beyermann 1 the protein ß2-microglobulin constitutes the noncovalently bound light chain of the major histocompatibility complex class i (mhc) and plays an essential role in the dialysisrelated amyloidosis. [1, 2] to examine the amyloid fibrils of the ß2-microglobulin (ß2-m) via infrared spectroscopy we intended to synthesize 13-c-labeled ß2-m. [3] due to the two cysteine residues in positions 25 and 80 we used the native chemical ligation (ncl) strategy for assembling the 99-mer protein. this necessitates the synthesis of three segments which was accomplished on solid phase using the fmoc/t-bu chemistry. the preparation of the segments had to be optimized with respect to aggregation, aspartimide and piperidide formation, trifluoracetylation, and s-tert-butylsulfonium formation. additionally, ncl steps had to be optimized, because of "internal" thioester formation, dimerization and the formation of side-products of the activated n-terminal segment peptaderm inc., krakowskie przedmie cie str. 13, warsaw, poland immunosuppressors, such as cyclosporine a (csa) and tacrolimus®, are routinely used in prevention of graft rejection after organ transplantation and in therapy of some autoimmune diseases, including skin inflammation. a naturally occurring in linseed oil cyclolinopeptide a (cla, c(-pro-pro-phe-phe-leu-ile-ile-leu-val) 1 possesses a strong immuno-suppressive activity, comparable at low doses with that of csa 2 , but is much less toxic. we synthesized new cla analogs, containing instead of one proline residue its six-membered mimics, pipecolic acid (pip): c(-pip-pro-phe-phe-leu-ile-ile-leu-val) (1) and c(-pro-pip-phe-phe-leu-ile-ile-leu-val) (2). the incorporation of pipecolic acid residue led to different conformational behavior of the nonapeptide cycle. nmr experiments in cdcl 3 solution showed that cla analogue 1 with the pipecolic acid residue in position 1 was much more flexible than cyclopeptide 2. the new peptides were devoid of toxicity up to 100μg/ml with regard to human peripheral blood mononuclear cells (pbmc), did not inhibit tumor necrosis factor alpha production in blood cell culture, but exhibited dosedependent, anti-proliferative actions for phytohemagglutinin a-activated pbmc. since peptide 1 was more potent it was tested for growth inhibition of l-1210 lymphatic leukemia. the peptide was found to strongly inhibit the cell growth even at low concentration (63% inhibition at 5μg/ml). hiv-1 has emerged as the largest and the most devastating public pandemic in our days, affecting approximately 70 million people worldwide 1 . development of an effective, safe and preventive hiv vaccine remains an urgently needed priority. epitopes for hiv-specific antibodies in elite controllers, a subgroup of long term non progressors, encompassing segments of mper of gp41 and for the v3 loop of gp120 were identified using the phage display technology 2 . immunization experiments with epitopes conjugated to an artificial sequential oligopeptide carrier (soc4), formed by four repeats of the tripeptide lys-aib-gly in tandem, or to the palmitoyl group are currently in progress. all syntheses were performed on a rink amide resin following the fmoc technology. conjugation of epitopes to the soc4 carrier was realized via a chemoselective ligation approach, which generates an oxime bond between the h2n-o-groups of the modified lysine residues and the aldehyde group of each epitope 3 institute of immunology and experimental therapy, polish academy of sciences, 53-114 wrocław, poland 4 peptaderm inc., krakowskie przedmieście 13, 00-071 warszawa, poland nonproteinogenic amino acids have been a tool to modify the structures of natural peptides since a long time 1 . bioactive peptides involved in a physiological and biochemical processes cannot be applied in the therapy because of their instability in physiological conditions. that's why the synthesis of their stable active analogues is a challenge for medicinal chemistry nowadays. 4-trans-hydroxyproline (hyp) is an important building block of natural collagen. it is responsible for the stabilization of collagen super helix, forcing the trans amide bonds configuration with preceding amino acids 2 . at the same time the impact of trans-4-hydroxyproline on the conformation other than the collagen peptide chains of biologically important compounds is little known. it is known that immunosuppressive activity of cla is comparable with cyclosporine a and is associated with the presence of the tetrapeptide fragment pro-pro-phe-phe containing pro-pro cis amide bond. 3 now we present synthesis, conformation and biological activity of new analogues of cyclolinopeptide a (cla), containing 4-transhydroxyproline instead of proline residues in position 6 or 7. we expected that the introduction of the hydroxyl group in the pyrrolidine ring might influence the biological activity and conformation of the native peptide due to its hydrophilic character and hydrogen bonding ability. the linear precursors of modified cla analogues were prepared manually by standard solid-phase procedure "step by step" on wang resin using fmoc/tbu strategy and tbtu as coupling reagent. the cyclizations of linear peptides have been made under high dilution conditions by means of edc/hobt coupling reagents. the biological activity of newly synthesized compounds as well as the conformational study will be evaluated. dip. di scienze ambientali, seconda università di napoli, caserta, italy nmr spectroscopy is a powerful method to perform structural studies on peptides. to completely fulfill the potential of nmr, peptides labeled with stable isotopes ( 15 n, 13 c, 2 h) are essential. 1 peptides are easily prepared on solid-phase but chemical synthesis becomes prohibitively expensive when applied to the incorporation of isotopes. an alternative cost-effective strategy is the recombinant expression of peptides in e. coli as fusion constructs with carrier proteins. 2 the main problem of this approach is the need of chemical reagents or proteases to cleave the target peptide from its fusion partner after purification. proteases may determine the heritage of extrasequence amino acids at the peptide n-or c-terminus, while chemical reagents require harsh reaction condition that may modify target peptides. an interesting solution is represented by the use of inteins as fusion partner. inteins are protein elements that can catalyze their self-excision from a flanking sequences in mild conditions, by adding nucleophilic agents such as thiols or simply by shift of ph and temperature, bypassing the use of proteases or chemical reagents. 3 we used the self-cleaving mxegyra mini-intein as fusion partner for the preparation by recombinant means of two isotope labeled peptides, hplw and qk. 4, 5 the two peptides target vascular endothelial growth factor receptor (vegfr) and have been described to modulate vegf-dependent angiogenesis. our expression and purification scheme allows to obtain homogeneously isotope labeled peptides. the availability of isotope labeled hplw and qk opens the way to nmr studies aimed to characterize the folding dynamics of the two peptides and their structures in complex with vegfr. an nmr method to discriminate between the fullyextended and different helical conformations in a spacer peptide c. peggion*, m. crisma, f. formaggio, c. toniolo icb, padova unit, cnr, department of chemistry, university of padova, 35131 padova, italy the ideal fully-extended, α-peptide conformation, also known as 2.05-helix, is characterized by φ = ψ = ω = 180°t orsion angles. the repeating motif of this foldamer is a pentagonal (pseudo)cyclic structure (called c5), stabilized by an intraresidue h-bond. the n-h and c=o groups in the 2.05-helix are not involved in intermolecular h-bonds. multiple c5 conformations were observed in homopeptides made up of c α,α -dialkylated glycines with both side chains longer than a methyl. this is the case for c α,αdiethylglycine (deg), the residue studied in this work. it is known that deg homo-peptides can adopt the 2.05-helix 1 or the 310-helix depending on environmental factors and nand/or c-terminal moieties. 2, 3 in this communication, we introduce an nmr method to discriminate between the 2.05-helix and the 310-helix based on the observation of cross-peak intensities in the noesy human serum amyloid a (saa) is a highly conserved apolipoprotein produced by the liver under inflammatory conditions accompanying e.g. atherosclerosis, cancer and amyloidosis [1] . it is also known that saa1α isoform has the amyloidogenic properties [2] . till now it is little known about structure of human saa, as it hampers structural studies due to its facile aggregation. the analysis of protein sequence and cd data together with theoretical studies revealed a typical globular structure of the protein [3] . the c-terminal sequence of saa contains three proline residues, which probably are responsible for the unordered structure. recent in vitro studies involving saa and human cystatin c (hcc) revealed direct interactions between the (86-104) fragment of saa and the (93-120) sequence of hcc. the results of elisa test for the (86-104) saa fragment have shown that it binds to hcc very well. the nmr studies for the wild (86-104) sequence found an unordered structure in phosphate buffer. based on these data we decided to check how the point mutations pro→ala in (86-104) saa fragment could influence the peptide's structures. we synthesized four peptides with pro→ala point mutations and we performed cd experiments at different conditions. the results show that two of them contain disordered structure and two α-helical structures. in this project we analyze the solution structures of these peptides at the atomic resolution using 2d nmr supported with molecular dynamics. design and conformational analysis of stapled peptides mimicking cullin3 binding region to kctd11. i. de paola, a l. pirone, a e. pedone, a s. di gaetano, a l. vitagliano, a r. fattorusso, b g. malgieri, b l. zaccaro*, a acknowledgement: this study was supported by eu within the european regional development fund (poig. 01.01.02-00-007/08-04). model of angiotensin ii bound to the at1 receptor in the lipid bilayer environment m.t. matsoukas, t. tselios* department of chemistry, university of patras, gr-26504, patras, greece the renin-angiotensin system () plays a major role in blood pressure regulation. a sequence of enzyme reactions leads to the release of angiotensin ii which interacts principally with the type-1 angiotensin ii receptor (at1), a 359-residue, which belongs to the g protein-coupled receptor family. in the present study, the human at1 3d model was constructed using modeler for the sequence alignment and loop refinement tools. on this basis, the crystal structure of bovine rhodopsin, (pdb code 1u19), was used as a 3d template. the gromacs software and amber99sb forcefield were utilized for molecular dynamics calculations [1] in order to evaluate the binding mode of angiotensin ii. the role of the critical amino acids of the binding site v108, n111, l112, a163, k199, s252, h256, n294 and y292 is being studied. moreover, newest information on the role of the 2 nd extracellular loop by unal et. al. [2] have been implemented on the model, therefore we propose the contribution mechanism of the residues f170-q187 for binding of angiotensin ii to the at1 receptor for activation and signaling. a. stavrakoudis department of economics, university of ioannina, greece one key step in the immune response against infected or tumor cells is the recognition of the t-cell receptor (tcr) by class i major histocompatibility complexes. it has been found [1, 2] that such peptide/mhc complexes can interact with antibodies as well. this happens mainly in the central part of the peptide in class i complexes [1] , or at the cterminal of class ii complexes [2] . in some cases, the same peptide/mhc complex has been found to interact with both tcr and antibodies [3] . in these study a series of supermolecular complexes have been studied with stateof-art molecular dynamics simulations [4] the dipeptide kyotorphin (tyr-arg, kyo) plays a role in pain modulation in the mammalian central nervous system (cns), and is one of the most investigated neuropeptides. the tyr-arg motif exists widely throughout the brain not only as kyotorphin, but also as the n-terminal part of several endogenous analgesic peptides 1,2 . also, this peptide is very rapidly degraded by aminopeptidases 3 . one of the successful strategies in the design of neuropeptides with enhanced stability and improved delivery to the cns is that with the use of non-protein amino acids, like canavanive (cav), a structural analogue and antimetabolite of arginine (arg trichogin ga iv (noct-aib-gly-leu-aib-gly-gly-leu-aib-gly-ile-lol, in which noct is n-octanoyl and lol is leucinol) is an antimicrobial lipopeptaibol, a unique group of membraneactive compounds of fungal origin, characterized by a high content of the nonproteinogenic ca,a-disubstituted glycine aib (a-aminoisobutyric acid). owing to the gem-dimethyl substitution on the c a atom, aib exhibits a strong propensity to induce β-turns and 310/α-helical conformations in peptides. we have previously reported on a fluorescent analog of trichogin ga iv, the primary structure (and acronym) of which are: fmoc-aib-gly-leu-aib-gly-gly-leu-toac-gly-ile-leu-ome (f0t8) where fmoc is fluorenyl-9-methyloxycarbonyl, toac is 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, and ome is methoxy. the double substitution of an energy donor (fmoc) at the n-terminus and an acceptor (toac) in the trichogin sequence enabled us to make use of time-resolved optical spectroscopies, spanning from the nanosecond to the microsecond time regime, to investigate the conformational propensity and the dynamical features of f0t8. experimental and computational results indicated that the 3d-structural and dynamical properties of f0t8 are characterized by a transition from an elongated helix to a more compact conformation mimicking a helix-turn-helix motif. to further investigate the role of the flexible gly5-gly6 central motif we synthesized a new trichogin analog having the gly6 residue substituted by aib: fmoc-aib-gly-leu-aib-gly-aib-leu-toac-gly-ile-leu-ome (f0a6t8) experimental and computational results indicated that also the f0a6t8 peptide populate two conformations, the dynamics of which were studied at different temperatures using time-resolved spectroscopic measurements. this replacement was demonstrated to stiffen the peptide backbone by reducing the flexibility around the crucial -gly5-gly6-dipeptide unit. the antigen α4β1, a member of the integrin family, is involved in the migration of lymphocytes through endothelium to the site of inflammation. 1 thus, α4β1 antagonists may be useful tools for the treatment of various inflammation disorders such as asthma and inflammatory arthritis. in addition, recent studies indicate that α4β1 integrin promotes angiogenesis by allowing the invasion of myeloid cells into tumors, while α4β1 antagonists prevent monocyte-induced angiogenesis, macrophage colonization of tumors and tumor angiogenesis. 2 aiming to the discovery of novel α4β1 antagonists, a series of new peptide analogues cyclized through cysteine disulphide bonds were synthesized and tested in vivo against angiogenesis in chicken embryo chorioallantoic membrane (cam model) 3 . sar results indicated that: yr-c(cdpc)-conh2 promoted angiogenesis at the higher studied concentration and showed slight inhibition at the lower one, sal-r-c(cdpc)-oh, sal=salicylic acid, showed important inhibition of angiogenesis at dose-dependent manner, yr-c(cdpc)-oh and sal-yr-c(cdpc)-oh both showed no activity on angiogenesis. nmr spectroscopy was applied for the sequential assignment as well as for the elucidation of specific conformational features. experimental noe data were further imposed as distance constraints to a thorough conformational search by applying molecular dynamics simulations. energy refined produced conformers were used as template for the generation of the pharmacophore model associated with the antagonistic activity. such studies are intended to drive a rationalized design and development of this class of inhibitors. hynes r. o. cell, 1992, 69, 11-25. scaffold discovery by phylomers: a novel cd40l specific scaffold derived from glycyl trna synthetase s.r. stone [1] , k. hoffmann [1, 2] , n. milech [1, 2] , p. t. cunningham [1] , m. kerfoot [1] , s. winslow [1] , y-f, tan [1] , m. anastasas [1] , c. hall [1] , m. scobie [1] , p.watt [2] , and r. hopkins [1, 2] [1] drug discovery technology unit, telethon institute for child health research, 100 roberts road, subicao, 6008, western australia [2] phylogica pty ltd, 100 roberts road, subiaco, 6008, western australia biopanning of phylomer 1 phage display libraries against human cd40l yielded a cluster of highly specific overlapping peptide fragments, from three bacterial genomes, corresponding to the highly conserved catalytic domain from the tetrameric gα2β2 class of glycyl trna synthetases. structural analysis of the overlapping peptide fragments described a scaffold consisting of a central βsheet, comprising 4 anti-parallel β-strands, flanked by nand c-terminal α-helices. further structural analysis revealed that these key structural features, which also encompass the crucial atp-binding motifs of the catalytic domain, are conformationally conserved across both tetrameric gα2β2 and dimeric gα2 glycyl trna synthetases, yet importantly, there is only limited sequence conservation across these classes. given the identical function of the described domain and it's structural conservation, we postulated that members of the dimeric gα2 class would display similar cd40l specific binding as the tetrameric gα2β2 class, despite the sequence dissimilarity. to test this hypothesis, structurally equivalent peptide fragments of representative bacterial, archaeal and eukaryotic genomes comprising the dimeric gα2 class were tested for cd40l binding in a process we termed ortholog scanning. the results showed that both archaeal (p. horikoshii) and eukaryotic (h. sapiens) structurally equivalent peptides bound to cd40l with reasonable specificity and inhibited the cd40:cd40l interaction with comparable ic50's to the primary gα2β2 class sequences. similar results were also observed for the representative bacterial gα2 class peptides. that the sequentially diverse orthologous peptides display cd40l specific binding has important implications to the affinity enhancement strategies to develop the scaffold as a therapeutic agent, and in improving its "drug-like" properties. we have initiated 1 an investigation related to the effect of radical species upon structures of some peptide segments. in the proposed experimental protocol, aqueous peptide solution was submitted to gamma ray irradiation in controlled 1-15 kgy doses. the generation of peptide analogues, possibly induced by reactive oxygen species were examined by electrospray triplequadrupole tandem mass spectrometry (collision induced dissociation approach) and amino acid analysis of crude and/or purified by-products. noteworthy, the gamma irradiation process induced, regardless of the peptide sequence, a non-linear and progressive degradation of all peptides assayed. furthermore, these peptides could be classified in some different classes according to their halflife dose. for instance, the vasoactives angiotensin ii (aii), ang (1-7), bradykinin (bk) and some related peptides were more stable than the melanocyte-stimulating hormone α-msh, substance p or the bk 's (305-325) b2 receptor fragment (lvyvivgkrfrkksrevyqai). usually, the most prominent derivatives generated from this experimental protocol revealed that they are likely induced by oxidation process, yielding a variation of +16 da in their molecular weight. the main source of peptide modifications seems to lie either on the phe (hydroxyl group insertion at o-, m-or p-positions of its aromatic side chain) or met oxydation. in the former case, only phe 8 and not phe 5 is oxidized in the bk structure whereas substance p generates an analogue bearing metsulfoxide without modifying its phe 7,8 residues. thus, collectively, these findings clearly stress the complexity of factors involved in peptide structural modifications induced by gamma ray-type strong electromagnetic irradiation experiment. an additional target of this approach lies indeed, in the production of unusual peptides for further structure-function investigations. university of bern, bern, switzerland linear peptides are typically poor drug candidates due to their low bioavailability and rapid proteolysis. these limitations can be overcome by rigidifying their structure through head-to-tail or side chain-involving cyclizations. cyclic constraints may also increase biological activity by stabilizing secondary structures and by reducing the entropic penalty of binding to a protein target. the use of multiple branching amino acids in a peptide sequence, like diamino acids (as used in peptide dendrimers 1 ) or amino diacids, allows to design peptides resembling polycyclic alkanes, a type of topology only rarely found in nature (e.g. amatoxins and lantibiotics). bicyclic homodetic peptides such as "norbornapeptides" (bicyclo[2.2.1]heptapeptides) were prepared using an orthogonal protection scheme: the first cyclization is performed on resin after selective deprotection of a glutamic acid residue, whereas the second ring closure is achieved by amide bond formation at high dilutions. these peptides are structurally well-defined and cover an almost pristine area of peptide topological space. 2 their conformational rigidity was investigated by means of 2d-nmr and x-ray crystallography and may offer a platform to design drugs tackling protein-protein interactions. the interaction of peptide ligands with protein receptors face peculiar challenge in recognizing binding surfaces due to availability of a multitude of conformations. therefore it is essential to constrain the peptide conformations for the recognition of receptors and thus finding the bioactive conformation. the cell surface receptor protein family integrins recognize "rgd" sequence which is present in different proteins. to determine the bioactive conformation required to bind with receptor αiibβ3, the peptide sequence "riprgdmp" from kistrin was inserted into cdr 1 loop region of rei protein (rei-rgd34). it helps out in finding the possible bioactive conformation of peptide by restricting the sampling space. the activity of rei-rgd34 was studied and found that as the temperature increased rei-rgd34 showed a higher affinity towards the receptor αiibβ3. the proposed mechanisms for the increased activity of rei-rgd34 at higher temperature were justified in either of two ways. the modified complex forces the restricted peptide to adopt a bioactive conformation or it unfolds the peptide in a way that opens its binding surface with high affinity for receptor. in this study we model the conformational preferences of "rgd" sequence in octapeptide "riprgdmp" at two different temperatures (250 o c and 420 o c) using multiple md simulations. we found that at higher temperature "rgd" sequence from "riprgdmp" adopt turn conformation, while a bend conformation was observed at low temperature. the analysis of various pharmacophoric parameters hint that the turn conformation of "rgd" sequences adopted at higher temperature could be the potential bio-active conformation, and helps out in designing of antagonists for cell surface receptor αiibβ3. the 14-residue peptaibol antibiotic trichovirin i-4a (tv) of the linear, covalent structure ac-aib-asn-leu-aib-pro-ala-val-aib-pro-aib-leu-aib-pro-leuol (ac, acetyl; aib, α-aminoisobutyric acid; leuol, l-leucinol) has been synthesized1 and very thin (~25 μm) hair-like crystals were obtained from a methanolacetonitrile-water mixture. diffraction data were collected at 100 k at the diamond light source england, using the microfocus beamline 2 i24 and a x-ray beam focused to a size of 10 μm full-width-half-maximum. two independent molecules (a) and (b) were located in the crystal's asymmetric unit 3 . both chains assume 4 complete turns of a curved 310 right-handed helical conformation stabilized by intramolecular hydrogen bonds. up to now tv represents the longest right-handed 310-helix of a natural peptaibol sequence complementing those of synthetic, protected homooligo-aibinsulin is a protein hormone that plays a key role in regulation of blood glucose levels and, thus, has widespread impact on lipid and protein metabolism.insulin is known to act through binding to the insulin receptor (ir); however, the structure of the insulin-ir complex is not known. the crystal and nmr structures of insulin represent only inactive storage forms. it is widely acknowledged that insulin must undergo structural changes in the c-terminus of the b-chain upon binding to the ir. in addition, the n-terminus of the bchain may adopt two different conformations in hexamer, known as t-and r-states. the r-state of the n-terminus of the b-chain creates a long b1-b19 central a-helix. the t-state of n-terminus is in an extended conformation. however, the biological relevance of the t/r forms remains elusive 1 . in this study, we have focused on the synthesis of new insulin analogues modified at the nterminus of the b-chain and subsequently correlated their biological activities with their 3d-structures. the invariant residue glyb8 seems to be critical for the t/r transition. glycine can adopt wide range of dihedral angles (φ/ψ) and it occupies significantly diverse dihedral angles in t-and r-states. a-aminoisobutyric acid (aib) is an amino acid with a high helical propensity, which often folds into right-or left-handed α-helix. we have introduced aib at position b3, b5 and b8 with the aim to induce the r-state of the hormone. in contrast, as d-pro and nmeala are not able to adopt the φ/ψ angles of the right-handed α-helix we have introduced these amino acids at position b8 to obtain the t-state of insulin. peptide dendrimers are tree-like molecules formed by alternating functional amino acids with branching diamino acids such as lysine. 1 unfortunately these molecules have not yielded to structural characterization and little is known about their molecular-level structure. computational methods seem to be an adequate tool to address these issues.herein we present a comprehensive structural characterization of peptide dendrimers using molecular simulation methods. 2 multiple long molecular dynamics (md) simulations were used to extensively sample the conformational preferences of several third-generation peptide dendrimers, including some known to bind aquacobalamine. we used several conformational analysis procedures (clustering, energy landscapes and multivariate analysis) to analyze conformational changes that can be correlated with particular structural trends.the results point to a high conformational flexibility of these molecules, with no clear "folded state", although two markedly distinct behaviours were identified. some dendrimers favour mainly loose conformations, while others prefer more compact configurations. through a series of computational mutations we investigated the influence of the presence and placement of charged residues in dendrimer topology, finding that electrostatic interactions among charged residues are a major determinant in structure acquisition by peptide dendrimers. these conclusions bring new insight into the conformational behaviour of these systems and may provide better routes for their functional design. acid-mediated prevention of aspartimide formation in solid phase peptide synthesis t. michels, a r. dölling, b u. haberkorn a , w. mier*, a a department of nuclear medicine, university hospital heidelberg, 69120 heidelberg, germany; b biosyntan gmbh, robert-rössle-straße 10, 13125 berlin, germany aspartimide formation is one of the major obstacles that impede the solid phase synthesis of large peptides and proteins. the main reason for aspartimide formation is the piperidine-catalyzed fmoc cleavage of peptides containing aspartic acid. several side chain protecting groups have been developed 1 but the complete prevention of aspartimide formation can only be achieved using n-(2hydroxy-4-methoxybenzyl) (hmb) as backbone protecting group. 2 however, hmb-protected building blocks are difficult to synthesize and only the dipeptide containing glycine (fmoc-asp(tbu)-(hmb)gly) is commercially available. until now, no cost effective strategy to suppress this side reaction has been developed. formally, aspartimide is the result of an attack of an amidate species at the carbonyl carbon of the otbu protected side chain carboxylate of aspartic acid, which might be prevented by protonation of the amidates with piperidinium ions. in this work the suppression of aspartimide formation by adding small amounts of organic acids to the deprotection agent piperidine was studied. this procedure was shown to efficiently prevent the formation of aspartimide side products in several peptides, i.e. pres9-33-y, a 26-mer peptide derived from the hbv surface antigen and a peptide parathyroid hormone (pth) fragment. the testing of a series of 18 different acids covering a broad range of pka values showed that this effect is virtually independent of the acid strength. since aspartic acid is found in most oligopeptides, the authors recommend to generally add 5% (v/v) formic acid to piperidine based fmoc cleavage mixtures. decomposition of the resin linkers during tfa cleavage of peptides in fmoc-strategy leads to alkylation of sensitive amino acids 1 . this side product formation is a crucial drawback, especially during the synthesis of biologically important cys-containing peptides on wang support. through a battery of approaches (1h-nmr, uv and lc/esi-ms) we detected an unexpected alkylation of the sulfhydryl group of cysteine side-chain residues by the phydroxyl benzyl group from the wang resin linker. herein, we present the feasibility for s-alkylation of cys-containing peptides from wang linker decomposition. this sidereaction occurs during the final tfa cleavage of the peptide from the solid support, while the position of the cysteine residue within the peptide sequence as well as the resin's substitution influence the extent of cys-alkylation. the stephan angeloff institute of microbiology, bulgarian academy of sciences, sofia, bulgaria influenza viruses cause epidemics and pandemics all over the world. therefore, the development of virus resistance to drugs, leads to search for novel derivatives and approaches to chemotherapy for human influenza infection. antioxidant therapy is known to be one potential approach. the application of combination therapy of antioxidants with antiviral drugs could reduce the complications and lethal effects, caused by an influenza virus 1 . in our study, amino group of neuraminidase inhibitor -oseltamivir, which belong to second generation anti-flu drugs, was covalent conjugated with known antioxidantscysteine, histidine. tryptophan and etc. the study of the role of the modified by antioxidants oseltamivir on proliferation of influenza virus is in progress. recently we reported a short synthesis of 5 or 6-membered cyclic guanidine via intramolecular reaction of alkyl diamine with n,n'-cbz-methylisothiourea(1a). here we report a further application of synthesis two types of cyclo-peptide guanidine-bridged cyclopeptides utilizing this mechanism -n-terminus local cyclo-guanidine peptide and backbone guanidine-bridged marco-cyclic peptide. three n,n'protected diaminoacids (daa) including 2,4-diaminobutyric p334. antifreeze glycoproteins (afgps) are found in the deep sea teleost fish in arctic and antarctic oceans. these biomolecules are able to inhibit the growth of ice crystals and depress the freezing temperature of the blood serum in fish enough to keep them from freezing in their sub-zero environments while the melting temperature remains unchanged 1 . despite afgps have been consider as a potent cryopreservation, obstacles to develop afgps as medicinal and industrial application are mainly due to the lack of access to pure form from natural sources and the problem of understanding how afgps inhibit ice crystal growth. as a result, a considerable progress toward the design and synthesis of afgp analogues has been made several groups 2 . in the course of the studies on the structure-activity relationships of afgps, we are interested in peptoids as mimics of α-peptides and synthesized monoglycosylated peptoid analogues by substituting the glyco-thr residue as afgps mimics. in this presentation, we will show our studies on how the insertion of peptoid residue into afgp backbone affects the afgp activity by measuring both thermal hysteresis (th) and ice recrystalliztion inhibition (iri). [1] , both for diagnosis and endoradiotherapy. for this application the peptides can be attached with chelating agents that bind radioactive metals such as 67 ga, 68 ga or 111 in for imaging or therapeutic radiometals such as 90 y and 177 lu. the chelating agent most frequently applied is the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-n,n',n'',n'''-tetraacetate (dota), it is commonly introduced as the tris(tbu ester). the cleavage of the tbu protecting groups on dota is known to be sluggish [2] . several attempts have been made to synthesize dota with protecting groups that can be removed under mild conditions. however, these derivatives have not yet found widespread application. our new approach was to prepare a protecting group for dota-based prochelators that is convergently cleaved under the cleavage conditions of the amino acid protecting groups of the peptide. o-phenylisopropyl (opp) esters are more sensitive towards acid than tbu esters. deprotection occurs with 2% trifluoroacetic acid in dichloromethane [3] . therefore, a synthesis of the prochelator dota-tris(opp ester) was developed. the copper-catalyzed azide-alkyne cyclization (cuaac), the most commonly recognized variant of "click chemistry," has emerged as a powerful technique for ligation, conjugation, and cyclization reactions of peptides. it is known that cyclization can increase the metabolic stability of peptides, as well as enhancing potency or selectivity by stabilizing an active conformation. one application of the cuaac that has generated interest is the use of this reaction to replace a disulfide bridge with the product triazole, which among other complementary properties may prevent in vivo redox chemistry. in this poster, we synthesize a new analogue of the cyclic cancertargeting peptide cngrc where we replace the disulfide bond with a triazole linkage using click chemistry and a fully automated, on-resin method using the single-shot delivery feature on the prelude® peptide synthesizer. unnatural amino acids including d-amino acids are manufactured mainly by the enzymatic process. however, one enzyme can produce only one amino acid due to its high specificity and it takes a long time and a lot of expenses to develop the appropriate enzyme itself. arca (alanine racemase chiral analogue) is an organic catalyst1 which can overcome these drawbacks and can produce almost all kinds of amino acids efficiently. the amine functionality of l-threonine is freely reacted with the aldehyde group of arca to form the corresponding imine, which is easily epimerized in the presence of organic base due to the acidity of the alpha proton of imine. the difference in the stability between the imines of the optical epimers rendered them to be shifted to d-allo-threonine derivative dominantly. once the epimerization reaction reached equilibrium, the reaction mixture was hydrolyzed under acidic condition to give d-allo-threonine and arca, which could be recycled repeatedly without significant loss in yield or purity to produce more d-allo-threonine from lthreonine in excellent yields. optimization of the reaction conditions with various bases and solvents is discussed and mass production of optically active d-allo-threonine including optical purification is described. the manufacuring process for the preparation of arca will be shared as well. our group is interested in the development of efficient synthetic routes for the preparation of enantiopure atrifluoromethylated amino acids (a-tfm-aas) starting from chiral cf3-oxazolidines or imines 1 and their incorporation into a peptide chain. 2 these non-natural amino acids are very attractive compounds for the design of biologically active molecules, particularly peptides, due to the unique physical, chemical and biological properties impart by the cf3 group. 3 as conformationally constrained cyclic amino acids have recently gained considerable interest, we are particularly focused on the preparation of pyrrolidine-type a-tfm aas. 1, 4 incorporation of proline derivatives is known to restrict the amino acyl-proline cis/trans isomerization, to limit the protein folding and consequently to modulate the biological activity of peptides. based on these observations, mutter's group introduced pseudoproline building blocks (ψpro) into a peptide sequence as reversible protecting groups for ser, thr and cys. 5 the ψpro residues proved to be versatile tools for overcoming the aggregation caused by hydrophobic interactions encountered during solid-phase peptide synthesis (spps). they also turned out to be inducers of βturns containing predominantly cis-amide bond and useful tools in peptide cyclization. here, we report the results obtained for the preparation of various hydrolytically stable trifluoromethylated pseudoprolines (cf3-ψpro) as well as the methodological studies developed to optimize the synthesis of various c-and n-terminal cf3-ψpro containing dipeptides. rennes, france protein strructure and function rely on a still not fully understood interplay of energetic and entropic constraints defined by the permutation of the twenty genetically encoded amino acids. many attempts have been undertaken to design peptide-peptide interaction pairs and synthetic receptors de novo by using special building blocks. 1 a rational approach starting from hydrazine to create new building blocks based on a tailored metalchelating amino acid analogues was envisaged. to create chemical recognition units, which bind oligohistidine tags with high affinity and stability, several supramolecular entities containing one to three nitrilotriacetic acid analogue (ynta) moieties were synthesized. these new building blocks additionally contained an amino group or an acido group, which can be flexibly introduced into peptide in n or c-termini or into the peptidic chain by solid phase chemistry in fmoc/t-bu strategy. these multivalent chelators were characterized and the corresponding metalchelating peptides could act as metal sensors and synthetic receptors for histidine-tagged proteins. the potential of peptides as drug candidates is often limited by their pharmacokinetic properties. structural modification of the peptide backbone via n-methylation is a powerful medicinal chemistry tool that confers oral bioavailability to these molecules. n-methylation exerts a strong effect on the backbone conformation and, as a result, many n-methylated peptides show enhanced biological activity and higher receptor selectivity. another approach to increase the solubility of peptides is by conjugation of peg to a derivatizable functionality. by combining these two approaches we have developed n-oegylation. this novel form of peptide modification consists of the attachment of oligoethylene glycol (oeg) chains to the amide bonds. many bioactive peptides comprise one or more n-me amino acids which are essential for their activity. thus, we consider that replacement of a backbone n-me group by an oeg chain may imply a minimal structural perturbation and may lead to n-oegylated peptides with preserved biological activity. furthermore, our strategy is a promising way to improve the bioavailability of cyclic peptides that do not have any site where a peg could be attached. as a proof of principle, several n-oeg analogs of two bioactive cyclic peptides were synthesized in spps. first, we performed a full n-oeg scan of the sansalvamide a peptide. next, several analogs of cilengitide were prepared by replacing the n-me of valine by oeg chains of different length. depending on its size, the oeg residue was incorporated by using an n-oeg derivative as building block or, alternatively, using an n-substituted amino acid bearing an attachment site where a peg was conjugated post-synthetically.the biological activity of all the n-oegylated peptides was evaluated. some of the sansalvamide a peptide analogs exhibited cytotoxicity within the same range as the original peptide, which suggests that backbone amide groups may be useful oegylation sites in bioactive cyclic peptides. the modification of peptides is an important step in pharmacology to vary the affinity and the stability of peptidic drugs. whereas a wide range of strategies exists for the functionalization of the n-terminus and the side chains, facile variation of the c-terminus remains an important challenge. we consider peptidyl-phosphoranes as a promising platform to enable orthogonal and mild introduction of a great variety of chemical functionalities at the c-terminus. a convenient method for the synthesis of soluble peptidyl-phosphoranes has been presented by our group recently. 1 in this, 2-bromo-acetyl bromide was coupled to a wang-resin followed by alkylation of triaryl-or trialkyl phosphine moiety. deprotonation to the phosphorus ylide and subsequent acylation with an fmocamino acid created the basis for assembly of the peptide by spps. final acidic cleavage produced a decarboxylated and unprotected, soluble peptidyl-phosphorane. from this point, a variety of orthogonal modification reactions at the peptides c-terminus is possible, e.g. click reaction with azides allow for the incorporation of triazoles as peptide bond mimetics. the wittig reaction opens up another interesting portal for c-terminal modification, as vinyl ketones are formed by reaction with aldehydes. the described chemistry was applied to modify caspase-3 inhibitors. in order to address the s1 site of caspase-3, the commonly known devd inhibitor was varied at the c-terminus by introduction of different residues. the devd motif was synthesized as peptidyl-phosphorane and modified in wittig reactions. the resulting c-terminal vinyl ketones were obtained by the reaction of aliphatic and aromatic aldehydes. a small library was generated, and 12 novel compounds were tested for their potential to inhibit caspase-3. semmelweis university, department of biophysics and radiation biology, budapest, hungary considering the impact of uv irradiation on the structure and function of proteins 1 , it is a matter of utmost importance to resolve the conditions of photolysis more deeply. we think that a protein, as a complex unit, gives multiple responses to all impacts therefore the analysis of these responses is a rather complex problem. the main goal of our research is the deeper understanding the tryptophan-mediated photolysis of disulphide bridges in bio-active proteins upon near-uv irradiation using cyclic peptide models, as small protein units, to define the caused functional damage. cation -pi interaction is increasingly recognised as an important noncovalent binding interaction which plays a role in establishing the final structures of proteins. within a protein, cation -pi interactions can occur between the cationic side-chains of either lys or arg and the aromatic side-chains of phe, tyr or trp 2 . our earlier results with gla indicate that new covalent bonds are also formed between cys and lys during illumination, which is also a reason why the lys residue is planned to be included in the sequence of the models. our aim is to study whether the cation -pi interaction can have an influence on the ss-bridge splitting in small cyclic pentapeptide models. here we report about the conformational analysis, synthesis and spectroscopic investigation of lys-and arg-containing model peptides. the azide functionality is very popular mainly due to azidealkyne click chemistry 1 used in many peptide ligation strategies. azido-peptides are usually prepared by incorporation of azide containing residues or azide functionalization of aldehyde resins affording c-terminal azido-peptides. 2 alternatively, the n-terminus can be converted into an azide via a cu(ii)-catalyzed diazotransfer reaction using triflyl azide. 3 recently, a number of safer, shelf-stable and easily prepared diazotransfer reagents has been developed, of which imidazole-1-sulfonyl azide has been used to introduce azide moieties in proteins under copper-free conditions. typically, it has not been reported to be used on the solid phase. 4 we provide a very easy, fast and efficient method for conversion of amines into azides on a solid phase support which in our opinion has major benefits over earlier reported methods making using of less stable reagents that require a metal ion catalyst. we demonstrate how the diazotransfer reaction can be performed on a solid phase support using the imidazole-1sulfonyl azide reagent without the need for a metal ion catalyst. using a model peptide we studied the effect of stoichiometry, added base and solvent. in addition we examined the effect of the nature of the n-terminal residue on the efficiency the diazotransfer reaction. finally, we found that the optimal conditions to perform the reaction also depend on the nature of the solid phase support the reaction is performed on. the novo nordisk foundation center for protein research, university of copenhagen, copenhagen, denmark site-selective strategies for post-translational modification of peptides and proteins are essential tools for many areas of research in the life sciences, yet remain a chemical challenge due to the multiplicity of functional groups present. there are powerful chemoselective reactions, however, they aim at introducing only one functionality at each reaction site. here we present a one-pot, threecomponent dual-functionalization of peptides or proteins based on a 1,3-dipolar cycloaddition between a functionalized malemide, an n-hydroxylamine and a peptide or protein with an n-terminal serine residue at the n-terminus, which is selectively oxidized to a 2-oxoaldehyde. most common moieties for labeling, e.g. fluorophors and peg-chains, are commercially available as maleimides. nitrones were easily obtained by condensation of peptide-aldehydes and primary nhydroxylamines under aqueous conditions. the chemoselective 1,3-dipolar cycloaddition reaction between the peptide-nitrone, and a functionalized maleimide proceeded in aqueous solution at room temperature or with gentle heating, which provided the stable isoxazolidine product. we envision that this 'one site -two functions' method can be used widely to introduce two separate moieties. the method was used to introduce two separate ligands in a range of other peptides. for example, new multimodal molecular imaging techniques depend on facile chemical methods for site-selective dual-functionalization. we used our new methodology to synthesize a cyclic rgd-peptide for combined pet and optical molecular imaging. finally, the small protein ipb3 was successfully n-terminally modified, including with a peg-chain, using this new, general method. multiple sclerosis (ms) is the most known chronic, inflammatory, demyelinating disease of the central nervous system (cns), characterized by a progressive neurodegeneration, caused by an autoimmune response to self-antigens in genetically susceptible individuals. it is nowadays known that post-translational modifications may affect the immunogenicity of self-protein antigens, triggering an autoimmune response and creating neoantigens; in particular aberrant glycosylations affect various parts of the immune response and have profound effects on immune tolerance. in previous studies we demonstrated the value of the glycopeptide csf114(glc) which, by virtue of the particular type i' β-turn structure, optimally exposes the minimal epitope asn(glc) to autoantibody recognizing in elisa in multiple sclerosis patients' sera 1 . elisa assays allowed to conclude that the ability in detecting autoantibodies in multiple sclerosis sera was stricktly linked to saccharidic moieties and to conformation around minimal epitope of the antigenic glycopeptide. herein, taking advantage of such considerations, we focused our attention on the synthesis of a little library of lysine branched multiple antigen peptides (maps), containing the minimal epitope asn(glc), in an attempt to increase the antigenicity of linear peptide sequences 2 . with this aim, we performed the spps of glucosylated maps via the building block approach, studying the role of different long spacers on the dendrimeric core, and the role of different peptide sequences around the sugar moiety, in order to optimize the synthetic process and to evaluate the influence on the affinity and specificity in sp-elisa. environmentally induced co-or post-translational modifications of autoantigens are hypothesized to break immune tolerance leading to self reactivity in pbc. 1 it has been previously reported that the use of synthetic post-translationally modified peptides, introducing fmoc-l-lys(nε-(±)-α-lipoic acid)-oh, as peptidomimetics of natural neoantigens allowed to detect autoantibodies in the sera of patients affected by pbc, and they might be useful diagnostic tools that can be used in earlier stage patients and possibly to monitor disease activity. 2 only the r-(+)-enantiomer of α-lipoic acid exists in nature and is an essential cofactor of four mitochondrial enzyme complexes. 3 but it remains unclear if the tridimensional structure of the lipoic acid is of any importance in the interactions antibody-peptide during the indirect elisa tests. therefore, it is necessary to synthesize each peptide separately with one absolute configuration of the lipoic acid. herein, we describe the synthesis of the two diastereoisomers fmoc-l-lys(nε -(r)-α -lipoic acid)-oh and fmoc-l-lys(nε -(s)-α-lipoic acid)-oh that have to be used in fmoc/tbu spps as building blocks for the synthesis of post-translationally modified peptides. 2 recently it has been reported the introduction of a new generation of cd diagnostics based on a unique antigen approach, consisting on human ttg cross-linked with gliadin peptides coated on the elisa plates 3 . on the basis of experimental data obtained by mass spectrometry and indicating which are the fragments of these two proteins that are supposed to be involved in the antibody recognition, we were able to select the most representative ttg and gliadin fragments 4, 5 to design and synthesize by fmoc-spps nine cross-linked eoepitopes. aim of our study was the characterization of autoantigenic epitopes by testing, in celiac patients' sera, the reactivity of these nine synthetic peptides. these neoepitopes were tested in elisa to evaluate the iga and igg response against ttg-gliadin adducts in celiac patients' sera in order to develop a new elisa test based on peptides as an even more powerful diagnostic tool in terms of specificity and sensitivity. 1 more than 80 analogs have already been described, wherein the hydroxy acid and the amino acid constituents were replaced by d-amino acids and/or n-methyl amino acids with preserved or altered side chains. for certain types of cancer cells, several of these analogs were found to be more active than the natural product itself. 2 however, it does appear that many of these compounds have limited solubility in water. 2b here we report the synthesis of 5 novel analogs of the sansalvamide a peptide bearing an n-oligoethyleneglycyl (oeg) chain attached to the different backbone positions. attachment of this chain is aimed to enhance the hydrophilicity of the original peptide. our synthetic strategy to modify the backbone with the n-oeg group relies on the use of n-oeg amino acids, which were synthesized in solution and then used as building blocks in spps. as expected, couplings to the n-oeg residues were found to require special conditions. methods for the coupling to nmethyl amino acids were applied and this enabled to obtain the different linear pentapeptides, which were cyclized in solution. both the synthetic strategies of these demanding peptides as well as the preliminar evaluation of their biological activity will be deeply discussed. glycoconjugates such as glycoproteins and glycolipids have important roles in cell functions, for example, intercellular recognition, cell proliferation control, and information transmission. in order to study the structurefunction relationship, synthesis of these glycoconjugates is essential. glycoproteins and glycopeptides are classified into two categories: n-and o-glycosylated derivatives. the n-acetyl-α-d-galactopyranosylated ser or thr derivatives [ser/thr(α-d-galnac)] are important intermediates for o-glycopeptide synthesis. however, the synthesis of ser/thr(α-d-galnac) derivatives by chemical glycosylation is difficult because of the decreased nucleophilicity of hydroxy function in the glycosyl acceptor due to an unfavorable hydrogen-bonding pattern between the oh and α-nh groups 1 . several approaches to overcome this problem have been reported 1, 2 . in addition, the o-glycosidic bond is cleaved easily in acidic conditions. in this study, we assumed that the formation of a cyclic structure containing an α-nh group would increase the reactivity of oh function. thus, we focused on the n, n'isopropylidene derivatives of ser/thr containing dipeptides 3 . we found the reaction of mannopyranosyl trichloroacetimidate and the n, n'-isopropylidene dipeptide in the presence of tmsotf in dichloromethane produced the desired glycosylated dipeptide in good yield. however the selective intermolecular disulfide bond formation is a very difficult and complicated synthetic problem. in this work we report on synthetic approaches for the formation of conjugates with intermolecular thioether or disulfide bonds. for the disulfide bond formation, we use two activation approaches: i) activation of the four cys residues of the carrier testing two activating reagents, in both solid and liquid phase respectively and ii) activation of the cys containing bioactive molecule. as bioactive molecule we selected the r 997 ppleed 1003 sequence derived from the intracellular part of the αiibplatelet integrin receptor. this region is critically involved in platelet aggregation and is a target of intervention for developing antithrombotic agents 2 . the ac-[lys-aib-cys(ch2co-αiib997-1003)]4-nh2 and ac-[lys-aib-cys(cys-αiib997-1003)]4-nh2 conjugates were synthesized and examined for their ability to inhibit platelet aggregation. the biological assays indicated that the synthesized conjugates penetrate the platelet membrane and inhibit human platelet aggregation, in contrast to the corresponding free peptide analogues. the molecules were reported to exhibit broad-spectrum cytotoxicity against the tumor cell lines. although these peptides contained the novel β-methoxytyrosine, lipton et al. reported the synthesis and cytotoxicity of desmethoxycallipeltin b, in which substitution of d-tyrosine for β-methoxytyrosine did not substantially affect the cytotoxicity of callipeltin b 3, 4 . however, a structure-activity relationship study of the molecules has not been shown to date in detail. in the course of our recent research regarding the synthetic study of cyclic depsipeptides, we conducted studies on the synthesis of callipeltins supposed to be efficient structures for ccr5 inhibitors as anti-hiv drugs or anti-cancer agents. in the present study, we report the synthesis of cyclic depsipeptides of callipeltin b analogues consisting of l-, d-amino acids and/or n-methyl amino acids, for a structure-activity relationship study of linear-and cyclic depsi-peptides against hela cells5. in the assay of synthetic peptides, all of the synthetic callipeltin b analogues exhibited no cytotoxicity. we supposed that dimethylpyroglutamic acid of callipeltin b was essential structure to show the cytotoxicity against hela cells. monash university, melbourne, australia protein-protein interactions represent a significant portion (15-40%) of all interactions within the cell; 1 as such these interactions are ideal targets for drug discovery. while difficult to target using small molecules, these interactions can be disrupted using a small section of the protein's binding partner. these short peptides must retain the defined secondary structure associated with the protein binding interface in order to inhibit their protein targets. as the secondary structure adopted by the parent protein is not always exhibited by its derived peptides, constraints are introduced as necessary to help define the structure of the peptide. inducing secondary structure reduces the energy required for organisation, decreasing the energy of binding and has the potential to increase stability with respect to degradation by proteases. 2 solid phase peptide synthesis was used to make several small peptides corresponding to the structured sections of the binding partners for three protein-protein interactions. these peptides were designed to target heart disease, prostate cancer, and liver cancer respectively. secondary structure was introduced using lactam bridge constraints. for the αhelical peptides, a side chain constraint approach was used to nucleate helix formation. as hydrogen bonding between the c=o of the i th residue and the nh of the (i+4) th residue stabilises native α-helices, constraints were introduced linking the side chains such that the residues were held in close proximity. for β-pleated peptides, an antiparallel β-sheet arrangement was achieved by introducing turn regions into the peptide in such a way that the β-strands were aligned. constraints were again introduced using lactam bridges between lys and arg or glu side chains. these peptides were characterised by nmr and cd spectroscopy to verify the correct secondary structure had been induced. göttingen, germany different properties can be combined in a single molecule by using a scaffold arranging functional groups in a predefined topology. the tasp (template-assembled synthetic proteins) concept describes templates to reinforce and direct the folding of designed molecules into a predetermined topology. [1] due to their resistance to proteolytic degradation and their rigid basic structure, cyclic β -tripeptides are suitable carrier molecules for bioactive compounds; they are further known to form tubelike structures by stacking of the peptide rings leading to higher organization of functionalized peptides. [2] with this scaffold different inhibitory systems were synthesized that feature cell penetrating and fluorescent properties. the signal transducer and activator of transcription 3 (stat3) protein, which has been described as an oncogenic protein, was selected as the first target. [3] a peptide sequence which targets the sh2 domain of stat3 was used in two different approaches. it was either directly attached to the cyclic β-tripeptide via a huisgen [2+3]cycloaddition or the peptide was incorporated into the inhibitor loop of the cystine knot microprotein omcoti-ii, which was also attached to the cyclic-β -tripeptide. [4] further, sodium channels are addressed usingconotoxines. first, an alkyne functionalized conotoxin siiia was synthesized applying different folding methods. the alkyne linker will be used to attach a fluorophore or to functionalize a cyclic-β-tripeptide. using single molecule imaging the spatial distribution, local concentration and organization of the ion channels in neurons will be imaged. further, the cyclic-β -tripeptide templating effect will be used functionalizing with μ-conotoxines. those proteins are folding helper proteins. together with chaperones, they form receptor complexes. they catalyze the isomerization of prolyl bonds in various folding states of target proteins. indeed, their role has been implicated in refolding of denatured proteins, de novo protein synthesis and the biologically active conformation of proteins 1 . among them, the fkbp subclass comprises the small ppi calstabin 1 and 2. it is of interest to try to understand the way those proteins act, in order to help the overexpression of various types of membrane proteins, aiming at the renaturation, purification and crystallization attempts of receptors. we chose to work on calstabin 2 because this short (107 aa) protein has been described as a sub-family comprising 4 isoforms (from ~3 0 to ~1 00 aminoacids), some of them not being fully described to date. the relative shortness of those proteins together with the fact that the two higher molecular weight ones are catalytically active as prolyl isomerases, facilitate the characterization of the synthetic proteins.in order to obtain the full length calstabin 2, a native chemical ligation (ncl) approach was chosen 2 . an optimized stepwise elongation allowed the obtention of the c-terminal segment up to the cys 22. moreover, several methods were compared for the synthesis of peptide 1-21 opportunely functionalized at its c-terminus for the ncl.the ligation at thr site between peptide 1-21 featuring a bis(2-sulfanylethyl)amino 3 the chemical diagnostics of paintings is a relevant topic in the field of chemical sciences applied to the conservation and safeguard of cultural heritage. chromatography is a highly sensitive and suitable technique for accurate methods of analysis of the limited amount of sample material typically available from works of art. paint media deriving from proteins traditionally include egg, milk, animal and fish collagen glue. egg yolk (egg tempera), egg albumin (glair) and casein (a blend of related phosphoproteins commonly found in milk) are traditionally used as pigments binders. we propose the uplc-based amino acid analysis as diagnostics technique on non pre-treated or submitted to extraction processes model samples, showing that good results can be achieved with very scarce sample manipulation and great advantage. we applied the amino acids analysis carried out by the accq•tag™ ultra performance liquid chromatography to the standard and model samples. in particular, after protein hydrolysis (24h, 114 °c, 6m hcl) of the samples, the amino acid derivatization by 6-aminoquinolyl-n-hydroxysuccinimidyl carbamate allowed a reproducible amino acids analysis characteristic of the protein type. the results obtained confirmed the reliability of the data achieved and demonstrated that the accq•tag™ ultra uplc method could be a powerful technique to be applied to the relevant field of protein binders diagnostics for paintings conservation. moreover a multivariate analysis that offers a wide variety of tools and methods mainly concerned with mathematical models for the representation of multidimensional data has been proposed and the high model efficiency has been established for sample containing mixture of proteins. reactions performed on solid supports, such as resin, are commonly monitored by hplc-uv after cleaving the products from the support. however, uv-absorption coefficients may differ between compounds, and therefore the relation of the area percentage values of the peaks may not directly reflect the molar concentrations of the corresponding compounds. it is for this reason that, for example, in solid-phase peptide synthesis it is difficult to calculate the yield of the coupling of a fmoc-amino acid or the removal of the fmoc-group because of its high absorbance. recently, we reported the identification of minimal phosphopeptides that specifically interact with the pbd of human plk1, but not those of the closely related plk2 and plk3 1 . comparative analyses of the crystal structures of the plk1 pbd in complex with the minimal phosphopeptides revealed that the c-terminal spt dipeptide functions as a high-affinity to the interaction. in an attempt to obtain the adequate cellular permeability and stability in vivo, we have accomplished the peptide-peptoid hybrid or peptomers cyclization using various methods like formation of amide, thioether and triazole and screened the plk1 inhibition activity on the first cyclic peptomers liibrary using pbd-binding assay. based on our first screening results, we also carried out the detailed investigation to further increase the activity and also to understand the significance of peptoid mimics as plk1 inhibitors. the mode of interaction between the cyclic peptomers and pbd might provide a template for designing therapeutic agents that target plk1. a synthetic 83 amino acid long peptide corresponding to the minimal metacaspase catalytic domain induces cell death in leishmania major c. servis, h. zalila*, i. gonzalez, l. lozano, n. fasel department of biochemistry, university of lausanne, epalinges, switzerland despite a lot of controversy during the last decade, there is increasing experimental evidence that cell death (cd) is genetically programmed in lower eukaryotes.in the cd proteolytic cascade of plants and protozoa, caspases are likely replaced by metacaspases that are cysteine peptidases recognizing arginines or lysines in p1position. metacaspases have been found to control cell death in plants. the human protozoan parasite leishmania major expresses a single metacaspase (lmjmca) harboring a central domain with the catalytic dyad histidine and cysteine as found in caspases. metacaspase could therefore be one of the executioners of the death pathway in leishmania.in this work we showed that, in stress conditions, lmjmca precursor forms were extensively processed into soluble forms containing the catalytic domain and this domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion function. we tested different lengths of the lmjmca catalytic domain and found that the overexpression of the polypeptide corresponding to amino acids 136-218 was sufficient to sensitize l. major mitochondria to oxidative stress.we synthetized an 83aa long peptide corresponding to the minimal metacaspase catalytic domain (aa136-218) and showed that it has specific metacaspase activity in vitro.we are currently investigating its activity on possible target proteins, which have been identified in a yeast two-hybrid screen. identifying proteins involved in the metacaspase signaling pathway will shed light on the understanding of cd in leishmania and open new perspectives in drug target investigation to fight leishmaniasis and other major infectious diseases. s. alasibi, g. ashkenasy department of chemistry, ben-gurion university of the negev, beer-sheva, israel various factors can affect the conformations and folding states of protein molecules and as a consequence their activity. these factors include amino acid mutations, interactions with other macromolecules, binding to regulatory molecules, and also external changes such as ph jump or shining light. in order to control the folding states and to modulate the functions of peptides and proteins by light, photocleavable groups are usually incorporated into specific residues to mask critical interactions. for example, introducing caging groups into coiled-coil proteins recognition interface affects complex formation and template-assisted ligation reactions, in which the coiled-coils serve as templates to catalyze the condensation reactions between two short peptide fragments 1 . our research group has been studying peptides replication networks, which were made of coiledcoil peptides and analyzed the response of such networks to light as external trigger 1 . it was shown that even replicating networks made up of a small number of molecules can possess complex behavior, considering the wealth of catalytic pathways and transformations. hence, boolean logic operations can provide valuable means to analyze and interpret their behavior 2 . herein, we describe the use of chemical inputs and uv light to manipulate peptides folding and functionality within new synthetic networks. these networks perform complex behavior and, as a result, selective product formation is used to implement boolean operations that have not been achieved before. institute of bioorganic chemistry of ras, moscow, russia earlier, we have shown that n-acylated amino acid nitriles and amides react with ethylene derivatives forming the 3amino-and 3-hydroxypyridines and pyrroles [1] . a possible reaction mechanism is the geterodienic condensation of 5aminooxazole derivatives to dienophiles. the higher yields were observed when used the dicarboxylic acids as dienophiles and 5-amino or 5alkoxyoxazole as geterodienes. while the same reactions with the fullerene derivatives, as dienophiles, gave low yields. the nitrile groups of specified pyridines possess ability to react with amino groups of peptides and proteins even at room temperature. in view of high activity of nitrile groups such pyridines can form tetrapyridotetrazoporphyrins and self-condensation products giving appropriate dendrimers (possible due to mobile hydrogen in the 6th position). high molecular weight dendrimers were identified by massspectrometry, gel electrophoresis and dynamic light-scattering. catalytic oxidizing properties of tetrazaporphyrin derivatives and phtalocyanin were used in synthesis of cyclic peptides and for the s-s bonds formation. the transformation of peptides into heterocycles via an intramolecular reaction of nitrile groups was used to determine the sequence of some peptides, which favored the resistance of transformed compounds to hydrolysis and to the electron impact at mass spectrometry. diazotization of peptides and their derivatives facilitates identification of amino acid sequence by mass spectrometry due to the peculiarities of their fragmentation. in addition, an amino acid analysis of the diazotized peptide makes it easy to determine the n-terminal amino acid. we present here a multi-disciplinary approach combining x-ray crystallography, computational analyses, and immunological tests to identify epitopes of the oligopeptide-binding protein a (oppa bp) from the gramnegative pathogen burkholderia pseudomallei, the etiological agent of melioidosis. 3 computational analysis on oppa crystal structure was used to design potential consensus epitopes, that once synthesized as free peptides (comp 1-3) were found to be immunoreactive against sera from melioidosis patients. notably, one of the predicted peptides allowed to distinguish between seropositive, seronegative and recovered groups, underlining its potential for diagnostic purposes. parallel experimental epitope mapping, based on proteolysis and mass spectrometry, allowed us to identify linear peptide epitopes (exp 4-6) localized in similar protein regions as comp1-3. moreover, the match between theoretical and experimental mapping of epitopes was improved by expanding our computational approach, i.e. including an energy based decomposition procedure to divide oppa bp into separate fragments. overall, our results illustrate the successful development of a novel integrated structurebased approach for the discovery, design and preparation of epitopes. nonetheless, given antigen crystal structures, our method is expected to be broadly applicable in the design and generation of new epitope candidates, as being confirmed by on going experiments on different antigens. the application of peptide thioacids as reactive intermediates and building blocks has received considerable attention recently. the chemical ligation reaction between thioacids and azides has been reported for the synthesis of small to larger peptides as well as for the modification of proteins. 1 fmoc based methods for the preparation of peptide thioacids have to our knowledge not been extensively researched and a facile approach to their synthesis is desirable.we have recently shown that t-butyl thioesters are robuster than previously reported, and can be used for the fmoc based solid-phase preparation of peptide thioesters being also easily cleavable with thiolates. 2 peptides attached via a 4-mercapto 4-methylpentanol (mmp) resin can be cleaved using 3-mercaptopropionitrile to obtain protected thioacid peptides with a ß-elminable cyanoethyl group.the thioacid peptides could then be obtained in situ after treatment with dbu (6.5 % in dmf) and further reacted with sulfonyl azides in the presence of 2,6-lutidine in a one pot reaction. by treating the cyanoethyl peptide thioesters with (nh4)2s in a sodium phosphate buffer (ph = 9), various model penta-peptide thioacids could be obtained cleanly at room temperature in up to 75 % overall yield based on initial coupling. these peptides were then further ligated with electron deficient sulfonyl azide functionalized peptides.larger peptide thioacids could also be obtained using this protocol. a 16mer derivative of penetratin-1, a cell-penetrating peptide from the third helix of the homeodomain of the antennapedia protein, was prepared as a peptide thioacid in a 51 % yield (based on coupling of the first amino acid). in this report, a sensitive, selective and rapid uplc-ms method was developed for the determination of the [lys-gly]5-mog35-55 peptide in order to control the conjugation of mannan with the [lys-gly]5-mog35-55 peptide. the separation was performed on an acquity uplc system with a beh c18 column packed with 1.7 μm particles. the total run time was 3 min. calibration curve based on peak area ratio was linear at the concentration range of 20 -90 μg/ml, with a detection limit of 5 μg/ml. the method showed satisfactory reproducibility and confirmed the entire conjugation between oxidized mannan and peptide sequence. the development of simple, low-cost and fast methods for protein purification is of increasing importance both for academic and industrial applications. a very promising approach is inverse transition cycling (itc) that exploits the temperature dependent aggregation properties of elps. 1 elastin-like polypeptides (elp) are artificial polypeptides composed of pentameric repeats (val-pro-gly-xaa-gly) derived from mammalian elastin. 2 elps are characterized by a specific transition temperature (t t ) that depends on the amino acid composition of the pentarepeat; they are water-soluble below and aggregate reversibly above this narrow temperature range (t t ). 3 these properties are transferred to target proteins by n-or cterminal fusion with elps. 4 during itc these fusion proteins precipitate, while other components remain in solution. repeated cycles of heating and cooling allow simple recovery of the target protein.we synthesized various elps consisting of 5 to 10 pentameric repeats and including different guest residues. the transition temperature of all synthetic elps was determined using photometric assays and measuring turbidity. 3 in order to test if elp properties can be efficiently transferred, we fused elp to a small recombinant protein (ras-binding domain, rbd) by expressed protein ligation. 5 this approach will allow the incorporation of elps with unnatural amino acids and other chemical modifications into target proteins. currently we are focusing on biotechnologically relevant enzymes that constitute a major cost factor in industrial processes. the authors thank süd-chemie/clariant for their financial support. department of pharmacy, university of patras, rio, greece peptides penetrating the cell membrane, known as cell penetrating peptides (cpps), as well as their mimics, used as delivery agents to cells have been reported 1,2 . cpps can be natural sequences or artificial constructs designed to capture the features of natural formations. cpps are particularly important in the delivery of peptides, proteins, nucleic acids, small molecule drugs or imaging agents. incorporation of a heterocyclic motif into a peptide or peptide-like backbone introduces conformational constraints and/or latent reactivity related to the heterocycle's structural profile. heterocycle-based cpp mimics are, thus, promising candidates for therapeutics 3 protected synthetic non-ionic peptides, which are for example synthetic intermediates for the production of api's, are often very hydrophobic and not soluble in most common solvents. they are thus difficult to purify by preparative rp-hplc, classically used for industrial production. it is then challenging to develop alternative purification chromatographic processes using suitable solvents and providing good yields, high purity and sufficient productivity. the technique of support free liquid-liquid chromatography 1 , including both its hydrostatic (centrifugal partition chromatography or cpc) and its hydrodynamic (counter-current chromatography or ccc) declensions, are mainly involved in phytochemical studies 2 but has also been applied to peptide purification 3 . the previously developed biphasic solvent systems are not adapted to the purification of highly hydrophobic protected peptides. to overcome this problem, two new scales of biphasic solvents systems and a ternary biphasic solvent system were developed to overcome solubility problems often encountered with those peptides. the new systems composed of heptane/thf/ch3n/dmso/water, heptane/me-thf/nmp/water, and cmpe/dmf/water were efficiently used for the cpc purification of a 39mer protected exenatide and a 8mer protected peptide intermediate of bivalirudin synthesis. the developed scales show a wide range of polarity and should be useful for general use in cpc for the separation of hydrophobic synthetic free or protected peptides. the progressive aggregation of β-amyloid peptide (β-ap) into insoluble amyloid fibrils ultimately leading to formation of toxic amyloid plaques is widely considered to be the central pathogenic cause of alzheimer's disease. in the last decade accumulating evidence suggests that soluble oligomeric non-fibrillar forms of β-ap are neurotoxic as well. consequently, inhibiting the aggregation of β-ap is one of the therapeutic strategies against alzheimer's disease and a number of small molecules have been identified as inhibitors of β-ap aggregation and neurotoxicity. among these, curcumin, the phenolic yellow pigment and active ingredient of the turmeric herb, is receiving special attention because of its rich pharmacology that includes in vitro and in vivo inhibitory action against alzheimer's disease insults. in the current work the interaction of β-ap(1-40) with curcumin is investigated with fluorescence, cd, and nmr spectroscopies in water and water-methanol mixtures and at various β-ap(1-40):curcumin ratios. in nmr studies in 100% methanol curcumin behaves like a macromolecular species with a change in the sign of its noe signal providing direct indication of its association with β-ap(1-40). in 50% methanol the presence of β-ap(1-40) results in great broadening of the 1 h peaks of curcumin, indicative of a complete change in its solution state. additionally, the fluorescence of curcumin in 50% methanol shows a blue shift with enhanced intensity, observations consistent with a hydrophobic modification of curcumin environment upon interaction with β-ap. finally, in water the induced circular dichroism spectrum of curcumin in the near uv region provides clear evidence for the loss of symmetry of curcumin molecule due to changes in its microenvironment generated by interaction with β-ap(1-40). our experimental findings support the direct interaction of β-ap(1-40) with curcumin and establish its importance as a potential aggregation inhibitor of β-ap. [1] . based on its sequence, we synthesized h-tyr-d-trp-nh-1-ada (1-adamantane) (yo-14) and h-tyr-d-trp-nh-2-ada (2-adamantane) (yo-13) and reported they had potent antiproliferative activity on cancer cells (a-430 and sw480), which were comparable to tt-232 and cycloheximide. a structure-activity relationship analysis revealed that lipophylicity of yo-14 and -13 could be responsible for their antiproliferative activity. now, we described the substitution of tyr of yo-14 and -13 by tyr(bzl), phe, 1-nal(1-naphthylalanine), 2-nal (2-naphthyalanine) and the anticancer and dna polymerase inhibitory activities in order to explore the effect of hydrophobic substituent. among the compounds, yo-113 and -114 had the highest lipophilicity judging from their retention time and lipophilicity index (yo-113: 45.5 min, 59.9; yo-114: 44.6 min, 55.9). yo-113 and -114 exhibited strong dna polymerase inhibitory activity as well as antifroliferative activity on hct116 cells at 100 m. these activities were greater than those of yo-14 and -13. antiproliferative activity of the compounds containing 1-ada such as yo-14, -89, -109, -111 and -113, was comparable to that of the compounds containing 2-ada such as yo-13, -90, -110, -112 and -114. these findings suggest that the lipophilicity well correlates with dna polymerase inhibitory activity and antiproliferative activity on hct116 cells. further structureactivity relationship study is progressing in our group. multiple sclerosis (ms) is a chronic autoimmune disease of the central nervous system (cns) 1,2 . our aim was to immunologically control the attack of the myelin sheath in ms patients without the total suppression of the immune system. anthraquinones (mitoxantrone, ametantrone) are widely used in cancer therapy as immunosuppressants.mitoxantrone is also used to treat several forms of advancing ms, including secondary progressive ms, progressive relapsing ms, and advanced relapsingremitting ms 3 . more specifically, mitoxantrone is an inhibitor of the type ii topoisomerase, which disrupts dna synthesis and dna repair in both healthy cells and cancer cells. herein, we report the synthesis of an anthraquinone type compound conjugated to the immunodominant 35-55 myelin oligodendrocyte glycoprotein (mog35-55) for the selective immunosuppression of the encephalitogenic t cells in ms patients. the anthraquinone was synthesized by a friedel-crafts acylation of hydroquinone from phthalic anhydride, followed by reduction of the resulted quinizarine to its leuco form, addition of the appropriate diamine and air oxidation 4 . the synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and 1 h-nmr. the synthesis of the mog35-55 was performed under microwave irradiation 4 and its conjugation with the anthraquinone was performed in solution. the final analogue was purified by rp-hplc and identified by esi-ms. benzopyrans, diketopiperazines and 1,5benzodiazepin-2,4-diones are well-known and widely investigated scaffolds, e.g. the latter showing anxiolytic and antiarrhythmic effects. now, we propose a new potential "privileged structure" containing a triazole moiety mimicking the cis-amide bond within the 1,5-benzodiazepin-2,4-dione motif 2 .molecules based on this [1,2,3]-triazolo [1,5-d] benzo-1,4diazepin-2-one scaffold are synthesized and decorated via a modular approach on wang resin using α-amino acids, 2-ethynylaniline building blocks and n-alkylating agents resulting in five points of diversity. the methodology involves the attachment of α-amino acids onto a solid support, subsequent removal of the fmoc group followed by an optimized diazotransfer reaction of the resulting amine yielding a resin-bound azide. conversion of the latter into a 1,5-disubstituted 1,2,3-triazole moiety is achieved quantitatively by addition of a range of 2-ethynylaniline building blocks using a ru(ii)-catalyst. the desired scaffold can be obtained in high crude purities (>95%) in solution via an acid catalyzed one-step cyclisation-release strategy. solution-phase n-alkylation finally affords the fully diversified scaffold. interestingly, n-alkylation induces atropisomeric effects which can be studied via 1 h nmr spectroscopy.taking into account future screening results of the synthesized libraries, a well-thought decoration of this scaffold leading to discovery of new lead molecules is within reach. peptide symposium in the wonderful small seaside town of porto carras. maurice manning and lajos balaśpiri were nominated as captains of the two teams, the rest of the world and europe. similar matches were organized at subsequent european peptide symposia. now, in greece, the two captains would like to hand over their roles to younger scientists [professors gabor mezö(hungary) and laśzlóötvös (usa)] to continue this tradition at the coming european and possibly american peptide symposia. it seems best to play in the free time (in the evenings after the excursions). necessary conditions: good weather; a nice large soccer field; a soccer ball, preferably new; and jerseys and shorts (different colours), organized as always by the organizer commettee. the captain of the winning team will receive a trophy at the end of the 32 nd european peptide symposium. the teams will remember two earlier excellent referees: professor lajos kisfaludy in porto carras [1986] and the soccer professor + ferenc puskaś (hungary) in budapest (1998). in the poster session, the results from the past 25 years will be presented in about 15-20 pictures. these pictures may possibly be bought free, 0% can be saved at the poster session. all participants are welcome at the new party in athens. conclusion will be presented by the players and fans in athens. the human lactoferrin-derived peptide, hlf1-11, was proven to be highly active against antibiotic-resistant bacteria 1 . however, the clinical use of this antimicrobial peptide (amps) is hampered by the peptide low stability due to fast degradation or to peptide aggregation, as the use of higher peptide concentrations results on higher toxicity levels. amp immobilization onto a biomaterial surface could be the pathway to overcome these difficulties 2 . the aim of this work is the development of an antimicrobial surface by covalent immobilization of hlf1-11 onto the surface of chitosan thin films. chitosan ultrathin films were prepared through the spincoating of a 0.4% chitosan solution in gold substrates. hlf1-11 immobilization was performed through an ss bound between hlf1-11 terminal cysteine and an n-acetyl cysteine previously coupled at chitosan films. surfaces were characterized using ellipsometry (thickness), infrared reflection absorption spectroscopy (irras) and x-ray photoelectron spectroscopy (xps). bacterial adhesion studies were performed using methicillin-resistant s. aureus (atcc33591). chitosan films were incubated with this bacterial suspension at 37ºc for 6h and 24h. the viability of the attached bacteria was evaluated using live/dead® bacterial viability kit (baclight tm ) and fluorescence microscopy. hlf1-11 peptide was successfully covalently immobilized onto chitosan thin films. both soluble and attached peptide presented a higher antimicrobial activity than the control chitosan. identified as a potent vasoconstrictor that binds with high affinity to ut receptor. 1 the cysteine-linked cyclic region, hut-ii(4-11), is responsible for the biological activity and has been widely used to elucidate the structure-activity relationship of hut-ii. 2 with the aim to investigate the role of hydrogen bond and the effects of a peptide backbone constraint on binding key: cord-309043-dlmx12vt authors: von brunn, albrecht; teepe, carola; simpson, jeremy c.; pepperkok, rainer; friedel, caroline c.; zimmer, ralf; roberts, rhonda; baric, ralph; haas, jürgen title: analysis of intraviral protein-protein interactions of the sars coronavirus orfeome date: 2007-05-23 journal: plos one doi: 10.1371/journal.pone.0000459 sha: doc_id: 309043 cord_uid: dlmx12vt the severe acute respiratory syndrome coronavirus (sars-cov) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. the functions of a large number of viral orfs are poorly understood or unknown. in order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral orfeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. about 38% of interactions were subsequently confirmed by coip in mammalian cells. nsp2, nsp8 and orf9b showed a wide range of interactions with other viral proteins. nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. it was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. we show that also accessory protein orf9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in vero cells. however, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the sars proteome network, or play some unrealized role in regulating protein-protein interactions. the interactions identified provide valuable material for future studies. the observation of atypical pneumonias in the chinese province guangdong in november 2002 led to the identification of the severe acute respiratory syndrome (sars). within a few months, the disease spread to a large number of countries and caused more than 8,000 cases and almost 800 deaths. the causative pathogen identified was shown to be a new human coronavirus designated the sars-cov [1] . tight intervention strategies limited the further spread of the pathogen. sequence analysis of the first isolates of the newly identified sars-cov revealed characteristic features typical of the known three coronavirus groups [2] [3] [4] . according to several phylogenetic analyses the virus is grouped either a novel group iv or an early split-off of group ii coronaviruses [5, 6] . the genome of the sars-cov consists of a positive-stranded rna of approximately 29,700 nt in length. the replicase genes span the first two-thirds of the genome containing the two overlapping orf1a and orf1b, which are connected by a ribosomal frameshift. the two polyproteins expressed are predicted to encode and to be cleaved by a papain-like proteinase 2 (pl-pro = part of nsp3) and a 3c-like proteinase (3cl-pro = nsp5) to 16 mature replicase proteins [1, 6] . they are well conserved between sars-cov and other coronaviruses. it is suggested that they are required for the synthesis of the full-length genome and subgenomic rna synthesis as well as for virus replication [6, 7] . functions of the processed proteins include a single-stranded rna-binding protein (nsp9) an rna-dependendent rna polymerase (rdrp = nsp12) as well as a non-canonical rdrp (nsp8) synthesizing short primers for nsp12, a superfamily 1like helicase (hel1 = nsp13), and a uridylate-specific endoribonuclease (nendou = nsp15) [7] [8] [9] [10] [11] [12] [13] [14] [15] . nsp3, nsp14, nsp16 are thought to have adp-ribose 19-phosphatase, 39-.59 exonuclease and 29-o-ribose methyltransferase activities, respectively [6] . but many of the functions of the nsps are still unknown. at their 59terminus the subgenomic mrnas share a common leader sequence encoded at the 59-end of the genome, which is joined to the respective gene sequences at specific transcription regulatory sequences. their common 39-ends extend to the end of the genome. the last third of the genome encodes the s, e, m and n structural genes with the group-specific genes interspaced among them. former are encoded by mrnas 2, 4, 5, and 9, latter by transcripts 3, 6, 7, 8, and 9, respectively. these genes, orf3a/b, orf6, orf7a/b, orf8a/b, and orf9b are not found in other coronaviruses and their functions with respect to replication and pathogenesis are not well understood. there is evidence that some of the accessory orfs can be deleted individually or in combination with almost no impact on in vitro growth, rna synthesis, or on in vivo virus replication in a murine model [16] . also, the nsp2 replicase protein of murine hepatitis virus (mhv) and sars-cov is dispensable for virus replication in cell culture. its deletion results in attenuation of viral growth and rna synthesis [17] . there are reports that a number of mhv and sars-cov replicase proteins colocalize and eventually interact in cytoplasmic membrane bound complexes, in which viral rna synthesis occurs [18, 19] . direct interactions of nsp7 and nsp8 in a hexadecameric supercomplex could be demonstrated by crystallography [20] . interactions of the structural n and m proteins were demonstrated by a mammalian two-hybrid system [21] . for the elucidation of molecular mechanisms during the course of viral growth and propagation there is a need to systematically examine possible interactions of all viral proteins. we therefore cloned the sars-cov orfeome by recombinatorial cloning (gateway technology) and performed a genome-wide analysis for viral protein interactions by yeast-two-hybrid (y2h) matrix screen. we have designed a set of nested pcr primers to amplify all viral non-structural, structural and accessory orfs at the predicted protease cleavage sites or at the respective start and stop codons ( table 1) . for cloning reasons, nsp3 was subdivided into a nterminal (nsp3n, nt positions 2719-4431) and a c-terminal (nsp3c, nt positions 4885-8484) fragment containing the adpribose-1''monophosphatase domain and the papain-like proteinase, respectively. an accessory orf14 described only by marra et al. was also included [3] . primers were designed such that they contained gene-specific sequences for the amplification of the respective orfs. overhanging sequences made them compatible to the gatewayh recombinatorial cloning system allowing the cloning into a so-called pdonr207 vector with the subsequent subloning into the destination vectors pgadt7-dest (prey) and pgbkt7-dest (bait). the y2h bait and prey vectors pgadt7-dest and pgbkt7-dest containing the sars orfs were transformed into the haploid yeast strains ah109 and y187, respectively, and mated and grown under selective conditions on media lacking leucine, tryptophane and histidine. all orfs were tested pairwise against each other and 900 individual interactions were tested in quadruplicates. interaction of the viral proteins was indicated by colony growth on the selective plates. the result of the matrix screen is shown in figure 1 . positive interactions are indicated by (black and patterned squares). positive y2h interactions were validated by co-immunoprecipitation (coip) in mammalian 293 cells as a second interaction test (double-lined red squares). of 65 interactions detected by y2h 25 were corroborated by coip. four coips were detected in both directions: non-structural proteins nsp12 (rdrnap) and nsp13 (c/h, ntpase, dntpase, 59-to 39 rna helicase, dna helicase, rna 59-triphosphatase), nsp8 and accessory protein orf9b, nsp14 (c/h, 39-to 59 exoribonuclease) and orf9b, and accessory proteins orf8a and orf8b. six of the proteins, including nsp7, nsp8, nsp13, ''e'', orf9b and orf14 interacted with themselves indicating the formation of dimeric or multimeric complexes. four of the self-interactions including nsp8, ''e'', orf9b and orf14 self-interactions were also found in the coip assay. two non-structural proteins nsp2 and nsp8, and the accessory protein orf9b showed a rather large number of interactions (8, 14 and 15 interactions, respectively). six interactions of the non-structural proteins nsp2 could be confirmed by coip including the non-structural proteins nsp3n, nsp6, nsp8, nsp11 and nsp16 and orf 3a, which only recently had been described to be a novel structural protein [22, 23] . figure 2a shows ip and coip results with anti-ha and anti-c-myc antibodies: 293 cells were transfected with ha-tagged nsp2 and cmyc-tagged nsp2-, nsp3n-, nsp6-or nsp8. the lysates were split into two and immunoprecipitated in the presence of protein g with the anti-ha (left upper panel) or with the anti-c-myc (right upper panel) antibody. the bound proteins were separated by 12.5% sds-page western blot analysis. expressed ha-tagged proteins are indicated by stars and coprecipitated proteins by arrows. although the nature of the additional protein species with higher molecular weight in the anti-ha wb of the anti-c-myc coip is unclear, they might reflect a multimeric nsp2 band that is not present in the other lanes, further supporting the nsp2-nsp2 interaction. but they could also be a result of post-translational modification upon binding to other proteins. in the reciprocal analysis (lower left and right panel) nsp2 proteins coprecipitated with itself and with nsp3n. the second-most connected protein of the replicase complex is nsp8 ( figure 2a, 2b) . of the 14 interacting proteins (nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13, nsp14, e protein, orf8, orf8a, orf9b, orf14) found in the y2h system eight could be confirmed by coip (nsp2, nsp7, nsp8, nsp9, nsp12, nsp13, nsp14, e protein). nsp 8 and nsp12 proteins interacted with orf9b and nsp13, respectively, in both directions in the y2h. this was also the case for the interaction between nsp14 and orf9b, which could also be confirmed by coip. of the ''classical'' structural proteins s, e, m and n, only the e protein showed a number of interactions with the non-structural proteins nsp1, nsp8, nsp11, as well as with the accessory proteins orf3b, orf7b and orf9b. interestingly, m and s reacted with the only recently described accessory structural proteins orf 3a and orf7a [22, 24] . self-interaction of the e protein was seen in both assays. the interaction between the structural proteins n and m, which has been described in two recent reports [21, 25] , was found positive by coip only. the most dominant interactor of the accessory proteins was the orf9b. of the 16 orf9b y2h interactions detected, four could be confirmed by coip ( figure s1 ). 10 of the non-structural and five of the other accessory proteins showed interactions with other proteins. self interactions were seen for orf9b and orf14. these could be confirmed by coip in both directions as well as the reactivity of orf7b with the two e and orf 6 proteins. orf 8a and orf 8b were also reactive in both directions in the y2h system. the dominant interactor proteins nsp2 [17] and nsp8 (deming et al., submitted) are dispensable or essential for viral replication in vitro, respectively. to determine the importance of the dominant interactor orf9b accessory protein for viral growth a deletion mutant was made by synthesizing a dna fragment that included changes that ablate each of the orf9b atg start sites while maintaining the primary sequence of the n protein ( figure 3a ). the recombinant virus had normal plaque sizes and grew like wildtype virus in vero cells after infection at a moi of 0.1 pfu/ cell ( figure 3b ). thus, deletion of orf9b is not lethal. y2h matrix and coip interaction screens of the present study were performed similarly as in our recently published study on herpesviral protein networks describing intra-viral protein interactions in kaposi sarcoma-associated herpesvirus (kshv) and varicella-zoster virus (vzv) [26] . table 2 shows the comparison of network parameters of sars to kshv as well as other cellular protein interaction networks. the average degree and characteristic path length are slightly smaller than in the kshv network and significantly smaller than in the cellular networks due to smaller network size. the fraction of pairwise interactions confirmed among those tested is higher in the sars network with 65 out of ,450 possible non-redundant pairwise interactions ( = 14.4%) than in the kshv network with 123 out of ,4050 possible interactions ( = 3%). furthermore, the clustering coefficient is significantly higher than in all of the networks analyzed. when comparing against random networks of the same size, we found that the clustering coefficient is approximately as high as expected at random given the degree distribution. interestingly for the kshv network it is actually smaller, whereas for the cellular networks it is much higher. known virus-host and intraviral interactions of sars proteins were identified by a literature screen (see tables s1 and s2). based on ten known sars-host interactions as well as two interactions predicted from homologous proteins, the viral network was connected to the human network assembled from large-scale y2h screens [27, 28] , ortholog predictions [29] and literature mining ((ref-hprd) and [27] ). the sars-cov-host interaction network is shown in figure 4 . human proteins are included which are distant from the sars-cov proteins by at most two or three interactions as well as the interactions between these proteins. on the basis of the small number of known virus-host interactions the intraviral sars-cov network is separated from the bulk of the human interactome. proteins targeted by sars either directly or via other proteins are involved in various molecular functions and pathways such as apoptosis, cell communication and signalling pathways. the literature screen for intraviral sars interactions identified 23 interactions. 3 of these (13%) were also determined by the y2h screen. to systematically study the subcellular localization of viral proteins within eukaryotic hela cells the sars-cov orfs were transfected in eukaryotic vectors with either n-or c-terminal flag tags and detected with an anti-flag antibody. since artificial tagging of proteins often leads to an aberrant expression in cellular compartments, we tagged the sars-cov protein on both the nand c-terminus and only considered the cellular localization correct if consistent with both tags [30] . some of the proteins were not expressed if tagged either n-or c-terminally (see figure s2 ), and some were not expressed at all, and were not included in the analysis. the table shows network parameters for two viral and two cellular protein interaction networks. the sars network contains 65 interactions between 31 nodes with 6 of those interactions being self-interactions. for comparison purposes, network parameters are also shown for kshv [26] , s. cerevisiae and h. sapiens. interactions for the cellular networks were derived from the following sources: s. cerevisiae from dip (the database of interacting proteins) [45] , the yeast two-hybrid interactions of h. sapiens from the studies of stelzl et al. [28] and literature interactions from rual et al. [27] , predicted human interactions (core) from lehner and fraser [29] and interactions taken from the hprd (human protein reference database) [46] . parameters shown include the number of nodes and edges in the networks, the average degree, characteristic path length (average shortest path), diameter (maximum shortest path), clustering coefficient and for the clustering coefficient enrichment values compared to appropriate random networks (er and es). both types of random networks contain the same number of nodes and edges as the original network. er networks [47] are created by connecting edges randomly, whereas es networks are created by an edge swapping strategy which preserves the degree distribution (see [26, 48] orf3a, orf7a and m were detected in the golgi, orf3b in the nucleus, orf6, orf7b, nsp3n and nsp16 in the er ( figure 5 ). orf8b and orf14 showed a vesicular staining, and nsp2 was found both in cytoplasm as well as in the nucleus. orf3a and m were found to be interacting by y2h, and were both detected in the golgi. similarly, orf6 and orf7b were both found in the er. in this study we report the cloning of the complete orfeome of sars-cov and the results of a matrix-based yeast two-hybrid screen of pairwise viral protein-protein interactions. from a number of recent structural studies it is clear that during the viral life cycle large replication complexes are formed, which involve a large number of viral proteins [31] . sars-cov is a representative of the coronaviridae, the largest rna viruses known (27 to 32 kb, plus-stranded). sars-cov expresses at least 16 non-structural replicase proteins which are cleaved co-and post-translationally from two precursor polyproteins by two viral proteinases, four structural proteins and a set of eight accessory proteins specific for the individual virus groups [6] . since the polypeptide processing sites of non-structural proteins are well defined, we chose the strategy to subclone all individual orfs predicted, and not to use the precursor polyproteins. using this approach we expected to avoid problems in the expression, folding or targeting of the polypeptides due to incorrect processing. such problems had been reported for yeast two-hybrid assays performed with the plus-stranded rna viruses hepatitis c virus [32] and wheat streak mosaic virus (wsmv) [33] , where interactions had been observed only when random fragments, not mature proteins were used. but there are also reports on potato virus a (pva) and pea seed-borne mosaic virus (psbmv), which belong to the potyvirus (+) strand rna virus family similar to wsmv [34] , as well as on a subset of poliovirus proteins [35] , where interactions have been detected among cloned mature proteins. in our screen approximately 14% of the 450 possible nonredundant protein interactions tested were positive and approximately 38% of which were confirmed by coip. this result is in the same order of magnitude as the outcome of similar y2h matrix screens in kshv and vzv, indicating that this approach can also be applied for plus-strand rna viruses. the low numbers of y2h interactions detected in two directions are a common phenomenon in y2h assays and are probably due to steric constraints of either bait or prey fusion proteins. coronavirus replication complexes consist of intricate macromolecular structures in which many of the non-structural replicase proteins are involved. one of the most interesting interactor proteins found in our study is nsp8, which interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13, nsp14. the importance of this protein is supported by recently reported crystallization studies, which described the multimeric association of various of the non-strucutural proteins. nsp 8 seems to be one of the proteins involved in these complexes. nsp8 deletion or irreversible fusion to nsp7 or nsp 9 by mutagenesis of the corresponding cleavage site results in a lethal phenotype supporting the idea that nsp8 is absolutely essential for virus replication (deming et al., submitted). evidence has been presented for interaction with nsp9, a ssrna-binding protein, by analytical ultracentrifugation experiments and by a decrease of the disorder of the nsp8 n-terminal region after the addition of nsp9 [36] . furthermore, a hexadecameric nsp7-nsp8 supercomplex was described which was suggested to encircle rna where it may serve as a general processivity factor for the rna-dependent rna polymerase (rdrp) nsp12 (19) . a very recent report described nsp8 as a second rdrp of sars-cov. it was shown to initiate the synthesis of complementary oligonucleotides of ,6 residues in a low fidelity reaction which eventually might serve as primers for the primer-dependent nsp12 rdrp [15] . for mhv it was shown that rdrp co-immunoprecipitates with nsp8, nsp9, nsp5 and the helicase nsp13 [37] , and that it also colocalizes with nsp7, nsp9 and nsp10 [18, 37] . thus, the nsp8 interactions found by us are confirmed by a number of different studies and it seems to play an important role in the viral replication complex. in this manuscript, we demonstrate interactions between rdrp (nsp12) and nsp8, and with the helicase nsp13 in both directions of the y2h screen. it is likely that the rdrp interacts with more nsps than were found, but these interactions may require mediator proteins like nsp8. nsp2 interacted with seven other nsps including nsp8 and with one of the newly described structural proteins orf3a. as shown by coip, it also self interacts to a dimeric or multimeric complex. the relatively large number of interactions might imply a crucial role of nsp2 in the viral life cycle. however, it was shown by deletion mutants of sars-cov and mhv that neither the encoding genomic rna sequences nor the nsp2 proteins are necessary for the generation of infectious viruses in cell culture [17] . since these viruses displayed slightly reduced phenotypes in growth, rna synthesis but not protein processing, it was speculated that nsp2 might play a role in global rna synthesis, and possibly in virus-cell interactions or viral pathogenesis. the reported subcellular localization of individually expressed nsp2 in delayed brain tumor (dbt) cells [17] is similar to the diffuse cytoplasmic and nuclear immunofluorescence staining pattern found with our n-or c-terminally tagged nsp2 proteins. thus, the exogenously expressed nsp2 does not target specific membranes in the absence of infection. however, after coinfection with a mhv mutant virus lacking nsp2, the protein expressed in trans was reported to be recruited into distinct viral replication complexes. this relocalization of nsp2 to small vesicular foci in the cytoplasm was also confirmed in sars-cov-infected vero cells by immunofluorescence staining with anti-nsp2 antibodies [19] . in our study, only few interactions were found for the structural proteins, which might be biased by transmembrane sequences preventing the transfer of expressed prey (containing the gal4 activating domain) and/or bait (containing the gal4 dnabinding domain) fusion proteins to the nucleus of the yeast cell where protein-protein interaction leads to transcription. only the e and orf3a proteins showed a number of associations whose relevance is unclear. interactions of orf3a -m and orf7a-s fit to the recent finding that the two accessory proteins display structural functions as has been described [24, 38] . for the group-specific accessory proteins it has recently been shown that deletion of five of the eight orfs (orfs 3a, orf3b, orf6, orf7a and orf7b) alone or in combination did not influence dramatically the level of rna or the replication efficiency in vitro or in an in vivo mouse model [16] . the most interesting accessory protein with respect to interactions in our study turned out to be orf9b. y2h interactions with nsp8 and nsp14 were found bi-directionally and the self-interaction could also be confirmed by coip. latter result is confirmed by recent structure data [39] . the orf9b protein, which is encoded within the nucleocapsid gene, is an intertwined dimer with an amphipathic outer surface and a long hydrophobic lipid binding tunnel. this suggests that orf9b is targeted to er-golgi compartments via an unusual anchoring mechanism and acts as an accessory protein during virion assembly. although most of the accessory proteins do not seem to play pivotal roles in viral replication, they might still be important for the virus-host interplay and for pathogenicity. currently, there is no reasonable explanation for the large number of interactions found for orf9b by the y2h screen. as deletion of orf9b does not seriously reduce virus replication in vitro consistent with a luxury function, the 9b protein may function to enhance the global stability of the sars proteome network and play some unrealized role in regulating virus-host protein-protein interactions. it thus might be more important for enhancing in vivo virulence. immunofluorescence localization of flag-tagged viral proteins corresponded in most cases to published data on sars-cov and other coronaviruses. we found nsp2 proteins in the cytoplasm and to some extent in the nucleus which is in accordance with anti-nsp2 antibody stainings of stably dbt-nsp2 (mhv) expressing cells [17] . many of the non-structural proteins are involved in the replication of the virus and locate to virus-induced cytoplasmic double-membrane vesicular complexes as the sites of viral replication. it is therefore important to take into account that the localization patterns of nsps might be quite different when expressed individually in cells as compared to the situation of viral infection where various viral proteins might help to recruit each other to the sites of active replication. accessory protein 3a, for which a number of effects on cellular functions were described [40] , we located in our flag-tagged versions to the golgi complex as yuan et al. [41] observed using egfp-tagged constructs. as a structural protein orf3a interacts with the m protein [23] which was also clearly found in the golgi as flag fusion proteins. the nuclear localization of orf3b is also reasonable because it induces cell cycle arrest at the g0/g1 phase and apoptosis [42] . proteins orf6 and orf7b, interacting in y2h and coip, were both found in the er. to our knowledge this localization has not been described for orf7b before. not much is known about orf9b other than it is expressed in infected cells [43] and that antibodies to it are found in infected patients [44] . as opposed to meier et al. [39] , who located orf9b to intracellular vesicular structures (293t cells), we found it to be diffusely distributed within cytoplasm and nucleus (hela cells). analysis of network statistics showed that despite high clustering coefficients the sars interaction network is not higher clustered than expected at random. it, thus, appears as a single module such as the kshv network and is not subdivided into separate functional modules as cellular networks. based on currently known and predicted host-virus interactions, a joint virus-human network was derived in which the viral part of the network appears to be separated from the main host network. in this respect, the sars network differs from the kshv viral network which is incorporated into the host interactome. however, this may be due to the small number of virus-host interactions identified so far for sars. indeed for kshv, the predicted virus-host network was based on about twice as many interactions to the host. to better understand the role of the intraviral protein interactions it is necessary to gain more knowledge on the sars-cov with it's host during infection. we certainly missed a considerable number of intraviral protein interactions in our y2h screen as can be seen for m-n and nsp2-nsp2, nsp5-nsp5 self-interactions, which we could only detect by coip. although, it is generally acknowledged and certainly has to be taken into account that y2h assays are error-prone by producing false positives and false negative results, we identified a large number of interactions which have not been reported previously and which could be confirmed biochemically. these interactions will be of great help for further studies which are aiming at the elucidation of sars-cov replication and pathogenesis. future experiments with the mutant viruses lacking nsp2, nsp8 or orf9b will show the relevance of the interactions detected for virus replication, growth and pathogenicity in vitro and in vivo model systems. experimental procedures viral nucleic acids sars-cov orfs were derived from subcloned cdnas described by yount et al. [16] and thiel et al. [7] . orfs were amplified by nested polymerase chain reaction (pcr) using plasmids ptopo xl containing fragments a (nt 1-4436) and b (nt4344-8712), psmart containing fragments c (8695-12070), d (12055-18924), e (18907-24051) and f (24030-29736) as well as plasmids pmal-scov-mpro 5 (nsp5), pmal-scov-nsp8, pet-scov-pol 8 (nsp12), pmal-shel-nsp13, pet-scov-exon 3 (nsp14), pmal-scov-nsp15-7, pet-scov-mtr 12 (nsp16)and pbs-sarscov-s30. the nucleotide sequences of sars-cov urbani (genbank accession ay278741), frankfurt (genbank accession ay291315) and tor2 (genbank accession nc_004718) isolates were used to design primers for subcloning of all putative orfs and making them compatible to gatewayh recombinatorial cloning system (invitrogen). nested pcrs were performed with two separate sets of primers. for the first pcr internal forward (aaaaagcaggctccgccatgn [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] ) and reverse (agaaagctgggtcn [13] [14] [15] [16] [17] [18] [19] [20] primers containing the internal attb1 and attb2 recombination sites were used. the gene-specific 59 forward sequence (n) introduced an aug start codon prior to the predicted protease cleavage sites with further 14-27 nucleotides downstream, while the 39 reverse sequence (n) matched 13-20 nucleotides. there was no stop codon introduced at the specific ends of the predicted cleavage sites in order to allow the c-terminal inframe fusion of tag sequences. the second pcr was performed using forward (59-ggggacaagtt-tgtacaaaaaagcaggct-39) and reverse (59-ggggac-cactttgtaca agaaagctgggt-39) primers which included the external parts of the attb1 and attb2 recombination sites. for the putative peptide nsp11 two oligos were synthesized as the coding (59-aaaaagcaggctccgccatgtctgcgg atgcatcaacgtttttaaacgggtttgcggtggacc-cagctttct-39) and non-coding (59-agaaagctggg-tccaccgcaaacccgtttaaaaacgttgatgcatccg cagacatggcggagcctgcttttt-39) strands. primers for orfs n and s were designed such that the complete attb1 and attb2 sites were added to the gene specific sequences. pcr conditions were 20 mm of dntps, 0,2-0,4 mm forward and revers primers, 20 ng of template and 1 u of long expand taq polymerase enzyme (roche diagnostics gmbh). depending on size and nucleotide composition of the amplificates two standard conditions were used for amplification. pcr fragments were separated by agarose gel electrophoresis and purified utilizing nucleospin extraction kits (macherey& nagel). the resulting pcr-fragments, flanked by complete attb1 and attb2 sites, were cloned by gatewayh recombinatorial cloning into the entry vectors pdonr207 or pdonr221 (invitrogen) via the bp clonase reaction as described by the manufacturer (invitrogen). the overlaps of the vector and sars-cov-orf sequences were confirmed by dna sequencing using the big dye terminator kit (perkin elmer) on a 377 dna sequencer, a 310 genetic analyser (both applied biosystems) or the genome lab dtcs-quick start kit on the ceq tm 8800 sequencer (beckman coulter). two eukaryotic destination vectors were constructed allowing the inframe fusion of a his-flag tag to the n-terminus and of a flag-his tag to the c-terminus of a the viral orfs. for the nterminal tag a dna oligo adaptor molecule was synthesized (coding strand: 59-ttagtcaagcttgaaggagatagagc-caccatggcacaccatcaccatcaccatgactacaag-gacgacgatgacaaggcgatatcttaatctagatgat-a-39) and sub-cloned via hind iii and xbai restricition sites into plasmid pcr3. the c-terminal oligo adaptor molecule (coding strand: 59-tttatatgatatcgactacaaggacgacgatga caaggcacaccatcaccatcaccattaactcgagatt-aata-39) was subcloned via ecorv and xhoi into pcr3. both plasmids were converted to destination vectors by ligating an ecorv -ecorv dna fragment, containing the gate-wayh conversion cassette reading frame b (rfb cassette) into their individual ecorv sites. in the 59-and 39-tag vectors an inframe stop codon was introduced immediately after the ecorv site or after the his tag sequence, respectively. the y2h destination vectors pgbkt7-dest (bait) and pgadt7-dest (prey) were derived from pgbkt7 and pgadt7 (clontech) by introducing a gatewayh rfb conversion cassette into the smai sites as described recently (23) . into these vectors the sars-cov orfs were transferred from the donor plasmids via lr reaction. clones were checked by restriction enzyme analysis using ecorv for the his-/flag tag vectors and ecori and bamhi (neb) for the yeast vectors. the haploid saccharomyces cerevisiae strains ah109 (mata, trp1-901 m, leu2-3, 112, ura3-52, his3-200, gal4d, gal80d, lys::ga-l1 uas -gal1 tata -his3, mel1 gal2 uas -gal2 tata -ade2, ura3::mel1 uas -mel1 tata -lacz) and y187 ( mata, his3-200, trp1-901, ade2-101, ura3-52, leu2-3, 112, gal4d, met, gal80d, ura3::gal1 uas -gal1 tata -lacz, mel1) were chosen for the yeast-two hybrid assay. ah109 and y187 were transformed using 1 mg of prey (pdest-gadt7) or bait vector (pdest-gbkt7), respectively. yeast cells were incubated for 1 h in 750 ml peg/bicine solution (40% peg 1000, 200 mm bicine ph 8.35) at 30uc, followed by 5 min at 45uc. cells were pelleted and resupended in 1 ml np-buffer (0.15 m nacl, 10 mm bicine ph 8.35), pelleted a second time and resuspended in 200 ml npbuffer and plated on to sd medium (+2% agar) lacking either leucine (prey) or tryptophane (bait). colonies were visible after 2-3 days. the yeast strains ah109 and y187 containing proteins in prey and bait were arrayed in a 96-deep-well plates with sd liquid media lacking leu or trp according to the interactions to be tested. the liquid cultures were transferred on to sd medium plates lacking leu or trp using a 384-pin replica tool (nunc). colonies were grown for 2 days at 30uc and used directly for mating on ypd medium plates. each mating was performed in quadruplicates, and after 2 days at 30uc the colonies were stamped onto sd medium (-leu-trp) plates. the interactions were assessed by transfer to sd-leu-trp-his plates, and interactions considered positive if at least three out of four possible colonies grew. viral proteins acting as self-activating baits were analyzed on increasing amounts of +3 mm 3-amino-1,2,4-triazole (3at, sigma) (3 mm, 10 mm, 50 mm and 100 mm), and excluded from the results if no clear positive interactions could be determined. 293 cells were infected with recombinant vaccinia virus vtf-7 expressing the t7 rna polymerase (nih aids repository) at a moi of 10 in dmem/1% fcs. one hour post infection, viruscontaining medium was removed and substituted by dmem/1% fcs. ten micrograms (per dish) of the respective pgbkt7-sars-cov-orf and pgadt7-sars-cov-orf plasmids were then transfected into two 10 cm dishes of 293 cells using the calcium phosphate method. after 20 to 24 h, cells were lysed by incubation in np-40 lysis-buffer (1% np-40, 140 mm nacl, 5 mm mgcl 2 , 20 mm tris ph 7.6, 1 mm pmsf, one tablet of complete protease inhibtor cocktail (roche) per 50 ml on ice for 30 min. lysates were centrifuged at 20.5006 g for 10 min and precleared using 50 ml preequilibrated protein g-sepharose (amersham pharmacia). lysates were precipitated using either 5 ml (200 mg/ml) mouse monoclonal anti-myc (santa cruz biotechnology) or 10 ml (100 mg/ml) rat monoclonal anti-ha (roche diagnostics gmbh) antibodies in the presence of 50 ml protein g sepharose beads and incubated on at 4uc by overhead rotation. the beads were washed 3 times in ice-cold np-40 buffer and resuspended in 26sds protein sample buffer. precipitates were separated by sds-page using 12.5% or 15% polyacrylamide gels. proteins were transferred to nitrocellulose (schleicher & schuell) membranes in western blot chambers on at 4uc. filters were blocked with 5% milk powder in tbst (50 mm tris, ph7.6, 150 mm nacl, 0,05% tween-20) for 1 h. they were then incubated with the anti-myc and anti-ha antibodies at dilutions of 1:1000 on at 4uc. filters were washed three times for 10 min with tbst. incubation with the secondary, peroxidase-conjugated antimouse igg or anti-rat igg (1:3000 each) antibodies (jackson) was carried out for two to three hours. after three further washing steps filters were developed using the ecl tm western blotting detection kit (amersham biosciences). the coip was scored positive if a coprecipitate was detected in at least one direction. recombinant sars-cov technology was done as described by yount et al. [16] . the sequence of the mutated sars-cov orf9b knockout (icsarsdorf9b) was confirmed by sequencing cdna isolated from recombinant virus. immunofluorescence subcellular localization analysis of orfs was carried out in hela cells (atcc ccl-2). cells were transfected with the plasmids using fugene6 (roche) according to the manufacturer's instructions, for a total of 24 h. cells were then fixed in ice cold methanol prior to processing for immunofluorescence. primary mouse anti-flag (m2) (sigma) and anti-mouse-alexa488-conjugated secondary antibodies (molecular probes) were used to detect transfected cells, and coverslips were mounted in mowiol. images were acquired on a zeiss axiovert 200 microscope with a 636/1.4 na oil objective and standard filter sets. figure s1 coips of accessory proteins. 293 cells were infected with vaccinia virus vtf-7 and subsequently co-transfected with ha-and c-myc-tagged plasmids carrying the respective sars-cov orfs. after 20 hours half of the cell lysates 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of protein networks we are very grateful to dr. christian drosten and prof. j. ziebuhr for providing materials and a number of plasmids, and to prof. ulrich koszinowski for critically reading the manuscript. key: cord-306733-df36w6l7 authors: rosales-mendoza, sergio; márquez-escobar, verónica a.; gonzález-ortega, omar; nieto-gómez, ricardo; arévalo-villalobos, jaime i. title: what does plant-based vaccine technology offer to the fight against covid-19? date: 2020-04-14 journal: vaccines (basel) doi: 10.3390/vaccines8020183 sha: doc_id: 306733 cord_uid: df36w6l7 the emergence of new pathogenic viral strains is a constant threat to global health, with the new coronavirus strain covid-19 as the latest example. covid-19, caused by the sars-cov-2 virus has quickly spread around the globe. this pandemic demands rapid development of drugs and vaccines. plant-based vaccines are a technology with proven viability, which have led to promising results for candidates evaluated at the clinical level, meaning this technology could contribute towards the fight against covid-19. herein, a perspective in how plant-based vaccines can be developed against covid-19 is presented. injectable vaccines could be generated by using transient expression systems, which offer the highest protein yields and are already adopted at the industrial level to produce vlps-vaccines and other biopharmaceuticals under gmpc-processes. stably-transformed plants are another option, but this approach requires more time for the development of antigen-producing lines. nonetheless, this approach offers the possibility of developing oral vaccines in which the plant cell could act as the antigen delivery agent. therefore, this is the most attractive approach in terms of cost, easy delivery, and mucosal immunity induction. the development of multiepitope, rationally-designed vaccines is also discussed regarding the experience gained in expression of chimeric immunogenic proteins in plant systems. the coronaviruses (covs) are enveloped viruses having a positive-sense, single-stranded genomic rna [1] and are grouped into four genera: α-covs, β-covs, γ-covs, and δ-covs. the ones that affect mammals are αand β-covs, while the other two genera infect birds and could also infect mammals [2] . lately, the coronavirus has become a remarkable concern for global health after the diagnosis of a cluster of unknown pneumonia patients in december 2019 in wuhan, china. the outbreak was associated with workers in the wuhan wholesale seafood market, in which live exotic animals are sold [3] . the pathogen was named sars-cov-2 given its similarity (70% of genomic similarity) to sars-cov-1, which was responsible for the 2002-2003 severe acute respiratory syndrome epidemic (sars) . until now, the official and accepted name for this new coronavirus strain is covid-19 virus, an acronym for coronavirus infectious disease 2019 [1] . a systematic genomic analysis has revealed 380 amino acid substitutions between sars-cov-1 and sars-cov-2, which is a starting point to study its functional and pathogenic divergence [4] . presently, there is no specific treatment for covid-19. as an emerging pathogen, several knowledge gaps exist about the sars-cov-2 virus and the coming months will be critical to refine clinical management and advancement in the validation of possible treatments. a myriad of efforts is ongoing to perform clinical trials and determine the efficacy of preexisting drugs, with hydroxychloroquine and remdesivir as the most promising candidates [10] . receptor blockers are also under evaluation [11] , along with the assessment and development of monoclonal antibodies. moreover, transfusions using plasma from individuals recovered from sars-cov-2 infection (containing neutralizing antibodies) are under exploration with promising findings [12] . nonetheless, vaccination is the most effective approach to control and ultimately eradicate infectious diseases. since sars-cov-2 possesses high transmissibility (asymptomatic and presymptomatic virus shedding, which results in a high number of patients with mild symptoms), the development of vaccines is an urgent goal to fight against this pathogen. the straightforward path to generate vaccine candidates is the technology of inactivated vaccines, which can be formulated with sars-cov-2 virions previously inactivated by chemical or physical treatments. a vaccine based on live-attenuated virus is another possible approach [13] . for sars-cov-1 and mers-cov, inactivated vaccine candidates have induced robust humoral responses leading to virus neutralization. alternatively, the construction of a chimeric attenuated virus constitutes an interesting path. for instance, the influenza virus can be used as a scaffold to expose sars-cov-2 antigens and generate a bivalent vaccine targeting two relevant pathogens causing respiratory diseases [14] . similarly, adenoviral vectors could be applied in this field [15] . vaccines based on whole viruses are associated with concerns about antibody-dependent enhancement of viral infection, reactogenicity, and strain reversion to pathogenic forms, among other safety issues [16] . therefore, the path for vaccine development will require a detailed characterization, not only in terms of efficacy but also safety [17] . although inactivated vaccines will perhaps be the main avenue for generating the first experimental vaccines, alternative approaches should be explored in parallel, namely the development of subunit vaccines. since the coronavirus spike (s) glycoproteins initiate entry into cells; they are considered the primary target for neutralizing antibodies (figure 1 ). cytotoxic t-lymphocyte (ctl) responses are also of key relevance to protect against viral infections [18] . under these principles, vaccine development against covid-19 has been initiated and efforts announced in this field include the development of rna-based vaccines by curevac [19, 20] , and an rna vaccine candidate formulated with a novel lipid nanoparticle (lnp) carrying mrna encoding for a full-length, prefusion stabilized s protein [21] . another candidate (developed by shenzhen geno-immune medical institute [22] ) consists of a multiepitopic vaccine based on the generation of artificial antigen presenting cells through transduction as a way to express viral antigens and immune modulatory genes to ultimately activate t cells. vaccines 2020, 8, x 3 of 20 these principles, vaccine development against covid-19 has been initiated and efforts announced in this field include the development of rna-based vaccines by curevac [19, 20] , and an rna vaccine candidate formulated with a novel lipid nanoparticle (lnp) carrying mrna encoding for a fulllength, prefusion stabilized s protein [21] . another candidate (developed by shenzhen geno-immune medical institute [22] ) consists of a multiepitopic vaccine based on the generation of artificial antigen presenting cells through transduction as a way to express viral antigens and immune modulatory genes to ultimately activate t cells. figure 1 . structure of the sars-cov-2 virus. the virus is formed by an envelope membrane associated with the following structural proteins: spike protein (s), which mediates binding to the host cell receptors and considered a critical target for the induction of antibodies capable of neutralizing the virus; hemagglutinin-esterase dimer (he), which acts as a potent mediator of attachment and destruction of sialic acid receptors on the host cell surface; a membrane glycoprotein (m), which is important to generate the virus; and the envelope protein (e), which adheres to the m protein to form the viral envelope. the viral structure also comprises a nucleocapsid protein (n) that, along with the rna genome, produces the nucleocapsid. although the conventional developmental paths for vaccines take several years, the vaccine against hand, foot, and mouth disease (hfmd) that was approved in china; demonstrates how joint efforts can lead to new vaccines that are accepted in relatively short periods after a pathogen emerges [23] . similarities with the sars-cov-1 virus constitute a valuable reference for vaccine development, however it should be considered that the genetic variability of sars-cov-1 seems higher as more than two variants have been described. despite this, the cross protection of sars-cov-1 and mers experimental vaccines remains to be explored. thus far, there is evidence that some sars-cov-1 neutralizing antibodies cross react with sars-cov-2, however extensive research in this regard is needed. the epitopes conserved among sars-cov-1 and sars-cov-2 have been identified and proposed for vaccine development [24] . the race for generating fundamental knowledge on covid-19 is leading to rapid advances, which will be useful to refine vaccine design. for instance, walls et al. [25] , reported cryo-em structures of the ectodomain trimer, which will be of critical importance for designing vaccines and viral entry blockers. they also found evidence of potent inhibition of sars-cov-2 entry into host cells by using murine polyclonal antibodies against sars-cov-1, which suggests that conserved s figure 1 . structure of the sars-cov-2 virus. the virus is formed by an envelope membrane associated with the following structural proteins: spike protein (s), which mediates binding to the host cell receptors and considered a critical target for the induction of antibodies capable of neutralizing the virus; hemagglutinin-esterase dimer (he), which acts as a potent mediator of attachment and destruction of sialic acid receptors on the host cell surface; a membrane glycoprotein (m), which is important to generate the virus; and the envelope protein (e), which adheres to the m protein to form the viral envelope. the viral structure also comprises a nucleocapsid protein (n) that, along with the rna genome, produces the nucleocapsid. although the conventional developmental paths for vaccines take several years, the vaccine against hand, foot, and mouth disease (hfmd) that was approved in china; demonstrates how joint efforts can lead to new vaccines that are accepted in relatively short periods after a pathogen emerges [23] . similarities with the sars-cov-1 virus constitute a valuable reference for vaccine development, however it should be considered that the genetic variability of sars-cov-1 seems higher as more than two variants have been described. despite this, the cross protection of sars-cov-1 and mers experimental vaccines remains to be explored. thus far, there is evidence that some sars-cov-1 neutralizing antibodies cross react with sars-cov-2, however extensive research in this regard is needed. the epitopes conserved among sars-cov-1 and sars-cov-2 have been identified and proposed for vaccine development [24] . the race for generating fundamental knowledge on covid-19 is leading to rapid advances, which will be useful to refine vaccine design. for instance, walls et al. [25] , reported cryo-em structures of the ectodomain trimer, which will be of critical importance for designing vaccines and viral entry blockers. they also found evidence of potent inhibition of sars-cov-2 entry into host cells by using murine polyclonal antibodies against sars-cov-1, which suggests that conserved s epitopes among these viruses are relevant for immunization and perhaps preexisting immunization models will be useful in the fight against covid-19. the characterization of other key targets for drug design has been accelerated (e.g., the protease x-ray structure has been recently described [26] ). therefore, these recent advances will set the basis to accelerate the development of covid-19 subunit vaccines taking into consideration the sars-cov-1 vaccine development, especially the approach targeting the s protein. in parallel with these advances in vaccine design, defining vaccine platforms amenable for rapid and large scale production is required. these must keep in mind that low cost, safe, and easy distribution and delivery are the required attributes to implement broad coverage of vaccination campaigns; especially in the developing countries. genetically engineered plants constitute a consolidated platform for the manufacturing of biopharmaceuticals. plants have been used over the last three decades for this purpose. thus far, a diverse group of biopharmaceuticals has been functionally produced in plant systems that include antibodies, vaccines, growth factors, and cytokines [27] . remarkably, a recombinant enzyme produced in carrot cells has been already approved by the fda for gaucher's disease treatment [28] . the main advantages of plant-based platforms include the inability to replicate human pathogens (diminishing the risk of contamination and making the purification process less strident), use of unsophisticated bioreactors, and efficient synthesis of complex proteins (multimeric or glycosylated) [29, 30] . the current expression approaches for recombinant proteins using the plant cell as host comprise stably-transformed plants at the nuclear or chloroplast levels and transiently-transformed plants. these expression modalities are summarized in table 1 . the conventional approach for expression of transgenes in plants comprises transgene insertion into the nuclear genome. currently, agrobacterium-mediated transformation is the most popular method to achieve this modification since this bacterium has the ability to transfer large segments of dna with minimal rearrangement at high efficiency with low number of insertions. nonetheless, the transgene is randomly inserted into the genome, which often leads to positional effects that make expression levels unpredictable and interruption of endogenous genes a possibility. another limitation is the induction of silencing mechanisms that hamper productivity. nevertheless, it should be considered that new technologies are emerging to cope with these limitations by providing ways to achieve site-directed insertion through a number of mechanisms [31] . the n-terminal fragment of the sars-cov-1 s protein (s1) was expressed in stably transformed tomato and low-nicotine tobacco plants, which induced iga and igg responses in mice. [32] transient nuclear genome transformation rapid production; high productivity; implemented at the industrial level seed bank cannot be generated; requires purification of the antigen to eliminate toxic compounds from the host and ag-robacteria residues s protein; multiepitope vaccines a chimeric protein of gfp and amino acids 1-658 of the sars-cov-1 s protein (s1:gfp) was transiently expressed in tobacco leaves and stably transformed in tobacco and lettuce. no immunization assays were performed the sars-cov-1 n protein was transiently expressed in nicotiana benthamiana, which induced in mice high levels of igg1 and igg2a and up regulation of ifn-γ and il-10 in splenocytes. a chimeric protein of gfp and the sars-cov-1 s protein was transiently expressed in tobacco plants. no immunization tests were performed. the sars-cov-1 m and n proteins were transiently expressed in n. benthamiana. the n protein was antigenic but immunogenicity was not assessed. [ [33] [34] [35] transplastomic technologies high productivity; multigene expression is possible; improved biosafety since the transgene is inherited maternally; site-specific insertion through recombination; not affected by silencing or position effects complex post-translational modifications are not performed; transformation protocols available for few species and the generation of lines takes long time. a chimeric protein of gfp and amino acids 1-658 of the sars-cov-1 s (s1:gfp) was expressed in transplastomic tobacco plants. [32, 36] vaccines 2020, 8, 183 in the case of transplastomic technologies, they typically comprise the site-directed insertion of the foreign dna into the chloroplast genome in an event induced by homologous recombination. the main advantage of this technology is the high protein yield, which is a consequence of the high copy number of the transgene and the fact that silencing events have not been reported thus far, and position effects are avoided due to the site-directed insertion. in addition, transgene confinement is achieved given the maternal inheritance shown by most plant species and several proteins can be produced from a single transformation event through the use of operon-like arrangements [37, 38] . for more information regarding transplastomic technologies, readers are referred to reviews published by [36, 39] . another approach to express heterologous protein in plants relies on the use of viral-based vectors, which exploit the efficient promoters, utrs, and dna/rna replication mechanisms found in plant viruses. interestingly, the agrobacterium-mediated delivery of viral vectors has consolidated as a highly efficient strategy to achieve rapid production of biopharmaceuticals with yield up to 5 g of protein per kg of fresh plant biomass. the tobamoviruses, potexviruses, tobraviruses, geminiviruses, and comoviruses have served as a basis for the development of transient, efficient expression systems in plants [40] . this technology, however, demands the purification of the target protein given the presence of toxic compounds (e.g., alkaloids) in the typical host (nicotiana benthamiana), as well as bacterial residues, especially endotoxins. therefore, at present, this technology is applied for the formulation of injectable or nasal vaccines. thus far, some plant-vaccine candidates have entered clinical trials, including candidates against swine influenza, rabies, and hepatitis b [41] . the most promising candidates are the influenza vaccines developed by medicago inc. that rely on using a non-replicative vector carrying viral regulatory sequences to mediate the transient expression of hemagglutinin (ha) in n. benthamiana, which has led to injectable vaccine candidates [42, 43] . overall, these vaccines have been seen as safe and their immunogenic properties have been positively evaluated using in vitro assays with human and mice cells. the trials conducted in volunteers revealed proper immunogenicity with no serious adverse effects (see table 2 ). moreover, a group of respiratory diseases has been targeted through the plant-based production of antigens with promising immunogenicity in preclinical trials [44] . both homologous and heterologous antigen-specific cd4+ t cells were elicited. additionally, production of ifn-γ, il-2, and/or tnf-α was achieved upon ex vivo antigenic re-stimulation. ha from a/california/07/2009 h1n1. vlps were evaluated in vitro using human monocyte-derived macrophages. the plant-made vlps were efficiently captured and subjected to endosomal processing and cross-presentation. [47] ha from a/h1n1/california/07/09 (pdmh1n1). the inactivated h1n1 vaccine (iiv) was included as a reference vaccine. mice were i.m.-immunized twice. cd4+ (tnf-α, ifn-γ) and cd8+ (ifn-γ) t cell responses were higher for the plant-made vaccine than the iiv formulation. the plant-made vlp vaccine elicited stronger and more balanced immune responses than iiv. [48] ha from a/california/7/09 (h1n1) and a/indonesia/5/05 (h5n1). in vitro assays were performed using mouse and human dcs. mice were immunized by the i.m. route using alhydrogel as an adjuvant. human dcs exposed to plant-made vlps showed high stimulation in terms of secretion of il-6, il-10, and tnfα and cd83 expression, along with activation of cd4+ and cd8+ t cells. vlps induced accumulation of both cdc1s and cdc2 in the draining lymph nodes of test mice. lymphocytes from mice immunized with plant-made vlps secreted higher concentrations of pro-inflammatory cytokines (including il-12, il-6, and tnf-α) upon antigenic stimulation. [49] ha from a/california/7/2009 or a/indonesia/5/05 strains. in vitro assays were performed using mouse dendritic cells. the plant-made vlps trimmers were morphologically stable over time and interacted with activated antigen-presenting cells similar to the wild type virus. [50] nonetheless, the ultimate goal for low-cost vaccine development could be achieved by generating oral formulations not requiring purification and being composed of freeze-dried biomass encapsulated in gelatin pills or tablets. under this focus, the goal is to trigger specific immune responses via the gut-associated lymphoid tissues (galt). for this purpose, edible plants lacking toxic metabolites should be used. perhaps the main disadvantage of this technology is the long time required to generate transformed lines of edible crops efficiently expressing the antigen of interest (e.g., rice or corn transgenic lines or transplastomic lines) [51] . these oral vaccines will overcome the disadvantages of injectable vaccines such as painful administration and the risk associated with invasive delivery routes. in terms of costs, avoiding the requirements of sterile devices and trained personnel represent substantial savings. the fact that plant-based vaccine formulations will not require antigen purification will be, without a doubt, the main factor that will make them low-cost alternatives [52] , which is necessary to provide wide vaccination coverage in developing and low-income countries. recent promising reports in the viability of formulating oral vaccines include the case of the hepatitis b virus surface antigen [53] and models of immunotherapy against allergy with promising results [54] , among other diseases. therefore plant-based vaccines are becoming a reality, although it should be recognized that the progress has been slower than initially expected. this is particularly true for the development of oral vaccines, with the main drawbacks being poor characterization of antigen stability, bioavailability, and reproducibility [51, 55] . the currently available expression strategies for foreign proteins in plants allow projecting the generation of several vaccine candidates against covid-19 in the short term. the expression approach and targeted organelle will depend on the nature of the selected target antigen. in the following section, possible avenues for the development of plant-based vaccines are envisaged (table 3 and figure 2 ). the currently available expression strategies for foreign proteins in plants allow projecting the generation of several vaccine candidates against covid-19 in the short term. the expression approach and targeted organelle will depend on the nature of the selected target antigen. in the following section, possible avenues for the development of plant-based vaccines are envisaged (table 3 and figure 2 ). developmental paths for plant-based vaccines. vaccine antigens can comprise full length viral proteins or chimeric proteins rationally designed to serve as multiepitopic vaccines. this is based on the selection of t cell and b cell epitopes with the aid of immune bioinformatics tools and experimental data of their protective capacity. current genetic engineering technologies allow expressing target antigens either stably or transiently, which allow exploring distinct immunization approaches. transient expression typically requires purification of the antigen-which is useful for the formulation of injectable vaccines-whereas stable transformation, especially in edible crops, allows implementing oral immunization schemes without purification. combined schemes, using pure antigens for priming by parenteral administration and plant biomass containing the antigen for oral boosting, are likely ideal approaches capable of inducing long-term protection in several compartments, especially in mucous membranes. vaccine antigens can comprise full length viral proteins or chimeric proteins rationally designed to serve as multiepitopic vaccines. this is based on the selection of t cell and b cell epitopes with the aid of immune bioinformatics tools and experimental data of their protective capacity. current genetic engineering technologies allow expressing target antigens either stably or transiently, which allow exploring distinct immunization approaches. transient expression typically requires purification of the antigen-which is useful for the formulation of injectable vaccines-whereas stable transformation, especially in edible crops, allows implementing oral immunization schemes without purification. combined schemes, using pure antigens for priming by parenteral administration and plant biomass containing the antigen for oral boosting, are likely ideal approaches capable of inducing long-term protection in several compartments, especially in mucous membranes. one prominent approach for vaccine design is based on the use of virus-like particles (vlps), which are macromolecular complexes resembling viruses, but lacking their genome. in this way, vlps mimic the native structure of viruses, but are not infective. this avoids the disadvantages of vaccines formulated with attenuated or inactivated viruses that include reactogenicity and reversion to pathogenic forms [61, 62] . a myriad of reports on the production of vlps can be found in the literature that comprise the cases of the influenza virus, human papillomavirus, human immunodeficiency virus, foot and mouth disease virus, norwalk virus, rift valley fever virus, and hepatitis b virus [61] [62] [63] [64] . the precedents of sars-cov-1 and mers antigens expressed in recombinant systems leading to the formation of vlps constitute important guides for the topic of covid-19 vaccine development. mortola and roy (2004) produced coronavirus vlps applying baculovirus/insect cells expression (sf9 cells), which was based on the following structural proteins: s (spike), e (envelope), m (membrane), and n (nucleocapsid) of sars-cov, either individually or simultaneously. simultaneous expression at high levels was achieved for s, e, and m proteins, leading to an efficient vlps assembly and release, evidenced by electron microscopy and immunofluorescence. the authors demonstrated that the formed vlps were similar in morphology to the sars-cov-1 virions [65] . another group reported in the same year that m and e proteins were sufficient for the efficient formation of vlps in insect cells [66] . in 2007, the immunogenicity of sars-cov-1 vlps was first described by evaluating insect cell-made vlps based on the m, e, and s proteins. electron microscopy demonstrated vlps formation in co-infected insect cells. mice subjected to an immunization scheme consisting of four subcutaneous (s.c.) doses of vlps emulsified with freund's adjuvant showed high antibody titers against sars cov. furthermore, vlps elicited cellular immunity following increased production of ifn-γ and il-4 [67] . subsequent in vitro assays using vlps designed with the bat-isolated coronavirus protein s and the sars-cov-1 proteins e and m; demonstrated ability to stimulate dcs in terms of cytokine induction, evidenced by il-6 and tnf-alpha production. furthermore, the study indicated that ifn-γ+ and il-4+ cd4+ t cells increased in co-culture with dcs pre-exposed to the vlps tested [68] . given that immunization by mucosal routes is the most pertinent for vaccination, sars-cov-1 vlps were analyzed in a mouse model based on intraperitoneal or nasal immunization. both routes led to sars-cov-1-specific igg responses, igg levels in the groups immunized intraperitoneally were higher. nasal immunization usually results in the induction of secretory iga responses at the genital tract, saliva, and lungs; a type of response that is not efficiently induced by systemic administration. in the mentioned study, iga production at the intestinal tract was higher for intraperitoneal administration when compared to intranasal administration, however only genital tract secretions from the i.n.-immunized group showed neutralization activity [69] . another approach for the production of vlps consisted in co-expressing the sars-cov-1 s protein and the m1 influenza protein in the baculovirus/insect cell expression system. interestingly, the chimeric vlps showed similar size and morphology compared to the wild type sars-cov-1. immunogenicity and protective efficacy of these chimeric vlps were tested in a mouse lethal challenge model. complete protection from death was observed in mice vaccinated intramuscularly or intranasally with the chimeric vlps, which contained 0.8 µg of the sars-cov-1 protein s [70] . in the case of the middle east respiratory syndrome (mers), vlps with proteins s, e, and m were generated using the baculovirus/insect cell expression system by wang et al. (2017) [71] . electron microscopy and immunoelectron microscopy revealed high structural similarity between the mers-cov vlps and the native virus. rhesus macaques inoculated with mers-cov vlps and aluminum adjuvant showed final virus neutralizing antibody titers reaching 1:1280; inducing t-helper 1 (th1) cell-mediated immunity. in a more recent study, the structural proteins of mers-cov were expressed in silkworm larvae and bm5 cells for the development of vaccine candidates. however, protein s was not detected in the form of vlps despite the fact that e and m proteins were secreted to the culture supernatant. surfactant treatment and mechanical extrusion using protein s or bm5 cells expressing structural proteins allowed producing nanovesicles exhibiting protein s with a diameter ranging from 100 to 200 nm as revealed by immuno-tem [72] . although insect cells possess a protein processing capacity similar to that of higher eukaryotic cells, the protein processing pathways are not totally equivalent [73] . in terms of n-acetylglycosylation, insect cells have the ability to assemble n-glycans to a nascent polypeptide. however, they have unusual final processing activity mediated by β-n-acetylglucosaminidase (soluble or membrane-bound form), which trims an intermediate product common to mammalian and insect pathways resulting in the insect-specific paucimannose as a final product that avoids the galactosylation process occurring in mammals [74] . considering that vlps of enveloped viruses have been successfully expressed in plants by expressing the ha protein, it is anticipated that sars-cov-2 vlps could be assembled by expressing the s protein. in fact, although not published in the literature yet, medicago has described the generation of vlps in plants [75] . for this purpose, nuclear expression targeting the trans-golgi secretion route is proposed to yield a protein subjected to the glycosylation and secretion machinery that could make possible the production of vlps [76] . therefore, adoption of these works would be valuable during the development of covid-19 plant-based vaccines (see the section below). besides vlps resembling the sars-cov-2 virus, another possible approach is to adopt sars-cov-2 epitopes and express them in chimeric vlps. in this way, a vlp from an unrelated virus can serve as the scaffold to present the target sars-cov-2 epitopes. for this purpose, the hepatitis b core protein has been applied as scaffold to display unrelated antigens of some pathogens. in particular, this has been applied at the immunodominant c/e1 loop region, which allows displaying the target on the spike structures of the vlps [77] . a recent review revealed that at least 97 vaccine candidates have been developed based on plant viruses covering infectious agents, cancer, and autoimmune disorders [78] . the glycosylation patterns of proteins that form vlps can impact their immunogenicity and protective capacity. interestingly, glycoengineering strategies have been successfully implemented for plants, which allows diversifying their application as hosts for the production of biopharmaceuticals. this is a relevant aspect considering that plants lack the ability to perform glycosylation, which is a characteristic of mammalian systems. for instance, plants perform beta1,2-xylosylation, core alpha1,3-fucosylation, and the addition of a second n-acetylglucosamine (glcnac) to the mannose core. moreover, plant glycans lack β (1,4')-galactose and sialic acid, as well as bi-antennary n-glycans [79] . in some cases, such as antibody production, these differences on glycosylation can be associated to adverse effects that include generation of immunogenic activity, which can eventually lead to blocking antibodies against the therapeutic antibody. nonetheless, in the case of vaccines these differences could add more immunogenic potential and improve vaccine efficacy [80] . in any case, it is of interest to comment that glycoengineering has allowed developing knock down n. benthamiana plants for beta1,2-xylosyltransferase (xylt) and alpha1,3-fucosyltransferase (fuct) genes, which showed significant reduction in the production of xylosylated and/or core alpha1,3-fucosylated proteins with no phenotypic alterations [81] . these lines will be valuable tools to study the impact of glycosylation in the efficacy of covid-19 vlps, which will allow optimizing the plant-based vaccines. another approach that deserves attention is the development of multiepitopic vaccines, which offers the opportunity of achieving a fine rational vaccine design by selecting epitopes that potentially induce robust and protective immune responses. at the same time, this approach will discard those related to non-protective responses or even those inducing antibody-dependent enhancement of the disease. in this way, a highly efficacious and safe vaccine can be obtained. of special interest are the reports proving that specific s protein epitopes enhance the disease upon a pathogen challenge, which highlights the relevance of recurring to the rational design of vaccines to ensure not only immunoprotection, but safety [82] . for the case of multiepitope vaccines against infectious agents, several reports have focused on selecting the most promising th, b cell, and t cell epitopes that might allow the rational design of a vaccine inducing robust protective responses, while at the same time avoiding deleterious or non-relevant responses. one key factor that adds importance to the development of multiepitopic vaccines against viral disease is genetic variability. in fact, it has been suggested that the sars-cov-2 virus evolved into two major types, l and s. the l type (∼70%) predominates versus the s type (∼30%), with the latter being proposed as the ancestral version. the l type is more aggressive than the s type and the analysis performed by tang et al. [83] suggests that human interference influenced the prevalence of l and s types right after the outbreak appearance. another study has also suggested a rapid evolution of this coronavirus [84] . therefore, the design of multiepitopic vaccines must adopt the selection of epitopes conserved among the viral variants with the capacity to induce neutralizing humoral responses as critical criterion. it should also be considered that ctl responses are relevant to cope with covid-19 [85] . the selected epitopes are the targets to which adaptive immune responses should be induced, but they lack the required complexity to trigger robust immune responses. therefore, a carrier is required to increase antigen complexity and, through adjuvant effects, enhance potency of the induced immune response, making it properly polarized. among these carriers, the b subunit of the cholera toxin and the heat-labile enterotoxin from escherichia coli have been extensively used to carry unrelated antigens. the main advantage of these molecules is their adjuvant effects at the mucosal levels, as well as their ability to efficiently deliver the antigens to the submucosa thanks to a mechanism of translocation mediated by binding to the gm1 receptor at the epithelial cells in the mucosal surfaces [86] . once at the submucosa, the antigen is efficiently captured and processed by antigen presenting cells, which subsequently induce robust mucosal and systemic th1 immune responses at the lymph nodes. the design of ltb/ctb-based chimeras carrying sars-cov-2 epitopes is an interesting approach that should be explored. proline-containing linkers have been used to properly display unrelated antigens without altering the ability of those carriers to form the pentameric structure responsible for gm1 binding [57] . the accumulation of those chimeric proteins in the er is advisable, although toxicity has been reported for some plants accumulating high levels of those recombinant proteins [87] . given that plant systems have been successfully adopted for the production of multiepitope proteins, the generation of such vaccines against covid-19 is considered highly viable [88] [89] [90] . besides expressing chimeric proteins, multiple antigen expression can be achieved by transplastomic technologies (operon-like expression) or at the nuclear level using the picornaviral 2a sequence that allows releasing individual antigens from a single coding gene [91] . the production of immune complexes (ics) in plants is another approach that renders highly immunogenic agents. ics consist of antigens complexed to antibodies recognizing them, constituting macromolecular entities that are efficiently captured and processed by antigen presenting cells [92] . this results in the induction of robust humoral and cell-mediated immune responses [59, 93] . taking advantage of the machinery of plant cells for protein synthesis and processing, they have been exploited as antibodies and ics factories [94] . for instance, ics based on the tetanus toxin fragment c fused to a monoclonal antibody were produced in transgenic tobacco plants. these ics were highly immunogenic, inducing immunoprotective effects when administered without accessory adjuvants by the s.c. (subcutaneous) route in mice [95] . this approach has also been applied to the case of the ag85b and acr antigens from mycobacterium tuberculosis and the gp1 antigen from the ebola virus [96, 97] . perhaps the main disadvantage of this approach is the requirement of defined antibodies targeting the antigen, which is not available yet for sars-cov-2. however, it should be considered that the cross reactivity of anti-sars-cov-1 s protein antibodies with the counterpart of sars-cov-2 has been reported [3] . the purification of antigens is a laborious and expensive activity as part of the production of injectable vaccines [97] . one alternative to simplify purification relies on the fusion of elastin-like polypeptides (elp) that possess a unique property named reversible phase transition, which allows precipitating the protein of interest by changing the temperature. this approach is an alternative to the complex/expensive affinity chromatography [98] that has been applied to develop plant-based vaccines candidates with relevant findings. for instance, the m. tuberculosis antigens ag85b and esat-6 were fused to elp and expressed in transgenic tobacco [99] , and the plant-made antigens were subcutaneously (s.c.)administered to mice and able to trigger long-lasting humoral immune responses. the elp did not negatively impact the immunogenic properties of the antigens. the haemagglutinin (ha) antigen from the influenza virus has also been expressed under this modality in tobacco, where antigen purification was simplified and the obtained product induced neutralizing antibodies in mice after two s.c. immunizations. elpylation did not alter the immunogenicity of ha [99] . therefore, these precedents suggest that the elp technology is a possible approach to be explored for the production of antigens against the sars-cov-2 virus. the common steps to the above-mentioned antigen design will comprise determining antigen yields and antigenic activity, assessing the immunogenic activity in test animals under distinct administration routes, and validating the safety and stability of the vaccine. the latter will be critical to justify the application and get approval to conduct clinical trials. a key precedent for this field is the vaccine candidates already reported for sars-cov-1 and mers, which are both closely related to sars-cov-2. following nuclear expression approaches, the n-terminal fragment of the sars-cov-1 s protein (s1) was expressed in tomato and low-nicotine tobacco plants using an agrobacterium-mediated method. mice orally immunized with this transgenic tomato revealed significantly increased levels of sars-cov-1-specific iga. sera of mice parenterally primed with the transgenic tobacco showed the presence of anti-sars-cov-1-igg [32] . another study focused on expressing a chimeric protein of gfp and amino acids 1-658 of the sars-cov-1 spike protein (s1:gfp) by using tobacco leaves subjected to transient expression. after microscopy localization, the fusion protein was observed in the cytosol and the periphery of the nucleus. stable transgenic tobacco and lettuce plants were generated by agrobacterium-mediated transformation. the expression of the chimeric antigen was also achieved in tobacco chloroplasts. however, no immunization assays were performed [33, 100] . the sars-cov-1 nucleocapsid (rn) protein of 423-aa was transiently expressed in n. benthamiana. yields reached up to 79 µg/g of leaf fresh weight (corresponding to 0.8%-1% of the total soluble protein, tsp) at 3 dpi under silencing suppression conditions (p19). mice immunization revealed that three doses led to effective b-cell maturation and differentiation, achieving high levels of igg1 and igg2a. ifn-γ and il-10 were up-regulated in splenocytes, while the expression of il-2 and il-4 was not [33] . the nucleocapsid protein (n) and the membrane protein (m) were transiently expressed in n. benthamiana. yields reached up to 3-4 µg/g fresh leaf weight (0.2% tsp) for the n protein, while the m protein yields were 0.1%-0.15% tsp. the purified n protein reacted with human sera having n-specific antibodies. no immunization assays were performed [35] . these studies constitute a valuable guide to select the most convenient expression platforms for specific sars-cov-2 antigens. although most of the vaccines against respiratory diseases are administered by parenteral routes, it should be recognized that immune profiles deserve improvements, especially in terms of immunogenicity and efficacy in the elderly [101] . mucosal vaccines, especially intranasal-administered vaccines, are a promise for immunization against respiratory diseases given the protections induced in lungs and other mucosal tissues that are critical ports for pathogen entry. critical alternatives for the development of mucosal vaccines include guaranteeing antigen delivery and immunostimulating capacity at the mucosa, while at the same time being minimally reactogenic/toxic. among the immunopotentiator molecules that can be explored are bacterial toxin derivatives, toll-like receptor ligands, and cytokines [102, 103] . although some companies have already started the production of plant-based vlps vaccines [75, 104] using transient expression systems, these efforts are mainly directed to developing injectable vaccines. therefore, it is imperative to start developing alternatives related to mucosal immunization. for this purpose, generating plants stably expressing sars-cov-2 antigens will open new avenues for the exploitation of this technology. this approach will result in the production of edible plant material containing sars-cov-2 antigens that could be used for the development of oral boosting agents. interestingly, this concept has been assessed by some groups with relevant results. in the case of a vaccine targeting the hepatitis b virus, a scheme comprising intramuscular (i.m.) boosting with the pure hbsag antigen followed by double oral boosting with plant material containing hbsag at six week intervals led to a comparable response to that induced by the standard intramuscular (i.m.) vaccination scheme [53] . moreover, in the case of poliomyelitis vaccination, a plant-based vaccine based on transplastomic plants expressing the vp1 antigen fused to a carrier has been assessed as oral boosting agents after priming with the inactivated polio vaccine administered subcutaneously. boosting with the plant-made vp1-based antigen significantly enhanced vp1-igg1 and vp1-iga titers when compared to the response in animals not receiving boosts. the scheme also allowed inducing neutralizing antibodies for the three poliovirus sabin serotypes when two doses of ipv and plant-cell oral boosts were administered, whereas a single dose of ipv resulted in low neutralization potential [105] . in this way, freeze-dried plant material can be used as a low-cost vaccine rendering thermostability and easy to administer features. similarly, pure antigens produced in plants have been assessed with promising results in schemes where a conventional vaccine is used as a priming agent and the plant-made antigen is administered by the oral route as boosting [106] . exploring these schemes will be useful to achieve the best immune profile against sars-cov-2 in terms of safe and long-lasting protective immune responses. in fact, the potential of using prime-boosting schemes through different immunization routes has been proven as an efficacious approach to achieve the desired immune profiles [107] . the emergence of covid-19 has led to a global emergency that demands the development of new biologics, especially vaccines, to counteract against this threat. in this scenario, a plant-made vaccine is a viable approach to rapidly respond to this need. the current expression technologies offer relevant paths for developing anti-covid-19 vaccines. vlps constitute an attractive approach for the development of efficient and safe vaccines, which is associated with high immunogenicity, preservation of the antigenic determinants, and lack of replicative capacity. thus, vlps based on the main sars-cov-2 structural proteins is an attractive approach for vaccine development against coronavirus infections. the transient expression systems based on deconstructed viral vectors and n. benthamiana as host will allow for immediate exploitation of plants as efficient biofactories of injectable vaccine candidates, which are expected to be implemented and entered into clinical trials in a year. the development of vaccines based on transplastomic lines or edible plant species transformed at the nuclear level and intended to result in oral vaccines (especially boosting agents to provide mucosal immunity) are considered long term goals. however, they have special importance given their low cost and potential to serve as effective boosting agents, which could lead to attractive immune profiles characterized by proper humoral response in the mucosal compartments and long-lasting protection, especially for the elderly. in parallel to these developments, the production of monoclonal antibodies in plants will provide another strategy to generate alternatives to the convalescent plasma transfusion, in which plant-made antibodies will constitute a low cost and safer intravenous treatment for critically ill patients. perhaps the main challenge envisioned for the development of covid-19 plant-based vaccines will be, as is typical for all vaccines, testing their efficacy in large clinical trials to validate their safety while fulfilling the requirements of regulatory agencies. the fact that there are precedents of a plant-made biopharmaceutical approved for human use and plant-made vaccines against influenza under clinical trials (with promising safety and efficacy) is encouraging. therefore, plant-based vaccines have a realistic potential to contribute to the fight against covid-19. as the covid-19 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license key: cord-291727-4wfhuvww authors: ketteler, robin title: on programmed ribosomal frameshifting: the alternative proteomes date: 2012-11-19 journal: front genet doi: 10.3389/fgene.2012.00242 sha: doc_id: 291727 cord_uid: 4wfhuvww frameshifting results from two main mechanisms: genomic insertions or deletions (indels) or programmed ribosomal frameshifting. whereas indels can disrupt normal protein function, programmed ribosomal frameshifting can result in dual-coding genes, each of which can produce multiple functional products. here, i summarize technical advances that have made it possible to identify programmed ribosomal frameshifting events in a systematic way. the results of these studies suggest that such frameshifting occurs in all genomes, and i will discuss methods that could help characterize the resulting alternative proteomes. frameshifting is a process whereby the ribosome is guided toward a triplet nucleotide that is either shifted one nucleotide position upstream (+1 frameshift) or one nucleotide position downstream (−1 frameshift). such frameshifting occurs in all known organisms, from e. coli to mammals (namy et al., 2004; dinman, 2012a) . there are two main mechanisms that produce out of frame peptides: changes in the genome sequence that result in insertions or deletions (indels) and programmed ribosomal frameshifting as a consequence of the ribosome either slipping back one nucleotide (−1 frameshifting) or skipping one nucleotide (+1 frameshifting) (figure 1) . indels generally produce nonfunctional proteins and are associated with either spontaneous mutations across the genome or somatic genomic instability, for instance, as a consequence of tumour progression. by contrast, programmed ribosomal frameshifting can result in dual-coding genes that produce alternative functional proteins, which form an integral part of the organism's physiology. programmed ribosomal frameshifting has been historically associated with viruses and retrotransposons. retroviruses require frameshifting for replication and infection (maia et al., 1996; brierley and dos ramos, 2006; dulude et al., 2006) . for example, the hiv1 polyprotein gag-pol requires efficient −1 frameshifting for expression of the individual gag and pol gene products. this form of frameshifting usually depends on a combination of a "slippery sequence," a spacer sequence of 1-15 nucleotides, and a stem-loop secondary rna structure such as a pseudoknot (figure 1 ) (namy et al., 2006) . the slippery sequence is generally of the type x xxy yyz, where x denotes any nucleotide, y denotes a or u, and z is a, u, or c. pseudoknots are secondary rna substructures that contain two or more stemloop motifs with intercalated stems. the pseudoknot or stemloop structure in the mrna is thought to result in pausing of the ribosome, resulting in eventual frameshifting (namy et al., 2006) . structural evidence for this mechanism comes from the crystal structure of the mouse mammary tumor virus (mmtv) pseudoknot, which has an unpaired adenine that acts as a hinge to mediate frameshifting (chen et al., 1996) . generally, there is a correlation between the mechanical strength of an mrna pseudoknot and its frameshifting efficiency (hansen et al., 2007) : the stronger the pseudoknot the higher the frameshifting efficiency, although very strong pseudoknots can cause a road block that limits translation downstream (tholstrup et al., 2011) . other examples of mammalian genes that utilize −1 frameshifting are the mouse embryonic carcinoma differentiation regulated (edr) gene and its human ortholog peg10. a slippery sequence of g gga aac, in combination with a pseudoknot, mediates highly efficient −1 frameshifting, similar to viral frameshifting motifs (clark et al., 2007) . recently, a programmed ribosomal −1 frameshift has been identified in the adenomatous polyposis coli (apc) mrna in caenorhabditis elegans that is mediated by a slippery sequence a aaa aaa or a aaa aac (baranov et al., 2011) . the functional relevance of this frameshift is uncertain. although the slippery sequence and pseudoknot are the most common motifs for frameshifting identified thus far, there are alternative mechanisms that may result in the production of out-of-frame proteins. alternative splicing may contribute to frameshifting (hiller et al., 2005) , as can codon bias. for instance, rare trna codons can favor −1 and +1 frameshifting (gurvich et al., 2005; laine et al., 2008) , and rare arginine codons prime mitochondrial sequences for frameshifting (temperley et al., 2010) . moreover, cag repeats are prone to frameshifting, which results in poly-alanine proteins that may contribute to the pathogenesis of neurodegenerative diseases (toulouse et al., 2005) . the use of the peptidyltransferase inhibitor anisomycin reduces −1 frameshifting in these cases and reduces the toxicity associated with the expanded triplet repeats. importantly, out-of-frame proteins (compared to the standard orfeome annotation) can also result from alternative aug or cug start sites (ingolia et al., 2011) , thereby considerably increasing the size of an alternative "frameshifted" proteome. main sources of out-of-frame peptides and proteins are shown in figure 2 . an interesting example of programmed ribosomal +1 frameshifting is that of the ornithine decarboxylase (odc) gene that produces the antizyme from frameshifting of the mrna sequence (bekaert et al., 2008) . odc catalyzes the production of polyamines, such as putrescine, spermidine, and spermine from ornithine through decarboxylation. odc activity is terminated by the antizyme (murakami et al., 1992) -providing an elegant mechanism for shutting down the activity of an enzyme by producing an out-of-frame antizyme from the same mrna. this frameshifting is tightly regulated and can be enhanced by treatment of cells with polyamines (nilsson et al., 1997) . the odc antizyme mechanism is highly conserved throughout all eukaryotes (ivanov et al., 2000b) . the frameshifting requires the figure 2 | main sources for out-of-frame peptides. regular expression of this mrna with translation initiating trnamet will result in expression of peptide "aug(0)" (red). out-of-frame peptides could arise from alternative out-of-frame cug or aug start sites resulting in translation of peptide "cug(−1)" (green). alternatively, −1 or +1 frameshift signals within the original reading frame could result in expression of out-of-frame peptides "aug(−1)" (blue) or "aug(+1)" (yellow), respectively. uga stop codon and a 3 stem loop that forms a rna pseudoknot. these rna hallmarks are still the standard way to identify other +1 frameshifted proteins. it has been proposed that frameshifting is a common mechanism to increase protein-coding capacity of small genomes such as those of viruses and mitochondria. in agreement with this proposal, frameshifting is common in mitochondrial genes, and genome size seems to correlate with the abundance of frameshifting (seligmann, 2010) . in fact, some organisms display a high complexity in frameshifting: in the dinoflagellate perkinsus marinus, the mitochondrial gene that encodes cytochrome c oxidase subunit 1 can shift up to 10 times within the same mrna sequence in order to produce the correct gene product (masuda et al., 2010) . clearly, such complex frameshifting requires efficient regulatory control. there is increasing evidence that ribosomal frameshifting is a regulated event. several enzymatic and non-enzymatic mechanisms have been proposed that result in an enhancement of frameshifting. for instance, the production of antizyme by +1 frameshifting is enhanced by the end-products of odc-spermine, putrescine, and spermidine (ivanov et al., 2000a) . how polyamines regulate +1 frameshifting is not well understood, but one hypothesis is that polyamine binding to rna may enable read-through of the termination codon. similar to polyamines, amino-glycosides such as gentamicin allow read-through of stop-codons (martin et al., 1989; malik et al., 2010) . it is not clear at present whether there are normal regulator proteins that enhance frameshifting efficiencies. because −1 frameshifting is essential for retroviral gene expression, it has been proposed that chemical interference with frameshifting would be a good anti-viral strategy. recently, a genome-wide screen to identify regulators of hiv-1 frameshifting has identified erf1 as an essential host gene required for −1 frameshifting (kobayashi et al., 2010) , suggesting that erf1 may be a good therapeutic target in aids (brakier-gingras et al., 2012) . in addition, it has been proposed that hiv frameshifting can be modulated by protein kinase r, as well as by factors that modulate translation efficiency such as rapamycin (gendron et al., 2008) . interestingly, annexin a2 (anxa2) can bind the pseudoknot structure of avian coronavirus infectious bronchitis virus (ibv) and reduce −1 frameshifting (kwak et al., 2011) . as a consequence, anxa2 has been suggested as a more general antiviral regulator in eukaryotic cells. other potential anti-viral agents could either specifically bind to the frameshift signal (such as antisense oligonucleotides, non-coding rnas, or frameshift signal binding compounds) or interfere with peptidyltransferase activity (e.g., anisomycin or sparsomycin) or eef2 activity (e.g., sordarin) (dinman, 2012b) . for certain diseases, however, it may be benefitial to enhance frameshifting. for instance, in monogenetic diseases such as cystic fibrosis or duchenne's muscular dystrophy, where frameshift mutations result in premature translation termination, the deliberate induction of frameshifting may overcome the problem by skipping the affected sites (aurino and nigro, 2006) . it has been noted that aminoglycosides such as gentamicin can enhance frameshifting and stop codon read-through. gentamicin-induced read-through of stop codons has been evaluated as a treatment option for duchenne's muscular dystrophy (malik et al., 2010) . while the authors conclude from this phase i clinical trial that gentamicin may not be a good treatment option, they note that other read-through agents may have benefits. a phase i/ii clinical trial using a morpholino oligomer (avi-4658) to correct a frameshift mutation in the dystrophin gene has been completed with the conclusion that avi-4658 was well tolerated and had significant benefit in patients with duchenne's muscular dystrophy (cirak et al., 2011) . genome-wide analysis of the yeast genome (jacobs et al., 2007) and other genomes (hammell et al., 1999) suggests that frameshifting is more common than previously thought. however, it has been proposed that frameshifting may predominantly serve to modulate rna levels rather than to produce frameshifted proteins (plant et al., 2004) . evolutionary studies argue that the generation of out-of-frame proteins has been minimized by codon optimization that results in non-functional small peptides rather than functional proteins (bollenbach et al., 2007) . however, it is becoming increasingly recognized that programmed ribosomal frameshifting can result in peptides or proteins with physiological functions (dinman, 2012a) . one question is whether frameshifting compromises the function of the original frame. it has been proposed that dual coding limits the evolutionary flexibility of the underlying nucleotide sequence (firth and brown, 2006; rancurel et al., 2009 ). thus, one possibility may be that frameshifting occurs predominantly in highly conserved, essential genes. however, it has been argued that once a frameshift event is released from selective pressure, as occurs in gene duplication, it can evolve to produce a beneficial functional protein (raes and van de peer, 2005) . indeed, in some cases, novel genes seem to have emerged by frameshifting of a pre-existing coding sequence (ohno, 1984; ranz et al., 2003) . moreover, regulated frameshifting can allow the same gene to produce alternative beneficial proteins. in fact, evolutionary studies indicate a high abundance of frameshift events in human and mouse genomes that may be linked to an increased usage of the opal tga stop codon (okamura et al., 2006) . it has been proposed that at least 1% of the human genome consists of dual coding regions (michel et al., 2012) and that the number of out-of-frame peptides or proteins may be even higher than that. other studies have suggested that ∼10% of the genome contains −1 frameshift signals (belew et al., 2011) . i would argue that the number of frameshifted peptides or proteins is somewhere in the range of 1-10% of the genome. this is a very significant fraction of the genome, thus suggesting that out-offrame peptides are an inherent part of animal physiology and part of evolutionary selection processes. accordingly, it can be anticipated that dual coding is regulated and a common mechanism for producing additional gene products. the main questions are: 1. what is the sequence or structural motif for dual coding? 2. what is the identity of all dual-coding genes and out-of-frame proteins in the genome? 3. how is dual coding regulated? to answer these questions, we need to look at commonly used methods to identify frameshift events and out-of-frame peptides and proteins. soon after the discovery of programmed ribosomal frameshifting, it was proposed that frameshifting may be a common mechanism for dual decoding of genetic information (dinman, 2012a) . the existence of an alternative genome has been postulated, but it has remained difficult to identify. most methods aim to identify frameshifting events, but very few can determine which produce functional out-of-frame gene products. a summary of methods to identify frameshifting events in mrnas and out-of-frame products is schematically shown in figure 3 . computational prediction has proven very useful. first, one has to identify in mrnas the requirements for a productive frameshift peptide: one might argue that a slippery sequence and a pseudoknot are a good predictor of frameshifting events, but-as pointed out above-there are alternative mechanisms that result in frameshifting (see also figure 2 ). for identification of the frameshifted products, i propose three hallmarks: first, the presence of an initiation triplet (aug or cug) and a stop codon in +1 or −1 frame; second, a stable peptide that follows the 50-nt rule (nagy and maquat, 1998; hillman et al., 2004) ; and third, the validation that the peptide is endogenously expressed. validation can be done by low-throughput experimental methods, either using antibodies raised against the frameshifted protein or using genetic manipulations that ablate the frameshift protein without disrupting the zero frame protein, if possible. to identify frameshifting on a genome-wide scale, higher throughput approaches can be applied, such as genomic, phenotypic, or proteomic screening. several databases have been created to help predict frameshift sequences in the genome. such predictions are based on primary mrna sequence stretches or on secondary hairpins or pseudo-knots within the mrna sequence. for instance, hammell et al. searched the genome for slippery sequences and pseudoknot figure 3 | methods to detect frameshifting events and out-of-frame peptides. computational methods (1) using databases that interrogate the genome for −1 or +1 frameshift motifs can give information about frameshifting events. limitations of this approach are that only known frameshift motifs are taken into account. furthermore, this method does not give any information whether predicted frameshift events occur in vivo or the functional relevance of frameshifting events. experimental methods (2) can identify frameshift events that occur in vivo. most commonly, an antibody that is specific to the out-of-frame sequence of the frameshifted protein is used to detect frameshifted proteins. ribosome profiling methods such as ribo-seq (3) can be used to detect out-of-frame peptides on a genomic scale. cdna screening and the use of tandem luciferase reporter constructs (4) can be used to experimentally detect frameshift events. limitations of this approach are that overexpression may result in dysregulated expression and differences in translation compared to endogenous expression levels. proteomic methods (5) using mass spectrometry are suitable to detect endogenous out-of-frame peptides. however, the levels of frameshifted proteins may be low and escape the detection limit. a combination of these methods may provide most suitable to identify out-of-frame proteins on a genome-wide scale. see text for details. structures (hammell et al., 1999) . this approach identified over 200 putative programmed ribosomal frameshifting events. for identification of pseudoknots and slippery sequences in the genome databases such as recode, knotinframe, prfdb, and fsdb are very useful (table 1) . recode provides information about programmed frameshifting, read-through and bypassing, based on published results in the literature (baranov et al., 2001; bekaert et al., 2010) . the database utilizes information from ∼1500 known frameshifted gene products. a majority of the data on frameshifting comes from two frameshifted proteins, rf2 and antizyme, and is constantly updated using the respective prediction tools [arfa (bekaert et al., 2006) and oaf (bekaert et al., 2008) , respectively]. pseudoviewer is used for visualization of pseudoknot structures. (http://recode.ucc.ie) knotinframe is a program for predicting sites of −1 frameshifting based on the formation of rna pseudoknots (theis et al., 2008) . the authors have developed a specialized rnafolding program called pknotsrg-fs that compares the minimal free energy of an enforced pseudoknot structure to that of a freely folded structure such as that given by rnafold. the spacer region after the common slippery sequence x xxy yyz is between 1 and 12 nt long. (http://bibiserv.techfak.uni-bielefeld.de/knotinframe) prfdb (http://prfdb/umd.edu/) is limited to −1 frameshifting in eukaryotes only. again, the prediction if based on the presence of a heptameric slippery sequence in combination with a pseudoknot (jacobs et al., 2007) . the slippery sequence is modeled with a 1-8 nt spacer, and the pseudoknot is identified using rnamotif. the pseudoknot is then further confirmed with other secondary rna-structure-prediction tools, including pknots (rivas and eddy, 1999) , nupack (dirks and pierce, 2004) , and hotknots (ren et al., 2005) . the frameshift signal database (fsdb) is a compilation of all known frameshift motifs, plus some reported (predicted) frameshift sequences (moon et al., 2007) . based on commonalities between these sequences, the associated fsfinder allows mining of the genome for potential frameshift sequences. (http:// wilab.inha.ac.kr/fsdb) at present, the database contains a total of 63 experimental and 190 predicted sequences from viruses, prokaryotes and eukaryotes. fsfinder uses a combination of slippery sequences and pseudoknot or stem-loop prediction. the heptameric slippery sequences for +1 and −1 frameshifting are frontiers in genetics | genomic assay technology november 2012 | volume 3 | article 242 | 4 listed in table 2 . it is noteworthy that fsdb includes deviations from the standard x xxy yyz slippery sequence for prediction of −1 frameshifting, whereas +1 frameshift sequences often contain stop codons. although most prediction databases use similar principles (slippery sequence plus pseudoknot), they differ in prediction of frameshifting events. this may be due to variable thresholds applied or different rna folding algorithms. in order to identify expressed proteins, one has to consider the length of the predicted out-of-frame protein. most frameshifted proteins initiated by predicted slippery sites will terminate within 5-10 codons, thus producing truncated or non-functional peptides or proteins. alternative approaches that take out-of-frame protein length into account might be beneficial. for instance, mlogd (http:// guinevere.otago.ac.nz/aef/mlogd/) is a program for detection of overlapping coding sequences based on sequence alignments and analysis of mutation patterns (firth and brown, 2006) . the limitations of this approach are that less conserved coding sequences will not be identified, and -2 frame overlaps can be identified as false positives. in some organisms, including mammals, codon usage has evolved to minimize frameshifting. thus, it can be expected that certain codons may favour frameshifting. fscan is a program to identify +1 frameshift sequences based on codon usage in e. coli . fscan searches 16 nt sequences and calculates a score for aa-trna competition between the zero and +1 frame. accordingly, a stop codon, or a rare codon in the zero frame can be a predictor of +1 frameshifting. shah et al. suggested that selective pressure would lead to an under-representation of frameshift sites in protein-coding sequences relative to an organism's codon bias (shah et al., 2002) . they predicted the sequences cuu agg c and cuu agu u, which mediate +1 frameshifting of abp140 and est3, respectively, to be highly under-represented and predictive for frameshifting in s. cerevisiae. once frameshifting events have been identified, it is important to characterize the gene products. one could apply the filters mentioned above, such as the 50-nt rule, although it is quite possible that short peptides are stable and endogenously expressed (kondo et al., 2010; ingolia et al., 2011) . ultimately, all predictions of frameshifted peptides need to be validated with experimental methods. experimental methods include generation of frameshift-specific antibodies, reporter constructs, biophysical methods, and single molecule measurements. the most commonly used frameshift reporter is a tandem luciferase construct where the two luciferases with different substrate specificities are separated by a stretch of nucleotides of a length that shifts the downstream luciferase either one nucleotide up or down (grentzmann et al., 1998) . the downstream luciferase will be expressed only if the nucleotide stretch can mediate frameshifting, while the upstream luciferase serves as an expression control. alternative reporter genes include the use of fluorescent proteins (cardno et al., 2009) , although the dynamic range of luciferases is generally much higher. this reporter can be used experimentally to confirm predicted frameshift events. one hypothesis is that the −1 frameshifting efficiency correlates with the mechanical force required for pulling the rna pseudoknot apart. with biophysical single-molecule methods that measure these forces using optical tweezers, hansen et al. have confirmed that unfolding of a ibv-based pseudoknot required ∼500 kj/mol, compared to the theoretically determined 292 kj/mol (hansen et al., 2007) . chen et al. have determined that a 100% confidence in −1 frameshifting is reached by an unfolding force of ∼57 pn (chen et al., 2009) . such single-molecule biophysical approaches may help to identify potential frameshift sequences, but as they require immobilization of the rna, they will be technically challenging to implement on a genome-wide scale. an alternative is the use of single molecule foerster resonance energy transfer (smfret) (aitken and puglisi, 2010) . fret is a useful technique to measure proximity of biomolecules. a donor fluorophore attached to a molecule can transfer photons to an acceptor fluorophore on a different molecule when both molecules are close together (generally less than 10 nm). aitken and puglisi have used this technique to label individual trna molecules with donor and acceptor dyes that result in energy transfer when in close proximity and correct orientation. this technique enables to monitor relative trna positions and movement of trnas on ribosomes at a millisecond scale. it has been used to identify ribosomal translocation events on fluorescent ribosomes on immobilized rna sequences and predicted the slipperiness of various rna sequences. again, this will be technically difficult to implement on a genome-wide scale. all of the above methods will record mostly frameshifting events, but they will not validate the expression of an out-offrame protein. it is worth noting that frameshifting will most likely result in chimeric sequences composed of a stretch of zero frame peptides linked to out-of-frame peptides, which can be a small or large part of the overall protein, depending on where the frameshift site is. alternatively, frameshifting could lead to truncation of the original protein, where a very small fractionif any-of the overall protein is out-of-frame (figure 4) . those proteins that have a sufficient predicted length could then be validated by raising specific antibodies against that sequence. in order to raise an antibody, one has to know the precise figure 4 | protein composition as a consequence of frameshifting. the original zero frame is shown in gray, whereas out-of-frame sequences are shown in red or blue colour. a frameshifting event is marked by the arrow. frameshifting can result in chimeric peptides composed of the original frame and out-of-frame sequences that can form a small or larger part of the overall protein, depending on where the frameshift event takes place. it is also possible that more than one frameshifting event takes place within the same mrna, thus resulting in mosaic hybrid peptide sequences. (a, original frame; b, early frameshift with extended orf; c, early frameshift with truncation; d, late frameshift with extended orf; e, two frameshift events that switch back to the zero frame; and f, two frameshift events that produce a chimeric sequence of three different frames.) sequence of the frameshifted protein. therefore, this approach is useful for confirming a known out-of-frame protein, but it is not amenable to large-scale genome-wide screening. in addition, the detection of an endogenously expressed out-of-frame protein does not necessarily indicate a functional relevance for this protein. in order to validate a physiological role for the frameshifted gene product, the best method is to genetically ablate the expression of the frameshifted protein while preserving the in-frame sequence. this can be accomplished by gene targeting with a gene sequence that harbours multiple wobble base pair mutations or mutating the frameshift motif, thus altering the rna sequence but not the in-frame protein sequence. this is technically challenging and may not always be feasible. for instance, an oaz3 knockout mouse model has been generated, where both the zero frame and the +1 frame has been deleted (tokuhiro et al., 2009) . to my knowledge, no specific gene targeting of a frameshift protein has been done so far. genomic methods such as rna sequencing (rna-seq) have emerged as powerful tools to profile rna content in cells. rna-seq is based on high-throughput sequencing of a cdna library generated from cellular rna. in its standard form, rnaseq enables the identification of indels, but will fail to identify post-transcriptional phenotypes and will therefore fail to identify programmed ribosomal frameshifting or dual coded genes. nevertheless, ribosome profiling methods such as ribo-seq have been developed that allow the identification of active translation, based on sequencing of cdna libraries generated from ribosomeprotected fragments (ingolia et al., 2009) . in this case, mrna bound to ribosomes is first cross-linked and then isolated using, e.g., sucrose gradient density centrifugation. next, a nuclease digestion step results in removal of mrna sequence that is not bound ("protected") to ribosomes. the protected rna bound to ribosomes is then reverse transcribed into cdna and sequenced. therefore, the precise position of a ribosome can be matched to the site of active translation. an adaptation of this method using the drug harringtonine to cause ribosome accumulation at initiation codons has allowed the identification of translation start sites and confirmed that many proteins are initiated at non-aug or alternative aug sites (ingolia et al., 2011) . in this study, 44% of detected aug start sites were unannotated, and a large fraction of these encoded out-of-frame peptides. further uses of ribosome profiling have confirmed that the identification of frameshifts is possible using genomic technologies (michel et al., 2012) . although this approach is unbiased in the sense that it does not pre-filter genetic regions, one problem is the nonuniformity of ribosome-protected fragment reads. for instance, the preparation of cdna libraries generated from ribosomeprotected fragments can result in over-or under-representation of sequence reads. this can be overcome by a computational approach that measures the cumulative subcodon proportion difference of ribosome-protected fragments relative to local subcodon positions. the authors have therefore combined experimental data from ribo-seq with a computational approach in order to identify novel frameshifted protein sequences. using this approach, several new frameshifted protein sequences were identified, most of which were dual-encoded. the authors estimate that more than 1% of the genome may consist of dualcoding regions, and this is likely an underestimation. further improvements to the method, including deeper sequencing to get better coverage of ribosome-protected fragments, will help to identify these genes. most genome-wide screening approaches such as rnai-based knockdown methods will fail to identify dual-coding regions, as both gene products will be deleted. further, most commonly used cdna libraries such as the orfeome are designed to avoid expression of frameshifted peptides-for instance, by deletion of the 5 and 3 -utr. it may, however, be possible that certain phenotypes in cdna screening are exerted by out-of-frame proteins. in order to fish for phenotypic effects exerted by such peptides, cdna libraries can be designed in a way that genomic fragments are inserted downstream of an aug start codon in +1 or −1 frame so that all genomic fragments are deliberately frameshifted. a subsequent functional phenotypic screen can then identify phenotypes associated with expression of such deliberate frameshift fragments. however, there are limitations in such an approach, e.g., where to place the frameshift. also, deliberately expressed out-of-frame proteins may have little physiological relevance. an alternative may be to use agents such as gentamicin that enhance programmed frameshifting. in that case, cells expressing a cdna library would be treated with a frameshift inducer, and the occurrence of differential phenotypes plus/minus frameshift inducer would be recorded. it is imperative that cdna libraries with 3 -utr regions are used in such an approach to facilitate the out-of-frame peptide expression after stop codon read-through. a similar approach can be used to identify endogenous regulators of frameshifting. one could use the dual luciferase reporter construct (grentzmann et al., 1998) with a known frameshift motif and screen sirna or cdna libraries to identify genes that enhance or inhibit the frameshifting efficiency of this reporter. the existence of non-coding rnas that modulate frameshifting suggests that frameshifting is regulated by endogenous gene products. the dual luciferase reporter mentioned above can also be used to probe the sequence space for optimal frameshifting motifs (rakauskaite et al., 2011) . recently, an adaptation of this approach has been developed using fluorophores that are amenable to high-throughput screening applications (cardno et al., 2009 ). an in vivo adaptation for yeast cells has been proposed for use in high-throughput screening experiments (harger and dinman, 2003) . for instance, a random nucleotide sequence can be inserted between the in-frame and the out-of-frame luciferase to determine which sequence will result in high levels of out-of-frame expression. the main advantages of such a system are the broad linear range of the assay, the internal mrna expression control (luciferase 1), the possibility to normalize relative frameshift expression, and the ease of use. one problem with the approach is that the secondary structure is affected by the length of the sequence inserted in the dual luciferase reporter. thus, the context of the frameshift motifs needs to be taken into account. further, the high number of potential nucleotide combinations may outweigh the capacity of even highly automated processes. even though this will identify putative sequences with high potential for frameshifting, it is still not clear whether the out-of-frame peptides or proteins are stable and functional. their presence in open reading frames may hint that a frameshift is buried within the gene. subsequent low-throughput experimental methods (see above) need to be designed to confirm expression of the frameshifted protein. proteomic approaches have enabled the identification of expressed peptides under physiological conditions. in mass spectrometry (ms), peptides of endogenous proteins are first detected as a mass per ion. in the most common approach, recorded masses are matched to all potential masses in the respective database by search algorithms like sequest, mascot, andromeda, or peaks. in a next step, sequence information generated in tandem ms (ms/ms) experiments is employed to identify potential hits within the shortlisted peptide variants. commonly searched databases by mascot are swissprot, ncbinr, and embl est. these databases integrate cdna and est sequences and generally do not contain out-of-frame peptides. however, as ms database search algorithms compute the entire sequence space of potential peptide matches to identify peptides within analyzed samples, it is in principle possible to identify out-of-frame peptides. an interesting approach for the identification of frame-shift peptides is de novo sequence analysis by ms. this is based solely on the analysis of ms and ms/ms spectra, without amino acid sequence information from databases. however, identification rates are typically lower than in classical database-based proteomics experiments. a major difficulty is to assign frame-shifted peptides to a particular gene. decoding of the underlying nucleotide sequence is sometimes problematic, as, for instance, isoleucine and leucine have the same exact mass. in some cases, the identified peptides do not match the database for various reasons, including errors in gene/protein annotation and post-translational modifications in the peptides that are not accounted for. usually, unmatched peptides are not reported in publications and are disregarded from further analysis. it is possible that some of these unmatched peptides correspond to an alternative reading frame. in order to match these peptides, one would need to generate a database derived from the +1/−1 frames of the orfeome, similar to what okamura et al. have done (okamura et al., 2006) , and make these accessible to the proteomic community. one problem is the "breakpoint" of frameshifting, which would generate a peptide that is partially composed of the original frame and partially of the frameshifted peptide (see figure 4) . another problem in this approach may be the paucity of frameshifted peptides, as they are commonly expressed at lower levels (see above) and may not be easily detectable by ms approaches. however, ms is probably the most powerful method to identify out-of-frame peptides to confirm endogenous expression. an alternative is to study peptides presented by cell-surface, class i mhc proteins. they present peptides derived from intracellular proteins to enable immune tolerance and immune surveillance. mhc-presented out-of-frame peptides were discovered in the early 1990's (shastri et al., 1995) and have enabled the unbiased identification of endogenous frameshifting in mammalian cells long before the technical advances of ms. however, it is technically challenging to use this approach as a systematic tool for the identification of genomic out-of-frame peptides. in which cellular processes should we expect to see a high abundance of frameshifted proteins? in principle, such proteins might be involved in any cellular process, but may be correlated with certain cellular pathways. for instance, amino acid starvation can induce frameshifting in bacteria (barak et al., 1996) . one hypothesis is that this is due to a short supply of amino-acylated trna as a consequence of amino acid limitations. this is supported by the observation that antizyme expression (the +1 frame) is maintained under conditions of amino acid starvation, while expression of odc (the 0 frame) is reduced in rat intestinal epithelial cells (ray et al., 2012) . moreover, treatment with mtor inhibitors such as rapamycin also reduce 0 frame expression while maintaining antizyme expression. one could hypothesize that reduced fidelity in translation may enhance frameshifting. on the other hand, both translation inhibition and amino acid starvation are conditions that increase autophagy in eukaryotic cells, raising the possibility that the production of outof-frame proteins may be functionally coupled to the regulation of autophagy. there is increasing evidence for an alternative genome/proteome in both prokaryotes and eukaryotes, reflecting programmed ribosomal frameshifting. it is likely that a combination of computational, experimental, genomic, and proteomic methods will be needed to determine the entire frameshifted proteome, as required to understand fully gene expression in any organism. we need to identify the frameshift motifs that enable frameshifting, as well as all the genes that produce out-of-frame peptides. finally, as a low level of frameshifting can be considered "biological noise," we need to determine the physiological relevance of these frameshifted proteins. following the intersubunit conformation of the ribosome during translation in real time readthrough strategies for stop codons in duchenne muscular dystrophy on the mechanism of leftward frameshifting at several hungry codons recode: a database of frameshifting, bypassing and codon redefinition utilized for gene expression programmed ribosomal frameshifting in the expression of the regulator of intestinal stem cell proliferation, adenomatous polyposis coli (apc) arfa: a program for annotating bacterial release factor genes, including prediction of programmed ribosomal frameshifting recode-2: new design, new search tools, and many more genes ornithine decarboxylase antizyme finder (oaf): fast and reliable detection of antizymes with frameshifts in mrnas endogenous ribosomal frameshift signals operate as mrna destabilizing elements through at least two molecular pathways in yeast evolution and multilevel optimization of the genetic code targeting frameshifting in the human immunodeficiency virus programmed ribosomal frameshifting in hiv-1 and the sars-cov a homogeneous cell-based bicistronic fluorescence assay for high-throughput identification of drugs that perturb viral gene recoding and read-through of nonsense stop codons triplex structures in an rna pseudoknot enhance mechanical stability and increase efficiency of −1 ribosomal frameshifting a characteristic bent conformation of rna pseudoknots promotes −1 frameshifting during translation of retroviral rna exon skipping and dystrophin restoration in patients with duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study mammalian gene peg10 expresses two reading frames by high efficiency −1 frameshifting in embryonic control of gene expression by translational recoding mechanisms and implications of programmed translational frameshifting an algorithm for computing nucleic acid base-pairing probabilities including pseudoknots decreasing the frameshift efficiency translates into an equivalent reduction of the replication of the human immunodeficiency virus type 1 detecting overlapping coding sequences in virus genomes the presence of the tar rna structure alters the programmed −1 ribosomal frameshift efficiency of the human immunodeficiency virus type 1 (hiv-1) by modifying the rate of translation initiation a dualluciferase reporter system for studying recoding signals expression levels influence ribosomal frameshifting at the tandem rare arginine codons agg_agg and aga_aga in escherichia coli identification of putative programmed −1 ribosomal frameshift signals in large dna databases correlation between mechanical strength of messenger rna pseudoknots and ribosomal frameshifting an in vivo dual-luciferase assay system for studying translational recoding in the yeast saccharomyces cerevisiae creation and disruption of protein features by alternative splicing -a novel mechanism to modulate function an unappreciated role for rna surveillance genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes antizyme expression: a subversion of triplet decoding, which is remarkably conserved by evolution, is a sensor for an autoregulatory circuit conservation of polyamine regulation by translational frameshifting from yeast to mammals identification of a cellular factor that modulates hiv-1 programmed ribosomal frameshifting small peptides switch the transcriptional activity of shavenbaby during drosophila embryogenesis annexin a2 binds rna and reduces the frameshifting efficiency of infectious bronchitis virus ribosome can resume the translation in both +1 or −1 frames after encountering an aga cluster in escherichia coli fsscan: a mechanism-based program to identify +1 ribosomal frameshift hotspots gene expression from viral rna genomes gentamicin-induced readthrough of stop codons in duchenne muscular dystrophy aminoglycoside suppression at uag, uaa and uga codons in escherichia coli and human tissue culture cells extensive frameshift at all agg and ccc codons in the mitochondrial cytochrome c oxidase subunit 1 gene of perkinsus marinus (alveolata; dinoflagellata) observation of dually decoded regions of the human genome using ribosome profiling data fsdb: a frameshift signal database antizyme, a protein induced by polyamines, accelerates the degradation of ornithine decarboxylase in chinese-hamster ovary-cell extracts a rule for termination-codon position within intron-containing genes: when nonsense affects rna abundance a mechanical explanation of rna pseudoknot function in programmed ribosomal frameshifting reprogrammed genetic decoding in cellular gene expression polyamines regulate both transcription and translation of the gene encoding ornithine decarboxylase antizyme in mouse birth of a unique enzyme from an alternative reading frame of the preexisted, internally repetitious coding sequence frequent appearance of novel protein-coding sequences by frameshift translation a programmed −1 ribosomal frameshift signal can function as a cis-acting mrna destabilizing element functional divergence of proteins through frameshift mutations a rapid, inexpensive yeast-based dual-fluorescence assay of programmed-1 ribosomal frameshifting for high-throughput screening origin and evolution of a new gene expressed in the drosophila sperm axoneme amino acids regulate expression of antizyme-1 to modulate ornithine decarboxylase activity hotknots: heuristic prediction of rna secondary structures including pseudoknots a dynamic programming algorithm for rna structure prediction including pseudoknots an overlapping genetic code for frameshifted overlapping genes in drosophila mitochondria: antisense antitermination trnas uar insert serine computational identification of putative programmed translational frameshift sites major histocompatibility class i molecules can present cryptic hungry codons promote frameshifting in human mitochondrial ribosomes knotinframe: prediction of −1 ribosomal frameshift events mrna pseudoknot structures can act as ribosomal roadblocks oaz-t/oaz3 is essential for rigid connection of sperm tails to heads in mouse ribosomal frameshifting on mjd-1 transcripts with long cag tracts i would like to thank joern dengjel and martin raff for critically reading the manuscript. this work was supported by the medical research council. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-298736-9bvyp21d authors: gerold, gisa; bruening, janina; pietschmann, thomas title: decoding protein networks during virus entry by quantitative proteomics date: 2016-06-15 journal: virus research doi: 10.1016/j.virusres.2015.09.006 sha: doc_id: 298736 cord_uid: 9bvyp21d abstract virus entry into host cells relies on interactions between viral and host structures including lipids, carbohydrates and proteins. particularly, protein–protein interactions between viral surface proteins and host proteins as well as secondary host protein–protein interactions play a pivotal role in coordinating virus binding and uptake. these interactions are dynamic and frequently involve multiprotein complexes. in the past decade mass spectrometry based proteomics methods have reached sensitivities and high throughput compatibilities of genomics methods and now allow the reliable quantitation of proteins in complex samples from limited material. as proteomics provides essential information on the biologically active entity namely the protein, including its posttranslational modifications and its interactions with other proteins, it is an indispensable method in the virologist's toolbox. here we review protein interactions during virus entry and compare classical biochemical methods to study entry with novel technically advanced quantitative proteomics techniques. we highlight the value of quantitative proteomics in mapping functional virus entry networks, discuss the benefits and limitations and illustrate how the methodology will help resolve unsettled questions in virus entry research in the future. viruses need to enter permissive host cells in order to propagate and spread. the penetration of cells requires an initial interaction of viral and host surface structures. typical viral attachment proteins (vap) are transmembrane glycoproteins for enveloped viruses or capsid proteins for naked viruses. alternatively, some viruses incorporate phosphatidylserine into their envelope and use this lipid for host cell interaction (amara and mercer, 2015; jemielity et al., 2013; kondratowicz et al., 2011; mercer and helenius, 2008; moller-tank et al., 2013; shimojima et al., 2012) . for the majority of viruses, however, proteins bind to cellular receptors, i.e., proteins or carbohydrates, and the expression of a particular receptor is often determining tissue and species tropism of a virus. in this review we focus on the role of protein-protein interactions (ppi) during viral penetration as protein-lipid and protein-carbohydrate interactions in cell entry of viruses have been discussed elsewhere (amara and mercer, 2015; lozach et al., 2007; stencel-baerenwald et al., 2014) . historically, the first virus receptor found was a glycan. in 1981 helenius and coworkers described sialic acid as the influenza a virus receptor (matlin et al., 1981) . in the following years it became clear that most virus receptors are proteins or protein-linked entities, such as glycosaminoglycans. as a consequence, protease treatment rendered susceptible host cells non-susceptible for many viruses. with the discovery of cr2 as epstein barr virus (ebv) and cd4 as human immunodeficiency virus type 1 (hiv-1) receptor the hunt for virus receptors began (fingeroth et al., 1984; maddon et al., 1986) . since then technological developments like antibody based affinity purification (ap), mass spectrometry (ms) of proteins, dna mediated transformation and molecular cloning led to the discovery of dozens of receptors for human pathogenic viruses (fig. 1) . nevertheless, for many viruses the cognate receptor(s) remain enigmatic and a "one fits all" method for their discovery has not yet been described. virus entry factors, which interact with vaps directly, can be differentiated into attachment factors and entry receptors. attachsurface. virus receptors not only mediate cell attachment but the ppi of vap and receptor also guides subsequent entry steps, e.g. internalization ( fig. 2 and box 1). sometimes the vap-receptor interaction unleashes the fusogenic property of viral glycoproteins by inducing conformational changes in the vap ectodomains. a well-studied example is the priming of the hepatitis c virus (hcv) glycoprotein e2 by the cd81 receptor (sharma et al., 2011) . for viruses, which fuse in intracellular vesicular compartments, a drop in ph typically acts as primer for fusion. in some cases host proteases interact with and cleave vaps rendering them fusogenic. ppis between viral and cellular factors orchestrate viral attachment, trafficking, fusion and uncoating. classical virus infection pathways are illustrated for a generic enveloped virus. note that naked virions infect cells in a similar manner with viral capsid proteins mediating critical ppis with cellular proteins (not shown). ppis facilitating virus attachment, surfing, fusion and transport are depicted. in some cases proteolytic processing mediated by host factors triggers viral membrane fusion. moreover, secondary ppis can lead to assembly of multiprotein receptor platforms that allow internalization and ultimately membrane fusion. proteolytic processing of vaps either occurs during vap biosynthesis or during virus entry. the latter applies to ebola virus, which requires processing of its envelope glycoprotein by cathepsins in endosomes (chandran et al., 2005) . moreover, interactions of vaps with secondary receptors, occurring after membrane surfing of viruses towards cellular junctions may trigger internalization (coyne and bergelson, 2006) . after endocytosis, secondary ppis with endosomal receptors can in addition to the ph drop render viral glycoproteins fusogenic (carette et al., 2011; cote et al., 2011) . subsequently viruses replicating in the nucleus are transported along the cytoskeleton towards the nuclear pore, where the genome is released into the nucleus. transport relies again on ppi of virus structural proteins and microtubular motor proteins. all viruses additionally need to undergo uncoating, i.e., the viral nucleocapsid needs to disassemble to release the genome into the cell. uncoating is poorly understood for most viruses, but as detailed below, also requires interaction of viral capsids with host proteins. fig. 2 highlights the most important steps in virus entry and critical ppi for each step. in brief, most processes during virus entry are coordinated by ppis. in fact, eighty percent of the approximately 20,000 protein coding genes of a cell are estimated to perform their function through interactions with other proteins. roughly 10,000 distinct interaction types, i.e., structurally similar interactions, and 130,000-650,000 binary protein interactions can occur in a cell (aloy and russell, 2004; stumpf et al., 2008; venkatesan et al., 2009) . thus, knowledge on protein complexes is essential for the understanding of cellular mechanisms and for the targeted interference with a protein's function. in some instances this knowledge has been successfully translated into therapeutic approaches for treatment of viral infections. the best known examples are the hiv-1 ccr5 coreceptor antagonist maraviroc and the hiv-1 gp41 fusion inhibitors (dorr et al., 2005; wild et al., 1993) . importantly, many interactions are mediated by short 10 amino acid long unstructured peptide motifs, are transient, and have relatively low affinity. classical high stringency tandem ap protocols thus often missed peptide motif based protein interactions. novel shotgun proteomics of one step affinity enriched samples provides now the opportunity to unravel a multitude of previously missed interactions. typical host peptide motifs, which coordinate ppis and occur in several hundred human proteins, are the functional unit of src-homology 2 domain (sh2-domain), sh3-domain, the post synaptic density protein (psd95), drosophila disc large tumor suppressor (dlg1), and zonula occludens-1 protein (zo-1)-domain box 1: definitions and abbreviations in virus entry. susceptible cell: cell that expresses virus receptors and entry factors and thus allows entry of a virus. permissive cell: cell that expresses all host factors required for replication, assembly and release of a virus and thus can generate infectious progeny after infection. virus entry: process of delivery of the viral genome to the viral replication compartment in the cells. depending on the replication site and the virus entry route, virus entry includes cell attachment, endocytosis, membrane penetration, trafficking in the cytoplasm, nuclear import, release of viral structural proteins like tegument and genome uncoating. virus attachment factor: cell surface protein interacting with a virus. attachment factors facilitate entry by concentrating virions at the cell surface and can promote subsequent interactions with entry receptors. virus entry receptor: cellular protein, lipid or glycan that binds the virus particle and mediates virus uptake and/or triggers critical conformational changes crucial for infection. viral receptors are often localized to the plasma membrane, but can also reside in endosomal compartments. virus entry factor: operationally defined as any cellular factor that in some way participates in the virus entry process, but not necessarily interacts with the virus. viral attachment protein (vap): viral surface protein that binds to the virus receptor. vaps of enveloped viruses are transmembrane glycoproteins, while vaps of naked viruses are viral capsid proteins. capsid (also: core): protein shell of a virus arranged around the viral genome or complexed to it; build by repeat units of single proteins or protein complexes. the capsid proteins are encoded by the viral genome. nucleocapsid: complex of viral genome and capsid, which may for enveloped viruses be surrounded by a lipid bilayer. envelope (also: peplos): host cell derived lipid bilayer surrounding the nucleocapsid of enveloped viruses. the envelope typically displays viral genome encoded transmembrane proteins. these envelope proteins are vaps and in some cases enzymes (neuraminidases) that cleave cellular carbohydrates to facilitate virus shedding or ion channel proteins that prime viral uncoating. recently, it was shown that some classical naked viruses, like hepatitis a virus, could be enveloped as well. virion surfing: directional movement of virions within the cell plasma membrane, i.e. on the cell surface or cell projections like filipodia, towards the viral entry sites virus fusion: membrane mixing of the lipid bilayer of enveloped viruses with a host cell lipid bilayer, i.e. either the plasma membrane or the endosomal membrane, resulting in the release of viral nucleocapsids into the cytoplasm. uncoating: disassembly of the viral capsid to release the viral genome into the cytosol or nucleus of a host cell. large dna viruses additionally require tegument disassembly prior to disassembly of the capsid. (pdz-domain) and the so called ww-domains which carry two tryptophan residues as signature (tompa et al., 2014) . notably, viruses and other pathogens often make use of these and other peptide motifs to hijack cellular pathways during their life cycle including virus entry hagai et al., 2014) . while transcriptomics can reveal long-term alterations of the cellular state, virus entry usually occurs within minutes and typically relies on rapid changes of protein conformation, localization, interactions and post-translational modifications (ptm). thus studying the proteome state, preferentially in a time-resolved manner, is ideally suited for understanding the mechanisms of virus entry. proteomics has already led to the characterization of global cellular alterations induced by virus infection. for instance, virus induced changes in the protein composition of subcellular hcv replication compartments revealed several host replication and assembly factors (huang et al., 2007; mannova et al., 2006; paul et al., 2013; yi et al., 2006; yi et al., 2011) . interaction proteomics at a large scale was moreover performed for all 18 hiv-1 proteins outside of the context of hiv-1 infection and revealed important virus manipulations of cellular pathways (jager et al., 2012) . another global interaction proteomics study revealed host protein interactions of viral immune modulating proteins uncovering viral immune evasion strategies (pichlmair et al., 2012) . although in principle possible, global interaction proteomics during virus attachment and entry has not yet been extensively pursued. as virus entry is the process in the viral life cycle, which can most easily be synchronized by inhibitors or temperature shifts, we believe that proteomic analyses can close many gaps of knowledge in virus entry research. below we discuss successful applications of proteomics to understand virus uptake and highlight future perspectives. the various entry routes of viruses have been extensively reviewed (grove and marsh, 2011; mercer et al., 2010; amara and mercer, 2015) . similarly, we refer the reader elsewhere for methods to define protein interactions other than ms based proteomics, i.e., native page, yeast two hybrid, protein microarrays, fluorescence resonance energy transfer, surface plasmon resonance, structurebased approaches and phage display (ngounou wetie et al., 2014) . here, we focus on the ppis and ptms that mediate virus uptake and on past and present ms proteomics based techniques to study virus invasion. to gain access to a susceptible host cell the vast majority of viruses interacts with proteinacious receptors on the cell surface. consequently, biochemical protein-based approaches successfully discovered virus receptors in the past (table 1) . typically, these approaches build on virus overlay protein blot assays (vopba). vopba relies on probing of cell lysates or cell membrane extracts, separated by gel electrophoresis and transferred onto nitrocellulose membranes, with virus particles (boyle et al., 1987) . virus bound proteins are in this case either detected by radiolabelling of the virus or by specific antibodies against the virus glycoproteins. in both cases ms fingerprinting of a corresponding polyacrylamide gel band identifies the putative receptor. a prime example for vopba approaches is the identification of alpha-dystroglycan as receptor for lymphocytic choriomenigitis virus (cao et al., 1998) . the vopba qualitative proteomics approach has the advantage that the protein is left intact until the last step of identification and thus information on the molecular weight and posttranslational modifications like glycosylation is available. however, it suffers from low sensitivity, as retrieval of proteins or peptides from polyacrylamide gels is typically low. moreover interactions could be lost under denaturing conditions. an alternative to vopba-based receptor identification is the affinity purification (ap) of virus glycoprotein -receptor complexes. this method overcomes the caveat of protein denaturation and analyzes interactions in their native state. for instance, igfusion proteins of truncated soluble viral glycoproteins were successfully used to co-purify the new world arenavirus receptor transferrin receptor 1 (radoshitzky et al., 2007) . other examples for qualitative interaction proteomics are the identification of receptors for nipah virus (ephrin b2), kshv (ephrin a2) and sars (angiotensin-converting enzyme 2) (hahn et al., 2012; li et al., 2003; negrete et al., 2005) . here, glycoprotein−fc fusion proteins served as baits for ap of the receptor post cell lysis. in a third (yan et al., 2012) . generally, biotinylation of surface proteins prior to ap or amine-reactive crosslinking are strategies to increase specificity of the ap and help avoid detection of biologically irrelevant interactions after cell lysis. a refinement of both methods is the use of biotinylated ligands equipped with hydrazine groups for crosslinking of cell surface proteins glycans. to this end, a trifunctional reagent for ligand derivatization, termed triceps, has been developed by frei et al. (2012) . critical negative controls further include, non-susceptible cell lines, which do not bind the virus, and glycoprotein mutants or peptide variants, which are deficient for receptor binding. in principle, ms fingerprinting of the purified proteins is the sole method to unambiguously identify the prey after ap. it should however be noted that analysis of the prey with respect to its sensitivity to glycosidases, proteases, heparinases and lipid raft altering agents can lead to an educated guess, which upon further validation might identify a virus receptor. a prominent example is the identification of scavenger receptor class b type i for hcv (scarselli et al., 2002) . similar to vopba approaches, receptor identification after ap relied on qualitative proteomics in the past, i.e. gel purification of the co-precipitated proteins, followed by ms fingerprinting of a defined gel band. such interaction proteomics approaches typically require a truncated and epitope tagged virus glycoprotein for efficient affinity enrichment (ae) of cellular receptors. high affinity antibodies against the viral glycoprotein may supersede epitope tagging of the bait. both vopba-based and ap-based interaction proteomics are well suited for the identification of primary receptor interactions. however, interactions with late receptors, which require a conformational change of the glycoprotein and are more difficult to synchronize, cannot be captured easily with these techniques. also virus-triggered secondary ppis are not resolved by these classical methods. the development of shotgun proteomics techniques described below thus presents a major breakthrough, which holds the promise of detecting higher order, reduced affinity and even transient virus receptor interactions. in the past decade, the development of quantitative shotgun proteomics techniques revolutionized the protein interactions field. in contrast to previous qualitative approaches, shotgun techniques avoid extensive purification from gels and instead analyze a complex mixture of proteins, thereby tremendously increasing sensitivity and throughput. technological development of high performance liquid chromatography (lc) and ultra-high resolution ion trap ms together reduced measurement time, increased peptide sensitivity and measurement reproducibility and thus made shotgun techniques possible. moreover, mass accuracy of new generation ion trap mass spectrometers increased up to 0.001 da, guaranteeing high confidence peptide identification and analysis of posttranslational modifications (ptms). lc-ms thus emerged as prime in depth proteomics method leaving behind alternative techniques like two-dimensional gel electrophoresis, low-resolution maldi and protein arrays (mann and kelleher, 2008) . shortly, in shotgun interaction proteomics affinity enriched protein mixtures are as a whole digested to peptides and analyzed by lc-ms. to separate the specific binding partners from the excess of background proteins, specific and control purifications are directly compared (vermeulen et al., 2008) . this is achieved either by stable isotope labeling of amino acids in culture (silac) or by the more recent computational based label-free quantification methods (lfq) described in the subsequent paragraph. most recent techniques use one-step ae protocols, which preserve weak and transient interactions (see below for details). fig. 1 highlights the milestones in the development of ms-based proteomics techniques and box 2 describes common proteomics terms. while quantitative shotgun proteomics has been used in a variety of fields including signal transduction, cell adhesion and even viral immune modulation, we are just starting to appreciate its power for the study of the viral replication cycle and in particular virus entry (kruger et al., 2008; pichlmair et al., 2012; weekes et al., 2014; zanivan et al., 2013) . ms based methods are commonly used to identify and quantify proteins, to profile protein-interactions or whole proteomes. therefore, proteins are isolated and proteolytically digested, the resulting peptides are separated via lc, and then analyzed by tandem mass spectrometry (ms/ms) (bottom-up approach or shotgun proteomics) (vermeulen et al., 2008) . alternatively a top-down approach can be used, where the protein is fragmented during the mass spectrometric analysis (boeri erba, 2014). for quantitative proteomics, most often bottom-up high resolution ms techniques are used in combination with either labeling or label-free methods: a powerful and the most commonly used label-based method for the quantification of proteins is silac (ong et al., 2002) . in short, an isotope label is metabolically incorporated into peptides and proteins of a cell. typically, essential amino acids with heavy isotopes (e.g., 15 n, 13 c) are fed to cells over several passages until nearly all unlabeled amino acids are replaced. as the physicochemical properties of light and heavy amino acids are nearly identical, cells labeled with the isotopic analogs behave similar or even identical to unlabeled cells. based on the isotope label silac allows the direct comparison of two cell populations (heavy and light) from two distinct experimental conditions; for instance, from cells with surface-bound virus or cells without bound virus (gerold et al., 2015) . prior to proteomic analysis, i.e., whole cell lysate analysis or ap of protein complexes, the differently labeled samples are mixed. this is advantageous as all samples are processed equally excluding sample handling biases. proteins are then digested, peptides reduced and alkylated, separated by lc and analyzed by ms. peptides derived from heavy or light cells are subsequently differentiated by their characteristic mass offset. relative abundances of proteins in each sample are calculated based on the heavy and light signal intensities in the same spectrum. typically, silac is used for relative quantification of protein levels under two different cellular conditions. of note, medium labeled amino acids are additionally available and allow expanding a silac experiment to three cellular conditions (hilger and mann, 2012) . the high accuracy of silac typically requires analysis of only two experimental replicates. in addition, two replicates of a label swap experimental condition allow assessing label-specific effects. thus, a typical dual silac experiment starts with eight experimental samples mixed into four silac pairs. those are duplicates of the forward label pair (light-condition 1; heavy-condition 2) and duplicates of the reverse label pair (light-condition 2; heavy-condition 1). fig. 3a depicts the silac workflow. silac followed by lc-ms/ms can answer various questions in virology. for example, it can be used to compare the proteome of uninfected to that of virus infected cells (e.g., zhang et al., 2013) . also cells at different time points post infection can be analyzed to understand how viruses alter host cell protein expression (weekes et al., 2014) . for instance, berard et al. performed a temporal quantification of cells infected with herpes simplex virus type 1 (hsv1) and by this showed how hsv1 modulates the metabolic function of the host cell (berard et al., 2015) . beside this, silac followed by lc-ms/ms can also be used to identify cellular targets of viral enzymes, as it has been done for the identification of the hcv protease ns3-4a targets: by comparing whole proteomes from hepatoma cells with and without the ns3-4a protease, morikawa et al. discovered that 'probable glutathione peroxidase 8' is cleaved by the viral protease (morikawa et al., 2014) . silac is also suitable to analyze how viruses alter the proteome of distinct cellular compartments. for instance, xie et al. defined the modification of lipid raft composition in hbv infection (xie et al., 2012) . in combination with ap of a protein of interest, silac followed by lc-ms/ms can also discover protein-interactions and dynamic changes in interaction complexes (reviewed e.g., in (gingras et al., 2007; trinkle-mulcahy, 2012) ). high-throughput ap-ms has led to important findings regarding virus entry, for instance the identification of receptors or receptor-complexes. a successful example is the ap-ms analysis of the hcv receptor cd81, which revealed hras as a cd81-binding partner and signaling molecule needed for hcv entry (zona et al., 2013) . a clear benefit of silac is its high reproducibility, robustness and relatively easy execution. silac is applicable to most cell culture cells and sample conditions. even silac mice have been developed in the past for ex vivo proteomics analyses (zanivan et al., 2012) . box 2: definitions and abbreviations in mass spectrometry based proteomics. proteome: entity of proteins expressed at a given cellular state or in a given tissue. the term was coined in analogy to 'genome' in 1996. ms: mass spectrometry. esi: electrospray ionization; ionization technique based on spraying of a sample solution into a strong electric field. maldi: matrix-assisted laser desorption/ionization; ionization technique based on mixing of samples with a matrix, which absorbs ultraviolet light and converts it to heat energy leading to sample vaporization. bottom up proteomics: identification of proteins based on ms peptide fingerprints after digestion of a protein into peptides using a sequence-specific enzyme such as trypsin. top down proteomics: ms analysis of entire proteins. shotgun proteomics: bottom-up proteomics technique to identify proteins in a complex mixture. proteins are digested to peptides, peptides separated by liquid chromatography and then identified by tandem ms. quantitative proteomics: shotgun proteomics with the additional dimension of measuring the quantity of a protein in a complex sample. relative quantities in two or more samples are determined either by silac or lfq (see below). absolute quantification can be achieved using spike in standards or a proteomics ruler (see paragraph on protein complex stoichiometry). interaction proteomics: unbiased identification of protein-protein interactions by affinity enrichment followed by ms. silac: stable isotope labeling by amino acids in cell culture; first quantitative method for the comparison of cellular protein amounts in two to three different cellular states. silac is based on the isotope labeling of cellular proteins by feeding cells with heavy isotope amino acids (e.g. n-15 and c-13 labeled arginine and lysine). label-free quantification (lfq): quantitative proteomics technique based on either signal intensity measurement or spectral counting followed by computational matching of peptides from multiple samples and signal normalization. ppi: protein-protein interactions. ptm: posttranslational modification. common protein modifications include among others phosphorylation, lipidation, glycosylation, methylation, acetylation, ubiquitinylation and sumoylation. ap-ms: affinity purification of proteins using antibodies against endogenous epitopes or epitope tags followed by mass spectrometric identification or quantification of the bound proteins. ae-ms: affinity enrichment of proteins using fast single step methods, which preserve transient and low affinity interactions, followed by mass spectrometric identification or quantification of bound proteins. caveats of silac are the limited sample number of maximum three and the additional labeling step requiring culturing of cells for two to three weeks prior to analysis. thus some primary human cells, which rapidly dedifferentiate ex vivo, are not suited for silac. similarly, clinical samples cannot be labeled and are thus not amenable to silac analysis. increasingly popular alternatives to silac are lfq methods. these can determine the relative or absolute abundance of a protein in complex samples. the first semi-quantitative labelfree method developed, which has since then been continuously refined, is based on the measurement of ms/ms peptide intensities (bondarenko et al., 2002) . to this end, peak areas of all identified peptides from one protein are summed up and normalized to one or several proteins with constant concentration or even to all iden-tified proteins in the sample. bondarenko et al. showed that there is a linear correlation of the abundance of a protein and its total reconstructed peak area. an alternative semi-quantitative method used is spectral sampling (liu et al., 2004) . it was shown that highly abundant proteins are sampled more frequently by lc-ms/ms and that the spectral copy number obtained increases linearly with the concentration of a protein. accordingly, the number of ms/ms spectra gained for one protein can be counted and compared between different samples measured using the same experimental conditions. several computational approaches to predict specific protein interactions in label-free datasets have been developed, among them significance analysis of interactome (saint) and mass spectrometry interaction statistics (mist) (choi et al., 2011; jager et al., 2012) . these tools compute interaction probabilities based on spectral counts of all interactions that a prey-bait pair is involved in or based on abundance, reproducibility and specificity of a preybait pair, respectively. recent developments in ms technology and analysis algorithms now allow highly sensitive, efficient and robust intensity-based label-free protein quantification (reviewed e.g., in (nahnsen et al., 2013) ). for this, samples are processed in parallel and not mixed, as in silac. thus, an unlimited number of samples can be compared. normalization is achieved post lc-ms/ms analysis by comparing all peptide signals from all ms runs and assuming that most protein signals remain unaltered in the different sample conditions. thus, no external standards are needed . however, acquisition of accurate datasets requires the analysis of at least three, typically four, biological replicates (fig. 3b ). ms combined with lfq algorithms is suitable for virological research and has been used e.g., to show that subsets of dendritic cells (dcs) differ in their ability to sense single stranded rna (ssrna) viruses (luber et al., 2010) . briefly, luber at al. compared the whole cell proteome of dc subsets by high-resolution ms combined with lfq. they found, that dc subsets differently express proteins involved in pattern recognition pathways and that this correlates with their ability to sense ssrna viruses, such as sendai virus and influenza a virus. label-free methods also allow the analysis of protein complexes. importantly, extensive purification of complexes prior to lc-ms/ms is no longer necessary for lfq. instead one ae step is sufficient for precise complex profiling, when controls are designed appropriately . false positive interaction partners, which are proteins unspecifically binding to the affinity resin, are computationally eliminated by comparing datasets to those of a control ae sample from cells without bait. fig. 3b outlines the here described lfq workflow. when compared to label-based methods, lfq techniques have several advantages: nearly all kinds of samples can be measured, including clinical material and primary cells; sample number is unlimited; no special sample processing steps are needed; slight differences in sample preparation and measurement between the different samples are tolerated. both, labeling and label-free quantification methods identify and quantify proteins with high confidence and lead to highly accurate results. each step of the proteomics workflow can be quality controlled, e.g., by immunoblotting, computational analysis of peptide intensity distributions, and clustering analysis of biological replicate datasets (fig. 3c ). to study virus entry both silac and lfq are valuable tools. lfq is ideally suited to study steady state virus receptor interactions. to this end, virus receptor-complexes are purified from cells expressing the receptor and cells lacking the receptor. comparison of both datasets then allows to define receptor interacting proteins in a given cell line. silac on the other hand can sensitively distinguish low degree quantitative changes, as they occur when virus receptor complexes change upon virus binding. briefly, heavy labeled cells are incubated with virus and light cells are left untreated (forward label); and vice versa (reverse label). after sample mixing, ap of receptor complexes and lc-ms analysis, changes in interaction strength of each protein in the receptor complex can be determined. importantly, the untreated control should ideally contain all extracellular components as the virus sample, i.e. the ideal control is incubation with virus, which had been neutralized by blocking antibodies, or incubation with a "mock" virus preparation if such antibodies are not available. using silac, we recently identified transient, virus-triggered interactions of the hcv receptor cd81. among the 26 dynamic cd81 interactors we identified serum response factor binding protein 1 as a host factor aiding hcv entry (gerold et al., 2015) . of note, similar studies will soon be possible with advanced lfq methods. fig. 3 illustrates how quantitative proteomics can identify steady state and dynamic virus-triggered receptor interactions. in summary, silac and lfq are powerful tools for analyzing virus-host interactions and global proteomic changes upon virus infection. due to the recent improvements in high resolution ms and label-free analysis tools, the highly accurate, cost effective and broadly applicable lfq methods will soon become the method of choice for profiling proteins, their interactions and whole proteomes. steady state interactions of cellular proteins, which preexist before virus attachment, can be crucial for virus infection. this has been shown in the past mainly by biochemical assays. for instance, the interaction of moesin and cd46, which is needed for measles virus entry, was demonstrated by ap followed by immunoblot analysis (schneider-schaulies et al., 1995) . since msbased proteomics became the method of choice to analyze protein interactions, several studies used mostly non-quantitative proteomic techniques to characterize interactions of virus receptors. for example chakraborty et al. (2012) analyzed immuoprecipitates of the kaposiís sarcoma-associated herpesvirus (kshv) receptor and lipid raft protein integrin ␣3␤1 via sds-page and ms of selected gel bands. they identified the association of integrin ␣3␤1 with several proteins, including epha2. the identified associations were enriched in lipid rafts of kshv infected human dermal microvascular endothelial cells, compared to uninfected cells. with virological assays chakraborty et al. then showed that this enrichment is important for effective kshv infection, especially for macropinocytosis and trafficking of the virus. also quantitative ms-based techniques in combination with traditional virological assays can be used to decipher the role of steady state protein interactions in different steps of the viral life cycle. an excellent example is the identification of a ppi network consisting of the hcv receptors cd81 and claudin1 and the membrane proteins hras, rap2b and itgb1, which are required for efficient entry of the virus into hepatocytes (zona et al., 2013) . the authors identified the hcv receptor network by silac of human hepatoma cells with and without cd81 receptor expression. the role of the identified proteins in the hcv entry process was then studied by virological assays in conjunction with rna silencing and small molecule inhibitors. using a similar workflow, host restriction factors can likewise be discovered. one example is the association of ewi-2 and ␣-actinin-4 in t-cells. gordon-alonso et al. (2012) identified the complex by analyzing ewi-2 co-precipitated proteins via non-quantitative ms, and later on showed that the complex negatively regulates hiv 1 entry. similarly, the cleavage product of ewi-2, ewi-2wint, has been shown to interact with the hcv receptor cd81 and by this inhibit hcv entry (rocha-perugini et al., 2008) . as ewi-2wint is not expressed in the target cells of hcv, which are hepatic cells, this interaction could contribute to the viral cell tropism. in summary, the analysis of steady state virus receptor interactions can reveal protein complexes critical for virus uptake and in some cases help understand host and tissue tropism. upon binding of viruses to their receptors, additional secondary ppis are triggered, most of which lead to virus entry. these interactions have at least five distinct functions depending on the virus entry strategy. first, binding of the multivalent virus particle to the cell surface can induce receptor clustering, as has been shown for phleboviruses and influenza a viruses (eierhoff et al., 2010; lozach et al., 2011) . while it is unclear whether influenza a virus triggered clustering of sialylated receptor tyrosine kinases like egfr and c-met is required for endocytic virus uptake, phlebovirus induced clustering of dc-sign seems to trigger signaling needed for uptake. other viruses, which have been suggested to induce receptor clustering, include coxsackievirus b and lassa virus (coyne and bergelson, 2006; moraz et al., 2013) . hiv-1 entry also relies on clustering of its cd4 and cxcr4 receptors on the plasma membrane. specifically, the hiv gp120 glycoprotein signals for recruitment of actin adaptor proteins filamin and debrin, which regulate actin rich cap formation at the virus entry site (gordon-alonso et al., 2013; jimenez-baranda et al., 2007) . notably, actin depolymerization is induced at later stages of the hiv entry process, highlighting that actin remodeling during virus entry is highly dynamic and tightly regulated. while proteomes of detergent resistant membrane microdomains have been determined in naïve and virus replicating cells resulting in the identification of more than 200 proteins (foster et al., 2003; xie et al., 2012) , few quantitative proteomics dataset are currently available for receptor platforms upon virus binding to the cell surface. for the hcv receptor cd81, we recently described the virus triggered receptor interactome and identified cytoskeleton regulators, cell junction proteins and a clathrin adaptor protein (gerold et al., 2015) . second, viruses can induce signaling leading to actin cortex disassembly. this is particularly important for viruses, which directly penetrate the cell at the plasma membrane and need to overcome the physical barrier of the actin cortex. viruses entering by endocytosis might similarly require local actin cortex disassembly at the site of the incoming vesicle. although the concept that the cortex can prevent or delay transit of viruses has already been suggested by colleagues in 1997 (marsh and bron, 1997) , only few studies address virus induced actin cortex remodeling. a well-studied example is hiv, which upon env binding to the cxcr4 coreceptor induces galpha i signaling and subsequent cofilin activation leading to actin depolymerization thereby facilitating nuclear translocation of hiv particles (yoder et al., 2008) . of note, not only actin cortex but also actin stress fiber disruption can aid virus entry. for instance cytomegaloviruses binding to egfr and integrin alphavbeta3, induces cofilin dependent actin stress fiber disruption thereby facilitating nuclear translocation of incoming virions (wang et al., 2005) . third, viruses, which use multiple receptors for entry, can induce their lateral translocation along the plasma membrane towards the site of endocytic uptake. a prominent example of such surfing is coxsackievirus b, which after binding to the apical decay-accelerating factor (daf) on epithelial cells induces abl kinase signaling and subsequent rac-dependent actin reorganization. this leads to the lateral translocation of the coxsackievirus b-daf complex to tight junctions, where the secondary receptor coxsackievirus-adenovirus receptor (car) is localized. after car engagement coxsackievirus b is endocytosed. this endocytosis is also triggered by ppis as initial daf binding additionally induces fyn kinase, which phosphorylates caveolin thereby facilitating uptake (coyne and bergelson, 2006) . hcv seems to use a similar multistep strategy to enter after initial binding to the basolateral side of hepatocytes, where the receptors sr-bi and cd81 reside. through a yet insufficiently described mechanism hcv also triggers lateral translocation of the cd81-virus complex to tight junctions, where the two additional entry factors cldn1 and ocln reside and endocytosis occurs (brazzoli et al., 2008) . receptor tyrosine kinases like egfr are thought to provide the trigger for lateral membrane translocation through hras signaling (zona et al., 2013) . moreover, surfing of hcv and retroviruses on filopodia has been observed and is governed by ppi with the actin cell cortex (coller et al., 2009; lehmann et al., 2005) . fourth, viruses, which enter through endocytic routes, can induce endocytic uptake mechanisms including clathrin-mediated, caveolin-dependent, macropinocytic or alternative uptake routes like the clathrin-independent carriers in parvovirus entry (nonnenmacher and weber, 2011) . on the one hand, some viruses like human rhinovirus 2 and phleboviruses hijack endogenous constitutive receptor recycling pathways. after endocytosis phleboviruses, which hijack dc-sign for cell entry into dcs, however dissociate from the receptor in the early endosome thereby avoiding antigen processing and presentation (lozach et al., 2011) . on the other hand, viruses can actively trigger their endocytosis like influenza a virus. after interaction of influenza virions with sialylated membrane receptors, the clathrin adaptor epsin-1 is recruited in a ubiquitin-dependent manner towards the receptor complex and induces clathrin-coated pits (chen and zhuang, 2008) . pi3k activation was independently reported to be required for influenza uptake, thus more than one signaling pathway might coordinate uptake of a virus. this is in line with reports, showing that influenza virus can utilize more than one endocytosis pathway to gain access to host cells (rust et al., 2004; de vries et al., 2011) . larger viruses like poxviruses enter by macropinocytosis and actively induce this uptake pathway through rac-1 and p21-activated kinase 1 (pak-1) dependent actin remodeling (mercer and helenius, 2008) . formation of plasma membrane protrusions is however not specific to poxviruses, but occurs also during entry of herpes viruses, papillomaviruses and flaviviruses (clement et al., 2006; coller et al., 2009; smith et al., 2008) . lastly, several viruses need to interact with host enzymes to trigger their uptake. a prominent example is ebola virus, which requires proteolytic processing of its envelope glycoprotein gp1 by cathepsin b and l to render it fusion competent (chandran et al., 2005) . similarly coronavirus relies on cathepsin cleavage of its spike protein to permit fusion (simmons et al., 2005) . in addition to lysosomal enzymes, cell surface proteases can prime vaps for fusion, like shown for some influenza a virus strains and sars coronavirus (bertram et al., 2010; bertram et al., 2011) . host protein disulfide isomerases similarly regulate entry by catalyzing disulfide shuffling in viral envelope proteins, as shown for dengue virus, hiv-1 and newcastle disease virus (jain et al., 2008; ryser et al., 1994; stantchev et al., 2012; vega-almeida et al., 2013) . of note, high resolution proteomics can not only reveal transient interactions of vap with enzymes, but also has the potential to identify proteolytic cleavage sites and redox modifications in vaps. it is conceivable that virus induced protein interactions during entry not only serve to promote the virus uptake pathway, but can also help cloak viruses and lead to immune evasion. for instance, the hiv capsid recruits two cofactors, cleavage and polyadenylation specificity factor subunit 6 (cpsf6) and cyclophilins (nup358 and cypa), thereby preventing antiviral ifn responses (rasaiyaah et al., 2013) . more complex viruses can further express viral gene products on the infected cell surface, which then bind receptors on bystander cells and modulate immunity. for instance, hcmv protein pul11 binds to cd45 on t cells thereby reducing t cell proliferation (gabaev et al., 2011) . while we here focus on the role of virus -host ppi in entry, it should be noted that quantitative virus entry proteomics at the same time has the potential to reveal immunomodulatory mechanisms. similarly, ppis of viral tegument and capsid proteins with host proteins occurring after membrane fusion are amenable to quantitative proteomics. we refer the reader elsewhere for the detailed description of post-fusion ppis coordinating nuclear trafficking (dohner et al., 2005; yarbrough et al., 2014) . comprehensive proteomics analyses of virus induced ppis leading to actin remodeling and endocytic uptake of virions are lacking to date. however, several studies underline the value of proteomics to analyze stimulus specific interactions in cellular signaling pathways. for instance, the ligand-induced changes of the two wnt signaling pathway members adenomatous polyposis coli protein (apc) and axin-1 identified 28 and 18 dynamic interaction partners, respectively (hilger and mann, 2012) . for other virus life cycle steps, like e.g., cmv particle assembly, interaction proteomics uncovered important mechanistic insights (moorman et al., 2010) . further, interaction proteomics of viral immunomodulatory proteins revealed virus induced alterations in human protein interaction networks and underlying viral perturbation strategies (pichlmair et al., 2012) . similar studies hold the promise of uncovering common and specific viral strategies to alter and exploit host cell functions during cell entry. post-translational modifications (ptms) play two major roles during virus entry into a host cell. first, virus receptor ptms can be critical for the localization to specific membrane microdomains. for instance, palmitoylation of the hcv receptor cd81 and its associated proteins ewi-2 and ewi-2wint regulate their interaction with each other and the localization to cholesterol rich membrane domains thereby influencing hcv entry (montpellier et al., 2011; zhu et al., 2012) . second, viruses rely on cell signaling for their uptake and such cellular signals are often transmitted via ptms like reversible protein phosphorylation. a widespread example common to viruses of diverse families including herpesviruses, papillomaviruses and flaviviruses is the triggering of receptor tyrosine kinase signaling (hahn et al., 2012; lupberger et al., 2011; surviladze et al., 2013; zheng et al., 2014) . in most cases signaling through growth factor receptors regulates actin dynamics required for virus trafficking into the cell. some respiratory viruses however use egfr signaling to suppress antiviral signaling in the airway epithelium (ueki et al., 2013) . other phosphorylation signals that coordinate virus entry are the src kinase pathway in hcmv penetration (nogalski et al., 2013) and the lassa virus induced phosphorylation of its receptor dystroglycan (moraz et al., 2013) . certain kinase activities can be a hallmark of a specific uptake pathway, as for instance pak-1 for macropinocytic virus entry (amstutz et al., 2008) . in particular for large dna viruses not only host proteins are phosphorylated early during virus infection, but also viral gene products. for instance, herpes simplex virus type 1 (hsv-1) protein pul46 is heavily phosphorylated six hours post infection and interacts with several viral and host kinases as determined by quantitative interaction proteomics (lin et al., 2013) . apart from protein phosphorylation, other ptms like ubiquitination are thought to regulate virus uptake, but are poorly characterized. ubiquitination of viral proteins seems to induce viral uncoating in at least two instances. influenza a virus m1 protein is ubiquitinated by the e3 ligase itch and this is a prerequisite for endosomal escape (su et al., 2013) . vaccinia virus on the other hand uses ubiquitinated incoming viral core proteins to trigger proteasomal degradation of its protein coat . all aforementioned studies, except for the hsv-1 study, demonstrate ptms of receptors, downstream signaling molecules or viral proteins by immunoblotting, thus requiring prior knowledge of the signaling pathways. in contrast, ms-based identification and quantification of ptms upon virus binding to a host cell is completely unbiased and thus broadly applicable. another advantage over previous immunoblot based methods is the global and quantitative measurement of ptms by ms in conjunction with the ability to pinpoint the modified amino acid. clearly, high read depth is required for ptm proteomics as the search space for the peptides explodes when taking into account all possible peptide ptms and all of their combinations. still successful large-scale measurements of protein phosphorylation (ficarro et al., 2002; olsen et al., 2006) , n-glycosylation (kaji et al., 2007) , lysine methylation (ong et al., 2004) or acetylation (kim et al., 2006) , ubiquitination (peng et al., 2003) and sumoylation (andersen et al., 2009; impens et al., 2014) have been achieved. to overcome the caveat of low abundance of modified peptides, enrichment methods for instance using anti-phosphotyrosine antibodies are available. importantly, when measuring ptms in interaction proteomics samples, which are of much lower complexity than whole cell lysates, ptm identification is facilitated. for stimuli other than viruses, ptm proteomics has yielded comprehensive datasets. for instance, phosphoproteomics of epidermal growth factor (egf) stimulated hela cells unraveled 6600 phosphorylation sites on 2244 proteins, with 14% of the interactions showing an at least 2-fold modulation by egf. the majority of proteins further showed multiple ptm sites with different kinetics highlighting how one protein can integrate numerous signals (olsen et al., 2006) . this and other studies underline the complexity of receptor signaling. how extensively virus-receptor interactions alter the phosphoproteome of a host cell, was demonstrated in a first global description for hiv-1 entry (wojcechowskyj et al., 2013) . a total of 239 phosphorylation sites in 175 proteins changed just one minute after hiv-1 binding and the study disclosed several previously unknown hiv-1 entry factors. it is conceived that most viruses will trigger ptm-dependent signaling during their entry process and this is either critical for cell penetration or for modulating cellular immunity. recent developments in ptm proteomics now allow the global mapping of virus induced changes in host proteome ptms and can thereby disclose signaling pathways active during cell entry. the stoichiometric composition of receptor complexes is critical for membrane microdomain localization, signal coordination and ligand binding. interaction proteomics can provide information on the relative abundances of a protein in a complex. typically prey protein abundance in the pull down is corrected for the prey abundance in a control pull down and subsequently normalized to the bait protein. this approach was used to estimate the stoichiometry of the yap and taz protein complexes in the human hippo pathway (kohli et al., 2014) . beyond complex stoichiometry measurements, shotgun proteomics now allows to estimate which fraction of a protein is sequestered into a protein complex. mann and colleagues recently developed a method to estimate protein copy numbers in labelfree proteomes from whole cells. this "proteomic ruler" technique determines the absolute abundance of a protein in a cell, based on the fact that the total amount of histones, which is defined by the cell ploidity and genome size, can be used for normalization of any detected protein in a proteomic dataset of sufficient depth. the minimum depth of a whole cell proteome to guarantee reliable histone based scaling is 12,000 peptides. this scaling method is similarly accurate as previous spiked-in protein epitope signature tags (prests) based methods, which required cell counting and cell labeling (wisniewski et al., 2014) . thus, whole cell proteomic datasets can provide information on total protein copy numbers in a given cell. based on this knowledge the receptor complex stoichiometry can be estimated by comparing relative abundances of co-purified proteins in an ae proteomics dataset. success-ful receptor complex stoichiometry calculation has recently been demonstrated for subcellular fractionation proteomics (borner et al., 2014) . thus, shotgun proteomics goes beyond pure identification of virus entry receptor complexes, but is also suitable to estimate complex stoichiometry and degree of sequestration of a protein into a complex. the era of systems biology and proteomics puts the virologist into the unique position of drawing a comprehensive picture of all molecular interactions during virus infection of a host cell. large scale genome and transcriptome-based methods for the characterization of virus-host interactions, including mrna microarrays, rna interference and cdna library screens, led to the discovery of a multitude of host factors. we currently lack information on the physical interconnection of these host factors and thus it is difficult to identify critical players in virus entry pathways. open access databases on ppis like the search tool for the retrieval of interacting genes/proteins (string) integrate public databases like intact and reactome and can facilitate the mapping of host factors into known networks (croft et al., 2014; orchard et al., 2014; szklarczyk et al., 2015) . however, many virus host factors do not map to previously described networks. also, virus induced changes of cellular ppis are not deposited in current databases. some repositories, however, curate host-pathogen interactions, e.g., virhostnet and the hivcentric gpsprot site (fahey et al., 2011; guirimand et al., 2015) . importantly, viruses do not play according to the rule, i.e., they may induce ppi that do not normally exist in non-infected cells. for instance, they can change the subcellular localization of a host factor and thereby connect pathways, which in an uninfected cell operate independently. thus, existing databases on host ppi may have only limited predictive value for infected cells, in particular at steps of the life cycle that heavily depend on the cell's machinery like virus entry. again this calls for repositories that either specialize in or integrate ppi from infected cells. the above described technological advances now enable rapid acquisition of large sets of protein interaction data from one-step aes. thus, proteomics is now high throughput compatible and amenable to systems biology studies (hosp et al., 2015) providing the unprecedented chance to globally define protein-protein networks during virus infection. critical for high confidence network generation and data deposition is the analysis of a large number of biological replicates, experimental conditions and controls including immunoprecipitates from bait deficient cells. furthermore, several laboratories performed control pull-downs under various experimental conditions and established a "contaminant repository for ap" (crapome) (mellacheruvu et al., 2013) . however, background binders can critically differ between experimental setups and thus a universal crapome is problematic, making internal controls more reliable . clearly, newly acquired datasets on virus infection interactomes should be integrated into established host protein-protein networks. currently, the above mentioned virhostnet is probably the most comprehensive database for virus-host interactions, which directly links interaction partners to uniprot (guirimand et al., 2015) . as many pathogens share mechanisms for exploiting host cells, a central database for host-pathogen interactions, multiplexing datasets for viruses, bacteria, fungi and parasites would allow the rapid identification of critical nodes and putative broad spectrum drug targets. clearly, publically available databases should reliably catalogue virus-host interactions and make these easily accessible to the virologist community through user-friendly web interfaces. ultimately, genomics and proteomics techniques provide essential complementary datasets, which can be integrated to reveal a box 3: network term definitions. node: component of a network, e.g., a gene or a protein. hub: node, which is highly connected and therefore a point of control of a specific subnetwork. bottleneck: node, which connects subnetworks and thus critically restricts information flow. edge: connection of two nodes in a network, e.g., representing a physical interaction of two proteins. most complete picture of molecular events during virus infection. functional genomics screens can weigh the importance of a host factor in an interaction network and reveal redundant pathways. annotation clusters can highlight the enrichment of certain cellular processes, molecular functions or protein domains . novel open access bioinformatics solutions building on available resources like string and david can help harmonizing available datasets. scripting and visualization environments like cytoscape, r and bioconductor, which were previously developed for transcriptomics and genomics data integration, are also suitable for pathway mapping of proteomics data (cline et al., 2007; gentleman et al., 2004) . proteomics-specific and publically available programs like perseus provide an additional tool for data analysis and integration (cox and mann, 2012) . meta-analysis of omics datasets together with sharing of data in a timely manner will be critical to successfully model the complexity of virus-host interactions in the future (law et al., 2013) . such networks will then allow the identification of essential nodes, bottlenecks and hubs for selection of optimal drug targets. each node in a ppi network represents a protein, while the edges indicate interactions. bottlenecks are nodes in a network, which critically connect subnetworks and thus restrict information flow, like e.g. central kinases in a signaling pathway. hubs instead are highly interlinked and therefore central points of control, but typically only control one branch of a signaling pathway. while bottlenecks are less attractive drug targets as their inhibition would shut down complete endogenous pathways, hub inhibition can lead to an efficient block of infection (box 3). furthermore, drug targeting of pathogen relevant ppis using small molecule drugs or peptidomimetics seems an attractive strategy to combat infection (de chassey et al., 2012a; law et al., 2013) . in particular, targeting of interfaces between viral and host proteins, holds the promise of minimal side effects. but even host ppis, which are triggered or exploited during virus infection, represent attractive targets. in contrast to direct acting antivirals, which often lead to rapid emergence of viral resistance mutations, host proteins and thus their interactions represent a high evolutionary barrier (gerold and pietschmann, 2014; von hahn et al., 2011) . in particular, host proteases and kinases are attractive drug targets and both protein classes can be identified by advanced interaction proteomics and ptm proteomics, respectively. taken together, increasing knowledge on interactomes during virus infection and the compilation of interactome datasets can spur the development of novel antiviral therapeutics. the experimental validation of central network hubs, also as potential drug targets, is discussed below. protein interaction network analysis allows in the next step to hypothesize, which biological pathway is critical for virus uptake and whether certain proteins are central hubs in the network. the hypothesis can then be tested by disrupting the function of one of the interaction partners and studying the resulting phenotype (fig. 3d) . to this end, blocking antibodies have been used, but the method is limited to proteins with accessible extracellular domains. alternatively, small molecule inhibitors, if available, can block the protein of interest. due to their microreversibility and rapid mode of action, such molecular probes have in the past proven indispensable to dissect the kinetics of virus-host cell interplay. the most commonly used strategy to address a protein's function was in the past the disruption or reduction of its expression by rna interference (rnai) (elbashir et al., 2001; fire et al., 1998) . here, short rna molecules sequence-specifically target complementary mrnas and induce their degradation. rnai has been used to address the role of protein interactions identified by ms in virus entry (zona et al., 2013) . however, rnai approaches can have offtarget effects (reviewed e.g., in (mohr et al., 2014) ) and the slow kinetics of action of two to three days can lead to compensatory adaptation of cells. this might be the reason why large-scale rnai screens performed poorly in studies of virus entry, which operates at a minute timescale (de chassey et al., 2012b) . since 2012, targeted genome editing became possible making use of a bacterial adaptive antiviral immune mechanism. clustered regularly interspaced short palindromic repeats (crispr) in combination with the rna guided dna endonuclease crispr associated protein 9 (cas9) is able to induce specific dna double strand breaks (gasiunas et al., 2012; jinek et al., 2012) . therefore, the system can be used to generate site-specific modifications in genomes, e.g. to knock-out specific proteins (reviewed e.g. in (doudna and charpentier, 2014) ). the crispr-cas9 technology has already been applied to analyze the function of different proteins in the viral entry process. for example crispr-cas9 knock-out of pdzd8 verified its role in hiv-1 and murine leukemia virus infections (zhang and sodroski, 2015) . in a different approach, a genome wide lentiviral crispr-cas9 library was developed to screen for cellular factors essential for entry and cell-to-cell transmission of hcv (ren et al., 2015) . in this screen, the known hcv entry factors cd81, claudin-1 and occludin were identified. while knock-out strategies are limited to host factors with non-essential endogenous function and similar to rnai compensatory effects can occur, a genetically clean knock-out system holds the promise of unambiguously defining essential virus entry factors. future studies will show whether knockout screens have higher reproducibility than rnai screens and are suitable for virus entry research. both techniques, rnai and crispr-cas9, display specific advantages and disadvantages, as reviewed in (boettcher and mcmanus, 2015) . therefore, the appropriate technique, including small molecule inhibitors, dominant negative variants and blocking antibodies, has to be chosen carefully depending on the experimental setup and the target genes. after hypothesis testing, drug targets can be defined and searches for compounds interfering with critical network nodes initiated. alternatively, novel baits can be selected and fueled into a new round of ae-ms, thereby widening the interaction network. such iterative processes will ultimately complete the picture of ppi during virus invasion (fig. 4) . proteomics is the only method to directly characterize the biologically active entity of most biological processes, the proteins. in an ae setup, it provides information on primary protein interactions and higher order complexes depending on the stringency of purification. it can further yield the order of binding as well as interaction interfaces, when crosslinkers are used (chen et al., 2010; leitner et al., 2010) . as detailed above, interaction proteomics can reveal receptor complex stoichiometry as well as ptm of the analyzed proteins. with increasing instrument sensitivity not only stable protein interactions, but also transient and virus-induced interactions are accessible. protein networks between two cellular states, e.g., with and without receptor bound virus, can be compared in a quantitative manner. in the past, detection of subtle differences in protein abundance required isotope labeling, but label-free methods are nowadays sensitive enough for most applications. a clear advantage over rna interference or image-based screens is that the host cell is in its endogenous state without the requirement for labeling of proteins or change of their expression level. recent developments moreover make proteomics high throughput screen compatible (hosp et al., 2015) , so that virus entry processes could be studied in a large panel of cell lines as well as in a time resolved manner. as every method, proteomics has its own shortcomings. certain proteins are detected as common contaminants and these false positives have to be eliminated using proper controls and by comparison to contaminant databases. when searching for interactions of endogenous proteins, antibodies of high specificity are required. in the future, crispr knockin methods might overcome this caveat and allow expression of affinity tagged proteins from their natural locus. alternatively, controlled expression systems, although not under endogenous gene control, can help express tagged proteins at endogenous levels. clearly, interaction proteomics just highlights one biological function of a given protein, i.e., its interaction network. thus the method should be considered as complimentary tool in the virologist's toolbox. to address whether an interaction is critical for virus infection, follow up analysis using established (rnai, inhibitors, dominant negative mutants) or newly developed techniques (crispr) is needed. nonetheless, we believe that interaction proteomics will prove essential to model molecular networks during virus infection, assign critical nodes, generate and test hypotheses on the molecular function of the proteins in the network and thereby reveal essential host factors, which can be targeted by antiviral drugs. genetic screens identified numerous virus receptors, but their actual binding to the respective virus has often not been formerly proven. quantitative interaction proteomics approaches hold the promise of closing this gap of knowledge. due to technological and computational developments in the past decade proteomics is now on par with genomics and allows high throughout comprehensive analyses of complex samples. recently, a 96-well compatible and streamlined interaction proteomics protocol has been developed (hosp et al., 2015) . given the fact that for many viruses the target cells are either not known or are of multiple host and tissue origin, the parallel analysis of many biological samples will in the future allow proteomics screening approaches without prior knowledge of receptor expression or susceptibility. a second consequence of the vast increase in ms sensitivity is that patient material and material from animal experiments, which is typically limited, can be analyzed in depth. transcriptomics data only accounts for a subset of pathogen induced changes in a host as it lacks information on ptms and ppi. proteomics of ex vivo samples thus holds the promise of providing a more complete picture of the virus host interplay in an infected organism. it 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entry into neuronal cells significance of palmitoylation of cd81 on its association with tetraspanin-enriched microdomains and mediating hepatitis c virus cell entry hras signal transduction promotes hepatitis c virus cell entry by triggering assembly of the host tetraspanin receptor complex we thank stefan kunz for critical reading of the manuscript. key: cord-294677-l1b4mw9d authors: prashantha, c.n.; gouthami, k.; lavanya, l.; bhavanam, sivaramireddy; jain, ajay; shakthiraju, r.g.; suraj, v.; sahana, k.v.; sujana, h.s.; guruprasad, n.m.; ramachandra, r. title: molecular screening of antimalarial, antiviral, anti-inflammatory and hiv protease inhibitors against spike glycoprotein of coronavirus date: 2020-10-13 journal: j mol graph model doi: 10.1016/j.jmgm.2020.107769 sha: doc_id: 294677 cord_uid: l1b4mw9d the target spike protein docking with antimalarial, antimicrobial, anti-inflammatory and hiv-protease inhibitors. the docking processed using autodock vina and the binding affinity score is noted based on kcal/mol and estimated inhibitory constant (ki). [figure: see text] by coronavirus (cov) it belongs to the family of coronaviridae [1, 2] . initially most of the infected people reported in china, wuhan city in 2019 with a large extent of seafood and animal meat market [3, 4] . many researchers have reported that the disease is distributed among humans, mammals and birds with the large implications on respiratory, gastrointestinal and neurological infections [5] . in the beginning of january 2020, the disease is increasing in china and distributed throughout world with quantifiable speed [6] . [7] [8] [9] . in april 12, 2020, the disease infected patients 1,896,156; deaths 117,671 and recovered 438,205 cases were reported globally [10] . the most common symptoms observed with the patients are fever, runny nose, sore throat, diarrhea, tiredness, dry cough and some patients have observed aches and pains in the body [11] . some people become infected but have not develop any symptoms; about 80% of population was recovered from the disease without needing special treatment [12] . till date, there are no approved drugs and therapeutic protocols recommended to prevent the disease. it is the greatest challenge of both pharmaceutical companies and research organizations to develop novel anticorona viral drug. understanding the molecular structure of the virus is important to researchers to develop targeted therapies to the disease. based on the taxonomy of corona virus, sars-cov2 is causing disease in 2020 [13] . sars-cov2 belongs to the group of single stranded rna (++ssrna) virus associated with nucleoprotein within the capsid comprised of matrix protein. the virus has spherical or pleomorphic enveloped proteins surrounded by a fatty outer layer covered with a thin layer of crown like structure (spike protein) made of glycoprotein projections. it also associated with membrane glycoprotein, hemagglutinin-acetylesterase glycoprotein, and small envelope glycoprotein [14] [15] [16] . the target spike glycoprotein has two importance functions (1) receptor binding domain (rbd), (2) cleavage site. the rbd grapping hook that grip onto host receptor such as zinc peptidase angiotensin-converting enzyme 2 (ace2) [17] [18] , j o u r n a l p r e -p r o o f aminopeptidase n (apn) [19] [20] , and dipeptidyl peptidase 4 (dpp4) [21] [22] and the cleave site that can opens host receptor that allows the virus to enter host cells. the envelop (e) is small, integral membrane structural protein involved in virus' life cycle and pathogenesis, but still the complete structural and functional information remains unknown [23] . the membrane (m) protein is most abundant structural protein involved in viral envelop and integrate in pathogenesis. the integration of s with m proteins in necessary for viral envelope and cause pathogenesis [24] [25] . the nucleocapsid protein relating to viral genome involved in cov replication cycle and the host cellular response to viral infection [26] . although, the interaction between viral and human proteins is suggested as potential targets for identification of therapeutic protocols. the solidarity trails of world health organization (who) and indian council of medical research (icmr) proposed the fda approved drugs, trails with the combination of antimalarial drugs chloroquine and hydroxychloroquine can helps to prevent the disease. chloroquine prevents the viral attachment itself to the ace2 receptors but it causes several side effects in this regard the current trail made with the less toxic derivative hydroxychloroquine [27] [28] [29] . the effect of these two drugs is still being studied to understand the inhibition of sars-cov2. another set of trails include antihiv drug combination such as lipinovir-ritonavir regulates inflammation in the body that can split hiv proteins [30] , these combinations also recommended to inhibit sars-cov2. the another trailed drug remdesivir is a nucleotide analog originally created to inhibit ebola virus, but this drug also recommended to inhibit the novel coronavirus by targeting the action of a key enzyme that facilitates its replication [31] . some studies are looking at the investigation of viral protein structure and its behavior as a potential target for future drugs. in recent study suggested that the recommended drugs might help patients with mild cased of covid19, but that study has limitations. researchers also evaluating the results of the disease in severe condition go into overdrive with inflammation that can damage the lungs and other organs, but so far there is no proof that it has that effect. based on these limitations, the current research is designed to evaluate the drugs with target proteins. the major objective is to design the target proteins of both structural and non-structural proteins. second objective is to j o u r n a l p r e -p r o o f study the modeling and evaluating the target proteins to predict active site amino acids. third objective is focused o screening of recommended drugs based on pharmacophore and pharmacokinetic analysis. fourth objective is to predict the protein-ligand interaction and virtual screening to understand how strong the drug can interact with target proteins to predict the potential effect against sars-cov2. the insilico analyses were performed using hp z440 workstation with next-generation hp intel xeon e5-1630v4 3.70 hz processor. the antimalarial, antibiotic, anti-inflammatory and hiv-protease inhibitor drugs were retrieved from drug bank database [32] and the conformational structures were observed in chemsketch v12.0 [33] . pharmacophore properties are analyzed using molinspiration [34] . absorption, distribution, metabolism, excretion and toxicity (admet) properties of chemical structure were analyzed using admetsar tool [35] . further, the docking studies were carried out using autodock 4.2 [36] and virtual screening using autodock vina [37] . the intermolecular interactions were analyzed using pymol [38] and biovia discovery studio (ds) 2017 r2 [39] . based on data mining approaches and clinical practicing antimalarial drugs such as chloroquine the experimental spike protein sequence was retrieved from genbank database translated as a large polypeptide that is subsequently cleaved to s1 and s2 domains [40] [41] . using c-itasser pipeline to create three dimensional protein models based on deep convolutional neural-network guide to the i-tasser (https://zhanglab.ccmb.med.umich.edu/i-tasser/) fragment assembly simulations [42] . the 3d protein structure is evaluated using structure analysis and verification server (saves v5.0)36 (https://servicesn.mbi.ucla.edu/saves/) and ramapage37. saves is used to understand the complexity of protein structure based on atom-to-atom interaction of ψ versus φ conformational angels of 3d macromolecule measures the torsion angels of cα (ideal) -n-cβ (obs) and the results were represented in ramachandran plot. active site amino acids were predicted using the castp calculation server based on the delineating measures of surface regions such as surface area and surface volume of 3d protein structure. molecular docking studies used to find the binding affinity of the ligand molecule with target protein. the homology modeled protein structure docked with selected chemical structures using autodock 4.2 and virtual screening by autodock vina. the protein structure is selected to add gasteiger chargers and hydrogen atoms to the polar group of amino acids the macromolecule, whereas ligand structure by adding torsion counts of amide bonds rotatable and all active bonds non-rotatable and generated the pdbqt file for both protein and ligand structures. grid space was set in autogrid by selecting important residues with the grid box size x=54å, y=54å and z=54å and grid spacing of 0.886å that provides search space, the grid centre was selected at dimensions x=−22.885, y=-10.008, z=514.693 used to calculate grid parameters that help to understand the grid energy with equilibrated energy distribution. autodock was used to dock protein and ligand structures by adding lamarckian genetic algorithm (lga) with default parameters. the best docking conformation of protein-ligand interactions is predicted with the energy value in kcal/mol and followed by the analysis of hydrogen bonding interaction and the hydrophobic interaction. the best docking complex of protein-ligand was screened based on clustering analysis and visualized using pymol and biovia discovery studio (2017v). pharmacophore analysis is performed to understand the drug-like character of the chemicals based on lipinski and veber's rule with selected parameters such as logp, tpsa, j o u r n a l p r e -p r o o f molecular weight, hydrogen bond donor, hydrogen bond acceptor, volume, number of rotatable bonds and total number of atoms, bioactive properties such as gpcr, ion channel, kinase inhibitor, nuclear receptor, protease inhibitor and enzyme inhibitor properties are predicted using molinspiration. admet analysis is performed to the selected compounds using admetsar tool to screen the compounds based on absorption, distribution, metabolism, excretion and toxicity prediction. the most important parameters such as blood-brain barrier, acute toxicity, carcinogenicity, ld 50 , maximum recommended daily dose and mutagenicity are predicted by lazar toxicity predictions server. prediction of protein structure is important in molecular docking, using i-tasser to build three dimensional protein structure and used for homology modeling using swisspdbviewer (spdbv) software. ramachandran plot were predicted to understand the complexity of amino acids within allowed region, energy is minimized to the protein structure with e-value=-39051.781. using saves to predict the errat value of 89.2% and verify3d of 81.6%. castp is used to predict ligand binding sites with fmoc-amino acids lys353, arg355, arg403, lys417, ile418, asp424, pro426, asp427, phe429, tyr453, leu455, asn460, leu461, lys462, pro463, phe464, ser469, gln493, tyr495, gly496, phe515, leu517 amino acids ( fig. 1 ). spike protein structure predicted using i-tasser, the red label represents (1) spike receptor binding domain (330-583) which corresponds to immunogenic ace2 receptor binding domain. (2) coronavirus s2 glycoprotein (662-1270) is translated as a large polypeptide that is subsequently cleaved to s1 and s2 domains from molecular docking plays vital role in computer-aided drug discovery to predict best active molecules to the target protein. using autodock 4.2 and autodock vina to predict the best drug binding sites towards the affinity of active site amino acids are screened based on binding energy and number of hydrogen bonds formed to the target amino acids. the antimalarial drugs such as chloroquine, hydroxychloroquine, artemisinin, gly416 amino acids ( fig.3 ; table 2 ). j o u r n a l p r e -p r o o f (fig. 4, table 3 ). the antibiotic drugs is also docked with target receptors by forming 3-6 hydrogen bonds and the interaction energy ranges from -6.0 to -9.0kcal/mol. the azithromycin is strong interaction with target receptor by forming 2 hydrogen bonds within active site amino acids and leu492 amino acids ( fig. 5; table 4 ). table: 1). the outbreak of research is to finding multiple treatment plans for coronavirus infection perspectives for repurposing drugs for the coronavirus disease 2019 chapter 31 -coronaviruses, fenner and white's medical virology a novel coronavirus from patients with pneumonia in china who statement regarding cluster of pneumonia cases in wuhan first case of 2019 novel coronavirus in the united states a novel coronavirus emerging in china -key questions for impact assessment clinical features of patients infected with 2019 novel coronavirus in wuhan novel wuhan (2019-ncov) coronavirus korean society of epidemiology 2019-ncov task force team. an interim review of the epidemiological characteristics of 2019 novel coronavirus host factors in coronavirus replication incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan, china report 2: estimating the potential total number of novel coronavirus cases in wuhan city structure, function, and evolution of coronavirus spike proteins sars-associated coronavirus understanding the coronavirus novel coronavirus disease 2019 (covid-19): the importance of recognising possible early ocular manifestation and using protective eyewear angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry aminopeptidase-n is a major receptor for the enteropathogenic coronavirus tgev further characterization of aminopeptidase-n as a receptor for coronaviruses dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus coronavirus envelope protein: current knowledge coronavirus envelope (e) protein remains at the site of assembly a structural analysis of m protein in coronavirus assembly and morphology assembly of severe acute respiratory syndrome coronavirus rna packaging signal into virus-like particles is nucleocapsid dependent chloroquine and hydroxychloroquine in the treatment of covid-19 with or without diabetes: a systematic search and a narrative review with a special reference to india and other developing countries chloroquine and hydroxychloroquine as available weapons to fight covid-19 breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 remdesivir is a direct-acting antiviral that inhibits rna-dependent rna polymerase from severe acute respiratory syndrome coronavirus 2 with high potency drugbank: a comprehensive resource for in silico drug discovery and exploration acd/chemsketch 1.0 (freeware) acd/chemsketch 2.0 and its tautomers, dictionary, and 3d plug-ins application of pharmaceutical profiling assays for optimization of drug-like properties admet in silico modelling: towards prediction paradise? autodock4 and autodocktools4: automated docking with selective receptor flexiblity autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading the pymol-molecular graphics system discovery studio modeling environment the structure of a rigorously conserved rna element within the sars virus genome cloning and sequencing of the gene encoding the spike protein of the coronavirus ibv protein structure and sequence re-analysis of 2019-ncov genome refutes snakes as its intermediate host and the unique similarity between its spike protein insertions and hiv-1 • corona virus infection is a pandemic disease caused from coronaviridae. • the spike protein is the major target to attached to the host-receptor ace2 • computational approaches to build the 3d structure of protein • drug selection for preclinical trials to study the efficacy and disease treatment • molecular docking approaches to screen the compounds based on binding energy we thank dr.c.n.prashantha, assistant professor, department of biotechnology, school of applied sciences, reva university for providing overall guidance towards the docking studies. the authors declare no conflict of interest. the authors declare no conflict of interest. key: cord-305143-mqd4ioj4 authors: zmasek, christian m.; knipe, david m.; pellett, philip e.; scheuermann, richard h. title: classification of human herpesviridae proteins using domain-architecture aware inference of orthologs (daio) date: 2019-01-06 journal: virology doi: 10.1016/j.virol.2019.01.005 sha: doc_id: 305143 cord_uid: mqd4ioj4 we developed a computational approach called domain-architecture aware inference of orthologs (daio) for the analysis of protein orthology by combining phylogenetic and protein domain-architecture information. using daio, we performed a systematic study of the proteomes of all human herpesviridae species to define strict ortholog groups (sogs). in addition to assessing the taxonomic distribution for each protein based on sequence similarity, we performed a protein domain-architecture analysis for every protein family and computationally inferred gene duplication events. while many herpesvirus proteins have evolved without any detectable gene duplications or domain rearrangements, numerous herpesvirus protein families do exhibit complex evolutionary histories. some proteins acquired additional domains (e.g., dna polymerase), whereas others show a combination of domain acquisition and gene duplication (e.g., betaherpesvirus us22 family), with possible functional implications. this novel classification system of sogs for human herpesviridae proteins is available through the virus pathogen resource (vipr, www.viprbrc.org). herpesviruses comprise a large and diverse order (herpesvirales) of double stranded dna viruses that infect humans and a wide range of other hosts (pellet and roizman, 2007; virus taxonomy: the classification and nomenclature of viruses the online 10th report of the ictv, 2017). human diseases caused by herpesviruses range from vesicular rashes to cancer. the order herpesvirales is subdivided into three families, including the herpesviridae, which is further subdivided into three subfamilies, the alpha-, beta-, and gammaherpesvirinae. within subfamilies, groups of related herpesvirus species are classified into genera. the nine species of human herpesviruses are distributed across the three subfamilies and several genera (table 1) ; these viruses are the main focus of this work. prior studies found that the beta-and gammaherpesvirinae are more closely related to each other than to alphaherpesvirinae (montague and hutchison, 2000) . in contrast to some other human viruses, the human herpesviruses have a long evolutionary history, with evidence suggesting that the primordial herpesvirus diverged into the alpha-, beta, and gammaherpesvirinae approximately 180 million to 220 million years ago (mcgeoch et al., 1995) . coupled with their genome complexity and the availability of numerous complete genome sequences, this deep evolutionary history makes herpesviruses a tractable and informative model to study virus genome evolution at the levels of gene duplication and protein domain rearrangement. nehrt et al., 2011; rogozin et al., 2014) , due to its importance for computational sequence functional analysis (eisen, 1998; zmasek and eddy, 2002) and the significance of gene duplications for biological evolution (zhang, 2003) . orthologs (or groups/clusters of orthologs) have often been inferred by indirect methods based on (reciprocal) pairwise highest similarities [e.g. (remm et al., 2001; tatusov et al., 1997) ]. in this work, we used explicit phylogenetic inference combined with comparison to a trusted species tree for orthology inference, as this approach is likely to yield more accurate results eddy, 2002, 2001) . many eukaryotic proteins, and by extension, proteins of eukaryotic viruses, are composed of multiple domains, components that can each have their own evolutionary history and functional implications. the architecture of a protein is a product of the ordered arrangement of its several domains and their overall tertiary structure. evolutionarily, individual domains can combine with other partner domains, enabling formation of a vast number of domain combinations, even within the same species (moore et al., 2008) . assembling multiple domains into a single protein creates a distinct entity that can be more than the sum of its constituent parts. the emergence of proteins with novel combinations of duplicated and then diverged domains is considered to be a major mechanism for rapid evolution of new functionality in eukaryotic genomes (itoh et al., 2007; peisajovich et al., 2010) . it is especially important in the evolution of pathways, where novel linkages between existing domains may result in the rearrangement of pathways and their behaviors in the cell (peisajovich et al., 2010) . the modular structure of eukaryotic proteins provides a mechanism that enables evolutionarilyrapid differentiation and emergence of a multitude of novel protein functions from an initially limited array of functional domains. proteins can gain (or lose) new domains via genome rearrangements, creating (or removing) domain combinations, in addition to modification of domains themselves by small-scale mutations (patthy, 2003; ye and godzik, 2004) . here we present a systematic classification of proteins catalogued in the ncbi refseq entries for each of the nine human herpesviruses plus selected comparisons with homologs from non-human herpesviruses based on phylogenetic inferencing and domain architecture analysis using domain-architecture aware inference of orthologs (daio). this analysis resulted in the classification of proteins into "strict ortholog groups" (sogs), in which all proteins are orthologous to each other (related by speciation events) and exhibit the same domain architecture. the sog classification also enabled the development of an informative name convention for each sog that includes information about the protein's function (if known) and a suffix indicating the taxonomic distribution of the protein. for example, an "abg" suffix would indicate that proteins of this group are found in some (but not all) human alphaherpesvirinae species (lowercase "a"), and all human beta-and gammaherpesvirinae species (uppercase "b" and "g"). such suffixes allow for the quick understanding of presumed conserved protein function and minimal common genome across the herpesviridae family. the sog classification results have been made publicly available through the virus pathogen resource (vipr) (pickett et al., 2012) at https://www.viprbrc.org. for this analysis, we developed a rational, phylogeny-and domain architecture-aware classification approach for human herpesvirus proteins, the domain-architecture aware inference of orthologs (daio) method, which produces strict ortholog groups (sogs) of proteins. before we present genome-wide findings, we show results for a few instructive sog examples, including protein groups that have evolved in a "simple" manner, recapitulating the herpesviridae evolutionary tree without gene duplications or domain rearrangements, and protein groups in which domain rearrangements (domain gains) and/or gene duplications have occurred. table 2 lists the 23 sogs common to all nine human herpesviruses. for every sog, a suggested name is provided, composed of a protein names and a suffix indicating the taxonomic distribution (a, b, g: present in all human members of the alpha-, beta-, gammaherpesvirinae, respectively; a, b, g: present in some but not all human members of the alpha-, beta-, gammaherpesvirinae, respectively). gene names/symbols (a forward slash is either part of the accepted gene name or is used to separate multiple gene names) and pfam domain architecture names are also included. the table is organized into three sections. the first section lists protein families that have apparently evolved without gene duplication or domain rearrangements [e.g., uracil dna glycosylase and the capsid scaffolding protein protease (cspp)]; the second section lists proteins that have evolved with domain rearrangements and or duplications [e.g., glycoprotein b (gb), dna polymerase, and multifunctional regulator of expression proteins (mre)], and the third section lists proteins that share some function (and even genome region) but have been formed from distantly or unrelated domains (e.g., gl, gn, and dna polymerase processivity factor). uracil dna glycosylases catalyze the first step -removal of the rna base uracil from dna -in base excision repair, the mechanism by which damaged bases in dna are removed and replaced (krusong et al., 2006) . uracil dna glycosylases are found in eukaryotes, bacteria, and archaea, as well as in herpesviruses and poxviruses (chen et al., 2002) . our phylogenomic analysis shows that for all nine human herpesviruses, uracil dna glycosylase is well conserved and contains one pfam domain, udg (uracil dna glycosylase superfamily). in addition, the gene tree for human herpesvirus uracil dna glycosylases ( fig. 1b ) precisely recapitulates the herpesvirus species tree (fig. 1a) ; therefore, this protein family can be inferred to have evolved from a single common ancestor and without any gene duplications or domain rearrangements (see table 2 for virus-specific gene names). capsid scaffolding protein proteases are essential for herpesvirus capsid assembly and maturation, and have an essential serine protease activity (liu and roizman, 1993) . these proteins contain one pfam domain, peptidase_s21. in contrast to uracil dna glycosylases, currently available data indicate that protease-scaffolding proteins with a peptidase_s21 domain are unique to herpesvirales. like uracil dna glycosylases, cspp evolved without domain architecture rearrangements or gene duplications (fig. 1c , table 2 ). other examples of herpesviridae genes that have evolved without any domain architecture rearrangements or gene duplications are listed in the first section of table 2 . herpesvirus virions have an envelope that consists of an outer lipid bilayer studded with 12 or more surface glycoproteins (originally defined in hsv). after virion glycoprotein engagement with cell surface receptors, the envelope fuses with the plasma membrane -a process which, for herpes simplex virus 1 (hsv-1), requires four of its 12 envelope glycoproteins, namely glycoproteins gb, gd, gh, and gl (cai et al., 1988; forrester et al., 1992; ligas and johnson, 1988; roop et al., 1993; spear and longnecker, 2003) . in contrast, for other herpesviruses, only glycoproteins gb, gh, and gl have been reported to be required for membrane fusion (alhajri et al., 2017) . gb and gh are highly conserved across all nine human herpesviruses (table 2) . a protein annotated as gl is also present in all nine human herpesviruses, yet its occurrences in members of the alpha-, beta-and gammaherpesvirinae are homologous within, but not between subfamilies. gls from different subfamilies contain unrelated protein domains (pfam: herpes_ul1, cytomega_gl, and phage_glycop_gl). gl is discussed in more detail below. detailed phylogenetic analysis of the human herpesvirus gb family ( fig. 2a) , including proteins from selected non-human members of the herpesviridae, shows a picture of a protein that has evolved without gene duplications (or, at the very least, duplicated genes have not been retained) and with nearly completely conserved domain architectures. the one exception to this is that human cytomegalovirus (hcmv) glycoprotein b (gb) has a short region of about 40 amino acids near its nterminus that comes in two forms that differ by approximately 50% at the amino acid level. this sequence variant was identified in hcmv strains isolated from chinese patients (shiu et al., 1994) and is identified in pfam as "hcmvantigenic_n domain". in our global hmmscan analysis (applying the same threshold of e = 10 −6 for every pfam domain) evalue support for presence of this domain in some strains is strong (e < 10 -22 ) and matching over the entire pfam model while other hcmv strains do not exhibit significant sequence similarity with this domain. it has been suggested that this domain polymorphism may be implicated in hcmv-induced immunopathogenesis, as well as in strainspecific behaviors, such as tissue-tropism and the ability to establish persistent or latent infections (pignatelli et al., 2004) . in our new systemic naming approach (see below) we term the sog of the protein with hcmvantigenic_n domain "glycoprotein b_ abg.b", whereas all other proteins fall into the "glycoprotein b_ abg.abg" sog. all members of the herpesviridae encode six conserved proteins that play essential roles at the replication fork during viral dna replication: a single-strand dna binding protein (major dna binding protein), a dna polymerase composed of two independently coded subunits (the catalytic dna polymerase subunit and a dna polymerase processivity factor encoded by three distantly related genes in members of the alpha-, beta-, and gammaherpesvirinae, see below), and a three subunit helicase/primase complex (dna replication helicase, dna helicase primase complex associated protein, and dna primase) (pellet and roizman, 2007) . our analysis shows that the catalytic dna polymerase subunits of all members of the herpesviridae contain two domains: an n-terminal dna polymerase family b exonuclease domain, and a c-terminal polymerase domain from dna polymerase family b (fig. 2b ). cellular family b dna polymerases are the main polymerases involved with nuclear dna replication and repair in eukaryotes and prokaryotes, and include dna polymerases ii and b, and polymerases α, δ, and ε (garciadiaz and bebenek, 2007) . family b dna polymerases are also found in other dsdna viruses, such as the insect ascoviridae, and members of the iridoviridae (e.g., fish lymphocystis disease virus) and phycoviridae (e.g., chlorella virus) (villarreal and defilippis, 2000) . in addition to these two large and ubiquitous domains, simplexvirus (which include human simplex virus 1 and 2) and mardivirus also possess a small c-terminal domain, called the dna polymerase catalytic subunit pol (dnapoly-mera_pol) domain in pfam (zuccola et al., 2000) , and are longer by about 45 aa on average than dna polymerase proteins from other herpesviridae. according to currently available genomic data, dnapo-lymera_pol is found in members of the simplexvirus genus of the alphaherpesvirinae. while varicella-zoster virus (human herpesvirus 3) and other members of the varicellovirus genus of the alphaherpesvirinae possesses dna polymerases that also tend to be longer, similarity of these protein regions to the dnapolymera_pol domain is low, using the current pfam model for dnapolymera_pol (pfam version 31.0). the function of this third domain is to mediate interaction between dna polymerase and its cognate processivity factor (bridges et al., 2000; loregian et al., 2000) based on the observation that a peptide corresponding to the 27 c-terminal amino acids of hsv-1 dna polymerase has been shown to inhibit viral replication by disrupting the interaction between dna polymerase and ul42 (digard et al., 1995; loregian et al., 1999) . in this context, it is interesting to note that the dna polymerase processivity factors are only distantly-related across the alpha-, beta-, and gammaherpesvirinae (see below). it is therefore conceivable that the interactions of beta-, and gammaherpesvirinae dna polymerase processivity factors with their corresponding dna polymerases (which lack a dnapolymera_pol domain) is different in nature than for alphaherpesvirinae. as for varicellovirus it is unclear whether they possess a functional dnapolymera_pol domain, and a definitive answer will require similar biochemical assays as have been performed for hsv-1. phylogenetic analysis of human herpesvirus dna polymerase proteins, plus related proteins from selected mammalian herpesviruses, shows that, similar to the glycoprotein b family, dna polymerases of the herpesviride evolved without gene duplication. nonetheless, in contrast to gb, dna polymerases acquired a new domain early in alphaherpesvirinae evolution. this domain might have been lost again, or underwent significant mutations, during varicellovirus evolution. the presence of the longer domain in varicelloviruses suggests that the longer domain emerged prior to the varicellovirus/simplexvirus split. multifunctional regulator of expression (mre; also known as immediate-early protein ie63, infected cell protein 27, icp27, and α27) is a protein with homologs in all human herpesviruses (for gene names see table 2 ). multifunctional regulator of expression is a regulatory protein that plays a role in the prevention of apoptosis during hsv1 infection (aubert and blaho, 1999) . multifunctional regulator of expression interacts directly with a number of proteins in performing its many roles. in particular, multifunctional regulator of expression protein contributes to host shut-off by inhibiting pre-mrna splicing by interacting with essential splicing factors, termed sr proteins, and affecting their phosphorylation (sciabica et al., 2003) . furthermore, the mre protein c.m. zmasek et al. virology 529 (2019) [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] has been shown to associate with cellular rna polymerase ii holoenzyme in a dna-and rna-independent manner and to recruit rna polymerase ii to viral transcription/replication sites (dai-ju et al., 2006; zhou and knipe, 2002) . mre also competes with some transport receptors, resulting in the inhibition of host pathways while supporting mrna export factor-mediated transport of hsv-1 mrnas (malik et al., 2012) . all of the multifunctional regulator of expression proteins analyzed here have a single copy of a pfam "herpesvirus transcriptional regulator family" (herpes_ul69) domain that is specific to members of the herpesviridae. in addition to the herpes_ul69 domain, human simplexvirus mre have an additional n-terminal domain, the "herpes viral adaptor-to-host cellular mrna binding domain" (hhv-1_vabd) (tunnicliffe et al., 2011) . besides human simplexvirus, architectures with c-terminal hhv-1_vabd and n-terminal herpes_ul69 domains are also found in chimpanzee herpesviruses (e.g. ncbi reference sequence: yp_009011042 (severini et al., 2013) ), while other non-human simplexviruses lack the hhv-1_vabd domain. using currently available genomic data, we were unable to detect hhv-1_vabd domains outside of the simplexvirus genus. phylogenetic analysis of human herpesvirus mre proteins, including proteins from selected herpesviruses of other mammals, shows that multifunctional regulator of expression proteins evolved without observable gene duplications (since this gene tree recapitulates the herpesvirus species tree). nine groups of human herpesviruses are annotated as performing the same, or very similar function, in the absence of discernable protein sequence similarity (table 2, fig. 3 ). as mentioned above, dna polymerase processivity factor is one of the six proteins that play essential roles at the replication fork during viral dna replication. processivity factors, also called clamp proteins, help to overcome the tendency of dna polymerase to dissociate from the template dna, and thus greatly enhance dna polymerase processivity (weisshart et al., 1999; zhuang and ai, 2010) . in contrast to the protein families discussed so far, dna polymerase processivity factors are only distantly-related across the alpha-, beta-, and gammaherpesvirinae. in the alphaherpesvirinae, the protein is composed of two tandem herpes_ul42 domains; betaherpesvirinae have a single herpe-s_pap domain; gammaherpesvirinae have a single herpes_dnap_acc domain (fig. 3a, b, c) . these three domains are very distant homologs and are members of the dna clamp superfamily (pfam clan cl0060). gl (fig. 3d , e, f) is another example of a protein function performed by different, probably non-homologous domains present in different herpesviridae subfamilies (pfam domains herpes_ul1, glyl_c, cyto-mega_gl, and phage_glycop_gl). interestingly, the open reading frames for these seemingly unrelated proteins are located in analogous conserved genomic contexts, including open reading frame sizes and orientations relative to the surrounding conserved coding regions. the remaining seven groups with these characteristics are: cytoplasmic egress tegument protein, cytoplasmic egress facilitator-1, cytoplasmic egress facilitator-2, encapsidation chaperone protein, glycoprotein n pfam clan herpes_glyco, cl0146), ltp binding protein, and small capsid protein (table 1 and supplementary table 1) . in contrast to the protein families discussed so far, the evolutionary history of human herpesviridae proteins with 7-transmembrane receptor domains is more complex (fig. 4) (spiess et al., 2015) . by comparing this gene tree with a species tree for human herpesviridae (fig. 1a) , we can infer three gene duplication events (marked as red squares in fig. 4) , resulting in four groups of orthologous genes: ul33/u12, us27, u51/orf74, and us28. in our new nomenclature (see below), we call the first group "gprotein coupled receptor homolog ul33/u12_b" because it is found in all four human betaherpesvirinae species (uppercase b suffix). the second group is called "g-protein coupled receptor homolog us27_b" as it is found in some human betaherpesvirinae (lowercase b suffix). the third group is called "g-protein coupled receptor homolog u51/orf74_bg" because it found in some human betaherpesvirinae and in some human gammaherpesvirinae (lowercase "bg" suffix). the fourth group is called "envelope protein us28_b". no orthologous genes were found in the human alphaherpesvirinae. whenever available, we base our names preferably on (mocarski, 2007) or the "recommended name" (under "protein names") from the uniprotkb database (bateman et al., 2017) . for reasons of consistency and objectivity, we used an automated approach to root all trees by mid-point rooting. it is possible, that the true root for the 7-transmembrane domain proteins tree is at the base of the u51-orf74 subtree. in this case there would be only two duplications in the tree, but still the same four ortholog groups: u51/orf74, us28, us27, ul33/u12. functionally, all these proteins appear to be hijacked human proteins that are being used by the virus to modulate the host immune system. in particular, many of them appear to act as chemokine (orphan) receptors (casarosa et al., 2003 (casarosa et al., , 2001 isegawa et al., 1998; murphy, 2001; zhen et al., 2005) (fig. 5) . proteins with us22 domains have the most complex evolutionary history of all herpesviridae proteins, even though among the human herpesviruses, the us22 domain has been found only in c.m. zmasek et al. virology 529 (2019) 29-42 betaherpesviruses (hanson et al., 1999) . us22 domain proteins are also present in gallid herpesvirus 2 (a member of the alphaherpesvirinae), in members of the alloherpesviridae family, in other dsdna viruses (e.g., poxviridae and iridoviridae), and in some animal species. most proteins with us22 domains carry two copies of the domain. us22 is a member of a large group of distantly homologous proteins (the sukh superfamily, pfam clan cl0526), which, for example include bacterial syd proteins. it has been suggested that a function of the us22 family is to act against various anti-viral responses by interacting with specific host proteins (zhang et al., 2011) . here we summarize the results of our phylogenetic analysis of us22 domain proteins of the human bataherpesviruses. unfortunately, the phylogenetic signal across this group of protens is weak, thus some support values are low. two groups of us22 orthologs span all four human betaherpesviruses: cmv tegument protein ul23 is likely to have orthologs in hhv-6a, hhv-6b, hhv-7 (roseolovirus) protein u3 ("tegument protein ul23/protein u3_b"). similarly, cmv tegument protein ul43 is likely to be orthologous to hhv-6a, hhv-6b, hhv-7 (roseolovirus) protein u25 ("tegument protein ul43/protein u25_b"). u3 and u25 are paralogous towards each other, as they are connected by a gene duplication, as are hcmv ul23 and 43. four groups of orthologs specific to roseolovirus are tegument protein dr1, tegument protein dr6, protein u7, and protein u17/u16. in u17/u16 proteins, it is unclear whether they possess a second us22 domain, as the similarity to this domain is weak to the point of insignificance. in contrast, u7 proteins possess at least three us22 domains and an additional c-terminal herpes_u5 domain. proteins u7 are most closely related to cmv ul29, but differ in their domain architecture (lack of herpes_u5 domain). thus cmv ul29 forms its own species-specific group of orthologs. numerous proteins with us22 domains are specific to cmv (and thus all paralogous to each other) given current data: apoptosis inhibitor ul38, early nuclear protein hwlf1, tegument protein ul26, us24, protein ul24, ul29, ul36, us23, us26, protein irs1, and protein trs1. c.m. zmasek et al. virology 529 (2019) 29-42 2.8. the inferred minimal proteomes of the human herpesviruses as described above, we classified viral proteins into "strict ortholog groups," requiring that all proteins exhibit the same domain architecture and are orthologous to each other. we attempted to give an informative name for each of these groups including a suffix that indicates the taxonomic distribution of a protein. for example, an "ag" suffix would indicate that proteins of this group are found in some (but not all) members of human alphaherpesvirus species (lowercase "a"), and members of both human gammaherpesvirus species (uppercase "g"). families which have a (some) domain(s) in common but differ in their domain architectures, are more difficult to rationally name (we found 17 of these cases). an example of such a family is dna polymerase. in such cases, the suffix is split by a period into two parts. the first part indicates overall presence of common domain(s) for all members of this sog, the second part (after the period) relates to specific domain architectures. thus, "dna polymerase_ abg.abg" refers to the simpler dna_pol_b_exo1--dna_pol_b domain architecture present in nearly all alphaherpesvirinae species. "dna polymerase_ abg.a" refers to the dna_pol_b_exo1--dna_pol_b-dnapolymera_pol da that is present in a smaller subset of alphaherpesvirinae species. the rationale behind this approach for labeling members of protein families that have different domain architectures is that it gives users a choice between "traditional" ortholog groups, which do not consider domain architectures (by ignoring the part after the period), and sogs (taking the full name into account). in total, we were able to establish 169 sogs (supplementary table 1 ). of these, 40 (23 +8 +9) functionally similar groups (table 2) are present in all 9 human herpesviridae species and represent the core proteins of human herpesviruses. besides proteins with clearly defined pfam domains, we found 29 protein families for which pfam domains have not been defined. classification of these proteins was based on manual blast searches. an example of such a family is the virion host shutoff protein ul41. another unusual case is the hsv1 ul13 serine threonine protein kinase. all nine human herpesviruses have homologs of this protein, but its associated pfam domain ul97 only matches sequences in betaherpesviruses. extension of the family to alpha-and gammaherpesviruses is thus based on manual blast searches. finally, two protein families could not be classified due to lack of phylogenetic signal: protein b8 of hhv-6a and hhv-6b (associated gene names u92, u93, hn1, hn92d, b8) and protein ul28/ul29/u8 of hhv-6a, hhv-6b, and hhv-7. proteins which are species or strain specific are listed in supplementary table 2. in order to make the results of daio classification available to all herpesvirus researchers for experimental hypothesis testing, we incorporated sog data into the virus pathogen resource (vipr) at https://www.viprbrc.org (pickett et al., 2012) . through vipr, scientists can search, sort, and download sog names (including taxonomic distribution), pfam domain architecture data, and individual protein sequences belonging to selected sogs. fig. 6a shows an example of a search result table, which includes data for some of the protein families discussed above, namely glycoprotein b family members (associated with two distinct sogs: "glycoprotein b_ abg.b" and "glycoprotein b_ abg.abg"), dna polymerase ("dna polymerase_ abg.a" and "dna polymerase_ abg.abg"), and multifunctional regulator of expression ("multifunctional regulator of expression_abg.a" and "multifunctional regulator of expression_abg.abg"). by clicking on the "total # of proteins" table entries, users can view and download the individual protein sequences belonging to a given sog. fig. 6b shows how sog data, including domain architecture information, is part of protein annotations in vipr (simplexvirus "dna polymerase_abg.a" example). as new genome sequence data become available, the sog data in vipr is continuously updated in order to keep current with the ever expanding universe of herpesvirus protein sequences. in addition, sog annotations in vipr will be expanded to include non-human herpesviruses in the future. sog data is also available for pox-and coronaviruses in vipr, and will be applied to other virus families in the future. in this work, we used domain-architecture aware inference of orthologs (daio) to provide a classification for proteins of human herpesviruses, based on domain architecture and phylogenetic history. while the work presented here is limited to human herpesviruses, and thus does not take full advantage of all the sequence data that is currently available, we plan to extend our daio approach to all herpesviruses with a known phylogenetic history. a major contribution of our classification system to herpesvirus biology is that it provides a series of testable hypotheses for further experimental investigations. for example, it informs experimental reconstruction of minimal genome viruses. such synthesized minimal genomes could prove useful for identification of genes responsible for pathogenic and other biological differences between viruses. of particular interest in the field of molecular biology is the relationship between domain architecture and protein function. the detailed analysis of domain architectures presented here suggests studies that investigate the functional effects of removing or swapping domains in viral multidomain protein architectures the fact that simplexvirus dna polymerases contain the extra dnapolymera_pol domain and that this domain architecture is conserved among simplexvirus isolates suggests that it may provide some unique function necessary for efficient replication of simplexviruses. this hypothesis could be explored experimentally. similarly, what would be the consequence of adding a cterminal glyl_c domain to the gl protein of vzv (which contains one herpes_ul1 domain), and so making it similar to the gl protein found in hsv-1 and hsv-2 (which has a herpes_ul1--glyl_c architecture)? interestingly, while it has been noted that domain loss is an important mechanism in eukaryote evolution (probably equally-and possibly even more-important than domain gain) (zmasek and godzik, 2011) ; and references therein), in herpesvirus evolution domain loss seems to play a lesser role, as most of the events we were able to detect are domain gains (according to the parsimony principle). another implication of this work relates to the observation that in some cases proteins that share the same name are composed of either unrelated (e.g. gl) or very distantly related domains (e.g. dna polymerase processivity factor) in different herpesvirus species. this raises the question -are such share named truly justified for proteins composed of unrelated domains? and to what extent has their putative shared function been experimentally validated. our approach is also expected to facilitate the detection and subsequent experimental study of species-(and strain-) specific proteins (listed in supplementary table 2). whereas hsv1 and hsv2 do not have any species specific proteins given current data, vzv has six, and cmv has by far the most with 130 proteins which are not found in any other species. interestingly, many of these 130 proteins are specific to one strain (or isolate) of cmv. unsurprisingly, many of these species-and strain-specific protein do not yet have a pfam domain (and thus were analyzed by manual blast searches in this work). an example of such a protein is the orf45 protein of kshv (zhu and yuan, 2003) . our automated approach provides a starting point for the systematic computational and experimental study of these species-and strain-specific proteins-studies, which eventually will provide answers to such questions as: are these species-and strain-specitic proteins essential under certain conditions? do they result in altered pathology or clinical symptoms? do they function in host interaction? do they possess as of yet undiscovered, but shared protein domains? in summary, we developed a computational approach called domain-architecture aware inference of orthologs (daio) for the classification of viral proteins into groups of orthologous proteins with identical domain architecures (sogs). in addition, we established a nomenclature for sogs that provides the user with information about the biological function and taxonomic distribution for the member proteins of a sog. we applied this classification and nomenclature to the proteomes of all human herpesviridae species and made the results publicly accessible via the vipr database. the acquisition and retention of novel domain architectures suggests that some herpesviridae proteins may have acquired novel functional characteristics, which can now be explored experimentally. we developed a semi automated software pipeline to analyze amino acid sequences for their protein domain based architectures and to infer multiple sequence alignments and phylogenetic trees for the molecular sequences corresponding to these architectures, followed by gene duplication inference. this pipeline contains the following five major steps: (1) sequence retrival; (2) domain architecture anlysis, including the inference of the taxonomic distributions of domain architectureseach of which corresponding to one preliminary sog, and manual naming of domain architecures/preliminary sogs (to be automated in future versions of this pipeline); (3) extraction of molecular sequences corresponding to domain architectures/preliminary sogs; (4) multiple sequence alignment and phylogenetic inference; (5) gene duplication inference, to determine which preliminary sogs contain sequences related by gene duplications and thus need to divided in multiple, final sogs. links to all custom software programs developed for this work are available here: https://sites.google.com/site/cmzmasek/home/ software/forester/daio. in the following the tools and methods used are described in more detail. individual protein sequences were downloaded from the vipr database (pickett et al., 2012) , while entire proteomes were downloaded from uniprotkb (bateman et al., 2017) . multiple sequence alignments were calculated using mafft version 7.313 (with "localpair" and "maxiterate 1000" options) (katoh and standley, 2013; kuraku et al., 2013) . prior to phylogenetic inference, multiple sequence alignment columns with more than 50% gaps were deleted. for comparison we also performed the analyses based on alignments for which we only deleted columns with more than 90% gaps. protein domains were analyzed using hmmscan from hmmer v3.1b2 (eddy, 2011) and the pfam 31.0 database (finn et al., 2016) . c.m. zmasek et al. virology 529 (2019) 29-42 phylogenetic trees were calculated for individual domain architectures (not full-length sequences) except for us22 domain proteins, because us22 domain alignments lack phylogeneticly sufficient signal. distance-based minimal evolution trees were inferred by fastme 2.0 (desper and gascuel, 2002) (with balanced tree swapping and "gme" initial tree options) based on pairwise distances calculated by tree-puzzle 5.2 (schmidt et al., 2002) using the wag substitution model (whelan and goldman, 2001 ), a uniform model of rate heterogeneity, estimation of amino acid frequencies from the dataset, and approximate parameter estimation using a neighbor-joining tree. for maximum likelihood approaches, we employed raxml version 8.2.9 (stamatakis et al., 2005) (using 100 bootstrapped data sets and the wag substitution model). tree and domain composition diagrams were drawn using archaeopteryx [https://sites.google.com/site/cmzmasek/home/ software/forester]. rooting was performed by the midpoint rooting method. unless otherwise noted, pfam domains are displayed ith a e = 10 −6 cutoff. gene duplication inferences were performed using the sdi and rio methods eddy, 2002, 2001) . automated genome wide domain composition analysis was performed using a specialized software tool, surfacing version 2.002 [zmasek cm (2012) , a tool for the functional analysis of domainome/genome evolution [available at https://sites.google.com/site/cmzmasek/home/software/ forester/surfacing]. all conclusions presented in this work are robust relative to the alignment methods, the alignment processing, the phylogeny reconstruction methods, and the parameters used. all sequence, alignment, and phylogeny files are available upon request. the processes for defining and naming strict ortholog groups were formalized into a set of "rules" and then implemented into a semi-automatic domain-centric phyloinformatics pipeline. any unique arrangement of single or multiple pfam domains is considered a domain architecture (da) godzik, 2012, 2011) . most proteins of members of the herpesviridae have das consisting of only a single domain. for example, the udg domain of uracil dna glycosylase is a single domain da, whereas the combination of n-terminal dna_-pol_b_exo1 and c-terminal dna_pol_b (denoted as dna_pol_b_ex-o1--dna_pol_b) of dna polymerases is a da with two domains. in this analysis, we consider a given da "present" in a given herpesviridae species s if the da is present under a set of thresholds in at least one strain of the species s. the rationale for this is that it is possible to miss a da in a genome, due to incomplete or erroneous sequences, erroneous assembly and gene-predication (false negatives), and even recent, actual gene loss. the opposite (false positive), on the other hand, is far less likely. for this work, we used two thresholds: a minimal domain length of 40% of the length set forth in the pfam database (domain fragments are unlikely to be functionally equivalent to full length domains) and a hmmscan e-value cutoff of e = 10 −6 . for every domain architecture, a set of bootstrap resampled phylogenetic trees (gene trees) was calculated by raxml (stamatakis et al., 2005) using protein sequences from one representative for each of the nine human herpesviridae species. for comparison and validation, we also calculated phylogenetic trees that included non-human hosted herpesviridae. for illustrations, gene duplications were inferred by comparing the consensus gene trees to the species tree ( fig. 1) for herpesviridae using the sdi (speciation duplication inference) algorithm (zmasek and eddy, 2001) . to obtain confidence values on orthology assignments (bootstrap support values), we employed the rio approach (resampled inference of orthologs) to compare sets of bootstrap resampled phylogenetic trees with the species tree for herpesviridae (zmasek and eddy, 2002) . in this work, we define a strict ortholog group (sog) as sequences related by speciation events and exhibiting the same domain architecture (based on pfam domains from pfam 31.0, a length threshold of 40%, and e-value cutoff of e = 10 −6 ). based on this approach for defining sogs, we developed the following naming syntax. for protein families such as uracil dna glycosylase, which exhibit the same da in all nine human herpesviridae, and which are related by speciation events only, we base our names on (mocarski and edward, 2007) as the base name and add a case-sensitive suffix that indicates the taxonomic distribution -"abg" in this case, since uracil dna glycosylase appears in each human alpha-, beta-, and gammaherpesvirinae species. therefore, the full name is "uracil dna glycosylase_abg". to indicate presence in some, but not all members of a subfamily, we use lower-case suffixes. "replication origin-binding protein_ ab" implies that members of this sog are present in all human alphaherpesvirinae species ("a"), and in some (but not all) betaherpesvirinae ("b"). while most of the human herpesviridae protein families fall into these basic cases, families which have a (some) domain(s) in common but differ in their da, are more difficult to rationally name. an example of such a family is glycoprotein b described above. because members of this family have different das, namely "glycoprotein_b" and "hcmvantigenic_n-glycoprotein_b", it is composed of two sogs (named "glycoprotein b_ abg.abg" and "glycoprotein b_ abg.b"). in such cases, we split the suffix into two parts, separated by a period. the first part ("abg") indicates overall presence of common domain(s) for all members of this sog, glycoprotein_b in this case. the second part (after the period) relates to entire das. ". abg" of "glycoprotein b_ abg.abg" means that the glycoprotein_b da is present in all human alpha-and gamma-, and some betaherpesvirinae. ".b" of "glycoprotein b_ abg.b" implies that the "hcmvantigenic_n-glycoprotein_b" da is present in some betaherpesvirinae. ovine herpesvirus 2 glycoproteins b, h, and l are sufficient for, and viral glycoprotein ov8 can enhance, cell-cell membrane fusion resolving the ortholog conjecture: orthologs tend to be weakly, but significantly, more similar in function than paralogs the herpes simplex virus type 1 regulatory protein icp27 is required for the prevention of apoptosis in infected human cells uniprot: the universal protein knowledgebase secondary structure and structure-activity relationships of peptides corresponding to the subunit interface of herpes simplex virus dna polymerase role of glycoprotein b of herpes simplex virus type 1 in viral entry and cell fusion constitutive signaling of the human cytomegalovirus-encoded chemokine receptor us28 constitutive signaling of the human cytomegalovirus-encoded receptor ul33 differs from that of its rat cytomegalovirus homolog r33 by promiscuous activation of g proteins of the gq, gi, and gsclasses roles of uracil-dna glycosylase and dutpase in virus replication the ortholog conjecture is untestable by the current gene ontology but is supported by rna sequencing data icp27 interacts with the cterminal domain of rna polymerase ii and facilitates its recruitment to herpes simplex virus 1 transcription sites, where it undergoes proteasomal degradation during infection herpesvirus systematics evolution of the herpesviruses fast and accurate phylogeny minimum-evolution principle specific inhibition of herpes simplex virus dna polymerase by helical peptides corresponding to the subunit interface accelerated profile hmm searches phylogenomics: improving functional predictions for uncharacterized genes by evolutionary analysis the pfam protein families database: towards a more sustainable future distinguishing homologous from analogous proteins construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein h coding sequences deleted multiple functions of dna polymerases transcriptional analysis of the murine cytomegalovirus hindiii-i region: identification of a novel immediate-early gene region human herpesvirus 6 open reading frame u12 encodes a functional beta-chemokine receptor evolutionary history and functional implications of protein domains and their combinations in eukaryotes orthologs and paralogs -we need to get it right mafft multiple sequence alignment software version 7: improvements in performance and usability a comparative study of uracil-dna glycosylases from human and herpes simplex virus type 1 aleaves facilitates on-demand exploration of metazoan gene family trees on mafft sequence alignment server with enhanced interactivity a herpes simplex virus mutant in which glycoprotein d sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells characterization of the protease and other products of aminoterminus-proximal cleavage of the herpes simplex virus 1 ul26 protein intranuclear delivery of an antiviral peptide mediated by the b subunit of escherichia coli heatlabile enterotoxin the catalytic subunit of herpes simplex virus type 1 dna polymerase contains a nuclear localization signal in the ul42-binding region herpes simplex virus icp27 protein directly interacts with the nuclear pore complex through nup62, inhibiting host nucleocytoplasmic transport pathways molecular phylogeny and evolutionary timescale for the family of mammalian herpesviruses toward a comprehensive phylogeny for mammalian and avian herpesviruses comparative analysis of herpesvirus-common proteins gene content phylogeny of herpesviruses arrangements in the modular evolution of proteins viral exploitation and subversion of the immune system through chemokine mimicry testing the ortholog conjecture with comparative functional genomic data from mammals modular assembly of genes and the evolution of new functions rapid diversification of cell signaling phenotypes by modular domain recombination. science. (80-.) herpesviridae: a brief introduction vipr: an open bioinformatics database and analysis resource for virology research genetic polymorphisms among human cytomegalovirus (hcmv) wild-type strains automatic clustering of orthologs and in-paralogs from pairwise species comparisons gene family level comparative analysis of gene expression n mammals validates the ortholog conjecture a mutant herpes simplex virus type 1 unable to express glycoprotein l cannot enter cells, and its particles lack glycoprotein h tree-puzzle: maximum likelihood phylogenetic analysis using quartets and parallel computing icp27 interacts with srpk1 to mediate hsv splicing inhibition by altering sr protein phosphorylation genome sequence of a chimpanzee herpesvirus and its relation to other primate alphaherpesviruses sequence variation of the amino-terminal antigenic domains of glycoprotein b of human cytomegalovirus strains isolated from chinese patients herpesvirus entry: an update identification and functional comparison of seven-transmembrane g-protein-coupled bilf1 receptors in recently discovered nonhuman primate lymphocryptoviruses raxml-iii: a fast program for maximum likelihood-based inference of large phylogenetic trees a genomic perspective on protein families. science (80-.) structural basis for the recognition of cellular mrna export factor ref by herpes viral proteins hsv-1 icp27 and hvs orf57 a hypothesis for dna viruses as the origin of eukaryotic replication proteins the classification and nomenclature of viruses the online (10th) report of the ictv herpes simplex virus processivity factor ul42 imparts increased dna-binding specificity to the viral dna polymerase and decreased dissociation from primer-template without reducing the elongation rate a general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach comparative analysis of protein domain organization a novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and dna viral systems evolution by gene duplication: an update the human herpesvirus 6 g protein-coupled receptor homolog u51 positively regulates virus replication and enhances cell-cell fusion in vitro association of herpes simplex virus type 1 icp8 and icp27 proteins with cellular rna polymerase ii holoenzyme the orf45 protein of kaposi's sarcoma-associated herpesvirus is associated with purified virions processivity factor of dna polymerase and its expanding role in normal and translesion dna synthesis rio: analyzing proteomes by automated phylogenomics using resampled inference of orthologs a simple algorithm to infer gene duplication and speciation events on a gene tree this déjà vu feeling-analysis of multidomain protein evolution in eukaryotic genomes strong functional patterns in the evolution of eukaryotic genomes revealed by the reconstruction of ancestral protein domain repertoires the crystal structure of an unusual processivity factor, herpes simplex virus ul42, bound to the c terminus of its cognate polymerase the authors thank sanjay vashee for critical review of the manuscript. we also thank the primary data providers for sharing their data in public archives, including vipr and uniprotkb. this work was funded by the national institute of allergy and infectious diseases (nih/dhhs) under contract no. hhsn272201400028c to rhs. supplementary data associated with this article can be found in the online version at doi:10.1016/j.virol.2019.01.005. key: cord-269011-230p8rsf authors: de haan, cornelis a.m.; rottier, peter j.m. title: molecular interactions in the assembly of coronaviruses date: 2005-08-31 journal: adv virus res doi: 10.1016/s0065-3527(05)64006-7 sha: doc_id: 269011 cord_uid: 230p8rsf this chapter describes the interactions between the different structural components of the viruses and discusses their relevance for the process of virion formation. two key factors determine the efficiency of the assembly process: intracellular transport and molecular interactions. many viruses have evolved elaborate strategies to ensure the swift and accurate delivery of the virion components to the cellular compartment(s) where they must meet and form (sub) structures. assembly of viruses starts in the nucleus by the encapsidation of viral dna, using cytoplasmically synthesized capsid proteins; nucleocapsids then migrate to the cytosol, by budding at the inner nuclear membrane followed by deenvelopment, to pick up the tegument proteins. viruses are multimolecular assemblies that range from small, regular, and simple to large, pleiomorphic, and complex. they consist of virus-specified proteins and nucleic acids and, in the case of enveloped viruses, of host-derived lipids. in infected cells the assembly of these different components into virions occurs with high precision amidst a huge background of tens of thousands of host compounds. two key factors determine the efficiency of the assembly process: intracellular transport and molecular interactions. directional transport ensures the swift and accurate delivery of the virion components to the cellular compartment(s) where they must meet and form (sub)structures. some viruses achieve this goal relatively simply when genome production occurs in close proximity to the virion assembly site (e.g., picornaviruses). many viruses, however, have evolved more elaborate strategies. this is illustrated, for instance, by the a-herpesviruses. assembly of these viruses starts in the nucleus by the encapsidation of viral dna, using cytoplasmically synthesized capsid proteins; nucleocapsids then migrate to the cytosol, by budding at the inner nuclear membrane followed by deenvelopment, to pick up the tegument proteins. subsequently, the tegumented capsids obtain their final envelope by budding into vesicles of the trans-golgi network (tgn), where the viral envelope proteins have congregated after their synthesis in the endoplasmic reticulum; the assembled viral particles are finally released by fusion of the virion-containing vesicles with the plasma membrane. to achieve their transport goals viruses provide their components with address labels that can be read by the transport machinery of the cell. once brought together, formation of the viral (sub)structures is governed and driven by their interactions. whereas the assembly of nonenveloped viruses is generally restricted to the cell cytoplasm, although often in association with membranes, that of enveloped viruses involves multiple cellular compartments, as exemplified already for herpesviruses. this review deals with the assembly of coronaviruses. we first describe what is known about the structure of the coronavirion and about the relevant properties of the structural components. we summarize the limited ultrastructural information about coronavirus assembly and budding. the main body of the review describes the interactions between the different structural components of the viruses and discusses their relevance for the process of virion formation. this review has a limited scope; for further information about other aspects of coronavirus biology the reader is referred to other reviews (de vries et al., 1997; enjuanes et al., 2001; gallagher and buchmeier, 2001; holmes, 2001; holmes et al., 2001; lai, 1997; lai and cavanagh, 1997; lai et al., 1994; masters, 1999; perlman, 1998; rossen et al., 1995; sawicki and sawicki, 1998; siddell, 1995; ziebuhr et al., 2000) . coronaviruses are a group of enveloped, plus-stranded rna viruses presently classified as a genus, which, together with the genus torovirus, constitutes the family coronaviridae. these viruses are grouped with two other families, the arteriviridae and the roniviridae, into the order nidovirales. this classification is not based on structural similarities-in fact, structure and composition of the viruses from the different families differ significantly-but on common features of genome organization and gene expression (de vries et al., 1997; lai and cavanagh, 1997) . coronaviruses infect a wide variety of mammals as well as avian species (table i ). in general they cause respiratory or intestinal infections, but some coronaviruses can also infect other organs (liver, kidney, and brain). until recently, these viruses were mainly of veterinary importance. this situation has changed quite dramatically because of the emergence of severe acute respiratory syndrome(sars-cov) in late 2002, which emphasized the potential relevance of coronaviruses for humans. on the basis of antigenic and genetic relationships the coronaviruses have been subdivided into three groups (table i) ; the taxonomic position of sars-cov has not been formally assigned. coronavirus particles have a typical appearance under the electron microscope. by the characteristic, approximately 20-nm-long spikes that emanate from their envelope the viruses acquire the solar image to which they owe their name (fig. 1) . the 80-to 120-nm virions have a pleiomorphic appearance that, whether artifact or real, reflects a pliable constellation, a feature that has severely hampered the ultrastructural analysis of these viruses. hence, our knowledge about the structure of coronaviruses is still rudimentary. the schematic representation of the current model of the coronavirion drawn in fig. 1 is based on morphological and biochemical fig 1. electron micrographs of mouse hepatitis virus strain a59 (mhv-a59) virions without (a) and with (b) the hemagglutinin-esterase (he) envelope protein (viruses kindly provided by r. de groot, virology division, utrecht university, the netherlands; image courtesy of j. lepault, vms-cnrs, gif-sur-yvette, france). large, club-shaped protrusions consisting of spike (s) protein trimers give the viruses their corona solis-like appearance. viruses containing the he protein display a second, shorter fringe of surface projections in addition to the spikes. (c) schematic representation of the coronavirion. the viral rna is encapsidated by the nucleocapsid (n) protein forming a helical ribonucleoprotein (rnp), which is in turn part of a structure with spherical, probably icosahedral, configuration. the nucleocapsid is surrounded by a lipid bilayer in which the s protein, the membrane glycoprotein (m), and the envelope protein (e) are anchored. in addition, some group 2 coronaviruses contain the he protein in their lipid envelope as illustrated on the right side of the particle. observations. as this picture illustrates, the particle consists of a nucleocapsid or core structure that is surrounded by a lipid envelope. anchored in this envelope are the three canonical coronavirus membrane proteins: the membrane (m) protein, the envelope (e) protein, and the spike (s) protein. viruses from group 2 have an additional, fourth membrane protein, the hemagglutinin-esterase (he) protein. as a consequence these viruses display a second, shorter (5 nm) fringe of surface projections in addition to the spikes (fig. 1b) (bridger et al., 1978; king et al., 1985; sugiyama and amano, 1981) . the ribonucleoprotein (rnp) core contains one copy of the viral genomic rna. this rna is packaged into a helical structure by multiple copies of nucleocapsid protein (n). size estimations of the flexible cylindrical structures varied quite considerably, ranging between 7 and 16 nm in diameter and up to 0.32 mm in length (see laude and masters, 1995) . the ribonucleoprotein helix appears in turn to be contained within a spherical, probably icosahedral, configuration as indicated by various ultrastructural approaches using purified transmissible gastroenteritis virus (tgev) and mouse hepatitis virus (mhv) (risco et al., 1996 (risco et al., , 1998 . the molar ratio of the major structural proteins, s:n:m, has been variously estimated to be approximately 1:8:16 (sturman et al., 1980) , 1:6:15 (cavanagh, 1983a) , 1:8:8 (hogue and brian, 1986) , and 1:11:10 (liu and inglis, 1991) , although an m:n molar ratio of 3 has also been reported (escors et al., 2001a) . the s:he molar ratio was estimated to be 4 (hogue and brian, 1986) . the e protein is only a minor virion component and was calculated to occur in infectious bronchitis virus (ibv), tgev, and mhv virions at a rate of approximately 100, 20, and 10 molecules per particle, respectively (godet et al., 1992; liu and inglis, 1991; vennema et al., 1996) . the lipid composition of coronaviral envelopes has been studied only to a limited extent. comparison of the phospholipid composition of mhv with that of its host cell showed increased levels of sphingomyelin, phosphatidylserine, and phosphatidylinositol and a decrease in the level of phosphatidylethanolamine (van genderen et al., 1995) . whether the lipid composition of mhv is an accurate reflection of its budding compartment or whether certain lipids become enriched in the virus during budding is not known. what follows is a general description of the individual virion components and their properties. this description is by no means complete as it is restricted to the information that is of relevance to the main topic of this review. for a schematic representation of the coronavirus life cycle see fig. 2 . the coronavirus life cycle. the replication cycle starts with attachment of the virion by its s protein, that is, through the s1 subunit thereof, to the receptors on the host cell. this interaction leads to fusion of the virus envelope with a cellular membrane, coronaviruses contain a single-stranded positive-sense rna genome of some 27 to 31 kilobases, the largest nonsegmented viral rna genomes known. the rna has a 5 0 -terminal cap and a 3 0terminal poly(a) tract. both genomic termini contain untranslated regions (utrs) of some 200-500 nucleotides that harbor several cis-acting sequences and structural elements functioning in viral replication and transcription. coronaviruses have a typical genome organization characterized by the occurrence of a distinctive set of genes that are essential for viability and occur in a fixed order: 5 0 -polymerase ( pol)-s-e-m-n-3 0 (fig. 3) . the pol gene comprises approximately twothirds of the genome, from which it is translated directly. it encodes two large precursors (pol1a and pol1ab), the many functional cleavage products of which are collectively responsible for rna replication and transcription (for reviews on coronavirus transcription and replication see de vries et al., 1997; lai, 1997; lai and cavanagh, 1997; lai et al., 1994; sawicki and sawicki, 1998; ziebuhr et al., 2000) . the more downstream pol1b gene is translated by translational readthrough, for which the s2 subunit is responsible. from the genomic rna that is released by disassembly of the incoming particle the pol1a and pol1b genes are translated, resulting in the production of two large precursors (pol1a and pol1ab), the many cleavage products of which collectively constitute the functional replication-transcription complex. genes located downstream of the pol1b gene are expressed from a 3 0 -coterminal nested set of subgenomic (sg) mrnas, each of which additionally contains a short 5 0 leader sequence derived from the 5 0 end of the genome (shown in red). transcription regulatory sequences (trss) located upstream of each gene serve as signals for the transcription of the sgrnas. the leader sequence is joined at a trs to all genomic sequence distal to that trs by discontinuous transcription, most likely during the synthesis of negative-strand sgrnas. in most cases, only the 5 0 -most gene of each sgrna is translated. multiple copies of the n protein package the genomic rna into a helical structure in the cytoplasm. the structural proteins s, m, and e are inserted into the membrane of the rough endoplasmic reticulum (rer), from where they are transported to the er-to-golgi intermediate compartment (ergic) to meet the nucleocapsid and assemble into particles by budding. the m protein plays a central role in this process through interactions with all viral assembly partners. it gives rise to the formation of the basic matrix of the viral envelope generated by homotypic, lateral interactions between m molecules, and it interacts with the envelope proteins e, s, and he (if present), as well as with the nucleocapsid, thereby directing the assembly of the virion. virions are transported through the constitutive secretory pathway out of the cell-the glycoproteins on their way being modified in their sugar moieties, whereas the s proteins of some but not all coronaviruses are cleaved into two subunits by furin-like enzymes (see text for references). using a ribosomal frameshift mechanism for which a "slippery" sequence and a pseudoknot structure are required. the genes located downstream of pol1b are expressed from a 3 0 -coterminal nested set of subgenomic (sg) rnas, each of which additionally contains a short 5 0 leader sequence derived from the 5 0 end of the genome. transcription regulatory sequences (trss) located upstream of each gene serve as signals for transcription of the sgrnas. the leader sequence is joined at a trs to all genomic sequence distal to that trs by discontinuous transcription, most likely during the synthesis of negative-strand sgrnas (sawicki and sawicki, 1998) . besides the characteristic genes encoding the replicative and structural functions, coronaviruses have a more variable collection of additional genes that are located in two clusters in the 3 0 -terminal onethird of the genome. the genes differ distinctly in their nature and genomic position among the coronavirus groups, but they are specific for each group. these so-called group-specific genes appear not to be essential as shown by the occurrence of natural mutants defective in some of them (brown and brierley, 1995; herrewegh et al., 1995; kennedy et al., 2001; luytjes, 1995; shen et al., 2003; vennema, 1999; vennema et al., 1998; woods, 2001) and by the observed viability of engineered deletion mutants lacking some or all of these genes (de haan et al., 2002b; fischer et al., 1997; haijema et al., 2004; ortego et al., 2003; sola et al., 2001) . except for the group 2-specific he protein and, possibly, the poorly characterized i protein (fischer et al., 1997; senanayake et al., 1992) , the latter encoded by an open reading frame completely contained within the n gene, the groupthe single-stranded, positive-sense rna genome contains 5 0 -and 3 0 -terminal untranslated regions (utrs) with a 5 0 -terminal cap and a 3 0 -terminal poly(a) tract. the leader sequence (l) in the 5 0 utr is indicated. all coronaviruses have their essential genes in the order 5 0 -pol-s-e-m-n-3 0 . the pol1a and pol1b genes comprise approximately twothirds of the genome. the more downstream pol1b gene is translated by translational readthrough, using a ribosomal frameshift mechanism. transcription regulatory sequences (trss) located upstream of each gene, which serve as signals for the transcription of the subgenomic (sg) rnas, are indicated by circles. the genes encoding the structural proteins he, s, e, m, and n are specified. gray boxes indicate the accessory, group-specific genes, in the case of group 2 coronaviruses genes 2a, he, 4, 5a, and i. specific proteins do not appear to occur in virions. although their functions have not yet been resolved, mutant studies indicate that they play important roles in the interaction of coronaviruses with their host (de haan et al., 2002b; fischer et al., 1997; haijema et al., 2004; ortego et al., 2003) . the n protein is the most abundantly expressed viral protein in infected cells (for a review, see laude and masters, 1995) . its size varies considerably between viruses from different groups (377-455 amino acids, i.e., molecular masses ranging between 45 and 60 kda), n proteins from group 2 coronaviruses (table i) being the largest. whereas the amino acid sequences of n proteins are quite similar within the groups, the homology between proteins from different coronavirus groups is rather limited (30-35%). an exception is a region spanning about 50 residues within the amino-terminal one-third of the n molecule, where high sequence identity has been conserved across the different groups. despite the overall sequence variation the n proteins have a number of common characteristics. consistent with their role as nucleic acidbinding proteins they are all highly basic because of the abundance of arginine and lysine residues. these are clustered mainly in two nearby regions in the middle of the molecules. the abundance of basic residues is reflected in the calculated overall isoelectric points of the n proteins, the values of which are in the range of 9.7-10.1. these numbers are the more significant in view of the acidic nature of the very carboxyterminal domain; pi values ranging from 4.3 to 5.5 were calculated for the terminal 45 residues (parker and masters, 1990) . another general characteristic of the n proteins is their high content (7-11%) of serine residues, which are potential targets for phosphorylation. although these residues occur all over the n molecule, their relative abundance within the first of the two basic regions is notable. little is known about the three-dimensional structure of the n protein. of the sars-cov n protein the amino-terminal domain (residues 45-181) was analyzed by nuclear magnetic resonance spectroscopy. it appeared to consist of a five-stranded b sheet with a folding distinct from that of other rna-binding proteins (huang et al., 2004) . in coronavirus-infected cells the n protein can often be detected as one major and several minor forms, the latter polypeptides having a slightly lower molecular weight. the major species appeared to comigrate in gels with the n protein observed in virions, indicating that only the full-len gth n spec ies is incorp orated in to partic les. how th e minor n spec ies ar ise and whet her they are of partic ular significan ce for infection is unc lear. they are most likely derived by proteoly tic proc essing from the major n species. this is supp orted by stud ies from eleo uet et al. (2000 ) , who show ed the tge v n prot ein to be cleav ed by casp ases. cas pase cleava ge sites wer e also predicted in the carboxy terminus of several other coronavirus n proteins (eleouet et al., 2000; ying et al., 2004) . these features are in agreement with observations showing that antibodies directed against the carboxy terminus of the mhv and tgev n proteins were not reactive with the faster migrating electrophoretic forms. furthermore, these smaller n protein forms appeared to be derived from the major species as judged from pulse-chase analyses (for a review see laude and masters, 1995) . the n protein is the only coronavirus structural protein known to become phosphorylated (for references see laude and masters, 1995) . both the major and minor n species appear to be phosphorylated as shown for mhv-a59 in sac(ã�) cells (rottier et al., 1981b) and for tgev in llc-pk1 cells (garwes et al., 1984) . of the many potential target serines only a few are actually modified in the case of mhv (stohlman and lai, 1979; wilbur et al., 1986) . n protein phosphorylation does not seem to play a critical role in the regulation of virus assembly. in contrast, it has been hypothesized that dephosphorylation of the protein might facilitate disassembly during mhv cell entry (kalicharran et al., 1996; mohandas and dales, 1991) . immunofluorescence microscopy has shown the n protein to be localized in a particulate manner throughout the cytoplasm of coronavirus-infected cells. although the protein lacks a membrane-spanning domain it was found in association with membranes (anderson and wong, 1993; sims et al., 2000; stohlman et al., 1983) . for mhv, the n protein was found to colocalize partly with the membrane-associated viral replication complexes van der meer et al., 1999) . in addition to its cytoplasmic localization, the n proteins of ibv, mhv, and tgev have also been demonstrated to localize to the nucleolus both in coronavirus-infected cells and when expressed independently wurm et al., 2001) . putative nuclear localization signals were identified in these proteins. the ibv n protein was found to interact with nucleolar antigens, which appeared to occur more efficiently when the n protein was phosphorylated, and to affect the cell cycle (chen et al., 2002) . however, because mhv is able to replicate in enucleated cells (brayton et al., 1981; wilhelmsen et al., 1981) the nucleolar localization of the n protein does not appear an essential step during infection. although the primary function of the n protein is the formation of the viral ribonucleoprotein complex, several studies indicate the protein to be multifunctional. as indicated by its intracellular localization, the n protein is a likely component of the coronavirus replication and transcription complex. its presence is not an absolute requirement for replication and transcription because a human coronavirus (hcov) rna vector containing the complete pol1ab gene appeared to be functional in the absence of the n protein (thiel et al., 2003) . however, the efficiency of the system was much enhanced when the protein was present. furthermore, using an in vitro system, it was demonstrated that antibodies to the n protein, but not those against the s and m proteins, inhibited viral rna synthesis by 90% (compton et al., 1987) . interactions that have been observed between the n protein and leader/trs sequences nelson et al., 2000; stohlman et al., 1988) and between n protein and the 3 0 utr (zhou et al., 1996) suggest a role for the n protein in the discontinuous transcription process. furthermore, the n protein was also shown to interact with cellular proteins that play a role in coronavirus rna replication and transcription (choi et al., 2002; shi et al., 2000) . in addition, the n protein was reported to function as a translational enhancer of mhv sgrnas (tahara et al., 1998) . the m protein (previously known as e1 protein) is the most abundant envelope protein. it is the "building block" of the coronavirion and has been shown to interact with virtually every other virion component, as detailed in section iv. the m protein is 221-230 residues in length, except for the group 1 m proteins, of which the amino terminus is about 30 residues longer. despite large differences in primary sequences between m proteins from different antigenic groups, their hydropathicity profiles are remarkably similar. the m protein is highly hydrophobic. it has three hydrophobic domains alternating with short hydrophilic regions in the amino-terminal half of the protein, with the exception of the aforementioned group 1 m proteins, which have at their amino terminus a fourth hydrophobic domain that functions as a cleavable signal peptide. the carboxy-terminal half of the protein is amphipathic, with a short hydrophilic domain at the carboxy-terminal end (fig. 4) . in the center of the protein, directly adjacent to the third hydrophobic domain, is a stretch of eight amino acids that is well conserved (swwsfnpe). the conservation of the overall chemical features suggests that there are rigid structural constraints on the m protein as a result of functional requirements (for a review on the m protein, see rottier, 1995) . biochemical and theoretical studies led to a topological model for the mhv m protein rottier et al., 1984 rottier et al., , 1986 , in which the polypeptide spans the lipid bilayer three times, leaving a small amino-terminal domain (15-35 residues) in the lumen of intracellular organelles (or outside the virus), whereas the carboxyterminal half of the protein is located on the cytoplasmic side of the membrane (or inside the virion). the lumenal domain and the hydrophilic carboxy terminus are susceptible to protease digestion and are thus exposed. the bulk of the carboxy-terminal half of the m protein is protease resistant, indicating that the amphipathic part of the protein is either folded tightly or embedded in the polar surface of the membrane. indeed, a mutant lacking all three transmembrane domains was found to be associated with membranes (mayer et al., 1988) . the model for the disposition of the m protein in the membrane was confirm ed for ibv (cavanag h et al., 1986) . interest ingly, the fig 4. membrane topology of the coronavirus envelope proteins. the he and s proteins are both type i membrane proteins, with short carboxy-terminal cytoplasmic tails. the he protein forms disulfide-linked homodimers, whereas the s protein forms noncovalently linked homotrimers. the s1 subunits presumably constitute the globular head, whereas the s2 subunits form the stalk-like region of the spike. the m protein spans the lipid bilayer three times, leaving a small amino-terminal domain in the lumen of intracellular organelles (or on the outside of the virion), whereas the carboxy-terminal half of the protein is located on the cytoplasmic side of the membrane (or inside the virion). in tgev virions some of the m proteins have their cytoplasmic tail exposed on the outside (not shown). the m protein is glycosylated at its amino terminus (indicated by a diamond). the amphipathic domain of the m protein is represented by an oval. the hydrophilic carboxy terminus of the e protein is exposed on the cytoplasmic side of cellular membranes or on the inside of the virion. the e protein may span the bilayer once (b) or twice (a). m protein of tgev was shown to adopt an additional conformation. in virions about one-third of the m molecules have their carboxy terminus exposed on the virus surface rather than buried inside the particle (escors et al., 2001a; risco et al., 1995) . this appears to have immunological consequences (risco et al., 1995) but the real significance of the dual topology is unclear. in mhv the m protein was found to assume only one defined membrane topology . the coronavirus m protein is almost invariably glycosylated in its exposed amino-terminal domain. this provides the virion with a diffuse, hydrophilic cover on its outer surface. whereas the group 1 and 3 coronaviruses and sars-cov all contain m proteins with only n-linked sugars, the m proteins of group 2 coronaviruses are o-glycosylated (for a review see rottier, 1995 ). an exception is mhv-2, the m protein of which carries both o-and n-linked sugars (yamada et al., 2000) . n-glycosylation is initiated in the endoplasmic reticulum by the cotranslational linkage of a large oligosaccharide structure to the polypeptide at asparagine residues within the consensus sequence nxs/t (where x is any amino acid). in contrast, mucin-type o-glycosylation starts posttranslationally with the addition of an n-acetylgalactosamine (galnac) monosaccharide to a hydroxylamino acid. o-glycosylation is subsequently completed by stepwise addition of other monosaccharides such as galactose, n-acetylglucosamine, fucose, and sialic acid. mhv m proteins carry a well-conserved ss(x)ttxxp sequence at their extreme amino terminus. despite the apparent presence of multiple hydroxylamino acids as potential oligosaccharide acceptor sites the m protein of mhv-a59 was found to be modified by the addition of only a single oligosaccharide side chain (de haan et al., 1998b) . this side chain, when studied in ost7-1 cells, appeared to be attached to the threonine at position 5. mutation studies, however, revealed that alternative acceptor sites can also be used. no unique sequence motifs for o-glycosylation of mhv m could be identified, which is probably related to the occurrence in cells of multiple galnac transferases (de haan et al., 1998b) . as the expression of these enzymes varies in cells, conservation of the ss(x)ttxxp motif in mhv m protein may serve to increase opportunities for the protein to become glycosylated in different cell types. the distinct conservation of n-and o-glycosylation among the m proteins of the different groups of coronaviruses suggests that the presence and the particular type of carbohydrates are somehow beneficial to the virus, most likely in its interaction with the host. glycosylation of the m protein appeared not to be required for envelope assembly (de haan et al., 1998a,) or for interaction with the s protein (de haan et al., 1999) , nor did it influence virus replication in vitro (de haan et al., 2002a laude et al., 1992) . coronaviruses are able to induce interferon a (ifn-a) by their glycoproteins (baudoux et al., 1998a,b) . for tgev (charley and laude, 1988; laude et al., 1992) and mhv (de haan et al., 2003) , the oligosaccharides linked to the m protein were demonstrated to be important for efficient ifn induction in vitro. the glycosylation status of the mhv m protein was found to influence the ability of the virus to replicate in the liver but not in the brain . thus, viruses with n-glycosylated m proteins replicated to a significantly higher extent in liver than otherwise identical viruses carrying o-glycosylated m proteins. mhv with unglycosylated m proteins replicated to the lowest extent. the mechanism behind these observations remains to be elucidated. when expressed in cells independently from the other viral proteins, the m proteins of mhv, ibv, tgev, and feline coronavirus (fcov) accumulate in the golgi compartment, that is, beyond the site of virus budding locker et al., 1992; machamer and rose, 1987; machamer et al., 1990; rottier and rose, 1987) , which is the intermediate compartment between the er and the golgi (ergic). the fine localization of the different proteins is, however, not the same. for instance, whereas the mhv m protein is concentrated in the trans-most golgi compartments, the ibv m protein localizes to the cis side of the golgi complex. signals for localization appear to reside in the hydrophilic part of the cytoplasmic tail and in the transmembrane domains. the extreme carboxy-terminal tail of mhv m was shown to be necessary, although not sufficient, for golgi localization (armstrong and patel, 1991; locker et al., 1994) . mutant proteins lacking this domain were transported to the plasma membrane. also, mutation of a single tyrosine in this domain, which occurs in the context of a potential internalization signal, resulted in plasma membrane localization (c. a. m. de haan and p. j. m. rottier, unpublished results). the first transmembrane domain of the ibv m protein was shown to be required and sufficient for localization to the cis-golgi region (machamer and rose, 1987; machamer et al., 1990 machamer et al., , 1993 swift and machamer, 1991) . this is not the case for the mhv m protein of which mutants with only the first transmembrane domain did not leave the er (armstrong et al., 1990; locker et al., 1994; rottier et al., 1990) . moreover, insertion of the first transmembrane domain of mhv m into a reporter protein resulted in a chimeric protein that was transported to the cell surface (armstrong and patel, 1991; machamer et al., 1993) , unlike a similar chimeric protein containing the first transmembrane domain of ibv that was retained in the golgi compartment . other mhv m mutant proteins lacking the first and second transmembrane domains were also not efficiently retained in the golgi compartment and were diverted to endosomal structures (armstrong et al., 1990; locker et al., 1994) . the mechanism by which golgi retention of m proteins is regulated has not yet been resolved. however, oligomerization of the proteins, mediated by the transmembrane domains, seems to play an important role, perhaps in combination with retrieval mechanisms maceyka and machamer, 1997) . formation of oligomeric complexes has been demonstrated to correlate with golgi retention of a reporter protein containing the first transmembrane domain of ibv m (weisz et al., 1993) while also the golgi-resident mhv m protein was found to occur in large, homomeric complexes (locker et al., 1995) . the lumenal domain of the m protein does not appear to contribute to localization; its deletion from mhv m did not affect the intracellular destination of the protein (mayer et al., 1988; rottier et al., 1990) . in infected cells the m proteins of ibv and mhv were observed to occur in the membranes of the budding compartment as well as in the golgi compartment. under these conditions their cis-trans distribution in the golgi compartment was the same as when these proteins were expressed independently machamer et al., 1990) . the e protein (previously known as sm protein) is a small protein (76-109 residues) and a minor component of the coronaviral envelope. although the primary structures of e proteins are quite conserved within the different coronavirus groups, they share little homology between the groups. however, the proteins have several structural features in common. the e protein contains a relatively large hydrophobic region in its amino-terminal half, followed by a cysteine-rich region, an absolutely conserved proline residue, and a hydrophilic tail. e is an integral membrane protein, which is assembled in membranes without the involvement of a cleaved signal peptide . its membrane topology has not been firmly established. although the opposite was proposed initially for the tgev e protein (godet et al., 1992) , there seems to be consensus about the hydrophilic carboxy terminus being exposed on the cytoplasmic side in cells or on the inside of the virion (corse and machamer, 2000; raamsman et al., 2000) . the amino terminus of the mhv e protein was not detectably present on the virion outside but appeared to be exposed cytoplasmically when it was extended with an amino-terminal epitope tag (maeda et al., 2001) , consistent with a topological model in which the hydrophobic domain spans the bilayer twice. for the ibv e protein evidence was provided indicating that the amino terminus is exposed lumenally in cells, consistent with a single spanning topology (corse and machamer, 2000) (fig. 4) . the e protein is not glycosylated but appears to become palmitoylated. this was shown most convincingly for the ibv e protein by labeling with [ 3 h]palmitate (corse and machamer, 2002) , both in ibv-infected cells and when the protein was expressed. mutagenesis revealed that one or both of the two conserved cysteines became modified. the result is consistent with the observed increase in electrophoretic mobility of the mhv e protein after treatment with hydroxylamine, an agent that cleaves thioester-linked acyl chains (yu et al., 1994) . others, however, were not able to confirm this posttranslational modification (godet et al., 1992; raamsman et al., 2000) . in coronavirus-infected cells the e protein has been observed by immunofluorescence studies to occur at intracellular membranes as well as at the cell surface (godet et al., 1992; smith et al., 1990; tung et al., 1992; yu et al., 1994) . when expressed exogenously from cdna, the e protein was detected only in intracellular organelles, although at different locations. the mhv e protein localized to pre-golgi membrane compartments, as was demonstrated by its colocalization with rab-1, a marker for the endoplasmic reticulum and the ergic, by electron microscopy . the ibv e protein, tagged at its amino terminus with an epitope, was also localized to pre-golgi compartments (lim and liu, 2001) . in another study, however, the ibv e protein was shown to accumulate in the golgi apparatus, being distributed throughout the complex machamer, 2000, 2002) . while the former study identified an ertargeting signal in the extreme carboxy terminus of the e protein (lim and liu, 2001) , the latter studies, using carboxy-terminal truncations, mapped the golgi-targeting information to a region between tail residues 13 and 63 (corse and machamer, 2002) . in addition, these authors showed the ibv e cytoplasmic tail to be necessary and sufficient for golgi targeting. the e protein was identified as a virion component relatively late, due to its low abundance and its small size (godet et al., 1992; liu and inglis, 1991; yu et al., 1994) . it was estimated to occur in ibv, tgev, and mhv virions at a rate of about 100, 20, and 10 molecules per particle, respectively (godet et al., 1992; liu and inglis, 1991; vennema et al., 1996) . because of its low abundance the e protein may not have a genuine structural function in the virion envelope. rather, it may have a morphogenetic function by taking strategic positions within the m protein lattice to generate the required membrane curvature. alternatively, it may serve to close the neck of the budding particle as it pinches off the membrane (vennema et al., 1996) . expression of the e protein alone induced the formation of characteristic membrane structures also observed in infected cells, which apparently consist of masses of tubular, smooth convoluted membranes (david-ferreira and manaker, 1965; raamsman et al., 2000) . in addition, it resulted in the formation of vesicles containing the e protein, shown to be released from the cells (corse and machamer, 2000; maeda et al., 1999) . mhv infection induces caspase-dependent apoptosis in some, but not all, cells. by expressing the viral structural proteins separately in cells, the activity could be attributed to the e protein . apoptosis induction has not been reported for e proteins from other coronaviruses. coronavirus e proteins share structural similarities with small hydrophobic membrane proteins found in other enveloped viruses. examples are the vpu protein of hiv-1, the 6k protein of alphaviruses, and the m2 protein of influenza virus. these proteins, also known as viroporins (gonzalez and carrasco, 2003) , were demonstrated to modify membrane permeability and to help the efficient release of progeny virus. the s protein (previously known as e2) constitutes the spikes, the hallmark of coronaviruses under the electron microscope. it is the major determinant of host range, tissue tropism, pathogenesis, and virulence. it is a relatively large, 1160-to 1452-amino acid-long type i glycoprotein with a cleavable n-terminal signal sequence and a membrane-anchoring sequence followed by a short hydrophilic carboxy-terminal tail of about 30 residues (fig. 4) . when comparing primary sequences, the s protein shows two faces: an amino-terminal half with hardly any sequence similarities and a carboxy-terminal half in which regions with significant conservation can be observed (de groot et al., 1987a,b ; for a review see cavanagh, 1995) , consistent with the distinctive functions of these domains (see later). the s protein is synthesized as a heavily glycosylated polypeptide as demonstrated by the susceptibility of the glycans to endoglycosidases and by the dramatic effect of the n-glycosylation inhibitor tunicamycin. the number of potential n-glycosylation sites ranges from 21 (mhv) to 35 [feline infectious peritonitis virus (fipv)]. the s protein has not been reported to contain o-linked sugars. cotranslational n-glycosylation is an essential requirement for proper folding, oligomerization, and transport of the s protein, as has also been shown for other (viral) glycoproteins (doms et al., 1993) . growth of coronaviruses in the presence of tunicamycin resulted in the production of spikeless, noninfectious particles mounir and talbot, 1992; rottier et al., 1981a; stern and sefton, 1982) . these particles were devoid of s protein, which was found to aggregate in the endoplasmic reticulum when glycosylation was inhibited (delmas and laude, 1990) . folding of the s protein is a relatively slow process. besides the addition of oligosaccharides it involves the formation and rearrangement of many intramolecular disulfide bonds. for the s protein of mhv-a59, the lumenal domain of which contains 42 cysteine residues, the major conformational events appear to take about 20 min during which the protein passes through a continuous spectrum of folding intermediates (opstelten et al., 1993a) . folding of s is probably the rate-limiting step in the process of oligomerization. sufficiently folded s protein monomers associate in the endoplasmic reticulum to form trimers (delmas and laude, 1990; lin et al., 2004) , with a half-time of approximately 1 h (delmas and laude, 1990; vennema et al., 1990a,b) . trimerization is likely to be required for export out of the endoplasmic reticulum. in infected cells s protein trimers interact with m protein and perhaps also with e protein, and migrate to the virus assembly site. a fraction of the s protein is transported to the plasma membrane where it can cause cell-cell fusion, a feature formally attributed to the s protein by its individual expression in cells (de groot et al., 1989; pfleiderer et al., 1990) . under such expression conditions the bulk of the s protein remains intracellularly (vennema et al., 1990a) in the endoplasmic reticulum . retrieval signals have been identified in the cytoplasmic tail of the s proteins from coronavirus groups 1 and 3 as well as in the tail of the sars-cov s protein, but not in the group 1 mhv s protein (lontok et al., 2004) . during its transport to the cell surface, either alone or as part of virions, the s protein undergoes further modifications. the n-linked sugars are modified and become mature during passage through the golgi complex. the mhv s protein was shown to become palmitoylated, a modification that may already take place in the endoplasmic reticulum (van berlo et al., 1987) . as a late step the s protein can be cleaved. a basic amino acid sequence resembling the furin consensus sequence motif (rxr/kr) occurs approximately in the middle of the protein and was shown to be the target of a furin-like enzyme in the case of mhv-a59 (de haan et al., 2004) . cleavage has been demonstrated for s proteins from coronavirus groups 2 and 3, but not for s proteins from group 1 viruses (cavanagh, 1995) or from sars-cov (bisht et al., 2004) . the resulting amino-terminal s1 subunit and the membrane-anchored s2 subunit remain noncovalently linked. it has been suggested that the s1 subunit constitutes the globular head, whereas the s2 subunit forms the stalk-like region of the spike (cavanagh, 1983b; de groot et al., 1987a,b) . the coronavirus s protein has two functions, which appear to be spatially separated. the s1 subunit (or the equivalent part in viruses with uncleaved s protein) is responsible for receptor binding, and the s2 subunit is responsible for membrane fusion. for several coronaviruses the receptor-binding site in s1 has been mapped. for mhv strain jhm (mhv-jhm), for instance, it was located in the domain composed of the amino-terminal 330 residues of the s molecule (kubo et al., 1994) , residues 62-65 and 214-216 being particularly important (saeki et al., 1997; suzuki and taguchi, 1996) . this amino-terminal domain also determined ceacam1 receptor specificity of various mhv strains (tsai et al., 2003) . for tgev (godet et al., 1994) , hcov-229e breslin et al., 2003) , and sars-cov (babcock et al., 2004; wong et al., 2004 ) the receptor-binding domains have also been mapped to the s1 subunit, although in different regions. in several cases neutralizing antibodies were demonstrated to bind the receptor-binding domains and to prevent the interaction with the receptor (godet et al., 1994; kubo et al., 1994; sui et al., 2004) . the interaction between the s protein and its receptor is the major determinant for virus entry and host range restriction. nonpermissive cell lines can be rendered susceptible by making them express the receptor (see later references). coronaviruses can also be retargeted to specific cells by exchanging the ectodomain of the s protein for that of an appropriate other coronavirus, as was demonstrated for mhv (kuo et al., 2000) and fipv (haijema et al., 2003) . receptors have so far been identified for the group 2 coronavirus mhv (ceacam; dveksler et al., 1991 dveksler et al., , 1993 williams et al., 1991) ; the group 1 coronaviruses tgev and porcine respiratory coronavirus (prcov) (papn; delmas et al., 1992 t resnan et al., 1996) , and hcov-229e (hapn; yeager et al., 1992) ; and for sars-cov (ace2; li et al., 2003) . the s proteins of the group 2 coronaviruses have been observed to exhibit hemagglutinating activities. although for bovine coronavirus (bcov), hcov-oc43, and hemagglutinating encephalomyelitis virus (hev) 9-o-acetylated sialic acids were identified as a receptor determinant (krempl et al., 1995; kunkel and herrler, 1993; schultze and herrler, 1992; schultze et al., 1991a; vlasak et al., 1988b) , specific receptors for these viruses have not been identified. also, the mhv s protein appears to bind sialic acid derivatives in addition to its specific receptor ceacam, which may suggest that sialic acids function as an additional receptor determinant for mhv-like coronaviruses (wurzer et al., 2002) . the ectodomain of the s2 subunit, which is involved in the fusion process, contains two heptad repeat (hr) regions (de groot et al., 1987a,b) , a sequence motif characteristic of coiled coils. mutations in the first (i.e., membrane-distal) hr region of the mhv s protein resulted in fusion-negative phenotypes (luo and weiss, 1998) or in a low-ph dependence for fusion (gallagher et al., 1991) , whereas mutations in the second hr region caused defects in s protein oligomerization and fusion ability (luo et al., 1999) . a fusion peptide has not yet been identified in any of the coronavirus spike proteins, but is predicted to be located at (bosch et al., 2004b; chambers et al., 1990) or within (luo and weiss, 1998) the amino terminus of the first hr region. binding of the s1 subunit to the (soluble) receptor, or exposure to 37 c and an elevated ph, has been shown to trigger conformational changes that are supposed to facilitate virus entry by activation of the fusion function of the s2 subunit (breslin et al., 2003; gallagher, 1997; lewicki and gallagher, 2002; matsuyama and taguchi, 2002; miura et al., 2004; sturman et al., 1990; taguchi and matsuyama, 2002; zelus et al., 2003) . this conformational change is thought to lead to exposure of the fusion peptide and its interaction with the target membrane, further changes resulting in the formation of a heterotrimeric six-helix bundle, characteristic of class i viral fusion proteins, during the membrane fusion process. indeed, peptides corresponding to the hr regions of mhv (bosch et al., 2003; xu et al., 2004) and sars-cov (bosch et al., 2004b; ingallinella et al., 2004; liu et al., 2004; tripet et al., 2004; zhu et al., 2004) were found to assemble into stable oligomeric complexes in an antiparallel manner, which in the natural situation would result in the close colocation of the fusion peptide and the transmembrane domain. these peptides were further shown to be inhibitors for viral entry (bosch et al., 2003 (bosch et al., , 2004b liu et al., 2004; yuan et al., 2004; zhu et al., 2004) . besides the hr regions, other parts of the s protein are also likely to be important for the fusion process. all coronavirus s proteins contain a highly conserved region (de groot et al., 1987a) , rich in aromatic residues, downstream of the second hr region, part of which may form the start of the transmembrane domain. the function of this domain is unknown, but a similar region in the hiv-1 env protein was demonstrated to be important for viral fusion and env incorporation into virions (salzwedel et al., 1999) . immediately downstream of the transmembrane domain all s proteins contain a cysteine-rich region (de groot et al., 1987a) . using a mutational approach including deletions, insertions, and substitutions, both the transmembrane domain and the cysteine-rich region immediately downstream thereof, but not the carboxy-terminal part of the cytoplasmic tail, were shown to be important for mhv s protein-induced cell-cell fusion (bos et al., 1995; chang and gombold, 2001; chang et al., 2000) the cleavage requirements of the s proteins for the biological activities of the coronavirus spike remain enigmatic. whereas the s proteins of group 1 coronaviruses, such as fipv (vennema et al., 1990a) , are not cleaved, those of other coronaviruses, particularly of groups 2 and 3, are cleaved to variable extents, depending on the viral strain and the cell type in which the viruses are grown (frana et al., 1985; reviewed by cavanagh, 1995) . cleavage of the s proteins is not required to expose the internal fusion peptide. whereas cleavage of the mhv s protein generally correlates strongly with cell-cell fusion (cavanagh, 1995) , virus-cell fusion appeared not to be affected by preventing s protein cleavage, indicating that these fusion events have different requirements (de haan et al., 2004) . similarly, whereas trypsin activation of sars-cov s protein was required for cell-cell fusion, it did not enhance the infectivity of cell-free pseudovirions (simmons et al., 2004) . for mhv-4, the spikes of which are able to initiate fusion without prior interaction with the primary mhv receptor (gallagher et al., 1992) , the stability of the s1-s2 heterodimers after s protein cleavage is low, allowing receptor-independent fusion. during cell culture adaptation, however, selected mutant viruses carried deletions in the s1 subunit, downstream of the receptor-binding domain, which resulted in stabilized s1-s2 heterodimers and receptor-dependent fusion activity (krueger et al., 2001) . virions of group 2 coronaviruses generally contain a fringe of shorter surface projections in addition to the characteristic spikes (bridger et al., 1978; king et al., 1985; sugiyama and amano, 1981) . these viruses express and incorporate into their particles an additional membrane protein, he (for a review see brian et al., 1995) . although all group 2 viruses contain an he gene, the protein is not expressed by all mhv strains (luytjes et al., 1988; yokomori et al., 1991) , indicating that he is a nonessential protein also in these viruses. the he gene encodes a type i membrane protein of 424-439 residues that contains a cleavable signal peptide at its amino terminus (hogue et al., 1989; kienzle et al., 1990 ) and a transmembrane domain close to its carboxy terminus, leaving a short cytoplasmic tail of about 10 residues (fig. 4) . the ectodomain contains 8-10 putative n-linked glycosylation sites. the putative esterase active site (fgds) is located near the (signal-cleaved) he amino terminus. the coronavirus he protein has 30% amino acid identity with the he-1 subunit of the he fusion protein of influenza c virus and the he protein of torovirus (cornelissen et al., 1997) . it has been suggested that coronaviruses have captured their he module from influenza c virus or a related virus (luytjes et al., 1988) . however, influenza c virus, toroviruses, and coronaviruses may well have acquired their he sequences independently, not from each other but from yet another source (cornelissen et al., 1997) . the he protein becomes cotranslationally n-glycosylated when expressed in cells, giving rise to a polypeptide of approximately 60-65 kda that rapidly forms disulfide-linked dimers (hogue et al., 1989; kienzle et al., 1990; king et al., 1985; parker et al., 1989; yokomori et al., 1989; yoo et al., 1992) . the he dimers (or a higher order structure thereof) become incorporated into virions, while a proportion is transported to the cell surface (kienzle et al., 1990; pfleiderer et al., 1991) . little is still known about the function(s) of the coronavirus he protein. the protein contains hemagglutinin and acetyl esterase activities (brian et al., 1995) . while the he proteins of bcov, hev, and hcov-oc43 hydrolyze the 9-o-acetyl group of sialic acid and therefore appear to function as receptor-destroying enzymes (schultze et al., 1991b; vlasak et al., 1988a) , the he proteins of mhvlike coronaviruses function as sialate-4-o-acetylesterases (klausegger et al., 1999; regl et al., 1999; wurzer et al., 2002) . although inhibition of the esterase activity of bcov resulted in a 100-to 400-fold reduction in viral infectivity (vlasak et al., 1988a) , it was shown both for bcov and for an mhv strain expressing an he gene that the s protein is required and sufficient for infection (gagneten et al., 1995; popova and zhang, 2002) . in view of these results it has been proposed that the he protein might play a role at an even earlier step and may mediate viral adherence to the intestinal wall through the specific yet reversible binding to mucopolysaccharides. the process of binding to sialic acid receptors followed by cleavage and rebinding to intact receptors could theoretically result in virus motility and even allow migration through the mucus layer covering the epithelial target cells in the respiratory and enteric tracts (cornelissen et al., 1997) . several studies have indicated the he protein to play a role in pathogenicity. the he protein of bcov (deregt and babiuk, 1987) , but not that of mhv, was able to induce neutralizing antibodies. however, passive immunization of mice with nonneutralizing, mhv he-specific antibodies protected the animals against a lethal mhv infection (yokomori et al., 1992) . furthermore, intracerebral expression of the he protein in mice was found to affect the neuropathogenicity of mhv (yokomori et al., 1995; zhang et al., 1998) . strikingly, he protein-defective mhv mutants were rapidly selected during viral infection in the mouse brain (yokomori et al., 1993) , which may suggest that the he protein plays a more critical role during the infection of other tissues. early electron microscopic studies demonstrated that coronavirus morphogenesis takes place at intracellular membranes and identified the cisternae of the endoplasmic reticulum as the site of budding of ibv and hcov-229e (becker et al., 1967; chasey and alexander, 1976; hamre et al., 1967; oshiro et al., 1971) . later studies revealed that early in infection particle formation occurs predominantly at smoothwalled, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and the golgi complex (ergic). this so-called intermediate compartment was shown to be used as the early budding compartment by mhv, ibv, fipv, tgev, and sars-cov (goldsmith et al., 2004; klumperman et al., 1994; tooze et al., 1984) . at later times during infection the rough endoplasmic reticulum was seen to gradually become the major site of mhv budding in fibroblasts (tooze et al., 1984) . as already mentioned, ultrastructural studies localized the mhv and ibv m proteins in the budding compartment(s) but also in the golgi complex, that is, beyond the site of budding tooze et al., 1984) . apparently, accumulation of m protein alone is not sufficient to determine the site of budding; other viral and/or cellular factors are required as well. for mhv the e protein, which was found to localize to the intermediate compartment by immunoelectron microscopy (immuno-em), was suggested to be such a candidate , but more players are likely to be involved. whether the helical nucleocapsids, visible as electron-dense cytoplasmic elements adjacent to budding profiles (david-ferreira and manaker, 1965; dubois-dalcq et al., 1982; massalski et al., 1982; risco et al., 1998 ; and references given previously), are a determining factor is unclear. in this respect, knowledge about the budding location of coronavirus-like particles (see later) might be informative. coronavirions are subject to an intracellular postbudding maturation process that occurs while they are on their way through the constitutive exocytic pathway by which they are exported out of the cell (risco et al., 1998; salanueva et al., 1999; tooze et al., 1987) . indications of this had already been noticed in early morphological studies with hcov-229e (becker et al., 1967; chasey and alexander, 1976; hamre et al., 1967; oshiro et al., 1971) and mhv (holmes and behnke, 1981; , but were described in somewhat more detail for mhv by tooze and coworkers (1987) . the pictures show "immature" virions in pre-golgi compartments and golgi cisternae that appear as spherical structures with the ribonucleoprotein core immediately below the viral envelope and with an "empty" center. by contrast, virions in the trans-golgi network and beyond have the mature morphology showing a fairly uniform, high internal electron density. an extensive analysis of the structural maturation of coronavirions was reported for tgev (risco et al., 1998; salanueva et al., 1999) . budding was shown to yield relatively large virions with an annular, electron-dense internal periphery and a clear central area. smaller particles, with the characteristic morphology of extracellular virions, that is, having a compact, dense inner core with polygonal contours, were seen to accumulate in secretory vesicles in the periphery of the infected cell. both types of particles appeared to coexist in the golgi complex (fig. 5) . obviously, the larger particles are the precursors of the smaller mature virions (salanueva et al., 1999) and probably undergo their morphological maturation during their transport through the golgi complex. the reorganization of the particle gives rise to the supposedly icosahedral core shell and is accompanied by a dramatic, approximately 50% reduction of the particle volume. it is presently unknown what triggers the morphological reorganization in the golgi complex. application of drugs affecting the state of the organelle did not give clues. as virions encounter an increasingly acidic ph on passage through the golgi stack, studies addressing this parameter were done with lysosomotropic agents. thus, chloroquine and nh 4 cl were applied to mhv-infected cells to elevate the ph at the trans side of the golgi complex, but no effect on the maturation of mhv was observed (tooze et al., 1987) . monensin, a drug that reversibly disorganizes the golgi complex and blocks transport along the exocytic pathway, led to the accumulation of the large, annular tgev virions; after reversal of the blockade formation of the small, compact particles was again restored (salanueva et al., 1999) . these observations confirm that the golgi complex is necessary for tgev structural transformation. nocodazole treatment of cells causes a reversible fragmentation of the golgi complex. under these conditions tgev virions were still able to undergo normal structural maturation. in contrast, still another golgi-disrupting compound, brefeldin a, prevented their maturation (risco et al., 1998) . this compound leads (risco et al., 1998) . (b) for a direct comparison of size and morphology a small, dense particle and a large particle are shown (salanueva et al., 1999) . pictures were kindly provided by c. risco. to a redistribution of golgi membranes to the er, leaving no definable golgi system. interestingly, mhv particles accumulated in infected cells under these conditions appeared to be infectious when liberated by sonication (j. meertens and p. j. m. rottier, unpublished results), leaving us with an intriguing question about the function of the maturation process. a. nucleocapsid assembly helical nucleocapsids are assembled in the cytoplasm of coronavirus-infected cells. they have been recognized by their tubular appearance in electron microscopy studies with several viruses including ibv, hcov-229e, tgev, and mhv (becker et al., 1967; chasey and alexander, 1976; david-ferreira and manaker, 1965; dubois-dalcq et al., 1982; hamre et al., 1967; massalski et al., 1982; oshiro et al., 1971; risco et al., 1998) . large inclusions of nucleocapsids were seen to accumulate late in the infection of cells with hcov (caul and egglestone, 1977) and mhv-jhm (duboisdalcq et al., 1982) . the structure of the nucleocapsid as it occurs in infected cells has not been studied in any detail. ribonucleoprotein particles supposed to represent nucleocapsids have been isolated from mhv-infected cells and were shown to consist of genomic rna and n protein (perlman et al., 1986; robb and bond, 1979; spaan et al., 1981) . the particles sedimented as edta-resistant structures of 200-230s in sucrose gradients. during the active phase of viral replication the majority (90%) of the intracellular genome-size rna was found in these structures (spaan et al., 1981) . ultrastructural studies of nucleocapsids derived from purified virion preparations have shown quite a variety of helical structures, depending on the virus and the experimental conditions used. the overall feature, however, was that of a thread-like coil, sometimes appearing to be hollow, with a diameter varying between 9 and 16 nm and a length ranging from 0.32 mm up to 6 mm (caul et al., 1979; davies et al., 1981; kennedy and johnson-lussenburg, 1975; macnaughton and davies, 1978) biochemical analysis of nucleocapsids prepared by detergent disruption of purified coronaviruses revealed the presence of genomic rna and n protein. interestingly, however, particles obtained by treatment of virions with nonidet p-40 appeared to be spherical when viewed under the electron microscope and, in addition, to contain m protein, as was observed with tgev (garwes et al., 1976) , hev (pocock and garwes, 1977) , and mhv-jhm (wege et al., 1979) . the presence of the m protein was found for mhv-a59 to depend on the preparation conditions. whereas the protein was absent when the virions had been disrupted with nonidet p-40 at 4 c, solubilization at 37 c resulted in copurification of m protein with the nucleocapsid (sturman et al., 1980) . the higher temperature was found to cause a conformational change in the m protein, leading to its aggregation and association with the viral rna in the nucleocapsid. similarly, nucleocapsid structures essentially lacking the m protein were also reported for ibv when virions were treated with detergent at low temperature (davies et al., 1981) . although there is no direct evidence yet, it seems reasonable to assume that the helical nucleocapsids seen accumulating in the cytosol of infected cells constitute the reservoir that feeds into the viral budding system. the location where these nucleocapsids are assembled has not been defined. their production may take place either free in the cytoplasm, where the n protein is synthesized, or, alternatively, in association with the membrane-bound structures where genomic rna is produced. the observed colocalization of n protein with the replication complexes (bost et al., 2000 (bost et al., , 2001 denison et al., 1999; van der meer et al., 1999) is consistent with the latter possibility. coronavirus replication appears to occur on double-membrane vesicles (gosert et al., 2002) , which utilize components of the cellular autophagy pathway (prentice et al., 2004) . whereas early in infection the replication complexes were shown to be almost entirely discrete from sites of m protein accumulation, at later times of infection helicase and n proteins appeared to colocalize with the m protein (bost et al., 2000 (bost et al., , 2001 . it was proposed that the translocation of helicase-n protein complexes to sites of virus assembly may serve as a mechanism to deliver the newly synthesized rna and nucleocapsids and to facilitate the retention of the m protein in the intermediate compartment. encapsidation of genomic rna into a nucleocapsid is presumably initiated by an interaction of the n protein with a specific nucleotide sequence, the packaging signal, which is subsequently followed by the polymerization of n proteins around the rna molecule in a nonsequence-specific manner. the selective incorporation of genomic rna into virions would predict the packaging signal to be located in sequences unique to this rna, that is, within the approximately 20-kb region comprising the 5 0 utr and open reading frame 1 (orf1) with the exception of the leader sequence. although the data obtained so far support this prediction, no consistent picture has emerged yet. the approach generally used to map the packaging signal involved the study of helper virus-assisted encapsidation of natural and artificially obtained defective rna genomes. thus, a 650-nucleotide region located at the 3 0 end of the pol1b gene was initially identified for mhv (van der most et al., 1991) , which was subsequently narrowed to an area of 190 nucleotides (fosmire et al., 1992) . within this area a stable stem-loop of 69 nucleotides was predicted. mutation studies revealed that the integrity of this secondary structure was important and that the sequence of the packaging signal could be trimmed further to a minimum stretch of 61 nucleotides (fosmire et al., 1992) . the signal appeared to be sufficient for rna packaging as its inclusion allowed a synthetic subgenomic mrna of mhv-a59 to be packaged specifically; the encapsidation efficiency of the mrna was, however, significantly lower than that of the defective genomic rna from which it was transcribed . even a nonviral rna was found to be packaged into mhv particles when provided with the packaging signal (woo et al., 1997) . buoyant density analysis of the particles revealed that the rna was not assembled separately but copackaged with helper virus rna. studies of the corresponding pol1b region of another group 2 coronavirus, bcov, indicated that within this group the packaging signal is structurally and functionally conserved. a 69-nucleotide sequence with significant homology (74%) to that of mhv was identified within a cloned 291-nucleotide segment sharing 72% homology overall . when this segment was fused to a noncoronavirus reporter gene sequence, the resulting rna appeared to be packaged not only by the homologous helper virus bcov but also by mhv. conversely, when the mhv packaging signal was fused to the reporter gene sequence, the rna was found to be encapsidated also in the context of a bcov-infected cell . mapping studies of packaging signals in the genomes of group 1 and group 3 coronaviruses have yielded quite different results. for ibv, deletion mutagenesis of a defective rna led to the conclusion that only the sequences in the 5 0 utr and/or a region of the 3 0 utr were specifically required for packaging, although parts of the pol1b sequence, but not any part in particular, also enhanced the efficiency (dalton et al., 2001) . somewhat similar conclusions could be drawn from a study with tgev (izeta et al., 1999) . by comparing packaging efficiencies of different defective genomes it was inferred that information for packaging was present both at the genomic 5 0 end (about 1.0 kb) and in parts of pol1b. a packaging signal was subsequently mapped to a fragment representing the 5 0 terminal 649 nucleotides of the genome by inserting a series of overlapping segments covering a stretch of about 2300 nucleotides from the 5 0 end of the genome into an mrna reporter expression construct contained within a defective genome (escors et al., 2003) ; only the 5 0 terminal sequence conferred to the mrna the ability to become packaged by helper virus. it is too early to conclude that the apparently contrasting results reflect true, fundamental differences in encapsidation strategies between the different (groups of ) coronaviruses. although the overview may suggest the existence of different cis-acting signals, the data still allow a scenario in which multiple domains in the genome are involved cooperatively, each one contributing differently to (the efficiency of) the encapsidation process. such contributions would not necessarily concern n protein binding only; the exceptional complexity of the coronaviral genome might call for additional provisions, related perhaps to the structuring of the encapsidation complex. several observations indeed imply the involvement of multiple domains. the efficient rescue, for instance, of a bcov defective genome (drep) that completely lacks the putative 69-nucleotide packaging signal entails the participation of additional sequence(s) (chang and brian, 1996; . another example is the strongly increased rescue of an otherwise poorly packaged defective tgev genome (m22) due to the presence of about 4.1 kb of sequences derived from the pol1b gene (m62) (izeta et al., 1999) . there is no direct evidence yet for the actual functioning of the presumed packaging signals in the initiation of nucleocapsid assembly. binding of n protein to these signals, the first step in the process, has so far been addressed only for the 69-nucleotide sequence of mhv. specific binding to rna transcripts containing this sequence was indeed demonstrated biochemically with mhv n protein derived from infected cells, from virions, and from cells expressing the protein (molenkamp and spaan, 1997) . the binding efficiency, however, appeared to be relatively weak as was shown by comparing n protein binding to different parts of a packageable defective genome (midi-c) . the highest binding efficiency was observed with an rna transcript representing about 1 kb from the 5 0 end of pol1a. remarkably, not even removal of the packaging signal from midi-c rna affected binding to the n protein as measured in the filter-binding assay used. the observations indicate that the domain containing the 69-nucleotide sequence does not function as a packaging signal in the conventional sense, adding further support to the notion that the intricacies of coronaviral nucleocapsid assembly are complex. apart from the studies mentioned, the occurrence of n-rna interactions has been amply documented. this is not surprising as the n protein has been implicated in several other processes that involve interaction with rna, such as replication, transcription, and translation (lai and cavanagh, 1997 ; see also section ii.c). as the relevance of these interactions for viral assembly is generally unclear, a brief survey of the available information is included here. in addition, an overview of data on the mapping of rna interactions on the n polypeptide is schematically presented in fig. 6 . a high-affinity interaction between the n protein and the 5 0 leader was demonstrated for mhv-a59 . using an rna overlay protein blot assay and various in vitro rna transcripts, the binding of n protein was localized to a stretch of nucleotides (nucleotides 56-65) at the 3 0 end of the leader . the stretch included the pentanucleotide repeat ucuaa now known to be critical for transcription. biochemical analyses of the interaction measured a dissociation constant (k d ) of 14 nm for bacterially expressed mhv n protein to the leader rna (nelson et al., 2000) . consistent with the presence of a leader at the 5 0 end of all viral rnas, an n protein-specific monoclonal antibody coimmunoprecipitated genomic rna as well as the subgenomic rnas from mhv-infected cells . similar observations were made for bcov . packaging of subgenomic rnas has been reported for tgev (sethna et al., 1991) , bcov (hofmann et al., 1990) , and ibv (zhao et al., 1993) but not for mhv, suggesting that their incorporation is not mediated by n protein-leader interaction. whereas for tgev and ibv the relative packaging of subgenomic rnas was found to be inefficient, genomic rna appearing in virions at a more than 10-fold molar excess over any subgenomic rna species, the bcov subgenomic n and m mrnas appeared to be packaged as abundantly as the genome (hofmann et al., 1990) . a reevaluation for tgev revealed that the detection of subgenomic rnas in virions was related to the purity of virus preparations, indicating that mrnas were not specifically encapsidated (escors et al., 2003) . besides the leader, the n protein has been found to bind to other parts of the coronaviral genome. in addition to the binding site in the mhv fig 6. structural organization of the coronavirus n protein. common features and their distribution along the polypeptide chain are shown schematically. the hatched box indicates the most conserved part of the n protein, with a high proportion of aromatic residues. the n protein contains many basic residues throughout the polypeptide, but with particular clustering in two regions (ã¾ã¾ã¾). the upstream cluster contains a serine/arginine-rich region (s/r). the carboxy terminus, which contains a high proportion of acidic residues, is also indicated (ã�ã�ã�). the bars indicate parts of the n protein that have been implicated in n-n and n-rna interaction. furthermore, the location of the deletion in the mhv-a59 mutant alb4 (d) is indicated as well as the domain where second-site mutations in revertant viruses of alb4 are mapped. finally, the parts of the n protein that could not be transferred from bcv into the mhv genome are marked. references are included in the figure. pol1a gene mentioned previously, a high-efficiency binding site was identified by the same authors in the 3 0 half of the n gene of both mhv and bcov . the ibv n protein was shown to bind sequences in the 3 0 terminal utr of the genome (zhou et al., 1996) . coronavirus n proteins do not contain sequence motifs typically found in other rna-binding proteins. they appear to bind rna both nonspecifically (masters, 1992; robbins et al., 1986) and in a sequencespecific way nelson and stohlman, 1993; nelson et al., 2000; stohlman et al., 1992) . non-sequence-specific rna binding has been mapped to a large central domain of the mhv n molecule (fig. 6) (masters, 1992) . also, the leader-binding property was assigned to this domain; this activity was initially mapped to the area containing the two highly basic regions (nelson and stohlman, 1993) , but was later narrowed to a 55-residue segment containing the serine/ arginine-rich basic region (nelson et al., 2000) . interestingly, this particular region could not be interchanged with its bcov counterpart in a study on the functional equivalence of the n proteins from these related viruses (peng et al., 1995b) . another domain in the mhv n protein implicated in viral rna binding was mapped to an area that partly overlaps with the second basic region. the assignment was based on an analysis of second-site revertants of mhv mutant alb4, the virions of which are extremely thermolabile because of a 29-residue deletion located between the central and carboxy-terminal domain of the n protein (koetzner et al., 1992) . the reverting mutations correlated with restoration of the disturbed rna-binding capacity of the mhv n protein and were found clustered close to the basic region some 80 residues on the amino side of the deletion (fig. 6) (peng et al., 1995a) . although all these studies consistently attribute a major role in rna binding to the central portion of the coronaviral n protein, the interaction of the ibv n protein with the 3 0 utr of ibv rna mentioned previously was mapped to the amino-and carboxy-terminal domains of the molecule (zhou and collisson, 2000) . 3 0 utr rna-binding activity was also assigned to the amino-terminal domain of the sars-cov n protein on the basis of studies using nuclear magnetic resonance spectroscopy (huang et al., 2004) . it is obvious that the wrapping of the 30-kb coronaviral genome into the compact helical nucleocapsid is largely driven by n protein interactions. as there are no indications for packaging of the rna into a preformed capsid, these interactions can be described by the following model. packaging is initiated by binding of the n protein, either as a monomer or in a multimeric form, to the rna. by analogy to other rna viruses, this sequence-specific interaction may induce a conformational change in the n protein, thereby creating a nucleation site for the cooperative stacking of n protein units along the entire length of the rna, now in a non-sequence-specific way. these n units can again be monomeric or consist of defined multimers. finally, helix formation is driven by interactions between n molecules separated along the ribonucleoprotein chain but that become adjacent in neighboring helices. this model predicts multiple nonequivalent interactions between n molecules. n-n interactions have been experimentally demonstrated for mhv, bcov, and hcov-oc43. high molecular weight species of the n protein, possibly trimers, were detected by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) of virion preparations under nonreducing (but not under reducing) conditions, which is indicative of intermolecular disulfide bonds between the n subunits (hogue et al., 1984; narayanan et al., 2003b; robbins et al., 1986) . these complexes are likely to be additionally stabilized by noncovalent interactions as coronavirus n protein cysteines are not well conserved, the sars-cov n protein even lacking any cysteine residues. both monomeric and oligomeric n species were able to bind rna (robbins et al., 1986) . multimeric forms of the n protein were also found in association with intracellular genomic rna in mhv-infected cells as shown after the selective isolation of this ribonucleoprotein through coimmunoprecipitation with the m protein (narayanan et al., 2003b) . high molecular weight forms of the n protein corresponding to dimers and trimers were also demonstrated in vitro after ultraviolet (uv) cross-linking of bcov n protein to rnas . few studies have addressed the identification of the n-n interaction domains. the results so far are inconsistent (fig. 6 ), but this might as well reflect the predicted occurrence of nonequivalent interactions. interaction sites were mapped to the amino-terminal part of the mhv n protein (wang and zhang, 1999) . using an in vitro binding assay in which the full-length n protein was incubated with bacterially expressed fusion proteins containing different segments of the n protein, interaction was observed with a polypeptide derived from the amino-terminal one-third (residues 1-162) of the protein and with a polypeptide representing the central part (residues 163-292). the latter domain contains the serine/arginine-rich region implicated in the binding to the leader/trs-specific sequences (nelson et al., 2000) . this domain could not be replaced by the corresponding domain from bcov without loss of viral viability, from which the authors indeed inferred an involvement in protein-protein interactions (peng et al., 1995b) . the same domain was also shown to be essential for the homotypic association of the sars-cov n protein . using a mammalian two-hybrid approach, the n-n interaction appeared to be abolished completely when the serine/arginine-rich region had been deleted. however, in another study using the yeast two-hybrid system this interaction was not confirmed. a polypeptide consisting of the amino-terminal two-thirds of the sars-cov n protein, that contains the serine/arginine-rich region, exhibited no association with the full-length protein (surjit et al., 2004) . rather, self-association was attributed to the carboxy-terminal 209 residues of the molecule, which lacks the motif. unlike most other enveloped viruses, coronaviruses have the remarkable feature of being able to independently assemble their envelope. indications for this were already noticed in early electron microscopy studies of viral preparations and infected cells showing the occurrence of apparently "empty" particles (afzelius, 1994; chasey and alexander, 1976; macnaughton and davies, 1980) . incomplete virions with the typical coronavirus morphology but lacking the n protein and the genome could indeed be separated from normal ibv particles by their lower density in sucrose gradients (macnaughton and davies, 1980) . the definition of virus-like particles (vlps) and the requirements for their formation were established by the coexpression of the coronaviral structural proteins in mammalian cells (vennema et al., 1996) . membranous particles were assembled when the mhv envelope proteins m, e, and s were coexpressed, without the need for an n protein or genomic rna. the particles were released from the cells and, when examined under the electron microscope, appeared to be morphologically indistinguishable from authentic virions, that is, they had the characteristic shape and dimensions of normal virions. also, their membrane protein composition was similar to that of mhv, with a high abundance of m protein and only trace amounts of e protein. quite surprisingly, only the m and e proteins were required for particle assembly. both s and n proteins were dispensable for particle formation but, whereas the s protein became incorporated when present, this was not the case for the n protein (vennema et al., 1996) , except in combination with (defective) genomic rna, in which case a nucleocapsid was coassembled (bos et al., 1996; kim et al., 1997) . individual expression of the m or the e protein in cells did not give rise to formation of vlps although e protein synthesis by itself led to the secretion of e-containing vesicles (corse and machamer, 2000; maeda et al., 1999) . the nature of these particles has not been characterized in much detail; the vesicles induced by mhv e sedimented slightly slower than virions (maeda et al., 1999) whereas the particles obtained with ibv e had about the same density as virions (corse and machamer, 2000) . besides for mhv and ibv, vlps have so far been described for bcov (baudoux et al., 1998b) , fipv , and tgev (baudoux et al., 1998b) . the observations demonstrate the unique budding mechanism of coronaviruses, which is dependent solely on the envelope proteins m and e but independent of a nucleocapsid. somewhat similar observations have been described for the flavivirus tick-borne encephalitis virus and for hepatitis b virus, which also produce proteolipid particles on expression of their envelope proteins prem and e (allison et al., 1995; mason et al., 1991) and s (patzer et al., 1986; simon et al., 1988) , respectively, but these particles are much smaller than the corresponding virions. in contrast, particles with the typical, large size of coronaviruses are acquired by the concerted action of just the proteins m and e (baudoux et al., 1998b; vennema et al., 1996) . budding of enveloped viruses generally requires a nucleocapsid (for a review see garoff et al., 1998) . for retroviruses the gag protein, the precursor to the nucleocapsid, is all that is needed to obtain particles resembling immature virions; the env protein is dispensable. budding of alphaviruses, on the other hand, requires both the envelope proteins and the nucleocapsid. interestingly, the same appears to hold true for arteriviruses (r. wieringa, a. a. f. de vries, and p. j. m. rottier, unpublished observations), which are closely related to the coronaviruses, share with them a triple-spanning envelope protein, and bud into early membranes of the secretory pathway, like coronaviruses but unlike alphaviruses. it is unknown how the coronavirus m and e proteins cooperate in budding. as the extensive electron microscopy work with m proteins from various coronaviruses gave no indications that this protein causes membrane bulging by itself, it is believed that the function of the e protein in coronavirus budding is in the induction of curvature in the m protein lattice (see later) and the subsequent budding of the membrane (vennema et al., 1996) . by its low abundance in the virion, the e protein does not seem to serve a genuine structural function in that it occupies frequent, regular positions in the m protein framework. consistent with an important role of the e protein in particle morphogenesis, mutations in its hydrophilic carboxy-terminal part, introduced by targeted recombination into the mhv genome, yielded thermolabile viruses one of which showed aberrant virion morphology with pinched and elongated shapes when viewed in the electron microscope (fischer et al., 1998) . revertant analyses revealed that a single second-site amino acid change within the e protein was able to reverse the phenotypic effect of the original mutations, providing support for possible interactions between e protein monomers during budding (fischer et al., 1998) . unexpectedly, complete deletion of the e gene from the coronaviral genome does not abolish virion formation, demonstrating that the protein is not essential for budding. whereas this deletion dramatically (at least 1000-fold) reduced the release of infectivity from infected cells in the case of mhv-a59 , knockout of the tgev e gene resulted in a lethal phenotype (curtis et al., 2002; ortego et al., 2002) . remarkably, however, in the latter case virions still assembled but these appeared to be unable to leave the cells (j. ortego and l. enjuanes, personal communication). the vlp system offers a convenient assay to study many aspects of coronavirus envelope assembly. it was thus used to analyze the primary structure requirements of the m and e proteins for particle formation. for the m protein such studies demonstrated each of its different domains to be important. in general, mutations (deletions, insertions, and point mutations) in the lumenal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain of the mhv m protein strongly affected its ability to form vlps (de haan et al., 1998a) . the assembly process was particularly sensitive to changes in the carboxy terminus of the protein. truncation by only one residue reduced the efficiency severely whereas removal of two residues fully abolished particle formation. these effects appeared to be less severe in the context of a normal coronaviral infection, probably because additional interactions can compensate. the single-residue deletion, when introduced into the mhv genome, was without measurable phenotype and also a mutant virus with a truncation of two residues could be obtained, although with difficulty, as it was severely affected in its growth (de haan et al., 1998a; kuo and masters, 2002) . the importance of the m protein cytoplasmic and transmembrane domains was confirmed by vlp studies in the ibv system; mutant proteins lacking portions of either of these domains were unable to support particle assembly (corse and machamer, 2003) . studies of the primary structure requirements of the e protein for vlp formation revealed that the sequence of its hydrophobic domain was not critical. the assembly capacity of the protein was maintained when its transmembrane region was partly or completely replaced by the corresponding domain of the vesicular stomatitis virus (vsv) g protein (corse and machamer, 2003) . however, when its small aminoterminal ectodomain was additionally replaced by the large vsv g protein counterpart, the chimeric protein became nonfunctional. deletion of the transmembrane, but not the amino-terminal, domain rendered the e protein essentially assembly incompetent (lim and liu, 2001) . deletions in the cytoplasmic carboxy-terminal half of the e protein mapped the cysteine-rich region as the most important part for vlp assembly (lim and liu, 2001) . although the interaction between m and e proteins is amply demonstrated by their interdependence for vlp formation, direct evidence for their interaction has actually been provided only for the ibv proteins (corse and machamer, 2003; lim and liu, 2001) . the two proteins could be cross-linked to each other in ibv-infected cells and in cells coexpressing the m and e genes (corse and machamer, 2003) . it appeared that the cytoplasmic tails of both proteins were required, suggesting they are involved in the interaction. in another study m-e interaction was demonstrated by a coimmunoprecipitation assay. also in this assay the cytoplasmic domain of the e protein, comprising the cysteine-rich region, was found to be important as its deletion affected m-e interaction to the greatest extent when compared with other deletion mutant e proteins (lim and liu, 2001 ). the results from both studies also showed that the ability of mutant e or m proteins to interact did not correlate with their assembly competence. apparently, other requirements such as homotypic e or m interactions or interactions with host cell components must be met. the specificity of the interaction between the m and e proteins during particle assembly was further demonstrated by the poorly successful attempts to generate chimeric vlps. no particles were observed when heterologous combinations of tgev and bcov m and e proteins were coexpressed (baudoux et al., 1998b) and the same was true for heterologous combinations of fipv and mhv m and e proteins (h. vennema and p. j. m. rottier, unpublished results) . in both studies chimeric m and e proteins were also tested, demonstrating that, except in one case, exchanges between corresponding domains rendered the proteins assembly incompetent. only when the tgev m protein amino-terminal ectodomain was replaced with that of bcov did the chimeric polypeptide support vlp formation in combination with the tgev e protein; vlp formations was also supported, but to different extents, with tgev/bcov chimeric e proteins and-poorly, however-with the bcov e protein (baudoux et al., 1998b) . the reciprocal m construct, the bcov m protein carrying the tgev ectodomain, was nonfunctional, even in combination with tgev e protein. consistently, replacement of the ectodomain with that of fipv m also abolished the productive partnership of mhv m protein with mhv e (de haan et al., 1999) . as the disproportionate amounts of m and e proteins in vlps already imply, homotypic interactions between m molecules must constitute the energetic basis underlying the formation of the coronaviral envelope. in mhv-based vlps generated by coexpression of m and e proteins, for instance, the sheer excess of m protein-the relative molar presence of e in the particles is less than 1%-is evidence for the strong interactive forces between m molecules. hence, envelope assembly is thought to be driven primarily by laterally interacting m molecules that form a two-dimensional lattice in intracellular membranes (opstelten et al., 1993b . large multimeric complexes of m protein have indeed been demonstrated biochemically after individual expression of the mhv protein in cells. when the association of the m molecules was maintained by the careful selection of cell lysis conditions, sucrose gradient analysis revealed the existence of large heterogeneous (up to about 40 molecules) complexes, which accumulated in the golgi compartment (locker et al., 1995) . somewhat smaller complexes were obtained when the cytoplasmic tail of the protein was removed; these complexes were no longer retained in the golgi apparatus but transported to the cell surface. apparently, the tail domain is not essential for the lateral interactions between m proteins, but it is critically required for budding (de haan et al., 1998a . similar higher order complexes of the m protein have also been demonstrated in mhv-infected cells as well as in mhv virions (opstelten et al., 1993b . further support for the existence of homotypic m protein interactions and additional insight into the domains involved in these interactions came from work with mutant m proteins that are unable to assemble into vlps. in these studies mhv m proteins with deletions in either the transmembrane regions, the amphipathic domain, or the extreme carboxy terminus or with substitutions of the lumenal domain were tested for their ability to associate with other m proteins and to be rescued into vlps formed by assembly-competent m proteins (de haan et al., 1998a . it appeared that the mutant proteins maintained these biological activities despite the often severe alterations; actually, the only mutant protein that had lost these abilities was one in which all three transmembrane domains had been replaced by a heterologous transmembrane domain. it was concluded that m protein molecules interact with each other through multiple contact sites, particularly at the transmembrane level. it was furthermore hypothesized that the full complement of interactions between the m molecules is required for efficient particle formation; possibly, all these interactions are required to provide the free energy to generate and stabilize the budding envelope. the failure of m protein mutants capable of associating with assembly-competent m protein to assemble into vlps by themselves (de haan et al., 1998a indicates that additional interactions with viral (e) and/or host proteins is required. in this respect it is of note that the ifn-inducing capacity of the m protein, demonstrated for tgev and bcov, also requires the presence of the e protein, which suggests that the induction of ifn is dependent on a specific, probably regularly organized structure of the m protein (baudoux et al., 1998a,b) . it is unclear how the presence of the e protein alters the m protein lattice to achieve this effect. coronavirus envelope assembly is not dependent on the s protein or the he protein. this is obvious from work with vlps as well as with viruses showing that bona fide particles were produced when these proteins were either simply absent or unavailable for assembly. availability can be compromised under conditions in which proper folding of the proteins is affected. inhibition of n-glycosylation by the drug tunicamycin, for instance, can lead to aggregation and retention of membrane proteins in the endoplasmic reticulum and has been shown to prevent the incorporation into virions of both the s protein mounir and talbot, 1992; rottier et al., 1981a; stern and sefton, 1982) and the he protein (mounir and talbot, 1992) . the same effect has been observed with temperature-sensitive mhv mutants carrying defects in their s gene, which, when grown at the restrictive temperature, gave rise to spikeless particles ricard et al., 1995) . both s and he proteins are assembled into the coronaviral envelope through interactions with the m protein. such interactions have been demonstrated for mhv and bcov m and s proteins and for bcov m and he proteins, in infected cells, in cells coexpressing the proteins, and in virions (nguyen and hogue, 1997; opstelten et al., 1995) . complexes of the proteins were shown by coimmunoprecipitation and cosedimentation analyses as well as by immunofluorescence studies in which the intracellular transport of s and he proteins to the plasma membrane was found to be inhibited by coexpressed m protein, the proteins being retained in the golgi apparatus, the natural residence of the m protein. the kinetics with which the proteins engage in heteromeric complex formation appeared to be different for the different proteins. this effect is due to their different rates of folding and oligomerization. for the s protein these rates are low, involving the formation of multiple intramolecular disulfide bonds and the addition of numerous oligosaccharide side chains (delmas and laude, 1990; opstelten et al., 1993a; vennema et al., 1990a,b) . in contrast, folding of the mhv and bcov m proteins is independent of disulfide bonds and glycosylation, as a result of which they are, for instance, swiftly transported out of the endoplasmic reticulum (opstelten et al., 1993a) . as a consequence, m molecules enter into m-s and m-he complexes immediately after their synthesis whereas for newly synthesized s and he molecules it took 15-30 min before they started to appear in these heterocomplexes (nguyen and hogue, 1997; opstelten et al., 1995) . the importance of folding as a major rate-limiting step was illustrated by the inability of the s protein to interact with m protein when its folding had been inhibited by in vivo reduction; only completely oxidized s molecules were association competent (opstelten et al., 1993a . whether the m protein interacts with s and he proteins while they are still in their monomeric form or only after their oligomerization remains to be elucidated. it is, however, clear that the proteins engage in interaction with each other in early compartments, most likely the endoplasmic reticulum, as judged from the oligosaccharide maturation states of freshly formed protein complexes (de haan et al., 1999; nguyen and hogue, 1997; opstelten et al., 1995) . only dimers of he were associated with he-m-s complexes that were observed in bcov-infected cells; because the appearance of he in these complexes correlated with the kinetics of he dimerization it was concluded that proper oligomerization is most likely a requirement for its association (nguyen and hogue, 1997) . interestingly, such heterotrimeric complexes were not observed on coexpression of the three proteins in cells. under these conditions only the heterodimeric m-s and m-he associations were detected. the structural domains of m and s proteins that are involved in the formation and stabilization of their complex have been identified. using the coimmunoprecipitation and colocalization assays referred to previously, the essential domains in the mhv m protein were mapped by a mutag enetic appro ach (de ha an et al ., 1999 ) . it appear ed tha t m-s complex formation was sensitive to changes in all membrane-associated parts of the m molecule. interactions between m and s proteins were found to occur at the level of the transmembrane domains and of the amphipathic domain, which is located on the cytoplasmic face of cellular membranes. in contrast, neither the lumenally exposed amino terminus nor the hydrophilic cytoplasmic tail of the m protein was required; even the deletion of these parts-known to abrogate the ability of the protein to form vlps-did not prevent association with the s protein. chimeric s proteins were used to show that the large ectodomains of the spikes are not involved in interaction with m proteins. such chimeric proteins were constructed from the mhv and fipv s proteins and consisted of the ecto-or lumenal domain from the one and the transmembrane plus endodomain from the other. these proteins, which seemed biologically fit as they were still fusion active, were initially tested in coexpression studies with the m and e proteins from either virus for their ability to be incorporated into vlps. they were found to assemble only into viral particles of the species from which their carboxy-terminal domain originated . the chimeric s genes were subsequently incorporated into the proper coronavirus genomic background, creating the chimeric viruses fmhv and mfipv, the spike ectodomains of which are from the feline and murine coronavirus, respectively; these studies provided the basis for the development of a novel targeted recombination system for reverse genetics of coronaviruses (haijema et al., 2003; kuo et al., 2000) . further fine mapping of the carboxy-terminal parts of the s protein involved in m-s protein interaction revealed the importance of the cytoplasmic tail. again using coimmunoprecipitation and vlp incorporation assays, it appeared that increasing truncations gradually abolished the association with the m protein (b. j. bosch, c. a. m. de haan, and p. j. m. rottier, unpublished results) . the significance of the tail domain was demonstrated most convincingly by showing the coimmunoprecipitation and vlp assembly of a chimeric vsv g protein the cytoplasmic tail of which had been replaced by that of mhv s. tail truncations were tolerated in the context of the coronavirus; recombinant mhvs were generated that lacked 12 or 25 (but not 35) residues from the s protein carboxy terminus, but their growth was impaired by about 10-and 10 4 -fold, respectively. also, tail extensions were tolerated, allowing the construction of a recombinant mhv with a spike protein extended at its carboxy terminus by the green fluorescent protein (gfp), yielding green fluorescent virions (bosch et al., 2004a) . the extension was, however, lost quite rapidly on serial passaging of the virus. molecular details of the interaction of m and he proteins and the requirements of he for incorporation into viral particles have not been described. one study reported that he protein mutants lacking part of their ectodomain were not assembled into particles (liao et al., 1995) . most likely, however, this observation was due to folding or maturation defects of the mutant proteins. in another study the bcov s and he proteins were shown to be incorporated into mhv particles when coexpressed in mhv-infected cells. apparently, homology between the proteins of these related group 2 coronaviruses is sufficiently high for heterologous m-s and m-he interactions to occur (popova and zhang, 2002) . numerous electron microscopy studies have pictured the process of virion assembly in the coronavirus-infected cell. they show the close apposition of-presumably preassembled-tubular nucleocapsids to intracellular membranes, the appearance of membrane curvature at the contact sites, the "growth" of these buds into particle-sized vesicles, and the ultimate detachment of virions from the membranes by pinching off. it has become clear that the m protein is the central player, which, through its interactions with every known component of the virion, orchestrates the entire assembly process (see fig. 7 ). in the process two levels of interaction can be distinguished. one is the level of the membrane where, as detailed in the previous section, the m protein interacts (1) with itself, to generate the basic molecular framework of the viral envelope, (2) with the e protein, to induce curving and budding of the m protein-modified membrane, and (3) with s and he, to coassemble these spikes into the viral envelope. the other level at which the m protein operates involves the incorporation of the nucleocapsid into the virion. here, two types of interactions have been described: interactions of the m protein with the n protein and with the viral genome. an instrumental role of the m protein in drawing the nucleocapsid into the budding particle is indicated by their demonstrated interaction in studies with virion preparations. the m protein has been shown to remain associated with subviral particles obtained after treatment of virions with detergent that removes the spikes (escors et al., 2001a,b; garwes et al., 1976; lancer and howard, 1980; wege et al., 1979) . the association was shown for mhv to be temperature dependent (sturman et al., 1980) . in the case of tgev the association of m with the spherical structure, termed the core, was stabilized by basic ph and divalent cations but lost at high salt concentration, resulting in disruption of the core structure and release of the helical nucleocapsid (escors et al., 2001a,b; risco et al., 1996) . in an elegant study of the binding of in vitro-translated m polypeptides to purified nucleocapsids the ionic interaction was mapped to a 16-residue sequence in the hydrophilic carboxy-terminal tail domain (escors et al., 2001b) . also in infected cells, interaction of the m protein with ribonucleoprotein structures, presumably nucleocapsids, has been demonstrated. using m-specific antibodies, structures containing both n protein and genomic rna were coimmunoprecipitated with m protein from mhv-infected cell lysates (narayanan et al., 2000) . conversely, m protein was coprecipitated when an n-specific antibody was used, while in this case all viral mrnas copurified because of their known leader-mediated affinity for the n protein. these interactions did not require an s or an e protein (narayanan et al., 2000) . although interactions between the m and n proteins might intuitively be expected to drive the process of attachment of the nucleocapsid to the intracellular target membrane, direct experimental evidence for this interaction is strikingly lacking. significantly, mhv m and n proteins coexpressed in cells were found not to interact (narayanan et al., 2000) nor did purified tgev n protein interact with in vitrosynthesized m protein (escors et al., 2001b) . because the n protein occurs in coronavirus-infected cells in various configurations-as a free protein and in association with an array of partners including the viral genome-it is obvious that a selection mechanism must act to ensure that only nucleocapsids are assembled into particles. consistently, coexpressed n protein is not incorporated into vlps, but its inclusion depends on the presence of viral rna (bos et al., 1996; vennema et al., 1996) . thus, unless the selection process is performed by a mechanism not involving the n protein, its association with genomic rna to form a nucleocapsid seems required to generate the unique conformation that enables it to interact with the m protein. the only evidence to date for an interaction between m and n proteins is indirect and comes from genetic studies. analysis of second-site revertants of a constructed mhv-a59 mutant virus lacking the two carboxyterminal residues of its m protein revealed that the highly defective growth phenotype of this virus could be restored, among others, by mutations in the carboxy-terminal domain of the n protein (kuo and masters, 2002) . in two independently obtained revertants the n protein had lost 15 residues of this-among different strains of mhv highly conserved-domain because of a frameshifting 10-nucleotide deletion. the results argue strongly for a direct cooperation of the carboxy-terminal regions of the m and n proteins during virion formation. other indications supporting the occurrence of m-n interactions come from studies of complexes of m protein with ribonucleoprotein from mhv-infected cells and with tgev cores (escors et al., 2001b; narayanan et al., 2000) . when such complexes were treated with rnase the association of m and n proteins was not destroyed, suggesting a direct interaction. however, the presence of short rnas inaccessible to the rnase but sufficient to bridge the m-n interaction could not be excluded. the most unusual interaction that the coronaviral m protein seems to engage in involves genomic rna. this interaction has so far been reported only for mhv, by makino and co-workers. these workers had shown earlier that the 69-nucleotide packaging signal located in the pol1b gene could mediate the incorporation into virions of rnas of even noncoronaviral origin (woo et al., 1997) . they subsequently showed that this incorporation is most likely effected by a direct and specific interaction of the signal with the m protein. when defective genomic rnas or nonviral rnas were introduced into helper mhv-infected cells, they could subsequently be isolated as ribonucleoproteins from lysates of the cells by immunoprecipitation with an m-specific antibody, but only if the rnas contained the packaging signal (narayanan and makino, 2001) . coexpression experiments using noncoronaviral vectors showed the interaction to be independent of the n protein. a reporter gene transcript generated in cells expressing the m protein could be coimmunoprecipitated with an anti-m monoclonal antibody provided that the rna carried the packaging signal (narayanan et al., 2003a) . moreover, when the e protein was additionally coexpressed, the signal-containing rna-but not an identical rna lacking this sequence-was found to be coincorporated into vlps, irrespective of the presence of the n protein. altogether these observations reveal a hitherto unknown type of interaction between a viral envelope protein and genomic rna. although its significance remains to be further established the m-rna interaction seems to provide additional selectivity to the assembly of the coronaviral nucleocapsid. assembly of viruses is a process of generally high specificity. directed by specific targeting signals, the viral structural components colocalize to distinct places in the cell where unique and complex molecular interactions control their assembly. these rules hold particularly for naked viruses and many of the smaller enveloped viruses; there are, however, many examples where the process is considerably less selective and where "nonself " (host or viral) components are coassembled (see, e.g., garoff et al., 1998) . interestingly, formation of the large, pleiomorphic coronaviruses appears to combine aspects of both great selectivity and extreme flexibility. with the m and e proteins as the fixed minimal requirement, coronaviral particles appear to tolerate the presence of all other viral components in practically every possible combination. a nucleocapsid is not required but, if available, it can take almost any length as defective (including chimeric) genomes of largely varying sizes have been accommodated. rnas need not necessarily be packaged into a nucleocapsid; whether of viral or nonviral origin, if provided with the proper packaging signal they can be taken in even in the absence of an n protein. also in the composition of their viral envelope these viruses are highly flexible. spikes seem to be incorporated in variable numbers depending on availability. they tolerate severe manipulation, both of their ectodomain and of their endodomain. thus, swapping of ectodomains between unrelated coronaviruses (i.e., from different groups) creates viable chimeric viruses whereas a foreign protein such as gfp appended to the s protein endodomain is accommodated in the particle, although reluctantly. some coronaviruses have an he protein but continue growing well if for any reason the gene is not (properly) expressed as happens among different mhvs. consistently, s and he proteins are incorporated independent from each other. direct interactions between s and he were not observed when the proteins were coexpressed in cells (nguyen and hogue, 1997) . this result is consistent with the idea that these proteins are separately drawn into the m protein lattice by their distinctive interactions with m molecules. it is also consistent with the concept that these proteins assume different positions within this lattice, a hypothesis based on the presumed different geometric requirements for the incorporation of trimeric and di-or tetrameric s and he complexes, respectively. how, in the face of this enormous flexibility in accommodating all these various numbers and combinations of viral components, do coronaviruses manage to maintain specificity? host proteins have not been noticed to occur in virions, although this may simply not have been looked at carefully enough. by probing the specificity, using viral and nonviral membrane proteins, it appeared that foreign proteins are effectively excluded from coronaviral particles . however, some missorting was found to occur, consistent with earlier observations (yoshikura and taguchi, 1978) . the picture of coronaviral envelope formation is one that is directed entirely by lateral interactions between the envelope proteins. in infected cells, membrane proteins-viral and cellular-are sampled for fit into the lattice formed by m molecules. the specificity of the molecular interactions acts as a quality control system to warrant the formation of the two-dimensional assemblies that contain the full complement of viral membrane proteins but from which cellular proteins are segregated. for each cellular protein the efficiency of this exclusion process is determined by its lack of interaction with the m protein, its lack of fit in the m protein framework, and its success in competing with the s and he oligomers for the (geometrically different) vacancies within this framework. the precise location of coronavirus budding and the factors that govern it have not been established. although it is clear that particle formation occurs at membranes early in the secretory pathway, up to the cis-golgi compartment, the precise site has not been identified for any coronavirus. several considerations may explain this lack of knowledge. one is that these early compartments are themselves rather complex and highly dynamic and have hence been difficult to define structurally. another is the possible alteration of the structural integrity of these compartments by infection; studies of these effects have not been described. a third complication may be that coronaviruses do not behave uniformly, different viruses possibly preferring different membranes for budding. in this respect it may be of note that differences have been observed, for instance, in the intrinsic localization of the m proteins from ibv and mhv; they appeared to accumulate on the cis and trans side of the golgi apparatus, respectively machamer et al., 1990) . it has long been assumed that the m protein determines the site of coronavirus budding. when, however, this protein appeared to localize beyond this site, the idea became attractive that the association of the envelope proteins may create the novel targeting signals that direct these multimeric complexes to the budding site. in support of such a notion is the fact that the s and he proteins, when coexpressed with the m protein, are retained in the golgi apparatus rather than being transported to the plasma membrane. the critical question now is whether and how the e protein affects the localization of the m protein. as the e protein by itself does not seem to localize to the virion budding site it will be of great interest to determine the membranes at which vlps assemble. as the envelope proteins can direct particle formation by themselves, it may seem that the nucleocapsid is not leading the assembly process. still, besides giving rise to virions rather than vlps, its involvement in assembly might have important consequences. first of all, nucleocapsids may enhance the efficiency of the budding process. the physical yields of vlps obtained by coexpression of the envelope proteins in cells are generally poor. although there may be many reasons for this, in infected cells the availability of nucleocapsids is likely to facilitate particle production. empty particles, considered to be vlps, have nevertheless been observed during natural infection (afzelius, 1994; macnaughton and davies, 1980) . their formation might simply serve as a means to dispose of excess viral membrane proteins from infected cells if required. another effect of the nucleocapsid could involve the localization of budding. it is conceivable that, unless a defined budding station is created by a specific interplay between viral and host proteins (for which no indications yet exist), preassembled nucleocapsids dock at those intracellular membrane sites where sufficiently sized patches of m protein-based envelope structure have accumulated. early in infection such patches might start to form only after the envelope proteins have left the endoplasmic reticulum and become concentrated in intermediate membranes on their way to the golgi complex. later, when viral protein synthesis increases, this density might be reached earlier, perhaps explaining the observed late budding in the endoplasmic reticulum (goldsmith et al., 2004; klumperman et al., 1994; tooze et al., 1984) . it will again be interesting to learn where vlps independently bud and how this relates to the local density of the envelope proteins because this information will shed light on the role of the nucleocapsid in the localization of virion assembly. the picture of coronavirus assembly that the available literature allows us to draw in this review is still a rough draft. we know the identity and some characteristics of the key elements of the picture, we know the relative positions and orientations of most of them, but we are unable to fit them all into a sensible composition. although this may seem like a discontented retrospective, it certainly is not. in the 25 years that the senior author has been in coronavirology research, enormous progress has been made in practically all its aspects including virion assembly. however, the rewarding act of compiling and ordering the available information and trying to abstract from it actual knowledge was at the same time a sharp and recurrent confrontation with the unknown. we want to conclude this work by summarizing what in our opinion will be the main issues for the near future. with the obstacle of reverse genetics technology solved, "structure" will be the dominating issue of the next decade. biology has taught that molecular insight into processes will eventually depend critically on detailed structural information. for coronavirus assembly this means data on the individual structural components and, particularly, on the virion. with respect to the former, this will be most challenging for the membrane proteins, notably m and e. virions, by their apparent elasticity, have eluded structural analysis. here, despite the still limited resolution to be expected, cryoelectron microscopy should provide the urgently required insight into the structural organization of the particle. another issue will be the cell biology of assembly. this actually refers to a number of poignant problems at every stage of the process. starting with nucleocapsid formation we must admit that we know practically nothing. by which interactions and where on the genome the packaging is initiated, how the wrapping of the rna proceeds, how the condensation of the ribonucleoprotein structure takes place, and where in the cell these activities take place are all unresolved questions. although we seem to know more about the budding process, several fundamental issues are still unresolved. obvious issues are the site of budding and the determinants of its location, and the inclusion of the 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glycosylation site human aminopeptidase n is a receptor for human coronavirus 229e neuropathogenicity of mouse hepatitis virus jhm isolates differing in hemagglutinin-esterase protein expression hemagglutininesterase specific monoclonalantibodies alter the neuropathogenicity of mouse hepatitis virus heterogeneity of gene expression of the hemagglutinin-esterase (he) protein of murine coronaviruses biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus the detection and characterization of multiple hemagglutinin-esterase (he)-defective viruses in the mouse brain during subacute demyelination induced by mouse hepatitis virus synthesis and processing of the haemagglutinin-esterase glycoprotein of bovine coronavirus encoded in the e3 region of adenovirus mouse hepatitis virus strain mhv-s: formation of pseudotypes with a murine leukemia virus envelope mouse hepatitis virus gene 5b protein is a new virion envelope protein suppression of sars-cov entry by peptides corresponding to heptad regions on spike glycoprotein conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 c either by soluble murine ceacam1 receptors or by ph 8 expression of hemagglutinin/esterase by a mouse hepatitis virus coronavirus defective-interfering rna alters viral pathogenesis presence of subgenomic mrnas in virions of coronavirus ibv the amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with 3 0 genomic rna the infectious bronchitis virus nucleocapsid protein binds rna sequences in the 3 0 terminus of the genome following the rule: formation of the 6-helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors virus-encoded proteinases and proteolytic processing in the nidovirales we thank henk halsema for making most of the drawings. we are grateful to cristina risco for providing the electron micrographs demonstrating the structural maturation of virions; to raoul de groot, berend jan bosch, and jean lepault for their combined efforts in providing the electron micrographs of the purified virions; and to raoul de groot for critical reading of the manuscript. key: cord-002842-4evbeijx authors: pandey, rajan kumar; bhatt, tarun kumar; prajapati, vijay kumar title: novel immunoinformatics approaches to design multi-epitope subunit vaccine for malaria by investigating anopheles salivary protein date: 2018-01-18 journal: sci rep doi: 10.1038/s41598-018-19456-1 sha: doc_id: 2842 cord_uid: 4evbeijx malaria fever has been pervasive for quite a while in tropical developing regions causing high morbidity and mortality. the causal organism is a protozoan parasite of genus plasmodium which spreads to the human host by the bite of hitherto infected female anopheles mosquito. in the course of biting, a salivary protein of anopheles helps in blood feeding behavior and having the ability to elicit the host immune response. this study represents a series of immunoinformatics approaches to design multi-epitope subunit vaccine using anopheles mosquito salivary proteins. designed subunit vaccine was evaluated for its immunogenicity, allergenicity and physiochemical parameters. to enhance the stability of vaccine protein, disulfide engineering was performed in a region of high mobility. codon adaptation and in silico cloning was also performed to ensure the higher expression of designed subunit vaccine in e. coli k12 expression system. finally, molecular docking and simulation study was performed for the vaccine protein and tlr-4 receptor, to determine the binding free energy and complex stability. moreover, the designed subunit vaccine was found to induce anti-salivary immunity which may have the ability to prevent the entry of plasmodium sporozoites into the human host. increase the drug efficacy and kills the remaining parasites. the most widely used combination therapies are artemether-lumefantrine (commercial name: coartem) and amodiaquine-artesunate (commercial name: coarsucam) 10 . while the recently approved combination therapies are artesunate-pyronaridine (commercial name: pyramax) and dihydroartemisinin-piperaquine (euartesim) 10 . the recent emerging resistance against artemisinin urges to develop some new strategy to prevent the malaria diseases condition 11 . therefore, in this study, we applied a novel immunoinformatics approach to design multi-epitope based subunit vaccine that may prevent the disease by maintaining the host hemostasis by the inhibition of anticoagulant and anti-inflammatory proteins present in mosquito saliva. it will also inhibit the entry of parasite within the host body by a similar mechanism. apart from this, if any way parasite enters into the host body, vaccine candidate will stop the salivary protein-mediated induction of parasitic growth. among different anopheline vectors, anopheles stephensi is a sub-tropical species that most abundantly present in the indian subcontinent and also distributed across the middle east and south asia region 12 . a. stephensi transmit the malarial infection by injecting various plasmodium species to the human host typically via bites. salivary gland of mosquitos implement various functions for the survival of the vector and complement their feeding behavior by producing a large array of a biochemically active molecule that has immunomodulatory, anti-coagulant and anti-inflammatory properties that disable the host hemostatic response for successful blood feeding 13, 14 . salivary proteins are antigenic and immunogenic in nature which helps the infectivity of parasite 14, 15 . d7 protein and salivary apyrase are two different salivary proteins that help in binding, and inhibition of the platelets aggregation, respectively. salivary peroxidase helps in heme binding and peroxidase activity while putative til domain polypeptide functions as trypsin inhibitor 16 . recently, it was reported that hamadarin is a 16 kda protein present in anopheles stephensi saliva which inhibits the activation of plasma contact system 17 and ultimately blood coagulation. another protein from anopheles gambie saliva namely gsg6 plays essential blood feeding 18, 19 . recently, vijay s. et al. reported that salivary proteins might be utilized to develop novel antimalarial control strategies via innate immune protection against malaria 16 . these proteins could likewise evoke a host igg response in natural conditions 20, 21 . mosquito salivary gland surface (sgs) proteins are the prevalent immunogenic component present in saliva having ability to induce immunogenic responses 22 . this is the reason why we chose the a. stephensi salivary proteins from the national center for biotechnology information (ncbi) and subjected to design multi-epitope subunit vaccine. allergenicity, antigenicity and physiochemical properties were also obtained for the vaccine protein. moreover, tertiary structure prediction followed by refinement was performed to get a refined 3d model having a higher number of residues in the favored region of ramachandran plot. molecular docking and molecular dynamics simulation of vaccine constructs with tlr4 were also performed to check the binding energy and complex stability. finally, disulfide engineering and in silico cloning was performed to increase the stability of vaccine construct and ensuring its effective expression in the microbial system, respectively. this study finally represents a novel approach to develop malaria vaccine using salivary protein instead of parasitic protein, which could be helpful to prevent the plasmodium infection to human host. sequence retrieval of salivary protein and assurance of antigenic conduct. in order to design an immunogenic multi-epitope subunit vaccine, the sum of 33 a. stephensi salivary protein sequences was retrieved from the ncbi protein database. major proteins name is salivary lysozyme, a salivary protein precursor, salivary galectin, salivary lipase, anti-thrombin anopheline, salivary protein sg3, salivary apyrase, salivary secreted serine protease inhibitor, salivary defensin and salivary cecropin. among 33 salivary protein sequences, only 14 proteins were found to be antigenic as predicted by antigenpro. these 14 sequences were selected based on their score obtained for the probability of antigenicity and all these proteins having a score of ≥08 23 . obtained score for antigenicity probability clearly denoting the antigenic nature of selected protein sequences which can be used for the subunit vaccine designing 24 . subset of t-cell responses to kill those target cells having intracellular viral, bacterial or protozoan infection 25 . during infection, whenever they encounter to the mhc-i mounted antigen specific to their receptor, they enter the cell cycle and perform several mitotic divisions followed differentiation into the effector cells 26 . here, we tried to predict the ctl receptor specific immunogenic epitopes using the netctl 1.2 server and total 83 ctl epitopes of 9mer length were obtained for the input of 14 salivary protein sequences 27 . in the next step, the immunogenicity of epitopes was determined and as per the instruction of iedb 28 , higher score indicate greater probability to elicit an immune response; therefore total 21 ctl epitopes with high immunogenicity score were selected and subjected to the vaccine designing (table 1) . helper t-lymphocyte is the key player of both humoral and cell-mediated immune response 29 . therefore, htl receptor specific epitopes are probably going to be a crucial part of the prophylactic and immunotherapeutic vaccine 30 . all 14 salivary protein sequences were subjected to iedb mhc-ii epitope prediction module and 8751 epitopes of 15mer length were obtained. in order to become highest immunogenic epitopes, they must have a lower percentile rank and ic 50 value 24 . only 14 epitopes with lowest percentile rank ranging from 0.03-0.3 were selected for the vaccine designing (table 2 ). their ic 50 value ranging from 368-959 denoting that out of 14 epitopes with lowest percentile rank, 7 epitopes have intermediate affinity while remaining to have low affinity for the htl epitopes. on the other side, all 14 epitopes were found to have ifn-γ inducing capability that was obtained from their positive score on the ifnepitope server output 23, 31 (supplementary table 1 ). all these 14 epitopes were used for the vaccine construction. construction of multi-epitope subunit vaccine. a final vaccine construct of 541 amino acid residues was designed using 21 ctl and 14 htl epitopes as described elsewhere 23, 24 (supplementary figure 1) . in order to attain maximum immune response tlr-4 agonist (rs09) was used as an adjuvant at the n-terminal site of the vaccine construct 32 . each joint was occupied by the suitable linkers as described by nezafat et al. 32 , for example, adjuvant and ctl epitopes were combined together by eaaak linker, intra-ctl and intra-htl epitopes joint by aay and gpgpg linker, respectively. finally, vaccine construct was obtained having adjuvant, linker, ctl, and htl epitopes in a sequence moving from n-terminal to c-terminal. as this designed subunit vaccine consisting of immunogenic ctl and ttl epitopes along with suitable adjuvant and linker, it may have the ability to inhibit the entry of malaria parasite within the human host body 23,24 . b-cell epitope mapping. b-cells are a key player of humoral immunity. an epitope corresponding to the b-cell receptor plays an important role in vaccine design following antibody production 33 . therefore, bcpreds server was used to reliably predict the linear b-cell epitopes where bcpred was the selected prediction method 23 . total 14 b-cell epitopes of 20mer length were predicted among the primary input sequence of final vaccine construct. among them, only 11 epitopes were selected and finalized because of their high score of 1.0 ( fig. 1a ). due to the selection of highest scoring b-cell epitope among designed subunit vaccine, our vaccine may have the ability to enhance humoral immunity as well as cell mediated immunity 23 . discontinuous epitopes of 60 amino acids long were also predicted from the final 3d model of vaccine construct with the probability scoring of 0.791 (fig. 1b) . the obtained probability score also confirming the immunogenic behavior of the designed subunit vaccine 34 . antigenicity and allergenicity prediction of designed vaccine. a vaccine given to human host must be immunogenic in nature and capable to trigger significant humoral immune response which ultimately leads to the memory cell formation against the pathogenic epitopes. the antigenicity of designed vaccine construct was determined by using an alignment-free antigenpro server and found that it has the antigenicity probability of 0.86, which represent the antigenic nature of vaccine construct 24 . the antigenicity score obtained for this vaccine construct is comparable with the antigenicity of subunit vaccine reported elsewhere 24 . allergy is an overreaction by our immune system to the previously encountered, ordinarily harmless substance that results in sneezing, wheezing, skin rash, and swelling of the mucous membrane 35 . allergenicity of predicted vaccine construct was determined using allertop online server and found that the vaccine protein is nonallergic in nature and safe for the human use 23,35 . physiochemical properties assessment. the physiochemical properties of vaccine construct were characterized by using protparam server and evaluated for seven parameters. the molecular weight of vaccine protein was found to be 58 kda which will favor the antigenicity of the vaccine construct 23 . the theoretical pi was found to be 5.61 showing its slightly acidic nature while the total numbers of negative and positive charge residues were 48 and 40, respectively 24 . the estimated half-life in mammalian reticulocytes was 4.4 hours, in vitro; while 20 and 10 hours in yeast and e. coli, in vivo. the extinction coefficient was found to be 119530 m-1 cm-1, at 280 nm measured in water, assuming that all cysteine residues are reduced. the score obtained for instability index was 29.56, showing the stable nature of vaccine construct. the value of the aliphatic index and grand average of hydropathicity (gravy) was 71.24 and −0.276, respectively. the estimated value of aliphatic index represents the thermostable nature of designed subunit vaccine because higher the value of aliphatic index, greater will be the thermo stability 24 . while, negative value of gravy for the input subunit vaccine represents the hydrophilic nature of vaccine 24 . conclusively, the designed vaccine is immunogenic, thermostable and hydrophilic in nature. tertiary structure prediction, refinement, and validation. the tertiary structure was predicted by using the raptorx server and 3d model was obtained as described elsewhere 24 (fig. 2a) . the best template used for the homology modeling was crystal structure of a legionella phosphoinositide phosphatase (pdb id: 4fye). total 541 amino acid residues were modeled as a single domain with 1% disorder. secondary structure information resulting in the presence of 53% helix, 4% beta sheet, and 41% coiled structure. p-value is a parameter of homology modeling where low p-value defines the good quality of modeled structure 23 . the p-value obtained for the modeled structure was 6.14e-04 which is low and significant. further, protein refinement using galaxyrefine leads to the increase in a number of residues in the favored region 24 . initially, 87% of residues were in the rama-favored region while after refinement the number of residues in the rama-favored region reached to 92.4%. the refinement output was also validated by plotting ramachandran plot and found the same that 92.4% residue in rama-favored region, 5.4% residues in allowed region and only 2.2% residues in outlier region (fig. 2b ). disulfide engineering for vaccine stability. in order to stabilize the modeled structure of final vaccine constructs disulfide engineering was performed using disulfide by design v2.0 36 and found that there are total 63 pairs of residues that can be used for the purpose of disulfide engineering. but after evaluation on other parameters like energy and chi3 value, only four pairs of residues were finalized because their value comes under the allowed range i.e. the value of energy should be less than 2.2 and chi3 should be in between −87 and +97 degree 37 . therefore, total 8 mutations were created at the residues pairs namely ala95-gln414, tyr96-gly422, trp133-glu173, and gly135-phe255 (fig. 3) . codon adaptation and in silico cloning. the main purpose of in silico cloning was to express the vaccine protein epitope of anopheles mosquito origin into e. coli expression system 23 . therefore, it was necessary to adapt the codon respective to subunit vaccine construct as per the codon usage of e. coli expression system. we adapted the codons as per e. coli k12 strain using jcat server and found that the gc-content of the improved sequence was 58.16% while the value of codon adaptive index was 0.97 which is near to 1.0 that was satisfactory 23 . later on, xhoi and ndei restriction sites were created and cloned into the pet28a(+) vector (fig. 4) . the target sequence in the clone is represented in blue color in between aforementioned restriction sites 23 . the target sequence is also enclosed between 6-histidine residues on both ends that will be helpful to the purification purposes. the total length of the clone was 6.9kbp. and tlr-4 receptor was performed using the cluspro 2.0 and total 30 models were generated 38 . among them, only that model was selected which properly occupied the receptor and having lowest energy score and found that model number 0.00 fulfill the desired criteria that's why selected as the best-docked complex (fig. 5 ). the energy score obtained for the model 0.00 was found to be −1187 which is lowest among all other predicted docked complex showing highest binding affinity. formed using gromacs 5.1.5 using a gromos9643a1 force field 39 . the potential energy obtained for the complex was −9.9 kj/mol, while the value of temperature, pressure, and density was obtained as 299.77 k, 2.54 bar and 1016.4 kg/m 3 ( supplementary figure 2a-c) . the radius of gyration obtained for the docked complex showing that the distance in rotating complex from the center of mass is 4.3 nanometers that decreases up to 4.25nanometers at the time duration of 10 nanoseconds (supplementary figure 2d) . the rmsd value of protein backbone was 0.4 nanometers (fig. 6a) while rmsf score obtained for the protein side chain was found to be 0.2 nanometers (fig. 6b ). both these scores are satisfactory showing strong complex stability 23, 24 . malaria is severe infection characterized by the high fever with irregularity but may also lead to brain injury and coma. it affects 212 million people from 91 countries, worldwide. lack of effective vaccine and emergence of resistance against artemisinin created a disastrous condition among the people living in the endemic zone. therefore, it's the need of time to search for the new options to tackle this severe problem. the vector for malaria is the anopheles stephensi in the indian subcontinent leads to the transfer of malaria parasite. literature survey reveals that salivary proteins of anopheles mosquito not only supports the pathogenesis but are also immunogenic in nature. therefore, this study was designed to reach a step ahead in the path of vaccine development. we used the primary amino acid sequence of anopheles mosquito salivary protein to design a subunit vaccine construct. the constructed vaccine has ctl, htl and bcl epitopes of varying length. it has antigenic properties in the absence of allergenic properties. it was stable and having a good binding affinity for the tlr-4 receptor. collectively, this study applied a series of immunoinformatics tools in a sequential manner to find an effective vaccine that may fight against the malaria infection. however, this study needs experimental validation to prove this computational work. the experimental work may include the synthesis of designed subunit vaccine followed by the in vitro and in vivo analysis to determine the immunogenicity and safety concern of the same. anopheles stephensi mosquito is the vector of malaria transmission to the human being in the indian subcontinent. while the salivary gland proteins of anopheles mosquito was reported for their role in parasite pathogenesis and having the ability to induce igg response in the natural host 20, 21 . therefore, total 33 salivary proteins of a. stephensi were obtained from the national center for biotechnology information (ncbi) protein database (retrieval date 25/08/2017) and subjected to multi-epitope vaccine designing. as the main purpose of vaccination is to induce an immunogenic response within the host body, all the retrieved protein sequences were subjected to their antigenicity prediction using antigenpro. based on the antigenicity result, only 14 proteins were found to have an antigenic probability of ≥0.8 were selected and used in the next step. cytotoxic t-lymphocyte were shown to inhibit malaria parasitic growth and development, inside the hepatocytes cells 40 . to get an immunogenic ctl epitopes having the ability to elicit cell-mediated immunity and form the memory cells, all 14 salivary protein sequences were subjected to the netctl 1.2 server 41 . netctl 1.2 is an online web server intended for predicting ctl epitopes among input protein sequences based on the training dataset. netctl was selected to predict the ctl epitopes because of its higher prescient execution on all execution parameters as compared to the recently developed servers namely mhc-pathway, mappp, and epijen. all the salivary protein sequences were submitted in the fasta format to predict the ctl epitope at the threshold score of 0.75 (default). those epitopes having a combined score of greater than 0.75 were selected as ctl epitope and further subjected to the immune epitope database (iedb) mhc class i immunogenicity prediction module. predicted ctl epitopes for each salivary protein was used as an input sequence and the result was obtained in the form of the score, where higher score determines that greater will be the probability of eliciting an immune response. helper t-lymphocyte (htl) epitope prediction. helper t-cell response is the major part of cell-mediated immunity and helps in pathogen clearance by the help of various cytokines and immune cells 42, 43 . they have the ability to induce both ctl and humoral immune response by the secretion of lymphokines like il-2, il-4, il6, granulocyte-macrophage colony-stimulating factor (gm-csf), and ifnγ. in view of that, we can say that htl epitopes mainly of th1 type are most likely going to be a crucial part of the prophylactic and immunotherapeutic vaccine. therefore, iedb mhc-ii epitope prediction module was used to predict the htl epitopes for all 14 anopheles salivary protein sequences 44 . the available parameters were kept default except for allele selection where the nominated alleles were h2-iab, h2-iad, and h2-ied. output epitopes were ranked based on their percentile rank score where lower percentile rank representing that greater will be the binding affinity for htl receptor. secondly, to prove our work that the predicted htl epitopes will have ability to activate th1 type immune response followed by the ifn-γ production, top 14 predicted htl epitopes were subjected to the ifn epitope server using predict option. all 14 epitopes were submitted in the fasta format followed by the approach selection and model of prediction. motif and svm hybrid was selected as the approach and ifn-gamma versus other cytokine as model of prediction. designing of multi-epitope subunit vaccine. so as to plan an appropriate vaccine candidate, it must have the ability to induce ctl and htl immune response. in other words subunit vaccine must contain both ctl and htl epitopes along with suitable linkers. keeping in mind the end goal to effectively activate both innate and adaptive immune response, subunit vaccine must consist of a strong immunostimulatory adjuvant. in the previous decades, there is a huge headway in the adjuvant engineering, for instance, toll-like receptor (tlr) agonists have made its contribution as a part of peptide-based subunit vaccine as a functional option for present-day immunotherapy 45 . recently, junqueira et al. have shown that cpgs oligodeoxynucleotides (cpg odns) and glycoinositolphospholipids (gipl) gotten from trypanosome cruzi having the ability to activate tlr-4 and tlr9 leads to actuate potent pro-inflammatory reaction 46 . secondly, proteo-glycolipid complex (p8glc) derived from leishmania parasite has shown its affinity for the tlr-4 receptor and recognized as ligand 47 . moreover, tlrs having the capability to recognize the plasmodium ligands, for example, plasmodium falciparum primes the human tlr-4 response towards high proinflammatory cytokine profile 48 . shanmugam a. et at. has reported that synthetic tlr-4 agonist namely rs-09 (sequence: apphals) can be used as a novel class of adjuvant 49 , therefore, it was added as an adjuvant and linked with epitopes (ctl and htl) by using eaaak linker 50 . linkers assume an imperative part in simulating the vaccine construct to work as an independent immunogen and producing higher antibody titer than that of single immunogen 51 . total three linkers namely eaaak, aay, and gpgpg, were used to construct the final vaccine. aay and gpgpg linkers were added at the intra-epitope position to link the ctl and htl epitopes, respectively. b-cell epitope prediction. b lymphocytes, a type of white blood cells, are the key player of humoral immunity by antibody production. the identification of b-cell epitopes is an essential part in vaccine designing. bcpreds server was used to predict the linear b-cell epitopes of 20 amino acids long. the amino acid sequence of final vaccine construct was used as an input sequence in plain format followed by the selection of fixed length epitope prediction method and length of the epitope. bcpreds (default method) was selected as the prediction method for the epitope of 20 amino acids long 52 . the specificity threshold was set to be by default at 75% to get the result in a user-friendly format. while conformational epitopes were predicted using ellipro server for the input of tertiary protein structure of vaccine construct. antigenicity and allergenicity prediction of designed vaccine. antigenicity determines the ability of an antigen to binds with the b-and t-cell receptor that may lead to the immune response and memory cell formation. therefore, the antigenic nature of predicted vaccine construct was determined to ensure its ability to interact with the immune receptor. antigenpro is a sequence based, pathogen independent and alignment-free prediction method that was used to check the antigenic behavior of vaccine protein. it uses svm classifier to summarize the probable antigenic or non-antigenic nature of proteins. antigenpro uses the existing protein antigenicity microarray files of eight feature sets for five pathogens to construct two-stage architecture; among them, the first one is multiple representations of the primary protein sequence and the second one is five machine learning algorithms. allergenicity is the potential of a material to cause sensitization and allergic reactions associated with the ige antibody response. therefore, the predicted vaccine construct must be free from the allergenic nature. allertop v. 2.0 was used to check the allergenicity of the vaccine construct based on the method that uses auto cross-covariance (acc) transformation of protein sequences into uniform equal-length vectors. input protein sequence of vaccine protein was classified by the k-nearest neighbor algorithm (knn, k = 1) which is based on the training set of 2427 known allergen from different species and 2427 nonallergen from similar species. physiochemical properties assessment. the main purpose of vaccination is to induce an immune response after injecting the vaccine into the body. therefore, it is necessary to define the physical and chemical parameters associated with the vaccine. protparam 53 web server, a part of expert protein analysis system (expasy), was used to define various physicochemical properties of predicted vaccine construct. the primary protein sequence of the vaccine was used to predict the various parameters including molecular weight (kda), estimated half-life, theoretical pi, aliphatic index, grand average of hydropathy (gravy) and so on. tertiary structure prediction. protein molecule achieves maximum stability in its lowest energy state by proper bending and twisting to form a tertiary structure. it is the interaction between the amino acids side chain residue which is responsible to stabilize the protein structure. the 3-dimensional structure of predicted vaccine construct was obtained by utilizing raptorx structure prediction server. raptorx is a pure ab initio method that can be used to build a 3d model in a template-free manner. it is the degree of likeness between the target and available template structure that determines the quality of protein model structure created by contemporary protein structure prediction techniques 39, 54 . therefore, it was necessary to improve the template based predicted model beyond the accuracy by utilizing the template information. to fulfill this thought, output model of raptorx server was subjected to the galaxyrefine web server 55 , which is based on the casp10 tested refinement method. galaxyrefine performs rehashed structure perturbation followed by overall structural relaxation by performing molecular dynamics simulation. disulfide engineering for vaccine stability. before proceeding to the next step, it was necessary to improve the stability of refined protein model. disulfide bonds are covalent interactions that emulate the stabilizing molecular interaction and provide a considerable stability to protein model by confirming precise geometric conformations. disulfide engineering is a novel approach for creating disulfide bonds into the target protein structure. therefore, the refined model of final vaccine construct was subjected to the disulfide by design 2.0 36 to perform disulfide engineering. initially, the refined protein model was uploaded and run for the residue pair search that can be used for the disulfide engineering purpose. total 4 residue pairs were selected to mutate them with cysteine residue using create mutate function of the disulfide by design 2.0 server. in silico cloning. codon adaptation is a way to attain major expression rate of foreign genes in the host when the codon usage of the host differs from that of the organism where the gene stems from. unadapted codon may lead to the minor expression rate in the host. therefore, the primary sequence of vaccine protein was submitted to the java codon adaptation tool (jcat) to adapt their codon usage to most sequenced prokaryotic organisms (e. coli k12) 23 . cai value and gc content of the adapted sequence was also obtained. later on, the adapted nucleotide sequence corresponding to the designed vaccine construct was cloned into the e. coli pet28a(+) vector by using the restriction cloning module of snapgene tool 24 . used to predict the preferred orientation of ligand molecule to the receptor molecule in their stable complex form 56 . it can be also used to predict the binding affinity between these two molecules in terms of scoring function. as mentioned in the previous section, tlrs having the capability to recognize the plasmodium ligands and p. falciparum primes the human tlr-4 response towards high proinflammatory cytokine profile 48 . therefore, tlr-4 was selected as receptor and its pdb file (pdb id: 4g8a) was obtained from rcsb-protein data bank while the refined model of vaccine protein was used as a ligand. protein-protein docking was performed using the cluspro 2.0: protein-protein docking server, to check the binding affinity between them 57 . widely accepted computational approach which is used to determine the stability of protein-ligand complex at the microscopic level 58 . the protein-protein docked complex output of cluspro was used as an input to perform the molecular dynamics simulation using gromacs v5.1.5. initially, the crystal water of complex was removed followed by the topology generation using a gromos9643a1 force field. in the next step, protein complex was centered in a cubic boundary box and filled by water molecule using 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nicotine vaccine design and use predicting linear b-cell epitopes using string kernels protein identification and analysis tools in the expasy server molecular modeling and virtual screening approach to discover potential antileishmanial inhibitors against ornithine decarboxylase protein structure refinement driven by side-chain repacking febrifugine analogues as leishmania donovani trypanothione reductase inhibitors: binding energy analysis assisted by molecular docking, admet and molecular dynamics simulation the cluspro web server for protein-protein docking high-throughput virtual screening and quantum mechanics approach to develop imipramine analogues as leads against trypanothione reductase of leishmania structure-based virtual screening, molecular docking, admet and molecular simulations to develop benzoxaborole analogs as potential inhibitor against leishmania donovani trypanothione reductase r.k.p. is thankful to department of science and technology for providing inspire fellowship. v.k.p. is thankful to the central university of rajasthan for providing computational facility. we acknowledge the scientific help of ms. rani soni from department of biotechnology, central university of rajasthan for this manuscript. protocol designed by r.k.p., t.k.b., v.k.p. methodology performed by r.k.p., v.k.p. manuscript was written by r.k.p., t.k.b.,v.k.p. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-19456-1. the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-284648-yznlgzir authors: varanko, anastasia; saha, soumen; chilkoti, ashutosh title: recent trends in protein and peptide-based biomaterials for advanced drug delivery date: 2020-08-29 journal: adv drug deliv rev doi: 10.1016/j.addr.2020.08.008 sha: doc_id: 284648 cord_uid: yznlgzir nan they also increase the occurrence of dose-dependent side effects. the lack of target specificity made it impossible to deliver a therapeutic to the organ of interest without impacting other cells, which caused off-target toxicity. in the 1950s, scientists began to focus on developing drug delivery systems (figure 2) . in the next thirty years, they established the principles of drug diffusion, dissolution, and pharmacokinetics [11, 12] . research then shifted toward prolonging drug release and increasing the drug's retention time in circulation [11] . scientists also focused on how to specifically deliver drugs to a disease site by engineering systems for local drug release, passive drug accumulation in the diseased tissue, and active targeting [12] . this research led to the first fda approval of a drug delivery system -liposomal amphotericin b-in 1990 for treatment of fungal infections [13] . since then, controlled delivery has been leveraged to improve the bioavailability and efficacy of numerous therapeutics [14] . biomaterials have been critical to the success of drug delivery systems. many drug formulations employ biomaterials to extend the therapeutic window by both sustaining its release from the formulation and slowing its elimination from the body. beginning in the late 1960s, scientists used synthetic materials, especially polymers, as drug carriers [11, 12] . these systems successfully extended the drug's circulation time by increasing its molecular weight to slow renal excretion. biodegradable systems were also engineered to prolong drug release, thus reducing the frequency of administration [15] . despite these accomplishments, synthetic materials can have high immunogenicity or toxic degradation products [16] . furthermore, there is limited control over the stereochemistry, structure, and molecular weight of synthetic polymers, which impacts the drug's biodistribution and pharmacokinetics [17, 18] . the production of synthetic polymer drug carriers can be difficult and expensive to scale up [19] . figure 6 : conjugation of the cyclic pentapeptide crgdfk and the photodynamic agent chlorin e6 (ce6) to sf was achieved using a simple acid-amine coupling reaction and the resulting conjugate was doped with 5-fluorouracil (5-fu) using genipin peptide as a crosslinker. adapted with permission from [84] . the adaptable mechanical properties and thermal stability of sf make it an ideal candidate material for forming hydrogels and scaffolds. its degradation can be tuned by controlling the self-assembly of sf which can stably encapsulate the therapeutics and release them on demand. kaplan and colleagues have extensively studied the abilities of sf and its hybrids to form hydrogels. they developed a method to synthesize a thixotropic silk nanofiber hydrogel from aqueous solution, which otherwise requires an organic co-solvent [85, 86] . the injectable nanofiber hydrogel stably entraps dox, solidifies in situ, and demonstrates ph-triggered sustained release of dox [86] . kaplan et al. also used an injectable sf-hydrogel system to sustain the delivery of anti-vascular endothelial growth factor (anti-vegf) therapeutics [87] , and small molecule drugs [88] . however, the prolong gelation time (weeks-months) of sf hydrogel acts as a major roadblock for their practical application. to decrease the gelation time gong et al. synthesized another class of thixotropic hydrogels by blending regenerated sf and hydroxyl propyl cellulose (hpc), a natural cellulose ether approved by fda [89] . hpc has lower critical solution temperature (lcst) of about 40 °c above which it precipitates in water. the j o u r n a l p r e -p r o o f hpc-sf blend gelled at 37 °c within 1 h. results from confocal laser scanning microscopy (clsm), raman spectroscopy, and 13 c-nmr spectroscopy suggested that the conformational transition of sf from random coil to β-sheet during phase separation resulted in gel formation through β-sheet crosslinking and immobilization of the molecules of sf and hpc in the dispersed phase. the blended hydrogel encapsulated mice fibroblasts and protected them against high shear force during injection, which suggests that the hydrogel can be used for cell delivery [89] . germershaus et al. developed heuristics to decipher protein interactions with sf using protamine and polylysine as model proteins [90] . the author concluded that the interaction between protein and sf arises primarily from entropy-driven complex coacervation, which depends on the ionic strength of the solution and presence of kosmotropic and chaotropic salts. sf-hydrogels have also been fabricated with various nanoassemblies including sfnps, carbon nanotubes and hybrid nanoparticles of different materials. mao and colleagues encapsulated curcumin-loaded cationic nanoparticles of rrr-α-tocopheryl succinate-grafted-εpolylysine conjugate into a sf-hydrogel, which promoted the penetration of curcumin into the thickening corneum of psoriatic mice and thus inhibited skin inflammation. compared to 49% of curcumin released from the nanoparticle, only 30% was released from the mixed hydrogelnanoparticle system. the author concluded that this slow release of curcumin might be due to adherence of the nanoparticles into the sf hydrogel, making it difficult for the embedded curcumin to diffuse out of the gel. this delayed release profile resulted into significant accumulation of curcumin in the stratum corneum at the 48 h [91] . wu et al. incorporated salinomycin and paclitaxel-loaded sf-nanoparticles into a sf hydrogel to inhibit cancer stem cell and tumor growth. the dual drug-loaded hydrogel had homogeneous drug distribution and exhibited increased tumor inhibition compared to the single drug-loaded hydrogel, as evidenced by fewer cd44+cd133+ tumor cells in vivo. because paclitaxel and salinomycin interacted differently with sf, their release profiles were different, with paclitaxel showing sustained release and salinomycin an initial burst release [92] . gangrade et al. introduced carbon nanotubes into sf hydrogels to construct an on-demand, tumor-targeting system [93] . they synthesized folic acid functionalized, dox-loaded, single-walled carbon nanotubes (swcnt) and incorporated them into an sf hydrogel matrix. only 7% of dox was released from the composite material over a period of five days under physiological conditions. however, intermittent exposure to near-infrared light stimulated on-demand dox release (~15%) due to the photothermal property of swcnt. he et al. developed an injectable silk fibroin nanofiber hydrogel system complexed with upconversion nanoparticles and nano-graphene oxide (sf/ucnp@ngo) for upconversion luminescence imaging and photothermal therapy [94] . the naluf 4 :er 3+ ,yb 3+ upconversion j o u r n a l p r e -p r o o f nanoparticles were complexed with the nano-graphene oxide and then doped into an aqueous sf solution to form a hybrid hydrogel system. sf/ucnp@ngo hydrogels efficiently ablated 4t1 breast cancer cells via the photothermal effect both in vitro and in vivo. recombinant techniques can also be utilized to modulate the properties of silk-based scaffolds and hydrogels. anderson et. al. recombinantly synthesized slp segments conjugated to a human fibronectin segment and cell attachment domain [95] . crystal structure characterization of (gagags)-based slps revealed the hydrophobic domains of silk collapsed to form anti-parallel β-sheets that assembled into crystallite whiskers at the nanometer scale. this self-assembled material is mechanically tough, can undergo processing in the presence of bioactive molecules, and can be processed for into thin films, hydrogels, and three-dimensional scaffolds. schacht et al. fabricated highly porous foams made from recombinant spider silk protein eadf4(c16) and a variant containing an rgd motif using a salt-leaching technique [96] . in contrast to other salt-leached silk scaffolds, the swelling behaviors of these scaffolds as measured by discovery v20 stereomicroscope were low, and the mechanical properties were suitable for soft tissue engineering. the compressive moduli of the foam in a hydrated state was 3.24 ± 1.03 kp at a protein concentration of 8% (w/v). the pore size and porosity of the foams were optimized by altering the salt crystal size, thus rendering them suitable to adhere and culture fibroblasts. as silk fibroin can self-assemble into hydrogels from an aqueous solution, it has been widely used for ocular delivery of various drugs ranging from small molecules to antibodies and other therapeutic proteins. in one such example, silk hydrogel formulations of bevacizumab, a clinically used antibody as angiogenesis inhibitor showed sustained release of the antibody over a three months' period in an intravitreal injection model in dutch-belted rabbits [87] . the bevacizumab concentration in the vitreous humor on day 90 using hydrogel formulation at both standard (1.25 mg /50 µl injection) and high dose (5.0 mg /50 µl injection) was equivalent to the levels achieved with positive control on day 30 (1.25 mg bevacizumab/50 µl injection). [87] . this concentration is estimated to be the therapeutic threshold based on the current dosage of 1 injection/month. these gels also got degraded after 3 months, indicating a repetitive dosing may be possible. additionally, the propensity of sf to bind positively charged molecules through electrostatic interaction can be exploited for topical drug delivery on the eye surface. dong et al. electrostatically coated an ibuprofen-encapsulated liposome of cationic lipids with sf for ocular drug delivery [97] . the sf being a potential mucoadhesive biopolymer aided in retention of the drug on eye surface and facilitated its sustained release. recently a phase ii clinical trial within the protein. additionally, disulfide bonding, electrostatic interactions, and hydrogen bonding provide keratin with mucoadhesive properties, which make it a useful material for delivery of drugs to the gastrointestinal tract [101] . furthermore, keratins have been employed as -smart‖ materials for stimulus responsive drug delivery. the large number of carboxyl groups within their sequence makes them phsensitive so that in response to an increase in ph, release their cargo in a controlled manner as these groups become deprotonated [102] . additionally, their rich cysteine content renders them redox-responsive. tuning the number or level of crosslinking of disulfide bonds within the protein alters the duration of drug release from a keratin-based material. similarly, keratin is responsive to changes in glutathione (gsh) concentration. this is particularly useful for delivery of chemotherapeutics to metastatic cancer cells, which have significantly higher concentrations of gsh than healthy cells. moreover, the high lysine and arginine content in some keratins enables their cleavage by high concentrations of trypsin [103] , which is frequently overexpressed in inflamed tissue [104] . thus, keratin could be a useful material when targeting injured or tumorous tissues. due to its unique material properties, keratin has been used to create nanoparticles that encapsulate and sequester a therapeutic cargo before releasing it in response to biological stimuli. these nanoparticles can stably carry therapeutics via electrostatic interactions, hydrogen bonding, disulfide bond formation, or chemical conjugation. keratins are durable and remain stable in the bloodstream, which increases the half-life of its cargo [100] . positively charged drugs electrostatically adsorb to the surface of negatively charged keratin nanoparticles; this interaction provides long-term, sustained release of the drug from its carrier. zhi et al. first explored this strategy by complexing the model drug chlorhexidine (chx) with keratin nanoparticles generated by ionic gelation [102] . carboxylate groups on the nanoparticle surface stabilized the polyanion complex with chx. this interaction provided a remarkable chx encapsulation efficiency of 91.2% and a loading content of 9.2%. zhi et al. demonstrated that for 140 hours, the drug was released in a ph-dependent manner with greater release observed at neutral and slightly acidic ph, which indicates the utility of this system to deliver chemotherapeutics to the acidic tumor microenvironment [102] . can be loaded with dox via electrostatic interactions [105] . these nanoparticles were designed to exploit keratin's responsiveness to ph and glutathione concentration to release the drug, which is a useful strategy to treat solid tumors, which have a ph of 6.2-6.9 and gsh concentration of 0.5-10 mm, which is approximately 10-fold greater than the gsh concentration of healthy tissue [105] . kdnps were created with a desolvation method that destabilizes keratin with ethanol and causes it to aggregate into nanoparticles that are crosslinked with glutaraldehyde. in an environment with a low ph or high concentrations of glutathione, kdnps experienced a stark increase in zeta potential and underwent a negative-to-positive charge conversion. the positive charge facilitated internalization by cells and electrostatically repelled the drug, thus accelerating its release (figure 7) . error! reference source not found.moreover, the charge conversion destabilized the nanoparticles, causing them to aggregate and accumulate at the tumor due to the enhanced permeation and retention (epr) effect, which further enhanced their anti-cancer potency [105] .the stimulus-responsive properties of kdnps were expanded by li et al., whoe engineered triple stimuli-responsive kdnps via drug-induced ionic gelation. in addition to releasing dox in response to changes in ph and glutathione concentration, these nanoparticles broke down in the presence of high concentrations of trypsin, which digested the peptide bonds within the keratin [106] . similarly, keratin graft poly(ethylene glycol) nanoparticles have also been explored as glutathione-responsive vehicles; dox-hcl entrapped in the disulfide-crosslinked keratin core was released in response to increased intracellular glutathione concentrations [107] . due to its exquisite and versatile loading capacity, keratin has also been harnessed to create bimodal nanoformulations that combine chemotherapeutics and photodynamic therapy. martella et al. functionalized high molecular weight keratin with the photosensitizer chlorin-e6 (ce6) and induced spontaneous nanoparticle formation by mixing the protein with paclitaxel, a chemotherapeutic that aggregates with the hydrophobic residues in keratin [108] . this is an attractive bottom-up nanoparticle fabrication strategy, as it did not require any toxic crosslinkers or downstream purification steps for nanoparticle fabrication. when administered to an osteosarcoma cell line, the nanoparticles localized to the cell lysosome. although ce6 typically experiences a drop in fluorescence in acidic conditions, the keratin protected the photosensitizer and no decrease in fluorescence was observed. further, the nanoparticle formulation j o u r n a l p r e -p r o o f transported the paclitaxel in a three-dimensional tumor model system without reducing the drug's potency [108] . keratin nanoparticles have also been engineered for mucoadhesive drug delivery. kerateine (ktn) and keratose (kos) are two forms of keratin that have been extracted and processed in its reduced and oxidized forms, respectively. cheng et al. demonstrated that, by altering the ktn:kos ratio, the mucoadhesive properties and thus drug release, gastric retention time, and bioavailability of keratin nanoparticles could be tuned [109] . an evaluation of the hydrophobicity, surface charge, and terminal groups of the nanoparticles revealed that the mucoadhesive properties of ktn were dominated by electrostatic interactions, whereas those of kos were primarily due to hydrogen bonding with gastric mucin. moreover, cheng et al. discovered that gastric retention time decreased with an increase in kos, and release of the model drug, amoxicillin, increased with a larger proportion of ktn due to its ph responsiveness [109] . keratin-based films have been widely explored for biomedical applications due to the presence of cell attachment sites, their biocompatibility, and their large surface area. these mechanically and chemically stable materials have been useful for delivering a variety of drugs and peptides. in an early study of keratin films, fuji et al. extracted keratin from human hair in the absence of surfactant, thus creating a water-soluble film comprised primarily of α-keratins [110] . alkaline phosphatase, a model enzyme, was incorporated into the film by mixing it with the keratin prior to gelation. the biochemical properties and bioactivity of alkaline phosphatase were maintained for two weeks after loading [110] . while keratin films have excellent biocompatibility, they lack mechanical strength. thus, many studies combine keratin with other materials or treat the protein with a crosslinking agent to improve its rigidity, stiffness, and stability. in one example, keratin was blended with sf to create a composite film for the delivery of bowman-birk inhibitors (bbi), synthetic peptides designed to inhibit elastase in wound healing applications [111] . sf provided structural stability to the film while keratin governed the degradation and bbi release rates. using fitc -tagged bovine serum albumin (bsa) as a model protein, vasconcelos et al. demonstrated that, although the sf was compact and rigid, increasing the percentage of hydrolytic keratin could increase the rate of fitc-bsa release by film degradation and diffusion [111] . another study used transglutaminase (tgase) to crosslink keratin films to improve the mechanical strength and chemical stability of the material [112] . treatment with tgase resulted in more compact network correlated with the amount of iodoacetamide used, though it was not directly proportional due to the binding affinity of the model drug to the keratin [116] . a disulfide shuffling strategy was used by cao et al. to explore how disulfide bond formation impacts drug release. they cleaved intramolecular disulfide bonds with a reductive agent (e.g., cysteine) to free thiol groups that could then form intermolecular disulfide bonds ( figure 8a ) error! reference source not found. [117] . this tactic increased the mechanical strength of the hydrogel, reduced its gelation time, and required a lower amount of keratin for gelation. the release rates of ciprofloxacin and dox were inversely proportional to the level of cysteine in the hydrogels. hydrogels that entrapped ciprofloxacin also demonstrated zero-order release kinetics in pbs (figure 8b & c) . moreover, when these hydrogels were exposed to increasing concentrations of glutathione, the degradation rate increased in response to the more rapid degradation of disulfide bonds ( figure 8d & e) [117] . these studies indicate that the degradation rate of keratin hydrogels can be altered for specific disease states and drugs. ph-responsive keratin hydrogels have also been designed to reversibly swell in response to their environment to modulate the release of their cargo. in many of these examples, keratin has been combined with itaconic acid, n-isopropyl acrylamide, or methacrylic acid to achieve ph responsiveness [118] [119] [120] . more recently, peralta ramos et al. reported a stimulus-responsive hydrogel that did not depend on chemical grafting to achieve its mechanical properties and phresponsivity [121] . this was credited to a novel synthesis strategy during which disulfide bridges were broken and the α-keratin reorganized. at an acidic ph, the carboxyl groups were protonated and formed hydrogen bonds, which created a higher fraction of β-sheet conformations and kept the gel in a collapsed state. at a basic ph, hydrogen bonds broke to create more sites for water adsorption, thus forcing chain reorganization and allowing the material to swell [121] . villanueva et al. expanded upon this work by incorporating antimicrobial zno nanoplates into the ph-responsive hydrogel [122] . upon administration to a chronic wound, the hydrogel swelled in response to the basic environment and locally released the biocidal agent. as the wound healed and the presence of microbes decreased, the gel collapsed and inhibited the release of zno [122] . this study demonstrates how keratin hydrogels can be used to adjust the delivery of a therapeutic agent in response to alterations in ph. albumin is the most abundant protein in human plasma and has a set of properties that make it a unique molecular carrier for drugs: (i) it is a natural physiological carrier of native ligands and nutrients; (ii) it bypasses systemic clearance and degradation by the body's own innate mechanisms, so that it has an exceptionally long half-life of 19 days in humans, and similarly long half-lives in most animal species [123] [124] [125] [126] ; (iii) it preferentially accumulates at sites of vascular leakiness; (iv) it is highly internalized and metabolized by rapidly growing, nutrient-starved cancer cells; and (v) it is biodegradable and has no known systemic toxicity. because of these properties especially its extraordinarily long plasma circulation, albumin has attracted a lot interest as a carrier for diverse drugs. three naturally derived albumin molecules have been used to address the delivery challenges associated with small molecule drugs. ovalbumin (ova), a highly functional food protein with a molecular weight of 47 kda and isoelectric point (pi) of 4.8, is a monomeric phospho-glycoprotein consisting of 386 amino acid residues [127] . each ova molecule has one internal disulfide bond and four free sulfhydryl groups. ova was originally chosen as a carrier for drug delivery because of its availability, low cost, ability to form gel networks and stabilize emulsions and foams, and ph-and temperature-sensitive properties [128] . bovine serum albumin (bsa), which has a molecular weight of 69 kda and a pi of 4.7 in water (at 25 °c), has also been widely used for drug delivery because of its abundance, low cost, ease of purification, unusual ligand-binding properties, and wide acceptance in the pharmaceutical industry [129] [130] [131] [132] [133] [134] . however, due to possible human immunologic response to ova and bsa in vivo, human serum albumin (hsa) is now exclusively used for drug delivery [50] . hsa, which has a mw of 66.5 kda, is the most abundant serum protein with a concentration of 30-50 g/l in human serum. it is long-circulating with an exceptionally long in vivo half-life of ~19 days [123] [124] [125] 135] . about 10-15 g of albumin is synthesized by liver hepatocytes daily and released into circulation [136] . when albumin extravasates into tissue, it is naturally recycled and returned to the vascular space via the lymphatic system. the same approximate mass of 10-15 g of albumin entering the intravascular space is also catabolized daily. hsa is a carrier of a wide variety of endogenous and exogenous compounds and facilitates the colloidal solubilization and transport of j o u r n a l p r e -p r o o f hydrophobic molecules, such as long chain fatty acids, and variety of other ligands, including bilirubin, hormones, amino acids, metal ions, and drugs [136, 137] . hsa is a soluble, globular, monomeric protein consisting of 585 amino acid residues. it contains 35 cysteinyl residues that form one sulfhydryl group and 17 disulfide bridges [123, 135, 138] . figure 9a shows the crystal structure of albumin and the sites where ligands can bind [139] . three distinct features of albumin make it ideal for use in drug delivery applications: binding, trafficking, and recycling. binding: each class of drugs has a distinct binding affinity to different binding sites of albumin. dicarboxylic acids and sterically demanding anionic heterocyclic molecules (e.g., warfarin) bind to sudlow's site i (figure 9a ), whereas aromatic carboxylic acids with a single negatively charged acid group separated by a hydrophobic center (e.g., diazepam, ibuprofen) bind to sudlow's site ii [140] . hsa has seven long-chain fatty acid binding sites (fa1-7 in figure 9a , asymmetrically distributed throughout its three domains) that promote binding of up to two moles of fatty acid per mole of hsa under normal physiological condition [141] [142] [143] . compounds with p-isothiocyanate (p-scn) or nhs ester (n-hydroxysuccinimide) functional moieties covalently react with albumin's lysine, with lys199 residue being the most reactive [144, 145] . however, due to multiple other (at least ten) surface accessible lysine residues in hsa, reaction with lysine residues leads to poorly defined conjugates with a range of stoichiometries [146, 147] . alternatively, the solvent accessible cysteine34 in hsa can be selectively modified with a prodrug containing a maleimide functionality, as (i) 70% of circulating serum albumin possess one free cysteine; (ii) other major serum proteins lack a solventaccessible free cysteine; and (iii) the 34 th cysteine residue of albumin (pk a ~ 7) has the most reactive thiol group in human plasma [144, 148] . trafficking: albumin naturally transcytoses across the vascular endothelium, which normally creates an impermeable barrier to most plasma proteins. this process is attributed to the 60 kda vascular endothelium receptor sialoglycoprotein (gp60). albumin binds to gp60 and forms a cluster in association with cav-1, the main protein critical to caveolae formation. clustered albumin-gp60 receptors and compounds bound to albumin are then internalized and transported to the basolateral membrane to complete transcytosis [149] . interestingly, modified albumins show preferential binding to gp18 and gp30 [150] . preferential internalization of albumin in cancer cells has also been correlated with albumin binding to sparc (secreted protein acidic and rich in cysteine). for example, immunohistochemical staining detected stromal j o u r n a l p r e -p r o o f sparc in ∼70% of non-small cell lung cancer (nsclc) cases, and increased chemotherapeutic efficacy of albumin-bound paclitaxel was correlated with high stromal sparc reactivity of nsclc cells [151] . recycling: the long half-life of albumin is attributed to the neonatal fc receptor (fcrn), a widely distributed intracellular receptor responsible for salvaging albumin from cellular catabolism [143] . fcrn binds to albumin in the acidic endosome, diverts it from the lysosomal degradation pathway, and the complex is exocytosed. albumin is released from fcrn at extracellular ph and enters circulation through the lymphatics, thus prolonging its half-life. albumin also avoids renal clearance by reabsorption through megalin and cubilin receptormediated endocytosis in the renal proximal tubule. in situ albumin-binding drugs have been designed to covalently react with or noncovalently bind to endogenous albumin, which capitalizes on the long-circulating property of the protein to enhance the pk and hence pd of the drug. indeed, exploitation of endogenous serum albumin has several advantages over the use of exogenous albumin: first, although commercial exogenous albumin can be isolated with high yield and purity, it is often contaminated with pathogens. second, the cost, effort, and time required for manufacturing exogenous albumin is altogether avoided. third, as no external macromolecular carrier is involved, the quality control associated with endogenous albumin is comparable to small molecule drug candidates. irreversible covalent bond formation between endogenous albumin molecules and a therapeutic payload can be utilized to enhance the pharmacokinetics of the latter. kratz et al. pioneered this strategy by synthesizing a prodrug that selectively reacts with the 34 th cysteine residue of circulating serum albumin after systemic administration, which improves the drug's plasma half-life and protects it from premature degradation. the therapeutic cargo (e.g., dox) was anchored with a thiol reactive maleimide group via a ph-sensitive hydrazone bond and a carefully optimized alkyl spacer. upon intravenous injection, the highly reactive maleimide group formed an irreversible thioether bond with the thiol group of the albumin. the ph-sensitive hydrazone bond facilitated the liberation of the covalently attached dox from albumin after en endocytosis by cells. aldoxorubicin, also known as dox-emch (figure 9b & c) is currently j o u r n a l p r e -p r o o f under various phases of clinical trial for the treatment of soft tissue sarcomas (nct01673438 and [152] ) and small cell lung cancer (nct02235688). inspired by dox-emch, many other albumin-binding prodrugs have been developed. these prodrugs often consist of an anticancer drug, a maleimide group as the thiol-binding moiety, and a cleavable linker [144] . native albumin-binding ligands such as fatty acids can be directly conjugated to therapeutics, which results in docking of the drug to endogenous albumin upon systemic administration. a notable example of this class is semaglutide, an fda approved drug that is a glucagon-like peptide-1 (glp-1) analog with an octadecyl fatty diacid conjugated to the epsilon amine of a lysine residue in the peptide via a glutamyl ethylene glycol spacer [153] . the plasma half-life (t 1/2 , seven days in humans) and glp-1 receptor (glp-1r) binding affinity (k d ~ 0.38 nm) of semaglutide make it suitable for once-weekly administration for treatment of type 2 diabetes [153, 154] . this was an important advancement, as its predecessor liraglutide -which contains a hexadecyl monoacid conjugated to the same lysine residue of glp-1 via a γ-glutamic acid spacer-has a t 1/2 of only 0.5 days in humans, and is hence only approved for once-daily treatment of type 2 diabetes, despite having a three-fold higher affinity for glp-1r (k d ~ 0.11 nm) than semaglutide. this dramatic improvement was attributed to both the spacer and ligand chemistry of the glp-1 analog in semaglutide, which influenced in vivo albumin binding [153] . other therapeutic payloads are bound to endogenous hsa by conjugation of the drug to fatty acids through various cleavable linkers [125, 144] . synthetic small molecules can also bind endogenous albumin [144] . most drugs and small molecules that interact with hsa are anionic, although a few cationic drugs have detectable affinity [142] . evans blue (eb), an aromatic dye with four anionic charges, reversibly binds to serum albumin with a micromolar affinity. the affinity of eb for albumin allows 100% of the injected dose to be retained in the blood; thus, it can be used to quantify total plasma volume of a test subject [155] . albumin-bound eb can also be used to assess blood brain barrier (bbb) permeability, as the bbb is impermeable to the complex in healthy, non-pathological conditions [156] . eb derivatives with various affinities to albumin have been used to deliver therapeutic cargo [144, 157] . yamamoto et al. developed an o-toludine eb analogue that chelates gadolinium (iii), as a t 1 -weighted mri contrast agent for imaging of blood vessels [158] . labeling with different pet isotopes (e.g., 68 ga, 64 cu) [159] [160] [161] . chen et al. evaluated labeled neb in mice as a blood pool imaging agent under various pathological conditions, including myocardial infarction (mi) and serum leakage from permeable or abnormal blood vessels. sentinel lymph nodes (slns) were visualized in all 24 pathologically diagnosed breast cancer patients within 4.0−10.0 (5.6 ± 1.4) min [161] . by analyzing 68 ga-neb pet/ct images. to identify molecular features required for albumin binding, the neri group screened a dnaencoded chemical library comprising >600 oligonucleotide-conjugates of potential albumin-binding molecules coded with unique six-base-pair sequences for identification [162] . the hsa-binding tags were chosen through systematic evolution of ligands by exponential enrichment (selex). the selection, amplification, and microarray analysis of the pool suggested that following structural features are important for albumin binding: (i) the presence of a 4-phenylbutanoic acid moiety is essential; (ii) a butanoyl acid moiety increases albumin affinity as propanoyl and pentanoyl acid residue was not enriched in the pool; and (iii) substitution at the para position of the phenyl ring with a hydrophobic functionality increases the affinity towards albumin. albumin-binding domains (abds) are small protein domains that non-covalently associate with serum albumin. abds are structurally robust and stable, as evidenced by their ability to withstand denaturation at extremely low ph (2.4) and high temperatures. and have also been explored for drug delivery. abds are used for drug delivery by either fusion with a therapeutic protein at the gene level if the protein is recombinantly produced or are chemically synthesized to conjugate small molecule drugs for systemic administration. sjöbring and colleagues isolated a 14-kda albumin binding protein fragment from streptococcal protein g. this 46-amino acid native abd (abdn) is a 3-helix bundle and has nanomolar affinity for hsa [163, 164] . using abdn as a template, jonsson et al. identified an engineered version of abdn from a phage library. specifically, they targeted 15 residues in a combinatorial protein engineering strategy to identify an albumin -binding sequence with improved hsa affinity. the high -affinity abd (abdh) has superior thermal stability and binds hsa with femtomolar affinity. chilkoti and colleagues recombinantly fused abdn and abdh to the n-terminus of a hydrophilic, thermally responsive chimeric polypeptide (cp) that contained multiple c-terminal cysteine residues [126] . the recombinant polypeptides formed highly monodisperse micellar nanoparticles upon covalent conjugation of multiple copies of hydrophobic dox molecules to the cysteine residues through the ph-sensitive hydrazone bond (using maleimide-thiol chemistry) ( figure 10a) . the dox-loaded abdn/abdh-decorated polypeptide micelles -termed abd-j o u r n a l p r e -p r o o f journal pre-proof cp-dox-bind both human and mouse serum albumin with high affinity, as seen by native page and isothermal titration calorimetry (figure 10b & d) . hence, upon systemic injection, they instantaneously bind endogenous albumin with high affinity and are coated with an albumin corona. the albumin decorated abd-cp-dox nanoparticles had significantly superior pk in both murine and canine models compared to a negative control, cp-dox nanoparticles that do not present abd domains on the surface of the nanoparticle (figure 10e & f) the long plasma circulation of the abd-cp-dox nanoparticles also resulted in higher accumulation in tumors than the cp-dox nanoparticles, and these nanoparticles also showed better tumor regression, and a wider therapeutic window than the cp-dox nanoparticles. these results clearly show the enhancement in pk and pd conferred by decorating a drug-loaded nanoparticle with endogenous albumin. furthermore, the same group took advantage of the native sortase reaction to directly install dox onto the abd (abd-dox) through a ph-sensitive linker, without forming nanoparticles [59] . abd-dox bound to both human and mouse serum albumin with nanomolar affinity, had a terminal t 1/2 of 29.4 h in mice, and increased tumor accumulation by ~120 fold compared to free dox ( figure 11a ). in multiple mouse xenograft models, abd-dox resulted in greater tumor regression than aldoxorubicin, dox-prodrug under clinical development that was designed to covalently bind endogenous serum albumin. other notable examples of albumin-based delivery systems involve the genetic fusion of abd to various therapeutic proteins including affibodies [165, 166] , human soluble complement receptor type 1 [167] , single chain antibody-drug conjugates [168] , insulin-like growth factor ii [169] , immunotoxins [170] , and respiratory syncytial virus subgroup a (rsv-a) g protein (g2na) [171] . j o u r n a l p r e -p r o o f albumin-bound therapeutics can also be formulated ex vivo with purified albumin molecules prior to administration. in contrast to other physiological proteins, albumin tolerates a wide range of ph (stable in the range of 4-9), temperatures (can be heated at 60 °c for up to 10 h), and organic solvents [50] . these properties have motivated researchers to covalently and non-covalently append various payloads, including radioisotopes (e.g., 18 f, 68 ga, 111 in) and anticancer drugs (e.g., dox, curcumin, methotrexate), to albumin for imaging and delivery purposes [125, 144] . however, modification of exogenous albumin with drugs is often associated with suboptimal drug loading, as denaturation and albumin molecule crosslinking potentially compromise its physiological behavior. smith et al. addressed these challenges by synthesizing an albumin-polymer-drug conjugate with high drug loading efficiency [172] . using a convergent, reversible addition−fragmentation chain transfer (raft) polymerization reaction, they synthesized a polymeric prodrug of panobinostat, an fda-approved anticancer agent, with hpma (n-2-hydroxypropylacrylamide). multiple copies of panobinostat were attached to the polymeric prodrug, which was then conjugated to albumin using nhs chemistry under physiological conditions, thereby minimally affecting the albumin molecule ( figure 11b ). the first albumin-drug conjugate that entered phase i/ii trial was a methotrexate-albumin (mtx-hsa) conjugate synthesized using covalent coupling between carboxylate of the mtx and lysine residues of hsa. seventeen patients not amenable to standard care were treated with up to eight injections given in weekly intervals. tumor responses were seen in three with no sign of toxicity and drug accumulation. the mtd for weekly administration was found to be 4 × 50 mg/m 2 and a injection of 50 mg/m 2 every 2 weeks was recommended for a further study [173] . however, in a subsequent phase ii study no objective responses were seen, although eight patients did not have disease progression (stable disease) for up to 8 months (median 121 days) [174] . a phase ii study showed combination of mtx-hsa with cisplatin as an effective treatment modality against urothelial carcinomas with an acceptable toxicity profile [175] . patients were treated with a loading dose of 110 mg/m 2 of mtx-hsa followed by a weekly dose of 40 mg/m 2 starting on day 8. tumor response was observed in 7 patients. complete response (cr) and partial response (pr) was observed in 1 patient each (overall response rate: 29%) but no follow up clinical investigation was undertaken. paclitaxel, an fda approved paclitaxel formulation for the treatment of multiple cancer types [176, 177] . abraxane was developed by american bioscience using the so-called nab-technology in which paclitaxel and human serum albumin are passed through a jet under high pressure to form nanoparticles with a mean diameter of 130 nm. several albumin nanoparticle of small molecule drugs has been developed using nab technology and are under various phases of clinical trials [178] . nab-rapamycin is currently under phase i clinical trial to treat non-muscle invasive bladder cancer (nct02009332) and under phase ii clinical trial to treat progressive high-grade glioma (nct03463265). nab-docetaxel proved to be effective against hormone refractory prostate cancer (nct00477529) and metastatic breast tumors (nct00531271). nab-5404, a novel albumin formulation of thiocolchicine dimer exhibits dual inhibition of tubulin polymerization and topoisomerase i activities. it showed antiangiogenic and vascular targeting activities and was found to be effective against solid tumors and lymphomas (nct01163071). lin et al. reported a method to simultaneously encapsulate paclitaxel by denaturing albumin with sodium borohydride at high urea concentration, followed by addition of hydrophobic drugs to the denatured protein, and diluting the mixture to spontaneously refold albumin and form nanoparticles [179] . this method avoids chemical crosslinkers and high-pressure emulsification techniques. by adding a cell-penetrating peptide to the dual drug-loaded albumin nanoparticles, they achieved bbb penetration through interaction with sparc, which resulted in greater survivability in a u87 orthotopic glioma model [179] . albumin has also been derivatized into a diblock format in which a hydrophilic albumin block gets exposed at the surface of the hybrid nanoparticle and a synthetic hydrophobic polymer, such as polycaprolactone, forms the core. to achieve this, jiang et al. synthesized two different polymers based on ring-opening polymerization: poly(oligo-(ethylene glycol) methyl ether acrylate)-poly(ε-caprolactone) (poegma-pcl) and maleimide-functionalized polycaprolactone (mi-pcl). mi-pcl was conjugated to the free cysteine on bovine serum albumin (bsa-pcl) [180] . the authors formulated hybrid nanoparticles by co-assembling poegma-pcl, bsa-pcl, and curcumin with increasing albumin content. cellular uptake of the nanoparticles in cancer cell lines increased with albumin content [120] . additionally, albumin nanoparticles can be decorated with targeting ligands, such as mannose [181] , folic acid [182] , antibodies [183, 184] , and aptamers [185] , to provide greater receptor specificity. recombinant albumin is another alternative to animal-derived albumin and is increasingly being used in exogenous formulations. direct genetic fusion to recombinant albumin enables one-step fabrication of therapeutic proteins. for instance, rlx-fp, a genetically encoded fusion protein linking recombinant human albumin with human coagulation factor ix, has a 5-fold longer j o u r n a l p r e -p r o o f plasma half-life than unmodified coagulation factor ix (fix) products. an advanced phase iii clinical trial (prolong-9fp) demonstrated the long-term safety, tolerability, and efficacy of rix-fp for prophylactic and on-demand treatment of bleeding episodes in children [186] . li et al. synthesized an albumin-lidamycin conjugate by recombinant dna technology. lidamycin, a cyclic enediyne antibiotic is ~1000 fold more potent than dox against multiple cultured cancer cell line and it consists of an apoprotein (ldp) and an enediyne chromophore (ae) that can be separated and reassembled in vitro [187] . a dna fragment encoding hsa-ldp was constructed and the fusion protein was purified from pichia pastoris using imac. the hsa-ldp conjugate was then reconstituted with the ae to form the albumin-lidamycin conjugate that had a sub nanomolar ic 50 value in various cancer cell lines, significant tumor retention , and potent in vivo tumor regression efficacy against mouse hepatoma h22 model [188] . other recombinant albumin proteins include genetic fusions with interleukin-2 [189] and interferons [190, 191] . albuferon® aka albinterferon-α-2b was developed as a fusion protein of albumin and interferonα-2b (infα-2b) and is under phase iii for the treatment of hepatitis c infection (nct00724776). several other phase iii trials are under progress aimed at evaluating the efficacy and safety of albinterferon-α-2b [177] . however, high-yield synthesis of pure recombinant albumin and albumin fusions requires a complex procedure and specialized yeast expression system that are not readily available to most researchers. nguyen et al. made significant progress by purifying recombinant hsa from a bacterial expression system using maltose-binding protein and protein disulfide isomerase [192] . but an optimized bacterial expression system to purify recombinant albumin is still needed. collagen is the most abundant protein in mammals, making up nearly one-third of all proteins in the body. a diverse family of structural proteins, collagen is a major component of the extracellular matrix (ecm) and connective tissue. it has a broad spectrum of functions, including cell adhesion, cell migration, tissue scaffolding, and repair [193] . to date, thirty different classes of collagen have been identified and characterized [194] , though not all are relevant for this discussion. the most abundant class of collagen -fibrillar collagen-represents more than 90% of human collagen and includes types i, ii, iii, v, and xi. this class is a major component of skin, hair, ligament, tendon, cartilage, bone, and placenta. other common types of collagens, like iv and viii, construct the network structure of basement membranes [193, 194] . before discussing their molecular architecture and biomedical applications, it is important to define the term -collagens.‖ gelatin, a thermally degraded, collagen-derived product, is also termed j o u r n a l p r e -p r o o f ‗‗collagen'' in the literature despite losing the characteristics of real collagen, and will hence be discussed separately [195] . although the molecular architectures and functions of collagens vary greatly, they all share the same tertiary structure -the collagen triple helix [196] . under the electron microscope, native collagen shows a thread-like structure -fibrilsconsisting of collagen molecules organized in three interlocking polytripeptide chains that form a triple helix , with each chain having the general sequence giy-x-y, where x and y are occupied by proline (pro) and (4r)hydroxyproline with the highest statistical frequency. each collagen polypeptide chain is twisted around a threefold screw axis and exists in a secondary structure analogous to the left-handed polyproline ii-helix. the three polyproline ii-helices are held together by hydrogen bonding, which repeats periodically between the glycine amide in one helix and the carbonyl group of the amino acid residue of the neighboring helix. the three polyproline ii-helices form a right-handed triple helix stabilized by hydrogen bonds [194, 196, 197] . with tunable self-assembly, gel-forming ability, biodegradability, and responsiveness to external stimuli, collagens have been increasingly used for drug delivery and tissue regeneration [195, [198] [199] [200] . current research is focused on developing short, bioinspired model collagens, termed as collagen-mimetic peptide (cmps) or collagen-like peptides (clps) [201, 202] . clps are short, synthetic peptides arranged in the same triple-helical conformation as native collagens. clps have been used: (i) to elucidate the triple helix structure and the molecular forces responsible for this architecture; (ii) to target pathological collagen in vivo through triple helix hybridization; and (iii) as a bioactive domain to construct smart biomaterials through hierarchical self-assembly. each of these applications will be discussed separately as they are relevant for drug delivery. to exploit clps to fabricate biomaterials, it is critical to elucidate the intrinsic parameters affecting the sequence-based thermal stability of the triple helix. in contrast to native collagen, clps exhibit reversible phase transition behavior. their slow folding rate upon denaturation and small size allow researchers to thermodynamically characterize the folding and melting processes of clps by x-ray crystallography, atomic force microscopy (afm), light scattering, and circular dichroism (cd) and nuclear magnetic resonance (nmr) spectroscopy. many researchers have synthesized and studied polypeptides with gly-x-y sequences that fold into a triple-helical structure. persikov et al. investigated the role of guest amino acid residues at the x and y positions on the thermal stability of the triple helix [203] . they documented that the guest j o u r n a l p r e -p r o o f triplets gly-x-hyp and gly-pro-y can be used to quantitate the conformational propensities of all 20 amino acids to form a triple helix at the x and y positions in a (gly-pro-hyp) 8 persikov proposed that the triple-helical structure of clps is the direct result of the propensity of amino acids to adopt a polyproline ii-like conformation, which is driven by the high propensity of ionizable residues in the x position for interchain hydrogen bonding and a low propensity of bulky residues in the y position for effective solvation. building upon this concept, many studies have deciphered the structure-function relationship of the clp phase transition [201, 202, 204] . to improve the stability of the triple-helical structure of clps, c-terminal covalent crosslinking was performed using an interchain cystine knot derived from collagen type iii [205] [206] [207] . such covalent modification also includes the crosslinking of three α-chains at the cterminus using various templates such as cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid (kta) and tris(2-aminoethyl)amine-(succinate-oh) 3 (tren) [208, 209] . comparative analysis using cd and nmr spectroscopy revealed that the flexibility of the tren scaffold is superior to that of the kta scaffold for the induction of triple helicity [208] . however, to avoid cumbersome covalent crosslinking, simple noncovalent crosslinking strategies have been developed based on: (i) interchain metal bridging by functionalization of the terminus of clps with a chelating ligand [210, 211] and (ii) hydrophobic interactions using a single saturated hydrocarbon or lipid tail [212] [213] [214] . the thermal stability of the triple helix of lipid-modified clps increased as the monoalkyl chain length increased from c 6 to c 16 [214] . furthermore, introducing amino acids with guest residues that have a cγ substitution with a highly electronegative atom, such as fluorine and chlorine, in place of hydroxyproline also increased triple helix stability [215] [216] [217] [218] . it is important to note that the triple-helical structure restricts the sequence space available for synthetic clps, and until recently, was thought to be intolerant to substitution of glycine in the gly-x-y triplet. multiple recent reports have shown that modification of glycine with a thioamide, nitrogen atom, or azaglycine (azgly) yielded comparable or better stability of clps compared to their non-substituted counterparts. another recent approach incorporated metal-binding sites at the ends of clps to drive thermodynamically stable, nanomicro scale self-assemblies of various shapes [219, 220] . zheng et al. elucidated the role of surface electrostatics and hydrogen bonding on the stability of the triple helix and provided computational tools for de novo design of clps beyond the gly-x-y triplet [221] . taken together, these structure-function studies indicate that nature may not have optimized the stability of the collagen triple helix, and that there is plenty of room for further improvement through precise design of clps beyond tandem arrays of the gly-x-y triplet sequence. although the major impediment in clp research is deciphering the triple helix conformation and supramolecular assembly formation (discussed in the next section), biomedical applications of monomeric clps have gained attention in recent years using denatured collagen. degradation of the ecm is a crucial element governing the progression of tissue remodeling in many life-threatening diseases, including cancer, cardiovascular diseases, and organ fibrosis, as well as prevalent debilitating conditions, such as arthritis and intervertebral disc degeneration. during tissue remodeling, collagen molecules within the collagen fibers and networks are degraded by proteases, such as mmps or cathepsin, and denature at body temperature. in a seminal work, yu and coworkers demonstrated that unfolded, single-stranded clps with a sequence (gpo) n (where n=6-10) have a high propensity for binding denatured collagen through a -strand hybridization process‖, which is similar to the binding of complementary dna strands [222, 223] . due to the high serum stability of such collagen -hybridizing peptides [224] and their affinity for native collagen [222] , they have been used to selectively stain native collagen both in vitro and in vivo by fluorescently labeled clps [225] [226] [227] [228] [229] . fluorescently labeled clps can also target solid tumors through the triple helix hybridization process, as high mmp-9 activity in tumor tissue exposes denatured collagen [230] . because homotrimeric clps have little driving force for collagen hybridization, clps must be thermally dissociated by heating at 80 °c to the monomeric state before binding to a collagen substrate. to avoid the preheating step, the yu group developed a sequence (gfo) 9 by replacing the hydroxyproline with a fluoroproline (f) [231] . this new sequence cannot self-trimerize at body temperature but maintains the ability to hybridize with natural collagen chains. by appending an octa-glutamic acid residue at the nterminus of the (gpo) 9 , the authors attracted vascular endothelial growth factors (vegfs) to the collagen-binding site of the endothelial cells through charge-charge interactions, which resulted in tubulogenesis [232] . triple helix hybridization has also been used to impart collagen-targeting properties to nanoparticles [233, 234] and for sustained release of nucleic acids from collagen films and depots [235, 236] . recently, the hubbell lab recombinantly fused a collagen-binding domain (cbd) to interleukin-12 (figure 12) , a potent cytokine that stimulates the innate and adaptive immune system (cbd-il-12). intravenously administered cbd-il-12 preferably accumulated in the tumor stroma and provided a sustained intratumoral level of interferon-γ, thereby eliciting superior anti-tumor effects and fewer off-target toxicities when compared to naked il-12 [237] . in the past few decades, the wealth of knowledge accumulated about the structure of collagen has driven a surge in research focused on the supramolecular assembly of collagenlike peptides. in the previous section, we described how various molecular interactions can stabilize the formation of a triple helix. in this section, we delve deeper into the molecular determinants of higher-order self-assembly of clp--based biomaterials -beyond the triple helix-and how they can be used in drug delivery. we have organized this section by the specific molecular stimuli that trigger self-assembly. cysteine knot in clp self-assembly: in an early example, raines and coworkers used two cysteine knots to covalently link three clps, one of which protruded out to produce a sticky end during triple helix assembly [238] . the sticky ends forced the trimers to configure themselves in a head-to-tail fashion, which resulted in a long, single collagen triple helix. such collagen-like fibrils were much longer (400 nm) than the native type i collagen (300 nm). in a similar approach, the koide group developed a synthetic clp system in which three clps were preorganized in a staggered configuration that was locked in place by two cysteine knots [239] . cd, ultrafiltration, and laser diffraction analysis indicated that the staggered trimers formed large supramolecular architectures through intermolecular triple helix formation. however, hydrogels made of these collagen fibers did not successfully form due to concentration-dependent aggregation. to address this limitation, yamazaki et al. appended a hydrophilic arginine residue at the cysteine knotted end of the synthetic clps [240] . the disulfide-linked trimers of the gly-x-y triplet repeats formed hydrogels by spontaneous intermolecular triple helix formation upon cooling. the thermal sol-gel transition is reversible, and the design of the peptides can tune the transition temperature. koide's group also incorporated an integrin-binding sequence gfoger into one clp strand of the same knotted trimeric base unit. the supramolecular structure exhibited adhesion to human dermal fibroblasts that was comparable to natural collagens [241] . to further tune the gel properties, ichise et al. appended multiple end-to-end disulfides through crosslinking of chemically synthesized triple-helical clp. rheology showed that gel stiffness is controlled by the number of cysteine residues up to three, at which point it plateaus. with the incorporation of an integrin α 2 β 1 -binding sequence, the peptide polymer showed receptorspecific cell binding in vitro. moreover, cell signaling activity and biodegradability of such a system can be tuned by altering the content of integrin binding ligand in the peptide polymer and by varying the weight percentage of clp [242] . π-π interaction in clp self-assembly: the maryanoff group introduced an aromatic-aromatic recognition motif at either the n-or c-terminus of each peptide chain of the clp triple helix to drive the supramolecular assembly of clps [243, 244] . the aromatic π-π interaction facilitated head-to-tail stacking of clps into a micrometer-sized fibrillar structure, as evidenced by cd, 1 h nmr, dynamic light scattering (dls), tem, afm, and computational energy dynamics. the fibrillar clps induced the aggregation of human blood platelets, an ability lacking in less organized clps, with nearly the same potency as native type i collagen [245] . kar et al. investigated how the presence of aromatic residues on neither end, one end, or both ends of a collagen model peptide affected the kinetics of self-association. clps with aromatic residues at both termini self-assembled and aggregated too quickly to be monitored by turbidimetry at a concentration of 7 mg/ml [246] . similar interactions, such as ch-π interaction between amino acids (proline and hydroxyproline) and aromatic residues (phenylalanine and tyrosine) and cation-π interaction between a positively charged n-terminal arginine and a c-terminal phenylalanine, were also introduced to fabricate collagen-like fibrillar structures in a head-to-tail manner [246, 247] . hydrophobic interaction in clp self-assembly: hydrophobic interactions also contribute to clphybrid self-assembly and collagen-mimicking properties. the field and tirrell research groups independently synthesized clp-hybrids by conjugating collagen peptides with single or double hydrocarbon tails. lipidation of clps increased the triple helix stability, triggered self-assembly of the clp-hybrid in spherical micelles, induced the formation of lipid film, and promoted cell adhesion [248, 249] . to induce even higher-order assembly, the tong research group designed a series of clps consisting of four segments: an n-terminal palmitoyl segment, a β-sheetforming domain, a polylysine spacer, and a bioactive clp domain [250] . a clp with the sequence (gpo) 3 gfoger(gpo) 3 and a c-terminal c 16 hydrocarbon tail self-assembled into micrometer-long fibers with a 16 nm diameter in aqueous solution. self-assembled peptide nanofibers made of triple-helical clp constructs with bioactive gfoger sequences recognized and adhered to several integrin receptors and promoted cell spreading, thereby mimicking native collagen [250] . electrostatic interaction in clp self-assembly: the conticello and chaikof research groups first presented evidence of a self-assembled, fibrous, collagen-like structure with a well-defined dperiodicity using lateral electrostatic interaction through a sticky-ended nucleation strategy, j o u r n a l p r e -p r o o f reminiscent of that commonly used in the assembly of dna [251] . they used a synthetic peptide sequence (prg) 4 (pog) 4 (eog) 4 consisting of three distinct segments: a central tetrameric pog sequence flanked by an arginine-containing positively charged domain at the n-terminus and a glutamic acid-containing negatively charged domain at the c-terminus (figure 13 ). tem suggested that in the absence of thermal annealing, non-banded fibers are formed. in contrast, fiber growth was observed within several hours after thermal annealing. a nucleation-growth mechanism was followed by the self-association of multiple triple helices, which was driven by electrostatic interactions between oppositely charged terminal residues and hydrophobic interactions between central domains from neighboring triple helices. building upon this concept, o'leary et al. proposed a new sequence (pkg) 4 (pog) 4 (dog) 4 by replacing arginine and glutamic acid with lysine and aspartic acid, respectively [252] . they argued that arginine has a higher propensity to hydrogen bond with a carbonyl group of a neighboring strand compared to lysine, which inhibits its ability to form a salt bridge with the carboxylate group of the glutamic acid and thus forms homogenous collagen mimetic nanofibers. the modified peptide sequence formed both homogeneous fibers and hydrogels, and the hydrogel was degraded by collagenase at a rate similar to that for natural collagen. raines's group adapted this approach to generate extended fibers a few microns in length with a highly symmetrical and uniform -tesselated‖ geometry that was dictated by the precise placement of lysine-aspartate salt bridges in the staggered triple helix building blocks. the extended geometry of this construct maximizes the surface area available to support the growth and maturation of cells [253] . notably, the banding periodicity of these peptide fibers (18 nm) is significantly longer than the length of the peptide itself (10 nm) [251] . this elongation suggests that the units driving self-assembly are not the individual triple-helical peptides but oligomers of multiple triple helices that are preorganized such that their assembly into mature fibers leads to periodic packing and charge distribution, as visualized by tem. this contrasts with the native type i collagen fiber, in which staggered assembly of individual collagen molecules is responsible for the periodic banding pattern and suggests that true mimicry of the natural collagen assembly process has yet to be achieved. hydroxyproline (o-propionate hydroxyproline, p e ) that shows ph-sensitive folding [254] . melting and folding experiments using cd indicated that introducing 1-3 units of the unnatural amino acid p e into the backbone had little effect on triple helix stability at acidic and neutral ph, although the rate of folding into the triple helical structures were different. they also observed that a peptide in which all seven hydroxyproline residues were substituted with p e underwent ph-triggered folding with an even lower transition temperature than the parent clps [254] . research group led by chmielewski and hsu also placed metal-binding ligands at various points along the clp backbone for chelation-controlled higher-order assembly. they appended histidine or iminodiacetic acid to the termini of the (pog)n backbone and upon adding divalent metal ions, such as ni 2+ , zn 2+ , and cu 2+ , the clps co-assembled into petal-like microstructures with a periodic banding at the nanometer scale [255] . subsequent work from the chmielewski group demonstrated that a diverse range of distinct structures, including microflorettes, stacked sheet microsaddles, and fiber-like meshes, could be made by varying the length of the (pog)n core of the peptides and the metal ion concentration. [256] . as demonstrated by hsu and coworkers, a similar metal-promoted supramolecular assembly can also be obtained by introducing histidine residues at both termini as well as into a clp backbone lacking hydroxyproline. a variety of higher-order structures were obtained, ranging from the nano-to micro-scale [219] . metal-induced self-assembly is advantageous over lateral electrostatic interaction because diverse supramolecular structures can be obtained under mild physiological conditions. a repertoire of unsatisfied metal-ligand interactions are also available in these selfassembled structures that enable incorporation of functional peptide sequences [257] [258] [259] [260] for biomedical applications, including 3d culture of human endothelial cells, protein purification, and fluorescence tagging. temperature -triggered self-assembly of clps: kiick and co-workers recently reported the temperature -triggered self-assembly of clp-hybrid materials [261] [262] [263] [264] . they appended a synthetic polymer, poly(diethylene glycol methyl ether methacrylate) (pdegmema) or human tropoelastin -derived elastin-like polypeptide (elp, to be discussed in a later section) with clps. both pdegmema and elps are thermally responsive and exhibit lower lcst-phase behavior. above a transition temperature (t t ), they undergo reversible phase transition from a soluble phase to an insoluble coacervate, which drives the self-assembly of the clp-hybrid into vesicular-or platelet -like structures. conversely, triple helix formation of the clp chains drives the formation of a bilayer of the supramolecular assembly (figure 14 ) [265, 266] . it is important to note that the t t of the short elp sequence is drastically reduced in the clp-elp hybrid due to formation of a clp-triple helix that increases the local concentration of the elp. above the j o u r n a l p r e -p r o o f melting temperature of the clps, these nanostructures disassemble into unimers. moreover, the clp-elp vesicle can target type ii collagen, which is overexpressed in various diseases, including cancer, through triple helix hybridization. these vesicles exhibited sustained release of an encapsulated fluorophore over a period of three weeks, and complete release was triggered by destroying the clp -helical structure with heat [267] . gelatin is a well-known, biodegradable, biocompatible, and nonimmunogenic biomaterial obtained either by partial acid or alkaline hydrolysis or by thermal or enzymatic degradation of type 1 collagen [268] . gelatin is composed primarily (85-92%) of proteins, with minerals and water constituting the rest. gelatin is used in a wide range of food, cosmetic, and pharmaceutical applications [268, 269] . the triple helix conformation of gelatin endows it with high thermal stability in solution [270] . the use of gelatin has gained popularity for the following reasons: (i) gelatin is readily available and inexpensive; (ii) gelatin is highly soluble and easy to use; (iii) gelatin's structure is highly similar to that of collagen and contains peptide binding motifs for cell attachment; (iv) gelatin is biocompatible, biodegradable, and does not induce antigenicity or at the sequence level, gelatin molecules contain repeating (gly-x-pro)n triplets, where x represents the amino acid (usually lysine, arginine, methionine, or valine), that is responsible for the triple helical structure of gelatin. gelatin is a polyampholyte that is ~13% positively charged (lysine and arginine), ~12% negatively charged (glutamic and aspartic acid), and ~11% hydrophobic (leucine, isoleucine, methionine and valine) [271] . another important trait shared by collagen and gelatin is the low frequency of aromatic amino acids -tryptophan, tyrosine, and phenylalanine-in their primary sequence. the low frequency of these amino acids is the primary reason for the low antigenicity and toxicity of both gelatin and native collagen [272] . commercially, two different types of natural gelatin -cationic type a & anionic type b-are j o u r n a l p r e -p r o o f available and differ based on the details of the manufacturing process -collagen hydrolysisused to obtain them [273] . gelatin a is obtained from porcine skin following acid pre-treatment, which minimally hydrolyses the amide groups of glutamine and asparagine and results in a macromolecule with a pi ranging from 7 to 9. conversely, gelatin b is extracted from osseincut bovine hide-and is pre-treated at alkaline conditions, which leads to hydrolysis of asparagine and glutamine to aspartate and glutamate, respectively, thus resulting in a lower pi of 4-6. recombinant gelatin, which can serve as a substitute for the natural protein, is also commercially available [274, 275] . however, unlike collagen, gelatin suffers from mechanical instability and has high rate of degradation. it is also highly susceptible to several proteases. gelatin nanoparticles (gnps) prepared without crosslinking were unstable and formed aggregates [271, 273] . for these reasons, crosslinking of gelatin-based materials is required to give it mechanical stability, shape, and enhanced circulation time in vivo [8, 271, 276] . crosslinking of gelatin can improve its thermal and mechanical stability, as well as lower its degradation in vivo. crosslinking, which modifies cleavage sites of gelatin molecules, inhibits enzyme-substrate interactions and hence increases the resistance to degradation [277] . various crosslinking agents, including aldehydes, genipin, carbodiimide/n-hydroxysuccinimide, and enzymes, have been explored to covalently crosslink gelatin [268, 271] . the crosslinking density, defined as bloom number, of the gelatin biomaterials can be altered by the crosslinking reaction conditions and/or increasing the concentration of crosslinker, thus regulating its physicochemical and biomedical properties. in addition, the mechanical properties of gelatin can also be tuned by temperature and ph. at a sufficiently high concentration, gelatin forms a semisolid hydrogel at lower temperatures. as the gelatin solution cools to below 30°c, a random coil to triple helix transition, promoted by stable hydrogen bond formation, occurs [278] . changes in ph affect the melting temperature of gelatin and hence the propensity for gel formation. moreover, changes in ph modify ionic interactions between gelatin monomers and consequently affect hydrogen bondmediated crosslinking [279] . physical crosslinking is also possible and can reversibly modulate gel strength and has the advantage that it does not need enzymes or chemical reagents to effect crosslinking [280] . various methods such as desolvation, coacervation, emulsification, nanoprecipitation, and layer-by-layer coating have also been used to fabricate gelatin-based systems for drug and gene delivery [268, 271] . gelatin nanoparticles (gnps) range from 200 to 400 nm in size [281] . colloidal stability, drug release, drug encapsulation, tissue distribution, and cellular uptake are strongly dictated by the charge and size of gnps. the effects of various parameters, such as temperature, ph, j o u r n a l p r e -p r o o f cosolvents, degree of crosslinking, and type of gelatin, on the physicochemical properties of gnps have been investigated by several groups [282] [283] [284] . a temperature of 40°c was found to be optimal to attain a narrow size distribution of gnps because at q high temperature (50 °c), gelatin forms a random coil, which destroys the self-assembly and the nanoparticle while at room temperature, gelatin forms a highly viscous semi-solid. in contrast, at 40 °c, a controlled uncoiling of the triple-helical structure results in monodisperse gnps. the size and dispersity of gnps is highly dependent on solution ph, and ph 3 and ph 11 were found to be optimal for nanoparticle formation of cationic type a and anionic type b gelatins, respectively. increasing the degree of crosslinking results in greater polydispersity and a reduction in particle size. gnps and their hybrids have been explored to encapsulate both hydrophobic and hydrophilic bioactive molecules including antimalarial drugs [285] , such as chloroquine phosphate; anti-cancer drugs [271, 286] , such as paclitaxel, dox, cisplatin, curcumin, and methotrexate; growth factors [287] , such as osteogenic bone morphogenetic protein-2 (bmp-2), and angiogenic basic fibroblast growth factor (bfgf); anti-inflammatory drugs [288] , such as ibuprofen; photo-responsive dyes [289, 290] ; anti-hiv drugs [291] such as didanosine; and therapeutic nucleic acids [271, 292] . in all cases, an on-demand release profile was achieved by controlling the degree of crosslinking, ph, temperature, and fabrication method and thus the physicochemical properties of the gnps. for example, amjadi et al. designed a ph-responsive nanocarrier by decorating the gnps with (methoxy poly (ethylene glycol)-poly ((2dimethylamino) ethyl methacrylate-co-itaconic acid) (pgnps) (figure 15a ) [286] . the nanoparticles (162 nm diameter) were encapsulated with dox and betanin with high efficiency (loading capacities of 20.5% and 16.25%, respectively), and demonstrated ph-and temperature -responsive drug release, and synergistically inhibited the growth of mcf-7 breast cancer cell lines. in another example, rose bengal -conjugated gelatin nanoparticles (rb-gnps) were developed for antimicrobial photodynamic therapy ( figure 15b ) [290] . rb-gnps generated singlet oxygen that damaged the microbial cell membrane, which demonstrated their potential for treating multi-drug-resistant microbial infections. gelatin microparticles (gmps) have been extensively investigated for many biomedical applications. solorio et al. engineered self-assembled, gelatin microsphere-incorporated human mesenchymal stem cell (hmsc) sheets [293] . the microspheres were loaded with growth factor tgf-β1, distributed within the hmsc sheets, and released chondrogenic growth factor to the j o u r n a l p r e -p r o o f interior of the sheets, thus enabling spatially homogenous differentiation of the hmscs. the differentiation rate could be tailored by adjusting the microsphere crosslinking levels, which controls the release kinetics of the growth factor. this system not only produces hmsc sheets with superior mechanical properties but also decreases the pre-implantation culture time. in addition, the hmscs are continuously exposed to growth factor by microparticles, which prevents loss of the chondrogenic phenotype in vivo. similar controlled release of growth factor and proteins from gmps to stimulate tissue regeneration have been achieved for vascular endothelial growth factor (vegf) [294] , bone morphogenetic protein-2 (bmp-2) [295] , basic fibroblast growth factor (bfgf) [296] , and matrix metalloproteinases (mmps) [297] . in all cases, the release kinetics were dependent on the degree of crosslinking of gmps. it is well known that the use of most hydrophobic drugs is limited by their poor aqueous solubility, short circulation time, low bioavailability, and narrow therapeutic window. gmps have been used to address some of these challenges. for instance, the antibacterial activity of curcumin -a hydrophobic polyphenol derived from turmeric with a wide range of biological properties-is negligible at concentrations up to 100 mg/ml. however, curcumin encapsulated in gmps at 4 mg/ml reduced the microbial population by 2.08, 1.67, 2.70, and 2.18 log counts (cfu/ml) for l. monocytogenes, s. enterica, s. aureus, and e. coli, respectively [298] . gmps encapsulated with noscapine, an anticancer drug that targets human nsclc, demonstrated first order release kinetics and increased the plasma half-life of the drug by ~ 10-fold with an accumulation of ~50% drug in the lungs [299] . kim et al. used a microfluidic approach to fabricate monodisperse gmps with a microshell structure for embolization, a minimally invasive, nonsurgical procedure that deliberately blocks a blood vessel [300] . uniform gelatin emulsion precursors were fabricated using the microfluidic technique and consecutively crosslinked by inbound diffusion of glutaraldehyde from the oil phase to the suspending droplets of gelatin precursor. the authors also performed a model micromechanics study in an artificial blood vessel, which revealed that the microshell structure results intorapid degradation of the gmps within a narrow time period leading to burst release of the drug under chemoembolic conditions. gelatin fibers are used for various biomedical applications such as tissue-engineering and wound-healing, due to their morphological resemblance to the structure of the native ecm. gelatin fibers can be fabricated by several methods, such as melt spinning, wet spinning, gel spinning, electrospinning, centrifugal spinning, and solution/melt blow spinning. multiple processing parameters, including solution properties, humidity, and temperature, can influence the morphology of nanofibers and release rate of the entrapped drug. for instance, nanofibers fabricated by electrospinning of poly (d, l-lactide-co-glycolide) (plga) and gelatin were loaded with fenbufen. the hydrophilicity of the fibers was enhanced by increasing the gelatin content, which in turn increased the release rate of fenbufen. in contrast, the addition of a crosslinker (glutaraldehyde vapor) reduced the drug release rate [41] . vitamins a and e incorporated in gelatin nanofibers via electrospinning promoted the proliferation of fibroblasts, increased the expression of collagen-specific genes, and supported wound healing [301] . another research group demonstrated that genipin-crosslinked gelatin fibers loaded with human vegf sustained viability, promoted endothelial differentiation, attracted human mesenchymal stem cells (hmscs) and stimulated early angiogenesis [302] . chemically modified gelatin has been extensively used as hydrogel because of its tunable mechanical and biochemical properties. gelatin-methacryloyl (gelma), a synthetic, photosensitive crosslinkable derivative of gelatin, is widely used as a precursor to fabricate gelatin hydrogels and is commercially available as a bio-ink for 3d printing. various crosslinking methods, such as ultraviolet stereolithography, gamma irradiation, and two-photon polymerization, have been used to fabricate gelatin-based hydrogel and scaffolds [303] . light-activated crosslinking of methacrylated gelatin in air or in an aqueous solution resulted in an injectable hydrogel that supported hmsc growth and tgf-b3-induced chondrogenesis [304] . mazaki et al. developed furfurylamine-conjugated gelatin (gelatin-fa) that was rapidly crosslinked by visible light with rose bengal [305] . hybrid gelatin hydrogels can also be made from various synthetic polymers, including plga, poly (hydroxyethyl methacrylate), and polyvinyl alcohol, or natural polymers, such as chitosan and alginate. composite hydrogels consisting of gelatin, polyvinyl alcohol, and the anticancer drug cisplatin were synthesized as a controlledrelease drug delivery system. in vivo, inhibition of tumor growth was similar for hydrogels containing a low dose of cisplatin and conventional intraperitoneal administration of high doses of free cisplatin [306] . kinsella and colleague fabricated various alginate-gelatin bio-printable composite hydrogels that resemble the microscopic architecture of native tumor stroma [307] [308] [309] . 3d-printed hydrogels embedded with breast cancer cells, and fibroblasts promoted the self-assembly of breast cancer cells into multicellular tumor spheroids (mcts), which were viable for more than 30 days [307] . gelatin -based tissue bioadhesives, fabricated alone or with other synthetic and natural polymers, have also been used in drug delivery. composite tissue adhesives, developed by carbodiimide -mediated crosslinking of gelatin and alginate, were used to entrap the antiinflammatory drugs bupivacaine and ibuprofen, and the resulting adhesive was used for local pain management [310, 311] . hydrophilicity of the adhesive and its swelling properties control the release of drugs from this matrix. the incorporation of a drug -bupivacaineimproved the bonding strength of the adhesive, whereas incorporation of ibuprofen adversely affected the bonding strength. this is due to the inert nature of bupivacaine and the reinforcing effect of its fibrous crystals. in contrast, ibuprofen reacts with the crosslinking reagent -carbodiimideand decreases the crosslinking density and hence bonding strength of the adhesive networks [311] . to address this issue, n-hydroxysuccinimide can be added during crosslinking of gelatin and alginate to decrease the carbodiimide content without compromising the bonding strength. the resulting patch is less cytotoxic (due to low carbodiimide content) and can be used for controlled release of various antibiotics such as clindamycin, vancomycin, and ofloxacin [310] . like collagen, elastin is a key protein found in the ecm and connective tissues. it is abundant in organs that require elasticity and resilience for their function, including skin, ligaments, lungs, and blood vessels [312] . its inherently disordered structure provides elastin with unique properties that impact its recombinant production and purification, drug-loading capacity, and in vivo behavior. the soluble precursor to elastin, tropoelastin, is composed of alternating hydrophobic and hydrophilic domains that undergo enzymatic crosslinking via their lysine residues in the extracellular space. the resulting elastin fibrils are durable and insoluble. although these characteristics are critical for its function in biological tissue, they were a hindrance during early efforts to manipulate elastin as a biomaterial [313] . consequently, soluble and recombinant forms of elastin were explored. the most widely studied elastin-based materials used for drug delivery are elastin-like polypeptides (elps) [314] . elps are biopolymers with the val-pro-gly-x-gly pentapeptide motif, in which x represents any amino acid besides proline [315] . perhaps the most interesting property of elps is their thermal responsiveness, as they demonstrate lcst phase behavior. in an aqueous solution, they are soluble below a characteristic transition temperature (t t ). above their t t , elps reversibly phase transition and forms an insoluble, polymer-rich phase. this behavior is thermodynamically driven by the j o u r n a l p r e -p r o o f favorable entropy of demixing above the t t (figure 16a & b) [316] . spontaneous mixing occurs when the gibbs free energy change is negative. for an elp to be solubilized, water molecules must be ordered along the polypeptide chain. this interaction results in a negative entropy term. a small temperature term compensates for this hit in entropy, which keeps the gibbs free energy change negative and allows the elp to stay in solution. however, as the temperature term increases, elp-water mixing becomes more energetically unfavorable. the entropy term becomes dominant, causing the elp to aggregate into a -coacervate‖ phase. the temperature at which this phenomenon occurs is dependent on multiple features of the elp and the solution environment [317, 318] the elp's molecular weight and concentration in the aqueous solution are inversely related to its t t , which is also impacted by the guest residue. high concentrations of hydrophobic residues, such as lysine or valine, depress the t t and high concentrations of hydrophilic residues, such as alanine or glycine, increase the t t [319] . elps retain their lcst behavior when recombinantly fused to a drug, targeting moiety, or other molecule, but the t t can be similarly affected by the surface hydrophobicity and charge of its fusion partner [320] . the t t of an elp solution can be easily quantified by optical turbidity measurements of an elp solution at 350 nm (od 350 ) as a function of solution temperature. as the temperature is increased, the elp will transition from an optically transparent solution to an opaque mixture within a narrow, 1-2°c temperature window. the t t can be graphically determined at the inflection point of an od 350 vs temperature plot, which is typically a sigmoidal curve (figure 16c ) [321] . because the t t is concentration dependent, it should be analyzed over a range of application -relevant concentrations. the lcst behavior of elps can also be used in a chromatography-free purification process termed inverse transition cycling (itc). initially developed by meyer and chilkoti, this procedure cycles cell lysate through a series of hot and cold centrifugation steps [322] . after insoluble cell debris is removed from the lysate via centrifugation, the elp-containing supernatant is heated above the t t of the elp. kosmotropic salts, which interact more favorably with the water along the elp chain, may be added to facilitate the phase transition and promote aggregation. the solution is centrifuged to pellet the elp while soluble contaminants remain in the supernatant. the elp pellet is then dissolved in cold buffer and centrifuged below the t t , which causes insoluble contaminants to precipitate while the elp remains in solution. this process is rapid and inexpensive, avoids the use of harsh chemicals, and can be scaled up [322] . elps have hence been used as a purification tag for other proteins [323] . after itc, the elp can be cleaved from the protein of interest using a proteolytic cleavage site or self-cleaving intein that is incorporated between the elp and protein [324, 325] . j o u r n a l p r e -p r o o f though small molecule drugs and peptides demonstrate potency and -in the case of peptides-specificity, their efficacy is frequently hindered by their rapid clearance from circulation or poor solubility (kalepu & nekkanti, 2015) , necessitating frequent dosing [326, 327] . [328] scientists have addressed these limitations by conjugating macromolecular carriers to these therapeutics to improve their circulation time, target-site accumulation, and solubility [327, 329] . soluble elp unimers have been recombinantly fused to therapeutic peptides and chemically conjugated to small molecule drugs for these purposes [330] [331] [332] . elp fusions benefit from the large molecular weight of the elp, which extends the circulation time of the fusion partner, and enhance the solubility of the drug, and improve their biodistribution, and pharmacokinetic profiles. compared to synthetic drug carriers, elps benefit from monodispersity and a lack of toxicity [315] . despite the advantages of employing elps as soluble macromolecular carriers, they lack the stealth properties provided by some hydrophilic polymers, such as poly(ethylene glycol) and zwitterionic polymers [333] . these -stealth‖ polymers create a hydration sphere around the payload and polymers, which improves the pharmacokinetic profile of the conjugate by protecting it from opsonization and premature clearance from circulation [334] . recently, banskota and coworkers modified the canonical elp sequence to impart stealth behavior to biopolymers [335] . they designed zwitterionic polypeptides (zipps), which are composed of repeat units of the val-pro-x1-x2-gly motif, where x1 and x2 are positively and negatively charged amino acids, respectively ( figure 17a) . like elps, zipps can be recombinantly expressed in e. coli, and exhibit the favorable properties of elps including monodispersity, ability to recombinantly be fused to a therapeutic peptide, and sequence-level control of their properties. additionally, zipps can be purified without chromatography by inverse transition cycling, similar to elps. zipps provide greater solubility to their payload than elps as a result of their charged residues [335] . most importantly, zipps had a 3-fold longer half-life and 2-3-fold greater bioavailability compared to an uncharged elp control. consequently, when glp-1 was j o u r n a l p r e -p r o o f fused to either a zipp or a mw-matched uncharged elp control, the zipp achieved 1.5-fold longer blood glucose control, which demonstrates that the stealth behavior of zipps can increase the efficacy of their therapeutic cargo (figure 17b ) [335] . owing to their ease of production and thermal phase behavior, elps are useful materials for design of self-assembling nanostructures and nanoparticles to deliver a variety of therapeutic agents. elp nanoparticle self-assembly is driven by hydrophobic interactions. depending on the design, the hydrophobic component might be either an elp segment or its conjugated hydrophobic cargo. elp amphiphiles, which contain two connected elp blocks with different hydrophobicities and hence t t , form micelles at temperatures between the t t of each block. at phenylalanine and valine were the guest residues. due to the presence of ionizable residues in the sequence, the size and shape of the self-assembled nanoparticles were ph-sensitive. above the cmt, these elp fusions self-assembled into spherical and cylindrical morphologies [336, 337] . conticello et al. later designed triblock elps, which were made of a hydrophilic block between hydrophobic end blocks [338] . the hydrophobic blocks phase transitioned and aggregated to act as -crosslinks‖ in an elastomeric network. j o u r n a l p r e -p r o o f subsequently dreher and coworkers systematically explored the design parameters that controlled elp nanoparticle self-assembly by creating a library of ten diblock elps [339] . they discovered that the temperature at which unimers assemble into micelles depends upon the length of the hydrophobic domain. in contrast, micelle diameter is controlled by the length of the copolymer and the ratio of hydrophobic to hydrophilic blocks. dreher et al. also incorporated a functional ligandthe tumor-homing ngr peptide sequence at the terminus of the hydrophilic segment of a diblock elp. these micelles were readily internalized by cells that recognized the displayed ligand, which demonstrates that the elp diblock design can be engineered for targeted drug delivery [339] . several models have contributed to our understanding of diblock elp self-assembly. elps based on the t t values of each elp domain. they found that although the cmt was dependent on the hydrophobic domain, the bulk transition temperature was dependent on the hydrophilic block [340] . a second, theoretical model developed by hassouneh et al. used polymer physics to study micelle assembly. upon examination of six diblock elps, they found that these copolymers form -weak‖ micelles with dense cores and unstretched coronas, which is unique for diblock polymer micelles [341] . the initial studies on diblock elp micelle targeting were expanded by the development of dynamic affinity modulation, a strategy in which local hyperthermia induces micelle formation to increase the avidity of a low-affinity ligand that is fused to the n-terminal end of the hydrophilic segment of diblock elp for site-specific accumulation and targeting. simnick and coworkers first used this strategy in a proof -of -concept study in which a low-affinity ligand specific to α v β 3 integrin was recombinantly fused to the n-terminus of an elp [342] . cysteine residues were incorporated at the c-terminus for drug conjugation. at temperatures below the cmt of the elp, the fusion protein existed as a soluble unimer and the low-affinity ligand interacted weakly with the α v β 3 integrin. in the presence of local hyperthermia, the elp underwent temperature -triggered self-assembly into spherical micelles, thus increasing the valency and avidity of the ligand such that it could efficiently bind to and be taken up by cells [342] . this strategy was then modulated to achieve more specific cell internalization of cell-penetrating peptides (cpps) [343] . cpps are arginine -rich sequences that promote uptake by electrostatically interacting with the cell membrane of multiple cell types. cpps have been conjugated to nanoparticle surfaces to improve their internalization but lack specificity. macewan and chilkoti addressed this issue by incorporating five arginine residues onto a diblock elp. they found that below the cmt, the number of arginine residues was below the threshold of six necessary to induce cell uptake [344] . local hyperthermia drove the formation of micelles that displayed a high valency of arginine on their surfaces, resulting in 8-fold increase in cell internalization [343] . the design of these nanoparticles and their thermal -switch‖ allow for control over the site of action of elp nanoparticles, which can reduce off-target drug delivery and mitigate systemic toxicity. furthermore, adding these targeting moieties did not disturb micelle formation. hassouneh and coworkers showed that when proteins ~10 kda in size are incorporated at the end of the hydrophilic block, the diblocks still self-assembled [345] . diblock elps have also been designed for applications beyond cancer, including specific targeting to liver cells [346] and lacrimal gland cells [347] . to improve tumor-specific drug delivery of diblock elp micelles, callahan and coworkers engineered a diblock elp that disassembled in the acidic tumor microenvironment [348] . histidine, which has a pka near physiological ph, was incorporated into the hydrophobic block. these micelles assembled at 37°c, but upon exposure to the low ph conditions mimicking the tumor microenvironment (ph ~6), the histidine became ionized. consequently, the t t of the hydrophobic block increased, and it was no longer energetically favorable for elps to selfassemble. the micelles broke down into elp unimers, resulting in better penetration and accumulation of the elp in the tumor compared to ph insensitive elp micelles. this study demonstrated that diblock elps can be engineered to overcome the diffusion barrier many nanocarriers face when delivering drugs to tumors [348] . mackay and coworkers have adapted diblock elps nanoparticles for two-phase rapamycin release by nonspecifically entrapping the drug in the nanoparticle core as well as specifically displaying the drug on the nanoparticle corona [349, 350] . this type of display was achieved by appending fk506 binding protein12 (fkbp12), the cognate protein target of rapamycin, to the hydrophilic block of the elp fusion. the entrapped rapamycin was rapidly released upon administration, while the release of fkbp12-bound rapamycin was prolonged. the two-phase release increased the terminal t1/2 of rapamycin to 57.8 h compared to 2.2 h for nanoparticles without fkbp12-bound rapamycin [350] . the nanoparticle formulation also reduced off-target toxicity compared to the free drug. in the aggressive breast cancer model mda-mb-468, treatment with the nanoparticle resulted in more potent reduction in tumor volume and extended survival time [350] . in a model for the chronic autoimmune disease sjögren's syndrome, the treatment downregulated inflammation in the endocrine gland and reduced lymphocytic infiltration into the lacrimal gland [349] . other elp nanoparticle designs fuse an elp segment with a peptide or protein to drive self-assembly. in an early example of this method, mcdaniel and coworkers incorporated repeats of the sequence (xg y ) z onto the elp c-terminus, where x is a hydrophobic residue [351] . relative to the elp, the (xg y ) z domain is only a few percent by mass in the diblock. despite the low mass fraction of the second segment, it was surprising and notable that these highly asymmetric amphiphiles self-assembled into cylindrical micelles with lengths that could be controlled by altering the identity of x or the value of y-the number of glycine spacers [351] . mackay et al. further explored how small proteins and peptides impact nanoparticle assembly. they discovered that fusing an scfv -a single chain antibody-to a hydrophilic elp block drives the fusion to self-assembly into a worm-like micelle, with the elp forming the corona [352] . despite being entrapped in the core, the scfv remained active due to the low packing density of the corona. these nanoworms outperformed their antibody equivalent in an in vivo xenograft model of lymphoma [352] . another study by mckay and coworkers was driven by the amphipathic nature of l4f, whose four phenylalanine residues orient face-to-face to create vesicles with 8 nm thick lamellae and a 49 nm radius [353] . l4f maintained its anti-inflammatory properties despite being in the vesicle lamellae. this study demonstrates how amphipathic peptides may be used to drive elp vesicle formation, which can be leveraged to encapsulate soluble therapeutics into the aqueous vesicle interior. hydrophobic drug conjugation can also trigger elp self-assembly into nanoparticles, masking the drug inside the core, thus increasing the aqueous solubility of the drug and protecting it from premature release and degradation. this results in improved drug half-life, biodistribution, tumor accumulation, and efficacy while reducing off-target toxicity. an early example of this strategy was developed by chilkoti and coworkers. in their bottom-up nanofabrication method, termed attachment directed assembly of micelles (adam), a hydrophobic (cgg) 8 domain was conjugated to the c terminus of a hydrophobic elp [354] to create a -chimeric polypeptide‖ (cp). the cysteine-rich domain of the cp enabled the attachment of up to eight maleimide -functionalized small molecules (figure 18) . when the hydrophobicity of the small molecule exceeds a threshold (log ≥ ~1.5), it provides the conjugate sufficient amphiphilicity to drive self-assembly [354] . this platform was adapted for drug delivery by chemical conjugation with dox to create cp-dox nanoparticles, which self-assemble at physiological ph at concentrations above 3 µm. dox is rapidly released upon exposure to an acidic ph (5) , which mimics the conditions of late endosomes and lysosomes. a single injection of cp-dox had 4-fold higher maximum tolerated dose and reduced tumor size nearly 13-fold compared to free drug, indicating that this nanoparticle formulation can be harnessed to j o u r n a l p r e -p r o o f enhance potency while reducing off-target effects such as cardiotoxicity. adam has also been leveraged to deliver the hydrophobic chemotherapeutics paclitaxel [355] and niclosamide [356] . while the original adam strategy was successful at enhancing the efficacy and safety of hydrophobic chemotherapeutics, many cancer drugs are hydrophilic and cannot be encapsulated by this strategy. to address this, the chilkoti and coworkers developed a new strategy by the design of an asymmetric triblock polypeptides [357] . atbps consist of three segments-a hydrophilic elp segment that is fused to a hydrophobic elps segment that in turn is fused to a drug attachment (ggc)8 segment. the atbp is deigned to retain its temperature triggered amphiphilicity even upon conjugation of hydrophilic small molecules with a logd ≤ 1.0 that the drug-conjugated atbp self-assembles into rod-shaped micelles above its cmt, which is designed to be below body temperature. they showed that conjugation of hydrophilic drugs, such as gemcitabine, to the cysteine residues of the atbp did not abrogate the formation of micelles, and these drug-loaded atbp micelles had greater efficacy in an aggressive in vivo model of colon cancer compared to free drug [357] . while cysteine-maleimide covalent conjugation can incorporate a structurally diverse set of drugs, molecules, and imaging agents into the nanoparticle's core, this strategy relies on functionalization of a naturally occurring amino acid. these residues can be promiscuously distributed across other peptides that may be appended to the elp such as targeting peptides or proteins, and, consequently, site-specific drug attachment for these constructs is challenging. to address this concern, costa and coworkers inserted p-acetylphenylalanine, an unnatural amino acid, onto an elp micelles that displayed a functional nanobody [358] . the nanobody contains a disulfide bond that is essential for its stability and functionality, so that site specific covalent conjugation of a drug by introduction of cysteine residues in a drug attachment was not possible. the p-acetylphenylalanine residue provided a biorthogonal ketone moiety to sitespecifically conjugate dox via a ph-sensitive bond without affecting the disulfide bond of the nanobody. as a result, this platform enabled site-specific drug conjugation while enabling the display of a functional nanobody on an elp nanoparticle [358] . [354] . fatty acids have also been incorporated by a post-translation modification into elps to drive nanoparticle assembly. luginbuhl and coworkers incorporated a myristoyl group at the nterminus of an elp that presented a peptide substrate for n-myristoyltransferase. the enzyme was co-expressed with the elp from a bicostronic plasmid in e. coli that grown with exogenous myristic acid added to the culture medium [359] . this led to close to 100% myristoylation of the elp in e. coli, and the myristoylated elp (m-elp) formed spherical or rod-like micelles depending on the elp chain length. chemotherapeutic drugs could be physically loaded into the micelles via hydrophobic interactions with the fatty acid, which resulted in a greater encapsulation efficiency than that possible with diblock elps [359] . this strategy thus provides a useful mechanism for entrapping drugs without covalent conjugation, making it possible to deliver drugs that do not contain an active functional group for conjugation, or drugs that lose potency upon conjugation. elps have been similarly combined with silk to form silk-elastin like protein (selp) nanoparticles [71, 360] . nanoparticle self-assembly is driven by both hydrophobic interactions and hydrogen bonding between the silk blocks. the radius of the nanoparticle is controlled by altering the elp guest residue as well as the ratio of silk blocks to elp blocks [361] . xia and coworkers demonstrated that these nanoparticles can be used to deliver hydrophobic small molecule drugs by loading dox into the nanoparticles [362] . dox was entrapped in the core by hydrophobic interactions with the silk blocks. the selp nanoparticles were endocytosed into the cell, thus allowing drug to accumulate in the cytoplasm. the cytotoxicity of the selp-dox nanoparticles was 1.8-fold greater than that of free dox [362] . elps provide a useful platform for sustained drug release due to their lcst behavior. their t t can be precisely tuned by modulating the molecular weight and guest residue of the sequence, making it possible to engineer the elp with a t t below physiological temperature. the elp thus remains soluble at room temperature, enabling injection through narrow-bore syringe, but rapidly coacervates upon exposure to body temperature, forming a subcutaneous depot. because the t t is concentration -dependent and the boundary layer of the coacervate is diluted by interstitial flow, the depot slowly undergoes surface-to-core dissolution. as it dissolves, the therapeutic cargo is released into circulation. this sustained -release platform, coupled with the ability of elps to impede renal filtration, extends the half-life of small molecules and peptides to which the elp is fused. prolonged -release systems are advantageous for diseases that require long-term treatment, such as cancer or chronic illnesses. liu et al. systematically altered elp parameters to adapt the molecule for in situ radiotherapy [363, 364] . they recombinantly fused a hydrophobic elp, comprised of 60 repeats of a val-pro-gly-val-gly pentapeptide, to a tyrosinerich domain that could be radiolabeled with 131 i. an intratumoral injection of the 131 i-elp was retained in the tumor for more than one week. the elp shielded the radionuclide from dehalogenase degradation and increased its tumor retention time [363] . liu and coworkers found that doubling the molecular weight of the elp increased tumor retention time by 14-fold; however, the effect plateaued with the t t once the elp surpassed 120 repeats. similarly, increasing the injection concentration of 131 i-elp 16-fold resulted in 5-fold greater tumor retention [364] . furthermore, 131 i-elps with seven repeats of the hydrophobic, tyrosine-rich peptide domain self-assembled into rod-shaped micelles and formed long-lasting -seeds‖. the seed-like particles exhibited potent anti-tumor efficacy, remarkable intratumoral retention, and minimal toxicity [364] . the coacervate was further stabilized by radiation-induced crosslinking of the tyrosine residues in the hydrogel, which resulted in retention of 52% and 70% of its radioactivity over 60 days in a prostate and pancreatic tumor model, respectively [365] . more recently, wang and coworkers engineered an elp depot for the sustained release of interferon-α, an anti-cancer protein that has been clinically hindered due to its short half-life and dose-dependent toxicity [366] . fusion of the protein to 90 repeats of a val-pro-gly-val-gly pentapeptide resulted in near zero-order release of ifn-α over the course of a month. compared to free ifn-α and ifn-α fused to a soluble elp, the depot-forming ifn-α-elp exhibited greater antitumor efficacy in a melanoma model. the fusion was well tolerated and demonstrated little renal toxicity, key advantages over free ifn-α [366] . the enhanced pharmacokinetics of glp1 are attributed to the increased residence time of depot-forming glp1-elp compared to the soluble control or glp1 alone. adapted with permission from [368] . elp depots have also been extensively engineered for the treatment of diabetes. antidiabetic peptides such as insulin and glucagon-like peptide-1 (glp-1) have short half-lives that necessitate frequent and repeated injections to achieve long-term blood glucose control. there is hence a need for sustained -release systems that prolong the efficacy of these peptides, and which reduce the need for multiple injections and improve patient compliance. glp-1 to and elp that was designed to coacervate at body temperature. the depots were retained in the subcutaneous space and lowered blood glucose levels for up to 5 days [367] . luginbuhl and coworkers expanded upon this work and optimized the elp parameters for depot retention and glp-1 half-life [368] . they discovered that elps with t t values of approximately 30°c and molecular weights greater than 36 kda achieved near zero-order release kinetics for glp-1. the optimized glp1-elp fusion formed a depot in the subcutaneous space upon injection as a solution and provided glycemic control in three different diabetic mouse models for up to 10 days after a single injection (figure 19) . in a cynomolgus monkey model, circulating levels of the elp fusion were detected for 17-21 days post-injection. glp-1 delivery has also been achieved by introducing protease cleavage sites between oligomeric repeats of glp-1 on an elp. once these so-called -protease-operated depots‖ are injected subcutaneously, proteases under the skin free glp-1 from the elp in the depot. this system extended blood glucose control for 5 days [367] . the antidiabetic protein fibroblast growth factor 21 (fgf21) has also been fused to an elp by gilroy et al. to improve its release, half-life, and production [369] . fusion to the elp enhances fgf21 solubility, thus allowing it to be expressed in the bacterial cytosol rather than inclusion bodies and consequently avoiding costly, complex refolding steps during purification. the elp-fgf21 depot was retained in the subcutaneous space for up to 4 days and achieved glycemic control in an ob/ob mouse model for up to 5 days. in a long-term study, treatment with an elp-fgf21 fusion resulted in 40% reduction in mean serum insulin and triglyceride levels when mice received subcutaneous injections every 5 days for up to 60 days [369] . other elp depots have been engineered for the treatment of dry eye disease [370] , infectious illnesses [371] , and inflammation [372] . resilin is another elastomeric protein that has been extensively engineered for biomedical applications. natural resilin is a rubbery protein with remarkable elasticity that is derived from the exoskeletons of insects and arthropods, where it is a critical component of locomotive systems such as jumping and flight [375] . it possesses high resilience and extensibility, low stiffness, and the ability to efficiently store energy [376] . these unique mechanical properties, coupled with its biodegradability, make resilin an attractive protein for regenerative medicine and drug delivery [377] . after ardell and anderson identified tentative resilin gene sequences in drosophila melanogaster [378] , elvin and coworkers engineered the first example of a recombinantly expressed resilin-like polypeptide (rlp) in 2005 [379] . the resulting polypeptide, termed rec1resilin, is composed of the first exon of the d. melanogaster cg15920 gene, which contains seventeen copies of the ggrpsdsygapgggn motif [379] . rec1-resilin possesses the favorable mechanical properties of native resilin with the added benefit of customizability provided by its recombinant synthesis. upon photochemical crosslinking of the tyrosine residues, rec1-resilin demonstrated up to 92% resilience that is comparable to that of many synthetic rubbers [379] . dros16. an16 is composed of sixteen repeats of an eleven amino acid motif found in anopheles gambiae while dros16 is composed of sixteen copies of a fifteen amino acid motif in the first j o u r n a l p r e -p r o o f exon of the cg15920 gene from which rec1-resilin is derived [380] . similar to rec1-resilin, these rlps were highly resilient, elastic, and capable of photo-crosslinking [380] . rlps are intrinsically disordered polypeptides that contain high concentrations of glycine, which provides them with high flexibility and makes secondary structure formation entropically unfavorable. the high proline content of rlps also prevents secondary structure formation, as the bulky residue reduces hydrogen bonding [381] . further, unlike other elastin-mimetic proteins, rlps contain high frequencies of polar and charged residues and are thus hydrophilic. rlps are responsive to a variety of stimuli, including temperature [382, 383] . rlps demonstrate dual phase behavior (dpb), an unusual property characterized by both a lcst and upper critical solution temperature (ucst), the temperature above which the rlp is fully miscible in solution. the dpb of rlps is fully reversible and largely dependent on the molecular composition and architecture of the rlp. their ucst behavior is also strongly impacted by ph [384] . at ph values greater than the pi, the charged residues enhance rlp solubility and the ucst increases. at low ph values, carboxylic acid residues on the rlp become protonated, which reduces the molecule's solubility and drives up the ucst [383, 385] . the tunable dpb of resilin is useful to fabricate modular drug delivery systems, described in the next section. the unique, temperature-driven phase behavior of rlps provides numerous opportunities for designing nanoparticles, whose size, shape, and aggregation number can be tuned to create drug delivery vehicles for a variety of applications. li et al. initially described the factors that impact rlp nanoparticle formation using a novel rlp based on twelve repeats of a fifteen amino acid consensus motif derived from d. melanogaster [385] . they replaced the tyrosine residues with phenylalanine and methionine, which can be leveraged for subsequent chemical modification. to form nanoparticles, the polypeptide was heated to its lcst of 65°c, at which point it irreversibly aggregated [385] . li and coworkers found that the lcst was concentration-and ph-dependent; transition temperature decreased with increasing concentration or decreasing ph (figure 20) . interestingly, the presence of salt improved rlp solubility and increased the transition temperature, which is the opposite of elps (figure 20) . this phenomenon is attributed to the ionizable residues in the rlp. the resulting nanoparticles can be tuned to have diameters ranging from 20 nm below the transition temperature up to nearly 400 nm when heated beyond their transition temperature [385] . this study demonstrates how the temperature-triggered phase behavior of rlps can be exploited to design nanoparticles with precision. block copolypeptides that incorporate rlps have also been used to create nanoparticles. the self-assembly of diblock copolymers containing both ucst and lcst blocks is strongly impacted by temperature. at low temperatures, the ucst block is relatively hydrophobic and aggregates at the core while the lcst block remains soluble. as the temperature increases, the ucst block becomes hydrophilic and relocates to the nanoparticle corona as the lcst block aggregates [386] [387] [388] . this phenomenon motivated weitzhandler et al. to combine rlps with elps to better characterize temperature-triggered phase behavior of ucst-lcst block co-polypeptides with the goal of nanoparticle formation [389] . using an rlp composed of repeat units of the gln-tyr-pro-ser-asp-gly-arg-gly octapeptide and an elp, they identified two principles that affect nanoparticle formationthe rlp:elp mass ratio and elp hydrophilicity. with an increase in the rlp:elp ratio, they observed an increase in the nanoparticle size. in contrast, the hydrophobicity of the elp impacted morphology; a hydrophobic elp resulted in spherical micelles, while a hydrophilic elp promoted the formation of worm-like structures [389] . [385] . dzuricky and coworkers expanded upon this work to observe how nanoparticle morphology impacted cell uptake [55] . a fibronectin type-iii (fn3) -binding domain that binds the α v β 3 integrin was fused to the c-terminus of an rlp-elp diblock copolymer. they found that the fn3 domain did not perturb self-assembly of the rlp-elp diblock copolymer, so that it was displayed on the nanoparticle surface upon self-assembly (figure 21) . the tunable morphology and high avidity of the rlp-elp-fn3 nanoparticles significantly improved the binding affinity to the α v β 3 integrin via multivalency. they found that shape played an important role in promoting multivalency though the avidity effect. while a spherical rlp-elp micelle that presented a fn3 a) j o u r n a l p r e -p r o o f domain showed a respectable 10-fold increase in affinity for the α v β 3 integrin compared to the monovalent rlpelp-fn3 unimer, the worm-like rlp-elp-fn3 nanoparticles showed a remarkable 1,000-fold increase in affinity for the integrin (figure 21 ) [55] . the combination of morphology and high avidity yielded a picomolar k d , a remarkable achievement considering that a therapeutic antibody against the same integrin exhibits a k d of only 20 nm. consequently, the worm-like rlp-elp-fn3 was more readily taken up by cells than the antibody [55] . these studies demonstrate that rlp-elp nanoparticles can be harnessed for robust, specific cellular targeting and uptake of therapeutic agents. because of their exquisite stimulus responsiveness and mechanical properties, rlps have been engineered as hydrogels for many biomedical applications. although many of the hydrogels were designed for use in tissue engineering applications, the principles derived from these studies can are also useful for the design of rlp-based controlled drug delivery systems. charati and coworkers reported an early example of a rlp hydrogel [390] . to test its utility for biomedical applications, they incorporated several biologically active domains into the sequence to control cell adhesion, hydrogel degradation, and biocompatibility. upon purification from e. coli, the protein was crosslinked with [tris(hydroxymethyl) phosphine] propionic acid (thpp), which linked the lysine residues in the rlp sequence. the resulting hydrogel was mechanically stable and capable of supporting hmscs and mouse nih-3t3 fibroblasts [390] . li and coworkers later evaluated the mechanical properties of rlp hydrogels more precisely and showed that the extent of thpp crosslinking impacts factors such as swelling ratio, elastic shear modulus, and resilience [391] . su and coworkers adapted rlps for cell adhesion by combining cell binding domains from fibronectin with a consensus sequence derived from a. gambiae. after it was crosslinked with tris(hydroxylmethyl)phosphine, the novel rlp scaffold exhibited mechanical properties consistent with those of rec1-resilin and a compression modulus comparable to that of cartilage [392] . the material supported mscs at 95% viability for up to 3 days, and the cells exhibited extensive spreading on the material surface [392] . while this hydrogel was primarily engineered j o u r n a l p r e -p r o o f for tissue engineering applications, it provides an example of how peptides can be appended to the rlp sequence for other biomedical applications such as drug delivery. other moieties, such as heparin [390, 391] , vegf-mimicking peptides [393] , and chitin -binding domains [394] , have similarly been appended onto rlps. in a reducing environment (b) , dextran release was not mw-dependent. in a non-reducing, pbs environment (c), dextran release was dependent on mw. adapted from [395] . taking advantage of the recombinant nature of rlps, su et al. engineered their novel rz10-rgd hydrogel to respond to changes in redox [395] . they used 3,3'dithiobis(sulfosuccinimidyl propionate) (dtssp), a redox-responsive crosslinker, to create rlp hydrogels that degrade in a reducing environment ( figure 22a) . various molecular weights of fitc-labeled dextran were loaded into the crosslinked rz10-rgd hydrogel. in reducing conditions, there was no observed difference in the release rates of dextrans with different molecular weights. in non-reducing conditions, release rate decreased with an increase in dextran mw (figure 22b ) [395] . the redox-responsiveness of this novel hydrogel makes it a promising candidate for tumor-specific drug delivery. other -smart‖ rlp hydrogels have been designed to degrade in response to elevated mmp concentrations [396] . when an mmp-sensitive domain was incorporated into a rlp sequence, the hydrogel underwent 90% degradation in the presence of physiological levels of mmps over the course of 48 rlp hydrogels without the mmp-sensitive domain did not degrade [396] . because the concentration of mmp-sensitive domains within the rlp hydrogel may be modulated to control degradation, this design can be adapted for drug delivery to tissues with elevated mmp concentrations, such as tumors or wound sites. resilin has also been combined with other proteins, including elastin, collagen, silk, and peptide amphiphiles (pas), to enhance the properties of rlps. bracalello and coworkers designed a chimeric polypeptide comprised of an rlp, elp, and clp in tandem, which they termed the rec polypeptide [397] . the purified material formed highly aligned fibrils that, over time, self-assembled into large fibers and fiber bundles. the fibers and bundles possessed a young's modulus of 0.1-3 mpa, which demonstrated that the material retained the elastic properties of resilin and elastin [397] . more recently, okesola and coworkers developed a highly tunable, multicomponent hydrogel using a covalent co-assembly strategy to combine an rlp with a peptide amphiphile (pa). this approach enabled control over the hierarchical self-j o u r n a l p r e -p r o o f assembly of the rlp hydrogel and enhanced its mechanical properties. the hydrogels were synthetized using a thiol-ene photoclick reaction and, depending on the pa concentration, assembled into a variety of nanostructures. in the absence of pa, the rlp hydrogels formed nanospheres upon photo-crosslinking, a low concentration of pa resulted in co-assembled beaded strings, and a high concentration of pa created co-assembled nanofibers. the photoclicked hydrogels maintained the remarkable elastic and energy-storing properties of rlp hydrogels, thus demonstrating that the co-assembly strategy can improve the mechanical properties of more brittle proteins such as pas. these results further support that rlp materials can be precisely tuned to achieve desirable properties for a drug release system. silk-elastin-like polypeptides, or selps, are block co-polymers consisting of tandem repeats of silk-like and elastin-like blocks [360] . these hybrid polypeptides combine the mechanical strength and chemical stability of silk with the unique thermal responsivity of elps, resulting in a material that can assemble into a variety of architectures ranging from nanomaterials to hydrogels [350] . like many other biopolymers, these materials are recombinantly produced, offering precise control over their length, the ratio of slp to elp blocks, and the ability to introduce biologically active molecules into the sequence [398] . an interesting characteristic of selps is their ability to undergo an irreversible sol-gel transition under physiological conditions, creating a matrix in situ that is useful for local delivery [360] . selps have been engineered to self-assemble into micellar structures that can be used as nanocarriers for drugs. the core of the micelle is formed by the slp blocks which form intermolecular hydrogen bonds to stabilize the micelle while the relatively hydrophilic elp forms the corona [361] . the hydrodynamic radius of the micelle can be tuned by altering the slp to elp block ratio or by altering the choice of guest residue in the elp block [361] . the self-assembly of selp nanoparticles may also be triggered by encapsulation of a hydrophobic drug. xia et al first explored this concept by mixing dox with selps with a slp to elp block ratio of 1:8, 1:4 or 1:2 [362] . the dox hydrophobically interacted with the silk j o u r n a l p r e -p r o o f domains, driving the formation of nanoparticles with hydrodynamic radii between 50 and 142 nm ( figure 23a) . these nanoparticles are in the appropriate size range to be passively targeted to solid tumors via the epr effect [399] . this approach is attractive as it avoids the need for harsh solvents and provides a respectable 6.5% drug loading efficiency [362] . dox-loaded selp nanoparticles enter hela cells via endocytosis rather than diffusion like free dox (figure 23b ). this allowed for potent accumulation of the drug in the cytoplasm and nearly 2-fold greater cytotoxicity than the free drug [362] . more recently, parker et al developed mucoadhesive selp nanoparticles for transmucosal drug delivery [400] . to identify potential mucoadhesive sequences, they generated a series of selps with a 1:4 slp to elp block ratio with four different guest residues -cys, arg, lys, and glu. these amino acids were chosen due to the potential of charged residues and thiol groups to interact with mucus. ans, a hydrophobic, solvatochromatic dye, was loaded into the nanoparticles via simple mixing to serve as both a model compound and reporter. in an in vitro biosimilar mucus model, the selp with cys guest residues demonstrated the strongest affinity for mucus, followed by selps containing lys, arg, or glu as the guest residue, respectively. interestingly, when incubated with mucus-secreting ht29-mtx cells, the cys-containing selp demonstrated little cell retention, suggesting it may have mucolytic properties. meanwhile, selps with charged guest residues were well retained by the cells, indicating their potential for transmucosal delivery. perhaps the most interesting property of selps is their ability to undergo a sol-gel transition under physiological conditions. these materials can be engineered to be soluble at room temperature, at which point drugs and other therapeutic molecules can be loaded by simple mixing without the use of toxic or denaturing solvents. upon injection, the material coacervates to form a polymeric matrix in situ. this property, along with the biocompatibility and j o u r n a l p r e -p r o o f sequence control of selps, makes them an attractive material for engineering local, controlled drug delivery systems. this approach was first explored by cappello et al in 1998 . in this study, fluorescently labeled macromolecules of varying molecular weights were loaded into selp hydrogels and their release rates were monitored [401] . for all compounds, release from the hydrogels was near first-order; however, there was no correlation between the release kinetics and the molecular weight of the molecule. this was attributed to the large size of the hydrogel pores, which are not small enough to impede diffusion [401] . the release rate decreased with an increase in selp concentration. the hydrogels were biocompatible when implanted in vivo with no sign of immunological activity or inflammation [401] . dinerman et al further evaluated the diffusion behavior of molecules out of selp hydrogels by using cytochrome c, theophylline, and vitamin b12 as model compounds [402] . they observed fickian, diffusion-controlled release behavior wherein diffusion slowed with an increase in molecular weight and polymer volume fraction [402] . due to the simplicity of drug loading and their gelation behavior, selps are also attractive materials for gene delivery. nucleic acids can be incorporated into the selp hydrogel by physical mixing, and upon injection, the selp hydrogel protects its cargo from rapid degradation while controlling the rate and location or release. this concept was first explored by megeed et al in 2002 using an selp containing positively charged residues. the negatively charged dna molecules electrostatically adsorbed onto the selp before injection [403] . they discovered that the release of dna was inversely related to the selp concentration and gelation time and that the ionic strength of the buffer directly correlated with dna release rate; as ionic strength increases, the greater concentration of counter ions disrupt the electrostatic interaction between the selp and dna, facilitating the release of the dna [403] . in a later study, megeed et al discovered that the release rate of plasmids from the selp hydrogel was inversely proportional to plasmid mw. furthermore, plasmid conformation impacted release rate, j o u r n a l p r e -p r o o f with linearized plasmids diffusing from the hydrogel fastest followed by supercoiled and circular plasmids, respectively [404] . the hydrogel maintained the stability and bioactivity of plasmid dna for 28 days and adenoviruses for 22 days. in an in vivo model with athymic nude mice bearing subcutaneous mda-md-435 tumors, mice treated with selps loaded with a plasmid for luciferase exhibited greater gene expression than mice treated with naked dna [404] . these studies aid the groundwork for selp design for dna delivery to treat cancer. hatefi et al loaded selp-47k, an selp comprised of four slp and 7 elp blocks with one elp block containing a lys substitution, with the adenoviruses ad.gfp or ad.cmv.lacz that encode green fluorescent protein (gfp) or β-galactosidase, respectively [405] . in an in vitro study, the selp hydrogel maintained the release of the adenoviruses over a 28 day period with only a 30-40% loss in virus activity ( figure 24a ) [405] . the ad.gfp-loaded selp was intratumorally showed significantly lower gfp expression than tumors receiving the selp formulation ( figure 24b ) [405] . this study demonstrated the potential of selps for adenoviral delivery to solid tumors. the ability of selp-815k to deliver adenoviruses was further tested in a head-to-head comparison with poloxamer 407, a commercially available synthetic copolymer used for gene delivery. both platforms were used to deliver a genetically encoded prodrug with an antiviral vector to a xenogenic model of hnscc. compared to poloxamer 407 and free virus, selp-815 exhibited the highest initial gene expression in tumors followed by prolonged expression for up to three weeks [406] . this high and sustained level of gene expression resulted in the greatest tumor size reduction and survivability across all groups. furthermore, selp-815k showed superior safety than poloxamer 407, as adipocytic necrosis was observed in animals treated with the latter [406] . a subsequent study demonstrated that, compared to free virus, virus-loaded selps also had reduced systemic toxicity [407] . more recent studies have provided new insights into the stability of viral vectors in selp hydrogels. two different selps were loaded with the oncolytic adenovirus ad-δb7-kox and evaluated with afm imaging to visualize the interaction between the adenovirus and selp j o u r n a l p r e -p r o o f nanofibers. afm showed that as adenovirus concentration increased, the density of the selp nanofibers also increased despite their fixed concentration [408] . this is likely attributed to the brownian motion of the adenovirus during assembly, as it acts as a stimulus for selp network formation. although most adenoviruses exhibited a round morphology for the first half-hour of being attached to selp, they began to deform after approximately 1 h. this is likely due to multiple strong interactions forming between adenovirus and different selp fibers [408] . it was also discovered that selp analogues with shorter elastin units exhibited lower temperature responsivity and more rapid elastase degradation [408] . with this fundamental understanding of selp degradation and adenovirus complexation, better gene delivery systems can be engineered from this material. selp hydrogels have also been used for sustained release of polysaccharides. the ghandehari group synthesized positively charged selp hydrogels that could electrostatically interact with an anionic polysaccharide -semisynthetic glycosaminoglycan ether (sage)-that is an anti-inflammatory drug for mucosa restoration. unfortunately when sage is administered, it does not achieve a high enough therapeutic concentration due to its insufficiently long residence time [409, 410] . to solve this problem for treatment of radiationinduced proctitis (rip), the ghandehari group designed an in situ gelling enema ( figure 25a ) [410] . in a murine model of rip, sage was present in the rectal tissue of mice 12 h after receiving a sage-loaded selp enema, while it was undetectable in mice receiving sage with saline ( figure 25b ) [410] . the ghanedhari group further adapted the selp platform for the sustained release of intravesically delivered sage for treatment of interstitial cystitis (ic) model. they found that employing a softer selp with a slower gelation time could provide an analgesic effect for 24 h without exacerbating the discomfort associated with ic, whereas stiffer gels could reduce the capacity of or obstruct the bladder [409] . j o u r n a l p r e -p r o o f more recently, researchers have focused on the design of de novo artificial polypeptides as drug carriers. these genetically engineered polypeptides are conformationally disordered, hydrophilic, and inert. like other protein polymers, they are recombinantly expressed and purified in bacterial systems, providing exquisite control over their length and sequence. they can also be genetically fused to therapeutic peptides or proteins to increase the half-life, stability, and biodistribution of their therapeutic partner. in addition to their stability and nonimmunogenicity, these hydrophilic biopolymers have long plasma circulation, making them an intriguing alternative to pegylation. in the last decade, two polypeptide sequences in particular-xten and pashave gained traction as biopolymeric drug carriers. the first xten molecule was designed and synthesized by schellenberger et al in 2009 [411] . to create a stable, soluble and unstructured stealth polymer, they eliminated hydrophobic amino acids known to contribute to aggregation or secondary structure formation, positively charged molecules that could bind cell membranes, and amide-containing residues that reduce stability. these criteria limited their pool of potential amino acids to sixalanine, glutamate, glycine, proline, serine and tyrosine. schellenberger et al then screened a library of nonrepetitive, randomized sequences to obtain an 864-residue biopolymer with optimal expression levels, yield, solubility, and stability [411] . this original xten molecule was genetically fused to exenatide, an antidiabetic peptide that has an in vivo half-life of 2.4 h. fusion to xten increased the peptide's half-life to 60 h in cynomolgous monkeys, with a predicted half-life of 139 h in a 75 kg human (figure 26 ) [411] . xten similarly enhanced the terminal half-life of gfp, human growth hormone, glucagon and factor vii. since this initial study, different mw variants of xten have been produced [412] and different functional groups for drug conjugationincluding azide, maleimide, and alkyne groups -have been introduced into the polypeptide [413] . xten has been similarly employed to counteract nocturnal hypoglycemia in diabetic patients. geething et al fused the biopolymer to gcg, a peptide hormone that converts glucagon stores into glucose but suffers from a minutes-long half-life and poor stability in liquid formulations [415] . in addition to improving the stability of gcg 60-fold, fusion to xten prolonged the efficacy of gcg in a fasted beagle dog model. gcg-xten provided strong resistance to a glucose challenge 6 h after prophylactic administration without affecting baseline blood glucose level [415] . the effect tapered off at 12 h post-administration, whereas free gcg was ineffective 2 h post-administration. xten also remarkably enhanced the pharmacokinetic profile of teduglutide, a glucagon-like peptide 2 analog used to treat an inflammatory malabsorption disorder in the small intestine with a half-life of ~3 h in humans [416] . the teduglutide-xten fusion achieved a 120 h half-life in cynomolgous monkeys, with a predicted half-life of 240 h in humans, further indicating how xten can be employed for once-monthly administration. the formulation also reduced the occurrence of ulcerations and presence of inflammatory cytokines j o u r n a l p r e -p r o o f in the small intestine [416] . additionally, this study was the first instance where the payload was chemically conjugated to xten; in this case, teduglutide was chemically conjugated via a cterminal cysteine residue. since then, xten has been chemically conjugated to other therapeutics including antivirals [412] and imaging agents such as annexin 5 [417] to improve their circulation time. xtenylation has been used to improve th p to human growth hormone (hgh), which typically suffers from an hours-long half-life and requires daily administration. somavaratan , an xtenylated formulation of hgh, achieved a half-life of 131 hr, a 30-60 fold increase compared to recombinant hgh in a phase i clinical trial (nct01718041) [418] . interestingly, fusion to xten reduced the potency of hgh 12-fold, yet the prolonged tissue exposure xten provided a 3 to 5-fold increase in efficacy compared to recombinant hgh [418] . in a phase ii clinical trial vista study (nct02068521), patients treated with somavaratan demonstrated increasing height velocity with increasing doses of the drug. and dose-dependent adverse events were minimal [419] . unfortunately , somavaratan failed to achieve the primary endpoint of non-inferiority versus genotropin, a recombinant formulation of hgh, in a phase iii velocity clinical trial (nct02339090) for pediatric patients with growth hormone deficiency. a phase ii clinical trial (nct02526420) has also been completed for growth hormone deficiency in adults. these studies demonstrate the promise of xten in improving drug half-life, bioavailability, and efficacy. [420] . more recently, a von-willebrand factor independent fviii-xten fusion, termed bivv001, has demonstrated good safety and efficacy in early stage clinical trials (nct03205163) and is currently recruiting for phase 3 (nct04161495) [421, 422] . xtenylation of fviii increases its plasma half-life three to four-fold relative to recombinant fviii, achieving a half-life of up to 34 h in cynomolgous monkeys [423] . this resulted in four-fold longer hemostatic control, suggesting that this formulation could provide clotting protection for patients for up to one week [423] . xten's large size has also been harnessed to take advantage of the epr effect to passively deliver anticancer therapeutics to tumors. to demonstrate its utility, haeckel et al designed a multicomponent fusion protein to improve the delivery of killin, a cytostatic and cytotoxic protein [424] . despite the promising antitumor effects of killin, its expression in bacterial systems was impossible due to its toxicity. c-terminal fusion to xten deactivated the protein so it could be stably expressed and purified. upon administration, xten would further serve to enhance the circulation time of killin and exploit the epr effect for tumor-specific targeting. a matrix metalloproteinase cleavage site was incorporated between the xten and killin, allowing the killin to be released and activated upon tumor penetration. to improve cell membrane penetration and dna binding, a cell penetrating peptide (cpp) was also added onto j o u r n a l p r e -p r o o f the c-terminus of killin [424] . the fusion protein was preferentially taken up into cancer cells with high mmp-2 or mmp-9 expression. this xten-killin-cpp fusion also arrested growth and induced concentration-dependent apoptosis in killin-sensitive cell lines, with the effect peaking at 48 h after treatment [424] . this study demonstrates the potential of xten as a carrier for anticancer therapeutics. this concept has recently been adapted by amunix to develop xtenylated protease-activated t-cell engagers, or xpat. in this technology, the bispecific t-cell engager blincyto, which engaged cd3 and and cd19, is xtenylated to reduce its immunogenicity. tandem protease recognition sites are incorporated between the xten chain and tandem scfvs of the bispecific t-cell engager to promote its release upon protease exposure. this technology has been adapted to target her2+ (amx-818) and egfr+ cells [425] . in recent pre-clinical studies, these fusions achieved picomolar affinity ec50s against cell lines expressing their target proteins while reducing t-cell toxicity 15,000-fold. they also induced significant proteasedependent tumor regression in xenograft models. her2-and egfr-xpat were also well tolerated by cynomolgous monkeys, with a 1000-fold greater c max for the her 2-xten fusion and a 100-fold greater c max for the egfr-xpat than their non-xtenylated counterparts [425] . a second recombinant polypeptide, termed pas, was engineered by xl-protein gmbh company in 2013 and is conceptually similar to xten [426] . both are intrinsically disordered, hydrophilic polypeptides that are designed to enhance the plasm half-life of fused proteins and peptides [426] . pas are reported to be non-immunogenic, biodegradable, and are produced in bacterial systems [426] . their recombinant synthesis enables precise control over their sequence and length enable gene-level fusion to a therapeutic peptide or protein of interest in order to enhance the pharmacokinetic profile of the fusion partner [427] . whereas xten incorporates negatively charged residues and consists of pro, tyr, glu, gly, ala and ser, pas do not contain charged amino acids and their sequence is limited to pro, j o u r n a l p r e -p r o o f journal pre-proof ala and ser [427] . in the initial study published by xl-protein gmbh company, pas was recombinantly fused to three proteins -a recombinant fab region of a humanized anti-her2 antibody, human interferon a2b, and human growth hormone-to evaluate its utility as a drug delivery carrier. fusion to pas increased the hydrodynamic volume of the cargo up to 26-fold and extended the blood circulation of the fusions [426] . the plasma half-life and auc of pasylated ifn was 32-fold and 100-fold greater than the non-pasylated ifn and the protein's binding affinity for its receptor was not affected by fusion to pas (figure 27a) . similarly, pasylated hgh exhibited a 94-fold greater plasma half-life than control hgh and exhibited a 3fold greater growth promoting effect than native hgh on an equimolar basis ( figure 27b ) [426] . since this initial study, pasylation has been employed to enhance the half-life and pharmacokinetics of numerous therapeutics. like other biopolymers, many applications that harness pasylation require long-lasting therapeutics to reduce dosing frequency and side effects. as such, xl-protein gmbh company has developed several formulations to improve the therapeutic efficacy of several peptides for the treatment of many diseases, including exendin for type ii diabetes, hgh for pediatric growth hormone deficiency, and anti-inflammatory agents for rheumatoid arthritis [428] . many of these fusions are currently in preclinical trials. other groups have also explored pasylation as a strategy to improve the circulation and efficacy of interferons for the treatment of multiple sclerosis. in a study conducted by harari et al, a superagonistic analog of ifnβ, ynsα8, was fused to the c terminus of a 600 amino acid pas polypeptide chain [429] . when administered to a transgenic mouse model harboring humanized ifnar receptors (hybnar model), pas-ynsα8 exhibited a 10-fold increase in half-life compared to ynsα8 alone without impacting the biological activity of the superagonist. despite being administered at an overall 16-fold lower dosage than ifnβ alone, pas-ynsα8 outperformed the control group in an experimental autoimmune encephalomyelitis model, as indicated by a decrease in infiltrating macrophages in the central nervous system and an increase in regulatory t cells [429] . in a separate study by zvonona et al, pas was fused to the j o u r n a l p r e -p r o o f journal pre-proof c-terminus of ifn-β1b and resulted in a 4-fold increase in hydrodynamic radius compared to free ifn-β1b [430] . the fusion protein also notably increased ic50 2-fold compared to free ifn-β1b, an accomplishment considering pegylation of the same interferon decreases its activity. furthermore, this formulation was stable when stored for 22 months at -20°c, indicating how pasylation can contribute to the maintenance of biological stability [430] . similarly, pasylation has contributed to the improved half-life and efficacy of numerous proteins including leptin [431, 432] , erythropoietin [433] , conversin [434] and clotting factor viii [435] . pasylation has also been harnessed to improve the delivery of nanocarriers, most notably for the treatment of cancer. in one study, pas 40 or 75 residues long were recombinantly fused to ferritin, an iron storage protein that self-assembles into a hollow sphere and binds internalizing transferrin receptor i, which is upregulated on many cancer cells [436] . pasylation stabilized the ferritin during purification and drug loading, resulting a greater yield of nanocarriers and 3-fold higher dox loading. the half-life of the pasylated nanocarrier was 5fold greater than that of the control nanocarrier and 55-fold greater than that of free dox [436] . the same research group further developed this platform by incorporating an mmp-cleavable linker between pas and ferritin. this design resulted in a 5-6 fold decrease in affinity between ferritin and its receptor, transferrin receptor i. thus, healthy cells with low expression of transferrin receptor i were not affected by the encapsulated dox [437] . upon exposure to high mmp concentrations in the tumor microenvironment, the pas shield was cleaved from the ferritin, increasing the affinity of the nanocarrier for its target. in mice bearing xenogeneic paca-44 pancreatic tumors, treatment with the pasylated formulation led to 4-fold greater tumor regression than a non-pasylated nanocarriers and 8-fold greater regression than seen with free dox, respectively [437] . these studies indicate how pasylated nanocarriers can be used to improve drug potency while reducing off-target effects. pasylation is also useful for nucleic acid delivery by providing stealth behavior to the delivery system. morys et al conjugated short pas sequences only four repeats long to polyethylenimine, a cationic polymer frequently employed in nucleic acid delivery systems [438] . the pas provided enough hydrophilicity to prevent aggregation and dissociation of the pei/dna polycomplexes. furthermore, pasylation of the pei-dna complex prevented interaction with blood serum proteins, preventing polyplex opsonization and clearance in vivo [438] . although this system needs further optimization for in vivo gene transfer, this study indicates that pasylation is potentially a useful strategy for improving the stability and delivery of nucleic acids. gliadin is a gluten protein extracted from wheat. its monomeric form, which has a mw of 25-100 kda, is up to 70% soluble in alcohol, and its polymeric form, which has a mw of 106 kda, contains a high level of disulfide bonds that renders it largely insoluble [439] . as such, gliadin materials show excellent stability without the need for toxic crosslinking agents. gliadin is rich in neutral residues that provide it with mucoadhesive properties and make it an ideal platform for oral drug delivery [440] . gliadin also contains many lipophilic residues that allow it to protect its cargo for prolonged release and hydrophobic interaction with tissues, including skin. due to its apolarity, gliadin has been most useful as a carrier for hydrophobic or amphiphilic drugs [441] . the solubility, hydrophobicity, and bioadhesive properties of gliadin contribute to its ability to sustain the release of therapeutics in drug delivery applications. polymeric gliadin has been widely explored as a material for nanoparticulate systems targeting the gastrointestinal mucosa. arangoa et al. demonstrated that upon oral administration of the lipophilic molecule carbazole in a gliadin nanoparticle to mice, the gliadin delivery vehicle improved drug adsorption through the stomach mucosa [442] . this phenomenon resulted in a sustained carbazole concentration in circulation and improved bioavailability. further research evaluated gliadin as a nanoparticulate carrier for the anti-cancer drug cyclophosphamide [443] . after loading cyclophosphamide onto the nanoparticles with a remarkable 72% loading efficiency, gulfam et al. demonstrated that the drug was released in a two-step manner. after a brief period of rapid release from the nanoparticles, cyclophosphamide was steadily released over the course of 48 h and induced apoptosis in breast cancer cells, which highlighted the utility of this system utility as a carrier for anti-cancer therapeutics [443] . because gliadin nanoparticles improve drug penetration into the gastric mucosa, it has been extensively engineered to protect and deliver antibiotics to the stomach to treat helicobacter pylori infections [51] . ramteke et al. loaded gliadin nanoparticles with clarithromycin and omeprazole via a desolvation technique [51] . the nanoparticles showed sustained release of the drugs at an acidic ph that mimicked conditions in the stomach and were more effective in eradicating h. pylori in vitro than free drug. in a similar study, umamaheshwari et al. demonstrated that gliadin nanoparticles increased the gastrointestinal residence time of amoxicillin, thus necessitating a lower dose to eradicate h. pylori infection than the free drug in a mongolian gerbil model [444] . these studies indicate that the bioadhesive properties of gliadin can be leveraged to improve gastric absorption and drug availability. another abundant source of plant protein is the soybean, from which soy protein isolate (spi) is extracted. spi contains glycinin and β-conglycinin, which compose nearly 60% and 40% of the protein, respectively [445] . these proteins exist as globular structures in an aqueous environment and aggregate upon the addition of a crosslinking agent or solvent [446] . this can drive the formation of hydrogels, polymer blends, or microspheres. moreover, because spis are enriched in glutamine, aspartic acid, and leucine that offer a variety of secondary bonding interactions, many types of drugs can be incorporated onto the proteins for delivery purposes. [447] . engineered soy nanoparticles to sequester and relase curcumin [448] . they prepared the nanoparticles using a desolvation technique and achieved an encapsulation efficiency of 97%. curcumin release from the nanoparticles followed a biphasic trend. initial burst release was observed as the nanoparticles swelled, followed by sustained release [448] . the pharmacokinetics, gastrointestinal uptake, and encapsulation efficiency of soy nanoparticles have also been improved by combining the protein with other materials such as folic acid [449] , zein [450, 451] , or alginate [452] . spi films have also been engineered to release a variety of drugs to treat bacterial infections and promote wound healing. in one example, peles et al. incorporated the antibiotic gentamicin into the matrix of an spi film prepared by solvent-casting. after an initial period of burst release, gentamicin exhibited prolonged release over the course of four weeks [453] . other studies have explored various crosslinking agents to improve the structural integrity and degradation kinetics of spi films. to circumvent the toxicity associated with chemical crosslinkers, song et al. employed genipin, a natural crosslinking agent, to control the swelling ratio and enhance the strength of the film [454] . films crosslinked with genipin exhibited slowed release rates of bsa, the model protein drug, compared to spi films without genipin. furthermore, the film exhibited ph-sensitivity that could be employed for delivery to the gastrointestinal tract [454] . chen et al. also designed spi films to release drug in the gastrointestinal tract by using formaldehyde as a crosslinking agent [455] . formaldehyde crosslinking increased the tensile strength of the film. methylene blue -a model drug-and rifampicin-and antibiotic -interacted strongly with the spi such that they were not released until bulk erosion occurred, which was accelerated by gastric enzymes [455] . another ph-sensitive spi film was engineered by vaz et al. using glyoxal as a crosslinking agent for spis produced via a melt extrusion technique [456] . because the pi of soy protein isolate is between 4.3-4.8, rapid release of the model drug theophylline was observed at a ph of 5, as there was little interaction between the drug and soy. at physiological ph, however, soy protein isolate was negatively charged and more strongly interacted with the drug than in acidic conditions , thereby slowing its release [456] . spi fibers have also been investigated as a drug delivery scaffold. xu et al. studied the sorption, kinetics and thermodynamics of drug release from spi fibers for three different drugs [457] . as the temperature was increased, the diffusion coefficient of drug sorption onto the fibers increased. for metformin and diclofenac, sorption and release relied on the ionic interactions j o u r n a l p r e -p r o o f between the drug and spi scaffold. conversely, these parameters were governed more strongly by van der waals forces for drugs such as 5-fluorouracil. for all model drugs, burst release was controlled by altering the drug-loading concentration. because release relies on the affinity of the drug for the spi, a lower loading concentration resulted in less burst release [457] . collectively, these studies demonstrate that spi vehicles can be designed and tuned to deliver a variety of therapeutics. zein, the major storage protein in corn, is a low-molecular weight protein in maize endosperm [458] . depending on its molecular weight and extraction process, zein is classified asα-zein, β-zein, γ-zein, or δ-zein. the most abundant form, α-zein, is a 22-to 24-kda protein that forms a triple super-helix [459] . though the structure is not fully elucidated, small-angle xray scattering data suggests that nine or ten homologous, helical repeating units arrange into an anti-parallel helix that is stabilized by hydrogen bonding [460, 461] . all classes of zein are rich in alanine, leucine, proline, and glutamine residues, but α-zein is the most frequently used class in drug delivery applications due to its abundance. though zein contains both hydrophilic and hydrophobic regions, its large fraction of non-polar residues makes it insoluble in water without the addition of alcohol, urea, or anionic or alkaline surfactants [459] . the hydrophobicity of zein also renders it mucoadhesive [462] . zein has been incorporated into -smart‖ hydrogels to control the release of drugs in response to a biological or environmental stimulus. liu et al. generated complex hydrogel beads composed of zein and pectin [463] . the hydrophobic zein suppressed bead swelling in physiological conditions, while pectin protected the zein from protease degradation in the gastrointestinal tract, thus promoting colon-specific drug delivery [463] . zein has also been engineered into an injectable, biodegradable gel that forms in situ. gao [464] . when administered to venous malformations as a sclerosant, the zein-saib hydrogel provided four days of prolonged drug release [464] . zein hydrogels have also been designed for sustained release of chemotherapeutics and reduce their off-target effects. cao et al. demonstrated that an intratumoral injection of an in situ zein hydrogel extended the release of entrapped dox, resulting in increased anti-tumor efficacy [465] . they also showed that dox release could be modulated by adjusting the zein concentration [465] . most zein-based materials for drug delivery have been microspheres or nanoparticles. zein is rich in nonpolar residues, which allows it to stably complex with drugs and entrap them in j o u r n a l p r e -p r o o f the nanoparticle core. the first reported zein microparticle was designed by matsuda et al. to improve the therapeutic index of polysaccharide-kureha (ps-k), a protein polysaccharide that stimulates anti-cancer immune activity [466] . monodisperse microspheres were generated by sonication and produced 1-µm microspheres that could be phagocytosed by macrophages [466] . subsequent studies by this group involved combinations of zein with three other anti-cancer drugs to improve their pharmacokinetics, which demonstrates the versatility of the platform [467] . since then, zein microparticles have been engineered to deliver antibiotics such as ciproflaxin [468] , anti-parasitic drugs such as ivermectin [458] , and corticosteroids such as prednisolone [469] . muthuselvi et al. investigated how the solvent used for microsphere fabrication impacts drug release [470] . using the cardiotonic glycoside gitoxin as a model drug, they fabricated zein microspheres with ethanol, methanol, and isopropanol mixtures. while microspheres made with methanol and isopropanol rapidly released the drug over four days, microspheres prepared with ethanol exhibited biphasic release. following an initial phase of burst release, drug release was sustained for 80 h before tapering off into a slower, final stage of release [470] . their ability to harness the epr effect provides another reason for the use of zein nanoparticles in chemotherapeutics. in one example, lai et al. loaded zein nanoparticles with 5fluorouracil for delivery to the liver [471] . when labeled with rhodamine-b intravenously administered, the zein nanoparticles increased liver-targeting efficiency 31% vs. free rhodamine b and 5-fluorouracil uptake in the liver was ~3-fold higher than that of free drug [471] . dong et al. loaded zein nanoparticles with dox and achieved 90% loading efficiency [472] . dox release was ph-dependent, with more rapid release occurring in acidic conditions. confocal laser scanning microscopy in hela cells showed that the zein nanoparticles were effectively endocytosed into the cytoplasm and that smaller nanoparticles could enter the nucleus [472] . improve the anti-cancer potency of statins using lovastatin as the model drug [473] . lovastatin-loaded zein nanoparticles promoted anti-proliferative activity in hepg2 cells by accumulating in the cytoplasm during the g2/m and pre-g phases of mitosis [473] . further, zein nanoparticles have improved the pharmacokinetics, solubility, and potency of anti-tuberculosis drugs-including rifampicin, pyrazinamide, and isoniazid [474] -and vitamin d3 [475] . caseins are milk proteins that evolved from a family of secreted calcium (phosphate)binding phosphoproteins [476] . accounting for nearly 80% of all bovine milk proteins, caseins j o u r n a l p r e -p r o o f exist in four different forms -αs1, αs2, β-, and κ [477, 478] . though they are all amphiphilic and proline-rich, the four caseins differ in their overall amino acid, phosphorous, and carbohydrate content [479] . caseins are naturally unstructured, which endows them with the flexibility to exhibit intermolecular interactions. consequently, they assemble into micelles 50-500 nm in diameter with negatively charged surfaces [479] . the size distribution of these micelles is readily impacted by environmental factors, including ph, ionic strength, and hydrostatic pressure, due to their amphiphilicity and lack of tertiary structure [480, 481] . although they withstand high temperatures of up to ~71°c, they are quickly destabilized by acidic environments, which makes them a good option for oral delivery [482] . because casein naturally takes on a micellar conformation, it has been readily adapted as a drug nanocarrier. an early example of this idea was demonstrated by semo et al. they encapsulated the fat-soluble vitamin d2 in the core of casein micelles and demonstrated that the casein barrier protected vitamin d2 from uv degradation [483] . soon after this finding was published, casein micelles were used to entrap poorly soluble chemotherapeutics, such as curcumin. in this study, sahu and coworkers used steady state fluorescence spectroscopy to show that curcumin shows hydrophobic interactions with casein. the complexes self-assembled into micelles ~166 nm in diameter, which were efficiently internalized by hela cells [484] . the ph-sensitivity of casein nanoparticles also makes them useful for gastrointestinal delivery. the livney group loaded β-casein nanoparticles with the chemotherapeutic drug mitoxantrone to create stable nanoparticles 100-300 nm in diameter that clustered to trap the drug between micelles as well as within their cores [485] . these micelles rapidly disintegrated in an acidic microenvironment, suggesting their potential for targeting gastric cancer [486] . casein micelles have similarly been adapted to improve the bioavailability and targeting of other drugs, such as dox [487] , folic acid [488] and flutamide [489] . casein films have also been applied to tablets to slow drug release. these films, which are typically prepared with transglutaminase as the crosslinker, exhibit remarkable tensile strength, and are gradually hydrolyzed by chymotrypsin [490] . abu diak and coworkers coated diltiazem-hcl tablets with casein using four different plasticizing agents and ultimately derived a continuous tablet coat with oleic acid as the olasticizer [491] . at ph 1.2, casein-coated tablets slowed the release of diltiazem-hcl compared to uncoated tablets, which released 80% of drug within 2 h. the rate at which the coated tablets released the drug decreased with increasing post-coating heat treatment temperature. similarly, at ph 6.8, only coated tablets that underwent j o u r n a l p r e -p r o o f post-coating heat treatment at 100°c successfully showed sustained drug release, which was likely due to casein crosslinking under these conditions [491] . the favorable hydrophilicity and biocompatibility of casein, coupled with the availability of reactive sites for chemical modification within its sequence, make it an ideal material for forming hydrogels. in an early example, song and coworkers created genipin-crosslinked casein hydrogels to encapsulate bsa [492] . hydrogel swelling and subsequent bsa release was governed by the environmental ph. at ph 1.2, there was minimal hydrogel swelling and the bsa remained entrapped in the gel. when the ph was increased to 7.4, the hydrogel swelled and released greater amounts of bsa, making this hydrogel useful for intestinal delivery. the swelling behavior was similarly influenced by the degree of crosslinking by genipin [492] . in a later study, the same group employed microbial transglutaminase to crosslink casein hydrogels into a fractal network structure compared to uncrosslinked casein hydrogels [493] . vitamin b 12 , was loaded into the hydrogel under mild conditions and was slowly released from the hydrogel at ph 7.4. the rate of release slowed with increased crosslinking [493] . casein-drug composites have also been designed to improve the solubility and dissolution of various drugs. millar and corrigan first demonstrated that freeze-dried compacts made from sodium caseinate and acidic casein enhanced the dissolution rates of chlorothiazide and hydrochlorothiazide 35-fold and 1.5-fold, respectively. the improvement in dissolution was related to the complexation with casein and processing of the drug [494] . millar et al. further characterized the dissolution rate of the compacts over a range of compositions and found that the dissolution rate of acidic casein was half that of sodium caseinate [495] . using ibuprofen as the model drug, they showed that acidic casein compacts significantly slowed drug dissolution compared to sodium caseinate compacts. they attributed the difference to the changing form of casein that took place due to microenvironmental ph differences in the boundary layer. acidic casein was also more viscous and rigid than sodium caseinate, which decreased its solubility and reduced drug diffusivity [495] . a subsequent research group demonstrated that acidic casein and sodium caseinate could be mixed with other materials, such as hydroxypropylmethocellulose matrices, to slow drug dissolution [496] . iron binding proteinsmost notably transferrin and ferritinhave been widely explored as drug delivery vehicles due to the increased need for iron in many diseases, including cancer. in these applications, iron binding proteins serve to both improve the pharmacokinetics and stability of j o u r n a l p r e -p r o o f their cargo, but also to target the drug by taking advantage of the body's natural iron transport mechanisms. transferrin is an abundant glycoprotein that plays a critical role in the systemic transport of iron. as a natural iron-chelating agent, it reversibly binds one or two ferric ions to maintain their solubility and prevent toxic free-radical generation [497] . it also promotes iron endocytosis by binding the transferrin receptor 1, which is upregulated on malignant cells to meet their increased iron needs [498] . transferrin receptor 1 is also expressed on brain endothelial cells, making transferrin an attractive option for delivering therapeutic cargo across the blood brain barrier [499] . it should be noted that, due to its unique targeting capacity, transferrin has been used as a targeting moiety to deliver both small molecule chemotherapeutics and peptides/proteins. transferrin-drug conjugates have been employed to target chemotherapeutics to tumors delivery and reduce the off-target toxicity of the drug. szwed et al coupled dox to transferrin and showed that transferrin could overcome anthracycline resistance by impairing the pglycoprotein activity in several anthracycline-resistant cell lines such as dox resistant k562 cells [500] . in a subsequent study, they demonstrated that a dox-transferrin conjugate successfully induces apoptosis in leukemia cells while avoiding toxicity to non-malignant cells [501] . transferrin was also used to target cytochrome c to lung tumors by conjugating it to transferrin using sulfo-lc spdp as a crosslinker [502] . this strategy resulted in nanocarriers ~10 nm in diameter, allowing them to avoid rapid kidney filtration. upon endocytosis by cells, the redox-sensitive bond in the linker was cleaved, releasing cytochrome c from transferrin and initiating apoptosis [502] . transferrin based nanoparticles have also been engineered to deliver photothermal and photodynamic agents for cancer therapy. wang et al treated transferrin with dtt, leading to exposure of hydrophobic residues that could interact with the near-infrared dye ir780 through hydrophobic interactions [503] . these dye-loaded nanoparticles had a half-life of 20 h and were j o u r n a l p r e -p r o o f readily endocytosed by malignant cells within 2 h. they accumulated in which tumor with a high tumor-to-background ratio and, when pulsed with an 808 nm laser, caused a temperature increase to 49.5°c that caused tumor regression, whereas tumors treated with pbs only showed a temperature increase to 36.3°c, which had no effect on tumor growth [503] . this study demonstrates how transferrin could be used to enhance the pharmacokinetics and delivery of hydrophobic agents. transferrin has similarly been fused to glp-1 to improve the with improved pharmacokinetics of this peptide drug for treatment of type 2 diabetes [504] . this technology, developed by biorexis and later acquired by pfizer, consists of non-glycosylated transferrin genetically fused to glp-1, creating a fusion protein that is expressed in yeast cells. fusion with transferrin did not affect the insulinotrophic properties of glp-1 and yielded a half-life of 1-2 days, compared to a half-life of minutes for native glp-1 [504] . furthermore, the glp-1transferrin fusion promoted greater β-cell proliferation and β-cell area compared to transferrin alone. two days after receiving an intraperitoneal injection of glp-1-transferrin, treated mice demonstrated a significant increase in β-cell proliferation with nearly 10% of total islet cell possessing brdu+ nuclei. meanwhile, β-cells in animals treated with transferrin alone did not show signs of proliferation after two days [504] . as such, glp-1-transferrin may provide a novel strategy for restoring β-cell function in diabetics while maintaining blood glucose control. transferrin has also been exploited to treat autoimmune diseases such as myasthenia gravis (mg). in patients with mg, autoantibodies recognize and block nicotinic acetylcholine receptors (achr). by fusing the α-subunit of achr to transferrin, keefe et al created shg2210, a fusion protein that is recognized and bound by anti-achr autoantibodies and is internalized by cells via the transferrin receptor [505] . results from in vitro binding and internalization studies show that anti-achr autoantibodies bind shg2210 with an ic50 of 0.37 µm, while no binding to transferrin alone was observed [505] . when the shg2210:antibody complex was added to hela cells in culture, the complex was readily internalized via the transferrin receptor, as seen by the j o u r n a l p r e -p r o o f negligible uptake by the cells in the presence of saturating levels of a soluble achr inhibitor. after internalization, the complex was trafficked to the lysosome and was degraded [505] . transferrin delivery platforms were further optimized by chen et al, who evaluated the effect of various linkers on the binding affinity and pharmacokinetics of transferrin fusion proteins. in this work, growth hormone (gh) or granulocyte colony-stimulating factor (g-csf) were genetically linked to transferrin via a dipeptide linker, a helical peptide linker, or a thrombincleavable, disulfide cyclopeptide linker [506] . for both gh-tf and g-csf-tf, the longer -helical or cyclopeptide linkers-linkers resulted in less steric hindrance and thus provided greater binding affinity between gh or g-csf and their receptors as well as transferrin and its receptor. fusion proteins with the highest affinity for their targets also had the greatest plasma half-life [506] . the fusion containing the cyclopeptide linker had the greatest affinity for the transferrin receptor (ic50 of 0.9 nm) and achieved a half-life of 5.7 hr. in contrast, the fusion with the dipeptide linker had an 8-fold lower affinity for the transferrin receptor and exhibited a shorter half-life of 4.2 hr [506] . this study demonstrates how delivery of a protein drug via a transferrin carrier can be modulated by tuning the length and type of linker between the transferrin and its cargo. like transferrin, ferritin has also been exploited to target and carry drugs. ferritin is an iron-storage protein composed of 24 subunits that assemble into a spherical nanocage [507] . while this nanocage naturally houses up to 4500 iron atoms, it can also be engineered to house a variety of therapeutics [508, 509] . its loading capacity depends on several factors, including mw of the drug, the encapsulation strategy, and choice of co-solvent for entrapping drugs. metal salts achieve the highest encapsulation efficiency with nearly 5000 particles of agno 3 fitting in a single cage, whereas small, hydrophobic moleculesincluding many chemotherapeutics, exhibit lower loading efficiency [510, 511] . taking advantage of ferritin's natural ability to traverse the blood brain barrier, fan et al loaded a ferritin nanocage with an infrared dye and administered it to mice bearing u87mb j o u r n a l p r e -p r o o f orthotopic gliomas [512] . accumulation of the ferritin nanocage in the glioma-bearing mice was 10-fold greater than in the control group that received the dye alone, with maximum accumulation 4-6 hr after administration. interestingly, nanocage accumulation was limited to the tumor and no significant accumulation was observed in healthy brain tissue [512] . fan et al then loaded the nanocages with dox. and administered them intravenously. the dox-loaded nanocages nearly doubled survival time compared to treatment with free dox and significantly reduced tumor volume. additionally, mice treated with the dox-loaded nanocages exhibited slowed weight loss, indicating that the formulation reduced toxicity compared to free dox [512] . in a separate study, dox loaded ferritin nanocages increased the half-life of dox 12-fold and the auc by over 100-fold [513] . ferritin nanocages have also been used to deliver the poly adp ribose polymerase inhibitor olaparib to treat multiple forms of breast cancer. a study conducted by mazzucchelli et al demonstrated that the encapsulation of the drug in a ferritin nanocage led to 1000-fold greater uptake of the drug by triple negative breast cancer cells compared to the free drug [514] . the nanocage enhanced the cytotoxicity of the drug in cancer cells and reduced off-target toxicity in healthy cells. these studies demonstrate that ferritin nanocages can be harnessed to improve the delivery of chemotherapeutics in a variety of cancers. in another application, jiang et al synthesized a cobalt nanozyme -a nanomaterial with intrinsic enzyme-like activity -within a ferritin nanocage [515] . synthesis of the nanozyme within the confines of the nanocage provided better control over the size and structure of the nanozyme compared to nanozymes synthesized outside of the nanocage. the hepatocellular carcinoma targeting peptide sp94 was recombinantly fused to ferritin such that it was displayed on the nanocage exterior for optimal targeting [515] . though this design was used for diagnostic purposes, the remarkable targeting specificity it achieved demonstrates how it can be readily adapted as a drug delivery vehicle. ferritin nanocages can also be employed to deliver nucleic acids. li et al successfully encapsulated sirna into the nanocage, protecting it from rapid in vivo degradation [516] . this j o u r n a l p r e -p r o o f system exhibited highly efficient transfection into primary human tumorigenic cell lines, pmbcs, and mesenchymal stem cells. notably, up to 85% gene knockdown was achieved by the nanocage, while lipofectamine -the gold standard-only achieved 40% gene knockdown [516] . at the low sirna concentration of 10 nm used in this study, lipofectamine is relatively inefficient at transfection as it requires a higher sirna concentration for optimal activity, whereas the nanocage is more effective at gene knockdown at this low sirna concentration. because the biophysical properties and encapsulation of sirna are sequence-independent, this approach could be adapted for sirna delivery for numerous applications [516] . the research discussed in this review highlights the versatility and multifunctionality of protein-based materials for drug delivery. these carriers benefit from exceptional biocompatibility and biodegradability and minimal toxicity. most importantly, protein-based materials are highly customizable. their chemical, physical, and biological properties can be tailored for a specific application with sequence-level precision, thus providing the researcher with full control over the formulation's pharmacokinetics, pharmacodynamics, biodistribution, and in vivo interactions. much of this work has been made possible by continuing advances in genetic engineering and synthetic biology, which have made it possible to incorporate new chemistries and structural motifs into the polypeptide sequence to improve drug conjugation, targeting, stimulus responsiveness, and self-assembly. researchers continue to explore new ways to engineer proteins to possess desirable chemical and mechanical properties. one such strategy is to create hybrid materials by combining a protein with other proteins or synthetic components to generate a material with the favorable physicochemical properties of its building blocks. another method focuses on conferring new functionalities onto proteins through chemical processing, post-translational modification, or the incorporation of unnatural amino acids into the protein sequence. this approach expands the range of drugs that can be recombinantly fused to, chemically conjugated to, or physically encapsulated by the material. new protein-based materials also continue to be developed from existing proteins and by de novo design of peptide sequences that can introduce new attributes to protein materials to improve drug delivery. as researchers continue to make progress in adapting existing and developing new protein materials, there is also great opportunity for biological and materials science research that will maximize the clinical success of protein drug carriers. despite the progress made in this field, formulations using protein-based materials rarely reach or survive clinical trials. they are hindered by poor in vivo stability and performance, which results from an insufficient understanding of how to create materials that remain stable and functional in the harsh environments present during storage, administration, and circulation. this is further highlighted by the fact that, even though the half-life of a drug can be significantly increased by its protein carrier, the half-life of the formulation still falls short of that of the native protein. alterations to the chemical and physical structure can change how a material and drug are endocytosed by their target, degraded and cleared from circulation, or recognized by immune cells. as new chemistries and hierarchical structures are explored, this knowledge becomes more critical. through increased understanding of these fundamental principles, we can better design and predict the in vivo behavior of protein-based drug delivery vehicles. as new materials are developed, enhanced biological and materials science research can improve tools used to predict their behavior and improve their design. for example, experimental results will boost theoretical models and molecular simulations, which will in turn enhance de novo design of protein architectures. this will inform our understanding of structurefunction relationships such that new morphologies can be engineered to control the delivery of therapeutics. it is also 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antioxidant and antimicrobial properties of curcumin by means of encapsulation in gelatin through electrohydrodynamic atomization, food hydrocoll sterically stabilized gelatin microassemblies of noscapine enhance cytotoxicity, apoptosis and drug delivery in lung cancer cells monodisperse microshell structured gelatin microparticles for temporary chemoembolization electrospun gelatin nanofibers loaded with vitamins a and e as antibacterial wound dressing materials induction of angiogenesis using vegf releasing genipin-crosslinked electrospun gelatin mats gelatin-based hydrogels for biomedical applications cartilage tissue engineering application of injectable gelatin hydrogel with in situ visible-light-activated gelation capability in both air and aqueous solution a novel, visible light-induced, rapidly cross-linkable gelatin scaffold for osteochondral tissue engineering a cisplatin slow-release hydrogel drug delivery system based on a formulation of the macrocycle cucurbit[7]uril, gelatin and polyvinyl alcohol bioprintable alginate/gelatin hydrogel 3d in vitro model systems induce cell spheroid formation engineering bioprintable alginate/gelatin composite hydrogels with tunable mechanical and cell adhesive properties to modulate tumor spheroid growth kinetics directing the self-assembly of tumour spheroids by bioprinting cellular heterogeneous models within alginate/gelatin hydrogels in vitro microbial inhibition, bonding strength, and cellular response to novel gelatin-alginate antibioticreleasing soft tissue adhesives novel gelatin/alginate soft tissue adhesives loaded with drugs for pain management: structure and properties fibrous proteins: coiled-coils, collagen and elastomers elastin-based materials applications of elastin-like polypeptides in drug delivery elastin-like polypeptides: biomedical applications of tunable biopolymers molecular description of the lcst behavior of an elastin-like polypeptide ucst and lcst behavior in polymer blends a unified 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enhance intratumoral spatial distribution a rapamycin-binding protein polymer nanoparticle shows potent therapeutic activity in suppressing autoimmune dacryoadenitis in a mouse model of sjögren's syndrome elastin-based protein polymer nanoparticles carrying drug at both corona and core suppress tumor growth in vivo noncanonical self-assembly of highly asymmetric genetically encoded polypeptide amphiphiles into cylindrical micelles a hybrid protein-polymer nanoworm potentiates apoptosis better than a monoclonal antibody an amphipathic alpha-helical peptide from apolipoprotein a1 stabilizes protein polymer vesicles self-assembly of thermally responsive nanoparticles of a genetically encoded peptide polymer by drug conjugation a paclitaxel-loaded recombinant polypeptide nanoparticle outperforms abraxane in multiple murine cancer models niclosamide-conjugated polypeptide nanoparticles inhibit wnt signaling and colon cancer growth encapsulating a hydrophilic chemotherapeutic into rod-like nanoparticles of a genetically encoded asymmetric triblock polypeptide improves its efficacy active targeting of cancer cells by nanobody decorated polypeptide micelle with bioorthogonally conjugated drug recombinant synthesis of hybrid lipid-peptide polymer fusions that self-assemble and encapsulate hydrophobic drugs silk-elastin-like protein biomaterials for the controlled delivery of therapeutics tunable self-assembly of genetically engineered silk--elastin-like protein polymers hydrophobic drug-triggered self-assembly of nanoparticles from silk-elastin-like protein polymers for drug delivery injectable intratumoral depot of thermally responsive polypeptide-radionuclide conjugates delays tumor progression in a mouse model brachytherapy using injectable seeds that are self-assembled from genetically encoded polypeptides <em&gt injectable polypeptide micelles that form radiation crosslinked hydrogels in situ for intratumoral radiotherapy one-month zero-order sustained release and tumor eradication after a single subcutaneous injection of interferon alpha fused with a body-temperature-responsive polypeptide injectable protease-operated depots of glucagon-like peptide-1 provide extended and tunable glucose control one-week glucose control via zero-order release fusion of fibroblast growth factor 21 to a thermally responsive biopolymer forms an injectable depot with sustained anti-diabetic action a thermo-responsive protein treatment for dry eyes sustained release of antibiotics from injectable and thermally responsive polypeptide depots development and characterization of a fusion protein between thermally responsive elastin-like polypeptide and interleukin-1 receptor antagonist: sustained release of a local antiinflammatory therapeutic phasebio-announces-global-license-of-pb1023-forthe-treatment-of-sarcopenia-related-diseases-to-immunoforge.html phasebio announces case study highlighting pb1046 hemodynamic data presented at the 14th pulmonary vascular research institute world congress mechanism of resilin elasticity resilin-like polypeptide hydrogels engineered for versatile biological functions resilin-based materials for biomedical applications tentative identification of a resilin gene in drosophila melanogaster synthesis and properties of crosslinked recombinant pro-resilin comparisons of recombinant resilin-like proteins: repetitive domains are sufficient to confer resilinlike properties structural ensembles reveal intrinsic disorder for the multi-stimuli responsive bio-mimetic protein rec1-resilin purification of recombinant protein by cold-coacervation of fusion constructs incorporating resilininspired polypeptides a genetically engineered protein responsive to multiple stimuli a ph-responsive interface derived from resilin-mimetic protein rec1-resilin temperature-triggered phase separation of a hydrophilic resilinlike polypeptide ph-responsive schizophrenic diblock copolymers hydration changes during thermosensitive association of a block copolymer consisting of lcst and ucst blocks switching the inside and the outside of aggregates of water-soluble block copolymers with double thermoresponsivity micellar self-assembly of recombinant resilin-/elastin-like block copolypeptides hydrophilic elastomeric biomaterials based on resilin-like polypeptides tunable mechanical stability and deformation response of a resilin-based elastomer characterization of resilin-based materials for tissue engineering applications targeting angiogenesis: structural characterization and biological properties of a de novo engineered vegf mimicking peptide expression, cross-linking, and characterization of recombinant chitin binding resilin redox-responsive resilin-like hydrogels for tissue engineering and drug delivery applications resilin-like polypeptide hydrogels engineered for versatile biological function design and production of a chimeric resilin-, elastin-, and collagen-like engineered polypeptide genetically engineered silk-elastinlike protein polymers for controlled drug delivery the enhanced permeability and retention (epr) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting design of silk-elastin-like protein nanoparticle systems with mucoadhesive properties in-situ self-assembling protein polymer gel systems for administration, delivery, and release of drugs solute diffusion in genetically engineered silk-elastinlike protein polymer hydrogels controlled release of plasmid dna from a genetically engineered silk-elastinlike hydrogel in vitro and in vivo evaluation of recombinant silk-elastinlike hydrogels for cancer gene therapy adenoviral gene delivery to solid tumors by recombinant silk-elastinlike protein polymers comparison of silk-elastinlike protein polymer hydrogel and poloxamer in matrix-mediated gene delivery silk-elastin-like hydrogel improves the safety of adenovirus-mediated gene-directed enzyme−prodrug therapy direct observation of interactions of silk-elastinlike protein polymer with adenoviruses and elastase temperature-responsive silk-elastinlike protein polymer enhancement of intravesical drug delivery of a therapeutic glycosaminoglycan for treatment of interstitial cystitis/painful bladder syndrome silk-elastinlike protein polymers enhance the efficacy of a therapeutic glycosaminoglycan for prophylactic treatment of radiation-induced proctitis a recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner multivalent antiviral xten-peptide conjugates with long in vivo half-life and enhanced solubility extension of in vivo half-life of biologically active molecules by xten protein polymers safety, pharmacokinetics, and pharmacodynamics of a single subcutaneous dose of vrs-859 in patients with type 2 diabetes gcg-xten: an improved glucagon capable of preventing hypoglycemia without increasing baseline blood glucose glp2-2g-xten: a pharmaceutical protein with improved serum half-life and efficacy in a rat crohn's disease model xten-annexin a5: xten allows complete expression of long-circulating protein-based imaging probes as recombinant alternative to pegylation vrs-317), a long-acting rhgh, in pediatric growth hormone deficiency safety and efficacy of somavaratan (vrs-317), a long-acting growth hormone, in children with growth hormone deficiency (ghd): 2.5-year results from the vista trial prolonged half-life and improved recovery of recombinant factor ix-xten fusion proteins in hemophilia b mouse model safety and prolonged activity of recombinant factor viii fc fusion protein in hemophilia a patients long-term safety and efficacy of recombinant factor viii fc fusion protein (rfviiifc) in subjects with haemophilia a bivv001, a new class of factor viii replacement for hemophilia a that is independent of von willebrand factor in primates and mice xten as biological alternative to pegylation allows complete expression of a protease-activatable killin-based cytostatic off-tumor toxicity with potent efficacy in vitro and in vivo and large safety margins in nhp pasylation: a biological alternative to pegylation for extending the plasma halfactive proteins influence of size and charge of unstructured polypeptides on pharmacokinetics and biodistribution of targeted fusion proteins fusion proteins for half-life extension of biologics as a strategy to make biobetters enhanced in vivo efficacy of a type i interferon superagonist with extended plasma half-life in a mouse model of multiple sclerosis pasylation technology improves recombinant interferon-β1b solubility, stability, and biological activity pasylation of murine leptin leads to extended plasma half-life and enhanced in vivo efficacy treatment of diet-induced lipodystrophic c57bl/6j mice with long-acting pasylated leptin normalises insulin sensitivity and hepatic steatosis by promoting lipid utilisation molecular design, expression and evaluation of pasylated human recombinant erythropoietin with 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[1] key: cord-299413-3o6mdx3e authors: wang, hui; liu, rihe title: advantages of mrna display selections over other selection techniques for investigation of protein–protein interactions date: 2014-01-09 journal: expert rev proteomics doi: 10.1586/epr.11.15 sha: doc_id: 299413 cord_uid: 3o6mdx3e mrna display is a genotype–phenotype conjugation method that allows for amplification-based, iterative rounds of in vitro selection to be applied to peptides and proteins. mrna display can be used to display both long natural protein and short synthetic peptide libraries with unusually high diversities for the investigation of protein–protein interactions. here, we summarize the advantages of mrna display by comparing it with other widely used peptide or protein-selection techniques, and discuss various applications of this technique in studying protein–protein interactions. the functions of many proteins are realized by interacting with other proteins. often, such protein-protein interactions only occur under specific conditions. one of the most effective strategies to obtain a thorough understanding of a protein of interest is through mapping its protein-protein interaction network on a proteomewide scale, while simultaneously searching the peptide sequence space to find what random sequences it can interact with. a number of protein-selection techniques, including yeast twohybrid, phage display, ribosome display, mrna display, in vitro compartmentalization (ivc) and bacteria/yeast cell-surface display, have been developed to address the challenge [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . for any protein-selection techniques, the most important feature is the linkage of a functional peptide or protein sequence with its coding nucleic acid sequence. this linkage between the phenotype and the genotype can be virus or micro organisms as in phage display, cell surface display and n-hybrid systems, aqueous micro-droplet as in ivc, complex of protein and nucleic acid as in ribosome display and simply a covalent bond as in mrna display. in these methods, the proteins are directly or indirectly coupled with either dna or mrna. many rounds of enrichment and selection can be performed, thus enabling the selections for sequences with desired properties from a very large library. here, we briefly review the advantages of mrna display over other selection techniques for the investigation of protein-protein interactions. it should be noted that some important works on mrna display are not discussed, owing to the focus on protein-protein interactions of this article. mrna display is a completely in vitro selection technique that allows for the identification of polypeptide sequences with desired properties from both a natural protein library and a combinatorial peptide library [5] [6] [7] 12, 13] . the central feature of this method is that the polypeptide chain is covalently linked to the 3´ end of its own mrna (figure 1 ). this is accomplished by synthesis and in vitro translation of an mrna template with puromycin attached to its 3´ end via a short dna linker. during in vitro translation, when the ribosome reaches the rna-dna junction and translation pauses, puromycin, an antibiotic that mimics the aminoacyl moiety of trna, enters the ribosome 'a' site and accepts the nascent polypeptide by forming a peptide bond. this results in tethering the nascent polypeptide to its own mrna. when the initial mrnas are composed of many different sequences, the corresponding protein or proteome library will be generated. since the genotype coding sequence and the phenotype perspective polypeptide sequence are covalently combined within the same molecule, the selected proteins can be revealed by dna sequencing after reverse transcription and pcr amplification. therefore, mrna display provides a powerful means to effectively 'reverse translate' a peptide or protein for reading and amplifying after it has been functionally isolated from a library with high diversity. multiple rounds of selection and amplification can be performed, enabling enrichment of rare sequences with desired properties. so far, mrna display has been successfully applied in the identification of drug-binding targets, mapping of the protein-protein interaction network and dna-protein interaction network, elucidation of the enzyme-substrate interactions, improvement of the binding affinities of existing affinity molecules, synthesis of peptides containing unnatural amino acids and evolution of enzymes from partially randomized protein scaffolds [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] . there exist many other protein screening platforms that allow for studying protein-protein interactions (figure 2 ). here, we briefly summarize the principles of some widely used screening techniques and their limitations. among the techniques available, co-immunoprecipitation (co-ip) is a widely used conventional method for identifying and verifying protein-protein interactions. co-ip can be performed when a protein of interest is present at the physiological concentration and in the correct conformation with all the necessary post-translational modifications and physiological binding partners [25] . the combination of co-ip with mass spectrometry turns it into a powerful perspective approach for screening protein-protein interactions. however, the success of co-ip is highly dependent on the availability of highquality antibodies. one solution is to fuse an epitope or affinity tag to the protein of interest. tandem affinity purification is one of the most robust immunoprecipitation techniques in which the protein of interest is fused with a tandem affinity purification tag that allows for highly efficient detection and purification [26] . however, this approach is limited to one round of purification and enrichment and the signal detection relies on highly sophisticated mass spectrometric techniques, making it challenging to identify weak interacting partners, particularly when the abundance is low. the yeast two-hybrid system is widely used to screen for protein-protein interactions with numerous successes [1, [27] [28] [29] . the method is based on the modular nature of the activating and dna-binding domains of eukaryotic transcription factors that can function in close proximity to each other without direct binding. in yeast two-hybrid, the dna-binding domain is fused with a target protein of interest as the bait, while the activation domain is fused with a library of known or unknown proteins as the prey. the two plasmids are then transformed into engineered yeast cells. if the bait and prey fusion proteins physically interact with each other, the activation domain will be brought in proximity to the transcription start site, resulting in transcription of reporter gene(s) and subsequent phenotypic changes of yeast cells [28, 29] . the libraries of the proteins used in yeast two-hybrid are typically from natural sources, such as those based on cdnas of humans or model organisms [29] . the major advantage of yeast two-hybrid is that it is an in vivo method in which both the protein expression and the selection for protein-protein interaction occur in eukaryotic yeast cells. the challenge is that the bait-prey interactions take place within the yeast nuclei, which are highly regulated and difficult to manipulate. in addition, the complicity of the intracellular environment, including numerous positive and negative feedbacks, compensating pathways and other cellular regulations, could result in false-positive clones whose origins are difficult to track [30] . another well-established technology for the selection of peptide or protein sequences with desired properties is phage display. this method uses bacteriophage to noncovalently couple perspective a protein with its coding cdna by displaying the peptide of interest as a fusion with a coat protein on the surface of filamentous phage [2] . this technology has been widely used to display randomized short peptide or antibody fragment libraries. selected phages containing fusion proteins with desired properties are used to infect a suitable bacterial host, from which the phagemids can be collected and the relevant dna sequences amplified for the next round of selection or sequenced and analyzed [31] [32] [33] . in phage display, the proteins in the library are expressed in host bacteria, thus transformation is mandatory. the selection for protein-protein interaction is performed under in vitro conditions, typically using a technique called biopanning [31, 32] . phage display is most effective in the selection of short synthetic sequences, which are often poorly correlated with cellular proteins [34] . other limitations of phage display in screening cdna libraries include the different codon usage of bacteria from mammalian cells, the lack of post-translational modifications, and the limited and biased folding capacity of proteins in bacteria [35] . bacteria and yeast cell-surface displays are similar to phage display in many ways. the library proteins are fused to a membrane protein, which acts as an anchor to display the library proteins on the surface of bacteria or yeast cells [36] [37] [38] . in this method, in addition to biopanning that can be used for the selection, the targets can be coupled with fluorescent probes, allowing the use of facs for target-binding selections [36] [37] [38] . the most impressive advantage of this technique is the lower nonspecific background by using facs when compared with that of biopanning. however, the sequence space that can be screened is limited due to the much lower throughput of the cell-sorting procedure. in addition, the displayed proteins should be compatible with the transport machinery of bacteria or yeast to be displayed on the cell surface correctly. ribosome display is an entirely in vitro protein-selection technique that utilizes stalled ribosome-mrna-polypeptide complexes to couple a protein of interest with its coding mrna [3, 4] . this method relies on maintaining the integrity of the ternary complex of mrna, ribosome and nascent polypeptide chain, which can be stabilized to some extent by removing the stop codon, lowering the temperature and adding cations such as mg 2+ [39, 40] . since the transformation step is not necessary, both synthetic peptide and natural proteome libraries with very high diversities can be used. the selected peptide or protein sequences can be directly amplified by pcr or sequenced for the next round of selection or ana lysis. the protein expression is performed by using an in vitro cell-free translation system, such as cell lysates from bacteria, wheat germ or rabbit reticulocyte. the major limitation of ribosome display is that the selection for protein-protein interactions should be carefully performed under in vitro conditions that maintain the integrity of the fragile ribosome/mrna/protein complex [39, 40] . in vitro compartmentalization is an emulsion-based in vitro protein-selection method that noncovalently couples genotype and phenotype within the same cell-like vesicle [8, 9] . each compartment, typically water-in-oil emulsion, contains no more than one gene, whose transcription and translation result in the corresponding protein being trapped inside the compartment together with its encoding cdna. in principle, any protein libraries, including synthetic peptide and natural proteome libraries, can be used. the in vitro transcription, translation and protein-protein interaction should be performed within the vesicles when the compartments are intact [8, 9, [41] [42] [43] . to recover the sequences, emulsion will be broken by successive steps of removing mineral oil and surfactants and the cdnas are amplified for the next round of selection or ana lysis [41] [42] [43] . it is critical to design an appropriate selection scheme that allows for tracking each protein product to its coding gene. while this method could be very useful for directed evolution of enzymes, its application in protein-protein interactions is not general because of the difficulty in designing a selection scheme that permits the conjugation of phenotype and genotype after the compartment is destroyed. while performing protein selection using these techniques under in vitro conditions provides great flexibility, in vitro selections could generate false positives that do not function inside the cells due to degradation, loss of solubility and conditions different from the physiological environment. therefore, in vivo selection has unique advantages that are difficult to achieve using in vitro approaches. mrna display is similar to ribosomal display except the linkage between the mrna and the protein is a highly stable covalent amide bond instead of noncovalent coupling through an unstable ternary complex. compared with other protein-selection techniques, mrna display provides the advantages listed in the following section. the success of any protein-selection techniques for investigating protein-protein interactions relies on a selection scheme that allows specific enrichment of sequences with desired targetbinding properties, while minimizing nonspecific sequences that might be coselected due to various biases. however, the nature of the selection techniques often restricts the use of highly specific selection conditions. in yeast two-hybrid, for example, the selection conditions cannot be readily controlled because the interaction takes place in the cell nuclei. ribosome display shares many important features with mrna display, such as use of cell-free translation system and being applicable to protein libraries with very high diversity. however, the fragile noncovalent conjugation between genotype and phenotype in ribosome display requires that the selection should be performed under very mild conditions. by contrast, the phenotype-genotype linkage in mrna perspective display is covalent and highly stable, making it possible to perform many types of selection under very stringent conditions. optimal selection conditions that allow specific binders to be distinguished from nonspecific ones can be readily applied. the selection stringency can be carefully controlled and tuned, including using detergents, chelating agents, unusual ph, temperature and ionic strength, in addition to nonspecific or specific competitors, cofactors and metal ions, as long as they do not affect the functions of the peptides or proteins. most protein-selection methods that rely on an in vivo step are typically limited to relatively small libraries with low complexity, owing to the low efficiency of transforming or transfecting the starting cdna library into the organism of choice. the phage display allows a complexity in the range of approximately 10 9 -10 10 , but typically around 10 8 [33]. the library size for cell-based selections, such as the yeast two-hybrid system, bacteria and yeast surface display, is typically limited to approximately 1 million [30]. in ivc system, water-in-oil micro-droplets with a mean diameter of 2-3 µm are typically used, corresponding to 10 10 droplets/ml of emulsions [42, 43] . since no more than one cdna molecule is contained in each compartment where the transcription and translation are coupled, the diversity of the library is typically limited to less than 10 9 [43] . mrna display is an entire in vitro selection method that takes advantage of cell-free translation systems for the generation of polypeptide sequences. both the expression and the selection are performed in vitro. since no transformation is required, the upper limit of the library size is dictated by the genetic materials one can reasonably handle in the laboratory, including the amount of dna oligonucleotides that can be synthesized, the volume of pcr that can be performed to generate the cdna library and the volume of in vitro translation reaction to express the protein library [13] . peptide or protein libraries containing as many as 10 12 -10 14 unique sequences can be readily generated and selected, while a few orders of magnitude higher than that can be achieved using phage display and other selection techniques [13] . for totally or partially randomized peptide or protein libraries, the much higher diversity provides a big advantage for exploring the sequence space to a greater depth for the identification of target-binding partners. according to lancet and colleagues' model of olfactory receptors and antibodies [44] , the affinity of the best binders can increase by 300-fold as the library size increases 10,000-times [13, 44] . therefore, the larger library size means the higher possibility of isolating target-binding sequences with higher affinity and specificity. compared with totally synthetic protein libraries, natural proteome libraries have much lower diversities. the human genome contains only 25,000-30,000 protein-coding genes [45, 46] . in principle, the full-length proteins in the proteome can be displayed. however, it requires amplification of the genes of interest using gene-specific primers, which significantly reduces the throughput and increases the costs of the selection. one compromise is to construct a cdna library from isolated mrnas by using a random reverse-transcription primer to cover all the possible regions of the open reading frames in the genome. hence, a much larger cdna library is required for the comprehensive coverage of the protein sequences with desired function, which is enabled by the large library size feature of mrna display. in addition, each gene and each domain of a particular gene can have numerous copies in an mrna-displayed proteome library. therefore, both the likelihood of isolating rare sequences and the diversity of the sequences isolated in a given selection are significantly increased. in many other selection approaches, the context in which an expressed protein is present can profoundly influence the nature of the library generated. in phage and cell-surface display, the candidate protein sequences in the library are expressed in the context of a fusion protein on the surface of viruses or cells. in ribosome display, the proteins are displayed on very large ribosomes, hence there would be unpredictable interactions between the huge ribosome complexes with the target. not being restricted by these factors, mrna display allows the use of any candidate libraries with the random regions present in isolation or within the context of a desired fusion protein. from the point of view of protein expression, phage display relies on using the translational machinery in bacteria for protein expression, while yeast two-hybrid is limited to yeast cells, although mammalian cells can be used but with lower efficiency. it is well known that some sequences are expressed poorly in bacteria or in yeast [47, 48] . very often, in vivo selection biases will be raised against certain proteins and scaffolds, due to folding, transport, membrane insertion and complexation problems. this is particularly true for partially unfolded proteins, which are usually rapidly degraded within cells [47] . these problems can result in the loss of functional molecules or restrict the nature of the selection procedures that one can apply. in mrna display-based selection, the translation efficiency is the limiting factor. the proteins are expressed in cellfree eukaryotic translation systems, such as the rabbit reticulocyte lysate, allowing the synthesis of proteins with reasonable post-translational modifications [49] . one major challenge in most protein-selection platforms is that some abundant sequences, either specific or nonspecific binders, could dominate the pool as selection goes on. this is a particular problem for the selections using natural cdna libraries, in which the copy number of the most abundant mrna species could be more than 10,000-times higher than the least abundant ones [50] . some protein sequences with desired target-binding properties have more copies than others in the initial cdna library and could be preferentially enriched, thus interfering with the isolation of other lower abundance sequences and generating false positives. in mrna display, since the nucleic acid coding sequences are always linked with the selected proteins, those most abundant sequences could be efficiently removed at the mrna level. specifically, after perspective obtaining the identity of the abundant genes by sequencing a couple of hundred clones, biotinylated antisense oligonucleotides are designed against the common region mapped by aligning the abundant protein sequences derived from the parental proteins [18, 21, 51] . these abundant sequences can be effectively captured and removed by the complementary oligonucleotides immobilized on streptavidin-agarose beads. this unique feature significantly increases the chance of discovering nonabundant sequences. typically, we use an mrna displayed proteome library whose length of polypeptides is in the range of 50-300 residues. longer protein sequences with desired length distribution can also be obtained by appropriate fractionation (e.g., by gel electro phoresis). peptides or proteins of this size (5.5-33 kda) are much more likely than short synthetic peptides to adopt native conformations or structures. the high diversity also allows the identification of residues or motifs that play a range of major or minor roles in mediating protein-protein interactions. random mutagenesis is now widely used to generate mutations in directed evolution for the isolation of sequences with desirable functions [52] . most random mutagenesis approaches are pcr based, using methods such as error-prone pcr, dna shuffling, random insertion and deletion, and random insertional-deletional strand exchange [53] . the in vivo screening approaches do not readily allow the incorporation of these methods directly because pcr products have to be ligated to a plasmid and transformed into the host cells. nevertheless, mrna display uses pcr to amplify cdna in every round of selection. the resulting pcr products are directly used to perform in vitro transcription and translation, making it highly compatible with pcr-based mutagenesis and recombination techniques. indeed, fukuda and colleagues used mrna display to improve an antifluorescein single-chain variable fragment antibody from a library generated by error-prone dna shuffling and identified five consensus mutations, with the offrates decreased by more than one order of magnitude and the dissociation constants improved by 30-fold [22] . tsuji and colleagues used mrna display to screen for gtp-binding proteins from a dna shuffling library constructed from the human estrogen receptor ligand-binding domain [54] . after three rounds of selection, they successfully identified a novel gtp-binding protein with k d of approximately 224 nm. schultz and colleagues have developed an approach for the incorporation of unnatural amino acids into proteins by introducing an engineered trna that can recognize a stop codon [55, 56] . this approach can be used to introduce affinity tags, biophysical probes and substrate analogues to the library proteins without post-translational modifications. mrna display is compatible with this approach and can be used to screen for proteins or peptides with unnatural amino acids. li and colleagues undertook the first attempt to combine these two systems [57] . they used the engineered thg73 amber suppressor trna, which can incorporate biocytin, a biotin derivative of lysine, to the the uag stop codon. after two rounds of selection against immobilized streptavidin, the dna sequences with uag codon dominated the library. in addition to the nonsense suppression system, novel in vitro translation systems utilizing unnatural base paring or four-base codon-anticodon system can incorporate a much larger set of amino acids to construct polypeptide libraries similar to synthetic small molecule libraries [58, 59] . mrna display has been used to synthesize polypeptides that contain many unnatural amino acids and to evolve de novo proteins with enzymatic activities. using a reconstituted escherichia coli translation system, szostak and colleagues demonstrated that the reprogramed genetic code could be used to direct the ribosomal synthesis of polypeptides containing up to 13 different unnatural amino acids, including n-methyl amino acids, which allow for further post-translational cyclization and chemical derivitization [23, [60] [61] [62] . significantly, this system is compatible with mrna display, enabling the synthesis of unnatural peptide libraries of 10 14 unique members for in vitro selection of unnatural peptides with desired properties. seelig and szostak used mrna display to evolve synthetic proteins with genuinely new enzymatic activities without the need for prior mechanistic information [24] . starting from an mrna-displayed, partially randomized noncatalytic zinc finger scaffold library of very high diversity, they successfully evolved novel rna ligases that exhibit multiple turnover with rate enhancements of more than 2 millionfold. this system has the potential to be further developed for the evolution of novel enzymes that bind protein substrates and catalyze biochemical reactions [24] . despite its unique advantages in addressing a wide variety of biological problems, mrna display has limitations like most other protein-selection platforms do [51] . one major concern is whether the covalently attached mrna portion interferes with the function of the displayed protein or with the interaction between the target molecule and the displayed protein. it appears that proteins displayed on the mrna retain the binding specificity of the free proteins very well [7] . the interference of interaction is possible when the mrna is present as the flexible single-stranded form, particularly for proteins that nonspecifically bind to nucleic acids. in order to minimize the problem, the mrna can be reversely transcribed into an mrna/dna hybrid, which has a very rigid structure and is less likely to interfere with other molecules. mrna display works well when the displayed proteins are less than 300 residues in size. large proteins might also be displayed, but typically with lower efficiency [51] . mrna display cannot be used to address those proteins whose biological functions are strictly dependent on the formation of complexes with their binding partners. furthermore, mrna display is not suitable for displaying membrane-bound proteins due to the difficulty in expressing such proteins using in vitro translation systems [29] . owing to the simultaneous manipulation of proteins and nucleic acids at a low-nanomolar scale in an rnase-free environment, mrna display-based selection is relatively difficult to perform. the efficiency with which a particular sequence is selected depends perspective on several factors, including its mrna abundance, efficiencies of pcr amplification, in vitro protein expression, fusion formation and functional selection. in addition, all the target proteins used in the selection must be highly purified without contamination from rnases and proteases. because of the quick degradation of rna by nucleases, mrna display is not favorable for screening against the targets on the surface of living cells. another limitation of mrna display is that the solid surface-based biopanning procedure often results in nonspecific binding, even after preblocking using bovine serum albumin and trna. thus, it takes quite a few rounds to enrich a library with a diversity of 10 12 -10 13 [20, [63] [64] [65] . furthermore, the mrna portion of the fusion molecules is highly negatively charged, so it might be problematic when applied to a selection where the target is highly positively charged (e.g., a target protein with an isoelectric point of >9) [65] . there have been several attempts to address these limitations. to increase the stability of the fusion molecules, conversion of the single-stranded mrna in the mrna-protein fusion into an mrna/dna hybrid significantly increases the stability of the nucleic acid portion. a variant of mrna display, called cdna display, has also been developed [66] [67] [68] . in this technique, puromycin is introduced by the reverse-transcription primer, thus the polypeptide will be covalently linked to the cdna that is presumably more stable. the purification and enrichment efficiencies by using the target protein immobilized on microsphere beads are typically low. this prompted the use of other more efficient separation methods such as microfluidic systems [64] . tabata and colleagues showed that a much higher enrichment efficiency could be achieved by combining mrna display with a gold microfluidic chip on which the target protein was immobilized to minimize nonspecific binding [64] . using this approach, the washing and elution can be monitored in real-time and the target-binding sequences are enriched after two rounds of selection from an original diversity of approximately 10 12 . however, the cost for higher enrichment efficiency is the loss of ability to obtain binding partners with low affinity, which could be very useful for many applications. thus, this modified version is appropriate for applications such as the generation of antibody-like affinity molecules, where only the binders with the highest affinities are desired. to investigate protein-protein interactions, an ideal protein-selection method should allow the identification of the target-interacting sequences from both natural protein and synthetic peptide libraries under the conditions that are specific for such interactions. mrna display is particularly suitable for such purpose [69] . to perform the selection, the mrna-displayed peptide or protein library is first incubated with a target protein of interest (figure 1) . the target protein/binding partner complexes are isolated from the reaction mixture using affinity purification on a solid surface. after washing away sequences that bind to the target protein nonspecifically, sequences that bind to the target protein in a desired manner are eluted from the solid surface under mild and highly specific conditions. the selected mrna-protein fusion molecules are then amplified by pcr to regenerate the cdna library for the next round of selection. after several rounds of selection and amplification, the final enriched library is cloned and sequenced for ana lysis. the design of the selection scheme is the most critical issue. if designed appropriately, specific target-binding partners will be rapidly enriched after several rounds of selection, whereas a poorly designed selection scheme will either take many more rounds in order to get desired sequences or enrich for irrelevant sequences that quickly overwhelm the regenerated library pool. in general, capture or immobilization of target/binding partner complexes on a solid surface followed by competitive elution using a free target is an effective approach for specific enrichment. selection approaches based on 'binary' events such as conditional binding in the presence or absence of small molecules or a third regulatory partner can also be effective in identifying proteins with desired properties. selections based on binding events where conditions that achieve specificity are not known are challenging and may take more rounds of selection to enrich the desired sequences. to facilitate capture and isolation of the target/binding partner complexes from the reaction mixture, the target protein should be appropriately tagged. a terminal epitope tag such as polyhistisine (his × 6), hemagglutinin, e and flag tags can be engineered. however, the binding affinities between these affinity tags and the corresponding capturing molecules (ni 2+ -nitrilotriacetic acid or antibody) are typically in the nanomolar range. such binding strength does not allow for extremely tight immobilization of the target protein on the solid surface, making it difficult to isolate highly specific and tight binders that often require a very stringent washing procedure. one effective approach to addressing the problem is to biotinylate the target protein, which allows for immobilization of the target almost irreversibly on the solid surface coated with streptavidin. both chemical and enzymatic biotinylation can be used. the first approach is easy to perform but suffers from lack of control of the extent and sites of biotinylation. the latter is achieved by engineering a terminal short peptide called avitag™ that can be recognized by a biotin protein ligase (bira) for highly efficient and site-specific biotinylation, either during recombinant expression in an appropriate bacterial strain or after expression using a purified bira enzyme [21] . for all the selections, special procedures should be designed to minimize the enrichment of nonspecific sequences. the wash step before elution is important. in the target-binding selection, a stringent wash effectively removes sequences that bind the target nonspecifically, but it might also result in loss of the desired sequences if they bind to a target relatively weakly. typically, an appropriate wash condition should be determined prior to selection by using both positive and negative control sequences. for most selections in protein-protein interactions, gentle and competitive elution should be applied whenever possible. the various applications of mrna display in studying protein-protein interactions can be classified based on the type of the initial libraries. the first type of libraries that can be used for mrna display-based selection are synthetic combinatorial peptide libraries with varying length of randomized residues or protein domain libraries containing totally or partially randomized perspective amino acids (e.g., single-chain variable fragment antibody library, fibronectin type iii [fn3] domain library or zinc finger scaffold library). the second type of libraries are natural proteome libraries derived from the mrnas of organisms (or tissues) of interest. direct comparison of these two types of libraries against the same target using mrna display has been reported [69] . wilson and colleagues used mrna display to identify nonconstrained peptides that bind to streptavidin [70] . starting with a library of approximately 10 13 random peptides, 20 streptavidinbinding peptides were isolated. these linear peptides contain a histidine-proline-glutamine consequence motif, with dissociation constants as low as 5 nm, which are three to four orders of magnitude higher than those isolated by phage display from lower complexity libraries of shorter random peptides. xu and colleagues used mrna display to isolate affinity molecules that bound to tnf-α with the highest k d of 20 pm after ten rounds of selection from a library of more than 10 12 unique sequences by randomizing three solvent exposed loops of the tenth fn3 domain [71] . ja and colleagues applied mrna display to map the epitope of an antipolyhistidine monoclonal antibody with a random, unconstrained 27-mer peptide library [72] . after iterative rounds of selection and refinement, the resulting peptides bound to the antibody with 10-75-fold higher affinities than a hexahistidine peptide. the highest affinity peptides encoded both a short histidine track and the arrxa motif, suggesting that the motif and other flanking residues make important contacts adjacent to the core polyhistidine-binding site. olson and colleagues used mrna display to isolate affinity molecules that bind to phosphorylated ikbα from an fn3 domain library with 3 × 10 13 unique sequences. one of the affinity molecules they isolated bound to phosphorylated ikbα with an affinity of approximately 18 nm and was more than 1000-fold specific compared with the nonphosphorylated peptide [73] . the same library was also used to screen for inhibitors of nucleocapsid protein of severe acute respiratory syndrome coronavirus [74] . six n-terminal binders and two c-terminal binders of severe acute respiratory syndrome nucleocapsid protein were isolated after six rounds of selection. the best sequence has an affinity of approximately 1.7 nm and seven of the isolated binders can inhibit the virus replication from 11-to 5900-fold. matsumura and colleagues used mrna display to select bcl-xl inhibitors from a 16-mer peptide library [75] . the best inhibitor has an ic 50 of 0.9 µm, approximately ten-times better than the natural inhibitor: bh3 domain of bak protein. kosugi and colleagues used mrna display to define consensus patterns and properties of importin α-dependent nuclear localization signals (nlss) [76] . starting from an mrna-displayed random peptide library, they were able to identify six classes of nlss that specifically bound to distinct binding pockets of importin α, including two noncanonical classes that specifically bound the minor binding pocket and two classical monopartite nlss that bound to the major binding pocket. austin and colleagues demonstrated that mrna display is a powerful technology that can be used to evolve short peptide ligands that target protein families with high class specificity and state specificity [77] . starting from an mrna-displayed peptide library based on the short gα-modulating peptide r6a-1, they were able to identify and further maturate r6a-1 variants that target a convergent protein-binding surface of gαs/gdp. interestingly, the selected peptides made specific contacts with the effector-binding region of gα. the optimized matured gαs(s)-binding peptide-1 peptide showed remarkable selectivity and affinity, exhibited little or no binding to nine homologous gα subunits or human h-ras, and discriminated the gαs splice variant gα. there have also been a lot of successful cases using mrna display on natural proteomic libraries. hammond and colleagues used mrna display to screen for natural proteins that can bind to the anti-apoptotic protein bcl-xl from a mixture of four human cdna libraries derived from kidney, liver, bone marrow and brain tissue [14] . after four rounds of selection, more than 70 different proteins were obtained, including known bcl-xl-binding partners such as bim, bax and bak. horisawa and colleagues used a similar approach to identify the binding partners of transcription factor jun from a mouse brain proteome library [17] . a total of 20 sequence groups of proteins were isolated after five rounds of selection, among which 16 sequence groups had already been known and four of them were hypothetical jun-interacting proteins. mcpherson and colleagues used mrna display to examine drug/receptor interactions by identifying the binding target(s) of the small molecule therapeutic agent fk506 [16] . starting with an mrna-displayed proteome library from human liver, kidney and bone marrow, fkbp12, the well-known fk506-binding protein, was found to be the dominant clone after three rounds of drugbinding selection. they further demonstrated that the method allowed mapping of the minimal drug-binding domain within fkbp12. shen and colleagues used mrna display to screen a human proteome library derived from brain, heart, spleen, thymus and muscle for ca 2+ -dependent calmodulin binding partners [18] . after two rounds of selection, a large number of proteins that can bind calmodulin in a ca 2+ -dependent manner were identified, with the dissociation coefficients ranging from nanomolar to micromolar. a similar study has been performed by using the adult caenorhabditis elegans proteome library. after two rounds of selection, nine known and 47 novel ca 2+ /calmodulin-binding proteins were enriched [19] . a similar strategy was used to identify the associating proteins of tyrosine phosphatase nonreceptor type substrate-1 (shps-1) from a cdna library constructed from vascular smooth muscle cell mrna [78] . a number of proteins that were not known to bind to phosphorylated shps-1 were isolated. miyamoto-sato and colleagues used mrna display to map the transcription regulation network [79] . they used 68 proteins to represent 50 human transcription factors as the baits and screened for the sequences that can bind to these proteins from a human brain cdna library. after verifying the interactions by pull-down assays, they analyzed the interaction networks and generated a domain-based protein interaction database. mrna display has also been used to study the specificity of antibodies. shibui and colleagues used a monoclonal antihuman p53 antibody as the target and screened binders from a cdna library constructed using mrnas from several tissues and cell lines [80] . five proteins were found to bind to the antibody, including p53 protein, with a consensus motif of sdlxkl. • mrna display has many advantages over other widely used peptide or protein-selection techniques, including: -easy and robust recovery of the selected polypeptide sequences -freedom of using arbitrary selection conditions -making use of very large libraries of candidate sequences -minimizing context dependence in both expression and selection -easy removal of the abundant sequences from the candidate pools -applicable to the selection of protein sequences of long length -compatible with most diversity-generation methods • researchers have used mrna display to study various protein-protein interactions from both natural protein and synthetic peptide libraries, including: -generation of affinity molecules against target proteins from synthetic polypeptide libraries -mapping protein-protein interactions network from proteomic libraries -mapping dna-protein interactions network from proteomic libraries -studying the specificity of antibodies -mapping the consensus-binding motifs of proteins -identifying the downstream substrates of kinases and proteases -evolution of enzymes from partially randomized protein scaffolds mrna display has also been used to identify the natural downstream substrates of enzymes such as kinases and proteases. cujec and colleagues used mrna display to study the substrates of the v-abl tyrosine kinase from both a synthetic peptide library and a natural proteome library [15] . when the synthetic peptide library was used, a consensus sequence closely matching that previously reported for the v-abl tyrosine kinase was isolated. selections from a proteomic library derived from cellular mrnas revealed several novel targets of v-abl, including a new member of a class of sh2 domaincontaining adaptor proteins. ju and colleagues designed a general strategy to identify the downstream substrates of highly specific proteases [21] . in this method, the substrate library was immobilized on streptavidin agarose. upon incubation with a protease of interest, the substrates were cleaved and the corresponding mrnas released. they used this method to identify the downstream substrates of human caspase 3 and obtained 115 positive sequences [21] . currently, biopharmaceuticals based on drug target-binding affinity molecules are considered to be the future of pharmaceutical industry. indeed, targeted protein therapeutics based on humanized anti-egf receptor, anti-her2 and anti-vegf monoclonal antibodies have been demonstrated with clinical efficacy in several cancers, including breast, lung and colon malignancies. many target-binding biopharmaceuticals rely on a full-length humanized monoclonal antibody that is difficult to optimize through a directed protein evolution approach. currently, there is an urgent need for the development of small protein domains that are amenable for directed protein evolution to create the next-generation affinity molecules exhibiting desirable therapeutic or targeting features. recently, the patent rights to mrna display technology were acquired by bristol-myers squibb as a major protein design engine for the systematic development of biopharmaceuticals with high potency and specificity [101] . ct-322, an fn3 domain-based antagonist of vegf receptor-2 developed by using mrna displaybased protein-protein interaction selections, is currently under clinical trials in oncology, including in patients with recurrent glioblastoma multiforme, metastatic colorectal cancer and nonsmall-cell lung cancer. the success of this product will further justify the potential high power of mrna display in the development of targeted therapeutic or diagnostic agents for the treatment of many human diseases. currently, mrna display is a totally in vitro peptide or protein selection method. owing to its unique features, we expect that this technique will be widely applied to investigate many different types of protein-protein interactions that are difficult to address using other protein-selection techniques. in particular, it is ideal for the investigation of conditional protein-protein interactions that rely on the present of a third partner. we expect that improvement will be made to make mrna display suitable for selections using bioactive receptors on the surface of living cells. we also expect that mrna-displayed full-length proteins, such as all the proteins involved in a particular signaling pathway, will be used to understand their comprehensive protein-protein interaction networks and the regulatory mechanisms governing them. the mrna display work in the liu laboratory was supported by start-up funds from the carolina center for genome sciences and eshelman school of pharmacy at the university of north carolina at chapel hill (nc, usa) and by nih grants ns047650, ca119343, da025702 and ca151652, and american cancer society grant rsg-tbe-110472. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no writing assistance was utilized in the production of this manuscript. advantages of mrna display in studying protein-protein interactions a novel genetic system to detect protein-protein interactions filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface an in vitro polysome display system for identifying ligands from very large peptide libraries in vitro selection and evolution of functional proteins by using ribosome display rna-peptide fusions for the in vitro selection of peptides and proteins in vitro virus: bonding of mrna bearing puromycin at the 3´-terminal end to the c-terminal end of its encoded protein on the ribosome in vitro optimized synthesis of rna-protein fusions for in vitro protein selection man-made cell-like compartments for molecular 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estrogen-receptor fragments adding new chemistries to the genetic code beyond the canonical 20 amino acids: expanding the genetic lexicon in vitro selection of mrna display libraries containing an unnatural amino acid four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids four-base codon mediated mrna display to construct peptide libraries that contain multiple non-natural amino acids • describes a combination of a four-base codons in vitro translation system and mrna display enzymatic aminoacylation of trna with unnatural amino acids an expanded set of amino acid analogs for the ribosomal translation of unnatural peptides ribosomal synthesis of n-methyl peptides photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins rapid antibody selection by mrna display on a microfluidic chip describes the use of a combination of mrna display and microfluidic surface plasmon resonance system to achieve ultra-high enrichment efficiency chemical and genetic wrappers for improved phage and rna display cdna-protein fusions: covalent proteingene conjugates for the in vitro selection of peptides and proteins an in vitro dna virus for in vitro protein evolution cdna display: a novel screening method for functional disulfiderich peptides by solid-phase synthesis and stabilization of mrna-protein fusions describes the use of a modified version of mrna display called cdna display for the selection of functional disulfide-rich peptides comparison of mrna-display-based selections using synthetic peptide and natural protein libraries the use of mrna display to select high-affinity protein-binding peptides directed evolution of high-affinity antibody mimics using mrna display describes the use of mrna display to develop single-domain antibody mimics that tightly and specifically bind to a potential drug target epitope mapping using mrna display and a unidirectional nested deletion library mrna display selection of a high-affinity, modification-specific phospho-ikbαbinding fibronectin describes the use of mrna display to develop single-domain antibody mimics that bind to peptides in a phosphorylation-dependent manner mrna display design of fibronectin-based intrabodies that detect and inhibit severe acute respiratory syndrome coronavirus nucleocapsid protein mrna display selection of a high-affinity, bcl-x(l)-specific binding peptide six classes of nuclear localization signals specific to different binding grooves of importin alpha evolution of class-specific peptides targeting a hot spot of the gαs subunit identification of novel shps-1-associated proteins and their roles in regulation of insulin-like growth factor-dependent responses in vascular smooth muscle cells a comprehensive resource of interacting protein regions for refining human transcription factor networks isolation of cross-reacting antigen candidates by mrna-display using a mixed cdna library key: cord-287477-aios0h8s authors: sicari, daria; chatziioannou, aristotelis; koutsandreas, theodoros; sitia, roberto; chevet, eric title: role of the early secretory pathway in sars-cov-2 infection date: 2020-07-28 journal: j cell biol doi: 10.1083/jcb.202006005 sha: doc_id: 287477 cord_uid: aios0h8s similar to other rna viruses, sars-cov-2 must (1) enter a target/host cell, (2) reprogram it to ensure its replication, (3) exit the host cell, and (4) repeat this cycle for exponential growth. during the exit step, the virus hijacks the sophisticated machineries that host cells employ to correctly fold, assemble, and transport proteins along the exocytic pathway. therefore, secretory pathway–mediated assemblage and excretion of infective particles represent appealing targets to reduce the efficacy of virus biogenesis, if not to block it completely. here, we analyze and discuss the contribution of the molecular machines operating in the early secretory pathway in the biogenesis of sars-cov-2 and their relevance for potential antiviral targeting. the fact that these molecular machines are conserved throughout evolution, together with the redundancy and tissue specificity of their components, provides opportunities in the search for unique proteins essential for sars-cov-2 biology that could also be targeted with therapeutic objectives. finally, we provide an overview of recent evidence implicating proteins of the early secretory pathway as potential antiviral targets with effective therapeutic applications. as obligatory parasites, viruses must find a suitable host cell, deliver into it their genome (either dna or rna), and hijack the endogenous molecular machineries for their own replication (su et al., 2016) . thus, some scientists exploit viruses as trojan horses to deliver genes into target cells, whereas others search for the achilles heels of those causing diseases. to find a suitable host, viruses bind to receptors expressed on the cell surface. selective binding activates membrane fusion, allowing the viral genome to enter the target cell. fusion can occur directly at the plasma membrane (e.g., human immunodeficiency virus) or within endocytic compartments (e.g., influenza). in the cytoplasm, the viral genome is replicated while enough structural and nonstructural proteins are produced. all the essential components must then be assembled and surrounded with an envelope composed of viral proteins in combination with host cell-derived membranes. viruses exploit the ability of human cells to produce, assemble, and transport to the cell surface receptors, transporters, and other complex proteins (su et al., 2016) . for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and other coronaviruses (covs), these steps take place in organelles of the early secretory pathway, the er, the er-golgi intermediate compartment (ergic) , and the golgi apparatus. sars-cov-2, a member of the sars-cov family, is responsible for a global pandemic that started in 2019 (lake, 2020; myint, 1994; andersen et al., 2020) . because sars-cov-2 shares 79% of the genome with sars-cov, one can use knowledge obtained on the viruses that caused prior pandemics, such as sars and middle east respiratory syndrome (lake, 2020; lim et al., 2016) . cov-2 infection starts when its spike (s) protein binds to angiotensin i-converting enzyme 2 (ace2) receptors on the host cell membrane (lake, 2020; letko et al., 2020) . cov-2 does so with higher affinity than sars-cov (wan et al., 2020; wu et al., 2020) . in the cytosol, the virus is uncoated, and its rna genome is used to produce replicases and nonstructural proteins (nsps). replicases generate subgenomic rna capable of producing the structural proteins spike (s), nucleocapside (n), membrane (m), and envelope (e) proteins. the group of proteins helping the assembly and transport of infective viral particles are referred to as accessory proteins (orfs). a large number of these proteins are cotranslationally translocated into the er to be glycosylated, folded, and assembled in preparation for virus budding and release via the secretory pathway. thus, virion spread critically depends on recruiting the most efficient secretory machineries of host cells (su et al., 2016; proteins of the early secretory pathway bound by sars-cov-2 as the entire world asks for ways to stop cov-2, many laboratories are investigating the virus's achilles heel(s). because the virus is an obligatory parasite, a powerful strategy to identify endogenous molecules essential for its replication starts with the expression of suitably tagged cov-2 proteins in human cells. the host interactors are identified by biochemical methods and grouped on the basis of their known functions. experiments along these lines have been performed in a cooperative effort led by the krogan laboratory in a study aiming to reposition approved drugs against the virus (gordon et al., 2020) . taking advantage of affinity purification mass spectrometry assays, the authors, using hek293t cells as a model, highlighted 66 putative targetable cov-2-host protein-protein interactions among the 332 identified (gordon et al., 2020) . many (∼24%) involved proteins from the host secretory pathway (fig. 2, a and b ). these observations are in line with those of previous analyses of other viruses but also revealed a number of proteins specific for cov-2, the most representative being orf8 (43%), orf9c (12%), nsp7 (11%), nsp13 (10%), and m (7%; fig. 2 c and table 1) . hereafter, we comment on the interactions involving elements of the secretory pathway and how these could be targeted to combat cov-2. this approach has great potential but also some limitations. perhaps the most relevant caveat is that each viral protein was expressed independently of the other sars-cov-2 proteins in human cells. on the one hand, this is key to identifying specific interactors in sufficient quantities. on the other hand, however, it might yield artifacts due to the absence of other physiological partner(s) or accessories, nonstructural proteins encoded in the viral genome or produced by the host cell upon viral infection. overexpression per se might elicit er stress responses that do not necessarily reflect what happens in an infected cell. in addition, considering that sars-cov-2 can infect different human cell types, the hek293-based interactome might miss tissue-specific factors and introduce biases. for instance, similar experiments performed in a549 cells produced slightly different results in terms of interactors (stukalov et al., 2020) , because protein-protein interaction assays depend on the gene expression patterns specific for each cell line (table 2) . nonetheless, the interactome data obtained in hek293 and a549 cells clearly identify commonly enriched pathways, including er-to-cytosol trafficking, protein quality control, inflammatory response, and dna damage (stukalov et al., 2020) . the role of glycosylation and protein quality control in sars-cov-2 infections most covs bud at the ergic level ( fig. 1) and are then transported along the exocytic pathway (klumperman et al., 1994; stertz et al., 2007) . the domains facing the er lumen figure 1 . the journey of sars-cov-2 in the host cell. coronavirus binds to cognate receptors on target cells via the spike proteins (dark red). this drives conformational changes that promote fusion of the virus with the host cell's plasma membrane (entry by endocytosis and green membrane-containing virion, bottom left). in the cytoplasm, viral capsids are uncoated, and the viral rna genome is translated, producing two poly-proteins (pp1a and pp1ab). these polypeptides are then proteolytically processed by both host and viral proteases, thereby generating nonstructural proteins (nsps) and leading to the formation of the replicase-polymerase complex (rtc). the latter is responsible for the replication of the viral genome and for the production of subgenomic rnas, which are translated into the structural proteins nucleocapsid (n), spike (s), membrane (m), and envelope (e). in addition to these genomic elements shared by other covs, the sars-cov-2 genome also contains eight open reading frames (orfs) that drive the production of accessory proteins. s, m, and e structural proteins and some accessory proteins are co-translationally translocated into the er, where they undergo diverse post-translational modifications, including disulfide bond formation and n-linked glycosylation. structural proteins concentrate in the ergic, where they assemble around the newly formed genome-nucleocapsid complexes. mature virions are further modified (e.g., o-glycosylated) as they proceed through the golgi complex and later stations of the secretory pathway before being released in the extracellular milieu (release by exocytosis and pink membrane-containing virions, top left). the membrane of the virus derives from the host cell, which synthetizes it in the er. undergo n-glycosylation, disulfide bond formation, and other posttranslational modifications necessary for viral tropism and host cell specificity. addition of n-glycans occurs cotranslationally in the er, and their processing continues in downstream compartments (fung and liu, 2018; liang et al., 2019; oostra et al., 2006) . the glycosylation profiles of the cov-2 s protein revealed 86% conservation of the n-sites with sars-cov strains (watanabe et al., 2020) . it has been proposed that conservation of the n-glycans in the receptor-binding domain is important for recognition by a neutralizing antibody (cr3022) raised against sars-cov. indeed, the lower affinity of cr3022 for cov-2 than for other sars strains might reflect the absence of a specific glycan in the n370 residue (yuan et al., 2020) . interestingly, two o-glycosylation sites are instead present only in cov-2 s protein. these o-glycans are thought to hinder s protein epitopes from host immune system responses and to improve ace2 receptor binding (watanabe et al., 2020) . cov-2 protein orf8 might support o-glycosylation by binding poglut2, poglut3, two o-glucosyltransferases, and pofut1, an o-fucosyltransferase (table 1 ). these enzymes have been shown to be pivotal for the proper glycosylation and presentation on the plasma membrane of the notch1 receptor (takeuchi et al., 2018; yang et al., 2019; okamura and saga, 2008) , confirming once more the importance of glycan processing in the maturation and transport of cov-2 s and other proteins. accordingly, many viral accessory proteins interact with components of the host cell's secretory pathway. we still have a limited understanding of how the assembly of viral structural proteins is coordinated. in sars-cov, copi binds the kxhxx motif found in the cytosolic domain of the s protein. this motif sicari et al. journal of cell biology is necessary for s to accumulate in ergic and interact with m and is hence crucial for assembly (mcbride et al., 2007) . cov-2 s protein presents the same motif. in both hek293 and a549 cells, it interacts with two golgi-localized proteins, zdhhc5 and golga7 (gordon et al., 2020; stukalov et al., 2020) , which form a complex localized to the plasma membrane (ko and dixon, 2018; ohno et al., 2006) . zdhhc5 is an acyltransferase that can localize to the er, golgi apparatus, and plasma membrane and controls golgi trafficking and palmitoylation of anthrax toxin (sergeeva and van der goot, 2019). golga7 is a golgin subfamily member that assists zdhhc5 activity on the plasma membrane (ko et al., 2019) . the zdhhc-golga7 complex may help s protein to reach the ergic compartment and favor assembly. interestingly, erp44, slc30a6, and slc30a7 were found to interact with orf8, orf9c, and m proteins, respectively. erp44 cycles between the er and the golgi to retrieve select clients back to the er (anelli et al., 2002; otsu et al., 2006) . this process is regulated by the ph and zinc gradients between the two compartments (vavassori et al., 2013; sannino et al., 2014; watanabe et al., 2017) . two golgi-resident transporters (slc30a6/znt6 and slc30a7/znt7) provide sufficient zinc to activate erp44 (watanabe et al., 2019) . moreover, erp44 binds the lectin ergic53/lman1, which promotes the production of infective covs (klaus et al., 2013) . lman2 (vip36) and ergic1, two ergic53like lectins, also interact with orf7 and nsp10, respectively (table 1) . vip36 has been shown to control transport of high-mannose proteins (gupta, 2012; mages et al., 2008) . thus, it could be that cov-2 takes advantage of erp44-and lectin-dependent er-to-golgi transport processes to accumulate in the ergic for budding. the formation of proper disulfide bonds is key for the folding, assembly, and recognition of ace2 receptors by spike (oostra et al., 2007) . cov-2 orf8 protein binds to the er-resident oxidase ero1β (table 1) . orf8-mediated ero1β interaction may be important for disulfide bond formation in protein s or for the formation of orf8 multimers. considering that ero1β is a powerful oxidase induced by the unfolded protein response (upr), this interaction might also reflect a response to er stress. cov-2 orf8 protein was also found to bind fkbp10 and fkbp7 (table 1) , two er-resident peptidyl-prolyl cis-trans isomerases, and uggt2 (table 1) , a key element in the glycoprotein quality table 1 . proteins of the early secretory pathway found to interact with sars-cov-2 list of host proteins (columns) related to the secretory pathway that interact with different viral proteins (first line). proteins derived from the input signature (gordon et al., 2020) have been divided based on the cellular compartment localization (fig. 2 a) . in fig. 2 b, candidates related to er and golgi compartments have been sorted based on the interacting viral protein. sicari et al. journal of cell biology 5 of 13 a closer look at a sars-cov-2 achilles heel https://doi.org/10.1083/jcb.202006005 table 2 . conserved features in sars-cov-2 and sars-cov interactomes (continued) hek293 (gordon et al., 2020) a 5 4 9 ( stukalov et al., 2020) secretory pathway components proteins listed in table 1 were matched with human protein-protein interactions obtained in a549 cells expressing sars-cov or sars-cov-2 proteins (stukalov et al., 2020) . the comparison highlights cell type-and virus-specific differences within an overall similarity of the interactome. control machinery and part of a large multichaperone in the er lumen. other cov-2 protein interactors are part of the sophisticated machineries that guarantee protein homeostasis within the exocytic compartment; that is, the balance between protein production, secretion, and degradation, a process generally referred to as "proteostasis" (klaips et al., 2018) . for instance, orf8 binds numerous proteins involved in erassociated degradation (erad), a key pathway for limiting protein condensation and aggregation in the early secretory compartment (sun and brodsky, 2019) . orf8 binds edem3 (er degradation enhancing α-mannosidase like protein 3) and os9 (table 1) , two erad-related proteins that are abundant in coatomer complex ii-independent vesicles. these structures are referred to as "edemosomes" and are key elements in proteostasis adjustments because they normally control the levels of erad components (araki and nagata, 2011) . interestingly, during sars-cov infection, edemosomes are used for forming double-membrane vesicles (dmvs) that recruit the replicationtranscription complexes (reggiori et al., 2010) . dmvs are also important to protecting the viral genome from sensors in the cytosol and from ifns (zinzula and tramontano, 2013) . finally, emc1, a subunit of the er membrane protein complex, was found to interact with orf8 (table 1) . with emc4 and emc7, emc1 is thought to engage the rab7 gtpase in late endosomes (bagchi et al., 2020) . bridging late endosomes and the er, this interaction might enable efficient entry and/or excretion of viral particles. emc proteins also associate with sec61 (chitwood and hegde, 2019), a component of the translocon mediating the entry of nascent polypeptides into the er, and have been presented by gordon et al. (2020) as a sars-cov-2 modulator. the above lines of evidence suggest that the biogenesis of effective viral proteins requires efficient assembly and transport lines in the host's secretory protein factory. how can cov-2 divert them to its advantage? in general, viral infection induces er stress via the strain imposed by the massive flux of viral proteins on the cell's folding and transport machineries (zhang and wang, 2012) . er stress induces activation of three er resident receptors, inositol-requiring enzyme 1α (ire1), protein kinase r-like er kinase (perk), and activating transcription factor 6 (atf6), which in turn promote a transcriptional program named the "upr" (hetz et al., 2015) . these receptors have all been implied in cov infection. for instance, s protein from sars-cov presents a upr-activating domain mapped in the central region of the s1 subunit (between 201 and 400 aa) that acts in an n-glycosylation-independent manner (siu et al., 2014) and is highly conserved among covs. sars-cov orf3a activates atf4 and chop promoter activities, suggesting the involvement of the perk branch during the infection (minakshi et al., 2009) . perk activation induces phosphorylation of the α-subunit of the eukaryotic initiation factor α (eif2α), leading to translation suppression and activation of a downstream signaling pathway known as the "integrated stress response." under the integrated stress response, translation of a majority of cellular mrnas is suppressed while mrnas containing small upstream orfs in their 59utr are translated, among them the proapoptotic transcription factor atf4. during cov infection, eif2α phosphorylation, global host mrna translation inhibition, and atf4 translation were detected (bechill et al., 2008) . however, it remains unknown how virus mrnas can still be translated normally when eif2α is phosphorylated. an attractive hypothesis, based on recently published data, is that sars-cov-2 infection impacts protein phosphorylation profiles. for instance, larp1, which interacts with the n viral protein, has been found to be less phosphorylated in cov-2-infected cells. this modification increases the affinity of larp1 for the 39utrs of mrnas encoding ribosomal proteins, thus attenuating protein synthesis stukalov et al., 2020) . er stress induces the cytosolic kinase and rnase activities of ire1α. this leads to the nonconventional splicing of x-box binding protein 1 (xbp1) mrna, rna degradation via ire1αdependent rna decay (ridd), and jnk phosphorylation. xbp1 spliced (xbp1s) is a transcription factor that promotes transcription of genes encoding proteins involved in entry into the er, folding, glycosylation, erad, lipid biogenesis, and vesicular trafficking (huh et al., 2010) . ridd activity has been described as either adaptive or terminal (maurel et al., 2014) . it has been shown to degrade er-associated mrna to facilitate er homeostasis (adaptive) or mrna encoding prosurvival proteins, thereby contributing to er stress-induced cell death (terminal). sars-cov infection failed to induce xbp1 splicing, ridd, or jnk phosphorylation (dediego et al., 2014; versteeg et al., 2007) , suggesting that the ire1 branch of the upr remains inactive during sars-cov infection. however, cov-2 nsp6 protein binds sigma receptor 1. this er-resident integral membrane protein, together with sr2 (tmem97), which interacts with cov-2 orf9c, modulates calcium fluxes (table 1 ) and controls ire1 activation (mori et al., 2013; rosen et al., 2019) . thus, cov-2 may affect ire1 activity through its interaction with sigma receptor 1. last, er stress induces atf6 cleavage and its er-to-golgi translocation and finally an atf6 transcriptional program, whose main target genes consist of er chaperones (i.e., grp78/bip or grp94) and erad components (shoulders et al., 2013) . thus, activation of the atf6 branch enhances the er protein-folding capacity and homeostatic balance (adachi et al., 2008) . overexpression of the sars-cov s protein leads to the activation of both grp78 and grp94 promoters (siu et al., 2014) , suggesting atf6 activation. notably, the small amounts of bip found on the cell surface have been proposed to help cov entry and have been suggested as a target to impede sars-cov-2 infection (ha et al., 2020) . atf6 cleavage and nuclear translocation of the released cytosolic fragment were observed following sars-cov orf8ab transfection. in addition, orf8ab has also been shown to localize to the er and to interact with the luminal domain of atf6 (sung et al., 2009 ). these observations are consistent with the fact that many proteins that interact with cov-2 orf8 are encoded by atf6 target genes. moreover, the cytosolic atf6 fragment has been shown to be phosphorylated and activated by p38 mapk (luo and lee, 2002) , one of the most active kinases upon sars-cov-2 infection , supporting a role for atf6 in cov-2 infection. collectively, these studies indicate the importance of evaluating the precise impact of cov-2 proteins on the activation of er stress receptors and induction of the upr. interestingly, cov-2 m and nsp7 proteins were found to bind molecules involved in the regulation of er morphology. among them, receptor expression-enhancing proteins are implicated in formation and restructuring of the er network, as well as intracellular transport of receptors to the plasma membrane. yif1a recycles between the er and the golgi with a major ergic localization, maintaining homeostasis of the early secretory pathway . rab7a modulates er morphology by controlling er stress responses (mateus et al., 2018) . thus, these cov-2 interactions may be crucial to controlling er morphology and homeostasis, limiting induction of er stress and apoptosis. other links between er structure and cov-2 infection are the orf9c-scap interaction and nsp13/gorasp1 or golga2 binding (table 1 ). in particular, scap regulates the er-to-golgi translocation and activation of sterol regulatory elementbinding protein (srebp). srebp target genes are involved in lipid biosynthesis and metabolic homeostasis, two key processes during virus infection . orf9c-mediated scap binding may be used to promote lipid synthesis via srebp activation for generating the envelopes needed for cov-2 replication. thus, available scap-targeting drugs and pre-srebp inhibitors that have shown some inhibitory effects on middle east respiratory syndrome-cov replication, together with treatment using the cholesterol metabolite 25-hydroxycholesterol, could be interesting to test . nsp13 binds gorasp1 and golga2 and other proteins regulating golgi structure and vesicular transport (table 1) . especially during er stress, the gorasp1-golga2 complex mediates the unconventional trafficking of select glycoproteins to the cell membrane. together, these results suggest that cov-2 may control er-golgi homeostasis maintenance by acting on diverse er-golgi-related structures and pathways. targeting er homeostasis taken together, the above observations suggest that cov-2 reshapes the entire secretory pathway to its own advantage. therefore, repositioning available compounds to target the secretory pathway and upr components could represent a strategy against cov-2. inhibitors for er stress receptors are available and employed in numerous pathologies, including cancer and metabolic diseases. perk/eif2α inhibitors are gsk2606414, salubrinal/guanabenz, and integrated stress response inhibitor. the use of these drugs may lead to exaggerated protein load in the er lumen and increased er stress induction by preventing eif2α phosphorylation and translation inhibition (matsuoka and komoike, 2015; hetz et al., 2013) . ire1 rnase inhibition with kinase-inhibiting rnase attenuators (kiras; kira6 and kira8), toyocamycin, stf-083010, 4µ8c, mkc series, and b-i09 could be used to dampen xbp1 mrna splicing and ridd activity (cubillos-ruiz and glimcher, 2015) . although no drugs directly targeting atf6 are available, indirect inhibition was obtained using nelfinavir. this compound targets human immunodeficiency virus proteases and also inhibits the proteases responsible for atf6 cleavage (guan et al., 2015; sicari et al., 2019) . to slow down viral spread, it could be ideal to target the protein quality control machinery, which in turn would dampen viral assembly and maturation. for instance, compounds that target glycosylation enzymes, such as kinefusine and iminosugar derivative 1-deoxynojirimycin, which, respectively, inhibit mannosidase and α-glucosidase, are used in the clinic to dampen the morphogenesis of enveloped viruses. in addition, these drugs alter ace2 receptor glycosylation and transport to the cell surface, thus contributing to prevention of virus entry (winchester, 2009) . exacerbating er stress to induce apoptosis could be obtained by targeting er enzymes or chaperones. several protein disulfide isomerase inhibitors are available, including pacma31, which specifically targets the cysteine residue present in the active site of protein disulfide isomerase proteins and blocking their capability to bind cargoes (kaplan et al., 2015) . finally, downstream stations of the secretory pathway could be targeted using golgicide a (ga), an inhibitor of er-golgi transport vesicles. it was already shown that ga is able to inhibit protein secretion, shiga toxin trafficking in the endosomal compartment, and replication of coxsackievirus (alborzinia et al., 2018) . in this perspective, ga could be used to block virus spreading and maturation, acting on golgi proteins that interact with nsp13. eeyarestatin i targets erad by binding p97, thus eliciting er stress. interestingly, this drug interferes with the trafficking of a shiga-like toxin (wang et al., 2010) . moreover, er/golgi/sec pathway-related compounds have been proposed by gordon et al. (2020) . among the 66 u.s. food and drug administration-approved drugs, they suggested ps30613 (er protein processing, sec61 inhibitor); ihvr-19029 (er protein processing); fk-506 (fkbp binder); zotatifin (eif4e2/h, eif4a inhibitor); rapamycin and rapalogs (larp1, fkbp15, fkbp7/10, mammalian target of rapamycin inhibitor); diverse sigmar1/2 modulators, such as chloroquine, pb28, and haloperidol e-52862, pd-144418, and rs-ppcc; as well as diverse sigmar1/2 direct modulators, such as the antagonist pd-144418 and the agonist rs-ppcc (gordon et al., 2020) . however, owing to the huge numbers of substrates of some of the above targets, these protocols might lack the specificity needed to guarantee a reasonable therapeutic window. to evaluate whether the select interaction of cov-2 with components of the secretory pathway could represent an actionable strategy, we sought to identify therapeutic targets through signature-driven pharmacogenomic analysis using three distinct ontological vocabularies (mgi mammalian phenotype, reactome, and gene ontology). to this end, the whole cov-2 human host interactome (gordon et al., 2020) was imported into bioinfominer (bioinfominer.com; lhomond et al., 2018) . this allowed mapping the viral interactome to a consensus semantic tree graph that provides a systemic functional overview. this was used to interrogate the l1000 connectivity map repository, statistically selecting nontrivial network perturbagens, namely compounds that, when administered in cellular models, elicit perturbation in gene subsets of the input signature (table 3) . this approach differs in the sense that it exploits the whole vector of the derived signature, which in our case integrates core regulatory proteins linking broadly perturbed modules of the host cellular physiology, in order to prioritize compounds that affect a critical subset of them, ordered according to the significance of the enrichment score, measuring their overlap with the proposed targeted gene sets of the l1000 repository. furthermore, because bioinfominer enables delineation of the systemic tree snapshot of the mode of host infection, we exploited this feature to provide a detailed comparison of 12 viral interactome functional profiles. this network-aided phylogenetic analysis measured the enrichment of the vertices of this graph, estimated the functional similarities of the semantic tree graphs of the viral interactomes, and applied agglomerative clustering for the pathogens under analysis. thus, cov-2 is robustly found to functionally share the highest similarities with enteroviruses (rhinovirus c15 and coxsackievirus a10; fig. 3 ). the ontological terms obtained from the whole cov-2 interactome with the three bioinfominer vocabularies (mgi mammalian phenotype, reactome, and gene ontology) revealed many proteins related to the secretory pathway among the perturbed genes (last columns in table 2 ). starting from the idea that cov-2 exploits host-derived secretory pathway components for assembly, budding, and spreading, we repeated the same bioinformatic analysis using the secretory pathway component list (table 1) . we observed enrichment for five host-derived virus-interacting proteins (golgb1, pde4dip, tor1a, hmox1, and hyou1) involved in different processes and related to quality control and er-golgi homeostasis maintenance. specifically, golgb1 interacts with cov-2 nsp13, and it is key for golgi organization and long intercisternal communication. interestingly, it is also an antigen in chronic rheumatoid arthritis. on the basis of our analysis, golgb1 could be a target of diverse classes of compounds, such as catechins, ganciclovir, and cetirizine-dihydrochloride. catechins, already proposed as potential anti-influenza virus agents, inhibit neuraminidase and specifically interact with ha (müller and downard, 2015) . moreover, because catechins also alter the viral membrane, they the table highlights compounds, ordered according to their statistical scoring, which perturb gene subsets of the input signature (last column), when administered in the indicated cell line. the input gene signature has been derived from the bioinfominer analysis, using the mgimp ontology, for the cov-2 secretome (87 protein interactions). may also interfere with particle maturation. cetirizines present anti-inflammatory effects and decrease expression of leukocyte intercellular adhesion molecule 1 membrane receptors in nasal epithelial cells (ciprandi et al., 1995) . this receptor is a major target for respiratory viruses and reasonably also for sars-cov-2. finally, ganciclovir is an anti-herpesviridae family drug (cytomegalovirus, epstein-barr virus, or herpes simplex virus); it blocks dna replication, and it has already been tested in patients with cov-2 infection in combination with two other antiviral drugs to minimize the potential for septic shock and inflammation (yan et al., 2020) . a second possible therapeutic target is pde4dip, which anchors components of the campdependent pathway involved in cell movement and migration to the golgi stacks (robinson et al., 2014; andruska et al., 2015 (andruska et al., 2015 ). nsp13 interaction with pde4dip may help viral particle movement during the exit step. among pde4dip-targeting drugs, estrone has been employed against influenza virus infection and other respiratory diseases (robinson et al., 2014) . a third target that came up in our analysis is tor1a, a member of the aaa+ atpase superfamily. because its depletion impairs herpes simplex virus 1 replication and induces improper envelope formation (maric et al., 2014) , tor1a might facilitate envelope formation of sars-cov-2. hmox1 and hyou1 are er residents triggered by oxygen deprivation that interact with orf8 and orf3a, respectively. their presence in the top hits supports the involvement of hypoxia and er stress in cov-2 infection, possibly reflecting an antiapoptotic role. hmox1 and hyou1 are targets of radicicol, an hsp90 inhibitor, which blocks vesicular stomatitis virus replication (born et al., 2013) . the sars-cov-2 genome encodes for nonstructural and accessory proteins, each with different specific host-protein binding patterns. although none of them is essential for replication in other covs, some of them help with virus assembly and virulence, recruiting elements of the host cell's secretory pathway compartments. indeed, these represent a considerable part of the sars-cov-2 host-protein interactome, whose pathophysiological significance in the context of sars-cov-2 infection requires further functional studies. our bioinformatic analyses unveiled that some of those proteins, such as tor1a, golgb1, or hmox1, could represent attractive targets for diverse classes of compounds (table 3) . however, we still do not know how these proteins impact cov-2 biology. in-depth studies on their structure and localization during cov-2 infection are necessary to better characterize the related phenotypes. synergistic effects may be obtained when canonical treatments (protease and/or rna polymerase inhibitors) are accompanied by drugs targeting secretory pathway components, the rationale being that viral propagation depends on the efficiency of secretory organelles. given the strong interconnections with apoptosis and innate immunity, the upr elicited by cov-2 may modulate host antiviral responses from different points of view. reducing er stress and enhancing protein folding, it may promote chaperone production and massive production of suitably glycosylated s proteins, helping cov replication. the expansion of the er (likely facilitated by scap-srebp) may provide a source of membranes for dmv production and budding. erad may be important to guarantee stoichiometric virus assembly, though its role during infection remains to be understood. accumulation in apoptotic bodies might shield viruses from immune recognition (hay and kannourakis, 2002) . cov-2 highlights once more the jekylland-hyde dualism of the upr. on the one hand, the upr attenuates global translation and activates innate immunity. on the other hand, exaggerated immune response activation is associated with tissue damage and immunopathogenesis, typical in figure 3 . network-aided phylogenetic analysis of 12 viral pathogen infection models. the graphs depict functional comparisons of 12 virus-host protein interactomes, using bioinfominer with the indicated vocabularies (gene ontology, mgi mammalian phenotype, and reactome). for each graph, the comparison estimates the degree of their semantic similarities via agglomerative clustering to construct the phylogenetic tree. the similarity of two viruses was calculated by averaging the values derived from three different semantic similarity measures (resnik, 1999 ; aggregate ic [song et al., 2014] ; and xgrasm [mazandu et al., 2016] ) in conjunction with the average best matches approach (mazandu et al., 2016) . cov infection (dandekar and perlman, 2005) . for all these reasons, studies on the interconnections between covs, inflammation, and upr ramifications are needed. to conclude, identification of new secretory pathway-related targets for antiviral compounds may help the development of more specific anti-cov treatments but also may help to draft a roadmap for future studies on virus biogenesis. atf6 is a transcription factor specializing in the regulation of quality control proteins in the endoplasmic reticulum golgi stress mediates redox imbalance and ferroptosis in human cells the proximal origin of sars-cov-2 anticipatory estrogen activation of the unfolded protein response is linked to cell proliferation and poor survival in estrogen receptor α-positive breast cancer erp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family protein folding and quality control in the er selective emc subunits act as molecular tethers of intracellular organelles exploited during viral entry coronavirus 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membrane-associated protein and induces the activation of atf6 two novel protein o-glucosyltransferases that modify sites distinct from poglut1 and affect notch trafficking and signaling a ph-regulated quality control cycle for surveillance of secretory protein assembly the coronavirus spike protein induces endoplasmic reticulum stress and upregulation of intracellular chemokine mrna concentrations receptor recognition by the novel coronavirus from wuhan: an analysis based on decade--long structural studies of sars coronavirus the erad inhibitor eeyarestatin i is a bifunctional compound with a membrane-binding domain and a p97/ vcp inhibitory group structural basis of ph-dependent client binding by erp44, a key regulator of protein secretion at the er-golgi interface zinc regulates erp44-dependent protein quality control in the early secretory pathway sitespecific glycan analysis of the sars-cov-2 spike iminosugars, from botanical curiosities to licensed drugs a new coronavirus associated with human respiratory disease in china the first 75 days of novel coronavirus (sars-cov-2) outbreak: recent advances, prevention, and treatment pofut1 promotes endometrial decidualization by enhancing the o-fucosylation of notch1 yipf5 and yif1a recycle between the er and the golgi apparatus and are involved in the maintenance of the golgi structure srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target a highly conserved cryptic epitope in the receptor binding domains of sars-cov-2 and sars-cov virus-induced er stress and the unfolded protein response strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit we thank dr. van der goot and dr. saraste for their critical comments on fig. 1 .e. chevet was funded by the institut national de la santé et de la recherche médicale, agence nationale de la recherche submitted: 2 june 2020 revised: 3 july 2020 accepted: 7 july 2020 key: cord-293798-qc22cps9 authors: gómez-mascaraque, laura g.; fabra, maria j.; castro-mayorga, jinneth l.; sánchez, gloria; martínez-sanz, marta; lópez-rubio, amparo title: nanostructuring biopolymers for improved food quality and safety date: 2018-04-06 journal: biopolymers for food design doi: 10.1016/b978-0-12-811449-0.00002-5 sha: doc_id: 293798 cord_uid: qc22cps9 food-grade biopolymers, apart from their inherent nutritional properties, can be tailored designed for improving food quality and safety, either serving as delivery vehicles for bioactive molecules, or as novel packaging components, not only improving the transport properties of biobased packaging structures, but also imparting active antibacterial and antiviral properties. in this chapter, the potential of different food-grade biopolymers (mainly proteins and carbohydrates but also some biopolyesters) to serve as encapsulating matrices for the protection of sensitive bioactives or as nanostructured packaging layers to improve transport properties and control the growth of pathogenic bacteria and viruses are described based on some developments carried out by the authors, as well as the most prominent works found in literature in this area. molecular chains (santiago and castro, 2016) . fig. 2 .1 shows the chemical structure of several polysaccharides that have been widely explored as encapsulation matrices. other commonly used carbohydrates, such as pectins or some gums are a heterogeneous group of polymers and thus its chemical structure has not been represented. another interesting feature of polysaccharides is the great stability of their structure, even under harsh temperature conditions, in contrast with proteins, which can be denatured during material processing at high temperatures. furthermore, a number of polysaccharides are considered dietary fibers, being resistant to enzymatic degradation during oral, gastric, and small intestine digestion but vulnerable to microbial fermentation in the colon (fathi et al., 2014) . most of the commonly used polysaccharides for encapsulation have a plant origin, such as starch, pectin, cellulose, alginate, or guar gum, but animal-derived polysaccharides, such as chitosan or those from bacterial origin, such as dextran are also widespread for this application. an exhaustive review highlighting the advantages and disadvantages of each of them as delivery vehicles in food systems has been recently published (fathi et al., 2014) . food proteins are excellent candidates as microencapsulation matrices because, in addition to the protective effects they might exert on the bioactive ingredients, they have a high nutritional value and generally exhibit functional properties themselves (ma et al., 2014) , being considered the most nutritionally beneficial delivery systems (mohan et al., 2015) . moreover, as livney (2010) points out, some proteins are natural vehicles whose main function is to store, transport, and/or deliver essential compounds throughout the organism and, thus, they could also be exploited to deliver other molecules of interest. especially taking into account that proteins are capable of binding both hydrophobic and hydrophilic small molecules (considine and flanagan, 2009) , and they have self-assembling properties, surface activity, and good gelation characteristics (livney, 2010) . in general, proteins from animal origin, especially dairy proteins, but also gelatin and egg proteins, are more commonly employed than plant proteins for encapsulation purposes (karaca et al., 2015) . some of the advantages of milk proteins over plant proteins include their higher solubility over a broader range of ph, their lower molecular weight, and their greater flexibility. on the other hand, plant proteins have a better acceptance from some groups of consumers and are generally less expensive (karaca et al., 2015) . the most widely used milk proteins are caseins and whey proteins, and their application as encapsulation vehicles has been recently reviewed by tavares et al., (2014) . regarding plant proteins, soy proteins are the predominant choice as wall materials, being those obtained from pulses (such as pea, lentil, or chickpea) and cereal grains (for instance zein or barley protein) the most relevant alternatives (karaca et al., 2015) . table 2 .1 shows some examples of food ingredients that have been encapsulated using proteins. α-linolenic acid (gómez-mascaraque and lopez-rubio, 2016), lycopene (pérez-masiá et al., 2015) , cardamom essential oil (mehyar et al., 2014) , folic acid (pérez-masiá et al., 2015) , probiotics (gomez-mascaraque et al., 2016; lopez-rubio et al., 2012 ) caseins animal (milk) garden cress seed oil (umesha et al., 2015) , w-3 fatty acids (santhanam et al., 2015) , curcumin (pan et al., 2014) , indonesian propolis (sahlan and supardi, 2013 ) gelatin animal green tea polyphenols (gómez-mascaraque et al., 2015 , holy basil essential oil (sutaphanit and chitprasert, 2014) , sunflower oil (piacentini et al., 2013 ) soy proteins vegetal polyphenols and anthocyanins from pomegranate (robert et al., 2010) , w-3 fatty acids (santhanam et al., 2015; gómez-mascaraque and lopez-rubio, 2016) pea proteins vegetal conjugated linoleic acid (costa et al., 2015) , α-tocopherol (pierucci et al., 2007) , oils (gharsallaoui et al., 2007) chickpea proteins vegetal flaxseed oil (karaca et al., 2015) , folate (ariyarathna and nedra karunaratne, 2015) , probiotics lentil proteins vegetal flaxseed oil (karaca et al., 2015) , probiotics (wang et al., 2015) zein vegetal fish oil (moomand and lim, 2014) , green tea polyphenols as inferred from the previous discussion, both proteins and polysaccharides are generally good candidates for the encapsulation of bioactive ingredients. however, there is no consensus in the literature regarding the best choice among them. indeed, each particular compound to be protected has unique characteristics and distinct physical-chemical behavior, and thus the choice of an optimal matrix for encapsulation should be done taking into account the properties of both components. bearing in mind that the choice of wall material will have an impact not only on the encapsulation efficiency and protection of the bioactive ingredient of interest, but also in its release behavior (dias et al., 2015) , the selection of the most adequate encapsulation matrix is of outmost importance in the development of microencapsulation structures. therefore, the purpose of the following sections is to offer a nonexhaustive summary of some relevant findings regarding the selection of carbohydrate or protein matrices for some of the most relevant groups of bioactive ingredients. natural polyphenols are a group of bioactive molecules that attract a great deal of interest in the food industry due to their great antioxidant activity and their many attributed health benefits. however, these compounds are very unstable and some of them have poor bioavailability and unpleasant tastes (munin and edwards-lévy, 2011) . thus, many scientific works have dealt with microencapsulation of polyphenols to overcome these limitations, using different matrices and processing techniques (fang and bhandari, 2010) . robert et al., (2010) reported that the encapsulation efficiency for polyphenols extracted from pomegranate was higher when a soy protein isolate (spi) rather than a maltodextrin (md) was used as wall material, while the md matrix provided greater protection during storage at high temperature (60°c). higher encapsulation efficiencies and enhanced protection for (-)-epigallocatechin gallate (egcg, a green tea polyphenol) were also obtained when encapsulated in a protein matrix, namely gelatin, than when entrapped within the carbohydrate chitosan, both by spray-drying (gómez-mascaraque et al., 2016b) and electrospraying (gómez-mascaraque et al., 2015 . similar encapsulation efficiencies as those obtained for chitosan (∼85%) had been previously obtained when egcg was encapsulated within other carbohydrate-based matrices . the better results obtained when polyphenols were encapsulated within protein matrices in these works might be related to the fact that these compounds generally exhibit complexing properties toward proteins (munin and edwards-lévy, 2011) . furthermore, the bioaccessibility of egcg after in vitro digestion was reported to be higher when gelatin was used as delivery vehicle as compared to the chitosan carriers (gómez-mascaraque et al., 2016a,b). attempts have been made to determine which encapsulation matrices (or blends thereof) are suitable for lipid encapsulation. for instance, matsuno and adachi (1993) proposed a method that is based on the determination of the drying rate of the emulsions to measure the ability of the wall material to form a dense protective network during spray-drying. however, this method does not take into account the chemical nature of the wall materials, not being able to distinguish between materials with similar drying curves (pérez-alonso et al., 2003) . given that many protein and carbohydrate matrices are water soluble or water dispersible, and taking into account that processing in food-grade conditions (i.e., avoiding organic solvents) is imperative in the food industry, the encapsulation of bioactive oils and other lipophilic molecules usually involves the preparation of oil-in-water (o/w) emulsions prior to capsule formation. thus, the ideal wall material for oil encapsulation should have good emulsifying properties (jafari et al., 2008) . in this sense, the amphiphilic nature of proteins is advantageous as they behave as good emulsifiers (mcclements, 2004) , allowing improved encapsulation efficiencies and oxidative stabilities (karaca et al., 2015) . for this reason, proteins have been the preferred matrices for the encapsulation of polyunsaturated fatty acids (gómez-mascaraque et al., 2016a,b; moomand and lim, 2014; perez et al., 2014) and essential oils (abd el-salam and el-shibiny, 2015; baranauskienė et al., 2006; sutaphanit and chitprasert, 2014) in many works. in general, carbohydrates have low surface activity (barrow et al., 2013) and usually yield poor retention efficiencies (jafari et al., 2008) , although modification of some polysaccharides has also been proposed to overcome this limitation (tesch et al., 2002; trubiano and lacourse, 1988) . also, some gums, such as gum arabic, contain small protein fractions that provide them with emulsifying properties (gharsallaoui et al., 2007) . still, the suitability of some polysaccharides as encapsulation matrices for sensitive oils has been questioned. for instance, kolanowski et al. (2006) reported that encapsulation of fish oil within modified cellulose by spray-drying did not improve its oxidative stability. in another work, gallardo et al. (2013) claimed that microencapsulation of linseed oil within spray-dried gum arabic did increase its thermal stability, but a great loss of α-linolenic acid was observed upon fortification of bread with these capsules, suggesting limited application of the polysaccharidebased formulation in real food systems. on the other hand, addition of a whey protein isolate to the wall material further improved the thermal stability of the encapsulated oil. more recently, santhanam et al. (2015) encapsulated fish oil within sodium caseinate, md, and soy protein and supplemented cakes with the capsules. their results showed that, among the different matrices, the milk protein was the matrix that yielded the best oxidative stability, which also demonstrated desirable organoleptic characteristics. indeed, milk proteins are usually smaller and more flexible than vegetal proteins, being more efficiently absorbed at the w/o interface and hence better stabilizing the emulsions (karaca et al., 2015) . umesha et al. (2015) also proved that milk proteins (specifically sodium caseinate and whey protein concentrate) achieved higher encapsulation efficiencies and provided enhanced oxidative stability to cress seed oil as compared to a carbohydrate-based matrix (blend of md and gum arabic). conversely, al-ismail et al. (2015) concluded that gum arabic yielded better encapsulation efficiency and oil retention during storage than whey protein isolate for a cardamom essential oil. in summary, although controversy exists among different works, proteins seem to generally yield better results than polysaccharides as wall materials for the encapsulation of oils, which can be explained in light of their excellent emulsifying properties, forming a protective film at the o/w interface. carotenoids are a group of natural pigments with many attributed health benefits when consumed in sufficient levels, some of which also exhibit provitamin a activity (qian et al., 2012) . together with polyphenols, carotenoids are the most abundant phytochemicals (kaulmann et al., 2016) . however, they have a very low water solubility and poor chemical stability, resulting in low bioaccessibility upon consumption. dissolution of carotenoids in edible oils has shown to increase absorption in vitro and in vivo (ribeiro et al., 2010) , and thus o/w emulsions are often prepared before capsule production as a suitable strategy to both increase their bioaccessibility and disperse them in aqueous biopolymer solutions or dispersions. therefore, some of the previous discussion regarding the encapsulation of bioactive oils could be applied to carotenoids. in this sense, pérez-masiá et al. (2015) showed that higher encapsulation efficiencies were obtained when lycopene (previously dissolved in a soy bean oil) was microencapsulated using a whey protein matrix as compared to carbohydrate-based matrices, such as dextran or chitosan. robert et al. (2003) also obtained higher encapsulation efficiencies for the main carotenoid pigments present in an oleoresin from rosa mosqueta upon spray-drying within gelatin capsules than using starch as wall material. furthermore, gelatin provided a greater protective effect on the main carotenoids, especially on trans-β-carotene, than starch. however, information on the bioavailability of these microencapsulated carotenoids during in vitro digestion is scarce (donhowe and kong, 2014) . being the bioaccessibility of carotenoids one of their main limitations to exert their bioactivities, this feature should be carefully addressed when selecting a wall material for encapsulation. for instance, alginate has been proposed as a desirable encapsulation matrix for its ability to protect carotenoids from the acidic conditions during gastric digestion, and indeed, zhang et al. (2016) have recently shown that it effectively protected β-carotene from degradation during digestion. however, the encapsulation within alginate beads caused a reduction in β-carotene bioaccessibility due to insufficient release. the bioaccessibility of carotenoids relays on their incorporation into mixed micelles (fernández-garcía et al., 2009; qian et al., 2012) , and research suggests that some polysaccharides, such as alginates or pectins, significantly inhibit micelle formation; and hence carotenoids absorption (riedl et al., 1999; yonekura and nagao, 2009 ). on the other hand, soluble proteins have also been shown to impede incorporation of carotenoids into the oil phase of the gastric emulsion (rich et al., 2003) , although this limitation might be overcome if carotenoids are previously incorporated in o/w emulsions prior to their encapsulation within protein capsules. probiotics are a particular group of bioactive ingredients. they are defined as "live microorganisms which, when consumed in adequate amounts, confer a health benefit to the host" (pineiro and stanton, 2007) , so they must be alive and metabolically active at the time of consumption. however, a number of studies report low levels of viable probiotic cells in commercial products (al-otaibi, 2009; lin et al., 2006; shah et al., 2000; weese, 2002) , emphasizing the need for their microencapsulation. the choice of potential matrices for this group of ingredients is more restricted, as the processing conditions must be adequate to warranty their survival. moreover, to adequately protect the cells from the acidic environment of the stomach and efficiently deliver them to the intestine, the ideal matrix for their encapsulation should be stable at low phs and disrupt at phs above 6 ( gbassi and vandamme, 2012) . for these reasons, one of the preferred matrices for the encapsulation of probiotic bacteria is alginate (dong et al., 2013) . other anionic polysaccharides with gelling capacity, such as carrageenans have also been used, although their processing at mild temperatures is problematic (mangione et al., 2003; yuguchi et al., 2002) . proteins, especially milk proteins, are relevant alternatives to alginate for the microencapsulation of probiotics (el-salam and el-shibiny, 2015). rodrigues et al. (2011) assessed the viability of probiotic bacteria when immobilized in different food-grade polymers and found that whey proteins, together with alginate, were the most adequate materials for most of the strains they tested. nevertheless, the results were strain-dependent, emphasizing the need for designing the encapsulation system for each specific bioactive ingredient. lopez-rubio et al. (2012) also observed that a whey protein concentrate (wpc) exerted enhanced protection to bifidobacterium animalis subsp. lactis bb12 during storage at high relative humidity as compared with a polysaccharide, namely pullulan, both processed by electrospraying. electrosprayed wpc-based capsules were also used to protect a strain of lactobacillus plantarum (cect 748 t), which experienced a reduced viability loss during in vitro digestion (∼3 log cfu g −1 ) (gomez-mascaraque et al., 2016) . in contrast, alginateencapsulated l. plantarum (strain ufrgs bl011) suffered a viability loss of almost 3 log cfu ml −1 when exposed to simulated gastric juice, and an additional drop of 3 log cfu ml −1 when exposed to simulated intestinal juice (coghetto et al., 2016) . however, these results are not directly comparable, since the protected l. plantarum strain is different and the protocols employed for simulation of gastrointestinal conditions also differ. it is worth noting that alginate microcapsules have already been successfully incorporated into real food matrices, such as ice creams, frozen yogurts, and mayonnaise (corona-hernandez et al., 2013) . the microencapsulation of probiotics has been specifically examined in a number of recent review articles (de prisco and mauriello, 2016; el-salam and el-shibiny, 2015; iravani et al., 2015) . as discussed, both polysaccharides and proteins have advantages and limitations as encapsulating agents. therefore, combinations of them have also been studied as wall materials with the aim of improving the attributes of the individual biopolymers (bastos et al., 2012; fernandes et al., 2014; smilkov et al., 2014) . for instance, addition of proteins to mds, which are considered to be one of the best thermal defenders (munin and edwards-lévy, 2011) for microencapsulation by spray-drying, allows it to increase the powder recovery of the materials (muzaffar and kumar, 2016; shi et al., 2013) , while incorporation of dextrins to proteins as encapsulant material for fish oil increased its oxidative stability (kagami et al., 2003) . aberkane et al. (2014) observed that the combination of pectin with pea protein for the microencapsulation of pufa-rich oil enhanced its oxidative stability as compared with the pea protein alone. however, protein-polysaccharide combinations do not always perform better than the individual components. mehyar et al. (2014) obtained better overall results by encapsulation of cardamom oil within a spithan using mixtures of the protein with either guar gum or carrageenan. also, complexation of proteins with polysaccharides (benichou et al., 2002) represents a widely used strategy for the encapsulation of bioactive ingredients (gutiérrez et al., 2013; jain et al., 2015; nori et al., 2011; piacentini et al., 2013) , being coacervation the second most exploited encapsulation technique in the food industry (dias et al., 2015) . furthermore, proteins and carbohydrates can be conjugated by dry heating through the maillard reaction (martins et al., 2000) , and these conjugates have also been recently applied to the encapsulation of food ingredients. davidov pardo et al. (2015) developed caseinate-dextran coated zein nanoparticles and used them to encapsulate resveratrol, enhancing its bioaccessibility. coating the nanoparticles with these maillard complexes resulted in a greater stability to aggregation under simulated gastrointestinal conditions as compared to those coated with plain caseinate. ifeduba and akoh (2016) used the maillard reaction to crosslink gelatin-gum arabic coacervates to improve the oxidative stability stearidonic acid soybean oil. in addition, maillard reaction products have been described to exhibit antioxidant properties, but the last stages of the reaction can lead to undesirable color and aromas, and even to toxic compounds in certain cases (tessier and niquet, 2007) , facts that should be considered when designing this type of carriers. evaluating the release and stability of encapsulation systems is crucial to assess their efficiency and to optimize the developed structures in terms of achieving the desired release rates when subjected to particular conditions. several factors, such as the composition and chemical structure of the encapsulating material (also referred to as "matrix") and the encapsulated bioactive compound, the interactions established between the bioactive and matrix materials and the morphology of the encapsulating system (size distribution, shape, surface area, porosity, etc.) have a strong impact on the stability of the encapsulating systems and, therefore, they should be taken into account when designing a system for a particular application. moreover, understanding the mechanisms controlling the release of the encapsulated compound from the matrix when subjected to specific conditions (relative humidity, ph of the medium, temperature, etc.) is essential to maximize the material performance for its intended application. a wide range of techniques is currently available to investigate the properties and release mechanism in different encapsulating systems. the selection of the characterization methods for a particular encapsulating structure has to be made on the basis of the system composition, release rate and medium. in general, a combination of several techniques, covering from the molecular to the microscale structural levels, is the most efficient approach to thoroughly study the system. the following sections provide a brief overview of the main tools available to characterize encapsulating materials, with special emphasis on the potential of small angle scattering techniques to provide valuable structural information and to exhaustively investigate the release mechanisms under a wide range of conditions. imaging techniques based on microscopy instruments are one of the most widely used methods to characterize the structure of encapsulating systems. they may provide qualitative information on the shape, size, and aggregation state of the materials under study. electron, optical, and laser microscopy methods may be selected depending on the composition of the matrix and the size range of the generated materials. although optical microscopy is the most easily accessible and low-cost imaging technique, its main drawback is its relatively low resolution, which is limited by the diffraction of light to about 1000 diameters magnification (luykx et al., 2008) . as an alternative, electron microscopes offer much higher magnifications, which enable the characterization of particles with size ranges from tenths of nm to tenths of µm. among the electron microscopy techniques, transmission electron (tem) and scanning electron microscopy (sem) are the most widely used ones. tem is a technique that is based on the transmission of a beam of electrons through an ultrathin sample. the major limitation from this technique lays in the tedious sample preparation that is often required. first of all, the samples have to be thin enough to be electron transparent and must be dry to be subjected to the high vacuum present inside the instrument. additionally, negative staining is often required to increase the contrast between the sample and the background. this involves placing a small amount of the sample on a tem grid and staining with a solution (such as uranyl acetate), which provides high contrast. to obtain information about the particle internal structure, the sample can be subjected to the freezefracture technique. in this case, the sample is placed on the tem grid, which is then vitrified by rapid freezing and subsequently fractured under constant cooling and vacuum. it is evident that these preparation procedures are likely to have a strong impact on the structure of the sample, especially in the case of biological samples and moisture sensitive materials. sem presents lower resolution than tem, but it has a greater depth of view (thus providing more information on the particle surface structure) and the sample preparation is usually less complex. this technique is based on scanning a beam of electrons across the surface of the sample, providing information on the sample surface topography and composition. sem requires high vacuum and sample conductivity, which is again translated into possible structural modifications induced by the drying and coating processes applied to the sample specimens. as an alternative, atomic force microscopy (afm) is a more recent technique in which a physical sharp probe is scanned over a specimen that has been previously immobilized onto a surface. the surface topography of the sample is resolved by determining the reaction of the probe to the forces that the sample imposes on it when scanned. one of the main advantages of afm, apart from its high resolution (ca. 0.1 nm) is the fact that, unlike sem, the samples do not need to present a conductive surface. this type of microscopy is, however, not suitable for surfaces that are soft or sticky. in those cases, the sample may need to be dried prior to the analysis. although microscopy techniques are routinely used to characterize the structure of encapsulating systems, the results have to be carefully interpreted and any possible structural alterations induced by sample preparations have to be taken into account. therefore, although microscopy methods may be useful, they should be preferably combined with complementary techniques to ascertain the morphology of the encapsulated system in its native state. chromatography methods can be routinely used to separate the components in encapsulating systems, that is, the matrix and the encapsulated compound and, subsequently, quantify the amount of the active compound to, for instance, evaluate the encapsulation efficiency. the separation of different constituents in a sample is done on the basis of the different speeds at which distinct compounds travel through a structure (usually a column) containing a specific material (known as stationary phase) when dissolved in a certain fluid (known as mobile phase). the final step after separating the components in the sample consists in the quantification of each component by using a suitable detector, such as an ultraviolet-visible (uv-vis) light absorbance detector, a fluorescence detector, or a diffractometer. the advantage of chromatographic techniques is that they offer a range of possibilities (mobile and stationary phases, type of separation technique, detector type) that can be adjusted depending on the particular composition and properties of the encapsulated system. depending on the physical state of the mobile phase, we can differentiate gaschromatography (cg) and liquid chromatography (lc) techniques. in particular, highperformance liquid chromatography (hplc) is a widely used technique in the food analysis field. in this particular case, the sample has to be dissolved in a liquid, which is then pressurized and injected into a column containing a stationary phase. depending on the type of stationary phase, the mechanism for the separation of compounds in the sample will be different, giving rise to a range of chromatographic separation methods. for instance, the separation may occur on the basis of the molecular mass of the different compounds [size exclusion chromatography (sec) or gel permeation chromatography (gpc)] or the charge (cation or anion exchange chromatography). in addition to hplc, gc coupled with mass spectrometry (gc-ms) is also one of the most widely used techniques to separate and quantify compounds that can be vaporized without affecting their molecular structure. in this case, after the compounds in the sample travel through the chromatographic column and are released (with different retention times), the coupled mass spectrometer breaks down the molecular structure of each component into ionized fragments, separates the ions of differing masses, and determines their relative abundance by measuring the intensity of the ion flux. some examples of applications of chromatographic techniques include the encapsulation efficiency assessment of gallic acid/cyclodextrin complexes incorporated into electrospun polylactic acid (pla) nanofibers (aytac et al., 2016) or resveratrol encapsulated in chitosan-sodium tripolyphosphate microspheres (cho et al., 2014) by using hplc coupled with a uv detector, or the use of gc-ms to evaluate the release of essential oils, such thyme essential oil in β-cyclodextrin capsules (del toro-sánchez et al., 2010) . although these techniques are useful to determine the encapsulation efficiency of a certain system, they do not provide information on the structure of the system, the interactions established between the matrix, and the encapsulated compound or the release mechanismtaking place under specific conditions. a wide range of techniques can be utilized to investigate the structural changes and the mechanisms controlling the release and stability of encapsulated systems. among the most commonly used methods, spectroscopy and scattering techniques are particularly interesting to study structural changes taking place at the molecular and nano-/microstructural levels. in particular, scattering techniques present an enormous potential, although, to date, their application within this research field is still very limited. this is mainly due to the unfamiliarity of many researchers within the food science field and unawareness of the valuable information that can be extracted from these techniques. however, several works have already demonstrated the great advantages of combining small angle scattering techniques with traditional methods to provide unique insights into the characterization of food systems (blazek and gilbert, 2011; lopez-rubio and gilbert, 2009 ). spectroscopy methods rely in the study of interactions established between a source of radiation (most commonly electromagnetic radiation) and the particles composing a sample and, subsequently, measuring changes in the intensity or frequency of the radiation energy. in particular, nuclear magnetic resonance spectroscopy (nmr) and fourier transform infrared spectroscopy (ft-ir) are widely used to characterize the molecular structure and molecular interactions between the components in encapsulating systems. nmr is based on the measurement of the electromagnetic radiation, which is absorbed by an active nucleus, such as 1 h or 13 c, at a certain frequency. the absorption of an atomic nucleus is conditioned by the surrounding atoms and, thus, detailed information on the molecular structure of a certain compound can be obtained. this technique can be applied to any kind of sample, in the solid or solution state, which contains nuclei possessing spin. in the particular case of encapsulating systems, 1 h-nmr and 13 c-nmr techniques have been used to prove the formation of stabilizing inclusion complexes between β-cyclodextrins and essential oils (del toro-sánchez et al., 2010; rǎileanu et al., 2013) or to determine the molecular state of curcuminoids encapsulated in poly(butyl) cyanoacrylate nanoparticles (mulik et al., 2009) . another interesting fact is that this technique is sensitive to dynamics, that is, it is possible to discern molecular domains with distinct mobility. this can be exploited to determine the level of molecular interaction established between different components in encapsulating materials. for instance, the level of interaction between silica matrices and encapsulated ibuprofen molecules has been estimated based on the ibuprofen molecular mobility deduced from 13 c and 1 h spectra (babonneau et al., 2004) . in addition, relaxation experiments, that is, measuring how the signal changes with time, have been applied to study the water-polymer interactions in whey protein/alginate beads containing riboflavin (wichchukit et al., 2013) . the ft-ir technique, as any other absorption spectroscopy technique, consists on measuring the amount of light absorbed by a sample at different wavelengths. the peculiarity of ft-ir is that instead of using a monochromatic beam of light, the sample is radiated by a beam composed of many wavelengths at once and data are subsequently processed to determine the absorption taking place at each measured wavelength. ft-ir can be utilized to confirm the encapsulation of the active compound into the matrix by identifying characteristic absorption bands. for instance, ft-ir has been used to determine the presence of vitamin b12 and vitamin c encapsulated into different biopolymers by spray-drying (estevinho et al., 2016) , tea tree oil in sodium alginate/chitosan capsules (chen et al., 2016) , xylitol in multicomponent microcapsules obtained by complex coacervation (santos et al., 2014) , and resveratrol in chitosan-sodium tripolyphosphate microspheres (cho et al., 2014) . scattering techniques are based on the analysis of the scattered radiation produced after a source, such as light, x-rays, and neutrons, interacts with the particles present in a sample. they represent a powerful tool to obtain information about the size, shape, and orientation of components in ordered (long-range or crystalline order), but also disordered systems. in practice, the radiation scattered by the sample of interest is typically measured on a two-dimensional detector; for isotropic scatterers, after being radially averaged, the intensity is plotted versus the magnitude of the scattering vector q, which describes the relationship between the incident and the scattered wave vectors, being defined as: where θ is half the angle through which the radiation is scattered and λ is the wavelength of the incident radiation. combining eq. (2.1) with bragg's law (λ = 2d sin θ), one can determine the real-space dimension of the scattering object, through the relationship: according to this, scattering methods are classified into wide angle and small angle scattering techniques depending on the range of scattering angles (and corresponding length scales) covered. therefore, whereas wide-angle scattering techniques are used to probe subnanometer dimensions, small angle scattering techniques, are able to cover a size range from 1 nm to several hundreds of nanometers. coupling small angle with ultrasmall angle scattering techniques it is possible to study larger structures, hence extending the size range up to ca. 10 µm. it is important to select the proper source of radiation based on the sample composition and structural features to be probed. while x-rays are scattered by the electrons, being saxs sensitive to variations in electron density, neutrons are scattered by the atomic nuclei and, thus, sans depends on the nuclear structure of the atom. as a result, whereas x-ray scattering intensity is a function of the atomic number of the scattering elements, in the case of neutron scattering, different isotopes of an element may present significantly different scattering intensity. conversely, light is scattered by fluctuations in the dielectric constant of a material. it is worth noting that, because the wavelength of visible light is in the order of hundreds of nanometers, light scattering techniques typically probe larger structural features than x-ray and neutron scattering techniques (i.e., low q values are covered in light scattering experiments). light scattering techniques are widely used for the characterization of colloidal suspensions, such as those typically found in food systems. static light scattering (slc), also known as laser light scattering (lsc), measures particle size based on light scattering intensity, which changes with variation in scattering angle, size of particles, refractive indices of particles, and the medium. slc measures the average intensity of scattered light arriving at the detector, although actually, the intensity shows fluctuations over short time scales. moreover, in the case of solutions, the brownian motion of particles causes fluctuations in the intensity of the scattered light arriving at the detector. dynamic light scattering (dls) is based on the measurements of these fluctuations as a function of time. by analyzing these fluctuations through correlation functions, it is possible to extract information regarding the particle size. dls is commonly used to characterize the particle size of encapsulating systems based on microemulsions (flanagan et al., 2006; gaysinsky et al., 2008; yin et al., 2012; zhong et al., 2009; ziani et al., 2012) . the main issue of slc and dls methods is that highly diluted samples are often required to avoid multiple scattering (i.e., the photons are scattered more than once between the light source and the detector). in addition, to ensure a proper data interpretation, the particles should present a narrow size distribution and they must present the same refractive index (which is not true in the case of multicomponent systems). the issue of multiple scattering is often avoided when using x-rays and neutrons, because these radiation sources do not scatter as strongly as light. hence, these techniques are more suitable for the characterization of a wide range of food-based encapsulating systems. the most widely used techniques, classified on the basis of the source of radiation and the covered size ranges, are designed as wide angle and small angle x-ray scattering (waxs and saxs) and small angle neutron scattering (sans). it should be mentioned that waxs is often regarded as identical to the x-ray diffraction technique (xrd), which is not completely true. while waxs is conducted in the transmission geometry, xrd may be performed in the reflection or in the transmission geometry. xrd is the most commonly used method to characterize the crystalline structure in materials presenting long-range order. in addition to the previously mentioned techniques, ultrasmall angle scattering techniques (usaxs and usans) can be utilized to probe larger structural features. fig. 2 .2 shows the relevant size ranges covered by these techniques and complementary methods, as related to the different structural features found in typical encapsulating systems. the suitability of saxs or sans to characterize a particular encapsulating system depends on the scattering length density (sld) contrast between the sample and the surrounding element materials or between different sample components (i.e., the difference in the scattering intensity of the molecules in the different components). the radiation source should be thus chosen to enhance the sld contrast between the sample components. specifically for sans, the scattering length difference existing between hydrogen (−0.3742 × 10 10 cm -2 ) and its heavier isotope, deuterium (0.6671 × 10 10 cm -2 ), is the basis of the contrast variation method. this is an extraordinary valuable approach used to enhance the sld of hydrogencontaining samples or to selectively match desired components in a multicomponent system by using different h 2 o/d 2 o mixtures. apart from the nondestructive nature of small angle scattering techniques, they have further advantages, such as enabling the study of representative samples with relatively larger volumes as compared to methods, such as tem, in which small sample areas of the order of hundreds of square microns are analyzed. on the contrary, the main drawback of small angle scattering techniques is that they are indirect methods and mathematical models have to be applied to describe the experimental scattering curves. these models require prior knowledge of the system to be built and, therefore, it is of outmost importance to combine small angle scattering techniques with complementary methods, such as microscopy and spectroscopy techniques. a multitechnique approach is the most successful path to obtain detailed knowledge of the sample properties based on realistic physical-chemical parameters (martínez-sanz et al., 2015 , 2016 . as previously mentioned, the potential of saxs and sans techniques for the characterization of encapsulating systems has not been fully exploited and very few studies, which are relatively recent, report on the use of these techniques within this particular research field. nevertheless, the existing works highlight the adequacy to investigate the release mechanisms and stability of encapsulating materials. as an example, some of the most relevant works are summarized later. gerelli et al. (2008) reported on the structural characterization of soybean lecithin/chitosan nanoparticles loaded with a lipophilic positively charged drug used in breast cancer therapy by combining cryo-tem, static light scattering, and saxs and sans techniques. by applying proper models to describe the experimental scattering data, it was possible to determine the structure of the encapsulating vesicles, as well as the interactions established lopez-rubio, a., gilbert, e.p., 2009 . neutron scattering: a natural tool for food science and technology research. trends food sci. technol. 20(11-12), 576-586; martínez-sanz, m., gidley, m.j., gilbert, e.p., 2015. application of x-ray and neutron small angle scattering techniques to study the hierarchical structure of plant cell walls: a review. carbohydr. polym. 125, [120] [121] [122] [123] [124] [125] [126] [127] [128] [129] [130] [131] [132] [133] [134] between the matrix and the encapsulated drug. the unilamellar structure of liposomes loaded with antibiotics has also been investigated by means of saxs characterization (colzi et al., 2015) . the internal structure of poly(lactide-co-glucolide)-block-poly(ethylene glycol) (plga-peg) nanoparticles, commonly used as drug carrier systems, was resolved by finding a proper model (consisting of fractal structures with polydisperse spherical building blocks) to simultaneously fit the sans experimental data from the nanoparticles dispersed in two different h 2 o/d 2 o mixtures (yang et al., 2015) . this allowed identifying the release mechanism of a hydrophilic anticancer drug, such as carboplatin, when incorporated into the plga-peg nanoparticles. the distinct sld contrast generated when using neutrons and x-rays was exploited to characterize microemulsions embedded in alginate hydrogels (josef et al., 2013) . whereas x-rays provided a low sld contrast between the microemulsion and the alginate gel (thus probing the structure of the whole system), the use of neutrons enhanced the sld contrast between the two phases, hence highlighting the structure of the microemulsion droplets embedded within the gel. in another study, fractal analysis and evaluation of the particle distance distribution function from saxs experiments evidenced the structural changes undergone by bovine serum albumin when combined with chitosan and poly(sodium-4-styrene) sulphonate to produce core-shell nanoparticles loaded with ibuprofen for controlled release of the drug (varga et al., 2014) . liu et al. (2011) investigated the release of an enzyme encapsulated in freeze-dried chitosan/xanthan gum hydrogels and observed that the release was triggered by lowering the ph of the medium. on the other hand, the addition of montmorillonite (mmt) nanoclay into the hydrogels was seen to stabilize the system, lowering the enzyme release rate. the use of waxs and saxs was crucial to explaining the stabilizing effect of mmt. although waxs evidenced that a high degree of mmt intercalation within the chitosan/xanthan gum blend was achieved, saxs showed that the incorporation of mmt reduced the structural changes undergone by the hydrogels when the ph of the medium was modified. the encapsulation of a vitamin into spray-dried protein-stabilized emulsions was evaluated and the vitamin stability was linked to structural changes by combining light scattering and differential scanning calorimetry with saxs characterization (relkin et al., 2014) . the formation of lamellar structures as a result of the packing of the fatty phase, evidenced by the presence of a bragg peak in the saxs patterns, was seen to reduce the vitamin degradation. due to the severe environmental pollution caused by plastic food packaging, there has been a growing amount of interest in the production of biodegradable films, which, at least, partially replace nonrenewable conventional petroleum-based packaging materials (faruk et al., 2012; jiménez et al., 2012) . among the most widely researched biodegradable thermoplastics, sustainable biopolymers for monolayer packaging applications are starch, polyhydroxyalkanoates (pha), and pla. specifically, starch and pla biopolymers are, undoubtedly, the most interesting families of biodegradable materials, as they have become commercially available, being produced in a large industrial scale. of particular interest in food packaging is the case of pla due to its excellent transparency and relatively good water resistance. however, as already commented, the main challenge for these specific biomaterials is to improve their properties so that they perform (in terms of barrier and thermal properties) like polyethylene terephthalate (pet). biopolymer materials extracted from biomass resources, such as proteins (e.g., zein, wheat gluten), polysaccharides (e.g., starch, chitosan), and lipids (e.g., waxes) have excellent potential as gas and aroma barriers in dry state but they have very strong water sensitivity arising from their hydrophilic nature, which leads to a strong plasticization and the subsequent deterioration of film properties as relative humidity and water sorption in the material increase. in general, one of the main disadvantages of these biopolymers is that they are permeable, in more or less extent, to low molecular weight compounds (i.e., water vapor, oxygen, and aroma compounds), which can compromise packaged food quality and safety. in the particular case of starch, although native starch is not an inherently thermoplastic material, it can be processed like conventional polymers, giving raise to what is known as thermoplastic starch (tps), after some specific processing steps. however, tps shows several drawbacks that reduce their applicability as packaging material, such as their highly hydrophilic character, limited mechanical properties, and the retrogradation phenomenon that occurs during ageing (jiménez et al., 2012) . to avoid this problem, several studies have been focused on the optimization of the physicochemical properties of packages and the minimization of food-package interactions, such as gas or aroma migration, by developing nanocomposites and multilayer food packaging structures. one of the most used approaches to improve the performance of packaging materials is the use of blends (e.g., plasticizers, other biopolymers) or the addition of nanomaterials (e.g., nanoclays) to achieve the desired properties (bordes et al., 2009) . however, it is well known that the most efficient structure to constitute high barrier materials using two element components is for them to be disposed in a layered form (fabra et al., 2013) . this methodology has been widely used in synthetic polymers but it has been barely developed in biodegradable food packaging systems because it is difficult to assemble materials thermodynamically immiscible without the addition of synthetic adhesives. in fact, one of the main challenges in the development of multilayer biobased and biodegradable films is to achieve good adhesion between layers. interestingly, both electrospinning and layerby-layer (lbl) assembly can at the same time be used to enhance physicochemical and barrier properties of packaging materials and for the entrapment of functional compounds to develop active packaging structures (fabra et al., 2013 (fabra et al., , 2015 (fabra et al., , 2016d . in this regard, fabra et al. (2014a fabra et al. ( ,b, 2015 have successfully developed hydrophobic coatings or interlayers with bioadhesive properties to improve the barrier and functional performance of biodegradable polymers combining biomass-derived nanocomposites and the electrospinning process. fig. 2.3 shows an example of the microstructure reached in multilayer films obtained with pla (outer layer) and electrospun zein interlayer. recently, it has been reported that electrospun zein interlayers have the potential to generate multilayered biopolymeric structures with improved barrier properties in pla (busolo et al., 2009 ) and pha-based films (fabra et al., 2013 (fabra et al., , 2014a with minimum changes in the optical properties, which could be ascribed to the light scattering generated by the arrangement of the biopolymeric components distributed throughout the inner layer. specifically, fabra et al. (2014b) demonstrated that biopolyester-based multilayers prepared with the electrospun zein interlayers significantly improved oxygen permeability values (up to 70%) of pla and pha biopolymers, although the water vapor barrier properties depended on the biopolymer used as outer layers. as opposed to the pha's materials performance, pla did not improve the water vapor permeability values of these films. however, not only the nature of the outer layers but also the morphology, thickness, and the inherent barrier of the electrospun interlayer materials determine the oxygen and water vapor barrier properties of the overall multilayer. for instance, fabra et al. (2014a) reported that fibrillar structures obtained for zein and pullulan electrospun interlayers significantly contributed to improve barrier performance of biopolyester-based multilayer systems whereas bead microstructures obtained by electrospraying a whey protein isolate did not have an impact on it. the methodology already described has also been proven to avoid the moisture uptake of hydrophilic-based thermoplastic materials (i.e., wheat gluten or starch). to this end, biopolyester coatings have been directly electrospun onto both sides of hydrophilic wheat gluten or starch based films (fabra et al., 2015) . as opposed to the earlier mentioned structures obtained by using electrospun bioadhesives, the structures developed by sandwiching thermoplastic proteins and polysaccharides-based films with electrospun hydrophobic coatings were not high barrier materials and they could be used for some fresh produce (e.g., mushroom, strawberry) with high respiration rates that require packaging films with high oxygen and carbon dioxide permeability values. in this type of multilayer structures, proteins and polysaccharides have to be first plasticized to become thermoplastic and form a continuous film, a fact that made them suitable in terms of barrier properties, for fulfilling the packaging requirements of fresh produce. however, water and oxygen permeability values of the neat thermoplastic wheat gluten or tps films could be detrimentally altered by the presence of plasticizers and these biopolymers could be disintegrated or even dissolved at high relative humidity conditions, limiting their real application. to overcome this issue, electrospun hydrophobic coatings were incorporated as outer layers, avoiding swelling problems of the plasticized wheat gluten or starch-based film. in this regard, fabra et al. (2016c) have observed that barrier properties of the developed multilayer systems can be tailored by using different biodegradable electrospun coatings (such as pla, polycaprolactone, pcl, or pha) and by modulating the thickness of the outer layers. in fact, they have found a positive linear relationship between the amount of the electrospun coatings deposited onto both sides of a thermoplastic cornstarch film and the thickness of the electrospun biopolyester coating. interestingly, the addition of electrospun coatings led to an exponential oxygen and water vapor permeability drop as the amount of the electrospun coating increased. another interesting approach to enhance the overall functionalities of tps-based film, with special focus on their barrier performance, is the development of multilayer systems in which the inner layer is a nanobiocomposite starch-based film containing bacterial cellulose nanowhiskers (bcnw). thereafter, the optimized nanobiocomposites were successfully hydrophobized by coating them with electrospun pha or electrospun pha-bcnw fibers. to this end, hybrid electrospun pha fibers reinforced with highly dispersed crystalline bcnw in solutions concentrations up to 15 wt.% were directly electrospun onto both sides of starch based nanobiocomposites containing 15 wt.% bcnw. results showed that the incorporation of bcnw in one of the layers of a multilayer system enhanced oxygen barrier properties to a great extent (fabra et al., 2016e) . therefore, by means of the technology here proposed, one can tailor the barrier properties of food-packaging materials according to the final intended use. interestingly, another advantage arising from this procedure is that this technology also offers the possibility to develop active and bioactive packaging multilayer structures producing simultaneously interlayers or coatings with encapsulation performance of the active/bioactive compounds thus preserving their functional properties. in this sense, fabra et al. (2016d) have developed active multilayer food-packaging materials by means of electrospinning. specifically, α-tocopherol, as a model antioxidant, was encapsulated in three different protein-based matrices (whey protein isolate, spi, and zein), which were directly applied as coatings onto a thermoplastic wheat gluten film. the antioxidant activity of the active compound was preserved during the encapsulation process (up to 95%) and it was seen that the electrospun shell materials were able to protect the antioxidant from degradation during a typical sterilization process. another technique with great potential in the development of nanoscopically structured new materials is the sequential lbl adsorption of polymer of solid substrates (pinheiro et al., 2012; weis et al., 2006) . by means of this procedure, several biopolymers with opposite charges can be used to produce nanolayered films assembled through lbl. the lbl technique is quite simple and enables using a wide range of materials (e.g., proteins, polysaccharides, lipids, and nanoparticles). these materials are able to interact either by electrostatic interactions, hydrogen bonding, covalent bonds, complementary base pairing, or hydrophobic bonding. for instance, two polysaccharides with opposite charges, chitosan and sodium alginate, were deposited onto aminolyzed/charged pet (a/c pet) and the resulting multilayer nanofilm showed lower water vapor permeability that could be expected for the materials with which the film was built (carneiro-da-cunha et al., 2010) . the use of the lbl technique has also been studied for the development of active coatings. in this line, fabra et al. (2015) developed lbl films by alternating layers of alginate and zein/carvacrol nanocapsules with improved barrier properties and antifungal activity against alternaria sp. the globally increasing demand for minimally processed, easily-prepared, and readyto-eat "fresh" food products has encouraged manufacturers to develop new packaging technologies. these demands are strongly influenced by the market trends, which together with globalization of food trade and distribution from centralized processing facilities, are the driving forces for continuous development and upgrade of food packaging (appendini and hotchkiss, 2002; ozdemir and floros, 2004) . active packaging is an innovative solution to meet the continuous changes in current consumer demands and market trends because it extends shelf life and improves safety of the food by scavenging of oxygen, moisture, or ethylene, and by promoting emission of ethanol, flavors, and antimicrobial agents (lópez-rubio et al., 2004) . in particular, antimicrobial packaging is attracting increased attention from the food and packaging industry, because the use of preservative packaging films offers several advantages compared with the direct addition of preservatives into food products (suppakul et al., 2003) . the incorporation of antimicrobial agents into polymeric films allows industry to combine the preservative functions of antimicrobials with the protective functions of the preexisting packaging concepts (appendini and hotchkiss, 2002) . antimicrobial activity can be achieved by including pads containing volatile antimicrobial agents into packages, incorporating antimicrobial agents directly into polymers, coating antimicrobials onto polymer surfaces, immobilizing antimicrobials by chemical grafting, or using polymers that are antimicrobial by themselves (malhotra et al., 2015) . in this context, the application of engineered nanomaterials is considered a promising tool to improve the functionality of biopolymers used in antimicrobial food packaging and antimicrobial food contact surfaces. it has been demonstrated that the high surface-to-volume ratio of nanostructured materials enhance the antimicrobial activity at low concentrations and, additionally, provides exceptional chemical and physical properties as an additive for material for the uv-shielding, deodorizing, antiseptic, and so on. in fact, antibacterial nanoparticles composed of metals, metal oxides, metal salts, metal hydroxides, organic nanocarriers loaded with antibacterial agents, hybrid materials, and polymers exhibiting antimicrobial properties themselves have been recently developed, being the metal nanoparticles of zinc oxide and silver, the most common nanoparticles used (mauriello, 2016; moritz and geszke-moritz, 2013) . metal nanoparticles can be used as antimicrobial agents because they have numerous advantages over other antimicrobials. their broad spectrum against foodborne pathogens (even at very low concentration) (lara et al., 2010; nocchetti et al., 2013) and their negligible toxicity toward human cells at the same concentration ranges, as well as their improved stability and low volatility in comparison with organic antimicrobial agents (dutta et al., 2012) , make them a promising materials for food packaging and food contact surfaces applications. however, the main challenge today in producing this kind of antimicrobial is the synthesis of stable nanoparticles in a polymer solution, as their antimicrobial effectiveness not only depends on their size but also on their size distribution and agglomeration state (castro-mayorga et al., 2016b) . in this regard, biopolymers have emerged as suitable platforms for metal nanoparticles because they can act as carriers for stabilizing metal nanoparticles against agglomeration and simultaneously constitute a biodegradable active material. the effectiveness of nanoclays and nanosilver as antimicrobial agents and, hence, their possible use in active packaging systems, has been demonstrated in various in vitro experiments as summarized by kuorwel et al. (2015) . in the case of silver, several research works have investigated the effect of incorporating silver fillers and nanofillers into different biopolymer matrices, such as poly(vinyl alcohol) (fortunati et al., 2013; krklješ et al., 2007; mbhele et al., 2003) pla (martínez-abad et al., 2014) but these silver particles were not synthesized and stabilized into the matrices, thus causing significant changes in the optical properties and reducing the antimicrobial efficiency. in this way, the pioneering studies made by castro-mayorga et al. (2014) examined not only the role of a pha as stabilizing agent for the in situ synthesis of silver nanoparticles (castro-mayorga et al., 2014) but also the use of this material as masterbatch for the preparation of active nanocomposites films by direct blending with a commercial poly(3-hydroxybutyrate-co-3-hydroxyvalerate). the so obtained films led to a surprising oxygen permeability drop of ca. 56% compared to the neat polymer and had a high and prolonged bactericidal activity against two of the most common food borne pathogens, salmonella enterica and listeria monocytogenes . however, the highest potential of metal nanoparticles as antimicrobial agents can hardly be achieved in most cases because they have low solubility/compatibility with the polymers, leading to the agglomeration of nanoparticles, and the prepared antibacterial polymeric materials present low surface-to-mass ratios. to solve this problem, the metal nanoparticles can be alternatively preincorporated into submicro-or nanofibers by means of electrospinning to generate masterbatches, which are subsequently melt mixed with polymers pellets, or even better, used as active coatings over polymer surfaces (amna et al., 2014) . the incorporation of metal nanoparticles in electrospun fibers has enabled the development of novel materials with useful features, such as fibrous membranes with antibacterial properties for water filtration (botes and cloete, 2010) , protective clothing (pant et al., 2011) , wound dressings, implant materials, or tissue engineering (navalakhe and nandedkar, 2007) . specifically, in the active food packaging area, the electrospinning technique successfully avoids the agglomeration of metal nanoparticles and greatly increases their antimicrobial activity. for instance, zno nanoparticles showed a more effective and prolonged biocide activity against foodborne pathogens when they were applied as a coating of annealed electrospun pha/zno fiber mat (fig. 2 .4a) over a compression molded pha film ( fig. 2 .4b) than their counterparts prepared by melt-mixing. this indicated that the biocide potential of metal nanoparticles could be more efficiently exploited when they were incorporated in electrospun coatings, as this processing technique improved the dispersion and distribution of the nanoparticles within the polymer matrix (castro-mayorga et al. 2016a). beside the metal nanoparticles, the incorporation of other natural compounds, such as essential oils by electrospinning is being considered an interesting option for active food packaging, especially taking into account that these compounds are thermally sensitive and, thus, cannot be directly incorporated during typical processing methods used for polymeric materials. for example, wen et al. (2016a,b) carried out in vitro studies with cinnamon essential oil, β-cyclodextrin, and polyvinyl alcohol (pva) nanofibrous films, which demonstrated an excellent antimicrobial activity against gram-positive and grztive bacteria. in addition, they also proved that these nanofibrous films were able to prolong the shelf life of strawberries and pork. likewise, munteanu et al. (2014) incorporated allyl isothiocyanate (aitc) and β-cyclodextrin into pva nanofibers via electrospinning and demonstrated that the evaporation of aitc was hindered by ciclodextrin inclusion complexation and thus, their antimicrobial effect against escherichia coli and staphylococcus aureus was enhanced. additionally, natural polymers, such as chitosan and cellulose, have also been used to make nanostructured antimicrobial materials. the original antimicrobial chitosan was used by torres-giner et al. (2008) for the preparation of active fiber using electrospinning and by cárdenas et al. (2009) as polymer matrix for the incorporation of nanocopper, which exhibited antimicrobial activity against s. aureus, and salmonella typhimurium with a 3-4 log cfu ml −1 bacterial reduction. a cellulose-derived polymer, that is, methylcellulose has been utilized to yield good mechanical properties and barrier properties but also to synthesize silver nanoparticles with high antimicrobial activity (maity et al., 2012) . another interesting application was one developed by munteanu et al. (2014) , who combined the antimicrobial properties of silver nanoparticles with the antioxidant activity of vitamin e within multifunctional electrospun pla/agnp/vitamin e nanofibers, which may be quite applicable for the fabrication of active packaging structures for fruits and juices. enteric viruses are those human viruses that are primarily transmitted by the fecal-oral route, either by person-to-person contact or by ingestion of contaminated food or water, although they may also be shed in vomitus. food may be contaminated by enteric viruses during all stages of the food supply chain, and transmission can occur by consumption of food contaminated during the production process (primary production, or during further processing), or contaminated by infected food handlers. data on viral foodborne diseases are still fragmented, and epidemiological studies have focused either on particular countries or on particular pathogens. epidemiological evidence indicates that enteric viruses, in particular human noroviruses (nov), which cause acute gastroenteritis, are the leading causes of foodborne illnesses in industrialized countries (cdc, 2013; efsa and ecdc, 2015) while hepatitis a virus (hav) has recently been considered as a reemerging foodborne public health threat in europe due to the number of foodborne outbreaks associated with imported foods (sprenger, 2014) . recently, the world health organization estimates the global burden of foodborne diseases, reporting that infectious agents that cause diarrhoeal diseases accounted for the vast majority (550 million cases per year), in particular norovirus (120 million cases per year) (who, 2015) . in the eu, foodborne viruses were identified as the most frequently detected causative agent of foodborne outbreaks in 2014, accounting for 20.41% of the reported outbreaks (efsa and ecdc, 2015) . moreover, the cost of foodborne illness in the usa is estimated to be around 3 billion dollars a year (scharff, 2012) . other enteric viruses, including sapoviruses, rotaviruses, coronavirus, astroviruses, and hepatitis e virus (hev), are not frequent causes of foodborne disease but can occasionally be transmitted by contaminated foods. although there is an increasing awareness of the importance of foodborne diseases caused by enteric viruses, few studies have confronted the task of evaluating materials with antiviral activity against enteric viruses. in a recent innovative study, an active renewable packaging material with virucide properties was synthesized by the incorporation of silver ions into polylactide acid films. these films showed strong antiviral activity on feline calivicirus (fcv), a norovirus surrogate, using the japanese industrial standard (jis z 2801) (martínez-abad et al. 2013) . when films were applied to vegetables, antiviral activity was very much dependent on the food type and temperature. likewise, bright et al. (2009) evaluated the antiviral activity of active packaging, reporting that fcv titers were reduced by 5 log 10 when in contact with plastic coupons impregnated with 10% silver-copper zeolites. moreover, the antiviral activity of adding grape seed extract (gse), green tea extract (gte), or cinnamaldehyde (cnma) into films has also been reported. amankwaah (2013) developed chitosan edible films incorporated with gte or gse, which showed significantly antiviral activity against murine norovirus (mnv). fabra et al. (2016a) showed that the incorporation of an active electrospun interlayer based on zein and 9.7% of cnma to a polyhydroxybutyrate packaging material (phb) in a multilayer form completely inactivated fcv according to iso 22196:2011, while mnv titers were reduced by 2.75 log 10 . when the developed multilayer films were evaluated after 1 month of preparation or at 25°c, the antiviral activity was reduced as compared to freshly prepared multilayer films evaluated at 37°c. these studies show the excellent potential of polymers with virucide activity for food contact applications, as well as for active packaging technologies to maintain or extend food quality and safety. summarizing, this chapter, which includes an overview of relevant developments on biopolymer-based materials for encapsulation and packaging applications in the food area, shows the potential of nanostructuring these biopolymers for advanced applications. not only nanostructuring the biopolymers, but also combining them 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dynamic thermogravimetric degradation of gamma radiolytically synthesized ag-pva nanocomposites review of mechanical properties, migration, and potential applications in active food packaging systems containing nanoclays and nanosilver mode of antiviral action of silver nanoparticles against hiv-1 viable counts, characteristic evaluation for commercial lactic acid bacteria products enzyme encapsulation in freeze-dried bionanocomposites prepared from chitosan and xanthan gum blend milk proteins as vehicles for bioactives overview of active polymer-based packaging technologies for food applications neutron scattering: a natural tool for food science and technology research electrospinning as a useful technique for the encapsulation of living bifidobacteria in food hydrocolloids a review of analytical methods for the identification and characterization of nano delivery systems in food effect of spray drying and freeze drying on the immunomodulatory activity, bitter taste and hygroscopicity of hydrolysate derived from whey protein concentrate synthesis of methylcellulose-silver nanocomposite and investigation of mechanical and antimicrobial properties antimicrobial food packaging: potential and pitfalls thermoreversible gelation of k-carrageenan: relation between conformational transition and aggregation characterization of transparent silver loaded poly(llactide) films produced by melt-compounding for the sustained release of antimicrobial silver ions in food applications evaluation of silver-infused polylactide films for inactivation of salmonella and feline calicivirus in vitro and on fresh-cut vegetables application of x-ray and neutron small angle scattering techniques to study the hierarchical structure of plant cell walls: a review multi-scale model for the hierarchical architecture of native cellulose hydrogels a review of maillard reaction in food and implications to kinetic modelling lipid encapsulation technology-techniques and applications to food control of microbial activity using antimicrobial packaging -barros-velázquez fabrication and characterization of silver-polyvinyl alcohol nanocomposites protein-stabilized emulsions stability of cardamom (elettaria cardamomum) essential oil in microcapsules made of whey protein isolate, guar gum, and carrageenan encapsulation of food protein hydrolysates and peptides: a review oxidative stability of encapsulated fish oil in electrospun zein fibres the newest achievements in synthesis, immobilization and practical applications of antibacterial nanoparticles development of curcuminoids loaded poly(butyl) cyanoacrylate nanoparticles: physicochemical characterization and stability study encapsulation of natural polyphenolic compounds: a review polylactic acid (pla)/silver-np/vitamin e bionanocomposite electrospun nanofibers with antibacterial and antioxidant activity effect of soya protein isolate as a complementary drying aid of maltodextrin on spray drying of tamarind pulp application of nanotechnology in biomedicine ag/agcl nanoparticle decorated layered double hydroxides: synthesis, characterization and antimicrobial properties microencapsulation of propolis extract by complex coacervation active food packaging technologies self-assembling polymer systems for advanced treatment of cancer and inflammation ph-driven encapsulation of curcumin in self-assembled casein nanoparticles for enhanced dispersibility and bioactivity electrospun nylon-6 spider-net like nanofiber mat containing tio2 nanoparticles: a multifunctional nanocomposite textile material β-lactoglobulin heat-induced aggregates as carriers of polyunsaturated fatty acids estimation of the activation energy of carbohydrate polymers blends as selection criteria for their use as wall material for spray-dried microcapsules morphology and stability of edible lycopene-containing microand nanocapsules produced through electrospraying and spray drying microencapsulation of oil droplets using cold water fish gelatine/gum arabic complex coacervation by membrane emulsification comparison of αtocopherol microparticles produced with different wall materials: pea protein a new interesting alternative probiotic bacteria: legislative framework-requirements to evidence basis interactions between k-carrageenan and chitosan in nanolayered coatings-structural and transport properties nanoemulsion delivery systems: influence of carrier oil on β-carotene bioaccessibility a way for improving the stability of the essential oils in an environmental friendly formulation spray dried protein-stabilized emulsions as vitamin matrix carriers: contribution of protein aggregates and lipid nano-and micro-structures to vitamin long-term protection encapsulation of carotenoids solubilization of carotenoids from carrot juice and spinach in lipid phases: i: modeling the gastric lumen some dietary fibers reduce the absorption of carotenoids in women stability of spray-dried encapsulated carotenoid pigments from rosa mosqueta (rosa rubiginosa) oleoresin encapsulation of polyphenols and anthocyanins from pomegranate (punica granatum) by spray drying epigallocatechin gallate-loaded polysaccharide nanoparticles for prostate cancer chemoprevention on the viability of five probiotic strains when immobilised on various polymers encapsulation of indonesian propolis by casein micelle delivery of omega-3 fatty acids into cake through emulsification of fish oil-in-milk and encapsulation by spray drying with added polymers novel technologies for the encapsulation of bioactive food compounds microencapsulation of xylitol by double emulsion followed by complex coacervation economic burden from health losses due to foodborne illness in the united states populations of lactobacillus acidophilus, bifidobacterium spp., and lactobacillus casei in commercial fermented milk products effect of addition of whey protein isolate on spray-drying behavior of honey with maltodextrin as a carrier material optimization of the formulation for preparing lactobacillus casei loaded whey protein-ca-alginate microparticles using full-factorial design more can be done to stop "silent disease"of hepatitis active packaging technologies with an emphasis on antimicrobial packaging and its applications optimisation of microencapsulation of holy basil essential oil in gelatin by response surface methodology milk proteins as encapsulation devices and delivery vehicles: applications and trends stabilization of emulsions by osa starches the metabolic, nutritional and toxicological consequences of ingested dietary maillard reaction products: a literature review development of active antimicrobial fiber-based chitosan polysaccharide nanostructures using electrospinning emulsion-stabilizing starches: use in flavor encapsulation enrichment of biscuits with microencapsulated omega-3 fatty acid (alpha-linolenic acid) rich garden cress (lepidium sativum) seed oil: physical, sensory and storage quality characteristics of biscuits bsa/polyelectrolyte core-shell nanoparticles for controlled release of encapsulated ibuprofen entrapment, survival and release of bifidobacterium adolescentis within chickpea protein-based microcapsules encapsulation of bifidobacterium adolescentis cells with legume proteins and survival under stimulated gastric conditions and during storage in commercial fruit juices microbiologic evaluation of commercial probiotics functional materials on food nanotechnology fabrication of electrospun polylactic acid nanofilm incorporating cinnamon essential oil/β-cyclodextrin inclusion complex for antimicrobial packaging encapsulation of cinnamon essential oil in electrospun nanofibrous film for active food packaging who estimates of the global burden of foodborne diseases: foodborne disease burden epidemiology reference group whey protein/alginate beads as carriers of a bioactive component small angle neutron scattering studies on the internal structure of poly(lactide-co-glycolide)-block -poly(ethylene glycol) nanoparticles as drug delivery vehicles stable nano-sized emulsions produced from soy protein and soy polysaccharide complexes soluble fibers inhibit carotenoid micellization in vitro and uptake by caco-2 cells structural characteristics of carrageenan gels: temperature and concentration dependence. food hydrocoll encapsulation of β-carotene in alginate-based hydrogel beads: impact on physicochemical stability and bioaccessibility formation and characterization of mint oil/s and cs/water microemulsions encapsulation of functional lipophilic components in surfactantbased colloidal delivery systems: vitamin e, vitamin d, and lemon oil this work was supported by the spanish ministry of economy and competitiveness (mineco) (ryc-2012-09950, agl2015-63855-c2-1 and inia grant rta2014-00024-c04-03). gs and mjf were supported by the "ramón y cajal" young investigator from the mineco. jlc-m was supported by the administrative department of science, technology and innovation (colciencias) of the colombian government. key: cord-284208-8fsqgkw5 authors: zolla, lello title: proteomics studies reveal important information on small molecule therapeutics: a case study on plasma proteins date: 2008-11-07 journal: drug discov today doi: 10.1016/j.drudis.2008.09.013 sha: doc_id: 284208 cord_uid: 8fsqgkw5 the most abundant proteins in serum, such as albumin and igg, act as molecular sponges that bind and transport low molecular weight proteins/peptides and drugs. in the near future, pharmacoproteomics, the use of proteomic technologies in the field of drug discovery and development, and interactomics, the branch of proteomics which is concerned with identifying interactions between proteins, will allow researchers to (i) know the specific protein changes that occur in biological compartments in response to drug administration; (ii) design small novel therapeutic molecules that can have extended half-lives if carried by plasma protein in the blood stream. advances in these fields will open new avenues of tailor-made molecular therapy, reducing present limitations on treatment arising from toxicity and inefficiency. in this short review we report and discuss the most recent developments arising from the use of proteomic tools in blood plasma protein research, looking at the identification of proteins found in plasma as well as their interactions with small molecules such as drugs, peptides, organic chemicals and metals. we believe this research demonstrates that proteomic technologies, and in particular pharmacoproteomics, interactomics and post-translational modification analysis, could be instrumental in the design of new tailor-made drugs leading to substantial improvements in molecular therapy. proteomics studies reveal important information on small molecule therapeutics: a case study on plasma proteins lello zolla department of environmental science, university of tuscia, piazzale università, viterbo, italy the most abundant proteins in serum, such as albumin and igg, act as molecular sponges that bind and transport low molecular weight proteins/peptides and drugs. in the near future, pharmacoproteomics, the use of proteomic technologies in the field of drug discovery and development, and interactomics, the branch of proteomics which is concerned with identifying interactions between proteins, will allow researchers to (i) know the specific protein changes that occur in biological compartments in response to drug administration; (ii) design small novel therapeutic molecules that can have extended half-lives if carried by plasma protein in the blood stream. advances in these fields will open new avenues of tailormade molecular therapy, reducing present limitations on treatment arising from toxicity and inefficiency.in this short review we report and discuss the most recent developments arising from the use of proteomic tools in blood plasma protein research, looking at the identification of proteins found in plasma as well as their interactions with small molecules such as drugs, peptides, organic chemicals and metals. we believe this research demonstrates that proteomic technologies, and in particular pharmacoproteomics, interactomics and post-translational modification analysis, could be instrumental in the design of new tailor-made drugs leading to substantial improvements in molecular therapy. blood plasma is the most complex human-derived proteome. because of this complexity, and the enormous range of concentration encountered across the population of protein components, spanning in excess of ten orders of magnitude, whole blood plasma is the most difficult specimen to analyze, and this creates serious challenges for proteomics. much progress has already been made in this field and new directions have been put forward and discussed as part of the hupo plasma proteome project (ppp), to focus efforts on the remaining challenges [1] . the ppp initiative has three stated long-term goals (i) to make a comprehensive analysis of the protein constituents of plasma; (ii) to determine the extent and source of variation in an individual's plasma over time; and (iii) to determine the extent of variation in plasma between individuals within and across populations [2] . blood plasma is known to contain proteins derived from blood cells and other body tissues that may have ended up there through cell death or damage (causing proteins to be released from normal cells), or they may come from aberrant protein secretions from tumor cells. in a recent investigation [3] , the examination of the plasma protein component categories revealed that many of the proteins detected in plasma are normally associated with cells (i.e. they are not known plasma proteins). these 'cellular leakage proteins' were categorized according to their original location and function. intracellular proteins accounted for up to 42% of the proteins identified, while membrane-associated proteins, including those proteins that are membrane-based but not known to be released in plasma (i.e. receptors, coreceptors and adhesion molecules) [3] accounted for another 13%. another 5% of the proteins were found to be of cellular origin, and are either secreted or occupy an extracellular location, and 3% were identified as specific cytokines or cytokine-related proteins. all these proteins are generally considered passenger proteins (some more transient than others) that utilize plasma for transportation, localization and mediation of cellular responses. overall, this group is the least characterized but possibly the most interesting one in terms of potential to yield biomarkers, with protein concentrations believed to range from low mg/ml to pg/ml levels, and possibly extending to levels below the detection limits of traditional elisa assays (1 pg/ ml). the 'classic' plasma proteins, those whose activity is specifically localized in the plasma, such as human serum albumin (hsa), complement components and apolipoproteins, make up only 4% of the total protein found in plasma. approximately 34% of the proteins identified, however, had no known function [3] . hence, with such a diverse population of proteins derived from a range of sources we might expect that the analysis of the extracellular proteome of proteins circulating in the plasma and the cell-based proteome are necessary and complementary for an exhaustive plasma investigation. one strategy applied in several recent examples involves using a secondary tissue or fluid of interest to first identify potential candidates for biomarkers and then screening the complementary plasma sample for their presence. such an investigation is highly desirable because disease markers present in plasma may include proteins with significant potential for early disease diagnosis, containing information that directly reflects pathophysiological states and represents an invaluable source of diagnostic information for a variety of different diseases [4] . thus, a broad inventory of plasma proteins (both qualitative and quantitative) could be used for the identification of putative protein markers for any diagnosable disease as well as for the development of new therapeutic products [4] . quantitatively speaking, the core plasma protein is albumin, representing about 50% of the total plasma protein content (in the order of 30-50 g/l). immunoglobulins (igs) represent 20-25% of the total protein mass [3] . low-abundance plasma proteins from tissue leakage and cytokines are present in the range of picograms to nanograms per milliliter. the most abundant proteins in serum, such as albumin, igg and transferrin, are known to act as the carriers for hormones, lipoproteins and many other proteins, lipids and metals. they may, in fact, be described as molecular sponges, which bind and transport low molecular weight (lmw) protein/peptide species preventing their rapid clearance by the renal system, thereby extending their half-life in the blood stream. although it has been estimated that 32% human plasma proteins do not display associations with the major proteins, about 48% have been found in association with igg and 20% of proteins have also been found with hsa codepleted groups, suggesting the possibility of weak overlapping interactions between some proteins and both hsa and igg (see fig. 1 ). moreover, because proteins in the circulatory system are exposed to a variety of proteases or chemical oxidations [5, 6] , plasma is rich in peptides, the so-called 'peptidome' (see box 1) from which about 5000 peptides have been revealed [7] , prevalently associated with the carrier proteins. if igs are retained in the sample during peptide-based proteomic studies, they can act as a generous source of random peptide sequences contributing to a large library of 'background' species that, because of their abundance (20 mg/ml sample concentration) and variety, greatly complicate the process of peptide detection and identification. plasma protein interactions appear to exhibit little dependence on protein size. in fact, small plasma proteins such as the basic proline-rich peptide p-e (ib-9, mw = 6020 da, igg codepleted protein) show more interaction specificity than large proteins such as cardiac titin isoforms (mw = 2,991,181 da) that are typically found in more than one protein interaction group [3] . this kind of information, concerning the interactions between human plasma proteins, should be useful for further studies in human blood systems/network biology, such as evaluating possible protein contaminants in therapeutic protein products prepared from human plasma, and for the design of analytical approaches to deplete high abundance plasma proteins effectively. additionally, it could be used to help design proteins with a reduced rate of clearance from the circulatory system, particularly important when small proteins are used as therapeutic agents [8] . normally, small uncomplexed proteins and peptides (i.e. less than 40 kda) are rapidly cleared from the circulation through enzymatic degradation, uptake by the reticuloendothelial system or by glomerular filtration, which discriminates on the basis of molecular size and charge [8] . it is believed that the circulation half-life of this lmw fraction is directly related to its binding affinity to large high abundant carrier proteins. drug discovery today volume 13, numbers 23/24 december 2008 box 1 the array of peptides normally present within the circulatory proteome is termed the 'peptidome' , and could be a rich source of cancer-specific diagnostic information because it provides a record of the cellular and extracellular enzymatic events that take place at the level of the cancer-tissue microenvironment. this new information archive seems to show that most peptides in vivo are bound to high-abundance proteins such as albumin. measuring panels of peptidome markers might be more sensitive and specific than conventional biomarker approaches. a biomarker is a biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or a disease. a biomarker may also be used to see how well the body responds to a treatment for a disease or condition, to measure the progress of disease or the effects of treatment. the use of proteomic technologies in the field of drug discovery and development. toxicoproteomics is the use of global protein expression technologies to better understand environmental and genetic factors, both in the episodes of acute exposure to toxicants and in the long-term development of disease. primary aims in toxicoproteomics are the discovery of key-modified proteins, the determination of affected pathways, and the development of biomarkers for eventual prediction of toxicity. the use of proteomic technologies and tools, such as phosphorylation-site-specific antibodies, to assess and monitor the phosphorylation state(s) of various proteins involved in signaling pathways in cells. a method that allows for the quantitative measurement of proteins in two separate populations by mass spectrometry via the differential tagging of proteins in each population at cysteine residues with heavy or light isotope reagents. protein microarrays are composed of a series of immobilized spots, containing a homogeneous or heterogeneous 'bait' molecule. a spot on the array can represent an antibody, a cell or phage lysate, a recombinant protein or peptide or a nucleic acid. the array is queried with either a probe (a labeled antibody or ligand), or an unknown biological sample (e.g. a cell lysate or serum sample) containing analytes of interest. by directly or indirectly tagging the query molecules with a signal-generating moiety, a pattern of positive and negative spots is generated. a combinatorial ligand library is composed of millions of diverse hexapeptide baits, able to capture aspecifically all peptides/ proteins in any given proteome. in this way they concentrate the 'low-abundance' proteome, while drastically cutting the concentration of the most abundant compounds. it is based on the concept of a 'one-bead, one-peptide' approach. free-flow electrophoresis (ffe) is electrophoresis carried out in an aqueous medium without using any solid matrix, such as acrylamide. it is useful for the separation of a wide variety of charged analytes like low-molecular weight organic compounds, peptides, proteins, protein complexes, membranes, organelles and whole cells in aqueous media under native and denaturing conditions. the analyte is injected into a thin, laminar separation buffer film (which defines the electrophoretic mode such as zone electrophoresis, ief or isotachophoresis) and is deflected by an electric field perpendicular to the flow direction. differential scanning calorimetry differential scanning calorimetry measures the difference in the amount of heat required to increase the temperature of a changed biological sample with respect to an unchanged one (reference) as a function of temperature. the difference in thermograms of blood plasma between normal and diseased individuals is not related to the concentrations of the most abundant plasma proteins but, rather, seems to arise from binding interactions involving as yet unknown biomarkers with the more abundant plasma proteins, particularly albumin. such a behavior is consistent with the 'interactome' hypothesis. interactomics is a fusion science of biology, informatics and engineering which provides a global view of protein family interaction networks. it involves the study of both the interactions and the consequences of those interactions between and among proteins, and other molecules within a cell and can be used to compare networks of interaction between and within species to see how the traits of such networks are varied and conserved. the interactome network includes certain calculated parameters that weigh the reliability of a given interaction (i.e. the 'edges' of the interactome network) between two proteins, and also qualify the functional environment around any given protein (i.e. the 'nodes' of the interactome network). some of the strategies devised to retard the clearance of therapeutic proteins include the covalent attachment of polyethylene glycol or dextran chains or by protein-protein crosslinking. genetic modification has also been used to create chimeras of the therapeutic protein of interest with long-lived plasma proteins like albumin or igg [9] . over 10,000 different proteins have been estimated to be commonly present in the plasma, most of which are at very low relative abundances [10] . the dynamic range of currently available proteomic techniques such as mass spectrometry (ms) or 2d gel electrophoresis (2-de) spans three to four orders of magnitude and as such does not approach the ten orders of magnitude represented by the plasma proteins. thus, a significant challenge for proteomic analysis of plasma is how to reveal the low-abundance proteins. the strategies that have been most frequently used to overcome the dynamic range problem are to fractionate the plasma proteome into smaller subsets, and/or to deplete one or more of the major proteins. immunoaffinity is the most efficient way to deplete proteins and so these methods are most widely used [11] [12] [13] . immobilized antibodies packed in columns or cartridges capture several the most abundant proteins lowering the protein content down to 1% of the initial amount. although nontarget proteins are removed in this process, it has been shown to be reproducible [14] . multiple orthogonal separation steps have also been used, such as strong cation exchange chromatography followed by reverse phase-liquid chromatography (rp-lc) [8] or insolution isoelectric focusing (ief) [15] . recently, a novel separation approach has been proposed, using 2d free-flow electrophoresis (ffe), separating proteins and peptides in solution according to their pi before lc-ms/ms [16] . a commercial combinatorial ligand library (proteominer) is now available, enabling researchers to pick out the low-abundance proteins [17] . another innovation is the microflow mf10, a device to prefractionate complex low volume, low-abundance samples that can also enrich for very specific species of proteins based on charge and/or size either in native or in denaturing format [18] . all these strategies concomitantly remove proteins/peptides associated with the highly abundant proteins targeted for depletion. a recent study [19] focused on the binding properties of six of the most abundant serum proteins -to investigate the small peptides/proteins that may be bound to them. each of the proteins targeted was found to be capable of binding several different peptide/proteins (210 proteins from a total of 378 unique peptides), many of which are clinically useful biomarkers. these results suggest that, on the one hand, albumin or igg depletion before protein identification may actually eliminate many valuable biomarkers but, on the other hand, the identification of biomarkers, through the selective isolation of protein bound to the more abundant proteins in serum, could be a novel proteomic strategy. because the general and specific sample losses increase with each separation stage, the best solution to this dilemma is to maximize the efficiency of each separation stage and minimize several dimensions required for the characterization of complex mixtures. as stated above, proteomic approaches may be utilized for disease classification as well as for the development of novel biomarkers related to prognosis, diagnosis and choice of therapeutic regimen. it is generally accepted that these biomarkers will not originate from classic plasma secretions but, most probably, from leakage, secretion or shedding of proteins from the specific affected tissue, cell type or cellular pathway. nevertheless, blood is still the logical choice of biospecimen and has become the most frequently used biomarker discovery matrix to date, because the driving force of biomarker discovery is the development of blood-based assays for early detection and prediction of therapeutic response [20] . it follows that if protein biomarkers have been identified in biopsied tissues, researchers will then be able to examine the blood to determine whether these biomarkers are in circulation. the great advantage being that blood tests are much less invasive than biopsies. the downside is that once such proteins are released into around 6 liters of plasma, their concentration is extremely low. hence, the need to extend the dynamic range of protein detection and this is where proteomics comes into its own. although blood is a very convenient and noninvasive fluid to monitor for biomarkers, it poses many challenges from the perspective of protein detection. probably the greatest potential application for proteomics lies in investigating pathways that are easily targeted by small molecules or therapeutic antibodies. among the challenges facing clinical proteomics is the ability to link protein expression profiles to specific disease phenotypes and the identification of relevant biomarkers to develop diagnostic tools [21] . in this sense, proteomics is an expansion of reductionist biology, where single proteins are analyzed in a high throughput fashion to arrive at an understanding of the entire system. the intention is that, once the human blood proteome has been fully described, several biomarkers will emerge for each particular disease state that can be used to provide a 'fingerprint' of that disease and here, there are obvious implications for blood donor testing [22] . there are already systematic searches underway to look for plasma proteins that are biological indicators or biomarkers for cancer (see refs. [23, 24] ). if a suite of biomarkers were to be available for early detection, stratification into distinct subtypes and monitoring of progression or response to therapy we could expect significant improvements in clinical outcomes for cancer patients. the intention is to develop panels of biomarkers that will allow early detection of cancer and prediction of the probable response to therapy, possibly detectable in a single proteomics experiment. despite the recent progress in proteomic technologies based on ms, however, the discovery of novel clinical assessment tools has been slow. this is partly because of the inherent difficulties in working with blood. it is hoped that a better understanding of the limitations of blood for comparative protein profiling and an appreciation of the advantages of cancer tissue or cancer cell secretomes will greatly enhance the progress [25] . a comparative proteome study of the body fluids of schizophrenia patients has been carried out to look for biomarkers or associated proteins in an effort to understand the etiology of schizophrenia. it was found that protein expression of the ttr tetramer and apolipoprotein e (apoe) was downregulated by up to 1.68 and 3.62 times, respectively, in schizophrenia patients compared to normal controls [26] . a study that revealed potential diagnostic cancer biomarkers used blood from sjl (selected by james lambert) mice, in which the growth of rcsx lymphoma cells induces an inflammatory response by stimulating v b 16+t cells [27] . after the depletion of albumin and immunoglobulin, 1079 mouse nr proteins were identified in sjl mouse plasma in a single experiment. most were found to be upregulated (e.g. acute phase reactants) but some proteins were found to be unique to the tumor-bearing mouse plasma (i.e. haptoglobin, proteosome subunits, fetuin-b, 14-3-3 zeta and mage-b4 antigen). while it remains to be seen whether a similar unique profile occurs in human lymphomas, recent studies have demonstrated that in sepsis and cancer, the interalpha inhibitor proteins (iaips), a family of structurally related serine protease inhibitors, are present in relatively high concentrations in human plasma, suggesting their suitability as potential biomarkers [28] . it would seem logical to assume that a better understanding of the biological mechanisms of drug toxicity and the development of therapeutic resistance would lead to the development of improved therapeutics with significant utility in clinical research. in this context, pharmacoproteomics, the use of proteomic technologies in the field of drug discovery and development (see fig. 2 ), is an emerging science. protein drug-activity biomarkers can be defined as specific protein changes in total protein content in biological compartments that occur in response to drug administration [29] . in contrast to genomic approaches, the application of pharmacoproteomics, by virtue of being stimulus-induced, may be more amenable to systematic experimental manipulation that could ultimately result not only in validation of a biomarker, but also in characterizing its behavior under specific conditions [30] . in association with chemical compound library screening strategies, pharmacoproteomics is expected to accelerate the discovery of new lead compounds for future drugs. it should also play an important part in preclinical studies by providing information on the mode of action and the eventual deleterious side effects of drugs. hopefully, ongoing and future proteomic studies will open up new avenues of tailor-made molecular therapy, reducing present limitations associated with treatment toxicity and efficiency. ms-based proteomics is ideal in this respect, because it offers a targeted way to identify hundreds of protein or peptide biomarkers simultaneously, with the obvious potential for discovering drugactivity markers because there is often no a priori knowledge of the particular proteins that are likely to change [31] in response to drug administration [32, 33] . while sample enrichment narrows the dynamic range over which molecules of interest must be detected, the improvements in separation technology and ms have extended the ability to find and identify proteins over an ever wider range of relative protein concentrations. ion trap instrumentation has been superseded by the greatly improved linear ion trap mass spectrometers. at the same time, there has been a steady decrease in the diameters of lc-capillary columns that has resulted in increased sensitivity and ability to quantify proteins. this has improved analysis by increasing, by orders of magnitude, the detectable dynamic range of a protein that would not have been picked up previously in samples of similar size. as such, at this time, lc-fticr-ms (fourier transform ion cyclotron drug discovery today volume 13, numbers 23/24 december 2008 theoretical example of differential screening of plasma proteins from healthy, ill and treated (responding and resistant) patients by 2d gel electrophoresis. the appearance or disappearance of protein spots as well as quantitative variations of protein expression can be observed. resonance ms) technology provides the most sensitive and powerful measurement platform and though some significant potential exists for further increasing its dynamic range, this applies to those cases limited by sample complexity, rather than detection limits. recently, however, differential scanning calorimetry has provided a new window into the plasma proteome. thermograms of plasma from diseased individuals not only were found to differ dramatically from those of normal (control)_individuals, but also differed between sufferers of different illnesses, at least for the three diseases examined (arthritis, lyme disease and lupus). each disease appears to have a distinctive and characteristic thermogram. these radically different thermograms seen for different diseased states seem to arise from binding interactions involving as yet unknown biomarkers with the more abundant plasma proteins, particularly albumin. such behavior is consistent with the 'interactome' hypothesis, suggesting a novel use for calorimetry as a diagnostic tool [34] . for a careful approach to clinical design and data analysis it is important to maximize the chance of discovering meaningful drug-activity markers. the first step is to establish a link between drug administration and the resulting biologically quantified change [35] . this strong foundation should set the stage for any data-driven hypothesis generation and testing, and further establish the utility of such a multidisciplinary tool in expanding the frontiers of clinical research. too often there is no information available on the tissue under investigation, in these circumstances protein-profile changes in response to drug treatment can serve as evidence of cell pharmacodynamic activity, and such data can be used to establish a minimum dose for early clinical studies. more broadly, because targeted proteomics can also be hypothesis generating, detecting a drug-induced change can also lead to new lines of research and inquiry in the field of drug development or pathophysiology. a clear understanding of proteomic variability under controlled conditions is crucial to enable future clinical studies to be appropriately designed and to improve the contextual interpretation of data obtained from pharmacoproteomic studies. it is also important to establish a suitable time period between drug administration and analysis primarily to allow sufficient time for a tissue-targeted drug to reach a pharmacokinetic steady state before exploring protein profile changes [36] . this is to ensure minimal interference from extraneous factors on endogenous variability, thus maximizing the probability of identifying even subtle drug-induced changes. the variability between individual subjects could potentially affect observations in unpaired comparison studies, such as diseased versus healthy volunteers, and should be taken into account [37] . in other words, we cannot assume that pharmacodynamic responses to identical chemical or biochemical stimuli will be similar among healthy individuals and that these responses will differ significantly and consistently in patients with pathological disorders, because a variety of factors will influence the profile of each individual [37] . this aspect requires further investigation to ascertain how useful biomarkers will ultimately prove to be and what consequence this will have for drug development. pharmacoproteomics is not limited to the extracellular proteome analysis of blood plasma, because cell-based proteome analysis also has an important role in this field. in this context, post-translational modifications (ptm) of proteins have a significant impact. some 300 potential modifications are known, and they include phosphorylation, glycosylation, acetylation, myristoylation, palmitoylation, methylation, sulfation, prenylation and ubiquitylation (http://www.abrf.org/index.cfm/ dm.home). during the past two decades, sensitive ms methods have been refined to determine the type and site of protein modifications that can seldom be predicted from a genomic sequence [38] . some of the protein modifications are regulatory and reversible, most notably phosphorylation which controls protein functions such as localization, complex formation, stability and activity through different mechanisms [39] . as a result of extensive research, we now know that about 30% of all human proteins can be modified by phosphorylation, while only 0.05% of the phosphorylated residues are tyrosine [40] . protein phosphorylation is one hallmark of the protein-protein interactions (ppis) underlying signaling networks and, in many cases, it is aberrant protein kinase activity that drives diseaseassociated derangements in signaling pathways. the aim would be to design drugs that effectively disrupt this aberrant proteinphosphorylation-based enzymatic activity and epigenetic phenomena. pharmacoproteomics, or the tailoring of therapy based on proteomic knowledge, is expected to take a central role in this process [41] . a recent study describes a cell-based drug discovery platform based on phosphospecific flow cytometry, or phosphoflow, with which researchers were able to screen for inhibitors of multiple endogenous kinase signaling pathways in heterogeneous primary cell populations at the single-cell level [42] . protein microarrays that examine protein-protein recognition events (i.e. phosphorylation) in a global, high-throughput manner have been used to profile cellular signal pathways in a way not possible with gene arrays [43, 44] . the activity levels of the proteins in a particular pathway can thereby be assessed in 'real time', to tailor treatment to each patient's cellular 'circuitry' [45] [46] [47] . the advantage of protein microarrays lies in their ability to provide a 'map' of known cellular signaling proteins that generally reflect the state of information flow through protein networks in individual specimens. the identification of crucial nodes or interactions within the network (see later) is a potential starting point for drug development and the design of individual therapeutic regimens [48] [49] [50] . accurate annotation of peptide modifications through unrestrictive database searches can reveal post-translational modifications, as well as sequence polymorphisms [51] . monitoring different phosphoprotein levels will also help to identify treatment-acquired resistance to chemotherapy. the effect of imatinib on the tyrosine phosphoproteome in bcrabl positive leukemia cell lines has been examined by proteomic approaches. the investigation revealed 64 sites of tyrosine phosphorylation corresponding to 32 different proteins [52] . affinity column chromatography using an immobilized pyrido(2,3-d)pyrimidine derivative has been successfully used to select protein kinases that bound to the matrix and were then identified using ms, demonstrating the fact that pyrido(2,3-d)pyrimidine is a kinase inhibitor with low specificity [53] . a similar methodological approach was applied to study the p38 kinase inhibitor sb203580, demonstrating that cyclin g-associated kinase (gak) and ck1 were almost as potently inhibited as p38a [53, 54] . protein expression subsequent to treatment with the hdaci trichostatin-a has been studied in human pancreas ductal carcinoma cell lines using 2-de and maldi-tof ms [55, 56] . trichostatin-a appears to upregulate proteins which promote cell death and downregulate proteins that favor cell growth. similarly rc307 modulates proteins that are involved in proliferation, cell cycle regulation, apoptosis and gene expression in colon carcinoma cells [57] . the pharmacoproteomic approach was found to be particu-larly useful for the identification of molecular alterations implicated in type 2 diabetes, and for further characterization of existing or new drugs [58] . research has shown that several physiological factors lead to changes in both the plasma proteome and the peptidome, including stress, sleep, sport training, eating meals and pregnancy. exposure to toxic agents, including drugs, can also be detected using proteomics leading to a distinct field of study known as toxicoproteomics [59] . recently, the proteins in the plasma of workers exposed to benzene were analyzed [60] . two drug discovery today volume 13, numbers 23/24 december 2008 human erythrocyte protein-protein interaction network (interactome). nodes of the network are the known rbc proteins and links connecting the nodes correspond to the known interactions [70] . significantly upregulated protein profiles were found in workers exposed to the toxic chemical: the t cell receptor b chain and matrix metalloproteinase-13. thus, the plasmatic t cell receptor b chain may be a useful indicator for the early detection of exposure to benzene. comparative proteome analysis of human plasma allowed qian et al. [61] to identify 32 proteins that were significantly increased after lipopolysaccharide administration. a study of the plasma of workers occupationally exposed to polycyclic hydrocarbons revealed several proteins as markers of such an exposure [62] . using the same technique, truncated forms of a1-antitrypsin were discovered in serum samples of patients presenting with severe acute respiratory syndrome [63] . in another study involving patients with the same syndrome, samples revealed a total of 38 differential spots, most of them corresponding to acute phase proteins [64] . the authors also identified proteins such as peroxiredoxin ii, not previously detected in plasma 2-de. the investigation of plasma protein interactions with metals remains a relatively new and challenging arena for toxicoproteomics. current studies include the identification of the protein carrier and how to determine toxic metal concentrations, which requires sensitive analytical techniques and powerful equipment. thankfully, the search for suitable quantitative techniques in the field of drug design and proteomics may be almost over with the development of inductively coupled plasma ms (icp-ms) [65] . another relevant field for pharmacoproteomic applications is that of elucidating the biological mechanisms of drug resistance. in drug-resistant cells alternative protein forms appear that prevent the drug binding to active sites and/or executing signaling effects independent of the regulated native protein forms. the methodology of proteomics seems highly applicable to the search for drug-resistant protein forms (drug-resistance proteomics). in this context the plasma proteome in aspirin (acetylsalicylic acid [asa])-sensitive and asa-resistant coronary ischemic patients has been analyzed. the expression of one isotype of the fibrinogen g chain and three isotypes of haptoglobin was increased in asa-resistant patients as well as that of three vitamin d binding protein (dbp) isotypes. it has been suggested that dbp regulates the inhibitory effect of asa on platelets, by reducing the inhibitory effect of asa on thromboxane a2 production [66] . in the field of oncology, proteomics is becoming widely used for the identification of tumor-specific protein markers, and pharmacoproteomics has found a place in the evaluation of chemotherapy, particularly for the characterization of drugresistance mechanisms [58] . identifying important interactions between blood proteins is emerging as another focus of blood-based proteomic research. many proteins circulate in blood, not as single entities but as multicomponent complexes, and as such comprise the blood 'interactome' [4] . protein-protein interactions (ppi) information can be extracted from the currently available databases of interactions (in silico approaches). little is currently known about the interactions of any given protein in the blood or whether these interactions are biologically relevant. examining protein complexes will, however, often yield information about protein function. the identification of such interactions may bring to light important information for designing new small molecule therapeutics. this conceptual framework contrasts with the single protein (or single pathway) approach that has previously dominated biology. the combination of proteomic and in silico approaches allows one to not only identify disease and/or drug-related changes in the proteome but also predict the changes in the protein interactome network that are associated with disease-related change of function. it can also predict modifications associated with ptm changes occurring as a result of a specific disease or stage of disease, as well as drug or gene therapy treatment. these linked approaches represent the future of clinical identification of a specific disease, its current stage or severity, and the effects and side effects of various treatments. clearly, an interactome of plasma proteins is far from reality at present, because of its complexity, while the simplicity of the human erythrocyte cell structure has instead made it an optimal cell for proteomic study. in fact, while nucleated cells contain 20,000-30,000 proteins [31, 67, 68] , red blood cells (rbcs), which lack nuclei and other organelles, contain far fewer. a comprehensive list of rbc proteins known to date contains 751 entries obtained using proteomics technology [69] . the resulting ppi network is depicted in fig. 3 [70] . the network presented here was derived from protein interaction data obtained from the unified human interactome (unihi) [71] . the correlation reported by unihi is derived from gene expression experiments and represents a measure of confidence for the interaction [72] . as the knowledge of ppis continues to increase owing to advances in proteomics research we expect that, in addition to a deeper understanding of the erythrocyte protein complement and how it relates to function, it will also be possible to understand how changes in the rbc proteome and interactome affect the development of erythrocyte disorders [69] . the promise of proteomic-based profiling, as opposed to gene transcript profiling alone, is that the resulting prognostic signatures are derived from drug targets (proteins) not genes, so the pathway analysis provides a direction for therapeutic mitigation. the only dark cloud on the horizon may be that extending the use of pharmacoproteomics (beyond molecular network analysis for patient-tailored therapy to include risk stratification or 'predispostional testing') is likely to raise a larger societal issue, particularly in a climate of cost containment. the powerful combination of pharmacoproteomics and interactomics may win through despite costs, furnishing new targeted therapies for disease prevention as well as for posttherapy monitoring. in short, these techniques not only provide a window on the presence or severity of a specific disease, and drug and gene therapy treatment, but can also be used to identify disease-and/ or drug-related changes in the proteome. they can also predict those changes in the protein interactome network that are associated with disease-related changes of function [73] . without doubt, these linked approaches represent the future for the cost effective and noninvasive identification of specific clinical diseases, their prognosis and effective treatment. hupo plasma proteome project: challenges and future directions the human proteome organization plasma proteome project pilot phase: reference specimens, technology platform comparisons, and standardized data submissions and analyses characterization of the human blood plasma proteome utilizing human blood plasma for proteomic biomarker discovery oxidation of proteins: basic principles and perspectives for blood proteomics proteomic analysis of rbc membrane protein degradation during blood storage composition of the peptide fraction in human blood plasma: database of 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electrophoresis and mass spectrometry the application of inductively coupled plasma mass spectrometry in pharmaceutical and biomedical analysis relationship between vitamin d binding protein and aspirin resistance in coronary ischemic patients: a proteomic study the abc's (and xyz's) of peptide sequencing proteomics of organelles and large cellular structures the human red blood cell proteome and interactome centrality measures for the human red blood cell interactome unihi: an entry gate to the human protein interactome a graph theoretic approach to testing associations between disparate sources of functional genomics data transfusion medicine in the era of proteomics this work was supported in part by the italian national blood centre, istituto superiore di sanità, rome, italy (directed by dott. giuliano grazzini). the author would like to thank dr g.m. d'amici for his help and dr j. scarpa for english revision. key: cord-300429-b0zev8zb authors: sobocińska, justyna; roszczenko-jasińska, paula; ciesielska, anna; kwiatkowska, katarzyna title: protein palmitoylation and its role in bacterial and viral infections date: 2018-01-19 journal: front immunol doi: 10.3389/fimmu.2017.02003 sha: doc_id: 300429 cord_uid: b0zev8zb s-palmitoylation is a reversible, enzymatic posttranslational modification of proteins in which palmitoyl chain is attached to a cysteine residue via a thioester linkage. s-palmitoylation determines the functioning of proteins by affecting their association with membranes, compartmentalization in membrane domains, trafficking, and stability. in this review, we focus on s-palmitoylation of proteins, which are crucial for the interactions of pathogenic bacteria and viruses with the host. we discuss the role of palmitoylated proteins in the invasion of host cells by bacteria and viruses, and those involved in the host responses to the infection. we highlight recent data on protein s-palmitoylation in pathogens and their hosts obtained owing to the development of methods based on click chemistry and acyl-biotin exchange allowing proteomic analysis of protein lipidation. the role of the palmitoyl moiety present in bacterial lipopolysaccharide and lipoproteins, contributing to infectivity and affecting recognition of bacteria by innate immune receptors, is also discussed. geranylgeranyl cysteine thioether h-and n-ras (28) rab proteins (28) attachment of glycosylphosphatidylinositol anchor or phosphatidylethanolamine (29) to the c-terminus of proteins is also a form of lipidation but is not shown here. a n-myristoylation is in most cases co-translational, but during apoptosis caspases can cleave some proteins, such as bid, exposing their n-terminal glycine residue, which is then modified by attachment of myristate (30) . b hedgehog proteins are additionally modified by covalent attachment of cholesterol to their c-terminus (31) . c o-acylation of wnt proteins is reversed by notum of the α/β hydrolase superfamily (31) . exceeds the consumption of other saturated fatty acids and, in the usa it accounts for about 60% of the total intake of saturated fatty acids (2) . a growing body of experimental and clinical evidence points to a link between a westernized diet, including a high intake of saturated fatty acids, and chronic inflammatory diseases (3) (4) (5) . as dietary saturated and unsaturated fatty acids apparently modulate activity of immune cells, their influence on the immune responses triggered upon infection is also beginning to be investigated (6) . these facts drive the interest in palmitic acid with an aim of elucidating the molecular mechanisms of its immunomodulatory properties. in this review, we focus on s-palmitoylation of proteins crucial for the interactions of pathogenic bacteria and viruses with the host. we emphasize novel data on the role of s-palmitoylated proteins in the invasion of host cells by pathogens and those involved in the host innate immune responses to the infection, which have been obtained thanks to the application of new technical approaches. recently, substantial progress in the understanding of protein palmitoylation was made possible by the development of methods allowing high-throughput analysis of cellular/tissue palmitoyl proteomes. we begin, however, by showing how unique protein s-palmitoylation is among other protein lipidations. s-palmitoylation is a posttranslational modification of proteins consisting in a potentially reversible covalent attachment of palmitoyl chain to a cysteine residue(s) of proteins through a thioester bond ( table 1) . thus, s-palmitoylation resembles other reversible regulatory posttranslational protein modifications, including phosphorylation or acetylation, well-established factors affecting protein structure and functions. in particular, s-palmitoylation modifies cellular localization of proteins and their stability. the most dramatic changes of localization concern cytosolic proteins which upon s-palmitoylation acquire a hydrophobic anchor facilitating their docking into membranes (figure 1) . however, several integral membrane proteins also undergo s-palmitoylation. it often occurs on cysteine residue(s) located in the proximity of the junction of the transmembrane and cytoplasmic domains of the protein. s-palmitoylated transmembrane proteins occupy various cellular compartments, such as endoplasmic reticulum, golgi apparatus, and the plasma membrane. in accordance, for some proteins, such as transmembrane adaptor proteins in leukocytes, s-palmitoylation was found secondary to the length and hydrophobicity of the transmembrane domain as a determinant of plasma membrane destination (7) . s-palmitoylation also contributes to the compartmentalization of proteins to distinct domains of membranes-rafts and tetraspanin-rich microdomains. in fact, the interest in s-palmi toylation was boosted when it was found to be required for the targeting of some signaling proteins to rafts. rafts are nanodomains of the plasma membrane and some intracellular membranes, mainly of the trans-golgi apparatus, rich in sphingolipids, glycerophospholipids with saturated fatty acid chains, and cholesterol (32) . the plasma membrane nanodomains are sites of signal transduction by distinct receptors of immune cells involved in both acquired immune reactions, such as t cell receptor (tcr), fcε receptor i, fcγ receptor ii, and in innate immune responses, such as toll-like receptor 4 (tlr4) (33, 34) . rafts are also sites of virion assembly and budding, as established, e.g., for influenza a virus and human immunodeficiency virus-1 (hiv-1) (35, 36) . peripheral membrane proteins acylated with saturated fatty acids are likely to anchor preferentially between the ordered saturated lipids of rafts rather than between the disordered lipids of the surrounding membrane. it has been shown that, owing to their raft localization, s-palmitoylated kinases of the src family interact with raft-associating plasma membrane immunoreceptors and initiate signaling cascades fundamental to acquired immunity (15, 37, 38) . it is worth noting that also the acyl chains attached to proteins can affect the membrane structure. studies on model membranes have revealed that palmitic and myristic acids facilitate formation of ordered lamellar membrane regions (39, 40) . in accordance, s-palmitoylation of erythrocyte peripheral membrane protein called membrane-palmitoylated protein 1 (mpp1) was found to be required for the proper lateral organization and fluidity of erythrocyte membrane. in the absence of mpp1 s-palmitoylation, raft assembly was disturbed and erythrocyte functioning compromised leading to hemolytic anemia in patients deficient in the enzyme catalyzing this reaction (41, 42) . preferential raft association is a feature of some s-palmitoylated transmembrane proteins, e.g., adaptor proteins pag, lat, and ntal, which collaborate with the abovementioned immunoreceptors. in fact, palmitoylation is required for the raft association of most integral raft proteins (8, 43, 44) . on the other hand, s-palmitoylation does not obligatorily confer raft localization on transmembrane proteins. certain s-palmitoylated proteins, such as transferrin receptor, glycoprotein g of vesicular stomatitis virus (vsv), and anthrax toxin receptor, tumor endothelial marker 8 (tem8), are actually excluded from rafts. apparently, a combination of s-palmitoylation and the properties of the transmembrane domain of the protein contribute to its destination to the raft or non-raft environment (43, 45) . it has also been proposed that the attachment of a fatty acyl chain at the juxtamembrane cysteine(s) of a protein can induce tilting of its transmembrane fragment, determining in which part of the membrane it will accommodate to avoid a hydrophobic mismatch potentially caused by the thickness of the bilayer (46) . that not all s-palmitoylated proteins associate with rafts has been shown convincingly for macrophage-like raw264 cells, where only about half of those proteins were found in the triton x-100-resistant membrane fraction enriched in rafts (47, 48) . in accordance, proteomic data on the distribution of s-palmitoylated proteins in prostate cancer cells have revealed that several such proteins are recovered in the non-raft (triton x-100-soluble) fraction and are likely localized to microdomains enriched in scaffold proteins called tetraspanins (49) . the tetraspanins are small integral membrane proteins found in the plasma membrane and other cellular membranes, having four transmembrane helices and undergoing s-palmitoylation at several conserved cysteine residues. the tetraspanins interact with each other and with various transmembrane and cytosolic partners, often also s-palmitoylated, forming microdomains ("tetraspanin web") (50) . it has been suggested that the amino acid composition of the s-palmitoylation site in some transmembrane proteins, such as the adaptor proteins involved in acquired immune responses, determines the association of those s-palmitoylated proteins with rafts or with the tetraspanin-enriched microdomains (44) . an intriguing and still poorly addressed question concerns the relation between rafts and the tetraspanin-enriched microdomains, apparently of functional significance, e.g., during virus budding from host cells (35) . this uncertainty stems partially from the fact that s-palmitoylation of tetraspanins governs their interactions with cholesterol and gangliosides leading at certain conditions to the recovery of tetraspanins in detergent-resistant membrane fractions enriched in rafts (51, 52) . besides its involvement in targeting proteins to rafts or tetraspanin-enriched microdomain, s-palmitoylation has been found to govern accumulation of the transmembrane chaperone protein calnexin in the perinuclear domain of endoplasmic reticulum (53) . s-palmitoylation also affects protein stability through its interplay with ubiquitination or phosphorylation, as found for the anthrax toxin receptor tem8, antiviral interferon-induced transmembrane protein ifitm1, calnexin, and zdhhc6, one of palmitoyl acyltransferases described below (54) (55) (56) (57) . possibly the most intriguing is the reversible character of s-palmitoylation. enzymes catalyzing palmitoylation and depalmitoylation of proteins have been characterized (58, 59) . palmitate is transferred onto the thiol group of cysteine from cytosolic palmitoyl-coa by palmitoyl acyltransferases, enzymes containing the zinc finger dhhc domain named after the highly conserved asp-his-his-cys peptide (figure 1 ). this is a twostep reaction comprising transient autoacylation of zdhhc enzymes and transfer of the fatty acyl chain from this intermediate to a protein substrate (60) . in mammals, the zdhhc enzyme family consists of 24 proteins, and zdhhc proteins are also found in other eukaryotes but not in bacteria nor are they encoded by viral genomes. mammalian zdhhc enzymes, each having at least four transmembrane helices, are located in the plasma membrane, endoplasmic reticulum, and golgi apparatus (58) . they display some specificity toward their protein substrates and also selectivity toward fatty acyl moieties other than palmitate, which contributes to the heterogeneity of lipids attached to proteins, such as viral glycoproteins described below (61) . in the opposite process, the thioester bond is cleaved by acyl-protein thioesterases (apts) (apt1 and apt2) and palmitoyl protein thioesterases (ppts) (ppt1 and ppt2), which are localized in the cytosol and in lysosomes, respectively. apt1 and apt2 likely govern the dynamic functional changes of s-acylation of proteins (62) while ppt1 and ppt2 depalmitoylate proteins during their degradation (63, 64) . recently, serine hydrolases of the abhd17 family have also been identified as depalmitoylating enzymes, and their specific substrate proteins determined (65, 66) . of note, the zdhhcs, apt1/apt2, and abhd17 proteins are s-palmitoylated themselves, and palmitoylation of zdhhcs and depalmitoylation of apt1/2 can occur in a cascade manner (57, 62) . the dynamic cycles of palmitoylation/depalmitoylation detected for several peripheral membrane proteins are often synchronized with intracellular trafficking of those proteins. they circulate between the plasma membrane and the golgi apparatus or endosomes, as exemplified by n-and h-ras, r7-regulator of g protein and apts. in fact, it is proposed that palmitoylationdependent anchoring of apt1 in the plasma membrane allows it to depalmitoylate h-ras at this location, while subsequent autodepalmitoylation releases apt1 guiding it, alongside h-ras, for another round of palmitoylation at the golgi apparatus (62, (67) (68) (69) . cycles of palmitoylation/depalmitoylation are crucial for signaling by distinct plasma membrane receptors and for their distribution (69) (70) (71) . activation of tcr receptor or fas receptor in t cells was found to trigger quick and transient palmitoylation of lck kinase of the src family (72, 73) , but the exact meaning of the dynamic protein s-palmitoylation for processes triggered during the host-pathogen interaction awaits elucidation. it is worth mentioning that although the zddhc enzymes catalyze bulk protein palmitoylation in eukaryotic cells (74) , some proteins have a unique autopalmitoylation activity. these include bet3, a component of a multisubunit transport protein particle complex involved in vesicular trafficking, tea domain transcription factors, and also bacterial evf protein (75) (76) (77) (78) . the palmitic acid residue is attached constitutively to a specific cysteine residue of those proteins, remains buried inside a hydrophobic pocked in their core thereby affecting the tertiary structure and, thus, interactions with other proteins (75, 77) . an exhaustive discussion on the physiology of s-palmitoylated proteins in eukaryotic cells can be found in several recent reviews (46, 79, 80) . it has been established that, in addition to palmitate, various other fatty acyl moieties, such as saturated stearate (c18:0) or protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january 2018 | volume 8 | article 2003 monounsaturated palmitoleate (c16:1), and oleate (c18:1) can be attached via the thioester linkage to proteins. the early reports on the heterogeneity of the fatty acyl moieties attached to cysteines obtained by analysis of selected immunoprecipitated proteins (15, 16, (81) (82) (83) have recently been complemented by a comprehensive proteomic analysis of fatty-acylated proteins of macrophage-like raw264 cells (14) . the latter study showed that an enrichment of culture medium of cells with monounsaturated fatty acids leads to their incorporation into a similar set of proteins as those normally modified with palmitate. among them, several proteins relevant to innate immune responses were found. all these data justify the use of a broader term s-acylation rather than s-palmitoylation ( table 1 ). the physiological consequences of s-acylation of proteins with individual fatty acids are slowly being revealed. modification of fyn kinase with polyunsaturated fatty acid residue, such as arachidonate (c20:4), disturbed its raft localization and, thereby, tcr signaling (15) . a heterogeneity of s-acylation was also found in viral spike proteins, such as hemagglutinin (ha) of influenza a virus, and e1 and e2 of semliki forest virus, which are modified in host eukaryotic cells by attachment of both palmitate and stearate (9) . in ha, stearate is attached at the transmembrane cysteine while palmitate is attached to two cysteine residues in a membrane-proximal region of the protein. the stearoyl chain seems to accommodate into a groove formed by amino acids of the transmembrane helix shaping the domain in a way that facilitates its fitting into rafts (84) . s-stearoylation of human transferrin receptor 1 at the juxtamembrane cysteine residues(s) is a key factor of the signaling cascade controlling mitochondrial morphology and functioning (13) . of interest, the latter study also showed that dietary supplementation of stearic acid reversed the deleterious effects of a genetically determined mitochondria dysfunction in drosophila. taking into account that unsaturated fatty acids affect the profile of s-acylation of proteins in vitro (14, 15) , it is of outmost interest whether a similar effect of unsaturated and saturated (palmitic) fatty acids could be achieved in vivo with respect to proteins of immune cells. beside s-acylation, less frequently palmitate can also be attached to the amine group of various amino acids (glycine, cysteine, and lysine) giving n-palmitoylation or to the hydroxyl group of serine or threonine in a process called o-palmitoylation ( table 1) . as during s-palmitoylation, also other fatty acids can be utilized in these processes named then n-and o-acylation. thus, a type of protein n-acylation is n-myristoylation, a frequent modification contributing to membrane anchoring of peripheral proteins. the saturated myristate (c14:0) is transferred to the protein from myristoyl-coa by n-myristoyl transferase (two isozymes in mammals). in a vast majority of cases, myristate is attached cotranslationally to the n-terminal glycine residue (after removal of the initiator methionine) via an amide linkage ( table 1) . like most lipidations, this modification is irreversible. several viral proteins are n-myristoylated, such as gag of hiv-1 crucial for budding of newly formed virions from plasma membrane rafts of host cells, and proteins of parasitic protozoa plasmodium falciparum, trypanosoma brucei, and leishmania donovani (causing malaria, african sleeping sickness, and leishmaniosis, respectively). for this reason, n-myristoyl transferase is considered a potential drug target in the therapy of these diseases (17-19, 85, 86) . data on the n-and o-palmitoylation of proteins involved in the host-pathogen interactions are limited, but interesting conclusions can be drawn from the information concerning proteins taking part in other processes. n-palmitoylation of the n-terminal glycine of the α-subunit of a heterotrimeric g protein (gαs) has been described (20) ( table 1 ) besides the wellknown s-palmitoylation of this pivotal signaling protein. the n-palmitoylation of gαs is irreversible, and the enzyme responsible for this modification is unknown. it has been speculated that s-to n-palmitoyl migration can occur both in vivo and also in vitro during mass spectrometry analysis (20, 87) . this suggests that caution is needed in interpreting results of this methodological approach, which is used with increasing frequency to study fatty acylation of proteins in immune cells (see next sections). probably the best-characterized is the n-palmitoylation of the n-terminal cysteine residue of hedgehog proteins (sonic, indian, and desert in mammals). it determines secretion of these proteins, which regulate embryonic patterning ( table 1) . secreted wnt and ghrelin proteins are examples of o-acylation of serine residues with unusual fatty acid residues such as palmitoleate (c16:1) and octaonoate (c8:0) ( table 1 ). the fatty acylation of hedgehog, wnt, and ghrelin is catalyzed by enzymes from the multipass membrane-bound o-acyl transferases family (31) . besides these unusual fatty acid residues, attachment of palmitate to serine and threonine residues is found in secreted venom toxins of the spider plectreurys tristis, which selectively target neuronal ion channels (88) . also histone h4 is o-palmitoylated at a serine residue in the nucleus by acyl-coa:lysophosphatidylcholine acyltransferase (25) ( table 1 ). the latter is of special interest in the context of innate immune responses since histone h4 o-palmitoylation regulates transcriptional activity, which is the final outcome of the pro-inflammatory signaling pathways triggered by receptors of the innate immune system. special attention should be devoted to ε-n-acylation consisting in the attachment of a fatty acid residue to the side chain of lysine by amide linkage ( table 1) . ε-n-myristoylated are interleukin 1α (il-1α) and tumor necrosis factor α (tnfα), the pro-inflammatory cytokines crucial in combating bacterial infections (22) . the enzyme(s) catalyzing this reaction is unknown, but it has been established that sirtuins reverse this modification (89) . the ε-n-acylation affects the release of tnfα by immune cells (90, 91) . surprisingly, this rare modification is also found in toxins of so-called rtx (repeats-in-toxin) class released by some pathogenic gram-negative bacteria (23, 24) . we describe these cases in more detail in the following sections. besides s-palmitoylation and n-myristoylation, s-prenylation is another common lipidation that endows proteins with a hydrophobic moiety and contributes to their association with membranes. this modification relies on the posttranslational and irreversible attachment of either farnesyl or geranylgeranyl chains to a cysteine residue in the c-terminal caax box (alternatively also cc and cxc motifs) via a thioether linkage. the process is catalyzed by protein prenyl transferases that use polyprenylpyrophosphate as the donor of the isoprenoid group ( table 1) . in peripheral membrane proteins, the s-palmitoylation site is often located in proximity of n-myristoylation or s-prenylation sites or a polybasic motif, which all are likely to mediate initial weak binding of a protein to a membrane and thereby facilitate subsequent attachment of palmitate to the protein by the integral membrane zdhhc enzymes (31) . in contrast to s-palmitoylation, data on the role of s-prenylation of proteins key to the host-pathogen interactions are scarce (92) . however, since s-prenylation is typical for the ubiquitous small gtpases of ras superfamily, it is vital for proper functioning of b and t cells (93, 94) . a glance at table 1 indicates that palmitate can be covalently bound via oxyester, amide, and thioester linkages to respective amino acid residues creating an array of possible modifications. o-and n-palmitoylation of proteins seems to be stable, resembling in this regard the other common protein lipidations, n-myristoylation and s-prenylation. by contrast, there exist enzymes cleaving the thioester bond formed during s-palmitoylation. for a long time, our understanding of protein s-palmitoylation and its dynamics was poor in comparison with other reversible protein modifications due to technical difficulties. only recently have these difficulties been overcome with the introduction of methods allowing high-throughput identification of palmitoylated proteins, also those involved in the immune response to microbial pathogens, as discussed in the next sections. one of the basic problems hindering studies on protein s-palmitoylation lies in the fact that there is no identifiable consensus sequence for the palmitoylation site that could facilitate its prediction. from the technical point of view, the progress in a comprehensive survey of protein s-palmitoylation was also hampered by a lack of antibodies detecting this modification, with the sole exception of an antibody specific to palmitoylated psd-95 (95) . a classical method used to demonstrate protein palmitoylation is based on metabolic labeling of living cells with [ 3 h]-palmitic acid, subsequent immunoprecipitation of a selected protein and detection of the incorporated tritiated fatty acid by autoradiography (96) . a major disadvantage of this method is its low sensitivity. only a minute fraction of the radioactive palmitate is bound to proteins, the majority being incorporated into lipids, which requires lengthy film exposure (counting in days). a methodological breakthrough in the identification of palmitoylated proteins came with the development of two nonradioactive methods based on so-called click chemistry (97) (98) (99) and acyl-biotin exchange (abe) (74, 100) . these techniques have paved the way for high-throughput mass spectrometry-based proteomic analysis of protein palmitoylation in various cells and tissues and facilitated identification of new palmitoylated proteins of both pathogens and host cells involved in the innate immune responses. in the click chemistry-based method, cells are metabolically labeled with a palmitic acid analog bearing an alkyne group at the ω carbon of the fatty acyl chain, such as 17-octadecynoic acid (17odya) or alk-16 (figure 2a) , and this step resembles the classic labeling of cells with [ 3 h]-palmitic acid. however, in the click chemistry-based assay, the labeling and lysis of cells is followed by in vitro coupling of the function group of the palmitic acid analog to a reporter tag, which greatly enhances the sensitivity of detection of labeled proteins (98, 99) . thus, after cell lysis, the labeled proteins are subjected to cu (i) -catalyzed cycloaddition known as "click" reaction with an azide-bearing detection tag. in this step, a triazol is formed between the alkyne group in the palmitic acid analog and the azide of the tag (figure 2a) . the azide-bearing tags can be either fluorescent, such as tetramethylrhodamine or dyes with infrared fluorescence, or carry a biotin moiety. depending on the tag used, subsequent sds-page separation of proteins allows global visualization of palmitoylated proteins by simple in-gel fluorescence or by blotting with a streptavidin-conjugated reporter (98, 101, 102) . notably, proteins biotinylated via the click reaction can also be enriched on streptavidin-coated beads and then subjected to on-bead tryptic digestion (or in-gel digestion if eluted from the beads) followed by identification by mass spectrometry. such comprehensive click chemistry-based proteomic analysis has brought about identification of an array of palmitoylated proteins in dendritic cells (10, 103) , macrophage-like raw264 cells (14) , and t cells (99, 104, 105) . some of the s-palmitoylated proteins newly identified in those studies, such as ifitm3 and tlr2, are involved in the host-pathogen interactions regulating innate immune responses (10, 103) , while many others are known to contribute to adaptive immunity (99, 105) , as described below. recently, global profiling of toxoplasma gondii (the causative agent of toxoplasmosis) has been performed revealing that many components of the parasite's motility complex are palmitoylated (106) . similar studies on cryptococcus neoformans (the fungus causing cryptococcal meningitis) have revealed a contribution of specific zdhhc palmitoyl acyltransferase, called pfa4, to its virulence (107) . moreover, application of analogs of various saturated and unsaturated fatty acids confirmed the heterogeneous nature of the fatty acylation of proteins in raw264 cells and suggested that dietary unsaturated fatty acids, after incorporation to proteins, can change their properties and thereby affect the functioning of immune cells (14) . the major advantage of the click chemistry-based method is that it can reveal the time course of protein s-palmitoylation. by using click chemistry-based labeling in the pulse-chase mode, one can follow the dynamics of protein palmitoylation. with such an approach, it was found that the palmitate turnover on lck, an src-family tyrosine kinase, is accelerated by t cell activation (72) . additional introduction of stable isotope labeling by amino acids in cells (silac) has provided quantitative proteomic data , and after cell lysis, the click reaction is conducted with azido-tagged biotin or fluorescent probes allowing enrichment and detection of labeled proteins in various ways. biotinylated proteins can be bound on a streptavidin resin and then released using, e.g., high concentrations of urea and sds (108) . when a cleavable derivative of biotin, azido-azo-biotin, is used the labeled proteins are eluted from streptavidin beads with sodium dithionite, which cleaves the diazobenzene moiety in the linker arm of azido-azo-biotin, and analyzed by mass spectrometry or immunoblotting (109) . (b) abe method. cells or tissues are lysed, free thiol groups of proteins are blocked by alkylation, and palmitoyl moieties are released with hydroxylamine. the newly exposed protein thiol groups are subjected to labeling with biotin-hpdp allowing selective binding, elution, and analysis of the originally s-palmitoylated proteins. the proteins can also be captured without biotinylation through a direct interaction of their thiol residues with a thiol-reactive resin (acyl-rac technique). on the dynamics of protein palmitoylation in the cell (104, 110) . this approach revealed, rather unexpectedly, that in unstimulated t cell hybridoma, the palmitoylation of most protein species does not undergo turnover (104) . another advantage of the click chemistry-based assay is its high specificity, because the alkyne group introduced in the analog of palmitic acid is not normally found in cells (98, 102) . the click chemistry-based methods can also be used to follow the cellular localization of palmitoylated proteins by immunofluorescence when combined with the proximity ligation technique (111, 112) . palmitoylation of individual proteins can also be studied after their immunoprecipitation (11, 72, 73, 98 ). despite its unquestionable success, the click chemistry-based methods have limitations. they will detect only those proteins that undergo palmitoylation during the period of the metabolic labeling of cells. one should also bear in mind that the palmitic acid analog can be incorporated at s-, n-, and o-palmitoylation sites alike (111, 112) . in addition, although 17odya (alk-16) is preferentially used to mimic palmitoylation of proteins, it can also be incorporated with low efficiency at n-myristoylation sites of proteins (98, 99) . another group of proteins that will be labeled with the palmitic acid analog but are not s-palmitoylated are those bearing the glycosylphosphatidylinositol (gpi) anchor (85, 113) . most of these limitations can be overcome using various fatty acid reporters, inhibitors, and by exploiting the sensitivity of the thioester bond to hydroxylamine treatment. given the large variety of chemical reporters preferentially mimicking distinct fatty acids, recent years have witnessed a plethora of chemistry-based proteomic studies not only on palmitoylated but also myristoylated proteins and proteins bearing the gpi anchor, including those of pathogens and immune cells (10, 14, 85, 86, 114) the abe method reveals protein the abe method can be used as a complement to the click chemistry-based approach in cell studies but unlike the latter it is uniquely suitable for studying whole tissues. abe does not require metabolic labeling of proteins in living cells, thus some of the abovementioned limitations and difficulties do not apply. the abe method relies on in vitro exchange of thioester-linked palmitate to a derivative of biotin which allows subsequent affinity purification of the resulting biotin-labeled proteins on streptavidin-coated beads ( figure 2b) . the first step of the abe involves lysis of cells or tissues followed by irreversible blockage of free thiol groups in the solubilized proteins by alkylation, most often with n-ethylmaleimide. subsequently, the thioester bonds existing in s-palmitoylated proteins are broken with hydroxylamine, releasing palmitoyl moieties. the newly exposed thiol groups can now be tagged with sulfhydryl-reactive derivatives, such as biotin-hpdp, forming disulfide bonds with thiols. the biotinylated proteins are subsequently captured on streptavidin-coated beads and eluted with agents that reduce the disulfide bond between the protein and biotin-hpdp, such as β-mercapthoethanol, dtt, or tcep (49, 74, 115, 116) . as an alternative to biotinylation, in the so-called acyl-rac technique, the newly exposed protein thiol groups in hydroxylamine-treated cell lysates are captured on a resin containing sulfhydryl-reactive groups (117) . in both abe and acyl-rac, the eluted proteins can be separated by sds-page and visualized by gel staining or immunoblotting, or identified by mass spectrometry. furthermore, when the hydroxylaminereleased palmitoyl moieties are exchanged for a polyethylene glycol-maleimide derivative of a distinct molecular weight, a shift in-gel migration of tagged proteins is observed reflecting the number of fatty acyl residues originally s-bound to the protein (118, 119) . the abe method has so far been used successfully for proteomic profiling of s-acylated proteins in immune cells, such as raw264 cells (48), several types of blood cells, such as platelets, primary t cells, and immortalized b cells (120) (121) (122) , pathogenic microorganisms such as t. brucei and t. gondii (123, 124) , and tissues (125, 126) . to quantify the aberrations in protein palmitoylation in a mouse model of huntington's disease, whole animal stable isotope labeling of mammals (silam) was applied followed by tissue isolation and abe procedure (127) . in another approach, for quantitative analysis of the t-cell palmitoylome, abe was combined with labeling of proteins with various oxygen isotopes during their digestion with trypsin before mass spectrometry analysis (122) . in addition, preselection of tryptic peptides obtained by abe on streptavidin-coated or sulfhydrylreactive resins greatly facilitates the identification of s-acylation sites by mass spectrometry (49, 110, 117) . some aspects of the abe method deserve a comment. since the assay relies on the sensitivity of thioester bonds to hydroxylamine, abe detects all s-acylation without distinguishing between s-palmitoylation and the other cases. furthermore, there is a possibility of false-positive detection of proteins bearing a thioester linkage with compounds other than fatty acyl residues, such as ubiquitin in the e2 ubiquitin conjugase ubc1 (115) . another source of false-positives is proteins in which free thiol groups were not completely alkylated before biotinylation. on the other hand, insufficient deacylation of bonafide fatty-acylated proteins with hydroxylamine results in their absence in the final sample (116) . in summary, the click chemistry-based method relies on metabolic labeling of cells with a palmitic acid analog which incorporates into proteins and next tagging it with reporter molecules greatly enhancing the sensitivity of detection. it only reveals proteins undergoing s-palmitoylation during metabolic labeling of cells and allows revealing turnover of this modification. by contrast, the abe method is based on direct binding of sulfhydryl-reactive derivatives to thiol groups of cysteines unraveled by hydroxylamine treatment after lysis of cells or tissues. it allows the investigation of the whole but static palmitoylome. a comparative proteomic study of protein palmitoylation in p. falciparum found that the sets of proteins identified using these two approaches overlapped in 57.2% (113) , indicating that they provide complementary data on the cellular palmitoyl proteome. thanks to the application of the click chemistry-and abe-based methods numerous new palmitoylated proteins have been identified. in 2015, a swisspalm database was launched, (128) which provides an excellent, manually curated resource of information on palmitoylated proteins, palmitoylation sites, etc., available at http://swisspalm.epfl.ch/. all these efforts have greatly furthered our knowledge on molecular mechanisms regulating diverse aspects of cell functioning, including host-pathogen interactions and progress of infectious diseases, as highlighted below. bacteria lack protein palmitoyl acyltransferases of the zdhhc family and, therefore, are essentially devoid of s-palmitoylated proteins. yet, they have developed unique mechanisms utilizing fatty acids, such as palmitic acid, to modify their glycolipids and proteins. these modifications augment infectivity and help bacteria evade recognition by the host innate immune system. for example, the vast majority of gram-negative bacteria produce lipopolysaccharide (lps) as a part of their outer membrane. lps is composed of the variable polysaccharide o-antigen and more-conserved lipid a containing two glucosamine residues hexa-acylated with hydroxymyristic, myristic, and lauric acid. lipid a is recognized by cd14 protein and tlr4 receptor complexed with md2 protein on the plasma membrane of the host immune and some non-immune cells. activation of tlr4 triggers strong pro-inflammatory reactions aiming at eradication of the bacteria, but when exaggerated, eventually leading to sepsis (129) . incorporation of an additional palmitoyl chain into lipid a markedly diminishes its ability to activate tlr4 and to induce the host pro-inflammatory responses, which is correlated with an increased survival of bacteria forming a biofilm (130, 131) . this strategy is utilized among others by salmonella typhimurium, a causative agent of gastroenteritis, by bordetella bronchiseptica, a respiratory pathogen of human and other mammals, and by protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january 2018 | volume 8 | article 2003 yersinia pestis causing plague (132, 133) . the formation of the extra-acylated lps relies on the transfer of palmitate from phospholipids onto the hydroxymyristate chain at position 2 of glucosamine of lipid a. the reaction is catalyzed by lipid a palmitoyltransferases (pagp in salmonella and its homologs in other bacteria) localized in the outer membrane of these pathogens (134, 135) . in addition to causing steric hindrance preventing the binding to the tlr4/md2 complex, the hepta-acylation of lps also protects bacteria from the lytic activity of cationic antimicrobial peptides, most likely by reducing the fluidity of the bacterial outer membrane (136, 137) . apart from being incorporated into lps in diverse bacteria, palmitate has also been found to modify a virulence factor of gram-negative erwinia carotovora, the evf protein. the palmitoyl chain is linked via a thioester bond to the cys209 residue at the center of evf, plausibly by a self-palmitoylating activity of the protein. e. carotovora is a phytopatogen using insects such as drosophila as vectors for dissemination between plants. the palmitoylation of evf is required for infectivity of e. carotovora and its persistence in the insect gut, however, its mode of action of unknown. it has been speculated to be linked with an ability of evf to associate with lipid bilayers, but the lack of similarities between evf and any other bacterial protein of known function makes prediction on this subject difficult (79) . a number of bacterial toxins of so-called rtx class released during infection of mammals by pathogenic gram-negative bacteria undergo ε-n-acylation of the side chain of internal lysines. these toxins include adenylate cyclase of bordetella pertussis, acylated with palmitic acid, and α-hemolysin of extraintestinal (uropathogenic) escherichia coli, acylated with myristic acid and also 15-and 17-carbon fatty acids. the acylation is catalyzed by an endogenous bacterial acyltransferase which, unlike its eukaryotic counterparts, transfers the acyl chain not from acyl-coa but from acyl-carrier protein. the acylated toxins secreted by the bacteria bind to the plasma membrane of the host cells, oligomerize and form pores causing cell lysis. in the case of the toxin of b. pertussis, essential is also the delivery of the adenylate cyclase moiety to the cell interior. acylation is required for virulence possibly being involved in oligomerization of the toxins (23, 24, 138) . although lacking s-palmitoylated proteins (with the single known exception of evf), bacteria express a wide range of membrane-bound proteins modified by a complex lipidation at the n-terminus, with palmitate frequently being a component of the lipid moiety (139, 140) . the bacterial lipoproteins are synthesized in a multistep process catalyzed by a unique set of lipoprotein processing enzymes, lgt, lspa, and lnt, absent in eukaryotic cells. the formation of these lipoproteins begins with the attachment of a diacylglycerol via a thioester bond to a cysteine residue located in the so-called lipobox motif of the signal sequence of the transmembrane lipoprotein precursor. the signal sequence is then cleaved next to the lipid-modified cysteine leaving it at the n-terminus of the mature protein (141) . in gramnegative and less frequently also gram-positive bacteria, a third fatty acid residue is additionally attached via an amide linkage to the amino group of the cysteine in a reaction analogous to the n-acylation of hedgehog proteins (see table 1 ). this di-and tri-lipidation ensures membrane anchoring of the lipoproteins. all such lipoproteins of gram-positive bacteria are exposed to the milieu while in gram-negative bacteria some face the periplasm. the lipoproteins of gram-positive bacteria, e.g., streptococcus pneumoniae (causing pneumonia), mycobacterium tuberculosis (tuberculosis), and gram-negative bacteria, such as neisseria meningitidis (meningitis), y. pestis (plague), the spirochaete borrelia burgdorferi (lyme disease) and treponema pallidum (syphilis) are crucial for their virulence. they control several aspects of the host-pathogen interactions, like adhesion and entry to host cells, protection against proteolysis and oxidative stress in the host cell, and regulation of expression of genes encoding cytokines both during initiation and progress of the disease (140) (141) (142) . the surface exposure of the lipoproteins allows their involvement in the host cell invasion while on the other hand forming the so-called pattern signal recognized by the tlr2 receptor, which triggers the pro-inflammatory responses helping to combat the bacteria (143) . of interest, tlr2 is s-palmitoylated, as discussed below. the involvement of lipoproteins in pathogenesis fuels studies on their properties. one such recent work employing click chemistry to profile the lipoproteins of e. coli identified 88 lipoproteins with high/medium confidence, 70% of them predicted before by bioinformatics analysis (144) . notably, in that study a 14-carbon alkynyl fatty acid analog alk-14 rather than alk-16 was preferentially incorporated into the lipoproteins, contradicting earlier studies using gas chromatography and tlc, which found that palmitate was predominantly used for bacterial protein modification (139) . further studies are required to establish whether the fatty acid found in lipoproteins varies depending on culture conditions or is species specific. for example, 17odya labeling for click reaction confirmed incorporation of palmitate into pallilysin (tp0751), a lipoprotein of t. pallidum. pallilysin is a metalloprotease that degrades human fibrinogen and laminin. it is suggested that its exposure on the bacteria surface enables degradation of host structural proteins to facilitate rapid dissemination of this highly invasive pathogen (140) . bacteria occasionally high-jack the palmitoylation machinery of host cells to modify the environment so as to favor their internalization, survival, and replication inside the cells. bacillus anthracis (the causative agent of anthrax) is an example of such bacteria that modify s-palmitoylation of host proteins to their ends. the anthrax toxin produced by this pathogen binds to the tem8 and cmg2 (capillary morphogenesis protein-2) proteins which, under physiological conditions, are involved in cell-cell and cell-extracellular matrix interactions. they are s-palmitoylated at multiple (two to four) cysteines (54) . the s-palmitoylation of tem8 was found to inhibit its association with plasma membrane rafts preventing its ubiquitination by the raft-associated e3 ubiquitin ligase cbl. the binding of anthrax toxin drives association of the receptor-toxin complexes with rafts possibly correlated with depalmitoylation of the receptor. this allows subsequent ubiquitination of the receptor, an uptake of the receptor/toxin complexes in a clathrin-dependent manner and eventual delivery of the toxin to endosomes. these events are facilitated by s-palmitoylation of partner(s) of the receptors, most likely including kinases of the src family (54, 145, 146) . while b. anthracis utilizes palmitoylated host proteins to induce its internalization, a growing body of data suggests that protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january 2018 | volume 8 | article 2003 also bacterial proteins can undergo s-palmitoylation inside the host cells. this type of modification concerns so-called effectors, bacterial proteins that are injected into the host cell cytoplasm either across the plasma membrane or the membrane of vesicles enclosing internalized pathogens, with the help of their secretion systems. these are secretion systems type iii and type iv, homologs of which have been described for pathogens and symbionts of mammals, insects, and plants (147, 148) . the bacterial effectors can be s-palmitoylated to reach host cell membranes and thereby accumulate at a location most suitable for their activity. application of the click chemistry-based method utilizing an analog of palmitic acid (alk-16) for cell labeling has revealed s-palmitoylation of two effector proteins of salmonella enterica, such as ssph2 and ssei (149) . s. enterica invades gut endothelial cells and is a leading cause of gastroenteritis and typhoid fever. ssph2 carries an e3 ubiquitin ligase domain while ssei shows sequence homology to bacterial proteins that have a deamidase activity, and inhibits migration of salmonella-infected cells. the latter activity requires s-palmitoylation of ssei. both proteins are stably s-palmitoylated, most likely by zdhh3 and zdhh7 of the host and bind to the plasma membrane in a palmitoylationdependent manner (149) . also two effector proteins of the ipah family of shigella spp. were found to be s-palmitoylated in that study, suggesting that this modification can control the activity of effector proteins of other pathogens as well (149) . indeed, gobx and lpda, effector proteins of legionella pneumophila, the causative agent of legionnaires' disease invading macrophages and lung endothelial cells, are s-palmitoylated as was found recently using click chemistry. lpda is a phospholipase hydrolyzing various phosphatidylinositols while gobx is an e3 ubiquitin ligase. gobx is targeted in a palmitoylation-dependent manner to the golgi apparatus, and lpda to the plasma membrane and a subset of intracellular vesicles (150, 151) . thus, the diversified subcellular localization of bacterial effector proteins reflects that of eukaryotic proteins. it is worth noting that global profiling of acylated proteins with the application of click chemistry and an alkyne-functionalized analog of myristic acid, alk-14, for cell labeling was effective in reveling the mechanism of action of shigella flexneri effector protein ipaj of type iii secretion system. this is a unique protease that cleaves off the n-terminal myristoylated glycine. this proteolytic demyristoylation activity of ipaj is specific toward golgi-associated arf/arl family of gtpases regulating cargo transport through the golgi apparatus, inhibition of which is apparently pivotal for virulence of the bacteria causing diarrhea in humans (152) . in addition to the s-palmitoylation of the effectors of pathogenic bacteria of mammals mentioned earlier, double acylation, n-myristoylation and s-palmitoylation, has been reported of the so-called avirulence (avr) proteins (effectors of type iii secretion system) of pseudomonas syringae, a causative agent of diverse plant diseases. among them, avrrpm1 and arvb are n-myristoylated and s-palmitoylated by host acyltransferases at neighboring glycine and cysteine residues localized at the n-terminus of the proteins (similarly to eukaryotic kinases of the src family), while in avrpphb and two avrpphb-like effectors-orf4 and nopt, the double acylation motif is exposed after auto-cleavage of the proteins (similarly to some eukaryotic proteins cleaved by caspases). the acylation of the avr proteins ensures their anchoring in the host plasma membrane, which is required for their functioning. in disease-susceptible plants avr proteins contribute to successful infection; however, in plants expressing host resistance (r) genes they trigger plant defense signals, in both cases engaging plasma membrane-associated host proteins (153, 154) . the importance of palmitoylation of bacterial effector proteins for their infectivity is only beginning to be uncovered, in no small part owing to the development of the click chemistry-based method for detection of this protein modification. however, the strategy of high-jacking the host palmitoylation machinery to modify own proteins seems to be much more commonly employed by viruses. viruses do not encode palmitoyl acyltransferases but exploit extensively the host palmitoylation machinery to modify their proteins essential for infection of host cells and own replication. in fact, s-palmitoylation of proteins was discovered in 1979 as a modification of envelope glycoproteins of sindbis virus and vsv. in those studies [ 3 h]-palmitic acid was used for metabolic labeling of virus-infected cells and labeled proteins were identified by autoradiography (12, 155) . subsequently, a number of other viral proteins have been found to be palmitoylated using this approach. the most-studied group of viral palmitoylated proteins is those found in enveloped viruses, i.e., viruses covered by a lipid bilayer obtained during their replication from a membrane of the host cell, such as the plasma membrane or endoplasmic reticulum. influenza virus, hiv-1, hepatitis c virus (hcv), and herpes simplex virus (hsv) are the best known enveloped viruses. the envelope is rich in transmembrane, often s-palmitoylated, glycoproteins called spikes, which can bind to cognate receptors on the host cell plasma membrane triggering endocytosis of the virion, mediate subsequent fusion of the viral and cellular membranes allowing entry of the viral genome to the cytoplasm, and are also involved in the budding of newly formed virus particles from the cell. an example of such multifunctional palmitoylated transmembrane glycoproteins is ha present in the envelope of influenza virus together with another palmitoylated transmembrane protein, the matrix protein m2, which forms a proton channel earning the protein the name viroporin. as mentioned earlier, ha of influenza a virus is s-stearoylated and s-palmitoylated, respectively, at one cysteine residue located in the transmembrane domain of ha and two cysteines found in the cytoplasmic (intraviral) tail in close proximity to the membrane (156) . on the other hand, m2 is s-palmitoylated on the amphiphilic helix located in the cytoplasmic part of the protein. due to the s-palmitoylation and the presence of a cholesterol-binding motif the helix bends toward and associates with membranes (157, 158) . during infection, ha binds to sialic acid residues of glycans localized on the surface of airway and alveolar epithelial cells. the bound virions are endocytosed and next the viral and endosome membranes fuse. the membrane fusion is driven by ha, which undergoes conformational changes induced by low ph of endosomes. acidification of endosomes activates also the m2 proton channel activity, protons entering viral core facilitate dissociation of the viral genome which then moves to the nucleus where rna replication occurs. the s-palmitoylation of ha is required for the fusion of the viral and endosome membranes at least in some subtypes of the virus while the ion channel activity of m2 is not dependent on its s-palmitoylation (159) . newly synthesized viral proteins and rna are assembled into virions in the plasma membrane rafts which merge into lager platforms crucial for the virion assembly and budding off. the triple fatty acylation of ha is required for its targeting to plasma membrane rafts (160, 161) . besides s-palmitoylation, also the amino acid sequence of the transmembrane domain of ha determines its association with rafts (45) . on the other hand, among the amino acids of the cytoplasmic tail of ha no other than the two s-palmitoylated cysteines are required for viral assembly and replication, although it is still not clear whether raft targeting (in cooperation with the transmembrane fragment) is the only mechanism of their participation. it is proposed that they affect conformation of the ha tail controlling its interaction with structural matrix protein m1 lying beneath the viral envelope (162, 163) . the budding off of the virion is facilitated by m2 which localizes at the edges of rafts as a result of a combination of its s-palmitoylation, cholesterol binding, and properties of the transmembrane fragment. m2 protein can create a "wedge" altering membrane curvature thereby facilitating membrane scission and release of the virion (157, 164) . the influenza virus s-palmitoylated proteins are the archetype for many other viral proteins. thus, s-palmitoylated spike glycoproteins include s-protein of coronaviruses (e.g., severe acute respiratory syndrome virus), the fusion (f) protein of paramyxoviruses (e.g., measles virus), env of retroviruses [e.g., hiv-1, feline immunodeficiency virus (fiv)], and filoviruses (e.g., ebola). other viral proteins modified with palmitate are viroporins, such as e protein of coronaviruses, and also peripheral membrane proteins or nucleocapsid proteins absent in influenza virus. it has been found that s-palmitoylation of f13l, a peripheral protein of the envelope of vaccinia virus, controls the association of the protein with intracellular membranes, thereby the formation of the envelope (165) . the core protein of the nucleocapsid of hcv resides on the surface of lipid droplets and binds in a palmitoylation-dependent manner to membranes of the droplet-associated endoplasmic reticulum. subsequently, it recruits viral proteins and newly synthesized rna for viral particle formation (166) . besides the interest in the role of viral protein s-palmitoylation for infectivity and possible use of host zdhhc enzymes as targets of anti-influenza drugs (167) , viral proteins often serve as a model to study the consequences of fatty acylation for protein functioning and localization in distinct membrane domains (see s-palmitoylation of proteins and its influence on protein localization, trafficking, and stability of this review). readers are referred to recent exhaustive reviews that consider these topics (36, 84, 168) while we will focus here on the recent advances in the field of viral protein palmitoylation brought about mainly by proteomic studies. the click chemistry-based approach has led to the identification of s-palmitoylation in the cytoplasmic domain of the transmembrane spike protein env of fiv, considered to be the cat equivalent of hiv-1. env comprises three transmembrane gp41 glycoproteins and three associated gp120 which bind to cd4 receptor and coreceptors on the surface of t lymphocytes allowing fusion of the viral envelope and the plasma membrane and entry of viral capsid. four cysteines in fiv env are s-palmitoylated vis-a-vis two found in the env of hiv-1. the two most membrane-proximal cysteines, 804 and 811, are required for the fiv membrane-fusion activity and incorporation of env into virions (169) , in agreement with the importance of env s-palmitoylation for virion assembly of some hiv-1 strains (170) (171) (172) . the assembly of hiv-1 virions takes place in plasma membrane rafts and is driven by n-myristoylated gag protein which anchors and oligomerizes preferentially in these plasma membrane domains due to the presence of the fatty acyl chain (18) . the development of click chemistry-based methods allowed for the first time global profiling of acylated proteins in virusinfected cells. in addition to identifying acylated viral proteins this approach has also revealed how the viral infection modulates the acylation pattern of the host cell proteins. thus far, click chemistry has been used to study protein myristoylation and palmitoylation in cells infected with hiv-1 and with hsv. in the latter case, the standard metabolic labeling with alkynefunctionalized myristic and palmitic acid analogs followed by click chemistry and mass spectrometry was combined with silac to discern between the changes in the extent of protein acylation and those in their abundance following viral infection. this approach allowed an elaborate quantitative analysis of host protein acylation and has revealed an overall downregulation of the level of both host protein modifications in infected cells. while the decreased content of myristoylated proteins resulted mainly from suppression of host protein synthesis, the drop in several s-palmitoylated proteins ensued from the inhibition of their palmitoylation in infected cells. the affected proteins were localized mainly to the plasma membrane and the golgi apparatus and were involved in vesicle-mediated transport and ion transport. in addition, the study has expanded the list of hsv-encoded acylated (mostly palmitoylated) proteins that play different functions in the viral cycle, such as ge, gi, gk, us2, and us3 (110) . similar results pointing to global changes of host protein acylation were obtained upon analysis of protein myristoylation and palmitoylation in cells infected with hiv-1. in that study, the cells were labeled with analogs of palmitic or myristic acid tagged with an azide moiety for click chemistry reaction; however, the following mass spectrometry analysis did not address the relation between changes of protein acylation vs. alteration of protein level. the study identified 17 palmitoylated and 7 myristoylated proteins significantly differing in abundance between hiv-1 infected and uninfected cells. several of the proteins affected by the infection were of host origin. the abundance of myristoylated proteins was in general increased while that of the palmitoylated ones-decreased in infected cells (173) . in other words, the two studies have revealed that hsv and hiv-1 not only encode proteins that are acylated in the host cell but protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january 2018 | volume 8 | article 2003 also alter the palmitoylation of host proteins, likely to adapt the cellular environment to favor their replication and budding. the majority of the acylated proteins affected by hiv-1 or hsv infection had not been described earlier in this context; therefore, further studies on these proteins could be crucial for better understanding of viral infection. thus, the click chemistry-based approach has been highly effective in revealing changes of the host protein palmitoylation and opening new possibilities for the identification of novel antiviral drug targets. the innate immune responses are the first line of active defense against microbial infections. the application of click chemistrybased and abe methods and their use for large-scale analysis of protein palmitoylation in murine dendritic cd2.4 cells (10, 103) , and murine macrophage-like raw264 cells (14, 48) complemented by proteomic analysis of the raft fraction of those cells (47) have contributed significantly to the understanding of the role of palmitoylation of host receptors and signaling proteins involved in innate immune responses. thus, the palmitoyl proteome analysis of murine dendritic cells unraveled s-palmitoylation of tlr2, a receptor expressed in cells of myeloidal lineage, which heterodimerizes with tlr1 or tlr6 to bind bacterial tri-or diacylated lipoproteins, respectively, and also other microbial components, such as glycolipids (e.g., lipoarabinomannan) of mycobacterium and yeast zymosan (174) . besides tlr2, two other human tlrs out of 10 ectopically expressed in hek293 cell, flagellin receptor tlr5, and tlr10, a unique tlr negatively regulating the pro-inflammatory activity of tlr2, were also found to be palmitoylated. the s-palmitoylation site of human tlr2 was mapped to cys609 adjacent to its transmembrane domain. the modification was present in unstimulated cells and was linked with up-regulation of the cell surface localization of tlr2. mutation of cys609 abolished the ability of the receptor to induce pro-inflammatory signaling in response to microbial ligands of tlr2 (10) . further studies are needed to reveal whether s-palmitoylation of tlr2 controls its association with rafts as sites of tlr2 activation (175) and/or affects endocytosis of the receptor, as found for the anthrax toxin receptor (54) . one of the most extensively studied tlrs, tlr4 activated by bacterial lps, is not palmitoylated. yet, saturated fatty acids have been indicated to trigger pro-inflammatory signaling of tlr4. thus, the tlr4/md2 receptor complex is involved in the pro-inflammatory outcome of a diet rich in palmitic acid, as was found when analyzing markers of inflammation in the heart and adipose tissue of high fat diet-fed mice (176, 177) . the molecular mechanisms underlying the pro-inflammatory properties of palmitic acid can involve its influence on the plasma membrane lipid order, hence raft organization, in a way that facilitates translocation of tlr4 (and tlr2) toward rafts (178, 179) . palmitic acid also directly binds to the tlr4-associated md2 protein (177, 180) . an influence of palmitic acid on sphingomyelin/ceramide metabolism, which enhances the lps-induced responses, has also been considered (181) . recent proteomic studies based on 17odya labeling of raw264 macrophage-like cells followed by click chemistry have revealed that stimulation of cells with lps induces profound changes of the abundance of palmitoylated proteins (182) . the data are in agreement with earlier findings showing that lps induces accumulation of s-palmitoylated lyn kinase in the raft-enriched fraction of cells, allowing it to downregulate tlr4 signaling (11) . one of the upregulated s-palmitoylated proteins was type ii phosphatidylinositol 4-kinase iiβ, which phosphorylates phosphatidylinositol to phosphatidylinositol 4-monophosphate. it was shown that palmitoylation determines the involvement of the kinase in lps-induced signaling (182) . these data suggest that s-palmitoylated proteins, including enzymes catalyzing phosphatidylinositol synthesis and turnover, are important factors affecting the pro-inflammatory responses triggered by lps. notably lps induces production of tnfα, a pro-inflammatory cytokine that is s-palmitoylated itself. tnfα is synthesized as a transmembrane 27-kda precursor (tmtnfα) transported from the endoplasmic reticulum to the plasma membrane through the golgi apparatus and recycling endosomes (183) . human tmtnfα is s-palmitoylated at cys30 located at the boundary between its transmembrane and cytosolic fragments, as was found independently by radiolabeling and by labeling with 17odya followed by click chemistry (184, 185) . poggi et al. (185) arrived at a complex model explaining how the s-palmitoylation of tnfα affects its activity ( figure 3a) . the modification was shown to favor the association of tmtnfα with rafts. upon cell activation, the extracellular domain of tmtnf is cleaved by adam17 metalloproteinase whereupon the soluble tnfα (stnfα) is released to the extracellular milieu and activates tnf receptor (tnfr) 1 and tnfr2. as adam17 localizes to both non-raft and raft regions of the plasma membrane, the s-palmitoylation of tmtnfα does not affect its cleavage and production of the soluble cytokine. however, s-palmitoylated tmtnfα interacts with tnfr1 in rafts thereby reducing the binding of stnfα and consequently reducing the sensitivity of the cell to this cytokine. in addition, the fragment of tmtnfα which remains after the release of stnfα in rafts if further processed by intramembrane sppl2a and 2b proteases giving rise to icd (intracellular domain) of an own biological activity. by contrast, the non-raft fragment of the adam17-cleaved tmtnfα is rapidly degraded (185) . the transport and maturation of tnfα are also regulated by another posttranslational acylation, ε-n-myristoylation (22) . as shown in figure 3b , myristic acid residues are attached to two lysines (lys19 and 20) of human tmtnfα. this modification is reversed by sirtuin 6 catalyzing the demyristoylation. depletion of sirtuin 6 decreases the release of stnfα since the ε-n-acylated tnfα precursor is redirected to and accumulates in lysosomes (90, 91) . it is worth noting that exogenous palmitic acid stimulates the ε-n-myristoylation of tmtnfα, thereby reducing the release of stnfα in favor of accumulation of tmtnfα in lysosomes (90, 91) . this somehow surprising anti-inflammatory effect of palmitic acid can be explained by competitive binding between long-chain fatty acids (in this case, palmitic) and myristoylated substrates of sirtuin 6 found in vitro(89) and adds a new dimension to the potential effects of palmitic acid. (a) non-palmitoylated tmtnfα is localized outside rafts while that s-palmitoylated on cys30-in rafts of the plasma membrane. tmtnfα is cleaved by adam17 protease in both these plasma membrane environments giving rise to stnfα, which subsequently activates tnf receptor (tnfr) 1 receptor leading to activation of nfκb and erk1/2. however, only the raft-residing tmtnfα is further processed by sppl2b protease to yield icd, which activates the promoter of interleukin (il)-1β and expression of il-12. on the other hand, a pool of s-palmitoylated tmtnfα interacts in rafts with tnfr1 preventing its activation by stnfα. (b) tmtnfα is transported from the endoplasmic reticulum via golgi apparatus and recycling endosomes [1, 2] to the plasma membrane [3] . in the plasma membrane, tnfα is cleaved by adam17 giving rise to stnfα [4] or is internalized [5] and either returns from the endosomes to the plasma membrane [6, 3] or is directed to lysosomes for degradation [7] . ε-n-myristoylation of tmtnfα at lys19 and lys20 facilitates its degradation [5, 7] at the expense of processing to stnfα [4] . oligomerization of tmtnfα and tnfr1 is not shown. s-palmitoylation of host proteins is also vital in antiviral defense. viral nucleic acids, which are recognized by several tlrs and also cytoplasmic pattern-recognition receptors, induce robust production of type i interferons (ifns), mainly infα and ifnβ. the ifnα and ifnβ released from cells which first encounter viruses, e.g., dendritic cells, induce an antiviral reaction in an autocrine and paracrine manner upon binding to plasma membrane ifnα/β receptor (ifnar) consisting of subunits 1 and 2. both human ifnar subunits are s-palmitoylated, as has been found by classical radiolabeling. the s-palmitoylation of ifnar1 on cys463, localized near the cytoplasmic end of the transmembrane domain, is required for downstream activation of stat1 and stat2 and the following transcription of ifnα-activated genes (186) . among the ifn-induced proteins, some have been shown to be palmitoylated, using click chemistry and abe. they include the immunity-related gtpase irgm1, bst2 also known as tetherin, and ifitm1 and 3 (10, 104) . ifitms are potent restriction factors against a wide range of enveloped viruses, e.g., influenza, west nile, dengue, and zika viruses (187, 188) . ifitms localize primarily to endolysosomal membranes where they inhibit viral replication by blocking their fusion with these membranes and also facilitate virus degradation (187) . the exact mechanism of this antiviral activity is not clear, but it seems to rely on a perturbation of the organization of endolysosomal membranes. this can be linked with the intramembrane topology of ifitms and their s-palmitoylation. ifitm1 and 3 likely possess two loops embedded in but not spanning the membrane with both the n-and c-termini facing the cytoplasm (55, 189) . s-palmitoylation of conserved cysteine residues adjacent to these loops, cys71, 72, and 105 in murine ifitm3, contributes to the membrane binding, similarly as found earlier for caveolins (119, 189) . the s-palmitoylation also facilitates clustering of ifitm3 in the membranes, which is of potential significance for its antiviral activity (103) . in support of the latter, the antiviral capacity was markedly reduced for non-palmitoylated mutant forms of ifitm3 (103, 119) . however, s-palmitoylation did not affect the endolysosomal localization or stability of ifitm3. subsequent studies have revealed that the localization and degradation of murine ifitm3, both shaping its antiviral capacity, are orchestrated by numerous posttranslational modifications comprising polyubiquitination, tyrosine phosphorylation by the src-family kinase fyn, and methylation (189, 190) . by contrast, s-palmitoylation alone of the closely related murine ifitm1 endowed it with an antiviral activity and enhanced stability by preventing proteasomal degradation (55) , which indicates diverse effects of this modification on individual ifitm isoforms. the presented data are only beginning to fill the gap which existed in our understanding of the role of protein palmitoylation in innate immune responses. for a long time, it was lagging behind that on acquired immune responses, in which a plethora of s-palmitoylated proteins have long been known to be involved. they include receptors (cd4 and cd8), tyrosine kinases of the src family, transmembrane adaptor proteins (e.g., lat, ntal, and pag/cbp), and α subunits of heterotrimeric g proteins. their s-palmitoylation in most cases targets them to rafts and is a prerequisite for their involvement in the signaling pathways triggered by immunoreceptors [tcr, b cell receptor (bcr), and fcγ and fcε receptors] crucial for the acquired immune responses. an association of some components of these signaling pathways with tetraspanin-enriched domains has also been considered. these topics are discussed in several earlier reviews (44, 79, 191, 192) . it is worth noting that large-scale proteomic analyses of fatty-acylated proteins of t cells (99, 104, 105, 122) and b cells (121) , identifying numerous new palmitoylated proteins, have been published recently. further studies will shed light on the possible engagement of those proteins in acquired immune responses and/or in the cross talk between the innate and the acquired immune system, in which phagocytic cells, such as macrophages and dendritic cells, are essential (193) . protein s-palmitoylation affects their localization, trafficking, and stability. it has long been known as an important factor controlling signal transduction by the bcr and tcr receptors involved in acquired immune responses. it is now becoming evident that palmitic acid is also a key lipid affecting the diverse processes at the host-pathogen encounter. palmitate is a component of bacterial lps and lipoproteins; s-palmitoylation of viral, some bacterial, and numerous host proteins is recognized as a crucial factor affecting both the virulence of pathogens and the innate immune reactions of the host. our understanding of the latter has benefited greatly from the development of novel methods of detection of this protein modification. their application has led to the identification of numerous proteins involved in the host-pathogen interaction. the methods have also allowed highthroughput proteomic analysis of palmitoylation of proteins in infected cells, showing widespread changes of the host cell palmitoylome. future studies will tell whether complex feedback loops comprising palmitoyl acyltransferases and acylthioesterases, 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accessory protein md2 modification of pro-inflammatory signaling by dietary components: the plasma membrane as a target mechanisms for the activation of toll-like receptor 2/4 by saturated fatty acids and inhibition by docosahexaenoic acid palmitic acid is a toll-like receptor 4 ligand that induces human dendritic cell secretion of il-1β acid sphingomyelinase plays a key role in palmitic acid-amplified inflammatory signaling triggered by lipopolysaccharide at low concentrations in macrophages lps upregulates palmitoylated enzymes of the phosphatidylinositol cycle. an insight from proteomic studies cytokine secretion in macrophages and other cells: pathways and mediators transmembrane tnf (pro-tnf) is palmitoylated palmitoylation of tnf alpha is involved in the regulation of tnf receptor 1 signalling palmitoylation of interferon-α (ifn-α) receptor subunit ifnar1 is required for the activation of stat1 and stat2 by ifn-α ifitm-family proteins: the cell's first line of antiviral defense the ifitms inhibit zika virus replication s-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein 3 (ifitm3)-mediated resistance to influenza virus phosphorylation of the antiviral protein interferon-inducible transmembrane protein 3 (ifitm3) dually regulates its endocytosis and ubiquitination greasing their way: lipid modifications determine protein association with membrane rafts protein acylation and localization in t cell signaling (review) patterns, receptors, and signals: regulation of phagosome maturation key: cord-018963-2lia97db authors: xu, ying; liu, zhijie; cai, liming; xu, dong title: protein structure prediction by protein threading date: 2010-04-29 journal: computational methods for protein structure prediction and modeling doi: 10.1007/978-0-387-68825-1_1 sha: doc_id: 18963 cord_uid: 2lia97db the seminal work of bowie, lüthy, and eisenberg (bowie et al., 1991) on “the inverse protein folding problem” laid the foundation of protein structure prediction by protein threading. by using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. their follow-up work (elofsson et al., 1996; fischer and eisenberg, 1996; fischer et al., 1996a,b) and the work by jones, taylor, and thornton (jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term “protein threading.” these computational tools have played a key role in extending the utility of all the experimentally solved structures by x-ray crystallography and nuclear magnetic resonance (nmr), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now. the seminal work of bowie, liithy, and eisenberg (bowie et ai., 1991) on "the inverse protein folding problem" laid the foundation ofprotein structure prediction by protein threading. by using simple measures for fitness ofdifferent amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. their follow-up work (elofsson et ai., 1996; fischer and eisenberg, 1996; fischer et ai., 1996a,b) and the work by jones, taylor, and thornton (jones et ai., 1992) on protein fold recognition led to the development of a new brand ofpowerful tools for protein structure prediction, which we now term "protein threading." these computational tools have played a key role in extending the utility of all the experimentally solved structures by x-ray crystallography and nuclear magnetic resonance (nmr), providing structural models and functional predictions for many ofthe proteins encoded in the hundreds of genomes that have been sequenced up to now. what has made protein threading particularly attractive as a protein structure prediction tool is the observation that the number ofunique structural folds in nature is a few orders ofmagnitude smaller than the number ofproteins in nature (finkelstein andptitsyn, 1987; bairochetai., 2005) . although this is still not a fully resolved issue, both theoretical and statistical studies (murzin et ai., 1995; brenner et ai., 1996; li et ai., 1996 li et ai., , 1998 li et ai., ,2002 wang, 1996; orengo et ai., 1997; holm and sander, 1996a; zhang and delisi, 1998) suggest that the number ofunique structural folds in nature ranges from a few hundred to a few thousand. clearly this is a significantly smaller number than the number ofproteins in nature -as we understand now, the number of different living organisms on earth could range from millions to hundreds ofmillions (may, 1988) . since each organism often has at least thousands of different proteins encoded in its genome, the total number of different proteins in nature is at least in the tens ofbillions or possibly significantly higher, even without considering protein variants such as alternatively spliced proteins. this disparity suggests an effective paradigm for possibly solving all protein structures through combining experimental and computational approaches, that is, to solve structures of proteins with unique structural folds using the expensive and time-consuming experimental techniques and computationally model the rest ofthe proteins using the experimental structures as templates. this is the key strategy currently being employed by the worldwide structural genomics efforts (gaasterland, 1998; skolnick et ai., 2000; baker and sali, 2001) . the basic idea of protein threading is to place (align or thread) the amino acids of a query protein sequence, following their sequential order and allowing gaps, into structural positions of a template structure in an optimal way measured by fitness scores outlined above. this procedure will be repeated against a collection of previously solved protein structures for a given query protein. these sequencestructure alignments, i.e., the query sequence against different template structures, will be assessed using statistical or energetic measures for the overall likelihood of the query protein adopting each ofthe structural folds. the "best" sequence-structure alignment provides a prediction of the backbone atoms of the query protein, based on their placements in the template structure. currently, protein threading is being widely used in molecular biology and biochemistry labs, often for initial studies of target proteins, as it may quickly provide structural and functional information, which could be used to guide further experimental design and investigation. as a prediction technique, protein threading has a number of highly challenging computational and modeling problems. these include (a) how to effectively and accurately measure the fitness of a sequence placed in a template structure, (b) how to accurately and efficiently find the best alignment between a query sequence and a template structure based on a given set of fitness measures, (c) how to assess which sequence-structure alignment among the ones against different template structures represents a correct fold recognition and an accurate (backbone) structure prediction, and (d) how to identify which parts of a predicted structure are accurate and which parts are not. as researchers find more effective solutions to these and other challenging problems, we expect that protein threading will play an increasingly significant role in structural and functional studies of proteins. as of now, over a million protein sequences have been determined (bairoch et ai., 2005) , among which~30,000 have had their tertiary structures experimentally solved (dutta and berman, 2005) . given that there could be at least tens of billions of different proteins in nature as discussed above, one interesting question, particularly relevant to the idea of protein threading, is how many unique protein structures or structural folds these proteins might have adopted. to answer this question, we need to first look at the basic structural units of proteins, called protein domains (wetlaufer, 1973; richardson, 1981) . protein domain is extensively discussed in chapter 4 of this book. here we describe briefly the concept of a domain from the perspective of threading. a structural domain is a distinct and compact structural unit that could fold independently of other domains. while many proteins are single-domain proteins, there are proteins with two, three, or even more structural domains (ekman et ai., 2005) . our study shows that in the fssp (fold classification based on structure-structure alignment of proteins) nonredundant set (holm and sander, 1996b) of the pdb, 67% of the proteins have single domains, 21% have two domains, 7% have three domains, and the remaining 5% have four or more domains (xu et ai., 2000a) . this distribution may not necessarily reflect the actual domain distribution for all the proteins in nature as the set of known protein structures in pdb might have overrepresented small proteins due to the relative ease in solving these structures. for eukaryotic organisms, it was estimated that at least two-thirds oftheir proteins are multidomain proteins (gerstein and hegyi, 1998; gerstein, 1997 gerstein, , 1998 doolittle, 1995; apic et ai., 2001a,b) . this number is somewhat smaller for bacterial and archaeal organisms but still represents a significant percentage of all their proteins. previously domain partition of multidomain proteins was typically done manually. to keep up with the rate at which protein structures are being solved, there is a clear need for more automated domain-partitioning methods to process the newly solved structures. currently computer programs are being used for partitioning a protein structure into individual domains. popular programs for this purpose include dali (dietmann and holm, 2001) , domainparser (xu et ai., 2000a) , and pdp (alexandrov and shindyalov, 2003) . a protein domain could be part of different protein structures, through combining with other domains. figure 12.1 shows an example of a domain in different proteins. both proteins, rna 3'-terminal phosphate cyclase and glutathione s-transferase, have the thioredoxin fold domain, which has two layers, one with two a-helices and one with four antiparallel r3-strands, although some details differ in the two proteins. the parts other than the thioredoxin fold domain in the two proteins have no structural relationship. since domains are the basic structural units of proteins, current studies on the number of unique structural folds in nature have been carried out on protein domains rather than whole proteins (hereafter, the term "proteins" will refer to single-domain proteins for simplicity of discussion). to estimate the number of unique folds of proteins, one popular approach is through examining all protein families and the relationships between protein families and unique structural folds. using the definition of scop classification scheme (murzin et ai., 1995; brenner et al., 1996) , a proteinfamily represents a group of orthologous proteins (makarova et ai. 1999; gerlt and babbitt, 2000; tatusov et ai., 2000; gelfand et al., 2000) . the number ofprotein families in nature could possibly be estimated through finding orthologous gene groups covering all the genes in the genomes that have been sequenced. one such estimate suggests that there are 23,100 such protein families (orengo et al., 1994) . this number has been used in later studies on estimating the number of unique structural folds in nature. other studies estimate this number to be in the tens of thousands (koonin et al., 2002) . whether it is 23,100 or some other number ranging from 10,000 to 50,000, the idea is that all proteins in nature fall into one of these families. the estimation on the number of unique structural folds is obtained through estimating the number of families that each structural fold covers and studying the coverage distribution by all the known structural folds. one of the estimates by zhang and delisi (1998) suggests that the number n of unique structural folds is probably around 700. this estimation is based on the observation that the number of structural folds covering x number of protein families follows a power-law distribution, withxbeing a variable. in essence it says that a few structural folds each cover many families (e.g., tim barrel fold covers 31 protein families) while many structural folds each cover only a small number of families ; more generally, the number of structural folds decreases as their coverage of protein families increases . specifically, zhang and delisi proposed a formula which matches well with the known protein families and structural folds. let m and n represent the number of families and the number of unique folds in nature, respectively. the probability that a fold covers exactly x families is given by let m, and n, be the numbers of protein families and unique structural folds currently having solved structures, respectively. through a simple algebraic transformation, zhang and delisi showed that which is used to estimate the number of unique structural folds. using the known numbers of m, = 736 and n, = 361 at the time of the estimation, they estimated that n is roughly about 700, which many researchers will argue to be too small (see following for more discussion). similar estimations have been made by other researchers, estimating the size of n ranging from a few hundred to a few thousand (orengo et ai., 1994; wang, 1996) , based on somewhat different assumptions. coulson and moult (2002) recently developed a new model for estimating n, based on the work of zhang and delisi. using the more recent data on the numbers of genes, gene families, and structural folds, they argued that there are two "special" cases that have not been treated well by previous estimation models. based on their argument, they consider that there are three classes of structural folds, which are termed unifolds, mesofolds, and superfolds. unifolds represent structural folds that each covers only one family of proteins, superfolds represent structural folds, each ofwhich covers many structural folds, and mesofolds represent structural folds in between. for example, tim barrel covers 31 families, while many unifolds exist in sco~based on their observation, they argued that previous models such as zhang and delisi did not fit well with the data of unifolds and superfolds. so a new piecewise model was then developed which treats unifolds, mesofolds, and superfolds, separately. using this new model, coulson and moult (2002) estimated that less than 20% of the protein families belong to unifolds, while 20% of the families belong to a few dozen superfolds and the rest of the protein families belong to mesofolds. considering that the estimated number of protein families ranges from 10,000 to 50,000 (or 23,100 as one of the popular estimates suggests), we can infer that the number of unifolds ranges from 2,000 to 10,000. the number of mesofolds could be estimated using the zhang and delisi model, based on the sizes of m, and n s , after excluding the unifolds and superfolds. hence, coulson and moult concluded that the most probable size for the number ofmesofolds is about 400. the number of superfolds is believed to be very small, possibly in the range of low dozens. overall this model suggests that over 80% ofthe protein families fold into a little over 400 structuralfolds, the majorityofwhichare alreadyknown, whilethe restoftheprotein families each belongs to a unique unifold. the implication ofthis estimation is that about 80% ofthe protein families are amenable for structural modeling using protein threading techniques, assuming that at least one protein in each of the meso-and superfolds has its structure solved. if experimental facilities for structure solution will strategically select their solution targets to maximally cover all the meso-and superfolds, we could expect that at least 80% of the protein families will be structurally modelable in the near future. this is exactly the strategy that the national institute of health (nih) is using in its protein structure initiative (http: //www.nigms.nih.gov/psi/) . for the remaining 20% of protein families, it might take some time to have at least one solved structure in each of the unifolds. hence, the threading technique will be less applicable for this class of proteins, at least in the near future. there are a number of popular schemes and associated databases for classification of proteins into structural folds, including scop (murzin et ai., 1995) , cath (orengo et ai., 1997) , and fssp (holm and sander, 1996b) . these classification schemes classify all solved protein structures into different structural folds and subclasses of structural folds. the classification of protein structures is essentially achieved through grouping protein structures into clusters ofsimilar structures, which can be done computationally through structure-structure alignments (holm and sander, 1996a) . scop (murzin et ai., 1995; brenner et ai., 1996; andreeva et ai., 2004 ) groups all protein structures essentially into a three-level classification tree. at the top level, scop (scop1.65) currently consists of about 800 structural folds, each ofwhich is further divided into superfamilies and then into families. while a family represents a group of orthologous proteins, a superfamily represents a group of homologous proteins, possibly made ofmultiple families. currently scop consists ofabout 1300 superfamilies and about 2400 families. among the 800 structural folds, 489 have only one family each, which might represent unifolds; and 9 cover a large number offamilies each, which are considered as superfolds by coulson and moult. one thing worth noting is that among the 800 scop folds, only 36 represent membrane proteins. this is a reflection of the fact that only 2% of all the solved protein structures are membrane proteins (http: //blanco.biomol.uci.edu/membraneyroteins...xtal.html) . this suggests that threading is generally not applicable to structure prediction of membrane proteins, at the present time. scop's hierarchical classification ofstructural folds provides a convenient tool for applications ofthreading methods, as query proteins falling into a scop protein family are generally expected to have accurate structure predictions, while proteins with structural homologues in a scop superfamily will have a good chance to have the correct structural folds identified and some portions oftheir backbone structures predicted accurately. in general, it still represents a challenge to correctly identify the structural fold by a threading method if a query protein only has a structural analogue (i.e., similar structure but not homologous) in sco~ the realization that protein structures are clustered into structural folds in the structure space and the number ofsuch clusters is possibly quite small has led to a new way ofpredicting protein structures in a more efficient and effective manner. the general belief is that different proteins fold into similar 3d shapes because at some level, they share similar interaction patterns among their residues and between the residues and the environments. it has been shown that these interaction patterns could possibly be captured using simple statistics-based energy models as exemplified by the earlier work ofeisenberg and colleagues (bowie et al., 1991; fischer et al., 1996a,b; fischer and eisenberg, 1996) , the work of sippl and colleagues (sippl, 1990) , and others (jones et al., 1992; rost et al., 1997) . these simple statistics-based energy functions have been used, for many cases, to distinguish the correct structural folds from the incorrect ones and to distinguish the correct placements of the residues in a query protein into the structural positions of a correct structural template from the incorrect ones. placing the (backbone atoms of) residues of a query protein into the correct structural positions in a correct structural fold gives a prediction of the backbone structure of the query protein. to accomplish this, one would need two capabilities: (a) an energy function whose global minimum will correspond to the correct placement of residues into the correct structural template, and (b) a computational algorithm that can find the global minimum of the given energy function. we explain the basic idea of developing such energy functions in this section and leave the algorithmic issues to the next one. in their earlier work (bowie et al., 1991; fischer et al., 1996a,b, fischer and eisenberg, 1996) , eisenberg and colleagues demonstrated that simple residue-based, instead of atom-based, energy functions could provide substantial discriminating power in separating good from poor placements of individual residue types into different structural environments, justifying the usage ofresidue-based energy functions. in their work, structural environments are simply defined in terms oftwo parameters, solvent accessibility sol and secondary structure ss. specifically the quantity sol of solvent accessibility is discretized into a number of intervals, say 30--40% exposed to the solvent. a secondary structure could be a helix, a beta-strand, or a loop, or it could be defined in terms of more refined categories of secondary structures, say including different types oftums. then a structural environment for each residue in a template structure could be defined using (sol, s), say (0-10% exposed, alpha-helix). statistics could be collected from a collection of solved protein structures about how frequent a particular type of amino acid appears in a particular structural environment as we just defined. this can be done by going through all protein structures under consideration to count the number of occurrences of each amino acid type in each encountered structural environment. if we consider, say, three levels of solvent accessibility, {exposed, intermediately exposed, buried}, and three types of secondary structures, we will have nine types of structural environment. under this assumption, the result of counting the numbers of occurrences above will be a 20 by 9 table, with each of the 20 rows representing an amino acid type and each of the 9 columnsrepresenting a structuralenvironment. based on the collected statistics, we can build a simplepreference model to measure how preferred a particular amino acid type is to a particular structural environment. this can be done using the following measure: where oi,} represents the observed frequency of amino acid type i in structural environment j and ei,l represents the expected frequency of amino acid type i in structuralenvironment j. in the work of eisenberg and colleagues, ei,l is estimated using the frequency of amino acid type i in all proteinsunder consideration. hence, if an aminoacidtype i has a higherfrequency in a particularstructuralenvironment j than its frequency overall, it willbe assigneda negative score-in(oi,}i ei,l); otherwise it will get a positivescore or zero (when oi,l = ei,j). the higher oi,l is comparedto ei.j» the more negative the corresponding energy is. a popular name for this type of energyfunction is singletonenergy.by performingstatistical analysis on a database, one can get the scoring functionusing the above formulation for the 20 amino acid types appearing in the nine structuralenvironments. when building such statistics-based energy functions, one needs to be careful in selectingthe data set for statistics collection. forexample, someproteinsin scop (or in pdb) havemore homologous structuresthan the others,which could possibly leadto biased statistics. toremove this type of statisticbias in our data set, one needs to remove homologous structuresin the data set for statisticscollection. there are a numberof databases forthis purpose,suchas the nonredundant sequencerepresentativesin fssp, pdb-select (hobohmet al., 1992) , and pisces (wang and dunbrack, 2003) ,whereno two proteinshavehigher than a certainlevelof sequencesimilarity. another statistics-based energy functionwidelyused in threadingprogramsis often calledpairwise interaction energy. it measures the preference of having two particulartypes of amino acids that are spatiallyclose. one particular form of such an energy function was developed by sippl (1990) . the basic idea of this energy function is to compare the observed frequency of a pair of amino acids within a certain distancein solvedprotein structureswith the expectedfrequency of this pair of aminoacid types in a protein structure. the basic idea of such an energyfunction comes from statistical mechanics where the probability pi} of having a pairwise interaction betweenresidues i and j has the boltzmanncorrelationto its energygij (gibbs free energy), definedas wherek is the boltzmannconstant, t is the temperature, andz is a partitionfunction. when using a residue-averaged state as a reference state g(p), a knowledge-based potential can be derivedusing . specifically, if n o(i, j) and ne(i, j) represent, the observedand expectednumbers of amino acid types i andj within a certain distance, respectively, we can use the following to measurethe preferenceof havingthese two types of amino acids close to each other: while no(i, j) canbe collectedby goingthroughtheproteinstructuresin the sample set, an accurate estimation of ne(i, j) represents a challenge. there have been a number of proposed models for estimatingthis quantity. among these models, the simplestone is the "independentreference state" model (xu et al., 1998) ,in which ne(i, j) is estimatedas follows: table 12 .2 shows a scoring function for the preference between 20 types of amino acids using the above formulation (xu et ai., 1998) . there are more sophisticated models for defining the reference state, one of which is the uniform distribution model (sippl, 1990; dewitte and shakhnovich, 1996; lu and skolnick, 2001; samudrala and moult, 1998) , as discussed later. these more complex models take more factors into consideration in building the reference state, hence making the energy models more likely to be accurate. in addition to using physics-or statistics-based energy functions, researchers have incorporated evolutionary information into energy model building. one of the earlier major improvements in energy function modeling is the incorporation of sequence profile information (panchenko et ai., 2000; zhou and zhou, 2005) derived from homologous proteins into the energy models outlined above. it was noticed that when using a sequence profile ofa protein family (or superfamily) rather than a single (query) protein sequence, threading accuracy could be significantly improved (panchenko et ai., 2000; zhou and zhou, 2005) . the very basic idea ofthis generalized approach is that rather than asking the question "will protein sequence a fit well with structural fold b?" we ask the more general question "will the whole family of protein a fit well with structural fold b?" clearly if done properly, this approach could iron out some ofthe spurious predictions, caused by the appearance ofspecific individual sequences. now a threading problem becomes a fitting problem between a sequence profile and a structural fold. a sequence profile is defined in terms ofa multiple sequence alignment ofthe members ofthe query protein's family (or superfamily), with each element being the frequency distribution ofthe 20 amino acid types in this aligned position rather than a specific amino acid. to generalize the aforementioned energy functions to take into account the profile information, we can simply use the relative frequency of each amino acid in the position-dependent distribution as a weight factor when calculating the energy values for each amino acid or amino acid pairs, and then sum over all possible amino acids or amino acid pairs. specifically,let pibe the relative frequency ofamino acid type i in a particular aligned position with l pi = 1.0. then the eisenberg type of energy can be calculated as similarly, pairwise interaction energy could be generalized as follows: other types of energy functions have also been used in existing threading programs, including fitness scores between specific amino acids and the secondary structures in the template structure and threading alignment gap penalties. typically these energy scores are combined using a simple weighted sum while often the scaling factors are empirically determined, based on some training data. it has been observed that distance-dependent pairwise interaction energy could provide more accurate threading results than distance-independent models as outlined above. a distance-dependent energy could be estimated as follows: where r is the distance between residues i and j (possibly measured between their c-beta atoms), no(i, j, r) is the observed number of pairs of residues (i, j) within a distance bin from r -~r12 to r +~r/2 in a database of folded structures for some bin width sr, and ne(i, j, r) is the expected number of pairs (i, j) within the same distance bin. the challenging issue in accurately estimating the interacting energy u(i,j,r) is how to estimate ne (i, j, r) . under the assumption that we are dealing with an ideal infinite liquid-state system within a volume v and residues are distributed uniformly (sippl, 1990; dewitte and shakhnovich, 1996; lu and skolnick, 2001; samudrala and moult, 1998), ne(i, j, r) can be estimated using where n, and n, are the numbers of amino acid types iand j in the protein database, respectively. researchers have realized that this model needs to be corrected when dealing with finite systems like a protein structure, to make the model more accurate when used in threading programs. twoparticular corrections are made in the dfire (distance-scaled ideal gas reference state) energy model (zhou and zhou, 2002) , a popular energy function for threadirtg. in the first correction, dfire used r" instead of r 2 , considering that the number of interaction pairs in a finite system could not actually reach the level of r 2 as in an infinite system, where a < 2 is determined throughminimizing the distribution fluctuation of interaction distanceon a set of trainingdata. in the secondcorrection, dfireassumes that only short-range interactions need to be considered. that is, interaction energy becomes zero when the distance betweenthe interacting pairs is beyonda cutoffdistancercut. afterthese corrections, the interactionenergy could be estimatedusing the following formula: where constant11 is related to the systemtemperature and can be determinedempirically. these simple energy models have played key roles in making threading programs as popular and as useful as we have seen today. while they have been used to help to solve many structurepredictionproblems, the limitations of these simple models have also become quite clear as we have seen from the recent casp prediction results-the improvement in predictionaccuracyhas been only incremental in the past few casps (kinch et ai., 2003) . one of the key reasons for the incremental improvement comes from the crudeness of the threading energy functions. currently, the algorithmic techniques for protein threadinghaveadvanced to a stage that should be able to handle more sophisticated energy models in the threading framework, which could lead to more accuratepredicted structures. we can expect that more detailedand more physics-based energyfunctions will emergein the near future as the field is clearly in need of more accurate energy models for protein threading. the general form of threadingenergy function could be written as follows: wherees measuresthe overall fitness of puttingindividual residuetypesinto specific structuralenvironments, e p measuresthetotalinteraction energyamongpairswithin the cutoffdistance, and egap represents the total penalty for the gaps in a sequencestructurealignment. the scalingfactorsfi andj3 are typicallydeterminedempirically through optimizingthe performance of a threadingprogram on a representative set of proteinpairs. withthe optimizedfi andj3values, the goal of threadingis to findan alignment(or placement) betweena queryprotein sequence and a templatestructure that optimizesthe energy function. in a sense,proteinthreadingis like sequence-sequence alignmentas it finds an alignmentbetweena sequence of aminoacids and a sequence of structuralpositions in a 3d structure. what makes threading more difficult than a sequence alignment problem is the pairwise interaction energy term ep. for a sequence alignment problem, a simple dynamic programming can guarantee to find the global optimal alignment between two sequences as the problem formulation follows the principle of optimality (brassard and bratley, 1996) . this type of simple dynamic programming algorithm does not work for a threading problem as the global optimal threading alignment could not be easily reduced to a small number of optimal threading alignments for the partial problems as in a sequence alignment problem. intuitively, a simple dynamic programming approach, like the one used for sequence alignment that goes from the starts of the sequences to their ends and extends partial optimal alignments to include more elements at each step, will not work for protein threading as at each current point, we do not know what residues will be available to be assigned to future structural positions, which might have interactions with the previously aligned positions. such interactions will have a global impact on a sequence-structure alignment. it is such a global nature of the problem that makes the threading problem more challenging from the algorithmic point of view. there have been a number of studies attempting to understand the "intrinsic" difficulty, or computational complexity, ofthe threading problem. under the assumption that all pairwise interactions need to be considered, the threading problem was proved to be an np-hard problem by a number of authors (lathrop, 1994; calland, 2003) . while these mathematical proofs provide some evidence that the problem is computationally difficult, they might not be particularly relevant to the true difficulty of a threading problem as previous studies have shown that pairwise interactions beyond certain cutoffdistances (e.g., 10-12 a between c-beta atoms) do not contribute to fold recognition and threading alignment (lund et al., 1997; melo and feytmans, 1997; xu et al., 1998; zhang and skolnick, 2004) , and hence need not be considered. as of now, it remains an open problem regarding whether the threading problem, using a distance cutoff for pairwise interactions, is polynomial-time solvable. the challenging issue in studying the "intrinsic" computational complexity of a threading problem with a cutoff for pairwise interactions is that we do not have a good and realistic characterization for the overall interaction patterns for such a threading problem. because of the algorithmic challenges, earlier threading programs have employed various heuristic strategies for solving the optimal sequence-structure alignment problem. one particular strategy is called "frozen approximation" (westhead et al., 1995) . the basic idea is that it uses a dynamic programming approach to find a sequence-structure alignment and uses an approximation scheme to calculate the interaction energy. when the algorithm assigns an amino acid to a specific structural position from the beginning to the end of the query sequence during the dynamic programming procedure, it calculates the relevant interaction energy using the amino acids in the template structure rather than assigned amino acids from the query protein, for all the unassigned structural positions up to the current point of the dynamic programming procedure, within a certain cutoffdistance. intuitively the algorithm should work to some degree in capturing some ofthe interaction "patterns" encoded in the query protein sequence as some of the position-equivalent residues between the native structure and the native-like template structure should have similar physicochemical properties, suggesting the validity ofthe frozen approximation scheme. practical applications have also confirmed that the frozen approximation, while not guaranteeing global optimal threading alignment, does have an advantage over threading programs that do not consider pairwise interactions (westhead et al., 1995; skolnick and kihara, 2001; zhang et al., 1997) . a number ofrigorous threading algorithms have been developed that guarantee to find the global optimal threading alignments, measured in terms of energy functions that consider pairwise interactions. these include a divide-and-conquer algorithm employed in the prospect threading program and an integer programming algorithm used in the raptor program (xu et ai., 2003a,b) . it was convincingly demonstrated, through applications ofthese programs at the casp contests (xu et ai., 2001; xu and li, 2003) , that threading programs with guaranteed global optimality do have an advantage over programs without this property. one particular advantage of such programs is that they can be used to rigorously benchmark a proposed energy function. when using programs without such a guarantee to assess an energy function, it will be difficult to decide whether it is the energy function or the lack of rigor in the threading algorithm that has resulted in a subpar performance by a particular energy function. the following provides some detailed information about three types of threading algorithms. the basic idea of the divide-and-conquer algorithm in prospect can be outlined as follows. the algorithm first divides the query protein sequence into two subsequences and also divides the template structure into two substructures by cutting at one of its loop regions. then it tries to find the globally optimal threading alignments between the first subsequence/substructure pair and between the second subsequence/substructure pair, respectively. when calculating pairwise interaction energy for each "half" of the sequence-structure alignment problem, we might need information about which amino acids are assigned to which structural positions in the other "half" of the problem. to facilitate this calculation, the algorithm uses a simple data structure for each structural position l in the current substructure, that keeps a list of structural positions in the other substructure that are close enough to l to have interactions with the amino acid to be assigned to it. figure 12 .2 depicts schematically the situation where each of the two substructures has a number ofstructural positions, which are close enough to structural positions in the other substructure so that their alignments with amino acids need to be considered when calculating the interaction energy in the other substructure. these structural positions can be considered as extended parts of the other substructure (shown as extended arms for each substructure in fig. 12.2) . the difference between these extended parts and the original positions ofa substructure is that when doing alignment between the substructure and the corresponding subsequence, we do not have any knowledge about which amino acids are assigned to these positions in the other substructure. hence, the optimal threading alignment for each substructure/subsequence pair depends on the optimal threading alignment for other substructure/subsequence pair. this codependence relationship makes the problem challenging. in prospect, this problem is overcome using the following strategy : consider all possible combinations of amino acids possibly assigned to these extended positions (in the other "half" of the problem); and then solve an optimal threading alignment problem for each substructure/subsequence pair under each possible combination of amino acid assignments to these extended structural positions. assume that we can solve the optimal threading problem for each pair of (extended) substructure/subsequence and for each combination of such an assignment. then it can be checked that the global optimal threading alignment for the whole structure and sequence must be the union of two optimal threading alignments for the two extended subproblems, under one specific combination of the amino acid assignment to the extended part of each subproblem. this realization lays the foundation for the divide-and-conquer algorithm of prospect as it allows reducing a whole threading problem to two smaller threading problems. if we can solve the smaller threading problems, the whole threading problem can be solved by simply going through all combinations of the amino acid assignments to the extended parts for each subproblem to find the one that gives the overall best combined score. during the "conquer" step, it needs to make sure that the two optimal solutions, to be combined, to the subproblems is consistent. to solve a smaller threading problem, we can apply the same divide-and-conquer strategy to reduce it to even smaller problems. this procedure can continue until the size ofthe problem is small enough that it can be solved using a brute force exhaustive search strategy. the trick is how to make this algorithm run efficiently.note that ifnot done carefully, the number of possible combinations of assignments needed to be considered could be very large. in prospect, (sub)structures are divided in such a way which minimizes such a number ofcombinations, through cutting at the "weakest" link with the least interactions between two substructures (xu et ai., 1999) . the overall computational complexity of the divide-and-conquer algorithm is dominantly determined by the thickest link among all weakest links throughout the bipartitioning of a protein structure into a series of small structures during the whole divide-and-conquer scheme. intriguingly, we found that this thickest link is generally a small number for the vast majority ofthe solved protein structures (xu et ai., 1999) , making the actual computing time of prospect threading practically acceptable. this observation has also raised an interesting question: "is there something special about the topology of protein structures, which could be further and more rigorously exploited for efficient threading algorithm development?" 12.4.2 raptor raptor uses a more general framework to rigorously solve the threading problem (xu et ai., 2003a,b) than prospect. it formulates a threading problem as a linear integer programming (lip) problem. it uses an integer variable ("0" or "1") to represent if a particular residue in the query protein sequence is assigned to a particular structural position. then the set ofall feasible solutions to a threading problem could be defined in terms of a set of equalities or inequalities (called constraints), each of which is defined in terms of the above and other integer variables. the global optimal threading problem is then defined to find a feasible threading alignment that optimizes a given energy function. generally a linear integer programming problem requires an exponential computing time to find an optimal solution, and hence intractable for large-size problems. branch-and-bound represents a popular technique for solving linear integer programming problems. typically, an integer programming problem is first relaxed to a linear programming problem, i.e., variables could take real values as possible solutions. there are efficient algorithms for solving linear programming problems as they are polynomial-time solvable (papadimitriou and christos, 1998) . if by chance the solution to the relaxed linear programming problem is all integral, a solution to the original linear integer programming is found. otherwise the linear solution will be used to constrain the search space, through fixing one variable to "0" or"1" and then the algorithm iterates this process until all solutions have integral values. an interesting observation made is that for the vast majority of threading problems, this relaxation procedure stops after a few iterations, solving the threading problem efficiently and also indicating that threading problems seem to have a special structure in terms of the integer programming formulation. such special characteristics could possibly be utilized for developing more efficient threading algorithms, using more specialized algorithmic techniques. one particularly interesting technique which is being actively investigated by a number of researchers is based on the idea of tree decomposition of an interaction graph representing possible alignments between a query sequence and a template structure (song et al., 2005; xu et al., 2005) . in a sense this type of technique is a generalization of the divide-and-conquer outlined above. tree decomposition technique has been widely used for various graph-related optimization problems, for example finding the maximum independent set and dominating set (arnborg and proskurowski, 1989) . we now provide a detailed description of one such algorithm for solving the protein threading problem. using a tree decomposition algorithm, both the template structure and the query sequence are represented as graphs; vertices denote core secondary structures (or simply cores) and edges represent interactions between cores (two cores are considered to be in interaction if their shortest distance is within a predefined cutoff distance). a sequence-structure alignment problem essentially corresponds to finding an isomorphic mapping from the structure graph to a subgraph of the sequence graph. the efficiency of the alignment hinges on the tree width of the structure graph. intuitively, the tree width of a graph measures how much the graph is "treelike." a graph can be represented as a "tree having thick trunks," where the "trunk thickness" is quantified by the tree width of the graph. this technique of "treelike" representation for graphs is called tree decomposition. in a tree decomposition of a graph, vertices of the graph are grouped into possibly overlapping subsets, each of which is associated with a node in the tree. the maximum size of such a subset corresponds to the tree width ofthe tree decomposition. given a tree decomposition of a structure graph with tree width t, a dynamic programming algorithm can be employed to find the optimal sequence-structure alignment in time oik: n 2 ) , for some small integer parameter k and n being the number of amino acids in the template structure. the alignment algorithm is very efficient since the tree width for such structure graphs is small in general (by the nature of protein structures). for example, among 3890 protein tertiary structure templates compiled using pisces (wang and dunbrack, 2003) only 0.8% of them have tree width t > 10 and 92 % have t < 6, when using a 7.5-a c~-c~distance cutoff for defining pairwise interactions [see fig. 12 .5(a)]. we now provide the details of a tree-decompositionbased threading algorithm. a sequence-structure alignment can be formulated as a generalized subgraph isomorphism optimization problem, for which both the template structure and the query sequence are represented as mixed graphs that contain both directed and undirected edges. we use v(g), e(g), and a(g) to denote the vertex set, the undirected edge set, and the directed edge (arc) set of a mixed graph g, respectively. the graph h for the template structure is constructed as follows: each vertex in v(h) represents a core, each undirected edge in e(h) represents the interaction between two cores, and each directed edge (arc) in a(h) represents the loop between two consecutive cores (from the n-terminal to the c-terminal). for technical convenience, both n-and c-terminals are presented as vertices. figure 12 .3 gives a protein tertiary structure and its corresponding structure graph representation. a query sequence is preprocessed so that for each core in the template, all substrings (called candidates) of the query sequence that align well with the core are identified (xu et ai., 2000) . by representing each candidate as a vertex, a query sequence can also be represented as a mixed graph. that is, each edge in e(g) connects a pair of candidates that may possibly interact but do not overlap in the sequence, and each arc in a( g) connects two candidates (from the n-terminal to the c-terminal) that do not overlap. as in a structure graph, both n-and c-terminals are represented as vertices in the sequence graph . figure 12 .4 illustrates the sequence graph with a simple example. the relationship between a core v in the template and its candidates in the query sequence can be constrained using a mapping function m such that m(v) contains all possible candidates of v. the less restricted m is, the more accurate the alignment is expected to be and the more time it may take to compute. xu et a1. (1998) used a similar approach in their divide-and-conquer threading algorithm, which can find all suitable candidates for each core. the maximum size k = 1m(v) lover all cores v is called the map width ofm, an important parameter for the alignment algorithm. now a sequence-structure alignment problem can be formulated as a problem offinding an isomorphism mapping f between the structure graph and a subgraph of the sequence graph g such that the following sum ofthe alignment energy functions l ecore (u, f(u) achieves the minimum, where e eore is the alignment energy score (singleton type of energy) between a core u in the template and its candidate feu) in the sequence, e pair represents interaction energy (pairwise interaction energy) between residues (f(u),f(v)) assigned to cores (u, v) , and el oop is the alignment score between the loop < u, v > in the template and the corresponding <feu), i(v» in the sequence. for a structure graph h, its tree decomposition (t, x) is defined by a tree topology t, which is a binary tree, and a collection x = {xl, x 2 , ... , x m } , where each xi is a subset of v(h),called a bag for tree node i in t. any tree decomposition (t, x) of a graph h should satisfy the following conditions: (a) uxi = v(h); (b) for any u, v forming an (un) directed edge in the graph, u and~are contained in the same bag; and (c) the tree nodes whose bags contain the same vertex should form a connected subtree of t. intuitively, a tree decomposition groups locally connected vertices together into bags, and the set of vertices in the bag for each tree node can actually separate the graph into two disconnected components. the tree width of a tree decomposition is the maximum size ofthe bag associated with a tree node minus one. the tree width of the graph is the minimum tree width over all possible tree decompositions for the graph (robertson and seymour, 1986; bodlaender, 1996) . there are algorithms oftime 0 (c' n) that can find an optimal tree decomposition for a graph of n vertices, provided that the graph has a tree width t. however, the constant c may be too large for the algorithm to be practically useful. for structure graphsformulated in ourcurrentcontext, wefoundthat fastheuristic algorithms may producetree decompositions with a tree widthreasonably closeto the minimum tree width. the following describes one such heuristic algorithm for tree decomposition. given a structure graph, two undirected edges (representing two interactions between cores)crosseachotherifthefourcoresinvolved interleave intheirsequential positions. tofind a tree decomposition of the graph, an edgethat crosses the greatest number of other edges is selected and removed from the graph (but the endpoints of the edge are kept). this process is repeated until the remaining graph does not contain any crossing edges. the remaining graph is called an outerplanar graph (frederickson, 1991) that has tree width 2, whose optimal tree decomposition can based on the tree decomposition for the derived outerplanar graph, a tree decomposition for the original graph can be constructed by adding u, for every removed edge (u, v) , to everybag on the shortestpath from the bag containing u to the bag containing v in the tree. forexample, fig. 12 tree width (b) fig.12 .5 (a) tree width distribution of the structure graphs for 3890 protein tertiary structure templates compiled using pisces (wang and dunbrack, 2003) . (b) a tree decomposition for the structure graph given in fig. l2 .3(b) , constructed by first removing edge (3,5), building a tree decomposition for the remaining graph, and then putting vertex 3 (in the bold font) in node {i ,5,4} on the path from node {1,4,3} to {1,8,5} . of the structure graph in fig. 12.3(b) . in fig. 12 .5(b) the crossing edge (3,5) is first removed and a tree decomposition is constructed for the derived outerplanar graph, which is then extended to a tree decomposition for the original graph by placing vertex 3 (in the bold font) in tree node bag {i ,5,4} on the path from node {i ,4,3} to node {1,8,5}. this heuristic strategy produces a tree decomposition of size at most 2 + c if there are c crossing edges removed. in actual applications, the obtained tree decomposition in general has a much smaller tree width than 2 + c. once a tree decomposition (t, x) and a mapping constraint m are given, a dynamic programming can be designed to traverse the tree to find an optimal alignment between the template structure graph and a subgraph of the sequence graph g, as follows. in a bottom-up fashion, the algorithm establishes one table for each tree node i, such a table consists of t + 3 columns, where t is number of vertices (i.e., cores) contained in the bag for the tree node. each row in the table is a combination f of candidates for the t cores constrained by m. the additional three columns store the validity, the score, and the optimality for each such combination. the computation ofthese three columns is performed by considering the cores in the present tree node bag that also occurs in its child node bags and in its parent node bag. in particular, the validity asserts that the combination f ofthe cores' candidates is legal not only for all cores in the present tree node bag xi, but also consistent with at least one valid combination in the tables of its two child nodes (if it has children). the score for each combination f in the present table mk is calculated as the sum of the following three scores: 1. the alignment score of the core-candidate correspondences defined by the combination f for the cores in xi only; 2. the maximum score over all combinations in the table ni; for the left child node k which are consistent with combination f; and 3. the maximum score over all combinations in the table m j for the right child node j which are consistent with combination f. the optimality column indicates if the score for each combination f is optimal over all combinations with the same candidate choices as f for the subset of cores of xi that also occur in its parent node bag. figure 12 .6 illustrates the computation for a row in the table of an internal node that has two child nodes. the optimal score over all combinations computed in the table for the root of the tree t is the best alignment score. a recursive routine can be used to trace back the corresponding optimal alignment. there are three steps to accomplish the structure template-target sequence alignment. the time complexity for each step is analyzed in the following. the running time for the tree-decomposition-based structure-sequence alignment is otk' t 2 n), where k is the maximum number of candidates of a core, t is the width of the tree decomposition, and n = iv(h)i. this is because the tree decomposition contains at most n nodes, each maintaining a table of at most o(k t ) rows. for each row in the table, the combination of core-eandidate correspondences needs to be validated and a score is computed according to the objective function given in section 12.4.3.1 (by looking up precomputed values of the three alignment energy functions e eor e , epair, and e loop ) ' the former step needs 0(t 2 ) and the latter 0(t 2 + 2t log~k) (note that the table for a child node can be ordered so as to facilitate binary search by the computation for its parent node). it takes o(nn) time to preprocess the target sequence of length n to construct the sequence graph. simultaneously, this step precomputes the values of alignment functions e eor e and epair' the values of function e loop can then be precomputed, where ii is the length of the segment (between two candidates of cores) to be aligned to the ith loop in the structure, and i is the total number of loops in the structure. putting together the time needed by the preprocessing, tree decomposition, and the alignment gives us a loose upper bound oik' nn), or oik' n 2 ) , for the total time needed by the whole algorithm for the structure-sequence alignment part of the threading. based on preliminary computational results, the tree-decompositionbased threading algorithm seems to run faster than both the divide-and-conquer and the integer programming-based threading programs. protein threading is used mainly for two purposes: (a) identification of the correct structural fold or folds from a collection ofprotein structures for a query protein and (b) prediction of the backbone structure through identifying the correct assignment (or alignment) of the amino acids of a query sequence to the structural positions in the correct structural fold. in a way, protein structure prediction through protein threading could be considered as a problem to find a global optimal threading result among many sequence-structure alignments. the threading algorithms outlined in the previous section could only provide the best alignment between a query sequence and a particular template structure, i.e., a local optimum. the problem of finding a global optimum from the many local optima for the threading problem represents a highly challenging problem. the key reason is that the alignment scores between a query sequence and different template structures are in general not comparable directly with each other. some structures might tend to have higher baseline threading scores than the others, no matter what query sequences are used. we can think of this problem using the following analogy from local sequence-sequence alignment problems. suppose we have two "template" dna sequences (consisting of four different letters). we assume that one sequence is significantly longer than the other one. for simplicity of discussion, we can regard the length of the longer one to be infinite. it is not hard to convince ourselves that for a randomly selected dna sequence, its best (local) alignment with the longer sequence will have a higher probability to have a better score than with the shorter sequence. the reason is that the longer sequence, by chance, should have a higher probability to have portions of its sequence similar to the query sequence, compared to the shorter sequence. this indicates that the background alignment-score distributions for the two "template" sequences are different. hence, alignment scores against these two sequences could not be compared with each other directly in a meaningful way. similar can be said about structural templates for protein threading. to compare threading alignment scores, we need to first "normalize" the scores so that the quality ofthreading alignments against different templates could be compared with each other. for sequence-sequence alignments, there have been a number of methods developed for assessing the quality of an alignment through assessing the statistical significance of its alignment score. the p-value calculation for blast represents a popular method for such a purpose (altschul and gish, 1996) . developing rigorous and effective statistical models for threading has proven to be more challenging than sequence alignment problems. hence as of now, there has not been a dominating method, like the p-value calculation for blast, in estimating the statistical significance of a threading alignment score. early studies on statistical significance analysis ofthreading results were mostly empirical in nature. a representative of these studies was by bryant and co-workers (bryant and altschul, 1995; panchenko et ai., 2000) . in their model, they assume that threading scores between a template structure and a large set of sequences of the same length and with the same amino acid composition follow a gaussian distribution, and have estimated the parameters of the gaussian distribution through .calculating these threading scores. specifically, for a query sequence and a template structure, they have generated a large number of sequences with the same length and the same amino acid composition as those of the query sequence by randomly reshuffling the query sequence and have used these sequences to estimate the parameters ofthe gaussian distribution. with the estimated gaussian distribution, they then calculate the probability (p-value) that a random sequence would have a better threading score than the score between the template and the query sequence. the effectiveness of this p-value calculation was demonstrated through their successful performance at the casp3 contest (panchenko et ai., 1999) . while effective, the approach has a number of limitations which have limited its wider range of applications. these limitations include (a) its high demand for computing time as it requires solving a large number of threading problems involving the reshuffled sequences and (b) its not well justified assumption that the threading scores follow a gaussian distribution. it has been shown that the scores ofoptimal sequence-sequence and structurestructure alignments generally follow extreme-value distributions (levitt and gerstein, 1998) . based on the assumption that sequence-structure alignment scores also follow an extreme-value distribution, sommer et al. (2002) have recently developed another scheme for p-value estimation, in which parameters of the model are estimated by fitting the threading scores against an extreme value distribution. a key characteristic of the work is that it attempts to have a unified model for threading problems involving different lengths and compositions of the proteins involved. based on the random energy model (rem), shakhnovich and co-workers systematically studied the distribution ofthreading alignment scores (chen et ai., 2003; mimy et ai., 2000) . rem provides an adequate description ofequilibrium properties of random heteropolymers, which assumes that for a query sequence, the energy of a set ofalignments, including the "native" alignment with energy en and m decoys, followed a gaussian distribution with an average energy e av and standard devia-tion~. assisted by a null hypothesis where the optimal native sequence-template alignment should be the highest ranked, they proved that the threading scores for random sequence-structure pairs follow an extreme-value distribution. a critical energy e; was derived, which was a quantity in the rem, for characterizing the probable lower boundary ofthe density of states and it represents the most probable energy value of an optimal random sequence-structure alignment, which is determined by eq. (12.1) . they used another parameter e for the statistical significance [eq. (12. 2)] based on e e , corresponding to the relative energy gap for an alignment. this e score has been tested for both gapless threading and limited-gap threading results. they found that when e > 1, especially when e > 1.2, there was a significant energy gap between the optimal alignment and the decoy alignments. hence, this quality could be used as a measure for assessing the significance of a threading alignment. a good method for assessing the statistical significance of a threading score should not only allow comparing threading results on the same footing but also provide a way to indicate if a particular fold is possibly the correct fold for a query sequence, without using other reference information. a typical threading program consists of four key components from the implementation perspective: (a) a database oftemplate protein structures, (b) an energy function, often residue-based, (c) a threading algorithm that can find the optimal threading alignment between a query sequence and a template structure, and (d) a method for calculating "significance" scores of threading alignments. from the prediction accuracy point of view, the larger a template structure database is, the more accurate we can expect the threading prediction will be. however, it might not always be realistic to use the whole pdb database as the template database due to the amount of time required to thread a query sequence against each pdb structure. often a template structure database consists of a representative subset of all the structures in pdb, say pdb-select, which consists of pdb structures with the "redundant" structures removed. here "redundant" refers to structures that have high sequence similarities to other structures in the database. to make prediction more accurate, some threading programs employ a two-stage strategy: (a) thread a query sequence against a representative structure database and to identify a few possible native-like structures, and (b) thread the query sequence against all family/superfamily members ofthe identified structures in (a). certain preprocessing of the template database might be needed for some threading programs. for example, as we discussed in section 12.4, protein structure needs to be represented as structure graphs as required by the threading algorithm. the majority ofthe current threading programs employ the types ofenergy functions outlined in section 12.3 or their variations. these energy functions are statistics, rather than physics, based. they are used to distinguish correct structural folds from the incorrect ones and to distinguish accurate alignments against the correct structural folds from inaccurate alignments. threading programs have been using these simple energy forms, mainly because of the consideration of computational efficiency and also partly due to the constraints of limited available structural data for more sophisticated energy forms when such statistics-based energy functions were first developed. as those simple energies began to reach their limits, we began to see more physics-based energy functions developed, as we discussed in section 12.3. we expect that as the threading algorithms become more efficient, we will see more physics-based energy functions. we expect that one particular type ofextension to the existing energy functions is to consider multibody interactions, which have been mostly ignored by existing threading programs. recent studies have shown that multibody interactions could help to improve the performance ofthreading programs (munson and singh, 1997; li and liang, 2005) , and hence should be considered. existing threading programs employ various algorithmic techniques for solving the sequence-structure alignment problem, including dynamic programming with enhanced heuristics (westhead et ai., 1995; skolnick and kihara, 2001; zhang et ai., 1997) , divide-and-conquer algorithm , and integer programming (xu and li, 2003; xu et ai., 2003a,b) . in this chapter, we presented a new class of threading algorithm based on a tree decomposition of sequence and structure graphs. while integer programming might represent the most general framework for handling sequence-structure alignments, particularly so for threading problems considering multibody interactions, tree-decomposition-based algorithm could prove to be more popular down the road because of its conceptual simplicity and computational efficiency. we expect that a class of more general threading algorithms will begin to emerge to deal with more complex threading problems as the existing threading algorithms become faster and faster. this general class of threading algorithms should be able to handle simultaneous backbone threading and side-chain packing problems, leading to significantly more accurate capabilities in fold recognition and protein structure prediction. existing threading programs use various ideas and techniques to assess the "significance" of threading results. these methods include z-score calculation (sommer et ai., 2002) , normalized threading scores using techniques such as support vector machines or neural network (xu et ai., 2002; ding and dubchak, 2001) , and (panchenko et al., 2000; bryant and altschul, 1995) . while useful to some degree, none ofthese methods have reached the level of performance comparable to p-value calculations for blast sequence alignments (altschul and gish, 1996) . this is possibly due to a combination of the inadequacy of the existing threading energy functions for accurate threading prediction and the lack of general understanding about distinguishing characteristics between correct and incorrect native folds and between correct and incorrect placements of amino acids into structural positions. overall, compared to other areas of protein threading, this is a somewhat underdeveloped area. new ideas and techniques are clearly needed to fill the holes in this area. because of the importance of solving protein structures for functional studies and the power of threading techniques, many protein threading programs have been developed. using these programs, a large number of protein structures have been predicted prior to the solution of their experimental structures, providing highly useful information for guiding experimental design in investigation ofthese proteins. examples ofsuch predictions include an obese gene (madej et al., 1995 ), vitronectin (xuet al., 2001 , and a sars protein (von grotthuss et al., 2003) . table 12 .3 provides a list of popular threading programs and urls for accessing these prediction tools. we now summarize the highlights of some of these threading programs which use different energy functions and different computational techniques, each ofwhich has its strengths and limitations. prospect kim et al., 2003) : the prospect program employs a divide-and-conquer algorithm for rigorously solving the global optimal threading problem, which employs a somewhat standard threading energy function, including a singleton energy term and a pairwise interaction energy term plus a secondary structure fitness score and a gap penalty score. for a typical threading problem, it can find the best alignments against a template structure database of 2000+ within a couple of days on a single cpu while it can virtually get a linear speed-up using multiple cpus when the number ofcpus is smaller than the number of structures in the template structure database. it achieves its computational efficiency by taking advantage of the fact that protein structures generally have small topological complexities (xu et ai., 1998) and through using a filtering procedure to filter out "improbable" alignment positions for each core secondary structure in the template structure. while this heuristic filtering works well for the vast majority of the threading cases, it might filter out the correct alignment positions for some cases. prospect normalizes the threading scores, along with various parameters of the template structure and the query sequence, using a support vector machine. then a z-score is calculated based on the "normalized" threading scores. raptor (xu and li, 2003; xu et ai., 2003a,b) : the raptor program formulates a threading problem as a linear integer programming problem, and solves the problem using a branch-and-bound method plus a standard integer programming solver. raptor employs the same energy functions ofprospect and uses a similar approach for assessing the "statistical significance" of threading results to that of prospect. a unique feature of the program is that its threading algorithm is more rigorous than that of prospect as it does not use a heuristic filter to filter out "improbable" alignment positions. for a typical threading problem, it takes minutes to hours to thread the query sequence against 2000 structures in its structure database. since the program is data-parallelizable, its speed-up is virtually linear when running on multiprocessor computers. genthreader (jones, 1999b) and an improved version mgenthreader (mcguffin and jones, 2003) use psi-blast profile (altschul et ai., 1997) and predicted secondary structures by psipred (jones, 1999a) for threading. it employs a double dynamic programming strategy (jones et ai., 1992) in its threading program. the algorithm does not treat pairwise interactions rigorously but its performance has been among the top threading programs, indicating the effectiveness ofthis strategy. a web server for this program has been set up at http: //bioinfcs.ucl.ac.uklpsipred/. a user in general can expect the return of a threading prediction in minutes. the prediction program runs fast enough that it can be used for genome-scale protein structure predictions. prospector (skolnick and kihara, 2001 ) recognizes native-like structural folds using a hierarchical strategy for obtaining sequence profiles. it uses two types of sequence profiles, one type derived using close homologous sequences whose sequence identity lies between 35% and 90%, and another type constructed using more remote homologous sequences with a fasta e-value less than 10. both types of sequence profiles are incorporated into a typical threading energy as described in section 12.3 to screen a structural database. the program uses a dynamic programming algorithm to find the best threading alignment, and employs z-scores for assessing the significance of each threading alignment. fugue (shi et ai., 2001 ) uses a typical threading energy function as described in section 12.3, with some unique features: (1) its structural environment singleton term includes a term for hydrogen bonding status, and the singleton term was derived from structural alignments in the homstrad database (de bakker et ai., 2001; http://www-cryst.bioc.cam.ac.uk/homstrad/); (2) its gap penalties are structure-dependent based on solvent accessibility, its position relative to the secondary structure elements, and the conservation ofthe secondary structure elements; and (3) its alignment is based on multiple sequences against multiple structures to enrich the conservation/variation information. fugue uses dynamic programming as its threading algorithm and employs z-scores to assess the statistical significance of a threading result. since each of these threading programs has its own strengths and limitations, a popular strategy for predicting a protein structure is to use multiple prediction programs and combine their prediction results. further discussion on this topic is given in chapter 17. we now use prospect as an example to illustrate how to use a threading program for predicting structures of sars-co v proteins (wan et ai., 2005) , which playa role in the development ofthe sars disease. we used the prospect pipeline to survey all ofthe 11 open reading frames (orfs) in sars-coy strain urbani (genbank id: 30027617), one of the first sars-cov genomes. among the 11 orfs, the sand m proteins playa key role in the virus infection process. interestingly, both the m protein and the s2 domain in the s protein are predicted to adopt the fold ofig-like beta sandwich. the structural similarity suggests that the s2 domain and the m protein may be evolutionarily related through gene fusion and duplication, although their sequences do not have significant similarity after a long period of evolution. the threading results might explain how the m protein interacts with the s2 domain, for the virus assembly: since the s2 domain with the fold ofig-like beta sandwich can interact with the s1 domain, the m protein with the same fold could possibly interact with the s1 domain. this suggests that the si domain may act as an on/off switch between the s2 domain and the m protein. such a mechanism may suggest that the m protein could also be involved in the virus-host cell interaction. this hypothesis was supported by a recent study in the murine hepatitis coronavirus study, which showed that glycosylation ofthe m protein affected the interferogenic capacity of the virus (de haan et al., 2004) . threading programs have been used for genome-scale applications. a recent study (guo et ai., 2004) performed structure prediction for all ofthe orfs ofpyrococcusfuriosus, which is found in the marine sand surrounding sulfurous volcanoes and can grow at temperatures above 100 a e.the microbe utilizes peptides, proteins, and some carbohydrates as carbon sources. its entire genome is about 2 mb in length with 2195 annotated orfs. out of a total of 2195 orfs, 540 are predicted to be membrane proteins, and 753 proteins can be predicted with structures in high confidence, among which 190 orfs cannot be detected using psi-blast. recent prediction results in casps indicate that even when the correct structural folds are identified, the threading alignments could often be off. from the same statistics, we found that the overall alignment accuracy has not been improved over the past few casps. in addition, the alignment accuracy of the best casp models using templates with <30% sequence identity ranges from 60% to 90% (venclovas et al., 2003) . all these statistics suggest that there is significant room for improvement in threading alignments. the prediction accuracy of the current threading programs is mainly limited by the inaccuracy of statistics-based and residue-based threading energy functions. while improving the threading energy functions represents one direction to take for improving threading performance, other approaches may also help, which include (a) application ofpartial experimental data as constraints in the threading process and (b) refinement of threaded structures using molecular dynamics and energy minimization. we refer the reader to chapter 11 for related discussions. often partial structural data is available for specific proteins before the determination ofthe detailed structure ofa protein. these partial structural data might be in the form of (a) residue-residue distances such as disulfides between specific cysteines, (b) specific residues involved in particular active sites, binding sites, or other functionally important sites, (c) specific residues known to be on the surface ofa protein structure, or any other information providing geometric information about specific residues in a protein structure. in addition to such information about specific residues, there are experimental techniques that can be used to generate geometric information in a systematic manner. nmr represents one such technique. nmr methods solve a protein structure through generating either distance restraints [called noe, or nuclear overhauser effect, distances (prestegard, 1998) ] between different residues (or more specifically different atoms) or orientations of certain chemical bonds in a protein, called residual dipolar coupling (tolman et ai., 1995) . then a protein structure is solved through finding structural models that are consistent with the collected geometric constraints and have their energy minimized. to accurately solve a protein structure using nmr technique, it typically requires 15-20 distance restraints per residue (clore et ai., 1993) , which will require multiple nmr experiments. partial distance restraints could possibly be collected using fewer nmr experiments, possibly involving labeling of specific amino acid types. similar can be said about orientation information collected through residual dipolar coupling experiments. while these partial nmr data might not be sufficient for solving a protein structure accurately, they provide highly useful constraints for protein fold recognition and backbone structure prediction by a threading method. chemical cross-linking experiments provide another systematic approach to generating partial structural information for a protein. in such experiments, chemical cross-linkers, with customized arm lengths, are designed to link specific types of amino acids within a certain distance range (cohen and sternberg, 1980) . such experiments followed by tandem mass spectrometry experiments and data interpretation could provide distance information between certain amino acids. such an approach has been used for structural data collection for both soluble and membrane proteins (yan et ai., 2005) , which have then been used for protein structure prediction (young et ai., 2000) . one way to use such structural information, such as distances or structural locations, in a threading program is to add an energy term in the threading energy function, which measures the consistency between the collected structural data and a threading alignment. for example, if residues a and b are known to be within a certain distance, then an energy term could be specifically designed to penalize threading alignments which violate this particular geometric constraint. the energy term could be designed so that the bigger the violation, the larger the penalty. similarly, if a residue x is known to be on the surface of a protein structure, an energy term could be specifically designed for this knowledge so that it penalizes threading alignments that do not put x into a surface position in the template structure, and the amount of penalty could be designed to reflect the degree of violation of this particular knowledge. to deal with all geometric constraints, we can design a new energy term eg ,which is the sum of the individual penalty functions for all the specific geometric constraints. the overall scaling factor for this new energy term in a threading energy function could be empirically determined based on a training data set, for which actual partial experimental data is available. the effectiveness of applying such partial structural data in a threading program has been documented in a number of studies (xu et ai., 2000b,c; young et ai., 2000; qu et ai., 2004a,b) . table 12 .4 shows a systematic study on improving the "query" and "temp" represent the pdb codes of the query and template proteins, respectively. "rmsd/rank vs. percentage of assigned ss" are the ca-rmsd between the experimental structure and the predicted structure using mod eller based on threading alignments for the alignable portions in the structure-structure alignment between the query protein and the template, and the rank of the correct template structure among 667 templates. 0%, 20%, 40%, ... , 100% represent the percentage of residues with secondary structure assignment, respectively. the highlighted numbers show improvement between using no secondary structure information and using full secondary structure assignments. threading performance by incrementally increasing the number ofnmrlnoe distance constraints used in a threading process. it is seen that distance constraints help both fold recognition and threading alignment accuracy. in the best scenario, a threading program can provide an accurate prediction of the backbone atoms of a protein structure, which is still a long way from having a detailed all-atom structure. in the most general situation, a threading program could provide a somewhat accurate structure for the backbone atoms in the core secondary structures while predictions for the loop regions are often not accurate. the reason is that threading predicts a structure based on a known template structure. while the core secondary structures among homologous proteins are generally "well" conserved, loops are often not. hence, template-based loop predictions are generally not accurate. fortunately, existing methods for short loop prediction « 14 residues) have reached a level that the predicted loops could be as accurate as the predicted core structure. for example, tile recent work by jacobson et al, (2004) has achieved prediction, accuracy of 0.43 a for 5-residue loops, 0.84 a for 8-residue loops, and 1.63 a for i l-residue loops, using an accurate all-atom energy function and hierarchical refinement protocol. potentially the predicted full backbone structure, after adding loop structures, could be refined using an energy-based approach. to do this, one needs to put all the atoms, backbone and side chains, into a structural model. one can use alignments with the selected templates in fold recognition to produce a 3d atomic model through homology modeling tools, such as modeller (sali and blundell, 1990) , which runs a protocol ofenergy minimization and molecular dynamics simulation to refine a structural model. after a structure model is generated, one can apply structure assessment tools such as whatif (vriend, 1990) and procheck (laskowski et ai., 1993) to evaluate the packing and backbone conformations, the inside/outside occupancies ofhydrophobic and hydrophilic residues, and stereochemical quality of a predicted structure. based on this assessment, a user can pick the best among the multiple structures derived from an alignment. while widely used, the potential of protein threading as a protein structure and function prediction technique is far from being fully realized. there are a number of factors that have limited its wider range of applications. first, fold recognition for structural analogues and some remote homologues is still challenging (kinch et al., 2003; sippl et ai., 2001) . such proteins might account for about 40% of all proteins encoded in a typical genome according to our studies (xu et ai., 2003; guo et ai., 2004) . these structures are theoretically modelable using comparative modeling techniques such as protein threading, but the predictions typically gave a low confidence level and the results may be wrong. second, even when a correct fold is identified, the accuracy ofthreading alignment has been about 60-90% for proteins with less than 30% sequence identity with their template structures (venclovas et al., 2003) . novel ideas and new techniques are clearly needed now to make a major jump in improving the prediction capability of the existing threading methods; this has become quite clear based on the slow and incremental improvements in threading performance in the past few casp contests (venclovas et al., 2003) . the current energy functions are generally coarse gained mainly to achieve fast predictions. given the significant advances in computer hardware and algorithm development, it may be the time to use more sophisticated energy functions. for example, multibody interactions and more physical energy functions may help improve the threading prediction accuracy. although many theoretical studies have been carried out for threading algorithms, there is still significant room for further improving the computational efficiency of threading programs. better search methods against structural templates using advanced database techniques have not been explored thoroughly. more work can be done at the implementation level in a similar way to the implementation of blast, where many low-level operations were implemented in a highly efficient manner. in addition, algorithmic development needs to address new types of energy functions, such as new threading algorithms that could handle simultaneous backbone threading and side-chain packing to fully take advantage of more detailed energy function forms. threading algorithms that could handle multibody interactions and energy functions capturing more global properties (e.g. , compactness) ofproteins are clearly underdeveloped. existing confidence assessments are either too time-consuming in computation or not sufficiently accurate. more rigorous and faster assessment techniques for threading are clearly needed to achieve comparable performance to that of blast. assessments of different alignments using the same fold were basically not studied. furthermore, identification of"reliable" versus "unreliable" parts ofa threaded structure, and quantitative assessment of the structural deviations in terms of rmsd for regions of predicted structures have not been achieved. it has been found that using multiple fold recognition programs to build consensus of structural template is an effective way to increase the prediction accuracy (lundstrom et al., 2001) . furthermore, one can thread subdomain structures and use these substructures from different templates to build a new structure through a shotgun approach (fischer, 2000 (fischer, , 2003 . currently a consensus was built by a simple scheme of majority vote. much statistics can be done to do this in a more scientifically sound way. in addition, how to piece different substructures together to form a global protein structure is another challenging issue. further discussion on consensus building and subdomain threading can be found in chapter 17. as a structure prediction technique, threading potentially applies to at least 80% of all protein families. however, the application ofthreading to membrane proteins has been very limited due to the lack of available structural templates. threading techniques have been widely used for various purposes in biological studies, including (a) functional studies of proteins and experimental design (e.g., targeted mutagenesis) (madej et ai., 1995; xu et al., 2001; von grotthuss et al., 2003) , (b) genome annotation (xu et ai., 2003; mcguffin et al., 2004) , (c) helping solve experimental structures (ye et al., 2004) , (d) modeling protein complex structures (lu et al., 2003) , (e) prediction ofmisfolded structure (see chapter 9), and (f) protein design (sorenson and head-gordon, 1999) . to further increase the utility of threading techniques to meet the needs for genome-scale protein structure prediction to keep up with the rate of genome sequencing and gene prediction, we clearly need a new generation of threading energy functions, threading algorithms, methods for assessing the statistical significance of threading results, and refinement of threaded structures. there are several comprehensive reviews and books on various aspects ofthreading. recent reviews related to threading include fetrow et al. (2002) and godzik (2003) . a number of books also provide some general coverage of threading and protein structure predictions (tsigelny, 2002; jiang et al. 2002; bourne and weissig, 2003) . for scoring function, the readers can find more information in chapters 2 and 3 ofthis book. for more information about general protein structure and structure-function relationship, we recommend branden and tooze (1999) and lesk (2001) . pdp: protein domain parser local alignment statistics gapped blast and psi-blast: a new generation of protein database search programs scop database in 2004: refinements integrate structure and sequence family data domain combinations in archaeal, eubacterial and eukaryotic proteomes an insight into domain combinations linear time algorithms for np-hard problems restricted to partial k-tree the universal protein resource (uniprot) protein structure prediction and structural genomics a strategy for the rapid multiple alignment of protein sequences. confidence levels from tertiary structure comparisons flexible protein sequence patterns. a sensitive method to detect weak structural similarities a linear time algorithm for finding tree-decompositions of small treewidth structural bioinformatics a method to identify protein sequences that fold into a known three-dimensional structure introduction to protein structure fundamentals of algorithmics understanding protein structure: using scop for fold interpretation statistics of sequence-structure threading on the structural complexity ofa protein fold recognition with minimal gaps exploring the limits of precision and accuracy of protein structures determined by nuclear magnetic resonance spectroscopy on the use of chemically derived distance constraints in the prediction ofprotein structure with myoglobin as an example a unifold, mesofold, and superfold model of protein fold use homstrad: adding sequence information to structure-based alignments of homologous protein families cleavage inhibition ofthe murine coronavirus spike protein by a furin-like enzyme affects cell-eell but not virus-eell fusion smog: de novo design method based on simple, fast, and accurate free energy estimates .1. methodology and supporting evidence identification of homology in protein structure classification multi-class protein fold recognition using support vector machines and neural networks the multiplicity of domains in proteins large macromolecular complexes in the protein data bank: a status report multi-domain proteins in the three kingdoms of life: orphan domains and other unassigned regions a study of combined structure/sequence profiles the protein folding problem: a biophysical enigma why do globular proteins fit the limited set of folding patterns? hybrid fold recognition: combining sequence derived properties with evolutionary information 3d-shotgun: a novel, cooperative, fold-recognition metapredictor protein fold recognition using sequence-derived predictions assessing the performance of fold recognition methods by means of a comprehensive benchmark assigning amino acid sequences to 3-dimensional protein folds planar graph decomposition and all pairs shortest paths structural genomics: bioinformatics in the driver's seat prediction of transcription regulatory sites in archaea by a comparative genomic approach can sequence determine function? a structural census ofgenomes: comparing bacterial, eukaryotic, and archaeal genomes in terms of protein structure how representative are the known structures of the proteins in a complete genome? a comprehensive structural census comparing genomes in terms ofprotein structure: surveys of a finite parts list fold recognition methods prospect-pspp: an automatic computational pipeline for protein structure prediction selection of representative protein data sets mapping the protein universe the fssp database: fold classification based on structure-structure alignment of proteins a hierarchical approach to all-atom protein loop prediction current topics in computational molecular biology protein secondary structure prediction based on position-specific scoring matrices genthreader: an efficient, and reliable protein fold recognition method for genomic sequences a new approach to protein fold recognition prospect ii: protein structure prediction program for genome-scale applications casp5 assessment of fold recognition target predictions the structure of the protein universe and genome evolution procheck: a program to check the stereochemical quality of protein structures the protein threading problem with sequence amino acid interaction preferences is np-complete introduction to proteinarchitecture: the structuralbiology ofproteins a unified statistical framework for sequence comparison and structure comparison emergence of preferred structures in a simple model of protein folding are protein folds atypical? designability of protein structures: a lattice-model study using the miyazawa-jernigan matrix a distance-dependent atomic knowledge-based potential for improved protein structure selection geometric cooperativity and anti-cooperativity of threebody interactions in native proteins multimeric threading-based prediction of protein-protein interactions on a genomic scale: application to the saccharomyces cerevisiae proteome protein distance constraints predicted by neural networks and probability density functions peons: a neuralnetwork-based consensus predictor that improves fold recognition threading analysis suggests that the obese gene product may be a helical cytokine comparative genomics ofthe archaea (euryarchaeota): evolution of conserved protein families, the stable core, and the variable shell how many species are there on earth improvement ofthe genthreader method for genomic fold recognition protein structure prediction by protein threading the genomic threading database: a comprehensive resource for structural annotations of the genomes from key organisms novel knowledge-based mean force potential at atomic level statistical significance of protein structure prediction by threading statistical significance of hierarchical multibody potentials based on delaunay tessellation and their application in sequence-structure alignment scop: a structural classification of proteins database for the investigation of sequences and structures protein superfamilies and domain superfolds cath-a hierarchic classification of protein domain structures a local alignment method for protein structure motifs threading with explicit models for evolutionary conservation ofstructure and sequence combination ofthreading potentials and sequence profiles improves fold recognition combinatorial optimization: algorithms and complexity new techniques in structural nmr-anisotropic interactions protein fold recognition through application of residual dipolar coupling data protein structure prediction using sparse dipolar coupling data the anatomy and taxonomy ofprotein structure graph minors .2. algorithmic aspects of tree-width protein fold recognition by predictionbased threading definition of general topological equivalence in protein structures. a procedure involving comparison of properties and relationships through simulated annealing and dynamic programming an all-atom distance-dependent conditional probability discriminatory function for protein structure prediction fugue: sequence-structure homology recognition using environment-specific substitution tables and structuredependent gap penalties calculation ofconformational ensembles from potentials ofmean force. an approach to the knowledge-based prediction of local structures in globular proteins assessment of the casp4 fold recognition category structural genomics and its importance for gene function analysis defrosting the frozen approximation: prospec-tor: a new approach to threading confidence measures for protein fold recognition tree decomposition based protein threading redesigning the hydrophobic core of a model beta-sheet protein: destabilizing traps through a threading approach the cog database: a tool for genome-scale analysis of protein functions and evolution protein structure alignment nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution protein structure prediction: bioinformatic approach assessment of progress over the casp experiments mrna cap-l methyltransferase in the sars genome what if: a molecular modelling and drug design program application of computational biology in understanding emerging infectious diseases: inferring the biological function for s-m complex ofsars-co~in progress in bioinformatics pisces: a protein sequence culling server how many fold types of protein are there in nature protein fold recognition by threading: comparison of algorithms and analysis of results nucleation, rapid folding, and globular intrachain regions in proteins model for the three-dimensional structure ofvitroneetin: predictions for the multi-domain protein from threading and docking characterization of protein structure and function at genome scale with a computational prediction pipeline sequence-structure specificity of a knowledge based energy function at the secondary structure level a tree decomposition approach to protein structure prediction assessment of raptor's linear programming approach in cafasp3 raptor: optimal protein threading by linear programming. 1 bioinform protein threading by linear programming a polynomial-time algorithm for a class of protein threading problems protein threading using prospect: design and evaluation a computational method for nmr-constrained protein threading protein threading by prospect: a prediction experiment in casp3 protein structure determination using protein threading and sparse nmr data protein domain decomposition using a graph-theoretic approach a practical method for interpretation ofthreading scores: an application of neural network an efficient computational method for globally optimal threading a graph-theoretic approach for the separation ofb and y ions in tandem mass spectra probabilistic cross-link analysis and experiment planning for high-throughput elucidation of protein structure high throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry similarities and differences between nonhomologous proteins with similar folds: evaluation of threading strategies estimating the number ofprotein folds scoring function for automated assessment of protein structure template quality distance-scaled, finite ideal-gas reference state improves structure-derived potentials of mean force for structure selection and stability prediction fold recognition by combining sequence profiles derived from evolution and from depth-dependent structural alignment offragments this research was sponsored in part by the u.s. department of energy's genomes to life program (www.doegenomestolife.org) under project "carbon sequestration in synechococcus sp.: from molecular machines to hierarchical modeling" (www genomesllife.orgj. yxandzjl'sworkwasalsosupportedinpartbynsfidbi-0354771, nsf/itr-iis-0407204, and a "distinguished cancer scholar" grant from the georgia cancer coalition. dx's work was also partially funded by nsf/eia-0325386. key: cord-280878-1kt51viz authors: to, janet; surya, wahyu; torres, jaume title: targeting the channel activity of viroporins date: 2016-01-07 journal: adv protein chem struct biol doi: 10.1016/bs.apcsb.2015.12.003 sha: doc_id: 280878 cord_uid: 1kt51viz since the discovery that certain small viral membrane proteins, collectively termed as viroporins, can permeabilize host cellular membranes and also behave as ion channels, attempts have been made to link this feature to specific biological roles. in parallel, most viroporins identified so far are virulence factors, and interest has focused toward the discovery of channel inhibitors that would have a therapeutic effect, or be used as research tools to understand the biological roles of viroporin ion channel activity. however, this paradigm is being shifted by the difficulties inherent to small viral membrane proteins, and by the realization that protein–protein interactions and other diverse roles in the virus life cycle may represent an equal, if not, more important target. therefore, although targeting the channel activity of viroporins can probably be therapeutically useful in some cases, the focus may shift to their other functions in following years. small-molecule inhibitors have been mostly developed against the influenza a m2 (iav m2 or am2). this is not surprising since am2 is the best characterized viroporin to date, with a well-established biological role in viral pathogenesis combined the most extensive structural investigations conducted, and has emerged as a validated drug target. for other viroporins, these studies are still mostly in their infancy, and together with those for am2, are the subject of the present review. the field of viroporins was born about 20 years ago from the realization that viruses inflict injuries in the membranes of cells during infection, which results in increased membrane permeability (carrasco, 1995) . this early observation heralded the current interest in viroporins, along with the therapeutic opportunities afforded by the modulation of their channel activity using small molecules (fischer, wang, schindler, & chen, 2012; scott & griffin, 2015) , and the deeper understanding of the roles of viroporin ion channel activity on viral replication and pathogenesis (nieto-torres, verdia-baguena, castano-rodriguez, aguilella, & enjuanes, 2015) . this interest will likely increase with the discovery of new viroporins along with their ever-growing roles in the virus life cycle. indeed, the number of medically important viroporins known is expected to rise as new human viruses continuously emerge from animal hosts. it is also possible that new viroporins may have novel features and structural motifs which will contribute in updating their classifications in terms of topology and number of transmembrane α-helical domains (nieva, madan, & carrasco, 2012) . in vertebrates alone, the number of viruses was estimated to be around one million (morse, 1993) . more recent estimates quantify the viral diversity in mammals, which are the reservoir hosts of the majority of emerging zoonoses, as 320,000 (anthony et al., 2013) . this strongly contrasts with the few thousand viruses identified to date, which means that more than 99% of viruses-and their viroporins, if any-are still unknown. most viroporins have been identified as virulence factors that lead to viral attenuation when deleted. this attenuation is attributed only in part to their channel activity, but nevertheless small-molecule channel inhibitors have been sought after. the vast majority of these channel inhibitors have been developed against the influenza a virus m2 (iav m2 or am2) protein (reviewed recently in du, cross, & zhou, 2012; gu, liu, & wei, 2013) , which is the first viroporin discovered. this is not surprising since am2 is the best characterized viroporin to date, with a well-established biological role in viral pathogenesis combined the most extensive structural investigations conducted, and has emerged as a validated drug target. for other viroporins, these studies are still mostly in their infancy, although a highresolution structure of the hepatitis c virus (hcv) p7 protein has been recently described (ouyang et al., 2013) that may be useful for the rational design of p7 channel inhibitors in the future. however, attempts to rationally design small-molecule inhibitors against viroporins will encounter several challenges. one is the potential for sequence variability in the viroporin. indeed, it has been argued that since the number of genes susceptible to deleterious mutations increase with genome size, mutation rates are higher for small genomes (drake, 1969) . a related problem is that viral proteins, when compared to prokaryotic and eukaryotic proteins, have a higher degree of structural flexibility that may represent adaptation mechanisms to increase resistance to random mutations, especially in the case of rna viruses, which mutate and evolve faster than dna viruses. a less dense packing, and a concomitant smaller number of network interactions, translates not only to a smaller difference in energy between folded and unfolded states, but an also smaller percentual contribution of each mutation to stability (tokuriki, oldfield, uversky, berezovsky, & tawfik, 2009 ). additionally, this enhanced flexibility may have an impact on the ability of a single protein to perform a variety of tasks, consistent with the economical use of resources in viruses. adding to this intrinsic flexibility, another problem in structure-based drug discovery concerning viroporins is the strong dependence of the protein structure on environment. indeed, solution nmr can be applied to study small-membrane proteins in membrane-mimicking environments that allow rapid reorientation, but these artificial systems may also alter the intrinsic properties of the proteins (cross, dong, sharma, busath, & zhou, 2012; cross, sharma, yi, & zhou, 2011) . last, some viroporins seem to form a protein-lipid complex rather than a purely proteinaceous pore, as shown for the vp4 protein in the triatoma virus (sanchez-eugenia, goikolea, gil-carton, sanchez-magraner, & guerin, 2015) , and similar observations have also been reported for coronavirus envelope proteins (verdia-baguena et al., 2012) . in the quest to discover small molecules targeting viroporins, amantadine (amt) can be considered to be the first viroporin channel inhibitor, and one of the first antivirals licensed for use in humans. although it was known that this adamantane was active against iav (davies et al., 1964; wingfield, pollack, & grunert, 1969) , the mechanism of inhibition was described only in 1992 (duff & ashley, 1992; pinto, holsinger, & lamb, 1992) . the target itself, am2, was not known until 1985 (hay, wolstenholme, skehel, & smith, 1985) . even today, amt, together with its methylated derivative rimantadine (rim) which was licensed in the 1980s, are the only licensed antiviral drugs that target viroporins. this contrasts with the availability of hundreds of compounds that exert their effects with subnanomolar affinities on nonviral ion channels. although this phenomenon could be explained in principle by a lack of structural data for viroporins, this is not the case for am2, where precise structural data has been obtained, even for some of its amt-resistant mutants wang, qiu, soto, & degrado, 2011) . unfortunately, an effective alternative to adamantanes has not yet been licensed. with our current knowledge, the am2 channel provides yet another paradigm valid for other viroporins. it has a very simple structure compared to ion channels from higher organisms (such as k + and ca 2+ channels), but the mechanism of proton conduction and drugs binding and inhibition are not yet fully understood. indeed, the structural and functional properties of the am2 protein vary widely in changing experimental conditions, such as peptide length, drug binding, lipid composition, lipid thickness, or ph (acharya et al., 2010; cady, wang, & hong, 2011; cross et al., 2012; kovacs, denny, song, quine, & cross, 2000; thomaston et al., 2013; zhou & cross, 2013 ). an important factor in this sensitivity is the direct contact of its tm domains with the lipid molecules, in contrast to higher organism ion channels (eg, k + channels) (cuello, jogini, cortes, & perozo, 2010) , where pore-forming α-helices are surrounded and protected by additional transmembrane helices. however, while the am2 channel exists in a variety of conformational states in the apo-form, inhibitor-binding significantly reduces the conformational flexibility of the channel. the structural dynamics of the channel represents another drawback in the use of high-resolution structures for rationally designed drug discovery. amantadine is just one of the four antivirals against iav available: two neuraminidase inhibitors (oseltamivir and zanamivir) and two am2 channel blockers (amt and rim) (vanderlinden & naesens, 2014) . the seasonal flu caused by iav is associated with high medical burden (molinari et al., 2007) and sudden pandemics with high mortality rates (hay, gregory, douglas, & yi, 2001; neumann, noda, & kawaoka, 2009 ). currently, most circulating strains are amt resistant (bright, shay, shu, cox, & klimov, 2006; bright et al., 2005; deyde et al., 2007; hayden & de jong, 2011) and therefore the use of amt and rim has been discontinued for the human population (fiore et al., 2011) . the am2 channel is a homotetramer (sakaguchi, tu, pinto, & lamb, 1997) , where each monomer has 97 amino acids containing one α-helical tm domain (residues 25-46). the extramembrane c-terminal domain (residues 47-97) is exposed to the cytoplasmic side (lamb, zebedee, & richardson, 1985) and contains an amphipathic helix (residues 51-59). four of these amphipathic helices stabilize the tetrameric channel, forming a base on the membrane surface almost perpendicular to the tm bundle. the end of this c-terminal tail is disordered and interacts with the viral matrix protein 1 (m1). the m2 protein performs several critical roles for virus replication (pinto & lamb, 2006) . for instance, m2-mediated acidification of the interior of the virus is required for the uncoating of genetic material inside the cell (pinto et al., 1992) . m2 is also required to regulate the intralumenal ph of the golgi apparatus preventing the premature conformational change of hemagglutinin (takeuchi & lamb, 1994) . influenza b, the closest relative of the influenza a virus, accounts for about 50% of all influenza disease in recent years (according to the us centers for disease control and prevention website, www.cdc.gov) and has a similarly essential ph-activated proton channel m2 viroporin, the bm2 (hatta, goto, & kawaoka, 2004; pinto & lamb, 2006) . although am2 and bm2 share almost no sequence identity, both have a single tm domain and form homotetramers wang, pielak, mcclintock, & chou, 2009 ). in comparison, bm2 is completely insensitive to amt or rim (mould et al., 2003; pinto & lamb, 2006) and no bm2 inhibitors have been identified yet. the location of the binding site for the adamantanes amt and rim in am2 was initially controversial. a crystallographic structure suggested a pore-binding model in the amino-terminal part of the tm (22-46), ie, the p-binding site (stouffer et al., 2008) (fig. 1a -c). in the same and in the following year, a solution nmr-based structure of a longer construct of am2 (18-60) (pielak, schnell, & chou, 2009; schnell & chou, 2008) proposed a surface-binding site (the s-binding site) for rim, on the carboxy-terminal, lipid-facing surface of the helices (fig. 1d-g) , suggesting an alternative allosteric mechanism of inhibition. in this allosteric binding site, the adamantane group interacted favourably with the hydrophobic side chains of leu-40, leu-43, and ile-42, whereas the positively charged ammonium group interacted with the polar patch formed by figure 1 p-and s-binding sites for amt and rim to iav m2. (a-c) am2 tm domain crystallized in detergent, with the most critical residues identified by site-directed mutagenesis lining the am2 pore (a); omit map showing electron density in the amt binding region (b); structure of amantadine (nitrogen in cyan (light gray in the print version) and carbon in white) inside the binding site showing the surface associated with residues val-27 (red (light gray in the print version) surface), ala-30 (green (dark gray in the print version)), ser-31 (blue (light gray in the print version)), and gly-34 (orange (light gray in the print version)) (c); (d and e), an ensemble of 15 low-energy structures derived from nmr restraints, with tm α-helices (residues 25-46) and amphipathic helices (residues 51-59) superimposed separately (d); ribbon representation, showing the drug rim (colored in red (light gray in the print version)) (e); (f and g), surface representation of the rim-binding pocket, showing the asp-44, the indole amine of trp-41, and arg-45, which form the polar patch, as well as the hydrophobic wall composed of solution nmr structures of the (am2-bm2)tm channel in the absence and presence of rim; ensembles of 15 low-energy structures of drugbound chimera channels. rim is highlighted in red (light gray in the print version) (h); ribbon representation of drug-free (am2-bm2)tm tetramer (left), and overlay of its am2 and bm2 regions (green (dark gray in the print version)) with the corresponding regions (yellow (light gray in the print version)) of the am2 (pdb code: 2rlf) and bm2 (pdb code: 2kix) structures (right) (i); overlay of the drug-free (white) and the drugbound (cyan (light gray in the print version)) chimera structures, showing substantial differences in helical packing (j). adapted by permission from macmillan publishers ltd.: stouffer, a. l., acharya, r., salom, d., levine, a. s., di costanzo, l., soto, c. s., et al. (2008) . asp-44 and arg-45 from the adjacent subunit (fig. 1g) . a dilemmatic feature of allosteric sites is that viral mutations at these sites are easily accommodated by viruses without dramatic loss of fitness. in hcv, for example, protease inhibitor monotherapy can rapidly select resistant viral populations within a few days (schmidt et al., 2014) . however, clear support for the p-binding site was obtained from solidstate nmr in lipid bilayers (pdb id: 2kqt) (cady et al., 2010) ; the inhibitor was found in a hydrophilic pocket formed by ala-30, ser-31, and gly-34, ie, occluding the am2 channel. in this case, hydrophobic interactions were detected between the adamantane group and val-27 side chains, whereas the ammonium group was hydrogen-bonded to pore-facing residues and water molecules. the latter work revealed that the s-binding site was in fact just a low-affinity site, and only observed at high concentrations of the drug in the membrane. importantly, amt was found to undergo significant motion in the n-terminal lumen, suggesting that its structure can be improved for a better fit to the am2 channel. this pore-location (p-binding) was also further validated by chou et al. by solution nmr (pielak, oxenoid, & chou, 2011) using an m2 chimeric sample, where the n-terminal part of the m2 tm domain corresponded to that of the am2, whereas the c-terminal part was from the amt-insensitive bm2 ( fig. 1h -j). in that model, methyl groups of val-27 and ala-30 from four subunits form a hydrophobic pocket around the adamantane, with the amino group in polar contact with the backbone oxygen of ala-30. this ended the controversy, with the acceptance of the initially proposed p-binding site. there are 12 structures of the wild-type and drug-resistant mutant m2 channels available in the protein data bank solved by different techniques and conditions (see summary in gu et al., 2013) . among these, structure 2rlf solved in micelle (schnell & chou, 2008) and structure 2l0j in the bilayer environment contain, in addition to the tm domain, short c-terminal intracellular amphipathic helices. in 2rlf, the c-terminal helices were connected to the transmembrane helices via a short loop (residue 47-51) and were exposed to the solution. however, in 2l0j, the c-terminal helices were connected to the tm helices through a rigid turn (residue 47) and were positioned on the bilayer surface (sharma et al., 2010) , and this conformation was stabilized by extensive hydrophobic interactions among these helices, and polar residues asp-44 and arg-45 were buried by hydrophobic residues of the intracellular amphipathic helices, making the s-binding site more hydrophobic than that in micelles. therefore, the s-binding site was absent in 2l0j (sharma et al., 2010) . molecular dynamics simulations of the m2 proton channel with inhibitors binding at different sites have shown that the p-binding site is more stable for drug binding, but a higher energy barrier needs to be overcome for binding to occur (gu et al., 2011) . the s-binding site was less stable for drug binding but it was nearly barrier less and was easily accessed. therefore, the p-binding site represents the thermodynamic binding site where the drug molecule binds slowly and stably and dissociates even more slowly, whereas the s-binding site is a kinetic binding site where the drug molecule binds readily but less stably and dissociates easily. over the last four decades, systematic studies of amantadine analogues and library screening have elucidated structure-activity relationships (sars) and helped to identify potent wild-type (wt) am2 channel blockers (de clercq, 2006; lagoja & de clercq, 2008) . several other molecules were not based on adamantanes. for example, the spirene guanidine analogue, 2-[3-azaspiro(5,5)undecanol]-2-imidazoline (bl-1743) ( fig. 2a ) was discovered during a high-throughput screen based on the ability of inhibitors to reverse the toxicity associated with m2 channels expressed in the yeast saccharomyces cerevisiae (kurtz et al., 1995) . based on this nonadamantane, a sar study using a combination of viral plaque assays and two-electrode voltage clamp (tevc) (wang, cady, et al., 2009 ) generated 3-azaspiro[5,5]undecane hydrochloride (fig. 2b) , which lacks the (wang, cady, et al., 2009). imidazoline group of bl-1743. this compound showed an ic 50 of 0.92 ae 0.11 μm against am2, ie, more than an order of magnitude more potent than amantadine (ic 50 ¼ 16 μm) and more than 45-fold increase in potency relative to the parental bl-1743. compared to amt, this spiro-piperidine induced a more homogeneous conformation of the am2 peptide, reducing dynamic disorder near the water-filled central cavity of the helical bundle, suggesting a more extensive binding to the am2 channel, thus leading to stronger inhibitory potency. however, this compound was only effective against the wild-type am2. this is crucial because of the current prevalence of amt-resistant variants of am2, eg, l26f, v27a, and s31n, which comprise more than 99% of the reported resistance mutants (furuse, suzuki, & oshitani, 2009; suzuki et al., 2003) . of these three mutations, v27a is the only one known to originate from drug selection pressure (furuse et al., 2009) , and this mutant is the only one among the three that is completely resistant to amt and rim ). on the other hand, s31n is found in more than 98% of the mutated m2 channels in the iav subtype h3n2 viruses (bright et al., 2005) , and causes a 10-20 times decrease in the ic 50 , from low micromolar to 200 μm (amt) and more than 2 mm (rim) . the first non-adamantane inhibitor of the v27a mutant was a polycyclic pyrrolidine, a dual inhibitor of wt (ic 50 of 3.4 μm) and v27a (ic 50 of 0.29 μm) (rey-carrizo et al., 2013). later, a compound with triple inhibitor efficacy was reported by the same authors, active against the wt, v27a, and l26f mutants, with ic 50 of 18, 0.7, and 9 μm, respectively (rey-carrizo et al., 2014) . the inhibitory activity of the compounds was tested on am2 channels expressed in xenopus laevis oocytes using the two-electrode voltage clamp (tevc) technique, but most of the compounds were cytotoxic (more than amt and rim), as tested in cell-based antiviral plaque reduction assays. therefore, despite the encouraging results obtained in vitro, the toxic properties of these compounds still hinder their therapeutic potential. the spiro-piperidine inspired the generation of another compound ( fig. 3c ) that could inhibit not just the wt am2 (ic 50 ¼ 18 μm) but also mutants l26f (ic 50 ¼ 6 μm) and v27a (ic 50 ¼ 0.3 μm) (wang, ma, fiorin, et al., 2011) . direct interaction of this compound with the v27a mutant was assessed by solid-state nmr, and inhibition was further demonstrated in plaque reduction assays. this compound, however, was inactive against the s31n mutant. such observation was rationalized by the fact that the s31n mutant is more dynamic and hydrated in the pore than the corresponding wt protein , as shown by nmr studies. this lead to a disruption in the size and polarity of the pore in precisely the region that accommodates the adamantane group, thereby hampering the discovery of inhibitors of s31n. this led to attempts to increase the polarity or the dimensions of the adamantane core in order to capture more interactions with the backbone of the channel. a variety of scaffolds have been used to substitute for the hydrophobic adamantane group (hu et al., 2010; wang, ma, balannik, et al., 2011) . these compounds showed excellent activity against the wt am2, and some were highly active against v27a and l26f (wang, ma, fiorin, et al., 2011) . however, none was better than amt against s31n. attempts to introduce additional groups to the amine to enhance affinity for both s31n and wt via additional electrostatic interactions led to the discovery of benzyl-substituted amantadine derivatives ( fig. 3d ) as dual wt and s31n inhibitors with ic 50 of 35 and 59 μm for s31n and wt, respectively. the degrado group later discovered small-molecule drugs, eg, m2wj332, that locked the dynamic s31n mutant into a well-defined conformation, enabling high-resolution structure determination by solution nmr that showed the drug bound in the homotetrameric channel, threaded between the side chains of asn-31. these compounds were formed by conjugation of a-ch2-heteroaryl group to the amine of amt ( fig. 3e and f, ic 50 of 16 μm) and inhibited s31n with potencies greater than amt against am2 . the charged ammonium binds as a hydrate to one of three sites aligned along the central cavity that might stabilize hydroniumlike species formed as protons diffuse through the outer channel to the proton-shuttling residue his-37 near the cytoplasmic end of the channel. ; (e and f) compounds with ic 50 of $16 μm ; (g) compound found in wu et al. (2014) . based on structural and molecular dynamics data, the degrado group realized that inhibitors that are active against both wt and s31n channels bind in opposite orientations: the aromatic headgroup faces toward the c-terminus in wt, and toward the n-terminus in s31n. investigation of substitutions led to the identification of halide-substituted thiophene compounds. the most potent of which, n-[(5-bromothiophen-2-yl) methyl]adamantan-1-amine (fig. 3g ), inhibited both wt and s31n mutant with comparable affinities as amt against wt (wu et al., 2014) . it would be desirable to develop the same paradigm found in iav am2 and their inhibitors for other viroporins, so that their channel inhibitors can be validated. for example, many amantadine-resistant influenza viruses can be selected in cell culture (grambas, bennett, & hay, 1992; hay et al., 1985) , and some of these are also found in infected patients undergoing treatment with amantadine (shiraishi et al., 2003) . in turn, engineered viruses harboring these pore-lining mutations, while competent to replicate in mouse (abed, goyette, & boivin, 2005) , give rise to attenuated viruses that tend to revert to the wt in the absence of drug pressure suzuki et al., 2003) . another very important viroporin is p7, found in the hepatitis c virus (hcv), a member of the flaviviridae family that has six genotypes (gts). hcv is a positive-strand rna virus that causes severe liver disease and is the major cause of hepatocellular carcinomas in the developed world. hcv has chronically infected 170 million people worldwide and is responsible for more than 300,000 yearly deaths, while no prophylactic or therapeutic vaccine is available. hcv infection can be cured with drugs targeting viral enzymes and proteins, as well as host cellular proteins. one host-targeted antiviral agent (htas) is the type i ifn-α (hoofnagle et al., 1986) , which exerts antiviral activities against both rna and dna viruses through ifn-stimulated gene (isg) products. pegylated ifn has been used in combination with ribavirin (rbv), a synthetic guanosine analogue (herrmann, lee, marinos, modi, & zeuzem, 2003) , but this antiviral therapy can only last up to 1 year, associated with side effects, and is not always effective (manns, wedemeyer, & cornberg, 2006; pawlotsky, chevaliez, & mchutchison, 2007) . other drugs target hcv proteins with high potency, such as those against the hcv ns3/4a protease, the ns5a protein or the ns5b rna-dependent rna polymerase (rdrp) (sulkowski et al., 2014) [see pawlotsky, 2014; schmidt et al., 2014 , for recent reviews]. however, drug resistance creates a burden for patients, worsened by the rapid turnover of hcv replication, which produces up to 10 12 virions daily (neumann et al., 1998) . this is compounded by the error-prone activity of the hcv rna polymerase, which leads to the high genetic diversity of hcv and the formation of "quasi-species." in this complicated context, the clinical niche of p7 inhibitors is unclear. the viroporin hcv p7 is encoded within a single polyprotein precursor (simmonds, 2013) processed by host and viral proteases (moradpour & penin, 2013) . it separates the structural proteins (core and envelope glycoproteins e1 and e2) from the nonstructural proteins ns2, ns3, ns4a, ns4b, ns5a, and ns5b (lindenbach & rice, 2005) . p7 results from cleavage of e2-p7-ns2 and e2-p7 by a signal peptidase at the er (lin, lindenbach, prágai, mccourt, & rice, 1994; mizushima et al., 1994) . the p7 protein is 63 residues long with two tm domains, found mainly at the er membrane. although p7 is not necessary for rna replication (lohmann et al., 1999) , it is essential for productive hcv propagation in vivo . however, the precise role of the p7 in virus production was only determined when it was possible to replicate a genotype 2a isolate from a japanese patient suffering from fulminant hepatitis (designated jfh-1) (kato et al., 2003; wakita et al., 2005) . this isolate efficiently replicated in human hepatoma cell lines and produced infectious virus particles. this was used to demonstrate that p7 is essential for virus particle assembly and release (jones, murray, eastman, tassello, & rice, 2007; steinmann et al., 2007) . the bovine viral diarrhea virus (bvdv) (harada, tautz, & thiel, 2000) and the hepacivirus gb virus b (gbv-b) (takikawa et al., 2006) , hcv's closest relatives, also have a p7 protein crucial for virus replication. the channel activity of p7 was examined more than 10 years ago in experiments involving artificial lipid bilayers. it was suggested that p7 has preference for cations vs anions, with permeability ratios in the range 7-11 (griffin et al., 2003; montserret et al., 2010; premkumar, wilson, ewart, & gage, 2004; pavlovic et al., 2003) and more recently in xenopus oocytes (ouyang et al., 2013) . this selectivity is rather low compared to other channels, eg, for cystic fibrosis transmembrane conductance regulator (cftr) cl à channels, selectivity for anions over cations is 10-30 (anderson et al., 1991; bear et al., 1992) . for potassium channels, which conduct k + ions near the diffusion limit, the selectivity for k + over na + is about 10,000-1 (doyle et al., 1998) , and in aquaporins water to ion flux ratio is about 10 9 (pohl, 2004) . however, the biological importance of p7 cation channel activity may be relative, in the light of its recently found role to permeabilize membranes to protons, and acting to prevent acidification of intracellular vesicles (wozniak et al., 2010) . this proton channel activity was found to be crucial for the production of infectious viruses, and was observed in intracellular vesicles harboring p7. recently, this proton channel activity has been directly confirmed in vitro using purified nontagged p7 protein in a liposome-based assay (gan, surya, vararattanavech, & torres, 2014) , where the lipid composition used was the mixture paesc (pa/pe/ ps/pc 5:2:2:1 w/w) (gervais et al., 2011) . several initial papers also reported the inhibition of channel activity by some compounds. for example, his-tagged p7 from genotype 1b was inhibited by 1 μm amantadine (griffin et al., 2003) , whereas synthetic p7 from genotype 1a (strain h77) was inhibited by 100 μm hexamethyleneamiloride (hma, fig. 4a ), although this peptide was largely unpurified (premkumar et al., 2004) . another synthetic p7, also unpurified, was inhibited by 50-100 μm of the long-alkyl-chain imino sugar derivatives nn-dgj, nn-dnj (fig. 4b ) and n-7-oxanonyl-6-deoxy-dgj (pavlovic et al., 2003) . however, the channel activity of purified synthetic p7 hcv-j genotype 1b in artificial lipid bilayers was inhibited by just 50% in the presence of 200 μm hma, whereas amt did not show any effect even at 1 mm concentration (montserret et al., 2010) . in fact, the efficacy of amt in hcv culture systems is low (steinmann et al., 2007) , and no clinical benefit of amt-based combination therapy was found in comparison to standard of care (peg-ifnα plus ribavirin) (castelain et al., 2007; mihm et al., 2006) . some of these compounds have been studied in infected cells, where they reduced virus production up to 10-fold in a genotype-dependent manner (foster et al., 2014; gottwein et al., 2011; steinmann et al., 2007) , reducing secretion of hcv particles by inhibition of the acidification of viruscontaining compartments (wozniak et al., 2010) , without impairment of the infectivity of intracellular virions (foster et al., 2011; griffin et al., 2008) . to discover more p7 channel inhibitors, a liposome-based dye (carboxyfluorescein, cf) release assay has been proposed that involves the addition of p7 protein to preloaded liposomes (stgelais et al., 2007) . the method has been claimed to be sensitive to inhibitors such as amt and rim, and proposed as a high-throughput functional assay to test drug sensitivity of p7 mutants, or to discover new p7 channel activity inhibitors (foster et al., 2011; gervais et al., 2011) . these dye release assays have been previously performed with tagged p7 protein, either with flag (li et al., 2012; stgelais et al., 2009) or flu-antigen (foster et al., 2011) . however, this method has been put into question since just the c-terminal half of p7, p7(27-63) was as efficient as full-length p7 in releasing cf, while a properly reconstituted α-helical sample of p7 was not able to elicit cf release (gan et al., 2014) . nevertheless, p7 inhibitors may be able to inhibit both the proton channel activity and dye release if the mechanisms are similar, eg, via an allosteric effect that increases the rigidity of the c-terminal half (tm2) of p7. this is supported by the fact that tm2 of p7 interacts with amt and is the site of inhibition by adamantanes. indeed, amt interacts with leucines 51-57, and these leucines have been shown unequivocally to be part of a membrane inserted α-helix (cook & opella, 2009; ouyang et al., 2013) . binding of amt to p7 in dihexanoylphosphatidylcholine (dhpc) micelles led to resonance shifts in several residues, some of which are the leucines 51-57 (but not 53), ie, located in tm2 (cook & opella, 2009) . consistent with this, leu to ala mutations at this region produced an amt-resistant p7 mutant revealed using the cf release assay (stgelais et al., 2009) . the imino sugar nn-dnj and rim inhibited secreted infectivity in amt-resistant jfh-1 and con-1/jfh-1c3 viruses, whereas mutation l20f conferred resistance to rimantadine (foster et al., 2011) , consistent with rim resistance was found in gt1b patients not responding to the triple therapy ifn/rib/amt (mihm et al., 2006) . using an oligomerization assay in dhpc gels, the gt1b j4 p7 oligomerization was abolished in presence of imino-sugars, whereas rim did not affect oligomerization, suggesting two different mechanisms of action for these two compounds (foster et al., 2011) . the p7 of gt3a, but not gt1b, was found to be resistant to nn-dnj both in vitro and in culture, and this was attributed to a f25a mutation (foster et al., 2011; griffin et al., 2008) . however, direct interaction between hma or imino sugar derivatives with p7 reconstituted in a detergent or membrane has not been reported. addition of rim to gt1b j4 p7 monomer in methanol (foster et al., 2014) showed disruption at several residues, but not especially at leu-20. thus, this observation is either inconsistent with the proposed role of l20f in acquired resistance, or the methanol environment is not a good system to test this interaction. structural data for p7 was obtained initially in solvents and micelles by solution nmr. for example, solution nmr data was obtained for synthetic p7 (c27a mutant) in 50% of the helix inducer trifluoroethanol (tfe) (montserret et al., 2010) , and for recombinant p7 (c27s mutant) in dhpc micelles ). the prevalent model for the p7 monomer according to these studies was that of an α-helical hairpin with two α-helical tms kinked in the middle (cook, zhang, park, wang, & opella, 2010; montserret et al., 2010) , where the n-terminal tm helix (tm1) would face the lumen of a channel (carrere-kremer et al., 2002; chew, vijayan, chang, zitzmann, & biggin, 2009; patargias, zitzmann, dwek, & fischer, 2006 ) formed by either six or seven monomers (clarke et al., 2006; griffin et al., 2003; luik et al., 2009; montserret et al., 2010) . in a more recently published structural model of p7 (ouyang et al., 2013) , the oligomeric size is still ambiguous since this data could not be obtained in the same environment used in electron microscopy. from that model, the "old" tm1 and tm2 domains would correspond to three helical segments h1-h3, ie, h1 and first half of h2 (tm1) and second half of h2 and h3 (tm2). there are concerns, however, that this model can be present in a physiological context since the folding implied is difficult to explain by our current understanding of protein folding in the cell (madan & bartenschlager, 2015) . in the ouyang et al. paper, the authors systematically tested p7 amino acid sequences from various hcv genotypes and found that the sequence from gt5a (euh1480 strain) generated samples adequate for structure determination. although the oligomeric size of p7 in dodecyl phosphocholine (dpc) micelles was not determined, this was combined with data from negative stain electron microscopy (em), which showed hexameric, flower-shaped particles. the latter were similar to those p7 hexamers (of gt2ajfh-1) observed in em in the presence of dhpc micelles used earlier for single-particle reconstruction (luik et al., 2009) . the nmr-based model showed that the c-terminal helix, p7(27-63), is not the pore-lining sequence but instead forms a "lipid facing" part of the molecule, whereas the n-terminal half would orient lining the lumen (ouyang et al., 2013) . this is in agreement with previous models based on the partial inhibition of p7 channel activity by cu 2+ , but not by mg 2+ , which suggested exposure to a his residue located in the n-terminal half to the lumen of the channel . although this interpretation may be correct, it is unfortunate that synthetic peptides corresponding to the n-terminal half "tm1," eg, p7(1-34) or the c-terminal half "tm2," eg, p7(35-63) aggregate easily, such that individual channel activity of these peptides cannot be properly measured (montserret et al., 2010) . in fact, neither of these helices separately forms α-helical structure in micelles or membranes, and peptide p7(1-26) was totally insoluble in tfe or in detergent (gan et al., 2014) , a difficulty also encountered for the n-terminal fragment of p7(1-34) (montserret et al., 2010) . in contrast, tm domains of other viroporins are completely α-helical, eg, the iav m2 (torres, kukol, & arkin, 2000) , severe acute respiratory syndrome coronavirus (sars-cov) e , or respiratory syncytial virus (rsv) sh (gan et al., 2012) produce sharp amide i bands in the infrared spectrum consistent with $100% α-helix. nevertheless, the amt binding site in the p7 channel was determined by identifying nuclear overhauser enhancements (noes) between the protein backbone amide protons and drug protons (ouyang et al., 2013) (fig. 5) . although the full-scale structure determination of the complex could not be achieved, amt (10 mm) showed noe cross-peaks between the adamantane protons and the amide protons of val-26, leu-55, leu-56, and arg-57, ie, between the pore-forming and peripheral helices, suggesting a pocket formed by leu-52, val-53, and leu-56 from h3, and phe-20, val-25, and val-26 from h2, with the polar amino group of amt pointing to the channel lumen, with similar results also obtained for rim. amt and rim have also been reported previously to interact with some residues in the n-terminal half (cook & opella, 2009; stgelais et al., 2009) . isothermal titration calorimetry and nmr chemical shift perturbation analyses of p7(gt5a)-rim interaction produced a binding constant (kd) from 50 to 100 μm at 3 mm detergent concentration. the authors proposed that large differences in drug efficacies observed between different hcv genotypes are probably due to variations in the hydrophobicity of the binding pocket among p7 variants, and that the adamantane derivatives inhibit channel activity by restricting the structural rearrangement of the channel, ie, an allosteric mechanism. perhaps the most efficient effect for a p7 inhibitor has been reported for bit225 (n-[5-(1-methyl-1h-pyrazol-4-yl)-napthalene-2-carbonyl]guanidine) (fig. 4c ). this amiloride was identified in a bacterial assay that involved expression of p7 in e. coli; only in presence of inhibitors the normal ionic gradients are maintained and cells are able to grow despite the presence of p7. this compound had antiviral activity against the hcv model pestivirus bovine viral diarrhea virus (bvdv) with an ic 50 of 314 nm. the addition of 100 μm of bit225 blocked ion channel activity of synthetic hcv p7 protein of gt1a h77, although no data was reported in the context of hcv infection . while some docking efforts have been performed (bichmann, wang, & fischer, 2014) , direct interaction of bit225 with p7 has not been proven using biophysical or structural assays, and therefore rational optimization of the compound is still not possible. in addition to hcv p7, the human immunodeficiency virus (hiv) viroporin vpu is also targeted; bit225 was tested against the vpu tm domain using a black lipid membrane (blm) system, where the target was exposed to 40 μm of the drug (khoury, ewart, luscombe, miller, & wilkinson, 2010) . although direct interaction with vpu was not observed, this was assumed since the drug was not active against hiv-2, which has no vpu gene. one early clinical trial (phase ib/iia) involving the treatment of 24 naïve gt1 hcv patients indicated a significant reduction of viral load after 12 weeks treatment with bit225 in combination with ifnα/ribavirin (www.biotron.com.au: biotron 2011). currently, a phase 2a/2 clinical trial is in progress and the first results indicate activity against several hcv genotypes, including the difficult-to-treat gt3a and gt1a (www.biotron.com. au; may 29, 2015). since it is also active against the hiv-1 vpu, bit225 might be suitable for treatment of hcv/hiv coinfected patients. in a jul. 2015 update of a 3-month dosing trial with bit225, there was a recommendation that future trials would focus on patients infected with hcv gt3 and to further study bit225 in combination with other direct-acting antiviral drugs. in these trials, patients received 400 mg of bit225 twice daily for 3 months. a phase 2a trial on hcv demonstrated that 100% of the patients infected with hcv gt1 who received bit225 (400 mg) in combination with current standard of care therapies, ifn/rbv, had undetectable virus after 48 weeks. a phase 2 trial in hiv/hcv coinfected patients showed that all hcv gt3 patients completing 28 days of treatment with bit225 in combination with ifn/rbv have undetectable hcv load for 12 weeks (sustained viral response, svr12) after completing all therapy. bit225 is also in development for treatment of hiv, and is the pioneer in a new class of antiviral drugs that may provide a new approach to the eradication of this virus. it has shown clinical efficacy against hiv in reservoir cells, and has the potential to be combined with new or existing antiretroviral drugs to eradicate long-lived pools of virus that are not successfully eliminated with current treatments. despite these promising results, however, it remains to be demonstrated that bit225 targets p7 or vpu in infected cells. the most studied viroporin in coronaviruses is the envelope (e) protein. coronaviruses (cov) typically affect the respiratory tract and gut of mammals and birds. covs belong to the subfamily coronavirinae in the family coronaviridae, and are organized into four genera (enjuanes et al., 2000) : α, β, γ, and δ. approximately 30% of common colds are caused by two human coronaviruses-oc43 and 229e. of particular medical interest is the virus responsible for the severe acute respiratory syndrome (sars), which produced a near pandemic in 2003 (holmes, 2003) , and the recently emerged middle east respiratory syndrome coronavirus (mers-cov), which after 3 years has caused hundreds of deaths (raj, osterhaus, fouchier, & haagmans, 2014) . other coronavirus viroporins, eg, the sars-cov3a (lu et al., 2006) , a 274-amino acid protein with three putative tm domains, are also attracting increasing interest (chien et al., 2013; hsu & fischer, 2012) , but no inhibitors or structural data is available so far. currently, no effective licensed treatment exist against coronavirus infection (kilianski & baker, 2014; kilianski, mielech, deng, & baker, 2013; lou, sun, & rao, 2014) , although live vaccines consisting of attenuated viruses is a promising strategy (enjuanes, nieto-torres, jimenez-guardeno, & dediego, 2011; graham et al., 2012) , along with fusion inhibitors (reviewed in heald-sargent & gallagher, 2012) . even so, the possibility of reappearance of virulent phenotypes, drug side effects, and resistance calls for continued antiviral development. the envelope (e) proteins are 76-109 residues long with one tm domain (li, surya, claudine, & torres, 2014; parthasarathy et al., 2008 parthasarathy et al., , 2012 pervushin et al., 2009; torres et al., 2006; torres, wang, parthasarathy, & liu, 2005) , and most of them have a cytoplasmic c-terminal domain and a lumenal n-terminus (corse & machamer, 2000; nieto-torres et al., 2011; raamsman et al., 2000; ruch & machamer, 2012) . most cov e proteins are present at low concentrations in virions (corse & machamer, 2000; godet, l'haridon, vautherot, & laude, 1992; liao, yuan, torres, tam, & liu, 2006; yu, bi, weiss, & leibowitz, 1994) , but found abundantly in internal membranes of infected hosts (corse & machamer, 2003; liao et al., 2006; raamsman et al., 2000; tung et al., 1992) , eg, the er-golgi intermediate compartment (ergic), where virions assemble (lopez, riffle, pike, gardner, & hogue, 2008; nieto-torres et al., 2011) . cov e proteins have been found to be critical for viral pathogenesis, while having a protective antiapoptotic effect on infected cells which may help the virus evade premature death of host cells, thereby allowing viral replication. interestingly, deletion of e protein reduced pathogenicity and mortality in animal models through a reduced inflammation (dediego et al., 2014)-as discussed later-and this has led to the development of live attenuated vaccines based on e-deleted or e-truncated virions (almazán et al., 2013; lamirande et al., 2008; netland et al., 2010) . the only structural data available for a cov e protein is for sars-cov e, where the tm domain (e-tm) has been characterized in some detail in lipid membranes . later, solution nmr of the selectively labelled synthetic e-tm (residues 8-38) in dpc micelles produced a similar pentameric left-handed parallel bundle (pervushin et al., 2009) . in these models, asn-15 is facing the lumen of the channel whereas val-25 is involved in helix-helix interactions with other subunits (pervushin et al., 2009) . mutations at these residues, n15a and v25f, abolished channel activity in vitro (torres et al., 2007) , and introduction of these mutations in a recombinant sars-cov resulted in in vivo attenuation in a mouse model. the integrity of the tm domain, and preservation of channel activity, were shown to be important for inflammasome activation and elevated production of the proinflammatory cytokine il-1β (nieto. in the same study, revertant mutants of the channel-inactive e that regained fitness and pathogenicity also regained channel activity, as measured in black lipid membranes. both e-tm and the full-length e protein form homopentameric channels with poor ion selectivity that can be partially inhibited by the drug hma with micromolar affinity (verdia-baguena et al., 2012; wilson, gage, & ewart, 2006) . the noes between hma and e-tm suggested the presence of two binding sites at both ends of the tm domain. at the n-terminal domain, leu-19 exhibited the largest chemical shift, and the relative intensities of the hma proton cross-peaks indicated an hma:e-tm stoichiometry of 1:5 at the n-terminal binding site near asn-15, suggesting one hma molecule per e-tm pentamer. this is consistent with a pore-blocking inhibition mode similar to amt in am2, where in this case the hma molecule may be stabilized by a hydrogen bonding network to the asn-15 side chains of the sars-cov e, with the cyclohexamethylene ring pointing away from the center of the channel (fig. 6a) . at the c-terminal of the tm domain, near arg-38, the hma:e-tm stoichiometry was 1:2, suggesting for this site a rapid exchange, in the chemical shift timescale, between e-tm-bound and micelle-bound forms of hma. at this location, the amiloride group of hma is likely to be involved in interactions with the guanidinium groups of arg-38 (fig. 6b) . these results were confirmed using the extended peptide e (8-65) in mixed sds/dpc (1:4 molar ratio) micelles , where the addition of hma perturbed residues clustered near the n-terminal end of the tm domain, eg, . at the c-terminal end of the tm, leu-37 was the most affected, suggesting that the interaction of hma at the hε of arg-38 reported previously may have been an artifact due to the use of an e-tm (8-38) peptide. in any case, these results suggest a similar behaviour of hma-to-e protein and amt-to-am2 protein, ie, the presence of two binding sites, one pore-binding and one surface-binding in the tm domain. this structure provided a possible rationale for inhibition and a platform for future structure-based drug design of this potential pharmacological target. the small hydrophobic (sh) protein is found in the human respiratory syncytial virus (hrsv), an enveloped pneumovirus in the paramyxoviridae family. hrsv is the leading cause of bronchiolitis and pneumonia in infants and elderly (dowell et al., 1996) , and the most frequent cause of hospitalization of infants and young children in industrialized countries. in developing countries, hrsv is a significant cause of death, with global estimates of more than 70,000 deaths in young children. hrsv is the third most important cause of deadly childhood pneumonia after streptococcus pneumoniae and haemophilus influenzae (nair et al., 2010) . several compounds target the rsv fusion (f) protein, which facilitates viral entry through the host cell membrane (zhao, singh, malashkevich, & figure 6 binding of hma to the sars-cov e-tm pentameric channel (pervushin et al., 2009 ). (a) side view of the binding of hma to the vicinity of asn-15. the side chains of amino acids interacting with hma are shown using a stick representation; (b) binding of hma to the c-terminal binding site of the channel, in the vicinity of thr-35 and arg-38. for clarity, one of the e-tm monomers has been removed. kim, 2000) through formation of a 6-helix bundle (douglas et al., 2005; lambert et al., 1996; pastey, gower, spearman, crowe, & graham, 2000; razinkov, gazumyan, nikitenko, ellestad, & krishnamurthy, 2001; roymans et al., 2010; shepherd et al., 2006) . other approaches have involved gene transfer to expose viral proteins to cells (kumar et al., 2002) , or sirna against specific viral proteins (zhang et al., 2005) . vaccines have been recently designed based on a stabilized rsv-f form which preserves a highly antigenic site in its prefusion state, yielding rsv-specific neutralizing antibodies in mice and macaques (mclellan, chen, joyce, et al., 2013; mclellan, chen, leung, et al., 2013) . recently, a vaccine candidate based on the extracellular domain (c-terminal) of the rsv viroporin, the small hydrophobic (sh) protein, has been reported (schepens et al., 2014) . in addition, the prevention of nasopulmonary infection in mice caused by rsv has been reported using stapled peptides targeting the fusogenic f-protein 6-helix bundle . however, despite all these efforts, new fda-approved drugs have yet to emerge. the only licensed drug for use in infected individuals is ribavirin, a nucleoside analogue, but its efficacy is very limited (hall et al., 1986) . the hrsv genome transcribes 11 proteins (collins & melero, 2011) . one of these is the viroporin sh protein, which is 64-65 residues long (depending on subgroup, a or b) and has a single α-helical tm domain with n-and c-terminal extramembrane domains oriented cytoplasmically and lumenally/extracellularly, respectively (collins & mottet, 1993; gan et al., 2012) . most sh protein accumulates at the membranes of the golgi complex in infected cells, but it has also been detected in the er and plasma membranes (rixon et al., 2004) . rsv that lacks sh (rsvδsh) is still viable, and still forms syncytia (bukreyev, whitehead, murphy, & collins, 1997; karron et al., 1997; techaarpornkul, barretto, & peeples, 2001) , but is attenuated in vivo. in mouse, rsv δsh replicated 10-fold less efficiently in the upper respiratory tract (bukreyev et al., 1997) , whereas chimpanzees developed significantly less rhinorrhea than those infected with wild-type rsv (whitehead et al., 1999) . other reports have shown that the lack of sh protein leads to an attenuated phenotype in children and in rats (jin et al., 2000; karron et al., 1997) . overall, these results indicate involvement of hrsv sh protein in replication and pathogenesis. in common to the sars-cov e protein, sh protein blocks or delays apoptosis in infected cells (fuentes, tran, luthra, teng, & he, 2007) , and a similar antiapoptotic effect of sh protein has been observed for other members of the paramyxoviridae family, eg, j paramyxovirus (jpv) (jack, boyle, eaton, & wang, 2005; li et al., 2011) , mumps virus (muv), and the parainfluenza virus 5 (piv5), formerly known as simian virus 5 (sv5). in all these systems, the sh protein seems to block apoptosis during infection-at least in part-through inhibition of the tnf-α pathway (fuentes et al., 2007; li et al., 2011; lin, bright, rothermel, & he, 2003; wilson, fuentes, et al., 2006) . the mutual orientation of the tm α-helices that form the ion channel was determined in lipid bilayers using site-specific infrared dichroism (gan, ng, xin, gong, & torres, 2008) . a description of the full-length rsv sh protein monomer has been obtained by solution nmr in dpc micelles (gan et al., 2012) and later in bicelles, only for the extramembrane domains . like sars-cov e, the rsv sh protein forms homopentameric channels (gan et al., 2008 (gan et al., , 2012 that have low ion selectivity . the tm domain of sh protein has a funnel-like architecture (gan et al., 2012) , as observed in other viroporins, eg, the iav m2 (schnell & chou, 2008) , sars-cov e (pervushin et al., 2009 ) and hcv p7 (ouyang et al., 2013) . a narrower region (gan et al., 2012) in the tm domain is lined with hydrophobic side chains whereas the more open n-terminal region is lined by polar residues, ie, his-22, thr-25, and ser-29. the tm α-helix extends up to his-51 in the c-terminal region, followed by a loop, whereas the n-terminal cytoplasmic extramembrane domain forms a short α-helix (residues 5-14) present both in micelles and in bicelles . only one compound, pyronin-b, has been reported to inhibit the channel activity of the sh protein. this compound was obtained from liposome-based fluorescence assay, and was tested against purified sh protein reconstituted in planar lipid bilayers (blm). a concentration of 10 μm led to a $60% inhibition of channel activity, with a kd of $7 μm. the effect of pyronin-b was tested against rsv replication in vero cells, where the tissue culture infectivity (50% infective dose, tcid50) was zero at a drug concentration of 0.25 μm. in the nmr studies, the binding of pyronin-b to the sh protein revealed backbone resonance perturbations at both ends of the tm domain. at the n-terminal cytoplasmic end, residues more affected were ile-6 and ile-21. at the lumenal c-terminal end, the residue most affected was ala-39, and a group of nearby residues, ile-38, ile-40, leu-41, and lys-43 (fig. 7a) . interestingly, most of these juxtamembrane residues (residues 38-43) form a conserved motif in the sh protein "a 39 ilnkl 43 ," which suggests that this is a critical region for sh protein with a high barrier to resistance. determination of intermolecular nuclear overhauser effects (noes) for the drug-protein complex was not possible, probably due to weak interaction. docking studies of pyronin-b to the sh pentameric model obtained in dpc micelles (gan et al., 2012) produced two predicted binding sites at both ends of the tm domain. indeed, a majority of structures showed the drug located near residues with largest chemical shift perturbation, ie, ile-6, ile-21, and ala-39 (fig. 7) . the binding site near the n-terminus is formed by phe-14, ile-21 in one monomer (i + 1), and ile-6 of the previous (i) monomer. the one near the c-terminus is formed by residues ile-40, leu-41, and lys-43 in one monomer (i + 1) and ile-38 and ala-39 of the previous (i) monomer ( fig. 7a-c) . similar "druggable pockets" on the sh pentamer surface were identified using dogsitescorer (volkamer et al., 2012) (fig. 7d) . however, the c-terminal end of the tm domain (residues 38-41) is sufficient for inhibition, since a conservative mutation a39s almost completely prevented inhibition ($10% inhibition). only two monomers of sh protein pentamer (i and i + 1) are shown for simplicity; (d) druggable pockets (green (dark gray in the print version) mesh) predicted by dogsitescorer (volkamer, kuhn, rippmann, & rarey, 2012) . the main residues that showed largest nmr chemical shift changes are shown in red (dark gray in the print version). two monomers have been removed from the pentamer for clarity. vpu is a 81-residue small-membrane protein encoded by the singlestranded positive-sense rna human immunodeficiency virus 1 (hiv-1), and some simian immunodeficiency virus (siv) isolates. vpu is one of four hiv-1 accessory proteins involved in the release of new virions from host cell membranes (cohen, terwilliger, sodroski, & haseltine, 1988; strebel, klimkait, & martin, 1988; terwilliger, cohen, lu, sodroski, & haseltine, 1989) . without vpu, hiv particles fail to detach from the plasma membrane and accumulate in large numbers in the cell surface (klimkait, strebel, hoggan, martin, & orenstein, 1990) . the initial structural studies of vpu were performed more than 20 years ago by solution nmr in tfe/water using synthetic peptides of various overlapping lengths, which led to a model of an n-terminal hydrophobic transmembrane helix (the tm domain) and a c-terminal cytoplasmic domain with a helix-loop-helix arrangement [reviewed recently in opella, 2015] . structural similarity of vpu with am2 suggested that vpu could form pores (maldarelli, chen, willey, & strebel, 1993) and was later shown to have cation-selective channel activity (ewart, sutherland, gage, & cox, 1996; schubert et al., 1996) , but a unique structural model for the vpu oligomer cannot be defined [reviewed in radoicic, lu, & opella, 2014 ]. an analysis of the vpu tm (residues 1-40) by solid-state nmr in membranes, combined with analytical centrifugation measurements in detergent, produced data compatible with a variety of oligomers coexisting in micelles and phospholipid bilayers (lu, sharpe, ghirlando, yau, & tycko, 2010) . this led to the proposal that regulation of virus release by vpu might involve an alteration of the plasma membrane potential, and this was supported by (i) the reduced rate of hiv-1 virus release after membrane depolarization (hsu, han, shinlapawittayatorn, deschenes, & marbán, 2010) , (ii) the observed reduction in viral load and viral pathogenicity when vpu tm domain is scrambled in the context of a simian-human immunodeficiency virus (shiv) (hout et al., 2005) , and (iii) the substitution of ala-18 by a histidine (a18h) rendered hiv-1 infections susceptible to rim (hout et al., 2006) . however, recent studies challenge this model and argue against a critical involvement of vpu ion channel activity in vpu-enhanced virus release. indeed, mutations in the vpu tm domain that affect channel activity do not impair the ability to enhance virus release, whereas mutations that affect virus release retain ion channel activity (bolduan et al., 2011) . thus, the functional significance of vpu ion channel activity for hiv replication, and the role as a target for channel inhibitors, remains unclear. there are several other viroporins of which only very limited structural data is available, and no channel inhibitors have been reported. the following section will briefly describe three of such viroporins: the alphavirus 6k protein, polyomavirus agnoprotein, and the 2b protein found in picornaviruses. the 6k protein is 58-61 amino acids long and has one predicted α-helical tm domain. this viroporin is found in several members of the alphavirus genus in the positive-sense rna family togaviridae. this includes, for example, equine encephalitis viruses, chikungunya virus, sindbis virus, semliki forest viruses (sfv), ross river virus (rrv), and barmah forest virus (bfv). in these viruses, structural proteins are translated in the er from a subgenomic mrna as a single polyprotein later cleaved by proteases, in the order capsid-pe2-6k-e1 strauss & strauss, 1994) . the 6k protein was first described as early as 1980 (welch & sefton, 1980) . its expression in e. coli caused an increase in membrane permeability, whereas in eukaryotic cells it promoted viral budding (sanz, perez, & carrasco, 1994) . further, 6k proteins of rrv and bfv formed cationselective ion channels in planar lipid bilayers (melton et al., 2002) . this suggested a role for viroporins in alphavirus infection, since an increased permeability of cells to monovalent cations is followed by budding of virions. whatever the mechanism, 6k proteins have an important role in virus assembly and budding. for example, in sindbis virus, cysteine-less mutants dramatically reduced the release of virus particles, leading to aberrant enveloped particles containing multiple nucleocapsids (gaedigk-nitschko, ding, levy, & schlesinger, 1990) . other results are consistent with an indirect role in the packing of the virus spike glycoproteins; mutation c39s resulted in defective budding and a revertant contained two additional mutations at the ectodomain of the virus glycoprotein (ivanova, lustig, & schlesinger, 1995) . further, deletion of 6k in a full-length cdna clone of sfv resulted in a dramatic reduction in virus release (liljestrom, lusa, huylebroeck, & garoff, 1991) , and bhk cells expressing a δ6k strain produced thermolabile particles, suggesting a disrupted glycoprotein packing (loewy, smyth, von bonsdorff, liljestrom, & schlesinger, 1995) . despite these investigations, the specific role of 6k channel activity in these processes is still uncertain. agnoprotein is a 71-amino acids long membrane protein with one predicted α-helical tm domain required for the productive replication of the human polyomavirus jcv (john cunningham virus). jcv causes progressive multifocal leukoencephalopathy, a fatal demyelinating disease resulting from lytic infection of oligodendrocytes. agnoproteinis also found in other polyomaviruses, including bk virus, and simian virus 40 (sariyer, saribas, white, & safak, 2011) . agnoprotein was shown to play important regulatory roles in the jcv replication cycle (ellis, dang, norton, & koralnik, 2012; ellis, norton, dang, & koralnik, 2013; saribas et al., 2013) , and possesses properties commonly associated with viroporins (suzuki et al., 2013; suzuki, orba, malcino, et al., 2010; suzuki, orba, okada, et al., 2010) . for example, a deletion mutant is defective in virion release and viral propagation. also, agnoprotein localizes to the er during early stages of infection, but is also found at the plasma membrane late in infection. finally, agnoprotein, an integral membrane protein, forms homo-oligomers that enhance permeability of cells to hygromycin b and induce the influx of extracellular ca 2+ , leading to membrane dysfunction and enhancement of virus release. only a solution nmr structure of a synthetic peptide corresponding to the tm domain (thr-17 to glu-55) is available, but with an uncertain oligomeric size in lipid bilayers (coric et al., 2014) . as with the 6k protein, characterization of this viroporin is still in its infancy. protein 2b is a $100 residues long nonstructural protein found in enteroviruses [see ao, sun, & guo, 2014 for a recent review], eg, poliovirus (agirre, barco, carrasco, & nieva, 2002) . enteroviruses belong to the single-stranded positive-sense rna picornaviridae family, and include poliovirus, coxsackie virus and human enterovirus 71 (ev71). the single open reading frame encodes a peptide chain divided into three regions, p1 to p3. the 2b protein is one of the products from the polypeptides in the p2 region, and has two tm domains, one more hydrophobic than the other (de jong et al., 2003; nieva, agirre, nir, & carrasco, 2003) . the c-and n-termini of the poliovirus 2b protein are exposed to the er and golgi lumen when expressed in cells (de jong et al., 2003) , but the 2b protein in coxsackie virus adopts an opposite orientation (abed et al., 2005 , van kuppeveld, galama, zoll, van den hurk, & melchers, 1996 . poliovirus 2b protein was found to increase the concentrations of free calcium in the cytosol, which has been proposed to promote viral replication and repression of the antiviral immune response of host cells (aldabe, irurzun, & carrasco, 1997) . however, precise structural data is not yet available for this protein or its oligomers. one common, specific effect of viroporin channel activity may be associated with the observed ability of viruses to activate the host inflammasome, which activates caspase-1 to produce proinflammatory cytokines, eg, il-1β. these effects have been shown in many cases to be caused by the disruption of cellular ion homeostasis by allowing ion leakage from intracellular organelles into the cytosol, through the expression of viroporins [reviewed in guo, jin, zhi, yan, & sun, 2015] . for example, the iav activates the nlrp3 inflammasome as a result of h + or ion flux from golgi mediated by its m2 ion channel (ichinohe, pang, & iwasaki, 2010) . the 2b protein from several picornaviruses, including the encephalomyocarditis virus (emcv), poliovirus, enterovirus 71, and human rhinovirus (triantafilou, kar, van kuppeveld, & triantafilou, 2013) were shown to induce nlrp3 cytoplasmic relocalization and inflammasome activation in an intracellular ca 2+ -mediated manner (ito, yanagi, & ichinohe, 2012) . the channel activity of the sars-cov e protein is required for the processing of il-1β (nieto, which requires caspase-1 activation. channel activity of the sh protein may also result directly or indirectly in activation of the nlrp3 inflammasome (segovia et al., 2012; triantafilou, kar, vakakis, kotecha, & triantafilou, 2013) . in these cases, therefore, development of channel inhibitors should improve the pathological effects of host inflammation caused by these viruses. apart from the effects of their channel activity, viroporins are involved in many protein-protein interactions (ppis) that are susceptible for therapeutic intervention, and may be increasingly important once these interactions are properly characterized [see recent reviews fischer, li, mahato, wang, & chen, 2014; fischer et al., 2012; nieva & carrasco, 2015] . for example, the cytoplasmic regions of am2 and bm2 are important for proper virus assembly and interaction with m1 (chen, leser, jackson, & lamb, 2008; imai, kawasaki, & odagiri, 2008; imai, watanabe, ninomiya, obuchi, & odagiri, 2004; mccown & pekosz, 2006) , which can be proposed as an attractive drug target. also, the hcv p7 interacts with other viral structural and nonstructural proteins that are important to promote virus assembly and release (gentzsch et al., 2013; haqshenas, dong, ewart, bowden, & gowans, 2007; pietschmann et al., 2006; sakai et al., 2003; yi, ma, yates, & lemon, 2007) , and functions related to capsid assembly and localization of several viral proteins are not affected by either channel-inactivating mutations or treatment with rim (tedbury et al., 2011; wozniak et al., 2010) , eg, the transfer of hcv core protein from lipid droplets to the er depends on the interaction between p7 and ns2 (boson, granio, bartenschlager, & cosset, 2011) . therefore, the disruption of such interactions would require the action of ppi modulators rather than channel inhibitors. the interaction partners of e and sh proteins have been reviewed recently (torres, surya, li, & liu, 2015) . in the case of cov e proteins, the interaction with m protein has long been reported to contribute to m localization and virion formation (corse & machamer, 2003; hogue & machamer, 2008; ilk, egelseer, & sleytr, 2011; lim & liu, 2001; siu et al., 2008; vennema et al., 1996) . recent reports have highlighted interactions with protein associated with lin seven 1 (pals1) (teoh et al., 2010) and syntenin through pdz domains of e proteins, which are consistent with alterations of lung epithelia integrity and inflammation observed during cov infection. disruption of these pathways may have clear therapeutic implications, since in sars-cov-infected patients it is an exacerbated inflammatory response that leads to epithelial and endothelial damage, edema, and acute respiratory distress syndrome (smits et al., 2010) . noticeably, several other viruses, eg, human papillomavirus and influenza a, have been found to enhance pathogenesis through proteins containing pdz binding motifs (reviewed in javier & rice, 2011) , which suggests that this is a particular case of a widely used viral strategy. vpu is known for its ability to degrade the hiv-receptor cd4 (bour & strebel, 2003) , which requires interaction between vpu and cd4 cytoplasmic domains (mcnatt, zang, & bieniasz, 2013; singh et al., 2012) and probably tm domains (do et al., 2013; magadán & bonifacino, 2012) of the two proteins. this may not even require formation of oligomers, and even less channel activity. vpu also affects virus release via interaction with interferon-induced protein tetherin (bst-2) (miyagi, andrew, kao, & strebe, 2009; neil, zang, & bieniasz, 2008; skasko et al., 2012; van damme et al., 2008) , an interferon-induced host protein that inhibits the release of many enveloped viruses by tethering virions to the cell surface [reviewed in strebel, 2014] . in another case, the interaction between rsv sh and g proteins has been reported previously in infected cells (low, tan, ng, tan, & sugrue, 2008; rixon, brown, murray, & sugrue, 2005) , although the significance is not clear yet. the b-cell receptor-associated protein 31 (bap31) has been recently reported to interact with the sh protein . although this interaction has not been reported in infected cells, it could prevent the cleavage of bap31 and the formation of proapoptotic fragment p20, thus delaying apoptosis of infected cells. interaction with bap31 was also observed in the e5 viroporin from the high-risk human papillomavirus hpv-16 and hpv-31, where it is similarly thought to regulate apoptosis (regan & laimins, 2008) . jcv agnoprotein has been shown to interact with a number of cellular and viral proteins (gerits & moens, 2012; gerits et al., 2015) . however, despite all these intense research, the exact regulatory function of agnoprotein in viral replication is not fully understood. jcv agnoprotein specifically interacts with the adaptor protein complex 3 through its delta subunit. this interaction interrupts adaptor protein complex 3-mediated vesicular trafficking. the protein is then not targeted to the lysosomal degradation pathway, while allowing transport of agnoprotein to the plasma membrane. the findings suggest that the viroporins of other viruses may also be highly regulated by specific interactions with host cell proteins (suzuki et al., 2013) . these pathologically relevant interactions between viroporins and their associated viral and host proteins present an attractive therapeutic target for disruption by small molecules, as well as peptides stabilized by internal covalent linkage (stapled peptides) (walensky & bird, 2014) . in principle, both the transmembrane and extramembrane domains of viroporins can be potential templates for peptide-based modulators. for example, one could design a stapled peptide mimicking the α-helical cytoplasmic domain of the sh protein to disrupt the interactions between sh and a variant of the death effector domain (vded) of bap31, which would lead to enhanced apoptosis and cell death of infected cells. one could also mimic the transmembrane domain to disrupt monomer-monomer interactions, as shown recently for halobacterium salinarum where efflux by a small multidrug resistance protein was inhibited with peptides targeting its tm domain (bellmann-sickert, stone, poulsen, & deber, 2014) . in the case of viroporins, the disruption of the oligomeric status would abolish channel activity, a classical strategy revisited with a different approach. however, more detailed mechanistic information should be available before these conceptual strategies can be widely used. generation and characterization of recombinant influenza a (h1n1) viruses harboring amantadine resistance 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implications for membrane protein structure and function: influenza a m2 protein this work has been funded by singapore moe tier 1 grant rg 51/13 (j.t.). key: cord-307227-x6xketcn authors: martin, william r.; cheng, feixiong title: repurposing of fda-approved toremifene to treat covid-19 by blocking the spike glycoprotein and nsp14 of sars-cov-2 date: 2020-09-10 journal: j proteome res doi: 10.1021/acs.jproteome.0c00397 sha: doc_id: 307227 cord_uid: x6xketcn [image: see text] the global pandemic of coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has led to the death of more than 675,000 worldwide and over 150,000 in the united states alone. however, there are currently no approved effective pharmacotherapies for covid-19. here, we combine homology modeling, molecular docking, molecular dynamics simulation, and binding affinity calculations to determine potential targets for toremifene, a selective estrogen receptor modulator which we have previously identified as a sars-cov-2 inhibitor. our results indicate the possibility of inhibition of the spike glycoprotein by toremifene, responsible for aiding in fusion of the viral membrane with the cell membrane, via a perturbation to the fusion core. an interaction between the dimethylamine end of toremifene and residues q954 and n955 in heptad repeat 1 (hr1) perturbs the structure, causing a shift from what is normally a long, helical region to short helices connected by unstructured regions. additionally, we found a strong interaction between toremifene and the methyltransferase nonstructural protein (nsp) 14, which could be inhibitory to viral replication via its active site. these results suggest potential structural mechanisms for toremifene by blocking the spike protein and nsp14 of sars-cov-2, offering a drug candidate for covid-19. as of august 4, 2020, there are over 18 million documented cases of covid-19 (over 675,000 resulting in death), with nearly one-third of all cases occurring in the united states (over 150,000 deaths 1 ). as of this writing, there are no fda approved treatments or vaccines for covid-19, both of which are sorely needed. a search on clinicaltrials.gov on june 1 for covid-19 as the disease and the additional term "drug" yields 806 results, providing evidence for the need for an intervention. drug repurposing allows an acceleration of the drug discovery pipeline; drugs which have already been fda approved to treat another disease are repositioned as therapeutics for diseases for which they have not yet been used. 2, 3 in our initial network-based drug repurposing study, 4 we identified toremifene, another selective estrogen receptor modulator (serm), as a strong candidate for the potential treatment of covid-19. a drug repurposing study for sars-cov-1 5 indicated a low 50% effective concentration (ec 50 ) for toremifene, and noted that estrogen signaling may not be involved in the inhibitory pathway, similar to that of inhibition of ebola. 6 indeed, a crystal structure of the ebola virus with bound toremifene indicates the interaction lies between the attachment (gp1) and fusion (gp2) protein subunits. 7 in a study using human organs-on-chips, 8 toremifene was found to significantly inhibit entry of a pseudotyped sars-cov-2 virus. however, the mechanism of action for toremifene in the inhibition of sars-cov-2 is not yet known. the actual target for toremifene has not yet been elucidated in coronaviruses as it has in ebola, which was determined to have an 50% inhibitory concentration (ic 50 ) of roughly 1 μm. 6 dyall et al. 5 found the ec 50 of toremifene for mers-cov and sars-cov-1 to be 12.9 μm and 11.97 μm, respectively; while these results are not different, the story is different for tamoxifen, which differs from toremifene by a substitution of hydrogen for the chlorine on toremifene. while the ec 50 for tamoxifen is slightly lower in mers-cov (10.1 μm), the ec 50 for sars-cov-1 is worse than for toremifene (92.9 μm), which could potentially indicate that the inhibition is related to a viral, and not human, protein. importantly, a recent study indicated an ic 50 of 3.58 μm for toremifene with sars-cov-2. 9 there have been numerous studies recently targeting singular proteins in sars-cov-2 using virtual screening techniques, such as 3-chymotrypsin-like protease 10−12 and the papain-like protease. 13 further studies have been done using large databases, such as zinc, 14 to test large compound libraries across multiple viral proteins. in an effort to determine potential interactors with toremifene specifically, 15 we used inverse virtual screening to test 13 of the 29 viral proteins encoded by the sars-cov-2 genome. proteins which did not have a crystal structure at the time of this study were modeled using homology modeling. we believe this to be a comprehensive study to combine virtual screening with molecular dynamics and molecular mechanics/poisson− boltzmann surface area (mm/pbsa) calculations to determine potential protein−ligand interactions in sars-cov-2 proteome. here, we have discovered two potential targets for toremifene from the entire sars-cov-2 proteome. proteins for which crystal structures were not available were constructed using homology modeling. each sequence was accessed from ncbi by its accession number. the protein sequence was submitted to a blast 16 search within ucsf chimera, 17 and the best matching structure was chosen for homology modeling. each homology model was constructed using a single template using modeler 9.21 18 within ucsf chimera. the best model as determined by z-score was used for docking. proteins which do not have potential templates with high homology were not modeled. generally, sequence identity of greater than 40% will yield an acceptable homology model. 19 prior to docking, all homology models were subjected to a short minimization and equilibration period using molecular dynamics simulations. in short, each system was constructed using standard tools in gromacs 2018.2. 20 systems were submerged in a water box with edges no less than 10 å from any part of the protein, and neutralized using sodium and journal of proteome research pubs.acs.org/jpr article chloride ions to an ionic strength of 0.15 m. parameterization for the protein, ions, and water was done using the charmm36 force field. 21 each protein was minimized using a steepest descent algorithm for 5000 steps, followed by a short 200 ps equilibration. the toremifene structure was downloaded from the zinc database, 14 while all crystal structures used for docking were obtained from the rcsb protein data bank. 22 all docking was done using autodock vina 23 within ucsf chimera. the highest scoring binding pose (kcal/mol) was selected for further analysis where appropriate ( figure 1 ). each search generated 10 binding modes with the exhaustiveness set to the maximum value. proteins without a clear binding region (for example, accessory protein 7a) were not included. the search grid for each protein was selected to encompass the entire protein. each system was constructed using the solution builder in charmm-gui. 24−26 following a processing step involving the addition of hydrogens not added in docking and parametrization of the ligand, a water box with edges at least 10 å from any part of the protein was added. the system was neutralized and brought to an ionic strength of 0.15 m using sodium and chloride ions. the charmm general force field (cgenff) 27 was used to parametrize toremifene, while the charmm36m force field was used to parametrize the protein, ions, and tip3p water molecules. all systems were simulated using gromacs 2020.1 on the aimos supercomputer at the rensselaer polytechnic institute center for computational innovations in a three-step process. initial minimization of the systems was run until changes in the potential energy of the system reached machine precision. following minimization, an nvt equilibration step was completed with a 2 fs time step for 500,000 steps using 400 kj mol −1 nm −2 and 40 kj mol −1 nm −2 positional restraints on the backbone and side chains, respectively. a 500 ns production step was completed using the npt ensemble with no position restraints and a 2 fs time step. hydrogen atoms were constrained using the lincs 28 algorithm. temperature for the system was held at 300 k using a nose-hoover thermostat 29 with a 1 ps coupling constant. for the production simulation, pressure was coupled isotropically using a parrinello−rahman barostat 30 with a 5.0 ps coupling constant and compressibility of 4.5 × 10 5 bar −1 to maintain a pressure of 1 bar. the pair-list cutoff was constructed using the verlet scheme 31 with a cutoff distance of 1.2 nm. particle mesh ewald electrostatics 32 were used to describe coulombic interactions with a 1.2 nm cutoff, while van der waals forces were smoothly switched to between 1.0 and 1.2 nm using a force-switch modifier to the cutoff scheme. linear center of mass translation was removed every 100 steps for the entire system. sars-cov-2 has a roughly 30 kb genome which encodes 29 proteins. 34 these 29 proteins include 16 nonstructural proteins (nsp), 4 structural proteins, and 9 accessory proteins. the nonstructural proteins include proteinases (nsp3, nsp5), rna polymerases (nsp12), helicases (nsp13), ribonucleases (nsp14, nsp15), and methyltransferases (nsp14, nsp16), while structural proteins are involved in viral assembly (envelope, membrane) and binding with the host protein (spike glycoprotein). molecular docking over the entire protein was carried out on 13 of the 29 possible viral proteins in sars-cov-2. these 13 were selected based on a combination of criteria: (1) whether a crystal structure for the protein exists; (2) if a crystal structure does not exist, is there a template protein (generally from sars-cov-1) available to use for homology modeling; (3) based on either the crystal structure or homology modeling, is there a potential binding pocket. proteins which did not meet these criteria (for example, the membrane protein has partial homologous coverage at ∼20% homology; protein 3a does not appear to have a potential binding pocket) were not chosen for the docking study. the best scoring poses are tabulated in table 1 , with a larger negative number indicating a better binding affinity. unsurprisingly, the affinity was not high for a few of the smaller systems (nsp1 and the nucleocapsid, for example), which were included as a sort of negative control; it was not expected that good binding would be achieved with these systems. interestingly, the spike glycoprotein and nsp14 had the highest binding affinities based on molecular docking. to determine which systems would be selected for further analysis via simulation to better determine if a particular protein−ligand binding pose maintains its integrity, we visually inspected the binding of each system. while a hard cutoff was not selected, certain systems were rejected for further analysis. systems which did not have strong binding within a clear pocket were not simulated, including nsp1, nsp7, nsp9, the helicase, and the nucleocapsid. as an example, the interaction with nsp9 does not involve a binding pocket, but sits on the surface of the protein. we have no expectation that such an interaction would be maintained throughout a simulation, and therefore did not include it in the next step. each of the above systems with a strong predicted binding was simulated according to the protocol listed in the methods. here, we have monitored the protein−ligand interaction over the entire 500 ns trajectory. unsurprisingly, due to the low binding affinities, the interaction between the ligand and the protein was not maintained throughout the trajectory in most systems. table 2 indicates the length of simulation before a particular protein−ligand system lost its interaction. both the interaction between toremifene and nsp14, as well as that between toremifene and the spike glycoprotein, were maintained (and within the original binding pocket) throughout the entire 500 ns trajectory. these two systems were analyzed further to determine the nature of the protein− ligand interactions. to ensure that the results were not simply a result of poor initial docking poses, all systems for which toremifene did not maintain contact with the initial docked region were redocked using swissdock 42 selecting the "accurate" setting under "extra parameters", with flexible residues within 5 å of the docked ligand. the implementation of autodock 23 used sacrifices the inclusion of flexible residues in the binding pocket for significantly improved speed, while swissdock allows us to implement a different algorithm, while also including flexible residues (at significantly higher computational cost). a specific region of interest was not defined. these newly docked systems were also simulated in the same fashion as those docked with autodock, and yielded similar results; all simulations resulted in a residence time less than 200 ns. the initial docking position (figure 2a ) lies at the interface between two separate domains in the spike glycoprotein, with one domain having its receptor binding domain (rbd) in the "up" position, while the other has its rbd in the "down" position. in the b domain of pdb id 6vsb, the interaction with the spike glycoprotein is with the helical region between the loop separating the s1 and s2 subunits and the fusion peptide, shown in sars-cov-1 to mediate membrane fusion in a calcium-dependent manner, 43 while the interaction in the a domain involves the n-terminal region of the rbd, as well as heptad repeat 1 (hr1), a key component of the fusion core (figures 2b and 4a) . while the nonhelical linker between t941 and l945 did extend to k947 in the b and c domains, the remainder of this helical region remained unperturbed when compared to the crystal structure, with a long helix between l948 and s967. however, the interaction with toremifene resulted in a helical region between s943 and q954, with a short helical region from a958 through t961 and the remainder unstructured. an interaction between the dimethylamine region of toremifene and q954 and n955 appears to be key in perturbing the secondary structure of hr1. in an effort to determine the strength of the interaction, we carried out an mm/pbsa binding affinity calculation between the entire protein and toremifene. our calculation resulted in a final binding energy of −91.036 (±0.933) kj/mol. the main contributors (figure 2c ) to this binding energy were v772 and l861 in the b domain due to large nonpolar interactions (−9.7 and −4.0 kj/mol, respectively), and y313 in the a domain due to a chlorine−π interaction (−3.7 kj/mol). the chlorine−π interaction could indicate a potential mechanism by which toremifene has a stronger inhibitory action than tamoxifen as seen in sars-cov-1. nsp14 has both exoribonuclease and methyl transferase activity; here, we have found a strong interaction with the n7-methyl transferase domain (figure 3a ). throughout the molecular dynamics simulation, very little movement of the ligand was observed. the docked position appears as though it would potentially be inhibitory to interaction with the functional ligand s-adenosyl methionine, while clearly being inhibitory to interaction with its substrate, gppp-rna (figures 3c and 4b) . as with the spike protein, we assessed the binding affinity using mm/pbsa, finding a significant π−π interaction with f426 (−8.0 kj/mol), a chlorine−π interaction with f506 (5.0 kj/mol), a strong hydrophobic interaction with c309 (−4.2 kj/mol), and a total binding energy of −119.805 (±1.013) kj/mol. many of the residues identified here as interacting with toremifene ( figure 3b) were identified in the crystal structure used to generate our homology model as interacting with gppp-rna. we have demonstrated two plausible targets for toremifene in sars-cov-2. previous work has indicated that, as noted earlier, toremifene is likely to be inhibitory to viral entry. 6,7 a proposed mechanism for ebola posited a mechanism by which fusion between the viral and endosomal membranes is disrupted. 7 the interaction with the spike glycoprotein proposed here could prevent such a fusion through its disruption of the hp1 helix. the interaction with nsp14 elucidated here would indicate an inhibition of viral reproduction by interfering with interaction with the substrate. toremifene, a first generation nonsteroidal serm, shows striking activity in blocking multiple viral infections, including ebola 6,7 (ic 50 ≈ 1 μm), mers-cov 5 (ec 50 = 12.9 μm), sars-cov-1 5 (ec 50 = 11.97 μm), and sars-cov-2 9 (ic 50 = 3.58 μm), in established, virus-infected human cell lines. the inhibition in ebola by serms has been found to not be via the estrogen receptor pathway, but instead it disrupts endolyso44 toremifene has been approved for the treatment of breast cancer for over 25 years, 45 and has been investigated as a treatment for prostate cancer. 46 in this study, we have determined two potential targets for toremifene, the spike glycoprotein and nsp14. toremifene has been used in postmenopausal women with breast cancer, premenopausal women, and men with prostate cancer in clinical trials involving thousands of participants since its inception in 1997. 47 liver toxicity has occurred in only long-term studies and is not shown in short-term studies of <8 weeks. 48 therefore, there is a low risk of safety concern with the use of toremifene 60 mg daily for short-term treatment of covid-19, such as 2 weeks. based on human studies, the mean plasma concentration of toremifene during administration of 60 mg/ day was 0.88 mg/l (2.17 μm) in postmenopausal breast cancer patients. 49 the peak plasma concentration of toremifene (60 mg/day) was over 10 μm at the 4 h administration in patients, 50 which is ∼3-fold of the antiviral effect on sars-cov-2 (ic 50 = 3.58 μm). an animal model study showed that toremifene (peak plasma concentration of 2.98 μm) was needed to protect 50% of mice from death caused by ebola virus infection, a more fatal virus than sars-cov-2. the combination of consistent findings from in vitro antiviral data supporting the antiviral effects of toremifene against coronaviruses, along with reasonable tolerability, provide the basis to pursue toremifene as a viable candidate therapeutic against sars-cov-2. caution, however, must be exercised when interpreting these results. there are several limitations for molecular docking approaches. for example, molecular docking scores could yield false positives when proteins have significant conformational changes. although we employed molecular dynamics simulation to verify our findings from molecular docking, further experimental validation is warranted in the near future. it should be noted that, to this date, there are no crystal structures published for the spike glycoprotein with an inhibitory ligand. similarly, no inhibitors have been crystallized with nsp14, nor has a structure for nsp14 in sars-cov-2 been crystallized, though the homology with sars-cov-1 is indeed high. however, work on the flavivirus methyltransferase 38 has indicated there is a potential for inhibition. while the proposed mechanisms for sars-cov-1 in toremifene are posited to not involve human proteins, further study would be required to confirm the mechanism. while in vitro studies on toremifene do exist for sars-cov-2, there are no studies to date that allow a comparison with other serms, as has been done with sars-cov-1 and mers-cov. future work will be needed to confirm these results; optimally, the determination of a cocrystal structure with journal of proteome research pubs.acs.org/jpr article nsp14 and/or the spike glycoprotein from sars-cov-2 with toremifene would be solved. alternatively, other functional validation analyses (in vitro assays, for example) could be carried out to determine the correct protein target. the final poses could also be used to inform further work to refine a potential inhibitor using medicinal chemistry techniques. the present study has demonstrated two potential targets for toremifene in sars-cov-2. we have demonstrated a potential mechanism for inhibition via the spike glycoprotein, through which an interaction with q954 and n955 in heptad repeat 1 appears to disturb the secondary structure, resulting in what is normally a long α-helix instead having a lack of secondary structure. additionally, the interaction we found with nsp14 appears as though it could be inhibitory in two ways: first, it appears as though there would be steric hindrance between toremifene and any functional ligands (such as s-adenosyl methionine), as well as interfering with substrate interaction in the catalytic pocket. further experimental and functional studies would be needed to validate these findings, such as a cocrystal structure of the spike glycoprotein or nsp14 with toremifene. ■ associated content the supporting information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jproteome.0c00397. initial docked conformations and conformations at the time listed in table 2 for nsp3 (pl-pro), nsp4, nsp5, nsp12, 375 nsp15, and nsp16 are provided in figures s1-s6. rmsd plot for all proteins stimulated is provided in figure s7 . (pdf) an interactive web-based dashboard to track covid-19 in real time drug repurposing: new treatments for zika virus infection? g systems biology-based investigation of cellular antiviral drug targets identified by gene-trap insertional mutagenesis network-based drug repurposing for novel coronavirus 2019-ncov/ sars-cov-2 repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection fdaapproved selective estrogen receptor modulators inhibit ebola virus infection toremifene interacts with and destabilizes the ebola virus glycoprotein human organs-on-chips as tools for repurposing approved drugs as potential influenza and covid19 therapeutics in viral pandemics identification of antiviral drug candidates against sars-cov-2 from fda-approved drugs structural basis of sars-cov-2 3clpro and anti-covid-19 drug discovery from medicinal plants prediction of the sars-cov-2 (2019-ncov) 3c-like protease (3clpro) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates structure-based design of antiviral drug candidates targeting the sars-cov-2 main protease potential inhibitors against papain-like protease of novel coronavirus (sars-cov-2) from fda approved drugs zinc: a free tool to discover chemistry for biology how to discover antiviral drugs quickly gapped blast and psi-blast: a new generation of protein database search programs chimeraa visualization system for exploratory research and analysis comparative protein structure modeling using modeller all are not equal: a benchmark of different homology modeling programs high performance molecular simulations through multi-level parallelism from laptops to supercomputers optimization of the additive charmm all-atom protein force field targeting improved sampling of the backbone ϕ, ψ and side-chain χ 1 and χ 2 dihedral angles the protein data bank autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading charmm-gui ligand reader and modeler for charmm force field generation of small molecules simulations using the charmm36 additive force field a webbased graphical user interface for charmm charmm general force field: a force field for drug-like molecules compatible with the charmm all-atom additive biological force fields lincs: a linear constraint solver for molecular simulations a configurational temperature nose-hoover thermostat polymorphic transitions in single crystals: a new molecular dynamics method particle mesh ewald: an n · log(n) method for ewald sums in large systems tool for high-throughput mm-pbsa calculations novel β-barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus crystal structure of the c-terminal cytoplasmic domain of non-structural protein 4 from mouse hepatitis virus a59 structure of the rnadependent rna polymerase from covid-19 virus delicate structural coordination of the severe acute respiratory syndrome coronavirus nsp13 upon atp hydrolysis structural basis and functional analysis of the sars coronavirus nsp14-nsp10 complex crystal structure of nsp15 endoribonuclease nendou from sars-cov-2 cryo-em structure of the 2019-ncov spike in the prefusion conformation swissdock, a proteinsmall molecule docking web service based on eadock dss the sars-cov fusion peptide forms an extended bipartite fusion platform that perturbs membrane order in a calcium-dependent manner selective inhibition of ebola entry with selective estrogen receptor modulators by disrupting the endolysosomal calcium toremifene in the treatment of breast cancer toremifene, a selective estrogen receptor modulator, significantly improved biochemical recurrence in bone metastatic prostate cancer: a randomized controlled phase ii a trial association between serum insulin-like growth factor-i levels and thyroid disorders in a population-based study pharmacokinetics of toremifene and its metabolites in patients with advanced breast cancer a review of its pharmacological properties and clinical efficacy in the management of advanced breast cancer the authors declare no competing financial interest. we acknowledge support from the ohio supercomputer center, the oak ridge leadership computing facility, the ibm researchai hardware center, and the center for computational innovation at rensselaer polytechnic institute for computational resources. key: cord-284990-klsl1nzn authors: zhang, dapeng; iyer, lakshminarayan m.; aravind, l. title: a novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and dna viral systems date: 2011-02-08 journal: nucleic acids res doi: 10.1093/nar/gkr036 sha: doc_id: 284990 cord_uid: klsl1nzn the use of nucleases as toxins for defense, offense or addiction of selfish elements is widely encountered across all life forms. using sensitive sequence profile analysis methods, we characterize a novel superfamily (the sukh superfamily) that unites a diverse group of proteins including smi1/knr4, pgs2, fbxo3, skip16, syd, herpesviral us22, irs1 and trs1, and their bacterial homologs. using contextual analysis we present evidence that the bacterial members of this superfamily are potential immunity proteins for a variety of toxin systems that also include the recently characterized contact-dependent inhibition (cdi) systems of proteobacteria. by analyzing the toxin proteins encoded in the neighborhood of the sukh superfamily we predict that they possess domains belonging to diverse nuclease and nucleic acid deaminase families. these include at least eight distinct types of dnases belonging to hnh/endoviiand restriction endonuclease-fold, and rnases of the endou-like and colicin e3-like cytotoxic rnases-folds. the n-terminal domains of these toxins indicate that they are extruded by several distinct secretory mechanisms such as the two-partner system (shared with the cdi systems) in proteobacteria, esat-6/wxg-like atp-dependent secretory systems in gram-positive bacteria and the conventional sec-dependent system in several bacterial lineages. the hedgehog-intein domain might also release a subset of toxic nuclease domains through auto-proteolytic action. unlike classical colicin-like nuclease toxins, the overwhelming majority of toxin systems with the sukh superfamily is chromosomally encoded and appears to have diversified through a recombination process combining different c-terminal nuclease domains to n-terminal secretion-related domains. across the bacterial superkingdom these systems might participate in discriminating `self’ or kin from `non-self’ or non-kin strains. using structural analysis we demonstrate that the sukh domain possesses a versatile scaffold that can be used to bind a wide range of protein partners. in eukaryotes it appears to have been recruited as an adaptor to regulate modification of proteins by ubiquitination or polyglutamylation. similarly, another widespread immunity protein from these toxin systems, namely the suppressor of fused (sufu) superfamily has been recruited for comparable roles in eukaryotes. in animal dna viruses, such as herpesviruses, poxviruses, iridoviruses and adenoviruses, the ability of the sukh domain to bind diverse targets has been deployed to counter diverse anti-viral responses by interacting with specific host proteins. the use of toxins as a defensive, offensive or selfish addictive strategy is observed across the tree of life. interestingly, a diverse set of protein toxins from distantly related organisms have a propensity to catalyze nucleic acid modifying or cleaving reactions in their target cells. well-known examples are currently known from across the phylogenetic spectrum: plants deploy toxins such as ricin, abrin and modeccin to protect their seeds, which are rna n-glycosidases that remove a specific purine *to whom correspondence should be addressed. tel: +1 301 594 2445; fax: +1 301 480 9241; email: aravind@ncbi.nlm.nih.gov base from eukaryotic 28s rrna to render it non-functional (1, 2) . in a similar vein, the fungal toxin a-sarcin, produced by fungi such as aspergillus giganteus, acts as a specific endonuclease that cleaves the 28s rrna at a position close to the site of action of the above plant toxins (3) . among animals the use of nucleic acid-targeting enzymes is observed in the venoms of snakes (4) . several animals, including vertebrates, are known to deploy cytotoxic rnases, such as rnase a, which potentially target rna from bacteria and viruses (5) . bacteria are a particularly rich source of nucleic acid-targeting toxins, which are deployed in various contexts. pathogenic bacteria secrete rna n-glycosidases that target the 28s rrna of eukaryotic hosts similar to the ricin-like plant toxins (6) . bacteria are also known to deploy rnase and dnase bacteriocins in intra-and possibly inter-specific competition that target molecules such as trna and genomic dna (7) . the best known are the plasmid-borne toxins of the model bacterium escherichia coli, which kill closely related competing strains. of these colicins e3, e4 and e6 cleave rrna, colicins e5 and d cleave trna and colicins e2, e7, e8 and e9 cleave dna (8) . additionally, bacterial genomes are also colonized by systems such as the toxin-antitoxin systems and restriction-modification systems which produce enzymes that function as nucleic acid-targeting toxins (9) (10) (11) (12) . in these systems the primary function of the toxin is to kill the host bacterial cell if the toxin encoding system is genetically disrupted in some way (10, 11) . thus, they act as selfish elements that forcibly 'addict' the host to maintain them in genomes or plasmids. in many of these cases, organisms or genetic elements that produce the toxin also produce an antitoxin or immunity protein that renders the 'self' resistant to the action of the toxin. the study of these toxins and antitoxins or immunity proteins has not only expanded our understanding of the evolution of inter-species competition but also thrown considerable light on the biochemistry of nucleic acids and other molecules that interact with them (9) (10) (11) (12) . in practical terms these nucleic acid-targeting toxins and antitoxins/immunity proteins are potential reagents that could be utilized in numerous biotechnological contexts ranging from chemical analysis of nucleic acids to bio-defense. availability of an enormous wealth of genomic sequence data provides opportunities to identify novel versions of such toxins and associated immunity and delivery systems through computational analysis, thereby opening the door for new investigations on nucleic acid-modifying enzymes. the first step in this process requires detailed case-by-case analysis of protein sequences and structures using the best available methods for detecting sequence and structure similarity. results from such an analysis of protein structures needs to be further combined with in-depth analysis of genomic contexts and domain architectures to glean novel functional associations. finally, these results need to be placed in the context of phyletic patterns of the occurrence of various components of the system in order to reconstruct a total picture of their natural history and predict aspects of their biochemistry and biological functions. indeed, such a strategy has allowed the prediction of novel biochemical activities and has laid the foundations for further systematic investigations of the toxin-antitoxin and peptide-modification systems of prokaryotes (9, (13) (14) (15) . in this article we present the results of such a strategy that helped us uncover and characterize a remarkable, diverse class of nuclease toxins, whose immunity appears to depend primarily on a protein superfamily prototyped by the saccharomyces cerevisiae protein smi1/knr4. the smi1/knr4 protein was first recovered in a screen for s. cerevisiae mutants that confer resistance to the killer toxin produced by the competing yeast species hansenula mrakii (16, 17) . smi1/knr4 was shown to physically interact with the tyrosyl trna synthetase and it appears to functionally interact with the non-ribosomal peptide ligase dit1, with a trna-synthetase-like catalytic domain, in the efficient synthesis of dityrosine a peptide metabolite that is typical of fungal spore-walls (18) . interestingly, it also shows synthetic lethal and physical interactions with a great number of proteins (19) . nevertheless, its exact significance and biochemical action has remained poorly understood (20) . parallel studies recovered other smi1/knr4 eukaryotic homologs namely fbxo3, a subunit of a scf-type e3 ubiquitin ligase in vertebrates (21) , and pgs2, a subunit of the tubulin polyglutamylase, which is a non-ribosomal peptide-ligase that links multiple glutamates to the g-carboxyl group of target proteins (22, 23) . exploratory sequence surveys suggested that smi1/knr4 homologs are also abundantly represented in bacteria (smi1/knr4 domain, pfam: pf09346). furthermore, our preliminary contextual analysis of conserved gene neighborhoods of these representatives suggested that they might be functionally linked to potential nucleases. very recently, a novel contact-dependent inhibitory (cdi) toxin system has been reported in proteobacteria that delivers multiple nuclease toxins into target cells (24, 25) . our observations indicated that smi1/knr4 homologs are potential immunity proteins in a subset of these cdi systems. together, these observations prompted us to systematically investigate both the bacterial and eukaryotic smi1/knr4 homologs and explore their potential connection to nuclease toxins, their delivery and immunity against them. as a result we were able to identify a diverse group of previously unknown nuclease toxins and immunity proteins that are present across all the major bacterial lineages with considerable significance for intra-specific and host interactions. this investigation also allowed us to uncover diverse, previously unknown nuclease and deaminase domains in bacterial toxins and predict their folds and biochemical mechanisms. we also show that the smi1/knr4 homologs, which were ultimately derived from bacterial toxin-immunity systems, have been recruited by eukaryotic double-stranded dna viruses to perform multiple roles in intracellular survival and morphogenesis of these viruses. finally, we present evidence that the ability of the conserved domain in the smi1/knr4 superfamily of proteins to bind structurally diverse protein partners has been re-used in eukaryotes as a means to recruit targets to peptide-modifying systems such as the ubiquitin and the polyglutamylase systems. iterative sequence profile searches were run using the psi-blast program (26) against the non-redundant (nr) protein database of national center for biotechnology information (ncbi). similarity based clustering for both classification and culling of nearly identical sequences was performed using the blastclust program (ftp://ftp.ncbi.nih.gov/blast/documents/blast clust.html). the hhpred program was used for profileprofile comparisons (27) . structure similarity searches were performed using the dalilite program (28) . multiple sequence alignments were built by muscle (29) , promals (30) , kalign (31) and pcma (32) programs, followed by manual adjustments on the basis of profile-profile and structural alignments. the consensus for alignments were calculated and colored by the chroma program (33) . secondary structures were predicted using the jpred and psipred programs (34, 35) . for earlier known domains the pfam database (36) was used as a guide, though the profiles were often augmented by addition of newly detected divergent members that were not detected by the original pfam models. clustering with blastclust followed by multiple sequence alignment and further sequence profile searches were used to identify other domains that were not present in the pfam database. signal peptides and transmembrane segments were detected using the tmhmm and phobius programs (37, 38) . contextual information from prokaryotic gene neighborhoods was retrieved by a perl custom script that extracts the upstream and downstream genes of the query gene and uses blastclust to cluster the proteins to identify conserved gene-neighborhoods. phylogenetic analysis was conducted using an approximately-maximum-likelihood method implemented in the fasttree 2.1 program under default parameters (39) . the modeller9v1 program (40) was utilized for homology modeling of the structure of the irs1 n-terminal domain. structural visualization and manipulations were performed using vmd (41) and pymol (http://www.pymol.org) programs. the in-house tass package, a collection of perl scripts, was used to automate aspects of large-scale analysis of sequences, structures and genome context (anantharaman, v., balaji, s., and aravind, l., unpublished data sequence profile searches and structural comparisons reveal a vast superfamily of smi1-related proteins as a first step to computationally characterize the smi1/knr4 protein, we analyzed it using the seg program to identify potential globular regions in it (42) . this indicated the presence of a single globular domain that was then used as a seed in iterative sequence profile searches of the nr database with psi-blast and jackhmmer from the hmmer3 package. in addition to recovering other eukaryotic proteins with a homologous region, such as fbxo3 from animals, skip16 from plants and pgs2, a subunit of tubulin polyglutamylase complex, the search also recovered a large number of bacterial proteins such as the bacillus subtilis yobk. given the great diversity of sequences recovered prior to convergence from bacteria, we initiated transitive sequence profile searches with several distinct bacterial starting points to achieve maximal coverage in terms of detection. we also noted that a crystal structure for yobk has been solved by the joint structural genomics initiative (pdb: 2prv). we used this structure as a query for structure similarity searches using the dalilite program and recovered hits to four other homologous structures (3ffv, 2pag, 2icg, 3d5p; z > 7.5). of these, 3ffv was the structure of the earlier characterized protein syd from e. coli which interacts with secy, a key component of the sec-dependent protein secretion system that traffics proteins across the bacterial inner membrane (43) (44) (45) . consistent with this, we also found that syd homologs were recovered with borderline e-values (e $ 0.03-0.05) in the above jackhmmer and psi-blast searches. hence we included the syd homologs in the profiles to further expand the relationships of the group of proteins homologous to smi1/knr4. at convergence, some of these searches also recovered with borderline e-values proteins (e $ 0.05) from certain dna viruses such as fpv250 (gi: 9634920) from the fowl poxvirus, and the us22 family of proteins (e.g. us22, ul26, irs1 and trs1) from herpesviruses. to confirm the relationship of these proteins to smi1 we used them in a profile-profile comparison search with the hhpred program against a library of hmms created using the sequence of polypeptides in the pdb database as a query. these searches recovered the structures 2prv, 3ffv and 2icg as the best hits with significant p-values (p = 10 à4 to à8 ). furthermore, examination of the hits produced by the viral proteins in profile-profile comparisons showed that most of the versions from herpesviruses possessed two tandem repeats of the domain homologous to smi1. additional transitive searches with these viral proteins revealed that homologous proteins are present in a number of distantly related or unrelated dna viruses. finally, the above searches also recovered hits to two distinct groups of proteins each with over 100 representatives in the nr database, predominantly from bacteria, typified respectively by ca_c3700 (gi: 15896931) from c. acetobutylicum and sgr_4389 (gi: 182438182) from s. griseus. profileprofile comparisons with the hhpred program using alignments of each of these groups of proteins also confirmed their relationship to the smi1-like proteins via recovery of significant hits (e = 10 à4 to à6 ) to hmms generated using the sequences of 2prv and 3ffv as best hits. thus, it became clear that smi1/knr4 defines a large superfamily of conserved domains that is widespread in bacteria, eukaryotes and various dna viruses but practically absent in currently sequenced archaeal genomes. we accordingly named it the sukh (for syd, us22, knr4 homology) domain superfamily. despite the low average pairwise sequence similarity across this superfamily, all representatives are known or predicted to possess a similar core fold comprising of four conserved helices and six strands ( figure 1 , supplementary data). strands 1 and 2 form a b-hairpin and the strands 3-6 form a 4-stranded b-meander; however, the b-hairpin and the b-meander show only limited or no hydrogen-bonding along their length, despite being spatially beside each other. thus, the structural core of the sukh domain can be described as a split b-sheet with only weak interaction between its two parts. this structural peculiarity could potentially be critical for the functional interactions of the domain (see below). based on sequence-similarity-based clustering and phylogenetic analysis five major groups can be recognized within the sukh domain superfamily (figure 1 , supplementary data). the first of these, and the most widespread, is the one typified by smi1/knr4, fbxo3, skip16, pgs2 and yobk (that entirely includes the pfam model pf09346, 'smi1/knr4 family', and additional proteins not detected by that model within it) and is seen in both bacteria and eukaryotes. this ensemble, which we term smi1-like or sukh-1 group includes the majority of the sukh domains. we term the second group, prototyped by syd, the syd-like or sukh-2 group. this group is largely restricted to the gammaproteobacteria and firmicutes. the sukh-3 group prototyped by ca_c3700 (gi: 15896931) is widely distributed across most bacterial lineages. the group prototyped by sgr_4389 (gi: 182438182), the sukh-4 group, is again seen in several bacteria and sporadically in fungi. the sukh-5 or us22-like group is present in fowl adenoviruses, various vertebrate iridoviruses, archosaur poxviruses (crocodilepox virus and fowlpox virus), and in multiple copies in several herpesviruses (representatives of the alphaherpesvirus, betaherpesvirus and alloherpesvirus clades). members of this group are also encoded by genomes of the early-branching chordate branchiostoma, the salmon, the frog rana catesbeiana and the duckbilled platypus, where they appear to have been acquired from the genomes of integrated herpesviruses (46) . phylogenetic analysis of each group, along with the phyletic patterns, strongly suggests that sukh domain proteins have been widely disseminated both within and across the superkingdoms via extensive lateral transfer (supplementary data). in light of this pattern, the near complete absence of this superfamily in archaea suggests that there could be certain specific functional barriers that prevent acquisition of the sukh domain by that superkingdom. phylogenetic analysis strongly suggests that the groups sukh-2-5 are monophyletic clades. the largest group, sukh-1 is likely to represent the ancestral group from within which the above clades have diversified through rapid sequence divergence. contextual information gleaned from gene neighborhoods in prokaryotes and domain architectures of proteins, when combined with sequence analysis, can be a powerful means of discerning protein function (47) . indeed, this method has proven particularly effective in both function prediction and identification of new analogous systems, using the organizational syntax of tightly linked genes, in case of toxin-antitoxin and restrictionmodification systems (9, 13, 14, 23, 48) . to better understand the role of the sukh domain we performed a detailed analysis of the gene-neighborhoods of all bacterial genes encoding a protein with this domain ( figure 2 ). consequently, we were able to identify at least three striking themes among the gene-neighborhoods of this superfamily. firstly, across the bacterial phylogenetic tree we found numerous genomic neighborhoods that linked two or more adjacent genes encoding sukh domain proteins. in certain cases, e.g. b. grahamii (gi: 240850988), we found tandem arrays with up to six paralogous sukh superfamily genes ( figure 2 ). we found that in several instances these paralogous versions are not closely related and in certain cases adjacent paralogs might belong to completely different sukh groups. for example, we found combinations of genes encoding proteins belonging to the smi1-like (sukh-1), syd-like (sukh-2), sukh-3 and sukh-4 groups in the same neighborhood in several bacteria such as b. cereus mm3 and various streptomyces species (figure 2 ). this observation suggested that there appears to be selective pressure for the diversification of the linked sukh domain proteins encoded in a gene neighborhood either via sequence divergence, or independent assembly of neighborhoods from distantly related paralogs of different groups. this situation, wherein multiple paralogous genes are linked together as tandem arrays in a neighborhood, is relatively rare in bacteria (49) . given that products of genes linked in conserved gene-neighborhoods physically interact, it is possible that these paralogs interact to form a single complex (47) . on the other hand, the multiple paralogs could also represent different alternative versions of the same component of a system which is under selection to display diversity. given the great variability in the numbers and types of paralogous versions of the sukh superfamily encoded by these neighborhoods, we favor the later explanation in this case (details see below). the second major feature that emerged from the analysis of gene neighborhoods was the linkage of genes encoding diverse sukh superfamily members to genes encoding different types of nucleases ( figure 2 ). among these, we observed multiple linkages in distantly related bacteria, such as b. thuringiensis and m. marina and s. griseoflavus, to genes for nucleases of the metal-dependent nuca family, which includes the well-studied s. marcescens secreted endonuclease (50) and the anabaena non-specific endonuclease nuca, which degrades both rna and dna (51) . another prominent linkage observed in several bacteria, such as m.infernorum, various bacillus species and n. mucosa, was to genes encoding proteins with a hnh superfamily nuclease domain ( figure 3 ). sequence analysis showed that several of the hnh domains were related to similar nuclease domains found in previously studied bacteriocins such as pyocin ap41 of p. aeruginosa, klebsiella klebicin b and colicin e8 of e. coli (52) . these linkages involved members of both the smi1-like and syd-like groups; thus, despite their diversity, potential functional interactions with different types of nuclease domains are a common feature of the bacterial representatives of the sukh superfamily. the third major linkage we observed was between sukh superfamily genes and those encoding gigantic bacterial surface proteins with repetitive motifs such as the hemagglutinin-repeats, rhs repeats (yd) and another previously uncharacterized a-helical repeat motif. all these proteins showed a characteristic feature of possessing a highly variable but globular domain at the extreme c-terminus of the protein, downstream of the repetitive region. these proteins also usually contain 'h' in red, a-helix). the numbers in bracket are indicative of the excluded residues from sequences. 'hash' indicates the residues involved in metal ion-binding, 'percent' symbol indicates the conserved histidine which is required for activation of the water molecule for hydrolysis and 'asterisk' indicates the conserved asparagines. on the right, structures of hnh and endog families are shown as cartoon representations with the central structural core colored by structural element type (a-helices in purple, b-sheets in yellow), and key catalytic residues highlighted. for those newly identified families, inferred topology diagrams of their core nuclease domains are shown with conserved catalytic residues. certain domains related to adhesion and the two-partner secretory (tps) system n-terminal to the repetitive region, such as paar (pfam: pf05488) and the tpsa-secretion domain (tpsa-sd, also known as the filamentous hemagglutinin fhab secretory domain; pfam: pf05860) with a pectate lyase-like fold (53) (54) (55) . some of these proteins with repetitive domains, which were recovered in our analysis of sukh superfamily neighborhoods, are representatives of toxins of the cdi systems ( figure 2 ) that were reported even as this study was being prepared for submission (24, 25) . like the above proteins, the cdi toxins are characterized by multiple n-terminal tpsa-sd domains and hemagglutinin-repeats combined with polymorphic c-terminal domains that vary greatly between different cdi toxins. in all these cdi proteins the polymorphic c-terminal domain is separated from the repetitive region by either or both of two small a-helical domains annotated as domains of unknown function in the pfam database (duf638 or duf637). furthermore, it was shown that the protein encoded by the gene following the cdi toxin was an immunity gene, whose product provided resistance against the toxin to the cell that was producing it (25) . by this criterion it became clear that the sukh superfamily genes in the cdi operons were actually immunity proteins for the toxins encoded by the upstream genes. however, in contrast to the pan-bacterial distribution of the sukh superfamily, the cdi operons were only observed in proteobacteria (25) . furthermore, we observed that polymorphic c-terminal domains of the cdi toxins, which are found linked to the sukh superfamily immunity proteins in cdi systems, are also seen in bacterial lineages outside of proteobacteria, where too they are linked to sukh superfamily genes. in these cases they are linked to other n-terminal domains that are distinct from the tpsa-sd and hemagglutinin repeat domains. studies on cdi systems indicated that the toxin function resides in the polymorphic c-terminal domains and at least two of these domains are nuclease toxins that cleave both trnas and dna (25) . our above observations indicate that outside of cdi systems, the sukh superfamily genes are linked to genes encoding the hnh and nuca nucleases; hence, it is likely that even these nucleases function as distinct but analogous toxins that cleave nucleic acids in target cells. together, the above observations raised the possibility that the sukh superfamily protein might serve as immunity proteins, not just in certain proteobacterial cdi systems, but also more generally function, across all major bacterial lineages, to protect against linked genes, which are predicted to act as toxins. interestingly, in addition to gene-neighborhoods with multiple tandem divergent sukh superfamily genes, in several bacteria, we also observed notable lineage-specific expansions of sukh domain proteins (e.g. 21 paralogs in gemmata obscuriglobus, 20 paralogs in c. gingivalis and 15 in s. albus). these observations also make sense in light of the above toxin-immunity protein hypothesis: while the sukh superfamily gene adjacent to a nuclease or cdi toxin gene is likely to provide immunity to the 'self' toxin, the supernumerary sukh superfamily genes, which occur as tandem arrays or as isolated versions, might provide immunity against other 'non-self' toxins delivered by competing bacteria in the environment. such associations of multiple distinct immunity genes have also been observed in the case of plasmid-borne colicin gene operons (8) . other features of the genomics of the sukh superfamily also support this proposal. gene neighborhoods encoding sukh proteins and linked nucleases or cdi toxin are highly variable in terms of being present or absent between different strains of the same species or between different closely related species which share an otherwise similar genomic organization. secondly, there appear to have been recent duplications of entire loci encompassing these gene-neighborhoods within the same genome in several bacteria (supplementary data). this kind of phyletic and genomic polymorphism is also typical of loci involved in inter-and intra-genomic competition such as toxin-antitoxin, restriction-modification and virulence toxin systems (6, 9, 10, 15) , suggesting that even systems with sukh superfamily proteins might have comparable roles. to test this proposal further, as the first line of investigation, we aimed at exploring further the link between nucleases and the sukh domain proteins. while the polymorphic c-terminal domains of two cdi toxins have been characterized as nucleases, the c-terminal domains of those cdi toxins which are found linked to the sukh superfamily immunity proteins have not be characterized. we speculated that these domains, along with some of the other uncharacterized domains in proteins encoded by conserved gene-neighborhoods containing a sukh superfamily gene, might be as yet uncharacterized nuclease domains. as a second line of investigation we sought to uncover those among the associated uncharacterized domains, which might have a role in distinct toxintrafficking mechanisms, comparable to the two-partner system used by the proteobacterial cdis. therefore, to accomplish these two objectives and identify other components of these systems we resorted to systematic sequence analysis of the uncharacterized proteins recovered in the above gene-neighborhood analysis. sequence analysis reveals the presence of 11 distinct families of nuclease toxins encoded by genes adjacent to those of the sukh superfamily sequence analysis indicated that at least 11 distinct families of domains recovered in our searches in proteins encoded by genes adjacent to one encoding a sukh domain protein are potential nucleases. while some of these, as noted above, belong to the earlier characterized families, several of those identified here belong to entirely new families or are highly distinctive previously unrecognized versions of previously known families (figures 3-5 and supplementary data). identification of this diverse panoply of nuclease domains as being functionally linked to the sukh domain lends critical support to the proposal that this domain functions primarily as an immunity protein against nucleic acid-targeting toxins in bacteria. we briefly describe below these newly identified nuclease domains. nuclease toxins of the hnh/endovii fold. the hnh or the endovii fold is a version of the treble-clef fold. the treble-clef fold is one of the most prevalent zn-binding motifs across the three superkingdoms of life (56) . classical hnh nucleases, like the restriction endonuclease (rease) mcra and the t4 endonuclease vii, contain the four conserved, zn-chelating cysteines of the treble-clef fold (52) . however, these cysteines are lost in several forms, such as the rease mboii, colicin e8 and the nuca family, but these domains still retain the characteristic structural geometry of the treble-clef (52, 56) . the active site of these enzymes is formed at the interface of the characteristic helix and b-hairpin and contains a divalent cation, which is chelated by three polar residues usually from the first strand of the b-hairpin and the c-terminal helix of the treble-clef fold. the residues chelating the metal are typically histidine, aspartate and asparagine but their exact configuration can greatly vary between different members of this fold making them difficult targets for identification through sequence analysis (52) . among the nucleases of this fold occurring in the neighborhood of the sukh superfamily we observed eight distinct families spanning the entire gamut ranging from conventional hnh nucleases to certain highly derived forms that have not be identified before. the conventional hnh versions (e.g.am1_0143, gi: 158333371 from the cyanobacterium a. marina) retain all the four cysteines of the treble-clef fold and a typical arrangement of residues chelating the catalytic metal. others, like the nuclease domains of the pspto_3229 protein from p. syringae (gi: 28870395) and some cdi proteins, belong to the colicin e7/e8/e9 family (figure 2) . a highly derived version is represented by the nuca family (57) , where structural analysis reveals that a treble-clef domain which has lost the characteristic cysteines is inserted between two copies of a three-stranded domain with distinct loop-like c-terminal extensions (figure 3 ). we uncovered several divergent, earlier unrecognized nuca family nuclease domains in both the sukh superfamily neighborhoods and cdi systems, such as those typified by the b. subtilis protein yeef (gi: 251757354). the structural organization of the nuca domain suggests that it arose from an ancestral hnh/endovii domain, which 'carried' these duplicated three-stranded units along with it to form a more complex domain. consistent with this proposal, we discovered a family of novel hnh fold nucleases in our gene-neighborhoods, which contain an active site similar to the nuca nucleases, but are standalone versions without the two flanking three-stranded units. we called this family gh-e after the three conserved residues associated with the active site typical of these domains. interestingly, a subset of the gh-e family preserves the conserved cysteines of the treble-clef suggesting that they indeed represent the potential evolutionary intermediate from a classical hnh domain to the derived nuca-like forms (figure 3) . we also recovered three other novel families of domains, which are respectively typified by nearly absolutely conserved tripeptide sequence motifs lhh, whh and ahh (figure 3 ). most cdi operons, which encode a sukh domain immunity protein, have proximal toxin genes with a lhh domain as the polymorphic c-terminal unit of their products ( figure 2) . additionally, the lhh domain is found in products of genes adjacent to the sukh superfamily gene outside of proteobacteria in several other bacterial lineages such as firmicutes, actinobacteria, bacteroidetes and planctomycetes ( figure 2 ). although we also found the whh domain as the polymorphic toxin unit of a subset of proteobacterial cdi systems, none of these have a sukh superfamily immunity protein. however, we found several non-cdi gene neighborhoods, which are likely to define distinct but analogous toxin systems, in proteobacteria, firmicutes, actinobacteria, synergistetes and bacteroidetes that combine genes for whh and sukh domain proteins (figure 2 ). the ahh domain is also found in similarly organized gene-neighborhoods from the same bacterial lineages as those in which the whh and lhh domains are found. profile-profile comparisons with multiple alignments of all these three novel domains indicated that the best matches are families of the hnh fold. indeed, a visual examination of the conservation patterns of these three domains showed that the hh dyad shared by them corresponds to the hh or dh dipeptide found in the first strand of treble-clef fold of the classical hnh domains (52). the first h forms one of the catalytic metal-chelating ligands and the second h contributes to the active site that directs the water for phosphoester hydrolysis (58) . further, the sequence alignments of the lhh, whh and ahh motifs revealed two further conserved histidines, which were associated with the helix of the treble-clef fold and aligned with the two c-terminal metal-chelating residues in the profile of the classical hnh domains (58, 59) . these observations indicated that the lhh, whh and ahh domains are highly derived versions of the hnh fold. the eighth family of hnh fold enzymes emerging from this analysis comprises of proteins typified by the protein dd586_1447 (gi: 271499995) from d. dadanti found in predicted toxins in sukh neighborhoods and also in cdi operon products which do not contain a sukh-type immunity protein (figure 2) . a subset of these domains constitutes the pfam model for a 'domain of unknown function', duf1994, that does not define the boundaries of this domain precisely. we were able to define the proper boundaries of this domain by using the diversity of distinct architectural contexts in which we detected it and used the refined alignment for profile-profile comparisons. this comparison revealed the representatives of hnh domains as the best hits and indicated a perfect match between the polar residues conserved in this domain and catalytic and active-site metal chelating residues of the classical hnh domains. we named this family of hnh domains as dh-nnk after the conserved dh dyad in the strand-1 and the two asparagines and lysine which are conserved in the helix of the core treble-clef fold (figure 3 ). while all these above versions have lost the cysteines of the ancestral treble-clef, they nevertheless, retain the catalytic configuration typical of those nucleases. hence, we predict that these domains are likely to be nucleases with a similar catalytic mechanism. practically all characterized hnh fold nucleases, barring those of the nuca family, which show a distinct active metal chelating site (51), have a preference for dna substrates. hence, it is likely that most of these domains are the active components of toxins that hydrolyze dna in the target cells. nuclease toxins of the endou fold. the endou nuclease domain is typified by the nuclease domain previously identified in the u-specific, metal-dependent endonuclease, which in eukaryotes processes intron-encoded u16 and u86 snornas and generates products with 2 0 -3 0 cyclic phosphate and 5 0 -oh termini (60) . a related endonuclease was identified in nidoviruses, such as the severe acute respiratory syndrome coronavirus where it appears to process rnas as a part of the replication complex (60, 61) . our structural analysis revealed that the catalytic domain of these enzymes contains two elements each comprised of a single helix followed by a three-stranded unit. this suggests that it is likely to have emerged through duplication of the simple helix-three-strand structural element, followed by flipping of the sheet in one of the units ( figure 4a ). the catalytic residues, i.e. two histidines, appear to have emerged asymmetrically in a peculiar hairpin insertion within the helix of the first repeat. this hairpin insertion appears to be mobile and adopts different conformations in structures (60,61)-this mobility might have a role in accommodating the substrate between the helix and the sheets formed by the three-stranded units of the repeats ( figure 4a ). we found that the bacterial members of the endou family are linked to genes of the sukh superfamily mainly in firmicutes and proteobacteria ( figure 2 ). other than sukh superfamily gene-neighborhoods, related versions also comprise the polymorphic c-terminal domain of the cdi toxins from moraxella and mannheimia that, however, lack a sukh superfamily immunity gene. a further set of bacterial nucleases of this family are predicted secreted versions encoded by intracellular symbiotic and pathogenic bacteria, such as wolbachia (gi: 310643370) and ehrlichia (gi: 73666818). most bacterial versions that we identified are extremely divergent relative to the eukaryotic and viral forms and are not recognized by the previously available hmm models for this nuclease (pf09412). hence, the identification of these relationships represents a significant extension of this superfamily ( figure 4a , supplementary data). versions within these gene-neighborhoods show considerable variability including loss of strands from the first unit. this variability suggests that the endou fold is rather flexible to accommodating drastic modification, which in turn might help it recognize a diverse spectrum of substrates. on the precedence of the eukaryotic endou and the nidoviral nuclease and their genomic organization we suggest that the majority of the bacterial endou homologs are nuclease toxins that cleave rnas in the competitor cells. those secreted by intracellular bacteria could be deployed as toxins or regulators to manipulate host physiology by cleaving specific transcripts. with the identification of these new endou homologs it becomes clear that the bacteria contain the greatest diversity of this superfamily, with certain versions closer to the eukaryotic and nidoviral versions and others that are more divergent (supplementary data). this suggests that the original radiation of this superfamily probably happened within the bacterial toxin systems and were subsequently acquired, perhaps from intracellular symbiotic bacteria, by eukaryotes and viruses. in the latter they appear to have been recruited as rna processing enzymes. nuclease toxins of the rease fold. the rease fold is a highly versatile fold that accommodates considerable structural diversity and has, not surprisingly, been used as the primary fold from which reases of restriction-modification systems are derived (48, 52) . we also found several proteins with this fold to be encoded by genes that are neighbors of sukh superfamily genes ( figure 2 ). these versions were originally identified as a distinct conserved domain of unclear affinities-both psi-blast and hmmer searches failed to identify any relationships with previously known domain. however, we observed that the multiple sequence alignment of this domain showed a characteristic signature of conserved residues of the form ge-d-exk-q ( figure 4b ) that matched the pattern of similar conserved residues in the lambda exonuclease and the recb family of the rease fold (52, 62) . the predicted secondary structure pattern of these domains also closely matched the rease fold with conserved d and exk motif falling on a b-hairpin as is typical of the rease fold ( figure 4b ). these observations induced us to use the alignment of this domain in a profile-profile comparison with the hhpred program, and we recovered a composite profile made of diverse rease fold superfamilies such as the vrr-nuc, lambda exonuclease, the archaeal holliday junction resolvase and recb as the best hits (p = 10 à5 ). this suggested that this family defines a novel group of rease-fold nucleases. given that the majority of the rease-fold enzymes are dnases, we predict that these toxins are likely to cleave the dna of the target cells. nuclease toxins of the cytotoxic rnase fold. the last family of nucleases that we found encoded by genes linked to the sukh superfamily genes was the cytotoxic rnase family (63) . this nuclease domain was first characterized as the toxin domain of the colicins e3 and e6 and is typified by a conserved active site configuration with an aspartate followed by a glutamate sandwiched between two histidines (supplementary data). the version of this domain in colicin e3 has been demonstrated to function as an endornase that specifically cleaves the phosphoester bond between bases 1493 and 1494 of 16s ribosomal rna (63) . given that versions detected in systems characterized in our current study are closely related to the version found in colicin e3 and e6, we posit that these nuclease domains act as rnases that similarly cleave rna in the target cells. other domains with a possible role in nucleic acid modifications. we found three other families of domains in proteins that were encoded by genes which occupied positions adjacent to sukh superfamily genes in certain predicted operons, equivalent to positions of the genes encoding the above nucleases. additionally, these families of domains are also found as representatives of the polymorphic c-terminal module of the proteobacterial cdis. together these observations hinted that they are potentially uncharacterized enzymatic domains operating on nucleic acids. psi-blast and jackhmmer searches showed that the first of these families belonged to the nucleotide deaminase superfamily that includes rna-editing enzymes, such as the apobecs and dna-modifying enzyme aid of vertebrates. hence, like the nucleases, these enzymes are likely to function as toxins that mutate nucleic acids in the target cells. we discuss the natural history of these enzymes in a separate article (iyer lm, zhang d, aravind l, manuscript in preparation). the second of these families prototyped by the b. cereus protein bcere0017_55840 (gi: 229119351) is characterized by a conserved signature [ns]hh followed by another conserved histidine (supplementary data). although we were unable to unify this family with any of the other nuclease folds, the presence of the hh motif typical of many of the above families of hnh/endovii fold nucleases might point to a divergent relationship with those proteins. the third of these families, typified by the cdi system from p. luminiscens (gi: 37524545) includes a globular domain of 170-200 amino acids that might define yet another uncharacterized nucleic acid-modifying domain (cdiac in figure 5 , supplementary data). earlier characterized toxin systems such as the classical plasmid-encoded bacteriocins and the recently characterized cdi systems use thematically comparable, albeit biochemically distinct mechanisms for trafficking of nuclease toxins. while these systems have been used as models to understand bacterial protein trafficking, the complete set of events starting from the extrusion of the 'pro-toxin' by the producing cell to its recognition at the target cell surface and delivery into the target cell are only partially understood (8) . classical plasmid-borne colicins and cognate bacteriocins from other bacteria do not have secretory mechanisms and their release appears to occur primarily through cell-lysis mediated by the colicin-release proteins (8) . colicin-like bacteriocins are multidomain proteins with an extreme c-terminal toxin module, which is either a nuclease or a membraneperforating domain (e.g. colicin e1 and a) (8) . they typically possess two additional n-terminal modules, of which the first facilitates translocation across the target cell membrane and the second (i.e. the central module) facilitates binding to a membrane receptor on the target cell. these colicins hijack either the tol or the tondependent molecular import systems to enter the target cells (8) . the chromosomally encoded proteobacterial cdi system toxins do not require lysis; instead they are trafficked out of the cell which produces them via the twopartner-system that depends on the cdib proteins belonging to the tpsb class of outer-membrane trafficking proteins (25) . these latter proteins contain n-terminal periplasmic polypeptide-transport-associated (potra) domains linked to a c-terminal b-barrel transmembrane domain. they recognize the secretory domains such as the tpsa-sd in the extreme n-terminal region of the cdi 'pro-toxins' to deliver them across the outer membrane of proteobacteria (64) . this n-terminal region is separated from the c-terminal regions by repetitive regions with rhs-or filamentous hemagglutinin-type repeats. their uptake by the target cell is less-clearly understood. in the well-studied examples, the first step of this process appears to depend on the outer membrane-biogenesis protein bama recognizing a conserved a-helical domain immediately n-terminal to the toxin module, with a venn signature that overlaps with the pfam model termed 'duf638 0 . subsequently the inner-membrane protein acrb, a transporter, appears to be necessary for uptake into the target cell cytoplasm (25) . additionally, it is posited that a proteolysis step at the cell surface releases just the c-terminal nuclease module for uptake by the target cell (25) . thus, despite the differences between the cdi and classical colicin-like systems they share a common feature of the toxin activity being borne by the extreme c-terminal domain in a multidomain polypeptide. further, the modules located immediately-n-terminal to the nuclease domain (e.g. the a-helical domain with the venn motif $pfam duf638) are involved in association with receptors on the target cell. hence, we term these domains collectively the pre-toxin (pt) domains. the extreme n-terminal domains appear to play a critical role in export from the host cell in the cases where lysis is not involved, i.e. typically chromosomally borne versions. these observations accordingly presented the organizational logic for these systems, wherein there are usually three functionally distinct sets of modules in the pro-toxin going from the n-to the c-terminus of the protein. analysis of the domain architectures of the nuclease domain-containing proteins encoded in the sukhsuperfamily neighborhoods revealed that the majority of the proteins followed an architectural logic which was consistent with the above-described organization of these earlier studied toxin systems ( figure 5) . however, only a relatively small subset of the sukh domain-associated systems overlaps with the cdi systems. further the sukh superfamily proteins and functionally linked toxins are also found outside of proteobacteria, in lineages lacking outer membranes and cdib-like delivery systems. we reasoned that analysis of these distinct pre-nuclease and extreme n-terminal domains might reveal features pertaining to the trafficking of toxins in non-cdi systems and point to alternative delivery mechanisms. identification of multiple distinct trafficking systems for toxins encoded in sukh superfamily neighborhoods. we observed that in gram-positive bacteria, proteins with the c-terminal nuclease typically possessed one of a set of several distinct domains at the extreme n-terminus of the protein ( figure 5) . a significant subset of these could be unified using sequence profile searches with the psi-blast and jackhmmer programs to the wxg/esat6 superfamily of a-helical domains (65) . these domains are a specific signal recognized by the yuea-like atpases of the hera-ftsk superfamily that secrete them in an atp-dependent manner (65, 66) . this indicated that the wxg/esat-6 domain-containing toxins in gram-positive bacteria are extruded by yuea-like pumps using an atp-dependent mechanism. a significant subset of toxin proteins from firmicutes possessed a distinctive n-terminal domain that could not be unified with any earlier known domain (a subset of these have been included in the erroneously annotated model transposase_30 of pfam; pf04740). sequence searches showed that this domain possessed a conserved [lf]xg sequence motif and it was predicted to assume an a-helical bundle fold based on the multiple sequence alignment (supplementary data). we accordingly termed it the lxg domain ( figure 5 ) and were able to unify it with the wxg domain by means of profile-profile comparisons with the hhpred program (p = 10 à7 ). contextual analysis indicated that this domain is encoded by certain conserved gene-neighborhood across firmicutes, where it is associated with genes coding for a yuea-like hera-ftsk superfamily protein pump and a small protein related to the s. aureus esac protein (gi: 282917938, supplementary data). through profile-profile comparisons we showed that the esac-like superfamily is a bacterial version of the eukaryotic evh1 peptide-binding domains with the ph-like fold (hhpred p-value: 10 à4 ) (67). these observations suggest that the lxg domain is comparable to the wxg/esat-6 domain, and is likely to utilize the atp-dependent yuea pumps and the potential peptide-binding esac domain as partners for extrusion from the producing cell. the protein srot_0310 (gi: 296392744) from the actinobacterium s. rotundus contains two copies of a distinct domain n-terminal to the gh-e nuclease domain ( figure 5 ). this domain is also widely found in several actinobacteria at the n-termini of putative cell-surface proteins. profileprofile comparisons suggested a possible relationship between these n-terminal domains and the wxg domain suggesting that it might be yet another representative of the wxg-like superfamily (p = 10 à4 ) and might utilize a similar atp-dependent mechanism for its extrusion. a fourth group of proteins, restricted to certain firmicutes (e.g. s. aureus sacol0281 protein; gi: 57652555), is typified by yet another n-terminal a-helical domain (ldxd in figure 5 ) that is also found in domain architectural contexts very similar to the wxg and lxg domains. it is conceivable that this domain is comparable to them and functions similarly as a mediator of export via the hera-ftsk superfamily pumps. thus, a notable mode of export of nuclease toxins in gram-positive bacteria appears to be via the atp-dependent extrusion system, which while biochemically distinct from the tps of the proteobacteria, is thematically comparable. in actinobacteria, but not firmicutes, we observed several large proteins with architectures similar to the cdis of the proteobacteria. these typically contain rhs repeats; however, their extreme n-terminal domains did not bear any close relationship to the proteobacterial tpsa-sd. instead they were found to contain an n-terminal signal peptide and some of these proteins (e.g. gi: 256812841, a protein from s. griseus) contain multiple lamining domains embedded within repetitive regions. the protein dip1652 (gi: 38234225) from c. diphtheria shows another distinct low complexity repeat n-terminal to the nuclease domain ( figure 5 ) and like in the above case it also possesses a conventional signal peptide. likewise, a distinctive signal peptide, which is highly conserved in multiple proteins only within the genus planctomyces, is seen in predicted nuclease toxins from this organism (e.g. gi: 149178028). another group of large toxin proteins with rhs repeats, which predominantly occur in proteobacteria, are defined by the presence of repeats of the paar domain (pfam: pf05488) n-terminal to the rhs repeats. all these proteins are typified by the presence of a conserved transmembrane domain with two tm segments ( figure 5 and supplementary data) just n-terminal to the paar domains. we propose that these tm segments are required for their trafficking to the cell membrane, following which they might be processed in the periplasm for release via the outer membrane in a process that might depend on the paar domains. we also noticed a comparable domain with two tm segments in few firmicutes (e.g. gi: 125974537 from c. thermocellum) and in chlamydiae (e.g. 189219187 from m. infernorum, which is a rare case of the nuclease domain occurring n-terminal to the two tm domain; figure 5 ). these proteins lack paar domains but the firmicute versions have additional hedgehog-intein (hint) peptidase domains (see below) that could aid in their release on the cell-surface ( figure 5 ). these observations suggest that at least some nuclease toxins in bacterial lineages such as actinobacteria, bacteroidetes and planctomycetes with conventional signal peptides, and those in proteobacteria, chlamydiae and firmicutes with two-tm domains are probably delivered to the cell using the conventional sec-dependent system (68) . in the context of the above cases, it is of interest to note that e. coli syd, an archetypal member of the sukh superfamily, was first identified as a possible proof-reading component of the sec-dependent export system (43) (44) (45) . in this context it is possible that the binding of certain members of the sukh superfamily (at least the syd-like group) in the producing cell might not only help in conferring immunity to 'self' but also in guiding the 'pro-toxin' to the sec-dependent export machinery. both actinomycetes and firmicutes do not display proteins with a pt domain with the venn motif (pt-venn). however, we observed that in both these lineages there was a conserved a-helical domain that frequently occurred just to the n-terminus of several distinct nuclease modules in different predicted toxins. this domain had a conserved tg motif and we accordingly named it the pt-tg domain ( figure 5 another domain, which we found frequently associated with several unrelated or distantly related nuclease domains from gram-positive bacteria, was the nuclease_n domain ( figure 5, supplementary data) . it is predicted to be an a-helical domain and might also play a role in the delivery of the toxin module into the host cells. toxins in the sukh superfamily neighborhoods, irrespective of the type of the nuclease domain, can also be distinguished into two major architectural groups: one comprised of relatively small proteins with no notable stretches of repetitive sequence separating the n-from the c-terminal regions, and the second in which such repetitive sequences, such as the rhs and the filamentous hemagglutinin are present ( figure 5 ). this might reflect a mechanistic difference in their mode of action: the smaller proteins could be soluble toxins that diffuse away from the cell producing it. in contrast, the large proteins with repetitive elements might form filamentous appendages that stick out from the cell-surface and depend primarily on contact with target cells for delivery [hence, the latter group includes the recently characterized cdis (25) ]. alternatively, this difference might reflect the differences in the cell-wall structures of the bacterial lineages, with the smaller toxin proteins being more prevalent in the firmicutes. a subset of the smaller proteins with nuclease domains lack noticeable trafficking-related (n-terminal) domains. the corresponding genes could represent cassettes for alternative toxin modules that are linked by recombination to the larger full-length genes ( figure 5 , see below). other auxiliary domains which might play a role in resistance, trafficking or processing of toxins. several other domain families were found to be encoded by genes having persistent association with the sukh superfamily neighborhoods across distantly related bacterial species. one of these is the sufu superfamily ( figure 2 and supplementary data) prototyped by the suppressor of fused protein from drosophila (69). in addition, we also detected members of this superfamily to be encoded by cdi-like operons, such as the one from n. gonorrhoeae that encodes a toxin with a distinct version of the hnh fold nuclease domain (toxin ngo1392, gi: 59801740; supplementary data). in these cases the sufu superfamily gene occupies a position equivalent to that of the sukh superfamily gene, suggesting that they might be functionally comparable. we also found several examples wherein the sufu and sukh domains are combined in the same polypeptide (figures 1 and 5) . based on these associations we propose that the sufu domain represents a second widely conserved domain that function as an immunity protein for diverse nuclease toxins. two other conserved protein families are encoded in the toxin neighborhoods (sukh-neighborhood conserved family 1 and 2; sncf1 and sncf2, supplementary data) that occupy positions similar to the sukh and sufu superfamily genes ( figure 2 ). they were not found in multi-domain architectures typical of the nuclease toxins and always occurred as proteins with standalone domains. this suggested that they were unlikely to be novel toxins but act as alternative immunity proteins just like the sufu and sukh superfamily proteins. the hint domain, prototyped by the peptidase domains of the animal hedgehog proteins and protein-splicing inteins, is also frequently associated with sukh superfamily neighborhoods (70) (71) (72) . these versions of the hint domain are closer to those found in several bacterial surface proteins and the secreted animal proteins such as hedgehog and the c. elegans hog proteins (70) . when present in a multidomain 'pro-toxin' protein, the hint domain always occurs sandwiched between the pt domains such as pt-venn and pt-tg and the nuclease toxin domain. this location of the hint domain suggests that it is likely to serve as a peptidase that undergoes autoproteolytic cleavage, similar to what is observed in hedgehog and the inteins (70) , to release the c-terminal nuclease domain for uptake by the target cell. it is conceivable that this cleavage step is regulated by the interaction of the pt domains with the surface receptor on the target cell. eukaryotic/dna viral members and structure-function analysis of the sukh superfamily while sukh superfamily neighborhoods are very widespread in bacteria, they are largely absent in archaea. although we uncovered potential extruded nuclease toxins in certain halophilic archaea such as h. borinquense (gi: 312291883, with a gh-e nuclease domain), which are delivered by means of a distinctive n-terminal metallopeptidase domain, we did not find any immunity proteins of the sukh or sufu superfamilies. although the exact reason for this exclusion is unclear, it is conceivable that these immunity proteins are ineffective in the context of the distinct archaeal secretory systems. however, several eukaryotes possess one or more sukh superfamily members. phylogenetic analysis and phyletic patterns suggest that there are two major eukaryotic lineages of the sukh superfamily that are nested within the radiation of the bacterial versions (supplementary data). they are respectively prototyped by the polyglutamylase subunit pgs2 (22) , and the vertebrate scf ubiquitin e3 ligase subunit fbxo3 with yeast smi1/knr4 (21, 73) . the pgs2 version is found in basal eukaryotes such as giardia and spironucleus, animals and chlorophyte algae suggesting that it was likely to have been acquired prior to the last eukaryotic common ancestor (leca) and subsequently lost in several lineages. the fbxo3 lineage is present in animals, fungi, plants, stramenopiles and ciliates. however, it does not group with the pgs2 lineage, instead grouping with other bacterial forms. hence, it was probably acquired relatively early in eukaryotic evolution via an independent transfer from bacteria. in both plants and animals the fbxo3 version is fused to an n-terminal f-box domain and a distinctive c-terminal immunoglobulin superfamily domain (overlaps with the pfam model duf525), suggesting that it was recruited as an e3 subunit prior to the radiation of these eukaryotic groups. in addition to these versions, there appear to have been other sporadic transfers of sukh superfamily members to eukaryotes. for example, land plants contain a version typified by the arabidopsis protein at3g50340 (gi: 15229727) which seems to have been independently acquired by them from a bacterial source. another sporadic transfer is seen in certain filamentous fungi, which acquired a version of the sukh-4 group that has been independently fused to an n-terminal f-box domain (e.g. a. oryzae gi: 169782758). dna viral versions show no specific relationship with eukaryotic forms; instead, they share specific sequence motifs with the sukh-3 group, recover them as best hits in profile-profile comparisons, and group with them in the phylogenetic tree (supplementary data). within viruses they are most widespread and abundant in herpesviruses, with the versions from adenoviruses, poxviruses and iridoviruses being nested within the herpesviral radiation of the family (supplementary data). thus, they appear to have been acquired first by an ancestral herpesvirus, similar to that inserted in the amphioxus genome (46) , from a bacterial source and subsequently disseminated across diverse dna viruses. although there has been gene loss in several eukaryotic lineages, at least the two ancient versions, namely pgs2 and fbxo3 appear to have been largely vertically inherited and show no lineage-specific expansions within eukaryotes. this is in sharp contrast to the high propensity for lateral transfer and for lineage-specific expansions of the sukh superfamily that is observed in bacteria. this feature, together with the available functional evidence suggests that these conserved eukaryotic versions have acquired a biological role distinct from that in the toxin-immunity systems of bacteria. nevertheless, there were several features that suggested to us that biochemically the eukaryotic versions might be exploiting an ancient functional template provided by the sukh domains in bacterial nuclease toxin systems. firstly, the studies on yeast smi1/knr4 have shown that it interacts with a large number of structurally and functionally distinct proteins (19) . in fbxo3, and independently in the above-mentioned fungal proteins, it appears in a domain architectural context corresponding to the part of the e3 f-box subunit that recognizes the substrate for ubiquitination (74) . this suggests that it might be deployed as a recognition domain to recruit particular substrates for ubiquitination. in bacteria the sukh superfamily domains are one of the most widespread immunity proteins that appear to function in conjunction with a repertoire of nuclease toxins that are extremely diverse in sequence and structure (figures 3 and 4) . taken as a whole, these observations indicate that the sukh domain contains a scaffold that has been adapted to recognize a diverse set of protein partners. a possible clue for the structural basis of this capability is offered by studies on the e. coli syd protein: it has been shown to contain a prominent negatively charged cleft with which it could interact with partner proteins (45) . examination of the structure of this protein indicates that this cleft is formed by the space between the conserved helix h3 and the fissure in sheet between the two-stranded n-terminal unit and the c-terminal 4-stranded meander ( figure 1 ). given that this unusual feature is seen across the fold, we examined the surface renderings of different sukh superfamily members and a corresponding cleft is observed in most of them ( figure 1 ). although this cleft is not necessarily negatively charged as in syd, and might vary in depth and shape, its widespread presence suggests that it might be the means by which the sukh superfamily is able to accommodate different protein partners. in support of this hypothesis we observed that in the case of two distantly related members of the sukh superfamily, namely syd (pdb: 3ffv) and yobk (pdb: 2prv), this cleft is used in protein-protein interactions. in both these crystal structures one of the monomers is bound in the cleft of the other monomer resulting in an asymmetric dimer (supplementary data). these dimers are unlikely to represent biologically native dimeric states, but in any case illustrate the ability of the conserved cleft of the sukh fold to accommodate other proteins. interestingly, the sufu superfamily also shows a comparable kind of sheet with a fissure between two sets of strands (69) . experimental studies on the drosophila sufu shows that it also functions as protein tether which holds the zn-finger transcription factor gli in the cytoplasm in the absence of the hedgehog signal (75) . in vertebrates the sufu ortholog has been shown to bind gli2 and gli3 and prevent their degradation due to ubiquitination by f-box e3 ligases (76) . thus, the presence of comparable binding interfaces that have the flexibility to recognize a wide range of protein ligands might be a common feature shared by both the sukh and the sufu superfamilies of immunity proteins. it is this feature that appears to have resulted in them being utilized as adaptors for recruiting other proteins in eukaryotic regulatory systems. the extensive spread of the us22 group of the sukh superfamily across unrelated or distantly related dna viruses of animals suggests that it confers an important advantage to these viruses. this is also supported by the lineage-specific expansion in betaherpesviruses of the sukh superfamily in the form of multigene arrays similar to what is seen in bacteria ( figure 2 ). indeed multiple studies suggest that distinct copies of the proteins in herpesviruses are required for effective survival and replication of the virus in their hosts. for instance, mutagenesis of two sukh superfamily paralogs m142 and m143 in the murine cytomegalovirus was shown to be essential for survival of the virus itself, whereas mutagenesis of other paralogs m139, m140 and m141 specifically prevents its replication in macrophages (77) . other studies indicated that m142 and m143 form a heterotetrameric complex which counters the action of the host protein kinase r (pkr) in shutting down viral protein synthesis (78) (79) (80) (81) . the human cytomegalovirus sukh superfamily proteins trs1 and irs1 have been shown to similarly counter the pkr and the dsrna dependent arm of the anti-viral response (78, (82) (83) (84) (85) (86) . another paralog ul38 inhibits the host cell stress responses by antagonizing the tuberous sclerosis protein complex in the endoplasmic reticulum (87, 88) and counters apoptosis in conjunction with yet another paralog ul36 (89, 90) . in light of these observations it appears that the viral versions of the sukh superfamily are deployed to counter different facets of the host anti-viral and stress response. by analogy to the bacterial versions, which function as immunity proteins, we propose that the viral sukh domain proteins in general bind diverse host proteins that are used against the virus. here again the special ability of the sukh scaffold to bind diverse proteins appears to have been exploited by the virus as a flexible binding interface to neutralize a diverse group of host anti-viral defenses. identification of the sukh superfamily and associated nucleic acid modifying toxin systems has considerable implications for understanding bacterial genetic conflicts, evolutionary forces acting on strongly linked multi-gene loci, and potential biotechnological applications. we briefly discuss some of these implications that emerge directly from our observations. relationship of toxin systems to genetic conflicts in the bacterial world. classical colicins and earlier characterized cdis act primarily on related bacterial strains of the same 'species'. although the systems identified in our studies are abundantly represented in extracellular pathogenic bacteria, they are rare in intracellular symbionts or pathogens. this might be because intracellular bacteria are much less likely to encounter a heavy load of competing cells in the same niche. the bacterial toxin systems which we uncovered in this study and the related cdis are also different in certain features from the classical colicin-like systems. classical colicins are in large part encoded on plasmids, which might be either single copy, medium-sized conjugative plasmids or small multi-copy small plasmids that depend on the conjugative plasmids for their transmission (8) . such bacteriocins are relatively rare on chromosomes. in contrast, 99.25% of the systems recovered in our study are chromosomally encoded. majority of the plasmid-encoded classical colicin-like toxins are accompanied by a gene encoding a lysis protein and their release is concomitant with the lysis of the host cell. however, none of the systems identified in this study or the cdis have lysis genes in their neighborhoods (25) . this difference suggests that, while both the plasmid-borne bacteriocins and these systems might be directed at close relatives, they appear to be geared toward distinct genetic conflicts. the lysis of the cell nullifies the fitness of the chromosome; hence, it would be largely deleterious for the chromosome to encode systems that require lysis. the plasmid being a selfish element is not completely affected by loss of fitness of the host as long as it can offset it by holding on to, or spreading in the host population (i.e. the plasmid's own fitness is enhanced or maintained). cells of the host type without the bacteriocinogenic plasmid are competitors that affect the plasmid fitness, especially under stationary phase or starvation conditions. hence, the plasmid-borne colicin would be primarily selected to act against host cells that have lost the plasmid or lack it by default under these stress conditions. further, the plasmid toxins are unlikely to have ready access to trafficking by the host because, given the large amounts in which the colicins are produced (8) , their export is likely to impair host fitness. further, it has been shown that under starvation only $3% of the cells produce colicin (91) . although the loss of the cells producing the colicin would endanger the resident plasmid, a relatively small fraction of the host population is affected. by the principle of inclusive fitness of kin (92), the plasmid could still have an enhancement of fitness from the copies in the surviving cell along with the elimination of competitors by the released toxin. on the other hand, the toxin domains of many of the chromosomal versions like the cdis and those identified in this study appear to be borne on filamentous structures that are primarily geared toward to elimination of competitors that come in physical contact with the cell-surface (25, 93) . therefore, these systems are likely to be critical in the context of the formation and organization of biofilms and solid substrate colonies. when bacterial cells are aggregating in the above contexts it would benefit to eliminate resource sharing with non-kin competitors. hence, presence of a chromosomally encoded toxin that acts at a short range is likely to be selected, resulting in the proliferation of systems such as those described here. nevertheless, it would also benefit 'cheater cells' to evade such defensive mechanisms. hence, they would be selected to maintain a wide diversity of immunity proteins to counter different non-self toxins, which might explain the arrays of diverse sukh genes in several bacterial genomes. potential evolutionary processes in diversification of toxins and immunity proteins. imprints of the evolutionary arms race arising from the above processes are readily observed in our systems. the toxin proteins appear to show a rather peculiar pattern of diversification. the n-termini, which are typically associated with trafficking, tend to be relatively conserved while c-terminal nuclease domains show major diversity ( figure 5 ). this is consistent with a recent study on the diversification of rhs proteins in enterobacteria which showed that the rhs proteins undergo c-terminal polymorphism due to rampant recombination with invading cassettes that encode alternative c-terminal modules (94) . this type of recombination or gene-conversion with polymorphic c-terminal cassettes might explain the presence of smaller loci found in the gene-neighborhoods characterized here that encode just a nuclease domain by itself or with an additional small n-terminal extension (figures 2 and 5) . hence, we extend the original proposal for rhs diversification to suggest that, more generally, recombination with cassettes with distinct c-terminal modules is the primary proximal mechanism for diversification of the toxin proteins across all bacterial lineages ( figure 5) . furthermore, the presence of nuclease and nucleic acid deaminase domains as the primary toxin modules of these systems raises the possibility that their nucleic acid cleaving or mutating activity is involved in triggering recombination events. this appears plausible given the observations that most of these nucleases are likely to be endonucleases, which like their counterparts in the restriction-modification systems could cleave at specific sequences. similarly, deaminase-induced mutations have been implicated in the triggering of class-switching recombination events in vertebrates (95) . more generally, this ties in with earlier studies which have demonstrated the role for both recombination and positive selection in the evolution of plasmid-borne bacteriocins (96) . it has been proposed that pore-forming versions have predominantly utilized recombination for diversification whereas nucleases have mainly evolved through positive selection. in our systems, the evidence points to both these forces being active at different levels in the evolution of the toxin proteins (96) . while the basic architectures evolve through recombination generating c-terminal polymorphism, the c-terminal nucleases themselves show evidence for considerable sequence diversification within each family. indeed, much of the diversification of the hnh/endovii fold appears to have happened within the context of these systems, with several structurally distinct forms evolving amidst the nuclease toxins ( figure 3 ). phyletic and phylogenetic analysis of the sukh superfamily indicates three salient features, namely rampant lateral transfer between different branches of the bacterial tree, gene loss and lineage-specific expansion followed by divergence of the lineage-specific paralogs (supplementary data). this suggests that there is a notable trend for maintaining diversity within the sukh superfamily that probably arises from selection for recognition of a diverse range of nucleic acid-modifying toxins. although there are multiple distinct types of immunity proteins known from plasmid-borne bacteriocins and cdi systems, most show very limited phyletic patterns. for example the cdii toxin seen in several cdi systems is entirely limited to proteobacteria (25) . we observed that it is a protein with two tm segments that is likely to form a membrane channel (supplementary data) and have a mode of action very distinct from the sukh superfamily. as only the sukh superfamily and, to certain extent, the sufu superfamily show a pattern of wide dissemination across bacteria it is likely that only these scaffolds can support sufficient diversification that goes hand in hand with the polymorphism of the toxin domains. implications for eukaryotic and viral functions. our observations also suggest that the biochemical diversity generated within these bacterial toxin systems has been taken up and utilized for very different functions by eukaryotes and their viruses. both the sukh and the sufu superfamily domains have been utilized as adaptors that regulate recognition of different substrates by protein modification systems such as ubiquitination and polyglutamylation. in a completely different context, the hint domains derived from such bacterial toxin systems appear to have been used to release peptide messengers in animal signaling pathways, like the hedgehog pathway (70) . the nuclease domains ultimately derived from various toxins also appear to have been used for different functions by eukaryotes and their viruses. the endou nuclease domain, which ultimately emerged from these toxin systems, has been recruited by the nidoviruses for the replication of their negative-strand rna genome, whereas a related domain was recruited by eukaryotes for processing of certain snrnas. we also observed that a hnh/endovii fold nuclease found in the bacterial toxin typified by the n. gonorrhoea protein ngo1392 is found in several eukaryotic lineages such as animals, plants, stramenopiles and apicomplexans (supplementary data). given its conservation and relatively lower divergence, it is unlikely that the nuclease functions as a toxin in eukaryotes. however, it is possible that it has been recruited as a dna-repair enzyme, as has been previously observed in the case of certain nucleases of bacterial restriction-modification and phage replication systems (97) . in general terms, these observations suggest that the origin of key systems in eukaryotes, including those related to the emergence of certain lineages, such as animals (i.e. the hedgehog pathway), appear to have extensively benefited from the availability of 'pre-adaptations' in the form of components whose ultimate origins lay in these toxin systems. the current study points to the remarkable flexibility of sukh domains in mediating different protein-protein interactions. in a sense, this situation resembles what has earlier been observed with certain scaffolds like the immunoglobulin domain and the leucine-rich repeats of various immunity-related proteins of eukaryotes (98, 99) . the ability of the sukh scaffold to accommodate diverse binding partners makes it a potential candidate as a template for protein engineering to generate novel binding 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the population inclusive fitness theory from darwin to hamilton bacterial contact-dependent delivery systems evolutionary diversification of an ancient gene family (rhs) through c-terminal displacement the aid/apobec family of nucleic acid mutators nucleotide polymorphism in colicin e2 gene clusters: evidence for nonneutral evolution the hiran domain and recruitment of chromatin remodeling and repair activities to damaged dna conservation of folding and stability within a protein family: the tyrosine corner as an evolutionary cul-de-sac structure of a lamprey variable lymphocyte receptor in complex with a protein antigen supplementary data are available at nar online. conflict of interest statement. none declared. key: cord-297324-me5ff1pb authors: zeng, rong; yang, rui-fu; shi, mu-de; jiang, man-rong; xie, you-hua; ruan, hong-qiang; jiang, xiao-sheng; shi, lv; zhou, hu; zhang, lei; wu, xiao-dong; lin, ying; ji, yong-yong; xiong, lei; jin, yan; dai, er-hei; wang, xiao-yi; si, bin-ying; wang, jin; wang, hong-xia; wang, cui-e; gan, yong-hua; li, yu-chuan; cao, ju-tian; zuo, jiang-ping; shan, shi-fang; xie, en; chen, song-hua; jiang, zhi-qin; zhang, xi; wang, yuan; pei, gang; sun, bing; wu, jia-rui title: characterization of the 3a protein of sars-associated coronavirus in infected vero e6 cells and sars patients() date: 2004-07-30 journal: j mol biol doi: 10.1016/j.jmb.2004.06.016 sha: doc_id: 297324 cord_uid: me5ff1pb proteomics was used to identify a protein encoded by orf 3a in a sars-associated coronavirus (sars-cov). immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. elisa indicated that sera from sars patients have significant positive reactions with synthesized peptides derived from the 3a protein. these results are concordant with that of a spike protein-derived peptide. a tendency exists for co-mutation between the 3a protein and the spike protein of sars-cov isolates, suggesting that the function of the 3a protein correlates with the spike protein. taken together, the 3a protein might be tightly correlated to the spike protein in the sars-cov functions. the 3a protein may serve as a new clinical marker or drug target for sars treatment. coronaviruses are a large and diverse family of enveloped, positive-stranded rna viruses. members of the coronavirus family can be divided into three groups according to serologic properties. 1 although an individual coronavirus naturally infects distinct animal hosts and causes particular diseases, the whole-genome sequencing of several coronaviruses such as avian infectious bronchitis virus (ibv), murine hepatitis virus (mhv), human coronavirus 229e (hcov-229e), and porcine transmissible gastroenteritis virus (tgev), revealed similar genomic structure among these different coronaviruses. 2 -5 a novel coronavirus has recently been identified, which causes the latest worldwide outbreak of severe acute respiratory syndrome (sars). the analysis of the complete nucleotide sequence of sars-associated coronavirus (sars-cov) shows that its genomic organization is similar to that of other known coronaviruses. 6, 7 the genome of sars-cov is approximately 30 kb in size and has 14 open reading frames (orfs). 6 -8 it looks easy to analyze the entire genome of coronaviruses, but their protein component identification has proven to be a difficult task. so far, only five types of structural proteins have been identified as members of the coronavirus family. 1 the spike (s) glycoprotein, together with small envelope (e) protein and membrane (m) glycoprotein, consist of the viral envelope, while the nucleocapsid (n) protein interacts with genomic rna of the virus to form the viral nucleocapsid. 1 -9 in addition, a hemagglutinin-esterase (he) glycoprotein has been found in the virion surface of some coronaviruses such as turkey coronavirus. 1 the analysis of mrnas of sars-cov revealed many transcription units covering unknown orfs of the genome. 6, 8, 10 although some researchers speculated that these unknown orfs encode nonstructural proteins, 6 there is no evidence to support this assumption. analysis of the genomic organization of sars-cov showed that a gene locus containing orf3a and 3b located between the s and e genes, 6 -8 is frequently found in different members of the coronavirus family (see figure 4a ). it has been hypothesized that orf 3/3a and orf3-1/3b may be related to viral virulence and pathogenesis, 11 -13 whereas some suggested that orf3a of tgev is not essential for enteric virulence. 14 however, these previous studies are not conclusive, since all data are based on the analysis of genomic sequences or genes. here, we detected with proteomics a protein derived from orf3a of the sars-cov genome. immunoblotting revealed interchain disulfide linkages between the spike protein and the 3a protein. mutation analysis of sars-cov isolates and elisa of sars patients' sera indicated that the orf3a product might function together with the s protein in vivo. the "shot-gun" strategy using two-dimensional liquid chromatography electrospray tandem mass spectrometry (2d-lc-ms/ms) is a newly developed proteomic approach in which a protein mixture of adenovirus can be directly analyzed with mass spectrometry without separation by twodimensional gel electrophoresis. 15 in the present study, the cytosol of vero e6 cells infected with sars-cov was collected and subjected to 2d-lc-ms/ms. the tryptic-digested peptides of s, m, n, e and other known proteins of sars-cov were identified (not shown). in addition, two novel peptides were detected, corresponding to the residues 180-193 and 182-193 of a predicted orf3a protein, respectively (table 1 ; supplementary material). according to the genomic sequence information for sars-cov, orf 3a encoded a putative 31 kda protein containing 274 amino acid residues. 6, 7 in order to confirm the result by shot-gun assay, the collected cytosol of sars-cov-infected vero e6 cells was separated with sds-page, and then the gel slice around the 31 kda region was excised and analyzed by capillary liquid chromatography electrospray tandem mass spectrometry (mlc-esi-ms/ms). the ms results showed four peptides of this orf 3a protein including one n-terminal peptide from position 7 -19, one peptide from 180 -193 and two identical c-terminal peptides from 236 -274, encompassing 24.08% of the whole 3a protein sequence (table 1 ; supplementary material). the results from two complementary proteomic studies indicate the existence of a 31 kda protein encoded by orf 3a of sars-cov. the predicted amino acid sequence of the 3a protein of sars-cov isolate bj-01 is shown in figure 1 †. this information combined with dnastar analysis (dnastar inc. usa) predicted the existence of three transmembrane regions of the 3a protein, which is in agreement with that of the tor2 isolate. 7 the membrane topology of this protein might belong to type 3b: its n terminus (about 40 amino acid residues) is on the outer surface of the viral particle, which contains a putative signal peptide from residues 1 to 15 predicted by signalp. 17, 18 its c-terminal domain of 160 amino acid residues is located in the interior of the viral particle, of which there is a potential cytoplasmic region of ca atpase (gi: 737940) from residues e209 to d264 (also see marra et al. 7 ). we noticed that a cysteine-rich region overlaps the third membrane-spanning domain and the cytoplasmic tail of the 3a protein ( figure 1 ). moreover, three cysteine residues within this cysteinerich region are arranged every three residues (cwlcwkc from residues c127 to c133, figure 1 ). if these cysteine residues exist in one helix, they would be located in the same surface, suggesting that the 3a protein containing these cysteine residues is able to form interchain disulfide linkages with other viral structural proteins. interestingly, comparison with s protein sequences of the coronavirus family also shows a cysteinerich region overlapping the junction of the membrane-spanning domain and the cytoplasmic tail, which is a conserved motif of spike proteins in all analyzed coronaviruses (see figure 3 of marra et al. 7 ). this feature is not seen in other known structural proteins of sars-cov. therefore, we hypothesize that the protein encoded by orf3a forms the interchain disulfide bonds with s protein of sars-cov though their "cysteine-rich" regions. in order to address whether the 3a protein forms interchain disulfide bonds with s protein, we generated a polyclonal antibody against a peptide (3a1) derived from the cytoplasmic region of the 3a protein of sars-cov (see materials and methods). western blotting detected 3a proteins in the cytosol of infected vero e6 cells (figure 2a , lane 2), which agrees with the results of 2d-lc-ms/ms analysis. moreover, the 3a protein was also detected in the crude virions of sars-cov ( figure 2a , lane 3), suggesting that the 3a protein might be a structural protein of sars-cov, although further validation is required employing more purified sars-cov virions. if according to sequence analysis, the 3a protein forms interchain disulfide linkages with s protein, a protein complex would be formed of around 210 kda in sars-cov virions. to test this hypothesis, the crude virions were analyzed by immuno-blotting under both reducing and nonreducing conditions. a polyclonal antibody against a peptide (s2) derived from the sequence of s protein detected two bands around 210 kda and 180 kda under non-reducing conditions ( figure 2b , lane 2 of left panel), whereas only one band corresponding to 180 kda was detected under reducing conditions ( figure 2b , lane 1 of left panel). furthermore, the immuno-blotting with the anti-3a1 antibody also showed a band at 210 kda under non-reducing conditions ( figure 2b , lane 2 of right panel). in order to further confirm the observations, the antibodies were stripped out from these blotted pvdf membranes, and then the membranes were re-probed by the exchange of the antibodies (s2 for 3a1 and 3a1 for s2, respectively). the same patterns were detected again (not shown). these results suggest that the 3a protein binds to the spike protein with the interchain disulfide bonds on the interior side of the viral envelope of sars-cov. since two bands corresponding to s protein co-existed in the gel under the non-reducing conditions ( figure 2b , lane 2 of left panel) and no band was detected by the anti-3a1 antibody at 31 kda in the gel under the non-reducing conditions (not shown), it suggests that all molecules of the 3a protein are used for the formation of interchain disulfide linkages with the s proteins of sars-cov. if the 3a protein binds to s protein with disulfide bonds, it is possible that the 3a protein-specific antibodies may be detected in the sera of sars patients. to address this question, two synthetic peptides, 3a1 and 3a2 corresponding to the cytoplasmic region and the extracellular region of the 3a protein, respectively, were used as antigens to probe for 3a protein-specific antibodies in the sera from clinically diagnosed sars patients. elisa results showed that the sars patients could be divided into two groups corresponding to high responders and low responders based on the a values, whereas the control produced basal low characterization of sars-cov 3a protein levels of antibodies against 3a1 and 3a2 ( figure 3a and b). as a negative control, a peptide derived from mice il-12 b2 receptor was used and no peptide-specific antibodies were detected in all negative controls and sars patients (no shown). the results suggest that when the 3a protein was exposed to patients during sars-cov infection, 3a protein-specific antibodies were produced. since s protein is a major structural protein of sars-cov, the peptide-specific antibodies against s protein in the patients' sera should also be detected by the same approach. the same serasamples of sars patients that were analyzed with 3a2 peptide, were measured again with s1 peptide derived from the extracellular region of s protein. as expected, the pattern of elisa assay with s1 peptide was similar to that of 3a1 and 3a2 peptides ( figure 3c ). in addition, statistical analysis of the correlation between the responder of s1 and 3a2 with sigmaplot (spss inc. usa) indicated that the response of the 3a2 was associated with that of s1 (r ¼ 0:60; p , 0:01; figure 3d ). our data strongly suggest that the 3a protein and s protein are equally exposed to the human immune system, and then the induced antibodies are expressed at comparable levels to each other in the same sars patient. until now, there have been 60 complete or partial genomic sequences of sars-cov isolates from human and from four available himalayan palm civets. according to these sequencing data, we analyzed the 3a protein mutations of these isolates. the results showed that the 3a protein sequences of 48 sars-cov isolates were identical, which is referred to as the wild-type, whereas the rest of the isolates showed different mutations on their 3a protein sequences, of which 15 isolates showed point-mutations, including six synonymous mutations, and one isolate showed a frame-shift mutation. interestingly, the analysis of s protein sequences showed that the sequences of 44 isolates were identical, of which 43 isolates also belong to the "wild-type" of the 3a protein, whereas another 19 isolates had mutations on their spike sequences. statistical analysis with sigmaplot indicated that the mutations of the 3a protein were associated with that of s protein (r ¼ 0:79; p , 0:05). we did not find similar correlation with other structural proteins of sars-cov isolates (not shown). further analysis indicated that the mutation patterns in the s protein sequences from himalayan palm civets were identical only in three human sars-cov isolates (gd01, ay278489.2; gz43, ay304490.1; gz60, ay304491.1), whereas the same mutant site (19 t ! a) of 3a protein sequences from himalayan palm civets was also only found in those three human sars-cov isolates. this kind of mutation-correlation between s protein and 3a protein suggests that there is some relationship between the sars-cov isolates from himalayan palm civet and those from human being, at least, gd01, gz43 and gz60. taken together, these results suggest that the biological function of the 3a protein is tightly correlated with s protein of sars-cov. it has been shown that orf 3a is located within the region between the s and e genes in the sars-cov genome. 6, 7 by analyzing the genome organizations of other known coronaviruses, we found that the pattern of a potential gene locus between the s and e genes is conserved in all three groups of coronavirus genomes ( figure 4a ). therefore, we designate this locus as sne (s neighbor e). in general, the sne locus consists of one to three orfs in different species of coronaviruses, which encodes putative proteins containing at least one predicted transmembrane region ( figure 4a ). figure 4a shows that only the genomes of sars-cov and pedv have one complete orf in the sne locus, whereas the putative genes corresponding to orf 3 in snes of five other coronaviruses are truncated and short orfs are produced due to a mutation. however, if the entire sequence of the sne locus was used for phylogenetic analysis, the topology of the resulting phylograms of sne would be remarkably similar to that of s proteins ( figure 4b ). 6, 7 in other words, the phylogenetic analysis of sne sequences indicates that sars-cov is not closely related to any of the three known groups of coronaviruses, which is in agreement with the phylogenetic analyses of all other known structural proteins of sars-cov. 6,7 figure 1 . sequence analysis of the 3a protein of sars-cov. the orf 3 gene encodes a putative protein of 274 amino acid residues. predicted transmembrane regions are demarcated by arrows and the direction is from the outer surface to the interior of virion envelope. cysteine residues are indicated with triangles and the cysteine-rich region is underlined. amino acid residues in italics have mutations among the isolates of sars-cov, especially g marked with dots has two different mutations. the 3a protein might be a minor structural protein though proteomics identified the 3a protein, an orf3a gene product of sars-cov, it is a minor structural protein on the surface of sars-cov viral envelope. this is apparent because: (1) western blotting with anti-3a1 antibody detected the 3a protein in the crude virions ( figure 2 ). (2) since the conserved motif of the cysteine-rich region of the s protein is located between the end of the membrane-spanning region and the cytoplasmic tail, this motif may provide a frame for formation of interchain disulfide bonds (see figure 3 of rota et al. 6 ), the spatial orientation of the 3a protein might determine the usage of its cysteine-rich region with the frame of the s protein in the virus envelope interior. (3) it is well known that the . detection of orf3a peptide-specific igg antibodies in sars patients. the igg antibodies in the sera of sars patients were measured by elisa with: a, 3a1 peptide; b, 3a2 peptide; c, s1 peptide. the low-response group consists of individual patients who had low a values, which were below the average of the normal control, whereas the high-response group had a values that were above the average of the normal control. d, the correlation of antibodies induced by the 3a protein and s protein in the same patient in response to 3a2 and s1-peptides with sigmaplot software. reducing environment of the cytosol in cells decreases the likelihood of disulfide bond formation. therefore, the formation of the interchain disulfide bonds between the 3a protein and s protein must be in the interior of the viral envelope, in which there is a non-reducing environment. however, it might be necessary to further confirm this assumption with the more purified sars-cov virions, since the crude virions may be contaminated with cellular proteins and non-structural viral proteins. spike glycoprotein of coronaviruses contains many cysteine residues. in addition, there is a cysteine-rich region, overlapping the junction of the membrane-spanning region and the cytoplasmic region of s proteins, and this conserved structure of s protein exists in all three groups of coronaviruses. 6 the molecular function of the cysteine-rich domain in s protein is largely unknown. recently, some evidence showed that the cysteine-rich domain of s protein was required for coronavirus-induced membrane fusion. 19, 20 the sequence analysis showed that the 3a protein of sars-cov has a similar cysteinerich region that is also located at the junction of the putative transmembrane and cytoplasmic region ( figure 1 ). (4) here for the first time we show that the 3a protein might form interchain disulfide bonds with s proteins, probably through the cysteine-rich domains (figure 2 ). since no such kind of cysteine-rich domain exists in any other known sne loci of coronaviruses, interchain disulfide bonds might not form between the proteins encoded by sne loci and s protein in other coronaviruses. therefore, we postulate that the interaction between the 3a and s proteins occurs through their cysteinerich regions, which is a special case in the coronavirus family, which may play an important role in affecting sars-cov virulence. the 3a protein probably evolves from the sne locus an obvious question to ask is: what is the origin of the 3a protein of sars-cov during the evolutionary process of the coronaviridae family. the analysis on the sne locus may provide some clues to answer this question, since the orf gene encoding the 3a protein is located in this locus ( figure 4) . first of all, the sne locus exists widely in all three groups of coronavirus family besides sars-cov, which consists of one to three orfs ( figure 4 ). it is likely that the sne locus in the primitive coronavirus has only one orf, and many short orfs within the sne locus may be produced in different strains of coronaviruses due to high mutation rates in this locus during the evolutionary process. based on complete genome comparison, phylogenetic analysis of sars-cov showed that sars-cov is closer to group 1 of coronaviruses, 21 which is in agreement with our phylogenetic tree of the sne locus ( figure 4b ). since it was proposed that pedv and h229e diverged later than tegv from their common ancestor in the group 1 according to the analysis of sne locus, 10 we speculate that the 3a protein of sars-cov evolved from the sne locus of pedv or a pedv-like ancestor. it is not clear whether the sne is necessary or dispensable for virulence of coronaviruses. it has been hypothesized that orf 3/3a and orf3-1/3b may be related to viral virulence and pathogenesis, 11 -13 whereas some suggested that orf3a of tgev was not essential for enteric virulence. 14 according to the data collected by elisa (figure 3 ), we postulate that the 3a protein plays an important role in the virulence of sars-cov, although it might not be the same case in other coronaviruses. therefore, the 3a protein may serve as a new clinical marker or drug target in the treatment of sars. african green monkey kidney cells (vero e6, atcc) were maintained in dulbecco's modified eagle's medium (dmem, gibco-brl) supplemented with 10% (v/v) fetal bovine serum (fbs, gibco-brl) at 37 8c in a 5% (v/v) co 2 atmosphere. for virus infection, vero e6 cells were treated for one hour with the dmem (2% fbs) containing sars-cov virions (bj-01 isolate, provided by academy of military medical sciences). the virus-medium was removed after the infection and the infected cells were cultured in dmem with 2% fbs at 37 8c in 5% co 2 . after 48 hours post-infection, the supernatant medium of the infected cells was collected and centrifuged at 12,000 rpm for 30 minutes (eppendorf 5415d) to eliminate the cell debris as crude sars-cov virions, of which tcid 50 (tissue culture infectious dose) was identified as 10 6 dilution. all the experiments using the virus were carried out in a bio-safety level 3 laboratory. vero e6 cells were infected with sars virus for 24 hours. the infected cells were lysed with a solution containing 40 mm tris (ph 8.3) and 0.5% (v/v) nonidet p-40 at room temperature for five minutes. the lysate was centrifuged at 8000 rpm for five minutes (eppendorf 5415d), and then the supernatant was collected and heated at 100 8c for five minutes. the peptide sequences were determined based on the prediction of epitopes of the spike protein (gi:29836496) and the 3a protein (gi:30275670) as described by rammensee et al. 22 combined with analysis of antigenicity (dnastar, †). the s1 peptide (amino acid residues 332 -351, tkfpsvyawerkkisncvad) and the s2 peptide (amino acid residues 758-780, rntrevfaqvkq-mykt ptlkyfg) correspond to the spike protein; the 3a1 peptide (amino acid residues 176-199, stpklke-dyqiggysed rhsgvkd) and the 3a2 peptide (amino acid residues 7 -26, fftlgsitaqpvkidnaspa) correspond to the 3a protein. these peptides were synthesized using conventional solid-phase chemistry and purified by gl biochem (shanghai) ltd. a peptide from the sequence of mice il-12 2 receptor (amino acid residues cnrldlginlspd laesprfi) was synthesized as a negative control. in addition, the nucleocapsid protein was expressed in escherichia coli and purified as an antigen for generating a polyclonal antibody. 23 rabbits were immunized with 200 mg of peptide-bsa or protein in 0.4 ml of emulsion mixed 1/1 (v/v) with complete freund's adjuvant (cfa) supplemented with mycobacterium tuberculosis to a final concentration of 1 mg/ml. the rabbits were boosted twice with freshly prepared emulsion of the conjugated peptide or protein and freund's incomplete adjuvant at three week intervals. blood was drawn from the rabbits at seven weeks following immunization, the blood was allowed to clot at 4 8c, and the antiserum was recovered by centrifugation. the antiserum was then purified with a bsa affinity column (cnbr-sepharose 4b) according to the manufacture's instruction (amersham pharmacia). the sera of 56 sars patients, who were diagnosed by clinical symptoms and laboratory examination, were collected from different hospitals in beijing. as normal control, the sera of 36 healthy people were collected. the sera dilution was 1 : 10 (v/v) for elisa assay. elisa assay was done as described. 24 in brief, 96-well microtiter plates were coated with the peptides (s1, 3a1 and 3a2, respectively) in 0.1 m carbonate buffer (ph 9.6) at 4 8c overnight. after blocking with pbs containing 3% (w/v) gelatin and 0.1% (v/v) tween 20, the plates were incubated with diluted sera from individual sars patients or healthy controls at 37 8c for two hours. bound antibodies were detected with horseradish peroxidase-coupled goat anti-human igg (bio-rad) and the absorbance values were measure by microplate autoreader (bio-tek) at 450 nm. the cellular fractions or viruses were lysed either in the non-reducing loading buffer (50 mm tris (ph 6.8), 2% (w/v) sds, 10% (v/v) glycerol, 0.1% (w/v) bromophenol blue) or reducing loading buffer containing 100 mm dtt. the mixtures were subjected to sds-page, and then transferred to immobilon-p membrane (millipore). immunoblotting was carried out with the anti-3a1 antibody (against the 3a protein) or anti-s2 antibody (against s protein). the proteins were detected by enhanced chemiluminescence (ecl, amersham pharmacia biotech). the cytosol fraction of infected vero e6 cells was digested with trypsin and analyzed with 2d-lc-ms/ms system (proteomex, thermo finnigan) as described. 15 protein identification was performed with bioworks version 3.1(thermo finnigan) and the sequest algorithm. since the vero-e6 cells were from monkey, both the human database and the sars database from ncbi were merged. the ms results were searched against either the combined database or the sars database alone. protein results were further filtered with xcorr (1 þ . ¼ 1.5, 2 þ . ¼ 2.0, 3 þ . ¼ 2.5). the cytosol fractions of infected vero e6 cells were subjected to page and stained with coomassie brilliant † http://www.dnastar.com/ characterization of sars-cov 3a protein blue. gel slices were excised and in-gel digestion was carried out with trypsin as described. 25 the digested peptides were analyzed by mass spectrometry (lcq deca xp plus, thermo finnigan). data analysis was carried out as described for the shot-gun approach. coronaviridae: the viruses and their replication completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus the complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and rna polymerase molecular analysis of the human coronavirus (strain 229e) genome complete genomic sequence of the transmissible gastroenteritis virus characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group 2 lineage virology. the sars coronavirus: a postgenomic era sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf pathogenicity and sequence analysis studies suggest potential role of gene 3 in virulence of swine enteric and respiratory coronaviruses sequence comparison of porcine respiratory coronavirus isolates reveals heterogeneity in the s, 3, and 3-1 genes genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus characterisation of a recent virulent transmissible gastroenteritis virus from britain with a deleted orf 3a analysis of the adenovirus type 5 proteome by liquid chromatography and tandem mass spectrometry methods tmbase-a database of membrane spanning proteins segments identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites prediction of signal peptides and signal anchors by a hidden markov model coronavirus-induced membrane fusion requires the cysteine-rich domain in the spike protein murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein phylogeny of sars-cov as inferred from complete genome comparison syfpeithi: database for mhc ligands and peptide motifs identification of an epitope of sarscoronavirus nucleocapsid protein pertussis toxin enhances th1 responses by stimulation of dendritic cells femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry we thank drs jing-wu zang ( key: cord-103430-x6zzuu7v authors: contu, lara; balistreri, giuseppe; domanski, michal; uldry, anne-christine; mühlemann, oliver title: characterisation of the semliki forest virus-host cell interactome reveals the viral capsid protein as an inhibitor of nonsense-mediated mrna decay date: 2020-10-12 journal: biorxiv doi: 10.1101/2020.10.12.335497 sha: doc_id: 103430 cord_uid: x6zzuu7v the positive-sense, single-stranded rna alphaviruses pose a potential epidemic threat. understanding the complex interactions between the viral and the host cell proteins is crucial for elucidating the mechanisms underlying successful virus replication strategies and for developing specific antiviral interventions. here we present the first comprehensive protein-protein interaction map between the proteins of semliki forest virus (sfv), a mosquito-borne member of the alphaviruses, and host cell proteins. among the many identified cellular interactors of sfv proteins, the enrichment of factors involved in translation and nonsense-mediated mrna decay (nmd) was striking, reflecting the virus’ hijacking of the translation machinery and indicating viral countermeasures for escaping nmd by inhibiting nmd at later time points during the infectious cycle. in addition to observing a general inhibition of nmd about 4 hours post infection, we also demonstrate that transient expression of the sfv capsid protein is sufficient to inhibit nmd in cells, suggesting that the massive production of capsid protein during the sfv reproduction cycle is responsible for nmd inhibition. suggesting that the massive production of capsid protein during the sfv reproduction cycle is 13 responsible for nmd inhibition. 14 15 as we live through the current sars-cov2 pandemic, the world is reminded of the unpredictable 17 nature of viral epidemics and the importance of studying potential emerging viral threats. recent 18 studies present valid arguments for the worldwide epidemic threat of alphaviruses (among other 19 arboviruses) that currently circulate endemically in particular regions 1,2 . the outbreak potential of 20 alphaviruses has already been showcased by the two worldwide epidemics caused by chikungunya 21 virus (chikv) that affected more than 8 million people in over 50 countries and could be attributed to 22 a single point mutation leading to a 100-fold increase in infectious virus in the salivary glands of urban 23 mosquitoes 1,2 . this demonstrates that small genetic alterations can cause dramatic changes in human 24 transmissibility and infection. semliki forest virus (sfv) is closely related to chikv, both evolutionarily 25 grouped within the semliki forest (sf) clade of the old world alphaviruses (family: togaviridae) 3 . sfv 26 causes lethal encephalitis in mice 4 . though mostly associated with mild febrile illness or 27 asymptomaticity in humans, sfv is endemic to african regions 1 and a handful of studies indicate 28 serious disease relevant symptoms associated with sfv in humans, including encephalitis, myalgia and 29 arthralgia 5-8 . 30 31 sfv is a small (~70 nm in diameter), enveloped virus comprising a nucleocapsid core made up of 240 32 copies of capsid protein that surrounds its positive-sense single-stranded rna genome (~11.8 kb). the 33 genome contains a 5´ cap (n7mgppp) and poly(a) tail and is organised into two distinct open reading frames (orfs). the first orf encodes the non-structural proteins (nsp1, nsp2, nsp3 and nsp4) ( figure 1 1a), which are translated as one polyprotein (p1234) immediately upon exposure of the viral mrna-2 genome to the cytoplasm [9] [10] [11] [12] . the polyprotein is then proteolytically cleaved by the protease activity 3 of nsp2 to yield functional viral replicase complexes 13 . the first protein to be cleaved from the 4 polyprotein is nsp4, comprising rna-dependent rna polymerase activity. the resulting p123 5 polyprotein in complex with nsp4 forms the viral replication complex (rc), responsible for synthesizing 6 minus strand template rna from the genomic viral (v)-rna early during infection 9 . the ensuing 7 double-stranded vrna intermediates can trigger the activation of protein kinase double-stranded 8 rna-dependent (pkr), resulting in phosphorylation of the α-subunit of the eukaryotic translation 9 initiation factor 2 (eif2) and thus causing a decrease in global translation of host cell messenger rnas 10 (mrnas) 10,14,15 . as proteolytic cleavage of p123 by nsp2 progresses, individual nsps form new viral rcs 11 of altered composition, resulting in a shift from synthesis of the minus strand template, to synthesis 12 of new viral genomes and viral subgenomic rna (sgrna) from the 26s promoter ( figure 1a ) 9, 16 . 13 alphavirus replication occurs in membrane invaginations called 'spherules', where high 14 concentrations of rcs are present 9, 11 . binding of host cell proteins to rcs has been reported, though 15 the abundances and the functions thereof are still not fully understood 11,17, 18 . in addition, individual 16 sfv proteins localise independently of the rc to perform functions separate from viral replication 17 9, 11, 19, 20 . one example is the nsp2 protein, which translocates to the nucleus 21 here, we investigated the virus-host protein interactome of sfv. a greater understanding of the 1 repertoire of host proteins that may be exploited by viruses is a vital first step toward developing 2 antiviral strategies aimed at targeting or interfering with interactions that may be critical for the 3 infection. while previous studies have reported host interactors of sfv from isolations of rcs from 4 lysosome fractions, as well as affinity purifications and localisation studies of nsp3-tagged 5 recombinant virus 18, [29] [30] [31] , there are so far no sfv studies that assess the complete set of viral-host 6 protein-protein interactors (ppi). using affinity purifications followed by high-throughput quantitative 7 mass spectrometry, we identified host protein interactors of individual sfv proteins in human cells. in 8 addition, using a genome-wide sirna screen we assigned pro or antiviral functions to some of the 9 identified sfv interactors. gene ontology (go) enrichment analyses of protein complexes that could 10 form between the identified host interactors revealed highly significant go terms related to 11 translation and nonsense-mediated mrna decay (nmd). nmd is known to restrict infection of 12 alphaviruses, but whether and how the virus counteracts this cellular intrinsic defence is still not clear 13 32 . here we show that during the course of infection sfv suppresses nmd. we present evidence that 14 the capsid protein of sfv is sufficient to suppress nmd independently of translation inhibition. 15 16 17 as obligatory parasites, all viruses exploit the host cell to favour their own replication. in turn, cells 19 have evolved mechanisms to protect against viral infections. to gain insight into the repertoire of host 20 proteins that could be exploited by sfv, we systematically mapped the interactions between the 21 individual sfv proteins, nsp1-4, c, and the envelope polyprotein, env (which includes e3, e2, 6k and 22 e1), and the host cell proteome using high-throughput quantitative mass spectrometry (figure 1a) . 23 the sfv proteins were n-terminally tagged with 3xflag and transiently expressed in hela cells, a cell 24 type susceptible to infection by sfv 33 . the proteins were then affinity purified from the respective 25 lysates using anti-flag antibodies, with and without treatment with rnase a to distinguish rna-26 mediated from protein-mediated interactions (figure 1b) . western blot analysis of the eluates from 27 each anti-flag affinity purification revealed the successful pulldown of all six transiently expressed 28 sfv proteins (figure 1c) . 29 the protein compositions of the eluates, from three biological replicates of each affinity purified sfv 31 protein, were analysed by quantitative mass spectrometry (figure 2a and suppl. figure 1 ). significant 32 interactors (see methods) (figure 2b, purple circles and table 1 ) were further filtered by abundance, 33 such that proteins whose abundance made up at least 0.5 % of the relevant sfv bait protein were 5 1 2 figure 1 . strategy for creation of semliki forest virus (sfv) -host protein-protein interaction map. a, schematic illustration 3 of the genomic organisation of sfv. the first orf encoding the non-structural proteins (nsp1, nsp2, nsp3, nsp4) is highlighted 4 in green. the zsg tag inserted within the nsp3 protein is also depicted. the second orf encoding the structural proteins 5 (capsid, e3,e2,6k,e1) is highlighted in orange. other viral features depicted include the 5´ cap, poly(a) tail, as well as the 6 position of the 26s subgenomic viral promoter. b, flowchart outlining the experimental approach to transiently express n-7 terminally flag-tagged (yellow rectangle) sfv proteins in mammalian cells in order to construct a sfv-host protein-protein 8 interactome. nsp3-z refers to the nsp3 protein with the zsg tag, c refers to the capsid, and e3 e2 6k e1 refer to the envelope 9 proteins (env) that were expressed as one polyprotein. c, anti-flag western blot of sfv proteins after transient transfection 10 and affinity purification from hela cells (without rnase a treatment). red asterisks indicate 3xflag tagged sfv proteins at 11 their expected sizes. untransfected cells (untr) and cells transfected with a plasmid encoding only the 3xflag tag with no 12 additional coding region (empty) were included as controls. the expected sizes of the 3xflag-tagged proteins were: empty 13 ~8kda; nsp1 ~63kda; nsp2 ~92kda; nsp3-z ~82kda; nsp4 ~72kda; capsid ~33kda and env (polyprotein ~111kda, cleavage 14 intermediates ~57kda / ~64kda). the affinity purifications were conducted in triplicate (± rnase a treatment), and eluates 15 analysed by mass spectrometry. table 1 ). in the case of the nsp3 bait (here fused with the 18 fluorescent protein zsgreen, nsp3-z), which was very lowly abundant in the sample as it proved 19 difficult to elute from the beads (suppl. figure 2) , we retained proteins whose abundance made up at 20 least 5 % of the bait (table 1 ). the heat map in figure 2c summarises the most abundant significant 21 interactors of each sfv protein in the -rnase a samples, with their corresponding abundance in the 22 +rnase a samples alongside them. many of the host interactors identified in the -rnase a sample 23 were lost upon treatment with rnase a, indicating that these interactions were likely mediated by 1 rna. this was clear for many nsp2, nsp3-z and capsid interactors, where the heat map ( figure 2c ) 2 corroborated the observations in the analytical silver stain gel (figure 2a) . in both the heat map and 3 the gel, we noted proteins in the nsp2 eluate that were enriched in the +rnase a sample compared 4 to the -rnase a sample. also reflected in the heat map were proteins observed in the gel that were 5 more than or as abundant as nsp2 (~92 kda) (figure 2a and c, +rnase a samples). these agreements 6 between the quantified lists obtained to create the heat map and the observations in the analytical 7 silver stain gel gave us confidence in the strategy employed to collect host protein interactors from 8 the mass spectrometry datasets. since many sfv-host protein interactions were dependent on rna, 9 we chose to focus on the lists of interactors from the -rnase a datasets going forward. a summary of 10 these revealed that a large fraction of the interactions for nsp2, nsp3-z and capsid consisted of 11 ribosomal proteins (figure 2d ). 12 these stringent lists of interactors are displayed as sfv-host interactome networks (figure 3a and 14 suppl. figure 3 ). affinity purified sfv proteins are displayed as black circles, while host cell proteins 15 are displayed as smaller, colour-coded circles. host proteins that were identified as unique interactors 16 to one of the sfv proteins are connected to the relevant host protein with a grey line. many of the 17 host proteins were identified as interactors to more than one of the sfv proteins. for simplicity, these 18 non-unique interactors are grouped into grey boxes with grey lines connecting the whole group of 19 proteins to the sfv proteins for which they were identified as interactors (figure 3a ). considering this 20 overlap, the total number of host proteins that were identified as interactors was 251 (figure 3a and 21 suppl. figure 3) , 77 of which were ribosomal proteins and are shown separately (suppl. figure 3 ). host 22 proteins displayed in the networks were manually curated and categorised into colour-coded groups 23 based on descriptions gathered from both gene ontology (go) and string analyses ( figure 3a) . 24 interactions that stood out included subunits of the chaperonin-containing t-complex polypeptide 1 25 (cct complex) (pink) that interacted with both nsp2 and nsp4, a number of cytoskeletal proteins or 26 proteins involved in cytoskeletal signalling (grey) interacting with nsp2 and nsp1 (tubulins), er 27 chaperones (pink) bound uniquely to env, and a large number of rna binding proteins (violet) 28 interacting with nsp2, nsp3-z and capsid. in addition, a striking presence of rrna processing / 29 ribosome biogenesis factors emerged as interactors, many of which were found bound uniquely to 30 the capsid (dark pink) (figure 3a ). previously reported human protein-protein interactions were 31 analysed by string and additionally displayed on the networks (pink dashed lines). the dense 32 network of edges (pink dashed lines) that emerged among the rrna processing / ribosome biogenesis 33 factors (dark pink) reflects the known protein-protein interactions that have been reported between depicted. c, using the list of -rnase a interactors, a threshold of abundance of at least 0.5 % of the bait protein (in the case 5 of nsp3-z, at least 5 % of the bait protein) was applied. a heat map summarising either the top10 most abundant interactors 6 (or all if there were fewer than 10 interactors identified) for each sfv bait protein without rnase a treatment (-) is shown. the corresponding abundance as % of bait of the interactors that were statistically significantly enriched when treated with 8 rnase a (+) is also shown in the heat map. grey blocks indicate proteins that did not appear or were not statistically 9 significantly enriched in the +rnase a samples. note that due to the low abundance of nsp3-z, the abundance as % of bait 10 of nsp3-z and all its interactors are presented as a factor of 10 less than what was calculated (for better visual representation 11 of the heat map as a whole). for all values and the complete list of interactors, see table 1 . d, bar graph summary of the 14 15 them ( figure 3a ). this indicates that the capsid protein (and to a lesser extent nsp2/nsp3-z) may 16 interact with a complex of proteins involved in rrna processing and/or ribosome biogenesis. 17 18 we next set out to determine whether the identified host proteins have pro-or antiviral effects in the 19 context of infection. to systematically address this question, we used a genome-wide fluorescence 20 microscopy-based sirna screen 33 to identify host proteins that affected sfv replication ( table 2 ). in product, compared to the control, non-specific sirnas (set as 1). a low multiplicity of infection (moi) 27 of 0.3 was used to allow for detection of both reduced or increased infection levels. the maximum ii 28 obtained in the screen was 1.85 (table 2) . we therefore chose to set an ii threshold of 1.3 to identify 29 proteins having a potential antiviral role against sfv, and an ii threshold of 0.5 to indicate proteins 30 having a potential proviral role for sfv (table 2 ). when we compared the proteins identified in the 31 sirna screen with the sfv-host protein interaction networks, a sizable fraction of the interactors 32 overlapped. those with potential pro-or antiviral roles during infection are depicted with turquoise 33 or green outlines, respectively (figure 3a and suppl. figure 3 ) and collected in two lists: 'proviral' and 34 'antiviral', with their ii values shown alongside them ( figure 3b ). as expected, all the identified 35 ribosomal proteins affecting sfv replication had a proviral effect (suppl. figure 3 ) and are listed 36 separately ( figure 3b ). in addition to ribosomal proteins, many other rna binding proteins were also 37 identified as having a potential role in sfv replication (figure 3a (nsp1, nsp2, nsp3-z, nsp4, capsid and env) as well as for the merged list (all baits) was also applied. 28 this allowed us to compare the significance of the go terms collected in figure 4a for the different 29 sfv proteins (figure 4b ). we observed that the most significantly enriched go terms were those 30 related to translation, nmd and ribosome biogenesis. in addition, it became clear that it was mainly 31 the interactors of nsp2, nsp3-z and capsid that contributed to the enrichment of these go terms 32 because of the enriched go terms, we chose to investigate the effect of sfv on translation, nmd and 9 ribosome biogenesis. we reported previously that the nmd machinery could target the sfv genome 10 independently of the 3´ utr 33 . half-life measurements of the genome of a replication incompetent 11 sfv mutant suggested that this occurred early during infection, upon entry of the viral genome into 12 cells 33 . since viruses are known to evade cellular defence responses against viral infection, we 13 wondered whether the virus could inhibit nmd at later stages of infection. since nmd depends on 14 translation 34 and viruses are known to inhibit translation, it was important to carefully analyse the 15 time course of infection in our system in an attempt to disentangle these two tightly linked cellular 16 processes. we used anti-zsg or anti-nsp3 antibodies to detect nsp3-z, as a representative for the 17 presence of early produced non-structural proteins expressed from the grna, and anti-capsid 18 antibodies to detect the capsid, as a representative for the presence of structural proteins that are 19 expressed from sgrnas later during the virus replication cycle (figure 5a and b). the nsp3-z protein 20 was reproducibly detected at 3-4 hours post infection (p.i.) and as early as 2 hours p.i., while the capsid 21 was reproducibly detected at 4 hours p.i. (figure 5a and b). we measured the presence of 22 phosphorylated (p-)eif2α compared to total eif2α as an indication of virus-induced translation 23 inhibition and showed that a virus-dependent accumulation of p-eif2α was reproducibly detected at 24 3-4 hours p.i. (figure 5a ). in addition, we performed time course puromycin incorporation assays to 25 assess global translation activity using a more direct method 35 . this assay involves a puromycin pulse 26 for 10 minutes, which causes the release of nascent polypeptides and results in many puromycin-27 labelled polypeptides of different lengths that can then be visualised by western blotting using anti-28 puromycin antibodies. the decrease in puromycin 3-4 hours p.i. is therefore indicative of a decrease 29 in global translation, in agreement with the observed increase in p-eif2α ( figure 5b ). 30 we set aside samples from the time course infections in figure 5a to assess nmd activity by measuring 32 relevant rna levels by rt-qpcr. to assess nmd activity, we adapted an assay described in 36 , which 33 measures the relative amounts of a nmd-sensitive splice isoform (nmd target) versus a nmd 34 insensitive protein coding isoform (non-nmd target) of the same gene. we showed that at 4 hours started to accumulate at 3 hours p.i., whereas the increase of bag1_nmd only became apparent at 4 1 hours p.i. (suppl. figure 4 ). in addition, we measured the rna levels of the well-known endogenous 2 nmd targets, rp9p, ire1α and gadd45, which also accumulated 3-4 hours p.i. (figure 5c and suppl. 3 figure 4 ). together, these data are indicative of reduced nmd activity 3-4 hours p.i., suggesting that 4 sfv can indeed inhibit nmd at later stages of infection. since the timing of the nmd inhibition 5 correlated with that of eif2α-dependent inhibition of cellular mrna translation, we were unable to 6 pull apart the effect the viral infection had on the two cellular processes independently. translation 7 inhibition by sfv, and rna viruses in general, is well described to occur through induction of p-eif2α, 8 which occurs upon host cell detection of the double-stranded viral rna intermediate that arises during 9 its replication cycle 10,14,15 . we therefore reasoned that, if one of the sfv proteins was responsible for 10 the nmd inhibitory phenotype, we would be able to disentangle the effect of the virus on the two 11 cellular processes. taking the mass spectrometry data analysis ( figure 4b ) into account, we reasoned 12 that nsp2, nsp3-z or capsid could be responsible for the nmd inhibitory phenotype. first, in order to 13 confirm the interactions identified between nsp2, nsp3-z and capsid with cellular upf1 (figure 3 were identified in the upf1 eluates of the infected sample ( figure 5d ). it should be noted that their 23 abundance was higher than that of the nmd factors, upf3a and smg6. taken together, we were 24 therefore able to confirm the rna-mediated interactions of nsp3-z and capsid with upf1. 25 the results above suggested that nsp3-z and capsid were the most likely candidates to influence nmd 27 activity in cells. nevertheless, we decided to analyse the effect of all individual sfv proteins on nmd 28 activity. to do this, the sfv proteins (from plasmids described in figure 1b suppress nmd in cells. since translation-related go terms were also enriched for capsid interactors 1 (among other sfv proteins) (figure 4b) , it was important to investigate whether expression of capsid 2 influenced translation in cells, as this would in turn influence nmd activity. we showed that 3 expression of capsid did not induce p-eif2α (figure 6c ) nor, as judged from the puromycin 4 incorporation assays, effect changes in global translation (figure 6d ). in addition, polysome profile 5 gradients revealed that cells expressing the sfv capsid retained intact polysomes (suppl. figure 5) , 6 indicative of unperturbed translation. these three independent sets of data convincingly show that 7 expression of the capsid protein did not influence global translation in cells. we therefore concluded 8 that the sfv capsid suppresses nmd through a mechanism independent of translation inhibition. since 9 ribosome biogenesis related go terms were most highly enriched among capsid interactors compared 10 to the other sfv proteins, we used capsid expressing cells to look for any indication of altered 11 ribosomes/rrna that could give us any hints on the mechanistic action of capsid. though capsid could 12 be trapped in the nucleus upon blocking of export, we were unable to find any phenotypic changes in 13 polysome gradients (suppl. figure 5) (figure 2a and c) . as such, a large number of rna-binding proteins were 28 identified as host interactors of these three sfv proteins (figure 3a) , raising the question of whether 29 they could play a role in altering and exploiting the compositions of mrnps during infection. some of 30 the identified rna-binding proteins that were previously found in sfv rcs include hnrnpc, hnrnpa1, 31 sfpq, dhx9, ddx3x, pabpc1, g3bp1 and g3bp2 18 . in addition to being found in rcs, the nsp3: g3bp 32 interaction has been well characterised 29-31 . sfv nsp3 has been reported to bind g3bp and suppress 33 the formation of stress granules, thought to have antiviral activity 30 . consistent with these previous findings, we identified g3bp1, g3bp2 and usp10, a deubiquitinase protein known to bind g3bp 30 , as 1 interactors of nsp3-z. usp10 was identified as a unique interactor of nsp3, while g3bp1 and g3bp2 2 were identified as also interacting with nsp2 and capsid. thanks to a nuclear translocation signal, nsp2 3 shuttles between cytoplasm and nucleus during infection and interferes with transcription 22 . in line 4 with this, and as observed by others, we observed localisation of the 3xflag-nsp2 in the nucleus of 5 hela cells at steady state (data not shown). our study identified a number of nuclear proteins 6 interacting with nsp2, many of which are splicing regulators, including hnrnpc, hnrnpa1, hnrnpa3, 7 hnrnpf, hnrnph3 and sfpq. hnrnpc was the most abundant significantly enriched protein in the 8 nsp2 pulldown. it was also significantly enriched and abundant in the rnasea-treated nsp2 sample, 9 indicating that this interaction may be either direct or mediated by another protein. interestingly, 10 sfpq was found to be one of the most abundant interactors not only of nsp2, but also of capsid ( figure 11 2c). sfpq was additionally identified through the sirna screen, along with the mrna export factor, 12 nxf-1, as being among the strongest proviral interactors (i.e. depletion of these factors inhibited viral 13 infection). the binding of nsp2 and capsid to these nuclear proteins could influence the regulation of 14 rna processing or mrna modification steps in the nucleus or re-localise nuclear proteins to the 15 cytoplasm in order to achieve a productive infection. some cytoplasmic viruses, for example, hijack 16 nuclear proteins including splicing factors (hnrnps and sfpq) from the nucleus to the cytoplasm, 17 increasing infectivity 40-42 . we also identified rna-binding interactors exhibiting strong antiviral effects 18 activity, including rps27a, which was interestingly the only ribosomal protein to be found bound to 28 (in addition to nsp2) nsp1, nsp4 and env (figure 2c ). we were surprised by the presence of newly 29 identified nuclear interactors involved in rrna processing and ribosome biogenesis, many of which 30 exhibited antiviral activity. interestingly, many of these were uniquely bound to capsid (figure 3a) . 31 evidence of capsid in the nucleus has previously been reported 47 and we were able to trap 3xflag-32 capsid in the nucleus of hela cells upon blocking of export (data not shown). even so, little is known 33 about the role of the capsid in the nucleus and how this could affect the cellular ribosome. we therefore assessed polysome gradients (suppl. figure 5) and measured 18s, 28s and 45s precursor 1 rrnas in nuclear fractions of capsid expressing cells (data not shown), but found no obvious 2 phenotypic changes compared to cells expressing the 'empty' vector. perhaps the effects of these 3 interactions on the ribosome are more subtle or only affect a small pool of 'specialised' ribosomes, 4 making changes difficult to detect. since viruses rely on the host cell ribosome for translation of their 5 own genomes, a better understanding of the involvement of the viral proteins in recruiting or 6 potentially altering the host cell ribosomes through interaction with specific ribosomal proteins and 7 this novel set of ribosome biogenesis factors definitely warrants further investigation. 8 9 to obtain additional hints about possible functional consequences of the detected interactions 10 between sfv and host cell proteins, we used mcode to analyse the protein complexes that could form 11 between all host interactors, including the ribosomal proteins (figure 4 and suppl. figure 3 ). go 12 enrichment analyses of the protein complexes reinforced many of the cellular processes discussed 13 above and revealed that the most highly enriched go terms were related to translation and nmd 14 (figure 4a and b) . as a counter defence strategy, viruses are known to inhibit cellular mrna decay 15 factors that can degrade viral rnas and restrict infection 32, 48, 49 . we therefore hypothesized and 16 decided to investigate, whether sfv was able to inhibit the nmd pathway, which has antiviral activity 17 against alphaviruses 33 . indeed, we found that starting from 3-4 hours after infection, sfv antagonises 18 the nmd pathway, with consequent stabilisation of bona fide nmd mrna transcripts (figure 5c and 19 suppl. fig. 4) . viruses known to inhibit mrna decay pathways do so by different mechanisms. often a 20 viral protein counteracts a key cellular regulator. here, we show novel data that in the case of sfv, it 21 is the capsid protein that inhibits nmd (figure 6b) . therefore, inhibiting this function of the viral capsid 22 could lead to novel avenues for therapeutic intervention. 23 24 using both sfv protein affinity purifications in transient expression experiments and upf1 ips in sfv 25 infected cells, we show that the core nmd factor upf1 binds to sfv capsid among other sfv proteins 26 in an rna-dependent manner. together, this indicates that the capsid, and potentially other sfv 27 proteins, associate with mrnp molecules that also contain upf1. the large number of ribosomal 28 proteins pulled down by capsid (figures 2d, s3, and 4b) reported 50,51 . it was also postulated that the capsid or 'core' protein of hepatitis c virus (hcv) may 33 be responsible for the nmd inhibitory phenotype that was reported upon hcv infection 52 . our findings therefore add sfv as the first alphavirus to a growing list of viruses of which the capsid protein 1 is responsible for an nmd inhibitory effect. though the stability of the sfv genome has thus far been 2 attributed to evasion of deadenylation through binding to hur 53 , the virus may require additional 3 strategies to protect itself in order to ensure efficient translation of viral genes and packaging of 4 genomes into new progeny viruses. perhaps the sfv capsid plays a protective role against degradation 5 of its rnas by nmd. 6 7 in summary, we present here two valuable resources that will aid in the study of sfv: a sfv-host 8 protein interactome as well as a genome-wide sirna screen for host factors influencing sfv infection. the coding sequences for the sfv proteins were cloned into pcdna5_frt_to_3xflag(n), to yield 4 pcdna5.3xflag-nsp1, pcdna5.3xflag-nsp2, pcdna5.3xflag-nsp3-zsg, pcdna5.3xflag-nsp4, 5 pcdna5.3xflag-capsid and pcdna5.3xflag-env, which were used for all subsequent transfections. 6 pcdna5_frt_to_3xflag(n) was linearised using bamhi. pcr products for each of the sfv proteins, 7 were generated from either sfv-zsg(-3'utr) 33 , sfv-capsid or sfv-envelope 58 plasmids using the 8 following primers: buffer and 500 μl of the cleared cell lysate was added, and the mixture was then incubated at 4 °c for 22 one hour with rotation. beads were collected on a magnet and washed three times with lysis buffer. 23 at the third wash, each sample was split into two (for treatment or no treatment with rnase a). for 24 +rnase a samples, rnase a treatment was performed on the beads. the supernatant was removed 25 using the magnet, and 50 μl of rnase a (0.8 mg/ml, sigma aldrich) containing lysis buffer was added 26 to the beads and incubated at 25 °c for 15 minutes, shaking. thereafter, the rnase a treated beads 27 were washed with lysis buffer, and then samples eluted from the beads using 3xflag peptide 28 (sciencepeptide.com): 20 μg of flag peptide was incubated with the beads at 25 °c for 15 minutes, 29 shaking. the flag peptide elution was also performed for the -rnase a samples. thereafter, the 30 eluates were collected and 10 μl of loading buffer (4xlds + dtt [75 mm]) was added to each eluate, 31 while 30 μl of loading buffer was added to the remaining beads samples. the samples were then 32 incubated at 75 °c for 10 minutes, ready for analysis by western blot, silver stain, and coomassie gel 33 for mass spectrometry sample preparation. samples were electrophoresed on 26-well nupage™ 4-12 % bis-tris gradient gels (thermo fisher 3 scientific) in 1xmops running buffer at 200v for approx. 1 hour. the gels were fixed in 50 % methanol 4 / 12 % acetic acid for one hour at room temperature, followed by three 5 minutes washes in 35 % 5 ethanol. the gels were sensitised for 2 minutes in 0.02 % sodium thiosulfate, followed by three 5 6 minutes washes in milli-q h2o. the gels were stained in 0.2 % silver nitrate / 0.03 % formaldehyde for 7 20 minutes, followed by two 1 minute washes in milli-q h2o. the gels were developed in 0.57 m 8 sodium carbonate + 0.02 % formaldehyde / 0.0004 % sodium thiosulfate and then incubated in 50 % 9 methanol / 12 % acetic acid for 5 minutes. thereafter, the gels were placed in 1 % acetic acid for short 10 term storage at 4 °c. images were taken using the gel documentation system (www.vilber.com) 11 12 the protein compositions of the eluates from the sfv affinity purifications were analysed by label-free 14 quantitative mass spectrometry. the eluates (20 μl) were electrophoresed in 1xmops running buffer 15 about 1 cm into the 26-well nupage™ gels and then stained with coomassie-blue (10 % phosphoric 16 acid, 10 % ammonium sulfate, 0.12 % coomassie g-250, 20 % methanol) as described previously 59 . 17 images were taken using the gel documentation system. rectangular segments (10 mm x 3 mm) for 18 each lane were cut from the gels using sterile blades. the gel pieces were reduced, alkylated and 19 digested by trypsin as described elsewhere 60 to custom nsp1, nsp2, nsp3-z, nsp4, capsid and env sequences. the following maxquant settings were 32 used: separate normalisation groups for the +rnase a and -rnase a samples, mass deviation for 33 precursor ions of 10 ppm for the first search, maximum peptide mass of 6000da, match between runs activated with a matching time window of 0.7 min only allowed across replicates; cleavage rule was 1 set to strict trypsin, allowing for 3 missed cleavages; allowed modifications were fixed 2 carbamidomethylation of cysteines, variable oxidation of methionines, deamination of asparagines 3 and glutamines and acetylation of protein n-termini. protein intensities are reported as maxquant's 4 itop3 63 values (sum of the intensities of the three most intense peptides). peptide intensities were 5 first normalised by variance stabilisation normalisation and imputed. imputation values were drawn 6 from a gaussian distribution of width 0.3 centred at the sample distribution mean minus 1.8x the 7 sample standard deviation, provided there were at least 2 evidences in the replicate group. in order 8 to perform statistical tests, itop3 values were further imputed at the protein level, following the rule: 9 'if at least two detections in at least one group' and using the following protein impute parameters: 10 imputation values were drawn from a gaussian distribution of width 0.3 centred at the sample 11 distribution mean minus 2.5x the sample standard deviation. potential contaminants and proteins 12 marked 'only identified by site' were removed prior to performing differential expression (de) tests, 13 which were done by applying the empirical bayes test 64 followed by the fdr-controlled benjamini and 14 hochberg 65 correction for multiple testing. a significance curve was calculated based on a minimal 15 log2 fold change of 1 and a maximum adjusted p-value of 0.05. proteins that were consistently 16 reported as de throughout 20 imputation cycles were flagged as "persistent". in addition, saint 17 analysis was performed according to 66 and a significance threshold of fdr<0.05 was applied. 18 significant interactors for each sfv bait protein were defined as those that were significantly 21 differentially expressed (enriched) compared to the untransfected control and the significance 22 persisted throughout the imputation cycles. in addition, the proteins taken as "significant interactors" 23 had to be considered "true interactors" as determined using saint analysis (with a threshold fdr of 24 ≤ 0.05). to simplify the lists and attempting to retain potentially biologically relevant interactors, we 25 further filtered the lists, retaining proteins whose abundance made up at least 0.5 % of the relevant 26 bait protein. in the case of the nsp3-z bait, which was very lowly abundant in the sample as it proved 27 difficult to elute from the beads, we retained proteins whose abundance made up at least 5 % of the 28 sfv bait. 29 30 the final lists of proteins were used to create sfv-host protein interaction networks using 32 cytoscape_v3.7.2. string analysis (string-db.org, version 11.0) was performed using a minimum 33 required interaction score of highest confidence (0.900) for both database and experimental evidence and the known protein-protein interactions were overlaid onto the sfv-protein interactome 1 networks, using cytoscape_v3.7.2. were infected for 6 hours with sfv-zsg at a concentration giving an infection rate of approx. 30 % in 10 control sirna-treated cells. following fixation in 4 % formaldehyde and hoechst staining, nine images 11 per well were acquired using high-content automated fluorescence microscopes (imagex-press, 12 molecular devices). infected cells were detected using cell profiler (www.cellprofiler.org) and 13 advanced cell classifier (www.acc.ethz.ch/acc.html) and the % of infected cells per well was 14 determined. an "infection index" value was calculated for each gene, indicating the fold change of 15 infection upon depletion of the gene product, compared to the control sirnas, which was set as 1. an 16 infection index threshold of 1.3 was chosen to indicate proteins having a potential antiviral role against 17 sfv, and an infection index threshold of 0.5 to indicate proteins having a potential proviral role for 18 sfv ( table 2 , raw data). the proteins identified in the sirna screen were overlaid with the sfv-host 19 protein interaction networks. 20 21 protein-protein interaction enrichment analysis was performed using the metascape online tool 23 (www.metascape.org) according to 67 . metascape allows for the input of multigene lists. for each 24 given list (ie: interactors of nsp1, nsp2, nsp3-z, nsp4, capsid and env) as well as for the merged list, 25 protein-protein interaction enrichment analysis was carried out using the following databases: identified for the merged list of interactors. go / pathway and process enrichment analysis was carried 1 out with the following ontology sources: kegg pathway, go biological processes, reactome gene 2 sets, canonical pathways and corum, using the metascape tool. all genes in the genome were used 3 as the enrichment background. from the top10 most significant go term descriptions gathered for 4 each mcode network (table 3 , supplementary information) , between 1-4 terms were chosen to 5 assign biological meaning to each mcode term (figure 5a ). the log(q-value) of the terms indicates the 6 significance calculated using the merged list of interactors (table 3, total rna was extracted from tri-reagent by isopropanol precipitation and resuspended in disodium 5 citrate buffer (ph 6.5). contaminating dna was degraded by treatment with turbo dnase (ambion). 6 thereafter, reverse transcription was carried out using affinityscript multiple temperature reverse 7 transcriptase (agilent), followed by qpcr using brilliant iii ultra-fast sybr® green qpcr master mix 8 (agilent), according to manufacturer's instructions. the following primers were used: 9 hnrnpl_prot 5'-caatctcagtggacaaggtg -3' for one hour at 4 °c. the coupled beads were washed three times with lysis buffer and then 500 μl of 16 the cleared cell lysate was added to the beads pellet. the cleared lysates and coupled beads were 17 incubated at 4 °c for one hour with rotation. the beads were collected on a magnet and washed three 18 times with lysis buffer. at the third wash, each sample was split into two (for treatment or no 19 treatment with rnasea). the supernatant was removed and the beads were resuspended with 20 µl 20 lysis buffer + 10 µl loading buffer (4x lds+dtt). the samples were incubated at 75 °c for 10 minutes. 21 using the magnet, the supernatants (eluates) were transferred to new tubes, ready to load onto gels 22 for coomassie staining and mass spectrometry sample preparation. the compositions of the eluates 23 were quantified by mass spectrometry, following the same protocol as above. triplicate samples were 24 processed against the same sequence database as above, by transproteomics pipeline (tpp) 68 tools. 25 four database search engines were used (comet 69 , xtandem 70 , msgf 71 and myrimatch 72 , with search 26 parameters as above. each search was followed by the application of the peptideprophet 73 tool; the 27 iprophet 74 tool was then used to combine the search results, which were filtered at the false discovery 28 rate of 0.01; furthermore, the identification was only accepted if at least two of the search engines 29 agreed on the identification. protein inference was performed with proteinprophet 75 . for those 30 protein groups accepted by a false discovery rate filter of 0.01, a normalized spectral abundance 31 factor (nsaf) 76 was calculated based on the peptide to spectrum match count; shared peptides were 32 accounted for by the method of 77 , giving normalised spectral abundance factor (dnsaf) values. the dnsaf values were used to calculate abundance as a % of the upf1 bait protein, which were reported 1 for the nmd factors and sfv proteins that were detected in the samples. 2 3 small-scale sfv protein expression and puromycin incorporation assays 4 1.5x10 5 hela cells per well were seeded into 6-well plates. the following day, 1.25 μg of the relevant 5 pcdna5.3xflag plasmids were transfected using dogtor transfection reagent (oz biosciences), as per 6 manufacturer's recommendations. cells were harvested 48 hours later, after which 1x10 6 cells per 7 condition were collected to make lysates for western blot analysis, while the remaining cells were 8 collected for rna analysis. the cells were harvested at 4 °c for 5 minutes at 250 x g, washed once with 9 1x pbs, and resuspended in either 2x sds-page loading buffer (for protein analysis) or 900 μl of tri 10 reagent (for rna analysis). for the puromycin incorporation assays, cells were transfected as above 11 and prior to harvesting, the medium was aspirated and medium containing either dmso or 100 µg/ml 12 cycloheximide (chx) was added to the cells and incubated for 2 hours at 37 °c. thereafter, the cells 13 were pulse labelled for 10 minutes with puromycin (10 μg/ml). after the 10 minutes pulse, as a 14 recovery step, medium containing either dmso or chx was re-added to the cells for 30 minutes at 37 15 °c. cells were harvested as above and 1x10 6 cells per condition were resuspended in 2x sds-page 16 loading buffer and incubated at 95 °c for 5 minutes for western blot analyses. 17 18 lysates were loaded into either 10 % sds-page gels or pre-casted 4-12 % bis-tris 26-well nupage™ 20 or 10-well bolt™ gels (invitrogen, thermo fisher scientific) and electrophoresed in either 1x sds-page 21 running buffer or 1x mops running buffer. proteins were transferred onto nitrocellulose membranes 22 using either the biorad semi-dry blot system (30 minutes in 1xbjerrum buffer + 20 % methanol) for 23 sds-page gels or the iblot2® (p0, 7mins, using iblot® 2nc regular stacks) (invitrogen, thermo fisher 24 scientific) for pre-casted gels. membranes were blocked for 1 hour at room temperature (rt) in 5 % 25 milk-tbs-t (0.1 % tween20), or in the case of rb anti-eif2α, ms anti-eif2α and rb anti-p-eif2α blots, 26 5 % milk-tbs, and then incubated with the indicated primary antibodies at 4°c overnight. primary 27 antibodies were diluted in 5 % milk-tbs-t, apart from rb anti-eif2α, ms anti-eif2α and rb anti-p-28 eif2α, which were diluted in 5 % bsa-tbs-t (0.1 % tween20). after three 10 minutes washes in tbs-29 t, membranes were incubated with the indicated secondary antibodies in 5 % milk-tbs-t for 1.5 hours 30 at rt, followed by three 10 minutes tbs-t washes. the blots were then visualised using the odyssey 31 system (licor). 32 the protocol for polysome fractionations was adapted from 78 . hela cells were transfected with the 2 'empty' (pcdna5_frt_to_3xflag) or 'capsid' (pcdna5.3xflag-capsid) plasmids as described for the 3 large scale transfections above. the cells were treated with 100 µg/ml chx for 4 mins at 37 °c prior 4 to harvesting. cells were washed once with ice-cold pbs containing 100 µg/ml chx, scraped off the 5 surface of the dish with 750 µl per 15cm dish of pbs-chx and then transferred into tubes. cells were 6 collected by centrifugation at 4 °c for 5 minutes at 500 x g and lysed in 600 µl of lysis buffer (10 mm 7 tris-hcl [ph 7.5], 10 mm nacl, 10 mm mgcl2, 1 % triton x-100, 1 % sodium deoxycholate, red asterisks indicate 3xflag tagged sfv proteins (or the 3xflag alone) at respective sizes. 'untr' refers to an 3 untransfected control condition that underwent the affinity purification procedure, while 'empty' denotes transfection with 4 a plasmid construct containing only the 3xflag tag (with no additional coding region) followed by affinity purification. the 5 expected sizes of the proteins (3xflag included) were: empty ~8kda; nsp1 ~63kda; nsp2 ~92kda; nsp3-z ~82kda; nsp4 6 ~72kda; capsid ~33kda and env ~111kda. the left side of the wbs indicate the eluates (after flag peptide elution from the 7 beads) of each sfv affinity purification, which were sent for mass spectrometry analysis. the right hand side of the wbs 8 indicate the sfv proteins that were still present on the beads after the flag peptide elution step. identified as interactors to more than one of the sfv proteins. in these cases, the solid grey lines connect the grouped set of 5 host proteins to the sfv proteins for which they were identified as interactors. host-host ppi ascertained through string mosquito-3 borne arboviruses of african origin: review of key viruses and vectors arthritogenic alphaviruses: a worldwide emerging threat? the molecular pathogenesis of semliki forest 9 virus: a model virus made useful? semliki forest virus: cause of a fatal case of human encephalitis an outbreak of human semliki forest virus infections in central african 13 isolation of a 15 newly recognized alphavirus from mosquitoes in vietnam and evidence for human infection 16 and disease me tri virus: a semliki forest virus strain from vietnam? microtubule-dependent transport of semliki forest virus replication complexes from the plasma membrane to modified lysosomes alphavirus infection: host cell shut-off and inhibition of antiviral 23 responses polyprotein processing as a 25 determinant for in vitro activity of semliki forest virus replicase the alphaviruses: gene expression, replication, and evolution semliki forest virus-specific non-structural polyprotein by nsp2 protease the eif2α kinases: their structures and 32 functions eukaryotic aspects of translation initiation brought into focus independent mechanisms are involved in translational shutoff during sindbis virus infection alphavirus rna synthesis and non-5 structural protein functions magnetic fractionation and proteomic dissection of cellular organelles occupied by the late replication complexes of semliki forest virus novel functions of the alphavirus nonstructural protein 10 nsp3 c-terminal region induces filopodia and rearrangement of actin filaments nuclear localization of semliki forest 14 virus-specific nonstructural protein nsp2 evasion of the innate immune response: the old 16 world alphavirus nsp2 protein induces rapid degradation of rpb1, a catalytic subunit of rna 17 a structural and functional perspective of alphavirus 19 replication and assembly translation of sindbis virus mrna: effects of sequences 21 downstream of the initiating codon translation of sindbis virus mrna: analysis of sequences 23 downstream of the initiating aug codon that enhance translation alphavirus vectors and vaccination the regulation of translation in alphavirus-28 infected cells the alphavirus exit pathway: what we know and what 30 we wish we knew the c-terminal old world alphaviruses bind directly to g3bp viral and cellular proteins containing fgdf motifs bind g3bp to block 38 structure and function of a protein folding machine: the eukaryotic cytosolic chaperonin cct function and regulation of cytosolic molecular chaperone cct hnrnps relocalize to the cytoplasm following 23 infection with vesicular stomatitis virus exploitation of nuclear functions by 25 human rhinovirus, a cytoplasmic rna virus mapping of chikungunya virus interactions with host proteins identified 2 nsp2 as a highly connected viral component karyophilic properties of semliki forest virus nucleocapsid protein cytoplasmic viruses: rage against the (cellular rna decay) machine the role of stress granules and the nonsense-mediated mrna decay pathway in antiviral defence interplay between 11 coronavirus, a cytoplasmic rna virus, and nonsense-mediated mrna decay pathway the cellular nmd pathway restricts zika virus infection and is targeted 14 by the viral capsid protein a combined proteomics/genomics approach links hepatitis c virus 16 infection with nonsense-mediated mrna decay sindbis virus usurps the cellular hur protein to stabilize its transcripts 18 and promote productive infections in mammalian and mosquito cells tracking and elucidating alphavirus -host protein interactions new world and old world alphaviruses have evolved to exploit different 23 components of stress granules, fxr and g3bp proteins, for assembly of viral replication 24 eastern equine encephalitis virus nsp3 redundantly utilizes multiple cellular proteins for 27 new world alphavirus 29 protein interactomes from a therapeutic perspective two-helper rna system for production of recombinant semliki 31 blue silver: a very sensitive colloidal coomassie g-250 staining for 33 proteome analysis proteome 1 remodelling during development from blood to insect-form trypanosoma brucei quantified 2 by silac and mass spectrometry maxquant enables high peptide identification rates, individualized p.p.b.-4 range mass accuracies and proteome-wide protein quantification uniprot: a worldwide hub of protein knowledge detecting significant changes in protein 11 abundance controlling the false discovery rate: a practical and powerful 13 approach to multiple testing analyzing protein-protein interactions from affinity purification-mass 15 metascape provides a biologist-oriented resource for the analysis of systems-17 level datasets a guided tour of the trans-proteomic pipeline a deeper look into comet-implementation and features a method for reducing the time required to match protein sequences 23 with tandem mass spectra ms-gf+ makes progress towards a universal database search tool for 25 proteomics highly accurate tandem mass spectral peptide identification by multivariate hypergeometric analysis statistical validation of peptide identifications in large-30 scale proteomics using the target-decoy database search strategy and flexible mixture 31 multi-level integrative analysis of shotgun proteomic data improves peptide and protein identification rates and error estimates interpretation of shotgun proteomic data quantitative shotgun proteomics using a 4 protease with broad specificity and normalized spectral abundance factors quantitation: how to deal with peptides shared by multiple proteins studying the translatome with polysome profiling methods in molecular biology key: cord-294945-hcf7gsv8 authors: lin, k.h.; chen, l.f.o.; li, s.d.; lo, h.f. title: comparative proteomic analysis of cauliflower under high temperature and flooding stresses date: 2015-02-12 journal: sci hortic (amsterdam) doi: 10.1016/j.scienta.2014.12.013 sha: doc_id: 294945 cord_uid: hcf7gsv8 high-temperature and waterlogging are major abiotic stresses that affect the yield and quality of cauliflower. cauliflower cultivars ‘h41’ and ‘h69’ are tolerant to high temperature and flooding, respectively; however, ‘h71’ is sensitive to both stresses. the objectives of this study were to identify the proteins that were differentially regulated and the physiological changes that occurred during different time periods in ‘h41’, ‘h69’, and ‘h71’ when responding to treatments of flooding, 40 °c, and both stresses combined. changes in the leaf proteome were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (maldi-tof-ms) and identified by mascot peptide mass fingerprint (pmf) and database searching. stress treatments caused significant reductions in electrolyte leakage, chlorophyll fluorescence fv/fm, chlorophyll content, and water potential as stress times were prolonged. by the comparative proteomic analysis, 85 protein peaks that were differentially expressed in response to combination treatments at 0, 6, and 24 h, 69 (33 in ‘h41’, 29 in ‘h69’, and 9 in ‘h71’) were identified, of which were cultivar specific. differentially regulated proteins predominantly functioned in photosynthesis and to a lesser extent in energy metabolism, cellular homeostasis, transcription and translation, signal transduction, and protein biosynthesis. this is the first report that utilizes proteomics to discover changes in the protein expression profile of cauliflower in response to heat and flooding. cauliflower (brassica oleracea var. botrytis), a member of the brassicaceae family, is an economically and nutritionally important cole crop. the optimum mean temperature range for growing cauliflower is 18-25 • c. high temperatures cause cauliflower to form uneven and loose heads, puffy buds, yellow eyes and leaves in the head, narrow leaves, reduced leaf growth, delayed initiation of heading, and increased petiole-to-blade ratio, all of which lower the quality of or even make the heads unmarketable (wurr et al., 1996; nowbuth and pearson, 1998) . however, heat-tolerant cultivars are able to form heads at mean temperatures higher than 25 • c. to expand the area of food production, crops are grown under stressful environments that likely lead to lower yields. the main contributing factors in the reduction of yield and quality in these areas are variations in climatic conditions such as flooding caused by rains (lin et al., 2010) . heavy rainstorms and standing water can leave soils saturated for days before draining, making waterlogging a problem in many parts of the world. air pockets in the soil become filled with water during saturation, thus creating hypoxic conditions followed by anoxia. flooding causes oxygen starvation, which is a consequence of the relatively slow diffusion of gases in water and from oxygen consumption by plant roots (takeshi and julia, 2004) . rapid leaf chlorosis is seen when cauliflower is subjected to waterlogging (shih et al., 2013) . flooding from large rainfall events is a major risk to fresh-market cauliflower production in taiwan, and most cauliflower cultivars are unable to tolerate flooding during typhoon-caused heavy summer rains. waterlogging and increasing temperatures associated with global warming are a growing concern, as they limit plant growth and productivity, especially in temperate species. over the past decade in the tropics and subtropics, there have been considerable increases in production because of the availability of new, tropically adapted cultivars, resulting in increased farmer incomes. in the field, the co-occurrence of several abiotic stresses rather than individual stresses are most damaging to crop production (mittler, 2006) . many physiological changes occur during high temperature and flood stressing that result in increased heat and flood http://dx.doi.org/10.1016/j.scienta.2014.12.013 0304-4238/© 2014 elsevier b.v. all rights reserved. (hf) tolerance. the physiological mechanisms of hf tolerance are extensively studied in various plant species; however, our current understanding of the molecular biology of acquired tolerances to high temperature and waterlogging are still limited, and relatively little is known about proteins that are critical for controlling these dual mechanisms (wahid et al., 2007) . understanding these processes requires the identification and analysis of major proteins that underlie stress-regulatory networks. plant adaptations to environmental stresses depend on the activation of cascades of molecular networks involved in stress perception, signal transduction, and the expression of stress-related proteins. knowledge of hf-responsive proteins is critical for further understanding of the molecular mechanisms of stress tolerance. plants respond to stress in part by modulating protein regulation, which eventually leads to restoration of cellular homeostasis, neutralization of toxins, and recovery of growth. however, there are no reports on the effect of hf stresses on the functioning of cauliflower. proteins are the direct effectors of the response of plants to stress. they not only include enzymes that mediate changes in the levels of metabolites but also serve as components of the transcription and translation machinery. in recent years, methods for analyzing the proteome have advanced considerably, and together with emerging sequence information in crops, plant proteomics has become increasingly useful for understanding gene functioning and networks in response to environmental stimuli. proteomics is a powerful tool for the quantitative analyses of different biochemical pathways including plant stress-related responses. comparative proteomic analyses of plants subjected to specific stress conditions allow the exploration of various defense-related mechanisms. for instance, proteomics approaches for the comparative analysis of protein abundance between untreated and stress-treated or tolerant and intolerant rice plants have greatly facilitated the study of plant cellular stress responses (komatsu et al., 2003) . therefore, identifying novel proteins and studying their differential display patterns in response to hf stresses will provide the molecular and physiological bases for improving tolerance to hf by cauliflowers. understanding the basis of hf stress signaling and tolerance mechanisms in cauliflowers is required for engineering local cauliflower genotypes that are more tolerant to heat and flood stressing. this goal can be achieved by deciphering the physiological and proteomic responses of cauliflower genotypes, heat-tolerant 'h41', flood-tolerant 'h69', and hf-sensitive 'h71' to flood stress at 40 • c. the objective of this study was to identify the leaf proteins that are differentially regulated in response to hf stress using a pro-teomelab pf-2d aided by improved databases. our hypothesis was that both hf stresses trigger plant responses that result in quantifiable changes in the proteins of 'h41', 'h69', and 'h71'. proteomic characterization and functional analysis should facilitate a better understanding of the hf response mechanisms in brassica so that effective strategies for the genetic improvement of hf-tolerant plant cultivars can be established. to the best of our knowledge, there are no published reports addressing the identification of hfresponsive proteins in brassica using a proteomic approach. 2.1. plant materials, culturing, and heat-and flood-stress treatments seeds of cauliflower (b. oleracea var. botrytis) 'h41', 'h69', and 'h71' were obtained from chin-long seed co. (tainan, taiwan). 'h41' is a heat-tolerant cultivar used especially in warm-subtropical regions such as southern taiwan, where average day temperatures reach as high as 40 • c during the summer (june-august). 'h69' is a popular cultivar grown in taiwan and is flood-tolerant during the rainy season, particularly in rain-fed lowlands. 'h71' is a heat-and flood-sensitive cultivar, and mostly grown during winter in taiwan due to its cold tolerance. seeds were sterilized with 1.5% (v/v) sodium hypochlorite, rinsed with distilled-deionized (dd) h 2 o, and sown in a commercial potting soil mixture, and germinated seedlings were transplanted into 12.7-cm diameter plastic pots and raised in a growth chamber under 300 mol m −2 s −1 light with a 14 h photoperiod and 22/18 • c day/night temperatures at a relative humidity (rh) of 80%. plants were watered three times a week and fertilizer (17:36:39, n:p:k) applied once a week to maintain optimal growth for 30 days before the imposition of stress treatments. pots containing 'h41' and 'h69' plants were subjected to four treatments: non-flooding at 20 • c (nfc, as control), flooding at 20 • c (fc, control temperature), non-flooding at 40 • c (nfh, high temperature), and flooding at 40 • c (fh), for periods of 0, 6, 12, 24, 48, 72, and 96 h in four growth chambers having a 14 h photoperiod at 300 mol m −2 s −1 radiation and 80% rh (lin et al., 2010) . in the case of flooding treatments, pots were randomly placed in 28 cm × 14 cm × 14 cm plastic buckets and subjected to flooding by filling the buckets with tap water to 5 cm above the soil surface. pots were removed from the buckets at different times following flooding, and plants were removed and their leaves from each plant clipped, frozen in liquid nitrogen, and stored at −80 • c in an ultrafreezer until used. three replicates of each time period for the four treatments were randomly placed in a growth chamber. the experiment was performed twice independently in a randomized design for growth environment, sampling day, and physiological analyses. 2.2. determination of electrolyte leakage (el), chlorophyll fluorescence (cf), chlorophyll content (cc), and water potential (wp) cell membrane stability was estimated by measuring leaf ion leakage according to the method of huang and guo (2005) . leaves were excised and immersed in 15 ml of distilled water in test tubes overnight at room temperature. the initial conductivity of the water was determined using a conductivity meter (model cdm 210, radiometer, cedex, france). tubes were placed in boiling water for 15 min and then cooled to room temperature, and conductivity was again determined. the relative el (%) was calculated as the ratio of conductivity before boiling to that after boiling. cf components were quantified with a portable modulated fluorometer (mini-pam photosynthesis yield analyzer, walz, effeltrich, germany). the measurement of variable fluorescence to (fv)/maximum fluorescence level in light-adapted leaves (fv/fm) was previously described (lin et al., 2007) . relative cc per unit leaf area was determined using a spad (soil plant analysis development) analyzer (spad-502 chlorophyll meter, konica minolta, tokyo, japan). wp (in bars) was measured on the third leaf from the top of each plant using a pressure chamber (plant water system, skye skpm 1400, tokyo, japan) (sairam et al., 1998) . the data shown in tables 1-4 represent the means of at least two independent sets of experiments with similar results. measurements of physiological parameters were analyzed by a three-factor completely randomized anova that compared cultivars, treatments, and time periods. for significant values, means were separated by a least significant difference (lsd) test at p ≤ 0.05 using pc sas 8.2 (sas institute, cary, nc, usa). because the negative effects of flood stressing on hf-sensitive 'h71' at 40 • c were observed after 48 h of stress treatments (see section 4), 'h41', 'h69', and 'h71' plant leaves subjected to hf conditions for 0, 6, and 24 h were used for subsequent proteomics analysis. proteins were extracted according to a previously published method (yan et al., 2013) with some modifications. briefly, two grams of plant leaves were ground in liquid nitrogen and crude protein extracts solubilized in 7 ml of extraction buffer containing 0.5% sds (sodium dodecyl sulfate), 5% ␤-mercaptoethanol, 10% glycerol, 0.2% polyvinylpyrrolidone, and 50 mm tris-hcl, ph 8. after 30 min of incubation at 4 • c, samples were centrifuged for 30 min at 15,000 × g at 4 • c; these two steps were done twice. the extraction buffer was further replaced by 25 ml of start buffer (50 mm tris-hcl, ph 8) with a pd-10 column (ge healthcare) according to manufacturer (proteomelab pf-2d kit, beckman coulter) protocols. protein concentrations in the samples were determined using the bradford assay (bio-rad, hercules, ca, usa) and bovine serum albumin (bsa) was used to generate a standard curve. a 200 g aliquot of the total protein sample was passed through a 0.45 m filter before injection into the chromatography column. the high performance chromatofocusing (hpcf) column was treated according to manufacturer instructions (proteomelab pf-2d protein fractionation system, beckman coulter, fullerton, ca, usa). briefly, the column was washed with 10 volumes of water at a flow rate of 0.2 ml/min for 45 min and then equilibrated with 30 volumes of start buffer for 130 min at 0.2 ml/min. after equilibration, each sample was introduced with a manual injector into the column and absorbance of the column effluent was monitored at 280 nm. in the first dimension, proteins were bound to a strong anion exchanger and eluted with a continuously decreasing ph from 8.0 to 4.0. fractions were collected at ph intervals of 0.3 in a 96 deepwell plate. proteins eluted in the gradient were then separated in the second dimension using high performance reversed phase (hprp) chromatography. fractions were separated from the second dimension with an rp-hplc column using two solvents: 0.1% trifluroracetic acid (tfa) in hplc water (solvent a) and 0.08% tfa in acetonitrile (acn) (solvent b) (lee et al., 2008) . separation was performed at 50 • c with a flow rate of 0.75 ml/min and protein fractions were detected by uv absorbance at 214 nm. equilibration was achieved with solvent a for 10 min followed by solvent b for 5 min prior to each injection. from the selected first dimension fractions, 0.2 ml were injected, run for 2 min, and the column eluted with a linear gradient of 0-100% solvent b for 25 min. thereafter, solvent b was continued for 5 min, followed by re-equilibration with 100% solvent a for 10 min (irar et al., 2010) . fractions from the second dimension were analyzed with karat tm version 7.0 (beckman coulter). protein extraction and two-dimensional gel electrophoresis (2-de) were carried out on three independent technical replicas of the bulk samples. proteins were in-gel digested with trypsin in the automatic investigator progest robot of genomic solutions. briefly, excised gel bands were washed sequentially with 50 mm ammonium bicarbonate (nh 4 hco 3 ) buffer and acn. proteins were reduced and alkylated, respectively, by treatment with 10 mm dithiothreitol (dtt) solution for 1 h at 60 • c and treatment with a 50 mm solution of iodine acetamide. after sequential washings with buffer and acn, proteins were digested overnight at 37 • c with 120 g/ml of trypsin. tryptic peptides were extracted from the gel matrix with 10% formic acid and acn, and extracts were then pooled and dried in a vacuum centrifuge. digested peptide solutions were desalted with zip-tip pipet tips (nikkyo technos, tokyo, japan) and redissolved in 10 l of 0.1% tfa in 50% acn according to manufacturer instructions. proteins excised from two-dimensional gels were analyzed in matrix-assisted laser desorption ionization time-of-flight (maldi-tof, 4700 proteomics analyzer, applied biosystems) mass spectrometers. a 1 l aliquot was mixed with the same volume of a matrix solution (5 mg/ml ␣-cyano-4-hydroxycinnamic acid in 0.1% tfa in 50% acn) and spotted on a maldi plate. ms spectra were acquired in the positive reflector mode, with 500 shots per spectrum being accumulated. three major peaks were selected for further characterization by ms/ms analysis. ms spectra were acquired using collision-induced dissociation (cid) with atmospheric air as the collision gas, the ms 1 kv positive mode being used. the peak lists for ms/ms spectra from same-replicate samples were merged into a single file prior to database searching. the pkl and mgf files were searched against the swiss-prot database using mascot (http://www.matrixscience.com; london, uk). search parameters were set as follows: 1 missed cleavage, fixed modification, peptide charge ±1, and variable modifications were carbamidomethyl of cysteine and oxidation of methionine. trypsin was specified as the proteolytic enzyme. peptide tolerance and ms/ms mass tolerance were 50 ppm and 0.25 da, respectively. peptide mass fingerprinting (pmf) match confidence was based on the mowse score and confirmed by accurate overlapping of matched peptides with mass spectrum major peaks. scores greater than 67 (p < 0.05) were considered positive. peaks with multiple proteins detected by ms were not considered. only significant hits, as defined by mascot probability analysis (p < 0.05), were accepted. characteristic physiological responses of plants to different temperature and waterlogging treatments were evaluated. table 1 illustrates the comparison of fv/fm values under four treatments at seven different times in leaves of three genotypes of cauliflower. levels of fv/fm in all plants progressively decreased as flood stress and heat stress (nfh and fh) durations were extended. fv/fm in 'h41' plants showed significantly lower values after 6 h with table 5 identification of differentially expressed proteins found in cauliflower 'h41' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for 0 and 6 h. peak ( table 4 lists the different points in time that waterlogging and high temperature treatments were monitored by measuring the changes in wp. when different treatments across time were compared, wp with stress treatments was significantly lower (more negative) than those with nfc treatment from 12 to 96 h for all genotypes, indicating that high temperature and/or flooding induced a decrease in leaf water level, subsequently affecting leaf wp. the trends in wp differed in all stressing treatments from 12 to 96 h in all plants. thus, the wp of the different genotypes responded differently to specific stresses. the lowest value in wp (−2.27 mpa) was detected at 48 h with exposure to nfh treatment in 'h71'. proteome alterations in the three genotypes in response to short-term exposures to flood and high-temperature conditions were compared relative to treatments and controls. the proteome-lab system uses two-dimensional liquid chromatography based on high-performance chromatofocusing in the first dimension followed by high-resolution reversed-phase chromatography in the second dimension, and has become available for sample fractionation and more resolution at extreme ph values (soldi et al., 2005; pirondini et al., 2006) . protein peaks were identified and their expression patterns analyzed. protein fractions were separated according to isoelectric point (pi) and hydrophobicity and detected in a ph range of 8.0-4.0 by proteomelab pf-2d. proteins were analyzed by maldi-tof-ms and identified via pmf and database searching by their calculated molecular weight, pi score, and percent coverage, and their database accession numbers and alteration values among stress treatments are represented in tables 5-8 . changes in the protein levels of 'h41' exposed to fh stresses at 0 and 6 h and their differentially expressed protein peaks are listed in table 5 . compared to nfc control treatment, two protein peaks (291 of phosphoglucosamine mutase and 292 of rotidine 5 -phosphate decarboxylase) under fc treatment for 6 h were up-regulated (supplementary fig. s1a) . furthermore, the expression level of s-adenosylmethionine synthetase (peak 294) was increased during high-temperature stressing over 6 h (supplementary fig. s1b ). two identified peaks, 301 of 16 kda calcium-binding protein and 302 of acetyl-coenzyme a carboxylase carboxyl transferase subunit beta, were also up-regulated when 'h41' plants were treated with fc for 6 h in comparison to nfc treatment at 0 h (supplementary fig. s1c) . nine protein expression patterns were detected in 'h71' plants under high-temperature and flood stress at 6 h, and the dynamic changes in the level of each protein are displayed in table 6 . compared to nfc treatment at 0 h, nfh treatment for 6 h showed that the abundances of peaks 271 (phosphoserine aminotransferase), 272 (imidazole glycerol phosphate synthase subunit hisf), table 6 identification of differentially expressed proteins found in cauliflower 'h71' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for 0 and 6 h. peak ( 273 (phosphoribosyl formyl glycin amidine synthase 1), and 295 (probably hydrogenase nickel incorporation protein hypa) were up-regulated (supplementary fig. s2a) . moreover, the abundance of peak 296 (putative holliday junction resolvase) in 'h71' plants was also up-regulated during 6 h of fc treatment as compared to nfc treatment at 0 h (supplementary fig. s2b ). the last comparison includes four proteins (peaks 281, 282, 297, and 304, namely trna-methyltransferase, arginyl-trna synthetase, elongation factor g, and methionyl-trna formyltransferase, respectively) whose abundances were consistently up-regulated during flood stressing for 6 h at 40 • c. interestingly, all peaks were up-regulated after all treatments for 0 h and 6 h in both 'h41' (table 5) and 'h71' (table 6) plants. the identified proteins showed altered expressions by upregulation or down-regulation in 'h41' plants at the 0 h and 24 h time points when combination treatments were compared (table 7) . only six protein peaks (231, 232, 241, 251, 252, and 253 ; supplementary fig. s3 ) from comparative proteomic analyses between fh and nfc for 24 h were down-regulated, all others being up-regulated. there were 18 peaks found in fh vs nfc for 24 h, with the remaining 12 peaks being among other comparisons, including fh vs nfh, fc vs nfc, nfh vs nfc, nfh vs fc, and fh at 24 h vs nfc at 0 h. the carbohydrate metabolism related protein, fructose-1,6-bisphosphatase (peak 232), was down-regulated under fh treatment compared to nfc treatment. nevertheless, the photosynthetic-related proteins ribulose bisphosphate carboxylase (rubisco) small chain (peak 236) and large chain (peak 255) were increased in abundance in 'h41' under flooding for 24 h. additionally, dna mismatch repair protein muts was found in peak 253 with down-regulation (fh vs nfc) and peak 254 with up-regulation (fc vs nfc) in protein abundance through a homologue search. differentially expressed proteins identified in 'h69' plants at 0 h and 24 h under stressing are shown in table 8 . the protein abundances of eight peaks (258, 259, 2511, 2512, 2513, 264, 265 , and 266 of ribose import atp-binding protein, phenylalanyl-trna synthetase, rubisco large subunit, gmp synthase, atp synthase subunit, and phosphoribosylformyl glycinamidine synthase) were consistently down-regulated during the fh condition when compared to nfc treatment for 24 h, but the other 33 peaks were up-regulated in protein abundance. mitochondrial inner membrane protease (peak 221) and rubisco small subunit (peaks 222 and 223) were up-regulated in 'h69' upon fc treatment compared to nfc after 24 h ( supplementary fig. s4a ). peaks 2514 and 2515, respectively identified as uncharacterized 37.2 kda and sec14 cytosolic factor, were increased under heat stress at 24 h ( supplementary fig. s4b) . meanwhile, rubisco small subunit (peak 268) was also increased in 'h69' under fh at 24 h as compared to nfc at 0 h ( supplementary fig. s4c) . notably, the majority of peaks found in 'h69' at 24 h represented rubisco down-regulation (peaks 2511, 264, and 266) and up-regulation (peaks 2312, 2314, 2318, 222, 223, 2310, 2311, 246, 247, and 268) in protein abundance. because flood and heat often co-occur in stress-prone environments, this study compared the combined effects of flood and heat with those of the single stresses on plant physiology. in general, the changes in plant physiology were greater under the combined treatment than under heating or flooding alone. the differences in responses to flood and heat in fv/fm, cc, el, and wp seen between cultivars suggested that the three genotypes have unique mechanisms for coping with environmental stresses. the heat-tolerant 'h41' genotype showed significantly higher fv/fm, cc, and wp values (tables 1, 2 and 4) and lower el% (table 3 ) than the sensitive 'h71' genotype when heated for 24 h under nfh treatment, which reflects its tolerant nature. the exposure to flooding for at least 6 h under fc treatment provoked greater reductions in fv/fm and cc values in 'h71' plants than 'h69' plants (tables 1 and 2 ). in addition, flooding also significantly decreased the el in 'h69' plants relative to 'h71' plants after 48-96 h ( table 3 ), indicating that 'h69' is flood-tolerant. similar losses of fv/fm, cc, and wp and an increase in el were observed in 'h71' under flooding and high temperature stresses (fh), but responded at different levels over time (tables 1-4) . table 7 identification of differentially expressed proteins found in cauliflower 'h41' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for 0 and 24 h. peak ( a soil water potential of approximately −2 mpa was used in greenhouse experiments to simulate field situations under severe stress conditions (ashoub et al., 2013) . the leaf wp of 'h71' plants under high-temperature treatment was significantly lower than in 'h41' and 'h69' plants after 12 h, indicating that 'h71' plants suffered from heat exposure. a lower osmotic potential is responsible for maintaining turgor when water loss occurs due to high temperature. thus, high electrical conductivity due to a high concentration of electrolytes in the leaf sap of 'h71' plants should lead to alterations in membrane permeability and a reduced ability to retain solutes and water during high temperature and waterlogging. however, the ability of 'h41' and 'h69' plants to tolerate heat and flood stress, respectively, strongly depends on adjustments in water balance and ionic leakage. a decline in wp and an increase in el during heat and flood stress were associated with leaf water deficits as well as increases in ionic leakage. at the physiological level, the many effects of flooding and heat stresses indicate the importance of protecting plants from oxidative damage caused by the overproduction of ros that is elicited by increased ion leakage (kangasjarvi et al., 2008) . the chlorophyll fluorescence emission parameter, fv/fm, which is widely used as a proxy for the maximum quantum efficiency of ps ii photochemistry, was correlated with flooding and high temperature tolerance. in healthy leaves, the fv/fm value is close to 0.8, which is a typical value for uninhibited plants. a lower table 8 identification of differentially expressed proteins found in cauliflower 'h69' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for 0 and 24 h. peak ( value indicates that some proportion of the psii reaction centers are damaged, which is often observed in plants under stress (camejo et al., 2006) . flooding and high temperatures inhibit photosynthetic co 2 fixation and damages photosynthetic electron transport at the site of psii where there exists a very sensitive photosynthesis apparatus. this reduction in the co 2 assimilation rate observed in 'h71' plants was generated by effects on the calvin cycle and also on psii functioning. exposure of 'h71' plants to flooding and high temperature may lead to reductions in net photosynthetic rate, stomatal closure, and cell activities that damage photosynthetic membranes (jiang and huang, 2001) . these results suggested that the chlorophyll fluorescence parameters were stress specific and were not expressed solely in response to an increasing excess of photon energy. chloroplast development in 'h71' plants may be particularly sensitive to high temperature and flooding. alternatively, the pigments of the sensitive cultivar might have been destroyed because of a high sensitivity to oxidative stress. 'h71' plants suffered greater cc losses than 'h41' and 'h69' plants after 24-96 h of exposure. flooding and heat may induce stomatal closure and consequently reduce cc levels and wp as well. typically, the amount of cc is reduced by stress. this is consistent with our observations that chlorophyll loss was more pronounced in waterlog-and heat-sensitive 'h71' plants in accordance with the more pronounced and increased visible symptoms of leaf injury. leaf curling or folding were the initial and most obvious changes observed. most 'h71' leaves progressively became necrotic, epinastic, or wilted over the course of time; however, most 'h41' and 'h69' leaves visually appeared to be green and healthy after 72 h of flooding and high temperature (photos not shown). along with visual symptoms, reduced cc could be used to monitor flooding and heat damage in green or senescent leaves. attempts have been made to breed for increased flooding tolerance and modify brassica cultivation or management practices to avoid injury from flooding and heat. environmental stresses represent the most limiting conditions for horticultural productivity and plant exploitation worldwide. important factors among them are water and temperature. flooding and high temperature are major abiotic stresses resulting in serious problems for the growth and yield of flood-and heat-sensitive plants. it is necessary to identify the physiological characteristics that reflect their complex underlying genetic make-up. these easily measured physiological biomarkers could be used as generic tools to develop a reliable method for selecting cauliflower cultivars having flood-and heat-stress resistance. tables 1-4 demonstrate that the chlorophyll losses in 'h71' plants were in concert with increasing el accumulations after 6 h of flooding and heat treatments, which is an index of oxidative damages to cell constituents in general. this suggests that fv/fm and cc can be effectively used as reporter signals in screening for flooding and high temperature tolerances. these easily measured physiological biomarkers could be used as generic tools for developing a reliable method to select 'h41' and 'h69' cultivars having flooding and heat-stress resistances. these findings are important for farming in high-temperature areas and wetlands or other areas subject to short and intense rainfall events. proteomics techniques are becoming indispensable for understanding how plants adapt to abiotic stresses, and have promoted the identification of numerous stress-related proteins and the pathways in which they function (kosová et al., 2011) . changes in protein accumulation under stress are directly related to the physiological phenotypic responses of plants to stress. the proteomic responses to high-temperature or flood stresses are known for several plants, including rice (lin et al., 2005) , wheat (majoul et al., 2004) , barley (süle et al., 2004) , soybean (komatsu et al., 2013) , radish (zhang et al., 2013) , arabidopsis thaliana (palmblad et al., 2008) , agave americana (shakeel et al., 2013) , and populus euphratica (ferreira et al., 2006) . in the present study, we analyzed physiological and proteomics data in different genotypes of cauliflower under conditions of high-temperature and waterlogging stresses over different time periods. approximately 250 peaks were detected at the tested time points, whereas 85 peaks were successfully observed to be differentially regulated (14 down-regulated, 71 up-regulated) and expressed at a minimum of one time point. among them, 33, 29, and 9 proteins were identified in 'h41', 'h69', and 'h71' plants, respectively, indicating that they were regulated in a genotype-specific manner ( supplementary fig. s5 ). rubisco small and large chains, 16 kda calcium-binding protein, molybdenum cofactor biosynthesis protein a, and phosphoserine aminotransferase were observed in both 'h41' and 'h69' plants. phosphoribosylformyl glycinamidine synthase and arginyl-trna synthetase were both found in 'h71' and 'h69' plants ( supplementary fig. s5 ). moreover, in 'h41' plants, peaks 253 and 254 were both identified as dna mismatch repair protein muts, while 291 and 263 were identified as phosphoglucosamine mutase (tables 5 and 7 ). these sister peaks may represent close homologues, but this could not be resolved based on mass spectrometric data and might instead be isoforms resulting from differential post-translational modifications (rollins et al., 2013) . as stress times increased, additional responsive proteins continued to be identified. for instance, 5 (table 5 ) and 30 (table 7) peaks were observed in 'h41' under stress treatments for 6 h and 24 h, respectively. in this study, some of the proteins identified were well characterized in terms of response to stressing, while others were not. the biological functions of differentially regulated proteins include roles in photosynthesis, energy metabolism, cellular homeostasis, response to stimuli, transcription and translation, protein biosynthesis, mediation of signal transduction, and other functions. for survival, plants must respond to flood and heat stresses in a different manner from regulating protein expressions for biochemical and physiological adaptations. photosynthesis is one of the systems that is most sensitive to high-temperature stress. changes in environmental temperatures are primarily reflected in photosynthesis, triggering a response that is aimed toward the best possible performance under new conditions. for this, a balance is sought between the energy of absorbed light, carbon assimilation, and consumption by metabolic sinks. several studies have shown that high-temperature stress can significantly inhibit the rate of photosynthesis (yan et al., 2013; salvucci and crafts-brandner, 2004) . in our study, rubisco units (small and large chain) related to photosynthesis were differentially expressed and regulated by combination treatments and among genotypes. for example, the up-regulation of rubisco was detected in 'h41' (peaks 236 and 255, table 7 ) and 'h69' (peaks 2312 (peaks , 2314 (peaks , 2818 (peaks , 222,223, 2310 (peaks , 2311 . however, the level of expression of the rubisco large subunit was down-regulated in 'h69' during fh treatment for 24 h (peaks 2511, 264, and 266, table 8 ). rubisco proteins resist high-temperature and flood stresses by inhibiting photosynthesis and other nonessential metabolic processes. in contrast, to increase energy metabolism, the expression of proteins related to redox homeostasis and response to stimuli were up-regulated, thereby maintaining physiological balance during stress. rubisco catalyzes the first major step of carbon fixation in photosynthesis. affinity of rubisco for co 2 decreases with increasing temperature (yan et al., 2013) . the diminished capacity of rubisco and its low affinity for co 2 suggest that carbon fixation or assimilation was highly inhibited when 'h69' plants were exposed to stress, especially under the high temperature and flooding stresses ( table 8 ) that lead to reduced rates of photosynthetic co 2 assimilation. photorespiration serves as an energy sink, preventing the over-reduction of the photosynthetic electron transport chain and photoinhibition. lee et al. (2007) reported that high temperature over 2 h significantly inhibits the activity and quantity of rubisco subunits in rice seedlings. the effects of drought stress on rubisco represent reductions in rubisco activity (parry et al., 2002) . exposure to salt stress brings about a reduction in the overall growth and productivity of plants by disturbing the function of vital components of photosynthesis, such as psii and rubisco (ghaffaria et al., 2014) . consequently, a strong reduction in fv/fm value (table 1 ) and cc content ( table 2 ) while maintaining photosynthesis under stress after 24 h was seen in all plants. some researchers report its up-regulation (ge et al., 2012; budak et al., 2013) , whereas others find downregulation (gaoa et al., 2011) or even both (guoa et al., 2012) in response to abiotic stresses. as a result, an up-regulation in rubisco levels may also indicate an increase in the photorespiration rate. molecular chaperones, including heat shock proteins (hsps), are key components of innate immunity in plants. they have major roles in protein folding and signal-transduction networks, cellcycle control, protein degradation, and protein trafficking (wang et al., 2004) . hsp chaperone pathways require energy in the form of atp hydrolysis in order to function. the atp-dependent clp proteases (hsp100) represent a class of hsp. in addition to their function as molecular chaperones, they function in protein disaggregation and protein degradation when removal of potentially harmful polypeptides arising from misfolding, denaturation, or aggregation are important for the maintenance of cellular homeostasis. the mechanism for rescuing proteins from aggregation is proposed to include the cooperation hsp100 (kregel, 2002; sarkar et al., 2011) . hsps are typically induced when cells are exposed to various types of environmental stresses. the synthesis of hsps under high temperatures is reported to be related to thermotolerance in plants, and plants with a decreased expression of hsps show compromised tolerance to acquired thermo-tolerance (charng et al., 2006) . hsps are closely related to the acquisition of heat resistance in plants. choi et al. (2012) identify several high molecular weight hsps whose expression was up-regulated significantly in pyropia tenera under high-temperature stress for 1 h and 3 h. furthermore, lee et al. (2007) report that several hsps are expressed in rice leaves during heat stress. high temperature might increase the potential risk of protein misfolding, and an active protein quality control system inside cells plays an important role in plant tolerance to heat stress. hsps are also increased by drought stress in the sugar beet (hajheidari et al., 2005) , wheat (demirevska et al., 2008) , and sugarcane (jangpromma et al., 2010) . in our study, peak 261 of atp-dependent clp protease proteolytic subunit 1 was up-regulated in response to heat stress for 24 h in 'h41' plants (table 7) . this suggests that the increased level of hsp100 under high-temperature stress allowed the heat-tolerant 'h41' to produce more energy through atp hydrolysis and the degradation or reactivation of damaged proteins and prevented protein misfolding, resulting in reestablishing normal protein conformations and cellular homeostasis. s-adenosylmethionine (sam) synthetase catalyzes the synthesis of sam that serves as a methyl group donor in transmethylation of proteins, nucleic acids, and a precursor in the biosynthesis of polyamines, biotin, and nicotianamine in plants (roeder et al., 2009) . sam in shoots underwent increased protein synthesis in a heat-tolerant barley cultivar and showed a reduced trend in an abiotic stress-susceptible cultivar (süle et al., 2004) . in our study, the expression of sam synthetase was significantly up-regulated (peak 294, table 5 ) in heat stressed 'h41' over 6 h. therefore, it is most likely that an increased amount of sam synthetase (as a result of the increased formation of sam methyl donors) in 'h41' contributes to its stronger heat tolerance compared to controls. in addition, peak 238 of adenylate kinase (adk) was observed in 'h69' under heat stress for 24 h (table 8) . adk catalyzes the salvage synthesis of adenine monophosphate from adenosine and atp (wang et al., 2010) . heat and flood treatments markedly increased the abundance of two elongation factors (efs): (1) elongation factor g was increased in 'h71' (peak 297) under flooding at 6 h ( table 6) and (2) transcription elongation factor grea was increased in 'h69' (peak 2816) under fh at 24 h ( table 8 ). the elongation of polypeptide chains during translation is a conserved process among prokaryotes and eukaryotes. efs are the critical regulators of protein synthesis, which is influenced by high temperature and drought stress in arabidopsis (rizhsky et al., 2004) and rapeseed (mohammadi et al., 2012) . regulation of the transcriptional and translational machinery is considered to be an important component of cellular stress response. the increased expression of ef grea in 'h69' and ef g in 'h71' likely led to increased protein synthesis, which served to reduce the effects of treatment stress. plants under stress can respond by sensing and transferring stress signals through signal transduction networks. the response to stress is likely mediated through signaling pathways that regulate the expression levels of a range of proteins (hirayama and shinozaki, 2010) . in our study, gtp-binding protein lepa (peak 233, table 7 ) involved in signal transduction was identified. the up-regulation of the lepa protein in response to flooding at 40 • c might reflect the role of this protein in cell signaling under stress. ca 2+ -binding protein is mainly resident in the endoplasmic reticulum where it serves as a calcium modulator and chaperone of newly synthesized glycoproteins (nam et al., 2012) . the latter have functions in plant growth and development as well as biotic and abiotic stress responses. aghaei et al. (2008) indicated that ca 2+ -binding protein was up-regulated by salt stress in potato leaves. salinity induces ca 2+ accumulation in the cytosol that starts the signaling pathway. the modulation of intracellular ca 2+ levels is regulated by calcium-binding proteins, which, after activation, induce specific kinases (capriotti et al., 2014) . a 16 kda calcium-binding protein was identified as an up-regulated peak (301) under flooding (table 1) , suggesting the involvement of intracellular calcium homeostasis and signal transduction in 'h41' during waterlogging. atp synthase is a large multi-subunit transmembrane enzyme complex. the catalytic site for atp synthesis is localized primarily on the beta subunit. atp synthase is known to be involved in energy metabolism and was found in 'h69' plants upon fh stressing for 24 h (table 8) . down-regulation of the catalytic subunit of atp synthase subunit beta (peak 2513) suggested that the energy production systems of 'h69' were highly affected by heat and flood stresses. huseynova et al. (2007) also found that the atp synthase complex was less accumulated in drought-sensitive wheat 'giymatli-2/17' than in drought-tolerant azamatli-95 under water stress. in addition, impaired atp synthesis under drought is regarded as a major constraint to photosynthesis (flexas and medrano, 2002) . fructose-1,6-bisphosphatase (fbpase) is involved in carbohydrate metabolism during gluconeogenesis, and also an important enzyme in the calvin cycle that plays a crucial role in plant responses to some abiotic stimuli (fan et al., 2009) . the downregulation of fbpase in 'h41' under fh at 24 h (peak 232, table 7) indicated that gluconeogenesis was inhibited under stressing. an increase in ribosomal protein is a candidate for initiating a translation site, and the phosphorylation of ribosomal proteins is important in the regulation of protein synthesis (fatehi et al., 2012) . the 50s ribosomal subunit catalyzes the peptidyl transfer reaction of mrna-directed protein biosynthesis (sobhanian et al., 2010) . up-regulation of the 50s ribosomal protein l11p (# 2914) was observed under high temperature stress at 24 h in 'h69' (table 8) , indicating the resistance of 'h69' plants to the inhibitory effect of high temperature on protein biosynthesis. in addition, pentatricopeptide repeat (ppr) proteins are characterized by tandem arrays of degenerate 35 amino acid motifs (o'toole et al., 2008) and are thought to be rna binding proteins involved in posttranscriptional processes in mitochondria and chloroplasts (schmitz-linneweber et al., 2006) . meanwhile, ppr proteins are also essential for rna editing in chloroplasts (emi et al., 2005) . peak 2313 (pentatricopeptide repeat containing protein) of 'h69' was up-regulated under fh within 24 h ( table 8 ), suggesting that 'h69' was able to increase rna maturation and protein function in chloroplasts under fh. taken together, genetic differences are supported by the high number of proteins differentially regulated between genotypes. our data suggest that the early response of cauliflower to flooding and high temperature might be an important stress adaptation for survival following not only hypoxia and heat, but also direct damage to cells by flooding and high temperature. all of the identified proteins likely work cooperatively to reestablish cellular homeostasis under stress. the long term goal of our work is to help breed a competitively higher flood-and heat-tolerant cauliflower to be grown in lowlands during summer. the identification of the unique stress-responsive proteins will allow further dissection of the genetic basis of this transgressive performance in offspring. our results not only provide information for selecting lines having better tolerance to waterlogging and heat stresses, but also provide a basis for understanding cauliflower metabolic pathways and their cross-talk under stress. further verification of the correlative stress-responsive protein by gene expression analyses of these stressed-responsive genes, such as rubisco, efs, hsp100, fbpase, adk, atp synthase, sam synthetase, ppr protein, gtp-binding protein lepa, ca +2 -binding protein, and ribosomal protein l11p, would facilitate our understanding of the heat-and flood-response mechanism in cauliflowers. this study provides information on mechanisms underlying the contrasting responses to hf stresses. different genotypes showed differing physiological responses to combinations of stress treatments. all dynamics of the peaks were analyzed across all treatments, and a comparative analysis identified 85 stressresponsive proteins in cauliflower under short-term stressing. the majority of these stressed-responsive proteins were genotype specific. these identified proteins emerged as key participants in stress tolerance. most of the changes in the expression levels of these proteins in response to stressing were involved in photosynthesis. 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2014.12.013. key: cord-300418-s4wt5gim authors: bedford, lynn; lowe, james; dick, lawrence r.; mayer, r. john; brownell, james e. title: ubiquitin-like protein conjugation and the ubiquitin–proteasome system as drug targets date: 2010-12-10 journal: nat rev drug discov doi: 10.1038/nrd3321 sha: doc_id: 300418 cord_uid: s4wt5gim the ubiquitin–proteasome system (ups) and ubiquitin-like protein (ubl) conjugation pathways are integral to cellular protein homeostasis. the growing recognition of the fundamental importance of these pathways to normal cell function and in disease has prompted an in-depth search for small-molecule inhibitors that selectively block the function of these pathways. however, our limited understanding of the molecular mechanisms and biological consequences of ubl conjugation is a significant hurdle to identifying drug-like inhibitors of enzyme targets within these pathways. here, we highlight recent advances in understanding the role of some of these enzymes and how these new insights may be the key to developing novel therapeutics for diseases including immuno-inflammatory disorders, cancer, infectious diseases, cardiovascular disease and neurodegenerative disorders. protein homeostasis is essential for most cellular processes. the ubiquitin-proteasome system (ups) is responsible for much of the regulated proteolysis in the cell, and has non-degradative functions as well. ubiquitin is a small 76 amino-acid protein that can be reversibly attached to other proteins and lies at the core of an elaborate post-translational modification pathway. several ubiquitin-like proteins (ubls) have also been identified, including nedd8, sumo and isg15, which share a characteristic three-dimensional fold with ubiquitin but are otherwise distinct. the ups and ubl conjugation pathways have multiple essential biological roles and it is not surprising that their function, and often malfunction, are important factors in various human diseases 1 , including numerous cancer types [2] [3] [4] , cardiovascular disease 5 , viral diseases 6 and neurodegenerative disorders 3 . these diseases may feasibly be targeted by modulating components of the ups and ubl conjugation pathways using small-molecule inhibitors. indeed, the therapeutic potential of intervention in the ups in cancer has been demonstrated by the proteasome inhibitor bortezomib (velcade; millennium pharmaceuticals), which was approved by the us food and drug administration in 2003. recent and ongoing research to elucidate the roles of other components of the ups and ubl conjugation pathways has identified several enzymes that could be additional targets for therapeutic intervention using small-molecule inhibitors. in this review, we first provide an overview of the enzyme classes in the ups and ubl pathways that represent potential therapeutic targets, highlighting considerations that are important for drug discovery and recent progress in the development of small-molecule inhibitors. we then review recent developments in our understanding of the role of the components of the ups and the ubl pathways in disease and their potential for therapeutic intervention. ubiquitin and ubls typically modulate protein function following covalent attachment to a primary amino group within a substrate protein, usually by forming an isopeptide bond with a lysine side-chain. the ubiquitin pathway is representative of this process and is shown in fig. 1. the enzymatic cascade that results in protein ubiquitylation and degradation involves several distinct steps, each requiring a different class of enzyme. in the first step, an e1 activating enzyme (primarily ubiquitin-activating enzyme (uae; also known as uba1), although ubiquitin-like modifier activating enzyme 6 (uba6) can also activate ubiquitin 7, 8 ) consumes atp and forms a high-energy thioester bond with the carboxyl terminus of ubiquitin. ubiquitin is then passed to one of several e2 conjugating enzymes through a transthiolation abstract | the ubiquitin-proteasome system (ups) and ubiquitin-like protein (ubl) conjugation pathways are integral to cellular protein homeostasis. the growing recognition of the fundamental importance of these pathways to normal cell function and in disease has prompted an in-depth search for small-molecule inhibitors that selectively block the function of these pathways. however, our limited understanding of the molecular mechanisms and biological consequences of ubl conjugation is a significant hurdle to identifying drug-like inhibitors of enzyme targets within these pathways. here, we highlight recent advances in understanding the role of some of these enzymes and how these new insights may be the key to developing novel therapeutics for diseases including immuno-inflammatory disorders, cancer, infectious diseases, cardiovascular disease and neurodegenerative disorders. figure 1 | overview of the enzymatic cascade involved in ubiquitin-like protein (ubl) conjugation and the ubiqitin-proteasome system (ups). a | ubiquitin-activating enzyme (uae) binds atp and ubiquitin (ub) to form a ternary complex consisting of e1-ubiquitin thioester with ubiquitin-amp bound (see text for details). the thioester-bound ubiquitin is then passed to one of several e2 conjugating enzymes through a transthiolation reaction. the ubiquitin-charged e2 then forms a complex with an e3 ligase and a protein substrate to transfer ubiquitin to a lysine residue on the substrate. to mark substrates for degradation, multiple ubiquitins are similarly recruited to produce a k48-linked polyubiquitin chain. following release from the e3, the proteasome recognizes the polyubiquitin chain, and the substrate is deubiquitylated and destroyed. substrates marked with ubiquitin chains linked through lysines 6, 11, 27, 29 and 33 also seem to be primarily destined for degradation. alternatively, substrates marked with monoubiquitin, linear ubiquitin chains or k63 ubiquitin chains are involved in signalling functions that are independent of the proteasome. a second e1 (ubiquitin-like modifier activating enzyme 6 (uba6)) also activates ubiquitin, but the function(s) of this pathway are unknown. b | nine classes of ubl and eight e1 activating enzymes participate in diverse biological pathways in humans. e1s, e2s and ubls are structurally and mechanistically related but are unique to each pathway. ubiquitin and some e2s are exceptions in that they can be used by both the uae and uba6 pathways. ufl1 and c20orf166 were recently identified and reported by tatsumi et al 139 . dub, deubiquitylating enzyme; nae, nedd8-activating enzyme; pp i , inorganic pyrophosphate; sae, sumo-activating enzyme. the 26s proteasome is a protease complex that degrades polyubiquitylated proteins. it is composed of two subcomplexes: a barrel-shaped 20s core particle containing the protease active sites and two 19s regulatory particles that cap the barrel and control access of substrates to the core. nuclear factor-κb (nf-κb) . a transcription factor with a key role in regulating the immune response. nf-κb is involved in cellular responses to stimuli, including stress, cytokines, free radicals, ultraviolet irradiation and bacterial or viral antigens. misregulation of nf-κb has been linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infection and improper immune development. reaction. the ubiquitin-charged e2 then binds an e3 ligase and its protein substrate to transfer ubiquitin to an acceptor lysine residue on the substrate. to mark substrates for degradation, multiple ubiquitins are similarly attached to produce a k48-linked polyubiquitin chain. on release of the substrate from the e3 ligase, the proteasome recognizes the polyubiquitin chain and the substrate is destroyed. alternatively, substrates marked by monoubiquitylation or by polyubiquitin chains using different ubiquitin lysine sites are involved in functions such as protein trafficking, signalling and enzyme activation 2, [9] [10] [11] . deubiquitylating enzymes (dubs) regulate the function of these various ubiquitin modifications and may, for example, rescue a substrate from degradation by removing a degradative ubiquitin signal or by changing or removing a nondegradative ubiquitin signal 12 . although all ubl conjugation pathways are catalysed by structurally and functionally related enzyme cascades, ubls participate in biologically distinct pathways (table 1) . ubiquitylation is by far the most thoroughly studied ubl pathway and is 'information rich' in that numerous downstream receptors recognize and process differentially ubiquitylated proteins. substrates that are polyubiquitylated with k48-linked ubiquitin chains are delivered to the 26s proteasome to be degraded into small peptides, with the ubiquitins released to be used again. proteins that are polyubiquitylated with k63linked ubiquitin chains are generally not degraded but are essential components of signalling pathways, for example, nuclear factor-κb (nf-κb)-dependent expression of inflammatory and immune genes. the function of ubiquitin chains that are linked through lysines 6, 11, 27, 29 and 33 (ref. 12) and of chains containing 'mixed' linkages is still emerging, although there is increasing evidence that non-k63 chains target proteins for degradation by the 26s proteasome 12 . with the exception of the proteasome (box 1; table 2) , the enzymes in the ups and ubl conjugation pathways represent unprecedented drug targets and target classes. indeed, significant advances in identifying small-molecule inhibitors of the ubl pathways have recently been achieved by targeting e1s, e2s, e3s, dubs and the proteasome itself. each ubl pathway enzyme class is mechanistically distinct. also, e1s are highly specific for their respective ubls and match individual ubls with respective e2s. e3s are mostly associated with the ubiquitin pathway and are primarily responsible for substrate specificity and in some cases directly facilitate ubl transfer 13 . similarly, dubs also demonstrate a high degree of substrate specificity 14 . therefore, there are a number of discrete e1-e2-e3 cascades that could provide opportunities to specifically target the aberrant signalling that is associated with particular diseases, and these are outlined below. there are eight structurally and functionally related e1 activating enzymes that act at the apex of each of the nine classes of ubl conjugation pathways 15 16 . ubl-amp then reacts with the e1 active-site thiol to form an e1~ubl thioester (~ denotes a high-energy bond). a second atp and ubl then bind the enzyme as in the first step to form a ternary complex that contains two ubl a domain comprising ~70 amino acids that possesses a tertiary structure resembling the ring finger domain. the major difference is that the u-box lacks the characteristic zinc-chelating cysteine and histidine residues of the ring finger. consequently, u-box e3s use intramolecular interactions other than zinc chelation to maintain the ring finger motif. a domain present in most e3s that is defined by the consensus sequence c-x 2 -c-x -c-x [1] [2] [3] -h-x [2] [3] -c-x 2 -c-x -c-x 2 -c (where x means any amino acid). the ring domain coordinates two zinc ions. molecules bound to the e1. this form of e1 is competent for transthiolation of the thioester-bound ubl to a pathway-specific e2 and is required for the downstream function of ubl conjugation. as the enzymatic mechanism of e1s suggests, there are multiple points at which a small molecule could potentially inhibit ubl activation (box 2) . for example, the inhibition of ubl-acyl adenylate formation could be achieved by preventing atp from binding to e1 using an atp-competitive small-molecule inhibitor similar to those now commonly used to inhibit kinases and other atp-dependent enzymes. alternatively, blocking e1~ubl thioester formation could be achieved by targeting the e1 active-site thiol. blocking transfer of an e1 thioester-bound ubl to an e2 using molecules that compete with e1-e2 binding is yet another potential mechanism of inhibition 17 . several strategies for inhibiting e1 function are now available and are beginning to demonstrate the feasibility of selective e1 targeting as a means to further understand ubl pathway biology . e2s are responsible for transferring ubls to substrate proteins and often function with a single or limited number of e3 ligases, although in some cases no e3 is required. in total, there are approximately 40 e2s, most of which associate with uae and are involved in ubiquitin conjugation. the other e1s typically associate with one or sometimes two e2s 15 (fig. 1; table 1 ). ubiquitin pathway e2s mediate ubiquitin chain assembly, determine ubiquitin-chain linkage topology and switch between chain initiation and elongation 18, 19 . together these factors determine the fate of ubiquitylated substrate proteins depending on whether they are mono-or polyubiquitylated and on the site(s) to which ubiquitin is conjugated. although several studies have linked e2s from different ubl pathways to cancer and other diseases, a fundamental question remains as to how the limited number of e2s pair with the much greater number of putative e3s. unfortunately, despite structural and biochemical analyses of e2-e3 interactions, it is not yet possible to predict which e3 functions with a specific e2 and, as potent and selective e2 inhibitors are yet to be reported, our knowledge about their functions in cells is limited [20] [21] [22] . despite these challenges, there are several emerging strategies that may yield inhibitors of e2s [20] [21] [22] and these are described in box 3 substrate selectivity of the ups relies primarily on the specificity of the hundreds of e3 ubiquitin-protein ligases in the human genome. e3s are represented by four main classes: hect domain e3s, u-box e3s 13 , monomeric ring finger e3s 23 and multisubunit e3 complexes that contain a ring finger protein (fig. 3) . these classes differ in their protein interaction domains for binding to e2s, as well as in other domains related to substrate binding. the ring finger and u-box domains have an adaptor function in bringing ubiquitin-loaded e2 and the substrate together to promote ubiquitylation, whereas hect e3s form a thioester intermediate with ubiquitin before transfer to a substrate. individual e3s within these classes are responsible for the recruitment of specific substrate proteins to be tagged with ubiquitin or, in some cases, another ubl. selective e3 inhibition may therefore enable direct targeting of an aberrant signalling pathway in cancer or other diseases. in spite of the tremendous experimental and bioinformatics efforts that have taken place in the past 15 years, the annotation of e3 ligases that are associated with human disease is still in its infancy. the ring finger ligases alone constitute one of the largest enzyme groups in the cell (exceeding the kinases). the molecular mechanisms of e3 function have been elucidated primarily from structural studies. identifying the substrate-binding surface or related sites mediating ubiquitylation activity, including sites of assembly for components of the e3 complexes, may facilitate the discovery of small-molecule inhibitors of e3s. indeed, recent reports of inhibitors of cullin-ring ligases (crls; of which scf is an archetype, see ref. 23 for review) represent significant advances. in one example, a chemical genetics screen in yeast for enhancers of rapamycin identified an inhibitor of scf met30 that directly bound to the f-box adaptor met30 and diminished its binding to the core cullin-ring complex in vivo. therefore, inhibition was achieved through the apparent failure to assemble a functional scf complex 24 . in principle, the inhibition of ubiquitin-like protein pathway enzymes would provide specificity for the individual pathways and substrate proteins targeted. by contrast, inhibition of the proteasome blocks the final common step of the degradative pathways and is therefore a relatively nonspecific target. nevertheless, the first clinically validated drug to target the ubiquitin-proteasome system (ups) was the proteasome inhibitor bortezomib (velcade; millennium pharmaceuticals), which was approved in 2003. owing to its success, several second-generation proteasome inhibitors are currently in development. each of the three types of proteasome active site -the caspase-like (β1), trypsin-like (β2) and chymotrypsin-like (β5) sites -use an amino terminal threonine as the catalytic amino-acid residue. the interactions of these active sites with various types of inhibitor are now known in atomic detail 98 . bortezomib, as well as the newer inhibitors that are under clinical evaluation, use different chemical 'warheads' 73 to covalently modify the n-terminal threonine residue in one or more of the three sites. the properties of these agents, including enzyme inhibition kinetics and pharmacological differences, are summarized in table 2. for the most part, these compounds inhibit the β5 sites of both the constitutive proteasome and the immunoproteasome isoforms. differences in enzyme kinetics may differentiate these proteasome inhibitors with regards to activity and safety; the irreversible nature of carfilzomib 99 , onx0912 (refs 100, 101) and npi-0052 (ref. 102 ), compared with the slowly reversible binding of bortezomib 103 and cep18870 (ref. 104 ), and the more rapidly reversible binding of mln9708 (ref. 105 ), might result in differences in tissue distribution 106 , and consequently in levels of ups inhibition within different tumour types. similarly, differences in specificity for the three enzymatic sites of the proteasome between these compounds 99,100,102-104,106 might also result in differences in activity in the different tumour types and in the safety of these proteasome inhibitors 105 . furthermore, a number of selective inhibitors of the chymotrypsin-like subunit of the immunoproteasome have been reported, including pr957 (ref. 107) and ipsi-001 (ref. 108 ). because of their selectivity, these compounds or similar inhibitors might have potential as therapeutic agents for autoimmune disorders. (crls). crls are a large family of multi-component e3s consisting of a core cullin protein bound to a ring finger protein (rbx1/2), and an interchangeable substrate-binding adaptor protein. there are seven cullins and ~600 adaptors in the human genome. modification of the cullin subunit by nedd8 is required for activation of crl e3 ligase activity. scf complexes are cullin ring ligases (crls) that catalyse the ubiquitylation of proteins targeted to the proteasome for degradation. scf core subunits include the structural protein cullin 1, the ring-finger protein rbx1/2 and the adaptor protein skp1. this core complex binds to one of the approximately 100 f-box proteins that are responsible for recruiting substrates. f-box proteins are named for the conserved 50 amino acid f-box domain that binds to skp1. all crls, including scfs, require nedd8 modification of the cullin subunit for ligase activity. in a second example, an inhibitor of scf cdc4 was identified in a biochemical screen that disrupted binding of the f-box protein cdc4 and its substrate, phosphorylated sic1. structural analysis showed that the compound bound cdc4 at a site 25 å from the sic1 binding site, resulting in a distortion of the substrate binding pocket and therefore achieving inhibition through an allosteric mechanism 25 . a third recent example demonstrates how thalidomide, a compound that was discovered over 50 years ago and was originally prescribed as a sedative until its use was discontinued, exerts its now notorious teratogenic effects. a search for primary targets of thalidomide identified cereblon (crbn) as a thalidomide-binding protein 26 . crbn forms a crl complex with cullin 4 and the adaptor dna damage-binding protein 1 (ddb1) that has e3 activity and is inhibited by thalidomide. interestingly, a mutant of crbn that does not bind thalidomide but retains e3 activity confers resistance to the teratogenic effects of thalidomide in animal models of embryonic development. this suggests that thalidomide exerts its teratogenic effects through crbn. together, these results not only shed light on the mechanism of thalidomide teratogenicity, but are also an example of the modulation of an e3 in a clinical setting using a small molecule (although with unintended and tragic consequences). thalidomide is currently used clinically under strict control to treat multiple myeloma and leprosy, although the molecular target(s) in either of these contexts are unknown. collectively, these results indicate the feasibility of obtaining selective inhibitors of at least one important class of e3. however, difficulties remain given the overall protein subunit arrangements combined with the need for small-molecule inhibitors (generally a molecular mass of less than 1,000 da) to bind and disrupt protein interfaces or otherwise allosterically affect ligase activity. the hect ligases may ultimately prove to be more amenable to small-molecule intervention as these enzymes form covalent thioester intermediates with ubiquitin before transfer to lysine residues of target proteins. table 3 provides several examples of e3s with disease implications along with inhibitors or other interventions that have been studied. dubs are proteases that perform three major functions in ubl conjugation (see ref. 12 for a review). first, as ubls are often translated as pro-proteins or as linear fusion proteins, dub activity is required to cleave the c termini of ubls and generate their mature forms. second, dubs can remove ubls from modified substrates, attenuating ubl signalling functions and recycling free ubls. third, in the case of ubiquitin, dubs have a polyubiquitin chain editing or 'proof reading' function. in mammals, there are nearly 100 dubs belonging to five different families, the majority of which are cysteine proteases; the remainder consists of a small number of zinc metalloproteases. historically, it has been problematic identifying drug-like inhibitors of cysteine proteases, whereas the general class of metalloproteases has been more amenable to inhibition using small-molecule drugs (box 4) . recently, a global proteomic analysis identified 774 candidate interacting proteins associated with 75 dubs, allowing placement of this class of protease uses a cysteine thiol group in its catalytic mechanism. deprotonation of the cysteine sulphydryl by an adjacent residue (usually histidine) is followed by nucleophilic attack on the peptide carbonyl carbon. a thioester linking the new c terminus to the cysteine thiol is an intermediate of the reaction. a class of protease for which the active sites include two histidine residues that coordinate a zinc ion. during catalysis, the zn 2+ promotes attack of the peptide carbonyl carbon by the oxygen atom of a water molecule at the active site. an active site base facilitates this reaction by extracting a proton from the attacking water molecule. previously unstudied dubs within cellular protein complexes as well as putative biological pathways 27 . however, analogous to the challenges mentioned above for e3s, it is unclear at present whether defining the dub interactome will reveal additional drug targets. nonetheless, the biological roles of dubs make them attractive targets for pharmaceutical intervention. the clinical utility of proteasome inhibitors and our increasing understanding of the enzyme mechanisms of ubl conjugation and deconjugation hint at the increasing likelihood of identifying drug-like, small-molecule inhibitors of these novel targets. ongoing research to elucidate the roles of specific enzyme components of the ups and ubl conjugation pathways has identified several potential targets in specific disease settings. in the next sections, we detail several examples in which therapeutic opportunities may exist. the degree to which diverse biological processes are regulated by the ups and ubl conjugation pathways is extraordinary. it is therefore not surprising that misregulation of these pathways is implicated in a growing number of diseases. oncology is an area for which targeting this system is particularly exciting and for which proteasome inhibition is a valid approach. here, we focus on additional examples in which ups and ubl pathway enzyme targets are tightly linked with specific pathologies. the complexity of protein ubiquitylation in signal transduction is illustrated by the fundamental role of ubiquitin in the activation of the transcription factor nf-κb, which has a key role in inflammation and immunity 28, 29 . there are differences in the activation of nf-κb by different ligands, that is, binding to receptors for tumour necrosis factor (tnf), interleukin-1 (il-1) and t cell receptor, which include the use of linear and k63-linked ubiquitin chains. the study of the multiple roles of ubiquitin in the enzymatic cascade from receptor-ligand binding to the activation of the expression of nf-κb-dependent genes is complicated by several issues (fig. 5) . first, multiple enzymes catalyse ubiquitylation at the same target lysine in a protein. for example, the a20 (also known as tnfα-induced protein 3 or tnfaip3) and itch ligases trigger ubiquitylation and degradation of the rip kinase, a vital component of the ligand-dependent activation of nf-κb. second, the lability of ubiquitylated species is a problem due, for example, to regulatory deubiquitylation. third, there are different types of polyubiquitin chain formation at single ubiquitylation sites (lysine residues), such as k63-linked or linear chains, and some proteins also have multiple ubiquitylation sites. fourth, there are experimental difficulties in studying ubiquitylation reactions, such as the low abundance of ubiquitylated proteins and the need to experimentally use mutated ubiquitins with non-physiological conformational changes 29 . the active-site thiol that is present in e1s is essential for ubiquitin-like protein (ubl) activation and offers a potential opportunity for inhibition using small molecules (fig. 2a) . pyr41 is a pyrazone derivative identified in an in vitro high-throughput screen for inhibitors of hdm2-dependent p53 ubiquitylation. characterization of the inhibitor revealed that the nitrogen dioxide group on the furan ring of pyr41 covalently modified the ubiquitin-activating enzyme (uae) active site cysteine 109 . importantly, the compound did not demonstrate inhibitory activity against other thiol-dependent enzymes, including several e2s 109 . the effects in cells treated with pyr41 included inhibition of cytokine-induced nuclear factor-κb (nf-κb) activation, and stabilization of p53 coupled with the induction of p53-dependent transcription 109 . the related dioxopyrazolidine, pyzd4409, showed similar uae inhibitory properties in vitro and also demonstrated antileukaemic activity in a mouse cancer model 110 . the potential therapeutic value of targeting uae is further underscored by the unexpected finding that the nitric oxide (no)-producing prodrug js-k inhibited uae~ubiquitin thioester formation (~ denotes a high-energy bond) through an interaction between no and the uae active-site thiol 111 . consistent with other uae inhibitors, the downstream effects of js-k treatment included decreased levels of total ubiquitylated proteins and increased p53 expression. acyl-adenylate analogues of ubl-amp are known to be potent and selective e1 inhibitors but are not suitably drug-like for delivery to intracellular targets. a new approach using small molecules to form inhibitory ubl-amp mimetics in situ overcomes this limitation. inhibition of the nedd8 pathway was recently demonstrated using the small-molecule nedd8-activating enzyme (nae) inhibitor, mln4924 (refs 112, 113) . mln4924 is an adenosine sulphamate analogue that binds to the nucleotide-binding pocket of the nedd8 thioester form of nae and forms a covalent adduct with nedd8 by a mechanism referred to as 'substrate-assisted inhibition' (fig. 2b) . because nedd8 conjugation of cullin proteins is required for the ubiquitin ligase activity of the cullin-ring finger ligases (crls) 23,112,114,115, (fig. 3) , blocking nae results in the inhibition of crl activity and the stabilization of crl substrates, some of which are important for cancer cell growth and survival (table 3) . for example, nae inhibition results in the stabilization of cdt1, a substrate of the crl1 skp2 and crl4-ddb1 cdt2 ligases, leading to dna re-replication and apoptosis in proliferating cells 112 . studies using nf-κb-dependent human cancer models have demonstrated increased levels of the crl1β trcp substrate piκbα and inhibition of nf-κb activity and apoptosis 116, 117 , suggesting the feasibility of nae inhibition for the treatment of disease that is associated with constitutively active nf-κb signalling 112 the recent discovery of the importance of linear ubiquitin chains in nf-κb activation extends the complexity of the regulation of the system. a linear ubiquitin chain-assembly complex (lubac) conjugates head-totail-linked linear polyubiquitin chains to target proteins. lubac is involved in the physiological regulation of the canonical nf-κb pathway by binding to nemo (nf-κb essential modulator; also known as ikkγ) and in the conjugation of linear polyubiquitin chains on to specific lysine residues of nemo 30 . the recruitment of lubac to the tnf receptor 1 (tnf-r1) signalling complex (tnf-rsc) is stimulation-dependent and requires tradd, traf2 and cellular inhibitors of apoptosis (iaps), but not nemo or rip1. it seems that lubac is recruited to the tnf-r1 by cellular iap-generated ubiquitin chains and generates linear ubiquitin chains to increase the efficiency of nf-κb and jun n-terminal kinase activation. activation of nf-κb increases tnfdependent gene expression, whereas activation of jun n-terminal kinase inhibits tnf-mediated apoptosis. the attraction of nemo to the tnf-rsc is increased by lubac, and the enzymatic activity of lubac seems to stabilize the tnf-rsc 31 . this biochemical mechanism is facilitated by the fact that the uban (ubiquitin binding in nemo) motif of nemo selectively binds linear ubiquitin chains. the specific amino-acid residues of nemo that are involved in binding linear ubiquitin chains have been reported to be mutated in humans and result in x-linked ectodermal dysplasia and immunodeficiency 32 . the binding of polyubiquitin chains to nemo is interesting in that the c-terminal zinc finger of nemo can bind ubiquitin as well as uban. although neither uban nor the zinc finger show any preference for k63-linked chains, together the domains form a high-affinity k63-specific ubiquitin-binding domain. this suggests that the main function of the c-terminal half of nemo is to specifically bind k63-linked polyubiquitin chains. the binding of nemo to linear polyubiquitin chains is dependent on the uban alone and does not require the presence of the zinc finger 33 . furthermore, unanchored k63-linked polyubiquitin chains can directly activate tak1 and ikk kinases. this indicates a new role for free ubiquitin chains in activating kinases. cleavage of the unanchored k63-linked ubiquitin chains prevents tak1 and ikk activation 34 . further research may show that unanchored ubiquitin chains are generated as 'second messages' , as with camp. whatever the mechanism(s), protein ubiquitylation/ deubiquitylation is at the heart of the inflammatory and immune responses, together with protein phosphorylation/dephosphorylation. therefore, ubiquitylation should be amenable to therapeutic intervention for inflammatory and immune disorders, but at what level? although specific e3s such as cellular iaps are obvious targets, the e2s that are involved in k63 ubiquitin chain synthesis (for example, ubc13/uev1a or ubch5) or in linear chain assembly (for example, ubch5, e2-25 kda and ubch7), might be targeted with the provision that multiple signalling pathways may be compromised by inhibitors of these enzymes. it seems that e3s use different e2s to catalyse distinct types of ubiquitin tagging. for example, ring finger iaps seem to bind to ubch5a, ubch5c, ubc7, ubc8 and ubc13/uev1a 35 . it is also generally thought that, although e3s are the ultimate arbiters of substrate selection for ubiquitylation, the e2s determine what type of polymeric ubiquitin chain will be formed. it remains to be seen how e3s select appropriate e2s for customized polymeric ubiquitin chain assembly on specific target proteins. in addition, the e3s are key regulators of immune functions. several e3s, including c-cbl, cbl-b, grail, itch and nedd4, have been shown to negatively regulate t cell activation by contributing to t cell apoptosis. however, the hect ligase aip2 positively regulates t cell activation by enhancing t cell proliferation and il-2 production to suppress apoptosis. aip2 interacts with and promotes ubiquitin-mediated degradation of egr2, a zinc finger transcription factor, to suppress egr2-mediated fasl expression and activation-induced death of t cells 36 . careful selection of the appropriate e3 target for inhibition might ameliorate or block t cell activities. the taxing biological issue with disease-related consequences is what aspect of nf-κb signalling should be clinically interrupted. for example, normal nf-κb functions are essential for the immune response against microbial infections, but continuous nf-κb activation can lead to inflammation and cancer. for this reason, a | hect domain e3s use a unique mechanism in which ubiquitin (ub) is transferred from an e2 to a conserved cysteine residue within the e3 via transthiolation and is then transferred from the e3 to a substrate amino group. all other types of e3 facilitate transfer of ub from a charged e2 directly to a substrate. b | the ring finger motif comprises a zn 2+ binding domain that is required for e2 binding. the u-box also binds e2s and is structurally similar to the ring motif, but does not bind metal ions. monomeric ring finger e3s and u-box e3s bind to both the substrate and the e2. in multimeric ring finger complexes, the ring finger protein binds the e2 while other proteins in the complex bind the substrate. these multimeric complexes include the anaphase promoting complex/cyclosome and cullin-ring ligases (crls). a unique aspect of crls is the requirement for modification of the cullin subunit by nedd8 for ubiquitin ligase activity . rbx, ring-box protein. tight negative regulation of nf-κb activity occurs through mechanisms involving dubs. the first dub that was shown to be involved in the nf-κb system was cyld, a tumour suppressor involved in a neoplasm called cylindromatosis. the enzyme has a great specificity for k63-linked ubiquitins 37 and the catalytic site involved in the cleavage of k63-linked chains is often mutated in this cancer. additionally, cyld (like isopeptidase t) preferentially cleaves unanchored k63linked polyubiquitin chains and therefore supports the role of these chains in protein kinase activation in the nf-κb pathway 34 . another dub with a key role in the negative regulation of the nf-κb response is a20. this enzyme has dual functions in removing k63-linked chains from rip1 and traf6, and uses its ubiquitin ligase activity to attach k48-linked chains to rip1 for proteasomal degradation 38 . the inhibition of cyld and/or a20 could enhance the inflammatory or immune response, but the prolonged consequences are unclear and await assessment in animal models and in early clinical trials. alternatively, prevention of tnf-dependent nf-κb activation by linear chains through the inhibition of one or more of the e2s (ubch5, e2-25kda or ubch7) may suppress nf-κb activity and inflammatory and immune responses -but again, with what long-term clinical price? for context, the use of steroids for the long-term suppression of various inflammatory conditions is routine, with generally acceptable side effects. there might be multiple mechanisms to normally ensure the termination of nf-κb activation. however, it is clear that the intracellular ubiquitin-editing protein a20 has a key role in the negative feedback regulation of nf-κb signalling (through tnf and toll-like receptors (tlrs)) in response to multiple stimuli. the importance of a20 in the negative regulation of nf-κb signalling through tlrs is illustrated by recent findings that a20 disrupts the ubiquitin ligase activities of traf6, traf2 and ciap1 by preventing interactions with the e2s ubc13 and ubch5c and with another regulatory protein, tax1bp1, to trigger the a20dependent ubiquitylation of the e2s and their proteasomal degradation 39 . recent genetic studies demonstrate that several mutations at the human a20 locus relate to immunopathologies such as crohn's disease, rheumatoid arthritis, systemic lupus erythematosus, psoriasis and type 1 diabetes. the gene encoding a20 seems to be a susceptibility gene for many different immune disorders 40 . finally, and a caveat to any inhibition of a20, is the role of a20 as a tumour suppressor 41 . acute transient inhibition of a20, by promoting nf-κb activation, may be useful in resolving infections, but chronic inhibition may promote cancer. during the innate immune response to some microorganisms, the binding of lipopolysaccharide to the tlr receptor complex tlr4-md2-cd14 leads to the recruitment of the adaptors mal, myd88, traf6 and several irak kinases. traf6 is activated by irak to cause the attachment of k63 chains to both traf6 and irak. these chains are recognized by tab2 and nemo, leading to tak1-mediated phosphorylation and activation of the ikk kinase complex. the a20 dub activity is directed at traf6 to downregulate nf-κb activities 40 . it is possible that inhibitors of the dub activity of a20 may enhance antimicrobial activity in response to intracellular bacterial infections, such as salmonella, listeria and typhimurium. there are further consequences of manipulating the nf-κb system other than the regulation of inflammatory and immune responses. the system is intricately linked to cell death. for example, internalization of the tnfr1 complex can give rise to further complexes in the cytosol that are dependent on the tradd component of the tnfr1 complex (to give complex iia) or rip1 (to give complex iib). complex iia formation involves the recruitment of the protein fas-associated death domain (fadd) and caspase 8 to trigger apoptosis, whereas complex iib is formed when the fadd pathway is blocked by, for example, viral proteins or chemical inhibitors of apoptosis, and mediates cell death by several studies have linked e2s from different ubiquitin-like protein (ubl) pathways to cancer and other diseases, including ube2q2 in head and neck squamous cell carcinoma 119 ; the sumo e2 ubc9 in ovarian carcinoma, melanoma, lung adenocarcinoma and breast cancer [120] [121] [122] ; ube2t in lung cancer 123 ; and ubch10 in chromosomal instability and tumour formation 124, 125 . in all cases, an e2 must engage its respective e1 to be charged with a ubl through transthiolation. in the ubiquitin pathway, e2s usually require an e3 for substrate ubiquitylation and therefore must also bind to an e3-substrate complex. for conjugation to a protein substrate to occur, a ubl is transferred from the e2 thiol active site to the amino group of a substrate acceptor lysine residue, which must be positioned for nucleophilic attack on the carbonyl group in the e2-ubl thioester bond (fig. 4) . each of these e2 interactions offers potential opportunities for selective inhibition. for example, a synthetic peptide corresponding to the 26-residue amino terminus extension of ubc12 (called ubc12n26) has been shown to compete for binding of ubc12 to the nedd8-activating enzyme (nae), thereby blocking transfer of nedd8 to downstream targets 17 . alternatively, recent published findings indicate that e2-e3 recognition, although necessary, is not sufficient for ubiquitin transfer 126 . in one example, the g2br domain of the ring finger e3 gp78 binds ube2g2 with high affinity and allosterically activates ubiquitylation activity on formation of the e2-e3 complex 127 . targeting allosteric activation of an e2 would prevent subsequent ubiquitylation of substrate proteins, thereby blocking a specific ubl pathway. finally, a conserved asparagine residue (exemplified by asparagine 79 in ubc13) in e2s has been shown to be crucial for ubl transfer and is thought to form the oxyanion hole required to stabilize the e2 thioester-substrate transition state intermediate 128 . targeting this site with a small-molecule effector may offer yet another approach for inhibition. literally means 'self-eating'; a highly regulated catabolic process in which cellular proteins and organelles are sequestered in a characteristic double-membrane vesicle called an autophagosome and are then degraded following vesicular fusion with a lysosome. endosomes are membranebound vesicles that are involved in protein transport between the plasma membrane, golgi and lysosomes. in the endocytic pathway, internalized molecules are delivered to early endosomes, where efficient sorting occurs. some molecules, including recycling receptors, are shunted back to the plasma membrane to be reused. others, including downregulated receptors, are transported to late endosomes and lysosomes for degradation. necroptosis. in this pathway, complex iib contains both rip1 and rip3, which by mutual co-phosphorylation can activate enzymes involved in metabolism. activated rip3 increases the activities of enzymes involved in glycolysis and mitochondrial oxidative phosphorylation and, in some cases, can cause the production of reactive oxygen species to mediate necrotic cell death. it is assumed that complex iib contains deubiquitylated rip caused by cyld and a20. necroptotic cell death may therefore be compromised by inhibition of these dubs, for example, in virally infected cells, although complex iia-dependent apoptotic cell death may still operate 42, 43 . again, to show the complexity of these cellular responses to various stresses, the necrostatin inhibitors of necroptosis 44 can reduce tissue damage in brain ischaemia and myocardial infarction 45 . therefore, it is possible that cyld and a20 inhibitors may prevent tissue necrosis in appropriate clinical indications, such as following stroke and heart attack, for which it is currently difficult to limit tissue damage. bone disorders. the roles of nf-κb in inflammation and the immune response, and therefore the usefulness of ubiquitin pathway inhibitors, could be extended to osteolytic disorders. osteoclasts are responsible for bone resorption and have a pivotal role in the pathogenesis of osteolytic disorders. nf-κb signalling pathways are strictly regulated to maintain bone homeostasis through cytokines, such as rankl, tnf-α and il-1, which differentially regulate classical and/or alternative nf-κb pathways in osteoclastic cells. abnormal activation of nf-κb signalling in osteoclasts has been associated with excessive osteoclastic activity, and is frequently observed in osteolytic conditions, including periprosthetic osteolysis, arthritis, paget's disease of bone and periodontitis. nf-κb modulators such as parthenolide and nemobinding domain peptide demonstrate therapeutic effects on inflammation-induced bone destruction in mouse models 46 . therapeutic intervention in the activities of the ups may slow disease progression of osteolytic disorders that cause debilitating disease in the ageing population, for example, paget's disease of bone. the ups and heart disease. the role of the ups in cardiac function and disease is again complex and sometimes conflicting. proteasome inhibitors have been used in the experimental manipulation of heart disease. the outcomes so far indicate, as might be expected, that the effects of proteasome inhibitors are concentration dependent, with lower concentrations mediating cytoprotective effects and higher concentrations being toxic. for example, studies on non-toxic proteasome inhibition in neonatal rat cardiac myocytes show upregulation of antioxidative enzymes, which confers cardioprotection. this upregulation is a response to mild proteasome inhibition by antioxidative transcription factor nf-e2-related factor 2 (nrf2)-dependent transcriptional activation of an antioxidant response element (are) in the superoxide dismutase 1 (sod1) promoter. the induction of antioxidative enzymes and cytoprotection was evident in cardiomyocytes from wild-type mice, but was completely abolished in cells from nrf2-knockout animals 47 . more understanding of the ups in the pathophysiology of heart disease is needed to contemplate new routes for therapy. it seems that pressure overload, ischaemic heart disease or genetic mutations in contractile proteins that cause heart failure are accompanied by elevation in levels of misfolded proteins, which may be removed by the ups and through autophagy. however, proteasome inhibitors may be useful for the treatment of cardiac hypertrophy and ischaemic heart diseases 48 . a complication is that impaired proteasome function is commonly associated with myocardial ischaemic injury, but recent evidence also supports a cardioprotective role for proteasome inhibitors in myocardial ischaemia 49 . the loss of cardiomyocytes is a key problem in the development of cardiovascular disease. two main processes mediate cardiomyocyte loss: necrosis and apoptosis. in contrast to necrosis, apoptosis is a relatively well-understood, regulated process that is essential in normal development and tissue homeostasis. tight regulation of both processes is crucial, especially in post-mitotic cells lacking regenerative capacity, such as cardiomyocytes and neurons. the ups is involved in the regulation of cardiomyocyte apoptosis 50 and possibly necrosis. as in cerebrovascular accidents in the brain, two different processes might be controlled by the proteasome: upregulation of the expression of gene products that ameliorate oxidative damage in surviving cardiomyocytes by proteasome inhibition, and the activation of cardiomyocyte death pathways. the clinical outcome will be dependent on the balance between these proteasome (and general ups) activities. caution is needed clinically, as cardiotoxicity has been reported in patients treated with the proteasome inhibitor bortezomib 51 . in spite of these complexities, consideration should be given to the use of proteasome inhibitors and other modulators of the ups for viral myocarditis. the primary intracellular protein degradation systems, both the ups and autophagy, seem to regulate successive stages of viral infectivity. viral myocarditis, such as that caused by coxsackievirus b3, progresses in three distinct stages: acute viral infection, immune cell infiltration and cardiac remodelling. the ups has a central role in each of these stages of viral infection and might be modulated to slow viral disease progression and heart disease 52 . chronic neurodegenerative disease. as expected, ubiquitin-dependent processes, including the degradation of ubiquitylated proteins by the 26s proteasome, by autophagy and in the endosome-lysosome pathway, have central roles in neuronal development, homeostasis and disease. these catabolic systems are essential for neuronal activities, including synaptogenesis, cellcell interactions (for example, neuromuscular junction formation 53 ) and synaptic plasticity 54 . in the adult central and peripheral nervous systems, which are based predominately on non-dividing cells, protein ubiquitylation and deubiquitylation are essential for neuronal survival. most age-related chronic neurodegenerative diseases involve the accumulation of proteins in aggregates, frequently as paranuclear inclusions (fig. 6) . in every case, proteins within the inclusions are ubiquitylated. a | e2s engage in a sequence of highly specific interactions to faithfully transfer a ubiquitin-like protein (ubl) to a substrate. e2s first bind their respective e1 to receive an activated ubl through a transthiolation reaction. in most cases, the e2-ubl thioester then binds a specific e3 or e3-substrate complex. during catalysis to protein substrates, the ubl is transferred from the e2 thiol active site to the amino group of a substrate acceptor lysine residue that is positioned for nucleophilic attack on the e2-ubl thioester bond. each of these e2 interactions offers potential opportunities for selective inhibition as illustrated by the following examples. b | targeting e2~ubl thioester formation by blocking e1-e2 protein-protein interaction. a synthetic peptide called ubc12n26 corresponds to the 26-residue amino terminus extension of ubc12 that specifically binds the nedd8-activiating enzyme (nae). ubc12n26 competes for binding of ubc12 to nae, thereby blocking transthiolation of nedd8 to ubc12 and inhibiting downstream nedd8-dependent events. c | targeting e3-dependent e2 allosteric activation. in some cases, e2-e3 binding is necessary but not sufficient for optimal ubl transfer to a substrate. for example, the e3 gp78 binds the e2, ube2g2, through a ring finger and a second domain called g2br that binds the backside of ube2g2, which is opposite from the catalytic cysteine and distal from the ring binding surface. blocking g2br interaction with ube2g2 would inhibit allosteric activation of the e2 and prevent subsequent ubiquitylation of substrate proteins. d | targeting catalysis of ubl transfer. a conserved asparagine residue in e2s (exemplified by asparagine 79 in ubc13) has been shown to be crucial for ubl transfer and is thought to form the oxyanion hole required to stabilize the e2 thioester-substrate transition state intermediate. targeting lewy bodies are abnormal protein aggregates that develop inside nerve cells in parkinson's disease and alzheimer's disease and some other disorders. they are identified when histology is performed on the brain and appear as spherical masses that displace other cell components. the reasons are incompletely understood but seem to be the result of malfunction or overwhelming of the activities of neuronal 26s proteasomes, autophagy and the endosome-lysosome pathway. this is shown by experimental genetic findings. parkin encodes an e3 that ubiquitylates itself and the α-synuclein interacting protein, synphilin 1. familial associated mutations in park2/parkin that are defective for e3 activity are linked to autosomal recessive parkinson's disease 55, 56 . regional ablation of neuronal 26s proteasomes in the brain by deletion of a 26s proteasomal atpase gene causes the neuropathological hallmarks of parkinson's disease and dementia with lewy bodies 57 . proteasomal dysfunction may be the primary cause of neurodegenerative disease 58 . genetic deletion of autophagy genes in the brain causes neurodegeneration with the accumulation of deposits of ubiquitylated proteins, although not with the human hallmark neuropathology 59, 60 . the degradation of ubiquitylated proteins by the 26s proteasome and autophagy is essential for neuronal homeostasis. when either or both protein-catabolic processes are compromised, neurodegeneration ensues 61, 62 . the normal functioning of the endocytic and multivesicular body (mvb) pathway for receptor internalization and degradation is also essential for neuronal homeostasis, as it seems to contribute to normal autophagic activity. mutations in the endosomal sorting complex required for transport (escrt)-iii subunit chmp2b are associated with frontotemporal dementia and amyotrophic lateral sclerosis. efficient autophagic protein degradation requires functional mvbs. the escrt machinery delivers ubiquitylated proteins into invaginations of endosome membranes. the escrt machinery then mediates the breaking off of cargo-containing intraluminal vesicles from the perimeter membrane to form mvbs, which may fuse with lysosomes to cause degradation of their protein content. defects in this pathway would explain the observed neurodegenerative phenotype seen in patients with chmp2b mutations 63, 64 . to extend the cell biological context of neurodegeneration further, it is apparent that the unfolded protein response (upr) in the endoplasmic reticulum (er) is connected to autophagy through the key upr transcription factor xbp1. genetic xbp1 deficiency causes a significant decrease in the toxicity of mutant sod1 aggregates (which cause amyotrophic lateral sclerosis) due to an enhanced clearance by autophagy. these data indicate extensive cross-talk between the er-associated upr and autophagy to provide protection against neurodegeneration 65 . the malfunction of the upr in the er probably triggers autophagy to remove distended er from the neuron. finally, chain-specific protein ubiquitylation has a role in neurodegeneration as k63-linked polyubiquitylation has been detected within pathological lesions of the brains of patients with alzheimer's, huntington's and parkinson's disease. immunoreactivity to k63 chains is also a feature of inclusions in neurons of proteasomedepleted mice, suggesting a proteasomal contribution to the degradation of k63-linked polyubiquitylated proteins in vivo or that k63 polyubiquitylation has a role in inclusion biogenesis 66 . chronic neurodegenerative diseases, including alzheimer's disease, parkinson's disease, dementia with lewy bodies and amyotrophic lateral sclerosis, are considered to be 'proteinopathies' associated with the intraneuronal accumulation of insoluble protein aggregates in surviving neurons (inclusions) and extracellular amyloid deposits. in alzheimer's disease, the aggregates contain intraneuronal tau protein and extraneuronal enzymes that reverse the action of the ubiquitin conjugation cascade, the deubiquitylating enzymes (dubs), are attractive targets for drug discovery because of the various ubiquitin-mediated biological processes that they regulate 14, 27, 129, 130 . in mammals, there are nearly 100 dubs belonging to five different families. four of these families -the ubiquitin c-terminal hydrolases (uchs), ubiquitin-specific proteases (usps), ovarian tumour domain proteins and machado-joseph disease protein -are all papain-like cysteine peptidases. many potent inhibitors exist for enzymes of this general mechanism, but historically there has been limited success in turning these leads into drugs. nevertheless, a desire to clear this hurdle persists because of the many biological roles that dubs have. individual cysteine peptidase dubs have been associated with pathways of importance in specific diseases, such as usp1 with the fanconi anaemia dna repair pathway 131 ; usp6 with aneurysmal bone cysts and with a wider role in mesenchymal tumours 14 ; and usp7 with non-small cell lung adenocarcinoma 14, 132 . loss of the deubiquitylating activity of the dub cyld is associated with cylindromatosis 133 , and uch-l1 has been associated with parkinson's disease 134 . despite the difficulty so far in turning these leads into drugs, there are glimmers of hope. for example, a non-covalent inhibitor of the papain-like protease from the sars coronavirus, which acts as a dub for the virus, has been shown to block virus replication; importantly, this compound demonstrates specificity, inhibiting the pathogenic dub but not host dubs 2,135-137 . the fifth family of dubs, the jamm (jab1/mpn domain-associated metalloisopeptidase) domain proteins, are zinc metalloisopeptidases 14 . historically, the general class of metalloproteases has been more amenable to small-molecule drugs. several members of the jamm class, including poh1, csn5, amsh and brcc36, might make good drug targets. poh1 functions as part of the 19s cap of the 26s proteasome to trim polyubiquitin chains from substrates that are destined for degradation. targeting this enzyme could yield new chemical classes of proteasome inhibitor (box 1) . csn5 is a subunit of the cop9 signalosome, and it functions to regulate nedd8 attachment to cullins, thereby regulating cullin-ring ligase activity and stability. the biological consequences of csn5 inhibition might be similar to the inhibition of nedd8-activating enzyme (box 2) . amsh is associated with the endosomal sorting complex required for transport, where its activity regulates trafficking of receptor tyrosine kinases and g protein-coupled receptors 138 . finally, brcc36 is part of two complexes, brca1 a and brisc, and has a role in the dna damage response. in parkinson's disease and dementia with lewy bodies, there are intraneuronal deposits of α-synuclein. however, the seminal feature of all these diseases is regional extensive neuronal loss in the brain and spinal cord. it has been difficult to recapitulate these features of the diseases using mouse transgenesis to overexpress amyloidogenic proteins in the brain 67, 68 . these approaches have led to intraneuronal aggregates and extraneuronal deposits but not extensive neurodegeneration. by contrast, gene targeting to knock out a proteasomal gene and autophagy genes has resulted in the accumulation of intraneuronal inclusions containing ubiquitylated proteins (some resembling human disease) and extensive regional neuronal loss or neurodegeneration. currently, it seems that age-related chronic neurodegenerative disease can be directly attributed to some dysfunction of the ups and/or autophagy. any malfunction of these key neuronal processes for regulated removal of proteins results in molecular neuropathological features of neurodegenerative disease. promotion of the degradation of ubiquitylated proteins by 26s proteasomes (for example, with 26s proteasome activators such as inhibitors of the proteasomal deubiquitylating enzyme usp14 (ref. 69)) or by autophagy (for example, with mtor (mammalian target of rapamycin) inhibitors 70 ) would facilitate slowing or prevention of chronic neurodegenerative disease. the regional genetic ablation of a proteasomal atpase gene causes a reduction in neuronal 26s proteasomes, which is a neuropathological feature of both parkinson's disease and dementia with lewy bodies, and extensive neuronal death accompanied by some features of neuronal apoptosis. the characterization of the neuronal signal transduction pathways that control cell death in these models by approaches such as analyses of gene expression changes by microarray hybridization and proteomics will delineate these pathways and the downstream consequences of changes in these signalling pathways. therefore, further studies of the consequences of genetic deletion of neuronal 26s proteasomes (and autophagic functions) will identify receptor and enzyme targets for novel therapeutic developments to slow neuronal death and neurodegeneration. an example of a novel signalling target is the discovery that mutations in optineurin cause amyotrophic lateral sclerosis 71 . optineurin antagonizes the activity of nemo by similarly binding to ubiquitin chains. there is a feedback loop with nf-κb increasing although it is tempting to represent a general consensus, some enzymological steps are controversial due, for example, to enzymological redundancy. the role of ubiquitin (ub) in signalling to nf-κb needs considerably more investigation. see the main text for more details. dub, deubiquitylating enzyme; lubac, linear ubiquitin chain-assembly complex. 72 . because optineurin mutations prevent this inhibition it is possible that drugs that inhibit nf-κb activity may be useful in the treatment of this debilitating and essentially untreatable disease 71 . brain tumours. brain tumours (including aggressive paediatric tumours) are essentially intractable to current therapies without major side effects (for example, developmental disorders). there is therefore a particular need for novel therapeutic advances in this area. as it is clear that the regional targeted ablation of a neuronal proteasomal atpase gene causes a depletion of neuronal 26s proteasomes and extensive neuronal loss, it is worth considering intervention in the ups in brain tumours. there are nearly 100 types of brain tumour, many of which develop from glial cells or astrocytes. anaplastic astrocytoma (also called grade 3 astrocytoma) and glioblastoma multiforme (or grade 4 astrocytoma) are the most common brain tumours in adults and are often found in children. about half of all primary brain tumours are gliomas (astrocytomas, ependymomas and oligodendrogliomas). current treatments include surgery, radiotherapy and chemotherapy, all with some degree of success but with considerable side effects. given that deletion of a proteasomal atpase gene causes neuronal death 57 , inhibitors of proteasomal atpases (which belong to the aaa (atpases of alternative activities) superfamily) might be useful for the treatment of brain neuronal tumours and, as all cells contain 26s proteasomes, glial tumours and other tumour cell types too. such an approach offers a new 26s proteasome target that is independent of inhibiting proteasomal catalytic activities, for which there is already evidence of drug resistance 73 . the anticancer potential of proteasomal atpase inhibitors might be increased if other cellular atp-dependent proteases are inhibited. the blood-brain barrier could represent a delivery challenge, but drug-coated 'wafers' incorporating proteasomal atpase inhibitors might offer neurosurgeons a powerful adjunct to existing drugs for localized chemotherapy 74 . such proteasomal atpase inhibitors might also be useful for treating other types of tumour. viruses and bacteria use several strategies to block the ups and to manipulate specific aspects of the system for infection and replication. as exemplified by the classical example of human papillomavirus encoded e6 oncoprotein, which binds and redirects the e3 ligase e6-ap to target p53 to the proteasome 75 , pathogens can be adept at manipulating hostencoded enzymes. alternatively, pathogen-encoded enzymes that are specific for ubiquitin or ubls, such as proteases, seem to be a common feature shared by many viruses, bacteria and protozoa. for example, pathogens can code for orthologues of enzymes of the ups, such as an e2 (ref. 76 ), or express their own novel analogues of ups enzymes, such as ubiquitin protein ligases 77 . some of the proteases share a common origin with mammalian cell-encoded enzymes, but most of them have ancient intrinsic functions, such as processing pathogen protein components, and may have acquired the specificity for ubiquitin or ubls by interacting with mammalian hosts (and their immune system) throughout evolution. many of the pathogen-encoded proteases are different from their mammalian counterparts and are therefore attractive targets for drug development to combat infectious diseases 78 . one example of viral manipulation of the ups is provided by the ebola zaire virus vp35 protein. this viral protein interacts with the transcription factor irf7, which is required for interferon gene expression, and also interacts with the sumo e2 enzyme ubc9 and the e3 ligase pias1. this interaction increases pias1-mediated sumoylation of irf7, which represses expression of the interferon gene. by contrast, vp35 does not interfere with the activation of nf-κb, which is required for the induction of many pro-inflammatory cytokines that are needed for viral neuronal proteins are normally degraded by the activities of the ubiquitin-proteasome system (ups), chaperone-mediated autophagy (cma) and macro-autophagy (collectively referred to as autophagy), and the endosome-lysosome pathway. unfolded proteins, proteins altered by mutation or post-translational modifications and proteins that are damaged (for example, by oxidative stress, irradiation or toxins) are recognized by molecular chaperones and delivered to the ups and autophagy pathways. age-related and chronic neurodegenerative diseases, including alzheimer's disease, parkinson's disease, dementia with lewy bodies and amyotrophic lateral sclerosis, are 'proteinopathies' associated with the intraneuronal accumulation of insoluble protein aggregates resulting from the malfunction of the neuronal ups and/or autophagy pathways (see main text for details). the mechanisms of how neurodegeneration results from abnormal protein accumulation due to impaired function of the ups and autophagy pathways are not known. infection. these sumo-related events are part of the mechanism that causes rapid overwhelming infection and eventually pathology such as septic shock 79 . a second example is provided by the hiv virus, which uses mvb proteins to egress from the cell 80 and capitalizes on different stages of the autophagic process to mature. early, non-degradative stages of autophagy increase hiv yield. hiv gag-derived proteins co-localize and interact with the autophagy factor lc3 to cause autophagypromoted productive gag processing. later, the hiv protein nef acts as an anti-autophagic maturation factor through interactions with the autophagy regulatory protein beclin 1, thereby protecting hiv from degradation 81 . the isg15 protein was one of the first gene products shown to function as an innate immune protein with broad-spectrum antiviral activity. protein ubiquitylation might be productive for some rna viruses but isgylation is antiviral. a better understanding of the antiviral activities of isg15 could provide useful insight into the development of novel therapeutic approaches to improve the immune response against such pathogens 82 . observations of intracellular bacteria, protein ubiquitylation and autophagy have revealed that autophagy of ubiquitylated proteins requires the p62 protein (also known as sqstm1), which is an adaptor protein with a c-terminal ubiquitin-associated domain for binding to ubiquitylated proteins and an lc3 interaction region for binding the autophagosome protein lc3. ubiquitylated proteins on the surface of the intracellular bacterium salmonella enterica serovar typhimurium recruit p62 and cause the autophagic engulfment of the bacteria. this work demonstrates that the detection of bacteria (and almost certainly of misfolded proteins) occurs by a conserved pathway and that p62 has a role in innate immunity 83 . additionally, the ndp52 protein, not previously known to contribute to innate immunity, recognizes ubiquitincoated s. typhimurium in human cells (in a similar way to p62) and binds the adaptor proteins nap1 and sintbad to recruit tank-binding kinase 1, a non-canonical member of the ikk kinase family of enzymes. the ndp52 protein also recruits lc3 to activate autophagy against bacteria attempting to colonize the cytoplasm 84 . bacteria have evolved mechanisms to evade autophagy. for example, the bacterium listeria monocytogenes efficiently escapes autophagy by recruiting the host arp2/3 complex and ena/vasp via the bacterial acta protein to the bacterial surface to disguise the bacteria from ubiquitylation, p62 binding and autophagic sequestration. significantly, the ability of acta to mediate protection from ubiquitylation was elegantly demonstrated by generating a gfp-acta-q79c chimera, consisting of gfp (green fluorescent protein), the acta protein and segments of aggregate-prone polyq (from the huntington's disease protein). gfp-acta-q79c forms aggregates in the host cell cytoplasm. however, these acta-containing aggregates are not targeted for ubiquitylation and p62 recruitment 85 . one or more steps in these pathways may be subjected to therapeutic intervention to slow or prevent viral and bacterial replication in human cells. although few bacteria have bona fide proteasomes and none has ubiquitin, one very important bacterial pathogen, mycobacterium tuberculosis (mtb), does contain a proteolytic system that is analogous and orthologous to the ups 86 . with estimates of 1.3 million deaths per year attributable to this pathogen 87 , there is an urgent unmet medical need for new drugs to target it. because the prokaryotic ubiquitin-like protein (pup)proteasome system of mtb is essential for pathogenesis 88, 89 , the various components of the system present excellent opportunities for therapeutic intervention. these include the enzymes involved in 'pupylation' of substrates 90 , analogous to the ubiquitin e1-e2-e3 cascade, as well as the 'depupylase' activity that was recently described 91 . the aaa atpase complex that regulates the mtb proteasome 92 and the mtb proteasome itself 88 could also be targeted. considerable work has already gone into characterizing active site specificity of the mtb proteasome 93,94 to find inhibitors that could selectively block the function of the mtb proteasome 95,96 without inhibiting the proteasome of the (human) host. the fundamental role of the ups and ubl conjugation pathways in normal cell function and in disease prompts the search for inhibitors that selectively disrupt pathway function. despite our limited understanding of the molecular mechanisms of pathway targets, the inhibition of pathway enzymes is an attractive, and increasingly tractable, approach to targeting aberrant signalling pathways in multiple cancers and other diseases. therapeutic intervention in the ups, the endosome-lysosome system, the autophagic system and in ubl signalling is an emerging area for the treatment of acute and chronic human diseases, including the treatment of viral and bacterial infections. some cancers and other disease settings in which these pathways are constitutively active have been shown to be more sensitive to inhibition, potentially limiting the side effects of inhibiting all or part of these key cellular systems. the precedent for ups inhibition in cancer has been established by the proteasome inhibitor bortezomib, and second-generation inhibitors are now in clinical development with the aim of improving drug pharmacology. beyond its use in multiple myeloma and mantle cell lymphoma, the most exciting new therapeutic application of bortezomib is as a drug to prevent antibody-mediated rejection of renal allografts in transplant patients by depleting normal antibodyproducing plasma cells 97 . in addition, mln4924 has established the precedent for inhibiting a specific e1 target, with mechanistic studies indicating the possibility of such an approach for other e1s. understanding the molecular mechanisms involved in the interactions seen at each step of the ups and ubl conjugation pathways, between e1s, e2s, e3s, substrates, dubs and the proteasome itself, is increasing through the use of tools such as small-molecule inhibitors and small interfering rna, improved compound screening strategies and crystal structure studies. consequently, there are opportunities present and emerging for other novel therapeutics that target numerous specific pathways in the ups and ubl conjugation pathways. the ubiquitinproteasome pathway: the complexity and myriad functions of proteins death targeting the ubiquitin system in cancer therapy the ubiquitin proteasome system in neuropathology role of the ubiquitin proteasome system in renal cell carcinoma the ubiquitin-proteasome system in cardiovascular diseases-a hypothesis extended the ubiquitin system, disease, and drug discovery dual e1 activation systems for ubiquitin differentially regulate e2 enzyme charging e1-l2 activates both ubiquitin and fat10 distinct monoubiquitin signals in receptor endocytosis ubiquitylation and cell signaling proteasomeindependent functions of ubiquitin in endocytosis and signaling breaking the chains: structure and function of the deubiquitinases targeting e3 ubiquitin ligases for cancer therapy deubiquitylating enzymes and disease ubiquitin-like protein activation by e1 enzymes: the apex for downstream signalling pathways conservation in the mechanism of nedd8 activation by the human appbp1-uba3 heterodimer a unique e1-e2 interaction required for optimal conjugation of the ubiquitin-like protein nedd8 building ubiquitin chains: e2 enzymes at work lingering mysteries of ubiquitinchain assembly the structure of k63-linked and linear ubiquitin dimers, and the specificity of various dubs and ubds the e2 ubiquitin conjugating enzymes direct polyubiquitination to preferred lysines uses a systematic approach to evaluate the role of e2 enzymes in determining the topology of the different polyubiquitin chains. the analysis shows that e2s are capable of directing chain formation to distinct subsets of ubiquitin lysines independently of an e3 enzyme the family of ubiquitin-conjugating enzymes (e2s): deciding between life and death of proteins function and regulation of cullin-ring ubiquitin ligases chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an scf family e3 ubiquitin ligase an allosteric inhibitor of substrate recognition by the scf(cdc4) ubiquitin ligase identification of a primary target of thalidomide teratogenicity this systematic investigation of dub function uses proteomic analysis of dubs and their associated protein complexes to identify over 750 candidate interacting proteins associated with 75 dubs a tangled web of ubiquitin chains: breaking news in tnf-r1 signaling nonproteolytic functions of ubiquitin in cell signaling involvement of linear polyubiquitylation of nemo in nf-κb activation nf-κb is a key transcription factor for inflammatory, anti-apoptotic and immune processes and is regulated by the ubiquitin pathway. the lubac e3 ligase complex is composed of two ring finger proteins (hoil-1l and hoip), forms head-to-tail-linked linear ubiquitin chains and activates nf-κb, suggesting a physiological function for this recently identified form of polyubiquitin recruitment of the linear ubiquitin chain assembly complex stabilizes the tnf-r1 signaling complex and is required for tnf-mediated gene induction specific recognition of linear ubiquitin chains by nemo is important for nf-κb activation nemo specifically recognizes k63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain direct activation of protein kinases by unanchored polyubiquitin chains rings and ubiquitylation the hect-type e3 ubiquitin ligase aip2 inhibits activation-induced t-cell death by catalyzing egr2 ubiquitination the structure of the cyld usp domain explains its specificity for lys63-linked polyubiquitin and reveals a b box module de-ubiquitination and ubiquitin ligase domains of a20 downregulate nf-κb signalling inhibition of nf-κb signaling by a20 through disruption of ubiquitin enzyme complexes the ubiquitinediting enzyme a20 (tnfaip3) is a central regulator of immunopathology a20: from ubiquitin editing to tumour suppression phosphorylation-driven assembly of the rip1-rip3 complex regulates programmed necrosis and virus-induced inflammation rip3, an energy metabolism regulator that switches tnf-induced cell death from apoptosis to necrosis identification of rip1 kinase as a specific cellular target of necrostatins rip kinases at the crossroads of cell death and survival nf-κb modulators in osteolytic bone diseases nrf2-dependent upregulation of antioxidative enzymes: a novel pathway for proteasome inhibitor-mediated cardioprotection functional alterations of cardiac proteasomes under physiological and pathological conditions proteasome inhibition during myocardial infarction regulatory roles of the ubiquitin-proteasome system in cardiomyocyte apoptosis unexpected cardiotoxicity in haematological bortezomib treated patients protein degradation systems in viral myocarditis leading to dilated cardiomyopathy the proteasome-associated deubiquitinating enzyme usp14 is essential for the maintenance of synaptic ubiquitin levels and the development of neuromuscular junctions roles of ubiquitination at the synapse the role of parkin in familial and sporadic parkinson's disease parkin and defective ubiquitination in parkinson's disease depletion of 26s proteasomes in mouse brain neurons causes neurodegeneration and lewy-like inclusions resembling human pale bodies is malfunction of the ubiquitin proteasome system the primary cause of alphasynucleinopathies and other chronic human neurodegenerative disease? suppression of basal autophagy in neural cells causes neurodegenerative disease in mice loss of autophagy in the central nervous system causes neurodegeneration in mice the ups and autophagy in chronic neurodegenerative disease: six of one and half a dozen of the other -or not? does impairment of the ubiquitin-proteasome system or the autophagylysosome pathway predispose individuals to neurodegenerative disorders such as parkinson's disease? the escrt machinery in endosomal sorting of ubiquitylated membrane proteins escrting autophagic clearance of aggregating proteins xbp-1 deficiency in the nervous system protects against amyotrophic lateral sclerosis by increasing autophagy immunoreactivity to lys63-linked polyubiquitin is a feature of neurodegeneration a decade of modeling alzheimer's disease in transgenic mice parkinson's disease: a rethink of rodent models enhancement of proteasome activity by a small-molecule inhibitor of usp14 usp14, a proteasome-associated dub, is shown to inhibit the degradation of ubiquitin-protein conjugates by trimming ubiquitin chains. a small-molecule inhibitor of usp14's dub activity enhances the degradation of proteasome substrates in cells, including some implicated in neurodegenerative disease potential therapeutic applications of autophagy mutations of optineurin in amyotrophic lateral sclerosis optineurin negatively regulates tnfalpha-induced nf-kappab activation by competing with nemo for ubiquitinated rip proteasome inhibitors in cancer therapy: lessons from the first decade targeted therapy for brain tumours the e6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53 a ubiquitin conjugating enzyme encoded by african swine fever virus structure of a shigella effector reveals a new class of ubiquitin ligases ubiquitin and ubiquitin-like specific proteases targeted by infectious pathogens: emerging patterns and molecular principles ebola zaire virus blocks type i interferon production by exploiting the host sumo modification machinery retrovirus budding autophagy pathway intersects with hiv-1 biosynthesis and regulates viral yields in macrophages antiviral activity of innate immune protein isg15 the adaptor protein p62/sqstm1 targets invading bacteria to the autophagy pathway the tbk1 adaptor and autophagy receptor ndp52 restricts the proliferation of ubiquitin-coated bacteria ndp52, a factor previously not known to contribute to innate immunity, is shown to activate autophagy against invading bacteria by collaborating with the ubiquitin system listeria monocytogenes actamediated escape from autophagic recognition prokaryotic ubiquitin-like protein provides a two-part degron to mycobacterium proteasome substrates fact sheet n.°104: tuberculosis. who website the proteasome of mycobacterium tuberculosis is required for resistance to nitric oxide ubiquitin-like protein involved in the proteasome pathway of mycobacterium tuberculosis pup is specifically conjugated to proteasome substrates, revealing a bacterial system that uses small-protein modifiers to control protein stability. pup is essential for the pathogenesis of mtb, suggesting that the various components of the pupylation system may present opportunities for therapeutic intervention molecular analysis of the prokaryotic ubiquitin-like protein (pup) conjugation pathway in mycobacterium tuberculosis depupylation" of prokaryotic ubiquitin-like protein from mycobacterial proteasome substrates structural insights on the mycobacterium tuberculosis proteasomal atpase mpa mycobacterium tuberculosis prcba genes encode a gated proteasome with broad oligopeptide specificity structure of the mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate distinct specificities of mycobacterium tuberculosis and mammalian proteasomes for n-acetyl tripeptide substrates inhibitors selective for mycobacterial versus human proteasomes relates the initial experience of using proteasome inhibition as first-line therapy to treat refractory antibody-mediated rejection (amr). the results suggest that proteasome inhibitor-based combination therapy may provide a means for rapid donor-specific anti-human leukocyte antigen antibodies elimination in early acute amr 20s proteasome and its inhibitors: crystallographic knowledge for drug development antitumor activity of pr-171, a novel irreversible inhibitor of the proteasome design and synthesis of an orally bioavailable and selective peptide epoxyketone proteasome inhibitor (pr-047) selective inhibition of chymotrypsin-like activity of the immunoproteasome and constitutive proteasome in waldenstrom macroglobulinemia a novel orally active proteasome inhibitor induces apoptosis in multiple myeloma cells with mechanisms distinct from bortezomib evaluation of the proteasome inhibitor mln9708 in preclinical models of human cancer cep-18770: a novel, orally active proteasome inhibitor with a tumor-selective pharmacologic profile competitive with bortezomib building on bortezomib: second-generation proteasome inhibitors as anticancer therapy discovery of a potent, selective, and orally active proteasome inhibitor for the treatment of cancer a selective inhibitor of the immunoproteasome subunit lmp7 blocks cytokine production and attenuates progression of experimental arthritis targeted inhibition of the immunoproteasome is a potent strategy against models of multiple myeloma that overcomes resistance to conventional drugs and nonspecific proteasome inhibitors inhibitors of ubiquitin-activating enzyme (e1), a new class of potential cancer therapeutics the ubiquitin-activating enzyme e1 as a therapeutic target for the treatment of leukemia and multiple myeloma nitric oxide prodrug js-k inhibits ubiquitin e1 and kills tumor cells retaining wild-type p53 an inhibitor of nedd8-activating enzyme as a new approach to treat cancer the nedd8 conjugation pathway controls the activity of the cullin-ring ligases and regulates the turnover of a large subset of proteins targeted for degradation by the proteasome. mln4924, a selective inhibitor of the nedd8-activating enzyme, blocks cullin-ring ligase-mediated protein turnover leading to apoptotic death in human tumour cells by the deregulation of s-phase dna synthesis targeting nedd8-activated cullin-ring ligases for the treatment of cancer cullin-based ubiquitin ligase and its control by nedd8-conjugating system structural insights into nedd8 activation of cullin-ring ligases: conformational control of conjugation pre-clinical antitumor activity of mln4924, an inhibitor of nedd8-activating enzyme (nae), in a novel primary human dlbcl xenograft model inhibition of nedd8-activating enzyme: a novel approach for the treatment of acute myeloid leukemia substrate-assisted inhibition of ubiquitin-like protein activating enzymes: the nedd8 e1 inhibitor mln4924 forms a nedd8-amp mimetic in situ ubiquitin-conjugating enzyme ube2q2 suppresses cell proliferation and is downregulated in recurrent head and neck cancer targeting the sumo e2 conjugating enzyme ubc9 interaction for anti-cancer drug design targeting ubc9 for cancer therapy ubc9 promotes breast cell invasion and metastasis in a sumoylation-independent manner elevated expression of ube2t in lung cancer tumors and cell lines overexpression of the e2 ubiquitinconjugating enzyme ubch10 causes chromosome missegregation and tumor formation association of clinicopathological features with ubch10 expression in colorectal cancer e2-c-cbl recognition is necessary but not sufficient for ubiquitination activity allosteric activation of e2-ring fingermediated ubiquitylation by a structurally defined specific e2-binding region of gp78 a conserved catalytic residue in the ubiquitin-conjugating enzyme family regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes mechanisms, biology and inhibitors of deubiquitinating enzymes a uaf1-containing multisubunit protein complex regulates the fanconi anemia pathway the hausp gene plays an important role in non-small cell lung carcinogenesis through p53-dependent pathways cyld is a deubiquitinating enzyme that negatively regulates nf-κb activation by tnfr family members the uch-l1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and parkinson's disease susceptibility targeting ubiquitin specific proteases for drug discovery structure-based design, synthesis, and biological evaluation of a series of novel and reversible inhibitors for the severe acute respiratory syndrome-coronavirus papain-like protease a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication amsh interacts with escrt-0 to regulate the stability and trafficking of cxcr4 the role of the jamm domain deubiquitinating enzyme amsh in the regulation of the endocytic sorting and down-regulation of the chemokine receptor cxcr4 was investigated. reversible ubiquitylation modulates the activity of the endocytic machinery, suggesting that amsh may directly regulate endocytic adaptor protein function by specifying the fate of endocytosed receptors a novel type of e3 ligase for the ufm1 conjugation system signalling to nf-κb: regulation by ubiquitin without ub i am nothing": nemo as a multifunctional player in ubiquitin-mediated control of nf-κb activation ubiquitin and ubiquitin-like proteins in protein regulation origin and function of ubiquitin-like proteins mdm2 is a negative regulator of p21waf1/cip1, independent of p53 in vivo activation of the p53 pathway by small-molecule antagonists of mdm2 small molecule rita binds to p53, blocks p53-hdm-2 interaction and activates p53 function in tumors parthenolide promotes the ubiquitination of mdm2 and activates p53 cellular functions wrenches in the works: drug discovery targeting the scf ubiquitin ligase and apc/c complexes the human f box protein beta-trcp associates with the cul1/skp1 complex and regulates the stability of beta-catenin nedd8 modification of cul-1 activates scf(beta(trcp)-dependent ubiquitination of iκbα iaps: what's in a name? xiap as a ubiquitin ligase in cellular signaling x-linked inhibitor of apoptosis protein and its e3 ligase activity promote transforming growth factor-β-mediated nuclear factor-κb activation during breast cancer progression triptolide sensitizes aml cells to trail-induced apoptosis via decrease of xiap and p53-mediated increase of dr5 simultaneous activation of p53 and inhibition of xiap enhance the activation of apoptosis signaling pathways in aml method validation and preliminary qualification of pharmacodynamic biomarkers employed to evaluate the clinical efficacy of an antisense compound (aeg35156) targeted to the x-linked inhibitor of apoptosis protein xiap the mrna decay factor tristetraprolin (ttp) induces senescence in human papillomavirus-transformed cervical cancer cells by targeting e6-ap ubiquitin ligase de novo truncating mutations in e6-ap ubiquitin-protein ligase gene (ube3a) in angelman syndrome vhl inactivation in renal cell carcinoma: implications for diagnosis, prognosis and treatment hif-1alpha and epas ubiquitination mediated by the vhl tumour suppressor involves flexibility in the ubiquitination mechanism, similar to other ring e3 ligases rnf4 and vhl regulate the proteasomal degradation of sumo-conjugated hypoxia-inducible factor-2α oxygen-independent degradation of hif-alpha via bioengineered vhl tumour suppressor complex cell death: protein misfolding and neurodegenerative diseases the e3 ubiquitin-ligase bmi1/ ring1a controls the proteasomal degradation of top2alpha cleavage complex -a potentially new drug target the proteasome: a suitable antineoplastic target the development of proteasome inhibitors as anticancer drugs effects of ps-341 on the activity and composition of proteasomes in multiple myeloma cells potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma salinosporamide a: a highly cytotoxic proteasome inhibitor from a novel microbial source, a marine bacterium of the new genus salinospora the authors thank s. hill and n. beavan of firekite for assistance in the development of this manuscript. l.b., j.l. and r.j.m. thank the alzheimer's research trust and parkinson's uk for generous support of some of the work reported here. the authors declare competing financial interests: see web version for details. key: cord-283096-qm7h4qui authors: jeon, young joo; yoo, hee min; chung, chin ha title: isg15 and immune diseases date: 2010-02-12 journal: biochim biophys acta mol basis dis doi: 10.1016/j.bbadis.2010.02.006 sha: doc_id: 283096 cord_uid: qm7h4qui isg15, the product of interferon (ifn)-stimulated gene 15, is the first identified ubiquitin-like protein, consisting of two ubiquitin-like domains. isg15 is synthesized as a precursor in certain mammals and, therefore, needs to be processed to expose the c-terminal glycine residue before conjugation to target proteins. a set of three-step cascade enzymes, an e1 enzyme (ube1l), an e2 enzyme (ubch8), and one of several e3 ligases (e.g., efp and herc5), catalyzes isg15 conjugation (isgylation) of a specific protein. these enzymes are unique among the cascade enzymes for ubiquitin and other ubiquitin-like proteins in that all of them are induced by type i ifns or other stimuli, such as exposure to viruses and lipopolysaccharide. mass spectrometric analysis has led to the identification of several hundreds of candidate proteins that can be conjugated by isg15. some of them are type i ifn-induced proteins, such as pkr and rig-i, and some are the key regulators that are involved in ifn signaling, such as jak1 and stat1, implicating the role of isg15 and its conjugates in type i ifn-mediated innate immune responses. however, relatively little is known about the functional significance of isg15 induction due to the lack of information on the consequences of its conjugation to target proteins. here, we describe the recent progress made in exploring the biological function of isg15 and its reversible modification of target proteins and thus in their implication in immune diseases. since the discovery of type i interferons (ifnα and ifnβ) in 1957, they have widely been used as clinical drugs [1, 2] . for example, ifnα has been used for the treatment of chronic hepatitis b and hepatitis c viruses and of several cancers, such as leukemia, and ifnβ is effective for treating multiple sclerosis. type i ifns exert their effects through the activation of janus tyrosine kinase (jak)/signal transducer and activator of transcription (stat) signaling pathway [3] . upon binding of the ifns to their cell surface receptors (ifnar), the receptorassociated kinases jak1 and tyrosine kinase 2 (tyk2) become activated and phosphorylate tyrosine residues in the cytoplasmic tails of the receptor subunit, ifnar1. the phosphorylated subunit provides specific docking sites for the activation of stats by jak1/tyk2mediated phosphorylation [4, 5] . activated stats dissociate from the receptor and translocate into the nucleus, where they act as transcription factors that bind to the promoter regions of ifn-stimulated genes (isgs) [6] . stat1/stat2 heterodimers associate with ifn regulatory factor 9 (irf-9) to form the transcription complex ifn-stimulated gene factor 3 (isgf3), which binds to ifn stimulatory response elements (isres) within the promoters of isgs [7] . on the other hand, homodimers and heterodimers of stat1 and stat3 bind to gammaactivated sequence (gas) response elements [8] . the isg proteins generated by these pathways play key roles in the induction of innate and adaptive immune responses [9, 10] . protein modifications by ubiquitin and ubiquitin-like proteins (ubls), including sumo and nedd8, have emerged as critical regulatory processes, such as in the control of cell cycle, stress response, signaling transduction, and immune response [11] [12] [13] [14] [15] . moreover, deregulation of the modification systems gives rise to numerous human diseases, such as cancers, neurodegenerative diseases, and immune diseases. conjugation of ubiquitin and ubls to target proteins involves threestep cascade enzymes: ubiquitin-and ubl-activating e1 enzymes, ubiquitin-and ubl-conjugating e2 enzymes, and ubiquitin-and ubl e3 ligases. protein modifications by ubiquitin and ubls are reversible processes that are catalyzed by isopeptidases, called deubiquitinating enzymes (dubs) and ubl-specific proteases (ulps), respectively. isg15, a member of the ubl family, shows a significant sequence homology to ubiquitin. like ubiquitin, isg15 is conjugated to numerous cellular proteins via isopeptide bonds. this isg15 conjugation (isgylation) utilizes ube1l as e1 enzyme, ubch8 as e2 enzyme, and some ubiquitin e3 ligases, such as efp and herc5, as e3 enzymes (fig. 1) . on the other hand, ubp43 (also called usp18) acts as a major isg15-specific deconjugating enzyme. isg15 is not present in yeast, nematode (caenorhabditis), or insect (drosophila), indicating that the functions of isg15 and its modification are restricted to higher animals with evolved ifn signaling. isg15 is strongly induced by type i ifns [16, 17] . viral infection also strongly induces isg15 [18, 19] because one of its major host responses is the production of type i ifns. a number of proteins that are involved in antiviral signaling pathways, including rig-i, mda-5, mx1, pkr, stat1, and jak1, have been identified as target proteins for isgylation. moreover, recent studies have explored the biological functions of isg15 and its conjugation to target proteins. isg15 and its conjugation impair viral replication in vivo [20] [21] [22] [23] [24] [25] [26] . conversely, certain viruses induce viral specific proteins that can deconjugate isg15 from its target proteins or prevent the genera-tion of isgylated proteins, thus abrogating the antiviral response [18, 27, 28] . however, functional consequences of reversible isg15 modification of most target proteins, which are induced by viral infection, are still largely unknown. one of the key components of the innate immune response in regulating cancer development is the activation of type i ifn signaling pathways [29, 30] . type i ifns suppress cell proliferation and promote apoptosis and, therefore, have been used in the treatment of several cancers, such as leukemia [31] . notably, isg15 appears to function as an oncogenic protein as well as a tumor suppressor protein [32] . the findings that cancer chemotherapeutics cause an increase in the level of isg15 conjugates suggest the role of isg15 as a tumor suppressor [33] [34] [35] [36] [37] . conversely, the observations that deregulated overexpression of isg15 and enhanced isgylation are positively correlated with carcinogenesis implicate the oncogenic potential of isg15 protein [38] [39] [40] . however, since ifns are induced on the development of many cancers and isg15 is an interferon-inducible protein, the enhanced expression of isg15 and its conjugation could be a side product of ifn response to cancer and may not play a general role in carcinogenesis. thus, further studies are required for clarification of the functional relationship between isg15 and carcinogenesis. 2. isg15, the product of interferon-stimulated gene 15 isg15 was originally identified by farrell et al. [16] . because some antibodies directed against ubiquitin also react with isg15, it was initially named as an ubiquitin cross-reactive protein (ucrp) [17] . this cross-reactivity is explained by the fact that the 15-kda isg15 protein consists of two domains, each of which bears high sequence homology to ubiquitin. the primary sequences of the two ubiquitinlike domains that correspond to the n-and c-terminal regions of isg15 share 29% and 31% identities with ubiquitin, respectively (fig. 2) . furthermore, both the n-and c-terminal domains of isg15 show a striking similarity in their tertiary structures to ubiquitin (fig. 3 ) [41] . like isg15, fat10, which is also a member of the ubl family, fig. 1 . protein modification by isg15. isg15-specific proteases cleave off the c-terminal extensions from isg15 precursors to generate matured isg15 molecules. isg15 is activated by ube1l (e1) at the expense of atp and is subsequently linked to the activating enzyme via thiosester bond. isg15 linked to ube1l is transferred to ubch8 (e2) and then to a target protein with the aid of an isg15 e3 ligase, such as efp and herc5. ubp43 functions in the reversal of the isgylation process by cleaving off isg15 molecules that are conjugated to the substrate proteins via isopeptide bonds. comprises two ubiquitin-like domains [42] . fat10 is conjugated to a limited number of cellular proteins [43, 44] . like ubiquitin, isg15 proteins in some species are synthesized as precursors that need to be processed before conjugation to target proteins (fig. 2) [45] . while isg15 molecules in human, mouse, and rat are translated as precursors containing the extensions of5-8 amino acids in their c-termini, isg15 proteins in most other species, including fish and bovine, are synthesized as matured forms [46] . the c-terminal lrlrgg sequence of isg15, as that of ubiquitin, is essential not only for its conjugation to substrates but also for its recognition by the relevant processing proteases. however, this hexapeptide sequence is absent in the analogous position of the n-terminal domain. instead, a pro residue is present at the position 81 of isg15, thus preventing the incorrect processing that might generate the two cleaved ubiquitinlike domains of the 15-kda polypeptide [47] . the c-terminal ubiquitin-like domain is sufficient for its activation by ube1l and subsequent thioesterification of ube1l and ubch8 [48] . when overexpressed, the c-terminal domain of isg15 can be conjugated to cellular proteins. however, the level of cellular proteins conjugated by the c-terminal domain alone is much lower than that by full-length isg15, suggesting that both domains of isg15 are required for efficient conjugation to cellular proteins. interestingly, the replacement of arg153 in human isg15 (arg151 in mouse) by ala ablates the binding of isg15 to ube1l and subsequent transesterification of ubch8 [49] . accordingly, the ability of the mutant isg15 to form protein conjugates in 293t cells is markedly diminished. furthermore, while expression of wild-type isg15 protects ifnar −/− mice from lethality after sindbis virus (snv) infection, expression of the arg-to-ala mutant form of isg15 confers no survival benefit. this mutation also attenuates the ability of isg15 to decrease snv replication in ifnar −/− mice or prolong the survival of isg15 −/− mice. thus, arg153 appears to serve as a binding site for ube1l. type i ifns are capable of inducing isg15 [47] . notably, the induction of isg15 shows biphasic kinetics. after ifnβ treatment, the increase in the level of free isg15 molecules is first observable within 2 h, continues for the next 10 h, and becomes maximal by about 18 h. on the other hand, isg15 conjugates are observable at least 12 h after the exposure to ifnβ, and their level dramatically increases between 12 and 18 h and continuously increases thereafter although at a slower rate. this delayed induction of isg15 conjugates indicates the requirement of isg15-conjugating machinery that is expressed in the later periods after the treatment with type i ifns. isg15 is strongly induced by viral infection [18, 19] . like other ifn-stimulated genes, the isg15 gene has a 5′-cis-regulatory sequence called isre (interferon-stimulated response element) [50] . a number of irfs, including irf-3 and irf-9, bind to the isre for isg15 induction (fig. 4) [50] [51] [52] . irf-3 forms a complex with cbp/p300 coactivators for isg15 induction [53, 54] . on the other hand, irf-9 interacts with stat1 and stat2, leading to the formation of isgf3 complex that also induces isg15. upon dsrna treatment, however, irf-3 induces isg15 independently of ifns [55] . isg15 is also strongly induced by lipopolysaccharide (lps) [56] [57] [58] . when macrophages are stimulated by lps, isg15 can be detected as early as 1 h and its level becomes maximal at about 4 h [59] . infection of mice with bacille calmette-guérin (bcg) markedly induces isg15 in macrophages. moreover, this bacterial infection leads to isgylation of serpin2a (serine protease inhibitor 2a), which was the first identified target protein, although its biological significance remains unknown. isgylation of serpin2a may protect macrophages from lysosomal enzymes because a variety of lysosomal proteases are upregulated by ifnγ for the destruction of ingested bacteria and other pathogens. in addition, it has been shown that trif/ irf-3 signaling pathway is responsible for lps-mediated induction of isg15 and its conjugation to target proteins [60] . isg15 is a target gene of nf-κb. lps induces isg15 in 70z/3 cells that recapitulate aspects of the pre-b to immature b-cell transition in response to nf-κb activation, but not in 1.3e, which is a nf-κb signaling defective mutant cell line, indicating that nf-κb activation is required for isg15 induction [56] . ataxia telangiectasia (at) is a multifaceted autosomal recessive genetic disorder, characterized by the loss of coordination, progressive neuronal degeneration, immunodeficiency, and cancer proneness [61] . nf-κb is constitutively activated in human fibroblasts derived from at patients [62] . moreover, the level of isg15 is constitutively elevated in the at cells [63] . because activated nf-κb is capable of inducing the expression of type i ifns, it appears that the elevation of isg15 level in the at cells is due to abnormal production of type i ifns. isg15 also is a target gene of p53 [64] [65] [66] . activation of temperaturesensitive p53 leads to isg15 induction in the absence of exogenous stimuli. since cycloheximide treatment does not influence the increase in the level of isg15 mrna, isg15 induction appears to be a primary response to p53. while type i ifns induce the accumulation of isg15 mrna in both wild-type or p53-deficient cells, dsrna induces it only in wild-type cells, indicating that p53 is required for isg15 induction by dsrna but not for that by type i ifns. in addition, sequence analysis of the human isg15 gene has led to the identification of a putative p53-responsive element that is located adjacent to the isg15-specific isre [50] . thus, in virus-infected cells, p53 seems to exert its antiviral function by the induction of isg15 in addition to the induction of apoptotic signaling [67] . jnk induces the expression of isgs, such as isg15, isg12, and igtp [68] . swiss 3t3 cells expressing constitutively active mkk7-jnk1β fusion protein show increased resistance to apoptosis induced by vesicular stomatitis virus (vsv) infection, suggesting the involvement of jnk signaling pathway in antiviral response. recently, pi3k has been shown to control both ifnα-and ifnγ-induced expression of isgs, including isg15, at both transcriptional and translational levels [69] . ifn-mediated antiviral response is defective in cells lacking p85α/p85β, the regulatory subunits of pi3k, suggesting the involvement of pi3k signaling pathway in innate immune response. isg15 is induced by certain genotoxic stresses. for example, treatment with camptothecin, an inhibitor of topoisomerase i, leads to an increase in the level of isg15 mrna, and this increase requires protein synthesis and a functional p53 protein [35] . interestingly, ifns and jak/stat are dispensable for camptothecin-mediated induction of isg15. isg15 conjugates generated by camptothecin are distinct from those produced by type i ifns, suggesting that different protein substrates are targeted for isgylation. however, camptothecin markedly increases ifn-induced isgylation of cellular proteins. furthermore, treatment with both ifns and camptothecin causes synergistic killing of colorectal cancer xenografts in nude mice, suggesting that the combination of the drugs can be an effective therapeutic strategy [36] . retinoic acid upregulates the levels of isg15, its conjugates, and ube1l in acute promyelocytic cells [33, 34] . the retinoic acid-mediated accumulation of isg15 and its conjugates occurs in retinoic acid-sensitive leukemic cells but not in retinoic acid-resistant cells and the pattern of accumulated isg15 conjugates is similar to that observed by the exposure to type i ifns. in addition, several of the type i ifninduced proteins, such as irf-1 and (2′-5′) oligoadenylate synthetase, are induced by retinoic acid [70] [71] [72] . treatment with retinoic acid also leads to an increased secretion of type i ifns into culture media. blockade of ifnar with a neutralizing antibody prevents retinoic acidmediated accumulation of isg15 and its conjugates. thus, retinoic ns1b inhibits the thioesterification of ube1l and thereby the generation of isg15 conjugates that are required for antiviral response. plpro, otu protease, and viral e3 protein mediate the removal of isg15 from its conjugates. acid seems to elevate the levels of isg15 and its conjugates by stimulating cells to secrete ifns. ube1l is a 112-kda protein that shows a 45% identity in amino acid sequence to the human ubiquitin-activating e1 enzyme (ube1) [73] . ube1l expressed in baculovirus forms a thioester bond with isg15 but not with ubiquitin [18] , indicating that it is a specific isg15activating e1 enzyme. in addition, ube1l has a c-terminal ubiquitinfold domain that is required for the transfer of isg15 from ube1l to ubch8 as well as for the binding of ube1l to ubch8 [74] . as expected, ube1l-deficient mice are not capable of producing isg15 conjugates upon stimuli [75] . in ube1l −/− macrophages treated with lps and in ube1l −/− mouse embryonic fibroblasts (mefs) treated with ifns, ubiquitination of cellular proteins occurs normally, but isgylation of proteins is completely abolished, confirming the strict requirement of ube1l for isgylation. the ube1l gene is located in the d3f15s2 locus of chromosome 3q21 [76] . ube1l can be detected in the jejunum, colon, lung, liver, tonsils, testis, and skin but not in the spleen and pancreas. significantly, ube1l is not detectable in all tested human lung cancer cell lines [73, 77] , implicating a tumor-suppressive role of ube1l. this repressed expression in lung cancer cell lines is intriguing because a region of chromosome 3q, in which the ube1l gene resides, is often deregulated in preneoplastic or neoplastic epithelial tissues [78] . however, upon generation of ube1l −/− /k-ras la2 mice, it has recently been shown that the loss of isgylation does not affect tumor spectrum, tumor pathology, or survival of k-ras la2 mice [79] . nonetheless, it is possible that ube1l deficiency and k-ras mutation are two separate pathways in lung cancer development. additional works with other lung cancer mouse models are necessary to clarify the potential tumor suppressor function of ube1l in k-ras mutation-independent human lung cancers. ubch8, an isg15-conjugating e2 enzyme, is induced by type i ifns and viral infection [80] [81] [82] . ubch6 is also induced by type i ifns and forms a thioester intermediate with isg15, suggesting that ubch6 has the potential to function as an isg15-conjugating e2 enzyme [83] . however, the amount of the thioester intermediate formed by ubch6 is much lower than that by ubch8, indicating that ubch8 is a major e2 enzyme for isg15. ubch8 was originally identified as an ubiquitin-conjugating e2 enzyme by a yeast two-hybrid screening for its interaction with e6ap [84] . ubch8 was later shown to also interact with other ubiquitin e3 ligases, such as parkin, dorfin, staring, efp, and rlim and function as an ubiquitin-conjugating e2 enzyme [85] [86] [87] [88] [89] [90] . thus, ubch8 serves as an e2 enzyme for both ubiquitin and isg15. however, although ubch7 is the most closely related e2 to ubch8, it does not function in isg15 conjugation [74] . moreover, ubch8 shows a higher affinity to ube1l than ubch7, while ubch7 binds more strongly to ube1 than ubch8. in addition, it has been shown that two structural elements within the e2 n-terminal region are responsible for the differential interaction of ubch8 and ubch7 with ube1l. thus, ubch8 may preferentially, but not exclusively, function as an e2 enzyme for isg15. interestingly, ubch8 and isg15 can act as functional regulators of rnf125, an ubiquitin e3 ligase of rig-i [91] . in the absence of isg15, ubch8 interacts with rnf125 and interferes with rig-i ubiquitination. upon overexpression of isg15, however, the interaction of ubch8 with isg15 leads to the dissociation of ubch8 from rnf125, thus allowing the association of rnf125 with ubch5 for rig-i ubiquitination. the overlapping function of ubch8 as an e2 enzyme for both ubiquitin and isg15 raises a possibility that some ubch8-interacting ubiquitin e3 ligases can also function as isg15 e3 ligases. indeed, the ubch8-interacting protein efp (estrogen-responsive finger protein) that has a ring finger domain serves as an isg15 e3 ligase [92] . in the absence of isg15, ubch8 and efp may function as e2 and e3 enzymes for ubiquitination, respectively. upon isg15 induction, however, isg15 may compete with ubiquitin for binding to ubch8, allowing ubch8 and efp to serve as the enzymes for isgylation of 14-3-3σ. replacement of the active site cys residue in the ring domain disrupts efp-mediated isgylation of 14-3-3σ. like ubch8, efp is a type i ifninducible protein [93] . interestingly, efp can isgylate itself on the lys117 residue and this auto-isgylation negatively regulates the isg15 e3 ligase activity of efp toward 14-3-3σ, suggesting that a feedback loop is operating for the control of the protein isgylation [94, 95] . an additional ring-containing ubiquitin e3 ligase, called hhari (human homolog of drosophila ariadne), also serves as an isg15 e3 ligase for 4ehp [96] . herc5 (hect domain and rcc1-like domain containing protein 5) is an isg15 e3 ligase that contains the hect domain [97] [98] [99] . like efp, herc5 is a type i ifn-inducible protein. knockdown of herc5 by using sirna prevents ifn-mediated increase in the total level of isgylated cellular proteins, unlike that of efp, which blocks the isgylation of 14-3-3σ with little or no effect on the total level of isgylated proteins. thus, it appears that herc5 serves as a general ifn-induced isg15 e3 ligase. like efp, herc5 itself is a target for isgylation, but its functional significance is unknown. 3.4.1. ubp43 and other isg15-specific proteases ubp43 cleaves off isg15 from its protein conjugates that are linked via isopeptide bonds [100] . ubp43 can also cleave the peptide bonds immediately after the lrlrgg sequence in isg15 fusions [100] . thus, ubp43 appears to also function in the processing of isg15 precursors to generate matured isg15 molecules. however, apart from ubp43, additional isg15-specific proteases must exist because isg15 precursor is processed to its matured form in ubp43-deficient mice [101] . the overlapping function of some e2 and e3 enzymes in the conjugation of both isg15 and ubiquitin also implies the existence of promiscuous dubs that can serve as isg15-specific proteases. indeed, several dubs, including usp2, usp5, usp13, and usp14, have been identified as the candidates that can function as isg15-processing and/or deconjugating enzymes [102] . in addition, it has been reported that recombinant isg15 precursors can be properly processed by a 100-kda enzyme in the extracts of human lung fibroblasts cell line a549, whose activity is unaffected by type i ifn stimulation [103] . this 100-kda enzyme is a cysteine protease and shows a partial similarity in its amino acid sequence with that of the human ortholog of yeast ubp1 or ubp1-related protein. however, deletion of the ubp43 gene in mouse leads to a massive increase of isg15 conjugates in tissues without affecting the level of ubiquitin conjugates, indicating that ubp43 is the major isg15-specific protease that deconjugates isg15 from its target proteins. thus, it appears likely that the above-mentioned dubs as well as the 100-kda cysteine protease function primarily in the processing of isg15 precursors to generate matured isg15 molecules. ubp43 is induced by type i ifns, and this induction requires a functional jak/stat signaling pathway [104] . ifnβ induces ubp43 more strongly than ifnα and dsrna, but ifnγ barely induces it [105] . ubp43 is also induced by lps [58] . irf-3 is responsible for lps-mediated induction of ubp43, while irf-2 is involved in its induction to a basal level. however, both irf-2 and irf-3 are required for optimal responsiveness to lps. interestingly, ubp43 induction is upregulated by the acute myelogenous leukemia (aml1)-eto fusion protein that is created by t(8;21), suggesting a possible involvement of ubp43 in hematopoiesis [106] . however, it is unknown how aml1-eto affects the up-regulation of ubp43 induction. in addition, ubp43 has been identified as a substrate for skp2 [107] . skp2 promotes the ubiquitination of ubp43 and subsequent degradation by the proteasomes, suggesting that the scf skp2 may be involved in the regulation of type i ifn signaling by controlling the stability of ubp43. the human ubp43 gene maps to the chromosome 22q11.2 [108] . this region, known as digeorge syndrome critical region, is consistently deleted in digeorge syndrome and related disorders, suggesting that ubp43 may be involved in the development of thymus or differentiation of t cells. ubp43-deficient mice are viable and resistant to the fatal lymphocytic choriomeningitis and myeloencephalitis that develop in wildtype mice upon intracerebral inoculation of lymphocytic choriomeningitis virus (lcmv) and vsv, respectively [101, 109] . furthermore, survival of ubp43 −/− mice after lcmv infection correlates with severe inhibition of lcmv replication as well as with an increase in the level of isg15 conjugates in the brain. none of the ubp43 −/− mice infected with lcmv died or developed clinical symptoms by day 11 after infection, whereas all lcmv-infected wild-type mice died by day 7 infection. these findings strongly suggest that ubp43 deficiency causes an unfavorable environment for lcmv replication. in addition, ubp43 −/− mef cells exhibit enhanced type i ifn-mediated resistance to cytopathic effect caused by vsv and snv. these findings strongly suggest that ubp43 serves as a negative regulator of innate immune response against viral infection. however, the enhanced resistance to viral infection in ubp43 −/− mice cannot be rescued in ubp43 −/− / isg15 −/− or ubp43 −/− /ube1l −/− double knockouts, indicating that the phenotypic alterations are not associated with the protein modification by isg15 [75, 110] . thus, ubp43, independently of its isopeptidase activity, may have another biological function. ubp43 −/− cells are hypersensitive to type i ifns, resulting in a dramatic increase in the level of isgylated proteins, which is associated with the enhanced and prolonged jak/stat signaling [111] . however, ubp43, independently of its catalytic activity, also functions as a negative regulator of type i ifn signaling [112] . this ubp43 action is achieved through a direct interaction between ubp43 and ifnar2, a subunit of type i ifn receptor. binding of ifnar2 to ubp43 interferes with the interaction between jak and the receptor, leading to the inhibition of downstream phosphorylation cascade and other signaling events. in addition, complementation of ubp43 −/− cells with a catalytically inactive mutant of ubp43 leads to the inhibition of stat1 phosphorylation to a level seen by that with wild-type ubp43. moreover, down-regulation of the total level of isg15 conjugates by sirnamediated knockdown of ube1l in ubp43 −/− cells shows little or no effect on stat1 phosphorylation in comparison with that seen in ubp43 +/+ cells, indicating that the deisgylating activity of ubp43 is not required for its inhibitory effect on type i ifn signaling. this conclusion is further corroborated with the findings that phenotypic alterations in ubp43 −/− mice are not influenced by the lack of isg15 in ubp43 −/− /isg15 −/− double knockout mice [75, 110] . the isopeptidase-independent action of ubp43 is also involved in the replication of hbv (hepatitis b virus) [113] . ubp43 −/− cells show an increased induction of isgs in response to type i ifns, indicating that the lack of ubp43 results in a strengthened immune response. consistently, the steady-state level of hbv dna is substantially reduced in ubp43 −/− mice in comparison with that in ubp43 +/+ mice. thus, approaches that modulate ubp43 level could be used as therapeutic potentials in treating viral infection, especially for viruses sensitive to type i ifn signaling. in addition to the protection against hbv, ubp43 deficiency increases the resistance to oncogenic transformation by bcr-abl (breakpoint cluster region abelson leukemia protein), a fusion protein consisting of the n-terminal portion of bcr joined to most of the abl tyrosine kinase [114] . this resistance to leukemia development is heavily dependent on the activation of type i ifn signaling pathway. loss of type i ifn signaling through the loss of ifnar results in a reversal of the original resistance to the leukemia development observed in mice that received a transplant of bcr-ablexpressing ubp43-deficient donor cells. thus, it appears that inhibition of the negative effect of ubp43 on type i ifn signaling could enhance innate immune response against cancer development. by using a combination of affinity purification and mass spectrometry, hundreds of target proteins for isgylation have been identified. some of them are type i ifn-induced proteins, including pkr, mxa, hup56, and rig-i [115] . some are key regulators that are involved in type i ifn signaling, such as plcγ1, jak1, erk1, and stat1 [116] . most other targets are constitutively expressed and function in diverse cellular pathways, including translation, glycolysis, cell motility, protein modification, intracellular protein trafficking, rna splicing, chromatin remodeling, cytoskeletal organization, and stress responses [97, 115, 117] . these findings implicate the role of protein isgylation in type i ifn-induced immune responses as well as in the control of numerous fundamental cellular pathways. isg15 is synthesized in many cell types and secreted from human monocytes and lymphocytes [118] . both native and recombinant isg15 induce the synthesis and secretion of ifnγ from b-cell-depleted lymphocytes, implicating the role of isg15 as a cytokine that modulates immune response [119] . treatment with human isg15, but not its precursor form, leads to an increase in dna synthesis in cultured primary blood lymphocytes in a dose-dependent fashion, suggesting that isg15 can act as a mitogen and that the processing of isg15 precursor is required for the generation of biologically active isg15 [120] . furthermore, isg15 has been identified as a member of the cytokine cascades. upon viral infection, type i ifns produced in infected cells induce the synthesis of isg15. these isg15 molecules can be secreted or released by lysis of the infected cells. they may then induce the production of ifnγ from t cells, augment nk cell proliferation, activate non-major histocompatibility complex-restricted cytolytic lymphocytes, and activate monocytes and macrophages via the induced ifnγ. filamins are nonmuscle actin-binding proteins that comprise a family of three members: filamin a, b, and c [121, 122] . these filamin isoforms are large cytoplasmic proteins that play important parts in cross-linking cortical actin filaments into a dynamic three-dimensional structure. recently, it has been shown that filamin b functions as a scaffold that links between activated rac1 and a jnk cascade module for mediating type i ifn signaling [123, 124] . filamin b interacts with rac1, mekk1, mkk4, and jnk and enhances their sequential activation in response to type i ifns, thereby promoting jnk activation and apoptosis. this acceleration of jnk-mediated apoptosis provides a biological basis for antitumor and antiviral functions of type i ifns. furthermore, it has been revealed that type i ifns induce isgylation of filamin b, which leads to dissociation of rac1, mekk1, and mkk4 from the scaffold protein and thus to the prevention of prolonged activation of type i-induced jnk signaling pathway [124, 125] . in contrast, blockade of filamin b isgylation by substitution of the isg15 acceptor site lys2467 with arg or by sirna-mediated knockdown of ubel1 prevents the release of the signaling molecules from filamin b, resulting in persistent promotion of jnk activation and jnk-mediated apoptosis. these findings indicate that isgylation of filamin b serves as a negative feedback regulatory gate for desensitization of type i ifn-induced jnk signaling, thus providing an important mechanism for the survival of uninfected bystander cells. pp2cβ dephosphorylates tak1 and suppresses tak1/tab1mediated iκbα degradation, thus controlling nf-κb signaling pathway, which plays a critical role in innate and adaptive immunity and cancer [126] [127] [128] [129] . pp2cβ is a target for isgylation, and this modification blocks the suppressive function of the protein phosphatase against tak1/ tab1-mediated nf-κb activation [130] . this conclusion is based on the observation that overexpression of ube1l, ubch8, and isg15 blocks the suppressive effect of pp2cβ on nf-κb activation, but not that of its mutant, in which the isg15 acceptor sites lys12 and lys142 are replaced by arginine. thus, isgylation of pp2cβ seems to play a role in the control of nf-κb pathway. ubc13, an ubiquitin-conjugating e2 enzyme, is a target for isgylation [131, 132] . isg15 is conjugated to lys92 of ubc13, which is very closely located to its active site, thus preventing the formation of a thioester bond with ubiquitin. since mms2, which forms a heteromeric complex with ubc13, interacts with both unmodified and isgylated ubc13, the inhibitory effect of ubc13 isgylation on the thioesterification of ubiquitin seems to be achieved by the inability of isgylated ubc13 to accept ubiquitin to its active site or by blocking the recognition of ubiquitin e1 enzyme and subsequent transfer of ubiquitin from the e1 enzyme to ubc13. ubc13 is known to mediate the generation of lys63-linked poly-ubiquitin chains that are conjugated to a variety of signaling molecules [133, 134] . thus, it is possible that isgylation of ubc13 may play an important role in the control of signal transduction pathways, such as nf-κb pathway, which are associated with lys63-linked poly-ubiquitination [132] . ubch8 and ubch6 also are targets for isgylation [83] . isgylation of ubch6 occurs in response to type i ifns and blocks the thioesterification of ubiquitin, like that of ubc13, suggesting that isgylation of ubch6 may also lead to the suppression of its ubiquitin-conjugating activity. initial efforts that intend to determine the role of isg15 in antiviral responses appeared unsuccessful. ube1l −/− mice are fertile and exhibit normal antiviral responses against vsv and lcmv infection, indicating that ube1l and protein isgylation may not be essential for ifn signaling [75] . furthermore, isg15 −/− mice are also fertile and display no obvious abnormalities [135] . lack of isg15 does not affect the development and composition of the main cellular immune system. the ifn-induced antiviral and immune responses directed against vsv and lcmv are not significantly altered in the absence of isg15. in addition, ifn-or endotoxin-induced stat1 phosphorylation as well as the expression of typical stat1 target genes remain unaffected by the lack of isg15, indicating that isg15 is dispensable for stat1-mediated ifn signaling. however, the function of isg15 as an antiviral effector has come to the front. unlike the infection by vsv and lcmv, ube1l −/− mice display markedly increased susceptibility to influenza b virus infection, with only 25% survival of ube1l −/− mice for 21 days, compared to 86% survival of ube1l +/+ mice [20] . furthermore, both of the kinetics of lethality and the overall survival of ube1l −/− mice are identical to those of isg15 −/− mice, indicating that the antiviral activity of isg15 against influenza b virus is mediated by its conjugation to target proteins. the predominant site of isg15 action during influenza virus infection resides within a stromal cell population. a likely candidate is the respiratory epithelium, since it is the site of influenza virus replication. this is the first phenotype described for ube1l −/− mice, which were previously found to have no defect in response to vsv or lcmv infection [75] . in addition, it has recently been shown that both the synthesis of influenza a virus protein and the early rate of the virus replication are inhibited by isg15 conjugation of cellular proteins [21] . isg15 −/− mice also exhibit increased susceptibility to infection by a number of other viruses, such as influenza a/wsn/33 and b/lee/40 viruses, herpes simplex virus type 1 and murine gamma herpes virus 68, and snv [22] . in addition, isg15 is induced after snv infection, and this induction is markedly attenuated in ifnar −/− mice, indicating that induction of isg15 by snv infection is dependent on type i ifns [136] . isg15 induction protects against snv-induced lethality and decreases the virus replication in multiple organs. the increased susceptibility of ifnar −/− mice to snv infection can be rescued by the expression of wild-type isg15 having the c-terminal diglycine residues but not by that of an isg15 mutant, of which the c-terminal diglycine is replaced by dialanine, again indicating that the antiviral action of isg15 requires its conjugation to target proteins. type i ifn-mediated inhibition of human immunodeficiency virus (hiv) assembly and release has been shown to correlate with the induction of isg15 [23] . furthermore, overexpression of isg15 mimics the ifn effect and inhibits the release of hiv-1 virions without having any effect on the synthesis of the viral proteins in infected cells [24] . in cells infected with hiv-1 provirus, overexpression of isg15 and ube1l causes a complete inhibition of hiv-1 replication. on the other hand, coexpression of ubp43 can partially rescue the release of hiv-1 in isg15-expressing cells. intriguingly, isg15 expression specifically inhibits the ubiquitination of gag and tsg101 and disrupts their interaction, thereby preventing assembly and release of virions from infected cells. expression of isg15 alone (i.e., without ube1l) does not block the viral release, indicating that isgylation of target proteins, but not isg15 itself, is required for the inhibition of gag ubiquitination, although isgylation of either gag or tsg101 could not be detected. thus, isgylation of certain unknown viral and/or host proteins appears to play a critical role in the ifn-mediated inhibition of hiv-1 assembly and release. overexpression of isg15 or ube1l with ubch8 has been shown to inhibit budding of ebola virus vp40 virus-like particles (vlps) [25, 26] . ebola virus is a member of the filoviral family of negative-sense rna viruses. the vp40 matrix protein is a key structural protein that is critical for the virion release. the efficient budding of vlps requires the interaction of overlapping l-domains (late-budding domains) in the vp40 protein with nedd4, an ubiquitin e3 ligase, as well as with other members of the escrt pathway (e.g., tsg101) [137] [138] [139] [140] . isg15 overexpression decreases not only the level of vp40 detected in vlps but also the levels of endogenous nedd4 incorporated into budding vp40 vlps. nedd4 interacts with and is conjugated by isg15. moreover, isg15 overexpression blocks the ability of nedd4 to ubiquitinate vp40, leading to the prevention of l-domain-mediated budding of vp40 vlps. in addition, it has been shown that free isg15 specifically binds to nedd4 and inhibits the interaction of the e3 ligase with ubiquitin e2 enzymes (e.g., ubch6), thus preventing the transfer of ubiquitin from the e2 enzymes to nedd4 [26] . these findings reveal a mechanism for the antiviral function of isg15 that involves the isgylation and inactivation of the host nedd4 e3 ligase. irf-3 is a target for isgylation, which can be induced by both type i ifns and viral infection [141] . this isgylation prevents the ubiquitination and degradation of irf-3 in ndv (newcastle disease virus)-infected human fibrosarcoma 2ftgh cells, and promotes the translocation of irf-3 to the nucleus, where it binds to the ifnβ promoter. the relative level of irf-3 is significantly lower in ndvinfected isg15 −/− cells than in isg15 +/+ cells, indicating that the subversion of antiviral response is mediated by proteolysis of irf-3. moreover, the degradation of irf-3 can be counteracted by the induction of isg15. this finding provides a positive feedback mechanism for the induction of host antiviral response by isgylation-dependent stabilization of irf-3. however, isg15 is typically conjugated to only a small fraction of target proteins. therefore, an important issue is how the small fractions of isgylated proteins, including irf-3, can exert their biological functions. it has been proposed that if the small fractions of proteins that are modified by ubls (e.g., sumo and isg15) are localized to some functionally unique cellular site or the transient modification is sufficient to switch the protein into new state, their functions could efficiently operate [124, 142, 143] . intriguingly, overexpression of isg15 leads to the appearance of irf-3 as punctates in the nucleus [141] . this nuclear retention could allow irf-3 to exert its profound antiviral function, although only a small portion of irf-3 is isgylated upon the viral infection. an additional example is that upon overexpression of filamin b, ubch8 is recruited to membrane ruffles where filamin b is also concentrated with actin [123, 125] . this recruitment of ubch8, which otherwise resides throughout the cytoplasm and the nucleus, should allow the e2 enzyme to efficiently function in filamin b isgylation and thus in desensitization of type i ifn-induced jnk signaling (see above). the inducible nitric oxide synthase (inos) is not expressed under normal conditions, but like isg15, it is induced by various stimuli, such as exposure to cytokines, microbes, or microbial products, resulting in the sustained production of no [144] . upon stimuli, no as well as the products generated by its interaction with ros, such as peroxynitrite and peroxynitrous acid, are accumulated and utilized for the antibacterial or antiviral effects [144] [145] [146] . no is also covalently attached to the thiol group of cysteine residues of proteins, causing their s-nitrosylation. this posttranslational modification serves as an important mechanism that mediates antibacterial and antiviral effects through the alteration in enzymatic activity, protein-protein interaction, and signal transduction [147] . interestingly, the cysteine residue in isg15 can also be modified by no and this s-nitrosylation decreases the dimerization of isg15, thereby increasing the availability as well as the solubility of monomeric isg15 for its conjugation to cellular proteins [148] . moreover, treatment with s-ethylisothiourea, an inos inhibitor, reduces the level of isg15 conjugates, whereas overexpression of inos increases it. thus, inos may play a role in the enhancement of innate immune responses by mediating isg15 s-nitrosylation. 4ehp is an mrna 5′cap structure-binding protein and acts as a translation suppressor by competing with eif4e for binding to the cap structure [149] . 4ehp is a target protein for isgylation and this modification substantially increases its cap structure-binding activity [96] . this is the first report that shows "gain of function" by ifn-induced isgylation, thus providing a mechanism by which a small fraction of any isgylated protein can generate profound biological effects in response to ifns or pathogen infections. interestingly, 4ehp fused with isg15 at either of its n-or c-terminus dramatically enhances the cap structure-binding activity, indicating the isg15 fusion protein can mimic the isgylated 4ehp. since ifns inhibit the translation of viral mrnas while allowing normal translation of the majority of cellular mrnas [150] , it is possible that isgylated 4ehp acts as a viral mrnaspecific translation inhibitor in a cap binding-dependent manner. rig-i senses intracellular virus-specific nucleic acid structures and as an early antiviral response induces the production of type i ifns, which in turn activates the expression of rig-i, thus generating a positive feedback loop for further accumulation of rig-i [151] [152] [153] [154] [155] . intracellular dsrna activates nf-κb and irf-3/irf-7 through rig-i and the mitochondrial adaptor protein ifn promoter stimulator 1 (ips-1) [156] [157] [158] [159] . furthermore, rig-i is a target protein for isgylation [115, 160] . however, isgylation of rig-i leads to a reduction in the levels of both basal and virus-induced ifn production. consistently, the basal mrna and protein levels of rig-i are significantly higher in ube1l −/− cells than in ube1l +/+ cells. based on these observations, it has been proposed that a negative feedback loop is operating for fine-tuning the strength of rig-i-mediated signaling to maintain a balance between innate immune response and hypersensitivity during antiviral responses. in addition, ubiquitination of rig-i by trim25 is required to mediate antiviral signaling responses, whereas that by rnf125 results in the proteasomal degradation of rig-i [161] . thus, multiple positive and negative mechanisms appear to contribute to the maintenance of optimal rig-i level and thus to the control of rig-i-mediated signaling. viruses escape from the antiviral activity of isg15 by using different mechanisms. influenza b virus strongly induces isg15 during infection but specifically blocks isgylation of host proteins [18] . this inhibition is mediated by the viral ns1b protein, which interacts with isg15 and prevents the generation of isg15 conjugates under in vitro conditions as well as in infected cells (fig. 4) . purified ns1b inhibits the formation of isg15-ube1l-like thioester intermediate. isg15 conjugates accumulate in ifn-treated cells, but not in cells infected by influenza b virus, although similar amounts of isg15 are synthesized in both cells. moreover, isg15 conjugates are not detectable in cells infected with the virus expressing full-length ns1b but are markedly accumulated in cells infected with the virus expressing truncated ns1b, which cannot bind to isg15, indicating that the interaction of ns1b with isg15 is responsible for the inhibition of isg15 conjugation and thus of the antiviral function of isg15. sars-coronavirus (sars-cov) produces a papain-like protease, called plpro, which can generate an authentic isg15 molecule by cleaving off a protein that is fused to the c-terminus of isg15 [162] . it is possible that the activity of plpro may mimic the isg15-deconjugating activity of ubp43, thus favoring viral replication by counteracting protein isgylation. vaccinia virus (vacv) e3 protein is an early protein that interacts with isg15 through its c-terminal domain [28] . whereas sirnamediated knockdown of isg15 enhances the viral replication, complementation of isg15 to isg15 −/− cells attenuates it. notably, the level of isg15 conjugates is much higher in isg15-complemented isg15 −/− cells infected with the e3-deficient virus than that with the wild-type virus, suggesting that vacv e3 protein is directly or indirectly involved in deconjugation of isg15 from cellular proteins. moreover, the virus lacking e3 protein that is unable to grow in isg15 +/+ cells can replicate in isg15 −/− cells. these findings suggest a new strategy for poxviruses to evade the host antiviral response through the viral e3 protein-mediated control of protein isgylation. the ovarian tumor domain (otu)-containing proteases from nairoviruses and arterviruses, two unrelated groups of rna viruses, are capable of deconjugating ubiquitin and isg15, but not sumo, from cellular target proteins [27] . purified otu protease cleaves off ubiquitin from both lys48-and lys63-linked poly-ubiquitin chains in vitro. remarkably, the expression of otu protease antagonizes the antiviral effects of isg15 and enhances the susceptibility to snv infection. it has been shown that approximately 70% of ifnar −/− mice survive after infection of the virus expressing both isg15 and an otu variant, of which the catalytic cys residue is replaced by ala. in contrast, only 20% of the mice survive when infected with the virus expressing both isg15 and wild-type otu protease, indicating that the antiviral response is mediated by isg15, but not by other isgs, and that the immune-evading effect of otu protease requires its catalytic activity. these findings indicate that snv uses otu protease as a unique strategy to evade the host antiviral response. hepatitis c virus (hcv) is an enveloped virus that causes liver diseases, including cancer [163, 164] . rig-i signaling induces a host response that controls hcv rna replication [165] . however, hcv can evade this response in part through its ability to antagonize the relay of rig-i signaling to irf-3 [166] . ns3/4a, a major serine protease expressed by hcv [164] , cleaves ips-1, causing the release of cleaved ips-1 from the mitochondrial membrane [167, 168] . this cleavage results in the subcellular redistribution of ips-1 and loss of its interaction with rig-i, thereby preventing downstream activation of irf-3 and ifnβ induction. intriguingly, in liver tissues chronically infected by hcv, the ips-1 cleavage and its subcellular redistribution are associated with the lack of isg15 and its conjugates. among the hcvinfected tissues, isg15 and its conjugates can be detected only in the liver from patients, which has predominantly uncleaved, full-length ips-1 protein. these findings indicate that ns3/4a plays a critical role in evading the host antiviral response by attenuating the ifn amplification loop of rig-i and thus the expression of isgs, including isg15, which are normally induced by rig-i-and irf-3-dependent pathways. 4.6. isg15 in tumorigenesis 4.6.1. isg15 as a tumor suppressor ube1l has been shown to play an important role in the suppression of lung cancer growth. ube1l overexpression inhibits the growth of human bronchial epithelial cells and lung cancer cells. furthermore, ube1l promotes cyclin d1 isgylation and this modification leads to the destabilization of cyclin d1, indicating that ube1l confers growth suppression by targeting cyclin d1 [169] . these findings provide a mechanism for the tumor-suppressive role of ube1l [37, 73, 77, 170] , although it is unknown how the stability of cyclin d1 is affected by its isgylation. acute promyelocytic leukemia (apl) is characterized by the accumulation of oncogenic pml/rarα fusion protein [171, 172] . retinoic acid induces ube1l and subsequent isgylation of pml/rarα [33, 34, 37] . moreover, ube1l-mediated isgylation of pml/rarα leads to a decrease in the level of pml/rarα, thus overcoming the oncogenic effects of the fusion protein [173, 174] . on the other hand, treatment with n-acetyl-leu-leu-norleucinal, a proteasome inhibitor, prevents the degradation of isgylated pml/rarα, indicating that the proteasomes are involved in the control of pml/rarα stability. these findings implicate an important role of ube1l in the suppression of leukemia. in many tumors and tumor cell lines, isg15 is highly elevated and extensively conjugated to cellular proteins [39] . the level of isg15 mrna is significantly higher in cancerous mammary epithelial cells lines, such as bt20, mda-mb468, mda-mb231, t47d, and mcf7, than in nonmalignant mammary cell lines, including hmec and mcf10a. the level of isg15 protein is also higher in breast tumors than in normal tissues [175] . in addition, a correlation between increased isg15 level and unfavorable prognosis in the survival of patients has been reported, suggesting a potential role of isg15 in breast cancer development, although the role of isg15 in breast carcinogenesis is unknown. significantly, the increased level of isg15 in tumor cells is associated with the decreased levels of ubiquitinated proteins, suggesting that isg15 plays an antagonistic role against ubiquitin-mediated proteolysis and that deregulation of ubiquitin-proteasome pathway is related with tumorigenesis [39] . since some of the e2 (e.g., ubch8) and e3 enzymes (e.g., efp) can utilize both ubiquitin and isg15 for conjugation to target proteins, it seems possible that isg15 may potentially interfere with the ubiquitination pathway at the level of e2 and e3 enzymes, thus decreasing the level of ubiquitinated proteins in tumor cells. however, it has been shown that the level of ubiquitin conjugates in ubp43 −/− cells is nearly the same as that in ubp43 +/+ cells [116] , despite the fact that treatment with type i ifns leads to a dramatic accumulation in the level of isg15 conjugates in ubp43 −/− cells as compared with that in ubp43 +/+ cells. furthermore, treatment with lactacystin, an inhibitor of the proteasome, shows little or no effect on the level of isg15 conjugates in either of ubp43 +/+ or ubp43 −/− cells whether or not exposed to ifnβ, while as expected the level of ubiquitinated proteins markedly increases in both cells. thus, it appears that isg15 and its conjugates by themselves do not interfere with the ubiquitination of cellular proteins and their subsequent degradation. the reason for the tumor-specific overexpression of isg15 in different tumors is unclear. one possibility is that the elevated expression of isg15 may be due to constitutively activated nf-κb in many tumor cells. this possibility is supported by an observation that isg15overexpressing zr-75-1 cells show a greater nf-κb activity than isg15-underexpressing bt474 breast cancer cells [176] . alternatively, the deregulated expression of ube1l and ubp43 in certain tumors could contribute to the variation in the levels of isg15 conjugates in tumor biopsy samples. androgen receptor has been implicated in the initiation and progression of prostate cancer [177, 178] . significantly, ube1l and ubch8 are upregulated in prostate cancer lesions as compared to those in corresponding nonmalignant tissues [179] . moreover, overexpression of ube1l in lncap cells leads to a marked increase in the mrna and protein levels of androgen receptor in addition to an increase in the rate of cell proliferation. on the other hand, sirna-mediated knockdown of isg15 and ube1l in lncap cells results in a significant decrease in the transcript and protein levels of androgen receptor. these findings suggest that isgylation machinery participates in a positive control of androgen receptor expression during the onset of prostate cancers, thereby promoting the tumor growth. since the discovery of ifns, a vast knowledge on the role of the cytokines in innate immune responses has been accumulated. isg15 is one of the most strongly induced isgs upon exposure to type i ifns, virus, lps, and other stresses, including certain genotoxic stresses [16, 17, 35, 56, 64, 180] . considering that type i ifns play critical roles in innate immune responses regulating the antiviral responses as well as the cancer development, there is no doubt that isg15 and its conjugation to target proteins play critical roles in the type i ifninduced immune responses. however, the biological significance of protein modification by isg15 has been established only in several cases. therefore, much studies are required for clarification of the role of isg15 in innate immune responses against viral infection and cancer and thus for the control of immune diseases as well as for resolving the contradictory findings, such as the role of isg15 as a tumor suppressor versus an oncogenic protein. clinical uses of interferons virus interference: i. the interferon jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins contribution of stat sh2 groups to specific interferon signaling by the jak-stat pathway a single phosphotyrosine residue of stat91 required for gene activation by interferon-gamma stat proteins and transcriptional responses to extracellular signals the jak-stat pathway: cytokine signalling from the receptor to the nucleus stats and gene regulation antiviral actions of interferons type i interferons in host defense evolution and function of ubiquitin-like protein-conjugation systems isg15, not just another ubiquitin-like protein the ubiquitin system protein regulation by monoubiquitin ubiquitin and its kin: how close are the family ties? accumulation of an mrna and protein in interferon-treated ehrlich ascites tumour cells interferon induces a 15-kilodalton protein exhibiting marked homology to ubiquitin influenza b virus ns1 protein inhibits conjugation of the interferon (ifn)-induced ubiquitin-like isg15 protein induced expression of the endogenous beta interferon gene in adenovirus type 5-transformed rat fibroblasts mice lacking the isg15 e1 enzyme ube1l demonstrate increased susceptibility to both mouse-adapted and non-mouse-adapted influenza b virus infection interferon-induced isg15 conjugation inhibits influenza a virus gene expression and replication in human cells virgin, ifn-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses role of interferon-stimulated gene isg-15 in the interferon-omega-mediated inhibition of human immunodeficiency virus replication innate antiviral response targets hiv-1 release by the induction of ubiquitin-like protein isg15 isg15 inhibits ebola vp40 vlp budding in an l-domain-dependent manner by blocking nedd4 ligase activity isg15 inhibits nedd4 ubiquitin e3 activity and enhances the innate antiviral response ovarian tumor domain-containing viral proteases evade ubiquitin-and isg15-dependent innate immune responses vaccinia virus e3 protein prevents the antiviral action of isg15 interferons alpha and beta as immune regulators-a new look apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis cancer immunotherapy: the interferon-alpha experience the interferon regulated ubiquitin-like protein, isg15, in tumorigenesis: friend or foe? involvement of ube1l in isg15 conjugation during retinoid-induced differentiation of acute promyelocytic leukemia ube1l is a retinoid target that triggers pml/ raralpha degradation and apoptosis in acute promyelocytic leukemia camptothecin induces the ubiquitinlike protein, isg15, and enhances isg15 conjugation in response to interferon interferon potentiates antiproliferative activity of cpt-11 against human colon cancer xenografts ube1l represses pml/rar{alpha} by targeting the pml domain for isg15ylation stage-associated overexpression of the ubiquitin-like protein, isg15, in bladder cancer elevated expression of isg15 in tumor cells interferes with the ubiquitin/26s proteasome pathway exploration of global gene expression patterns in pancreatic adenocarcinoma using cdna microarrays crystal structure of the interferon-induced ubiquitin-like protein isg15 a mhcencoded ubiquitin-like protein (fat10) binds noncovalently to the spindle assembly checkpoint protein mad2 the ubiquitin-like protein fat10 forms covalent conjugates and induces apoptosis fat10, a ubiquitinindependent signal for proteasomal degradation a 15-kda interferon-induced protein is derived by cooh-terminal processing of a 17-kda precursor molecular cloning of the fish interferon stimulated gene, 15 kda (isg15) orthologue: a ubiquitin-like gene induced by nephrotoxic damage the interferon-inducible 15-kda ubiquitin homolog conjugates to intracellular proteins different roles for two ubiquitin-like domains of isg15 in protein modification virgin, isg15 arg151 and the isg15-conjugating enzyme ube1l are important for innate immune control of sindbis virus interferoninduced transcription of a gene encoding a 15-kda protein depends on an upstream enhancer element gene induction pathways mediated by distinct irfs during viral infection identification of a member of the interferon regulatory factor family that binds to the interferonstimulated response element and activates expression of interferon-induced genes characterization of specific dna-binding factors activated by double-stranded rna as positive regulators of interferon alpha/beta-stimulated genes interferon regulatory factor 3 and crebbinding protein/p300 are subunits of double-stranded rna-activated transcription factor draf1 direct induction of interferon-gamma-and interferon-alpha/beta-inducible genes by double-stranded rna novel nemo/ikappab kinase and nf-kappa b target genes at the pre-b to immature b cell transition bacterial lipopolysaccharide and gamma interferon induce transcription of beta interferon mrna and interferon secretion in murine macrophages lipopolysaccharide activates the expression of isg15-specific protease ubp43 via interferon regulatory factor 3 serpin 2a is induced in activated macrophages and conjugates to a ubiquitin homolog enhanced antibacterial potential in ubp43-deficient mice against salmonella typhimurium infection by up-regulating type i ifn signaling ataxia-telangiectasia: an inherited disorder of ionizing-radiation sensitivity in man. progress in the elucidation of the underlying biochemical defect correction of radiation sensitivity in ataxia telangiectasia cells by a truncated i kappa b-alpha elevation of interferon beta-inducible proteins in ataxia telangiectasia cells 14-3-3 sigma is a p53-regulated inhibitor of g 2 /m progression a model for p53-induced apoptosis role for p53 in gene induction by doublestranded rna integration of interferon-alpha/beta signalling to p53 responses in tumour suppression and antiviral defence differential gene regulation by specific gain-offunction jnk1 proteins expressed in swiss 3t3 fibroblasts dual regulatory roles of phosphatidylinositol 3-kinase in ifn signaling gene expression profiling during all-trans retinoic acid-induced cell differentiation of acute promyelocytic leukemia cells gene expression networks underlying retinoic acid-induced differentiation of acute promyelocytic leukemia cells stat1 is induced and activated by all-trans retinoic acid in acute promyelocytic leukemia cells a gene in the chromosomal region 3p21 with greatly reduced expression in lung cancer is similar to the gene for ubiquitin-activating enzyme the basis for selective e1-e2 interactions in the isg15 conjugation system ube1l and protein isgylation are not essential for alpha/beta interferon signaling deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes the ubiquitin-activating enzyme e1-like protein in lung cancer cell lines alteration of tumor spectrum by isgylation in p53-deficient mice deficiency of a potential 3p21.3 tumor suppressor gene ube1l (uba7) does not accelerate lung cancer development in k-rasla2 mice proteome analysis reveals ubiquitin-conjugating enzymes to be a new family of interferon-alpharegulated genes interferon-inducible ubiquitin e2, ubc8, is a conjugating enzyme for protein isgylation the ubch8 ubiquitin e2 enzyme is also the e2 enzyme for isg15, an ifn-alpha/beta-induced ubiquitin-like protein link between the ubiquitin conjugation system and the isg15 conjugation system: isg15 conjugation to the ubch6 ubiquitin e2 enzyme physical interaction between specific e2 and hect e3 enzymes determines functional cooperativity staring, a novel e3 ubiquitin-protein ligase that targets syntaxin 1 for degradation parkin suppresses unfolded protein stress-induced cell death through its e3 ubiquitin-protein ligase activity the histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of hdac2 a novel centrosomal ring-finger protein, dorfin, mediates ubiquitin ligase activity efp targets 14-3-3 sigma for proteolysis and promotes breast tumour growth parkin functions as an e2-dependent ubiquitin-protein ligase and promotes the degradation of the synaptic vesicle-associated protein, cdcrel-1 ubch8 regulates ubiquitin and isg15 conjugation to rig-i the interferon-inducible ubiquitin-protein isopeptide ligase (e3) efp also functions as an isg15 e3 ligase novel growth and death related interferon-stimulated genes (isgs) in melanoma: greater potency of ifn-beta compared with ifn-alpha2 a ubiquitin e3 ligase efp is up-regulated by interferons and conjugated with isg15 negative regulation of isg15 e3 ligase efp through its autoisgylation isg15 modification of the eif4e cognate 4ehp enhances cap structure-binding activity of 4ehp identification and herc5-mediated isgylation of novel target proteins herc5 is an ifn-induced hect-type e3 protein ligase that mediates type i ifn-induced isgylation of protein targets herc5, an interferon-induced hect e3 enzyme, is required for conjugation of isg15 in human cells ubp43 (usp18) specifically removes isg15 from conjugated proteins role of isg15 protease ubp43 (usp18) in innate immunity to viral infection screen for isg15-crossreactive deubiquitinases precursor processing of pro-isg15/ucrp, an interferon-beta-induced ubiquitin-like protein rnase-l-dependent destabilization of interferon-induced mrnas. a role for the 2-5a system in attenuation of the interferon response cloning and characterization of human ubiquitin-processing protease-43 from terminally differentiated human melanoma cells using a rapid subtraction hybridization protocol rash a novel ubiquitin-specific protease, ubp43, cloned from leukemia fusion protein aml1-eto-expressing mice, functions in hematopoietic cell differentiation the isg15 isopeptidase ubp43 is regulated by proteolysis via the scfskp2 ubiquitin ligase cloning and characterization of a novel human ubiquitin-specific protease, a homologue of murine ubp43 (usp18) dysregulation of protein modification by isg15 results in brain cell injury reexamination of the role of ubiquitin-like modifier isg15 in the phenotype of ubp43-deficient mice protein isgylation modulates the jak-stat signaling pathway ubp43 is a novel regulator of interferon signaling independent of its isg15 isopeptidase activity the level of hepatitis b virus replication is not affected by protein isg15 modification but is reduced by inhibition of ubp43 (usp18) expression ubp43 regulates bcr-abl leukemogenesis via the type 1 interferon receptor signaling human isg15 conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways high-throughput immunoblotting. ubiquitiin-like protein isg15 modifies key regulators of signal transduction proteomic identification of proteins conjugated to isg15 in mouse and human cells ifn-induced 15-kda protein is released from human lymphocytes and monocytes a human 15-kda ifn-induced protein induces the secretion of ifn-gamma immunoregulatory properties of isg15, an interferon-induced cytokine filamins as integrators of cell mechanics and signalling structural and functional aspects of filamins filamin b serves as a molecular scaffold for type i interferon-induced c-jun nh2-terminal kinase signaling pathway filamin b: a scaffold for interferon signalling isg15 modification of filamin b negatively regulates the type i interferon-induced jnk signalling pathway regulation of the tak1 signaling pathway by protein phosphatase 2c protein phosphatase 2cbeta association with the ikappab kinase complex is involved in regulating nf-kappab activity signaling to nf-kappab nf-kappab: linking inflammation and immunity to cancer development and progression negative regulation of protein phosphatase 2cbeta by isg15 conjugation isg15 modification of ubiquitin e2 ubc13 disrupts its ability to form thioester bond with ubiquitin isg15 modification of ubc13 suppresses its ubiquitinconjugating activity molecular insights into polyubiquitin chain assembly: crystal structure of the mms2/ubc13 heterodimer crystal structure of the human ubiquitin conjugating enzyme complex isg15, an interferonstimulated ubiquitin-like protein, is not essential for stat1 signaling and responses against vesicular stomatitis and lymphocytic choriomeningitis virus virgin, identification of interferon-stimulated gene 15 as an antiviral molecule during sindbis virus infection in vivo a ppxy motif within the vp40 protein of ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies role for amino acids 212klr214 of ebola virus vp40 in assembly and budding overlapping motifs (ptap and ppey) within the ebola virus vp40 protein function independently as late budding domains: involvement of host proteins tsg101 and vps-4 isg15 enhances the innate antiviral response by inhibition of irf-3 degradation modification of proteins by ubiquitin and ubiquitin-like proteins protein modification by sumo inos (nos2) at a glance perspectives series: host/pathogen interactions. mechanisms of nitric oxide-related antimicrobial activity reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens protein snitrosylation: purview and parameters nitrosylation of isg15 prevents the disulfide bond-mediated dimerization of isg15 and contributes to effective isgylation a new paradigm for translational control: inhibition via 5′-3′ mrna tethering by bicoid and the eif4e cognate 4ehp mechanisms of type-i-and type-ii-interferon-mediated signalling pathogen recognition and innate immunity species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 small anti-viral compounds activate immune cells via the tlr7 myd88-dependent signaling pathway 5′-triphosphate rna is the ligand for rig-i tlr3 in antiviral immunity: key player or bystander? ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 visa is an adapter protein required for virus-triggered ifn-beta signaling negative feedback regulation of rig-imediated antiviral signaling by interferon-induced isg15 conjugation negative regulation of the rig-i signaling by the ubiquitin ligase rnf125 the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme global epidemiology of hepatitis c virus infection unravelling hepatitis c virus replication from genome to function regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i regulation of interferon regulatory factor-3 by the hepatitis c virus serine protease control of antiviral defenses through hepatitis c virus disruption of retinoic acid-inducible gene-i signaling viral and therapeutic control of ifnbeta promoter stimulator 1 during hepatitis c virus infection ube1l causes lung cancer growth suppression by targeting cyclin d1 proteasomes modulate conjugation to the ubiquitinlike protein, isg15 pulsed-field gel electrophoresis analysis of retinoic acid receptor-alpha and promyelocytic leukemia rearrangements. detection of the t(15;17) translocation in the diagnosis of acute promyelocytic leukemia chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses rar alpha with a novel putative transcription factor accelerated degradation of pml-retinoic acid receptor alpha (pml-rara) oncoprotein by all-trans-retinoic acid in acute promyelocytic leukemia: possible role of the proteasome pathway caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia pml/raralpha fusion protein the ubiquitinlike molecule interferon-stimulated gene 15 (isg15) is a potential prognostic marker in human breast cancer isg15 as a novel tumor biomarker for drug sensitivity prostatic intraepithelial neoplasia in mice expressing an androgen receptor transgene in prostate epithelium androgeninduced differentiation and tumorigenicity of human prostate epithelial cells expression, regulation and function of the isgylation system in prostate cancer identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays we thank mr. sangman michael kim for his critical reading of this article. this work was supported by grants from the krf (krf-2005-084-c00025) and kosef (m10533010001-05n3301). we apologize for the event that any relevant publications were inadvertently omitted. key: cord-010776-ura14oci authors: hosseini, elahe seyed; zeinoddini, mehdi; saeedinia, ali reza; babaeipour, valiollah title: optimization and one-step purification of recombinant v antigen production from yersinia pestis date: 2020-01-02 journal: mol biotechnol doi: 10.1007/s12033-019-00234-x sha: doc_id: 10776 cord_uid: ura14oci the purpose of this study was to develop an efficient and inexpensive method for the useful production of recombinant protein v antigen, an important virulence factor for yersinia pestis. to this end, the synthetic gene encoding the v antigen was subcloned into the downstream of the intein (int) and chitin-binding domain (cbd) from the ptxb1 vector using specific primers. in the following, the produced new plasmid, ptx-v, was transformed into e. coli er(2566) strain, and the expression accuracy was confirmed using electrophoresis and western blotting. in addition, the effects of medium, inducer, and temperature on the enhancement of protein production were studied using the taguchi method. finally, the v antigen was purified by a chitin affinity column using int and cbd tag. the expression was induced by 0.05 mm iptg at 25 °c under optimal conditions including tb medium. it was observed that the expression of the v-int–cbd fusion protein was successfully increased to more than 40% of the total protein. the purity of v antigen was as high as 90%. this result indicates that v antigen can be produced at low cost and subjected to one-step purification using a self-cleaving int tag. yersinia pestis has evolved from gastrointestinal pathogens and its antibiotic resistance can cause a dangerous disease, i.e., plague. therefore, plague is still considered as a major threat to human health [1] [2] [3] . the human plague vaccine (usp) is a killed whole-cell plague bacilli that prevents plague infections through subcutaneous injection [4] . however, some reports have recently demonstrated the cytotoxic effects of whole-cell vaccines and their poor protection against virulent strains without capsules [5, 6] . the low calcium response (lcr) of v antigen (lcrv) and the component 1 (f1) capsular antigen are the two important virulence factors which have been considered as vaccine candidates tested for their efficacy on humans and primates. lcrv is known as the virulence and multifunctional protein. this crucial protein has been shown to act at the level of secretion control by binding to other proteins in order to modulate the host immune response by altering cytokine production [7, 8] . genetic engineering can be used to produce recombinant vaccines using different parts of yersinia, such as v antigen and f1 [9, 10] . to this purpose, the selection of effective methods to recombinant protein purification is a key factor of production. highly purified recombinant proteins with the lowest and shortest time are the major problems for biotechnology researchers. intein (int) is the internal part of the protein that is derived from immature proteins during protein splicing process [11] [12] [13] . since the discovery of the int feature in 1990, this fusion tag has been used in many biotechnology applications. one of its wonderful applications is in the int-tagged self-cleaving step to purify recombinant proteins [14, 15] . the int tag is separated from the target protein by thiol induction, as well as by temperature and ph changes for one-step protein purification. the advantage of this technique over other protein purification procedures is that the protease phase is eliminated, making the method economically affordable [16] [17] [18] . thus, in the commercial ptxb1 and ptwin vectors, intein is fused to the chitin-binding domain (cbd). the expressed fusion protein can be ligated to the chitin beads and then, ph, 1 3 temperature changes, or thiol induction leads to cleavage and separation of the target protein from int tag and cbd. finally, the target protein can be purified from the column without any additional amino acid [19, 20] . on the other hand, the traditional reaction optimization method involves the influence of one variable factor at a time. this method requires costly, time-consuming, and laborious experiments. statistical methods including response surface and taguchi methodologies are usually used to optimize the effective factors in increasing the production of recombinant proteins. in response surface method, the relationship between the independent factors and the effective response can be evaluated in designed experiments to predict, with more tests than taguchi approach [21, 22] . in turn, in taguchi methodology, more variables and qualitative factors can be investigated [23, 24] . generally, when the cost and time limitations make it difficult to perform more experiments in the optimization process and also when discrete or qualitative factors such as media culture types are investigated, taguchi design is preferred in bench-scale fermenter [25] . in this work, the int tag was used to purify v antigen from y. pestis in order to develop a new purification strategy for this important protein. in addition, detailed studies were conducted to find optimal conditions of temperature, medium, inducer concentrations, and overexpressed v-int-cbd fusion protein using the taguchi method. to amplify the v antigen encoding sequence, specific primers were designed based on the v antigen gene sequence retrieved from gene bank (accession no. af167310.1). the ndei enzyme restriction site sequence was added to the forward primer (5ʹ-ggt ggt cat atg att agag cct ac gaac-3ʹ) and sapi enzyme restriction site sequence was also added to the reverse primer (5ʹ-ggt ggt t gctct tccgc att tac cag acg tgt cat c-3ʹ). the pet-v (pet28a containing the v antigen encoding sequence) was amplified using the polymerase chain reaction (pcr). the pcr mixture (25 μl) contained 1 × pcr buffer, 4 mm magnesium sulfate, 300 mm of each dntp, 40 pmol per primer, 5 μl (1 ng) v antigen in pet28 vector, and 0.2 unit pfu dna polymerase (fermentase). the amplification was performed using the techne thermocycler, with initial denaturation at 94 °c for 4 min, 35 cycles at 94 °c for 60 s, 30 s at 53 °c, and 90 s at 72 °c, and the final extension was performed at 72 °c for 10 min. the pcr product was analyzed using 1% agarose gel electrophoresis. the pcr product was purified using a gel purification kit (bioneer). the pcr product and the ptxb1 vector (neb #n6707, biolab) were double-digested with sapi and ndei enzymes and then ligated together with t4 dna ligase. the cloning of v antigen in the ptxb1 vector was verified by restriction enzyme mapping. after confirming the ptx-v construct (fig. 1c) , the plasmid was transformed into the competent er 2566 strain of e. coli, prepared by the calcium chloride method [24] . the transformed bacteria were grown in a 1-l lysogeny broth (lb) containing 100 μg/ml ampicillin at 37 °c in an air shaker (250 rpm) until the optical density (od600) at 600 nm reached 0.5-0.6. then, the t7 promoter was induced by 0.3 mm isopropyl β-d-1-thiogalactopyranoside (iptg) at 16 °c for 16 h (to increase the soluble protein). the expression of v antigen was assessed by sds-page and western blotting at different times using specific antichitin-binding domain serum antibodies (neb #s6654, new england biolabs inc.). the cells were harvested and resuspended in 100 ml of column buffer (20 mm tris-hcl, ph 8.0, 0.5 m nacl) and the crude cell extracts were prepared by sonication for 10 cycles of 30 s (hielscher ultrasonic, germany). the supernatant was separated from the cell debris by centrifugation at 12,000×g for 30 min at 4 °c and passed through a 1 × 10 cm column (bio-rad, hercules, ca) containing 10 ml of chitin beads (neb #s6651). the flow rate was 0.5 ml/min. after loading the supernatant on the column, the flow rate was increased to 2 ml/min, and the column was thoroughly washed with the column buffer until the eluted non-specific protein content reached a minimum. thereafter, a column buffer containing 50 mm dithiothreitol (dtt) was slowly passed through the column, the flow was stopped, and the column was incubated at room temperature for 16 to 40 h. each fraction (1 ml) containing v antigen was obtained by eluting the column with the column buffer. all samples were analyzed by sds-page using 12% tris-glycine gel. the protein concentration was estimated using the bradford method. after purification, dtt was removed from the buffer and the protein was concentrated using a millipore centricon tube. the enhancement of recombinant protein production and subsequently, the purification of the target protein, the effects of temperature on the induction (15, 20, and 25 °c), media (terrific broth or tb, contains tryptone 1.2%, yeast extract 2.4%, and glycerol 0.5%; supper broth or sb which contains tryptone 3%, yeast extract 2%, mops 1%, and glucose 2%; and 32y which contains tryptone 0.8%, yeast extract 3.2%, and nacl 0.58% in 10 mm tris-hcl, ph 7.6), and inducer concentration (0.05, 0.1 and 0.15 mm) were investigated using the taguchi statistical method [20] ( table 1) . the taguchi method is used to evaluate the factors considered. this method allows the simultaneous study of different effective factors and their interactions. for this purpose, the experiments were designed on the l9 orthogonal array ( table 2 ) based on which each proposed test condition was repeated twice. then the results (final concentration of the recombinant protein) were analyzed using minitab-16 software. taguchi uses analysis techniques that do not depend on a model that relates the response to the factor effects. rather, it uses a calculated response average to identify factors and their levels that yield maximum quality or quantity of the considered response (in this research the amount of recombinant protein). to compute the average performance or mean effect of any factor, amount of recombinant protein of experiments related to any level of each parameter are added and divided by the number of such trials. after the pcr reaction, the product was confirmed by the emergence of a 981 bp band on 1% agarose gel electrophoresis at about 981 bp (fig. 1a) . to clone the v antigen in ptxb1, the pcr product of the previous step and ptxb1 were digested by sapi and ndei restriction enzymes, and the product was ligated into the ptxb1. two colonies were randomly selected and ptxb1-v double digestion was performed using psti and ndei enzymes (we could not use sapi enzyme because this site was disrupted after cloning, and the restriction site of psti is present on ptxb1 but not in the sequence of the cloned v antigen). according to fig. 1b , the emergence of a 1772 bp band on 1% agarose gel electrophoresis confirmed the production of a new plasmid ptxb1-v (fig. 1c) . the ptxb1-v plasmid was transferred into e. coli er2566 and induced by iptg. the protein expression was assessed by using sds-page and western blotting at 0 and 4 h postinduction. a protein size of about 65-kda confirmed the expression of v antigen together with int and cbd (fig. 2) in which the molecular weight of int and cbd was about 34 kda and the molecular weight of v antigen was about 33 kda. the v-int-cbd fusion protein was purified using chitin beads affinity chromatography. to this end, the lysed cells were passed through a column packed with chitin beads. after washing and removal of non-specific proteins, most of the fusion precursors were attached to the resin due to the high affinity of cbd to chitin beads. a lysis buffer containing 50 mm dithiothreitol (dtt) as a cleavage disulfide bond was added into the column and incubated at room temperature for 16 h to release v antigen. in this step, int and cbd were bound to chitin column. finally, to remove int-cbd from the resin, a 2% sds stripping buffer was flowed through, and cbd and int (34 kda) were eluted from the chitin resin. figure 3 shows the purification step of v antigen with an int tag on sds-page. the results of recombinant protein optimization using the taguchi method are shown in table 3 . table 4 shows the analysis of variance (anova) for the responses of recombinant protein production carried out according to the factors' contribution (4), the output after passing the cell lysis from column (5), output of washing column with buffer (6), initial output after induction by cleavage buffer (7), purified protein after 40 h (8, 9, and 10), the chitin beads after cleavage step (11), output after striping step (12) by the taguchi method. in addition, the optimal conditions were obtained by analyzing the final concentration of the recombinant fusion protein and the variance of productivity (anova). the average effect of the investigated four factors has been plotted in fig. 4 for visual inspection. the higher the slope of the graph of each factor in these figures indicates the amount of factor effect on the amount of produced protein. hence, it can be concluded that the type of medium had the greatest influence on the production of the recombinant fusion protein. also, the induction temperature had the greatest effect impact on the protein production, while iptg concentration had the least effect (fig. 4) . furthermore, the results showed that test 7 had the optimum conditions for the production of an int-v antigen fusion protein in e. coli. figure 5 shows the effect of different conditions including (1, 9) , test level one (2), two (3), three (4), protein size marker (5) , test level four (6) , five (7), six (8) , seven (10), eight (11) and nine (12) temperature, iptg concentration, and medium on the v antigen expression. after optimizing the production conditions, the cells were incubated in the cleavage buffer after incubation at 4 °c for 16 h to obtain a protein with a high purity of 90% and a suitable efficiency, as shown in fig. 6 . there is no safe or effective plague vaccine. the world health organization classifies the plague as a recurrent disease and as a class a pathogen causing an epidemic disease [26] . killed whole-cell vaccines can only prevent pests and live attenuated vaccines, making guinea pigs and mice immune to plague, although this vaccine has health problems for humans [27, 28] . today, researchers emphasize subunit vaccine development. for decades, humans have been focusing on two recombinant proteins, yersinia pestis, f1 and v proteins, as vaccine candidates [9, 10] . in order to facilitate the purification of recombinant proteins, soluble and affinity tags, such as schistosomiasis glutathione s-transferase (gst), escherichia coli maltose-binding protein (mbp), transcription termination anti-termination factor (nusa), and peptide tags, such as poly his and poly arg, are often used [29] [30] [31] [32] . since the affinity tag can affect the activity and/or structure of the target protein, it is typically cleaved from the target protein by a protease. therefore, the isolation of protease and target protein fusion markers requires some additional purification steps [33] . in addition, proteases are generally inefficient and non-specific, resulting in partially cleaved products. in an intein (int)-based purification system, a modified int fusion that induces a cleavage reaction at one end is catalyzed between the chitin-binding domain (cbd) and the recombinant target protein [34] . using the natural self-splicing mechanism of int, genetic engineers are able to produce commercial int vectors to obtain recombinant proteins that do not require enzymatic or cofactor removal labels [35] . the most important feature of int tag in recombinant protein production is its self-cleavability with the addition of amino acids at the end of the target protein [36] . furthermore, this method does not require proteases enzyme and time consuming steps for the removal of target proteins. wood and co-authors believed that this method combined with other methods not only reduces the time and cost of recombinant protein production but also improves the efficiency of the final product [17, 37, 38] . co-expression of v antigen with a suitable carrier protein results in the protease being less susceptible to degradation during purification [39] . for this purpose, different expression systems are used to increase the production of v antigen. v antigen with gst and int-cbd was produced in a relatively fused form in 2000 using three expression systems and after cleavage and purification of the target protein, its final concentration was 25 (pgex-5x-2), 31 (pgex-6p-2), and 0.75 mg/l [40] . in the present study, we first cloned and optimized the expression and purification of v antigen in the ptxb1 vector containing the 28-kda-int tag and 6-kda-cbd. v antigen production was examined using the int tag to assess the self-cleavage properties of int and to optimize culture conditions to increase protein production. the results of the taguchi method showed that the yield increased to 40% (1.28 g/l). recent studies have also shown that the molecular weight of hybrid proteins increases by 34 kd compared to the native protein, but it has no negative effect on protein expression. on the other hand, intthiol induction demonstrated suitable v antigen purification (fig. 5 ) and this strategy was used for large-scale production of proteins and int self-cleavage can be performed at 4 °c without dtt. in summary, the ptx-v expression system can be used as a suitable substitute for the industrial production of vaccines against sub-units of plague. the yersiniae: a model genus to study the rapid evolution of bacterial pathogens yersinia pestis-etiologic agent of plague genome sequence of yersinia pestis, the causative agent of plague vaccination against bubonic and pneumonic plague plague vaccine development: current research and future trends plague vaccines: current developments and future perspectives map of f1 and v antigens from yersinia pestis astride innate and adaptive immune response lcrv plague vaccine with altered immunomodulatory properties expression of a recombinant form of the v antigen of yersinia pestis, using three different expression systems recombinant v antigen protects mice against pneumonic and bubonic plague caused by f1-capsule-positive and -negative strains of yersinia pestis protein splicing involving the saccharomyces cerevisiae vma iintein: the steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system protein splicing: its discovery and structural insight into novel chemical mechanisms inteins: structure, function, and evolution intein-mediated purification of a recombinantly expressed peptide single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element a genetic system yields self-cleaving inteins for bioseparations inteins and affinity resin substitutes for protein purification and scale up overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems utilizing the c-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step the cyclization and polymerization of bacterially expressed proteins using modified self-splicing inteins the taguchi methodology as a statistical tool for biotechnological applications: a critical appraisal a primer on the taguchi method laccase production by pleurotusostreatus 1804: optimization of submerged culture conditions by taguchi doe methodology taguchi's experimental design for optimizing the production of novel thermostable polypeptide antibiotic from geobacillus pallidus sat4 enhanced production of insulin-like growth factor i protein in escherichia coli by optimization of five key factors molecular and cell biology aspects of plague yersinia pestis (plague) vaccines developing live vaccines against yersinia pestis enhancement of soluble protein expression through the use of fusion tags inteinmediated protein purification of fusion proteins expressed under high-cell density conditions in e. coli 6xhis-ni-nta chromatography as a superior technique in recombinant protein expression/purification expression and production of recombinant scorpine as a potassium channel blocker protein in escherichia coli gene fusion expression systems in escherichia coli intein as a novel strategy for protein purification cloning and expression of aequorin photoprotein using intein tag purification of antibacterial chap k protein using a self-cleaving fusion tag and its activity against methicillinresistant staphylococcus aureus the potential role of self-cleaving purification tags in commercial-scale processes new trends and affinity tag designs for recombinant protein purification active immunisation with recombinant v antigen from yersinia pestis protects mice against plague expression of a recombinant from of the v antigen of yersinia pestis, using three different expression systems publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank the research council of malek-ashtar university of technology for the financial support of this investigation. key: cord-298369-66ifwtlp authors: smith, sherri a.; waters, nigel j. title: pharmacokinetic and pharmacodynamic considerations for drugs binding to alpha-1-acid glycoprotein date: 2018-12-28 journal: pharm res doi: 10.1007/s11095-018-2551-x sha: doc_id: 298369 cord_uid: 66ifwtlp according to the free drug hypothesis only the unbound drug is available to act at physiological sites of action, and as such the importance of plasma protein binding primarily resides in its impact on pharmacokinetics and pharmacodynamics. of the major plasma proteins, alpha-1-acid glycoprotein (aag) represents an intriguing one primarily due to the high affinity, low capacity properties of this protein. in addition, there are marked species and age differences in protein expression, homology and drug binding affinity. as such, a thorough understanding of drug binding to aag can help aid and improve the translation of pharmacokinetic/pharmacodynamic (pk/pd) relationships from preclinical species to human as well as adults to neonates. this review provides a comprehensive overview of our current understanding of the biochemistry of aag; endogenous function, impact of disease, utility as a biomarker, and impact on pk/pd. experimental considerations are discussed as well as recommendations for understanding the potential impact of aag on pk through drug discovery and early development. according to the free drug hypothesis only the unbound drug is available to act at physiological sites of action, whether it is the intended pharmacological target, or action at an undesired site with potential toxicological consequences. the importance of plasma protein binding primarily resides in its impact on pharmacokinetic properties such as clearance (cl) and volume of distribution (v ss ), with serum albumin, lipoproteins and alpha-1 acid glycoprotein (aag) being the major proteins involved in sequestering drugs in plasma (1) . aag also known as orosomucoid (orm), is a member of the acute phase protein (app) family, first described in 1950 (2) . it has a single polypeptide chain consisting of 183 amino acids with five glycan chains, accounting for~45% of its total molecular weight (~41-44 kda) (3, 4) . aag is formed primarily in the liver and circulates from 0.5 to 1.0 mg/ml in the plasma of healthy humans (3) . levels in healthy animals are generally lower compared to healthy humans (table i) . in most disease states including inflammation, infection, and cancer, aag levels increase from 2 to 6-fold in humans (3) , and show a much broader fold of induction in animals from 2 to 20-fold depending on animal species and disease (table i) . while the biological role of aag remains unclear, it has been demonstrated to regulate immunity and play a role in both pro-and anti-inflammatory response (37, 38) . aag has long been used as a clinical biomarker, and the potential to expand its 2-3 c a n c e r 1.43 ± 0.65 (0. 71-2.27) infection 0.50 ± 0.14 (0.28-0.92) (32) application for disease diagnosis, prognosis, and characterization has grown given the recent advances in proteomics and high resolution mass spectrometry (39) (40) (41) (42) . while aag represents a relatively small portion (~1-3%) of the total plasma proteins, compared to~60% composition of albumin, it can play a significant role in drug binding and pharmacokinetics (pk) (43) . aag is considered a high affinity/low capacity plasma protein, whereas albumin is considered low affinity/high capacity. aag is a highly acidic protein with a very low isoelectric point (pi) ranging from 2.8 to 3.8 (37) . this property enables aag to bind mainly basic drugs (i.e. lidocaine, propranolol, verapamil) but it may also bind to neutral lipophilic molecules (i.e. steroid hormones) and to acidic drugs (i.e. phenobarbital), whereas albumin is mostly implicated in binding to the latter charge type (44, 45) . most drugs bind to both plasma proteins with varying degrees of affinity. the extensive and variable sialylation of aag is what drives the low and wide pi range, a property that can impact drug affinity, and ultimately pk (3). since aag levels increase in most disease states (46) , drugs with a high affinity may demonstrate higher binding (lower fraction unbound, f u ) and altered pk properties (e.g. lower total cl), lower v ss . given the known species differences in aag abundance and drug affinity there is a growing body of work where pk in preclinical species did not accurately predict pk in human, and several of these case studies are discussed herein. incorporating the ontogeny of aag may also enable more accurate predictions of pk in neonate and infant patients (47) . we provide a rationale for testing the extent and affinity of drug binding to aag and albumin in the drug discovery process to aid in prospective human pk prediction efforts (fig. 1) . research is still lagging in the characterization of higher species aag which could help better predict pk in human. furthermore, there are experimental factors that are emerging as critical to the accurate determination of aag-drug binding in vitro. these aspects are also discussed in this review. albumin, aag, and lipoproteins are considered the most relevant plasma proteins in terms of drug binding (1). the range of albumin levels is slightly lower in animals compared to human. while levels of aag are also relatively lower in most animals compared to human in the healthy state, levels are much more variable in the diseased setting as described below and in table i . human albumin and aag levels in table ii provide units of mg/ml and μm to serve as a quick reference when considering stoichiometry with drug concentrations, assuming a single drug binding site model. generally, in rodents (0.1-0.3 mg/ml) and mini-pigs (0.3-0.5 mg/ml) aag values are lower compared to human (table i ). an exception to this generalization was reported in mice, however no explanation was suggested by the authors (9) . in dogs, aag demonstrates highly variable levels (0.04-1 mg/ml) (25) , though generally lower mean values (0.25-0.5 mg/ml) have been reported (26) (27) (28) . there is little characterization of monkey aag in the literature, though it has also been reported to be lower in cynomolgus monkey (0.1 mg/ml) compared to human (30) . the conventional pig is the only species where aag levels have been reported to be higher (1.1-2.5 mg/ml) (14, 18) compared to human. however, this finding has not been consistently observed, lower aag values of 0.24 mg/ml (16) and 0.34 mg/ml (15) have also been reported in the conventional pig. in most species, aag behaves as a positive acute phase protein (app), increasing in response to stimuli, including infection, inflammation, and cancer (table i) . the acute phase response (apr) is considered part of the innate (non-specific) defense system that offers protection prior to the adaptive (specific) immune response. the magnitude of response can differ across species and disease setting. minor, moderate, and major aaps are characterized by increases in protein expression by 0.25-1, 1-10, and > 10-fold, respectively. in mice aag is considered a major positive app as levels increase up to 20-fold following stimuli (7, 8) , whereas in human the response is relatively moderate with increases of~2-6 fold (3, 31) . in the domestic cat, aag increases are moderate (~2-3 fold) in the oncology setting, whereas a more robust response has been reported with infections (up to 17-fold) (20) (21) (22) . mixed responses have been reported in the conventional pig and the minipig. in the conventional pig, aag has been shown to behave as both a negative app with levels declining to~0.6-0.7-fold normal values (14) and as a positive app in response to infection and inflammation, with levels increasing 3-6-fold (15) . in both conventional pigs (18) and minipigs (16, 48, 49) there was no aag response following acute stimuli (lps or turpentine injection), despite increases in other apps in the conventional pig (tnfα and il-6) and in minipigs (c-reactive protein (crp), serum amyloid a (saa), haptoglobin (hp), pig major acute phase protein (pmap)). christoffersen et al. (19) hypothesized the aag response in the minipig may have different sensitivity in the acute versus chronic setting, an observation previously reported for other apps (saa and hp) in cattle (49) . in a chronic inflammatory setting, obese and mildly diabetic minipigs fed a high fat diet had elevated (~2-fold) aag levels (19) . changing housing conditions also caused up to a 2-fold increase in aag in minipigs (as well as increases in crp, saa, hp and pmap), and it is unclear when levels return to baseline, a finding that should be considered in acclimation, experimental design and data interpretation (19) . neither the negative app response nor lps insensitivity of porcine aag have been reported in any other species to date. however, it is not unprecedented for a given app to behave differently across species. for example, while crp and saa are considered major positive apps in human and pig, crp and saa are not affected in mice and rats, respectively. another example of differential species response is with transferrin, which acts as a negative app in human, and a positive app in mice (19, 48) . there have been mixed reports regarding gender differences in aag levels. while there may be statistical differences, they are relatively modest compared to the differences observed in disease or developmental settings. booker et al. (50) reported statistically significant differences in aag levels between newborn human males (0.42 mg/ml) and females (0.33 mg/ml) ( table iii) . the opposite trend was observed in adult humans; aag levels of 0.39 and 0.50 mg/ml in males and females, respectively (51) . similarly, blain et al. (36) reported slightly lower levels in human males (0.74 mg/ml) compared to female (0.84 mg/ml). female minipigs generally have slightly lower, though overlapping ranges, of aag levels compared to males (19) . no gender difference was observed in dog aag levels (0.32 mg/ml) (25) . ontogeny aag levels range from undetectable in the developing human fetus, to 0.1-0.2 mg/ml in cord blood (47, 51, 53) , up to 0.3 mg/ml at birth (34, 51, (54) (55) (56) (57) , steadily increasing to 0.4-0.7 mg/ml at 2-3 months (34, 50, 55) , and achieving adult levels (0.6-0.9 mg/ml) by 10-12 months of age (33,34,58) (table iv) . similarly, aag is undetectable (<0.04 mg/ml) in the cord blood of dogs, thus significantly lower compared to adult dogs (0.32 mg/ml) (25) . the opposite trend was observed in conventional pigs, with 12.7 mg/ml reported in the fetal pig, 14.3 mg/ml in 1 day newborns, 0.70 mg/ml in 4 week olds, and 0.24-0.34 mg/ml in adults (15, 16) . consistent with this pattern, heegaard et al. (14) reported aag levels of 6.6 mg/ml in newborn pigs (5-6 days old) that declined to 1.1 mg/ml in adults. aag comprises about 50% of the total plasma proteins in the newborn pig, whereas only about 0.3% in the adult pig. this property has not been reported in mini-pigs or any other laboratory animal to date. limited data have been published on fetal and neonatal plasma levels of aag in other species. however, liver aag levels in rat have also been shown to vary in development (60) . pregnancy can also impact aag levels. in human aag levels are lower in the pregnant female and continue to decline throughout pregnancy until birth when they begin to climb back to pre-pregnancy values (53, 56, 57) . wood and wood (51) reported the same values in female non-pregnant healthy volunteers and pregnant women, however the study size was relatively small (n = 10). the opposite has been reported in the pregnant dog (25) and in rhesus monkey (59) , with aag levels about 2-fold and 4-fold higher in the pregnant animal, respectively. given the striking differences between fetal, newborn, and adult aag levels, it may be important to understand placental transfer and the milk to plasma ratio (m/p) for drugs that bind to aag. fleishaker and mcnamara (61) described a diffusional model to assess drug distribution in milk, showing that the in vitro drug binding to serum and milk protein reasonably predict m/p drug ratio in vivo. the same authors tested the model in lactating rabbits using propranolol, a compound known to bind with high affinity to aag. to mimic the disease setting, rabbits were dosed with bovine aag and propranolol pk parameters were evaluated. the diffusional model was able to accurately predict the decrease in propranolol m/p from 2.13 to 1.23 before and after aag administration. importantly, a roughly proportional reduction in total plasma cl (35%) counteracted the decrease in f u (22%), maintaining consistent cl u rate and total drug levels in milk. to improve the prediction of f u in human infants mcnamara and alcorn (47) considered the ratio of aag and albumin in cord blood of newborns and adult blood. the corresponding ratios for aag and albumin employed were 0.38 (0.24 mg/ml cord divided by 0.60 mg/ml adult) and 0.81 (36 mg/ml cord divided by 45 mg/ml adult). prediction of f u in newborns was better for drugs that predominantly bind to albumin. the average predicted and observed ratios (newborn/adult), of f u were 1.20 and 1.38, respectively, for drugs that predominantly bind to albumin (n = 28 drugs in study set). the average predicted and observed ratios (newborn/adult), of f u were 1.61 and 2.50, respectively, for drugs that predominantly bind to aag (n = 11 drugs in study set). for the majority of drugs, the f u in newborns was under-predicted, 10/11 and 22/28 drugs that predominantly bind to aag and albumin, respectively. possible explanations for the disparity suggested by the authors include changes in drugligand affinity associated with age as well as increased free fatty acids and bilirubin in the newborn that can contribute to decreased drug binding. in addition, the under prediction may be due to inaccurate (falsely high) aag and albumin newborn levels employed in the model. while aag levels are generally more variable (higher dynamic range) compared to albumin, this alone cannot explain the trend in under prediction. three genes (aag-a, aag-b, and aag-b′), located on chromosome 9, encode human aag (haag) (37) . aag-a encodes orm1 and is expressed in the liver at >100-fold that of aag-b and aag-b′. aag-b and aag-b′ are identical in structure, differ from aag-a by 22 amino acids, and encode orm2 (62) . aag shares significant homology with human immunoglobulin g (igg) and the epidermal growth factor (egf)-binding domain of the egf receptor (63, 64) . aag-a (orm1) is polymorphic with three closely related genetic variants: f1, f2, and s, differing by <5 amino acid residues, and generally referred to as f1*s in table v (65, 66) . aag-b and aag-b′ (orm2) encode the genetic variant a. most individuals possess a mixture of these variants (67) . f1 + s + a is the most common phenotype (50%), followed by f1 + a (35%) and s + a (15%). the molar ratio of f1*s to a is~2-3:1 in healthy individuals (67) . the ratio can increase up to 8:1 in the disease setting since the f1*s variant is inducible (68) . no gender related differences have been observed in expression of these variant forms (32) . x-ray crystallography showed two common binding pocket lobes between the f1*s and a variants, while the f1*s variant possesses a unique third lobe making it more promiscuous for drug binding (69, 70) . drug binding properties have been shown to differ amongst these variants (71) . for example, the basic drug imipramine was shown to bind more strongly to a variant, whereas warfarin more strongly to the f1 and s variants. for most drugs, binding to genetic variants has not been well characterized since protein binding studies are routinely conducted on a pooled supply of healthy human plasma or in the whole plasma of individual subjects/patients. proteins routinely undergo post-translational modifications that can impact physiological function and half-life (t 1/2 ). glycosylation, the addition of oligosaccharide chains (glycans) is one of the most abundant post-translational modifications, with an incidence of~50% in eukaryotic proteins (72) . aaps are particularly susceptible to glycosylation. glycosyltransferases and glycosidases are responsible for building the precursors to glycans, a process highly vulnerable to changes in disease state (73) . oligosaccharyltransferases then transfer glycans to the polypeptide chain at asparagine (n-linked) or serine/threonine (o-linked) residues, the former of which exhibit a common pentasaccharide core. the glycan bonds occur in either α or β configuration allowing for more structural diversity. the antiinflammatory and immunomodulatory properties are directly impacted by glycan composition which, in turn, changes throughout the various stages of inflammation. the heavily sialylated glycans make aag one of the most acidic plasma proteins. there is a high level of heterogeneity resulting in a very low but wide pi ranging from 2.8 to 3.8 which in turn can impact drug binding and aag t 1/2 (37) . desialylation can result in an increase in pi range from 4.2 to 4.7 (74, 75) . human aag contains 5 n-linked glycans of the polypeptide backbone, each of which can form a variety of bi, tri, or tetra-branches all potentially further expressing sialic acid moieties (65) . despite thousands of potentially unique glycan combinations associated with aag, only about 12-20 glycan combinations are observed in the plasma of healthy humans (37, 76) . however, in the disease state many more glycan modifications have been detected under the regulation of inflammatory cytokines, tumor necrosis factor (tnfα), interleukin-1 (il-1) and il-6 and its utility as a biomarker will be described later (76) . aag offers two drug binding sites for basic drugs, one for acid drugs (44) , and up to 7 for steroids (45) . drug binding to aag is reportedly mediated predominantly via hydrophobic interaction with some data suggesting potential for electrostatic interaction. evidence to support the latter includes the observation of stereoselective binding in propranolol isomers (77) . desialylation and lower plasma ph have been shown to decrease drug binding to aag (78, 79) . propranolol binding was reduced and progesterone binding unchanged with desialylated aag. homologous aag genes have been observed across mammals including rodents, cats, dogs, pigs, monkeys and humans (table vi) . mouse, rat, rabbit, and pig aag genes sharẽ 44%, 59%, 70% and 70% homology, respectively, with human aag (37, 80) . while there are three human aag genes, there is only one gene reported in rat and two to three in mouse depending on strain or source. despite the presence of multiple (81) . two disulfide bridges have been described in human and pig aag, with only one in rat. various binding sites have been characterized across species; in human acidic, basic, and steroid binding sites have been reported, whereas cows and dogs lack the acidic binding site (4). aag belongs to a family of apps mainly generated in liver parenchymal cells at elevated levels within 12-24 h of injury (i.e. infection, inflammation, burns, cancer). apps by definition are proteins that change in response to injury by >25% in plasma (89) . examples of positive apps include ceruloplasmin, aag, and serum amyloid a (saa), all increasing levels in humans by about 50%, 3-fold, and > 1000-fold, respectively, in the diseased state (90) . negative apps include albumin, transferrin, and insulin-like growth factor i, which modestly decline in plasma in the diseased state. aag is a member of lipocalins, a superfamily of extra-cellular transporters that bind and transport small hydrophobic endogenous and exogenous chemicals. upregulation of apps enhances local inflammation by aiding in recognition of microbes, directing leukocytes, and increasing blood flow to the site of insult while minimizing inflammatory responses elsewhere (91) . the rapid and high magnitude of response, as well as the short t 1/2 , are properties characteristic of apps and hypothesized to be key elements in innate immunity. homeostasis is kept in check with redundancy such that a given app may impact multiple pathways while multiple apps may provide overlapping biological function. the inflammatory cytokines, tnfα, il-1 and il-6, have been shown to regulate aag, in addition to the other apps including c-reactive protein (crp), haptoglobin (hp), saa and hemopexin (92) . the function of aag is still poorly understood, however, as part of a cytokine mediated feedback mechanism it has been implicated in both anti-and pro-inflammatory modulation (37, 38) . monocyte activation and induction of t-cell proliferation (93) as well as activation of tnfα, il-1 and il-6 secretion (94) (95) (96) have been associated with the pro-inflammatory role of aag. su et al. (97) proposed a positive feedback mechanism of apps whereby inflammation is amplified in response to tnfα-mediated synthesis of aag-stimulated monocytes and vice-versa. the anti-inflammatory role of aag has also been reported. aag inhibits neutrophil chemotactic response associated with stimulation of n-formylmethionyl-leucyl-phenylalanine (fmlp) and the inflammatory peptide complement component c5a (98, 99) . aag was shown to modulate release of free radicals regardless of treatment time, whether aag was introduced prior to or post neutrophil activation (100) . multiple in vivo septic shock models in rodents have demonstrated the protective effect of aag when dosed prophylactically to animals challenged by tnfα or endotoxin (101) . the role of aag in angiogenesis was studied using an ex vivo rat model (102) . following aortic excision, macrophages respond by rapidly (within minutes) increasing tnfα levels, peaking at 24 h, and remaining elevated throughout angiogenesis. tnfα guides overexpression of aag within 24 h, with levels peaking after 2-3 days and sharply declining thereafter. as with inflammation, aag (and tnfα) has both pro-and anti-angiogenic effects depending on the context. early in the angiogenesis process, aag plays an inhibitory role via modulation of mitogen-activated protein kinases, whereas later in the process aag promotes angiogenesis via vascular endothelial growth factor regulation. as an app, levels of aag typically increase within 24 h of injury and begin to decline several days post amelioration. increased aag levels have been reported in the serum of breast, lung, and ovarian cancer patients (103) . in a study comparing aag levels in lung cancer patients versus individuals with no known cancers, results showed 89% sensitivity and 85% specificity and aag levels correlated with relapse-free survival (104) . in the case of hepatocellular carcinoma (hcc), diagnosis can be challenging due to other liver conditions (i.e. cirrhosis) presenting similar abnormalities, however increased aag levels are more pronounced with hcc providing a potential basis to differentiate these diseases (105) . the proportion of breast cancer patients with increased aag levels increases with disease progression, for example 25% and 81% of stage ii and iv patients respectively, had elevated levels compared to 12% in healthy donors (106, 107) . in most disease states, aag is modified both quantitatively as described above, as well as qualitatively, a phenomenon described by alminquist and lausing (39) after comparing serum glycoproteins of cancer patients versus healthy donors. relative to the associated polypeptide backbone, the heterogeneity in glycan composition and structure make it a good system for characterization and correlation with disease state (73) . some commonly exploited glycoproteins used in clinical cancer biomarker tests include carcinoembryonic antigen (cea), cancer antigen 125 (ca-125), ca-19-9, and prostate-specific antigen (psa), for the diagnosis of colorectal, ovarian, pancreatic and prostate cancers, respectively. technological advances in mass spectrometry and proteomics have led to an improved understanding of glycan structure and function. glycans are relatively more abundant compared with their associated proteins, often times multiple copies per glycoprotein, as is the case for aag known to have 5-6 associated glycans depending on species (table vi) . not only are they associated with the cancerous tissue but they can also be detected in serum, making its use as a biomarker more feasible in the clinical setting. in addition, the same modified glycan may be associated with more than one glycoprotein affording multiple opportunities/patterns for detection. modifications of the glycans associated with these biomarkers are relatively more specific and selective than the epitope itself, allowing for more accurate means for distinguishing healthy versus diseased tissue, and disease progression (40) . for example, testing serum levels of modified glycans associated with psa enable the distinction between benign prostatic hypertrophy and cancerous prostate (41) . in breast cancer, the serum glycosylation pattern not only distinguishes healthy from diseased tissue but also differentiates between malignant and non-malignant tumors and disease stage (73) . in inflammatory diseases including rheumatoid arthritis and asthma, aag glycans are more branched compared to healthy subjects (42) . patients suffering from acute inflammation, infection, burns, and tissue damage all showed an asialylated carbohydrate-deficient variant of aag (37) . human aag t 1/2 is relatively short,~2-5 days (108,109), compared to albumin, 14-21 days (110) . aag turnover is dependent on sialic acid residues and terminal galactose groups. mccurdy (111) studied the impact of glycosylation on in vivo cl of human derived aag in rabbit following intravenous injection. the terminal t 1/2 of native human aag was 58 h (consistent with the t½ value of 69 h reported by regoeczi et al. (112) . altered/reduced or absence of glycosylation lowered the t½ to 50 h and 42 h, respectively. cl of native aag was 2.2 ml/h/kg, whereas much higher values were observed for aag forms with altered (11 ml/h/kg) or absent glycosylation (100 ml/h/kg). the steady-state volume of distribution (v ss ) was 160 ml/kg for native aag, whereas much higher values were observed with altered (550 ml/kg) or absent glycosylation (2000 ml/kg). the absence, reduction or alteration of n-linked glycosylation resulted in a marked increase (>10-fold) in renal elimination compared to native aag. these studies support that the dispositional properties of aag are dependent on disease state since the biochemistry of aag is altered with disease as described earlier. therefore, if a given drug binds to aag to a high extent, it is possible underlying differences in the pk of the drug between healthy and diseased populations may be attributable to aag. drug binding to plasma, tissue(s) and intended target are critical parameters to predict pk and pharmacodynamics (pd). however, optimizing protein binding to plasma in the drug discovery setting is scientifically unsound (113, 114) . nearly 30% of the 260 fda approved drugs prior to 2003 are classified as highly bound (>95% or f u < 0.05), and this trend has increased in recent years with 45% of new drugs classified as highly bound, 24% of which have f u < 0.01 (115) . while the free drug hypothesis describes that the free concentration drives activity as it can cross cellular membranes to reach its target, the focus should be to optimize cl u and permeability. as described above, albumin, aag, and lipoproteins are considered the most important plasma proteins involved with drug binding. while albumin has a higher drug capacity due to its relative abundance in plasma, aag levels are lower and with high affinity drugs saturation may occur. the saturation of aag may or may not be buffered by albumin depending on the drug binding affinity for albumin. plasma protein levels can change with disease state, a decrease in albumin and an increase in aag are generally observed, which may impact protein binding and pk. both albumin and aag levels are significantly lower in the newborn, with newborn:adult ratios of about 0.81 and 0.38, respectively (47), a factor that should be considered when predicting pk in the very young pediatric population. a proposed flowchart to ascertain the impact of aag as a potential covariate for pk variability early in drug development is shown in fig. 1 . routine screening in human and laboratory animal species at a single low concentration (1-2 μm) is typically performed in early drug discovery as pk data in preclinical species is acquired. as a program advances towards development candidate selection with preliminary projections of human pk and dose, there is value in assessing concentration dependency and the identity of the major plasma proteins involved in drug binding. if the extent of binding is high (>90% bound) under single concentration assay conditions, further characterization of the binding constants for aag and albumin is warranted particularly if human free fraction is lower relative to animals or if concentration dependency is observed. if there is high affinity to human aag it may be worthwhile to assess k d in other species to build additional confidence in human pk predictions. multiple routine in vitro analytical procedures exist to assess the extent and affinity of drug binding to proteins. a recently published industry white paper (di et al., 2017) provides a comprehensive review of commonly used protein-binding practices, challenges, and recommendations (116) . the intention in this section of the manuscript is to suggest use of control compounds and to describe a source for erroneous fraction unbound values that has been overlooked. as with any assay it is good practice to include control compounds that are assessed along with test compounds to ensure a properly functioning assay. if data for control compounds are not available for a given assay/species one can still monitor the value for the control over time to ensure its consistency. when conducting definitive assays the use of multiple control compounds is advised since multiple factors can influence binding and some are compound specific. literature values for propranolol and warfarin are summarized across species in table vii . the intent here is to provide references for acceptable free fraction values for control compounds across species. propranolol was selected because it has moderately high binding to human plasma proteins, however the affinity is higher for aag relative to albumin by approximately two orders of magnitude (125) , therefore, propranolol can serve as a control for compounds that preferentially bind to aag. the reported propranolol f u values range from 0.10 to 0.29 in human plasma, a 3-fold difference, and outside what is typically deemed normal assay variability. the acceptable assay f u value for propranolol should be within 0.10 to 0.20 in healthy human plasma. warfarin was selected because it is highly bound to both aag and albumin (126, ge life sciences application note 29263246aa). it is helpful to include a highly bound control compound since they generally require longer incubation time to achieve equilibrium. while the reported warfarin f u values range from 0.005 to 0.022 in human plasma, more than a 4-fold difference, the absolute difference is low. the majority of references indicate a narrower range, therefore the acceptable assay f u value for warfarin should be within 0.005 to 0.015, a range similar to that reported in the recently published white paper (116) . it is not advised to select a compound with moderate or low protein binding to serve as a control because the f u values are generally more variable. based on recent studies (127, 128) it is possible the large range in human f u values is due to the blood collection and storage procedure. in the clinical setting, f u is typically measured in the plasma or serum of patients from blood collected in vacutainers. the collection procedure and storage of blood can have a significant impact on protein binding results. it has long been reported that plasticizers can disrupt the binding of drugs to aag (129) (130) (131) . for example, the plasticizer tris (2-butoxyethyl) phosphate (tbep), used to soften rubber stoppers in vacutainers, was shown to disrupt aag binding to the basic drugs lidocaine and quinidine (132) . polyvinyl chloride (pvc) bags containing the plasticizer diethylhexyl phthalate (dehp) are routinely used in blood collection. butler et al. (127) reported an average of a two-fold increase in f u for drugs known to bind to aag when blood was collected in these pvc bags versus blood collected in vacutainers. more recently, experiments were conducted to show the correlation between dehp levels and change in f u with drugs that bind to aag (128) . when blood was immediately transferred from terumo® bags to vacutainers the dehp levels were low (1-10 μm) but steadily increased with storage after 7 days (up to 300 μm) and 28 days (300-1000 μm). dehp can easily leach into the contents of bags since it is not chemically bound to the pvc. as expected, f u was higher (2-5-fold) with drugs known to bind to aag when tested using plasma containing high levels of dehp. the shift in f u was significantly reduced, though not eliminated, when the blood was immediately transferred to vacutainers. results generated using blood products collected/stored in plasticizer containing bags should be interpreted with caution as the f u values may over-estimate true in vivo values. in most instances when protein binding is measured clinically, vacutainers (no/minimal dehp exposure) are used since small (<10 ml) blood volumes are collected. this is in contrast to the relatively larger volumes (up to 350 ml) collected in bags for donation and/or non-clinical research purposes. it should be noted that the blood from animals is typically collected in smaller vessels not containing dehp, therefore the over-estimation of f u is less likely to occur in animal blood. studies may be warranted to assess effects on drug binding with other commercially available vacutainers as a precaution. despite what has been reported in the literature for decades, bags containing plasticizers known to disrupt aag-drug binding continue to be widely used for blood collection with the intended use in both research and in the clinical setting. dehp is essential in maintaining the shelf life of blood products up to 42 days as it protects the membrane of the red blood cell (133) . transfusion recipients routinely receive blood that has been collected and stored in these bags. clinical effects with regard to drug displacement have not been reported. other blood collection bags have been developed though it is unclear if they have an effect on aag-drug binding. multiple classes of drugs have been reported to bind to aag. examples of the relationship between aag binding and lipid solubility and/or electrostatic interactions have been reported for benzodiazepines, phenothiazine neuroleptics, beta blockers, anthracycline derivatives, antihistamines, and analgesics (3, 126) . here we focus on several well-studied examples where the drug-aag binding affected pk and/or pd in the clinical oncology setting. vismodegib was approved for the treatment of metastatic basal cell carcinoma by the food and drug administration (fda) in 2012. pk characteristics exhibited in the phase i clinical trial were unexpected based on preclinical allometric scaling. following a single oral dose, exposure was higher than predicted with a very low apparent cl and a long t 1/2 of about 10-12 days (134) . cross species in vitro data showed high plasma protein binding (≥95% bound) and low metabolic turnover in hepatocytes of all species except monkey, which agreed well with in vivo cl values (135) . mechanistic pk modeling was employed to explain the roles of plasma protein binding, solubility-limited absorption, and low metabolic cl in contributing to the unusual clinical pk properties (136) . in vitro studies revealed far lower vismodegib solubility, 0.0001 mg/ml at higher ph range 6.5-7.4, compared to~1.0 mg/ml at ph 0.1 (136) . the impact of solubility was manifested in saturation of oral absorption. there was no increase in mean steady-state concentration (c ss ), 22.6, 21.3 and 22.0 μm, with increase in oral dose from 150 to 270 and 540 mg, respectively (137) . the free fraction of vismodegib remained constant at 0.5 ± 0.1% across all dose groups (137) . however, in a separate study, a 2.6-fold increase in free fraction of vismodegib was reported between a single 150 mg dose (0.25 ± 0.14%) versus repeat daily 150 mg doses (0.65 ± 2.9%) (138) . vismodegib plasma protein binding properties were further characterized by isothermal titration calorimetry (itc) and surface plasmon resonance (spr) with both procedures showing higher vismodegib-aag affinity to the human isoform relative to the rat isoform (139) . by itc the k d values of vismodegib-aag were 1.1 and 118 μm in human and rat, respectively. by spr the k d values of vismodegib-aag were 13 μm and not detectable in human and rat, respectively, whereas the k d values for vismodegib-albumin were similar in human and rat (120 and 140 μm, respectively). in vitro experiments showed a negative correlation between aag concentration and target engagement, whereby supplementing physiologically relevant concentrations of aag resulted in a dampening of hh signaling via gli1-luciferase reporter assay (139) . there was a high correlation (r 2 = 0.73, slope 0.48) between aag and total vismodegib c ss in plasma samples from cancer patients, suggesting the role of plasma protein binding in vismodegib drug disposition (136) . perhaps even more compelling was the intra-patient parallel changes in total vismodegib concentrations with changes in aag. no correlation was found with albumin levels and vismodegib concentrations (139) . saturation of aag has been proposed to be a key determinant in the non-linear pk of vismodegib given that aag is a high affinity-low capacity protein and near stoichiometric levels of vismodegib and aag are reported (138) . despite the high affinity and resultant low free fraction, there remains sufficient unbound vismodegib available to interact with target to demonstrate pharmacological effect. in order to understand the mechanism(s) of non-linear pk, healthy human subjects received either a single oral dose or 7 daily oral doses of 150 mg vismodegib, followed by an iv microtracer dose of 10 μg [ 14 c]-vismodegib 2 h post first or last (day 7) oral dose (140) . aag levels were within close range in the two dose groups to eliminate need for correction. cl and v ss values after a single iv dose were 43.4 ml/h and 16.4 l, roughly 10-65-fold lower (depending on number of species employed in model) and 3-fold lower, respectively, compared to preclinical allometric scaling predictions (135) . relative to day 1, cl and v ss increased 81% and 63%, respectively on day 7, while t 1/2 remained unchanged at~10-11 days. mean free fraction increased 2.4-fold after 7 days oral dosing (0.79 ± 0.23%) compared to a single dose (0.33 ± 0.12%), a finding consistent with the observations previously described (138) . correcting for protein binding of vismodegib, the unbound cl and v ss values were relatively similar on day 1 and 7. absolute bioavailability was 31.8% following a single oral dose and declined to 7.4% after 7 days of repeat dosing, a finding attributed to slow absorption and limited intestinal solubility (140) . given the non-linear pk with long t 1/2 , a phase ib clinical study was conducted to determine if vismodegib steady-state concentrations could be maintained with less frequent dosing (138) . three oral dosing schedules were evaluated: 150 mg once daily (qd), once weekly (qw) or three times per week (tiw), all after having received a loading dose of vismodegib (150 mg qd, 11 days). after steady-state was achieved for the alternate dose schedules, vismodegib levels declined in the qw and tiw groups relative to qd, however the decline in unbound vismodegib (50% and 80%, respectively) was greater than the decline in total vismodegib (24% and 45%, respectively). only the qd dose schedule maintained unbound vismodegib concentrations sufficient to achieve target pathway inhibition (ic 95 ) of glioma-associated oncogene (gli1) previously described (141) , therefore the recommended dose and schedule was maintained at 150 mg qd. one additional point of consideration is the discrepancy between in vitro and ex vivo free fraction values reported for vismodegib in human plasma,~3-4% and < 0.25-0.79%, respectively (135, 137, 138, 140) . this discrepancy may be attributed to the collection and storage of blood products as described herein and in recent publications (127, 128) . free fraction values increased sharply for aag-binding drugs when human blood was exposed to plasticizer dehp; for example, vismodegib free fraction increased from 0.2% when collected in vacutainer (no/minimal plasticizer), to 0.4% and 1.4% when collected and stored in terumo® bags containing plasticizer for <1 and 7 days, respectively. additionally, vismodegib free fraction increased from 0.3 to 2.9% when human plasma was spiked with 800 μm plasticizer, a concentration one could expect to measure in blood after several weeks storage in terumo® or similar bags. ucn-01 (7-hydroxystaurosporine) is a small molecule protein kinase inhibitor. single agent clinical trials were initiated for multiple oncological indications in the late 1990s, followed by combination studies with other anti-cancer agents. the pk parameters in preclinical species (mouse, rat and dog) ranged as follows: moderate to high cl roughly 30 to 80% hepatic blood flow, high v ss 6 to 17 l/kg, and moderate t 1/2 from 3 to 12 h (142) . clinical pk were not predicted by allometry and exhibited low cl (17 ml/h), low v ss (12 l) and very long t 1/2 (>200 h) (143) . while ucn-01 (1 μg/ml) is considered highly bound to plasma proteins in preclinical species with free fraction ranging from 0.5 to 1.8%, the free fraction was substantially lower in human plasma at <0.02% (144) . ucn-01 free fraction was <0.02% or 6.2% when incubated with physiologically relevant levels of haag (1 mg/ml) or albumin (40 mg/ml), respectively, showing the preferential binding to aag. in vitro studies showed marked increase in ucn-01 free fraction with increases in concentration approaching stoichiometric levels of aag (145) . the association constant (k a ) was 799 × 10 6 l/mol in haag (roughly equivalent to k d of 1.25 nm), whereas in dog the k a was~60-fold lower at 13.2 × 10 6 l/mol (145, 146) . sparreboom et al., further characterized the role of aag in the pk of ucn-01 (147) . with an increase in dose ranging from 3.6 to 53 mg/m 2 /day iv infusion over 72 h, there was an increase in cl, 4.13 ml/h to 24.1 ml/h, respectively, a linear increase in v ss , 0.113 l to 0.276 l, respectively, and less than proportional (3.5-fold) increase in auc ∞ , (area under the curve extrapolated to infinity) 7460 to 26,140 mg*h/l. cl trended (r 2 = 0.264) with pre-dose aag levels, despite a relatively small data set (n = 39). it is proposed the increase in cl in humans is due, at least in part, to the increase in free fraction once aag becomes saturated. cl in dogs had demonstrated no dependence on dose from 0.81 to 6.48 mg/kg (142, 148) , a finding consistent with the notable difference in ucn-01-aag k d in human versus dog. the k d of haag-ucn-01 is roughly 4 orders of magnitude lower compared to k d haag-vismodegib (139) . in addition, ucn-01 does not appear to bind significantly to albumin given the comparable k d values of ucn-01 to aag vs human plasma, 799 vs 802 × 10 6 l/mol, respectively (145) . saturation of aag and the differential affinity to aag and albumin are likely to contribute to the non-linear pk of ucn-01. as with vismodegib the haag-unc-01 k d differed considerably from that of the preclinical species leading to poor predictive accuracy with allometric methods. ucn-01 has a slow dissociation rate which may reduce v ss , further hindering free drug from target interaction (139) , in contrast to the preclinical observations of high v ss , high tumor:plasma ratios, and decline in tumor volume (142) . ucn-01 has not advanced in the clinic due to unpredictable pk and off-target kinase inhibition (146) . imatinib is a selective inhibitor of bcr-abl, platelet-derived growth factor receptors, and c-kit receptor tyrosine kinases (149) . approval was granted for the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors by the fda in 2001 and 2002. imatinib-aag binding is concentration-dependent with a reported k a of 1.7 × 10 6 l/ mol, roughly equivalent to k d of 0.6 μm, while imatinibalbumin binding is considerably weaker with a k a of 3.0 × 10 4 l/mol, roughly equivalent to k d of 33 μm (139, 150) . adding to the complexity is the differential binding of imatinib to various human aag isoforms, the k a reported above is for the f1-s variant, whereas binding was much weaker for the a variant (unpublished data) (151) . separately incubating 24 μm of the aag variants f1-s and a with 5 μm imatinib resulted in 6% and 18% free fraction, respectively. imatinib exhibits linear pk in patients (152) with low oral cl ranging from 8 to 12 l/h (150, 153, 154) , long t 1/2 of 18 h and high oral bioavailability >90%. a proportional increase in auc is observed with oral doses from 25 to 1000 mg. pk parameters are similar between single and repeat doses, showing 1.5-to 2.5-fold accumulation at steady-state. correlations between imatinib pk and abcb1 genotype, body weight and aag levels was shown in patients (150, 155) . despite linear pk in total imatinib, a non-linear relationship exists between free fraction and total imatinib concentrations in plasma as a result of high affinity to aag and~55-fold weaker affinity to albumin (156) . elevated levels of aag in patients have been linked with delayed or lack of response to imatinib treatment as well as potential resistance mechanism (157, 158) . in a clinical study with cml patients, approximately half exhibited elevated aag levels positively correlating with disease progression and white blood count. in the chronic, accelerated, and blast crisis phases of disease, 33, 83 and 75% of these patients, respectively, were increasingly likely to have higher aag levels. the impact of the plasticizer dehp on imatinib free fraction in human plasma was also assessed (128) . imatinib free fraction increased from 3.5% when collected in vacutainer (no/minimal plasticizer), to 4.9% and 14.7% when collected and stored in terumo® bags containing plasticizer for <1 and 7 days, respectively. additionally, imatinib free fraction increased from 3.5 to 15.3% when human plasma was spiked with 800 μm plasticizer. the plasticizer-free free fraction values reported by ingram et al. (128) are similar (~5%) to those reported in the package insert (novartis pharmaceuticals corporation, reference t2017-101). pinometostat (epz-5676) is a first-in-class, small molecule inhibitor of dot1l and was the first member of the novel histone methyltransferase inhibitor class to enter phase 1 clinical trials in both adult and pediatric mll-r leukemia patients. consensus preclinical predictions across multiple diverse methods suggested pinometostat would be a moderate-to-high cl compound in human with estimates ranging from 8 to 18 ml/min/kg, and species-invariant time approaches showed cross-species congruence in time-concentration profile. however, during early development, the observed cl in human was shown to be markedly lower than that determined in preclinical species. the majority of interspecies scaling and allometric methods over-predicted human cl of pinometostat with fold errors ranging from 4 to 13 (159) , characterized by 'vertical allometry'. the 3-5-fold difference in free fraction between rat and human provided the basis for the improved prediction using the free fraction corrected intercept (fcim) method. the unambiguous species difference in cl was not related to qualitative differences in metabolic pathways or routes of elimination, but instead to cross-species differences in plasma protein binding. concentration dependence in protein binding was observed in human plasma, over a relevant concentration range, which was less apparent in the preclinical species. this, along with in vitro kinetic determinations, suggested the saturable binding of pinometostat to aag. the equilibrium dissociation constant (k d ) for pinometostat binding to human aag was measured as 0.24 μm indicating a high affinity interaction. by comparison, prototypical aag ligands such as dipyridamol, disopyramide and thioridazine have k d values of 15.5, 1.0 and 63 μm respectively. furthermore, there is the disproportionately higher expression of aag in human plasma relative to preclinical species, which is likely a contributing factor alone, irrespective of potential speciesspecific differences in aag affinity. it has been suggested when the k d for a given drug-aag binding is low, and more than a log order lower relative to the k d for albumin, the pk may exhibit non-linearity (136) . if the drug also has a high affinity to albumin, a high capacity protein, fluctuations in free fraction will be minimal. if the drug has a low affinity to albumin, the non-linear effect may be exacerbated when drug levels are near stoichiometric with aag, since aag is a low capacity protein and may become saturated. given the known differences in abundance and homology across species for aag, allometric scaling may not be suitable for human pk prediction when there are differences in k d , something that can easily be measured in vitro now that aag of preclinical species are commercially available, though still limited in supply. monitoring aag and/or free drug concentrations, as well as phenotyping the genetic variants of aag in patients may be warranted in special circumstances to better understand pk and pd. in fig. 1 , we propose a flowchart for incorporation of protein binding assessment in drug research and development. investigations are ongoing to propose more reliable procedures for the collection and storage of blood for future use in drug free fraction measurements (personal communications with q 2 solutions holdings, llc). special consideration should be given to pregnancy and pediatric populations since aag levels are substantially lower until about 10 months post-natal. pbpk models have shown predictive utility with incorporation of aag and albumin levels and drug binding parameters. quantitative and/or qualitative analysis of aag may prove useful as a biomarker for disease diagnosis and prognosis, with the potential to serve as discerning criteria to improve likelihood of successful treatment. since aag behaves as a positive 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note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-290472-w77cmljm authors: sharon, donald; chen, rui; snyder, michael title: systems biology approaches to disease marker discovery date: 2010-06-09 journal: dis markers doi: 10.3233/dma-2010-0707 sha: doc_id: 290472 cord_uid: w77cmljm our understanding of human disease and potential therapeutics is improving rapidly. in order to take advantage of these developments it is important to be able to identify disease markers. many new high-throughput genomics and proteomics technologies are being implemented to identify candidate disease markers. these technologies include protein microarrays, next-generation dna sequencing and mass spectrometry platforms. such methods are particularly important for elucidating the repertoire of molecular markers in the genome, transcriptome, proteome and metabolome of patients with diseases such as cancer, autoimmune diseases, and viral infections, resulting from the disruption of many biological pathways. these new technologies have identified many potential disease markers. these markers are expected to be valuable to achieve the promise of truly personalized medicine. disease markers are of vital importance to clinicians and their patients as early detection, accurate prognosis/diagnosis and monitoring of therapy can lead to increased overall survival and cure rates. as our knowledge of diseases quickly expands, the field of disease marker discovery will play increasingly important roles in the delivery of improved diagnosis and treatment. these markers, such as protein (including autoantibodies, which are antibodies specific to self-antigens [43] ), hormonal markers (such as lack of insulin in type i diabetic patients [89] ), and genetic/genomic markers (such as brca1 mutation in breast cancer patients [52] ), enable clinicians to diagnose the disease while it is still at early stages, to ensure appropriate surgical intervention, efficient drug treat-ment and monitoring, and to predict an individual's risk of developing specific diseases before they experience symptoms. traditionally, discovery and detection of these disease markers relied on low throughput technologies such as enzyme-linked immunosorbant assay (elisa) or 2d-gel plus edman degradation for protein markers, reverse transcription-polymerase chain reaction (rt-pcr) for mrna markers, and restriction enzyme digestion, cloning and sanger sequencing for dna markers. before the dawn of high-throughput technologies these methods played important roles in marker identification and yielded significant discoveries in various diseases such as systemic lupus erythematosis, rheumatoid arthritis and breast cancer [32, 52, 107] , which greatly enhanced the diagnostic efficiency in these diseases. during the past two decades, high-throughput technologies emerged and have displayed great potential in large-scale studies for marker discovery. these technologies include protein microarrays [42, 119] , mass spectrometry for large-scale shotgun studies [116] , and, more recently, high-throughput parallel sequencing (including rna-sequencing) [9, 63, 67] . this article will review disease marker discoveries using these systems biology approaches, with a focus on high-density protein microarray technologies. we will also briefly review current progress in disease marker identification using parallel sequencing and mass spectrometry technologies. currently, high-density protein microarrays contain hundreds to thousands of proteins that are arrayed on coated glass microscope slides (e.g. nitrocellulosecoated slides) in an addressable format [23] . these arrays are usually probed with fluorescently labeled molecules and the signals are then acquired with a confocal laser scanner. a number of surface chemistries have been employed for their ability to bind proteins efficiently although there is often a trade-off between retention and decreased protein function or improper folding. there are several broad categories of protein microarrays: arrays composed of cell or tissue lysates or protein fractions isolated from crude lysates [33, 74] , antibody or analytical arrays that contain types of antibodies directed specific analytes [13] as well as socalled functional protein arrays [86, 118] . functional protein arrays contain full length proteins with intact catalytic function and proper epitope folding, which are often generated by arraying purified proteins produced individually prior to printing [118] or proteins produced in situ by in vitro transcription and translation of dna that is printed directly on the surface of the array [86] . our group has been developing the last type of protein microarrays by arraying purified proteins on nitrocellulose-coated glass slides. this type of functional protein microarray has clear advantages over the other alternatives: the ability to specifically identify individual proteins compared with cell lysate arrays and the ability to ensure the quality of each arrayed protein compared to the in situ transcription-translation arrays. after development of the first protein microarray that contains 5800 full-length proteins of the budding yeast saccharomyces cerevisiae [118] , our group produced a number of protein arrays including the yeast n-terminal and c-terminal arrays, a 500 protein arabidopsis array, and a coronavirus array [119] . in conjunction with protometrix corporation (now part of invitrogen corporation) we also collaborated in the development of a human protein array that currently holds more than 9,000 proteins expressed individually using a baculovirus/sf9 expression systems. these various arrays were used for a variety of applications including assaying for protein-protein, protein-lipid and proteinnucleic acid interactions as well as probing for substrates of protein kinases [41, 45, 118] . we also developed algorithms for positive signal calling and large dataset processing [50] . recently, we have applied this technology in a novel proteomics-based approach to screen for human antibodies that react with foreign and self-antigens [64, 65, 79] . particularly notable in this review is our use of these protein microarrays to analyze the immune response to coronaviruses [119] as well as our screening projects to analyze ovarian cancer [43] , myeloma, multiple sclerosis and asthma. in this section we will review disease marker identification in several fields using high-density protein microarrays. currently, tests for the detection of microbial infections are the only clinical tests that rely on measuring antibody responses. elisa-based detection methods are often used to detect a patient's antibody titer to epitopes of the microorganisms for diagnosis of the infection. in late 2003, an outbreak of a novel coronavirus (cov), the severe acute respiratory syndrome (sars) virus, resulted in that killed over 900 deaths. novel diagnostic tests were required to identify and monitor this disease, and elisa, immunofluorescence and nucleic acids tests were employed. it was shown that protein array-based methods proved to be more accurate than any of the existing antibody based methods [119] . our lab developed a coronavirus proteinmicroarray that contained 82 coronavirus proteins including all sars-cov proteins and proteins from five additional coronaviruses. the microarray was used to probe sera obtained from 399 canadians and 203 chinese during the sars outbreak, including samples from confirmed sars-cov cases, other respiratory disease patients, and healthcare professionals. after detection with cy3-labeled anti-human igg antibodies, the bound reactive antibodies to coronavirusencoding proteins from sera were visualized and quantified. the reactivity results of the different proteins were analyzed using a variety of computational methods, and we developed computer algorithms based on the reactivity results to predict which patients were infected with sars [119] . the protein microarray platform displayed a very high sensitivity, and reliably detected sars-cov reac-tive antibodies even when serum was diluted at 16,000 fold. the assay showed good reproducibility with less than 10% variance in signal intensity between duplicate slides. importantly, the method requires less than one microliter of serum for detection, which is desirable when serum samples are limited. moreover, probing of the coronavirus protein microarrays with sars infected serum samples also shows high specificity to sars-cov-specific proteins, as very little crossreactivity with proteins of other coronaviruses has been observed in sars infected serum samples. to determine the best classifiers to distinguish sars-positive from sars-negative sera, we used one unsupervised clustering method and two supervised methods: k-nearest neighbor (k-nn) and logistic regression (lr). both supervised models showed high sensitivity (90% for k-nn and 89% for lr) and specificity (93% for k-nn and 94% for lr) with a panel of 5 (k-nn) or 4 (lr) best classifiers, and these numbers were greater than 97% when the assay was performed in triplicate. the prediction methods were then tested on 56 sera from chinese fever patients for sars-infection prediction and were determined to have 100% sensitivity and 95% specificity, which are superior to two elisa-based detection methods that were used during the sars outbreak. our study in sars-cov infection diagnosis via detection of sars-cov-specific antibodies by protein microarrays demonstrated that this approach is sensitive (50-fold more sensitive than elisas), specific (little crossreactivity with other coronavirus proteins), and rapid (performed in a few hours). nevertheless it should be noted that tests based on immune responses to foreign antigens are more likely to achieve higher accuracy than those based on autoantibody-autoantigen responses since there is no self-tolerance to the foreign antigens and the presence of pathogen associated molecular patterns (pamps) significantly increases the immune response. overall, this study demonstrated for the first time that protein microarrays could be used to diagnose and monitor human antibodies as protein markers that are generated during the course of a disease. moreover, it demonstrated the power of using a panel of multiple classifiers for diagnostics. while protein microarray technology can efficiently and accurately detect antibodies generated against foreign antigens from infectious organisms, perhaps the most intriguing application of this technology is in the discovery of novel protein markers for the early detection of various cancers. the identification of disease markers holds the promise of increasing the effectiveness of clinic therapies and marker-based routine screening programs and can potentially enable diagnosis at the earliest stages of the disease, before the development of clinically recognizable cancers that are usually at advanced stages. for instance, in heavy smokers autoantibodies recognizing mutant forms of the tumor suppressor p53 have been detected prior to the diagnosis of lung cancer [103] . early detection and treatment would result in markedly improved survival rates, especially for patients whose cancers do not present symptoms during early stages such as pancreatic and ovarian cancer [25, 29, 91] . oncoproteomics is a rapidly expanding field aimed at applying high-throughput proteomics approaches to understanding the mechanisms involved in cancer. proteomic approaches to discover cancer markers have been an area of strong interest in recent years. in the past, these projects often involved serum screening with phage expression libraries prepared from cancer tissues, or serex (serological analysis of cdna expression libraries), or by immunoblotting cancer cell lysates after two dimensional polyacrylamide gel electrophoresis (2de-page). these approaches have yielded some promising candidate markers but suffer from particular issues, such as the fact that phage expression libraries often contain out of frame and truncated protein targets and protein candidates discovered by 2de-page are difficult to identify since the proteins are unknown. mass spectrometry is often required in order to identify the candidate autoantigens [20, 46, 53, 90 ]. an additional problem with these approaches is that the samples are usually limited in amount and are difficult to reproduce. protein microarrays overcome those difficulties as all of the spotted proteins are derived from known, well-characterized clones. additionally, even a small amount of purified protein is sufficient to print hundreds of arrays for patient screening [4, 7, 16, 47, 81] . as most traditional disease markers are proteins that have become over-or under-expressed during the course of disease, there is much interest in the potential use of autoantibodies as a novel class of disease markers. recently, detection in serum of circulating autoantibodies targeting tumor-associated antigens (taas) has emerged as an effective approach for identifying cancer early detection markers (e.g. breast, lung and ductal pancreatic cancer [5, 80, 102] ). this approach is based on the fact that the immune system produces an-tibodies against abnormal/mutated proteins generated from apoptotic/necrotic cancer cells. these autoantibodies can then be detected with immunosorbant assays like elisa. because the levels/stability of autoantibodies are potentially much greater than those of the original autoantigens, they would be more easily detected. by comparing autoantibody profiles between different groups (cancer patients versus controls), it is possible to identify markers that are significantly differentially expressed. this method is expected to be superior to dna array-based methods since changes in rna expression levels do not necessarily correlate with protein expression. the area of research in autoantibody marker discovery using protein microarrays has rapidly expanded over the last several years as the protein array platform continues to mature. the recent availability of high content protein microarrays allows for global profiling of autoantibodies to cancer antigens in both highthroughput (thousands of protein candidates) and high sensitivity ( 10 fg of protein) [43, 118] . improvements in printing techniques and increases in protein spot quantity have made these arrays promising vehicles for exploring the repertoire of autoantibodies in human disease. this approach has been applied, by various groups, for the discovery of autoantibody markers in breast cancer [5] , lung cancer [80] and ovarian cancer [43] , as well as a smaller study in pancreatic cancer [74] . here we will review past and ongoing research in immune response profiling using protein microarrays relating to a number of disease states. while self-tolerance usually abrogates the antibody response to self-proteins it is possible to elicit an autoimmune response under certain conditions present in cases of disease. the antigenicity of self proteins may result from overexpression of normal proteins such as in the case of her-2 in breast cancer subtypes and prostate specific antigen (psa) in prostate cancer, from aberrant post-translational modification such as different mucin-1 glycoforms in breast cancer, or from mutations in the proteins as has been found to be the case with the tumor suppressor p53 in multiple cancer types [2, 14, 17, 55] . additionally, proteins that are usually restricted to expression in germ line cells or are expressed only in the early stages of development may be aberrantly expressed in cancer. this is the case with the testis antigen ny-eso-1 and carcinoembryogenic antigen (cea) respectively. because many of the proteins mentioned above are detected only at very low levels in serum, even in late stages of disease, they would be of little utility for screening purposes. however, even slight increases in the expression of those antigens can lead to detectable increases in the corresponding autoantibody. generally, we find the existence of a basal autoantibody level to many self-antigens, however, this response has been shown to be markedly increased in cases of diseases such as those mentioned above [2, 26, 87] . ca-125 is currently the only clinically approved marker for ovarian cancer screening. unfortunately, although ca-125 serum levels are significantly elevated in advanced stages of the disease, its positive predictive value for the detection of early stage ovarian cancer is less than 10% [66] . for this reason the identification of new markers for this disease is of critical importance. scientists, such as the group led by gil mor at yale university, recruited proteomics-based approaches using antibody-based protein microarrays to identify new serum biomarkers, which, in combination with ca-125, may enhance the early detection of ovarian cancer [48, 66, 110] . our group also launched a pilot study to profile ovarian cancer-associated autoantibodies with protein microarrays containing 5,005 fulllength human proteins [43] . we compared the autoantibody profiles in 30 cases of epithelial ovarian cancer patients and 30 healthy controls, and after statistical analysis, identified 90 proteins to have significantly different immune reactivity in the patient group versus the control group. the results were validated by immunohistochemistry (ihc) and demonstrated high sensitivity (95%) and specificity (97.5%) when the top two markers (lamin a/c and ssrp1) were combined. however, further validation is required before the candidate markers can be adopted in a clinical setting. therefore we carried the top ranking candidates through to the validation phase in which a much larger set of samples will be tested to evaluate the performance of the potential markers. we have generated focused protein microarrays containing these candidates as well as control proteins such as ca-125 (fig. 1) . these arrays are printed in twelve blocks per slide allowing as many as twelve samples to be screened per array. this approach will allow hundreds to thousands of samples to be screened in order to determine which markers or combination of markers demonstrate the best receiver operator characteristic (roc) performance [34, 43] . autoantigens in breast cancer subtypes such as her-2/neu positive tumors have been shown to correlate with increased autoantibody responses in patients. her-2/neu autoantibodies in those patients demonstrate approximately 18% sensitivity and 94% specificity. other groups have adapted similar approaches to multiplexing immune responses using high-density protein microarrays in breast cancer [15, 117] . among these studies, anderson et al. used nucleic acid programmable protein arrays (nappa) for sera screening in breast cancer [5] . these arrays were generated by printing individual genes as plasmid dna along with capture antibodies to gst tags on the fusion proteins. the arrays were then incubated with in vitro transcription and translation coupled cell-free lysates to produce the proteins and anchor them to the array surface. each array consisted of 1700 cancer associated candidate proteins including p53 as well as the epstein bar nuclear antigen (ebna) as a control. sera from four breast cancer patients and four healthy controls were used to probe the arrays and they found anti-p53 autoantibodies in cancer patients. because the proteins are not produced until the arrays are ready to be probed they do not suffer from degradation during periods of storage. however, the protein quantity is more variable between spots compared to directly printed arrays. we have found that spot to spot variation in protein amount may be overcome by evaluating the autoantibody response relative to the protein amount (i.e. the ratio of autoantibody signal to the signal from an epitope tag on the autoantigens). in our protein array immune response profiling studies we have found that this approach results in decreased signal variance between replicate spots (unpublished data). to date, no studies that attempt to identify novel breast cancer markers have been performed using high-density protein microarrays. pre-vious high-throughput serum screens in breast cancer have relied on serex and 2de-page and involved relatively small sample sets [53, 90] . a more interesting immune response profiling study may be the autoantibody responses to self-antigens in cancers of the blood and lymph, such as multiple myeloma. our lab has been involved in a protein microarray based screening project aimed at elucidating the autoantigen repertoire in multiple myeloma. multiple myeloma is a cancer of the bone marrow system resulting from the uncontrolled proliferation of monoclonal plasma cells (precursors of the bcells responsible for the production of antibodies) [60, 83] . monoclonal gammopathies of unspecified significance (mgus) is a precursor disease to multiple myeloma characterized by bone marrow plasmacytosis and increased m-protein levels in the blood [84] . we have probed high-density protein arrays using plasma from dozens of cases of mgus, multiple myeloma, and healthy controls. by comparing igg responses to individual antigens on the arrays between the healthy and diseased groups we have identified multiple autoantigens that are significantly differentially targeted by igg autoantibodies in early stage disease. this study was unique as it employed the highest density protein arrays for multiple myeloma immune response profiling to date and the patient samples were from a prospective collection. because samples were drawn prior to disease onset there are no artifacts resulting from medical treatment of the patients. by querying such early stage samples we have a better chance of identifying markers that will be effective for diagnosing patients at early stages when they are more treatable and will experience better outcomes. the markers identified in this study may yield insight into the biological processes and mutational events that contribute to the development of aggressive forms of multiple myeloma. in addition to early detection, cancer markers may also enable clinicians to offer personalized treatment. recent advances using the anti-cancer drugs herceptin and iressa illustrate this point. these drugs target specific patient populations: herceptin is effective against those tumors expressing the her2 receptor and iressa is effective for patients with specific mutations in the epidermal growth factor receptor [58, 109] . these drugs offer limited benefits to patients with the same cancer types when these markers are not present. thus, determining the marker profile of an individual's disease can enable the identification of distinct patient populations, allowing tailored and more effective treatment. one issue that we would like to note is that no single autoantibody response to an autoantigen has been confirmed to have sufficient sensitivity and specificity for screening purposes in early stage disease. however, by evaluating the antibody responses involving a panel of autoantigens, accuracy has been markedly improved [15, 43, 66] . thus, future protein microarray immune response screening tests will likely combine multiple autoantigens. numerous approaches have been applied to combining disease markers. some of the common methods for combining multiple autoantibody responses are linear regression, split-point analysis, and k-nearest neighbor (k-nn) [24, 119] . still there are currently no clinical screening or diagnostic tests that rely on a panel of protein markers,although multi-parameter dna microarray tests are becoming commonplace in diseases such as breast cancer [106] . clearly, more work is needed before autoantigen based microarray tests can be implemented in a clinical setting. the use of protein microarray technology for biomarker discovery in autoimmune diseases seems a natural extension of the technique as autoantibody responses have already been shown to contribute to disease progression in diseases such as lupus [59] . while antinuclear antibody tests are sometimes used to confirm diagnosis of certain autoimmune diseases, they are not disease specific [59, 78] . multiple sclerosis (ms) is a debilitating disease of the central nervous system characterized by rounds of axonal demyelination and repair. it affects mostly younger people and is more common in women than men. the underlying cause remains unknown, though there is mounting evidence that antibody responses to self proteins play a role in both demyelination and repair [28] . previous efforts have identified a number of myelin specific proteins that demonstrate increased autoantibody responses in ms. these autoantibodies have been detected in both serum and cerebrospinal fluid (csf). recently, protein microarray screening has been applied to evaluate autoantibody responses to subsets of myelin proteins that are known to be associated with ms and other neurodegenerative diseases [78, 82, 97] . the antigens that were shown to elicit autoantibody responses include classical ms antigens such as myelin-basic protein (mbp), myelin associated glycoprotein (mag) and myelin oligodendrocyte glycoprotein (mog) as well as proteins that have not been demonstrated to play a significant role in ms previously. these studies have suffered from the fact that the arrays were focused on a relatively small number of previously known candidate autoantigens. our group is currently conducting larger-scale screenings based on high-density protein microarrays. with this platform, we have tentatively identified a number of novel candidate markers in multiple sclerosis. we are currently working to validate these markers in a larger sample set. this less biased approach may result and the identification of novel autoantigens leading to a better understanding of the underlying mechanisms in the etiology of ms and result in improved screening and diagnostic tests. asthma, a common disease with a prevalence of 11% for all ages [75] , and 13% in children under 18 years of age in the united states [11] , is another disease involving an autoimmune mechanism [88, 121] . this heterogeneous inflammatory disease of the airways is marked by recurrent episodes of airway obstruction and wheezing [95, 114] , and is anatomically characterized by bronchoconstriction, inflammation and thickening of the airway walls [37] . considering asthma as an aberrant chronic wound healing process [36] , it would not be surprising that some of the released/leaked cellular contents from the airway epithelium due to damage and remodeling, similar to necrotic cancer cells, may elicit autoimmunity. in fact, aberrant autoantibodies have been detected in asthmatic sera by autologous serum skin tests as compared to normal controls [44] . autoreactive antibodies have also been detected in the asthmatic sera against the high-affinity ige receptor fcîµri [100, 101] . a few specific autoantigens have been identified in the serum of asthmatic patients, including the autoigg-reactive î²-adrenergic receptor [35, 108] , cytokeratin 18 [68] , dfs70 [105] , and î±-enolase [54] . moreover, studies on atopic dermatitis, which often occurs with asthma, revealed autoreactive ige antibodies against hom s 1-5 (hom s 1 = sart1; hom s 2 = î±-nac; hom s 3 = bcl7b; hom s 4 = a protein with calcium-binding motif; hom s 5 = a type ii cytokeratin) [70, 104, 105] and dfs70 [105] . however, these studies focused on small patient groups with no cross validation, as well as limited number of potential targets investigated. since both autoreactive igg and ige may be involved in the pathogenesis of asthma, we conducted a large-scale screening for asthma-associated auto-igg and ige reactive autoantigens using protein microarrays with more than 8,000 protein candidates (unpublished data). this is the first large-scale study to profile asthma-associated autoantigens, and the results will greatly improve our understanding of the role of autoimmunity in the etiology of asthma. one unique feature of this study is that, in order to maintain uniform probing conditions, we multiplexed the detection for both autoreactive igg and autoreactive ige in the serum samples simultaneously on the same array, with a mixture of anti-human igg and ige secondary detection antibodies labeled with distinct fluorescent dyes. our result suggested that the protein array is capable of detecting both igg and ige reactive signals in distinct emission channels with high specificity and no/detectable signal bleeding across the channels (fig. 2) . similar applications of protein arrays have been performed in studies of other autoimmune diseases. song et al. discovered 3 novel autoantigens, namely rps20, alba-like and dutpase for autoimmune hepatitis (aih) using a protein microarray containing 5011 nonredundant proteins [98] . horn et al. profiled the repertoire of igg autoantibodies in plasma samples from dilated cardiomyopathy (dcm) patients with a redundant protein microarray containing 37,200 total proteins and identified 26 autoreactive proteins to igg (with 6 of them reactive specifically to the igg3 subclass) [39] . autoantigens were also identified for the chronic disease alopecia areata by protein microarray technology [61] . these examples demonstrate the great potential of protein microarray technology in the application of autoantigen marker identification in autoimmune diseases. although protein microarray technology provides a high-throughput method with high sensitivity and specificity for protein marker discovery, there are particular limitations that investigators need to be aware of before applying the technology to their research. first of all, as probing protein microarrays for autoantibodies are in vitro studies with all the protein targets arrayed in a 2-d platform, one has to take into consideration off-target binding. therefore findings from protein microarray screenings should be validated in larger sample sets, and the autoantigens have to be confirmed by direct detection methods such as western blotting, elisa, or ihc in patient samples before an autoantigen can be confidently associated with the disease. secondly, microarrays that contain fulllength, folded proteins may not be recognized by autoantibodies that are directed against misfolded or degraded proteins expressed in disease cases, contributing to false negative detections. patwa et al. developed a method to chemically digest the proteins with cnbr before printing them on the arrays, which may help to overcome this problem [74] , however, as the digestion rate is hard to control, the final complex mixture of digested proteins at different levels may complicate normalization efforts and experimental control. normalization of the array data is another important consideration. as we have already discussed, normalization of protein spot morphology and quantity can be achieved by probing with a labeled antibody directed against an epitope-tag appearing on all of the arrayed proteins (e.g. gst). various software based methods have been adopted to adjust for regional defects and background that has traditionally been an issue for protein and dna based microarrays [120] . nevertheless, analysis and interpretation of the acquired large-scale data is still a challenge to both biologists and statisticians, therefore the development of improved algorithms is an ongoing effort. under real probing conditions, uncontrollable events such as scratches on the slides, deposition of salt and non-homogeneous local concentrations can further complicate the analysis of the array data, although internal controls are often included to help overcome these defects and improved array surface chemistries have significantly decreased local and regional background defects. in recent years it has become feasible to sequence entire genomes and transcriptomes using massively parallel sequencing platforms such as the 454 and solexa genome analyzer. these platforms use a highly sensitive light sensor (such as cmos sensors or ccd cameras) to capture fluorescent signals emitted from each deoxynucleotide as they are added to the dna chain simultaneously in up to millions of parallel reactions in a flowcell [38] , thus performing sequencing in a high-throughput manner to obtain short sequences (vary from 30 to 450 bp depending on the platform) from one or both ends. currently, related platforms and products are available through illumina ig, applied biosystems solid, roche 454 life science, and the helicos biosciences tsms [112] . another company, pacific biosciences, will manufacture a new sequencer that will perform single molecule sequencing by the end of 2010 [22] . a typical parallel sequencing procedure consists of the following steps: dna/rna isolation, fragmentation and dna/cdna library construction, highthroughput sequencing and read assembly and mapping. this method has many advantages compared to the traditional tiling microarray hybridization-based methods, or the more traditional rt-pcr and sanger sequencing method. these new platforms achieve single-base resolution in a high-throughput manner, have low background, no cross-hybridization noise, low dependence on the availability of existing genomic sequence, high reproducibility and low cost per base, and there is no upper limit for quantification [112] . in this section we will briefly review recent efforts in genetic marker discovery with next-generation parallel sequencing. this new generation of sequencing technology is shaping a new paradigm in disease marker research, in which massive amounts of sequence information from genomic dna and expression libraries are screened for linkages and associations of genetic and genomic markers to specific diseases by comparing disease patients and healthy individuals [1, 21, 31, 62] . genomic dna sequencing provides rich information on genetic variations (such as single nucleotide polymorphisms, insertions and deletions) and structural variations (such as copy number variations, transposition and transloca-tion) of the investigated genomes and is a powerful tool to reveal novel disease-associated markers. genome sequencing can also detect integrated viral sequences which may help address studies of virus-associated diseases. whole genome sequencing has already been applied in organisms with small genomes, such as acinetobacter baumannii [96] , toxoplasma gondii [12] , and drosophila melanogaster [77] , however, due to the large size of human genome and the high cost of parallel sequencing, human whole genome sequencing is still in its infancy. ley et al. were the first to sequence the entire genome of one type of cancerous tissue, the acute myeloid leukemia (aml) cells (32.7x haploid coverage), as well as corresponding normal tissue, the patient's skin tissue (13.9x haploid coverage) [57] . due to the unbiased nature of the sequencing methods, they were able to use read frequency to establish how rates of mutations vary within the cancer tissue. this concept is important for future works as we seek to understand the progression of mutational events that lead to the development of diseases like cancer. the researchers found that 59,209 single nucleotide variations were unique in the cancer tissue sample. these mutations resulted in changes to the coding regions in ten genes, two of which were previously implicated in cancer. nonetheless, as sequencing costs continue to decrease with the maturation of the platforms, whole genome sequencing of larger sample sets is shedding light on new venues of genetic and genomic marker identification in various diseases. both biologists and clinicians are preparing for this coming revolution, and projects have already been conceived such as clinseq, a pilot project led by green et al. which currently enrolls about 1000 participants for whole genome sequencing [10] . whole transcriptome sequencing by rna sequencing (rna-seq) is another promising application of parallel sequencing technology and has already been applied to marker discovery in various cancers. gene expression profiling has been shown to predict the outcome of breast and other types of cancer [40, 76] . shah et al. used paired end rna-seq to canvas the transcriptomes of four granulose-cell tumors (gct) from ovarian cancer patients [93] . they found a common missense mutation in the foxl2 gene (c402g) in those tumors that was not present in 11 other ovarian cancer transcriptomes that they also sequenced. additionally, this mutation was confirmed to be present in 97% of other gct cancers that they tested. leven et al. performed illumina sequencing on tiling-array-hybridization enriched transcripts representing 467 cancer associated genes from the k-562 chronic myeloid leukemia cell line and detected a wide range of dna and rna sequence alterations in the targeted transcripts [56] . these alterations included fusion transcripts such as bcr-abl1 and nup214-xkr3, as well as snps within and splice isoforms of these transcripts. while whole genome sequencing is still a relatively expensive proposition, transcriptome sequencing can be performed at much lower cost, facilitating the discovery of any mutations in the transcriptome that may contribute to the development of disease. in addition, rna sequencing also provides information that would not be obtained through whole genome sequencing, such as information on the expression level of each transcript, alternative splicing and rna editing, as well as trans-splicing events. we should expect to see genetic and genomic biomarkers identified as causative or contributing factors in human disease in the near future thanks to the utility of rna sequencing. one other promising application for transcriptome sequencing is to identify viruses in the host sample. in a study carried out by sorber et al., the authors successfully detected hepatitis b virus (hbv) sequences in the serum sample from a patient with hbv infection [99] . similarly, palacios et al. used rna-seq and identified a novel old world arenavirus in rna samples extracted from the liver and kidney of three deceased patients who had transplantation-related infection [72] . nakamura et al. obtained 20-460 reads of influenza virus sequence in nasopharyngeal aspirates and 484-15,260 reads of norovirus sequence in fecal specimens from patients suffering from influenza or norovirus infections [69] . meanwhile, rwahnih et al. sequenced the rna from a grapevine with the roche 454 system and revealed infection of 32 plant viruses as well as one novel virus in the diseased grapevine [3] . our group has also carried out an rna-seq experiment to detect west nile virus sequences in infected macrophages (unpublished data). we obtained 4700 reads (0.06% of the total mapped reads) mapped to the west nile virus genome from the infected cells and very few reads (30, â�¼0.0003% of the total mapped reads) mapped to viral sequences in rna isolated from mock control cells. after analysis recheck of the few reads that did map to the virus genome in the control cells, we found that they were redundant with sequences in the human genome. these studies proved that rna-seq can achieve high sensitivity and specificity to identify and profile infecting viruses, or the "virome", us-ing rna samples isolated from the host. moreover, rna-seq will also provide information on virus-host interactions by monitoring expression changes of the host's genes. while mutations in protein-coding sequence are well known to contribute to multiple diseases, rna-seq is limited to only those actively transcribed sequences of the genome. this bias results in overlooking variations in non-transcribed regions of the genome that can be important contributors to disease as mutations in these regions can result in aberrant gene regulation. besides whole genome sequencing, chip-seq, or chromatin immunoprecipitation-sequencing, is another approach to address mutations in functional non-transcribed regions of the genome. this technology sequences the genomic regions bound by transcription factors or other dna-binding proteins (such as histones), and provides information on the position of these binding sites as well as possible mutations in these sites. the binding site profiles of multiple transcription factors, as well as the identified sequence variations within, may act as new markers for diseases such as leukemia [8, 73] . sono-seq is a related technology developed in our lab, which parallel sequences sonicated formaldehyde cross-linked chromatin dna via illumina sequencing, and identifies the chromatin regions that are open and accessible (nucleosome-free therefore susceptible to sonication) [6] . with this technology we identified multiple highly accessible chromatin regions including actively transcribed promoter regions as well as the ctcf insulator protein binding sites. this technology is similar to another open chromatin finding technology, termed faire (formaldehyde-assisted isolation of regulatory elements), which selects open chromatin regions for dna microarray hybridization by phenolchloroform extraction of sonicated cross-linked samples [27] . when interrogated in the background of a disease compared with healthy controls, the identified profiles of these nucleosome-free regions may provide a new type of disease marker for future studies. while next-generation sequencing holds great promise for the discovery of novel disease markers, there are issues with the current technology, such as artifacts due to sample preparation (both reverse transcription and polymerase chain reaction can generate biases) and data processing (assembly of short reads can result in errors, especially in regions of repetitive sequence). newer technologies from companies like helicos biosciences and pacific biosciences are on the horizon that could overcome these issues by direct rna sequencing and long single molecule sequencing, fulfilling the promise of personalized medicine in the post genome era [22, 71] . mass spectrometry technology (ms) has been growing rapidly in the past several decades. since john bennet fenn and koichi tanaka developed new soft desorption methods that made mass spectrometric analyses of biological macromolecules possible, this technology has been widely used in proteomic studies [19, 49] . the ability to identify and quantify target molecules (e.g. peptides) makes mass spectrometry methods a popular tool for disease marker discovery. disease marker discovery with mass spectrometry is usually combined with varied sample separation methods such as 2de (2-dimentional electrophoresis) and 2d-dige (2-dimentional differential in-gel electrophoresis) [18] . in a typical procedure, mixed proteins from pooled disease samples and pooled controls are separated with 1d or 2d electrophoresis, and individual protein bands or spots are visualized and differential bands or spots are then excised followed by enzyme digestion (e.g. trypsin). the digested peptides are then subjected to mass spectrometry analysis for protein identification. with this method, shen et al. identified 40 potential markers for pancreatic adenocarcinoma, and the spectrum of these markers covered antioxidant proteins, chaperones, calcium-binding proteins, catalytic enzymes, signal transduction proteins and extracellular matrix proteins [94] . similarly, wang et al. identified 52 differentially expressed proteins (including 8 novel markers) associated with oral squamous cell carcinoma, and validated one of the eight markers named rack1 using immunostaining and gene silencing studies [113] . even though 2d electrophoresis can improve protein separation and assist in further identification by mass spectrometry, the discovery of low-abundance disease markers has been greatly limited by the poor resolution and sensitivity of 2de or 2d-dige methods. furthermore, the pooled samples will not only lose important information such as personal variation of the disease markers among different individuals, but also miss protein that are only present in a subset of the sample population. recent improvement of mass spectrometry techniques as well as data analysis algorithms has enabled analysis of complex protein samples [116] . currently, liquid chromatography (lc)-ms/ms using electrospray ionization (esi) is one of the commonly used methods for large-scale shotgun proteomic studies. gel-free methods, such as mudpit (multidimensional protein identification technology) has been more and more popular and greatly enhanced detection limits [115] , and the improved resolution and accuracy of mass spectrometers makes this technology more useful in disease marker discovery [111] . with 2d lc-ms/ms, ralhan et al. identified 811 nonredundant proteins in head-and-neck squamous cell carcinoma from 15 individual cancer samples compared with one pooled normal control, and the panel of the three best performing markers achieved a sensitivity of 92% and specificity of 91% in cancer classification [85] . a most recent study to identify ovarian cancer biomarkers from patient ascites samples with 2d lc-ms/ms also yielded a panel of 25 known and 52 novel protein markers [51] . these studies demonstrated that the improved ms techniques not only enabled researchers to search for biomarkers in unpooled samples, but also could lead to the identification of a larger number of potential markers due to improved sensitivity. in addition to protein marker identification, mass spectrometry is also capable of identifying metabolom-ic markers [92] . metabolomics is a novel field that studies the global profiles of all metabolites in a given sample. since diseases such as cancer usually have unique metabolomes [30] , the over-or under-presented metabolites could serve as potential markers of the disease. moreover, certain metabolites in the cells will also influence the activity of larger biomolecules such as kinases (unpublished data), therefore identification of the cancer-specific metabolomes would be of great value. currently, multiple metabolites have been associated with various tumors, such as alanine, saturated lipids, ccms, glycine, lactate, myo-inositol, nucleotides, pufas and taurine [30] , and it will not be surprising if this list will grows dramatically in the coming years. although mass spectrometry is a powerful tool in molecular marker identification, the clinical application of this technology for diagnostic purposes is still limited. mass spectrometry may one day become the platform of choice for detection of disease-associated markers, however, the high cost of mass spectrometers as well as lack of standardized methods are preventing it from being adopted by clinicians as a diagnostic tool. moreover, the high level of molecular complexity of biological samples is still a large obstacle in both marker identification and application. there remains great space for improvement before mass spectrometry realizes its ultimate potential in the clinic. disease markers are important for the efficient diagnosis, prognosis and treatment of a disease, therefore identifying these markers is crucial especially for diseases with high mortality rates such as cancer. each technology reviewed in this article has a unique niche in disease marker discovery. protein microarray technology excels in finding protein markers, especially antibody markers; next-generation parallel sequencing is designed for rna and genetic/genomic marker discovery; and mass spectrometry specializes in the identification of protein markers as well as metabolomic markers. each technology has its unique advantages and limitations, and the "-omics" information obtained with the help of these technologies may complement each other and leading to a comprehensive view of disease (fig. 3) . this information will greatly expand our understanding of the etiology and course of human diseases, resulting in more efficient diagnosis and treatment of disease. moreover, by adopting a comprehensive "-omics" view of human diseases, it will be interesting to discover the extent to which these systems interact with each other. will we find that a disease associated with certain genetic markers also develops specific autoantibodies? or a certain autoantigen response is actually due to the dysregulation of a specific metabolite or trans-spliced mrna? is it possible that diseases such as multiple sclerosis and asthma actually are caused by the coordinating effect of genetic susceptibility and, say, viral infection? while systems biology approaches to disease marker discovery are still in their infancy they have already led to the discovery of many promising genetic and protein markers in various diseases. additionally, new dimensions in the development and application of these approaches may further revolutionize this field by interrogating additional "-omes", such as the methyl-genome, the "kinome" and the "virome". these systems may be as important as the systems reviewed above to establish a complete understanding of, and efficient treatments for, many diseases. comprehensive genomic characterization defines human glioblastoma genes and core pathways immune responses in cancer deep sequencing analysis of rnas from a grapevine showing syrah decline symptoms reveals a multiple virus infection that includes a novel virus oncoproteomic profiling with antibody microarrays application of protein microarrays for multiplexed detection of antibodies to tumor antigens in breast cancer mapping accessible chromatin regions using sono-seq identification of tumor-associated 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mass spectrometry-based shotgun proteomics autoantibodies as potential biomarkers for breast cancer global analysis of protein activities using proteome chips severe acute respiratory syndrome diagnostics using a coronavirus protein microarray procat: a data analysis approach for protein microarrays asthma and autoimmunity: is there a connection? we cordially thank our collaborators, drs. gil mor (ovarian cancer studies), geoffrey chupp (asthma studies) and ruth montgomery (west nile virus studies) at yale university, and dr. jonas bergqvist at uppsala university, sweden (multiple sclerosis study) for their contributions to the projects outlined in this review. key: cord-294125-v2dr4hm0 authors: albert, manuel; bécares, martina; falqui, michela; fernández-lozano, carlos; guerra, susana title: isg15, a small molecule with huge implications: regulation of mitochondrial homeostasis date: 2018-11-13 journal: viruses doi: 10.3390/v10110629 sha: doc_id: 294125 cord_uid: v2dr4hm0 viruses are responsible for the majority of infectious diseases, from the common cold to hiv/aids or hemorrhagic fevers, the latter with devastating effects on the human population. accordingly, the development of efficient antiviral therapies is a major goal and a challenge for the scientific community, as we are still far from understanding the molecular mechanisms that operate after virus infection. interferon-stimulated gene 15 (isg15) plays an important antiviral role during viral infection. isg15 catalyzes a ubiquitin-like post-translational modification termed isgylation, involving the conjugation of isg15 molecules to de novo synthesized viral or cellular proteins, which regulates their stability and function. numerous biomedically relevant viruses are targets of isg15, as well as proteins involved in antiviral immunity. beyond their role as cellular powerhouses, mitochondria are multifunctional organelles that act as signaling hubs in antiviral responses. in this review, we give an overview of the biological consequences of isgylation for virus infection and host defense. we also compare several published proteomic studies to identify and classify potential mitochondrial isgylation targets. finally, based on our recent observations, we discuss the essential functions of mitochondria in the antiviral response and examine the role of isg15 in the regulation of mitochondrial processes, specifically oxphos and mitophagy. the innate immune response is the first line of defense against microbial and viral infections. invading microorganisms produce danger-and pathogen-associated molecular patterns that interact with host pattern-recognition receptors, triggering several intracellular signaling cascades that activate nuclear factor kappa-b (nf-κb), mitogen-activated protein kinases (mapks) and interferon (ifn) regulatory factors (irfs), resulting in the expression of a broad array of proteins involved in host defense such as type-i ifns and proinflammatory cytokines [1, 2] . the release of type-i ifns has both autocrine and paracrine effects via ifnα/β receptors (ifnars) on the cell surface. binding to ifnars leads to the activation of the janus kinase-signal transducer and activator of transcription proteins (jak-stat) signaling pathway and the formation of the interferon-stimulated gene factor 3 (isgf3) complex, with the subsequent expression of ifn-stimulated genes [3] that establish an antiviral state and play important roles in determining the host innate and adaptive immune responses [4] . one of the most highly induced genes in the type-i ifn signaling cascade is isg15 (interferon-stimulated gene 15), which encodes a small ubiquitin-like protein involved in a post-translational modification (1) . intracellular isg15 can be processed into its mature form and conjugated to de novo synthesized proteins in a process termed isgylation. isg15 processing exposes its carboxy-terminal lrlrgg motif, allowing its conjugation to lysine residues in target proteins to modulate their function. in addition, isgylation is reversible due to the action of the protease usp18, which also regulates ifnar-mediated signaling (2) . isg15 can remain unconjugated within the cell, regulating protein activity (3), or be secreted as a cytokine, acting as a chemotactic and stimulating factor for immune cells (4) . binding of isg15 to lfa-1 integrin receptor on the surface of nk cells promotes the activation, production and release of ifn-γ il-10 after il-12 priming. moreover, extracellular isg15 is able to form dimers/multimers through cysteine residues, to modulate cytokine levels. isg15 conjugation to target proteins is a covalent and reversible process through the action of a 43-kda deisgylase enzyme, ubiquitin-specific protease 18 (usp18) [9, 10] . interestingly, both isg15 and its conjugating and deconjugating enzymes are upregulated by type-i ifn [9] , as well as by other stimuli such as type-ii and type-iii ifns [11] [12] [13] , lipopolysaccharide [14] , retinoic acid [15] , dna damage or genotoxic reagents [16] . usp18 not only acts as a deconjugating enzyme, but also as a negative regulator of the type-i ifn pathway (figure 1) , with important implications in antiviral and antibacterial responses, immune cell development, autoimmune diseases and cancer [17] . in humans, isg15 binds to usp18, increasing its stability and leading to a decrease in ifn-α/β signaling. consequently, isg15 deficiency results in low usp18 levels, and therefore a sustained elevation in figure 1 . intracellular and extracellular activities of isg15. different stimuli trigger the expression of isg15, which is produced as a precursor of 17 kda with two ubiquitin-like domains linked by a hinge region (1) . intracellular isg15 can be processed into its mature form and conjugated to de novo synthesized proteins in a process termed isgylation. isg15 processing exposes its carboxy-terminal lrlrgg motif, allowing its conjugation to lysine residues in target proteins to modulate their function. in addition, isgylation is reversible due to the action of the protease usp18, which also regulates ifnar-mediated signaling (2) . isg15 can remain unconjugated within the cell, regulating protein activity (3), or be secreted as a cytokine, acting as a chemotactic and stimulating factor for immune cells (4) . binding of isg15 to lfa-1 integrin receptor on the surface of nk cells promotes the activation, production and release of ifn-γ il-10 after il-12 priming. moreover, extracellular isg15 is able to form dimers/multimers through cysteine residues, to modulate cytokine levels. isg15 conjugation to target proteins is a covalent and reversible process through the action of a 43-kda deisgylase enzyme, ubiquitin-specific protease 18 (usp18) [9, 10] . interestingly, both isg15 and its conjugating and deconjugating enzymes are upregulated by type-i ifn [9] , as well as by other stimuli such as type-ii and type-iii ifns [11] [12] [13] , lipopolysaccharide [14] , retinoic acid [15] , dna damage or genotoxic reagents [16] . usp18 not only acts as a deconjugating enzyme, but also as a negative regulator of the type-i ifn pathway (figure 1) , with important implications in antiviral and antibacterial responses, immune cell development, autoimmune diseases and cancer [17] . in humans, isg15 binds to usp18, increasing its stability and leading to a decrease in ifn-α/β signaling. consequently, isg15 deficiency results in low usp18 levels, and therefore a sustained elevation in isg expression. this role for isg15, which is absent in mice, seems to be predominant in humans, since patients appear not to be more susceptible to viral infections [18, 19] . beyond the above-mentioned forms of isg15-conjugated to target proteins or unconjugated within the cell-isg15 is also secreted into the serum, mainly by granulocytes via their secretory pathway [20] . lymphocyte function-associated antigen 1 receptor (lfa1) has recently been identified as the cellular receptor for isg15 ( figure 1 ). isg15 binding to lfa1 triggers the activation of src family kinases, promoting ifn-γ and interleukin-10 (il-10) secretion in natural killer (nk) cells and, likely, also t-lymphocytes [21] . the role of isg15 as an inductor of ifn-γ secretion seems to be the basis for the increased susceptibility to mycobacterial diseases in patients lacking a functional form of isg15 [20] . secreted isg15 has also been described to promote nk [22] and dendritic cell [23] maturation, and to act as a chemotactic factor for neutrophils [24] . along this line, a recent study highlighted the presence of dimeric and multimeric forms of extracellular isg15 important for its cytokine activity during parasite infection, and speculated on the existence of an unknown isg15 receptor on dendritic cells that mediates chemotaxis of these cells to the site of infection and il-1β production [25] . although there are several features of isg15 that are shared with ubiquitin, specially its structure, conjugation and deconjugation mechanisms [26] , isgylation has not been shown to stimulate proteasomal degradation of its substrates [10] . furthermore, some of the isgylation consequences are exerted by restricting the ubiquitin system, what might be mediated through the conjugation of isg15 to different e2 and e3 ubiquitin-conjugating enzymes [27] , or even through the formation of mixed ubiquitin-isg15 chains [28] . as a result, isgylation can decrease the polyubiquitylated proteins levels and downregulate protein turnover by the proteasome system [28] . additionally, unlike ubiquitin, no poly-isg15 chains or specific isg15-interacting motifs have been identified yet. in the following sections, we discuss the antiviral mechanisms mediated by isgylation of both viral and cellular proteins, with a focus on mitochondrial proteins, as we recently showed that isg15 modulates essential mitochondrial metabolic processes such as respiration and mitophagy in macrophages, with important implications for innate immune responses [29] . the antiviral activity associated with isg15 and/or isgylation has been widely described since the first observation that isg15-/mice were more susceptible to viral infections than their wild-type counterparts, albeit the role of isg15 and isgylation in viral life cycles is specific to the virus involved [30] . early studies using isg15-/mice demonstrated that isg15 has a protective effect against lethal infection by influenza virus, herpes simplex virus (hsv-1) and sindbis virus (sinv) [31] . similarly, mice deficient in ube1l-the e1 enzyme of isg15-were also more susceptible to lethal infection by sinv [32] . moreover, exogenous expression of wild-type isg15 by recombinant chimeric sinv protected ifnar-/-mice against systemic and lethal infections, whereas expression of isg15 mutants unable to conjugate to proteins did not show this protective effect [33] , indicating an intrinsic antiviral role for isgylation. it should be noted that such an antiviral effect could be due to the conjugation of isg15 to viral and/or cellular proteins. by contrast, free isg15, but not isgylation, has been described to promote antiviral responses against chikungunya virus (chikv) infection [34] . to date, an antiviral effect mediated by isg15 or isgylation has been described using in vitro and/or in vivo systems for many other dna and rna viruses, including hepatitis b virus [35] , vesicular stomatitis virus [36, 37] , respiratory syncytial virus [38, 39] , human immunodeficiency virus type 1 (hiv-1) [40] , and ebola virus [27] . the antiviral effect of isg15 and isgylation has also been described against viruses of the genera novirhabdovirus, birnavirus and iridovirus in zebrafish, an example of the evolutionary conservation of the antiviral role of isg15 among vertebrates [41] . given the importance of the antiviral response governed by isg15, it is not surprising that viruses have evolved strategies to counteract its antiviral effects. for example, influenza b virus (ibv) ns1 protein [42] , vaccinia virus e3 protein [43] , and human cytomegalovirus (hcmv) ie1 and pul26 proteins obstruct isg15 antiviral action by preventing isgylation [44] . similar mechanisms are also described for orthonairovirus and arterivirus otu-domain-containing proteases [45] and for coronavirus papain-like proteases (plpro), which cleave isg15 from target proteins. remarkably, a plpro inhibitor was shown to protect mice from lethal infection in vivo [46] . surprisingly, it has been reported that isgylation is necessary for robust production of hepatitis c virus (hcv), conferring a novel role for isg15 as a proviral factor that promotes virus production. indeed, in human hepatocytes, sirna silencing of isg15 was sufficient to both inhibit hcv replication and increase ifn expression [47] . several reports have now highlighted a role for isg15 in the monitoring of hcv replication in cell cultures, as well as in the maintenance of hcv in liver, and pinpoint isg15 as among the predictor genes for non-response to ifn therapy [48] . regarding the direct antiviral effect of isgylation via conjugation to viral proteins, perhaps the best-known example is the influenza a virus (iav) ns1 protein. this non-structural protein is abundantly expressed in infected cells and acts in multiple stages of the viral cycle, with important roles in ifn antagonism including sequestering double-stranded rna (dsrna), inhibiting dsrna-activated protein kinase (pkr) and contributing to the nuclear export of viral mrnas while blocking the splicing and export of cellular mrnas [49] . seven lysine (k) residues in the ns1 protein were identified as potential target sites of isgylation [50] . specifically, isg15 binding to k41, which is part of the ns1 nuclear-localization signal, prevents its interaction with importin-α, inhibiting the translocation of ns1 to the nucleus and therefore repressing iav replication and viral rna processing [51] . moreover, isgylation of the iav ns1 protein blocks its ability to counteract the innate immune response, prevents its interaction with pkr and, therefore, restores ifn-induced antiviral activities against iav [50] . beyond ns1, influenza virus nucleoprotein (np) and matrix protein (m1) have also been reported as targets of isg15 conjugation. isgylated np hinders the oligomerization of the more abundant unconjugated np, acting as a dominant-negative inhibitor of np oligomerization, impeding the formation of viral ribonucleoproteins and causing decreased viral protein synthesis and virus replication [52] . interestingly, this study also identified a new role for influenza b virus ns1 in the sequestration of isgylated viral proteins, especially isgylated nps, which is perhaps an evolutionary mechanism to block the antiviral effect of isgylation. another example of isgylation of a viral protein with antiviral effects is the 2a protease (2apro) of coxsackievirus b3 (cvb3). isg15 conjugation to 2apro inhibits its ability to cleave the eukaryotic initiation factor eif4g in cardiomyocytes, hindering the translational shutoff induced by cvb3 infection [53] . consequently, isg15 conjugation to cvb3 leads to a reduction in virus titers and limits inflammatory cardiomyopathy, heart failure and lethality [53] . similarly, isgylation of the hcmv scaffold protein pul26 interferes with the viral modulation of the innate immune response. specifically, isgylation of pul26 at k136 and k169 inactivates its function in the downregulation of tnfα-mediated nf-κb activation, suppressing hcmv growth [44] . finally, another example of an isgylated viral protein is the human papillomavirus (hpv) l1 capsid protein. isgylated l1 proteins were shown to be incorporated into hpv pseudoviruses, resulting in a reduced infectivity; the precise mechanism that mediates this inhibitory effect remains elusive [8] . knowledge about the impact of host protein isgylation in virus replication and cell homeostasis is still scant. in contrast to ubiquitylation, the molecular effect of isg15 conjugation on target proteins is not always clear. protein isgylation has been reported to increase protein degradation by selective autophagy [54] , but there are also many examples where isgylation inhibits ubiquitylation, frustrating proteasome-mediated degradation of target proteins [55] [56] [57] . with regard to proteins involved in antiviral response, many effectors of ifn signaling such as pkr [58] , retinoic acid-inducible gene-i (rig-i) [59] and myxoma resistance protein 1 (mxa) [60] have been reported to be targets of isgylation. pkr isgylation at k69 and k159, both located in the dsrna-binding motif, triggers its activation. this modification occurs in the absence of viral rna and leads to the phosphorylation of eif2α, preventing protein translation [58] and suggesting that isgylation might mediate the activation of pkr in response to stressful stimuli beyond viral infection. further, isg15 conjugation to rig-i decreases rig-i cellular levels and downregulates rig-i-mediated signaling. accordingly, isgylation of rig-i represents a negative feedback loop that might control the strength of the antiviral response [59] . interestingly, free isg15 also regulates rig-i levels by promoting the interaction between rig-i and the autophagic cargo receptor p62, mediating rig-i degradation via selective autophagy [61] . the interferon-induced mxa protein is also a target of isgylation, though the effect of this modification is not clear. other proteins involved type-i ifn signaling and regulation, such as components of the jak-stat pathway or regulators of signal transduction (e.g., jak1 and extracellular signal-regulated kinase 1 [erk1]), are also bound by isg15, although the functional consequences of isgylation remain unknown [9, 62] . moreover, interferon regulatory factor 3 (irf3), stat1 and the actin-binding protein filamin b are also targets for isg15 conjugation, with implications in the development of the innate immune response. irf3 is isgylated at k193, k360 and k366, which attenuates its interaction with the peptidyl-prolyl isomerase pin1, preventing irf3 ubiquitylation. thus, isgylation of irf3 sustains its activation and enhances irf3-mediated antiviral responses by inhibiting its degradation [63] . in a similar manner, isgylation of phosphorylated stat1 (pstat1) inhibits its polyubiquitylation and further proteasomal degradation, supporting sustained stat1 activation [57] . isgylation of filamin b, which acts as a scaffold of ifn signaling mediators, negatively regulates ifnα-induced c-jun n-terminal kinases (jnk) signaling, preventing apoptosis induction [64] . beyond antiviral response, isgylation has been described to block the process of virus budding by interfering with the endosomal sorting complexes required for transport (escrt) machinery. for example, isgylation of chmp5 triggers its aggregation and the sequestration of the vps4 cofactor lip5, impairing the membrane recruitment of vps4 and its interaction with the gag budding complex of avian sarcoma leukosis virus and hiv-1, leading to the inhibition of virus release from the cell [65] . similarly, isgylation of tumor susceptibility gene 101 protein (tsg101), another component of the escrt sorting complex, inhibits the trafficking of viral hemagglutinin to the cell surface during iav infection [66] , blocking virus release. isg15 has also been described to inhibit the interaction of hiv-1 gag protein with tsg101, underscoring a critical role of isg15 in the ifn-mediated inhibition of hiv-1 budding and release [40] . this sorting mechanism is also used in the generation of exosomes, which are small vesicles secreted to the extracellular environment by most cell types. interestingly, isgylation of tsg101 has been recently reported to inhibit exosome secretion [67] . the above examples serve to illustrate the relevance of isgylation in the induction and regulation of the antiviral response (for a more complete review of isgylated cellular proteins see reference [30] ), and highlight the complexity of fully understanding the consequences of isgylation in the regulation of biochemical processes where it is involved. although the significance of isgylation of host proteins has been elucidated for only a small set of cellular proteins, isgylation has a broad target specificity, and there is increasing evidence for its role in regulating many cellular functions. to address this concept, several proteomic studies have been performed to determine isgylated host proteins. zhao et al. [60] transfected a tagged isg15 protein into ifn-stimulated hela cells, and used affinity selection to identify 158 isgylated proteins. in a similar approach, giannakopoulos et al. [68] used ifn-stimulated usp18-/-mouse embryonic fibroblasts and human u937 cells to detect up to 76 proteins conjugated to endogenously-expressed isg15. a third proteomic study [69] identified 174 isgylated cellular proteins in ifn-stimulated a549 human lung adenocarcinoma cells stably expressing flag-isg15. more recently, peng et al. [70] examined isgylated proteins in influenza virus-infected a549 cells, identifying a total of 22 cellular proteins in addition to viral ns1 protein. we have surveyed the proteins identified by these four studies, which rendered up to 330 cellular proteins. identified proteins include, as previously outlined [8] , abundant constitutively expressed proteins as well as diverse interferon-induced proteins. interestingly, there is only a low degree of overlap between the studies, and only four proteins are common to all four analyses (the glycolytic enzymes aldo1 and eno1, the peroxiredoxin prdx1, and stat1). these discrepancies may reflect the different transcriptional/translational patterns of the different cell lines included in each study, as it is believed that the biological effects of isgylation are dynamic and cell type/tissue-specific [5] . we used david bioinformatics resources [71, 72] to determine the subcellular localization of the proteins identified as isgylation targets in the aforementioned studies, with the aim to obtain a comprehensive picture of the broad range of actions of isg15. in agreement with a previous report [73] , our analysis ( figure 2) shows that isg15-targeted proteins are found almost throughout the cell, including nucleus, perinuclear space, cytosol, mitochondria, rough endoplasmic reticulum and cell membranes [73] . moreover, a similar percentage of isgylation targets were predicted to be located in the nucleus, cytoplasm, extracellular space or as secreted proteins (figure 2) . interestingly, proteins associated with cytoskeleton and cell junctions represent a significant percentage of the isg15 target proteins. other cell structures such as the melanosome or myelin sheath were also represented in the study, perhaps accounting for a specific role of isgylation in these organelles. the potential role of isg15 in mitochondria seems to be relevant, as a recent study predicted that 17% of free isg15 was localized to mitochondria [13] . in our own analysis of the above proteomic studies, fifty-two isgylated proteins were predicted to localize to mitochondria, representing about 5% of the total isg15 target proteins ( figure 2 ). further examination of these potentially isgylated proteins indicate that different mitochondrial processes could be affected by isg15 conjugation (table 1) . remarkably, several subunits of the atp synthase (complex v of the respiratory chain) appear to be isg15 targets, which may be of relevance as mitochondrial atp production is the main source of energy for the cell. in line with these observations, our recent work linked isg15 to the control of the mitochondrial oxidative metabolism in macrophages in the context of viral infection [29] . based on the evident association between isg15 and mitochondria, we will briefly review the role of mitochondria as antiviral mediators and targets of ubiquitin-like modifiers, focusing on the current knowledge about isg15-and isgylation-mediated regulation of these multifunctional organelles. included in each study, as it is believed that the biological effects of isgylation are dynamic and cell type/tissue-specific [5] . we used david bioinformatics resources [71, 72] to determine the subcellular localization of the proteins identified as isgylation targets in the aforementioned studies, with the aim to obtain a comprehensive picture of the broad range of actions of isg15. in agreement with a previous report [73] , our analysis ( figure 2) shows that isg15-targeted proteins are found almost throughout the cell, including nucleus, perinuclear space, cytosol, mitochondria, rough endoplasmic reticulum and cell membranes [73] . moreover, a similar percentage of isgylation targets were predicted to be located in the nucleus, cytoplasm, extracellular space or as secreted proteins (figure 2) . interestingly, proteins associated with cytoskeleton and cell junctions represent a significant percentage of the isg15 target proteins. other cell structures such as the melanosome or myelin sheath were also represented in the study, perhaps accounting for a specific role of isgylation in these organelles. the potential role of isg15 in mitochondria seems to be relevant, as a recent study predicted that 17% of free isg15 was localized to mitochondria [13] . in our own analysis of the above proteomic studies, fifty-two isgylated proteins were predicted to localize to mitochondria, representing about 5% of the total isg15 target proteins ( figure 2 ). further examination of these potentially isgylated proteins indicate that different mitochondrial processes could be affected by isg15 conjugation (table 1) . remarkably, several subunits of the atp synthase (complex v of the respiratory chain) appear to be isg15 targets, which may be of relevance as mitochondrial atp production is the main source of energy for the cell. in line with these observations, our recent work linked isg15 to the control of the mitochondrial oxidative metabolism in macrophages in the context of viral infection [29] . based on the evident association between isg15 and mitochondria, we will briefly review the role of mitochondria as antiviral mediators and targets of ubiquitin-like modifiers, focusing on the current knowledge about isg15-and isgylation-mediated regulation of these multifunctional organelles. [60, [68] [69] [70] predicted to locate to mitochondria. proteins are grouped according to biological functions. acyl-coa thioesterase 8 (acot8) [60] complement c1q binding protein (c1qbp) [69] receptor for activated c kinase 1 (rack1) [60] solute carrier family 25 member 5 (slc25a5) [69] solute carrier family 25 member 6 (slc25a6) [69] staphylococcal nuclease and tudor domain containing 1 (snd1) [69] negative regulation of apoptotic process nme/nm23 nucleoside diphosphate kinase 2 (nme2) [69] annexin a1 (anxa1) [69, 70] glutathione s-transferase pi 1 (gstp1) [60] heat shock protein family a (hsp70) member 5 (hspa5) [69] interferon-induced protein with tetratricopeptide repeats 3 (ifit3) [60] positive regulation of protein insertion into mitochondrial membrane involved in apoptotic signaling pathway stratifin (sfn) [69] tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta (ywhab) [69] tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein épsilon (ywhae) [69] tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (ywhag) [69] tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta (ywhaq) [69] tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (ywhaz) [69] atp biosynthetic process atp synthase, h+ transporting, mitochondrial f1 complex, alpha subunit 1, cardiac muscle (atp5a1) [60, 69] atp synthase, h+ transporting, mitochondrial f1 complex, beta polypeptide (atp5b) [60, 69] atp synthase, h+ transporting, mitochondrial fo complex subunit g (atp5l) [70] oxidation-reduction process aldehyde dehydrogenase 18 family member a1 (aldh18a1) [70] fatty acid synthase (fasn) [60, 69] glutathione-disulfide reductase (gsr) [69] lactate dehydrogenase b (ldhb) [69] malic enzyme 1 (me1) [68] peroxiredoxin 1 (prdx1) [60, 69, 70] peroxiredoxin 4 (prdx4) [69] sorbitol dehydrogenase (sord) [68] superoxide dismutase 1, soluble(sod1) [69] thioredoxin reductase 1 (txnrd1) [60, 69] thioredoxin (txn) [69] aminoacyl-trna synthetase alanyl-trna synthetase (aars) [68] glycyl-trna synthetase (gars) [68] phenylalanyl-trna synthetase 2, mitocondrial (fars2) [60] tricarboxylic acid cycle malate dehydrogenase 1 (mdh1) [69] malate dehydrogenase 2 (mdh2) [69] glycolisis oxoglutarate dehydrogenase (ogdh) [60] pyruvate kinase, muscle (pkm) [60, 69, 70] chaperone chaperonin containing tcp1 subunit 7 (cct7) [69] heat shock protein 90 alpha family class b member 1 (hsp90ab1) [60, 69, 70] heat shock protein family a (hsp70) member 1a (hspa1a) [60, 69] heat shock protein family d (hsp60) member 1 (hspd1) [60, 69] ion channel chloride intracellular channel 1 (clic1) [60, 69] annexin a6 (anxa6) [69] other functions creatine kinase, mitochondrial 1b (ckmt1b) [69] ubiquitin-like modifier activating enzyme 1 (uba1) [69] leucine aminopeptidase 3 (lap3) [60] 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/imp cyclohydrolase (atic) [60, 69] clathrin heavy chain (cltc) [60, 69] queuine trna-ribosyltransferase accessory subunit 2 (qtrt2) [60] enoyl-coa hydratase and 3-hydroxyacyl coa dehydrogenase (ehhadh) [69] atp binding cassette subfamily f member 2 (abcf2) [60] mitochondria have myriad functions in the cell although they are best known for providing energy in the form of atp and for controlling metabolism to maintain energy homeostasis. owing to their endosymbiotic origin, mitochondria have their own genome, a single 16-kb circular dna which codes for 13mitochondrial proteins, 2 ribosomal rnas and 22 transfer rnas [74] . the remainder of mitochondrial proteins are encoded by nuclear dna and are then transported to the mitochondria through the recognition of amino acid sequences known as mitochondrial targeting signals [75] . as double-membrane organelles, mitochondria have an outer mitochondrial membrane (omm), where proteins responsible for transport of different molecules are embedded [76] ; an intermembrane space (ims) and an inner mitochondrial membrane (imm), where electron transport chain (etc) proteins are localized and oxidative phosphorylation (oxphos) and atp production takes place [77] , and a mitochondrial matrix (mm), compartment, where many metabolic pathways occur, such as the tricarboxylic acid cycle, fatty-acid oxidation, synthesis of biomolecules and regulation of apoptosis [78] . the proper development of mitochondrial processes is critical for immune response, as the susceptibility to microbial infections and the risk of systemic inflammatory responses increases considerably when these organelles malfunction [79, 80] . mitochondria are important for antiviral signaling. during rna-virus infection, viral rnas are initially recognized by cytoplasmic sensors, mainly rig-i-like receptors (rlrs) [2] , whose interaction with mitochondria is essential for the coordination and development of an adequate antiviral response. the common structure of rlrs consists of a carboxy-terminal regulatory domain, a central rna helicase domain and amino-terminal caspase recruitment domains (cards) [81] . after binding to viral rna, rlrs trigger ifn-mediated antiviral responses through their interaction with mitochondrial antiviral-signaling protein (mavs), a card-containing omm protein [82] . the card-card interaction between rlrs and mavs causes mavs polymerization and consequent recruitment of a variety of downstream effectors, including tumor necrosis factor receptor-associated factor family proteins, ikb kinase epsilon (ikkε) and tank binding kinase 1, among others [83] . this "mavs signalosome" activates nf-κb, irf3 and irf7, promoting the expression of type-i ifn and antiviral molecules [84] . given the central role of mavs in mitochondrial antiviral signaling, mavs and both upstream and downstream molecules are under tight regulation to ensure an adequate response [85, 86] . mitochondria are dynamic organelles that undergo constant fusion and fission to regulate their morphology, activity and turnover according to the metabolic needs of the cell [87] , and these mitochondrial dynamics are involved in the regulation of mitochondrial immune functions. mitofusins and optic atrophy protein 1 are responsible for mitochondrial fusion, whereas the cytosolic gtp-ase dynamin-related protein 1 (drp1) mediates mitochondrial fission through its interaction with adaptor proteins in the omm [88, 89] . interestingly, these proteins have been shown to be implicated in the regulation of various mitochondrial immune-relevant processes, such as rlr signaling [83, 90, 91] , apoptosis [92, 93] , autophagy and mitochondrial bioenergetic conditions [94] , which are important mechanisms to combat viral infections. mitophagy is a selective autophagic process in which defective mitochondria are engulfed in autophagosomes and eliminated by fusion with lysosomes [95] . damaged mitochondria constitute a signal for the recruitment of pten-induced putative kinase protein 1 (pink1), which surrounds the mitochondrial surface. the accumulation of pink1 and its kinase activity promote the translocation of the e3 ubiquitin ligase parkin from the cytosol to dysfunctional mitochondria, triggering the ubiquitylation of omm proteins. the formation of ubiquitin chains by parkin favors the binding of adaptor proteins (e.g., p62 and optineurin), which mediate the interaction with autophagosomes and the further degradation of dysfunctional mitochondria [96, 97] . mitophagy is closely related to mitochondrial dynamics, as mitochondrial fragmentation promotes mitophagy whereas mitochondrial fusion hinders this process [98, 99] . interestingly, defective mitochondria can play either positive or negative roles against viruses. for example, alterations in mitochondrial respiration trigger the production of reactive oxygen species (ros) which, in addition to being harmful for the cell in high levels, play an important role as second messengers in diverse intracellular signaling pathways [100] . in the context of antiviral signaling, ros are involved in the regulation of the rlr pathway, potentiating rlr-mavs signaling and the production of type-i ifn [101] . because healthy mitochondria are required for an adequate metabolic state and the activation of apoptotic processes [94, 102, 103] , mitophagy must be finely regulated to modulate the mitochondria-mediated antiviral response. the implications of mitochondria in innate immunity are enormous. we have briefly discussed the interplay between different mitochondrial pathways, such as rlr signaling, mitochondrial dynamics, mitophagy, ros production and apoptosis, in the protection against viruses. however, mitochondria perform a plethora of functions in the establishment of a defensive state of the cell, which have been thoroughly reviewed by others [85, [104] [105] [106] [107] [108] . moreover, the regulation of mitochondrial function is not only carried out by the host, but also by viruses with the aim to shut down defense mechanisms, complete their life cycle and spread [109] , underscoring the relevance of mitochondria in antiviral response. such critical roles of mitochondria in the control of pathogen invasion and maintenance of cellular homeostasis must be strictly coordinated. as we previously discussed, ubiquitin and ubiquitin-like ptms are key regulatory processes of the innate and adaptive immune response against viruses, and both processes are finely regulated by mitochondria. in this regard, there is a broad spectrum of ptms [110] , some of which occur within mitochondria, which are responsible for modifying their internal state and function [111, 112] . thus, mitochondria are targets of ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifiers (sumos) and isg15. ubiquitin is a highly conserved 8.6-kda protein known as a master regulator of cellular processes. its covalent conjugation to target proteins has proteolytic and non-proteolytic or regulatory outcomes, which fine-tune protein function and recycling [113] . indeed, ubiquitylation is essential for the regulation of many mitochondrial processes, such as mitophagy [95, [114] [115] [116] , mitochondrial dynamics [117, 118] , and mitochondria-related immune signaling [86, 119, 120] , establishing the importance of this modifier in the homeostasis of these organelles. sumos are a family of highly conserved 12-kda proteins that are essential in eukaryotic cells. similar to ubiquitin, sumo conjugation to specific lysine residues of target proteins (sumoylation) alters their function, their interaction with other proteins, and their stability [121, 122] . although the major role of sumoylation is in the regulation of nuclear processes [123] , it also targets mitochondria. sumos have been proven to be involved in the regulation of mitochondrial dynamics by binding to drp1 [124] , with an important implication in programmed cell death [125] [126] [127] . furthermore, sumoylation of the mitochondrial oxidative stress sensor dj-1 results in its stabilization and full activation, reinforcing its protective role against parkinson's disease [128] , where mitochondrial dysfunction has great significance. although isg15 has been associated with mitochondria, its functions, both free or conjugated to mitochondrial proteins, are still being examined. one exception is the protein parkin. while not strictly a mitochondrial protein, its translocation to the omm from the cytoplasm is essential for parkin-mediated mitophagy [96] . isg15 conjugation to parkin enhances its e3 ubiquitin ligase activity and its cytoprotective effect in parkinson's disease [129] , an example of how isgylation affects mitochondrial processes. isg15 and isgylation for the regulation of mitochondrial metabolism [29] . we undertook a comprehensive analysis of bone marrow-derived macrophages (bmdms) from wild-type and isg15-/mice to interrogate how isg15 and isgylation could modulate the regulation of mitochondria in the context of stressful stimuli. monomeric isg15 and isgylated proteins were observed in mitochondrial fractions from wild-type bmdms after type-i ifn pre-treatment, and these proteins were preferentially located to the ims and imm (figure 3) . given their localization, we hypothesized that isg15 and isgylation could impact mitochondrial respiratory metabolism, and we focused our study on oxphos and atp production. this analysis revealed that oxygen consumption and atp production were lower in isg15-/-bmdms than in equivalent wild-type cells, indicative of defective oxphos. in accord with these observations, a clear difference in the distribution of etc supercomplexes was observed between the two groups, pointing to a possible role for isg15 in the correct assembly of etc proteins ( figure 3 ). as recently reported by yoshizumi et al. [130] oxphos activity is required for rlr-mediated antiviral signaling, and mice with oxphos defects showed increased susceptibility to viral infections. similarly, knockout mice for isg15 or the isg15-activating e1 enzyme (ube1l) were more susceptible to infection with many viruses than wild-type mice [30] . since the lack of isg15 seems to cause alterations in oxphos, such increase in the sensitivity to viral infections in isg15-/and ube1l-/-mice might be explained as a result of defects in rlr-mediated antiviral responses, supporting the role of isg15 as a regulator of mitochondrial functions. regarding mitochondrial respiration byproducts, isg15-/-bmdm also produced lower levels of ros. because ros production is tightly controlled by mitochondrial membrane potential [131] , low levels of ros in isg15-/-bmdm might be the result of abnormalities in the transmembrane proton gradient due to the absence of isg15, and could affect the immune response against viral infections, as discussed earlier. mitochondrial ros also participate in the regulation of macrophage polarization [132] and, interestingly, isg15-/-bmdms displayed mixed features of m1 and m2 phenotypes, suggesting that alterations in mitochondrial oxphos could drive changes to immune cell function. finally, bmdms lacking isg15 accumulated non-functional mitochondria with an absence of parkin, suggesting that isg15 is also implicated in the regulation of mitophagy, perhaps through the control of parkin translocation from the cytosol (figure 3) . taken together, these findings establish a relevant role for isg15 and isgylation in the control of mitochondrial oxphos and recycling, at least in murine bmdm, expanding the range of functions of this ptm and underscoring its importance in the regulation of essential cellular processes. viruses 2018, 10, x for peer review 11 of 17 figure 3 . impact of isg15 on mitochondrial activities. mitochondria are targets of isg15 and isgylation in murine bone marrow-derived macrophages (bmdms). isgylated proteins can be found in all mitochondrial localizations, mainly in the mitochondrial intermembrane space (ims) and inner mitochondrial membrane (imm), where free isg15 is also present. isg15 and isgylation are involved in the regulation of mitochondrial metabolism. absence of isg15 leads to alterations in oxphos, with lower oxygen consumption rates and atp production levels, in addition to aberrant etc supercomplexes assembly. such disruption of oxphos mechanisms decreases ros production, with repercussions for macrophage polarization. mitophagy is also altered in cells lacking isg15. finally, isg15-/-bmdm accumulate defective mitochondria and parkin cannot be found in mitochondrial extracts, suggesting that isg15 is important during the translocation of parkin from the cytoplasm to mitochondria. the functional significance of ptms in disease etiology, and the pathologic response to their disruption, is the subject of intense investigation. many of these reversible modifications act as regulatory mechanisms in mitochondria and show promise for mitochondria-targeted therapeutic strategies. with the advent of mass spectrometry-based screening techniques, there has been a vast increase in our current state of knowledge on mitochondrial ptms and their protein targets. detecting isgylated proteins in different organelles remains challenging, as it typically occurs in only a small portion of the total protein pool of the cell, albeit with essential roles in regulating protein fate and function. understanding the consequences of isgylation of mitochondrial proteins will require much work, but should be rewarding not only for developing new strategies to combat viral infections, but also for future applications in other biomedically relevant processes/diseases, for example inflammation, cancer and neurodegeneration. . impact of isg15 on mitochondrial activities. mitochondria are targets of isg15 and isgylation in murine bone marrow-derived macrophages (bmdms). isgylated proteins can be found in all mitochondrial localizations, mainly in the mitochondrial intermembrane space (ims) and inner mitochondrial membrane (imm), where free isg15 is also present. isg15 and isgylation are involved in the regulation of mitochondrial metabolism. absence of isg15 leads to alterations in oxphos, with lower oxygen consumption rates and atp production levels, in addition to aberrant etc supercomplexes assembly. such disruption of oxphos mechanisms decreases ros production, with repercussions for macrophage polarization. mitophagy is also altered in cells lacking isg15. finally, isg15-/-bmdm accumulate defective mitochondria and parkin cannot be found in mitochondrial extracts, suggesting that isg15 is important during the translocation of parkin from the cytoplasm to mitochondria. the functional significance of ptms in disease etiology, and the pathologic response to their disruption, is the subject of intense 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pathways regulation of mitophagy by the ubiquitin pathway in neurodegenerative diseases building and decoding ubiquitin chains for mitophagy dynamic survey of mitochondria by ubiquitin regulation of mitochondrial dynamics by proteolytic processing and protein turnover ubiquitin in the activation and attenuation of innate antiviral immunity post-translational modification control of innate immunity protein modification by sumo regulation of cellular processes by sumo: understudied topics mediated regulation of nuclear functions and signaling processes sumo1 conjugates mitochondrial substrates and participates in mitochondrial fission senp3-mediated desumoylation of dynamin-related protein 1 promotes cell death following ischaemia mapl sumoylation of drp1 stabilizes an er/mitochondrial platform required for cell death sumo-modified fadd recruits cytosolic drp1 and caspase-10 to mitochondria for regulated necrosis sumo-regulated mitochondrial function in parkinson's disease covalent isg15 conjugation positively regulates the ubiquitin e3 ligase activity of parkin rlr-mediated antiviral innate immunity requires oxidative phosphorylation activity use the protonmotive force: mitochondrial uncoupling and reactive oxygen species the metabolic prospective and redox regulation of macrophage polarization we thank diego sanz for his technical assistance and kenneth mccreath for reviewing and correcting the manuscript. the authors declare no conflicts of interest. acknowledgments: we thank diego sanz for his technical assistance and kenneth mccreath for reviewing and correcting the manuscript. the authors declare no conflicts of interest.viruses 2018, 10, 629 key: cord-314751-i9rxesrg authors: oh, jongsuk; lee, kyung-won; choi, hwan-won; lee, changhee title: immunogenicity and protective efficacy of recombinant s1 domain of the porcine epidemic diarrhea virus spike protein date: 2014-07-10 journal: arch virol doi: 10.1007/s00705-014-2163-7 sha: doc_id: 314751 cord_uid: i9rxesrg porcine epidemic diarrhea virus (pedv) is a highly contagious enteric pathogen of swine. acute pedv outbreaks have continually emerged in most swine-producing asian countries and, recently, in the united states, causing significant economic losses in the pig industry. the spike (s) protein of pedv is a type 1 transmembrane envelope glycoprotein and consists of the s1 and s2 domains, which are responsible for virus binding and fusion, respectively. since the s1 domain is involved in a specific high-affinity interaction with the cellular receptor and induction of neutralizing antibody in the natural host, it is a primary target for the development of effective vaccines against pedv. in this study, a codon-optimized pedv s1 gene containing amino acid residues 25–738 was synthesized based on a multiple alignment of the s amino acid sequences of pedv field isolates and used to establish a stable porcine cell line constitutively expressing the pedv s1 protein. the purified recombinant s1 protein was found to mediate highly potent antibody responses in immunized rabbits. the antibodies strongly recognized the recombinant s1 protein from cell lysates and supernatants of s1-expressing cells, whereas they bound weakly to the authentic s protein of pedv vaccine strain sm98-1. furthermore, a serum neutralization test revealed that the rabbit antisera completely inhibit infection of the pedv vaccine strain at a serum dilution of 1:16. we then tested the ability of vaccination with the recombinant s1 protein to protect piglets against pedv. late-term pregnant sows were inoculated intramuscularly with the purified s1 protein, and the outcome was investigated in passively immunized suckling piglets after a virulent pedv challenge. the results showed that vaccination with s1 protein efficiently protected neonatal piglets against pedv. our data suggest that the recombinant s1 protein shows potential as an effective and safe subunit vaccine for ped prevention. porcine epidemic diarrhea (ped) is a devastating swine disease that is characterized by acute enteritis and lethal watery diarrhea followed by severe dehydration leading to death, with a high mortality rate in piglets [6, 26, 28] . the disease was initially recognized in england in 1971 [23] , but the causative agent of this disease, ped virus (pedv), was later identified in 1978 [25] . ped epidemics were first reported in asia in 1982, and since then, ped has continued to threaten swine health, causing substantial economic losses in the asian swine industry [4, 19, 27, 33] . in 2013, ped outbreaks suddenly occurred in the united states and have swept through the pork industry across the country, raising concerns about control measures for ped prevention [21, 30] . in korea, pedv appeared in 1992 [14] ; however, a retrospective study indicated that the virus had been present as early as 1987 [24] . although periodic vaccination strategies have been implemented nationwide to control ped in korean swine herds, pedv has continually emerged, causing tremendous harm to the productivity of korean pig farms. pedv, a member of the genus alphacoronavirus within the family coronaviridae of the order nidovirales, is a large, enveloped virus possessing a single-stranded, positive-sense rna genome of approximately 28 kb with a 5 0 cap and a 3 0 polyadenylated tail [25, 28] . the pedv genome is composed of the 5 0 untranslated region (utr), at least seven open reading frames (orf1a, orf1b, and orf2 through 6), and the 3 0 utr [13] . the two large orfs 1a and 1b make up the 5 0 two-thirds of the genome and encode the non-structural replicase genes. the remaining orfs in the 3 0 terminal region code for four major structural proteins: the 150-220-kda glycosylated spike (s) protein, the 20-30-kda membrane (m) protein, the 7-kda envelope (e) protein, and the 58-kda nucleocapsid (n) protein [8, 28] . the s protein of pedv is a type i membrane glycoprotein composed of 1,383 to 1,386 amino acids (aa), depending on the strain. it contains a putative signal peptide (aa 1-24), a large extracellular region, a single transmembrane domain (aa 1,334-1,356), and a short cytoplasmic tail. although pedv has an uncleaved s protein because it lacks a furin cleavage site, the s protein can be divided into s1 (aa 1-735) and s2 (736-the last aa) domains based on homology with s proteins of other coronaviruses [7, 11, 16, 31] . like other coronavirus s proteins, the pedv s protein is known to play a pivotal role, interacting with the cellular receptor to mediate viral entry and inducing neutralizing antibodies in the natural host [2, 3] . more precisely, previous studies have shown that the s1 domain includes the main neutralizing epitopes and the receptor-binding region [17, 32] . furthermore, along with the full-length s gene, the s1 portion is known to be a suitable region for determining genetic relatedness among the different pedv isolates and for developing differential diagnostic assays [5, 16] . considering these molecular and biological features of the s1 domain, it would be an appropriate target for developing effective vaccines against pedv. in the present study, therefore, we first synthesized a full-length, codon-optimized pedv s1 gene and then generated a stable porcine-origin cell line constitutively expressing the recombinant s1 protein. the protective efficiency of a recombinant-s1-protein-based vaccine against pedv was then evaluated in the natural host. the recombinant s1 protein was capable of inducing an efficient antibody response in immunized rabbits. moreover, immunization with the pedv s1 protein was found to elicit specific neutralizing antibodies in sows and to protect passively immunized suckling piglets against pedv challenge. cells, virus, antibodies, and plasmid hek-293t cells (crl-1573) were purchased from the american type culture collection (atcc, manassas, va) and cultured in dulbecco's modified eagle medium (dmem) with high glucose (invitrogen, carlsbad, ca) with 10 % fetal bovine serum (fbs, invitrogen) and antibiotic-antimycotic solutions (1009; invitrogen). pk-15 cells were grown in rpmi 1640 medium (invitrogen) supplemented with 10 % fbs and antibiotic-antimycotic solutions. vero cells were cultured in alpha minimum essential medium (a-mem, invitrogen) with 10 % fbs and antibiotic-antimycotic solutions. the cells were maintained at 37°c in a humidified 5 % co 2 atmosphere. the pedv vaccine strain sm98-1 was obtained from the korean animal and plant quarantine agency and propagated in vero cells as described previously [10] . challenge pedv was prepared from the small intestine of a 4-day-old suckling piglet orally inoculated with small intestine homogenate containing the field virus. small intestine tissues were collected and homogenized in a 10 % suspension with a-mem using a magna lyser (roche diagnostics, mannheim, germany) with three repetitions of 15 s at a speed of 7,000 rpm, and suspensions were clarified by centrifugation at 4,500 9 g (hanil centrifuge fleta5, incheon, korea). the clarified supernatant was filtered through a 0.22-lm-pore-size syringe filter (millipore, billerica, ma), aliquoted, and stored at -80°c until use as crude challenge virus. all of the horseradish peroxidase (hrp)-conjugated secondary antibodies were purchased from santa cruz biotechnology (santa cruz, ca). the pedv s-protein-specific monoclonal antibody (mab) was a kind gift from sang-geon yeo (kyungpook national university, daegu, korea). a plasmid encoding the s1 fragment of severe acute respiratory syndrome coronavirus (sars-cov), pcdm8-sars-cov-s1-ig, was kindly provided by hyeryun choe (harvard medical school, boston, ma). dna manipulation and cloning were performed according to standard procedures [29] . the e. coli strain dh5a (rbc bioscience, taiwan) was used as the host for general cloning. the plasmid encoding the full-length s1 gene of the pedv field strain knu-0801, pcdm8-pedv-s1-ig, was described previously [17] . to construct the plasmids expressing s1 and its variants, the consensus sequence of pedv s1 was identified based on a multiple alignment of the s aa sequences of pedv field isolates [16] and utilized to synthesize a full-length, codon-optimized pedv s1 gene (encoding aa 24-735) according to a method described previously [1] . the codon-optimized pedv s1 gene was cloned into a modified expression vector, pcdm8-fc, which contains the cd5 signal sequence and the fc domain of human igg1 [9] , thereby producing a human fc-tagged fusion protein, rs1-ig. all of the pedv s1-truncated variants used in this study were generated using this template with the previously described primer sets [17] . an fctagged pedv rs1-ig fragment obtained from pcdm8-fc-rs1-ig was then subcloned into a pfb-neo retroviral vector (stratagene, la jolla, ca) using the sali and xhoi restriction sites to construct a pedv rs1-ig gene expression plasmid, pfb-neo-pedv-rs1-ig. all of the constructed plasmids were verified by nucleotide sequencing. generation of a stable pk-15 cell line expressing pedv rs1-ig the retrovirus gene transfer system (stratagene) was applied to generate a cell line constitutively expressing the recombinant pedv rs1-ig gene or an empty retroviral vector as described elsewhere [16, 22] . antibiotic-resistant continuous cell clones were examined by rt-pcr to verify the presence of the full-length rs1-ig gene, and the positive clones (pk-rs1-ig) were then amplified for subsequent analyses. immunofluorescence assay (ifa) pk-rs1-ig cells were grown on microscope coverslips placed in 6-well tissue culture plates. at 48 h post-seeding, the cells were fixed with 4 % paraformaldehyde for 10 min at room temperature (rt) and permeabilized with 0.2 % triton x-100 in pbs at rt for 10 min. the cells were subsequently blocked with 1 % bovine serum albumin (bsa) in pbs for 30 min at rt and then incubated with a goat anti-human igg antibody conjugated with fluorescein isothiocyanate (fitc) (santa cruz biotechnology). finally, the cells were counterstained with 4 0 ,6-diamidino-2-phenylindole (dapi; sigma, st. louis, mo), and cell staining was visualized using a fluorescent leica dm il led microscope (leica, wetzlar, germany). fluorescence-activated cell sorting (facs) analysis rs1 expression in pk-rs1-ig cells was analyzed by flow cytometry. briefly, cells were trypsinized at 48 h postseeding and centrifuged at 250 9 g (hanil centrifuge fleta 5) for 5 min. the cell pellet was washed with cold washing buffer (1 % bsa and 0.1 % sodium azide in pbs) and 1 9 10 6 cells were resuspended in 1 % formaldehyde solution in cold wash buffer and fixed at 4°c in the dark for 30 min, followed by incubation in 0.2 % triton x-100 in pbs for permeabilization at 37°c for 15 min. following centrifugation, the cell pellet was resuspended in normal mouse igg1 antibody (santa cruz biotechnology) and incubated at 4°c for 30 min. the cells were washed and reacted with fitc-conjugated anti-human or anti-mouse igg secondary antibody at 4°c for 30 min in the dark. the stained cells were washed again and analyzed by facscan flow cytometry. western blot analysis hek 293t cells were transfected with each plasmid using lipofectamine 2000 (invitrogen) according to the manufacturer's protocol and solubilized in lysis buffer at 48 h post-transfection as described previously [22] . cell lysates were also prepared from vero cells infected with pedv sm98-1 at a multiplicity of infection (moi) of 0.1 at the indicated time points using lysis buffer. the protein concentrations of the cell lysates were determined using a bca protein assay (pierce, rockford, il). pk-rs1-ig cells were grown at 5 9 10 5 cells/well in a 6-well tissue culture plate, and the protein-containing culture supernatants were harvested on days 1, 2, and 3. soluble proteins were immunoprecipitated with protein a sepharose cl-4b beads (ge healthcare, piscataway, nj) in the presence of protease inhibitors at 4°c for 16 h. the beads were collected by centrifugation at 5,000 9 g (eppendorf centrifuge 5415r, hamburg, germany) for 5 min at 4°c and washed three times with 0.5 m nacl in pbs. the cell lysates or the beads were mixed with 49 nupage sample buffer (invitrogen) and boiled at 70°c for 10 min. the proteins were separated on a nupage 4-12 % gradient bis-tris gel (invitrogen) under reducing conditions, and electrotransferred onto immunobilon-p (millipore). the membranes were then blocked with 3 % powdered skim milk (bd biosciences, belford, ma) in tbs (10 mm tris-hcl [ph 8.0], 150 mm nacl) with 0.05 % tween-20 (tbst) at 4°c for 2 h and reacted directly with the goat anti-human igg hrp-conjugated secondary antibody, the anti-s1 rabbit serum or the anti-pedv s mab, followed by the corresponding hrp-labeled secondary antibody at a dilution of 1:5,000 for 2 h at 4°c. finally, the proteins were visualized using enhanced chemiluminescence (ecl) reagents (ge healthcare) according to the manufacturer's instructions. protein purification pk-rs1-ig cells were grown at 5 9 10 5 cells/well in a 6-well tissue culture plate in serum-free medium (optipro sfm; invitrogen). at 72 h post-seeding, the protein-containing culture supernatants were harvested and soluble proteins were immunoprecipitated with protein a sepharose cl-4b beads in the presence of protease inhibitors at 4°c for 16 h. the beads were collected and washed as described above. the samples were subsequently eluted with 50 mm sodium citrate/50 mm glycine (ph 2.0) and neutralized with 1 m tris-hcl (ph 8.0). the purified proteins were concentrated with amicon ultra centrifugal filters 100k (millipore). protein concentration was measured using a bca protein assay (pierce, rockford, il) and the final products were analyzed by western blotting to confirm target protein purification. two new zealand white rabbits were immunized intradermally with 250 lg of purified rs1-ig resuspended in pbs in the presence of freund's complete adjuvant and boosted four times with a freshly prepared emulsion of 250 lg immunogen and freund's incomplete adjuvant at 2-week intervals. pre-immune sera were collected before starting the immunization, and antisera were collected at each boost. the in vivo swine experiments described here were performed at the choongang vaccine laboratory animal facility under the guidelines established by its institutional animal care and use committee. a total of eight pregnant sows were obtained from a pig farm with no outbreaks or vaccination with pedv and randomly divided into four groups of two sows. all animals were determined to be free of antibodies to pedv. the design for the present immunogenicity study involving eight pregnant sows is outlined in table 1 . the sows in group 1 were immunized intramuscularly with attenuated pedv live vaccine and inactivated pedv vaccine obtained from choongang vaccine laboratory, in order at 2-week intervals prior to farrowing. the pigs in group 2 were immunized intramuscularly with pedv live vaccine and 400 lg of purified rs1-ig resuspended in pbs in the presence of an oil-in-water adjuvant, in order at 2-week intervals before farrowing. both the pedv live and inactivated vaccines were administrated according to the manufacturers' manuals. the sows in group 3 and group 4 were inoculated intramuscularly three times at 2-week intervals with 400 lg of purified rs1-ig mixed with the oil adjuvant or with cell culture medium as a negative control. paired blood samples and colostrum were collected at 3-week intervals before farrowing and at farrowing, at delivery, respectively. one 4-to 5-day-old piglet (8 piglets in total) was selected randomly from each farrowing sow in the immunized and control groups for challenge exposure with virulent pedv. piglets from all groups were challenged orally with 1 ml of small intestine homogenate containing 10 5 tcid 50 /ml of pedv field virus, equivalent to 10 9.8 viral genome copies per ml, determined using real-time rt-pcr as described previously [12] . clinical signs of diarrhea and death in challenged piglets were monitored daily throughout the study. stool samples from all groups were collected every day with 16-inch cotton-tipped swabs and subjected to rt-pcr using a tge/ped detection kit (intron biotechnology, seongnam, korea) according to the manufacturer's protocol. all piglets from the vaccinated and control groups were euthanized at 5 days after challenge for post-mortem examination. small-intestinal tissue specimens collected from each piglet (\3 mm thick) were fixed with 10 % formalin for 24 h at rt and embedded in paraffin according to standard laboratory procedures. the formalinfixed paraffin-embedded tissues were cut into 5-to 8-lmthick sections on a microtome, floated on a 40°c water bath containing distilled water, and transferred onto glass slides. the tissues were then deparaffinized in xylene for 5 min and washed in decreasing concentrations of ethanol (100, 95, 85, 70, and 50 %) for 3 min each. the deparaffinized intestinal tissues sections were subjected to immunofluorescence assay using an n-specific mab and alexa fluor 594-conjugated goat anti-mouse antibody as described above. the presence of pedv-specific neutralizing antibodies in serum and colostrum samples collected from sows in all groups was determined using a serum neutralization (sn) test in 96-well microtiter plates using pedv vaccine strain sm98-1 as described previously [15] . briefly, individual virus stocks were diluted in serum-free a-mem to make 200 pfu in a 50 ll volume. the diluted virus was then mixed with 50 ll of twofold dilutions of individual pedv s1 immunogen a pedv s1 immunogen pedv s1 immunogen 4 placebo placebo placebo a placebo (cell culture medium) and protein (pedv s1) immunizations were given intramuscularly with the oil-in-water adjuvant b commercial pedv live and inactivated vaccines were administrated intramuscularly according to the manufacturers' instructions with the oil-in-water adjuvant inactivated sera in 96-well plates and incubated at 37°c for 1 h. next, the mixture was incubated at 37°c for 1 h, approximately 1 9 10 4 vero cells in 100 ll of a-mem were added to each well, and the mixture was maintained at 37°c in a 5 % co 2 incubator for 3 to 4 days. the neutralization titer was calculated as the reciprocal of the highest dilution of serum that inhibited the virus-specific cytopathic effect in both of the duplicate wells. results were also visualized by staining the wells with a crystal violet-formaldehyde staining reagent (0.013 % crystal violet, 2.5 % ethanol, and 10 % formaldehyde in 0.01 m pbs) for 1 h at rt. the student's t test was used for all statistical analyses, and p-values of less than 0.05 were considered statistically significant. generation of stable porcine-origin cell lines expressing the full-length, codon-optimized s1 protein we have previously constructed a series of expression plasmids encoding the full-length s1-ig (s1 ). although expression of this gene could be detected in transiently transfected cells by western blot analysis, its expression level was relatively low and insufficient for further protein purification study (fig. 1, lane 2) . thus, to enhance s1 protein expression, we first synthesized the full-length, codon-optimized s1 gene and constructed a panel of expression plasmids encoding the codon-optimized rs1-ig (s1 ) and two c-terminally truncated s1 variants: rs1 25-543 -ig and rs1 25-432 -ig. these constructs were independently transfected into hek-293t cells, and expression of each construct was robustly detectable by western blot analysis using an anti-human igg secondary antibody (fig. 1, lanes 3 to 5) , compared with a normal s1 protein (lane 2). this result indicates that codon optimization greatly increases the expression level of s1 upon transient transfection. subsequently, to easily produce preparative amounts of the s1 protein, sublines of pk-15 cells were established to stably express the recombinant codon-optimized s1 under the control of a retroviral ltr promoter. ten generated cell clones were initially collected and subjected to rt-pcr and western blotting to identify s1 gene expression at the mrna and protein level, respectively (data not shown). based on the results of western blot analysis, one pk-rs1-ig cell clone that consistently expressed the highest level of s1 was chosen for subsequent studies. to characterize pk-rs1-ig cells, intracellular and extracellular expression levels of s1 were examined by immunofluorescence, facs analysis, and western blotting. as shown in fig. 2a , specific cell staining was clearly evident when pk-rs1-ig cells were reacted with the antihuman igg antibody, confirming a consistent high expression level of the s1 protein. the majority of the cells consistently exhibited specific fluorescent signals, indicating a homogenous population of cells expressing s1 (fig. 2b) . time-course western blot analysis using culture supernatants revealed that the pk-rs1-ig cells stably express and cumulatively secrete high levels of approximately 160-kda s1 (fig. 2c ). in addition, the overall growth kinetics of s1-gene-expressing pk-15 cells were found to be similar to those of the parental pk-15 cells, indicating that s1 expression has no effect on cell growth (data not shown). further, the recombinant s1 protein tagged with the human igg fc domain expressed in the supernatants of stable pk-rs1-ig cells was purified using protein a sepharose beads. the purified s1 protein was detectable at a high level by ponceau s staining, and this was confirmed by immunoblotting with anti-human igg antibody (fig. 2d ). using our purification and concentration procedures, we were able to obtain approximately 15 lg of the s1 protein from 1 ml culture supernatant of pk-rs1-ig cells cultivated in one well of a 6-well plate for 72 h. antibody response of the recombinant s1 immunogen in rabbits rabbit antisera were collected before immunization (preimmune) and at each boost at 2-week intervals. the serum samples at 1:10,000 to 1:100,000 dilutions were examined for binding to the recombinant fusion protein rs1-ig or the authentic s protein by western blot analysis. the antiserum collected at the second boost reacted with the purified rs1 protein in a concentration-dependent manner (fig. 3a, top panel) , similar to the binding capacity of the anti-igg secondary antibody, which can directly recognize the protein fused to the fc region of human igg1 (middle panel). the antisera were found to retain nearly equivalent reactivity at the third and fourth boosts (data not shown). in contrast, the rabbit antiserum bound weakly to the authentic s protein prepared from cells infected with a pedv vaccine strain (top panel), whereas an mab raised against the vaccine strain reacted strongly with the authentic s protein (bottom panel). the rabbit antisera were further tested for their neutralizing activity against the pedv sm98-1 vaccine strain, which can be grown in vero cells in the absence of trypsin. as shown in fig. 3b , the antisera collected at the final boost at 1:16 dilution fully protect vero cells from sm98-1 infection (left panel), whereas pig hyperimmune sera raised against the vaccine virus were highly effective in inhibiting virus infection with neutralizing antibody titers of [1:256 (right panel). the rabbit sera at the second and third boosts possessed comparable neutralizing activities against the vaccine strain (data not shown). taken together, our data indicated that the recombinant s1 immunogen elicits potent antibody responses in immunized rabbits. however, the rabbit antisera strongly recognized the homologous s1 protein representing the s protein of field isolates but recognized the heterologous s protein of the pedv vaccine strain inefficiently, suggesting that there are antigenic differences between the vaccine strain and field pedvs. to determine the immunogenicity of the s1-based subunit vaccine, pregnant sows assigned to four groups were immunized intramuscularly, as outlined in table 1 . serum samples were collected at 6 weeks and 3 weeks prior to farrowing and at delivery and were subjected to a serum neutralization test against the pedv vaccine strain. nonimmunized sows showed only minimal neutralizing antibody titers, whereas immunized sows exhibited gradually increased neutralizing antibody titers, which increased considerably after the final vaccination (fig. 4) . briefly, sows immunized with two doses of pedv live and inactivated vaccines at 2-week intervals (group 1) had neutralizing titers of [1:256 at delivery. in contrast, sows immunized with two doses of pedv live and s1 protein vaccines (group 2) or three doses of s1 protein vaccine (group 3) at 2-week intervals produced relatively lower neutralizing antibody titers of 1:16 to 1:32 compared to those in group 1. in addition, colostrum samples from each group were found to have neutralizing antibody titers comparable to those in the corresponding serum samples (fig. 4b) . these results were coincident with the rabbit study described above, since the rabbit antisera generated by immunization with the recombinant s1 protein also contained low levels of neutralizing antibodies to the heterologous pedv vaccine strain. lastly, to assess the efficacy of immunization with the s1 protein, eight 4-to 5-day-old neonatal piglets from each sow were arbitrarily selected and challenged orally with wild-type pedv. clinical observations of death, diarrhea, and virus shedding in challenged piglets are summarized in table 2 . in the control group, one piglet died and the other piglet experienced severe watery diarrhea post-challenge. although none of the piglets from any of the groups of immunized sows died during the challenge experiment, the number of piglets exhibiting diarrhea after challenge varied depending on the group. all piglets delivered from group 1 sows showed mild-to-severe diarrhea lasting for the entire experiment at 2 days post-challenge. in contrast, piglets from sows in groups 2 and 3 experienced only mild diarrhea for 1 or 2 days after challenge or throughout the challenge experiment. likewise, all piglets from immunized sows (groups 1, 2, and 3) exhibited mild intestinal lesions, and viral antigens were detected only in their small intestines, whereas the majority of enterocytes over the entire villi in the control piglets (group 4) were affected by pedv, showing moderate-to-severe villous atrophy (fig. 5) . altogether, all immunization methods used in this study were capable of protecting passively immunized neonatal piglets against mortality and severe disease after challenge. however, immunizations involving at least one dose of pedv s1 protein vaccine (groups 2 and 3) were more efficacious than immunization of sows with two doses of pedv live and killed vaccines (group 1) in reducing the overall degree of diarrhea, in terms of the duration and severity, in the suckling piglets. pedv continues to have a severe economic impact in swine-raising countries in asia and, more recently, in the united states. vaccination against pedv is an important and effective prevention measure. pedv entry into host cells is mediated by the s glycoprotein on the viral surface, which interacts with the cellular receptor and induces direct membrane fusion. the pedv s protein is also responsible for inducing neutralizing antibodies in the natural host and hence is a logical target in the development of effective vaccines. furthermore, the s1 domain is a key functional portion of the s protein, which is associated with viral binding to host-cell receptors and contains neutralizing epitopes [17, 32] . therefore, the s1 protein of pedv is considered to be a potential candidate antigen for vaccination attempts. in the present study, the first aim was to stably express the full-length, codon-optimized s1 gene of pedv in porcine-origin cells and to evaluate the immunogenicity and efficacy of the recombinant s1 protein. our codon-optimization approach dramatically enhanced the expression level of the gene of interest upon transient transfection using a mammalian expression system. subsequently, we were able to successfully generate a stable pk cell line continuously producing large amounts of the codon-optimized s1 protein. following the purification and concentration processes, approximately 15-20 lg of the recombinant s1 protein could be consistently harvested from pk-rs1-ig cells in each well of a 6-well plate. since the antibody response is a critical indicator to assess the effect of a vaccine, we immunized rabbits with the s1-based immunogen prepared from culture supernatants of pk-rs1-ig cells and determined whether or not they developed humoral immunity. pedv-specific antibodies were strongly detectable in rabbit sera collected from the second boost, even at the highest dilution (1:100,000), when reacted with the recombinant pedv-s1 protein purified from s1-expressing pk-rs1-ig cells. in contrast, the binding capacity of the antiserum was dramatically reduced when the authentic s protein in wholecell lysates prepared from cells infected with sm98-1 was used as the antigen for western blotting. moreover, the rabbit antisera could completely block infection of sm98-1 only at a serum dilution of 1:16, whereas the pig sera raised against sm98-1 contained high levels of neutralizing antibody against the homogeneous virus ([1:256). since the pedv field isolate propagated in the current culture system is unavailable to us, we were unable to test the neutralizing capacity of those antisera against the field virus in this study. however, it is conceivable that rabbit antisera directed against the s1 protein may be more effective in neutralizing infection with the field virus than with sm98-1. on the other hand, the weak interaction and neutralizing activity of the anti-s1 rabbit serum against the sm98-1 s protein may be attributed to genetic variations between the s proteins of the vaccine strain and field isolates. indeed, the korean field isolates, including the challenge strain used in this study, were found to display a high degree ([10 %) of genetic heterogeneity, especially in the s1 domain, compared with the pedv vaccine strain sm98-1 [16, 18] . nevertheless, western blot and virus neutralizing assays showed that the recombinant s1 protein efficiently elicits humoral immune responses against pedv. in korea, several management strategies, including vaccination, have been employed to control pedv in pig farms. the most highly recommended immunization schedule involves two doses of attenuated live and inactivated killed vaccines in gilts at 2-to 3-week intervals before mating and in pregnant sows at 12 and 14 weeks of gestation. in the present study, we compared the efficacy of this common vaccination protocol and s1 protein-based vaccination. although all of the immunized sows developed neutralizing antibodies against sm98-1, only the sows immunized with live and killed vaccines (group 1) had high neutralizing antibody titers, ranging from 1:32 to 1:1024. however, consistent with the rabbit immunization study, sows in groups 2 and 3, immunized with at least one dose of the s1-protein-based subunit vaccine, developed weak neutralizing responses to the vaccine virus, with titers ranging from 1:8 to 1:32. successful protection against pedv is based on the presence of specific neutralizing antibodies in immune sows that are passively transferred to their piglets through colostrum and milk. our data showed that, regardless of vaccination group, all neonatal piglets from immunized sows survived after challenge with virulent pedv, suggesting that the s1 subunit vaccine provides effective lactogenic immunity to prevent mortality comparable to whole-virus-based vaccines. however, the s1based vaccination strategies, which produced relatively low neutralizing antibody responses, exhibited more-efficient protection, with respective to the duration and severity of diarrhea, than the common live and killed vaccination procedure. it is therefore plausible that the actual levels of neutralizing antibodies raised against the s1 subunit vaccine are underestimated by the vaccine-virus-based neutralization test. thus, improved diagnostic tools are needed to differentiate vaccinal antibodies from those resulting from natural infection with the field virus. for this purpose, the recombinant s1 protein purified from pk-rs1-ig cells could be further used as the diagnostic antigen in an enzyme-linked immunosorbent assay (elisa), and this aspect is currently under investigation. additionally, it is possible that the full-length s protein may induce stronger immune responses than s1 alone because it contains multiple functional domains and neutralizing epitopes. in fact, meng et al. [20] have recently reported that the full-length pedv s gene induces a better immune response than the n-terminal half alone, using the recombinant dna plasmid in a mouse model. however, production of the full-length s protein may be unachievable in our system because its expression may cause cytotoxicity due to the presence of potential fusion activity in the c-terminal portion of the s protein. in conclusion, to the best of our knowledge, this is the first evaluation of the immunological and protective effects triggered by recombinant s1 protein in rabbit and pig models. the results presented here indicate that the recombinant s1 protein can elicit a specific antibody response and induce neutralizing antibodies, suggesting its excellent immunogenicity in the natural host. furthermore, challenge experiments revealed that the s1protein-based vaccine protected passively immunized suckling piglets against field pedv. despite the nationwide use of commercial live and killed pedv vaccines, swine herds in korea have continued to experience repeated outbreaks, leading swine practitioners and researchers to question their protective efficacy. based on the present and previous studies, we hypothesize that antigenic and genetic variation between the vaccine virus and field pedvs responsible for periodic outbreaks in herds may be the cause of the low efficacy or failure of vaccination. accordingly, current pedv vaccines manufactured from cell-adapted viruses should be reassessed to determine their efficacy and improved if necessary. although further studies with a larger number of animals will be needed to better evaluate the efficacy of the s1 protein vaccine and to optimize immunization procedures, the recombinant s1 protein has potential for use in improving or developing effective and safe vaccines for ped prevention. amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states experimental infection of pigs with a new porcine enteric coronavirus, cv777 sequence of the spike protein of porcine epidemic diarrhea virus sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf tyrosine sulfation of the amino terminus of ccr5 facilitates hiv-1 entry propagation of the virus of porcine epidemic diarrhea in cell culture spike glycoprotein cleavage recognition site analysis of infectious bronchitis virus multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus completion of the porcine epidemic diarrhea coronavirus (pedv) genome sequence isolation of porcine epidemic diarrhea virus (pedv) in korea mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea the n-terminal region of the porcine epidemic diarrhea virus spike protein is important for the receptor binding outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea new variants of porcine epidemic diarrhea virus, china evaluation on the efficacy and immunogenicity of recombinant dna plasmids expressing spike genes from porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus deadly pig virus slips through us borders contribution of the porcine aminopeptidase n (cd13) receptor density to porcine epidemic diarrhea virus infection letter to the editor retrospective study of porcine epidemic diarrhea virus (pedv) in korea by in situ hybridization a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs chinese-like strain of porcine epidemic diarrhea virus coronaviruses molecular cloning: a laboratory manual emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences proteolytic cleavage of peplomeric glycoprotein e2 of mhv yields two 90k subunits and activates cell fusion spike protein region (aa 636789) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan acknowledgments we gratefully thank hyeryun choe from harvard medical school for providing reagents. this research was supported by basic science research program through the national research foundation of korea (nrf) funded by the ministry of education, science and technology (2010-0002318). key: cord-306261-yc2y2xak authors: van tricht, ewoud; de raadt, pascal; verwilligen, annemiek; schenning, martijn; backus, harold; germano, marta; somsen, govert w.; sänger-van de griend, cari e. title: fast, selective and quantitative protein profiling of adenovirus-vector based vaccines by ultra-performance liquid chromatography date: 2018-12-28 journal: journal of chromatography a doi: 10.1016/j.chroma.2018.10.045 sha: doc_id: 306261 cord_uid: yc2y2xak abstract a method for the quantitative determination of the protein composition of adenovirus-vector based vaccines was developed. the final method used rp-uplc with uv absorbance detection, a c4 column (300 å, 1.7 μm, 2.1 × 150 mm), and a wateracetonitrile (acn) gradient containing trifluoroacetic acid (tfa) as ion-pairing agent. the chromatographic resolution between the various adenovirus proteins was optimized by studying the effect of the tfa concentration and the column temperature, applying a full factorial design of experiments. a reproducible baseline separation of all relevant adenovirus proteins could be achieved within 17 min run time. samples containing adenovirus particles could be directly injected into the uplc system without sample pretreatment. the viruses reproducibly dissociate into proteins in the uplc system upon contact with the mobile phase containing acn. the new rp-uplc method was successfully validated for protein profiling and relative quantification of proteins in vaccine products based on adenovirus vector types 26 and 35. the intermediate precision of the relative peak areas of all proteins was between 1% and 14% rsd, except for the peak assigned to protein v (26% rsd). the method proved to be stability indicating with respect to thermal and oxidation stress of the adenovirus-vector based vaccine and was successfully implemented for the characterization of adenovirus-based products. adenoviruses are non-enveloped icosahedral-shaped viruses of 70-90 nm, consisting of a protein capsid containing a double stranded dna genome [1, 2] . adenoviruses are promising vectors for intracellular delivery of dna [3] . the advac ® technology in combination with the per.c6 ® technology [4, 5] allows for the production of adenovirus vectors in which the viral dna can be modified to encode for an antigen of interest. for example, in the case of the ebola vaccine the adenovirus vector is modified to encode for a certain glycoprotein from the ebola virus [6] . upon vaccination, this glycoprotein is expressed in the host cell and presented to the immune system, thereby expectedly eliciting a protective immune response against the ebola virus. the adenovirus proteome is composed of different proteins in varying stoichiometries [1, 7] . the adenovirus proteins are identified by numbers, in order of decreasing molecular weight as observed by sdspage [8] . the virus capsid consists of 240 trimers of protein ii (hexon) and 60 copies of protein iii (penton base). the fiber protein (iv) is extending outwards from the penton bases and is considered essential in the binding of the adenovirus to the cell surface. four minor proteins (iiia, vi, viii and ix) are located on the inner side of the capsid and are thought to act as cement proteins. the core of the particle contains six different proteins: v, vii, x, iva2, the terminal protein (tp), and the adenovirus protease (avp) [1] . the viral protein identity, heterogeneity and content in the adenovirus particles determine the identity of the particle, its interaction with the cell and, possibly, its stability. if the incorrect proteins are expressed or the virus particles are not correctly assembled, then this could lead to adenoviruses with decreased or no biological activity, and, therefore, a vaccine with decreased potency. in addition, degraded or modified proteins in the adenovirus vector [26] and liu et al. [10] . particles could also result in decreased biological activity or adverse effects upon vaccine administration. it is a challenge to develop a suitable analytical method for the analysis of the adenovirus proteins due to the great dynamic range needed to measure all protein subunit concentrations simultaneously, and due to the large variability of protein sizes and charges [9] . the molecular masses of the adenovirus proteins range between 7 and 108 kda, and the copy numbers per virus particle are between 2 (for the tp) and 720 (for the protein ii monomer) [1, 10, 11 ]. an additional layer of complexity is added by the need to disassemble the adenovirus into stable and analyzable protein before analysis. typically, an extensive sample treatment is required to denature the viruses into proteins, to remove matrix components (e.g. surfactants) from the samples, or to concentrate the samples [12, 13] . intact proteins in biopharmaceutical products are commonly analyzed by spectrometry [14] , electrophoresis [15] , liquid chromatography [16, 17] , or mass spectrometry [14, [18] [19] [20] . more specifically, viral proteins are typically analyzed by sds-page [10, 21, 22] , capillary gel electrophoresis (cge) [23] [24] [25] or rp-hplc [12, 13, 26] . sds-page has been extensively used for adenovirus protein analysis [27, 28] . the key proteins of the adenovirus are separated from each other by sds-page, and ms can be used after in-gel trypsin digestion for band assignment. such methods do not allow for accurate quantification of the adenovirus proteins due to the poor separation on the gel. in addition, protein modifications (for example, detected by ms) could be introduced by the harsh sds-page and ms pretreatment conditions. capillary gel electrophoresis was applied for the analysis of recombinant gb virus-c proteins [29] , the analysis of the four major influenza virus proteins [25] , and the characterization of proteins from western, eastern, and venezuelan equine encephalitis (wevee) virus like particles (vlps) [23] . the downside of the capillary gel electrophoresis methods is the limited molecular weight range. the method has a low efficiency for proteins with sizes lower than 15 kda or larger than 100 kda resulting in insufficient resolution. another drawback is the extensive sample treatment that is required to denature, reduce and alkylate the virus samples before analysis by cge. liquid chromatography-based methods for analysis of intact proteins in virus and virus-based vaccines have been reported in the literature. although rp-lc is already an established technique for protein analysis, recent technology developments especially improved the stationary phases (i.e. particle sizes down to 1.7 m with pore sizes of 300 å) allowing for even better, more reproducible separation of intact proteins [14] . another technical improvement is the introduction of low-binding materials in the lc equipment that decreased artifacts such as adsorption to injectors, tubing etc. [14] . rp-hplc was used for analysis of the nucleocapsid protein (n-protein) from the severe acute respiratory syndrome (sars) coronavirus [13] . separation was on a reversed phase c18 column (5 m particle size and 300 å pore size) with acn/water and trifluoroacetic acid (tfa) as mobile phase. prior to analysis, the samples were dialyzed, lyophilized, and concentrated. the method did not achieve baseline resolution between the analyte of interest and interfering peaks from the sample matrix. the n-protein could only be identified after fraction collection and maldi-tof analysis. rp-hplc [12, 30, 31] was also used for quantification of the influenza hemagglutinin protein (ha) from the influenza virus. ha is cleaved by trypsin into a hydrophilic ha1 and a hydrophobic ha2 fragment. with rp-hplc, fragment ha1 is well-resolved from other sample components and a quantitative method with external calibration has been reported in the literature [31] . the sample preparation in this method is laborious and time-consuming and the other major proteins from the influenza virus, ha2, m and np, could not be detected. an rp-hplc with uv absorbance detection, based on lehmberg et al. [26] and liu et al. [10] , is being used for quality control of the protein profile of adenovirus vaccine products, see fig. 1 for an example chromatogram. disadvantages of the rp-hplc method are its inadequate resolution for certain proteins (e.g. protein ii and ix) and the insufficient repeatability of the retention times and peak areas. therefore, reliable quantification of all proteins by rp-hplc is not possible. another drawback of the rp-hplc method is its very long run time of 130 min, which limits the throughput to 10 samples per sequence (including controls and blanks) as sample instability is observed after sample storage for 18 h in the auto sampler. this paper describes the development and validation of an rp-uplc method for protein profiling of adenovirus serotype 26 (ad26) and 35 (ad35) in adenovirus vector-based vaccine products. the method has two purposes: confirm identity of the test sample and detect protein modifications or degradation products of the adenovirus vector. for identity purposes, the protein profile of the test sample is compared to the profile of a reference sample based on the relative retention times and relative peak areas. by calculating the relative peak areas of all peaks in the chromatogram and identifying new peaks in the chromatogram, the method also allows the detections of degradation products in the sample additional requirements for the method development were: a chromatographic run time of less than 30 min, baseline separation of all relevant adenovirus proteins allowing for their quantification, and an increased method robustness. acetonitrile uplc/ms grade (pn 01204101) from biosolve (valkenswaard, the netherlands), acetonitrile gradient grade (sn 34851-2.5l), trifluoroacetic acid 99+% (pn 302031) and cytochrome-c (pn c2506) from sigma-aldrich (zwijndrecht, the netherlands), methanol (pn 106008) and milli-q water from merck millipore (amsterdam-zuidoost, the netherlands). formulation buffer, ad35 reference material (with transgenes b and c), ad26 reference material (with transgenes a and b) and ad26 process samples were from janssen vaccines (leiden, the netherlands). a waters acquity h-class uplc system equipped with a quaternary solvent manager, autosampler, and pda detector (waters corp., ma, usa) was used. data processing was performed by empower 3 software. various settings were tested in method development; the settings for the final, validated method were as follows. the analytical column was an acquity uplc protein beh c4 column (300 å, 1.7 m, 2.1 × 150 mm). new analytical columns were preconditioned with 3 injections (30 l each) of 10 mg/ml cytochrome-c followed by 3 injections (30 l) of milli-q water to prevent unwanted protein adsorption with the stationary phase. solvent a was 5% v/v acetonitrile (acn) in milli-q water, solvent b was 100% acn, and solvent c was 1% w/v tfa in 5% acn. from a, b and c a linear elution gradient was formed from 20% acn to 60% acn containing 0.175% w/v tfa in 17 min. the flow rate was 0.6 ml/min, the injection volume was 30 l, the column temperature was 50 • c, the auto sampler temperature was 8 • c, and for detection uv-absorbance was monitored at 280 nm. after analyses, the system was flushed with 30% v/v acn in milli-q, the needle wash was with 20% v/v acn in milli-q, and pump seal wash was with 10% v/v methanol in milli-q. for preparative runs intended for peak assignment, samples of selected advac vaccines were first concentrated in 1.5 ml eppendorf ® lobind microcentrifuge tubes protein (hamburg, germany) by centrifugation in an eppendorf 5417r microcentrifuge at 17000 g for 30 min at 4 • c and removal of the supernatant. concentrated samples were analyzed by the rp-uplc method on a waters acquity h-class uplc system (method as described in section 2.2). fractions were collected every 10 s for 16 min in eppendorf twin.tec pcr plates lobind using the fraction collector of a triversa nanomate ® (advion, ithaca, ny, usa). the fractions were dried in a vacuum centrifuge without heating for 4 h. the pellets were resuspended in 6 l of 30% acn, then 12 l of trypsin solution (20 g/ml in 50 mm ammonium bicarbonate) was added, and samples were incubated overnight at 37 • c. the tryptic digests were again dried in a vacuum centrifuge and resuspended in 18 l of 20% acn with 0.1% formic acid. the trypsin digests were analyzed by lc-ms e on a waters acquity h-class uplc system coupled to a waters synapt g2-si mass spectrometer (waters corp., ma, usa). the analytical column was an acquity uplc protein beh c18 column (300 å, 1.7 m, 2.1 × 150 mm). solvent a was 0.1% formic acid in milli-q water and solvent b was 0.1% formic acid in 100% acn. elution was by a linear gradient from 2% b to 35% b in 45 min. the flow rate was 0.2 ml/min, the injection volume was 10 l, the column temperature was 40 • c, the autosampler temperature was 6 • c. for ms e conditions were: collision energy, 20 40 v (ramped); lock-mass solution, leucine enkephalin (2 ng/ml) in 1:1 acn-milli-q water with 0.1% formic acid; lock-mass flow, 5 l/min; capillary voltage, 3.0 kv, sample cone temperature, 25 • c; source temperature, 120 • c; desolvation gas, 800 l/h; cone gas flow rate, 20 l/h; employing resolution mode with 1 scan per s. samples of adenovirus vector type 26 based vaccine were first diluted to 2.0 × 10 11 virus particles (vp)/ml in formulation buffer and then the following stress conditions were applied: thermal stress by incubation at 50 • c for 45 or 120 min; oxidative stress by addition of 30% v/v hydrogen peroxide and incubating for 4 h. the stressed samples were analyzed by capillary zone electrophoresis (cze) according to van tricht et al. [32] , by potency assay according to ma et al. [33] , and by rp-uplc as described above (section 2.2.). to compare the different techniques recoveries between stressed samples and non-stressed samples were used (i.e. total peak area recovery for rp-uplc, potency recovery for the potency assay or virus particle concentration recovery for cze). specificity was demonstrated during development for ad26 and ad35 proteins by ms-based peak assignments and by demonstrating absence of the viral proteins in the formulation buffer. the method repeatability, accuracy, intermediate precision, and linearity were determined for 5 concentration levels in the range of 0.5 × 10 11 -2.0 × 10 11 vp/ml with 3 replicates at each concentration level, repeated on 3 different days by two operators on two analytical columns, and two lc instruments. the method repeatability requirements were ≤ 15% rsd for relative peak areas and ≤ 1% rsd for relative retention times. the intermediate precision was determined based on the method repeatability and linearity data on three different days and the limits were ≤20% rsd for relative peak areas and ≤ 2.0% rsd for relative retention times. the linearity requirements were r 2 ≥ 0.98 and the sum of residuals less than 5%. the accuracy requirement was recoveries of 80-120% for relative peak areas. the limit of quantification (loq) was determined as the lowest concentration level that met the requirements for accuracy and precision. robustness was derived from the results of method development obtained with design of experiments. in the original rp-hplc method, based on lehmberg et al. [26] and liu et al. [10] , the adenovirus proteins were separated on a c4 column (5 m, 2.1 × 250 mm) with a water-acn gradient, with three segments of different slopes, from 20% to 60% acn containing 0.1% tfa in 110 min, see fig. 1 . the adenovirus samples are directly injected into the uplc system without sample pre-treatment (e.g. denaturation, reduction or dilution) and the viruses dissociate into proteins upon coming in contact with the water-acn mobile phase in the uplc system. dissociating the viruses into proteins prior to the uplc analysis resulted in irreproducible chromatograms (data not shown) [26] . this method was transferred to an uplc xbridge beh 300 c4 column (1.7 m, 2.1 × 150 mm). the linear gradient with three slopes was replaced by a single-slope linear gradient to improve elution reproducibility and the flow rate was increased the critical method parameters of the rp-uplc method were studied in a screening design of experiments to assess their impact on the method's run time and the resolution between the adenovirus proteins.: the following conditions were evaluated: gradient start composition (0-20% solvent b), gradient end composition (45-65% solvent b), gradient run time (17-25 min), tfa concentration (0.04-0.12%), and column temperature (40-70 • c). jmp statistical software was used to fit a model based on standard least squares using the main effects of the parameters. the response that was chosen to study the effect of the tested parameters was the best resolution between the maximum number of protein peaks with the shortest run time. from a pareto-analysis with standardized parameter estimates it was concluded that the tfa concentration and the column temperature had the strongest impact on the response. the peaks between approximately 6 and 8 min (proteins v, vii and viii) and between 13 and 15 min (proteins iiia and ix) were most influenced by the column temperature and the tfa concentration ( fig. 2a -c and g-j). the best resolution between the different peaks was obtained at the highest concentration of tfa (0.12%) and a column temperature of 50 • c (fig. 2b) . the gradient start and end composition and the gradient time were not critical regarding resolution or elution order, provided the gradient start composition was above 10% acn to assure complete dissociation of the virus particles and the end composition was above 45% acn to allow elution of all adenovirus proteins. based on this feasibility experiment, the gradient start composition was fixed at 20% b and the end composition at 50% b, with a gradient time of 17 min. these settings resulted in the shortest run time with the best resolution possible. initially, solvents a and b were prepared with 0.04-0.12% tfa, however, allowing this range of tfa concentration caused dayto-day variability (i.e. shifts in retention time), indicating that the tfa concentration is a critical method parameter which needs to be tightly controlled. fig. 2a-c shows that tfa has a significant effect on the separation even with slight changes from 0.09% to 0.12% tfa. the method robustness with respect to this parameter was examined specifically by design of experiments (see below, section 3.1.1). in addition, to increase the reproducibility of the mobile phase composition, the gradient mobile phase preparation method was changed as follows: solvent a was 5% acn in water, solvent b was 100% acn, and solvent c was 1% tfa in 5% acn. the uplc-system was then used to mix the three solvents to reproducibly obtain constant concentrations of tfa in the mobile phase throughout the gradient. the effects of the tfa concentration in the mobile phase and the column temperature were studied by a 3 1 4 1 full-factorial design (1 factor at 3 levels and 1 factor at 4 levels) with the aims to reduce the total run time of the analytical method and to improve the separation of critical peak pairs: v-viii(1), viii(1)-vii, vii-vi(2) (ad35 only), vi(2)-vi(3) (ad26 only), ii-ix, and ix-iiia. the selected factors were: tfa concentration, 0.150, 0.175, or 0.200%; column temperature, 40, 50, 60, or 70 • c. the study was executed by analyzing both ad26 and ad35 samples. the responses that were considered to evaluate the effects were the retention time of protein ii (used as indicator of the method's run time) and the separation of the critical peak pairs (see above). as the protein peak widths did not significantly change under the tested conditions, the differences in retention times of the critical peak pairs were used instead of the resolution as indicator for separation. a protein retention difference of 0.3 min or higher corresponded with baseline separation. the data were processed by jmp statistical software based on the factor main effects and interaction effects. for both adenovirus type 35 (fig. 3a) and type 26 (figs. 2e and 3 b) critical peak pairs were baseline separated in 17 min using 0.175% tfa and a column temperature of 50 • c. the contour plot in fig. 4 summarizes the results of this design of experiments. peak pairs ii-ix and vviii (1) were baseline separated at all tested conditions, with a retention time difference of > 0.6 min (fig. 2d-f) . for column temperatures between 40 and 55 • c, the separation of peak pair vi(2)vi(3) (ad26 only) was compromised (resolution 0.8) at a tfa concentration of 0.2% (fig. 2f ), but remained acceptable (baseline resolution) for the other peak pairs. column temperatures of 55 • c or higher resulted in insufficient separation for three out of five peak pairs at all tfa the robustness of the method for small changes in the tfa concentration was studied by assessing the reproducibility of the retention times and peak areas. an ad26 sample was analyzed at four different tfa conditions (with three independent analysis per tested tfa concentration): 0.170, 0.175, 0.180, or 0.185% tfa in the mobile phase (fig. 5) . the retention times and peak areas for 1.70, 0.180 and 0.185% tfa were compared to the retention times and peak areas for the default conditions (0.175% tfa). the critical peak pairs were always baseline separated and the peak areas and retention times at 1.70, 0.180 and 0.185% tfa did not deviate more than 5% as com-pared to the optimal system employing a tfa percentage of 0.175% in the mobile phase. based on the results it was concluded that the method is robust within the concentration range of 0.170 to 0.185% tfa, provided the mobile phase is prepared as described above in section 3.1. peaks were assigned to the respective adenovirus proteins by separate lc ms analysis. due to the presence of tfa in the mobile phase, it was not possible to attain accurate mass data form the intact adenovirus proteins by online coupling to ms. therefore, fractions were collected from the rp-uplc chromatograms of ad26 and ad35, which were digested by trypsin and subsequently analyzed by rp-hplc-ms e in order to identify the proteins by peptide mapping. peaks observed for both ad26 and ad35 (figs. 3 and 5) could be assigned to proteins ii, iii, iiia, v, vi, vii, viii, ix, and x. multiple fragments from cleavage of proteins vi and viii were identified, marked vi(1), vi(2), vi(3) (only in ad26), viii(1), and viii(2) (only in ad26). indeed, maturation of adenovirus into complete, infectious virus particles involves proteolytic cleavage of capsid and core proteins by the adenovirus proteinase (avp), and the results of peak assignment indicate that the ad26 sample contains mature adenovirus particles, the fiber protein (iv) was also identified and had a retention time of 12.0 min, but the peak height was very close to the limit of detection of the rp-uplc-uv method. the terminal protein could not be identified in the rp-uplcuv chromatogram, probably because of its low copy number (n = 2) in the virus (for comparison: the copy number for protein ii is 720). proteins 52.55k (1) and (2) , assigned in the last two peaks of the rp-uplc chromatogram (fig. 3b) , are associated with maturation of the virus particles and reported only present in immature virus particles [34, 35] . preliminary data showed that the peak area ratios of 52.55 k/iii, viii(1)/iii and viii(2)/iii are a measure of the relative concentration of immature adenovirus particles, which is in line with observations by takahashi et al. [27] and vellekamp et al. [28] . summary of method repeatability and intermediate precision of the previous method rp-hplc [26] and rp-uplc. the precision is given as overall precision in rsd% including all concentration levels and all tested proteins. the optimized rp-uplc-uv method is intended for protein profiling of ad26 and ad35 vector-based vaccine products. the protein profile of the test sample was compared to the profile of a reference sample based on the relative retention times and relative peak areas. the first purpose of the comparison was to confirm the identity of the adenovirus vector (type 26 or 35) based on matching relative retention times of the detected adenovirus proteins in the test sample versus the reference (i.e. protein fingerprinting). the second purpose was to detect potential protein modifications and degradation products of the vaccine product, by monitoring the relative peak areas of all peaks in the chromatogram, including any previously unidentified peaks. to validate the method for these two purposes, the specificity, precision, accuracy, linearity, and loq were assessed. the specificity of the method for the adenovirus proteins was demonstrated by ms during method development (see paragraph 3.2) and by demonstrating absence of peaks assigned to the viral proteins in the chromatogram of the formulation buffer (blank). precision was evaluated as method repeatability and intermediate precision of the relative peak areas and relative retention times. the method repeatability of the relative retention times of all proteins was between 0.1 and 1% rsd. the method repeatability of the relative peak areas was between 1 and 14% rsd, which was a significant improvement compared to the current rp-hplc method with a method repeatability of 6-26% rsd ( table 1 ). the intermediate precision of the relative retention times was 0.1-2.0% rsd and 1-14% rsd for the relative peak areas (table 1) , which met the acceptance criterion for method validation (≤ 20% rsd), except for the relative peak area for protein v (9-26% rsd), which was higher than obtained for other proteins and did not meet the acceptance criterion for method validation. linearity was demonstrated for samples diluted with formulation buffer within a range of 1.0 × 10 11 to 2.5 × 10 11 vp/ml. the determination coefficients for the peak areas were between 0.98 and 1.00, based on five concentration levels with three replicates at each level. the accuracy of the absolute peak areas could not be determined by spiked recovery due to unavailability of pure adenovirus protein standards. therefore, the accuracy was determined as percentage recovery of the measured relative peak area against the expected relative peak area at all concentration levels, based on dilutional linearity of the highest concentration level. the recoveries of the relative peak areas were between 79-108% for all adenovirus proteins. the loq of the method, the lowest concentration level within the linear range with acceptable precision and accuracy, was 1.0 × 10 11 vp/ml. the rp-uplc-uv method was not validated for the quantification of protein v since the peak area repeatability was higher than the criterion. protein iv, vi(3), viii(2), 52.55 k (1), 52.55 k (2) could not be quantified because their peak areas were below the limit of detection. in summary, the method was validated for the quantification of the relative peak areas of adenovirus proteins ii, iii, iiia, vi(1), vi(2), vii, viii(1), viii(2), ix, and x and for the detection of adenovirus proteins iv, v, vi(3), viii(2), 52.55 k (1), 52.55 k (2) in adenovirus type 26 and type 35 vector-based vaccine products. to assess if the rp-uplc-uv method can pick up changes in the protein composition that may occur during product storage, ad26 samples were stressed under different conditions and characterized by several physicochemical and biochemical methods. the stress conditions comprised oxidation with peroxide or temperature stress at 50 • c for 45 and 120 min. the stressed samples and a non-stressed control sample were analyzed by rp-uplc-uv and the chromatograms of the stressed samples were compared to the control sample based on the relative retention times and relative peak areas. the same stressed samples were also analyzed by capillary zone electrophoresis [32] and by a potency assay, for determination of the concentration of virus particles and the infectivity, respectively. thermal stress at 50 • c for 45 min resulted in overall rp-uplc peak area decreases of approximately 17%. thermal stress for 120 min resulted in 56% decrease in overall peak areas and, consequently, some proteins were close to the limit of quantification. the relative peak areas of the proteins, however, were the same in both temperature-stressed samples compared to the non-stressed sample. the cze analysis of the temperature-stressed samples showed a decrease in virus particle concentration of 25% at 45 min and 74% at 120 min, which was in line with the peak area decrease of the rp-uplc chromatograms (fig. 6 ). this suggests that temperature stress resulted in loss of virus particles rather than specific degradation of adenovirus proteins. for the same samples, the infectivity decreased by 22% and 91% for stress times of 45 and 120 min, respectively. oxidative stress resulted in the loss of almost all adenovirus proteins (approx. 91% overall chromatogram peak area decrease) and a number of new peaks were observed in the chromatogram, which were tentatively assigned to oxidized adenovirus proteins. the relative peak areas did change after oxidation stress due to the new peaks. the virus particle concentration measured by cze decreased by 88% and the infectivity decreased by 99%, which indicated that almost all the adenovirus particles were degraded in the oxidized sample. the rp-uplc method is sensitive for temperature stress and oxidative stress. the validated method was implemented for the analysis of adenovirus-based products. some examples are provided in fig. 7 and further described below. fig. 7a shows the chromatogram of an ad26 process intermediate sample which was stored for 9 months at 5 • c (normal storage condition of final product is −80 • c). all peak areas decreased by a factor of 3 compared to the reference sample (fig. 7d) . moreover, a degradation product was observed as shoulder of the peak of protein ii at 12.9 min. this peak was identified by ms as intact protein ii with oxidation and deamidation modifications. fig. 7b shows a partially purified sample from downstream processing. additional peaks were detected in the rp-uplc chromatogram at 2 and 9 min. the dad spectra of these peaks did not show the typical protein spectrum characteristics. fractions containing these peaks were collected and analyzed by gc-ms and lc-ms. the peaks at 2 min and 9 min were assigned to 2,4dimethylbenzaldehyde and 2,4 di tert-butylphenol, respectively. these could be leachables from the disposable container bags used in the process. trace c in fig. 7 is from another partially purified process intermediate sample with high concentrations of host cell protein impurities. the dad spectra of the additional peaks showed typical protein-spectra, however the relative peak areas of the adenovirus proteins were unchanged compared to the reference sample. in this case, fractions of the additional peaks were collected and analyzed by lc-ms. the unknown peaks between 11 and 13 min were assigned to host cell proteins, which were present at high concentrations and could therefore be detected by the rp-uplc method. an rp-uplc method was developed and validated for protein profiling of adenovirus type 26 and type 35 proteins. compared with a currently used rp-hplc method, the method's run time was decreased from 130 min to 17 min, allowing for the analysis of up to 50 samples per day instead of 10. protein profile changes were observed upon oxidative stress of adenovirus samples, indicating that the method can be used to monitor stability of adenovirus vector-based vaccines. analysis of process intermediate samples showed the ability of the rp-uplc method to detect and possibly quantify protein degradants, leachables, and host cell protein impurities. future method development will be directed towards enhancing the sensitivity of the rp-uplc method to be able to accurately quantify protein iv (the fiber protein). for example, alternative detection modes such as fluorescence will be explored. the new rp-uplc method is a useful addition to the analytical toolbox for the analysis of adenovirus-based products, as it allows for the rapid and accurate quantification of the adenovirus protein profile and any protein degradation products. adenovirus composition, proteolysis, and disassembly studied by in-depth qualitative and quantitative proteomics adenovirus-based vaccines for fighting infectious diseases and cancer: progress in the field adenoviruses as gene/vaccine delivery vectors: promises and pitfalls replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups b and d safety and immunogenicity of novel adenovirus type 26-and modified vaccinia ankara-vectored ebola vaccines: a randomized clinical trial localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center latest insights on adenovirus structure and assembly analytical method validation for biotechnology proteins, peptides, and antibodies proteomic study of recombinant adenovirus 5 encoding human p53 by matrix-assisted laser desorption/ionization mass spectrometry in combination with database search cryo-em structure of human adenovirus d26 reveals the conservation of structural organization among human adenoviruses optimization and qualification of a quantitative reversed-phase hplc method for hemagglutinin in influenza preparations and its comparative evaluation with biochemical assays proteomic analysis on structural proteins of severe acute respiratory syndrome coronavirus intact protein analysis in the biopharmaceutical field capillary electrophoresis-mass spectrometry for the analysis of intact proteins ultra-performance liquid chromatography/mass spectrometry of intact proteins intact-protein-based high-resolution three-dimensional quantitative analysis system for proteome profiling of biological fluids investigation of intact protein complexes by mass spectrometry ce-ms for the analysis of intact proteins capillary electrophoresis-mass spectrometry for the analysis of intact proteins narcissus mosaic virus found in nerine bowdenii. identification aided by anomalies in sds page application of deglycosylation to sds page analysis improves calibration of influenza antigen standards development of high-throughput and high sensitivity capillary gel electrophoresis platform method for western, eastern, and venezuelan equine encephalitis (wevee) virus like particles (vlps) purity determination and characterization purification of recombinant rotavirus vp7 glycoprotein for the study of in vitro rotavirus-like particles assembly new capillary gel electrophoresis method for fast and accurate identification and quantification of multiple viral proteins in influenza vaccines reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome quantitation of adenovirus type 5 empty capsids empty capsids in column-purified recombinant adenovirus preparations capillary electrophoresis for purity estimation and in-process testing of recombinant gb virus-c proteins rapid and selective characterization of influenza virus constituents in monovalent and multivalent preparations using non-porous reversed-phase high performance liquid chromatography columns hplc-based quantification of haemagglutinin in the production of egg-and mdck cell-derived influenza virus seasonal and pandemic vaccines one single, fast and robust capillary electrophoresis method for the direct quantification of intact adenovirus particles in upstream and downstream processing samples rapid determination of adenoviral vector titers by quantitative real-time pcr virus assembly and disassembly: the adenovirus cysteine protease as a trigger factor adenovirus l1 52-and 55-kilodalton proteins are present within assembling virions and colocalize with nuclear structures distinct from replication centers key: cord-290445-vb53bih9 authors: ahmed, shiek ssj; paramasivam, prabu; raj, kamal; kumar, vishal; murugesan, ram; ramakrishnan, title: interplay of host regulatory network on sars-cov-2 binding and replication machinery date: 2020-04-23 journal: biorxiv doi: 10.1101/2020.04.20.050138 sha: doc_id: 290445 cord_uid: vb53bih9 we dissect the mechanism of sars-cov-2 in human lung host from the initial phase of receptor binding to viral replication machinery. we constructed two independent lung protein interactome to reveal the signaling process on receptor activation and host protein hijacking machinery in the pathogenesis of virus. further, we test the functional role of the hubs derived from both interactome. most hubs proteins were differentially regulated on sars-cov-2 infection. also, the proteins of viral replication hubs were related with cardiovascular disease, diabetes and hypertension confirming the vulnerability and severity of infection in the risk individual. additionally, the hub proteins were closely linked with other viral infection, including mers and hcovs which suggest similar infection pattern in sars-cov-2. we identified five interconnecting cascades between hubs of both networks that show the preparation of optimal environment in the host for viral replication process upon receptor attachment. interestingly, we propose that seven potential mirnas, targeting the intermediate phase that connects receptor and viral replication process a better choice as a drug for sars-cov-2. coronavirus disease 2019 (covid-19, sars-cov-2) became a pandemic, spread almost 150 countries worldwide [1] . sars-cov-2 causes lower respiratory infections leading to pneumonitis and multi-organ failure [2] . till date, 1991562 cases were reported as on 16th april 2020 and expected to increase every day by a human-tohuman transmission that occurred due to close contact with one another [3] . an increase in patient number increases the fatality rate because of non-specific treatment against sars-cov-2. today covid-19 became a serious threat in both developed and under-developing countries [4] . there is an urgent need to accelerate work on specific treatments to decrease disease, morbidity and mortality [5, 6] . repurposing of existing drugs has been considered as an immediate remedy rather than waiting for a new molecule to pass through human clinical trials. in addition, sars-cov-2 screening at the early stage may benefit the vulnerable population with the risk factor of diabetes, hypertension [7] [8] [9] , and cardiovascular disease [10] . however, understanding the mechanism of sars-cov-2 and host involvement will provide knowledge that guides towards developing biomarkers and drugs to treat this pandemic disease. sars-cov-2 is a single-stranded rna virus-derived from the human sars-cov-2 group of ß-genus with the genome size of 30kbp. sars-cov-2 genome encodes most structural proteins at 3' orfs and non-structural proteins (nsp) at 5' end orfs [8, 11] . the nsp proteins are categorized as nsp1-16, which plays a potential role in replicase / transcriptase activity whereas the orfs at 3' encode nine putative accessory factors along with four structural proteins, nucleocapsid (n), envelope (e), membrane (m), and spike (s) proteins [8, 11] . the envelope (e), membrane (m), and spike (s) proteins localized at vial surface suggested having host binding capacity. on host attachment, sars-cov-2 initiates the infectious cycle and undergoes rna replication inside the host and assembles their substrate into their progeny virus. in general at the initial phase of the host binding, virus may create an optimal environment for its replication by interacting with the host proteins. simultaneously, the host alters its gene expression to react against the virus for self-defense leading to the overexpression of inflammatory genes and cytokine storm [12] . due to the viral genome complexity and 20% sequence dissimilarity with other pathogenic hcovs [13] . notably, the sequence variations at replicase complex (orf1ab), envelope (e), spike (s), nucleocapsid (n) and membrane (m) suggest the possibility of different mechanisms adopted by sars-cov-2 that brings the question of repurposing the existing drugs. the current knowledge of host molecular components utilization by the sars-cov-2 is still at an early stage compared with other known human infecting rna viruses. understanding the regulatory behavior will enhance our knowledge of the virus-host mechanism, which may through light on diagnosis and treatment. in this study, we first time report a systems biological framework (fig 1) to investigate the molecular interplay between sars-cov2 and human lung tissue. the novel approach uses high through-put experimental data and human protein interactome to reveal the sars-cov2 driven host mechanism. our approach provides (i) functional hubs that activated upon viral attachment to the receptor and viral genome evasion to the host. (ii) association of viral modulated functional hubs with diabetes, hypertension, and cardiovascular disease. (iii) an interdependency between the functional hubs that demonstrates the utilization of a host environment created upon receptor activation for the viral replication process. (iv) hubs representing mirna for therapeutic intervention. overall, our framework accesses the information such as human tissue proteome, transcriptome, text mining, and mirna data to postulate a mechanism in host on sars-cov2 infection. firstly, we analyzed whether sars cov-2 protein interacting with the endogenous human proteins (host). for which we searched and collected sars-cov-2 host proteomics data derived from affinity purification mass spectrometry [14] . the data provide 332 human endogenous proteins interacting with 27 sars cov-2 proteins. further, these human endogenous proteins were categorized as set-a and set-b, respectively. set-a composed of 38 human proteins showing physical affinity with structural proteins like as envelope (e), spike (s), and membrane (m) proteins localized at sars-cov2 surface. whereas, the set-b contains 294 host proteins confirming its affinity with 23 sars-cov2 non-structural and one nucleocapsid (n) proteins. then, we investigate host proteins that favor the attachment of sars-cov2. so, the cellular localization of set-a proteins was annotated to have atp6v1a, ap3b1, stom, and zdhhc5, localized at the host cell surface that may enable sars-cov2 attachment. further, the proteins in the set-a and b were mapped with an in-house lung-expressed gene database to confirm their expression in the lungs that relate the mechanism that occurs in lung tissue. next, we investigate the high-through-put protein-protein interaction networks that reveal the sars-cov2 mechanism in the host. two independent protein interactome networks were generated from set-a and set-b, respectively. the first interactome, receptor-mediated network from set-a contains four cell surface seed proteins that extended to have 45 neighboring proteins with every node directly connected to its seed proteins. the receptor-mediated network describes the molecular signaling initiated on sars-cov2 attach to the host. secondly, the viral replication machinery network from set-b with 332 seed proteins extended to 1486 neighboring proteins with 11438 interacting edges which representing the mechanism attributed to evasion of the sars-cov2 genome into the host. the proteins involved in the networks were mapped to the in-house lung-expressed-gene database. as a result, all 49 proteins with four seed in the receptor-mediated network were expressed in the lungs (fig 2) . similarly, the viral replication machinery network was acquired with 1522 proteins with 9747 interacting edges showing the complex sars-cov-2 mechanism in the human lungs. here, we looked for densely connected protein, which provides essential functional hubs within the network. molecular complex detection (mcode) algorithm [15] used to extract hubs that define the core regulatory signaling and cellular processes within the network. eleven core functional hubs were obtained from the viral replication machinery network (s1-11 fig) . in a receptor-mediated network, the implementation of the mcode algorithm was exempted for its limited number of nodes and edges, which by itself forms hubs. hence, the interacting proteins for all four distinct receptors were considered as hubs for the receptor-mediated network (fig 2) . all hub proteins were mapped to differential expressed genes from rna-seq data. of 374 hub proteins from both the network, 76 were differentially regulated in primary human lung epithelium (nhbe) on sars-cov2 infection (rna-seq dataset gse147507). for instance, eight differentially expressed hub proteins were noticed in the receptor-mediated network (s1 table) . similarly, among 325 hub proteins of viral replication machinery network, 68 were altered significantly, which confirms the hijacking of endogenous human proteins for the viral replication process (s1 table) . next, we investigated why sars-cov2 infection is susceptible to metabolic diseases like cardiovascular disease, diabetes, and hypertension. for that, we tested the associations of hub proteins with these metabolic diseases using natural language processing in our indigenous r-code. among 374 hub proteins from the network, 186, 155, and 103 proteins were associated with cardiovascular disease, diabetes, and hypertension, respectively (s2 table) . we adopted a similar mining process to determine the relevance of hub proteins with lung diseases such as asthma, chronic bronchitis, pneumonia, chronic obstructive pulmonary disease (copd), and emphysema. simultaneously, the relevance of hub proteins was tested in viral infections such as adenovirus, influenza, metapneumo, parainfluenza, respiratory syntical virus, rhinovirus, hcov-nl63, hcov-229e, hcov-hku1, hcov-oc43, sars-cov and mers-cov. these results suggest that 101 hub proteins have relevance to lung disease (s3 table) , and 117 proteins were associated with the known viral infection (s4 table) . these associating hubs convey the likelihood of vulnerability in the risk population, symptoms related to lung diseases, and similar modes of viral infection. subsequently, the protein enrichment analysis was executed to check the molecular involvement of hubs on sars-cov2 infection. the enrichment analysis of hubs showed several over representing pathways for the hubs of receptor-mediated ( fig 3) and viral replication machinery network (fig 4) . the receptor-mediated network hubs exhibit several molecular signaling and cellular events upon sars-cov2 attachment (s5 table) . notably, most hub proteins showed significant association with immune mechanism, signal transduction, metabolism of proteins, metabolism of rna, transcription machinery, vesicle-mediated transport, metabolism, transport of small molecules cell cycle and homeostasis were noticed (s5 table) . also, a moderate involvement was seen in cellular response mechanism, cell-cell communication, organelle biogenesis and maintenance. similarly, the enrichment analysis of hubs derived from viral replication machinery network (fig 4) showed a major involvement of immune mechanism, signal transduction, metabolism of proteins, metabolism of rna, transcription machinery, dna repair, dna replication, programmed cell death, and cellular responses to external stimuli were noticed (table s5) . interestingly, several common mechanisms were noticed that occur due to the existence of 34 interconnected proteins between the hubs of both the network (fig 5-9 ). these common molecules represent the inter-connecting mechanism involved in the transcription machinery, immune response, cell growth and/or maintenance, transport, metabolism, protein metabolism, cell communication and signal transduction that activated upon virus binding and has been subsequently utilized for viral replication process (s5 table) we further investigate these 34 interconnecting proteins that may use for a therapeutic process. for which, the mirna target for interconnecting proteins (genes) was identified. among 34 proteins, 26 shown targeted by 119 mirna. of which, mir-124-3p, let-7g-5p, mir-133a-3p, mir-133b, mir-218-5p, mir-22-3p and mir-506-3p were noticed to target more than three interconnected proteins (fig 11) . these mirnas may show its utility for therapeutic intervention. we implement a comprehensive systems biological framework ( fig.1 ) that utilizes a variety of datasets to illustrate the critical mechanism mediated by sars-cov2 in the lungs. two independent lung proteins interactome were constructed. the first interactome termed as a receptor-mediated network that revealed the molecular interaction that mediates cellular and signaling processes on sars-cov2 attachment to the host. in the receptor-mediated network, atp6v1a, ap3b1, stom, and zdhhc5 are the seed proteins localized at the host surface showed significant affinity to the outer surface envelope (e), spike (s), and membrane (m) proteins of sars-cov2. on bounding with atp6v1a, ap3b1, stom, and zdhhc5, the host activates multiple signaling and cellular events that demonstrated in the receptor-mediated protein network (fig 3) . for instance, viral envelope (e) bound to the ap3b1 cell-surface protein that initiates the interaction with its neighboring proteins, ap1g1, ap1m1, ap3d1, ap3m2, ap3s1, ap3s2, arf1, arf6, arrb1, arrb2, bub1, bub1b, cltc, and csnk1a1. similarly, spike (s) interacts with the zdhhc5 that connects cldnd1, flot2, fxyd1, fyn, lrrc8a, and pif1. likewise, the sars-cov2 membrane (m) interacts with atp6v1a and stom host surface protein that signals 25 human endogenous proteins (fig 2) . each host cell surface proteins and it's interacting neighbors form a closed hub with no extensive interconnection with another hub. these hubs were pathways enriched that takes part in wide verities of molecular pathways that include endosome transport, vesicle-mediated transport, protein modification, regulation of cell cycle, immune response, kinase activity, signal transduction, protein metabolism, cell communication, energy and metabolic pathway, cell growth, and maintenance, regulation of nucleic acid metabolism. to our knowledge, most of this mechanism is very well connected with the viral pathogenesis machinery mechanism. notably, at an early phase of surface attachment, the host initiates inflammatory signaling such as interleukin signaling (il1, il2, il5, il23, il8), tnf receptor, and nf-kappa b signaling pathways that may initiate cytokine storm in lung cells [12, 16] . simultaneously, the involvement of transcription and translational regulatory processes were noticed in the pathway enrichment of receptor hubs. these results suggest that on surface receptor activation host prepares an optimal environment in the host for the viral replication. the second lung protein interactome was named as viral replication machinery network. in this network, we demonstrated the complex interconnectivity of human endogenous proteome with nucleocapsid (n) and 23 non-structural sars-cov2 proteins. the viral replication machinery network conveys the utilization of host factors for sars-cov2 replication and self-defense mechanism. using the mcode algorithm, eleven hubs (s1-11fig) were extracted from the viral replication machinery networks, which play a significant role in cellular and molecular mechanisms (s5 table) . further, mapping the differently expressed genes with the hubs showed approximately 20% of hub proteins were altered upon sars-cov2 infection (s1 table) . this result confirms the hijack of host hub proteins and their molecular pathways for sars-cov2 machinery. some hub proteins were proven linked to diabetes, hypertension, and cardiovascular disease (s2 table) . these results suggest that the sars-cov-2 alter and utilize the disease proteins for its replication machinery that may be the considerable cause for susceptibility, severity, and complication. besides, similar observations were noticed while mapping hub proteins with proteins related to lung disease and other viral infections. on mapping, 101 hub proteins were shared between the lung diseases suggesting a common mechanism relating disease symptoms and/or vulnerability (s3 table) . also mapping with other viral infection dataset, 50 hub proteins of the replication machinery network have noticed in influenza virus infection (s4 table) , which suggests sars-cov2 and influenza may have a similar mode of host infection machinery [17] . as apart, several common pathways between the hubs of two networks were noticed (s5 table) . we obtained these common pathways as an intersection of the 34 interacting molecules between the hubs (fig 5-9 ). for example, ap3b1 hub derived from the receptor-mediated network interact with the "hub 4" of viral replication machinery network by gtf2f2, polr2e, arrb1, and ap3b1 interconnecting proteins ( fig 5) . overall, five interacting hubs were noticed between receptor-mediated and viral replication machinery network (fig 5-9 ). the molecular pathways of interconnecting protein hubs could be the intermediate phase that connects the receptor activation mechanism and viral replication process (fig 10) . such intersecting proteins will aid in developing a drug for sars-cov2. here, we suggest a few mirnas that regulates the proteins of interconnecting hubs, which may have therapeutic potential. the mirna has been regarded as drug molecules for various diseases [18 19 ]. of 111 mirna, mir-124-3p, let-7g-5p, mir-133a-3p, mir-133b, mir-218-5p, mir-22-3p and mir-506-3p targets minimum of three interconnecting proteins (fig 11) . these seven mirna molecules are needed to be viewed in a better perspective for future research to screen and treat the current sars-cov2 pandemic. overall, this study displays several advancement and advantages, 1) establishes interplay between sars-cov2 and human lung host mechanism by integrating experimental evidence arrived from high throughput multi-omics observation on sars-cov2. 2) our approach provides a possible clue for susceptibility and severity in an individual with metabolic disease complications and suggests possible proteomic relevance with lung diseases and other viral infections. 3) our study increases knowledge of molecular interconnectivity between receptor binding mechanism and viral replication machinery that guide towards drug target research. although our approach provides the multi-dimensional view on sars-cov2 and host interaction, two major limitations need to be considered, 1) the interaction between sars-cov2 spike protein with the host angiotensin (ace2) receptor is not defined in affinity purification mass spectrometry data. hence, no direct ace2 interaction has been established in our interactome. 2) we propose a few mirnas from our approach, are needed to be validated further for clinical application. in summary, our systems biological approach provides an extensive investigation on sars-cov2 host interaction by constructing interactome based on experimental evidence. we identified crucial functional hubs that relate the mechanism on activation sars-cov2 attachment through receptor and subsequent utilization functional hubs for viral replication in the host. enrichment analysis supports our hypothesis showed the common mechanism associated with transcriptional and protein translation processing activated upon in host attachment. hub proteins showed linked with diabetes, hypertension, and cardiovascular disease proteins that provide the clue for severity and susceptibility in the diseased population. the relationship between hub proteins with lung diseases like copd, asthma, pneumonia establishes the likelihood of similar symptoms. also, assessing the hubs with the reported proteins of other viral infection establishes the similarity in the mode of viral infection. interestingly, we propose mir-124-3p, let-7g-5p, mir-133a-3p, mir-133b, mir-218-5p, mir-22-3p, and mir-506-3p from the genes of interconnecting proteins from its molecular pathways as a mirna drug for sars-cov2. however, further work is needed to confirm its utility for clinical application. we believe our results will add knowledge to the existing information that may open up mirna as a drug for sars-cov2 infection. the dataset relating severe acute respiratory syndrome coronavirus 2 (sars-cov2) and human proteins interactions searched electronically. of extensive search, the experimentally proven dataset reporting the physical association of 26 sars-cov2 proteins with human proteins determined using affinity purification mass spectrometry (ap-ms) [14] was retrieved. we converted all collected interacting human proteins to an official gene/protein symbol using the uniport database (https://www.uniprot.org/). next, we collected lung protein expression data from the hprd proteins. subsequently, in order to determine the involvement of each hub in viral pathogenesis, the expression data of sars-cov2 infection was analyzed, and the differential expressed genes were mapped hub proteins. we searched the gene expression dataset in ncbi gene expression omnibus were retried from ncbi, sra database. after quality assessment, the reads were aligned to the human genome (hg19) following the rna-seq analysis pipeline to determine the differential expression genes using deseq with p-value < 0.05. further, the significant differentially expressed genes in the host on sars-cov2 infection were collected and mapped to the hubs. r-program was employed to find out the associated genetic hubs risk factors genes reported in the literature for diabetes, hypertension, and cardiovascular diseases. in brief, the r-code collects the abstract from the ncbi, pubmed, for the keywords related to each risk factor. all abstracts were automatically examined for the presence of risk factors (examples: "diabetes") and hub proteins using a natural language processing method. further, the co-occurrence of risk factors with each protein symbol in the abstract was assessed using a point-wise mutual information method. a similar data mining process was carried out for lung diseases (asthma, chronic bronchitis, pneumonia, and emphysema) and other viral pathogens such as adenovirus, influenza, metapneumo, parainfluenza, respiratory syntical virus, rhinovirus, hcov-nl63, hcov-229e, hcov-hku1, hcov-oc43, sars-cov and mers-cov. simultaneously, all hubs were enriched using kyoto encyclopedia of genes and genomes (reactome; https://reactome.org//) database to evaluate the relevance of the pathway of each hub in sars-cov2 infection and replication process. in particular, the pathway enrichment of receptor-mediated network hubs shows the mechanism initiated upon sars-cov2 receptor activation. whereas, the pathways of viral replication machinery network hubs explain the mechanism attribute on evasion of sars-cov2 genome into the host for its replication process. this cluster is termed as hub 3 that represents the closed interconnected proteins (rectangular box) showing no direct connectivity with sars-cov2 proteins. however, these proteins involved in the host inflammatory process, protein serine/threonine kinase, dna topoisomerase, transcription factor, transcription regulator, chaperone, and ubiquitin-specific protease activity. this cluster is termed as hub 4 that represents the closed interconnected proteins this cluster is termed as hub 11 that represents the closed interconnected proteins transport. over-representation of these molecular events suggests host self-defense mechanism and preparation of the optimal environment in the host for the viral replication process on receptor attachment. additionally, the red highlighting node represents the differentially expressed genes on sars-cov2 infection derived from rna-seq analysis. interconnecting hub proteins are represented. these mirnas will be the potential molecule as a drug that helps in inhibition of connective between host attachment and the viral replication process. who strategic and technical advisory group for infectious hazards. covid-19: towards controlling of a pandemic hypothesis for potential pathogenesis of sars-cov-2 infection-a review of immune changes in patients with viral pneumonia coronavirus disease 2019 (covid-19) situation report -87 managing covid-19 in low-and middle-income countries covid-19 -navigating the uncharted real estimates of mortality following covid-19 infection covid-19 and diabetes: knowledge in progress a new coronavirus associated with human respiratory disease in china china hypertension survey investigators. status of hyperten-sion in china: results from the china hypertension survey european centre for disease prevention and control. novel coronavirus disease (covid-19) pandemic: increased transmission in the eu/eea and the uk -sixth update genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan hlh across speciality collaboration, uk. covid-19: consider cytokine storm syndromes and immunosuppression network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 a sars-cov-2-human protein-protein interaction map reveals drug targets and potential drug an automated method for finding molecular complexes in large protein interaction networks how to reduce the likelihood of coronavirus-19 (cov-19 or sars-cov-2) infection and lung inflammation mediated by il-1 sars-cov-2 detection in patients with influenza-like illness mirnas in alzheimer disease -a therapeutic perspective microrna therapeutics: towards a new era for the management of cancer and other diseases all authors thank chettinad academy of research and education (care) for the support. we specially thank professor ram murugesan, research director (care) for the critical comments and feedback on the manuscript. key: cord-295351-0zr2e8lh authors: mohd ropidi, muhammad izzuddin; khazali, ahmad suhail; nor rashid, nurshamimi; yusof, rohana title: endoplasmic reticulum: a focal point of zika virus infection date: 2020-01-20 journal: j biomed sci doi: 10.1186/s12929-020-0618-6 sha: doc_id: 295351 cord_uid: 0zr2e8lh zika virus (zikv) belongs to the flavivirus genus of the flaviviridae family. it is an arbovirus that can cause congenital abnormalities and is sexually transmissible. a series of outbreaks accompanied by unexpected severe clinical complications have captured medical attention to further characterize the clinical features of congenital zikv syndrome and its underlying pathophysiological mechanisms. endoplasmic reticulum (er) and er-related proteins are essential in zikv genome replication. this review highlights the subcellular localization of zikv to the er and zikv modulation on the architecture of the er. this review also discusses zikv interaction with er proteins such as signal peptidase complex subunit 1 (spcs1), er membrane complex (emc) subunits, and er translocon for viral replication. furthermore, the review covers several important resulting effects of zikv infection to the er and cellular processes including er stress, reticulophagy, and paraptosis-like death. pharmacological targeting of zikv-affected er-resident proteins and er-associated components demonstrate promising signs of combating zikv infection and rescuing host organisms from severe neurologic sequelae. zika virus (zikv) is a mosquito-borne virus that belongs to the flaviviridae family together with other notable flaviviruses such as dengue virus (denv), west nile virus (wnv), japanese encephalitis virus (jev), and yellow fever virus (yfv). zikv was first isolated from a febrile rhesus monkey in april 1947 and was subsequently isolated from aedes africanus mosquitoes 9 months later in zika forest of uganda [1] {dick, 1952 #15}. despite its discovery more than a half-century ago, zikv received little attention due to sporadic cases of human infection with mild and self-limiting symptoms [2] . zikv was put under scrutiny following its first outbreak in the yap island of micronesia in 2007. during this outbreak, 185 suspected cases of zikv infection were reported, and at least 24% of these patients were either serologically or molecularly confirmed for zikv infection [3] . the same study estimated that 5005 island residents (73%) were infected, of which 919 were symptomatic [3] . zikv-infected patients typically present mild clinical symptoms such as fever, maculopapular rash, conjunctivitis, and arthralgia [2] . nevertheless, the second zikv outbreak in french polynesia in 2013 provided the first compelling relationship between zikv infection and a neurological complication, where a woman was diagnosed with guillain-barré syndrome (gbs), an autoimmune disease typically affecting motor neuron functions, a week following the onset of zikv-like symptoms [4] . this epidemic also recorded a 20-fold increase of gbs incidence, where 41 gbs-diagnosed patients (98%) were serologically positive for zikv [2, 5] . the sudden spike of microcephaly cases among newborns in brazil following the 2015-2016 zikv outbreak triggered the brazilian ministry of health and the world health organization to declare zikv as a national and international public health emergency [6, 7] . the causative link between zikv and congenital neurological anomalies was established based on several evidence including the detection of zikv rna within the amniotic fluid acquired from zikv-infected mothers with confirmed fetal microcephaly case [8, 9] . subsequent clinical and pre-clinical findings further supported the causal relationship between zikv infection and microcephaly in newborns [10] [11] [12] . zikv is believed to infect cells through receptormediated endocytosis. these putative receptors include cluster of differentiation 209 (cd209), tyrosine-protein kinase receptor tyro3, and axl, where overexpression of these receptors in zikv-impervious hek293t cells rendered the cells susceptible to zikv infection [13] . in particular, the role of axl in zikv infection has been extensively investigated due to its crucial role in dengue virus infection [13] . during zikv infection, axl mediates zikv entry indirectly whereby the phosphatidylserine extension on zikv lipid membrane binds to growth arrest-specific 6 (gas6), one of the ligands for axl that serves as a bridge for zikv and axl interaction, resulting in clathrin-mediated virus internalization [14, 15] . the acidic microenvironment within the endosome promotes fusion between the virus envelope proteins and the endosomal membrane resulting in the release of zikv genome into the host cell cytosolic space [15, 16] . the zikv genome is a positive-sense, single-stranded rna ((+)ssrna) of approximately 11,000 bases in length [2] . zikv genome contains a single open reading frame that encodes three structural and seven non-structural (ns) proteins. these structural proteins consist of capsid (c), pre-membrane (prm), and envelope (env) proteins, which are predominantly involved in viral pathogenesis and virion structure. the seven non-structural proteins, ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5 proteins, largely contribute towards the purposes of viral pathogenesis, replication, and immune evasion [17] . zikv utilizes host translational machinery to produce a single polyprotein that is further cleaved by viral ns2b-ns3 serine protease and host cell protease into functional viral proteins [18] . these viral proteins are then distributed to cellular compartments for various functions [19] . zikv proteins localize to distinct subcellular compartments zikv proteins are primarily distributed within and in close proximity to several endomembrane compartments including the endoplasmic reticulum, golgi apparatus, endosomes, lysosomes, autophagosomes, and nucleus [20] . molecular cloning and expression of individual zikv proteins reveal distinct and specific organellar localization. zikv capsid localizes to several compartments including the nucleus and the golgi [19] . in addition to capsid, ns2b and ns4a also localize to the golgi. three zikv proteins namely env, prm, and ns2a are distributed to the er as indicated by their co-localization with calreticulin expression. ns5, which contains nuclear localization signals, forms punctate distribution in the nucleus [19] . however, these data should be interpreted with caution as the subcellular localization of these cloned viral proteins may differ in zikv-infected cells. for example, individual expression of zikv ns3 localizes to the mitochondria, but ns3 is instead localized at the er when co-expressed with ns2b [21] . similarly, although ns5 protein is primarily detected in the nucleus of zikv-infected cells [22] , zikv ns5-rna polymerase has been shown to interact with zikv ns3-helicase during viral rna replication in the er [23] . inhibiting this interaction reduces ns3helicase activity. subcellular localization of viral proteins is affected by viral proteins interaction, which is crucial for productive infection. importantly, understanding the mechanisms of viral protein transport and viral protein-protein interaction may provide novel therapeutic targets. the subcellular distribution of certain zikv proteins was corroborated in a separate study where interatomic analyses using proximity-dependent biotin-identification (bioid) labeling and flag-based immunoprecipitation (ip) coupled with mass spectrometry (ms) uncover indepth molecular interactions between zikv proteins with various host organelles and proteins. in general, zikv proteins mainly interact with host proteins that are involved in protein processing, vesicle trafficking, rna processing, and lipid metabolism [20] . zikv capsid interacts with multiple nucleolar proteins, whereas ns5 protein targets and disrupts the cajal bodies in the nucleus [20] . this observation is consistent with the aforementioned experimental study that showed nuclear localization of zikv capsid and ns5 protein [19] . the study also identified numerous interactions between viral proteins (prm, env, ns2a, ns2b, ns4a, and ns4b) with various er proteins, which highlights the importance of the er in zikv life cycle [20] . zikv has been reported to be dependent on several er proteins including er-associated signal peptidase complex (spc) proteins, er translocon, and er membrane complex (emc) proteins. spc, especially spc subunit 1 (spcs1), is crucial for zikv pathogenesis as knocking out spcs1 in 293 t cells significantly reduced zikv infection and drastically impaired the production of infectious zikv particles [24] . a study using pooled crispr/cas9 cell survival enrichment assay identified zikv strongly depends on er membrane complex (emc) during early-stage zikv replication [25] . emc is a highly conserved oligomeric complex residing on the er membrane and is crucial for transmembrane protein folding and lipid trafficking [25] . the dependency on six emc subunits (emc1-emc6) was verified with sirna assays, where depletion of these proteins significantly impaired the replication of several zikv strains [25] . another study reported that zikv ns4b physically interact with emc1 subunit and depletion of this subunit markedly reduced the level of zikv ns4a and ns4b protein [26] , indicating that emc proteins are required for viral protein biogenesis. further investigation using denv ns4b identified two marginally hydrophobic domains at the n-terminal of ns4b to be crucial for ns4b dependence on emc [26] . since zikv ns4b shares moderate sequence identity, high sequence similarity, and similar topology with other flaviviruses [27] , it is plausible that the two weak hydrophobic domains of zikv ns4b are the specific determinants for zikv dependency on emc proteins. in addition to its direct interaction with viral proteins, emc proteins also associate with er sec61 translocon and oligosaccharyltransferase (ost) complex proteins, both of which are also important for zikv infection [24, 25] . sec61 is a major component of the er translocon that facilitates the entry of nascent polypeptides into the er lumen for protein processing. zikv dependency on sec61 translocon was validated in a separate study wherein myolactone treatment, a sec61α inhibitor, dramatically reduced zikv expression [28] . zikv replication was restored in cells expressing mutant sec61α that conferred resistance against myolactone inhibition [28] . zikv dependency on ost complex, an integral part of the translocon consisting of eight er-transmembrane protein subunits that catalyzes co-translational n-glycosylation, is based on emc1, emc2, emc4 and emc5 interaction with ost complex subunits namely stt3a/b, rpn1/2, and ddost [25] . additionally, zikv proteins including ns4b have been reported to directly interact with these ost subunits [29] . zikv dependency on ost complex is corroborated by marceau et al. that reported a significant abrogation of zikv rna replication in stt3a knockout cells [30] . importantly, treatment with ngi-1, a small molecule ost complex inhibitor, significantly reduced zikv rna replication with an ec 50 value of 2.2 μm [31] . the inhibitory activity of this non-toxic molecule (cc 50 = 34.9 μm) is independent of n-linked glycosylation activity of the ost complex as zikv replication was drastically impaired in hap1 cells containing wild-type or catalyticinactive stt3a [31] . the specific mechanism of zikv dependency on stt3a is unclear, but immunoprecipitation and electron microscopy studies showed physical interactions between denv proteins and stt3a/b, forming viral rna replication complexes that reside within the er in close proximity to denv2-vesicle packets [30] . since zikv proteins also directly interact with stt3a and zikv infection induces the formation of vesicle packets [29, 32] , it is plausible that zikv depends on ost complex in a similar manner. in short, zikv proteins interact with various host proteins and exploit their functions to accommodate the progression of the viral life cycle. er and er-related proteins are especially important in the virus genome replication, which may be useful in developing antiviral therapies. the following sections of this review will discuss the structural and molecular changes in the er following zikv infection. throughout the course of infection, viruses induce various modifications on the host cells' metabolisms and their cytoarchitecture to support the progression of virus life cycle. one of the common targets is the er [33] . among the important functions of the er include protein processing, synthesis of essential biosynthetic compounds (primarily lipids and proteins), metabolism of steroids and carbohydrates, and storage of calcium ions (ca 2+ ) for intracellular signaling [34] . structurally, the er is made of a continuous network of membraneenclosed flattened sacs and tubules. the dynamic and fluid nature of these structures enable this organelle to accomplish tubule extension, membrane curvature and shape alteration in response to stress-induced or factordemanding conditions such as cell division and differentiation [34] . likewise, zikv infection is also capable of exploiting the organelle's plasticity and remodels the er structure for their replicative benefit. recent transmission electron microscopy analyses on zikv-infected cells frequently observed significant expansion of the er [32, 35, 36] . this structural modification typically consisted of proliferating er lamellae and dilated er lumen, which cumulatively increases the overall er size and volume. enlargement of the er is believed to occur in response to virus-induced er stress, due to a supply-demand imbalance that will be elaborated in the next section. zikv infection also forms distinct aggregates of intricate er tubular network that resembles sponge-like matrix called the convoluted membranes (cm) (fig. 1 ) [32, [35] [36] [37] . cm aggregates are frequently seen bordering zikv virions and other zikv-induced structures, with occasional virions also found within the cm itself [32, 36] . the function of the cm in zikv infection is unclear; nevertheless, evidence from other flaviviruses studies suggest that the cm possesses roles closely associated to protein synthesis and processing [18] . interestingly, cm aggregates are not observed in zikv-infected neural progenitor cells, suggesting cell-type-specific factors are required to develop the cm [32] . no discernible difference in the cm structure is observed between the african and asian zikv strains [36, 37] . next, vesicle packets (vps) are also commonly observed in zikv-infected cells ( fig. 1 ) [32, [35] [36] [37] . vps refers to an aggregate of closely packed single-membrane vesicles that are encapsulated within an er cisterna. individually, these vesicles measure between 60 and 100 nm in diameter with a narrow channel of approximately 10 nm that connects the vesicle lumen and the cytosol [32, 35] . the radial dimension of these vesicles varies according to the host cell type, thereby hinting possible involvement of cell-type-specific factors in its formation [36] . modest radial difference was also noted between zikv lineages [32] ; however, more morphologically pronounced difference between the zikv lineages is observed in the vesicle's shape whereby african strain tend to form ovoid vesicles, whereas the asian strain's vesicles are generally more spherical [37] . nevertheless, these differences, especially the latter, remain contentious due to limited experimental measurements and warrants additional investigations across various cell types to support these observations. despite these differences, the vesicles' pore maintain a comparable diameter of 10 nm regardless of the virus strain and the host cell type, suggesting that a conserved cellular or viral machinery mediates pore formation [32] . zikv exploits the er dynamic characteristic and remodels the er structure to generate virus-induced structures, vesicles (ve), vesicle packet (vp), convoluted membrane (cm) and zippered er (zer), for the benefit of virus replication. blue and green box depicts schematic representation of host and viral factors involved in vesicle and virus particle (vi) formation, respectively. zikv ns4a utilizes the host reticulon 3.1a (rtn3.1a) to facilitate membrane curvature during vesicle invagination into the er lumen (blue box). viral genome replication takes place within this vesicle. neosynthesized viral rna genome is released into the cytosol and could undergo either translation, virus particle assembly, or another round of genome replication. virus particle assembly takes place in apposed er leaflet. separate zikv ns2a independently recruits viral genome rna, ns2b-ns3, and unprocessed c-prm-env complexes, and subsequently congregates at the virus particle assembly site through ns2a oligomerization (green box). once assembled, ns2b-ns3 proteolytical activity cleaves the recruited c-prm-env complex to generate individual capsid, prm and env protein. cleaved capsid protein interacts with capsid-dense lipid droplets and viral rna to generate nucleocapsid core followed by env and prm proteins encapsulation of the nucleocapsid core invagination of zikv-induced vesicles into the er lumen is mediated through the host reticulon 3.1a (rtn 3.1a) that facilitates er membrane curvature (fig. 1 , blue box) [38] . silencing of rtn3.1a impairs ns4a protein stability and results in significant reduction of virusinduced vesicles and virus replication [38] . typically, these vesicles contain the virus' replication complex and therefore carries out the virus rna synthesis or viral replication [32, 36] . following viral replication, neosynthesized viral genome rna is released into the cytosol through the vesicle's narrow channel, where it would consequently either undergo another round of genome replication, translation or virion assembly [17, 30] . in the latter case, zikv virion assembles and buds into the er lumen directly apposed the narrow pore of the replication vesicle (fig. 1 , green box) [32] . although detailed mechanistic insights of virion assembly remains uncertain, recent study identified that zikv ns2a independently recruits viral (+)-ssrna, ns2b-ns3, and unprocessed c-prm-env complexes to the virus particle assembly site, possibly through ns2a oligomerization [39] . the same study postulates that virus particle assembly proceeds through ns2b-ns3 protease complex cleaving the recruited c-prm-env complex. cleaved capsid protein form nucleocapsid core with the viral rna and capsid-dense lipid droplet; and subsequently, it is encapsulated by prm and env proteins, which leads to eventual virion budding into the er lumen [39] . ultrastructural analyses within the dilated er lumen consistently identified virus particles arranged in a two-dimensional paracrystalline array [32, 37] . this array is located in close proximity to the replication vesicles and is therefore largely comprised of fully assembled virions and viral-like particles (enveloped virions without viral genome) [37] . moreover, zikv-infected huh7 cells also exhibited a unique structural alteration of collapsed er cisterna or more commonly known as zippered er (zer) (fig. 1 ) that reminisce the observation reported in avian infectious bronchitis virus (ibv) infection, a (+)-ssrna virus from the coronaviridae family [32, 40] . this finding presents the first identification of such structure among flavivirus-infected cells and has only been observed in zikv-infected huh7 cells, suggesting that cell-typespecific factors are possibly required to generate this structure [32] . the role of zer in zikv infection is unclear; however, zer are frequently found adjacent to vps suggestive of functions associated with vesicles formation, which is consistent with observations made in ibvinfected cells [32, 40] . nonetheless, this unique finding warrants further research to elucidate the mechanistic details and biological relevance of the zer. in addition to the direct modulation of the er structure, zikv also induces the formation of viroplasm-like structures, which is incidentally the first reported observation of such structure in flavivirus infection [41] . contrary to other zikv-induced structures, viroplasms are cytoplasmic inclusion of viral replication components and various relevant host factors associated with virus replication that are formed through reorganization of cell membrane or cytoskeletal elements [42] . these structures are significantly larger than vps and can measure up to 500 nm in diameter [41] . viroplasms are primarily located in the perinuclear region and in close proximity to the er, mitochondria, and microtubules to facilitate viral genome replication [41] . the spatial segregation of virus-induced enclosed structures (viroplasms and vesicles) is postulated to create a subcellular microenvironment concentrated with factors required for virus genome replication, and simultaneously provides physical barriers against rna nucleases and the host immune responses [32, 42] . the latter, in particular, is consistent with an investigation on wnv that virusinduced structures confer protection, albeit partially, against the host immune mechanism [43] . in summary, zikv and several zikv proteins localize to the er and eventually leads to the remodeling of the reticular architecture and exploitation of the organelle's unique characteristics. these modifications create an ideal environment for viral genome replication, but simultaneously introduce additional burden and stress on the host cell leading to the activation of several cellular responses, which will be discussed in the following sections. one of the fundamental roles of the er is the folding of secreted and membrane proteins, which depends on a multitude of factors including supply of atps, stable ca 2+ concentration, and a balanced redox environment [44] . disruption of this specialized environment within the er, whether through glucose deprivation or viral infection, may lead to overwhelming accumulation of misfolded and unfolded proteins-a condition described as er stress [45] . under normal conditions, glucose regulated protein 78 (grp78), one of the er molecular chaperones, binds to er stress sensor transmembrane proteins: protein kinase rna-activated (pkr)-like er resident kinase (perk), activating transcription factor 6 (atf6), and inositolrequiring enzyme 1 (ire1). in the presence of misfolded or unfolded protein, grp78 will bind to exposed hydrophobic residues of these proteins to induce proper protein folding [45, 46] . accordingly, during er stress, the accumulation of misfolded or unfolded proteins saturates the free pool of grp78 and titrates the bound grp78 away from the stress sensors leading to activation of respective sensor's molecular pathways [45] . these molecular initiatives and their corresponding downstream effects are collectively known as the unfolded protein response (upr). this evolutionarily conserved countermeasure induces pro-survival responses including global arrest of protein synthesis, upregulation of protein degradation factors, and enhanced protein folding capability [45] . however, under severe er stress conditions, upr will instead trigger apoptosis. following zikv infection, the accumulation of misfolded virus polyproteins in the er lumen overwhelms the er protein-folding capacity leading to er stress and triggers the activation of the upr (fig. 2) [47] . additional evidence of er stress and upr activation are demonstrated in the elevated expression of grp78 and other chaperones such as calnexin, calreticulin, and protein disulfide isomerase (pdi) in zikv-infected neural cells in vitro and in vivo [35, 48, 49] . increased expression of these er stress markers was accompanied by impaired indirect neurogenesis and microcephalic phenotype in mice [49] . this finding is consistent with a previous report wherein the induction of upr in cerebral apical progenitor cells tipped neuronal differentiation towards direct neurogenesis at the expense of indirect neurogenesis, leading to depleted intermediate progenitors, reduced overall cortical neuron output, and diminished cerebral volume, which ultimately caused microcephaly in vivo [50] . in addition to elevating the expression of er chaperone proteins, zikv has been shown to induce upr by activating the stress sensors as summarized below (fig. 2) . atf6 is a type ii er transmembrane protein that contains a transcription activation domain (tad) on its cytosolic-facing n-terminal and two independent golgilocalization signals (gls) on its luminal-facing cterminal [51] . following the dissociation of grp78 that unmasks the gls, atf6 translocate to the golgi apparatus and undergoes sequential proteolytic processing by fig. 2 zikv-induced er stress initiates host cell unfolded protein response (upr). zikv infection induces er stress due to the increased amount of unfolded/misfolded viral (red strand) and host cell (grey strand) protein aggregates in the er lumen. the accumulation of these partially processed proteins leads to the overwhelming demand of correct protein folding. to facilitate protein folding, grp78 (orange) dissociates from the stress sensors (ire1, atf6, and perk) and binds to the misfolded/unfolded proteins. expression of other chaperones is also elevated to address the overwhelming protein folding demand during zikv infection. binding of misfolded protein to perk (green) activates the sensor and triggers phosphorylation of eif2 that in turn stimulates 1) global translational block except for selective mrna involved in upr, and 2) formation of stress granules. however, zikv bypasses global translation block and inhibits stress granules formation, but the mechanistic details of these viral interference are still unclear. activation of atf6 (yellow) promotes the protein proteolytical processing in the golgi apparatus into atf6n, atf6n nuclear translocation, and expression of upr target genes including xbp1. activated ire1 (purple) splices xbp1 transcript which produces an active transcription factor (xbp1s) that stimulates expansion of the er volume, and expression of chop and erad factors such as edem-1. alternatively, ire1 can trigger ask1-p38 mapk pathway that enhances chop apoptotic activities and promotes other apoptotic-related activities under severe er stress condition. blue pointed arrows denote activation, blue blunt-end arrows denote inhibition, and red pointed arrows denote increased expression/activity site-1 and site-2 proteases, liberating tad-containing atf6 n-terminal (atf6n) [51] . subsequently, atf6n enters the nucleus and binds to er stress response elements in the promoter region of upr-target genes, including x-box binding protein 1 (xbp1) transcription factor, a downstream effector of ire1 stress sensor pathway, and grp78 to autoregulate the upr [51] [52] [53] . zikv infection has been shown to activate the upr via atf6, where atf6 proteolysis and atf6n nuclear translocation were induced in vitro and in vivo [48] . ire1 is a single-pass transmembrane protein. the protein's er luminal domain serves as a stress sensor, whereas the cytosolic domain is sub-divided into kinase and ribonuclease (rnase) domains. under er stress, grp78 releases from ire1 luminal domain and binds onto the misfolded/unfolded proteins. this dissociative event frees the luminal domain and in turn, permits binding of the misfolded/unfolded protein onto the liberated domain [54, 55] . consequently, ire1 oligomerizes at its luminal domain, which brings the kinase domains in close proximity leading to auto-phosphorylation of the kinase domains [56] . the phosphorylation event at the activation loop is postulated to induce conformational changes of the rnase domain that permit a more efficient binding of rna substrates [57] . activated ire1 cleaves off 26 nucleotides from xbp1 transcripts, resulting in a translational frameshift to produce a potent transcriptional activator (xbp1s) that elevates the expression of upr-target genes to promote protein folding, processing, and secretion [53, 58] . xbp1s also modulates the expression of factors involved in er-associated protein degradation (erad), a cellular process of eliminating aberrant proteins through retro-translocation and proteasomal degradation [45] . alternatively, ire1 protein can also mediate er stressinduced apoptosis following chronic stress damage by triggering activation of apoptotic signaling kinase 1 (ask1), which consequently activates p38 mitogen-activated protein kinase (p38 mapk) and jun n-terminal kinase (jnk) leading to apoptosis [59] . jnk mediates apoptosis primarily by inhibiting the anti-apoptotic b-cell lymphoma 2 (bcl-2) and activating the pro-apoptotic bim protein [59] , whereas p38 mapk promotes apoptosis by phosphorylating ccaat-enhancer-binding protein (c/ebp) homologous protein (chop) at serine residues 78 and 81 to enhance its transcriptional activity and induce apoptosis [60, 61] . chop is expressed downstream of all three er stress sensors, indicating overlapping upr pathways during severe er stress [62] . chop forms heterodimers with other c/ebp family transcription factors; therefore, the basic domain in chop renders the transcription factors incapable of binding to their respective dna binding sites, which reduces the expression of their target proteins including bcl-2 [63] . chop also contains a tad and thus, can bind to a unique sequence to induce the expression of target genes including bim [63, 64] . zikv infection has been shown to promote xbp1 splicing and xbp1s translocation into the nucleus, leading to elevated expression of xbp1 downstream effectors such as er degradation-enhancing α-mannosidase-like 1 (edem-1) and chop, indicating the activation of the ire1 arm of the upr [48] . edem-1 is an enzyme involved in the erad process that recognizes and facilitates retrotranslocation of misfolded proteins to the cytosol for subsequent proteasomal degradation and thus, alleviating stress damage [65] . instances of aggravated er stress were also demonstrated in zikv-infected cells, which lead to elevated expression of chop protein and initiation of erstress-induced apoptosis [49] . pharmacological intervention using ire1 inhibitor, 4μ8c, prevented microcephaly and impaired zikv replication in mouse fetal brains [49] . modulation of ire1-ask1 pathway during zikv infection is still unclear. previous study reported that ectopic expression of xbp1 promotes expansion of the er, a visual sign often manifested in response to er stress [66] . this morphological change is hypothesized to reduce the local concentration of misfolded/unfolded proteins and accommodate the overwhelming demand for protein folding-a significant portion of which is neosynthesized viral polyproteins [47, 67] . consistent with increased expression of xbp1, ultrastructural characterization of zikv-infected cells reported significant er enlargement extending across the cytoplasm and is related to zikv-induced er stress [32, 35, 36, 47] . similar to ire1, release of grp78 proteins enable misfolded/unfolded proteins binding onto exposed perk luminal domain leading to oligomerization and autophosphorylation of perk protein to activate its downstream pathway [55, 68] . perk phosphorylation, in turn, induces eif2α phosphorylation (p-eif2α) that mediates three interlinked upr mechanisms: expansion of protein folding capacity, suppression of nascent protein production and induction of apoptosis in the event of severe stress [69] . phosphorylation of α subunit in the eif2 complex hinders the assembly of preinitiation complex (pic) by blocking the activity of eif2b guanine exchange factor, leading to global suppression of mrna translation [69] . however, selected mrnas, typically transcripts for upr machinery, can bypass this translational block [69] . interestingly, viruses have evolved various mechanisms to overcome this translational block, but the mechanism of this process in zikv infection is unclear. recent analyses of zikv infection in vitro and in vivo reported a substantial increase of eif2α phosphorylation and elevated expression of several downstream perk effectors such as atf4, atf3, chac1, and chop [48, 49] . importantly, intracerebroventricular administration of pharmacological perk inhibitor in zikv-infected mice restored appropriate neurogenesis balance and rescued infected mouse embryos from microcephalic phenotype. however, unlike ire1 inhibitor, perk inhibitor did not affect zikv replication [49] . perk inhibitor also prevented microcephaly in placental zikv inoculation model, which mimics natural zikv vertical transmission [49] . in summary, zikv localizes to the er for viral genome replication. these additional transcriptional and translational processes impart significant burden on the er leading to er stress and upr induction. during the early stage of upr, upr effectors elicit adaptive responses to mitigate er stress. these responses include the upregulation of chaperones and protein processing enzymes to promote and rectify protein folding, induction of erad to degrade misfolded/unfolded proteins, global translational arrest, and activation of autophagy and/or reticulophagy. beside upr, er stress also concomitantly induces several other cellular processes. specifically, stress granules assembly and er autophagy impart fundamental importance in regulating translational arrest and er homeostasis, respectively. zikv have been reported to subvert these processes to allow the progression of viral replication. er stress signal can be transmitted to other cellular components to rescue cell survival. a clear example to illustrate stress signal transmission is the generation of stress granules (sgs) in the cytoplasm to stall initiation of translation [70] . sgs assembly are generally triggered through two distinct categories of mechanisms: 1) eif2α-independent and 2) eif2α-dependent mechanisms [71] . in particular, the latter category, or more specifically eif2α-phosphorylation, is mediated by pkr, perk, general control non-derepressible-2 (gcn2), and heme-regulated inhibitor kinase (hri) in response to diverse cellular stress signals [71] . sgs are ribonucleoprotein (rnp) structures primarily composed of non-translating mrnas, stalled translation initiation complexes, and rna-binding proteins [70] . the formation and components of sgs are reviewed in details here [71] . sgs assembly during global translational arrest negatively impact viral genome translation as the sgs reduce the accessibility of translational machinery complexes [72] . following the relief of translation suppression, the sgs are disassembled via several mechanisms, one of which involves eif2α dephosphorylation by growth arrest and dna-damage-inducible 34 (gadd34) protein [73] . this allows the pic to resume protein translation at the surface of the sgs, resulting in sgs shrinkage and disappearance [71] . viruses have adopted several mechanisms to repress the assembly of stress granules and utilize sg protein components for viral polyprotein synthesis instead [74] . for example, herpes simplex virus genome encodes γ 1 34.5 protein that functionally mimics the activity of gadd34 and directs the dephosphorylation of eif2α [75] . alternatively, coronaviruses repress the assembly of stress granules by asserting an inhibitory effect on pkr activation and upregulating the expression of gadd34 [76] . likewise, zikv also suppresses the formation of stress granules in favor of virus replication by upregulating the expression of gadd34 [77] . consistent with this finding, pharmacological inhibition of gadd34-mediated eif2α dephosphorylation rescued sgs assembly and decreased zikv particles production [77] . additionally, another study reported that zikv proteins, namely capsid, ns3, ns2b-ns3, and ns4a proteins, suppressed sgs assembly; however, this modulation was observed in an eif2α-independent manner [78] . zikv capsid inhibited sgs assembly by forming stable complexes with sg core proteins, ras gtpase-activating proteinbinding protein 1 (g3bp1) and caprin-1, but not with t-cell-restricted intracellular antigen 1 related (tiar) protein [78] . zikv utilized these sg core proteins for viral protein and virion production. beside regulating eif2α phosphorylation and hijacking key sg proteins, rna viruses were reportedly capable of interfering sgs assembly through cleaving and redistributing sgs nucleating factors [74] . it was previously reported that zikv ns2b-ns3 protease could cleave host antiviral factors to impair intrinsic host defense; intriguingly, zikv did not mediate the cleavage of sg factors even though sgs assembly was affected by zikv ns3 and ns2b-ns3 protease [77] [78] [79] [80] [81] . nonetheless, zikv infection was found to facilitate the redistribution of tiar to viral replication sites, which correlates to viral replication output [77, 78, 81] . hence, it is unclear how zikv ns3, ns2b-ns3, and ns4a proteins block sgs formation and whether these viral proteins affect eif2α phosphorylation. interestingly, a recent study also identified a key sg-interacting protein, human antigen r, exhibits substantial anti-zikv effect potentially by destabilizing the zikv rna [81] . as previously discussed in section 4, the induction of er stress initiates several adaptive countermeasures such as upregulation of upr pro-survival factors and er enlargement to repair stress-induced damages and restore normal cellular functions. interestingly, initial studies in mammalian and yeast cells revealed that a fraction of cells also contain autophagosome-like structures following er stress induction [82, 83] . these autophagic vacuoles expel selectively excised er membrane containing aberrant protein aggregates [84] . this process, known as reticulophagy, is an additional mechanism that occurs in parallel with upr to regulate er volume and homeostasis [85] . autophagy, including reticulophagy, is also a host innate defense mechanism as these autophagosomes have been shown to incorporate viral proteins for degradation [86] . reticulophagy, also known as er-phagy, is mediated by er-phagy receptors such as family with sequence similarity 134 member b (fam134b) and reticulon-3 (rtn3) proteins that reside on the er membrane. these autophagy receptors sequester er fragments via its lc3-interacting-region (lir) domain interaction with autophagosomal-presenting microtubuleassociated protein 1 light chain 3 (map 1lc3) [87] . similar to other er-shaping proteins, fam134b also possesses a reticulon homology domain (rhd), a motif that promotes membrane curvature [87] . in zikv-infected cells, depletion of fam134b protein renders notable er expansion and significant upregulation of zikv replication activity [80] . this is due to the proteolytic activity of zikv ns2b-ns3 that cleaves exposed rhd located on fam134b protein, which impedes the oligomerization of fam134b proteins and consequently, prevents er membrane excision and reticulophagy from taking place [80] . this efficiently blocks host cell innate defense mechanism from degrading zikv proteins. to summarize, zikv virus bypasses the upr by inhibiting stress granules assembly and reticulophagy to ensure continuous viral protein translation and virion production while simultaneously protecting the virus from host cell defense mechanisms. prolonged er stress exacerbates the stress condition and leads to the activation of another arm of the upr: cell death. countless studies showed that zikv infection causes cell death through several cell death mechanisms such as paraptosis and apoptosis [12, 88] apoptotic events in zikv-infected cells occur through both the intrinsic and extrinsic pathways based on the activation of pathway-specific caspase-9 and caspase-8, respectively [88, 89] . interestingly, zikv env protein is sufficiently capable to induce pro-apoptotic expression profile that represents intrinsic apoptosis including elevated expression of tumor protein p53, which correspondingly mediates the expression and activity of its downstream targets by suppressing anti-apoptotic bcl-2 and facilitating expression of pro-apoptotic bcl-2associated x (bax) [90] . this modulation consequently reduces mitochondrial membrane permeability; thus, aiding the release of cytochrome c, formation of apoptosome, and initiation of caspase cascade, which eventually leads to cell death [62] . additionally, increased level of several apoptotic markers, such as tumor necrosis factor alpha and receptor-interacting protein 1, in zikv-positive microcephalic neural specimens are suggestive of extrinsic apoptotic activation [91] . as previously mentioned, er stress elevated the expression of chop to initiate apoptosis in zikvinfected cells [48, 49] . prolonged er stress could also trigger non-apoptotic cell death as demonstrated by an investigation using time-lapse and electron microscopy that revealed the formation of extensive erderived vacuoles within the cytoplasm of zikvinfected cells, which eventually resulted in paraptosislike death [28] . this observation is consistent with earlier report that showed cytoplasmic vacuolization in zikv-infected human skin biopsy specimens [13] . vacuole formation was inhibited with class i pi3k/akt inhibitor treatment. more importantly, pan-caspase inhibitor, zvad-fmk, only slightly rescued cell survival but did not affect vacuolization [28] , implying that certain zikv strains, namely hd78788, pf13 and nc14, induce cell death primarily through paraptosislike death. the role of pi3k/akt pathway in zikvinduced cell death was verified by a recent study that reported treatment with ar-12, a celecoxib derivative kinase inhibitor, significantly inhibited the replication of zikv in a129 mice and improved mice survival mainly through akt down-regulation [92] . zikv, like other viruses, is an intracellular parasite that largely depends on the host biosynthetic, energetic and structural resources for successful viral replication and propagation. in particular, exploitation of these resources is manifested through the manipulation of the endoplasmic reticulum architecture and the processes that take place within or in proximity to the er as summarized in fig. 3 . briefly, zikv infection leads to structural changes of the er such as er enlargement due to the accumulation of misfolded/unfolded zikv proteins (fig. 3d ) and the formation of convoluted membranes, vesicle packets, zippered er, and viroplasm-like structures for viral rna replication (fig. 3e) . the accumulation of misfolded/unfolded proteins induce er stress and triggers the upr (fig. 3f) . in parallel, er stress also initiate several response mechanisms such as stress granules assembly to impede global protein translation (fig. 3g ) and reticulophagy to remove damaged er (fig. 3h ). however, zikv are able to bypass these processes through various strategies, which eventually lead to paraptosis-like cell death (fig. 3i) . although the number of zikv cases have subsided, zikv remains a significant threat due to the sporadic and unpredictable nature of its outbreak. in addition, zikv shares the same vectors with another widespread flavivirus, denv [93] , which is projected to increase exponentially in the future [94] . thus, the potential re-occurrence of outbreaks, coupled with devastating neurological complications, warrants for extensive research to completely understand the virus pathophysiology for antiviral drug development. this is effectively demonstrated by several studies included in this review that employ multiple omics technologies to identify and target er-associated zikv dependency factors and er stress sensors. this strategy could pave the way in developing anti-zikv drugs. other cell receptors such as dc-sign can also serve as zikv entry receptor. b) acidic endosomal microenvironment facilitates endosomal fusion thereby, releasing zikv rna genome that is immediately bound onto cytosolic ribosomes. c) translation of the viral polyprotein (orange) likely initiates in the cytosol and continues with cotranslational insertion into the er membrane via sec61 translocon complex (blue). post-translational processed zikv proteins induce er structural alterations such as d) enlargement of the er, and e) formation of convoluted membrane (cm), vesicle packets (vps), zippered er (zer) and viroplasms. simultaneously, accumulation of misfolded and unfolded zikv proteins in the er lumen results in f) the induction of er stress and thus, initiates the host unfolded protein response (upr) and other intrinsic defense mechanisms including reticulophagy and stress granule formation. zikv proteins g) impede formation of stress granules and h) inhibit reticulophagy to sustain viral replication. zikv infection also induces i) er-derived cytoplasmic vacuolization, a visual characteristic of paraptosis. blue pointed arrows denote activation, blue blunt-end arrows denote inhibition. ve, virus-induced vesicle; vi, virus particle zika virus. i. isolations and serological specificity zika virus zika virus outbreak on yap island, federated states of micronesia zika virus infection complicated by guillain-barre syndrome -case report guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study pan american health organization / world health organization. epidemiological alert: increase of microcephaly in the northeast of brazil zika virus and microcephaly: why is this situation a pheic? zika virus and birth defects -reviewing the evidence for causality detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study zika virus associated with microcephaly zika virus impairs growth in human neurospheres and brain organoids zika virus disrupts neural progenitor development and leads to microcephaly in mice biology of zika virus infection in human skin cells axl mediates zika virus entry in human glial cells and modulates innate immune responses infection by zika viruses requires the transmembrane protein axl, endocytosis and low ph ph dependence of zika membrane fusion kinetics reveals an off-pathway state zika virus structure, maturation, and receptors role of host cell secretory machinery in zika virus life cycle molecular cloning and characterization of the genes encoding the proteins of zika virus global interactomics uncovers extensive organellar targeting by zika virus zika ns2b is a crucial factor recruiting ns3 to the er and activating its protease activity zika virus targets human stat2 to inhibit type i interferon signaling zika virus ns3 is a canonical rna helicase stimulated by ns5 rna polymerase a crispr screen defines a signal peptide processing pathway required by flaviviruses identification of zika virus and dengue virus dependency factors using functional genomics the er membrane protein complex promotes biogenesis of dengue and zika virus nonstructural multi-pass transmembrane proteins to support infection predicting zika virus structural biology: challenges and opportunities for intervention zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells an orthogonal proteomic survey uncovers novel zika virus host factors genetic dissection of flaviviridae host factors through genome-scale crispr screens a small-molecule oligosaccharyltransferase inhibitor with pan-flaviviral activity ultrastructural characterization of zika virus replication factories endoplasmic reticulum: the favorite intracellular niche for viral replication and assembly the endoplasmic reticulum: structure, function and response to cellular signaling cytoarchitecture of zika virus infection in human neuroblastoma and aedes albopictus cell lines zika virus induced cellular remodelling a novel sheet-like virus particle array is a hallmark of zika virus infection the host protein reticulon 3.1a is utilized by flaviviruses to facilitate membrane remodelling zika virus ns2a-mediated virion assembly infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes structural investigation of c6/36 and vero cell cultures infected with a brazilian zika virus virus factories, double membrane vesicles and viroplasm generated in animal cells west nile virus-induced cytoplasmic membrane structures provide partial protection against the interferon-induced antiviral mxa protein protein folding and modification in the mammalian endoplasmic reticulum the role of endoplasmic reticulum stress in human pathology affinity panning of a library of peptides displayed on bacteriophages reveals the binding-specificity of bip crosstalk between endoplasmic reticulum stress and the unfolded protein response during zika virus infection zikv infection activates the ire1-xbp1 and atf6 pathways of unfolded protein response in neural cells stress-induced unfolded protein response contributes to zika virusassociated microcephaly a dynamic unfolded protein response contributes to the control of cortical neurogenesis er stress regulation of atf6 localization by dissociation of bip/grp78 binding and unmasking of golgi localization signals role of the unfolded protein response regulator grp78/bip in development, cancer, and neurological disorders xbp1 mrna is induced by atf6 and spliced by ire1 in response to er stress to produce a highly active transcription factor unfolded proteins are ire1-activating ligands that directly induce the unfolded protein response endoplasmic reticulum stress sensing in the unfolded protein response structure of the ire1 autophosphorylation complex and implications for the unfolded protein response phosphoregulation of ire1 rnase splicing activity from endoplasmic-reticulum stress to the inflammatory response er stress-induced cell death mechanisms stress-induced phosphorylation and activation of the transcription factor chop (gadd153) by p38 map kinase attenuation of chop-mediated myocardial apoptosis in pressureoverloaded dominant negative p38alpha mitogen-activated protein kinase mice the c/ebp homologous protein (chop) transcription factor functions in endoplasmic reticulum stress-induced apoptosis and microbial infection stress-induced binding of the transcriptional factor chop to a novel dna control element er stress triggers apoptosis by activating bh3-only protein bim edem1 recognition and delivery of misfolded proteins to the sel1l-containing erad complex xbp1, downstream of blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response the luminal domain of the er stress sensor protein perk binds misfolded proteins and thereby triggers perk oligomerization translational control in stress and apoptosis eukaryotic stress granules: the ins and outs of translation mechanistic insights into mammalian stress granule dynamics translation inhibition and stress granules in the antiviral immune response growth arrest and dna damageinducible protein gadd34 targets protein phosphatase 1 alpha to the endoplasmic reticulum and promotes dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 viral regulation of rna granules in infected cells the gamma(1)34.5 protein of herpes simplex virus i complexes with protein phosphatase 1 alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded rna-activated protein kinase inhibition of protein kinase r activation and upregulation of gadd34 expression play a synergistic role in facilitating coronavirus replication by maintaining de novo protein synthesis in virus-infected cells zika virus inhibits eif2 alphadependent stress granule assembly zika virus hijacks stress granule proteins and modulates the host stress response species-specific disruption of sting-dependent antiviral cellular defenses by the zika virus ns2b3 protease dengue and zika viruses subvert reticulophagy by ns2b3-mediated cleavage of fam134b zika virus subverts stress granules to promote and restrict viral gene expression autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response differential effects of endoplasmic reticulum stress-induced autophagy on cell survival retention of mutant alpha(1)-antitrypsin z in endoplasmic reticulum is associated with an autophagic response er-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery autophagy in immunity and inflammation regulation of endoplasmic reticulum turnover by selective autophagy zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells loss of the tam receptor axl ameliorates severe zika virus pathogenesis and reduces apoptosis in microglia zika virus envelope protein induces g2/m cell cycle arrest and apoptosis via an intrinsic cell death signaling pathway in neuroendocrine pc12 cells correlation between apoptosis and in situ immune response in fatal cases of microcephaly caused by zika virus the celecoxib derivative kinase inhibitor ar-12 (osu-03012) inhibits zika virus via downregulation of the pi3k/akt pathway and protects zika virus infected a129 mice: a host-targeting treatment strategy an overview of mosquito vectors of zika virus the current and future global distribution and population at risk of dengue publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions mimr and ask contributed to the topic conception and writing of the manuscript together. mimr prepared the figures. mimr, ask, nnr and ry edited and revised the manuscript. all authors read and approved the final manuscript. this work was supported by the grant [frgs fp007-2017a] from the ministry of higher education, malaysia.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests.received: 3 september 2019 accepted: 14 january 2020 key: cord-296794-ml2luc1t authors: sollner, johannes; grohmann, rainer; rapberger, ronald; perco, paul; lukas, arno; mayer, bernd title: analysis and prediction of protective continuous b-cell epitopes on pathogen proteins date: 2008-01-07 journal: immunome res doi: 10.1186/1745-7580-4-1 sha: doc_id: 296794 cord_uid: ml2luc1t background: the application of peptide based diagnostics and therapeutics mimicking part of protein antigen is experiencing renewed interest. so far selection and design rationale for such peptides is usually driven by t-cell epitope prediction, available experimental and modelled 3d structure, b-cell epitope predictions such as hydrophilicity plots or experience. if no structure is available the rational selection of peptides for the production of functionally altering or neutralizing antibodies is practically impossible. specifically if many alternative antigens are available the reduction of required synthesized peptides until one successful candidate is found is of central technical interest. we have investigated the integration of b-cell epitope prediction with the variability of antigen and the conservation of patterns for post-translational modification (ptm) prediction to improve over state of the art in the field. in particular the application of machine-learning methods shows promising results. results: we find that protein regions leading to the production of functionally altering antibodies are often characterized by a distinct increase in the cumulative sum of three presented parameters. furthermore the concept to maximize antigenicity, minimize variability and minimize the likelihood of post-translational modification for the identification of relevant sites leads to biologically interesting observations. primarily, for about 50% of antigen the approach works well with individual area under the roc curve (aroc) values of at least 0.65. on the other hand a significant portion reveals equivalently low aroc values of < = 0.35 indicating an overall non-gaussian distribution. while about a third of 57 antigens are seemingly intangible by our approach our results suggest the existence of at least two distinct classes of bioinformatically detectable epitopes which should be predicted separately. as a side effect of our study we present a hand curated dataset for the validation of protectivity classification. based on this dataset machine-learning methods further improve predictive power to a class separation in an equilibrated dataset of up to 83%. conclusion: we present a computational method to automatically select and rank peptides for the stimulation of potentially protective or otherwise functionally altering antibodies. it can be shown that integration of variability, post-translational modification pattern conservation and b-cell antigenicity improve rational selection over random guessing. probably more important, we find that for about 50% of antigen the approach works substantially better than for the overall dataset of 57 proteins. essentially as a side effect our method optimizes for presumably best applicable peptides as they tend to be likely unmodified and as invariable as possible which is answering needs in diagnosis and treatment of pathogen infection. in addition we show the potential for further improvement by the application of machine-learning methods, in particular random forests. the applicability of peptides for the generation of preventive vaccines, therapeutics and diagnostics is an actively investigated field. although historically disfavored the application of peptides in vaccine design is currently experiencing a renaissance [1] . while the focus is often on tcell responses especially the generation of b-cell responses is of relevance against certain pathogens such as hiv to prevent initial infection [2] . it is thus not surprising that since the early days of computational biology scientists have attempted to predict the relevance of protein domains and peptides in several areas of application. initial hallmarks of the field are represented, among many others, by work of hopp and woods [3, 4] . during the following and more recent years various methods and problems concerning the prediction of continuous b-cell epitopes have been proposed [5] [6] [7] [8] [9] [10] [11] [12] . recently the usability of amino acid scales for the prediction of b-cell epitopes has been profoundly questioned [13] and common standards regarding the validation of epitope predictions have been discussed [14] . generally, b-cell antigenicity predictions should probably be understood as a measure of the likelihood to develop antibodies against a particular determinant or part of a surface, rather than another. in addition, most proposed classifiers of continuous epitopes are ultimately a composite of accessibility and charge-interaction potential prediction with a strong focus on delivering a few experimentally applicable peptides rather than an overall complete probability distribution for raising antibodies. in addition, continuous epitopes make up an undefined but presumably small part of the complete "epitope space" of an antigen. this even so when assuming distinct epitopes rather than a continuous surface and accepting dominant continuous elements of structural epitopes as continuous epitopes. in this study we extend previous work by investigating a subgroup of continuous b-cell epitopes, namely protective continuous epitopes. this aspect has to the best of our knowledge not been systematically tackled so far. from a biological point of view several principles should govern the availability and evolutionary behavior of protective amino-acid sites on proteins. one of the questions we ask is whether these principles or constraints lead to signals which can be used for predicting or rather detecting candidate epitopes. we assume that an antibody (and hence it's epitope) is protective because the function of the antigen (target protein) is inhibited and the activity of the organism is thus reduced. or alternatively because immunological processes are activated leading to the destruction of the organism. the prior (protectivity class i) might most likely be expected in adhesion molecules or pathogenicity factors like matrix degrading proteases and toxins. the second category (protectivity class ii) would primarily refer to downstream events of antibody induced complement activation such as pore formation and opsonization leading to phagocytosis. it can be considered likely that the two mechanisms would often lead to differently characterized epitopes. consequently as different functional constraints can be expected separate strategies for detecting them may be required. basically any protein of high expression and density on the surface with at least one good epitope can be the target of class ii protectivity. we define a "good" epitope as a surface area of high interaction potential, shape complementarity to the basic layout of an antibody [15] and dissimilar to self. predicting that class therefore also requires to assess (or estimate) the density of a protein on the cell during pathogenesis, optimally experimentally or by inference from related organisms. a practically applicable continuous epitope could then be any exposed, possibly evolutionarily highly variable loop with an amenable antigenicity and solvent-accessibility profile. class ii protectivity may often be comparably straightforward to predict as soon as a target protein has been identified because selection of high scoring b-cell epitope scores often seems to be relatively straightforward and selection routines primarily falter in the domain of suboptimal scores as has been indicated by sollner and mayer [9] . however, immunologically subdominant or even cryptic b-cell epitopes can be of special interest regarding protectivity and inter-strain cross-reactivity [16] and are sometimes consistently immuno-silent during natural infection [17, 18] . as a consequence we focus on conserved and therefore presumably functionally constrained epitopes without post-translational modifications which still exhibit amenable antigenicity scores. regarding the previous definition these epitopes may often fall into class i of protective continuous epitopes. such principles are primarily valid for pathogens already adapted to the host. organisms in the process to adapt to new hosts or receptors can undergo significant alterations in their antigenic structure as has been demonstrated for sars virus [19] [20] [21] and hiv [22] , respectively. we speculate that by means of functional constraints centers of biological activity can exert conserving pressure on closely associated potential epitopes while less relevant regions can be more variable. it may also be viable to suggest that conservation of posttranslational modification patterns may be different when comparing highly variable exposed loops and sites of functional relevance as modifications can play a major role in the masking of protective epitopes [23, 24] but may often be undesired near functional centers. as class ii protectivity is to a certain degree already approached by standard b-cell epitope prediction (the maximization of antigenicity) and on the other hand depends on a bioinformatically more elusive factor (expression levels of pathogen protein) we see reason to focus on the prediction of conserved, functionally constrained epitopes (class i protectivity). this work assesses in how far correlation between antigenicity, variability, post-translational modifications and protectivity/functional relevance can be put to use in a predictive model without the availability of 3d data. to compensate for the lack of 3d data multiple alignments of selected proteins are harnessed to derive information regarding the conservation of post-translational modification motifs as well as sequence variability per-se. we believe that understanding evolutionary "movement" of pathogen proteins allows insights into the importance of potential epitopes and we interpret such importance as indicator of protectivity. classification into presumably protective or non-protective epitopes is conducted using three independently determined parameters: predicted b-cell antigenicity, sequence variability and conservation of post-translational modification motifs. as described in the methods section used antigenicity, variability and motif-conservation scores are based on multiple-alignments i.e. each sequence contributes to a composite value. all values are determined within 10-mers which slide over the alignments and overlap by 9 amino-acids. b-cell antigenicity and sequence variability are averaged within these 10mers. we chose this size to use an intermediate between common assumptions about sizes of continuous epitopes (usually between 7 and 15 amino-acids). the maximum ratio of post-translational modifications over all constituent alignment columns is used within the same area. in other words, for the prior two the 10-mer values are calculated as the averages over the averages calculated from individual alignment columns. the latter seeks the maximal ratio of possibly modified amino-acids over all alignment-columns in the area because it presumably is most indicative of actual modifications. an overview over the major steps in the workflow has been highlighted in figures 1 and 2. logistic regression models based on pca of 505 amino acid propensity scales we examined regression models based on 505 amino acid propensity scales. as described in methods each of these scales characterizes amino acid residues regarding specific properties by assigning a value. from this representation, the information of 505 amino acid propensity scales was transformed into 19 principal components applying principal component analysis. based on these 19 principal components, a logistic regression model was derived. it is in the following mentioned as pca19. briefly, a dataset of 197 proteins was obtained by clustering all bcipep sequences with at least 30% identity. this step removed redundancies potentially biasing validation procedures. in a second step non-antigenic amino-acids were randomized (maintaining the original amino-acid composition) to avoid the misclassification of unknown epitopes. each amino-acid of those proteins was then parameterised using all of the described 19 components. validation on the training set by bootstrap analysis in combination with a logistic regression indicated an aroc value of 0.60. to obtain an unbiased impression of the performance of the pca19 classifier compared to an accepted gold standard such as abcpred [25] an independent validation-dataset published by blythe and flower was used. to make methods compatible abcpred predictions were run with standard settings except that the threshold was lowered to 0.1. scores reported for peptides by abcpred were assigned to each comprising amino-acid where larger values superseded the prediction of an overlapping peptide. aroc values were calculated. both methods performed close to random (as is not too astonishing concerning the findings by blythe et.al), with aroc values of 0.55 and 0.52 for pca19 and abcpred, respectively. these results are relativated later in this work when using only potentially relevant domains of a protein antigen, indicating systematic problems of the way b-cell epitope prediction validation is usually conducted. to assess the effect of domain accessibility filtering (masking) from protectivity prediction aroc values of the described linear parameter combination before and after filtering were compared. whereas the median aroc over all protein was determined as 0.56 before masking of presumably inaccessible trans-membrane or cytoplasmic domains it increased to 0.65 afterwards. while the aroc before masking is comparable to the one obtained by antigenicity prediction on the blythe and flower validation dataset the improvement to 0.65 strongly indicates the benefit of the procedure. domain accessibility filtering can be considered an aspect of fair evaluation in b-cell epitope classification as a whole as it can be assumed that the figure shows the first part of the overall workflow applied in this project figure 1 the figure shows the first part of the overall workflow applied in this project. the figure shows the second part of the overall workflow applied in this project figure 2 the figure shows the second part of the overall workflow applied in this project. continuous epitopes of accessible domains are more likely mapped or otherwise reported than others, besides the protectivity aspect. while it may be argued that inclusion of uniprot data into the process adds an aspect of human intervention we see that many data-sources can and are used for the annotation of putative ectodomains. among those are also experimental data, which is in itself not a problem for bioinformatical validation strategies as long as the validation dataset was not engineered to fit these data particularly well. that is not the case. the domain filter was simply built by manually collecting different datasources according to simple rules as we considered automated harvesting for 57 proteins and unnecessary effort. to evaluate the prediction of protective linear epitopes a new validation dataset was generated as described in methods and data. briefly, the iedb resource was queried for pathogen proteins with linear antibody determinants which lead to a biological effect upon interaction. in viral polyproteins commonly only the dominant surface proteins were used as could be expected for a newly sequenced pathogen without in detail knowledge. to limit predictions to candidate regions (i.e. possibly immunologically accessible regions) domain accessibility filtering was applied. to do so domain data regarding polyproteins, trans-membrane structures or signal-peptides (predicted or experimentally determined) were taken from uniprot [26] or predicted using the tmhmm v.2.0 [27] and signalp 3.0 servers [28] . domains which were not considered relevant for protective b-cell responses were intracellular domains of trans-membrane proteins, the first 10 amino-acids of a putative extra-cellular domain after a trans-membrane region and leader-peptides. briefly, proteins were completely scored for antigenicity/protectivity but amino-acid scores in regions outside domains assumed to be surface exposed were set to 0 thus leading to a generic classification as non-protective. masked amino-acid stretches were still considered for roc calculation to reflect the impact of the analysis as a whole. aroc measures were thus based on completely scored proteins partially set to 0. for each amino-acid of proteins in the protectivity dataset variability and percentage of modifications (ratio of sequences which carry a modification motif indicating this specific amino acid versus all aligned sequences) based on multiple alignments were calculated as described earlier. finally the average predicted antigenicity was calculated for each alignment column. each of the three sub-scores was then rescaled between 0 and 1 for easier comparability. to asses the power of score-combinations antigenicity, 1 -variability and 1 -ptm ratio were summed for all overlapping 10-mers where the score was assigned to the central amino-acid. see table 1 for aroc values of individual proteins using any of the three sub-scores alone as well as the linear combination (sum score). the last row of the table indicates the overall aroc when analyzing all unmasked aminoacids together. considering the aroc merged value (resulting from the concatenation of all putatively accessible domains after scoring) antigenicity alone outperforms all other scores, including the combined one. yet, although table 1 indicates overall better performance of antigenicity, the distribution of aroc values for individual proteins indicates a different view. table 2 shows that for the combined score fewer proteins fall in the very low aroc area (7 versus 10 are < = 0.35) whereas substantially more (30 versus 21) fall in the aroc area we considered good (> = 0.65). eight of the 57 proteins used for protectivity prediction are similar or identical to sequences used for training the pca19 classifier (as identified using blastp). to evaluate this bias/contamination the aroc value medians of truly independent versus dependent (contaminated) proteins has been listed in table 2 . interestingly and against expectation contaminated proteins underperform when measured by median compared to independent entries leading to the observation that no pre-emptive separation of shared sequences is necessary in this case. for an overview of aroc distributions see the histograms in figure 3. taken together 25 or 30 (44 or 53%) of 57 investigated proteins reveal aroc values > = 0.65 depending on whether contaminated proteins are neglected or not. this population is characterised by mean and median aroc values of 0.77 and 0.74, respectively. we interpret this as a roughly 50% probability that protectivity prediction will significantly enhance selection of peptides for the stimulation of protective immune responses for a particular protein, given that a relevant protein has been identified beforehand. we also want to point out that overall up to 65% of proteins show aroc values < = 0.35 or > = 0.65. as described in methods a "synthesis score" representing the number of peptides required for likely experimental success is a relevant readout concerning minimization of experimental cost. for vaccine design it is crucial to limit the number of synthesized peptides which enter experimental validation to a practically feasible amount. how many peptides can be synthesized depends on budget and resources as well as ethical considerations regarding the number of lab-animals, entities which are tied to the number of proteins (antigens) to be screened as well as available time. to provide such a measure we defined the size of peptides to be synthesized as 17-mers (a size commonly used by us) and the minimal overlap with protective epitopes to call it a hit as five amino-acids. selected peptides could overlap, but each new epitope had to be centered on a hitherto uncovered amino-acid. these central amino acids were selected by maximizing the described sum score of antigenicity + (1-variability) and (1-maximal modification ratio). following this procedure we determined how many peptides had to be selected per protein to provide a likely working selection for at least 50% of screened proteins. the presented combination method required six peptides to be selected compared to eight for random picking. this compares to five versus eight peptides when only the 30 proteins with aroc > = 0.65 were looked at. note that random picking of central-amino acids was also restricted to the regions not filtered out by domain-exclusion to warrant fairness in the comparison. proteins highlighted in bold are markedly homologous or identical to sequences used for the training of the pca19 antigenicity classifier. values derived from those proteins are denoted as "contaminated". the last five rows compare overall (global) classification performances of concatenated (merged) proteins as well as mean and median values between individual parameters and the combined score. note that "merged" does not mean averaged and is thus biased by the length of individual proteins. conserved, optimally unmodified, continuous and protective or at least functionally altering epitopes. to assess the relationship of antigenicity, variability and modifications pearson correlation-coefficients were calculated. after domain-filtering all distinct (non-overlapping) protective regions were analyzed separately to obtain an idea whether a common trend could be identi-fied. in summary, overall average and median correlation were not significant between antigenicity, variability and modification ratio (generally < = 0.30 and > = -0.18). for each combination a subset of epitopes showed high correlation, however. results have been summarized in table 3 . note that no pair of features shows significant positive or negative correlation for more than 30% of the total the figure shows the distribution of aroc values for protectivity predictions solely based on antigenicity, ptm (post transla-tional modifications), variability as well as the sum score of the three figure 3 the figure shows the distribution of aroc values for protectivity predictions solely based on antigenicity, ptm (post translational modifications), variability as well as the sum score of the three. note that like for the prediction of protectivity the used modification and variability scores (features) have been used as 1-value, so actual associations have to inverted as well. each field in the table contains a pair of numbers where the first and second indicate the number of epitopes associated with a pearson coefficient of < = -0.5 and > = 0.5, respectively. high numbers therefore indicate a strong degree of feature association. number of 110 considered epitopic regions. however, for all pairs of sub-scores strong positive and negative associations exist. in the case of antigenicity and variability more than 50% of analyzed epitopes show strong correlation between the two, but at practically equal numbers positive and negative association. this means that roughly 25% of epitopes are either markedly antigenic and conserved or non-antigenic and variable. furthermore another 25% are markedly antigenic and variable or nonantigenic and conserved. protective epitopes seem to be tendencially positively correlated considering antigenicity and conservation of motifs for posttranslational modification while variability and the evolutionary constraint index (eci) are often negatively associated, as could be expected. unfortunately no association between protein functional class and type of correlation could be detected (data not shown). preliminary experiments evaluating the potential power of the correlation coefficient as a new parameter for protectivity prediction indicated close to random performance (data not shown). the previously described observation that for about 65% of proteins either substantially good or bad predictive power can be seen suggests that the described 30% correlated epitopes are distinct sets depending on the parameter-combination. alternatively it may also be that weaker correlations than -0.5 and +0.5 can still positively influence aroc. an unweighted linear combination score is a simple way to combine parameters without optimizing a model, i.e. as a first approach strategy or if non independent optimization dataset exists. to extend this strategy, albeit without the option to analyse the unbiased effect on the entire protectivity dataset, machine-learning procedures were applied. in particular, to analyse the validity of using all three parameters as well as the relative merit of the sum-score the protectivity dataset was converted into a format amenable for machine learning procedures such as decision trees. as described in the methods section the entire protectivity set of 57 proteins was subjected to domain filtering to restrict to possibly accessible residues which were then compiled into the cml set. the class separation baseline of this set is 93,01%, due to the massive domination of non-protective residues. using weka standard settings a c4.5 decision tree achieved 96.12% separation on the same set. because relative merits of machine learning are difficult to assess on strongly biased datasets such as cml, balanced and stratified training and validation sets (mts and mvs, respectively) were randomly sampled from cml to make independent validation possible. using the training set (mts) and again applying c4.5 different parameter combinations were assessed. results can be seen in table 4 and figure 4 . the decision tree algorithm was applied because for single parameters this should basically be a search for the entropically optimal class-split whereas for parameter combinations also non-linear relationships (particularly the presence of distinct groups) can be captured. the table shows that each parameter contributes significantly and the best model comprises all three attributes as well as the sum-score as a fourth attribute. independent of variability alone shows the best class-separation among individual parameters. interestingly ptm alone yields no improvement over random classification while the combination with the remaining features significantly boots its impact. to assess the performance of a somewhat more sophisticated machine-learning technique validation using the validation set and cross-validation are compared in table 5 for a c4.5 tree and a random forest. obviously there is much potential for improvement over a single decision tree as the random forest achieves a class separation power of up to 83-84% compared to 70-73% using a single tree. analysis of the c4.5 tree generated from the validation set indicates the merit of all four attributes including the sum-score as an additional parameter as all four were incorporated by the algorithm (tree not shown). in addition the resulting tree is astonishingly complicated (109 leaves) when considering that only four parameters were used, again indicating the existence of different clusters. preliminarily, these groups may well be interpreted as distinct epitope signatures pointing towards different types of protective epitopes. in other words, different but recognizable epitope profiles should be considered. it also becomes obvious that a single unweighted combination score has its merit but is outperformed by variability alone and both of them by decision tree based multi-parameter models, at least in the stratified data representation. also, the sum-score proves to be an important additional component in the decision models as can be seen by the increase from 63% class separation to 70% class separation by inclusion of this extra feature. to exemplarily show how the described methods may be used for the prediction of continuous, protective epitopes a recently published relevant epitope on borrelia burgdorferi ospc has been used [29] . the protein was selected because it was the first to show up in a new iedb query for functionally altering epitopes and because it shows no significant homology to any protein in the protectivity dataset. figure 5 indicates the experimentally determined epitope (fat red bar) together with a masked signal peptide (fat gray bar) together with results from three predictive methods. the top-most plot shows the sum-score which does not obviously correlate strongly with the epitope. even after removal of isolated, positively predicted amino-acids (which can be considered as noise) both the c4.5 prediction and the random forest predict amino acids inside the 15-mer epitope. as the random forest performed best during validation it is used for the selection of five 17-mer peptides each centered on a cluster of a least two amino-acids. one of these peptides covers more than 50% of the protective epitope. while the proposed methods do not excel on this independently chosen protein five selected peptides may be enough to sufficiently cover the relevant site. as a remark it is also of interest that the consensus prediction of the c4.5 tree and the random forest would obviously reduce the selection to two peptides with the same epitope coverage. this work describes the generation of a novel b-cell classifier based on the logistic regression of amino acid parameters derived from principal component analysis of a commonly used parameter database. we extend the classical application of antigenicity classifiers from the prediction of continuous b-cell epitopes to the prediction of protectivity by introducing measured variability and predicted modification patterns into the concept. we could show that validation on the data-set by blythe and flower revealed close to random performance for both the gold standard abcpred as well as for pca19, although our method exhibited marginally better performance in comparison. this result is clearly relativated as we could show far better performance on a different dataset when considering only potentially relevant domains. in detail, to assess the benefit of excluding presumably irrelevant domains from the prediction of protective continuous determinants a manual selection of protein regions was created based on standard bioinformatical tools. for this selection we used a curated set of 57 proteins with known protective or otherwise functionally altering, continuous epitopes. on this compilation protectivity prediction using pca19 in combination with variability and modification likelihood performed significantly better after domain-accessibility filtering as measured by aroc values, while without filtering performance was comparable (although again slightly better) to antigenicity validation results on the blythe et.al validation-set. even when disregading eight proteins which contaminate the validation process due to similarity with the pca19 training data the difference persists and is even enhanced. another primary observation is the gross variance in aroc values observed for various proteins. approximately 65% of sequences exhibited aroc values < = 0.35 or > = 0.65, indicating a pattern substantially different from random noise. it may be wise to investigate the per-formance on a per-protein or per-epitope basis and to register how many percent of known, distinct sites were found with high reliability. that may help to avoid an averaging effect leading to the underestimation of the predictive power of classifiers. if a method performs well on every second protein or on certain epitopes that may be sufficient for many practical applications in the life-sciences field. this is especially so where high throughput is involved. interestingly no correlation between the functional category of a protein or pathogen class (bacterial or viral) and the aroc could be established, indicating a more complex situation than just two types of epitope each associated with a distinct functional class. by determining the number of theoretically required synthetic peptides to achieve satisfying protectivity in a vaccine or mab approach we conclude that up to six peptides would be required to achieve success in every second protein. such success naturally requires the existence of protective, largely conserved and possibly unmodified epitopes. we also want to point out that the selected validation set presumably represents a combination of differently well mapped proteins. in addition, each of these can contain one or several protective b-cell epitopes of both class i and ii. as we tried to detect only one of these in the set, neglecting the other, validation is skewed against our method. it may also be good to remember that calculated aroc values are averaged over entire proteins, not distinct epitopes. the application of machine-learning procedures potentially combines the prediction of different epitope classes and allows an estimation of the information content in the data. a random forest model achieved 83% class separation in an equilibrated model, pointing towards the potential to significantly improve upon the single-score method. although the selection of pathogen proteins relevant for the stimulation of protective immune-responses can be enhanced by bioinformatics that is a topic distinct from the prediction of likely protective epitopes on these antigens [30, 31] . by combining in-silico ranking of likely protective targets and likely protective peptides from these targets completely automatized screening for applicable peptides is possible. although methods do exist now for both aspects of in silico protectivity screening caution is still necessary. for example, the method published by doytchinova and flower predicts 95% of the proteins in our dataset as protective antigens (or at least antigens). on the other hand 65% of the proteome of staphylococcus aureus col (1727 of 2618 proteins) are also predicted to be relevant antigens which although possible seems to be a very high number suggesting a higher false positive rate than approximated by validation procedures in the publication. on the other hand all proteins are ranked by a score, so an order of predicted relevance is available, in our view essential for practical use. unfortunately 701 proteins would be selected to cover all four proteins for which s. aureus protectivity data is available in the iedb and which have close homologues also in strain col (fibronectin-binding protein a and b, enterotoxin b and enterotoxin type a), yet not all may be required for a protective immune response and others may contain unmapped or discontinuous epitopes [32] . these findings indicate that several predictive methods and experimental data should be combined when selecting candidates for the generation of protective immune responses. our work solely focuses on the prediction of candidate peptides after such a selection has taken place, independent whether by means of experimental data, literature mining or purely bioinformatical/biological considerations. we can show that prediction of protective or at least functionally relevant continuous b-cell epitopes can be efficiently done for approximately 50% of 57 analyzed proteins of pathogen origin. by minimizing sequence variability and probability of post-translational modification it can also be assumed that selected peptides are particularly suited for vaccine or monoclonal antibody generation. exclusion of rationally selected domains strongly enhances the prediction of protective sites, indicating the relevance of a filtering step to restrict to immunologically likely accessible regions. furthermore, analysis of correlation between variability, conservation of modification profiles and predicted antigenicity shows different and opposed categories of correlation thus indicating the existence of distinct epitope types. at this point it is difficult to verify or falsify our basic assumption of the twoclass nature of protective epitopes. on the other hand the high percentage of epitopes with either positive or negative feature correlation indicates the existence of at least two types. also, decision tree based models significantly outperform the single score-model pointing towards more complex relationships as well as possibly several distinct epitope signatures. future paths may lie in the detailed unraveling of parameter-associations and the utilization of more sophisticated classification methods such as regression trees for the assessment of biological relevance and protectivity in the selection of peptides for biotechnological application, as indicated by initial random forest classification. the set of 505 unique amino acid propensity scales taken from the aaindex database [33] forms a 20 ã� 505 descriptor matrix of rank 19. calculation of sequence properties from propensity scales can be expressed as a matrix multiplication of the amino acid sequence in matrix representation with the descriptor matrix. hence, the resulting property matrix is of rank 19 as well, corresponding to 19 linearly independent vectors. from this only 19 coefficients plus the intercept can be estimated in regression models. therefore principal component analysis (pca) was employed to transform the propensity scales from the 505-dimensional space to a 19-dimenstional subspace spanned by all 19 principal components with non-zero eigenvalues, comparable to how parameter reduction has been done before [34, 35] . the 20 ã� 505 descriptor matrix was centered and scaled to unity variance before the application of pca. the full information contained in the original 20 ã� 505 descriptor matrix is retained, because no component is omitted. from this pca-transformation, a new 20 ã� 19 descriptor matrix was built. these 19 pca-derived propensity scales were used in turn to calculate sequence characteristics for the data set. the values were averaged over a sliding window of nine amino acids. logistic regression employing the logit function was used to build models for the classification of single amino acid residues as epitopic or non epitopic. in order to estimate the generalization error, bootstrap validation was employed. error estimates were acquired from out-of-bag validation -i.e. using those residues that are not part of the bootstrap sample as validation data. 50 replicates were calculated for the validation of each investigated model. to numerically represent variability of a protein at a certain amino acid position an information-entropy measure has been applied. for each protein where variability should be determined the sequence was blasted against the non-redundant protein database (nr) and hits were selected manually. the aim was to choose a diverse but not too diverse set of sequences to optimally represent the degree of evolutionary freedom of each amino acid position. those proteins were downloaded and multiply aligned using clustalw. for each alignment column a variability value was calculated as follows: randomly draw 100 samples of size 30 (i.e. 100 times 30 amino acids which is a combination with repetition) from each column, independent of how many sequences have been aligned. that way the evaluation should be less dependent on the number of homologues as for some proteins only five elements can be found whereas for others hundreds are available. determine the most abundantly found amino-acid in the column. then calculate the shannon-entropy weighted by the emboss [36] eblosum variant of the blosum62 [37] substitution scores between each amino acid and the most abundant one in the column. after averaging over all 100 samples to obtain the mean variability the final variability score for each alignment column computes as where f(j) is the score at alignment column j, f x is the frequency of the most abundant letter x in column j, f i is the frequency of letter i in column j and w ix is the substitution weight between letters i and x. for each alignment variability scores are independently rescaled between 0 and 1. the variability score ultimately used for protectivity scoring is 1 -rescaled f(j). note that each individual gap is regarded as a new character which occurs only once (thus extending the 20 letter alphabet to a potentially high number leading to the perception of strongly gapped positions as highly variable). as an independent strategy an evolutionary constraint index (eci) was calculated for each alignment column. this constraint is essentially the difference between the standard shannon entropy and the entropy after reducing the amino acid alphabet according to the same substitution matrix as above. briefly, amino acid identities were grouped as follows: e < = e, d, q, k, r ; i < = i, l, m, v; y < = y, w, f, h; s < = s, t, a. other amino-acids remained ungrouped. the eci is primarily discussed when parameter correlations are analyzed. to predict posttranslational protein modifications prosite [38] patterns were used. in particular we considered patterns ps00001, ps00002, ps00003, ps00004, ps00005, ps00006, ps00007, ps00008, ps00009, ps00010, ps00012, ps00013, ps00294, ps00409. all sequences in the previously created multiple alignments were searched for the occurrence of these patterns. for each hit the amino acid putatively carrying the modification was marked and for each column in the alignment the ratio m of modified amino-acids was calculated. as for sequence variability the value used for protectivity scoring is 1-m. the advantage of using motif predictions on aligned sequences is the possibility to derive the degree of conservation of the motif. combined with the assumption that conserved motifs of post-translational modification are more reliable and do otherwise carry a high false positive rate this increases the weight of the prediction. a reference data set was generated from the antibody binding site repository bcipep [39] . this database holds a collection of experimentally determined b-cell epitopes. the bcipep data set is highly redundant with plenty of entries showing relation to more than one source protein. to realize a non-redundant data set, homologue proteins of this collection were grouped. members of two different groups differed by at least 30% in sequence identity. after this partitioning, the number of epitopes with length between 6 to 30 amino acids that could be localized on each protein was determined. finally, the proteins bearing the largest number of epitopes were selected as the representative for their group. this procedure leads to a diverse set of protein sequences with experimentally determined immunogenic regions for each protein. this data set holds in total 197 proteins with an average length of 449 amino acids. the prevalence of amino acids being part of an epitope is 7.6%. the mean length of an epitope is 16 residues. out of the 197 proteins 60 originate from bacteria, 55 from viruses, 14 from fungi, 11 from human, 3 from allergens, and 54 from other sources (e.g. eukaryotic parasites). the data set holds in total 401 continuous epitopes, i.e. about 2 epitopes per protein. since no systematic epitope mapping of all the proteins in the data set has been performed, categorizing sequence regions as non-antigenic is problematic, as these could be categorized false negative. epitopes classified as nonepitopes do exist, yet it is problematic to discern whether those are the results of organism or individual (i.e. responses varying from individual to individual) or yet other biases. we chose to declare the non-defined regions of our reference dataset as non-epitopes. regions not part of an epitope were randomized while maintaining the average amino-acid frequency of bcipep. the resulting was used for training b-cell epitope classifiers. each amino-acid functioned as the central amino acid of a 9-mer (peptide), inheriting its class (antigenic or nonantigenic) to the peptide which was then used for parameterization and training/validation. nine-mers were used due to the desire to use an intermediate between common assumptions about sizes of continuous epitopes (usually between 7 and 15 amino-acids). each sequence in the generated multiple alignments was independently scored for its antigenicity using the pca19 classifier. pca19 classification resulted in the assignment of an antigenicity value for each amino acid in the multiple alignment with the exception of the flanking 5 amino acids due to a window effect. for each alignment column overall antigenicity was calculated as the average antigenicity over the corresponding amino acids of individual sequences. proteins with known b-cell determinants were downloaded from the "the immune epitope database and analysis resource" (iedb) [40, 41] . the iedb allows filtering by various criteria. we applied the following step-wise exclusion filters to obtain a protectivity-related dataset: 8. remove entries from non-pathogens (including pathogenic plants). src6129 and src6623 were removed because no uniprot or genbank ids were specified. a neutral protease (gi 30260755) of bacillus anthracis str. ames was manually added from a literature source [42] . all proteins were then clustered and identified groups multiply aligned using the standard tools blastclust and clustalw, respectively. epitopes of all sequences present in the alignment were manually mapped to the homologous sequence where the fewest remapping steps were necessary, or where all epitopes could be represented as can be the case for large deletions or proteins with precursor variants. the process is thus similar to the one applied at the los alamos national laboratory hiv database where all reported epitopes are remapped to the reference strain hxb2 [43] . after removal of all redundancies and impractically short proteins 57 entries (31785 amino acids), with an average peptide coverage (and thus protectivity prevalence) of 7.25% remained, which are from now on referred to as "protectivity dataset". it has to be cautioned that the functional effect of antibodies directed against these determinants is classified only as "leading to biological activity", not necessarily protectivity. for our purposes we consider this close enough an approximation. this dataset can be found in the supplementary materials [see additional file 1]. validation results were analyzed using the rocr package) [44] where specifically aroc (area under the curve of true-positive rate versus false-positive rate plots) calculation has been most relevant. the weka package [45] was used where machine-learning functions were needed, in particular a c4.5 and a random forest implementation. from a practical point of view predictions of continuous epitopes should be measured by the number of synthesized peptides required to cover known epitopes. the synthesis score is defined as the number of peptides required to cover at least five epitopic amino-acids in the protectivity validation dataset. five has been selected as a minimum requirement for an epitope. to analyse the relevance of the used parameters simple machine learning techniques were applied as implemented in the weka package. for these analyses a dataset was generated based on the entire protectivity dataset (i.e. not the antigenicity dataset) after exclusion of likely inaccessible regions. essentially all residues which were likely immune-accessible according to the rules mentioned earlier were represented by the sum score (individually rescaled between 0 and 1 for each protein), antigenicity, ptm pattern conservation and variability. this dataset of dimension 21293 with 1485 antigenic (protective) and 19808 non-antigenic residues (baseline prediction 6.97%) will be termed complete machine learning set (cml set). in a second step for each antigenic (protective) residue a non-antigenic residue was randomly sampled to obtain an equilibrated set. this set was then randomly resampled into two stratified sets representing 80% and 20% of cml for training and validation, respectively. the training set (2376 instances) and validation set (594 instances) are termed mts and mvs, respectively. more than one reason to rethink the use of peptides in vaccine design cellular immunity elicited by human immunodeficiency virus type 1/ simian immunodeficiency virus dna vaccination does not augment the sterile protection afforded by passive infusion of neutralizing antibodies prediction of protein antigenic determinants from amino acid sequences a computer program for predicting protein antigenic determinants a semi-empirical method for prediction of antigenic determinants on protein antigens predictive estimation of protein linear epitopes by using the program people. vaccine bepitope: predicting the location of continuous epitopes and patterns in proteins selection and combination of machine learning classifiers for prediction of linear b-cell epitopes on proteins machine learning approaches for prediction of linear b-cell epitopes on proteins predicting antigenic determinants in proteins: looking for unidimensional solutions to a three-dimensional problem? mapping epitope structure and activity: from one-dimensional prediction to four-dimensional description of antigenic specificity improved method for predicting linear b-cell epitopes benchmarking b cell epitope prediction: underperformance of existing methods towards a consensus on datasets and evaluation metrics for developing b-cell epitope prediction tools identification of discontinuous antigenic determinants on proteins based on shape complementarities protective immunity to rabbit oral and cutaneous papillomaviruses by immunization with short peptides of l2, the minor capsid protein universal influenza b vaccine based on the maturational cleavage site of the hemagglutinin precursor formalin inactivation of the lactate dehydrogenase-elevating virus reveals a major neutralizing epitope not recognized during natural infection receptor and viral determinants of sars-coronavirus adaptation to human ace2 crosshost evolution of severe acute respiratory syndrome coronavirus in palm civet and human adaptive evolution of the spike gene of sars coronavirus: changes in positively selected sites in different epidemic groups cryptic nature of a conserved, cd4-inducible v3 loop neutralization epitope in the native envelope glycoprotein oligomer of ccr5-restricted, but not cxcr4-using, primary human immunodeficiency virus type 1 strains evolutionary dynamics of the glycan shield of the human immunodeficiency virus envelope during natural infection and implications for exposure of the 2g12 epitope influence of n-linked glycosylation of porcine reproductive and respiratory syndrome virus gp5 on virus infectivity, antigenicity, and ability to induce neutralizing antibodies prediction of continuous b-cell epitopes in an antigen using recurrent neural network uniprot: the universal protein knowledgebase a hidden markov model for predicting transmembrane helices in protein sequences machine learning approaches for the prediction of signal peptides and other protein sorting signals characterization of a unique borreliacidal epitope on the outer surface protein c of borrelia burgdorferi vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines spaan: a software program for prediction of adhesins and adhesin-like proteins using neural networks a novel staphylococcus aureus vaccine: iron surface determinant b induces rapid antibody responses in rhesus macaques and specific increased survival in a murine s. aureus sepsis model aaindex: amino acid index database peptide quantitative structure-activity relationships, a multivariate approach a new set of amino acid descriptors and its application in peptide qsars emboss: the european molecular biology open software suite amino acid substitution matrices from protein blocks prosite: a dictionary of sites and patterns in proteins bcipep: a database of b-cell epitopes an ontology for immune epitopes: application to the design of a broad scope database of immune reactivities curation of complex, context-dependent immunological data effective antiprotease-antibiotic treatment of experimental anthrax rocr we acknowledge emergentec biodevelopment gmbh for funding our research. the author(s) declare that they have no competing interests. js designed the study, assembled the protectivity dataset, performed all analysis based on this data and drafted the manuscript. rg and rr prepared the bcipep dataset (removed redundancies) and developed the pca19 antigenicity classifier. rg, rr, pp and al worked on the validation of the pca19 classifier and investigated the contribution of individual sequence features. bm conceived of the study, and participated in its design and coordination. key: cord-301827-a7hnuxy5 authors: uversky, vladimir n title: a decade and a half of protein intrinsic disorder: biology still waits for physics date: 2013-04-29 journal: protein science doi: 10.1002/pro.2261 sha: doc_id: 301827 cord_uid: a7hnuxy5 the abundant existence of proteins and regions that possess specific functions without being uniquely folded into unique 3d structures has become accepted by a significant number of protein scientists. sequences of these intrinsically disordered proteins (idps) and idp regions (idprs) are characterized by a number of specific features, such as low overall hydrophobicity and high net charge which makes these proteins predictable. idps/idprs possess large hydrodynamic volumes, low contents of ordered secondary structure, and are characterized by high structural heterogeneity. they are very flexible, but some may undergo disorder to order transitions in the presence of natural ligands. the degree of these structural rearrangements varies over a very wide range. idps/idprs are tightly controlled under the normal conditions and have numerous specific functions that complement functions of ordered proteins and domains. when lacking proper control, they have multiple roles in pathogenesis of various human diseases. gaining structural and functional information about these proteins is a challenge, since they do not typically “freeze” while their “pictures are taken.” however, despite or perhaps because of the experimental challenges, these fuzzy objects with fuzzy structures and fuzzy functions are among the most interesting targets for modern protein research. this review briefly summarizes some of the recent advances in this exciting field and considers some of the basic lessons learned from the analysis of physics, chemistry, and biology of idps. a bit more than ten years ago, protein science published a review entitled "natively unfolded proteins: a point where biology waits for physics" (protein sci 2002 11(4):739-756). 1 the major goal of that article was to bring an intriguing protein family of natively unfolded proteins (which are recognized now to constitute a subset of a very broad class of intrinsically disordered proteins, idps) out of shadow, to emphasize their lack of ordered structure under physiological conditions (at least ordered structure that could be detected by traditional low resolution techniques), to systemize their major structural properties, and to highlight their biological significance. the introduction of such biologically active but essentially unstructured proteins was used to challenge the hitherto dominant structure-centric viewpoint (structure-function paradigm), according to which a specific function of a protein is determined by its unique and rigid three-dimensional (3d) structure. the title of the review ("a point where biology waits for physics") was inspired by the observations that many of such "structure-less" proteins analyzed by that time acted as "binders" that did undergo at least partial folding after interaction with their binding partners. these observations provoked an idea that these biologically important proteins with little or no ordered structure have to wait to become more folded (and functional) as a result of binding to their specific partners. in other words, for these proteins, "biology," that is, the ability to have biological functions, seemed to wait for "physics" which is manifested in their ability to undergo binding-induced folding (at least partial), which is necessary to bring the functional state of these proteins to life. 1 at the beginning, the idea that structure-less proteins can be biologically active was taken as a complete heresy by many researchers, since it was absolutely alien to then dominated structure-function paradigm which represented a foundation of the long-standing belief that the specific functionality of a given protein is determined by its unique 3-d structure. this structure-function paradigm that describes reasonably well the catalytic behavior of enzymes was based on the "lock-and-key" hypothesis formulated in 1894 by emil fischer. 2 this viewpoint was solidified by the successful solution of x-ray crystallographic structures of many proteins (as of february 26, 2013 there were 81,922 protein structures in the protein data bank, 3 with 72,761 of these structures being determined by xray crystallography). these many crystal structures reinforced a static view of functional protein, where a rigid active site of an enzyme can be viewed as a sturdy lock that provides an exact fit to only one key, a specific and unique substrate. 4 despite numerous limitations, this lock-and-key model was an extremely fruitful concept that was responsible for the creation of modern protein science. 1 figure 1 (a) shows some of the most obvious scientific consequences of the application of structure-function paradigm which is deservedly placed at the center of the "big bang" model that gives rise to the protein science universe. 1 obviously, the consideration of a protein as a rigid crystal-like entity is an oversimplification, since even the most stable and well-folded proteins are dynamic systems that possess different degrees of conformational flexibility. this is because of the simple fact that so-called conformational forces, that is, forces stabilizing the secondary structure of a protein and its tertiary fold, are weak and can be broken even at ambient temperatures due to thermal fluctuations. 4 the breaking of these weak interactions releases the groups that were involved in these interactions and gives them the possibility to be involved in the formation of new weak interactions of comparable energy. 4 since these structural rearrangements are of relatively small scale and since they occur typically in a time scale that is faster than the time required for structure determination by x-ray crystallography and many other physical techniques, the 3-d structures of proteins determined by these techniques represent averaged pictures. 6 furthermore, one should keep in mind that not all proteins structures which are deposited to pdb are structured throughout their entire lengths. instead, many pdb proteins have portions of their sequences missing from the determined structures (so-called regions of missing electron density) 7, 8 due to the failure of the unobserved atom, side chain, residue, or region to scatter x-rays coherently caused by their flexible or disordered nature. such flexible/disordered regions are rather common in the pdb, since only about 30% of protein structures deposited in the pdb do not have such regions of missing electron density. 9 in addition to ordered proteins possessing disordered regions of varying length, the literature contains numerous examples of biologically active proteins with flexible structures. 4 therefore, there is another class of functional proteins and protein regions that contain smaller or larger highly dynamic fragments, and some proteins are even characterized by a complete or almost complete lack of ordered structure under physiological conditions (at least in vitro) which appears to be a critical aspect of these proteins' function in vivo. 4, [10] [11] [12] [13] [14] [15] these proteins and protein regions (which are known now as idps and idp regions (idprs)) have no single, well-defined equilibrium structure and exist as heterogeneous ensembles of conformers such that no single set of coordinates or backbone ramachandran angles is sufficient to describe their conformational properties. these proteins were independently discovered one-by-one over a long period of time and therefore they were considered as rare exceptions to the general rule. although the phenomenon of biological functionality without stable structure was repeatedly observed, for a long time it was unnoticed by a wide audience because the authors frequently invented new terms to describe their protein of interest. 16 in fact, an incomplete list of terms coined in the literature to describe these proteins includes floppy, pliable, rheomorphic, 17 flexible, 18 mobile, 19 partially folded, 20 natively denatured, 21 natively unfolded, 12, 22 natively disordered, 15 intrinsically unstructured, 11, 14 intrinsically denatured, 21 intrinsically unfolded, 22 intrinsically disordered, 13 vulnerable, 23 chameleon, 24 malleable, 25 4d, 26 protein clouds, 27 dancing proteins, 28 proteins waiting for partners, 29 and several other names often representing different combinations of "natively/naturally/inherently/intrinsically" with "unfolded/unstructured/disordered/denatured" among several others. therefore, the majority of the names used in the early literature express that the "unfolded, unstructured, disordered, and denatured" state is a "native, natural, inherent, and intrinsic" property of these proteins. 16 although protein intrinsic disorder is considered now as an established concept and pubmed contains hundreds and hundreds of papers talking about different aspects of idps/idprs, the route to recognizing these proteins as a novel functional entity was complex and lengthy. as it is often the case for new scientific concepts, the idea of structure-less functionality went through the stages of passive ignorance and active denial to scrupulous examination and enthusiastic acceptance. for example, it took me more than a year to publish my first paper dedicated to the systematic analysis of such proteins, and the manuscript was successively rejected by 14 journals before it was finally accepted by proteins. 12 however, time showed that the concept of protein intrinsic disorder was a useful invention and could be considered as a universal lock-pick that helps in solving many of the seemingly unsolvable figure 1 . a: protein structure-function paradigm is the "big bang" created universe of the modern protein science. some major directions based on the consideration of protein function as lock-and-key mechanism are shown. modified from ref. 1 . b: paradigm shift caused by the introduction of the protein intrinsic disorder concept opened a wide array of new directions in protein science. in essence, introduction of this concept can be considered as a scientific revolution that, according to kuhn, 5 "occurs when scientists encounter anomalies that cannot be explained by the universally accepted paradigm within which scientific progress has thereto been made" (http://en.wikipedia.org/wiki/paradigm_shift). uversky problems in protein science. one could say that this idea gave a new boost to the development of the protein science, generating a wide array of principally novel research directions [see fig. 1(b) ]. the goals of this review are: (i) to outline some recent advances in the field of idps/idprs; (ii) to illustrate the usefulness of intrinsic disorder for protein function; (iii) to show that intrinsic disorder can affect different levels of protein structural organization; (iv) to indicate intimate involvement of intrinsic disorder in pathogenesis of various maladies; (v) to emphasize the exceptional structural heterogeneity of idps/idprs and to show that idps are definitely much more structurally complex than random coillike polypeptides; (vi) to accentuate that although this structural heterogeneity is very important for protein functionality, it represents a crucial hurdle for structural characterization of idps; (vii) to stress that new experimental and computational approaches and new theories and models are crucially needed for future progression of this field and protein science in general. these and other points highlight the current state of the field, where further advances in understanding of the "biology" of idps still waits for "physics," with "physics" now being new theories, instrumentation, and analytical approaches. identification of idps as unique entities belonging to a new protein tribe is directly related to the recognition that their amino acid sequences are dramatically different from those of ordered proteins. 10, 12, 13, [30] [31] [32] for example, it has been pointed out that the low content of hydrophobic residues combined with the high load of charged residues that often gives rise to high net charge of a polypeptide chain represents a characteristic feature of some idps (so called extended idps or natively unfolded proteins with coil-like or close to coil-like structures, see below). 12 therefore, compact proteins and extended idps can be distinguished based only on their net charges and hydropathies using a simple charge-hydropathy (ch) plot, where the idps are specifically localized within a specific region of ch phase space and are reliably separated from compact ordered proteins. 12 more detailed comparison of amino acid sequences revealed that in comparison with ordered proteins and domains, the idps/idprs are significantly depleted in order-promoting amino acids (trp, tyr, phe, ile, leu, val, cys, and asn), 10, 33 being instead enriched in disorder-promoting residues, such as ala, arg, gly, gln, ser, glu, lys, and pro. 13, 31, 32, 34, 35 difference between ordered and disordered proteins goes far beyond these differences in their amino acid compositions. in fact, based on the comparison of the 265 amino acid physico-chemical property-based scales (such as hydropathy, net charge, flexibility index, helix propensities, strand propensities, aromaticity, etc.) 34 and more than 6000 composition-based attributes (e.g., all possible combinations having one to four amino acids in the group) 36 it has been concluded that ordered and disordered proteins and regions can be discriminated using many of these attributes. 13 based on the analysis of 517 amino acid scales, a novel amino acid scale, top-idp (trp, phe, tyr, ile met, leu, val, asn, cys, thr, ala, gly, arg, asp, his, gln, lys, ser, glu, and pro), was built to provide ranking for the tendencies of the amino acid residue to promote order or disorder. 30 the fact that the sequences of ordered and disordered proteins and regions are noticeably different suggested that idps clearly constitute a separate entity inside the protein kingdom, that these proteins can be reliably predicted using various computational tools, [37] [38] [39] [40] [41] [42] and structurally, that idps should be very different from ordered globular proteins since peculiarities of amino acid sequence determine protein structure. natural abundance of idps: touching the tip of the iceberg initial systematic analyses revealed that intrinsic disorder in proteins is a rather common phenomenon. in fact, as of 2002, the list of experimentally validated natively unfolded proteins with chain length greater than 50 amino acid residues contained more than 100 entries. 1 it was also pointed out that this list would probably be doubled if shorter polypeptides 30-50 residues long were included, 1 and that these 100 experimentally validated natively unfolded have at least 250 homologues, which are also expected to be natively unfolded. 1, 12 it happened that these "large" numbers (which actually were large enough to make a crucial point that biologically active structure-less proteins represent the new rule and not mere rare exceptions) constitute just a small tip of an iceberg. in fact, using computational tools developed for sequence-based intrinsic disorder prediction the wide spread of idps and hybrid proteins containing idprs was convincingly shown. [43] [44] [45] [46] for example, more than 15,000 out of 91,000 proteins in the thencurrent swiss protein database were identified as having long idprs. 47 the published in 2000 analysis of 31 whole genomes that span the 3 kingdoms of life revealed that many proteins contained segments predicted to have 40 consecutive disordered residues and that the eukaryotes exhibited more disorder by these measures than either the prokaryotes or the archaea. 43 other studies on the abundance of intrinsic disorder in various evolutionary distant species supported these findings and consistently showed that the eukaryotic proteomes had higher fraction of intrinsic disorder than prokaryotic proteomes. 44, [48] [49] [50] [51] [52] this conclusion is in line with the results of a comprehensive bioinformatics investigation of the disorder distribution in almost 3500 proteomes from viruses and three kingdoms of life, results of which are shown in figure 2 as the correlation between the intrinsic disorder content and proteome size for 3484 species from viruses, archaea, bacteria, and eukaryotes. 46 surprisingly, figure 2 shows that there is a well-defined gap between the prokaryotes and eukaryotes in the plot of fraction of disordered residues on proteome size, where almost all eukaryotes have 32% or more disordered residues, whereas the majority of the prokaryotic species have 27% or fewer disordered residues. 46 therefore, it looks like the fraction of 30% disordered residues serves as a boundary between the prokaryotes and eukaryotes and reflects the existence of a complex step-wise correlation between the increase in the organism complexity and the increase in the amount of intrinsic disorder. a gap in the plot of fraction of disordered residues on proteome size parallels a morphological gap between prokaryotic and eukaryotic cells which contain many complex innovations that seemingly arose all at once. in other words, this sharp jump in the disorder content in proteomes associated with the transition from prokaryotic to eukaryotic cells suggests that the increase in the morphological complexity of the cell paralleled the increased usage of intrinsic disorder. 46 the variability of disorder content in unicellular eukaryotes and rather weak correlation between disorder status and organism complexity (measured as the number of different cell types) is likely related to the wide variability of their habitats, with especially high levels of disorder being found in parasitic host-changing protozoa, the environment of which changes dramatically during their life-span. 53 the further support for this hypothesis came from the fact that the intrinsic disorder content in multicellular eukaryotes (which are characterized by more stable and less variable environment of individual cells) was noticeably less variable than that in the unicellular eukaryotes. 46 it was pointed out that idps possess noticeable amino acid biases, and many idps/idprs are characterized by sequence redundancy and low sequence complexity, containing long stretches of various repeats and being completely devoid of some (often many) types of amino acid residues. these observations seem to indicate that the sequence space of idps/idprs should be simpler than that of ordered proteins. however, the reality is more complex than conventional wisdom might suggest, and the sequence space attainable by simple idps/idprs is more diversified than that of the structurally more sophisticated ordered proteins. in fact, a 100 residue-long protein in which any of the normally occurring 20 amino acids can be found has a sequence space of 20 100 (10 130 ) sequences. 54 obviously, not all random amino acid sequences can fold into unique structures. in other words, a sequence space of a foldable protein (or "foldable" sequence space) is noticeably smaller than the entire sequence space available for a random polypeptide chain. for decades, the actual size of "foldable" sequence space continues to be unsolved mystery despite a large body of theoretical, biochemical, and computational work that aims to unravel the relationship between a protein's primary sequence and its resulting 3d structure. 55 however, the actual number of different amino acid residues in a given foldable sequence can be dramatically reduced, 54 since all twenty residues are not necessary for protein folding and the actual physicochemical identity of most of the amino acids in a protein is irrelevant. [56] [57] [58] [59] [60] [61] [62] [63] in other words, folding alphabet can be noticeably reduced, 55, 64 and amino acids can be clustered based on some shared features such as homolog substitution frequency, 65 local structural environments, 66 or peculiarities of the tertiary structural environments. 67 this simplified folding code further reduces the available "foldable" sequence space. 68 figure 2 . correlation between the intrinsic disorder content and proteome size for 3484 species from viruses, archaea, bacteria, and eukaryotes. each symbol indicates a species. there are totally six groups of species: viruses expressing one polyprotein precursor (small red circles filled with blue), other viruses (small red circles), bacteria (small green circles), archaea (blue circles), unicellular eukaryotes (brown squares), and multicellular eukaryotes (pink triangles). each viral polyprotein was analyzed as a single polypeptide chain, without parsing it into the individual proteins before predictions. the proteome size is the number of proteins in the proteome of that species and is shown in log base. the average fraction of disordered residues is calculated by averaging the fraction of disordered residues of each sequence over the all sequences of that species. disorder prediction is evaluated by pondr-vsl2b. modified from ref. 46. simply by virtue of their existence, idps/idprs add a new level of complexity to the sequence-structure relationship, dividing the population of protein sequences into two categories, sequences that yield natively ordered, and sequences that code natively disordered proteins. 55 idps/idprs cannot fold spontaneously and some of them require specific partners to gain more ordered structure. therefore, they do not possess an entire folding code that defines the ability of foldable proteins to fold spontaneously into a unique biologically active structure. the missing portion of the idp folding code (or at least part of it) is supplemented by binding partner(s). this defines a principal difference between structured proteins and idps/idprs: foldable proteins fold first and then bind to their partners whereas idps/idprs remain disordered until they interact with their partners. 68, 69 furthermore, many idps/idprs do not require folding to be functional, 1, 4, 13, 14, [70] [71] [72] [73] and some of them form fuzzy complexes, in which they preserve significant amount of disorder. 74, 75 all this suggests that the sequence space of idps (at least those which either do not fold at all or do not completely fold at binding) is noticeably greater than the "foldable" sequence space due to the removal of restrictions posed by the need to gain ordered structure spontaneously. 68 this represents one of the conundrums of intrinsic disorder, where the apparent sequence redundancy and simplicity are combined with the lack of structural restrains leading to the increase in the dimensions and complexity of the available sequence space. also, the existence of a noticeable sequencestructure heterogeneity of idps should be emphasized. 68 since the unique 3d-structure of an ordered single-domain protein is defined by the interplay between all (or almost all) of its residues, one could expect that the structure-coding potential is homogeneously distributed within its amino acid sequence. on the other hand, a sequence of an idp/idpr contains multiple, relatively short functional elements and therefore represents a very complex structural and functional mosaic. 68 this important feature defines the known ability of an idp/idpr to interact, regulate, and be controlled by multiple structurally unrelated partners. 76 such functional "anatomy" of idps/idprs is determined by the extremely high level of their sequence heterogeneity, which is further increased due to the ability of a single idpr to bind to multiple partners gaining very different structures in the bound state. 77 one of the crucial consequences of an extended sequence space and non-homogeneous distribution of foldability (or the structure-coding potential) within amino acid sequences of idps and idprs is their astonishing structural heterogeneity. in fact, a typical idp/idpr contains a multitude of elements coding for potentially foldable, partially foldable, differently foldable, or not foldable at all protein segments. 68 as a result, different parts of a molecule are ordered (or disordered) to a different degree. this distribution is constantly changing in time where a given segment of a protein molecule has different structures at different time points. as a result, at any given moment, an idp has a structure which is different from a structure viewed at another moment. 68 another level of structural heterogeneity is determined by the fact that many proteins are hybrids of ordered and disordered domains and regions, and this mosaic structural organization is crucial for their functions. 16 also, even when they do not possess ordered domains, idps are known to have various levels and depth of disorder. 78 over a few past years, an understanding of the available conformational space of idps/idprs underwent significant evolution. in fact, for a long time, idps were considered mostly "unstructured" or "natively unfolded" polypeptide chains. this was mostly due to the fact that the majority of idps analyzed at early stages of the field contained very little ordered structure, that is, they were really mostly unstructured or unfolded. finding and characterization of such "structure-less" proteins was important to build up a strong case to counter-point the dominant view represented by the classical sequence-to-structureto-function paradigm, especially since such fully unstructured, yet functional proteins clearly represented the other extreme of the protein structurefunction spectrum. 16 the top half of the figure 3 illustrates this situation by opposing rock-like ordered proteins and cooked spaghetti-like idps. however, already in some early studies, it was indicated that idps/idrs could be crudely grouped into two major structural classes, proteins with compact and extended disorder. 1, 4, 12, 13, 73 based on these observations, the protein functionality was ascribed to at least three major protein conformational states, ordered, molten globular, and coil-like, 13, 79 indicating that functional idps can be less or more compact and possess smaller or larger amount of flexible secondary/tertiary structure. 1, 4, 12, 13, 73, 79 roughly at the same time, it was emphasized that the extended idps (known as natively unfolded proteins) do not represent a uniform entity but contain two broad structural classes, native coils and native pre-molten globules. 1 currently available data suggest that intrinsic disorder possesses multiple flavors, can have multiple faces, and can affect different levels of protein structural organization, where whole proteins, or various protein regions can be disordered to a different degree. 68 this new view of structural space of functional proteins can be visualized to form a continuous spectrum of differently disordered conformations extending from fully ordered to completely structure-less proteins, with everything in between (fig. 3, bottom half) . here, functional proteins can be well-folded and be completely devoid of disordered regions (rock-like scenario). other functional proteins may contain limited number of disordered regions (a grass-on-the rock scenario), or have significant amount of disordered regions (a llama/camel hair scenario), or be molten globule-like (a greasy ball scenario), or behave as pre-molten globules (a spaghetti-and-meatballs/sausage scenario), or be mostly unstructured (a hairball scenario). notably, in this representation, there is no boundary between ordered proteins and idps, and, the structure-disorder space of a protein is considered as a continuum. it is important to remember that even the most ordered proteins do not resemble "solid rocks" and have some degree of flexibility. in fact, a protein molecule is an inherently flexible entity and the presence of this flexibility (even for the most ordered proteins) is crucial for its biological activity. 80 also, another important point to remember is that due to their heteropolymeric nature, proteins are never random coils and always have some residual structure. 68 protein biophysicists/biochemists working on different aspects of ordered proteins (e.g., analyzing their structural properties, functions, folding, etc.) would find biophysical properties of functional idps/idprs to be rather unusual since these highly dynamic proteins do not follow the well-accepted wisdom that a protein has to be well-folded to be biologically functional. however, the unusualness is a subjective feature, and from the viewpoint of polymer physics the extended idps/idprs possess the expected behavior . structural heterogeneity of idps/idprs. top half: bi-colored view of functional proteins which are considered to be either ordered (folded, blue) or completely structure-less (disordered, red). ordered proteins are taken as rigid rocks, whereas idps are considered as completely structure-less entities, kind of cooked noodles. bottom half: a continuous emission spectrum representing the fact that functional proteins can extend from fully ordered to completely structure-less proteins, with everything in between. intrinsic disorder can have multiple faces, can affect different levels of protein structural organization, and whole proteins, or various protein regions can be disordered to a different degree. some illustrative examples includes ordered proteins that are completely devoid of disordered regions (rock-like type), ordered proteins with limited number of disordered regions (grass-on-the rock type), ordered proteins with significant amount of disordered regions (lhama/camel hair type), molten globule-like collapsed idps (greasy ball type), pre-molten globule-like extended idps (spaghetti-and-sausage type), and unstructured extended idps (hairball type). of flexible and charged polymers, whereas the behavior of an ordered protein is rather unexpected (i.e., due to the existence of the native ensemble that for well-folded, ordered proteins can be approximated as a harmonic well around a unique, welldefined equilibrium structure). therefore, one definitely should keep in mind that the "unusual" biophysics of extended idps/idprs has its roots in the usual polymer physics of highly charged and flexible polypeptides. each protein is believed to be a unique entity that has quite unique primary sequence which governs its 3d structure (or lack thereof) and ensures specific biological function(s). therefore, understanding the effect of sequence variance on the biological performance presents a challenging task. however, natural polypeptides have originated as random copolymers of amino acids, which were adjusted or "selected" over evolution based on their functional capacities. 56, 81 despite their differences in primary amino acid sequences, protein molecules in a number of conformational states behave as polymer homologues, suggesting that the volume interactions can be considered as a major driving force responsible for the formation of equilibrium structures or structural ensembles. 82 for example, ordered globular proteins and molten globules (both as folding intermediates of globular proteins or as examples of collapsed idps) exhibit key properties of polymer globules, where the fluctuations of the molecular density are expected to be much less than the molecular density itself. extended idps (both intrinsic coils and intrinsic pre-molten globules) and ordered proteins in the pre-molten globule intermediate state possess properties of squeezed coils, since water is a poor solvent for a polypeptide. in fact, even high concentrations of strong denaturants (e.g., urea and gdmcl) are very likely to be bad solvents for protein chains, resulting in the preservation of extensive residual structure even under these harsh denaturing conditions. 82 based on these and related observations, and taking into account the fact that many idps/idprs are characterized by significant amino acid composition biases, the overall polymeric behavior of these proteins and regions can be mimicked reasonably well by the behavior of low-complexity polypeptides (e.g., homopolypeptide and block copolypeptides). following these ideas, it was shown that water is a poor solvent for polypeptide backbone alone and for the idps containing long tracts of polar amino acid residues since polar homo-polypeptides without hydrophobic groups (e.g., polyglutamine or glycineserine block copolypeptides) were shown to prefer collapsed ensembles in aqueous media. [83] [84] [85] [86] [87] [88] furthermore, even polyglycine was shown to have a tendency to form heterogeneous ensembles of collapsed structures in water. 88 a systematic analysis of the conformational behavior of protamines, arginine-rich idps involved in the condensation of chromatin during spermatogenesis, and protamine-like peptides revealed that there is a charge-driven coil-to-globule transition in these highly charged polypeptides, where the net charge per residue serves as the discriminating order parameter. 89 overall, the increase in the hydrodynamic dimensions of a polypeptide chain with increase in its net charge per residue can be attributed to the increase in the intramolecular electrostatic repulsions between similarly charged sidechains and the favorable solvation of these moieties. 89 based on these premises, at least three different classes of globule-forming polar/charged idps were proposed. the first class is comprised by polar tracts which collapse due to water being a poor solvent for a backbone and non-charged side chains. the second class is represented by weak polyelectrolytes and weak polyampholytes, which have low per residue net charge and low fractions of positively and/or negatively charged residues. these idps/ idprs form collapsed structures since the driving force responsible for the collapse is not overcome by the intramolecular electrostatic repulsion between the charged side-chains and by their favorable free energies of solvation. furthermore, if such idps/ idprs possess polyampholytic nature, their globular state could be additionally stabilized by electrostatic interactions between the oppositely charged sidechains. finally, idps/idprs from the third class are strong polyampholytes characterized by high fractions of positively and/or negatively charged residues but have low per residue net charge. such intrinsically disordered protein can form collapsed structures stabilized mostly by multiple electrostatic interactions between solvated side-chains of opposite sign. 89 the extended idps/idprs were used as a model system for the analysis of the effect of electrostatic interactions on conformational properties of unfolded proteins, and for testing the quantitative descriptions and predictions of polymer theory related to the influence of charged amino acids on chain dimensions. 90 for example, based on the analysis of the conformational equilibrium of coarse-grained polypeptides as a function of sequence hydrophobicity, charge, and length it has been concluded that the variations in sequence hydrophobicity and charge define a coil-to-globule transition comparable to that seeing in the empirical ch-plot, 12, 91 suggesting that a minimal, polymer physics-based model can capture the elements of global protein conformation. 92 idps/idprs with very high net charges are expected to be more extended and behave more similar to random coils (i.e., similar to conformations adopted by proteins in the denaturant gdmcl). the analysis of the gdmcl-induced expansion of the unfolded states suggested that protein charge density plays a crucial role in defining the hydrodynamic behavior of the unfolded polypeptide chain. 90 here, highly charged proteins can exhibit a prominent expansion at low ionic strength that correlates with their net charges. 90 it has been also hypothesized that the pronounced effect of charges on the dimensions of unfolded proteins might have important implications for their cellular functions. 90 similarly, a comprehensive analysis of the hydrodynamic dimensions of fg-nucleoporins containing large idprs with multiple phenylalanineglycine repeats (fg-domains) revealed that under the physiologic conditions in vitro these domains adopt distinct categories of disordered structures, such as molten globule, pre-molten globule, relaxedcoil, extended-coil (as in urea), or very extended-coil (as in gdmcl). 93 the category of intrinsically disordered structure in a given fg-domain was related to its amino acid composition, namely to the content of charged residues, where more charged fg-domains possessed larger hydrodynamic dimensions. 94 furthermore, fg-nucleporins with higher charge density were shown to be more dynamic than the collapsed-coil fg-domains, being also prone to repel other fg-domains. on the other hand, the collapsedcoil fg-domains were prone to oligomerize. these observations suggested that different types of fgdomains with different aggregation propensities provide molecular basis for two different gating mechanisms operating at the nuclear pore complex at distinct locations; one acting as a hydrogel, and the other as an entropic brush. 94 therefore, the abundance and peculiarities of the charged residues distribution within the protein sequences might determine physical and biological properties of extended idps and idprs. also, simple polymer physics-based reasoning can give reasonably well-justified explanation of the conformational behavior of extended idps. in general, the conformational behavior of idps is characterized by the low cooperativity (or the complete lack thereof) of the denaturant-induced unfolding, lack of the measurable excess heat absorption peak(s) characteristic for the melting of ordered proteins, "turned out" response to heat and changes in ph, and the ability to gain structure in the presence of various binding partners. 95 the analysis of the temperature effects on structural properties of several extended idps revealed that native coils and native pre-molten globules partially fold as the temperature is increased. 1, 73, [95] [96] [97] [98] these heating-induced structural changes in extended idps were attributed to the increased strength of the hydrophobic interaction at higher temperatures, leading to a stronger hydrophobic attraction, which is the major driving force for folding. similarly, extended idps/idprs are characterized by the "turned out" response to changes in ph, 96,99-102 where a decrease (or increase) in ph induces their partial folding due to the minimization of their high net charges viewed at neutral ph, thereby decreasing charge/charge intramolecular repulsion and permitting hydrophobicdriven collapse to the partially folded conformation. 95 every disordered protein is disordered in its own way data accumulated so far indicate that intrinsic disorder exists at multiple structural levels and might differently affect different regions/domains of idps. this defines noted structural complexity and heterogeneity of idps/idprs which are further enhanced by the way different proteins/protein regions respond to their environments. furthermore, since intrinsic disorder is crucial for many biological functions and therefore must prevail in different environments, the amino acid sequences and compositions of idps and idprs are specifically shaped by the peculiarities of their global and local environments. all this makes the protein intrinsic disorder phenomenon to be so broad that one can even assume that every disordered protein (or at least every family of disordered proteins) is disordered in its own way. this hypothesis has far-reaching consequences since it implies that a general disorder predictor has limited accuracy and cannot predict with equally high accuracy disorder status of all protein sequences due to their heterogeneity. it also implies that some environmental factors definitely should be taken into account when assessing intrinsic disorder in proteins. several examples are presented below to support the overall validity of these statements. the first example is given by transmembrane (tm) proteins, in which disorder is widely observed (e.g., 40% of human integral plasma proteins were predicted to contain long idprs). [103] [104] [105] [106] [107] furthermore, disorder is unevenly distributed between the cytoplasmic and the external surfaces of these proteins, with cytoplasmic domains being up to threefold more disordered than extracellular domains. 105 although these analyses gave interesting hints on the abundance of disorder in tm proteins, the obvious weakness of such evaluations is in the fact that they were performed using the disorder predictors developed from structured and disordered regions found in water-soluble proteins. 108 however, the major physico-chemical properties of water-soluble and integral membrane proteins are very different due to the differences in their environments. for example, similar to typical water soluble proteins, the tm regions of membrane proteins are often highly structured, containing a-helices 109 or b-structure, 110 which are especially likely to occur due to the low dielectric constant values within the membrane bilayers. 111, 112 on the other hand, the exterior regions of tm proteins are much more apolar than the exteriors of water-soluble proteins. [113] [114] [115] therefore, the peculiarities of the membrane environment, with its highly nonpolar nature originating either from lipids or from protein interiors, are especially unfavorable for intrinsic disorder, since propensity for intrinsic disorder is typically encoded in a high content of polar and charged residues. therefore, the idprs found in integral membrane proteins would be expected to be generally localized within the regions external to the membrane bilayer. 108 also, the distinctive environment of the membrane bilayer imposes constraints on the amino acid composition of integral membrane proteins, even on the regions external to the membrane bilayer. 116, 117 comprehensive bioinformatics analysis revealed that integral membrane proteins commonly possess idprs defined as regions of missing electron density in their crystal structures. 108 comparison of the idprs found in the a-helical and the b-barrel bundle integral membrane proteins with the idprs viewed in typical water-soluble proteins revealed the existence of statistically distinct amino acid compositional biases characteristic for these three protein classes. therefore, the use of specific amino acid signatures of idprs found in tm helical bundles and b-barrels can potentially lead to significantly more accurate disorder predictions for these two classes of integral membrane proteins. 108 another illustrative example of the specific disorderrelated and environment-dependent sequence features is given by archaeal proteins. 46, 51 based on the levels of predicted disordered residues, archaeal proteins can be grouped into three classes, with ranges of the disordered residue content of 12-21%, 21%-32%, and 32%-38% (see fig. 2 ). the archaeal proteomes with the highest disorder contents are halophiles and methanophiles. 46, 51 similar to tm proteins, the estimation of intrinsic disorder in the extremophilic proteins of the microorganisms surviving under hypersaline conditions using predictors developed for the "normal" non-halophilic proteins existing under the normal physiological conditions of 100-150 mm nacl may not be accurate. 46 in fact, one of the strategies used by the halophilic archaea, which are salt-loving extremophilic organisms that grow optimally at high salt concentrations, to maintain proper osmotic pressure in their cytoplasm is a so-called "salt-in" strategy that involves accumulation of molar concentrations of potassium and chloride in their cytosoles. 118 this strategy requires extensive adaptation of the intracellular proteins to the presence of near-saturating salt concentrations. the proteomes of such "salt-in" organisms are highly acidic, 46, 51 and their proteins are characterized by remarkable instability at conditions of low salt concentrations and by maintaining soluble and active conformations in hypersaline conditions that are generally detrimental to the non-halophilic proteins. [118] [119] [120] [121] [122] [123] [124] [125] [126] [127] finally, peculiarities of disorder distributions in viral proteins can be used to further support the importance of considering environmental factors. 46, 51 here, the comprehensive analysis of intrinsic disorder in various completed proteomes revealed that the viral proteomes have the largest variation of disorder content, which ranges from 7.3% disordered residues in the human coronavirus nl63 to 77.3% disordered residues in the avian carcinoma virus proteome (see fig. 2 ). 46 the high predicted intrinsic disorder content in viruses has multiple functional implications, where some idprs are used in the functioning of viral proteins and help viruses to highjack various pathways of the host cells, others likely have evolved to help viruses accommodate to their hostile habitats, and still others evolved to help viruses in managing their economic usage of genetic material via alternative splicing, overlapping genes, and anti-sense transcription. 128 these findings are in agreement with another study revealing that in comparison with archaea and bacteria, viral and bacteriophagic proteins were significantly more enriched in polar residues and depleted in hydrophobic residues and were close to eukaryotic proteins in terms of their amino acid compositions and the reduced content of the order-promoting residues. 129 functional protein clouds: major functional advantages of being intrinsically disordered the high natural abundance of idpds/idprs and their specific structural features indicate that these proteins and regions might carry out important biological functions. this hypothesis has been confirmed by several comprehensive studies, 1, [11] [12] [13] [14] [71] [72] [73] 78, [130] [131] [132] [133] [134] which revealed that these structure-less members of the protein kingdom are abundantly involved in numerous biological processes, where they are frequently found to play different roles in regulation of the functions of their binding partners and in promotion of the assembly of supra-molecular complexes. 1, 4, [11] [12] [13] [14] [15] 31, [70] [71] [72] [73] [76] [77] [78] [79] 131, 132, [134] [135] [136] [137] [138] [139] [140] [141] [142] [143] [144] [145] [146] [147] [148] [149] the conformational plasticity of idps/idprs provides them with a wide spectrum of exceptional functional advantages over the functional modes of ordered proteins and domains. 4, 10, 11, 13, 32, 71, 72, 77, 78, 131, 132, 134, 141, 142, 150, 151 some of these advantages are: 1 increased speed of interaction due to greater capture radius and the ability to spatially search through interaction space; 2 increased interaction (surface) area per residue; 3 strengthened encounter complex allows for less stringent spatial orientation requirements; 4 efficient regulation via rapid degradation; 5 the ability to be involved in one-to-many binding, where a single disordered region binds to several structurally diverse partners; 6 the ability to be involved in many-to-one binding, where many distinct (structured) proteins may bind a single disordered region; 7 the ability to overcome steric restrictions, enabling larger interaction surfaces in protein-protein and protein-ligand complexes than those obtained with rigid partners; 8 the ability to fold upon binding (completely or partially); 9 the ability of some idps/idprs to form very stable intertwined complexes; 10 the ability of some idps/idprs to stay substantially disordered in bound state; 11 binding fuzziness, where different binding mechanisms (e.g., via stabilizing the binding-competent secondary structure elements within the contacting region, or by establishing the longrange electrostatic interactions, or being involved in transient physical contacts with the partner, or even without any apparent ordering) can be employed to accommodate peculiarities of interaction with various partners; 12 binding plasticity, where an idpr folds to specific bound conformations (which can be very different) according to the template provided by binding partners; 13 high accessibility of sites targeted for posttranslational modifications (ptms); 14 efficient structural and functional regulation via ptms such as phosphorylation, acetylation, lipidation, ubiquitination, sumoylation, and so forth, allowing for a simple means of modulation of their biological functions; 15 efficient functional control via regulatory proteolytic attack sites of which are frequently associated with idprs; 16 ease of regulation/redirection and production of otherwise diverse forms by alternative splicing (given the existence of multiple functions in a single disordered protein, and given that each functional element is typically relatively short, alternative splicing could readily generate a set of protein isoforms with a highly diverse set of regulatory elements 152 ); 17 the possibility of overlapping binding sites due to extended linear conformation; 18 decoupled binding affinity and specificity, where, due to the induced folding, idp/idpr can be involved in the formation of specific but weak complexes. in other words, idp/idpr might possess high specificity for given partners combined with high k on and k off rates that enable rapid association with the partner without an excessive binding strength. this combination of high specificity with low affinity defines the broad utilization of intrinsic disorder in regulatory interactions where turning a signal off is as important as turning it on; 19 diverse evolutionary rates with some id proteins being highly conserved and other id proteins possessing high evolutionary rates. the latter ones can evolve into sophisticated and complex interaction centers (scaffolds) that can be easily tailored to the needs of divergent organisms; 20 flexibility that allows masking (or not) of interaction sites or that allows interaction between bound partners; 21 the ability to be involved in the cascade interactions, where idp binding to the first partner induces partial folding generating a new binding site suitable for interaction with the second partner, and so forth. many disorderrelated functions (e.g., signaling, control, regulation, and recognition) are incompatible with well-defined, stable 3-d structures. 1, [11] [12] [13] [14] 31, 73, 78, 79, 132, 134, [138] [139] [140] 142, 144, 153 functions of many idps/idprs rely on interactions with specific binding partners, and many idps/idprs tend to undergo disorder-to-order transitions as a result of binding to their specific targets. 12 functionally, idps/idprs were grouped in at least six broad classes based on the mode of action. 14,136 these broad classes included protein and rna chaperones, entropic chains, effectors, scavengers, assemblers and display sites, 14,136 and 28 separate functions, including molecular recognition via binding to other proteins, or to nucleic acids, were assigned for idprs in early studies. 71, 72 later, a rich spectrum of biological functions associated with idps/idprs was found based on a comprehensive computational study of a correlation between the functional annotations in the swiss-prot database and predicted intrinsic disorder. [138] [139] [140] the approach was based on the hypothesis that if a function described by a given keyword relies on intrinsic disorder, then the keyword-associated protein would be expected to have a greater level of predicted disorder compared to the protein randomly chosen from the swiss-prot. this analysis revealed that 44% and 34% of swiss-prot functional keywords were associated with ordered and disordered proteins, respectively, whereas 22% functional keywords yielded ambiguity in the likely function-structure associations. [138] [139] [140] interestingly, most of the structured protein-associated key words were shown to be related to enzymatic activities, whereas the majority of the disordered protein-associated keywords were related to signaling and regulation. these results agree well with the notion that enzymatic catalysis requires ordered structure and that effectiveness of signaling is dependent on binding reversibility, a property directly associated with the thermodynamics of disorder-to-order transition induced by binding. [138] [139] [140] many idps and idprs undergo a disorderto-order transition upon functioning. 11, 13, 15, 71, 72, 78, 79, [130] [131] [132] 134, [154] [155] [156] [157] when disordered regions bind to signaling partners, the free energy required to bring about the disorder to order transition takes away from the interfacial, contact free energy, with the net result that a highly specific interaction can be combined with a low net free energy of association. 13, 155 high specificity coupled with low affinity is a useful pair of properties for a reversible signaling interaction. furthermore, a disordered protein can readily bind to multiple partners by changing shape to associate with different targets. 13, 158, 159 all this clearly suggests that there is a new twopathway protein structure-function paradigm, with sequence-to-structure-to-function for enzymes and membrane transport proteins, and sequence-to-disordered ensemble-to-function for proteins and protein regions involved in signaling, regulation, and control. 1, 13, 71, 73, 79 one of the first generalization of this concept was given by the protein trinity hypothesis, which suggested that native proteins can be in one of three states, the solid-like ordered state, the liquid-like collapsed-disordered state, or the gas-like extended-disordered state. 79 function is then viewed to arise from any one of the three states or from transitions between them. this model was subsequently expanded to include a fourth state (pre-molten globule) and transitions between all four states. 1 in reality, based on the outlined above idea of the continuous spectrum of protein structures, functional proteins contain various amounts of intrinsic disorder and this continuous structural spectrum of protein defines their limitless functional variability. among intriguing protein functions relying on intrinsic disorder are moonlighting activities, 137 actions of hub proteins, 78, 93, 134, [160] [161] [162] [163] [164] and scaffolding functions. 141, 165 since all these functions illustrate the notions that the intrinsic disorder concept represents a universal skeleton key (or lock-pick) that helps unlocking seemingly unresolvable mysteries of protein science and therefore can be considered as a new ariadne's thread that helps navigate the unusual twists of the sophisticated relationships between protein sequence, structure, and function, they are considered in some detail below. moonlighting proteins. moonlighting is the ability of a protein to fulfill more than one function. often, these functions are unrelated or at least are not obviously related to each other. 137, [166] [167] [168] the capability of a protein to be involved in moonlighting or multi-tasking activities represent one of the solutions used by the nature to increase the organism's complexity without the expansion of the genome size, where by acting differently at distinct points of metabolic networks proteins increase network complexity without increasing the actual size of the network. 137, [166] [167] [168] among various molecular mechanisms used by the moonlighting proteins to switch between functions are changes in cellular localization, changes in ligand binding, expression in different cell types, and variations of the oligomerization state. 137 in addition to these mechanisms that can be explained within the frames of the traditional structure-function paradigm, consideration of the intrinsic disorder phenomenon opens new possibilities. 137 in fact, one of the peculiar functional advantages of idps/idprs is their binding promiscuity and ability to be involved in one-to-many signaling, whereby an idp/idpr binds structurally different partners in a template-induced folding process. 11, 77, 132, 169 therefore, idps/idprs can use the same region or overlapping interaction regions/surfaces to exert distinct effects and employ the disorderbased mechanisms to switch function that relies on their capability to form different conformations upon binding. 137 such structural malleability of idps/ idprs defines their ability to participate in unprecedented moonlighting events, where these disordered moonlighting proteins or regions produce the opposing effects (inhibition and activation) on different partners or even the same partner molecule. 137 hub proteins. signaling interactions inside the cell can be described as specific and complex networks that can be considered as "scale-free" or "small-world" networks, which have hubs, with many connections, and ends, that have the only connection to just one neighbor. 170, 171 such scale-free networks combine the local clustering of connections characteristic of regular networks with occasional long-range connections between clusters, as can be expected to occur in random networks. in other words, the distance between nodes in these scalefree networks follows a power-law distribution. 172 based on their spatiotemporal peculiarities protein hubs were grouped into two broad categories, "date hubs" that binds their numerous partners sequentially, and "party hubs" simultaneously interacting with their partners. 173 since many idps are known to be involved in interaction with large number of distinct partners, they clearly can be considered as hubs in the scale-free protein-protein interaction networks. 78, 134 based on the systematic analysis of several know hub proteins 134 followed by a series of robust bioinformatics studies, 93,160-164 it was concluded that hubs commonly use disordered regions to bind to multiple partners and that there are at least two primary mechanisms by which disorder is utilized in protein-protein interaction networks where one disordered region binds to many partners or many disordered region bind to one partner. 134 scaffold proteins. scaffold proteins constitute an important subclass of hubs that typically have a modest number of interacting partners and that are commonly found at the central parts of functional complexes, where they interact with most of their partners at the same time and therefore act as party hubs. 160 besides being responsible for bringing together specific proteins within a signaling pathway and providing selective spatial orientation and temporal coordination to facilitate and promote interactions among interacting proteins, some scaffolds can influence the specificity and kinetics of signaling interactions via simultaneous binding to multiple participants in a particular pathway and facilitation and/or modifying the specificity of pathway interactions, 174 other scaffold can change conformations of individual proteins and thus modulate their activities, 174 still other scaffold proteins may modulate the activation of alternative pathways by promoting interactions between various signaling proteins. 141 analysis of several well-characterized signaling scaffold proteins reveled that their large idprs are crucial for the successful scaffold function. 141 a more global bioinformatics analysis revealed that a typical design of a scaffold protein includes a set of short globular domains (80 amino acids on average) connected by long linker regions (150 residues on average) with crucial binding functions. 165 this gave further support to the notion that signaling scaffold proteins utilize the various features of highly flexible id regions to obtain more functionality from less structure. 141 disorder and transcription regulation. conformational plasticity and adaptability associated with intrinsic disorder are crucial for various protein functions. among the proteins whose functional life is strongly disorder-dependent are transcription factors (tfs) 175, 176 and other proteins involved in transcriptional regulation, such as the mediator complex, 24,177 core and linker histones, 178 and ribosomal proteins. 179 for example, from 83 to 94% of tfs might possess long idprs, with the degree of disorder in eukaryotic tfs being significantly higher than in prokaryotic tfs. 175, 176 also, tfs were shown to be depleted in order-promoting residues and enriched in disorder-promoting residues, and were characterized by high levels of a-molecular recognition feature (morf). 175 furthermore, disorder is unevenly distributed within the tfs, with the degree of disorder in their activation regions being much higher than that in dna-binding domains. however, the at-hooks (which are dna-binding motifs present in many proteins which binds to the (ataa) and (tatt) repeats of dna) and basic regions of tf dna-binding domains are highly disordered suggesting that eukaryotes with their well-developed gene transcription machinery require transcription factor flexibility to be more efficient. 175 a number of interesting and important roles were also ascribed to intrinsic disorder in tfs related to the regulation of heat shock response (so called heat shock factors, hsfs) 180 and in the reprogramming tfs (the yamanaka factors, namely sox2, oct3/4 (pou5f1), klf4, and c-myc, and the thomson factors, namely sox2, oct3, lin28, and nanog) overexpression of which is known to generate induced pluripotent stem (ips) cells from terminally differentiated somatic cells. 181 disorder in the regulation of cellular pathways. of special interests are the vital roles of intrinsic disorder in regulation and orchestration of various cellular pathways. one of the illustrative examples of this regulatory role of intrinsic disorder is the canonical wnt-pathway that involves five proteins, axin, cki-a, gsk-3b, apc (adenomatous polyposis coli, also known as deleted in polyposis 2.5 protein), and b-catenin (all shown to contain long idprs). this pathway is known to play a number of crucial roles in the development of organism, and the malfunctions of which might lead to various diseases including cancer. 182 the comprehensive analysis of published data revealed that idprs found in wntpathway proteins orchestrate protein-protein interactions, and facilitate ptms and signaling. 182 furthermore, the scaffold protein axin and another large protein, apc, are heavily enriched in disorder and act as flexible concentrators in gathering together all other proteins involved in the wnt-pathway. 182 intriguingly, the multifarious roles of highly disordered apc in regulation of b-catenin function were established by showing that disordered apc helps the collection of b-catenin from cytoplasm, facilitates the bcatenin delivery to the binding sites on axin, and controls the final detachment of b-catenin from axin. 182 another important illustration of the involvement of intrinsic disorder in regulation of crucial pathway is given by the process of the programmed cell death (pcd), which is one of the most intricate cellular processes where the cell uses specialized uversky cellular machinery and intracellular programs to kill itself and which enables metazoans to control cell numbers and eliminate cells that threaten the animal's survival. 183 pcd includes several specific modules, such as apoptosis, autophagy, and programmed necrosis (necroptosis). these modules are not only tightly regulated but also intimately interconnected and are jointly controlled via a complex set of protein-protein interactions. recently, several large sets of pcd-related proteins across 28 species were analyzed using a wide array of modern bioinformatics tools to understand the role of the intrinsic disorder in controlling and regulating the pcd. 183 this analysis revealed that proteins involved in regulation and execution of pcd possess substantial amount of intrinsic disorder and idprs were implemented in a number of crucial functions, such as protein-protein interactions, interactions with other partners including nucleic acids and other ligands, were shown to be enriched in post-translational modification sites, and were characterized by specific evolutionary patterns. 183 unique catalytic function of a protein is believed to be dictated by its unique 3d structure. this axiom constitutes a cornerstone of the lock-and-key paradigm and it seemed to be able to sustain the furious attack on protein structure-function relationship initiated by the discovery of idps and hybrid proteins with ordered domains and idprs. in fact, from the vast majority of experimental and computational studies a general conclusion was drawn over and over again, where the functional repertoire of idps complemented the functional arsenal of ordered proteins, with ordered proteins being mostly responsible for catalysis and transport and with idps doing the majority of other jobs in the cell. on the other hand, all proteins (even the most ordered and tightly folded ones) are intrinsically flexible molecules that undergo conformational changes over a wide range of timescales and amplitudes. 184 in fact, the combination of active site reactivity with the dynamic character of proteins allows enzymes to be promiscuous and remarkably efficient at the same time. 185 furthermore, in general, dynamic fluctuations are crucial for enzyme catalysis, since they can influence substrate binding and product release, and may even adjust the effective barriers of the catalyzed reactions. [186] [187] [188] [189] [190] often, dynamic changes in the enzyme during the catalytic reaction can be described using the induced-fit model, where a conversion of one tight conformational ensemble (free enzyme) to another distinct ensemble (bound enzyme) takes place through a series of local substrate-mediated structural rearrangements. 191 despite this crucial role of local flexibility in the enzymatic catalysis, enzymes are still relatively stable molecules whose dynamic character is restricted to a small set of tightly folded conformations and whose unique (albeit locally flexible) structures are needed for efficient catalysis. from this viewpoint, the presence of intrinsic disorder is expected to be poorly compatible with enzymatic catalysis, which requires a well-organized environment in the active site of the enzyme in order to facilitate the formation of the transition state of the chemical reaction to be catalyzed. 192 in a sharp contrast to this common wisdom supported by a wide array of specific examples, several enzymes were shown to be much more dynamic than the catalytic machines are expected to be, clearly possessing, in their precatalytic states, many characteristic properties of molten globules and retaining unusually high flexibility in structurally defined enzyme-ligand complexes. one of the best characterized examples of such molten globular enzymes is the engineered monomeric form of chorismate mutase from methanococcus jannaschii (mjcm). 184, [193] [194] [195] here, a functional monomer (mmjcm) was created by inserting the hinge-loop sequence into the long, dimer-spanning n-terminal helix. 193 in its unbound form, mmjcm was shown to exists as a native molten globule that was described as a dynamic ensemble of a-helical conformers rapidly interconverting on the millisecond timescale. 193 interaction with natural ligand induced global conformational changes in the molten globular mmjcm promoting formation of a defined enzyme-ligand complex, which, however, preserved unusually high flexibility. 184 catalytic mechanism of the molten globular mmjcm was described as follows: "though probably stochastic in nature, internal motions in the complex may generate a collective dynamic matrix that samples catalytically active conformation(s) often enough to achieve rapid turnover in the presence of the true transition state." 184 therefore, some enzymes can represent a highly dynamic heterogeneous conformational ensemble which is still compatible with efficient catalysis. in agreement with this hypothesis, a molten globular character was described for circularly permuted dihydrofolate reductase (dhfr), 196, 197 and urease g from bacillus pasteurii (bpureg). [198] [199] [200] of these three enzymatic molten globules ureg is the only natural molten globular enzyme known to date, since both circularly permuted dhfr and monomeric mjcm were obtained as a result of some genetic manipulations. although the number of known native molten globules with enzymatic activity is small, their existence provides an interesting hint on early protein evolution. in fact, simple logics suggests that well-ordered enzymes appear as a result of long evolutionary process, whose very likely starting point was a partially folded polypeptide with some general properties of the molten globule. idps/idprs can form highly stable complexes, or be involved in signaling interactions where they undergo constant "bound-unbound" transitions, thus acting as dynamic and sensitive "on-off" switches. the ability of these proteins to return to the highly flexible conformations after the completion of a particular function, and their predisposition to gain different conformations depending on the environmental peculiarities, are unique physiological properties of idps which allow them to exert different functions in different cellular contests according to a specific conformational state. 4 due to their lack of rigid structure, combined with the high level of intrinsic dynamics and almost unrestricted flexibility at various structure levels in the non-bound state, as well as due to the unique capability to adjust to structure of the binding partner, idps are characterized by a very diverse range of binding modes, creating a multitude of unusual complexes, many of which are not attainable by ordered proteins. 201 some of these complexes are relatively static, resemble complexes of ordered proteins, and, therefore are suitable for the structure determination by x-ray crystallography. among these static complexes are: morfs, wrappers, chameleons, penetrators, huggers, intertwined strings, long cylindrical containers, connectors, armature, tweezers and forceps, grabbers, tentacles, pullers, and stackers or b-arcs. 201 these binding modes are shown in supporting information figure 1s and briefly described in the supporting information materials. in addition to the static complexes, where bound partners have fixed structures, some idps/idprs do not fold even in their bound state, forming so-called disordered, dynamic, or fuzzy complexes with ordered proteins, 97, [202] [203] [204] [205] [206] other disordered proteins, [207] [208] [209] or biological membranes. 210, 211 in complexes of some of these idps with their binding partners, the disordered regions flanking the interaction interface but not the interface itself remain disordered. such mode of interaction was recently described as "the flanking fuzziness" in contrast to "the random fuzziness" when the disordered protein remains entirely disordered in the bound state. 75, 212 it is also expected that the similar binding mode can be utilized by disordered protein while interacting with nucleic acids and other biological macromolecules. 201 physically, binding is considered as joining objects together and suggests spatial and temporal fixation of bound partners. the formation of protein complexes with specific binding partners is expected to bring some fixation (at least at the binding site). therefore, disordered complexes where interaction of a disordered protein with the binding partners is not accompanied by a disorder-to-order transition within the interaction interface clearly cannot be described by the classical binding paradigm. this contradiction can be resolved assuming that the ordered binding partner and/or disordered protein contain multiple low affinity binding sites. the existence of several similar binding sites combined with a highly flexible and dynamic structure of disordered protein creates a unique situation where any binding site of disordered protein can interact with any binding site of its partner with almost equal probability, in a staccato manner. the low affinity of each individual contact implies that each of them is not stable and can be readily broken. therefore, such disordered or fuzzy complex can be envisioned as a highly dynamic ensemble in which a disordered protein does not present a single binding site to its partner but resemble a "binding cloud," in which multiple identical binding sites are dynamically distributed in a diffuse manner. in other words, in this staccato-type interaction mode, an disordered protein rapidly changes multiple binding sites while probing binding site(s) of its partner. 201 an additional factor which can help holding a dynamic complex together could be a weak longrange attraction between protein molecules. 213 this long-range attraction is universal for all protein solutions and has a range several times that of the diameter of the protein molecule, much greater than the range of the screened electrostatic repulsion. 213 the most common outcome of these function-related structural changes is the overall increase in the amount of ordered structure. however, functions of some ordered proteins require local or even global unfolding of a unique protein structure. 68 among specific features of these structural alterations are their induced nature and transient character combined with a wide range of molecular mechanisms by which they can be promoted. 68 these functional unfolding-activating factors include light; mechanical force; changes in ph, temperature, or redox potential; interaction with membrane, ligands, nucleic acids, and proteins; various ptms; release of autoinhibition due to the unfolding of autoinhibitory domains induced by their interaction with nucleic acids, proteins, membranes, ptms, and so forth. 68 among rather unusual factors used by nature to activate proteins via functional unfolding are light and mechanical force. for example, exposure to blue light results in the activation of the photoactive yellow protein (pyp), which is an ordered, water-soluble 14 kda protein that contains a thioester linked uversky p-coumaric acid cofactor and serves as a photosensor in ectothiorhodospira halophila. 214, 215 pyp is a bacterial blue light sensor that undergoes conformational changes upon signal transduction. the absorption of a photon triggers substantial protein unfolding and leads to the formation of the transient signaling state that interacts with the partner molecules. this allows the swimming bacterium to operate the directional switch that protects it from harmful illumination. comprehensive analysis combining double electron electron resonance spectroscopy (deer), high resolution nmr, and timeresolved pump-probe x-ray solution scattering (tr-saxs/waxs) revealed that the transiently activated and short-lived signaling state of the pyp possessed a large degree of disorder and existed as an ensemble of multiple conformers that exchange on a millisecond time scale. 216, 217 this unusual behavior is illustrated in figure 4 that shows structures of inactive folded pyp and its light-activated functional form, which is highly disordered. 68 some proteins undergo local unfolding induced by the mechanical force and therefore can serve as force sensors. 68 among these natural force sensors are mechanosensitive ion channels that recognize and respond to the membrane tension, which is the mechanical forces applied along the plane of the cell membrane, rather than to the hydrostatic pressure perpendicular to the membrane plane. 220 these ion channels are activated via partial unfolding of some of their functional parts induced by membrane tension. 221 for a long time, the fact that idps/idprs undergo disorder-to-order transitions either during their functions or in order to be functional was used as one of the strongest arguments against the idea of protein intrinsic disorder. it was stated that most idps (those which are not the artifacts of current methods of protein production) are in fact proteins waiting for a partner (pwps) that serve as parts of a multi-component complex and that do not fold correctly in the absence of other components. 29 therefore, when folded after binding to their partners, these proteins are not too different from typical ordered proteins. however, one need to keep in mind that a portion of "folding code" that defines the ability of ordered proteins to spontaneously gain a unique biologically active structure is missing for idps/idprs since they cannot fold spontaneously. this missing portion of the "folding code" (or a part of it) can be supplemented by binding partner(s). as a result, ordered and disordered proteins can be discriminated on a simple basis of temporal correlation between their folding and binding: ordered proteins fold first and then bind to their partners while the idps/idprs remain disordered until they bind their partners and often preserve substantial disorder in the bound state. 69 furthermore, numerous cases of functional unfolding (or transient disorder, or upside-down functionality) represent further support to the concept of functional disorder by clearly showing that many proteins possess dormant disorder that needs to be awakened in order to make these proteins functional. it is clear now that the idps and idprs are real, abundant, diversified, and vital. the highly dynamic nature of idps and idprs is a visual illustration of the chaos. however, the evolutionary persistence of these highly dynamic proteins (see below), their unique functionality, and involvement in all the major cellular processes evidence that this chaos is tightly controlled. 147 to answer the question as to . ground state structure was determined by multidimensional nmr spectroscopy. 218 this structure is in agreement with an earlier published 1.4 å crystal structure, 219 and modeled structure based on combined deer, tr-saxs/waxs, and nmr data. 217 it consists of an open, twisted, 6-stranded, antiparallel b-sheet, which is flanked by four ahelices on both sides. [217] [218] [219] on the contrary, the light-activated form is highly disordered. this structure satisfies deer, saxs/ waxs, and nmr data simultaneously. 217 how these proteins are governed and regulated inside the cell, gsponer et al. conducted a detailed study focused on the intricate mechanisms of idp regulation. 222 to this end, all the saccharomyces cerevisiae proteins were grouped into three classes using one of the available disorder predictors, dis-opred2 44 : (i) 1971 highly ordered proteins containing 0-10% of the predicted disorder; (ii) 2711 moderately disordered proteins with 10-30% predicted disordered residues; and (iii) 2020 highly disordered proteins containing 30-100% of the predicted disorder. then, the correlations between intrinsic disorder and the various regulation steps of protein synthesis and degradation were evaluated. this analysis revealed that the transcriptional rates of mrnas encoding idps and ordered proteins were comparable. however the idp-encoding transcripts were generally less abundant than transcripts encoding ordered proteins due to the increased decay rates of the transcripts of genes encoding idps. 222 furthermore, idps were shown to be less abundant than ordered proteins due to the lower rate of protein synthesis and shorter protein half-lives. as the abundance and half-life in a cell of certain proteins can be further modulated via their ptms such as phosphorylation, 223 the experimentally determined yeast kinase-substrate network was also analyzed. idps were shown to be substrates of twice as many kinases as were ordered proteins. furthermore, the vast majority of kinases whose substrates were idps were either regulated in a cell-cycle dependent manner, or activated upon exposure to particular stimuli or stress. 222 therefore, ptms may not only serve as important mechanism for the fine-tuning of the idp functions but possibly they are necessary to tune the idp availability under the different cellular conditions. 222 in addition to s. cerevisiae, similar regulation trends were also found in schizosaccharomyces pombe and homo sapiens. 222 based on these observations it has been concluded that both unicellular and multicellular organisms appear to use similar mechanisms for regulation of the intrinsically disordered protein availability. overall, this study clearly demonstrated that in eukaryotes, there is an evolutionarily conserved tight control of synthesis and clearance of most idps. this tight control is directly related to the major roles of idps in signaling, where it is crucial to be available in appropriate amounts and not to be present longer than needed. 222 it has been also pointed out that although the abundance of many idps is under strict control, some idps could be present in cells in large amounts or/and for long periods of time due to either specific ptms or via interactions with other factors, which could promote changes in cellular localization of idps or protect them from the degradation machinery. 13, 70, 138, 223, 224 overall, this study clearly showed that the chaos seemingly introduced into the protein world by the discovery of idps is under the tight control. 147 in an independent study, a global scale relationship between the predicted fraction of protein disorder and protein expression in e. coli was analyzed. 225 this study showed that the fraction of protein disorder was positively correlated with both measured rna expression levels of e. coli genes in three different growth media and with predicted abundance levels of e. coli proteins. 225 when a subset of 216 e. coli proteins that are known to be essential for the survival and growth of this bacterium were analyzed, the correlation between protein disorder and expression level became even more evident. in fact, essential proteins had on average a much higher fraction of disorder (0.36), had a higher number of proteins classified as completely disordered (19% vs. 2% for e. coli proteome), and were expressed at a higher level in all three media than an average e. coli gene. 225 the manual literature mining for a group of e. coli proteins that had high levels of predicted intrinsic disorder revealed that the disorder predictions matched well with the experimentally elucidated regions of protein flexibility and disorder. 225 a direct link between protein disorder and protein level in e. coli cells could be because the idps may carry out the essential control and regulation functions that are needed to respond to the various environmental conditions. another possibility is that idps might undergo more rapid degradation compared to structured proteins, which cells can counter by increasing mrna levels of the corresponding genes. in this case, higher synthesis and degradation rates could make the levels of these proteins very sensitive to the environment, with slight changes in either production or degradation leading to significant shifts in protein levels. 225 even more support for the tight control of idps inside the cell came from the analysis of cellular regulation of so-called "vulnerable" proteins. 23 the integrity of the soluble protein functional structures is maintained in part by a precise network of hydrogen bonds linking the backbone amide and carbonyl groups. in a well-ordered protein, hydrogen bonds are shielded from water attack, preventing backbone hydration and the total or partial unfolding of the soluble structure under physiological conditions. 226, 227 since soluble protein structures may be more or less vulnerable to water attack depending on their packing quality, a structural attribute, protein vulnerability, was introduced as the ratio of solvent-exposed backbone hydrogen bonds (which represent local weaknesses of the structure) to the overall number of hydrogen bonds. 23 it has been also pointed out that structural vulnerability can be related to protein intrinsic disorder as the inability of a particular protein fold to protect intramolecular uversky hydrogen bonds from water attack may result in backbone hydration leading to local or global unfolding. since binding of a partner can help to exclude water molecules from the microenvironment of the preformed bonds, a vulnerable soluble structure gains extra protection of its backbone hydrogen bonds through the complex formation. 226 to understand the role of structure vulnerability in transcriptome organization, the relationship between the structural vulnerability of a protein and the extent of co-expression of genes encoding its binding partners was analyzed. this study revealed that structural vulnerability can be considered as a determinant of transcriptome organization across tissues and temporal phases. 23 finally, by interrelating vulnerability, disorder propensity and co-expression patterns, the role of protein intrinsic disorder in transcriptome organization was confirmed, since the correlation between the extent of intrinsic disorder of the most disordered domain in an interacting pair and the expression correlation of the two genes encoding the respective interacting domains was evident. 23 because of the fact that idps are highly abundant and play crucial roles in numerous biological processes, it was not too surprising to find that some of them are involved in human diseases. for example, a number of human diseases originate from the deposition of stable, ordered, filamentous protein aggregates, commonly referred to as amyloid fibrils. in each of these pathological states, a specific protein or protein fragment changes from its natural soluble form into insoluble fibrils, which accumulate in a variety of organs and tissues. [228] [229] [230] [231] [232] [233] [234] several unrelated proteins including many idps are known to be involved in these protein deposition diseases. 234, 235 an illustrative examples of human neurodegenerative diseases associated with idps includes alzheimer's disease (deposition of amyloid-b, tau-protein, a-synuclein fragment nac) [236] [237] [238] [239] ; various taupathies (accumulation of tau-protein in the form of neurofibrillary tangles) 238 ; down's syndrome (nonfilamentous amyloid-b deposits) 240 ; parkinson's disease and other synucleinopathies (deposition of asynuclein) 241 ; prion diseases (deposition of prp sc ) 242 ; and a family of polyq diseases, a group of neurodegenerative disorders caused by expansion of gac trinucleotide repeats coding for polyq in the gene products. 243 furthermore, most mutations in rigid globular proteins associated with accelerated fibrillation and protein deposition diseases have been shown to destabilize the native structure, increasing the steady-state concentration of partially folded (disordered) conformers. [228] [229] [230] [231] [232] [233] [234] the maladies given above have been called conformational diseases, as they are characterized by the conformational changes, misfolding, and aggregation of an underlying protein. however, there is another side to this coin: protein functionality. in fact, many of the proteins associated with the conformational disorders are also involved in recognition, regulation, and cell signaling. for example, functions ascribed to a-synuclein, a protein involved in several neurodegenerative disorders, include binding fatty acids and metal ions; regulation of certain enzymes, transporters, and neurotransmitter vesicles; and regulation of neuronal survival (reviewed in ref. 241) . overall, there are about 50 proteins and ligands that interact and/or co-localize with this protein. furthermore, a-synuclein has amazing structural plasticity and adopts a series of different monomeric, oligomeric, and insoluble conformations (reviewed in ref. 24) . the choice between these conformations is determined by the peculiarities of the protein environment, suggesting that asynuclein has an exceptional ability to fold in a template-dependent manner. therefore, the development of the conformational diseases may originate not only from misfolding but also from the misidentification, misregulation, and missignaling of the related proteins. analysis of so-called polyglutamine diseases gives support to this hypothesis. 244 polyglutamine diseases are a specific group of hereditary neurodegeneration caused by expansion of cag triplet repeats in an exon of disease genes which leads to the production of a disease protein containing an expanded polyglutamine, polyq, stretch. nine neurodegenerative disorders, including kennedy's disease, huntington's diseases, spinocerebellar atrophy1, 22, 23, 26, 7, 17 , and dentatorubral pallidoluysian atrophy are known to belong to this class of diseases. [245] [246] [247] [248] in most polyq diseases, expansion to over 40 repeats leads to the onset. 248 it has been emphasized that such molecular processes as unfolded protein response, protein transport, synaptic transmission, and transcription are implicated in the pathology of polyq diseases. 244 importantly, more than 20 transcription-related factors have been reported to interact with pathological polyq proteins. furthermore, these interactions were shown to repress the transcription, leading finally to the neuronal dysfunction and death (reviewed in ref. 244) . these results suggest that polyq diseases represent kind of transcriptional disorder, 244 supporting our misidentification hypothesis for at least some of the conformational disorders. disorder is very common in cancer-associated proteins too. in a 2002 study, it was found that 79% of cancer-associated and 66% of cell-signaling proteins contain predicted regions of disorder of 30 residues or longer. 130 in contrast, only 13% of a set of proteins with well-defined ordered structures contained such long regions of predicted disorder. 130 in experimental studies, the presence of disorder has been directly observed in several cancer-associated proteins, including p53, 249 p57 kip2 , 250 bcl-x l and bcl-2, 251 c-fos, 252 a thyroid cancer associated protein tc-1, 253 ews-fli1 fusion protein that includes a potent transcriptional activator, the ews domain, alongside the highly conserved dna-binding domain fli1, 254,255 among many other examples. the best characterized example of the important cancerrelated idp is the tumor suppressor protein p53, which occupies the center of a large signaling network. p53 regulates expression of genes involved in numerous cellular processes, including cell cycle progression, apoptosis induction, dna repair, as well as others involved in responding to cellular stress. 256 when p53 function is lost, either directly through mutation or indirectly through several other mechanisms, the cell often undergoes cancerous transformation. 257, 258 cancers showing mutations in p53 are found in colon, lung, esophagus, breast, liver, brain, reticuloendothelial tissues, and hemopoietic tissues. 257 p53 is regulated by several different mechanisms including inhibition of its activity by interaction with e3 ubiquitin ligase mdm2, which binds to a short stretch of p53 located within the transactivation domain. mdm2-bound p53 cannot activate or inhibit other genes. mdm2 ubiquitinates p53 and thus targets it for destruction. mdm2 also contains a nuclear export signal that causes p53 to be transported out of the nucleus. 259, 260 the possibility of interrupting the action of diseaseassociated proteins (including through modulation of protein-protein interactions) presents an extremely attractive objective for the development of new drugs. since many proteins associated with various human diseases are either completely disordered or contain long disordered regions, 261, 262 and since some of these disease-related idps/idprs are involved in recognition, regulation, and signaling, these proteins/regions clearly represent novel potential drug targets. 27 due to failure to recognize the important role of disorder in protein function, current and evolving methods of drug discovery suffer from an overly rigid view of protein function. in fact, the rational design of enzyme inhibitors depends on the classical view where 3d-structure is an obligatory prerequisite for function. while generally applicable to many enzymatic domains, this view has persisted to influence thinking concerning all protein functions despite numerous examples to the contrary. this is most apparent in the observation that the vast majority of currently available drugs target the active site of enzymes, presumably since these are the only proteins for which the "unique structure-unique function" paradigm is generally applicable. idps often bind their partners with relatively short regions that become ordered upon binding. [263] [264] [265] targeting disorder-based interactions should enable the development of more effective drug discovery techniques. there are at least two potential approaches for the inhibition of the disorder-based interactions, where small molecule either bind to the binding site of the ordered partner to outcompete the idps/idprs or interacts directly with the idp/ idpr. the principles of small molecule binding to idprs have not been well studied, but sequence specific, small molecule binding to short peptides has been observed. 266 an interesting twist here is that small molecules can inhibit disorder-based proteinprotein interactions via induction of the dysfunctional ordered structures in targeted idpr, that is, via the drug-induced misfolding. in agreement with these concepts, small molecules "nutlins" have been discovered that inhibited the p53-mdm2 interaction by mimicking the inducible a-helix in p53 (residues 13-29) that binds to mdm2. 259, 260 although x-ray crystallographic studies of the p53-mdm2 complex revealed that the mdm2 binding region of p53 forms an a-helical structure that binds into a deep groove on the surface of mdm2, 267 nmr studies showed that the unbound n-terminal region of p53 lacks fixed structure, although it does possess an amphipathic helix part of the time. 249 a close examination of the interface between the proteins reveals that phe 19 , trp 23 , and leu 26 of p53 are the major contributors to the interaction, with the side chains of these three amino acids pointing down into a crevice on the mdm2 surface. 259, 260 the structure of nutlin-2 was shown to mimic the crucial residues of p53, with two bromophenyl groups fitting into mdm2 in the same pockets as trp 23 and leu 26 , and an ethyl-ether side chain filling the spot normally taken by phe 19 . [268] [269] [270] nutlins and related small molecules increased the level of p53 in cancer cell lines. this drastically decreased the viability of these cells, causing most of them to undergo apoptosis. when one of the nutlins was given orally to mice, a 90% inhibition of tumor growth compared to the control was induced. 260, [268] [269] [270] this successful nutlin story marks the potential beginning of a new era, the signaling-modulation era, in targeting drugs to protein-protein interactions. importantly, this druggable p53-mdm2 interaction involves a disorder-to-order transition. principles of such transitions are generally understood and therefore can use to find similar drug targets, which are inducible a-helices. 271 in addition to nutlins inhibiting p53-mdm2 interaction, several other small molecules also act by blocking proteinprotein interactions. 272, 273 some of these interactions involve one structured partner and one disordered partner, with disordered segments becoming a-helix upon binding. 271 therefore, the p53-mdm2 complex is not a unique exception and many other disorderbased protein-protein interactions are blocked by a small molecule. all this suggest that there is a cornucopia of new drug targets that would operate by blocking disorder-based protein-protein interactions. for these p53-mdm2-type examples, the drug molecules mimic a critical region of the disordered partner (which folds upon binding) and compete with this region for its binding site on the structured partner. these druggable interaction sites operate by the coupled binding and folding mechanism. they are small enough and compact enough to be easily mimicked by small molecules. 25 methods for predicting such binding sites in disordered regions have been developed 274 and the bioinformatics tools to identify which disordered binding regions can be easily mimicked by small molecules have been elaborated. 271 a complementary approach for small molecules to inhibit the disorder-based protein-protein interactions relies on the direct binding of drugs to the idps/idprs, which is illustrated by the c-myc-max story. 275 in order to bind dna, regulate expression of target genes, and function in most biological contexts, c-myc transcription factor must dimerize with its obligate heterodimerization partner, max, which lacks a transactivation segment. both c-myc and max are intrinsically disordered in their monomeric forms. upon heterodimerization, they undergo coupled binding and folding of their basic-helix-loophelix-leucine zipper domains (bhlhzips). since the deregulation of c-myc is related to many types of cancer, the disruption of the c-myc-max dimeric complex is one of the approaches for c-myc inhibition. several small molecules were found to inhibit the c-myc-max dimer formation. 275 these molecules were shown to bind to one of the three discrete sites within the 85-residue bhlhzip domain of c-myc, which are composed of short contiguous stretches of amino acids that can selectively and independently bind small molecules. 275 inhibitor binding induces only local conformational changes, preserves the overall disorder of c-myc, and inhibits interaction with max. 275 furthermore, binding of inhibitors to c-myc was shown to occur simultaneously and independently on the three independent sites. based on these observations it has been concluded that a rational and generic approach to the inhibition of protein-protein interactions involving idps may therefore be possible through the targeting of intrinsically disordered sequence. 275 recently, a functional misfolding concept was introduced to describe a mechanism preventing idps from unwanted interactions with non-native partners. 276 idps/idprs are characterized by high conformational dynamics and flexibility, the presence of sticky preformed binding elements, and the ability to morph into differently-shaped bound configurations. however, detailed analyses of the conformational behavior and fine structure of several idps revealed that the preformed binding elements might be involved in a set of non-native intramolecular interactions. based on these observations it was proposed that an intrinsically disordered polypeptide chain in its unbound state can be misfolded to sequester the preformed elements inside the noninteractive or less-interactive cage, therefore preventing these elements from the unnecessary and unwanted interactions with non-native binding partners. 276 it is important to remember, however, that the mentioned functional misfolding is related to the ensemble behavior of transiently populated elements of structure. in other words, it describes the behavior of a globally disordered polypeptide chain containing highly dynamic elements of residual structure, so-called interaction-prone preformed fragments, some of which could potentially be related to protein function. 276 this ability of idrps/idprs to functionally misfold can be used for finding small molecules which would potentially stabilize different members of the functionally misfolded ensemble, and therefore prevent the targeted protein from establishing biological interactions. 277 this approach is very different from the discussed above direct targeting of short idprs since it is based on a small molecule binding to a highly dynamic surface created via the transient interaction of preformed interaction-prone fragments. in essence, this approach can be considered as an extension of the well-established structure-based rational drug design elaborated for ordered proteins. in fact, if the structure of a member(s) of the functionally misfolded ensemble can be guessed, then this structure can be used to find small molecules that are potentially able to interact with this structure, utilizing tools originally developed for the rational structure-based drug design for ordered proteins. 277 ideally, a drug that targets a given protein-protein interaction should be tissue specific. although some proteins are unique for a given tissue, many more proteins have very wide distribution, being present in several tissues and organs. how can one develop tissue-specific drugs targeting such abundant proteins? often, tissue specificity for many of the abundant proteins is achieved via the alternative splicing of the corresponding pre-mrnas, which generates two or more protein isoforms from a single gene. estimates indicate that between 35 and 60% of human genes yield protein isoforms by means of alternatively spliced mrna. 278 the added protein diversity from alternative splicing is thought to be important for tissue-specific signaling and regulatory networks in the multicellular organisms. the regions of alternative splicing in proteins are enriched in intrinsic disorder, and it was proposed that associating alternative splicing with protein disorder enables the time-and tissue-specific modulation of protein function. 152 since disorder is frequently utilized in protein binding regions, having alternative splicing of pre-mrna coupled to idprs can define tissue-specific signaling and regulatory diversity. 152 these findings open a unique opportunity to develop tissue-specific drugs modulating the function of a given id protein/region (with a unique profile of disorder distribution) in a target tissue and not affecting the functionality of this same protein (with different disorder distribution profile) in other tissues. wavy pattern of global evolution of intrinsic disorder idps/idprs are more common in eukaryotes than in less complex organisms. 43, 44, [48] [49] [50] [51] [52] this suggests that disorder, with its ability to be implemented in various signaling, recognition, and regulation pathways and networks, is important for the maintenance of life in eukaryotic and especially muticellular eukaryotic organisms. 4, 45, 78, 134 also, the finding that alternatively spliced regions of mrna code for idprs much more often than for structured regions suggests that there is a linkage between alternative splicing and signaling by idprs that constitutes a plausible mechanism that could underlie and support cell differentiation, which ultimately gave rise to the multicellular eukaryotic organisms. 152 therefore, one can assume that intrinsic disorder represents a relatively recent evolutionary invention. however, this hypothesis obviously would be wrong if earlier stages of evolution would be taken into account. in fact, the chances that the first polypeptides that appeared in the primordial soup of the primitive earth possessed well-developed and unique 3d structures are minimal. the earth formed about 4.5 billion years ago. scientists dated the first fossils to 3.85 billion years ago. there are still debates and different theories about what happened in those years between the time the earth was cool enough to spawn life and the time the first fossils were formed. at the beginning of the 20th century, oparin 279 and haldane 280 proposed that some organic molecules could have been spontaneously produced from the gases of the primitive earth atmosphere, assuming that this primitive atmosphere was reducing (as opposed to oxygen-rich), and there was an appropriate supply of energy, such as lightning or ultraviolet light. thirty year later, this hypothesis (that constitutes a cornerstone of the theory of molecular evolution) received strong support from the elegant experiments of stanley l. miller and harold c. urey who were able to synthesize various organic compounds including some amino acids from non-organic compounds which were believed to represent the major components of the early earth's atmosphere (water vapor, hydrogen, methane, and ammonia) by putting them into a closed system and running a continuous electric current through the system, to simulate lightning storms believed to be common on the early earth. 281, 282 however, the miller-urey experiment yielded only about half of the modern amino acids 281, 282 suggesting that the first proteins on earth may have contained only a few amino acids. these findings go in parallel with the biosynthetic theory of the genetic code evolution suggesting that the genetic code evolved from a simpler form that encoded fewer amino acids, 283 probably paralleled by the invention of biosynthetic pathways for new and chemically more complex amino acids. 284 furthermore, some additional support of the validity of this hypothesis can be found in the standard genetic code (that consists of 4 3 4 3 4 5 64 triplets of nucleotides, codons), which is redundant (64 codons encodes for 20 amino acids). in fact, with only two exceptions, codons encoding one amino acid may differ in any of their three positions. however, only the third positions of some codons may be fourfold degenerate, that is, any nucleotide at this position specifies the same amino acid and all nucleotide substitutions at this site are synonymous. using these observations as a reflection of the evolutionary development, it was proposed that there was a period during code evolution where the third position was not needed at all and a doublet code preceded the triplet code, giving rise to 4 3 4 5 16 codons encoding for 16 or fewer amino acids, if a termination codon is taken into account. 285 based on these and many other premises, one can discriminate evolutionary old and new amino acids. in 2000, eduard n. trifonov combined 40 different single-factor criteria into a consensus scale and proposed the following temporal order of addition for the amino acids: g/a, v/d, p, s, e/l, t, r, n, k, q, i, c, h, f, m, y, w. 286 even superficial analysis of this sequence reveals that many of the early amino acids (such as g, d, e, p, and s) are disorder-promoting, as they are very abundant in modern idps. on the other hand, the major orderpromoting residues (c, w, y, and f) were added to the genetic code late. this observation is further illustrated by figure 5 (a) which represents modern genetic code, contains information on the early and late codons (shown by light red and light blue colors, respectively), and on corresponding disorder-and order-promoting residues (shown by red and blue colors, respectively). codons with intermediate age and disorder-neutral residues are shown by light pink and pink colors, respectively. figure 5 uversky illustrates that there is relatively good agreement between the "age" of the residue and its disorderpromoting capacity, with early residues being mostly disorder-promoting, and with the majority of late residues being mostly order-promoting. this conclusion follows from the abundance of the matching colors (light red-red, light blue-blue, and light pinkpink). there are only two noticeable exceptions from these rule, valine and leucine, which are early but order-promoting residues. this strongly suggests that the primordial polypeptides were intrinsically disordered. it is very unlikely that these disordered primordial polypeptides possessed catalytic activity. 287 this hypothesis is in line with the rna world theory suggesting that during the evolution of enzymatic activity, catalysis was transferred from rna first to ribonucleoprotein (rnp) and only then to protein. 288 therefore, the first proteins in the "breakthrough organism" (the first to have encoded protein synthesis) would be nonspecific chaperone-like proteins rather than catalysts. 136, 287 such rna chaperone activities of early proteins conferred to their carriers a significant selective advantage in the rna world, where rna, which is especially prone to misfolding, 289, 290 was used for both information storage and catalysis. 291 since the variability of physicochemical properties of amino acids greatly exceeds that of figure 5 . peculiarities of disorder evolution. a: modern genetic code with information on the early and late codons (shown by light red and light blue colors, respectively) and disorder-and order-promoting residues (shown by red and blue colors, respectively). codons with intermediate ages (i.e., those located between early and late codons) are shown by light pink color, whereas disorder-neutral residues are shown by pink color. b: wavy pattern of the global disorder evolution. x-axis represents evolutionary time and y-axis shows disorder content in proteins at given evolutionary time point. here, primordial proteins are expected to be mostly disordered (left-hand side of the plot), proteins in lua likely are mostly structured (center of the plot), whereas many protein in eukaryotes are either totally disordered or hybrids containing both ordered and disordered regions (right-hand side of the plot). nucleotides and since protein structures are noticeably more stable than rna structures, the transition from rnas (ribozymes) to proteins as carriers of enzymatic activity was a logical evolutionary step. however, efficient catalysis relies on the proper spatial arrangement of catalytic residues which requires a stable structure. 292 therefore, grafting of the enzymatic activity to proteins generated strong evolutionary pressure toward the well-folded structures. in other words, the global evolution of intrinsic disorder is characterized by a wavy pattern [see fig. 5 (b)], where highly disordered primordial proteins with primarily rna-chaperone activities were gradually substituted by the well-folded, highly ordered enzymes that evolved to catalyze the production of all the complex "goodies" crucial for the independent existence of the first cellular organisms. due to its specific features crucial for the regulation of complex processes, protein intrinsic disorder was reinvented at the subsequent evolutionary steps leading to the development of more complex organisms from the last universal ancestor (i.e., the most recent organism from which all organisms now living on earth descend 293, 294 ) , and culminating in the appearance of the highly elaborated eukaryotic cells [see there is no simple answer to the question on the comparative evolutionary rates of ordered and idps and regions in modern organisms. in fact, it looks like everything is possible, and intrinsically disordered sequences may evolve faster, slower or similar to ordered sequences. for example, disordered and ordered domains of the same protein (e.g., papillomavirus e7 oncoprotein) were shown to possess similar degrees of conservation and co-evolution. 295 many other idps/idprs were shown to be characterized by high evolutionary rates 151,296,297 determined by the lack of specific structural restrictions. in fact, the analysis of calcineurins, 10 topoisomerase, 298 ribosomal protein s4, 299 b-subunits of the potassium channel kvb1.1, 300 and many other proteins showed that disordered regions in these proteins contained more amino acid substitutions, insertions, and deletions than the ordered regions of the same proteins. 151, 301 furthermore, based on the observation that a significantly higher degree of positive darwinian selection was observed in idprs of proteins compared to regions of a-helix, b-sheet or tertiary structures, it was hypothesized that idprs may be required for the genetic variation with adaptive potential and that these regions may be of "central significance for the evolvability of the organism or cell in which they occur." 302 on the other hand, some idps and idprs are highly conserved. human a-synuclein (a canonical idp of 140 residues 140,303 ) differs from its mouse counterpart by merely six residues (4%), and there are just 21 residue differences (12%, which include residue differences at 18 positions and 3 insertions/ deletions) between the human and canary a-synucleins. 304 in flagellin, the ordered central region has greater sequence diversity than its disordered termini. 305 functionally important conserved regions of predicted disorder were shown to be rather common in proteins from all kingdoms of life, including viruses. 306, 307 furthermore, many functional domains of a significant size were shown to be intrinsically disordered. 165 overall, a systematic study of several families of proteins with at least one structurally characterized disordered region revealed that their idprs are characterized by highly heterogeneous evolutionary rates, with some disordered amino acid sequences evolving slowly, and others evolving more rapidly than ordered sequences. 151 also, even different parts of the same disordered region can possess noticeable variability in their divergence during the evolutionary process. 308 finally, in some disordered proteins, peculiarities of the amino acid composition, and not the amino acid sequence might be conserved. 309, 310 some future directions the last 15 years witnessed a real revolution in our understanding of the protein structure-function relationships. the fact that there is an entire class of polypeptides which do not have rigid structures but possess crucial biological function was heavily underappreciated and ignored for a very long time despite numerous examples scattered in literature. the work which started in my group as an attempt to understand what is so special about several natively unfolded proteins produced a real explosion of interest to structure-less proteins with biological functions. a new field was created and a lot of intriguing information was produced related to structures and functions of idps/idprs. there is no need to list once again all the discoveries and findings made in this field-they are subjects of many recent reviews and some of them are briefly covered in this article. although the amount of data generated during the past decade and a half on specific features related to the structural properties of idps and idprs, their abundance, distribution, functional repertoire, regulation, involvement into the disease pathogenesis, and so forth is vast, it seems that this mass of data produced so far is just a small tip of a humongous iceberg. idps/idprs continue to 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interactomes disorder and sequence repeats in hub proteins and their implications for network evolution role of intrinsic disorder in transient interactions of hub proteins intrinsic disorder in yeast transcriptional regulatory network high levels of structural disorder in scaffold proteins as exemplified by a novel neuronal protein, cask-interactive protein1 moonlighting proteins multifunctional proteins: examples of gene sharing molecular mechanisms for multitasking: recent crystal structures of moonlighting proteins exploring the binding diversity of intrinsically disordered proteins involved in one-to-many binding collective dynamics of 'small-world' networks classification of scale-free networks emergence of scaling in random networks revisiting date and party hubs: novel approaches to role assignment in protein interaction networks axin is a scaffold protein in tgf-beta signaling that promotes degradation of smad7 by arkadia intrinsic disorder in transcription factors human transcription factors contain a high fraction of intrinsically disordered regions essential for transcriptional regulation malleable machines in transcription regulation: the mediator complex more than just tails: intrinsic disorder in histone proteins a creature with hundred of waggly tails: intrinsically disordered proteins in ribosome hsf transcription factor family, heat shock response, and protein intrinsic disorder protein intrinsic disorder and induced pluripotent stem cells the roles of intrinsic disorder in orchestrating the wnt-pathway resilience of death: intrinsic disorder in proteins involved in the programmed cell death structure and dynamics of a molten globular enzyme flexibility and reactivity in promiscuous enzymes dynamics of proteins multiple conformational changes in enzyme catalysis an nmr perspective on enzyme dynamics relating protein motion to catalysis dynamical contributions to enzyme catalysis: critical tests of a popular hypothesis the catalytic and regulatory properties of enzymes enzymatic activity in disordered states of proteins an enzymatic molten globule: efficient coupling of folding and catalysis kinetics and thermodynamics of ligand binding to a molten globular enzyme and its native counterpart relative tolerance of an enzymatic molten globule and its thermostable counterpart to point mutation circularly permuted dihydrofolate reductase possesses all the properties of the molten globule state, but can resume functional tertiary structure by interaction with its ligands circularly permuted dihydrofolate reductase of e. coli has functional activity and a destabilized tertiary structure ureg, a chaperone in the urease assembly process, is an intrinsically unstructured gtpase that specifically binds zn21 intrinsically disordered structure of bacillus pasteurii ureg as revealed by steady-state and time-resolved fluorescence spectroscopy insights in the (un)structural organization of bacillus pasteurii ureg, an intrinsically disordered gtpase enzyme multitude of binding modes attainable by intrinsically disordered proteins: a portrait gallery of disorder-based complexes polyelectrostatic interactions of disordered ligands suggest a physical basis for ultrasensitivity dynamic equilibrium engagement of a polyvalent ligand with a single-site receptor protein dynamics and conformational disorder in molecular recognition structure/ function implications in a dynamic complex of the intrinsically disordered sic1 with the cdc4 subunit of an scf ubiquitin ligase the intrinsically disordered cytoplasmic domain of the t cell receptor zeta chain binds to the nef protein of simian immunodeficiency virus without a disorder-toorder transition homooligomerization of the cytoplasmic domain of the t cell receptor zeta chain and of other proteins containing the immunoreceptor tyrosine-based activation motif binding of intrinsically disordered proteins is not necessarily accompanied by a structural transition to a folded form quantitative observation of backbone disorder in native elastin membrane binding mode of intrinsically disordered cytoplasmic domains of t cell receptor signaling subunits depends on lipid composition lipid-binding activity of intrinsically unstructured cytoplasmic domains of multichain immune recognition receptor signaling subunits limitations of induced folding in molecular recognition by intrinsically disordered proteins effective long-range attraction between protein molecules in solutions studied by small angle neutron scattering properties of a water-soluble, yellow protein isolated from a halophilic phototrophic bacterium that has photochemical activity analogous to sensory rhodopsin measurement and global analysis of the absorbance changes in the photocycle of the photoactive yellow protein from ectothiorhodospira halophila structural and dynamic changes of photoactive yellow protein during its photocycle in solution the short-lived signaling state of the photoactive yellow protein photoreceptor revealed by combined structural probes solution structure and backbone dynamics of the photoactive yellow protein ) 1.4 a structure of photoactive yellow protein, a cytosolic photoreceptor: unusual fold, active site, and chromophore a mechanosensitive ion channel in the yeast plasma membrane an improved open-channel structure of mscl determined from fret confocal microscopy and simulation tight regulation of unstructured proteins: from transcript synthesis to protein degradation cdk-inhibitory activity and stability of p27kip1 are directly regulated by oncogenic tyrosine kinases the importance of intrinsic disorder for protein phosphorylation protein disorder is positively correlated with gene expression in escherichia coli insufficiently dehydrated hydrogen bonds as determinants of protein interactions keeping dry and crossing membranes the alternative conformations of amyloidogenic proteins and their multi-step assembly pathways protein misfolding, evolution and disease biological activity and pathological implications of misfolded proteins protein deposits as the molecular basis of amyloidosis. i. systemic amyloidoses protein deposits as the molecular basis of amyloidosis. ii. localized amyloidosis and neurodegenerative disordres amyloid fibrillogenesis: themes and variations conformational constraints for amyloid fibrillation: the importance of being unfolded pathways to amyloid fibril formation: partially folded intermediates in fibrillation of unfolded proteins alzheimer's disease and down's syndrome: sharing of a unique cerebrovascular amyloid fibril protein neuronal origin of a cerebral amyloid: neurofibrillary tangles of alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels a68: a major subunit of paired helical filaments and derivatized forms of normal tau molecular cloning of cdna encoding an unrecognized component of amyloid in alzheimer disease alzheimer's disease in down's syndrome: clinicopathologic studies part ii: alpha-synuclein and its molecular pathophysiological role in neurodegenerative disease shattuck lecture-neurodegenerative diseases and prions polyglutamine diseases: protein cleavage and aggregation polyglutamine diseases: a transcription disorder? fourteen and counting: unraveling trinucleotide repeat diseases molecular genetics: unmasking polyglutamine triggers in neurodegenerative disease beyond the qs in the polyglutamine diseases polyglutamine expansion neurodegenerative disease local structural elements in the mostly unstructured transcriptional activation domain of human p53 intrinsic structural disorder and sequence features of the cell cycle inhibitor p57kip2 identification of a novel regulatory domain in bcl-x(l) and bcl-2 intrinsic structural disorder of the c-terminal activation domain from the bzip transcription factor fos tc-1 is a novel tumorigenic and natively disordered protein associated with thyroid cancer multiple aromatic side chains within a disordered structure are critical for transcription and transforming activity of ews family oncoproteins oncogenic partnerships: ews-fli1 protein interactions initiate key pathways of ewing's sarcoma signaling to the p53 tumor suppressor through pathways activated by genotoxic and nongenotoxic stress mutations in human cancers activation and activities of the p53 tumour suppressor protein inhibition of the p53-hdm2 interaction with low molecular weight compounds inhibition of the p53-mdm2 interaction: targeting a protein-protein interface intrinsically disordered proteins in human diseases: introducing the d2 concept unfoldomics of human diseases: linking protein intrinsic disorder with diseases predicting binding regions within disordered proteins mining alpha-helix-forming molecular recognition features with cross species sequence alignments coupled folding and binding with alpha-helix-forming molecular recognition elements fluorescent, sequence-selective peptide detection by synthetic small molecules structure of the mdm2 oncoprotein bound to the p53 tumor suppressor transactivation domain small-molecule antagonists of p53-mdm2 binding: research tools and potential therapeutics targeting the p53-mdm2 interaction to treat cancer in vivo activation of the p53 pathway by small-molecule antagonists of mdm2 rational drug design via intrinsically disordered protein small-molecule inhibitors of protein-protein interactions: progressing towards the dream protein-protein interactions and cancer: small molecules going in for the kill antagonists of protein-protein interactions multiple independent binding sites for small-molecule inhibitors on the oncoprotein c-myc intrinsically disordered proteins may escape unwanted interactions via functional misfolding intrinsically disordered proteins and novel strategies for drug discovery function of alternative splicing the origin of life (in russian) the rationalist annual for the year 1929 a production of amino acids under possible primitive earth conditions organic compound synthesis on the primitive earth the origin of the genetic code a co-evolution theory of the genetic code possibilities for the evolution of the genetic code from a preceding form consensus temporal order of amino acids and evolution of the triplet code the path from the rna world relics from the rna world beyond kinetic traps in rna folding the ubiquitous nature of rna chaperone proteins origin of life-the rna world proteins, rnas and chaperones in enzyme evolution: a folding perspective uprooting the tree of life a formal test of the theory of universal common ancestry sequence evolution of the intrinsically disordered and globular domains of a model viral oncoprotein the relationships among microrna regulation, intrinsically disordered regions, and other indicators of protein evolutionary rate proportion of solvent-exposed amino acids in a protein and rate of protein evolution the hydrophilic, protease-sensitive terminal domains of eucaryotic dna topoisomerases have essential intracellular functions structural preordering in the n-terminal region of ribosomal protein s4 revealed by heteronuclear nmr spectroscopy nmr structure and functional characteristics of the hydrophilic n terminus of the potassium channel beta-subunit kvbeta1.1 evolution and disorder proteome-wide evidence for enhanced positive darwinian selection within intrinsically disordered regions in proteins alpha-synuclein misfolding and parkinson's disease the synucleins terminal regions of flagellin are disordered in solution conservation of intrinsic disorder in protein domains and families. i. a database of conserved predicted disordered regions conservation of intrinsic disorder in protein domains and families. ii. functions of conserved disorder intrinsically disordered regions of p53 family are highly diversified in evolution dynamic behavior of an intrinsically unstructured linker domain is conserved in the face of negligible amino acid sequence conservation chemical composition is maintained in poorly conserved intrinsically disordered regions and suggests a means for their classification this work would be impossible without numerous collaborators whose enthusiasm and help drove the studies on intrinsically disordered proteins for many years. over the years i collaborated with more than 550 colleagues around the globe, and i am grateful to all former and current colleagues for their priceless contributions, assistance and support. key: cord-306904-8iteddug authors: uversky, vladimir n title: unreported intrinsic disorder in proteins: building connections to the literature on idps date: 2014-12-12 journal: intrinsically disord proteins doi: 10.4161/21690693.2014.970499 sha: doc_id: 306904 cord_uid: 8iteddug this review opens a new series entitled “unreported intrinsic disorder in proteins.” the goal of this series is to bring attention of researchers to an interesting phenomenon of missed (or overlooked, or ignored, or unreported) disorder. this series serves as a companion to “digested disorder” which provides a quarterly review of papers on intrinsically disordered proteins (idps) found by standard literature searches. the need for this alternative series results from the observation that there are numerous publications that describe idps (or hybrid proteins with ordered and disordered regions) yet fail to recognize many of the key discoveries and publications in the idp field. by ignoring the body of work on idps, such publications often fail to relate their findings to prior discoveries or fail to explore the obvious implications of their work. thus, the goal of this series is not only to review these very interesting and important papers, but also to point out how each paper relates to the idp field and show how common tools in the idp field can readily take the findings in new directions or provide a broader context for the reported findings. disorder. this series serves as a companion to "digested disorder" which provides a quarterly review of papers on intrinsically disordered proteins (idps) found by standard literature searches. the need for this alternative series results from the observation that there are numerous publications that describe idps (or hybrid proteins with ordered and disordered regions) yet fail to recognize many of the key discoveries and publications in the idp field. by ignoring the body of work on idps, such publications often fail to relate their findings to prior discoveries or fail to explore the obvious implications of their work. thus, the goal of this series is not only to review these very interesting and important papers, but also to point out how each paper relates to the idp field and show how common tools in the idp field can readily take the findings in new directions or provide a broader context for the reported findings. recent years evidence an exponential increase in the number of papers on intrinsically disordered proteins (idps), indicating that the phenomenon of protein intrinsic disorder is becoming a popular research topic. this point is also reflected in the articles of the "digested disorder" series published in this journal. [1] [2] [3] to make this point stronger some interesting numbers are provided below. pubmed search (as of august 03, 2014) for ((intrinsically disordered) or (natively unfolded) or (intrinsically unstructured) or (intrinsically unfolded) or (intrinsically flexible protein)) returned 2,490 hits. restricting this search to the past one and a half years only (i.e., searching for ((intrinsically disordered) or (natively unfolded) or (intrinsically unstructured) or (intrinsically unfolded) or (intrinsically flexible protein)) and ("2013/01/ 01"[date -publication]: "3000"[date -publication])) gives 659 hits. curiously, although papers on idps (2,490) constitute just 0.045% of all the protein-related papers (5, 569, 481) published during the past 115 years, the fraction of idprelated papers increases to 0.18% if only publications of the past year and a half are taken into account. it is of interest to compare these trends with the related tendencies of the research on proteins in general. protein-related papers (5, 569, 481) constitute »23% of all the papers published during the past 115 y and annotated in pubmed (24, 068, 783) . however, this number decreases to »21% if only papers published during the past year and a half are taken into account (in 2013-2014, the overall number of published papers is 1,719,568; the number of papers dealing with proteins is 362,612). this suggests that an idp is a new rising star in the field of protein science. despite this steady and sure increase in the appreciation of protein intrinsic disorder, there are still numerous instances when this concept is overlooked, or missed or simply ignored. unfortunately, such "missed disorder" phenomenon continues to be rather common in the modern literature. the original goal of the first article in the new "unreported disorder in proteins" series was to find several papers published in different journals that talk about idps (or hybrid proteins containing both ordered and disordered regions) not recognizing that they are talking about such proteins, to show how consideration of intrinsic disorder can be used to strengthen conclusions and draw relationships to prior discoveries in the field. however, analysis of recent publications in just 2 journals revealed that the "missed disorder" happened to be essentially more abundant than it was originally expected. in fact, as of august 3, 2014, of 677 papers published during 2013-2014 in the journal of molecular biology, 609 were dealing with proteins, with 87 papers being dedicated to the idps or hybrid proteins. papers were considered to be dealing with intrinsic disorder if their texts contained at least a single mention of disorder, flexibility, conformational flexibility, or unfoldedness in relation to the protein of interest or any of its regions. obviously, this is a very relaxed and inclusive approach, since simple mentioning of disorder or conformational flexibility is not sufficient for the detailed elucidation of the functional role of disorder/flexibility. the direct pubmed search for the idprelated papers published in the journal of molecular biology using the search criteria ((intrinsic disorder) or (intrinsically disordered) or (natively unfolded) or (intrinsically unstructured) or (intrinsically unfolded) or (intrinsically flexible protein)) and ("2013/01/01" [date -publication]: "3000"[date -publication]) and ("journal of molecular biology"[journal]) generated just 17 hits. analysis of the table of content of this journal combined with a brief computational analysis of related proteins revealed that there are at least 22 more papers, research subject of which are idps. analogous analysis of the publications in biochemistry during the 2013-2014 provided comparable data: of 1455 published papers, 25 were "officially" dedicated to intrinsic disorder or to intrinsically disordered/natively unfolded/intrinsically unstructured/ intrinsically unfolded/intrinsically flexible proteins. mining of papers published in biochemistry during 2013-2014 revealed that some 189 papers contained at least a single mention of "conformational flexibility" or "intrinsic disorder" or "disordered protein" or "disordered peptide" or "disordered region" or "natively unfolded" in relation to the protein of interest or any of its regions. furthermore, based on the combination of literature mining and a brief computational analysis 20 "hidden gems" were found; i.e., papers that missed protein disorder. the mentioned 42 papers dealing with the unreported intrinsic disorder and their related idps or hybrid proteins containing ordered and intrinsically disordered regions are briefly outlined below. table 1 contains all the proteins considered in this review. proteins are arranged according to the increase in the extent of their disorder evaluated as percentage of the residues predicted to be disordered (i.e., possessing disorder scores above 0.5) by pondr ò vsl2, which is among the more accurate disorder predictors. 4 table 1 shows that the extent of unreported intrinsic disorder in 143 proteins ranges from 10% to 100%, indicating that all these proteins belong to the category of moderately or highly disordered proteins; i.e., proteins with the disorder content ranging from 10% to 30% and from 30% to 100%, respectively. table 1 also shows that a very significant fraction of such proteins with overlooked disorder (>75%) belongs to the "highly disordered" category. the order of sections in this review crudely follows the order of proteins in table 1 . when several proteins are discussed in a paper, the corresponding section within the text is placed at a position ascribed by table 1 to a protein with the lowest disorder content. the corresponding sections are organized in the following way: first, a brief description of the paper is provided; then, the biological importance of the related proteins is discussed; next, the results of the computational and bioinformatics analyses of the disorder status of a given protein (or set of proteins) are represented to show how consideration of intrinsic disorder can enhance conclusions of a paper. eukaryotic translation initiation factors 4a, 4b and 4g (eif4a, eif4b, and eif4g) andreou & klostermeier investigated the peculiarities of the combined action of 3 eukaryotic translation initiation factors, eif4a, eif4b, and eif4g, in rna unwinding needed to resolve secondary structure elements from the 5 0 -untranslated region of mrnas to enable ribosome scanning. 5 a set of eukaryotic translation initiation factors (eifs) is involved in the complex, multi-stage process of initiation of mrna translation in eukaryotes. in the first step of this process, the eif4f complex binds to the m7 gppp cap on the 5 0 -end of the mrna. this eif4f complex consists of the cap-binding protein eif4e, the scaffolding protein eif4g, and the deadbox helicase eif4a. 6, 7 at the next steps of the translation initiation, the eif4f complex recruits the 43s ribosomal preinitiation complex which scans the 5 0untranslated region in 5 0 -to-3 0 direction in search for the translation start codon. 8, 9 one of the important points in this process is the resolution of secondary and tertiary structures in the mrna, which is typically done by the dead-box helicase eif4a, which possesses rna-dependent atpase and atp-dependent rna helicase activities. 10 these 2 activities of eif4a are enhanced by some auxiliary proteins, such as eif4b and eif4g that act synergistically stimulating the eif4a helicase activity in the mrna scanning process. 5 figure 1 illustrates the disorder propensities of the 3 proteins related to the mrna unwinding. disorder was evaluated by a family of pondr predictors. here, scores above 0.5 correspond to disordered residues/regions. pondr ò vsl2b is one of the most accurate standalone disorder predictors, 11 pondr ò vl3 possesses high accuracy in finding long idprs, 12 pondr ò vlxt is not the most accurate predictor but has high sensitivity to local sequence peculiarities which are often associated with disorderbased interaction sites, 13 whereas pondr-fit represents a metapredictor which, being moderately more accurate than each of the component predictors, is one of the most accurate disorder predictors. 14 the various predictors often give different predictions of disorder for the same protein, perhaps leading so some confusion when trying to understand the implications of this and the following figures. these differences arise because of different computational methods and especially because different training sets were used. despite the differences, the overall disorder prediction trends are typically similar for each protein. in agreement with the notion that catalytic functions require specific and ordered structure, eif4a, with its atpase and atp-dependent rna helicase activities, is predicted to be mostly ordered protein (see fig. 1a ; uniprot id: p10081). the mostly ordered nature of this enzyme is further supported by the fact that almost entire sequence is seen in the crystal structure (see red structure at fig. 1b , pdb id: 2vso), except to the residues 1-11, 126-135, 351-356, and 394-395. on the contrary, the auxiliary proteins eif4b and eif4g are predicted to possess long disordered regions (see figs. 1c and 1d; uniprot ids: p34167 and p39935, respectively). eif4g is also predicted to have an ordered region that coincides with its middle or eif4a interacting domain (residues 571-854). this region was cocrystallized with the eif4a factor (see blue structure at fig. 1b) . curiously, several eif4g regions were missing in structure of the eif4a-eif4g complex (e.g., residues 571-576, 583-596, 686-688, 717-729, 803-811, and 853-854). long intrinsically disordered regions are important for functions of eif4b and eif4g. in fact, according to anchor, 15 human plasminogen activator inhibitor 1 (pai-1) florova et al. investigated the mechanisms of the stabilization of human plasminogen activator inhibitor 1 (pai-1, also known as serpin e1) during the formation of the transient ternary "molecular sandwich" complex (msc) containing pai-1, vitronectin (vn), and the target enzyme. 17 major players involved in the formation of this complex are pai-1, which is a major endogenous inhibitor of plasminogen activators, a cell adhesive glycoprotein vn, and a proteinase inhibited by pai-1. an important feature of pai-1 is its ability to exist in 2 forms, a metastable active conformation with solventaccessible reactive center loop (rcl) and thermodynamically stable, inactive, latent conformation, where rcl is spontaneously inserted to the middle of the b-sheet b of pai-1. 17 this "magic" chameleonlike rcl is located in the 331-350 region. figure 2a shows that although human pai-1 (uniprot id: p05121) is predicted to be mostly ordered, its rcl is located within grayish area with a mean disorder score close to 0.5, suggesting that this region is characterized by noticeable intrinsic mobility. perumal et al. reported that a specific interaction between the bacteriophage t4 uvsw helicase and the t4 singlestranded dna (ssdna) binding protein gp32 is required for enhancement of the uvsw dna unwinding function. 18 curiously, uvsw interact with gp32, both in the presence and absence of dna, through the c-terminal acidic tail of the gp32 protein. in the absence of this interaction, the ssdna annealing and atp-dependent translocation activities of uvsw are severely inhibited when gp32 coats the ssdna lattice. however, when uvsw and gp32 do interact, uvsw is able to efficiently displace the gp32 protein from the ssdna. the tail-less gp32 inhibits activities of uvsw on dna in the presence of gp32. 18 the uvsw helicase, a member of the sf2 superfamily of helicases 19 (i.e., enzymes that use atp to carry out mechanical work related to the unwinding of duplex dna structures and reorganization of rna secondary structures 20 ) , is one of the 3 helicases encoded by the bacteriophage t4, 21 where it plays a number of roles in a variety of dna repair and recombination pathways. [22] [23] [24] [25] [26] [27] the involvement of this helicase in dna replication is achieved via the uvsw-mediated unwinding of r-loop structures generated at the replication origin by the host rna polymerase. 22 uvsw (uniprot id: p20703) activity involves the generation and consumption of single stranded dna (ssdna). in agreement with the overall low disorder predisposition (see fig. 3a ), almost entire sequence of uvsw was successfully crystallized (fig. 3b , pdb id: 2oca). naked ssdna rarely occurs within t4infected cells as they are immediately coated with gp32 ssdna binding protein, which serves to protect it against degradation by endonucleases. the gp32 is also thought to coordinate the many activities necessary to carry out dna replication, recombination and repair by recruiting and modulating the activity of the enzymes involved in these processes. 28 the bacteriophage t4 gp32 (uniprot id: p03695) is a 301 residue-long protein. the crystal structure of the gp32 central region (residues 22-239) is known (see fig. 3c ). 29 gp32 binds preferentially to ssdna and destabilizes double-stranded dna. it is involved in dna replication, repair and recombination, dinds ssdna as the replication fork advances and stimulates the replisome processivity and accuracy. the n-terminal region of gp32 is important for the cooperative binding of gp32 monomers on ssdna, whereas the c-terminal acidic tail is involved interaction with the replicative dna polymerase, the primase and helicase loader protein. [30] [31] [32] also, this c-terminal region (residues 254-301) was shown to play a crucial role in controlling the d-loop unwinding capability of uvsw, since the presence of a mutant gp32 protein lacking the acidic tail (delta254-301, gp32-a) led to a significant reduction in the unwinding of the d-loop substrate. 18 fig. 3d shows that a significant portion of the c-terminal half of this protein is predicted to be mostly disordered (residues 200-301). curiously, this disordered region includes the functionally important c-terminal tail that defines the ability of gp32 to modulate the uvsw activity. pilf and pilq of the pseudomonas aeruginosa type iv pilus system some bacteria and archaea contain specific cell envelope-spanning biomolecular machines, type iv pili (t4p), which are used by bacteria and archaea to interact with the environment. pilus biogenesis is controlled by the assembly of the outer membrane pilq secretin channel through which the pili are extruded. koo et al. investigated the roles of the pseudomonas aeruginosa type iv pili (t4p) pilotin protein pilf in the outer membrane pilq secretin channel targeting, oligomerization, and function. 33 pilf belongs to the class 1 pilotins, which are a-helical proteins containing several tetratricopeptide (tpr) motifs, that are comprised of approximately 34amino acid and are able to form a superhelical fold that mediates proteinprotein interactions in both prokaryotes and eukaryotes. 33 secretins (including pilq sectretin) possess a conserved c-terminal region, containing the secretin domain that is putatively embedded into the outer membrane, and a variable, system-specific n-terminal region. 34, 35 figure 4a shows that the p.aeruginosa pilf (uniprot id: q51385) is predicted to be a hybrid protein possessing both ordered and disordered regions. curiously, about 2/3 of this protein possesses disorder scores close to 0.5. this grayish area is likely to be characterized by high conformational plasticity and is predicted to contain 4 aibss, residues 5-16, 41-46, 76-81 and 111-115. similarly, pilq (uniprot id: p34750) is predicted to be a hybrid protein (see fig. 4b ) with several disorder-based binding sites (residues 101-107, 120-122, 245-251, 502-505, and 533-547). bonet et al. analyzed the molecular mechanisms of interaction of the 14-3-3z protein with several integrin proteins. 36 the authors provided a detailed biophysical characterization of the cytoplasmic tails of a4, b1, b2 and b3 integrins binding to 14-3-3z and showed that binding affinities and interaction modes of different integrins with this 14-3-3 are rather different. furthermore, although many structural features of these interactions are similar to other known 14-3-3 complexes, the binding exhibits specific features involving secondary sites. particularly, in addition to a canonical binding mode for the a4 phospho-peptide, some residues outside the consensus 14-3-3z binding motif of this integrin were shown to be essential for an efficient interaction. although a short b2 phospho-peptide is sufficient for high-affinity binding to 14-3-3z, the authors also found novel 14-3-3z/integrin tail interactions that were independent of phosphorylation. 36 the members of the 14-3-3 family are highly conserved acidic proteins of »30 kda that are abundantly expressed in all eukaryotic cells. in human, there are 7 14-3-3 isoforms (b, g, e, h, s, t and z) forming homodimers or heterodimers. these proteins regulate and control many signaling pathways, such as cytoskeletal dynamics programmed cell death, and cell cycle progression, and are involved in the pathogenesis of several human diseases, such as cancer and neurological disorders. 37,38 although 14-3-3 proteins are considered as phosphor-serine/threonine binding modules possessing 2 consensus recognition motifs, rsxpsxp and rxf/ yxpsxp, 39 some other binding modes are known including interaction with some unphosphorylated motifs. 40 ,41 x-ray structures of various 14-3-3 isoforms and their numerous complexes are known providing considerable knowledge on the various binding modes. 42, 43 curiously, a detailed analysis of more than 200 binding partners of 14-3-3 proteins showed neither structural nor functional relatedness in this group of proteins. 44 however, bioinformatics analysis established that >90 % of the 14-3-3 interactors contain disordered regions and that almost all 14-3-3-binding sites are located inside disordered regions. 44 among various partners of 14-3-3 proteins are membrane-spanning receptors, integrins, which are involved in numerous biological functions, such as cell adhesion, migration and differentiation and are associated with a wide-range of diseases. 45, 46 integrins are heterodimers formed by aand b-subunits composed of large extracellular (ecto) domains, trans-membrane domains and short (13-70 residues) c-terminal cytoplasmic domains, 47 which are flexible tails acting as hubs for numerous integrin-based protein-protein interactions. [48] [49] [50] these cytoplasmic tails of the 2 integrin subunits also mediates the bidirectional inside-tooutside and outside-to-inside signaling, where signals transmitted by integrins from outside to inside the cell promote cell survival and proliferation, and where integrin affinity for the extracellular ligands can also be controlled by intracellular factors. 47 figure 5a represents a structure of the complex between the 14-3-3z protein (blue cloud) and a phosphorylated peptide from the b2 integrin tail (red chain). 51 it is seen that similar to many other 14-3-3 complexes, the b2 integrin tail is bound in a highly extended form. to illustrate interactivity of the b2 integrin, figure 5b shows the results of the analysis of this protein by string. here, settings were chosen to find binding partners with the highest confidence (0.9). finally, figure 3c shows some disorder-based alignments of the c-terminal tails used in the work of bonet et al., 36 namely residues 1001-1032 of human integrin a4 (black line, uniprot id p13612), residues 752-801 of human integrin b1 (black line, uniprot id p05556), residues 724-769 of human integrin b2 (black line, uniprot id p05107), residues 742-788 of human integrin b3 (black line, uniprot id p05106), and residues 747-798 of human integrin b7 (black line, uniprot id p26010). figure 5c clearly shows that all these integrin tails involved in the specific interaction with the 14-3-3z protein are mostly disordered. in the review by rajsbaum et al., an important family of proteins, tripartite motif (trim) proteins is introduced together with the multitude of their functional roles in the innate antiviral immunity. 52 since there are more than 70 distinct members in the family of human trim proteins, it is physically impossible to consider all of them even very superficially. the feature that links all these proteins together is the fact that they share 3 conserved n-terminal domains: a really interesting new gene (ring) domain, one or 2 b-boxes (b1/b2) and a coiledcoil domain. [53] [54] [55] [56] in addition to immunerelated functions many trim proteins are involved in a wide range of biological activities, such as transcriptional regulation, apoptosis, cell differentiation, development, oncogenesis, ubiquitin e3 ligases, and e3 ligases for other ubiquitinlike molecules such as sumo and the ifn-inducible protein isg15. 57 computational analysis (see table 1 ) revealed that all members of the human trim family are extensively disordered. helbig and beard overviewed the ability of viperin to modulate conditions within the cell and to interfere with proviral host proteins in order to create an unfavorable environment for viral replication. 58 viperin is one of the few products of numerous interferon-stimulated genes (isgs) possessing direct antiviral activity, being able to limit a broad range of viruses and to play an emerging role in modulating innate immune signaling. this is a highly species conserved protein consisting of 3 distinct domains: a variable n-terminal domain that contains an amphipathic helix and a leucine zipper region, a highly conserved central domain containing a "radical sam domain," and a conserved c-terminal critical for the antiviral properties of viperin against a number of viruses. 58 the authors showed that viperin plays a role in innate immune signaling, limits different viruses through both direct inhibition of replication and interference with viral budding/release, and disrupts the actin cytoskeleton to increase infectivity of human cytomegalovirus (hcmv) in a known example of evolutionary escape of hcmv from the antiviral properties of viperin. 58 viperin is able to interact with the 5 host proteins fpps, tfp, irak1, vap-a and traf6 and the 3 viral proteins denv ns3, hcv ns5a and hcmv vmia in order to accomplish these diverse biological functions. 58 figure 6a represents the results of the string 59 -based evaluation of viperin interactivity. although it has been emphasized that "it is unusual for viperin to be able to interact with such a divergent range of other proteins and to potentially mediate quite distinct cellular functions", 58 the mystery of such "unusual" polyfunctionality is naturally solved by the presence of extensive disorder in this protein (see fig. 6b ; uniprot id: q8wxg1). sze et al. overviewed available information on the sterile a motif and histidineaspartic domain (hd) containing protein 1 (samhd1), which is a member of the unique group of host restriction factors that limit retroviral replication at distinct stages of the viral life cycle. 60 samhd1 is a deoxynucleoside triphosphate triphosphohydrolase responsible for the degradation of the deoxynucleoside triphosphates (dntp) into their deoxynucleosides (dn) and inorganic triphosphates, thus depleting the cellular dntp pool required for cellular dna polymerase. 61 activity of this protein is modulated by post-translational modifications, cell-cycle-dependent functions and cytokine-mediated changes, as well as via interaction with the vpx accessory protein. 60 samhd1is a predominantly nuclear protein composed of 2 functional domains, the sterile a motif (sam) domain involved in protein-protein and samhd1-nucleic acid interactions, 62 and the hd domain containing the enzymatic sites crucial for its triphosphohydrolase activity, rna binding and nuclease activity. 63 another functionally important region is located at the c-terminus of samhd1, where a v-domain capable of interaction with the hiv-2/sivsm vpx accessory protein is located (residues 595-626). 64 based on the intrinsic disorder propensity analysis (fig. 2b) , samhd1 (uniprot id: q9y3z3) is expected to be a predominantly ordered protein possessing disordered n-and c-terminal tails (residues 1-100 and 580-626). obviously, intrinsic disorder in the c-terminal tail is important for the samhd1 interaction with the hiv-2/sivsm vpx accessory protein. interaction between the major bacterial heat shock chaperone groesl and an rna chaperone cspc in their recent paper, lenz & ron described a novel interaction between the major heat shock chaperone groesl of e. coli (hsp60) with an rna chaperone (cspc) leading to the cspc proteolysis needed for the transient nature of the heat shock response. 65 protein chaperones and proteases have multiple functions in controlling the wellbeing of a cell, acting as protein quality control that enables the cells to cope with the unfolding and aggregation of proteins. 66, 67 cspc is a member of the cold shock protein (csp) family that consists of 7 small homologues with high affinity to single-strand nucleic acid and that serve as rna chaperones. [68] [69] [70] [71] it has been shown that cspc is degraded during heat shock and that interaction of cspc with the major protein chaperone groesl plays a role in the enhanced, temperature-dependent proteolysis of cspc. 65 the fact that cspc is able to interact with groesl (which is known to bind partially unfolded and misfolded protein species) is a clear indication that cspc does not possess rigid structure, at least under the conditions favoring such interaction. the likely explanation for this binding is in the potential intrinsically disordered nature of cspc. in fact, many protein and rna chaperones were shown to be intrinsically disordered or hybrid proteins possessing both ordered and intrinsically disordered regions/ domains. 72 in agreement with this observation, figure 2c represents the pondr plots for the cspc from e. coli (uniprot id: p0a9y6) and shows that a significant portion of this small proteins (up to 35%) is predicted to be disordered. kalli et al. used multiscale molecular dynamics simulations to define the interaction mechanisms of phosphatase and tensin homolog (pten; uniprot id: p60484) and of the pten domain of ciona intestinalis voltage sensitive phosphatase (ci-vsp; uniprot id: q4w8a1) with phosphatidylinositol phosphate (pip)-containing lipid bilayers. 73 the authors revealed that the association of the pten with such bilayers involves the formation of an initial electrostatics-driven encounter complex between the protein and bilayer followed by reorientation of the protein to optimize its interactions with pip molecules in the membrane. 73 pten is a cytosolic enzyme that can interact with the inner leaflet of the plasma membrane and, being bound to membrane, catalyzes dephosphorylation of pi(3,4,5)p 3 to ptdins(4,5)p 2 . 74 pten has 4 domains, an n-terminal pip 2 -binding module, a phosphatase domain (pd), a c2 domain, and a c-terminal tail. several recent studies indicated that both nand c-tails of this protein are intrinsically disordered. [75] [76] [77] furthermore, it has been emphasized that post-translational modifications, conserved eukaryotic linear motifs, and molecular recognition features are present in the disordered c-tail of pten, enhancing protein-protein interactions of this protein needed for the various cellular functions of pten. 75 domain swapped pukovnik xis singh et al. determined the structure of pukovnik xis by x-ray crystallography. 78 xis is the recombination directionality factor that serves as a dna bending machine that difines the outcome of integrase-mediated site-specific recombination by redesign of higher-order protein-dna architectures. 79 mycobacterium phage pukovnik (which is a relative of phage l5) contains a small xis protein (56 residues) that binds cooperatively to attr dna at specific x1-x4 binding sequences. similar to l5, pukovnik xis stimulates integrase-mediated excision and inhibits integration. 80, 81 the presence of both xis and intergrase is needed for the formation of an attr intasome. 78 the cooperative binding of several xis proteins to dna is driven by a wingedhelix motif and relies on the use of contacts between a central loop and the wing motif to mediate interactions between xis proteins when bound to dna leading to the formation of a micronucleoprotein filament with a modest bend. 78, 82, 83 singh et al. showed that in the dnabound form, 5 individual pukovnik xis subunits stack onto each other through an extensive array of protein-protein interactions forming a filament with left-handed superhelical twist. 78 stacking of xis subunits within the filament is rather regular, and each monomer exhibits a twist of 40 and an »60 bending angle. within the asymmetric unit, the filament region contains 4 xis protomers whereas the fifth protomer is positioned adjacent to the filament. this "outside" protomer forms the domain-swapped complex with the xis protomer 3. this domain-swapped xis 59 string produces the network of predicted associations for a particular group of proteins. the network nodes are proteins. the edges represent the predicted functional associations. an edge may be drawn with up to 7 differently colored lines -these lines represent the existence of the 7 types of evidence used in predicting the associations. a red line indicates the presence of fusion evidence; a green line -neighborhood evidence; a blue lineco-occurrence evidence; a purple line -experimental evidence; a yellow linetext mining evidence; a light blue line -database evidence; a black lineco-expression evidence. 59 (b) intrinsic disorder propensity of the human viperin. disorder propensity was evaluated by a set of predictors from the pondr family, pondr ò fit, vsl2b, vl3, and vlxt. scores above 0.5 correspond to disordered residues/regions. pair is created due to a dramatic rearrangement within the loop connecting b1 and b2 (residues 36-39) allowing the c-terminal residues 40-56 of one subunit to interact with the n-terminal 35 residues of a neighboring xis monomer. 78 it has been emphasized that the most important protein-protein interactions mediating the filament formation are formed by residues 51-54 of pukovnik xis that make extensive contacts with the wing of the adjacent monomer and deletion of which greatly diminishes dna binding affinity and abolishes excision. 78 therefore, although the xis filament is composed of identical subunits, the individual xis protomers are involved in very different interactions and form very different interfaces. to further illustrate this point, figure 7a represents a crystal structure of the pukovnik xis pentamer (pdb id: 4j2n) and 4 pairs of different dimers found within this pentameric filament. figure 7b shows that pukovnik xis (uniprot id: b3vgi6) is predicted to contain substantial amount of intrinsic disorder (note scale of the yaxis), including disordered n-and c-terminal tails. therefore, it is likely that the intrinsically disordered nature determines the ability of this small protein to be involved in a complex net of proteinprotein and protein-dna interactions, including its ability to form a wide array of regular and domain-swapped dimers. li et al. provided a detailed characterization of the functional mechanisms of interesting hsp90 co-chaperones, human aryl hydrocarbon receptor (ahr) interacting protein (aip) and aip like 1 (aipl1). 84 aip and aipl1 share 49% sequence identity, contain an n-terminal fkbp-like prolyl peptidyl isomerase (ppiase) domain (which is inactive in both proteins) followed by a tetratricopeptide repeat (tpr) domain. in addition, aipl1 harbors a unique c-terminal proline-rich domain (prd). 84 the authors showed that aip is inactive as a chaperone, whereas aipl1 exhibits chaperone activity and prevents the aggregation of non-native proteins, suggesting that prd is crucial for the chaperone function of this protein providing a means for efficient binding of aipl1 to non-native proteins. 84 since aipl1 possesses decreased affinity to hsp90, the c-terminal prd plays a role of a negative regulator of the aipl1-hsp90 interaction. 84 figure 8a shows that the major portion of the human aipl1 (n-terminal 75%) containing fkbp-like prolyl peptidyl isomerase and tpr domains is predicted to be mostly ordered (uniprot id: q9nzn9), whereas the c-terminal domain is predicted to be highly disordered. on the other hand, figure 8b indicates that the human aip (uniprot id: o00170) is expected to be mostly ordered. in agreement with these disorder predictions, 3d structures are known for the ppiase fkbp-type domain of human aip (residues 2-166, pdb id: 2lkn) and for its tpr domain (residues 173-330, pdb id: 4aif). the intrinsically disordered nature of the c-terminal prd of the human aipl1 possessing chaperone activity is in accord with earlier observations that chaperones are often either entirely disordered or contain long disordered regions. 72, 85 basic residues in the activated protein c (apc) exosite takeyama et al. provided a detailed description of the interaction between the activated protein c (apc) and factor (f) viiia. 86 the authors showed that the basic residues located within the 39-, 60-, and 70-80-loops of apc constitutes an exosite that contributes to the binding of fviii and therefore are important for the subsequent proteolytic inactivation of fviii. 86 apc, which is also known as vitamin k-dependent protein c, anticoagulant protein c, autoprothrombin iia, and blood coagulation factor xiv, is a single chain vitamin k-dependent zymogen for a plasma serine protease that upon activation by the thrombin-thrombomodulin complex down regulates the coagulation cascade by limited proteolysis of fva and fviiia. [87] [88] [89] analysis of the crystal structure of human apc (pdb id: 1aut) revealed that there are 3 surface loops (39, 60, and 70-80) , rich in basic residues, located in the protease domain of apc near the active site pocket. 86, 90 fviiia contains acidic c-terminal sequences that may potentially provide interactive sites for the basic exosite of apc. 86 figure 8c represents the results of the computational disorder analysis in human apc (uniprot id: p04070) and shows that mentioned loops enriched in basic residues are predicted to be disordered or very flexible, thereby providing an interesting mechanistic plane for the molecular basis of apc recognition and binding of fviii. the escherichia coli primosomal dnat protein molecular mechanisms of the oligomerization of e.coli primosomal dnat protein are uncovered in the study by szymanski et al. 91 the authors showed that the removal of the short c-terminal region dramatically affects the oligomerization process and instead of the trimer, the isolated n-terminal domain of dnat forms a dimer. 91 priming of the dna strand during the replication process is catalyzed by a multiprotein-dna complex known as the primosome. 92, 93 the translocation of this complex along the dna is fueled by ntp hydrolysis. besides being responsible for the synthesis of short oligoribonucleotide primers used to initiate synthesis of the cdna strand, primosome plays a role in the restarting of the stalled replication fork at the damaged dna sites. 93, 94 the assembly of the primosome is driven by an essential replication protein in escherichia coli, the dnat protein, where, the primosome assembly is initiated by recognition of a specific primosome assembly site (pas) of the replicating dna or the damaged dna site by the pria protein, or the prib protein-pria complex, followed by the association of the dnat and the pric protein. [92] [93] [94] this primary dna-protein complex is a scaffold, specifically recognized by the dnab helicase-dnac protein complex, which results in formation of the preprimosome. next, the preprimosome is recognized by the primase, and a functional primosome is formed. the dnat protein is crucial for the specific entry of the dnab helicase into the primosome complex. [92] [93] [94] the dnat monomer consists of the large, n-terminal core domain that includes the first 161 residues of the protein and a small c-terminal region containing the 18 remaining amino acids. 91 disorder analysis of this protein (uniprot id: p0a8j2) revealed that although the majority of the dnat is predicted to be ordered, the crucial for trimerization cterminal tail is expected to be completely disordered (see fig. 8d ). this observation is in agreement with recent notion that disordered protein tails are commonly involved in a wide array of important functions. 95 platelet endothelial cell adhesion molecule 1 (pecam-1) tourdot et al. dedicated their research to the analysis of the effect of the sequential phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (itims) on function of the platelet endothelial cell adhesion molecule-1 (pecam-1). 96 pecam-1 is a dual itim-containing receptor of the ig superfamily that is capable of inhibiting immunoreceptor tyrosine-based activation motif (itam)induced activation of b cells, t cells, and mast cells, as well as gpvi/fcrg chainmediated platelet activation. 97 the inhibitory properties of pecam-1 require phosphorylation of both of its itims located in the vicinity of tyrosine residues at positions 663 (vqy 663 tev) and 686 (tvy 686 sev). 96 curiously, the itims of pecam-1 are not phosphorylated in resting cells but are phosphorylated upon cellular activation. furthermore, these pecam-1 itims are phosphorylated sequentially, with phosphorylation of y 663 depending on prior phosphorylation of y 686 . 98 this sequential process of the pecam-1 phosphorylation starts with the phosphorylation of the c-terminal itim by src family kinases, which enables phosphorylation of the n-terminal itim of pecam-1 by other src homology 2 domain-containing nonreceptor tyrosine kinases (nrtks). 96 figure 8e shows that although human pecam-1 (uniprot id: p16284) is predicted to be mostly ordered, the residues, phosphorylation of which is crucial for its function (y 663 and y 686 ), are located within the highly disordered c-terminal tail. mouse serine/arginine-rich splicing factor 1 (srsf1) aubol et al. studied the molecular mechanisms of the phosphorylation of sr splicing factors, which are the proteins possessing regions enriched in arg-ser dipeptide repeats known as rs domains, by a specific serine kinase srpk1. 99 phosphorylation of rs domains is crucial for the regulation of the activities of the sr splicing factors. rs domains of sr proteins can range from 50 to over 300 residues in length, and the arg-ser dipeptide repeats can vary in both length and position. srpk1 phosphorylates the prototype sr protein, serine/arginine-rich splicing factor 1 (srsf1), via a directional mechanism where 11 serines flanked by arginines are sequentially fed from a docking groove in the large lobe of the kinase domain to the active site. 99 figure 8f shows that srsf1 (uniprot id: q6pdm2) is predicted to be a highly disordered protein. the disordered nature of this splicing factor is in agreement with recent bioinformatics studies, which showed that human 100 and yeast spliceosomes 101 are enriched in intrinsic disorder, and abundant disordered regions are crucial for functions of numerous auxiliary protein factors involved in the formation of this ribonucleoprotein complex. 100, 101 the potential functionality of the disordered regions found in mouse srsf1 was further supported by anchor-based analysis, 15 102 replication of viruses requires breaching the plasma membrane of the host cell. the entry of the enveloped viruses relies on the specialized fusion proteins. there are 3 major classes of viral fusion proteins, all containing a fusion peptide that inserts into the cytolemma, thereby anchoring the 2 membranes side by side. 106 human ifitms block the entry of viruses from each of the 3 classes of viral fusion proteins, 102 by preventing viral-host membrane fusion subsequent to viral binding and endocytosis. 104, 107 in humans, there are 5 ifitms (ifitm1, 2, 3, 5, and 10; corresponding uniprot ids: p13164, q01629, q01628, a6nnb3, and a6nmd0). these proteins each contain 2 hydrophobic membrane-associated domains separated by a conserved intracellular loop. all of them also contain the cytosolically-located n-terminal domain (ntd) of various length. 102 figure 9 shows that the ntds of all 5 human ifitms are predicted to be intrinsically disordered. the likely functional role of these disordered tails is reflected in the fact that the longest ntd of ifitm10 contains 3 aibss, residues 8-29, 51-64, and 71-82. shorter tails of other human ifitms might also be involved in protein-protein interactions since all of them contain characteristic "dips," which are commonly associated with the existence of specific molecular recognition features, morfs, which are short foldingprone fragments of disordered regions that are able to fold (at least partially) at interaction with specific binding partners. [108] [109] [110] [111] [112] chameleon region undergoing a-helix to b-strand transition in the mutant form of the type 2 ryanodine receptor domain a (ryr2a) linked with a heritable cardiomyopathy using the solution nmr spectroscopy and x-ray crystallography, amador et al. studied the effects of mutations associated with arrhythmogenic right ventricular dysplasia type 2 (arvd2) on the structure and dynamics of the n-terminal domain of the mouse ryanodine receptor ryr2a (uniprot id: e9q401). 113 ryanodine receptors (ryrs) are the largest tetrameric ca 2c release channels (»2.2 mda) of the sarcoplasmic reticulum in the electrically excitable cells. here, ryrs resides in close proximity to plasma membrane voltagegated dihydropyridine receptors (dhprs) and respond to the dhpr activity through a direct interaction and/or indirect ca 2c sensitivity, propagating sarcoplasmic reticulum luminal ca 2c release, thereby playing an integral role in excitation-contraction coupling in skeletal muscle cells and cardiomyocytes. 114, 115 mutations in the ryr genes are associated with several heritable human diseases, with most disease-associated mutations making ryrs hypersensitive to activating stimuli such as ca 2c on either the luminal or the cytosolic side of the receptor. in one of such mutations, the removal of exon 3 in ryr2 (ryr2ad3) results in a 35-residue deletion (n57-g91) that does not destabilize the protein. 116, 117 earlier structural analysis revealed that the b-trefoil fold of this domain is rescued by the transformation of the part of the preceding region of unknown structure (residues 88-109) into the missing b-strand leading to the noticeable increase in the thermal stability of the ryr2ad3 domain. 117 using solution nmr analysis, amador et al. showed that this "part of the preceding region of unknown structure" corresponds to a previously unresolved a 2helix (residues 95-104) in the wild-type ryr2a. 113 curiously, this a-helix undergoes structural transformation to a b-strand, thereby rescuing the b-strand deleted in the ryr2ad3 due to the removal of 3 in ryr2 gene. therefore, the authors revealed that this newly discovered a 2 -helix corresponds to the region of missing electron density in the earlier crystal structures of the protein, is rather dynamic, exhibits a greater backbone rmsd compared to the remaining secondary structure elements, and is able to undergo an a-to-b transition after the deletion of residues n57-g91 in ryr2ad3. 113 in other words, applying nmr to look at the crystallographically invisible region, an important chameleon region was discovered. although this chameleon region is relatively small, it is really mighty, with at least some mightiness being attributed to its intrinsically disordered nature. rodriguez et al. used complementary biophysical techniques for examining the atp-induced conformational switching of the isolated e subunit of the f 0 f 1 atp synthase from the thermophile bacillus ps3 (te). 118 the authors showed that atp binding induces large-scale conformational transition consistent with formation of stable helical structure in the isolated te. 118 based on the complimentary hydrogen/deuterium exchange (hdx) mass spectrometry analysis it has been concluded that this transition is accompanied by a pronounced stabilization in the vicinity of the atp-binding pocket. 118 structurally, the e subunit consists of a b-sandwich and 2 c-terminal helices, a1 and a2. 119 curiously, this protein is able to undergo dramatic structural rearrangements from a compact structure in the non-bound form (see fig. 10a ; pdb id: 1aqt) 119 to a highly extended open form in a complex with other subunits of the bacterial f 1 complex (see fig. 10b ; pdb id: 3oaa). the ability of the e subunit to undergo large conformational change associated with binding to the f 1 complex is determined by the presence of long idpr at the c-terminal region (see fig. 10c ; uniprot id: p0a6e6). simon et al. investigated functional peculiarities of the candida albicans cyclin capcl5. 120 the authors showed that the activation of the cyclin-dependent kinase pho85 that induces phosphorylation of the transcription factor cagcn4 is controlled via the specific binding of capcl5 to pho85. furthermore, it is shown that capcl5 induces its own phosphorylation at 2 adjacent sites in the n-terminal region of the protein and that this phosphorylation causes degradation of the cyclin in vivo, whereas in vitro studies indicated that this phosphorylation was accompanied by a loss of specific substrate recognition thereby representing a novel mechanism for limiting cyclin activity. 120 activity of cyclin-dependent kinases (cdks) is regulated via binding of ancillary subunits, the cyclins, 121 which also participate in targeting the kinase to specific substrates. 122 similar to other members of the cdk family, a yeast (saccharomyces cerevisiae) cdk pho85 can bind up to 10 different pho85 cyclins (pcls), 123 which defines the ability of this cdk to phosphorylate different substrates leading to a wide range of pho85 functions in cellular regulation. among various regulatory functions of pho85 complexed with different cyclins, is the pho85/pcl5-driven phosphorylation of the bzip transcription factor gcn4 leading to its degradation. 124 curiously, it was shown that cyclin pcl5 coevolved with its substrate gcn4, and in the pathogenic yeast candida albicans, degradation of cagcn4 depends on capcl5. 125 mutagenic analysis of the capcl5 (uni-prot id: q5ak08) revealed 2 potential cdk phosphorylation sites located in the n-terminal segment, at positions 38 and 43. 120 figure 11 shows that both of these phosphorylated sites are located within the disordered region, providing further illustration for an important notion stating that sites of posttranslational modifications in proteins are commonly located within the disordered regions. 126 nuclear localization of the alternatively spliced sirtuin-2 isoform rack et al. described a discovery of a new alternatively spliced isoform of the human sirtuin-2. 127 the new isoform results from skipping of exons 2-4 in the sirt2 gene. 127 sirtuin-2 is a member of a large family of protein-modifying enzymes, nad cdependent deacetylases, which are highly conserved throughout bacteria, archaea, and eukaryotes, and are heavily involved in modulation of various cellular processes ranging from metabolic homeostasis, to cell cycle control, to development, and to chromatin organization. [128] [129] [130] [131] [132] [133] in their turn, the numerous activities of sirtuins are regulated by alternative splicing, modulation of expression levels, posttranslational modifications or subcellular compartmentalization. [134] [135] [136] [137] in humans, there are 7 sirtuin homologues with distinct cellular locations. for example, sirt1, sirt6 and sirt7 are primarily localized to the nucleus, whereas sirt3, sirt4 and sirt5 are mitochondrial proteins, and sirt2 is believed to have a predominant cytosolic localization. 138 earlier, 4 variants of human sirt2 gene were reported, with alternative splicing affecting the n-terminal part of the sirtuin-2 protein in 2 isoforms (residues 1-37 are missing in the isoform-2 and the residues 1-38 (maepdpshpletqagkv-qeaqdsdsdseggaaggeadm) are substituted by residues mplaecpsc-rclssfrsv in the isoform-3), and with the isoform-4 possessing changes at the c-terminus, where residues 266-271 (vqpfas) are substituted by grglag, and residues 272-389 are missing. these isoforms contain a leucine-rich nuclear export signal (nes) within their n-terminal region which mediate their cytosolic localization 139 where they have numerous functions. a recent finding a new isoform-5 with the predominant nuclear localization helped to resolve an apparent contradiction between the predominant cytosolic localization of this protein and the existence of multiple nuclear functions. 127 the newly discovered alternative splicing event results in the substitution of the codons for amino acids 6-76 of the full-length orf of isoform-1 by an arginine codon in a new isoform. this isoform-5 is predominantly localized in the nucleus, does not exhibit detectable deacetylase activity, but is properly folded and retains the ability to bind p300. 127 it has been established that the protein segments affected by alternative splicing are most often intrinsically disordered, suggesting that alternative splicing enables functional and regulatory diversity while avoiding structural complications associated with the deletion of portions of the well-folded proteins. 140 in line with these observations, later studies revealed that the tissue-specific protein segments produced by alternative splicing often contain disordered regions, are enriched in posttranslational modification sites, and frequently embed conserved binding motifs. 141 also, it was proposed that alternative splicing of intrinsically disordered regions containing linear interaction motifs and/or post-translational modification sites results in complete rewiring of protein interactions. 142 figure 12 shows that in agreement with these earlier observations the alternatively spliced isoform-5 of human sirtuin-2 differs from the canonical isoform (uniprot id: q8ixj6) by lacking the intrinsically disordered nterminal tail (cf. figs. 12a and 12b) . curiously, the anchor analysis 15,16 of the human sirtuin-2 revealed that this alternative splicing event removed 2 aibss (located at the residues 1-15 and 36-49 of the isoform-1). therefore, functionality of siruin-2 is modulated by alternative splicing that removes potential binding sites from the disordered region of this protein. parker et al. investigated the role of the amino acid sequence at the c-terminal region of human semaphorin-3 in interaction of this protein with neuropilin-1. 143 the neuropilin-1 is a member of the family of type i transmembrane receptors that are essential in development, homeostasis, and pathogenesis being involved in the coordination of several important volume 2 issue 1 intrinsically disordered proteins signaling events in the cardiovascular and nervous system. the members of the class iii semaphorin (sema3) family of axon guidance molecules are among the ligands neuropilins (nrps). ligand binding of nrps is a subject of the intensive research due to the involvement of these transmembrane receptors in various pathogenic conditions. it has been established that all known nrp ligands require a c-terminal arginine (c r ) for binding to a conserved pocket in the nrp b1 domain. [144] [145] [146] [147] to better understand the mechanism of interaction between the nrps and their ligands and to understand the role of residues upstream of the c r , parker et al. synthesized a library of semaphorin-3 derived peptides. this study revealed that the c-1 residue (i.e., residue preceding the c r ) serves the critical role of positioning the c r and c-2 residues to promote concurrent nrp binding. 143 bioinformatics analysis revealed that the analyzed sequence (wdqkkprnrr) is a part of long intrinsically disordered tail that spans over the last 100 residues of human semaphorin-3 (uni-prot id: q13275, see fig. 9f ). malashkevich et al. reported the x-ray crystal structure of the unmodified 3 glu-ocn form of bovine osteocalcin. 148 this is an important contribution since it adds a crucial structural information on osteocalcin, which is a small, abundant noncollagenous protein synthesized by osteoblasts, and that typically contains 3 g-carboxyglutamic acid (3 gla-ocn) residues generated posttranslationally in a vitamin k-dependent process 149,150 by the vitamin k-dependent (vkd) carboxylase. 151 earlier, based on the circular dichroism study it has been concluded that in the presence of ca 2c , the 3 glu-ocn molecule contained significantly less a-helical structure than the 3 gla-ocn form. 152 furthermore, it was shown that ca 2c no longer binds to decarboxylated, unmodified osteocalcin 153 malashkevich et al. revealed that the crystal structure of the thermally decarboxylated bovine osteocalcin contained residues 17-47, was rather similar to 3 gla ca 2c -ocn, consisted of 3 a-helices surrounding a hydrophobic core, and contained c23-c29 disulfide bond between 2 of the helices but did not contain bound ca 2c . 148 this is an interesting finding since earlier work clearly showed that in their apo-forms, modified and non-modified osteocalcin from different sources are mostly disordered. 148 the explanation for this apparent contradiction can be derived from the analysis of conditions used for the 3 glu-ocn crystallization, where protein in 20 mm nacl and 10 mm cacl 2 (ph 7.0) was mixed with the reservoir solution containing 2.5 m ammonium sulfate and 0.1 m bis-tris propane (ph 7.0) in 1:1 ratio. 148 therefore, high ionic strength solution was used to partially neutralize the high anionic charge in the helical regions of osteocalcin. although this approach permitted better realization of the a-helix-forming potential of the nonmodified osteocalcin, the crystallization conditions are clearly non-physiological. sasi et al. investigated the peculiarities of the regulation of expression of the inducible heat shock protein hspa1a at the transcription level by several transcription factors. 154 hspa1a, together with hspa1b are the members of the human inducible heat shock protein family (hsp70), altered expression of which has been attributed to the pathogenesis of various diseases, such as cancer, cardiovascular diseases, and neurodegenerative disorders. [155] [156] [157] [158] it is known that the heat shock response-related proteins (including hsp70) can be expressed during normal conditions (e.g., during the cell growth and development) or can be induced by various pathological conditions, such as infection, inflammation, and protein conformation diseases. sasi et al. show that transcription factors hsf-1, creb, nf-y, and nf-kb synergistically regulate expression of the heat-shock-induced gene hspa1a. 154 the initiation of the heat shock response is manifested by the activation of the heat shock transcription factors (hsfs), a family of related transcription factors which, in mammals, is composed of hsf-1, -2, -3, -4, -5, -y and -x. 159 camp response element binding protein (creb) is the camp-regulated transcription factor that has been shown to stimulate target gene expression often via the associating with the coactivator paralogs p300 and creb binding protein (cbp). [160] [161] [162] complex formation between creb and cbp/p300 requires protein kinase a (pka)-mediated phosphorylation of creb at ser-133. 160 the nuclear transcription factor y (nf-y) is a sequence-specific dna-binding protein that recognizes the y box, which is a promoter element common to all major histocompatibility complex class ii genes. 163 also, nf-y is known to interact with a ccaat box, which is one of the most common elements in eukaryotic promoters, found in the forward or reverse orientation. 164 furthermore, nf-y has been reported to play a key role in the basal expression of many hsps through this ccaat box. 165, 166 nf-y is heterotrimeric transcription factor composed of 3 components, nf-ya (subunit a, uni-prot id: p23511), nf-yb (subunit b, uniprot id: p25208), and nf-yc (subunit g, uniprot id: q13952), where the dimerization of nf-yb and nf-yc is a prerequisite for the nf-ya association and dna binding. 167 finally, the transcription factor nuclear factor-kappa b (nf-kb; uniprot id: 19838) is a key player in an intracellular signaling cascade regulating many inflammatory mediators. 168 nf-kb is a common transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. nf-kb is a homo-or heterodimeric complex formed by the rel-like domain-containing proteins rela/p65, relb, nf-kb1/p105, nf-kb1/p50, rel and nf-kb2/p52, and the heterodimeric p65-p50 complex. figure 13 represents the results of the multiparametric evaluation of intrinsic disorder propensities of hsf-1 (a, uniprot id: q00613), creb (b, uniprot id: p16220), nf-ya (c, uniprot id: p23511), nf-yb (d, uniprot id: p25208), nf-yc (e, uni-prot id: q13952), and nf-kb (f, uniprot id: p19838) and shows that these proteins are predicted to be highly disordered. the high levels of functional intrinsic disorder is a "family signature" of the transcription factors in general. 169 furthermore, the recent analysis of the abundance and importance of idprs in functions of various hsfs clearly indicated that the heat shock response requires hsf flexibility to be more efficient. 170 also, the importance of intrinsic disorder in functions of various proteins involved in the innate immune response (including nf-kb) has been recently analyzed. 171 phosphorylation sites in melanopsin blasic et al. analyzed the peculiarities of phosphorylation of the c-terminal domain of the photopigment melanopsin possessing 37 serine and threonine sites that are potential sites for phosphorylation by a g-protein dependent kinase (grk). 172 melanopsin is a member of the g protein coupled receptor (gpcr) family that undergoes light-dependent phosphorylation that is involved in deactivation of the photoresponse. blasic et al. revealed that of the 37 phosphorylation sites, a small cluster of 6 or 7 sites in the proximal region of the c-tail is critical for mediating deactivation. 172 figure 14a shows that mouse melanopsin (uniprot id: q9qxz9) is predicted to have long disordered c-tail (residues 380-521). this is a rather expected output since phosphorylation sites are known to be preferentially located within the idprs. [173] [174] [175] human defensins a recent review of wisons et al. is dedicated to the antiviral activities of human defensins. 176 these peptides with a wide range of antimicrobial activities are important components of the innate immune system. among various antiviral mechanisms of human defensins are direct targeting of viral envelopes, glycoproteins, and capsids, inhibition of viral fusion and post-entry neutralization, inhibition of viral replication via disruption of host intracellular signaling and binding and modulation of host cell surface receptors, and augmentation and altering of adaptive immune responses. 176 of the 2 types of defensins, aand b-defensins, found in human, significant knowledge is accumulated about the molecular mechanisms of the a-defensin action, whereas molecular details on the b-defensin activities are more disperse. 176 defensins are small (»29-129 amino acids) cationic, amphipathic polypeptides with a predominantly b-sheet structure stabilized by 3 disulfide bonds. 176 human a-defensins are typically shorter than b-defensins, with a-defensins staying in a range of 29-39 residues and some b-defensins can being as long as 129 residues. search of uniprot for human defensins produces about 40 hits, of which 6 entries were a-defensins. defensins are produced in a form of proprotein containing a signal peptide and/or a propeptide. figure 15 represents disorder profile for several representative members of the human defensin family and shows that all proproteins possess noticeable amount of intrinsic disorder, the content of which varies significantly between different defensins. it is likely that the presence of noticeable disordered tails plays a role in the maturation of defensins since sites of the proteolytic attack are commonly located within regions of intrinsic disorder. 177 it is also possible that these disordered tails serve as entropic bristle domains 178 burke et al. investigated the roles of the phosphorylation of the c-terminal domain of the retinoblastoma protein (rbc) in interaction of rb with the e2f transcription factor and related regulation of the rb activities in growth suppression. 179 deregulation of the broad-functioning tumor suppressor retinoblastoma protein (rb) is associated with several human cancers. 180, 181 among numerous functions ascribed to this important protein is a negative regulation of cell division at the g 1 -s transition of the cell cycle, 182 where rb forms a growth-repressive complex with e2f transcription factors 183 in a phosphorylation-dependent manner. burke et al. showed that there are at least 2 different mechanisms by which rbc phosphorylation inhibits e2f binding. here, phosphorylation of s788 and s795 weakens the direct association between the rbc and the marked-box domains of e2f, whereas phosphorylation of s788, s795, s807 and s811 induces an intramolecular association between rbc and the pocket domain, which overlaps with the site of e2f transactivation domain binding. 179 figure 14b shows that human retinoblastoma-associated protein (uniprot id: p06400) is predicted to have numerous disordered regions, including long disordered tails. the longest disordered region is the c-terminal tail (residues 760-928) that contains all the phosphorylation sites discussed above. according to the anchor analysis, 15 www.landesbioscience.com e970499-23 intrinsically disordered proteins are typically found in regions of intrinsic disorder. 126, 174, [185] [186] [187] the tetratricopeptide repeat (tpr) motifcontaining protein lgn and its binding partner frmpd1 (ferm and pdz domain containing 1) pan et al. described structural peculiarities of the human lgn-frmpd1 complex. 188 to this end, a crystal structure of the complex between the 15-350 fragment of human lgn containing 8 tetratricopeptide repeat motifs (uniprot id: p81274) and the 901-951 fragment of human frmdp1 (uniprot id: q5syb0) was solved at 2.4 a resolution. 188 in this lgn-tpr/frmpd1 complex, almost the entire length of lgn-tpr was well resolved, and most residues of the frmpd1 fragment were clearly resolved adopting an extended conformation that occupied most of the concave channel formed by the 8 tpr motifs and buried a total of 2541 a 2 surface area. 188 the noticeable exceptions were the 11 residues in the loop connecting the aa and ab of tpr3 (amino acids 153-163) and the 6 residues at the c terminus (amino acids 345-350) in the lgn-tpr, and the nterminal 8 residues (amino acids 912-919) and the last 2 residues at the c terminus (amino acids 937-938) of the frmpd1 fragment, all of which were missing in the structure of the lgn-tpr/frmpd1 complex. 188 leu-gly-asn repeatenriched protein lgn/ ags3 in mammals (pins, or partner of inscuteable in drosophila neuroblasts) plays a number of crucial roles in regulation of cell polarity and spindle orientation during cellular differentiation and self-renewal in multicellular eukaryotes development. [189] [190] [191] structurally, lgn consist of 8 tetratricopeptide repeat (tpr) motifs in its n-terminal half and 3 or 4 goloco motifs (also referred to as g-protein regulatory or gpr motifs) in the c-terminal half. [192] [193] [194] both of these motifs are crucial for protein-protein interactions, 195, 196 with each goloco motif of pins/lgn being capable of binding to gdp-bound gai 197 leading to stable cortical localization of pins/lgn, 198 and with the tpr motifs possessing multiple binding partners. the canonical tpr motif is a 34-amino-acid protein-protein interaction module multiple copies of are found in a wide range of proteins with diverse functions such as cell cycle regulations, gene transcription and splicing processes, protein trafficking, and protein folding. 195, 199 figure 15a shows that human lgn protein is involved in a wide range of protein-protein interactions, clearly serving as an important hub protein. this interactivity analysis is done by string. 59 frmpd1 protein (ferm and pdz domain containing 1) is known to serve as a regulatory binding partner of ags3, with a short fragment of frmpd1 (amino acids 901-938) being shown to bind to the 8 tpr motifs of ags3. 200 also, a 50residue fragment (amino acids 901-951) of frmpd1 was shown to bind to lgn with a k d »1 mm. 188, 194 curiously, besides the mentioned above regions of missing electron density in the lgn-tpr/frmpd1 complex figures 15b and 15c shows that fulllength lgn and frmpd1 proteins both belong to the category of hybrid proteins possessing some ordered domains and significant number of intrinsically disordered protein regions (idprs). in fact, the goloco motif containing c-terminal half of human lgn (residues 350-684) and the major portion of frmpd1 (residues 511-1390) are predicted to be mostly disordered by all the computational tools used in this study. overall, more than 40% of the lgn residues and more than 50% of the frmpd1 residues are predicted figure 16 . (a) evaluation of the interactivity of the human lgn by string, which is the online database resource search tool for the retrieval of interacting genes that provides both experimental and predicted interaction information. 59 string produces the network of predicted associations for a particular group of proteins. the network nodes are proteins. the edges represent the predicted functional associations. an edge may be drawn with up to 7 differently colored lines -these lines represent the existence of the 7 types of evidence used in predicting the associations. a red line indicates the presence of fusion evidence; a green line -neighborhood evidence; a blue lineco-occurrence evidence; a purple line -experimental evidence; a yellow linetext mining evidence; a light blue line -database evidence; a black lineco-expression evidence. 59 to be disordered, which clearly places these proteins to the category of highly disordered proteins. furthermore, both proteins are expected to have numerous disorder-based interaction sites. these can be identified by anchor, 15,16 a computational tool that relies on the pair-wise energy estimation approach developed for the general disorder prediction method iupred, 201, 202 and is based on the hypothesis that long regions of disorder contain localized potential binding sites that cannot form enough favorable intra-chain interactions to fold on their own, but are likely to gain stabilizing energy by interacting with a globular protein partner. 15, 16 here the term anchor-indicated binding site (aibs) is used to identify a region of a protein suggested by the anchor algorithm to have significant potential to be a binding site for an appropriate but typically unidentified partner protein. this analysis revealed that there are 10 aibss in lgn (residues 370-379, 436-452, 491-499, 508-513, 531-537, 545-553, 566-573, 596-607, 629-638 and 661-671), whereas frmpd1 contains 23 aibss (residues 534-554, 609-617, 622-636, 652-662, 704-715, 747-779, 792-804, 828-878, 896-909, 932-949, 958-967, 983-1019, 1023-1077, 1087-1100, 1106-1114, 1119-1129, 1138-1171, 1192-1220, 1245-1250, 1290-1297, 1312-1319, 1336-1342, and 1424-1431) . of special interest is the fact that the 901-951 fragment of human frmdp1 used in the mentioned structural analysis 188 is predicted to overlap with 2 aibss (residues 896-909 and 932-949). this is a clear indication that intrinsic disorder plays a role in the formation of the lgn-tpr/frmpd1 complex. davenport et al. reported a crystal structure of human sirtuin-1catalytic domain (residues 234-510) in complex with its c-terminal regulatory segment (ctr, residues 641-665). 203 in this structure (see fig. 17a , pdb id: 4ig9), the catalytic nad c -binding domain adopts the canonical sirtuin fold, whereas ctr forms a b-hairpin structure complementing the b-sheet of the catalytic domain and covering an essentially invariant hydrophobic surface. 203 biological significance of the sirtuin family was outlined in the previous section. human sirtuin-1 (sirt1) is implicated in a wide range of human diseases due to the fact that this enzyme plays multiple roles in cellular processes ranging from energy metabolism to cell survival via it ability to deacetylate a wide range of substrates, such as p53, nf-kb, foxo transcription factors, and pgc-1a. [204] [205] [206] deacetylation activity of sirt1 was shown to be affected by various regions located within the long n-and c-termini that flank the sirt1 catalytic domain. 207, 208 figure 17b illustrates that in agreement with a recent bioinformatics study revealing that n-and c-terminal segments of sirtuins in all known organisms are expected to be intrinsically disordered, 209 both termini of human sirt1 (uniprot id: q96eb6, residues 1-250 and 500-747) are predicted to be mostly disordered, whereas the central catalytic domain is mostly ordered. the anchor analysis 15, 16 revealed that human sirt1 contains 14 aibss located at the residues 1-33, 43-49, 53-125, 133-169, 184-193, 220-225, 498 -503, 521-528, 549-574, 588-593, 618-624, 637-672, 691-706, and 710-744. it is important to emphasize here that the ctr (residues 641-665) used in the mentioned above structural analysis of human sirt1 203 is completely embedded within one of the aibss (residues 637-672). the endosome-associated deubiquitinase amsh davies et al. performed a systematic mutational analysis to elucidate the molecular mechanisms of activation of amsh [associated molecule with a src homology 3 domain of signal transducing adaptor molecule (stam)], a deubiquitinating enzyme (dub) with exquisite specificity for lys63-linked polyubiquitin chains and recruitment of this protein to the endosomal sorting complexes required for transport (escrt) machinery. 210 the fact that the mutations amsh were implemented in microcephaly capillary malformation (mic-cap) syndrome in children 211 further reiterates the importance of this study. amsh is a member of the jamm (jab1/mpn/mov34) family of dubs involved in the regulation of ubiquitin signaling by catalyzing the hydrolysis of isopeptide (or peptide) bonds between ubiquitin and target proteins or within polymeric chains of ubiquitin. 212 the catalytic activity of amsh is stimulated upon binding to stam, which is a member of the escrt-0 complex. here, the amsh-stam interaction is conducted through the binding of the sh3 binding motif of amsh to the sh3 domain of the stam. 213 davies et al. used in their analysis the catalytic domain of amsh (residues 219-424). 210 figure 14c represents the results of the disorder analysis of human amsh (uniprot id: o95630) and shows that this protein contains a long idpr (residues 90-250) thereby illustrating that the n-terminal part of the analyzed catalytic domain is predicted to be disordered. tonb is the e.coli inner membrane protein, which possesses a polyproline motif (residues 70-81) that may span the length of the periplasmic space 216 and a globular c-terminal domain (residues that interacts with the transporters. 217, 218 figure 18a shows that the n-terminal half of the periplasmic domain of tonb protein (uniprot id: p02929) is predicted to be highly disordered, whereas its c-terminal domain possesses significant amount of order. figure 18b illustrates an important point that the dimer formed by the truncated periplasmic domain (residues 150-239) is a highly intertwined and elongated structure with noticeable domain swapping. a crystal structure of the tonb-fhua complex is shown in figure 18c . curiously, although the entire periplasmic domain of tonb was used in the crystallization experiment, residues 39-157 and 236-239 are not seen in the resulting structure, clearly indicating that these regions are likely to preserve mostly disordered structure even in the bound form of tonb. the disordered n-terminal and c-terminal tails clearly have functional importance since they contains aibss, 3 at the n-terminus (residues 39-65, 92-95, and 124-164) and one at the c-terminus (residues 232-239). one more aibs is predicted at residues 171-188. stark et al. performed functional analysis of the important human chaperone, dnaj homolog subfamily a member 1 (dnaja1), and solved solution structure of its j-domain (residues 1-67). 219 human dnaja1 has multiple important functions and act as protein chaperone, regulator of androgen receptor signaling, and activator of the dnak protein. furthermore, levels of the human dnaja1 serve as a biomarker for pancreatic cancer, since the expression of this protein in pancreatic cancer cells is downregulated fold5-. 220 nmr analysis revealed that the structure of the human dnaja1 j-domain consists of 4 a-helices, residues 17-21 (a1), 29-42 (a2), 52-65 (a3), and 68-75 (a4) (see fig. 19a ; pdb id: 2m6y). 219 on the contrary, the evaluation of intrinsic disorder propensity of the full-length protein suggested that dnaja1 (uniprot id: p31689) possesses significant amount of disorder throughout its n-and c-terminal tails (see fig. 19b ). curiously, by anchor analysis, there are 3 disorderbased binding sites in dnaja1, residues 26-31, 85-93, and 392-397, with binding site #2 being overlapped with a2. structure and functions of the purinerich element binding protein b (purb) in a recent comprehensive study, romora et al. showed that the purine-rich element binding protein b (purb) serves as a suppressor of myofibroblast differentiation and acta2 repression. 221 pura and purb are members of a small family of nucleic acid-binding proteins that interact with purine-rich ssdna or rna sequences homologous to the so-called pur element originally described in eukaryotic gene flanking regions and origins of dna replication. [222] [223] [224] figure 14d shows that human purb protein (uniprot id: o35295) is predicted to possess significant amount of functionally important intrinsic disorder. importance of the long disordered regions in this protein is supported by finding 10 aibss (residues 1-6, 18-27, 47-75, 87-95, 99-106, 135-137, 142-144, 186-201, 243-256, and 277-287) . in agreement with these predictions, the authors showed that several regions of purb were (residues 41-112, 125-210, and 125-303) were intrinsically unstable. 221 the host restriction factor tetherin hotter et al. provided a comprehensive review of the antiviral role of one of the host restriction factors (i.e., specific cellular antiviral factors that inhibit retroviral replication at different steps of the viral life cycle), human tetherin. 225 among various antiviral mechanisms ascribed to tetherin are the inhibition of the release of diverse enveloped viruses by tethering them to the cell surface, induction of an inflammatory response, activation of nf-kb, action as an innate immune sensor of viral infections, activation of immune response through interactions with the immunoglobulin-like transcript 7 (ilt7, lilra4). 225 as commonly seen in the virus-host arm race, effective antiviral strategies of the host are counterbalanced by the development of novel invasive strategies, and various viruses have evolved antagonists against this host restriction factor, such as accessory hiv-1 (human immunodeficiency virus type 1) vpu protein that counteracts the effects of tetherin. 225 tetherin is a type ii transmembrane protein that contains both an n-terminal transmembrane region and a c-terminal glycosyl-phosphatidylinositol (gpi) anchor. 226 structurally, human tetherin exists as a disulfide-linked parallel homodimer, which is formed via the extracellular part that is involved in the formation of a canonical coiled-coil structure, where a long a-helix contains 3 cysteines that form disulfide bonds with the corresponding cysteines of a second tetherin molecule. 227 it is believed that tetherin-caused viral retention relies on this unusual architecture, where one transmembrane anchor may remain in the cellular plasma membrane, whereas the other is able to stick to the viral membrane. 228, 229 figure 20a represents the results of the multi-tool disorder predictions for human tetherin (also known as bone marrow stromal antigen, uniprot id: q10589) and shows that this protein is expected to be mostly disordered. this is not a very surprising observation since coiled-coil proteins are expected to be intrinsically disordered in their monomeric states and fold to a helical structure at the formation of coiled-coil structure caused by the interaction with corresponding partners. 230 cyclin d1/cdk2 complex to clarify some uncertainties in previous studies regarding formation and activities of the cyclin d1/cyclin-dependent kinase 2 (cdk2) complexes jahn et al. utilized a novel p21-pcna fusion protein and p21 mutant proteins to understand the mechanisms of the cyclin d1/cdk2 complex formation. 231 the authors show that p21 serves as an important scaffolding protein, which is required for the formation of the functional cyclin d1/cdk2 complex, since cyclin d1 and cdk2 failing to complex in its absence. 231 figure 20b shows that the scaffolding protein p21 (which is a cyclin-dependent kinase inhibitor 1, uniprot id: p38936) is predicted to be mostly disordered and packed with disorder-based binding sites. in fact, there are 5 aibss in this relatively small protein, residues 36-41, 68-79, 100-105, 109-123, and 145-164. using this approach, the authors were able to identify a novel cdk2 substrate, polypyrimidine tract binding protein-associated splicing factor (psf). figure 20c shows that psf (uniprot id: p23246) is predicted to have highly disordered tails. anchor analysis 15, 16 revealed that intrinsic disorder is crucial for the psf human and mouse apolipoproteins a-i nguen et al. analyzed mechanistic peculiarities of the interaction between the high density lipoprotein (hdl) particles and human or mouse apolipoprotein a-i (apoa-i). 232 the authors established that the c-terminal domains (ctds) of human and mouse apoa-i proteins are the major players facilitating interactions of these proteins with hdl. curiously, human ctd, being a bit more hydrophobic than mouse ctd, binds to the hdl particle with higher affinity. on the other hand, the isolated n-terminal helix bundle domains (residues 1-190) of human and mouse apoa-i proteins binds hdl poorly. 232 apoa-i stabilizes discoidal hdl particles by forming a double-belt structure 233, 234 and plays a major role in the reverse cholesterol transport pathway. 235, 236 apoa-i contains a globular n-terminal domain (residues 1-43) and a lipid-binding c-terminal domain (residues 44-243). 234 in the belt-like structure of the smallest discoidal hdl, 2 apoa-i molecules wrap around a small patch of bilayer containing 160 lipid molecules. the c-terminal domain of each monomer is ring-like, curved, planar amphipathic a-helix with the hydrophobic surface curved toward the lipids. 234 figure 21 shows that both human and mouse apoa-i proteins (uniprot ids: p02647 and q00623) are predicted to be mostly disordered, with mouse protein possessing a bit more disordered structure. according to the anchor analysis, there are 3 aibss in both proteins (residues 15-21, 158-164, and 221-230 in human protein, and residues 13-18, 173-204, and 207-230 in mouse apoa-i). figure 21c represents a structure of the human apoa-i dimer in a complex with lipids (pdb id: 3k2s). this model was built by uniting several synergistic experimental techniques, such as small angle neutron scattering (sans) with contrast variation, isotopic deuteration of selected macromolecule components, and hydrogen/ deuterium exchange tandem mass spectrometry (hd-ms/ms). 237 in agreement with the results of disorder predictions, the apoa-i dimer represents a highly intertwined double superhelix with a minimal number of intrachain contacts, which is stabilized mostly by the interchain interactions. in other words, this complex is characterized by a very large interface area, and, therefore, according to gunasekaran et al. 238 it belong to the category of 2-state dimers, where the monomers are unfolded in the unbound state and fold simultaneously with the complex formation. function and regulation of mavs jacobs and coyne provided an important overview of the mitochondrial antiviral signaling protein (mavs, also known as ips-1/visa/cardif). 239 mavs is the mitochondrially-located innate immune signaling adaptor regulating and coordinating signals received from 2 independent cytosolic pathogen recognition receptors to induce antiviral genes. mavs can be regulated by host cell factors that inhibit mavs signaling by direct protein-protein interactions, by altering mitochondrial properties or dynamics, or by post-translational modifications. 239 the tip of the mavs c-terminus contains a mitochondrial intermembrane region (residues 535-540) needed for the interaction with the mitochondrial membrane. according to uniprot, human mavs (uniprot id: q7z434) is involved in a wide array of protein-protein interactions. among established mavs binding proteins are: ddx58/rig-i, ifih1/mda5, traf2, traf6, c1qbp, irf3, fadd, ripk1, chuk, ikbkb, hcv and hepatitis gb virus b ns3/4a proteases, hhav protein 3abc, nlrx1, psma7, trafd1, pcbp2, ips1, itch, cyld, src, dhx58/lgp2, ddx58/rig-i, ikbke, tmem173/mita, ifit3, tbk1 , and human respiratory syncytial virus (hrsv) ns1 protein (http://www. uniprot.org/uniprot/q7z434). mavs contains multiple alternatively spliced isoforms, sites of posttranslational modifications, and a proline-rich region (residues . in other words, mavs satisfies all the major criteria to be considered as an intrinsically disordered protein. figure 22a represents the vast interactome of mavs evaluated by string. figure 22b shows that human mavs is predicted to be a highly disordered protein. anchor analysis 15 khasnis et al. analyzed the mechanisms and functional outputs of the phosphorylation/dephosphorylation events at thr696 and thr853 sites of the mypt1 protein, which is a regulatory subunit of the myosin light chain phosphatase (mlcp). 240 mlcp is a cytoskeleton-associated protein phosphatase-1 (pp1) that serves as a rhoa/rock effector, regulating dynamic reorganization of the cytoskeleton crucial for cell motility. the authors also showed that the c-terminal domain of human mypt1 (residues 495-1030) was responsible for the binding to the n-terminal portion of myosin light meromyosin. 240 promiscuous interactability of mypt1 is illustrated by figure 23a representing the results of string analysis. figure 23b shows that the very significant part of mypt1 (uniprot id: o14974) is expected to be intrinsically disordered, with longest disordered region covering »72% of protein length (residues 290-1030) and containing 18 disorder-based binding sites (residues 318-338, 374-403, 406-419, 434-454, 472-477, 492-506, 548-564, 579-588, 626-649, 666-673, 697-712, 754-774, 780-815, 853-860, 887-892, 897-917-932-943, and 1021-1030) . clearly, intrinsic disorder is crucial for function of this protein. li et al. investigated structural peculiarities of the core polypeptide of the inner membrane gerd lipoprotein from geobacillus stearothermophilus. 241 some bacteria are able to form endospores in response to adverse growth conditions. 242, 243 spores formed as a result of such sporulation process are extremely resistant to various environmental insults thereby providing the bacteria with an important means to exist in the metabolically dormant state indefinitely and remain viable for hundreds of years without water or nutrients. 244, 245 restoration of the favorable conditions triggers spore germination leading to the fast (within minutes) "awakening" of the normal metabolism in bacteria followed by outgrowth to generate growing cells. 243, 244, 246 this awakening is controlled by the specific nutrient sensors, cognate germinant receptors (grs) located in the inner membrane of the spore. gerd is one of such bacterial cognate germinant receptors that trigger spore germination in the presence of specific nutrients called germinants in the environments of the spores. 241 li et al. have determined the crystal structure of the 121-residue core domain of the geobacillus stearothermophilus gerd protein (gerd ) that lacks the n-terminal signal and lipobox sequences (residues 1-28), the h01 and h02 helices (residues , and the c-terminal acidic tail (residues [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] [191] [192] [193] [194] [195] . 241 by a set of biophysical techniques, this gerd 60-180 was shown to form a stable, well-ordered 3helix bundle in solution. in crystal structure, gerd 60-180 trimer consists of 3 parallel polypeptide chains twisted into a superhelical, right-handed rope (pdb id: 4o8w). 241 in this elongated trimer, each of the individual gerd 60-180 chains forms 8 helices (h1 to h8) linked by short turns. structures of the individual chains can be superimposed on each other except to the loosely packed h8 helices. helices h1-h7 in each gerd 60-180 chain twist around the central axis to form 2 complete turns of a right-handed supercoil with a pitch of »44a . figure 24a represents crystal structure of the gerd 60-180 trimer as a molecular surface and ribbon diagram and also shows the highly extended structure of one of the gerd 60-180 monomers. 241 analysis of the overall shapes of the highly intertwined and extended monomers within the gerd 60-180 trimer suggests that the formation of this trimer represents an example of the folding-uponbinding process. in agreement with this hypothesis, figure 24b shows that the core domain of the geobacillus stearothermophilus gerd protein (uniprot id: q5l3q1) is mostly disordered. prokaryotic ubiquitin-like protein (pup), the pup ligase pafa and bacterial proteasome forer et al. investigated the interaction between the prokaryotic ubiquitin-like protein (pup), the proteasome regulatory subunit mpa (mycobacterium proteasome atpase), and proteasome accessory factor a (pafa), an enzyme that forms an isopeptide bond between the g-carboxylate of a glutamate at the c-terminus of pup and the e-amine of a substrate lysine. 247 the authors show that pup is involved in simultaneous interaction with both mpa and pafa, and that mpa forms a complex with pafa, suggesting that pafa and the proteasome acts as a modular machine for the tagging and degradation of cytoplasmic proteins. 247 figure 25a represents a crystal structure of 3 pup molecules bound to the mpa hexamer (pdb id: 3m9d), and the results of disorder prediction for the mycobacterium tuberculosis pup (uniprot id: p9whn5) are shown in figure 25b . pup is obviously a highly disordered protein that partially folds at binding. in fact, of 68 residues used in the crystallization experiment, only 31 are visible in structure, whereas remaining 37 residues (1-20 and 52-68) are located within the regions of missing electron density. according to the anchor analysis, pup possesses 2 disorder-based binding regions, residues 1-15 and 34-64. curiously, forer et al. suggested that pup has 2 binding sites for rafa located at residues 38-47 and 51-58, and that the first rafa binding site is overlapped with the region responsible for the mpa binding (residues 21-51). 247 the nucleolar pict-1/gltscr2 protein borodianskiy-shteinberg et al. studied self-association of the human protein interacting with carboxyl terminus-1 (pict-1), also known as the glioma tumor suppressor candidate region 2 gene product (gltscr2), a nucleolar protein with unknown function. 248 pict-1/ gltscr2 is conserved among eukaryotes and is assumed to be essential for preimplantation embryogenesis and embryonic stem cell survival and proliferation. 249 it was suggested that pict-1 might act as a potential tumor suppressor, being involved in interaction with the cterminal region of the tumor suppressor phosphatase and tensin homolog (pten), promoting its phosphorylation and stabilization. 250 it was also suggested that pict-1 can function as an oncogene through the inhibition of p53. 249 figure 26a represents the results of the evaluation of the interactivity of the human pict-1/gltscr2 (uniprot id: q9nzm5) by string and shows a dense interaction network of this intriguing protein. figure 26b provides an explanation for the binding promiscuity of pict-1 by showing that this protein is predicted to be highly disordered. recognition of modified trna by the hiv-1 nucleocapsid protein p7 and related peptides spears et al. studied the molecular mechanisms underlying specific recognition of highly post-transcriptionally modified human trna lys3 uuu (which is the primer for hiv replication) by the hiv-1 nucleocapsid protein p7. 251 it is known that p7 recruits htrna lys3 uuu from the host cell by binding to and remodeling the trna structure. an intrigue here is in the fact that htrna lys3 uuu is one of the most uniquely processed trnas that has a broad spectrum of chemically different post-transcriptional modifications playing crucial roles in regulation of the conformation and function of this trna during protein synthesis. 252 using phage display approach, spears et al. showed that p7 contains a specific htrna-lys3 uuu -binding region whose interaction with fully modified trna can be mimicked by the 15-and 16-amino acid peptides with the signature sequence of r-w-q/n-h-x 2 -f-pho-x-g/a-w-r-x 2 -g (where x can be most amino acids, and pho is any hydrophobic residue). 251 hiv-1 nucleocapsid protein p7 is a 55residues-long protein containing 2 zinc finger domains flanked by basic amino acids required for interaction with nucleic acids. 253, 254 in addition to the discussed above capability of p7 to specifically recognize htrna lys3 uuu , the major function of p7 is to bind specifically to the packaging signal of the full-length viral rnas and to deliver them into the assembling virion. 255 as it is a highly charged basic protein, p7 binds single-stranded nucleic acids nonspecifically. consequently, it coats the genomic rna protecting it from nucleases and compacting viral rna within the core. protein p7 also serves as an rna chaperone that enhances several nucleic acid-dependent steps of viral life, such as melting rna secondary structures, promoting dna strand exchange reactions during reverse transcription, [256] [257] [258] and stimulating integration. 259 in the nmr solution structure of p7, regions corresponding to the 2 zinc fingers (residues 15-28 and 36-49) possess wellresolved structures, whereas residues 1-13, 32-34, and 52-55 were highly dynamic and did not converge to the unique conformations. 260, 261 recent intrinsic disorder propensity analysis revealed that p7 is a highly disordered protein, with regions corresponding to the zinc fingers predicted to be more ordered than the remainder of the protein and identified as potential a-morfs. 262 this article represents a set of papers dealing with interesting and important proteins with diverse functions from various organisms. the only uniting theme for all these studies is the notion that they represent noticeable disorder overlooks. in fact, all proteins studied there contain intrinsic disorder. the amount of disorder ranges, and in some proteins only relative short regions are disordered or highly flexible, whereas some other proteins are almost entirely disordered. however, very often this disorder has functional implementations, and consideration of studied proteins in light of intrinsic disorder often provides interesting mechanical clues on the molecular mechanisms of their action. we hope that the readers will find this new series useful, since it might increase the awareness of the scientific community about importance of intrinsic disorder for protein function. we also hope to see submissions from readers who found some important intrinsic disorder overlooks in published articles. obviously, papers in this seris will range in their size, and we envision several types of submissions, such as comprehensive reviews of unreported disorder in papers published in particular journals (i.e., similar to this review), reviews of unreported disorder in specific areas of protein-related research, as well as brief notes about overlooked disorder in individual papers. the biggest hope though is that this series will become obsolete, and protein intrinsic disorder will find its way to be robustly present in the scientific literature. no potential conflicts of interest were disclosed. digested disorder: quarterly intrinsic disorder digest digested disorder, issue #2: quarterly intrinsic disorder digest digested disorder, issue #3: quarterly intrinsic disorder digest comprehensive comparative assessment of in-silico predictors of disordered regions eif4b and eif4g jointly stimulate eif4a atpase and unwinding activities by modulation of the eif4a conformational cycle new initiation factor activity required for globin mrna translation the mechanism of eukaryotic translation initiation and principles of its regulation molecular mechanism of scanning and start codon selection in eukaryotes mrna helicases: the tacticians of translational control the dead-box helicase eif4a: paradigm or the odd one out exploiting heterogeneous sequence properties improves prediction of protein disorder predicting intrinsic disorder from amino acid sequence sequence complexity of disordered protein pondr-fit: a meta-predictor of intrinsically disordered amino acids prediction of protein binding regions in disordered proteins anchor: web server for predicting protein binding regions in disordered proteins remarkable stabilization of plasminogen activator inhibitor 1 in a "molecular sandwich" complex interaction of t4 uvsw helicase and single-stranded dna binding protein gp32 through its carboxy-terminal acidic tail crystallographic and nmr analyses of uvsw and uvsw.1 from bacteriophage t4 dna helicases: new breeds of translocating motors and molecular pumps analysis of the dna translocation and unwinding activities of t4 phage helicases uvsw protein regulates bacteriophage t4 origin-dependent replication by unwinding r-loops dna helicase requirements for dna replication during bacteriophage t4 infection the t4 phage uvsw protein contains both dna unwinding and strand annealing activities processive and unidirectional translocation of monomeric uvsw helicase on single-stranded dna bacteriophage t4 uvsw protein is a helicase involved in recombination, repair and the regulation of dna replication origins the phage t4 protein uvsw drives holliday junction branch migration repetitive lagging strand dna synthesis by the bacteriophage t4 replisome crystal structure of a replication fork single-stranded dna binding protein (t4 gp32) complexed to dna the gene 59 protein of bacteriophage t4. characterization of protein-protein interactions with gene 32 protein, the t4 single-stranded dna binding protein characterization of bacteriophage t4-coordinated leading-and lagging-strand synthesis on a minicircle substrate identification and mapping of protein-protein interactions between gp32 and gp59 by cross-linking functional mapping of pilf and pilq in the pseudomonas aeruginosa type iv pilus system structure and function of pilq, a secretin of the dna transporter from the thermophilic bacterium thermus thermophilus hb27 secretins: dynamic channels for protein transport across membranes characterization of 14-3-3-zeta interactions with integrin tails 14-3-3 proteins-a focus on cancer and human disease 14-3-3 proteins as potential therapeutic targets cantley lc. the structural basis for 14-3-3:phosphopeptide binding specificity binding of 14-3-3 protein to the plasma membrane h(c)-atpase aha2 involves the three c-terminal residues tyr(946)-thr-val and requires phosphorylation of thr(947) atm-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins structural determinants of 14-3-3 binding specificities and regulation of subcellular localization of 14-3-3-ligand complexes: a comparison of the x-ray crystal structures of all human 14-3-3 isoforms structural basis of 14-3-3 protein functions intrinsic disorder is a key characteristic in partners that bind 14-3-3 proteins integrins: bidirectional, allosteric signaling machines integrin signalling at a glance the tail of integrin activation integrin cytoplasmic domain-binding proteins mechanisms that regulate adaptor binding to beta-integrin cytoplasmic tails the final steps of integrin activation: the end game beta2 integrin phosphorylation on thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding trimmunity: the roles of the trim e3-ubiquitin ligase family in innate antiviral immunity a unique bipartite cysteinehistidine motif defines a subfamily of potential zincfinger proteins a novel zinc finger coiled-coil domain in a family of nuclear proteins a novel protein sequence motif related to the zinc finger trim/rbcc, a novel class of 'single protein ring finger' e3 ubiquitin ligases the role of viperin in the innate antiviral response the string database in 2011: functional interaction networks of proteins, globally integrated and scored a molecular dynamics simulation-based interpretation of nuclear magnetic resonance multidimensional heteronuclear spectra of alpha-synuclein dopamine adducts hiv-1 restriction factor samhd1 is a deoxynucleoside triphosphate triphosphohydrolase nuclease activity of the human samhd1 protein implicated in the aicardi-goutieres syndrome and hiv-1 restriction contribution of sam and hd domains to retroviral restriction mediated by human samhd1 hiv/simian immunodeficiency virus (siv) accessory virulence factor vpx loads the host cell restriction factor samhd1 onto the e3 ubiquitin ligase complex crl4dcaf1 novel interaction between the major bacterial heat shock chaperone (groesl) and an rna chaperone (cspc) interplay between the heat shock response and translation in escherichia coli proteolysis in the escherichia coli heat shock response: a player at many levels the cold-shock response-a hot topic recent developments in bacterial coldshock response cold-shock response and cold-shock proteins rna remodeling and gene regulation by cold shock proteins the role of structural disorder in the function of rna and protein chaperones interactions of phosphatase and tensin homologue (pten) proteins with phosphatidylinositol phosphates: insights from molecular dynamics simulations of pten and voltage sensitive phosphatase the tumor suppressor, pten/mmac1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate intrinsic disorder in pten and its interactome confers structural plasticity and functional versatility the pten long n-tail is intrinsically disordered: increased viability for pten therapy pathological unfoldomics of uncontrolled chaos: intrinsically disordered proteins and human diseases the structure of xis reveals the basis for filament formation and insight into dna bending within a mycobacteriophage intasome determinants of directionality in lambda site-specific recombination control of directionality in l5 integrase-mediated site-specific recombination identification and characterization of mycobacteriophage l5 excisionase structure of the cooperative xis-dna complex reveals a micronucleoprotein filament that regulates phage lambda intasome assembly regulation of directionality in bacteriophage lambda site-specific recombination: structure of the xis protein unique proline-rich domain regulates the chaperone function of aipl1 flexible nets of malleable guardians: intrinsically disordered chaperones in neurodegenerative diseases variable contributions of basic residues forming an apc exosite in the binding and inactivation of factor viiia molecular events that control the protein c anticoagulant pathway regulation of blood coagulation by the protein c system the mechanism of inactivation of human factor v and human factor va by activated protein c the 2.8 a crystal structure of gla-domainless activated protein c the escherichia coli primosomal dnat protein exists in solution as a monomer-trimer equilibrium system assembly of the primosome of dna replication in escherichia coli requirements for replication restart proteins during constitutive stable dna replication in escherichia coli k-12 replication fork reactivation downstream of a blocked nascent leading strand the most important thing is the tail: multitudinous functionalities of intrinsically disordered protein termini immunoreceptor tyrosine-based inhibitory motif (itim)-mediated inhibitory signaling is regulated by sequential phosphorylation mediated by distinct nonreceptor tyrosine kinases: a case study involving pecam-1 signal transduction pathways mediated by pecam-1: new roles for an old molecule in platelet and vascular cell biology residues within a lipid-associated segment of the pecam-1 cytoplasmic domain are susceptible to inducible, sequential phosphorylation splicing kinase srpk1 conforms to the landscape of its sr protein substrate intrinsic disorder in the human spliceosomal proteome malleable ribonucleoprotein machine: protein intrinsic disorder in the saccharomyces cerevisiae spliceosome ifitms restrict the replication of multiple pathogenic viruses the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus ifitm1 is a tight junction protein that inhibits hepatitis c virus entry mechanism of membrane fusion by viral envelope proteins ifitm3 inhibits influenza a virus infection by preventing cytosolic entry predicting disordered regions from amino acid sequence: common themes despite differing structural characterization predicting binding regions within disordered proteins coupled folding and binding with alpha-helix-forming molecular recognition elements analysis of molecular recognition features (morfs) mining alpha-helix-forming molecular recognition features with cross species sequence alignments type 2 ryanodine receptor domain a contains a unique and dynamic alpha-helix that transitions to a beta-strand in a mutant linked with a heritable cardiomyopathy involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle interactions between dihydropyridine receptors and ryanodine receptors in striated muscle crystal structures of the nterminal domains of cardiac and skeletal muscle ryanodine receptors: insights into disease mutations van petegem f. the deletion of exon 3 in the cardiac ryanodine receptor is rescued by beta strand switching atpinduced dimerization of the ff epsilon subunit from bacillus ps3: a hydrogen exchange-mass spectrometry study crystal structure of the epsilon subunit of the proton-translocating atp synthase from escherichia coli phosphorylation of the cyclin capcl5 modulates both cyclin stability and specific recognition of the substrate principles of cdk regulation cyclin specificity: how many wheels do you need on a unicycle? a family of cyclin-like proteins that interact with the pho85 cyclin-dependent kinase regulation of the transcription factor gcn4 by pho85 cyclin pcl5 coevolution of cyclin pcl5 and its substrate gcn4 understanding protein non-folding constitutive nuclear localization of an alternatively spliced sirtuin-2 isoform crosstalk between components of circadian and metabolic cycles in mammals sirtuins as regulators of metabolism and healthspan sirt2 maintains genome integrity and suppresses tumorigenesis through regulating apc/c activity the mammalian sir2alpha protein has a role in embryogenesis and gametogenesis the conserved role of sirtuins in chromatin regulation impaired dna damage response, genome instability, and tumorigenesis in sirt1 mutant mice regulation of sirtuin function by posttranslational modifications characterization of the murine sirt3 mitochondrial localization sequence and comparison of mitochondrial enrichment and deacetylase activity of long and short sirt3 isoforms distinct regulation of mitochondrial localization and stability of two human sirt5 isoforms transcription levels of sirtuin family in neural stem cells and brain tissues of adult mice evolutionarily conserved and nonconserved cellular localizations and functions of human sirt proteins interphase nucleo-cytoplasmic shuttling and localization of sirt2 during mitosis alternative splicing in concert with protein intrinsic disorder enables increased functional diversity in multicellular organisms tissue-specific splicing of disordered segments that embed binding motifs rewires protein interaction networks alternative splicing of intrinsically disordered regions and rewiring of protein interactions effect of c-terminal sequence on competitive semaphorin binding to neuropilin-1 furin processing of semaphorin 3f determines its anti-angiogenic activity by regulating direct binding and competition for neuropilin structural basis for ligand and heparin binding to neuropilin b domains structural basis for selective vascular endothelial growth factor-a (vegf-a) binding to neuropilin-1 new prospects in the roles of the c-terminal domains of vegf-a and their cooperation for ligand binding, cellular signaling and vessels formation x-ray crystal structure of bovine 3 glu-osteocalcin direct identification of the calcium-binding amino acid, gamma-carboxyglutamate, in mineralized tissue characterization of a gamma-carboxyglutamic acid-containing protein from bone the vitamin k-dependent carboxylase calcium-dependent alphahelical structure in osteocalcin a method for decarboxylation of gamma-carboxyglutamic acid in proteins. properties of the decarboxylated gamma-carboxyglutamic acid protein from calf bone coordinated transcriptional regulation of hspa1a gene by multiple transcription factors: crucial roles for hsf-1 plasma levels of hsp70 and anti-hsp70 antibody predict risk of acute coronary syndrome heat shock protein 70 kda: molecular biology, biochemistry, and physiology heat shock proteins in breast cancer progression-a suitable case for treatment? impaired heat shock response in cells expressing full-length polyglutamineexpanded huntingtin the heat shock factor family and adaptation to proteotoxic stress phosphorylated creb binds specifically to the nuclear protein cbp activation of camp and mitogen responsive genes relies on a common nuclear factor nuclear protein cbp is a coactivator for the transcription factor creb a multiplicity of ccaat box-binding proteins a survey of 178 nf-y binding ccaat boxes a ccaat box confers cell-type-specific regulation on the xenopus hsp70 gene in oocytes hsp-cbf is an nf-y-dependent coactivator of the heat shock promoters ccaat boxes sequence-specific transcription factor nf-y displays histone-like dna binding and h2b-like ubiquitination role of the nf-kappab signaling pathway and kappab cis-regulatory elements on the irf-1 and inos promoter regions in mycobacterial lipoarabinomannan induction of nitric oxide intrinsic disorder in transcription factors hsf transcription factor family, heat shock response, and protein intrinsic disorder intrinsic disorder in proteins involved in the innate antiviral immunity: another flexible side of a molecular arms race identification of critical phosphorylation sites on the carboxy tail of melanopsin the importance of intrinsic disorder for protein phosphorylation intrinsic disorder-based protein interactions and their modulators the structural and functional signatures of proteins that undergo multiple events of post-translational modification antiviral mechanisms of human defensins identification of intrinsic order and disorder in the dna repair protein xpa sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression multiple mechanisms for e2f binding inhibition by phosphorylation of the retinoblastoma protein c-terminal domain cellular mechanisms of tumour suppression by the retinoblastoma gene molecular mechanisms underlying rb protein function cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product the retinoblastoma protein copurifies with e2f-i, an e1a-regulated inhibitor of the transcription factor e2f identification, analysis, and prediction of protein ubiquitination sites functional anthology of intrinsic disorder. 1. biological processes and functions of proteins with long disordered regions functional anthology of intrinsic disorder. 2. cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions functional anthology of intrinsic disorder. 3. ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins structural and biochemical characterization of the interaction between lgn and frmpd1 analysis of partner of inscuteable, a novel player of drosophila asymmetric divisions, reveals two distinct steps in inscuteable apical localization spindle orientation during asymmetric cell division mechanisms of asymmetric stem cell division lgn/minsc and lgn/numa complex structures suggest distinct functions in asymmetric cell division for the par3/minsc/ lgn and galphai/lgn/numa pathways inscuteable and numa proteins bind competitively to leu-gly-asn repeat-enriched protein (lgn) during asymmetric cell divisions structural basis for interaction between the conserved cell polarity proteins inscuteable and leu-gly-asn repeat-enriched protein (lgn) the tetratricopeptide repeat: a structural motif mediating protein-protein interactions return of the gdi: the goloco motif in cell division mammalian pins is a conformational switch that links numa to heterotrimeric g proteins distinct roles of galphai and gbeta13f subunits of the heterotrimeric g protein complex in the mediation of drosophila neuroblast asymmetric divisions tpr proteins: the versatile helix the pdz and band 4.1 containing protein frmpd1 regulates the subcellular location of activator of g-protein signaling 3 and its interaction with g-proteins the pairwise energy content estimated from amino acid composition discriminates between folded and intrinsically unstructured proteins iupred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content structural and functional analysis of human sirt1 aging and disease: connections to sirtuins sirtuins in aging and disease sirtuins in aging and agerelated disease peptide switch is essential for sirt1 deacetylase activity sirt1 contains n-and c-terminal regions that potentiate deacetylase activity genealogy of an ancient protein family: the sirtuins, a family of disordered members mechanism of recruitment and activation of the endosome-associated deubiquitinase amsh mutations in stambp, encoding a deubiquitinating enzyme, cause microcephaly-capillary malformation syndrome mpnc, a putative catalytic motif found in a subset of mpn domain proteins from eukaryotes and prokaryotes, is critical for rpn11 function activation of the endosome-associated ubiquitin isopeptidase amsh by stam, a component of the multivesicular body-sorting machinery the eukaryotic linear motif resource elm: 10 years and counting monomeric tonb and the ton box are required for the formation of a high-affinity transporter-tonb complex the proline-rich domain of tonb possesses an extended polyproline ii-like conformation of sufficient length to span the periplasm of gram-negative bacteria outer membrane active transport: structure of the btub:tonb complex structure of tonb in complex with fhua, e. coli outer membrane receptor structure and function of human dnaj homologue subfamily a member 1 (dnaja1) and its relationship to pancreatic cancer proteomic analysis of chronic pancreatitis and pancreatic adenocarcinoma structural basis of multisite single-stranded dna recognition and acta2 repression by purine-rich element binding protein b (purbeta) the hela pur factor binds single-stranded dna at a specific element conserved in gene flanking regions and origins of dna replication sequence of cdna comprising the human pur gene and sequencespecific single-stranded-dna-binding properties of the encoded protein the pur protein family: genetic and structural features in development and disease emerging role of the host restriction factor tetherin in viral immune sensing bst-2/hm1.24 is a raft-associated apical membrane protein with an unusual topology structural and biophysical analysis of bst-2/tetherin ectodomains reveals an evolutionary conserved design to inhibit virus release running in reverse: the structural basis for translocation polarity in hexameric helicases immunoelectron microscopic evidence for tetherin/bst2 as the physical bridge between hiv-1 virions and the plasma membrane intrinsic disorder of the extracellular matrix assembly, activation, and substrate specificity of cyclin d1/cdk2 complexes interactions of apolipoprotein a-i with high-density lipoprotein particles the structure of human lipoprotein a-i. evidence for the "belt" model a detailed molecular belt model for apolipoprotein a-i in discoidal high density lipoprotein high density lipoprotein structure-function and role in reverse cholesterol transport high-density lipoprotein heterogeneity and function in reverse cholesterol transport double superhelix model of high density lipoprotein analysis of ordered and disordered protein complexes reveals structural features discriminating between stable and unstable monomers mechanisms of mavs regulation at the mitochondrial membrane reconstituted human myosin light chain phosphatase reveals distinct roles of two inhibitory phosphorylation sites of the regulatory subunit, mypt1 structural and functional analysis of the gerd spore germination protein of bacillus species spores of bacillus subtilis: their resistance to and killing by radiation, heat and chemicals germination of spores of bacillales and clostridiales species: mechanisms and proteins involved spore germination how do spores germinate? the ger receptor family from sporulating bacteria bacterial proteasome and pafa, the pup ligase, interact to form a modular protein tagging and degradation machine the nucleolar pict-1/gltscr2 protein forms homo-oligomers regulation of the mdm2-p53 pathway and tumor growth by pict1 via nucleolar rpl11 regulation of pten phosphorylation and stability by a tumor suppressor candidate protein amino acid signature enables proteins to recognize modified trna human trna(lys3)(uuu) is pre-structured by natural modifications for cognate and wobble codon binding through keto-enol tautomerism mutations of basic amino acids of ncp7 of human immunodeficiency virus type 1 affect rna binding in vitro charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in rna packaging and infectivity hiv-1: fifteen proteins and an rna effect of mutations in the nucleocapsid protein (ncp7) upon pr160(gagpol) and trna(lys) incorporation into human immunodeficiency virus type 1 human immunodeficiency virus type 1 nucleocapsid protein promotes efficient strand transfer and specific viral dna synthesis by inhibiting tar-dependent self-priming from minus-strand strong-stop dna mutations in hiv reverse transcriptase which alter rnase h activity and decrease strand transfer efficiency are suppressed by hiv nucleocapsid protein human immunodeficiency virus type 1 nucleocapsid protein specifically stimulates mg2c-dependent dna integration in vitro nucleocapsid zinc fingers detected in retroviruses: exafs studies of intact viruses and the solution-state structure of the nucleocapsid protein from hiv-1 determination of the structure of the nucleocapsid protein ncp7 from the human immunodeficiency virus type 1 by 1h nmr protein intrinsic disorder as a flexible armor and a weapon of hiv-1 key: cord-300796-rmjv56ia authors: nan title: the signal sequence of the p62 protein of semliki forest virus is involved in initiation but not in completing chain translocation date: 1990-09-01 journal: j cell biol doi: nan sha: doc_id: 300796 cord_uid: rmjv56ia so far it has been demonstrated that the signal sequence of proteins which are made at the er functions both at the level of protein targeting to the er and in initiation of chain translocation across the er membrane. however, its possible role in completing the process of chain transfer (see singer, s. j., p. a. maher, and m. p. yaffe. proc. natl. acad. sci. usa. 1987. 84:1015-1019) has remained elusive. in this work we show that the p62 protein of semliki forest virus contains an uncleaved signal sequence at its nh2-terminus and that this becomes glycosylated early during synthesis and translocation of the p62 polypeptide. as the glycosylation of the signal sequence most likely occurs after its release from the er membrane our results suggest that this region has no role in completing the transfer process. iosynthesis of proteins at the er can be subdivided into several steps. these are (a) targeting of translation complexes to the er membrane; (b) synthesis and transfer (translocation) of the polypeptide chain across the lipid bilayer; and (c) protein maturation in the lumen of er (chain folding, disulphide bridge formation, glycosylation, and oligomerization). the mechanisms for these processes have been studied extensively during recent years (kornfeld and kornfeld, 1985; wickner and lodish, 1985; rapoport, 1986; lodish, 1988; rothman, 1989) . a most important finding has been that all proteins made at the er carry a signal sequence (also called signal peptide), a hydrophobic peptide which is usually located at the nh2-terminal region of the polypeptide chain. one function of the signal peptide is to achieve targeting of the polysome to the er membrane (rapoport, 1986) . when the signal sequence emerges from the ribosome it binds to the signal recognition particle, which mediates binding of the polysome to the docking protein in the er. after this another function of the signal sequence is expressed, that is to interact with some components of the er membrane and thereby initiate translocation of the polypeptide chain into the lumen of the er (gilmore and blobel, 1985; robinson et al., 1987; wiedmann et al., 1987) . further synthesis of the polypeptide then continues with concomitant chain translocation. an important but as yet unresolved question is whether the signal sequence has any role in the translocation process per se or whether its functions are limited to the targeting and translocation-initiation steps. for instance, singer and co-workers danny huylebroeck's present address is innogenetics, industriepark 7, box 4, b-9710, ghent, belgium. (1987a) have suggested a translocator protein model in which the signal sequence helps to keep the machinery open for chain transfer. it is specifically this last question we have addressed in the present work. we describe the characteristics and behavior of the uncleaved signal sequence of the p62 protein of semliki forest virus (sfv) l upon translocation across the er membrane in vitro. the p62 protein is one subunit of the heterodimeric spike protein of the sfv membrane (reviewed in garoff et al., 1982) . it is made as a precursor protein together with the other structural proteins of sfv, i.e., the nucleocapsid protein, c, and the other spike subunit, el. the three proteins are synthesized from a 4.1-kb long mrna in the order c, p62, and el, and separated by cleavage of the growing precursor chain. during synthesis of the p62 polypeptide at the er all but a 31 residue cooh-terminal portion and the membrane anchor is translocated across the membrane. the p62 signal sequence has so far been only roughly localized to the nh2-terminal third of the polypeptide chain (garoffet al., 1978; bonatti et al., 1984) . we show here that the signal sequence of p62 consists of a 16 residue peptide at its nh2-terminal region. this region includes one out of four glycosylation sites (asn~3) for n-linked oligosaccharide on the p62 chain. we also demonstrate that the glycosylation of the p62 signal sequence occurs early during chain translocation. as this modification of the signal region most likely correlates with its release into the lumen of er it follows that the signal sequence of p62 is probably only needed for an initial step in chain translocation and not to small scale plasrnid dna preparations were done using the alkali-sds method essentially as described by birnboim and dnly (1979) . large quantities of plasmids to be used for in vitro transcription were prepared by lysozyme-triton lysis of the bacteria, followed by csc1-etbr banding (kahn et al., 1979) . etbr was removed by several extractions with isopropanol and, after fivefold dilution, the dna was precipitated twice with ethanol and further purified over a biorad a-50m column. restriction endonucleases and dna-modifying enzymes were used according to the suppliers instructions. removal of the 3' sticky end from the sac i site in pgem2-alphag (zerial et al., 1986) with t4 dna polymerase was done at 15°c (2 h), dntps were added (end concentration 100 #m each), and the dna was subsequently filled in at 15°c for 1 h. all ligations were done at 24°c for 4 h except for linker ligations (4°c, 16 h). all other molecular biological manipulations were done using slightly modified standard protocols (maniatis et al., 1982) . in vitro transcription (0.3 #g supercoiled template dna per 10 #1 vol) in the presence of sp6 rna polymerase (6-8 u) and the cap structure was carried out as previously described (zerial et al., 1986) . in vitro translation reactions using a rabbit reticulocyte lysate were performed at 30°c essentially as described . 1.5 #1 of the in vitro synthesized rna was translated in a total volume of 15 #1. potassium, magnesium, and spermidine concentrations were 100, 1.2, and 0.375 mm, respectively. when indicated, 1 #1 of er membranes was included. in some translocations the membranes were pretreated with 200 #m peptide for 5 min on ice. the final peptide concentration in the total translation mixture here was, after addition of the pretreated membranes, adjusted to 100/~m. to obtain partial synchronization of translation, ata was added after a preincubation of 1.5-3.0 rain (borgese et al., 1974) . a final ata concentration of 0.075 mm was found to be sufficient for inhibiting initiation of chain synthesis (see control in fig. 6 , lane/). higher concentrations of ata inhibited first transloeation and then also chain elongation. for protease protection experiments, proteinase k was added to a final concentration of 0.1 mg/ml and the samples were incubated at 0*c for 30 min in the presence or absence of 1% triton x-100. proteolysis was stopped by the addition of pmsf (final concentration 2 mg/ml) and samples were kept at 0*c for 5 min before further processing for electrophoresis (cutler and garoff, 1986) . bands containing labeled protein were visualized by fluorography. quantitation of proteins was done by cutting the bands out of the dried gel, solubilizing them with protosol (from dupont de nemours, nen) according to the instructions of the manufacturer, and finally counting the 3~s radioactivity in a liquid scintillator (wallac lkb, turku, finland). the localization of the bands on the dried gel was done with the aid of the fluorograph in transillumination. 15-#1 translation mixtures were adjusted to ph 11-11.5 by adding an appropriate volume (pretitrated) of 0.1 n naoh. after a 10-rain incubation on ice the samples were separated into a pellet fraction and a supernatant fraction by centrifugation through a 100-#1 alkaline socmse cushion (gilmore and blobel, 1985) for 10 rain at 30 psi in an airfuge (beckman instruments, inc., palo alto, ca) using the a-100/30 rotor and cellulose propionate tubes precoated with bsa (1% solution). the entire supernatant was removed, neutralized with 1 n hci, diluted 2.5 times with water, and then precipitated by adding 3.5 vol of acetone. these precipitated proteins and pelleted membranes (obtained from the airfuge tube) were taken up in 4% sds by incubating at 56°c for 15 min and then processed for immunoprecipitation reactions as described below. total translation mixtures were adjusted to 4% sds, then boiled for 4 rain and diluted 1:2 with water. 4 vol of immunoprecipitation buffer (2.5 % triton x-100, 190 mm nac1, 60 mm tris-hc1, ph 7.4, 6 mm edta, and 20/~g pmsf/rni) and 2 #1 of antibody were added for 16 h at 40c. the mixture was briefly centrifuged (2-3 min in an eppendorf minifuge) and to the supernatant one fifth volume of a 1:1 slurry of protein a beads were added and incubated at 24"c for 2 h under constant agitation. the beads were collected and washed four times with 1 ml ripa buffer (gielkens et al., 1976) by centrifugation, followed by a single wash with a buffer containing 150 mm naci, 10 mm tris-hcl ph 7.4, and 20 t~g pmsf/ml. the beads were then taken up in excess gel loading buffer (cutler and garoff, 1986) , heated at 95°c for 5 min, and cleared by centrifugation before loading the immunoprecipitate on the gel. constructions of pgem2alphagx and pgem2dhfrx. for the construction of the final fusion protein-coding plasmids used in this study we first had to make plasmids pgem2alphagx, which are derived from pgem2alphag and pgem2dhfrx, which are derived from pgem2dhfr (zerial et al., 1986) . plasmid pgem2alphag contains a 548 bp-long nco i-pst i fragment encompassing the entire chimpanzee alpha-globin coding region between the hinc ii and pst i sites of the polylinker of the plasmid pgem1 (pmmega biotech). the nco i site contains the translation initiation codon from alpha-globin (zerial et al., 1986 ). an xho i site, allowing subsequent in-frame ligations of sfv sequences, had to be introduced in pgem2alphag. therefore, this plasmid was cut (upstream of the nco i site) with sac i, the 3' sticky ends removed with "1"4 dna folymerase, an xho i octamer linker introduced and, after cutting with xho i, the plasmid was religated at low dna concentration (1 #g/ml). plasmid pgem2alphagx then contains the 2,057 bp-long xbo i-pvu i fragment needed for the construction of the fusion protein-coding plasmid pc62alphag. an intermediate construct, analogous to pgem2alphagx, and also conraining a unique xho i site, was needed for the constructions of dhfrcontaining plasmids. for this purpose we inserted the xho i linker into partially xmn i cut pgem2dhfr (zerial et al., 1986) . after cutting the linkers, linear plasmid was purified on agarose gel and religated. since the second xmn i site in pgem2dhfr is located in the beta-lactamase coding region of the vector (snt~liffe, 1979) and insertion of an xho i site by an octamer linker will result in an ampicillin-sensitive e. coli phenotype after transformation, only the desired pgem2dhfrx construct was obtained. from this plasmid, an xho i-pvu' i fragment of at least 2,012 bp (the precise length of the cdna insert, i.e., the length of the 3' untranslated region of dhfr, is not known in pgem2dhfr) was used for the construction of pc62dhfr. construction of the fusion protein-coding plasmids pc62alphag and pc62dhfr. plasmid pgemi-sfv (also called pg-sfv-15/5; melancon and garoff, 1986 ) contains a re, engineered edna copy of the sfv 26s mrna sequences cloned as a barn hi fragment in the barn hi site of the polylinker downstream of the sp6 promoter in the plasmid pgem1 (promaga biotech). from the sfv plasmid, a 2,381 hp-long pvu i-xho i fragment, containing the coding sequences for the capsid protein and the nh2-terminai region of the p62 protein, was isolated. the xho i-pvu i fragments from pgemi-sfv, pgem2alphagx and pgem2dhfrx were isolated and ligated at a 1:1 molar ratio to obtain pc62alphag and pc62dhfr, respectively. plasmid dnas from ampicillin-resistant colonies were screened and compared to the starting vectors by restriction analysis. altogether, the sfv-alpha-globin edna fusion results in a complete c region and 40 codons from the 5' end of the p62 region fused to the whole of the alpha-globin coding sequence (see fig. 1 ). eight new codons have been introduced at the point of edna fusion. in the sfv-dhfr construction the c region and the 40 first codons of p62 are fused to the dhfr coding sequence such that one new codon is introduced and the first 31 codons of dhfr are lost. construction of plasmidp62dhfr. for engineering of a p62 protein signal sequence-dhfr fusion protein which is not derived from a c proteincontaining precursor we synthesized the whole p62 signal sequence region. two overlapping oligonucleotides were made (dna-synthesizer; applied biosystems, foster city, ca) :(1) 5' atacacagaattcagcaccatgt-ccgccccgctgattac tgccatgtgtgtcctiv~caatc_~tacct-tcccgtc~ttccagcccccgtgtgtacc~, (2) 5' gttatcct-cgagcatccgtagtgtggcctctgcgttgttttcatagcagca-aggtacacacgggggc tggaagcac gggaaggtagcattgcjca-aggac. they correspond to both strands of the p62 signal sequence region of the sfv edna. together they span the coding region of amino acid residues 1-40 of p62. oligo 1 (the coding strand) includes, in addition, the region coding for initiator methionine of the c protein plus its 5' flanking sequences (5' agcaccatg). at the extreme 5' end of this oligo we have added the recognition sequence for eco ri and its flanking sequences from the 5' end of the structural part of the sfv cdna (5' atacacagattc). oligo 2 ends at its 3' end with the xho i site which follows the signal sequence region on the p62 gene. the two oligonucleotides were hybridized (51 complementary bases), filled in using sequenase (united states biochemical co., cleveland, oh) and restricted with eco ri and xho i. the resulting dna fragment was then purified and inserted into pcp62dhfr instead of the c and p62 sequences. for this purpose the pcp62dhfr plasmid was eco ri and xho i restricted and the plasmid part with the dhfr sequences isolated. the resulting plasmid p62dhfr contains thus the coding sequences for the initiator methionine of c and the first 40 residues of the p62 protein, including the signal sequence, in front of the dhfr gene (see fig. 1 ). construction of pgem sfv d-4. this plasmid was constructed by ligatiag three fragments together. the first one was the major part of pgem1, cut just after the promoter region with hind iii and barn hi. the second fragment (hind i~-xho i) was isolated from the plasmid psvs-sfv . this fragment contains the sequences encoding the capsid and the nh2-terminal part of the 1962 protein of sfv. the third fragment was obtained by cleaving plasmid pl1 sfv d-4 (see below) with xho i and barn hi and isolating the fragment containing the 3' part of the coding sequence for the p62 protein. however, it should be noted that in the d-4 version there is an exchange of 15 codons at the 3' end of the 1062 gene for six aberrant ones. the corresponding p62 protein variant is called 1962 d-4 (see fig. 1 ). it should also be mentioned that pl1 sfv d-4 has been derived from pl1 sfv d-9, (cutler and garoff, 1986 ) by exchanging the xho i-cla i region containing the 3' part of the p62 coding region with the similar fragment from psv2 sfv d-4. this latter plasmid is described in garoff et al. (1983) . to define the p62 signal sequence we have studied the translocation phenotype of two reporter molecules, the rabbit alpha-globin and the mouse dihydrofolate reductase (dhfr), both of which have been extended at their nh:-termini with an nh2-terminal 40 residue peptide from p62. the hybrid molecules were tested in a microsome-supplemented in vitro translation system. the alpha-globin and the dhfr have earlier been shown to be translocation incompetent if not extended with a heterologous signal sequence at their nh2termini (zerial et al., 1986) . we first tested the expression of in vitro-made rna from the construction pcp62dhfr in an in vitro translation system. this would be expected to yield free c protein and p62reporter hybrid (p62-dhfr) through c-catalyzed autoproteolytic cleavage of the nascent c-p62-reporter precursor ( fig. 1 ) (aliperti and schlesinger, 1978; hahn et al., 1985; melancon and garoff, 1987) . furthermore, the p62-reporter hybrid should be translocated across microsomal membranes and possibly glycosylated at asn~3 of the p62 sequence if the 40 residues long nh2-terminal p62 peptide carries a signal sequence. is shown to be linked to asn residue 13 in the p62 part (garoff et al., 1982) . additional amino acids resulting from in frame translation of the multicloning region of pgem2 and the added xho i linker as well as the initiator met of p62dhfr are also indicated. analysis showing the translocation activity of the p62-dhfr protein. in the absence of membranes (lane/) two major protein species were translated from the sp6-directed transcript. one of these had the expected size of c (33 kd) and the other one that of the p62-dhfr hybrid molecule (21 kd). the coding region has apparently been translated faithfully and the precursor protein cleaved efficiently. the identity of the p62-dhfr was directly proven by immunoprecipitation with a dhfr specific antiserum (see fig. 3 ). the two weaker bands migrating faster than the capsid in fig. 2 , lane i were most likely derived from c coding sequences because they are found in all protein analyses of in vitro transcripfigure 3 . immunological identification of the p62-dhfr hybrid protein and analysis of its association with membranes. rna transcribed from pc62dhfr was translated in vitro in the presence of membranes which in some cases had been treated with an acceptor (acc) or nonacceptor (non) peptide. the samples were treated, after translation, at ph 11-11.5, and the proteins then separated into a membrane-bound pellet fraction (p) and a supernatant fraction (s) by centrifugation. in all samples the p62-dhfr polypeptides were isolated using an anti-dhfr antibody. the proteins were then analyzed by sds-page (10 %) and subsequent autoradiography. the slower migrating band corresponds to glycosylated and the faster one to nonglycosylated forms of p62-dhfr (compare fig. 3) . tion/translation mixtures involving cdnas with c regions (compare fig. 6 ). when microsomes were added to the c-p62-dhfr in vitro translation system a new band appeared which migrated somewhat slower than the p62-dhfr band seen in the analysis of the mixture lacking membranes (fig. 2, lane 2) . it almost comigrated with one of the two weak c derived bands. the new band apparently corresponds to iri2-dhfr hybrids that have been translocated into the lumen of the added microsomes and have become glycosylated. the immunoprecipitation analysis shown in fig. 3 confirmed the identity of this material. the protease digestions in the absence (fig. 2, lane 3) and presence of triton x-100 (lane 4) clearly demonstrated that the slower migrating p62-dhfr molecules were indeed translocated. about half of this material remains protected in the presence of intact microsomes whereas all is digested when the membranes are solubilized with detergent. in contrast, the other translated material did not show such a pronounced membrane-dependent protease resistance. note that protease treatment of all samples yielded a resistant protein of a small size. this most likely represents a protease-resistant c fragment. the glycosylation of the translocated p62-hybrid and its effect on the apparent size of the protein was shown in an experiment where a short peptide (asn-leu-thr), which competes for n-linked glycosylation, was included during translation. apparently only unglycosylated faster migrating p62-dhfr hybrids were formed in these conditions although chain translocation took place conferring protease resistance (fig. 2, lanes 5-7) . additional analyses (lanes 8-10) illustrate that a control peptide (asn-leu-athr) which cannot serve as an acceptor site for n-linked glycosylation, had no effect on the glycosylation of the p62-reporter hybrids when tested in an analogous way. similar studies as with pcp62dhfr were also performed with the pcp62globin coded proteins in vitro. the results (not shown) were analogous to those described above for the pcp62dhfr construct. c protein and p62-globin hybrid were synthesized in the absence of membranes. when membranes were added, a protease-protected form of the hybrid appeared. this hybrid was also glycosylated as deduced from an experiment involving the acceptor peptide for glycosylation. fig. 3 (lanes 1-6) shows the results of analyses in which we have tested whether the p62 signal sequence region confers stable membrane attachment to the p62-dhfr hybrid. microsome-supplemented translations were adjusted to ph 11-11.5 with naoh, incubated on ice for 10 min, and then separated into a membrane pellet and supernatant fraction by ultracentrifugation. in all samples the p62-dhfr polypeptides were isolated using an anti-dhfr antibody, sds-page shows that the hybrid protein segregates almost quantitatively into the supernatant fraction (compare lane i with lane 2). in similar conditions an integral membrane protein, the human transferrin receptor, was found to sediment with the membranes into the pellet fraction and a secretory protein, ig light chain, was only recovered in the supernatant (not shown). if the acceptor peptide for glycosylation was included in the in vitro translation and the mixture then analyzed we found that the now unglycosylated but still translocated p62-hdfr hybrids were again mostly found in the supernatant fraction (lanes 3 and 4) . lanes 5 and 6 show the analyses with the control peptide. to see whether the c protein exerts an influence on the translation phenotype of the p62-dhfr protein the p62dhfr plasmid (see fig. 1 ), lacking the c gene, was tested. the results shown in fig. 4 show clearly that the p62-dhfr hybrid is translocated and glycosylated in the same way as when expressed from pcp62dhfr. thus, apart from providing a free nh2-terminal end to the p62-dhfr protein by autoproteolysis of the c-p62-dhfr precursor the c protein has no role in the translocation process. we conclude that the 40 residue peptide from the p62 nh2-terminal region confers a translocation positive phenotype to the p62-globin and p62-dhfr polypeptides and therefore must contain a functional signal sequence. the translocated fusion proteins were also shown to be glycosylated. this must involve asn~3 of the p62 peptide as it is part of the only potential glycosylation site on the hybrid polypeptides (garoff et al., 1980 ; references on dhfr sequence in legend to fig. 1) , finally, we can also conclude that the p62 signal sequence does not provide a stable membrane anchor to the translocated chain. to define at what time point during p62-dhfr chain synthesis the asn13 becomes glycosylated we performed a time-course experiment essentially as described by rothman and lodish (1977) (fig. 5) . in this experiment a 150-#1 translation was initiated. after 1.5 rain ata was added (0.075 ram) to block additional starting of chain synthesis. then, at 0.5-rain intervals, two 7.5/~1 aliquots were removed; one for mixing with 40 #1 of hot page sample buffer (2 % sds) and the other one for further incubation after mixing with 0.75/~1 of 20% tx-100. the first sample from each time point was used for the determination of the time needed for chain completion, which is a function of the translation rate, and the other one allowed determination of the time course of glycosylation of the translocated chain. triton x-100 solubilizes the microsomal membranes and thereby inactivates glycosylation (but not chain elongation). therefore, only those p62-dhfr chains that have presented asnl3 to the glycosylation machinery before tx-100 addition have had the possibility to become glycosylated. in fig. 5, lanes 1-10, one can see that completed p62dhfr chains (197 residues with initiator met) appear after a 3-min incubation from the time point of ata addition. if one assumes constant chain initiation during the figure 5 . time course of p62 dhfr glycosylation. a 150-#1 translation was initiated. after a preincubation time of 1.5 min ata was added to inhibit further initiation of chain synthesis. then, at intervals of 0.5 min (indicated by 0.5', 1.0', 1.5', 2.0', 2.5', 3.0', 3.5', 4.0', 4.5', and 5.0') two 7.5-/zl samples were removed, one for mixing with page sample buffer and another one for mixing with tx-100 (final concentration 1%) and further incubation at 30°c (for a total time of 20 min after ata addition as indicated by the lower row of time points in the figure) . lanes 1-10 show the samples removed for mixing with the page sample buffer. from these results the approximate rate of translation can be derived. completed chains appear in the 3-min sample. lanes 11-20 show the samples in which the membranes have been solubilized with triton x-100 for inactivation of the glycosylation machinery. from these analyses it is possible to estimate when asn13 is modified during p62-dhfr synthesis. the first glycosylated forms are clearly visible in the 1.5-min sample, a time point where only about half of the p62-dhfr chain has been synthesized. the nature of the material in the two weak bands seen in lanes 1-5 is unclear. their transient appearance before the completion of the p62-dhfr chain suggests that they represent complexes of nascent p62-dhfr chains. figure 6 . time course of p62 d-4 glycosylation. seven translations in the presence of microsomal membranes were started in parallel. after a 3-rain initial incubation at 30°c, ata was added in order to inhibit further initiation of chain synthesis. incubation was then continued for 35 rain. at the indicated time points (5, 10, 15, 20, 25, 30 , and 35 rain) tx-100 (tx) was added to stop further chain glycosylation. at the same time one half of each sample was removed and put on ice in order to measure the extent of chain elongation at each time point. all samples were analyzed by sds-page (10%) and autoradiography. lanes 3-9 show the analysis of the samples incubatedwith tx-100 and lanes 10--16 the analysis of the portions put on ice at the different time points. the complete sequence of treatments for each sample is indicated by the labeling in each lane (upper row of time points indicate tx-100 addition and cooling on ice, respectively; lower row of timepoints indicate incubation in the presence of tx-100). lanes i and 2 represent controls. in the experiment shown in lane i, ata was added before starting a 40-rain membrane-supplemented translation. in the experiment shown in lane 2 a translation with membranes was allowed to proceed for 40 min. ata was added as in the time course samples but tx-100 was omitted. the c protein, the unglycosylated (p62) and the glycosylated (gp62) forms of p62 d-4 are labeled at right in the figure. arrowheads at left indicate (from above) the migration of the 53-kd igg heavy chain, the 46-kd ovalbumin, and the 30-kd carbonic anhydrase. note that somewhat different amounts of translation mixtures have been analyzed in the various lanes (compare intensities of c and c-derived bands). 1.5-rain preincubation without ata then the total time for chain synthesis is ,~3.75 rain (3 + 0.75 rain). this corresponds to a mean translation rate of 52.5 peptide bonds per rain. lanes 11-20 show that glycosylated chains appear in all those samples that have had the membranes intact for 1.5 min or more after ata addition. this means that p62dhfr chains that have been elongated for '~2.25 rain (1.5 min incubation after and 0.75 min before ata addition), to the length of ,'~118 residues already carry a sugar unit at asn13. as ~60 residues of the nascent chain are required to span the ribosome and the lipid bilayer we conclude that glycosylation occurs when the first 50-60 residues of p62-dhfr appear within the lumen of the er (malkin and rich, 1967; blobel and sabatini, 1970; bergman and kuehl, 1977; smith et al., 1978; glabe et al., 1980; randall, 1983) . we also studied the timing of the glycosylation of asn~3 in its normal background, i.e., during p62 chain synthesis. for this experiment we used the pgem sfvd-4 construct. this encodes the c and the p62 membrane protein variant, p62 d-4, in which a few residues of the cytoplasmic protein domain have been exchanged as compared to the wild type sequence (see materials and methods and fig, 1) . fig. 6 , lane 2, shows that rna, which has been transcribed from this construct, directs the synthesis of c and p62 d-4 chains. the protein has catalyzed correct c-p62 cleavage and the p62 signal sequence has catalyzed the insertion of about half of the p62 d-4 chains across the added microsomal membranes. these migrate as glycosylated 60-58 kd proteins in contrast to the noninserted molecules which have an apparent molecular mass of 'x,52 kd. the glycosylated and translocated nature of the 60-58 kd material was clearly demon-strated in experiments similar to those described above for the p62-dhfr hybrid molecule (not shown). altogether there are four glycosylation sites within the p62 d-4 sequence. these correspond to asn residues at positions 13, 60, 266, and 328 (see fig. 1 ). fig. 6 (lanes 3-16) shows the time course of the four glycosylation events during c-p62 d-4 translation. a slightly different protocol was followed in this experiment as compared to that with p62-dhfr. seven translations were initiated in parallel and after a 3-min incubation these were put on ice and ata was added. elongation of the already initiated chains was then continued for a total of 35 min, however, so that triton x-100 was added to individual samples at 5, 10, 15, 20, 25, 30, and 35 min. at these time points half of each sample was also removed and translation stopped by cooling on ice. lanes 3-9 show the sds-page of the samples that had received triton x-100 at different time points. we found the sequential appearance of p62 d-4 polypeptides with no carbohydrate (lane 3), with one and two units added (seen as two new bands with slower migration in lanes 4 and 5), with three units (lane 6), and all four sugar units (lanes 7, 8, and 9 ) attached to the protein backbone as the translation proceeded coordinately with time. note that the four glycosylation events result in different degrees of increase of the size of p62 d-4. the second event causes the largest increase and the third one the smallest. as the sugar unit added at each step should be the same we think that these differences reflect some conformational changes in the p62 folding which occur coordinately with glycosylation. in lanes 10--16 we have analyzed the samples that were withdrawn at the different times but were kept on ice. as expected, we see a sequential appearance of first the capsid the journal of cell biology, volume 111, 1990 protein (in the 10-min sample) and then the p62 d-4 protein (barely visible in the 20-min sample). the p62 d-4 protein is partly present in its glycosylated and partly in its unglycosylated form. using 21.5 min as a rough estimate for the translation time of the 746 residue long c-p62 d-4 chain (time point of p62 d-4 detection, 20 min, plus half of the 3-min preincubation time without ata) we have calculated the translation rate and derived the approximate earliest time points when the four glycosylation sites of p62 d-4 should be available for modification. according to these, asn~ and asn60 should be the only sites available for glycosylation in the 10-min sample, shown in lane 4, and the most abundant ones presented for modification in the 15-min sample, shown in lane 5. therefore, it appears reasonable to assume that those chains of these two samples which have obtained two sugar units carry these on the aforementioned two sites. thus, the peptide region with asn,a seems to be target for rapid modification also when present in its normal background, that is with the p62 protein. the fact that the 40 residue fragment of the nh2-terminal region of the p62 protein is able to translocate two different reporter molecules into microsomes constitutes in our mind convincing evidence for signal sequence activity in this protein fragment. a more precise location of the p62 signal sequence within the 40 residue p62 fragment can be done with the aid of the known consensus features of a signal sequence. the most typical characteristic of a signal sequence is a stretch of 10-12 uncharged residues, mostly hydrophobic ones (von heijne, 1985) . this part of the signal sequence probably forms an alpha helix in the er membrane (emr and silhavy, 1983; briggs et al., 1985 briggs et al., , 1986 kendall et al., 1986; batenburg et al., 1988) . the only possible candidate region within the 40 residue p62 fragment having these features is the 13 residue segment between pro 3 and pro 17 (see box in uppermost sequence in fig. 7) . the pro-rich region in the middle of the 40 residue fragment would not form an alpha-helix, and the cooh-terminal part of the p62 segment contains a high number of charged residues. as shown in fig. 7 these features are conserved in all those alphaviruses where the p62 protein has been sequenced. thus, we find the experimental results, together with the structural considerations discussed above, highly indicative that the 16 nh2terminal residues of p62 constitute its signal sequence. eventually, the signal sequence of the p62 protein becomes translocated across the membrane of the er into its lumenal space. in here it is found as a glycosylated peptide which is part of a 66 amino acid residues long "pro-piece of the p62 protein. this pro-peptide, called e3, is cleaved at a late stage during virus assembly (de curtis and simons, 1988) and is then either released into the extracellular medium as a soluble protein (sinbis virus) or remains as a peripheral protein subunit on the virus spike (sfv) (garoff et al., 1982; mayne et al., 1984) . our present tests of the p62-globin and p62 dhfr hybrids in the high ph wash assay of membrane supplemented in vitro mixtures also support the notion that the p62 signal sequence does not remain bound to the membrane where it has exerted its function as a translocation signal. in this work we like to use the glycosylation event at asn,3 of the signal sequence to mark the time point when the latter becomes released into the lumen of the er. the crucial question then becomes whether it is reasonable to assume that the signal peptide has to be released from the er membrane before it can become glycosylated. to answer this question we have to consider what is known about the topology of glycosylation as well as the way by which the p62 signal might interact with the er membrane. today there is no exact information about how a signal sequence might be inserted into the er membrane when exerting its function in chain translocation. however, the typical cytoplasmic orientation of the nh2-termini of membrane protein chains carrying a combined signal sequence-anchoring peptide suggests that signal sequences in general might direct their function in translocation through the insertion of their hydrophobic and uncharged stretch of amino acid residues into the membrane in such an orientation that the nheterminus of the signal remains on the outside of the er mem(garoff et al., 1980; rice and strauss, 1981; dalgarno et al., 1983; kinney et al., 1986; chang and trent, 1987) . amino acid residues are given using the one letter code and they are numbered from the nh2-towards the cooh-terminus. the boxes indicate that region in each sequence which best fulfills the consensus features of a signal sequence (the uncharged and hydrophobic region). the * symbols represent attachment sites for oligosaccharide and the (+) and (-) the presence of a charged amino acid side chain. proline residues are labeled with a dot. the sequences are aligned according to maximum amino acid sequence homology. brane (bos et al., 1984; lipp and dobberstein, 1986; spiess and lodish, 1986; zerial et al., 1986 ; see also shaw et al., 1988) . in addition, it is known from physical studies using synthetic signal peptides and artificial lipid membranes that the signal peptides readily insert into the membrane and there obtain an alpha-helical conformation (briggs et al., 1985 (briggs et al., , 1986 batenburg et al., 1988; cornell et al., 1989) . if the p62 signal sequence adapts such an orientation and conformation in the er membrane it would mean that the glycosylation site at asnt3 would locate inside the membrane (von heijne, 1985) . in this location the site can hardly be accessible for the glycosylation machinery. (note that in the related ross river virus, the venezuelan equine encephalitis virus and eastern equine encephalitis virus the corresponding glycosylation site is even closer towards the nh2terminus, that is at asn 11, see fig. 7 .) according to several recent studies, glycosylation requires the exposure of the glycosylation site in the lumen of er. firstly, it has been shown that the binding protein for the glycosylation site of n-linked oligosaccharides is a lumenal 57-kd protein of the er (geetha-habib et al., 1988) . secondly, one study with the asialoglycoprotein receptor and another one with the corona virus e1 membrane protein demonstrate that lumenally oriented glycosylation sites are not used on transmembrane polypeptides if they locate very close to the membranebinding segments of the chains (mayer et al., 1988; wessels and spiess, 1988) . in the case of the asialoglycoprotein receptor a site was not used if located 12 residues apart from the membrane anchor, however, if moved 8 more residues apart from the anchor it became glycosylated. in the case of the corona virus protein a site just adjacent to the combined signal sequence-anchor peptide remained unglycosylated, whereas an engineered site 24 residues further away was used for glycosylation. such restrictions in glycosylation are most likely to be explained by sterical problems in attaching the very spacious sugar unit (lee et al., 1984 ; see also wier and edidin, 1988) onto acceptor sites that are fixed in a position which is close to the membrane plane. therefore, we assume that the p62 signal sequence, with its glycosylation site at asn13, cannot become glycosylated before it has been released into er lumen. as this glycosylation event was shown to occur at an early stage of chain translocation it follows that this signal sequence can only interact with the er membrane during the beginning of chain translocation. in other words, the signal sequence of p62 can only function at the initiation stage of chain translocation and has no role in completing this transfer process. if the latter would be true we would have expected that the signal sequence glycosylation would have occurred first after all of the lumenal domain of the p62 d-4 chain would have been translated and translocated. the importance of our results in this work lies in the fact that they rule out translocation models in which the signal sequence would have a role throughout the whole process of chain translocation. for instance, if the translocation site is represented by a multisubunit protein complex forming an aqueous channel across the membrane for chain transfer (see signal hypothesis, blobel and dobberstein, 1975 ; amphiphatic tunnel hypothesis, rapoport, 1985; translocator protein hypothesis, singer et al., 1987a,b) , then the signal sequence could be involved in its assembly or "opening" but apparently not for keeping it together or open until chain transfer is completed (as suggested in singer et al., 1987a) . similarly, when considering models in which the chain transfer occurs directly through a lipid membrane (see the helical hairpin hypothesis, engelrnan and steitz, 1981; direct transfer model, von heijne and blomberg, 1979; phospholipid channel hypothesis, nesmayanova, 1982) the interaction of the signal sequence with the lipid bilayer could be of importance only at the stage of translocation initiation but not at the actual chain transfer step. the possibility that our results about p62 protein translocation would be unique to the viral system .and different from the general translocation process in the er we find most unlikely. several results from this and earlier works suggest that the signal sequence of the p62 protein functions much in the same way as cleavable ones do. firstly, studies with a temperature-sensitive mutant of sfv, ts3, have shown that the signal sequence of p62 requires a free nh2-terminal end for function (hashimoto et al., 1981) . at the nonpermissive temperature the ts3 mutant is defective in cleavage between the c and the p62 protein region of the protein precursor because of a mutation that inactivates the autoproteolytic activity of c. this defect results in a translocation negative phenotype for the p62 protein. secondly, the p62 signal sequence has been shown to be srp dependent. if the mrna for the structural proteins of sfv is translated in vitro in a wheat germ-derived system that is supplemented with saltwashed (and srp-deprived) membranes then p62 translocation is observed only in the presence of exogenous srp (bonatti et al., 1984) ~ if srp is supplemented without membranes then p62 translation is arrested. thirdly, our time course study about p62 synthesis and glycosylation in this work clearly demonstrates that the p62 chain is translocated cotranslationally across the er membrane. this was also suggested by earlier studies in vitro (garoff et al., 1978; bonatti et al., 1984) . in these studies it was shown that both microsomal membranes as well as srp have to be added to the synthesis mixture before extensive lengths (,,o100 amino acid residues) of the p62 chains have been translated. it is also possible to speculate on a mechanism in which the p62 signal sequence would be released from a putative translocation site by being replaced by another signal sequence-like structure in the p62 polypeptide. however, such a "rescue" mechanism appears improbable as the p62 signal sequence was found to be glycosylated early during translation of both the p62 polypeptide as well as the signal sequence-dhfr hybrid chain. evidence for an autoprotease activity of sindbis virus capsid protein characterization of the inteffacial behavior and structure of the signal sequence of escherichia coli outer membrane pore protein phoe addition of glucosamine and mannose to nascent immunoglobin heavy chains a rapid alkaline extraction procedure for screening recombinant plasmid dna transfer of proteins across membranes. i. presence of proteolytically processed and unprocessed nascent immunoglobin light chains on membrane-bound ribosomes of murine myeloma controlled proteolysis of nascent polypeptides in rat liver cell fractions. i. location of the polypeptides within ribosomes role of signal recognition particle in the membrane assembly of sindbis viral glycoproteins. fur ribosomemembrane interaction: in vitro binding of ribosomes to microsomal membranes nh2-terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation in vivo function and membrane binding properties are correlated for escherichia coli lamb signal peptides conformations of signal peptides induced by lipids suggest initial steps in protein export phenotypic expression in e. coli ofa dna sequence coding for mouse dihydrofolate reductase nucleotide sequence of the genome region encoding the 26s mrna of eastern equine encephalomyelitis virus and the deduced amino acid sequence of the viral structural proteins conformations and orientations of a signal peptide interacting with phospholipid monolayers structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma sis0 cells mutants of the membrane-binding region of semliki forest virus e2 protein. i. cell surface transport and fusogenic activity ross river virus 26 s rna: complete nucleotide sequence and decoded sequence of the encoded structural proteins dissection of semliki forest virus glycoprotein delivery from the trans-golgi network to the cell surface in permeabilized bhk cells importance of secondary structure in the signal sequence for protein secretion the spontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis solid phase peptide synthesis assembly of the semliki forest virus membrane glycoproteins in the membrane of the endoplasmic reticulum in vitro nucleotide sequence of edna coding for semliki forest virus membrane glycoproteins structure and assembly of alphaviruses expression of semliki forest virus proteins from cloned complementary dna. ii. the membrane-spanning glycoprotein e2 is transported to the cell surface without its normal cytoplasmic domain glycosylation site binding protein, a component of oligosaccharyl transferase, is highly similar to three other 57 kd luminal proteins of the er synthesis of ranscher murine leukemia virus-specific polypeptides in vitro translocation of secretory proteins across the microsomal membrane occurs through an environment accessible to aqueous perturbants glycosylation of ovalbumin nascent chains: the spatial relationship between translation and glycosylation sequence analysis of three sindbis virus mutants temperature-sensitive in the capsid protein autoprotease evidence for a separate signal sequence for the carboxy-terminal envelope glycoprotein e1 of semliki forest virus preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eucaryotic messenger rna dog pancreatic microsomalmembrane polypeptides analysed by two-dimensional gel electrophoresis plasmid cloning vehicles derived from plasmids cole1, f, r6k, and rk2 idealization of the hydrophobic segment of the alkaline phosphatase signal peptide nucleotide sequence of the 26 s mrna of the virulent trinidad donkey strain of venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins expression of semliki forest virus proteins from cloned complementary dna. i. the fusion activity of the spike glycoprotein assembly of asparagine-linked oligosaccharides binding of synthetic clustered ligands to the gal/galnac lectin on isolated rabbit hepatocytes structural and evolutionary analysis of the two chimpanzee alpha-globin mrnas signal recognition particle-dependent membrane insertion of mouse invariant chain: a membrane-spanning protein with a cytoplasmically exposed amino terminus transport of secretory and membrane glycoproteins form the rough endoplasmic reticulum to the golgi partial resistance of nascent polypeptide chains to proteolytic digestion due to ribosomal shielding molecular cloning: a laboratory manual membrane integration and intracellular transport of the coronavirus glycoprotein el, a class ill membrane glycoprotein biochemical studies of the maturation of the small sindbis virus glycoprotein e3 reinitiation of translocation in the semliki forest virus structural polyprotein: identification of the signal for the el glycoprotein processing of the semliki forest structural polyprotein: role of the capsid protease on the possible participation of acid phospholipids in the translocation of secreted proteins through the bacterial cytoplasmic membrane. febs (fed. fur structure and genomic organization of the mouse dihydrofolate reductase gene translocations of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation extensions of the signal hypothesis-sequential insertion model versus amphipathic tunnel hypothesis. febs (fed. eur protein translocation across and integration into membranes improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication nucleotide sequence of the 26s mrna of sindbis virus and deduced sequence of the encoded virus structural proteins identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane polypeptide chain binding proteins: catalysts of protein folding and related processes in cells synchronized transmembrane insertion and glycosylation of a nascent membrane protein evidence for the loop model of signal-sequence insertion into the endoplasmic reticulum on the translocation of proteins across membranes on the transfer of integral proteins into membranes nascent peptide as sole attachment of polysomes to membranes in bacteria an internal signal sequence: the asialoglycoprotein receptor membrane anchor complete nucleotide sequence of the escherichia coli plasmid pbr 322 subcellular location of enzymes involved in the n-glycosylation and processing of asparagine-linked oligosaccbarides in saccharomyces cerevisiae structural and thermodynamic aspects of the transfer of proteins into and across membranes trans-membrane translocation of proteins: the direct transfer model insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence multiple mechanisms of protein insertion into and across membranes a signal sequence receptor in the endoplasmic reticulum membrane constraint of the translational diffusion of a membrane glycoprotein by its external domains the transmembrahe segment of the human transferrin receptor functions as a signal peptide we thank ernst bause for constructive discussion; gunnar von heijne and michael baron for critical reading of the manuscript; johanna wahlberg for help with the figures; margareta berg, tuula marminen, and elisabeth servin for technical assistance; and ingrid sigurdson for typing. this work was supported by grants from the swedish medical research council (b88-12x-08272-01a), swedish natural science research council (b-bu 9353-300), and swedish national board for technical development (87-02750p).received for publication 7 march 1990 and in revised form 2 may 1990. key: cord-289026-v09m2fzw authors: sun, yan-gang; li, rui; jiang, longguang; qiao, songlin; zhi, yubao; chen, xin-xin; xie, sha; wu, jiawei; li, xuewu; deng, ruiguang; zhang, gaiping title: characterization of the interaction between recombinant porcine aminopeptidase n and spike glycoprotein of porcine epidemic diarrhea virus date: 2018-10-01 journal: int j biol macromol doi: 10.1016/j.ijbiomac.2018.05.167 sha: doc_id: 289026 cord_uid: v09m2fzw porcine epidemic diarrhea (ped) has caused huge economic losses to the global pork industry. infection by its causative agent ped virus (pedv), an alpha-coronavirus, was previously proven to be mediated by its spike (s) glycoprotein and a cellular receptor porcine aminopeptidase n (papn). interestingly, some recent studies have indicated that papn is not a functional receptor for pedv. to date, there is a lack of a direct evidence for the interaction between papn and pedv s protein in vitro. here, we prepared papn ectodomain and the truncated variants of pedv s protein in drosophila s2 cells. these recombinant proteins were homogeneous after purification by metal-affinity and size-exclusion chromatography. we then assayed the purified target proteins through immunogenicity tests, pedv binding interference assays, circular dichroism (cd) measurements, papn activity assay and structural determination, demonstrating that they were biologically functional. finally, we characterized their interactions by gel filtration chromatography, native-polyacrylamide gel electrophoresis (page) and surface plasmon resonance (spr) analyses. the results showed that their affinities were too low to form complexes, which suggest that papn may be controversial as the genuine receptor for pedv. therefore, further research needs to be carried out to elucidate the interaction between pedv and its genuine receptor. coronaviruses are enveloped single-stranded positive-sense rna viruses, comprising alpha-, beta-, gamma-, and delta-coronaviruses [1] . they are critical causative agents, whose infections are often associated with respiratory, digestive, and neurological diseases in humans and animals. among these viruses, two beta-coronaviruses severe acute respiratory syndrome coronavirus (sars-cov) [2] [3] [4] [5] [6] and middle east respiratory syndrome coronavirus (mers-cov) [7] [8] [9] [10] are lethal to humans, and an alpha-coronavirus transmissible gastroenteritis virus (tgev) has caused severe intestinal diseases to pigs [11] . the infection of a coronavirus is mediated by its spike (s) glycoprotein and the s protein is further cleaved into s1 and s2 subunits by endogenous and/or exogenous proteases [12] . the s1 subunit recognizes and binds to the corresponding host receptor, whereas the s2 subunit mediates membrane fusion between the virus and host cells [13] [14] [15] . since intervention of s1 subunit binding to host receptor shows a great antiviral potential, there are increasing studies to elucidate their interaction mechanisms. angiotensin-converting enzyme 2 (ace2) [16, 17] and dipeptidyl peptidase 4 (dpp4) [18] [19] [20] have been identified as the receptor for sars-cov and mers-cov, respectively. the viral receptor-binding domains (rbd) are located in the c-terminal domains (ctds) of the s1 subunits (s1-ctds) [17, 19, 20] . human coronavirus 229e (hcov-229e) [21, 22] and tgev [23, 24] utilize aminopeptidase n (apn) as the host receptor and their rbds are also located in the s1-ctds [22, 24] . porcine epidemic diarrhea (ped), characterized by vomiting, diarrhea and dehydration, has caused huge economic losses to the global pork industry [25] [26] [27] [28] [29] [30] . its causative agent, ped virus (pedv), was an alpha-coronavirus. previous studies showed that a porcine receptor apn (papn) mediated pedv infection [31] [32] [33] [34] and its density appeared to be an important factor in contributing to the virus efficient infection [35] . pedv s1-ctd (residues 505-629) was further demonstrated to interact with papn ectodomain (residues 63-963) [36] . interestingly, a few recent studies show that papn is not the functional receptor for pedv infection [37, 38] . however, there is a lack of direct demonstration of the interaction between papn and pedv s protein to clarify this discrepancy. in this study, we prepared the extracellular domain of papn, pedv s1 and its truncated variant (s1t) in drosophila schneider 2 (s2) cells. after purification, we assayed the target proteins for their biological functions. finally, we characterized their interactions by three canonical assays. ipec-j2 cell line (from porcine small intestines) was kindly provided by zhanyong wei of henan agricultural university, china. vero cell line (african green monkey kidney epithelial cell line), drosophila s2 cell line, pmt/bip/v5-his a vector, pcoblast vector [39, 40] and positive serum against pedv were kept in our laboratory. peasy-blunt-papn plasmid was constructed and kept in our laboratory. cellfectin ii reagent, blasticidin s, sf-900 ii serum-free medium (sfm) and trizol reagent were purchased from invitrogen (carlsbad, usa). escherichia coli strain trans5α competent cells were purchased from transgen biotech (beijng, china). fetal bovine serum (fbs), dulbecco's modified eagle's medium (dmem), roswell park memorial institute-1640 medium (rpmi-1640), and antibiotics were purchased from gibco (grand island, usa). schneider's insect medium, tryptose phosphate broth (tpb), tpck-trypsin, and l-alanine 4-nitroanilide hydrochloride were purchased from sigma-aldrich (st. louis, usa). the mouse anti-his epitope tag antibody was purchased from proteintech (wuhan, china). horseradish peroxidase (hrp)-conjugated anti-mouse immunoglobulin g (igg) was purchased from santa cruz biotechnology (delaware ave, usa). pre-stained protein ladder (10 to 180 kda) was purchased from thermo fisher scientific (waltham, usa). mlu i, bgl ii and peptide-n-glycosidase f (pngase f) enzymes were purchased from new england biolabs (massachusetts, usa). enhanced chemiluminescence (ecl) plus reagent was purchased from solarbio (beijing, china). the cdnas encoding pedv s1 (residues 21-793) and its truncated variants (residues 21-249, 253-638 and 505-629) were amplified by pcr using the codon-optimized s gene of the pedv ch/hnxc strain as template. the cdna encoding papn ectodomain (residues 63-963) was amplified by pcr from the peasy-blunt-papn plasmid. all pcr primers were listed in table 1 . the pcr products were isolated and inserted into the pmt/bip/v5-his a expression vector between the bgl ii and mlu i sites. all constructs were transformed into escherichia coli strain trans5α competent cells and the recombinant expression vectors were verified by sequencing at shanghai sangon biotech co. ltd. (shanghai, china). the drosophila s2 cells were kept in schneider's insect medium supplemented with 10% heat-inactivated fbs at 28°c in a humidified incubator. the vero cells were grown in dmem supplemented with 10% heat-inactivated fbs at 37°c in a humidified incubator with 5% co 2 . ipec-j2 cells were grown in rpmi 1640 medium with 15% heat-inactivated fbs at 37°c with 5% co 2 . all cell cultures were supplemented with antibiotics (100 u/ml penicillin, 100 μg/ml streptomycin). the recombinant expression vectors and the pcoblast vector were co-transfected into drosophila s2 cells by cellfectin ii reagent according to the manufacturer's instructions. the stably transfected s2 cells were selected in schneider's insect medium supplemented with 10% fbs, antibiotics, and 25 μg/ml blasticidin s. the stably transfected cell lines were then grown in sf-900 ii sfm and induced by 0.75 mm cuso 4 for 5 days. the culture supernatants were harvested by centrifugation at 2000 rpm at 4°c for 10 min. expression of recombinant proteins was checked by mouse anti-his epitope tag antibody and hrp-conjugated anti-mouse igg using western blot. the supernatant containing papn ectodomain, pedv s1 or s1t protein was adjusted to ph 8.0 by 1.5 m tris-hcl ph 8.8, followed by centrifugation at 10000 rpm at 4°c for 30 min. after filtration through 0.22 μm filter membranes, the supernatant was applied to histrap excel prepacked column (ge healthcare, fairfield, usa) or/and hitrap q hp prepacked column (ge healthcare) for purification. then, each target protein was subjected to size-exclusion column hiload 16/600 superdex 200 pg (ge healthcare) and superdex 200 increase 10/300 gl (ge healthcare) for further purification with 20 mm tris-hcl ph 7.6 and 150 mm nacl as the elution buffer. the purified proteins were treated with pngase f according to the manufacturer's instructions. the pngase f-treated and -untreated proteins were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) under reducing (with β-mercaptoethanol) or non-reducing condition (without β-mercaptoethanol). 2.6. function analyses of pedv s1 and s1t proteins 2.6.1. immunogenicity tests of pedv s1 and s1t proteins purified pedv s1 and s1t proteins were subjected to sds-page and transferred to pvdf (polyvinylidene fluoride) membranes. the immunogenicity was then tested by positive serum against pedv and ecl plus reagent. the pedv ch/hubei/2016 strain was propagated on vero cells as previously described [41] . briefly, when the confluence reached 80%, vero cells were washed three times and inoculated with 200 tcid 50 (50% tissue culture infective dose) pedv at 37°c for 1 h. after that, the viruses not entering were removed and the cells were cultured with growth medium consisting of 0.3% tpb, 0.02% yeast extract and 3 table 1 primers utilized in this study. sequence a a the boldface letters indicate restriction sites. μg/ml of tpck-trypsin. when obvious cytopathic effects (cpe) appeared, the cells were frozen and thawed three times, and centrifuged at 2000 rpm at 4°c for 10 min. finally, the supernatant was stored at −80°c for further use. 2.6.3. interference of pedv binding by purified s1 and s1t proteins the ipec-j2 cells were pre-incubated with indicated concentrations of the purified pedv s1 or s1t protein for 1 h at 4°c. cells were further inoculated with pedv ch/hubei/2016 strain at an moi (multiplicity of infection) of 0.1 for 1 h at 4°c. then, the virus and protein mixtures were washed away. total rna was isolated using trizol reagent according to the manufacturer's instructions and the pedv binding was represented by the copies of the pedv nucleocapsid (n) gene using absolute quantitative pcr (the primers were listed in table 1 ) [42] . 2.7. circular dichroism (cd) measurements of pedv s1 and s1t proteins cd measurements (180-260 nm) were carried out on aviv 420sf spectrometer (lakewood, usa) at room temperature (rt) using a 1 mm path-length quartz cell. the instrument was calibrated with 10 mm phosphate buffer (pb) ph 7.6, and freshly purified pedv s1 and s1t proteins were adjusted to 0.8 mg/ml and 1.9 mg/ml in 10 mm pb ph 7.6, respectively. five scans were accumulated at a scan speed of 1 nm/step with average time of 0.3 s. 2.8.1. activity assay of papn ectodomain 1 nm purified papn ectodomain was mixed with 100 μm to 10 mm l-alanine 4-nitroanilide hydrochloride as substrate in 100 μl 60 mm kh 2 po 4 buffer ph 7.2 at 37°c for 60 min. then, the product pnitroanilide was measured using a microplate reader (bmg labtech, offenburg, germany) at 405 nm [43] . the purified papn ectodomain was concentrated to 10 mg/ml in 20 mm tris-hcl ph 7.2, 150 mm nacl. crystallization of papn ectodomain was carried out by the sitting-drop vapor diffusion method with an equal volume of the target protein and various crystallization reagents from the crystallization screening kits (hampton, usa) at rt. the crystals were acquired with a reservoir solution containing 25% (wt/vol) peg3350, 0.2 m sodium fluoride and 100 mm hepes ph 7.2. the crystals were flash-frozen in liquid nitrogen using a crystal freezing buffer containing 20% ethylene glycol, 30% peg3350, 0.2 m sodium fluoride and 100 mm hepes ph 7.2. x-ray data sets of the crystals were collected at a wavelength of 0.979 å on the beam line bl19u1 at national center for protein sciences shanghai (ncpss) and shanghai synchrotron radiation facility (ssrf). the crystal structure was solved by molecular replacement [44] using the mammalian apn structure (pdb code: 4hom) [43] as the search model. the structure was refined by ccp4 program package [44] and manually adjusted by the molecular graphics program coot [45] until convergence of the refinement. solvent molecules were added using a fo-fc fourier difference map at 2.5σ in the final refinement step. statistics of data collection and final model refinement were summarized in table 2 . the coordinates of papn ectodomain were deposited in the protein data bank (pdb code 5z65, http://www.rcsb.org). the final structures were analyzed by software pymol [46] . papn ectodomain was mixed with excess pedv s1 or s1t protein for 1 h at rt in 20 mm tris-hcl ph 7.6, 150 mm nacl. then, pedv s1 or s1t protein, papn ectodomain and their mixtures were individually loaded on the superdex 200 increase 10/300 gl column (ge healthcare) in the same volume and superposed for uv absorption peaks. the eluted proteins were analyzed by sds-page. the 10% native-page gel and running buffer were prepared without sds. the papn ectodomain and pedv s1 or s1t protein were mixed in 30 μl 20 mm tris-hcl ph 7.6, 150 mm nacl at a molar ratio of 1:1/1:5. after incubation at rt for 30 min, pedv s1 or s1t protein, papn ectodomain and their mixtures were analyzed by native-page. spr analyses were performed on biacore s200 (ge healthcare) to determine the equilibrium binding constants (k d , m), the association rate constants (k on , m −1 s −1 ) and the dissociation rate constants (k off , s −1 ) of papn ectodomain binding to pedv s1 or s1t protein. pedv s1 or s1t protein was coupled to the cm5 sensor chips (ge healthcare) and papn ectodomain was injected with the indicated concentrations (187.5 nm, 375 nm, 750 nm, 1500 nm and 3000 nm). the running buffer was hbs-ep (10 mm hepes ph 7.4, 150 mm nacl, 3 mm edta, 0.005% surfactant p20). the binding kinetics were analyzed with the software biacore s200 evaluation 1.0, and the k on , k off and k d were calculated using a 1:1 interaction model as previously described [47] . the equations used for calculation are represented below with biacore spr terminology: where dr dt (response unit/s, ru/s) is the slope of the curve during association phase, c (m) is the concentration of injected papn ectodomain, rmax (ru) is the signal response for maximum absorption of pedv s1or s1t protein, and r (ru) is the signal response for papn ectodomain binding to pedv s1 or s1t protein. all experimental data were presented as group means and standard errors of the means (sem). the experimental data were analyzed using the unpaired, 2-tailed student t-test using graphpad prism software (graphpad). differences at the 95% confidence level (p b 0.05) were considered statistically significant. the recombinant papn ectodomain (residues 63-963), pedv s1 protein (residues 21-793) and s1 truncated variants (residues 21-249, 253-638 and 505-629) were produced in drosophila s2 cells. western blot analyses verified that papn ectodomain (fig. 1a) , pedv s1 protein (fig. 1b) and a pedv s1 truncated variant (s1t, residues 505-629, fig. 1c) were highly expressed. the expression of other pedv s1 truncated variants (residues 21-249 and 253-638) was too low to be detected (data not shown), which were consequently not considered in our subsequent experiments. the target proteins were purified through metal-affinity and sizeexclusion chromatography. after purification, these proteins achieved to a high purity (n99%, fig. 2 ). papn ectodomain (fig. 2, top) , pedv s1 (fig. 2 , middle) and s1t proteins (fig. 2, bottom) were eluted at 10.9 ml, 12.2 ml and 17.8 ml on the calibrated size-exclusion column, corresponding to approximately 240 kda, 110 kda, 15 kda, respectively. as shown in fig. 2 , there were differences between the bands of each target protein under reducing (lane 2) and non-reducing (lane 1) conditions, which implied that they contained disulfide bonds. in addition, the bands showed a significant change after de-glycosylation using pngase f (lane 2 and lane 3). therefore, the recombinant proteins were heavily glycosylated compared to their calculated molecular masses (papn ectodomain: 104 kda, pedv s1 protein: 85.5 kda, pedv s1t protein: 15.1 kda). based on the results above, recombinant papn ectodomain was obtained as dimers, and pedv s1 or s1t protein existed as monomers, which showed similar natures of mammalian apn [43] and other coronavirus s proteins [48] [49] [50] as previously reported. 3.3. immunogenicity tests, interference of pedv binding and cd measurements of purified pedv s1 and s1t proteins we carried out immunogenicity tests and pedv binding interference assays to analyze whether purified pedv s1 and s1t proteins were functional. as shown in fig. 3a and b, purified pedv s1 and s1t proteins could be recognized by positive serum against pedv, indicating that these recombinant proteins had a comparable immunogenicity to the native virus. since porcine small intestines are the major target organ for pedv infection, ipec-j2 cells from porcine small intestines were applied in interference of pedv binding by purified pedv s1 and s1t proteins. ipec-j2 cells incubated with pedv s1 or s1t protein showed the viral rna reduction in a concentration dependent manner (fig. 3c ). especially at a concentration of 200 μg/ml, pedv s1 or s1t protein pretreatment caused a significant reduction compared to that incubated with control protein buffer (p b 0.05), demonstrating that purified pedv s1 and s1t proteins were in functional form (fig. 3c) . we have taken efforts to crystallize functional pedv s1 or s1t protein, and determine their crystal structures. unfortunately, crystallization of these proteins was unsuccessful (data not shown). cd measurement is usual for determination of protein secondary structures [51] [52] [53] [54] [55] [56] and we performed cd measurements to study their secondary structures. as shown in fig. 4 , one minimum at about 215 nm was observed for both pedv s1 and s1t proteins, suggesting that the two proteins had β-sheets. these results were consistent with other coronavirus s1 proteins, which have a high content of β-sheets [49, 50, [57] [58] [59] [60] . mammalian apn is a member of zinc-dependent m1 metallopeptidases, which is widely distributed on the intestinal epithelia and the nervous system cells [61] . it plays multifunctional roles in tumor angiogenesis and metastasis, signal pathway, and degradation of enkephalins [62] . based on these functions, we performed activity assay for purified papn ectodomain. the enzymatic kinetics showed pedv s1 protein (residues 21-793). lane 1-2: pedv s1 protein; lane 3: mock. (c): pedv s1 truncated variant (s1t, residues 505-629). lane 1: pedv s1t protein. lane m: thermo pre-stained protein ladder (10 to 180 kda). that purified papn ectodomain could degrade the substrate l-alanine 4nitroanilide hydrochloride into the product p-nitroanilide. the k m and k cat were 0.18 ± 0.13 mm and 75.55 ± 7.9/s, respectively (table 2) . furthermore, we determined the crystal structure of papn ectodomain. purified papn ectodomain was crystallized with one molecule in each asymmetric unit and its crystal structure was determined at 2.65 å in a c121 space group (table 3) . as described previously [22, 24, 43, 63, 64] , papn ectodomain adopts a hook-like conformation or so-called seahorse shape ( fig. 5b and c) . it is consisted of four domains (i-iv) and a zinc ion in domain ii, characteristics of m1 metallopeptidases ( fig. 5a and b ). in addition, it is heavily glycosylated, and cys758 and cys765 in association with cys795 and cys831 bridge into two disulfide linkages in domain iv (fig. 5b) , which are consistent with our results stated above (fig. 2) . a close structural comparison of our current papn ectodomain and that determined by others (pdb code: 4hom) reveals that the root mean square deviation (rmsd) differences are slight (0.361 å for 853 matching cα atoms), demonstrating that they share almost identical structural fold (fig. 5c ). taken together, purified papn ectodomain was shown to be biologically active on the basis of its enzymatic activity and crystal structure. papn was previously demonstrated as a functional receptor for pedv [31] [32] [33] [34] . however, the vero cell lines, possessing no apn fig. 4 . cd spectra of functional pedv s1 (a) and s1t proteins (b). one minimum at about 215 nm was observed for both pedv s1 and s1t proteins. fig. 3 . immunogenicity tests and interference of pedv binding by purified s1 and s1t proteins. purified pedv s1 (a) and s1t proteins (b) were detected by western blot using the positive antiserum against pedv. (c) pedv binding interference with purified pedv s1 or s1t protein. data represent means ± sem of three independent experiments. *significantly reduced viral rna (p b 0.05). [32, 41] , are efficiently infected by pedv, and widely utilized for isolation and series propagation of the virus [41, [65] [66] [67] [68] . recently, no effects were reported on the susceptibility to pedv after knocking out apn in porcine swine testis cells and human cell lines (human hepatoma cells, huh7 cells and human cervical cancer cells, hela cells) by crispr/cas9 genome editing [38] . moreover, soluble papn ectodomain neither interacted with pedv nor inhibited its infection [37] . all these studies infer that papn may be not the receptor for pedv. to date, there are few direct evidences to clarify this discrepancy by characterization of the interaction between papn ectodomain and pedv s protein in vitro. we speculate that the lack of related studies is probably due to the difficulty in preparing the large pedv s protein, which contains many disulfide bonds and glycosylation sites as other coronaviruses [48, 49] . in the current study, three canonical assays were carried out to characterize the interaction between papn ectodomain and pedv s1 or s1t protein since these functional target proteins were successfully prepared. according to previous reports, coronavirus s-rbds and their host receptors have high affinities and yield stable complexes in vitro, which can be obtained by gel filtration chromatography [17, 20, 22, 24, 69, 70] . therefore, we firstly characterized the interaction between papn ectodomain and pedv s1 or s1t protein by gel filtration chromatography. the results showed that there were no complexes formed in these protein mixtures as no complex peaks appeared (fig. 6) . additionally, we performed native-page to detect whether they formed complexes. as shown in fig. 7a , there were no bands of the protein complexes compared to single proteins. interestingly, pedv s1t protein showed other forms in addition to monomers (figs. 6b and 7a) . in order to further corroborate the interaction between the proteins, the kinetics of papn ectodomain binding to pedv s1 or s1t protein were measured by spr using biacore s200. the profiles showed that there were no signal responses during their association and dissociation phases, which demonstrated that papn ectodomain neither interacted with pedv s1 nor s1t protein under physiological condition (fig. 7b ). in contrast, hcov-229e [22] and tgev [24] rbds had high affinities to their receptor apn. these results were similar to sars-cov rbd not binding to dpp4 or mers-cov rbd not binding to ace2, while the k d of dpp4 binding to mers-cov rbd was about 16.7 nm (k on : 1.79 × 10 5 m −1 s −1 , k off : 2.99 × 10 −3 m −1 s −1 ) [20] . in conclusion, our work characterized the interaction between recombinant papn ectodomain and pedv s protein in vitro. the results suggest that papn may be controversial as the genuine receptor for pedv because functional papn ectodomain neither binds to pedv s1 nor s1t protein. our current work sheds some light on the invasion mechanism of the virus, and supports a molecular basis for prevention and control of ped. as s protein recognizing and interacting with the host receptor are crucial for pedv infection, further research needs to be carried out to identify the genuine receptor of pedv and elucidate the specific interaction between them. the authors declare that they have no conflicts of interest. papn ectodomain is colored as described for (a). the zinc ion is represented as red sphere and n-linked oligosaccharides are shown in stick representation. the n and the c termini are also labeled. (c) structural comparison of papn ectodomain in the current study with that determined by others (pdb code 4hom) in cartoon diagrams. the papn ectodomain in the current study is colored in green and the papn ectodomain previously determined is in cyan. their n and c termini are labeled. fig. 7 . characterization of the interaction between papn ectodomain and pedv s1 or s1t protein. (a): native-page analysis. lane 1: pedv s1 protein; lane 2: papn ectodomain and pedv s1 protein mixtures (1:1); lane 3: papn ectodomain; lane 4: papn ectodomain and pedv s1t protein mixtures (1:5); lane 5: pedv s1t protein. (b): spr analyses of the interaction between pedv s1 (top) or s1t protein (bottom) and papn ectodomain. 6 . gel filtration chromatography analyses of pedv s1 or s1t protein (blue), papn ectodomain (green) and their mixtures (brown). the papn ectodomain, pedv s1 (a) or s1t protein (b) and their mixtures were loaded on a calibrated superdex 200 increase column (10/300 gl) individually. their chromatographs were superposed and shown in the figures. sds-page analyses of pooled samples (a: 1-2 and b: 1-3) were also presented. funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. coronavirus genomics and bioinformatics analysis a novel coronavirus associated with severe acute respiratory syndrome public health: broad reception for coronavirus coronavirus as a possible cause of severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome severe respiratory 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glycoprotein glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections the surfactant-induced conformational and activity alterations in rhizopus niveus lipase interplay of multiple interaction forces: binding of tyrosine kinase inhibitor nintedanib with human serum albumin synthesis, characterization and interaction studies of 1,3,4-oxadiazole derivatives of fatty acid with human serum albumin (hsa): a combined multi-spectroscopic and molecular docking study multispectroscopic and molecular modelling approach to investigate the interaction of riboflavin with human serum albumin binding of janus kinase inhibitor tofacitinib with human serum albumin: multi-technique approach biophysical and molecular docking insight into the interaction of cytosine β-d arabinofuranoside with human serum albumin cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding cryo-em structure of porcine delta coronavirus spike protein in the pre-fusion state the moonlighting enzyme cd13: old and new functions to target the structure and main functions of aminopeptidase n the x-ray crystal structure of human aminopeptidase n reveals a novel dimer and the basis for peptide processing allosteric inhibition of aminopeptidase n functions related to tumor growth and virus infection isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene isolation and characterization of a korean porcine epidemic diarrhea virus strain knu-141112 the dynamics of chinese variant porcine epidemic diarrhea virus production in vero cells and intestines of 2-day old piglets crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor we thank the staff of bl19u1 beamline at national center for protein sciences shanghai (ncpss) and shanghai synchrotron radiation facility (ssrf), shanghai, china, for assistance during data collection. we also acknowledge qiang wei and hongfang ma from henan academy of agricultural sciences for their assistance on crystallization, and baolin xiao from henan university for his assistance on cd measurements. this work was supported by the key: cord-291210-ghjseynl authors: arbely, eyal; khattari, ziad; brotons, guillaume; akkawi, mutaz; salditt, tim; arkin, isaiah t. title: a highly unusual palindromic transmembrane helical hairpin formed by sars coronavirus e protein date: 2004-08-13 journal: journal of molecular biology doi: 10.1016/j.jmb.2004.06.044 sha: doc_id: 291210 cord_uid: ghjseynl abstract the agent responsible for the recent severe acute respiratory syndrome (sars) outbreak is a previously unidentified coronavirus. while there is a wealth of epidemiological studies, little if any molecular characterization of sars coronavirus (scov) proteins has been carried out. here we describe the molecular characterization of scov e protein, a critical component of the virus responsible for virion envelope morphogenesis. we conclusively show that scov e protein contains an unusually short, palindromic transmembrane helical hairpin around a previously unidentified pseudo-center of symmetry, a structural feature which seems to be unique to scov. the hairpin deforms lipid bilayers by way of increasing their curvature, providing for the first time a molecular explanation of e protein's pivotal role in viral budding. the molecular understanding of this critical component of scov may represent the beginning of a concerted effort aimed at inhibiting its function, and consequently, viral infectivity. a previously unidentified member of the coronaviridae is the etiologic agent responsible for the recent severe acute respiratory syndrome (sars) outbreak. 1 viral genome sequencing, combined with protein phylogenetic analyses, have shown that sars coronavirus (scov) belongs to a new subfamily within the coronaviridae. 2, 3 in an effort to better understand the components contributing to the pathogenic mechanism of the virus, we have structurally analyzed the transmembrane domain of scov e protein using fourier transform infrared (ftir) spectroscopy, x-ray scattering, electron microscopy and global searching molecular dynamics simulations. functionally, coronavirus e protein's pivotal role in viral morphogenesis has been studied extensively. co-expression of mouse hepatitis virus (mhv) e and m proteins on their own has been shown to result in the production of virus-like particles. 4, 5 this fundamental observation proved that neither the nucleocapsid nor the viral spike are needed for viral budding. work on other viruses, such as transmissible gastroenteritis virus (tgev), 6 bovine coronavirus (bcov) 6 and infectious bronchitis virus (ibv) 7 has shown this to be a general phenomena of the coronaviridea. the interaction between the cytoplasmic domains of the two proteins is thought to take place in pre-golgi compartments. 8 the importance of the role played by e protein in virus budding and morphogenesis is further strengthened by the observation that in a number of coronaviruses, expression of m protein on its own does not produce virus-like particles. 4 -7,9 in contrast, expression of ibv or mhv e protein on its own causes the release of vesicles containing e protein, thereby pointing to the importance of 0022 coronavirus e protein in the budding process. 7, 9 interestingly, cells infected with mhv release vesicles containing e protein as well. 9 finally, mutations in mhv e protein cause marked morphological changes in the resulting viruses. 10 in the cell, evidence has been accumulating suggesting that ibv e protein localizes to the golgi apparatus, 7 the site from which coronaviruses are known to invaginate into mature virions. during the expression of e protein the golgi apparatus changes its morphology dramatically, 11 which could explain in part e protein's ability to induce apoptosis. 12, 13 structurally, e proteins are conserved within the different coronaviridea groups, yet exhibit little sequence similarity between the groups (figure 1 ). in line with its belonging to a new group within the coronaviridea, scov e protein differs substantially from other coronavirus e proteins. 3 whether this has any bearing upon the marked difference in pathogenicity of scov versus other coronaviruses remains to be seen. finally, scov e protein shares no significant sequence similarity with any other known protein. it is possible, however, to make the following generalizations regarding the sequence of all coronavirus e proteins: they are small proteins (, 75 residues), with an unusually long hydrophobic stretch (25 -30 residues) located between hydrophilic n and c termini (, 8 and , 40 residues, respectively). scov e protein shares the same overall characteristics, although only 20% identical with other coronaviridea e proteins: 3 74 residues in length with a stretch of 26 hydrophobic amino acid residues (figure 1 ). the length of the hydrophobic segment of scov e protein has posed a problem with respect to assigning a topology to the protein (figure 2(a) ). the average length of a transmembrane a-helix is 28 using a window size of 15 and a hydrophobicity cutoff of dg water!oil ¼ 227 kcal/mol. 29 the region of the synthesized peptide is boxed, while gray shading indicates the extent of the hydrophobic stretch of the protein. the location of the iodinated phe residue is indicated by dark shading. (b) inversion diagram of the scov e protein hydrophobic region outlining identity and similarity between the two inverted sequence elements, in continuous and broken lines, respectively. hydrophobic amino acid residues according to the ges scale 28 are indicated by gray shading. 21 residues, 14 far shorter than the hydrophobic stretch of e proteins. in contrast, if a hairpin were to be formed by the long hydrophobic stretch, the resulting helices would be much shorter than the average. protease digestion of mhv does not affect e protein's molecular mass, suggesting that no part of the protein protrudes beyond the viral membrane. 11 antibodies raised against the n terminus of the protein could detect the protein only after treatment of the cells with triton x-110, but not with digitonin. 7 both of the above studies concluded that the protein traverses the lipid bilayer once, with its c terminus located in the viral interior. 11, 7 more recent results with an epitope tagged mhv e protein are indicative of the protein traversing the lipid bilayer twice, whereby both termini of the protein reside in the virus lumen. 15 in the current study we present the results of a detailed structural examination of scov e protein, in which we determine the topology of the protein and the effects upon the lipid bilayer thereof. the results conclusively show that scov e protein has a highly unusual topology, consisting of a very short transmembrane helical hairpin. the hairpin forms an inversion about a previously unidentified pseudo-center of symmetry which seems to be unique to scov. electron microscopy studies indicate that the protein dramatically distorts lipid bilayers, causing tubulation. taken together, the structure of the protein facilitates a molecular explanation of the effects upon the bilayer curvature underpinning the function of the protein in vivo and may represent the beginning of a concerted effort aimed at inhibiting its function and consequently viral infectivity. in order to examine the secondary structure of the transmembrane domain (tmd) of scov e protein, peptides were made which encompassed the entire hydrophobic region of the protein (glu7-arg38), as depicted in figure 1 . the peptide was purified and reconstituted into dimyristoylphosphocholine (dmpc) lipid vesicles, to be used for ftir spectroscopy. the transmission ftir spectrum of the resulting proteo-liposomes focusing on the amide i vibrational mode (1600 -1700 cm 21 ) is presented in figure 3 (a). since the amide i vibrational mode arises mostly from the peptidic c ¼ o stretch, its frequency is well correlated with the secondary structure of the protein. 16 peaks resonating at 1655 cm 21 , 1630 cm 21 and 1645 cm 21 , correspond to a-helical, b-strand and random coil peptide segments, respectively. 16 the amide i mode of scov e protein tmd is centered at 1657 cm 21 with a peak-width at half height of 23 cm 21 , both indicative of the very high helical content of the protein, and the absence of any other secondary structure component. 16 e protein contains 26 residues embedded in the lipid bilayer ftir spectroscopy can be used to delineate the extent of membrane incorporation of transmembrane proteins. this is achieved by observing the reduction in any vibrational mode containing significant contributions from the amide proton (e.g ii mode : peptidic n -h deformation) upon exchanging the solvent from h 2 o to d 2 o. the reason is that the lipid bilayer protects the peptide from exchange, whereas amide groups exposed to the solvent undergo h þ /d þ exchange. any vibrational mode containing the "new" n -d group will resonate elsewhere and h þ /d þ exchange can be quantitated by measuring the reduction in the amide ii mode directly. 16 as shown in figure 3 (b), upon flushing the membrane with air saturated with d 2 o for several hours, the reduction in the amide ii peak, centered at 1545 cm 21 , is minimal. since the amide i peak at 1657 cm 21 is not expected to undergo any intensity changes upon exchange from h 2 o to d 2 o, it is possible to calculate the extent of exchange by normalizing both spectra (in h 2 o and d 2 o) on the amide i peak. the resulting exchange rate of 19^2% is indicative of only six out of 32 residues in the peptide undergoing h þ /d þ exchange. in other words, 26 residues of scov e protein are protected from exchange as a result of their being embedded in the lipid bilayer. note that this value corresponds exactly to the length of the hydrophobic stretch of the protein. after establishing the helicity of the peptide and determining that most of it is embedded in the lipid bilayer, experiments were undertaken to determine its orientation using polarized ftir spectroscopy of oriented membranes. attenuated total internal reflection (atr-ftir) spectra obtained using both parallel and perpendicular polarized light are depicted in figure 3 (c). the dichroism (ratio of absorption of parallel and perpendicular polarized light) for the amide i peak (centered at 1657 cm 21 ) is 3.76^0.52. from this dichroism it is possible to calculate an exceptionally high order parameter for the peptide of s ¼ 0:82^0:17, a value nearing the theoretical limit. 17 when taking into account sample disorder, it is possible to convert the maximal value of the measured order parameter to a maximal tilt angle. thus, the tilt angle of scov e protein helical elements from the membrane normal must be less than or equal to 6.38. so far three lines of evidence were obtained characterizing the transmembrane domain of scov e protein: (i) it is highly helical, (ii) it is composed of 26 amino acid residues and (iii) the helical elements are oriented normal to the membrane plane. thus, there are two ways in which to describe the structure of the protein: either as a single long transmembrane helix, or as two short helices forming a transmembrane helical hairpin. both models however, incur a hydrophobicity mismatch between the protein and the bilayer. in the case of a single helix, the hydrophobic stretch is too long relative to the bilayer thickness. † on the other hand, the helical hairpin would have to be comprised of two very short helices in order to enable the 26 residues to traverse the membrane twice. since the smallest possible loop is of three residues, each helix cannot contain more than 11-12 hydrophobic residues. this value is much smaller than the average of 21 residues per transmembrane helix. 14 in order to conclusively determine the topology of scov e protein we iodinated phe23, which is located at the center of the hydrophobic stretch of the protein (see figure 1) . ftir spectroscopy has shown that the iodinated and unlabeled peptides are indistinguishable in any of the experiments described above (data not shown). electron density profiles obtained using x-ray scattering should be able to pinpoint the position of the labeled electron-dense iodine. as shown in figure 4 , the electron density of lipid vesicles containing iodinated and unlabeled scov e protein transmembrane domain exhibit normal density profiles. 18 however, upon subtracting the densities form one another, clear density resulting from the iodine label is identified, located 16.5 å from the bilayer core, close to the lipid head-group region. thus, one can clearly state that the labeled phe23 is located adjacent to the head-group region, despite the fact that it is in the middle of the hydrophobic segment of the protein. taken together, our data prove conclusively that the transmembrane domain of scov e protein forms a hairpin with unusually short hydrophobic helices. after establishing that scov e protein forms a helical hairpin, close inspection of its sequence reveals an unusual feature. as shown in figure 2 (b), inverting the protein about itself at phe23 indicates that the protein is virtually symmetrical with respect to the inversion point, thereby forming a palindrome. the only deviation from palindromic symmetry is located at asn15, the significance of which is discussed below. in order to estimate the statistical significance of forming a palindrome within the helical hairpin framework, a monte -carlo simulation was performed. the sequence of each helix forming the hairpin was randomized, followed by assessing the extent of palindromic symmetry. the results indicate that the degree of inverted identity between the two helices of scov e protein (thereby forming a palindrome) is a rare event, observed at a frequency of ca 10 25 . this would explain why to our knowledge palindromic symmetry in helical hairpins has not been observed before. the scov e protein contains the shortest transmembrane hydrophobic helical hairpin known to date. its 26 hydrophobic amino acid residues not only traverse the lipid bilayer as two helices, but must form a connecting turn between the helices. an immediate question then arises: what affect does the unusual structure of scov e protein transmembrane domain have upon the lipid bilayer? in order to answer the above question we undertook two lines of experiments, aimed at (i) examining the molecular structure of the lipid in detail, alongside (ii) establishing the effects upon the membrane morphology as a whole. the order of the lipid bilayer acyl chains was examined using polarized ftir spectroscopy of oriented multilayered dmpc vesicles containing scov e protein. figure 3(d) presents the atr-ftir spectrum of the lipid ch 2 and ch 3 stretching modes of the aforementioned vesicles. the measured dichroism for the lipid ch 2 asymmetric stretching mode (centered at 2920 cm 21 ) is 1.30^0.13, yielding an order parameter of s ¼ 0:51^0:11. interestingly, the order parameter of the protein ðs ¼ 0:82^0:17þ is much higher than that of its ordering environment, the lipid bilayer. the reduced order of the lipid acyl chains is an inherent property of the system caused by scov e protein and cannot be attributed to incomplete sample deposition. the reason is that the exceptionally high order parameter obtained for the protein would not be possible under such circumstances. moreover, the mosaicity of the membranes was measured to be below 0.018, as determined by x-ray reflectivity rocking scans (not shown). thus, incorporation of scov e protein transmembrane domain in the lipid vesicle deforms the acyl chains of the lipid bilayer. the global change in the structure and morphology of the lipid bilayer upon incorporating scov e protein transmembrane domain was examined using negative staining electron microscopy. while vesicles without protein ( figure 5 (a)) exhibited normal globular structure, scov e protein-containing vesicles were markedly different ( figure 5(c) ), exhibiting extensive tubulation and deformation. vesicles containing a control transmembrane protein, the mhc class ii-associated invariant chain transmembrane domain, 19 were similar to vesicles without protein, and did not exhibit any tubulation ( figure 5(b) ). in summery, ftir spectroscopy has shown that the incorporation of scov e protein in the lipid bilayer results in a molecular disordering of the lipid acyl chains. this leads to the tubulation of the vesicles as we have shown using electron microscopy. thus, the molecular defects in the lipid molecule are translated into a distortion of the entire bilayer structure. the evidence obtained so far points conclusively to the fact that the scov e protein transmembrane domain forms an unusually short transmembrane helical hairpin: (i) the secondary structure of the transmembrane domain is highly helical. (ii) more than 80% of the protein is embedded in the lipid bilayer. (iii) the helical element of the protein is tilted from the membrane normal by no more than 6.38. (iv) phe23 residing in the middle of the hydrophobic stretch of the protein is located in the lipid head-group region. (v) the protein contains a center of symmetry upon which it can be inverted. what might the hairpin structure of scov e protein look like? the only deviation from hairpin symmetry is at asn15 (figure 2(b) ). the groups of engelman & degrado have recently shown that asparagine residues in transmembrane helices promote strong homo-dimerization due to h-bonding. 20, 21 in this instance there is only a single asparagine in the two helices and as such the hydrogen bonds that it might form are to a backbone amide group, thereby driving the two helices to close apposition. alternatively, an asparagine residue from a different protein might promote dimerization, forming a pseudo fourhelix bundle. another interaction that might take place between the helices is through a salt bridge between glu8 and arg38. the presence of opposing charged resides at both sites is highly conserved in the coronaviridea (figure 1) , thereby substantiating the possibility of an interaction between them. in order to derive a model for the protein based on its deduced topology and helix tilt angles, global searching molecular dynamics simulations were employed. 22 multiple starting structures were generated in which both helices of scov e protein (glu7-leu21 and val25-arg38) formed a hetero-dimer. the sizes of the helices reflected the high helical content of the protein. each structure was distinguished by the rotational angle of the two helices with respect to one another, in increments of 208, leading to a total of 18 £ 18 ¼ 324 structures. the tilt angle between the helices was set to 08, according to the high degree of orientation found experimentally. the stability of each structure was then determined by a molecular dynamics and energy minimization trajectory. the resulting structures were then compared in order to find regions of the configuration space to which structures from different starting configurations converged. the results of the global searching molecular dynamics simulations are shown in figure 6 , depicting two clusters to which structures have converged. the energy of the structures and the two clusters show a clear preference to lower rotational angles of the two helices (most likely due to the formation of a salt bridge, as discussed below). thus the cluster found at rotational angles of 508 and 108 (helix i and ii, respectively) was taken to represent a model for the interactions between the two helices (see figure 6 ). after the packing between the two helices forming the hairpin was obtained, the loop connecting the helices formed by amino acid residues ala22-val24 was constructed. the entire hairpin structure containing the loop was subsequently subjected to figure (7) , and exhibits three prominent features: (i) a salt bridge between glu8 and arg38, (ii) an inter-helical h-bond between the amide side-chain of asn15 and leu31 peptide carbonyl and (iii) three phenylalanine residues located at the tip of the hairpin pointing outwards forming an aromatic belt. finally, based on the evidence that ibv e protein is palmitoylated, 23 it is possible to speculate that scov e protein might be as well. in contrast, experiments attempting to label tgev e protein with palmitic acid have failed. 24 if present in scov e protein, palmitoylation will take place on one or more of the three juxtamembranous cysteine residues. it is difficult to estimate the effects, if any of palmitoylation on the scov e protein. one might speculate that the intercalation of the palmitic acid in the cytoplasmic membrane leaflet might further affect the local membrane structure. in any in vitro study the relevance of the results to those found under native conditions is obviously a concern. however, the following facts negate any suspicions of artifactuality: comparison to the full length protein. the protein under study encompasses all the hydrophobic domain of the scov e protein. the only thing known about the endogenous protein is that it traverses the lipid bilayer twice. 15 that is exactly what was found herein. thus it is highly unlikely that the full length protein and its membrane domain both traverse the lipid bilayer twice, but do so in different ways. structural similarity to that found in native conditions. the endogenous function of scov e protein is in viral budding by way of causing membrane tubulation. 4 -7,9 moreover, it is the only protein in the virus that is required for this process. 7, 9 thus, the finding that the membranous segment of scov e protein (analyzed in vitro) causes membrane tubulation in an indistinguishable manner to the wild-type protein, in vivo, is proof that the membranous segment is necessary and sufficient for scov e protein role in membrane tubulation. finally, the membranous segment of scov e protein was not studied in detergent micelles (as is customary), but rather in hydrated lipid bilayers, a system that is closest to the native conditions as possible. it was shown that the protein has a defined helical secondary structure and that it is highly embedded in the lipid bilayer. it is difficult to think of an example in which a small membrane protein was reconstituted in lipid vesicles, shown that it was helical as well as highly embedded in the lipid bilayer but is nevertheless in a non-native conformation. with a structural model of scov e protein at hand, is it possible to rationalize the effects of the protein upon the lipid bilayer? the hydrophobic stretch of both hairpin helices is only 11 residues long, which is much shorter than the average of 21 residues. 14 at the apex of the hairpin three amino acid residues have their amide carbonyl and amine groups unpaired, surrounded by an aromatic belt. at the other end of the hairpin a cluster of charged residues is located. the distance between these two polar elements is much shorter than the hydrophobic thickness of the lipid bilayer. the polarity of both elements is not equal however, whereby the charged residues at the opening of the hairpin predominate. thus, one can speculate that the hairpin opening is situated at the lipid headgroup region of one leaflet. therefore, the hairpin apex is situated in the hydrophobic region of the bilayer, with its aromatic belt (known for its affinity to the polar head group) and unpaired amide groups poised to attract the lipid head-group region. it is this attraction that exerts a deformation force on the bilayer, causing an increase in curvature. in comparison to other coronaviridea e proteins, only the length of the hydrophobic segment lined by opposing charged residues at the either end, is conserved ( figure 1) . thus, e proteins from different coronavirus subfamilies do not exhibit any sequence similarity to those from other subfamilies. it would seem therefore, that members of the coronaviridea have "found" at least four independent sequence motifs to form a functional e protein. the effects of the unique features of scov e protein-the aromatic belt, the inter-helical h-bond of asn15 and the inversion symmetry on the overall virus life cycle and resulting pathogenicity, remain to be seen. viral strains 7 to 38) were synthesized by standard solid-phase n-(9fluorenyl) methoxycarbonyl chemistry. the peptides were cleaved from the resin with trifluoroacetic acid (tfa, aldrich) and lyophilized. two different synthetic peptides were made: an unlabeled peptide and one containing a para-iodo phenylalanine at position 23 of the sequence, as shown in figure 1 . peptide purity was confirmed by mass spectrometry. the lyophilized peptide was dissolved in tfa (final concentration , 5 mg/ml), and immediately injected on a jupiter 5m c4 300 å column (phenomenex, chesire, uk), equilibrated with 80% data were recorded on a nicolet magna-560 infrared spectrometer (nicolet instrument corporation, usa) purged with dry air and equipped with an mcta detector, cooled with liquid nitrogen. a total of 1000 interferograms were collected at a resolution of 4 cm 21 . attenuated total reflection (atr) spectra were measured with a 25 reflections atr accessory from grasbey specac (kent, uk) and a wire grid polarizer (0.25 mm, graseby specac). three hundred microlitres of sample (ca 1 mg/ml of peptide and 10 mg/ml of lipid) were deposited onto a ge trapezoidal internal reflection element (50 £ 2 £ 10 mm) flowed by removal of bulk solvent. for the purpose of solvent exchange, air was followed over the sample that was bubbled through 2 h 2 o or h 2-o. transmission ftir spectra were collected by depositing 50 ml of sample (ca 1 mg/ml of peptide and 10 mg/ ml of lipid) on a caf 2 window. the dichroic ratios of the amide i band were calculated by integrating between 1670 cm 21 and 1645 cm 21 . all integrations were performed by using a straight baseline that contains points immediately before and after the band. reported standard deviations values represent a minimum of three data sets measured. order parameters for the transmembrane helices were calculated as described. 17 in brief, the order parameter s is defined as follows: whereby u represents the angle between the helix director and the z axis which is coincident with the membrane normal. the order parameter can be determined experimentally from the atr dichroic ratio r atr by the following equation: whereby the electric field components of the evanescent wave for a ge internal reflection element, e x ¼ 1:398, e y ¼ 1:516 and e z ¼ 1:625 are given by harrick, 25 and a is the angle between the helix director and the transition dipole moment of the vibrational mode: 398 for the amide i mode and 278 for the amide a mode. 26 lipid order parameters are obtained from the lipid methylene symmetric (2852 cm 21 ) and asymmetric (2924 cm 21 ) stretching modes using the same equation by setting a ¼ 908. these equations are based on the reasonable assumption that the thickness of the deposited film (. 20 mm) is much larger than the penetration depth (ca 1 mm) of the evanescent wave. 25 for x-ray reflectivity measurements, samples of varied molar peptide-to lipid ratio ðp=l ¼ 0; 0:002; 0:01; 0:05; 0:1; 0:13þ were deposited on silicon surfaces by spreading from hfip/chloroform solvent (0.1 ml of 20 mg/ml solution on wafers cut to 15 £ 25 mm 2 ). the orientational distribution (mosaicity) of the highly oriented membranes was measured to be below 0.018, as determined by a rocking scan. high resolution reflectivity scans up to vertical momentum transfer q z . 1 a 21 were taken at the d4 bending magnet beamline of the doris storage ring at hasylab/desy, at a photon energy of 11 kev, set by a si(111) monochromator. the curves were measured with a fast scintillation counter (cyberstar, oxford instruments), using motorized collimating slits on the incident and exit arms. the curves were corrected for ring current, sample illumination, and diffuse background (offset-scan). general aspects of reflectivity experiment and analysis are discussed in ref. 18 the samples were kept in an environmental chamber at controlled relative humidity of 98% and a temperature of t ¼ 45 8c to assure that the bilayers were in the fluid l a state. the electron density profile was obtained by the fourier synthesis method from the integrated peak intensities, using a lorentz correction factor 1=q z , and phases (þ, 2 , 2 , 2 , 2 ). the integrated intensity of the first bragg peak was normalized to 1 for the iodinated scov e protein and for the influenza a m2 curve, while the non-iodinated scov e protein curve was properly scaled to the iodinated curve corresponding to the respective scattering signals. full q z -range fits to determine the density profiles on an absolute scale and analysis of all samples at different r.h.% is in progress (z.k. et al., to be published). liposomes containing the transmembrane domain of the scov e protein (residues 7-38) and the mhc class ii-associated invariant chain (residues 29 -60) 19 where prepared as described. liposomes composed of pure dmpc were prepared in an identical manner, with no addition of peptide. unilamellar vesicles where obtained by two cycles of freeze-thaw sonication (40 seconds) using an inverted probe sonicator (vibra cell model vc130, sonics and materials inc., ct). the liposomes where passed twice through 200 nm polycarbonate filter using an avanti mini-extruder (avanti polar lipids inc., al). negative staining for transmission electron microscopy was done by mixing the liposome suspension at 1 : 1 ratio with 2% (v/v) phosphotungstic acid (electron microscopy sciences, hatfield, pa) and incubating for one minute at room temperature. the mixed sample was absorbed onto carbon-coated grids that had been rendered hydrophilic by glow discharge in air. excess solution was removed by filter paper absorbance. after drying, the samples were imaged with a tecnai 12 electron microscope (philips) operating at 100 kv. molecular modeling of the transmembrane domain of scov e protein was undertaken using the chi suite of macros 22 for cns 1.1. 27 briefly, bundles of helices were generated in which the angle of the helices about their director was rotated independently in increments of 208. the crossing angle of the helices was set close to that found experimentally: 08. each structure was subjected to the following simulated annealing and energy minimization protocol as follows: 600 steps of powell energy minimization followed by torsion angle molecular dynamics at 600 k for 5 ps and at 300 k for 10 ps. finally, an additional 250 steps of powell energy minimization were undertaken. throughout the simulations, harmonic restraints were employed to maintain helical geometry and distance restraints were employed to maintain the helix centers within 10.5 å one another. 22 the resulting 324 structures were compared, whereby clusters were defined as those that contained at least seven structures and a c a rmsd , 1.0 å . the clusters were than averaged and subjected to the same molecular dynamics/energy minimization protocol as above. are as follows, with the accession numbers in parentheses: aibv-avian infectious bronchitis virus beaudette us (cac39303) cec-canine enteric coronavirus strain insavc-1 (p36696) prc-porcine respiratory coronavirus strain rm4 (p24415) hcv1-human coronavirus strain 229e (p19741) bcv2-bovine coronavirus strain f15 (p15775) hcv2-human coronavirus strain oc43 (aar01017) mhv1-murine hepatitis virus strain a59 (np_068673) rsdcv-rat sialodacryoadenitis coronavirus strain sdav-681 (aaf97741) mhv2-murine hepatitis virus strain s (p29076) protein synthesis, purification and reconstitution peptides encompassing the predicted transmembrane domain of sars coronavirus e protein (figure 1, residues references 1 the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome the production of recombinant infectious di-particles of a murine coronavirus in the absence of helper virus nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles the missing link in coronavirus assembly. retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-golgi compartments and physical interaction between the envelope and membrane proteins release of coronavirus e protein in membrane vesicles from virus-infected cells and e protein-expressing cells analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly characterization of the coronavirus mouse hepatitis virus strain a59 small membrane protein e induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of e protein as an apoptosis inducer induction of apoptosis in murine coronavirus-infected 17cl-1 cells statistical analysis of predicted transmembrane alpha-helices membrane topology of coronavirus e protein fourier transform infrared techniques for probing membrane protein structure site-directed dichroism as a method for obtaining rotational and orientational constraints for oriented polymers x-ray reflectivity of solid supported, multilamellar membranes a structure for the trimeric mhc class ii-associated invariant chain transmembrane domain interhelical hydrogen bonding drives strong interactions in membrane proteins asparagine-mediated self-association of a model transmembrane helix computational searching and mutagenesis suggest a structure for the pentameric transmembrane domain of phospholamban the cytoplasmic tail of infectious bronchitis virus e protein directs golgi targeting tgev coronavirus orf4 encodes a membrane protein that is incorporated into virions internal reflection spectroscopy infrared dichroism and molecular conformation of a-form poly-g-benzyl-l-glutamate crystallography and nmr system: a new software suite for macromolecular structure determination identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins do more complex organisms have a greater proportion of membrane proteins in their genomes? transmembrane four-helix bundle of influenza a m2 protein channel: structural implications from helix tilt and orientation algorithm for ribbon models of proteins this research was supported in part by a grant from the israel science foundation (784/01) to ita, a grant from the deutsche forschungsgemeinschaft grant to ita, ma and ts and by a grant from niedersachsen to ita. ita wishes to thank professors a. panet, i. ohad and dr m. kosloff for helpful discussions. key: cord-286603-4p3t0vre authors: duan, zhiqiang; yuan, chao; han, yifan; zhou, lei; zhao, jiafu; ruan, yong; chen, jiaqi; ni, mengmeng; ji, xinqin title: tmt-based quantitative proteomics analysis reveals the attenuated replication mechanism of newcastle disease virus caused by nuclear localization signal mutation in viral matrix protein date: 2020-06-27 journal: virulence doi: 10.1080/21505594.2020.1770482 sha: doc_id: 286603 cord_uid: 4p3t0vre nuclear localization of cytoplasmic rna virus proteins mediated by intrinsic nuclear localization signal (nls) plays essential roles in successful virus replication. we previously reported that nls mutation in the matrix (m) protein obviously attenuates the replication and pathogenicity of newcastle disease virus (ndv), but the attenuated replication mechanism remains unclear. in this study, we showed that m/nls mutation not only disrupted m’s nucleocytoplasmic trafficking characteristic but also impaired viral rna synthesis and transcription. using tmt-based quantitative proteomics analysis of bsr-t7/5 cells infected with the parental ndv rss1gfp and the mutant ndv rss1gfp-m/nlsm harboring m/nls mutation, we found that rss1gfp infection stimulated much greater quantities and more expression changes of differentially expressed proteins involved in host cell transcription, ribosomal structure, posttranslational modification, and intracellular trafficking than rss1gfp-m/nlsm infection. further in-depth analysis revealed that the dominant nuclear accumulation of m protein inhibited host cell transcription, rna processing and modification, protein synthesis, posttranscriptional modification and transport; and this kind of inhibition could be weakened when most of m protein was confined outside the nucleus. more importantly, we found that the function of m protein in the cytoplasm effected the inhibition of tifa expression in a dose-dependent manner, and promoted ndv replication by down-regulating tifa/traf6/nf-κb-mediated production of cytokines. it was the first report about the involvement of m protein in ndv immune evasion. taken together, our findings demonstrate that ndv replication is closely related to the nucleocytoplasmic trafficking of m protein, which accelerates our understanding of the molecular functions of ndv m protein. paramyxoviridae family includes several enveloped viruses with non-segmented negative-sense rna genomes that can cause serious diseases in humans and animals, such as sendai virus (sev), measles virus (mev), mumps virus (muv), nipah virus (niv), hendra virus (hev), parainfluenza virus types 1-5 (piv 1-5), and newcastle disease virus (ndv) [1, 2] . as an important member in the genus avulavirus of the family paramyxoviridae, ndv is a highly infectious agent of avian species and may cause devastating losses in the poultry industry worldwide. the genome of ndv is approximately 15.2 kb in length and encodes eight proteins, including six structural proteins (nucleocapsid protein (np), phosphoprotein protein (p), matrix protein (m), fusion protein (f), hemagglutininneuraminidase protein (hn) and large polymerase protein (l)) as well as two non-structural proteins (v and w) derived from the rna editing of the p gene [1] . like most of the paramyxoviridae family members, although ndv completes its life cycle in the cytoplasm, both m protein and w protein of ndv can localize to the nucleus via a bipartite nuclear localization signal (nls) at specific times in virus-infected cells [3, 4] . however, unlike the nuclear localization and unknown function of w protein, the ndv m protein is demonstrated to be a nucleocytoplasmic shuttling protein and plays crucial roles in ndv life cycle [1] . in addition to participating in the assembly in the cytoplasm and budding of progeny virions at the cell membrane later in infection [5] , the ndv m protein is localized in the nucleus and nucleolus early in infection [6] , which is thought to inhibit host cell transcription and protein synthesis similar to the m protein of human respiratory syncytial virus (hrsv), vesicular stomatitis virus (vsv) and mev [7] [8] [9] . recent studies have shown that the nuclear import of ndv m protein is mediated by its nls region ( 247 kkgkkvifdkieekirr 263 ) interacting with importin β1 via the rangtp-dependent pathway [10] , and the nuclear export of ndv m protein is mediated by three nuclear export signals (ness) via the crm1-independent pathway [11] . but so far, there is limited information about the biological functions of m's nucleocytoplasmic trafficking of ndv and other paramyxoviridae family members. increasing lines of evidence have suggested that the nuclear localization and nuclear export of cytoplasmic rna virus proteins plays essential roles in successful virus replication by inhibiting antiviral response or promoting virus budding [12] [13] [14] [15] . for example, nuclear localization of porcine respiratory and reproductive syndrome virus (prrsv) nucleocapsid protein is found to benefit for optimal virus replication and inhibiting cellular antiviral processes [16] , and nuclear export of prrsv nonstructural protein 1α is necessary for type i ifn inhibition [17] . meanwhile, nuclear localization of hrsv m protein is important for inhibiting cell transcription and is associated with the pathogenesis of virus infection [7, 12] , and nuclear export-deficient of hrsv m protein fails to localize to regions of virus assembly and thereby absolutely inhibits virus replication [18] . in addition, the interaction of phospholipid scramblase 1 with influenza a virus (iav) np protein inhibits the incorporation of importin β into the importin α/β complex, which reduces the nuclear import of np and suppresses virus replication [19] , and inhibition of crm1mediated nuclear export of iav np protein and nuclear export protein impairs virus budding [20] . moreover, a previous study has also revealed that ubiquitinregulated nucleocytoplasmic trafficking of the niv m protein is associated with m's post-translational modification and plays a critical role in m-mediated viral budding [21] . in our recent studies, we demonstrated that a recombinant ndv with nls mutation ( 247 aagaavifdkieekiaa 263 ) in the m protein not only resulted in a pathotype change of virulent ndv but also significantly attenuated the replication and pathogenicity of ndv in chicken fibroblasts and spf chickens [10] . more importantly, a recombinant ndv carrying mutated m/ness can not be rescued by reverse genetics due to the core role of m protein in viral assembly and budding in the cytoplasm [11] . these results clearly indicated that nucleocytoplasmic trafficking of m protein was closely related to the replication and pathogenicity of ndv. proteomics analysis of host cells responding to virus infection are important tools in identifying cellular proteins involved directly or indirectly in virus replication and helping to understand how virus infection leads to host pathogenicity [22, 23] . in recent years, tandem mass tag (tmt) combined with liquid chromatography tandem-mass spectrometry (lc-ms/ms) analysis has become a powerful tool in the identification, characterization, and quantitation analysis of the proteomic profiles [24, 25] . this approach has been successfully applied to many virus-host interaction studies, such as in human immunodeficiency virus (hiv) [26, 27] , bombyx mori nucleopolyhedrovirus [28] , canine parvovirus [29] , hiv/tm co-infected patients [30] , and epstein-barr virus [31] . these studies have revealed the dynamic interactions between virus and host, and provided a better understanding of the pathogenesis involved in viral infection and replication. up to now, studies about proteomic changes in host cells or tissues infected with ndv mainly focus on the pathogenesis of ndv-infected chicken peripheral blood mononuclear cells [32] , ndv/infectious bronchitis coronavirus/h9 subtype avian influenza virus coinfected chicken tracheal [33] , ndv-infected chicken lung during heat stress [34] , and ndv-caused extracellular matrix degradation and immunopathology in chicken spleen [35] . however, there have been few comparative proteomics studies investigating the host interaction with the wild type ndv and its attenuated ndv caused by amino acids mutation in viral proteins. therefore, in this study, we employed a quantitative proteomics analysis based on tmt coupled with lc-ms/ms to screen the differential protein expression profiles of bsr-t7/5 cells upon infection with the parental ndv (rss1gfp) and the mutant ndv (rss1gfp-m/nlsm) carrying m/nls mutation. the results will provide valuable information for better understanding the attenuated replication mechanism of ndv caused by nls mutation in m protein and the potential biological functions of ndv m's nucleocytoplasmic trafficking, and also accelerate our understanding of the molecular mechanisms underlying the replication and pathogenesis of ndv. nucleocytoplasmic trafficking of m protein promotes the replication and cytopathogenicity of ndv and regulates viral rna synthesis and transcription we previously reported that m/nls mutation disrupted the nuclear localization of m protein and attenuated the replication efficiency and plaque formation ability of ndv in chicken fibroblasts [10] . to learn about the effect of m/nls mutation on the dynamic changes of m's intracellular localization in more detail, we compared the subcellular localization of m protein in rss1gfp-and rss1gfp-m/nlsm-infected bsr-t7/5 cells at different time points. as shown in figure 1 (a), at early time points, the m protein of rss1gfp was primarily concentrated in the nucleolus with a discrete punctuate staining pattern at 6 hour post-infection (hpi), and then was observed in the largest concentration in the nucleus and nucleolus at 12 hpi. while at later time points, the rss1gfp m protein was distributed diffusely in the cytoplasm, with some still localized in the nucleolus at 18 and 24 hpi. by contrast, most of the rss1gfp-m/nlsm m protein accumulated around the nucleus at 6 and 12 hpi, and then localized exclusively in the cytoplasm at 18 and 24 hpi (figure 1(a) ). interestingly, the appearance of large inclusion bodies induced by the membrane fusion of rss1gfp-infected cells was clearly observed through the fluorescence of gfp and dapi at 24 hpi (figure 1(a) ). the replication ability and cytopathogenicity of rss1gfp and rss1gfp-m/nlsm in bsr-t7/5 cells were then evaluated. the results of multicycle growth kinetics showed that the virus titers of rss1gfp-m/nlsm were remarkably lower than that of rss1gfp from 12 to 48 hpi (p < 0.001) (figure 1(b) ). in addition, the cytopathic effect (cpe) in rss1gfp-infected cells started at 12 hpi and cell monolayer began to be destroyed at 24 hpi, but the slight cpe in rss1gfp-m/nlsm-infected cells started to appear and cell monolayer destruction was not examined at 24 hpi (figure 1(c) ). moreover, the gfp fluorescence in rss1gfp-infected cells was also much brighter and more than that in rss1gfp-m/ nlsm-infected cells at the same time points (figure 1 (c)). these data suggested that m/nls mutation disrupted the nucleocytoplasmic trafficking of m protein and weakened the replication and cytopathogenicity of ndv. to investigate whether the attenuated replication and cytopathogenicity of rss1gfp-m/nlsm due to the reduced viral rna synthesis and transcription, the rna levels of np and p genes and the mrna levels of m and gfp genes in rss1gfp-and rss1gfp-m/ nlsm-infected cells were analyzed by quantitative realtime pcr (qrt-pcr). the results showed that the relative rna levels (corresponding to the np and p genes) between rss1gfp-and rss1gfp-m/nlsminfected cells had statistically significant differences at 6 hpi (p < 0.05), which continued to reduce in rss1gfp-m/nlsm-infected cells at 12 and 24 hpi (figure 1(d) ). similarly, the relative mrna levels of m and gfp genes in rss1gfp-m/nlsm-infected cells were more decreased than that in rss1gfp-infected cells at 6 and 12 hpi (p < 0.01), and significantly lower at 24 hpi (p < 0.001) (figure 1 (e)). consistent with the relative mrna expression levels of m and gfp genes, the expression levels of np, m and gfp proteins were also greatly reduced during the course of rss1gfp-m/nlsm infection when compared to rss1gfp infection (figure 1(f)). together, these results indicated that nucleocytoplasmic trafficking of m protein promoted the replication and cytopathogenicity of ndv possibly by affecting viral rna synthesis and transcription. rss1gfp and rss1gfp-m/nlsm exhibit great discrepancy in stimulating the cellular proteome to further investigate the mechanism underlying the attenuated replication of rss1gfp-m/nlsm, the global cellular protein expression profiles of bsr-t7/5 cells infected with these two viruses at 12 and 24 hpi was compared ( figure (2) ). after quality validation, a total of 530,600 (111,751 matched, accounted for 21.1%) spectra were obtained, of which 59,112 were identified peptides (56,897 unique peptides) and 5899 were identified proteins (5308 quantified proteins) ( table 1 ). the average peptides mass error was less than 10 ppm (figure 3 (a)), suggesting a high mass accuracy of the ms data. the length of most identified peptides mainly distributed from 7 to 20 amino acid residues (figure 3 (b)), which indicated that these samples met the required standard. the detailed information of identified proteins, including protein accession, protein description, gene name, coverage, peptide number, peptide-spectrum match, carried charges and so on, is shown in supplemental material table s1. in addition, significantly up-or downregulated proteins were determined by fold-change ratios >1.2 or <0.83 and p < 0.05. the results showed that 484 and 466 proteins from the rss1gfp group displayed significantly altered expression levels compared with the normal control at 12 and 24 hpi, respectively, including 306 up-regulated proteins and 178 down-regulated proteins at 12 hpi, and 190 up-regulated proteins and 276 down-regulated proteins at 24 hpi (figure 3 table s3 and s4). however, only seven deps were jointly shared by these two viruses at 12 and 24 hpi (figure 3 (d) and table 2 ). in addition, top 10 upregulated and down-regulated significantly deps induced by rss1gfp and rss1gfp-m/nlsm at 12 hpi and 24 hpi were listed in supplemental material table s5 and table s6 , respectively, showing that rss1gfp caused much higher ratio of up-regulated deps or much lower ratio of down-regulated deps than rss1gfp-m/nlsm. these results suggested that rss1gfp infection stimulated much greater quantities and more expression changes of deps than rss1gfp-m/nlsm infection. deps were then annotated to several gene ontology (go) analysis including cellular component, biological process and molecular function. go enrichment analysis showed that the most significantly enriched cellular components for rss1gfp group were the ribosome, extracellular region part and ribosomal subunit at 12 hpi, and the ribosome, intracellular ribonucleoprotein complex and ribonucleoprotein complex at 24 hpi; but the main enriched cellular components for rss1gfp-m/ nlsm group were the ribosome, ribonucleoprotein complex and intracellular ribonucleoprotein complex at 12 hpi, and the ribosome, non-membrane-bounded organelle and intracellular non-membrane-bounded organelle at 24 hpi (figure 4(a,b) ). as for the biological process enrichment, the peptide biosynthesis process, peptide metabolic process and amide biosynthesis process were commonly found in both rss1gfp group and rss1gfp-m/nlsm group at 12 and 24 hpi (figure 4(a, b) ). meanwhile, the structural constitute of ribosome and structural molecule activity were the enriched molecular functions commonly found in the two groups at 12 and 24 hpi (figure 4(a,b) ). to obtain more information about the biological pathways in which the deps may be involved, kegg pathway enrichment analysis was also compared. as shown in figure 4 (c,d), the ribosome pathway was the co-owned signaling pathway existed in rss1gfp group and rss1gfp-m/nlsm group at 12 and 24 hpi. but beyond that, the deps in rss1gfp group were also largely enriched in the glycolysis/gluconeogenesis, lysosome, glycosaminoglycan degradation and hif-1 signaling pathways 24 hpi (figure 4 (d)). it is remarkable that the deps were jointly enriched in the ribosome (cellular component), structural constitute of ribosome (molecular function) and ribosome pathway (kegg pathway) in rss1gfp group and rss1gfp-m/nlsm group at 12 and 24 hpi (figure 4 , indicated by arrow). therefore, the dynamic changes of ribosomerelated deps in bsr-t7/5 cells infected with rss1gfp and rss1gfp-m/nlsm at 12 and 24 hpi were compared. as shown in figure 5 (a), the hierarchical clustering heatmap of ribosome-related deps in rss1gfp group displayed more changes (most of deps were significantly down-regulated at 12 hpi and up-regulated at 24 hpi) than rss1gfp-m/nlsm group. in addition, the modeling of ribosome pathway showed that almost equivalent amount of up-regulated and down-regulated deps were present in rss1gfp group at 12 hpi, while a small number of down-regulated deps appeared in rss1gfp-m/nlsm group at 12 hpi ( figure 5(b) ). although the deps in both rss1gfp and rss1gfp-m/nlsm group exhibited up-regulated, the number of deps in rss1gfp group were much more than that in rss1gfp-m/nlsm group ( figure 5 (c)). clusters of orthologous groups (cog)/clusters of eukaryotic orthologous groups (kog) categories of the identified deps in rss1gfp and rss1gfp-m/ nlsm groups was further analyzed. the results showed that "general function prediction only" (group r, 58 deps), "posttranslational modification, protein turnover, chaperones" (group o, 51 deps), and "translation, ribosomal structure and biogenesis" (group j, 63 deps), "general function prediction only" (group r, 50 deps) represented the four largest groups in rss1gfp group at 12 and 24 hpi, respectively ( figure 6(a,b) ). however, "translation, ribosomal structure and biogenesis" (group j, 15 deps), "signal transduction mechanisms" (group t, 12 deps), and "translation, ribosomal structure and biogenesis" (group j, 16 deps), "signal transduction mechanisms" (group t, 9 deps) were the largest four groups in rss1gfp-m/nlsm group at 12 and 24 hpi, rss1gfp infection inhibits host cell transcription, rna processing and modification it has been reported that the m protein of several nonsegmented negative-sense rna viruses (nnsvs) including hrsv, vsv and mev has the ability to inhibit host cell transcription in various ways [7] [8] [9] . to determine whether the ndv m protein can inhibit cell transcription process, we mainly focused on the expression profiles of deps related to "transcription" and "rna processing and modification" according to the results of cog/kog categories analysis. a total of 53 and 39 deps associated with "transcription" and "rna processing and modification" were found in rss1gfp group, respectively, but 20 representative deps of each category were selected for further analysis. the results showed that the expression of most deps related to "transcription" and "rna processing and modification" was significantly decreased in rss1gfp group at 12 hpi, and was still slightly decreased at 24 hpi ( figure 7 (a,b)). on the contrary, the expression of many deps related to two processes showed up-regulated in rss1gfp-m/nlsm group at 12 hpi, and especially exhibited much higher up-regulation in "transcription" at 24 hpi (figure 7 (a,c)). to better understand how ndv interacts with the cellular proteins and affects cell functions, the deps were analyzed by searching the string database and the protein-protein interaction network. as shown in figure 7 (b), there was one group of proteins (brd2-tbp-mybbp1a-polr1e-abt1) participated in "transcription" process, which strongly interacted and was significantly regulated by rss1gfp infection. among them, brd2 [36] , tbp [37] , mybbp1a [38] , polr1e [39] , and abt1 [40] have been demonstrated to regulate cell transcription. in addition, two groups of proteins (pop1-nhp2-nop58-lsm6-txnl4a -nhp2l1, scaf4-ncbp2-snrpd1-sf3b4-cdc5 l -zcchc17-ddx39a) were found to interact strongly with each other and participated in "rna processing and modification" process ( figure 7 (d)). meanwhile, further investigation revealed that most of these deps in this interaction network are known to play important roles in rna processing and modification. to further validate the deps identified by tmtlabeled lc-ms/ms analysis, the expression of two deps in each category (eny2 and brd2 for "transcription", pop1 and swd2 for "rna processing and modification") was detected by qrt-pcr and western blotting, respectively. the results of qrt-pcr showed that the mrna expression levels of four selected genes in rss1gfp-infected cells were the lowest at 12 hpi, and were still obviously down-regulated compared with the rss1gfp-m/nlsm group at 24 hpi ( figure 7 (e)). in addition, the expression patterns analyzed by western blot analysis were consistent with the qrtthe host protein synthesis and processing machineries can be hijacked and usurped by viruses to synthesize, modify and transport viral proteins and also stifle host innate defense to facilitate viral propagation [41] [42] [43] . therefore, according to the results of cog/kog categories analysis, the expression profiles of deps related to "translation, ribosomal structure and biogenesis", "posttranslational modification, protein turnover, chaperones" and "intracellular trafficking, secretion, and vesicular transport" were analyzed. a total of 97, 88 and 56 deps associated with the three categories were found in rss1gfp group, respectively, but 20 representative deps of each category were selected for further analysis. we found that relatively lower expression profiles of deps associated with the three categories were observed in rss1gfp group compared to that of rss1gfp-m/nlsm group at 12 hpi (figure 8 (a,c,e)). however, the expression of deps related to "translation, ribosomal structure and biogenesis", "posttranslational modification, protein turnover, chaperones" and "intracellular trafficking, secretion, and vesicular transport" was significantly increased in rss1gfp group at 24 hpi, which was especially much higher in "translation, ribosomal structure and biogenesis" category ( figure 8(a,c,e) ). as for the rss1gfp-m/ nlsm group, the expression of most deps in "translation, ribosomal structure and biogenesis" and "posttranslational modification, protein turnover, chaperones" were decreased at 12 hpi, but then increased at 24 hpi (figure 8(a,c) ). by contrast, most of the deps in "intracellular trafficking, secretion, and vesicular transport" showed the expression from upregulated at 12 hpi to down-regulated at 24 hpi (figure 8(e) ). analysis of the protein-protein interaction networks indicated that complex and strong interactions existed in these ribosomal proteins, which contributed to "translation, ribosomal structure and biogenesis" process ( figure 8(b) ). in addition, two groups of proteins including (bag3-dnajb6-hsp90aa1-hspa8, and psmd10 and (vamp8-stx5-trappc3-tmed2, and nup153-kpna2-ran-ipo13) were found to interact strongly with each other and participated in "posttranslational modification, protein turnover, chaperones" and "intracellular trafficking, secretion, and vesicular transport" processes, respectively ( figure 8(d,f) ). moreover, literature search revealed that most of these deps in the three interaction networks play crucial roles in these biological processes. next, the expression of two deps in each category (rpl18 and rpl34, psmd3 and ubr5, rab12 and stx5) was confirmed by qrt-pcr and western blotting, respectively. the results of mrna expression of dep genes detected by qrt-pcr and expression of deps detected by western blotting remained basically consistent (figure 8(g,h) ), which were also consistent with the tmt-labeled lc-ms/ms analysis. thus, these data suggested that the nuclear aggregation of ndv m protein might participate in interfering with cellular protein synthesis, posttranslational modification and trafficking, but this kind of inhibition function could be weakened when most of the m protein was confined outside the nucleus. it is worth noting that traf-interacting protein with fha domain-containing protein a (tifa) was one of the jointly owned deps between rss1gfp group and rss1gfp-m/nlsm group at 12 and 24 hpi (table 2) . however, according to the tmt-labeled lc-ms/ms analysis, tifa was the highest and the lowest expressed dep in the two groups at 12 and 24 hpi, respectively ( table 2) . multiple research groups have demonstrated that the interaction between phosphorylated thr9 of tifa and tumor necrosis factor receptor-associated factor 6 (traf6) is the key mechanism for tifamediated nuclear factor-κb (nf-κb) activation [44] [45] [46] , which transactivates many cytokines and inflammation-associated transcriptional factors in response to immune response and inflammatory challenges [47, 48] . these results indicated that tifa/traf6/nf-κb signaling pathway might play crucial roles in the antiviral immune response. therefore, we further gained insight into the role of tifa/traf6/nf-κb in ndv replication. the mrna expression and protein expression of tira in rss1gfp-and rss1gfp-m/nlsm-infected cells were first analyzed by qrt-pcr and western blotting, respectively. as shown in figure 9(a,b) , the quantitative results trends of tifa in mrna and protein expression patterns were consistent with the tmtlabeled lc-ms/ms findings. to investigate whether the m protein alone and the subcellular localization of m protein can affect the expression of tifa, we cotransfected the plasmids pcmv-ha-tifa and pegfp-m or pegfp-m/nlsm into bsr-t7/5 cells, respectively. the fluorescent co-localization results showed that egfp-m mainly localized in the nucleus and nucleolus, and ha-tifa was present in the cytoplasm; whereas egfp-m/nlsm exhibited the clear co-localization with ha-tifa in the cytoplasm (figure 9(c) ). in addition, transfection of 0.5 μg pegfp-m or pegfp-m/nlsm nearly had no effect on the expression of endogenous tifa, but when the transfection dose of pegfp-m/ nlsm increased, the expression of tifa was obviously decreased in comparison to the pegfp-m transfection group (figure 9(d) ). overall, these results suggested that the m protein in the cytoplasm was able to reduce the expression of tifa in a dose-dependent manner. the expression changes of tifa, phosphorylated tifa (ptifa), traf6, nf-κb p65, phosphorylated nf-κb p65 (pnf-κb p65) and its transactivated downstream protein il-2 were then analyzed in rss1gfp-and rss1gfp-m/nlsm-infected cells. we found that the expression patterns of these proteins were increased during the course of rss1gfp and rss1gfp-m/nlsm infection at 12 hpi, but rss1gfp infection caused much higher increase than rss1gfp-m/nlsm infection ( figure 9 (e)). however, in contrast to the up-regulated expression of these proteins in rss1gfp-m/nlsm infection at 24 hpi, rss1gfp infection remarkably caused the decreased expression of these proteins (figure 9 (e)). in addition, plasmid pcmv-ha-tifa was transfected into bsr-t7/5 cells to perform the overexpression of ha-tifa, but the overexpressed ha-tifa greatly increased the expression of il-2 and reduced the virus titers of rss1gfp and rss1gfp-m/nlsm in virus-infected cells at 12 and 24 hpi (figure 9(f)). moreover, small interfering rna (sirna)-mediated knockdown of tifa (tifa sirna#1) in bsr-t7/5 cells obviously reduced the expression of il-2 and increased the replication efficiency of rss1gfp and rss1gfp-m/nlsm at 12 and 24 hpi (figure 9(g) ). together with the above the results, these data demonstrated that the cytoplasmic localization of m protein was able to reduce the expression of tifa and facilitate ndv replication by down-regulating the tifa/traf6/nf-κb-mediated production of cytokines (figure 9(h) ). viruses have co-evolved with their hosts for many years and developed effective approaches for hijacking and manipulating host cellular processes [41] . one of the important processes is that viruses take advantage of specific localization of viral proteins, which dynamically and temporally regulates their interactions with host proteins to ensure virus replication and proliferation [13, 49] . in recent years, increasing numbers of studies have reported that paramyxoviruses m proteins interact with cellular proteins at special orientation to achieve virus replication. for example, the ndv m protein localizes to the nucleolus through an interaction with b23, and knockdown of b23 results in the reduced cpe and virus replication [50] . the m protein of niv and hev transiently interacts with the beta subunit of the ap-3 adapter protein complex ap3b1 at the plasma membrane, which is effective for promoting viral particle assembly [51] . in addition, a recent study has demonstrated that annexin a2 mediates the localization of mev m protein at the plasma membrane by interacting with its n-terminal region, thereby aiding in mev assembly [52] . up to now, the presence of intrinsic nls and nes within m protein for its nucleocytoplasmic trafficking have been reported in most of the paramyxoviruses [53] . we previously showed that the replication and pathogenicity of ndv is significantly attenuated by m/nls mutation [10] and the recombinant ndv carrying mutated m/ness cannot be rescued [11] . in this study, we also found that the disruption of m's nucleocytoplasmic trafficking by mutating m/nls remarkably reduced the proliferative ability and cytopathogenicity of ndv. all of these findings suggested that the replication and pathogenicity of ndv was tightly associated with the viral rna synthesis, transcription and translation are the key steps in the process of nnsvs replication [2, 54] . although the m protein of nnsvs is known to play key roles in virus assembly later in infection [1, 12] , based on the findings from hrsv, vsv and sev, the m protein also modulates viral rna synthesis and can inhibit the transcriptase activity through m-np interaction early in infection, thereby repressing the signal to switch from transcription to packaging into the virion particles [12, 55, 56] . the relevant evidence is that the sev m protein is cross-linked to the np protein in generated progeny virions [57] , and the addition of m protein to sev and vsv nucleocapsids decreases their ability to transcribe viral rna [55, 58] . in addition, rna interference with the m gene efficiently increases viral transcription levels in mev-infected cells [59] , and in minigenome reporter gene assays, the m protein of mev inhibits viral rna synthesis only when it interacts with the np protein [60] . the ndv m protein is reported to be necessary and sufficient for virus budding, and the m-hn and m-np interactions are responsible for the incorporation of hn and np proteins into virion particles [5] . interestingly, we similarly found that viral rna synthesis and transcription efficiency were greatly decreased by m/nls mutation in rss1gfp-m/nlsm-infected cells, suggesting that precocious cytoplasmic localization of m protein had negative effects on viral rna synthesis and transcription. because the nnsvs m proteins can avoid the inhibition of viral transcriptase activity early in infection and mediate the association of the nucleocapsid with the nascent viral envelope later in infection, thus we concluded that the dominant nuclear accumulation of ndv m protein might ensure that viral rna synthesis and transcription in the cytoplasm proceeded smoothly until a certain level of viral rna and protein expression was synthesized, at which point the m protein entered the cytoplasm and plasma membrane to achieve virus assembly and budding. therefore, the reduced viral rna synthesis and transcription caused by m/nls mutation might be one of the reasons responsible for the attenuated replication of rss1gfp-m/nlsm. virus-host protein interactions based on quantitative proteomics analysis have become important methods in understanding cellular proteins involved in virus replication and pathogenesis [22, 23, 41, 42] . in this study, the quantitative tmt and lc-ms/ms approach were applied to compare the proteome of bsr-t7/5 cells in response to rss1gfp and rss1gfp-m/nlsm infection. we found that the two viruses showed great discrepancy in terms of stimulating host protein expression profiles (484 vs 109 deps and 466 vs 104 deps between rss1gfp and rss1gfp-m/nlsm at 12 and 24 hpi, respectively), and only seven deps were shared by these two viruses. go enrichment analysis of the deps showed that most of the deps were functionally related to the structural constituent of ribosome and structural molecule activity in rss1gfp group and rss1gfp-m/nlsm group, but rss1gfp infection caused more changes in go enrichment. the kegg pathway analysis further determined that these deps were mainly involved in important cellular pathways including ribosome, glycolysis/gluconeogenesis, lysosome signaling pathways in rss1gfp group. in addition, further cog/kog categories analysis revealed that rss1gfp virus highly activated more deps to participate in "translation, ribosomal structure and biogenesis", "signal transduction mechanisms", "posttranslational modification, protein turnover, chaperones", "intracellular trafficking, secretion, and vesicular transport", "transcription", "rna processing and modification", "cytoskeleton" and so on. therefore, these results suggested that rss1gfp infection could cause much more expression changes of deps, which were involved in numerous functional categories and kegg pathways, to assist virus replication in comparison to rss1gfp-m/nlsm infection. numerous studies have demonstrated that m's nuclear localization of nnsvs such as hrsv [7] , mev [9] , and vsv [61, 62] has the capability to inhibit host cell transcription independently of other viral components. supporting this conclusion is the fact that nuclear extracts from hrsv-infected cells have less transcriptional activity in vitro and also inhibit the transcriptional activity of nuclear extracts from mock-infected cells [7] . in addition, a recent study revealed that transient expression of mev m protein in plasmid-transfected cells binds to nuclear factors and is able to inhibit in vitro transcription in a dosedependent manner [9] . additionally, studies focusing on vsv m protein showed that the m protein directly inhibits host cell transcription by inactivating host rna polymerases ⅰ and ⅱ [62] , and also interacts with nuclear pore complexes to impair nuclear export of cellular mrnas [63] , thereby indirectly leading to a decrease and an increase in host cell and virus transcription [64, 65] . more recently, using microarray analysis of rss1gfp-infected chicken embryo fibroblasts, we found that nuclear localization of ndv m protein might inhibit host cell transcription, showing that the transcription repressor activity-and negative regulation of transcription-related genes were up-regulated, while the rna polymerase ⅱ transcription factor activityand transcriptional activator activity-related genes were down-regulated [65] . in addition, sirna-mediated knockdown of the representative upregulated gene or down-regulated gene significantly reduced or increased viral rna synthesis and virus replication, respectively [66] . consistent with this finding, the quantitative proteomics analysis also revealed that most of these deps, which had important functions in the process of "transcription" and "rna processing and modification" in rss1gfp infection, exhibited obviously down-regulated expression patterns. meanwhile, it is notable that in addition to the transcription activation function, both eny2 and thoc2 are also demonstrated to be involved in cellular mrna nuclear export [67, 68] . however, whether the ndv m protein can interact with eny2 or thoc2 to impair nuclear export of cellular mrnas remains to be explored. remarkably, several deps associated with rna transport, rna degradation, rna polymerase, basal transcription factors, spliceosome, mrna surveillance pathways in rss1gfp group showed much more and lower expression levels than that in rss1gfp-m/ nlsm group at 12 and 24 hpi (supplemental material table s7 ). based on these findings, we speculated that the inhibition of host cell transcription caused by the dominant nuclear accumulation of ndv m protein might occur through more diverse pathways than that of other nnsvs. together, these results indicated that the weakened inhibition of host cell transcription due to m/nls mutation possibly affected viral transcription and replication, which might be another reason responsible for the attenuated replication of rss1gfp-m/ nlsm. ribosomal proteins (rps) are the major components of ribosomes involved in the cellular process of protein biosynthesis. nowadays, increasing evidence has demonstrated that multifarious rps interact with viral proteins to participate in viral protein biosynthesis and regulate virus replication in host cells [69] . for example, 60s ribosomal protein l18 (rpl18) is found to be incorporated into ebola virions, and the reduced expression of rpl18 effectively represses ebola virus infection [70] . rice stripe tenuivirus nucleocapsid protein interacts with rpl18 of insect vector and silencing of rpl18 obviously reduces viral translation and replication [71] . in addition, rna interference of ribosomal protein rpl34 causes serious damages to abortive infection of autographa californica multiple nucleopolyhedrovirus, indicating that ribosomal components are essential for productive baculovirus infection [72] . meanwhile, recently relevant research results also suggested that many other rps including rpl24, rpl19, rpl4, rps6 and so on play critical roles in the life cycle of viruses. in our studies, we found that most of the rps showed significantly down-regulated in rss1gfp group at 12 hpi, but exhibited remarkably up-regulated at 24 hpi when compared to rss1gfp-m/nlsm group. this kind of changing trends of rps was also clearly observed in the hierarchical clustering heat map of ribosome-related deps and the modeling of ribosome signaling pathway in the two viruses-infected cells at 12 and 24 hpi. a previous study has demonstrated that ndv is the most effective paramyxovirus at inhibiting the production of host proteins [73] . one possible explanation for this was due to the inhibition of host cell transcripts and the drastic reduction of rps early in ndv infection. it is noteworthy that rpl18 has been reported to interact with multiple viral proteins and participate in viral protein biosynthesis [69] . here, rpl18 was the highest up-regulated dep during rss1gfp infection at 24 hpi (rss1gfp, 4.169-fold upregulation; rss1gfp-m/nlsm, 1.286-fold upregulation), and the viral proteins (such as np and m) were increasingly expressed in rss1gfp group at 24 hpi, indicating that rpl18 played crucial roles in promoting viral protein biosynthesis. however, further studies are necessary to investigate whether ndv m protein interacts with rpl18 and how this interaction regulates ndv viral protein biosynthesis. overall, these results suggested that the dominant nuclear aggregation of m protein was conducive to inhibit the expression of cellular rps, and viral protein synthesis could be enhanced when most of the m protein entered the cytoplasm. currently, accumulating studies have indicated that viruses employ various strategies to hijack and usurp host cellular machinery for their own benefit [41, 42, 74] . the representative studies revealed that many identified abundant proteins in iav-infected cells are associated with protein synthesis, chaperone-mediated responses, protein metabolism and posttranslational modification, including protein folding, proteolysis, and the ubiquitinproteasome system [75] [76] [77] . in this study, according to "posttranslational modification, protein turnover, chaperones" analysis in rss1gfp group, we found that in addition to the few deps related to proteasome and chaperones, most deps belonged to ubiquitin-protein ligases such as herc4, ubfd1, trim26, trim31, ubr5, ubxn1 and so on. importantly, these deps showed obviously down-regulated at 12 hpi, but exhibited remarkably up-regulated at 24 hpi during rss1gfp infection. studies have shown that angiomotin-like 1 is a piv5 m-interacting protein and serves as a linker between paramyxovirus budding and the escrt pathway [78] . the subsequent study found that angiomotin-like 1 links piv5 and muv m proteins to nedd4 family ubiquitin ligases, which represents a novel host factor recruitment strategy for paramyxoviruses to achieve viral particle release [79] . in addition, another study also demonstrated that ubiquitin-regulated nucleocytoplasmic trafficking of the niv m protein is associated with m's post-translational modification and plays important roles in m-mediated viral budding [21] . these results suggested that ubiquitin ligases might participate in the nucleocytoplasmic trafficking of m protein and the assembly and budding of ndv progeny virions, but whether ndv m protein interacts with some of these ubiquitin ligasesrelated deps and the exact role of their interaction in ndv budding are worth to be deeply studied. interestingly, rss1gfp infection also changed the expression patterns of deps involved in "intracellular trafficking, secretion, and vesicular transport". the possible explanation for these changing trends might be that some ndv proteins such as np, p, f, and hn interfered with the expression of deps in endoplasmic reticulum during the nuclear localization of m protein [80, 81] , but the interference effect was weakened when the m protein entered the cytoplasm to interact with these viral proteins. it is remarkable that many enveloped viruses including ndv encode late domain motifs that are able to hijack vps4a and/or vps4b to complete viral budding [11, 82] . compared to the expression level of vps4a in rss1gfp-m/nlsm group, vps4a showed highly up-regulated expression pattern in rss1gfp group at 24 hpi, suggesting that rss1gfp possessed much stronger budding ability than rss1gfp-m/nlsm. meanwhile, some deps such as stx5, kpna2, ipo13, ran, nup153, which showed upregulated expression in rss1gfp group, are associated with intracellular trafficking and vesicular transport and have been reported to regulate virus replication. therefore, together with the above results, we speculated that the relatively decreased expression of deps involved in ribosome structure, protein posttranslational modification and trafficking due to the disrupted nuclear accumulation of m protein affected viral protein synthesis and budding, which might be the third reason responsible for the attenuated replication of rss1gfp-m/nlsm. inflammatory responses are important aspects of the innate immune system during virus infection. in previous studies, several signaling pathways, such as irak1/traf6/nf-κb, tlr4/traf6/nf-κb, sirt1/ ampk/nf-κb, vegfa/erk1/2/nf-κb and so on, have been reported to participate in inflammatory responses. recently, alpha-kinase 1 (alpk1) controlling tifa/traf6-dependent innate immunity against bacterial infection is gradually reported [48, [83] [84] [85] . it has been demonstrated that tifa serves as a traf6 binding protein and plays a major role in the activation of nf-κb [44, 46, 48] , which transactivates various of cytokines, chemokines, and inflammation-associated transcriptional factors in response to immune response and inflammatory challenges. however, the role of tifa/traf6/nf-κb signaling pathway in the replication of viruses remains unknown. in our studies, we found that the protein expression of tifa in rss1gfp group was high up-regulation at 12 hpi, but showed very low down-regulation at 24 hpi when compared to that in rss1gfp-m/nlsm group. the quantitative results of tifa in mrna and protein expression patterns detected by qrt-pcr and western blotting were consistent with the tmt-labeled lc-ms/ms analysis. in addition, we also found that the m protein in the cytoplasm effected the inhibition of tifa expression in a dose-dependent manner. moreover, the expression of tifa, ptifa, traf6, nf-κb, pnf-κb and il-2 was increased at 12 hpi, but decreased at 24 hpi in rss1gfp-infected cells. furthermore, overexpression of tifa or sirna-mediated knockdown of tifa obtained the different results, which showed the obviously increased or decreased expression of il-2 and the remarkably raised or reduced virus titers in rss1gfp-and rss1gfp-m/nlsm-infected cells. it is reported that virulent ndv infection induces transcriptional up-regulation of numerous cytokines, such as ifn-α, ifn-γ, il-8, il-2, and il-1β [86, 87] . however, overexpression of il-2 or il-1β can lead to the decreased systemic viral load and pathogenicity of virulent ndv [88, 89] . thus, these results indicated that the inhibition of tifa expression could reduce nf-κb-mediated production of cytokines by downregulating tifa/traf6/nf-κb signaling pathway, which was beneficial to ndv replication. while the precocious cytoplasmic localization of m protein could not down-regulate tifa/traf6/nf-κb signaling pathway, which might be the fourth reason responsible for the attenuated replication of rss1gfp-m/nlsm. in summary, we demonstrated that nucleocytoplasmic trafficking of m protein played crucial roles in regulating viral rna synthesis and transcription, and participating in the hijack and despoil of host cellular machinery for ndv replication ( figure 10 ). however, m/nls mutation disrupted the nucleocytoplasmic trafficking of m protein and affected these important biological processes, which in turn caused the attenuated replication of ndv. our findings therefore reveal for the first time that the replication of ndv is closely associated with the nucleocytoplasmic trafficking of m protein, which provides a better understanding of the potential functions of m's nucleocytoplasmic trafficking in ndv life cycle and also aids in understanding the poorly understood molecular pathogenesis of ndv. bsr-t7/5 cells stably expressing the t7 phage rna polymerase were generated by buchholz et al. [90] , and were a kind gift from prof. xiufan liu (key laboratory of animal infectious diseases, yangzhou university, china). the parental ndv (rss1gfp) and the mutant ndv (rss1gfp-m/nlsm) carrying m/ nls mutation were generated in our previous study [10] , which were plaque purified three times in chicken embryonic fibroblasts and propagated once in specific pathogen-free (spf) embryonated chicken eggs. the rabbit polyclonal antibodies against ndv m protein and np protein was prepared in our laboratory. the rabbit polyclonal antibodies against eny2 (df12979), brd2 (df12857), rpl18 (df3700), rpl34 (df3708), psmd3 (df3645), rab12 (df12459), traf6 (af5376), nf-κb p65 (af5006), phospho-nf-κb p65 (ser536) (af2006), gapdh (af7021), gfp (t0006), ha (t0050) and il-2 (af5105) were purchased from affinity biosciences (usa). the rabbit polyclonal antibodies against pop1 (ab254978), swd2 (ab220240), ubr5 (ab70311), stx5 (ab217130), and rabbit monoclonal antibody against phospho-tifa (thr9) (ab214815) were purchased from abcam (usa). the rabbit polyclonal antibody against tifa (61358s) was purchased from cell signaling technology (usa). bsr-t7/5 cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs) and were cultured at 37°c under 5% co 2 . bsr-t7/5 cells grown in 6-well plates were infected with ndv strain rss1gfp or rss1gfp-m/ nlsm at a multiplicity of infection (moi) of 1. the cell culture supernatants were collected at different time points (6, 12, 24, 36, 48, 72 , and 96 hpi), and the virus titers were determined as 50% tissue culture infective dose (tcid 50 ) in bsr-t7/5 cells. in addition, the cpe and green fluorescence in virus-infected cells were observed under a inverted fluorescence microscope (nikon, japan) and the expression levels of np, m and gfp were detected by western blot at 6, 12 and 24 hpi, respectively. moreover, the brightness of green fluorescence in viruses-infected cells was quantitatively compared by image j software version 1.8.0. bsr-t7/5 cells cultured in 12-well plates were infected with ndv strain rss1gfp or rss1gfp-m/nlsm at an moi of 1 and prepared for indirect immunofluorescence analysis at 6, 12, 18 and 24 hpi, respectively. briefly, cells were collected at the stipulated times and rinsed thrice with phosphate-buffered saline (pbs), fixed with 4% paraformaldehyde for 20 min at room temperature, and then permeabilized with 0.25% triton x-100 in pbs for 5 min. cells were rinsed thrice with pbs and blocked with 10% fbs in pbs for 1 h, and then incubated with rabbit anti-m polyclonal antibody diluted in pbs containing 10% fbs for 1 h at 37°c. after three washes with pbs, the cells were incubated with cy3-labeled goat anti-rabbit igg (h + l) (beyotime biotechnology, china) for 1 h at 37°c. cells were rinsed thrice with pbs and then counterstained with dapi (sigma) to detect the nuclei. images were captured with a fluorescence microscope and processed with adobe photoshop cs5 software. quantification of viral rna synthesis and gene expression by qrt-pcr bsr-t7/5 cells grown in 6-well plates were infected with rss1gfp or rss1gfp-m/nlsm at an moi of 1. cells were collected at the indicated time points (6, 12, and 24 hpi), and total rna was extracted using trizol reagent (invitrogen) according to the manufacturer's instructions. the resulting rna samples (2 μg per sample) were reverse-transcribed as previously described [91] . quantification of viral rna synthesis (the rna levels of np and p genes) and gene expression (the mrna levels of m and gfp genes) by qrt-pcr was performed using previously reported methods [66, 92] . the relative gene expression levels were normalized to that of the gapdh gene. the threshold cycle 2 −δδct method was used to determine the fold change of gene expression levels. three biological replicates of two virus-infected groups and one mock group were prepared for tmt-based quantitative proteomics experiments ( figure 2) . briefly, bsr-t7/5 cells grown in 25-cm 2 flasks were infected with rss1gfp or rss1gfp-m/nlsm at an moi of 1. cell samples were collected at 12 and 24 hpi, respectively, and then sonicated three times on ice using a high intensity ultrasonic processor (scientz) in lysis buffer (8 m urea, 1% protease inhibitor cocktail). the remaining debris was removed by centrifugation at 13,000 g at 4°c for 10 min. the supernatant was collected and the protein concentration was determined using the bca kit (beyotime biotechnology, china) according to the manufacturer's instructions. for trypsin digestion, the protein solution was reduced with 5 mm dtt for 30 min at 56°c and alkylated with 11 mm iodoacetamide for 15 min at room temperature. the protein sample was then diluted by adding 100 mm teab to urea concentration less than 2 m. finally, trypsin was added at 1:50 (trypsin: protein) mass ratio for the first digestion overnight and 1:100 (trypsin: protein) mass ratio for a second 4 h-digestion. after trypsin digestion of protein samples, peptide was desalted by strata x c18 spe column (phenomenex) and vacuum-dried. peptide was reconstituted in 0.5 m teab and processed for 6-plex tmt kit according to the manufacturer's instructions. briefly, one unit of tmt reagent was thawed and reconstituted in acetonitrile (about 100 mg protein). 41 μl of tmt label reagent was carefully added to each 100 μl sample, and eighteen samples were differentially labeled with six tmt tags (control group: 126 label for 12 h and 129 label for 24 h; rss1gfp group: 127 label for 12 h and 130 label for 24 h; rss1gfp-m/nlsm group: 128 label for 12 h and 131 label for 24 h). the peptide mixtures were then incubated for 2 h at room temperature. then reactions were treated with 8 μl 5% hydroxylamine for 15 min to stop trypsin. finally, the six labeled peptides were combined in a new microcentrifuge tube and 1 ml mixed peptides were dried by vacuum concentrator. the labeled peptides were fractionated into 60 fractions by high ph reverse-phase high-performance liquid chromatography (hplc) using agilent 300extend c-18 column (5 μm particle size, 4.6 mm id, 250 mm length) with a gradient of 8% to 32% acetonitrile (ph 9.0) over 60 min. then, the 60 fractions were combined into 18 fractions and each fraction (volume of 800 μl) was dried by vacuum centrifuging pending for ms analysis. the lc-ms/ms analysis of the labeled peptides was performed as described previously [93] . the related technical support and data analysis were supported by jingjie ptm biolabs (hangzhou, china). the resulting ms/ms data were processed using maxquant with an integrated andromeda search engine (v.1.5.2.8). tandem mass spectra were searched against the uniprot cricetulus griseus database (cricetulus_griseus_uniprot_10029, 23885 sequences) concatenated with reverse decoy database. common contamination database was added to eliminate the influence of contaminated proteins in the identified proteins. trypsin/p was specified as the cleavage enzyme allowing up to 2 missing cleavages. the mass tolerance for precursor ions was set as 20 ppm in first search and 5 ppm in main search, and the mass tolerance for fragment ions was set as 0.02 da. carbamidomethyl on cys was specified as fixed modification and acetylation modification and oxidation on met were specified as variable modifications. gene ontology (go) annotation was derived from the uniprot-goa database (http://www.ebi.ac.uk/goa/). biological processes, cellular components, molecular functions, and kegg pathways analysis of the deps were conducted using david (database for annotation, visualization, and integrated discovery) version 6.7. the protein-protein interactions were analyzed by string software version 11.0 (http:// string.embl.de/). for tmt quantification, the ratios of the tmt reporter ion intensities in the ms/ms spectra (m/z 126-131) from rawdatasets were used to calculate fold changes between samples. false discovery rate (fdr) was adjusted to <1% at protein, peptide and peptide-spectrum match level, and minimum score for peptides was set at >40. only peptides unique for a given protein were considered for relative quantification. for each sample, the quantification was normalized using the average ratio of all the unique peptides. the two-tailed fisher's exact test was employed to test the enrichment of the deps versus all identified proteins. for data analysis, p-values were adjusted for multiple hypotheses testing based on the benjamini-hochberg fdr method, and protein quantification data with p-value <0.05 and fold change of >1.2 or <0.83 and was considered as significantly up-regulated or down-regulated proteins. the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride [94] partner repository with the dataset identifier pxd018098. based on the proteomics results, qrt-pcr was used to analyze the expression of 11 selected dep genes. the primers (table 3) for qrt-pcr were designed based on the target sequences using primer premier 5.0 software. total rna was isolated from virus-infected cells or normal cells using the trizol reagent according to the manufacturer's protocol. one microgram of total rna per sample was reverse-transcribed into cdna using superscript ⅳ reverse transcriptase (thermofisher scientific, usa). the qrt-pcr was performed using sybr® premix ex taq tm ⅱkit (takara biomedical technology, china). all of the reactions were performed in a 25 μl volume containing 12.5 μl of 2× sybr® premix ex taq tm ⅱ, 400 nm of each primer, 1.0 μl rox reference dyeⅱ, and 2.0 μl cdna. the cycling parameters were 1 cycle at 95°c for 30 s followed by 40 cycles at 95°c for 5 s and 60°c for 34 s. one cycle of melting curve analysis was added for all reactions to verify product specificity. the relative gene expression levels were normalized to that of the gapdh gene. the threshold cycle 2 −δδct method was used to determine the fold change of gene expression levels. bsr-t7/5 cells grown in 6-well plates were infected with rss1gfp or rss1gfp-m/nlsm at an moi of 1. at 12 and 24 hpi, cells were washed thrice with pbs and then lysed with 1× ripa buffer (beyotime biotechnology, china) for total protein extraction. equivalent amounts of cell lysate (30 μg) were dealt with sds-page loading buffer, resolved by sds-page and then transferred electrophoretically onto polyvinylidene fluoride (pvdf) membrane. the membranes were blocked for 1 h at 37°c with 5% nonfat dry milk in tris-buffered saline with tween (tbst) buffer and then incubated overnight at 4°c with the primary antibodies against the target deps. the blots were washed thrice in tbst buffer and incubated for 1 h at 37°c with horseradish peroxidase-conjugated anti-mouse or rabbit igg(h + l). rabbit anti-gapdh polyclonal antibody was used as an internal standard. the relative levels of the gaggcgttctctttctccagtttct egw09203 brd2 gcgaaagctcggactctgaggaa tgggatagggcagccagttgttc egw09104 pop1 accatgatctgtgtcccatccg gtatcaggctcttaaatgggtcgc egw04063 swd2 ggaccatttgcaacctttaagatgc gccgttggtggaaatgagtatgagt egw05670 rpl18 gtcccggatgatccgaaaaatga cttcagcttgggcacttcgagaa egw03967 rpl34 atttacttgcggggatgctgctt ggtaaacaatcctgttgccaggg egw10449 psmd3 ccaggatgtggagatgaaagagga ccaaggtgacagtgtccagctctc egw15211 ubr5 gtttacttctggacatcaagcccgt ggtgttggtcgtctggtggtctta egw09566 rab12 aagtatgcttcggaagatgctgagc aaccgcatcccagttatctgctgt egw05159 stx5 gcagagccgtcagaatggaatccaa ttccaatgcgcttggccataag egw04472 tifa caggtttcccgagttcagtttgc ttgaggtagccaagctcctgattg egw12881 gapdh tcaagaaggtggtgaagcaggcat catcaaaggtggaagagtgggagtc caa36368 selected proteins to control gapdh expression were determined by densitometry using image j software version 1.8.0. to investigate the effect of tifa overexpression on the replication of ndv, the constructed plasmid pcmv-ha-tifa or empty vector control pcmv-ha was transfected into bsr-t7/5 cells using fugene® hd transfection reagent (promega, usa) according to the manufacturer's instructions. after 36 hours post-transfection (hpt), the expression of ha-tifa was detected by western blotting using rabbit anti-tifa or anti-ha polyclonal antibody, respectively. bsr-t7/5 cells were infected with rss1gfp or rss1gfp-m/nlsm at an moi of 1 at 36 hpt, and the virus titers were examined at 12 and 24 hpi, respectively. three pairs of sirna (supplemental material table s8) were designed to knockdown tifa in bsr-t7/5 cells. for transfection with the sirna against tifa, bsr-t7/5 cells were transfected with 25 pmol tifa sirna using 7.5 μl lipofectamine® rnaimax (thermofisher scientific, usa) in opti-mem medium. the knockdown efficiency was measured by detecting endogenous protein expression by western blotting analysis after 48 hpt. to study the effect of tifa on the replication of ndv, rss1gfp or rss1gfp-m/nlsm was used to infect tifa sirna-or negative sirna-treated bsr-t7/5 cells at an moi of 1. the detection of virus titers was performed at 12 and 24 hpi, respectively. differences in the expression level of genes, proteins and virus titers between with rss1gfp-and rss1gfp-m/nlsm-infected cells were analyzed using spss 12.0 software. the independent-samples t test was used for data analysis. all experiments were repeated at least three times and the results were shown as the mean ± standard deviation (sd). a p-value of < 0.05 was considered significant. p-values are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). no potential conflicts of interest were disclosed. paramyxovirus assembly and budding: building particles that transmit infections paramyxovirus rna synthesis, mrna editing, and genome hexamer phase: a review w protein expression by newcastle disease virus the matrix protein of newcastle disease virus localizes to the nucleus via a bipartite nuclear localization signal requirements for the assembly and release of newcastle disease virus-like particles nuclear entry and nucleolar localization of the newcastle disease virus (ndv) matrix protein occur early in infection and do not require other ndv proteins the matrix protein of human respiratory syncytial virus localises to the nucleus of infected cells and inhibits transcription complexes of vesicular stomatitis virus matrix protein with host rae1 and nup98 involved in inhibition of host transcription measles virus matrix protein inhibits host cell transcription importin α5 negatively regulates importin β1-mediated nuclear import of newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts characterization of signal sequences determining the nuclear export of newcastle disease virus matrix protein central role of the respiratory syncytial virus matrix protein in infection nucleocytoplasmic transport of nucleocapsid proteins of enveloped rna viruses nucleocytoplasmic trafficking of dengue non-structural protein 5 as a target for 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quantitative mass spectrometry in proteomics: critical review update from 2007 to the present employing tmt quantification in a shotgun-ms platform tmt labeling for the masses: a robust and cost-efficient, in-solution labeling approach temporal proteomic analysis of hiv infection reveals remodelling of the host phosphoproteome by lentiviral vif variants proteomic profile associated with loss of spontaneous human immunodeficiency virus type 1 elite control comprehensive profiling of lysine acetylome in baculovirus infected silkworm (bombyx mori) cells changes in salivary analytes in canine parvovirus: a high-resolution quantitative proteomic study proteomic analysis of serum proteins from hiv/aids patients with talaromyces marneffei infection by tmt labeling-based quantitative proteomics identifying the cellular interactome of epstein-barr virus lytic regulator zta reveals cellular targets contributing to viral replication proteomic analysis of chicken peripheral blood mononuclear cells after infection by newcastle disease virus comparative proteome analysis of tracheal tissues in response to infectious bronchitis coronavirus, newcastle disease virus, and avian influenza virus h9 subtype virus infection integrated proteomic and transcriptomic analysis of differential expression of chicken lung tissue in response to ndv infection during heat stress quantitative proteomics identify an association between extracellular matrix degradation and immunopathology of genotype vii newcastle disease virus in the spleen in chickens the bet protein brd2 cooperates with ctcf to enforce transcriptional and architectural boundaries transcription initiation factor tbp: old friend new questions ribosomal stress induces processing of mybbp1a and its translocation from the nucleolus to the nucleoplasm rna polymerase i-specific subunits promote polymerase clustering to enhance the rrna gene transcription cycle mouse dfa is a repressor of tata-box promoters and interacts with the abt1 activator of basal transcription proteomic approaches to uncovering virus-host protein interactions during the progression of viral infection virus-host interactome: putting the accent on how it changes autophagy enhances the replication of peste des petits ruminants virus and inhibits caspase-dependent apoptosis in vitro identification of tifa as an adapter protein that links tumor necrosis factor receptor-associated factor 6 (traf6) to interleukin-1 (il-1) receptor-associated kinase-1 irak-1) in il-1 receptor signaling intermolecular binding between tifa-fha and tifa-pt mediates tumor necrosis factor alpha stimulation and nf-κb activation binding and enhanced binding between key immunity proteins traf6 and tifa tifa as a crucial mediator for nlrp3 inflammasome alpk1: innate attraction to the sweetness of bacteria the importance of the nucleus during flavivirus replication the nucleolar phosphoprotein b23 targets newcastle disease virus matrix protein to the nucleoli and facilitates viral replication matrix proteins of nipah and hendra viruses interact with beta subunits of ap-3 complexes annexin a2 mediates the localization of measles virus matrix protein at the plasma membrane evidence for ubiquitin-regulated nuclear and subnuclear trafficking among paramyxovirinae matrix proteins the rna synthesis machinery of negative-stranded rna viruses sendai virion transcriptase complex: polyeptide composition and inhibition by virion envelope proteins the matrix (m) protein of vesicular stomatitis virus regulates transcription protein-protein interactions within paramyxoviruses identified by native disulfide bonding or reversible chemical cross-linking role of the membrane (m) protein in endogenous inhibition of in vitro transcription by vesicular stomatitis virus rna interference with measles virus n, p, and l mrnas efficiently prevents and with matrix protein mrna enhances viral transcription the matrix protein of measles virus regulates viral rna synthesis and assembly by interacting with the nucleocapsid protein identification of a consensus mutation in m protein of vesicular stomatitis virus from persistently infected cells that affects inhibition of host-directed gene expression effect of vesicular stomatitis virus matrix protein on transcription directed by host rna polymerases i, ii, and iii vsv disrupts the rae1/mrnp41 mrna nuclear export pathway vesicular stomatitis virus matrix protein inhibits host cell gene expression by targeting the nucleoporin nup98 the matrix protein of vesicular stomatitis virus inhibits host-directed transcription of target genes via interaction with the tfiih subunit p8 nuclear localization of newcastle disease virus matrix protein promotes virus replication by affecting viral rna synthesis and transcription and inhibiting host cell transcription multifunctional factor eny2 is associated with the tho complex and promotes its recruitment onto nascent mrna thoc2 mutations implicate mrna-export pathway in x-linked 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protein interaction with host protein angiomotin-like 1 angiomotin-like 1 links paramyxovirus m proteins to nedd4 family ubiquitin ligases newcastle disease virus np and p proteins induce autophagy via the endoplasmic reticulum stress-related unfolded protein response hemagglutininneuraminidase and fusion proteins of virulent newcastle disease virus cooperatively disturb fusion-fission homeostasis to enhance mitochondrial function by activating the unfolded protein response of endoplasmic reticulum and mitochondrial stress mechanisms for enveloped virus budding: can some viruses do without an escrt? alpk1 controls tifa/traf6-dependent innate immunity against heptose-1,7-bisphosphate of gram-negative bacteria innate recognition of intracellular bacterial growth is driven by the tifa-dependent cytosolic surveillance pathway role of nod1 and alpk1/tifa signalling in innate immunity against helicobacter pylori infection virulent newcastle disease virus elicits a strong innate immune response in chickens immune responses of poultry to newcastle disease virus expression of chicken interleukin-2 by a highly virulent strain of newcastle disease virus leads to decreased systemic viral load but does not significantly affect mortality in chickens expression of interferon gamma by a highly virulent strain of newcastle disease virus decreases its pathogenicity in chickens generation of bovine respiratory syncytial virus (brsv) from cdna: brsv ns2 is not essential for virus replication in tissue culture, and the human rsv leader region acts as a functional brsv genome promoter transcription and replication initiate at separate sites on the vesicular stomatitis virus genome the glutamic residue at position 402 in the c-terminus of newcastle disease virus nucleoprotein is critical for the virus dynamic tmt-based quantitative proteomics analysis of ccritical initiation process of totipotency during cotton somatic embryogenesis transdifferentiation the pride database and related tools and resources in 2019: improving support for quantification data key: cord-318853-mxyxwkhx authors: sallie, richard title: replicative homeostasis ii: influence of polymerase fidelity on rna virus quasispecies biology: implications for immune recognition, viral autoimmunity and other "virus receptor" diseases date: 2005-08-22 journal: virol j doi: 10.1186/1743-422x-2-70 sha: doc_id: 318853 cord_uid: mxyxwkhx much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. rna polymerases (rna(pol)) generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. thus, rna(pol )causes more morbidity and premature mortality than any other molecule. the extraordinary genetic heterogeneity defining viral quasispecies results from rna(pol )infidelity causing rapid cumulative genomic rna mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking rnapol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral rna is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. this mechanism – "viral receptor disease (vrd)" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type ii diabetes mellitus, and some forms of obesity. viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations. many of the world's population suffer from acute and chronic viral infection. the two common types of chronic viral hepatitis (cvh), hepatitis b (hbv) and c (hcv) are major causes of death and morbidity; conservative esti-mates suggest 400 million people are persistently infected with hbv, while hcv may infect a further 200 million. annually, in excess of two million people will die from cirrhosis or liver cancer caused by cvh, and many more suffer chronic ill health as result. during the 20 years since the human immunodeficiency virus (hiv) was identified, perhaps 40 million people have become infected worldwide and each year about a million die from resulting immunodeficiency and consequent opportunistic infections, particularly tuberculosis, and other complications. poor countries bear a disproportionate burden of disease caused by these viruses that further exacerbate poverty through pervasive economic disruption and diversion of limited resources to healthcare and disease control. emerging viral pathogens including west nile virus (wnv), the sars coronavirus, endemic viruses like murray valley, japanese, and other encephalitis viruses, dengue and yellow fever, and seasonal influenza, hepatitis a (hav) and e (hev) cause enormous further morbidity and mortality, while pandemic outbreaks of virulent influenza strains remain a constant threat. together, these viruses probably kill more people every ten days than the boxing day tsunami. rna viral infections, including foot and mouth, bovine viral diarrhea virus (bvdv) and hog cholera virus (hchv), cause similar devastation of animal populations with enormous economic consequences. rna polymerases generate massive genetic variability of rna viruses and retroviruses that circulate within infected hosts as vast populations of closely related, but genetically distinct, molecules known as quasispecies. after translation, this genetic variability causes near-infinite antigenic heterogeneity, facilitating viral evasion of host defences. tuberculosis, malaria and other cellular pathogens also express broad cell-surface antigenic heterogeneity, generated by dna-dependent rna pol . thus, rna polymerases probably cause more morbidity and premature mortality in man, and other animals, and greater economic loss, than any other molecule. despite a depressing global epidemiology that strongly suggests otherwise, the immune system is thought to "control" viruses. what practical meaning does "immune control" have for the individual? there is no argument for hbv, and other viruses, high affinity antibody, generated by prior vaccination or other exposures and directed against neutralizing epitopes, will prevent hbv infection (excepting vaccine escape mutations [1, 2] ), in part by blocking viral ligand interaction with cell receptors, or that most patients exposed to hbv develop neutralizing antibodies (hbsab), clear hbsag from serum, and will normalize liver function long term. however, even patients who develop robust immune responses to hbv, defined by high-affinity antihbsab and specific antiviral cytotoxic t cell (ctl) responses, will have both "traces of hbv [3] ... many years after recovery from acute hepatitis" [3] and transcriptionally active hbv demonstrable in peripheral blood mononuclear cells (pbmcs) [4] . furthermore, occult hbv is detected in liver tissue of patients with isolated antihbc (i.e. hbsag/hbsab negative) [5] and in patients with hbsag-negative hepatocellular carcinoma [6] suggesting, at least some patients, hbv in may persist irrespective of any immune responses, implying long term latency and low level basal replication may be a survival/reproductive strategy for hbv. for most patients, acute hcv or hiv infection results in life-long viral persistence. although many patients develop immunological responses, including specific antibody and ctl reactivity to various viral antigens, these responses have little discernible impact on either hcv or hiv replication that occurs essentially unchecked at rates estimated between 10 10 and 10 12 virions per day [7, 8] , indefinitely, while progressive destruction of liver or immune cells proceeds, commonly resulting in cirrhosis or liver cancer (for hcv) or death from immune deficiency (for hiv). evidence that prior hcv infection confers no protective immunity against heterologous hcv infection in humans [9] or chimpanzees [10] or against either homotypic [11] or heterotypic [12] human reinfection, confirmation that active hcv infection persists long after either apparent spontaneous [13] or treatmentinduced [14] viral clearance, or that vaccines causing specific antiviral b and t cell responses fail to protect against infection in animals [15] , and that antibodies to hcv envelope protein e2 are only detected in animals with persistent infection [16, 17] , further undermines the potency of "immune control" and suggests, at least for patients with hcv, the definition of "control" may need to broadened significantly. based on observations that stronger specific cd4/cd8 immune responses with t-helper (th1) cytokine profiles are found more frequently in patients with self limiting viral infections than those who develop chronic viral carriage [18, 19] it is thought ability to mount robust adaptive immune responses predicts viral clearance while failure to do so results in chronic viral carriage [20] . however, detailed and very painstaking studies, albeit in small numbers of chimpanzees [21] and patients following antiviral therapy [22] , have failed to demonstrate any relationship between t cell responses and viral clearance. although development of th1and other immune responses are certainly temporally and, probably, causally related to reduced viral replication and viral clearance the assumed direction of causality (immune response -> reduced viral replication), is not proved by the fact those responses develop, post hoc ergo propter hoc, as comforting a conclusion as it may be to reach. the first part of this paper explores the impact of rna pol fidelity on quasispecies behaviour, specifically in mediating immune avoidance during acute hcv infection. we suggest the primary event causing reduction in viral replication is inhibition of rna pol processivity by variant viral proteins, specifically envelope and envelope-related proteins. we also suggest that immune responses to viruses are thwarted initially by broad antigenic diversity generated by low rna pol fidelity but develop, when they do, after viral replication falls (because of reduced rna pol processivity) and polymerase fidelity increases -linked events that occur because of replicative homeostasisthus restricting antigenic diversity sufficiently to permit focused immune recognition. we further suggest immune responses strategically exploit replicative homeostasis to force viruses to reveal critical dominant antigenic epitopes, facilitating progressively more focused immune responses. the second part explores the ineluctable consequence of viral rna quasispecies: that is, translation of rnas into protein quasispecies with a spectrum of phenotypes and unpredictable properties, among which may be disruption of the cell surface receptors that viruses co-opt for cell entry. this innate property of viral quasispecies may explain a wide variety of diseases apart from viral autoimmunity. acute hcv and hbv infection have characteristic kinetics of viral replication, adaptive immune responses, and cause predictable tissue injury, reflected in elevated serum aminotransferases. these kinetic and transaminase responses are summarized schematically for patients with persistent infection (figure 1) [23]. initial hcv replication is very rapid and viral load increases exponentially until about week 4, at which point viraemia increases more slowly, and asymptotically, towards ~10 7 genome equivalents (geq)/ml by weeks 7-8 (these kinetics alone suggesting competitive inhibition of rna pol ). this exponential increase of viral rna in serum reflects explosive dissemination of virus in tissues, detectable by in-situ hybridisation throughout hepatocytes, including the nuclei, within days of infection [24] . viral replication declines rapidly from weeks 10-11 to weeks 14-16 falling by 10 2-3 geq/ml but lower level (~10 5 geq/ml) fluctuating replication persists, generally indefinitely, thereafter. by contrast, neither hbv dna nor hbv antigens are detectable in either viral replication, immunological and tissue injury kinetics following acute hcv and hbv infection figure 1 viral replication, immunological and tissue injury kinetics following acute hcv and hbv infection. data summated from figure 1 [29] and modified to represent typical patients with chronic viral persistence. note: a) high level hcv replication for 6-8 weeks prior to any immune responses, b) onset of humoral immune response well after down-regulation of viral replication [34] , and c) transaminase peaks occurs ~ 2weeks later. both hbv and hcv are non-cytolytic and viral clearance from hepatocytes, as well as hepatocyte injury, thought to be immune mediated. however, for both hbv and hcv the brisk fall in viral replication following acute infection paradoxical hcv replication kinetics figure 2 paradoxical hcv replication kinetics. if host immune clearance forces (i c , black arrows) reduce viral replication acutely (point a), then they must exceed viral expansive forces (v e , grey arrows) at that point. at equilibrium (e.g. points b through d), viral concentrations (-) and, therefore, viral forces, have fallen by 10 2-3 hence, immune forces i c must fall by >10 2-3 from a to b for equilibrium to develop. there is no evidence this happens. http://www.virologyj.com/content/2/1/70 precedes the peak of transaminase rise by at least two weeks (figure 1). if falling viral replication is due to adaptive immune responses causing hepatocyte lysis the transaminase peak should either precede or be coincident with falling replication. this temporal relationship is also inconsistent with the belief immune factors cause the falling replication seen during acute hcv or hbv, and is analagous to non-cytolytic reductions of viral replication observed for both hbv and lymphocytic choriomeningitis virus (lcv) experimentally, that suggested either [unspecified] antiviral mechanisms are operative [30,31], or that auto-inhibition of rna pol by viral mechanisms (replicative homeostasis) occurs [28] . however, if other non-cytopathic host anti-viral mechanism(s) are responsible, the kinetic paradox implies their potency falls significantly between points a and b. hepatitis c replication kinetics and their relationship to immune responses are well documented [32,33] but reveal an unexplained paradox. despite high level viral replication, adaptive cellular immune responses to hcv are completely undetectable for at least 7-10 weeks [33] after infection, while humoral responses are rarely detected before 12-14 weeks [34] , and in some patients [35] , and some chimpanzees [36] , are never detected at all. an exhaustive and very careful review of the clinical and experimental data relating adaptive immune response and hcv replication kinetics has been published recently [29] . seeking to rationalize the enigma posed by a complete lack of immune responses to hcv replication of ~10 6-7 geq/ml at week 6 but [variable] immune responses to replication at ~10 5 geq/ml after week 14, the authors conclude "..[the data]...appear[s] to be consistent with the interpretation that hbv and hcv are ignored by the adaptive immune system for about 2 months after primary infection" and "[in hcv].. the adaptive response seems to really ignore for several weeks a substantial quantity of virus (at least 10 6 copies/ml)..". this is certainly an accurate synthesis of an extensive and highly complex literature but does it make any sense? if adaptive immune responses really ignore high level hcv replication for two months, as suggested, then the following mechanism(s) are implied: a) an accurate mechanism for prompt detection of infection; b) a timing mechanism; c) a trigger mechanism for immune responses independent of any viral factor (given levels of virus are greater before immune recognition than afterwards the trigger for immune response must be either non-viral or falling (!) viraemia); and, as cytomegalovirus (cmv)-specific cd4(+) t cell responses arise within 7 days of cmv infection [37] ; d) a mechanism allowing the immune system to differentiate hcv from cmv and other viruses (and reasons to do so). while possible, this seems unusually inelegant and pointlessly counterproductive, especially as events soon after infection probably determine whether virus is cleared or chronic infection develops. it is much more likely that adaptive cellular or humoral immune responses do not develop in the first 6-7 weeks of hcv infection simply because the virus isn't "seen". why should hcv replicating at 10 6-7 geq/ml at week 6 be invisible to the immune system but visible when replicating at 10 5 geq/ml long term? dissection of this problem requires explicit analysis of what is being measured and how. assay of hcv rna and detection of hcv by immune responses measure two quite different things. quantitation of hcv is typically performed by branch-chain cdna assay (bdna) or quantitative pcr (qpcr) using probes or primers complementary to conserved 5'untranslated (5'utr) hcv rna sequences. immune responses to hcv typically "measures" envelope proteins translated from envelope-encoding rna (eerna) sequences and are directed at specific antigenic amino acid sequences and polypeptide conformations, not total viral envelope protein concentrations. while concentrations of 5'utr rna will be proportional to eerna concentrations in any given sample, they may not be identical for two reasons; i) rna transcription may prematurely terminate making 5'utr rnas relatively more prevalent than eernas and ii) hcv 5'utr is highly conserved, while eerna s are less constrained, making hybridization efficiencies of pcr primers or bdna probes greater for 5'utr rnas than for the population of eernas, causing relative under-estimation of true envelope rna concentration 1 . nonetheless, as 5'utr hcv rna concentrations will be proportional to eerna concentration, the question remains; why should envelope proteins translated from eerna sequences present at concentrations corresponding to ~10 5 5'utr geq/ml at 16 weeks be visible immunologically, but envelope proteins derived from eerna sequences corresponding to ~10 6-7 5'utr geq/ml at 4-6 weeks remain unseen? quasispecies biology, specifically variable rna pol fidelity, replicative homeostasis, and sequence-specific requirements for both genetic and immunological detection suggest an answer. rna viruses replicate by copying antigenomic templates, a process catalysed by rna pol , an enzyme lacking fidelity or proof reading function [38] [39] [40] [41] . theoretically, an rna viral genome like hcv (about 9200 bases) could assume any of 4 9200 (about 8.95 × 10 5538 ) possible sequence combinations exceeding, by some margin, population estimates of protons in the known universe (about 10 80 ), meaning the potential complexity of rna viral quasispecies is infinite, for all practical purposes. an rna pol fidelity rate of 10 -5 errors per base copied predicts at least one and as many as 10 (estimated for hiv) [39] genomic mutations will be introduced during each cycle of replication. furthermore, as hcv replication results in synthesis of ~10 12 virions per person per day [8] , on average, mutations will develop at each genomic locus ~10 7 times/day, while the probability any two genomes synthesized consecutively will be identical is about 10 -6 . the sum effect is inexorable accumulation of genomic mutationsthat, by itself, should threaten replicative fitness because of muller's ratchet [42] -and progressive dilution of wildtype genomes (figure 3), processes that make long-term stability of rna virus quasispecies highly paradoxical [43] . as argued previously, a combination of selective genomic replication and variable rna pol fidelity, both mediated by replicative homeostasis, act together to prevent rna quasispecies extinction [28] . the phenotypic consequences of viral quasispecies biology may be more important. progressive divergence of genomic rna sequences away from wild-type sequences caused by rna pol infidelity generates a massive population of closely related, but genetically distinct, rna molecules (figure 3), an effect operative at all scales from each open reading frame (orf) to whole virus species. a quasispecies of orf rnas has but one inevitable outcome; translation of a quasispecies of viral proteins with a vast and highly variable spectrum of phenotypes, some subtly nuanced, others grossly defective. furthermore, mutations simplified, two dimensional clade diagram of hyperdimensional viral rna and protein sequence-space figure 3 simplified, two dimensional clade diagram of hyperdimensional viral rna protein sequence-space. because of rna pol (p) infidelity and müller's ratchet, mutations ( ) are introduced into each rna template synthesized, and progressively accumulate, resulting in an rna quasispecies with sequence progressively divergent from consensus sequence. translation results in a spectrum of proteins ( , , , etc.) with properties that also vary progressively from wild-type sequence ( ) to highly variant proteins ( , , etc.). some rnas will be so abnormal that translation or replication fails or is truncated ( ), while others will code for grossly defective proteins ( , etc.). that create new, or obliterate pre-existing, start or stop codons in a significant proportion of rnas, will cause translation of highly unusual and heterogeneous proteins, particularly during high-level viral replication, a phenomenon that may explain hbeag. viral quasispecies cannot, and will not, produce homogeneous proteins with predictable and consistent phenotypic and antigenic properties. while rna pol infidelity will cause progressive divergence of copied sequences away from wild-type or consensus sequences, the probability of any particular sequence arising will fall dramatically with increasing genetic distance from that consensus sequence (figure 4), allowing conceptual representation of the resulting genomic (and consequent phenotypic) diversity as a frequency distribution curve, with increasingly variant sequences surrounding a 'centre of gravity of replication', formed by wild-type sequences. viral quasispecies occupy hyperdimensional sequence-spaces, hence any physical representation is necessarily simplified, but because mutation away from wildtype sequences is equally probable in all directions, variant rna and protein frequencies will be normally distributed and the standard deviation (sd, σ) -insofar as 'normal' or 'standard' can be applied to a hyperdimensional space -of that distribution will be a function of rna pol fidelity; if rna pol is completely faithful, the rnas and proteins will be monoclonal and σ = 0; if rna pol has no fidelity, rna will be synthesised randomly, and all rna and consequent protein sequences will arise with equally probability, therefore σ = ∞. while viral rna and related protein sequences are theoretically unconstrained (at least before any consideration of functionality), the sequence specificities of any reagents used in their detection (bdna probes, pcr primers, mabs etc) are not, by definition, and their specificity and the efficiency with which they detect variant molecules will fall progressively the further those variant sequences are from the consensus sequence. a zone of 'reagent specificity' may therefore be defined probably encompassing wild type and some variant sequences, but there will exist some rna sequences and corresponding proteins of any quasispecies that are undetectable with these sequence-specific reagents. a threshold of detection of any assay (including immune detection) may similarly be defined; rna or protein sequences present at concentrations below this conceptual level being undetectable by that particular assay. the hcv "early replication" paradox now partially resolves; the 5'utr sequences are both highly conserved and common to virtually all rnas in the quasispecies, therefore, the 5'utr concentration -that is, the common measure of hcv viraemia -corresponds to the area under the frequency distribution curve. by contrast, envelope rna sequences (and related envelope proteins) are not so constrained and their relevant concentrations (i.e. whether or not that rna or protein sequence is detectable) corresponds to the frequency of that specific sequence in the quasispecies and that, in turn, depends on rna pol fidelity; if rna pol fidelity is low, the frequency or concentration of any particular rna or protein sequence will also be low and may be below the detection threshold, while increasing rna pol fidelity may increase sequence frequency [i.e. the concentration of specific proteins] above detection threshold. but why should specific eerna sequence frequencies -in other words, hcv rna pol fidelity -increase after week 8, facilitating adaptive immune responses? viral autoregulation, specifically replicative homeostasis, provides an answer. interactions among species, whether between humming birds and flowering plants, primitive viroids and prokaryotic cells or hcv and man, results in an unremitting process of adaptation and responsive counter-adaptation -in effect, a molecular arms race -for each species just to maintain ecological parity. the price of survival for a species is continual evolution. survival, for viruses, requires cell entry, a precondition long antedating necessity to evade more complex host defenses, including interferons and other cytokines and adaptive immune responses, while for cells, and complex cellular organisms, cell wall defenses, including receptor polymorphisms, form a principal barrier against viral invasion. viral survival -effectively meaning rna pol survival -on an evolutionary timescale, as argued previously [28, 44] , requires control of mutation and replication rates in a manner adaptively responsive to constantly changing biota and this implies dynamic linkage of rna pol fidelity and processivity with quasispecies phenotypic and antigenic diversity, meaning an autoregulatory linkage -replicative homeostasis -between rna pol fidelity and processivity and envelope proteins, as argued previously [28] . by definition, evolutionary co-adaptation occurs in response to adaptations in locally prevalent interacting species. natural selection for beak variation(s) in darwin's finches occurs as a consequence of concrete survival benefits these variations -mediating, for example, enhanced food harvesting interactions with other variable plant or animal species -confer to individual galapagos island birds, rather than any inexorable hypothetical 'improvement' in beak function for finches in general. if a species is widely distributed in space, but population mixing is slow or incomplete, locally prevalent interactions with other species will vary and regional genetic variations will arise and be maintained, hence progressive divergence from the original genotype (speciation) may result. for viruses, and their hosts, genetic variations -reflected in viral genotype and cell surface polymorphisms and resulting disease susceptibilities -would be predicted, and are observed [45] [46] [47] [48] [49] [50] , to have frequencies that vary geographically. consider the following; an enzyme (e) functioning in a closed system synthesizes either product a or b that both interact with e to influence output such that a:e interactions cause production of b, while b:e interactions pro-duce a. irrespective of starting conditions (excluding substrate exhaustion and product inhibition), an equilibrium will eventually develop ( figure 5 ) with the relative concentrations of a:b determined by the relative association constants (k) of a:e (k a:e ) and b:e (k a:b ) and the velocity (ν) of production of a from b:e (ν a ) and b from a:e (ν b ). removal or addition of either a or b will alter equilibrium conditions but not the fact equilibrium is reached; if a is removed, for example, the increased two-dimensional representation of hyperdimensional rna (or corresponding protein) frequency distribution curve (scale arbitrary) with conceptual centre of gravity of replication (wild type, green) and variant sequences (blue), zone of reagent spe-cificity (red shading) and threshold of detection (tod) of any assay figure 4 two-dimensional representation of hyperdimensional rna (or corresponding protein) frequency distribution curve (scale arbitrary) with conceptual centre of gravity of replication (wild type, green) and variant sequences (blue), zone of reagent specificity (red shading) and threshold of detection (tod) of any assay. as mutations ( , ) accumulate and rna sequence progressively diverges from consensus sequence (0) the probability of that rna sequence and corresponding protein (e.g. envelope, env.) arising falls rapidly. standard deviation (σ) of frequency distribution is proportional to rna pol fidelity. frequency genetic distance 0 threshhold of detection (tod) reagent specificity ♦ ♦ frequency of b:e interactions will cause compensatory increased a synthesis; in this sense enzymatic autoregulation occurs. intuitive analysis suggests that enzymes acting in a milieu of increasing concentrations of inhibitory molecules become progressively less processive until reduced enzyme output is insufficient to further inhibit enzyme activity, and an equilibrium state is reached. considering viral replication, if alteration of rna pol fidelity causes synthesis of either wild-type or variant rna sequences (simplified, as a continuum between these two must exist) that are subsequently translated into either wild-type or variant polypeptides that then interact with rna pol such that wild-type: rna pol are high affinity interactions that induce rapid, low fidelity rna pol replication while variant protein: rna pol interactions are low affinity and cause high fidelity rna pol replication at low rate then an equilibrium will eventually develop. hence, as relative concentrations of wild-type and variant viral proteins vary, alteration of both processivity and fidelity of rna pol results, permitting viruses to adaptively respond to environmental changes, including immune recognition and reaction to evolving cell receptors. stable, highly reactive equilibria not only develop as a result of rna pol /envelope interactions and viral autoregulation, there is no option but for this to occur. generation and maintenance of polymorphisms, that is, replacement of existing genes -that, by operational dar-winian definition, have proved their functionality and evolutionary fitness by surviving to reproduce -with variant genes (polymorphisms) of uncertain functionality, fitness or overall compatibility within an organism, is an evolutionary strategy that will only be sustained on a geological timescale if new polymorphisms confer survival benefits to organisms that exceeds the risks and metabolic costs of generating and sustaining those polymorphisms. for primitive cells, lacking functional humoral, cellular or cytokine defense mechanisms, development of cell-surface protein polymorphisms is an obvious adaptive strategy to thwart invasion by primitive viruses. like other adaptive strategies, cell-surface polymorphisms are strongly selected for, and have been highly conserved over deep time, and are found in all organisms from primitive prokaryotic cells [51] and thermophilic bacteria [52] through to plants [53] as well as mammalian cells, strongly suggesting a critical evolutionary function. the lock and key hypothesis, for which there is very considerable evidence [54] [55] [56] [57] , first proposed by jbs haldane [58] , contends polymorphisms arise, and are maintained, as protection against cellular parasitism, particularly by viruses 2 . while dna-encoded protein polymorphisms form necessary defenses against viral access, they may not be sufficient; a quasispecies of cells (e.g. the liver) expressing similar and static receptor variations renders those cells vulnerable to sustained attack from any virus that successfully invades any one cell, and further dynamic modification of cell receptors, triggered by viral infection and mediated at the transcriptional level by modulation of dna dependent rna polymerase fidelity in nearby uninfected cells, by a mechanism similar to replicative homeostasis would seem possible. a clear, unambiguous band at the "c" position on a sequencing gel, causes "cytosine" to be assigned to that genetic locus. but does this certitude reflect reality, at least for viral rna quasispecies? direct pcr sequencing is an "averaging" procedure revealing the most frequent nucleotide at any particular locus. however, nucleic acids and proteins cannot express 'an average', and discrete quanta of specific nucleotides or amino acids are present at every locus. a typical clinical serum sample, containing 4 × 10 5 geq/ml hcv and mutating at 10 -5 substitutions/base, will contain examples of each possible nucleotide at every locus, but most variations will remain undetected during sequencing or any other method of quasispecies analysis. analysis of cloned dna gives cleaner data than pcr sequencing but if 100 clones (and multiple hcv quasispecies clones are highly unlikely to be identical) provides definitive sequence, would we process the 101 st to reveal different and, potentially, critical sequence variations? and if we did, how would we recognise its importance? is important sequence likely to be present at frequencies of http://www.virologyj.com/content/2/1/70 < 1%? infectious virions containing, presumably, fulllength functional genome and corresponding wild-type proteins, are often outnumbered by ~6 × 10 4 :1 in serum by defective and non-infectious particles [53] that presumably do not, suggesting that important genetic sequence and associated phenotype may occasionally be extremely rare. how the immune system recognizes uncommon, nondescript, but important protein sequences in a featureless background of similar molecules is a non-trivial problem for which replicative homeostasis may suggest a solution. replicative homeostasis, described in detail elsewhere [28, 44] , is an epicyclic mechanism of viral autoregulation that results when viral proteins, notably envelope (env), influence rna pol fidelity and processivity. the predicted consequences of replicative homeostasis for rates of intracellular viral replication and mutation, cellular expression of viral proteins and immunological responses occurring because of replicative homeostasis is represented schematically (figures 6, 7). during early viral replication in a naive cell devoid of inhibitory molecules (panel a, a), high affinity wild-type envelope:polymerase interactions predominate, causing rapid low-fidelity polymerase activity resulting in rapid synthesis of variant viral rnas and subsequently proteins, hence causing a broad spectrum of viral proteins to be expressed on the cell surface, each at concentrations below the threshold of immune detection (tod). rna pol infidelity ensures synthesis of variant viral rnas and proteins predominates early, hence variant protein molecules progressively accumulate within cells relative to wild-type viral molecules (panels b-d) and increasing the probability of variant viral envelope:rna pol interactions. variant viral envelope:rna pol interactions causing progressive inhibition of rna polymerase processivity and increasing rna pol fidelity, reducing diversity of viral rnas synthesized and progressively restricting dynamic progression of rna pol functional properties, processivity () and fidelity () predicted by replicative homeostasis figure 6 dynamic progression of rna pol functional properties, processivity ( ) and fidelity ( ) predicted by replicative homeostasis. initial state (a, corresponding to panel a, figure 7 ): in a newly infected cell, high-affinity wild-type:rna pol interactions will predominate resulting in high rna pol processivity but low fidelity causing high-level viraemia with broad virus phenotypic spectrum, maximizing cell tropism. intracellular accumulation of variant viral proteins (b, c.f. panel b, figure 7 ) reduces rna pol processivity but increases fidelity reducing viral rna synthesis and consequently, viraemia before a dynamic, fluctuating equilibrium (c, c.f. panel c or d, figure 7 ) develops in which inhibition of rna pol by variant viral proteins is balanced by increases in rna pol fidelity (with consequent synthesis of wild-type viral products tending to cause high rna pol processivity). conceptual progression of intracellular viral replication events, including variable rna pol fidelity and processivity, restriction of antigenic diversity and immune recognition under influence of replicative homeostasis env viral protein diversity expressed on the cell surface (panels b to d), increasing cell-surface concentrations of individual viral proteins above the threshold of detection (panels c, c) at which point a polyclonal immune response develops. development of low-affinity polyclonal blocking antibodies, restricting cellular egress of viral proteins, further increasing intracellular concentrations of variant envelope proteins, still further increasing the probability of variant viral envelope:rna pol interactions and inexorably further restricting antigenic diversity increasing relative expression of wild-type proteins thus further exposing these epitopes to immune surveillance and facilitating specific high-affinity immune responses, including cytotoxic t cell responses, (d,d) to wild-type proteins. thus, the immune responses can strategically utilize replicative homeostasis to force viruses to reveal important and dominant wild-type epitopes, but those responses develop initially as a consequence of restriction of rna pol fidelity that occur because of replicative homeostasis. high-affinity responses further deplete intracellular concentrations of wild-type proteins, progressively reducing wild-type envelope:rna pol interactions, greatly reducing rna pol processivity to the point of viral latency (e,e), caused by variant viral envelope:rna pol interactions. the hepatitis c "early replication" paradox now resolves completely when considered in the context of replicative homeostasis; initial high level hcv replication (due to high rna pol processivity) remains immunologically undetectable for 6-8 weeks, or more, because of low rna pol fidelity causing a broad spectrum of hcv envelope proteins each expressed on cell surfaces at concentrations below the threshold of detection even while viraemia, reflected in concentrations of 5'utr rna common to each rna species, are present at 10 6-7 geq/ml. as replication progresses, intracellular accumulation of variant viral proteins increase rna pol fidelity but decrease processivity (replicative homeostasis), downregulating hcv replication and reducing viraemia but restricting antigenic diversity and increasing expression of hcv envelope proteins to beyond the threshold of immune detection. furthermore, the temporal tissue injury (aminotransferase) paradox also resolves in this light: focussed immune recognition (including cytotoxic t cell responses) doesn't develop until after viral antigenic diversity is restricted by replictive homeostasis the transaminase peak would not be expected until after viral replication falls due to autoinhibition of rna pol processivity. varying expression of viral proteins by modulating rna pol fidelity to facilitate immune escape would seem a useful evolutionary adaptation that might be retained by more complex organisms, including cellular pathogens like tuberculosis and malaria, to optimize their stability within hosts. this mechanism of immune avoidance might also explain maternal-foetal tolerance. the human foetus maintains a stable parasitic existence during gestation (and, i expect, to university age and beyond) that is tolerated despite normal maternal immune responsiveness in general and lack of specific tolerance to paternal antigens in particular, a situation made more problematic as expressed foetal antigens are predominantly of paternal origin [54] . while immunological isolation of foetal tissue by the placental trophoblastic layer [55] , and placental display of hla-g [56] , probably contribute to foetal stability in the face of a potentially robust immune attack, neither mechanism would explain persistence of viable foetal nucleated red blood cells within the maternal circulation [57] in quantities sufficient to permit clinical prenatal diagnosis [58] . is it possible foetal tolerance is mediated by regulating the fidelity of foetal dna dependent rna transcriptases to ensure any cell-surface antigens are expressed heterogeneously and at levels below the threshold of maternal immune responsiveness? for many classical autoimmune disorders, including primary biliary cirrhosis [59] , multiple sclerosis, and rheumatoid arthritis, convincing epidemiological evidence [60] , including cases clustering [61, 62] , strongly suggests these diseases are triggered by infectious agents in genetically predisposed individuals. in others, such as diabetes mellitus, tantalizing epidemiological [63] , clinical [64] and laboratory [65] evidence has implicated enteroviruses, but has suggested viral-triggered autoimmune processes, rather than cytolytic destruction of pancreatic betacells [66] . similar circumstantial evidence exists for myocarditis, demyelinating diseases, myositis and other post infectious inflammatory disorders. when macfarlane burnet wrote autoimmunity arises from "inability to distinguish self from non-self" hbv, hcv, hiv and other viruses, now established to cause diseases with clear autoimmune features were unknown. viral infections, particularly hepatitis c -and its treatment with interferon -are associated with many varied autoimmune phenomena [67] , and thyroid disease [68] [69] [70] , diabetes mellitus [71, 72] , membranous, membranoproliferative and cryoglobulinemic glomerulonephritis, vasculitis and peripheral neuropathy [73] , and autoimmune gastritis [74] are all very well documented, although the mechanism(s) are unknown and causality is certain. classical serological markers of autoimmunity, including rheumatoid factor, antinuclear antibodies (ana), anticardiolipin, antithyroid, anti-liver/kidney/microsomal antibodies (anti-lkm), as well as hcv/anti-hcv immune complex formation and mixed essential cryoglobulinemia are common accompaniments of chronic hcv infection [73] , raising the obvious question of whether all "autoimmunity" has a viral basis. indeed, zinkernagel's pragmatic and subtly anticipatory; "if we know the infection, we call the disease immunopathologically mediated; if we do not recognize or know it, we call the disease autoimmune [75] " fully reflects recent explosive growth of information and the deeper questions this information poses. rna virus quasispecies biology, specifically the generation of rna quasispecies by rna pol , and translation of these immensely variable rnas into protein quasispecies, suggests an immediate solution to the problem of viral autoimmunity and, by extension, to autoimmunity in general, as well as suggesting a unifying hypothesis to explain other diseases known to have multi-factorial aetiologies that include inflammatory components -such as coronary artery disease -in addition to other diseasesincluding schizophrenia and some forms of depressionthat currently lack rational and coherent pathogenic explanations. viruses are known to co-opt cell surface molecules, including lectins, hormone receptors and cell signaling molecules, to access cells. receptors, and other cell surface molecules, identified as "viral receptors"or to specifically interact with viral proteins include prostaglandins, catecholamines and acetylcholine receptors [76] , serotonergic neurotransmitters (5ht) [77] , endothelial cell glycoproteins [78] , insulin-like growth factor (igf-ir) and its major signaling molecules insulin receptor substrates irs-1 [79] and irs-2 [80] , epidermal growth factor (egf) [81] , neurotrophin receptor [82] , thyroid hormone receptor tralpha1 [83] , an immunoglobulin protein superfamily [84] , low density lipoprotein (ldl) receptors [85, 86] , transferrin receptor (tfr) [87] , asialoglycoprotein receptor (asgp-r) [88, 89] , and angiotensin-converting enzyme 2 [90] , to cite biologically diverse examples. of necessity, some receptor affinity studies have used cloned viral protein ligands, an artificial situation that cannot approach the phenotypic complexity of rna viral protein quasispecies. nonetheless, variable virus receptor affinities [91, 92] , evolutionary adaptation of receptor affinity [93] , emergence of escape variants with altered receptor affinities [94] , temporal alteration of receptor usage [92] and capacity to exploit alternative entry pathways [95] have all been confirmed, suggesting viruses are capable of generating highly plastic ligands with very broad receptor affinities. if a virus co-opts a receptor for cell entry, then wild-type envelope (consensus sequence) epitopes, coded for by wild-type rna sequences, will probably form the common viral ligand. however, any viral rna quasispecies also contain a vast spectrum of rnas derived from, and similar to, envelope open reading frame (orf) consensus sequence, but variant from it. as the envelope orf quasis-pecies sequences progressively diverge from wild-type, the quasispecies of envelope proteins translated from these variant orfs will also, and inexorably, diverge in sequence, structure and biological function from wildtype envelope sequence proteins. some of these envelope proteins will be functionally identical, but others, and probably the vast majority, will range from subtly different to grossly abnormal, either due to major differences of sequence and/or chemical or steric amino acid incompatibility, or because of premature introduction of stop codons. even minor amino acid differences, as sickle cell anaemia illustrates, and has been confirmed specifically for viral receptor usage [96, 97] , may catastrophically alter a proteins' function with respect to co-opted viral receptors, with some having no binding affinity, while others will bind strongly and act as agonists, antagonists or competitive inhibitors of normal receptor function. variant and defective viruses, and their polypeptides, will be in vast molar excess compared to wild-type [53] but will exhibit similarly high antigenic variability, permitting escape from immune and other scavenger mechanisms. as many variant viral polypeptides will bind tightly to "self" receptors, but contain immunogenic non-self motifs, a polymorphic (because variant viral proteins will themselves be highly polymorphic due to the quasispecies process) immune response, apparently directed against "self" antigens, but actually targeting virus protein-receptor complexes virtually indistinguishable from normal cell receptors, will result causing apparent 'autoimmune' tissue damage. this mechanism suggests an explanation for common autoimmune phenomena. if a virus enters cells because wild-type envelope motifs interact with insulin, insulin receptor substrate [79, 80] , tsh or related molecules [83] , or acetylcholine [76] receptors, many variant envelope polypeptides, generated by envelope orf quasispecies rnas, would have similar receptor binding affinity, but may effectively disrupt receptor function, predictably causing impaired glucose tolerance or diabetes mellitus, thyroid dysfunction, or myasthenia gravis with secondary resistance to, and elevation of, the normal hormone ligand (insulin, tsh etc.). the expected consequences disruption of receptor function by variant viral proteins might explain many common biochemical pathologies; for example, what effect would chronic blockade of parathyroid (pth) receptors by viral proteins have on pth levels, the parathyroid glands, or bone? leptin is a 16kda protein hormone secreted by adipocytes and carried across the blood-brain barrier by a rate-limiting transporter to act on hypothalamic receptors [98] where, among other functions, it regulates thyrotropinreleasing hormone (trh) genes and upregulates alphamelanocyte-stimulating hormone and other anorexigenic neuropeptides [99] important to appetite-regulation and energy balance [100] . leptin also regulates a broad spectrum of other processes and behaviours including thermogenesis, blood pressure and immune function. s=serum leptin concentrations and leptin resistance, are independent markers of obesity, weight gain, systemic hypertension [101] , diabetes mellitus [102] , obstructive sleep apnoea [103] and myocardial infarction [104] , while polymorphisms of the leptin gene are associated with insulin resistance [105] and long-term risk of developing diabetes mellitus [102] . predictably, variant envelope proteins generated by envelope orf rna quasispecies from viruses utilizing leptin receptors for cell access would have similar receptor affinity, but exhibit non-physiological leptin antagonist or agonist properties, thus disrupting leptin receptor function, altering energy regulation, and causing either excess caloric intake unrestrained by satiety responses, or inappropriate satiety signals with pathologically reduced caloric intake. as clear evidence exists for viral disruption of leptin function [106] and virus-associated weight gain in humans [107] and monkeys [108] , is it possible the global epidemics of type ii diabetes mellitus, insulin resistance, hyperlipidaemia and obesity now prevalent [109] [110] [111] [112] [113] [114] [115] [116] , are just that; epidemics fundamentally caused by viruses that co-opt insulin or leptin or other associated receptors for cell access and generate protein quasispecies that disrupt receptor function? could it also be that ethnically based epidemics of obesity, diabetes mellitus, hypertension and reno-vascular disease (the 'metabolic syndrome'), as seen in pima indians, nauruans and australian aborigines [115] have developed not primarily because of exposure to "western" foods and lifestyles -that, after all, are all-pervasive without necessarily having so dramatic an effect on other groups -but because of chronic or recurrent exposure to viruses, or genotypes of viruses to which their particular repertoire of receptor polymorphisms confer no protection? or that anorexia nervosa develops, in some patients, when variant viral proteins with aberrant leptin-agonist function arise during the course of viral infection, as the temporal relationship between infection and disease onset, very clearly documented in one study [117] , suggests. cardiovascular disease, the leading cause of premature death and disability in most western countries, has a wellestablished multi-factorial basis involving a complex interplay between genetic predisposition, environmental and personal risk factors -including systemic hypertension, diabetes mellitus, hyperlipidaemia, obesity and cigarette smoking -and more recently recognized mechanisms, including endothelial dysfunction [118] , vascular inflammation [119] and leptin levels [104] . systemic hypertension, diabetes mellitus and hyperlipidaemia have long-established, but complex, patterns of inheritance, a situation further compounded by evidence receptor polymorphisms -including those of angiotensin ii type 1 receptor [120] , irs-1 gene [121] and low density lipoprotein receptor (ldlr) [122] -both confer disease susceptibility and have regionally variable prevalences [123, 124] . the flaviviradae -including hcv -as a family, and the rous sarcoma virus, utilize low density lipoprotein receptors to enter cells [85, 125] , while angiotensin ii [90] , insulin receptor substrates (irs1 and irs 2) [79] , and endothelial cell glycoproteins [78] and other receptors widely distributed in vascular tissues are known to be permissive for virus cell entry establishing, in principle and in fact, viral-protein receptor affinity relevant to cardiovascular diseases. viruses accessing cells through these receptors will generate a quasispecies of variant proteins capable of disrupting receptor function potentially causing hyperlipidaemia, hypertension, hyperglycaemia and endothelial dysfunction, as well as immune-mediated endothelial cell damage, thus establishing the necessary and sufficient conditions and a chain of events that potentially link viruses and vascular diseases, including myocardial infarction. this hypothesis exists at the confluence of established risk factors for coronary artery disease, including genetic susceptibility, polymorphisms predisposing to hypertension [126] [127] [128] , diabetes [126] and hypercholesterolaemia and substantial new data implicating vascular inflammation [119, 129] , endothelial dysfunction [119, 130] , leptin dysregulation [104] and viral infection [131, 132] in the pathogenesis of vascular disease. furthermore, this final common pathway can account for that small, but significant, group of patients with vascular diseases but no clinically identifiable risk factors, as well as the non-random co-incidence of depression and coronary artery disease [133] (as discussed below) in addition to the anti-inflammatory action of hmg-coa reductase inhibitors (statins) [134] , and their effect in lowering cardiovascular mortality independent of cholesterol reduction [135] ; if statins compete with variant viral proteins for hmg-coa reductase receptor binding, and displace immunologically attractive molecules, inflammatory responses directed at viral product, but involving endothelial cell receptors, will be ameliorated (figure 8). human immunodeficiency virus hiv-associated dementia (hivd) occurs in 15% of hiv-infected adult patients, and as a major cause of dementia in the young represents "proof of principle" of virus-caused dementia, raising the possibility other forms of virus related dementia exist. although highly active antiretroviral therapy (haart) has reduced the incidence of hiv-d by 40-50% [136] , it remains a major cause of morbidity and the pathogenesis poorly understood. direct cytopathic effects of hiv or other viruses are unlikely, while active replication of virus, high-level viral protein expression [137] , and increased viral envelope sequence-diversity in blood and brain [138] are all important, clearly indicating viral proteins are pathogenically important. the clinical features of hivd, including psychomotor slowing, apathy, and altered gait and posture, strongly suggest a subcortical dementia with involvement of the basal ganglia and striatal dopamine receptor pathways. schizophrenia, depression and bipolar affective disorder, and anorexia nervosa are highly prevalent, chronic conditions of unknown aetiology that cause enormous morbidity and generate significant health care costs. each of these disorders have well documented, albeit regionally variable, associations with receptor -including dopamine -polymorphisms [124, [139] [140] [141] [142] [143] , as well as epidemiological evidence that viral infections are aetiologically important, either directly or as precipitating events [117, [144] [145] [146] [147] , although other sero-epidemiological studies [148] and work directly seeking viral nucleic acids in patients with schizophrenia have proved negative [149] . if a virus, or viruses, use dopamine, acetylcholine [76] , neurotrophin [82] , serotonergic (5-ht) [77] , or other neuro-transmitter receptors to access cells (and, given rna virus quasispecies biology, it would be surprising if some didn't), then the rna quasispecies will generate a quasispecies of variant polypeptides potentially reactive to these receptors. while it is difficult to imagine what effect perfusing a functional human brain with a solution of antigenic, inflammatory polypeptides that bind to, and are variably disruptive of, critical neurotransmitter receptor function, might have on cognition, perception, behaviour, attention span, abstract thought, fine motor or emotional control, it is unlikely to be beneficial. in this context, the welldocumented cognitive abnormalities -unrelated to depression -found in patients with early hcv and hiv infection [150] [151] [152] are unsurprising. virus receptor disease (vrd) is quite distinct from either immune complex deposition disease due to deposition of macromolecules in tight vascular arcades, or from disease related to altered cell tropisms and is also completely independent of the primary site of viral replication; both non-inflammatory receptor blockade and immune-mediated inflammation directed at viral protein-receptor complexes could cause pathology of tissues non-permissive for and remote from the primary site(s) of viral replication with "autoimmune" damage to the liver, pancreas, brain, skin or lungs arising, for example, from chronic small intestinal virus infection. viral quasispecies biology predicts vrd will have other characteristics. first, due to replicative homeostasis, the ratio of wild type to variant viral proteins of the quasispecies will both fluctuate with time and will alter dramatically after initial infection; if wildtype proteins are dominantly agonist in function with respect to their receptor, variant proteins, most likely, will predominantly exhibit antagonist function (and vice versa). furthermore, the net effect of viral proteins (because of viral autoregulation) will fluctuate initially between receptor agonist and antagonist function, before becoming predominantly antagonistic, thus providing a possible explanation for transient thyrotoxicosis during early thyroiditis (before hypothyroidism supervenes), for hypoglycaemia seen during early insulin-receptor antibody-mediated insulin resistance [153] , and for the contradictory functions ascribed to hiv nef [154] . a corollary of fluctuating phenotypic dominance of viral protein quasispecies is that receptor affinity of these proteins will also fluctuate, and any resulting inflammation may vary in both intensity and anatomical distribution over time. second, because viruses utilize alternate receptors for cell access, apparently homogeneous disease processes could result from multiple different viruses. similarly, because cell receptor (r) and normal ligand (l; insulin, pth, leptin etc.) relationship (1; unbound, 2; activated), receptor permis-sive for virus cell entry (3) or blocked by polymorphism (rp, 4) figure 8 cell receptor (r) and normal ligand (l; insulin, pth, leptin etc.) relationship (1; unbound, 2; activated), receptor permissive for virus cell entry (3) or blocked by polymorphism (rp, 4). receptor blockade by variant viral envelope proteins (green e, 5), blockade by antigenic envelope proteins stimulating "autoimmune"response apparently directed against self receptors (e, 6), competitive displacement of antigenic proteins by drug (d, e.g. statin, aspirin) abrogating immune response (7). virus quasispecies produce a broad spectrum of protein phenotypes, and the receptor polymorphisms permissive for cell entry for specific viruses will be variably distributed in host populations, pathology of widely variable tissues in different individuals could result from the same virus. third, as evolutionary co-adaptation results in progressive genetic co-divergence of interacting species, the receptor polymorphisms predisposing to (or protecting against) infection by any particular virus, and resulting vrd, and the common viruses causing them, would be predicted to vary geographically, an expectation multiply confirmed for disease associated polymorphisms. as a corollary this suggests individuals migrating from regions where hosts and virus strains are stably co-adaptated to other areas, where different viruses are prevalent, might experience increased rates of vrd -beaks optimally adapted for finch survival on the galapagos may be a liability elsewhere -a prediction again amply confirmed [155] [156] [157] ;. finally, if immune mechanisms are unable to clear rna viruses like hcv and do not cause the reduced viral replication seen during acute infection, are they any more likely to be effective against other rna viruses? is it possible that self-limiting infections like influenza and sars also autoregulate their replication, and, like hcv or hbv, become partially dormant, yet remain transcriptionally active, in the face of an active and powerful immune response? pcr amplification of influenza rna from convalescent samples makes this readily testable, while the documented relationship of influenza to myocardial infarction [132] and juvenile rheumatoid arthritis [61] makes the question important. if confirmed, the well-documented seasonality of some depressive illnesses [158] and schizophrenia, [146] and increased rates of schizophrenia during influenza epidemics [144] , and the increased incidence of both depression [146] and schizophrenia [144, 145] following in-utero exposure to influenza may be more rationally explained. if quantitative pcr (qpcr) assays of both 5'utr and envelope rnas are performed serially, and data expressed as [5' utr rna]/[env rna] for each sample, then a numerical expression describing changing quasispecies complexity over time may be obtained. in case prescient genius is unappreciated, haldane formulated the "lock and key" hypothesis on the basis of protein polymorphisms, defined by gel electrophoresis, and some general musing about predation and evolutionary struggle, two decades before the nature of dna was elucidated. i have no pecuniary interests, whatever, in this work and do not stand to gain financially or otherwise from it. publish with bio med central and every scientist can read your work free of charge a new immune escape mutant of hepatitis b virus with an asp to ala substitution in aa144 of the envelope major protein emergence of vaccine-induced escape mutant of hepatitis b virus with 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in villages of origin in india variability in the 5-ht(2a) receptor gene is associated with seasonal pattern in major depression i thank my wife sophie j coleman, and sons matt and tim, for everything important, my parents dick and janet for extraordinary opportunity, and some great physician-teachers -that most noble vocation -of the university of western australia medical school; professors mike mccall, dick joske, bill reed, bill musk, peter pullan, michael quinlan, dick lefroy and ted haywood. special thanks to karl ruckriegel for turning back-of-envelope sketches into first-class graphics. any remaining lack of clarity is my fault. key: cord-304040-64obh7i3 authors: sande, charles j.; mutunga, martin; muteti, jacqueline; berkley, james a.; nokes, d. james; njunge, james title: untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics date: 2018-09-14 journal: sci rep doi: 10.1038/s41598-018-32072-3 sha: doc_id: 304040 cord_uid: 64obh7i3 the upper airway – which consists mainly of the nasoand oro-pharynx is the first point of contact between the respiratory system and microbial organisms that are ubiquitous in the environment. it has evolved highly specialised functions to address these constant threats whilst facilitating seamless respiratory exchange with the lower respiratory tract. dysregulation of its critical homeostatic and defence functions can lead to ingress of pathogens into the lower respiratory tract, potentially leading to serious illness. systems-wide proteomic tools may facilitate a better understanding of mechanisms in the upper airways in health and disease. in this study, we aimed to develop a mass spectrometry based proteomics method for characterizing the upper airways proteome. nasoand oropharyngeal swab samples used in all our experiments had been eluted in the universal transport media (utm) containing significantly high levels of bovine serum albumin. our proteomic experiments tested the optimal approach to characterize airway proteome on swab samples eluted in utm based on the number of proteins identified without bsa depletion (total proteome: protocol a) and with its depletion using a commercial kit; allprep, qiagen (cellular proteome: protocol b, ci, and cii). observations and lessons drawn from protocol a, fed into the design and implementation of protocol b, and from b to protocol ci and finally cii. label free proteome quantification was used in protocol a (n = 6) and b (n = 4) while commercial tmt 10plex reagents were used for protocols ci and ii (n = 83). protocols ci and ii were carried out under similar conditions except for the elution gradient: 3 h and 6 h respectively. swab samples tested in this study were from infants and children with and without upper respiratory tract infections from kilifi county hospital on the kenyan coast. protocol a had the least number of proteins identified (215) while b produced the highest number of protein identifications (2396). when protocol b was modified through sample multiplexing with tmt to enable higher throughput (protocol ci), the number of protein identified reduced to 1432. modification of protocol ci by increasing the peptide elution time generated protocol cii that substantially increased the number of proteins identified to 1875. the coefficient of variation among the tmt runs in protocol cii was <20%. there was substantial overlap in the identity of proteins using the four protocols. our method was were able to identify marker proteins characteristically expressed in the upper airway. we found high expression levels of signature nasopharyngeal and oral proteins, including bpifa1/2 and amy1a, as well as a high abundance of proteins related to innate and adaptive immune function in the upper airway. we have developed a sensitive systems-level proteomic assay for the systematic quantification of naso-oro-pharyngeal proteins. the assay will advance mechanistic studies of respiratory pathology, by providing an untargeted and hypothesis-free approach of examining the airway proteome. the human naso-oro-pharynx constitutes the first line of mechanical and immunological defence against infectious pathogens and particulate pollutants in the air. pathogens and commensal microorganisms constantly interact with the cells of the upper airway [1] [2] [3] and in order to maintain homeostasis, humans have evolved complex and dynamic defence mechanisms that maintain a functional mucosal barrier 4 . this barrier regulates microbial colonisation and maintains a potent immunological defence against pathogenic micro-organisms 5 . the effector functions that underpin this regulatory role are a product of the airway proteome, which is derived mainly from epithelial cells as well as innate and adaptive immune cells [6] [7] [8] . as part of these regulatory mechanisms, incipient antigens are initially expelled by non-immunological mechanisms such as mucus entrapment, enzymatic degradation and through mechanical egress via mucociliary action [9] [10] [11] [12] . endosomal degradation by phagocytes such as neutrophils and macrophages as well as other non-specific immunological mechanisms such as complement activation may be employed for antigens that persist. the final layer of immunological defence in the upper airway consists of adaptive immune responses -such as antibody responses -that are specifically tailored to specific pathogens 9 . in spite of its crucial role in the maintenance of airway health, the proteome of naso-oro-pharynx remains poorly studied, particularly in infants and children with serious respiratory infections. the naso-oro-pharynx and its secretions provide an excellent resource for identifying potential biomarkers of disease and could contribute substantially to the understanding of mechanisms of airway diseases. mass spectrometry-based proteomics combined with computational analysis has become a powerful tool for large-scale systematic investigation of biological processes in an untargeted and hypothesis-free approach 13 . proteomics has been employed to profile the nasal cavity and mucous proteome in general [14] [15] [16] [17] [18] [19] , as well as investigate airway diseases such as allergic rhinitis 14, [20] [21] [22] [23] [24] [25] , chronic rhinosinusitis [26] [27] [28] , and cystic fibrosis 29, 30 . it is additionally notable that mass spectrometry platforms used to investigate protein expression in these studies may have previously lacked sufficient sensitivity necessary for extensive proteome coverage that would be helpful in elucidating important biomarkers of mechanisms or prognosis. in this study, we developed a high-throughput swab proteomics workflow that can be used to investigate the airway proteomes of infants and children with serious respiratory illnesses especially from hospitals and field samples. study population and sampling procedures. naso-oro-pharyngeal samples were collected using nasal and oropharyngeal (npop) swabs (copan diagnostics, usa) and were eluted in 3 ml of universal transport media (utm, copan diagnostics, usa) that had been supplemented with 500ul of a protein/dna/rna preservative, allprotect (qiagen, germany). upon collection, samples were stored at −80 °c. the study population consisted a total of 93 infants and young children admitted to kilifi county hospital, in coastal kenya, with respiratory infections and control infants who were sampled from home and did not exhibit any respiratory symptoms (protocol a: n = 6, protocol b: n = 4, protocol c: n = 83). diagnosis of respiratory infections was done using a multiplex pcr that was designed to detect 15 respiratory pathogens: respiratory syncytial virus (rsv -a & b), rhinovirus, parainfluenza virus (1, 2, 3 & 4) adenovirus, influenza (a, b & c), coronavirus (oc43 & e229), human metapneumovirus and mycoplasma pneumoniae. written informed consent was sought from the parents or legal guardians of the recruits prior to sampling while ethical clearance for the conduct of this study was provided by the kenya medical research institute (kemri) scientific and ethical review committee (seru). all methods were performed in accordance with good clinical laboratory practice (gclp) guidelines. overview of protocol development for analysis of the upper-airway proteome. our proteomic experiments tested the optimal approach to characterize airway proteome on swab samples eluted in utm based on the number of proteins identified. protocols were developed and tested in a stepwise sequence, with observations and lessons drawn from one protocol, feeding into the design and implementation of the next. the first protocol that was tested (a) was based on six randomly selected patient samples which were lysed with urea, peptides generated using a standard workflow and analysed by label free quantification. in the second protocol (b) nasal samples from four randomly selected children were selected and cells were obtained by centrifugation. the cells were then lysed, total protein extracted from the lysate using a commercial kit (allprep, qiagen), peptides generated and analysed by label free quantification. in the third and fourth protocols (ci and cii) samples were selected from 83 children. in protocol c, proteins and peptides were prepared in the same manner as protocol b, after which peptides were labelled with isobaric mass tags (tmt10plex). protocols ci and ii were carried out under similar conditions except for the elution gradient: 3 h and 6 h respectively. a schematic outline of all the protocols is shown in fig. 1 while a detailed description of the individual protocols is outlined in supplementary methods. mass spectrometry analysis. peptides (8 μl) were loaded using a dionex ultimate 3000 nano-flow ultra-high-pressure liquid chromatography system (thermo scientific, usa) on to a 75 µm × 2 cm c18 trap column (thermo scientific, usa) and separated on a 75 µm × 50 cm c18 reverse-phase analytical column (thermo scientific) at heated at 40 °c. for lfq protein quantification; elution was carried out with mobile phase b (80% acetonitrile with 0.1% formic acid) gradient (4 to 30%) over 180 min at a flow rate of 0.25 μl/min. for tmt protein quantification, two gradients were employed: in protocol c i, the gradient was similar to that used for lfq protein quantification, while the gradient employed in protocol c ii was modified to elute peptides for 310 min. each lc run was finished by washout with 98% b for 10 min and re-equilibration in 2% b for 30 min. five blanks of 40 min each were run on the column between each injection comprising of two wash cycles with 90% b and an equilibration phase of 15 min to avoid sample carryover. peptides were measured using a q exactive orbitrap mass spectrometer (thermo scientific, usa) coupled to the chromatography system via a nano-electrospray ion source (thermo scientific). on the q exactive, the ms^1 settings for peptides were: resolution, 70000; agc target, 3e6; maximum it, 120 ms; scan range, 400-1800 m/z; while the ms^2 settings for fragmentation spectra of peptides were: resolution, 17000 (35000 for labelled peptides); agc target, 5e4; maximum it, 120 ms; isolation scientific reports | (2018) 8:13814 | doi:10.1038/s41598-018-32072-3 window, 1.6 m/z. ms data were acquired by data dependent acquisition where the top 12 (15 for labelled peptides) most intense precursor ions in positive mode were selected for ms^2 higher-energy c-trap dissociation fragmentation which were subsequently excluded for the next 45 s following fragmentation event. charge exclusion was set to ignore peptide spectrum matches that were unassigned, singly charged, and those with ≥+8 charges. for lfq protein quantification. cysteine carbamidomethylation was set as a fixed modification and n-terminal acetylation and methionine oxidations as variable modifications. the false discovery rate (fdr) was set to 0.01 for both proteins and peptide-spectrum matches and was determined by searching a fasta protein database comprising target and reversed target sequeces (decoy) derived from the organism being studied, by switching the amino-carboxyl orientation of a protein's amino acids to generate sequences that do not exist in nature 33 . enzyme specificity was set as c-terminal to arginine and lysine with trypsin as the protease. a maximum of two missed cleavages were allowed in the database search. peptide identification was performed with an allowed initial precursor mass deviation of up to 7 ppm and an allowed fragment mass deviation of up to 20 ppm. a minimum peptide length of 7 amino acids and a maximum peptide mass of 4600 da was allowed for the searches. the label free quantification (lfq) algorithm in maxquant was used to obtain quantification intensity values. for labelled protein quantification. the 10plex tmt was specified under isobaric labels for reporter ion ms^2 and reporter mass tolerance was set at 0.01 da. carbamidomethylation(c) and tmt-10plex labeled n-terminus and lysine were set as a fixed modification while oxidation (m) and acetylation (protein n-term) as variable figure 1 . a schematic overview of the naso-oro-pharyngeal proteomics experiments: naso-and oropharyngeal swabs were collected from infants and eluted in universal transport media supplemented with a protein/dna/rna preservative. in one set of samples (protocol a) cells in utm were lysed with urea, reduced, alkylated and digested with trypsin. peptides were then separated by hplc and eluted in a 3 h gradient prior to ms analysis. in another set of samples (protocols b & c) cells in utm were pelleted by centrifugation, lysed and proteins extracted using a commercial extraction kit. proteins were then reduced, alkylated and digested with trypsin. in protocol b, peptides were separated by hplc and eluted on a 3 h gradient. in protocol c peptides were labelled using tmt isobaric mass tags and pooled together prior to hplc separation. in protocol ci, proteins were eluted on a 3 h gradient, while in protocol cii, proteins were eluted on a 6 h gradient prior to ms analysis. scientific reports | (2018) 8:13814 | doi:10.1038/s41598-018-32072-3 modifications with both types of modifications being used for protein quantification. the 10plex reporter ion intensity matrix for the study participants was extracted from the maxquant proteingroup matrix file and batch corrected using an in house platform. assay-performance. the performance of the four test protocols was based on the number of proteins identified in each protocol as well as by the presence of marker proteins that are known to be expressed by cells of the upper respiratory tract. of the four protocols, protocol b -in which proteins were extracted from the patient samples using a commercial protein/dna/rna extraction kit, followed by label free quantification -resulted in the highest number of protein identifications ( fig. 2a) while protocol a -where samples were lysed with urea and then reduced, alkylated and digested, followed by label-free quantification -resulted in the lowest number of protein identifications. protocol c, which comprised two sub-protocols, distinguished by the elution gradient, resulted in an intermediate number of identifications, although, protocol cii -where proteins were extracted using the commercial extraction kit, peptides labelled with tmt tags and subsequently eluted on a 310-minute gradient -yielded a substantial increase in protein identifications over protocol ci. of the three protocols that yielded the highest number of protein identifications (protocols b, ci & cii) there was a substantial overlap in the identity of proteins detected by the three methods as shown in fig. 2b . further analysis of assay stability and reproducibility was restricted to samples from protocol cii, which was deemed to be the most practical for routine application due to its capacity for multiplexing and superior performance in protein identification compared to protocol ci. the inter-batch correlation of the total proteome of the pooled control sample was carried out by calculating pairwise pearson's correlation coefficients between batches. as shown in fig. 3a , there was a high degree of correlation between batches, with a median pearson's r value of 0.85 (fig. 3b) . a random selection of 4 proteins (acat2, gcc1, rpl5 & dsp) whose median expression levels were spread out across the entire dynamic range of protocol cii, showed that the expression levels of these proteins remained relatively stable across all experimental batches (fig. 3c) . cell-specific marker protein analysis. in order to further characterise the proteomes identified in protocol cii, the expression of signature proteins that are typically expressed by upper-airway cells was assessed. high expression levels of the signature nasopharyngeal proteins bpifa1 and bpifa2 was noted (fig. 4a) . the coefficients of variation for these proteins as well as other proteins in the pooled control was less than 20% (fig. 4b) . in addition, mucins -muc5 and muc1 -major components of the mucinous secretions that ubiquitously coat mucosal surfaces of the respiratory tract, were expressed at high levels in the nasopharyngeal secretions of all infants in the study (fig. 4a & 4d) . the two most highly expressed proteins in the naso-oro-pharyngeal proteome were the signature mucosal epithelia keratins, keratin 13 (krt13) and keratin 4 (krt4) fig. 4d . in addition to these nasopharyngeal marker proteins, we also noted high expression levels of definitive oral/salivary proteins such as salivary amylase in all samples that were analysed fig. 4d . other proteins such as the polymeric immunoglobulin receptor (pigr), a protein that mediates active trans-cytosis of secretory immunoglobulins onto the luminal surface of the respiratory tract -were expressed at high levels in all patient samples analysed fig. 4d . in addition to proteins whose origins could be attributed to resident epithelial cells, we noted the presence of proteins secreted by non-epithelial cells. we evaluated the expression levels of different sets of proteins stratified by their putative origins, including acute phase proteins (app), antimicrobial peptides (amp), immunoglobulin proteins, milk proteins and proteins associated with the effector functions of phagocytic cells. there was a high degree of heterogeneity in the expression level of acute phase proteins, with the median expression level of haptoglobin (hp) being the highest in this category while c-reactive protein (crp) had the lowest median expression level among the apps analysed fig. 5a (first panel) . a similarly diverse pattern of expression was also observed among antimicrobial peptides, with cathepsin g (ctsg), being the most abundant amp while the alpha defensin, defa1, was expressed at much lower levels fig. 5a (second panel) . in addition to apps and amps, extremely high expression levels of immunoglobulin heavy chains (alpha, gamma and mu) were observed in the naso-oro-pharyngeal secretions of all the children in the study, although, the iga heavy chain, igah2, was expressed at much lower levels compared to the other immunoglobulin subclass heavy chains fig. 5a (third panel) . the final set of immune-related proteins was associated with the effector functions of phagocytic cells such as neutrophils and macrophages fig. 5a (fifth panel) . three proteins in this category, lactotransferrin (ltf), s100 calcium and zinc binding protein s100a9, a component of calprotectin (s100a8/9) and myeloperoxidase (mpo) were among the most highly expressed proteins in the entire naso-oro-pharyngeal proteomes fig. 4d -with their respective median expression levels being exceeded only by the cytoskeletal and microfibrillar mucosal keratins (krt4 and krt13) and beta-actin (actb). in addition to these host proteins, we also identified high levels of non-host proteins in the respiratory tract. the most highly expressed of these were three milk proteins: lactalbumin alpha (lalba), beta casein (csn2) and kappa casein (csn3) fig. 5a (fourth panel) . validation of whether these milk proteins were of human or bovine origin was not carried out. to determine the age correlates of these proteins, the respective expression levels of the top three proteins in each functional category was plotted against the age of the study participants fig. 5b . with the exception of milk proteins, no systematic changes in protein expression level could be associated with increasing age. in the case of the milk proteins, all three appeared to be most abundant in the first 6-12 months of life, after which a precipitous decline in later years was observed fig. 5b (fourth panel) . finally, we investigated the expression of selected t-cell associated pro-inflammatory proteins. interferons (ifn-α/β/γ), t-cell phenotypic markers (cd3/4/8) or their associated effector proteins (il4/5/13/17/10, granzyme) were not detected in the naso-oro-pharyngeal proteomes of any of the study subjects. using the data from protocol cii, we undertook ordination analysis to visualize whether the proteome of children with respiratory infections of varying severities and healthy controls sampled from home varied. using nonmetric multidimensional scaling, we found that the airway proteomes of children admitted to hospital with severe respiratory infections and those with mild infections that did not warrant hospitalisation, largely overlapped (supplementary figure 1) . however, the proteomes of healthy control children who were sampled from home, clustered separately from those with respiratory infections. we describe the development of a comprehensive method of analysing the naso-oro-pharyngeal proteome of infants and young children with respiratory illnesses. of the four protein identification methods reported, the highest number of protein identifications was achieved using protocol b, a method in which proteins in naso-oro-pharyngeal samples were extracted using a commercial extraction kit, followed by label-free quantification. however, unlike the methods described in protocol c, this method is limited by the fact that only one sample can be analysed at a time, limiting its practicality for high-throughput applications. in protocols ci and cii, peptides from different patient samples were labelled with tmt10plex reagents, allowing for sample multiplexing prior to ms analysis, followed in-silico de-multiplexing after analysis. this analytical design not only increases sample throughput, but concurrently limits the inter-sample variance that is inherent to single-sample analysis. for this reason, we limited further analysis of assay sensitivity, stability and reproducibility to protocols ci & cii. we found high levels of correlation between control samples run on different assay batches as well as limited inter-batch variance, suggesting that the methods developed were sufficiently robust for the quantitative analysis of the upper-airway proteome. in order to increase the sensitivity of protein identification, we experimented on the effect of different elution gradients on protein identification. in the initial set of experiments in protocol ci, peptides were eluted for 180 minutes, resulting 1,432 protein identifications, however when the elution time was increased to 310 minutes -(protocol cii) -a further 443 proteins were identified, bringing the total number of proteins identified in protocol cii to 1,875. compared to previous mass-spectrometry-based proteomics studies of the upper airway, the number of proteins identified in protocol cii, vastly exceeded the number of proteins reported in previously published reports 14, 20, 22, 23, 25, 26, 28, 29 . further characterisation the proteomes identified in protocol cii, was done by evaluating the expression levels of marker proteins of resident naso-oro-pharyngeal epithelial cells. in-line with recent studies of the upper airway transcriptome 34 we found consistently high expression of the signature upper-airway proteins bpifa1 and bpifa2. these proteins have a unique expression profile, which is restricted to the trachea, nasopharyngeal epithelia and salivary gland 35 and are therefore considered to be the signature proteins of the upper airway. in addition to these definitive upper-airway proteins, we found a consistently high level of expression of mucins (muc1 & 5) -the main component of respiratory tract mucus -and mucosal-associated keratins, krt4 and krt13. these keratins were the most abundantly expressed of all proteins detected in the naso-oropharyngeal proteome. krt4 and krt13 are both differentiation keratins of the oral mucosa 36 and therefore the high levels at which they were detected is consistent with the anatomical locations that were targeted for sampling. in addition to these upper-airway differentiation proteins, we found high expression levels of a number of antibody related proteins, including the polymeric immunoglobulin receptor, pigr. pigr is a type i, membrane-spanning antibody fc receptor that is expressed at high levels by mucosal epithelial cells and facilitates the active transcytosis of secretory immunoglobulins 37 . the expression of pigr was linked to the equally high expression of the immunoglobulin heavy chains alpha(iga), mu(igm) and gamma(igg), an association that aligns with the known biological function of pigr and likely indicates its role in maintaining high levels of these antibodies on the mucosal surface. we also examined the secretome of immune cells in the airway. we found a large number of proteins that are associated with innate immunity; which in most cases, were expressed at very high levels. for example, the expression levels of the neutrophil antimicrobial effector proteins ltf, s100a9 and mpo, were not only among the highest of the entire naso-oropharyngeal proteome, but they were also present in the upper-airway proteome of each child in this study. the observed overabundance of proteins these proteins, as well others also related to antimicrobial immunity like ctsg, camp,defa1/2 and htn1 is most likely related to the need to prevent bacterial overgrowth and maintain homeostatic balance in the upper respiratory tract. previous studies have provided compelling evidence of the functional role of proteins such as ltf, camp, ctsg and others in limiting bacterial replication 38, 39 and the presence of these proteins at the high levels at which they were detected, supports the notion, that they play a key role in regulating the levels of commensal microbiota hence preventing bacterial overgrowth that could result in pathology. interestingly, we also noted high levels of milk proteins such as lactalbumin alpha (lalba) and alpha & kappa caseins (csn2 & csn3). these proteins were present at the highest levels in the first 6 months of life and declined in the second half of the first year of life. most children over the age of 12 months had relatively low levels of milk proteins in their upper airways. taken together, these observations are consistent with the fact most infants under the age of six months are predominantly breast-fed and the decline milk-protein levels after six months is most likely associated with weaning. the precipitous decline observed after the first year of life most likely reflects the complete cessation of breast feeding. finally, using ordination analysis, we compared the airway proteomes of children with different clinical manifestations of respiratory illness to that of healthy children who were sampled from home. the proteomes of children with respiratory infections clustered separately from those of well children. this distinction is most likely attributable host innate inflammatory responses mounted by the host in order to address an ongoing infection. these responses would be absent in well controls, in whom the absence of any clinical symptoms at the time of sampling, indicates the absence of an intense inflammatory response. our search of t-cell associated marker and effector proteins, did not identify any such proteins in the upper-airway proteomes of any of the children in this study. t cell effector cytokines and chemokines such as il-6, ifn-g, il-10, il-8 and other mediators have been widely reported in studies of paediatric upper airway secretions using sensitive immunoassays such as luminex and msd mesoscale. the absence of these mediators in this study, suggest a possible limitation of the methods reported in this paper: the failure to identify very low abundance proteins in the upper airway proteome. in spite of this shortcoming, ms-based methods reported here offer great advantages over targeted approaches by providing an unbiased, hypothesis-free overview of the upper-airway proteome, thereby increasing the likelihood of identifying novel associations with disease pathology. data 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peptide identification rates, individualized p.p.b.-range mass accuracies and proteomewide protein quantification andromeda: a peptide search engine integrated into the maxquant environment target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry the healthy infant nasal transcriptome: a benchmark study characterisation of the human plunc gene, a gene product with an upper airways and nasopharyngeal restricted expression pattern the catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells the polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces cathepsin g and neutrophil elastase play critical and nonredundant roles in lung-protective immunity against streptococcus pneumoniae in mice this study was supported by fellowship funding to cjs from the wellcome trust (wt105882ma). conducted data analysis and interpretation. all authors has input into the manuscript and have approved the manuscript for publication. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-32072-3. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-309384-vlk8cebh authors: kolter, thomas title: ganglioside biochemistry date: 2012-12-19 journal: isrn biochem doi: 10.5402/2012/506160 sha: doc_id: 309384 cord_uid: vlk8cebh gangliosides are sialic acid-containing glycosphingolipids. they occur especially on the cellular surfaces of neuronal cells, where they form a complex pattern, but are also found in many other cell types. the paper provides a general overview on their structures, occurrence, and metabolism. key functional, biochemical, and pathobiochemical aspects are summarized. together with glycoproteins and glycosaminoglycans, glycosphingolipids (gsls) contribute to the glycocalyx that covers eukaryotic cell surfaces. gangliosides are sialic acidcontaining glycosphingolipids and provide a significant part of cell surface glycans on neuronal cells. gsls are lipids that contain a sphingoid base and one or more sugar residues [1] . sialic acids ( figure 1 ) are nine-carbon sugars biosynthetically formed from n-acetylmannosamine and phosphoenolpyruvate [2, 3] . with a mean pk a value of around 2.6, they are more acidic than the majority of carboxylic acids and negatively charged at most physiological ph values. the name "ganglioside" was coined by the german biochemist klenk (1896 klenk ( -1971 and assigned to a group of acidic gsls that he isolated from ganglion cells [4, 5] and from the brains of patients who suffered from the so-called amaurotic idiocy [6, 7] . sialic acid was first isolated from submaxillary mucin in 1936 [8] . its structure was elucidated in the nineteen fifties by different groups and it was found to be identical to that of the n-acetylneuraminic acid isolated by klenk and faillard. the first structure of a ganglioside was elucidated in 1963 by kuhn and wiegandt [9] . in 1962, svennerholm suggested a nomenclature of brain gangliosides [10, 11] . the biochemical defects underlying the diseases formerly known as amaurotic idiocy, gm1gangliosidosis [12] , tay-sachs[13] , and sandhoff disease [14] were identified by sandhoff and others in the 1960s. in their structures, gangliosides combine a glycan and a lipid portion and contribute to both, the cellular lipidome and the glycome/sialome [15] . a great variety of carbohydrate sequences are found within the gsls [16] , including the gangliosides [17] . although carbohydrate residues of different structure, linkage, and anomeric configuration occur in gsls, only a limited number of the so-called series with characteristic carbohydrate sequences are found within evolutionary related organisms (table 1) . within the gangliosides, sialic acids can be attached only to a few of the gsl series, in adult mammals especially to the ganglio series. among the sialic acids, n-acetylneuraminic acid is the most frequently found member in humans, but also nglycolylneuraminic acid is abundant in many other species (figure 1) . a total of more than 50 different sialic acids have been described [18, 19] . they can be o-acetylated in positions 4, 7, or 9 [20] , but also n-deacetylated, omethylated, sulfated, or modified by lactonization [21] (see figure 8 ). the nomenclature of gsls specifies the glycan part of these lipids. two ganglioside nomenclature systems are currently in use to assign names to the corresponding structures. most researches prefer the short-hand nomenclature according to svennerholm, which was initially based on the migration order of ganglio-series gangliosides in chromatography [10] . later on, it has been extended to other root structures. the more comprehensive iupac system [22] is less frequently applied. according to svennerholm, a core structure of neutral sugars define the name of a respective series, in which the pyranose forms of dgalactose (gal), d-n-acetyl-glucosamine (glcnac), or d-n-acetylgalactosamine (galnac) are attached in defined order and linkage to lactosylceramide (galβ1,4glcβ1cer) or β-galactosylceramide (galβ1cer). the names contain information about the series ("g" = ganglio, "l" = lacto), the number of sialic acids ("a" = 0, "m" =1, "d" = 2, "t" = 3, "q" = 4, "p" = 5, "h" = 6, "s" = 7), and, indirectly, on the number of uncharged carbohydrates: initially it has been assumed that this number cannot exceed 5, so that the name "ganglioside gm1" indicates that this ganglioside contains (5 − "1" = 4) neutral sugars of the ganglio series. this series is defined by the sequence galβ1-3galnacβ1-4galβ1-4glccer. sialic acids can be attached once, twice, or severalfold to different positions within the core structures. most often, they are found in α2,3-linkage to the "inner" or "outer" galactosyl residue, and in α2,8-linkage to other sialic acids. ganglioside gm1 bears one sialic acid moiety connected to the 3-oh-group of the galactosyl residue in position ii of the gangliotetraose moiety (see also figure 7 ). the corresponding iupac-iubmb short-hand name is ii 3 neu5acgg 4 cer. structures of ganglio-series gangliosides can also be derived from the scheme of ganglioside biosynthesis (see below; figure 12 ). in general, ganglio-series gsls of the 0-series bear no sialic acids on the galactose in position ii, of the a-series bear one, of the b-series bear two, and of the c-series bear three sialic acid residues. however, gm1b and gd1c have a "b" and "c" in their names, although both are 0-series gangliosides (see the scheme of ganglioside biosynthesis, figure 12 ). gm4 is a gala-series ganglioside, although the "g" suggests ganglio series. figure 2 shows the structure of ganglioside gq1b, one of the most abundant gangliosides in adult human brain (g = ganglio series, q = 4 sialic acids, 5 − 1 = 4 neutral carbohydrate residues, and bseries = 2 sialic acids attached to the "inner" galactose). ganglioside core structures can be additionally modified; they can be elongated, such as in gd1agalnac [25] ( figure 3 ). this ganglioside occurs, for example, on spinal neurons [26, 27] and can give rise to autoantibodies as a cause of variant forms of the guillain-barré syndrome [28, 29] and other neuropathies [30] . a modified gm2 derivative that contains taurine in amide linkage to the sialic acid carboxyl group has been identified in the brain of patients with tay-sachs disease [31] . hybrid-type gsls and gangliosides with postglycosylation modifications add further complexity to this substance class [32] . as an example, lacto-ganglio hybrid-type gangliosides have been identified in bovine brain [33] . most gangliosides found in adult mammals belong to the ganglio, gala, lacto, and neolacto series. ganglioside gm4 (figure 3 ), a member of the gala series, has the structure neuacα2,3galβ1cer and is often found with an α-hydroxyfatty acid within the ceramide moiety. during development, also gangliosides with other core structures are transiently formed, such as the stage-specific embryonic antigen ssea-4, a ganglioside of the globo series [34] (figure 4 ). in adults, globo-series gangliosides occur on human erythrocytes [35] , in human kidney [36] , and on various stem cells [37] . for example, ssea-4, but not ssea-3 or globo-h (figure 4 ), is expressed in cord blood-derived mesenchymal stem cells [24] . with the exception of echinoderms (marine organisms of typically pentaradial symmetry), gangliosides are usually absent from invertebrates. arthropods, for example, form acidic gsls with a manβ1,4glcβ1,1 cer core, which contain glucuronic acid instead of sialic acids. for gangliosides of echinoderms [38] [39] [40] [41] [42] , there is no systematic short-hand nomenclature. they show structural features uncommon to mammalian gangliosides, such as sialic acid residues within the oligosaccharide moieties (e.g., lg-2, figure 5 ), α2,11linked sialic acids (e.g., llg-5, figure 5 ), sialic acid methylation or sulfation, or a glycosyl inositolphosphoceramide core, for example, [43, 44] . in cultured neurons, echinodermal gangliosides show neuritogenic and growth-inhibitory activities. in this regard, they are more potent than other figure 2 : structure of gq1b, one of the most abundant gangliosides in adult human brain, which is involved in long term potentiation, synaptic plasticity, and improvement of cognitive function [23] . gangliosides [45, 46] and potentiate the neuritogenic effect of nerve growth factor. heterogeneity is not only found within the glycan part, but also within the ceramide moiety. this can consist of different sphingoid bases [51] , sphinganine, sphingosine, and phytosphingosine of different chain lengths ( figure 6 ), which can be further modified by o-acetylation [52] . in higher animals, c 18 -and c 20 -sphingosine are the most abundant sphingoid bases of gangliosides. the fatty acids found in the ceramide part of gangliosides are mostly saturated. α-hydroxylated fatty acids [53] are not frequently found in brain gangliosides, but are, for example, abundant in gangliosides from intestine, liver, or kidney, and in gm4. to specify the lipoform of a ganglioside, designations such as (d18:1/18:0)gm3 are used for a ii 3 neu5aclaccer with a sphingosine (d = dihydroxy, 1 = one double bond; see also figure 6 ) of 18 carbons and a stearoyl residue (18:0) within the ceramide portion. the functional consequences of the heterogeneities in the lipid component are largely unknown, but the lipid part can mask the receptor function of ganglioside glycans via interaction with membrane cholesterol [54, 55] . as another example, the ceramide portion of gm1 dictates retrograde transport of cholera toxin bound to gm1, and only gm1 with unsaturated acyl chains is sorted from the plasma membrane to the trans-golgi network and the er [56] . ganglioside profiling with respect to glycan and ceramide structures is more and more in the focus of ganglioside analysis. gangliosides are especially abundant in the brain, where their occurrence in the grey matter is about 5-fold higher than in white matter. in adult human brain regions, the β3galt-v β3galnact figure 4 : formation of ssea-4 from globotriaosylceramide (gb3cer). ssea, stage-specific embryonic antigen; t, transferase; fut, fucosyltransferase (modified from [24] figure 5 : examples for gangliosides from echinoderms: lg-2 from the starfish astropecten latespinosus [47] and llg-5 from the starfish linckia laevigata [48] [49] [50] . d-ribo-phytosphingosine values range from 2 to 14 μg lipid-bound sialic acid/mg protein [57] . in the brain, ganglioside expression correlates with neurogenesis, synaptogenesis, synaptic transmission, and cell proliferation [58, 59] . in cultured murine hippocampal neurons, axonogenesis, but not dendritogenesis, is accompanied by an increase in the formation of complex gangliosides and by a shift from the a-to b-series [60] . in extraneural tissues, the ganglioside content is one-to twoorders of magnitude lower than in the brain; relatively high concentrations of ganglio-series gangliosides are found in bone marrow, erythrocytes, intestine, liver, spleen, and testis, gm4 in kidney, and ssea-4 in embryonic stem cells. cellular gangliosides form in part complex, cell-type-and tissue-specific glycan patterns [61] . these are not stable with time, but change with physiological and pathophysiological processes such as cell growth, differentiation, viral transformation, ontogenesis, oncogenesis, embryogenesis [62, 63] , lactation, or tumor progression [64] . gangliosides of the ganglio-series are especially found in the nervous system, where they contribute to 10-12% of the lipid content [65] . during brain development, the ganglioside pattern changes from the prevalence of the simple gangliosides gm3 and gd3 to more complex ones such as gd1a and gt1b [66] (for structures, see figures 7 and 11) . ganglioside content and composition of the brain change also during aging: for example, the amount of lipid-bound sialic acid decreased from 1070 μg/g wet weight in a 25-year-old healthy proband to 380 μg/g wet weight in a 85-year-old individual. despite this, the concentrations of gq1b, gt1b, and gd1b increase with age at the expense of gm1 and gd1a [67] (for structures, see figures 2 and 7) . changes in ganglioside composition with age also occur in liver [68] . there are only indications on the functional consequences of such changes [23] . gangliosides are also found in serum. there, especially gm3, gd3, gd1a, gm2, gt1b, sialylneolactotetraosylceramide ( figure 10 ), gd1b, and gq1b are present, where about 98% of them are transported by serum lipoproteins, predominantly by ldl (66%), followed by hdl (25%) and vldl (7%) [69] . after the discovery of extracellular microvesicles (formerly called microparticles) [70] , which were not distinguished from lipoproteins in earlier experiments, it might turn out that these assignments have to be revised. experiments in rats have shown that after injection of [ 14 c]sialic acid-labeled gangliosides gm3 and [ 3 h]sphingosine-containing labeled ganglioside gm1, the gm1 and gm3 probes had serum half-lives of 1.4 and 1.8 h, respectively. after three hours, 75% of the gm1 and 38% of the gm3 probes were taken up by the liver, and a smaller extent in the central nervous system, kidneys, and lung [71] . subcellularly, the majority of gangliosides resides in the plasma membrane [72] . however, gangliosides also occur in organellar membranes such as in mitochondria, where gd3 regulates apoptosis [73] , and in the nucleus, where they are involved in ca 2+ balance [74, 75] . the glycans found in gangliosides are sometimes modified by the acylation of sialic acid residues in different positions [20] . o-acetylated sialic acids in gangliosides occur especially in growing cells and tissues and are regarded as oncofetal markers present on different tumors [76] . they also serve as receptors for influenza c viruses or coronaviruses [77] . another modified sialic acid is n-glycolylneuraminic acid (neu5gc) [42] . with the exception of certain tumors and in fetuses, it is found only in trace amounts in human tissues [78] . as a component of glycoconjugates, neu5gc is known as the hanganutziu-deicher antigen [79] . it is abundant in many species of the deuterostome lineage, including simians, mice, rat, beef, pork, or lamb, but is nearly absent from birds and reptiles [80] . neu5gc on glycoconjugates contributes to xenoantigenicity in pig-human xenotransplantation [81] , and in cats, neu5gc distinguishes the blood groups a and b: [neu5gc] 2 gd3 is found in feline blood group a erythrocytes, [neu5ac] 2 gd3 on blood group b, and feline blood group ab erythrocyte membranes contain [neugc] 2 gd3, [neu5ac,neu5gc]gd3, and [neuac] 2 gd3 [82] . humans cannot synthesize neu5gc due to an irreversible inactivation of the cmah gene on chromosome 6p21.32 encoding cytidine monophosphate-n-acetylneuraminic acid hydroxylase [83] . this enzyme converts cmpneu5ac to cmpneu5gc and its function is thought to be lost during a "sialoquake" in human evolution [84, 85] . determination of neu5gc and neu5ac-containing gangliosides is either achieved by classical chromatographic techniques combined with antibody staining [86] , or, with higher sensitivity, by combination of chromatography with esi-ms [87] . a potential application is the immunochemical detection of [neu5gc]gm3 as biomarker of nonsmall-cell lung cancer [88] . also ganglioside lactones ( figure 8 ) have been detected in various tissues, for example, gd3 lactone in mouse brain [89] and gd1b lactone in human brain [90] . ganglioside lactones are more immunogenic than gangliosides [91] and occur on tumor cells such as melanoma as tumor-associated antigens. in vitro, lactonization of gangliosides can be followed by a strong negative cotton effect at 235 nm in cd spectroscopy [92] . temporal and spatial differences are also observed for the ganglioside lipid part. in undifferentiated neuronal cell cultures, gangliosides with c 20 sphingosine are present only in trace amounts, but their content increases with the onset of cell differentiation [93] . in rat brain, the fraction of gangliosides containing c 20 sphingosine increases with age [94] in cerebellum [95] or forebrain [96] . spatial differences regarding the sphingoid base chain length have also been detected in mice: while gangliosides containing c 18 species were widely distributed throughout the frontal brain, c 20 species are selectively localized along the entorhinalhippocampus projections [97] . fatty acid and sphingoid base composition is also different between human motor and sensory nerves [98] . nutrition. since gangliosides are components of most vertebrate cell types, they are ingested with the nutrition, for example, with egg yolk (gm3, gm4, and gd3), meat, or in milk [99] . milk contains gangliosides, especially gd3 and gm3, in the membrane fraction of the fat globule. dietary gangliosides modify the intestinal microflora and prevent infections during early infancy [100] . in infants, more than 80% of dietary gangliosides survive the passage through the stomach, in part with acid-catalyzed lactonization, and are absorbed in the intestine [99] . ingestion of dietary gangliosides leads to an increase of gangliosides in serum. in human nutrition, sialic acid derived from gangliosides and other glycoconjugates is an essential nutrient for the rapidly growing brain in infants [59] . the pathophysiological consequences of nutritional neu5gc uptake are unknown. in the past, ganglioside structures and levels were obtained by comprehensive chemical analysis, while nowadays this is attempted within lipidomics using mass spectrometry as the key technology [101] . in general, gangliosides are isolated from tissues and body fluids by chloroform-methanol extraction [102, 103] . extraction efficiency can increase when small amounts of water are present in the extraction solvent [104] , for example, using the solvent system chloroform : methanol : water (5 : 5 : 1) [105] . when extraction is followed by a partition step such as that developed by folch et al. [106] , gangliosides-in contrast to the majority of other lipid classes-partition into the upper, aqueous phase. from there, they can be isolated by a solid phase extraction and separated from neutral gsls by anion exchange chromatography [107] , such as with deae (=diethylaminoethyl) sephadex [108] . o-acetylation of sialic acids and also ganglioside lactonization [90] are modifications that are lost under alkaline conditions. these are often applied to remove glycerophospholipids that contain fatty acids in ester linkage [109] . if information on these modifications is desired, gangliosides from tissues can be determined without alkaline treatment, for example, after chloroform/methanol extraction in a ratio of 1 : 2 and a subsequent partition step [110] . separation of gangliosides according to their glycan composition is achieved by thin layer chromatography (tlc) [111] and by hplc and other techniques that can be coupled to mass spectrometry [112] . this facilitates their identification by mass spectrometry and is required for their characterization by staining with suitable antibodies [113, 114] , lectins, or other binding proteins [115] . although not required for mass-spectrometric profiling, separated ganglioside classes can also be further separated according to their ceramide structure by reversed phase chromatography [116] [117] [118] . quantification can be achieved by staining and densitometry, or-if suitable standard substances are availableby mass spectrometry. since the biosynthetic machinery generates heterogeneities within both, the lipid and the glycan part, comprehensive ganglioside analysis is a highly demanding task within lipidomics [101] . in addition to glycoforms that are also well known from glycoprotein analysis, "lipoforms" [119] become increasingly important to understand ganglioside metabolism and function. various protocols for ganglioside determination by mass spectrometry have been developed [101] . they are largely based on electrospray mass spectrometry as ionization technique; but also maldi plays a role. the available methods range from preanalytics to bioinformatic data handling and include imaging methods using maldi and secondary ion mass spectrometry (sims) to determine the spatial distribution of the analytes [101] . in addition to their constitution, little is known about the conformation of gangliosides in their native, membrane-bound surroundings. x-ray data are not available for gangliosides, although isolated glycans have been investigated by various means. for a simulation of gm3 conformations in a bilayer, compare [120] , which, for example, shows that the glucose moiety of gm3 is buried within phosphatidylcholine head groups. the diversity of cell surface glycans, including that of gangliosides, is generated within the golgi apparatus [121] , and the heterogeneities within the ceramide part result from the biosynthesis of ceramide at the endoplasmic reticulum (er). de novo synthesis of gangliosides can be distinguished from salvage processes [122, 123] , in which sialic acids, sugars, fatty acids, and sphingoid bases are recycled. the latter process can predominate by far in differentiated cells. ganglioside biosynthesis starts with the formation of ceramide ( figure 9 ) at the cytoplasmic leaflet of the er membrane [124] [125] [126] . the first step, the condensation of l-serine and a coenzyme a-activated fatty acid is catalyzed by the pyridoxal phosphate-dependent serine palmitoyltransferase (spt) [127] . the incorporation of l-serine into gsls can be used to monitor their de novo biosynthesis using l-serine radiolabelled in the position 3 8 isrn biochemistry (the carbon in position 1 is lost as carbon dioxide). in the brain, the external supply of l-serine by astrocytes is essential for neuronal lipid biosynthesis and brain development [128] . in agreement with this observation, genetically engineered rodents with deficient phosphoglycerate dehydrogenase required for l-serine formation from d-glucose show drastically reduced ganglioside levels, defects in brain morphogenesis, and drastically reduced lifespan [129, 130] . the next step in sphingolipid biosynthesis is the nadph-dependent reduction of 3-ketosphinganine to sphinganine by 3-ketosphinganine reductase, followed by acylation of sphinganine to dihydroceramides of different chain lengths [131] . during salvage, also other sphingoid bases are acylated by n-acyltransferases of the lass family. lass 1 encodes ceramide synthase 1, which is expressed in the brain and involved in the formation of the membrane anchor of gangliosides. in mice, spontaneous recessive mutations in the lass1 gene are associated with cerebellar ataxia and purkinje cell degeneration [132] . although the ceramide part of brain gangliosides contains mostly nonhydroxylated fatty acids, apparently all members of the lass family are also able to transfer the corresponding 2-hydroxy-fatty acids [133] . dihydroceramides are dehydrogenated to ceramide by the dihydroceramide desaturase des1 [134] , or hydroxylated to phytoceramides by des2. ceramide is the common precursor of gsls and sphingomyelin and is transported to the golgi apparatus at least in part in a protein-dependent manner by the transport protein cert [135] [136] [137] . formation. gsl synthesis continues by the stepwise transfer of nucleotide-activated monosaccharide units first on ceramide and then on gsls with growing glycan chains. glycosidation is coupled to exocytosis through the golgi apparatus to the plasma membrane [138] at the rate of bulk vesicle flow [139] . the complex ganglioside and gsl glycoforms on eukaryotic cell surfaces are generated by only a few enzymes that act within a combinatorial biosynthetic pathway [140, 141] . the first glycosyltransferases involved in ganglioside biosynthesis have been characterized in the laboratories of roseman and basu [142] . according to the number of sialic acids connected to the "inner" galactosyl residue, ganglioseries gangliosides are classified into members of the 0-, a-, b-, and c-series ( figure 12 ). b-series gangliosides contain the neu5acα2,8neu5ac sequence, which is commonly not found in glycoproteins. higher members of these different subseries can be formed by the action of the same glycosyltransferases, which show less specificity than those acting early in the pathway [143] [144] [145] [146] [147] . the glycosyltransferases and sialyltransferases [148, 149] of the ganglioside biosynthetic pathway are expressed in a cell-type-and developmental-dependent fashion. ganglioside pattern changes during the development of the brain [150] , and after differentiation, differences in glycolipid composition have even been found between different neuronal cell types [151] . in addition, ganglioside patterns vary between different cell types and change with the differentiation of the cell. as an example, β1,3-n-acetylglucosaminyl-transferase expression, which leads to the formation of glycolipids of the lacto and neolacto series ( figure 10 ), is high during murine embryonic development and decreases after birth to undetectable levels in most cell types [152, 153] . in adult animals, expression is high in spleen [154] , and in cerebellum, it is restricted to purkinje cells [155] . gsls including gangliosides are formed biosynthetically at intracellular membranes from which they are transported to the plasma membrane by exocytotic membrane flow [138] . while many human diseases are known that are due to defects in gsl and sphingolipid degradation, the only known human disease caused by a defect glycosyltransferase of ganglioside biosynthesis is the human autosomal recessive infantile-onset symptomatic epilepsy syndrome, which is caused by a nonsense mutation in the gene encoding gm3 synthase [156] . a principal difference between ganglioside biosynthesis in the golgi apparatus and degradation in the endolysosomal compartment is that during gsl formation, membranebound glycosyltransferases interact with their membranebound glycolipid substrates by diffusion within the twodimensional plane of the lipid bilayer. therefore, reaction rates can become independent of the reaction volume and obey two-dimensional enzyme kinetics. this means that kinetic constants can be normalized on lipid surface area instead of reaction volume, for example, in terms of the amount of membrane protein [157] . as a consequence, glycosyltransferases that lack their transmembrane domain lose most of their activity towards membrane bound substrates [158] . during degradation in endosomes and lysosomes, the glycosidases are soluble enzymes, and the substrates are membrane-bound. this explains in part the requirement for endosomal and lysosomal lipid-transfer proteins for the degradation of gsls with short glycan chains, which is not the case in biosynthesis. in addition to ganglioside biosynthesis in the golgi apparatus, there are also indications ganglioside formation by plasma membrane-associated glycosyltransferases [159] . in the monoglycosylceramides glucosylceramide (glccer) and galactosylceramide (galcer), which are also called cerebrosides, the hexosyl residues are present in βanomeric configuration. galcers with α-configuration occur only in lower organisms [42] and are highly immunogenic for mammals [160] . most gangliosides are biosynthetically derived from glccer; only ganglioside gm4 is derived from galcer. ganglioside gm4 has been discovered as a minor component of human brain gangliosides [161] , where it is localized within myelin [162] . it also occurs, for example, on erythrocytes, kidney, and in the intestine and is abundant in some fish species. however, the most frequently found members of the gala series are galcer and sulfatide (galcer-3-sulfate) in oligodendrocytes, schwann cells, kidney, testis, and intestine. they are present in high concentrations in the multilamellar layers of the myelin where they are required for glial adhesion [163] , apparently via interaction between the carbohydrate head groups of sulfatide and galcer on different myelin layers [164] . myelin lipids contain the highest fraction of 2-hydroxy-fatty acids, which are formed by fatty acid hydroxylase-2 [165] . their presence in gala-series gsls contributes to carbohydrate-carbohydrate interactions between the gsls [166] . in contrast to galcer synthase, glccer synthase appears to be dispensable for oligodendrocytes [167] . while ceramide galactosylation catalyzed by udp-glucose:ceramide galactosyltransferase (galt3) [168] occurs at the er membrane, the later steps of gala-series gsl biosynthesis, formation of sulfatide [169] , digalactosylceramide [170] , and ganglioside gm4 take place in the lumen of the golgi apparatus. in contrast to most glycosyltransferases in ganglioside biosynthesis, which are type ii transmembrane proteins, ceramide galactosyltransferase is a type i transmembrane protein with the catalytic domain on the luminal side of the er [171] . according to data obtained in zebrafish and mice, gm4 can be formed by st3gal v, which can also make gm3. therefore, gm4 and gm3 formation appear to depend on the availability of their precursors, galcer and laccer [172] . little is known about the function of gm4. it can interact with the myelin basic protein, shows immunosuppressive properties, and can prevent experimental allergic encephalomyelitis in guinea pigs [173] . glucosylceramide. the first step in the biosynthesis of most gangliosides is the transfer of a glucose residue from udp glucose to ceramide catalyzed by udp-glucose:ceramide glucosyltransferase [174, 175] . although glccer and galcer synthases catalyze similar reactions, their cdnas share no sequence homology. ceramide glucosyltransferase is a type iii transmembrane protein. it forms noncovalent dimers or oligomers [176] with their cterminal catalytic domains in the cytosol [177] . since the formation of glccer occurs on the cytoplasmic face [178] and that of laccer on the luminal site of the golgi membrane [179] , glucosylceramide has to be translocated across a membrane. this is mediated by a flippase of unknown identity: the abc-transporters, abc-b1 and -c1, translocate short chain glccer analogs through the golgi membrane [180, 181] . transversal translocation can be carried out after transport of glccer by the cytoplasmic lipid-transfer protein fapp2 (four-phosphate adaptor protein 2) either to the er [182] , where it might be translocated by an uncharacterized flippase [183] , or at the trans-golgi [184] . a part of the glccer pool can reach the cytosolic leaflet of the plasma membrane where it can be degraded by the β-glucosidase gba2 [185] . candidate cytosolic glccertransporters are the glycolipid transfer protein gltp and fapp2. the biosynthesis of higher gangliosides occurs on the luminal face of the golgi apparatus [186] , so that their glycan chains are orientated extracytoplasmic. laccer is formed by galactosyltransferase i, which transfers a galactose residue from udp galactose to glucosylceramide [187] . further carbohydrate residues are transferred in a stepwise manner to the growing glycan chains. laccer and its sialylated derivatives, the hematosides gm3, gd3, and gt3 ( figure 11 ) serve as precursors for complex gangliosides of the 0-, a-, b-, and c-series. these different series ( figure 12 ) are characterized by the presence of no (0-series), one (a-series), two (b-series) or three sialic acids residues linked to the position 3 of the "inner" galactosyl residue. in adult mammalian brain, gangliosides from the 0-and c-series are found only in trace amounts, and gm1b and gd1α are transiently expressed during chick brain biogenesis [188] . 0-series gangliosides (gm1b, gd1c, and gd1α) are found in genetically engineered mice deficient in st3gal v (gm3 synthase), where they are present in amounts that correspond to the total ganglioside content of normal animals [189] . these mice are not able to form gm3 and higher gangliosides of the a-c-series. they display altered glucose homeostasis with an accelerated insulin receptor signalling pathway, a key finding that demonstrates the inhibition of the insulin receptor by gm3 or a higher ganglioside derived from it in vivo [189] . c-series gangliosides (figures 12 and 13 ) are formed during mammalian brain development where they are thought to be involved in growth, differentiation, and migration of neuronal cells. they are abundant in fish brain, and in adult rats; they occur in liver, kidney, and pancreas [190] and in tumors such as glioma. the transferases that catalyze the first steps in ganglioside biosynthesis show high specificity towards their glycolipid substrates. the relative amounts of laccer, gm3, gd3, and gt3 seem to determine the amount of 0-, a-, b-, and c-series gangliosides. the glycosyltransferases that act late in this pathway represent a kind of assembly line and transfer the respective carbohydrates to glycosyl acceptors that differ only in the number of sialic acid residues bound to the "inner" galactose residue. the complex "α-"gangliosides with sialic acid moieties in α2, 6-glycosidic linkage to n-acetylgalactosamine residues is specific for cholinergic neurons [191] and has been added later to the biosynthetic scheme [192] . in mice, the sialyltransferases that form gangliosides gd1a and gt1b have been identified as st3gal ii and st3gal iii [193] . a significant advance towards understanding the function of the complex ganglioside pattern found in eukaryotic cells is the development of mice with defects in distinct biosynthetic steps [194] . a mouse melanoma cell line deficient in glccer and glccer-derived gsls was viable and showed only minor changes in cellular morphology and growth rate. from these observations it was concluded that gsls including gangliosides are not essential for animal survival [195] . later, it was reported that mice with targeted disruption of the ceramide glucosyltransferase gene displayed no cellular differentiation beyond the primitive germ layers and died around day 7.5 of embryonic development [62] . mice deficient in b4galnt i (gm2synthase) are not able to form gm2, gd2, and higher gangliosides derived from them. although these animals show only subtle impairment of brain function [196] , they exhibit multiple defects, such as axonal degeneration, defects in myelination [197] and motor function [198] , or an impaired response of t cells to interleukin 2 [199] , only to mention a few. later studies showed that cd4-and cd8-positive t cells require different ganglioside subsets for activation [200] . the mutant male mice are sterile and also show morphological and functional defects in the testis [196] . further examination of galnac-transferase deficient mice revealed that gm1-deficiency is accompanied by parkinsonlike symptoms, which could be rescued by l-dopa or the membrane permeable gm1-analog liga20 (see figure 19 ) [201] . this is in agreement with a series of reports that gm1 can alleviate symptoms in models of parkinson disease, for example, [202] . in parkinson disease, anionic lipids and especially gm1 inhibit aggregation of α-synuclein to cytotoxic fibrils [203] . mice deficient in st8sia i (gd3-synthase) do not form gd3 and b-series gangliosides. they have a normal life span and are without detectable developmental defects [204] . when these mice were crossbred with mice carrying a disrupted b4galnt i gene, the resulting double mutant mice express only ganglioside gm3 as their major ganglioside. these "gm3-only-mice" are extremely susceptible to sound stimuli, develop lethal seizures, and display a sudden death phenotype [204] . double knockout mouse deficient in b4galnt i and st3gal v (gm3-synthase) are not able to form any ganglioside of the ganglio-series. these animals are severely diseased and show elevated levels of laccer, laccersulfate, and traces of other gangliosides that are present also in normal brain [205] . sphingolipid biosynthesis is a highly regulated process and also coordinated with sterol and glycerolipid biosynthesis. sphingolipids are major regulators of lipid metabolism and activate sterol-regulatory element binding proteins (srebps) [206] . the sphingomyelin synthaserelated synthase, the ceramide transporter cert, and proteins of the orosomucoid-(orm)-familie seem to play key roles in sphingolipid homeostasis [207] . ganglioside pattern are characteristic for a cell type in a certain differentiation state, and, for example, mice deficient in gm3-synthase that cannot form the typical brain gangliosides show a ganglioside content similar to that of normal animals [189] . how exactly the relative amounts of gangliosides are controlled is not clear [208] , but the transcriptional regulation of transferase genes seems to be a key point [208] . the picture gets more complicated by the fact that different transferase isoforms with different properties can be present: three murine gm3-synthase isoforms that arise from two transcripts have been characterized. one is resident in the er membrane, the two others in the golgi, but with different half-life [209] . in addition, the kinetic parameters of the transferases, their topological organization within the golgi apparatus, or spatial neighborhood to other transferases will influence the resulting ganglioside pattern. an attempt has been made to calculate glycolipid pattern on the bases of the kinetic constants of the transferases, that were estimated from the steady state concentrations of the glycolipid substrates in intact cells [210] . contradictory results have been reported on the subcellular localization of the glycosyltransferases involved in the biosynthesis of ganglioseries gangliosides [211] . an additional feature of ganglioside biosynthesis and its regulation [212] is the formation of functional complexes, as predicted by roseman [213] . in these complexes [140] , the glycosyltransferases do not only form functional platforms, but can also show altered activity and suborganellar localization [214] . one of these complexes characterized in certain cho cells [215] comprises b4galnt i and b3galt iv (figure 12 ), so that it can accept gm3 and release gm1. this might explain why the brain contains large amounts of gm1 and gd1a, but little gm2. also galt i, st3gal v, and st8sia i can form such a complex [216] . 6.1. general. the constitutive degradation of gangliosides takes place in endosomes and lysosomes. in addition, also the plasma membrane-associated sialidase neu3 [217, 218] can degrade gangliosides and is, for example, highly expressed on melanoma cells [219] . even the nuclear envelope contains sialidases, with neu3 in the inner and neu1 in the outer nuclear membrane [220] . lysosomal ganglioside degradation takes place after the endocytosis of parts of the plasma membrane at intraendosomal and intralysosomal membranes and related lipid aggregates. this requires the presence of suitable glycosidases [221] , of an appropriate ph, in some cases also of lipid-transfer proteins, and of an appropriate composition of the ganglioside-containing membranes [222] . as proposed in 1992, two different membrane pools are present in endosomes and lysosomes [223] (figure 15 ). they differ in lipid-and protein composition and function. while the luminal membrane pool that is derived from the plasma membrane or by autophagy is degraded, the perimeter membrane ( figure 15 ) is protected from degradation by various means [224] . this ensures the integrity of the compartment, which can be abolished during apoptosis [224] . a marker lipid that is exclusively found in luminal membranes [225] is bis(monoacylglycero)phosphate (bmp; figure 14) , chemically incorrect also named as lysobisphosphatidic acid (lbpa). bmp plays a key role for membrane degradation [226] and is formed from phosphatidylglycerol [227] . due to its sn1, sn1 configuration, it is only slowly degraded by lipases and persists on inner membranes, in which it can amount up to 70% of total phospholipids [228] . with a predicted pk a value of about 2, bmp is negatively charged even at lysosomal ph. in vitro studies show that negatively charged lipids are required for binding of lysosomal proteins to membranes. although other negatively charged lipids such as dolichol phosphate or phosphatidylinositol can be present on luminal membranes, bmp appears to be the key factor that distinguishes this membrane pool from the perimeter membrane. on the other hand, the perimeter membrane of endosomes and lysosomes shows an entirely different lipid and protein composition. it is protected by a glycocalyx formed by highly n-glycosylated integral membrane proteins [229, 230] , and ganglioside gm3 present in this membrane is resistant to degradation [231] . ganglioside degradation starts with the action of glycosidases that cleave off monosaccharide units from the non-reducing end of the ganglioside glycan chains. this happens in a sequential manner, which explains the different human diseases that are associated with defects in this pathway. the glycosidases are soluble enzymes in the lumen of endosomes and lysosomes. it turned out that their activity is not sufficient towards gsl substrates with cleavage sites in proximity to the intralysosomal membrane surface. although also other factors play a role, this can be attributed to steric hindrance by adjacent membrane components that impede the access of the soluble enzyme. for example, in wild-type and gm2-activator deficient fibroblasts, radiolabelled gd1agalnac (figure 3 ), which has the same terminal trisaccharide as gm2, is degraded in the absence of the gm2-activator protein, while the degradation of gm2 itself is strictly dependent on the presence of the activator [232] . as glycosidase substrates, gsls with four carbohydrate residues or less require the additional presence of small lipid binding glycoproteins, either the gm2 activator protein or one of the four saposins a-d. these act in part as lipid-transfer proteins that extract the membrane-bound substrates and present them to the hydrolases. they have different specificities and mechanisms of action [233] . in the case of gangliosides, at least the gm2-activator protein and saposin-b participate in the degradation of gm1, gm2, and gm3 ( figure 16 ). in vitro, in addition to enzymes and activator proteins, also an appropriate membrane-lipid composition of the ganglioside-containing membrane is required for degradation [222] . saposin-a [234] and saposin-b [235] extract membrane lipids much better from membranes that are rich in bmp and poor in cholesterol. bmp also increases the ability of the gm2 activator to solubilize lipids [236] and stimulates the hydrolysis of membrane-bound gm1 by gm1 β-galactosidase [237] and of ganglioside gm2 by β-hexosaminidase a [236] . bmp also stimulates hydrolysis of the kidney sulfatide with ganglio-series gsl-core sm2 (gangliotriaosylceramide-ii 3 sulfate) by β-hexosaminidases a and s in the presence of the gm2 activator [238, 239] . cholesterol, which is known to stabilize lipid bilayers, has to be transported from intraendosomal membranes to the npc1 protein resident in the endosomal perimeter membrane by the soluble lipid-transfer protein npc2. in vitro, this transfer is greatly stimulated by bmp and strongly inhibited by sphingomyelin [240] . saposins are small, water-soluble lysosomal lipid-binding and -transfer proteins of about 8-11 kda molecular weight. they are derived from a common precursor protein, prosaposin, by proteolytic processing. saposins belong to a family of proteins with conserved three-dimensional fold [241] and occur as homo-and heterodimers and -oligomers. the first saposin has been characterized in 1964 as the so-called sulfatide activator since it enables the degradation of sulfatide by arylsulfatase a [242] . today this protein is known as saposin-b or sap-b. saposin-b has many functions: it is a lipid-binding protein with broad specificity [243] and forms water-soluble lipidprotein complexes [244] . with respect to gangliosides, it is able to stimulate the degradation of ganglioside gm1 by gm1-β-galactosidase [237] . studies in cultured human skin fibroblast derived from saposin-b and prosaposin-deficient patients show that it is also required for the degradation of gm3 [245, 246] . it is important to note that glycosylation of saposin-b is essential for some of its functions and that human patients without this postranslational modification die, although the unglycosylated variant protein is present in lysosomes [235] . mechanistically, saposin-b dimers seem to act similar to the gm2 activator: x-ray data indicate that they can adopt two conformations, an open one and a closed one [247] . according to a model view, the open conformation interacts directly with the membrane and extracts the lipid ligand. this is accompanied by a change to the closed conformation in which the ligand is exposed to the degrading enzyme in a water-soluble activator-lipid complex. human patients with an inherited deficiency of saposin-b develop an atypical form of metachromatic leukodystrophy with the accumulation of sulfatides, digalactosylceramide, and globotriaosylceramide [248] (see figure 16 for structures). saposin-b knockout mice show enhanced levels of sulfatides especially in brain and kidney [249] . activator. the gm2 activator is a small glycoprotein of 17.6 kda in its deglycosylated form and is required for the degradation of ganglioside gm2 by βhexosaminidase a in vivo [250] . inherited deficiency of the gm2-activator protein leads to the ab variant of gm2 gangliosidoses [251] . based on the x-ray structure [252, 253] and data from photoaffinity labeling [254] , in some respects the gm2-activator acts in a way similar to saposin-b. a more detailed picture of the binding mode was derived from binding studies using a spin-labelled gm2 activator to phosphatidylcholine bilayers [255] . the protein can extract a variety of lipids, which has been exploited for assay development [256] . however, its major function is to form a water-soluble gm2-protein complex that is the native michaelis-menten substrate of β-hexosaminidase a [250] . negatively charged lipids such as bmp, dolichol phosphate, or phosphatidylinositol increase the extraction efficiency towards gm2 [236] , gm1 [237] , and other lipids [257] from liposomal membranes. binding characteristics of the gm2 activator are altered by the presence of a his tag [257] . in langmuir experiments, the gm2-activator protein is able to penetrate into a phospholipid monolayer, but only when the lateral pressure is below a critical value, which depend on the lipid composition and is in the range from 15 to 25 mn/m [258] . in addition to its function as a ganglioside-transfer protein, the gm2 activator binds also other lipids like phosphatidylcholine [259] and platelet activating factor (paf) and inhibits its action [260, 261] . it is not clear whether the gm2 activator displays inherent hydrolytic activity towards lipid substrates such as platelet activating factor [262] or phosphatidylcholine [259] . apparently unrelated to its gm2 transfer property is the function of the gm2 activator as adipokine [263] . gm2activator orthologs might serve different functions in other organisms, for example as a pheromone-binding protein in drosophila [264] , or an inhibitor of paf-induced chemotaxis in nematodes [265] . the saposins and the gm2-activator play major roles in the transfer of lipid antigens to membrane-resident cd1proteins [266, 267] . degradation. gm1-β-galactosidase is a protein of 64 kda, which is derived from an 88-kda precursor [268, 269] . an alternatively spliced, enzymatically inactive β-galactosidase form of 67 kda is an elastin/laminin-binding protein [270] . gm1-β-galactosidase is part of a lysosomal multienzyme complex, together with the so-called protective protein (carboxypeptidase a), sialidase, and n-acetylaminogalactose-6-sulfate sulfatase [271] . gm1-β-galactosidase catalyzes the hydrolytic cleavage of several β-galactosides. the hydrolysis of ganglioside gm1 to gm2 requires the presence of either the gm2-activator protein, or saposin-b [56] , or, in vitro, of an appropriate detergent. degradation. gm2 is degraded by the cleavage of the n-acetylgalactosaminyl residue by β-hexosaminidases. in mice, the substrate specificity of the murine lysosomal sialidase allows for a significant cleavage also of the sialic moiety in gm2 (to yield ga2) [272] . cleavage of the galnac residue requires the presence of the gm2-activator protein in vivo, or of an appropriate detergent in vitro. three gene products participate in gm2 hydrolysis, the β-hexosaminidase αand β-chains, and the gm2-activator protein. β-hexosaminidases are dimers that result from the combination of their αand β-subunits and differ in properties such as stability and substrate specificity. β-hexosaminidase a with subunit composition α,β cleaves terminal β-glycosidically linked n-acetyl-glucosamine and n-acetylgalactosamine residues from negatively charged and uncharged glycoconjugates by a retaining doubledisplacement mechanism. the enzyme has two active sites, one on the α-chain and the other on the β-chain [273] . β-hexosaminidase b (ββ) [274, 275] predominantly cleaves uncharged substrates such as ga2 and oligosaccharides with terminal n-acetyl-hexosamine residues (see also figure 16 ). β-hexosaminidase s (αα) is thermolabile and of secondary significance for gm2 degradation, but it contributes to the degradation of glycosaminoglycans and sulfated glycolipids [238] . defects in enzymes and other proteins required for lysosomal degradation of complex lipids and of oligomeric or polymeric biomolecules lead to inherited diseases, the lysosomal storage diseases [276] . they can be classified according to the stored substances, as sphingolipidoses [277] , mucopolysaccharidoses, mucolipidoses, glycoprotein-, and glycogen-storage diseases [278, 279] . ganglioside degradation is impaired in the gangliosidoses and secondarily also in other sphingolipid storage diseases [280] . the principles [281] governing pathogenesis [282, 283] and therapy of sphingolipidoses [284] are also valid for the ganglioside storage diseases. key factors are the residual activity of the degrading system, which determines the course of the disease [285, 286] , and the cell-type-specific expression of storage material. due to the cell-type-specific expression of gangliosides, the central nervous system is especially affected in the gangliosidoses. in sphingolipidoses in general, the storage lipids coprecipitate other hydrophobic substances present in the endolysosomal compartment, lipids and proteins, as secondary storage products [280] . in niemann-pick disease, type c, which is a primary defect of endosomal cholesterol transport, a secondary accumulation of sphingomyelin (therefore the name niemann-pick) and of gangliosides is observed that is also of therapeutic relevance [287, 288] ; for a remarkable treatment of niemann pick c1 fibroblasts with a histone deacetylase inhibitor, compare [289] . secondary storage of gangliosides gm2 and gm3 occurs also in hurler disease [290] (mucopolysaccharidosis type i; α-liduronidase deficiency). lipid storage produces a kind of traffic jam [291, 292] , which interferes with lipid transport and lysosomal function. primary and secondary storages substances can impair nutrient delivery via the endolysosomal system: as demonstrated in mouse models of gm1 gangliosidoses and in a variant form of the gm2 gangliosidoses, sandhoff disease, iron homeostasis is impaired in the animals, and supplementation of the animals with iron ions increased their life expectancy by nearly 40% [293] . since also autophagy can be impaired in lysosomal storage diseases [294] , both pathways may lead to a shortage of nutrients. ganglioside degradation is impaired in the gangliosidoses. in another disease, galactosialidosis, the primary defect of carboxypeptidase a (protective protein), leads to a secondary loss of β-galactosidase and sialidase neu1 accompanied by gm1 storage [271] . gangliosidoses are caused by defects in the genes encoding glycosidases or lipidtransfer proteins that are required for lysosomal ganglioside degradation. the theoretical basis for the therapeutic approaches towards gangliosidoses is the "threshold theory" [286] , which predicts that the ratio of substrate influx into the lysosomes and the degradation capacity determine the course of the diseases. both parameters can be addressed by different therapeutic approaches [281] . 7.1. gm1 gangliosidosis. gm1 gangliosidosis is caused by an inherited deficiency of gm1-β-galactosidase (acid βgalactosidase; glb1; ec3.2.1.23) [295] . after the description of the first patients [296] it became also known as landing diseases [297] . it is a rare disease with an autosomal recessive mode of inheritance and characterized by the accumulation of gm1 and ga1 (figure 16 ) in neuronal cells [12] . according to the substrate specificity of the variant enzyme in the patients, an inherited defect of the β-galactosidase can also lead to another disease, morquio disease, type b. three clinical forms of gm1 gangliosidosis can be distinguished, infantile (type 1) gm1 gangliosidosis with the developmental arrest and progressive deterioration of the nervous system in early infancy and a life expectancy of about 2 years, late infantile/juvenile form (type 2), and an adult/chronic form (type 3). dysmorphic changes characteristic for morquio disease type b are less prominent or completely absent in these clinical forms. in addition to gm1, other enzyme substrates accumulate, such as ga1 (figure 16 ) [12] , oligosaccharides from glycoproteins, and intermediates of keratin sulfate degradation [268] . these substances are stored in different organs, according to their major site of biosynthesis. lysosomal gm1 accumulation in neurons leads to the degeneration of the nervous system. like in other storage diseases, an inflammatory response [298] , neurorestorative properties of excess ganglioside gm1 [299] in the plasma membrane, and an unfolded protein response [300] contribute to pathogenesis. such as in other sphingolipidoses [281] , severity and progression of the disease correlate with the residual enzymatic activity in cells and body fluids. morquio type b disease clinically resembles a mild phenotype of morquio a disease, where keratan sulfate accumulates due to n-acetyl-galactosamine-6-sulfatase deficiency. like gm1 gangliosidosis, morquio type b is due to the inherited defect of gm1-β-galactosidase. it is characterized by the predominant storage of keratan sulfate and oligosaccharides with terminal β-galactosyl residues. patients show generalized skeletal dysplasia without involvement of the nervous system and without hepatosplenomegaly; for a clinical description, compare [268] . differences between gm1 gangliosidosis and morquio b disease can be attributed to a lower affinity and activity of β-galactosidase variants towards substrates with gal-β1,4-glcnac motifs in morquio patients compared to the gal-β1,3-galnac motive present in ganglioside gm1 [301] . there is no causal therapy available for gm1-gangliosidosis; however, progress is made towards the development of pharmacological chaperones also for this lysosomal disease [302] [303] [304] . the gm2-gangliosidoses are caused by defects in degradation of ganglioside gm2 [305] . the three variant forms of the gm2-gangliosidoses are named according to the hexosaminidase isoenzyme that remains intact. the b-variant, in its infantile course better known as tay-sachs disease, is caused by the deficiency of hexosaminidases a and s, but with normal hexosaminidase b. the 0 variant, or sandhoff disease, is caused by the deficiency of the β-chain and the resulting deficient activity of β-hexosaminidases a and b (therefore, none of the major enzymes is intact), however with the remaining activity of β-hexosaminidase s. the ab-variant-β-hexosaminidases a and b (and s) intact-results from mutations in the gm2-activator gene; so that tissue samples from the patients are able to degrade gm2 in detergent-containing enzyme assays. clinically, the b variant of gm2 gangliosidoses can be subclassified into infantile, juvenile, chronic, and adult onset forms. the infantile form, tay-sachs disease, has a higher prevalence among ashkenazi jews with a heterozygote frequency of 1 : 27. affected children are normal at birth and show first symptoms, such as mild motor weakness, a cherry red spot in the central retina, and increased startle reaction between 3 and 6 months of life. progressive deterioration with weakness, hypotonia, or poor head control leads to a vegetative state and death often between the second and fourth year of life. juvenile and adult course is observed in patients with a higher residual activity of the variant hexosaminidase a [285] . symptoms are very heterogeneous; for a clinical description, compare [305] . the b1 variant of gm2 gangliosidoses [309, 310] was very difficult to elucidate: synthetic uncharged substrates used for diagnosis such as mufglcnac (figure 17 ; for kinetic parameters see [311] ) were cleaved, suggesting the presence of β-hexosaminidase, and also the gm2 activator was present. as it turned out, the b1 variant differs enzymatically from the b variant by an altered substrate specificity of the variant β-hexosaminidase a. while uncharged substrates are cleaved, no activity is detected towards gm2 and towards sulfated, negatively charged [312] synthetic fluorogenic substrates. in the b1 variant, the function of the α-chain active site is defective, but subunit association, enzyme processing, and the activity of the β-chain are not impaired. homozygous patients with the b1 mutation show the course of the juvenile disease; compound heterozygotes with a b1 and a null allele show a late infantile course. disease. the 0 variant of gm2 gangliosidosis was the first gangliosidosis for which the underlying enzymatic defect was identified [14] . due to the deficiency of two enzyme activities, β-hexosaminidases a and b, storage of negatively charged glycolipids characteristic for tay-sachs disease and, in addition, of uncharged substrates such as ga2 in the brain and globoside in visceral organs (figure 16 ) is observed. in infantile sandhoff disease, patients show clinical and pathological manifestations of tay-sachs disease (infantile b variant) and in addition also organomegaly and slight bone deformations. for further symptoms and the description of juvenile and adult forms, compare [305] . the ab variant is due to the deficiency of the gm2-activator protein [251] , with intact β-hexosaminidases a and b (and s), therefore the name. the disease is characterized by accumulation of gm2 and ga2 (for structures, see figure 16 ). the clinical picture [305] resembles that of tay-sachs disease with a delayed appearance of symptoms; an animal model is available [313] . although lysosomal gm2 as the major storage compound in gm2 gangliosidoses is neither toxic nor immunogenic, its accumulation induces inflammatory responses as demonstrated for glycoconjugates in the murine model of sandhoff disease [108] . huge axon hillock enlargements, the so-called meganeurites, have been observed in neurons of patients with different lysosomal storage diseases, which might be attributed to the storage substance gm2 and contribute to synaptic dysfunction [314] . as in other sphingolipidoses [281] , the corresponding (more toxic) lysolipid, in this case lysogm2 (figure 18 ), is elevated [315, 316] and contributes to the pathogenesis. lysogm2 has been suggested as a biomarker for tay-sachs and sandhoff disease [317] ; for occurrence and role of lysogsls in acquired diseases, compare [318] . despite naturally occurring animal models of gm2 gangliosidoses in dogs, cats, and pig, murine models are used for therapy studies. since the mouse model of tay-sachs disease is largely asymptomatic, the mouse model of sandhoff disease is used for most studies [272] . despite some success in the experimental treatment of juvenile and adult patients as well as in the animal models, there is no causal therapy available for the severe forms of the gm2 gangliosidoses. the limitations of the substrate reduction approach, which reduces the gm2 influx into the lysosomal compartment, have been evaluated by a genetic experiment: sandhoff-disease mice were crossbred with mice defective in gm2 synthase. the lifespan of these animals was much longer than that of sandhoff-disease mice, but instead of gm2 storage they developed a oligosaccharide storage, neurological disease [319] . therapeutic approaches such as bone-marrow transplantation [320] , enzyme-replacement therapy with recombinant highly phosphomannosylated β-hexosaminidase a [321] , or transplantation of neural stem cells [322] have been investigated in the animal model of the 0 variant, substrate-reduction therapy with n-butyl deoxynojirimycin [323, 324] , and with pyrimethamine as pharmacological chaperone [325, 326] in adult patients and gene therapy in endothelial cells [327] . treatment of the accompanying inflammation is beneficial [328] . aspects. in addition to inherited diseases, ganglioside levels can also be altered in several acquired diseases [318] . for example, gangliosides play roles in neurological diseases such as alzheimer's [329] , parkinson, or huntington's disease [330] . in cancer, ganglioside expression can also be altered in tumor cells with an impact on signalling and tumor-host interactions [331] . [neu5gc]gm3 [332] , gd2, gd3, gm2, and fucosylgm1 are regarded as tumor-associated antigens [333] and are targets for the immunotherapy of cancer [334] . also several neuropathies including variant forms of guillain-barré and miller-fisher syndrome are caused by serum antibodies against gangliosides [335] . there are only a few therapeutic roles for gangliosides, especially since they can induce neuropathies. in the past, gangliosides isolated from bovine brain have been investigated and also applied to human patients to improve neural repair and for the treatment of stroke [336, 337] . also the direct application of ganglioside gm1 into the brain of patients with alzheimer disease has been evaluated [338] . as indirect roles, the inhibition of ganglioside biosynthesis for the treatment of insulin resistance [339] , the interference with microbial binding to gangliosides [340] , or the reduction of neurotoxicity with liga20 [341] has to be mentioned. a plethora of functions has been attributed to gangliosides [342] , for example, for gm3 [343] , the most abundant ganglioside in most mammalian cell types, but not in neurons, or for gm1 [344] . in general, gangliosides mediate their function via interaction with soluble or membranebound binding molecules outside the cell ("trans" interaction), or by influencing properties of proteins within the same membrane ("cis" interaction) [32, [345] [346] [347] [348] . "trans" interactions occur between the glycan part of gangliosides on the one side with lectins on the other side. also gangliosides contribute to the chemical high-density sugar code of cell surfaces [349] . for example, gm1 can be recognized by galectin-1 [350] and sialic acids in α2, 3 figure 19 : structure of liga20, a semitruncated and halogenated gm1 analog that can pass the blood-brain barrier. are recognized by the sialic acid binding immunoglobulin lectins siglec-4, α2,6-sialosides by siglec-2, and α2,8sialosides by siglec-7 [347] . also carbohydrate-carbohydrate interactions can play a role [351, 352] . although interactions between individual carbohydrate residues [353] are weak, clustering of gangliosides offer the possibility for multivalent interactions, if they are not buried under glycoprotein glycans. apparently, gm3 on mouse melanoma b16 cells can mediate cell adhesion to mouse lymphoma l5178 cells by binding to ga2 (gangliotriaosylceramide, galnacβ1,4galβ1,4glcβ1,1cer; see figure 12 or figure 16 ) [354] . within the nervous system, gangliosides act in a "trans" manner with the myelin-associated glycoprotein mag. mag recognizes neuacα2-3galβ1-3galnactermini on axonal gangliosides, an interaction that is essential for axon-myelin stability and axon regeneration [355] . "cis" interactions can happen via a direct interaction, or, indirectly, via the properties of the membrane or of putative-membrane domains [356] . this way gangliosides influence the activities of receptor-tyrosine kinases in the plasma membrane, such as the receptors of epidermalgrowth factor, nerve growth factor, and insulin and therefore cell signaling [357] . for example, gm1 enhances trka neurotrophin receptor-activation in a "cis"-manner [358] ; for a brief overview of proteins affected by certain gangliosides, compare, for example, [359] . since the characterization of lipid microdomains in living cells is difficult, some conclusions can be drawn from in vitro experiments. for example, gm3 inhibits the autophosphorylation of purified egfr reconstituted into the proteoliposomes of defined lipid compositions, but not the egf binding [360] . there are indications that gangliosides may not act only in an autonomous manner, but might also support the formation of distinct membrane phases, although it is a matter of debate to which extent this operates in vivo. it is believed that gangliosides are not homogeneously distributed on the cell surface, but segregate into membrane domains together with gpi-anchored proteins, sphingomyelin and cholesterol. such rafts have been supposed to be the physiological surroundings of many membrane proteins, although no rigid proof for their existence has been provided. ganglioside plays a largely unexplored role for membrane structure [356] . due to their large hydrated head groups, they stabilize membrane areas with positive curvature [361] . a multitude of reports propose a segregation of gangliosides and other gsls with cholesterol and gpi-anchored proteins into the lipid platforms known as "rafts" in the membranes of living cells [362] [363] [364] [365] . since most of the applied methods (detergent extraction, antibody, and toxin staining) constitute a bias towards the formation of such domains [366] , the existence, size, and lifetime of rafts are a matter of debate. from thermodynamic considerations, it is clear that procedures such as detergent extraction can (or have to) produce artificial results and are not suitable for raft characterization [367] [368] [369] [370] . although it has been demonstrated that the treatment of cellular membranes with detergents causes the redistribution of gangliosides and gpi-anchored proteins [371, 372] , these techniques are still applied and not always critically examined. experiments in living cells using sted microscopy [308] and other visualization techniques [373] point to an upper limit of lifetime and size of such domains in the range of 20 ms and 20 nm. in addition, sialic acids and oligosialic acids present on gangliosides can modulate membrane surface charge density, the ph at the membrane surface, and membrane potentials [374] . in planar lipid bilayers, ganglioside gd1a can increase the excitability of voltage-dependent sodium channels [375] . 8.1. infection. "cis" and "trans" interactions of gangliosides play multiple roles in the immune system [376] and in infectious diseases [377, 378] , where gangliosides act as cellular receptors and coreceptors for viruses, bacteria, and microbial toxins. the most prominent example is gm1 as the receptor for cholera toxin [379] ; other examples are the toxin of clostridium botulinum and the saba adhesin of helicobacter pylori [380] that bind to cell surface gangliosides of the host [381] . binding of sialylated cell surface glycoconjugates to siglecs [382] on white blood cells is used within innate and adaptive immune responses to distinguish between self and nonself and to dampen autoimmune responses [383] . many pathogens use sialic acids on cell surface glycoconjugates for cellular entry, for example, periodontal pathogens [384] . recent examples include ganglioside gt1b, which seems to be the host cell receptor for the merkel cell polyomavirus [385] . this virus has been identified as the cause of merkel cell carcinoma, an aggressive type of skin cancer. also sialidase-insensitive rotaviruses recognize sialic acid, for example, on ganglioside gm1, which is not substrate of all sialidases due to its branched structure [386] and the glycan present in ganglioside gd1a serves as host receptor for the adenoviruses that cause epidemic keratoconjunctivitis [387] . gangliosides are secondary gene products. their function can be analyzed by knockout experiments, where in cells, tissues, or organisms their formation or degradation is interrupted by genomic, posttranscriptional, or chemical strategies. especially valuable were genetically engineered mice with defects in ganglioside biosynthesis [388] , which revealed, for example, a role of gangliosides in calcium homeostasis [389] , neural repair [390] , or neurological diseases [330] . also investigations in human patients [277] , genetically engineered mammals [391] , and other organisms [392] allowed insight into various aspects of ganglioside metabolism and transport. also mutant and silenced cells have been applied for functional studies. in vitro systems such as liposomes or planar monolayers allow investigations that are to difficult to be carried out in cells. for example, when gm3 is incorporated into liposomes, a phase separation into gm3-rich and gm3-poor phases occurs above a certain gm3 content [393] . this would fit to reports on gm3-enriched microdomains in living cells. in experimental approaches, ganglioside biosynthesis can be modulated by inhibitors [394] . also the enhancement of ganglioside biosynthesis can be used, for example, chemically, or by the introduction of glycosyltransferase encoding cdna in cultured cells [395, 396] . an enantiomer of the glucosylceramide synthase inhibitor d-threo-pdmp (pdmp = 1-phenyl-2-decanoylamino-3-morpholino-1-propanol), lthreo-pdmp, acts as an enhancer of ganglioside biosynthesis by upregulating glycosyltransferases. this was accompanied by increased neurite outgrowth [397] . additional possibilities are the generation of mutant cells, for example for gm2 synthase [398] or by posttranscriptional silencing like rna interference. while even complex systems like cultured cells can survive without gsls, they are required for the development of multicellular organisms [399] . gangliosides. structurally homogenous gangliosides and ganglioside probes that are modified by isotopes, fluorescence, chemical reporter groups, photoaffinity, or affinity ligands are valuable tools for the analysis of ganglioside function, metabolism, and transport. these tools are available by total or partial chemical synthesis, or by biosynthetic incorporation of suitable-for example, photolabile-n-acylmannosamine precursors into gangliosides [400] using the methodology for biosynthetic sialic acid modification developed by kayser et al. [401] . for enzymological, transport, and crosslinking studies, tritium and 14 c are incorporated into different positions of gangliosides, but also radioiodination is possible in the presence of aryl residues [402, 403] . ganglioside total synthesis is a time-consuming and demanding task and usually performed only in specialized laboratories for examples, see [49, 50] . it relies predominantly on the sequential glycosidation of a 3-o-protected azidosphingosine [404] with suitably protected and activated glycosyl donors. this includes the trichloroacetimidates [405] as well as methods for α-selective sialylation reactions [406] . also chemoenzymatic procedures have been developed, where different glycosidation steps are catalyzed by glycosyltransferases [407] or glycosidases [408] , or where oligosaccharyl fluorides are coupled to native or fluorescent ceramide anchors using an engineered endoglycoceramidase (glycosynthase) [49, 409] . ganglioside oligosaccharides, such as those of gm3, gm2, gm1, gd3, and gt3, can be produced by genetically engineered bacterial strains. for the biotechnological production of ganglioside head groups, lactose can be internalized in e. coli as a precursor to be used as acceptor for glycosyltransferases [410] [411] [412] [413] [414] [415] . an application of the ganglioside biosynthetic machinery is the preparative production of neoglycolipids with ganglioside head groups [306] figure 21 : example of a gm1 analog [307] spin labelled with a 4,4-dimethyl-oxazolidine-1-oxyl-(doxyl-) residue and a fluorescent gm1 analog used for sted microscopy [308] . using a lung squamous-cell carcinoma line (rerf-lc-a) and 12-azidododecyl β-lactoside as a suitable primer [416] . gsls isolated from natural sources can be used for the preparation of chemically modified derivatives [417] like labelled gsls [418, 419] , or those of enhanced metabolic stability [420] . the chemical release of the ganglioside glycan chain can be achieved by osmium tetroxide/periodate treatment of protected gangliosides [421, 422] , or by ozonolysis of native gangliosides [423] . initially, both methods give rise to ganglioside aldehydes, which are not isolated but subsequently fragmented by alkaline treatment, or, if desired, are isolated for further applications [424] . the glycan part can also be released enzymatically by ceramide glycanase [425, 426] . lysogangliosides that lack the acyl moiety at the sphingosine nitrogen can be prepared by chemical procedures [427, 428] , or by enzymatic treatment of gangliosides with sphingolipid n-deacylase [429] [430] [431] . lysogangliosides (see figure 18 ) can be used for the introduction of fluorescence, spin, or radiolabels or other modifications into the lipid backbone of gangliosides. a semitruncated, dihalogenated gm1 analog that is able to pass the blood-brain barrier is liga20 [398] (figure 19 ). there are also several approaches to the synthesis of photoactivatable gsl derivatives [432] . it has to be noted that when gangliosides are added to the medium of cultured cells, they are largely present as oligomers in the form of micelles or vesicles, as monomers bound to proteins, and as free monomers. in aqueous surroundings, gangliosides form aggregates of different size and shape [361, 433] . in most cases these are micellar structures of 280-630 kda [361] , in the case of the gangliosides with small head groups, gm3 and gm4 also vesicles [361, 434, 435] . the critical micellar concentrations of gsls are in the range of 10 −9 -10 −5 m [436] and depend on temperature, ph, and, in part, on the method of determination. typical values are 3.4 × 10 −9 m for gm3 to 3.9 × 10 −8 m for gt1b [434] . uptake of exogenously added gangliosides by cells in culture [437] can proceed in different ways [306, 420] ( figure 20) . with the aid of radiolabelled [438] and spinlabelled gangliosides [307] (figure 21 ), three modes of adherence have been distinguished: 60-90% of the exogenous ganglioside consist in loosely associated micelles and also monomers, which can be removed by delipidated serum proteins. a second fraction is attached to cellular proteins in a trypsin-labile fashion, and, finally, a trypsin-stable fraction is presumably inserted into the plasma membrane of the cell. only the last fraction is in the topologically correct, native orientation. when bound to proteins, the offrate of gangliosides with native alkyl chain lengths can be very low. this is not the case for synthetic, semitruncated derivatives of higher solubility, which are frequently used, but show different intracellular transport behaviour compared to native gangliosides [420] . fluorescently labelled glycosphingolipids [439] have been applied, but also their properties can differ significantly from the ones of native glycosphingolipids. gangliosides can also be transferred from cultured donor to acceptor cells that are separated by a membrane [440] . by this process known as "shedding" [441] , tumor cells can release up to 0.5% of their gangliosides per hour [442] . fluorescent ganglioside probes that bear the fluorophore in at the membrane-water interphase ( figure 21 ) behave physicochemically more like native gangliosides. such compounds have been used as probes for sted microscopy [308] or to quantify the transfer capacity of gm2 activator in a liposomal fret assay system [443] . despite the fast development of analytical and biophysical tools, the analytical determination of ganglioside pattern, their spatial resolution, and their correlation with function is still a challenge. especially a convincing characterization of ganglioside-containing membrane domains in living cells and of their roles at the cellular level would constitute a considerable advance in the field. n-acetyl-neuraminic acid glc: glucose gal: galactose galnac: n-acetyl-galactosamine. the author has no direct financial relation with any commercial identity mentioned in the paper that might lead 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hy-cars glycosyltransferases both ga2, gm2, and gd2 synthases and gm1b, gd1a, and gt1b synthases are single enzymes in golgi vesicles from rat liver identity of g(a1), g(m1a) and g(d1b) synthase in golgi vesicles from rat liver identity of g(d1c), g(t1a) and g(q1b) synthase in golgi vesicles from rat liver substrate specificity of α2 → 3-sialyltransferases in ganglioside biosynthesis of rat liver golgi the c-series gangliosides g(t3), g(t2) and g(p1c) are formed in rat liver golgi by the same set of glycosyltransferases that catalyse the biosynthesis of asialo-, a-and b-series gangliosides current trends in the structureactivity relationships of sialyltransferases comprehensive analysis of sialyltransferases in vertebrate genomes gangliosides in human fetal brain differential distribution of major gangliosides in rat central nervous system detected by specific monoclonal antibodies cloning of a mouse β1,3 n-acetylglucosaminyltrans-fer-ase glcnac(β1,3)gal(β1,4)glc-ceramide synthase gene encoding the key regulator of lacto-series glycolipid biosynthesis molecular cloning and characterization of udp-glcnac:lactosylceramide β1,3-n-acetylglucosaminyltransferase (β3gn-t5), an essential enzyme for the expression of hnk-1 and lewis x epitopes on glycolipids multiple phenotypic changes in mice after knockout of the b3gnt5 gene, encoding lc3 synthase-a key enzyme in lacto-neolacto ganglioside synthesis n-acetylglucosaminyl transferase regulates the expression of the sulfoglucuronyl glycolipids in specific cell types in cerebellum during development infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of gm3 synthase model for the interaction of membrane-bound substrates and enzymes. hydrolysis of ganglioside g(d1a) by sialidase of neuronal membranes isolated from calf brain two soluble glycosyltransferases glycosylate less efficiently in vivo than their membrane bound counterparts neobiosynthesis of glycosphingolipids by plasma membraneassociated glycosyltransferases the stimulating adventure of krn 7000 further gangliosides from the human brain gangliosides of human myelin: sialosylgalactosylceramide (g7) as a major component oligodendrocyte development and myelin biogenesis: parsing out the roles of glycosphingolipids participation of galactosylceramide and sulfatide in glycosynapses between oligodendrocyte or myelin membranes fa2h is responsible for the formation of 2-hydroxy galactolipids in peripheral nervous system myelin a carbohydrate-carbohydrate interaction between galactosylceramide-containing liposomes and cerebroside sulfate-containing liposomes: dependence on the glycolipid ceramide composition absence of oligodendroglial glucosylceramide synthesis does not result in cns myelin abnormalities or alter the dysmyelinating phenotype of cgt-deficient mice ceramide udpgalactosyltransferase from myelinating rat brain: purification, cloning, and expression topography of cerebroside sulfotransferase in golgi-enriched vesicles from rat brain topology of sphingolipid galactosyltransferases in er and golgi: transbilayer movement of monohexosyl sphingolipids is required for higher glycosphingolipid biosynthesis udp-galactose: ceramide galactosyltransferase is a class i integral membrane protein of the endoplasmic reticulum zebrafish and mouse α2,3-sialyltransferases responsible for synthesizing gm4 ganglioside prevention of experimental allergic encephalomyelitis by ganglioside g(m4) purification and characterization of udp-glucose:ceramide glucosyltransferase from rat liver golgi membranes expression cloning of a cdna for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis oligomerization and topology of the golgi 28 isrn biochemistry membrane protein glucosylceramide synthase identification of active site residues in glucosylceramide synthase: a nucleotide-binding/catalytic motif conserved with processive β-glycosyltransferases topology of glucosylceramide synthesis in golgi membranes from porcine submaxillary glands lactosylceramide is synthesized in the lumen of the golgi apparatus mdr1 pglycoprotein is a lipid translocase of broad specificity, while mdr3 p-glycoprotein specifically translocates phosphatidylcholine abc lipid transporters: extruders, flippases, or flopless activators? pre-and post-golgi translocation of glucosylceramide in glycosphingolipid synthesis reconstitution of glucosylceramide flip-flop across endoplasmic reticulum: implications for mechanism of glycosphingolipid biosynthesis glycosphingolipid synthesis requires fapp2 transfer of glucosylceramide mutation of βglucosidase 2 causes glycolipid storage disease and impaired male fertility biosynthesis of glycolipids cdna cloning and expression of human lactosylceramide synthase isolation and characterization of extremely minor gangliosides, g(m1b) and g(d1α), in adult bovine brains as developmentally regulated antigens enhanced insulin sensitivity in mice lacking ganglioside gm3 tissue-specific expression of cseries gangliosides in the extraneural system structural characterization of a novel cholinergic neuron-specific ganglioside in bovine brain biosynthetic pathway for a new series of gangliosides, gt1aα and gq1bα biosynthesis of the major brain gangliosides gd1a and gt1b glycosphingolipid functions: insights from engineered mouse models a mouse b16 melanoma mutant deficient in glycolipids complex gangliosides are essential in spermatogenesis of mice: possible roles in the transport of testosterone mice lacking complex gangliosides develop wallerian degeneration and myelination defects a functional role for complex gangliosides: motor deficits in gm2/gd2 synthase knockout mice attenuation of interleukin 2 signal in the spleen cells of complex gangliosidelacking mice cd4 and cd8 t cells require different membrane gangliosides for activation mice lacking major brain gangliosides develop parkinsonism g(m1) ganglioside rescues substantia nigra pars compacta neurons and increases dopamine synthesis in residual nigrostriatal dopaminergic neurons in mptp-treated mice gm1 specifically interacts with α-synuclein and inhibits fibrillation mice expressing only monosialoganglioside gm3 exhibit lethal audiogenic seizures interruption of ganglioside synthesis produces central nervous system degeneration and altered axon-glial interactions sphingolipid synthetic pathways are major regulators of lipid homeostasis membranes in balance: mechanisms of sphingolipid homeostasis regulation of ganglioside biosynthesis in the nervous system the cytoplasmic tail of gm3 synthase defines its subcellular localization, stability, and in vivo activity multi-enzyme kinetic analysis of glycolipid biosynthesis golgi localization of glycosyltransferases involved in ganglioside biosynthesis regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases the synthesis of complex carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion glycosyltransferase complexes improve glycolipid synthesis physical and functional association of glycolipid n-acetylgalactosaminyl and galactosyl transferases in the golgi apparatus ganglioside glycosyltransferases organize in distinct multienzyme complexes in cho-k1 cells identification and expression of neu3, a novel human sialidase associated to the plasma membrane a close association of the ganglioside-specific sialidase neu3 with caveolin in membrane microdomains membrane sialidase neu3 is highly expressed in human melanoma cells promoting cell growth with minimal changes in the composition of gangliosides sialidase occurs in both membranes of the nuclear envelope and hydrolyzes endogenous gd1a degradation of glycolipids lysosomal degradation of membrane lipids activator proteins and topology of lysosomal sphingolipid catabolism regulation of apoptosis-associated lysosomal membrane permeabilization recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway biological function of the cellular lipid bmp-bmp as a key activator for cholesterol sorting and membrane digestion metabolism, function and mass spectrometric analysis of bis(monoacylglycero)phosphate and cardiolipin separation and characterization of late endosomal membrane domains lysosome biogenesis and lysosomal membrane proteins: trafficking meets function lysosomal membrane proteins: life between acid and neutral conditions hoppe-seyler's zeitschrift für physiologische chemie the human g(m2) activator protein. a substrate specific cofactor of β-hexosaminidase a principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids saposin a mobilizes lipids from low cholesterol and high bis(monoacylglycerol)phosphate-containing membranes: patient variant saposin a lacks lipid extraction capacity saposin b mobilizes lipids from cholesterol-poor and bis(monoacylglycero) phosphate-rich membranes at acidic ph: unglycosylated patient variant saposin b lacks lipid-extraction capacity degradation of membrane-bound ganglioside gm2 by β-hexosaminidase a. stimulation by gm2 activator protein and lysosomal lipids degradation of membrane-bound ganglioside gm1: stimulation by bis(monoacylglycero)phosphate and the activator proteins sap-b and gm2-ap physiological substrates for human lysosomal βhexosaminidase s kidney sulfatides in mouse models of inherited glycosphingolipid disorders: determination by nano-electrospray ionization 30 isrn biochemistry tandem mass spectrometry role of endosomal membrane lipids and npc2 in cholesterol transfer and membrane fusion a short guided tour through functional and structural features of saposin-like proteins hoppe-seyler's zeitschrift für physiologische chemie glycosphingolipid specificity of the human sulfatide activator protein the activator of cerebroside sulphatase. binding studies with enzyme and substrate demonstrating the detergent function of the activator protein the physiological role of activator proteins for lysosomal glycolipid degradation metabolism of g(m1) ganglioside in cultured skin fibroblasts: anomalies in gangliosidoses, sialidoses, and sphingolipid activator protein (sap, saposin) 1 and prosaposin deficient disorders crystal structure of saposin b reveals a dimeric shell for lipid binding characterization of a mutation in a family with saposin b deficiency: a glycosylation site defect neurological deficits and glycosphingolipid accumulation in saposin b deficient mice purification and characterization of an activator protein for the degradation of glycolipids g(m2) and g(a2) by hexosaminidase a deficiency of a factor necessary for stimulation of hexosaminidase a-catalyzed degradation of ganglioside gm2 and glycolipid ga2 crystal structure of human gm2-activator protein with a novel β-cup topology structural analysis of lipid complexes of gm2-activator protein photoaffinity labelling of the human gm2-activator protein mechanistic insight into ganglioside gm2 degradation interactions of the gm2 activator protein with phosphatidylcholine bilayers: a site-directed spin-labeling power saturation study a dansyl fluorescence-based assay for monitoring kinetics of lipid extraction and transfer ligand extraction properties of the gm2 activator protein and its interactions with lipid vesicles interaction of the gm2-activator protein with phospholipid-ganglioside bilayer membranes and with monolayers at the air-water interface crystal structure analysis of phosphatidylcholine-gm2-activator product complexes: evidence for hydrolase activity the gm2 activator protein, a novel inhibitor of platelet-activating factor gm2 activator protein inhibits platelet activating factor signaling in rats evidence for lipid packaging in the crystal structure of the gm2-activator complex with platelet activating factor adipokine ganglioside gm2 activator protein stimulates insulin secretion a drosophila protein family implicated in pheromone perception is related to tay-sachs gm2-activator protein functional characterisation of a nematode secreted gm2-activator protein saposins utilize two strategies for lipid transfer and cd1 antigen presentation lipid-binding proteins in membrane digestion, antigen presentation, and antimicrobial defense β-galactosidase deficiency (β-galactosidosis): gm1 gangliosidosis and morquio b disease crystal structure of human beta-galactosidase: structural basis of gm1 gangliosidosis and morquio b diseases the 67-kd elastin/laminin-binding protein is related to an enzymatically inactive, alternatively spliced form of β-galactosidase molecular mechanisms of pathogenesis in a glycosphingolipid and a glycoprotein storage disease mouse models of tay-sachs and sandhoff diseases differ in neurologic phenotype and ganglioside metabolism evidence for two different active sites on human β-hexosaminidase a. interaction of g(m2) activator protein with β-hexosaminidase a the x-ray crystal structure of human β-hexosaminidase b provides new insights into sandhoff disease crystal structure of human β-hexosaminidase b: understanding the molecular basis of sandhoff and tay-sachs disease clarifying lysosomal storage diseases multi-system disorders of glycosphingolipid and ganglioside metabolism lysosomal disorders: from storage to cellular damage lysosomal storage disorders in the newborn secondary lipid accumulation in lysosomal disease sphingolipid metabolism diseases pathogenic cascades in lysosomal diseasewhy so complex? the cellular pathology of lysosomal diseases treating lysosomal storage disorders: current practice and future prospects quantitative correlation between the residual activity of βhexosaminidase a and arylsulfatase a and the severity of the resulting lysosomal storage disease partial enzyme deficiencies: residual activities and the development of neurological disorders neurons in niemann-pick disease type c accumulate gangliosides as well as unesterified cholesterol and undergo dendritic and axonal alterations critical role for glycosphingolipids in niemann-pick disease type c histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in niemann-pick type c1 mutant human fibroblasts mucopolysaccharidosis type i: current knowledge on its pathophysiological mechanisms tarffic jam: a compendium of human diseases that affect intracellular transport processes traffic jams ii: an update of diseases of intracellular transport critical role of iron in the pathogenesis of the murine gangliosidoses lysosomal storage diseases as disorders of autophagy gm1 gangliosidosis: review of clinical, molecular, and therapeutic aspects gargoylism (hunter-hurler disease, dysostosis multiplex, lipochondrodystrophy); prenatal and neonatal bone lesions and their early postnatal evolution familial neurovisceral lipidosis. an analysis of eight cases of a syndrome previously reported as "hurler-variant," "pseudo-hurler," and "tay-sachs disease with visceral involvement central nervous system inflammation is a hallmark of pathogenesis in mouse models of gm1 and gm2 gangliosidosis enhanced susceptibility to kainate-induced seizures, neuronal apoptosis, and death in mice lacking gangliotetraose gangliosides: protection with liga 20, a membrane-permeant analog of gm1 gm1-ganglioside-mediated activation of the unfolded protein 32 isrn biochemistry response causes neuronal death in a neurodegenerative gangliosidosis imbalanced substrate specificity of mutant β-galactosidase in patients with morquio b disease chemical chaperone therapy for gm1-gangliosidosis the potential action of galactose as a "chemical chaperone": increase of beta galactosidase activity in fibroblasts from an adult gm1-gangliosidosis patient evaluation of nnonyl-deoxygalactonojirimycin as a pharmacological chaperone for human gm1 gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy the gm2 gangliosidoses a review and predictive models of ganglioside uptake by biological membranes incorporation of ganglioside analogues into fibroblast cell membranes. a spin-label study direct observation of the nanoscale dynamics of membrane lipids in a living cell variant of gm2-gangliosidosis with hexosaminidase a having a severely changed substrate specificity biochemical and molecular aspects of late-onset gm2-gangliosidosis: b1 variant as a prototype diagnosis and carrier detection of tay-sachs disease: direct determination of hexosaminidase a using 4-methylumbelliferyl derivatives of β-n-acetylglucosamine-6-sulfate and β-nacetylgalactosamine-6-sulfate tay-sachs disease: one-step assay of β-n-acetylhexosaminidase in serum with a sulphated chromogenic substrate mouse model of gm2 activator deficiency manifests cerebellar pathology and motor impairment distortion of neuronal geometry and formation of aberrant synapses in neuronal storage disease occurrence of lysoganglioside lyso-g(m2) (ii3-neu5ac-gangliotriaosylsphingosine) in g(m2) gangliodosis brain accumulation of lysosphingolipids in tissues from patients with gm1 and gm2 gangliosidoses lyso-gm2 ganglioside: a possible biomarker of tay-sachs disease and sandhoff disease a view on sphingolipids and disease a genetic model of substrate deprivation therapy for a glycosphingolipid storage disorder bone marrow transplantation prolongs life span and ameliorates neurologic manifestations in sandhoff disease mice highly phosphomannosylated enzyme replacement therapy for gm2 gangliosidosis neural stem cell transplantation benefits a monogenic neurometabolic disorder during the symptomatic phase of disease substrate reduction therapy with miglustat in chronic gm2 gangliosidosis type sandhoff: results of a 3-year follow-up substrate reduction therapy an open-label phase i/ii clinical trial of pyrimethamine for the treatment of patients affected with chronic gm2 gangliosidosis (tay-sachs or sandhoff variants) crystal structure of β-hexosaminidase b in complex with pyrimethamine, a potential pharmacological chaperone induced secretion of β-hexosaminidase by human brain endothelial cells: a novel approach in sandhoff disease? nsaids increase survival in the sandhoff disease mouse: synergy with n-butyldeoxynojirimycin pathological significance of ganglioside clusters in alzheimer's disease functional roles of gangliosides in neurodevelopment: an overview of recent advances aberrant glycosphingolipid expression and membrane organization in tumor cells: consequences on tumor-host interactions ngcgm3 ganglioside: a privileged target for cancer vaccines cancer vaccines and carbohydrate epitopes immunology in the clinic review series, focus on cancer: glycolipids as targets for tumour immunotherapy a common mechanism and a new categorization for anti-ganglioside antibody-mediated neuropathies the therapeutic role of gangliosides in neurological disorders gangliosides, ngf, brain aging and disease: a mini-review with personal reflections alzheimer disease-effect of continuous intracerebroventricular treatment with gm1 ganglioside and a systematic activation programme inhibition of ganglioside biosynthesis as a novel therapeutic approach in insulin resistance receptor mimicry as novel therapeutic treatment for biothreat agents semisynthetic sphingoglycolipid liga20 is neuroprotective against human immunodeficiency virus-gp120-mediated apoptosis gangliosides as modulators of cell function ganglioside gm3 and its biological functions in search of a solution to the sphinx-like riddle of gm1 functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains glycosynaptic microdomains controlling tumor cell phenotype through alteration of cell growth, adhesion, and motility gangliosides in cell recognition and membrane protein regulation glycolipid-mediated cell-cell recognition in inflammation and nerve regeneration beyond glycoproteins as galectin counterreceptors: tumor-effector t cell growth control via ganglioside gm1 galectin-1 is a major receptor for ganglioside gm1, a product of the growth-controlling activity of a cell surface ganglioside sialidase, on human neuroblastoma cells in culture carbohydrate-to-carbohydrate interaction, through glycosynapse, as a basis of cell recognition and membrane organization carbohydrate-carbohydrate interaction in basic cell biology carbohydrate-carbohydrate interactions in cell recognition specific interaction between gangliotriaosylceramide (gg3) and sialosyllactosylceramide (g(m3) as a basis for specific cellular recognition between lymphoma and melanoma cells brain gangliosides in axon-myelin stability and axon regeneration gangliosides and the multiscale modulation of membrane structure receptor modifications in glycobiology neural functions of glycolipids lipid rafts: keys to neurodegeneration regulation of human egf receptor by lipids gangliosides as components of lipid membrane domains membrane organization and lipid rafts revitalizing membrane rafts: new tools and insights lipid rafts as a membraneorganizing principle lipids and cholesterol as regulators of traffic in the endomembrane system principles of microdomain formation in biological membranes-are there lipid liquid ordered domains in living cellular membranes? pip2 signaling in lipid domains: a critical reevaluation rafts defined: a report on the keystone symposium on lipid rafts and cell function detergentresistant membranes should not be identified with membrane rafts triton promotes domain formation in lipid raft mixtures membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation imaging of mobile long-lived nanoplatforms in the live cell plasma membrane membrane oligo-and polysialic acids ganglioside gd1a increases the excitability of voltage-dependent sodium channels multifarious roles of sialic acids in immunity sphingolipids in infectious diseases viruses and sialic acids: rules of engagement cholera toxin-a foe & a friend helicobacter pylori and complex gangliosides sialic acids in human health and disease siglecs and their roles in the immune system siglecs as sensors of self in innate and adaptive immune responses sialic acid, periodontal pathogens and tannerella forsythia: stick around and enjoy the feast! ganglioside gt1b is a putative host cell receptor for the merkel cell polyomavirus sialic acid dependence in rotavirus host cell invasion the gd1a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis knockout mice and glycolipids cerebellar neurons lacking complex gangliosides degenerate in the presence of depolarizing levels of potassium regulatory mechanisms of nervous systems with glycosphingolipids functions of sphingolipid metabolism in mammals -lessons from genetic defects sphingolipids and membrane biology as determined from genetic models phase behavior in multilamellar vesicles of dppc containing ganglioside gm3 with a c18:1 sphingoid base and a 24:0 acyl chain (gm3(18,24)) observed by x-ray diffraction glycosphingolipids-nature, function, and pharmacological modulation enhancement of malignant properties of human osteosarcoma cells with disialyl gangliosides gd2/gd32012 gm1/gd1/ga1 synthase expression results in the reduced cancer phenotypes with modulation of composition and raft-localization of gangliosides in a melanoma cell line chapter 22 neurotrophic and neuroprotective actions of an enhancer of ganglioside biosynthesis mutant ng108-15 cells (ng-cr72) deficient in gm1 synthase respond aberrantly to axonogenic stimuli and are vulnerable to calcium-induced apoptosis: they are rescued with liga-20 biomolecule function: no reliable prediction from cell culture metabolism of diazirine-modified n-acetylmannosamine analogues to photocross-linking sialosides biosynthesis of a nonphysiological sialic acid in different rat organs, using n-propanoyl-d-hexosamines as precursors tubulin anchoring to glycolipid-enriched, detergent-resistant domains of the neuronal plasma membrane hplc-based procedure for the preparation of carbenegenerating photoreactive gm3 and gm1 ganglioside derivatives radioiodinated to high specific radioactivity with chloramine t as an oxidant azidosphingosine glycosylation in glycosphingolipid synthesis glycosyl trichloroacetimidates selective α-sialylation enzymatic approaches to o-glycoside introduction: glycosyltransferases enzymatic glycosylation by transferases glycosphingolipid synthesis employing a combination of recombinant glycosyltransferases and an endoglycoceramidase glycosynthase biosynthesis of conjugatable saccharidic moieties of gm2 and gm3 gangliosides by engineered e. coli the genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, campylobacter jejuni genetic engineering of escherichia coli for the economical production of sialylated oligosaccharides highly efficient biosynthesis of the oligosaccharide moiety of the gd3 ganglioside by using metabolically engineered escherichia coli a new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria large-scale in vivo synthesis of the carbohydrate moieties of gangliosides gm1 and gm2 by metabolically engineered escherichia coli large scale biosynthesis of ganglioside analogues by rerf-lc-ai cells cultured in hyperflask use of sphingolipid analogs: benefits and risks a simple and novel method for tritium labeling of gangliosides and other sphingolipids preparation of radiolabeled gangliosides uptake and metabolism of exogenous glycosphingolipids by cultured cells structure and function of glycosphingolipids and sphingolipids: recollections and future trends release of carbohydrates from sphingoglycolipid by osmium-catalyzed periodate oxidation followed by treatment with mild alkali carbohydrate components of extraneuronal gangliosides from bovine and human spleen, and bovine kidney structural and functional glycosphingolipidomics by glycoblotting with an aminooxy-functionalized gold nanoparticle a unique glycosphingolipid-splitting enzyme (ceramide-glycanase from leech) cleaves the linkage between the oligosaccharide and the ceramide preparation of homogenous oligosaccharide chains from glycosphingolipids synthesis of lysogangliosides lysogangliosides: synthesis and use in preparing labeled gangliosides facile method for the preparation of lyso-gm1 and lyso-gm2 design of a covalently bonded glycosphingolipid microarray anti-ganglioside antibodies bind with enhanced affinity to gangliosides containing very long chain fatty acids sphingolipid photoaffinity labels thermodynamicgeometric correlations for the morphology of self-assembled structures of glycosphingolipids and their mixtures with dipalmitoylphosphatidylcholine aggregative properties of gangliosides in solution lipid membrane domains in glycobiology micellar properties of glycosphingolipids in aqueous media shedding and uptake of gangliosides and glycosylphosphatidylinositol-anchored proteins characterization of the cellular binding of exogenous gangliosides chemical synthesis of fluorescent glycero-and sphingolipids synthesis, shedding, and intercellular transfer of human medulloblastoma gangliosides: abrogation by a new inhibitor of glucosylceramide synthase shedding and immunoregulatory activity of yac-1 lymphoma cell gangliosides shedding of human neuroblastoma gangliosides synthesis of novel nbd-gm1 and nbd-gm2 for the transfer activity of gm2-activator protein by a fret-based assay system support from professor dr. k. sandhoff, the dfg, and the european community (7th framework program "lipidomicnet", proposal no. 202272) is gratefully acknowledged. key: cord-302009-oqc21fah authors: geng, xindu; wang, chaozhan title: protein folding liquid chromatography and its recent developments() date: 2007-04-15 journal: j chromatogr b analyt technol biomed life sci doi: 10.1016/j.jchromb.2006.10.068 sha: doc_id: 302009 cord_uid: oqc21fah the ultimate goal of proteomics is to identify biologically active proteins and to produce them using biotechnology tools such as bacterial hosts. however, proteins produced by escherichia coli must be refolded to their native state. protein folding liquid chromatography (pflc) is a new method developed in recent years, and it is widely used in molecular biology and biotechnology. in this paper, the new method, pflc is introduced and its recent development is reviewed. in addition the paper includes definitions, advantages, principles, applications for both laboratory and large scales, apparatus, and effecting factors of pflc. in addition, the role of this method in the future is examined. one of the purposes of proteomics is to identify unknown biologically active proteins and use this information to develop novel drugs. some active proteins occur at very low levels in the human body and thus have to be produced by biotechnology. escherichia coli is one of the mostly used host cell in biotechnology. but when proteins are expressed in e. coli, they often form inactive protein aggregates called inclusion bodies. a step necessary in recovering active proteins from e. coli is protein refolding (it is simply called protein folding here) or protein renaturation; it is usually the key step during the production of therapeutic proteins by biotechnology, especially at the industrial scale. the yields from refolding by traditional methods are usually very low, typically 5-20%. application of liquid chromatography (lc) to protein folding is one of the most interesting and exciting methods to develop in recent years. when it is used in protein folding, the bioactivity recovery increases, the folded protein can be easily separated from misfolded forms, protein concentration after refolding is relatively high, and it is easy to scale up and automate, therefore it is regarded as an efficient, and close to ideal refolding method [1, 2] . additionally, it has the potential to be used at an industrial or large scale, today it has become a very popular technique for protein folding. protein refolding by liquid chromatography can be simply named as "protein folding liquid chromatography". it is defined as "a kind of liquid chromatography, with various kinds of biochemical and/or physicochemical processes originally accomplished in solution, which can result in either raising the efficiency, or shortening the time of protein folding" [3] . an ideal pflc should have the following four functions depicted simultaneously in fig. 1 [3] . they are the removal of denaturants, refolding of target proteins, separation from contaminant proteins including misfolded intermediates of the fig. 1 . scheme of an ideal protein folding liquid chromatography having four functions simultaneously [3] . four functions: removal of denaturants, refolding of target proteins, separation from contaminant proteins including misfolded intermediates of the target protein, and easy recovery of denaturants. target protein, and easy recovery of denaturants. it usually takes 20-40 min to complete a chromatographic run with simultaneous protein folding. in addition, by continuously changing the components of the mobile phase, different proteins can be separated with suitable folding conditions to refold and simultaneously purify in only one chromatographic run. by using the normal dilution method for protein folding, denaturants and contaminant proteins cannot be removed. some precipitates of target proteins will form during dilution; this not only results in a low recovery, but also requires centrifugation after an overnight incubation. therefore, the target protein must be further processed using coarse fractionation and fine fractionation. in addition, using the usual dialysis method for protein folding, it typically takes 24 h to refold a protein, with numerous changes of buffer during dialysis. this method can remove most of the denaturants, but cannot completely remove them, and cannot separate the target protein from contaminant proteins. in the past years, guo and geng [4] , li et al. [5] , jungbauer et al. [6] , middelberg [2] and two books [3, 7] separately introduced pflc and reviewed its development from different aspects. a comprehensive review of this field is presented in this paper, including their principles, recent developments and applications, apparatus, recent developments for pflc at large scale, and effecting factors were summarized and discussed. from a scientific point of view, pflc includes size exclusion chromatography (sec), hydrophobic interaction chromatography (hic), ion exchange chromatography (iec), and affinity chromatography (afc). as it is well known that there is no obvious interaction between denatured protein and the stationary phase of sec, but there are strong interactions between denatured proteins and the stationary phase in the other three chromatographic methods, therefore for convenience, the latter are referred to as adsorption liquid chromatography. the mechanisms for protein folding are discussed for the two kinds of lc. the principle of protein folding by sec was proposed by batas and chaudhuri [8] . they reported that denatured proteins have a much larger stokes radius than denaturants, so the former move much faster than the latter and result in a gradual decrease in denaturant concentration around denatured protein molecules, causing protein folding step by step, and as this happens their stokes radius decreases gradually. when protein folding is accomplished, their stokes radius is constant, and the protein is eluted out in its native state. they thought that their dispersion rate decreased when the denatured protein molecules entered gels, which reduced aggregation. they further tested the above mechanism using high performance sec [9] . 2.2.1. thermodynamic equilibria [3, 7] the key point here is that the chromatographic process for any kind of adsorption lc can be elucidated by thermodynamic equilibrium, providing a basis to describe the principle of protein folding for this kind of lc. in regular lc, only adsorption (p (n,mo,a) ) and desorption (p (n,mo,d) ) of the native protein in a monomeric state with the stationary phase involving the usual lc and any separation depends on the partition coefficient of proteins between these two phases, while protein folding in the usual buffer is mainly controlled by the primary structure of the protein. during protein folding in buffer, on the one hand, a series of aggregation processes, such as unfolding of the monomer, p (u,mo,d) to dimer, trimer, multimer, until protein precipitates may form, being unfavorable protein folding. on the other hand, the denatured state p (u,mo,d) , can also be converted to its native state, p (n,mo,d) under a suitable conditions, being favorable protein folding. however, with pflc, the stationary phase (see section 2.2.2) makes chemical equilibrium move from precipitate and/or its polymers to its monomer in the unfolded state, p (u,mo,d) to fold to its monomeric native state, p (n,mo,d) , until finally it elutes out of the column. in summary, pflc is the favorable chemical equilibrium to convert from aggregate to p (n,mo,d) , resulting in either an increase in the protein folding efficiency, or an acceleration in the folding process. as long as the chromatographic process can be described by thermodynamic equilibrium, the reported principle is suitable for each kind of lc listed above, iec, hic, and afc. from the standpoint of molecular interactions, the mechanism of protein folding for each kind of adsorption chromatography mentioned above should be different from each other. because the molecular mechanism of protein folding by iec and afc has not yet been reported, hic is taken as an example to demonstrate it. the mechanism of protein folding by hic was presented in 1992 [10] and reported in detail in 2002 [3, 11, 12] . the unfolded protein molecules are pushed by hydrophobic interaction forces from the mobile phase at a high salt concentration to move forward to stationary phase of hic (sthic) and tightly contact the sthic with apolar region of amino acid sequence residues to form a stable complex and the hydrophilic parts of the unfolded protein molecules face to the mobile phase. thus, the unfolded protein molecules cannot aggregate in this circumstance. the unfolded protein molecules take enough energy at the molecule level from the sthic and simultaneously carry out three functions: (i) the sthic can recognize a specific hydrophobic region of a polypeptide. (ii) squeezing out water molecules in a hydrated state from both the hydrated unfolded protein and the sthic. (iii) the microdomains of the protein molecules on the sthic are formed. the unfolded protein molecules desorb from the sthic as the salt concentration decreases, or as the water concentration in the mobile phase increases. protein molecules with incorrect microdomains would be corrected by the microdomains spontaneously disappearing in the mobile phase due to their unstable thermodynamics. after many rounds of adsorption and desorption of the protein during gradient elution, the incorrect microdomains would decrease, while the protein molecules with correct microdomains would increase, resulting in the protein attaining complete refolding. the complete refolded protein can be separated from its stable intermediates, or its incompletely refolded forms. several unfolded proteins can be simultaneously refolded and, at the same time, separated from each other. sec is most often applied to pflc due to its ease of operation, easy scale up and suitability to the refolding of a wide range of proteins. in 1992, one of the authors [10] used sec to refold three kinds of standard proteins. in 1994, werner et al. [13] prepared recombinant human ets-1, bovine ribonuclease a and integration host factor (ihf), and increase the capacity of the technique to the mg scale. in 1996, batas and chaudhuri [8] used sephacryl s 100 as sec gel, and refolded hen egg white lysozyme and bovine carbonate anhydrase (cab) at an initial protein concentration of 80 mg/ml, their bioactivity recovery was 63% (a specific bioactivity recovery of 104%) and 56% (a specific bioactivity recovery of 81%), respectively, they investigated the sec refolding method in detail. in order to provide a wild environment for protein folding, gu et al. [14] reported urea gradient sec, in which a gradually decreasing gradient of urea concentration from top to bottom was initially formed in the sec column, denaturants were removed linearly by this method and good results were obtained for denatured proteins with a high protein concentration. for denatured/reduced lysozyme at an initial concentration of 17 mg/ml, a bioactivity recovery as high as 90% could be obtained in 40 min. in addition, they developed a sec refolding method with dual gradients of ph and urea concentration, the results were relatively good. dong et al. [15] combined sec and the artificial molecular chaperone, they used cetyltrimethylammonium bromide (ctab) as a detergent and ␤-cyclodextrin as a stripping agent (i.e., eluent), denatured/reduced lysozyme with an initial concentration of 80 mg/ml was refolded using this method. the results showed that this method is favorable for protein folding under conditions of high flow rate of the mobile phase. chaperone solvent plug sec proposed by liu and chang [16] could obviously reduce precipitates formed before denatured protein entered the top of the column, and relatively high mass recovery was obtained. schlegl et al. [17] reported a continuous matrix assisted protein folding system based on sec refolding and continuous annular chromatography (cac), it forces denatured proteins to refold to their native states quantitatively and continuously. recently, simulated moving bed chromatography (smb) in sec mode [30] was also used to refold proteins [18, 19] , this gave new inspiration to the development of sec. table 1 shows some examples of protein folding by sec. as pointed out previously, in 1991-1992, one of the authors [10] refolded several denatured proteins and rhifn-␥ using hic. in 1997, hic was applied to the refolding and purification of several hiv protease mutants by du-pout-merck co. [31, 32] . so far, many proteins, such as recombinant human interferon-␥ (rhifn-␥) [10, [33] [34] [35] , bovine insulin [36] , lysozyme [37] , recombinant bovine prion protein [38] were successfully refolded using hic, and good results were obtained. additionally, in 2004 and 2006, the refolding with simultaneous purification of rhifn-␥ [34] and recombinant human granulocyte colony-stimulating factor (rhg-csf) [39] were reported, respectively, downstream processes for their production in biotechnology were simplified greatly. li et al. [40, 41] refolded the originally denatured lysozyme and recombinant staphylococcus aureus elongation factor g (ef-g) separately using hic with several urea gradients. recently, they refolded consensus interferon (c-ifn) using hic with a dual-gradient of decreasing guanidine hydrochloride concentration and increasing peg concentration [42] . compared with dilution refolding, the gradient hic process, in the presence of peg, gave about 2.6-folds of increase in specific activity, 30% increase in soluble protein recovery. urea denatured recombinant human stem cell factor (rhscf) produced by e. coli were renatured with simultaneous purification using a high performancehydrophobic interaction chromatographic (hphic) column packed with packing materials with an end group of peg400 [48] . the refolded rhscf had a good bioactivity and a high purity. table 2 shows some examples of protein folding by hic. protein folding by iec was introduced by creighton [49, 50] in 1986, he used a refolding system consisting of three buffers, in which a decreasing gradient of urea concentration was used to refold protein, then an increasing gradient of salt concentration was performed to elute the refolded protein. suttnar et al. [51] used 0.01 mol/l naoh solution to solubilize the inclusion body of papilloma virus hpv16 e7ms2 fusion protein, and successfully refolded the solubilized target protein using mono q strong anion exchange chromatographic column. stempfer et al. [52] fused a polycation tag containing hexa-arginine onto ␣-glucosidase, and refolded this fusion protein by iec with a polyanionic support. this method provides a novel protocol for proteins with very few charges, but the fusion and cleavage of the tag was relatively complex. kweon et al. [53] refolded cyclodextrin glycocyltransferase fused with c-ifn mass recovery was more than 80% 2006 [42] 10 lysine residues at the c-terminus (cgtk10ase) using strong cation exchange chromatography (scx) with sp sepharose gel, the refolded yield was approximately 100% and the protein concentration after elution was 2.5 mg/ml, the initial protein concentration was 7.5 mg/ml. this method is similar to that introduced by stempfer et al. [52] . li et al. [54] improved the refolded yield of lysozyme using urea and ph dual gradient scx with soft gel sp sepharose. in 2002, cho [55] used expanded bed adsorption chromatography (eba) packed with a weak anion exchange resin to refold proteins, it simplified the production procedure for proteins in inclusion bodies. in 2005, machold et al. [56] used preparative continuous annular chromatographic (p-cac) packed with deae sepharose to refold ␣-lactalbumin continuously. liu et al. [57] recently proposed a relatively versatile refolding method using iec with a silica-based weak cation exchanger, very high mass and bioactivity recoveries were obtained for denatured lysozyme. in this method, 4.0 mol/l urea was a constituent of the equilibration and elution buffers, and ammonium sulfate which is good for maintaining the stability of native proteins was selected as the elution agent. a similar method was also applied to rhg-csf [58, 59] . lu et al. [60] refolded recombinant dual human stem cell factor using iec similar to creighton's method, the target protein obtained had a purity of 90%, and a refolded yield of 19.46%. denatured/reduced bovine serum albumin (bsa) which contains 17 pairs of disulfide bonds was renatured using strong anion exchange chromatography (sax) with a q sepharose column [61] . bsa was eluted after an incubation of 40 h in the sax column, its refolded yield and mass recovery were 55 and 67%, respectively, this is one of the most complex proteins refolded by lc. examples for protein folding by iec are listed in table 3 . specific affinity interactions between ligands and target proteins are responsible for reducing aggregates between dena-tured protein molecules and increasing the refolded yield. afc applied to protein folding can be classified into three types according to their ligands: immobilized metal ion affinity chromatography (imac), immobilized liposome chromatography (ilc), and immobilized molecular chaperone chromatography (imcc). in 1994, phadtare et al. [69] immobilized the molecular chaperone groel on the surface of a stationary phase and used this column on tublin and glutamine synthetase. in 1997, altamirano et al. [70] immobilized histidine-fused groel (191-345) with 17 amino acid residues at the n-terminus on ni-nta resin through a chelating interaction, and used the column packed with this modified resin to refold indole 3-glycerol phosphate synthase (igps) mutants igps (49-252) denatured by 8 mol/l urea, its mass recovery was 92%, and the refolded protein had a bioactivity of 100%. they also used this affinity column to refold cyclophilin a, its mass recovery was 84% and its bioactivity recovery was 98%. in 1999, they immobilized molecular chaperone/disulfide isomerase/proline ppi on agrose resin and formed a tri-component stationary phase, they used this column to refold scorpion toxin cn5 [71] , which could not be refolded at all using other methods. its mass recovery was 87% and its bioactivity recovery was near 100%, and thus they called this method as refolding chromatography. they also used this method to refold another recombinant protein [72] . gao et al. [73] refolded rhifn-␥ using an immobilized molecular chaperone fragment. guan et al. [74] immobilized mini-molecular chaperone sht groel (191-345) on activated sepharose fast flow gel and used a column packed with this modified gel to refold rhifn-␥, the results showed that this method was very useful for the refolding of rhifn-␥. when 100 l of rhifn-␥ solution with a protein concentration of 17.8 mg/ml was injected into the column, the mass recovery of the target protein was 74.25% and its bioactivity was 6.74 × 10 6 iu/ml. imac is based on the affinity interaction between the ligands and the histidines tagged at the end of the target proteins. when protein molecules are adsorbed on the imac column, [75] [76] [77] . for the refolding of ykt6p snare by imac, its mass recovery was 88%, and its purity was 94% [78] . aequorin was adsorbed onto ni-nta agarose in batch mode, after that the agarose beads were packed into column, then the adsorbed aequorin was refolded by imac [75] , crowding of denatured proteins on the top of the column was avoided in this manner, and aggregates decreased. tien and co-workers [79] used the octapeptide repeated sequence in recombinant bovine prion as a natural affinity tag, and refolded the protein by ni-nta imac and the target protein with correct structure was obtained, its mass recovery was 11%. wild rhg-csf without any affinity tag was also refolded successfully using imac [80] . yoshimoto et al. [81] [82] [83] separately refolded bovine carbonate anhydrase, lysozyme and ribonuclease a using ilc. they thought that liposome could be regarded as a kind of aqueous two-phase system and can function as an artificial molecular chaperone, it has a high selectivity for different conformations of proteins denatured with different concentration of denaturants, and retention of proteins with different molecular conformations on the column has a relationship with the local hydrophobicity. during protein folding, the ilc can combine with protein folding intermediates, thus aggregates between denatured protein molecules were prevented and refolding yield was improved. some examples for protein folding using afc are listed in table 4 . for pflc, a series of problems exists, such as aggregates would form when loading the denatured protein solution. when aggregates formed, the back pressure of the column would increase significantly, even blocking the column. additionally, mass and bioactivity recoveries of the target protein would decrease. this problem is more important in large scale pflc. geng and co-workers [92, 93] designed and manufactured in a series of units for simultaneous renaturation and purification of proteins (usrpp), also called chromatographic cake, its length is very short, only 1.0-5.0 cm, but its diameter is up from 2.0 to 50 cm or even larger. its cross section is very large, so the increase in the column back pressure is not obvious when a few precipitates accumulate on the filter or on the top of column bed. this unit has very good resolution for proteins in laboratory (fig. 2a) and large scales (fig. 2b) , it has been applied to the refolding of many proteins, and was used for the refolding with simultaneous purification of rhifn-␥ [34] and rhg-csf [39] at an industrial scale. when the usrpp with a size of 10 mm × 200 mm i.d. was used, rhifn-␥ with a purity of more than 95%, and a specific bioactivity of 8.9 × 10 7 iu/mg can be obtained by using usrpp within 2 h, bioactivity recovery can reach 24-fold that of traditional methods, the time it takes was only half that of the traditional method. when using the usrpp with a dimension of 10 mm × 300 mm i.d., 700 ml of rhifn-␥ solution extracted by 7.0 mol/l guhcl containing 2.0 g of total protein could be processed in one run with a flow rate of 120 ml/min [34] . and the usrpp was also used to refold rhg-csf with simultaneous purification at large scales [39] . usrpp has a very good performance, but its price is relatively high due to packing with supports with small particles and using stainless steel for cake bodies. based on the fact that the retention of biopolymers in lc is dominated by the contact surface area between biopolymers and the stationary phase employed [7, 94, 95, 96] , a short column packing with small particles (i.e., less than 10 m) should theoretically have an identical resolution to that of a longer column packed with the same kind packing with large diameter particles (i.e., 50-100 m), but the latter is much cheaper than the former. thus, a series of simple and cheap columns were manufactured [3, 39, 97] , in which large particulates were packed and the hphic column packing materials are silica-based, it is much cheaper than the usrpp. compared to fig. 2a, although it still has a good resolution for protein (fig. 3) , its resolution is a little worse. it can be used either for investigating chromatographic conditions in laboratory scale, or as a pre-column for other uses. expanded bed adsorption chromatography (eba) is a newly developed chromatographic technique in recent years and it is a large-scale industrial chromatographic technique. it is an alternative to traditional clarification (centrifugation, tangential, micro-and ultrafiltration) and the first chromatography step. the work of refolding proteins using eba was initiated by mannen et al. [98] , they immobilized molecular chaperone on the chromatographic support and using eba to refold denatured proteins. in 2002, cho et al. [55] used eba column packed with streamline deae resin to refold the e. coli cell homogenate of the fusion protein of recombinant human growth hormone (rhgh) and glutathione s-transferase (gst) fragment, its refolding yield was up to 84%. choi et al. [67] compared the refolding efficiencies of an eba and a packing bed for recombinant lk68, the results indicated that both were comparable. cabanne et al. [68] refolded enhanced green fluorescent protein (egfp) expressed in e. coli using eba with an anion exchanger, good results were obtained. jin et al. [99] used eba packed with streamline sp strong cation exchange chromatographic resin to refold rhifn-␥ solubilized by 8 mol/l urea with simultaneous purification, its mass recovery was 52.7%, and its specific bioactivity was 8.18 × 10 6 iu/mg. applying eba to protein folding not only can reduce aggregates and improve refolded yield, but can also increase purity, reduce steps during refolding process, and lower production costs. eba provides pflc with an alternative routine to overcome the increase in column back pressure resulting from protein precipitation. continuous annular chromatography (cac) is also a new chromatographic technique. it can allow sampling and separation to be performed continuously, and it is a very important preparative chromatographic technique. schlegl et al. [17] reported a continuous matrix assisted protein folding system based on sec refolding and continuous annular chromatography (cac). this system consisted of a preparative cac and an ultrafiltration system, the cac was used for protein folding and separation, the ultrafiltration was used to recover protein aggregates formed during protein folding, and then the recovered aggregates were re-injected into the cac for refolding again. this system can help make denatured proteins be refolded into their native states quantitatively and continuously. when using superdex 75 prepgrade as a gel to refold bovine ␣-lactoalbumin, a refolded yield of only 30% was obtained by using the normal batch sec, but the refolded yield was increased to 41% by using the continuous system when the recycle rate was 65%. if the aggregates could be quantitatively solubilized and recycled to the sample solution, the refolded yield might have a potential to achieve nearly 100%. lanckriet and middelberg [100] also refolded lysozyme by using cac. machold et al. [56] refolded ␣lactoalbumin in continuous operation mode using a preparative continuous annular chromatographic (p-cac) column packed with deae sepharose resin. a solution containing 2 mol/l guhcl was used to dissolve protein aggregates and precipitates in the column during refolding. the re-dissolved proteins were recovered by ultrafiltration and were re-injected into the chromatographic system for refolding again; the refolded yield was enhanced to a certain extent. continuous operation of protein refolding by iec was achieved in this method, and it represents a very good idea for recovery of protein precipitates during pflc. however, p-cac is not stable, especially its flow rate is unstable, its peak wiggle, and it is not easy to operate. simulated moving bed chromatography (smb) is also a continuous chromatographic process. it has some advantages such as high reproducibility, low solvent cost, low product dilution ratio, therefore, its operational cost is relatively low, but its oneoff investment is relatively high. for separation at preparative and productive scales, low operational cost is in the highest flight compared to high one-off investment, therefore, the separation cost of smb is lower than batch chromatography [101] . this technique has been used for large scale separation in many fields. park et al. [18, 19] used four-zone smb in sec mode to refold denatured/reduced lysozyme continuously, both its bioactivity and mass recoveries were more than 90%. during the process, the productivity was high, cost of refolding buffer was low and support use rate was high. compared to batch sec, the concentration of lysozyme obtained by smb was high, and the cost of adsorbents was low. it can be seen from the above that pflc at a large scale and at an industrial scale has been performed from various points of view, and relatively good results were achieved. however, work in this field is only underway just now, and much research should be carried out to further develop this method. the expenses for manufacturing therapeutic proteins are very high and thus it is desirable to reduce these. besides selecting the kind of lc for protein folding, the optimization of the renatured condition for each lc is also significant. a suitable concentration of denaturant in sample solution and/or mobile phase can reduce a denatured proteins chances of aggregating to a minimum, or prevent precipitates appearing altogether facilitating protein elution during pflc, thus the refolded yield would be enhanced. urea is the mostly commonly used denaturant added to mobile phases. muller and rinase [22] refolded platelet-derived growth factor by using sec, it was found that aggregates increased from 10 to 60% when the mobile phase changed from 0.5 mol/l guhcl to 0.5 mol/l nacl. liu et al. [57] found that bioactivity recovery increased and then decreased with increasing urea concentrations during the refolding of lysozyme by high performance weak cation exchange chromatography (hpwcx), with the highest recovery at 4.0 mol/l urea. little bioactivity recovery was obtained when the urea concentration was less than 1.0 mol/l. similar results were obtained for the refolding with simultaneous purification of rhg-csf using sax with q sepharose ff gel, but the most suitable urea concentration is 3.0 mol/l [58, 59] . it can be seen that urea concentration is very critical for the pflc, especially for those proteins susceptible to aggregation, but different urea concentrations are required for different proteins. it is believed that the stationary phase adsorbs denatured protein molecules playing an important role not only in reducing aggregates during pflc, but also in associating protein folding by means of three functions [3, 12] , so the stationary phase plays a very critical role during protein folding by lc. fahey et al. [25] found that the types of stationary phase had a significant effect on the refolding of urokinase plasminogen activator by sec. it was thought that the fraction range of the stationary phase was responsible for the refolding results. gu et al. [26] obtained results similar to those above. wang et al. [38] refolded a recombinant bovine normal prion protein fragment [rbprp (104-242)] with simultaneous purification using a hphic with three different types of stationary phases (phenyl, peg600 and tetrahydrogen furfural), the results indicated that the types of stationary phase had a significant effect on the mass recovery and purity. denatured lysozyme was also refolded by hphic using the above three types of stationary phase [37] , it was found that the highest refolded yield was obtained when peg600, which has the weakest hydrophobicity, was used as the stationary phase. geng et al. [34] found that types of stationary phase also had a very significant effect on the refolding of rhifn-␥, the best results were achieved when using peg200 as the stationary phase, which has the weakest hydrophobicity among the seven investigated types of stationary phase. results obtained by li et al. [40] indicated that ligands with strong hydrophobicity were susceptible to causing misfolding of ef-g, thus resulting in irreversible adsorption and lower refolded yields. machold et al. [66] found that various refolded yields were obtained with different anion exchangers for denatured ␣-lactoalbumin, and the refolding time was much different with these exchangers. it also indicated that refolded yields of lysozyme depended sig-nificantly on the types of stationary phase during its refolding using hpwcx [102] . various proteins have various structures and characteristics, they require different stationary phases for their refolding, and therefore a suitable stationary phase should be selected from various stationary phases. additionally, stationary phases with new structures and characteristics should be developed to broaden the types of stationary phases, thereby accelerating the maturation of the pflc technique. flow rate can affect the contact time of denatured proteins to the stationary phase of a chromatographic column. it can also affect the rate of denatured protein molecules entering into the column and the rate of decreasing the denaturant concentration, thus resulting in different refolded yields. harrowing and chaudhuri [103] investigated the effect of flow rates on the refolded yields of ␤-lactamase by sec, it was found that the mass recovery increased with increasing the flow rate. fahey et al. [23] found the same phenomenon during the refolding of urokinase plasminogen activator using sec. but gu et al. [26] found that aggregates reduced and the bioactivity recovery increased with a decrease in flow rate. guan et al. [74] refolded rhifn-␥ using gels immobilized with mini-molecular chaperone sht groel (191-345), they found that both mass recovery and specific bioactivity increased with a decrease in the flow rate. liu et al. [29] thought that it was the key for protein folding using sec to reduce aggregates before denatured protein entered the column and to make protein stay for enough time in the column. they used higher flow rates during the process when the protein was moving from injection valve to the top of column, then lowering the flow rate to make the protein stay long enough in the column, the results demonstrated that the aggregates were reduced significantly and that the refolding results were very good. liu [102] refolded lysozyme using hpwcx, they found that the bioactivity recovery increased somewhat with an increase in the flow rate, approaching the highest refolded yield at a flow rate of about 2.0 ml/min, but stayed static after that. li et al. [54] found that bioactivity recovery increased initially and then decreased with an increase in the flow rate during the refolding of lysozyme using iec. geng et al. [34] found that the lower flow rate was favorable for improving mass of rhifn-␥ during its refolding using hphic. it can be seen that the effects of flow rate on the refolded yield is not consistent, this may have something to do with refolding methods, but it is affirmative that flow rate has an important effect on pflc. the kinds of salt in the mobile phase of hic have not been found to affect protein folding significantly [34] , but the kinds of salt in the mobile phase of iec affect it seriously [57] . however, some differences as well as some similarities between effects of ph on the protein folding using lc and dilution method were found to contribute to protein folding, because ph can affect both retention and elution of proteins during chromatographic processes. wang et al. [38] found that the most suitable mobile phase ph was 7.0 during the refolding of rbprp (104-242). kweon et al. [53] refolded cgtk10ase using scx, the results indicated that refolded yield and specific bioactivity decreased drastically when ph was below 7, and they approached approximately 100% between ph 7 and ph 8.5, but its mass recovery almost remained constant between ph 6 and ph 8.5. generally, mobile phase ph is selected in the range from 7 to 9 due to the fact that weak basic circumstances facilitate protein folding. additions of small molecular additives help proteins to refold into their native state; this method is also used in pflc. introduction of a detergent to a buffer for protein folding can reduce aggregates during refolding of exopolyphosphatase using imac [77] . li et al. [40] found that inclusion of 50% glycerol in the refolding buffer could markedly improve the refolded yield of lysozyme. a suitable concentration of glycerol could also benefit the refolding of rhg-csf using imac or sec [30, 80] . this is probably because the addition of glycerol favors the formation of correct refolded intermediates. the combination of pflc and refolding additives can have an additive effect, and thus hasten the protein folding; therefore more experiments should be performed in the future. similar to protein folding by dilution method, the injected total mass of unfolded protein can also affect protein folding. it was found that the refolded yields decreased by increasing the initial protein concentration during the refolding of carbonate anhydrase and urokinase plasminogen activator by using sec [23, 26] . guan et al. [74] obtained a similar result during the refolding of rhifn-␥ by using afc. li et al. [54] found that the bioactivity recovery decreased by increasing the injection mass during the refolding of lysozyme using iec, the bioactivity recovery decreased from 100 to 62% as injected mass increased from 2 to 30 mg. liu et al. [57] found that the bioactivity recovery increased gradually by increasing the initial protein concentration when the initial protein concentration was relatively low, but decreased by further increasing the initial protein concentration, this is consistent with the results obtained by stempfer et al. [52] . langenhof et al. [61] found that both refolded yield and mass recovery decreased by increasing the injection mass during the refolding of bsa using iec. this is because the higher injected mass of protein would affect the adsorption of the denatured protein molecules by the stationary phase, thus resulting in increasing aggregation of the partially refolded protein molecules. partial refolded intermediates also form at higher local protein concentration during elution process. but protein concentrations after elution and production efficiency would improve as injected protein mass increased; however, protein purity would decrease to a certain extent. therefore, several factors, such as refolded yield, production efficiency and protein purity must be considered together when investigating injection mass. pflc is a newly developed method which enables many biochemical and physicochemical processes, and performs protein folding which was originally in normal buffer by liquid chromatography. it has many advantages compared with other methods for protein folding, but it is not as simple to operate as lc. actually, many technical difficulties and theoretical problems must be overcome. although the presence of the stationary phase in an lc system brings many advantages for protein folding, because the mechanism of protein folding carried out in the usual buffer has not been fully understood yet, much more complicated theoretical problems stemming from pflc are required to be solved. thus, pflc is only at the starting point of its development cycle. however, because it has so many advantages, such as easy operation, high automatization, easy scale up, refolding of proteins at higher concentrations and also purifies target proteins simultaneously during protein folding, scientists may be encouraged to pay more attention to develop new theories and explore new technologies to facilitate this method. thus, it can be expected that pflc will have a very bright future. however, a lot of difficult problems are encountered during the folding of a denatured/reduced protein. it involves both thermodynamic problem (correct forming of many disulfide bonds existed in a protein molecule) and kinetic problem (rapid forming of disulfide bonds). an approach is to synthesize new chromatographic packing materials with good properties (having good capability of refolding and separating proteins) and low costs, such as to prepare stationary phases binding to a pair oxidized/reduced agents, such as glutathione in oxidation form (gssg) and its reduced form (gsh) to make the forming of disulfide bonds during chromatographic process. because ligands of many kinds of lc including hic, iec, afc, have hydrophobic region, the presence of hydrophobic region of an amino acid sequence can help to form a correct microdomain, resulting in each thiol, or at least most of thiols approaching a right place for subsequential thiol pairing. if a suitable catalyst can be found to accelerate the oxidation of each thiol, whole refolding of the protein can be accomplished during the process of elution. if several thiols existed in a denatured/reduced protein, some of these thiols could be rapidly oxidized to form correct disulfide bonds on a column, because a corrected domain has thermodynamically stable structure, the remaining thiols cannot freely move and can be permitted to stand enough time to continuously perform oxidation, or refolding for several hours, even overnight after being eluted out of column. an appropriate regenerization method for these types of stationary phase to make the gssg column regeneration should be established. it is also important to find out fast and low-cost disulfide pairing methods for industrial production, to avoid use of expensive agents, such as glutathione, molecular chaperones, and enzymes for protein folding. to find out more effective ways to dissolve the deposit formed by aggregates/precipitates on the packing bed, resulting in recovering the target protein and regenerating chromatographic column. further broadening the applications of pflc to various standard proteins and recombinant proteins in inclusion bodies, especially those that have large molecular weight or more than four disul-fide bonds, and to investigate factors effecting pflc to provide more data for the optimization of protein folding, to thoroughly develop pflc for the industrial scale, to investigate and to summarize rules for scaling-up pflc; to design a new and cheap equipment and to facilitate pflc, and to combine pflc and other refolding methods such as artificial molecule chaperones to develop new pflc methods. we do believe that pflc has a bright future. protein folding liquid chromatography stoichiometric displacement theory and its application chinese patent introduction to modern separation science ucla symposia on molecular and cellular biology, new series us patent 4 progress in biotechnology research masteral degree thesis key: cord-008777-i2reanan authors: nan title: ecb12: 12th european congess on biotechnology date: 2005-07-19 journal: j biotechnol doi: 10.1016/j.jbiotec.2005.06.005 sha: doc_id: 8777 cord_uid: i2reanan nan in the last 20 years biotechnology has made tremendous progress in its different application fields: red biotechnology, the use of biological methods for medical purposes, is firmly established in the development of new drugs. the use of plant or green biotechnology is under controversial discussion in politics and public. nevertheless, genetically modified herbicide and insect resistant crops are cultivated to a large extent. industrial biotechnology, now often named white biotechnology, seems widely underestimated in the public perception. it includes all industrial processes for the production of chemical products and enzymes, which fully or partly rely on the biological toolbox of nature. white biotechnology processes are carried out in a contained environment, typically in a bioreactor in a dedicated industrial plant. well-known examples are the fermentative productions of antibiotics, amino acids, vitamins and enzymes, products related to medical, food and feed applications. many products like the amino acids glutamic acid, lysine, threonine and tryptophane are exclusively produced using microbes in large scale industrial processes. in other cases, like the water soluble vitamin b2, biotechnological processes successfully replaced chemical productions, due to lower costs and improved ecoefficiency. in contrast to this, most industrial chemicals and polymers are produced by chemical synthesis based on oil and gas. however, there are some examples for bioproducts among industrial chemicals. the solvents acetone and butanol, for instance, were manufactured by fermentation for several decades in the last century. since the 1950s these fermentations have been replaced by more efficient and cheaper chemical synthesis. recently, new pilot and production processes for biopolymers like pha or biomonomers like 1,3-propanediol or lactic acid were announced by different companies. currently, ethanol is by far the largest white biotech product by volume. in brazil, where bioethanol is used as liquid transportation fuel, the annual production is in the range of 15 mio m 3 . bioethanol is of growing importance also in the united states. business consultants predict a tremendous growth of biotechnological products within the chemical industry. high prices for crude oil, dropping prices for renewable resources, and scientific progresses nourish the expectation that industrial biotechnology will replace many bulk chemicals. is this realistic? will we switch from a petrochemical industry to a biobased chemistry within the next years? based on economic considerations it can be stated that this is a long term goal. to achieve this it remains a scientific challenge to make renewable raw materials available for competitive bioproduction of bulk chemicals at low costs. conversion of lignocellulosic material to fermentation sugar may be a solution. also green biotechnology can contribute to the supply of cheap fermentation raw materials. innovative ideas for downstream processing or further chemical conversion of fermentation products are required to enter the chemical value chains. furthermore, the identification of new higher value bioproducts is a chance for short term successes in white biotechnology. enzyme and protein engineering has the potential to create new biomolecules, metabolic engineering can contribute to develop new metabolic pathways, may be even for unnatural compounds. by continuously increasing the efficiency and throughput of dna sequencing we, together with colleagues, have sequenced the human genome and the genomes of all the major model organisms. the challenge now centers on understanding these vast instruction sets. our ability to read these instructions must be enhanced through collection of key additional data sets. one productive path for delineating the functional sequences and inferring their function is comparative sequence. the mouse genome sequence, for example, led to estimates that only 5% of the human genome is functional. sequencing of an extensive set of additional mammalian genomes promises to define these functional sequences with a resolution of less than 10 base pairs. on a different course, we have sequenced the chimpanzee genome to learn what has changed in the evolution of humans. beyond providing for the first time a catalog of the differences between the two genomes, the comparison of the chimpanzee and human genomes reveals the patterns of neutral mutation and regions that deviate from that. the talk will summarize these and related findings. the sequence of additional primate genomes will help delineate what has changed specifically in humans and add power to the analysis. ultimately, capturing human sequence variation and correlating with phenotypic variation will be required to understand function. but learning what these functional elements do requires new sets of experimental data. for this, we have turned to the nematode c. elegans. in this simple system most of the ∼20k genes have been defined and experimentally confirmed. beyond the hundreds of genes with already known mutants, two centers are systematically producing gene knockouts or rnai can be used to inhibit any gene temporarily. sequences of three caenorhabditis species are already available, and two more are underway. expression data has been collected for all the genes under many conditions and time points through development. to enhance the resolution of expression data and to simplify phenotypic analysis of embryonic mutants, we are developing a system that will automatically trace the cell lineage and assign gene expression to precise cells with high temporal resolution. the latest results with the system will be described. in the longer term, this and similar datasets should provide an understanding of how the genome specifies the form and behavior of the worm. uhlen department of biotechnlogy, albanova university center, royal institute of technology (kth), stockholm, sweden here, we present a new protein atlas database (www.proteinatlas.org) showing the expression and localization of human protein in normal and cancer tissues. the atlas is based on the use of antibodies (agaton et al., 2003) to generate high-resolution immunohistochemistry images representing 48 normal tissues and 20 different cancer types (uhlen and ponten, 2005) . each antibody is used to generate more than 500 individual images and each image has been annotated by a pathologist (kampf et al., in press) . the database has been created by the swedish human proteome resource (hpr) and the program has been set-up to allow the exploration of the human proteome with antibody-based proteomics (nilsson et al., in press) . the basic concept is to generate, in a systematic and high-throughput manner (uhlen and ponten, 2005) , specific antibodies to all human proteins, and subsequently used these for functional analysis of the corresponding proteins in a wide range of assay platforms, including (i) a protein atlas for tissue profiles (kampf et al., in press) , (ii) specific probes to evaluate the functional role of individual proteins, and (iii) affinity reagents for purification of the specific proteins and their associated complexes for structural and biochemical analyses. ments, most effective source of variation was perturbation in growth medium, followed by perturbation in growth rate. effect of gene deletion on data variation was found to be less apparent when compared to other perturbations. a significant similarity in variation of metabolome and mrna data was observed, which may be used as the key point for integration of these two sets of data in functional analysis of genes. projection to latent structures (partial least squares, pls) is used for integration of transcriptome and metabolome data. comparison of pca and pls shows that linear model constructed via pls to predict the metabolome data does not make use of all the variation in transcriptome data. thus, pls allows the discrimination between the portion of gene expression change that affects the metabolome profile and the portion that is not directly effective on metabolome. both pca and pls can be used to detect the open reading frames (orfs) which are the main sources of variation in transcriptome data and/or effective on metabolome profile. extracellular metabolomics to accelerate the discovery of key genes involved in fibre degradation silas g. villas-bôas, geoffrey lane, graeme attwood, adrian cookson agresearch limited, grasslands research centre, tennent drive, private bag 11008, palmerston north, new zealand. e-mail: silas.villas-boas@agresearch.co.nz (s.g. villas-bôas) the genome of the hemicellulose-degrading microbe clostridium proteoclasticum is been sequenced and an array of candidate genes with diverse activity relevant to fibre degradation have been identified by automated gene annotation methods. c. proteoclasticum falls within the butyrivibrio-pseudobutyrivibrio assemblage of rumen bacteria which are though to play an important role in the degradation of plant hemicellulose-lignin complexes which limit fibre degradation in the rumen. for new zealand it makes strategic sense to invest in microbial genomics efforts applied to agriculture where the country holds a strong competitive advantage and where ruminants constitute the vast majority of farmed animals. in conjunction with dna sequencing, proteomics and transcriptomics (micro-array analysis) we are using metabolomics as an additional functional genomics tool for gene discovery. we have established a footprinting approach for microbial metabolome analysis focused mainly on metabolic intermediates of polysaccharide degradation to provide quantitative information on end products of fibre-degrading enzymes. a gc-ms method has been developed that is able to resolve complex biological mixtures containing mono-, di, and oligosaccharides, in addition to a series of organic acids. we are currently phenotyping a series of c. proteoclasticum mutants to validate our analytical methodology and we are going to fully characterize the fibrolytic ability of c. proteoclasticum to be compared with other fibre-degrading microbes. we believe that our metabolomics data will complement current proteomic analysis of fibre-degrading enzymes and micro-array analysis of gene expression from a series of mutants by providing direct evidence of the metabolic function of key genes involved in fibre-degradation processes. many gram-negative bacteria utilize cell-to-cell communication systems that rely on diffusible n-acyl homoserine lactone (ahl) signal molecules to monitor the size of the population in a process known as quorum sensing (qs). in human pathogens this form of gene regulation ensures that the cells remain invisible to the immune system of the host until the pathogen has reached a critical population density sufficient to overwhelm host defenses and to establish the infection. the qs regulon of pseudomonas aeruginosa and burkholderia cepacia, two important pathogens for patients suffering from cystic fibrosis, has been studied by proteome analyses. comparative twodimensional gelelectrophoresis of pre-fractionated protein mixtures (extra-, surface-, and intracellular proteins) coupled to mass spectrometry analysis or n-terminal sequencing has been employed to recognize and identify qs-controlled proteins. our findings strongly support the importance of ahl-mediated cell-cell-communication as a global regulatory system and suggest that qs control also operates via post-translational mechanisms. as qs has been proven to be a central regulator for the expression of pathogenic traits and biofilm formation in opportunistic human pathogens it represents a highly attractive target for the development of novel anti-infective compounds. functional genomics technologies (transcriptomics and proteomics) have been exploited to validate the target specificity of natural and synthetic qs inhibitors, thus having a great potential as alternative therapeutics for the treatment of bacterial infections. modeling cell cycle complex formation from high-throughput data sets lars juhl jensen european molecular biology laboratory, meyerhofstrasse 1, 69117 heidelberg, germany. e-mail: jensen@embl. de to analyze the dynamics of protein complexes during the mitotic cell cycle, we integrated data on protein interactions and gene expression. the resulting time-dependent interaction network for the first time places both periodically and constitutively expressed proteins in a temporal cell cycle context, thereby revealing novel components and modules. we discover that most complexes consist of both periodically and constitutively expressed subunits, suggesting that the former control complex activity by a mechanism of just-in-time assembly. consistent with this, we show that additional regulation through targeted degradation and phosphorylation by cdk1 (cdc28p) specifically affects the periodically expressed proteins. alessandra luchini, andrea callegaro, silvio bicciato department of chemical engineering processes, university of padova, padova, italy. e-mail: alessandra.luchini@unipd.it (a. luchini) since transcriptional control is the result of complex networks, analyzing dynamical states of gene expression is of paramount importance to detect the multivariate nature of biological mechanisms. although hundreds of studies fully demonstrated the relevancy of microarrays in describing different physiological conditions, to reconstruct complex interaction pathways it is necessary to analyze the temporal evolution of transcriptional states. however, a robust experimental design for identifying differentially expressed genes over a temporal window would require large amounts of microarrays. unfortunately, replicates for each time point and experimental condition are not always available, because of cost limitations and/or biological samples scarcity. in addition, common data analysis tools, like anova, require replicates and disregard correlation structure among times. we present a method for the identification of differentially expressed genes in un-replicated time-course experiments. the procedure does not assume any model or distribution function, takes into account the correlation of data, and does not require sample replicates at the various time points, other than the presence of an initial time point for all analyzed conditions. the identification of differentially expressed genes as the result of a system perturbation is formally stated as a hypothesis testing problem in which a defined statistic is used to rank transcripts in order of evidence against the null hypothesis. specifically, (i) data are structured so that measurements are correlated in time, within the same biological condition; (ii) the null hypothesis is formulated so that changes in expression levels at different time points are equivalent; (iii) time point t0 represents the system before the perturbation. therefore, modulated genes are detected testing the statistical significance of expression differences between physiological states at each time point, once corrected by the variability at t0, and given an empirical null distribution constructed using permutations. statistical significance is assessed by the q-value. the method has been tested on time-course microarray experiments aimed at studying the temporal changes of gene expression in: (i) skeletal muscle cells treated with a histone deacetylase inhibitor (iezzi et al., 2004) and (ii) immature mouse dendritic cells (dc) exposed to larval and egg stages of s. mansoni (trottein et al., 2004) . differentially expressed genes, identified using the proposed algorithm, have been compared with results obtained from anova model and sam paired test. the biological significance and soundness of selected transcripts was also verified using global functional profiling by means of ontotools. results demonstrate that this novel procedure allows the identification of biologically relevant genes using half of the replicates required by standard model-based approaches. carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y2 have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. the calcium-dependent antibiotic (cda) is a lipopeptide synthesised non-ribosomally and produced by streptomyces coelicolor a3(2). cda contains several non-proteinogenic amino acid residues. hydroxyphenylglycine (4-hpg) is one of the unusual amino acids in the structure of the cda and vancomycin groups of antibiotics. for the members of the vancomycin group of antibiotics, the 4-hpg residue plays crucial roles in the structure and function of the final glycopeptide antibiotic. to reveal the putative biosynthetic pathway of this amino acid in cda, a standard "double crossover replacement strategy" was used to delete 4-hydroxymandelic acid synthase (4-hmas, encoded by hpd) from s. coelicolor mt1110 and 2377, using the delivery plasmid pzmh3. there was no cda production in the disrupted strains. plates containing a gradient of hydroxymandelic acid were used to restore cda production in both s. coelicolor mt1110 hpd and 2377 hpd. exogenous supply of 4-hydroxyl phenylglyoxylate and 4-hydroxyphenylglycine reestablished cda production by the hpd mutant. feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues (mutasynthesis). a cxcl2 tandem repeat promoter polymorphism is associated with susceptibility to severe sepsis in the spanish population n. maca-meyer 1 , c. flores 1 , l. pérez-méndez 1 , r. sangüesa 2 , e. espinosa 2 , j. villar 1 : 1 research institute, hospital universitario n.s. de candelaria, s/c tenerife 38010, spain; 2 department of anesthesiology, hospital universitario n. s. de candelaria, s/c tenerife 38010, spain. e-mail: nmacame@ull.es (n. maca-meyer) sepsis describes a complex clinical syndrome resulting from a systemic inflammatory response to bacteria, and remains an important cause of mortality in the intensive care unit. cxcl2 chemokine (or mip-2) exhibits a pivotal role in the immune response, and several functional studies in animal models of sepsis have catalogued cxcl2 as a candidate gene for the development of sepsis. we have performed a case-control association study of cxcl2 gene variants and susceptibility to severe sepsis in 179 hospitalised patients and 364 healthy individuals. after the examination of linkage disequilibrium in the region, we analysed whether two promoter polymorphisms (snp rs3806792 and a newly described polymorphic short tandem repeat d4s3454) were associated with the syndrome. we found a significant association of common variants at d4s3454 with the development of severe sepsis (heterozygote carriers or 2.82; 95% ci 1.10-7.24, and homozygote carriers or 3.65; 95% ci 1.41-9.43; mantel-haenszel χ 2 test for linear trend p = 0.0006). the risks remained significant even after a genomic control adjustment, based on 20 additional genotyped polymorphisms not linked to the candidate gene. these preliminary results suggest that cxcl2 gene variants may contribute to the development of severe sepsis. kasper møller 1 , ana paula oliveira 1 , jens nielsen 2 , mark johnston 1 : 1 center for microbial biotechnology, biocentrum, technical university of denmark, denmark; 2 department of genetics, school of medicine, washington university, st. louis, usa glucose is the preferred carbon and energy source for most cells. in saccharomyces cerevisiae, a complex regulatory network ensures that s. cerevisiae ferments glucose to ethanol even in the presence of oxygen. to obtain a better understanding of this crabtree effect and the logic of the glucose signalling network in s. cerevisiae, we are analyzing glucose sensing and signalling in the related species saccharomyces kluyveri, which exhibits much less of a crabtree effect (it prefers not to ferment glucose when oxygen is available). we show that there are only two major glucose transporters in s. kluyveri, and that these are regulated in response to the availability of glucose via a glucose sensor and a signalling pathway similar to the glucose induction (rgt2/snf3-rgt1) pathway in s. cerevisiae. we have used dna-microarrays for s. kluyveri to find targets of the s. kluyveri glucose induction pathway, as well as to evaluate the global response to a change in environment from growth on ethanol to growth on glucose. this study identifies a number of differences in the regulation of glucose uptake and global responses to glucose between s. kluyveri and s. cerevisiae, which may contribute to their different glucose metabolism. detection and analysis of microrna using lna probes nana jacobsen, christian lomholt, peter mouritzen, peter stein nielsen, mikkel noerholm exiqon a/s, bygstubben 9, dk-2950 vedbaek, denmark. e-mail: mouritzen@exiqon.com (p. mouritzen) micrornas are a class of short endogenous rnas that act as post-transcriptional modulators of gene expression. growing evidence suggest that micrornas exhibit a wide variety of regulatory functions and exert significant effects on cell growth, development, and differentiation. recent studies have shown that human microrna genes are frequently located in cancer associated genomic regions and perturbed microrna expression patterns have been observed in many malignant tumors. we have exploited the significantly improved hybridization properties of lna oligonucleotides against rna targets to design lna-modified dna probes for detection of different micrornas in animal and plants by northern blot analysis, microarray hybridization and in situ hybridization. we will describe the results obtained from detection and analysis of different micrornas in c. elegans, zebrafish, mouse, and plants. in addition, we will describe a novel lna-based method for expression profiling of mature micrornas by quantitative rt-pcr. expression profile of the sty and paa genes in pseudomonas sp. y2 by means of dna microarrays david bartolomé-martín 1 , david juck 2 , m a teresa del peso-santos 1 , charles w. greer 2 , julián perera 1 : 1 departamento de bioquímica y biología molecular i, facultad de ciencias biológicas, universidad complutense de madrid, 28040 madrid, spain; 2 environmental microbiology group, biotechnology research institute, national research council canada, montréal, que., canada h4p 2r2. e-mail: perera@bio.ucm.es (j. perera) dna microarrays are a new and powerful tool to study gene expression in very diverse systems. environmental biotechnology and biodegradation are some of the fields of research where this technology may be very promising. pseudomonas sp. y2 is a bacterium able to grow in minimal medium plus either styrene (sty) or phenylacetic acid (paa) as the sole carbon and energy sources. this bacterium is the only organism where the genes that code for both the upper (sty genes) and the lower (paa genes) catabolic pathways for the styrene degradation have been described till now. it is unique in having two active copies of the genes encoding the lower pathway (paa1 and paa2 gene clusters). we have designed a dna microarray with the sty and paa genes in order to analyse their expression in the wild type pseudomonas sp. y2, in p. sp. y2 t2 (a paa2 deletion mutant) and in p. sp. y2 c1 (a crc gene mutant). this analysis has been performed on bacterial cultures grown in media with different carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y2 have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. dynamics in induced repression of phosphomannose isomerase pmi40 gene of saccharomyces cerevisiae anssi törmä 1,2 , juha-pekka pitkänen 1,2 , laura huopaniemi 2 , risto renkonen 2 : 1 medicel ltd., haartmaninkatu 8, 00290 helsinki, finland; 2 rational drug design program, department of bacteriology and immunology, haartman institute and biomedicum, university of helsinki, p.o. box 63, 00014 helsinki, finland. e-mail: juhapekka.pitkanen@medicel.com (j.-p. pitkänen) gdp-mannose is the precursor of cell wall biosynthesis in s. cerevisiae. to understand the system level role of gdp-mannose, we studied a conditional knock-out strain of the key enzyme in its synthesis; pmi40. the experimental procedure allowed us to study the order of mechanisms the cells launch in order to adjust to a sudden malfunction in the metabolic machinery. we collected 100 samples from continuous cultivations over 80 h and measured genome-wide gene expression levels, 10 enzyme activities, and concentrations of 30 intracellular metabolites. for sampling we have built a sample robot, which automatically takes and preserves the samples. in order to carry out this magnitude of experimentations and generated data, we have constructed a proprietary software platform to handle all the phases from project management in wet-lab to workflow and pathway management in in silico. after normalization and clustering, significantly changed genes and metabolites were searched for enrichment in biological processes and molecular complexes. further, gene expression levels, metabolite concentrations, and enzyme activities were searched against each other for causality over time. overall, we focused on thorough analysis of our own data and known database data in order to reward our efforts with knowledge. at the transcriptome level, repression of pmi40 led to two major types of activation profiles, one peaking at the time when pmi40p activity and gdp-mannose were depleted and the other later during recovery from the perturbation. the primary response was most enriched with genes known to play roles in mating and filamentous growth and associated with the transcription factors ste12p, tec1p, dig1p, and mcm1p, whereas the secondary response consisted of genes involved in carbon metabolism and associated with the general stress response regulators msn2p and msn4p. skn7p, a high-level transcription factor was associated with both the primary and the secondary response, consistent with its suggested role of coordinating environmental responses and developmental processes. transcriptome of pig ovarian cells: discriminant genes involved in follicular development bonnet a., le cao k.a., low-so g., san cristobal m., tosser-klopp g., hatey f. laboratoire de génétique cellulaire, centre inra de toulouse, castanet-tolosan 31326, france in order to identify genes and gene networks involved in pig ovarian follicular development, we built subtractive suppressive hybridization libraries (ssh) from granulosa cells of healthy follicles (small, medium or large). the rna isolated from these cells was used to hybridize cdna nylon micro-arrays. data analysis using a gaussian linear mixed model showed that 83% of the variability is due to the genes. two hundred fifty one regulated genes (from the 956 expressed) were identified and clustered into three groups according to the follicle size. moreover, we found previously identified genes such as aromatase, igfbp2 which supported the validity of our experimental model. ramdom forest analysis put forward the most discriminant 11 genes between the three follicle classes. this study put forward gene sets such as those involved in cell modeling, regulation of transcription, apoptosis during follicle growth. the next step will be to describe more precisely the spatio-temporal expression patterns at the mrna levels of the genes identified by these experiments. microalgae constitute a significant source of valuable natural products, e.g. sulfated polysaccharides, polyunsaturated fatty acids, and phycobiliproteins that find applications in wide range of industries, including food, pharmaceutical, agricultural and cosmetics. however, genomic and molecular genetic studies of microalgae lag far behind those of higher plants. in order to accelerate red microalgal genomic studies by taking advantage of current genomics and post-genomic technologies, we have generated expressed sequence tag (est) databases of two red microalgae porphyridium sp. and dixoniella grisea grown under various physiological conditions. to date we have sequenced 7210 and 6231 ests of porphyridium sp. and d. grisea, respectively. the sequence assembly resulted into, ca. 2000 non-redundant unigenes for each microalga, only 40% of which were identified by similarity to sequences in the public databases. porphyridium sp. and d. grisea unigenes were compared with the whole-genome predicted proteomes of three microalgae and those of representative eukaryotic and prokaryotic organisms. both microalgae have highest similarity to the red microalga cyanidioschyzon merolae. the order of sequence similarity to other organisms examined was arabidopsis thaliana, oryza sativa, chlamydomonas reinhardtii, thalassiosira pseudonana (diatom), saccharomyces cerevisiae, caenorhabditis elegans, archaea and cyanobacteria. although red microalgae are considered as phylogenetic bridge between prokaryotes and eukaryotes, our data show that the red microalgae have strong similarity to eukaryotes and only distant similarity to prokaryotes. gene expression profiles were studied by analyzing cdna and subtraction libraries constructed from algae grown under various physiological conditions. we observed that top three most abundant ests in the stationary phase of porphyridium sp. were adp ribosylation factor like-1, flavohemoglobin and adp ribosylation factor-1. in addition, we have identified several genes which were specific to nitrate-and sulfate starvation. the sarco(endo)plasmic reticulum ca 2+ -atpase (serca, "the calcium pump"), is responsible for pumping the ca 2+ released into the cytoplasm during muscle contraction back into the sarcoplasmic reticulum store while proton are pumped the opposite way as counter-cations. these transport processes go against the concentration gradients and are therefore energy consuming. the energy is derived from atp hydrolysis via formation and break-down of a phospho-enzyme intermediate. over the last year a number of new crystal structures have been published which have added to our understanding of how this task is accomplished, which provides an impressive insight to the mechanism of a molecular pump. rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. this work was supported principally by embo and the mrc. structure and target-specificity of thioredoxin h kenji maeda 1 , anette henriksen 2 , per hägglund 1 , christine finnie 1 , birte svensson 1 : 1 biochemistry and nutrition group, biocentrum-dtu, technical university of denmark, dk-2800 kgs. lyngby, denmark; 2 biostructure group, carlsberg laboratory, dk-2500 valby, denmark. e-mail: kenji@biocentrum.dtu.dk (k. maeda) thioredoxins are ubiquitous small proteins with protein disulphide reductase activity. thioredoxins can alter the structures and the activities of various target proteins by reducing their disulphide bonds. seeds of several plants are abundant in cytosolic thioredoxins referred as h-type. in barley, two thioredoxin h isoforms, hvtrxh1 and hvtrxh2 that share 51% sequence identity but differ in temporal and spatial distributions were previously identified and characterised. in the present study, the relationship between structures and targetspecificities of h-type thioredoxins are analysed. the 3d-structures of hvtrxh1 and hvtrxh2 are determined by x-ray crystallography as the first crystal structures of thioredoxin h. comparison of the structures shows that the majority of solvent exposed residues near the active sites are conserved between the two isoforms. this is in agreement with previously observed similarity in target-specificity of the two isoforms. thioredoxins from organisms distantly related to barley, such as e. coli, have highly similar folds but different surface charge distributions compared to barley thioredoxins. a comparison of the target-specificities of hvtrxh1, hvtrxh2, e. coli thioredoxin and several thioredoxin mutants will be attempted to reveal the structural features that influence specificity of barley thioredoxin h isoforms. enbrel is a dimeric fusion protein consisting of the extracellular ligand binding portion of the human 75 kda (p75) tumor necrosis factor receptor (tnfr) linked to the fc portion of human igg1. the cho-expressed molecule contains both n-and o-linked oligosaccharides with a total carbohydrate content of 20% by mass. the o-linked oligosaccharides were released by hydrazinolysis and their structure determined by exoglycosidase sequencing and maldi-tof mass spectrometry. to locate precisely the o-linked sites, the glycosylation heterogeneity of tnfr:fc was simplified by treatment with n-acetyl neuraminidase and n-glycanase. the remaining molecule, which only carries core o-linked glycan structures, was cleaved by trypsin and analyzed by lc-ms. precise localization of o-glycosylation sites was determined based on the concept of a specific modification of the o-glycosylated serine into 2aminopropenoic acid and o-glycosylated threonine into 2-amino-2butenoic acid. the deficit in mass resulting from this transformation was the marker used to localize the modified residues on the peptides by tandem mass spectrometry sequencing (ms-ms). ms-ms spectrum of enbrel glycopeptides were interpreted based on the presence of 2-aminopropenoic acid and 2-amino-2-butenoic acid, resulting in a complete map of o-linked glycans precisely located at 10 different sites. anu mursula, beatrix fahnert, sari krapu, eija-riitta hämäläinen, ritva isomäki, peter neubauer bioprocess engineering laboratory and biocenter oulu, university of oulu, oulu, finland. e-mail: anu.mursula@oulu.fi (a. mursula) wnt proteins form a highly conserved family of secreted glycoproteins important in cell-cell signaling events during embryogenesis and adult tissue maintenance. impairments within this complex signaling pathway can lead for example to developmental defects in embryos, degenerative diseases and cancer. respectively, wnt proteins can be used as tools in basic research concerning wnt function, developmental biology, screening for interacting compounds, and for medical applications (e.g. therapeutics, stem cells). hence, recombinant wnts provide a valuable basis for these purposes. however, production of recombinant wnt proteins is challenging, because they contain multiple disulfide bonds making the folding very difficult. a process for production of murine wnt-1 in e. coli has been developed in our laboratory. the knowledge obtained from this research has also been applied to the expression of other wnts, namely wnt-4 and wnt-6, and can be used to approach other cysteine-rich proteins as well. since the expression level of wnt proteins is rather low so far, tools for monitoring and optimizing the production process have been established. by means of this sandwich hybridization method the level of target (wnt) mrna can be measured. the technique has already been applied to analyzing wnt-1 mrna levels. probes also for wnt-4 and wnt-6 have been generated. thus, transcription of wnt genes in all kind of cells (e.g. tissue, recombinant hosts) in general as well as kinetics of transcription can be studied using these tools. in growth factor signaling, stimulation of cell-surface receptors first triggers activation of the receptor itself and then of a large number of intracellular effector molecules. the stimulus is integrated with a host of other cellular processes, leading to cytoskeletal changes, activating transcriptional programs in the nucleus and ultimately resulting in cell proliferation, differentiation or motility. classical signaling pathways and networks depict potential protein-protein interactions only in a static form. in the cell, these interactions are dynamic and occur in an ordered fashion. here, we apply a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance in order to study the global dynamics of signaling events. briefly, three cell populations are metabolically labeled with either normal arginine or a 13 c substituted form, or a 13 c 15 n variant (stable isotope labeling by amino acids in cell culture, silac). each population was then stimulated with egf for a different time period and tyrosine phosphorylated proteins were affinity purified with anti-phosphotyrosine antibodies. the proteins from the precipitated complexes were quantitatively analyzed and identified using lc-ms/ms. arginine containing peptides occurred in three forms, directly indicating protein activation at the corre-sponding time point. combination of two experiments sharing one common time point of activation then generated five-point dynamic profiles. from the 202 proteins quantified, we identified 81 signaling proteins, including virtually all known egfr substrates and 31 novel effectors, and the time course of their activation upon egf stimulation. discriminating proteins involved in the signaling network from unspecific binders was straightforward as these presented an activation profile. we have now further extended this study by directly measuring in vivo phosphorylation sites in response to growth factor stimulation and monitoring the time evolution of the phosphorylation events. finally, we determined and quantitatively compared the global egf and pdgf tyrosine phosphoproteomes in human mesenchymal stem cells and revealed a control point in their differentiation into bone-forming cells. such global activation profiles provide a novel perspective in cell signaling and will be crucial to model the highly dynamic signaling networks in a systems biology approach. klaus schneider 1 , dave g smith 2 , steven skaper 2 , alastair d. reith 1 : 1 discovery research, glaxosmithkline, coldharbour road, harlow, essex, uk; 2 neurology & gastrointestinal cedd, glaxo-smithkline, coldharbour road, harlow, essex, uk over the last 15 years, progress in signal transduction research has revealed an astonishing degree of complexity in cell signalling which is manifested in positive and negative regulations and feedback loops within signalling pathways and by cross-talks between pathways, all of which are highly cell-type dependent. it has become evident that protein phosphorylation by protein kinases plays a major role in this complex regulation of cell signalling (hunter, 2000) . due to the importance of signal transduction in disease processes, many protein kinases may constitute key targets for disease intervention. yet, the lack of a full understanding of the regulation, the activation and, importantly, of downstream substrates of particular protein kinases requires often more detailed studies before initiation of resource-intensive efforts to find disease-modifying molecules. technologies for the study of protein kinase signalling include 32 p labelling, mutational and knock-out studies and more recently rna interference. these tools are complemented by approaches that are based on proteomic technologies developed over the course of the last 10 years. in this presentation, the scope of proteomics technologies to contribute to an understanding of kinase signalling will be discussed. an overview of available technologies will be given and results will be presented from a proteomic study of glycogen-synthase kinase 3 (gsk3) signalling (coghlan et al., 2000) . novel findings will be presented on a study of gsk3 inhibition in a primary neuronal cell line by differential 2d gel electrophoresis, which resulted in the identification of more than 40 proteins that were significantly regulated. proteome analysis is typically done by nanospray lc/ms in order to achieve higher sensitivity and thus a greater number of protein identifications. however, nano-scale lc systems can be more challenging to use and maintain. to obtain the best chromatographic performance, connections must be made carefully to minimize band broadening. improved chromatographic performance can enhance the mass spectrometric results by tandem ms as a greater number of peptides can be detected. a microfluidic chip-based system has been developed (yin et al., 2004) that minimizes the number of connections and the delay volumes. this work evaluates the performance of this device against the traditional nanospray approach. a yeast extract sample was separated by sds-page and bands were excised from the gel for further analysis. after in-gel digestion, the sample was analyzed by both traditional nanospray and the microfluidicbased chip device. after protein database searching, the identified proteins and the protein sequence coverage's were compared for the two approaches. the microfluidic device was demonstrated to be equivalent or better compared to the traditional approach. yin, h., killeen, k., brennen, r., et al., 2004. anal. chem. 77, 527-533 . plant cytochromes p450 (p450s) play key roles in the biosynthesis of most bioactive compounds with agronomic and therapeutic applications. a collection of about 120 plant p450s was expressed in yeast. the cdnas were isolated from the model plant with a sequenced genome arabidopsis thaliana, some others from wheat, helianthus tuberosus or vicia sativa. they were expressed under the control of a galactose-inducible promoter in an engineered strain of saccharomyces cerevisiae in which the gene of the native p450 reductase was replaced with the gene of a p450 reductase from a. thaliana under the control of the same galactose-inducible promoter, in order to provide an optimal context for plant p450 expression and activity (pompon et al., 1996) . an original procedure was designed for the high-throughput functional screening of this enzyme collection. it is based on the detection of oxygen consumed during the catalytic reaction by a fluorochrome embedded in the bottom of the microwell plates. this method was validated using several recombinant p450s of known activity. it also allows for a very efficient screening for enzyme inhibitors. the advantages and limits of the method will be discussed. this work was carried out with the support of génoplante programme (no 2001004) . reference pompon, d., louerat, b., bronine, a., urban, p., 1996. methods enzymol 272, 51-64. 3 folding of a bacterial membrane protein studied by protein engineering daniel e.otzen, pankaj sehgal, peter a. christensen department of life sciences, aalborg university, sohngaardsholmsvej 49, dk -9000 aalborg. e-mail: dao@bio.aau.dk (d.e. otzen) we have carried out a kinetic analysis of the folding of the 4-helix transmembrane protein dsbb in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate and dodecyl maltoside. this analysis incorporates both folding and unfolding rates, making it possible to determine both the stability of the native state and the process by which the protein folds. the analysis also takes into account the composition of the mixed micelles, which is different from the bulk detergent composition. refolding and unfolding are consistent with a three-state folding scheme involving the sdsdenatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of sds. the temperature-dependence of the folding reaction displays an unusual decrease in heat capacity accompanying unfolding, which probably reflects the amphiphilic environment of the membrane protein. destabilization of dsbb by different short-chain alcohols correlates very well with the alcohols' respective hydrophobicities. data from a series of ala-scanning mutants tentatively identify a nucleus for folding, which is relatively diffuse and involves all four helices. we are currently complementing this work with studies of the association of peptides corresponding to individual transmembrane segments of dsbb. the reca protein of e. coli plays a crucial role in homologous recombination and dna repair. the recombination process takes place in a filamentous complex, in which the protein monomers are arranged in a helical manner around a single-stranded dna (ss-dna). in the presence of atp the filament can accommodate a second, double-stranded dna (ds-dna) and the strand exchange reaction can occur. the three-dimensional structure of reca itself and its complex with adp have been determined by x-ray crystallography. the active nucleoprotein filament, however, has only been studied at low resolution. both electron microscopy (em) and small-angle neutron scattering (sans) indicate significant differences between the structures of the active nucleoprotein filament and the compressed, inactive filament of only reca. we have presented a structural model of the reca protein in its active filament with ss-dna, using data obtained by linear dichroism (ld) polarized-light spectroscopy, based on a technique, we call "site-specific linear dichroism", which allows the orientation of a set of amino acids to be determined from ld data by systematic modification of the protein. here, we show that ld data of the nucleoprotein filament with ds-dna is over all similar to the data of the complex with ss-dna, indicating that the orientation as well as internal structure of reca in the active filament is not significantly altered when the bound dna is changed from single-stranded to double-stranded. this result supports the idea that the strand exchange reaction occurs without large conformational change of the reca protein. the choline-binding modules: a powerful biotechnological tool jesús m. sanz instituto de biología molecular y celular, universidad miguel hernández, elche, spain choline-binding modules (chbms) are present in some virulence factors of streptococcus pneumoniae (pneumococcus). the most extensively studied chbm is c-lyta, the carboxy-terminal domain of the pneumococcal cell-wall amidase lyta. the three-dimensional structure of choline-ligated c-lyta is built up from six loop-hairpin structures ("choline binding repeats", chbrs) forming a left-handed ␤-solenoid with four choline binding sites. although the structure of the ligand-free form is not yet known, our folding studies suggest that it is more loosely packed, with a partially unfolded amino-terminal region and a stable carboxy-terminal moiety that is extremely resistant to chemical denaturation (maestro and sanz, 2005) . the affinity of c-lyta for choline and other structural analogues allows its use as an efficient affinity tag for overexpression, immobilization and single-step purification of proteins of biomedical interest (c-lytag fusion protein purification system). this system presents many advantages when compared to current commercial methods, namely simplicity, compatibility with buffers and robustness. the availability of multiple supports that specifically bind chbms (such as multiwell plates) has recently allowed the development of a new procedure for the immobilization of c-lyta-containing hybrid proteins that may be used in proteomics, diagnostics and peptide display. in this communication, we present our last results about the stability, folding and engineering of c-lyta, together with a compendium of the current biotechnological potential of this protein, and highlight the productive link between basic molecular studies and their application. many of the modern approaches for studying disease compare steady state functions, such as repair, growth, and regulated gene expression within the various biological compartments organised by specialized function, be it mitochondria or blood vessels. the assignment of protein identities, which are linked to key biological mechanisms, which are associated with disease processes and disease progressions are an important area of this work (marko-varga and fehniger, 2004) . today, the technology available for studying proteome expression and resolving exact protein and peptide identities in complex mixtures of biological samples allows global protein expression within cells, fluids, and tissue to be approached with confidence. this confidence is due in part to reproducible repetitive sampling and analysis technologies including robotics data acquisition and high level mass spectrometry including both laser-desorbtion and electro spray ionisation. the precision in defining differences between normal and diseased steady states is aided by the creation of compiled reference and master data sets and by new methods for multiplexing the analysis of samples in groups. the establishment of key representative reference proteome systems representing the dynamic changes in protein expression during disease will be vital to the interpretation of changes observed in specific samplings of disease states and specific cells obtained from these samples. the creation of reference databases of proteins linked to disease pathways will play an important role in furthering our understanding of the "proteome of disease". examples will be given where protein expression patterns have been generated from compartments within tissue sections. marko-varga, g., fehniger, t.e., 2004. j. proteome res. 3, 167178. 2 adaptation of the saccharomyces cerevisiae proteome to nutrient limitations studied by metabolic stable isotope labeling and mass spectrometry albert j.r. heck netherlands proteomics centre and utrecht university, the netherlands. e-mail: heck@npc.genomics.nl. url: www.netherlandsproteomicscentre.nl one of the major aims of proteomics is to provide quantitative data on differential protein expression levels. recently, mass spectrometry-based methods have been introduced that can provide quantitative data on differential protein expression, mostly using stable isotope labeling (goshe and smith, 2003) . we opted for metabolic labeling as this provides efficient means to quantify differential protein expression, and has the advantage that all proteins are labeled universally (romijn et al., 2003) . in their natural habitat microorganisms encounter non-optimal growth conditions and often growth is limited by one nutrient. microorganisms need to respond rapidly to changes in the environment in order to survive. in the present study, we investigate the proteome response of chemostat cultivated wildtype saccharomyces cerevisiae to two different nutrient limitations, namely carbon and nitrogen limitation. yeast was metabolically labeled in well-controlled chemostat cultures. 14 n and 15 n labeled proteins were separated using 1d gel electrophoresis followed by rp-lc-esi-ms on a lc-q. relative quantification was performed by using relex software (maccoss et al., 2003) . we quantified 759 proteins, using on average 8 peptide peak pairs per protein. this analysis revealed that 419 proteins showed a significant increase/decrease in expression level. the functional annotation of these proteins revealed that the yeast cells change expression levels of enzymes involved in metabolism of the growth-limiting compound. the protein expression ratios were compared with corresponding transcript levels. moreover, we compared the accuracy of quantifica-profiles mainly reflected differences in cellular origins in addition to different functional roles. mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute phase proteins, antioxidant proteins and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization and in lipid metabolism. several proteins not previously implicated in nerve regeneration were identified, e.g. translationally-controlled tumor protein, annexin a9/31, vitamin d-binding protein, ␣-crystallin b, ␣-synuclein, dimethylargininases and reticulocalbin. real-time pcr analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. in conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants. yeasts plasma membrane macromolecular components involved in stress resistance paola branduardi, paola paganoni, danilo porro dipartimento di biotecnologie e bioscienze, università degli studi di milano-bicocca, piazza della scienza, 2-20126 milano, italy. e-mail: paola.branduardi@unimib.it (p. branduardi) the plasma membrane is a universal structure of living cells constituting an essential barrier dividing and defining the intracellular from the extracellular environment. it is consequently easy to deduce the crucial role played by said structure for any cell of any living organism, and especially for unicellular organisms, since all the information deriving from the external environment as well as many of the consequent cellular responses have to pass through this barrier. unicellular organisms, thanks to easy manipulation and cultivation techniques, can represent a very useful model for studying the plasma membrane function and response. in addition microorganisms, and among them yeasts, can be considered advantageous cell factories for recombinant productions. in this contest, the implementation of any process of production has to take into account, among others, the response and the tolerance of the host to the external environment. from these considerations derives the interest of our group to analyse the main macromolecular components of yeasts plasma membranes (proteins, lipoproteins and lipids), isolated from cells grown under different stress conditions, with particular attention to acidic environments. here, we present our recent data about separation and identification (by sequencing analyses) of lipoproteins isolated from the conventional yeast saccharomyces cerevisiae as well as from the non-conventional and acid tolerant yeast zygosaccharomyces bailii cell cultures grown in different conditions. in parallel, the protein fraction is under evaluation through a differential 2d proteomic approach and consequent analyses. effect of fungal polysaccharides on the expression of pancreatic proteins in streptozotocin-induced diabetic rats sang woo kim 1 , hye jin hwang 1 , kwang bon koo 2 , jang won choi 2 , jong won yun 1* : 1 department of biotechnology; 2 department of bioindustry, daegu university, kyungsan, in an attempt to search novel biomarkers for monitoring diabetes prognosis, we examined the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of pancreatic proteins in streptozotocin-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited excellent hypoglycemic effect, lowering the average plasma glucose level of the diabetic rats to 52.3%. pancreatic proteome were analyzed by 2-de system, which separated more than 2000 individual spots. the 2-de analysis demonstrated that thirty-four proteins from a total of about 500 matched spots were differentially expressed, of which 26 spots were identified as the proteins whose expression has previously been associated with diabetes. twenty-two overexpressed and twelve underexpressed proteins were significant (p < 0.05) between the healthy and diabetic rats, and the altered proteins were restored (p < 0.05) upon eps treatment. it was first found that carbonyl reductase (18.6-fold, p < 0.001) and mawdbp (31.4-fold, p < 0.01) were surprisingly upregulated upon diabetes induction, and then those two protein concentrations were completely restored by eps treatment. moreover, we obtained eight unidentified proteins that have not been reported to be related with diabetes mellitus. these results evidenced the effect of fungal eps on searching potential markers for diagnosis and therapeutic manipulation of diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. the model established in our experiment is expected to mimic human diabetic status, which will help us to interpret the roles of biomarkers in diabetic state. the use of polyol-responsive monoclonal antibodies in immunoaffinity chromatography and as a probe for unfolding of wild-type and altered (t103i) amidase from pseudomonas aeruginosa s. martins 1 , j. andrade 1 , a. karmali 1 , a.i. custódio 2 , m.l. since immunoaffinity chromatography is a powerful protein purification technique of interest in proteomics, monoclonal antibodies (mabs) against mutant (t103i) amidase from p. aeruginosa were raised by hybridoma technology. in order to identify mabs that bind t103i amidase tightly but release under gentle conditions, hybridoma clones secreting polyol-responsive mabs (pr-mabs) were previously screened. nearly 10% of elisa assay-positive hybridoma produced clones secreting pr-mabs with potential application as ligands for immunoaffinity chromatography. to select the optimal conditions for amidase elution, an elisa-elution assay was carried out, with two of these clones (f6g7; e2a6). the dissociation of ag-ab complex required 10% of propylene glycol and either 0.25 m (nh 4 ) 2 so 4 or 0.25 m nacl. the binding of purified mab of igm class (e2a6) to wild-type and mutant amidases was investigate by direct elisa, which revealed that it recognised specifically a common epitope on both amidases. conformational changes on antigen molecule were studied. mab e2a6 showed a higher affinity for heat denatured forms than for native forms as revealed by affinity constants suggesting that the mab recognizes a cryptic epitope. the effect of mab e2a6 on amidase activity was also investigated. the binding of mab to wild-type and mutant amidases exhibited an inhibition and activation of 60% as a function of time, respectively. this pr-mab is useful as a probe to detect conformational changes in native and denatured amidases as well as a ligand in immunoaffinity chromatography, which is of great interest in protein purification and proteomics. fragility and solubility of non-classical inclusion bodieš s. peternel 1 , a. ristič 1,2 , v. gaberc-porekar 1 , v. menart 1,2 : 1 national institute of chemistry, ljubljana, si-1000; 2 lek pharmaceuticals d.d., ljubljana, si-1000. e-mail: spela.peternel@ki.si (š. peternel) human granulocyte colony stimulating factor (g-csf) is a pharmaceutically important cytokine. when overexpressed in escherichia coli, it is usually accumulated in the form of inclusion bodies (ibs) . when produced at 42 • c classical insoluble ibs are formed while at 25 • c non-classical ibs containing a high amount of correctly folded g-csf are formed. as higher fragility and solubility of non-classical ibs were noticed, we decided to check whether bacterial cell disruption method has any influence on their mechanical stability and solubility. enzymatic lysis, sonication and homogenization, methods often used for disruption of bacterial cells during the isolation of ibs were compared. lysozyme treatment of bacterial cells appears to be mild enough disruption method not influencing the integrity of ibs. homogenization of bacterial cells at high pressure (100.000-120.000 kpa) shows no impact on classical ibs while some loss of target protein from the non-classical ibs is observed. sonication seems to be most harmful as even at rather low sonication altitudes, noticeable disassembling and solubilization of non-classical ibs occurs while no effect on classical ibs is perceived. our studies show that non-classical ibs are much more fragile and soluble than classical ones. therefore, one should extremely carefully choose the method for cell disruption to avoid undesirable loss of the target protein. the danish tick ixodes ricinus parasitize three different hosts both mammals and birds during the 3-year life cycle. the aim of this study was to identify the last blood host being the host, which the nymph had parasitized before molting to the adult instar. the reason for the study was to reveal the origin of the host contributing the most to the life cycle of the tick and thereby the maintenance of tick-borne diseases in denmark. the most common tick-borne diseases are lyme borreliosis and tick-borne encephalitis (tbe) causing illness in both animals and humans. we analyzed adult ticks, which were collected from known hosts. the analysis was performed at different heat stable proteins, which could be detected during the off host period by elisa. we found that heat stable proteins could be used as identification markers for host recognition. mushroom polysaccharides alter the expression of diabetesassociated proteins in the liver of streptozotocin-induced diabetic rats hye-jin hwang 1 , sang-woo kim 1 , kwang-bon koo 2 , jang-won choi 2 , jong-won yun 1* : 1 department of biotechnology, daegu university, kyungsan, kyungbuk 712-714, korea; 2 department of bioindustry, daegu university, kyungsan, kyungbuk 712-714, korea. e-mail: jwyun@daegu.ac.kr (j.-w. yun) in the present study, we investigated the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of liver proteins in streptozotocin (stz)-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited an excellent hypoglycemic effect, lowering the average plasma glucose level in eps-fed rats to 52.3%. in the next step, we analyzed the differential expression patterns of rat liver proteins from each group, to discover potent candidates for diabetesassociated proteins. a total of 69 proteins of the 2-de gel were expressed differentially between diabetic and healthy rats. among them, 34 proteins were upregulated and 35 proteins were downregulated upon diabetes induction. many of these changes were in accordance with observations in previously published studies. surprisingly, the altered levels of most proteins in diabetic group were fully or partially restored to those of non-diabetic control group by eps treatment. moreover, we obtained 13 unidentified proteins that have not been reported to be related with diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. potential is still limited by the differences observed in the structure of plant and mammalian n-glycans. indeed, theses differences and particularly the presence of ␤1,2-xylose and ␣1,3-fucose glycoepitopes are responsible for the immunogenicity of plant n-glycans. in order to reduce the structural differences between plant and mammalian n-glycans, current strategies are to knock out plant-specific glycosyltransferases or to humanize plant n-glycans by expression of mammalian glycosyltransferases in plants. in the present study, we have expressed a human ␤1,4-galactosyltransferase in alfalfa. in order to further increase the efficiency of the human ␤1,4-galactosyltransferase in the plant golgi apparatus, we have exchanged the endogenous targeting signal of this human glycosyltransferase for the ones from plant glycosyltransferases recently characterized in our laboratory. we will illustrate this approach of targeted expression with the results obtained by fusion of the catalytic domain of human ␤1,4-galactosyltransferase with the n-terminal sequence of a plant glycosyltransferase that targets the fusion to the very early compartments of the golgi apparatus. the efficiency of natural versus targeted expression of human ␤1,4galactosyltransferase in alfalfa will be compared in term of n-glycan humanization. altogether, our results clearly illustrate that we are now on the way to get perfect copy of mammalian glycoproteins in alfalfa plants. construction of recb-recd gene fusion and analysis of fusion enzyme activities oytun portakal 1 , gerald r. smith 2 , pakize dogan 1 : 1 department of biochemistry, hacettepe university medical school, 06100, ankara, turkey; 2 divisions of basic sciences, fred hutchinson cancer research center, seattle, wa 98109-1024, usa. e-mail: oytun@hacettepe.edu.tr (o. portakal) protein folding is a fundamental process to gain protein function. in an oligomeric protein, the interaction between polypeptides affects the folding process and assembly to the holoenzyme. recbcd is a heterotrimeric and multifunctional enzyme that plays an essential role for major pathway of homologous recombination in e. coli. it is composed of recb, recc and recd gene products. recd is the fast motor unit of the recbcd enzyme. recd also plays a role for high affinity dsdna binding, nuclease activity and chidependent regulation. this study was designed to test the hypothesis that recd polypeptide regulates the essential reca loading activity. the approaching of the study was to fuse recd gene to subsequent recb gene and to observe the changes in enzyme activity and structure. for these purpose two genetic fusion mutations, two-nucleotide deletion and three-codon substitution were created at the overlap sites (ta) of recb and recd genes. fusion mutations were constructed by phage-mediated recombination system, which is called recombineering. this technology requires red function but not host reca protein function. here, we showed the recbd fusion polypeptides in crude extracts. genetic characterization tests were revealed that both fusion enzymes are recombination proficient and have wild-type phenotype. biochemical assays demonstrated that recbdc fusion heterotrimers have dsdna exonuclease, unwinding and chi cutting activities. they were also resistant to dna damaging agents. western blot analysis also detected a wild type length recd polypeptide together with recbd fusion polypeptides and a decreased heterotrimer compared to wild type. our findings suggest that recb-recd genetic fusions may affect recd assembling to the heterotrimer, but not affect it's native folding. sandwich immunoassay-a simple strategy for enhancement of the sensitivity and the specificity in prostate specific antigen detection based on surface plasmon resonance cuong cao, sang jun sim department of chemical engineering sungkyunkwan university, 300 chunchun-dong, jangan-gu suwon, prostate cancer is a deadly disease in men. prostate specific antigen (psa) has been proved to be the most reliable and specific biomarker in preoperative diagnosis, monitoring and followup of patients with prostate cancer. in this study, a biochip based on surface plasmon resonance was fabricated to detect psa at concentrations ranging from 1 to 1000 ng/ml. to reduce nonspecific binding, the chemical surface of sensor was constructed by using various ethyleneglycol mixtures of different molar ratios of hs(ch 2 ) 11 (och 2 ch 2 ) 6 cooh and hs(ch 2 ) 11 (och 2 ch 2 ) 3 oh. we also biotinylated the sams surface to enhance the orientation of protein immobilization. by using this surface, spr-based psa detection gave a positive ru value at the fist response in the whole range of psa concentrations. however, this ru value could get better and more reliable by simply applying a secondary interactant, the psa polyclonal antibody, in sandwich immunoassay. the results shown this approach could satisfy our purpose without modify the secondary interactant, which has usually been done by the other report. expression of epitopic domains of human coagulation factor viii in escherichia coli amir amiri yekta 1,2 , naser amirizadeh 3 , alireza zomorodipour 1* , fariba ataei 1 : 1 department of mol genet. national institute for genet eng & biotechnol tehran-iran p.o. box: 14155-634, tehran, iran; 2 islamic azad university of jahrom, jahrom, iran; 3 department of hematol, faculty of med, tarbiat modarres university, tehran, iran. e-mails: amir amiriyekta@yahoo.com (a.a. yekta), * zomorodi@nrcgeb.ac.ir (a. zomorodipour) human factor viii (hfviii) plays major role in the intrinsic pathway of blood coagulation and is used to treat individuals with hemophilia a for bleeding episodes. many researches have been focused on the molecular aspects of this protein. in this regard, epitopes of hfviii as well as their corresponding antibodies have many important applications. bacterially produced fviii-epitopes are capable to neutralize the alloantibodies that inhibit hfviii activity. the purpose of present study was to over-express two epitope-containing fragments of fviii in e. coli under t7 promoter (novagen). two dna fragments from light-and heavy-chains of hfviii (942bp-c1c2 and 1644bp-a1a2, respectively) were subcloned in the expression vector. the use of his 6 -tagged tail was also considered for detection and purification purposes. in each of the examined clones, a protein of expected size was detectable. in the c1c2-expressing clone the specificity of the over-expressed protein was confirmed by its reaction with the rabbit serum directed against native hfviii as well as anti-his-tag antibody. in the heavy chain-related-expressing clone, the expression level was low, but it was detectable by immunoblotting experiments. manipulations of the growth as well as induction may be required. the over-expression of the other epitopes reported in the heavy chain may be achievable by the expression of (a) sub-fragment(s) of this region. the over-expressed his-tagged c1c2-related protein was appeared to be trapped in the cell as non-soluble inclusion bodies. therefore, after homogenizing of the induced recombinant cells, the nonsoluble fraction was dissolved in a solution of denaturant (guanidine hydrochloride) and subjected for the purification, using a ni-nta resin (qiagen) followed by protein measurement. accordingly, an expression level of 5 mg/l (of culture) of the purified c1c2-related peptide was obtained. the recombinant hfviii c1c2-derived peptide has provided useful mean for further experimental and medical applications. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red 646 and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed 2d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with 0.6% w/v epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than 75 consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph 8.1) or chloride (ph 8.5) as leading ion and -amino-caproic acid (ph 8.9) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic 2danalysis. development of strategies for heterologous expression of glucose dehydrogenase from the halophilic archaeon halobacterium sp. nrc-1 juan carlos cruz-jiménez 1 , lorenzo saliceti-piazza 1 , rafael montalvo 2 : 1 chemical engineering, university of puerto rico-mayagüez campus, mayagüez 00680, puerto rico; 2 biology, university of puerto rico-mayagüez campus, mayagüez 00680, puerto rico. e-mail: juancruzj@hotmail.com (j.c. cruz-jiménez) halophilic archaea are excellent model organisms and valuable for biotechnology applications; they are easy to culture in the lab, genetically tractable, and exhibit a variety of interesting and useful characteristics. most halophilic archaea require 1.5 m nacl to sustain growth and structural integrity. among the 2.630 genes in halobacterium, we are studying the gene encoding a glucose dehydrogenase, gene id is 446, located between the 345205 and 346272 bases (halobacterium sp. nrc-1 genome project). this extremozyme is bioengineerable, and its use as a model for studying biocatalysis in aqueous/organic and nonaqueous media has not been explored to date. the utilization of enzymes in organic solvents has several potential advantages over aqueous systems. a major benefit is the increased solubility of many substrates, resulting in higher concentrations of reactant and products, hence reducing and purification costs and simplifying recovery protocols. cells were grown aerobically during seven days at 37 • c in a complex medium, harvested by centrifugation and their genomic dna extracted. for cloning of the gene, primers were designed based on the sequence recently published by the halobacterium sp. nrc-1 genome project. the forward (5 -ccgcatgcgcc cacagtccc-3 ) and reverse (5 -ccggcctctagaacggcctgg-3 ) primers were designed to incorporate restriction sites for sph i and xba i, respectively (in bold). we are pcr amplifying the genomic dna and developing methods for the heterologous expression using the mesophilic escherichia coli, as well as purifying the enzyme. the purification procedure will be carried out using high resolution methods based on the protein's halophilicity. bioinformatics methods will be used to facilitate conforming of protein function and for comparison with a native enzyme. quantitative measurements by mass spectrometry of hundreds of proteins simultaneously using the new proteinchip systemseries 4000 p. iversen, e. fernvik ciphergen biosystems inc., symbion research park, fruebjergvej 3, dk-2100 copenhagen, denmark. e-mail: piversen@ciphergen.com (p. iversen) most mass spectrometry methods used in proteomics allow for the identification of multiple proteins in a limited number of complex samples, but lack the ability to assess the quantity of the proteins and their modifications. however, mounting evidence shows specific cleavage of well-known proteins as being strong candidates for specific biomarkers, and in order to discover these biomarkers one has to be able to monitor the quantity and mass of hundreds of proteins from hundreds of complex samples reproducibly. the new series 4000 instrument in connection with proteinchip arrays ® from ciphergen biosystems enables this. the new 4000 series instrument is optimized for sensitivity, reproducibility and quantitation. new ion optics allows the use of higher acceleration voltages thus increasing the sensitivity, but without lowering the resolution. a new method of blanking the detector in connection with a non-linear gain of the detector also increases the sensitivity to the effect that igg can be detected down to 0.2 fmol. furthermore, the unique design of the instrument permits the detection of proteins with great variation in both mass and concentration and thus making it ideal for proteomics studies. a unique feature of the 4000 series instrument is the possibility to normalize the output by controlling the laser and detector so that results can be read with equal precision on different instruments, which is not often possible in mass spectrometry where individual instruments yield different results. this feature is vital in the validation of research results beyond individual laboratories. the coupling of liquid chromatography with mass spectrometry is now firmly established as a routine method for the identification of proteins that have been subjected to enzymatic digestion. in an on-line lc-ms experiment, the column eluent is coupled to the electrospray source via an emitter and any tryptic peptides present in the mixture are mass analyses as they elute from the hplc column. should there be any co-eluting species in the eluent, these will be separated in the mass analyser by their mass-to-charge ratio. it has become increasingly clear that relative quantification of protein expression changes is important in modern biology and medicine. several current approaches have been developed that utilise stable isotope labelling of samples in combination with separation and subsequent analysis by mass spectrometry. however, we have recently described an lc-ms strategy where quantification is achieved via normalisation of the ms datasets and comparison of the peptide intensities (of the observed tryptic peptides) across samples is performed. in this case, it is desirable to perform replicate injections and hence reduce statistical errors. this approach places a requirement upon good chromatography, especially in terms of retention time reproducibility. in addition exact mass measurement of the eluting ions is required as well as the ability to generate reproducible and reliable peak intensity, or area, calculations for the eluting tryptic peptides. the ability to measure the mass to charge ratios of ions accurately, across injections and across samples, increases confidence that the same ions have been matched from each sample injection. in this presentation our current strategy for the relative quantification of proteins will be discussed using, as examples, complex protein mixtures from salmonella enterica, eschericia coli and human serum. proteomic analysis for the production of rhctla4ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen 4-immunoglobulin (rhctla4ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using 2-d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy2, cy3 and cy5 dyes and run within a single dige gel. using decyder tm software, 2218 spots were detected with two-fold thresholds with 95% confidence and it was found that 60 proteins underwent significant change during the production of rhctla4ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla4ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. study of substrate specificity of rnr-exoribonucelases using hybrid proteins ana barbas, mónica amblar, cecília m. arraiano instituto de tecnologia química e biológica, ean, 2784-505 oeiras, portugal. e-mail: ab@itqb.unl.pt (a. barbas) the ribonucleases are essential enzymes responsible for the regulation of gene expression and have shown to be important for biotechnology purposes. for instance, commercial mutants deficient in ribonucleases have been quite relevant for the over-production of recombinant proteins. escherichia coli rnase ii is a processive 3 -5 exoribonuclease prototype of the rnr family that has homologues widespread in the majority of the sequenced genomes. by sequence alignment it has been proposed for the rnr type proteins the existence of three different domains: an n-terminal cold shock nucleotide binding domain (csd), a rnb catalytic domain, and a c-terminal s1 nucleotide binding domain. we have constructed several rnase ii deletion mutants to enable the characterization of each domain. these studies have allowed us to determine that both csd and s1 are involved in the binding of the enzyme to the rna substrate, being the s1 domain the most important. in rna-binding proteins it has been shown that the s1 domain's conformation is highly conserved. however, it is not known whether the substrate specificity is s1-dependent. in order to characterize the s1 domain and verify if it is directly related to substrate specificity, we have constructed rnase ii hybrid proteins in which the s1 domain was substituted by the s1 of two other exoribonucleases, rnase r (rnii-rnr) and pnpase (rnii-pnp). preliminary results have demonstrated that both quimeric proteins are capable of binding and degrading various rna substrates. in addition, studies are currently being carried out to verify the possibility that s1 domain of pnp in the hybrid protein might be involved in multimerization and/or interaction with other proteins. the murine monoclonal antibody igg1, anti-digoxin was produced in a rolling bottle fermentor. purification was performed on a protein g column. cd spectra were recorded on a jasco-810 spectropolarimeter. protein concentrations of 20-50 g/ml and path length of 1 cm were used for measurements in a far uv region. all measurements were performed in a cell holder thermostand with an accuracy of ±0.2 at 25 • c. at this temperature the predominance of ␤-strands is indicated. large conformational changes occur at 78 • c. at this temperature the spectra tense to irregular ␤-strands and unordered structures. these evidences confirming temperaturedependent conformational changes of protein and also high thermal stability of mentioned monoclonal antibody. generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling janni kristensen, kim kusk mortensen, hans peter sørensen 1 laboratory of biodesign, department of molecular biology, aarhus university, gustav wieds vej 10 c, dk-8000 aarhus c, denmark. e-mail: hans.peter.sorensen@teknologisk.dk (h.p. sørensen) we recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of escherichia coli. recombinant proteins were covalently coupled to the e. coli ribosome by fusing them to ribosomal protein 23 (rpl23) followed by expression in an rpl23 deficient strain of e. coli. this allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpl23 and the target protein. to assess the efficiency of separation of target protein from ribosomes, by site specific proteolysis, we required monoclonal antibodies directed against rpl23 and gfp. we therefore purified rpl23-gfp-his, rpl23-his and gfp from e. coli recombinants using affinity, ion-exchange and hydrophobic interaction chromatography. these proteins could be purified with yields of 150, 150 and 1500 g per gram cellular wet weight, respectively. however, rpl23-gfp-his could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. the purified rpl23-gfp-his fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by elisa using rpl23-his and gfp to select for monoclonal antibodies specific for each protein. this resulted in 20 antibodies directed against rpl23 and 3 antibodies directed against gfp. antibodies were screened for isotypes and their efficiency in western immunoblots. the most efficient antibody against rpl23 and gfp were purified by protein g sepharose affinity chromatography. the purified antibodies were used to evaluate the separation of ribosomes from gfp, streptavidin, murine interleukin-6, a phagedisplay antibody and yeast elongation factor 1a by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. proteomic analysis for the production of rhctla4ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen 4-immunoglobulin (rhctla4ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using 2-d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy2, cy3 and cy5 dyes and run within a single dige gel. using decydertm software, 2218 spots were detected with two-fold thresholds with 95% confidence and it was found that 60 proteins underwent significant change during the production of rhctla4ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla4ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. similarity searches and multiple alignment of s 1 and s 2 protein of sars-cov for modeling 3d structure and its evolution (origin) mohammad soltany rezaee rad, iman tavassoly, negar mottaghi, banafsheh rezaee. e-mail: mohammad.soltany@gmail.com (m.s.r. rad) aims: the exact origin of the cause of severe acute syndrome (sars) is still an open question. nowadays 8 recombinant origins for this virus have been found. s 1 and s 2 subunit of spike protein of this virus are the most important proteins responsible for severe acute respiratory syndrome. in fact they are glycoproteins of this virus exist on its surface. they are responsible for mediating fusion of viral and cellular membrane. the classification and modeling 3d structure of this virus can help us to suggest new ideas about its charististics and function, which may lead to new therapeutic and preventing modalities. methods: we used nucleotide sequence of s 1 and s 2 subunit of s (spike) protein for multiple alignments. we have done multiple alignments with different bioinformatics software (clusterx, entrez) for comparing the sequence with the other viruses and, we used weblab view software for modeling and identifying 3d structure of these proteins. findings: the similarity searches on nucleotide sequence of this protein with the 30 single strands rna (ssrna) shows the virus belong to a known classification named coronaviridae. these 3d structures show the responsibility of s protein in this syndrome. another findings based on these alignments is an important similarity between these subunits and genome of hiv-1 showing they have familiar mechanism in pathogenesis. discussion: multiple alignments are powerful tool in classification of new recombinational virus and emerging infection. 3d structure model of this virus is an important guide to understand the mechanism of this virus. the shape of glycoprotein that modeled with bioinformatics software can help us in understanding mechanism of binding this virus to human cells. this fact can be used in designing drug and vaccine to cure and prevent the sars. blocking these origins and sites leads to inhibiting the virus attachment. also the similarity between this virus and hiv-1 shows us that both of them have similar proteins that cause pathogenesis of these viruses. the simulated moving bed (smb) technology is a continuous countercurrent chromatographic separation technique that has been applied successfully in the last years to a number of significant problems. an smb consists of a series of fixed bed chromatographic columns connected in a loop, and outperforms column chromatography in terms of productivity and solvent consumption. the use of smb instead of batch processes for bioseparations, i.e. separations involving large and rather complex molecules with multiple 3d configurations depending on parameters such as ph, temperature, etc., is becoming of greater and greater interest. examples of these are therapeutic proteins, antibodies and plasmid dna among others. for all chromatographic processes in this field, one of the most crucial issues is the cleaning of the chromatographic media with a special solvent system, an operation usually referred to as cleaning in place (cip). in single column chromatography this is easily done off-line, but this is not compatible with standard smb operation. in order to overcome this limitation, the standard smb configuration has been modified by adding a dedicated section plus an additional section for the re-equilibration of the freshly cleaned column with the working solvent before it is re-inserted into the smb loop. in such a way, cip according to gmp criteria can be incorporated into the smb unit and operation, which is then called cip-smb. this new smb configuration is also related to the three fraction separation unit called 3f-smb that has been recently introduced and applied to the separation of nucleosides. in this work we apply cip-smb using a size exclusion stationary phase to the separation of plasmid dna from the filtered cell lysate solution. plasmid purification has become a key issue in the last years as a result of the advances in gene therapy, whereas traditional laboratory methods are not always suitable for therapeutic purposes. we report about separation performances, which are then discussed in the light of smb design criteria and compared to column chromatography performance. computer guided optimization of adsorptive bioseparation processes bernt nilsson department of chemical engineering, lund university, 221 00 lund, sweden. e-mail: bernt.nilsson@chemeng.lth.se separation processes like chromatography can be highly nonlinear and the behavior can sometimes be hard to predict. optimization of preparative chromatography is often done experimentally, which is both time consuming and expensive. a model-based approach to optimization is therefore an attractive and challenging way to overcome some drawbacks in the traditional working procedure in biotechnical industry. efficient model-based optimization for industrial needs requires three parts; models, methods and tools. model-based methodology requires a set of chromatography column model structures, which can capture the phenomenon of interest. for instance they have to capture column load variations, elution profile changes, operation condition disturbances, column configurations and stationary phase properties. to derive a reliable model for optimization it has to be calibrated and validated to experimental data, which requires an efficient calibration procedure. different calibration procedures are discussed and compared. after validation the model can be used in the design of a separation step. to do a robust design a set of requirements have to be fulfilled. the column size and operation conditions are used to optimize the performance of the step, which requires a constraint nonlinear optimization method. the choice of objective function for optimization and corresponding constraints are not obvious and the resulting operation conditions are often not robust. therefore there have to be additional methods available for analysis of the performance, like sensitivity and robustness analysis. optimization of the purification of antibodies is discussed and exemplified. the work with mathematical models and numerical methods has to be supported by a set of computer tools of different kinds in order to solve industrial problems effective. there is a need for different kinds of tools; customized tool to solve specific problems by a non skilled user and general toolbox for the expert. an example of a toolbox is presented. tina tarmann, alois jungbauer department of biotechnology, university of natural resources and applied life sciences, vienna, austria plasmids and viruses are the contemporary vehicles for genetherapy and genetic vaccination. extremely promising results have been reported from in-vitro, in-vivo and clinical studies. currently a lot of these compounds are manufactured with a technology which has been directly transferred from laboratory to pilot scale without further engineering. membrane based separations, chromatographic separations and precipitation have been employed for separation of plasmids and viruses. chromatographic separation have been designed with aim of protein separation. thus such processes suffer from either mass transfer limitations or low capacity. monoliths without intraskeleton mesopores and chromatography particles with giga pores are excellently suited for adsorption and separation of plasmids and viruses. low mass transfer resistance and high capacity compared to conventional beaded materials can be observed. adsorption kinetics were derived from infinite and finite bath methods and isotherms were constructed. these data also suggest that a conformational change of the plasmids takes place upon adsorption. discussion of the mass transfer properties and an example of scale up of a chromatographic separation process using these novel materials will be shown and discussed in respect to already existing processes. the recent developments in molecular therapies such as non-viral gene therapy and dna vaccination have fostered the development of efficient plasmid dna (pdna) purification processes. the separation of supercoiled (sc) and open circular (oc) isoforms is one of the key steps in the large scale purification of pdna vectors intended for a therapeutic use. although escherichia coli produces mainly the more compact sc pdna isoform, oc, linear and denatured pdna isoforms are usually present and are likely to be less efficient in transferring gene expression. for this reason, regulatory agencies specify that more than 90% of pdna in a therapeutic product is in the sc isoform. in this work histidine-base recognition is explored as a mean to separate pdna isoforms. the agarose gel used here combines the mild hydrophobic characteristics of an epoxy spacer arm with a pseudo-affinity histidine ligand. chromatographic profiles were obtained by injection of native plasmid (sc + oc) samples in the histidine-agarose support showing an efficient and baseline separation of both isoforms. the high resolution obtained with this support indicates that the method is potentially applicable to the separation of pdna at preparative and analytical scale. affinity ligand development with a novel encoded bead screening technology ib johannsen, versamatrix a/s, gamle carlsberg vej 10, dk-2500 valby, denmark. e-mail: www.versamatrix.com the presentation describes a new invention for fast development of affinity ligands, where up to 20,000 ligands can be screened onbead and identified in a few hours. combinatorial synthesis by the split and mix procedure is a powerful technique for generating vast numbers of diverse chemical compounds on polymer beads with relatively little effort. traditionally, the technique is hampered by the laborious spectroscopic and chemical analysis, needed to determine the exact structures of the ligand on selected beads. in this way, 6-12 month analysis time could easily be spent just to analyze a tiny fraction of the library. in the versaffin tm technology each bead is encoded, individually tracked, and identified during the synthesis and screening. this decreases the whole ligand development time from months to weeks and increases the amount of information significantly. the bead code further enables evaluation of the ligandprotein binding under varying binding and elution conditions. the instrument for reading the encoded beads and for quantifying the amount of bound protein is presented. the encoded beads we use are based on functional cross-linked polyethylenglycol (peg), which is compatible with water as well as most organic solvents. thus, the combinatorial synthesis can be carried out in organic solvents and the resulting compounds can be evaluated, still bound to the parent beads, under aqueous conditions. a further advantage of using peg based beads for on-bead screening is the fact that peg is biologically inert and therefore does not interfere in a bioassay. in the biopharmaceutical industry, pressure is mounting to shorten development times and thereby time to market, e.g. in the field of monoclonal antibodies, generic processes have been established which allow for more rapid development from gene to production of pre-clinical and proof of concept/phase i material. for non-mab products from various expression systems, productspecific approaches still prevail. however, for most product types similar issues like clearance of process-and product-related impurities, overall purity and yield, or manufacturing issues have to be dealt with in downstream process development. integrated and timely approaches based on process science and developed orthogonal analytical tools are often hampered by tight time frames and limited resources available. on the other hand, thorough understanding and analytical characterization of product characteristics but also of (process-related) impurities are pre-requisites for fully exploiting separation power and for achieving final purities way above 95% in a robust and cost-effective manner. from primary separation to polishing steps, we have made several attempts to improve the efficiencies of process steps themselves but also of ways to develop them. the strategies applied comprise implementation of innovative processing tools, rational streamlining and optimization of a sequence of unit operations, tech transfer and scale up considerations. also in this context, the applicability of scale down and ultra scale down models for process development and optimization purposes, their potential for speeding up and their limitations will be discussed. chromatographic monoliths are rather new chromatographic stationary phases. they consist of a single piece of a highly porous material. the pores are interconnected forming a network of channels. since the transport mechanism is predominantly based on convection, mass transfer between mobile and stationary phase is significantly enhanced resulting in short separation times. because of that they seem to be an ideal support for separation and purification of extremely large molecules like proteins, dna or even viruses. in this talk, various features of the monoliths like high porosity, fast mass transfer, surface accessibility and dynamic binding capacity will be described. effect of each feature on the separation and purification efficiency will be discussed in terms of molecular size and properties. while chromatographic monoliths are already widely accepted in microchip fluid devices, capillary columns as well as analytical columns, very few reports about preparative monolithic columns can be found. reasons for lack of preparative chromatographic columns will be elucidated and preparation strategy for construction of several liter volume methacrylate based monoliths will be presented. finally, several examples of biomolecule purification like large proteins, plasmid and genomic dna and viruses on cim convective interaction media ® monolithic columns will be given. further, their application as bioreactors and supports for solid state synthesis will be demonstrated. hubbuch institute of biotechnology, forschungszentrum juelich, 52425 juelich, germany. e-mail: m.schroeder@fz-juelich. de (m. schroeder) the intraparticle diffusion coefficient is an important parameter for modeling of chromatographic separation processes. a new method based on dynamic measurements of intraparticle concentration profiles of proteins in process chromatographic media with a confocal laser scanning microscope is presented. the diffusion coefficient is determined by fitting experimental data to a spherical diffusion model. excellent agreement of experimental data with simulation results is obtained. the diffusion coefficient is measured for seven proteins in sepharose 6 ff, spanning molecular weights from 14.3 to 160 kda. in addition, multicomponent diffusion processes for combination of differently sized proteins are analyzed and the influence of adsorbed proteins on the diffusion coefficient is measured in sp or q sepharose ff. taken together the presented method allows measuring the diffusion coefficient of proteins in process chromatographic media in a packed column. use of automated docking for predicting chromatographic behavior of proteins in hydrophobic interaction chromatography andrea mahn, m. elena. lienqueo contreras university of chile, santiago, chile in the present work, we have extended and automated the methodology proposed by mahn et al., 2005 for predicting protein behavior in hydrophobic interaction chromatography (hic). this methodology is based on the good correlation level between the average surface hydrophobicity of the interfacial zone (local hydrophobicity lh) and protein retention time in hic, for only three different ribonucleases. for determining the lh it is necessary to select the most probable protein-ligand conformation. in this work, we have determined the most probable conformation, of more than 12 proteins, using (i) first, the module insight ii affinity by accelrys for providing automated docking (grid method) of ligands (phenyl) to the proteins (100 conformations); (ii) then, the different probable docked protein-ligand conformations were automatically scored using the module insight ii ludi by accelrys; (iii) after that, each conformation was clustered and each cluster was scored by using the average score of each cluster (iv) finally, the most probable conformation was selected using a function based on the number of cluster components, and the average score value. then, when the most probable conformation was selected, the local hydrophobicity (lh) was calculated using the graphical representation and analysis of structural properties (grasp) program. the results have shown an acceptable correlation level (r > 0.90) between lh and the experimental dimensionless retention time (drt). in view of these results, we consider that this methodology could be used to adequately represent the chromatographic behavior in hic for all kinds of proteins (with a heterogeneous and homogeneous surface hydrophobicity distribution) and without a large number of tedious experiments, but only using computational simulation and adequate score criterion. potato tuber proteins are nutritious and show potential as functional ingredient in food systems. however, the present bulk processing technology can only recover byproduct protein for animal feed use. an expanded bed adsorption (eba) process for isolating functional food-grade protein from crude potato starch effluent was previously developed. moderate capture efficiency (20-25%) of the total crude protein was most likely caused by diffusion limitations and aggregated protein, inaccessible for adsorption. we employed the same adsorption ligand attached to agarose-tungsten carbide beads to create stable beds of 2.0-2.7× expansion using flow rates at 400-750 cm h −1 . a pilot scale eba process was run in a commercial processing plant over a three month campaign of starch production from potatoes of mixed variety. fresh crude effluent (150-300 l/cycle) was applied to a column (20 cm × 2 m) containing 20 l of resin. protein capture by eba was reliable in operation, producing a refined protein material, which after dewatering and gentle drying, showed improved functionality over heat-coagulated protein produced at the same plant. overall productivity increased. however, finding a robust operating window of predictable productivity is challenging since the potato fruit water is complex and deteriorates easily. from breakthrough curves, it is observed that the major bulk protein, patatin, displays non-langmuir adsorption behavior. this may indicate a range of interactions for different species of the same protein. chlorogenic acid (ca), the main polyphenolic substance in potato tuber, causes enzymatic browning and undesirable flavor changes, but polyphenols can also react with protein. assessing the effects of interacting cell components therefore applies to the bioprocessing of plant material. at present the acceptance of biochip technology for on site use, e.g. diagnosis or environmental control is hindered by rather expensive and complex instrumental systems. there is a need to provide reliable and cost-effective systems that can be operated with minimal training. the construction of electronic biochip microarrays using semiconductor technology enables the construction of compact systems with high integration at acceptable production costs. the key feature of the fully electrical biochip technology are micro arrays made in advanced si-technology and carrying several array positions with interdigitated nanometer gold electrodes on its surface. the chips are fabricated by standard silicon fabrication methods allow-ing high volume production and to minimise the cost per chip. the advantage of fully electronic microarrays is the intrinsic high spatial resolution and direct signal coupling of the biosensing element and the transducer. the function of fully electronic biochips is also based on the electrochemical transduction and quantification of the formation of affinity complexes on the chip surface. a portable device for field applications and point of care diagnosis have been designed and manufactured. the amperometric device enables the recognition of biomolecular interactions by measuring the redox recycling products of elisa enzyme labels. the highly sensitive signal transduction is achieved with a 16-channel interdigitated ultramicroelectrode array. one major advantage of fully electronic microarrays is the direct signal coupling of the biosensing element and the resulting robustness and opportunity for miniaturisation. those electrical biochip arrays have been adapted for the detection of all types of affinity complexes, such as for dna, rna, proteins and haptens. self assembling of capture oligonucleotides via thiol-gold coupling have been used to construct the array chip nucleic acid interface. thus e.g. pathogenic micro organisms have been identified and quantified via their genomic dna or ribosomal rna respectively. another application based on immobilized antibodies is shown to sense extreme low concentration of bioagent toxins. for processing the assay formats and the electrical read out of the detection of affinity complexes a modular fully automated measurement system has been developed. it is manufactured in industrial lines and available at market. dynamics and self-assembly of organic molecules on surfaces revealed by high-resolution, fast-scanning stm flemming besenbacher interdisciplinary nanoscience center (inano), university of aarhus, dk-8000 aarhus c, denmark. e-mail: fbe@inano.dk in the interdisciplinary area of nanoscience and nanotechnology, the adsorption and self-assembly of organic molecules on singlecrystal surfaces have attracted much attention lately due to the potential applications in fields ranging from molecular electronics to biocompatible interfaces. the supramolecular structures formed upon deposition of molecular species on solid surfaces depend on the molecular architecture and the distribution of functional groups on one hand, which determines the thermodynamically stable molecular arrangement, and on the other hand, on kinetic factors like thermal diffusion, spontaneous rotations and conformational dynamics. i will show how the unique aspect of our aarhus stm and the time-resolved, high-resolution stm imaging can be used to obtain important new insight into the dynamics, and can provide very important new information on the atomic-scale realm and on the dynamics of molecular nanostructures. the time-resolved stm data are visualized in the form of stm movies (see www.inano.dk/spm) which can subsequently be analyzed in order to extract quantitative information on the activation energy, the prefactors and the adsorbate-promoted diffusion. i will specifically discuss: (i) the self-assembly of guanine quartets on au(1 1 1) and the influence of cooperative hydrogen bonds, and (ii) the molecular recognition in binary mixtures of dna bases. g molecules are found to self-assemble into a hydrogen-bonded network of g-quartets, whose structure corresponds perfectly with the quartet structure of telomeric dna determined by x-ray crystallography. the strong preference of g molecules to form quartets can be explained by a cooperative effect that strengthens the hydrogen bonds within the g-quartet network over the hydrogen bonds in isolated dimers. by means of a combination of stm experiments and dft calculations we compare the 2d molecular networks formed on deposition of the binary mixtures g-c (purine-pyrimidine pair of complementary bases) and a-c (purine-pyrimidine pair of non-complementary bases). we find that the non-complementary bases segregate into islands of pure a and a network of pure c, whereas the complementary bases g and c form a network that cannot be separated by annealing up to the desorption temperature for c. high-resolution stm images allow us to identify the structures for the enhanced thermal stability as structures that contain g-c bonds, possibly with the same structure as the watson-crick pairs in dna molecules. kühnle, r., et al., 2002 . nature 415, 891. otero, r., et al., 2004 . angewandte chemie int. ed. 43, 2092 . otero, r., et al., 2004 . nat. mater. 3, 779. otero, r., et al., 2005 . angewandte chemie int. ed. 44, 2. rosei, f., et al., 2002 . science 296, 328. schunack, m., et al., 2002 . biomedical and pharmaceutical companies are using an increasing number of carbohydrate polymers in the formulation of drugs. one such polymer with highly attractive features is chitosan that can be produced from crustacean shells. chitosan is non-toxic, biocompatible and biodegradable. chitosan can be formulated as nanoparticles or membranes and have enhanced several bioprocesses. among the well-documented features are enhanced drug uptake by tight junction relaxation and enhanced in vivo uptake and protection of nucleic acid formulations. chitosan research has increased throughout this decade. research programs are addressing the potential of chitosan applications but preparations with variable molecular size and charge are not easily available. specifically companies working with the development of new drugs and enhancement of drug functionality are in need of formulation technology that provides well characterized biocompatible material. in the chitosan innovation consortium the danish companies, coloplast, novozymes biopolymers, pipeline biotech, and zgene, together with the research center inano (aarhus university) and bioneer a/s have developed a series of chitosan preparations suitable for research of biopharmaceutical applications (www.chitosan.dk). the ability to obtain functional formulations is currently being tested in both in vitro and in vivo experiments. the consortium participants have established a platform from which chitosan processing, characterization and formulation technology can be extracted. by providing specified chitosan preparations the polymer feature can be adjusted to fit specialized biopharmaceutical applications. high throughput bioprocessing govind rao center for advanced sensor technology, umbc, baltimore, md 21250, usa the post genome era holds a great deal of promise. an enormous number of new proteins await study. these will require sophisticated culture techniques, as cells will have to be grown under large numbers of environmental conditions to elucidate expression triggers. unfortunately, unlike molecular biology, bioreactor technology is little changed since its inception. the primary reason has been a lack of sensor technology that can be readily employed to monitor the cellular environment. we will take a look at the current status of the technology and report on promising optical sensor technology that permits low-cost high throughput cell culture and fermentation. noninvasive sensors that monitor ph, po 2 and pco 2 and high sensitivity solutions for glucose and glutamine measurements will be presented. in addition, the mixing characteristics that determine bioreactor performance will be examined at the small scale and their relevance to the large scale will be demonstrated. on-line monitoring and fed-batch operation in shake flask and micro titre plate cultures jochen büchs 1 , frank kensy 1 , markus jeude 1 , tibor anderlei 2 , barbara dittrich 3 , doris klee 3 : 1 biochemical engineering, rwth aachen university, aachen, germany; 2 ac biotec, jülich, germany; 3 textile chemistry and macromolecular chemistry, rwth aachen university, aachen, germany although methods of molecular biology has led to rational design of micro-organisms to suit our requirements, screening of large numbers of strains and media is still one of the most important tasks in biotechnology. batch operation of shaken bioreactors and absence of on-line monitoring is still the general state of the art for that purpose. it is also a very common practice in screening projects that only the final product titre is measured at the end of the culture for the evaluation of the "best performers". in the recent years several approaches were introduced to follow microbial cultures also in shaken bioreactors like shake flasks or micro titre plates (mtp's). it became obvious that the cultures can behave quite unexpected and most relevant and essential information is lost, if only the final product titre at the end of the cultures is utilised for evaluation. as a result, the screening may be directed to an unknown and non-intended direction or may even fail. this is demonstrated with some examples in this contribution. new methods and techniques are introduced to measure the oxygen and carbon dioxide transfer rate and the respiratory quotient in shake flasks and the optical density, nadh fluorescence, ph and do 2 in mtp's. if the desired product can be fused to a fluorescence protein, like gfp or yfp, also the product formation can be monitored on-line in mtp's. it is of utmost importance that the operating conditions of the applied shaking bioreactors are suitable and shaking motion is not stopped during the measurement. otherwise, e.g. power input, mixing and oxygen supply is interrupted and the micro-organisms will ongoingly rearrange their metabolism to cope with these disturbances of their environmental conditions. another problem of screening is the commonly applied batch operation mode. a lot of microbial systems display an overflow metabolism, substrate or osmotic inhibition or are characterised by a catabolite repressed product formation. in all these cases, batch operation is not the preferred operation mode and, therefore, these cultures are run in fed-batch in larger scales. in particular, it is nearly impossible to screen systems, which are catabolite repressed by the carbon source, in batch mode in defined mineral media. after initial growth has led to a nearly complete consumption of the carbon source, the product formation is derepressed. but then no more carbon source is available to continue with production. it is quite questionable whether in batch operation mode suitable strains can be selected for later fed-batch operation or not. this consideration has resulted in the development of a new technique which allows to run the screening in fed-batch operation mode. this technique is applicable in shake flasks as well as in mtp's. dramatic increases in product titre between 4-and 400-folds were observed under these conditions compared to conventional batch screenings. mikkel nordkvist, john villadsen center for microbial biotechnology, technical university of denmark, dk-2800 lyngby. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) efficient mixing and mass transfer are highly important in the chemical industry and in the fermentation industry. poor mixing can result in low yield and variable product quality in a number of cultivation processes, and mass transfer can easily become the limiting step in aerobic cultivations, especially at high cell density. we have tested a new tank reactor system, where liquid is withdrawn from the bottom of a tank, rapidly circulated, and injected back into the bulk liquid through the nozzles of rotary jet heads. liquid feed as well as gas is added in the recirculation loop and thereby distributed via the rotary jet heads. solid feed in powder form can also be added in the loop with advantage, and heat is efficiently removed in a plate-type heat exchanger, which is part of the loop. the system has a very simple design with no internal baffles or heat exchange area, and between batches the rotary jet heads are used for cleaning in place. a number of applications ranging from dispersion of liquid and powder to mass transfer will be presented. mass transfer applications include baker's yeast cultivation and oxidation of lactose to lactobionic acid by a carbohydrate oxidase. fast and accurate analytical information that can be used to rapidly evaluate the interactions between biological systems and bioprocess operations is essential for optimization of biological production processes. we have researched and developed a multiplexed microbioreactor system for the parallel operation of multiple microbial fermentations. the microbioreactors have working volumes from 5 to 150 l, and are instrumented for real-time monitoring of dissolved oxygen, ph and optical density. the growth profiles obtained with escherichia coli compare favorably to results obtained from conventional 500 ml batch bioreactors. we also demonstrate the use of our microbioreactors coupled to dna microarray analy-sis, as a tool for accelerated discovery and elucidation of metabolic pathways and gene expression profiles. the multiplexed system represents a significant step towards high-throughput data acquisition and has the potential to replace current instrumented bioreactors, which are bulky, expensive to run, and require many mechanical manipulations. design of a laboratory scale bioreactor to study solid-state tobacco fermentation m. di giacomo, l. nappi, d. silvestro, m. paolino, d. parente r&d biology department, british american tobacco italia, naples 80126, italy italian toscano cigar production is based on the fermentation of dark fire-cured tobacco. the process starts with the rise of leaf moisture to levels of water activities assuring development of the wild phylloplane microflora in the absence of free water. the intense growth of microorganisms modifies leaf characteristics (ph rise from acidic to alkaline condition) contributing to define the toscano typical smoke profile. tobacco fermentation takes place in great bulks of 500 kg which cause considerable amount of heat evolution as a function of the metabolic activities of the microorganisms. this heat accumulates and temperature can rise to as high as 70 • c. a laboratory cylindrical packed-bed bioreactor was designed to work under isothermal conditions. the reactor was ideal to ferment small quantities of tobacco (200 g) and was made up of a column aerated from the bottom with humidified air and placed in a thermoregulated room. experiments were conducted with constant temperature and air flow. moreover, bioenrichment experiments were conducted in the presence of different microbial starter cultures. fermentation courses were monitored by measuring microbial counts and chemical/physical modification of the substrate. with this laboratory scale system we obtained different kinds of information on the role and dynamics of the microorganisms involved in the fermentation process and on the influence of different environmental conditions. for the future, the design of an adiabatic device for tobacco fermentation is planned. on-line liquid chromatography as a process analytical technology for monitoring and control of biotech processes rick e. cooley 1,2 : 1 process analytics center of excellence, dionex corporation, sunnyvale, ca, usa; 2 eli lilly and company, indianapolis, in, usa (retired) biotech processes, used to produce an active pharmaceutical ingredient (api), generally differ from small molecule api manufacturing processes in that the starting materials tend to be more variable, more complex, and the product of interest in lower concentration due to the fact that they originate from a biological rather than a chemical process. this complexity and low starting concentration has generated a high level of interest in developing technologies that can be utilized to increase yield and reduce variability in the initial bioreactor phase, as well as, downstream isolation and purification operations. the use of process analytics, or on-line analytical measurements, is a technology approach that can contribute to increased process understanding and control. this presentation will provide examples of how various analytical measurement technologies have been utilized to monitor typical bioprocess unit operations leading to increased automation and control. examples of the use on-line liquid chromatography to monitor bioreactors, process scale chromatography columns, and enzymatic reactions will be presented in more detail. application of advanced monitoring strategies for recombinant protein production karl bayer department of biotechnology, university of natural resources and applied life sciences, vienna a-1190, austria high yield in combination with the required quality are the key objectives of large-scale production of recombinant proteins using different host/vector systems. however, in the past the efficient production of recombinant proteins was frequently limited due to inadequate exploitation of the cell factory and deficiencies in process design. to achieve optimal exploitation of microbial cell factories the key requirement is to enhance the monitoring capabilities to improve the insight into the host metabolism dynamics and to cope with limited understanding of the interaction of recombinant protein synthesis with host cell metabolism. since each protein exerts an individual influence on the host/vector system, the selection of appropriate analytical methods is even more important. in order to overcome these problems high throughput technology platforms, such as dna microarrays for transcriptome and differential 2-d electrophoresis (dige) for proteome analysis provide extensive data to screen for significant analytes and provide an appropriate basis for in silico modelling and reverse engineering of regulatory networks. taking advantage from such an iterative process experimental design will be improved and further aid to increase the performance of modelling. in addition, transcriptome data are used to screen fast stress responsive promoters to set up gfp based reporter gene fusions for in-situ monitoring of the metabolic load due to recombinant gene expression. moreover chemometric methods are frequently applied to model complex data from easily obtainable on-line data sets to overcome the limited monitoring capabilities due to the high complexity and nonlinearity of biological reactions and reaction networks. this strategy has been successfully applied to model bdm (bacterial dry matter), pcn (plasmid copy number) and the amount of recombinant protein using data sets acquired from off-gas analysis (o 2 consumption, co 2 evolution), alkaline consumption rate, in-situ capacitance and multi-wavelength fluorescence measurements. at-line monitoring of bioprocess-relevant marker genes using an electric dna-chip britta jürgen 1 , daniel pioch 1 , le thi hoi 1 , jörg albers 2 , rainer hintsche 2 , stefan evers 3 , karl-heinz-maurer 2 , michael hecker 4 , thomas schweder 1 : 1 institut für pharmazie, ernst-moritz-arndt-universität, 17487 greifswald, germany; 2 ebiochipsystems, 25524 itzehoe, germany; 3 vtb-enzymtechnologie, henkel kgaa, 40191 düsseldorf, germany; 4 institut für mikrobiologie, ernst-moritz-arndt-universität, 17487 greifswald, germany. e-mail: britta.juergen@uni-greifswald.de (b. jürgen) the gram-positive bacteria bacillus licheniformis and bacillus subtilis represent important industrial hosts for the production of enzymes (e.g., proteases and amylases) or antibiotics (e.g., bacitracin). both organisms are attractive for this purpose because of their apathogenity and their classification as gras organisms (generally regarded as save). moreover, their easy cultivation and their high natural capacity to secrete proteins into the growth medium qualify them for the industrial overproduction of homologous or heterologous proteins (simonen and palva, 1993) . for the control of the physiological state and the productivity of the production cells efficient analysis tools are of great interest. a prerequisite for the evaluation of the physiological state of cells during industrial fermentation processes is the analysis of so-called process-relevant marker genes, the expression of which indicates unfavorable growth and production conditions (schweder and hecker, 2004) . by means of proteome and transcriptome analyses we have identified critical process-relevant genes of b. licheniformis and b. subtilis cells under different nutrient limitation conditions and during industrialclose bioprocesses. dna-chips with probes for such process-relevant marker genes could be valuable diagnostic tools for the monitoring of the cellular physiology during microbial bioprocesses. in order to provide reliable tools for the monitoring of the cell physiology during microbial bioprocesses, we have developed a fast mrna analytical approach, which allows an at-line monitoring of the transcriptional activity of selected marker genes during bioprocesses. this approach is based on an easy, fast and reliable rna isolation procedure and the measurement of specific mrnas by means of an electric dna-chip barken et al., 2004 a robust bioprocess is crucial to ensure the consistent process performance and provide the high quality of product for drug manufacturing in biopharmaceutical industries. existing methodologies for bioprocess design do not involve establishing mechanisms to achieve the desirable robust bioprocesses and have low capacity in handling uncertainty in the product manufacturing. also, the solutions are often obtained step wise and do not account for interactions between the steps. despite its importance, the robustness of a bioprocess has not been properly defined and studies carried out in statistic sense are often retrospective. in addition, the computational cost is expensive due to using a line search algorithm for finding an optimal operating solution. finally, the existing methodologies are difficult to apply to the whole bioprocess in biopharmaceutical industries. this paper attempts to define rigorously a measure for process robustness and presents a new methodology for evaluating the robustness of bioprocess operations and their performance. the methodology is based on the concept of 'windows of operation' which shows the whole range of possible operating regions. the methodology also establishes a lower bound for the largest variation of a design variable to ensure the performance. these bounds are achieved by min-max optimization techniques. a direct search algorithm has been developed and its computational cost is much lower than the line search algorithm. results include visualization of robust operating regions and a set of indices which compare the performance of different operating strategies. the capabilities and efficiency of this methodology are illustrated by applying it to the centrifuge selection for the clarification of high solids density cell broths. the research work will impact considerably upon robust bioprocess operation. when grown on methanol, pichia pastoris is able to synthesize proteins to high titres as well as secreting and glycosylation, thereby making this organism a very interesting host for the production of recombinant drugs at large scale. the methanol residual concentration has been reported to strongly influence the specific productivity, the optimum concentration being around 3 g/l. a suitable monitoring and control technique is therefore necessary to study and improve the productivity of p. pastoris fermentations. the current research aims at showing how a mid-infrared spectrometer (atr-ftir) can be calibrated in-situ in order to monitor and control p. pastoris fermentations. this method is simple and fast, and eliminates the need of both standards preparation and off-line calibration. it is based on the observation that during fed-batch processes, only substrate and biomass concentrations vary significantly. the method therefore consists in adding a known amount of methanol at the beginning of the process, just after inoculation, and subsequently calibrating the instrument. financial support for the swiss national science foundation is gratefully acknowledged. implantable biomaterials are subjected to inflammatory responses mediated by adherent phagocytes such as monocytes and macrophages. these cellular responses and behavior have been shown to be dependent on the type of protein that adsorbs to the surface. surface modification is necessary to control and prevent protein adsorption, and thus modulate the inflammatory responses. hydrophilic surfaces that adsorb minimal amounts of protein are considered useful for minimizing the inflammatory reactions to biomaterials. in this study we have used two routes to modify polyethylene terephthalate (pet) films: (1) a wet-chemical method for attachment of linear polyethylene glycol chains (mpeg); and (2) gas-phase plasma polymerisation of diethylene glycol vinyl ether (degve) to generate peg-like surfaces. the surface chemistry was assessed by x-ray photoelctron spectroscopy (xps), fourier transform infrared spectroscopy (ftir) and time-flight secondary ion mass spectrometry (tof-sims). the two pegylated surfaces were compared for their ability to minimise both fibrinogen adsorption and the adhesion and activation of macrophage-like human leukocytes. adsorbed fibrinogen has been shown to be one of the key proteins in stimulating inflammatory responses to biomaterials. adsorption was investigated quantitatively using 125 i-radiolabeled human fibrinogen. in addition, the conformation of the adsorbed protein was tested using an antifibrinogen monoclonal antibody in an enzyme-linked immunosorbent assay. the results showed that pegylated surfaces adsorbed up to 90% less fibrinogen, and that unfolding of adsorbed fibrinogen was more pronounced on the linear mpeg layers than on the peg-like plasma polymer surfaces. adhesion of in-vitro differentiated macrophage-like u937 cells was reduced on both the peg-like plasma polymer surfaces and the linear mpeg layers compared to the unmodified pet surface, but cells adhering to the peg-like plasma polymer surfaces secreted less tumor necrosis factor-␣ (tnf-␣) than cells adhering to the linear mpeg layers. thus, the linear mpeg surface is relatively efficient at reducing adhesion of macrophage-like cells, but those cells that do attach are in a more activated and proinflammatory state. analysis of ceramides from biological samples m. budvytiene 1 , j. liesiene 1 , b. niemeyer 2 , n. ceramides in human skin play an important role in the regulation of cell growth, differentiation and apoptosis. moreover, they are involved in the numerous signaling pathways. the growing interest in the investigations of ceramides physiological functions requires efficient separation methods of ceramides from biological resources and sensitive analytical methods. in this work some sensitive and selective methods, involving thin layer chromatography (tlc), high performance liquid chromatography (hplc), mass spectrometry (ms) and nuclear magnetic resonance spectroscopy (nmr) were employed for the separation and characterization of ceramides from human foreskin. epidermal lipids were extracted from human foreskin for 24 h at room temperature using three solvent mixtures (chloroform/ethanol/water 1:2:0.5, v/v/v); (chloroform/ethanol 1:1, v/v), and (chloroform/ethanol 2:1, v/v). ceramides retention char-acteristics in tlc and hplc were compared with the retention of commercial standards. the best separation was obtained using normal phase column packed with hilic silica 3 m. the elution was performed using mixture of chloroform and ethanol 50/50 (v/v) as an eluent. two commercial standards n-stearoyl-sphingosine cer(ns), r f = 0.51, and n-palmitoyl-sphingosine cer(np), r f = 0.50, were selectively separated with hplc system in above mentioned conditions. the retention time of cer(np) and cer(ns) was 4.31 and 5.19 min, respectively. the same lipids were detected by hplc in the human foreskin extracts. the structure of the lipids from collected fractions was confirmed by means mass spectrometry and nmr. the physiological functions of the separated ceramides are investigated. production recombinant human thymosin-␣ 1 overexpressed as intein fusion protein in e. coli roman s. esipov, vasily n. stepanenko, anatoly i. miroshnikov, shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya 16/10, moscow, 117997 russia. e-mail: esipov@ibch.ru (roman s. esipov) medicines based on polypeptides consisting of 30 and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin-␣ 1 production system intein mediated purification with affinity chitin (impac system)-binding tag has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin-␣ 1 . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl 2 and we have found that, in case of intein-thymosin-␣ 1 , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of 0.5-1 mm zinc chloride in buffers on all stages of thymosin ␣ 1 isolation. the structure of recombinant thymosin-␣ 1 of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. we present a microbioreactor with thermoelectric cooling to inactivate cellular metabolism by cell culture freezing. small-scale cultivation methods have gained increased importance and their development has been supported by advances in bioprocess monitoring methods. yet efficient sampling methods for off-line analysis remain important where in-situ real-time measurements are difficult, for example for intracellular metabolite concentration or enzyme activity measurements, and for all methods which are invasive by nature, such as protein purification. freeze-stop measurements of metabolite levels are ideal, because they are inert, i.e. do not require the addition of a chemical. in large systems, the chilling time is often the limiting factor, and alternative methods for cell metabolism inactivation are required, such as the spraying of the cell suspension in 60% methanol at a temperature of −40 • c, or the use of boiling buffered ethanol. due to the small thermal mass of microsystems, shorter chilling times can be expected. sample cooling to 4 • c in a microbioreactor has been presented previously. in this contribution, we investigate sample freezing to completely inactivate the cell metabolism from microbioreactor working volumes of approximately 100 l. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic 1 , alvin w. nienow 1 , ian w. taylor 2 , ryan hicks 2 , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf-21 cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf21 infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp486 coding for am-cyan coral protein, which emits natural green fluorescence. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w3110). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. when choosing an expression host for production of a specific recombinant protein, one can essentially select from a multiplicity of different systems. while e. coli bacterium is usually the starting point for any cloning and expression effort, there is no universal expression host that would work optimally for all proteins. practical issues to consider include e.g. need for post-translational modifications and protease activity of the host. we have produced recombinant hiv-1 nef in different host cell systems: e. coli, p. pastoris yeast and stable drosophila s2 insect cells. using strain/cell line development, production and purification data from practical experiments, we were able to conduct a techno-economical comparison of the different host cell systems. the annual production goal was set at 100 mg of high-purity nef. this was supposed to be produced campaign-wise in 1-2 batches using laboratory-scale bioreactors and other equipment. in this study, it was shown that although the production costs of the different systems were in the same range, the production in e. coli was most inexpensive, and the s2 cell system was the most expensive. regardless of the selected host system, the labour costs incurred the most expenses. when comparing different stages of the work (strain/cell line development, bioreactor production and down-stream processing), the strain development, the most man-hours demanding stage, involved approximately half of the costs of every production system. although e. coli was the most inexpensive host system for producing nef, it has some definite disadvantages: e.g. the production of endotoxins and the disability to perform post-translational modifications. if these disadvantages are of importance, the production must be done using more expensive system. modelling and control of industrial fermentation j.k. rasmussen, s.b. jørgensen capec, department of chemical engineering, technical university of denmark, dk-2800 lyngby, denmark. e-mail: jkr@kt.dtu.dk (j.k. rasmussen) fed-batch processes play a very important role in chemical and biochemical industry. fermentations in biochemical industry are most often carried out as fed-batch processes. present control schemes do not utilize the full potential of the production facilities and may fail to achieve uniform product quality and optimal productivity. the introduction of model based control strategies is considered difficult because suitable models are not readily available. first principle engineering models can be used but the usually limited knowledge of the regulatory network in the micro-organism makes model development very time consuming. another strategy is to use a purely data-driven approach where only limited prior knowledge of the process is required. a framework for generation of such blackbox models is used in this project. this framework is called "grid of linear models" (golm), it uses a large number of linear models which each describes the behaviour of the process within a certain time interval. the combination of these models results in a model which covers the entire time span of the fermentation and approximates the nonlinear time varying behaviour. a procedure for deriving golm models from operational data has been developed and because they consist of a large set of linear models it makes them suitable for model predictive control implementation with iterative learning capabilities from batch to batch. iterative learning model predictive control (ilmpc) based on a golm model is being implemented on a fermentor at novozymes a/s. the results will be evaluated in terms of the controller's capability to ensure uniform product quality and reject both intra and inter batch process disturbances. the model based approach renders optimization of the process recipe possible by using the ilmpc capability. this opportunity provides a great potential for increase of productivity and reduction of cost. j.m. viader-salvadó, j.a. fuentes-garibay, l.j. galán-wong, m. guerrero-olazarán institute of biotechnology, biological science school, autonomous university of nuevo león, san nicolás de los garza, n.l., méxico, the production of recombinant trypsin and trypsinogen has been reported as difficult due to a probably toxicity on host or its instability. in an effort to attain high-level production of shrimp trypsin for aquaculture applications, we have evaluated shrimp trypsinogen production by recombinant pichia pastoris strains in 5-l bioreactors. a p. pastoris gs115 mut+ containing in its genome the litopenaeus vannamei trypsinogen cdna fused in frame to saccharomyces cerevisiae ␣-factor secretion signal, previously constructed in our laboratory, was used. four three-step fermentations (glycerol batch, glycerol and methanol fed-batch) were carried out. the glycerol batch step (2 l of basal salts medium, 50 g/l glycerol, 8.8 ml biotin 0.02%, 8.8 ml ptm1, 250 l 289 antifoam, ph 5 adjusted to with 28% nh 4 oh) was carried out until glycerol was completely exhausted (21 h). the glycerol fed-batch step was carried out feeding with 50% glycerol (12 ml/l biotin 0.02% and ptm1) at 0.8 ml/min by 9 h and 45 min of posterior starvation. the methanol fed-batch step was carried out feeding with 100% methanol (12 ml/l biotin 0.02% and ptm1) by 133 h using a methanol concentration on/off feedback control to maintain constant the methanol concentration in the culture medium to 1 g/l. in all the fermentations the air flow rate and the agitation were set at 5 l/min and 800-1000 rpm, respectively. with the four fermentation assays, the influence of the ph and temperature in the production phase to the recombinant shrimp trypsinogen production were evaluated. in the four fermentations, at the end of the second step a biomass of 250 g/l wet weight were obtained (od600 230). the methanol demand in the four fermentations surprisingly was not increasing rather initially it was 0.37 ml/min, after 32 h decreased 2.5 times for 29 h, increased to 0.49 ml/min for 23 h and afterwards it was decreased manually to a constant value of 0.3 ml/min for that the dissolved oxygen will not decrease to values less than 20% (last 48 h). the total protein amount in the culture medium supernatant increased during the production step until values of 1.6 g/l (assay at ph 6), 6.5 times more than the worst assay (ph 3) recombinant shrimp trypsinogen production was confirmed by sds-page (about 500 mg/l) and trypsin enzymatic activity was detected using bapna as substrate after trypsinogen activation with shrimp hepatopancreas extract. large conformational change on giant dna induced by ascorbic acid: a nobel scheme on its antioxidative activity yuko yoshikawa, emi sakai, yoshiko oda department of food and nutrition, nagoya bunri college, nagoya 451-0077, japan ascorbic acid is often regarded as an antioxidant in vivo, where it protects against dna damage by scavenging reactive oxygen species. in the present, we will show another potent scenario on the protective effect of ascorbic acid through a significant structural change of giant dna. recently, we examined the effect of ascorbic acid on the higher order structure of dna through single molecular observation with fluorescence microscopy, and found that ascorbic acid generates a pearling structure in giant dna molecules, where elongated and compact parts coexist along a molecular chain. the results of observations with atomic force microscopy indicate that the compact parts assume a loosely packed conformation. as the extension, here we study the protective effect against double-strand breaks by reactive oxygen at different concentrations of ascorbic acid, in relation to the change of the higher order structure of giant dna. we have performed a real time observation on the doublestrand breaks on individual dna molecules by use of fluorescence microscopy. we have found that the double-strand break is markedly protected when ascorbic acid exists over millimolar concentrations. it is found that such a protective effect of ascorbic acid corresponds well to the above mentioned change on the higher order structure of dna. it has been reported that human circulating immune cells, such as neutrophils, monocytes and lymphocytes, accumulate ascorbic acid in millimolar concentrations. therefore, it is expected that the ascorbic acid concentration that induces the large conformational change on dna may be of physiological significance. , y., et al., 2004 . febs lett. 566, 39-42. yoshikawa, y., et al., 2003 . plant proteins are increasingly being used as an alternative to proteins from animal sources and substantially contribute to the human diet in several developing countries. there are many process both industrial and food based which employ protein hydrolysis and hydrolytic products have a wide variety of applications from industrial fermentation media to food additives. traditionally, proteins are hydrolysed by chemical means. acid hydrolysates of protein are used to produce food ingredients and flavour compounds. however, hydrolysis by chemical reagents produce potentially hazardous byproducts and these non-selective hydrolysis products cannot easily be defined. the use of enzymes allows for selective hydrolysis of protein and thus produces a potentially safer and more defined material. the present investigation describes the effects of substrate, enzyme and hydrolysate concentration on the hydrolysis of corn gluten. the corn gluten was hydrolysed by a commercial protease preparation neutrase. the protein hydrolysis reactions were carried out in 0.2 l of aqueous solutions at the temperature of 50 • c and ph 7 and were monitored by using ph-stat method. the degree of hydrolysis (%) and soluble protein concentration depending on time were investigated by using 1, 2, 4, 6, 8 and 10% (w/v) substrate concentrations; and 0.25, 0.4, 0.5, 0.6 and 0.75, 1% (v/v) enzyme concentrations; and 25, 50, 75 and 100% (v/v) this paper describes medium optimization for urease production by aspergillus niger ptcc 5011 by one-factor-at-a-time and orthogonal array design methods. the one-factor-at-a-time method was used to study the effects of carbon and nitrogen sources on urease production. among various carbon and nitrogen sources used, sucrose and yeast extract were the most suitable for urease production, respectively. subsequently, the concentration of sucrose, yeast extract and mineral sources were optimized using the orthogonal array method in two stages. the effects of nutritional components for urease production by a. niger ptcc 5011 in the first and second stages were in order of urea > niso4 > sucrose >kh 2 po 4 > k 2 hpo 4 > cacl 2 > yeast extract > mgso 4 and yeast extract > sucrose > k 2 hpo 4 > kh 2 po 4 > urea > cacl 2 > niso 4, respectively. the optimal concentrations of nutritional components for improved urease production were determined as 20g/l sucrose, 0.85 g/l urea, 3.4 g/l yeast extract, 0.03g/l niso 4 ·6h 2 o, 0.5 g/l mgso 4 ·7h 2 o, 0.04 g/l cacl 2 , 0.35 g/l kh 2 po 4 , and 0.35 g/l k 2 hpo 4 . these results showed that urea, niso 4, yeast extract and sucrose had significant effect on urease production by a. niger ptcc 5011. tween 80 and mgso 4 had negligible effect on urease production by this strain. the subsequent confirmation experiments determined the validity of the models. maximum urease activity in optimized media by onefactor-at-a-time and orthogonal array methods were about 1.14 and 2.74 times greater than with the basal medium, respectively. carbon sources create fingerprint fermentation characteristics pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 : 1 bre lab, department of chemical engineering, ankara university, 06100 ankara, turkey; 2 ib lab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e. glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e. serine alkaline protease (sap; ec 3.4.21.62),) and ␤-lactamase (ec 3.5.2.6), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. 1.5-2-fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide ç alık bre lab, department of chemical engineering, ankara university, 06100 ankara, turkey ␣-amylase (e.c. 3.2.1.1) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣-1,4 glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b-14396) which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in 0.5 dm 3 airfiltered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs1). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled 1.0 and 3.5 dm 3 systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc28s ultracentrifuge, ␣-amylase activity was measured by the dns method (bernfeld, 1955) . amino acid concentrations were determined with a hplc (waters), protein and organic acid concentrations were measured with a hpce (waters, quanta 4000e) (ç alık et al., 1998) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method (rainer, 1990) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources i.e., glucose, fructose, maltose, lactose and soluble starch; n sources i.e., (nh 4 ) 2 hpo 4 , (nh 4 ) 2 so 4, and nh 4 cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and byproduct concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar ç alık iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc18::bal carrying recombinant e. coli on a defined medium with 8.0 kg/m 3 glucose were investigated in order to finetune the bioreactor performance, in v = 3 dm 3 batch bioreactors at five different conditions with the parameters at, i.e. q 0 /v r = 0.5 vvm and n = 250, 375, 500 and 750 min −1 and; q 0 /v r = 0.7 vvm and n = 750 min −1 . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph 0 7.25 are optimum for maximum bal activity, i.e. 860 u/cm 3 at 12 h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains 102 metabolites and 133 reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e.coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. the detection and diagnosis of pathogenic bacteria causing many diseases to the human body is an area of important research to public welfare. food is the most important energy source to humans, but it can give rise to disease caused by pathogenic bacteria not performing adequate detection tests. oligonucleotide-based microarrays are becoming increasingly useful for the analysis of expression profiles and polymorphisms among interested genes. here, we checked the possibility of development of oligonucleotide-based microarrays for detection and diagnosis of foodborne pathogenic bacteria. the oligonucleotide chip technology was applied to one control strain and seven foodborne pathogenic bacteria strains. it was designed repeated spots of eight hyperspecific and two highly conserved (control) capture probes from 16s rdna sequences. in order to validate experimental quality and to certificate specificities among specific spots at a glance by 2d and 3d views, quantitative visualization tool was developed. using the proposed oligonucleotide chip, we could classify and diagnose species and even subtypes of some pathogens. induction of in vitro neuro-muscular junctions using neuroblastoma and fibroblast cell lines for facilitating receptor-binding studies with botulinum toxin arindam chaudhury, bal ram singh department of chemistry and biochemistry, university of massachusetts dartmouth, 285 old westport road, north dartmouth, ma 02747-2300, usa. e-mail: g achaudhury@umassd.edu (a. chaudhury) botulinum toxin (bont), the most potent biological toxin known, is responsible for botulism, a fatal paralytic disease of the neuromuscular transmission. it blocks the release of acetylcholine at the neurotransmitter junction of the synapse. the objective of the current study was to induce in vitro neuro-muscular junctions through co-culturing of nerve and precursor-muscle cell lines. presently no known primary cultures or cell lines are available for nerve-muscle co-culture, thus validating the current work. j2-3t3 fibroblast cell line was first adapted to grow in media conducive for growth of sh-sy5y neuroblastoma cell line. the two cell lines were then splitted and co-cultured and observed for junction formations. light and fluorescent microscopic studies revealed en plaque (twitch-type) and en grappe (bulbous nerve endings) nerve-muscle junctions. growth rate of j2-3t3 cells decreased substantially when the media was initially changed. structurally they were more spindle-shaped than the normal reticular shapes of j2-3t3 cells, when grown in a media tailor-made for them. the formation of nerve-muscle junctions were confirmed using markers specific for each cell type. future work is focusing on receptor identification for the botulinum toxin in the established in vitro neuro-muscular junctions and also the transcellular translocation of the toxin. fourier-transform infrared (ftir) spectrometers have recently enjoyed widespread popularity in bioprocess monitoring applications due to their non-invasiveness and in-situ sterilizability. their online applicability creates an interesting opportunity for process control and optimization. however, the precision and accuracy of the predicted analyte concentration values directly depend on the quality of the measured signal and the robustness of the calibration model. instability and time drift in the measured spectra are currently one of the main obstacles in ftir monitoring. the intensity of the detected signal is influenced both by random noise and structural drifts and offsets. as a result, it is often necessary to scale the measured spectrum with respect to a constant reference spectrum, a technique similar to the internal standard approach used in analytical assays, such as hplc. applying this technique has lead to a noticeable decrease in the standard error of prediction in the monitoring of an anaerobic s43 fermentation of the saccharomyces cerevisiae yeast. in order to test the robustness of the calibration model and to increase its resistance to signal instability, random spikes of known amounts of analytes were introduced into the measured medium. this approach can be used to fine-tune the calibration model on-line and is currently one of the aspects investigated in this laboratory. the effect of the stringent response induction on l-valine biosynthesis by corynebacterium glutamicum ilze denina 1,2 , longina paegle 2 , liga zala 1,2 , maija ruklisha 2 : 1 faculty of biology, university of latvia, kronvalda blvd. 4, riga lv-1586, latvia; 2 institute of microbiology and biotechnology, university of latvia, kronvalda blvd. 4, riga lv-1586, latvia. e-mail: ilzede@hotmail.com (i. denina) the present study was focused on methods of the stringent response induction and on investigation of its effect on valine overproduction by isoleucine auxotrophs of corynebacterium glutamicum. the intracellular level of guanosine 5 -diphosphate 3diphosphate (ppgpp) increased and bacterial growth rate (µ) decreased during the short-term experiments when the exponentially growing cells were exposed to isoleucine limited conditions. the induction of the cellular stringent response resulted in a drastic increase in the activity of acetoxydroxy acid synthase (ahas), also by a significant increase in valine production. in contrast, an increase in ahas activity and valine synthesis by c. glutamicum was not achieved when bacterial growth was down-regulated in a ppgpp-independent manner. these results demonstrated that induction of the ppgpp-mediated stringent response might be significant in order to increase valine overproduction by c. glutamicum. infections with human cytomegalovirus (hcmv) continue to be an important health problem in certain patient populations, such as newborns, graft recipients of solid organs, or bone marrow and aids patients. in these groups, hcmv is a major cause of morbidity and mortality. the complex biology of hcmv necessarily begins with an initial interaction between the envelope of the infectious virus and the host cell. glycoprotein b (gb) is the major antigen, on the envelope of hcmv, for the induction of neutralizing antibodies. the region between aa 552 and 635 of hcmv gb (termed antigenic domain 1, ad-1) has been identified as the immunodominant target for the humoral immune response following natural infection. screening methods for detection of neutralizing antibodies have not been used because they are costly and labor intensive and thus far are not feasible for use on a large scale. for the development of reliable and inexpensive serodiagnostic tests the ad-1 of hcmv glycoprotein gp58, which are known to bind neutralizing antibodies, was expressed in prokaryotic systems. in this work, one prokaryotically expressed fusion protein which codifies ad-1 with ␤-galactosidase was used. the influence of different process conditions, on the production of the fusion protein containing the ad-1 as well as sugars addition to the fermentation medium was investigated. in order to analyze the expression of fusion protein, the ␤-galactosidase activity was followed throughout the fermentation. lysis process was also optimized and some final confirmation tests about protein antigenicity were performed. polyenzymic systems for the preparation of drugs based on modified nucleosides d. chuvikovsky, r. esipov, t. muravyova, a. miroshnikov shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya 16/10, moscow 117997, russia. e-mail: esipov@ibch.ru (r. esipov) considerable progress in the preparation of nucleoside analogues was achieved by combination of chemical and biochemical transformations. enzyme-catalyzed chemical transformation is now widely recognized as practical alternative to traditional organic synthesis in pharmaceutical and chemical industries. pentofuranosyltransfer reaction catalyzed by nucleoside phosphorylases was successfully employed for the synthesis of a variety of base-and sugar-modified nucleosides. enzymes involved in the metabolism of ribose phosphate and deoxyribose phosphate, such as ribokinase and phosphopentomutase, were used for the preparation of sugar-modified nucleosides. nucleosides phosphorylases (thymidine phosphorylase (tp), uridine phosphorylase (up) and purine nucleoside phosphorylase (pnp)), ribokinase and phosphopentomutase from e. coli have been cloned and overexpressed in e. coli. fast and efficient methods for the purification of nucleosides phosphorylases have been developed. the amount of purified protein was about 140 mg/l of cell culture, corresponding to 6300, 16,800 and 4200 units, respectively, of the up, tp and pnp. synthesis of medicinal drugs ribavirin (1-(␤-d-ribofuranosyl)-1,2,4-triazole-3-carboxamide), cladribine (2-chloroadenine-9-␤-d-2 -deoxyribofuranoside) and fludarabine (2-fluoroadenine-9-␤-darabinofuranoside) with the use of recombinant enzymes were studied. several important factors affecting the modified nucleosides production (ph, temperature, enzyme concentration, donor/acceptor ratio) were investigated and optimized. under optimum conditions ribavirin, cladribine and fludarabine produced in the reaction mixture in yields of 84, 85 and 80%, referred to 1,2,4-triazole-3-carboxamide, 2-chloroadenosine and 2-fluoroadenosine, respectively. aggregation and adsorption of fibroin molecules in aqueous solution won hur school of biotechnology and bioengineering, kangwon national university, chunchon 200-701, korea. e-mail: wonhur@kangwon.ac.kr fibroin, the structural protein from bombyx mori, is composed of heavy chain (generally called 'fibroin') and light chain polypeptides of about 370 and 25 kda, respectively. this study investigated the aggregation of fibroin and the adsorption between fibroin and surfaces. the variations of particle size and zeta potential were investigated by electrophoretic light scattering spectrophotometer (els). the adsorption of fibroin on surface was investigated in a continuous flow system by biacore applied surface plasmon resonance(spr) technique. the particle size and zeta potential range of aqueous fibroin were 140 nm, ±20 mv, respectively. iso-electric point(ph iep )of fibroin was ph 4.29. the amount of fibroin adsorbed on a gold surface was less than 0.1 g/ml even in the presence of high concentration of fibroin. the modification of gold surface was accomplished by applying chemicals known to form self-assembled monolayer, those are carrying nh 3 + , coo − , benzene ring and peptide that similar structure with fibroin. the adsorbed amount of fibroin on the self-assembly monolayers (sams) increased in the following order: nh 3 + > benzene ring > coo − > peptide surface. the deposition of fibroin in aqueous solution on non-waven fabric was affected by nacl and high temperature. ph influences metabolite profiling of ␤-lactamse producing b. licheniformis nazari̇leri 1 , pınar ç alık 1 , ali şengül 2 : 1 ib lab, department of chemical engineering, metu, 06531 ankara, turkey; 2 gülhane sch med, dept immunol, 06018 ankara, turkey. e-mail: e115715@metu.edu.tr (n.i̇leri) ph conditions in the bioreactor affect product and by-product formations by influencing metabolic pathways and changing metabolic fluxes, based on its influence on, i.e. dna transcription, protein synthesis, transport mechanism, atp generation and cellular energetics. whereupon, some fermentation processes favours uncontrolled-ph conditions while others favours controlled-ph conditions. on the bases of the interactions between the cell and the bioreactor through a process, carried out at either uncontrolled-or controlled-ph conditions, intracellular ph can be widely different and variable during the fermentation process. consequently, if one aims towards a quantitative understanding of the cell metabolism, one has to take into account the time variations of the intracellular ph and its effects on the in-vivo kinetics of the metabolic steps involved. moreover, since the presence of dormant or dead cells in the cultivation medium have negative effect on the synthesis of the production of desired product; the physiological state of the culture has great importance. in this context, the effects of ph on the regulation of intracellular ph, transport mechanism, and metabolic activity of b. licheniformis during production of ␤-lactamase (ec 3.5.2.6), an industrial enzyme catalyzing the hydrolysis of beta-lactam ring in beta-lactam antibiotics, was investigated. in addition, the physiological state of the organism and its effect on the production were observed. the optimal controlled-ph operation was found to be ph 6.75 with 54 u/cm 3 enzyme activity. the intracellular and extracellular na + , k + ion concentrations increased significantly throughout the process with increasing ph. on the other hand, the intracellular nh 4 + ion concentration was relatively constant. isolation and characterization of angiotensin-i converting enzyme inhibitory peptides by use of anti-peptide antibody fida hasan 1 , megumi kitagawa 2 , yoichi kumada 1 , naoya hashimoto 1 , masami shiiba 2 , shigeo katoh 1 , masaaki terashima 2 : 1 graduate school of science and technology, kobe university, nada, kobe 657-8501, japan; 2 department of human science, kobe college, okadayama, nishinomiya 662-8505, japan inhibitory peptides against angiotensin-i converting enzyme can be promising bio-functional peptides as natural alternatives for the non-peptide ace inhibitory drugs. these peptides are inactive within sequences of parent proteins and can be released during enzymatic digestion or food processing. immunointeraction is very effective for the purification of proteins and peptides with high purity. in this study, ace inhibitory peptides from hydrolysate of bonito meat were isolated by an anti-peptide antibody column and hplc, and kinetics of production of these ace inhibitory peptides was studies. an anti-peptide antibody against an ace inhibitory peptide, which was found by kohama et al. from tuna was obtained by immunization of the antigen peptide pc-iace (kkpthikwgd). water extract of bonito meat was digested at 37 • c in a modified gastric juice, 1.76 mg/ml nacl containing 312 g/ml pepsin (ph 2). peptides in hydrolysates were purified by use of an affinity column coupled with the antipeptide antibody and separated by hplc equipped with a reverse phase column (cosmosil 5c18-ms-ii, 4.6 cm × 150 cm). amino acid sequences and ic 50 values of the potent ace inhibitory peptides were determined. sds-page and western blotting experiments clarified that bonito protein contained peptides having similar sequence to the antigen peptide. a fraction of retention time 18-28 min in hplc purification samples showed high inhibitory activity, and several peptides in this fraction were separated. after 48 h digestion, two major inhibitory peptides, herdpthikwgd and pthikwgd, were found to be relatively stable in the gastric juice. kluyveromyces marxianus physiology on several levels of carbon, nitrogen sources and oxygenation during inulinase production silva-santisteban yépez, o. bernardo, francisco maugeri department of food eng./unicamp, 13081-970, campinas, sp-brazil. email: maugeri@fea.unicamp.br (f. maugeri) inulinase produced by yeasts is an interesting alternative compared with the one produced by filamentous molds, as culture conditions can be better controlled. during the assays, it was observed that inulinase production levels varied with nutritional conditions in batch culture. kluyveromyces marxianus atcc 16045 culture is described by two main phases, the first one being the growth phase, where substrate consumption and basal inulinase production were performed, and the second one being the phase where some metabolites are uptaken and high inulinase production is observed. the metabolic fluxes analyses were used to describe the cell physiology in the first phase, in a variety of conditions of sucrose and ammonium sulfate concentration and aeration condition. the metabolic network included the main metabolic pathways such as glycolisis, pentose phosphate pathway, krebs cycle, oxidative phosphorylation and biomass biosynthesis. the physiology in this phase was correlated with high inulinase production in the second phase. it was also noticed that inulinase production diminished when sucrose was in high concentration, leading, additionally, to ethanol production. in these terms, it was unveiled a kind of crabtree effect performed by this strain. forward extraction of l-aspartic acid from fermentation broths by reverse micelles and backward extraction by gas hydrate methodö. aydogan 1 , e. bayraktar 1 ,ü. mehmetoglu 1 , m. parlaktuna 2 , t. mehmetoglu 2 : 1 department of chemical engineering, faculty of engineering, ankara university, tandogan, ankara 06100, turkey; 2 department of petroleum and natural gas engineering, middle east technical university, ankara 06531, turkey. e-mail: mehmet@eng.ankara.edu.tr (ü. mehmetoglu) recently gas hydrate method has been applied as a technique for backward extraction of amino acids from reverse micelle systems. in this study, backward extraction of l-aspartic acid was investigated by gas hydrate method. at the first stage, production of l-aspartic acid was carried out using 50 ml of 0.5 m ammonium fumarate (ph 9.5) as substrate at 37 circc in an orbital shaker at 150 rpm for 4 h. e. coli (atcc 11303) was used as biocatalyst. at the end of reaction excess fumaric acid was extracted in reverse micelle phase. then forward extraction of l-aspartic acid was carried out with injection method in reverse micelle phase. for back extraction, co 2 is used to form gas hydrates crystalline structure. back extraction experiments were carried out between 35-37 bar g pressure and at 2 circc. at the end of the back extraction l-aspartic acid was obtained in crystalline form. the results indicate that recovery of l-aspartic acid from reverse micelles by forming gas hydrate can be achieved with a yield of 55.3%. consequently, gas hydrate method can be used as a new technique for backward extraction of amino acids from reverse micelles. aerobic and anaerobic cultivations of aspergillus niger on different nitrogen sources susan meijer, gianni panagiotou, lisbeth olsson and jens nielsen center of microbial biotechnology, biocentrum-dtu, technical university of denmark, dk-2800 lyngby, denmark in this study, we aim at creating a succinic acid producing strain of a. niger. a. niger is known to be a strictly aerobic organism, meaning it is not able to use the reductive part of the tca cycle to produce succinate. during aerobic growth a. niger uses oxygen as electron acceptor in the respiratory chain, thereby reoxidizing the produced nadh to nad + . however, under anaerobic conditions other compounds than oxygen, such as no 3 − are required as electron acceptor (denitrification). this process consists of no 3 − reduction to nh 4 + coupled to substrate-level phosphorylation that supports growth under anaerobic conditions. in the present study, our aim was to investigate the effect of different nitrogen sources on the physiology of a. niger during growth under aerobic and anaerobic conditions. aerobic growth experiments on three different nitrogen sources; ammonium, nitrate and nitrite, showed that ammonium and nitrate could be consumed by the filamentous fungus. nitrite on the other hand could not facilitate growth, indicating the absence of a nitrite uptake system. however, under anaerobic conditions notable growth was only observed on nitrate. these data support the hypothesis of the existence of an alternative electron acceptor that might facilitate anaerobic growth of a. niger. among the therapeutic proteins derived from mammalian cells, recombinant antibodies received a great deal of attention as a prominent product through biotech pipelines toward the marketplace. they now occupy about 25% of the estimated medicines in clinical development and many more antibodies which lead the value of the market going forward are reported. there are various environmental factors affecting rcho cell cultures such as medium components, temperature, ph, and byproducts (ammonia, lactate, and, etc.) . because most of mammalian cells are very sensitive to their environmental change, appropriate control of environmental parameters is a very important consideration to enhance cell growth and production of target proteins. balanced addition of limiting medium components plays an essential role on improvement of cell density and product concentration. temperature and ph are easily adjustable process parameters, being reported to influence cell growth and recombinant protein production. ammonia and lactate are well-known byproducts which have an inhibitory effect on cell growth when their concentrations exceed a specific level. in this work, effects of various environmental factors including temperature, ph, amino acids, vitamins, hormones, and metabolic byproducts on cell growth and recombinant antibody production were investigated in the cultivation of recombinant chinese hamster ovary cells. the most suitable condition of each environmental condition was proposed for enhancement of the cell growth and the productivity of recombinant antibody. the present study was carried out in order to assess the protective effects of calycosin-7-o-␤-d-glucopyranoside isolated from astragali radix (ar) on hyaluronidase (haase) and the recombinant human interleukin-1␤ (il-1␤) induced matrix degradation in human articular cartilage and chondrocytes. we isolated the active component from the n-butanol soluble fraction of ar as haase inhibitor and structurally identified as calycosin-7-o-␤-d-glucopyranoside by lc-ms, ir, 1 h nmr, and 13 c nmr analyses. the protective effect of arbu on the matrix gene expression of immortalized chondrocyte cell line c-28/i2 treated with haase was investigated using a reverse transcription polymerase chain reaction (rt-pcr). its effect on haase and il-1␤-induced matrix degradation in human articular cartilage was determined by using a staining method and calculating the amount of degraded glycosaminoglycan (gag) from the cultured media. pretreatment with calycosin-7-o-␤-d-glucopyranoside effectively protected against matrix degradation of the human chondrocytes and articular cartilage. therefore, it would appear that calycosin-7-o-␤-d-glucopyranoside from ar is a potential natural ant-inflammatory or anti-osteoarthritis agent and can be effectively used to protect against proteoglycan (pg) degradation. catechol-o-methyltransferase (comt) is an enzyme that catalyses a variety of endogenous and exogenous catechol substrates by transferring a methyl group from s-adenosylmethionine (sam) to either the meta-or the para-hydroxyl group of the catechol ring. the enzyme has a physiological role in the metabolism of the catechol estrogens, inactivation of the catecholamine neurotransmitters such as dopamine and epinephrine and detoxification of a variety of xenobiotic catechols. comt activity has been identified in various tissues; however with the developments in molecular biology and gene technology, the production and purification of large amounts of recombinant comt is a good option for biochemical, pharmacological and structural studies. in this work, cultures of recombinant e. coli harbouring a model plasmid were grown in a 500 ml shake-flask containing 125 ml of complex medium. the influence of medium composition and induction time on comt production, recovery and clarification by sonication, ammonium sulphate precipitation and purification by hydrophobic interaction chromatography onto a butyl-sepharose column will be presented and discussed. bioactive bacterial exopolysaccharides corinne sinquin 1 , karim senni 2 , jacqueline ratiskol 1 , farida guéniche 2 , jean guézennec 1 , gaston godeau 2 , sylvia colliec-jouault 1 : 1 ifremer, 44311 nantes cedex 3, france; 2 ea2496 université rené descartes, 92120 montrouge, france. e-mail: corinne.sinquin@ifremer.fr (c. sinquin) interest in mass culture of microorganisms from the marine environment has increased considerably, representing an innovative approach to the biotechnological use of under-exploited resources. marine bacteria associated with deep-sea hydrothermal conditions have demonstrated their ability to produce in an aerobic carbohydrate-based medium, unusual extracellular polymers (guezennec, 2002; colliec-jouault et al., 2004) . these exopolysaccharides (eps) present original structural features that can be modified to design innovative bioactive compounds and improve their specificity. with the aim of promoting biological activities, chemical modifications (depolymerization and substitution reactions) of one eps produced by vibrio diabolicus have been undertaken (raguenes et al., 1997) . the structure of the native eps has been described (rougeaux et al., 1999) the potential of the eps derivatives as therapeutical agents will be presented. physiological responses of e. coli to glucose and oxygen shifts in fed-batch fermentations jaakko soini 1 , christina saarimaa 1 , arne matzen 2 , peter neubauer 1 : 1 bioprocess engineering laboratory, department of process and environmental engineering, university of oulu, oulu fi-90014, finland; 2 sanofi-aventis, germany. e-mail: jaakko.soini@oulu.fi (j. soini) in high-cell density fermentations e. coli cells are often subjects of transient changes in microenvironment around them. these changes can be, for example, medium component gradients or differences in oxygen availability. we have studied the physiological response of e. coli w3110 cells to simultaneous oxygen limitation and overfeeding of glucose. the aim is to obtain more information of physiological changes for better understanding of the bottlenecks in such processes. the response of the cells for glucose and oxygen shifts was studied by analyzing key metabolites and proteins and mrna transcript levels. the transcript levels were measured using a sandwich hybridization technique (rautio et al., 2003) proteomic analysis was carried out by 2d-electrophoresis and the metabolite analysis by hplc. the main focus of this study is to monitor the expression patterns of marker genes involved in mixed acid fermentation, glycolytic pathway and tricarbonic acid cycle. rautio et al., 2003 . sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates. microb. cell fact. influence of ner on genetic instability of the (ctg/cag) tracts in bacterial chromosome sylwia szwarocka 1 , paweł parniewski 2 : 1 department of microbiology and immunology, university of łódź, 90-237 łódź, banacha 12/16, poland; 2 centre for medical biology, polish academy of sciences, 93-232 łódź, lodowa 106, poland many human hereditary neurological diseases, including fragile x syndrome, myotonic dystrophy and friedreich's ataxia, are associated with expansions of triplet repeat sequences (trs) (cgg/ccg, ctg/cag and gaa/ttc) in or near specific genes. mechanisms that mediate the expansions and deletions of trs include dna replication, repair and recombination. many investigations suggest that the structural properties of the trs play a consequential role in their genetic instabilities. nucleotide excision repair (ner) is the major cellular system in both prokaryotes and eukaryotes and recognises damages due to distortion of the dna helix. involvement of ner in the hairpin loop repair that can form within ctg tracts has been reported. the participation of this repair systems in the trs instability was investigated in e. coli only on multicopy plasmids. the results showed that deficiency of some ner functions dramatically affects the stability of long (ctg/cag) inserts. in this work we present a chromosomal model to study the instability of the trs in e. coli. we introduced the (ctg/cag)n tracts into the chromosome of e. coli and used strains with some deficiency of the ner and investigated genetic stability of these tracts after multiple recultivations. in general, our results show that the (ctg/cag)n repeats are much more stable in the chromosome than in plasmids. these data may suggest that instability of trs in plasmids is associated with interaction between repetitive tracts on different plasmid molecules inside the cell. however, mutations of ner genes may increase (uvra and uvrb mutants) or decrease (uvrc and uvrd mutants) stability of the trs in the e. coli chromosome. this study was partially funded by the kbn grant 2 p05a 01927. performance analyses of a multi-stage integrated fermentation process for lactic acid production hsun-tung lin, feng-sheng wang department of chemical engineering, national chung cheng university, chia-yi 621-02, taiwan. e-mail: chmfsw@ccu.edu.tw (f.s. wang) in this work, we considered a multi-stage integrated continuous fermentation process for producing lactic acid. each stage consists of a mixing tank, a fermenter, a cell recycle unit and an extractor. the generalized kinetic model is first applied to formulate the integrated process. we have compared the overall productivity and conversion of the integrated process with those of two simplified processes. from the design equations, we obtain that three processes have the identical overall conversion. however, the proposed process has the greatest overall productivity. the specific kinetic model for lactic production (youssef et al., 2000) was applied to the integrated process in order to find the maximum overall productivity. two optimization problems are respectively considered to determine the optimal stages, operating conditions and design variables. the first problem supposes that the integrated process has the equal working volume ratio for each fermenter. such a process requires four stages to yield the maximum overall productivity and the nearly complete overall conversion. however, if the working volume ratio for each stage is considered as the decision variables in the second optimization problem, three stages is enough to achieve the identical overall productivity. youssef, c.b., guillou, v., olmos-dichara, a., 2000. contr. eng. pract. 8, 1297-1307. modelling of the binding of ligands to macromolecules jørgen m. mollerup department of chemical engineering, building 229, dtu, 2800 lyngby, denmark a variety of factors that govern the properties of proteins are utilized in the development of chromatographic processes for the recovery of biological products including the binding and release of protons, the non-covalent association with non-polar groups (often hydrophobic interactions), the association of small ions (ion exchange) and the highly specific antigen-antibody interaction (affinity interactions). the fulcrum point in the understanding and modelling a chromatographic separation is the adsorption isotherm that determines the peak shape at preparative load. to enable an efficient chromatographic process development strategy it is necessary to conduct theoretical and experimental investigations of the adsorptive behaviour of proteins. thermodynamically consistent models for ion exchange chromatography and hydrophobic interaction chromatography have been developed and can be utilised in the simulation of a chromatographic separation. besides, measurements on hic media can be utilised to determine the cohn salting-out coefficient. the lig-and binding process can frequently be coupled to associated structure changes in the protein, the ligand or both. this gives rise to nonlinear adsorptive behaviour known as cooperativity which cannot be modelled using conventional models which displays convex behaviour. examples of cooperative behaviour are the reversible binding of oxygen and carbon monoxide to haemoglobins and the binding of nad + to yeast glyceraldehydes 3-phosphate dehydrogenase. in the paper we discuss the modelling of reversible binding of mobile as well as immobilised ligands to macromolecules and compare modelling to experiment. comparative analysis of the temperature policy for processes with a deactivating native enzyme i. grubecki, m. wojcik department of chemical and biochemical engineering, university of technology and agriculture, 85-326 bydgoszcz, ul. seminaryjna 3, poland a comparative analysis of the temperature policy for an enzymatic reaction with michaelis-menten kinetics in a batch reactor has been carried out. both isothermal and optimal temperature policies for processes with deactivating native enzyme have been considered. in the model, the thermal deactivation was described by a first-order reaction, and the arrhenius-type dependence between rate parameters and temperature was assumed. as an indicator for a direct comparison between the isothermal and optimal temperature policies the quotient of conversions under identical initial and final condition was used. a method was presented to calculate this indicator, which is based on the analytical and numerical solutions. this method can be of great importance for the industrial practice. application of changeable temperature policy could result in significant increase in conversion when ratio of activation energy for deactivation and activation energy for reaction is high. studies on the impact of mixing during brewing using near and mid-infrared spectroscopy georgina mcleod 1 , alvin w. nienow 1 , graham poulter 2 , reg wilson 3 , henri tapp 3 , christopher j. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w3110). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. template refolding utilizing biospecific interactions shigeo katoh 1 , yoichi kumada 1 , nanae maeshima 1 , daisuke nohara 2 : 1 graduate school of science and technologykobe university, kobe 657-8501, japan; 2 department of biomolecular sciencegifu university, gifu 501-1193, japan. e-mail: katoh@kobe-u.ac.jp (s. katoh) recombinant proteins over-expressed in e. coli are often accumulated as insoluble particles called inclusion bodies. proteins in inclusion bodies must be solubilized by a denaturing agent, such as urea and guanidine hydrochloride, and refolded to recover their native structures having biological activities. in bioprocesses it is important to obtain high refolding efficiencies and high throughputs at high protein concentrations. in refolding operation, a denatured protein solution is usually added batch-wise into a large volume of a refolding buffer in order to start refolding by reducing the concentration of a denaturant and to prevent aggregate formation of renaturing molecules. thus, a large volume of a stirred tank is required, and the concentration of protein after renaturation becomes low. biointeractions between a pair of biomolecules, such as enzyme-inhibitor, antigen-antibody and hormone-receptor, are highly specific and have been used for detection and separation of biomolecules. these interactions may be used as templates for refolding of target molecules, which can be captured with the templates and are prevented from aggregate formation and, in the case of proteases, from autoproteolysis. the specific interaction might promote refolding of the target molecules. these might improve the refolding efficiency. the biointeractions between antigen-antibody and enzyme-inhibitor were used for efficient refolding in packed columns, in which template ligands (antibody, inhibitor) were coupled on gel support. denatured solutions of target molecules (carbonic anhydrase and s. griseus trypsin) were mixed with refolding buffer and supplied to the affinity column coupled with the template ligands for refolding. with refolding in the column, higher refolding efficiencies were obtained than those by the batch dilution method with relatively low concentrations of denaturants. by increasing the adsorption capacity of the column, throughput of refolding can be increased without decrease in the refolding efficiency. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic 1 , alvin w. nienow 1 , ian w. taylor 2 , ryan hicks 2 , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf-21 cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf21 infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp486 coding for am-cyan coral protein, which emits natural green fluorescence. carbon sources create fingerprint fermentation characteristics pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 1 bre lab, department of chemical engineering, ankara university, 06100 ankara, turkey; 2 ib lab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e., glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e., serine alkaline protease (sap; ec 3.4.21.62),) and ␤-lactamase (ec 3.5.2.6), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. 1.5-2-fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. kinetic resolution of racemic benzoin with different lyophilized microorganisms ç . babaarslan 1 ,ü. mehmetoglu 1 , a.s. demir 2 : 1 ankara university, faculty of engineering, department of chemical engineering, 06100, ankara, turkey; 2 middle east technical university, department of chemistry, 06531 ankara, turkey. e-mail: barslan@eng.ankara.edu.tr (ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the 2-hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at 30 • c and 150 rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs 112-07 as ee s = 16% and ee p = 30% (conversion = 35%) using thf as solvent and vinyl acetate as acyl donor. otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box 6121, campinas, cep 13083-862, são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during 3, 5 and 7 days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the mediun was composed by, cwb:cr were mixed with distilled water and transferred into 250 ml capacity erlenmeyers flasks and autoclaved at 120 • c for 20 min. the medium was then inoculated with spores (5.0 × 10 7 ) and the flaks were incubated at 32 • c. tannase was assayed according to the methodology of mondal et al. (2001) . acording to the statist analyses, the optimum conditions to produce tannase was the range of temperature (29-34 • c); tannic acid (8.5-14%); residues % (coffe: wheat bran) (50:50) and 5 days fermentation time. the enzyme production increased 8.6 times more enzyme production than that was obtained before this optimization. how to cope with fda's pat-initiative with respect to fermentation process monitoring and control marco jenzsch 1 , andreas luebbert 1 , rimvydas simutis 2 : 1 institute of bioengineering, martin-luther-university halle-wittenberg, halle (saale), germany; 2 process control department, kaunas university of technology, kaunas, lithuania with its pat initiative, fda forces drug manufacturers to increase their activities in innovative manufacturing techniques, and, more than previously, to focus on quality assurance. the agency particularly places emphasis on making use of modern process supervision and control techniques such as up-to-date process analytics, multivariate data acquisition and analysis tools in order to improve process monitoring and control. in this contribution we show by means of practical examples how this guidance can be applied to cultivations of genetically modified microorganisms. a comparison of different multivariate state estimation techniques will be presented and compared with more knowledge-based techniques such as the extended kalman filter. the comparison was made for the model system gfp expressed from e. coli bacteria (bl21/de3/gfp) for which more than 40 full data sets are available. all these techniques have already been used during real protein formation at productionscale fermenters, with the same success. hence, the requirements expressed in the pat initiative can immediately be put into practice. feedback control of the recombinant protein production processes based on such estimations is show for several cultivation systems. simple parameter adaptive controllers are compared with model supported controllers, for instance, generic model controllers and model predictive controllers. the results clearly show that we have at hand a rather extended arsenal of feedback control procedures that can be used successfully to tightly control the processes even along set-point profiles of physiological variables such as the specific growth rate (µ). again, fda's suggestion with respect to "control in the engineering sense" can be applied immediately to reduce batch-to-batch variances and thus to increase process quality. extending life by alternative respiration? alexander kern, franz hartner, anton glieder institute of biotechnology, graz university of technology, a-8010 graz, austria. e-mail: a.kern@tugraz.at (a. kern) alternative oxidase transfers electrons directly from the ubiquinol pool in mitochondria to oxygen, allowing cell respiration in presence of complexs iii and iv inhibitors like antimycin a or cyanide. electron transfer by alternative oxidase is not coupled with proton transfer across the mitochondrial membrane, thereby uncoupling the supply of small metabolic intermediates by the central metabolic pathway from energy production in the cell. alternative oxidase is present in mitochondria of plants, many fungi and a few, mostly crabtree-negative yeasts, but not in p. angusta (hansenula polymorpha) and s. cerevisiae. alternative oxidase has multiple functions in different organisms. it is involved in stress answers, in programmed cell death, maintenance of the cellular redox balance, and also citric acid accumulation in a. niger. we isolated the alternative oxidase gene from the methylotrophic yeast p. pastoris in order to study its effects on the cellular energy content, respiratory activity, its protective role against oxidative stress. our results indicate the importance of an exact regulation of the alternative oxidase due to its impact on many cellular functions. new types of energy efficient fermenters with better mass transport, mixing and cooling properties than the current crop of rushton turbine derived tank bioreactors are likely to be required in the future. such fermenters will be needed in order to meet the increasing pressure on costs for low price commodity type products such as single cell protein or food and technical grade enzymes, and to meet the demands of the new wave of white biotech, in which bio-produced chemicals must be made at prices competitive with those of the traditional chemical industry. with this in mind, a prototype pilot scale (500 l) u-loop fermenter has recently been commissioned at biocentrum-dtu. in this fermenter, liquid circulation is driven by a propeller pump through a vertical u-shaped pipe, which is connected at the top with a de-gassing tank. we present here a study of liquid mixing and dispersion in the prototype u-loop fermenter. sub-sequently we show that the results can be described with the tanks in series model. mixing was characterised using pulses of nacl tracer, which were detected with a conductivity probe in various parts of the fermenter. bodenstein numbers (bo) were determined for flow rates corresponding to a linear fluid velocity of 1.05 m/s in the 'legs' of the reactor and showed that the majority of the mixing occurred in the top degassing part (bo = 29) rather than in the u-loop section (bo = 422). it was also observed that the time for mixing to 90% homogeneity after tracer pulse addition was a function of the number of cycles through the reactor (3-3.5) within the range of flow velocities (u) studied (u = 0.41 m/s to u = 1.74 m/s). the mixing time to 90% homogeneity was between 44.6 s (at u = 1.74 m/s) and 181 s (at u = 0.41 m/s). today many biotechnological processes are operated at suboptimal conditions and according to best practice. however, the current industrial development is towards analyzing more parameters and in particular there is a large interest in analysis of biological/biochemical variables. the quality of products and also the possibility to optimize production in submerged cultivations would be greatly enhanced if more on-line/real-time information were at hand. the present investigation was undertaken with the aim of evaluating the potential in using multi-wavelength fluorescence for monitoring and control of filamentous fungi fed-batch cultivations. a recombinant a. oryzae expressing a heterologous lipase was applied as model system. spectra of multi-wavelength fluorescence were collected every five minutes with the bioview ® system (delta, denmark) and both explorative and predictive models, correlating the fluorescence data with the important biological parameters cell mass and lipase activity, were built. the models will be presented, furthermore, advantages and disadvantages of multiwavelength fluorescence for monitoring of cultivation processes will be discussed. moving from r&d to pharmaceutical development is a costly process. it is therefore of paramount importance to design a manufacturing process that combines robust and well-documented technological platforms. therapeutic recombinant proteins designed for human administration should be as close to the authentic product as possible. here, the use of a scalable process and an economically sound affinity tag can be a relevant choice. the tagzyme tm system has been designed to allow for the precise removal of amino terminal affinity tags. the system is based on the use of recombinant aminopeptidases including dipeptidyl peptidase i (dapase tm ). dapase tm is currently produced under cgmp providing a suitable strategy for its use in pharmaceutical production. the tagzyme tm system is superior to other methods since: (1) it is based on exopeptidases, precluding, e.g., unspecific protein cleavage reported when using so-called site-specific endoproteases. (2) it has been tested for production of more than 200 recombinant proteins. (3) it is easily scalable from lab scale to kg of processed protein. (4) it allows the use of his-tags for commercial production without patent infringment, due to our ipr position. (5) the commercial use of tagzyme tm does not require any licensing, only purchase of the enzyme(s). (6) the use of aminopeptidases for pharmaceutical production has been extensively documented for approved drugs. (7) a number of therapeutics is currently being developed using tagzyme tm . (8) unizyme can assist in the optimization of the dsp to enable further cost reduction in the process. these aspects will be discussed and illustrated in the presented poster. website sphingolipids are biologically active molecules involved in the regulation of a large quantity of biological responses. they function in cell proliferation, survival and death (apoptosis) as messengers. dysregulation of apoptosis has significance in numerous pathological conditions including cancer. several anticancer agents act by increasing tumor cell ceramide (a kind of sphingolipid) content. so, a novel approach to cancer therapy would be the pharmacological manipulation of sphingolipid metabolism. in this study, sphingolipid metabolism in baker's yeast s. cerevisiae is used as a model system as many of its sphingolipid related genes and proteins have been characterized. gepasi-biochemical kinetics simulator was used for metabolic control analysis (mca) of the above-specified system. the concentration control coefficients (ccc), flux control coefficients (fcc) and elasticity coefficients were calculated, and their significance in identification of anticancer drug targets is determined. elementary flux modes were also identified and metabolic pathway analysis (mpa) was performed. quantitatively, control effective flux (cef) values were used for potential drug target identification. the results from mca and mpa indicate almost the same potential drug targets: serine palmitoyl transferase, ceramide synthase and ceramidase. drugs against these targets are in preclinical and clinical development. for the identification of new potential drug targets, the cccs, fccs, cefs and elasticity coefficients were examined with an objective function of maximizing the cell ceramide concentrations. it was found that manipulation of inositol-1-phosphate synthase and phosphoinositide kinase activities have considerable effects on ceramide concentrations. if a drug targeting the two enzymes at the same time is designed, it might give a better outcome in terms of cancer therapy. in recent years, there is a growing interest in utilization of airlift reactors (alrs) to biotechnological processes. nevertheless, their industrial application still remains limited because of a lack of reliable studies on transfer phenomena and mixing enabling a suggestion of suitable scale-up procedure. the way to more widely utilization of alrs to biological processes lies in experimental research (on a model medium as well as on a real fermentation medium) followed by mathematical modelling and scaling-up of the processes. this paper deals with a modelling of a glucose-gluconic acid fermentation by a. niger in an internal loop airlift reactor. knowledge of the stoichiometric relationship in the key reaction provides a good opportunity for estimation of substrate and product concentration. the model is based on material balance equations and has been adjusted to experimental data obtained from three internal loop airlift reactors (10.5, 40 and 200 l) . in the model, the alr is divided into ideal stirred tanks in series. in each zone (tank) of the alr the material balance is calculated in two phases (the gas and the liquid phase). this work was supported by the slovak scientific grand agency, grant number vega 1/0066/03 alkaline phosphatase (ap, e.c. 3.1.3.1) is a thermolabile enzyme which is indigenous to all dairy products. it has an inactivation temperature slightly above the value that is required to destroy the most resistant pathogenic microorganism likely to be found in milk. due to that feature, this enzyme is used as an indicator of proper pasteurization. the effect of temperature treatment on the activity of ap was investigated in raw cow's and goat's milk. the stability of alkaline phosphatase in raw milk was compared with the stability of this enzyme in a 0.1 m potassium phosphate buffer with ph 6.6. the ph value of the buffer was approximately the same as that of raw milk. the inactivation curves were measured in the temperature range from 54 to 69 • c. ap in cow's milk was completely inactivated at 69 • c during 60 s but approximately 30% of activity remained at 54 • c after 100 min of treatment. the time required for a complete inactivation of the enzyme in the raw cow's milk was reduced from 90 to 1 min as the temperature increased by 11 • c. heat treatment of goat's milk caused the decrease of activity of the enzyme in the same temperature range as in the case of cow's milk. the increase of temperature from 58 to 68 • c reduced the inactivation time from 35 min to 40 s. the study of thermal stability of the alkaline phosphatase in the buffer solution showed that the time required for inactivation of enzyme was significantly shorter than in milk. milk thus had a protective effect on the activity of alkaline phosphatase. the experi-mental curves were fitted simultaneously using kinetic models where the initial heating period was considered. this work was supported by a grant of 6th framework program of eu, project foodpro, no. sme-2003-1-508374. during the process of separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale, the chromatographic unit operations have an important role. three different protein-binding modes are employed: ion-exchange, hydrophobic and affinity binding. two adsorbent properties are of uppermost importance: a high selectivity and adsorption capacity. in the case of ion-exchange/hydrophobic chromatography, the binding of charged proteins can be affected by ph and ionic strength. in this work, the adsorption capacity of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros 50a, prosep-va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was measured as a function of ph. as a model mab and contaminant proteins, human immunoglobulin (igg), human serum albumin (hsa) and horse skeletal muscle myoglobin (myo) were used. the resin properties were investigated within the range of ph 4-8. the experiments were conducted in a batch-mode, individually for each model protein. the results showed that ion-exchange and hydrophobic resins provided the best selectivity for igg at ph 6. the selectivity of affinity adsorbents was essentially unaffected by ph, however, the highest capacity for igg was at ph 7. another investigated aspect was the dynamics of protein binding. the solution of individual protein in contact with tested adsorbent was circulated through an uv spectrophotometer, what enabled the measurement of time-dependent decrease of protein concentration. the results indicated that affinity adsorbents with a rigid matrix needed approximately four times shorter time to reach the adsorption equilibrium with igg in comparison with a gel. the gels, however, provided higher adsorption capacity. at ion-exchange resins, the time necessary to adsorb 99% of total amount of igg was about 0.5-2 h. the affinity adsorbents were highly selective and therefore they adsorb very small amount of tested contaminant proteins (hsa, myo). the adsorption capacity was saturated by 50% in less than 25 min in all cases of dynamic adsorption measurements. this work was supported by a grant of 6th framework program of eu, project aims, no. nmp3-ct-2004-500160. microtechnology has for several years been applied within chemical reaction engineering. the advantages of microtechnology are that it makes it possible to develop light weight and compact systems, and the systems enable large surface-to-volume ratio, which results in low mass-transfer distances. in addition, parameters like pressure, temperature, residence time, and flow rate are more easily controlled. the use of microtechnology is also beginning to find its ways into the field of biotechnology. what we are aiming at is the development of a microreactor that can be applied as a production tool in industry as an alternative to conventional enzymatic reactors. our strategy is to use a small plate of a suitable material with microchannels fabricated into its surface, and the approach is to covalently couple enzymes into the microchannels. substrate can then be pumped through the channels and the enzymatic conversion will take place within the channels. as model enzyme in the development of the microreactor we are applying celb, a thermostable ␤-glycosidase from pyrococcus furiosus. kinetic resolution of racemic benzoin with different lyophilized microorganisms ç . babaarslan 1 ,ü. mehmetoglu 1 , a.s. demir 2 : 1 ankara university, faculty of engineering, department of chemical engineering, 06100, ankara, turkey; 2 middle east technical university, department of chemistry, 06531, ankara, turkey. email: barslan@eng.ankara.edu.tr (ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the 2-hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at 30 • c and 150 rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs 112-07 as ee s = 16% and ee p = 30% (conversion = 35%) using thf as solvent and vinyl acetate as acyl donor. chromatography is one of the most important unit operations at separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale. since these proteins belong to the group of immunoglobulins, their molecular weight (about 150,000 g/mol) or hydrodynamic radius (10.7 nm), respectively, is relatively large. the adsorbents used in ion-exchange/affinity chromatography of these biomolecules should thus provide a high pore accessibility coupled with a high value of specific surface area in order to ensure a sufficient ligand density and a high binding capacity. in this study, the pore accessibility of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros 50a, prosep -va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was investigated via size exclusion of standard-sized molecules (glucose, sucrose and dextrans with molar weight range 1200-40 × 10 6 g/mol) at nonbinding conditions. the experiments were conducted in a batch and column-mode. the batch experiments provided absolute partition coefficients, which were calculated from a mass balance and represent the fraction of pore water accessible to a solute. it was found that several adsorbents contained a small fraction of very small pores (less than 1 nm), whereas some adsorbents contained a significant fraction of pores larger than 100 nm. the column measurements provided relative partition coefficients, which were calculated from the retention volumes of solutes and represented the relative accessibility of pores, scaled between the accessibility of the smallest and largest solute used. when the absolute partition coefficients were recalculated into the relative form, it was found that the coefficients obtained by both methods correlated very well. the relative partition coefficients of solutes with the hydrodynamic radius of 10 nm (corresponding to mabs) was about 0.2-0.4 at ion-exchange and hydrophobic adsorbents and 0.6-0.8 at affinity adsorbents. the relation between hydrodynamic radius of the solutes and their partition coefficients was successfully described with the giddings random plane model. this work was supported by a grant of 6th framework program of eu, project aims, no. nmp3-ct-2004-500160. the transfer of laboratory results to a larger scale is often a critical step in process development to industrial application. the objective of this study was to scale-up the bioreactor for the fructosyltransferase production based on the results obtained in a 3 dm 3 stirred bioreactor. the investigations made in this bioreactor provided a clear picture of the effect of medium composition on the obtained fructosyltransferase (ftase) activity but the influence of mixing intensity was unequivocal. the increase of agitation rate had a positive effect up to a certain level where both fructosyltransferase and biomass production increased. the final optimal yield factor of ftase per dry cell mass obtained in the laboratory bioreactor was 10,300 u g −1 . we studied the effect of oxygen transfer on the process of ftase production at a larger scale, in 12 and 100 dm 3 mechanically stirred bioreactors and in air-lift bioreactors, 20 and 60 dm 3 whilst the medium composition was kept constant. the yield factors of ftase were comparable in both mechanically stirred bioreactors and they were about 7500 u g −1 . this decrease compared to that in the laboratory bioreactor could be explained by a slower cell growth. this fact was also confirmed by that glucose was not depleted till the end of fermentation and free fructose concentration was also lower. the yield factor of ftase was 7400 u g −1 in the 60 dm 3 air-lift reactor and 6100 u g −1 in the 20 dm 3 air-lift bioreactor. the lower yield of ftase in 20 dm 3 bioreactor was caused by a better biomass growth. this work was supported by slovak scientific grand agency, grant numbers vega 1/0066/03 and 1/0065/03 and by science and technology assistance agency, grant number apvt-20-025704. the poster gives an overview of the objectives and achieved results of an interdisciplinary project on direct product isolation from crude feedstocks using magnetic micro adsorbents in combination with suitable magnet technology. the project was funded by the deutsche bundesstiftung umwelt and was running between august 2002 and november 2004. in the course of the project several milestones could be met, which can be looked at as critical key points on a route towards an industrial realization of the process. among these milestones are: (i) the production of magnetic micro adsorbents with high capacity and selectivity in batches up to 50-100 g; (ii) the proof that the micro adsorbents can be reused many times; (iii) generation of a variety of recombinant tagged, active enzymes; and (iv) the design, assembly and operation of a fully automated pilot plant capable of generating approx. 5 g/h (≈95% purity) protein. the process was also simulated by help of the software tool superpro designer and simple mass balance and sorption equilibrium approaches were used to derive rules for estimating optimum process parameters and productivities. finally an environmental performance evaluation was conducted externally by the german dechema. in this study the effect of fed-batch cysteine addition to a culture of a high-gsh-accumulating yeast strain on the metabolism of glutathione was investigated. it is known that cysteine is the rate limiting amino acid in the biosynthesis of gsh. the influence of the consumption rate of cysteine on glutathione metabolism and growth of s. cerevisiae mt-32 was determined. the results show that for rates of consumption below a critical value the microorganism growth is similar to a culture without feed of cysteine, but glutathione production is increased two-fold. on the other hand, if cysteine consumption rate is above the critical value the changes of cell metabolism implies ethanol accumulation in the extracellular media which diminishes biomass synthesis. the maximum specific glutathione production in this case is maintained at two-fold; however, gamma-glutamylcysteine accumulation is increased. cysteine present in culture media directs cell metabolism to a greater synthesis of ammonia and amino acids. hydrophobic interaction chromatography (hic) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. in contrast to reversed phase chromatography this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. when proteins are injected on hic columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. the higher the salt concentration the lower is the amount eluted protein. the rest can be desorbed with a buffer of low salt concentration or water. it has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. this has been also corroborated by physicochemical measurements. the retention data of five different model proteins and 10 different stationary phases were evaluated. partial unfolding of proteins upon adsorption on surfaces of hic-media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. furthermore we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain. stationary phases for bioseparation of glycoproteins j. aniulyte 1 , j. liesiene 1 , b. niemeyer 2 : 1 department of chemical technology, kaunas university of technology (ktu), radvilenu pl. 19, 50254 kaunas, lithuania; 2 institute of thermodynamic, helmut-schmidt-university/university of the federal armed forces hamburg, holstenhofweg 85, hamburg, germany d-22043 nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated.three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of 18 atoms) and carbonyldiimidazole activation.cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg per ml support and a high recovery (up to 93%). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affinity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately 1 × 10 −6 m, and 0.4 × 10 −5 m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. cell disruption and chromatography are key unit operations in the downstream processing of an intracellular product. the cost involved in the extraction and purification of intracellular products can be reduced by selective release of proteins and reduction in the number of steps involved in the purification. the extent of disruption can be varied to provide a selective release, limiting the release of the contaminant proteins. the particle size distribution of the cell debris in the resulting suspension depends on the extent of disruption. expanded bed adsorption chromatography allows for the direct capture of the proteins from an unclarified suspension. this technique allows for the integration of solid-liquid separation, concentration and preliminary purification in one unit operation. a perfectly stable expanded bed can be obtained by choosing the appropriate flow conditions and a suitable adsorbent. the difference in the density between the adsorbent and the cell debris in the suspension, permits the cell debris to flow through the column without blocking, whilst the protein molecules in the suspension are adsorbed onto the adsorbent. after sample application, the bed is washed with buffer and the proteins eluted from the column in the packed bed mode. the presence of the cell debris in the feedstock influences the expansion of the bed and the adsorption of protein molecules. the physical properties of the suspension obtained after cell disruption depends on the extent of disruption. the particle size distribution of the cell debris, the viscosity and the release of soluble proteins and other intracellular components are influenced by the extent of disruption. the influence of the extent of disruption of e. coli on the expansion of the bed and the adsorption of ß-galactosidase is presented in the current study. e.coli cells were disrupted at different operating pressure using a high pressure homogenizer. the resulting crude homogenate is subjected to expanded bed adsorption chromatography using streamline deae as adsorbent. the disrupted suspension was characterised in terms of viscosity, density, particle size distribution of the cell debris and the extent of protein and ß-galactosidase released. the interaction between cell debris and adsorbent was quantified as the cell transmission index (ratio of the amount of cells present in the sample before and after passing through the bed). the expansion of the bed at a constant settled bed height and flow rate was measured. the influence of the cell debris on the extent of adsorption of ß-galactosidase has been quantified in terms of dynamic binding capacity (dbc) at 10% of the inlet concentration. the dbc of ß-galactosidase that was released by disruption at 7500 psi (5%, w/v, w/w, 1 pass) was found to be 100 u/ml of adsorbent while dbc of samples disrupted at 2500 psi (5% w/v, w/w, 1 pass) was 67 u/ml of adsorbent. the extent of disruption of e. coli over a wide range and its effect on the expansion and adsorption will be presented. study of dna binding during expanded bed adsorption and factors affecting adsorbent aggregation ayyoob arpanaei 1 , niels mathiasen 1 , timothy hobley 1 , owen rt thomas 1,2 : 1 center for microbial biotechnology, building 223, biocentrum-dtu, technical university of denmark, 2800, lyngby, denmark; 2 department of chemical engineering, university of birmingham, edgbaston, b15 2tt, uk. e-mail: aa@biocentrum.dtu.dk (a. arpanaei) the adsorption of sonicated calf thymus dna (as a model dna molecule) to biosepra q hyper z adsorbents was evaluated in batch and expanded bed modes. stability of the expanded bed during feedstock loading was also studied. two batches of prototype q hyper z (batch 1 and 2) were examined, which had ionic capacities measured to be 122 and 147 mmol cl − /ml support respectively. in all adsorption experiments a 50 mm tris-hcl ph 8 buffer was used. maximum static binding capacities of adsorbent batches 1 and 2 were determined to be 20.9 and 23.8 mg dna/ml particle, respectively. dynamic binding capacity at 10% breakthrough (dbc 10% ) was measured in a 1-cm diameter eba column containing 6.7±0.5 cm settled bed with a feed of 20 g/ml dna. dbc 10% of the adsorbents were 7.4 and 12.7 mg dna/ml support for batches 1 and 2, respectively in buffer containing no salt. however, the maximum dbc 10% for batch 1 (10.1 mg dna/ml support) and 2 (18.9 mg dna/ml support) were obtained in buffers containing 0.25 and 0.35 m nacl, respectively. further increases in salt concentration led to a decrease in dbc 10% for both adsorbent batches. the bed compression during loading that was observed in experiments at high conductivities (achieved by adding salt) was less than that seen with low conductivity (2 ms/cm) solutions. aggregation of adsorbent particles and channeling of flow were not observed in the presence of salt concentrations more than 0.1 m. the effect of different concentrations of dna during loading in the presence of 0.15 m nacl was studied. it was found that increasing dna concentrations in the feed from 20 to 40 g/ml, 60 to 80 g/ml resulted in a decrease of dbc 10% by 16, 24 and 30%, respectively. the bed compressed slower during loading of feedstock with low dna concentrations compared to that for higher concentrations. the expanded bed showed a partly reversible compression behavior during feedstock loading. this is attributed to the electrostatic interaction between dna adsorbed on the particles surface and rearrangements of dna strands as the number of free ligands on the adsorbent surfaces decrease during loading. large-scale production of plasmid dna for gene therapy and dna vaccination applications has become necessary as a result of the increasing number of approved protocols using non-viral vectors for gene delivery. a major challenge of large-scale plasmid production is to establish a robust cgmp manufacturing capable of producing hundreds of milligrams or grams of a pharmaceutical grade product. alcohol and salt precipitation are operations largely used in the early steps of plasmid downstream processes. however, there are few systematic studies on the influence of these precipitation agents in the final plasmid recovery and purity. in this work, alcohol and salt precipitation steps used in a plasmid purification process developed by our group have been optimized aiming at large-scale production. the optimization of alcohol precipitation indicated that almost 100% of the pdna precipitated when 0.6 vol. of isopropanol were used. the studies also indicated that the precipitation profile was strongly influenced by pdna initial concentration. finally, the final plasmid recovery and purity after a sequential alcohol and salt precipitation were strongly dependent on the concentrations of these precipitation agents. thus, a commitment between high recovery and purity level should be made during the development of the downstream processes. comparison of novel and conventional processes for protein refolding and initial purification h. ferré 1,2 , u. jørgensen 1 , l. scale down of downstream processing unit operations is convenient for assessing process alternatives, particularly if feedstock is scarce. in this study it was imperative to use the smallest possible scale for comparison of a new system for continuous protein refolding and direct expanded bed adsorption (eba) capture with a traditional process composed of discrete operations of batch renaturation, centrifugation, microfiltration and packed bed chromatography (pbc). minimisation of the scale was restricted by the eba step: the smallest practical scale being a 1cm diameter column with 5-6 cm of settled bed, expanded two fold. in order to permit a fair comparison a similar column diameter and adsorbent volume was used in the packed bed process. in both alternatives, chelating media charged with cu 2+ was used and a feedstock of denatured hat-tagged human beta-2 microglobulin (hat-h␤2m). following batch refolding and clarification, the performance of the packed column was severely hampered due to fouling of the top adapter. reducing the protein loaded to the packed bed to 50% of dbc working lead to a recovery of 68.5% at a purity of 87% and 4.6-fold concentration. the eba-based process performed unimpeded and productivity was calculated to be 8% higher than for that employing a packed bed. however, due to the severe scale restrictions placed on the eba process, which limited optimisation, significant productivity improvements of eba over packed bed are expected at larger scale. high gradient magnetic filtration has the potential for rapid processing of large volumes of crude bioprocess liquors when magnetic adsorbents are employed. the binding of a protein to a superparamagnetic solid support provides a unique selective 'handle'. typically the focus is placed on using the magnetic handle for direct capture of a protein from a fermentation broth. however, magnetic adsorbents may provide solutions to a range of downstream processing problems and in this presentation we illustrate this with a number of case studies. using whey as a model system, we show that the extent of the tryptic hydrolysis (ca. 0.2 mg/ml added enzyme) of proteins could be controlled by adding benzamidine-linked magnetic adsorbents after a given period (4-15 min), followed by removal of the loaded adsorbents using a magnetic filter. hydrolysis was stopped effectively and approx. 50% of the added trypsin could be recovered. a coupled process was devised for the refolding and purification of inclusion body proteins. solubilised (in 8 m urea) inclusion bodies of recombinant histidine affinity tagged human beta 2 microglobulin (hat-h-beta2m), were refolded by dilution in a pipe reactor (14 s), then captured directly on cu(ii) charged magnetic immobilised metal affinity adsorbents in a second pipe reactor (10 s residence time). loaded adsorbents were retained in a magnetic filter, then washed and the protein eluted. a generic framework for the prediction of scale-up when using compressible chromatographic packings r. tran 1 , j. joseph 1 , a. sinclair 2 , y. zhou 1 , n. packed bed chromatography is the pre-eminent technique in the downstream purification of many biological products. the aspect ratio of a packed bed has a significant effect on the column pressure drop by virtue of wall support which is reduced at low aspect ratios. this can result in unexpectedly high pressures during manufacturing caused by the compression of the matrix via drag forces due to fluid flow through the bed. the need for an accurate model to predict flow conditions at increasing scale is essential for the scaling-up of chromatographic processes and for avoiding bed compression during operation so that maximum throughput can be achieved. several studies have generated correlations which allow for the prediction of column pressure drops but have either been mathematically complex, which makes their practical use unfeasible, or they have used highly specific empirical constants and hence require a large amount of experiments to be performed before they can be used. in this study, we have established relationships to link the critical velocity of operation, to bed height (l), column diameter (d c ), feed viscosity (µ) and also to the matrix rigidity through the level of agarose cross-linking (a%). the correlation is straight forward to use and involves very few system-specific constants thus significantly reducing the need for any preceding laboratory-scale experimentation. this paper describes the series of experiments that were performed to establish the correlation, using a range of cross-linked agarose matrices (2-10%), at various aspect ratios, fluid flow rates and varying viscosities (0.9-1.85 mpas). a mathematical model was developed where parameter estimation for multi-variables was achieved by least squares optimisation. the model can be used to predict the extent of compression in industrial chromatography applications and will be useful in the development of chromatographic operations and for column sizing. institute of process engineering, swiss federal institute of technology, zurich. e-mail: makart@ipe.mavt.ethz.ch (s. makart) simulated moving bed (smb) technology receives increasing attention in biotechnology and in the biopharmaceutical industry as it enables an increase in productivity per unit mass of stationary phase, reduced solvent consumption, and fast and reliable scale-up. combining continuous chromatographic separation unit and reactor should enable the production of biopharmaceuticals and fine chemicals with high purity and yield at the same time. due to the increasing demand of enantiopure intermediates in the pharmaceutical industry, biocatalytic processes gain more and more importance because of their excellent enantioselectivity. yet the application of biocatalytic carbon-carbon bond formation on process scale is often hampered by an unfavourable equilibrium position and difficult downstream processing due to substrate/product mixtures. coupling a continuous separation unit to such a process would improve the feasibility by driving the reaction to completion and thus increasing the overall yield. we will discuss the design of such an integrated biocatalytic/smb process, taking the formation of l-allo-threonine from glycine and acetaldehyde, catalysed by the glycine-dependent aldolase glya from e. coli, as a model reaction. the enzyme exhibits absolute stereoselctivity at the c-alpha atom, whereas selectivity is less strict at c-beta. in situ product removal, by the integration of an smb unit, would aid to maintain a high diastereomeric excess as it shortens the residence time of the products in the reactor, in addition to shifting the reaction to the product side. the in-line coupling of the chromatographic unit to the enzyme reactor requires the use of the same solvents for reaction and separation, so the choice is limited to aqueous solutions close to physiological ph, limiting in turn the possible stationary phase materials. in a screening of different cation exchangers, amberlite cg-120 ii gave promising results: threonine is more retained than glycine, acetaldehyde is poorly and the cofactor plp is not retained. adsorption isotherms were determined by the retention time method and a smb under process conditions was simulated. by improving the packing of the column, i.e. achieving a more even particle size distribution, we tried to further increase the efficiency of the separation step. the application of enzymes for the synthesis of optically active substances is nowadays of growing importance in the pharmaceutical industry. this requires a proper cultivation of the microorganism as well as a posterior isolation process yielding a constant catalyst quality at high purity. goal of this project is the development of an integrated process for the production and isolation of a lipase from trichosporon beigilie and its posterior application for the enantioselective synthesis of pharmaceutical products. the cultivation of the microorganism is optimised in a laboratory and pilot-scale fermenter in a fed-batch mode. parameters like media composition, temperature, ph and aeration rate are set up. taking advantage of the localisation of the enzyme (covalently attached to the cell membrane) the first step of product isolation consists of a continuous cell disruption. optimal results are achieved with the continuous bead mill disruption process (68% enzyme release with a specific activity of 0.8 u/mg of protein). the non disrupted cells are recycled as inoculums for a new cultivation, increasing the yield of the overall process (by 5-times in the pilot-scale fermenter). in order to isolate the product two different process sequences are considered. the first one consists of an extraction (peg and phosphate buffer) coupled to an ion-exchange chromatography (q-sepharose ff). the second one applies a precipitation step with ammonia sulphate followed by a hydrophobic interaction chromatography (sepharose-hic) provid-ing a lipase yield of 72% (8-times higher than the one provided by combining extraction-chromatography). an ultrafiltration process is used in order to concentrate the lipase and its final properties (molecular weight, isoelectric point, activity, stability and kinetic data) are studied using p-nitrophenylacetate as model substrate. the relevance of the obtained product for its application in the pharmaceutical industry is proven by transforming (r,s)-naproxen-methylester into (s)-naproxen acid with an enantiomeric excess of >99% (after 24 h). biotensides (sugar fatty acid esters, sfaes) find nowadays a wide range of applications in pharmaceutical, personal care and food industry because of their biocompatibility, biodegradability and special surfactant properties. goal of this project is the development and optimisation of an integrated process for the enzymatic synthesis of sfaes from renewable sources to be used in cosmetic formulations. the following figure shows the scheme of the overall process. commercial and also new screened lipases are applied in the reaction between sugar and fatty acid. the mixture grade of the initial reaction system is increased by ultrasounds taking into account the influence on the catalyst characteristics and also the necessity of an organic solvent as adjuvant. the reaction takes place in an enzymatic membrane reactor (emr) equipped with an ultrafiltration membrane which retains the catalyst. the separation of the by-product (water) from the rest of the components can be achieved by means of a pervaporation unit which coupling to the emr allows the semi-batch process. in order to separate the esters from the fatty acid a stepwise elution chromatography method is developed using silica as adsorbent and ethyl acetate and methanol as eluents. with this system 91% of the dimer is isolated with purity (hplc) of 93%. the application of a dialysis membrane technique allows the separation of 80% of the fatty acid by building ester micelles changing the polarity of the organic solvent used as eluent. solubility and crystallisation properties of recombinant bacillus halmapalus ␣-amylase cornelius faber centre for microbial biotechnology, biocentrum dtu, building 223, 2800 lyngby, denmark a comprehensive knowledge of solubility properties is a prerequisite for the efficient design and operation of bulk enzyme recovery processes, however, complete phase diagrams are only available for very few proteins, in particular lysozyme of high purity. here, we present the results of detailed solubility studies in aqueous solutions of an industrially relevant ␣-amylase of technical grade. experiments were conducted in small scale batch mode (working volume of 1 ml). the influence of selected cations and anions from the hofmeister series on the stability of the ␣-amylase was examined. the hofmeister series for anions was followed in the correct order at all salt concentrations studied, i.e. from 0 to 1 m, whereas the series was reversed for monovalent cations at concentrations up to 0.5 m, with the exception of lithium. to further investigate why the position for lithium was different to the hofmeister series established for lysozyme, the zeta potential of protein solutions at low concentrations of selected salts was determined. the results of these measurements indicate a pronounced effect of lithium on the zeta potential, as compared to other salts. in particular, the ph of zero zeta potential (i.e. the pi) was shifted approximately 0.5 ph units towards alkaline conditions in the presence of lithium, whereas the pi stayed almost constant for sodium and potassium. since the solubility exhibits a minimum at ph-values at or near the protein's pi, shifts in ph caused by salt addition are important to identify and quantify to avoid uncontrolled phase separation. the measurement of the zeta potential of proteins in solution holds significant promise as an attractive tool for understanding and controlling processes that are operated close to the solubility limit and which are often plagued by uncontrolled precipitation or crystallisation and thus rely on carefully chosen operating conditions. polyphenolic interactions with potato proteins during industrial expanded bed adsorption processing sissel løkra 1 , knut olav straetkvern 1 , bjørg egelandsdal 2 , gerd vegarud 2 : 1 department of natural science & technology, hedmark university college, n-2317 hamar, norway; 2 norwegian university of life sciences, 1432 as, norway. e-mail: sissel.lokra@lnb.hihm.no (s. løkra) in plant extracts it has been shown that polyphenols have a tendency to react with proteins, either by covalent or non-covalent interactions. these reactions can induce changes in the surface properties of the proteins, and, e.g. cause proteins to be insoluble and precipitate at ph-values below their isoelectric point. potato proteins have a high nutritional quality and show interesting functional properties in food systems. moreover, chlorogenic acid (ca) and caffeic acid constitute about 90% of the total polyphenol content of potato tuber. we have experienced expanded bed adsorption (eba) chromatography to be a method well suited for recovering industrial proteins from potato starch effluent. the process separates proteins from polyphenolic pigments, fiber and minerals. during the adsorption step, patatin, the major potato tuber protein shows complex binding kinetics demonstrated by breakthrough curves. in addition to diffusion limitations in the eba resin, changes in protein structure and surface properties probably are likely to affect this adsorption behavior. reactions between ca and patatin might result in a range of interactions for different species of the same protein. this project therefore aims to assess the interactions between ca, patatin and other major tuber protein fractions and how these changes affect the protein capture in eba. changes in size and charge are screened in 2-d electrophoresis and analyzed further. samples of different protein fractions are taken from breakthrough curves and dynamic binding capacity experiments in model systems with real feedstock. sandwich hybridisation assay for analysis of brewery contaminants s. huhtamella 1 , m. leinonen 1 , t. nieminen 1 , a. breitenstein 2 , p. neubauer 1 : 1 bioprocess engineering laboratory, university of oulu, finland; 2 scanbec gmbh, halle, germany. email: peter.neubauer@oulu.fi (p. neubauer) here we describe the development of a sensitive, cultivationindependent analytical method for the analysis of brewery contaminants which can be performed within three hours in crude sample extracts. the method is based on 16s rrna detection by a paramagnetic bead based sandwich hybridization assay (sha) with two oligonucleotide probes designed to either detect the species or a group of contaminants. the signals were read out either by a fluorimeter (rautio et al., 2003; leskelä et al., 2005) or potentiometrically with an electric biochip instrument (ebiochip systems) . this assay is advantageous over rt-pcr becasue it only detects viable cells and the method can be directly applied to crude cell extracts without prior purification. we describe the principle of designing and evaluating a series of groupspecific lactobacillus probes and the optimisation towards effective cell lysis and high assay sensitivity. the applicability of the sha was evaluated with real brewery samples and the results were compared to routine tests. in all steps of the evaluation the reliability and usability of the method was prioritised. the optimised method combined with a 24 h pre-enrichment period gave reliable results, had a detection limits of about 10 4 -10 5 cells per assay and was easily applicable in a brewery environment. biodesulfurization is one of the possibilities studied by the researchers to attain the maximum sulfur levels imposed for a near future by governments (european directive, 2003) . rhodococcus erythropolis igts8 is a natural and strictly aerobic microorganism able to remove the sulfur atom from dibenzothiophene (dbt) in a selective way (4s route (oldfield et al., 1997)), obtaining 2hydroxibifenyl (hbp) and sulfate. growth is carried out using the experimental procedure performed in previous works dealing with the inoculum built up, media composition and operational conditions (del olmo et al., 2005a (del olmo et al., , 2005b . this work is focused to determine the oxygen uptake rate during the production of the biocatalyst. four experiments were carried out at a biostat b fermentor (braun biotech.) using as only variable the constant stirrer speed used: 150, 250, 400 and 550 rpm. oxygen uptake rate have been determined by means of two methods: dynamic technique at different times during growth for few seconds to ovoid influences and from oxygen profile when the term dealing with oxygen transfer rate is known (predicted by the model proposed in a previous work (garcía-ochoa and gómez, 2004)). our values obtained from the techniques used present the same tendency in all the runs carried out: our values from dynamic technique is always lower than our values obtained from the oxygen profile. these values are modeled and the difference observed is explained due to the cellular economy principle: during the seconds employed in the dynamic technique determinations microorganism do not produce 4s route enzymes. it was studied different methods to recover a p. salmonis antigenic protein from recombinant e. coli cells. this protein has shown be highly effective in vivo vaccine. it has the ability to stimulate salmon immune system protecting them against of aggressive disease salmonid rickettsial syndrome resolving by this way a great problem of salmonid aquaculture. biomass obtained from iptg induced e. coli bl21 (de3) codon plus culture was used for soluble and insoluble antigenic protein recovery. it was evaluated recuperation by glass bead mill, freezing and thawing, osmotic shock and lisozyme/edta treatments, all of them applied in single or combined way. biomass was measured by dry weight of cells, soluble protein concentration was quantified according to bradford method, and antigenic protein was identified by sds-page and western-blot analysis. cells treated with lisozyme/osmotic shock and then glass bead mill the soluble protein was a 62.9% of the dry weight cell mass whereas using lisozyme/edta and glass bead mill as a single treatment only a 24.4 and 22.6% were obtained respectively. the freezing and thawing disruption treatment released less than 5% of soluble protein, as well as the osmotic shock procedure too. the sds-page and west-ernblot analysis revealed that the antigenic protein must be purified from the insoluble cell fraction when physical or mechanical disruption methods were employed and from the soluble cell fraction when chemical or enzymatic treatments were used. we propose investigate in further studies the inclusion bodies formation to design an efficient purification procedure for the target protein. the iso-peroxidase pox2 from garlic bulb allium sativum that represented the major peroxidase activity was purified to homogeneity. the enzyme is monomeric and has a molecular mass of 36 kda, and a pi around 9. the optimum temperature ranged between 25 and 40 • c, while optimum ph was around 5. pox2 appeared remarkably thermostable since it retained 50% of its activity at 50 • c for at least 6 h. in addition, the enzyme was stable at a ph range from 3, 5 to 11. kinetic constants were calculated, the apparent k m values were 500 and 150 m for gaïacol and h 2 o 2 , respectively. the high thermostability of pox2 may represent clear advantages in a number of processes including immobilizing peroxidase and use it as a biosensor to detect oxidant component as h 2 o 2 and other peroxides. immobilization of pox2 was achieved by binding covalently the enzyme to a sepharose matrix (bead and membrane va epoxy). the immobilized peroxidase showed great stability at heat and storage than the soluble enzyme. the native enzyme retained 55% of its activity at 60 • c for 10 mn while the immobilized pox2 retained full activity for 35 mn at the same temperature. in other side, the free enzyme retained full activity for at least one month and a half during storage at 25 • c, and lost 50 m of its activity after 2 months. the immobilized form of pox2 retained complete activity for 2 months at the same temperature. the immobilized enzyme was used to detect h 2 o 2 in some food components such as milk and fruit juices. in a second study, same experiments were performed in order to detect the smaller quantity of added h 2 o 2 to the farming milk. purpose: a new research field has been created to begin to address protein function at level of regulation of enzyme activity. this new area has been given the name chemical proteomics, or activity-based proteomics (abps), and makes use of small molecules that can covalently attach to catalytic residues in an enzyme active site. the selectivity of the chemically reactive group allows specific proteins or protein subset to be tagged, purified and analyzed. methods: this molecule (abps) has three subsets: tag, linker, and warhead. warhead is a nucleophile and attach to active site. linker is a polypeptide that makes a simple connection between warhead and tag. tag is fluorcent or radioactive material that facilitates the detection of drugs. findings: several diseases such as cancer, rheumatoid arthritis and osteoporosis are associated with elevated levels of protease activity. serine hydrolyses abps have been used to profile enzyme activity in a diverse range of cancer cell lines. in studies comparing metastatic and non-metastatic human breast cancer models, it was shown that the former exhibited a higher activity of a ␥-glutathione-s-transferase, an enzyme that has not previously been associated with breast cancer. discussion: additionally, abps can be used to develop robust screens for small molecule inhibitors of a specific enzyme target within a large family of related enzymes. this method of inhibitor screening allows compounds to be assayed for both potency and selectivity against a set of related in complex biological samples. this technique is able to identify novel enzymatic proteins and drugs and has the potential to accelerate the discovery of new drug target. a cyclodextrin glycosyltransferase (cgtase) from a new isolated strain from bacillus clausii e16, was purified through q-sepharose, gel filtration chromatography and deae-sephadex a-50. the mw of the pure enzyme was 75 kda with sds-page. the enzyme displayed optimum ph value and ph stability at ph 6.0 and in range of 6.0-11.0, respectively. the optimum temperature and thermostability were at 55 • c and up to 60 • c by 1 h, respectively. the k m and v max were 2.85 mg/ml and 80.0 mol/min mg and 0.83 mg/ml 13.45 mol/min mg using maltodextrin and soluble starch, respectively. the isoeletric point was 4.8 and the n-terminal region of the pure enzyme was sequencing by maldi-tof-ms. the ratio of ␣-, ␤and ␥-cd was 0.29:1.00:0.79 and 0:1:0 with maltodextrin and soluble starch at 2.5%. application of magnetic separation technology for recovery of immobilised lipases nadja schultz 1 , anke neumann 1 , george metreveli 3 , matthias franzreb 2 , christoph syldatk 1 1 chair of technical biology, university of karlsruhe, engler bunte ring 1, d-76131 karlsruhe; 2 forschungszentrum karlsruhe, institute of technical chemistry, water-and geotechnology; 3 inst. für wasserchemie, engler bunte ring 1,76131 ka. e-mail: nadja.schultz@ciw.uni-karlsruhe.de (n. schultz). url: www.fzk.de/itc-wgt (m. franzreb) first results on the development of the magnetic separation technology for the recovery of immobilised lipase from a 2-phase-system, which should be suitable for a large scale use in future, known as high gradient magnetic separation (hgms) are presented. the application of immobilised lipases makes the reuse of the enzyme in a process possible and is therefore interesting for industrial applications. in this study immobilised lipase is used in a 2-phase-system. here the new approach to recycle and reuse the lipase, immobilised on magnetic particles, from a 2-phase-system with the help of the new high gradient magnetic separator (hgms) is examined in 30 ml scale. as model enzyme for the immobilisation on magnetic microparticles (polyvinyl alcohol (pva), 1-2 m) the commercially available (novonordisc) lipase a (cala) from candida antarctica was used. necessary for screening of immobilisation methods and characterisation of the immobilised lipase (candida antarctica) was the development of a robust, simple and rapid chromophoric activity assay. therefore the pnpp-lipase assay was optimised for direct application on immobilised lipases (in preparation schultz et al., 2005) . further more a ph-stat-assay for measuring the activity of free and immobilised cala in a 2-phase-system of tributyrate and buffer was optimized for this system. another important basis for the realisation of the recovery of immobilised lipases was to optimise the immobilization technique of the lipase. furthermore approaches for the explication of generally empirical based immobilization techniques on insoluble support were made. hereby we successfully applied the zeta potential measurement on the immobilization behaviour of the lipase cala. for to determine the operating temperature for the biomagnetic separation procedure we studied stability analysis of free and immobilised lipase cala at different temperatures (37, 25, 4, −20 • c) and at different ph values (ph 6, 7 and 8). the optimal temperature and ph value for the free and immobilised lipase was determined. presently and constructively on the so far developed methods and techniques we intensively work on the demonstration and feasibility of the recovery of immobilized lipase from a 2-phase-system. challenging approaches and first results on the recovery of immobilized lipase from a 2-phase-system in 30 ml scale were shown already. nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated. three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of 18 atoms) and carbonyldiimidazole activation. cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg per ml support and a high recovery (up to 93%). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affin-ity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately 1 × 10 −6 , and 0.4 × 10 −5 m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. quantitative methods in high throughput screening of aqueous two phase systems matthias bensch, björn selbach, jürgen hubbuch institute of biotechnologie 2, forschungszentrum jülich, germany purification of biopharmaceuticals is one of the most expensive and at the same time least understood steps in bioprocesses. during the process development for protein production, short time to market and the demand for cheap processes dominate today's process development. one way of reducing process costs is to implement integrative processes. aqueous two phase systems (atps) combine the advantages of removing cell debris and simultaneously purifying and concentrating the target protein, however, to the cost of highly complex systems which are difficult to predict and optimize. using high throughput screening techniques in the development of atps processes thus seems to be an ideal candidate for achieving both a reduced development time and an economical process without the need for preliminarily well characterized systems. in this study, we use the robotic system tecan freedom evo tm as an automation platform for the evaluation of aqueous two phase systems. central to this workstation are the integrated hardware as liquid handler, gripper, reader and centrifuge. we have created high throughput methods for rapid parameter estimations. as a first step, pipetting and mixing had to be calibrated for the use of highly viscous polymer solutions which are common in atps. the focus of the current work lies on the integration of the automatic preparation and analysis of two phase systems in microtiter plate scale. the robotic platform can now automatically create aqueous two phase systems and measure characteristic values such as binodal curves, protein concentrations and protein distributions between the two phases. the major bottleneck of hts processes, namely the rapid analysis of impure systems, is tackled by using automated elisa tools. depending on the intended use, the high number of measured partition coefficients and yields can be used for modelling or rapid process design. today, most optimisations of chromatography separations are based on experimental work and rule of thumb. the pat initiative has opened up for a model-based approach for downstream processing of pharmaceutical substances. this work uses a nonlinear chromatography model to optimize an ion exchange separation step. the general rate model with langmuir mpm kinetics described the behaviour of the components in the column. the optimal operating points using both productivity and yield as objective functions were found. the optimizations were run with both igg and bsa as target proteins respectively to compare their optimal operating points. the requirement on the optimal operating point was a purity of at least 99%. this requirement was added to the optimization problem as a nonlinear inequality constraint. flow rate, loading volume, start salt concentration in elution, elution gradient and cut points were used as decision variables in the optimization. the more retained component, bsa, was much easier to separate from igg with a gradient elution than igg from bsa while still retaining a high productivity and yield. the higher load volume at the optimal operating point, with bsa as target protein, causes a displacement of igg and thereby improving the separation. a high productivity at the yield optimum was still possible with bsa as target protein. both a lower productivity and yield was obtained with igg as target protein. optimisation and robustness analysis of a hydrophobic interaction chromatography step niklas jakobsson, marcus degerman, bernt nilsson department of chemical engineering, lund university, p.o. box 124, se-221 00 lund, sweden process development, optimisation and robustness analysis for chromatography separations are often entirely based on experimental work and generic knowledge. the present study proposes a method of gaining process knowledge and assisting in the robustness analysis and optimisation of a hydrophobic interaction chromatography step using a model-based approach. factorial experimental design is common practice in industry today for robustness analysis. the method presented in this study can be used to find the critical parameter variations and serve as a basis for reducing the experimental work. in addition, the calibrated model obtained with this approach is used to find the optimal operating conditions for the chromatography column. the methodology consists of threes consecutive steps. firstly, screening experiments are performed using a factorial design. secondly a kinetic-dispersive model is calibrated using gradient elution and column load experiments. finally the model is used to find optimal operating conditions and a robustness analysis is conducted at the optimal point. the process studied in this work is the separation of polyclonal igg from bsa using hydrophobic interaction chromatography. department of biochemistry and microbiology, ict prague, technicka 3, prague cz-166 28, czech republic the display of novel metal binding sites on the surface of the biosorbent represents potent tool to increase its binding capacity and improve selectivity. in this study, the 15.8 kda transcriptional regulator merr of mercury-inducible mer operon of tn21 exhibiting high affinity and selectivity towards hg 2+ , was displayed on the surface of s. cerevisiae. to achieve this, merr was genetically fused with gene encoding c-terminal domain of ␣-agglutinin which resulted in covalent attachment of the of the fusion protein on the cell wall glucan via glycosylphosphatidylinositol anchor. to evaluate the performance of such modified whole-cell biosorbent with specific regard to hg 2+ , we constructed a new biosensor e. coli strain, which utilizes kanamycine resistance gene as a reporter under the control of mer promoter. it allowed determination of hg 2+ in a range of 5-100 nm by simply monitoring the growth in the hg 2+ /kanamycine-containing media. the effect of genetic engineering of s. cerevisiae surface by merr became significantly pronounced in biosorption experiments with solutions containing 10 m hg 2+ when modified cells accumulated 2.5-fold more hg 2+ than the control strain expressing mere anchoring domain. sensitivity analysis of amino acids in simulated moving bed chromatography ju weon lee, chong ho lee, yoon mo koo center for advanced bioseparation technology, inha university, inchon 402-751, korea. e-mail: ymkoo@inha.ac.kr (y.m. koo) the difficulty of simulated moving bed (smb) design is that the optimization of the operation conditions relies on the determination of accurate adsorption isotherms. most smb chromatograph is carried out under nonlinear conditions, and the nonlinear behavior should be considered properly in the equilibrium isotherms. the other difficulty is the smb operation which has the characteristics of continuous process, all flow rates and switching time of valves should be maintained during the operation of smb. if the disturbances of operating conditions and isotherm parameters are occurred, it affects the zone flow rates and the migration velocity of the solutes, and these effects change the internal profiles of the solutes. therefore, it is the reason of decreasing the purity and the yield of products the objective of this work is to consider the sensitivity of isotherm parameters and operating parameters in smb chromatography process. two amino acids, phenylalanine and tryptophan, separation by smb process is selected as control system. application of ph and po 2 probes during bacillus caldolyticus fermentation: an additional approach in improving a feeding strategy johannes bader 1 , boris neumann 2 , karima schwab 1 , milan popovic 1 , rakesh bajpai 3 : 1 studiengang biotechnologie, fachbereich v, tfh-berlin, seestr., 13347 berlin, germany 2 proteome factory ag, dorotheenstr. 94, 10117 berlin, germany; 3 department of chemical engineering, university of missouri-columbia, w2061 ebe, columbia, mo, usa. e-mail: popovic@tfh-berlin.de (m. popovic) bacillus caldolyticus,a thermophilic microorganism, is a good producer of thermostable liquefying ␣-amylase. during optimisation of initial and feeding media for fed-batch fermentation a two component feeding containing starch and casitone was found advantageous. to approach the optimal feeding rate the method published by akesson et al., 2001 was extended to two component feeding. the key idea, discussed in this presentation, was using the po 2 and ph probing signals to determine if the feeding of one or the other component should be increased or decreased. each of the probes offers information of different areas of feeding condition. to prevent excessive feeding of starch the ph probe is preferable. in case of excessive casitone feeding the po 2 probe responds in very authentic way enabling together with the ph signal reliable and reproducible evaluation of feeding strategy. however a congruent response of po 2 and ph probes means the approaching of the optimum feeding rate for both components. akesson, m., hagander, p., axelsson, j.p., 2001. probing control of fed-batch cultivations: analysis and tuning. contr. eng. pract. 9, 709-723. antibody immobilization by using the plasma polymerized acrylic acid r. jafari, m.tatoulian, f. arefi-khonsari laboratoire de génie des procédés plasmas et traitement de surface, enscp, upmc, 11 rue pierre et marie curie, 75005 paris, france the objective of this work is therefore to produce a surface containing a high density of cooh functions on the polymer beads (ps) for the covalent immobilization of antibodies. we have investigated the plasma polymerization of acrylic acid in a fluidized bed reactor the polystyrene (ps) beads. for such application, there is a strong need to obtain stable plasma polymerized acrylic acid (ppaa) coating, resistant to washing with water. different physico-chemical analyses have been used (water contact angle measurements (wca), xps and sem analysis) to characterize the ppaa coating deposited on ps beads under different experimental conditions. the xps results showed that the pretreatment of surface of the beads before deposition of acrylic acid plays an important role on the stability of ppaa layer. the instability of the coating is partially due the fact that under certain conditions the coatings are soluble in water and secondly due to the bad adhesion of the polymer beads which are hydrophobic to the growing ppaa coatings. xps as well as tof-sims gives evidence of the immobilization of the antibody. xps results as well as static sims allows to detect nitrogen on the surface of the treated beads which proves the presence of the immobilized antibodies. under optimum condition the ppaa coatings provides the possibility to show a nitrogen uptake which varies between 6.5 and 9% of the apparent stoicheiometry of the surface. holst department of medical physiology, the panum institute, university of copenhagen, dk-2200 copenhagen, denmark glp-1 (glucagon-like peptide-1), a peptide of 30 amino acids secreted by endocrine cells in the gut in response to meal ingestion, was discovered during a systematic search for gut factors capable of enhancing insulin secretion. it turned out to be the most efficacious insulin releaser known, and unlike other factors, was shown to retain its insulinotropic activity also in patients with type 2 diabetes. subsequent research has documented that the peptide not only releases insulin from the beta cells, but also enhance all steps of insulin biosynthesis, up-regulates beta cell gene transcription, and has trophic effects on the beta cells. the latter includes both proliferation of existing cells, neogenesis from ductal precursor cells, and inhibition of apoptosis. the peptide also inhibits glucagon secretion, reduces gastric emptying and reduces appetite and food intake. because of these actions, glp-1 administered to patients with type 2 diabetes dramatically lowers blood glucose as well as glycated hemoglobin levels, and reduces body weight. however, natural glp-1 is extremely rapidly metabolized in the body, and the problem has been how to convert the unstable peptide into a clinically useful agent. the two main problems are its susceptibility to enzymatic degradation by ubiquitous dipeptidylpeptide peptidase iv (dpp-iv) and its rapid renal elimination. a related peptide, isolated from the saliva of a lizard, exendin-4, was found to be a full agonist of the glp-1 receptor, to be resistant to dpp-iv and to be cleared more slowly by the kidneys. this peptide was highly effective in clinical studies and has now (30/4) been approved for diabetes treatment by the fda. other approaches include acylation of glp-1 whereby it attaches to albumin in the body and acquires resistance to dpp-iv as well as a slow renal elimination. also this analogue (liraglutide) has favourable clinical effects. fusion proteins of glp-1 and larger, slowly eliminated proteins in the body are currently being evaluated. small molecule, orally available inhibitors of dpp-iv have been demonstrated to protect endogenous glp-1 from degradation and to be efficacious in both experimental and clinical diabetes, and numerous inhibitors are currently in clinical development. the incretin hormones are released from gut endocrine cells upon meal ingestion. they enhance glucose-induced insulin secretion and nay be responsible for up to 70% of postprandial insulin secretion. the incretin hormones are glucagon-like peptide-1 (glp-1) and glucosedependent insulinotropic polypeptide (gip). in patients with type 2 diabetes (2dm) the incretin effect is severely reduced or absent. in 2dm patients the secretion of gip is normal, but its effect on insulin secretion is almost completely lost. glp-1 secretion, on the other hand, may be impaired, but its insulinotropic actions are preserved and it may restore insulin secretion to near normal levels. substitution therapy with glp-1 might therefore be possible. glp-1 is a product of the glucagon gene and its actions include: (1) potentiation of glucose-induced insulin secretion; (2) stimulation of the expression of ␤-cell genes essential for insulin secretion, including the insulin gene; (3) stimulation of ␤-cell proliferation and neogenesis (by enhancing endocrine differentiation of duct cells) and inhibition of ␤-cell apoptosis; (4) inhibition of glucagon secretion; (5) inhibition of gastrointestinal secretion and motility, notably gastric emptying; and (6) inhibition of appetite and food intake. these actions make glp-1 particularly attractive as a therapeutic agent for 2dm. thus, continuous subcutaneous administration of glp-1 for 6 weeks resulted in a 5 mmol/l reduction in mean plasma glucose and a reduction in hgba1c of 1.3%; a weight loss of 2 kg; improved insulin sensitivity; improved ␤-cell function; and the treatment was associated with no significant side effects. unfortunately, glp-1 is rapidly destroyed in the body by the ubiquitous enzyme, dipeptidylpeptidase iv (dpp-iv). clinical strategies therefore include: (1) the development of metabolically stable analogues of glp-1 viz. activators of the glp-1 receptor; and (2) inhibition of dpp-iv. orally active dpp-iv inhibitors have proven successful in experimental diabetes and several companies are now trying to develop clinically suitable inhibitors. so far the clinical experience is limited, but recent clinical studies have provided proof of concept. metabolically stable analogues/activators include the structurally related lizard peptide, exendin-4 or analogues thereof, as well as glp-1 derived molecules that bind to albumin and thereby assume the pharmacokinetics of albumin. these molecules are effective in animal experimental models of type 2 diabetes, and have been employed in clinical studies of up to 52 weeks' duration. on the basis of these studies it can be concluded that a therapy of type 2 diabetes mellitus based on stimulation of glp-1 receptors is likely to be effective and to become a clinical reality within the not too distant future(1-4). recombinant activated coagulation factor vii (rfviia) was developed to treat bleedings in hemophilia patients, who have developed inhibitors against fviii or fix, and has been demonstrated to have an efficacy rate of 80-90% in major surgery as well as in serious bleedings in such patients. to use rfviia as a hemostatic agent in severe hemophilia is a new concept of treatment, not being a substitution therapy, but using a pharmacological dose of exogeneous rfviia to compensate for the lack of fviii or fix. the administration of extra rfviia has been found not only to bind to tissue factor (tf), but also to the negatively charged phospholipids surface of thrombin activated platelets. hemostasis occurs on surfaces being initiated on the tf-expressing cells as a result of exposure of tf, not normally exposed to the circulating blood, following an injury to the vessel wall. tf is a true receptor protein with an intramembraneous part and an intracellular tail. its ligand is fvii/fviia. as soon as tf is being exposed to the blood, it forms complexes with fviia already present in the circulation. these complexes activates fx and provide the initial limited amount of thrombin molecules activating the co-factors, fviii and fv, as well as fxi and platelets. following the thrombin activation of platelets, negatively charged phospholipids are being exposed on the outer surface of the platelets. on this surface most coagulation proteins bind tightly, facilitating the conversion of fx into fxa and the full thrombin burst, necessary for the formation of a tight fibrin hemostatic plug resistant against premature lysis. in hemophilia patients the initiation of hemostasis is essentially normal, but, since they lack fviii or fix, they do not form the fviiia-fixa complex necessary for full thrombin generation on the activated platelet surface. as a consequence the fibrin plug formed in hemophilia is loose, fagile and easily dissolved resulting in continuous bleeding. pharmacological doses of rfviia have been demonstrated to mediate direct binding of rfviia to the negatively charged thrombin activated platelet surface, thereby generating thrombin formation in the absence of fviii/fix. through this mechanism hemostasis is generated in hemophilia patients independent of fviii/fix. furthermore, by generating more thrombin at an increased rate the formation of stable, tight fibrin hemostatic plugs are facilitated. such fibrin plugs are more resistant against premature lysis and help not only to initiate but also to maintain hemostasis. based on its capacity of enhancing thrombin generation locally on the activated platelet surface, rfviia has been used to ensure hemostasis also in other situations than hemophilia, such as platelet defects including thrombocytopenia. recently, rfviia was shown to be hemostatically effective in patients with profuse bleedings as a result of vast trauma and tissue damage. in these patients with a complex hemostasis pattern including a host of changes leading to an impaired hemostatic function, extra rfviia seems to help generate a burst of thrombin resulting in the formation of a stable hemostatic plug more resistant against the ongoing lysis. in patients with intracerebral haemorrhage, one single dose of rfviia recently was found to limit the expansion of the haemorrhage and thereby leading to improved functional outcome. institute for medical microbiology and immunology, panum 18.3.12, blegdamsvej 3, dk-2200 copenhagen n, denmark. email: s.buus@immi.ku.dk complete genomes from several species including many pathogenic microorganisms are rapidly becoming available along with the corresponding "proteomes". even at the peptide level, the diversity of proteome is enormous and easily represents a unique imprint of the originating organism. it is perhaps not surprising that the immune system considers peptides as key targets. recent immunological advances have shown that mhc molecules act as peptide selectors for immune recognition. we have proposed to generate accurate predictions of peptide binding to mhc and used these to identify immunogenic epitopes directly from genomic data. we have developed an iterative data-driven immunobioinformatics approach where data is used to generate predictors, and predictors are used to select new and complementary data for the next iteration. we have demonstrated the superior performance of this approach compared to a random data selection approach. we have also developed an efficient approach to select the most informative mhc molecules to investigate. the resulting, immunobioinformatics resource represents an immediate and powerful application and interpretation of genomic data, and will enable a rational approach to immunotherapy in the future. allergen specific immunotherapy is a causal treatment for igemediated allergic diseases such as hay-fever, and it has relied traditionally on preparations derived from aqueous extracts of various natural allergenic source materials. the cloning and production of an increasing number of allergens through the use of dna technology has not only facilitated the characterisation and analysis of the allergenic proteins, but also provided the opportunity to use these recombinant proteins instead of natural allergen extracts for the diagnosis and therapy of allergic disease. detailed physicochemical, biochemical and immunological characterisation are essential for the comparison of natural and recombinant proteins, and also provide a basis for developing derivatives. chemically modified allergens with attenuated ige-reactivity are currently used for immunotherapy in order to enable high doses to be achieved with a minimized risk of inducing allergic side reactions. dna technology provides the opportunity to develop and produce hypoallergenic allergen variants using strategies including gene mutation. the design of such variants must ensure that t cell reactivity and immunogenic activity are retained in order to preserve therapeutic potential. the recombinant allergens and their derivatives have several advantages over natural allergen extracts. they are relatively easy to produce in consistent pharmaceutical quality; the problems of natural extract standardisation can be avoided completely; the relative concentrations of the individual allergens can be controlled to obtain optimal dosages; nonallergenic proteins are excluded; the possible risks of contamination are avoided. the first clinical trials with grass pollen allergens and birch pollen hypoallergenic variants have yielded very encouraging results. the use of recombinant polyclonal antibodies (pabs) may improve the treatment of disease caused by complex targets such as infectious agents, when compared to monoclonal antibody therapy. symphogen has developed a method for reproducible production of target-specific fully human pab compositions, so-called symphobodies. the antibody genes are first isolated from donors with an immune response against the target and antibodies are screened for specificity. subsequently, the pabs are expressed in mammalian cells using the sympress technology, which is based on site-specific integration. this procedure ensures that each of the expression constructs encoding the antibody genes are stably integrated at the same site in each of the host cells, thereby eliminating genomic position effects and differential growth and production rates. further, the sympress technology comprises the generation of a polyclonal working cell bank (pwcb) which is used as inoculation material for the manufacturing. these cells display sufficient genetic stability to enable a controlled gmp production of recombinant polyclonal antibodies. symphogen's first product, sym001, is a recombinant human polyclonal rhesus d-specific symphobody preparation consisting of 25 different anti-rhesus d antibodies. this product is intended to be used for treatment of idiopathic thrombocytopenia purpura and prevention of hemolytic disease in newborns. recombinant anti-rhesus d symphobodies were produced and shown to be biologically active against rhesus d. the expression technology provided a compositional reproducibility between batches which is sufficient for manufacturing of such a polyclonal product for clinical use. scaledup production for clinical trials is currently ongoing. stem cells play an important role in renewing tissues such as skin and cornea. they are responsible for the continuous generation of the differentiated epithelium. we have characterized stem cells of the skin and cornea in situ and their fate in vitro in human skin reconstructed by tissue engineering using keratin (k)19. in the outer root sheath of the hair follicle, stem cells (label-retaining cells) present in the basal layer of the bulge area express k19 and present a loosely arranged keratin filament network and low levels of k14 protein in their cytoplasm. in addition, another stem cell population (also labelretaining) is present in the first suprabasal layers. these cells exhibit a very dense keratin network and express k17. three-dimensional tissue constructs (dermis and epidermis) obtained by the self-assembly approach of tissue engineering allow the preservation of k19 positive stem cells in the basal layer of the epithelium. in the eye, the stem cells are located in the limbal part but not in central cornea and they express k19. the epithelium of reconstructed cornea is thinner compared to reconstructed skin and more transparent. the characterization of stem cells in reconstructed tissue is essential to evaluate the long-term survival of these tissues in vitro but also after grafting. these human reconstructed tissues are developed for fundamental (physiological, toxicological studies) and clinical applications such as transplantation for the permanent replacement of damaged organs. lg is holder of the canadian research chair (cihr) on stem cells and tissue engineering. alessandra gliozzi physical department, university of genoa, 16146 genoa, italy hollow nanometer-sized capsules can be prepared by means of different techniques. first "nanocapsules" were liposomes, however they are too unstable for many medical or pharmaceutical applications. in contrast, recently developed polyelectrolyte capsules prepared by means of the layer-by-layer technique are much more stable and seem to be a very promising way for coating living cells or tissues in order to prevent or reduce their immune rejection after implantation. several observations on single living cells encapsulated by the alternative adsorption of oppositely charged polyelectrolytes will be presented. the most relevant result is that cell preserve their metabolic activity, are still capable of dividing and performing specific functions. moreover, a technique to immobilize in defined arrays coated cells expressing green fluorescent protein by using a microcontact printing of polyelectrolytes will be presented. finally, tests performed to study the induction of fibrosis and vascularization by nanocapsules implanted in rat kidney and liver will be presented. over the past decade we have developed methods to generate spontaneously and synchronously beating tissue equivalents from neonatal rat heart cells in the culture dish. these tissue equivalents display the key morphological and functional features of intact myocardium and have been termed engineered heart tissue (eht). to generate ehts, heart cells are mixed with freshly neutralized, liquid collagen i, matrigel and growth supplements and grown in a circular casting mold around a central cylinder, which subjects the cells to a continuous mechanical load. this process is enforced by cyclic mechanical stretch. we use eht mainly for two purposes, as a test bed for the effects of pharmacological or genetic manipulations and for cardiac repair. as a cell culture model, ehts compare with standard 2d monolayer cultures of neonatal rat cardiac myocytes and freshly isolated adult cardiac myocytes. advantages of ehts are their functional similarities with intact heart muscles, the ability to easily measure force of contraction under mechanical load, the pos-sibility to transfect cardiac myocytes inside ehts with adenovirus at high efficiency and the reproducibility in large series. a disadvantage is that contractile function as measured at the end of the culture period also integrates influences on tissue development, cell-cellconnections, extracellular matrix production and on non-myocytes. at present we are working on downscaling the eht method to a 96well format for screening purposes. to use ehts for cardiac repair we created multi-looped ehts from five circular ehts large enough to cover the infarct scar 14 days after coronary artery ligation in rats. ehts survived and formed a layer of muscle tissue on top of the infarct scar. ehts restored undelayed anterograde impulse propagation over the scar, prevented further ventricular dilatation, normalized enddiastolic pressure and relaxation, and partly restored contraction of the scar. thus, the study provides evidence that implanting ehts onto infarcted hearts can improve cardiac contractile function after myocardial infarction. the goal of tissue engineering is the development of skin, bones and even organs to restore, maintain and improve tissue function within the body. the current paper focuses on the investigation of invitro growth of osteoblast cells in different types of scaffolds. three of the scaffolds were made of pcl (polycaprolactone) 10% glycerol and 10% hca(hydroxylapetite). two of the scaffolds were made by compression molding, and one was made by fused deposition modeling utilizing the stratasys. the fourth cerabio was a commercially available product totally ceramic. the pores in compression molding were obtained by putting in 75% volume of sugar either 150 and 595 m which was later removed by leaching. the fused deposition scaffold was made by placing the filaments in a predetermined arrangement. the stratasys system was a computer designed model. the scaffolds were seeded with hfob 1.19 human fetal osteoblast cell line with vigorous shaking overnight and incubating at 37 • c and 5% co 2 . observation of the seeded scaffolds was made after 3 days and 9 days of incubation. the seeded cells were stained with bcip/nbp at 37 • c overnight. the cell proliferation in the 595 and 150 m scaffolds appeared approximately the same with a possible advantage of 595 m. the cerabio sample demonstrated the greatest proliferation among the four scaffolds studied and the stratasys sample exhibited a different type of cell adhesion with the cells were clustered in the interstices of the structure. denise freimark, ruth freitag, valérie jérôme chair of process biotechnology, university of bayreuth, d-95448 bayreuth, germany. e-mail: denise.freimark@uni-bayreuth. de (d. freimark) tissue engineering is emerging as an alternative to bone grafts for the regeneration of defects that do not heal spontaneously. ultimately, the development of an optimum carrier and the identification of ideal inductive factors and cells may enable tissue engineering to provide an improvement over bone grafts in the future. bone formation and repair require a complex cascade involving growth factors, cytokines and angiogenesis. at present the complexity of the molecular mechanisms that control gene expression in bone forming cells in embryo as well as in adult is not fully understood. several factors like bone morphogenetic proteins (bmps), transforming growth factor beta (tgf-␤), vascular endothelial growth factor (vegf) and insuline-like growth factor (igf) have been identified and their ability to stimulate bone formation in vitro and in vivo has been investigated. while much is known about these factors per se, less is known about genetic regulation of artificially stimulated osteogenesis. interestingly, some in vitro investigations showed that only optimal growth factors concentrations lead to effective bone formation whereas higher concentrations had deleterious effects which suggest some variation in the activated regulation pathways. therefore, one of our goals is to analyze kinetics, dose-dependence and synergistic effects of growth factors and cytokines on regulation pathways of bone formation. moreover, the optimal vascularization of the scaffold is a major hurdle in the development of engineered bone. it is well known that: (i) vascular invasion precedes bone growth and (ii) osteogenesis takes place in the vicinity of newly formed vessels. thus, inadequate bone vascularization is associated with decreased bone formation. further analysis of the intercommunication between endothelial cells and osteoprogenitors in co-culture systems could provide key information that could be thereafter used to solve this problem. we propose to add some new knowledge to this complicated puzzle. a first step in our investigation is the production of some of the growth factors mentioned above in recombinant form. these factors are expressed in a novel vector (ptriex tm ; novagen) which allows recombinant protein production in prokaryotic or in eukaryotic systems with a single plasmid. afterwards, we analyze potential synergistic effects of these factors as well as kinetic and dose-depend parameters on the genetic regulation of downstream pathways in osteoblasts culture. in parallel, we develop an in vitro system allowing us to investigate the intercommunication between endothelial cells and osteoprogenitors. there has been significant interest in the therapeutic and scientific potential of human embryonic stem (es) cells since they were first isolated in 1998. if human es cells could be differentiated into suitable cell types, stem cells might be used in cell replacement therapies for degenerative diseases such as type i diabetes and parkinson's disease, or to repopulate the heart following myocardial damage. however, there is a significant shortage of high quality human es cell lines and few research groups have experience in the propagation and manipulation of these cells. we are addressing this important issue using the combined expertise of the stem cell biology laboratory and the assisted conception unit at king's college, london. with local ethical approval and under licence from the uk human fertilisation and embryology authority, we have been establishing high quality human es cell lines from a novel source of human embryos. to date, we have derived three human es cell lines and are now focused on the generation of therapeutically important cell populations, including cells that may have clinical application in degenerative and traumatic injury to the brain and spinal cord, heart, retina and other target organs. wallenberg neuroscience center, department of physiological sciences, lund university, bmc a11, s-221 84 lund, sweden cell replacement therapy for parkinson's disease is based on the idea that implanted dopamine neurons may be able to substitute for the lost nigrostriatal neurons. in rodent and primate models of parkinson's disease it has been shown that transplanted dopamine neuroblasts can re-establish a functional innervation and restore dopaminergic neurotransmission in the area of the striatum reached by the outgrowing axons; that the grafted neurons are spontaneously active and release dopamine in an impulse-dependent manner, at both synaptic and non-synaptic sites; and that they can reverse or ameliorate some of the parkinson-like motor impairments induced by damage to the nigrostriatal system. clinical trials in patients with advanced parkinson's disease have shown that dopamine neuroblasts obtained from fetal human mesencephalic tissue can survive and function also in the brains of pd patients, restore striatal dopamine release, and ameliorate impairments in motor behavior. the principal limitation of this approach is the problems associated with the use of tissue derived from aborted human fetuses, and the large numbers of donors needed to obtain good therapeutic effects. until now, transplantation of dopamine neurons has focused primarily on differentiated neuroblasts and young postmitotic neurons, at the stage of neuronal development that is optimal for survival and growth of the grafted cells. however, progenitors taken at earlier stages of development might prove more effective. efforts are now made to expand multipotent neural stem-or progenitor cells in vitro, and control their phenotypic differentiation into a dopaminergic neuronal fate. initial results suggest that in vitro expanded cells can survive and function after transplantation to the striatum in the rat pd model, but the overall yield of surviving dopamine neurons has been very low. with further development, expanded progenitors or dopamine neuron precursors, possibly in combination with cell engineering techniques, may offer new sources of cells for replacement therapy in pd. stem cell therapy has been very much in vogue for several years now. like gene therapy before it, it has raised unrealistic hopes of cures being available imminently. unlike gene therapy, it can cite proof of principle in the well established practice of bone marrow transplantation which is actually a good example of stem cell therapy. however, most of the uses that are now touted as targets for stem cell therapy, envisage the conversion of the stem cells into lineage restricted progenitor cells or more commonly, the final differentiated cell type. such conversions are extremely difficult to initiate and control. the procedures involve the manipulation, differentiation, and expansion of cell cultures in the laboratory, with unknown long term effects on the genetics and physiology of the cells. these issues are compounded when one considers as source material, human embryonic stem cells, where not only the final cell product requires significant scrutiny, but also there are safety issues surrounding the persistence of undifferentiated cells. on top of all these challenges are commercial (for stem cell companies), clinical, and regulatory pressures which will impact heavily on the pace of progress. nonetheless, despite all these hurdles, various academic groups and companies are making significant progress and examples of such developments in diabetes and cardiovascular repair will be given stem cells have the unique ability to perpetuate themselves while continually replenishing tissues throughout the life of an organism. the era of cellular and tissue regeneration for the treatment of disease and the effects of aging has indeed begun. it has been known that mechanical factors play an important role in the regulation of cell physiology. it is therefore reasonable to believe that mechanical factors also play a significant role in the metabolic activity and differentiation of mscs. in this study, we investigated the viscoelasticity of individual bone marrow-derived adult human mesenchymal stem cells (hmscs), and the role of specific cytoskeletal component -f-actin microfilaments on the mechanical properties of individual hmscs. the mechanical properties of hmscs were determined using the micropipette aspiration technique coupled with a viscoelastic solid model of the cell. for the hmscs under control conditions the instantaneous young"s modulus e 0 was found to be 886 ± 289 (pa), the equilibrium young"s modulus e ∞ 372 ± 125 (pa), and the apparent viscosity 2714 ± 1626 (pas). after exposed to 2 m of chemical agent-cytochalasin d that disrupt the f-actin microfilaments, the young's moduli of hmscs decreased by up to 72% and the apparent viscosity increased by 167%. these findings suggest that microfilaments are crucial in providing the viscoelastic properties of the hmscs, and changes in the structure and properties of them may influence the mechanical properties of hmscs significantly. pharmacologic transcription control of desired transgenes is essential for gene-function analysis, drug discovery, biopharmaceutical manufacturing, design of complex artificial regulatory networks, precise and timely reprogramming of key cell characteristics for gene therapy and engineering of preferred cell phenotypes for tissue engineering. capitalizing on our recent advances in the design of small molecule-responsive transcription control modalities we have used conditional molecular interventions for (i) improvement of specific productivity in biopharmaceutical manufacturing, (ii) transdifferentiation of therapeutically relevant cell phenotypes and (iii) design of artificial microtissues. we will also report on a completely new dimension of transgene control as well as engineering of hysteretic and epigenetic gene networks in mammalian cells. chronic diseases are a growing burden for the individual and society alike. one key factor driving this increase is an ageing population. currently, there are 600 million persons aged 60 years or over and this number is predicted to triple by the middle of the 21st century. effective prevention of irreversible damage to major organ systems requires early diagnosis and treatment yielding significant quality of life to the individual and sparing valuable health care resources. even when safe and effective medicines are available, a remaining problem to successful therapy are issues of patient compliance. historically, vaccines have been one of the major advances towards the longevity we enjoy today, with compliance rates close to 100%. hence, vaccines for early treatment of chronic diseases are ideally positioned for long-term therapy, and will take away the burden of self-medication associated with orally active drugs. here we will discuss a new generation of therapeutic vaccines based on virus-like particles (vlps). by displaying target molecules in a highly repetitive manner on vlps, it is possible to break b cell unresponsiveness in experimental animals as well as in humans. using such vaccines in animals, chronic diseases such as hypertension, alzheimer's disease, obesity and rheumatoid arthritis could be treated. furthermore, vaccination against nicotine resulted in high nictotine-specific antibody titers in humans, greatly facilitating smoking cessation in immunized individuals. interdependence of the impact of methanol and oxygen supply on protein production with recombinant pichia pastoris n.k. khatri, f. hoffmann martin-luther-university halle-wittenberg, institute for biotechnology, halle d-06120, germany. e-mail: f.hoffmann@biochemtech.uni-halle.de (f. hoofmann) the methylotrophic yeast pichia pastoris is a potent expression system for secretion of recombinant proteins. methanol as inductor of the foreign gene expression is also a substrate with high oxygen demand, which can lead to sudden oxygen depletion upon induction. thus, supply rates of methanol and oxygen are major process parameter during protein production with recombinant pichia pastoris. limiting dosage of methanol allowed maintenance of oxygen sufficient conditions during production of a single chain antibody fragment, but the product was degraded from the c-terminal end. in contrast, full-length product accumulated with controlled methanol concentrations despite oxygen limitation. the volumetric methanol uptake rate are limited by the oxygen transfer capacity of the reactor. higher methanol concentrations decreased the biomass yield and thereby increased the specific methanol uptake rate. this enabled prolonged production and yielded fivefold higher product concentrations. at the same time, the accumulation of small molecular weight contaminants was reduced. dosage of pure oxygen accelerated methanol uptake, grow and production. switching to dostat mode upon oxygen depletion led to an arrest of product accumulation, in contrast to persistent methanol feeding. the productivity was tenfold higher than without oxygen. combined with high methanol concentrations, however, fast methanol uptake led to toxication of the cells and early stop of production. flow cytometry revealed that perturbation of oxygen metabolism was followed by partial lysis of the culture. recombinant production of therapeutic proteins poses severe challenges due to their complexity (cystines, subunits, size). formation of the correct disulphide bridges is a prerequisite for activity but difficult to achieve in a prokaryotic host. there proteins mainly fold post-translationally as opposed to eukaryotic co-translational folding. thus the recombinant products are often not soluble and/or active. that is why different production parameters (e.g. host, induction conditions, temperature, compartment, proteinaceous fusion partners, co-expression of chaperones, foldases) are applied in order to gain functional recombinant proteins. the impact of all these strategies cannot be predicted and every problem of the production (expression, solubility, activity) might need to be solved for every target protein separately. nevertheless, much effort has been put into this for almost 3 decades, because they are important targets for the pharmaceutical industry. human growth factors influencing cellular proliferation and/or differentiation are one example. this case study gives an overview of strategies tested within the development of two processes leading to an optimised yield of active protein. murine wnt-1 (wnt family) possesses 23 conserved cysteines (most likely all involved in disulphide bridge formation and one in posttranslational modification) and could only be successfully produced in e. coli (fahnert, 2004) recently despite various attempts for many years. the other target protein is human collagen prolyl-4-hydroxylase being a heterotetramer consisting of two ␣-subunits and ␤-subunits each. the ␣-subunit strongly aggregates if produced separately whereas the ␤-subunit is pdi and is suggested to have a chaperone function. therefore a sequential induction strategy was proposed for this protein . the moss physcomitrella patens has been recently recognized as an ideal producer of recombinant proteins with respect to glycosylation. due to the elaborated post-translational capabilities of moss cells, the glycosylation patterns have been manipulated to obtain similar proteins to those found in animal cells. the protein expression using moss in suspension offers important advantages in comparison to other systems e.g. cho cells. the recombinant proteins can be targeted into the mineral medium, simplifying the down stream processing. moreover, there are neither known moss viruses nor plant viruses that are pathogenic for humans. the moss are cultivated axenically in a filamentous stage, the so called protonema. a pilot 30 l tubular photoreactor is used to characterize the response of p. patens to variations on the culture conditions. the phototrophic culture in bioreactors is systematically investigated, where light quantity and quality, stress, concentration of phytohormones, and moss morphology influence the differentiation, growth, and protein expression. a tight control of the moss morphology in suspension, quantified by image analysis, has shown to be advantageous in order to delay the cell differentiation and maintain the carbon dioxide uptake in long bioreactor runs. the introduced perfused culture system by means of cross flow filtration allowed for a continuous product separation and concentration, and feed back of the productive cells. the characterization of this highly controlled culture system is presented and the potential of p. patens as an alternative tool for molecular farming is discussed. (rnai) is an evolutionarily conserved, endogenous mechanism for sequence-specific gene silencing that uses small double-stranded rnas (called short interfering rnas or sirnas) to direct cleavage or prevent translation of homologous mrnas. harnessing rnai for therapy presents an opportunity for potentially treating a wide variety of diseases. the main obstacle is delivering sirnas into the cytosol of target cells in vivo. although we were able to protect mice from autoimmune hepatitis by hydrodynamic tail vein injection of sirnas targeting fas, this delivery method is unlikely to be adaptable for human use. alternate strategies to deliver sirnas in vivo as small molecule drugs using currently available, clinically acceptable injection methods that have shown promise in mouse models will be discussed. these include local delivery to mucosal surfaces and delivery into specific cells via cell surface receptors using an antibody fragment fused to protamine. these sirna complexes silence gene expression in vivo only in cells bearing the targeted receptor. experiments showing efficient, effective and cell-specific delivery in a mouse tumor model will be discussed. in addition, encouraging preliminary data using rnai for a microbicide to prevent sexually transmitted infection will be presented. rna interference (rnai) holds significant progress as a therapeutic approach to siolence disease-causing genes, particularly those that encode "non-druggable" targets. the key hurdle for rnai therapeutics is in vivo delivery. a critical requirement for achieving systemic rnai in vivo is the introduction of "drug-like" properties, such as stability, cellular delivery and tissue biodistribution, into synthetic sirnas. our progress in achieving in vivo silencing of endogenous genes with chemically modified sirnas will be discussed. hiv-1 replication in human t cells can be inhibited by stable expression of a short hairpin rna targeting the viral nef gene (shrna-nef). however, hiv-1 escape variants emerge after prolonged culturing, and all but one escape mutant acquire a mutation in the shrna-nef target sequence. we observed single and multiple nucleotide substitutions, but also partial or complete deletion of the target sequence. these results demonstrate the sequencespecificity of this antiviral approach. we observed an inverse correlation between the level of resistance and the stability of the shrna/target-rna duplex for most of the escape mutants. however, two escape variants did not follow this pattern, including an escape mutant with a single point mutation at position −7 upstream of the target sequence. these mutants provide a much higher level of resistance than expected based on duplex stability, which is obviously not affected in the −7 mutant. we demonstrate that these mutants adopt an alternative rna secondary structure that occludes the target sequence. this results in reduced shrna-nef binding and provides a novel mechanism for rnai-resistance. to avoid viral escape, one should ideally target hiv-1 with multiple effective shrnas against conserved genome sequences. we performed a large scale screening to identify such targets, and we have identified at least nine genome segments that can be targeted effectively with shrnas. these potent antivirals are currently being assembled in a lentiviral vector for gene therapy applications in hiv-infected individuals. furthermore, we will describe approaches to forecast viral escape routes and to effectively block such evolutionary paths with additional rnai measures. in this study we analyzed the effect of antibodies against electronegative ldl on the development of atherosclerotic lesions in low-density receptor-deficient (ldlr −/− ) mice. two groups of (ldlr −/− ) mice (eight females) fed 0.5% cholesterol-enriched chow were used. the first group received a monoclonal antibody against electronegative ldl (100 g) and the second one received pbs (controls). additionally, other two groups (eight males) of (ldlr −/− ) mice were treated with a polyclonal antibody against electronegative ldl (100 g) or pbs (controls). antibodies were administered by intravenous route one week before starting the hypercholesterolemic diet and then every week over an experimental time of 21 days. afterwards, quantification of atherosclerotic plaque area of heart and aortic arch was done by analysis of the slices stained with oil red/hematoxolin/light green with the image propus software. the passive immunization with either monoclonal or polyclonal antibodies against electronegative ldl significantly reduced the atherosclerotic plaque areas in atherosclerosis-prone ldlr −/− mice. in conclusion, antibodies against electronegative ldl administered by intravenous route may play a protective role in atherosclerosis. supported by fundação de amparoà pesquisa do estado de são paulo (fapesp, scholarships to d.m.g., l.s. and a.b. and grants to m.h.k. and d.s.p.a.). the enzyme asparaginase from the procaryote escherichia coli or erwinia crysanthemi is used for the treatment of lymphoblastic leukaemia. the drug causes immunological reactions in despite of the treatment efficiency. asparaginase may also be obtained from saccharomyces cerevisiae and this enzyme could provide an alternative to its bacterial counterparts. in this study, the periplasmic nitrogen regulated asparaginase ii from s. cerevisiae, that is coded by the asp3 gene, was cloned and expressed in the methylotrophic yeast pichia pastoris under the control of the aox1 gene promoter. the recombinant p. pastoris strain was cultured in shake flasks and in a 2 l instrumented bioreactor. in both cases it was observed specific enzyme yields seven fold higher in comparison to that using a nitrogen derepressed strain of s. cerevisiae, reaching 800 u/g dry cell mass. high cell density cultures carried out in the 2 l bioreactor, in which it was attained 107 g dry cell mass/l, resulted in a dramatic improvement in asparaginase fermentation. as such, it was measured enzyme yields of 85,600 u/l and productivities of 1083 u/l h. department of biochemistry and food chemistry, biotechnology, university of turku, tykistokatu 6, biocity 6th floor, 20540 turku, finland. e-mails: lorenzo.galluzzi@utu.fi; deadoc@libero.it; deadoc@aliceposta.it (l. galluzzi) the bacterial luciferase operon from the bacterium photorhabdus luminescens has been used, since its first description, for exceptionally different applications. these ranged from the environmental monitoring to the cell tagging, from the analysis of cellular metabolism to the high-throughput screening of novel compounds. the wild-type luxcdabe operon has been engineered in countless ways (for instance by changing the order of the constituent genes, by optimizing the codon usage and by coupling it to many promoters) and has been expressed in prokaryotic and eukaryotic organisms in order to meet precise research and commercial needs. upon the operon expression light is emitted as the side product of a chemical reaction catalyzed by the luciferase enzyme, an ␣␤ heterodimer encoded in luxa and luxb genes. the reaction involves the oxidation of a long-chain aliphatic aldehyde and reduced flavin mononucleotide (fmnh 2 ) with the liberation of excess free energy in the form of a blue-green light at 490 nm. the luxcde genes code for the polypeptides (transferase, synthetase, and reductase) forming the fatty acid reductase complex that catalyzes the conversion of fatty acids into the long-chain aldehyde required for the luminescent reaction. noteworthy is that for the production of the substrates for the luciferase both atp and nadph are required, while neither is involved in the actual light emitting reaction (wilson and hastings, 1998) . recently, the coupling of the luciferase operon to regulated promoters lead to the construction of genetically modified bacteria able to sense the presence of chemicals and to respond, in a dosespecific manner, with light emission. this approach has been applied to the detection of antibiotics in samples from the food industry as well as to the detection of heavy metal ions in environmental samples (kurittu et al., 2000; bechor et al., 2002) . in addition, it opened the possibility of screening wide libraries of new compounds looking for molecules with pre-determined features, able to induce the bioluminescent response by de-repressing the lux operon transcription when incubated with the appropriate bacterial sensor. the high throughput and low costs are the main advantages of this system, which shows as well a certain degree of specificity (galluzzi and karp, 2003; galluzzi et al., 2004) . nevertheless, since the in vivo bioluminescence relies upon a complex network of biochemical reactions, under certain circumstances the light emission is not a direct consequence of the lux operon transcriptional induction but it more likely originates at a posttranslational stage. as a matter of fact, one can suppose that a change in the light emission will be observed whenever the concentration of one or more substrates for the lux␣␤ reaction occurs. consequently, all the molecules sharing the ability to impair the delicate chemical equilibrium regarding the compounds involved in bioluminescence will be sensed by the bacteria as inducing compounds. this, in turn, will result in a loss of specificity of the assay. we investigated this aspect of the whole-cell assays based upon the bacterial luciferase operon for drug discovery by means of a reporter plasmid in which the luxabcde genes, rearranged and optimized for the after 60 min (black downward arrow) of incubation at 37 • c under vigorous shaking the following concentrations of trimethoprim were added to the growing cells: 25 g/ml (triangles), 50 g/ml (circles) and 250 g/ml (rumbles). water was administered to control cultures (squares). light emission was quantified every 30 min by means of the wallac victor 2 multilabel counter (perkin-elmer, turku, finland) and normalized to the absorbance, measured at 600 nm with the same device. multi-96 white-walled transparent-bottomed plates were used for the assays (nalge nunc, usa). between the measurements the plates were kept at +37 • c under vigorous shaking. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). expression in gram + organisms, are under the control of the qacr regulatory region from staphylococcus aureus . non-pathogenic s. aureus rn4220 cells bearing the pqaclux plasmid (cultivated in lb broth supplemented with 0,5% d-glucose and 10 g/ml chloramphenicol) emit light upon the specific transcriptional induction with quaternary ammonium compounds, widely used as surface disinfectants and in many over-the-counter drugs . however, the incubation of the same cells with an inhibitor of the dihydrofolate reductase enzyme, trimethoprim (sigma-aldrich chemie, steinheim, germany), enhanced in a dosedependant manner the light emission observed upon the induction with the optimal concentration (1 g/ml) of benzalkonium chloride (bc). the extent of this increase in luminescence varied from 5-10% to more than 200%, according to the trimethoprim concentration and to the measurement time. interestingly, when the same plasmid is carried by escherichia coli xl1 cells, the lux operon is expressed constitutively (since the qacr regulatory region is not functional in xl1 cells) and at much higher levels than in induced rn4220 cells. also in this experimental system the incubation with trimethoprim results in a dramatic increase of the luminescent signal from the cultures. the explanation for these observations can be found in the molecular mode of action of trimethoprim. the inhibition of dihydrofolate reductase, indeed, directly leads to the accumulation of its substrates, among which is nadph, deeply entangled in the biochemical network of reactions centred on the light emission from the lux operon. nadph provides the reducing power to restore the reduced flavin mononucleotide pool and it is as well involved in fig. 2 . xl1/pqaclux growing cells were incubated with the following concentration of trimethoprim: 25 g/ml (triangles), 50 g/ml (circles) and 250 g/ml (rumbles). water was administered to control cultures (squares). light emission and absorbance measurements were performed as previously described for rn4220 cells. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). the diverse level of growth observed for rn4220 and xl1 cells can be accounted by the different antimicrobial activity exerted by trimethoprim towards gram− and gram+ cells and by the lower activity of the promoter which in pqaclux plasmid drives the transcription of the selection marker (chloramphenicol acetyl transferase). the production of the long-chain aldehyde, both substrates of the lux␣␤ heterodimer (wilson and hastings, 1998) . in conclusion, here we demonstrate that the use of light emission from the bacterial luciferase as a transcriptional reporter has to be very carefully controlled, since some molecules (here trimethoprim) could mimic to some extent a specific promoter activation by impairing the delicate intracellular biochemical equilibrium. on the reverse side of the coin, fig. 3 . simplified scheme depicting the bacterial folate metabolic pathway. only part of the reactions and compounds are reported. trimethoprim inhibits the nadph-dependant reduction of dihydrofolic acid into tetrahydrofolic acid catalyzed by dihydrofolate reductase. this results in the accumulation of both substrates, which become available for other reactions, and in the depletion of tetrahydrofolate, the major c 1 carrier in the synthesis of purines, thymidine, glycine, methionine, and pantothenate in bacteria. for antimicrobial purposes trimethoprim is often associated with sulfonamides, with which it displays a synergistic activity (since they act on the same pathway but at an earlier reaction) (scholar and pratt, 2000) . however, it has to be noted that the lux reporter system could be used in many different in vivo experimental setups, where the change in the intracellular concentration of nadph, atp or other metabolites would be sensibly detected. we have carried out the construction of five pichia pastoris strains harboring in their respective genome three different growth hormones cdna's (22 and 20 kda human growth hormones and bovine growth hormone), a shrimp (litopenaeus vannamei) trypsinogen cdna and a bacterial (bacillus subtilis) phytase gene. in all the cases, the same kind of expression vector and the same transformation technique were used. each dna was fused in frame to saccharomyces cerevisiae alpha-factor secretion signal to lead the secretion of the foreign protein into the culture medium. the induction of each recombinant strain, all with his + and mut + phenotype, was carried out in shake flaks using methanol buffered minimal medium (bmm) and growth rates on methanol determined. production level of secreted proteins was evaluated by protein analysis by sds-page. furthermore, the expression with three of the constructed strains was performed in a 5-l bioreactor and the level of secreted protein determined in each case. both human growth hormones (hghs) were secreted into the culture medium with a high degree of purity obtaining up to 64% of hghs of total proteins in crude fermentation medium and 3-18 mg/l of hghs. neither bovine growth hormone, shrimp trypsinogen or bacterial phytase were detected in methanol induced cultures of the respective strains, in spite of the same culture conditions were used for all p. pastoris strains. the growth rates on methanol were different between some strains. in fermentor cultures the hghs production were increased and shrimp trypsinogen was produced and secreted into the culture medium after improving the culture conditions. bovine growth hormone was detected only when the culture conditions were modified. the protein production level for each strain was affected by the proprieties of each recombinant protein produced. competitive advantages of the diagnostic method for invasive amoebiasis using preserved antigenic extracts without using enzymatic inhibitors m.s. flores 1,2* , e. tamez we have patented a method to diagnose invasive amoebiasis using a novel assay that preserves antigenicity of extracts with high protease content without using enzymatic inhibitors (ic:mc). the available tests for serologic diagnosis of invasive amoebiasis like elisa and indirect haemaglutination (iha) do not have consistent results in endemic zones. here we show the advantages of the assay and the validation of this diagnostic test for invasive amoebiasis. we demonstrated the reduction of proteolytic activity of ic:mc compared with the proteolytic activity of crude extract and crude extract with enzymatic inhibitors using assays. we displayed the ic:mc sds-page pattern and the western blot (wb) pattern useful for diagnosis. to search the clinical utility of this test we examined the wb obtained with sera from patients with different liver diseases; 90 patients had invasive amoebiasis and 45 patients had other liver diseases. the results were compared with those of iha test. also we have tested the accuracy of wb using sera from people with multiple intestinal parasites, like giardia lamblia, hymenolepis nana, blastocystis hominis, entamoeba coli, etc. the sensibility of the wb using the preserved amoebic antigens was 99%, specificity was 100%, positive predictive value was 100%, negative predictive value was 98% and accuracy was 99%. the wb did not exhibit cross reactions with sera from persons with intestinal parasites. our test was better than the (iha) test commonly used in endemic zones. these results show the improvement of using the preserved amoebic antigens in diagnostic tests. also they prove the diagnostic accuracy of our new wb test. antibacterial and antifungal activity of heracleum sphondylium subsp. artvinense yasemin kaçar 1 , sema tan 2 , aysun ergene 2 , perihan güler 2 , semra mirici 3 , ergin hamzaoglu 2 , ahmet duran 4 , sinem yildirim 2 : 1 mersin university, faculty of science and literature, department of biology, 33800 mersin, turkey; 2 kırıkkale university, faculty of science and literature, department of biology, 71450 yahsihan-kırıkkale, turkey; 3 akdeniz university, faculty of education, department of biology education, 07400 antalya, turkey; 4 selcuk university, faculty of education, department of biology education, 42300 konya, turkey turkey is covered yearly with a huge number of plant species. about 9222 species are condenced on the region that between asia and europe. many plant species have been used in folkloric medicine to treat various ailments. heracleum l (apiaceae) is include over than 70 species on the world. this variety is represent with 17 species in turkey that seven species are endemic. heracleum sphondylium subsp. artvinense is endemic species for turkey. ethanolic and aquous extract of heracleum sphondylium subsp. artvinense were investigated for their antimicrobial activities against six bacterial species (e. feacalis, e. coli, s. aureus, p. aeruginosa, l. monocytogenes, shigella) and two yeast (c. albicans, c. krusei). both ethanolic and aqueous extract of heracleum sphondylium subsp. artvinense showed antimicrobial activity against the gram negative bacterium (shigella) and gave the best activity against c.albicans. to develop artificial vectors allowing nucleic acid to transfect into mammalian cells are crucial for extending gene therapy. synthetic vectors based on lipid molecules are particularly attractive because of their potential safety. however, the low encapsulation efficiency of nucleic acid is one of the problem to be solved. recent advances in dna-lipid complex have improved this drawback, and now some lipid molecules are used as transfection agents. in particular, cationic lipids interact with negatively-charged cell surfaces and nucleic acids. the former interaction results in delivery of the nucleic acids directly across the cell membrane. on the other hand, the latter interaction improves the efficiency of nucleic acids entrapment. in this study, we developed a preparation method of "nanovesicle" containing nucleic acids by using reverse micellar solubilization. since dna interacts spontaneously with cationic amphiphiles, complete extraction of the dna molecules into an organic phase using dimethyl distearyl ammonium bromide (2c18ab), an oil-soluble cationic surfactant, is achieved. the re-encapsulation of the reverse micellar droplets solubilizing dna by water-soluble surfactants facilitates the formation of nanovesicles. a high salt concentration at the re-encapsulation step promotes the production of nanovesicles containing dna molecules. eventually, more than 80% of dna was encapsulated in this nanovesicles under the condition of 6m nacl and 5% (w/v) cetyltridecyl ammonium bromide (ctab). in addition, the nanovesicles prepared at 45 • c was smaller than that prepared at room temperature. the resultant 2c18ab/ctab nanovesicle was ca. 16 nm. asymmetric and chemically-modifiable vesicle surface are effective to design gene delivery system. moreover, such a small dna (or rna) carrier has a potential for novel gene therapy application from skin. the key players in clinically important inflammatory diseases, endothelial cells and leukocytes, communicate through membranebound cell adhesion molecules (cam's). obvious strategies for therapeutic intervention include specific means to affect the expression of cam's on the cells involved. rna-interference (rnai) is a well known means to achieve specific gene-inhibition. for this study, two cam's were chosen, namely vascular cell adhesion molecule-1 (vcam-1) as a target for inhibition, and intercellular adhesion molecule-1 (icam-1) as a non-target reference. we designed short interfering rna (sirna) oligos for vcam-1 in order to selectively inhibit the expression of this cam in cultured human vascular endothelial cells (huvec). real-time rt-pcr showed an 80% down-regulation of vcam-1 mrna while the expression of icam-1 remained unaffected. neither cam was affected by non-specific sirna. in order to further substantiate the potential use of sirna for therapeutic purposes, we have set out to investigate two things: (1) does vcam-1-specific sirna affect the amount of vcam-1 on the surface of huvec? (2) is adhesion between leukocytes and endothelial cells affected by vcam-1-specific sirna? the manufacturing of plasmid dna (pdna) is crucial to obtain a consistent product for gene therapy applications. although flowsheets for pdna production are established on the basis of experience, simulation tools provide a valuable help for evaluating alternatives. a process designed to produce 23 kg pdna/year is analysed with the superpro designer tool. the target pdna is amplified in escherichia coli. after harvest, alkaline lysis is used to disrupt cells and release pdna and impurities. precipitations with isopropanol and ammonium sulphate are performed to concentrate/pre-purify pdna prior to hydrophobic interaction chromatography. experimental data is used as input for simulation. inventory analysis identified water, yeast extract, tryptone, isopropanol and ammonium sulphate as the major raw materials. major raw material costs (50%) are related to fermentation components. economic analysis indicates a unit production cost of $ 375/g pdna. for a selling price of $ 1667/g, the payback time was 1.2 years and the roi was 88.6%. an environmental analysis highlighted the replacement of the isopropanol precipitation for a microfiltration step as a benefit which would: (i) reduce the cost of raw materials (13.8%), (ii) reduce the environmental impact associated with isopropanol (70%) and (iii) reduce costs associated with the treatment/disposal of liquid waste (32.3%). preparation of chitosan microspheres for controlled release of somatotropin s. simsek 1 , j. introduction: proteins and peptides have received extensive interest for their therapeutic applications in clinical applications. in order to achieve high administration efficacy of proteins, polymeric particulate carriers have been developed as an effective way to control the drug release profile and to protect the protein molecules from degradation. somatotropin also known growth hormone is a protein hormone of about 190 amino acids. growth hormone is of considerable interest as a drug used in both humans and animals. chitosan a natural linear biopolyaminosaccharide is obtained by alkaline deacetylation of chitin. properties such as biodegradability, low toxicity and good biocompatibility make it suitable for use in biomedical and pharmaceutical formulations. the aim of this study was to prepare chitosan microspheres containing somatotropin and to investigate these microsphere formulations in-vitro release properties. methods: somatotropin-chitosan microspheres were prepared as follows: chitosan was dissolved in acidic solution (2%) containing polysorbate 80. sodium sulphate solution (20% w/v) containing somatotropin was added into the chitosan solution and mixed 500 rpm for a hour. resulting suspension was centrifuged 15,000 rpm 15 min at 4 • c. the formed microspheres were freeze-dried and sieved. in-vitro release studies were performed in ph 7.4 phosphate buffer and time interval samples were removed and analysed by bradford protein assay method. results: microspheres were obtained by using chitosan. protein encapsulation efficiency was between 95 and 99%. average particle size of microspheres was between 48 and 57 m. during to in-vitro release studies burst effect was observed with chitosan microspheres. for decreasing the burst effect gluteraldehit, betacyclodextrin and poly ethylene oxide were added to formulations. conclusion: according to our results modified chitosan microspheres are promising vehicles for controlled release somatotropine delivery. cancer immunotherapy using hyperthermia with magnetic nanoparticles and dendritic cells k. tanaka, a. ito, t. kobayashi, t. kawamura, s. shimada, k. matsumoto, t. saida, h. honda department of biotechnology, school of engineering, nagoya university, nagoya, aichi 464-8603, japan. e-mail: h041306d@mbox.nagoyau.ac.jp (k. tanaka) our hyperthermia system utilizes magnetic nanoparticle covered with lipid layer including cationic lipid (magnetite cationic liposomes, mcls) as a heating mediator and necrotic cell death is induced by means of locally generating heat. in this process, heat shock proteins (hsps) are strongly induced and released. dendritic cells (dcs) are potent antigen-presenting cells (apcs) that play a pivotal role in regulating immune responses in cancer, which is in carrying antigens to apcs and in the maturation of dcs by acting as a danger signal. in the present study, we investigated the therapeutic effects of dc therapy combined with mcl-induced hyperthermia on b16 melanoma. in an in vitro study, when immature dcs were pulsed with b16 cells heated at 43 • c for 30 min, mhc class i/ii, costimulatory molecules cd80/cd86, and chemokine receptor ccr7 in the dcs were up-regulated, thus resulting in dc maturation. c57bl/6 mice bearing a b16 melanoma nodule were subjected to combination therapy using hyperthermia and dc immunotherapy. mice were divided into four groups: group i (control), group ii (hyperthermia), group iii (dc therapy), group iv (hyperthermia + dc therapy). complete regression of tumors was observed in 60% of mice in group iv, while no tumor regression was seen among mice in the other groups. increased ctl and nk cell activity was observed on in vitro cytotoxicity assay using splenocytes in the cured mice treated with combination therapy, and the cured mice rejected a second challenge of b16 melanoma cells. this study has important implications for the application of mcl-induced hyperthermia plus dc therapy in patients with advanced malignancies as a novel cancer therapy. fermentation of a marine bacterium for the production of cytotoxic compounds vicky webb 1 , els maas 1 , eiichi akaho 2 , hiroto kambara 2 , debbie hulston 1 , anna kilimnik 1 : 1 marine biotechnology, national institute for water and atmospheric research ltd, kilbirnie, wellington new zealand; 2 faculty of pharmaceutical sciences, kobe gakuin university, kobe 651-2180, japan marine bacteria are a potential source of novel compounds for the pharmaceutical industry. new zealand marine bacteria were isolated from a variety of sources and initially bacterial supernatants were screened using a cell based mtt cytotoxic assay. two bacteria were chosen for further fermentations in different media to optimise cytotoxic compound production. the media used were selected for their diverse ingredients. they were either carbon rich, nitrogen rich, starch rich or a basic seawater medium. the fermentations were extracted using ethyl acetate and methanol prior to assaying. the results showed that cytotoxic compound production was enhanced ten fold in the starch rich medium compared to the other media. the levels of cytoxicity appeared to be depended on the cell line used, with the epithelial lung carcinoma cell line a549 being more sensitive to the cytotoxic compounds than the human fibroblast cell line mrc-5. isolation and identification of marine bacteria from deep-sea sediments els maas 1 , cara brosnahan 1 , vicky webb 1 , helen neil 2 , phil sutton 2 : 1 marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington new zealand; 2 oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand polystyrene micro plate activated by utilizing a common co-60 gamma source with a crotonic acid. the well surface have been studied in term of optical quality, protein (h-igg) binding capacity and stability. a significantly enhanced total capacity and strength of binding to grafted surfaces was demonstrated as compared to passive adsorption of the proteins to untreated surfaces.the majority routine laboratory tests for the measurement of rheumatoid factor (rf) are semi quantitative and in order to achieve accurate, sensitive and specific rf assay, we are developed enzyme linked immuno sorbent assay (elisa) for human igm-rf kit. stable and reproducible binding of antigen (human-igg) to well of micro titer plate is a prerequisite for rf-elisa kit. the principle of the assay was that the antibodies in serum of patients with rheumatoid arthritis (ra), commonly rheumatoid factor (rf) are directed to the fc part of human igg. in most cases rf belong to the igm class, the well of micro titer plate are coated with antigen (h-igm), and antibodies (h-igg) binding to immobilized antigen is detected by adding enzyme conjugate (anti human-igm-hrp-enzyme) to the wells, substrate was used for color reaction.the performance characteristics of the assay was, the coefficient of variation (c.v) of intra and inter assay was 4.4% and 2.2% respectively, the linearity is ranging from 86 to 112%, and the recovery for three different sera is ranging from 89 to 106%. the data presented in this paper indicated that the activation of polystyrene micro titer plate by the gamma rays could be use for preparation of igm-rf kit and may be others immunoassay techniques. overcoming the nuclease barrier to gene expression during trafficking of plasmid dna vectors a.r. azzoni 1 , a. tavares 2 , g.a. monteiro 1 , d.m.f. prazeres 1 : 1 centro de engenharia biológica e química (cebq), instituto superior técnico, 1049-001 lisboa, portugal; 2 instituto gulbenkian de ciência, rua da quinta grande 6, 2780-156 oeiras, portugal. e-mail: azzoni@ist.utl.pt (a.r. azzoni) inefficient nuclear delivery of plasmid dna (pdna) vectors is thought to be a bottleneck to gene transfer in gene therapy and dna vaccination utilizing non-viral delivery systems. one of the main barriers found by pdna vectors during trafficking to the nucleus is degradation by a population of endo/exo-nucleases. this barrier may be partially circumvented by shielding the pdna from the nucleaserich cell environment with adjuvants or by using nuclease inhibitors. a different approach that has been studied at the cebq is the generation of pdna vectors that are more resistant to nuclease action a priori. in this work, the nuclease barriers to gene expression are being studied aiming at the generation of pdna with improved resistance to nucleases and thus higher transfection efficiency. by engineering the plasmid labile sequences, new plasmid vectors with an improved resistance to physical-chemical and biological degradation are being generated. this was indicated by an extended half-life of the supercoiled isoforms during storage and when the plasmids were exposed to nucleases present at eukaryotic cell lysates and mice plasma. the intracellular trafficking of the new plasmid vectors through the cytosol of mammalian cells was then assessed by fluorescence in situ hybridisation (fish) and the expression of the reporter protein (egfp) was detected by fluorescence microscopy. identification and evaluation of antibacterial phytochemicals of fishbone fern (nephrolepis cordifolia) rikhia chakraborty 1,2 , promod kumar verma 2 : 1 department of cancer biology, lerner research institute, 9500 euclid avenue cleveland, oh 44195, usa, 2 department of biotechnology, guru nanak dev university, amritsar, punjab 143005, india. e-mail: riar5400@rediffmail.com (r. chakraborty) in this study, different aqueous and organic extracts from the fern, nephrolepis cordifolia, were used for screening tests for antibacterial effects. protein and lipid extracts were first tested for antibacterial activity. subsequently, crude extracts of leaves, roots, and stems were prepared in methanol, ethanol, chloroform, hexane, petroleum ether, diethyl ether, and water using optimized standard protocols. each fraction was tested for anti-microbial effect through agar well-diffusion assay, and paper disc method. the antibacterial spectrum against which the fern is active was thus determined. dosagedetermination for optimum activity was also determined for each of the extracts. bacillus and staphylococcus were used as the indicator test-organisms. the results were very encouraging; being effective even at the 54th day, thus showing that the antibacterial properties were not due to any changes in external factors and physiological effects. ethanol, methanol and chloroform extracts from the subaerial portions had strong anti-microbial properties. agar-well diffusion assay and paper-disc diffusion assay done for different solvent fractions were giving comparable results. 2.5 mg was adequate for maximum effect against b. circulans, s. aureus, s. epididermis, and streptococcus sp. 7.5 mg was adequate as effective dosage for kleb-siella pneumoniae. 10 mg was required for mycobacterium bovis, e. coli. pseudomonas was not showing any susceptibility. given the broad spectrum of activity, especially towards the gram-negative bacteria, nephrolepis cordifolia is definitely a promising plant having pharmacological importance. for a full interpretation of the present results further investigations are necessary to elucidate the different physical and chemical parameters of the active principles and also to determine the mechanism of action. the present work highlights n. cordifolia as a plant having a broad spectrum of antimicrobial activity, a phenomenon very rarely observed in the plant kingdom. bacteria (escherichia, salmonella, proteus, staphylococci and bacillus) were isolated from hospital soil using selective enrichment and growth on selective and differential medium, viz. macconkey's agar, clyed medium and baird parkers medium. confirmation and species identification was carried out by biochemical and serological tests. from these isolates, three different pathogens were used to study multiple antibiotic resistances. e. coli bj 83 showed resistance to ampicillin, streptomycin and cefurixime. s. typhi and s. aureus showed resistance to ampicillin and cefuroxime. assay using octadisc using e. coli and s. typhi showed a broader resistance pattern to antibiotics including amoxicillin, clavulanic acid, cephalexin, chloramphenicol, ciprofloxacin, and cotrimoxazole. the r plasmid profile was studied to understand the mechanism of drug resistance. to combat the problem of drug resistance, a strategy of combined antibiotic response of cultures were studied. such a synergistic combination would possibly have the effect of overcoming multiple antibiotic resistances. for e.g. kanamycin resistance strains were inhibited in presence of kanamycin and cefotaxime. further, the bactericidal activities of antimicrobials in honey and garlic were also tested. the mic of honey was observed to be 4%, while that of garlic was between 0.1 and 1%. honey and garlic were also found to inhibit the growth of organisms in the presence of antibiotics. kanamycinresistant e. coli was unable to grow in presence of kanamycin and 0.08% garlic. these traditional agents, long used in ayurvedic system of medicine in india, could be further explored for potent antimicrobial properties. hepatitis b virus (hbv) infection is s global health problem. assays for hbv antigens and antibodies are widely available and standardized. extremely sensitive qualitative pcr kits are also available for detection of hbv in serum. hbv pcr kit may be useful in assessment of occult hepatitis b in hbcab positive alone subjects and carriers. a positive pcr results show presence of virus articles in serum without considering serologic results. but there are differences in efficiency of hbv dna amplification kits. in this study we compare two commercially available hbv pcr kits for evaluation viremia of hbsag positive carriers and hbcab alone positive subjects. material and methods: of the 368 randomly selected subjects serologically examined for hbv, 49 and 43 were positive for hbsag and hbcab alone respectively. both later groups were tested for hbv dna by two commercial kits, hbv pcr detection kit (cinnagen, iran) and hbv pcr test (pazhohesh azma, iran). dna extraction kits recommended by each manufacturer were used for hbv dna extraction. amplicons in both kits were a highly overlapped fragment in 5 conserved sequence of viral genome. results: of the 49 hbsag positive carriers, hbv dna was detected in 37.4 and 65.3% using kit1 and kit2 respectively. only 28.6% were positive in both kits. in hbcab positive subjects (n = 43), 23.3% were positive by kit 2 and all samples were negative when tested by kit 1. there was a significant difference between two kits. sensitivity of kit 1 and kit 2 were 48.6 and 91.4%, respectively. overall, kit 2 increased the detection rate of hbv dna by 88.2%. discussion: our study show there is a significant variation between these two commercial kits especially in hbcab positive subjects that the copy of virus is very low. from these results, it can be concluded that the unstandardized kits have not compatible results, and pcr test interpretation should be done with great care. . the antibacterial activity of the extracts was evaluated based on the inhibition zone using plate diffusion method. most of the extracts were active against both gram positive and gram-negative bacteria, but pseudomonas aerugenosa, bacillus subtilis and sarcina marcescens, were more susceptible to almost all the extracts. finally, further research will be done to elucidate the nature of the active compound and investigate for peptides by using protein gel immobilization bioassay. short interfering rna delivery and gene silencing using polymeric nanocarrier systems k.a. howard 1 , x. liu 1 , d. oupicky 2 , f. besenbacher 1 , j. kjems 1 : 1 inano, university of aarhus, denmark, 2 department of pharmaceutical sciences, wayne state university, detroit, usa the effectiveness of a drug is determined by the ability to migrate through the body and reach target sites in therapeutically relevant levels. nanocarriers for delivery of bioactive agents are being developed at inano to maximise drug payload at target sites. the inclusion of "biological triggers" into the nanocarrier design is used for modulation of cellular nucleic acid trafficking and increased target interaction. chitosan and peptide-based polymers were used to formulate nanocarriers in the size range 30-250 nm containing small interfering rnas (sirnas) for gene silencing applications. page analysis showed the structural integrity of the sirna was maintained during particle formation. in systems composed of bioresponsive polymers, nanocarrier disassembly and sirna release under cellular conditions were shown, using atomic force microscopy. the time course for sirna uptake into nih cells was visualised using confocal microscopy. in addition, sirna localisation within cells could be modulated by the composition of the polymer used. the ability of the nanocarrier system to mediate gene expression was investigated in a cell line stably expressing enhanced green fluorescent protein (egfp). furthermore, the various delivery systems were tested in a mouse model stably expressing the egfp protein using both nasal and intravenous delivery routes. the use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. however, the presence of xenoreactive antibodies in humans directed against swine gal antigen present on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute immunological reaction. the graft of genetically modified organ of a swine depleted of enzyme ␣1,3-galactosyltransferase that is responsible for gal antigen origin, would be tolerated with simultaneous administration of medicines decreasing other less severe immunological reactions. to prevent hyperacute rejection it is also possible to modify swine genome by human genes controlling enzymatic cascade of complement or modifying the set of donor's cell surface proteins. for this purpose genetic constructs containing inactivated ␣1,3-galactosyltransferase gene, human cd59, cd55 and cd46 genes controlling complement activation and human genes encoding ␣1,2-fucosyltransferase and ␣-galactosidase enzymes modifying cell surface proteins were prepared. these genetic constructs were transfected into the pig foetal fibroblast using lipofection method. after selection, molecular and cytogenetic characteristic of cells with transgene integrated into the host genome were performed. introduction: protease 2a(2a-pro) of coxsackievirus b3 (cvb3) plays major role in viral replication. in case of infection, viral proteins are being synthesized from viral mrna using host biosynthesis machinery. 2a-pro of virus, after being synthesized, exhibit two critical functions, cleavage of viral proteins and breaking eif4g (eukaryotic initiation factor 4g-formerly called p220) which leads to host cell translational system shot-off. the enzyme plays essential role in viral replication and cellular damage. to understand pathogenicity of infection and also developing potent and selective inhibitors against picornavirus infection, it is necessary to prepare pure 2apro enzyme. in this study an expression system with efficient and high yields was obtained. methods: cdna of 2apro was synthesized using in vitro infection of permissive host through reverse transcription process and was cloned in pet22b(+) and since 2a-pro is a toxic product, naturally before induction its expression will act on the host and damage the cells. for this different hosts were checked and finally, blr(de3) plyss which carries an extra-plasmid for lysozyme expression that minimizes unwanted target protein production (leakage) was selected. on the other hand for biological activity assay, polyclonal antibodies against antigenic sites of p220 was prepared by synthesizing small peptides, corresponding to antigenic site of p220 coupling to klh and injecting subcutanously to rabbit. then, the enzyme and its substrate (hela cells lysate that contain p220) were incubated together for different times intervals. results: the recombinant product was confirmed by sds polyacrylamide gel electrophoresis and immunoblot analysis. also p220 cleavage by 2apro was assessed by sds-page and western blot analysis. cleavage of p220 by r2a-pro was prominent after 24 h. so recombinat 2a-pro with good activity was prepared. application of bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system on production of glycoprotein in larvae of silkworm ayano kageshima, tatsuya kato, misun kwon, enoch y. park department of applied biological chemistry, shizuoka university, ohya 836, suruga-ku, shizuoka 422-8529, japan bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system was applied on production of glycoprotein in larvae of silkworm. the bacmid system of autographa californica nuclear polyhedrosis virus (acnpv) has already been established and widely used. since the acnpv does not have a potential to infect silkworm we developed the first practical bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system directly applicable for the protein expression of silkworm. by using this system, the green fluorescence protein and glycoprotein, human ␤1,3-n-acetylglucosaminyltransferase 2 were successfully expressed in silkworm larvae not only by infection of its recombinant virus but also by direct injection of its bacmid dna. three different kinds of signal sequences were tested for the secretion of glycoprotein into hemolymph of silkworm. signal peptides of prophenoloxidase-activating enzyme and bombyxin: insulin-like brain secretory peptide showed the highest secretion ratio, 99% of total expressed ␤1,3-n-acetylglucosaminyltransferase 2 was secreted into hemolymph of silkworm. using bacmid system 50 mu/ml of ␤1,3-n-acetylglucosaminyltransferase 2 was expressed in hemolymph of silkworm, which was 2-three times higher than that of insect cell. silkworm is one of the most attractive hosts for large-scale productions of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. this method provides the rapid protein production in silkworm, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses. we have established an efficient system for foreign gene expression in lily (lilium longiflorum) pollen in a transient mode. pollen was transformed using agrobacterium via vacuum filtration for 20 min. the pollen germinated for 24 h was analyzed to confirm its foreign gene expression in molecular analysis. mouse fed the transgenic pollen culture for 8 weeks showed immune reaction specific for pollen-derived recombinant protein. and the igg level was highly elevated by one time boosting injection afterwards. the lily pollen system may be suggested as a novel type of biofactory for producing edible vaccine with rapidity. anti-apoptosis engineering with the 30kc6 gene obtained from silkworm shin sik choi, won jong rhee, tai hyun park school of chemical and biological eng., seoul national university, seoul 151-744, korea the chinese hamster ovary (cho) cell line producing recombinant human erythropoietin (epo) was manipulated to express the 30kc6 gene, which was originally obtained from a silkworm. the expression of 30kc6 inhibited serum deprivation-induced apoptosis and increased the cell density and epo expression level per unit cell by five-and two-folds, respectively. an increase in these two factors resulted in a 10-fold increase in the volumetric productivity of epo. compared with the controls, the oligosaccharide structures of the epo synthesized by the cells expressing 30kc6 showed greater homogeneity. the terminal sialylation of the glycans of epo were promoted by the expression of 30kc6. the positive effects of 30kc6 expression on the cell viability and productivity were attributable to the stable maintenance of the mitochondrial activity. these results demonstrate that the cho cell line genetically engineered with the 30kc6 gene has a great potential for use in the production of therapeutic proteins. evaluation of fucoidan-chitosan hydrogels on superficial dermal burn healing in rabbit: an in vivo study a.d. sezer 1 , f. hatipoglu 2 , z. ogurtan 3 , a.l. baş 4 , j. introduction: healing of dermal wounds with macromolecular agents such as natural polymers is one of the research areas of the pharmaceutical biotechnology. fucoidan is a sulphated polysaccharide which is commonly obtained from seaweeds. although a great number of studies on the different pharmacological properties of fucoidan are present, there is very limited information on the fucoidan-based system used in dermal burns. the aim of this study was to prepare chitosan hydrogel containing fucoidan and to investigate this hydrogel formulation for treatment of dermal burns on rabbits. methods: fucoidan-chitosan hydrogels were prepared as follows: the polymers were dissolved in acidic solution and sonicated for removing the air-bubbles then the gels were stored at +4 • c for in vivo studies. seven adult male new zealand white rabbits (mean weight, 3.8 ± 0.6 kg) were used for the evaluation of the gels on superficial dermal burns. the back of the rabbits were depilated and sedated. the wounds were made by circular stamp aluminium caps (3.8 cm 2 ) at 80 • c. four wounds were formed for each rabbit; (a) was treated with fucoidan-chitosan gel, (b) was treated with fucoidan solution, (c) was treated with chitosan gel (without fucoidan) and (d) as a negative control group. biopsy samples were taken at 7, 14 and 21st days at the beginning of the study and each wound site was macroscopically observed and evaluated histopathologically. results: oedema was not observed in all groups after 3 days treatment except controls. after 7 days treatment, fibroplasia and scar were observed on wounds treated with fucoidan-chitosan gel and fucoidan solution. the best regenerate dermal papillary formation and the fastest closure of wounds were observed in group a after 14 days treatment. the wound epithel elongation and thickness values were measured; a (5566 and 179 m), b (3586 and, 146 m), c (3666 and, 162 m), d (3533 and, 134 m) at the end of the study. conclusion: re-epithelization and contraction of the wound area which was treated with fucoidan-chitosan hydrogel were faster than the other groups. the fucoidan-chitosan hydrogel formulations can be suitable for the treatment of dermal burns. aim: to analyze the neutralizing -related activity of antibodies against e1 region of hcv, specific polyclonal antibody was raised by immunized rabbits with synthetic peptide that had been derived from e1 region of hcv with the amino acid sequence e1 antibody [ghrmawdmm). materials and methods: hyper-immune hcv e1 antibodies were incubated over night at 4 • c with serum samples from patients positive for hcv rna, with different viral load, ranged 7-11 million copes/ml, then incubated 90 min to hepg2 cells. rt-pcr and flow cytometry were used to study the inhibition binding and entry effect of e1 antibody. direct immunostaining of e1 antibody conjugated with fitic and flow cytometry analysis showed reduced the mean fluorescence intensity in the samples pre-incubated with e1 antibody compared with samples without e1. of 18 positive serum samples, 13 (72%) samples showed completely inhibition of infectivity as detected by rt-pcr. conclusion: in house produced e1 antibody, blocks binding and entry of hcv virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. isolation of these antibodies that block virus binding and entry will be useful in providing potential therapeutic reagents and for vaccine development. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his7tnf␤ and n19tnf␤ were expressed in e. coli. high solubility of n19tnf␤ was expected, however, both analogs his7tnf␤ and n19tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n19tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his7tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with 0.2% nls, while his7tnf␤ and n19tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. enzymatic modification of sphingomyelin long zhang, lars hellgren, xuebing xu biocentrum-dtu. e-mail: lz@biocentrum.dtu.dk (l. zhang) due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. currently, chemical synthesis of ceramide is a costly process, and developments of alternative cost-efficient, high yield production methods are of great interest. in the present study, the potential of producing ceramide through enzymatic hydrolysis of sphingomyelin (sm) have been studied. sm is a ubiquitous membrane-lipid and rich in dairy products or by-products. in present study, we have optimized the production of ceramide from sm using phospholipase c from clostridium perfringens. water and enzyme amount had the biggest influence on sm hydrolysis in the system. botulinum neurotoxins constitute a family of bacterial toxins for botulism syndrome in human. the toxins bind with high affinity to nerve cells where they cause a complete inhibition and release of neurotransmitters and thereby produce flaccid paralysis. in this work we have reported isolation of the binding domain of type e neurotoxin by pcr and expressed in a proper expression vector. the output of this investigation can be used as a tool to study the mechanism of binding of holotoxins and also can be useful to study the antibody production against botulism syndrome. the synaptobrevin (vamp2) a protein which play a key role in the fusion and exocytosis of the vesicle of mammalian nerves terminals this protein is substrate different serotypes of botulinum neurotoxins. light chain of clostridium botulinum type b, d, f and g cleave end of neurons. due to intraction of clostridium botulinum neurotoxin with vamp2, this protein can be used as one of the toolsin the detection of poisings cause by this bacteria in the clinical laboratory using the enzyme with proof reading activity the above gene amplified by pcr technique for the production of the recombinant protein, the prokaryotic expression vectors (pet system) was used. a recombinant protein developed on poly acrylamide gel analyzed by western blotting and eliza. most children with down syndrome (ds) are born to younger mothers (<35 years). recent reports linking down syndrome (ds) to maternal polymorphisms at the methylenetetrahydrofolate reductase (mthfr) gene locus have generated great interest among investigators in the field. in this study, forty mothers with their affected outcomes and 100 control mothers were included. all mothers were subjected to complete medical and nutritional history with special emphasis on folate intake through food or oral supplementation. estimation of blood homocysteine level was done. also we examined the two polymorphisms in genes encoding the folate metabolizing enzyme methylenetetrahydrofolate reductase (mthfr), namely, 677c > t and 1298a > c. folic acid intake from food and from vitamin supplements was significantly low (below the recommended daily allowance) in the group of mothers with ds children compared to control mothers (p < 0.01) using student t-test. blood homocysteine was normal in both control and ds mothers. the prevalence of the two polymorphisms, namely, 677c > t and 1298a > c in mothers of ds children (case mothers) (n = 40) was compared with controls (n = 100). frequencies of mthfr genotypes (cc, ct, and tt) at position 677 demonstrated no difference between the case and control groups. genotype frequencies of mthfr at position 1298a (aa, ac, and cc) were different among the case and control mothers. we here report the first study on a possible relation between ds with mthfr 1298a > c genotypes in egypt. our results showed that mthfr 1298a > c polymorphism is a remarkable genetic entity among egyptian females with d.s. children. sufficient folate intake and supplementation is an important preventive strategy in overcoming the risk of nondisjunction. homologous recombination (hr) is the mechanism that permits the creation of genetically engineered strains through gene targeting. in order to further develop gene targeting techniques, notably for higher eukaryotes such as filamentous fungi, it is of crucial importance to fully understand the molecular mechanisms behind mitotic hr. in saccharomyces cerevisiae, a dna double strand break (dsb) is an essential intermediate in meiotic hr. however, as hr occurs at a low rate in mitotic cells, it has been difficult to determine the nature of the event(s) that triggers it. rad52 is a key protein involved in hr and is evolutionarily conserved from yeast to human. to shed light on the molecular events in hr, we have generated a large collection of defined rad52 mutant strains in the yeast s.cerevisiae. a screen of these mutants led to the identification of strains that fail to repair dna dsbs, yet are proficient for homologous recombination. this result strongly suggests that dsbs may not be the major cause of spontaneous mitotic hr and gives new perspectives in respect to novel potential gene targeting substrates. we have analyzed these separation of function mutants in a variety of new assays to obtain a more detailed understanding of their controversial phenotype. our latest results will be presented. the outcome of interferone plus ribavirine treatment of hepatitis c virus (hcv) genotype 4 is unfortunately poor. development of alternative therapy for this genotype is of a paramount importance. inhibition of hcv gene expression in vitro by the use of antisense phosphorothioate oligodeoxynucleotides (s-odn) against internal ribosomal entry site (ires) elements were associated with favorable results. to assess s-odn activity, previous studies utilized viral subgenomic or full cdna fragments linked to reporter genes and transfected into adhered cells or in a cell free system. in the present study we utilized hepg2 cells infected with native hcv rna of genotype 4. the culture system presented herein was shown to support hcv replication on the following bases (1) consistent detection of both plus and minus rna strands for 4 weeks in cells and in fresh culture supernatent, (2) ability of supernatent to infect naive hepg2 cells (3) consistent expression of core and e1 envelope proteins in infected cells throughout the 4 week culture. s-odns against aug translation start site (s-odn-1, nt 326-348) of the viral polyprotein precursor and stem loop iiid within the ires region (s-odn2, nt 264-282) were added to infected cells. intracellular viral replication was monitored by nested rt-pcr of plus and minus strands. the results of these experiments demonstrated that intracellular replication of hcv genotype 4 was completely arrested after 48 h in culture using either s-odn molecule (with more efficacy of s-odn1 than s-odn2) at concentrations as low as 1 m. the inhibitory effect of s-odn appeared to be specific to hcv replication since equal levels of human glyceralehyde 3-phosphate dehydrogenase (gapdh) gene expression were noted pre and post supplementation of s-odns. in conclusion, the present study provides evidence antisense phosphorothioate oligonucleotides have potent inhibitory effect on genomic replication of hcv genotype 4, the most common type in egypt. a sensitive and specific pcr-elisa was developed to detect shigella dysentery in food. the assay was based on the incorporation of degoxigenin-labeled dutp and a biotin-labeled primer specific for shiga toxin genes during pcr amplification. the labeled pcr product were bound to streptoavidin-coated wells of a microtiter plat and detected by an elisa. the elisa detecting system was able to increase the sensitivity of the pcr assay by up to 100-fold, compared with a conventional gel electrophoresis. the detection limit of the pcr-elisa was 0.1-10 cfu dependent upon shigella dysentery serotypes and genotypes of shigatoxin. the entire procedure took about 4 h. isolation, cloning, expression and purification of snap-25 as a substrate of botulinum neurotoxin type a and e m.l. mossavi 1 , f. ebrahimi 1 , j. amani 1* , h. basiry 1 , z. ahmadi 2 : 1 department of biology, faculty of science, imam hussein university, tehran, iran; 2 department of biology, faculty of medicine, bageatallah university, tehran, iran. e-mail: kpjamani@ihu.ac.ir (j. amani) clostridial neurotoxin inhibit neurotransmitter relase by selective and specific intracellular proteolysis of synaptosomal associated protein of 25 kda (snap-25); synaptobrevin/vamp-2 and syntaxin. snap-25 is one of the components that form docking complex in synaptic ends. this protein is substrate for botulinum neurotoxins type a and e. each of these toxin serotypes specifically cleave snap-25 in particular position and there by block docking and synaptic vesicle membrane fusion and finally prevent neurotransmitter exocytosis and transition of neurotic signals. in order to use the protein as a substrate for detection of different type of clostridium neurotoxin in vitro test, the protein was produced by recombinant technique. the dna encoding snap-25 was isolated from rat brain by pcr using the two primer the amplified fragment colonel into expression vector pet32a.the expression protein was purified by affinity chromatography. confirm by different method the his tag fusion protein was digested with entrokinase. the present work deals with the preparation of pure alpha-1antitrypsin (aat) protein from healthy subjects, which can be used in preparing its corresponding monospecific antibody in albino rabbits. this antibody was found very useful in the immuno-diagnosis of pulmonary emphysema. this study has been also concerned with the biochemical changes associated with aat deficiency in pulmonary diseases. to fulfill this work, a group of healthy blood donors was selected for separation of pure aat antigen from blood. the pure aat was used for the preparation of anti-aat, the purity and potency of antibody was checked by titration methods. the biochemical changes were studied in thirty subjects clinically divided into three groups including, control, heavy cigarette smokers with pulmonary emphysema, and non-smoking subjects with pulmonary emphysema. the activity of elastase and hydroxyproline (hp) level as a marker of elastin and collagen breakdown were assayed in bronchoalveolar lavage (bal) fluid. the activity of aat and to inhibit proteases as represented by tryptic inhibitory capacity (tic) was evaluated. serum ceruloplasmin, transferrin and iga level as well as thiobarbituric acid reactive substances (tbars) were estimated in all groups. our data revealed that aat and its tic showed very highly significant decreased levels in all patients with emphysema as compared to control, while the elastase activity and hp level in bal fluid were significantly increased in these patients. serum tbars was significantly increased in such patients associated with increasing level of both ceruloplasmin and transferrin. while, serum iga was significantly increased. furthermore, the biochemical changes were markedly changed in smokers with emphysema than non-smoking subjects. in conclusion, the preparation of anti-aat on the local level is very important where less expensive and less time consuming and can be useful in immuno-diagnosis and prognosis of pulmonary diseases. a new method combined with boosting and projective adaptive resonance theory for analysis of gene expression data from cancer patients hiro takahashi, yasuyuki murase, hiroyuki honda department of biotechnology, school of engineering, nagoya university, nagoya 464-8603, japan. e-mail: h041305d@mbox.nagoyau.ac.jp (h. takahashi) an optimal and individualized treatment protocol based on accurate diagnosis is urgently required for the adequate treatment of patients. for this purpose, it is important to develop that a sophisticated algorithm that can manage large amount of data, such as gene expression data from dna microarray, for optimal and individualized diagnosis. in our previous study, we developed the projective adaptive resonance theory (part) as a gene filtering method and boosted fuzzy classifier with sweep operator (bfcs) as a modeling method. in the present study, we applied the combination of part and bfcs (part-bfcs method) to microarray data of brain tumor (central nervous system tumor) obtained from the website. the method enabled the selection of 14 important genes related to the prognosis of the tumor, i.e., sensitivity for combined therapy with surgery, radiotherapy, and chemotherapy mainly with vincristine, cisplatin, and cytoxan. the constructed model showed about 20% higher accuracy than that of the conventional method. genomic signal analysis of hiv variability based on rt gene p.d. cristea, rodica tuduce d. otelea biomedical engineering center, university "politehnica" of bucharest, romania. e-mail: pcristea@dsp.pub.ro (p.d. cristea) dna sequences genotyped from 60 clade f hiv-1 isolates from romanian patients in the laboratory of the national institute of infectious diseases "matei bals", bucharest, romania, have been studied. the symbolic sequences have been converted into digital genomic signals by using a complex quadrantal representation of the nucleotides described earlier. the cumulated phase and unwrapped phase of a complex genomic signal reflect the statistical distribution of bases and base-pairs, respectively. independent component analysis of the genomic signals has been used to characterize the variability of the f subtype hiv strains isolated in romania. the sequenced segment is of (about) 1302 base pairs, approximately aligning with the standard sequence of hiv-1 (accession nc001802 in genbank) over the interval 1799-2430 bp. this segment, which is currently used for the standard identification and assessment of hiv-1 strains, comprises the protease (pr) gene and two thirds of the reverse transcriptase (rt) gene. only results referring to the analysis of the rt gene region are presented here and used for extracting features of virion isolates and for establishing phylogenetic trees of the studied strains. the analyzed rt encoding segment has the length 1005 bp and is located in the second interval (298-1302 bp) of the analyzed dna segment, respectively along the 2096-3100 bp region of nc001802. taking into account the mutations identified in these sequences, the samples were classified in three groups from the point of view of their resistance to current antiretroviral compounds: sensitive, resistant and multiresistant. the paper presents results for the isolates in which mutations leading to multiple drug resistance have been identified. over expression of secb protein in e. coli enhances the periplasmic expression of human growth hormone m. ghafari 1,2 , a. zomorodipour 2 : 1 islamic azad university of jahrom, tehran, iran; 2 national institute for genet eng and biotechnol., tehran, iran. email: maryamghafari2001@yahoo.com (m. ghafari) among several proteins involved in the secretion pathway of proteins in e. coli, secb plays a key-important role in solubilization of preproteins before processing. in order to increase processing of a human growth hormone precursor (pelb::hgh) which appeared to have problem in processing efficiency, as a possible solution a regulated co-expression of a secb gene was considered. in this regard, we designed an arabinose-regulated secb expressing plasmid compatible with an iptg/lactose-regulated pelb::hgh expressing plasmid. for the construction of the secb expressing plasmid the origin of replication and antibiotic resistant gene (amp) of a pbad vector was replaced by a p15a-ori and a kanamycin resistant gene, respectively. the expression and processing of pelb::hgh preprotein in the two-plasmid containing bacteria in a secb over-expression state was compared to that of the pelb::hgh expression in normal bacteria. although a decline in total expression level of hgh during the overexpression of secb was observable, probably due to presence of two different expressing plasmids, but both the processing efficiency of pelb::hgh and the transport of mature protein into the periplasmic space was enhanced during prolonged arabinose induction. current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts. natural allergen extracts contain a mixture of allergenic and non-allergenic components that are difficult to standardize. recombinant allergens can improve diagnosis and de-sensitization against single component. major cat allergen is a heterodimer composed of disulfide linked 7.8 and 10.1 kda polypeptide chains. both chains of feldi protein were obtained in e.coli system. purification of recombinant proteins was performed in denaturing conditions using immobilized metal affinity chromatography specific for proteins with histidine tag. from 1000 ml culture approximately 13 mg protein for feldi chain 1 and 43 mg of feldi chain 2 were obtained. after purification histidine tag was removed by hydrolysis with thrombin. immunological activity of feldi against serum of patients allergic to cat was narrowed to subgroup of patients allergic to feldi protein by surface plasmon resonance. immunological activity of each chain and renatured heterodimer was also tested using immunoprecipitation techniques against serum of population group. subdoligranulum variabile -a novel member of the human gut micro flora with a high prevalence kim holmstrøm, trine møller bioneer a/s, hørsholm, dk-2970, denmark in 2003 we isolated and cultured for the first time a bacterium from a human fecal sample representing a hitherto unknown member of the clostridium leptum rrna supra generic cluster. the c. leptum rrna supra generic cluster represents one of the 3 major phylogenetic lineages within the human gut microbiota, and is characterized by having only a small proportion of its members actually identified by cultivation compared to the estimated numbers of bacteria contained in this group from culture-independent gut flora analyses. s. variabile is an obligate anaerobe gram negative bacterium with a characteristic pleiomorphic coccoid-droplet-like cellular morphology. its closest previously cultivated relative based on a 16s rdna phylogenetic analysis is faecalibacterium prausnitzii, a rod-shaped and therefore easily distinguishable gram negative bacterium present in high numbers in the human fecal micro flora. based on 16s rdna sequence we designed a s. variabile specific oligonucleotide probe for use in fish analysis to estimate the prevalence of this "new" bacterium in fecal samples collected from healthy human beings. interestingly, we observed a high proportion of s. variabile present in all tested samples, and in some instances we observed a higher prevalence than the more well-known group of bifidobacteria equally estimated by fish analysis. documentation of these results will be presented. several species of sea cucumbers, long an incumbent of traditional medicines were selected as the source of animal based antibiotic compounds. swabs of the inner surface and coelomic fluid (inner fluid) samples from sea cucumber (holothuria atra jaeger) were taken. thirty strains of bacteria were isolated. these strains were grown in different antibiotic production media. nine human pathogenic bacterial species were used as test agents and they are, k. pneumoniae, mrsa, m. luteus, s. thyphimurium, s. epidermitis, s. saprophyticus, b. subtillis, p. aeruginosa and s. pyogenes; only four bacterial strains showed mild antibiotic activity against s. pyogenes and s. thyphimurium. similar testing on two other species, h. scabra and s. variegatus will be carried out. different media, especially antibiotic production enrichment media will also be used. characterization will be done upon obtaining an antibiotic compound, which shows moderate to high activity against at least one of the nine human pathogens used. for plant based medicines, three rhizomes, "cekor," "jerangau" and "bonglai" were analyzed. solvent extraction using ethanol, methanol and acetone was carried out, at a concentration of 20-50 mg per ml solvent. the filtrates were used for the antibiotic testing stage. all the three plant species showed moderate antibiotic activity against m. luteus, s. epidermitis, s. pyogenes and s. saprophyticus. interestingly, the antibiotic activity increased when combinations of the herbal extracts were used. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx 5cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most 1% of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx 5cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited 5% to 8% of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax7 and the musclespecific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. metabolic engineering design of an extracellular hgh synthesis system birgül şentürk 1 , pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 : 1 bre lab, department of chemical engng, ankara university, 06100 ankara, turkey; 2 iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.özdamar) metabolic engineering design of an extracellular human growth hormone (hgh) synthesis system is based on cloning the dna encoding human therapeutic protein together with the signal dna sequence of an extracellular enzyme gene into a host-vector system. in this context, extracellular protease (subc) signal dna sequence, i.e. pre-signal dna sequence, was fused into the frame in front of hgh mature dna sequence, by the use of four primers designed using pcr-based gene splicing by overlap extension method. b. licheniformis chromosomal dna and plasmid carrying hgh cdna were used as templates in pcrs, respectively, for the amplification of the subc signal dna sequence and hgh mature peptide sequence. for the fusion of two target genes, i.e. mature peptide sequence of hgh and, signal dna sequences were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their 5 ends that are complementary to 3 portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at 3 end. extension of this overlap by dna polymerase has yielded the recombinant hybrid-gene; and hybrid-gene serve as template for the continuation of reactions for the increase of the concentration in the microreactors. the hybrid gene fragment was first cloned into puc19, and then sub-cloned to pmk4 e.coli-bacillus shuttle plasmid. thus, a new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis 168 (spo − ). the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the proposed metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, hgh, amino acids and organic acids concentrations as the constraints. on the basis of the intracellular bioreaction rates and the interactions with the bioreactor operation parameters, an in-depth insight will be provided for further metabolic engineering design for the extracellular hgh production in r-b.subtilis. medicines based on polypeptides consisting of 30 and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin ␣ 1 production impac system (intein mediated purification with affinity chitin-binding tag) has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin ␣ 1 . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl 2 and we have found that, in case of intein-thymosin ␣ 1 , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of 0.5-1 mm zinc chloride in buffers on all stages of thymosin ␣ 1 isolation. the structure of recombinant thymosin ␣ 1 of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-d) or 1 mg/l naphtalenacetic acid (naa). the developed calli and regenerated plants were maintained on 2,4-d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + 2,4 d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his7tnf␤ and n19tnf␤ were expressed in e. coli. high solubility of n19tnf␤ was expected, however, both analogs his7tnf␤ and n19tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n19tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his7tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with 0.2% nls, while his7tnf␤ and n19tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. high level expression of recombinant growth hormones in e.coli faces common problems such as protein aggregation and inclusion body formation. discussions are raised whether it is more beneficial to obtain soluble protein but to loose expression rates. here we describe an experiment based on the hypothesis that slower expression should result in at least partially soluble recombinant protein. experiments were performed on bovine, chicken and mink growth hormones. expression rate was controlled externally by adjusting cultivation temperature, media, inducer amount, and both induction and cultivation times. another approach to the problem was performed through genetic manipulation. we changed strong t7 promoter to e. coli promoter consensus sequence thus reducing expression rate. recombinant growth hormone was still found to form aggregates, even when expressed at extremely low levelsseveral (2-8) percent of total intracellular protein. we developed optimization scheme of insoluble protein production and showed that expression rate minimization is not influencing recombinant growth hormone solubility in vivo thus suggesting an idea of sequence specific aggregation. to optimize the recombinant protein production in small-scale shake-flask system and high cell density fermentation, new tools have been developed at our laboratory which helps to get knowledge about the physiological state of the cell culture. these tools include (i) a quantitative monitoring system for cellular mrnas based on a sandwich hybridization technique, and (ii) a wireless online monitoring tool (senbit), applicable for standard sensors such as ph, po 2 and temperature for the continuous data collection from shake flasks. the senbit system is a new tool supplying valuable information for the optimisation of the expression of recombinant genes in shake flasks and allowing conclusions towards the reproducibility of shake flask cultures. the presentation will focus on use of the sensitive sandwich hybridization technology for the quantitative analysis of process relevant marker genes in different kind of microbial cell cultures with a focus on the production of recombinant proteins. samples from shake flask cultures and high cell density fed-batch fermentations of the yeast pichia patoris, have been analysed. additionally the mrna analysis was combined with the application of the senbit wireless system to study the production of a recombinant protein in shake-flask cultures of p. pastoris. aside from p. pastoris, mrna sandwich hybridization also was used to monitor product expression in fed-batch fermentation of e. coli for the production of a protein with two subunits by sequential induction. quantitative rna analysis as a tool for optimization of tetrameric collagen prolyl 4-hydroxylase production in e. coli a. neubauer 1 , m. bollok 2 , j. myllyharju 1 , p. collagen prolyl 4-hydroxylase (p4h) involved in the biosynthesis of collagens is an ␣ 2 ␤ 2 tetramer. recombinant expression of p4h in e. coli was described recently . the construct for cytoplasmic expression contains the genes of both subunits in one plasmid under control of different promoters. the ␣ subunit forms inactive aggregates, when expressed separately. in mammalian cells the ␤ subunit is available in large excess and keeps the ␣ subunit in a soluble active form. to mimic this in the bacterial system, we induced both genes sequentially. after induction of the ␤ subunit with iptg, expression of the ␣ subunit was initiated with anhydrotetracycline. here we use the analysis of the product mrnas with a bead based sandwich hybridisation assay (sha) (rautio et al., 2003) for optimization of the fermentation procedure. a high p4h activity was obtained if a high mrna level of the ␣ subunit could be maintained over a longer time. the obtained results illustrate the importance of the second induction for a high level expression of the p4h tetramer. the cells need to be in a "healthy state" with low metabolic load to react efficiently to the second induction. the data illustrate the optimization of a fermentation process by monitoring mrna levels which is of general interest for optimization of products which are difficult to detect. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx 5cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most 1% of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx 5cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited 5-8% of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax7 and the muscle-specific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. collagen and its derived product gelatin are attractive mammalian proteins to be used as model for the production of complex heterologous proteins in plants. the availability of a recombinant product will provide a safer, more homogeneous product than the current animal-derived material. the aim of the project is to investigate the feasibility of a production system for the accumulation of recombinant collagen for conversion to gelatin using barley. the 5 -end of the cocksfoot mottle virus (cfmv; genus sobemovirus) genomic rna sequence, called cfmv -element, has been shown to enhance recombinant protein synthesis in barley (wo 01/55298, mäkinen et al., 1995) . the -element will be used to study whether accumulation levels of complex mammalian proteins can be further increased, using collagen as a model that will serve as basis for exploring the expression of other complex proteins. this system can be study the production of barley-derived recombinant collagen for conversion to gelatin. classical swine fever virus (csfv) is an animal pestivirus which can be used as a surrogate model to elucidate the role of envelope glycoproteins of closely related human hepatitis c virus (hcv). the necessity to use the surrogate models for hcv is due to the fact that this virus cannot be grown in vitro cultures. csfv genome codes for three major antigenic glycoproteins which are located in the same cluster of genes; they are designated as e2 and e0 (e rns ) and e1. glycoproteins form heterodimeric and homodimeric complexes on the external part of viral particles. it is generally accepted that envelope glycoproteins play a major role in the initial stages of viral infection both for csfv and hcv. formation of complexes is needed to effectively infect host cells. we have investigated the formation of glycoprotein dimers by immunoperoxidase monolayer assay and by immunoblotting (western blotting). immunoblotting is a very useful technique in these studies because the complexes are formed via cysteine-cysteine disulphide bonds and they are retained during sds-page under non-reducing conditions. by modifying the glycoprotein genes and by arresting n-glycosylation of e2 and e0 we have investigated which factors influence the formation of complexes. it has been found that some glycosylation inhibitors which act at the early stages of glycan chain processing influence, not only glycosylation, but also the stability of e2 protein, effectively inhibiting the formation of glycoprotein complexes and the yield of the virus. these inhibitors are potential agents for arresting the multiplication and spread of csfv, and its relative -human hcv. recombinant proteins have been produced in a variety of heterologous protein expression systems. eukaryotic unicellular algae have distinct advantages, e.g. it can synthesize complex protein that requires post-translational modification. furthermore, microalgae can be grown in confined environment and thus prevents leakage of genes to the environment. our group has developed a platform technology for the production of recombinant proteins in red microalga porphyridium sp. we have constructed algal transformation plasmid vectors containing a camv 35s promoter and polya signal site. a streptoalloteichus hindustanus bleomycin-resistant gene was used as the selective marker. we have expressed ovalbumin and hepatitis-b surface antigen (hbsag) as model proteins. transformation was carried out by agitating algal cells and vector dna with glass bead. transgenic lines were selected by growing algal cells on agar plate containing 6 g/ml zeocin. positive transgenic lines were selected by screening the colonies by pcr and confirmed by dna sequencing. expression of ovalbumin and hbsag protein was examined by western blot analysis. ovalbumin was found to be expressed inside the algal cells while small hbsag was secreted into the medium due to presence of signal peptide. these findings indicate that red microalgae are capable of producing heterologous proteins. life-material exhibition as ethical interpretation chang shih-lung biotechnology industry study center, taiwan institute of economic research, taipei 106, taiwan. email: schang@tier.org.tw in this thesis we aim to explore the medical-related life-material exhibitions within ntu hospital-the humanity building, and taipei mackay hospital-the historical showroom as abecedarian clues to understand the burgeoning phenomenon-medical museums in taiwan. following these clues, we treat the whole context of medical museums, which transform values through situational construction, as the background to interpret the ethical implications of group val-ues that are transformed in the medical profession. in this thesis, we see medical museums, as the social prescription transforming medical profession, format the ritual context of situation ethics with the cultural construction of life-material. multi-disciplinary interactions and visiting itinerary can be transferred to the exploring horizon of research approach through description and interpretation. among them, we observe that humanistic elements have become essential equipment (or mat'eriel) of medical profession. though humanistic equipment (or mat'eriel) has its bottleneck in the museum situation, it can also unblock new possibilities for museum exhibitions, ethical practice or life ethics. as part of a wide research program aimed at developing new antitumoral agents, we present herein a series of stereoisomeric derivatives of fused tetrahydrofuranes (fthf) substituted with diverse protecting groups either at the primary or secondary hydroxyl groups. unprotected derivatives were also synthesised to investigate the influence of substituents on the in vitro activity of fthf. data on the synthesis, chemosensitivity and apoptosis induction by this series of fthf will be correlated to substitution pattern, stereochemistry and protecting groups, as an aid to the rational design of novel antitumour drugs. establishment of in vitro test systems for pulmonary edema resorption by peptide drug candidates dominik geiger, aswin mangerich, rudolf lucas, klaus p. schäfer, inge mühldorfer 1 department of biotechnology, altana pharma ag, konstanz, germany; 2 imc university of applied sciences, krems, austria tnf-␣ was found to up-regulate the rate of lung liquid clearance (llc) in several animal models by the activity of its lectin-like tip domain. this activity can be mimicked by a circular peptide designated tip. tip was shown to induce llc and consequently pulmonary edema resorption in different animal models. in order to study the mechanism of action of tip, we established two in vitro test systems for edema resorption with the human lung epithelial cell line calu-3: (1) the ability of calu-3 cells for spontaneous dome formation within confluent monolayers was utilized for quantitative examination of tip's activity on active transepithelial fluid transport ("dome assay"). (2) the effects of tip on bioelectrical properties of polarized cell monolayers were studied by using the transepithelial electrical resistance (teer) technology ("teer assay"). dome assay experiments confirmed that dome formation is a sodium dependent process and that tip is able to increase this process. teer assay experiments proofed that tip acts in a polarized and dose dependent manner. in conclusion, there is strong evidence that the dome and the teer assays are suitable systems for in vitro activity testing of tip and other anti-edema peptide drug candidates and are useful for studies on their mechanism of action. increasing safety concerns in gene therapy result in more stringent regulatory requirements. those cover the complete process chain of cell banking, fermentation, and purification. a wide range of applications for pdna requires gram to kilogram amounts for clinical trials and market supply. economic, productive and robust processes are a prerequisite for low cost of goods (cogs). therefore manufacturer of biopharmaceuticals need to address these considerations by developing new production processes meeting the new standards. boehringer ingelheim austria developed a novel pdna production process suitable for large-scale cgmp production. the process is based on e. coli fermentation. the process contains no components from animal origin. the optimized fermentation process yields up to 1 g pdna/l fermentation volume. for isolation of pdna from e. coli alkaline lysis in glass bottles or stirred tanks is commonly used. cell wall structure is destroyed by a combination of alkaline ph and detergents. in our process alkaline lysis is operated in a closed, continuous system directly connected to clarification without using enzymes. we developed a scalable process for pdna purification based on 3 different chromatographic principles. the capture step is carried out by hydrophobic interaction chromatography followed by anion exchange chromatography as intermediate step. final polishing is carried out by conventional size exclusion chromatography in a group separation mode providing also buffer exchange and desalting for the final formulation. the process results in a pdna drug substance of highest quality containing a low level of impurities (genomic dna, rna, proteins endotoxines) suitable for therapeutic applications. depending on the conditions during fermentation and the used host homogeneities of greater than 95% or even 98% are possible, while a high over all yield can be achieved (∼50%). during the development monolithic chromatography supports (cim ® ) were compared with conventional resins and evaluated as potential alternatives. the complete process is monitored by a set of analysis covering cell banking to final purification. new sensitive methods based on hpce, hpiex and fluorometric measurement were developed. whereas many natural amino acids are currently produced by very cost efficient biological processes, the manufacture of methionine is still performed by traditional chemical synthesis. this was mainly due to the poor performances of the currently available producing strains that inhibited the development and commercialization of a biological process to l-methionine. metabolic explorer has recently reinvestigated and developed an efficient biological process that employs an engineered microorganism and utilizes a renewable starting material (corn sugar) as its feedstock and converts glucose into l-methionine. we will describe (a) the general scheme for the engineering of the host organisms and (b) the general approach to maximize carbon and reducing equivalent throughput to l-methionine. to highlight this effort, we will present the global approach developed to improve the process combining metabolic flux analysis, traditional protein biochemistry, molecular biology and fermentation optimisation. saccharomyces cerevisiae is an established 'work horse' of the fermentation industry and modern biotechnology has led to a spectacular expansion of the range of products that can be produced by this yeast. however, for the large-scale sustainable production of chemicals, it is equally important that the range of carbohydrate feedstocks be expanded. especially relevant in this respect is the ability to consume the pentose sugars d-xylose and l-arabinose, which make up a substantial part of plant carbohydrates. wild-type s. cerevisiae strains cannot metabolise d-xylose, but are capable of slowly metabolising its keto-isomer, d-xylulose. therefore, efficient conversion of d-xylose into d-xylulose has long been a key issue in yeast metabolic engineering. non-saccharomyces yeasts that is capable of growing on d-xylose use two enzymes, xylose reductase and xylitol dehydrogenase for this purpose. while both enzymes have been successfully expressed in s. cerevisiae, this is not always compatible with efficient product formation. for example, in the case of ethanol production, the different cofactor specificities of these two oxidoreductases cause massive byproduct formation. theoretically, introduction of a xylose isomerase, which catalyses the interconversion of xylose and xylulose, might circumvent these problems. however, it is notoriously difficult to express bacterial and archaeal xylose isomerases in s. cerevisiae and, until recently, activities of heterologous xylose isomerases expressed in s. cerevisiae were vanishingly low, at least under physiological conditions. a breakthrough was reached when, in 2003, a xylose isomerase gene from the anaerobic fungus piromyces was expressed in s. cerevisiae. while this led to high activities of xylose isomerase, these were not enough to enable fast growth or product (ethanol) formation. in this presentation, we will discuss how a combination of metabolic and evolutionary engineering led to fast and efficient xylose utilization by engineered saccharomyces cerevisiae strains under aerobic as well as anaerobic conditions. furthermore, we will illustrate how evolutionary approaches can be applied to facilitate the utilization of mixed substrates. hyaluronic acid (ha) is a natural and linear polymer composed of ␤-1,3-n-acetyl glucosamine and ␤-1,4-glucuronic acid repeating disaccharide units with a molecular weight (mw) up to 6 mda. it is a major constituent of the extracellular matrices and the synovial fluid. in the last decades, various fields of application including cosmetics, ophthalmology, rheumatology, tissue engineering and drug delivery have been explored, owing to the many important biological functions of ha and its unique physico-chemical properties. however, for some specific applications, the relatively high mw of ha is a limiting factor and the availability of low mw fractions would be highly desired. for food applications, low mw ha has been shown to penetrate the gastrointestinal barrier, thereby increasing the ha bioavailability. moreover, low mw ha fractions are able to re-establish the ha content in the skin and can thus be used in cosmetics as anti-aging and anti-wrinkle agents. finally, low mw ha has shown to prevent oxygen free radical damage in granu-lation tissue during wound healing. a range of methods has been described for the depolymerization of ha to low mw fractions. these techniques involve heat treatment, ultrasonication, uv/gamma irradiation, chemical and enzymatic degradation. we present results on a degradation process of ha originating from bacillus subtilis fermentation into well-defined low mw fractions. the process developed in lab scale is safe, well-controlled and produce low mw ha fractions with narrow polydispersity. moreover, the process is readily up-scalable. the low mw ha fractions are being evaluated in various cosmetic applications. metabolic pathway manipulation for improving the properties and productivity of microorganisms is becoming an established concept. metabolic engineering can be defined as the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or introduction of new ones with the use of recombinant dna technology. a detailed analysis of the physiological means of the different pathways is needed to be able to introduce modifications aimed to the production of not only important metabolites, but also to understand the fundamentals of cell biology. aimed to produce single compounds, metabolic engineering necessarily includes the modification of the cellular pathway(s) as well as the redirection of the energy toward the production itself. the existing metabolic engineering applications are the culmination of more than two decades of global experience developing processes for the production of fine chemicals, vitamins, nutraceuticals and animal nutritional aids such as amino acids. based on the relative low complexity, the first biotechnological applications have been developed from microorganisms. our laboratory has been engaged in this field since different years. yeasts like saccharomyces cerevisiae, kluyveromyces lactis and zygosaccharomyces bailii have been developed for the production of fine chemicals like lactic and ascorbic acids from d-glucose. in this contribution, we will present the last data obtained. since 40 years amino acid production is in the focus of industrial microbiology. l-glutamate and l-lysine are produced with corynebacterium glutamicum, while escherichia coli is used for l-threonine production. the worldwide market of threonine drastically increases: in 1996 the amount of threonine produced worldwide was 15,000 t and raised to 30,000 t in 2000 and 45,000 t in 2004. concerning predictions of experts the demand of threonine will rise with a two-digit rate of economic growth within the next few years. meanwhile the prices declined. these conditions enforce very efficient production processes. beside the strain development and an optimised downstream procedure the fermentation process is an important target for productivity improvement. strain development is dependent on detailed knowledge of the production strains. with innovative methods we are able to get a close look inside the cells under different culture conditions. these methods have been called 'omics' in recent literature. knowledge about genome, transcriptome, proteome, phosphoproteome, metabolome and fluxome leads to new ideas for strain improvements. data generated by these methods must be based on clearly defined culture conditions. therefore, highly parallel fermentations have to be performed to generate biological parallel samples. cutting-edge technical equipment is the basic requirement for experiments like this. other requirements are optimised sampling for different analysis, technical parallels of analytical steps and a detailed statistical analysis of data. these procedures guarantee distinction between real data and data noise. integration of all these data to a holistic model of the cell is the challenge for the future. by combination of a new strain, process and downstream improvements the plant productivity was increased drastically. we ended up in an optimized, fast and high yield process to scope challenges worldwide market comes up with. rieping, m., hermann, t. (2002); fermentation process for the preparation of l-threonine; wo/0218543. ) are potentially quite useful biocatalysts, as they allow for the regioselective and stereoselective hydroxylation of activated as well as non-activated carbon atoms. in addition, the large number of members of the p450 superfamily exhibits a wide diversity of specificities from which a useful biocatalyst may be selected. from a technical point of view, however, they have significant drawbacks. thus, they usually cannot be produced in large quantities nor recovered or stored without a severe loss in activity. their catalytic activity is mostly quite low, and their operational stability leaves much to be desired. most p450 enzymes require a complex protein/phospholipid machinery for activity, and the final electron donor in the reaction cascade, usually nadph, does require extensive recycling to arrive at a commercially satisfactory process. recently, the use of bacterial cytochromes as hydroxylation biocatalysts has received considerable attention. some of them are natural fusion proteins, which contain the heme and the reductase domains on a single polypeptide chain. they are catalytically much more active compared to, e.g. cytochromes occurring in human tissue. we thus have set out to further improve the technical applicability of these enzymes, and have centered our activities around several bacterial cytochromes. it proved very useful to apply rational mutagenesis and directed evolution to these enzymes, leading to a surprising compatibility of mutant enzymes with a wide variety of substrates. mechanisms for the limited stability of the enzymes were explored, leading to hybrid enzymes with enhanced stability, and the cofactor problem was alleviated using auxiliary enzymes or mediator-based technologies. as a result, a bioreactor based on microbial cytochromes was built and operated for several days. baeyer-villiger monooxygenases represent useful biocatalytic tools as they can catalyze reactions, which are difficult to achieve using chemical means. however, so far only a limited number of these monooxygenases were available in recombinant form kamerbeek et al. (2003) . using a recently described protein sequence motif fraaije et al. (2002) and the available genome sequence information, we were able to identify and overexpress a number of novel bacterial bvmos. one of the overexpressed bvmos was found to be relatively stable as it originates from thermobifida fusca, which grows at ∼60 • c. the enzyme was shown to be active on a broad range of substrates, preferring aromatic ketones fraaije et al. (2005) . the best substrate discovered so far is phenylacetone, hence its name: phenylacetone monooxygenase. we have solved the crystal structure of phenylacetone monooxygenase, which represents the first structure of a bvmo malito et al. (2004) . the crystal structure provides insight into the complex mechanism of catalysis mediated by fadcontaining bvmos. by site-directed mutagenesis we have probed the role of several active-site residues. a crucial role is played by an arginine residue. as phenylacetone monooxygenase shares significant sequence identity (>40%) with all known nadph-dependent bvmos, many of the observed structural features seem to be conserved within this class of atypical monooxygenases. by homology modeling using the phenylacetone monooxygenase structure, catalytic properties of other baeyer-villiger monooxygenases can be explained or predicted. screening for fungal baeyer-villiger monooxygenases l. butinar 1 , j. friedrich 1 , v. alphand 2 : 1 laboratory of biotechnology, national institute of chemistry, ljubljana si-1001, slovenia; 2 groupe biocatalyse et chimie fine cnrs fre2712, université de la méditerranée, marseille, france the asymmetric form of the baeyer-villiger (bv) oxidation (transformation of ketones into lactones) is an important challenge for organic chemistry since the obtained lactones are valuable building blocks for synthesis of countless biologically active products. to date, enzymatic or microbial bv oxidations appears as more successful than their chemical counter-parts. (ten brink et al.) whereas most active bv monooxygenases are produced by bacteria (among which the well-studied enzyme of acinetobacter calcoaceticus), only a few fungal strains expressing bvmo were described (alphand et al., carnell and willetts) . in order to increase the number of available biocatalysts which perform such an asymmetric biotransformations, a screening of fungi belonging to major groups of zygo-, ascoand basidiomycetes was conducted using bicycloheptenone as testsubstrate. surprisingly, a large number of the tested fungi were able to transform the substrate into one to four different lactone isomers. the yields, the enantio-and regio-selectivity of the reaction depended on the fungal strain. alphand, v., furstoss, r., 2000. j. mol. catal. b 9, 209-17. carnell, a., willetts, a.,1992 . biotechnol. lett. 14, 17-21. ten brink, g.j., et al., 2004 . pyranose oxidase (p2ox) is a periplasmic enzyme that widely occurs in basidiomycetes. it catalyses the c-2 oxidation of several aldopyranoses to the respective 2-keto derivatives, transferring electrons to molecular oxygen to yield h 2 o 2 . p2ox is of interest for carbohydrate conversions, as its reaction products (2-keto sugars) can be attractive intermediates in the production of food ingredients. we cloned the gene encoding p2ox, and subsequently amplified a cdna clone by rt-pcr. the cdna was inserted into a bacterial expression vector and successfully expressed in e. coli. properties of the heterologous protein were compared to those of the native enzyme showing that they are essentially identical. both the native as well as the recombinant enzyme were used in biotransformations of sugars. recently, the 3d-structure of this tetrameric enzyme was elucidated. based on structural information, several enzyme variants containing point mutations were constructed and further characterised. two of these variants (e542k and e542r) displayed improvements in stability and certain kinetic properties thus making them attractive for biocatalytic applications. lactones are important compounds for the fragrance and flavour industry. right now the production of lactones is dependant on the import of crude materials from tropical countries. in this project, we want to tackle the manufacture of lactones via a biocatalytic route using p450 monooxygenases. cytochrome p450 monooxygenases catalyse the oxyfunctionalization of non-activated c-atoms. cyp102a1 from bacillus megaterium, cyp102a2 and cyp102a3 from bacillus subtilis are soluble fusion proteins comprising p450 monooxygenase and fad/fmn reductase domains in one polypeptide chain. all three enzymes are highly homologous fatty acid hydroxylases. especially, cyp102a1 also known as p450 bm-3 is well characterized and shows high activity compared to other p450 monooxygenases. the aim of the work is to change selectivity but conserve the high activity that is typical for those enzymes. using methods of structure modelling, rational protein design and directed evolution new mutants of these enzymes with changed regioselectivity are obtained. products of conversion with monooxygenases are intermediates in the production of lactones. the interface of biology and materials science has led to new materials with unique structural and functional properties, and new process technologies with the ability to produce, from "bottoms up", a wide range of biomimetic structures. these materials and their designs have broad application as catalysts, sensors, and devices for use in synthesis, cell and tissue engineering, bioanalysis and screening, and nanoelectronics. we have focused on the generation of sugar-based nanostructures, complete with tailored selectivities and biocatalytic activities at the molecular and nanoscales. these include biocatalytically-generated carbohydrate derivatives that selfassemble with high precision to give novel architectures with functional and responsive properties. izumoring: a strategy for total production of rare sugars ken izumori rare sugar research center, kagawa university, kagawa 761-0795, japan. e-mail: izumori@ag.kagawa-u.ac.jp we found a new enzyme, d-tagatose 3-epimerase (dte), that epimerize all ketohexoses at c-3 position. this epimerase catalyze not only between d-tagatose and d-sorbose, but also d-fructose = dpsicose, l-sorbose = l-tagatose, and l-psicose = l-fructose. this new enzyme offered us a useful key tool to connect all ketohexoses using hexitols as intermediates. the figure shows that all eight ketohexoses can be connected with dte and polyol dehydrogenases (pdh) in a ring. using this ring, we can easily find the pathway to transfer d-fructose to d-tagatose via d-psicose using dte and pdh. various aldose isomerases transform ketohexoses to the corresponding aldohexoses. so, we can connect all 16 aldohexoses with 8 ketohexoses using the enzyme. finally, all hexoses, 8 ketohexoses, 10 hexitols and 16 aldohexoses are connected using enzyme reactions in a ring structure (not shown). this kind of strategy is effective also on transformation of tetroses and pentoses. now, we can produce all monosaccharides; tetroses, pentoses and hexoses by enzyme reac-tions. the bioproduction strategy of all rare sugars (monosaccharides that are rare in nature) is illustrated using ring form structures named as izumoring. we have already succeeded to produce d-psicose in large scale and are now in the progress of mass production of various rare sugars from natural and cheap sugars using izumoring. bioprocess development for chiral intermediates christian wandrey institute of biotechnology, forschungszentrum jülich gmbh, jülich d-52425, germany chiral alcohols, diols, amino alcohols and chiral acids (e.g. hydroxy acids and amino acids) play an important role in pharma and agro synthesis. in the past such chiral intermediates were obtained by racemic resolution via chiral reductions using prochiral precursors. here the problem of cofactor regeneration arises. this problem could be solved by enzyme-coupled or substrate-coupled cofactor regeneration using formate or isopropanol as reducing agent. alternatively, whole cell bioreductions were developed where glucose is used as the reducing agent. in recent years "designer microorganisms" were developed in which oxidoreductases (e.g. alcohol dehydrogenases) were over-expressed in escherichia coli. in such cases, cofactor regeneration was achieved intracellularly with isopropanol as the reducing agent or by coexpression of a formate dehydrogenase, so that once again format could be used for reduction. another route to obtain chiral intermediates is a fermentative approach using classical pathways (like the aromatic amino acid pathway). here, the pathway is interrupted after the intracellular production of chorismate. new chiral intermediates can be obtained by over expression of additional genes, which catalyzed the production of chorismate derivatives leading to cyclohexadiene-transdiols and the corresponding amino cyclitols. the last example can be regarded as an example of industrial biotechnology where glucose is used as starting material (white biotechnology). here bioprocess development is carried out in an integrated approach, in which molecular biochemical engi-neering cares for the optimal intracellular metabolic network and the classical biochemical engineering cares for the optimal environment of the cell in a fermenter. examples will be given which reach (in cooperation with industrial partners) up to kilogram scale. biotransformations are usually involved in just one or very few separate reactions in organic syntheses. the development of a cell-free "system of biotransformations" (sbt), in which a set of enzymes acts in a coordinated fashion in a one-pot synthesis, lead to increased catalytic complexity, selectivity and yield, as well as facilitated operation at reduced costs. the example chosen to prove the usefulness of the sbt-approach is the production of dihydroxyacetone phosphate (dhap). dhap, a c3-compound from glycolysis, is an important precursor for asymmetric c-c-bond formation. so far, the production of dhap is difficult and expensive. for the construction of the dhap-producing sbt, e. coli's glycolysis is isolated from the metabolism to an as large as possible extent by the construction of a multi-ko-mutant. a culture is grown in an appropriate medium, homogenized in the production buffer, and used as the catalytic system. high production yields can be achieved since the production pathway is almost completely isolated from the metabolic network. the employed dhap-producing sbt provides not only a path from glucose to the product, but also an integrated atp-regeneration and nad-recycling system. in first experiments with a tpi-ko-mutant, a dhap-production yield on glucose of 32% could be achieved, without optimizing the system. the system remained active for more than 24 h. up to now, atp cannot be applied in catalytic concentrations, but has to be present in equimolar amounts to glucose. the production yield could be increased by 10% through the addition of phosphate ions as substrate to the reaction, enabling the system to utilize atp more efficiently. these experiments indicate that the sbt-approach is viable and a large potential remains to improve the dhap-producing sbt. for some years novozymes have manufactured a pectate lyase for scouring of textile as an ecological alternative to the traditional harsh chemical treatment, and recently, we at novozymes discovered additional applications for pectate lyases. however, to be commercially attractive more robust pectate lyses had to be developed. in this paper, we will demonstrate how we for two different pectate lyases have improved their stability significantly. as the two enzymes are quite similar in sequence and structure, it was a new discovery for us to find that different concepts of protein engineering had to be used in our attempt to stabilize each individual pectate lyase. the stability of one enzyme was improved by substitutions in the internal of the structure whereas the stability of the other pectate lyase was primarily improved by changing surface residues. starting with knowledge from structural analysis, we have applied rational based protein engineering resulting in few selected variants. also random based protein engineering combined with screening of hundreds of thousands variants was used. in conclusion: the project team showed that by synergistic use of the two approaches, we were able to move faster towards a solution and eventually we succeeded finding new stabilized pectate lyase variants, applicable for new business areas. the importance energy independence as a national goal equals or exceeds that of the moon landing in 1990. the development of a new industry to produce fuel ethanol from woody biomass would increase national security, improve employment and the environment, and provide substantial relief from the debt of imported petroleum. costs associated with the rapid development of this new industry (∼$1.4 billion per year) could be paid by re-assigning 1 cent per gallon from existing federal gasoline taxes, a small price to pay for future energy independence. the corn-to-ethanol industry continues to make a remarkable contribution to our liquid fuel needs through expansion. today, one row of every six rows of corn is converted into fuel ethanol in the u.s. however, this expansion will be limited to 3-4% of total automotive fuel by the economics of corn costs and production. corn can do more! corn stover is the single most abundant agricultural residue in the us and can be used as a feedstock to produce 60-80 gallons of ethanol per dry ton. further expansion with other biomass feedstocks such as agricultural and municipal residues (lignocellulose, woody biomass) could produce over 100 billion gallons of fuel ethanol annually according at a recent joint report by the usda and doe (april, 2005) . current technology has been demonstrated at pilot scale for the production of fermentable sugars from hemicellulose by dilute acid hydrolysis and for the hydrolysis cellulose using fungal cellulose enzymes. biocatalysts such as recombinant escherichia coli have been developed and demonstrated for the efficient conversion of all sugar constituents of biomass to ethanol. a national goal for the full-scale deployment of current technology to produce biomass-based fuel ethanol will allow the us to reduce imported petroleum by 50%. together with increased efficiencies of hybrid vehicles, energy independence could be achieved within 10-20 years. similar gains could be realized by many nations around the world to provide new manufacturing and employment, redistributing wealth and ensuring a cleaner, healthier environment. bioethanol production using thermophilic bacteria marie just mikkelsen, birgitte k. ahring emab, biocentrum-dtu, 2800 lyngby, denmark the industry of bioethanol production is facing the challenge of redirecting the process from fermentation of relatively easily convertible but expensive starchy materials, to complex but inexpensive lignocellulosic biomass. on lignocellulosic hydrolysates, gram-positive thermophilic bacteria have unique advantages over the conventional ethanol production strains. the primary advantages are their natural broad substrate specificities, and in some strains, a high tolerance to lignocellulosic hydrolysates. moreover, ethanol fermentation at high temperatures also has the advantages of high productivities and substrate conversions, low risk of contamination and facilitated product recovery. some thermophilic bacteria naturally produce primarily ethanol from most sugar monomers present in lignocellulosics, but modifications are still necessary to increase ethanol yields. the release of useable sugars from lignocellulose biomass for industrial fuel-ethanol fermentation is often facilitated by a weak acid hydrolysis step. as a consequence, inhibitors such as furfural and 5-hydroxymethylfurfural (hmf) are formed as degradation products of xylose and glucose, respectively. moreover, the fermentative end-product of ethanol is also inhibitory. these, and other inhibitors present an environment, which elicits the expression of stress-related genes in saccharomyces cerevisiae. recently, 65 s. cerevisiae genes have been identified as important in furfural stress tolerance. when furfural is present, yeast with these genes disrupted grows poorly compared to wild-type yeast. a sub-class of these genes suggests that yeast grown under furfural-induced stress may rely upon similar pathways as cells grown under various other stresses, including oxidative, heat, and sorbate. to investigate this link further, we analyzed stress-induced phenotypes such as ros activity, dna damage, and membrane damage in wild-type and mutant yeast exposed to furfural or hmf stress. moreover, we investigated whether overexpression of this sub-class of genes would provide protection from furfural-induced stress and oxidative damage. micro-organism to be used in fermentation of lignocellulose hydrolyzates should preferably have three characters: (a) high ethanol tolerance, (b) resistance to inhibitors found in the hydrolyzate, and (c) a broad substrate utilization range, since the hydrolyzate contains several sugars. in addition to the possibility of controlling the level of potential inhibitors, fed-batch fermentations also permit the parallel uptake of several different monomeric sugars. two strains of saccharomyces cerevisiae, cbs 8066 (a commonly used laboratory strain) and tmb 3000 (a strain isolated from a spent sulfite liquor fermentation plant), were characterized in batch and fed-batch fermentation of a dilute-acid hydrolyzate from spruce. the strains had different abilities to ferment spruce hydrolyzate. the study suggests that the furan reduction capacity of a yeast strain is a key factor for its performance in fermentation of lignocellulosic hydrolyzate. polyketides constitute a structurally highly diverse group of natural products that possess broad ranges of pharmacological properties and represent a major source for novel cancer therapeutics. however, these compounds may be sub-optimal in regard of activity, selectivity, availability and unwanted side effects. in addition, the sustainable production of these valuable metabolites can be a challenge. studying the molecular basis of the biosynthetic pathways may set the basis for improving the production and for rationally engineering derivatives with altered bioactivity profiles, e.g. through targeted knockouts, mutasynthesis ziehl et al. (2005) , and swapping of pathway genes. our results in elucidating and manipulating the biosynthesis of selected antitumoral polyketide metabolites from bacteria (aureothin, chartreusin) and fungi (cytochalasines, rhizoxin) are presented. analyses at the genetic and biochemical levels provided new insights into several unusual biosynthetic features, e.g. non-linear polyketide assembly for the nitroaryl-substituted polyketide aureothin he and hertweck (2003, 2005) , an oxidative rearrangement cascade in the chartreusin pathway xu et al. (2005) , and a fungal iterative pks-nrps hybrid synthase schuemann and hertweck (2005) involved in cytochalasin biosynthesis. the most surprising result was obtained from elaborating the biogenesis of the antimitotic agent rhizoxin from rhizopus sp., which allowed for a significant improvement in large-scale production partida-martinez and hertweck (2005). he, j., hertweck, c., chem. biol. 2003 , 10, 1225 -1232 chem. bio. chem. 2005, 6, glycopeptides such as vancomycin and teicoplanin are the drugs of last resort for the treatment of severe infections caused by antibiotic resistant gram-positive bacteria. glycopeptides inhibit the peptidoglycan biosynthesis by binding as dimers to the d-ala-d-ala termini of the cell wall precursors. amycolatopsis balhimycina synthesizes the vancomycin-type glycopeptide balhimycin, whose structure and biological properties greatly resemble vancomycin and which only differs by its glycosylation pattern. using a "reverse genetics" approach we have identified the 66-kb gene cluster encoding the biosynthesis of balhimycin. by a combination of genetics, biochemistry and analytical organic chemistry, we were able to elucidate the biosynthetic pathway and to assign functions to almost all genes of the cluster. the biosynthesis starts with the pathway-specific provision of the non-proteinogenic amino acids ␤-hydroxytyrosine (␤-ht), hydroxyphenylglycine (hpg) and dihydroxyphenylglycine (dpg) which form together with (n-methyl)-leucine and asparagine the heptapeptide backbone of balhimycin. dpg is synthesized via a polyketide synthase mechanism (pksiii) similar to that known from plant chalcon/stilben synthases (pfeifer et al., 2001) . for the ␤-ht synthesis three genes are essential which form an operon (puk et al., 2004) : bpsd, an nrps binds a tyrosine molecule, which is then hydroxylated by the p450 monooxygenase oxyd. the perhydrolase bhp is required for the release of ␤-ht. subsequently bhaa, a nadh/fad-dependent halogenase catalyzes the chlorination of ␤-ht to form chloro-␤-hydroxytyrosine (puk et al., 2002) , which is needed to stabilize the dimerization. the amino acids are linked by non-ribosomal peptide synthetases (recktenwald et al., 2002) , and the aromatic side chains are interconnected by p450 monooxygenases; a series of reactions which lead to the first antibiotically active intermediate. inactivation of the oxygenase genes revealed the order of the cyclization steps (bischoff et al., 2001) : the oxygenases act in a stepwise fashion in the sequence oxyb, oxya and oxyc. the resulting cross-linked heptapeptide is then finally modified by methylation and glycosylation. the biosynthesis is regulated by the strr-type regulator bbr, which was shown to bind in front of different operons of the balhimycin gene cluster. this ensures the coordinated expression of the biosynthetic genes. the non-producing mutants, defective in the supply of the non-proteinogenic amino acids, were used as recipients in cloning experiments as well as in approaches of precursor-directed biosynthesis by feeding chemically synthesized alternative precursors. thus, novel balhimycin derivatives were generated (weist et al., 2002 (weist et al., , 2004 . bischoff et al., 2001 . angew. chem. int. ed. 40, 4688-4691. pfeifer et al., 2001 . j. biol. chem. 276, 38370-38377. puk et al., 2004 . j. bacteriol. 186, 6093-6100. puk et al., 2002 . chem. biol. 9, 225-235. recktenwald et al., 2002 . microbiology 148, 1105 -1118 . weist et al., 2002 . angew. chem. int. ed. 41, 3383-3385. weist et al., 2004 spectroscopy guided discovery of novel bioactive microbial natural products thomas ostenfeld larsen, michael edberg hansen cmb biocentrum-dtu, technical university of denmark, 2800 lyngby, denmark the task of finding novel bioactive natural products is usually bioassay driven. often a certain type of compound (e.g. polyketide, alkaloid) turns out to be active in an assay. when having generated a promising hit in a bioassay the normal procedure in the drug discovery process usually is to produce a large number of structurally analogous compounds either by traditional chemical synthesis or by combinatorial chemistry in order to study structure activity relation-ships and to find even more active lead compounds. alternatively to chemical synthesis of analogues nature can be explored for structurally similar compounds by uv-spectroscopy guided screening. this work will present a new method for the systematic and automated computer assisted search of full uv spectra in large number of datafiles for both dereplication of known and discovery of new natural products based on the use of the new mathematical algorithm x-hitting. exploring the substrate spectrum of the antibiotic producing bacteria saccharopolyspora erythraea p. krabben, p. oliveira, f. baganz, j. ward department of biochemical engineering, university college london, london wc1e 7je, uk knowledge of substrate utilisation capabilities play an important role in the development of genome scale metabolic models (borodina et al., 2005) and refinement of first generation annotations. furthermore, knowledge of the product formation during catabolism of different substrates provides valuable information about the distribution of metabolic fluxes and thereby forms a basis for rational strain improvement. we present here, data on the substrate utilisation capabilities and the corresponding product formation of s. erythraea. this analysis will help in improving the production of erythromycin and provide clues to the activation of the cryptic secondary metabolic pathways present in the s. erythraea genome. reference borodina, i., et al., (2005) . genome-scale analysis of streptomyces coelicolor a3 (2) modeling of growth and product formation on complex media containing multiple substitutable substrates is a challenge. complex media offers the organism multiple choices of carbon and nitrogen substrates including free amino acids, peptides, soluble and insoluble proteins in addition to the defined sources such as glucose and ammonium sulfate. we present a structured model that accounts for growth and product formation kinetics of rifamycin b fermentation in a multi-substrate complex medium. the model considers the organism to be an optimal strategist with a mechanism to regulate the uptake of the substrate combinations. further, we assume that the uptake of a substrate depends on the level of a key enzyme, which may be inducible. the model also considers control parameters as fraction of flux through a given metabolic branch. the control parameters are obtained using a simple multi-variable constrained optimization. the model parameters were rigorously estimated via a specifically designed experimental plan. the model correctly predicts the experimentally observed growth and product formation kinetics and the regulated simultaneous uptake of the substitutable substrates under different fermentation conditions. the model and the model parameters provide useful insights into the growth and product formation strategy of this industrially important process. this presentation will describe the experimental results, the model development and the relevant model parameters for a. mediterranei s699. the recent surge in oil price and the increasing concern on our environment have generated much interest in the production of chemicals from renewable resources. succinic acid, also called as amber acid, is a four-carbon dicarboxylic acid, which can be used as a precursor of numerous products including biodegradable polymers, green solvents, pharmaceuticals, and bulk and fine chemicals. a new capnophilic bacterium named mannheimia succiniciproducens mbel55e was isolated from the rumen of korean cow. this bacterium can produce large amounts of succinic acid along with some other metabolites such as lactic, formic and acetic acids. we have completely sequenced the genome of m. succiniciproducens and charaterized its genome content in the context of metabolic pathways. we then constructed the genome scale in silico metabolic network for metabolic flux analyses, and carried out metabolic flux analysis under varying environmental conditions. based on the in silico analyses results, we selected several target genes to be manipulated for enhanced succinic acid production. detailed results of metabolic engineering based on genome-scale information will be reported. we have been developing tools for inverse metabolic engineering in order to identify gene targets that improve the phenotype of industrial strains and cells for medical applications. to this end, we create genomic fragment libraries from a source organism and use it to transform the host organism. cells are properly selected in environments that favor the phenotype of interest and genes enriched in these cells are sequenced and used in follow up transformations of cells with specific genetic backgrounds. this overall strategy is complemented with additional tools for modulating gene over-expression, gene deletion, and high throughput clone isolation. we will demonstrate applications of this strategy to the identification of gene targets for improved xylose assimilation in recombinant saccharomyces cerevisiae and improved lycopene production in escherichia coli. based on assumed reaction network structures, nadph availability has been proposed to be a key constraint in ␤-lactam production by penicillium chrysogenum. in this study, nadph metabolism was investigated in glucose-limited chemostat cultures of an industrial p. chrysogenum strain. enzyme assays confirmed the nadpspecificity of the dehydrogenases of the pentose-phosphate pathway and the presence of nadp-dependent isocitrate dehydrogenase. pyruvate decarboxylase/ nadp-linked acetaldehyde dehydrogenase and nadp-linked glyceraldehyde-3-phosphate dehydrogenase were not detected. although the nadph requirement of penicillin-gproducing chemostat cultures was calculated to be 1.5-1.7-fold higher than that of non-producing cultures, activities of the major nadph-providing enzymes were the same. isolated mitochondria showed high rates of antimycin a-sensitive respiration of nadph, thus indicating the presence of a mitochondrial nadph dehydrogenase that oxidizes cytosolic nadph. the presence of this enzyme in p. chrysogenum has important implications for stoichiometric modelling of central carbon metabolism and ␤-lactam production and may provide an interesting target for metabolic engineering. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf-21 cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf21 infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp486 coding for am-cyan coral protein, which emits natural green fluorescence. complete elucidation of the genetic control of a metabolic flux requires the availability of fine-grained expression levels of the gene(s) of interest. we developed a collection of promoters of varying strength for tuning gene expression in the yeast s. cerevisiae. engineered promoters were obtained through random mutagenesis of the constitutive tef1 promoter. eleven mutated promoters were selected by fluorescence-activated cell sorting (facs) spanning gradually increasing activities between about 8 and 120% compared to the native tef1 promoter. data were also confirmed at the level of mrna via rt-pcr. by introducing selectable markers in front of the different tef1 promoter mutations, we provided plasmid collections, which can be directly used to amplify promoter replacement cassettes for genomic integration of the fine-grained promoter collection in front of any yeast gene. l-arabinose, widely distributed in plant kingdom, is a component of plant cell wall. l-arabinose does not abundantly exist in free state in plants, but usually in corn hull, sugar beet pulp, gum arabic, mesquite gum, as the polysaccharide such as arabinoxylan and arabinogalactan. to produce arabinose from agricultural wastes, we screened arabinogalactan degradable strain from compost. thereafter, putative arabinase gene from this strain was cloned (b1029 ts2-8). as a result of spectrometric assay using -nitrophenyl ␣l arabinofuranoside, recombinant showed 3-4-fold higher activity than wild type e. coli strain. after enzymatic reaction with corn fiber, b1029 ts2-8 produced 2.15 g/l of l-arabinose, which was detected on hplc. however, the enzyme activity was very low. so, we are transferring the gene into expression vector system. further characterization study and enzyme engineering to enhance the activity toward corn fiber will be presented in poster. there are only a limited number of hypersaline areas all over the world, which include several locations in turkey such as van lake located in eastern region of turkey. isolation and identification of halophilic and hyperhalophilic microorganisms from such locations is essential for the determination of biodiversity in turkey. high-level production of extremozymes from these microorganisms has also many economical advantages due to their stability at extreme reaction conditions. proteolytic enzymes are the most important group of enzymes produced commercially. of these, proteases produced by alkalophilic microorganisms are investigated not only in scientific areas such as protein chemistry and protein engineering but also find wide application in food, pharmaceutical, leather and detergent industries. in this study, 24 microorganisms isolated from van lake were screened for the presence of extracellular alkaline protease activity. the optimum screening temperature and ph were determined as 37 • c and ph 9.5, respectively. one of the isolates that could grow at 0-20% salinity reached highest levels of extracellular alkaline protease activity. this best producer, which was identified as the halotolerant bacillus pumilus, was found to produce alkaline protease both in the presence and absence of nacl. to improve enzyme production yields, culture conditions such as medium composition, growth ph and temperature were optimized. the effect of different carbon sources, organic and inorganic nitrogen sources on the production of alkaline protease was studied. whereas a mixture of inorganic and organic nitrogen sources induced high protease production, use of only an organic nitrogen source supported poor enzyme production. halotolerant bacillus pumilus produced maximum alkaline protease activity when maltose, yeast extract and sodium nitrate were used as carbon source, organic and inorganic nitrogen sources, respectively. this project was supported by tubitak through project tbag 2321-103t069. in the market of biochemical products a very important role is played by heterologous proteins production, and despite recent advances in mammalian cells exploitation, yeasts can still present advantages as host systems. among them, the spoilage yeasts belonging to the zygosaccharomyces genus have become, due to some peculiar properties, significantly attractive. in particular, z. bailii is characterized by acid resistance, osmotolerance to high sugar and ethanol concentration combined with high biomass yield. despite still little is known about its genetics and cellular biology, our group is working on its development and exploitation for recombinant productions with an integrated approach coupling physiological study with the creation of molecular tools for heterologous proteins production. we previously described and did a patent application regarding the first techniques necessary to transform this yeast and to express and secrete different proteins derived from different sources. here we present and discuss the last advances in optimization of heterol-ogous protein expression in particular, on one side we present a reproducible strategy for target gene deletion, leading to the first z. bailii auxotrophic mutant, and on the other we show the improvement of gene dosage and plasmid stability by building a set of multicopy expression vectors based on the sequences of the z. bailii 2 -like endogenous plasmid psb2 and an integrative plasmid. all the known ␥-butyrolactone autoregulator receptors are highly conserved in the dna binding motif present in their n-terminal portions and have been proposed to play roles as transcriptional regulators in antibiotic production and/or morphological differentiation. previously, kim et al. reported that the cloned scar in streptomyces clavuligerus has several characteristics of the autoregulator receptors in the genus streptomyces. in this study, to clarify the in vivo function of scar, a scar-disrupted strain was constructed by means of homologous recombination after introducing a scar-disruption construct via transconjugation from e. coli. no difference in morphology was found between the wild-type strain and the scar disruptant. however, the scar disruptant showed a 1.8-fold higher production of clavulanic acid than the wild-type strain. the phenotype was restored to the original wild-type phenotype by complementation with intact scar. therefore, the autoregulator receptor, scar, acts as a negative regulator of biosynthesis of clavulanic acid but plays no role in cytodifferentiation of s. clavuligerus. lactate dehydrogenase catalyses the production of lactate from pyruvate. it is the first target for many researches on lactic acid producer microorganisms like rhizopus oryzae. in the present study based on the known sequences of r. oryzae ldha and ldhb genes skory (2000), they were cloned and expressed in a citric acid producer fungus aspergillus niger. the aspergillus niger strains expressing ldha or ldhb gene resulted in increased production of lactate in aspergillus niger. among 50 transformants tested 4 ldha and 5 ldhb expressing strains were found to have higher lactate dehydrogenase activity compared to wild type in the conditions tested. the highest specific activity obtained with ldha transformants was only 2.5 times of the wild type while this was 10 times for one of the transformants expressing ldhb. in addition to increased lactate production citric acid production was also increased. however, gluconic acid production ceased in the ldha or ldhb expressing a. niger strains. the production of lactic acid in a. niger transformants and lactate dehydrogenase a and lactate dehydrogenase b enzymes are being investigated in the chosen strains. selection of n source suitable for production of rhodococcus sp. biomass for the purposes of microbial transformation of 5␣h-epoxypregnanolone (5␣h) and 5 -epoxy-pregnenolone ( 5 ) into their 9␣-hydroxy-derivatives was carried out. three dehydrated and three non-dehydrated n sources were tested. the transformation reaction was carried out in phosphate buffered medium containing 1 g l −1 of the steroid substrate and 0.1 g l −1 cells. the steroids were determined by hplc. the transformation resulted in formation of three derivatives appearing in the reaction medium in the sequence: 4 -3-keto-; 9␣-hydroxy-and 9␣,20␤-hydroxy-epoxy-pregnenolone. a strong influence of the n source on the hydroxylating activity of the biomass was observed. triptose (difco) gave a cell depot actively hydroxylating 5␣h without any significant accumulation of the 4 -3-keto-derivative. the most effective accumulation of hydroxylated derivatives of 5 was observed with biomass grown on freshly prepared meat extract while the commercial products triptose (difco), meat extract (difco) and lactalbumin (flika) gave valuable information about the dynamics of the transformation process. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene 1 , nazif kolankaya 2 : 1 kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; 2 hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth (1% soytone, 4% d-glucose and 0.5% cellulose pulp). maximal extracellular ligninase production was detected after7 day (7 nkat). the optimum biobleaching conditions are 30 • c and ph: 4.8, with 10 days. in this condition p. versicolor decreased the kapa number from 38.55 to 19.42 and increased brigthness from 28 to 32.7 in 10 day treatment. xylanase production, purification and characterization from a soil isolate, bacillus m-13 ayşegül ersayin 1 , aytaç kocabaş 1 , b. zümrüt ogel 2 , ufuk bakir 3 : 1 biotechnology department, middle east technical university, ankara, turkey; 2 food engineering department, middle east technical university, ankara, turkey; 3 chemical engineering department, middle east technical university, ankara, turkey, 06531. e-mail: ubakir@metu.edu.tr (u. bakir) xylan is a major component of the plant cell wall, representing up to 35% of the dry weight. xylan molecule is a complex polymer consisting of a ␤-d-1,4-linked xylanopyranoside backbone substituted with acetyl, arabinosyl and glucuronosyl side chains. hydrolysis of the xylan backbone is mainly catalysed by endo-␤-1,4-xylanases (ec 3.2.1.8). many bacterial and fungal species are able to utilize xylan as a carbon source. interest in the enzymology of xylan hyrdolysis has increased because use of xylanases in bioconversion of agricultural wastes to valuable products like single cell protein, xylo-oligosaccharides and fuel, in bio-bleaching processes, food and animal feed industries. in this study, xylanolytic nature of a soil isolate bacillus spp., bacillus m-13, has been shown. bacillus m-13 produced multiple xylanases when grown on a liquid medium containing agricultural wastes as the sole carbon source. various agricultural wastes including corn-cobs and cotton waste, with and without pretreatments were used to maximize enzyme production. the major xylanase having molecular weight of 20 kda upon sds-page and a pi of 9.1 upon ief was partially purified by liquid chromatographic techniques 150-fold with 40% recovery, including gel filtration, ion exchange and hydrophobic interaction chromatography. enzymes are important constituents in the laundry detergents due to their contribution to shortening washing times, reduction of energy and water consumption by lowering washing temperatures, provision of environmentally friendlier wash-water effluents and fabric care. however, they can loose a significant part of their activity in the chemically-hostile detergent matrix over a time period of several weeks. therefore, improving the storage stability of enzyme granulates is the main challenge in the development of a new product. the complexity of the detergent matrix implies the presence of a complicated mechanism involved in the inactivation of the enzymes. a combination of factors, such as oxidation by h 2 o 2 released by the bleaching agents, humidity, high temperature, autolysis of enzymes, high local ph in a granule, oxygen, and other detergent components, plays a role in the activity loss. an experimental investigation on the inactivation of the solid-state enzyme during storage has been initiated. the release rate of h 2 o 2 from the bleaching agent, sodium percarbonate, was determined using a simple and accurate method for measuring the gas phase h 2 o 2 concentration. the deactivation kinetics of pure enzyme was determined as a function of gas phase h 2 o 2 concentration and humidity. the preliminary results indicate that humidity plays a significant role in the inactivation mechanism of the detergent enzyme due to a possible increase in the mobility of the enzyme molecule and the surface area exposed to destructive agents. the effect of main granulate ingredients on the stability of the enzyme was investigated and the extent of the protection of each component was estimated. the study is important for the revealing of the phenomena occurring in the detergent matrix during storage. understanding the inactivation mechanism provides a valuable tool for the development of more effective protective coatings and stabilizers. the use of biosurfactants in cosmetic industry has attracted great attention of biotechnological researchers because they consist of two inexpensive, renewable and easily accessible starting agricultural materials: sugar and oil/fat. carbohydrate based products are non-toxic, biocompatible and biodegradable. in addition, the enzymatic processes present many advantages in comparison with the chemical methods, which employ high temperatures in the presense of alkalin catalysts, high-energy consuption and low selectivity of products. sugar esters present vast application, such as for antibiotics, biomaterials, surfactants, cosmetics and so on. we investigated the synthesis of sugar vinyl esters, using protease-catalysed transesterefication method applying protease from bacillus subtilis. sucrose 0.125 m and vinyl ester 0.5 m has been mixed in dimethylformamide at 160 rpm of agitation. at first, we have studied the effects of protease from bacillus subtilis concentrations (5, 10, 20 and 40 mg/ml) as catalyst. afterwards, the influence of the temperature (30 and 50 • c). after that, the influence of the molar ratios (1:1; 1:2; 1:4 m/m) between vinyl laurate (ch3(ch2)10cooch ch2) and sucrose. subsequently, we investigated the effects of water amount, using 0, 5, 10 and 20% of water in dmf. the conversion ratios of sucroseto-sucrose esters were determined decreasing sucrose measurement with hplc. the results showed that the best conditions to produce high activity on the enzymatic reaction was by using 40mg/ml of protease from bacillus subtilis at 30 • c, molar ratio of 1:4 (vinyl laurate:sucrose) and adding 10% of water in dmf. finally, we succeeded in the characterization of vinyl sugar ester, which was produced after 25 hours of reaction by 13 c nmr. the results confirmed the c1substituted sugar mono-ester (1 -o-vinyl lauroyl sucrose). effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide ç alık bre lab, department of chemical engng, ankara university, 06100 ankara, turkey ␣-amylase (e.c. 3.2.1.1) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣-1,4 glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b-14396), which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in 0.5 dm 3 air-filtered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs1). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled 1.0 and 3.5 dm 3 systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc28s ultracentrifuge, ␣-amylase activity was measured by the dns method bernfeld (1955) . amino acid concentrations were determined with a hplc (waters), pro-tein and organic acid concentrations were measured with a hpce (waters, quanta 4000e) ç alik et al. (1998) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method rainer (1990) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources, i.e. glucose, fructose, maltose, lactose and soluble starch; n sources, i.e. (nh 4 ) 2 hpo 4 , (nh 4 ) 2 so 4, and nh 4 cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and by-product concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. bernfeld, p., 1955 . methods in enzymol. 1:149-159. ç alık, p., ç alık, g.,özdamar, t.h., 1998 . enzyme microb. technol. 23, 451-461. rainer, b.w., 1990 . geldanamycin is a benzoquinone ansamycin produced as a secondary metabolite by the actinomycete, streptomyces hygroscopicus var. geldanus, in submerged culture. it is a broad-spectrum antibiotic and exhibits an anti-tumour activity through its interaction with the heat shock protein 90 family of chaperone proteins. the optimal recovery of geldanamycin from fermentation broths is the focus of the presented work. the application of adsorbent resins was assessed and the viability of developing a solid phase extraction process for geldanamycin was determined. it was found that recovery of geldanamycin from fermentation broth was possible using adsorbent resins and the use of resins facilitated the recovery of a product stream of high purity. the composition of the fermentation broth had an impact on the performance of the resins and it was found that assessing performance on the basis of experimentally derived data was more apt than studying the kinetics of adsorption alone. adsorptive processes are, by their nature, difficult to optimise and this was found to be the case when optimising the recovery of geldanamycin from partially clarified fermentation broth. considerable effort was required to optimise geldanamycin adsorption, via examining the effect of environmental conditions and process system configuration, and geldanamycin desorption, via examining the effect of environmental conditions and investigating selective elution patterns. it is well known that halophilic eubacteria synthesize compatible solutes in order to face the high ionic strength environment in which they proliferate. these biomolecules are gaining more and more importance as biotechnological tools in a wide array of applications, and the recently developed novel bioprocesses enabled large-scale production of these compounds and therefore commercial distribution. however, there is still interest in the optimization of the production process of sole hydroxyectoine that was demonstrated to have a superior stabilization capacity. in this research project, we optimized growth conditions of marinococcus m52 to obtain high yield of hydroxyectoine. their production proved faster in the batch experiments at a higher oxygen supply, even if the stationary phase was comparable in all cases. the mf experiments showed a final biomass which was 5-fold that obtained in the corresponding batch process. in addition the monitoring of compatible solutes production showed that in the last 24h experiment hydroxyectoine accounted for 80-90% of the total content, accumulating up to 13-15% of the cell dry weight. studies for improving downstream process for ectoine and hydroxyectoine recovery showed that short permeabilization cycles in water are effective in a temperature range between 45 • c and 55 • c using a ratio 1:4/biomass:water. moreover, we evaluated the ability of ectoine to stabilize lactic acid bacteria during freeze-drying and to protect human cells from heat stress. in particular, the compatible solutes were added to the medium of confluent keratinocytes before subjecting the cells to heat stress, or lps insult. rt-pcr and western blot analysis demonstrated the hsp70b' gene over-expression in heat stressed human keratinocytes treated with ectoine. finally, we demonstrated that even at low concentration (50 mm) these compatible solutes are able to diminish cell death in lactic acid bacteria due to lyophilization procedure. among all existing alternative energy sources, biomass-derived bioethanol is especially advantageous since it is clean, sustainable and potentially inexpensive. the actual production of bioethanol is divided into a pre-treatment step, an enzymatic hydrolysis step and a fermentation step. while fermentation has been practiced by humans for centuries, our knowledge of enzymatic hydrolysis is still limited. nevertheless, it is well accepted that hydrolysis is a synergism among three classes of enzymes, ␤-glucosidase, endoglucanase and cellobiohydrolase. furthermore, a complete and efficient hydrolysis is only achieved when the enzymes are in the correct proportion. the common enzyme proportions have so far been based on the natural enzyme abundance as produced by the microorganism or mainly been determined by a trial-and-error approach. in this study, however, we used metabolic control analysis (mca) as a modelling tool to gain fundamental knowledge about enzymatic hydrolysis and to design an optimal enzyme mixture. using gepasi, a free software, the degree of control of each reaction step or each enzyme towards the overall hydrolysis can be calculated. our hypothesis is that the amount of each enzyme used for hydrolysis should be proportional to the degree of control of the enzyme. with mca, a significant amount of time, labour and reagents can be saved on developing hydrolysis enzyme mixture. furthermore, this study should demonstrate the usefulness of mca on understanding enzyme-catalyzed reactions outside the cell. process optimization for fed-batch fermentation of bacillus thuringiensis subsp. israelensis arindam chaudhury, gopinathan c department of biotechnology, university of calicut, calicut, kerala 673635 india. e-mail: g achaudhury@umassd.edu (a. chaudhury) bacillus thuringiensis (bt) is a desirable biopesticide because of its low cost and lack of toxicity. the use of bt in developing countries is limited due to process complications and economic non-feasibility of the fermentation process. in the present study, we have shown how regional production, using inexpensive alternatives for carbon and protein sources, can effectively reduce the cost of mass production of bt. while using alternative media supplements, the biomass production, nor the larvicidal activity was hampered. in addition, the positive effects of sparged aeration and the indispensable role of yeast extract were also proved. this work provides the first experimental proof of delineating the sporulation process and delta-endotoxin production. the role of various buffering agents and additives in increasing biomass production and early sporulation were also investigated. for the production of coenzyme q 10 (coq 10 ), an electron carrier in the respiration chain with antioxidant activity. with decrease of dissolved oxygen level from 20 to 5%, the intracellular coq 10 content increased about 4-fold, yielding 2 mg per g-dry cell weight at 5% dissolved oxygen level. azide significantly increased the intracellular coq 10 content, with the highest value of 5.3 mg per g dry cell weight in the presence of 0.45 mm of sodium azide. however, dnp (up to 200 m) and h 2 o 2 (up to 10 m) did not affect the intracellular coq 10 content, indicating proton gradient release and oxidative stress do not affect the synthesis of coq 10 . these results show that restricted electron flux by limited oxygen supply and the addition of azide increases the intracellular coq 10 content. fourier transform infrared spectroscopy (ft-ir), combined with in situ heat sterilizable attenuated total reflection (atr) probes, constitutes a promising and versatile technique for on-line monitoring of bioprocesses. the ft-ir enables rapid determinations of the medium composition without the requirement of sample withdrawal and preparation. in this work the concentration levels of the substrates glycerol and methanol were monitored on-line in pichia pastoris cultivation. partial least squares (pls) models were used for obtaining the concentration readings. the glycerol concentration measurement proved to be very reliable and reproducible in the glycerol batch phase. however, the on-line information regarding the glycerol concentration was not utilized for any process control purposes. on the other hand, the availability of on-line information about the methanol concentration proved to be crucial for the successful implementation of the cultivations. the temperature strategy in the methanol fed-batch phase utilized temperatures as low as 10 • c. in order to keep the metabolic activity at a reasonable level the culture was therefore pushed towards the maximal substrate consumption rate, rather than being a conventional substrate limited fed-batch. as a consequence methanol accumulation occurred on occasions. without on-line information about the concentration this accumulation, if sustained, would have resulted in a poisoning of the culture, either from methanol itself, or perhaps more importantly from formaldehyde. therefore, it can be concluded that the ft-ir/atr instrument was very useful in this application. jørgensen department of chemical engineering, denmark technical university, building 229, dk-2800 lyngby, denamrk. e-mail: fpd@kt.dtu.dk (f.p. davidescu) modeling biochemical reaction network in microorganisms still represents a challenge due to the very large number of enzyme catalyzed biochemical reactions, to the very complex system and to the many feed-forward and feedback regulation mechanisms. the presented approach to model such a system is based on the stochastic grey-box modeling framework proposed by kristensen et al. (2003) . this methodology consists of parameters estimation based on a prediction error method followed by different statistical tests for parameter significance and for model (in-) validity. the methodology furthermore allows estimation of unknown functional relations, e.g. kinetic rates. a set of experimental data zangirolami (1998) obtained during continuous cultures of a high enzyme producing aspergillus oryzae strain. the oxygen concentration was decreased stepwise and the substrate concentration was modified from one experiment to other. a model proposed by agger et al. (1998) is investigated on these data. the primary interest is to develop a physiologically feasible model, also at the low oxygen concentrations often found in industrial practice. microbially produced secondary metabolites such as antibiotics have tremendous economic importance. streptomyces spp. have long been identified as sources of antibiotics and chemotherapeutic compounds, synthesising over 4000 bioactive compounds. geldanamycin is a novel chemotherapeutic agent produced by streptomyces hygroscopicus var. geldanus in submerged fermentation. initial studies have focused on optimisation of media design through understanding and controlling metabolic routes of biosynthesis within the cell. geldanamycin is a by-product of the shikimate or aromatic amino acid biosynthesis pathway. stimulation of this pathway and concomitant production of geldanamycin is achievable through amino acid control. increasing concentrations of primary carbon source greatly influence biomass generation and product formation, as does the inclusion of cations such as magnesium and calcium to the fermentation media. optimisation of production media through balancing minerals, nitrogen, and carbon sources has significantly improved antibiotic yields in shake flask cultures and the development process will be extended into pilot scale through the use of bioreactors. microbiology and biotechnology research group, school of life sciences, napier university, edinburgh, eh10 5dt, scotland. email: m.el-mansi@napier.ac.uk (m. el-mansi) synopsis: during growth of corynebacterium glutamicum on glucose or other glycolytic intermediates, pep carboxylase fulfils an anaplerotic function as it replenishes intermediary metabolism with biosynthetic precursors that are essential for growth and glutamate production. under these conditions, pep carboxylase plays a central role and this in turn is characterised by a high flux control coefficient thus rendering this enzyme an ideal target for metabolic interventions. further analysis in silico revealed that any increases in the concentration of the enzyme was accompanied by increases in flux through the enzyme itself as well as glutamate formation, presumably as a consequence of sustaining a high intracellular level of ␣-ketoglutarate; the immediate precursor for glutamate biosynthesis. a combined approach to enhance periplasmic expression of human growth hormone in escherichia coli, using a modified signal peptide from alpha amylase gene of bacillus licheniformis s.k. falsafi 1,2 , a. zomorodipour 2 : 1 islamic azad university of jahrom, iran; 2 department of mol genet. national inst for genet eng & biotechnol., tehran, iran. e-mail: soheil falsafi@yahoo.com (s.k. falsafi) the alpha amylase gene signal peptide, originated from a strain of bacillus licheniformis, was shown to be able to transport its native protein, when expressed in e. coli the competence of the fusion protein being processed and translocated through the inner membrane is highly dependent on the amino acid sequences in the signal peptide. therefore, in order to increase the expression efficiency of bla signal peptide, we reconstructed the bla signal peptide coding fragment with the following modifications. two rare codons of arg 6 (cgg) and arg 10 (cga) and codons for leu 15 (tta) and pro 23 (cct), in the signal peptide were substituted with their corresponding e. coli major codons. two other changes, including phe 20 (ttc) → leu 20 (ctg) and ala 28 (gcg) → met 28 (atg), were also introduced to increase the processing efficiency. the hgh-expressing plasmid equipped with the modified bla (blaf2) was subjected for further expression analysis in a t7-based expression system. the results obtained from the protein patterns of the induced bacteria indicates in high expression level of hgh preprotein (hgh::blaf2) followed by efficient transfer of the mature hgh to e. coli periplasm. (ip6) has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip3) and inositol tetrakisphosphate (ip4) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip6 is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia1 was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia1 converted ip6 into ip5 (myoinositol 1,2,3,5,6 pentakisphosphates) and another isomer, which is yet to be elucidated. characterization of the novel ␤-peptidyl aminopeptidase (bapa) from sphingomonas sp. 3-2w4 that cleaves synthetic ␤peptides birgit geueke, hans-peter e. kohler environmental microbiology, eawag, 8600 duebendorf, switzerland. e-mail: birgit.geueke@eawag.ch (b. geueke) non-natural peptides, which are capable of evoking a specific biological response, are currently receiving much attention. oligomers of ␤-amino acids (␤-peptides) are representing a group of pharmaceutically interesting peptides because of their very high stability towards enzymatic degradation and their ability to mimic the structure of naturally occurring biologically active peptides. the pharmaceutical potential on the one hand and the high stability on the other hand aroused interest for studies on the environmental fate and the degradation behaviour of this class of compounds. a novel bacterial strain (sphingomonas sp. 3-2w4) that was capable of degrading short ␤-peptides was isolated from an enrichment culture. the ␤peptide degrading enzyme was purified and its gene sequence was determined (bapa). the gene encodes a ␤-peptidyl aminopeptidase (bapa) of 402 amino acids that is synthesized as preprotein with a signal sequence of 29 amino acids. it belongs to the n-terminal nucleophile (ntn) hydrolase superfamily and is the first peptidase that is capable of cleaving amide bonds in ␤-peptides composed of synthetic ␤-amino acids. the biochemical properties of recombinant bapa were investigated regarding its substrate specificity and possible application in the synthesis of ␤-peptides. to produce efficient strains of agaricus bitorquis (quel.) saccardo, which are resistant to high temperatures p. guler, a. ergene, s. tan kirikkale university, faculty of science and literature, department of biology, yahsihan-kirikkale in this study, the culture mushroom agaricus bitorquis (quel.) sacc. the growth of the mycelium and the fructifications under high temperature is examined. the spores taken from the mushrooms that were collected from nature were grouped as a, b, c, d, e. the spores were inoculated into malt extract agar and incubated at 30 • c and primer mycelium was produced. the mycelium discus taken from primer mycelium in 8 mm diameter were inoculated into the center of malt extract agar and incubated at 30, 32, 34, 36 and 38 • c separately. during the incubation period the growth of the mycelium were measured. during the growing period the radial growth speed of the mycelium were taken as criteria. the best mycelium growth for all groups was seen at 30 • c. at 36 • c the e group mycelium and at 38 • c other group's mycelium did not grow. these temperatures were determined as thermal lethal point for the groups. from all the mycelium produced from all temperatures spawn was prepared and with the results taken from these, spawn calendar is prepared. in this research, the spawn was inoculated to compost with mixing system and separately put in culture rooms, temperatures as 30 and 32 • c. at this level the culture mushroom production techniques were used. the harvested mushrooms were inspected morphologically. at this morphological inspections the cap width, cap tissue thickness, stalk thickness and stalk long ness was taken as criteria. in the study the best growth was seen at d group mushrooms and this group mushrooms tyrosinase's activities were measured and graphics were made. introduction: viral contamination of biological products; cause many problems in viral diagnostic laboratories, blood transfusion organizations, and biological producers. bovine viral diarrhea virus (bvdv), from the pestivirus genus, is the most common viral contamination in (fetal) bovine serums (fbs). also, bvdv used as a module, for study hepatitis c virus inactivation due to its similarity in structure and genome. pulsed uv lights (puvls) have this potential to inactivate known and unknown or reemerging viruses as well as prions. two puvl with the wavelengths of 355 and 266 nm, were produced by q-switched nd 3+ :yag laser in its third and forth harmonic, respectively. the energy of each pulse for 355 nm was 12.7 mj/cm 2 and for 266 nm was 35.2 mj/cm 2 . bvdv were produced and titrated in mdbk cell line. mdbk and fbs were already checked for non-cytopathic or cytopathic pestiviruses, using related ag-elisa kit. bvdv suspended in solution with the dilution of 1:2 before exposure. the quartz tube with the minimum uv-absorption in compare with air, used as a container for exposed solutions. calculation of the virus titer, 10 4.3 tcid 50 /ml, was done based on the reed and muench method. bvdv suspended in pbs was exposed into the 3.52-352 j/cm 2 of puvls with the wavelength of 355 nm and also, was exposed into the 1.27-92.25 j/cm 2 of puvls with the wavelength of 266 nm. furthermore, bvdv suspended in fbs was exposed into the 88, 176, 352 and 704 j/cm 2 of puvls with the wavelength of 355 nm and also, was exposed into the 6. 35, 25.4, 63.5, 127 and 190 .5 j/cm 2 of puvls with the wavelength of 266 nm. results: the minimum dose for inactivation of bvdv suspended in pbs with the 355 and 266 nm wavelengths of puvls, were 352 and 92.25 j/cm 2 , respectively. also, the minimum dose for inactivation of bvdv suspended in fbs with the 355 and 266 nm wavelengths of puvls, were 704 and 127 j/cm 2 , respectively. to evaluate the fbs quality to support cell culture, treated fbs with the dose of 190.5 j/cm 2 of 266 nm puvls was used to grow vero cell line in 12 successive passages. the viability of cells in two study groups was identical. the statistical evaluation of two treated groups showed no significant difference, in 12 passages. conclusion: because inexpensive equipment can be used to produce puvls capable of handling different volumes of biologics with operational ease, this viral inactivation technique is cost effective for relevant industries. the procedure has the potential to be combined synergically with other inactivation method. puvls offer a new, nonadditive and chemically safe alternative for the treatment of fbs to inactivate adventitious viruses and to preserve the biological activity necessary for the propagation of cell culture. characterization and gene cloning of the g-resorcylic acid decarboxylase for application to selective production of g-resorcylic acid y. iwasaki 1 , y. ishii 2 , k. kino 1 , k. kirimura 1 : 1 dept. appl. chem., sch. sci. eng., waseda univ., tokyo, japan; 2 bme, asmew, waseda univ., tokyo, japan. e-mail: iwasaki@moegi.waseda.jp (y. iwasaki) for selective production of ␥-resorcylic acid (␥-ra, 2,6-dihydroxy-benzoic acid) from resorcinol (re, 1,3dihydroxybenzene) under mild conditions, we screened various microorganisms and found the reversible ␥-ra decarboxylase (rdc) as a novel enzyme applicable to carboxylation of re to form ␥-ra, in a bacterial strain rhizobium radiobacter wu-0108 1) . rdc catalyzed the decarboxylation of ␥-ra, and regio-selective carboxylation of re to form ␥-ra, without formation of ␣-ra and ␤-ra. the molecular weight of rdc was estimated to be 130 kda by gel-filtration, and that of the subunit was determined to be 34 kda by sds-page, suggesting that rdc is a homotetrameric structure. the gene encoding rdc was sequenced, and a site-directed mutagenesis study revealed that the two histidine residues at positions of 164 and 218 in rdc are essential for the catalytic activity of rdc. through the reactions using e. coli cells highly expressing rdc, 6.7 mm ␥-ra was produced from 15 mm re at 30 • c for 16 h, with a yield of 45%. ishii, y. et al., 2004. biochem. biophys. res. commun., 324, 611-620. laccase biosynthesis in stirred fermenters teresa jamroz, stanislaw ledakowicz, barbara sencio department of bioprocess engineering, technical university, lodz pl 90-924, poland industrial applicability of enzymes is closely related to development of efficient methods of their production. currently, significant interest in lignolytic enzymes, including laccase, has been observed. laccase is an enzyme applied in various industrial branches and environmental processes. broad laccase applicability induces researchers to develop urgently efficient methods for its commercial production. laccase (ec.1.10.3.2. p-diphenol oxidase) is produced by cap mushrooms from the class basidomycetes. this is the so-called white rot fungi which in natural conditions appears on both living and dead wood. as shown in the practice of biotechnological processes, high-efficient strains have low resistance to destructive factors in bioreactors. hence, to preserve a proper morphology and physiological state of an organism, strictly determined culture conditions must be obeyed. this is very important in the case of basidomycetes for which submerged culture in the liquid phase is not a natural habitat. results of studies on laccase production from cerrena unicolor family are discussed. cultivation of active biomass was carried out in stirred tank and rotating disc bioreactors of different volume (b. braun of working volume 12 dm 3 ; fas-01 of working volume 3.5 dm 3 ). experiments in both fermenters were made at impeller revolutions 200 and 300 min −1 , on a modified lindberg substrate. significant differences in the rate and yield of laccase production were reported. an almost three times higher values of laccase activity were obtained in the b. braun fermenter, at rotational speed 300 min −1 . to retain suspended cells in bioreactor a filtration process can be used. the biomass is concentrated by withdrawing cell-free culture broth. if the desired product is dissolved in the broth (extracellular production), the procedure enables the continuous harvest in the cellfree permeate. an application test of filtration system for suspended biomass of aspergillus niger in submerged single stage continuous culture was presented in this report. the system is easy to construct and there is a possibility of its sterile exchange during cultivation. the culture medium contained the following substances (g/dm 3 ): white sugar, 150.0; nh 4 no 3 , 1.5; mgso 4 ·7h 2 o, 0.2; kh 2 po 4 , 0.2; feso 4 ·7h 2 o, 0.05. fermentations were carried out in the lab bioreactor biomer 10. the bioreactor was a standard cstr (continuous stirred tank bioreactor) with working volume of 5 dm 3 . high citric acid concentration in culture medium (p = 108.9 g/dm 3 ), high yield of citric acid (y p/s = 72.6%) and high efficiency coefficient (k ef = 71.1) were observed in single stage continuous culture with biomass retention. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar ç alık, iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc18::bal carrying recombinant escherichia coli on a defined medium with 8.0 kg/m 3 glucose were investigated in order to fine-tune the bioreactor performance, in v = 3 dm 3 batch bioreactors at five different conditions with the parameters at, i.e. q o /v r = 0.5 vvm and n = 250, 375, 500, 750 min −1 and; q o /v r = 0.7 vvm and n = 750 min −1 . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph o = 7.25 are optimum for maximum bal activity, i.e. 860 u/cm 3 at 12 h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains 102 metabolites and 133 reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e. coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. saprophytic mycobacterium strains belong to the best known microorganisms which have been applied to the pharmaceutical industry for the production of steroid drugs. the mycobacterial cell wall is the permeation barrier to chemical compounds, including lipophiles. using isoniazid (inh), the inhibitor of the mycolic acids biosynthesis, we were able to demonstrate increased ad production and susceptibility to antimicrobial agents. the process of sterol transformation and products accumulation was monitored using gas chromatography. isoniazid was shown to intensify ␤-sitosterol side-chain degradation by mycobacterium sp., and accumulation of 4-androstene-3,17-dione (ad) and 1,4-androstadien-3,17-dione (add), which are the starting materials in the biotechnology of medically important steroids. to confirm these results, the sensitivity of the bacteria to antimycobacterial drugs was performed. the minimum inhibitory concentration mic 50 of rifampicin and erythromycin decreased markedly in the presence of inh. this work was supported by grant nr 3p04c 06923 of the committee for scientific research. for the purpose of high-throughput screening and to reduce experiments with animals in pharma biotechnology biosensor systems gain importance. the principle of a biosensor is the combination of cultured cells and a sensorchip device, which allows the monitoring of cellular activity. in contrast to traditional analytics with a biosensor you can measure on-line cellular activity change caused by an effector as well as the restored activity after privation of the effector (re-native activity). cmos technology can be used for the realisation of various biological sensorchips such as adhesion sensorchips, metabolical sensorchips and electrophysiological sensorchips. the standard cmos technology allows a high reproducibility of the chips, the integration of electronic components on the chip, which reduces the amount of external devices and the combination of different sensors on one chip. in cooperation with the semiconductor company micronas and the biotech company bionas we have realised different types of cmos sensorchips to measure adhesion of a cellular monolayer with interdigitated electrodes (ides), metabolical activity via acidification with ion-sensitive field effect transistors (isfet) and sponteneous neuronal network activity with passive palladium electrodes. microbial agents have been applied to the different stages of pulp and paper processing. the work presented describes a study on the effect of applying ligninolytic enzymes, such as a laccase plus mediator system, on a variety of different types of pine and eucalyptus pulps and subsequently subjecting these to different ageing processes. industrial pulps were obtained from different portuguese pulp and paper companies. the pulps used were (1) unbleached pine pulp from portucel tejo; (2) unbleached eucalyptus pulp from portucel setúbal; (3) bleached eucalyptus pulp from portucel setúbal; and (4) pulp made from recycled paper from renova s.a. several types of handsheets were produced with 2 different grammage namely, 60 and 180 g/m 2 . the prepared handsheets were subject to an aging sequence in three different chambers: ultraviolet radiation (wavelength of 280 nm), temperature (19 • c) and moisture (70%); and thick saline fog at a concentration of 1% and temperature of 35 • c. in order to evaluate the effect of moisture cycles and temperature, two aging sequences were used for each type of handsheet. in the first, the moisture varied (60, 80 and 100%), while the temperature was held constant (25 • c); in the second the temperature varied (60, 70 and 80 • c) and the moisture was held constant (50%). following the aging phase, the handsheets were subject to several chemical (viscosity and index kappa) and physico-mechanical (colour, tensile breaking strength, stretch and the bursting strength) tests in order to characterize the effect of the aging conditions. results will be presented describing the effect of application of the laccase-mediator system on the optical and mechanical properties of the prepared handsheets. fundação para a ciência e a tecnologia, project pocti/ agr/47309/02. aspergillus niger is a filamentous fungi widely used in industry. its growth as freely dispersed hyphae leads to an increase in the medium viscosity and to problem of mass transfer, especially oxygen transfer. oxygen acts both as final electron acceptor in the mitochondrial chain and as nutrient for the biosynthesis of unsaturated fatty acids and sterols. thereby, a lack of oxygen affects the nadh/nad ratio, the atp production, the growth and has a strong influence on the physiology of the microrganism. in the present study, the metabolic changes of a. niger in response to a lack of oxygen was investigated using oxygen limited chemostats combined with nitrogen pulse. under these conditions, the main consequence of a sudden decrease of oxygen availability is an increase in the mannitol production. this work showed that the mannitol biosynthesis, involving the enzyme mannitol-1-p dehydrogenase, helps the reoxidation of nadh when the final electron transport acceptor, oxygen, is limiting. investigation of the lipase activity of the bacteria isolated from olive mill wastewater sevgi ertugrul 1 , nur koçberber 1 , gönül dönmez 1 , serpil takaç 2 1 department of biology faculty of science ankara university 06100 beşevler ankara turkey; 2 department of chemical engineering faculty of engineering ankara university 06100 tandogan, ankara, turkey the bacteria that could grow on media containing olive mill wastewater (omw) were isolated and their lipase production capacities were investigated. the strain possesing the highest lipase activity among 17 strains grown on tributryin agar medium was identified as bacillus sp. the effect of ph on the lipase activity of the strain was investigated in tributryin medium and ph 6 was found to be the optimal. the liquid medium composition was improved by adding different carbon sources and fatty acids into tributryin medium -omitted tributryin -to increase the enzyme activity. the cultivations were performed at 30 • c and ph 6. lipase activity of the bacillus sp. was measured spectrophotometrically through the hydrolysis of p-nitrophenol palmitate. among the media containing different compositions of tricapryn, trimyristin, tributyrin, triacetin, tween 80, glycerol-trioleate, glycerol-trioctanoate, glycerol-tridodecanoate, omw, glucose, and whey; the medium consisted of 20% whey + 1% glycerol-trioleate was found to give the highest lipase activity. cultivation of bacillus sp. in the optimum medium at ph = 6 and 30 • c for 64 h was resulted in the extracellular and intracelluar lipase activities of 15 and 168u/ml, respectively. this study was supported by ankara university biotechnology institute (project no: 2004-151 and 2005-164) . under different abiotic stresses, cell growth and metabolic activity are highly influenced in all types of living organisms medium osmolality is usually one of those factors affecting different types of biological systems in different ways. however, even in the same organ of higher eukaryotes the degree of osmoregulation mechanism is highly variable in different types of cells. therefore, studying the effect of osmotic stress on mammalian cell is very important subject for particular cell line. the effect of hyperosmotic pressure on the kinetics of cell growth of and metabolic activity of mesenchymal stem cells (mscs) and two industrially important cell lines, hybridoma cells and human embryonic kidney cell (hek) were investigated in batch cultures at different osmotic pressures in the range from 325 to 500 mosm kg −1 . in case of mscs cells, the maximal specific growth rate [] of 0.029 [h −1 ] associated with the highest specific glucose consumption rate [-q gluc ] of 0.1129 × 10 −6 [mol cells −1 h −1 ] was obtained in medium of 375 mosm kg −1 . in case of hybridoma cells, osmotic pressure showed not only influence on the kinetics of cell growth and metabolism but also on the monoclonal antibody production. the maximal mab production was obtained in case of cells cultivated under osmotic pressure of 375 mosm kg −1 . further increase in osmotic pressure resulted in significant reduction in growth rate as well as mab production. on the other hand, hek cells were more sensitive to osmotic pressure in industrially used serum free medium and the addition of serum decreased the inhibitory effect of high osmotic pressure on the cells. gustavo g. fonseca 1,2 , andreas k. gombert 2 , elmar heinzle 1 , christoph wittmann 1 : 1 biochemical engineering, saarland university, saarbrücken, germany; 2 chemical engineering, são paulo university, brazil kluyveromyces marxianus cbs 6556 is a potentially interesting yeast strain characterized by a high capacity of conversion of substrate into biomass. however, this yeast has been only marginally studied so far. therefore, we performed a metabolic characterization in batch and chemostat cultures at dilution rates of 0.10, 0.25 and 0.5 h −1 . the specific rate of o 2 consumption (qo 2 ) increased with dilution rate from 2.87 to 11.09 mmol (g dw) −1 h −1 . the respiratory coefficient remained almost stable around 1.0 for all metabolic states investigated. even at the dilution rate of 0.5 h −1 , which is close to the maximum growth rate of the strain of 0.56 h −1 , no significant overflow metabolism was observed. the concentration of extracellular metabolites increased with the dilution rate, but remained below 6% of the carbon consumed as glucose. all carbon balances closed near 100% underlining the consistency of the data. in contrast to s. cerevisiae the respiratory capacity of k. marxianus cbs 6556 is not strongly influenced by the dilution rate in aerobic chemostat or batch cultures, indicating its high potential for biomass-directed applications. a thermostable l-arabinose isomerase for enzymatic production of d-tagatose o. hansen, f. jørgensen, p. stougaard department of enzyme technology, bioneer a/s, kogle allé 2, dk-2970 hørsholm, denmark. e-mail: och@bioneer.dk (o. hansen) d-tagatose, an isomer of d-fructose, is a low-calorie bulk sweetener with a sweetness equivalent to sucrose. d-tagatose has obtained gras approval for use as a food ingredient, and is currently produced by chemical isomerization of d-galactose, which may readily be obtained by hydrolysis of lactose. structurally, d-galactose is closely related to l-arabinose, and it has previously been shown that some variants of l-arabinose isomerase (araa) may catalyze the conversion of d-galactose to d-tagatose, in addition to the metabolic conversion of l-arabinose to l-ribulose. we have screened a number of bacterial araa enzymes for their ability to catalyze the isomerization of d-galactose to d-tagatose. the best enzyme was found in the thermophilic bacterium thermoanaerobacter mathranii (dsm11426). the araa gene of t. mathranii was cloned, sequenced and expressed in e. coli. amino acid sequence comparisons of the t. mathranii sequence and other known araa sequences showed a relatively low sequence identity of about 30%, indicating a distant phylogenetic relationship to the other members of the l-arabinose isomerase group. the t. mathranii enzyme was thermostable with optimal activity at 65 • c and it required manganese ions. unlike other araa variants, the t. mathranii enzyme showed k m values in the same order of magnitude for l-arabinose and d-galactose, suggesting that this enzyme is a versatile isomerase capable of isomerizing structurally related aldoses. the enzyme was immobilized by chemical cross-linking of a crude e. coli cell homogenate, and the immobilized enzyme efficiently converted d-galactose into d-tagatose. currently, we are developing an enzymatic method for industrial production of d-tagatose using the immobilized enzyme. the agricultural production can be negatively affected by different pest insects (pi) and the use of chemical insecticides (chi) has been the traditional method for controlling pi during decades. nevertheless, there are various ecological implications due to the extensive application of chi. a viable alternative for the use of chi in certain agro-systems, is the use of entomopathogenic nematodes (epn) of the genera steinernema and heterorhabditis that are natural pathogens for different pi; besides, the presence of a symbiotic bacterium is necessary for an effective entomopathogenic activity can take place. the nematode/bacterium complex does not represent a risk for the environment. different authors propose that the best alternative for the massive production of epn is the submerged culture within bioreactors; nevertheless, more research is required to have really robust processes. particularly, information regarding the actual hydrodynamics during epn production and its relation with the epn productivities are scarce, among other aspects. the present study deals with the hydrodynamic characterisation during the production of the epn steinernema carpocapsae and its symbiont bacterium xenorhabdus nematophilus, in submerged monoxenic culture in an internal-loop air-lift bioreactor (v l = 4.5 l) using two culture media: one of them containing whey and the other one, agave juice, aguamiel, (agave spp.). process viscosity of the culture broth was determined along the time, exhibiting a maximum value of 20 mpa s. moreover, it was determined that the hydrodynamic conditions were always located within the laminar region (re < 500). at the experimental conditions tested, it can be inferred that the epn productivity are more sensitive to changes in the culture medium composition than on the prevailing hydrodynamic conditions during the fermentations. bacillus thuringiensis is a gram-positive bacterium used as a biological pest control agent. moreover, it is able to produce, several biologically active molecules such as bacteriocins and hydrolytic enzymes among which chitinases that play double roles, fungicide and improving the insecticidal effect of b. thuringiensis deltaendotoxins. a newly isolated b. thuringiensis subsp. kurstaki strain bupm4, was shown to produce a novel bacteriocin named bacthuricin f4. the highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. upon purification of bacthuricin f4, the specific activity was increased 100-fold. this bacteriocin was heat-stable up to 70 • c and resisted up to ph 3.0. its molecular mass, determined by mass spectrometry was 3160.05 da. direct n-terminal sequencing of bacthuricin f4 revealed the following sequence: dwtxwsxl. the latter was unique in the databases. bacthuricin f4 was active against bacillus species while it had little or no effect on gram-negative bacteria. the bacteriocin produced by the b. thuringiensis strain bupm4 respond to both criteria of thermostability and stability to low phs. thus, it could be used as a source of bacteriocin active against related species of bacillus harmful for agricultural products and as food preservative. the other example of antimicrobial compound produced by b. thuringiensis is a chitinase. we describe the selection of b. thuringiensis high chitinase-producing strain bupm255, and the characterization and the heterologous expression of a novel chitinase encoding gene. the cloning and sequencing of the corresponding gene named chi255 showed an open reading frame of 2031 bp, encoding a 676 amino acid residue protein. both nucleotide and amino acid sequences similarity analyses revealed that the chi255 is a new chitinase gene, presenting several differences from the published chi genes of b. thuringiensis. the identification of chitin hydrolysis products resulting from the activity, exhibited by chi255 through heterologous expression in e. coli revealed that this enzyme is a chitobiosidase. the addition of the sequence of chi255 to the few sequenced b. thuringiensis chi genes might contribute to a better investigation of the chitinase "structure-function" relation. cloning and characterization of s-adenosyl-l-methionine synthetase from pichia ciferrii dscc 7-25 kwon-hye ko 1 , gee-sun yoon 1 , gi-sub choi 1 , joo-won suh 2 , yeon-woo ryu s-adenosyl-l-methionine(sam) has an important role for dna methylation and cell signaling. sam was synthesized from methionine and atp by sam synthetase and play an pivotal function in the primary and secondary metabolism of cells. recent studies have revealed in the effect of sam in case of morphological differentiation in both eukaryotes and prokaryotes. the p. ciferrii produces large quantities of sphingoid base. tetraacetylphytosphingosine(taps), which is a precursor of sphingolipid, could be used for the production of pharmaceuticals and cosmetics. we isolated sam gene from p. ciferrii and cloned it into expression vector for e. coli and p. pastoris, respectively. an 1.2 kb sam-s gene fragment was isolated by low-strigency pcr using degenerated primer. by the analysed primary sequence deduced from dna sequence, this gene included conserved domains similar with other well-known sam synthetase. first of all, sam synthetase gene cloned pgem-t vector and subcloned into histidine tagging system to purify the expressed protein using metal chelating resin. typical characteristic analysis of this enzyme is underway. metabolic networks offer a large variety of different synthesis pathways starting from cheap substrates and leading to interesting high-value compounds, i.e. metabolites. in case an interesting pathway can be disconnected from the remaining metabolic network, the perforated cell or the crude extract could be used for a one-pot multi-step synthesis of the desired compound. pathway isolation, achieved by deletion of genes encoding gene products enabling side reactions, interferes with the viability of the organism, which is a requisite for the production of the system of biotransformation (sbt). in this work, a rational systems biology-derived approach is presented for the design of a sbt. it is illustrated for a sbt allowing for production of dihydroxyacetone phosphate. the design procedure comprises three steps: (i) a production pathway is identified in the metabolic network of e. coli. the e. coli pathway is complemented by two additional enzymes in order to obtain a fully energy and redox balanced production pathway. (ii) an optimal combination of gene knockouts is designed and a suitable growth medium composition is identified, both by a model-based approach: flux balance analysis of a genome-scale metabolic network is used to predict enzyme expression within the wild-type organism on different media, while a mixed-integer optimisation is employed to identify viable mutants as this approach strongly depends on the quality of the fba prediction, available regulatory information on the usage of metabolic pathways and thermodynamic constraints were taken into account. (iii) thermodynamic analysis of the obtained, partially branched sbt reaction cascade revealed the extent of loss in yield by the remaining side reactions. in summary, this systems biology-driven approach potentially enables the substitution of a elaborative multi-step synthesis process by a one-pot enzyme reaction cascade. institute of industrial biotechnology, inha university, incheon 402-751, korea. e-mail: leecg@inha.ac.kr (c.-g. lee) algal biotechnology is drawing increasing interest due to its potential as a source of valuable pharmaceuticals, pigments, carbohydrates, and other fine chemicals. currently, its application is being extended to the areas of wastewater treatment and agriculture. however, lack of suitable photobioreactors (pbrs) makes the cost of algally-derived compounds higher than those derived by chemical synthesis and thus has prevented widespread use of algal cultures. the culture of algae prior to the late 1940s was apparently restricted to laboratory scale operations. experiments on outdoor algal mass production began in the late 1940s with nearly concurrent development of experimental culture facilities in germany and the united states. for the next two decades, outdoor mass culture of algae was undoubtedly the hottest topic in the algal biotechnology area. recent developments of high-density pbrs enable the production of valuable biologically active compounds by algal mass cultures. however, light is almost always the limiting factor in high-density photobioreactors. key factors for successful photobioreactors will be discussed and various photobioreactors will be analyzed and compared for their advantages and disadvantages. the new techniques, such as pigment redcution and application of flashing light and lumostatic operation, will be discussed for possible solutions to overcome the light limitation in high-density microalgal cultures. a quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications, for example when one tries to develop a transformation system for a new fungal host. southern analysis is laborious and time consuming. several colony hybridisation methods have been developed for the analysis of a large number of transformants. unfortunately these methods suffer from different disadvantages such as non-specific binding, a limited usability to screen for specific integration and the fact that these procedures always take a few days (van zeijl et al., 1998) . recently, methods for pcr-based analysis of fungal transformants have been described. most of these methods require high quality dna. many methods for dna extraction from fungi have been described in the past few years. these methods often are tedious, time consuming, costly or limited to a small number of samples each time. most of the available protocols include the growth of mycelium in a liquid culture, followed by freeze-drying or maceration in liquid nitrogen and grinding of the frozen material to break the cell walls (cassago et al., 2002) . lately a few methods have been described to isolate dna from fungi suitable exclusively for pcr and appropriate for the simultaneous treatment of a large number of samples. some methods also describe the direct use of mycelium (cooke et al., 1997) in the pcr-reaction mixture. we compared the applicability of a few rapid dna extraction methods for myrothecium gramineum and tested the resulting dna samples on there suitability for pcr-applications. myrothecium gramineum is a filamentous ascomycete used in ongoing research as a new cloning and expression host. five methods were tested. in four of these methods dna was extracted from mycelium (goodwin et al., 1993 and aljanabi et al., 1997) or spores (ferreira et al., 1997 and xu et al., 1995) prior to pcr. a fifth assay used mycelium straight in the pcr-reaction mixture. only this last method seemed useful for myrothecium to isolate dna suitable for pcr. fragments up to 2000bp were amplified. cheese whey is a liquid effluent from cheese-making processes. there is an increased interest in the economic utilization of whey produced by the dairy industries, because the whey is a pollutant, due mainly to its lactose content. the goal of this work was to find the most suitable values of some fermentation parameters for lactic acid production from whey by a lactic acid bacterium, lactobacillus helveticus (atcc 15009). the effects of lactose content, temperature, ph and the supplementation with yeast extract were investigated using surface response methodology. a composite central design was used with three center points, making a total of 27 operational conditions. the region of maximum production is outlined by the following intervals: temperature around 40 • c; lactose concentration between 70 and 85 g/l; concentration of yeast extract between 20 and 25 g/l; ph between 7 and 7.5. fermentation studies on a continuous fermentative process coupled to a vacuum flash evaporator were carried out in lab scale equipment. the phases of this work consisted in an assembly and instrumentation of the prototype and elaboration of a supervisory system coded in labview 6.1, which allows the data acquisition and control through personal computers. the experiments in continuous fermentation used saccharomyces cerevisiae and sugar cane molasses as substrate. the analytical follow up was done through analysis of total reducing sugars, ethanol, glycerol, dry mass and viable cells. the system worked for months uninterruptedly, producing an alcoholic solution at the condenser with 50 • gl. the fermentation operated with concentrations of ethanol at 5 • gl, which is a weakly inhibitory value for the yeast of the process, even when fed with concentrated cane molasses, containing up to 330 g/l of sugar. the result meets the initial goal, which was to operate the system with low level of ethanol and to guarantee high productivity, even in high concentrations of sugar in the feeding. the results showed that system productivity was superior to that of the conventional continuous process. lactic acid (la) is a versatile chemical, used as an acidulant, flavor and preservative in the food, pharmaceutical, leather and textile industries, and for production of biodegradable poly lactic acid (pla). l(+)lactic acid is the only optical isomer for use in pharmaceutical and food industries because human body is only adapted to assimilate this form. in this research, lactic acid production was improved on 20 l fermentor. in our experience, among six strains of lactobacillus were examined for the production of l(+) lactic acid, lactobacillus casei ssp. casei atcc 39392 was selected as a highest l(+) lactic acid producer. optimized medium used for lactic acid production contained (per l) 80 g glucose, 50 g whey powder and 20 g corn steep powder. for a homofermentative process, ph 6.0 was found to be optimal. in order to avoid product inhibition, the produced lactic acid was neutralized using calcium hydroxide. maximum production and productivity of lactic acid in batch system, were 81 g and 1.35 g/lh, but in fed batch system, after 3 feeds of glucose, production and productivity increased up to 360 g and 4 g/lh. saleh a. mohamed molecular biology dept., national research centre, cairo, egypt an extracellular polygalacturonase (pgii) from trichoderma harzianum was purified to homogeneity by two chromatography steps using deae-sepharose and sephacryl s-200. the molecular weight of t. harzianum pgii was 31,000 da by gel filtration and sds-page. pgii had isoelectric point of 4.5 and optimum ph of 5.0. pgii was very stable at the ph 5.0. the extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (de). pgii had very low activity toward nonpectic polysaccharides. the apparent km value and kcat value for hydrolyzing polygalacturonic acid (pga) were 3.4 mg/ml and 592 s −1 , respectively. pgii was found to have temperature optimum at 40 • c and was approximately stable up to 30 • c for 60 min of incubation. all the examined metal cations showed inhibitory effects on the enzyme activity. 1, 10 phenanthroline, tween 20, tween 80, triton x-100 and sds had no effect on the enzyme activity. the rate of enzyme catalyzed reduction of viscosity of solutions of pga or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. the storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to one year. these properties of t. harzianum pgii with appreciable activity would be potentially novel source of enzyme for food processing. tarek m. mohamed, biochemistry division, chemistry department, tanta university, tanta, egypt preparation of peroxidase from horseradish, which could be used for commercial applications such as diagnostic kits, was occurred through a simple reproducible method consisting of extraction, ammonium sulphate precipitation, filtration through non-binding protein filter and lyophilization. the purification method was developed allow the preparation of 33 mg of enzyme from 1 kg of horseradish roots. the one mg of enzyme contains 900 units of peroxidase. this value is similar to that produced by sigma (50-1000 unit mg −1 powder). the final preparation is salt free reddish brown powder with free ammonia content less than 0.01 g −1 units. the rz value (a400/a280) of the enzyme, which is a good criterion of purity and heme content, was 2.6. the lyophilized enzyme was stable at −20 • c for at least one year. the liquid form of the enzyme in presence of 0.1% sodium azide was stable up to 25 days at 4 • c, while it lost most of activity at room temperature in the same period. the properties of horseradish peroxidase including km, optimal ph and temperature, activation energy, thermal stability and effect of different compounds were studied. the applicability of this enzyme in determination of serum glucose was performed. the analysis of glucose in human sera gave results using the kit containing the prepared peroxidase similar to those obtained with a commercial glucose kit. lactobionic acid production using lactose oxidase: from laboratory to 600 l scale mikkel nordkvist 1 , per munk nielsen 2 , peter budtz 3 , john villadsen 1 : 1 center for microbial biotechnology, technical university of denmark, dk-2800 lyngby, denmark; 2 novozymes a/s, dk-2880 bagsvaerd, denmark; 3 chr. hansen a/s, dk-2970 hørsholm, denmark. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) currently, lactobionic acid is mainly a high-price specialty product used, e.g. in solutions for organ stabilization. however, lactobionic acid can also be used as a biodegradable cobuilder in detergents, and it has several applications in food technology. with lower production costs it has the potential to become a bulk chemical. the kinetics for the oxidation of lactose to lactobionic acid by a new carbohydrate oxidase was studied in a 1 l bio-reactor with control of ph, temperature, and dissolved oxygen. the byproduct hydrogen peroxide has a negative influence on the lactose oxidase enzyme, and hence additional experiments were made with addition of catalase to remove hydrogen peroxide, thereby also providing extra oxygen. on the basis of the experiments in 1 l scale, experiments were performed in a 600 l reactor equipped with a new system for dispersion of air to supply the necessary oxygen for the oxidation. the aeration system in the large scale reactor was able to supply oxygen sufficiently fast to give the same production rate, at low values of the air flow rate and the energy input, as was obtained in the high-performance laboratory reactor. the non-characterized gene previously proposed as d-tagatose 3-epimerase from agrobacterium tumefaciens was cloned and expressed in escherichia coli. the expressed enzyme was purified by affinity chromatography on histrap hp, desalting chromatography on hiprep 16/60, and gel filtration chromatography on sephacryl s-300 hr with a final specific activity of 8.89 u/mg. using maldi-tof-ms, the native protein was estimated to have a molecular mass of 32,600 da and a monomeric structure. the purified enzyme exhibited maximal activity at 50 • c and ph 7.5 without the addition of metal ions and at 60 • c and ph 7.0 with 1.0 mm mn 2+ . among various metal ions, mn 2+ was the most effective divalent cation for d-fructose epimerization activity. the addition of mn 2+ significantly increased the thermal stability and the epimerization activity with other ketoses such as d-psicose, d-tagatose, d-ribulose, d-sorbose, and d-xylulose. the activity, substrate affinity, maximum velocity, and catalytic efficiency (k cat /k m ) of the enzyme for d-psicose were higher than those for d-tagatose, which suggests that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. the equilibrium ratio between d-psicose and d-fructose was 37:63 at 60 • c with 1.0 mm mn 2+ . when the enzyme was used at 14 u/ml, dpsicose was produced at 211 g/l from 700 g/l d-fructose containing 1 mm mn 2+ after 120 min, corresponding to a conversion yield of 30.2%. the role of ammonium ions in glucosamine formation during the citric acid fermentation process by aspergillus niger m. papagianni 1 , f. wayman 2 , m. mattey 2 : 1 department of hygiene and technology of food of animal origin, school of veterinary medicine, university of thessaloniki, thessaloniki 54006, greece; 2 department of bioscience, university of strathclyde, glasgow, g1 1xw, uk. e-mail: mp2000@vet.auth.gr (m. papagianni) stoichiometric modeling of the citric acid fermentation process by aspergillus niger, in 12-l stirred tank reactor, indicates that nh 4 + ions combine with a c-containing metabolite inside the cell to form a nitrogen compound which is then excreted by the mycelium. the close correlation between calculated and experimental profiles indicates that this metabolic process is rapid and takes place before the c-structure of the glucose has been greatly altered by glycolysis or the pentose phosphate pathway. hplc analysis identified glucosamine as the product of this relationship. a clear effect of medium concentration of nh 4 + on glucosamine formation was observed when fermentations carried out with optimal and sub-optimal ammonium concentrations. (nh 4 ) 2 so 4 addition when medium nitrogen was depleted, enhanced the formation of new cells from the tips of fragmented hyphae and led to glucosamine accumulation in amounts depending to pulse concentration. the fungus reacts in excess ammonium by converting it to glucosamine, to be utilized later when a regeneration process takes place with fragmentation of vacuolated hyphae and subsequent regrowth, depending always on the culturing conditions. however, depending on carbon and ammonium concentration in medium, glucosamine can be secreted in concentrations as high as 50 g/l. about 1000 microorganisms originated from traditional korean food origin were screened for efficient palatinose production. an isolate designated fmb1 was exceptionally efficient in sucrose-palatinose conversion activity. conversion of sucrose into palatinose by fmb1 was much faster than a reference strain of erwinia rhapontici. fmb1 is a gram negative, facultatively anaerobic, motile, noncapsulate, and straight rod-shaped bacterium producing acid from glucose. based on api and 16s rdna analyses, fmb1 was determined to be enterobacter sp. the maximum conversion of 10% sucrose to palatinose and trehalulose by enterobacter sp. fmb1 was achieved within 6 h. the preliminary dna sequencing result of the gene corresponding to sucrose isomerase of enterobacter sp. fmb1 revealed that it showed 87% similarity to that of klebsiella sp. (??). within the scope of an r&d project developed in collaboration with leather tanning portuguese industrial partners a screening of new proteases to be used in the industrial process was performed. a bacillus subtilis strain isolated from alkaline spent purge liquor was shown to be a promising protease producer. microorganism growth was studied for optimisation of temperature, agitation, ph and medium composition either for biomass or proteases production. optimal growth temperature is different for maximum biomass growth (40 • c) and optimal proteolytic activity (43 • c) yielding biomass specific growth rate of 1.6 and 1.4 h −1 , respectively. the achieved proteolytic activities were 5.7 and 7.2 u/ml of protease, respectively. optimised medium composition (7 g/l beef extract, 4 g/l yeast extract, 5 g/l peptone and 0.4 g/l cacl 2 ) yielded a specific growth rate of 1.5 h −1 and 13.9 ku/l of protease, in shake flask experiments. bioreactor experiments (from 1 to 16 l) with the selected medium were performed at 43 • c in order to test aeration rate (1 and 2 vvm), stirring (300-700 rpm) and ph (uncontrolled, controlled at 7 and 8). best protease activity was 64 u/ml in 2 l bioreactor without ph control at 500 rpm and 2 vvm. the proteolytic extract was characterized and compared to commercial bates. results indicate that these proteases can be employed in the purge phase of the leather tanning process in industry. the gram positive bacterium bacillus megaterium is known for its capacity to produce exoenzymes including amylases, proteinases, and penicillin amidase at industrial scale. here, we describe the development of various vectors for the production and export of recombinant heterologous proteins employing b. megaterium signal peptides. the target gene can be cloned directly adjacent to the signal peptide coding sequence (bart et al., 2005) . this arrangement allows for a correct n-terminal sequence of the mature protein after processing by the signal peptidase sipm. using this newly developed protein production and export system lactobacillus reuteri 121 levansucrase (van hijum et al., 2001) was secreted in significant amounts (∼4 mg/l) into the growth medium. fusion of the recombinant levansucrase to affinity tags allowed one-step purification of the recombinant protein from the growth medium. however, fused peptide tags resulted in a decreased secretion of the fusion protein. 1.4 mg his 6 -tagged levan-sucrase were purified per litre of culture. the system was further enhanced via coexpression of a gene for the signal peptidase sipm (malten et al., 2005a) and deletion of the gene for the extracellular protease nprm. developed new tools allow for various strategies of integrated high level production, export and purification of heterologous proteins in b. megaterium. methods for high-throughput screening of secreted enzymes are under development. the determined sequence of the b. megaterium genome, studies using high-cell density cultivations (malten et al., 2005b) and proteome data from batch fermentations implicate new targets for directed genetic optimization of b. megaterium production and secretion strains. novel strong and inducible promoters are currently under investigation. toru matsui 1 , naoya shinzato 1 , hisashi saeki 2 , hitoshi matsuda 2 : 1 center of molecular biosciences, university of the ryukyus, okinawa 903-0213, japan; 2 japan energy co., japan. e-mail: tmatsui@comb.u-ryukyu.ac.jp (t. matsui) optically active epoxides are considered as the potential intermediate for chiral drugs synthesis. although s-styrene oxide (so) have been extensively investigated using styrene monooxygenase from pseudomonas sp., microbial production of r-so with high enantiomeric excess was hardly examined. in this study, r-so producing bacteria from styrene was screened using various alkene assimilating bacteria. r-so with the highest ee (ca. 100%ee) was obtained using ethene utilizing bacteria, identified as mycobacteirum sp., while produced relatively lower at around 70%ee when using propene utilizing bacteria. the alkene monooxygenase gene homologue sequence amplified from the genomic dna revealed a significant similarity to that of etnabc. these bacteria also showed stereoselective degradation of racemic so, suggesting that the produced so might be further stereoselectively degraded to increase the ee. the ethene utilizing bacteira produced not only r-so but also s-epichrolhydrin at high ee.when using arylchloride as the substrate. this research was supported by nagase science and technology foundation. the secretion efficiency of the escherichia coli sec pathway is dependent on the growth phase but not on protein size f.j.m. mergulhão, g.a. monteiro centro de engenharia biológica e química, instituto superior técnico, av. rovisco pais, 1049-001 lisbon, portugal. e-mail: filipem@alfa.ist.utl.pt (f. mergulhão) the secretion efficiency of the escherichia coli sec pathway was evaluated through the expression of green fluorescent protein and human proinsulin fusion proteins. translocation to the periplasm is dependent on the growth phase of the bacterial culture and the highest secretion efficiency is attained in mid-exponential phase. secretion performance is independent of protein size (17-42 kda) and even when the amino acid composition of the secreted proteins is very similar, the amino acid distribution within the protein can affect translocation. in silico prediction analysis suggests that proteins that are prone to form ␣-helix structures are more efficiently translocated. culture medium composition plays an important role on secretion performance with the highest secretion results being obtained in minimal medium. streptokinase is a common fibrinolytic drug. that is used in thrombolytic therapy for long time. to compare with another thermbolytic drugs like tpa, it has lot of advantages. in this present research dna was extracted from s. equisimilis h46a for the first time in iran. streptokinase gene was amplified by using two forward primers and one reverse primer. a common restriction enzyme, bamh-i, was used for cloning. both ends of the pcr products (full length: 1323 bp and mature section: 1245 bp) and the restriction site on mcs of pqe-30 vector were digested. in this study, pqe-30 expression vector was used with high level expression ability for production of recombinant fusion streptokinase with simplifying the purification by employing affinity-metal chromatography method. in addition, the cloning results were controlled by double digestion and sequencing. takesono oxidation of short-chain iso-alkanes was studied with propanegrown resting mycelia of scedsporium sp. a-4. isobutane was oxidized to tert-butanol, but not to isobutanol. isobutanol was used for growth, but both isobutene and tert-butanol were not used for growth. isopentane was oxidized to 3-methyl-1-butanol, 2-methyl-2-butanol, and 3-methyl-2-butanol but not to 2-methyl-1-butanol. 2-methylpentane was oxidized to 4-methyl-1-pentanol, 2-methyl-2pentanol, and 4-methyl-2-pentanol but not to 2-methyl-1-pentanol or 2-methyl-3-pentanol. 3-methylpentane was not oxidized. oxidation of branched alcohols was also studied. application of nadph-dependent 2.5-diketo-gluconic acid reductase for production of l-ascorbic acid claudia pacher 1,2 : 1 division of food biotechnology, department of food sciences and technology, boku, university of natural resources and applied life sciences, muthgasse 18, a-1190 vienna, austria; 2 research centre applied biocatalysis, petersgasse 14, a-8010 graz, austria. e-mail: claudia.pacher@boku.ac.at ascorbic acid is an organic acid with various applications in the food and pharmaceutical industries. at present, the majority of commercially manufactured vitamin c is synthesized via the reichstein process, which is highly energy-consuming, involves considerable quantities of organic solvents and gives an overall yield of about 50%. therefore, during the past decades a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic or fermentative means, which show some advantages regarding costs and environmental friendliness. one of the fermentation routes runs via 2.5-diketo-d-gluconic acid (2.5-kdg), produced by pectobacter (erwinia) cypripedii. this compound has been reduced by a nadph-dependent 2.5-diketogluconic acid reductase (dkr) from corynebacterium glutamicum to the key intermediate 2-keto-l-gulonic acid (2-klg) before chemical rearrangement leads to the final product. for economical reasons we wanted to express dkr heterologously. based on our long term experience with coenzyme regeneration we wanted also to perform the reaction in homogeneous solution. the spent coenzyme of nadph-dependent dkr has been regenerated by a second isolated nadp-dependent enzyme like glucose dehydrogenase. we describe here the recombinant production, purification and characterization of dkr and the results of enzymatic 2-klg formation by using the recombinant enzyme in a homogeneous system with conjugated coenzyme regeneration. grp78 residing in endoplasmic reticulum (er) functions as a molecular chaperon by associating transiently with incipient proteins as they traverse the er and aiding in their folding and transport. furthermore, the protein can also be induced under various stress condition such as glucose starvation, inhibition of protein glycosylation by tunicamycin, blockage of vesicular trafficking by brefeldin a and er-calcium-atpase pump inhibition by thapsigargin. thus, substances that directly down and up-regulate grp78 transcription are expected to be useful for treatment of cancer and alzheimer's disease, respectively. in the course of our screening program to obtain substances, which regulate grp78 expression, we first constructed an assay system monitored by the expression of a reporter gene. hela cells, which are transformed with luciferase gene under the control of grp78 promoter designated as hela 78c6 cells, respond sensitively to luciferase grp78 induction by er stress such as treatment of tunicamycin. by using this screening system, we isolated pyrisulfoxin as an up-regulator of grp78, and valinomycin, citreoviridin and alternariol as down-regulators. detailed studies on other biological activities were now under way. pdh is an enzyme that was described only several years ago in a number of ecologically related litter-decomposing fungi (agaricales, gasteromycetales). it catalyzes the c-3 and/or c-2 oxidation of several aldopyranoses to the respective keto sugar derivates. pdh shows a very broad substrate range, oxidizing almost all major sugar components of wood polysaccharides, and is implicated to play a role in lignocellulose degradation. agaricus bisporus, the white button mushroom, is an economically significant agricultural crop. the cultivation, which is done by solid-substrate fermentation on straw-and hay-based composted substrate, is sometimes seen as one of only few economically feasible methods for bioconversion of lignocellulosic agricultural waste material. deeper insight in the physiological role of pdh may provide help for mushroom growers to increase yield, improve quality or make new sources of raw materials utilizable. we amplified a fragment of the pdh gene with degenerated primers derived from internal peptide sequences. the screening of a genomic library led to the isolation of the pdh gene. subsequently we amplified a cdna clone by rt-pcr and investigated the transcriptional regulation by different carbon sources on a defined minimal medium. optimization of monoclonal antibody production processes with simulation and scheduling tools demetri petrides, charles siletti intelligen inc., scotch plains, nj 07076, usa. e-mail: dpetrides@intelligen.com (d. petrides) this presentation will review the state of the art in batch process simulation and scheduling tools and their applications in the design and debottlenecking of integrated biopharmaceutical processes. a systematic methodology will be presented for identifying and eliminating size, time, and throughput bottlenecks that limit production in single and multi-product facilities. the methodology will be illustrated with an industrial case study dealing with the optimization of a multi-product facility that produces therapeutic monoclonal antibodies (mabs). mab processes are characterized by a long bio-reaction time (e.g., 1.5-2 weeks for fed-batch operation and 1-2 months for perfusion operation). the cycle time for processing a lot in the recovery and purification train typically takes 3-4 days. consequently, one way of increasing throughput is by installing extra bioreactors that operate in staggered mode and utilize the same recovery train. the result is that multiple batches may be at different stages of completion at any given time. since cleaning equipment (e.g., cip skids) and buffer preparation and holding tanks are shared by multiple steps across many batches, this type of operation leads to time/scheduling bottlenecks that constrain the cycle time and the throughput of a process. the problem becomes more challenging in the context of multi-product facilities and when constraints imposed by the limited availability of resources are considered. our methodology and its computer implementation will illustrate how to systematically identify and eliminate such bottlenecks. the industrial case study will provide a real world example of the methodology. application of two stage continuous cultures of aspergillus niger for citric acid biosynthesis jerzy j. pietkiewicz, malgorzata janczar, wladyslaw lesniak food biotechnology department, university of economics, wroclaw 53-345, poland. e-mail: jerzy.pietkiewicz@ae.wroc.pl (j.j. pietkiewicz) the aim of the work was application test of submerged two stage single stream continuous cultures of aspergillus niger for citric acid production from sucrose. studies were carried out in lab fermenters with working volume of 5 dm 3 . the bioreactors were standard cstrs. in two stage continuous cultures (tscc) high mycelium growth and high citric acid production were observed in the first bioreactor. there was almost four times lower growth of biomass rate and about three times lower citric acid production rate in the second bioreactor. studies on influence of dilution rate in race from 0.0098 to 0.0230 dm 3 /(dm 3 h) on course and efficiency of tscc showed, that the highest citric acid yield (y p/s = 86.3%), high volumetric rate of its production (r pc = 0.958 g/(dm 3 h)) and the highest biosynthesis efficiency coefficient (k ef = 82.7) were obtained with dilution rate d = 0.0148 dm 3 /(dm 3 h). there was also high citric acid concentration (p = 129.5 g/dm 3 ) and low residual sugar concentration (s k = 6.9 g/dm 3 ) in the medium flowing out the second bioreactor in those cultures. the beta-galactosidases in commercial use are of different origins and yeast and fungal lactases present the greatest interest. the yeast lactases present neutral optima ph and are suitable for the hydrolysis of lactose in milk. in this work, the aim was to study the influence of aeration in the production of beta-galactosidase in batch fermentations with kluyveromyces marxianus atcc 46537. the medium composition for culture was as follows (in g/l): lactose pa 50, yeast extract 5, (nh 4 ) 2 so 4 4 and kh 2 po 4 2. the fermentations was carried out at 30 • c, ph 5.5, at 200 rpm starting with an initial cellular concentration of 1 × 10 7 cels/ml, with different aeration rates. the cells were disruped with chloroform 2% (v/v) as solvent. the enzymatic activity was determined as initial rate of lactose hydrolysis at defined conditions. the studies have revealed the importance of aeration on kluyveromyces marxianus in the growth and beta-galactosidase synthesis. the enzymatic activity of fermented medium with 0.5 vvm was 50% higher than one without aeration. furthermore, the cellular growth was faster in the aerobic fermentation than in the anaerobic one. the aeration has taken an important place in the enzymatic synthesis and in the cellular growth, however the results have shown that the aeration rate increase of 0.5-1.5 vvm has not implied a increase in the cellular growth neither in the enzymatic reached activity. the lactose presents in the milk has a solubility of only 20% at 30 • c, and a high percentage of the world population presents intolerance to this sugar, due to the low or absence of the activity of the lactase enzyme in the organism. to minimize such problems, the most viable alternative for nourishing dairy products is the enzymatic hydrolysis of milk, although it is an expensive process due to the high cost of the beta-galactosidase enzyme. an alternative that has been greatly studied is the immobilization of this enzyme, originated from many different sources. there are several immobilization procedures for this enzyme, however, a procedure considered ideal was not obtained yet. the objective of this work was to study the immobilization process of beta-galactosidase from aspegillus oryzae in sodium alginate with commercial gelatin. in the immobilization process was studied the glutaraldehyde influence for 1, 3 and 5%, in the presence of commercial gelatin at the concentration of 2% at the immobilization medium. the activities of the immobilized enzymes were obtained in a stirred micro-reactor, at the temperature 30 • c, ph 4.5 with a 50 gl −1 lactose solution in acetate buffer. the experimental results showed that the immobilized biocatalyst that presented the larger initial activity was the one obtained at the immobiliza-tion medium that contained 5% of glutaraldehyde. after 20 daily determinations of enzymatic activities, a fall of 30, 40 and 24% was verified in the enzymatic activities for the immobilized biocatalysts using glutaraldehyde at 1, 3 and 5%, respectively, however, in all cases, the enzymatic activity reached the half of their initial activity after 10 determinations. hydrolysis of sucrose by immobilized beta-fructofuranosidase in silica eloízio júlio ribeiro, ubirajara coutinho filho faculdade de engenharia química, universidade federal de uberlândia, uberlândia 38400-902, brazil. e-mail: ejribeiro@ufu.br (e.j. invertase, known as beta-fructofuranosidase (ec 3.2.1.26), plays a catalytic role in the conversion of sucrose into glucose and fructose. it is largely used in the food industry to prevent the crystallization of sucrose in sugar mixtures and can be used in enzyme reactors for hydrolysis of sucrose. the objective of this work was to study the kinetic of sucrose hydrolysis by immobilized betafructofuranosidase in a continuous recirculation reactor, evaluated the enzyme stability and determine the effective half-life of immobilized enzyme. invertase was covalently immobilized on sillanized controlled pore silica. nonlinear fitting were used to determine the kinetic parameters for substrate and product inhibition observed in the enzymatic hydrolysis of sucrose. the kinetics studies of immobilized invertase were carried out in a continuous recirculating reactor. the half-time of enzyme inactivation (t 1/2 ) was calculated from the initial rates of the remaining enzyme activity. the model of inhibition by substrate and product adequately represented the enzymatic hydrolysis. the fructose effect was competitive inhibition (k f = 3.1022.10 −4 mol/ml) and the glucose effect was noncompetitive inhibition (k g = 2.2521.10 −4 mol/ml). the effective diffusivity of sucrose into the support was shown to be the same as for sucrose in dilute solution (0.75 × 10 −5 cm 2 /s at 40 • c). the half-time of enzyme inactivation (t 1/2 ) was 1656 h. controlled pore silica showed to be an excellent immobilizing support. the immobilized invertase was very stable at temperatures lower than 50 • c. the intrinsic parameters (k i , k f , k g and v m ) were shown to be similar to the apparent values. the low permeability of mycobacterial cell wall envelopes is a result of the unique composition and organization of the cell wall lipids. the permeability of mycobacterial cell wall can be changed by means of partial disintegration of its compounds. the aim of present work was to characterize the changes in the cell wall skeleton (cws) and non covalently bound free lipids under the influence of isoniazid, the inhibitor of mycolic acids biosynthesis. fatty acid (fames) and mycolic acid methyl esters (mames) obtained from all tested preparations were analyzed by gc/ms analysis. the analysis of free lipids and cws revealed distinct changes in the composition of the frac-tions obtained from the cells exposed to action of the isoniazid. the changes in the quantity of fatty acids in the inh-treated cells indicates that inh interferes with the synthesis of lipidic compounds of the mycobacterial cell wall also. the decreased amount of covalently bound mycolic acids in the cws is responsible for the enhanced penetration of hydrophobic compounds through the cell wall. this work was supported by grant nr 3p04c 06923 of the committee for scientific research. barbara sencio, teresa jamroz, stanislaw ledakowicz department of bioprocess engineering, technical university, lodz, poland the enzyme laccase (ec.1.10.3.2. p-diphenol oxidase) is a subject of research in many centres dealing with improvement of bioprocesses with the use of different white rot fungi species. most strains that produce this enzyme in vitro require inductors initiating its biosynthesis. when cerrena unicolor was applied, it was found that the strain produced laccase very efficiently without additional toxic compounds. to specify optimum conditions for laccase production in a submerged culture, research was undertaken to obtain the most efficient inoculum c. unicolor. the goal of this research was to determine the effect of form and incubation time of inoculum on enzymatic activity of the laccase producing strain. the experimental inoculum was the mycelium prepared on a solid and liquid substrate. basing on results obtained, it was found that the laccase yield was the highest in the cultures where the mycelium was grown on a solid substrate. maximum activity of the c. unicolor strain was achieved on the 16th day of culture, and the amount of laccase produced was higher by, ca. 30% as compared to the mycelium obtained from the liquid substrate. results of these experiments were used to continue studies on the impact of inoculum age. experiments were carried out using an inoculum incubated for 1-3 weeks at the temperature 30 • c in a certomat bs1 shaker at 110 rpm. the best results in the c. unicolor strain culture were achieved using a 7-day-old inoculum. effect of alcohol treatment on hydrolytic activity of candida rugosa lipase serpil takaç, a. ezgiünlü department of chemical engineering, institute of biotechnology, ankara university, 06100 tandogan, ankara, turkey candida rugosa lipase (crl) was treated with 20, 40, and 60% concentrations of methanol (m), ethanol (e), 2-propanol (2p) and 1-butanol (1b) to investigate the changes in its hydrolytic activity toward p-nitrophenylacetate. the treatment included the following steps at +4 • c: (i) stirring crl with phosphate buffer for 24 h; (ii) treating the solutions with alcohols; (iii) stirring treated-crl for 24 h; (iv) centrifugation at 10,000 rpm for 30 min; (iv) dialysis the supernatant against bidistilled water for 39 h. the activity of crls was followed for 120 h at 37 • c in the presence and absence of isooctane. the enzyme activity was measured spectrophotometrically and the protein concentration was measured by lowry's method. it was found that the recovered protein did not change considerably with the type of alcohol; however, decreased with alcohol concentration. in the presence of isooctane, specific activities of the untreated and treated-crls increased compared with those obtained in the absence of isooctane. 1b-crls and e-crls showed higher activities than m-crls and 2p-crls whereas untreated-crl exhibited higher activity than m-crls, e-crls and 2p-crls. the highest and the lowest activities were obtained with 20% 1b-crl and 60% 2p-crl, respectively. the changes occur in the structure of crl after treatments were investigated by electrophoretic analysis. this study was supported by ankara university biotechnology institute (project no: 89) . different genera, species and strains of microorganisms were found to posses different cryoresistance. optimal ways for cryopreservation of microorganisms-producers of antibiotics, microorganisms, used in food industry, agriculture and veterinary have been developed. it was demonstrated, that non-lethal damages could occur in cryopreserved microorganisms after their returning to physiological culture conditions, which were manifested in streptomyces' hypha fragmentation, that of cyanobacteria's, streptococci's chains as a result there was an increase in a number of colony-forming units, a reversible inhibition of microorganisms' proliferative activity in bacillus thuringiensis and lactic streptococci, stimulation of the enzyme processes and antibiotic production. non-lethal damages are repaired during microorganism culturing in the first passage. the cause of non-lethal damages is a reversible inhibition of biosyntheses of protein and nucleic acids respiratory activity. the repairing of non-lethal damages is accompanied by the production of stressproteins, different from heat shock proteins. effect of ph in the 2-propanol treatment of candida rugosa lipase on its enantioselectivity in the hydrolysis of racemic naproxen methyl ester serpil takaç, a. ezgiünlü department of chemical engineering, ankara university, 06100 tandogan, ankara, turkey candida rugosa lipase (crl) was treated with 2-propanol (2p) at the ph values of 1.5, 4, 6, 7.5, 9 and 12 to investigate the changes in its enantioselectivity in the hydrolysis of racemic naproxen methyl ester. the treatment included the following steps at +4 • c: (i) stirring crl with different buffer solutions to maintain the desired ph values for 24 h; (ii) treating the solutions with 40% 2p; (iii) stirring treated-crls for 24 h; (iv) centrifugation at 10,000 rpm for 30 min; (iv) dialysis the supernatant against bidistilled water for 39 h. hydrolyses of racemic naproxen methyl ester to form s-naproxen were performed in shaking flasks at 200 rpm and 37 • c for 192 h in isooctane-phosphate buffer solution biphasic system using treated-crls with the activity of 7.75 u. the concentrations of the enantiomers of naproxen methyl ester and naproxen were determined with hplc. it was found that the treatment ph has an important role on the enantioselectivity and conversion. the highest enantiomeric excess for the substrate, for the product, enantiomeric ratio, and con-version were obtained with crl treated at ph 1.5 as 39, 98, 181 and 29%, respectively. these values were followed with 2p treated crl at ph 12 as 35, 98, 121 and 27%. however, lower enantiomeric excesses, conversions and enantiomeric ratios were obtained at the treatment ph values between 1.5 and 12. the effects of fatty acids, nitrogen (as nh 4 no 3 ), phosphorus (as kh 2 po 4 ), ph value, manganese (mn 2+ ), iron (fe 2+ ) and methanol concentration on growth and production of oxalic acid from post refining fatty acids by a mutant of aspergillus niger xp in submerged fermentation experiments was studied. of the a. niger strains screened, a. niger xp was identified as the best oxalate producer on lipids. the influence of the ph on oxalic acid formation shows that the maximum production rate and higher concentration of product are observed at the ph ranging from 4 to 5. with a medium containing 50 g fatty acids/l, the production reached a maximum of 68 g oxalic acid/l after 7 days. the addition of 1.5% (w/v) methanol to seed culture increased the product yield and concentration of oxalic acid but decreased the amount of an undesired by-product (citric acid). under this condition, the maximum oxalate productivity (14-18 g/l days) was maintained for 2-4 days of fermentation. other results of the experiments show that supplementation of the production medium with manganese and iron enhances oxalate production. fatty acids proved to be a very good substrate for oxalic acid production by a. niger xp giving excellent yields and productivity at low ph. the results are very promising as they may lead to cheap alternative processes for oxalic acid production from renewable lipid resources. department of food engineering, middle east technical university, ankara 06531, turkey. e-mail: banuy@metu.edu.tr (b. yalcindag) laccase (e.c. 1.10.3.2, p-benzenediol:oxygen oxidoreductase), which is an enzyme belonging to the multi-copper oxidase family, catalyzes the oxidation of a broad variety of polyphenols with a preference for p-isomers, which are converted to p-quinones. fungi generally contain several laccases which have been found to be involved in delignification, melanin synthesis and pathogenesis. laccase has also important potential application areas especially in food and chemical industries. after aspergillus fumigatus genome data were released, research on functional analysis of laccases has been initiated in our laboratory. laccase genes of aspergillus nidulans, ya and tila, and laccase and multi-copper oxidase genes of aspergillus fumigatus, abr2 and abr1, were used to analyze a. fumigatus genome for laccases. this sequence analysis resulted in 4 probable laccase genes, one of which was the previously cloned abr2 gene. in this study, one of these genes (aflac1) was further characterized. after sequence alignment and characterization studies, aflac1 was predicted to have 2128 bp having six introns, which makes the protein 606 amino acid long, and the predicted protein sequence showed 63% homology with the dihydrogeodin oxidase of aspergillus terreus and 38% homology to the laccase 2 of botryotinia fuckeliana. aflac1 gene is found within an uncharacterized gene cluster containing genes with homology to glutathione-s-transferase, polyketide synthase, o-methyl transferase, and others. the information obtained from sequence analysis was employed in designing pcr-primers to amplify the aflac1 gene, followed by cloning onto pan52-1 and pan52-4 vectors for heterelogous expression in aspergillus sojae. in addition, by the use of rt-pcr, aflac1 cdna will be cloned and expressed in escherichia coli. furthermore, gene silencing studies will be performed to enlighten the function of aflac1 and associated gene cluster. stability of growth rate of photosynthetic cells is an important factor in designing of effective photobioreactors especially in long term operations. in our experiments, in order to keep operational parameters almost constant, a semi-continuous culture method was developed. in this method, a part of culture broth containing grown cells was repeatedly replaced by fresh medium at a predetermined time interval. the replacement of broth with fresh media could keep the cell concentration, volume of broth and distribution of light intensity constant at initial values throughout the cultivation. it was shown that in one side illumination with a halogen lamp, if the ratio of the light intensity at the front side of a flat plate photobioreactor to that at the rear side was kept lower than 4, the growth rates was sustained in constant levels. however, at higher ratios the growth was followed by rapid decrease after 5-6 h. supplemental illumination with a fluorescent lamp from the rear side of the flat plate photobioreactor could sustain almost stable growth rate. beside of the illumination conditions, increased ferrous ion concentrations in medium could keep the stability of growth rate even in unstable illumination conditions, while consumed ferrous ion was slight. glutathione (gsh) plays a pivotal role in protecting cells from by-products generated by oxidative metabolism. these characteristics make this active tripeptide an important drug for the treatment of liver diseases and is of interest in the food additive industry, therapeutics and sport nutrition. in the first part of the research, a screening was carried out among 48 yeast strains, to find out those able to accumulate higher gsh intracellular levels. two saccharomyces cerevisiae strains proved to be the best gsh producers (1.3%dw), in every samples the presence of s-adenosyl-methionine in traces (0.2% dw) was also evidenced. s-adenosyl-methionine (sam) plays a role in the immune system, maintains cell membranes, participates in detoxification reactions and in the manufacture of brain chemicals and cartilage. the second part of the research was aimed at increasing, in a post fermentative procedure, gsh levels present inside the cells at the end of the growth phase. moreover, time course of sam intracellular levels, to be related with accumulated gsh, was also monitored. cells were then suspended in an appropriate solution containing mineral salts, glucose and the aminoacids, precursors of the two studied molecules. according to this procedure, gsh intracellular levels reached 4.6% dw after 48 h incubation. moreover, gsh levels can be related to sam production (up to 1.3% dw). the presence, in several samples, of intermediate metabolites, such as cystathionine and omocysteine, proved the establishement of an intracellular equilibrium between gsh and sam; this behaviour represents a promising starting point for the set-up of a microbial process for the simultaneous production of the two studied molecules. three acetate mutants of y. lipolytica yeasts, which varied in colony morphology (rough and smooth), were employed for continuous citric acid production from glucose and fructose syrup in a membrane reactor with cell recycle. the strains were compared for their product yields, specific acid production rates and ratios of citric acid to isocitric acid. experiments shoved that glucose syrup was a better substrate for citric acid production by y. lipolytica. citric acid concentration in the effluent ranged from 80 to 120 g/l, depending on the yeast strain used. all y. lipolytica strains produced very low amounts of isocitric acid. its concentration did not exceed 3 g/l. based on the results of these experiments, smooth strain awg-7 was found to be the most suitable for citrate production both from glucose and fructose syrup during long time continuous processes (500 h). in the steady state, the highest citrate productivity (1.3 g/lh) was obtained with this strain, when the feed medium contained 200 g/l of glucose and dilution rate (d) was d = 0.013 1/h. supplementation of the feed medium with bacto-pepton improved the productivity, citric acid yield and stability of the continuous process in the cell recycle fermentation system. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. glycine oxidase is the product of the yjbr gene of bacillus subtilis that was predicted by sequence homology to be a flavoprotein similar to sarcosine oxidase. glycine oxidase catalyzes the oxidative deamination of various primary and secondary amino acids (e.g. sarcosine, n-ethylglycine, and glycine) and d-amino acids to form the corresponding ␣-keto acids and hydrogen peroxide. previous investigations reported on the cloning and production of the glycine oxidase gene in escherichia coli was up to 1 u/g cell. the present works has improved the expression of the recombinant his-tagged glycine oxidase by 15-fold by using pet28a and rosetta cells under the optimal iptg, temperature and time of induction. the protein obtained represented 30% of total soluble proteins in crude extract. the enzyme was purified to near homogeneity using imac with a 95% recovery and with and specific activity of 1.21 u/mg protein. the enzyme was active towards glycine, sarcosine and different d-amino acids, having in general, a basic ph optimum. the kinetic parameters were also studied, showing a km range from 0.3 to 300 mm. the enzyme was immobilized, and used to obtain pyruvic acid (␣-keto acid) from d-alanine with a good yield. the enzymatic synthesis of lipophilic derivatives of various natural antioxidants including flavonoid glycosides, as well as derivatives of cinnamic acid, was performed using various immobilized lipases in ionic liquids such as 1-butyl-3-methylimidazolium tetrafluoroborate (bmim-bf 4 ) and 1-butyl-3-methylimidazolium hexafluorophosphate (bmim-pf 6 ). the influence of various reaction parameters on the catalytic behavior and the selectivity of lipases was pointed out. a response surface methodology was applied for the optimization of the yield and the productivity of the biocatalytic process. the antioxidant activity of the biocatalytically prepared lipophilic derivatives of natural antioxidants, as expressed on cu 2+ -induced oxidation of low-density lipoprotein (ldl) and total serum, was investigated. process strategy for reduction of proteolysis in pichia pastoris fermentations jan weegar 1 , john dahlbacka 1 , noora sirén 2 , niklas von weymarn 2 , kaj fagervik 1 : 1 faculty of chemical engineering,åbo akademi university, finland; 2 laboratory of bioprocess engineering, helsinki university of technology, finland. e-mail: jan.weegar@abo.fi (j. weegar) the yeast pichia pastoris is a popular host organism for production of recombinant proteins. it is, however, common that the products are degraded by proteases towards the end of the fermentation, resulting in productivity and purity decreases. proteolysis of recombinant proteins in p. pastoris fermentations is affected by the temperature and ph of the growth medium. in this study, it was shown that decreasing the temperature from 30 to 10 • c during the induction phase effectively prohibited proteolysis. on the other hand, the temperature decrease resulted in a reduced maximal methanol consumption rate, which subsequently resulted in a culture highly sensitive to residual methanol. the temperature was slowly decreased according to a predetermined trajectory. as the temperature reached values below 12 • c, the methanol concentration had to be closely monitored and the substrate feed rate adjusted in order to prohibit methanol poisoning as well as to maintain the culture as a substrate limited fed-batch. measurement of protease concentrations revealed that proteases were present at 10 • c, but at this temperature the proteolysis rate was evidently effectively reduced. the recombinant protein produced could almost totally be recovered (i.e. high purity) with this process strategy compared to a constant high temperature culture (30 • c) where severe breakdown of the product was observed. with the growing of an ecological conscience in the public opinion, more and more industrial processes are analyzed for a possible ecologically beneficial alternative. for the production of ascorbic acid, which is nowadays done by the reichstein process, a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic means, which show some advantages regarding costs and environmentalfriendliness. besides of two-stage fermentation, our approach is to design a tailor-made organism that produces 2-keto-l-gulonic acid, which is the direct precursor of ascorbic acid from glucose or gluconic acid and which can easily be converted to the final product by conventional methods. erwinia (pectobacter) cypripedii, which is a natural producer of 2,5-diketo-d-gluconic acid, was selected as a suitable host for a 2,5-diketo-d-gluconate reductase from corynebacterium glutamicum. to increase the yield of the desired compound we investigate two 2-keto-reductases in the host organism that diminish the yield of 2-keto-l-gulonate by reducing the compound to l-idonic acid, or by metabolisation of intermediates. these two enzymes were investigated with vari-ous biochemical and molecular biological methods, which will be presented. overproduction of bioinsecticides by heat and salt stress and control of dissolved oxygen in cheap media of bacillus thuringiensis nabil zouari, dhouha ghribi, samir jaoua laboratoire des biopesticides, centre of biotechnology of sfax, tunisia, bp:k, 3038 sfax, tunisia bioinsecticides based on preparations of spores and insecticidal crystal-proteins (icps) produced by the bacterium bacillus thuringiensis (bt) proved to be a high tool for fighting some agricultural pests and vectors of diseases. however, the use of bt preparations as commercial insecticides would be prohibitively expensive because it is not easy to reach cheap overproduction of icps during large-scale fermentation. here, we report possibilities to improve delta-endotoxins production as a consequence of responses of bt strains to low levels of heat and salt stress. each stressor results differently in the improvement of delta-endotoxins production, but both were shown to be most efficient at the beginning or the midexponential phase of the cultures which become resistant at the stationary or the sporulation steps. heat stress caused increase of 84% of synthesis yields of the sporulating cells, in contrast, salt caused increase of 25% of spores counts, corresponding to 28% toxins production improvement. combined effects of both stressors lead to toxins production improvement of 66%, yield improvement of 40%. we focused on the overcome of carbon repression catabolite, closely related to oxidative metabolism, by an adequate control of dissolved oxygen in the cheap media we formulated for bt insecticides production. we showed that an equilibrium between the high density of vegetative cells and their ability to synthesize toxins during their sporulation was necessary to take into account. 40% increase of icps production was reached into 3 l fermenter combination of mutagenesis, heat and salt stress and oxidative metabolism control allowed more than 100% improvement of delta-endotoxins. these results are of great importance in practical point of view, since high bioinsecticides concentrations could be produced without decrease of the yields of their production. mechanism and function of the intramembrane-cleaving protease rhomboid marius lemberg 1 , javier menendez 2 , christopher koth 2 , matthew freeman 1 : 1 mrc laboratory of molecular biology, cambridge, uk; 2 ontario center for structural proteomics, toronto, canada rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. oligosaccharides and 3-keto-glycosides. availability of the enzyme is, however, hampered by the very slow growth and low production rates of the fungus. cloning of the encoding gene and production of the protein by heterologous expression are therefore a prerequisite not only for any biotechnological application, but further scientific investigations as well. on the basis of peptide sequences degenerated primers were designed, and the resulting pcr fragment was used as a probe to isolate the gene from a genomic library. two very similar genes encoding previously uncharacterized proteins were also found, and flanking regions were amplified using rage-pcr. furthermore cdna clones of all genes were isolated by rt-pcr. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. the behavior of phytate degrading enzymes isolated from malaysian zea mays root in rice bran media anis shobirin meor hussin 1 , abd-elaziem farouk 1 , hamzah mohd salleh 1 , ralf greiner 2 : 1 biomolecular engineering research group, department of biotechnology engineering, kulliyyah of engineering, international islamic university malaysia, jalan gombak, 53100 kuala lumpur, malaysia; 2 centre for molecular biology federal research centre for nutrition and foods, haid-und-neu-straße 9, d-76131 karlsruhe, germany phytate degrading enzymes catalyze the step-wise release of phosphate from phytate, the principle storage form of phosphorus in plant seeds and pollen. they are widespread in nature, occurring in plants and microorganisms, as well as in some animal tissues. phytate-degrading enzymes have been studied intensively in recent years because of the great interest in such enzymes for phytate degradation and their application for animal feed and human health. from over isolate 140 screened isolates of phytate degrading enzymes, three isolates from the root malaysian maize plantation have shown phytate degrading activity. the production of phytate degrading enzyme was studied using different concentrations [%, w/v] of rice bran media during different stages of cultivation. the dephosphorylation of phosphate from rice bran phytate has shown regulatory effect on the secretion of bacterial phytases. in this conference, we will present data for the characterization of the enzymes. in this paper we present the properties of a phytase purified from a bacterium isolated from malaysian wastewater, which might find application as an animal feed supplement. the phytase described herein is a periplasmic enzyme. the phytase was purified about 180fold to apparent homogeneity using ion-exchange chromatography and gel-filtration with a recovery of 10% referred to the phytatedegrading activity in the crude extract. the enzyme exhibits an activity of about 1106 u mg −1 . gel filtration of the native enzyme on a calibrated sephacryl s-200 column gave a molecular mass of the phytase of 42,000 ± 1500 da with elution position being measured by determination of enzyme activity. lower molecular mass species or higher molecular mass aggregates were not observed. the phytase appeared homogeneous by polyacrylamide gel electrophoresis under non-denaturing conditions at ph 8.3 and 4.8 and gave a single protein band upon sds gel electrophoresis after coomassie staining of the gels. these results indicate that the phytase could be regarded as homogeneous. the estimated molecular mass after sds-page indicated that the phytase having a molecular mass of 41,500 ± 2500 da. consequently, this enzyme is a monomeric protein. the purified enzyme had a single ph optimum at ph 4.5 and was virtually inactive above ph 7.0. at ph 3.0, 40% and at ph 2.5, 20% of the activity at optimal ph was observed. the effect on enzyme stability was studied in the ph range 1.0-9.0 at 4 • c. within 14 days the phytase did not lose any activity in the ph range from 3.0 to 8.0, but at ph values below 2.0 a rapid decline in activity was observed. at ph 1.5, 72% and at ph 9.0, 65% of the initial activity was lost during 24 h. in the range of temperatures studied, 10-80 • c, the optimum temperature for the enzyme was found to be 65 • c. the apparent activation energy was estimated at ph 4.5 from the slope of log v max versus 1/t. the data showed excellent linearity from 15 to 65 • c. the arrhenius activation energy for the hydrolysis of phytate was calculated to be 37.5 kj/mol. in order to check thermal stability, the purified enzyme was incubated at different temperatures, cooled to 4 • c and assayed using the standard phytase assay. the enzyme lost no activity in 10 min at temperatures up to 65 • c. when exposed for 10 min at 70 • c, it retained 50% and at 80 • c 12% of the initial activity. in order to determine the substrate selectivity of the purified phytase, several phosphorylated compounds in addition to phytate, were used for k m and v max estimation by detecting the release of the phosphate ion during hydrolysis using formation of a soluble phospho-molybdate complex in an acidic water-acetone mixture. only phytate was identified as a substrate. the kinetic parameters for the hydrolysis of phytate were determined to be k m = 0.15 mmol l −1 and k cat = 1164 s −1 at ph 4.5 and 37 • c. like other bacterial phytatedegrading enzymes, the purified enzyme showed substrate inhibition. the activity of the purified enzyme was inhibited at substrate concentrations >7 mm. the study of the effect of metal ions on enzyme activity showed that none of them had an activating effect when used at a concentration between 10 −4 and 10 −3 m. mg 2+ , ca 2+ , mn 2+ , co 2+ , ag + , hg 2+ , and cu 2+ had little or no effect on enzyme activity, while zn 2+ , fe 2+ , and fe 3+ showed strong inhibitory effects. the reduced phytate-degrading activity in the presence of fe 2+ and fe 3+ is attributed to a lower phytate concentration in the enzyme assay because of the appearance of a fe-phytate precipitate. when compounds which tend to chelate metal ions, such as o-phenanthroline, edta, oxalate, citrate or tartrate, were tested for their effect on enzyme activity, it was noted that none of them was inhibitory at a concentration from 10 −4 to 10 −3 m. fluoride, a known inhibitor of different phytate-degrading enzymes from bacteria and the hydrolysis product phosphate as well as its structural analogs molybdate, wolframate and vanadate were found to be strong inhibitors of the purified enzyme. flouride inhibited the hydrolysis of phytate with a k i value of 112 mol l −1 . several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (amps) in recombinant bacterial expression systems. in the present work, we investigated the use of the baculoviral polyhedrin (polh) protein as a novel fusion partner for production of a model amp (halocidin 18 subunit; hal18) in escherichia coli. the recombinant hal18 amp could then be hydroxylamine cleaved from the fusion protein and easily recovered by simple dialysis and centrifugation. this was facilitated by the fact that polh was soluble in the alkaline cleavage reaction but became insoluble during dialysis at a neutral ph. importantly, recombinant and synthetic hal18 peptides showed nearly identical antimicrobial activities against e. coli and staphylococcus aureus, which were used as representative gram-negative and -positive bacteria, respectively. these results demonstrated that baculoviral polh can provide an efficient and facile platform for production or functional study of target amps. extensive industrial and food additive applications of succinate have attracted much effort towards finding an environment-friendly alternative to the petrochemical production processes. it is very attractive to engineer s. cerevisiae for succinate production because of its generally regarded as safe (gras) status, ease of genetic manipulation and fermentation. we approached this metabolic engineering problem with a two-step methodology combining modern computational as well as molecular biology tools. in the first step we identified potential metabolic engineering targets leading to high succinate yield and productivity, with the aid of genome scale metabolic model and a bi-level optimization framework using flux balance analysis and quadratic programming. in the next step, various deletion mutants are being constructed and characterized for physiology and succinate production. results from these experiments then will be used to improve the predictions in computational models. so far, we have constructed a saccharomyces cerevisiae mutant deleted in sdh3, which encodes a major subunit of sdh-complex converting succinate to fumarate in mitochondria. the physiology of sdh3 mutant has been characterized in aerobic and anaerobic batch cultivations and in glucose limited chemostat at dilution rate as low as 0.027 h −1 . in aerobic batch fermentations, the mutant showed reduced maximum specific growth rate as compared to the wild-type, and it was incapable of growing on ethanol as sole carbon source, as predicted from the model. interestingly, the mutant showed much higher specific growth rate in anaerobic conditions, close to the wildtype strain. moreover, the chemostat cultivations indicate that the critical dilution rate of the mutant is below 0.027 h −1 . this opens further opportunities to investigate interesting behavior of the mutant and the underlying regulatory processes to improve our understanding of yeast mitochondrial metabolism. department of chemical engineering, yıldız technical university, davutpaşa campus, 34210 esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) lactose is the dominant carbohydrate in milks which are, in turn, the only significant natural sources of lactose. a large number of people do not digest lactose properly due to a lack or inactivity of the intestinal beta-galactosidase and they suffer from intestinal dysfunctions -gas, abdominal pain and diarrhea -if their diet contains lactose. moreover, lactose is a sugar with a high bod, low sweetness, low solubility, when compared to the products of its hydrolysis (glucose and galactose) and being a hygroscopic sugar has a strong tendency to adsorb flavours and odours. the hydrolysis of this sugar is very attractive towards the improvement of processes for the production of ice cream and other refrigerated dairy products and it could be very interesting for the development of additives for animal and human alimentation. the enzymatic hydrolysis of lactose is carried out by beta-galactosidases, enzymes that are widely distributed in nature, appearing in micro-organisms, plants and animal tissues. the present investigation describes the effects of the sonication process parameters on enzymatic hydrolysis of milk lactose and enzyme stability. bandelin sonopuls sonicator was used for the lactose hydrolysis experiments. ␤-galactosidase enzyme used is produced from kluyveromyces marxianus. the reactions were carried out in 250 ml of milk. the process variables for the sonicator are duty cycle, acoustic power and enzyme concentration. the amount of residual lactose concentration (g/l) and residual enzyme activity (%) against time were investigated versus process variables. beside of this; the mathematical models depending on the operating conditions were also derived by using the experimental data of lactose concentration and enzyme activity. kinözbek department of chemical engineering, yıldız technical university, davutpaşa campus, 34210 esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) over the last decade, the use of plant protein hydrolysates alternative to animal protein hydrolysates in human nutrition has broadly expanded. protein hydrolysates are often used in different nutritional formulations, such as supplementation of drinks to enhance their nutritional and functional properties, or special medical diets. there are many processes which employ protein hydrolysis and hydrolytic products. among these processes, the use of enzymes allows selective hydrolysis of protein and produces a potentially safer and more defined material. in the present study, the effect of the temperature, ph and viscosity on the hydrolysis of corn gluten was investigated using a stirred batch reactor system. the corn gluten was hydrolysed by using neutrase enzyme, a bacterial protease produced by a selected strain of bacillus amyloliquefacien. the reactions were carried out in 0.2 l of aqueous solutions containing 1% (w/v) corn gluten and 0.4% (v/v) enzyme. the degree of hydrolysis (%) and soluble protein concentration depending on the time were investigated at the temperatures 40, 45, 50, 55, and 60 • c; and at the ph values 6.5, 7, 7.5, and 8. to investigate the effect of viscosity, the various amounts of glycerol was added to the reaction solutions to produce viscosities in the range of 1.415-13.43 cp. the degree of hydrolysis (dh) was computed by using ph-stat method. for the soluble protein determination in the hydrolysates samples, the folin-lowry method (1951) was used. polyhydroxyalkanoates (pha), one of the most promising bioplastics for the partial replacement of synthetic polymers like polypropylene, are polyesters produced by bacteria as intracellular storage reserves of carbon and energy. the industrial production of pha is achieved by pure cultures in its natural state or using genetically engineered organisms. the main obstacle to the replacement of synthetic plastics by biopolymers is their great cost difference. research on the field of biopolymers synthesis using mixed cultures and waste organic carbon sources as substrates prove to decrease substantially the production costs of pha. the optimization of pha production under aerobic feeding conditions (adf) was achieved recently in our group, obtaining the highest value of pha content stored by mixed cultures (79.2% of cell dry weight). in this work only a homopolymer of polyhydroxybutyrate (phb) was obtained and since it is a highly crystalline and brittle material its application field is limited. the mechanical and thermal properties of pha can be varied to a great extend by adjusting the monomer composition. the incorporation of different monomeric units, other than hb, in the polymer chain, originates copolymers with improved mechanical properties. optimization of pha production from propionate by a mixed culture was studied varying the carbon and ammonia concentrations. propionate only, acetate alone or a mixture of acetate and propionate were tested. copolymers of hydroxybutyrate and hydroxyvalerate, p(hb/hv), with different compositions were obtained. consequently polymer properties could be manipulated by feeding the selected volatile fatty acid composition. the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of 6 weeks. mean transformation frequency ranged from 27% (for 8196 up to 31% (for 15834). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba 9402 tl-dna and the 35s gus gene showed an average of more than 35%. these obtained root cultures were additionly elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g.uralensis were obtained by infection of a. rhizogenes 8196 have produced gl at an yield of 4.5% dry weight on the period of culture as a 30 days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels (3.42 g/l) of the total flavonoids production have been identificated on the strains which transformed by lba 9402. this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. production, purification and characterization of scytalidium thermophilum phenol oxidases didem sutay 1 , ufuk bakir 1 , zumrut b. ogel 2 : 1 chemical engineering department, middle east technical university, inonu bulvari, 06531 ankara, turkey; 2 food engineering department, middle east technical university, inonu bulvari, 06531 ankara, turkey. e-mail: ubakir@metu.edu.tr (u. bakir) phenol oxidases (pos) are a group of enzymes which are responsible for oxidation of various phenolic compounds in the presence of molecular oxygen. there are different types of pos present in nature and three major groups of these enzymes are laccases (e.c. 1.10.3.2, p-benzenediol: oxygen oxidoreductase), catechol oxidases (e.c. 1.10.3.1, o-diphenol oxidoreductase) and tyrosinases (e.c. 1.14.18.1, monophenol monooxygenase). another group of enzymes, peroxidases (e.c. 1.11.1.7), can also be considered as a member of po family. pos have very wide substrate range and final oxidation products of these substrates are quinones, which are highly reactive molecules and polymerize into brown, red or black waterinsoluble compounds. pos are very common in nature, they can be found in almost all plants, animals and microorganisms. pigmentation and protection from the environment are main functions of these enzymes. pos have different applications in food, pharmaceutical, textile industries and waste-water treatment systems. the objective of this study was po production by the thermophilic fungus, scytalidium thermophilum, purification and characterization of the enzyme. for this purpose, enzyme production was performed either in a shaker-incubator or a temperature, ph and dissolved oxygen controlled 2 l bioreactor (probiotem) to optimize enzyme production medium composition and bioreactor parameters. as the carbon and nitrogen sources, 4% glucose and 0.4% yeast extract were determined as the optimal concentrations, respectively. copper, gallic acid and tannic acid were determined to increase enzyme production. purification was performed by using membrane and chromatographic techniques. hydrophobic, ion exchange and gel filtration columns were used by using a fplc system;äkta prime (amersham biosciences). especially the phenyl sepharose tm high performance column appeared to be very efficient for po purification from scytalidium thermophilum. purified po have been characterized by electrophoretic techniques and kinetic studies. isolation of lipolytic microorganisms from subtropical soils. cloning, purification and characterization of a novel esterase from strain pseudomonas sp. cr-611 núria prim, cristian ruiz, cristina bofill, f.i. javier pastor, pilar diaz department microbiology, university of barcelona. av. diagonal 645, microorganisms or their enzymes are used in a wide range of biotechnological activities such as polymer hydrolysis, synthesis of added-value compounds, sample decontamination, etc. thus, there is an increasing interest for isolating new enzymes and new enzymeproducing organisms for their use in industrial conversions (cherry and fidantsef, 2003) . among these enzymes, lipases, esterases, cellulases, xylanases and pectinases play an important role in many biotechnological processes like those related to pulp and paper processing. three samples of subtropical forest soil from puerto iguazú (argentina) were used for the isolation of autoctonous microorganisms growing in an organic matter-rich environment. a total of 724 pure cultures of bacteria and fungi were obtained and their hydrolytic activities on polysaccharide and lipidic substrates were assayed using olive oil, tributyrin, cholesterol esters, xylan, cellulose and pectin as substrates. among the isolates analysed, 449 were active on one or more of the substrates evaluated, and 43 of them degraded all substrates. nearly half of the strains displayed lipolytic activity, whereas the number of strains active on xylan, cellulose, pectin and cholesterol esters, was much lower. the 76 strains bearing the highest hydrolytic activities were selected and stored for further characterization (ruiz et al., 2005) . among them, strain cr-611, one of the most active isolates on tributyrin, was selected for identification and characterization of its lipolytic system. lipolytic strain cr-611 was identified by morphological, physiological and phylogenetic tests, as a pseudomonas sp., closely related to p. fluorescens. sds-page and zymogram analysis (diaz et al., 1999) of cell extracts and supernatants from the strain revealed a complex lipolytic system consisting of at least two lipolytic enzymes. sequence alignment and clustering of previously described pseudomonas lipases and esterases allowed the design of different sets of primers for the isolation of the lipase/esterase coding genes. a gene coding for an esterase with homology to family vi bacterial lipases (arpigny and jaeger, 1999) was isolated and cloned in escherichia coli. the cloned enzyme was further purified and characterized, showing preference for short fatty acid esters and displaying a typical michaelis-menten kinetics, with no interfacial activation. the substrate profile, together with the kinetic behaviour and sequence similarity of the cloned enzyme to family vi bacterial esterases, allowed to identify this enzyme as an esterase and was named esta6. maximum activity was achieved on muf-butyrate at 55 • c and ph 8.5, suggesting that it could be of interest for biotechnological purposes. microbial xylitol production from agricultural wastes has recently attracted much attention from industries because it has potentials to realize the cheaper production of xylitol with low environmental impact (tada et al., 2004) . in order to realize the effective xylitol production by a xylose utilizing yeast, the oxygen supply is a key for maximizing xylitol yield over consumed xylose (y xl ) because the intracellular xylitol metabolism is strongly influenced by the amount of available oxygen. in the present work, we tried to apply a metabolic reaction model in order to determine the optimal oxygen transfer rate (otr) in a fermentor for maximizing xylitol yield. corn cob hydrolysates containing 25 g-xylose/l was employed as medium for xylitol production by computer-controlled batch cultures using candida magnoliae (ferm p-16522, aist). a metabolic reaction model considering main xylitol metabolisms including glycolysis, pentose-phosphate pathway, tca cycle and cell synthesis was developed. the model allows to estimate various intracellular metabolic flux distributions including a xylitol production rate. the oxygen uptake rate to maximize the ratio of xylitol production rate over xylose consumption rate corresponds to the otr condition to maximize a xylitol yield over xylose consumed. based on the metabolic reaction model, the otr was optimized by a linear programming, the optimal otr and the maximum xylitol yield were estimated as 0.5 mmol o 2 /l h and 0.81 g-xylitol/g-xylose, respectively. the experimental verification using the optimal otr demonstrated that the xylitol yield was greatly improved to 0.75 g-xylitol/g-xylose from 0.6 g-xylitol/g-xylose in our previous study. expression of a bacterial sugar phosphate transporter in s. cerevisiae to release l-glycerol 3-phosphate accumulated by metabolic engineering almut popp, huyen thi thanh nguyen, ulf stahl, elke nevoigt department of microbiology and genetics, university of technology, 13353 berlin, germany. e-mail: a.popp@lb.tu-berlin. de (a. popp) in contrast to glycerol, its phosphorylated precursor l-glycerol-3phosphate (l-g3p) is retained by the plasma membrane. therefore, engineered yeast strains accumulate l-g3p in the cytosol resulting in low overall yield of the desired product and laborious downstream processing. a suitable sugar phosphate transporter in the yeast plasma membrane would overcome these limitations. the glycerol-3-phosphate transporter (glpt) of e.coli is an antiporter and naturally mediates the uptake of l-g3p in exchange with inorganic phosphate. we assume that this transporter would also mediate the excretion of accumulated l-g3p into a phosphate-rich medium if it was present in the plasma membrane of engineered yeast. despite many inconsistencies in codon usage, we were able to express the bacterial glpt gene in yeast. expression was monitored by a c-myc tag added to the c-or n-terminal hydrophilic tail, respectively. the quantity of the construct with n-terminal tag clearly exceeds the quantity of the construct with c-terminal tag. both gene products are located in the endoplasmic reticulum, as shown by immunofluorescence microscopy. obviously, yeast's transmembrane protein sorting machinery does not recognise it as a substrate for the secretory pathway. metabolic flux analysis of c-and p-limited shikimic acid producing e. coli gaspard lequeux 1 , louise johansson 2 , jo maertens 1 , peter vanrolleghem 1 , gunnar lidén 2 : 1 biomath, ghent university, coupure links 653, 9000 gent, belgium; 2 department of chemical engineering, lund university, p.o. box 124, 22100 lund, sweden. e-mail: gaspard.lequeux@biomath.ugent.be (g. lequeux) metabolic flux analysis (mfa) was applied to decipher why plimited e. coli fermentations are more optimal for shikimic acid production in comparison with glucose-limited fermentations. as mfa allows obtaining insight in the intracellular flux distribution over different pathways by only measuring net production and consumption rates of metabolites, under the condition that parallell pathways are removed. to this end a detailed metabolic network model was created and checked for consistency, dead-ends, and parallell pathways. several fermentations were performed at different dilution rates (ranging from 0.05 to 0.3 h −1 ) and different limitations (phosphate and glucose). the e. coli strain used was w3110 with genetic modifications in the aromatic amino acid pathway to enhance shikimic acid production. for each dilution rate, a metabolic model was solved. this way, the evolution of each flux can be followed with respect to the dilution rate. the p-limited cultures showed a better yield which can be explained by the diminished excretion of dehydro-shikimic acid (as is known from literature) and a reduction of the hydrolysis of atp. takasumi hattori, kuniki kino, kohtaro kirimura department of applied chemistry, school of science and engineering, waseda university, tokyo, japan. e-mail: takasumi@suou.waseda.jp (t. hattori) alternative oxidase is a terminal oxidase in respiration chain, which is a branched chain of cytochrome pathway, and inhibited by salicylhydroxamic acid (sham), but not by cyanide. the citric acid-producing fungus aspergillus niger wu-2223l has a cyanide-insensitive and sham-sensitive respiration catalyzed by the alternative oxidase (kirimure et al., 1999) and did not produce citric acid when cultivated with sham (kirimure et al., 2000) . in this study, the transcript levels of alternative oxidase gene (aox1) (kirimure et al., 1999) and activities of alternative oxidase under the conditions of citric acid production were examined during 9 days-cultivation. the amount of aox1 mrna was determined by northern blot analysis, and the specific activity of alternative oxidase as that of duroquinol oxidase. the transcript level and the activity were highest at 2 days at log-phase, decreased during 2 and 4 days, and thereafter maintained at low levels. however, the transcript and alternative oxidase activity was constitutively detected during whole the cultivation periods under the conditions of citric acid production. the sequence analysis of aox1 chromosomal dna revealed the presence of potential binding site of cyclic amp responsive element (cre), stress responsive element (stre) and heat shock factor (hsf) in its upstream region. these results indicated that the expression of alternative oxidase was regulated in the transcription level and alternative oxidase contributes as the main respiration chain during citric acid production. kirimura, k., et al., 1999 . curr. genet. 34, 472-477. kirimura, k., et al., 2000 . biosci. biotechnol. biochem. 64, 2034 -2039 . trichoderma strains are considered to be among the most useful fungi in industrial enzyme production, agriculture and bioremediation. metabolic versatility displayed by these fungi makes it a very amenable source of new gene products. functional analysis of candidate genes goes by two complementary ways: gene overexpression and loss-of-fuction mutants generation. a few expression systems are available mainly to direct constitutive gene expression in catabolite repression conditions. following a genomic approach, we have recently cloned some gene promoters with high expression in glucose in trichoderma harzianum cect2413. a main goal in a gene functional analysis is the generation of knock-out mutants. up to date, there is no reference about successful gene disruptions in t. harzianum mainly due to a very low homolog recombination frequency. rna-mediated gene silencing has been shown as an efficient tool to diminish or totally abolish specific gene expression. especially those strategies based on the use of hairpin constructs allow rapid and easy generation of strains with reduced levels of mrna from genes of interest. we have used the t. harzianum cect2413 tss1 promoter to direct the expression of a hairpin dna construct that induced the appearance of small-interfering rnas (sirnas) and the silencing of the uida reporter gene in a previous uida overexpressing strain. reduced levels of mrna and gus activity correlated with the presence of sirnas. this is the first report on rna-mediated gene silencing in trichoderma and constitutes a useful and promising tool for functional genomic studies in fungal systems. analysis of the metabolic response of escherichia coli to quantitative modulations of the glucose-6-phosphate dehydrogenase based on 13 c-labelling experiments cécile nicolas, fabien létisse, stéphane massou, philippe soucaille, jean-charles portais. e-mail: fabien.letisse@insa-toulouse.fr (f. létisse) microorganisms have an efficient capacity for adapting their metabolism in response to genetic or environmental changes, and understanding metabolic robustness has become an emergent issue. part of the robustness originates from the network organization of metabolic systems, where the interplay between all available biochemical reactions provides alternative mechanisms for compensating the perturbations. recently, 13 c-metabolic flux analysis ( 13 c-mfa) has been applied to escherichia coli knock-out mutants lacking key enzymes to determine the phenotypic effects of structural changes in the metabolic network, providing further evidences for compensatory phenomena. the aim of our on-going work is to understand how the central metabolism in e. coli responds to quantitative alterations at a specific key-point of the metabolic network. the glucose-6-phosphate dehydrogenase (g6pdh), a key enzyme in the central metabolism for which the effects of deleting the gene (zwf) has been already described (zhao et al., 2004) , was chosen as the target. to this aim we have generated a set of expression mutants, i.e. mutants having each a fixed level of expression of the zwf gene. four different levels of expression, leading respectively to g6pdh activity 2; 2.9; 5.7 and 14 times higher than in the wt strain, have been obtained. for each mutant, transcriptomics analysis will be carried out and compared to both the zwf-and wt strains to detect changes in the network structure, and the distribution of fluxes will be measured using 13 c-mfa. the flux maps obtained for the various strains will be compared to evaluate the quantitative response of the central metabolic network to imposed and increased g6pdh activity. metabolic control analysis will be applied to provide insights onto the sensitivity of the measurable metabolic fluxes to g6pdh activity. combination of transcriptomics and fluxomics approaches will provide information on the nature and extent of the compensatory mechanisms. because the activity of a single enzyme is tuned at different levels in knock-out and expression mutants, this investigation provides a situation that mimics gene-level regulation of metabolism. , j., et al., 2004, metab. eng., 6, 164 . with the depletion of the world's petroleum supply, there has been an increasing worldwide interest in ethanol as an alternative, nonpetroleum source of energy. this fact caused increased interest in the new ethanol technology fermentation process research. as reported before, bacteria zymomonas mobilis possesses more advantages than saccharomyces cerevisiae, microorganism used for ethanol production in industrial scale. for that reason, we have focused on the fermentation studies of this facultative bacterium in free and immobilized form. the immobilization of the cells into polyvinylalcohol (pva) hydrogel lens-shaped capsules lentikats, improved the batch fermentation process efficiency nine times. starch, the substrate considered as one of the best of renewable energy source is considered as fuel ethanol feedstock. due to z. mobilis disability of maltose, maltotriose and dextrin utilization, the starch has to be converted into glucose monomers. this pre-fermentation step can overcharged whole ethanol production process. this ineffective part of the process was resolved with immobilization of glucoamylase into lentikats. the system with immobilized enzyme and cell was stabile in continuous mode for 60 days without any significant change in the system efficiency. the combination of cell and enzyme immobilization can significantly improve the efficiency and the cost of ethanol production in industrial scale. fermentation of an inhibitory dilute acid-hydrolysate from spruce using a fed-batch procedure combined with cell-reuse andreas rudolf, gunnar lidén department of chemical engineering, box 124, lund university, se-221 00 lund, sweden. e-mail: andreas.rudolf@chemeng.lth.se (a. rudolf) a well-controlled addition of hydrolysate to the fermentation has proved very efficient in reducing yeast inhibition due to compounds formed during lignocellulose hydrolysis. furthermore, using high cell mass concentrations has been another way of avoiding the negative impact of the inhibitory compounds. if possible, the yeast should therefore be re-used in the process. in the present work a dilute-acid hydrolysate from spruce was fermented using a fed-batch procedure with reutilization of yeast. the fermentation procedure worked satisfactorily, with more than 98% of fermentable sugars consumed in each of the four consecutive fed-batch fermentations performed. the ethanol yields on fermentable sugars reached 0.45 g/g. there was continued cell growth in the repeated fed-batch experiments, with an average cell yield on fermentable sugars of 0.06 g/g. in contrast, only about 20% of the fermentable sugars were consumed within 24 h, when the fermentation of the hydrolysate was run in a batch process. the work shows the potential to re-use the yeast in a suitably designed process. metabolic engineering is defined by bailey in his seminal 1991 paper as "the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant dna technology". the manipulation of these functions ultimately results in the manipulation of metabolism, which is the purpose of many biotechnological processes. metabolic engineering has sought its methods and tools in mathematics and the physical sciences, and later in information technology (it) leading to the proliferation of bioinformatics. in this work we propose a novel approach to metabolic engineering that regards it as a business process reengineering (bpr) endeavour. hammer and champy in their celebrated 1993 book define bpr as "the fundamental rethinking and radical redesign of business processes to achieve dramatic improvements in critical contemporary measures of performance, such as cost, quality, service and speed". our thesis is that metabolic engineering with its goal of reengineering the metabolism of the microorganism, is equivalent to business process reengineering (bpr) in business and management. indeed this is essentially what metabolic engineering does to the cell through the use of recombinant dna technology, which can be viewed as a radical redesign of the metabolic process. after all, it causes changes that cannot be attained otherwise, and whose purpose is to achieve dramatic improvements in cellular activities such as the increase in production of some metabolites by orders of magnitude. the cost incurred by the process, which is metabolism in this case, can be, for example, energy requirements or change to a cheaper substrate. in this study we elucidate this parallelism between the two with emphasis on modelling of metabolism and how the concepts of business process modelling can be applied to it. the purpose is not to produce a model, but rather to introduce the modelling methodology and demonstrate its utilisation and benefits and outline its limitations and challenges. we believe that the novelty of our work lies in applying a new paradigm in approaching metabolic engineering that has not been considered previously. thermophilic ethanol production from wheat straw hydrolysate in continuous culture tania i. georgieva, birgitte k. ahring biocentrum-dtu, technical university of denmark, building 227, dk-2800 lyngby, denmark. e-mail: tig@biocentrum.dtu.dk (t. georgieva) ethanol production from lignocellulosic biomass has attracted widespread attention as an unlimited low cost renewable source of energy to transportation fuels due to increasing petroleum use and air pollution towards greenhouse gases. wheat straw available as agricultural residue has been considered as a potential lignocellulosic substrate for industrial bioethanol production. a major technical obstacle to commercialize bioethanol production form lignocellulose (such as wheat straw) is associated with a lack of microorganism able to rapidly and efficiently ferment both hexose and pentose sugars into ethanol and to tolerate the inhibitors present in undetoxified hydrolysates. currently used industrial mesophilic microorganisms (saccharomyces cerevisiae and zymomonas mobilis) are excellent ethanol producer from glucose, however, they are not able to ferment other sugars such as xylose, which is the second most abundant sugar in lignocellulose. thermophilic anaerobic bacteria have been considered for ethanol production from lignocellulosic biomass as an alternative to mesophilic ethanol producing strains, predominantly because of their abilities naturally to ferment the whole diversity of sugars found in lignocellulosic biomass. an increase attention to thermophilies for ethanol production have also arise from broad spectrum of advantages regarding industrial scale ethanol fermentation such as high growth and metabolic rates, low oxygen solubility, reduced risk of reactor contamination, and cost savings via mixing, cooling and facilitated product recovery. in addition, simultaneous co-fermentation of glucose and xylose in a single operation unit could substantially reduced the ethanol production cost. research has been attempted to study the potential of using a thermophilic anaerobic bacterium for continuous ethanol fermentation of lignocellulosic biomass, with particular emphasis on effectiveness of our strain to ferment undetoxified wet oxidized wheat straw hydrolysate with respect to sugar (glucose and xylose) conversion and ethanol yield. the experiment was carried out in a lab-scale reactor operated at 70 • c with wheat straw hydrolysate as a substrate in concentrations from 20 to 80 wt.% equivalent to total sugar mixture of 11-38 g/l. wheat straw hydrolysate (woh) [200 g/l wheat straw, 92.4% dry matter (dm)] was prepared using wet oxidation pretreatment process followed by enzymatic saccharification with commercial enzymes mixture of celluclast1.5, and novozym188 (novozymes, denmark). both xylose and glucose sugars were simultaneously converted to ethanol. the sugar utilization was higher than 90%, and high ethanol yields were achieved. reactor shows good long-term performance (124 days) in terms of operation stability and reactor contamination. maltotriose is the second most abundant fermentable sugar in wort and due to incomplete fermentation, residual maltotriose in beer may cause problems in the brewing industry. to study genes that might improve utilization of maltotriose we used a library with dna from brewer's strains and a laboratory strain and identified a new transporter encoded by mtt1. mtt1 gave lager strain a15 the ability to grow on yp/2% maltotriose in the presence of 3 mg/l of the respiratory inhibitor antimycin a. this transporter gene shares 74% similarity with mph2 and mph3, 62% similarity with agt1 and 91% similarity with mal61 and mal31. moreover, mtt1 shares even higher similarity (98%) with the s. pastorianus mty1 gene (m. salema-oom, unpublished, ncbi accession number aj491328). purified radiolabeled maltotriose and radiolabeled maltose were used to study sugar uptake of lager strains a15 and ws34/70, and of a15 containing mtt1 or mtt1alt, a more efficient, altered version of this gene lacking the 66 basepairs from the 3 end and containing 57 base-pairs of vector sequences. these transport studies show that mtt1 and, especially, mtt1alt encode maltose transporters with relatively high activity towards maltotriose compared to maltose. this study is part of a multi-disciplinary project, funded by the european union (contract no. qlk1-ct-2001-01066) focusing on the development of high-gravity resistant brewer's yeast strains. metabolic engineering of l-phenylalanine pathway in bacillus subtilis yasemin demirci 1 , pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 : 1 bre laboratory, department of chemical engineering, ankara university, 06100 ankara, turkey; 2 iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.özdamar) metabolic engineering design of a recombinant l-phenylalanine (phe) production system is based on coordinated overexpression of the flux-controlling genes in the aromatic-amino acid pathway. based on the insights gained by the work carried out in our laboratories (özçelik et al., 2004) , aroh for the reaction r96 at the branch-point chorismate that connects the preceding reactions of the aromatic group amino acid pathway to the proceeding reactions towards phe, was predicted as the first-, and aroa for dahp synthase (r89) predicted as second-metabolic engineering sites. aroa gene was cloned next to aroh, by the use of four primers designed using pcr-based gene splicing by overlap extension method. the genes were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their 5 ends that are complementary to the 3 portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at 3 end. extension of this overlap by dna polymerase has yielded the recombinant two-gene product; and the two-gene product serve as template for the continuation of reactions for the increase of the concentration in the micro-reactors. the two-gene fragment was first cloned into puc19, and then sub-cloned to pmk4 e. coli -bacillus shuttle plasmid. the new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis. the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, phe and other amino acids, and organic acids concentrations as the constraints. on the bases of calculated intracellular fluxes of recombinant b. subtilis carrying pmk4::aroa::aroh, an in-depth analyses of the metabolic engineering design will be presented. ozçelik,i̇., ç alık, p., ç alık, g.,özdamar,t.h., 2004. metabolic engineering of aromatic group amino acid pathway in bacillus subtilis for l-phenylalanine production. chem. eng. sci. 59 (22-23), 5019-5026. the 100% respiratory-deficient nuclear petite amylolytic saccharomyces cerevisiae npb-g strain capable of excreting a hybrid protein possessing both ␣-amylase and glucoamylase enzyme activities was generated and its employment for direct fermentation of starch into ethanol was investigated under both shake flask and controlled bioreactor cultivation conditions. when compared with a standard host strain, higher ethanol concentrations and yields were achieved with the nuclear petite strain under both cultivation conditions. further improvement in ethanol production was achieved by the use of an initial glucose supplement. comparison of the ethanol fermentation performances of the respiratory-deficient npb-g and the parental respiratory-sufficient wtpb-g strain showed an increase of, ca. 48% in both ethanol production yields and ethanol productivities with the respiratory-deficient strain. response surface methodology (rsm) was used as a statistical tool to optimize the initial yeast extract and starch contents of the medium, which resulted in a substantial increase in the stability of the expression plasmid in both strains with concomitant improvement in their amylolytic potentials. high ethanol yields on substrate values of the bioreactor cultures, that were very close to the theoretical yield, indicated that the amylolytic respiratory-deficient strain developed in this study was very effective in the direct fermentation of starch into ethanol. establishing a biotechnology educational framework to support a knowledge-based economy in puerto rico rosa buxeda, lorenzo saliceti-piazza industrial biotechnology program, university of puerto rico, mayagüez campus, mayaguez 00680, puerto rico. e-mail: rbuxeda@uprm.edu (r. buxeda) industrial biotechnology has been identified as a major thrust area of economic development within the past five years for the island of puerto rico. the portfolio of biotechnology manufacturing investments in the island has passed the two billion dollar mark. world known companies like amgen, abbott and eli-lilly lead these investments, which have catalyzed a strong technology transfer to the island. a strong collaboration between industry and academia was needed to provide a well-trained workforce for these company startups. as a result, the university of puerto rico, mayagüez campus (upr-m) developed four initiatives which are part of the educational pipeline in biotechnology. these are: (i) an industrial biotechnology program, a 5-year bs degree which contains a novel curriculum with courses from science and engineering with undergraduate research and industrial internships as part of the degree requirements; (ii) an industrial biotechnology learning center, which provides customized biotechnology and bioprocessing training modules, including lectures and hands-on experiences to train and develop the workforce needed in the biotechnology manufacturing plants; (iii) a biotechnology summer camp, which addresses the high school student population and its main purpose is to educate and advise on the different career paths that can be followed in the field of biotechnology; and (iv) a biotechnology center for research and training in bioprocessing, that will address the development of corporatesponsored research projects to strengthen links between industry and academia in order to build up a knowledge-based economy. our paper will describe each initiative in detail as well as its outcomes and impact on puerto rico's knowledge-based economy goals. isolation of acid phosphatase from sweet potato and immobilization using different adsorbent d. omay, y. güvenilir, n. deveci istanbul technical university, department of chemical engineering, maslak 34769, istanbul, phosphatase enzymes occur in a wide range of plant and animal tissues. they catalyze the hydrolysis of phosphate bonds in organic phosphates, between the phosphate group and rest of the molecule. immobilization of enzymes and biological compounds is currently gaining importance due to its wide variety of applications in the food and pharmaceutical industries and also its biomedical applications. it was reported that enzymes can be activated by complexation with polysaccharides such as chitin or chitosan. the aim of this experimental study was determined as partial purification and isolation of acid phosphatase enzyme and its immobilization. the purification was realized by applying centrifugation, ammonium sulfate precipitation and dialysis respectively. the specific activity of the supernatant was 0.1 u/mg and after 80% saturation this value increased 0.64 u/mg. furthermore, acid phosphatase was investigated using different adsorbent (chitin, chitosan, synthetic zeolite and raw zeolite) and evaluated the storage stability and re-usability of the immobilized acid phosphatase. it was estimated that, acid phosphatase activity was shielded the ratio of 94, 96, 99, and 92% by using raw zeolite, synthetic zeolite, chitin and chitosan respectively under 12 h operation condition. øyvind m. jakobsen 1,2 , michael c. flickinger 3 , svein valla 1 , trond e. ellingsen 2 , trygve brautaset 1 : 1 department of biotechnology, norwegian university of science and technology, norway; 2 sintef applied chemistry, sintef, norway; 3 biotechnol. institute, department of biochemistry, molecular biology and biophysics, university of minnesota, usa aerobic methylotrophs can utilize one-carbon (c 1 ) compounds such as methane and methanol as a sole c-source for growth and energy. the majority of research on these organisms has focused on their biochemical novelity and commercial viability. for the industrial production of bulk products such as the amino acids lysine and glutamate raw material costs and abundance are important, and c 1 sources are thus attractive compared to sugars. bacillus methanolicus can secrete up to 55 g/l of glutamate upon methanol growth at 50 • c (thermotolerant and methylotroph) and mutants producing 35-40 g/l of l-lysine have been selected. we study the genetics for conversion of methanol into biosynthesis of glutamate and lysine, and in the present report we unravel the regulation of genes impor-tant for the consumption and tolerance level for c 1 compounds by b. methanolicus. b. methanolicus has a methanol dehydrogenase gene (mdh) for oxidation of methanol into formaldehyde and a ribulose monophosphate (rump) pathway for assimilation of formaldehyde. we recently discovered that mdh and five rump genes are carried by natural plasmid pbm19 in this bacterium and this represented the first documentation of plasmid-dependent methylotrophy in any microorganism. we here use real-time pcr to analyse the regulation of plasmid-and chromosomally located rump genes, in cells upon methylotrophic and non-methylotrophic growth. high methanol concentrations in the growth medium is cell toxic and the mechanisms for this sensitivity of b. methanolicus is poorly understood. our results indicate that plasmid pbm19 plays a fundamental role for this trait as well and the impact of our results on the biotechnological applications of this bacterium is discusssed. department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, 3.9 (t1), 7.8 (t2) and 11.7 (t3) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period (15 days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t3 showed more less firm after 15 days of storage being 20.17 g/100 g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t2 and t3 of 433 and 479 mpa s respectively, compared with control of 299 mpa s at 15 days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t2 gained the highest scores (85 points) followed by the control (81.5 points) after 15 days of storage, while yogurt of t3 showed a low scoring being 75. from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of 7.8 units of plant proteinases/ml milk. the penicillium chrysogenum oat1 gene encoding a class iii omega-aminotransferase has been cloned and characterized. this enzyme that converts lysine into 2-aminoadipic semialdehyde is important in providing 2-aminoadipic acid, a precursor of penicillin and other ␤-lactam antibiotics. the enzyme is related to ornithine-5-aminotransferases and to lysine-6-aminotransferases encoded by the lat gene located in the bacterial cephamycin gene clusters. expression of oat1 is induced by lysine, ornithine and arginine and repressed by ammonium ions. area-binding consensus sequences and an 8-bp direct repeat associated with arginine induction in emericella (aspergillus nidulans) have been found in the oat1 promoter region. deletion of the oat1 gene resulted in the loss of omegaaminotransferase activity. the deletion mutants were unable to grow on ornithine or arginine as sole nitrogen sources and showed a reduced growth on lysine. complementation of the deleted mutant with the oat1 gene restored growth on ornithine, arginine and lysine to the levels of the parental strain and omega-aminotransferase activity. the strong expression of oat1 gene after induction by the basic amino acids may provide additional 2-aminoadipic acid for the formation of the 2-aminoadipyl-cysteinyl-valine tripeptide for ␤-lactam biosynthesis. morphological characterisation of two high producing strains of penicillium chrysogenum carrying a disruption in the nadph-dependent glutamate dehydrogenase k. rueksomtawin, j. thykaer, h. noorman, j. nielsen center for microbial biotechnology, biocentrum-dtu, technical university of denmark, dk-2800 lyngby, denmark. e-mail: kr@biocentrum.dtu.dk (k. rueksomtawin) metabolic engineering has proven to be useful in optimisation of ␤-lactam production, e.g. constructing superior strains with multiple copies of the ␤-lactam gene cluster. it is however, of equal importance to gain insight into other aspects of the metabolism to establish a general overview in order to apply metabolic engineering for further improvement of the production strains. in that context, the redox metabolism is essential, as it functions as a tightly controlled connection between the different parts of the metabolism. in order to investigate this role of the redox metabolism in more detail, the gdha-gene, encoding the nadph-dependent glutamate dehydrogenase, was disrupted in two industrial strains of penicillium chrysogenum. during physiological characterisation of the two strains it became apparent that considerable changes in the morphology had occurred due to the genetic alteration. since the morphology is an important parameter in process optimisation, an examination of the morphology of the two strains was undertaken. in this work, the morphological differences between the gdha-disrupted strains and the reference strains were comprehensively investigated both during growth on solid media and submerged growth in a flow-through growth chamber. with the advance development of computerized image analysis techniques, the key morphological properties of the individual hyphal elements were quantified. in comparison to the reference strains, the disruption of the gdha gene resulted in a morphological change from short hyperbranched hyphal elements to long elongated hyphal elements with less branches. in the parallel studies with aspergillus nidulans and its corresponding gdha-deleted strain, no difference in the morphology was observed. polyketides (pk) represent one of the largest groups of natural products and are found in fungi, bacteria and plants. since many useful polyketides either originate from sources that are difficult or even impossible to cultivate or are produced in inadequate amounts, we are interested in expressing polyketide synthases (pkss) in heterologous hosts. saccharomyces cerevisiae, aspergillus niger and aspergillus nidulans were chosen as initial hosts, because the techniques necessary to cultivate and manipulate these strains genetically are well established. 6-methylsalicylic acid synthase (6-msas) was chosen as a model pks. replicative plasmids carrying the genes encoding 6-msas from penicillium patulum and phosphopantetheinyl transferase (pptase), respectively, were transformed into s. cerevisiae. in addition, an integrative vector was designed and the gene encoding 6-msas was integrated in the yeast chromosome. batch cultivations on galactose minimal media were performed. the results are presented and in particular the effect of expression mode and type of pptase (bacterial versus fungal) is discussed. furthermore, the progress of the work on expressing 6-msas in a. niger and a. nidulans using an integrative vector system is presented and discussed. the valorisation of functionalized chemicals from biomass resources compared to the conventional fossil production route ben brehmer, wageningen ur agrotechnology & food sciences, workgroup: valorisation of plant production chains, p.o. box 17, 6700 aa wageningen, the netherlands. e-mail: ben.brehmer@wur.nl at some undisclosed point in the foreseeable future, cheap fossil fuels resources will become depleted and the industry will be forced to pursue more difficult reserves with increasingly high extraction costs. most alternatives available and under research do not consider price as the main motivation for replacement, but focus solely on sustainability and environmental benefits. sustainability is an interesting word as there are enough fossil resources scattered around the world to be sustainable in quantity but not sustainable in price. seeing that fossil fuels are derived from prehistoric biomass it is not at all presumptuous to assume that every application and product can be replaced by the biomass of today. in fact, many highly specialised pharmaceutical chemicals already have a biomass origin. yet, not all of the uses of fossil fuels need to be replaced by a comparable carbon based source, such as biomass. energy and transportation in particular do not necessary need to rely on carbon cleavage, whereas practically all of the petrochemicals contain a carbon backbone. the main stipulation in substituting fossil-based chemicals with bio-based chemicals is availability and cost. it is proposed that already today, by utilising existing, recently developed and developing technology, it is economically advantageous for many chemicals to derive from biomass, in particular the functionalized chemicals. the only way to validate this conjecture is to develop a complete comparative life cycle analysis. as opposed to a traditional lca, the "multicriterion" developed here will revolve around energy flows and process efficiency in terms of exergy. the aim is to assess the optimum route with the best production options along the whole production chain while determining any possible limiting factors. using this tool, a systematic production matrix relating several logical source crops and a few key chemicals of varying derivative levels can be created and compared to the conventional fossil routes. combined with economic considerations and some unambiguous environmental factors, the investigation will provide all the information relevant to the industry. the goal is to create an objective and reliable simulation system ratifying the economic and environmental feasibility of exploiting biobased chemicals today and indicate the steps necessary for further improvement. biosynthesis of multi-enzymatic preparation from aspergillus niger ibt-90 useful in textile fabric treatment rita pyc 1 , jadwiga sojka-ledakowicz 2 , tadeusz antczak 1 , joanna lichawska 2 : 1 institute of technical biochemistry, the technical university of lodz, lodz 90-924, poland; 2 textile research institute, lodz 92-103, poland among many methods of producing enzymatic preparations, i.e. by liquid surface fermentation -lsf, submerged fermentation -smf or solid state fermentation -ssf, this last is most advantageous. cultivation in solid state means fermentation on a matrix formed by industrial and agricultural wastes. most often filamentous fungi -due to the lack of available water in the foundation -are the efficient, competitive microorganisms applied in solid state bio-conversion. the aim of research works carried out at the institute of technical biochemistry of the technical university of lodz and at textile research institute, lodz was defining optimal conditions for biosynthesis and testing the possibility of applying multi-enzymatic preparation from aspergillus niger ibt-90 in the treatment of woven fabrics made of natural cellulose fibres. as the result of biosynthesis optimization process, malt sprouts, wheat barn and beet pulp were selected as the best media to obtain enzymes of high pectinolytic activity maintaining at the same time high activity of cellulolytic enzymes and xylanase. the highest obtained activities reached: 2000 0 pm for total pectinolytic activity, 7 j/ml for endoglucanase and 527 j/ml for endoxylanase and they were 2.4-3.0 times higher than those achieved before optimizing process. performed research works demonstrated that the optimum activity of applied enzymatic system is obtained in the range of ph 4.6-4.8. woven fabrics made of flax and cotton fibre blends, subjected to bio-pre-treatment, were evaluated with reference to their sorption properties. comparative evaluation of liquid sorption by woven fabrics allowed to notice efficient enhancement of fibres' sorption capabilities after pre-treatment using enzymes system from aspergillus niger ibt-90. this offers the possibility of substitution of alkali scouring of linen-cotton woven fabrics before their bleaching by bio-treatment. the phylum actinobacter includes many bacteria of industrial importance both for accumulation of primary and secondary metabolites. both primary and secondary metabolites are dependent on precursors and cofactors that are provided by the central carbon metabolism of microorganisms. there are alternative pathways for catabolism of carbon either via the embden meyerhof parnas (emp) and pentose phosphate (pp) pathway or through the entner doudoroff (ed) pathway. the emp pathway is energetically more favorable and has therefore been presumed to be the dominating route for carbon metabolism in bacteria producing secondary metabolites. however, primary metabolism is poorly studied for most actinobacter species as focus traditionally has been on secondary metabolism. with the aim to gain more knowledge about the diversity of central carbon metabolism within the phylum actinobacter, 17 different strains were collected from various sources and strain collections. the strains were grown in minimal medium with supplement of standard vitamin solution and [1-13 c] glucose as carbon source. the 13 c-labeling patterns of proteinogenic amino acids were determined by gc-ms analysis. through this method, the fluxes in the central carbon metabolism during balanced growth were estimated and pathways for carbon metabolism were determined. in particular the labeling patterns of alanine and valine were of interest as they are derived from pyruvate and therefore can be used to distinguish between whether glucose is metabolized through the ed pathway or the emp pathway. nobacter, amycolatopsis balhimycina produces the glycopeptide balhimycin. the balhimycin aglycone is identical to the aglycon of vancomycin, which is a commercial glycopeptide. as a. balhimycina appears to be accessible to genetic modifications and the biosynthetic cluster responsible for balhimycin production is published, this genetically well-characterised academic strain can serve as a platform strain for production of vancomycin analogues derived through combinatorial biosynthesis. the understanding of the physiology of this microorganism is essential for the efficient accumulation of potentially commercial secondary metabolites. the bacterium is capable of growth in a fully defined minimal medium, with the production of balhimycin. the strain was grown at either nitrogen or phosphate limitation and balhimycin accumulation was followed at these conditions. flux analysis of balhimycin production by amycolatopsis balhimycina based on 13 c labelling experiments was performed. the zygomycetes blakeslea trispora and phycomyces blakesleeanus accumulate beta-carotene, ubiquinone (coenzyme q), various different sterols, and other terpenoids, all of them produced via the mevalonate pathway. these fungi are used or could be used for the industrial production of these terpenoids, edible oil, chitosan, and various organic acids. by measuring the specific radioactivity of terpenoids made from radioactive mevalonate, leucine or acetate in the presence of excess glucose in wild types and mutant strains we have concluded that these fungi have separate subcellular compartments for the production of carotene, sterols and triacylglycerols. the terpenoid moiety of ubiquinone is synthesized in the same compartment as ergosterol. these compartments contain separate pools of all their common metabolites, beginning from acetyl-coa. mevalonate carbon atoms do not find their way back to general metabolism, i.e., these fungi lack the "shunt" pathway. the compartments are regulated independently. the very large variations in carotene content caused by many environmental and genetic changes are not accompanied by variations in the ubiquinone content. the ubiquinone content increases when the cultures grow on leucine or acetate as carbon sources and is not affected by illumination. phycomyces, but not blakeslea, increases the production of ubiquinone in presence of oligomycin. lincomycin, produced by streptomyces lincolnensis, is important, clinically used antibiotic. its gene cluster consists of 27 putative open reading frames with biosynthetic or regulatory functions (lmb genes) and three resistance genes (lmra, lmrb, lmrc). the organization of transcription units was determined. the analysis of the lincomycin biosynthetic gene transcripts in various cultivation stages revealed the genes with putative regulatory functions which are transcribed in early stages of cultivation. previous analysis of biosynthetic pathways of lincomycin and functionally different anthramycin antibiotics (anthramycin, sibiromycin, tomaymycin, mazetharmycin and porothramycin) indicates that the genetic information on the lincomycin and anthramycin biosynthesis should share common elements (genes), both biosynthetic and regulatory. hybridization experiments demonstrated presence of several analogues of lmb genes involved in the biosynthesis of anthramycin produced by streptomyces refuineus and porothramycin produced by streptomyces albus. effect of various calcium salts on the erythromycin production by saccharopolyspora erythraea m. rostamza 1 , a. noohi 1 , j. hamedi 2* : 1 department of biology, faculty of science, science and research branch, islamic azad university, tehran, iran; 2 microbial biotechnology lab., department of biology, faculty of science, university of tehran, tehran, iran. e-mail: jhamedi@khayam.ut.ac.ir (j. hamedi) calcium carbonate has the positive effect on the erythromycin, however, because of its low water solubility, caused to clogging the spargers of fermenters and fouling the microfilters. in this research, various soluble calcium salts was added to the fermentation medium and their effect the growth of saccharopolyspora erythraea and erythromycin production was studied in the complex medium consisted soy bean meal, dextrin and starch as major ingredients. the fermentation conditions were 220 rpm, 8 days at 30 • c. the results obtained showed that there is no significant difference between erythromycin concentrations in the medium containing calcium lactate and calcium carbonate. however, erythromycin concentrations in the other calcium salts containing media were less than to calcium carbonate containing medium. optimum concentration of calcium lactate for erythromycin production was 10 g/l. lincosamides and its derivatives are clinically important antibiotics. comparison of gene cluster coding for lincomycin biosynthesis and newly identified cluster of genes for celesticetin biosynthesis revealed new information on functions of several genes common for both biosynthetic pathways. the celesticetin gene cluster was identified by screening the cosmid library of streptomyces caelestis with heterologous probes based on lincomycin biosynthetic genes involved in a part of biosynthesis shared by both antibiotics. sequence analysis of the cluster revealed 24 putative orfs, out of which 18 are lincomycin biosynthetic genes analogues, four are specific for celesticetin biosynthesis and one codes for resistance. the gene cluster is bounded with transposase genes on both sides. in order to clarify function of three putative regulatory genes of lincomycin biosynthesis, the insertional inactivation with the pcr targeting system in streptomyces lincolnensis was done and resulted in differently reduced production of lincomycin. larsen cmb biocentrum-dtu, technical university of denmark, 2800 kgs. lyngby, denmark we present a new algorithm called "x-hitting" for automatic identification of novel bioactive compounds based on full spectroscopic characters of highly complex mixtures of natural product. one of the most dramatic advances in recent drug discovery has been the increase in screening capacity throughput and data handling. therefore, analysis has become the bottleneck in the drug discovery process. the algorithm presented here is investigated and demonstrated on identifying potentially new bioactive compounds. in addition method is shown to have a high performance for automatic identification of known structures. these tasks are referred to as new-hitting and cross-hitting, respectively. finally, the receiver operating characteristics (roc) is introduced to the research field as an important tool for evaluating the performance of the "compound predictor". through examples it is shown, that known cross-hits are identified with high proficiency, and that the new-hitting works on finding new targets represented by analogues and structurally new compounds. a gene encoding a ␥-butyrolactone autoregulator receptor that have a common activity as dna-binding transcriptional repressors, controlling secondary metabolism and/or morphological differentiation in streptomyces was cloned from a natamycin producer, streptomyces natalensis, and its function was evaluated by in vivo. pcr using the primers designed from two highly conserved regions of streptomyces autoregulator receptors gave a 102-bp band, the sequence of which revealed high similarity to the expected region of a receptor gene. by genomic southern hybridization with 102-bp insert as a probe, a 687-bp intact receptor gene (sngr) was obtained from s. natalensis. in vivo to clarify the function of sngr, a sngrdisrupted strain was constructed, and a phenotype was compared with the wild-type strain. the sngr disruptant started natamycin production 6 h earlier and showed a 4.6-fold-higher production of natamycin than the wild-type strain. in addition, sporulation was earlier and 10-fold abundance. the phenotype indicates that the autoregulator receptor protein of s. natalensis acts as a primary negative regulator of the biosynthesis of natamycin and is related to regulation of sporulation. exploring the biocatalytic potential of the novel thermostable baeyer-villiger monooxygenase: phenylacetone monooxygenase daniel e. torres pazmiño 1 , gonzalo de gonzalo 2 , gianluca ottolina 2 , giacomo carrea 2 , dick b. janssen 2 , marco w. fraaije 1 , 1 biochemical laboratory, groningen biomolecular sciences and biotechnology institute, university of groningen, nijenbaeyer-villiger monooxygenases (bvmos) represent useful biocatalytic tools as they can catalyze reactions which are difficult to achieve using chemical means. however, only a limited number of these atypical monooxygenases are available in recombinant form. using a recently described protein sequence motif, a putative bvmo was identified in the genome of the thermophilic actinomycete thermobifida fusca. the nadph-dependent and fad-containing monooxygenase is active with a wide range of aromatic and aliphatic ketones and sulfides. genetic and kinetic data suggest that phenylacetone is the physiological substrate of the enzyme. previously, it was reported that this bvmo exhibits only a moderate enantioselectivity with (r,s)-␣-methylphenylacetone. this poster we will show an overview of the biocatalytic potential of the enzyme as explored so far. interestingly the enzyme has been found to perform highly enantioselective oxidations with a range of ketones and sulfides. this again indicates that this novel thermostable oxidative biocatalyst can be a useful tool for the synthesis of chiral building blocks. the enzyme s-adenosylhomocysteine hydrolase (adohcyase) catalyzes hydrolysis of s-adenosylhomocysteine (adohcy), an inhibitor of transmethylation reactions, into adenosine (ado) and homocysteine (hcy). the catalysed reaction is reversible, and the equilibrium is strongly displaced in direction of the synthesis of adohcy when reaction occurs in vitro. nevertheless, its biotechnological application resides in the synthesis of antivirals. for it, we selected between different producing microorganisms of the enzyme, the gram positive bacterium corynebacterium glutamicum. after designing the specific oligonucleotides, the gene was expressed in escherichia coli using the expression system pet28a+ with iptg. the electrophoretic analysis under denaturing conditions, shows a clear induction and over-expression of a protein with a mw of 49 kda. on the other hand, the immobilization of this recombinant enzyme in a solid support allows to use it as a catalyst for the synthesis of adohcy. the enzyme was purified by imac thanks to the presence of n-terminal 6 × his tag end, and immobilized in eupergit c for the optimization of the production of adohcy, a product of high value. sequencing, cloning, expression, purification and characterization of a novel cytochrome p450 monooxygenase from rhodococcus rubber luo liu, rolf d. schmid, vlada b. urlacher institute of technical biochemistry, stuttgart university, allmandring 31, 70569 stuttgart, germany. e-mail: itbvur@itb.unistuttgart.de (l. liu) the cytochrome p450 monooxygenases are heme-containing proteins, which catalyze a wide range of oxidative reactions (werck-reichhart and feyereisen, 2000) . a monooxygenation activity was observed for the strain rhodococcus ruber dsm 44319. a p450-like gene fragment was amplified by pcr using degenerated primers. for identification of regions that flank this p450-like dna fragment, the method "directional genome walking using pcr" was applied (mishra et al., 2002) . the full size gene encoding a cytochrome p450 enzyme was amplified by pcr from genomic dna and cloned into the vector pet28a(+) for heterologous expression in escherichia coli bl21(de3) cells. the enzyme was purified using metal affinity chromatography. the primary protein structure suggests, that this enzyme is a natural self-sufficient fusion protein consisting of a ferredoxin, a reductase and a p450 monooxygenase. the reductase activity was determined using an exogenous electron acceptor cytochrome c. the reductase domain of this p450 monooxygenase showed a strong preference for nadph over nadh. the substrate spetrum was investigated. in the presence of nadph the p450 enzyme shows hydroxylation activity towards 7-ethoxycoumarin, naphthalene, indene, acenaphthene, toluene and fluorene. mishra, r.n., singla-pareek, s.l., nair, s., sopory, s.k., reddy, m.k., 2002 . directional genome walking using pcr. biotechniques 33, 830-834. werck-reichhart, d., feyereisen, r., 2000. cytochromes p450: a success story. genome biol. 1 (6), 3003.1-3003.9. selective production of monoglyceride consisted of conjugated linoleic acid by penicillium lipase yomi watanabe 1,2 , yoshie yamauchi-sato 3 , toshihiro nagao 1 , satoshi negishi 3 , tadamasa terai 4 , takashi kobayashi 1 , rolf d. schmid 2 , yuji shimada 1 : 1 osaka municipal technical research institute, osaka, japan; 2 stuttgart university, stuttgart, germany; 3 the nisshin oillio group, ltd, yokosuka, japan; 4 osaka institute of technology, osaka, japan conjugated linoleic acid (cla) is a group of c18 fatty acid (fa) containing two conjugated double bonds. it is expected to prevent cancer, adipositas, atherosclerosis etc, and is commertially available in the primary form of free fa, containing almost equal amounts of 9cis,11trans-and 10trans,12cis-cla. it is therefore desired to be converted to a palatable form. for this purpose, we have previously proposed two enzymatic ways to convert cla to monoglyceride, an emulsifier, with penicillium camembertii lipase; (1) sequencial esterification-glycerolysis, (2) esterification at low tem-perature. these methods, however, are time-and energy-consuming. esterification of cla with glycerol under ambient pressure by the lipase produces equal amounts of mono-and diglycerides. in contrast, it was newly found that the reaction under reduced pressure supressed the formation of diglycerides and achieved to produce 90% monoglyceride at 95% esterification. improving the thermal stability of cellobiohydrolases i (cel7a) from t. reesei by site directed evolution frits goedegebuur 1 , lydia dankmeyer 1 , peter gualfetti 2 , brad kelemen 2 , edmundo larenas 2 , paulien neefe 1 , pauline teunissen 1 , colin mitchinson 2 : 1 genencor international bv, archimedesweg 30, 2333cn leiden, the netherlands; 2 genencor international inc., 925 page mill road, palo alto, ca 94304, usa genencor international has been working to produce improved enzyme products for economic conversion of ligno-cellulosic biomass to fermentable sugars. most of this work was performed under a subcontract with the u.s. department of energy for cellulose cost reduction for biomass conversion. cellulolytic biomass conversion is performed in nature by a complex mixture of enzymes. cellobiohydrolases play a key role and all effective cellulase mixtures contain a large excess of cellobiohydrolases over endoglucanases, suggesting that it is the exoglucanase activity that is limiting. the fundamental dependence of reaction rate on temperature predicts that large increases in performance, and decreased enzyme cost, would be achieved if the enzymatic conversion could be operated at elevated temperatures. industrial strains of trichoderma reesei produce cellulases at very high levels and low cost. however, t. reesei cbh1 (hypocrea jecorina cel7a) does not have sufficient stability to survive and perform at high temperatures. this poster shows the thermal stability improvement in t. reesei cbh1 by site directed evolution. sites with increased thermal stability properties were combined and evolved in high temperature stable cbhi variants. the evaluation of lipases as biocatalysts for organic chemistry can be carried out, at laboratory scale, by using soluble enzymes for biotransformations in aqueous media. however, the industrial exploitation of such an enormous potential should require a suitable protocol for immobilization of lipases. the binding of lipases on suitable pre-existing supports should greatly improve the performance of industrial reactors allowing us a continuous use or re-use of such interesting biocatalysts. in addition, lipases, like most enzymes, are not perfect chemical catalysts. lipases may be unstable and they may not have the optimal activity nor the optimal enantio or regioselectivities. in this way, immobilization of lipases, together with its relevance for the performance of each different industrial reactor, could be also used as a tool to improve and optimize some of these parameters. that is, immobilization of lipases, far from an already solved problem, constitutes an exciting field of research in the promising area of industrial bio-organic chemistry. in this work we would like to present useful immobilization methods of several lipases. the lipase immobilised were used for enzymatic hydrolysis of peptidomimetics of structure a. type of immobilzation used can changed the enantioselectivity of biocatalyst prepared. the absolute configuration of products b and c obtained in enzymatic reactions were assigned by chemical correlation. two-component flavin-dependent monooxygenases form an interesting class of flavoenzymes. they consist of two separate proteins; a monooxygenase component, which catalyses an oxygenation reaction in the presence of reduced flavin, and a flavin reducing component, which reduces flavin (fad or fmn) using nad(p)h as an electron donor. a well-known example of this class of monooxygenases is styrene monooxygenase (otto et al., 2004) . due to the ability to form enantiopure epoxides, which are relevant building blocks for the pharmaceutical industry, styrene monooxygenases form a valuable class of enzymes for biocatalysis. while screening a metagenomic library for oxidative enzymes, an indigo-producing clone was found. sequencing the particular clone revealed an inserted fragment of environmental dna encoding a two-component monooxygenase ( many investigations over the recent years have been directed to the production of natural aroma compounds. through biotransformation and bioconversion, aroma compounds considered as "natural" can be produced starting from monoterpenes, generating high value products as rose oxide. rose oxide is found in small amounts in some essential oils such as bulgarian rose oil and geranium oil. (−)-rose oxide is an impacting flavor compound and has a small threshold: 0.5 ppb. application of agro-industrial residues in bioprocess on one hand provides alternative substrates, and on the other hand helps solving pollution problems, that might be caused by the disposal of this residue in nature. the liquid cassava waste, is originated by the pressing of cassava roots. it is considered as a "harmful" pollutant waste due to its high organic charge and presence of cyanide. on the other hand, can be considered rich in nutrients that can be used in other applications. it was found that sporulated surface cultures of penicillium sp. were able to convert citronellol into cis-and trans-rose oxides. other bioproducts were 3,7-dimethyl-5-octen-1,7diol and 3,7-dimethyl-6,7-epoxy-1-octanol. no chemical oxidation or auto-oxidation products were detected in liquid control broths. the experiments were conducted at 30 • c and 160 rpm. when the medium was cassava, the production of rose oxide, 3,7-dimethyl-5octen-1,7-diol and 3,7-dimethyl-6,7-epoxy-1-octanol were insignificant reaching trace amounts. but when the mycelium developed in cassava medium and than transferred to mineral medium (citronellol as c-source) the concentrations of rose oxide increased dramatically, reaching 70 mg/l for the cis-isomer and 30 mg/l for trans-isomer. a mechanistic mathematical model of enzymatic degradation of avicel and phosphoric acid swollen cellulose (pasc) has been proposed. the model is based on the degree of polymerization (dp) of starting substrate, and follows its decline with time, to the final end product -glucose. three enzyme classes, namely, endoglucanase (eg), cellobiohydrolase (cbh) and ␤-glucosidase (bg) are all individually incorporated in the model. the model is, additionally, taking into account cooperative action of the involved enzymes, as well as effects of enzyme inhibition by end-products, cellobiose and glucose. to be able to describe the complex process of enzymatic hydrolysis with a set of differential equations certain assumptions needed to be introduced. those assumptions represent the simplification of an up-to-date knowledge of both substrate and enzyme structure, but also enzyme mode of action. for example, one of the often asked questions is: "what is happening with shorter cellooligosacharides (dp 7 and up) laying on the surface of cellulose chain? are they being adsorbed to the core cellulose chain or partly solubilized to a hydrolysis broth?" to give answers to these questions and confirm mathematical modeling real enzyme hydrolysis data are needed. in this work, four well characterized, highly purified mono-component enzymes from humicola insolens (two eg and two cbh) and one bg from aspergillus niger were used to hydrolyze avicel and pasc. by careful choice of catalyst, some enzyme specific characteristics like presence or absence of cellulose binding domain will also be incorporated into the model. hydrolysis experiments were initially performed by distinct mono-component enzymes, to confirm the basic characteristics of each of the enzyme classes. soluble hydrolysis products (dp 1-6) were analyzed by hplc and detection of non-soluble, higher-dp polysaccharides was performed by technique of polysaccharide analysis using carbohydrate gel electrophoresis. optimisation of halogenase enzyme activity k. muffler 1 , m. retzlaff 2 , k.-h. van pée 3 , r. ulber 1 : 1 technische universität kaiserslautern, germany; 2 technische universität münchen, germany; 3 technische universität dresden, germany. e-mail: muffler@rhrk.uni-kl.de (k. muffler) halogenases provide the opportunity of a regioselective and stereospecific halogenation of organic subtrates in contrast to the class of haloperoxidases. these enzymes allow a gentle synthesis of halogenated organic molecules and are capable to halogenate in specific positions (hammer et al., 1997; keller et al., 2000) , whereas traditional organic synthesis often failures or mainly leads to byproducts (hasegawa et al., 1999) , e.g. halogenation of tryptophan in other positions than position 3. our current research is focussed on tryptophan-5-halogenases, because the 5-br/cl-tryptophan could be applied as a pharmacologically attractive precursor of serotonin. in our work we describe the optimization of an enzyme assay respectively the enzyme activity. for this purpose we use a genetic algorithm. the responsible gene of the fadh 2 dependent enzyme was cloned from streptomyces sp. origin into a pseudomonas fluorescens strain. however, the optimization procedure was done with the purified his-tagged tryptophan-5-halogenase, which was easily obtained from the crude extract of the lysed cells by application of immobilized metal affinity chromatography. the application of algorithms allows an optimization of the multidimensional search problem leading to a global optimum in the search space in contrast to the traditional used one-factor-at-a-time method. latter often failures, because this method does not reflect possible influences the parameters can have on each other. effect of organic solvent type on the enantioselectivity of candida rugosa lipase in the hydrolysis of racemic naproxen methyl ester in biphasic reaction system serpil takaç, deniz mutlu department of chemical engineering, institute of biotechnology, ankara university, 06100 tandogan, ankara, turkey hydrolysis of racemic naproxen methyl ester to produce s-naproxen was carried out in the biphasic system using isooctane, cyclohexane, hexane and toluene with candida rugosa lipase after stirring in the phosphate buffer (ph 7.5, 0.02 m) for 2 h at +4 • c and centrifuging. the hydrolyses were carried out in shaking flasks for 120 h at 200 rpm and 37 • c with the initial substrate concentration of 0.034 m. the concentrations of the enantiomers of racemic naproxen methyl ester in organic solvents and those of naproxen in buffer solution were determined with hplc. it was found that the enantiomeric excess for substrate (ee s ), enantiomeric ratio (e) and conversion (x) decreased in the following order: isooctane > cyclohexane > hexane > toluene. the enantiomeric excess for product (ee p ) was found to be the same for isooctane, cyclohexane and hexane where the lowest ee p was obtained in toluene. the highest ee s , ee p , e and x values achieved in isooctane at the residence time of 120 h were 91, 95, 130, and 49%, respectively. this study was supported by ankara university biotechnology institute (project no: 89). fusarium fujikuroi (gibberella fujikuroi mating group c) produces multiple secondary metabolites such as gibberellins and bikaverin. gibberellins are terpenoid hormones that induce growth and regulate various stages of development in plants. they have numerous applications in agriculture industry. bikaverins are polyketides that have toxicity against different organisms because they inhibit respiration. regulation of polyketide and gibberellin synthesis by nitrogen has been intensively studied in fusarium but little is known about their regulation by carbon source. our main interest is to understand the regulation of biosynthesis of these compounds in f. fujikuroi imi 58289. to investigate this regulation the organism was grown in high nitrogen medium under submerged conditions and then transferred to nitrogen-free media having various concentrations of different carbon sources. the gibberellin production was not affected significantly. on the other hand bikaverin was synthesized enormously when sucrose was used as the only carbon source. high production in sucrose required a minimal amount of the sugar, but did not change appreciably above this threshold along a wide range of concentration. the bikaverin synthesis was repressed when glucose coexisted with sucrose in the medium. the effect of the c source on the expression of key genes, cps/ks (copalyl diphosphate synthase/kaurene synthase) for gibberellin and pks4 (polyketide synthase) for bikaverin biosynthesis is currently under investigation. ment of dormancy and loss of viability in seeds with the passage of time, it lacks any systematic propagation from seeds and is typically propagated through rhizomes. this restricts large scale cultivation of this plant. in vitro propagation of plants is an effective means for rapid multiplication of species, in which conventional methods have limitations. in the pesent study we have analysed the role of various growth promoters and the effects of dark and light incubation on germination of n. alba seeds. the results indicate that in vitro propagation of n. alba from seeds can be applied as an efficient method of multiplication. the study was funded by the state planning commisson (dpt) turkey and the university of ankara vide projects no. 120640. this research was performed for developing of biological treatment process of odor gas such as mek, h 2 s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over 93% was obtained by biofilm formation. at 400 ppm of inlet odor gas concentration and 10 s of retention time, the removal efficiency was 76 and 93% in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over 97% at the operational conditions above 15 s of retention time. post soviet countries are going through the transition stage and are extremely sensitive to new technology, economics or social changes and globalization processes. that is why decision-making and system of regulation of the use of gmo's are very sensitive to number of factors. three levels of factors, the most influential to decision-making: global, regional and local; are identified at the research paper. global level depends on external policy of leaders of gmo regulations. usa and eu have the biggest influence on transition countries, though their positions completely differ. as usa was leader in inventing gmos, it is lobbing newly created biotechnological industry. in eu and other european countries lobbing of biotechnological industry was not as strong as in usa. thus, their national law is stricter. world trade organization and international agreements are also part of global level. regional level. geographical position of country is also very important because every regulation system depends a lot on regulation that is implemented in neighboring countries. countries do not exist in vacuum; they are linked territorially, politically, economically and socially with neighboring states. regulation systems of the transition countries in the eastern europe can be divided into three types: those who have no system of regulation of gmo (belarus, romania, hungary and ukraine); who approved some variety that are treated as safe to the market (poland, moldavia and georgia) and who approved all of the gmos (bulgaria, croatia and russia [before 2004]). but even if a country declares not to use gmo it is rather difficult to control import of such products, because of the lack of the testing laboratories, corruption of the state employees, agreements on intellectual property, and institutional country problems. in spite of global and regional tendency of gmos related regulation the most important part is the local level, namely the national regulation system. depending on national level we are choosing priorities at higher levels. as an example of post soviet countries ukraine is taken, as ukraine is one of the largest countries and one of the biggest exporters of agricultural products in europe. research includes the analysis of attitude to gm product, their potential risks and benefits of three categories that influence decision-making the most: farmers, gm experts and non-governmental organizations. recombinant microorganism development for extracellular benzaldehyde lyase production hande kaya 1 , pınar ç alık 1 , tunçer h. ozdamar 2 : 1 iblab, department of chemical engineering, metu, 06531 ankara, turkey; 2 bre laboratory, department of chemical engng, ankara university, 06100 ankara, turkey. e-mail: e119497@metu.edu.tr (h. kaya) in this study, the extracellular production of the benzaldehyde lyase (bal, ec 4.1.2.38) that catalyses the synthesis the enantiopure 2-hydroxy ketones for drug syntheses, by bacillus sp., was aimed. for this purpose, the signal dna sequence of an extracellular bacillus enzyme, i.e., serine alkaline protease, was fused in front of the bal gene (accession number ax349268) from pseudomonas fluorescens biovar i, using pcr-based gene splicing by overlap extension (soe) method. b. licheniformis (dsm 1969) chromosomal dna was used as sap gene (accession number x03341) template for the synthesis of sap signal sequence. bal gene was amplified by using the plasmid carrying bal gene, puc18::bal. thereafter, the signal peptide of sap with its own promoter was fused in front of the bal gene by soe method. the hybrid gene first cloned into puc19 plasmid, thereafter sub-cloned into pbr373, pmk4 and phv1431 shuttle vectors. the escherichia coli-bacillus plasmids carrying the hybrid gene pre(subc)-bal was transferred into bacillus subtilis npr− apr−, and bacillus licheniformis. the influence of the host bacillus species on bal production on a defined medium with glucose was investigated in bioreactor systems. for each of the recombinant (r-) bacillus species, effects of initial glucose concentration on cell growth and bal production were investigated; and, physiological differences and similarities between the wild-type and r-bacillus species are discussed. thereafter, the benzaldehyde lyase production capacities of recombinant e. coli and b. subtilis are compared in terms of cell concentration and bal volumetric and specific activities. for the comparison bal gene was cloned into prseta vector which is under the control of strong t7 promoter and expressed in e. coli bl21 (de3) plyss strain. the variations in by-product distributions with each recombinant organism and yields are also discussed. phosphoketolases (ec 4.1.2.9) are thiamine diphosphate (thdp)-dependent enzymes, that play a crucial role in the pentose phosphate pathway (ppp) of heterofermentative and facultative homofermentative lactic acid bacteria, and of the d-fructose 6phosphate shunt of bifidobacteria. reports affirm that cellulomonas flavigena can use the ppp when is cultured under anaerobic conditions. a genomic library of c. flavigena constructed in -zap express vector was screened. four positive clones were isolated, in vivo excised and the resulting pbk-cmv phagemids, each containing a 4.0 kb insert, were characterized by restriction analysis and dna sequencing. the open reading frame (orf) of the dxylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase gene, xpkl, was located from nucleotide 54 to 2466. the xpkl orf encoded a 804 amino acids-residue polypeptide (xpkl) with a calculated molecular mass of 89,000 da, a value coincident with that estimated by comparative sds-page (about 90,000 da). a putative ribosome binding site (gggagc) is present 11-5 nucleotide upstream of the translational start of the xpkl polypeptide. the c. flavigena xpkl polypeptide sequence was 66% identical to dxylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase from bifidobacterium sp., bifidobacterium gallinarum, gloeobacter violaceus and bifidobacterium adolescentis. this analysis also revealed highly conserved regions. lactococcus lactis is a main diacetyl-producing bacteria by citrate metabolism in dairy products. the transport of citrate in these bacteria is dependent on citrate permease that is encoded by citp gene. previous studies of the citqrp operon in escherichia coli mutants showed that citp message is considerably stabilized in rnase iii mutant. so, in the context of the citrate metabolism research, the characterization of the lactococcal rnase iii enzyme is very important for the dairy industry. rnase iii is an endoribonuclease which has an important role in rrna processing and control of gene expression. with the aim of studying lactococcal rnase iii we have cloned the rnc gene from l. lactis ssp lactis il1403 in the broad host range pls1rgfp vector. this plasmid includes the gfp gene, encoding the green fluorescent protein (gfp), cloned under the control of the pm promoter, that is inducible by maltose. maltose induction of the lactococcal rnc expression showed a 5-fold increase of rnc transcription from this plasmid. activity assays for lactococcal rnase iii were standardized using crude extracts and a substrate specific for b. subtillis rnase iii. the results showed that this substrate was specifically cleaved by lactococcal rnase iii and its activity induced by maltose. lac-rnc was cloned in pet15 vector and the corresponding six-histidine-tagged rnase iii protein was overproduced in e. coli bl21 (de3) strain by iptg induction. the protein was purified by affinity chromatography using hplc system and was shown to be active by in vitro activity assays using the lac-rnase iii specific substrate mentioned above. we have also cloned lactococcal rnc gene and studied its expression in an e. coli rnc deletion mutant ( rnc). complementation assays performed in e. coli demonstrate that the lactococcal rnase iii (lac-rnase iii) is able to process rrnas and to regulate the levels of polynucleotide phosphorylase (pnpase). these results demonstrate that the lactococcal enzyme is able to substitute the ec-rnase iii not only in the rrna processing, but also in the processing of mrnas. the amount of lactococcal rnc transcript in an e. coli rnc strain was 3.3-fold higher than in the wild type strain, suggesting that the e. coli rnase iii triggers the degradation of the heterologous rnc mrnas. the results obtained have shown that lac-rnase iii is an interesting enzyme for biotechnological purposes. objectives: the pharmaceutical and food industry has an increasing demand for selectively glycolized active agents. in our application isomaltose can be synthesized by immobilized dextransucrase, which transfers a glycosyl residue from sucrose (substrate) to glucose (acceptor). as the reaction proceeds, isomaltose can act as an acceptor and is converted into undesired follow-up products called isomalto-oligosaccharides, imos. we investigate on two approaches to avoid imo formation, selective adsorption of isomaltose (ergezinger et al., 2005) and the co-entrapment of dextranase adsorbate, which breaks imos down to isomaltose. results and conclusions: the first part of our research concerns the adsorption of dextranase on bentonite, which complies with langmuir model. at complete saturation (0.8 g g −1 ) our immobilisate exhibits an activity of 16,000 u g −1 . a kinetic analysis does not reveal significant differences between the adsorbed and free form of enzyme (k m,bentonit : 14.1 ± 0.7 m versus k m,free : 13.0 ± 0.7 m). thus, bentonite displays a high binding capacity paired with favorable kinetic properties. beyond that we investigate the activity of dextransucrase in co-immobilisates, which is reduced during coimmobilization due to interactions with the adsorbate. among various co-immobilisates, the one containing dextranase bound to preblotted bentonite imparts the highest activity (40% as compared to control: immob. dextransucrase). the molar yield coefficient of coimmobilisates y isomaltose/sucrose surpasses coefficient of control by 13%. further on we will characterize mass transfer of dextranase substrate into alginate matrix as well as bentonite-dextransucrase interactions. and kefir. a second approach is to use yeast as a production organism to produce natural folates for fortification. here we investigate and discuss the folate content in skq2n, a diploid strain of saccharomyces cerevisiae, when cultured in different media and at different stages of growth. the aim is to gain a basal knowledge of the folate production profile, forms of folate produced and degree of leakage to the surrounding medium, in relation to the culturing medium and physiological state of the cells. danisco innovation, danisco a/s, langebrogade 1, po box 17, dk 1001 copenhagen k, denmark we at danisco a/s (copenhagen, denmark) have revealed a new starch degrading pathway by the discovering several new enzymes and metabolites in fungi and algae. these new enzymes include glucan lyases, dehydratases and tautomerases, which proved to be useful in biocatalysis. these new metabolites proved to be useful as both antioxidants and antimicrobials for food and non-food applications. this pathway is named as anhydrofructose pathway of starch and glycogen degradation. this technology is referred to as the anhydrofructose technology. diet is evolving from nourishing populations via providing essential nutrients to improving health of individuals through nutrition. modern nutritional research focuses on health promotion and disease prevention, on protection against toxicity and stress, and on performance improvement. as a consequence of these ambitious objectives, the disciplines "nutrigenetics" and "nutrigenomics" have evolved. nutrigenetics asks the question how individual genetic disposition, manifesting as single-nucleotide polymorphisms (snps), copy-number polymorphisms (cnps) and epigenetic phenomena, affects susceptibility to diet. nutrigenomics addresses the inverse relationship, i.e. how diet influences gene transcription, protein expression and metabolism. the mid-term objective of nutrigenomics is integrating genomics (gene analysis), transcriptomics (gene expression analysis), proteomics (global protein analysis) and metabolomics (metabolite profiling) to define a "healthy" phenotype. the long-term deliverable of nutrigenomics is personalised nutrition for maintenance of individual health and prevention of disease. the major challenges for -omics in nutrition and health still lie ahead of us, some of which apply to -omic disciplines in general while others are specific for -omic discovery in the food context: (i) the integration of gene-and protein expression profiles with metabolic fingerprints is still in its infancy as we need to understand how to (a) select relevant sub-sets of information to be merged, and (b) resolve the issue of the different time-scales, at which transcripts, proteins and metabolites appear and act; (ii) the definition of health and comfort is less of a clear-cut case than the one of disease; (iii) -omics in nutrition must be particularly sensitive: it has to reveal rather many subtle than a few abundant signals to detect early deviations from normality; (iv) in the food context, health cannot be uncoupled from pleasure, that is, food preference and nutritional status are interconnected. transcriptomics serves to put proteomic and metabolomic markers into a larger biological perspective and is suitable for a first "round of discovery" in regulatory networks. metabolomics, the comprehensive analysis of metabolites, is an excellent diagnostic tool for consumer classification. the great asset of this platform is the quantitative, non-invasive analysis of easily accessible human body fluids like urine, blood and saliva. this feature also holds true to some extent for proteomics, with the constraint that proteomics is more complex in terms of absolute number, chemical properties and dynamic range of compounds present. proteomics in the context of nutrition and health has the potential to (a) deliver biomarkers for health and comfort, (b) reveal early indicators for disease disposition, (c) assist in differentiating dietary responders from non-responders, and, last but not least, (d) discover bioactive, beneficial food components. independent of the context of application, proteomics represents the only platform that delivers not only markers for disposition or condition but also targets of intervention: the only way to intervene in a biological condition and to modulate its outcome is interfering with the proteins involved. it is evident that not only comprehensive analyses with one discovery platform (lateral integration of information) are required but also vertical integration between different -omic levels are indispensable for a deeper understanding of disposition, health, environment and diet (desiere, 2004) . a major "vertical integration issue", to date unresolved, is given by different timescales of transcript production, protein expression and metabolite generation (nicholson et al., 2004) . the transcript machinery usually responds fast to an external stimulus (seconds to minutes), the proteins may be expressed within minutes to hours (and have a halflife from minutes to even months) and metabolites vary significantly during the day and depend on latest dietary input. this means that data, which seem to correlate qualitatively (e.g. reflecting the same pathway), may not necessarily be related time-wise. rather, they may represent different responses at different time points and, possibly, to different stimuli. comprehensive -omic analyses is an essential building block of "systems biology", which can be defined as follows (clish et al., 2004) : systems biology is the comprehensive analysis of the dynamic functioning of a biological system (cell, organ, organism or even ecosystem) at gene, protein and metabolite (or higher organizational) level, achieved by comparison of two defined biological states of this system, typically before and after perturbation. while a comprehensive list of components (genes, proteins, metabolites) of a given biological system is a pre-requisite for this kind of research, the main reasoning for the "system view" is that only information on the interactions between the components gives clues to function of the entire network. a systems biology approach has recently demonstrated the power of proteomics to dissect immunity and inflammation. toll-like receptor recognition and signalling was elucidated and showed, how bacterial "barcodes" are read and interpreted in order to trigger an adapted immune response (aderem and smith, 2004) . in order to address some of the challenging objectives of -omics-driven nutritional research, we have addressed (a) the effect of early antibiotic administration on the maturation of intestinal tissues, (b) protein discovery in human milk, (c) the effects of polyunsaturated fatty acids on gene expression and lipid profile in the liver, and (d) biomarkers for intestinal stress. (a) antibiotics and gut maturation: the effects of early administration of antibiotics on intestinal maturation were assessed at the gene expression level in a rat model. (b) human milk: rapid enrichment and iterative, consolidated identification of immunologically relevant milk proteins was achieved through the employment of restricted-access media and a tailored proteomic strategy (labéta et al., 2000; lebouder et al., 2003; panchaud et al., 2005) . (c) fatty acids and liver transcriptome/lipidome: epidemiological studies have correlated higher intakes of poly-unsaturated fatty acids (pufas) with lower incidence of chronic metabolic disease. the molecular mechanisms regulated by pufa consumption were examined assaying the liver transcriptome and lipid metabolome of mice fed a control and a pufa-enriched diet (mutch et al., 2005) . (d) gut stress markers: as a first step, we catalogued protein expression along the jejunum, ileum and colon of the rat intestine and found gut segment-specific proteins (marvin-guy et al., 2005) . the innovative combination of a neonatal separation model with proteomic analysis allowed us to study, whether early life psychological stress may impact the adult gut neuromuscular protein expression and the approach revealed specific protein biomarkers. omics for engineering lactic acid bacteria willem m. de vos wageningen center for food sciences, wageningen university, the netherlands. e-mail: willem.devos@wur.nl. url: http://www.wcfs.nl/ lactic acid bacteria (lab) are high at-rich gram-positive bacteria that have a well-established record in industrial food fermentations where they contribute to conservation, flavour and texture. in addition, several lab are used as food-grade hosts for the production of enzymes, peptides or metabolites. finally, lab are exploited in functional foods that contribute to the health and well-being of the consumer. a variety of metabolic engineering approaches have allowed for the improvement of many attributes of lab. these approaches have been facilitated by the possibility of uncoupling of growth and metabolite production in lab, the wealth of genetic tools that allow modulation of gene expression in a dynamic range, and the determination of several complete lab genomes (de vos et al., 2005) . we have developed lactobacillus plantarum as a paradigm for lab engineering by experimental and modelling approaches, the application of functional and comparative genomics, and the implementation of other post-genomics avenues (kleerebezem et al., 2003; smid et al., 2005; de vos et al., 2004) . examples of optimizing the production of vitamins and other cofactors, the impact of these engineering approaches on the global transcription and metabolite profiles, and determining the l. plantarum activity in the human host will be discussed. solanum tuberosum (potato) is the fourth major crop worldwide and used for food, feed and biotechnological applications. to fully realize the biosynthetic potential for production of starch, protein and metabolites, we conducted an extensive quantitative profiling of the expressed genes of mature potato tuber. a total of 58,322 sage (serial analysis of gene expression) tags of 19 nt representing 22,235 different tags were analyzed. the 695 tags seen 10 or more times were assigned a tentative function by comparison to homologous genes. contrary to the transcript profile of rice seedlings (gibbings et al., 2003) the storage organ of potato is not dominated by transcripts encoding storage proteins. transcripts for four types of protease inhibitors, a metallothionein and a lipoxygenase were more prominent than patatin isoforms. the lactic acid bacterium lactococcus lactis is used extensively in the production of fermented milk products. during cheese production the bacterium experiences many changes in its immediate environment, as a result of its own reactions. the most severe change is the accumulation of lactic acid, which changes the ph of the medium until growth is totally inhibited. we have focused upon a survey of these dairy related stress responses, as a means of constructing more robust strains. when l. lactis starter cultures are produced in rich media, they will experience an initial period with purine limitation after being added to the milk substrate, a stress condition that in several studies have been found to induce cross resistance towards a number of other stresses. we have analyzed both general purine nucleotide (atp and gtp) and specific gtp limitation in chemi-cally defined medium, using both proteomics and transcriptomics. the differential expression analyses were performed with a custom designed dna microarray of pcr amplified probes. the two stress conditions resulted in very different stress responses, both at the transcriptomic and proteomics level. from a new study on the temporal expression pattern of l. lactis during growth in milk, we present preliminary data showing differential expression of genes and proteins of the purine stress stimulon as well as other stress stimulons. cell physiology of the yeast saccharomyces cerevisiae glucose repression mutants ∆snf1, ∆snf4 and ∆snf1∆snf4 was studied in batch and glucose limited chemostat cultivations. detailed physiological studies were performed on cells grown in batch using glucose, galactose, or glucose-galactose mixture as a carbon source. during growth on glucose-galactose mixtures it was shown that after glucose was consumed, galactose consumption remained repressed for about 15 h in ∆snf1 or ∆snf4 mutants, and for more then 40 h in ∆snf1∆snf4 mutant, whereas it only lasted 6 h in wild-type cells. the global transcriptional response in the glucose repression mutants was studied using chemostat cultures. s. cerevisiae wild type and the mutants were grown in glucose limited aerobic chemostats at a dilution rate of 0.1 h −1 . biological triplicates were performed for each strain. to identify transcriptional responses of the glucose repression mutants, statistical tests, clustering method and a model-driven analysis method were used. the global transcription data analysis experiments showed that genes involved in hexose transport, carbohydrates metabolism, respiration, and signal transduction were differently expressed in ∆snf1 and ∆snf4 mutants comparing to wild type cells. combination of gene expression data and gene-scale metabolic model indicated changes in the metabolic sub-networks among studied glucose repression mutants. genomics technologies have recently been introduced into food and nutrition science for identifying targets of molecular actions of nutrients as well as non-nutrient components of foods. changes in the transcriptome, proteome and metabolome have been determined for assessing the molecular actions of zinc as an essential micronutrient and of flavonoids in processes such as colon carcinogenesis and atherosclerosis. zinc is essential for the structural and functional integrity of cells and plays a pivotal role in the control of gene expression. zinc deficiency effects in human cells and an animal model (rats) were analyzed by microarrays and showed that a low intracellular zinc concentration caused major alterations in the steady-state mrna levels of several hundred target genes-dependent on the tissues studied including liver, brain, muscle, intestine and kidney. proteome analysis from the same samples by 2d-page followed by peptide mass fingerprinting via maldi-tof-ms identified similarly a large set of proteins with altered expression level but allowed a common theme of action to be identified. although pleiotropic in first view, the obtained pattern of zinc-affected genes/proteins may represent a reference for defining the zinc regulon in mammalian cells. flavonoids occurring in large number in plant species are considered protective agents in a variety of processes including inflammation and cancer development. we have studied the effects of around 80 selected flavonoids in a screening program and identified for compounds such as flavon, genistein or quercetin by genomics technologies their putative mode of action in colon cancer models and endothelial cells. as part of a collaborative effort employing human endothelial cells and blood mononuclear cells from a human intervention trial with soy isoflavones (genistein/daidzein) the effects of the flavonoids on the stress-response to oxidized ldl and homocystein was analyzed. a set of markers of anti-inflammatory and anti-apoptotic activity was identified for genistein and daidzein and cell biological studies confirmed that both compounds prevented programmed cells death in stressed endothelial cells. in the food processing industry, unwanted presence of extremely heat-resistant bacterial endospores creates major problems due to their capability to survive classical thermal treatments and their ability to subsequently germinate and form actively growing vegetative cells. screening of spoilage isolates using genomic typing techniques to visualise putative genome-based biomarkers allowed us to classify strains according to the degree of thermal resistance of their spores. in addition, we showed that sporulation in the presence of ingredients rich in calcium ions promotes thermal resistance of developing spores and correlates with the expression of specific (marker) genes (see oomes and brul, 2004) . finally, the molecular program that forms the basis of spore germination has been assessed using genome-wide expression analysis. noticeably genes involved in dna-repair were transiently expressed in germinating wild-type spores. also, surprisingly, it was found that spores contain significant levels of ribosomal and messenger rnas. degradation of these rna molecules upon spore thermal injury was found to be characteristic for their thermal resistance and predictive for their subsequent outgrowth behaviour. this finding is currently being patented. the information on spore presence, predictions of their thermal resistance and process survival chances, is used to structure a process management system to facilitate optimal food quality assurance and allow for real time analysis in case of the need for quality control. the information on spore presence, predictions of their thermal resistance and process survival chances is now being integrated. this is used to formulate the conditions for a process management system with state of the art food production quality assurance, which allows for real time analysis in case of the need for quality control. the interplay of the pectinase spectrum of aspergillus niger as revealed by dna microarray studies elena martens, jac benen, johan van den berg, peter schaap fungal genomics group, laboratory of microbiology, wageningen university, dreijenlaan 2, 6703 ha wageningen, the netherlands. e-mail: elena.martens@wur.nl (e. martens) the saprobic fungus aspergillus niger is an efficient producer of extracellular enzymes many of which show carbohydrate modifying activities. these enzymes have gras status and therefore are widely used in the food and feed industry. after determination of the genomic sequence of a. niger by dsm, it was estimated that only a fraction of the potential of secreted enzymes is currently characterised. database mining using the proprietary genome sequence has resulted in the identification of more then 80 genes encoding enzymes involved in the depolymerisation of the back bone and the site chains of the complex polysaccharide pectin. additional enzymatic activities required to remove methyl and acetyl esters, present in pectin were also observed. by using dna microarrays we have sought to gain insight into the complex regulation of all the genes involved in pectin degradation. a. niger was cultivated on sugar beet pectin and on the monomeric constituents of pectin, viz. galacturonic acid, rhamnose and xylose. subsequently the corresponding transcriptomes were analysed. we will report on our findings concerning the regulation of the expression of the genes involved in the degradation of pectin and its main constituent-galacturonic acid and the consequences for the interplay of the encoded (novel) enzymes. since reactive oxygen species (ros) are formed in all living organisms a wide range of antioxidative enzyme systems are present to keep the system in balance. when an animal is slaughtered the cellular anti-oxidative capability is reduced, resulting in an accumulation of ros followed by an increased oxidation of dna, lipids and proteins. generally lipid oxidation is a well-known problem, causing increased rancidity during prolonged storage, of especially fatty fish. the implications of protein oxidation are, however, more unclear also in respect to quality decay of fish. protein oxidation causes a wide variety of amino acids modifications, where of the most studied is carbonylation of proline, argenine, lysine or threonine. these carbonyl groups can be labelled with 2,4-dinitro-phenylhydrazine. combining two-dimensional gel electrophoresis with immunoblotting enables the detection of carbonyl groups for each single protein. the results presented here, reveal that both during frozen storage and tainting of rainbow trout protein oxidation/carbonylation increases, furthermore there is an increase in oxidation/carbonylation for distinct proteins. anne-marie neeteson european forum of farm animal breeders, benedendorpsweg 98, 6862 wl oosterbeek, the netherlands society is concerned about food, animal welfare, food safety, new technologies, scientists and industry. these elements are all present in genomics for farm animals. therefore, it is important to build awareness in scientists and industry, start a dialogue with stakeholders and society, and to be transparent in a pro-active way. this paper will address the issues at stake for scientists and industry, when it comes to genomics and animal health. it will combine the results of imperical, ethical and sociological efforts in three eu funded projects. (c) the proper use of genomics in relation to infectious diseases in production animals, and the role of the scientist in the development in new technologies in this field, are being addressed in european animal disease genomics network of excellence (ead-gene, http://www.eadgene.org/). some observations are that: (1) genomics does not concern changing the gene. however, acceptability of any discovery dealing with living beings and edible products, is not obvious just like that! animals have a symbolic and emotional load. (2) genes are still related to eugenics, in the mind of people. genes as such cause reluctance, but it is seen as positive if the use of medication can be reduced, and if animals will be better resistant to disease. (3) consumers are in favour of consumer education, compulsory labelling and the imposition of minimum standards. the inclination to pay more for foods produced according to desired standards relates closely to income level. (4) animal welfare is the major issue citizens mention as a concern. the focus of breeding organisations on productivity should be counterbalanced by serious attention to the animal's needs in order to avoid unnecessary negative impact on the welfare of the animals. (5) when technical specialists and lay people communicate, they tend to use different languages: they use the same words, but with rather different interpretations. so transparency of breeding practices and clear definitions of terminology will be essential for effective communication among all stakeholders. (6) food safety and human health are the major concern for most people, when it comes to making a choice. during the latest decades research within the field of animal genomics has in general been following the same strategies as those used within the field of human genomics, although with much less resources. the porcine genome has been characterized intensively through the development of linkage maps, comparative maps and physical maps. until a few years ago it had not been anticipated that it would be possible to embark on whole genome sequencing of animals genomes. however, because of technological developments and much lower costs for sequencing, several animal genomes have now been assembled/are on the way to being assembled. the initial step towards sequencing the porcine genome was taken by the sino-danish pig genome project. the efforts within this project have now generated approximately 3.84 million genomic shotgun sequences and 700.000 expressed sequence tags (ests). the shotgun sequences have been included in a three-species alignment to make an initial evolutionary analysis. the results show that pig is much closer to human than mouse is. the ests represent 5 -end sequences from a total of 98 non-normalized cdna libraries. based on assembly and annotation of the ests the structure of the porcine transcriptome has been analysed. the relevance of assembling the porcine genomic sequence is justified both from the perspective of sustainable animal breeding and from the fact that the porcine model is an important research platform because of the anatomical, physiological, biochemical and metabolically similarities with man. examples of functional genomic studies both aimed at sustainable animal breeding and aimed at exploiting the pig as a model for medical studies will be discussed. genomics refers to global, systematic and high throughput approaches that allow collecting large amount of data and thus offer new possibilities for analysis and understanding biological processes. we will present some new knowledge related to reproduction in farm animals resulting from three different strategies. (1) functional analysis of gene and protein expression: the transcriptome and the proteome analysis allowed to identify new genes and proteins whose expression is associated with processes of ovarian follicular growth and atresia as well as oocyte maturation in bovine and porcine species, maturation of spermatozoa in the different compartments of epididymis. farm animals produce food as cost effectively as possible, however this may have negative side effects for their health and welfare. trade off processes between production on one hand and reproduction and health on the other hand play a crucial role. the principles of selective breeding for the best of naturally occurring variation has proven to be able to balance an increased level of production and quality of life for the animal. every year, the economic value of the genetic gain achieved by the breeders and carried over to the producers is 1.5% of the economic value of eu farm animal production. consequently, a conservative estimate of the gain from animal breeding is, every year d 1.2 billion in europe. recent developments, such as the sequencing of the genomes of the human, chicken and cow, together with high throughput laboratory techniques, means that there are new opportunities to enhance quality of life. the goal of this paper is to give an overview of the options offered by genomics for enhanced quality of life with focus on identifying relevant gene variants and technologies for large scale tracking and tracing. selective breeding for the best of naturally occurring variation remains the same as in traditional systems, but by pinpointing the relevant gene variants along genomics it is possible to identify directly the animals best selected for high production without comprising health and welfare. the combination of full genome sequences, software tools, study of functional physiological processes cost-effective high-throughput snp genotyping and comparative mapping have the (proven) potential to identify relevant gene variants, e.g. pork color, boar taint, general disease resistance. functional mutations have direct option of application in breeding programs. unfortunately this is not the case for genetic markers due to cost of genotyping and inconsistent phenotypic effects. new technologies for snp genotyping are cost effective and enable large scale genotyping (1.000 of animals/day). a selection of the best technology and strategic use of these opportunities enable tracking and tracing. the application of this technology offers new opportunities for quality of life, both for animal and humans. background: studies have shown that prebiotic and probiotic consumption alters the gastrointestinal flora, modulates the immune system, inhibits genotoxicity and has a protective effect on colon carcinogenesis. however, the effect of synbiotic consumption on these parameters in subjects at risk of colon cancer has not until now been investigated. aim: to determine if a synbiotic (prebiotic and probiotics together) modulates cancer risk biomarkers in human subjects at risk of colon cancer. methods: a 12-week randomised, double blind, placebo controlled, ethically approved trial of a food supplement containing lactobacillus gg, bifidobacterium bb-12 and raftilose synergy 1 (prebiotic) was performed in 37 colon cancer subjects who had undergone 'curative resection'. faecal and blood samples were obtained before (t1, week 0) midway through (t2, 6 weeks) and following intervention (t3, 12 weeks). rectal biopsies were obtained at t1 and t3. the effect of synbiotic consumption on the faecal flora was assessed using standard plate count techniques. genotoxic damage was measured in single cells derived from biopsies using the comet assay. fw was prepared by diluting faeces 1:1 in dmem, ultracentrifugation and sterile filtration. the genotoxic (comet assay) and cytotoxic potential (almar blue assay) of fw was determined. peripheral blood mononuclear cells were isolated from blood and cytokine production in vitro assayed by elisa. natural killer cell cytotoxic activity and the phagocytic and respiratory burst activity of monocytes and granulocytes in whole blood were determined by flow cytometry. results: in the synbiotic group faecal numbers of bifidobacteria significantly increased (p < 0.001) and lactobacilli increased although not significantly (p = 0.0674) while coliforms decreased (p < 0.05). enterococci, clostridium perfringens and bacteroides were unaffected. in the placebo group bifidobacteria decreased (p < 0.001), the other bacterial groups were unaffected. in biopsies genotoxic damage was increased in the placebo group at t3 versus t1 (p = 0.0301) but was unchanged in the synbiotic group. the genotoxic and cytotoxic potential of fw was unaltered. synbiotic consumption significantly increased (p < 0.05), ifn-␥ production by pbmcs but il-2, il-10, il-12, and tnf-␣ production was unaffected. natural killer cell, phagocytic and respiratory burst activities were unaltered. conclusion: synbiotic consumption did not have a strong immunomodulatory effect on the systemic immune system in this study, nor did it influence the genotoxic and cytotoxic potential of fw. however, synbiotic consumption altered the composition of the gut flora to a more beneficial composition as well as protecting against genotoxic damage in vivo, suggesting a protective effect of synbiotics against colon carcinogenesis. emmaårsköld, malin svensson, halfdan grage, peter rådström, ed w.j. van niel applied microbiology, lund institute of technology, lund university, p.o. box 124, sweden lactobacillus reuteri is used today in a variety of dairy products as a probiotic bacterium. several lactic acid bacteria have the ability to produce different kinds of exopolysaccharides (eps), which have the potential to be used as an alternative biothickener. however, the yield of eps is too low to be profitable in the food industry. to optimise the environmental conditions for eps formation a l. reuteri strain was chosen for a factorial design study. the factors used in this experiment were temperature (30-43 • c), ph (4.5-6.5) and sucrose concentration (50-150 g/l); for each factor three different values were chosen. the strain was grown in batch mode using a semi-defined medium at constant ph and sucrose as the carbon source. the results obtained with 27 fermentations revealed that the highest eps formation was found at 37 • c, ph 4.5 and 100 g/l of sucrose. sucrose did not further affect the eps formation above a concentration of 100 g/l. temperature and ph were significant for the eps formation, but only temperature was significant for growth. a central composite design study was chosen for further optimization of the ph and sucrose concentration for maximum eps formation. also the gene expression of the sucrase enzyme responsible for eps formation was investigated using qrt-pcr. the data were used to develop a model for growth and eps formation. dendritic cells (dc) play a pivotal immune regulatory role in the th1, th2 and treg cell balance. dc are present in the gut mucosa and may thus be target for modulation by gut microbes. here, we screened a large panel of human gut-derived lactobacillus and bifidobacterium spp. for dc polarizing capacity: bone marrow-derived murine dc were exposed to lethally irradiated bacteria and cytokine and dc surface markers were analyzed. substantial differences were found among strains in their capacity to induce proinflammatory cytokines, while the differences for anti-inflammatory cytokines were less pronounced. bifidobacteria were weak il-12, il-23 and tnf-␣inducers, while both strong and weak cytokine-inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12, il-23 and tnf-␣ production induced by otherwise strong cytokine-inducing strains, while il-10 production remained unaffected. those lactobacilli with greatest capacity to induce il-12 were also most effective in up-regulating surface markers. surface marker up-regulation was however reduced in the presence of weak il-12-inducing strains. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. cell surface-associated glycolytic enzymes from lactobacillus plantarum 299v mediate adhesion to human epithelial cells and extracellular matrix proteins s.m. madsen, j. glenting, a. vrang, p. ravn, h.k. riemann, h. israelsen, m.r. nørrelykke, a.m. hansen, m. antonsson, s. ahrné, h.c. beck bioneer a/s, hørsholm dk-2970, denmark among the main selection criteria of lactic acid bacteria for probiotic use, the ability to adhere to intestinal epithelial cells, mucus, or extracellular matrix proteins is considered important. using a proteom based approach we identified a group of novel surface proteins that are non-covalently bound to the cell wall of the probi-otic bacterium lactobacillus plantarum 299v. surface proteins were extracted and analysed by gel electrophoreses followed by mass spectrometry analysis. the surface proteins included glycolytic enzymes like, e.g glyceraldehyde 3-phosphate dehydrogenase, which usually is a typical intracellular enzyme. a collection of lactobacillus species was screened and the phenomenon of surface-associated glycolytic enzymes was found in many of the analyzed species. this is to our knowledge the first example of surface-associated glycolytic enzymes in probiotic bacteria. however, in pathogenic bacteria these enzymes are well known and their surface localization is involved in adhesion to human epithelial cells and invasion. we suspect that the presence of these enzymes on the surface of probiotic bacteria could prevent adhesion of pathogenic bacteria and possibly also involve other probiotic activities such as immune modulation. binding studies showed that the surface-associated glycolytic enzymes of lb. plantarum 229v were able to bind to caco-2 intestinal cells and extracellular matrix proteins like fibronectin. scientific and industrial interest on exopolysaccharides (eps) synthesized by microorganisms and on their chemico-physical properties has been quickly growing in the last years. many strains of lactobacilli produce eps allowing them to adhere to human mucosae and therefore to have probiotic effects such as the stimulation of immune response and even antitumoral activity of these molecules has been claimed. futhermore prebiotic actions of eps beneficially affect the human host health improving the properties of indigenous microflora. in this research we have studied two novel interesting lactobacilli strains: lactobacillus plantarum dsmz 12028 and a particular human isolated strain of lactobacillus crispatus that have probiotic potentialities and are good eps and l(+)-lactic acid producers. the aim of this work has been to characterize these strains and their metabolites (eps, organic acid and bacteriocins), to state them as probiotics and to study their adhering ability on human cells. we have studied the physiology of l. plantarum and l. crispatus in shaking flasks as well as their optimal fermentation conditions to obtain high cell density cultures suitable for use as starters in the food industry and eventually for probiotic preparations. fermentation experiments have been performed in a bioreactor equipped with microfiltration (mf) modules using a semidefined medium and various carbohydrates in different culture conditions (aeration, temperature). the kinetic of eps production has been followed and according to results both strains have shown a growth-related production ranging from 200 to 400 mg/l. in vitro studies concerning the ability of these strains to adhere to human mucosae are in progress as well as the structural characterization of the exopolisaccharides. previous studies have shown that compression coating improves the storage stability of freeze-dried lactobacillus acidophilus, although this stability is related to the degree of cell injury, which in turn is related to the compression pressure used. compression coating has also been found to improve the survival of freeze dried l. acidophilus during exposure to simulated gastric fluid (sgf). the aim of the present work is to create a compression coated l. acidophilus formulation, with targeted release at the terminal ileum and beginning of the colon in the human gastrointestinal tract. dissolution studies were performed using a phosphate buffer with a ph of 2 and 6.8, to simulate gastric fluid and intestinal fluid (sif), respectively. cell viability was monitored using multi-parameter flow cytometry (mpfc), together with traditional cfu/ml counts. mpfc was used to identify live, dead and stressed cell populations, using the fluorescent stains propidium iodide (pi), 3,3 -dihexylocarbocyanine iodide (dioc 6 (3)) and to-pro-3. results show that an enteric coating material, eudragit l100-55, is both suitable for compression coating, and enhancing the survival of cells when exposed to sif. pectin usp 100 has also been shown to promote targeted release of the cells. the opium poppy papaver somniferum contains more than 80 tetrahydrobenzylisoquinoline-derived alkaloids. it is the source of the narcotic analgesics codeine and morphine, which accumulate in specialized internal secretory cells called laticifers. in the aerial parts of the plant, the laticifer cells are anastomosed, forming an articulated network. laticifers are found associated with the vascular bundle in all plant parts. the morphinan alkaloids morphine, codeine and thebaine are found both in roots and in aerial plant parts and specifically accumulate in vesicles within laticifers. the benzo[c]phenanthridine alkaloids sanguinarine and 10-hydroxysanguinarine are found in root tissue. the syntheses of sanguinarine and of the tetrahydrobenzylisoquinoline latex alkaloid laudanine are completely understood at the enzyme level. nearly all enzymes of morphine biosynthesis have also been described. in more recent years, cdnas encoding enzymes of alkaloid biosynthesis in p. somniferum have been isolated and characterized. the cell-specific localization of several of the enzymes of morphine, sanguinarine and laudanine biosynthesis has also been described. with knowledge of many of the gene of alkaloid formation and their sites of expression, the metabolic engineering of p. somniferum for tailored alkaloid profiles is now being undertaken. an agrobacterium tumefaciens-based transformation and a regeneration protocol have recently been developed specifically for narcotic tasmanian cultivars. the various cdnas encoding genes of alkaloid biosynthesis in p. somniferum are being systematically reintroduced into the plant to achieve engineered plants with altered alkaloid profiles. the first results have now been obtained with sense and antisense genes stably expressed in a regenerated tasmanian cultivar. the ultimate goal of exploiting the genes of alkaloid biosynthesis is to produce transgenic medicinal plants of specific alkaloid content that would facilitate commercial production and improve our understanding of the factors that regulate biosynthesis as well as provide experimental systems with which to investigate the ecological role of alkaloids in planta. integrated transcript and metabolite profiling for gene discovery in plant natural product pathways richard a. dixon, lahoucine achnine, bettina deavours, mohammed farag, marina naoumkina, lloyd w. sumner plant biology division, samuel roberts noble foundation, ardmore, ok 73401, usa the rich diversity of chemical structures found in the plant kingdom arises in large part from a limited number of basic chemical scaffolds (e.g. terpene, polyketide) that are modified by a limited number of chemical substitution types (hydroxylation, glycosylation, acylation, prenylation, o-methylation, etc.) . much of the diversity is brought about by the substrate-and/or regio-specificities of the substitution enzymes. in contrast to the large collections of gene sequence and transcript level data available on-line, little detailed information exists on the plant (secondary) metabolome. promiscuity of substrate specificity in vitro may complicate attempts to assign functions to genes of secondary metabolism accessible to researchers through various cdna library collections. using the isoflavonoid and triterpene pathways in medicago species as examples, we describe how integrated metabolite and transcript profiling approaches can aid functional genomics, help explain metabolic regulation, and provide tools for assessing the impacts of genetic modifications in plant secondary metabolism. focused and non-targeted approaches were used to assess the impact associated with introduction of new high flux pathways in arabidopsis thaliana by genetic engineering. transgenic a. thaliana plants expressing the entire biosynthetic pathway for the tyrosine derived cyanogenic glucoside dhurrin as accomplished by insertion of cyp79a1, cyp71e1, and ugt85b1 from sorghum bicolor accumulated 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome and metabolome. in a similar manner, plants expressing only cyp79a1 accumulated 3% dry-weight of the novel tyrosine derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. in contrast, insertion of cyp79a1 plus cyp71e1 resulted in stunted plants, transcriptome alterations, accumulation of numerous new glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae specific uv protec-tants sinapoyl glucose and sinapoyl malate as well as kaempferol glucosides. the accumulation of new glucosides in the plants expressing cyp79a1 and cyp71e1, was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify novel xenobiotics. the pleiotrophic effects observed in plants expressing sorghum cyp79a1 and cyp71e1 were complemented by retransformation with s. bicolor ugt85b1. accordingly, insertion of high flux pathways directing synthesis and intracellular storage of high amounts of natural products is achievable in transgenic plants with marginal inadvertent effects. arabidopsis thaliana-distinct function in gene transcription dynamics? jeppe madura larsen, brian stougaard vad, søren mølgaard, kell andersen, mads n. davidsen, klaus d. grasser department of life sciences, aalborg university, 9000 aalborg, denmark in recent years it has been shown that introns to some extent can regulate expression in the eukaryotic cell. insertion of introns in the 5 utr in arabidopsis genes has shown to increase gene expression at both rna and protein level. in order to investigate if 5 utr introns have distinct characteristics, we analyse these in the well annotated arabidopsis thaliana genome published by the arabidopsis genome initiative. 12,898 loci annotated with full-length cdnas were analysed and 1989 loci (15.4%) containing 5 utr introns were isolated. we studied if the genes containing these introns showed different patterns in alternative splicing and gene function (gene ontology classification) compared to the remaining genes not containing this intron type. 1802 of the isolated loci (90.6%) contained only one 5 utr intron and these were used for further analysis, where it was investigated if the 5 utr introns had a characteristic size distribution. genes containing transcripts with 5 utr introns were more subjected to alternative splicing (9.63% versus 2.39%) and had a tendency to be more involved in cell regulatory functions compared to genes without this intron type. it was also found, that 5 utr introns was characteristically size-distributed. we identified thee predominant sizes of approximate 110, 270 and 390 bp compared to only one for orf introns. this suggests widespread multiple splicing events in 5 utr introns. the results presented here suggest that 5 utr introns have distinct characteristics and function in gene transcription dynamics. cytochrome p450 monooxygenases appear to be involved in the biosynthetic pathways of a large variety of primary and secondary metabolites in microbial, animal and plant cells. in particular, cytochrome p450 scc catalyzes the conversion of cholesterol into pregnenolone-the precursor of all steroid hormones in mammalian steroidogenic tissues. cytochromes p450 are also involved in the biosynthesis of different plant steroid derivates that play important role in regulation of plant growth and development. therein, investigation of possible influence of cytochrome p450 scc expression on plant regulatory system is of a great interest. this report devoted to the investigation of transgenic tobacco plants, which have been generated by the transformation with recombinant plasmid pgbp450f constitutively expressing cyp11a1 cdna of the bovine cytochrome p450 scc . the transgenic state of the plants was confirmed by southern blot analysis. transgenic plants are phenotypically different from the control ones. in particular, they obtained a substantially higher growth rate and are larger than wild type plants. we have demonstrated that incubation of fragments of the transgenic plants leaves in [ 14 c]-labeled cholesterol containing medium results in formation of the radioactively labeled product with chromatographic mobility corresponding to pregnenolone. the presence of this metabolite in the steroid fraction of lipid extracts obtained from the transgenic plants leaves was confirmed by gas chromatography mass spectrometry (gc-ms) method. the data obtained indicate that cytochrome p450 scc synthesized in transgenic plants displays its specific catalytic activity. biotechnological production of glucose isomerase enzyme with streptomyces olivochromogenes for production of fructose syrup from hydrol m. hashemiravan, a. sadat barikani department of food science and technology, azad university (pishva, varamin unit), institute of food science and agriculture, tehran, iran the use of glucose isomerase for isomerization and production of fructose syrup was performed by selected industrial strain of streptomyces olivochromogenes ptcc 1457. growth of microorganism and production of enzyme in different culture media was studied, and effects of different parameters such as phosphate and aeration was evaluated. the growth of microorganism at 28 • c, caused a production of high amount of enzyme. the production of enzyme was considered in two culture media (a and b). medium (a) was selected for the higher production amount of enzyme. the highest amount of enzyme production was seen in medium a, which was 36.4 giu/ml, after 80 h. the use of baffles in culture flasks, increased the amount of enzyme production, four times more. the production of enzyme was increased, 1.25 times more, in phosphate deficient medium. the cells containing enzyme (intra cellular glucose isomerase) was separated by centrifuge, and extraction and release of enzyme was performed by ultra sonication, that is a physical-mechanical method. this method released about 89.9% of total intracellular enzyme. the best length of time for sonication was found to be 4 min. experiments showed that optimum ph and temperature of the enzyme were 7.5-8 and 80 • c, respectively. the highest activity of the enzyme was observed at ph 5.8 for up 120 min. at the time of isomerization reaction the existence of magnesium ions showed to be necessary and omission of this ion cause a decrease of enzyme activity in isomerization process, but this effect was not necessary for the enzyme activity results showed that treatment of glucose syrup at temperatures of 40, 60 and 80 • c, by the enzyme, caused 48%, 50% and 52% of isomerization, respectively. efficiency increase of high acetic acid production with the use of acetobactereace iranian native strains mutation m. hashemiravan 1 , a. alirezasadat barikani 2 : 1 department of food science and technology, azad university (pishva, varamin unit) institute of food science and agriculture, iran; 2 young iranian researchrer's club, tehran, iran the first step in the present research is the isolation of acetobactereace native strains from fruits (such as grape or apple) and fresh vinegar. this separation has been done with the use of effective isolation methods and optimized mediums. ten strains were isolated effectively, the bio chemicals test were performed, each of them were detected, classified and nominated with a special code. two methods have been used for mutation: (a) mutation with the ultraviolet radiation; (b) mutation with nitrous acid. in the first method the microbial cells were treated with us radiation for different periods of 15, 30, 45 and 60 s, and the effect of the uv mutation was assessed. as a result, the period of 45 s was determined as the optimum mutation periods. in the second method, the microbial cells were first washed with the acetate buffer 0.2 m with the ph of 4.5, then 10 ml nitrous acid 0.07 m was added and was mixed for 2-10 min. finally the sampling was done in the periods of 2, 4, 6, 8 and 10 min and was transferred to a plate containing the medium of ethanol-phenol red-agar. the two methods have been compared with each other. each method has its own advantages and disadvantages. the mutation pathway in method (b) is more stable and conducted, while mutation with the uv radiation method, change the position of thiamine-cytosine bases absolutely randomly. finally, the best mutant site was inoculumed with the medium containing alcohol 16%, acetozyme gz 0.6 g/l, acetozyme d 1 g/l, acetic acid 1.5% and 1 l double distilled water. the acetic acid was produced in the rate of 16 g/100 ml. also the mutant strains were detected with scanning electron microscope (sem) and interesting photographs were taken from mutant cell. the performed experiments were planned with the use of taguchi statistical method (qualitek 4). this research has been performed in the irost as the thesis of ph.d. in food biotechnology industry. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red 646 and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed 2d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with 0.6% (w/v) epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than 75 consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph 8.1) or chloride (ph 8.5) as leading ion and -amino-caproic acid (ph 8.9) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic 2danalysis. genotypical differences affecting the response of pisum sativum to differing boron/iron applications e.e. hakki, u. zeynep, m. hamurcu, a. tamkoc, m.b. babaoglu, s. gezgin department of field crops, faculty of agriculture, selcuk university, kampus, konya 42079, turkey. e-mail: eehakki@selcuk.edu.tr (e.e. hakki) boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plantation areas of turkey. both defficiency and toxicity problems exist in a total of about 50% of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant aquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f9 plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. increase in sulfite production by accelerating sulfate uptake in brewing yeast t. fujimura, y. kodama, y. nakao, n. nakamura, w. miki institute for advanced technology, suntory ltd., mishima-gun, osaka 618-8503, japan sulfite plays a role as an antioxidant, which stabilizes beer flavor. therefore, it is important to control the sulfite concentration during fermentation. sulfite is produced as an intermediate in the sulfate assimilation pathway in yeast. we have already reported that over-expression of a lager yeast-specific ssu1 gene, encoding a sulfite efflux pump, leads to increase of sulfite production (fujimura et al., 2003) . in the present work, we have clarified that there are two types of sul2 genes (scsul2 and non-scsul2) each encoding a high affinity sulfate permease in lager brewing yeast. eighty percent and 86% identity are found by comparing the dna sequences and the deduced amino acid sequences, respectively. a comparative functional analysis of the two genes has been performed aimed at achieving further increases in sulfite production by accelerating sulfate uptake. over-expression of scsul2 and non-scsul2 has been achieved by transformation of lager brewing yeast, saccharomyces pastorianus. experiments have been done with and without expression of non-scssu1. the resultant transformants have been evaluated by fermentation tests in wort. over-expression of either scsul2 or non-scsul2 failed to show significant effect on sulfite formation. a combination of over-expression of non-scsul2 and non-scssu1 resulted in two-fold higher sulfite production compared with overexpression of only non-scssu1 and four-fold higher compared with the parental strain. these results suggest that the non-sc-gene types significantly contribute to sulfite production in lager brewing yeast. , t., et al., 2003. functional phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyse the release of phosphate from phytate (myo-inositol hexakisphosphate), the predominant form of phosphorus in cereal grains, oilseeds and legumes. possible applications of phytases have been suggested in animal nutrition to increase mineral bioavaliability and to decrease phosphate pollution in area of intensive life stock management and in human health. zea mays is one of the cereals that contain high amount of phytate as the major phosphate storage compound. over 200 bacteria were isolated and screened for phytases from the halosphere, rhizosphere and endophyte of malaysian maize plantation. the phytase activity of the isolates was screened by a modification of the ammonium molybdate method. the highest extracellular phytase activity was detected from bacteria that isolated from the endophyte of the maize root. in this paper, results for 24 isolates chosen for media, temperature and ph optimization will be presented. production of plant proteinase from jack fruit seeds (artocarpus integrifolis) and its influence on rheological and sensory characteristics of low fat yogurt el-sayed el-tanboly dairy sciences department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, 3.9 (t1), 7.8 (t2) and 11.7 (t3) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period (15 days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t3 showed more less firm after 15 days of storage being 20.17 g/100 g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t2 and t3 of 433 and 479 mpa s, respectively, compared with control of 299 mpa s at 15 days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t2 gained the highest scores (85 points) followed by the control (81.5 points) after 15 days of storage, while yogurt of t3 showed a low scoring being 75. from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of 7.8 units of plant proteinases/ml milk. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions 77 g/l from white distilled lees and 45.6 g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. toasted wine was traditionally produced in galicia, northwest of spain. nowadays this technique is being recovered. grapes after harvesting are air dried in order to concentrate sugars, acids and flavor compounds. raisings are pressed to obtain a must with high sugars concentrations. two different grape wines were prepared concentrating the sugars up to 37 and 57 brix, respectively. in order to get a better knowledge of the problems involved, synthetic media simulating the grape musts were prepared. theses musts were used to optimize the initial sugar concentration, the amount of nutrients required, the optimum temperature to carry out the fermentation and the influence of the type and amount of yeast. under the best conditions some fermentations with grape must were carried out to produce wines with intense aroma and flavor notes and high residual sugar concentrations. in this studies, bacillus sp. e1 strain was isolated from koreanstyle fermented soybean paste and it was producing the biological response modifier (brm). the brm activated the b cell selectively. it was identified the bacillus licheniformis e1. the brm was purified by ion-exchange chromatography and gel filtration. chemical properties of brm: molecular weight of brm was estimated to be about 1,594,000 da. sugar content of brm was 33.0% (w/w) and glucosamine (35.1 mol%) was the high level. protein content of brm was 4.3% (w/w) and serine (17.2 mol%) was the high level. infra-red absorption spectrum was showed the characterization of glycoprotein. biological properties of brm: the brm which isolated from fermented soybean paste was similar to that of bacillus licheniformis e1 by immuno-fluorescence assay. we confirmed that the brm was capsular substance of b. licheniformis e1. potato nitrogen concentrate (pnc) is a highly viscous liquid with high complex nitrogen content produced from the protein-fraction in potato starch extraction. the concentrated extract is rich in minerals and ␣-amino nitrogen. although ␣-amylase nowadays is mainly produced exploiting bacillus production systems there is still considerable demand for fungal ␣-amylase from aspergillus oryzae origin. the aim of the experiments to be reported here was to investigate, if pnc can replace commonly used complex nitrogen sources in the production of fungal ␣-amylase. the following data have been measured in pnc pretreated by diluting to 1/2 and clarifying by centrifugation. total-n: 8.4 g n/l; ␣-amino-n: 2.8% (w/v) (as glycine); soluble protein (bradford): 51.2 mg/l (as bsa); total carbohydrates: 110.0 g/l; reducing sugars: 5.6 g/l; dry weight: 57.3% (w/w). in the following experiments nitrogen sources were replaced on the basis of their ␣-amino nitrogen content. the carbon source for all experiments was maize starch. the formation of ␣-amylase by a. oryzae atcc 1011 in shake flasks -using pnc (centrifuged or not), yeast extract, malt extract, casein hydrolysate or meat extract -was compared to "standard" cultivation with corn steep liquor. the experiments showed only small differences in ␣-amylase titers using complex nitrogen sources. no remarkable differences were observed in the resulting biomass. in general no differences in enzyme productivity and biomass formation could be seen after 50 h of incubation. especially the bench top bioreactor experiments indicated an optimal fermentation time of about 100 h. cultivations of a. oryzae atcc 1011 were carried out in bench top bioreactors. comparing cultivations in a medium with pnc as the sole complex nitrogen source to one containing csl as such no significant differences both in the formation and amount of ␣-amylase and the fungal growth were observed. thus pnc might be able to replace complex nitrogen sources such as csl or even the more expensive yeast extract and casein hydrolysate in fungal amylase production systems. 1.19) is involved in the metabolism of inositol and catalyzes the conversion of d-glucuronic acid to l-gulonic acid with nadph as a cosubstrate posterior to the oxidation of inositol to glucuronic acid by the enzyme inositol oxygenase. although the yeast sporobolomyces oryzicola (nakase and suzuki, 1986) is not able to grow on inositol as the sole carbon source, intracellular glucuronate reductase can be found in cells grown in a medium containing d-glucuronic acid. the enzyme could be a useful tool in the design of a specific quantitative assay for glucuronic acid, e.g. in so called energy drinks. the organism was grown in media containing either glucose and glucuronic acid or only glucuronic acid and difco yeast nitrogen base. whereas growth on both media was similar in shake flask culture, hardly any growth in either medium was observed in bench top bioreactors. the influence of dissolved oxygen tension was investigated and the relevant data will be shown. the formation of intracellular glucuronate reductase activity by sp. oryzicola is inducible by media containing glucuronic acid. no activity is found in cells grown in a medium containing only glucose as the carbon source. besides the activity against d-glucuronic acid, activities against 5-ketogluconate and -at very low levelsagainst galacturonic acid and the lactone of glucuronic acid were detected. the enzyme activity is stable up to 35 • c. the ph has relatively low influence on the activity against glucuronate, whereas the reduction rate of 5-ketogluconic acid is optimal at ph 7.0-7.5 with significantly lower values at ph 6.0 and 8.0, respectively. data on the kinetics of the conversion of both glucuronate and 5-ketogluconate will be shown. nakase, t., suzuki, m., 1986. j. gen. appl. microbiol. 32, 149-155 . the multiple nutritional and functional impacts of food fermentation on human health have been widely accepted (reddy and pierson, 1994; hugenholtz et al., 2002) . however, the related role of the involved microorganisms to the nutritional effect from the fermented food is still not well defined and the mechanisms involved are still largely unknown. the present study was to investigate iron bioavailability in carrot juice fermented by two selected lab strains, l. pentosus fsc1 and ln. mesenteroides fsc2. after digestion by gi enzymes, the juice was supplied to fully differentiated caco-2 cells to study iron uptake and transepithelial transport by caco-2 cells from the digested juice. our data revealed strain specified changes in iron bioavailability in carrot juice fermented by these two strains. after in vitro digestion with pepsin and pancreatic-bile enzymes, the best yield of soluble iron was from ln. mesenteroides fsc2 fermented juice. surprisingly, the l. pentosus fsc1 fermented juice yielded about five times higher uptake iron as compared to fresh juice, while ln. mesenteroides fsc2 fermented juice was not significantly different from the fresh juice. interestingly, the transepithelial transferred iron across the cell line was however better from ln. mesenteroides fsc2 fermented juice than from l. pentosus fsc1 fermented juice. to summarise, our study showed that level of soluble iron after in vitro digestion does not necessary indicate iron absorption, especially in the case of lab fermented food. data on improved iron uptake from l. pentosus fsc1 fermented juice indicated exiting of promoter(s) for iron absorption in such juice that is not related to the production of organic acids and lowering ph effect. peng zhang, herve vanderschuren, martin stupak, wilhelm gruissem institute of plant sciences, 8092 zurich, the tropical root crop cassava (manihot esculenta crantz) is a major source of food for approximately 1 billion people worldwide. in sub-saharan africa, more than 200 million people rely on cassava as their major source of dietary energy. in many parts of africa and latin america, cassava leaves are a vegetable source for daily uptake. cassava is grown mostly by poor farmers under marginal environmental conditions and in areas where few other crops can sustain competitive yields. the crop is therefore fundamental for subsistence farming and food security, but it is also very susceptible to stresses common in the areas and conditions where it grows. in many parts of africa, reliable cassava production is strongly impacted by infections with the african cassava mosaic geminiviruses (cmgs), a rapidly spreading disease that causes large yield losses. in the coastal areas of east africa, cassava production now is threatened by another devastating disease, cassava brown streak disease (cbsd). cassava plants are also frequently attacked by many pests, such as cassava hornworm and stemborers. several reports also indicate that greater leaf longevity, especially under drought conditions, could be important for increasing yields and/or the stability of production in cassava, as well as improve the access to an important nutrient source. conventional breeding efforts have attempted to address the constraint to cassava production, but with limited success. the new tools of biotechnology can change this situation by offering new approaches to the challenges of cassava. these new technologies have the potential to make cassava much more productive, a better source of nutrients, and profitable to grow, hence, greatly contributing on the sustainable development of tropical agriculture. recently we have developed biotech cassava with value-add traits, including resistance to cassava mosaic virus, prolonged leaf life and insect resistance. new strategies are also explored to increase protein content of cassava storage roots. we are currently undertaking pilot studies with two teams of leading scientists and experts for projects to test acmv-resistant transgenic cassava lines in africa and lines with extended leaf retention at ciat, colombia under field conditions. this development of substantially equivalent improved transgenic cassava lines is part of a larger study to analyze the need, effectiveness and biosafety of biotech cassava for agricultural production. the goal of the pilot studies will be the development and coordination of a broader project that produces important and novel scientific results, valuable information on the need and impact of biotechnology at the subsistence farming level, and a sound scientific basis for the development of guidelines for biosafety assessments and release of transgenic organisms into the environment and agricultural production in africa and latin american countries. this study was conducted to reveal the effects of different pretreatments on obtaining haploid plants by using the anther culture in pepper capsicum annum l. cultivars demre sivrisi and sirena. buds were collected at uninucleate microspore stage. anthers collected from buds were cultured in ms medium containing different hormones and hormone concentrations. experiment results revealed that when sirena anthers were pre-treated cold at +4 • c for 24 h and kept in darkness at 25 • c for a period of 1 week gave good results. in the case of demre sivrisi anthers were pre-treated cold at +4 • c for 48 h and kept in darkness at 25 • c for a period of 1 week gave good results. on the other hand no cold pretreatment to anthers resulted with low embryo formation. similar results were also observed on the anthers kept at 35 • c for 1 week as callus was produced in some petri dishes but no regeneration was observed. as a conclusion, since no cold pretreatment to anthers resulted with low embryo formation it is possible to say that cold pretreatment should be applied to anthers in pepper another culture studies. the yeast d. hansenii ufv-170 was tested in this work in batch experiments in synthetic media at constant initial substrate concentration (100 g l −1 ) under variable oxygenation conditions. to get additional information on its fermentative metabolism, a stoichiometric network was proposed on the basis of the general knowledge available in the literature on xylose metabolism in pentose-fermenting yeasts and the specificities of xr and xdh activities in d. hansenii and checked through a bioenergetic study performed using the experimental data of product and substrate concentrations. it can be stressed that under strongly oxygen-limited conditions xylitol production was negligible, whereas under semi-aerobic conditions maximum xylitol production (p max = 76.6 g l −1 ) and yield (y p/s = 0.73 g g −1 ) were obtained. a progressive decrease in these parameters was observed under fully aerobic conditions, suggesting that xylitol-producing yeasts require limited oxygen conditions, which is species-dependent. the proposed model, which utilizes the experimental specific rates of substrate consumption and product formations, allows estimating the main bioenergetic parameters. besides, it proved to be an effective tool to investigate different metabolic situations and showed how they can influence the flux distribution of the carbon source and the bioenergetics of this biosystem. the effect of disinfectants on fungi anne svendsen, pernille skouboe bioneer a/s, hørsholm dk-2970, denmark prevention of mould spoilage of foods can only be carried out successfully, if the species, which are actually spoiling the food product, are known. a very limited number of fungal species has been associated with the spoilage of each food category. proper disinfection of production facilities is very important to avoid mould spoilage. resistance of moulds to disinfectant treatments are known and different species have shown different response to the same disinfectant. to obtain proper disinfection it is important to know the resistance of the spoilage fungi against different disinfectants. in this study the effect of disinfectants on the spoilage fungi of cheese, rye bread, liver paté and fruit juice was investigated. in collaboration with five food companies the dominating species responsible for spoilage of each food product were isolated and used for testing. commercial disinfectants and disinfectants "under development" were tested. tests were performed in suspension and on surfaces, the methods used were modified after en 1650 and en 13697. considerable variability in fungicidal effect among the species was observed. some disinfectants were ineffective at low temperature. some disinfectants showed different effect in suspension and on surfaces, resulting in an effective kill in suspension and almost no effect on surface. the identification of effective disinfectants in the food industry includes: (1) testing against the specific spoiling species of the food product, (2) testing on surface, not only in suspension, (3) test parameters adapted to the food manufacturing plant. intensified research efforts in recent years confirm the major importance of the microbial flora in the gastro-intestinal tract for human health. ingestion of prebiotic oligosaccharides increases the number of the desirable bacteria like bifidobacteria and lactobacilli in the colon. we are looking at beta-galactosidases from lacto-bacillus spp. for the production of galacto-oligosaccharides (gos) because we speculate that the enzymes of probiotics will form gos with high prebiotic potential. in this present study, purified betagalactosidases of selected lactobacillus strains were used for the production of gos from lactose. different enzyme reactor set-ups, both discontinuous and continuous, were tested and compared. temperatures up to 37 • c and ph values between 6 and 6.5 were required for satisfactory enzyme stability during the process. enzyme source, substrate concentration and the level of substrate conversion were found to be critical process parameters for gos yields and composition. yields of up to 40% (w/w) of total sugars were achieved when the initial lactose concentration was 200 g/l. capillary electrophoresis (ce) and hplc with pulsed amperometric detection were the analytical tools for investigating the influence of reactor type, enzyme source and conversion level on gos composition. the prebiotics market is increasing rapidly and is expected to more than double until 2010 to about 180 million d world-wide. therefore, the development of enzymatic processes on an industrial scale is a high priority goal of our research. starter addition does not always succeed in improving standardisation and quality of the complex sensory properties of traditional fermented foods. in many cases the added strains do not grow as well as the environmental strains present in the production plant. here, a method of geometric simplification (by dichotomy) of a complex ecosystem found on a raw milk livarot (82 strains) was tested on cheese curd. by a limited number of cultures, successively, 40 out of 82, 20 out of 40 and 10 out of 20 strains were selected on the basis of two criteria (i) respect of the taxonomic proportion, (ii) generation by the daughter ecosystems of an odour close to the one of the mother ecosystem. finally a sub-ecosystem of 10 strains gave an odour similar to the one of the more complex mixture. the use of molecular methods (pcr-sscp) permitted to follow the main species growing. mother and daughter ecosystems were characterized by sensory analysis and gc-ms. probably because of an important redundancy of the strain functions, the method was very efficient. this method may permit to improve a lot the set up of mixture of strains and species used in fermented food industry. effect of the dilution rate on the exopolysaccharide production by bifidobacterium longum atcc 15707 c. shene, m. rubilar, s. bravo universidad de la frontera, chemical engineering, av. francisco salazar 01145, casilla 54-d temuco, chile exopolysaccharides (eps) producing lactic acid bacteria are used in dairy industry (cheese and yogurt) due to the rheological properties that these compounds confer to the products. preliminary results also suggest the use of eps as health-promoting (anti-tumor and immunostimulatory actions) ingredients. bifidobacteria are grampositive bacteria natural inhabitants of the gut of warm-blooded animals and man. a number of investigations have shown that bifidobacteria promote host health mainly because of the reduction proliferation of some pathogenic bacteria through acid synthesis. in this work results obtained in the experiments carried out to test the capability of b. longum atcc 15707 to synthesize eps are presented. continuous culture fermentations were carried out at dilution rates between 0.04 and 0.44 h −1 . composition of the culture media was that of the mrs broth. biomass concentration presents higher values (2.9-3.2 g l −1 ) at dilution rates between 0.1 and 0.2 h −1 . biomass growing at these rates is difficult to pellet and adheres to the fermentor walls behavior that was not observed at other growth conditions. eps from cultures grown at these rates were preparated and fractionated. authors wish to thank the chilean conicyt for the economical assistance given through the project fondecyt 1050602. high pressure-low temperature (hplt) inactivation processes were performed on bacillus subtilis vegetative cells at various conditions. at atmospheric pressure, lowering the temperature to as low as −45 • c was found to have minor anti-microbial effects. upon application of high pressure various phase transitions occurred in the microbial suspensions under study. after pressure treatment at 150-450 mpa, cells were plated under optimal conditions to assess cell viability. treatments at 250-450 mpa and −25 • c were the most effective in inactivation. in these cases, ice i-iii solid-solid phase transition was observed. in addition, we hypothesised that intracellular thawing (solid-liquid phase transition) had already occurred while the extracellular surrounding was undergoing solid-solid phase transition. this double effect is suggested to be key in mediating the observed large drop in viability. we speculate that more cells survived after treatment at −45 • c compared to the same treatment at −25 • c because both the extra-and intracellular surrounding remained fully frozen. at −45 • c a solid-solid phase transition was observed when pressure was higher than 350 mpa. a metastable state of ice i was observed at 250 mpa treatment. results from the current study will be presented (see also shen et al., ifset in press) . the data call for a mechanistic evaluation of the effects of hplt as an anti-microbial treatment. such data are currently being gathered and will be used in defining optimal hplt process conditions for the food industry. the influence of saccharomyces cerevisiae, kloeckera apiculata and candida pulcherrima mixed cultures on the selected alcohols formation during model fermentation pawel satora, tadeusz tuszynski department of fermentation technology and technical microbiology, food technology faculty, agricultural university, cracow, poland. e-mail: psatora@ar.krakow.pl (p. satora) for the study five yeast species were chosen, isolated from successive stages of plum fruits spontaneous fermentation: from the beginning (candida pulcherrima, kloeckera apiculata, saccharomyces cerevisiae w4), middle (s. cerevisiae w54) and final fermentation (s. cerevisiae k1). to characterize the potential influence of yeast mixed cultures on the selected alcohols formation, wick-erham synthetic medium (10% glucose) was fermented by mixed cultures of two and three yeast species. after distillation, ethanol, propanol, isobutanol, isoamyl alcohols, hexanol and 2-phenylethanol were determined using gas chromatography. findings were compared with the results obtained after monoculture fermentations. the use of mixed cultures resulted in increasing of glucose utilization rate, ethanol and fusel alcohols formation (except propanol) and decreasing of methanol synthesis. the samples fermented using two yeast species characterized higher (about 10%) amount of volatile compounds in relation to monocultures. it takes note of especially high level of ethanol (av. 44.3 g/dm 3 ), methanol (16.7 mg/dm 3 ) and isoamyl alcohols (47.6 mg/dm 3 ). the positive feature of triple cultures using was limitation of methanol and fusel alcohols synthesis that was accompanied by relatively high ethyl alcohol production (av. 41.5 g/dm 3 ). the consumption of sugar syrup becomes increasingly significant in industrial processes due to economic advantages and the easy of use. the production of sucrose syrup using enzymatic hydrolysis represents the safest alternative, once the reaction does not produce any toxic or undesirable substance. this work consists on the production of sugar syrup by immobilized inulinase from kluyveromyces marxianus, with two alternatives process: (a) syrup enriched with fructooligosaccharides or solely with glucose and fructose. the process is comprised by the following stages: production and purification of the enzyme in optimized conditions, immobilization of the enzyme in solid support and the conversion of sucrose in a fixed bed bioreactor with the immobilized enzyme. the final composition of the product can be a mixture of glucose, fructose, sucrose and fructooligosaccharides or a mixture of fructose and glucose, according to the operational conditions. the bioreactor can be operated continually for approximately 5 months with the same biocatalyst. the product from this process is ideal for applications in the food products such as sweet, candies, chocolates, yogurts, etc. besides, the prebiotics properties of the fructooligosaccharides, is a beneficial stimulant of the intestinal flora, which gives to the product a functional property. studies on plant microbial interactions using azotobacter sp. as bio-inoculants towards soil fertility baljeet singh saharan faculty of biotechnology, jcdm college of engineering, sirsa 125055, india. e-mail: baljeet.saharan@gmx.de, baljeet br@yahoo.co.uk (b.s. saharan) high nitrogen fixing, phytohormone producing isolates of azotobacter, azospirillum, acetobacter and pseudomonas were used as inoculants on wheat and cotton with varying doses of nitrogen under field conditions. bio-inoculants were selected on the basis of yield, dry weight and survival rate of bacteria under field conditions. seeds of wheat variety wh 711 were treated with different biofertilizers using nitrogen level of 90, 120 and 150 kg ha −1 and one level of p, i.e. 60 kg ha −1 in field along with control. under field conditions, maximum yield was obtained with azotobacter chroococcum e 12 at 90 (2506 ± 0.04 kg ha −1 ) as well as 120 kg ha −1 (2817 ± 0.07 kg ha −1 ) followed by a. chroococcum ht 57 (2482 ± 0.16 kg ha −1 ) and avk 51 (2474 ± 0.37 kg ha −1 ). whereas, with 120 kg ha −1 highest yield was observed with mac 27 (2833 ± 2.59 kg ha −1 ) followed by e12 (2817 ± 0.91 kg ha −1 ) and avk 51 (2804 ± 0.16 kg ha −1 ). maximum height at 90 kg ha −1 was observed with mac 27 inoculation (71.1 ± 5.24 cm) followed by avk 51 (70.8 ± 4.70 cm) and ht 57 (70.3 ± 1.76 cm). various chosen strains were tested with desi (hd 123) and american cotton (h 1098) under similar pot and field conditions as for wheat in the following season. plant height and yield were determined at the time of harvesting whereas survival rate was monitored at various intervals of time. survival rate of inoculated bacteria was determined after 30, 80, and 135 days. highest survival rate was observed in mac 68 ((3.34 ± 2.56) × 10 6 ), which decreased after 80 and 135 days, respectively. (3.38 ± 1.48) × 10 5 , (1.53 ± 0.92) × 10 5 with mac 68 and (2.97 ± 2.01) × 10 5 and (1.26 ± 3.01) × 10 5 with ht 54, respectively. maximum boll weight was with avk 51 (76.2 ± 2.34 g boll wt. plant −1 ) followed by pseudomonas (71.3 ± 1.77 g), ac 18 (61.5 ± 1.73 g) and ala27 (61.4 ± 2.79 g) boll no. plant −1 was maximum with ala 27 and avk 51 (46 ± 2.59 plant −1 ) followed by pseudomonas (34 ± 0.07 plant −1 ). maximum height and dry matter was obtained with pseudomonas (179.7 ± 1.97 cm) and avk521 (146.7 ± 3.49 cm) with variety hd 123 under field conditions. net saving of 20% nitrogen was observed using a. chroococcum (e 12 and avk 51) bioinoculants for wheat and cotton, respectively. to characterize the antioxidative properties of tempeh-fermented food prepared from vicia faba (l. kontu) with the use of rhizopus oligosporus, the sulfhydryl groups content and surface aromatic hydrophobicity of albumins were investigated. the results obtained for tempeh albumins were compared with raw vicia faba and bovine serum albumin (bsa). these results indicate that tempeh fermentation increased antioxidative activity of albumins. the measurements of antioxidative activity were carried out with the use of 1,1-diphenyl-2-picrylhydrazyl (dpph) and 2,2 -azinobis-(3ethylbenzothiazoline-6-sulfonic acid (abts). the albumins of faba bean-tempeh have possessed much higher activity for scavenging free radicals as measured with the dpph and abts (49.4% and 41.1%) than raw seeds (27.9% and 23.2%) and bsa (5.8% and 13.4%), respectively. it has been also found that tempeh fermentation process increased 2.5 times sulfhydryl groups content (43.2 m/mg of albumins) as compared to raw seeds (17.5 m/mg of albumins). the tempeh albumins have possessed lower surface aromatic hydrophobicity than raw seeds (352.3 fi and 692.1 fi, respectively). orange peel characterization and generation of fermentable sugars solutions for the biotechnological production of food additives b. rivas 1 , j.m. domínguez 1 , p. torre 2 , j.c. parajó 1 : 1 department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, 32004 ourense, spain; 2 department of chemical and process engineering, genoa university, via opera pia 15, 16145 genoa, italy. e-mail: brivas@uvigo.es (b. rivas) the citrus processing industry generates in mediterranean area around 3 millions tonnes of orange peel as byproduct from the extraction of citrus juices in industrial plants. in order to avoid ecological problems and provide an extra profit, this residue was studied in order to generate a suitable substrate for the fermentation process oriented to the production of food additives. orange peels were characterized and the data collected allowed quantifying a 97% of this waste. soluble sugars (21.2%), cellulose (17.0%) and pectin (42.5%) were identified as more important fractions. this material was submitted to two hydrolysis techniques, prehydrolysis (with diluted sulfuric acid) and autohydrolysis (with water) under different experimental conditions. autohydrolysis was selected as the most appropriate technique for the production of suitable fermentation media. finally, the liquors obtained at 130 • c and liquid:solid ratio of 8 g/g, containing 38.2 g/l of sugars, without additional nutrients, were employed to citric acid production by aspergilus niger cect 2090 (atcc 9142, nrrl 599) . the influence of the addition of calcium carbonate and methanol were studied. under the best conditions an effective conversion of sugars into citric acid was attained, showing the viability of the production of fermentable solutions from this industrial waste. today there is an increasing interest in using high gravity fermentation in brewing. high-gravity fermentation involves production of beer wort of up to 18 • p or even higher and results in beer that has more consistent product quality. the main aim of this study is an increased understanding of how brewer's yeast respond to the various stress factors imposed during high gravity beer fermentation and the consequences these stress factors have on the gene regulation and its consequences on the metabolite levels (both intra-and extracellular). higher attenuation of the wort will be achieved by two different techniques: by the addition of highly fermentable adjuncts such as sucrose or glucose syrups and by mashing with addition of microbial enzymes such as pullulanases and glucoamylases. in the first part of the study model fermentation conditions are established, where the sugar uptake and product formation can be studied in details. characterization of the carbohydrate profile is analyzed by hplc. as flavour changes may occur at higher gravities, it is important to study changes in formation of secondary metabolites, especially esters. transcriptome and metabolome analysis will be used to establish how the stressful conditions prevailing under high gravity fermentations may influence the secondary metabolism in saccharomyces cerevisiae. furthermore, analytical aroma characterization of final beer will be studied by spme and gc-ms. detailed analysis of the effect of different stress factors on the cellular response using dna arrays and metabolite profiling will be carried out. dna arrays will be employed to evaluate if specific metabolic pathways are up-regulated or down-regulated as a consequences of the stress factors. naringin, a bitter compound that occurs in citrus fruit juices, may be converted to a nonbitter form by enzyme hydrolysis. the enzymatic complex naringinase was produced in aspergillus niger cect2088 cultures with naringin as inducer (pérez-mateos et al., 2004) . crude extracts from a. niger and purified naringinase from penicillium decumbens were immobilized into a polymeric matrix of polyvinyl alcohol (pva) hydrogel cryostructured in liquid nitrogen. the operating stability of the pva-naringinase beads was tested using synthetic citric juice (gray and olson, 1981) . immobilized enzymes reduced 40% the naringin content at 20 • c and ph 3.2. furthermore, immobilized preparations from aspergillus and penicillium could be re-used through six cycles (144 h) remaining 70% and 38% catalytic efficiency, respectively. financial support from "ministerio de ciencia y tecnología" and feder (no. agl2003-08006/ali). gray and olson, 1981 . j agric. food chem. 29, 1298 -1301 . pérez-mateos, et al., 2004 (1), 230. otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box 6121, campinas, cep 13083-862 são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was to identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during 3, 5 and 7 days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the medium was composed by, cwb:cr were mixed with distilled water and transferred into 250 ml capacity erlenmeyers flasks and auto-claved at 120 • c for 20 min. the medium was then inoculated with spores (5.0 × 10 7 ) and the flaks were incubated at 32 • c. tannase was assayed according to the methodology of mondal et al. (2001) . according to the statist analyses, the optimum conditions to produce tannase was the range of temperature (29-34 • c); tannic acid (8.5-14%); residues percent (coffe: wheat bran) (50:50) and 5 days fermentation time. the enzyme production increased 8.6 times more enzyme production than that was obtained before this optimization. yeast and lactic acid bacteria are two major microbial groups of the most fermented products. a large variety of fermented foods and beverage are made by the activities of both yeast and lactic acid bacteria, simultaneously or successively. during the spontaneous mixed fermentation of lactic acid bacteria and yeast population, it is extremely difficult to control microbial species due to the complexity of the microorganism involved. therefore, we have compared the antimicrobial activity of chitosan against two lactic strains, lactobacillus plantarum and lb. brevis, and yeast strains, saccharomyces cerevisiae to investigate the possible use of non toxic biopolymer chitosan for selective control in mixed culture. the lactobacilli were more sensitive to the inhibitory activity of chitosan than s. cerevisiae. the results suggest the possible use of low molecularweight-chitosan for the control of food fermentation in which both groups of organisms frequently occur together. the effect of vegetable oils on astaxanthin production of phaffia rhodozyma and xanthophyllomyces dendrorhous csaba vágvölgyi, gyöngyi lukács, miklós takó, árpád csernetics, tamás papp department of microbiology, faculty of sciences, university of szeged, p.o. box 533, astaxanthin (3,3 -dihydroxy-␤,␤-carotene-4,4 -dione) is one of the most important carotenoid product. it is used primarily as food colorant and animal feed additive. their effective antioxidant properties linked to a preventive action on various types of cancer and an enhancement of the immune response could lead to expanded commercial applications. among the natural microbial source available, the closely related red pigmented yeasts phaffia rhodozyma and xanthophyllomyces dendrorhous are of great biotechnological interest. these yeasts have desirable properties as biological sources of pigment, including rapid metabolism and producing high cell densities in fermentor, but the commercial production of astaxanthin is limited by the relatively low content in wild-type strains. the purpose of this study was to determine whether the different vegetable oils had an effect on the carotenoid production in p. rhodozyma. effects of media supplemented with corn germ oil, wheat germ oil, sesame-seed oil, palm oil, pumpkin-seed oil, coconut grease, olive oil (extra virgin), olive oil (sanza), sunflower-seed oil and cottonseed oil were tested. studies were performed on both a phaffia and a xanthophyllomyces strain. yeast was grown in yeast-pepton-glucose liquid medium complemented with the appropriate vegetable oil in different concentrations (0.5-2, v/v, %) . after four days the total carotenoid production was determined spectrophotometrically, and it was referred to dry cell mass. palm oil increased significantly the carotenoid production of the phaffia strain, while a similar effect on the xanthophyllomyces strain could be observed with coconut grease. composition of carotenoid compounds in the strains was determined by thin layer chromatography. lutein is considered a nutraceutic compound that has developed an increasing interest since it is one of the two carotenoids that are located in the macula of the human eye. its consumption is associated with the prevention of age related macular disease (amd). industrially, lutein may be produced using a saponification step of a mixture of lutein diesters that are previously extracted with hexane from natural sources. our proposal is to improve the process by catalyzing the same reaction using microbial lipases during the extraction step with hexane. additionally, the use of supercritical fluids represents an extension of enzymology in non-conventional media with process and environmental advantages. this work was developed using extracts from marigold flower (tagetes erecta) in hexane and supercritical carbon dioxide (sc-co 2 ) where the lutein esters were hydrolyzed by two commercial lipases: lipase b from candida antarctica (novozym 435) and lipase from mucor miehei (lipozyme rm 1m). in particular, we focused our interest in the role of water in the system. interestingly our results show an inverse dependence of the initial reaction rate with respect to the initial water activity (awi) for both lipases, a phenomena that seems to be related to the partition of substrates and products in the solid support)and the hexane phase as a function of water. when sc-co 2 was used as solvent an increase in the consumption rate of lutein diesters occurred, reaching conversions of 70% in 24 h. for hexane, the same conversion was reached after 160 h. this result suggests a significant effect of the media on the reaction that can be related to shifts in the partition of compounds that bring the substrates in closer contact with the enzyme. this work also demonstrates that lutein hydrolysis seems to be another potential application of commercial immobilized lipases in the food/nutraceutical market. the enzyme of interest in this work is ␤-galactosidase from lactobacillus sp. (ec 3.2.1.23). ␤-galactosidases catalyze the hydrolysis and transgalactosylation of ␤-d-galactopyranosides (such as lactose). an attractive biocatalytic application is found in the transgalactosylaction potential of these enzymes which is based on the catalytic mechanism of ␤-galactosidases. the products of transgalactosylation, galacto-oligosaccharides, are non-digestible carbohydrates which meet the criteria of 'prebiotics' and therefore have attracted increasing attention. to produce these 'prebiotic' galactooligosaccharides, an inexpensive and efficient process is desired. immobilization of the enzyme ␤-galactosidase on an insoluble support is an attractive tool to make the process of lactose conversion more economical because the enzyme can be recovered and reused during continuous operation. in this present study, we aimed at immobilizing ␤-galactosidase from lactobacillus sp. by covalent linkages on two solid supports which are commonly used for protein immobilization: chitosan and eupergit c. the protein-binding capacity, the immobilization yield, ph and temperature dependency of activity and stability, and the kinetic parameters of immobilized enzymes were studied. higher activity retention of the immobilized enzymes over a broader ph range and at higher temperatures compared to those of the free enzyme was observed. the immobilized enzymes were evaluated in terms of transgalactosylation activity and stability for a to introduce of foreign genes for the important crop plants such as rice, we need a reproducible efficient procedure for regeneration of the calli through somatic embryogenesis. for this intention, we established the best callus induction medium for tarom mahalli and deilamani cultivars and created the method that the regeneration frequency was reached to 48%. calli were induced from scutellar tissues of mature seeds on ms medium supplemented with three level of 2,4-d (2, 2.5 and 3 mg l −1 ) and n6 medium supplemented with five level of 2,4-d (1.5, 2, 2.5, 3 and 3.5 mg l −1 ). for deilamani cultivar the best medium was n6 with 1.5 mg l −1 2,4-d and for tarom mahalli the same medium with 2 mg l −1 were the best. in subculture media, sucrose was used instead of maltose. for regeneration analysis of plantlets, we used two-factorial experiment in base of crd; one factor was regeneration media with six levels (ms medium supplemented with five amount of kinetin and: naa (mg l −1 ) [(6:2), (4:2), (2:0.5), (3:1), (2:1), respectively] and 0.2 mg l −1 2,4-d and 2 mg l −1 bap). other factor was dehydration process with three levels (without dehydration, dehydration with two layers of filter paper for 30 min [prior to transfer to the regeneration medium], and third factor was factor of 2 with substitution of sucrose with maltose [after 2 weeks; 2 sucrose:1 maltose]). we conclude that maltose due to changing in osmolarity proceeding can elevate the regeneration frequency to 48%. therefore, type of carbon source is critical in callus induction and regeneration. márová ivana, hrdličková jana, kubešová jitka, kočí radka, vidláková tereza faculty of chemistry, brno university of technology, purkyňova 118, 612 00 brno, carotenoids are the most widespread natural pigments with important biological activities and applications mainly in food and feed industry. at present many ways including genetic engineering are developed to reach higher production of naturally formed carotenoids using microbial producers. in this work cloning and expression of crt gene cluster from pectobacterium carotovorum in recipient bacterial strain e. coli dh5␣ as well as in yeast strain s. cerevisiae was tested. plasmid vector phsg298 with inserted crt genes was used for transformation of chemically competent e. coli dh5␣ cells, while in s. cerevisiae shuttle vector paur135 was used. transformants were selected based on resistance to antibiotics, formation of orange-coloured transformant colonies, analysis of recombinant plasmid size and lc/ms analysis of carotenoids produced by recombinant cells. the yield of individual carotenoids (lutein, beta-carotene, lycopene) obtained from various bacterial transformants was several fold higher than in natural producer (lutein: 0.2-1.2 g/g of d.w., beta-carotene: 0.1-1.4 mg/g of d.w.). the highest yield obtained in transformed strain was 63.5 g/g of lutein and 15.4 g/g of beta-carotene. the yield of biomass and carotenoids in. transgenic s. cerevisiae was comparable to some industrial red yeast strains (4.5 mg of total carotenoids + 32 mg ergosterol/l; 36 g/l of biomass). so, transgenic yeasts could be suitable for large scale production of carotenoids and/or enriched biomass, while transgenic bacterial producers are perspective above al for high production of rare carotenoids as lutein or lycopene using transformation by specific genes of crt gene cluster. this work was supported by the project msm 0021630501 of the czech ministry of education, youth and sports. two forms of grape seeds, whole and powdered forms, were heated at four different temperatures-50, 100, 150 and 200 • c. after heating, grape seeds were extracted with 70% ethanol (0.1 g grape seed/10 ml of 70% ethanol), and total phenol contents (tpc), radical scavenging activity (rsa) and reducing power of the extracts were determined. thermal treatment of grape seed increased the antioxidant activity of extracts. the maximum tpc and rsa of whole grape seed extract (wgse) were achieved when the seeds were heat-treated at 150 • c for 40 min, while that of powdered grape seed extract (pgse) were at 100 • c for 10 min, and were greater than that of the non-treated control. according to the gc-ms analysis, several low-molecular-weight phenolic compounds were newly formed in the wgse heated at 150 • c for 40 min. these results indicated that antioxidant activity of gse was affected by heating conditions (temperature and time) and physical conditions of grape seeds at the time of heat treatments. analysis of the unexpected phenotypic consequences associated with plant transformation jonathan latham, allison wilson, ricarda steinbrecher econexus, 6, canon frome court, ledbury hr8 2td, uk. e-mail: jrlatham@gn.apc.org (j. latham) transgenic plants often exhibit unexpected phenotypes. such phenotypes could arise from pleiotropic effects associated with the transgene, or they could arise from other sources. a recent econexus report underlined the potential for the process of plant transformation to result in genetic damage to the transformed plant (genome scrambling-myth or reality? transformation-induced mutations in transgenic crop plants: http://www.econexus.info/). the report showed that mutations arising at the site of transgene insertion are often substantial, frequently resulting in loss or rearrangement of chromosomal dna and insertion of multiple superfluous dna fragments. unintended mutations were also documented at other locations in the plant genome. such transformation-induced mutations could provide an explanation for unexpected phenotypes in transgenic plants. we decided to survey regulatory documents and the scientific literature for instances of unexpected phenotypic consequences arising in transgenic plants. this poster documents the preliminary results of our survey. it is intended to assess the range and frequency of unexpected consequences and to examine whether there is sufficient data available to determine their origin. it is our belief that investigating the origin of these unexpected phenotypes should be a principal aim of biosafety research. biotechnological production of metabolites such as carotenoids could be of high interest because of their antioxidative and antimutagenic activities in human body. production of these metabolites by microbial cells is dependent on cultivation conditions. so, presence of exogenous stress in cultivation environment could stimulate biosynthetic pathways of desired metabolites. two non-conventional yeast strains, rhodotorula glutinis and sporidiobolus salmonicolor, were chosen for study of carotenoid production useful in feed industry. hydrogen peroxide, sodium chloride and/or their combinations were used as exogenous stimulators of carotenoid pathway. presence of exogenous stress led to important overproduction of pigments as well as of supplementary studied substances (ergosterol, glycerol). higher adaptability of yeast cells was observed not only in cultivations with one type of stress. combination of stress factors in cultivation media induced significant increase of pigment formation. moreover, under controlled conditions in laboratory fermentor s. salmonicolor produced about eight-fold amount of ␤-carotene (230 g/g) in medium with 2% nacl and 5 mm h 2 o 2 than in control sample. similar result was observed in r. glutinis cultivated in presence of 2% nacl in inoculation medium only (200 g/g of ␤-carotene). the use of stressed biomass of red yeasts in feed industry could have positive effect not only in animal and fish feeds because of high content of physiologically active substances, but it could influence nutritional value and organoleptic properties of final products for human nutrition. this work was supported by the project msm 0021630501 of czech ministry of education. the culture ph significantly affects mycelial growth and morphology, exopolysaccharide (eps) formation, and their molecular properties during submerged cultures of a medicinal mushroom ganoderma lucidum. when the culture ph shifts from 3 to 6, mycelial growth (12.5 g/l) and eps production (4.7 g/l) were favorable compared with other ph-control strategies. the mycelial morphology was also significantly varied upon culture ph: a feather-like pellets were found when the ph was controlled shifting from 6 to 3 at day 4, which was regarded as undesirable morphological form for eps production. compositional analyses revealed that the ratios and chemical compositions of the eps formed in bottom or top fractions of ethanolic precipitates were significantly different upon culture ph. the molecular characteristics of the eps were further investigated using a size exclusion chromatography/multi-angle laser light scattering (sec/malls) system. plant ␤-n-acetyl-hexosaminidase (hex) (ec 3.2.1.52) is reported to have diverse physiological roles like fruit ripening, degradation of reserved glycoproteins in germinating seed and chitin-elicited lignification. in this paper we report the purification and characterization of hex from korean ginseng roots. after extraction with citrate-phosphate buffer, hex was purified to homogeneity using ion exchanger chromatography, hydrophobic interaction chromatography and gel filtration. its molecular weight was determined using gel filtration and mass spectrometer. enzymatic parameters were studied with 4-methyl-umbelliferyl-n-acetylglucosaminide as substrate. the effect of heat stress and weak organic acids on escherichia coli and a comparison of its recovery by the plate count method and flow cytometry monica s. talsania due to the importance of microbiology for human health, methods have been developed to enumerate viable bacteria. dilution plating is seen to be the 'gold standard' for proof of a cells viability. however, the success of this method relies on post sampling growth, which is limited by our ability to grow cells in the laboratory. additionally, stressed or sub-lethally damaged cells, remain undetected. single cell measurements can provide rapid detailed physiological information, and the assessment of population heterogeneity. this work compared the recovery of stressed e. coli as measured by the number of cfu/ml and by multi-parameter flow cytometric analysis. weak organic acids and high temperature-short time processing (htst) were used to stress the cells both methods commonly used during food preservation. it was shown here that flow cytometry is a powerful tool for the enumeration and detailed analysis of any non-culturable microbial population, which is important because cytotoxic compounds and heat stresses used in food preservation often have a growth inhibiting effect but not necessarily a lethal one. paul g. kovalenko molecular biology & genetics nasu, zabolotnogo str. 150, 031473 kyiv, ukraine the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of 6 weeks. mean transformation frequency ranged from 27% (for 8196 up to 31% (for 15,834). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba 9402 tl-dna and the 35s gus gene showed an average of more than 35%. these obtained root cultures were additionally elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g. uralensis were obtained by infection of a. rhizogenes 8196 have produced gl at an yield of 4.5% dry weight on the period of culture as a 30 days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels (3.42 g/l) of the total flavonoids production have been identificated on the strains which transformed by lba 9402. this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. ayse gul nasircillar akdeniz university, biology, akdeniz univ. faculty of art-science, biological department, 07058 antalya, turkey mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-d) or 1 mg/l naphthalenacetic acid (naa). the developed calli and regenerated plants were maintained on 2,4-d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + 2,4-d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. the glycolytic enzyme triosephosphate isomerase (tpi), which catalyses the interconversion of the triosephosphates dihydroxyacetone phosphate (dhap) and glyceraldehyde-3-phosphate (gap), was studied for its control on glycolysis and mixed acid production in lactococcus lactis il1403. we constructed a number of l. lactis strains in which the tpi activity was modulated from 3% to 217% of the wild-type level. the enzyme was found to be present in high excess with 3% tpi activity supporting 30% of the wildtype glycolytic flux, and with 24% of the wildtype tpi activity the glycolytic flux was essentially unchanged. measurements of the upstream metabolites glucose-6-phosphate (g6p), fructose-1,6bisphosphate (fbp) and dhap were essentially unchanged for tpi activities from 24% to 217%, and only in the strain with 3% tpi activity we observed a significant increase in the intracellular dhap concentration. homolactic product formation was preserved throughout the interval of tpi activity studied, though a small increase in the amount of acetate and formate production was observed in the strain expressing tpi at the lowest level (3% tpi activity). the finding of an increased mixed acid pattern under intracellular conditions with a high dhap concentration is in contrast to earlier data from literature, which indicated that the triosephosphates play an important role in regulation of pyruvate metabolism in l. lactis with a negative effect on the mixed acid flux. we have recently shown that alcohols induce the adhesion of l. monocytogenes at low temperatures, presumably accompanied by enhanced exopolysaccharide (eps) production. however, little is known about the mechanisms involved in the formation of biofilm and eps by l. monocytogenes. in the present project, we show that deletion of selected regulatory and up-regulated genes did not abolish attachment, though the degree of alcohol-induction in some cases was affected. we are applying bioinformatics to search for homologues in l. monocytogenes of known eps genes from various gram positive bacteria. this has revealed candidate genes involved in the synthesis of eps, such as genes encoding glycosyltransferases. moreover, we are at present performing dna microarray analysis for the egde strain grown at 10 • c in the presence of 2.5% isopropanol. this data should, combined with the bioinformatic results, give us a good indication of the genes involved in alcohol-induced surface attachment. repetitive-pcr (rep-pcr) was applied in research on non-starter lactic acid bacteria (nslab) in cheese. we first showed that strains previously differentiated by pulsed field gel electrophoresis (pfge) also could be differentiated by rep-pcr. this was partially due to slight changes in the pcr conditions that allowed reproducible amplification of 7-9 kb bands. more than 20 bands were obtained for most strains. a clear differentiation was also obtained between lactobacillus paracasei, lactobacillus plantarum, lactobacillus curvatus and lactobacillus danicus (a new species found in danish and estonian cheeses and traditional starter cultures). we found that this technique is highly reproducible, e.g. identical profiles in three different pcr-machines, two different dna isolation procedures, and different trained personnel. we applied the developed rep-pcr technique to confirm that survivors after heat treatment, were the actual strains introduced and not due to post-pasteurization contamination. we also showed that when we added a cocktail combination of five strains as protecting cultures to cheese, two to three members of this cocktail was dominating the cheese nslab microflora. in control cheeses without the cocktail in most cases other strains dominated, but in a few cases we were able to show cross-contamination between cheese vats. these data indicate that the rep-pcr will be useful to follow development of adjunct cultures as well as provide a reproducible subspecies (e.g. strain) differentiation. rep-pcr is a much quicker and less labour requiring procedure than pfge, and is apparently a much more reproducible technique than what has been seen for rapd. dynamic modeling of lactococcus lactis metabolism and its dynamic behavior for lactate secretion and regulatory characteristics jinwon lee, ui sub jung, hye won lee department of chemical and biomolecular engineering, sogang university, seoul, south korea, 121-741. e-mail: jinwonlee@sogang.ac.kr (j. lee) dynamic metabolic model for lactococcus lactis has been developed in order to analyze a time-dependent behavior of lactate secretion mechanism and probe its regulatory roles. the model was used to compare and analyze the lactate metabolism through in silico simulation and in vitro experimental measurements most of all pyruvate branch point seems to play a major role in producing lactate, and the results of metabolic control coefficient analysis recommend to increase lactate dehydrogenase activity and to decrease nadh oxidase activity. for obtaining more realistic data, we have added some measured flux data including some intermediate metabolites. by combining the simulation results and experimental measurements, we could establish more reliable and robust systematic lactate secretion model. in addition, an efficient parameter estimation method was used to test the exactness of the reported kinetic parameters. what to choose -the fast or the detailed -strategy to get informative profiles of secondary metabolite produced by fungi in culture. chemo-diversity and lead discovery calls for high throughput techniques, but do we need columns will direct infusion esi-ms (dims) do the job. the latter may give matrix effects and lacks resolution resulting in loss of information, while lc-ms analysis takes time and challenge the data processing. results from nano-esi dims and lc-ms analyses of the same extracts important penicillium species are compared. these results illustrate advantages and problems using these techniques for rapid profiling of fungal secondary metabolites, reviling that matrix effects in dims do not seriously hampers detection of important metabolites while the specificity and certainty, for e.g. de-replication is much higher in lc-ms. phenotypic classification of fungi is essential in food biotechnology ulf thrane center for microbial biotechnology, biocentrum-dtu, søltofts plads 221, technical university of denmark, dk-2800 kgs. lyngby, denmark. e-mail: ut@biocentrum.dtu.dk fungi are of great importance in food and food production. the intended use of fungi as cell factories for production of food ingredients is an upcoming issue in food biotechnology; however, this brings up a possible contamination with mycotoxins as a major issue. a reliable identification of the producer strains is crucial as a correct identification at species level following an updated taxonomy is the key to information on functional characters, e.g. useful metabo-lites and potential mycotoxins, growth conditions, resistance, etc. unfortunately, many mycological reports do not specify the taxonomy used or do not pay sufficient attention to taxonomical systems based on classification by functional characters-in contrast they are using a nucleotide sequence based phylogeny, which conveys little -if anything -about function of the organism. this situation is a major challenge for biotechnologists and mycologists in the years to come and will be highlighted by illustrative examples. the commercial interest in functional foods containing sufficient amounts of living probiotics is paralleled by the increasing scientific attention to the beneficial effect in the digestive tract. a daily intake of viable cells is proposed to ensure probiotic effect on consumer's health. one of the approaches which seems to be feasible to enhance probiotic viability and stability is to improve the fermentation conditions. during batch fermentation the viability of lactobacillus gasseri 5714 decreases after reaching a maximal value apparently indicating cell death. in this work, the apparent loss of viability can be avoided during fed-batch fermentation. a three-fold increase in viability is obtained when nutrient concentration was controlled compared with the viability reached in batch cultures. as a consequence, higher biomass concentration and lower specific lactic acid production were obtained. a mathematical model was developed to simulate and describe the effect of nutrient limitation on growth, viability, glucose consumption and lactic acid production. contribution to the metabolic adaptation to food restriction in rabbits (preliminary results) s. van harten 1 , s. borges 2 , p. cravo 2 , l.a. cardoso 1 : 1 instituto de investigação científica tropical, cvz, lisboa, portugal; 2 instituto de higiene e medicina tropical, lisboa, portugal. e-mail: svharten@gmail.com (s. van harten) in order to understand metabolic differences between two breeds of rabbits (halop ab and oryctolagus cuniculus algirus) during food restriction, the activities and expression of key enzymes and hormones of the rabbit were studied. animals from each breed were divided in two groups (ad libitum and restricted), revealing the results a similar difference in glycemic levels between fed and underfed rabbits, with a restriction of 50% of ad libitum feeding in the wild animals (decrease of 23% lw) and 16% of that ingestion in the halop breed (decrease of 32% lw). the activities of glutamine synthetase and glutaminase show a higher reduction of these enzymes in the wild animals superior to that of the halop breed, compromising, in this way, the ammonium detoxification and the entry of residual carbonated groups of the protein catabolism into the krebs cycle. in the latter animals, a rapid mobilization capacity of triacylglycerols (tga) appears to exist, with a rapid catabolism of fatty acids leading to their oxidation. the wild breeds' results reveal a rise of circulating tga, reflecting difficulties in the lipolysis and mobilization of nefa for oxidation. in these underfed animals, phosphoenolpyruvate and pyruvate suffered a large increase and oxaloacetate a decrease. the halop breed revealed results that indicate a diminution of glycolisis, being glucoses' energy substituted by carbonated chains of lipolysis and protein catabolism. hormone results showed a higher decrease in insulin, t3 and igf-1 in the underfed halop animals. in order to confirm the biochemical results, relative quantification of enzyme expression was studied by real time-pcr. since the introduction of genetically modified (gm) crops in 1996, the area under their cultivation has globally increased from 1.7 million hectares in 1996 to 67.7 in 2003. the number of countries adopting gm crops also rose from one country, the usa, in 1996 to 20 in 2003. despite numerous successes public opinion still questions the ecological, moral, ethical considerations and issues concerning altering the natural state of the organisms. in this study, a survey of food shoppers' knowledge, attitudes and perceptions of gm foods was carried out in food outlets in nairobi. the food outlets were determined by simple random sampling. using systematic sampling, shoppers were interviewed at targeted imported food products. focus group discussions were also conducted with farmers at city markets. the survey reflected views of a systematic sample of 387 shoppers in seven food outlets between november and december of 2003. it revealed knowledge at 20%, with positive attitudes and good perceptions towards gm foods (χ 2 = 42.873, d.f. 9, p < 0.001). seventy nine percent of shoppers were willing to buy and consume gm foods (χ 2 = 61.321, d.f. 2, p < 0.001). cross-tabulation of shopper's position on various issues raised in the survey showed a strong correlation between the respondents' respective knowledge, attitudes, and perceptions (r = 0.84). nineteen percent of food sampled tested positive for gms. poisson statistics were used to calculate the number of sample sequences. the statistical tools were obtained from spss version 11.5. the results of this study will be of great interest in determining the use and adoption of gm crops in kenya. it will also guide the development of national foreign food policy on gm foods. the technology should be embraced as soon as it is acceptable to alleviate, drought, famine and hunger estimated to be affecting 3.3 million kenyans today, mostly children. consumers and gm foods: the case of turkeyözlenözgen 1 , mustafa yildiz 2 : 1 department of family and consumer sciences, school of home economics, university of ankara, ankara 06130, turkey; 2 department of field crops, faculty of agriculture, university of ankara, 06110 ankara, turkey the future development of food biotechnology depends on consumer acceptance. scientists are aware that consumer attitudes will have a crucial impact on the process of the food biotechnology. because, food is one of the central features in human life. consumers' attitudes and trusts in the institutions will determine how gene technology will be used in food sector, in the future. recently, research concerned with consumer aspects of gm foods accelerated. but in turkey, the literature that deals with this subject is very limited and sparse. therefore, this research was carried out on the turkish consumers with the purpose of analyzing the consumers' awareness, assessments about benefits-risks, market place and labelling, and trusts in institutions, towards gm foods. this study was based on interviews with consumers who have recently purchased from major malls, during shopping hours. of the four major malls, voluntary male and female consumers were included in the research if they had main or secondary responsibility for household shopping. the questionnaire form was applied to subjects through face-to-face individual interview. the data were analyzed by using statistical methods according to explanatory variables, including age, gender and educational level. findings indicated that consumers' awareness and views about gm foods were connected to selected demographic characteristics. the results of this study can be important for consumer educators, marketing managers and policy makers. benefit-risk perceptions and moral beliefs of turkish consumers towards transgenic productsözlenözgen 1 , haluk emiroglu 2 , mustafa yildiz 3 , ayşe sezen taş 1 : 1 department of family and consumer sciences, school of home economics, university of ankara, 06130 ankara, turkey; 2 faculty of law, university of bilkent, 06590 ankara, turkey; 3 department of field crops, faculty of agriculture, university of ankara, 06110 ankara, turkey the use of biotechnology in production has generated considerable debate involving the benefits-risks and moral beliefs associated with its use. consumer acceptance of genetically modified product is a critical factor will affect the future of this technology. this study was planned and conducted to determine the relationship between product/process related benefit perceptions, product/process related risk percentions and moral beliefs of consumers towards transgenic products. a total of 400 university educated consumers, consisting of 200 males and 200 females, employed at the ministries selected by random sampling method in ankara, were included into study. interview techniques were used in the gathering the research materials. the interview instrument had been prepared considering previous research and literature. answers given to sentences typed likert were scored, used "varimax analysis technique" for validity. in order to test the reliability of questionnaire were calculated "cronbach alpha" as inner consistency coefficient. the t-test were performed for determining the differences dependent on gender and age variables between product related benefit perceptions, process related benefit perceptions, product related risk perceptions, process related risk perceptions and moral beliefs of consumers. the examination of relationships between product/process related benefit perceptions, product/process related risk perceptions and moral beliefs of consumers was made by correlation analysis technique. it is thought that the results of this study are important both for scientists and social scientists. the application of the dna recombinant technology for food production is generating a great debate in our society with the participation of scientists trying to explain the way of obtaining these new foods and which are their implications; environmentalist groups and anti-biotechnology associations that systematically are against to the application of this technology; legislatives bodies and the public in general, represented by consumers' organizations that expresses their right to be informed. considering that university students will be the future professionals and consumers their opinion on this topic will be decisive in its success or failure, this research is aimed to performed a global and comparative study of the agrobiotechnology perception by students from different areas of knowledge and studies. this study was carried out during the academic years 2000-2004, being analyzed a total of 1516 valid surveys. the designed questionnaire included 30 questions relatives to: evaluation of the own knowledge and interest on the topic; evaluation of the information sources mainly used by university students to obtain nutritional information; the opinion about gm food labelling; risks/benefits perception; purchasing intention and support of biotechnology. results obtained showed that 37% of the university students interviewed have a clear positive perception of biotechnology, mainly the students of the health sciences area. these students understand the scientific terminology and they use the university as the main source of information. they support the development of the biotechnology and they consider that in a future it will report them benefits. other group (11%) has a clear negative perception, they are mainly students from law and art history. they do not understand the scientific terminology, they consider that biotechnology will cause them risks, and as a consequence they don't have intention of buying these foods. the technology of the dna recombinant can be also used to introduce in the plants genome the gene that codes a protein of interest for their use as antigen. the application of agrobiotechnology has allowed the development of a new generation of vaccines that try to reduce or to eliminate the inconveniences of the classic ones. for the new vaccines design, the detailed knowledge of the biology of the pathogen is considered. with this knowledge the genes implied in virulence can be inactivated or modified selectively. the term "edible vaccines" it is usually applies to the use of edible parts of the plants (tubers, fruits, leaves, etc.) genetically modified with the purpose to produce specific components (antigens) of a pathogen (virus, bacteria, etc.) against which is wanted to protect a person or animal. however, oral is not the best vaccination route since the quantity of antigen for an efficient immunization it is usually high, being also needed, the co administration of an adjuvant that stimulates the immune answer. on the other hand, it is also important to highlight that the levels of antigen accumulation in transgenic plants is usually lower than the necessary ones. another problem is the irregular accumulation of the antigen in the different parts of the plants, thus difficult the appropriate control of the doses. tannin is polyphenolic component having some antioxidant properties and exists in many plants and fruits. in pomegranate juice this component causes turbidity and haze. during fruit juice clarification by conventional gelatin method, all poly phenolic substances which are responsible for antioxidant activity are removed and as a result the quality of the product is reduced. in the present study tannase enzyme (tannin acyl hydrolase; ec 3.1.1.20) was used to decompose tannin to gallic acid and glucose and as the result the amount of turbidity of the juice is decreased, however, the antioxidant properties remain unchanged since tannin is not decomposed and not separated in the juice as it occurs in the gelatin method. the amount of gallic acid in pomegranate juice samples before and after addition of tannase was measured using hplc tests and the optimum temperature, the enzyme and juice contact time, ph, and solvent concentration for clarification of pomegranate juice were obtained as 45 • c, ph = 5.5, 2 h and 50 mm citrate buffer, respectively. the potential benefits of enzymatic clarification of pomegranate juice, that is preservation of antioxidant activity and hence increasing the quality of the fruit product, in comparison to that of conventional clarification method by gelatin introduce a new technique in turbidity and haze removed in tannin containing fruit juices. the objectives of this study are to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against escherichia coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. in lactococcus lactis the enzymes phosphofructokinase (pfk), pyruvate kinase (pk) and lactate dehydrogenase (ldh) are uniquely encoded in the las operon. we have applied metabolic control analysis to study the role of this organisation. earlier work showed that ldh at wildtype level has zero control on glycolysis and growth rate but high negative control on formate production (c j formate ldh = −1.3). we find that pfk and pk have zero control on glycolysis and growth rate at the wildtype enzyme level but both enzymes exert strong positive control on the glycolytic flux at reduced activities. pk has high positive control on formate (c j formate pk = 0.9 − 1.1) and acetate production (c jacetate pk = 0.8 − 1.0), whereas pfk has no control on these fluxes. decreased expression of the entire las operon resulted in a strong decrease in growth rate and the glycolytic flux. increased las expression resulted in a slight decrease in the glycolytic flux. at the wildtype level the control was close to zero on both glycolysis and the pyruvate branches. the sum of control coefficients for the three enzymes individually was comparable to the control coefficient found for the entire operon at the wildtype level; the strong positive control by pk almost cancels out the negative control by ldh on formate production. the analysis suggests that co-regulation of pfk and pk provides a very efficient way to regulate glycolysis, and co-regulating pk and ldh allows the cells to maintain homolactic fermentation during regulation of glycolysis around wildtype level. bovine chymosin is used extensively in cheese production because of its specificity and low proteolytic activity. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. we isolated and characterized the prochymosin cdna from the abomasum of milk-fed kid goats. this cdna predicts a polypeptide of 381 amino acid residues, with a signal peptide and a proenzyme region of 16 and 42 amino acids, respectively. the caprine preprochymosin has 99% and 94% identity with the corresponding lamb and calf sequences. the cdna fragment encoding prochymosin was fused in frame to the killer toxin signal sequence in a constitutive vector, and to the ␣-factor signal sequence-flag in an inducible expression vector. kluyveromyces lactis pm3-5c, k. lactis sel1, characterized by a "supersecreting" phenotype, and saccharomyces cerevisiae bj3505 were transformed with the recombinant plasmids. activated culture supernatants of yeast transformants showed milk-clotting activity. the flag-prochymosin fusion was purified from bj3505 culture supernatants by affinity chromatography. after activation at acid ph, proteolytic activity assayed toward casein fractions showed that the recombinant caprine chymosin specifically hydrolyzed -casein. the recombinant caprine enzyme could be an alternative milk coagulant in cheese making. lipid accumulation in schizochytrium g13/2s was studied under batch and continuous culture. different glucose and glutamate source concentrations were supplemented in a defined medium. during batch cultivation, lipid accumulation occurred towards the end of the growth phase but ceased when cell proliferation stopped. under continuous culture, as dilution rate decreased from 0.08 to 0.02 h −1 , both cell dry weight and total fatty acid content (tfa) of the cell increased. with a constant dilution rate of 0.04 h −1 , nitrogen limitation induced lipid synthesis (28% tfa) as described for other lipid-accumulating organisms. however, with carbon-limited conditions, some lipid accumulation was still possible, the tfa being 22%. finally, the batch and continuous culture methods are compared for docosahexaenoic acid (22:6, n − 6) production. the objectives of this study is to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against e. coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. nitrite-oxidizing bacteria catalyze an essential step of nitrogen elimination in biological wastewater treatment. recently, novel and yet uncultured nitrite-oxidizing nitrospira-like bacteria were found to be abundant in municipal and industrial wastewater treatment systems where they outcompete nitrobacter, which has long been considered as the organism responsible for nitrite oxidation in bioreactors. despite the importance of nitrospira-like bacteria for wastewater treatment and for nitrogen fluxes in natural ecosystems, little is known about their ecophysiology and interactions with other organisms. cultivation-independent molecular techniques were applied to investigate the diversity, distribution, and physiological and genetic features of nitrospira-like bacteria in nitrifying activated sludge and biofilm. a surprisingly high diversity of these organisms was found to exist in these engineered and in natural habitats. moreover, significant physiological differences could be identified among various phylogenetic sublineages in the genus nitrospira. quantitative co-localization analyses performed by novel image analysis software revealed that these metabolic features are reflected by the spatial organization of nitrifiers living in biofilm and activated sludge flocs. based on an environmental genomics approach the genome of a nitrospira-like bacterium found in activated sludge is being analyzed. results obtained so far point at unexpected physiological capabilities of this organism, and allow us to propose that nitrospira-like bacteria may also play roles in the bioremediation of (per)chlorate and chlorite. the activated sludge process is the most common way to remove organic matter, nitrogen and phosphorus from wastewater by microbiologically means. knowledge about the microorganisms involved is fundamental for optimisation of existing plants and development of new plants and process designs. many of the bacteria believed to be involved in nitrification, denitrification, biological phosphorusremoval, and removal of organic matter in full scale plants are now identified by use of molecular methods. recent developments in experimental approaches have allowed the study of the ecophysiology of these uncultured and potentially important bacteria, thus providing a better understanding of their function in full-scale activated sludge ecosystems. relatively few dominant species in each functional group (e.g. denitrifiers and polyphosphate accumulating organisms) seems to be present. some species appear to be very specialized regarding nutrient requirements while others are more versatile. a new method for mercury remediation from industrial wastewater based on the enzymatic reduction of mercury by live mercury resistant bacteria immobilized on the pumice particles has been developed in gbf, germany, and implemented in the industrial scale (unknown). the experience gained during operation of this instalation led to the idea, that the process of bioremediation may be integrated in one bioreactor with the sorption of mercury from wastewater, by immobilization of the bacteria directly on the activated carbon. for this it was necessary to define several significant parameters of the activated carbon used and the sorption process itself. the paper presents results of the equilibrium and kinetics investigations of the process of mercury sorption from aqueous solutions onto seven different types of activated carbon. the effective diffusion coefficients in the particles were obtained from the transient-state experiments and the sorption isotherms, saturation capacity of the sorbents and its dependence on the temperature and ph were identified. then the hydrodynamic and sorption characteristics of the activated carbon bed in a laboratory-scale fixed-bed bioreactor were investigated in different process conditions (mercury concentration, volumetric flow rate, temperature, ph). the results (effective capacity of the bed, dispersion and diffusion coefficients, mass transfer coefficient) enable implementation of this bioreactor for modified, integrated process of mercury bioremediation from industrial wastewaters. research supported by the grant kbn 4 t09c 013 25. bacterial cr(vi) reductases convert the very mobile toxic cr(vi) to the less toxic and less mobile cr(iii). the ability to reduce cr(vi) was studied on cell extracts of ochrobactrum tritici strain 5bvl1 and microbacterium sp. strain 3a. both microorganisms were isolated from the same sample of chromium-contaminated sludge, taken from a wastewater treatment plant. while in the first case activity was found to be associated with the intracellular soluble extract, in the second case it was a process occurring extracellularly. cr(vi) reduction by the intracellular soluble extracts of strain 5bvl1 required the presence of nadh or nadph as electron-donor, while the extracellular fraction of strain 3a only used nadph. several studies were made on strain 5bvl1 intracellular soluble extracts. a k m of 26.11 m cr(vi) and a v max of 5.75 ± 0.13 nmol cr(vi) min −1 mg −1 protein were estimated from the lineaweaver-burk plot and michaelis-menten non-linear regression. the temperature and the ph optima for cr(vi) reduction were 37.5 • c and 5.0, respectively. hyperthermus butylicus is an anaerobic hyperthermophilic crenarchaeon, isolated from the solfataric sea floor off sáo migel island, azores (zillig et al., 1990) . h. butylicus grows at up to 108 • c (optimally between 95 and 106 • c) at ph 7. it can utilize peptides, polysaccharides, and other substrates, as carbon sources to produce acetate, butyrate, and n-butanol. the capability to produce enzymes (e.g. hydrolases, dna and rna polymerases, etc.) that can tolerate and function at temperatures 20 • c higher than most other thermophilic archaea, renders h. butylicus of particular interest to the biotechnology industry. the complete genome sequence of h. butylicus was determined and it contains 1,667,186 bp on a single circular chromosome. 1695 protein encoding genes were identified which use a high level of uug and gug start codons. many of these were assigned functions on the basis of sequence comparisons. our analyses revealed some unusual metabolic properties in h. butylicus. several sugar transporters were identified, although the set of genes required for glycolysis is incomplete. moreover, genes encoding enzymes converting glucose to trioses are absent and no genes encoding enzymes of the pentose phosphate cycle or the kdpg pathway were detected. the h. butylicus genome encodes many proteases and peptidases although the lon proteases, encoded in all other archaeal genomes, are absent. although it was reported that h. butylicus does not utilize free amino acids in the media, genes for amino acid transporters were identified, and several proteins involved in di-or oligo-peptide transport are encoded. genes encoding signal peptidases are absent. we will summarize gene products of special biotechnological interest. reference zillig et al., 1990. j. bact. 172, 3959-3965. 2 hot genomics: insights in the thermophilic lifestyle of thermus thermophilus from its complete genome holger brüggemann 1,2 , anke henne 1 , gerhard gottschalk 1 : 1 göttingen genomics laboratory, institute of microbiology and genetics, university of göttingen, germany; 2 institut pasteur, unité de génomique des microorganismes pathogènes, paris, france thermus thermophilus is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment. recently completed genome sequences of two strains, hb27 and hb8, provide a solid foundation for investigating many aspects of thermophilic lifestyle; these range from molecular stability determinants to key elements of organismic physiology. in addition, the species has considerable biotechnological potential; many thermostable proteins isolated from members of the genus thermus are indispensable in research and in industrial applications. the closely related genera thermus and deinoccoocus belong to a distinct branch of bacteria called the deinococcus-thermus group. genome comparison of t. thermophilus and d. radiodurans, a mesophilic organism, which exhibits high resistant to radiation, oxidative stress, and desiccation, is of particular interest for the identification and exploration of thermophilic determinants. a large number of orthologs with a high degree of sequence identity are shared between the two species. this opens the opportunity for comparative studies of conformational and chemical thermostability of proteins, as well as for the identification of specific traits for each organism, explaining their unique physiological properties and their intriguing differences in stress tolerance. although strains hb27 and hb8 share a highly conserved chromosome, striking differences can be found between their megaplasmids, which encode a huge proportion of genes not found in the genome of d. radiodurans. possible contributions made by the megaplasmids to a thermophilic lifestyle will be discussed. microorganisms that can live in high temperatures, extreme ph and high salt concentration are called extromophiles. extromophilic microorganisms have extended our knowledge and understanding of fundamental questions such as the origin of life. the ability to grow in extreme conditions and to produce stable proteins makes extremopliles very attractive for the researchers and also for the industry. extremozymes from extremophiles have a great economic potential in many industrial processes, including agricultural, chemical and pharmaceutical applications. concurrent development of protein engineering will increase the application of enzymes from extremophiles in industry. turkey has vast and various ecologi-cal areas, and so it has a broad microbial diversity. based on the extremophilles which defined in the scope of this project, halophilic microorganisms produced industrially important proteins were isolated from ç amaltı saltern area in izmir, turkey. in this work, growth of isolates at different temperature, salinity and ph values were investigated to determine the effects of various growth conditions. eight isolates grow at ph between 6.50 and 8.50 and two isolates at 6.50-7.50. they grow at temperature between 37 and 55 • c and salt concentration between 3% and 25%. the results of some phenotypic characters showed that they are gram (−) and oxidase,ürease, dnase and nitrate reduction are (−), and catalase (+). they used d(+) glucose, maltose, lactose, sucrose, l(+) arabinose, d(+) mannose, glycerol and four of isolates used d(+) xylose as a carbon source. the isolates resistant to erythromycine, ampicilin sulbactam, cefoxitin, penicillin, bacitracin, novabiocin, amikacin and sensitive to ceftazidine, ciprofloxacin, amoxycillin/clavulanic acid, imipenem, chloromphenicol, ceftazidime/clavulanic acid, aztreonam, cefepime, cefotaxime, cefoperazone amoxicillin. this project was supported by tubitak through project tbag 2321-103t069. the technology of producing renewable energy sources such as ethanol, methane and hydrogen from biomass holds the potential of creating in-house energy resources while lowering the emission of greenhouse gasses as demanded by the kyoto protocol. recently, goals were defined for the european union determining that 5.75% of the transportation fuel has to come from biofuels in year 2010. a large-scale implementation of biofuels into the transportation sector will demand that lignocellulosic biomass, which is found in a surplus throughout the world is used as the raw material for the production process. the presentation will include a comprehensive description of the special bio-refinery concept developed in denmark for production of biofuels and other valuable products from straw. the concept includes several innovative steps such as a pre-treatment method using wet oxidation, on-site production of enzymes and a continuous fermentation process using a genetic modified thermophilic bacterium. by co-producing several biofuels in the plant optimal use of the biomass has been assured and the price of for instance of bioethanol is getting close to conventional oil-based fuels. optimizing each step in the bio-refinery, while having the full integration in mind, will be the way to make an economical viable biofuel production. in the presentation we will present our road map for achieving this goal in the nearest future. replacement of gasoline by liquid fuels produced from renewable sources is a high-priority goal in many countries worldwide. one such fuel, which has been found well suited, is ethanol. it may be produced from various lignocellulosic materials, such as forest and agricultural residues, which are fairly inexpensive. to compete with gasoline the production cost must be substantially lowered. ethanol production from lignocellulose comprises the following main steps: hydrolysis of hemicellulose, hydrolysis of cellulose, fermentation, separation of lignin, recovery and concentration of ethanol and wastewater handling. the enzymatic hydrolysis and fermentation can either be run separately (shf) or combined into a simultaneous saccharification and fermentation (ssf). the latter has been shown to result in higher ethanol yields than shf. some of the most important factors to reduce the cost are: efficient utilisation of the raw material by high ethanol yields, high productivity, high ethanol concentration in the feed to distillation and process integration in order to reduce capital cost and energy demand. in the last years we have performed several studies on the hydrolysis and fermentation of various forest and agricultural residues in a mini-pilot to improve the overall yield of ethanol and to reduce the energy demand and production cost. steam pretreatment, with small addition of acid catalyst, has resulted in sugar yields close to 90% of the theoretical for various types of raw materials, e.g. spruce, salix and corn stover. the ssf has been developed and optimized to give high yield of ethanol. for spruce an ethanol yield of about 80% of theoretical based on the composition of the raw material has so far been obtained using a two-stage steam-pretreatment of so 2 impregnated raw material followed by ssf. improvements of the ssf step, in the form of high dry matter content, recirculation of process streams and adapted yeast have resulted in ethanol concentrations around 45 g/l leading to substantial reduction in energy demand and production cost. these improvements have been assessed by techno-economic evaluation to determine the effect on the ethanol production cost. the process has been further optimised by process integration to further reduce the energy demand. the ethanol production cost was estimated to be around 0.38-0.46 euro/l ethanol assuming a yearly capacity of 200 000 tonnes raw material (dry matter). production of bioethanol from spent grain, a by-product of beer production sho shindo, tadanori tachibana, akita research institute of food and brewing, akita-city, akita 010-1623, japan. e-mail: shindo@arif.pref.akita.jp (s. shindo) the breweries generate one million tons of spent grain every year, and about 20% of the spent grain is recycled in japan. therefore, it is environmentally and economically significant to consider the production of ethyl alcohol as biomass energy using the spent grain from the breweries industry. ethyl alcohol production from spent grain with immobilized yeast cells was investigated. spent grains were liquefied by a steam explosion treatment to obtain liquefied sugar. when 1 kg of wet spent grain was treated under the 30 kg/cm 2 pressure for 1 min using a 5 l steam explosion reactor, 60 g of total sugar was obtained from the liquefied spent grain. furthermore, 1.3% (w/v) of glucose, 0.4% (w/v) of xylose, and 0.1% (w/v) of arabinose were produced when the liquefied spent grain was treated with glucoamylase, cellulase, and hemicellulase enzymes. ethyl alcohol production was carried out by immobilized sacchromyces cereviseae and immobilized yamadazyma stipitis simultaneously from liquefied spent grain. both yeast cells were immobilized on the glass beads carrier. xylose and arabinose were consumed after glucose was consumed completely during ethyl alcohol production. 5.8% (v/v) ethyl alcohol was produced from liquefied spent grain that was adjusted 17% of initial sugar concentration after 2 days. the vegetable oils constitute a resource of renewable potential for the production of fuels, becoming a viable alternative when compared to the diesel from petroleum. among the vegetable oils, the extracted oil of the castor plant seeds is a promising alternative source because it is constituted mainly of the ricinoleic acid (12-hydroxy-9octadecenoic) that represents 90% of the total constitution of the oil approximately. the biodiesel obtained from castor oil can be defined chemically as being a mixture of methyl esters or ethyl esters of carboxylic acids synthesized by transesterification reaction of the existent triglyceride and an alcohol of little chain through the use of alkaline or enzymatic catalysts. in this work, we described the results found in castor oil with different degrees of purity. initially, it was made a rheological characterization followed by structural characterization (rmn 13c, rmn 1h and infrared) and thermal characterization (dtg, dta and dsc) of the crude and refined castor oil. it has been also measured the hydroxyl tenor, acidity index, saponification index and iodine index in different oils. later, these results were used to evaluate possible differences in the quality of the biodiesel (ethyl esters) produced in the enzymatic alcoholysis of the castor oil catalyzed by lipases (novozym 435, liposyme rm im and lipozyme tl im). the degree of substitution of castor oil derivative was performed by titration with 0.1n hcl and confirmed by tlc analysis and the results showed conversion rates about 90%. has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip3) and inositol tetrakisphosphate (ip4) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip6 is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia1 was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia1 converted ip6 into ip5 (myoinositol 1,2,3,5,6-pentakisphosphates) and another isomer, which is yet to be elucidated. in a denitrifying pilot plant reactor, a new obligately anaerobic ammonium oxidation (anammox) process with great potential for nitrogen removal for high strength wastewater was discovered. after transfer of the complex microbial community to a laboratory sbr system, a highly enriched population, dominated by a single anaerobic chemolithoautotrophic bacterium related to the planctomycetes was obtained. the bacterium was purified via percoll centrifugation and characterized as 'candidatus brocadia anammoxidans'. survey of different wastewater treatment plants using anammox specific 16s rrna gene primers and anammox specific oligonucleotide probes revealed the presence of at least four other anammox bacteria, tentatively named 'candidatus kuenenia stuttgartiensis', 'candidatus brocadia fulgida', 'candidatus scalindua wagneri' and 'candidatus scalindua brodae'. a close relative of the two scalindua species, 'candidatus scalindua sorokinii' was found to be responsible for about 50% of the nitrogen conversion in the anoxic zone of the black sea and in the benguela upwelling system along the namibian coast, making anammox an important player in the global nitrogen cycle. electron microscopic studies of all five anammox bacteria showed that several prokaryotic membrane-bounded compartments are present inside the cytoplasm, which are surrounded by unique ladderane lipids. hydroxylamine oxidoreductase, a key anammox enzyme, was present exclusively inside one of these compartments, named the 'anammoxosome'. unique peptides fragments of the purified hao were used to locate the hao gene in genome assembly of 'candidatus kuenenia stuttgartiensis'. the implementation of the anammox process in the treatment of wastewater with high ammonium concentrations was started at the treatment plant in rotterdam, the netherlands, where it is combined with the partial nitrification process sharon. the estimated price for nitrogen removal with partial nitrification and anammox is about 0.75 euro/kg n. gas lift reactors could sustain the highest anammox capacity at 8.9 kg n removed/m 3 reactor per day. an alternative configuration of anammox is the oxygen-limited canon process in which aerobic ammonium-oxidizing bacteria protect anammox bacteria from oxygen and produce the necessary nitrite. maximum nitrogen removal with canon in gas lift reactors was 1.5 kg n/m 3 reactor per day. using several different conditions and parameters, the competition and co-existence of aerobic and anaerobic ammonium-oxidizing bacteria were modeled. in addition to ammonia, urea was also converted after a 2-week adaptation in the canon system. recently it was shown that anammox bacteria can use organic acids as additional energy source. murray moo-young, wa anderson department of chemical engineering, university of waterloo, waterloo, ont., canada n2l 3g1 bioreactors are central to the bioremediation of contaminated environments of water, air or soil. in all three areas of application, bioreactor design is critical to the development of new or improved processes. this overview focuses on the physical limitations of bioreactors caused by biological requirements. the information is based on our own research findings. the need for more applicationsoriented bioremediation research becomes apparent. for technoeconomic reasons, the airlift type has often been the bioreactor of choice for most bioremediations. however, lack of adequate understanding of the quantitative effects of operating conditions on its performance has been an ongoing concern. these effects have been characterized for engineering implementation. to enhance productivity, innovative pretreatment techniques of the polluted sources have also been developed using photocatalytic and chemical oxidation methods. case studies on petrochemical-contaminated water and soil reveal significant enhancement potentials. other studies on microbial biofilters for air bioremediation indicate that the active mass of the biological consortia is not sufficiently understood for rational design. analysis and retrofit design of wastewater treatment facilities using process simulation tools demetri petrides, alexandros koulouris, intelligen, inc., scotch plains, nj 07076, usa. email: dpetrides@intelligen.com (d. petrides) process simulators have been used in the petroleum and chemical industries for over four decades to facilitate the design of new processes and optimize the performance of existing ones. similar benefits can be derived from the use of such tools in the environmental arena, particularly in the field of physical and biological treatment of municipal and industrial wastewater. specifically, process simulators can be used to evaluate and improve options for: (1) more efficient removal of nutrients (e.g., organic nitrogen and phosphorous) that cause eutrofication, (2) estimation and control of volatile organic compound (voc) emissions from open tanks, and (3) more efficient removal and control of hazardous compounds. the potential benefits will be illustrated with cases studies involving both municipal and industrial wastewater facilities. the microbial reduction of metals has showed recent interest as these transformations can play crucial roles in the cycling of both inorganic and organic species in a range of environments and, if harnessed, may offer the basis for a wide range of innovative biotechnological processes. under certain conditions, however, microbial metal reduction can also mobilise toxic metals with potentially calamitous effects on human health. some effluents present heavy metals as soluble compounds, several microorganisms have the capacity to precipitate these metals as insoluble compounds, and this fact allows the collection and separation of these metallic precipitates from contaminated medium. sulfate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulfate as an electron acceptor and generate hydrogen sulfide. hydrogen sulfide reacts with heavy metal ions to form insoluble metal sulfides that can be easily separated from a solution. the purpose of this work was study the capacity of desulfovibrio sp. cultures to reduce mixtures of the heavy metals in presence or not of petroleum. for it the experimental design 2 k (k = 5) was carried out. the five studied factors were cr, cu, mn, zn and petroleum. the study was carry out with desulfovibrio sp. batch studies were performed in 50 ml sealed bottles with different concentrations (cr(iii)-10 ppm, cu(ii)-5 ppm, mn(ii)-10 ppm, zn(ii)-15 ppm) of metal sulfate and 2 g l −1 of petroleum. during batch incubation the dissolved concentration of metal studied in supernatant were decreased to undetectable levels for zn (70-100%), however with cu (40-60%), mn (40-70%) and cr (50-80%). the development of continuous process with sulfatereducing bacteria seems to be a suitable alternative to reduce metals in solution from contaminated media such as industrial or mine effluents. after these preliminary results, some experiments in course are focused to study that purpose. reduction of odour emissions from livestock buildings using a bioscrubbing system morten øgendahl, nawaf abu-khalaf, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, dk-5230 odense m, denmark. e-mail: tvede@bmb.sdu.dk (m. øgendahl) a bioscrubbing system for reducing odour emissions from livestock buildings is presented. the bioscrubbing system consists of two separate units; an absorption column and a water purification module. the absorption column is mounted in the ventilations stacks in the livestock buildings absorbing odorants in the effluent air flow. the odorants are absorbed in a spray of droplets formed by a grid of high pressure nozzles in the inlet of the absorption column. the spray of droplets is extracted from the air flow and pumped to a centrally located water purification module, an inverse three phase fluidised bed bioreactor, where the bio-degradation of the absorbed odorants occurs. the bioreactor features a split sparging system for maximum mixing and aeration. the cleaned water is recirculated to the absorption column. an electronic tongue will quantify key odorants in the bioreactor. the absorption column is designed to be retrofitted into existing livestock building ventilation systems. the water purification module is constructed in standard size units simplifying scaling to match the requirements of individual applications. the total bioreactor volume is increased by increasing the number of standard bioreactors. this work describes a "light off" toxicity bioassay sensor based on whole cell genetically modified bioluminescent bacteria. the biosensor was constructed by mating between the environmentally isolated phenol-degrading acinetobacter sp. strain df4 and the plasmid putk2 that is an inc p␤ plasmid with the bioluminescence genes luxcdabe inserted into a genetic region involved in plasmid replication and transfer. subsequently, the bioreporter designated df4/putk2 and used to investigate phenolics toxicity. among examined phenolics, pentachlorophenol, catechol and nitrophenol recorded the fastest effect on the bioluminescence of bioreporter df4/putk2 over incubation period of 350 min. the effect of various concentrations of phenol and its derivatives either in an individual, duple or triple mixture forms on the bioluminescence response of the constructed bioreporter df4/putk2 were also examined. significant reduction of the bioluminescence was observed whenever a mixture contained pentachlorophenol, catechol and nitrophenol, respectively. to develop a system appropriate to commercialize, the constructed bioreporter df4/putk2 was subjected for immobilization in microtiter plates using several entrapment gels. after a selection of materials was tried, lb/agar was chosen as the most suitable candidate material. characterization of key odour compounds in an air wet scrubber is presented. the key odour compounds represent five chemical groups, i.e. sulphide, alcohol, volatile fatty acids (vfas), phenol and indole. direct aqueous injection (dai) and solid phase extraction (spe) methods were used before injection of key odorants into the gas chromatography-flame ionisation detection (gc-fid). the dai and spe methods were efficient in the identification of odour compounds in the wet scrubber. the spe method had a high recovery and can be more effective in the identification of compounds at low initial concentration. however, dai showed a better linearity and a lower limit of detection (lod) than the spe method. the dai method was the method of choice for characterization, as it is cheaper, easier to handle and highly applicable. at least two odorants, phenol and 1-butanol, were quantified successfully using the dai method. their lod was less than their odour detection limit in the wet scrubber. dai method can be used as a reference measurement method for any further analytical application, e.g. electronic tongue. recent developments in biotechnology enabled the widespread use of microbial enzymes in textile, detergent, food and dairy industries and also in various environmental applications. microorganisms which live at extremes of temperature, ph and salinity, produce extremozymes that offer many exciting opportunities for their use in clean production. in this study, microorganisms were isolated from camaltı saltern area ini̇zmir, turkey. effect of medium salinity on the growth of these microorganisms was determined. seven out of 10 isolates required salt for growth. the salinity ranges at which growth was detected were: 5-25% for two isolates, 6-25% for one isolate, 7-25% for two isolates and 8-25% for one isolate. the isolates were also screened for their capability of producing industrially important enzymes such as amylase, protease, lipase, xylanase and cellulose which are widely used not only in textile, detergent, food and dairy industries but also in various environmental applications. all of the isolates were found to be producers of both amylase and xylanase enzymes at varying salinity array within 5-30% salt concentration range at ph 7.0. extracellular protease activity was detected in the medium of all isolates grown at 5, 10, 15, 20 and 25% salinity at both ph 7.0 (optimum growth ph) and ph 9.0. out of 10 isolates, 9, 10 and 9 were found to produce cellulase enzyme when the salt concentrations were 5, 10 and 15%, respectively. at 20% salt concentration, only one isolate was found to be cellulase enzyme producer. none of the isolates were found to produce lipase enzyme at 5-30% salt concentration range. this project was supported by tubitak through project tbag 2321-103t069. chemical engineering department, middle east technical university, ankara 06531, turkey. e-mail: ubakir@metu.edu.tr (u. bakir) glass and ceramic tiles are very widely used industrial materials. in most cases, periodical cleaning is required to maintain their optical properties such as transparency and visual aspects. because of the ever-growing demand for healthy living, there is a keen interest in materials capable of killing harmful microorganisms. the application of these tiles in care facilities to reduce the spread of infections, in public and residental places to improve hygienic conditions are of general interest. in this study the aim is developing methods to apply thin film coatings on glass tiles to make them anti-bacterial by utilizing photocatalysis and investigating their anti-bacterial properties. semiconductors because of their reasonable band gap energies find great attraction through this purpose. the photocatalytic property of semiconductors are used in this process. oxidising radicals are formed on the coated surfaces and these radicals attacks the organic pollutants and bacteria on contact with the surface. titanium dioxide (tio 2 ) coated surfaces are considered to be very effective against organic and inorganic materials, as well as against bacteria. in the experimental procedure coating solution is prepared by sol-gel technique. after pretreatment of surfaces, the coating solution is applied on the surfaces by dip-coating method. after appropriate thermal treatments, to achieve thin, dense and strong coatings, indicator microorganism is directly applied on the coated surfaces and illuminated under solar simulater light source. finally, the number of surviving microorganisms are determined. in this study, the effects of titanium dioxide (tio 2 ), tin oxide (sno 2 ) solutions and metal doping to these coating solutions on anti-bacterial function were investigated. as a result of this study, the number of escherichia coli that is used as indicator microorganism, on tio 2 and sno 2 coated glasses with respect to the control glass reduced by 80-85% and 40-45%, respectively. doping with metals increased the activity of the coatings, hence the number of surviving microorganisms decreased. activity of a methanogenic ecosystem during the primary contact with a solid support s. michaud, n. bernet, p. buffière, j.p. delgenès inra-lbe, avenue des etangs, f-11100 narbonne, france in this paper, the biological activity during the first initial contact between a methanogenic sludge and a solid support was investigated in batch experiments, at different solid concentrations, using two different granular solid materials and with glucose as the main organic substrate. in all cases, the introduction of a solid material in a methanogenic suspended biomass induced a response of the anaerobic microorganisms, after a lag phase during which biological activity was not detected. this lag phase could be the consequence of a physical stress induced by the first contact between microbial cells and the solid surface. this lag phase was not observed when the biomass used originated from a biofilm reactor, i.e. using a biomass previously exposed to a solid material. a change in the metabolism of organic matter from catabolism and methane production toward production of other compounds could be observed, characterised by a sharp decrease of the methane yield in the anaerobic system. analyses of the gas and liquid phases did not show the production of any new gaseous or soluble compound as the biological end product of this activity. this suggests the production of non-soluble compounds by an anabolic pathway, which could indicate the initiation of biofilm formation. this metabolic activity was shown to be directly correlated to the ratio between the solid surface introduced and the microorganism concentration in the anaerobic culture (m 2 g vs −1 ). from kinetic observations, it could be observed that acetogenic methanogenesis recovered more rapidly than syntrophic propionate and butyrate degradation. evaluating microbial diversity of hydrocarbon degrading bacteria cleantis braithwaite, howard rosser, tawfiq al-ibrahim, hussain, al-bandi research and development center, saudi aramco, dhahran, saudi arabia the analysis of microbial diversity with molecular methods is central to isolating and identifying new and potential biocatalysts resources for research and industry. the ability to degrade hydrocarbon components of petroleum is widespread among bacteria, and is an effective method for remediation of a variety of ecosystems. due to the high carbon content of oil and the low levels of other nutrients essential for microbial growth, treatment of oil with phosphorus and nitrogen is generally required to enhance the growth of hydrocarbon-degrading bacteria and to stimulate oil sludge degradation. in this research study, three types of oily sludges from a gas plant, refinery, and terminal facilities were treated with nutrients. to assess the microbial diversity, both biolog culture method and culture independent polymerase chain reaction (pcr,) denaturing gradient gel electrophoresis (dgge) methods were used. nutrient addition significantly improved oil sludge degradation. we identified and characterized several hydrocarbon degrading bacterial strains that have the ability to convert petroleum. these bacteria included representatives both gram positive and gram-negative genera. there were slight difference in the quantity and type of hydrocarbon degrading bacteria found in the three sites. this is the first molecular analysis of hydrocarbon degrading microbial population in saudi arabian operations. mussel adhesive proteins, including the 20-plus variants of foot protein type 3 (fp-3), have been suggested as potential environmentally friendly adhesives for use in aqueous conditions and in medicine. here we report the novel production of a recombinant mytilus galloprovincialis foot protein type 3 variant a (mgfp-3a) fused with a hexahistidine affinity ligand in escherichia coli, and its ∼99% purification with affinity chromatography. recombinant mgfp-3a showed a superior purification yield and better apparent solubility compared to those of the previously reported recombinant m. galloprovincialis foot protein type 5 (mgfp-5). the adsorption abilities and adhesion forces of purified recombinant mgfp-3a were compared with those of cell-tak (a commercial mussel extract adhesive) and mgfp-5 using qcm analysis and modified afm, respectively. these assays showed that the adhesive ability of recombinant mgfp-3a was comparable to that of cell-tak but lower than that of recombinant mgfp-5. collectively, these results indicate that recombinant mgfp-3a may be useful as a commercial bioadhesive or an adhesive ingredient in medical or underwater environments. cresol, a monomethylated phenol, is an aromatic compound. the environmental protection agency (epa) has determined the carcinogenic potential of cresol. various options are being examined for the degradation of cresol because of their unavoidable large scale production and toxicity. many aromatic hydrocarbons can be used as electron donors aerobically by species of pseudomonas, thus leading to the ring cleavage of these compounds. in the present study, pseudomonas strains were isolated from activated sludge collected from sewage treatment plant. it was repeatedly transferred onto nutrient agar plate to check the purity of the culture. the organism was grown aerobically in an inorganic medium with p-cresol as the solitary carbon source. pseudomonas was confirmed by the expression of green pigment, gram staining and biochemical tests including koh, catalase, nitrate reduction and carbohydrate fermentation reaction. inoculation status was used to determine the rate of degradation of p-cresol. the effect of temperature on p-cresol degradation was studied. moreover, the effect of different concentrations of the aromatic compounds on pseudomonas as well as substrate variability was also documented. phenolic intermediates were estimated colorimetrically using 4-aminoantipyrene, folin-lowry method, uv spectrophotometry and hplc. the results indicated that pseudomonas could degrade up to 300 mg/l of p-cresol within 10 h. pseudomonas sp. exhibited good metabolic versatility and degraded other aromatic compounds including m-cresol and p-hydroxybenzoic acid. we conclude that this strain of pseudomonas has excellent potential for bioaugmenting the degradation of p-cresol-containing waste water treatment units. a considerable amount of waste cooking oil is produced by the restaurant industry worldwide. this poses a significant environmental and economic problem, since high oil and grease concentrations in the sewage system could lead to pipes occlusion and decreased efficiency in water treatment operation plants. therefore, sending these wastes to recycling companies or hazardous waste processors is usually required. yarrowia lipolytica, a well-known lipase producer, requires the presence of lipidic compounds (i.e. vegetable oils) to boost enzyme biosynthesis. in this work, the suitability of waste cooking oil as lipase inducer in submerged cultures of this yeast has been assessed. if successful, this procedure could allow both the degradation of an abundant waste and its valorisation as a raw material for the production of a high added value product. the microorganism was grown in a liquid medium to which various amounts of waste cooking oil were added. biodegradation degrees up to 80% (measured as decrease in cod) were obtained after 3 days of treatment. also, initial glucose concentration in the basal medium seemed to influence the efficiency of the process. on the other hand, addition of waste oil led to a significant increase in lipase production (more than two-fold), compared to oil-free cultures. moreover, chain-length specificity of the produced enzymes was significantly different: high activity towards medium chain length esters was found, which hinted to the occurrence of both lipases and esterases. biodesulfurization: a documental review j. ferrer, simon bolivar university, environmental engineering lab. caracas, venezuela a documental review about larger interest aspects in biodesulfurization technique is showed. especifically, the investigation is related to general framework and the justification of this technique, degradatives pathways elucidated up to now, involved microorganism, important elements in development of bacterial desulfurization and progress areas, and future tendency. in situ bioremediation of a p-nitrophenol contaminated site and assessment of its community structure debarati paul, gunjan pandey, sumeet labana, rakesh k. jain institute of microbial technology, sector 39a, chandigarh 160036, india. e-mail: rkj@imtech.res.in (r.k. jain) biodegradation of p-nitrophenol (pnp), a priority pollutant, was studied as a model system for bioremediation of sites contaminated with nitroaromatic/organic compounds. bioremediation studies were carried out in pnp-spiked soil in small plots under natural field conditions using arthrobacter protophormiae rkj100. role of carrier material was examined by immobilizing the bacteria on corncob powder prior to adding them to soil. these studies demonstrated successful removal of pnp by immobilized cells that were able to deplete pnp completely in 5 days, whereas free cells were able to deplete 75% pnp in the same time period. monitoring the fate of released bacteria revealed fairly stable population of the cells when they were immobilized on corncob powder throughout the period of study. on the other hand, there was a decrease of 2.7 log units in colony forming units of free cells at the end of the study (30 days). bacterial community structure and diversity was also studied for the pesticidecontaminated site wherein the effect of addition of an exogenous strain on the existing soil community structure and on soil functionality was determined using molecular techniques. as revealed by restriction fragment length polymorphism (rflp) studies 45 different phylotypes could be identified on the basis of similar banding patterns. sequencing of representative clones of each phylopyte showed that the community structure of the pesticide-contaminated soil mainly constituted of proteobacteria and actinomycetes. terminal fragment length polymorphism (t-rflp) analysis showed only subtle changes in community structure during the process of bioremediation. bacteriocins encompass an array of structurally different molecules produced by a number of phylogenetically distinct bacterial groups and trigger the killing of the same or closely related species. the recombined escherichia coli strain harboring a bacterocin coding region of xanthomonas campestris pv glycines 8ra was disrupted to obtain cell homogenate. peptidic xanthomonas bacteriocins (pxb) were separated by lowering ph and adding salt. the resulting pxb's were partially purified using ion exchangers, gel filtration. two final active fractions, a and b, were obtained with a yield of 0.005% and 500-1000-fold purification. the activity of pxb was stable at the ph ranging from 7.0 to 10.0. andreja kresal, vanja kokol, vera golob textile department, university of maribor, 2000, slovenia wastewater from textile dyeing industries is characterized by high chemical and biological oxygen demands (cod and bod) and intense color due to the extensive use of synthetic dyes. as dyes of complex aromatic structures are resistant to removal by the typical microbial population and may be toxic to the microorganisms present in the treatment plants, discharge of the wastewater to the treatment plants may lead to its failure. beside, direct discharge of these effluents into municipal wastewater plants and/or environment may cause the formation of toxic carcinogenic and/or unhealthy breakdown products. different chemical and physical methods (adsorption, coagulation-flocculation, oxidation, filtration and electrochemical treatments) for color removal have been proposed, but due capital costs and slow operating speed as well as huge amounts of sludge creation there is still a great need to develop an economic and effective method. the use of lignin degrading white-rot fungi and their enzymes (laccase, lignin peroxidase, manganese peroxidase) has attracted increasing scientific attention due their ability to oxidative degrade a wide range of recalcitrant organic compounds. in the contribution, the decolorization efficacy of different commercial textile reactive dyes (anthraquinone, azo, triphenylmethane) will be investigated after the treatment by laccase from trametes versicolor. in order to examine the reuse of enzymatically decolorized liquors, the ecological suitability and the toxicity of the degradation products after different time of enzyme exposure will be studied. this work was carried out within the scope of research project e! 3100 cawab. influence of heavy metals on growth and extracellular enzyme production of a trichoderma harzianum strain with biocontrol potential l. hatvani 1 , l. kredics 2 , a. szekeres 1 , z. antal 2 , l. manczinger 1 , a. nagy 3 , c. vágvölgyi 1 : 1 department of microbiology, university of szeged, p.o. box 533, h-6701 szeged, hungary; 2 hungarian academy of sciences, university of szeged, microbiological research group, hungary; 3 pilze-nagy ltd. kecskemét, p.o. box 407, hungary. e-mail: kredics@bio.u-szeged.hu (l. kredics) trichoderma species are common soil inhabiting asexual filamentous fungi with teleomorphs belonging to the hypocreales order of the ascomycota division. besides the industrial and clinical importance of the genus, certain strains have been found to cause great losses in mushroom cultivation while other strains are well known to possess high antagonistic activity against several plant pathogenic fungi and therefore used as biocontrol agents. important mechanisms of antagonism include competition and mycoparasitism, which -among others -can be related to the fast growth of trichoderma strains and the production of several extracellular enzymes. the influence of certain, soil-occurring heavy metals on mycelial growth and the secretion of extracellular enzymes involved in competition and mycoparasitism was examined in this study regarding an effective, potential biocontrol isolate of trichoderma harzianum. the metal ions zinc, manganese, copper, iron, lead and mercury were applied at the concentrations of 8, 16, 24, 32, 40, 60 and 80 m, and dry mycelial weight as well as the activities of extracellular ß-glycosidase, cellobiohydrolase, trypsinand chymotrypsin-like protease and n-acetyl-glucosaminidase enzymes were determined. it was found that mercury totally blocked mycelial growth, while other metal ions exerted a much lower influence on growth. the presence of heavy metals did not have a significant effect on the activity of the examined extracellular enzymes with the exception of trypsin-like protease, which showed a four-to six-fold rise in activity in the presence of certain sublethal concentrations of copper. based on these results, our further aim is to develop copper-resistant derivatives by mutagenesis from trichoderma strains with biocontrol potential. since proteases play an important role in mycoparasitism, these strains could be applied within the frames of integrated pest management in combination with copper-containing fungicides, resulting in an enhanced level of crop protection even with reduced amounts of fungicides. this work was supported by grants f037663 of the hungarian scientific research fund and grant omfb-00219/2002 of the hungarian ministry of education. the significance of biocontrol agents (bcas) is that some of them possess good antagonistic abilities against plant pathogenic fungi. a significant number of the most prominent fungi for the purposes of agricultural application belong to the genus trichoderma. in previous studies, in vitro assays on agar plates were reported as the generally used method for the evaluation of antagonistic abilities, as the results of these assays are well transferable to the practical application. the aim of the present study was to develop an accurate, image analysis-based method for the evaluation of the biocontrol characters of bcas. randomly selected trichoderma isolates were tested against fusarium culmorum. in the currently developed method, the areas of the fungal colonies were calculated on petri dishes by measuring the occupied surface of the medium on digital images. the inhibition effect was recorded as the value of biocontrol index (bci), which was calculated from the ratio of the area of the trichoderma colony and the total area occupied by the colonies of trichoderma and the plant pathogen. the proposed method was tested for numerous parameters, and the results revealed that bci proves to be capable for the accurate measurement and scale of the biocontrol abilities of fungal isolates. this work was supported by grants f037663 of the hungarian scientific research fund and grant omfb-00219/2002 of the hungarian ministry of education. the effect of advanced oxidation processes and recirculation on biodegradation of leachates from aerobic landfills liliana krzystek, anna zieleniewska-jastrzębska, stanisław ledakowicz department of bioprocess engineering, technical university of lodz, 90-924 lodz, poland modern landfills are built and operated in a way which allows us to treat them as a special type of bioreactor. simulation of municipal waste biodegradation in lysimeters provides knowledge on basic processes that take place in an aerated landfill. the aim of aeration is to stabilise mainly biodegradable and nitrogen containing components and to reduce methanogenic potential. stabilised leachates from old landfills contain big quantities of refractive carbon compounds that cannot be removed by biological methods. in such case most advantageous is to apply advanced oxidation processes (aops). the objective of this study is an experimental simulation of a landfill aerobic stabilisation and the impact of aops and recirculation of leachate on the reduction of organic load. the performance of the processes was monitored by the reduction in time of basic indices of organic load (bod 5 , cod, toc, vfa, tkn, n-nh 4 + ) and changes in biogas composition. the simulation of aerobic landfill processes was carried out in lysimeters with a fixed bed of household solid waste stabilised during 8 months in anaerobic conditions. leachates taken from the lysimeters were recirculated and subjected to advanced oxidation processes, i.e. ozonation and uv radiation with the addition of h 2 o 2 . experimental studies showed that the aerobic waste stabilisation was a very quick process. during a month the bed was stabilised, reaching a significant reduction of organic load indices. aeration of the lysimeters caused a quick reduction of mainly degradable organic substance (in terms of bod 5 ) and n-nh 4 + and vfa. the reduction of methanogenic potential of the landfill was even faster. the composition of gas at the outlet from the lysimeter changed and after one day already its content was similar to atmospheric air. a more frequent recirculation of leachates enhanced greatly the aerobic biodegradation. it was found that application of advanced oxidation processes (especially ozonation) contributed to a growing reduction of the organic load in the leachates from aerated lysimeters. the application of leachate ozonation resulted in a very high degree of reduction of organic compounds (up to 77%). the objective of the experimental study was to assess the effect of temperature on the extent of aerobic batch biodegradation of potato stillage with a mixed culture of bacteria of the genus bacillus. the experiments were performed at 20, 30, 35, 40, 45, 50, 55, 60, 63 and 65 • c, at ph 7, in five l l working volume stirred tank reactor (str) (biostat ® b, b. braun biotech international). the duration of the process was 120 h. initial cod of the stillage amounted to 51.9 g o 2 /l, the main carbon sources being reducing substances (18.7 g/l), organic acids (determined as their sum) (12.2 g/l) and glycerol (3 g/l). at 65 • c, no cod reduction or biomass increment was found to occur. at the other investigated temperatures, the reduction in cod measured after suspended solids (ss) separation varied from 77.6% (55 • c) to 89.1% (35 • c). without ss separation, cod reduction ranged between 55.6% (20 • c) and 75.1% (35 • c). this indicates that, in terms of the extent of cod reduction, the optimal process temperature was 35 • c and that there was a local optimum at about 63 • c. according to the temperature applied, the content of reducing substances decreased by 84.3-96%, that the organic acids by 91.7-99.6%, and that of glycerol by 91.5-96%. the experiments also produced the following two findings: (1) the rise in temperature brought about a decrease of biomass concentration in the str (measured as ss and bacterial number), and (2) temperature was a factor affecting the demand for ammonia nitrogen (n-nh 4 ), which was the highest at 20 and 60 • c. the high n-nh 4 demand observed both over the higher and lover ranges of the investigated temperature should be attributed to the release of n-nh 4 and to the large amounts of the biomass produced, respectively. the results obtained imply that the extent of potato stillage biodegradation with a mixed bacterial culture was high over a wide range of the investigated temperature. polychlorinated compounds such as tetrachloroethylene (pce) have become serious environmental pollutants. considerable attention has been paid to these organochlorine compounds. this paper describes the molecular analysis of dechlorinating gene in halorespirating bacterium and efficient bioremediation process. an anaerobic bacterium, that dechlorinates pce to tce, was isolated and identified as a species of the genus desulfitobacterium. a novel pce reductive dehalogenase (prda) gene from the desulfitobacterium sp. strain kbc-1 was identified. these prd genes, including membrane anchor protein, were classified as a novel type of pce reductive dehalogenase (approximately 40% homology with the general pce dehalogenase). according to the substrate utility of this strain kbc-1 and phylogenetic analysis of prda, the type of this microorganism may be expected to play the role of a primary degrader of pce in the environment. high efficient bioremediation process so called the restricted aeration system which means microaerobic/aerobic reciprocal bioremediation process was developed. strong modifications take place, as ammonia production with a subsequent rise of the ph value and a rapid heat evolution leading to temperatures of up to 70 • c. little is known about the microbial community in the toscano cigar fermentation and its development as fermentation proceeds. the aim of this study is to investigate the microbial community composition, its dynamic and its influence on the toscano cigar production process. our results show that the fermentation could be divided into three different phases: initially yeasts are the predominant microorganisms while bacterial growth is partially inhibited; the middle phase is characterized by exponential growth of bacteria while yeasts disappear. in the final phase the microorganism population is mostly represented by sporigen microbial species. the occurrence of yeasts in the first phase could be attributed to their ability to grow at low temperature and low ph levels. the bacterial population flourishes after the yeast cells have reached a stationary phase and probably grows on residual nutrients and autolysing yeast cells. yeasts and bacteria involved in the fermentation process were isolated and characterized. the microbial community was investigated by a combination of phenotypic and molecular approaches. the phenotypic characterization was based on both colony and cell morphology. the isolates were then identified by rrna genes sequence analysis. finally, in order to clarify the role of the identified microorganisms in the production process, a preliminary biochemical characterization was carried out. biosensors have undergone rapid development over the last few years; in particular, in environmental field many biosensors using microorganisms and purified enzymes as biological component, were recently studied. benzene is present everywhere with high levels in the cities and sometimes, in petroleum processing plants. it is classified as carcinogenic compound of first class able to cause leukaemia. because the evaluation of benzene requires complex instruments and quite long analysis times, it is required to study alternative systems for benzene detection simple, fast and highly sensitive, such as biosensors. from pseudomonas putida mst, strain previously isolated in our laboratory and able to degrade benzene, we isolated genes encoding for benzene 1,2-dioxygenase and cis-1,2-dihydrodiolbenzene dehydrogenase to use in the development of two different hydrocarbon biosensors based on microorganisms and on purified enzymes. the genes isolated were cloned in pvlt33 and we developed three microbial systems carrying: (1) benzene dioxygenase, (2) dihydrodiol dehydrogenase and (3) benzene dioxygenase-dehydrogenase modified by pcr to obtain enzymes with histidine tag. the cloning was planned to construct recombinant strains able to overproduce the enzymes; the enzymatic activities will be evaluated both using whole cells and purified enzymes. study of operation condition of biofilter using fibril-form matrix for odor gas removal don-hee park, chonnam national university, this research was performed for developing of biological treatment process of odor gas such as mek, h 2 s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over 93% was obtained by biofilm formation. at 400 ppm of inlet odor gas concentration and 10 s of retention time, the removal efficiency was 76% and 93% in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over 97% at the operational conditions above 15 s of retention time. ozonated water is produced using an ozone generator in a container filled with cold water. it is useful for sanitizing the surfaces of various products for which heat or chemical treatment is inappropriate, such as fresh food products. in this study, we investigated the antimicrobial effects of ozonated water and electrolyzed ozonated water against escherichia coli, s. aureus, bacillus subtilis and yeast, saccharomyces cerevisiae for practical use in sanitizing various products. the results demonstrated that the electrolyzed ozone water was effective for the reduction of microbial population at relatively low concentration of ozone. also, the electrolyzed and the ozonated water showed synergistic antimicrobial effects. many xenobiotics can react spontaneously with thiol moieties of glutathione (gsh), forming gsh-conjugates, or via glutathione s-transferases (gst). these enzymes participate in detoxification of potentially harmful compounds from endo or xenobiotic origin. using saccharomyces cerevisiae as experimental model, we observed that cells mutated in the gtt1 or gtt2 genes showed twice as much cadmium absorption than the control strain. we proposed that the formation of the cadmium-glutathione complex is dependent on those transferases, since it was previously demonstrated that the cytoplasmic levels of this complex affect cadmium uptake. the addition of glutathione monoethyl ester (gme), a drug that mimics glutathione (gsh), to gtt1∆ cells restored the levels of metal absorption to those of the control strain. however, with respect to gtt2∆ cells, addition of gme did not alter the capacity of removing cadmium from the medium. taken together, these results suggest that gtt1p and gtt2p play different roles in the mechanism of cadmium detoxification. by analyzing the toxic effects of this metal, we verified that gtt2∆ and gsh1∆ cells showed, respectively higher and lower tolerance to cadmium stress than control cells, suggesting that although gsh plays a relevant role in cell protection, formation of the gsh-cd 2+ conjugate is deleterious to the mechanism of defense. furthermore, analyzing the harmful effects of other xenobiotic, menadione (2-methyl-1,4-napthoquinone), we have also observed that gtt1p and gtt2p isoforms play distinct functions in the process of cell protection as well as in drug remove, since both strains showed lethal phenotypes after direct exposure to 20 mm menadione. however, after adaptive treatments (mild-heat or exposure to a lower menadione concentration), cells acquired tolerance to menadione stress, although the gtt2∆ mutant had still shown a higher sensitivity against drug toxicity. by analyzing the malondialdehyde (mda) produced in response to menadione, we observed that gtt2∆ cells exhibited increased levels of lipid peroxidation, indicating that, during menadione exposure, gsh-conjugates are formed by the same transferase isoform, gtt2p, involved in cadmium stress. financial support: stint (sweden), cnpq and faperj (brazil). polycyclic aromatic hydrocarbons (pahs) are ubiquitous and persistent throughout the environment. they are generally distributed from both natural and industrial sources. many pahs can have a detrimental effect on the flora and fauna of affected habitats through uptake and accumulation in food chains, and in some instances, they induce serious health problems and/or genetic defects in humans. many research efforts have been expended to find a suitable method for remediation of soil and water environments contaminated with pahs. amongst them, the use of ligninolytic fungi is particularly suitable for the development of such processes, since they produce extracellular lignin-degrading enzymes (mnp, lip, laccase, . . .) which degrade a wide range of organic pollutants. coriolopsis rigida has been reported to produce extracellular laccase as the sole ligninolytic enzyme. this makes this fungus particularly suitable for the study of xenobiotics degradation by laccase. the purpose of this research was to obtain high laccase activities by c. rigida in solid state cultures and to determine their ability to degrade anthracene (typical pah). both in vivo and in vitro assays were performed. the former led to 60-80% degradation in 3 days depending on the culture conditions, whereas the latter showed a degradation percentage above 90% in 2 days when low mediator concentration (hbt) was added to the reaction mixture. focus will be given on pressure-driven membrane bioreactors, gastransfer membrane bioreactors and the novel ion exchange membrane bioreactor (iemb). the latter concept has been developed and currently studied by our group. this process, based on integration of donnan dialysis with bioconversion of one or more target pollutants to harmless products, has been modeled and experimentally verified for the removal of various charged inorganic pollutants such as nitrate, perchlorate and bromate by mixed microbial cultures under anoxic conditions. tests of up to 3 months showed a very good operational stability. the essential role of the microbial membraneattached biofilm, which develops naturally in this type of systems, will be also demonstrated and discussed. poly(lactic acid) (pla), which is one of biodegradable plastics, is depolymerized by hydrolysis and releases soluble monomer or oligomer of lactic acid. many bacteria can use the monomer and oligomer as an energy source or a carbon source. in this study, we applied pla to an electron donor for denitrification process of the previously developed bioreactor, which could remove ammonia from wastewater by simultaneously carrying out two biological processes, aerobic nitrification and anaerobic denitrification. a bench-scale bioreactor was constructed with a gel-plate containing pure-cultured cells of nitrosomonas europaea and paracoccus denitrificans and a pla-plate. the pla-plate was prepared by mixing three kinds of plas with different molecular weight and tricalcium phosphate to keep the constant release of the electron donor for a long term. batch treatment experiment with the bioreactor was repeated with an artificial wastewater containing ammonia for 100 days. the bioreactor could remove nitrogen from the artificial wastewater at nitrogenremoval rate of approximately 4 g n/day per square meter of gel-plate surface during the experiment period without an additional electron donor. the performance was equivalent to that obtained with our bioreactor using ethanol as electron donor for denitrification. the bioreactor using pla dose not need an additional pump for serving an electron donor (e.g., ethanol) and a hollow space for serving. therefore, the concept using solid electron donors like a pla would be effective our bioreactor to compact and simplify, and would be possible to develop a portable or disposable bioreactor. leucosporidium antarcticum as a source of enzymes for biotechnology arkadiusz wojtasik 1,2 , marianna turkiewicz 2 , jaroslaw dziadek 1 , pawel parniewski 1 : 1 centre for medical biology pas, 106 lodowa street, 93-232 lodz, poland; 2 faculty of biotechnology and food sciences technical university of lodz, stefanowskiego 4/10 street, 90-924 lodz, poland. e-mail: awojtasik@cbm.pan.pl (a. wojtasik) leucosporidium antarcticum is a psychrophilic yeast able to growth at low temperature. these microorganisms live in antarctic marine waters and are endemic to that cold environment. furthermore, l. antarcticum is also isolated from the digestive tract of antarctic krill euphausia superba. enzymes isolated from coldadapted microorganisms such as l. antarcticum having a specific activity at low temperatures ranging from 0 to 30 • c are considered for utilization at biotechnological applications such as bioremediation, production of polyunsaturated fatty acids of dietary significance and might be a source of industrially useful enzymatic proteins. the main goal of this study was to construct a cdna library of l. antarcticum. the partial cdna library was obtained and some of the clones were analysed. the sequencing analyses allowed us to find an approximately 450 base pair nucleotide sequence which displayed a very high homology to disulfide bond chaperone belonging to the hsp33 family from psychrobacter sp. high similarity of that heat shock protein was found on an amino acid sequence level and was reaching nearly 85%. the main object of our further research is to clone hsp33 family protein gene and to obtain its expression in a mezophilic host strain. also, further clones will be analysed to find other interesting genes encoding the psychrophilic proteins. this work was partially funded by the kbn grant i29/205/05. out of 9000 plant species found in the flora of turkey, about 3000 are endemic. beautiful flowering (geophytes) bulbous plants form an important part of this rich biodiversity. besides use as ornamental plants, these have great potential in perfume and pharmaceutical industry. genera of fritillaria, ornithogalum, muscari, bellevalia, tulipa, galanthus, sternbergia, crocus, arum and biarum have important and critically endangered species with high export potential that enters into this group. most of these are endangered and their collection from wild and export has been banned to conserve them. large scale production and conservation of these species could also be achieved by in vitro techniques. therefore bulb scale and immature embryo explants of sternbergia candida, s. fischeriana, muscari muscarimi, fritillaria imperialis and f. persica were cultured on different nutrient media supplemented with various concentrations of plant growth regulators using different culture applications. large numbers of bulblets were produced (over 100 bulblets/explants) from single immature embryos on nutrient media in most species tested after 12 months of culture initiation. regenerated bulblets were kept at 5 • c for 5 weeks and then transplanted to soil successfully. to our knowledge the present study is the first report for in vitro bulblet production from immature embryos of geophytes. the procedure described here provides a prolific bulblet production system that may form the basis of bioreactor culture and conservation of endemic and endangered geophytes. the commercial use of organofluorine compounds in industrial, pharmaceutical and pest-control applications has dramatically increased over the past few years, resulting in the introduction of numerous new organic compounds into the environment. organofluorine compounds are chemically very stable and are assumed to be resistant to biological degradation. given the chemical inertness of fluorinated organics, their bioactivity, and their potential for accumulation in the environment, it is important to understand their environmental fate and the mechanisms by which they might be degraded. examples of the biodegradation of fluorinated compounds in literature are scarce, being fluorobenzoic acids the most commonly reported. information on the cleavage of carbon-fluoride bonds in synthetic compounds is limited to fluoroacetate dehydrogenase. in this project we try to obtain more insight in the defluorination mechanisms by investigating the diversity of degradation routes for these compounds in several soil bacteria by making use of modern genetic tools. a gram-positive strain capable of aerobic biodegradation of 4-fluorophenol (4-fp) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. batch cultures were set up and substrate consumption, accumulation of intermediates and product formation were monitored. the consortium was able to use 4-fp up to concentrations of 448.4 mg l −1 and was able to utilize a range of other organic compounds. stoichiometric release of fluoride ions was measured in batch cultures suggesting that there is no formation of dead-end products during 4-fp metabolism. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene 1 , nazif kolankaya 2 : 1 kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; 2 hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth (1% soytone, 4% d-glucose and 0.5% cellulose pulp). maximal extracellular ligninase production was detected after 7 days (7 nkat). the optimum biobleaching conditions are 30 • c and ph 4.8, with 10 days. in this condition p. versicolor decreased the kapa number from 38.55 to 19.42 and increased brightness from 28 to 32.7 in 10-day treatment. boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plan-tation areas of turkey. both defficiency and toxicity problems exist in a total of about 50% of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant acquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f9 plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. butachlor is one of the selective systemic herbicide toxins that are act by inhibition of protein synthesis. this toxin is used exclusively in the rice, barely, cotton and wheat farmlands. butachlor is belonging to chloroacetanilide herbicide group, which are consisting of butachlor, alachlor, acetochlor, metolachlor and poropachlor. in the view of bioenvironmental, butachlor is degraded in the soil by microbial activity. its stability is about 6-10 weeks. it is converted to the water-soluble derivatives in soil or water, with a slow evolution of carbon dioxide. because of butachlor is one of the herbicide toxin, it is inhibitor factor against growth of bacteria and microorganisms. microorganisms can be continuing their activities in the limited concentration of butachlor. therefore treatment of industrial wastewater consist of concentrated butachlor by the biological treatment is impossible and it is necessary chemical or physico-chemical treatment are used. in this research, biological treatment methods are used. in the biological treatment, an activated sludge system with volume of 6.5 l is used. in this method, butachlor with concentration of 5 mg/l are treated. removal percent of butachlor for concentration of 2.5 and 3 mg/l are calculated to 41.20% and 41.67%, respectively. removal percent of cod is also calculated to 86%. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized purification and downstream process of xylitol obtained biotechnologically from hemicellulosic hydrolyzate of corncobs b. rivas 1 , p. torre 2 , j.m. domínguez 1 , j.c. parajó 1 , a. converti 2 : 1 department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, 32004 ourense, spain; 2 department of chemical and process engineering, genoa university, via opera pia 15, 16145 genoa, italy. e-mail: brivas@uvigo.es (b. rivas) biotechnological production of xylitol from lignocellulosic materials has been widely studied in the recent years with promising results that confirming the possible industrial application of this technology. xylitol purification from fermented broth is the limiting stage of this process. previous works suggest crystallization procedures in order to recovery xylitol from fermented synthetic solutions. the complexity of fermented hydrolyzate not allows direct crystallization. in this work, corncobs hydrolyzate obtained with autohydrolysis-posthydrolysis techniques, detoxificated with activated charcoal and concentrated was fermented to xylitol by d. hansenii. the fermented broth composition was 64.9% of xylitol (68 g/l), 13.3% of other sugars and 21.8% of other compounds that interferes in the crystallization process (as dry matter of the liquor). the fermented media was submitted to an absorption process with activated charcoal and concentrated until a xylitol concentration of 340 g/l. the liquor was then submitted to a second step of precipitation with ethanol, the best results achieved in this study were obtained with an ethanol/liquor ratio of 4. in these conditions this treatment allows to remove a 56.9% of the impurities. the resulting solution was evaporated and crystallized containing 60% of ethanol and a xylitol concentration of 443 g/l. crystallization was performed at t = 5 • c with slightly agitation. after 36 h were separated xylitol crystals with a recovery yield of 13% and a purity degree of 90%. numerous publications have documented that only a minor number of the indigenous prokaryotic organisms found in complex environments such as the human intestine, biogas reactors, and soil are known, and probably only a fraction of this diversity can be accessed using traditional culturing techniques. some of the reasons for this are the lack of knowledge of specific growth conditions, specific nutrients, and obligate coculture requirements. also growth on a solid surface directly exposed to the atmosphere puts a very strong selective pressure on single cells supposed to develop into visible colonies. therefore, the knowledge of these microorganisms is scarce and generally limited to the 16s rrna genes that have been extracted from different environments and cloned for phylogenetic analyses. an obvious approach to circumvent these problems was the development of techniques based upon micromanipulation for isolation of single cells from complex mixtures. continuous development of modern microscopes in combination with the precision of a servo-powered micromanipulator and the development of the modern microscopic micro injectors used in ivf techniques has further aided the manipulation of single cells. this technique, however, does not solve the problems of the non-culturable cells, and other approaches are needed to gain more information about these organisms. an approach to the non-culturable cells could be genomic analysis of isolated single cells without preceding cultivation. this pcr-based technique is widely used for genetic analysis of human cells, but due to the small amounts of dna present in prokaryotic cells it has so far not been possible to produce identifiable amounts of dna from single cell amplification using conventional polymerases. a promising alternative used for amplification of small amounts of dna is the f29 dna polymerase operating under isothermic conditions. applying random hexamer primers, this polymerase carries out a multiple displacement amplification (mda) of high molecular weight dna template. in this study we demonstrate the successful application of mda for selective amplification of genomic dna from a single prokaryotic cell. the yield was >20 mg of amplified genomic dna corresponding to about a 5 billion-fold amplification from a single cell. the technique was used to approach a large group of non-thermophilic archaea found in agricultural soil. our results show that combining mda with fluorescent in situ hybridization and cell isolation by capillary micromanipulation enables an unprecedented ability to investigate new species without cultivation. also this combination of techniques opens for studies of genetic heterogeneity within populations and processes such as horizontal gene transfer. precipitation of zn 2+ , cu 2+ and pb 2+ at bench scale using biogenic hydrogen sulphide produced from the utilization of volatile fatty acids by sulphate reducing bacteria maria teresa alvarez 1,2 , carla crespo 2 , bo mattiasson 1 : 1 department of biotechnology, center for chemistry and chemical engineering, lund university, p.o. box 124, s-22100 lund, sweden; 2 instituto de investigaciones fármaco bioquímicas, universidad mayor de san andrés, la paz, bolivia biological production of hydrogen sulphide (h 2 s) from sulphate using sulphate reducing bacteria (srb) is popular within environmental biotechnology. srb require absence of oxygen, presence of nutrients required for growth and oxidizable organic substrates (to supply hydrogen atoms for reduction of sulphate). many organic wastes have been used as electron donors for the sulphate-reducers in the treatment of acid mine drainage (amd) including straw, hay, sawdust, peat, spent mushroom compost and whey, however, other wastes such as municipal organic waste can be used. the aim of this work was to study the possibility of using srb for the treatment of amd at bench-scale. this process involved three stages: the volatile fatty acid (vfa) production by hydrolytic bacteria from the degradation of vegetables and fruits, the production of h 2 s through the utilization of the produced vfas by sulphate reducing bacteria and the precipitation of metals by using the biologically produced h 2 s. the substrates used for vfa production consisted of tomato, papaya, apple and banana. the h 2 s produced from the degradation of vfas was utilised for the precipitation of an artificial effluent simulating the heavy metal concentrations of a mine located at bolivian andean region, containing approximately 9 mg/l of zn +2 , 8 mg/l of cu +2 and 4 mg/l of pb +2 . the maximum concentration of hydrogen sulphide obtained was approximately 17 mm. removal efficiencies of 97%, 98% and 100% for zinc, cooper, and lead, respectively, were achieved in the present work. at 30 • c during 14 days. bacterial population was determined by counting in a neubauer chamber with optical microscope. sulfate concentration was measured by turbidity method and metal concentrations in the filtered supernatant were measured by icp-aes. the first part of study consists of determine the maximum concentration of each metal at which d. vulgaris and desulfovibrio sp. grow in similar way than control culture (without metal). both cultures tolerate: cr(iii) 15 ppm, ni(ii) 8.5 ppm, zn(ii) 20 ppm. the maximum precipitation percentages were approximately: 25% (15 ppm cr(iii)), 96% (8.5 ppm ni(ii)) and 99% (for d. vulgaris-10 ppm zn(ii) and desulfovibrio sp.-15 ppm de zn(ii)). time to reach the highest precipitation was minor for mixed culture (desulfovibrio sp.) y all the cases. the next part was focused to study the precipitation percentage when metals are present in combination in the same metal levels (cr(iii)-ni(ii), cr(iii)-zn(ii), ni(ii)-zn(ii) and cr(iii)-ni(ii)-zn(ii)). the combination of metals does not affect significantly the bacterial growth and precipitation percentage of metals. this fact supposes an importance advantage so metals are commonly found together in the environment. future experiments are focused in development of this process in continuous operation mode. biosolubilisation and depolymerisation of coal has potential to produce a clean energy source or high value organic products from low rank coals such as lignite or sub-bituminous coal. these complex soluble phenolic compounds are of value as starting materials for biotransformation to value-added compounds such as antioxidants and flavourants. the bioprocess is carried out at ambient temperature and pressure and is perceived to be environmental benign. in the evaluation of coal solubilisation an important quantity for the assessment of process feasibility is the yield, i.e. the determination of the mass of product obtained per unit mass of coal solubilised. to date, results for coal biosolubilisation reported in the literature are qualitative or at best semi-quantitative, indicating trends with operating variables. the process kinetics has not been determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. in this paper, the use of an indirect method for the estimation of the growth and metabolism of fungal biomass by measuring co 2 evolution and o 2 consumption using an off-gas analyser is reported in the study of fungal coal solubilisation. coal determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. biosolubilisation was carried out in a stirred tank slurry bioreactor with working volume of 1.0 l. complete suspension of the coal particles of 650-800 m mean diameter was achieved at an agitation rate of 560 rpm. growth yield coefficients based on coal and oxygen as well as maintenance coefficients were calculated from growth of the fungus under the same conditions using a non-coal carbon source such as glucose. these data were used to determine the stoichiometric coefficients for biomass growth, enabling the biomass production rate to be quantified in terms of co 2 production rate and o 2 consumption rate. a dna-chip platform for parallel detection of microorganisms related to biofilm in industrial systems and drinking water systems pernille skouboe, dorte lauritsen, kim holmstrøm bioneer a/s, kogle allé 2, dk-2970 hørsholm, denmark. e-mail: psk@bioneer.dk (p. skouboe) an oligonucleotide microarray for simultaneous detection and identification of pathogenic bacteria related to technical water systems as well as drinking water has been developed. the approach is based on the use of a tandem hybridization technique with two ribosomal 16s rdna-pcr products, 1000 bp and 500 bp long, generated from two consensus pcr reactions using conserved ribosomal primers end-labeled with cy3 and cy5, respectively. the tandem hybridization technique implies an internally quality control for discrimination between target and non-target signals. the current prototype of the dna-chip platform includes 20 oligonucleotide probes representing 11 different genera (and subgroups of species), e.g. legionella, mycobacterium, aeromonas, campylobacter, vibrio and enterococcus. the platform has been used for detection and identification of species from pure cultures, and initial experiments with water samples from industrial systems have been performed. the potential as well as the limitations of using a dna-chip based detection format in its present form will be documented. particularly, its potential application as a rapid method for initial screening of environmental or food samples will be addresses. the aim is to reduce and optimize the number of samples required for traditional microbiological identification tests. in the course of a project for the development of a novel kind of a mycotoxin inactivating feed additive, the aim of this study was to isolate and characterize microorganisms with the specific ability to enzymatically break down and detoxify fumonisins, a group of structurally related fungal toxins, with fumonisin b 1 (fb 1 ) being the most abundant and -with respect to toxicology -also the most important representative of this group. these toxins are produced as secondary metabolites by some fusarium species such as fusarium verticillioides and f. proliferatum and are naturally occurring contaminants of cereal grains worldwide. they are found especially in maize and maize based products, and are known to be hazardous to human as well as to animal health. a natural feed additive, based on microorganisms and/or enzymes, should ensure the detoxification of fumonisins during feed uptake and digestion via microbial or enzymatic break down of these compounds, by that protecting the animal from the harmful effects of these mycotoxins. besides an extensive screening of microbial strains derived from strain collections, various different natural habitats were investigated for the presence of fb 1 degrading microbial activity, such as intestinal contents of pigs, soil samples, and naturally fumonisin contaminated maize. while testing of nearly 150 organisms from strain collections did not show positive results, fumonisin transforming activity could be detected in one soil sample and a number of maize samples. trials in order to isolate the respective fumonisin degrading microorganisms resulted in a number of strains, whose fb 1 degrading activity could be proven. the most promising bacterial and yeast strains were further characterized with regard to a general taxonomic description, and to different aspects of their toxin degradation behaviour. approaching a more relevant in vivo situation, fb 1 degradation trials in food-and feed-stuffs were conducted. further on, the applicability of the respective organisms as stabilized lyophilisates was investigated. arsenic is one of the most important global environmental pollutants and the toxicological effects are related to its chemical form and oxidation state. arsenite [as(iii)] is reported to be on average 100 times more toxic than arsenate[as(iv)]. this work shows the ability of one strain of the species ochrobactrum tritici to grow in presence of several metals including arsenite, arsenate, selenite, selenate, tellurite and antimonite. its arsenite mic was determined as 50 mm, whereas for arsenate, this bacterium could resist to concentrations upper than 200 mm. we report the identification of two loci involved in high-level arsenic resistance. sequencing of the first locus identified four complete genes in the following order: arsr, arsd, arsa, arsb. the second locus containing genes for arsenic resistance was also characterized. each sequence has been compared with nucleotide and protein databank (blast programs) and significant homology with known orfs coding for arsenic resistance has been found. it is also possible that the phenomenon of high-level arsenic resistance in o. tritici could evolve other genes or loci. the ability of the ␣-proteobacterium o. tritici to tolerate high levels of arsenic in addition to other oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. this work is based on the mathematical modeling of kinetics of a thermophilic bacteria cultivation system. the cultivation proceeded by way of batch and continuous on the synthetic medium with the main carbon source-lactose. this medium simulated an industrial waste approximately. mixed thermophilic aerobic bacteria popula-tion, applied to the wastewater treatment (sludge v&k bystrice pod hostynem), was used to the inoculation. the cultivation system consisted of the laboratory fermentor biostat b (b. braun biotech) with working volume 2 l and the control unit connected with a computer. it is possible the temperature, ph, aeration, stirring and foaming regulation. physical and chemical cultivation conditions were optimized. the chemical oxygen demand (cod), generally expressive the impurity level, was selected as the main parameter for the cultivations run classification. but into the mathematical model also the kinetics of biomass growth, lactose consumption, production of choice metabolites (acetate, lactate, succinate) and dissolved oxygen concentration was included. the modeling was located to two head distinguishable growth phases of the microorganisms. an optimization and identification of mathematical model parameters was practised in the software language psi/c. the difference between simulated curves and experimental data is not statistically significant on the relevancy level 0.05 (f-test). it took place the cod degradation at 74.0% with the average yield coefficient y chsk/x 3.8 g cod/g biomass in the batch process with the air aeration only. there is a better way to the cod elimination (>90.0%)-the aeration of air enriched by the pure oxygen. in experiments with the continuous system was obtained the 69.0% cod decrease after the steady state stabilization. this work was supported by project msm 0021630501. fluorescence in situ hybridization (fish) of whole cells using oligonucleotide probes was applied to study the influence of low temperature and temperature reduction on the bacterial community of biofilm reactors for the removal of chlorophenols (cps). two packed bed reactors were set up for degradation of a mixture of 2-cp, 4-cp, 2,4-dicp, and 2,4,6-tricp as sole source of carbon and energy at 14 • c (ra) and 24 • c (rb) and were inoculated with bacterial consortia adapted to these respective initial temperatures. the performance of the reactors was studied under different conditions of pollutant loading, aeration rate, and hydraulic retention times over 7 months. total chlorophenol removal capacities of 1240 and 1420 mg l −1 day −1 were achieved in the bioreactors ra and rb, respectively, under a total pollutant load of 1440 mg l −1 day −1 . the population of ␤-proteobacteria was the major bacterial community of the biofilm (35-47%) followed by the ␥-proteobacteria (12-6.5%). two bacteria with the ability to mineralize 50 mg chlorophenols l −1 were isolated from the bioreactors and characterized as ralstonia basilensis and alcaligenes sp., both belonging to ␤-proteobacteria. decreasing the temperature by 10 • c (in two steps of 5 • c each) resulted in an increase in the population of ␥-proteobacteria and a decrease in the population of ␤-proteobacteria in both reactors. application of genus specific probes showed an increase in the pseudomonas population from 25% of the ␥-proteobacteria at 14 • c to 59% at 4 • c. the pollutant removal capacity decreased to 548 and 833 mg l −1 day −1 in ra (4 • c) and rb (14 • c), respectively. the ␣and ␦-proteobacteria, cytophaga-flavobacteria and actinobacteria the survey was carried out in urban and rural areas of two cities (ankara and isparta). this paper is only analysing urban people by excluding villagers. urban sample is consisted of 400 urban consumers and 200 professionals. professionals were selected amongst pharmacists, doctors, agricultural engineering's and industrialists that is thought are affective in the process of developing new technologies and also the development of biotechnology in the society. basic data was gathered by a questionnaire including both structured and open-ended questions besides deep interviewed. workforce development for life sciences-the scottish experience carol booth scottish entreprise, uk the presentation will look at, the background and definition of workforce development for scottish enterprise, examine information available to support scottish enterprises economic intervention and where and when to intervene. conclusions emerging from the evidence base will be used to outline scottish enterprises approach to workforce development and look at which actions might be required to address identified issues. moving on to reasons for integrating workforce development into business development and how the life sciences cluster team at scottish enterprise, stakeholders and partners in scotland have approached their current and future contributions to workforce development for life sciences using a variety of projects. the national institute for bioprocessing research and training (nibrt) in ireland is a proposal that will be a state-of-the-art training, research and pilot plant service facility that brings together institutions with complementary expertise and state-of-the-art research technology, and industry partners. these include university college dublin, trinity college dublin, institute of technology, sligo and dublin city university. nibrt is an innovative collaboration between academic institutions at the forefront of biotechnology, cell biology, engineering and pharmacy and industry. for training, two separate training labs, for upstream and downstream training, in addition to 5 research labs are planted. it will also include a state-of-the-art pilot plant fermentation facility for fermentation optimisation, fermentation scale-up, product separation and purification, regulatory aspects and automation. by aligning with industrial demands, the new institute will tailor its training programmes while remaining on the cutting edge of biotechnology research and technologies. the fermentation facility will offer hands-on training workshops and educational modules for outside researchers and companies. these workshops cover the fundamentals of small-scale fermenta-tion, scale-up considerations, and fermentor design and set-up. the training and educational philosophy underpinning the nibrt will focus on the needs of industry with an emphasis on providing training for accreditation of existing industry staff and prepare technicians and graduates for the technical, business, regulatory and professional aspects of the industry. the strategy is to provide specialised modules in nibrt in support of courses established in the higher education institutions, which will provide the certificates, diplomas and degrees. modules will be offered for all categories of students and will be given credits respected by other third level institutions in ireland. the role of professional graduate degrees in meeting current and future biotechnology industry workforce needs a. stephen dahms san diego state university, usa the presentation will review the status of new graduate training models designed to meet the unique needs of the biotechnology industry as it transitions to commercialization. emphasis will be on professional master's degree programs in biotechnology and their various versions, with a focus on operational and funding strategies and industry acceptance. discussion will also centre upon the creation and operation of industry-validated, specialized and highly targeted professional masters degrees in various refined aspects of the drug development process, including regulatory affairs, biomedical quality systems, clinical affairs, management of drug development, management of reimbursement affairs, bioinformatics, etc. the eurodoctorate in biotechnology, new combined mba/phd combined degrees in the molecular life sciences, the u.s. professional doctorate in chemistry and the proposed u.s. professional doctorate in biotechnology will be also discussed. data will also be presented on the current workforce and the industry's projected needs. genetic studies show that mankind is a rapidly expanded population of closely related individuals with very similar disease sensitivity. bad nutrition and infections dominate among the main health problems in the world. apart from malnutrition, the overeating habits of the developed world are now creating problems in the developing world as well. infectious diseases are also a global problem since new contagious agents like hiv, sars and avian flew do not recognize borders. thus, the global responsibilities of modern health care are obvious. many research scientists from and in developing countries find it nearly impossible to use their talents for the benefit of their own countries. some struggle to develop research and education programmes with poor facilities, some leave science completely, and others migrate to more developed countries. the talents of such people are either being wasted or lost completely to their home countries just at the time they are most needed to combat the great humanitarian challenges of hunger, illness and lack of knowledge. europe must strengthen programmes which allow third world scientists to work to their full potential in their home countries or regions. yang beijing genomics institute, chine academy of sciences, beijing, china europe has all the reasons to be proud of being the cradle of modern science and of its achievements and resources in life sciences. as a model of having solved many of the problems that many other counties are now facing, europe is expected by the whole world to make its further contribution to a better future of mankind and to play a more important role in the international community of life sciences. tropical diseases and public health basilio valladares director of the university institute of tropical diseases and, public health, university of la laguna, 38200 la laguna, tenerife, spain. e-mail: bvallada@ull.es the presentation will look at the main research interests of the university institute of tropical diseases. these are the following: 1. immunology and molecular biology of parasites. we express and purify recombinant proteins from leishmania sp. which have been shown to act as immunomodulators and protect against disease such as l25, hsp70, hsp83. the study of acanthamoeba pathogenic factors has also resulted in the isolation and silencing of extracellular proteins related to their pathogenecity, which has a great potential in the development of novel chemotherapeutics. 2. diagnosis of parasites. the immunological diagnosis of leishmaniasis has been one of the main research interests in our laboratory for several years. as a result, we have identified peptides which could be used to develop kits for the immunological diagnosis of leishmaniasis such as hsp70 c-end, l25 n-end, etc. we have also developed some dna based methods for the identification of acanthamoeba species from biological and environmental sources. 3. water quality. biological parameters. our water research group has the expertise to identify and characterise bacterial, viral and parasitic indicators of faecal contamination in diverse water sources including tap water, rivers, reservoirs, sea, etc. this research area has been developed in collaboration with the local sewage treatment plant and reservoir managing authorities. currently, we are establishing a conjoined project with the center for disease control and prevention (cdc) in atlanta, usa for the identification of water-borne emerging pathogens. 4. development and formulation of chemotherapeutic antiparasitic agents. in this field, we evaluate the leishmanicidal activity both in vivo and in vitro of natural and synthetic drugs and synthetic peptides. in a later stage, the drugs which have shown the highest antiparasitic activity have been subjected to cytotoxicity assays and their molecular targets dissected. some of the drugs tested in the last few years have been submitted to patent due to their outstanding activity. finally, in order to allow the commercialization of these drugs, both in vivo and in vitro assays are being carried out to predict their chemical stability and degradation pathways. this will be followed by the use of liofilization and controlled crystallisation strategies for the development of efficient and safe treatments. 5. human and population genetics. tachykinins and their receptors in different tissues and groups of patients and their association with molecular polymorphisms is another one of our research interests. the knowledge of ligand and receptor sequences and their similarities will allow the rational development of drugs with specific activity against these receptors. furthermore, we are also interested in the interspecific variation along the evolutionary scale of these markers. 6. nitrate assimilation group. research in our group is focused on understanding nitrate assimilation in the yeast hansenula polymorpha. several biotechnological companies use this yeast to produce heterologous proteins (hepatitis b vaccine). genetic manipulation techniques for h. polymorpha are available in our laboratory. head of the department of biotechnology, technological institute of canary islands, pozo izquierdo 35119 santa lucía, las palmas, spain single cell analysis by flow cytometry has proved to be a tool to perform simultaneous and rapid measurements related to cell morphology and physiological state. previous studies showed the possibility of quantifying neutral and polar lipids spectrofluorometrically using a lipid specific fluorescent dye, nile red (nr), however the existence of inter and intraspecific variations in the fluorescent response had not been clearly established. in this work, two strains of marine microalgae: crypthecodinium cohnii and tetraselmis suecica, characterized both by high contents of polyunsaturated long chain fatty acids (dha and epa, respectively) and an hypersaline microalgae: dunaliella salina, characterized by a high ␤-carotene production, were grown under different conditions and collected at different growth phases to be used for in vivo lipid quantification with nr by flow cytometry. our results showed a high correlation between the mean fluorescence signal of nr stained cells and the neutral and polar lipid content measured by gravimetry for each strain. in this respect, these data make feasible the development of a rapid method for lipid quantification in monoalgal cultures. however, differences in the dye uptake related to specificity were detected. in this communication we also assess the possibility of use this cytometric technique to select microalgal strains with high lipid and polyunsaturated long chain fatty acids content from mixed samples. performance of such technique would be a good alternative to the time-consuming traditional screening protocols based on gravimetry and gas chromatography and would optimise the search of new commercial strains of microalgae. claverie-martín head of research unit, biomedical research institute, hospital universitario, de n.s. de candelaria, santa cruz de tenerife, spain our group is involved in the cloning and production of proteins of interest to the food and pharmaceutical industry. we have recently expressed in yeast the cdna that encodes the precursor of caprine chymosin. chymosin is the enzyme responsible for the coagulation of milk in the abomasum of unweaned calves. this enzyme is secreted by gastric mucosa cells as an inactive precursor, known as prochymosin. in the acidic conditions of the lumen, prochymosin is converted into the active form by autocatalytic cleavage of the n-terminal prosequence. chymosin is used extensively in cheese production because it cleaves -casein in a specific manner with low proteolytic activity. several biotechnology companies are producing the bovine recombinant enzyme for commercial use in the process of cheese making. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. it is well known that the activity of these types of extracts varies depending on the age of the animal and the type of food ingested. these difficulties should be overcome using a recombinant caprine chymosin. the caprine mrna used for the synthesis of the cdna was obtained from the abomasum of milkfed kid goats. the cdna fragment encoding the mature portion of caprine prochymosin was fused in frame to a signal sequence in yeast expression vectors. culture supernatants of yeast cells transformed with the recombinant plasmids showed milk-clotting activity after activation at acid ph. proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed -casein (patent 200402025). the recombinant caprine chymosin could be an alternative milk coagulant in cheese making. work is underway to optimise the expression of the new recombinant prochymosin for further purification and characterization. smart molecules for health victor martín head of research, university institute of bio-organics "antonio gonzález", avda. astrofísico francisco sánchez 2, 38206 la laguna, tenerife, spain the instituto universitario de bio-orgánica "antonio gonzález" (iubo) is a multidisciplinary research centre that belongs to the university of la laguna. the iubo is located at the town of la laguna, inscribed on unesco's world heritage list in 1999, and former capital of the canary island of tenerife. the geographical location in addition to its mild climate has made the canary islands to posses a variety of ecosystems with unique plants and animals. the institute was started up in the 1960s with the need to study the natural products and secondary metabolites produced by those marine and terrestrial organisms, thus providing a new source of bioactive products. the main research lines that are being developed at iubo are summarised in the following paragraphs: anticancer agents from natural sources: several natural products and their semisynthetic derivatives are produced at the iubo in diverse joint projects for the development of new antitumour drugs with novel mechanisms of action. as an example we can mention natural products from the mevalonic, shikimic or polyketide pathways. some products have recently shown in vitro reversion of the resistance in multidrug resistant (mdr) tumour cell lines. genetic engineering: in vitro cultures of the plants atropa baetica, maytenus amazonica and m. macrocarpa are developed in order to manipulate their biosynthetic pathways and induce the production in large scale of the secondary metabolites for diverse applications, including arthritis, rheumatism, and back pain. marine organisms and toxins: dinoflagellates are marine organisms responsible for the red tides and food poisoning episodes. among others, okadaic acid and yessotoxin are the most common toxins present in european shellfish. the isolation of these products is best done from the microorganism cultures, since they are present in very low amounts in the natural sources. at the iubo we develop culture systems to provide us with amounts of toxins large enough to perform biological, metabolic and bioactive studies. insecticide and repellents: natural products are being isolated for their use against plagues, specially those affecting agriculture. these projects are run in collaboration with a number of public institutions and agrochemical companies throughout europe and latin america. fine chemicals and pharmachemicals: our institute possesses large expertise in the field of organic synthesis devoted to the synthesis of medicinal substances, with special focus on asymmetric processes. of particular interest is the development of new methodologies for the total synthesis of biologically active substances like polyether toxins, (un)natural amino acids, sphingosine analogs, alkaloids, etc. with an annual source of 100s of new compounds, the fine chemicals and medicinal chemistry branch at iubo have recently started and anticancer screening program in collaboration with the biomedical research unit at the hospital universitario n.s. de la candelaria. the program is committed to the discovery of novel drugs for application in cancer treatment. the outcome of this project in its first year has been outstanding, leading to the finding of several leads that form the basis for current and future projects. institute of canary islands, biotechnological department, playa de pozo izquierdo s/n, 35119 santa lucía, gran canaria, spain the presentation will look at the main research interests of the biotechnological department of the technological institute of canary islands. these are the following: nutrition and feeding in aquaculture: we conduct studies on digestion, absorption, transport, and utilization of the different nutrients applying also different techniques such as histology, enzymology, genetic or immunology among others. aquaculture feeding is the main important cost in fish farms, being higher that the prize of fries, personnel cost or energetic costs. thus, studies conducted on the improvement of diet formulation and the use of different dietary ingredients is one of our main research lines (such as vegetable oils and meals to be used as alternative to fish meal and oil, or carotenoid sources to improve fish colour). not only the use of the different ingredient is studied, but also the effect of these ingredients on fish health, flesh quality, flesh healthy aspects related with human consumption, are being studied. different formulae are being developed and patents of different diets are being obtained. studies on nutritional requirements are also conducted allowing us to patent different formulae in larvae studies, developing microdiets to substitute the high-cost processes associated with live prey in larval nutrition. development of immunostimulants, anti-stress diets, immune techniques to be applied as bio-indicators of fish health and welfare, as well as use of dietary ingredients derived from bio-reactors are other research lines in our group. genetics: genetic techniques applied to aquaculture are being an important tool. microsatellites are being used to determine genealogy of the fish, allowing to decreases important problems in aquaculture such as fish deformities. genetic techniques such as micro-arrays and gene expression are being applied to obtain indicators of stress and health in fish. these technologies also permit to make different selective breeding programs, increasing the accuracy to estimate genetic parameters and evaluating brood-stock. furthermore, this technology allows to obtain a procedure to increase the productivity and quality of fish hatcheries. new species for aquaculture. development of new culture techniques: the diversification of species cultured is one of the main objectives of european aquaculture, since nowadays only four marine species are commercially produced: gilthead sea bream. european sea bass, turbot and salmon. we have been developed rearing techniques for new species such as red porgy or canarian abalone and also we are conducting studies on different new species, such as different sparids species, octopus or yellowtail. new rearing technology: the election of adequate systems for fish growth for each species and site of production and the localization of more appropriate sites for farms, using gis technology are also of special importance in our research team. besides, new larval rearing techniques such as semi-extensive hatchery (mesocoms) were development and nowadays is being used to increase fry quality (survival, no-deformities, better growth). this technology is being exported to other countries in order to offer new technology easy to manage to be implanted in developing countries. alejandro cañeque project officer, canary islands special zone, ministry of finance, c/leon y castillo 431 -4 a planta, edificio urbis, 35007 las palmas, spain. e-mail: acaneque@zec.org the canary islands special zone is the newest tax instrument within the canary islands economic and fiscal regime (ref). it offers a reduced tax rate of between 1 and 5% of corporate income tax for companies setting up a business. the companies must cover the following minimum requirements: create employment and make a minimum investment. the zec offers other tax advantages such as the exemption from paying transfer tax and stamp duty and the canary islands general indirect tax (igic). this tax scheme particularly fosters the biotechnology and pharmaceutical sectors. references fahnert references bechor the antimicrobial drugs high-level production of human collagen prolyl 4-hydroxylase in sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates microbial processes and products press. biotech. bioeng. 218. van hijum microbial xylitol production from corn cobs using candida magnoliae the role of bacillus methanolicus citrate synthase gene, city, in 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integrative biological analysis of the apoe*3-leiden transgenic mouse innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble cd14 soluble forms of toll-like receptor (tlr)2 capable of modulating tlr2 signaling are present in human plasma and breast milk proteomics of the rat gut: analysis of the myenteric plexuslongitudinal muscle preparation an integrative metabolism approach identified stearoyl-coa desaturase as a target for an arachidonate-enriched diet the challenges of modeling mammalian biocomplexity rapid enrichment of bioactive milk proteins and iterative, consolidated protein identification by mudpit technology post-genomics of lactic acid and other food-grade bacteria to discover gut functionality genetics, metabolism and application of lactic acid bacteria. fems microbiol complete genome sequence of lactobacillus plantarum wcfs1 functional ingredient production: application of global and metabolic models metabolic engineering of lactic acid bacteria for the production of nutraceuticals reduction in antinutritional and toxic components in plant foods by fermentation the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no 2001k120240-41). the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no. 2001k120240-40) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aç ikel at gazi university. the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no 2001k120240-40) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aç ikel at gazi university. mrs. lynnette fernandez, johanna mäkeläinen, m.sc. and olli rämö, m.sc. are gratefully acknowledged for their help. this work was supported by the health science council, the academy of finland and the tekes-neobio program. supported by the mec of spain (ppq2002-04361-c04-02) and the canary islands government. jmp thanks icic for a postdoctoral fellowship. frpc thanks cajacanarias for a fpi fellowship. tm thanks the spanish mcyt-fse for a ramón y cajal contract. this work was supported by the korean systems biology research grant from the ministry of science and technology, lg chem chair professorship, ibm sur program and bk21 program. this study was supported by ankara university biotechnology institute (project no: 89). this work was partially supported by mec and feder (bio2004-00439), and fundación séneca carm (00842/pi/01). this work was supported by grants f037663 of the hungarian scientific research fund and grant omfb-00219/2002 of the hungarian ministry of education. keto sugars have long been implicated as attractive intermediates or substrates for further chemical or enzymatic reactions, to generate a number of synthetic sugar derivatives and fine chemicals. the quinone-dependent pyranose dehydrogenase (pdh) purified of the basidiomycete fungus agaricus meleagris catalyzes with high specificity the oxidation of the c-3 of glycosidically bound d-glucose, whereas in contrast it oxidizes simultaneously the c-2 and c-3 of free d-glucose. considering the broad substrate tolerance, pdh provides a new convenient tool for high yield production of 3-ketothis work was partially financed by the scientific and technological research council (cicyt, spain), grant ren2001-3224, by the iii pla de recerca de catalunya (generalitat de catalunya), grant 2001sgr-00143, and by the generalitat de catalunya to the "centre de referència en biotecnologia" (cerba). this work was supported by mega a.s. (czech republic) (www.mega.cz) and following vega grants: 1/2391/05 and 1/2390/05. this work was partially supported by mec and feder (bio2004-00439), and fundación séneca carm (00842/pi/01). financed by a marie curie re-integration grant merg-ct-2004-006378 and consejería de educación y ciencia de la junta de andalucía. this project is part of the collaborative research centre sfb 578 "development of biotechnological processes by integrating genetic and engineering methods", which is supported by the german research foundation dfg. this research was supported in part by grants from the hungarian scientific research fund (otka t37471, f46658 and d48537) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb00103/2005). this research was supported in part by grants from the hungarian scientific research fund (otka t37471, f46658 and d48537) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb00103/2005). this research was supported in part by grants from the hungarian scientific research fund (otka t37471, f46658, d48537) and gvop-3.1.1.-2004-05-0471. the authors thank dr. hamid narjiss (director of morocco inra) for the instruction to identify nadorcott mandarin by molecular markers and helpful discussions regarding this paper. this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). this work has been financed by xunta de galicia (pgidt03pxib30103pr). sevgil sadettin, gönül dönmez department of biology, faculty of science, ankara university 06100 beşevler, ankara, turkey this study was supported by ankara university biotechnology institute. demet ç etin 1 , sedat dönmez 2 , gönül dönmez 1 : 1 department of biology, faculty of science, ankara university, 06100 beşevler, ankara, turkey; 2 department of food engineering, faculty of engineering, ankara university, 06110 dışkapı, ankara, turkey sulfate-reducing bacteria (srb) that could grow on modified postgate c medium (pc) containing chromium(vi) were isolated from industrial wastewater and their chromium(vi) reduction capacities were investigated as a function of changes in the initial ph values, chromium, sulfate, nacl concentrations and carbon source. the optimum ph value at 50 mg l −1 initial chromium(vi) concentrations was determined as 8. chromium(vi) reduction by srb was investigated at 22.7-98.4 mg l −1 initial chromium(vi) concentrations. at the end of the experiments, the mixed cultures of srb were found to reduce more than 99% of the initial chromium(vi) levels which ranged from 22.7 to 74.9 mg l −1 within 2-6 days period. the effects of initial 0-9.0 g l −1 concentrations of sulfate and 0-6% (w/v) concentrations of nacl to chromium reduction were showed that, the lowest concentrations of sulfate and nacl, were the best for chromium reduction in the pc media including 50 mg l −1 chromium(vi). when the 25% whey was used as carbon source in the pc medium, 99.6% of the 65.9 mg l −1 initial chromium(vi) concentration was reduced within this study was supported by ankara university biotechnology institute. this study was carried out as a part of the project for analyzing and controlling the mechanism of biodegrading and processing entrusted by the new energy and industrial technology development organization (nedo). this work has been financed by xunta de galicia (pgidt03pxib30103pr). lead ions are considered a high pollutant of different waters. in our previous work were selected seven potential sorbent-strains rhodotorula mucilaginosa 1776, rh. aurantiaca 1195, rhodotorula sp. 4, williopsis californica 248, candida krusei 61t, cryptococcus sp. wt of lead ions. it was determined their stability to high concentration (up to 750 mg/l) of lead ions in medium. the ph changes and yeast physiologies of growth were studied in medium with these heavy metal ions. the influences of environmental factors such as ph of solution, age of microbial culture, biosorbent concentration in suspension, alive or living state of biosorbent, time of contact on sorption were investigated. the levels of maximal sorption ability and biomass affinity to heavy metal ions were established by experimentally received sorption isotherms with mathematical modeling of biosorption process separately for everyone researched yeast strain. the sorption isotherms obtained in these experiments for non-living yeast biomass showed that the maximal sorption capacity was 225 and 195 × 10 −6 mol (g sorbent) −1 for rh. aurantiaca 1195 and s. cerevisiae 1968, respectively. in the case of living biomass, the highthis work has been financed by the spanish ministry of science and technology and european feder (project ctm2004-01539). the authors wish to thank dra. m.j. martínez (cib, csic, madrid, spain) for providing coriolopsis rigida. this work was supported principally by embo and the mrc. fed-batch cultivation of haematococcus pluvialis under illumination with leds for production of astaxanthin abdolmajid lababpour, tomohisa katsuda, shigeo katoh department of molecular science and material engineering, kobe university, kobe, hyogo 657-8501, japan photosynthetic microalga haematococcus pluvialis is a most promising microorganism for production of astaxanthin, which has powerful antioxidant activity and is used both for human being as a food supplement and cosmetic; as well as animal farming such as salmon and poultry. the deficiency of nutrients in batch culture of h. pluvialis decreases the growth of cells and increases the induction of astaxanthin as an induction factor. therefore, it is impossible to reach to high cell concentrations in batch cultures. in previous experiments, the medium replacement increased the cell concentration, while accumulation of astaxanthin was not induced without other factors. in this work, the effects of fed-batch addition of culture medium on the cell growth and astaxanthin production in h. pluvialis cultures were studied. h. pluvialis was cultivated in 50 cm 3 culture medium containing sodium acetate, yeast extract, l-asparagines, mgcl 2 ·6h 2 o, feso 4 ·7h 2 o and cacl 2 ·2h 2 o (ph 6.8). light was supplied by panels of blue or red led lamps. the temperature was kept at 20 • c and the culture was mixed with a magnetic stirrer. in fed-batch cultures of h. pluvialis, the cell concentration and production of astaxanthin increased in comparison with those in batch culture. in addition, the operation in feb-batch manner is easier than medium replacement from industrial viewpoints for production of astaxanthin. simvastatin and similar compounds are wide used as antihypercholesterolemic agents. simvastatin is obtained by c-methylation of side chain of lovastatin. this process is not perfect and some unreacted lovastatin is present in the reaction mixture. simvastatin is separated from lovastatin using fungi clonostachys compactiuscula. using of living microbials has some disadvantages such as high cost, difficult product separation from the reaction mixture. we work on overcome these difficulties by separation and immobilization of enzyme that hydrolyses lovastatin ammonium salt in presence of simvastatin ammonium salt. our results of purification and immobilization lovastatin hydrolase will be presented. investigation of peptide antibiotics produced by trichoderma strains isolated from winter wheat rhizosphere a. szekeres 1 , l. kredics 2 , l. hatvani 1 , z. antal 2 , l. manczinger 1 , a. nagy 3 , c. vágvölgyi 1 : 1 department of microbiology, university of szeged, p.o. box 533, h-6701 szeged, hungry; 2 hungarian academy of sciences, university of szeged, microbiological research group, hungry; 3 pilze-nagy ltd. , kecskemét, p.o. box 407, species of the imperfect filamentous fungal genus trichoderma with teleomorphs belonging to the hypocreales order of the ascomycota division are of great economic importance as sources of enzymes, antibiotics, as plant growth promoters, decomposers of xenobiotics, and as commercial biofungicides. peptaibols and related peptaibiotics (prps) are secondary metabolites constituting a family of fungal peptide antibiotics which is constantly growing since alamethicin was isolated from cultures of trichoderma viride. these compounds are linear, amphipathic polypeptides composed of 5-20 amino acids and usually containing several non-proteinogenic amino acid residues, which are representing characteristic building blocks of the structure. one hundred and twenty trichoderma strains were isolated from roots of winter wheat grown in agricultural fields of southern hungary. the identity of species was examined based on morphological and molecular characters. the presence of prps-producing strains among the isolated trichoderma strains was detected by biological tests and the antibiotics were partially purified using a multistep chromatography procedure involving exclusion chromatography, adsorption chromatography and thin-layer chromatography. about 20% of the isolates proved to be able to produce prps. the antibacterial activity of the compounds was tested against staphylococcus aureus, bacillus subtilis, micrococcus luteus and escherichia coli, while the antifungal effect was recorded against fusarium oxysporum, f. culmorum, rhizoctonia solani and pythium debaryanum. ergezinger, m., bohnet, m., berensmeier, s., buchholz, k., 2005. integrierte enzymatische synthese und adsorption von isomaltose in einem mehrphasenbioreaktionsreaktor. cit 77, 167-171. glucansucrases from family 70 of glycoside-hydrolases are transglucosidases that produce ␣-glucans from sucrose, a very cheap substrate, without any use of nucleotide activated sugars. based on sequence analyses, these enzymes have been classified in two families, the family 70 and the family 13 of glycoside hydrolases. among the natural diversity existing in family 70 in which are found the glucansucrases produced by lactic acid bacteria, three enzymes have been selected for their distinctive specificities: dextransucrase from l. mesenteroides nrrl b-512f (dsr-s), which catalyses almost essentially the synthesis of ␣-1,6-linkages, alternansucrase from l. mesenteroides nrrl b-1355 (asr), which produces alternan polymer formed of ␣-1,6and ␣-1,3-alternated linkages and finally dextransucrase from l. mesenteroides nrrl b-1299 (dsr-e), which is responsible for the synthesis of a branched dextran composed of about 70% of ␣-1,6-linkages in the main chain and 30% of ␣-1,2-branched linkages. for all these enzymes, the natural polymerase activity can be shifted towards oligosaccharide production or gluco-conjugate syntheses by introducing acceptors in the reaction medium. a number of sugar acceptors have been successfully glucosylated with the view of developing new functional food products. acceptor glucosylation yield as well as acceptor reaction product structures were shown to be highly dependant on the enzyme specificity. consequently, using glucansucrases of distinctive specificities and varying the acceptors give access to a large variety of applications. amylosucrase, the sole glucansucrase found in family 13 of glycoside-hydrolases is also of great interest for functional food applications. this enzyme is able to synthesize highly resistant amylose from sucrose. again the reaction conditions can be used to modulate the yield and the size of amylose. the aim of our work is to further develop the applications of these enzymes via rational and combinatorial engineering. the most recent results obtained in this field will be discussed. novel food structure engineering concepts with enzymes johanna buchert vtt biotechnology, espoo, finland food structure is a very important quality attribute in food choice, since it affects not only the sensory perception of texture, but also release of flavour. enzymes offer specific means to engineer food structure by creating cross-links to food biopolymers, i.e. to proteins and/or carbohydrates. enzymatic cross-linking of food biopolymers can be exploited to create novel types of structures to foods without any need of added food ingredients. laccases and peroxidases can be used to crosslink ferulic acid containing carbohydrates, such as sugar beet pectin or arabinoxylan. proteins can be crosslinked by different oxidative or transferase type of enzymes. transglutaminases can crosslink protein via formation of isopeptide bond between glutamine and lysine residues. laccase and peroxidase can oxidize tyrosine residues to corresponding radicals, which in turn can further react with different groups in proteins. tyrosinases, on the other hand, oxidize tyrosine to a quinone, which can further react with aromatic ring, amine and thiol groups present in proteins. the biopolymer networks formed can be further engineered by combining adequate processing to the enzyme treatment. in this work the potential of enzymatic food structure engineering is reviewed. asparaginase-mediated reduction of acrylamide formation in baked, fried, and roasted products hanne vang hendriksen, beate kornbrust, steffen ernst, mary stringer, hans peter heldt-hansen, peter østergaard novozymes a/s, dk-2880 bagsvaerd, denmark. e-mail: hvhe@novozymes.com (h.v. hendriksen) in 2002, it was discovered that acrylamide is formed in several potato and grain-based foods (e.g. chips, french fries, toasted bread, biscuits, cereals) and in coffee, all of which have been prepared at high temperatures. the level of this potential carcinogen in the final food appears to range from 50 to 4000 ppb. later that year, the mechanism of acrylamide formation was unraveled, demonstrating that asparagine and reducing sugars are the precursors for acrylamide. this pointed to several potential enzymatic approaches to remove the root cause of the problem by degrading the precursors in situ. here, we demonstrate that asparaginase treatment leads to a more efficient reduction in acrylamide than alternative enzymatic treatments. asparaginase from aspergillus oryzae is used to reduce acrylamide formation significantly in laboratory models of a range of common food products. examples are french fries, biscuits, crisp bread, and fabricated chips. the sensory qualities appear to be constant. the implications for scaling up the processes for industrial food production are discussed. effect of cultivation conditions on folate content in yeast: exploring the potential of yeast as a bio-enrichment vehicle for folate in foods sofia hjortmo 1 , johan patring 2 , jelena jastrebova 2 , thomas andlid 1 : 1 department of chemical and biological engineering, chalmers university of technology, po box 5401, 402 29 gothenburg, sweden; 2 department of food science, swedish university of agricultural sciences, po box 7051, 750 07 uppsala, sweden. e-mail: sh@fsc.chalmers.se (s. hjortmo) over the past 10 years, the interest in health benefits of the b vitamin folate has increased considerably. a good folate status may hinder progression of several diseases such as neural tube defects and downs syndrome in foetus, as well as cancer, dementia, alzheimer's disease and cardiovascular disease in adults. it is however not easy to reach the recommended intake and new strategies have to be developed to increase the folate status. in this project we explore the use folate producing microorganisms for this purpose. many yeasts have the ability to synthesise folate de novo and can thus serve as a source for humans. folate enrichment in fermented foods could be much improved by using starter cultures better at producing folate compared to traditional strains. this is, e.g. applicable to bread making fb32 over-expression of isoprene biosynthetic enzymes in the ␤-carotene producer zygomycete mucor circinelloides tamás mucor circinelloides has been involved to study the carotene biosynthesis genesis of fungi. this fungus is more amenable to molecular techniques than the others traditionally used in carotenogenic studies (e.g. blakeslea trispora and phycomyces blakesleeanus). moreover, mucor has a great advantage: it is a dimorphic organism. this type of morphology is preferred by the fermentation industry because yeast-like growth allows the submerged culture, when usually higher biomass production can be achieved and cells can be more easily separated from the media. β-carotene is a terpenoid-type chemical compound likewise to sterols, quinones or chlorophylls. the production can be increased by improving the non-carotene specific terpenoid biosynthesis. this can be carried out by the overexpression of the genes responsible for the ratelimiting steps of these pathways. in this study, polyethylene glycol mediated transformations of m. circinelloides protoplasts were performed with autoreplicative expression vectors containing the known terpenoid genes of m. circinelloides (e.g. isoa encoding farnesyl pyrophosphate synthase and carg encoding geranylgeranyl pyrophosphate synthase). carotene production of the transformants and the wild-type strains were analysed by high-performance liquid chromatography (hplc). transformants harbouring plasmids with isoa or carg produce about 1.5 times more carotene than the recipient strain, while carotene production increased about two times in the co-transformants containing both type of plasmids. members of the genus rhizopus are important from biotechnological aspects in consequence of their effective extracellular enzyme, alcohol and organic acid production. moreover, rhizopus strains are used for fermentation of various foods, because they are capable of transforming soybeans into edible products. the high affinity iron permease (ftr1) contains both highly conservative and variable regions applicable for phylogenetic comparisons. the aim of this study was the comparative analysis of this gene of different rhizopus species in order to elaborate a simple and fast method to identify these fungi at a species and subspecies level. conserved regions of candida albicans and rhizopus oryzae ftr1 genes (fu et al., 2004) have been analysed to design degenerate primers for polymerase chain reaction. they were used to amplify the homologous regions from different strains of r. oryzae, r. microsporus, r. stolonifer and r. niveus. isolates of the similarly thermophilic rhizomucor miehei and r. pusillus, as well as a strain of m. rouxii were involved in the study as outgroups. deduced protein sequences were aligned and phylogenetic analysis was performed. surprisingly the r. oryzae isolates formed a group completely different with a significant distance from the r. microsporus isolate. r. niveus is currently not distinguished from r. stolonifer var. stolonifer because of morphological considerations. however, phylogeny of ftr1 gene sequences, in agreement with earlier results based on rapd data (vágvölgyi et al., 2004) , raise the need to handle r. niveus as a separate species. sequences and pcr primers useful for identification of all tested rhizopus strains were elaborated. potential application in a lactose conversion process. the prebiotics market is at high demand therefore the development of the process to produce 'prebiotic' galacto-oligosacharides efficiently and inexpensively is our particular interest. citrus, particularly mandarins and clementines, are among the most economically important fruit crops in morocco. besides morphological traits multiple molecular markers have been used for the caracterisation of citrus germplasm. the main aim of this study was to evaluate the moroccan mandarin germplasm and to identify specific polymorphisms among accessions sharing identical name. eighty mandarin and two sweet orange varieties were analyzed by dna markers. issr markers were amplified using 3 anchored primers and analysed by agarose gel electrophoresis. aflp markers analyses were performed using three primer combinations. the dice coefficient was used to estimate genetic similarities and the upgma algorithm was utilised to generate a phenogram depicting the genetic relationships among the acessions. the selection of 9 primers out of the 31 issr primers primarily assayed, allowed us to maximize the average number of amplified fragments analyzed per reaction (7.3), and the percentage of informative polymorphisms (49%). the three combinations of ecori/msei primers revealed 73 reliable aflp markers, 35 (47%) of witch were polymorphic. the range of fragment sizes varied from 100 to 650 bp. contrasting with the phenotypic diversity for agronomic and fruit quality traits, very low variability at the dna level has found among mandarins, which always showed a high (s > 0.81) coefficient of genetic similarity. the molecular marker analyses allowed the clarification of ambiguous denominations and the establishment of phenological relationships. the mandarin cultivars have been clustered into several different sub groups. this study allowed the identification of one issr marker, distinct and specific for the clementine sidi aissa and some aflp markers specific to maroc late and w. navel. many hybrids used in this study presented high coefficient of the similarity with one of their parents, such as siamelo and king of siam (s = 0.92), fortuna and clementine (s = 0.93), kara and king of siam (s = 0.92). this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). the new morocco mandarin variety nadorcott became very important in the international market because of high quality, good size, easy peeling and absence of seeds. also known as afourer or w. murcott, this variety was selected in 1981-1982 at the afourer experimental station, inra, located near to beni mellal city, as an original tree among several 18-year-old murcott honney (c. reticulata × c. sinensis) seedling trees. in order to shed additional light on the genetic origin of this variety, we have carried out isozyme and dna fingerprinting analyses. for better interpretation of the nadorcott molecular profiles, others mandarin cultivars, among which murcott honney, were also analyzed by molecular markers. three enzymatic systems (idh, pgm and pgi) permitted the discrimination between nadorcott and his female parent murcott honney. the molecular patterns displayed by these cultivars point out the sexual origin of nadorcott and discard the previously assumed hypothesis for its origin as a mutation of a nucellar zygote. the issr and rapd markers analyses allowed the identification of kinnow; du japon, vietnam; and swett lime as the genetically most closely related mandarins (s ∼ 0.95) to nadorcott. strong genetic similarity was also found with the clementine group, a possible male parent of nadorcott. the analysis by aflp markers confirmed the hybrid origin of nadorcott and the high genetic relatedness (s = 0.93) of this mandarin to its putative female parent murcott honey, and other cultivars as kinnow (s = 0.94) and clementine (s = 0.93). the possibility of an accurate molecular identification of nadorcott by specific molecular markers is of paramount importance for the protection and management of this original moroccan citrus variety. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions 77 g/l from white distilled lees and 45.6 g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. thermophilic cyanobacterial strains that could grow in the bg 11 media was isolated from hot springs and their reactive dye bioaccumulation was studied under thermophilic conditions in a batch system, in order to determine the optimal conditions required for the highest dye accumulation. in the experiments performed with newly isolated synechocystis sp. and phormidium sp., the optimum ph values at about 25 mg l −1 initial reactive dye concentrations was determined as 8. lipases are extremely versatile enzymes, that catalyze both hydrolysis and synthesis reactions. they have a wide range of industrial applications, among which the manufacture of detergents, pharmaceuticals and fine chemicals are outstanding. the fungus rhizopus oryzae has been reported to synthesize a number of commercially interesting enzymes. in this work, its ability to produce extracellular lipases when grown in solid state culture has been assessed. cultures were carried out in erlenmeyer flasks, using a complex medium and several supports, both synthetic (nylon sponge) and natural (barley bran, ground walnut or peanut). the latter appeared to be more suitable for lipase production. since the best results were initially obtained with lipid-containing supports, barley bran cultures were supplemented with a vegetable oil, in an attempt to optimise lipase production and design an efficient procedure for reusing this agroindustrial waste. surprisingly, this strategy did not improve enzyme synthesis. however, when a surfactant (triton x-100) was added to the basal medium, a dramatic increase in extracellular activity was detected (up to 20-fold). the results agreed with those previously obtained in submerged cultures of r. oryzae, in which addition of olive oil did not increase lipase production, while the presence of triton x-100 had a remarkably beneficial effect. also, enzyme concentration in solid state cultures was up to two-fold that of the submerged ones. est maximal sorption capacity was found for yeasts cryptococcus sp. wt and rh. aurantiaca 1195 . these cultures also demonstrated the high sorption affinity, which makes them especially efficient biosorbents at low concentrations of lead ions. the high efficiency of lead elution was shown with 0.1n edta. isolation and identification of marine bacteria from deep-sea sediments els maas 1 , cara brosnahan 1 , vicky webb 1 , helen neil 2 , phil sutton 2 : 1 marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand; 2 oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand marine sediments were obtained using a piston corer with associated trigger core (0.5 m, 0.06 m diameter). cores were collected from depths of 270 to 3911 m, along norfolk ridge and across challenger plateau. sediments ranged from coarse carbonate sands in the north to sandy and silty hemipelagic mud with increasing depth and latitude. all samples sites underlie subtropical surface water masses associated with, and south of, the tasman front. sediment samples were aseptically taken from the triggers cores upon recovery. samples were stored in sterile tubes at 4 • c on board the vessel for 3-17days. the core samples were plated on several different agar types and incubated aerobically for 4 weeks at 16 • c. individual colonies were sub-cultured and purified using standard microbiological techniques. morphological and molecular taxonomy revealed that the bacteria isolated from the sediments were closely related to novosphingomonas, halomonas, stappia, glaciecola, pseudoalteromonas and leeuwenhoekiella. phylogenetic trees constructed using 16s rrna gene sequence data showed that two other isolates were unrelated to known genera. the bacterial isolates are currently being investigated for their biotechnological potential. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by 20% was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were 90 and 50%, respectively. while the color and aromatocity decreased by 60 and 95%, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was 0.045 h −1 while the monod constant based on the consumed tp and cod (mg/l) were 370 and 6900, respectively. the control of water pollution has become of increasing importance in recent years. the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g −1 ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph. the maximum adsorption at 50 ppm was 88.52% equal to 11.8 mg of dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of 8-12). the effect of contact time was studied at initial concentration (50 ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after 15 min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. flocculation of saccharomyces cerevisiae (diastaticus) ifo1958 was studied. cells of ifo 1958 did not flocculate even in the stationary phase without mg 2+ ("mg 2+ -deficient cells") although they began to flocculate strongly 18h after inoculation in the presence of mg 2+ ("complete cells"). cycloheximide completely inhibited induction of floc-forming ability of "mg 2+ -deficient cells". co-flocculation between "complete cells" and "mg 2+ -deficient cells" was investigated by chemical modification. treatment of "mg 2+ -deficient cells" by proteolytic enzymes did not affect the co-flocculation with "complete cells". photo-oxidation or mercaptoethanol-reduction of "mg 2+ -deficient cells" failed to weaken the co-flocculation with "complete cells" while treatment of "mg 2+ -deficient cells" by periodate brought about a significant loss of the co-flocculation. on the contrary, "complete cells" deflocculated by proteolysis or chemical modification of proteinaceous component failed to co-flocculate with "mg 2+ -deficient cells". these findings suggest that "mg 2+ -deficient cells" are non-flocculent because of lack of proteinaceous component essential for flocculation of cells of ifo 1958. the industrial toscano cigar production starts with the dark firecured kentucky tobacco fermentation process. during this phase the present work is a trial to study the portal serum factors which stimulate the cell proliferation of the schistosomules, aiming to find ways to block or inhibit their effects. our previous studies showed that portal serum of human and hamster (highly susceptible hosts) and a 1-50 kd fraction separated from human portal sera by ultrafiltration stimulate cell proliferation in immature schistosomules (20 days old) in vitro. for further identification of the portal serum factors in the range of 1-50 kd that stimulate cell proliferation, schistosomules were incubated in vitro in medium containing 10% fetal calf serum, 10% portal human serum or 10% peripheral human serum or their fractions separated by native electrophoresis followed by electroelution, incubations were performed in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. the results showed that human portal sera enhanced cell proliferation of schistosomules compared to the peripheral serum. this stimulatory effect was substantially reproduced by fraction separated from human portal serum with molecular weight 20.8 kd. these results may help in designing a drug or antibody therapy to block the stimulating effect of the portal serum fraction and subsequently to disturb the life cycle of the parasite at early stage of development. center of molecular biosciences, university of the ryukyus, okinawa 903-0213, japan. e-mail: naoya-s@comb.u-ryukyu.ac.jp (n. shinzato)oil strage tank sludge, mainly composed of water and solid hydrocarbons (waxes) needs to be treated when harvesting the stored oil. although the sludge treatment by microbial surfactant or microbial cracking are considered as the feasible method, microbial degradation of the waxes (ca. solid n-alkanes) have been reported in a very limited species such as acinetobacter. in addition, long-chain n-alkanes (so called paraffin waxes) are one of the major components of oil, and their resistant properties to biological attack hold up the recovery of oil-polluted environments. in this report, we have screened n-tetracosane (c24) degrading bacteria from soils in okinawan island, an unique sub-tropical area in japan, to know the bacterial diversity and their degrading mechanism.16srdna phylogenetic analysis of the isolates, totally ca 40, showed they were not only acinetobacter and pseudomonas, but also other proteobacteria (alcaligenes), actinomycetes (gordonia, nocardia, and leifsonia), bacillus, staphylococcus, and unidentified ones. they also grew not only the solid n-alkanes but also iso-alkanes, mid-chain n-alkanes as the sole carbon source. results for the biosurfactant production will also be shown. the properties of 188 environmental enterococci were studied. the strains were isolated mainly from surface and waste waters and several strains from sheep manure were also included. species identification was provided by combination of phenotypic (micronaut system, merlin) and molecular detection methods (automated its-pcr, ddl-pcr). several discrepancies were observed when comparing molecular and biochemical identification. six enterococcal species were overall identified; e. faecium and e. hirae were the most abundant ones, almost 80% of isolates belonged to these two species. the distribution of selected genes conferring virulence to enterococci (cyla, gele and esp) was investigated, the positive signal was obtained mainly for e. faecalis strains. the strains were also characterized for the possession of enterocin genes (enta, entb, entp, ent31, entl50ab) and high frequency of enterocins was observed. biosorption of three different dyes (reactive black 5, cibacron brilliant yellow, cibacron brilliant red) onto immobilized scenedesmus obliquus a microalga was investigated in a batch sys-tem. the immobilized alga exihibited the highest dye uptake capacity at the initial ph value of 2.0 for all dyes. the effect of temperature on equilibrium sorption capacity indicated that maximum was obtained at 25 • c for rb5, cby and cbr biosorption. the freundlich, and langmuir adsorption models were used for the mathematical description of the biosorption equilibrium and isotherm constants were evaluated. biocontrol properties of microbially-treated sugar beet wastes in presence of rock phosphate n. vassilev, i. nikolaeva, m. vassileva department of chemical engineering, faculty of sciences, university of granada, c/fuentenueva s/n, granada-18071, spain. e-mail: nbvass@yahoo.com (n. vassilev) the effect of soil application of sugar beet wastes (sb) treated with aspergillus niger in the presence of rock phosphate (rp) on the control of fusarium wilt of tomato were studied. two treatments and a control were used: inoculation with glomus intraradices (am), further inoculation with a. niger grown on sb + rp medium, and the control (c). application of the am fungus increased plant growth, p and n uptake and reduced disease caused by fusarium oxusporum f. sp. licopersici (fol) as compared to non-mycorrhizal control plants. soil amendment with sb + rp + a. niger resulted in 347% and 467% (versus c) higher plant shoot biomass in plant-soil experiments contaminated or not with fol, respectively. in this case, disease severity and number of fol cfu reached the lowest levels while soil phosphatase and beta-glucosidase activities increased compared to all other treatments. fol negatively affected plant root mycorrhization determined in the am treatment while the difference between the mycorrhization of plants grown in the presence and absence of f. oxysporum in sb + rp + a. niger-amended soil was insignificant (53% versus 59%, respectively). in conclusion, the fermentation mixture containing mineralized organic matter, partially solubilized rp, and a. niger biomass could be efficiently used not only in improving plant growth, nutrient uptake and properties of degraded and polluted soils, as previously reported (vassilev and vassileva, 2003) , but also in environmentally-mild management of fusarium wilt. vassilev, n., vassileva, m., 2003. appl. microbiol. biotechnol. 61, 435-440 . biological treatment processes allow for the effective elimination of charged inorganic micropollutants, e.g. a number of oxyanions, heavy metals, etc. from contaminated drinking water supplies. however, dedicated technologies have to be implemented in order to eliminate the target pollutants without changing the quality of treated water, avoiding its secondary pollution by cells, nutrients and metabolic by-products. some innovative technologies, which combine the use of membranes with the bioconversion of charged micropollutants in order to deal with the secondary water contamination problem, will be presented and critically compared. a special on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by 20% was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were 90 and 50%, respectively. while the color and aromaticity decreased by 60 and 95%, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was 0.045 h −1 while the monod constant based on the consumed tp and cod (mg/l) were 370 and 6900, respectively. advanced start-up strategy of an anaerobic three-phase turbulent bed reactor treating winery wastewaters r. cresson, h. carrère, n. bernet, j.p. delgenès laboratoire de biotechnologie de l'environnement, institut national de la recherche agronomique (inra), avenue des etangs, 11100 narbonne, france. e-mail: cresson@ensam.inra.fr (r. cresson)the objective of our study was to compare two start-up strategies for an anaerobic biofilm process, to create an effective biofilm and increase the organic loading rate (olr) as quickly as possible. two methanogenic three-phase biofilm reactors have been started, using the same operational parameters (solid hold-up ratio, gas velocity of 1 mm s −1 ), in order to test two different strategies:• maximal load strategy (reactor a): the olr is increased as long as the global amount of removed cod (biogas production) increased. • maximal removal strategy (reactor b): the olr is increased stepwise as soon as the cod removal rate reaches 80%.both reactors have been operated for 90 days, until a volumetric olr of 20 g cod l −1 j −1 , with more than 90% of carbon removal. the total amount of cod removed and methane produced were higher in reactor b (19.6 and 32.2%, respectively). in both reactors, the short hydraulic retention time (hrt) applied during all the experiment caused a rapid wash-out of planktonic bacteria and an exclusive use of the substrate by the attached micro-organisms, which accelerates the biofilm growth. the lag-phase was reduced to approximately 7 days. the reactor submitted to repetitive disturbance by the maximal removal strategy appeared to be more robust when confronted to perturbation like organic overload or nutritional deficiency. experiments have demonstrated capability and the efficiency of the aggressive strategy for controlling anaerobic bioreactor start-up. sustainability is the generally accepted paradigm for future industrial development. the re-integration of waste products into production processes is a major aspect of environmental sustainability. in this study the use of sugar cane molasses is being investigated for the production of bioplastics by mixed microbial cultures, with the added possibility of parallel biohydrogen production. polyhydroxyalkanoates (phas) are polyesters synthesized by bacteria and accumulated as granules in the cytoplasm. studies conducted by this group have shown that mixed microbial cultures subjected to dynamic feeding conditions may accumulate phas up to 80% cell dry weight, a value close to that obtained for pure cultures. volatile fatty acids are good substrates for the production of phas by mixed cultures. on the other hand, sugar molasses, with a very high sugar content (about 50% dry weight), can produce organic acids by fermentation. the two-stage process being implemented in this study includes a molasses fermentation step, in which the high sugar content of the molasses is converted into volatile fatty acids (vfas), and a pha production step, in which the vfas serve as the precursors to the formation of phas under dynamic feeding conditions. moreover, hydrogen can be produced by anaerobic bacteria from carbohydrate-rich substrates giving organic fermentation end products, h 2 and co 2 . to optimize the production of both high-value products, design of experiments (doe) is being used to elaborate a set of experiments to study the effect of ph, hydraulic retention time and organic loading on both the organic acids distribution (which will serve as precursors for pha production in the second step) and h 2 production in the acidogenic fermentation reactor (a 1 l cstr). preliminary results show that the effluent of the acidogenic reactor fed with 10 g/l total sugars and operated at ph 7 and d = 0.1 h −1 (composed mainly of acetate and propionate) can be successfully fed to a polymer-accumulating mixed culture. under these conditions, the h 2 production yield has been estimated at 3.9 mol h 2 /mol sucrose. vanillin is a flavour compound used in food industry, fragrances and pharmaceutical preparations, which is nowadays mainly produced by chemical synthesis. the increased demand of natural products for the food industry as well as the high cost of natural vanillin extracted from vanilla pods has recently stimulated the research for alternatives to produce this compound by a natural way. the microbial transformation of ferulic acid, a phenolic compounds from lignin degradation, is recognized as being the most interesting alternative to produce natural vanillin. the combined effects of initial ferulic acid concentration (s 0 ) and biomass concentration (x 0 ) on vanillin production by resting cells of escherichia coli strain were investigated using response surface methodology. e. coli jm109/pbb1 a recombinant strain producing key enzymes of ferulate catabolic pathway from p. fluorescens bf13 (feruloyl-coa synthetase and feruloyl-coa hydratase/aldolase) was utilized in this work. a 3 2 full-factorial design was employed for experimental design. the results shown a possible inhibition phenomena at a vanillin concentration of about 0.1 g l −1 leading to the accumulation in the fermentation media of secondary compounds like vanillic acid and vanillin alcohol. removal of dissolved nutrients from wastewater using a microalgae biofilter line christensen, suvina sooknandan, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, odense, a microalgae biofilter can be used for treatment of wastewater from landbased fish farms in order to remove excess amounts of dissolved nutrients such as nitrate, ammonium and phosphate. a bubble column bioreactor has been developed for cultivation and characterization of microalgae. this type of bioreactor is equipped with a control system that enables online determination of the photosynthetic quotient and optimization of light intensity. furthermore the bioreactor has a dualsparging system simultaneously allowing adequate mixing and high gas-liquid mass transfer coefficients. different species of microalgae have been cultivated in batch and fed batch cultures to characterize growth and ability to take up the different dissolved nutrients. the specific growth rate and substrate uptake rate have been determined to compare and select the algal species most suited for use in a biofilter. additionally the composition of lipid, protein and carbohydrates has been measured to determine the nutritional quality of the algae when used as animal feed. at present, biological nitrogen-removal is mostly carried out through several complicated steps. to simplify the present systems for nitrogen-removal, we have investigated a new nitrogenremoval bioreactor using packed gel envelopes capable of simultaneous nitrification and denitrification. the envelope consists of two plate polymeric gels with a spacer in between. ammonia oxidizer, nitrosomonas europaea and denitrifier, paracoccus denitrificans are co-immobilized in the plate gels. when the envelopes are exposed to wastewater containing ammonia, the immobilized n. europaea oxidizes ammonia to nitrite in the outer aerobic surfaces of envelopes. at the same time, as ethanol solution is injected into the internal anaerobic spaces of envelopes, the immobilized p. denitrificans reduces the nitrite to nitrogen gas using the ethanol solution as an electron donor for denitrification. in this way, the envelopes can remove ammonia from wastewater in a single step. we have already reported advantages of our bioreactor in laboratory-scale experiments. in this study, we show our large-scale bioreactor (water volume 1.8 m 3 ) could treat three kinds of wastewater derived from coal power plants. ammoniacontaining wastewater that occurred regularly in a coal power plant was continuously treated with the bioreactor using thirty envelopes for over a year. the bioreactor could remove more than 90% of total nitrogen at hydraulic retention time (hrt) of 24 h. at hrt of 4 h, the bioreactor accomplished a maximum rate (the transformation of nh 4 + to n 2 ) of 6.0 g n/day m 2 of the envelopes' surface. the performance was equivalent to that obtained in the laboratory-scale experiments. furthermore, our bioreactor showed similar nitrogen-removal performances when it treated nitrate-containing wastewater occurring regularly and condensed ammonia-containing wastewater occurring at irregular intervals in coal power plants. these results show that our bioreactor can treat various wastewater containing nitrogen in coal power plants. thus, our concept is effective to simplify the large-scale systems in coal power plants and the other plants. in order to establish an environmentally friendly process for the treatment of metal containing waste, in a portuguese refinery a process involving sulphur oxidizing acidophilic microbes is being considered. bioleaching of metal containing bottom ash, from fluidised bed incineration of sludge resulting from the refinery water treatment station, was performed using a sulphur oxidising acidophilic culture isolated from an acid pool resulting from the weathering of sulphur piles from the claus plant. this sample served as inoculum for liquid medium cultures with 1% sterile sulphur flowers as source of energy. application of monod kinetics to adapted culture growth of free cells presented a value of µ = 0.124 day −1 . yield of sulphur conversion to sulfate after 17 days was η = 78%. in the presence of bottom ash from the incineration of refinery sludges µ = 0.141 day −1 and the yield of sulphur conversion was η = 67.5%. a η fe = 90% removal of iron is obtained from the treated ash. x-ray fluorescence spectroscopy of the solid residue revealed a total removal of metals namely, v, cu, ni, zn and most of the fe after 15 days of bioleaching. the present of heavy metals in the environment is a serious problem. they are commonly present in effluents from mining and industrial activities. usually, chemical conventional methods are very expensive and they have limitations when heavy metals are in low concentrations. at the moment the interest increases for processes that involve microorganisms as alternative method. some effluents present heavy metal sulphates which are soluble compounds. sulphate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulphate as an electron acceptor and generate hydrogen sulphide. hydrogen sulphide reacts with heavy metal ions to form insoluble metal sulphides that can be easily separated from a solution. the purpose of this work was to evaluate the ability of srb to reduce cr(iii), ni(ii) and zn(ii) in artificial contaminated solution. desulfovibrio vulgaris and desulfovibrio sp. strains has been tested in this study. batch cultures was carried out in 50 ml sealed bottles with different concentrations of studied metals (1-20 mg/l), with 10% of inoculum bacterial and adapted to postgate's medium c. a gaseous nitrogen current was employed to purge oxygen and obtain anaerobic conditions. the assays were incubated statically represented very low portion (less than 4-5%) of the total bacterial community at all temperatures tested. schistosomules of schistosoma mansoni (20 days old) were incubated in rpmi 1640 medium containing 10% fetal calf serum, 10% hamster portal venous or 10% hamster peripheral venous serum (highly susceptible host) or 10% rat portal venous or 10% rat peripheral venous serum (poorly susceptible host) in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. also the rate of cell proliferation of s. mansoni were assessed in vivo in hamster to study the cell proliferation in the natural ontogeny of the organism. the rates of cell proliferation as expressed by brdu labeling indices (blis) were determined as a function of time of incubation by immunohistochemistry using monoclonal antibody to brdu. compared to schistosomules cultured in presence of rpmi plus 10% fetal calf serum, blis were increased by 41% in the presence of hamster portal, but not in peripheral serum. while in case of rat, no significant changes were observed in the blis in both portal and peripheral sera. the experiment was repeated using hamster portal and peripheral sera containing different schistosomal igg antibody titres. the results showed decreased values of blis compared to sera which did not contain the schistosomal antibody(ies). the in vivo results revealed that there was no cell proliferation of s. mansoni schistosomules (6 days old) in the lungs. cell proliferation was detected in schistosomules of 17 days old and the results revealed a significant decrement in the brdu labeling indices (blis) with the increase of the age of schistosomules in vivo. the results indicated that hamster portal venous serum (highly susceptible host) could have stimulating factor(s) for schistosomule cell proliferation which is not found in rat (poorly susceptible host) and the presence of antibody(ies) greatly inhibit the cell proliferation. this could be due to the blocking of some portal serum factors, which stimulate the cell proliferation by the antibody(ies). the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g −1 ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph, the maximum adsorption at 50 ppm was 88.52% equal to 11.8 mg dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of 8-12). the effect of contact time was studied at initial concentration (50 ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after 15 min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. compared to other european nations, the austrian population shows a low level of knowledge in biosciences and a strong denial of gene technology (1). the austrian non-profit organisation dialog<>gentechnik, a scientific society, organizes various activities to raise awareness for the "hot topics" in the life sciences. according to its principle of independence, all activities are funded publicly. projects on behalf of the austrian authorities and international cooperations demonstrate credibility and trust in dialog<>gentechnik (2). a few examples will be presented. dialogue with the public: on the occasion of the first anniversary that the gmo labelling rules became effective, the action "gene technology on my plate" is performed austrian wide in shopping centres. here, consumers are informed about health and labelling aspects of gm food. two days of open discussions were organized in the context of the austrian genome research program gen-au (3): topics were "gene diagnosis" (2002) and "genome research-what is in it for me?" (2004) . an open lab is currently set up in vienna to offer "hands on" experience in life sciences for everybody. motivating students: in the very successful gen-au summer school (3), high school students spend 3-4 weeks in the lab and work with scientists. the best documentations are awarded. in an innovative project, student groups (age 16-18) work on the topics stem cells and cloning and develop units of an elearning course which will be accessible for all austrian schools in the near future. engaging stakeholders: dialog<>gentechnik manages interdisciplinary wor king groups that develop leaflets, brochures and questionnaires on various aspects of gene diagnosis. four products are currently distributed to the public and to health services. (1) european commission. eurobarometer 55.2, december 2001; (2) www.dialog-gentechnik.at; (3) www.genau.at. the aim of this paper is to compare the attitudes of the urban consumers with professionals towards to new technologies, especially to biotechnology. it was tried to find out in which area, medical, agriculture or industry, people can accept biotechnological developments and in which area not accept. key: cord-313694-p2sgaypq authors: west, christopher m. title: current ideas on the significance of protein glycosylation date: 1986 journal: mol cell biochem doi: 10.1007/bf00230632 sha: doc_id: 313694 cord_uid: p2sgaypq carbohydrate has been removed from a number of glycoproteins without major effect on the structure or enzyme activity of the protein. thus carbohydrate has been suggested to underly a non-primary function for proteins, such as in relatively non-specific interactions with other carbohydrates or macromolecules, stabilization of protein conformation, or protection from proteolysis. this non-specific concept is consistent with both the general similarity in carbohydrate structure on very diverse glycoproteins and the frequent structural microheterogeneity of carbohydrate chains at given sites. the concept is supported in a general sense by the viability of cells whose glycosylation processes have been globally disrupted by mutation or pharmacological inhibitors. in contrast to the above observations, other studies have revealed the existence of specific, selective receptors for discrete oligosaccharide structures on glycoproteins which seem to be important for compartmentalization of the glycoprotein, or the positioning of cells on which the glycoprotein is concentrated. sometimes multivalency in the carbohydrate-receptor interaction is crucial. there are additional possible roles for carbohydrate in the transduction of information upon binding to a receptor. the possibility of specific roles for carbohydrate is supported by the existence of numerous unique carbohydrate structures, many of which have been detected as glycoantigens by monoclonal antibodies, with unique distributions in developing and differentiated cells. this article attempts to summarize and rationalize the contradictory results. it appears that in general carbohydrate does in fact underlie only roles secondary to a protein's primary function. these secondary roles are simple non-specific ones of protection and stabilization, but often also satisfy the more sophisticated needs of spatial position control and compartmentalization in multicellular eukaryotic organisms. it is suggested that there are advantages, evolutionarily speaking, for the shared use of carbohydrate for non-specific roles and for specific roles primarily as luxury functions to be executed during the processes of cell differentiation and morphogenesis. new information about the significance of protein glycosylation is rapidly accumulating. this can be attributed to advancements in methods to detect and isolate new structures, to determine structure, and to modify carbohydrate structures experimentally. nevertheless, the field continues to experience profound unrest. many studies have failed to un-cover significant cellular or molecular consequences for dramatic changes in carbohydrate structures. other studies have pointed to isolated specific roles but no sweeping generalizations of function have been possible. interpreters of the former studies would attribute apparent specific effects to secondary consequences of changes in carbohydrate on protein structure. interpreters of the latter studies would argue that the former studies simply failed to ask the right questions of function. it is clear that there is much to be done to extend our knowledge for this particular type of posttranslational modification. the purpose of this article is to outline the scope of investigations presently being carried out in this subject area and to generalize the possible significance of these new findings. in particular, this article will attempt to compare and contrast the two prevailing views in the field. as intimated above, one view is that glycosylation plays a relatively non-specific role in maintaining and preserving polypeptide function. the alternate view is that carbohydrate structures participate in numerous specific interactions with discrete protein receptors, and that these interactions lead to predictable modifications in the localization or activity of the glycoprotein. these opposing views will be simply referred to as the non-specific and specific models. the importance of this issue is dramatized by the fact that nearly all secretory, extracellular matrix, cell surface and lysosomal proteins are glycosylated. there are many published comprehensive reviews on aspects of this subject (1-3, 14, 20, 43, 105, 110, 115, 130, 175 ) and the reader is referred to these for more detailed summaries and documentation. since this article will in part serve as an introduction and a background to the others in this volume, an overview of general features of the glycosylation process has been provided to precede the summary of existing and new ideas in the field which follows. oligosaccharides linked to asparagine occur in cellular slime molds, yeast, higher plants, insects, and humans, and several essential features of these structures are highly conserved throughout this phylogenetic range (14) . typically though not invariably, a glc3man9glcnac2 structure is synthesized on a polyisoprenoid derivative (dolichol-p-p) and transferred en bloc to asparagine residues on polypeptide acceptors forming an n-glycosidic linkage (hence n-linked). the three glcs are usually trimmed by a tandem pair of a-glucosidases to yield a high mannose n-linked structure. the highmannose structure is variably modified, beginning with removal of mannoses in a typical sequence by a tandem pair of o~-mannosidases. one pathway retains most mannoses but permits additional substitutions of e.g., sulfate and/or phosphate. a second pathway trims down to mansglcnac 2 and makes limited additions such as glcnac, and sometimes gal and core-linked fuc. these are referred to as hybrid structures. the third pathway continues the second pathway to build complex structures usually terminated by sialic acid. there can be two, three or four chains emanating from the branching mannose (and secondary branching mans) and these structures are accordingly referred to as biantennary, triantennary or tetraantennary complex nlinked oligosaccharides. the sialic acids, which appear not to precede echinoderms in their evolutionary appearance, comprise a diverse family of sugars (100) . numerous additional substituents including sulfate, phosphate, methyl groups and others have been documented (8, 9, 161) . each step in the glycosylation pathway is catalyzed enzymatically; there is usually a unique enzyme for each linkage position formed as monosaccharides are accreted or deleted (47) . n-linked glycosylation is not template driven. only the first step might be considered so, inasmuch there is a necessary but not sufficient requirement of an xasn-x-ser/thr-x sequence for the oligosaccharide acceptor. protein structure or corfformation has also been shown to influence whether a site becomes glycosylated as well as whether it becomes high mannose, hybrid, or complex (4, 7) or whether it receives a phosphodiester-linked glcnac-po4 (7) or galnac-so 4 (175). a relationship between position in the polypeptide chain (distance from n-to c-terminus) and whether an oligosaccharide becomes high mannose or complex has been observed (6) . finally these relations are not absolutely determinate, for spontaneous heterogeneity of carbohydrate structures at particular positions has been documented (12, 5, 37) . changes in structure also result from extracellular influences (see below). the other major structural class of oligosaccharides is o-linked on ser on thr (25, 26) . a largely distinct set of enzymes catalyzes the synthesis of these structures. these oligosaccharides are built by monomer addition from sugar-nucleotide donors directly on the polypeptide rather than on a lipid precursor. trimming does not appear to play a major role. gal and galnac are typical proximal sugars and man is rarely found. o-linked structures are found both in branched and straight chain forms. o-linked structures often resemble the oligosaccharides of glycolipids (45) , and in fact these may be built from shared enzymes. an experimental consideration of the role of these oligosaccharides on protein must inevitably involve an examination of homologous glycolipids. although the n-linked and o-linked structures discussed above account for 95-100% of incorporation of labelled sugar precursors into cellular glycoproteins, numerous additional small classes of oligosaccharides are also found. for example, galglc is found on hydroxylysine and hydroxyproline of collagens. glcnac-pon-ser is found on a lysosomal enzyme in d. discoideum (18) and glcnac-asn is found on certain glycoproteins of what is probably the nuclear envelope (19) . archeobacterial proteins are also glycosylated. a major class of these glycosylations shares some homology with n-glycosylation and may be evolutionarily related (10, 38, 11) . a polyisoprenoid lipid carrier is utilized prior to en bloc transfer. the ancient character of the mammalian n-linked glycan suggests important functional attributes for his posttranslational modification. several physical methods have been applied to ascertain the conformation of oligosaccharides on proteins (30, 31) . studies in model systems have implied that there is a preferred conformation at least for the core sugars of n-linked oligosaccharide chains. this conformation is stabilized by hydrogen bonding with the polypeptide chain, which orients the structure, and limited intrachain hydrogenbonding. the existence of preferred conformations implies that shape may be important for specific interactions of protein-linked carbohydrate with receptors. n-linked glycosylation appears to occur only in the rough endoplasmic reticulum (rer) and hence only to proteins which possess a signal sequence (150) . the process is co-or post-translational (14) . mannose trimming begins in the rer but can be concluded in golgi. modification of high-mannose forms or conversion to hybrid or complex forms occurs in the golgi. nearly all glycosylation is believed to be restricted to these two compartments. certain steps in glycosylation have, however, been postulated to occur in the ser, secretory vesicles, nuclear envelope and cell surface (e.g., 43, 139, 173) . of course, deglycosylation occurs in lysosomes as a part of protein turnover (102) . o-linked glycosylation appears to be initiated and concluded in the golgi (25, 26, but see 27). as for n-linked glycosylation, there has been some resolution of processes between the cis, medial and trans elements of the golgi apparatus. nearly all proteins which pass through the rer and golgi become glycosylated in some form. one exception is the secretory protein serum albumin (32) , which is synthesized in hepatocytes. there is no evidence for a glycosylated precursor. it should be recognized that at least some glycoproteins, e.g., cell surface transferrin receptor, can probably be reexposed to the golgi compartment subsequent to primary passage through this organelle (112) . this provides new opportunities for glycosylation subsequent to initial synthesis. occasional reports have appeared in the literature regarding the presence of glycoproteins in cytoplasmic compartments topologically discontinuous with the lumen of the rer and golgi. histones and other nucleoplasmic proteins (68, 77, 78) , a ribosomal protein (79) and certain mitochondrial proteins (132, 174) have been suggested to be glycoproteins. these findings contradict the dogma that glycosylation is strictly an rer and golgi dependent event unless it is postulated that modified proteins subsequently translocate across the membrane back to the cytoplasmic space. further work remains to be performed to confirm the existence of oligosaccharides on these proteins and to characterize their structure as a means for understanding their enzymatic basis. it must be appreciated that the description of the glycosylation pathway as we now understand it suggests many interesting kinds of regulatory mechanisms. for example, each step of the pathway can be influenced by enzyme synthesis and turnover, acceptor accessibility, as well as the supply of donor sugar, cofactors such as lipids, divalent cations, etc. the question of accessibility is particularly interesting because sequentially acting enzymes tend not to be mixed but are distributed vectorially through the rer and the multiple compartments of the golgi. movement of polypeptides through these compartments appears to be mediated by atp-dependent dissociative movement of vesicles (142) . thus glycosylation may conceivably be regulated by the pathway of movement of the acceptor protein. there is also an indication that some glycosyltransferases form supramolecular complexes. thus, it is possible that certain carbohydrate structures are dictated not only by vesicle movement, but also by which enzyme cluster initially captures the acceptor protein (122) . ideas about the role of protein glycosylation may be divided into two general groups: 1) those which imply specificity associated with particular carbohydrate structures, and 2) those which stress the general similarities of oligosaccharide structures, and the presence of microheterogeneity in the structure of particular oligosaccharides, in suggesting that combined carbohydrate bulk creates a common microenvironment for resident polypeptides much as the lipid bilayer provides an environment for integral membrane proteins. most effort has been devoted to the former category of ideas because of the greater ease of formulating hypotheses of this type. a non-specific role for oligosaccharides that is dependent on their generalized chemical properties has been suggested in several contexts. it was appreciated early on that carbohydrate afforded a stabilizing effect on protein solubility and against heat denaturation (see, e.g. 34, 38 and 35) . it was also realized that oligosaccharides afford resistance to proteolytic degradation (166) , presumably by a mechanism of steric hindrance, and these ideas have been reinforced in numerous studies up to the present (e.g., 12, 101, 102) . there are in addition several instances where carbohydrate is known to interfere sterically with protein-protein interaction not involving proteolysis (74, 75) . predating this understanding, it was known that carbohydrate is a major constituent of the extracellular matrix in the form of polysaccharides known as glycosaminoglycans (gags) (3). in the past 15 years, it has been discovered that most gags are covalently attached to polypeptides, resulting in the formation of glycoproteins which, due to the specialized nature of the attached carbohydrate, are known as proteoglycans. gags are anionic polymers which hydrate to form enlarged domains which can interact with one another, as well as with polycationic polymers such as collagen and with other glycoproteins (3) . a three dimensional space is thus defined by polysaccharide, protein, and the bound water which provides an environment for cells. convection and diffusion are modified, and fluctuations in ionic strength and ph are minimized. these structures depend on the polyanionic nature of the carbohydrate, and the general chemistry rather than the specific monosaccharide identities and sequences may be dominant in governing the properties of the system. carbohydrate oligomers are also known to selfassociate in the formation of mucous gels and in yeast and plant cell walls (152) (153) (154) 22) . in the former, the anionic nature of the carbohydrate appears to be important although in the algal and yeast gels the interactions are non-coulombic. the collective oligosaccharide density on cell surface proteins is sufficiently great so as to invite comparison with the carbohydrate density of the matrix space. this observation has led to the suggestion that cell surface carbohydrate might also establish a convection and diffusionqimited region around the cell in an area thick enough to be named, in certain cell types, the glycocalyx or fuzz. the polyanionic nature of the carbohydrate found on some cell surfaces probably results in marked cation and ph differences in this region relative to the surrounding medium (177). it has recently been proposed that the heterogeneity of carbohydrate structure found on proteins of this region might result in minor differences in protein function, leading to a broader range in the ionic strength and ph values optimal for protein function (36, 37) . as such, protein oligosaccharides might subserve a homeostatic process used by cells to maintain their viability in potentially changing milieus. it has also been suggested that a layer of non-immunogenic protein-associated carbohydrate may play an immuno-protective role for underlying cell surface antigens, especially on neoplastic cells (27) . plasma membrane carbohydrate, owing to intercarbohydrate associations, might also influence the lateral diffusion of certain glycoproteins. inter-molecular association at the cell surface has been suggested to be so extensive in an archeobacterial system that it can influence the shape of the included cell (11) . an extension of intermolecular association to contact between two cells in close mutual proximity leads to a possible mechanism for cell adhesion. interaction of oligosaccharides might be due to an apposition of the hydrophobic faces of two monosaccharides such as mannose (126) . implicit in this model is the idea of a large number of weak interactions equalling in strength a smaller number of strong interactions. this mechanism is feasible in polymeric systems with repreating similar subunits. cell surface oligosaccharides could simulate a polymer inasmuch as they are coanchored in the same membrane. the interactions involved in these phenomena may be relatively nonspecific. the notion of a 'non-specific' role for carbohydrate has received perhaps its strongest support from studies on cells whose glycosylation processes have been globally altered by mutation or drugs. the most dramatic studies have involved mutant cells selected on the basis of resistance to the toxic effects of certain lectins. many mutations leading to loss of lectin recognition have been identified in cho and other cells (20) . some have been characterized and in fact most steps in the pathway to complex, n-linked glycan formation are mutable. the striking finding is that mutant cells are largely viable in culture. most mutant cho cells remain adherent under appropriate conditions, proliferate, and are tumorigenic. mutants defective in early steps leading to the formation of high-mannose glycans are also viable (24) . this result was not, however, found in a yeast mutant (40) . selected activities of cells have also been investigated in some mutant strains. for example, the half-life of cell surface proteins was found not to be dramatically affected (23) . similar results were found following treatment of cells with sialidase (107) . a considerable literature details the consequences of carbohydrate modification on the secretory process. though several examples of apparently specific effects will be enumerated in a later section, the number of cases where little or no effect is found is noteworthy (82, 83, 98, 161) . in some cases detailed kinetic analysis has found only small effects on secretory rates (159) . similar results have been found regarding accxumulation of receptors in the plasma membrane and in their function at that site (88, 99, 90, 164) . genetic modification of the gene for the ldl receptor has deleted a sequence required for a cluster of o-linked glycans and even for this poorlystudied glycan type no consequence on function could be detected (163) . mutants in the enzymatic pathway of o-glycosylation still place the ldl receptor, albeit in a less stable form, on the cell surface (165) . these negative results have suggested that carbohydrate cannot underlie essential housekeeping functions in cells. this implies a non-specificity of function in the sense that obvious functions cannot specifically be attributed to the carbohydrate when it is present. this represents a shift in meaning from the above definition of nonspecificity inasmuch as specific receptors may be involved in functions non-essential in the tissue culture environment. thus it has alternatively been suggested that carbohydrate may be more important for particular differentiated functions, which are not expressed in cell culture systems such as the cho cell line. there are but few investigations into this possibility. for example, in the cellular slime mold d. discoideum, mutations have eliminated two developmentally regulated glycoantigens (28, 29) . nevertheless, the cells develop reasonably normally to produce fruiting bodies and viable spores. the result may imply that carbohydrate is either nonspecific or subserves subtle modulatory roles or that their roles are redundant with alternate (or back-up) mechanisms. alternatively, the value of these carbohydrate structures may only become evident under the competitive conditions of natural survival outside of the laboratory. although the above ideas have been grouped together under 'non-specific mechanisms', this is not meant to imply that carbohydrate structure per se is completely irrelevant. 'non-specific' should be interpreted relative to the types to be discussed below, were specific, saturable recognition mechanisms related in principle to enzyme-substrate or hormone-receptor interactions are apperently involved. in any case, the non-specific mechanisms tend to rely on the bulk chemical properties of the participating oligo(poly)saccharides. this phrase implies interactions between receptors and ligands (usually the oligosaccharide) of saturability and high selectivity and avidity. selectivity can be achieved by high affinity monovalent interactions or multiple weaker interactions. selective interaction is often equated with molecular or, on a more complex level, cellular recognition. the phrase also implies that a specific function can be attributed to the carbohydrate structure. perhaps the strongest indication for specific roles are the variety of structures which have been detected on protein carbohydrate. the following sections outline how several characterized structures are thought to function in specialized processes. in some cases, a receptor protein capable of recognizing a given structure is known. in 1965 eylar (80) presented the idea that proteil' glycans constitute a 'chemical passport' signalling export of a protein to the cell surface or for secretion. though this idea has since been supplanted by the 'signal hypothesis' of blobel and sabbatini (150) , it has served as a precursor to more refined ideas of chemical signals leading to the accumulation of glycoproteins in specific compartments (81) . for example, a mannose-6-phosphate moiety has been suggested to be responsible for associating proteins with receptors which in turn lead to protein accumulation in lysosomes (134) . the key to this understanding was the finding of mutant cells which appeared to secrete constitutively certain lysosomal enzymes that were distinct in their oligosaccharide structures: namely that normal enzymes possessed multiple phosphate residues 6-1inked to non-peripheral mans in high-mannose structures, and that mutant enzymes lacked these phosphate moieties. this suggested that there was a man-6-po 4 receptor in membranes of the golgi, and plasma membrane (since lysosomal enzymes can also be absorbed from the medium and translocated to lysosomes), which was responsible for binding the man-6-po4 recognition marker on certain glycoproteins. two such receptors, one of 215kd and the other of 46kd, have been discovered (157, 158) . these are located in the golgi and/or on the cell surface in concordance with their proposed functions. from studies on receptor movement and the effects of inhibitors there is fairly good evidence that the man-6-po 4 moiety, together with one or both of these receptors, plays a recognition role in the intracellular routing. however, not all lysosomal enzymes seem to be involved because some copies of the altered enzymes still accumulate in mutant cells (157) , and some lysosomal enzymes are not affected. it has been suggested that there is lysosomal heterogeneity with only one class of lysosome employing this recognition marker. it is nevertheless apparent that for some lysosomal enzymes in some cell types the man-6-po4 receptor does not function alone, even as an intermediate signal, for lysosomal routing. there must be other recognition signals on the enzyme polypeptide which lead to its potential lysosomal accumulation. it is interesting to note that the distinctive carbohydrate structure plays a compartmentalization role separate from the enzyme's function in catalytic degradation. furthermore, carbohydrate is employed to perform a common role shared by multiple proteins. a posttranslational modification such as glycosylation is well-suited to the demands of a mechanism for compartmentalization. the rer, golgi, smooth endoplasmic reticulum, and nuclear envelope are also intracellular destinations for proteins which traverse the rer cotranslationally. these compartments contain numerous characteristic and uniquely distributed glycoproteins, but there is not enough information to evaluate the possibility that carbohydrate structure is involved in routing or maintaining the positions of the glycoproteins. several cases of rer-specific glycoproteins have been considered. cells infected with rotavirus synthesize, under control of the virus, several proteins including vp7, an integral membrane glycoprotein whose distribution is restricted to the rer (95). this protein together with the membrane of the rer form a temporary coat for the virus. in a genetically modified form of vp7, a hydrophobic region, which presumably serves as a transmembrane anchor defining the proteins as an integral membrane glycoprotein, was deleted. the truncated gene encoding vp7 was introduced to cells by transfection. truncated vp7 was synthesized, but was then secreted despite this fact that glycosylation at its single site toward the c-terminus retained its high-mannose character. these results show that the key factor which retained the glycoprotein in the rer was the transmembrane sequence and was not related to the glycosylation site. the results would also imply that glycosylation is not essential to the process of secretion. several nuclear envelope glycoproteins have been found to express an unusual carbohydrate structure consisting of glcnac-asn (19) , which is an expected product of endo-glycosidic degradation. it is at present unknown where this structure is synthesized and thus whether glycosylation precedes or follows arrival into the nuclear envelope. the secretion of protein appears to involve at least two pathways: a constitutive pathway and a pathway which includes a secretory vesicle which is stored cytoplasmically (144) . a range of high mannose and complex bi-, tri-and tetra-antennary nlinked structures has been observed on both secretory and plasma membrane associated glycoproteins. most studies have been directed, however, toward n-linked glycans since these structures are easier to modify and detect than are olinked and other, unknown structures. one approach to evaluate the potential role of nlinked glycans has involved the use of pharmacological inhibitors of several steps, namely the initial steps of synthesis, and deglucosylation and demannosylation. tunicamycin blocks n-linked glycosylation completely (as well, possibly, as other types of glycosylation (15)), whereas deoxynojirimycin and swainsonine block certain steps leading from high mannose structures to the complex type. though the effects are not simple it tentatively appears that lack of n-linked glycosylation has a measureable affect on the rate of secretion of some proteins from hepatocytes or hepatoma cells (82, 85, 84) . secretion from these cells appears not to involve a stored secretory vesicle. modification of glycosylation by deoxynojirimycin or swainsonine appears in some cases to affect the rate of movement from the rer to the golgi and, in other cases, from the golgi to the extracellular space (162, 159, 84) . some glycoproteins and albumin are not affected at all. this result has led to speculation that there is a receptor which can recognize and retain glycoproteins bearing the high mannose marker. this mechanism would not be universal for all proteins. alternatively, these differences may reflect spurious differences in rates of dissociation from glycosylation enzymes. it seems reasonable to consider that glycans may be designed to play a modulatory role in the kinetics of secretion for only selected proteins. since secretion of these proteins is not regulated by storage in a secretory vesicle, this mechanism may be used by the cell as a finetuning mechanism for controlling release of protein. it is plausible that the passive constitutive pathway for secretion (95, 144) does not involve carbohydrate per se, but that modifications of this pathway or its kinetics do. further work remains to be done to compare carbohydrate structure on different glycoproteins and to consider the possible involvement of neighboring polypeptide regions in the interaction with a hypothetical receptor. entry of protein into stored secretory vesicles requires a specific signal as shown by the observation that some hormone polypeptides produced by pituitary cells are stored whereas others are constitutively secreted. it has been observed that peptide hormones which are stored in pituitary gonadotropes and thyrotropes are sulfated whereas related peptide hormones released from placental cells, but not stored there, are not sulfated. this sulfate has subsequently been shown to reside at one or more non-reducing termini of n-linked complex oligosaccharides in the position usually occupied by sialic acid. this substitution may be directed by a substitution of the galnac for underlying gal (156) . the functional significance of the relationship between galnac-so 4 and delivery to a vesicle which is stored cytoplasmically prior to release by a secretagogue remains to be explored. an exception to this structure-function correlation is posed by human fsh, which is stored in human pituitary gonadotropes but, unlike the lh stored in these cells, is not sulfated. possibilities consistent with a role for galnac-so4 are that fsh may segregate to a discrete vesicle population relative to lh, or that the two homones colocalize and that some intermolecular association substitutes for the lack of this carbohydrate modification. there is no concensus oligosaccharide structure discovered thus far which distinguishes plasma membrane from secreted glycoproteins. although this may suggest the possibility that plasma membrane and secretory glycoproteins may in fact share a common signal, on account of their topologically equivalent destinations, inhibitor experiments have shown that an absence of n-linked glycans does not consistently block the appearance of glycoproteins on the cell surface (88, 90) . there are, however, some instances of inhibition (86, 87, 89, (91) (92) (93) , which allow for the possibility that nlinked glycans may act as a signal or modulator for the export of specific proteins to the plasma membrane. alternatively, the failure of a few altered glycoproteins to be exported may be explained by enhanced proteolysis, by a secondary effect on conformation, or by the display of another signal elsewhere on the polypeptide (165) . a special class of extrinsic plasma membrane glycoproteins is known, however, for which a glycan structure appears to be significant for localization. this includes glycoproteins which are recognized by the cell surface protein ligatin, which recognizes a man-6-po4-1-glc, linked to a highmannose backbone (160) . this structure appears to be important for the cell surface association of these glycoproteins since glc-i-po4 can elute them from cells and a high concentration of ca + +, which can elute ligatin, also elutes the associated glycoproteins. glycoproteins associated by this mechanism were first recognized ultrastructurally on the surface of the intestinal mucosa of the suckling rat, but have since been discovered on the surfaces of a variety of cell types in sea urchin, chicken and mouse. it is unclear whether the gic-i-po 4 marker serves as a routing signal for export to the cell surface, using ligatin as an intermediate carrier, or whether it serves a recognition role for secondary association with the cell surface once secretion in soluble form has already occurred. though the identity of many of the ligatin associated glycoproteins is unknown, the first to be discovered in the intestinal mucosa was j3-nacetylhexosaminidase. in this case, the primary function of the glycoprotein can be viewed as an enzymatic one. the appended carbohydrate appears to serve a role in localization, contributing to the spatial organization of the system. consideration of the possible role of carbohydrate in other molecular associations with the plasma membrane will be considered below in sections on hormone-receptor interactions and cellsubstratum (e.g., extracellular matrix) and cell-cell adhesion. it has been proposed that specific carbohydrate/protein interactions might designate the localization of secreted glycoproteins to specific sites in the extracellular matrix (3). this idea has received support recently from the detection of animal lectins in matrices, basement membranes, and mucous secretions (140) . lectins, which are multivalent carbohydrate binding proteins with oligosaccharide specificity, are believed to crosslink other molecules to contribute to, e.g., the viscosity of certain mucous structures (44) . binding of matrix proteins to carbohydrate of gags has been reported (21, 32) . a recent report suggests that the neural adhesion protein n-cam has a binding site for heparan sulfate-like gags (171) . in the early 1970s, ashwell and morrell demonstrated that desialylation of serum glycoproteins, resulting in the exposure of a penultimate galactose in mammalian species, caused rapid removal of proteins by hepatocytes once these altered proteins were restored to the blood (reviewed in 105, 106) . this suggested that normal turnover of serum proteins is regulated by the structure of their glycans, which would be regulated by interaction with sialidases. though a carbohydrate binding receptor for (non-reducing) terminal gal/galnac has been characterized in hepatocytes, it has not been possible to prove this hypothesis directly. this mechanism is not universal because not all serum proteins, albumin being a notable example, are enzymatically glycosylated. further work must be done to determine whether this system actually functions in serum protein turnover. it is interesting to note that a family of serum proteins (iga1) binds this receptor by way of o-linked oligosaccharides, but only upon denaturation (175). it has also been recently shown that liver endothelial cells can desialate a serum protein, thus rendering it susceptible to uptake by hepatocytes via the gal/galnac receptor (178) . additional studies have revealed the presence of a distinct carbohydrate binding protein in kupffer and other macrophage-like cells (105, 108, 109, 175) . following recognition, these and other carbo-hs~drate binding proteins become internalized together with their ligands, which carry a man/glcnac/fuc specificity, but later return to the cell surface free of ligand. in addition, cell surface localized man-6-po4 receptors have been suggested to be responsible for hepatic uptake of enzymes supplied to the fetal blood from placental fluids (111). it has been suggested that receptors on the kupffer cell and other types of macrophages may also recognize cells which present the appropriate configuration of carbohydrate residues (110) . binding in these circumstances may lead to phagocytosis. this kind of binding has been detected in model systems (130) . several examples of a role for protein glycan in other forms of intermolecular recognition have been offered. these include glycans on the fc portion of igg being involved in the recognition by complement proteins and cell surface receptors (70, 129) , glycans of human chorionic gonadotropin being involved in activation of its receptor subsequent to the actual binding event (71) (72) (73) , and interaction between anti-thrombin iii and heparin leading to activation of inhibition of thrombin protease activity (176). as early as 1945, burnett (119) and his colleagues observed the importance of cell surface proteinbound sialic acid in virus binding and infection. this phenomenon is sufficiently complex, however, that it is necessary to discuss the kinds of evidence which are involved in concluding that carbohydrate plays a direct, rather than a regulatory, role in this and other examples of adhesion. any demonstration that carbohydrate comprises part of a physical bridge in cell adhesion (or any molecular binding) requires that four general criteria be satisfied: 1) removal or alteration of the carbohydrate diminishes cell adhesion; 2) reconstitution of the carbohydrate either on the cell or on an artificial surface re-11 stores cell adhesion; 3) a specific receptor for the carbohydrate is identified on the cell (complementary) surface; 4) inactivation of the receptor diminishes cell adhesion. satisfaction of these criteria can be complicated because cells appear to have multiple adhesion systems, rendering it difficult to assay one independently of the others. removal of carbohydrate can have other effects on cells. for example, cell viability can be reduced by drugs or mutations affecting glycosylation, and transport of polypeptides to the cell surface and other mechanisms of turnover and placement of glycoproteins can be altered. specific receptors may be difficult to detect because individual receptor-ligand interactions may have low affinity (e.g., 126). a rationale for this generalization is that, because of the large area of cell surfaces, cells can theoretically interact with other surfaces using a large number of low affinity associations. this may in fact be desirable for cell interactions which would need to be reversible. the results of several approaches where cell reassociation has been competed with model sugars have shown that the competing sugars must be crosslinked to form multivalent structures in order to be competitive (172) . a multivalency requirement has also been implicated in the man-6-po4 receptor system (155) . examination of cell-ceu and cell-substratum adhesion in several systems has identified candidate molecules which participate in the formation of the physical bridge (43, 135) . in some cases independent approaches have converged on the same candidate molecules. the candidate molecules are usually glycoproteins and in some cases the relative roles of carbohydrate and protein have yet to be resolved. in other studies, candidates have not yet been identified. the simplest adhesion systems involve parasite/host interaction. considerable evidence has been adduced that various parasites such as viruses, bacteria, protozoa, etc., possess carbohydrate binding proteins which can interact with sugar structures on the surfaces of cells with which the parasites associate (167, 168, (116) (117) (118) (119) . in some cases it is suspected that this sugar is associated with glycolipid but in other cases man is believed to be involved on the basis of competition studies (e.g., 114 ). this would imply the involvement of glycoprotein because this sugar is rare in glycolipid. the use of carbohydrate as attachment points on cells presumably reflects the stability of these structures on cell surfaces and their specific associations with target cells suitable for the parasite. these would of course be useful features for any mechanism of specific cell-cell interaction. species-specific cell-cell adhesion (the so-called secondary type) in the phylogenetically-primitive marine sponge is suspected to involve carbohydrate ligands (133) . ca-free sea water elutes a fraction from cells of microcionaprolifera which is required for their reaggregation. this fraction is the source of a purified proteoglycan which is referred to as aggregation factor. hypotonic shock elutes another molecular species (the so-caued baseplate or aggregation factor receptor) which is also required for reaggregation and may recognize the proteoglycan by a mechanism involving glucuronic acid on the proteoglycan. these preliminary findings were ascertained by competition studies with model sugars and enzymatic treatments, and it will be interesting to confirm these findings using purified molecules (179) . results of comparable studies in another marine sponge, geodia cydonium, are even more intriguing (133) . bound t3-glucuronic acid is also believed to be important in aggregation factor/baseplate recognition but its position is reversed so that the key sugar is on the baseplate glycoprotein. beta-glucuronidase and j3-glucuronyltransferase can be eluted from extracellular material suggesting a mechanism for the dynamic regulation of cell recognition. an investigation into the tissue specificity of cell recognition showed that ~-galactosidase treatment of nonaggregating cells permitted their aggregation in the presence of aggregation factor. a protein was then isolated which could inhibit aggregation of aggregation-competent cells in a fl-galactosidase sensitive fashion. finally, a t3-galactoside binding protein was isolated from extracellular material which rendered an effect similar to that of the glycosidase. though the significance of these observations is as yet unclear, it has been suggested that carbohydrate structures may underlie specific mechanisms of cell recognition in multiple ways in these species. cell-substratum adhesion in another primitive multicellular system, aggregating cellular slime mold cells, appears to involve a multivalent carbohydrate binding protein termed discoidin i. the involvement of discoidin i was initially recognized based on sequence homology between this protein and an oligopeptide sequence of vertebrate fibronectin which has been shown to contain its cell binding site. this oligopeptide, which blocks fibronectin binding to cells, also affects association of dictyosteliurn cells with their substrata (148) . the ceils become incapable of migrating properly on a surface. the effect can be mimicked by antidiscoidin i. the effect is also replicated if the cells are transformed with a gene which encodes an mrna which hybridizes with authentic discoidin i mrna and blocks its translation (149) . the evidence is very strong that this carbohydrate binding protein is involved in cell-substratum association. what is unclear, however, is the role of the carbohydrate binding activity. there is evidence that it does not associate with the cell via this site (103) . it is unclear whether the carbohydrate-binding site might associate the protein with elements of certain substrata, but it is apparent that this is not necessary. there is recent evidence that food bacteria are recognized by this lectin and hence discoidin i might assist in the adhesion of ceils to their food source (104) . perhaps the best documented instance of a direct role for carbohydrate in cell-cell adhesion occurs in higher organisms and involves the protein galactosyltransferase and bound glcnac, sometimes associated with oligolactosamine (120, 121, 74, 124) . this pair of molecules has been implicated in adhesion between mouse sperm and egg, neural crest cells and substrated, and embryonal carcinoma ceils. each of the four criteria cited above has been addressed by experimental approaches using antibodies, enzymes and drugs to inactivate the receptor and the ligand. a genetic approach to modify receptor or ligand has yet to be applied. though the latter approach with its inherent specificity is very important, the evidence using the approaches already employed is strong. the system is interesting because the receptor-ligand interaction can be readily reversed by supplying substrate for the galactosyltransferase enzyme activity. an independent approach to the problem of mouse sperm-egg recognition has resulted in the isolation, from the jelly coat of the egg, of a glycoprotein, zp3, which can, upon presentation to the sperm, block its ability to recognize eggs (125) . it was found that careful alkaline borohydride treatment to strip o-linked glycans without altering the polypeptide chain neutralized the inhibitory activity of zp3. finally, it was shown that the released glycans possessed inhibitory activity and in addition bound to sperm, suggesting that the carbohydrate itself served as the recognition site in the jelly coat for sperm. further work remains to be done to identify the sperm receptor (169) as well as to relate the findings to the above results implicating the galactosyltransferase. carbohydrate of a different type has also been implicated in cell-cell adhesion between mature mammalian cells. for example, it has been suggested that cell surface carbohydrate may be important for recognition between certain lymphocytes and certain high endothelial venule surfaces (hev) in lymphatic tissues (136) . pretreatment of lymphocytes with specific sugars renders them incapable of binding to hevs in an in vitro assay, and glycosidase treatment of hevs renders them incapable of being recognized by lymphocytes. there is conflicting evidence from these studies regarding the nature of the carbohydrate structure involved. additional results have indicated that one lymphocyte subclass recognition site involves a previously studied homing receptor, the ubiquitin-conjugated glycoprotein defined by the mel-14 antigen. recent work has found carbohydrate-derivatized polyacrylamide surfaces to which lymphocytes will specifically bind (137) . an effort is underway to compare the specificity of this interaction with that to hevs. the exciting prospect is that the manipulability of the artificial surface (130) will allow dissection of multiple adhesion mechanisms and permit more rapid investigation of the nature of the carbohydrate recognition system believed to be responsible for lymphocyte recognition preparatory to extravasation. carbohydrate has been suggested to perform an opposite kind of role in the function of the protein n-cam in associating neural cells (170) . fab fragments from anti-n-cam inhibit reassociation of dissociated neural cells and isolated n-cam can self associate. there are several forms of n-cam which differ in their degree of sialylation. the more heavily sialylated forms show less affinity for one another in the self association assays. thus it has been postulated that sialylation is a posttranslational modification which modulates the strength of adhesion mediated by this glycoprotein and in vivo evidence has now been provided (127) . recent-ly it has alo been suggested that n-cam can bind carbohydrate in the heparan sulfate proteoglycan (128) and that this recognition is involved in intercellular (171) and cell-substratum adhesion. it is too soon to speculate whether binding to a carbohydrate ligand may have secondary effects on cells as as been suggested for, e.g., binding of human chorionic gonadotropin (71) (72) (73) . this hormone does not depend upon its carbohydrate for binding to its receptor but the carbohydrate is important for activating the cellular response. the selected examples discussed here indicate that protein-linked (or possibly lipid-linked) carbohydrate may play a fundamental role in multicellular organization in a wide phylogenetic range of organisms. though in all of the examples cited there is a specificity in the adhesion, in some cases this is due to multiple possibly low-affinity binding interactions. in the extreme, each single binding event may approach non-specificity, with specificity accruing through multivalency. perhaps this implies that the formalization of non-specific carbohydrate-mediated events obscures an actual continuum of specificity in these events between these two extremes. it is unclear whether the involvement of carbohydrate in adhesion is universal. several other adhesion molecules, including glycoproteins such as gp80 in d. discoideurn (164) , cam 80/120 in mammalian cells, etc. and glycolipids (146) , are glycosylated but the role of carbohydrate is not known. if the use of carbohydrate is universal, then the specificity inherent in the examples discussed would suggest an underlying structural code which may be important for the affiliated processes of cell sorting and morphogenesis, to which adhesion makes a very important contribution. this may explain some of the interesting morphogenetic effects circumstantially suggested by many studies involving carbohydrate structure perturbations on developing cells. a role for carbohydrate in cell adhesion choice or sorting correlates with its postulated role in intracellular or extracellular compartmentalization of proteins. cell sorting and molecular compartmentalization can be regarded as sorting at different levels. it will be interesting to learn whether the proteins on which these carbohydrates are located exist only to place the carbohydrate or whether the carbohydrate piggybacks on another essential function of the protein. were the latter true, this would strengthen the analogy with the role of carbohydrate in routing or molecular compartmentalization. it may then become feasible to generalize carbohydrate's role as important for the three-dimensional compartmentalization of proteins, and the cells, with which they are associated. as such, carbohydrate may be the chemical structure cells used to address and organize space as they evolved from prokaryotes to unicellular eukaryotes, and continued to exploit as they evolved to form multicellular tissues and organs. numerous studies have implicated protein-linked carbohydrate in more complicated cellular phenomena (43, 122) . glycosylation is modulated coincidentally with numerous developmental processes (122, 53-67, 69, 96, 161) . carbohydrate interactions have been implicated in mammalian morula compaction (131, 122, 123) . proliferation has been reported to require glycosylation (41) or to be inhibited by glycopeptides and a defined fragment of heparan sulfate (143, 151) . oligosaccharides released from plant cell walls can stimulate cell proliferation and alter cell differentiation in plant cells (145) . myogenesis and chondrogenesis have been speculated to be affected by gags linked to proteoglycans (e.g., 113 ), but conclusions in this field are very controversial. attachment of cells to substratum-associated carbohydrate can result in its covalent modification (213, 130). neuritogenesis has been shown to be inhibited in vitro by tunicamycin (16) , though the specificity of the effect is difficult to assess, as tunicamycin-sensitivity does not convincingly demonstrate the involvement of protein-linked carbohydrate (15, 17) . a recent report suggests that alteration of processing of nlinked oligosaccharides with swainsonine, without affecting cell viablility, inhibits the ability of a line of melanoma cells to metastasize to the lung (52). this finding is consistent with another report documenting carbohydrate differences between metastatic and non-metastatic cell lines (49) . there is much new territory to explore in the field. the use of monoclonal antibodies and lectins which recognize carbohydrate-dependent epitopes continues to reveal an unexpected diversity of carbohydrate structures with surprisingly intricate distributions in embryonic and adult tissues. this has recently been appreciated in amphibian (53) and mouse (54, 55) embryos and in the simpler model system dictyostelium discoideum (96, 161) . tumor cells also differ in many cases in specific carbohydrate structures on both lipid and protein (44-46, 49-51,180) . several of the antibodies employed recognize carbohydrate epitopes whose corresponding structures have been determined. the antibodies provide unrivaled sensitivity and specificity relative to standard methods for detection of unique carbohydrate structures. caution must be applied, however, because 1) it is theoretically possible that unique structures at the monosaccharide level may present structures similar enough at the atomic level to be immunologically cross-reactive and 2) identical or similar structures may be immunologically distinguished by conformational restrictions imposed by the surrounding polypeptide environment. thus inference of structure based on antibody data must be confirmed directly by structural studies. in any case, antibodies and lectins may model potential receptors in cells which might specifically recognize carbohydrate-associated structures. the significance of the association of distinct carbohydrate structures with distinct embryonic or tumor cell types is absolutely unclear at present. ultrastructural localization has placed some epitopes intracellularly in specific compartments and others on the cell surface or extracellularly in the surrounding matrix. a cell surface localization is a necessary condition for a direct role in cell recognition and, on the strength of suggestions from other systems, this has been promiscuously interpreted to imply a role in cell recognition. this reflects the primitive status of our general understanding of the significance of protein glycosylation. thus the potential developmental roles of protein-linked carbohydrate in morphogenesis, differentiation, adherence and proliferation will likely be a primary target of investigation in this field in the future. interest in developmental involvement of carbohydrate is based not only on a large bank of indirect data but also on an intrigue in the special kinds of epigenetic regulatory mechanisms with which carbohydrate might be associated. for example, glycosylation offers the cell a mechanism to regulate the structure of multiple proteins without altering gene expression. in addition, monosaccharide residues may be processed independently of the associated polypeptide (141, 142) either by recycling through the golgi (112) or at locations near the site of funtion (130, 138) , far from the point of origin in the rer and golgi. it can be envisaged that the cell responded to the evolutionary requirements for cell differentiation by using epigenetic pathways such as glycosylation and other posttranslational modifications (13) . consistent with these speculations, there are, as cited above, numerous examples of glycosylation steps modified by differentiation (56) (57) (58) (59) (60) (61) (62) (63) (64) (65) (66) (67) 69) , as well as by the anchorage state of the cell and the ph and composition of the surrounding medium (39, 36) . an example of what the future may bear is exemplified by a group of developmental arrest mutants recently characterized in d. discoideum (147) . isolated on the basis of failure to accumulate an early regulated enzyme, many of these mutants appear to be altered in posttranslational modifications, including glycosylation. thus many potentially necessary roles played by glycosylation in development remain unexplored. 15 been shown that carbohydrate interaction is multivalent, composed of multiple weak associations which only in aggregate are strong (155, 123, 172) . it would appear that most protein-linked carbohydrate is not solely responsible for the essential housekeeping functions of cells required for life in the tissue culture environment. perhaps this is because specification of its structure, which is nontemplate-directed, is not precise enough. glycosylation presumably has instead afforded the cell, during the evolution of developmental mechanisms, an economy in allowing the structural modification of many preexisting proteins using the same enzymatic mechanism. it provides the cell with another control point over structure which can be exerted on many proteins at the site of function (away from the site of synthesis) without necessitating a change in gene expression. change in structure by glycosylation may be very suitable for the regulation of the 'luxury' functions of cell differentiation and morphogenesis. an economy of shared non-specifc and specific functions of carbohydrate would thus offer an unifying explanation for the often contradictory conclusions yielded from the varied experimental approaches summarized in this article. the nearly ubiquitous glycosylation of extracellular and cell surface proteins and proteins from certain organelles may reflect a primitive, nonspecific function supporting protein structure and protection. this carbohydrate carries out a shared function, however, when it becomes modified in subtle ways without affecting non-specific functions, to allow discrimination by receptors. recognition by discriminate receptors allows the carbohydrate to subserve specific functions. carbohydrate would appear to be used by cells to carry out secondary functions (both specific and non-specific) for proteins. these include specialized instances of compartmentalization, rate of transport through the cell, intermolecular association, conformation control and general protection. interaction of the cell with the environment, which is in a sense secondary to the life of the cell, would also appear to involve protein-linked carbohydrate. this might include compartmentalization or sorting of cells into tissues. in several instances it has structure, biosynthesis and functions of glycoprotein glycans carbohydrate moieties of glycoproteins complex carbohydrates of the extracellnlar matrix. structures, interactions and biological roles glycoprotein biosynthesis in yeast control of asparagine-linked oligosaccharide chain processing: studies on bovine pancreatic ribonuclease b correlation of glycosylation forms with position in amino acid sequence lysosomal enzyme phosphorylation occurrence of n-acetylglucosamine 6-phosphate in complex carbohydrates -0-methylation of mannose residues transient metbylation of dolichyl oligosaccharides is an obligatory step in halobacterial sulfated glycoprotein biosynthesis glycoproteins as ceil-surface components either high-mannose-type or hybrid type oligosaccharide is linked to the same asparagine residue in ovalbumin posttranslational covalent modification of proteins assembly of asparagine-linked oligosaccharides tunicamycin inhibits ganglioside biosynthesis in neuronal cells tunicamycin blocks neuritogenesis and glucosamine labeling of gangliosides in developing cerebral neuron cultures suppression of placental alkaline phosphatase biosynthesis by tunicamycin occurrence of nacetylglucosamine-l-phosphate in proteinase i from dictyostelium discoideum glycosylation mutants of animal cells laminin, proteoglycan, nidogen and collagen iv: structural models and molecular interactions purification, composition, molecular weight and subunit structure of ovine submaxillary mucin effect of altered oligosaccharide structure onithe cell surface number, distribution and turnover of the high molecular weight acidic glycoproteins of cho cells preliminary characterization of a chinese hamster ovary cell glycosylation mutant isolated by screening for low intracellular lysosomal enzyme activity temporal aspects of the n-and o-glycosylation of human chorionic gonadotropin o-linked oligosaccharides are acquired by herpes simplex virus glycoproteins in the golgi apparatus structural and functional aspects of tumor cell sialomucins antigenic determinants shared by lysosomal proteins of dictyostelium discoideum adhesion mutants of dictyostelium disclideum lacking the saccharide determinant recognized by two adhesion blocking monoclonal antibodies conformation of the glycopeptide linkage in asparagine-linked glycoproteins conformation of the complex oligosaccharides of glycoproteins multiple classes of heparan sulfate proteoglycans from fibroblast substratum adhesion sites effects of deglycosylation on the architecture of ovine submaxillary mucin glycoprotein preparation and properties of serum and plasma proteins. xxix. separation from human plasma of polysaccharides, peptides and proteins of low molecular weight. crystallization of an acid glycoprotein studies on fetuin, a glycoprotein of fetal serum. i. isolation, chemical composition, and physicochemical properties variation of the carbohydrates of glycoproteins of cells growing on different surfaces the function of protein-bound carbohydrates in normal and pathological cells asparaginyl-nacetylgalactosamine olden k: retinoic acid alters the proportion of high mannose to complex type oligosaccharides on fibronectin secreted by cultured chondrocytes a temperature-sensitive n-glycosylation mutant of s. cerevisiae that behaves like a ceil-cycle mutant nglycosylation of nascent proteins early in the prereplicative phase constitutes a process for controlling animal cell proliferation studies on the regulation of the biosynthesis of glucose-containing oligosaccharide-lipids carbohydrate structure, biological recognition, and immune function. in: ivatt rj (ed). the biology of glycoproteins glybopeptide changes and malignant transformation. a possible role for carbohydrate in maligant behavior demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens glycosphingolipids as differentiationdependent, tumor-associated markers and as regulators of cell proliferation biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides aggregation-deficient embryonal carcinoma cells: defects in peanut agglutinin (pna) receptors asparagine-linked oligosaccharides in murine tumor cells: comparison of a wga-resistant (wga r) nonmetastatic mutant and a related wga-sensitive (wga s) metastatic line wp: carbohydrates of the tumor cell surface oligosaccharide modification by swainsonine treatment inhibits pulmonary colonization by b16-f10 murine melanoma cells regional specificity of glycoconjugates in xenopus and axolotl embryos saccharide structures of the mouse embryo during the first eight days of development reactivity of five nacetylgalactosamine-recognizing lectins with preimplantation embryos, early postimplantation embryos, and teratocarcinoma cells of the mouse inhibition of glycoprotein synthesis in saccharomyces cerevisiae by mating pheromones ssea-1, a stagespecific antigen of the mouse, is carried by the glycoprotein-bound large carbohydrate in embryonal carcinoma cells trypanosoma cruzi cells undergo an alteration in protein n-glycosylation upon differentiation appearance of a class of cell-surface fucosyl-glyeopeptides in differentiated muscle cells in culture changes in protein glycosylation during chick embryo development changes in the expression of blood-group carbohydrates during oral mucosal development in human fetuses cell surface glycoconjugates as oncodifferentiation markers in hematopoietic cells glycoprotein synthesis and embryonic development decreased transfer of oligosaccharide from oligosaccharideqipid to protein acceptors in regenerating rat liver stimulation of lipid-linked oligosaccharide assembly duing oviduct differentiation cell-type-related segregation of surface galactosyl-containing components at an early developmental stage in hemopoietic bone marrow cells in the rabbit changes in asparagine-linked sugar chains of human promyelocytic leukemic cells (hl-60) during monocytoid differentiation and myeloid differentiation are glycoproteins and glycosaminoglycans components of the eukaryotic genome? n-linked glycoprotein biosynthesis in the developing mouse embryo biological significance of carbohydrate chains on monoclonal antibodies a role for glycosylation of the c~-subunit in transduction of biological signal in glycoprotein hormones role of carbohydrate in human chorionic gonadotropin antibodies against human chorionic gonadotropin convert the deglycosylated hormone from an antagonist to an agonist cell surface galactosyltransferase as a recognition molecule during development nelsestuen gl: deglycosylated prothrombin fragment 1 polylactosamine glycosylation on human fetal placental fibronectin weakens the binding affinity of fibronectin to gelatin investigations of the possible functions for glycosylation in the high mobility group proteins preferential association of glycoproteins to the euchromatin regions of crossfractured nuclei is revealed by fracture-label eukaryotic ribosomes possess a binding site for concanavalin a on the biological role of glycoproteins the significance of glycosylated proteins effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes differing requirements for glycosylation in the secretion of related glycoproteins is determined neither by the producing cell nor by the relative number of oligosaccharide units effect of the threonine analog/3-hydroxynorvaline on the glycosylation and secretion of c~l-acid glycoprotein by rat hepatocytes differential effects of 1-deoxynojirimycin on the intracellular transport of secretory glycoproteins of human hepatoma cells in culture the effects of processing inhibitors of nlinked oligosaccharides on the intracellular migration of glycoprotein e2 of mouse hepatitis virus and the maturation of coronavirus particles role of glycosylation in processing of newly translated insulin proreceptor in 3t3-l1 adipocytes influence of the nlinked oligosaccharides on the biosynthesis, intracellular routing, and function of the human asialoglycoprotein receptor glycosylation of the epidermal growth factor receptor in a-431 cells inhibition of n-linked 01igosaccharide trimming does not interfere with surface expression of certain integral membrane proteins evidence for a glycoprotein 'signal' involved in transport between subcellular organelles a single nlinked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus g protein to the cell surface the role of protein glycosylation in the compartmentalization and processing of mouse mammary tumor virus glycoproteins in mouse mammary tumor virus-infected rat hepatoma cells the formation of vesicular stomatitis virus (san juan strain) becomes temperature-sensitive when glucose residues are retained on the oligosaccharides of the glycoprotein deletions into a ni-i2-terrninal hydrophobic domain result in secretion of rotavirus vp7, a resident endoplasmic reticulum membrane glycoprotein glycoantigen expression is regulated both temporally and spatially during development in the cellular slime molds dictyostelium discoideum and d. mucoroides the identification of n-linked oligosaccharides on the human cr2/epstein-barr virus receptor and their function in receptor metabolism, plasma membrane expression, and ligand binding glycosylation and secretion of surfactant-associated glycoprotein a complete glycosylation of the insulin and insulin-like growth factor 1 receptors is not necessary for their biosynthesis and function chemistry, metabolism, and biological functions of sialic acids effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease swainsonine inhibits glycoprotein degradation by isolated rat liver lysosomes slime mold lectins bacterial glycoconjugates are natural ligands for the carbohydrate binding site of discoidin i and influence its cellular compartmentalization carbohydrate-specific receptors of the liver hepatic clearance of serum glycoproteins the repair of the surface structure of animal cells carbohydrate-mediated clearance of immune complexes from the circulation carbohydrate-mediated clearance of antibody-antigen complexes from the circulation surface carbohydrates and surface lectins are recognition determinants in phagocytosis the carbohydrate structure of porcine uteroferrin and the role of the high mannose chains in promoting uptake by the reticuloendothelial cells of the fetal liver intracellular movement of cell surface receptors after endocytosis: resialylation of asialotransferrin receptor in human erythroleukemia cells hyaluronic acid bonded to cell culture surfaces stimulated chondrogenesis in stage 24 limb mesenchyme cell cultures lectin activation in giardia lamblia by host protease: a novel hostparasite interaction carbohydrates as recognition determinants in phagocytosis and in lectin-mediated killing of target cells specificity of binding of a strain of uropathogenic escherichia coli to galcd-4gal-containing glycosphingolipids evidence for a malarial parasite interaction site on the major transmembrane protein of the human erythrocyte adherent bacterial colonization in the pathogenesis of osteomyelitis mucoproteins in relation to virus action the synthesis of complex carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion the receptor function of galactosyltransferase during cellular interactions the biology of glycoproteins a multivalent lacto-n-fucopentaose iii-lysyllysine conjugate decompacts preimplantation mouse embryos, while the free oligosaccharide is ineffective receptor function of mouse sperm surface galactosyltransferase during fertilization o-linked oligosaccharides of mouse egg zp3 account for its sperm receptor activity differential cell adhesion may result from nonspecific interactions between cell surface glycoproteins specific alteration of n-cam-mediated cell adhesion by an endoneuraminidase a heparin-bindifig domain from n-cam is involved in neural cell-substratum adhesion recognition of igg by fc receptor and complement: effects of glycosidase digestion cell surface carbohydrates in cell recognition and response feizi t: cell interactions in preimplantation embryos: evidence for involvement of saccharides of the poly-nacetyllactosamine series mitochondrial synthesis of glycoproteins and surface properties of mitochondrial membranes cell membranes in sponges the phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes cell-cell recognition in yeast, characterization of the sexual agglutination factors from saccharomyces kluyveri lymphocyte attachement to high endothelial venules during recirculation: a possible role for carbohydrates as recognition determinants lymphocyte adhesion to immobolized polysaccharides suggests multiple carbohydrate receptors for recirculation spontaneous glycosylation of glycosaminoglycan substrates by adherent fibroblasts immunocytochemical demonstration of ecto-galactosyltransferase in absorptive intestinal cells soluble lectins: a new class of extraeellular proteins effect of choroquine on the degradation of l-fucose and the polypeptide moiety of plasma membrane glycoproteins different half-lives of the carbohydrate and protein moieties of a 110000 dalton glycoprotein isolated from plasma membraned of rat liver structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells. ii. evidence for a pentasaccharide sequence that contains a 3-0-sulfate group pathways of protein secretion in eukaryotes structure and function of plant cell wall polysaccharides gangliosides support neural retina cell adhesion ~-mannosidase-1 mutants of dictyostelium discoideum: early aggregationessential genes regulate enzyme precursor synthesis, modification, and processing discoidin i is implicated in cell-substratum attachment and ordered cell migration of dictyostelium discoideum phenocopy of discoidin i-minus mutants by antisense transformation in dictyostelium mechanisms for the incorporation of proteins in membranes and organelles glycopeptides prepared from mouse cerebrum inhibit protein synthesis and cell division in baby hamster kidney ceils, but not in their polyoma virus-transformed analogs stereochemistry and binding behavior of carbohydrate chains associations of like and unlike polysaccharides: mechanism and specificity in galactomannans, intreacting bacterial polysaccharides, and related systems model for the structure of the gastric mucous gel freeze hh: interaction of dictyostelium discoideum lysosomal enzymes with the mammalian phosphomannosyl receptor differential processing of asn-linked oligisaccharides on pituitary glycoprotein hormones: implications for biologic function lysosomal enzyme bind!ng to mouse p388d l macrophage membranes lacking the 215-kda mannose 6-phosphate receptor: evidence for the existence of a second mannose 6-phosphate receptor identification and characterization of cells deficient in the mannose 6-phosphate receptor: evidence for an alternate pathway for lysosomal enzyme targetting variability in transport rates of secretory glycoproteins through the endoplasmic reticulum and golgi in human hepatoma cells glucose phosphotransferase and intracellular protein trafficking freeze hh: modifications of lysosomal enzymes in dictyostelium discoideum glucose removal from n-linked oligosaccharides is required for efficient maturation of cerain secretory glycoproteins from the rough endoplasmic reticulum to the golgi complex deletion of clustered o-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts absence of a carbohydrate modification does not affect the level or the subcellular 175. 176. 177. localization of three membrane glycoproteins in modb mutants of dictyostelium discoideum reversible defects in o-linked glycosylation and ldl receptor expression in a udp-gal/udp-galnac 4-epimerase deficient mutant groth sf: studies on mucoproteins. iii. the accesibility to trypsin of the susceptible bonds in ovine submaxillary land mucoproteins influenza c virus uses 9-0-acetyl-n-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells carbohydrate-binding sites of the mannose-specific fimbrial lectins of enterobacteria the mouse egg's receptor for sperm; what is it and how does it work? cell adhesion molecules neuronal cell-cell adhesion depends on interactions of n-cam with heparin-like molecules reconstitution of high cell binding affinity of a marine sponge aggregation factor by cross-linking of small low affinity fragments into a large polyvalent polymer localization of galactosyl-and sialyltransferase by immunofluorescence: evidence for different sites outer membrane terminal saccharide of bovine liver nuclei and mitochondria hendil kb: ion exchange properties of the glycocalyx of the amoeba chaos chaos and its relation to pinocytosis liver endothelium mediates the hepatocyte's uptake of ceruloplasmin the molecular basis of species specific cell-cell recognition in marine sponges, and a study on organogenesis during metamorphosis rous sarcoma virus-transformed baby hamster kidney cells express higher levels of asparagine-linked tri-and tetraantennary glycopeptides containing [glcnac-13(1,6)man-a(1,6)man] and poly-nacetyllactosamine sequences than baby hamster kidney cells i am grateful to drs. c. feldherr and g. erdos for their critisms of the manuscript during preparation. key: cord-307811-6e3j0pn7 authors: hao, wei; ma, bo; li, ziheng; wang, xiaoyu; gao, xiaopan; li, yaohao; qin, bo; shang, shiying; cui, sheng; tan, zhongping title: binding of the sars-cov-2 spike protein to glycans date: 2020-07-02 journal: biorxiv doi: 10.1101/2020.05.17.100537 sha: doc_id: 307811 cord_uid: 6e3j0pn7 the pandemic of sars-cov-2 has caused a high number of deaths in the world. to combat it, it is necessary to develop a better understanding of how the virus infects host cells. infection normally starts with the attachment of the virus to cell-surface glycans like heparan sulfate (hs) and sialic acid-containing oligosaccharides. in this study, we examined and compared the binding of the subunits and spike (s) proteins of sars-cov-2 and sars-cov, mers-cov to these glycans. our results revealed that the s proteins and subunits can bind to hs in a sulfation-dependent manner, the length of hs is not a critical factor for the binding, and no binding with sialic acid residues was detected. overall, this work suggests that hs binding may be a general mechanism for the attachment of these coronaviruses to host cells, and supports the potential importance of hs in infection and in the development of antiviral agents against these viruses. the 2019 novel coronavirus (cov) is the seventh human coronavirus. 1 it is a deadly virus that is affecting the whole world in an unprecedented way. the global impact of the coronavirus disease 2019 (covid19) pandemic is far beyond that of two other major coronavirus outbreaks in the past 20 years, the severe acute respiratory disease (sars) in 2003 2,3 and the middle east respiratory disease (mers) in 2012. 4 given that all three highly pathogenic covs were originated from bats and a large number of closely related covs are present in bats, future outbreak of this type of zoonotic virus remains possible. in order to avoid facing a similar pandemic in the future, it is necessary to develop a better understanding of these covs, especially with regard to effective ways that can help control the current covid-19 pandemic and prevent the second wave of outbreaks. studies showed that the genome of the sars-cov-2 has about 80% nucleotide identity with that of sars-cov. 5 the major differences are found in the regions encoding the structural proteins (envelope e, membrane m, nucleocapsid n, and spike s) and accessory proteins (orf3a/3b, 6, 7a/7b, 8 and 10), not the nonstructural proteins (nsp1 to nsp16). based on this genetic similarity, the 2019 novel cov was named by the international committee on taxonomy of viruses (ictv) as the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). 6 sars-cov-2 is more genetically distant from mers-cov and shares only about 57% genome homology with mers-cov. 5, 7 these similarities and differences are reflected in the viral binding receptors to cell surface receptors. sars-cov-2 and sars-cov use the same receptor angiotensin-converting enzyme 2 (ace2) for entry into target cells, [8] [9] [10] whereas mers-cov uses dipeptidyl peptidase 4 (dpp4, also known as cd26) as the primary receptor. 11 however, there are many important findings that cannot be explained by their genetic relations. for example, it was found that sars-cov-2 has a relatively lower mortality rate (2%) than sars-cov (15%) and mers-cov (35%), but is more transmissible among humans. [12] [13] [14] these findings suggest that more investigations other than genetic analysis need to be carried out to improve the understanding of sars-cov-2 infection. in order to efficiently infect host cells, sars-cov-2 must bind with cell surface molecules in the lungs and other organs to mediate viral attachment and entry into host cells. previous studies of many other viruses suggested that sars-cov-2 s protein may use other molecules on host cell surface as attachment factors to facilitate binding to the high-affinity receptor ace2. 15, 16 examples of such molecules include glycosaminoglycans (gags) and sialic acid-containing oligosaccharides. [17] [18] [19] [20] gags are primarily localized at the outer surface of cells. such a location makes them particularly suitable for acting as attachment factors to recruit viruses to cell surfaces. 21, 22 hs is one of the most prevalent types of gags in mammals. it is a linear and sulfated polysaccharide that is abundantly expressed on the surface of almost all cell types and in the extracellular matrix. 23, 24 the hs chains are mostly covalently linked as side chains to core proteins to form hs proteoglycans (hspgs) (fig. 1 ). a possible mechanism for sars-cov-2 entry and infection. at the early stage of the infection process, sars-cov-2 may first interact with the hspgs on the surface of susceptible cells using the s protein protruding from the virus particle. this initial attachment may promote the subsequent binding of the virus to the high-affinity entry receptor ace2. the serine protease tmprss2 on host cell surface and other host cell proteases may assist in viral entry by cleaving the s protein at the s1/s2 and/or at the s2' sites. hs is synthesized in the golgi apparatus by many different enzymes. during and after its assembly, hs undergoes extensive series of modifications including sulfation, acetylation and epimerization, which leads to glycan structures with high heterogeneity in length, sulfation, and glucuronate/iduronate ratio. 25 considerable variation in the sulfation pattern and degree of hs was noted in different species, organs, tissues, and even at different ages and disease stages. 23, 24, 26 the sequence and sulfation pattern of hs has been shown to be able to regulate the binding of many viruses to host cells during infection. 27, 28 a similar trend was also observed for sialylation patterns of cell surface oligosaccharides. 17, 29, 30 these findings implicate that a possible relationship may exist between the distribution of different types of hs/sialylated glycans and the viral tropism. [31] [32] [33] a better understanding of their relationship can potentially contribute to the design of new antiviral strategies. although currently the data on the viral tropism of sars-cov-2 are limited, results from recent studies suggested that its tropism may not be correlated with the ace2 expression. 34, 35 some other factors, such as proteases and glycans, may be the determinant of cellular susceptibility to the infection with this virus. 36 a recent study suggested that hs may bind to the receptor binding domain (rbd, the c-terminal region of the s1 subunit, fig. 2 ) of the sars-cov-2 spike protein and change its conformation. 37 the intriguing possibility that variation in hs and sialic acid characteristics could impact the tropism of viruses prompts us to investigate the binding of sars-cov-2 toward a series of hs and sialic acid containing oligosaccharides. 38 in this study, we systematically examined and compared the binding of the sars-cov-2 s protein subunits, full-length molecule and its trimer to different hs using microarray experiments (fig. 2) . our results suggested that all the tested protein molecules were capable of binding hs oligomers and had similar binding preferences, with higher affinity toward hs forms with higher degrees of sulfation. the binding was also shown to be positively related to the level of 6-o-sulfation. the hs chain length seemed to be not critical. a long heparin molecule (a highly sulfated hs) and a synthetic heparin pentasaccharide (fondaparinux sodium) were demonstrated to have similar binding affinity to these protein molecules. moreover, our study suggested that the sars-cov-2 s protein might not be able to bind sialic acid residues, or the binding might be too weak to be detected by the microarray technology, and the s proteins of sars-cov and mers-cov have similar hs and sialic acid binding properties as those of the sars-cov-2 s protein. overall, our study laid a foundation for future studies to explore whether the binding specificity to hs can serve as an important contributor to the viral tropism of sars-cov-2 and to explore the possibility of exploiting hs for therapeutic strategies. binding of the subunits and s proteins of sars-cov-2, sars-cov, and mers-cov to a hs microarray. an early study has suggested that the rbd of sars-cov-2 s protein might bind heparin. 9 in order to determine if there is any preference of the sars-cov-2 s protein for particular hs structures, we first investigated the binding of the rbd (here termed as sars-cov-2-rbd) to a hs microarray containing 24 synthetic heparan sulfate oligosaccharides. these oligosaccharides have systematic differences in their length, monosaccharide composition, and sulfation pattern (fig. 3) . the microarray experiment was performed using a previously established standard protocol. 29, 39, 40 briefly, the proteins were labeled with cy3 fluorescent dye and incubated with the microarray at different concentrations. after washing away the unbound hs molecules, a highly sensitive fluorescence method was used to detect the binding of the sars-cov-2-rbd to hs. under the experimental conditions, the binding can be detected at concentrations higher than 0.5 g/ml. increasing the concentration of the protein was not found to produce noticeable changes in binding (supporting information, fig. s1 ). quantification of fluorescence revealed that the sars-cov-2-rbd is able to bind to almost half of the molecules on the microarray, and not surprisingly, the binding is strongly affected by the sulfation level, which is a trend that has been previously noted for many hs-binding proteins. [41] [42] [43] as shown in figure 4a , the hs oligosaccharides with higher sulfation degree, hs020-hs024 (the number of sulfate groups per monosaccharide unit >1.00), exhibit higher fluorescence intensity. the highest fluorescence intensity is observed for hs023 (1.35 sulfate groups per monosaccharide), which is followed by those of hs021 and hs024 (1.25 sulfate groups per monosaccharide) and those of hs020 and hs022 (1.15 sulfate groups per monosaccharide). binding of sars-cov-2-rbd to hs seems to not be affected by the monosaccharide composition (compare hs020 with hs022, and hs021 with hs024). the oligosaccharides with relatively lower sulfation levels (hs001-hs019, the number of sulfate groups per monosaccharide unit <1.00) have lower or almost no fluorescence signals. an analysis of the effect of the variation in sulfation revealed that the position of sulfation is another factor that strongly influence the binding. as shown in the figure 4 , removal of the 6-o-sulfate group from the glucosamine units significantly reduced the binding (compare hs017 with hs020, hs018 with hs021, and hs019 with hs022), suggesting that the 6-o-sulfate in hs plays a crucial role in determining its interaction with sars-cov-2-rbd. the importance of the 6-o-sulfate for binding is further supported by comparing the binding of hs012, hs013, hs014, hs015, and hs016, which shows that the one-by-one addition of sulfate to the 6-oposition of the glucosamine residues gradually increased the binding of hs with sars-cov-2-rbd. the microarray study also indicates that the binding is not positively related to the length of the hs chains. short hs oligosaccharides could have comparable or even better binding properties (compare hs001-hs006, hs007-hs0012, and hs020-hs024). the binding results of the sars-cov-2-s1 follow a similar trend as those of the sars-cov-2-rbd, with only minor differences for a few hs molecules like hs001, hs007, hs016, hs021 and hs024 (fig. 4b) . this is consistent with the assumption that rbd is the major determinant for viral s protein binding to hs. examination of the sequence of the sars-cov-2 s protein revealed the presence of a potential cleavage site for furin proteases (rrars) at the s1/s2 boundary. 9 this is similar to the s protein of mers-cov, which also contain a furin consensus sequence rxxr (fig. 2) . furin cleavage can occur in the secretory pathway of infected cells and breaks the covalent linkage between the s1 and s2 subunits. 44 the s2 subunit functions to fuse the virus to the host cell. it may interact with hs on the cell surface to facilitate the fusion process. therefore, to determine the role of hs in sars-cov-2 infection, it is also important to investigate the binding of the s2 subunit to hs. very interestingly, our results indicate that the sars-cov-2-s2 can also bind to hs, with the binding preferences similar to those of the sars-cov-2-rbd and the sars-cov-2-s1 (fig. 4c) . this suggests that hs may also play an important role during viral membrane fusion after the s1 subunit is removed from the s protein. we also further investigated the binding full-length s protein and its trimer to hs using the same microarray. our data showed that, although the binding to hs020-hs024 still remains the highest, the full-length s protein has increased binding to hs with slightly less sulfation, particularly the molecules in groups hs007-hs012 and hs013-hs016 (fig. 4d, 4e) . additionally, we found that the binding preferences of the rbds, s1 subunits and full-length s proteins of sars-cov and mers-cov are similar to those of the subunits and s protein of sars-cov-2, although small differences are observed (fig. 4f-k) . because the mers-cov s protein also contains a furin cleavage site, we studied the binding of its s2 subunit to the hs microarray. once again, the results are similar to those of the s2 subunit of sars-cov-2. overall, these data support the involvement of hs in the binding with the subunits and s proteins of sars-cov-2, sars-cov and mers-cov and support the importance of hs for the infection of these coronaviruses. to determine the relative binding strength of the subunits and s proteins of sars-cov-2, sars-cov, and mers-cov, we measured and compared the dissociation constants (kd) of the protein molecules studied in the microarray experiment using a real-time spr-based binding assay. a commercially available porcine heparin from sigma-aldrich was used as the first binding partner for the measurement. it is a mixture of highly sulfated hs, with most chains in the molecular weight range of 17 to 19 kilodalton. this long heparin molecule is more similar to the hs molecules on the host cell surface than those on the microarray. in the spr assay, protein molecules were covalently linked to the surface of the cm5 (carboxymethylated dextran) sensor chips by amine coupling. the heparin in various concentrations was then flowed over the immobilized proteins. the changes in refractive index by molecular interactions at the sensor surface were monitored and the dissociation constants were obtained by fitting the results using the software available in the spr instrument (fig. 5) . the comparison of the dissociation constants revealed that all the tested protein molecules can bind to the heparin, but their binding affinities are relatively low (kds are at the micromolar level). this agrees well with previous observations that hs is a weak binder to viral s proteins. 45 the results also showed that rbd has the lowest binding affinity among the tested protein molecules of sars-cov-2 (fig, 5a) . the s protein trimer and s1 subunit have relatively lower binding affinity in comparison with the fulllength s protein and s2 subunit, respectively (fig, 5b-e) . very similar trends were also observed for differences in the binding affinities of the tested protein molecules of sars-cov and mers-cov (fig, 5f-l) . in addition to the long heparin molecule, we also measured the binding of the protein molecules to fondaparinux (arixtra®), which is an ultralow-molecular-weightheparin (ulmwh) containing five monosaccharide units (fig. 6 ). it has a well-defined chemical structure and is currently the only ulmwh that has been clinically approved as an anticoagulant. fondaparinux is very similar to hs023 in size, monosaccharide composition, and sulfation position and degree. the results showed that the kd values of fondaparinux binding to the tested protein molecules are in the same range as those for the long heparin. this agrees well with the observation in the microarray study, suggesting that the length of the hs chains is not a determining factor for binding. despite the similarity, there is a subtle but noticeable difference, which is that the binding affinities of the s1 subunit and rbd to fondaparinux are quite similar. can interact with sialic acid-containing glycans present on the cell surface. 17, 20 such an interaction is normally mediated by the n-terminal domain of the s1 subunit. 46 in order to find out if sars-cov-2 can bind to sialic acid residues, we carried out microarray analyses of its s protein and subunits. the first microarray used contains 100 different n-glycans that may be found on the surface of cells. 49 of them are terminated with α2,3-and α2,6-linked sialic acid, also known as n-acetylneuraminic acid (neu5ac), 8 with α2,3-and α2,6-linked n-glycolylneuraminic acid (neu5gc), and the rest with other glycan residues (supporting information, table s1 ). the experiment was performed in the similar way as described for the hs microarray study. the microarray results showed that both the sars-cov-2-rbd and sars-cov-2-s1 gave no binding signal, suggesting that they may not be able to interact with sialylated n-glycans or the binding signal is too low to be detected. in order to confirm this finding, we also investigated the binding of the full-length s proteins of sars-cov-2, sars-cov and mers-cov to more sialylated glycans, including sialylated n-and o-linked glycans and glycolipid glycans (supporting information, tables s2-4), but again no specific binding was detected. for a virus like sars-cov-2 to establish infection, it must first attach itself to the surface of target cells in different organs and tissues. the s protein plays an essential role in this attachment process. recently, the structure of sars-cov-2 s protein in the prefusion conformation was determined by the cryo-em technique. 10 it shows that the overall structure of the sars-cov-2 s protein is very similar to that of the closely related sars-cov s protein, which is organized as a homotrimer. each monomer can be divided into an n-terminal receptor-binding s1 subunit and a c-terminal fusionmediating s2 subunit. the s1 subunits are located at the apex of the spike, making them more accessible for binding to the proteinaceous receptor, ace2. although similar, there are some notable differences between the sars-cov-2 and sars-cov s proteins. 8, 10 for example, the key amino acid residues involved in the binding of the sars-cov-2 s protein to ace2 are largely different from those of sars-cov. 9, 47, 48 these differences may be related to the observed higher binding affinity of the sars-cov-2 s protein to ace2. another important difference between the sars-cov-2 and sars-cov s proteins is that the former protein contains a multibasic protease recognition motif (rrars) at the junction of s1 and s2. 9 a multibasic cleavage site (rsvrs) was also identified in the mers-cov s protein (fig. 2) . 49 the sars-cov s protein only has a monobasic amino acid (sslrs) at the same site. the multibasic site can be processed by furin or related proprotein convertases, which are widely expressed in different tissues, before the virus is released from the host cell. by contrast, the monobasic site can be cleaved by tmprss2 or other cell-surface proteases (whose expression is confined to certain tissues) only after the virus is released from the host cell. it was reported that the cleavage at the junction of s1 and s2 activates the s protein for virus-cell fusion. thus, the presence of the multibasic cleavage site may partially account for the enhanced infectivity and tropism of sars-cov-2 relative to sars-cov for human cells. 10, 50 however, what is vague is how the virus attaches to the host cells after losing the s1 subunit, which is responsible for the binding to the proteinaceous receptor. in addition to binding protein-based receptors, many viruses can interact with cell surface glycans, including gags and sialic acid-containing oligosaccharides. depending on the virus, the glycan molecules can act as attachment factors, coreceptors or primary receptors. 51 viruses typically bind gags through non-specific charge-based interactions. as one of the most abundant gags, hs appears to be the preferred binding partner for many viruses. 33, [52] [53] [54] [55] sialic acids are normally terminal monosaccharide residues linked to glycans decorating cell surface glycoproteins, glycolipids, or other glycoconjugates. 20, 56 in general, the interactions of viruses with hs or sialic acids are responsible for the first contact with host cells. such contact may serve to concentrate viruses on the surface of target cells, facilitate their binding to more specific high-affinity protein receptors and/or promote their entry into host cells. 57, 58 it has been demonstrated that virus binding and infection can be reduced by enzymatic removal hs or sialic acid from cell surface, or by treating virus with soluble hs or multivalent sialic acid conjugates. 30, [59] [60] [61] therefore, in order to better understand and treat covid-19, it is necessary to carry out research to investigate the possible interactions between sars-cov-2 and hs and sialic acid-containing glycans in the forms of separate subunits and full-length proteins, and to assess if such interactions could represent a target for therapeutic intervention. similar to studies that have been successfully conducted for many other viruses, we used the microarray and spr technology to study the binding of the rbds, s1/s2 subunits and full-length s proteins of sars-cov-2, sars-cov and mers-cov, and a trimer of the sars-cov-2 s protein to hs and sialic acid. 59 the microarray results showed that all the tested protein molecules can bind to about half of the oligosaccharides on the hs microarray. in contrast, only background levels of fluorescence were detected on various sialylated glycan microarrays. this observation suggests all the tested protein molecules are able to bind to hs 45,62 and may not bind to and/or have very low binding affinity to sialylated glycans. this well agrees with previous studies showing that sars-cov can bind to hs 62 and the binding of the mers-cov s protein to sialic acid-containing glycans can only be detected after the s1 subunit was attached to a nanoparticle to enhance its avidity via multivalent interactions. 17 our results also suggested that the binding of the tested proteins to hs is related to hs sulfation position and degree. it seems that more 6-o-sulfate groups and higher sulfation degree normally lead to better binding. because hspgs exhibit different sulfation patterns in different tissues, such a binding specificity may contribute to the tropism of sars-cov-2 for human cells. 63 the length of hs appears not to be a critical factor for the binding. short hs chains could have comparable binding specificity and signals. the comparison of the spr kd values obtained for the long heparin molecule and fondaparinux further support this finding. it implies that it may be possible to reduce the attachment of sars-cov-2 to the surface of host cells by low-molecularweight-heparin (lmwh). this is in agreement with a recent study showing that lmwh treatment may be associated with better prognosis in some severe covid-19 patients. 64 while these initial findings are encouraging, further research is required to determine if the binding to hs could affect the tropism and pathogenesis of sars-cov-2 and to determine if hs could be used for the inhibition of the infection of this virus. our spr data of the tested protein molecules also showed that the s2 subunits could have similar or better binding affinity for hs as compared to those of the s1 subunits. this finding suggests that the cleaved s proteins of sars-cov-2 and mers-cov may depend on hs for interaction with the host cells during viral membrane fusion. in parallel with our study, the linhardt and boons research teams also conducted studies to investigate the binding of s proteins to hs. 45, 65 the absolute values of the dissociation constants determined in our experiments are largely different from those presented by these two teams. this can be seen from the binding of the full-length s protein of sars-cov-2, which was studied by all three teams (supporting information, table s5 ). our kd is one order of magnitude higher than that reported by the boons team, which in turn is three orders of magnitude higher than that reported by the linhardt team. the differences in results may be due to the method of analysis and/or the experimental materials used in the studies. in our study, the tested proteins were immobilized on the surface of cm5 sensor chips, while in their experiments, biotinylated heparin molecules were immobilized on the chips. at the same time, because the s glycoproteins and the heparin molecules were obtained from different sources, their composition may be different from each other. in conclusion, through our study, we provided experimental evidence for whether or not the s protein of sars-cov-2 can bind to two types of cell-surface glycans, hs and sialic acid-containing glycans, which are commonly utilized by human viruses for attachment to target cells. our data revealed that the sars-cov-2 s protein can weakly bind to hs in a sulfation-dependent manner. no binding with sialic acid residues was detected using the microarray assay. the results suggest that hs may act as an attachment factor to concentrate the virus at the cell surface and affect its tropism. through comparison, we found that the s proteins of sars-cov and mers-cov have similar binding properties to hs as that of the sars-cov-2 s protein, indicating that hs binding may be a conserved feature for these three types of coronaviruses. our data also revealed that the s2 subunits could bind equally well as the s1 subunits to hs. this binding may be an important element for viral attachment to the host cell surface after the removal of the n-terminal receptor-binding domains by protease cleavage. overall, our findings support the potential importance of hs in sars-cov-2 infection and in the development of antiviral agents. expression and purification of sars-cov-2-rbd. dna containing the coding sequence for an n-terminal hemo signal peptide, the receptor binding domain (rbd, residues 319-541) of sars-cov-2 s protein and a c-terminal polyhistidine tag was amplified and inserted into a pfasebac1 vector for expression in high-5 insect cells using the bac-to-bac expression system (invitrogen). the resulting recombinant protein, termed sars-cov-2-rbd-his, was secreted into cell culture medium, and subsequently purified on a nickel-nitrilotriacetic acid (ni-nta) affinity column, followed by a superdex 200 gel filtration column (ge healthcare). the final buffer for the protein contains 10 mm hepes (ph=7.0) and 100 mm nacl. the purified sars-cov-2-rbd-his was concentrated to 3.5 mg/ml and flash frozen in liquid n2 and stored at -80 degrees celsius. binding of recombinant proteins to glycan microarrays. the tested protein molecules were first labeled with cy3 fluorescent dye (10 mg/ml in dmso). after dialysis, they were incubated at different concentrations with microarrays for 1 hour in the dark at room temperature. after incubation, the microarray slides were gently washed using washing buffer (20 mm tris-cl containing 0.1% tween 20, ph 7.4) to remove unbound proteins. finally, the slides were scanned with a microarray scanner luxscan-10k/aat an excitation wavelength of 532 nm and evaluated by the microarray image analyzer software. the spr measurements were performed using biacore t200 and s200 (ge healthcare). first, the carboxymethyl dextran matrix on cm5 sensor chip (ge healthcare) was activated by injection of a 1:1 mixture of 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (edc) and n-hydroxysuccinimide (nhs). recombinant proteins in 10 mm acetate buffer (ph 4.5, ge healthcare) was then injected over the chip surface at a flow rate of 30 μl/min to couple the amino groups of the recombinant proteins to the carboxymethyl dextran matrix. after the coupling reaction, the remaining activated ester groups were deactivated by ethanolamine. the binding study was carried out at 25°c in pbs-p buffer (ge healthcare). the heparin molecule or fondaparinux at different concentrations were flowed over the immobilized recombinant proteins at a flow rate of 30 μl/min with a contact time of 60 s and a dissociation time of 100 s. the surface was regenerated by injection of 10 mm glycine-hcl (ph 2.5) at a flow rate of 30 μl/min for 30 s. data was collected and analyzed by bia evaluation software (ge healthcare). all authors have given approval to the final version of the manuscript. a novel coronavirus from patients with pneumonia in china identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: 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title: immunogenicity of an s1d epitope from porcine epidemic diarrhea virus and cholera toxin b subunit fusion protein transiently expressed in infiltrated nicotiana benthamiana leaves date: 2016-08-23 journal: plant cell tissue organ cult doi: 10.1007/s11240-016-1059-5 sha: doc_id: 322231 cord_uid: jltk42dt porcine epidemic diarrhea virus (pedv) belongs to the coronaviridae family and causes acute enteritis in pigs. a fragment of the large spike glycoprotein, termed the s1d epitope (aa 636–789), alone and fused with cholera toxin b subunit, were independently cloned into plant expression vectors, yielding plasmids pmyv717 and pmyv719, respectively. plant expression vectors were transformed into agrobacterium tumefaciens and subsequently infiltrated into nicotiana benthamiana leaves. the highest expression level of s1d was found at 2 days post infiltration (dpi), reached 0.04 % of total soluble protein, and rapidly decreased thereafter. the expression and assembly of ctb–s1d fusion protein were confirmed by western blot and g(m1)-elisa. the highest expression level of ctb–s1d fusion protein was 0.07 % of tsp at 4 dpi, with a rapid decrease thereafter. in the presence of p19 protein from tomato bushy stunt virus, the s1d and ctb–s1d protein levels peaked at 6 dpi and were fourfold to sevenfold higher than in the absence of p19, respectively. after oral administration of transiently expressed ctb–s1d fusion protein, or with bacterial cholera toxin or rice callus expressing mutant cholera toxin 61f, mice exhibited significantly greater serum igg and siga levels against bacterial ctb and s1d antigen, peaking at week 6. transiently expressed ctb–s1d fusion protein will be administered orally to pigs to assess the immune response against pedv. vaccines have been used to prevent a wide range of infectious and non-infectious diseases (poland et al. 2002) . vaccine antigens are composed of antigenic proteins and are devoid of pathogenic genes, so cannot establish infection (strugnell et al. 2011) . vaccine antigens can be produced using a variety of host expression systems, including genetically engineered bacterial, yeast, mammalian and plant cells. plants are attractive expression vehicles for commercial production of vaccine antigens. plant-based vaccines hold great promise as they are cost-effective, easy-to-administer, easy-to-store, fail-safe and acceptable, particularly in developing countries (lal et al. 2007; sala et al. 2003) . novel approaches to improve the yield and biosafety of plantbased vaccines have been proposed (hernández et al. 2014) . agroinfiltration is a simple, efficient and robust method for transient protein expression . porcine epidemic diarrhea virus (pedv), classified as a member of group i of the genus alphacoronavirus, the family coronaviridae, and the order nidovirales, causes porcine epidemic diarrhea (ped) in pigs of all ages, especially in newborn pigs (pensaert and de bouck 1978) . pedv bamhi restriction site at the 5′-end and kpni at the 3′-end of the sequence was included to facilitate cloning. the s1d gene was amplified using ex taq (takara bio, shiga, japan) with the following pcr conditions: one cycle at 94 °c for 5 min; 30 cycles at 94 °c for 30 s, 58 °c for 30 s and 72 °c for 30 s, followed by one cycle at 72 °c for 5 min. the products were cloned into the pgem ® t-easy vector (promega, madison, wi, usa), creating plasmid pmyv712. gene identity was confirmed via sequencing using the universal primers t7 and sp6. the bamhi and kpni digested fragment containing the s1d gene from pmyv712 was introduced into the same sites of digested plant-expression vector pmyv497 under the regulation of the duplicated cauliflower mosaic viral 35s promoter (dp35s), ctb signal peptide, er retention signal (sek del) (munro and pelham 1987) and nos-t, yielding pmyv717. pmyv508 harboring the p19 protein of tomato bushy stunt virus (tbsv), which prevents post-transcriptional gene silencing (ptgs) in infiltrated tissues, was used for co-expression (voinnet et al. 2003) . to construct the ctb-s1d fusion gene, the bamhi and kpni-digested fragment containing the s1d gene from plasmid pmyv712 was inserted into the same sites of plasmid pmyv498 to generate plasmid pmyv719. this plasmid contains dp35s, ctb adjuvant fused with s1d at the n-terminus, an er retention signal (sekdel) and nos-t. plasmids pmyv717 and pmyv719 were transformed into agrobacterium tumefaciens strain lba4404 together with the helper plasmid prk2013 using the tri-parental mating method (horsch et al. 1985) . the plasmid pmyv98 containing the spike protein gene from pedv was used as a template for pcr. a pair of primers (forward primer 5′-ggatcc gac gtt tct ttt atg ac-3′ and reverse primer 5′-ggtaccttaaat act cat act aaa g-3′) was designed to amplify a pcr fragment containing the s1d gene and a stop codon (taa) upstream of the kpni enzyme site. a bamhi restriction site at the 5′-end and kpni at the 3′-end of the sequence was included to facilitate cloning. the s1d gene for expression in e. coli was amplified using ex taq (takara bio) with the pcr conditions described above and cloned into the pgem ® t-easy vector (promega), creating plasmid pmyv711. gene identity was confirmed via sequencing using the universal primers t7 and sp6. the bamhi and kpni-digested fragment containing the s1d gene from pmyv711 was introduced into the same sites of the e. coli expression vector pqe-30 (qiagen, hilden, germany), yielding pmyv714. the plasmid was confirmed by restriction enzyme mapping. plasmid pmyv714 was transformed into e. coli expression host disrupts villus enterocytes and causes villi atrophy within the jejunum and ileum, leading to a mortality rate of up to 95 % in infected suckling pigs (ducatelle et al. 1981) . ped was first detected in belgium (pensaert and de bouck 1978) and the uk (chasey and cartwright 1978) , and was designated cv777. outbreaks of this disease, which have been reported in many pig farming countries, have led to severe economic losses in canada (turgeon et al. 1980) , japan (takahashi et al. 1983) , china (li et al. 2012) , thailand (puranaveja et al. 2009 ), korea (lee and lee 2014) , the united states (stevenson et al. 2013 ) and, more recently, in austria (steinrigl et al. 2015) . the genome of pedv consists of a single molecule of linear positive-sense single-stranded rna. the complete sequence of the genome of strain cv777 was found to be 28,033 nucleotides in length, excluding the poly a-tail (k ocherhans et al. 2001) . the large spike glycoprotein peplomer (s), one of four structural proteins, stimulates production of neutralizing antibodies in the host. the predicted s polypeptide is 1383 amino acids long, contains 29 potential n-linked glycosylation sites and shows structural features similar to those of the coronavirus spike protein (duarte and laude 1994) . the pedv s protein can be divided into the s1 domain (aa 1-789) and s2 domain (aa 790-1383) based on the presence of the conserved nonamer and gxcx motifs at the proteolytic cleavage site of the s protein of other members of coronavirus group ii (follis et al. 2006) . within the s1 domain, two important epitope domains were determined. the first domain is the pedv-neutralizing epitope (co-26 k equivalent, coe gene), which is 139 aa long and spans the region aa 499-638 (chang et al. 2002) . sun et al. identified the second epitope domain, designated s1d (aa 636-789), and reported that it induces neutralizing antibodies (sun et al. 2008) . in this study, the s1d gene alone, and that fused with the cholera toxin b subunit (ctb) gene of vibrio cholerae, were cloned into plant expression vectors. these vectors were transiently expressed in nicotiana benthamiana (n. bentham iana) using agroinfiltration. the proteins were orally administered to mice to induce specific immune responses. to construct an s1d gene for transient expression in n. benthamiana, plasmid pmyv98 containing the spike protein gene of pedv was used as a template for pcr. a pair of primers (forward primer 5′-ggatcc gac gtt tct ttt atg ac-3′ and reverse primer 5′-ggtacc aat act cat act aaa g-3′) was designed to amplify the s1d gene. a n. benthamiana plants were a 16 h light/8 h dark cycle at 25 ± 0.5 °c in a greenhouse. six-week-old plants were used for agroinfiltration. transient expression of plasmids pmyv717 and pmyv719 was carried out using a syringe or vacuum agroinfiltration into n. benthamiana . for syringe agroinfiltration, a. tumefaciens harboring plasmids pmyv717, pmyv719 and pmyv508 was inoculated into 5 ml of lb medium containing kanamycin (50 mg/l) and rifampicin (50 mg/l) and incubated at 28 °c for 2 days. the cells were harvested by centrifugation and dissolved in mes infiltration buffer (10 mm mes, 10 mm mgso 4 , ph 5.5, 200 µm acetosyringone (bioworld, usa)) to an od 600 of 0.8. six-week-old n. benthamiana leaves were infiltrated using a 1 ml syringe lacking a needle. the infiltrated leaves were harvested at 2-8 days after infiltration (dpi) and protein expression was assessed. for vacuum agroinfiltration, agrobacterium strains were inoculated into 5 ml of lb medium containing the appropriate antibiotics at 28 °c for 2 days. the inoculated bacteria were transferred into 200 ml of lb medium supplemented with appropriate antibiotics and incubated at 28 °c for 1 day. the cells were harvested by centrifugation and dissolved in 10 l of mes infiltration buffer to a final od 600 of 0.2. a 6-week-old n. benthamiana plant was placed upside down on a shelf and its leaves were submerged in mes infiltration buffer in a vacuum chamber (fig. 2a) . vacuum at 0.1 mpa was applied for 2 min and the release valve was slowly opened to release the vacuum. the infiltrated plants were further grown in a greenhouse under 16 h continuous light per day (fig. 2b) . the infiltrated leaves were harvested at 6 dpi, lyophilized, ground and fed to mice. the infiltrated leaves were harvested at 2, 4, 6 and 8 dpi. total soluble protein was extracted from leaf tissues by grinding in liquid nitrogen with a mortar and pestle. the homogenate was suspended in arakawa buffer extraction buffer (arakawa et al. 1997) (1:2 w/v) (200 mm tris-hcl, ph 8.0, 100 mm nacl, 400 mm sucrose, 10 mm edta, 14 mm 2-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride (pmsf), and 0.05 % tween 20) and centrifuged at 13,000g for 15 min at 4 °c to remove insoluble cell debris. protein concentration was measured by bradford protein assay (bio-rad). aliquots of protein extracts containing 40 µg of tsp, along with prestained molecular weight markers, were separated by 10 % (non-boiled condition) or 12 % (boiled condition) using sds-page (bio-rad) at 120 v for 2-3 h in tris-glycine buffer, ph 8.3 (25 mm tris, 250 mm glycine and 0.1 % sds). the separated protein bands were transferred onto hybond c membranes (amersham pharmacia strain sg13009 (qiagen) for production of recombination protein. purification of the recombinant s1d protein synthesized in e. coli was performed under denaturating conditions in 8 m urea (k im et al. 2009 ). briefly, a bacterial colony harboring the s1d gene was inoculated into 5 ml of luria bertani (lb) medium containing ampicillin (100 mg/l) and kanamycin (5 mg/l), and incubated overnight at 37 °c. the culture was transferred to 200 ml of lb medium and incubation continued at 37 °c for 2 h to an od 600 of 0.6-0.8. expression of the recombinant proteins was induced by adding iso-propyl-β-d-thiogalactopyranoside (iptg) to a final concentration of 10 mm, followed by incubation for a further 6 h at 37 °c. the cells were harvested by centrifugation and lysed in 10 ml of 'buffer z' (8 m urea, 100 mm nacl, 20 mm hepes, ph 8.0) by sonication on ice (20 min; 20 s runs with 15 s breaks between each run). after centrifugation at 10,000 rpm for 10 min at 4 °c using a ja-14 rotor (beckman coulter, pasadena, ca, usa) to remove cell debris, imidazole was added to the bacterial lysate supernatant to a final concentration of 10 mm and the sample was loaded onto a 2 ml nickel column (ni-nta; invitrogen, carlsbad, ca, usa). the histidine-affinity column was washed with 15 ml of buffer z plus imidazole (10 mm) to remove weakly bound proteins of e. coli origin. the his-tagged recombinant proteins were eluted with buffer z plus 250 mm imidazole. the purified recombinant proteins were quantified by bradford protein assays (bio-rad, hercules, ca, usa) and dialyzed in phosphatebuffered saline (pbs) containing 8.0 g/l nacl, 0.2 g/l kcl, 1.44 g/l na 2 hpo 4 .2 h 2 o and 0.24 g/l k h 2 po 4 with ph 7.4 to remove urea and imidazole. after dialysis, the recombinant proteins were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) and western blotting using a mouse anti-his tag antibody and injected into mice for antibody production. for production of mouse anti-s1d antibody, 50 µg of purified s1d protein mixed with freund's complete adjuvant (sigma-aldrich, st. louis, mo, usa) were intravenously injected into the tail of mice. as a booster, 50 µg of purified s1d with freund's incomplete adjuvant (sigma-aldrich) was subcutaneously injected into mice twice at a 10-day interval (leenaars and hendriksen 2005) . blood serum was collected at 3 days after the final booster and tested for antibody-binding activity with purified s1d antigen by western blot analysis and subsequently used to detect expression of s1d protein in plants. nicotiana benthamiana seeds were germinated in small bottles for 2 weeks. the seedlings were transferred to bottles at one sapling per bottle. the optimal conditions for growth of incubated at 37 °c for 2 h, followed by three washes using pbst buffer (pbs containing 0.05 % tween 20). the wells were loaded with serial dilutions (100 µl/well) of extracts in arakawa protein extraction buffer and incubated for 2 h at 37 °c. the wells were washed three times with pbst, loaded with 100 µl/well of a 1:5000 dilution of rabbit anti-ct primary antibody or mouse anti-s1d antibody, and incubated for 2 h at 37 °c, followed by washing three times with pbst buffer. the plate was then incubated with 100 µl/ well of secondary antibody, a 1:5000 dilution of alkaline phosphatase-conjugated anti-rabbit igg (a-2556, sigma) or a 1:7000 dilution of anti-mouse igg conjugated with alkaline phosphatase (s372b, promega) for 2 h at 37 °c and washed three times with pbst buffer. the plate was finally incubated for 15 min at room temperature with 100 µl/well of phosphatase substrate (s0942, sigma). optical density was measured at 405 nm wavelength in an elisa reader (mra-006; packard instrument co., meriden, ct, usa). the ctb-s1d fusion protein, which was expressed at a high level in infiltrated leaves, was selected for mouse oral gavage. female balb/c mice were purchased from orientbio, inc. (seongnam, k orea) and randomly divided into four groups of five mice each (fig. 6a) . experimental mice were administered the powder through oral gavage every week for 6 weeks (huy et al. 2012) , as shown in fig. 6b . for feeding immunization, the mice were fasted 8 h before gavage with 2.0 ml of pbs buffer ph 7.0 containing lyophilized leaf powder, which harbored 100 µg of ctb-s1d fusion protein, or with bacterial cholera toxin-bct (sigma) or rice callus-expressed mutant cholera toxin 61 f (arg to phe)-rctx and wild-type (wt) n. benthamiana leaves. blood and fecal samples were collected before feeding and 3 days after feeding at weeks 4 and 6 (jespersgaard et al. 1999) . the collected blood samples were stored at room temperature for 1 h, chilled on ice for 1 h and centrifuged at 13,000 rpm for 10 min. sera were collected and stored at −70 °c for antibody assays. fecal samples (200 mg) were dissolved in 1 ml of pbs buffer containing 0.01 % sodium azide. insoluble materials were removed by centrifugation at 13,000 rpm for 10 min. fecal extracts were also stored at −70 °c for analysis of specific secretory iga (siga). for analysis of specific systemic antigen-specific igg and siga, an elisa was conducted according to a described protocol (k im et al. 2010 ) with minor modification. biotech, piscataway, nj, usa) in transfer buffer (50 mm tris, 40 mm glycine, and 20 % methanol) using a minitransblot apparatus (bio-rad) at 130 ma for 2-3 h. to prevent non-specific antibody reactions, membranes were blocked with 10 % non-fat milk powder in tbst buffer (tris-buffered saline with 0.05 % tween 20) with gentle agitation on a rotary shaker at 20 rpm overnight. membranes were incubated with a 1:5000 dilution of rabbit anti-ct antiserum (immunology consultants lab, portland, or, usa) or mouse anti-s1d serum in tbst antibody dilution buffer containing 3.0 % non-fat dry milk. membranes were then washed three times with tbst buffer and incubated for 2 h with a 1:5000 dilution of anti-rabbit igg conjugated to alkaline phosphatase conjugate (s3731, promega) or a 1:7000 dilution of anti-mouse igg conjugated to alkaline phosphatase (s372b, promega) in tbst buffer. membranes were washed twice with tbst buffer and once with n buffer (100 mm tris, ph 9.5, 5 mm mgcl 2 , and 100 mm nacl). color was developed using premixed bcip/nbt solution (sigma). immunoblot membranes were subjected to densitometry using alpha ease fc ™ software (ver. 3.3.3; alphainotech, san leandro, ca, usa) to estimate protein expression levels. the n-glycosylated structures of s1d protein produced in plants were determined using a deglycosylation enzyme. the infiltrated leaves were ground as described above. the homogenate was suspended in denaturing extract buffer, including 0.5 % sds and 1 % β-mercaptoethanol and subsequently centrifuged at 13,000g for 15 min at 4 °c to remove insoluble cell debris. the extracts were boiled for 10 min and chilled on ice for 5 min. the n-glycosylated sites of plant-produced protein were removed by pngase f (elpis biotech, taejeon, korea) in 35 µl of reaction mix, which comprised 3.5 µl of 10× reaction buffer, 1000 units of enzyme and 50 µg of protein extract for 1 h at 37 °c. the proteins were visualized using western blot analysis as described above. to examine the binding ability of the fusion proteins to gangliosides and to estimate the expression levels of ctb-s1d fusion protein in infiltrated leaves, a g m1 -ganglioside enzyme-linked assay was conducted. briefly, a 96-well microtiter plate was coated with 100 µl/well of monosialoganglioside g m1 (3.0 µg/ml) (g-7641, sigma) dissolved in bicarbonate buffer, ph 9.6 (15 mm na 2 co 3 and 35 mm nahco 3 ), covered with plastic wrap and incubated overnight at 4 °c. the wells were blocked by adding 300 µl/ well of 1 % bovine serum albumin (bsa) in pbs and the s1d gene alone was cloned into a plant expression vector, yielding plasmid pmyv717 (fig. 1a) , under the regulation of the duplicated camv 35s promoter, ctb signal peptide and terminator of nopaline synthase. to enhance the expression level, the kozak sequence (kang et al. 2004) was added upstream of the start codon and the er retention signal (sekdel) at the c-terminus of the s1d gene. to express the ctb-s1d fusion gene, the s1d gene was inserted into a plant expression vector containing the duplicated camv 35s promoter, the ctb gene and the nos terminator, yielding plasmid pmyv719 (fig. 1b) . plasmid pmyv508 contains the p19 gene, which encodes the suppressor of gene silencing under the control of the duplicated camv 35s promoter and the nos terminator (fig. 1c) . the plasmids pmyv717, pmyv719 and pmyv508 were transformed into a. tum efaciens lba4404 for agroinfiltration into n. bentham iana leaves. the conditions for vacuum infiltration with intact leaves were optimized in our laboratory (fig. 2) . vacuum agroinfiltration for production of transiently expressed proteins is highly scalable. agrobacterium tumefaciens lba4404 harboring the plasmid pmyv717 was co-infiltrated without or with the p19 construct (pmyv508) (fig. 3a) into n. benthamiana leaves. the infiltrated leaves were harvested at 2, 4, 6 and 8 dpi. immunoblot analysis was conducted to detect s1d protein in infiltrated leaves. the s1d protein was detected with a molecular briefly, the elisa plates were coated with 100 µl/well of antigen (80 ng/well) dissolved in bicarbonate buffer (15 mm na 2 co 3 , 25 mm nahco 3 , ph 9.6) covered with plastic wrap and incubated overnight at 4 °c. the wells were washed thrice with pbst (pbs plus 0.05 % tween 20), blocked by addition of 300 µl per well of 1 % bsa in pbs buffer, and incubated at 37 °c for 2 h, followed by three washes with pbst. the test sera and fecal extracts were diluted in pbs buffer at 1:100 and 1:4, respectively, which was loaded into the antigen-coated plates and incubated at 37 °c for 2 h. the plates were washed thrice with pbst, and then incubated with 100 µl per well of a 1:7000 dilution of anti-mouse igg (a-3688, sigma) or anti-mouse iga (a-4937, sigma) conjugated to alkaline phosphatase for 2 h at 37 °c and washed thrice with pbst. the plates were developed by addition of 100 µl per well of alkaline phosphatase buffer (10 % (v/v) diethanolamine, 0.1 % mgcl 2 , 0.02 % sodium azide, ph 9.8) plus one tablet of phosphate substrate (s0942-100tab, sigma) for 30 min at room temperature in the dark. the plates were read at 405 nm wavelength in an elisa reader (packard instrument co.). the data were analyzed using excel 2007 software (microsoft corp, redmond, wa, usa) and expressed as means ± sd of at least three independent experiments. the differences in the levels of total igg and siga antibodies were evaluated by one-way anova. statistical significance was determined using spss ® for windows software (ver. 21.0; spss inc., chicago, il, usa). p < 0.05 was considered to indicate statistical significance. nos-t nos-t nptii nos-p lb rb sp s1d dp35s nos-t nos-t nptii nos-p lb rb a -pmyv717 nos-t nos-t nptii nos-p lb rb p19 c -pmyv508 fig. 1 t-dna constructs used for agroinfiltration. binary vectors for transient expression of s1d protein (a), ctb-s1d fusion protein (b) and p19 protein (c). lb left border of t-dna; rb right border of t-dna; s1d s1d epitope; ctb cholera toxin b subunit; dp35s duplicated camv 35s promoter; nos-t terminator of nopaline synthase, nos-p promoter of nopaline synthase; sp ctb signal peptide; kozak kozak consensus sequence; sekdel er retention signal; npt ii neomycin phosphotransferase ii kanamycin resistance gene; p19 suppressor gene of the p19 protein of tomato bushy stunt virus and anti-s1d antibodies (fig. 3e, f) . the monomer of the ctb protein was detected at ~12 kda (fig. 3e) . the molecular weight of the monomer of ctb-s1d based on in silico analysis is ~30 kda. the high molecular weight of plantderived ctb-s1d (~51 kda) may be due to glycosylation of ctb (2 potential n-glycosylation sites) and s1d (seven potential n-glycosylation sites, fig. 3e, f) , resulting in retardation of migration. the densities of ctb-s1d fusion proteins were significantly increased in the presence (+p19) of p19 ( fig. 3c-f ). the biological function of the ctb-s1d fusion protein produced in infiltrated leaves was determined by g m1 -elisa. the g m1 -ganglioside-binding capacity of ctb-s1d fusion protein was determined using anti-ct (fig. 4a ) and anti-s1d (fig. 4b ) primary antibodies. these results indicated that the monomeric ctb-s1d fusion proteins were properly assembled into a biologically functional form and that s1d was conserved in the fusion protein. the immunoblot membranes were subjected to densitometry to estimate the level of s1d protein using the alpha ease fc ™ software. predetermined protein concentrations (20, 40 and 80 ng) were used to generate a standard curve. protein expression levels were estimated based on a timecourse analysis. the expression levels of transient proteins were expressed as percentages of total soluble protein (% of tsp). the highest expression level of s1d was found at 2 dpi, and reached 0.04 % of tsp (fig. 5a) . in the presence weight of ~34 kda (fig. 3a, b) in comparison with the bacterial s1d positive control, which was observed at ~17 kda ( fig. 3a-f ). no background signal bands corresponding to the s1d protein were detected in protein extracts from leaves infiltrated with p19 at 4 dpi, as a negative control. leaves infiltrated with s1d without p19 showed high-density bands at 2 dpi, whereas the s1d levels in the presence of p19 (+p19) were significantly increased, and peaked at 4 dpi (fig. 3a) . the s1d protein has seven potential n-glycosylation sites. to analyze n-glycosylation status, protein extracts were treated with pngase f (fig. 3b) to deglycosylate the glycan(s) from the core peptide in the denatured protein. the deglycosylated s1d protein band was lower than the untreated bands and migrated in a manner similar to the bacterial s1d protein (fig. 3b ). this indicates that s1d protein underwent glycosylation in infiltrated leaves. the ctb-s1d fusion protein was detected in both boiled and unboiled protein extracts from infiltrated leaves using anti-ct and anti-s1d antibodies. no signal band corresponding to the ctb-s1d protein was detected in protein extracts from leaves infiltrated with p19 at 4 dpi, as a negative control. in the unboiled condition, assembly of ctb-s1d fusion protein into oligomeric structures resembling pentamers with a molecular weight of over 170 kda was detected using anti-ct and anti-s1d antibodies (fig. 3c, d) , in comparison with the bacterial ctb positive control, which was observed at ~43 kda (fig. 3c) . a 17 kda band was observed for the bacterial s1d protein (fig. 3d, f) . when protein extracts were boiled for 5 min, the oligomeric fusion protein dissociated into ~ 51 kda monomers and was detected by both anti-ct thereafter. the ctb-s1d expression levels in the presence of p19 reached 0.49 % of tsp (fig. 5b) , and rapidly increased from 2 to 4 dpi, peaking at 6 dpi. this result indicates that p19 enhances the expression levels and assembly of ctb-s1d fusion protein into biologically functional molecules. of p19, expression levels significantly increased to 0.15 % of tsp at 4 dpi (fig. 5a) . g m1 -elisa was employed to evaluate the expression levels of the assembled ctb-s1d fusion protein. the highest amount of assembled ctb-s1d fusion protein was 0.07 % of tsp at 4 dpi, and decreased rapidly western blot analyses of transiently expressed s1d protein and ctb-s1d fusion protein in agroinfiltrated n. bentham iana leaves. transient expression of plasmid pmyv717 containing the s1d gene (a) co-infiltrated with (+ p19) or without plasmid pmyv508 (p19). infiltrated leaves were sampled at 2, 4, 6 and 8 dpi. protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and analyzed using a mouse anti-s1d primary antibody. analysis of n-glycosylation of s1d protein with pngase f(+) (b). transient expression of the ctb-s1d fusion protein in agroinfiltrated n. bentham iana leaves. n. bentham iana leaves were co-infiltrated with plasmid pmyv719 with or without p19 and sampled at 2, 4, 6 and 8 dpi. protein extracts were separated by sds-page and analyzed after boiling (e, f) or not (c, d) using an anti-cholera toxin (c, e) or anti-s1d (d, f) primary antibody. lane m, prestained protein ladder (fermentas, hanover, md, usa); bs1d, s1d antigen expressed in e. coli; bctb, commercial bacterial cholera toxin b subunit (sigma); p19-4, 4 dpi leaves infiltrated with plasmid pmyv508 used as a negative control. p and m indicate the pentamer and monomer of ctb-s1d fusion protein, respectively and anti-s1d igg and siga were significantly higher in all experimental groups at 4 weeks of feeding, and peaked at 6 weeks of feeding (fig. 7) . as expected, antibodies were not detected in the control group. anti-ctb and anti-s1d igg levels were significantly increased in mice administered ctb-s1d + bct and ctb-s1d + rctx in comparison with mice given ctb-s1d at 6 weeks ( fig. 7a, b) . moreover, anti-ctb and anti-s1d siga levels were significantly higher in mice given ctb-s1d + bct compared with those administered ctb-s1d and ctb-s1d + rctx at 6 weeks ( fig. 7c, d) . oral immunization with transient expression ctb-s1d fusion protein induced significantly higher anti-ctb, anti-s1d igg and siga levels compared with the control group (fig. 7 ). pedv causes acute enteritis characterized by vomiting and watery, fetid diarrhea in affected pigs of all ages. the mortality rate in piglets is 90-95 %, leading to significant economic losses (debouck and pensaert 1980; steinrigl et al. 2015) . ped was reported as a re-emergent disease in the united states and neighboring countries, such as canada and central american, european and asian countries (beam et al. 2015; li et al. 2012; steinrigl et al. 2015) . in late 2013, a severe outbreak of pedv infection occurred in k orea, causing economic losses to the pork industry (lee et al. 2015) . therefore, an effective vaccine to protect pigs against pedv infection is required. the neutralizing coe epitope (aa 499-638) in the spike glycoprotein was identified (chang et al. 2002) . based on optimized codon usage in plant, expression of the synthetic coe gene was fivefold higher than that of the native gene in transgenic tobacco plants (kang et al. 2005) . to enhance the immune mice were orally immunized with wt or transient leaf powder containing ctb-s1d protein (ctb-s1d) alone, ctb-s1d protein with rice-expressed mutant ct61f (ctb-s1d + rctx) and with bacterial ct (ctb-s1d + bct) adjuvants on six separate occasions using appropriate doses of antigen and adjuvant (fig. 6) . to determine the levels of anti-ctb and anti-s1d antibodies, sera and fecal extracts were subjected to elisa. the anti-ctb the biological activity of ctb-s1d fusion protein with the intestinal membrane g m1 -ganglioside receptor was confirmed using an anti-cholera toxin antibody (a) and anti-s1d antibody (b) transiently co-expressed proteins (mohammadzadeh et al. 2015; voinnet et al. 2003) . the s1d and ctb-s1d protein levels were enhanced from fourfold to sevenfold in the presence of p19 (figs. 3, 4) , respectively. moreover, accumulation of the ctb-s1d fusion protein was fourfold greater than that of the s1d protein (fig. 4) . the ctb portion plays a role in enhancing protein accumulation and as a mucosal adjuvant and carrier. in previous studies, the expression level of synthetic coe-co1 was 0.083 % of tsp in transgenic rice under the control of the strong ramy3d promoter at day 3 after induction in sucrose starvation medium (huy et al. 2012) . the highest expression level of ctb-coe fusion protein was 0.0065 % of tsp in transgenic lettuce plants (huy et al. 2011) . by performing transient expression in the presence of p19, the expression levels of s1d protein and ctb-s1d fusion protein were 2-and 75-fold higher than those of coe-co1 and ctb-coe in transgenic plants. the increased protein expression levels might result in elicitation of strong immune responses and prevent induction of immune tolerance by plant-based oral vaccines. the binding of functional ctb-s1d fusion protein to its biological receptor, g m1 -ganglioside, was confirmed by g m1 -elisa with anti-ct and anti-s1d antibodies (fig. 4) . these results indicated that the s1d protein of the ctb-s1d fusion protein was assembled into functional oligomeric structures and retained its antigenicity. to investigate the immunogenicity of the transiently expressed protein, the ctb-s1d fusion gene was transiently expressed in n. bentham iana leaves by vacuum agroinfiltration (fig. 2) . the infiltrated leaves were lyophilized and responses, the synthetic coe gene was fused with a mucosal adjuvant and carrier, ltb and ctb, respectively, and with the m cell-target peptide ligand co1, and expressed in various plant species including tobacco (kang et al. 2005) , lettuce (huy et al. , 2011 and rice callus (huy et al. 2012) . oral immunization with coe-co1 fusion protein expressed in transgenic rice callus induced systemic and mucosal immune responses against the coe antigen (huy et al. 2012) . recently, s1d (aa 636-789) has been seen as an alternative neutralizing epitope domain in the spike protein of pedv (sun et al. 2008) . in this report, the s1d epitope alone or fusion protein was expressed in infiltrated n. bentham iana by agroinfiltration. the immunogenicity of the transiently expressed proteins was investigated to develop an edible vaccine against pedv. plant cells have been widely used to express recombinant vaccine antigens. recombinant proteins can be expressed in plants by creating stable transgenic plants or by performing transient expression. transient expression offers several advantages: accumulation of high levels of protein, short timeline of protein production, scalability and elimination of transgene outflow . to optimize s1d expression, the gene was added to various expression vectors, including the agrobacterium vector ptrac (maclean et al. 2007 ), a viral pro-vector (marillonnet et al. 2004 ) and a binary vector. the target protein was expressed at a high level using the binary vector. however, the level of protein production was low (figs. 3a, 4a) , as it was limited by the ptgs factor. the p19 protein of tbsv prevents the onset of ptgs in infiltrated leaves and enhances the levels of a ntigen feeding dose adjuvant feeding dose wt -0 µg --0 µg ctb-s1d c tb-s1d 100 µg -0 µg ctb-s1d + rctx ctb-s1d 100 µg rice calli-expressed mutant ct61f (arg to phe) 5µg ctb-s1d+bct ctb-s1d 100 µg bacterial ct (sigma) 5 µg 1 2 3 4 5 6 7 week sampling: blood (igg); feces (iga) 0 b fig. 6 mouse feeding strategy. a female balb/c mice were randomly divided into four groups of five mice each. wild-type (wt) and infiltrated n. benthamiana leaves were lyophilized and ground. mice in the experimental groups were orally immunized with infiltrated leaf powder containing 100 µg of ctb-s1d fusion protein, 100 µg of ctb-s1d fusion protein with 5 µg of rice calli-expressed ct61f (rctx) and 5 µg of bacterial cholera toxin (bct) in 2 ml of pbs buffer. the mice in the negative control group were fed wt leaf powder. blood and fecal samples were collected 3 days after feeding at weeks 4 and 6 as well as 3 days before starting feeding ( fig. 7 induction of specific antibodies in mice. serum igg and siga against ctb antigen (a-c) and s1d antigen (b-d) in mice at weeks 0, 4, and 6. mouse sera and fecal samples were diluted 100-and fourfold, respectively, and used in elisas. results are mean od (405 nm) values of five immunized mice. error bars represent standard deviations. any two means in a column with the same letter in common are not significantly (p ≤ 0.05) different according to tukey's test. different letters within a column indicate significant differences (p ≤ 0.05) according to tukey's test ground, and the expression of assembled ctb-s1d fusion protein was confirmed before oral administration to female balb/c mice for 6 weeks at a 1-week interval (fig. 6b) . mice given the ctb-s1d fusion protein exhibited ctband s1d-specific systemic igg at week 4. the igg and siga levels were significantly increased at week 6 ( fig. 7a-d) . ct acts not only as a mucosal immunogen and mucosal adjuvant but also as induces immune responses to unrelated nasally and orally co-administered antigens (elson and ealding 1984; isaka et al. 2004 ). the ct61f mutant, which lacks adp-ribosylation activity but retains the mucosal adjuvant properties of native ct, was expressed in rice callus (unpublished paper). to enhance the immune responses, transiently expressed ctb-s1d was co-gavaged with bct and rctx into mice at appropriate doses (fig. 6a) . as expected, anti-ctb and anti-s1d ab levels, in mice given ctb-s1d with bct and with rctx at 6 weeks ( fig. 7a, b) , were significantly higher than those of mice fed ctb-s1d only. siga is the predominant antibody class in external secretions and the major humoral defense factor that prevents colonization of mucosal surfaces by infective agents, such as bacteria and viruses (haan et al. 1995; woof and mestecky 2005) . this siga may thus prevent ped. the levels of s1d-specific siga responses were significantly greater than those in the control group. the anti-s1d antibody levels in mice given ctb-s1d and bct were significantly increased in comparison with those fed ctb-s1d only (fig. 7d) . the transient expression of ctb-s1d fusion protein thus induced s1dspecific systemic and mucosal immune responses following oral administration to mice. bct and rctx enhanced s1dspecific antibody responses when orally co-administered with ctb-s1d protein. in conclusion, the s1d gene alone, and that fused with the ctb gene, were expressed in infiltrated n. benthamiana leaves. oral immunization of mice with ctb-s1d fusion protein elicited systemic and mucosal immune responses. the ctb-s1d fusion protein could be used to produce a plant-based vaccine against pedv. a porcine epidemic diarrhea virus outbreak in one geographic region of the united states: descriptive epidemiology and investigation of the possibility of airborne virus spread identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus virus-like particles associated with porcine epidemic diarrhoea agroinfiltration as an effective and scalable strategy of gene delivery for production of pharmaceutical proteins experimental infection of pigs with a new porcine enteric coronavirus, cv 777 sequence of the spike protein of the porcine epidemic diarrhoea virus three-dimensional sequential study of the intestinal surface in experimental porcine cv 777 coronavirus enteritis cholera toxin feeding did not induce oral tolerance in mice and abrogated oral 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porcine epidemic diarrhea virus strains similar to us strains isolation and characterization of a korean porcine epidemic diarrhea virus strain k nu-141112 critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations efficient agroinfiltration of plants for high-level transient expression of recombinant proteins new variants of porcine epidemic diarrhea virus, china optimization of human papillomavirus type 16 (hpv-16) l1 expression in plants: comparison of the suitability of different hpv-16 l1 gene variants and different cell-compartment localization in planta engineering of viral rna replicons: efficient assembly by recombination of dna modules delivered by agrobacterium acknowledgments this research was supported by the agriculture, food and rural affairs research center support program, ministry of agriculture, food and rural affairs, and nguyen-quang-duc tien was supported by the bk21 plus program, republic of korea.author contributions nguyen-xuan huy, nguyen-quang-duc tien and moon-sik yang conceived, designed and performed the overall study. mi-young kim generated the transformed rice callus expressing mutant cholera toxin 61f. tae-geum kim and yong-suk jang designed the mouse experiment. nguyen-xuan huy wrote the manuscript. key: cord-312741-0au4nctt authors: lin, panpan; wang, manni; wei, yuquan; kim, taewan; wei, xiawei title: coronavirus in human diseases: mechanisms and advances in clinical treatment date: 2020-10-01 journal: medcomm (beijing) doi: 10.1002/mco2.26 sha: doc_id: 312741 cord_uid: 0au4nctt coronaviruses (covs), a subfamily of coronavirinae, are a panel of single‐stranded rna virus. human coronavirus (hcov) strains (hcov‐229e, hcov‐oc43, hcov‐hku1, hcov‐nl63) usually cause mild upper respiratory diseases and are believed to be harmless. however, other hcovs, associated with severe acute respiratory syndrome, middle east respiratory syndrome, and covid‐19, have been identified as important pathogens due to their potent infectivity and lethality worldwide. moreover, currently, no effective antiviral drugs treatments are available so far. in this review, we summarize the biological characters of hcovs, their association with human diseases, and current therapeutic options for the three severe hcovs. we also highlight the discussion about novel treatment strategies for hcovs infections. f i g u r e 1 genome organization and structure of hcovs human covs generally cause only mild upper respiratory diseases, which is similar to common flu. 11, 12 a novel cov, named sars-cov-2 by the world health organization (who), has emerged again at the end of 2019, causing more infections and deaths worldwide than ever before. 13 the absence of effective antiviral treatments and serious consequences of these three covs have highlighted the urgent need for novel drug development to prevent the spread of covs. herein, this review mainly focuses on the biological characters of hcovs, their association with human diseases, and current therapeutic options for the three severe hcovs. we also highlight the discussion about novel treatment strategies for hcovs infections. covs possess a nonsegmented, positive, single-stranded rna genome of 26-32 kb. 2, 14, 15 all covs have a similar genome arrangement with a 5′-methylated cap structure along with 3′-polyadenylated tail. the replicase gene, occupying about 20 kb, two-thirds of the genome and comprising two open reading frames (orfs), orf1a and orf1b, is located at the 5′ end. 2 it encodes two large polyproteins (pp) 1a and 1ab that can be cleaved by papain-like cysteine protease (plpro) and 3c-like serine protease (3clpro) into nonstructure proteins, involving some proteases, several rna modification enzymes, as well as rna-dependent rna polymerase (rdrp) and helicase (hel) required for virus replication. 16 additionally, an untranslated region (utr) can also be identified at the 5′-end as same as the 3′terminal. structure proteins, encompassing the 3′-terminal one-third of the genome, are arranged in a certain order of hemagglutinin esterase (he) protein that is present in some beta-covs, spike protein (s), small membrane protein (e), membrane protein (m), and nucleocapsid protein (n). in brief, the arrangement of the cov genome can be shown as 5′-utr-replicase gene (orf 1a and orf 1b)-he protein (if have)-s protein-e protein-m protein-n protein-3′ utr-poly (a) 2 ( figure 1 ). cov is named for the club-shaped projections eradiating from the envelope, which forms its corona or crow-like morphology. the shape of the viral particles is roughly spherical with approximately 80-160 nm in diameters. [17] [18] [19] the nucleocapsid protein and the genome rna intertwine to form a helical structure located inside the envelope. for some covs, the spikes on the surface are not only formed by trimers of s protein, but also he proteins. m protein and e protein, two transmembrane proteins, also participate in the composition of the virus (figure 1 ). s protein, a transmembrane protein, mediates the initiation of cov infection by attaching to the specific receptors on the target cells. [20] [21] [22] for a prototypical cov, s protein is usually cleaved into an extracellular receptor binding subunit (s1) and a membrane-anchored subunit (s2), responsible for virus binding and membrane fusion, respectively. 23, 24 two heptad repeats (hr1 and hr2) and enriched alpha-helices are contained in the s2 domain, a feature typical of fusion protein. [25] [26] [27] the receptor-binding domain (rbd) of s protein specifically binds to target receptors, leading to the conformation change of s1/s2 complex that mediates virus entry. 26 furthermore, the rbd also induces potent neutralizing ab response, which turns s protein into an important antigenic determinant capable of protective immunity induction and provides a vital approach for the development of immunotherapies. [28] [29] [30] [31] [32] cov e protein is an integral membrane protein of 76-109 amino acids [33] [34] [35] and is present in small amount in virions. [36] [37] [38] it contains a short hydrophilic n-terminal followed by a single predicted hydrophobic domain and a hydrophilic c-terminal. although its membrane topology remains unclear, cov e protein is commonly referred to as a transmembrane protein with one transmembrane domain. [39] [40] [41] accordingly, the e protein mainly targets er and golgi-complex and participates in part of the life cycle of the virus, including virus assembly, budding, particles release, envelope formation, and viral pathogenesis. 33, 35, [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] in addition, cov e protein may have a crucial role in virus production and maturation, because recombinant covs lacking the e protein exhibit significantly reduced viral titers and toxicity. [51] [52] [53] [54] [55] the m protein is a small transmembrane protein (25) (26) (27) (28) (29) (30) with three transmembrane segments, an n-terminal ectodomain and a c terminal-endodomain, determining the shape of the virion. 56, 57 it is considered as the most abundant structural protein and plays a pivotal role in virion assembly via interacting with other structural proteins. [58] [59] [60] binding of s protein and m protein is essential to the virus assembly and the maintenance of s protein in the er-golgi intermediate compartment (ergic)/golgi complex. [61] [62] [63] virus-like particles (vlps) are assembled when the combination of m and e proteins occurs, which suggests that they are required for the formation of the envelope. 39, [64] [65] [66] additionally, when expressed alone, it can become a homomultimeric complex, the primary driving force of envelope formation. 43, 60, 67 furthermore, as to n protein, a stable nucleocapsid and internal core of virions can be achieved when combined with m protein. 68, 69 the n protein, combined with viral genomic rna to form a helical nucleocapsid inside the viral envelope, is a multifunctional protein. 70 it contains three highly conserved domains: the n-terminal domain (ntd), responsible for rna binding; a c-terminal domain (ctd) that is a hydrophobic and helix-rich terminal, capable of dimerization and oligomerization; and an intrinsically disordered region (rna-binding domain/domain 2) that is a serine/arginine-rich domain (sr-domain) with a significant phosphorylation potential. 15, [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] phosphorylation of the n protein can initiate structural modification leading to an increased rna-binding affinity. 72, 79, 87, 88 the n protein binds to the genomic rna through a beads-on-astring form. likewise, except for the interaction between n protein and nucleic, the ability of complex oligomerization is another pivotal activity required for the formation of the ribonucleoprotein complexes for viral assembly. 89 in addition to the role of the n protein in viral core formation and assembly, 69, [90] [91] [92] it is also involved in other critical processes of viral life cycle such as virus budding and envelope formation, 21,93-95 genomic mrna replication ,and genomic rna synthesis. 96, 97 3 although the pathogenic mechanisms of human covs have not yet been fully understood, the investigation of their unique characteristics of each cov enables to distinguish the various human covs including sars, mers, and sars-cov-2. the crucial early step of the infection of human covs into susceptible host cells is the interaction between viral s protein and cellular target receptors. one of the subunits of s protein, s1, containing rbd, is responsible for specific recognition and binding of the target receptors. the other subunit, s2, is in charge of the membrane fusion. 22, [98] [99] [100] [101] [102] the tissue tropism, as well as the susceptible host species, is mainly determined by the binding of s protein to target receptors. 102 based on lines of known evidence, human covs utilize multiple and different types of cellular receptors rather than use a common receptor. therefore, multiple cellular receptors have been identified as target receptors for the various human covs to date (table 1) . angiotensin-converting enzyme 2 (ace2), the first known human homolog of angiotensin-converting enzyme and the receptor for hcov-nl63, sars-cov, and sars-cov-2, is a vital component of the reninangiotensin system (ras). [103] [104] [105] [106] [107] [108] [109] it is a secreted enzyme with a transmembrane domain, a single metalloprotease active site and a signal peptide, 110 and predominantly expressed in heart, vascular endothelia, epithelia of the small intestine, kidney, and testis; alveolar macrophage and monocytes of the respiratory tract; and epithelial cells of the trachea, bronchi, and alveoli. 103, [110] [111] [112] in contrast to its homolog ace that contributes to the promotion of lung failure pathogenesis, induction of lung edemas, and impairment of lung function, ace2 plays a protective role in severe acute lung injury (ali). imai et al revealed that the deficiency of ace2 in the murine models of acute respiratory distress syndrome (ards) deteriorated the symptom in lung function, which could be recovered by hcov-nl63 hcov-hku1 the recombinant ace2. 113 thus, downregulation of ace2 expression in sars patients could be used as an indicator of severe clinical outcome. 114 besides lung damage caused by sars-cov infection could be attenuated by blocking the renin-angiotensin pathway. 114 overall, ace2 could serve as a novel therapeutic target for severe respiratory diseases. dipeptidyl peptidase iv (dpp4), a type ii transmembrane protein also known as cd26, is identified as a target receptor for mers. 115 it is a prolyl oligopeptidase expressed in various cells including endothelial cells, epithelial cells, and inflammatory cells in the lung and smooth muscle. [116] [117] [118] the multifunctional protein ddp4 is implicated in the activation of t-cell, the activity regulation of chemokines and growth factors, and the regulation of glucose metabolism. [119] [120] [121] [122] not only can it be embedded in the plasma membrane in the form of a homodimer, but it also presents in extracellular fluid like plasma as a soluble form. 118, 123 although ddp4 is expressed in epithelial cells of the upper respiratory tract in camels, it is principally found in alveoli but rarely in the nasal cavity or conducting airway. 115, 124 accordingly, in a murine mers model, though monocyte infiltration, alveolar edema and microvascular thrombosis were observed in the mers-cov-infected lungs, any symptoms were seldom found in the airways. 125 aminopeptidase n (apn), a type of ii metalloprotease also called cd13, is a ubiquitous enzyme expressed in various organs (lung, intestine, and kidney) and cells (epithelial cells, endothelial cells, leukocyte, and fibroblast). 126, 127 it serves as a target receptor for hcov-229e, 128 but not for hcov-oc43. hcov-oc43 shares the same specific target with hcov-hku1, namely 9-o-acetylated sialic acid. 129,130 virus entry is a finely regulated process requiring a series of interactions between the virion and host cell. [131] [132] [133] following the conjunction with the target receptor, cov fuse its envelope with the membrane of the host cell. these processes are forced by the conformational change of s protein, which is triggered by not only the target receptor binding but also ph acidification and proteolytic cleavage led by cell surface or endosomal proteases such as transmembrane protease serine 2 (tmprss2), furin, cathepsin l, elastase, and trypsin. [134] [135] [136] [137] [138] [139] [140] [141] [142] [143] cleavages of s protein are facilitated at two sites: the boundary between the s1 and s2 subunit (s1/s2) and the conserved site upstream of the fusion peptide (s2'). 138, 144 the former one is aimed at releasing rbd from the membrane fusion subunit, and the latter one is important for the exposure of the fusion peptide, hydrophobic in general, which acts as an anchor to target membrane. 138, 145, 146 then the fusion domain adopts two heptad repeats (hr1/hr2) to form a compact coiled-coil conformation called 6-helix bundle or 6hb. 144, 147 through direct interactions with lipid bilayers, the fusion domain disrupts two apposed membrane layers and fuses the viral envelope to host cell membrane. ultimately, the viral genome is successfully released into the cytosol of the target cell. in addition to the viral infection through the plasma membrane, the entry of covs into cells can be accomplished by the endocytic pathway, depending on the virus strains and host cells. 135,144 sars-cov, whose intermediary host is the palm civet, is a highly pathogenic respiratory virus that emerged in 2002, leading to global pandemic that affected more than 8000 people in 29 countries. 148, 149 patients infected with sars-cov initially present "flu-like" syndrome commonly showing high fever, headache, sore throat, myalgia, and dry cough. [150] [151] [152] during the disease progression, ali or ards is developed in a number of patients. 153 pathological manifestations can be described as three phases. the first step is the disturbance of gas exchange in the first week, owing to the extensive edema, shedding of ciliated epithelial cells, and deposition of hyaline-rich substances on the alveolar membrane. in the next step, pulmonary fibrosis occurs that is characterized as the deposition of fibrin at epithelial cells and the alveolar spaces, as well as the infiltration of inflammatory and fibroblasts. finally, fibrosis of lung tissue, collagen deposition, and proliferation of alveolar and interstitial cells represent the final step of disease, about 6-8 weeks. [154] [155] [156] [157] [158] diffuse alveolar damage (dad) accompanied by hyaline membrane formation as well as interstitial thickening is common characteristics of sars-cov inducing pulmonary damage. 159 although many investigators have devoted to inquiring into the pathogenesis of such virus, it has not yet been fully understood until now. the immune response is the earliest alert system of the host cells warning virus attacks. ironically, it also aids viral pathogenesis. pattern recognition receptors (prrs) that are retinoic acid-inducible gene i protein (rig-i, member of rlrs family) and melanoma differentiation-associated protein 5 (mda5) recognize viral pathogen-associated molecular patterns (pamps), such as viral components and replication intermediates, to initiate signaling cascades against virus infection. 160, 161 once the pamps from invaded viruses are detected, rig-i and mda5 interact with the mitochondrial antiviral signaling protein (mavs) that is a mitochondrial membrane-bound f i g u r e 2 escape mechanisms of innate immune response of sars-cov and mers-cov adaptor molecule, followed by the activation of several kinase complexes and multiple subsequent transcription factors (irf3, irf7, and nf-κb). activation of nf-κb induces the production of proinflammatory cytokines and chemokines in the nucleus that is a substantial cause of the ards. 162, 163 phosphorylation of irf3 and irf7 by the kinase complexes results in homo-or heterodimerization of irf3 and irf7. the dimerization initiates the transcription of type i interferons (ifns, ifn-α and ifn-β), activating the signal transducer and activator of transcription proteins (stats) to mediate antiviral response ( figure 2 ). [164] [165] [166] several defensive approaches are used by sars-cov to avoid the prrs defense system and, ultimately, host innate immune response. one approach is to replicate themselves within the double membrane vesicles (dmvs) that are protected from the prrs. 167, 168 the eukaryotic mrnas contain a 5′ cap that usually lacks in the viral mrnas. however, some viruses such as sars-cov are capable of building the rna cap through nsp14 and nsp16/nap10 complex, helping them bypass the recognition by prrs. [169] [170] [171] [172] in addition, a couple of more proteins encoded by the viruses participate in the suppression of the innate immune response by disrupting the ifn response. among the nonstructural proteins, nsp 1 mainly involves in the degradation of host mrnas, inactivation of host translational machinery as well as the inhibition of stat1 phosphorylating. [173] [174] [175] sars-cov plpro interferes phosphorylation of irf3 and disrupts nf-κb signaling probably via interacting with sting. [176] [177] [178] the nsp7 and nsp15 are also potential ifn antagonists though the mechanism is not clear. 177 structure proteins such as the n protein and m protein are likely to suppress the type i ifn path-ways. because it was known that n protein inhibits the ifn transcription, the n protein could have a strong potential influence on the viral rna. 179 m protein blocks the formation of signaling kinase complexes, and suppresses the irf3 and irf7 activities, suggesting the potential role of the n protein in the viral infection as well. 180 such accessory proteins as orf 3b protein are able to inhibit the rig-i activity and irf3 phosphorylation in addition to the transcription of ifn-stimulated genes (isgs) via the isre promoter, while orf6 blocks the nuclear translocation of stat1. 181, 182 in spite of the number of research findings in vitro, they have not been validated in vivo. therefore, it is urgent to examine the findings in vivo for a clear and solid understanding of the infectious process. besides the immune response of the host, ace2 also plays an essential role in the pathogenesis of sars-cov. as a negative regulator on the ras, ace2 has been closely linked to the pathogenesis of pulmonary diseases and considered as a protective factor for ali. 113 consequently, the downregulation of ace2 mediated by sars-cov binding might give an explanation for the progression of severe lung damage occurred on some sars patients. 114 a decade after sars, another novel cov was identified as the pathogen of mers that caused a higher mortality rate (30%-40%) compared with sars (around 10%). 183, 184 sars-cov and mers-cov are both emerged from bats, and are disseminated to human through palm civets and dromedary camels, respectively. [185] [186] [187] [188] mers-cov and sars-cov share some common clinical manifestations ranging from asymptomatic to severe pneumonia in multiple organs, 150, 184, 189 and pathological features including inflammatory cells infiltration and dad. 183 similar to sars-cov, mers-cov is also capable of causing immune dysregulation by attenuating the innate immune response ( figure 2 ). 190, 191 type i interferon is important for the inhibition of mers-cov replication in host cells probably via the suppression of the dmvs formation. 192, 193 capping viral mrna by nsp14 and nsp16/nap10 complex protects mers-cov, as well as sars-cov, from the prrs recognition since the structure of nsp16/nap10 complex in both viruses are analogous. 194 besides, several proteins of mers-cov are involved in immune escape mechanism by involving in the signaling cascades. it was reported that irf3 nuclear translocation and ifn promoter activation are inhibited by m protein, orf4a, orf4b, and orf5, former three of which also restrained the expression from an the isre promoter. 195 inhibition of the phosphorylation of irf3 might be the mechanism for ifr3 translocation inhibiting. 194 moreover, mers-cov orf4a can interact with ifn-inducible double-stranded dna (dsdna)dependent protein kinase activator a (prkra) and subsequently control the function of rig-i and mda5, resulting in the disruption of the ifn response. 196, 197 orf4a and orf4b are thought to participate in the nf-κb signaling by downregulating the gene stimulated by nf-κb and affecting the kinase complexes, respectively. 198 functions of plpro and nsp1 of mers-cov are analogous to the functions of those in sars-cov. 199, 200 like ace2, the entry receptor dpp4 for mers-cov has also a pivotal role in the disease pathogenesis and is considered as a key factor for the intraspecies variation shown in mers infection. 201, 202 it is usually expressed in type ii pneumocytes that cover 2% of the alveolar surface. 115, 124 approximately 95% of the surface area is occupied by the type i pneumocytes that are responsible for gas exchange. 203, 204 but the autopsy reports indicated that both type i and ii pneumocytes in patients died from mers-harbored dpp4 expression and these pneumocytes were infected by the virus. 205, 206 mers-cov infection of type i pneumocytes might lead to the damage of the cells in the alveoli subsequently causing the dad. 207 it suggests that type i pneumocytes expressing dpp4 might be included in the pathogenesis of the disease. this might explain why chronic obstructive pulmonary disease (copd) patients attacked by mers-cov had poor outcomes, since the expression of dpp4 was predominantly upregulated on type i pneumocytes in such patients. 202, 208 besides, owing to high expression of dpp4 shown in the kidney, the renal dysfunction might be caused by either the direct infection or the hypoxic damage. 116 evidence of tubular injury, such as cell debris and tubular dilation, could be observed in the late stage of the infection in mers-cov-infected mice. however, no virus could be detected in such animals in the early stage after infection, meaning such pathologic changes might be related to the hypoxia. 125 the outbreak of covid-19, whose pathogen is sars-cov-2, now poses a serious threat to the global public health. 209, 210 since the emergence of the virus, sars-cov-2 has affected more than 14 million people with more than 597 thousand deaths worldwide as of july 2020. next-generation sequencing of the novel virus has been developed soon after the outbreak, indicating that sars-cov-2 is closely related to the bat-derived sarslike covs. 107, 211 it is now believed that bats are likely to be the natural reservoir, 212, 213 and pangolins are regarded as intermediary host according to the later studies. typical clinical presentations of sars-cov-2-infected patients include fever, dyspnea, dry cough fatigue, myalgia, pneumonia, and ards symptoms, similar to those of sars and mers patients. [214] [215] [216] however, intestinal disorders such as diarrhea are less frequent in covid-19 patients than sars. 214, 215, 217 furthermore, in spite of the variation of amino acid at some residues, homology modeling informed that sars-cov-2 and sars-cov have an analogous rbd structure, and share the same target cell receptor, ace2, to mediate the viral entry. 107, 108, 216, [218] [219] [220] it is speculated that ace2 is involved in the pathogenesis of sars-cov-2. owing to the current sparsity of data on the pathological characters of sars-cov-2, it is poorly understood. a case report from an infected patient died of this disease showed that dad with hyaline membrane formation, infiltration of inflammatory cells, and pulmonary edema could be found in the samples taken from their lungs, which is notably corresponding with the symptoms of sars and mers patients. 221 additionally, lymphopenia is a common manifestation in covid-19 patients and might be an effective indicator to estimate the severity of hospitalized covid-19 patients. 222 lymphopenia is also supposed to be a vital factor related to the pathogenesis that has not been elucidated so far. 213 moreover, the concentration of some cytokines and chemokines detected in the plasma was higher in covid-19 patients compared with healthy individuals. moreover, higher plasma levels of gscf, il-2, il-7, il-10, mcp1, mip1a, ip10, and tnf-α were linked to the more severe disease. 215 all of the data reveal that immunopathology may occupy a crucial place in the development of the disease, and further researches about the pathogenesis of sars-cov-2 are urgently needed in the future. owing to the fact that no effective specific antiviral therapies are currently available for sars, mers, and covid-19, isolation and symptomatic support cares are the major management strategies for suspected and confirmed cases requiring hospital treatment including oxygen inhalation, fluid management, and rational use of antibiotics, to prevent organ failure and secondary bacterial infection and alleviate the symptoms. 189, [223] [224] [225] thus, the identification of effective agents against human covs is urgently needed in the response to the current covid-19 outbreak. all of sars-cov, mers-cov, and sars-cov-2 encode structure proteins (like s protein), nonstructure proteins (eg, plpro, 3clpro, rdrp, and helicase), and accessory proteins that are essential for the viral life cycle and that are considered as important targets for the development of antiviral agents ( figure 3 ). 226, 227 analyses of genomic sequences and protein structure indicated that the catalytic sites of four crucial enzymes and the key drug-binding pockets in viral enzymes were conserved across sars-cov, mers-cov, and sars-cov-2. 227 therefore, the therapeutic experience based on sars and mers is capable to guide the treatment of covid-19. the idea to disturb the normal life cycle of the virus provides significant insights into the clinical treatment strategy. the s protein is important for the discovery of antiviral agents due to its multifunction in virus infection. rbd located on the s1 subunit can bind to the host cell receptors (ace2 for sars-cov and sars-cov-2, dpp4 for mers-cov) initiating the conformational changes in s2 subunit to get viral and cell membranes closer and trigger membrane fusion. 228 consequently, the interaction between rbd and the host cell receptors serves as a key target for the production of neutralizing antibody followed by the vaccine development. 31, 229 monoclonal antibodies (mabs) and fusion inhibitors against s1 and s2 subunit, respectively, are potential antivirus drugs to conquer the viral infection, and the agents targeting the host receptors also play a similar role. [230] [231] [232] [233] [234] likewise, cleavage at the protease site of the s1/s2 complex by host proteases such as tmprss2 and furin is necessary for the membrane fusion and syncytium formation. 143, 235 the endosomal cysteine protease cathepsins promote the entry of covs into the host cell via the endosomal pathway. 236 inhibitors of these host proteases can potently block the cell entry, which has been proved in vitro and require further validation on animals studies. 237 once entering the host cells, covs release the nucleocapsid and genomic rna into the cytoplasm and start the translation of the replicase gene. the large replicase pp1a and pp1ab are cleaved by plpro and 3clpro to produce nonstructural proteins like rdrp and helicase, forming the replication-transcription complex. 238 numerous agents inhibiting these proteins have shown anti-cov effects in vitro. combination of the hydrophobic domains of the replication-transcription complex to the endoplasmic reticulum membrane can form the typical cov replication structures such as dmvs and convoluted membranes, protecting covs from the detection of prrs. 168, 239 viral rna synthesis produces genomic and subgenomic rnas. then the subgenomic rnas are translated to generate the structural and accessory proteins, participating in the assembly of the virion that is released into the extracellular compartment via exocytosis. 8 small interfering rnas (sirnas) disturbing these processes could be used in the treatment of covs infections. although covs are capable of disturbing the ifn response, they are still sensitive to the ifn treatment in vitro, indicating that augmented host innate ifn response can be an effective strategy to control the viral infection. 207, [240] [241] [242] in addition to the enhancement of inf response, several other cell signaling pathways are also regarded as potential anti-cov targets. these include calcineurin-nuclear factor of activated t cells (nfat) pathway and kinase signaling pathways such as erk/mapk and pi3k/akt/mtor pathways, the inhibitors of which have exhibited anti-cov activities as well. [243] [244] [245] since the discovery of new interventions may take months or even years, a more efficient approach is to repurpose existing antiviral agents approved for treating related viral infections. the followings are approved drugs or preclinical compounds that have potential antiviral abilities against sars, mers, and covid-19. virus-targeted therapeutic strategies all of the major proteases of the virus are attractive druggable targets since they are essential for viral transcription and replication ( table 2) . as a key part of replication-transcription complex, rdrp participates in the production of genomic rna and subgenomic rna. nucleoside analogues targeting rdrp is capable of inhibiting viral rna synthesis in a great variety of rna viruses including covs. [246] [247] [248] [249] [250] favipiravir (t-705), a guanine analogue approved in japan for influenza treatment, has been proven to effectively interfere the rna synthesis of rna viruses such as influenza virus, ebola virus, and other hemorrhagic fever viruses at rdrp level. [251] [252] [253] [254] [255] [256] several studies concluded that favipiravir could inhibit avian influenza a (h5n1) virus and ebola virus infection in mice. 256, 257 also, favipiravir has been proved with a notable effect increasing the survival rate of ebolainfected patients from 35.3% to 56.4% in sierra leone. 258 a recent study ended with the statement that favipiravir owns the ability against sars-cov-2 (ec50 = 61.88um, cc50 > 400um, si > 6.46). 259 covid-19 patients were enrolled in randomized trials for the evaluation of the efficacy of favipiravir plus inf-α or baloxavir marboxil (chictr2000029544 and chictr2000029600). another guanosine derivative with broad-spectrum antiviral activity, ribavirin, has been authorized for hcv and respiratory syncytial virus (rsv) treatment. 260 accurate mechanism of ribavirin against virus infection is not clear, but inhibition of mrna capping and viral rna synthesis could be pivotal to its antiviral activity. 261 although high dose of ribavirin has been applied to sars treatment, the anti-mers-cov effects were moderate at such dose in rhesus macaques infected by mers-cov and no obvious survival benefit has been observed in mers patients. 225, [262] [263] [264] [265] [266] [267] recently, an open-label, randomized phase ii clinical trial (nct04276688) has revealed that triple combination of ribavirin, interferon, and lopinavirritonavir in covid-19 treatment was safe and superior to lopinavir-ritonavir therapy alone in remission of symptoms, shortening virus shedding and promoting discharge of mild to moderate covid-19 patients. 268 remdesivir (gs-5734) is a small-molecule monophosphoramidate prodrug of an adenosine analog with the ability to interfere with the rna polymerase and the proofreading exoribonuclease and terminate the nonobligate chain. 269 on day 1 and 100 mg once-daily maintenance for 9 days. however, the first clinical trial (nct04252664) has been suspended so far and the second trial (nct04257656) with 237 covid-19 patients enrolled finally indicated that remdesivir hardly shown any statistically significant clinical benefits. 274 conversely, a research found that 36 of 53 (68%) hospitalized patients suffered from severe covid-19 and treated with compassionate-use remdesivir could achieve clinical improvement. 275 in addition, a phase iii, randomized, double-blind, placebo-controlled trial (nct04280705) conducted by beigel et al uncovered the fact that remdesivir prevailed over placebo in shortening the time to recovery in adults patients. 276 though remdesivir has been approved by the food and drug administration (fda) to treat severe covid-19 patients, further researches are urgently required to determine the efficacy and the indication of remdesivir therapy. a novel synthesized nucleoside analogue, bcx4430 (galidesivir), is designed to inhibit viral rna polymerase activity via terminating nonobligate rna chain. 277 bcx4430 exhibits a promising antiviral capability against a wide array of . 277 it is currently tested in phase i clinical trial (nct03800173) for marburg virus and can be a potential countermeasure against viral diseases that threaten the public health in the world. a recent study also concluded that penciclovir, another rdrp inhibitor that is approved for hsv, showed effects on reducing sars-cov-2 infection by high-dose administration (ec50 = 95.96 µm, cc50 > 400 µm, si > 4.17). 259 although resistance to nucleoside analogs has rarely been reported, it is worth noting that mutation in rdrp is probably responsible for the acquired resistance and should be monitored for the possible resistance. 278 covs plpro enzymes display proteolytic, deubiquitylating, and deisgylating activities. [279] [280] [281] plpro was first regarded as a druggable target for sars-cov, and then several compounds targeting sars-cov plpro were also found to be effective against mers-cov plpro, recently. 282, 283 though numerous plpro inhibitors have been identified, many of them only inhibit enzymatic activities and do not affect on the viral replication. 284, 285 a study from lin et al suggested that an fda-approved alcoholaversive drug, disulfiram could inhibit sars-cov and mers-cov plpro via different mechanisms. and the synergistic inhibition between disulfiram and known plpro inhibitors, like 6-thioguanine and mycophenolic acid, to mers-cov might offer the potential combination treatments against covs in clinical. 286 another essential protease that cleaves the viral polyproteins during viral replication is 3clpro. similar to plpro, a great many of inhibitors have been identified with the ability to target covs 3clpro. among the 3clpro inhibitors, the human immunodeficiency virus (hiv) protease inhibitors are the most comprehensively studied such as lopinavir, ritonavir, asc09f, as well as darunavir and cobicistat. lopinavir and ritonavir, applied in combination to treat hiv infection, have shown improvement in the outcome of sars patients in nonrandomized trials. 287, 288 though lopinavir alone hardly displayed antiviral activity against mers-cov in vitro, the combination of lopinavir and ritonavir ameliorated the outcome in mers-cov-infected nonhuman primates. 289, 290 therefore, the efficacy of the lopinavir-ritonavir combination in mers patients should be reappraised (nct02845843). however, no benefit was observed in lopinavir-ritonavir treatment compared to standard care in a randomized, controlled, open-label clinical trial (chictr2000029308) involving severe covid-19 patients. 291 further trials are still needed to confirm the therapeutic efficacy. in addition, several other clinical trials have been developed to confirm the efficacy of 3clpro inhibitors on covid-19 (nct04252274, nct04251871, nct04255017, chictr2000029539, nct04251871, nct04255017, and nct04261270), as well as darunavir and cobicistat (nct04252274), asc09f combined with oseltamivir (nct04261270). helicase acts on the duplex oligonucleotides and turns them into single strands in an atp-dependent manner in the cov replication cycle. that helicases in different covs are highly homologous making them potentially strong targets for the covs therapeutic options. based on the mechanism actions, the helicase inhibitors can be approximately classified into two groups. one is able to inhibit both the atpase and helicase activities represented by bananins derivatives, 5-hydroxychromone derivatives, and triazole derivatives (compound 16). 292, 293 the other group including ssya 10-001 and adks has the ability to inhibit the helicase activity with no or little effects on the atpase activity. 294 however, the toxicity of helicase inhibitors needs to be examined before being applied to human patients. the transmembrane glycoprotein, s protein, is also a promising target for antiviral agents' development ( table 2 ). one class of antiviral drugs targeting s protein mostly blocks the spike-mediated membrane fusion. a potent mers-cov inhibitor, nafamostat, has demonstrated to be inhibitive against the sars-cov-2 infection (ec50 = 22.50 µm, cc50 > 100 µm, si > 4.44). 259 griffithsin is a red-alga-derived lectin, which specially binds to oligosaccharides located on the surface of viral glycoproteins, for example, s glycoprotein of sars-cov and hiv glycoprotein 120. 295 a wide range of human covs infection could be inhibited by griffithsin, comprising hcov-229e, hcov-nl63, hcov-oc43, and sars-cov. 295, 296 but the value of griffithsin in the treatment or prevention of covid-19 is needed to be evaluated urgently. s2 subunit of s protein contains two heptad repeat (hr1 and hr2). antiviral peptides analogous derived from these regions exhibited inhibition to the spike protein-mediated cell-cell fusion and viral entry in viruses such as sars-cov, mers-cov, as well as hcov-229e. 231, 297, 298 hr2p, a peptide spanning hr2 sequences of mers-cov s protein, was capable of interacting with hr1 region to form a 6-hb complex, resulting in potent inhibition of viral fusion and replication. its analog hr2p-m2 exhibited an obvious improvement in stability, solubility, and antiviral activity after being modified with hydrophilic residues. 228 additionally, hr2p-m2 intranasal administration effectively prevented experimental mice transduced by adenoviral vectors conveying human dpp4 from mers-cov infection with a >1000-fold decrease in viral titers in the lung, and this protection was intensified via the combination of hr2p-m2 and ifn-β. 299 another newly designed fusion inhibitor from mers-cov called mers-five-helix bundle (mers-5hb), which is derived from the 6-hb, also displayed a potent suppression on s protein-mediated syncytial formation. compared with mers-6hb, mers-5hb lacks one hr2, which endows its capability to interact with viral hr2 to interrupt the membrane fusion. 300 besides, mers-5hb could effectively inhibit the entry of pseudotyped mers-cov with 50% inhibitory concentration (ic50) about 1 µm. 300 altogether, the resistance of these drugs can be overcome by combining antiviral peptides aiming at various domains of s2 subunit, which may also attain synergistic effects in vitro. as for sirnas, which displayed antiviral activities in vitro as well as in sars-cov-infected rhesus macaques, are still under preclinical development and demand further studies to seek out the reliable drug delivery methods in a human before the clinical application. [301] [302] [303] m, n, and e proteins and some accessory proteins are not only vital to the virion assembly but also involved in viral pathogenesis in which they function in the interruption of host innate immune response to assist viral infection. for instance, m protein as well as accessory proteins 4a, 4b, and 5 of mers-cov act as ifn antagonist, and n protein of sars-cov serves as an inhibitor of viral rna. researches carried out by he et al and akerstrom et al emphasized that sirnas silencing m, n, e, orf3a, and orf7a/7b play an important role in the inhibition of viral replication of the sars-cov. 304, 305 but the delivery methods still limit their application in human being. nevertheless, various agents related to these proteins are discovered via functional analysis. one example is resveratrol, a natural stilbene derivative demonstrated to reduce inflammation and exert antiviral activity against multiple viruses. [306] [307] [308] [309] [310] [311] in addition, it exhibited significant inhibition of mers-cov infection and prolonged cellular survival after virus challenge in vitro via deregulating the expression of n protein and the apoptosis induced by mers-cov. 312 alternatively, hexamethylene amiloride, a viroporin inhibitor, is capable to suppress the ion channel activity of e protein of covs such as hcov-229e and sars-cov. 313 identified as dna metabolism inhibitor, gemcitabine hydrochloride is a deoxycytidine analog inhibiting both sars-cov and mers-cov with micromolar ec50 and low toxicity, which suggests its potential antiviral capacity for other human covs. 314 lj001 and jl103, two novel lipophilic thiazolidine derivatives, could induce several changes in membrane properties including the decrease in membrane fluidity, contributing to inhibition of membrane fusion, which made them become broad-spectrum enveloped virus entry inhibitors and potential anti-cov agents. 315 host-cell-targeted therapies the host innate immune response is vital to the interruption of viral replication. recombinant interferons have been applicated in treating various viruses as well as many covs (table 3) . though the host interferon response can be inhibited by the covs, they are still proved effective against covs infection such as sars-cov and mers-cov in vitro and several animal models. 242, 264, 289, 299, 316 recombinant interferons are usually combined with other antiviral agents including ribavirin or lopinavir/ritonavir to treat sars-cov and mers-cov infections, 290, 317 and the anti-covs efficacy of interferons is enhanced when added with ribavirin. 318 a combination of interferon-α2b with ribavirin reduced virus replication and improves clinical outcomes in a rhesus macaque model of mers. 264 however, the effectiveness of combination treatments comprising interferons and ribavirin is still controversial in clinical researches. a study of five patients received interferon-α2b and ribavirin showed no survival, but the finding might be not reliable owing to the delayed administration. 266 contrarily, in another study (n = 44), mortality rates of individuals receiving interferon-α2b and ribavirin exhibited a significant reduction in day 14, compared with the individuals received standard supportive care, but no significant difference was observed in day 28. 265 moreover, no significant difference in mortality rates between interferon-α2b and ribavirin treatment group and interferon-β1a and ribavirin ta b l e 3 host-targeted agents for hcovs treating group was observed in a retrospective study. 267 on the other hand, interferon-β1b displayed stronger inhibition to the mers-cov replication in vitro compared with other interferons, and combined use of interferon-β1b with other antiviral compounds should be evaluated in further research. 289, 290 nitazoxanide, originally identified as an antiprotozoal agent, was later considered as a broad-spectrum antiviral agent able to inhibit the replication of numerous dna and rna viruses such as rsv, parainfluenza, rotavirus, hbv, hcv, hiv, yellow fever, as well as covs in vitro. 319 several clinical trials have confirmed its potential value in treating influenza, chronic hbv, and hcv. [319] [320] [321] moreover, recent research indicated that nitazoxanide was capable of inhibiting sars-cov-2 infection at a low micromolar concentration (ec50 = 2.12 µm; cc50 > 35.53 µm; si > 16. 76) , and the in vivo evaluation of this efficacy is demanded. 259 another type i interferon inducer, polyinosinic:polycytidylic acid (poly(i:c)), is an analog of dsdna with a powerful ability to reduce mers-cov load in animal models. 192 and phase ii clinical trials demonstrated that it was well tolerated by malignant gliomas patients when injected intramuscularly. 322, 323 overall, the use of interferons or interferon inducers may be a valuable strategy against covs infection and should be furtherly accessed in animal models. several host factors are considered essential to covs entry, such as the host receptors that bind to covs s protein, and host proteases that facilitated covs entry through the cell surface or endosomal pathway. thus, these factors become attractive targets for anti-cov agents' development (table 3) . antibodies, peptides, and some functional inhibitors targeting the host receptors can effectively interrupt the binding between s protein and the host cells. one example is that the n-(2-aminoethyl)-1-aziridineethanamine (naae), a small molecular inhibitor, is able to target ace2 leading to the block of s protein-mediated membrane fusion, so does the synthetic peptides analogous, p4 and p5. 324, 325 similar agents were also found in mers treatment, one of which, ys110, was confirmed to be well tolerated in patients with advance solid tumors. 326 owing to their specificity to appointed receptors, they were regarded as narrow-spectrum drugs. however, the efficacy of these receptor-targeted agents have never been evaluated in any covs-infected patients and the safeties of these agents also remain unclear. host protease such as cathepsins and tmprss2 play a key role in the cleavage of s protein and the suppression of these proteases with potent inhibitors can obstruct the virus entry through either endosomal pathway or cell surface pathway. k11777 is a cathepsin inhibitor with broad spectrum against enveloped rna virus including sars-cov and mers-cov. 237, 327, 328 however, the camostat mesylate, applied in chronic pancreatitis treating, is a tmprss2 inhibitor that interrupts the tmprss2mediated cell surface entry. 329, 330 the combination of cathepsin inhibitors and tmprss2 inhibitors is crucial to the complete suppression of mers-cov in vitro. inhibition of another host protease, furin, which is vital to furinmediated s cleavage for covs, also can block membrane fusion and the viral entry like mers-cov. 143 notably, inhibition of host proteases may result in some side effects and need further evaluation. some approved antipsychotic agents (chlorpromazine, triflupromazine, and fluphenazine) and cardiotonic steroids (ouabain and bufalin) can inhibit clathrinmediated endocytosis via affecting the assembly of clathrin-coated pits at the plasma membrane and binding sodium/potassium-transporting atpase subunit α1 (atp1a1), respectively. 314, 331, 332 thus, they are considered as clathrin-mediated endocytosis inhibitors. alternatively, endosomal acidification also has a profound impact on endocytosis. increase of endosomal ph shows an inhibitive effect on virus infection, which has been confirmed by chloroquine, a widely used autoimmune disease and antimalarial agents. 333 chloroquine proves to be active against a wide range of rna viruses including hcov-229e, hcov-oc43, sars-cov, and mers-cov. [334] [335] [336] [337] [338] recently, chloroquine is identified to function at both entry and postentry phase of the sars-cov-2 infection with the ec90 value of 6.90 µm in vero e6 cells. 259 additionally, as an immunoregulator, its antiviral activity may be synergistically intensified in vivo. 259 therefore, chloroquine is suggested as a potential option against the emerging sars-cov-2. significantly, higher dose of chloroquine should not be recommended for severe covid-19 patients owing to the its drug safety, particularly while simultaneously accepting azithromycin and oseltamivir treatment, which was presented by a randomized clinical trial (nct04323527). 339 hydroxychloroquine, a chloroquine analog with lower toxicity, was listed as a potential anti-sars-cov-2 agent and recommended to be applied in covid-19 treatment by chinese national guideline and fda. 340 however, evidence of the benefits and harms of hydroxychloroquine therapy is still insufficient and conflicting. some small studies show that hydroxychloroquine was capable of decreasing sars-cov-2 shedding that could be enhanced by the combination of azithromycin and achieving a shorter time to clinical recovery. 341, 342 but almost no clinical benefit was observed in other studies. 340, 343 therefore, therapeutic efficacy of hydroxychloroquine is still needed to be reconfirmed. moreover, there are several factors required to be reconsidered before efficacy evaluation, such as the severity of illness, definition of the endpoints, and effects of the comorbidities. except for the innate immune response, host receptors, and other factors affecting the endocytosis, some signaling pathways have also been suggested as useful approaches for discovering anti-cov reagents (table 3) . cyclophilins are peptidyl-prolyl isomerases with physiological functions showing as modulating the calcineurin/nfat pathway via reacting with covs nsp1, which is important for the immune cell activation. 244 in addition, they are also required for the replication of numerous viruses including hiv, hcv, as well as covs. 344 consequently, inhibition of cyclophilins by cyclosporines, such as cyclosporin a (csa) and its derivatives, has shown to block the replication of a wide range of covs. 244, 245, 345, 346 however, the obvious immune suppressive properties of csa limit its application in antiviral therapy. but alisporivir, a nonimmunosuppressive cyclosporin a-analog, also displayed the inhibition to the covs replication at a lowmicromolar concentration. 347 additionally, the combined use of cyclosporine and interferon or ribavirin in vitro was beneficial to inhibit sars-cov or mers-cov infection, which needed to be furtherly evaluated in animal models. 347, 348 furthermore, some antiviral agents inhibiting the cellular signaling pathways, in particular, the erk pathway and pi3k/akt pathway, also interrupt the replication of covs. 243, 314, 349 however, the efficacy and safety against covs infection of these agents still need to be reconsidered. corticosteroids, which were used in sars and mers treatment, have been linked to several complications such as psychosis, diabetes, and avascular necrosis. 350, 351 they also were pointed out to be associated with viral replication prolongation in mers patients. 351 however, an update on the efficacy of dexamethasone based on a press release publicized recently indicated that severe covid-19 patients given 6 mg dexamethasone once daily shown a lower mortality (about 8-26%) compared to patients with standard care. 352, 353 besides, another agent, methylprednisolone, also exhibited potential capacity in reducing the mortality rate and achieving better clinical outcomes in severe covid-19 patients. 354, 355 thus, it is wise to apply corticosteroids to the right patients at the right time. but physicians also need to monitor the corticosteroid-related complications. clinical trials of corticosteroid treatments are shown in table 3 . potential immunotherapeutic options s protein of sars-cov proves to be highly immunogenic during infection and responsible for eliciting a humoral immune response in the host. 356 antivirus antibodies could be detected in the plasma of convalescent patients' infected sars-cov and mers-cov. 357, 358 convalescent plasma therapy has been applicated in treating patients infected by numerous viruses involving ebola virus, junin virus, machupo virus, and lassa fever. [359] [360] [361] [362] as for sars-cov, higher day-22 discharge rate and lower mortality rate have been observed among sars patients who received convalescent plasma transfusion before day 14 of the illness. 363, 364 this is consistent with another small cohorts research concluding infected patients with severe conditions who failed to respond to current therapies but finally survived after transfused with convalescent plasma, with no obvious side effects. 358 similar results were found in mers patients. 365 additionally, plasma adoptive therapy with anti-mers-cov antibodies could block the virus adhesion and accelerate the viral elimination from mers-cov-infected animal models. 192 but the efficacy and safety of convalescent plasma therapy in covid-19 patients still need to be reevaluated. although convalescent plasma therapy proves to be a potentially effective therapeutic option for emerging covs, several factors still limit its extensive use in clinical, one of which is enough titers of serum neutralizing antibodies. the development of mabs targeting the s protein of covs is regarded as a remedial strategy (table 4 ). potent mabs against s protein of human covs can be attained via transgenic mice immunization, convalescent b cells immortalization, and cloning of small chain variable regions from naïve and convalescent patients. 366 the majority of mabs interact with the rbd of s protein leading to the interruption of rbd-receptor binding and block the viral attachment. a few mabs react with other regions of s protein besides the rbd. 366 although binding to different epitopes, these mabs exhibit capacity to reduce the viral titers. two rbd-specific neutralizing mabs, mers-4 and mers-27, which were derived from single-chain variable regions, revealed suppressive effects against both mers-cov and pseudotyped mers-cov infection at nanomolar concentrations and were recommended as promising candidates for therapeutic interventions to mers. 232 based on similar mechanisms, other human mabs for mers-cov were also capable of competing with dpp4 for rbd binding and neutralizing the virus. 233, 234, [367] [368] [369] [370] when administrated to individuals at risk, some of the mabs were capable of preventing viral replication and contributing to block the transmission of mers-cov among human. 368 thus, such antibodies could be served as prophylactic strategies in clinical and valuable tools to guild the development of effective anti-covs vaccines. 234,368 from sars-cov to sars-cov-2, the emergence of severe human covs have taught us many lessons about the importance of rapid diagnostics and effective vaccines to control the outbreak caused by these viruses. due to the persistence of zoonotic sources in endemic areas, lethal covs remain existing in human society and may lead to the epidemic at any time. thus, a priority is to develop vaccines targeting conserved alleles and providing broad-spectrum protection against varied viral strains. since the emergence of sars-cov and mers-cov, several strategies were applicated in vaccine design, including inactivated virus vaccines, live-attenuated virus vaccines, viral vector vaccines, nanoparticles, recombinant protein subunits vaccines, and dna vaccines (table 4) . 371, 372 and clinical trials have also been developed to test the efficacy of the novel vaccines (table 5) . effective vaccines are pivotal in blocking the virus spread from animals' reservoirs to human hosts. inactivated virus vaccines, preserving the viral structure and antigenicity but eliminating the infectious ability, could elicit neutralizing antibodies in animal models of sars-cov and show protection against viral replication when administrated with or without adjuvants. 372 which may be related to the disseminated infection observed in immunocompromised patients. in addition, live-attenuated virus vaccines can induce an innate and adaptive immune response and the protective value can last for a long time. 371 besides, other strategies for vaccines development are also evaluated in animal models. based on the experience of sars-cov and mers-cov, the development of novel sars-cov-2 vaccine is currently underway and requires more research. however, several concerns should be addressed about the vaccination. the first is the disease deterioration caused by vaccination. although this situation only appears in a small subset of sars vaccine studies, it is still a significant problem that needs to be properly solved. 372 second, the variability of s protein can mediate covs escape from neutralization, suggesting that recombinant protein subunits vaccines based on s protein may demand multivalent approaches. 376 last but not least, how to define ta b l e 5 important clinical trials with vaccines for sars-cov, mers-cov, and sars-cov-2 the emergence and prevalence of highly pathogenic cov severely threaten public health. a task of top priority is to make clear the viral structural and epidemiological characteristics and block the viral dissemination as well as the progression of the disease, at the first case. to date, further understanding of the life cycle and the pathogenesis of emerging human covs makes current therapeutic strategies of antiviral infection more rational. repurposing existing antiviral drugs is an effective short-term strategy to deal with emerging cov such as the ongoing sars-cov-2. various agents with different targets have been evaluated in vitro and in vivo. but not all antiviral agents are capable of achieving better efficacy than in vitro, and in vivo studies are needed to select optimal agents. suitable animal models are particularly significant. however, there are only a few effective animal models available for the studies of covs treatment, which may postpone the clinical evaluation of drugs. besides, due to the diversity of viruses and the capacity of rapidly mutating, some antiviral reagents available for existing covs may become invalid. accordingly, sequencing more natural specimens and combination therapy with two or more synergistic agents have become promising solutions. furthermore, the efficacy of current antiviral therapies needs to be estimated in larger scale, well-organized randomized clinical trial the efficacy and safety of the most practicable strategies for the ongoing covid-19 are urgently needed to be further assessed in clinical trials, involving remdesivir (gs-5734), lopinavir/ritonavir, lopinavir/ritonavir combined with ifn-β, convalescent plasma, and mabs, which prove to be potentially effective options against sars-cov-2. additionally, to control the future cov pandemics, the development of novel broad-spectrum antiviral agents covering numerous covs will become the ultimate goal. the authors declare no conflict of interest. wei https://orcid.org/0000-0002-6513-6422 coronavirus diversity, phylogeny and interspecies jumping coronavirus pathogenesis is the discovery of the novel human betacoronavirus 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structure-guided design of potent and permeable inhibitors of mers coronavirus 3cl protease that utilize a piperidine moiety as a novel design element synthesis and evaluation of phenylisoserine derivatives for the sars-cov 3cl protease inhibitor identification, synthesis and evaluation of sars-cov and mers-cov 3c-like protease inhibitors engineering a novel antibodypeptide bispecific fusion protein against mers-cov. antibodies (basel) identification of a peptide derived from the heptad repeat 2 region of the porcine epidemic diarrhea virus (pedv) spike glycoprotein that is capable of suppressing pedv entry and inducing neutralizing antibodies a novel peptide with potent and broad-spectrum antiviral activities against multiple respiratory viruses antiviral activity of k22 against members of the order nidovirales purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice key: cord-288101-pij16jaa authors: li, jun-yu; yong, yan-hong; gong, dong-liang; shi, lin; wang, xiao-min; gooneratne, ravi; yadnyavalkya, patil; ju, xiang-hong title: proteomic analysis of the response of porcine adrenal gland to heat stress date: 2019-02-28 journal: research in veterinary science doi: 10.1016/j.rvsc.2018.11.004 sha: doc_id: 288101 cord_uid: pij16jaa abstract heat stress (hs) and its associated pathologies are major challenges facing the pig industry in southern china, and are responsible for large economic losses. however, the molecular mechanisms governing the abnormal secretion of hs-responsive hormones, such as glucocorticoids, are not fully understood. the goal of this study was to investigate differentially expressed proteins (deps) in the adrenal glands of pigs, and to elucidate changes in the immune neuroendocrine system in pigs following hs. through a functional proteomics approach, we identified 1202 peptides, corresponding to 415 proteins. of these, we found 226 deps between heat-stressed and control porcine adrenal gland tissue; 99 of these were up-regulated and 127 were down-regulated in response to hs. these deps included proteins involved in substrate transport, cytoskeletal changes, and stress responses. ingenuity pathway analysis was used to identify the subcellular characterization, functional pathway involvement, regulatory networks, and upstream regulators of the identified proteins. functional network and pathway analyses may provide insights into the complexity and dynamics of hs-host interactions, and may accelerate our understanding of the mechanisms of hs. rising global temperatures have been accompanied by increased interest in researching the detrimental effects of heat stress (hs) on the swine industry. pigs enter a state of hs when the ambient temperature exceeds their thermal neutral zone (16-22°c for adult pigs) (coffey et al., 1995) . due to their high production of metabolic heat, accelerated fat deposition, and lack of sweat glands, pigs there are more sensitive to hs than many other mammals (d'allaire et al., 1996) . heat stress in pigs not only decreases food intake and body weight gain, but also has immunosuppressive effects, all of which may result in large economic losses for the swine industry (cruzen et al., 2015; pearce et al., 2013) . for instance, hs is estimated to cost the us swine industry losses of over $300 million each year (st-pierre et al., 2003) . understanding the stress-associated mechanisms involved in immune system function and the increased susceptibility of livestock to heat-related illness is more important now than ever, as the majority of emerging animal diseases are zoonotic and can potentially threaten public health. when exposed to a high-temperature environment, the central nervous system of mammals, including livestock, engages in physiological responses that result in the activation of the hypothalamic-pituitary-adrenal (hpa) axis and the sympatho-adrenal axis. the predominant hormone regulating the synthesis and secretion of adrenal glucocorticoids is adrencocorticotropic hormone (acth) (minton, 1994) . in pigs, cattle, and sheep, both corticotropin-releasing hormone and vasopressin regulate the secretion of acth, suggesting that these two proteins interact to enhance acth secretion. acth acts on the adrenal gland with the circulation, it to induces the expression and secretion of glucocorticoids, which suppress the production of cytokines and other pro-inflammatory mediators, including tnf-α, interferon-γ, il-1β, il-11, il-12, il-8, and prostaglandins (wilckens and rijk, 1997) . glucocorticoids also facilitate the release of anti-inflammatory mediators, such as transforming growth factor-α, il-10, and il-4, and have apoptotic effects and strong anti-proliferative properties in immune cells (visser and nagelkerken, 2002) . ultimately, cytokines activate the release of glucocorticoids, which in turn suppress cytokine synthesis fin a negative feedback loop (barrat et al., 2002; haddad et al., 2002) . thus, it is of interest to understand how higher core temperatures alter adrenal function. isobaric tag for relative and absolute quantification (itraq) is a powerful quantitative proteomics technique. in recent years, several proteomic studies have explored protein expression in a number of porcine cells and tissues, including pulmonary alveolar macrophages (lu et al., 2013) , mesenchymal stem cells (huang et al., 2015) , liver (liu et al., 2016) , heart (cabrera et al., 2012) , and intestine (colladoromero et al., 2015) . nevertheless, no large-scale proteomic analysis to date has examined the molecular complexes or pathways involved in the pathogenesis of the hpa axis. the purpose of the present study was to investigate protein expression in the adrenal gland of pigs in response to hs, and to elucidate potential changes in that occur in the endocrine system under hs. pigs were maintained and studied in accordance with the national institutes of health (nih) guidelines for the care and use of laboratory animals, and all protocols were approved by the guangdong ocean university animal care and use committee, china. six castrated bama miniature pigs (sus scrofadomestica) 3 months, weighing 30-40 kg were obtained from the bama miniature pigs breeding farm in the guangxi zhuang autonomous region of china. the six pigs were randomly divided into a heat stress group (3 barrows, ha) and a control group (3 barrows, ca). control pigs were housed with an ambient temperature of 28 ± 3°c, and the relative humidity was kept at approximately 90%. pigs assigned to the heat stress treatment were kept at 35 ± 1°c(maintained using an artificial climate chamber) in a manmade climate room, with a relative humidity of approximately 90%. all pigs were given access to water ad libitum. diet (see diet composition table) was formulated according to the recommended nutrient allowances for this breed of pig and the feeding was done twice a day, in the morning as well as in the evening. pigs were euthanized by a head-only electric stun tong apparatus on the 7th day under heat stress, followed by manual exsanguination. immediately after slaughter, adrenal tissue was removed and weighed. subsequently, tissues were washed with pbs to remove any blood and contaminants on the tissue surface. adrenal tissue was placed into sterile tubes and snap frozen in liquid nitrogen three pigs were used in the control group and three pigs were used in the heat-stressed group. the adrenal tissues of the 3 pigs in each of the groups were pooled together to form one pooled sample and utilized as one sample for further analyses. once in the laboratory, frozen specimens were stored at −80°c until biochemical and molecular analyses were performed. frozen samples of adrenal tissue from all pigs in the two groups were crushed in a mortar containing liquid nitrogen. the powder (approximately 100 mg per sample) was transferred to a sterile tube containing 1 ml lysis buffer (lb; containing 7 m urea, 2 m thiourea, 4% chaps, 40 mm tris-hcl, ph 8.5, 10 mm dithiothreitol, dtt). tissue homogenate was further disrupted using an ultrasonic cell disruptor (vcx130, usa) at 20% power output for 10 min, cycling between 2 s on and 4 s off. afterwards, the lysate was centrifuged at 25,000 ×g for 30 min at 4°c, and the supernatant was collected for protein quantification. protein concentration was measured using the pierce bca protein assay kit (thermo scientific, usa). protein digestion was performed as per the fasp procedure described by wisniewski, zougman et al. (wiśniewski et al., 2009) ; and the resulting peptide mixture was labeled using the itraq reagent-4plex multiplex kit (ab sciex, framingham, usa), according to the manufacturer's instructions. after 2 h of incubation at room temperature, labeled samples were mixed at equal ratios. subsequently, labeled peptides were combined and fractionated by strong cation exchange (scx) chromatography (han et al., 2015) and desalted on c18 cartridges (66872-u; sigma, st. louis, mo, usa). the dried peptide mixture was reconstituted and acidified with 2 ml buffer a (10 mm kh 2 po 4 in 25% of acn, ph 3.0) and loaded onto a column (4.6 × 250 mm). peptides were eluted at a flow rate of 1 ml/ min with a gradient of 0%-5% buffer b (2 m kcl, 10 mm kh 2 po 4 in 25% of acn, ph 2.7) for 5 min, 5-10% buffer b for 10-15 min, 10%-30% buffer b for 25-35 min, and 30%-50% buffer b for 35-50 min. the elution was monitored by absorbance at 214 nm, and fractions were collected every 1 min. the tryptic peptides were extracted, and the peptide mixtures were concentrated by speed vac centrifuge to dryness, and were again dissolved with 2% acetonitrile (acn) in 0.1% formic acid before lc-ms/ms analysis. the fractions from 2.3 were subjected to liquid chromatography-tandem mass spectrometry (lc-ms/ms) analysis. initially, samples were loaded onto pre-columns (180 μm × 20 mm; 5 μm-c18; waters, usa). peptide mixtures were separated on analytical columns (100 μm × 100 mm; 1.7 μm-c18; waters, usa) at a flow rate of 300 nl/ min over 60 min. thermo easy-nlc is a binary buffer system used for high performance liquid chromatography (hplc), consisting of 0.1% formic acid (buffer a) and 80% acetonitrile (acn) in 0.1% formic acid (buffer b). the related liquid phase gradient was as follows: 0-40 min with 5% to 35% buffer b; 40-45 min with 35%-80% buffer b; and 45-50 min with 80% buffer b. peptides eluted by hplc were directly injected into a q-exactive mass spectrometer (thermo fisher scientific). data were acquired in the positive ion mode with a selected mass range of 300-1800 mass/charge (m/z). q-exactive survey scans were obtained at 70,000 (m/z 200) and 17,500 (m/z 200), with the resolution for higher-energy collisional dissociation (hcd) spectra and maximum ion injection times fixed at 20 ms and 60 ms, respectively. dynamic exclusion (40.0 s duration) was used. ms/ms data were collected using the top 10 most abundant precursor ions. the normalized collision energy was 30 ev, and the underfill ratio defined as 0.1%. the instrument was operated with peptide recognition mode enabled. protein identification was performed using the mascot 2.3.02 search engine (matrix science, london, uk). according to the relative abundance of different itraq tags, the peptides derived from different groups were quantified by the scaffold software, and represent the ratio of one group to another. the relative quantification of the protein is calculated by using the relative quantification of the peptide, which is expressed as the average ratio. to determine the deps in the adrenal gland between the hs and the control groups, the average ratio of identified proteins was calculated by proteinpilot based on the weighted average log ratios of the peptides. the deps were further analyzed for significant down-or upregulation, which was not determined by the size of the ratio but was calculated by software perseus. a cutoff level of significance of 5% (or p < 0.05) was chosen as a criterion. gi numbers of all significantly regulated proteins and some unaltered proteins were imported into the ingenuity pathway analysis software (ipa, www.ingenuity.com) for bioinformatics analysis based on published reports and databases such as gene ontology, uniport and trembl. the canonical pathways and proteins interaction network of the deps were analyzed using the ipa. statistical analysis was performed using spss statistics 22.0. differences analysis in physiology indices between the ha group and the ca group were performed using a t-test, and p < 0.05 was taken to indicate statistical signifcance. we measured the body temperature, heart rate and respiratory rate of pigs at days 1, 3 and 6. it was found that body temperature and heart rate were increased significantly in heat-stressed pigs at days 1 and 6. (p < 0.05), however, the difference between body temperature and heart rate was not significant at 3 days. compared to controls, the respiratory rate, too increased substantially in pigs under heat stress at days 1, 3 and 6 (p < 0.01) (see table 1 ). the sample size used for this was 3 pigs each in control and hs groups respectively. we used the itraq method to identify proteins differentially expressed in the adrenal gland between the heat stress and control groups. we identified 226 differentially expressed proteins (deps), of which 99 were up-regulated and 127 were down-regulated in the ha group vs. the ca group. table 2 provides information about 18 key deps, 9 upregulated and 9 downregulated (see table 2 ). all the deps have been provided in the supplementary table 1. all protein and peptide identifications were obtained by database searching and stringent data filtering. the lc-ms/ms analysis produced 21,879 spectra, corresponding to 1202 unique peptides; 415 the effect of heat stress on the body temperature, heart rate and respiratory rate at days 1, 3, and 6. we found that heat stress increased the body temperature (bt), heart rate (hr) and respiratory rate (rr) of pigs significantly. the sample size used for this was 3 pigs each in control and hs groups respectively. the asterisk '*' and double-asterisk '**' denote a significant difference between stressed pigs and control pigs on the same day (p < 0.05 and 0.01, respectively). the units of the above indices are centigrade (°c), beats per minute and breaths per minute, respectively. proteins were identified at a false discovery rate (fdr) of ≤0.01 (fig. 1a) . the molecular weights of the identified deps ranged from 0 to 20 kd (n = 39), 20-40kd (n = 57), 40-60kd (n = 48), 60-80kd (n = 38), 80-100kd (n = 14), or > 100 kd (n = 30) (fig. 1b) . in addition, the identified deps had high peptide coverage; 85% of deps had > 10% sequence coverage, and 52% of deps had > 20% sequence coverage (fig. 1c) . 79.65% of the identified deps were represented by three or more peptides (fig. 1d) . to gain functional insights into the cellular proteome, gene ontology annotation was used to determine the subcellular localization of the 226 identified deps. the 226 deps identified from adrenal gland tissue altered by heat stress localized to various subcellular regions: high density lipoprotein particles (1.9%), extracellular area (15.0%), endoplasmic reticulum (7.9%), protein-lipid complexes (2.3%), cytoplasmic lipoprotein particles (2.3%), triglyceride-rich lipoprotein particles (1.9%), serosa (2.8%), platelet alpha particles (2.8%), endoplasmic reticulum inherent (1.9%), extracellular matrix (4.2%), chromatin (2.3%), pigment granules (5.6%), extracellular space (2.3%), serosa integrity (2.3%), neuronal processes (10.7%), secretory granules (5.1%), cytoplasmic vesicles (15.4%), and cytoskeleton (13.3%). (fig. 2) . gene identifications of the identified deps (supplementary table 1 ) were converted to human geninfo identifier (gi) numbers, since the pig genome database has poor annotations compared to the human genome, and because many proteins were unassigned or uncharacterized. to better understand these 226 deps, we used ingenuity pathways analysis (ipa) tool to examine canonical pathways; the top 20 enriched pathways are shown (fig. 3) , including pathways related to inflammation and immunity, such as 'acute phase response signaling' and 'il-12 signaling and production in macrophages'. the deps identified in adrenal gland tissue during heat stress by itraq were clustered according to different functions. four functional groups were found: diseases and disorders, molecular and cellular functions, physiological system development, and toxicity functions were significantly enriched (p ≤ 0.05; fig. 4 ). the 226 deps from adrenal gland under heat stress, which correspond to 24 diseases and disorders (fig. 4a) , included proteins that are related to neurological disease, psychological disease, metabolic disease, skeletal and muscular disorders, hereditary disorders, hematological disease, immunological disease, inflammatory disease, inflammatory response, respiratory disease, dermatological disease and conditions, connective tissue disorders, infectious disease, cardiovascular disease, cancer, and endocrine system disorders. these 226 deps were assigned to 25 molecular and cellular function groups (fig. 4b) , including cell death and survival, molecular transport, cellular growth and proliferation, cellular assembly and organization, cellular function and maintenance, cellular movement, lipid metabolism, small molecule biochemistry, free radical scavenging, protein degradation, protein synthesis, and cell morphology. these deps were also enriched in 18 physiological system developmental functions groups (fig. 4c) , including tissue development, nervous system development and function, organ morphology, organismal development, embryonic development, hematological system development and function, immune cell trafficking, and tissue morphology. finally, these deps were enriched in 9 toxicity function groups (fig. 4d) , including renal necrosis/cell death, liver hyperplasia/ hyperproliferation, kidney failure, cardiac inflammation, and heart failure. proteins that changed significantly in the adrenal gland under heat stress were mapped to 13 functional networks (fig. 5) . the main networks of interest correspond to (1) cell assembly and tissue, cellular function and maintenance (fig. 5a); (2) cell migration, intercellular signal interactions (fig. 5b) small molecule biochemistry (fig. 5d ). proteins that are present in these pathways and identified as up-regulated deps in our analysis are depicted in shades of red; those that were identified as down-regulated deps are shown in green. proteins in the network but not identified as deps in our study are depicted in white. we also predicted the upstream regulators of adrenal deps by ipa analysis and found that cytokines, kinases, chemical agents, chemical kinase activators, mature micrornas, and growth factor were activators of these deps, as an example, chemokine(c-c motif)ligand 5, curcumin and brain derived neurotrophic factor while cytokines, mature microrna, auxins, transcription regulators, and chemicals were inhibitors of these deps, such as, il-6, ccaat enhancer-binding protein β and dexamethasone etc. these predicted upstream regulators of deps responsive to heat stress may have an important role in regulating hormone secretion and signal transduction in pigs. . the previous research of our laboratory found that: the analysis of plasma cortisol levels in bama miniature pigs revealed that the levels increased with the duration of heat stress. although there was no significant difference in the cortisol levels of control and heat-stressed pigs on the first day. however, at subsequent time points, cortisol levels were significantly higher in heat-stressed pigs compared with those in the control animals on the 7th day (ju et al., 2014) . in the present study, we used a method combining proteomics and bioinformatics to identify proteins that are differentially expressed in the adrenal gland of pigs under heat stress, and to elucidate molecular pathways and cellular functions that might mediate the heat stress response through these deps. we identified a total of 226 deps in the pig adrenal gland under heat stress conditions. ipa analysis software was used to analyze the cell localization, molecular function, signal pathway, regulatory network, and upstream regulators of these deps, which laid the foundation to elucidate mechanism of heat stress and stress-induced immunosuppression. the function of tubulin is mainly to interact with microtubule-associated proteins and related proteins that activate microtubule structures and maintain microtubule polymerization and depolymerization, modulate cell morphology, and are involved in cell division, cell movement, and transport of intracellular substances. cytoskeletal changes are associated with trans-cellular membrane trafficking. together, β-tubulin and α-tubulin (table 2 ) participate in the formation of microtubules, the integrity of which is essential for the segregation of chromosomes during cell division, the maintenance of cell shape, and the intracellular trafficking of macromolecules and organelles. changes in β-tubulin and vimentin levels have been detected in sars-cov (jiang et al., 2005) and infectious bursa disease virus (ibdv) (zheng et al., 2008a) . β-tubulin was one of the deps identified in this study, and its expression was approximately 2.2-fold up-regulated in adrenal tissue under heat stress, suggesting that differentially expressed cytoskeletal proteins could promote stress response in the adrenal gland. further large-scale studies are required to understand the roles and interrelation of β-tubulin and vimentin in porcine heat stress response. the heat shock protein (hsp) response is a highly conserved cellular response to external stress in all species. hsps take part in antigen presentation, intracellular trafficking, and apoptosis, and acting as molecular chaperones by assisting nascent polypeptides in assuming their proper conformations (khar et al., 2001) . several hsps were identified as down-regulated deps in this study, including hsp27 and hsp60. several members of the hsp family are expressed on the surfaces of cells; these can stimulate immune effector cells directly or can play crucial roles in antigen cross-priming. in this situation, hsps act as shuttle molecules for exogenous antigens and can directly stimulate t cells by prompting apc cytokine secretion (kotsiopriftis et al., 2005) . hsps appear to play a part of the innate immune response since the emergence of phagocytes in early multicellular organisms, and were commandeered for adaptive immune responses with the appearance of immune specificity (srivastava, 2002) . hsp70 is an endogenous ligand for the toll-like receptors (tlrs) that bind to microorganism-and tumor-specific antigens, then combine with major histocompatibility complexes (mhc) i and ii, which activate tumor-specific pathogens and t cells (han et al., 2009 ). mortaz found that high temperature induced mouse bone marrow mast cells to release hsp70, and the secretion of hsp70 in turn activated the toll-like receptor 4(tlr4) pathway (mortaz et al., 2006) . the expression of hsp70 is enhanced under stress conditions, and is generally considered to act through its role as a molecular chaperone, but recent reports indicate that hsp70 also modulates inflammatory responses by inhibiting activation of the inflammatory transcription factor, nuclear factor-kappa b (zheng et al., 2008b) . in addition, hsp70 may directly interfere with cell death pathways, such as those involved in apoptosis and necrosis (yenari et al., 2005) . in this study, hsp70 was up regulated(1.52 fold reduction) in porcine adrenal gland tissue under heat stress, further indicating the role of hsp70 in adrenal gland injury and highlighting its relevance to inflammatory responses. hsp60 has direct modulatory effects on inflammatory cells, and can activate monocytes and granulocytes to produce inflammatory cytokines, tumor necrosis factor-α (tnf-α), il-12 and il-6 (wells and malkovsky, 2000) . hsp60 can also specifically bind tlrs, especially tlr4, which is involved in human and rat atherosclerotic lesions (grundtman et al., 2011) . hsp27 belongs to a family of small heat shock proteins, can affect protein assembly, and may also participate in protein degradation. this conclusion follows directly from data suggesting that heat stress reduces the expression of heat shock proteins, handicapping their ability to induce a protective immune response when immunocytes are confronted with foreign entities. antigen presentation by hsps activates the innate and adaptive immune systems to initiate an acute response to stress factors, and suppression of hsps obstructs this response. these properties allow hsps to be used in immunotherapy in novel ways, which could lead to a greater understanding of how heat stress modulate the immune response, and why heat stress induces immunosuppression in pigs afflicted by post weaning multi-system wasting syndrome (pmws). histone h2a was significantly down-regulated in porcine adrenal gland under heat stress. ipa analysis shows that histone h2a plays a fig. 5 . ingenuity pathway analysis of proteins significantly altered in heat stressed pigs. red, up-regulated proteins; green, down-regulated proteins significantly; white, proteins known to be in the network but were not identified in our study. the colour depth shows the magnitude of the change in protein expression level. the shapes are suggestive of the molecular class(i.e., protein family). lines connecting the molecules indicate the relationship between molecules. dashed lines demonstrate indirect interactions, and solid lines demonstrate direct interactions. the arrow styles demonstrate specific molecular relationships and the directionality of the interaction. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) role in cancer, infectious diseases, reproductive system diseases, liver diseases, and immune diseases. however, in a bovine proteome study, histone h2a was considered a new natural immune molecule, and was down-regulated in neutrophils of immunosuppressed dairy cows. the immunological function of neutrophils releases a series of dna, histones, and antimicrobial peptides, forming a microbial kill 'trap' (lippolis and reinhardt, 2008; john and lippolis, 2005; kimura et al., 2006) . histone h2a belongs to a group of conserved eukaryotic cationic proteins that are involved in antibacterial activity, and function in dna folding. further studies are needed to determine if down-regulation of h2a expression in the adrenal gland under heat stress conditions is related to immunosuppression. annexin (anxa) is a calcium-dependent phospholipid-binding protein widely found in eukaryotes. phosphatidylserine (ps) is transferred from the inside of cell membrane to the outside after cell apoptosis. in the presence of calcium ions, anxa5 has a high affinity for ps, and can specifically bind to ps exposed on cell membrane. thus, anxa5 specifically recognizes apoptotic cells, and acts as a novel molecular probe to detect apoptosis. anxa5 is one of the most widely distributed and abundant members of the anxa family, and is involved in the anti-coagulation activity, calcium channel activity, protein kinase inhibitory activity, and many other important physiological processes. anxa5 is also closely related to inflammatory responses and cell stress (gauer et al., 2013; lokman et al., 2011) . anxa1, 2, and 11, were down-regulated deps identified in our study. these proteins participate in cell function and maintenance, intercellular communication and interaction, cell survival and death, cell migration, neurological diseases, and other processes. whether these down-regulated proteins are involved in the regulation of heat stress-induced apoptosis is still unclear (gu et al., 2014) , and must be explored in further studies. cui performed similar studies on heat stressed pig livers and found that chronic heat stress caused a reduction in the weight of the liver. they also found 45 proteins in the liver that were differentially abundant between heatstressed pigs and control pigs. proteins found were responsible for heat shock and oxidative stress response, cellular apoptosis, metabolism, signal transduction, cytoskeleton and immune system responses. their research found that heat caused an innate immune response whereas the voluntary diet restriction resulted in stress and cellular apoptosis (cui et al., 2016) . in summary, in this study we identified deps in the porcine adrenal gland under heat stress were characterized, and we constructed a comprehensive network of protein regulation in the porcine adrenal gland in response to heat stress. although many significantly differentially regulated proteins and pathways are closely related to the symptoms or pathological responses of heat stress, further functional investigations ought to be carried out to facilitate our understanding of the pathogenic mechanisms and molecular responses of pigs to heat stress. further work may identify novel therapeutic targets or lead to the development of new methods of preventing heat stress. in conclusion, we identified 226 deps, of which 99 were up-regulated and 127 were down-regulated, from pig adrenal gland tissue under heat stress. among these 226 deps, tubulin and heat shock protein (hsp) such as hsp70, hsp60, hsp27, and histone h2a might be involved in the regulation of heat stress. ingenuity pathways analysis (ipa) tool examined canonical pathways related to inflammation and immunity, such as 'acute phase response signaling' and 'il-12 signaling and production in macrophages'. our expression profiles provide new insights into the key proteins involved in heat stress in the pig. supplementary data to this article can be found online at https:// doi.org/10.1016/j.rvsc.2018.11.004. in vitro generation of interleukin 10-producing regulatory cd4+ t cells is induced by immunosuppressive drugs and inhibited by t helper type 1 (th1)-and th2-inducing cytokines altered expression of mitochondrial electron transport chain proteins and improved myocardial energetic state during late ischemic preconditioning feeding growing-finishing pigs to maximize lean growth rate quantitative proteomics and bioinformatic analysis provide new insight into the dynamic response of porcine intestine to salmonella typhimurium effects of long term heat stress in utero or during finishing on pork carcass composition chronic heat stress induces immune response, oxidative stress response, and apoptosis of finishing pig liver: a proteomic approach sow mortality associated with high ambient temperatures membrane modulates affinity for calcium ion to 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cancer progression twodimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (itraq) labeling approac function of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system in models of acute stress in domestic farm animals acetylsalicylic acid-induced release of hsp70 from mast cells results in cell activation through tlr pathway the effects of heat stress and plane of nutrition on metabolism in growing pigs interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses economic losses from heat stress by us livestock industries 1 expression of the il-10 receptor on human monocytes is moderately suppressed by glucocorticoids heat shock proteins, tumor immunogenicity and antigen presentation: an integrated view glucocorticoids and immune function: unknown dimensions and new frontiers universal sample preparation method for proteome analysis antiapoptotic and anti-inflammatory mechanisms of heat-shock protein protection proteomics analysis of host cells infected with infectious bursal disease virus anti-inflammatory effects of the 70 kda heat shock protein in experimental stroke not applicable. the authors of this study have no conflicts of interest. jl conceived the study designed the experiments, prepared the samples for mass spectrometry, analyzed the mass spectrometry data, conducted the experimental work created the figures and wrote the manuscript. yy participated in the enrichment analysis, created the pathway figure, aided in revising the manuscript. dg, ls and pt analyzed the mass spectrometry data, participated in the enrichment analysis, participated in writing and revision of the manuscript. rg obtained and analyzed flow cytometry data and aided revising the manuscript. xwang was responsible for animal care xj provided the financial support, and designed the experiments and revised the manuscript. all authors have confirmed the final version of the manuscript. key: cord-306624-1mjmttec authors: wodrich, harald; henaff, daniel; jammart, baptist; segura-morales, carolina; seelmeir, sigrid; coux, olivier; ruzsics, zsolt; wiethoff, christopher m.; kremer, eric j. title: a capsid-encoded ppxy-motif facilitates adenovirus entry date: 2010-03-19 journal: plos pathog doi: 10.1371/journal.ppat.1000808 sha: doc_id: 306624 cord_uid: 1mjmttec viruses use cellular machinery to enter and infect cells. in this study we address the cell entry mechanisms of nonenveloped adenoviruses (ads). we show that protein vi, an internal capsid protein, is rapidly exposed after cell surface attachment and internalization and remains partially associated with the capsid during intracellular transport. we found that a ppxy motif within protein vi recruits nedd4 e3 ubiquitin ligases to bind and ubiquitylate protein vi. we further show that this ppxy motif is involved in rapid, microtubule-dependent intracellular movement of protein vi. ads with a mutated ppxy motif can efficiently escape endosomes but are defective in microtubule-dependent trafficking toward the nucleus. likewise, depletion of nedd4 ligases attenuates nuclear accumulation of incoming ad particles and infection. our data provide the first evidence that virus-encoded ppxy motifs are required during virus entry, which may be of significance for several other pathogens. many viruses use the microtubule network of the host cell for transport to their site of replication (i.e. the nucleus) [1] . access to the microtubule network is achieved through recruitment of cytoplasmic dynein motor proteins followed by efficient retrograde transport towards the nucleus [2, 3] . virus-induced cellular signaling cascades help stimulate the directionality and efficacy of the transport [4] . viral interaction with dynein motor proteins occurs either directly through capsid proteins or indirectly via hijacking of adapters from existing transport pathways [5] . most dna viruses accumulate transiently at the microtubule organizing center (mtoc) prior to nuclear translocation [1, 3, 6] . how they release from the microtubules or the mtoc and transport to nuclear pores is poorly understood. mtoc release may involve a switch from dynein to kinesin mediated transport, the cellular ubiquitin/ proteasome system and/or nuclear transport receptors [1, 3, [5] [6] [7] [8] . indirect evidence that the host's ubiquitylation machinery participates in parts of the viral entry process comes from studies using pharmacological inhibitors of the ubiquitin/proteasome system. for example, translocation of a murine coronavirus from the endosome to the cytoplasm is facilitated by the ubiquitinproteasome system [9] . similarly, influenza viruses appear to be trapped in an endosomal compartment upon pharmacological inhibition of the proteasome [10] . in contrast, blocking the proteasome increases the transduction efficiency of adenoassociated virus vectors and this correlates with ubiquitylation of capsid proteins [11, 12] . the semliki forest and the vesicular stomatitis virus, however, do not seem to be affected by proteasome inhibition during their entry suggesting different host factor requirements [10] . a role for the ubiquitylation machinery during egress of enveloped viruses is better understood. egress involves the transport of assembled capsids, subviral structures or individual capsid proteins to assembly and budding sites at the cell surface or at intracellular membranes [1] . budding, and potentially trafficking, to the egress site requires an intact class e vesicular sorting pathway (vsp, [13, 14] . the vsp is believed to involve the consecutive activity of three distinct heteromeric complexes termed endosomal sorting complexes required for transport (escrt-i, -ii and -iii, [15] ). the capsid proteins of several enveloped viruses encode 'late domains' that specifically interact with escrt components and redirect them towards the site of viral egress [13] . some late domains of the ppxy motif type (where x can be any amino acid) require the binding of ubiquitin ligases of the nedd4 family of hect-e3 ubiquitin ligases (homologous to e6-ap carboxyl terminus) for efficient escrt recruitment [13] . nedd4.1 and its close relative nedd4.2 are prototypic members of this family, which is conserved from yeast to mammals [16] . they encode a n-terminal c2 domain for ca 2+ -dependent lipid interaction, a catalytical hect domain and three to four wwdomains for protein-protein interactions with proline-rich domains such as the ppxy motif [16, 17] . the exact role of ppxyrecruitment of nedd4 ligases in vsp-mediated viral budding is still unclear. a possible link was recently shown by enhanced escrt ubiquitylation through nedd4.2 overexpression [18, 19] . late domains, including the ppxy type, have also been found in some nonenveloped reoviruses but a general function in virus release remains to be shown [20] . ppxy type late domains where also described for the ad capsid protein penton, which can interact with ubiquitin ligases of the nedd4 family. however, its role in ad infection is unclear [21] . at least in vitro, ad infects cells by first attaching to primary receptors, including car, cd46 and sialic acid, via the fiber protein [22] . in some cells endocytosis of ad may be triggered by penton base-mediated signaling through alpha(v) integrins [23] [24] [25] . in epithelial cells, ad serotype 5 (ad5) particles undergo stepwise disassembly during entry, starting with detachment of the fiber at or near the cell surface and followed by a passage through endosomal compartments in which acidification serves as additional disassembly trigger for membrane penetration and cytosolic translocation [26, 27] . partial disassembly releases the internal capsid protein vi, which can lyse membranes in vitro via its predicted n-terminal amphipathic helix [28] . in the cytosol, the particle engages in microtubule-directed transport towards the mtoc and is translocated to the nuclear pore complex (npc) for nuclear import of the genome [29] [30] [31] . in this study we address the mechanisms of ad cell entry. we demonstrate that the internal capsid protein vi is rapidly exposed to antibodies during cell entry, possibly at the cell surface or immediately after endocytosis. we further determine that protein vi remains partially associated with ad capsids as they traffic to mtocs and the npc. we identify a functional ppxy motif within protein vi that mediates the association of protein vi with nedd4 e3 ubiquitin ligases and facilitates its ubiquitylation. recombinant ad5 in which the protein vi ppxy motif is mutated have normal capsid morphology, escape from endosomes with similar efficiency as wildtype viruses, but are defective in genome delivery to the nucleus. we show that the ppxy motif in protein vi is involved in its efficient microtubule-mediated transport and mutating it in the virus alters the intracellular targeting of ads towards the mtoc region concomitant with a post-entry block in viral infectivity. furthermore, nedd4.1 and nedd4.2 are involved in ad infection and intracellular targeting of incoming virions to the mtoc. we propose that the ppxy motif, in other viral systems, may also function during entry and interact with novel cellular pathways for efficient viral entry. protein vi is exposed during ad infection and partially remains associated with the viral particle the fate of ad particles immediately after internalization is only partially characterized. in the context of endosome escape of ad5, very little is known about how this occurs and which, if any, cellular proteins are involved. from in vitro studies it was proposed that the internal capsid protein vi mediates ad endosome escape [28] . previous reports showed that protein vi dissociates from the ad capsid very early after attachment [26, 27] . to delineate the fate of protein vi during ad entry we performed infection assays and followed the intracellular distribution of the viral capsid and protein vi as a function of time. to this end, fluorescently labeled ad particles were adsorbed to either human retina epithelia pigment cells (htert-rpe1, figure 1 ) or human osteosarcoma cells (u2os, figure s1 ) at 4uc and then transferred to 37uc to synchronize internalization. cells were fixed at various times and analyzed by confocal microscopy (figure 1 ). protein vi was detected using an affinity purified polyclonalantiserum and the location of the mtoc was marked by detecting the primary cilia, which originates at the mtoc using antibodies against acetylated tubulin [32] . at 4uc viral particles accumulated at the cell periphery showing sporadic positive staining with the protein vi antiserum (,1%) possibly due to the recognition of protein vi from damaged particles (figure 1 first row). in contrast, 5 min after the temperature shift, ad particles were still localized close to the cell periphery but approximately 40% of them gave a signal with the protein vi antibody indicating that more protein vi was accessible ( figure 1 , second row). after 15 min, particles had entered the cell with some localized at the mtoc region ( figure 1 , third row) and some at the nuclear rim as described previously [6] . about 10% of the particles remained protein vi positive, including particles at the nuclear rim. after 45 min the majority of the particles were concentrated at the mtoc region ( figure 1 , bottom row) as previously reported [3] . protein vi staining was also concentrated at the mtoc region but most of the signal was not particle associated. similar results were obtained in u2os cells ( figure s1 ). together these data suggested that structural rearrangements leading to protein vi exposure take place at or close to the cell surface during ad entry. in addition, the data showed that protein vi trafficked to the mtoc region and partially remained associated with the capsid. to identify possible trafficking determinants, we analyzed the sequences of protein vi from several ad serotypes and identified a highly conserved ubiquitin-ligase interacting motif present in ppxy-type viral late domains (ppxy, figure s2 ). to examine the role of this ppxy motif in ad cell entry, we used an e1/e3-deleted ad5 that had the protein vi ppsy motif mutated to pgaa (ad5-vi-m1, detailed in figure s3 ) [33, 34] . this mutation, when viruses exploit cellular functions during entry and exit of cells. to redirect cellular functions for their own purpose, viruses encode high-affinity binding sites for key-cellular factors. one such domain is the ppxy motif, which is present in structural proteins of several, mainly enveloped viruses. this motif binds to ubiquitin ligases of the nedd4 family and recruits their function to sites of virus budding from cells. here we show that adenoviruses also encode a ppxy motif in the internal structural protein vi and that the ppxy motif has an unprecedented function in virus entry. adenoviruses with mutations in the protein vi ppxy motif undergo normal endosomal uptake and membrane penetration but have reduced infectivity, altered intracellular targeting and lack efficient gene-delivery. we also find that protein vi is ubiquitylated by nedd4 ligases in a ppxy dependent manner following partial capsid disassembly and displays rapid intracellular movement. depletion of nedd4 ligases also alters virus movement within cells during entry and reduces viral infectivity. given that ppxy motifs are important for virus exit our findings might have uncovered an additional function for ppxy motifs in virus entry, potentially expanding the significance of ppxy motifs and functionally related domains for viral replication. introduced into mason-pfizer monkey virus, was previously shown to abolish late domain functions with no apparent structural changes, which would impair virus assembly [35, 36] . to control for unintended mutations introduced during the cloning, we reverted the pgaa sequence back to ppsy (ad5-vi-wt). because the ,360 copies of protein vi appears to be in contact with several proteins in the mature capsid, modifications that disrupt the tertiary structure could also affect the capsid composition. in large-scale preparations, mutant and wt virus banded at identical densities and gave similar yields of particles as determined by genome and protein quantification. a biochemical analysis of the capsid composition of purified viral particles showed no apparent differences between wt and mutant viruses (figure 2a and data not shown). to confirm that viral capsid integrity between mutant and figure 1 . timecourse of protein vi release during ad entry. ad5-488 was pre-bound to htert-rpe1 cells at 4uc (top row) and shifted to 37uc for 5 min (second row), 15 min (third row) and 45 min (bottom row) as indicated. protein vi was detected using anti-protein vi antibodies (left column), adenoviral particles by detecting the alexa-488 fluorescent signal (second column) and the mtoc by staining the primary cilia (third column). a composite of all three signals including the nucleus (in grayscale) is shown to the left. the inset shows an inverted magnification of representative virus, protein vi and the primary cilia signals from the small dashed box. in the composite protein vi signals are shown in red, ad is shown in green, the primary cilia in grey and the nucleus in blue. colocalization of protein vi and ad appears as yellow. the scale bar is 10 mm. doi:10.1371/journal.ppat.1000808.g001 figure 2b capsid integrity and morphology of the mutant virus was indistinguishable from the wt virus. in contrast, the infectious versus physical particle ratio of ad5-vi-m1 was ,20-fold lower than ad5-vi-wt as assayed by plaque formation on monolayers of 911 cells ( figure 2c ). because infectious vs. physical particles can vary between preparations, we assayed plaque size, which is more informative measurement of propagation rate. plaques were significantly smaller for ad5-vi-m1 versus ad5-vi-wt (see below), suggesting that the altered ppxy domain affects some stages of virus propagation. to determine whether the m1 mutation influences ad cell entry, we performed a fluorescent focus forming assay and stained cells at 8, 12 and 24 h post-infection for expression of the e2a protein, which marks the appearance of viral replication centers ( figure 2d and data not shown). compared to ad5-vi-wt, the ad5-vi-m1 virus produced approximately 20-fold fewer fluorescent foci when equivalent numbers of viral particles were used for infections. this suggested that steps prior to replication (i.e. internalization) require an intact ppxy motif in protein vi. to address trafficking using a different approach, we inserted a gfp expression cassette into the frt site in the e1-deleted region of the wt and the m1 mutant virus (see figure s3 for details). the gfp expressing viruses showed no difference in the quantitative yield and biochemical composition after large-scale purification. we repeated plaque forming assays ( figure 2e ) and single round infection assays ( figure 2f ), this time using gfp expression as the quantification method in non-complementing u2os cells. we observed a reduction in infectivity in the same order of magnitude as previously (compare figures 2c with 2e and 2d with 2f). in addition the gfp expression allowed us to follow the plaque formation over time. as shown in figure s4 the spread of the m1 mutant virus was significantly slower and led to fewer and much smaller plaques ( figure 2e and figure s4 ). next we asked whether ad5 endosomal lysis efficiency following internalization is affected by the mutation in the ppxy motif of protein vi. we infected a549 cells with 30, 300 or 3000 particles per cell of either ad5-vi-wt or ad5-vi-m1 in the presence of alpha-sarcin, a membrane impermeable toxin that inhibits translation when it enters the cytoplasm. alpha-sarcin enters the cell by virus-mediated endosomal membrane lysis thus providing a quantifiable marker for endsome membrane lysis ( figure 2g , [28] ). we measured the incorporation of radiolabeled amino acids over time as a means to determine translational efficiency. as control we used the temperature sensitive ad mutant ad2ts1 that is closely related to ad5. ad2ts1 mutants poorly lyse the endosomal membrane when the virus is grown at non-permissive temperatures due to an increased capsid stability that lacks intraendosomal disassembly. ad2ts1 mutants grown at permissive temperatures lyse the endosome with the same efficiency as the wt virus. incorporation of radiolabeled amino acids was compared to alpha-sarcin treated cells. when we added alpha-sarcin and ad5-vi-wt, ad5-vi-m1 or the ad2ts1 mutant grown at a permissive temperature translation was diminished in a dosedependent manner over at least two orders of magnitude, showing efficient cytoplasmic delivery of the toxin. ad2ts1 grown at the non-permissive temperature did not inhibit translation in this assay ( figure 2g ). we observed no difference between the ppxy-mutant virus and the wt controls. this indicated that an attenuating effect of the ppxy-mutation occurs after endosomal lysis and prior to the onset of replication. we next characterized the release of protein vi from fluorescently labeled ad-vi-m1 and ad5-vi-wt following synchronous infections of u2os cells using the infection assay as described for figure 1 . ad5-vi-wt released protein vi within 5 min ( figure 3a , left panel). in contrast, we observed a delayed release of protein vi from ad5-vi-m1 (15% of m1 after 5 min compared to 38% of wt, figure 3a ) and an increase in colocalization of viral particles with protein vi at the nucleus ( figure 3a ). this observation suggests a delayed accessibility of protein vi within the virus or a defect in protein vi dissociation from the virion ( figure 3a , images to the right). in addition to the increase of protein vi capsid association, ad5-vi-m1 appeared to be more evenly distributed throughout the cell and did not efficiently accumulate at the mtoc region ( figure 3b , images to the left). quantification revealed that 45 min post-infection approximately 60% of the wt viral particles in proximity of the mtoc could be found within a 10 mm radius around the mtoc and 40% within 10-20 mm. in contrast, the localization for the m1 virus was 50% for each region showing a decreased targeting towards the mtoc ( figure 3b , right panel). in summary these data suggest that the ppxy motif in protein vi is required for proper uncoating and normal nuclear targeting. to understand the accumulation defect of ad5-vi-m1 at the mtoc, we expressed wt (vi-wt), mutant (vi-m1) and protein vi deleted in the amphipathic helix (vi-dw) fused to mrfp in cells and analyzed protein vi localization in relation to microtubules ( figure 4 ). we found vi-wt in a punctuated distribution throughout the cell suggesting association with a vesicular compartment or tubulo-vesicular structures associated with microtubules ( figure 4 , top row). in contrast, the ppxy motif mutant vi-m1 localized to a more central compartment and was rarely associated with the microtubules (figure 4 , middle row). deletion of the amphipathic helix, in contrast, abrogated purified ad capsids. electronic microscopy images of purified ad5-vi-wt capsids (left panel) and purified ad5-vi-m1 (right panel) are shown. the inset in each figure shows a magnification of individual capsids. the scale bar is 100nm. c) plaque assay comparison of ad5-vi-wt vs. ad5-vi-m1. quantification of plaques at day 12 for different physical particle per cell ratios for ad5-vi-wt and ad5-vi-m1. shown is the average plaque number per 6 well (+/2 standard deviation) of three individual experiments. d) focus forming assay. e1 complementing 911 cells were infected with ad5-viwt or ad5-vi-m1 and replication centers were stained with e2a antibodies 24 h post-infection and at different particle per cell ratios. .5 random fields with .200 cells were counted, standard deviation represents field-to-field variations. e) plaque forming assay comparison of ad5gfp-vi-wt vs. ad5gfp-vi-m1. quantification of gfp positive plaques at day 12 at different physical particle to cell ratios. the values show the average number of gfp positive plaques per 6-well (+/2 standard deviation) of two independent experiments. f) single round infection assay comparison of ad5gfp-viwt vs. ad5gfp-vi-m1. u2os cells were infected with increasing amounts of physical particle to cell ratios as indicated. gfp expression was quantified using facs. values correspond to the average percentage of gfp positive cells of two experiments done in triplicates (+/2 standard deviation). note that the ,100% infection of wt infected cells at 100 physical particles per cell is saturated and not comparable to m1. g) analysis of membrane penetration. a549 cells were infected with ad5-vi-wt, ad5-vi-m1, ad2ts1 grown at 32uc or at 38uc at different physical particle per cell ratios as indicated and in the presence of alpha-sarcin and translational efficiency was determined by measuring the incorporation of radiolabeled amino acids over time. values are given in percentage normalized to 100% translation measured in the presence of the toxin but in absence of virus. conditions are indicated below each bar and are the mean of at least three independent experiments done in triplicates. doi:10.1371/journal.ppat.1000808.g002 owing to its association with membranes, microtubules and the viral capsid, we next asked whether protein vi displayed intracellular dynamics that could explain virus trafficking. we therefore performed live-cell imaging (lci) using cells expressing mrfp-vi-wt or mrfp-vi-m1. we found that vi-wt was fast moving with short-and longrange movements whereas vi-m1 was essentially motionless ( figure 5a and video s1). the length of the trajectories and the movement of .300 particles were plotted ( figure 5b ). we found that protein vi-m1 motility was greatly reduced compared to protein vi-wt. we next asked whether vi-wt motility depends on intact microtubules and/or actin filaments. disrupting actin filaments with cytochalasin b had no apparent effect on vi-wt localization or motility ( figure 5b , right panel) suggesting that actin was not involved in the movement. in contrast, protein vi motility in nocodazole-treated cells was strongly reduced resembling the reduced motion observed for the m1 mutant ( figure 5b , right panel, see also video s2 in the supporting information). we asked whether vi-m1 showed only plus-end microtubule directed movement. we applied nocodazole to vi-m1 transfected cells, followed by washout of nocodazole. during treatment and after removal of nocodazole no movement or relocalization towards the cell center was observed for vi-m1. in contrast viwt rapidly stopped and restarted bidirectional movement under these conditions (data not shown). together these data suggested that protein vi is a highly mobile protein that moves along microtubules, presumably in association with vesicular structures whose motion depends on the ppxy motif. ppxy motif is essential for protein vi ubiquitylation upon partial ad disassembly ppxy domains are the physiological targets of ubiquitin ligases of the nedd4 family [16] . therefore we asked whether protein vi ubiquitylation depends on the ppxy motif. we adapted an in vitro ad disassembly assay mimicking the partial capsid disassembly believed to occur during ad entry by exposing virions to 48uc. projection. two concentric circles with 10 mm and 20 mm in diameter were positioned around the mtoc as shown in the left panel. virus particles were then counted inside the 10 mm radius and in the region between 10-20 mm. the graph to the right shows the relative abundance within the two regions. distribution for the wild type virus is shown on the left of the graph and for the ppxy mutated virus to the right. the analysis shows that mutated particles are less likely to accumulate at the mtoc then wt particles. the error bar represents cell-to-cell variation (n.15, p,0.05). doi:10.1371/journal.ppat.1000808.g003 figure 4 . subcellular localization of protein vi. u2os cells were transfected with protein vi fused to mrfp, either using wt protein vi (vi-wt, top row), with mutated protein vi (vi-m1, middle row) or with deleted amphipathic helix (vi-dw, bottom row). cells were co-stained for microtubules. the protein vi-signal is shown in the left column, the microtubule stain is shown in the second column and an overlay of the signals is shown in the third column. protein vi fusions appear in red, microtubules in green and the nucleus in blue. the right column shows the field of cells from which the insets (small white square) was taken. the scale bar is 10 mm, please note that the lower panel is a higher magnification. association of protein vi with microtubules in tubulo-vesicular structures is indicated by arrows in the top row. further examples of tubulo-vesicular structures can also be seen in video s1 showing life-cell imaging of vi-wt transfected cells. doi:10.1371/journal.ppat.1000808.g004 this assay dissociates the vertices including fiber, penton, protein vi and peripentonal hexons, but leaves the remainder of the capsid intact ( figure s5 ; [28] ). heat and mock-treated samples were subjected to in vitro ubiquitylation reactions, using free ubiquitin, recombinant e1 and e2 enzymes and purified cytosol as source for the e3 ubiquitin ligase(s), and analyzed by western blot (figure 6 , [28] ). western blot analysis showed that partial capsid disassembly resulted in the appearance of protein vi reactive signals with discrete size increments suggesting predominant modification with two to three ubiquitin as well as some higher molecular weight bands (lane 2, figure 6a ). in contrast, the lack of capsid disassembly (lane 1) or cytosol (lane 3) showed no additional protein vi reactive bands. we also tested ubiquitylation of the capsid proteins fiber, protein iiia and penton base as internal control. we only detected ubiquitylation of the penton base (which also harbors two ppxy domains at its n-terminus), while the fiber and protein iiia (which lack ppxy motifs) were not modified ( figure s5 ). the ubiquitylation of protein vi was also confirmed by using gst-ubiquitin in the above assay, followed by gstpulldown to show covalent modification of protein vi with ubiquitin confirming the predominant modification with two to three ubiquitin-moieties (data not shown). to address the role of the ppxy motif in protein vi ubiquitylation, we repeated the in vitro ubiquitylation assay using wt or m1 mutant protein vi purified from e. coli followed by western blot analysis. we detected protein vi-reactive bands, consistent with protein vi modified with two to three ubiquitin ( figure 6b , lane 2). in contrast, no modification was observed when the ppxy motif was mutated ( figure 6b , lane 1) or in the absence of atp ( figure 6b, lane 3 and 4) . using viral particles in in vitro ubiquitylation reactions (following partial capsid disassembly) protein vi of ad5-vi-m1 was not ubiquitylated ( figure 6c , figure 5 . subcellular dynamics of protein vi. a) u2os cells were transfected with vi-wt (top row) or vi-m1 (bottom row) fused to mrfp and analyzed by live-cell imaging. frames were taken at 1 sec intervals using a cascade 512b 2 camera. the left image in all rows shows maximum image projections of the full frame depicting the cell with scale bar of 10 mm. the four inverted images to the right show magnifications from the boxed inset of four consecutive frames (in the case of vi-wt) and every fifths frame (in the case of vi-m1). in the top panel a moving particle is depicted by a grey arrow within each frame using the departure point depicted by a white arrow as reference. the lower panel shows vi-m1 not moving. b) analysis of trajectory length and relative particle speed of vi-wt or vi-m1 (left side of both panels) or trajectory length and relative particle speed of vi-wt using drugs as indicated (right side of both panels). movies were processed and all recorded trajectories were analyzed for length and relative speed using the imaris tm software package. individual particles were plotted for length of trajectories (in mm, left panel) and the net movement (in mm/ 20sec, right panel) under the conditions tested (indicated below each chart). significant differences are indicated by bars. doi:10.1371/journal.ppat.1000808.g005 lane 3 and 4), while protein vi from ad5-vi-wt was ( figure 6c , lane 1 and 2). thus, the ppxy motif in protein vi is inaccessible in intact capsids but can recruit ubiquitin ligase activity from cytosol when protein vi is released from the capsid interior. together our results show that protein vi ubiquitylation depends on i) virus disassembly, ii) an intact ppxy domain and iii) the presence of a cytosolic ubiquitylation activity. to identify the ligase responsible for protein vi ubiquitylation, we focused on the nedd4-family members nedd4.1, nedd4.2, aip4/itch, wwp1 and wwp2 because they can interact with viral late domains that harbor ppxy motifs [38] . we first coexpressed the vi-wt or vi-m1 mrfp fusion protein together with each of the e3 ligases fused to gfp in u2os cells. when expressed alone, most ligases localized primarily to the cytoplasm (data not shown, wwp1 localized to the plasma membrane and wwp2 accumulated in an uncharacterized intracellular membrane compartment). in contrast, when vi-wt is coexpressed with nedd4.1, nedd4.2 or aip4/itch, the ligases are recruited to the same membrane compartment as protein vi ( figure 7a, row 1-3 ). wwp1 appears to sequester protein vi at the plasma membrane ( figure 7a, row 4) . wwp2 does not colocalize with vi-wt ( figure 7a , row 5). we did not detect significant colocalization between vi-m1 and the e3 ligases, consistent with a ppxydependent interaction ( figure s6 ). to determine whether any of the ligases specifically interact with protein vi, we used purified cytosol from cells overexpressing gfp-tagged ligases and performed pull-downs with beads coated with recombinant protein vi-wt or vi-m1. two ligases, nedd4.1 and nedd4.2, were highly enriched on vi-wt beads while none of the other ligases showed strong binding to vi-wt-or to vi-m1beads ( figure 7b ). taken together, these data suggest a preferential interaction between nedd4.1 and nedd4.2 and the ppxy motif in protein vi, which leads to relocalization of the ligases from the cytoplasm to a membrane compartment. to further characterize the interaction between protein vi and the ligases, we knocked down nedd4.1, nedd4.2, aip4/itch, wwp1 and wwp2 using sirnas ( figure 8a ). the cells were then incubated with an ad5 vector harboring a gfp expression cassette (adgfp) at a low multiplicity of infection (30 physical particles per cell) for 3 h to achieve approximately 20% transduced cells and limit the time of virus exposure. the following day the percentage of gfp-positive cells was quantified by flow cytometry. most ligase knockdowns had no significant effects on transduction, but nedd4.2 knockdown diminished transduction by 50% ( figure 8a ). because nedd4.2 showed the strongest effect on ad transduction we determined whether bacterially expressed and purified nedd4.2 could ubiquitylate purified protein vi in vitro. a minimal system where cytosol was replaced by recombinant nedd4.2 was sufficient for protein vi ubiquitylation ( figure 8b, lane 1) . the ubiquitylation pattern was similar to that obtained with cytosol anti-protein vi antibodies is shown with ubiquitylated proteins marked with grey arrows (vi-ubi) and unmodified protein vi (vi) is marked with a black arrow. c) reactions as in a) followed by detection of protein vi by western blot using ad5-vi-wt (lane 1 and 2) and ad5-vi-m1 capsids (lane 3 and 4). disassembly of capsids is indicated above each lane and protein vi reactive bands are marked as in b to the right. doi:10.1371/journal.ppat.1000808.g006 ( figure 6 ) and required an intact ppxy and a catalytically active nedd4.2 ( figure 8b, lane 2 and 3) . nedd4.1 and nedd4.2 both showed strong interaction with protein vi in pulldown assays. to further investigate the role of each ligase we transduced cells with lentiviral vector expressing shrnas against either nedd4.1 or nedd4.2 or luciferase as a control. we transduced cells in a dose-dependent manner to achieve different levels of knockdown. transduction efficiency was monitored using a gfp expressing lentiviral vector in control cells. seven days post-transduction shrna treated cells were infected with adgfp virus as described above and the transduction rate was determined by flow cytometry. we observed a dose-dependent decrease in infectivity for two different shrnas against nedd4.2, which was similar to what we observed when we used sirnas ( figure 8c ). the results for shrnas against nedd4.1 were less clear. one shrna also reduced viral infection at very high transduction rates but to a lesser extent than shrnas against nedd4.2 while a second shrna showed no effect on ad transduction ( figure 8c) . a combined treatment of cells with either sirnas or shrnas against nedd4.1 and nedd4.2 did not further decrease ad-transduction indicating that the effects of nedd4-ligase knockdowns on ad transduction may be complex (data not shown). a hallmark of ad5 infection is its transient accumulation at the mtoc during entry [5] . the mutation of the ppxy in the ad5-vi-m1 virus seemed to alter this localization (figure 3) . similarly, the ppxy motif was required for nedd4.2 dependent ubiquitylation of protein vi. because knockdown of nedd4 ligases also diminished transduction with adgfp vectors we examined accumulation at the mtoc region in cells depleted with control shrna and nedd4.1 and nedd4.2 specific shrnas. we used the same strategy as in figure 3 by quantifying viral particles in proximity to the mtoc, which was identified by stain for pericentrin. mtoc accumulation for nedd4.1 and nedd4.2 shrna treated cells was reduced when compared to cells treated with control shrna indicating that both ligases might be involved in proper targeting of viruses towards the mtoc ( figure 8d ). in summary, these data provide evidence that release of protein vi during entry and a possible interaction between the ppxy motif of protein vi and nedd4-family ligases are determinants of ad5 trafficking during infection. in this study we show that the ads internal capsid protein vi harbors a ppxy-motif that is involved in virus entry and infectivity. for ads, reaching the nucleus requires a series of sequential steps: receptor-mediated endocytic uptake, partial capsid disassembly, endosomal rupture, microtubule based transport to the mtoc and nuclear trafficking. the link between these steps has been best exemplified in the case of the thermostable temperature-sensitive mutant ad2ts1. this mutant enters cells by receptor-mediated endocytosis, but remains in an endosomal compartment due to increased capsid stability. therefore, ad2ts1 particles are directed to lysosomes for destruction and/or recycled back to the surface thus precluding accumulation at the nuclear pore complex [26, 28, 39] . the role of facilitating endosomal escape during ad entry was initially assigned to the penton base [40] . later, wiethoff and co-workers showed that most membrane lytic activity of ad viral capsids comes from the predicted n-terminal amphipathic helix of the internal capsid protein vi, and that membrane lytic activity required partial capsid disassembly to release protein vi [28] . here we present several lines of evidence that protein vi plays an additional and previously unidentified role in nuclear targeting of the ad capsid. we show that protein vi exposure from ad5 capsids occurs within minutes when pre-adsorbed ad5 is shifted from 4uc to 37uc. this is consistent with the loss of the fiber prior to internalization and the rapid accumulation of ad5 in the cytosol [41, 42] . we found that significant amounts of protein vi remains partially associated with the viral capsid after the initial exposure and until the viral particle accumulates at the mtoc or the nuclear rim. we show that protein vi is engaged in rapid intracellular trafficking that depends on intact microtubules and requires the nterminal amphipathic helix for microtubule association and the ppxy motif for motion. to our knowledge protein vi is the first ad capsid protein described that possesses its own microtubuledependent dynamics and future work has to address if other capsid proteins have similar properties. inactivation of the ppxy in the viral context (ad5-vi-m1) resulted in a post-entry delay that reduced infectivity and prevented efficient accumulation of the entering virus particles at the mtoc. while we cannot exclude the possibility that the ppxy motif in protein vi also plays a role in adenoviral replication, assembly or egress, our single round infection assays showed that the majority of the titer reduction for the mutant was related to steps prior to the initiation of replication or the delivery and expression of a reporter gene. moreover, our data show that the efficiency with which a toxin is delivered to the cytosol during ad infection is similar for mutant and wt virus. this provides strong evidence that the ppsy motif in protein vi has a function during cell entry, but probably only after endosomal membrane lysis has occurred. current structural data place protein vi inside the assembled capsid, therefore potentially precluding it from functions during egress at least when capsid associated [43] [44] [45] . our observations show that protein vi is exposed after entry and that capsid disassembly is required for its ubiquitylation, which is consistent with our hypothesis that the ppxy motif is accessible only during ad entry following partial capsid disassembly. furthermore, it is currently not clear whether late domains containing ppxy motifs present in other viral systems, which are required for viral egress, have additional functions. mutational inactivation of ppxy motifs in the vp40 structural protein of ebola virus and matrix protein of rabies virus both showed an attenuation of the virus and a reduction of infectivity [20, 46] . interestingly, for ebola virus, the ppxy mutants showed no budding defect, but virus production was reduced, which could indicate a disruption earlier on in the life cycle than previously thought [46] . for rabies virus, wirblich et al. describe a figure 7 . protein vi interacts with nedd4 ligases via the ppxy motif. a) protein vi (vi-wt) was n-terminally fused to mrfp and co-transfected with gfp-nedd4.1 (first row), gfp-nedd4.2 (second row), gfp-aip4/itch (third row), gfp-wwp1 (fourth row) and gfp-wwp2 (last row). confocal images of representative cells are shown and the mrfp signal for protein vi (left column), the gfp signal for the ligases (center column) and the merged signals together with dapi stain of the nucleus (right column) is indicated above each column. transfected plasmids are indicated. colocalization of nedd4 and protein vi results in a yellow signal. the scale bar is 10mm. b) diverse gfp-tagged nedd4 ligases were over-expressed in cells and cytosolic extracts were used for pulldown experiments using recombinant protein vi-wt or vi-m1 coupled to beads. 10% of the input material (ip) is shown in the first lane. bound material for protein vi-wt (lane 2) and vi-m1 (lane 3) was detected with respective antibodies as indicated to the right. co-eluted protein vi detected with a protein vi specific antibody is shown as loading control in the lower lane. doi:10.1371/journal.ppat.1000808.g007 budding defect of the late-domain mutant but also noted a reduction of early mrna production [20] . earlier studies have shown that ppxy type late domains bind to ubiquitin ligases of the nedd4 family rather then directly to class e vsp proteins [13] . our study showed that the ppxy motif in protein vi can interact with all nedd4 ligases tested but preferentially binds to nedd4.1 and nedd4.2 and re-targets them to membranes. owing to topological constrains, recognition of the ppxy domain should require membrane rupture because of the cytosolic localization of nedd4 ligases, which according to our data does not seem to be affected by the ppxy mutation in the m1 virus. the ppxy motif in protein vi seems to favor interaction with nedd4.1 and nedd4.2 although we observed interactions with aip4/itch and wwp1 as well. it is possible that an interaction between protein vi and wwp1, as we observe in transient transfections, is circumvented by exposure of the ppxy domain after the virus has entered the endosomal compartment. a role for nedd4.1 or nedd4.2 in ad entry is underscored by our observation that nedd4.2 can directly ubiquitylate protein vi via the ppxy motif and its depletion (and to a lesser extent also depletion of nedd4.1) reduces ad transduction. in addition this depletion also reduced mtoc accumulation of viral particles following infection, which is similar to the effect of the ppxy mutation in the m1 virus. however the effects were modest, indicating that additional mechanisms contribute to ad entry. further studies will be needed to identify a specific role for each ligase in ad entry and trafficking towards the mtoc and to determine whether other ligases like aip4/itch and wwp1 are involved. how ubiquitylation of protein vi or interaction with nedd4 ligases directs accumulation of ads at mtocs remains unknown. ubiquitylated protein vi could be specifically recognized by cellular factors. alternatively, recruitment of nedd4.1 and/or nedd4.2 by protein vi could result in the ubiquitylation of other cellular factors that constitute an efficient transport means used by the virus. recent work has shown that some members of escrt-i become ubiquitylated when nedd4.2 is overexpressed [18, 19] . therefore, it is possible that nedd4.2 (or other nedd4-ligases) binding to protein vi could activate the escrt pathway via ubiquitylation. whether membrane compartments or the escrt pathway plays a direct role in ad virus transport during entry remains to be addressed. however, escrt components can be found at the endosomal compartments as well as associated with the centromeric region [47] . it is noteworthy that endosomal escape is also required for interferon induction by ad via yet unknown mechanisms [48] . this pathway may also be related to protein interaction between protein vi and nedd4-family ligases. release and separation of protein vi from the capsid is clearly defective in the ad5-vi-m1 mutant virus. a lack of functional ppxy may therefore block disassembly steps, preclude efficient endosomal escape following the membrane lysis event or fail in the recruitment of subsequent factors required to efficiently link the virus with retrograde transport pathways. penton base also encodes ppxy motifs [49] . because eliminating the ppxy motif from protein vi had only modest effects on mtoc accumulation, there may be functional overlap between the ppxy in protein vi and penton base. this is further supported by the observation that ubiquitylation of penton could be abolished by depleting the cytosol with protein vi indicating that maybe the same ligases are involved ( figure s5 ). in conclusion, our study is the first demonstration of a ppxy motif, previously described for viral late domains, present in a non enveloped dna virus that exerts a function during entry. mastadenovirus suggests that protein vi fulfils a key function. we suggest that after endocytosis cell or serotype specific capsid disassembly cues regulate exposure, membrane-interaction and ubiquitylation of protein vi (and possibly other capsid proteins). this could facilitate the recruitment of a common cellular microtubule-dependent pathway for retrograde trafficking. this mechanism could be more important in vivo in highly polarized epithelia or neurons where long-range movement is crucial and might be less important in cell culture models [50] . given the prevalence of viral late domains, ubiquitylation and trafficking towards the mtoc our results may have uncovered a more general mechanism by which viruses and other cargos achieve intracellular delivery and provide a rational to look for further ''early'' functions of ppxy motif containing late-domains in other viral systems. cytoplasmic extracts for pulldowns were prepared from 293t cells (human embryonic kidney cells). all cells except htert-rpe were grown in dmem glutamax tm (gibco) supplemented with 10% of fetal calf serum (fcs) (biowest). htert-rpe1 cells were a kind gift from m. bonhivers (university of bordeaux 2) and grown in dmem/hamsf12 media supplemented with 10% fcs according to the suppliers instructions. prior to infection experiments, cells were serum starved for 24h to induce primary cilia growth [32] . recombinant ad5-vi-wt and ad5-vi-m1 viruses and their gfp expressing counterparts were constructed as described in the supplemental material. amplification of viruses was done in 293 cells and purified using double cscl 2 -banding. virus particle to cell ratios were calculated based on the estimated copy numbers of viral genomes. copy numbers were calculated according to mittereder et al. [51] . briefly, purified particles were diluted 1:10 in virus lysis buffer (0.1% sds, 10 mm tris/hcl ph 7.4, 1 mm edta) and incubated for 10 min at 56uc to release the viral genomes and the od 260 was determined. calculations were based on 1 od 260 = 1.1610 12 particles/ml [51] . lentiviral vector production for shrna encoding vectors was done by the service platform for lentiviral vector production of the indicated (from 25-100%) and transduced with adgfp. gfp expression levels were determined 24 h later using flow cytometry. gfp expression levels within each condition were normalized to transduction controls of cells treated with shrna expressing lentiviral vectors against luciferase (arbitrarily set to 1). values are the mean (+/2 standard deviation) of at least two experiments done in triplicates d) accumulation of ad at the mtoc region following nedd4 depletion. fluorescently labeled ad was used to infect cells following control depletions (shluc) or depletion of nedd4.1 or nedd4.2 using (sh4.1 (1) and sh4.2 (1) as in c). subcellular localization of viral particles was determined at 45 min. post infections. cells were fixed and stained for the mtoc using a pericentrin antibody. particles were counted and scored for their relative proximity to the mtoc using two concentric circles with 10 and 20 mm diameter around the mtoc (compare also figure 3 ). the graph shows percentage of viral particles within 10mm or 10-20 mm proximity to the mtoc as indicated. note that in nedd4.1 and nedd4.2 depleted cells the relation is inverted compared to control depleted cells showing less viral particles accumulating at the mtoc. the error bar represents cell-to-cell variation (n.15, p,0.05). doi:10.1371/journal.ppat.1000808.g008 institute federative de recherche 66 of the bordeaux 2 university. prevalidated lentiviral vectors encoding shrnas in the vector backbone plko.1 against nedd4.1 and nedd4.2 were purchased from the mission tm shrna collection from sigma. for downregulation of nedd4.1 we used nm_006154.1-1753s1c1 (sh4.1 (1), ccggccggagaattatgggtgtcaactcga-gttgacacccataattctccggttttt) against the coding sequence and nm_006154.1-3522s1c1 (sh4.1 (2), ccggg-cctttctcttgcctgcatatctcgagatatgcaggca-agagaaaggcttttt) against the 39 utr. for downregulation of nedd4.2 we used nm_015277.x-2772s1c1 (sh4.2 (1), ccgggcgagtacctatgaatggattctcgagaatcca-ttcataggtactcgcttttt) against the coding sequence and nm_015277.x-3959s1c1 (sh4.2 (2), ccggcctgtt-tgtatgcgtttgctactcgagtagcaaacgcatacaa-acaggttttt) against the 39 utr. control vectors for shrnas encoded for shrna against luciferase (sigma) and control vectors for transduction and estimation of the titer encoded for gfp. all sequences for protein vi were derived from ad serotype 5 (ad5) and cloned into the gateway tm compatible entry vector pdonr221. sequence verified donr plasmids were used for recombination into gateway tm compatible destination vectors for n-terminal fusion of mrfp (l30-mrfp, kindly provided by e. bertrand). bacterial expression vectors for protein vi are based on pet15b. site-directed mutagenesis was used to change amino acids 148-ppsy-151 to 148-pgaa-151 in protein vi. n-terminal tagged expression vectors for nedd4.1, and nedd4.2 were provided by e. bertrand [52] and tagged expression vectors for aip4/itch and wwp1 were a kind gift of paul bieniasz (rockefeller university, new york). bacterial expression vectors for catalytically active murine gst-nedd4.2 and the inactive gst-nedd4.2-dn was kindly provided by s. kumar [53] . sirnas were purchased as duplexes from eurogentec (only the reverse strand is shown): scramble (59-cgcaauucgauguccc-gugdtdt), nedd4.1 (59-aaacaacccagccaggcucdtda), nedd4.2 (59-cugugacuuuguguuguggdtda), were previously described by segura-morales et al. (2005) , aip4/itch sirnas (59-ucaucauucugagaagcacdtdt, [54] , and wwp1 sirna (59-cuucuacgaucaucaacucdtdt) was previously described by chen et al. (2005) . the wwp2 sirna was a smartpool from dharmacon. depletions were performed in 12-well dishes using 2610 5 u2os cells. cells were transfected after 24 and 48 h with 20 pmol of each sirna duplex per well. forty-eight hours after the first transfection cells were transduced using 30 physical particles per cell of ad5-gfp virus for 3 h without prior pre-adsorption. cells were harvested 24 h later and analyzed by flow cytometry for gfp expression and further processed for western blot analysis to verify knock-down efficiency. acquisitions were done with facscali-burh or facscantoiih cytometer (bd biosciences) and the data were processed and analyzed by the cellquesth pro and facsdivah software (bd biosciences). purified ad particles were labeled using the alexa-488 microscale protein labeling kit (invitrogen) using the manufacturers protocol. infectivity of labeled virus preparations was determined by quantification of gfp-transduction. only preparations with .90% activity where used. for time course experiments, u2os cells were seeded at semiconfluency on coverslips. pre-binding was done with 5000 physical particles per cell in 100 ml at 4uc on a shaking platform for 1 h. at t 0 coverslips where rinsed in cold dmem and transferred to pre-equilibrated (37uc, 5% co 2 ) dmem. at indicated time points the cells where fixed and processed for if analysis. cells grown on coverslips were rinsed in pbs and fixed with 4% pfa in pbs and blocked/permeabilized with if-buffer (10% fcs in pbs and 0.1% saponin). primary and secondary antibodies where applied to the coverslip in if-buffer for 1 h each. cells were mounted in dako mounting media containing dapi and analyzed by confocal-or epifluorescence microscopy. for if involving microtubule staining cells were treated with crosslinkers prior to fixation. the following primary antibodies were used in this study: mouse anti-actubulin (kind gift from c. janke, montpellier), mouse anti e2a (kind gift of t. dobner, hamburg), rabbit anti pericentrin (abcam) and rabbit anti-protein vi antibodies raised against recombinant protein vi and affinity purified (see supplemental material). secondary antibodies alexa546 anti mouse was from affinity research and atto647 anti rabbit was from sigma. confocal pictures were taken on a zeiss lsm 510 meta confocal microscope or a leica sp5 confocal microscope and epifluorescence pictures were taken on a zeiss axiolmager z1 microscope with coolsnap hq photometrics camera both equipped with metamorph tm software. confocal stacks where taken every 0.5 mm with a pinhole setting of 1 for all channels to achieve high local resolution. images were processed using imagej and adobe photoshop tm . counting of viral particles was performed using the semi-automated cell counting tool from imagej. colocalization analysis: stacks from confocal images where combined as z-projection using maximum intensity, converted into 8-bit images for each channel. colocalization between protein vi and ad was then determined using the colocalization finder plugin from imagej. live-cell imaging: u2os cells were seeded in 3.5cm glass-bottom dishes and transfected with 1.5 mg of protein vi-expressing vectors. twenty-four h later the medium was replaced by pre-warmed opti-mem (gibco). movies were acquired on a nikon te 2000 microscope with cascade 512b 2 camera using metamorph tm software for data acquisition (120 frames, 1 frame/sec). in some cases, 2 h before the acquisition, cells were incubated with either nocodazole (sigma) or cytochalasin b (sigma) diluted respectively at 0.4 mg/ml and 5 mg/ml in opti-mem. drugs were not removed during acquisition. acquired movies were further processed using imagej to enhance protein vi particle detection by background subtraction and bleach correction. particles were tracked using the spot-tracking tool of imaris tm software to determine the length of their trajectories and the speed of their movement. particle detection size was scaled to 0.75 mm and tracks were built with a maximum displacement of 1.5 mm between consecutive frames, a maximum gap size of 3 frames and a minimal track length of 20 s. at least 5 cells were analyzed for each condition that equals a minimum of 300 analyzed tracks per condition. three microliter of purified sample virus was adsorbed to a carbon-coated film (200 mesh grids). the grids with adsorbed virus were floated onto a solution of the negative stain (1% solution of uranyl acetate). the film was picked up by a copper em grid and then air-dried. specimens were examined under a hitachi h7650 electron microscope operating at 80 kv and images were further processed using imagej software. affinity purified rabbit anti-protein vi antibodies were used at a dilution of 1:2000. other antibodies used for the study were: rabbit polyclonal anti-nedd4.1 and anti-nedd4.2 antibodies that were a kind gift of o. staub (lausanne, switzerland) (dilution 1:1000), rabbit polyclonal anti-wwp2 antibody (sc-30052, santa cruz biotechnology) (dilution 1:200), goat polyclonal anti-wwp1 antibody (sc-11893, santa cruz biotechnology) (dilution 1:200), mouse monoclonal anti-aip4/itch antibody (sc-28367, santa cruz biotechnology) (dilution 1:100) and mouse monoclonal anti-gfp antibody (roche) (dilution 1:500). sds-page was done using 12% poly-acrylamide gels and transferred to nitrocellulose membranes. membranes were blocked in tbs containing 10% of dry-milk and 0.01% of tween 20 (sigma), followed by over-night detection of antigens using primary antibodies diluted in tbs containing 10% of dry-milk and 0.01% of tween 20 (sigma). primary antibodies were detected using hrp-conjugated secondary antibodies against rabbit, goat or mouse (sigma) at a dilution of 1:5000. specific signals were revealed using the enhanced chemiluminescence detection system (ecl) (perkinelmer). data are presented as mean, error bars as std. statistical analysis if not indicated otherwise was done using unpaired students t-test (*:p,0.05; **:p,0.01; ***:p,0.005). figure s1 protein vi release in u2os cells during ad entry. ad5-vi-wt-488 was pre-bound to cells at 4uc (top row) and shifted to 37uc for 5min (second row), 15min (third row) and 45min (bottom row). protein vi was detected using affinity purified antiprotein vi antibodies (left column) and ad by detecting the alexa-488 fluorescent signal (middle column). a composite of both signals including the nucleus (in greyscale) is shown in the left column. the inset shows a magnification of representative virus and protein vi signals in the small white box. protein vi signals are shown in red, ad is shown in green and colocalization of protein vi and ad is shown in yellow. the scale bar is 10 mm. the rabbit polyclonal serum against protein vi was generated against recombinant purified his-tagged protein vi. rabbit serum that reacted positive and specific against protein vi in western blots of purified viruses was used for further affinity purification for use in immunofluorescence applications. affinity purification was done using recombinant purified protein vi coupled to cnbr + -activated sepharose beads. bound antibodies were eluted with 0.1 m glycin ph2, neutralized with 2m tris ph 8.8 and dialyzed against pbs. affinity purified antibodies were used at 1:250 dilutions in immunofluorescence. found at: doi:10.1371/journal.ppat.1000808.s001 (2.69 mb tif) figure s2 alignment of the ppxy motif in the sequence of protein vi from different human and non-human adenovirus serotypes. a partial alignment of protein vi sequences from different human adenoviral serotypes as well as non-human adenoviruses from the genus mastadenovirus is shown. the conserved ubiquitin ligase-recruiting motif is boxed. sequences were retrieved from public databases with the following accession numbers; canine (cav-2, ap_000621), bovine (boad3, ap_000031), huad3 (serotype b, abb17802), huad35 (serotype b, ap_000584), huad4 (serotype e, yp_068031), huad17 (serotype d, ap_000149), huad2 (serotype c, ap_000174), huad5 (serotype c, ap_000210), huad12 (serotype a, ap 000120), huad40 (serotype f, np_040861), murine (muad1, ap_000350). found at: doi:10.1371/journal.ppat.1000808.s002 (0.45 mb tif) figure s3 construction of mutant ad5 with altered ppxy motif in protein vi using bac technology. a) to construct a bacterial artificial chromosome (bac) carrying an infectious ad5 genome, we cloned an adeasy system (stratagene) based virus genome into pksb2 vector as described previously (warming et al. [57] ; ruzsics et al. [34] ). this recombinant ad5 lacked the e1 and e3 regions and carried an frt site in the place of its e1 region, which was introduced through an frt containing pshuttle (stratagene) clone. the resulting bac, was termed pad5-frt and can be reconstituted to fully infectious recombinant ad5 viruses after transfection of e1 complementing cell lines such as 293 cells. to construct protein vi-modified viruses, pad5-frt was transformed into the e. coli strain sw102, which encodes the l-red recombination system from the bacteriophage under a heatinducible promoter [57] . we next amplified a kanamycin resistance cassette using primers with 50 nt 59 extensions homologous to protein vi coding regions. the forward primer was flanked with a homology located upstream to the ppsy motif and introduced a clai site into the protein vi orf without affecting its amino acid sequence. the homology region attached to the reverse primers carried the same clai site and overlapped with the ppsy motif. two different reverse primers were generated: one carried an unaltered ppsy motif and another that encoded the amino acids pgaa instead of ppsy and introduced an additional pst i site by the new coding sequence. the pcr products were transformed into the sw102 bacteria harbouring the ad5-bac following heatshock to induce red-recombination. chloramphenicol and kanamycin double resistant clones were selected and bac dna was prepared from individual clones. the isolated bac dna was digested with clai and subsequently religated. this procedure eliminated the kanamycin cassette and reconstituted the protein vi orf concomitant to the recircularisation of the clai treated bacs, because there was no other clai site present in the rest of the pad5-frt. if the reverse primer with an intact ppsy motif was used for amplification, the wild type protein vi amino acid sequence was reconstituted with a silent genetic tag introducing a clai site. if the reverse primer with pgaa motif was used for amplification, the protein vi coding sequence was modified at two positions i) a silent genetic tag was inserted introducing a clai site as above and ii) the ppsy motif was replaced by the pgaa motif introducing a new psti site. after the clai treatment and re-ligation the modified genomes were retransformed in e. coli dh10b. kanamycin negative colonies were identified by replica plating and the resulting mutants were analysed by restriction digestions and verified by sequencing. the mutant ad genomes were released from the respective bacs by pac i digestion and transfected into 293 cells. following the appearance of cytopathic effects the virus was amplified and purified by double cscl banding, dialyzed into pbs/10% glycerol and snap frozen. b) to verify the identity of the purified virions and analyse whether detectable reversion occurred during reconstitution and propagation of the virus stocks viral dna was extracted from purified virions and was pcr amplified with protein vi specific primers. the pcr products were digested with pst i (left) and cla i (right) to identify the recombinant viruses with or without the altered protein vi sequences. to insert a gfp expression cassette we used bacterial flp-recombination using the frt site in the e1-deleted region of the ad5-vi-wt and ad5-vi-m1 bacs. we cloned the left end of the ad5 (nt 1-341) flanked by a pac i restriction site into the plasmid porir6k-ie [55] ; genbank acc. ay700022) upstream of its frt site. we also replaced its zeocin resistance marker with an pgps1.1(neb) derived kanamycin cassette and cloned an egfp orf from pegfp-n1 (clontech) in its expression locus and termed this plasmid po6-a5-gfp. the porir6kie derived plasmids can only be maintained in special e. coli strains such as pir1 (invitrogen) because they are dependent on the presence of r6kc phage replicase [55] . to carry out the recombination, e. coli strain dh10b (invitrogen) was co-transformed with pad5-frt derived bacs and pcp20 encoding the flp-recombinase [56] and cultured at 30uc. the flp recombinations were carried out as described in bubeck et al. [55] . briefly, the e. coli cell carrying the target bacs and the flp expression plasmid pcp20 were transformed with the po6-a5-gfp and selected for chloramphenicol and kanamycin resistance upon induction of the flp expression by a temperature shift to 43uc. this treatment induced a recombination between the two frt sites (one in the pad5-frt derivative and one on the po6-a5-gfp) and induced unification of the bac and the po6 construct. only the recombined construct survived the double selection because po6 constructs are not maintained in dh10b. this approach essentially replaced the old left end of the ad5 bac by a cmv promoter driven gfp-expression cassette containing fragment, which also possessed all the cis elements needed for virus reconstitution as described above. found at: doi:10.1371/journal.ppat.1000808.s003 (0.45 mb tif) figure s4 the ppxy-mutant ad5gfp-vi-m1 forms smaller and fewer plaques. a) shown is a comparison of the growth of individual plaques starting from a single infected cell. e1 complementing 911 cells were infected at low multiplicity of infection with ad5gfp-vi-wt and the ppxy mutant virus ad5gfp-vi-m1 for 24 h and then washed and overlayed with agarose. virus growth was monitored by the appearance of gfppositive cells and images of representative cells/plaques were taken on days 1, 3, 9 and 12. the images in the left row show the plaque formation of the wild type virus 1, 3, 9 and 12 days after the initial infection (top-to-bottom). at day 9 and 12 significant large plaques with lesions of the cell monolayer can be observed. in contrast the mutant virus to the right shows a slow expansion of gfp-positive plaques with less damage to the cell monolayer. images are superimpositions of the gfp signal and the phase contrast image of the monolayer. b) the image shows the damage in the cell monolayer caused by plaque formation on day 12. the arrow indicates the average size of the plaque. the scale bar is 50 mm. found at: doi:10.1371/journal.ppat.1000808.s004 (2.12 mb tif) figure s5 penton is a target for ubiquitylation following partial disassembly of the virus. a) localization and sequences of conserved ppxy-motifs in protein vi and penton for ad5 (black box). note that processed protein vi is shown as it is present in the capsid during viral entry. b) schematic representation of the in vitro ubiquitylation assay. virus disassembly was induced by mild heattreatment, in vitro ubiquitylated using ubiquitin or recombinant gst-ubiquitin, recombinant e1 and e2, an energy regenerating system and purified cytosol as source for the e3-ligase and analyzed by western blot. controls lack the mild heat-treatment. c) western blot analysis of viral capsid proteins penton, fiber and protein iiia following capsid disassembly and in vitro ubiquitylation. heat-treatment is indicated above each lane. antibodies are indicated to the right of each blot. specific bands are labeled accordingly. grey arrows indicate band shifts due to ubiquitylation, black arrows indicates the unmodified protein. d) western blot analysis of in vitro ubiquitylation reactions of heat-treated viral particles. heat treatment is indicated above each lane. for individual reactions the cytosol was depleted with recombinant fiber beads (control), recombinant vi-wt beads or recombinant vi-m1 beads as indicated above each lane. reactions were blotted with anti-penton. the assay shows that the ubiquitylation activity can be depleted from cytosol with recombinant wt protein vi but not when the ppsy motif is mutated. the same assay also abolishes protein vi ubiquitylation showing that similar ubiquitylation activities are responsible (data not shown). e) protein vi depleted cytosol renders nedd4.2 active for penton ubiquitylation. in vitro ubiquitylation reactions using catalytically active or inactive nedd4.2 substituted with cytosol depleted by protein vi-wt (as indicated above each lane) and analyzed by western blot with antipenton antiserum. black arrows indicate ubiquitylated proteins, grey arrows the unmodified protein. this assay shows that additional cytosolic factors are required for full nedd4.2 activity for penton ubiquitylation. found at: doi:10.1371/journal.ppat.1000808.s005 (0.73 mb tif) figure s6 protein vi with altered ppxy motif does not colocalize with nedd4 ligases. protein vi with ppsy motif mutated to pgaa was n-terminally fused to mrfp and cotransfected with gfp-nedd4.1 (top row), gfp-nedd4.2 (second row), gfp-aip4/itch (third row), gfp-wwp1 (forth row) and gfp-wwp2 (bottom row). confocal images of representative cells are shown and the mrfp signal for vi (left column), the gfp signal for the ligases (centre column) and the merged signals together with dapi stain of the nucleus (right column) is indicated above each column. transfected plasmids are indicated left of each row. note that the cytoplasmic localization of each ligase is similar to that in cells without cotransfection of protein vi (data not shown). found at: doi:10.1371/journal.ppat.1000808.s006 (3.90 mb tif) video s1 intracellular dynamics of protein vi. u2os cells were transfected with vi-wt, fused to mrfp. the movie was acquired using a nikon te 2000 microscope equipped with cascade 512b 2 camera at 1 frame per second. the movie shows rapid intracellular movement of vi-wt depicting compartments resembling vesicular, tubulo-vesicular and reticular structures. video s2 comparison of intracellular dynamics for vi-wt vs. vi-m1 vs. vi-wt (+ nocodazole). u2os cells were transfected with vi-wt (left and right panel) or vi-m1 (middle panel) fused to mrfp. the cell to the right was treated with nocodazole (5 mg/ml) prior to image acquisition. movies were acquired using a nikon te 2000 microscope equipped with cascade 512b 2 camera at 1 frame per second. the movies were labeled and assembled using imagej and converted into quicktime tm movies. all three displayed movies were taking at the same frame rate. the movies show rapid intracellular movement of vi-wt at nearly real-time (left). vi-m1 shows strongly reduced movement (middle). treatment of vi-wt transfected cells with nocodazole, strongly reduces the vi-wt movement resembling vi-m1 dynamics (right). found at: doi:10.1371/journal.ppat.1000808.s008 (1.21 mb mov) virus trafficking -learning from single-virus tracking a superhighway to virus infection intracellular trafficking of adenovirus: many means to many ends signalling in viral entry viral strategies for intracellular trafficking: motors and microtubules association of adenovirus with the microtubule organizing center microtubule-dependent plus-and minus end-directed motilities are competing processes for nuclear targeting of adenovirus nuclear targeting of adenovirus type 2 requires crm1-mediated nuclear export the ubiquitin-proteasome system facilitates the transfer of murine coronavirus from endosome to cytoplasm during virus entry the ubiquitin-vacuolar protein sorting system is selectively required during entry of influenza virus into host cells endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors late budding domains and host proteins in enveloped virus release the emerging shape of the escrt machinery functional reconstitution of escrt-iii assembly and disassembly the nedd4 family of e3 ubiquitin ligases: functional diversity within a common modular architecture regulation of functional diversity within the nedd4 family by accessory and adaptor proteins efficient and specific rescue of human immunodeficiency virus type 1 budding defects by a nedd4-like ubiquitin ligase nedd4l overexpression rescues the release and infectivity of human immunodeficiency virus type 1 constructs lacking ptap and ypxl late domains ppey motif within the rabies virus (rv) matrix protein is essential for efficient virion release and rv pathogenicity adenovirus protein involved in virus internalization recruits ubiquitin-protein ligases adenovirus receptors adenovirus endocytosis hautala t (1999) rab5 gtpase regulates adenovirus endocytosis integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment stepwise dismantling of adenovirus 2 during entry into cells the role of the adenovirus protease on virus entry into cells adenovirus protein vi mediates membrane disruption following capsid disassembly nuclear import of adenovirus dna in vitro involves the nuclear protein import pathway and hsc70 import of adenovirus dna involves the nuclear pore complex receptor can/nup214 and histone h1 adenovirus core protein pvii is translocated into the nucleus by multiple import receptor pathways a bbsome subunit links ciliogenesis, microtubule stability, and acetylation recombineering: a powerful new tool for mouse functional genomics transposon-assisted cloning and traceless mutagenesis of adenoviruses: development of a novel vector based on species d a proline-rich motif (pppy) in the gag polyprotein of mason-pfizer monkey virus plays a maturation-independent role in virion release the mason-pfizer monkey virus pppy and psap motifs both contribute to virus release switch from capsid protein import to adenovirus assembly by cleavage of nuclear transport signals hect ubiquitin ligases link viral and cellular ppxy motifs to the vacuolar protein-sorting pathway infectious adenovirus type 2 endocytosis of adenovirus and adenovirus capsid proteins role of vesicles during adenovirus 2 internalization into hela cells the first step of adenovirus type 2 disassembly occurs at the cell surface, independently of endocytosis and escape to the cytosol visualization of alphahelices in a 6-angstrom resolution cryoelectron microscopy structure of adenovirus allows refinement of capsid protein assignments adenoviruses: update on structure and function a quasi-atomic model of human adenovirus type 5 capsid ebola virus vp40 late domains are not essential for viral replication in cell culture human escrt and alix proteins interact with proteins of the midbody and function in cytokinesis key role of splenic myeloid dcs in the ifn-alphabeta response to adenoviruses in vivo novel partner proteins of adenovirus penton carassociated vesicular transport of an adenovirus in motor neuron axons evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy tsg101 and alix interact with murine leukemia virus gag and cooperate with nedd4 ubiquitin ligases during budding regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases nedd4 and nedd4-2 the e3 ubiquitin ligase itch controls the protein stability of p63 comprehensive mutational analysis of a herpesvirus gene in the viral genome context reveals a region essential for virus replication gene disruption in escherichia coli: tcr and kmr cassettes with the option of flp-catalyzed excision of the antibiotic-resistance determinant simple and highly efficient bac recombineering using galk selection we thank e. bertrand, p. bienasz, m. bonhivers, t. dobner, e. everitt, s. kumar, a. lieber, g. nemerow and o. staub for their generous gifts of reagents. we thank j. chroboczek and m. piechazcyk for helpful discussions during the course of this work and michael kann for critical reading of the manuscript. we thank the members of the kremer lab for discussion. we would like to thank k. rogowski and l. doelken for valuable help. we further thank the montpellier rio imaging platform mri. we like to acknowledge the excellent technical support from e. gontier at the electron microscopy platform of the bordeaux imaging center (bic) and the help of n. dugot-senant, v. guyonnet-duperat and v. pitard from the imaging, lentiviral vector production and cytometry platforms at the institute federative de recherchã© 66 (ifr66) at the university of bordeaux 2. conceived and designed the experiments: hw csm oc zr cmw ejk. performed the experiments: hw dh bj zr. analyzed the data: hw dh bj zr cmw. contributed reagents/materials/analysis tools: hw csm ss oc zr cmw. wrote the paper: hw zr cmw ejk. key: cord-319809-33i6lzjd authors: drew, elliot d.; janes, robert w. title: identification of a druggable binding pocket in the spike protein reveals a key site for existing drugs potentially capable of combating covid-19 infectivity date: 2020-07-01 journal: bmc mol cell biol doi: 10.1186/s12860-020-00294-x sha: doc_id: 319809 cord_uid: 33i6lzjd background: following the recent outbreak of the new coronavirus pandemic (covid-19), the rapid determination of the structure of the homo-trimeric spike glycoprotein has prompted the study reported here. the aims were to identify potential “druggable” binding pockets in the protein and, if located, to virtual screen pharmaceutical agents currently in use for predicted affinity to these pockets which might be useful to restrict, reduce, or inhibit the infectivity of the virion. results: our analyses of this structure have revealed a key potentially druggable pocket where it might be viable to bind pharmaceutical agents to inhibit its ability to infect human cells. this pocket is found at the inter-chain interface that exists between two domains prior to the virion binding to human angiotensin converting enzyme 2 (ace2) protein. one of these domains is the highly mobile receptor binding domain, which must move into position to interact with ace2, which is an essential feature for viral entry to the host cell. virtual screening with a library of purchasable drug molecules has identified pharmaceuticals currently in use as prescription and over the counter medications that, in silico, readily bind into this pocket. conclusions: this study highlights possible drugs already in use as pharmaceuticals that may act as agents to interfere with the movements of the domains within this protein essential for the infectivity processes and hence might slow, or even halt, the infection of host cells by this new coronavirus. as these are existing pharmaceuticals already approved for use in humans, this knowledge could accelerate their roll-out, through repurposing, for affected individuals and help guide the efforts of other researchers in finding effective treatments for the disease. coronaviruses are a family of envelope viruses which are hosted primarily by mammals and by birds. their general structure comprises of a single-stranded positive sense rna genome which creates four viral proteins, the s (spike), n (nucleocapsid), m (membrane) and e (envelope) proteins. each has at least one key role: m and e make up the primary protein components of the viral envelope defining its shape and having a major role in virus propagation, respectively. n is involved in stabilising the nucleocapsid binding directly to the rna viral material. the spike protein (s), is pivotal to viral infection as it is this that binds to receptors on the host cells enabling subsequent fusion between the host and viral membranes such that the interior rna material can then invade the host cell [1] . the s protein is comprised of three identical polypeptide subunits arranged as a trimer structure. it is this protein that gives the virus its name as the end of the spike has the appearance of a small "crown" in shape. in this end region is a mobile domain which moves from an inaccessible (down) state to an accessible (up) state which makes it available for interaction with angiotensin converting enzyme 2 (ace2), the transmembrane protein through which coronavirus infection usually proceeds. it is the spike protein therefore, that is critical for infectivity and in this regard it can be considered as the optimal target for vaccine and drug intervention, being the most prominent on the surface of the virion. in recent years these types of viruses have posed a major threat due to their ability to cross the species barrier, causing infection in the human population from a virus innately from another source, mammal or bird. such cross-species events have resulted in severe acute respiratory syndrome (sars) [2] and middle eastern respiratory syndrome (mers) [3] which arose from bats and then jumped to humans from civet and camel, respectively, as intermediaries [4] . such viruses that arise in this way within the human population are potentially very problematic in that we have no innate antibodies to these virions and so there is always the possibility for severe illness and death to occur as a result. a new crossspecies event has recently arisen in wuhan city in china, now termed covid-19. the source is still being debated, although it is likely this virus again originated in bats and then crossed to humans probably again using a mammalian intermediary. the spike (s) protein from the sars and mers coronaviruses have been studied in detail and a number of x-ray and cryo-electron microscopy (cryo-em) structures have been produced. gaining detailed information about these structures offers ways both of understanding how the spike protein is used to infect host cells, and of combating this infectivity. to date, two structural models of this new coronavirus spike protein have been produced, one [5] developed using c-i-tasser [6] , the other [7] produced by swiss modeller [8] using as templates homologous structures from the sars and mers spike proteins. both models were used for evaluating the possibility of its binding to ace2, and zhang et al. [5] also dispelled suggestions of the presence of novel sequence inclusions in this protein. of key importance here, is the recently reported cryo-em structure, solved to 3.5 å resolution, of the spike protein of covid-19 [9] and the coordinates for this structure are available under the protein data bank (pdb) code 6vsb; it is this structure that prompted the investigations reported here to look for sites where it might be possible to bind drugs. the structure here is interesting as one subunit chain (a) is in the "up" accessible state, while the other two (b and c) are in the "down" inaccessible state. the structure is shown in fig. 1 where the differences in conformation of the up and down states are also shown. figure 1b shows chain a in the up state, whilst fig. 1c shows the same view of fig. 1 a the structure of the spike (s) protein from covid-19 (pdb code 6vsb) showing its three subunit conformation. in red is chain a in the "up" position, and chains b and c (blue and green respectively) are in the "down" position (see text for specific details). b chain a in the up position and c chain b in the same orientation as a, in the down position. these are coloured according to the structural differences between them where red indicates the most significant of these differences, using 2struccompare [10] . structure coloured grey only exists in chain b drew and janes bmc molecular and cell biology (2020) 21:49 page 2 of 13 chain b but in the down state; the mobile domain is coloured red in both cases, while structure that is in dark grey only exists in the one chain, b in this case. the protein retains its three fold symmetry over all regions of its structure that do not interact with this mobile domain. the aims were to see if pharmaceutical products currently available and approved for human use might be able to bind to the spike protein with the chance that this might disrupt, stall, or even prevent, the infection process. in order to infect, the spike protein has to be exposed and then become accessible within the host so it can contact the receptors on the host cell. if infectivity were able to be slowed down because of disruption of the process, this would leave the spike protein still exposed but in the inaccessible state and this would enable the host to register the virion as being foreign and so antibodies would be raised against it. results from the dogsitescorer server [11] identified a total of 106 possible pockets within the cryo-em structure, with druggability scores ranging from 0.125 to 0.849 over a scoring range between 0 and 1, where 1 would be a perfect druggable site. of these 106 possible sites, 79 had a druggability score less than 0.7, and a further 7 did not have a three-fold symmetry where it should be expected, leaving 20 remaining sites. removing pockets with a small overall volume (less than 500 å 3 ) as these were considered as unlikely to be successfully druggable [11] , left 12 potential candidate pockets. analysis of these revealed an~800 å 3 pocket with a high druggability score of 0.79 which was in the 90th percentile of those identified (table 1 and fig. 2 ), and its location, between the mobile domain (in the down position) of one subunit and a second subunit, suggested it could be of potential interest regarding infectivity. comparison between the residues of covid-19 lining this pocket and the matched sars spike protein residues is given in fig. 3 . figure 4 shows the structural comparisons between chain a and b in the up and down states with selected pocket residues identified to highlight the positional changes. the results from the three methods, autodock vina [12] , smina [13] and ledock [14] , were used to generate a list of 4358 poses of commercially-available drugs capable of binding into this covid-19 spike protein pocket. none of the pharmaceutical agents that were successfully docked into the site displayed any steric hindrance within the site and all of these had favourable empirically calculated binding energies. from the extensive spreadsheet of data, obtained from the results of the autodock vina, smina and ledock methods, together with related information associated with the drugs within the list (supplied as supplementary information), analyses were undertaken to establish the presence of any correlations between, or statistical significance within these results to aid further investigation. correlations were obtained between various properties, looking for the differences/similarities between them over the whole data set of results. specifically, the chemical and geometrical properties of the top 150 conformers, as ranked by combopc score, were compared against the full dataset to see if any pattern emerged concerning the top performing compounds. to assess the significance of any difference found, a t-test was employed and a ratio of the top 150 set average versus the whole set average was obtained to assess the direction and magnitude of any difference. table 2 lists the top 100 drug poses ordered by the combo percentile score. in this table 14 drugs are actively used for treatment and one further drug is highlighted, as it might be available, which is used in the treatment of various pulmonary diseases. these 15 molecules are shown in their poses binding into the pocket in fig. 5 . six of these are shown in fig. 6 as selected examples of how these drugs interact with the lining pocket residues. the figures are using the ledock poses in both cases. our criteria for pursuing a pocket site were that the druggability score should be high, that in the parts of the protein where a three-fold symmetry should be present, the same pocket should be found in each of the subunits, that the site was of a likely suitable volume [11] , and that, if possible, the site had a high interest regarding either structure or function within the s protein. the pocket identified in this study is of particular interest as it is located at an interface between two chains, b and c, of the protein and is not present between the comparable residues in a and b or a and c. this is because chains b and c have their mobile domains in the down position while chain a has its mobile domain in the up position. the pocket is between two domains, one flanking region in chain c of the protein (between residues 1 and 290) and the other, the underside of the mobile domain in chain b (fig. 2 ). the comparison of the pocket residues of covid-19 and the matched sars spike protein residues (fig. 3) shows a very high degree of conservation between them, adding weight to the suggestion they might be important regarding the function of the protein. the domain in chain b is the region between residues around 335 and 526 in the covid-19 spike protein structure, but in chain a, this same domain region has hinged away losing all contacts between residues 328 and 530 (in beta strands n-and c-terminal to the domain, respectively) as this domain is in the "up" position. in related spike proteins like that from human sars-cov (pdb code: 6ack), when this domain is in the up position it interacts with ace2 facilitating entry to the cell [16] . the movement of this domain, therefore, appears to be required for ace2 interaction, as in the sars-cov structure an ace2 is present and bound to the spike protein. critically, in the covid-19 cryo-em spike protein structure, when in the up position (and when no ace2 protein is present) then residues 460 to 473 in chain a are unobserved in the structure; they are too flexible to be detectable (grey coloured structure in fig. 4b ), whereas the equivalent residues are present in the sars-cov structure with the ace2 bound. this implies they are flexible when there is no contact with the ace2 protein, only being stabilised in this up position once contact has been established. however, residues 464 to 466 are structured and visible in the covid-19 cryo-em b chain and form part of the side of the druggable pocket (table 1 and fig. 4 ) because this chain is in the down position. if the inter-chain interactions within and around this pocket formed between the two domains could be stabilised by the binding in of an appropriate drug molecule, it might prevent the domain from moving to the up position and this could be crucial in preventing or hindering the infectivity of the virus. the location of this pocket near such a functionally important domain, and its high predicted druggability score were the reasons behind this site being chosen for the virtual screening studies. the averaged pairwise root mean square deviation (avrmsd) calculations between each of the docking methods were used as a guide to the quality of each drug pose being in a well-established position within the druggable pocket. a small rmsd value indicated that each method had placed the specific drug pose into a similar position within the pocket. in vina for example, due to the non-deterministic nature of the docking algorithm [12] , both it and smina [13] use a random seed approach to initiate the search process. the results of the poses from all three methods would ideally produce "comparable" but not "identical" results. for all three methods the poses obtained retain information of rotamer space, and overall structural space, which can be employed to find good candidates for fitting the pocket, but from slightly different start positions. so it would be expected and anticipated that, for a given drug molecule, good poses would be comparable, but not the same, from each of the methods used, which would be reflected in small and similar rmsd pairwise values being obtained. however, given that the scoring of the two methods, vina and smina, was "expected" to be different, but in fact proved to be highly correlated in their results, we removed the smina scoring from the overall combopc equation as we did not want to bias in favour of these two methods over all three. for the top 150 poses ranked by combopc the number of hydrogen bond acceptors was found to be higher than expected (1.16, p < 0.001). the number of rings/heterocycles per compound was also significantly higher (1.29, p < 0.001). compounds were also much more likely to contain halogen atoms (1.74, p < 0.001), particularly fluorine atoms (2.16, p < 0.001). compounds with so 2 groups were also overrepresented (2.97, p < 0.001), as exemplified by ladarixin, diflumidone and quinethazone. moieties of this kind are capable of many electrostatic interactions which need not be associated only with hydrogen-bonding. given the statistical significance associated with the data above it appears that the properties of the pocket, its shape and lining residue composition, have generated a focused list of drugs with notable component properties of their own. in the top 100 drug poses, of interest there are 20 antiinflammatory drugs of which 8 are members of the "profen" family, derivatives containing the 2-arylpropionic acid moiety. many of the highlighted drugs in table 2 could be capable of successfully binding into this pocket and hence alter the infectivity profile. clearly, these would need to be examined experimentally because at such a sensitive site in the spike protein there is a possibility of promoting the mobile domain into the up, infective conformation. however, with the fact that these drugs interact with the residues lining this pocket they might strengthen the interaction forces in this region and prevent the mobile domain from moving. this study provides a suggested list of pharmaceutical agents, identifying some of them as being from related structure families, that are available on the market and have been sanctioned for use in humans that have been shown to be capable of binding into a druggable pocket in the spike protein of covid-19. it must be stated that whether they do or do not actually bind in cannot be ratified here, that would have to be determined experimentally. some might even bind into the site and increase infectivity as a result. however, the aims of this study were to present and show that the members of this list might have the capacity to bind in, thereby providing open suggestions to experimentalists to establish whether many, some, or few, of these agents do actually bind into the spike protein. equally, it is not possible to state whether these drugs would be in any way efficacious towards suppressing the infectivity of covid-19 because that is not the remit of this work. again, that would be for those in the field to establish whether the listed drugs show any effect on reducing virus titre levels, thereby indicating that they are indeed interfering with the infectivity of the virion. the primary aim has been to show that pharmaceutical agents already available and approved might be usable as drugs to interfere with the process of infection, thereby providing time for the host generation of antibodies to combat this latest of cross-species coronavirus events. we utilised the cryo-em spike protein structure (pdb code: 6vsb), as a source for our studies to locate the presence of "druggable" pockets where small molecules fig. 4 images showing the differences between paired c-alpha residues from chain a and chain b of the spike protein coloured according to their extent of difference using 2struccompare [10] . residues coloured in red indicate differences in paired positions greater than 5 å from each other between the two chains. a. shows the shape and position of chain a in the "up" position. b. shows the same orientation for chain b in the "down" position. some residues are labelled to indicate approximately where the pocket residues are in the two positions of these chains, and in b three of these residues lie on the strand coloured dark grey indicating that this is not seen in chain a drew and janes bmc molecular and cell biology (2020) 21:49 page 6 of 13 could bind. the protein atomic coordinates were uploaded to the dogsitescorer server [11] to identify potential pockets within the structure. the server provides a druggability score that ranges from 0 to 1, 1 being the most druggable. three approaches to docking the pharmaceutical agents into the identified druggable pocket in the covid-19 spike protein were employed to act to corroborate the output produced. these were autodock vina [12] , smina [13] and ledock [14] . the centre position of the pocket was calculated and a search grid space, defined by those coordinates ±12 å in the x, y and z coordinate directions from that centre, was created and supplied to each of the docking methods. by default in each of the packages used, the drugs were free to explore their rotamer space to optimise their binding into the pocket. however, whilst it is customary to allow side chain flexibility in the residues lining the pocket when undertaking in silico docking studies, given that the resolution of the structure used here was only 3.5 å, no flexible side chains were defined. the number of poses considered by all three methods used was left as default because this was thought to be optimal for this study. for ledock, a maximum of 20 docking poses was returned per conformer, with an additional pose root mean square deviation (rmsd) cutoff of 1.0 å applied to reduce redundancy of returned poses. for autodock vina and smina, a maximum of 9 poses with a maximum energy range of 3 kcal/mol between best and worst were returned. a 1049-member library of compounds was derived from the drugs-lib dataset available from the virtual screening webserver mtiopenscreen [17] from an initial study we undertook on the s protein model from i-tasser. this dataset consists of approved drugs and research chemicals refined from the "drug" subset of the chembl database [18] , the "approved" subset of drug-bank version 5.0.10 [19] , the drugcentral online compendium [20] and the "approved" superdrug2 database version 2.0 [21] . initially autodock vina and ledock were the methods of choice as these have been considered as the best non _commercial docking packages available [22] . however, after pairing the poses between the two methods according to their structural similarity when in the docking site (as in the rmsd calculations below) the corresponding docking scores showed only a weak correlation (0.36, p < 0.0001)) and so smina, which is reported as having a different scoring scheme from autodock vina [13] , was used to see if this improved the correlation of the docking scoring to ledock. in fact, the scoring correlation between smina and autodock vina proved to be very high and so only autodock vina and ledock data were used in the docking scoring as a result. for a given drug, poses were obtained that fit the pocket. to establish the best superposition of these different poses from each method, firstly, pairwise rmsds were calculated using the following equation: where x i and y i are comparable atoms from two of the methods, and n is the number of atoms in the given drug. the best overall superposition from the three methods was then calculated by taking the average of these pairwise rmsd superpositions. from these calculations, the poses from each of the methods with the best overall superposition, and hence, highest degree of similarity, would have the lowest average rmsd values (avrmsd). an extensive amount of data was generated tabulating the output information from the docking packages of the druggable compounds with their structural characteristics. in generalised overview these correspond to the docking output scores from each of the methods, avrmsd values between the poses chosen for each compound, hydrogen bonding data, and the joelib native descriptor set, calculated using the chemmine webserver [23] , which include molar refractivity, polar surface area, and frequencies of atom and selected group types among other geometric and chemical properties. drugs within the search group were also classified by chemical compound class and, where possible, by subclass through classification using the classyfire webserver [24] . a compound with a pose that scored highly in the two scoring regimes, and with good superposition agreement between docking methods, as determined from their avrmsd values, was considered more likely to be a meaningful data point. as these individual terms were on different scales, their significant feature was their explicit "order" rather than "value". as a result each component, scoring value, and avrmsd, was ranked according to their percentile position within their individual scales. this therefore ordered the results by position rather than by value. thus, the following combined score, combopc, was created to rank the compounds applying equal weight to the estimated docking scores from each docking method and to the agreement between the superpositions from each of the methods, as measured by avrmsd: where ledockpc is the percentile rank of the docking score of the pose as docked by ledock, vinapc is the coronavirus envelope protein: current knowledge molecular epidemiology, evolution and phylogeny of sars coronavirus a comparative analysis of factors influencing two outbreaks of middle eastern respiratory syndrome (mers) in saudi arabia and south korea genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan protein structure and sequence re-analysis of 2019-ncov genome does not indicate snakes as its intermediate host or the unique similarity between its spike protein insertions and hiv-1 i-tasser server: new development for protein structure and function predictions evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission swiss-model: homology modelling of protein structures and complexes cryo-em structure of the 2019-ncov spike in the prefusion conformation 2struccompare: a webserver for visualizing small but noteworthy differences between protein tertiary structures through interrogation of the secondary structure content combining global and local measures for structure-based druggability predictions autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading lessons learned in empirical scoring with smina from the csar 2011 benchmarking exercise enriching screening libraries with bioactive fragment space ligplot+: multiple ligand-protein interaction diagrams for drug discovery cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 mtiopenscreen: a web server for structure-based virtual screening chembl: a largescale bioactivity database for drug discovery drugbank 5. 0: a major update to the drugbank database for 2018 drugcentral: online drug compendium superdrug2: a one stop resource for approved/ marketed drugs comprehensive evaluation of ten docking programs on a diverse set of protein-ligand complexes: the prediction accuracy of sampling power and scoring power chemmine tools: an online service for analyzing and clustering small molecules classyfire: automated chemical classification with a comprehensive, computable taxonomy we thank prof. b.a. wallace for her discussions throughout the preparation of this manuscript.authors' contributions rwj instigated this study and both edd and rwj carried out this work. both authors have read and approved the manuscript. ethics approval and consent to participate not applicable. the authors give consent to publication. none to declare. percentile rank of the docking score of the pose as docked by vina, and rmsdpc is the percentile rank of the avrmsd between the poses from each docking method. only the scores from ledock and vina, but not smina, were chosen for inclusion in the combopc score as the scores from vina and smina were found to be highly correlated (0.97, p < 0.001)). supplementary information accompanies this paper at https://doi.org/10. 1186/s12860-020-00294-x.additional file 1. supplementary_data_drug_scores.xlsx -the spreadsheet of data used and analysed in this paper.additional file 2. binding_pocket_residues.pdb -the binding pocket residues found in this study from the covid-19 spike protein structure (pdb code: 6vsb).additional file 3. ledock_top100.mol2 -the top 100 ledock poses listed in table 2 .additional file 4. smina_top100.mol2 -the top 100 smina poses listed in table 2 .additional file 5. vina_top100.mol2 -the top 100 autodock vina poses listed in table 2 .additional file 6. readme.txt -a readme.txt file to provide information to assist in using the structure data provided in additional files 2-5. key: cord-319658-u0wjgw50 authors: guven-maiorov, emine; tsai, chung-jung; nussinov, ruth title: structural host-microbiota interaction networks date: 2017-10-12 journal: plos comput biol doi: 10.1371/journal.pcbi.1005579 sha: doc_id: 319658 cord_uid: u0wjgw50 hundreds of different species colonize multicellular organisms making them “metaorganisms”. a growing body of data supports the role of microbiota in health and in disease. grasping the principles of host-microbiota interactions (hmis) at the molecular level is important since it may provide insights into the mechanisms of infections. the crosstalk between the host and the microbiota may help resolve puzzling questions such as how a microorganism can contribute to both health and disease. integrated superorganism networks that consider host and microbiota as a whole–may uncover their code, clarifying perhaps the most fundamental question: how they modulate immune surveillance. within this framework, structural hmi networks can uniquely identify potential microbial effectors that target distinct host nodes or interfere with endogenous host interactions, as well as how mutations on either host or microbial proteins affect the interaction. furthermore, structural hmis can help identify master host cell regulator nodes and modules whose tweaking by the microbes promote aberrant activity. collectively, these data can delineate pathogenic mechanisms and thereby help maximize beneficial therapeutics. to date, challenges in experimental techniques limit large-scale characterization of hmis. here we highlight an area in its infancy which we believe will increasingly engage the computational community: predicting interactions across kingdoms, and mapping these on the host cellular networks to figure out how commensal and pathogenic microbiota modulate the host signaling and broadly cross-species consequences. rather than existing as independent organisms, multi-cellular hosts together with their inhabiting microbial cells have been viewed as "metaorganisms" (also termed superorganisms or holobionts) [1] . millions of commensals, symbiotic, and pathogenic microorganisms colonize our body. together, they comprise the "microbiota". microbiota are indispensable for the host, as they contribute to the functioning of essential physiological processes including immunity and metabolism. hosts co-evolved with the microbiota. while some commensals are beneficial (symbionts), others may become harmful (pathobionts) [2, 3] . microbiota immune system development. the immune system recognizes antigens of microorganisms e.g. dna, rna, cell wall components, and many others, through pattern recognition receptors, such as toll-like receptors (tlrs) and downstream intracellular signaling circuitries are activated to generate immune responses [4] . however, like self-antigens, antigens from commensal microbiota are tolerated with no consequent inflammatory responses. this makes gut microbiota accepted as "extended-self" [5] . still, under some circumstances, commensals may act as pathogens. for example, staphylococcus aureus [6] or candida albicans [7] are commensals of human, but in "susceptible" hosts, they can undergo commensal-to-pathogen transition. thus, identifying microorganisms that reside in the host, and within these, those that are responsible for distinct host phenotypes, and the host pathways through which they act are significant goals in host-microbiota research. microbiota survival strategies within the host are likely to be limited. analysis of their repertoire may reveal core modules, thereby helping in classification, mechanistic elucidation and profile prediction. here we provide an overview of structural host-microbiota interaction networks from this standpoint. the host interacts with microbiota through proteins, metabolites, small molecules and nucleic acids [8, 9] . the microbiota employs a range of effectors to modulate host cellular functions and immune responses. they have sophisticated relationships with the host, and network representation enables an effective visualization of these relationships [10] . most proteins of bacterial and eukaryotic pathogens are not accessible to bind to host proteins; but some of their proteins either bind to host surface receptors [11] or enter the host cell and interact with host cytoplasmic proteins. various bacterial species have a secretion system-a syringe-like apparatus-through which they inject the bacterial effectors directly into the host cell cytoplasm [12] . via hmis, they specifically hone in on key pathways, alter host physiological signaling, evade the host immune system, modify the cytoskeletal organization [13, 14] , alter membrane and vesicular trafficking [2, 11, 13] , promote pathogen entry into the host, shift the cell cycle [15, 16] , and modulate apoptosis [17] . all are aimed to ensure their survival and replication within the host. host signaling pathways that are targeted by microbiota and turned on or off may change the cell fate. unraveling the hmis for both commensals and pathogens can elucidate how they repurpose the host signaling pathways and help develop new therapeutic approaches. hmis have complex and dynamic profiles. studies often focus on individual protein interactions and try to explain the pathogenicity of a microorganism with a single interaction. however, considering host-microbiota interactions one-at-a-time may not reflect the virulence scheme [18] . for instance, replication of vaccinia virus necessitates the establishment of a complex protein interaction network [19] and hence focusing on only one hmi is incomplete and may be misleading. at any given time, hundreds of different species reside in the gut. different microbial compositions and hence effector protein combinations from these microbial species may have additive (cross-activation) or subtractive (cross-inhibition) [4] impacts on the host pathways, which lead to signal amplification or inhibition, respectively (fig 1) . since numerous bacteria will be sensed by the host immune system at any given time, more than one signaling cascade will be active in a cell. communication and crosstalk among active, or active and inhibited, pathways determine the ultimate cellular outcome [4] : to survive, die, or elicit immune responses. the combinatorial ramifications of all active (or suppressed) host pathways and hmis will be integrated to shape the type and magnitude of the response, and thus the cell state. to tackle the pathogenicity challenge, it is reasonable to concomitantly consider all host pathways and hmis. the transkingdom (metaorganism) network analysis is a robust research framework that considers host and microbiota as a whole [1] . systems biology approaches that integrate the hmis with host endogenous protein interaction networks reveal the systematic trends in virulence strategies of pathogens. here we ask how interspecies (superorganism) networks can facilitate the understanding of the role of microbiota in disease and health. we focus on host-microbiota protein interaction networks since many bacteria or virus-induced pathological processes require physical interactions of host and microbial proteins [20] . the availability of genome-wide high throughput omics data makes it possible to associate microbiota with certain host phenotypes at multiple levels and construct host-pathogen interaction networks at the transcriptome [21], proteome combinatorial effects of microbial effectors and the active host pathways determine the cell response. (a) composition1 has certain microorganisms that secrete effector protein combination1. these effectors activate pathway1 in the host, which produces pro-inflammatory cytokines. (b) composition2 secretes effector combination2 and activates pathway2 in addition to pathway1. additive effects of these two pathways amplifies the signal and promotes inflammation (cross-activation). (c) microbial composition3 utilize effector combination3 to activate both pathway 1 and 3, which have opposing outcomes. subtractive effects of these pathways result in no inflammation (cross-inhibition). https://doi.org/10.1371/journal.pcbi.1005579.g001 [22], and metabolome levels [23] . steps toward the construction of host-microbiota networks of gene [1] , mrna [24], protein-protein interaction (ppi) [25] [26] [27] [28] , and metabolic networks [29] have already been taken. within this framework we highlight molecular mimicry, a common strategy that microorganisms exploit to bind to host proteins and perturb its physiological signaling. mimicry of interactions of critical regulatory nodes in core network modules in the immune system, may be a major way through which pathogens adversely subvert-and commensal microbiota may beneficially modulate-the host cell. microbiota developed several strategies to interact with host proteins and modulate its pathways. one efficient way is molecular mimicry, which has been extensively reviewed in our recent study [9] . molecular mimicry can take place at four levels: mimicking (i) both sequence and 3d structure of a protein, (ii) only structure without sequence similarity, (iii) sequence of a short motif-motif mimicry, and (iv) structure of a binding surface without sequence similarity-interface mimicry. interface mimicry (protein binding surface similarity) seems to be the most common type of molecular mimicry. global structural similarity is much rarer than interface similarity both within and across species. thus, employing interface mimicry instead of full-length sequence or structural homology allows microbes to target more host proteins. molecular mimicry follows the principle suggested over two decades ago that proteins with different global structures can interact in similar ways [30] [31] [32] . interface mimicry is frequently observed within intra-[33-35] and inter-species [18, 36] (fig 2) (intra-species interface mimicry: distinct proteins from the same species having the same/similar interfaces; inter-species interface mimicry: proteins from different species hijack the same interface architectures). interface similarity allows proteins to compete to bind to a shared target. if an interface is formed between proteins from the same species, it is an 'endogenous interface'. if it is formed by proteins from two different species, it is an 'exogenous interface' [18, 36] . endogenous (intra-species) interfaces mimic each other [33] [34] [35] , and exogenous (inter-species) interfaces mimic endogenous interfaces (fig 2) [18, 36]. by mimicking endogenous interfaces, exogenous interfaces enable pathogenic proteins to compete with their host counterparts and hence rewire host signaling pathways for their own advantage [9] . they can either inhibit or activate a host pathway. for example, the helicobacter pylori secreted protein caga interacts with human tumor suppressor tp53bp2, inhibits apoptosis and allows survival of infected host cells [37] . however, map protein of e. coli and sope protein of salmonella bacteria bind and activate human cdc42, a rho gtpase, and trigger actin reorganization in the host cell, facilitating bacterial entry into the host [38]. one of the most significant pattern recognition receptor families in the innate immune system is the tlr family. its members detect diverse bacterial compounds, like peptidoglycan, lipopolysaccharide, and nucleic acids of bacteria and viruses. they induce pro-inflammatory or anti-viral responses. once activated, they recruit other tir-containing proteins such as mal and myd88 or tram and trif through their cytoplasmic tir domains, forming the myd88-and trif-dependent tir domain signalosomes, respectively [39]. myd88 also assembles into a myddosome structure through its death domain together with irak4 and irak1/2 death domains. the myddosome then recruits e3 ubiquitin ligases-either traf6 or traf3 -to catalyze the addition of k63-linked ubiquitin chains to themselves, which serve as a docking platform for other proteins to bind, such as tak1. subsequently, nf-κb and mapk pathways are activated. in the nf-κb pathway, tak1 phosphorylates and activates ikk. activated ikk in turn phosphorylates iκb, which is the inhibitor of nf-κb. phosphorylated iκb is then ubiquitylated by other e3 ubiquitin ligases (k48-linked ubiquitin chain) and targeted for proteosomal degradation. this liberates the p65 subunit of nf-κb to translocate to nucleus and initiate transcription. in the mapk pathway, tak1 serves as a map3k that activates erk1/2, p38 and jnk pathways. the trif-dependent downstream path of tlrs recruits traf3 and leads to activation of interferon regulatory factors (irfs) and production of key antiviral cytokines, interferons (ifns). the tlr pathway is regulated by several endogenous negative regulators to prevent excess inflammation [40] . since this is one of the major immune pathways, its signaling is targeted by diverse microorganisms at various steps (fig 3) , compete with endogenous tir-containing proteins and interfere with the assembly of the tir-domain signalosome and prevent downstream signaling. since these microbial proteins do not enzymatically modify the endogenous proteins, elucidation of their inhibition mechanism requires structural information. the availability of the structures of their complexes with the orchestrators of the tlr pathway can clarify how they inhibit downstream signaling. microbial proteases prevent both tlr-induced mapk and nf-κb signaling and lead to proteosomal degradation of the key orchestrators in these pathways: nled of e. coli cleaves jnk and p38, inhibiting mapk pathway; and nlec cleaves p65, inhibiting nf-κb [46] . there are also bacterial acetyltransferases ( [57, 58] inhibit this protein to limit ifn production [59] . here, we listed only a couple of microbial proteins targeting tlr pathway as examples. there are many others. the tlr pathway does not constitute the whole innate immune system; other immune pathways also need to be considered as well as how these microbial proteins affect them as a whole. this can help foreseeing what kind of responses the coordinated actions of these pathways together with tlrs would generate. most cellular processes are elicited by proteins and their interactions. graph representations of ppi networks, where proteins are the nodes and their interactions are edges, are helpful for delineating the global behavior of the network. topological features of networks, such as degree (number of edges), betweenness-centrality (how a node affects the communication between two nodes), lethality-centrality, hubs (proteins with high node-degree, i.e. several a, b, c, d are host proteins and p is pathogenic protein. protein a has two interfaces: through blue interface it binds to b and through grey interface it binds to c and d. c and d proteins employ similar interfaces to bind to a. so, endogenous interfaces mimic each other. pathogenic protein p has similar interface as b and competes to bind to the blue interface on a. in this case, an exogenous interface mimics an endogenous interface. (b) the f1l protein of variola virus interacts with human bid protein (5ajj:ab.pdb) and inhibits apoptosis in the host cell by hijacking the interface between human bid-bclxl (4qve:ab.pdb): an exogenous interface mimicking an endogenous one. human mcl1 protein binds to human bid (5c3f:ab.pdb) in a very similar fashion that bclxl does: endogenous interfaces mimicking each other. https://doi.org/10.1371/journal.pcbi.1005579.g002 interaction partners), non-hubs (with only a few partners), and bottlenecks (nodes with high betweenness-centrality) help characterization of the importance of the nodes, i.e. the contribution of the node to network integrity [60, 61] . early on, hubs were classified as either party or date hubs. while party hubs interact with many partners at the same time since they use distinct interfaces, date hubs interact with their partners one at a time due to their overlapping interfaces. to infer whether a hub is party or date hub, structural information (interface residues) [62] or gene expression data (co-expressed proteins have higher chances of interacting with each other) [63] were used. later on, this definition was questioned. among the reasons were the many examples where a protein node can serve concomitantly as a party and date hub. large assemblies typically fall into this category. biological networks are often scale-free, with many non-hubs and fewer hubs [64, 65] . not all nodes have the same effect on the network: random node attacks do not harm the network as much as removing hubs from scale-free networks [66] . degree and betweenness-centrality are measures of the contribution of nodes to network integrity. there are also "essential" nodes, knock-out of which leads to lethality: a feature also known as "lethality-centrality". attack of a hub by microbiota is likely to influence the cell, either resulting in lethality, or in beneficial modulation. thus, integrated superorganism interaction networks may suggest candidate host and microbial node targets. structural interspecies networks and their topological features can shed light on how microbiota alter the host signaling and what will the outcome in different settings be. available hmi networks demonstrate that different bacteria often hijack the same host pathway in distinct ways [12] , like the tlr pathway subversion by numerous microbial species (fig 3) . however, importantly, the same host pathway is often targeted at several nodes, which was suggested to guarantee modulation of cellular function [12] . although there are a number of examples of constructed networks of host-pathogen superorganism interactions [12, 19, [67] [68] [69] [70] [71] [72] [73] [74] [75] , there are many fewer attempts of integrating 3d structural data with the hmi networks [18] . traditional network representation has low resolution, missing important details. however, structural interaction networks provide a higher resolution with mechanistic insights. they can decipher and resolve those that are not obvious in binary interaction networks [36] . the potential of structural networks in unraveling signaling pathways was demonstrated earlier [39, 40, 76, 77] . they are essential to fully grasp the mechanisms exerted by pathogens to divert the host cell signaling and attenuate immune responses. fig 4 displays an example of a structural hmi network, showing how host ppis can be affected by hmis. structures can detail which endogenous host ppis are disrupted by the hmis, possible consequences of mutations on either host proteins or pathogenic proteins, and whether variants of a virulence factor in different strains of the same species have distinct hmis. for instance, the pro-35 residue on hiv accessory protein vpr is at the interface with human cypa and its mutation to alanine abrogates the interaction [78] . the structure of the cypa-vpr complex shows that pro-35 is at the interface. if the structure of the vpr-cypa complex was unknown, it would have been difficult to understand why, or how, this mutation disrupts the ppi. previously built structural hmi networks demonstrated that endogenous interfaces that are hijacked by pathogens are involved in multiple transient interactions [18, 36] . these endogenous interfaces exhibit 'date-like' features, i.e. they are involved in interactions with several endogenous proteins at different times [18, 36] . hub and bottleneck proteins at the crossroads of several host pathways were suggested to be the major targets of viral and bacterial proteins [26, 28] and interface mimics allow transient interactions with the hub [79] . this allows them to interfere with multiple endogenous ppis. it was proposed that microorganisms causing acute infections, which are dramatic for the host, are likely to interfere with the hubs, whereas others that lead to persistent infections tend to target non-hubs [80] . during acute infection, pathogens replicate very quickly and are transmitted to new hosts. however, during chronic infections, they adapt to the host environment, which allows them to reside there for a long period of time. thus, how microbiota target certain proteins and pathways at the molecular level is of paramount importance. detecting the hmis, mapping them onto networks and determining their 3d structures as a complex are the major steps to construct structural hmi networks. despite the progress in experimental techniques, it is still challenging to determine structures of ppi complexes, particularly hmis. since large-scale experimental characterization of host-pathogen ppis is difficult, time consuming, and costly, experimentally verified hmi data is scarce. it is important to note that available endogenous protein structures are biased towards permanent, rather than transient interactions. if majority of the hmis are transient, this presents another hurdle since they will be under-represented in the structural space. several hmi databases have been developed, such as phisto [81] , hpidb [82] , proteopathogen [83] , patric [84] , phi-base [85] , phidias [86] , hopaci-db [87] , virhostnet [88] , virbase [89] , virusmentha [90] , hcvpro [91] , and likely some others as well. however, these databases cover only a limited number of pathogens and their interactions. given that thousands of species residing in the host, thousands of hmis are yet to be identified. computational approaches are becoming increasingly important in prioritizing putative hmis and complementing experiments. hence, construction of comprehensive metaorganism networks and increasing the coverage of the host-microbiota interactome will still mostly rely on computational models in the near future [92] . computational modeling of intra-species interactions is a well-established area; detection of inter-species interactions is relatively new. available computational tools to predict host-pathogen interactions have been recently reviewed by nourani et al. [93] . current methods mostly depend on global sequence and structure homology. sequence-based methods focus only on orthologs of host proteins. however, sequence by itself is insufficient to detect the targets of pathogenic proteins because several virulence factors do not have any sequence homologs in human. for instance, the vaca protein of helicobacter pylori, the most dominant species in gastric microbiota, has a unique sequence that does not resemble any human protein [94] . still, it alters several host pathways [95] . with sequence-based methods, it is impossible to find hmis for vaca. as noted above, global structural mimicry is much rarer than interface mimicry. hence, utilizing interface similarity, rather than global structural similarity in a computational approach would generate a more enriched set of hmi data together with atomic details [9] . several studies suggested that the available interface structures are diverse enough to cover most human ppis [96] [97] [98] [99] . therefore, success of template-based methods for prediction of human ppis is very high [34] . since exogenous interfaces mimic endogenous ones, both available endogenous and exogenous interface structures can be used as templates to detect novel hmis. thanks to the rapid increase in the number of resolved 3d structures of human-pathogen ppis in recent years [100] and advances in structural and computational biology, the performance of interface-based methods is expected to increase. both experimental and computational approaches have false-positives and false-negatives with varying rates depending on the approach. although the coverage of interface-based methods is higher, their false-positive rate is also higher. despite this, attempts to complete the host-microbiota interactome will improve our knowledge of microbiota and their roles in health and disease. advances in host-microbiota research will revolutionize the understanding of the connection between health and a broad range of diseases. building the rewired host-microbiota multiorganism interaction network, along with its structural details, is vital for figuring out the molecular mechanisms underlying host immune modulation by microbiota. topological features of such networks can reveal the selection of host targets by the microbiota. structural details are essential to fully grasp the mechanisms exerted by microbiota to subvert the host immunity. identification of the hmis will also help drug discovery and integrated superorganism networks would suggest how inhibition of an hmi can influence the whole system. here we highlighted the importance of building structural hmi networks. however, not only hmis are important; although to date data are scant, crosstalk among microorganisms is also emerging as critical. alterations in their population dynamics may lead to dysbiosis. signals from gut microbiota resulting from population shifts can affect profoundly several tissues, including the central nervous system. dysbiosis of microbiota is involved in several diseases, such as inflammatory bowel disease [101] , autoimmune diseases (e.g. multiple sclerosis) [102] , neurodegenerative diseases (e.g. parkinson's) [103] , and cancer [104, 105] . identifying bacterial effectors, or effector combinations, which are responsible for specific phenotypes, is challenging. in line with this, recently, parkinson's disease (pd) patients are found to have altered gut microbiota composition [106, 107] . transplanted microbiota from pd patients, but not from healthy controls, induce motor dysfunction and trigger pd in mice. it is not clear however whether dysbiosis triggers pd or it arises as a consequence of the disease [103] . the role of microbiota in host health and disease might be even more complex than thought: commensals once being benign can convert to disease-causing pathogens; different compositions of microbial communities trigger different phenotypes; more than one host pathway is targeted by more than one effector; the same microbial effector/antigen is sensed by several pattern recognition receptors (back-up mechanism, compensatory microbial sensing [4] ) and genetic variation in hosts results in different responses (i.e. some commensals transition to pathogen only in "susceptible" individuals). current knowledge on microbiota and their interactions with the host is still in its infancy, but given the advances that are accomplished so far and the attention this field started to attract these days, it is likely that many unknowns and questions will be uncovered soon. investigating a holobiont: microbiota perturbations and transkingdom networks cellular hijacking: a common strategy for microbial infection diet, microbiota and autoimmune diseases integration of innate immune signaling self or non-self? the multifaceted role of the microbiota in immune-mediated diseases differential expression and roles of staphylococcus aureus virulence determinants during colonization 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helicobacter pylori caga and vaca modulate host pathways that impact disease the helicobacter pylori's protein vaca has direct effects on the regulation of cell cycle and apoptosis in gastric epithelial cells structure-based prediction of protein-protein interactions on a genome-wide scale proceedings of the national academy of sciences of the united states of america structural space of protein-protein interfaces is degenerate, close to complete, and highly connected templates are available to model nearly all complexes of structurally characterized proteins structural models for host-pathogen protein-protein interactions: assessing coverage and bias the microbiome in inflammatory bowel disease: current status and the future ahead alterations of the human gut microbiome in multiple sclerosis gut microbiota regulate motor deficits and neuroinflammation in a model of parkinson's disease commensal bacteria control cancer response to therapy by modulating the tumor microenvironment gastrointestinal cancers: influence of gut microbiota, probiotics and prebiotics colonic bacterial composition in parkinson's disease gut microbiota are related to parkinson's disease and clinical phenotype key: cord-319614-4qi59pbz authors: benej, martin; danchenko, maksym; oveckova, ingrid; cervenak, filip; tomaska, lubomir; grossmannova, katarina; polcicova, katarina; golias, tereza; tomaskova, jana title: quantitative proteomics reveal peroxiredoxin perturbation upon persistent lymphocytic choriomeningitis virus infection in human cells date: 2019-10-25 journal: front microbiol doi: 10.3389/fmicb.2019.02438 sha: doc_id: 319614 cord_uid: 4qi59pbz experimental data indicate that during persistent infection, lymphocytic choriomeningitis virus (lcmv) may both directly or indirectly modulate regulatory cellular processes and alter cellular functions that are not critical for survival, but are essential for cell homeostasis. in order to shed more light on these processes, two-dimensional differential in-gel electrophoresis (2d-dige) and maldi-tof tandem mass spectrometry were used to determine the proteome response of the hela cell line to persistent lcmv infection. quantitative analysis revealed 24 differentially abundant proteins. functional analysis showed that lcmv-responsive proteins were primarily involved in metabolism, stress, and the defense response. among identified proteins, we discovered significant changes for peroxiredoxins, a family of antioxidant enzymes. decreased amount of these antioxidant proteins correlated with elevation of reactive oxygen species (ros) in infected cells. increased levels of ros were accompanied by changes in the pattern of telomere restriction fragments (trfs) in infected cells and mediated activation of hypoxia-inducible transcription factor-1 (hif-1) and phosphatidylinositol 3-kinase (pi3k)/akt signaling pathways. moreover, treatment with antioxidants resulted in reduced levels of viral nucleoprotein, indicating a connection between ros-dependent signaling and viral replication. persistent viral infections are currently one of the most important global health problems in the world. understanding how viral persistence is initiated and maintained, and the pathological consequences of ongoing virus replication, is therefore of high importance. the prototypic mammarenavirus, lymphocytic choriomeningitis virus (lcmv), provides a suitable model system for investigation of the mechanisms of persistent viral infection, virushost interactions, and pathogenesis. studies using this viral model have resulted in significant progress in virology and immunology that is applicable to other human microbial and viral infections (oldstone, 2002; zinkernagel, 2002) . in addition, lcmv is a neglected human pathogen of clinical significance that may lead to severe disease and consequences after prenatal infection (barton and mets, 1999; mets et al., 2000; barton et al., 2002) , and life-threatening conditions in immunosuppressed individuals (fischer et al., 2006; palacios et al., 2008; macneil et al., 2011) . moreover, several mammarenaviruses, such as the lassa, junin, and machupo viruses, are associated with severe hemorrhagic fever disease with significant morbidity and mortality in humans, representing serious public health concerns in their endemic regions (geisbert and jahrling, 2004; buchmeier et al., 2007) . currently, supportive care and ribavirin (nucleoside analogue) are the only treatment options available (damonte and coto, 2002; parker, 2005) . therefore, considerable interest focuses on the development of therapeutic strategies against mammarenavirus infection and disease. understanding how viruses and the hosts interact and how their interactions change over time would help in the rational design of targeting compounds. mammarenaviruses are enveloped viruses with a bisegmented negative-strand rna genome and a non-lytic life cycle restricted to the cell cytoplasm. the s segment of the genome encodes the nucleoprotein (np) that encapsidates viral rna and the glycoprotein precursor (gpc). after post-translational modification, gpc yields gp1 and gp2 that mediate virus entry. the l segment encodes the viral rna-dependent rna polymerase (l) responsible for viral rna synthesis, and the zincbinding protein (z) important for viral budding (buchmeier et al., 2007) . mammarenaviruses are able to establish persistent infection in their natural hosts and in a number of mammalian cells in vitro. during persistence, mammarenaviruses continue to replicate and express viral proteins. at the same time, they interfere with normal host homeostasis, resulting in possible disease without the destruction of the infected cell. additionally, they actively suppress the host's immune response in order to avoid recognition and to allow the spread of infection (oldstone, 2006) . although research on lcmv has led to many insights into viral persistence, the answers to several crucial questions related to the principles, by which persistence is initiated and maintained, are still lacking. a highly suitable tool to study virus-host cell interactions is proteomics, since viral infection fundamentally affects host cell proteins. it does so at many functional levels, such as affecting the cell signaling pathways, cytokine and growth factor production, apoptosis, phagocytosis, protein degradation, and cytoskeletal rearrangement (coiras et al., 2008) . so far, several large-scale analyses of host proteome (bowick et al., 2006 (bowick et al., , 2009 (bowick et al., , 2010 , kinome (bowick et al., 2007) , and transcriptome (djavani et al., 2007 (djavani et al., , 2009 muller et al., 2007) have been performed to study the consequences of an acute mammarenavirus infection. moreover, a number of studies employed the proteomic approach to identify interacting partners of mammarenavirus proteins in human cells (khamina et al., 2017; king et al., 2017; iwasaki et al., 2018; loureiro et al., 2018; ziegler et al., 2018) . on the other hand, none of them has focused on the host cellular response during persistent infection. herein, we analyzed proteomic changes in a human cell line hela upon persistent infection with the mx isolate of lcmv using quantitative gel-based proteomics. our data reveal significant changes in the proteins associated with metabolism, antioxidant activity, protein folding, rna binding, and immune response. many of the differentially abundant proteins have not been reported previously as virus-responsive upon lcmv infection. thus, these proteins represent potential targets for further in-depth investigations. understanding their roles in persistent lcmv infection would reveal more about the complexity and dynamics of interactions between virus and host cells, and would benefit antiviral research. this study thus offers useful basis for further research into the function of human proteins in persistent infection. human cervical carcinoma cells (hela), human alveolar adenocarcinoma cells (a549) (atcc ccl-185), and baby hamster kidney fibroblast cells (bhk-21) (atcc ccl-10), were grown in dulbecco's modified eagle medium (dmem) containing 10% fetal calf serum (fcs) supplemented with 40 µg/ml gentamicin in a humidified atmosphere with 5% co 2 at 37 • c. lcmv mx isolate was continuously propagated in persistently infected hela cells (designated hela-mx). the mx isolate represents a persistent form of the virus capable of propagation through cell-cell contacts without releasing infectious virions (gibadulinova et al., 1998; reiserova et al., 1999; tomaskova et al., 2008) . a549 cells were infected with mx isolate via cell-free extract from persistently infected bhk-21 cells (bhk-mx) as described in laposova et al. (2017) and subsequently passaged several times to allow virus spread. whole-proteome samples for 2d-dige analysis were prepared according to standard protocol. briefly, the cells were washed with ice-cold pbs, and 600 µl of lysis buffer (150 mm nacl; 1 m tris, ph 7.5; 1% triton x-100; and 0.1% sds) were added to the culture, followed by 20 min incubation on ice. the lysed cells were scraped off, transferred to tubes, passaged through an insulin syringe and centrifuged at 17,000 × g for 15 min at 4 • c. next, nine volumes of precipitation solution (acetone:methanol 9:1) were added, and the resulting solutions were incubated overnight at −20 • c. the samples were then centrifuged at 9000 × g for 30 min at 4 • c. obtained pellets were air-dried and resuspended in 300 µl of utc solution (7m urea, 2m thiourea, 4% chaps). final protein concentrations were determined using the bradford method (bio-rad). for the preparative gel, 100 µg of each sample were mixed with the utc solution to the final volume of 225 µl. an equal volume of loading/reswelling buffer [65 mm dithiothreitol (dtt); 2% (v/v) immobilized ph gradient (ipg) buffer, ph 3-10 nl (ge healthcare), 2.8% destreak reagent (ge healthcare); adjusted with utc solution to the final volume of 500 µl] was added to the mixture. eighteen centimeters ipg strip with non-linear 3-10 ph range (ge healthcare) was passively reswelled in 450 µl of the sample mixture overnight. isoelectric focusing was performed using the multiphor ii unit (ge healthcare), with the following 6-step protocol: (1) 100 v, 2 h; (2) 150 v, 2 h; (3) 600 v, 2 h; (4) 1000 v, 1 h; (5) 5000 v, 2 h; and (6) 5000 v, 16 h. the current limit was set to 2 ma, with a ramping voltage gradient between the steps. after separation in the 1st dimension, the strip was equilibrated in sds equilibration solution (6m urea; 2% sds; 20% glycerol; 37.5 mm tris-hcl ph 8.8) containing 0.5% (w/v) dtt for 15 min; followed by 15 min incubation with sds equilibration solution in the presence of 4.5% iodoacetamide (iaa). the equilibrated strip was placed on top of a 12% polyacrylamide gel cast between low-fluorescence glass plates (the backing plate being pretreated with bind silane solution; 4 ml 96% ethanol, 1.8 ml mq water, 0.2 ml acetic acid and 10 µl of bind silane, 1 h incubation at room temperature) and fixed by adding 0.5% agarose gel dissolved in mq water with a trace of bromophenol blue. separation in the 2nd dimension was carried out using the protean xl (bio-rad) chamber. the resulting glass-backed 2d gel was incubated 2 × 30 min in fixing solution (500 ml ethanol, 70 ml acetic acid, 430 ml mq water), the solution being replaced between individual fixing steps. total proteins were stained by overnight incubation with sypro ruby protein gel stain (molecular probes) in the dark. the gel was washed 2 × 30 min in wash solution (100 ml methanol, 70 ml acetic acid, 830 ml mq water) and 2 × 10 min in mq water prior to scanning. resulting 2d map of the proteome was visualized by the pharosfx molecular imager (bio-rad), using 50 µm resolution and appropriate laser/filter wavelengths. in the first step, sample ph was adjusted to fit the range of 8.0-9.0 using 1 m tris. fluorescent dyes cy2, cy3, and cy5 were used for minimal sample labeling (ge healthcare). fifty micrograms of each sample were labeled with 400 pmol of either cy3 or cy5, in biological triplicates, using the dye-swap technique between individual replicate pairs. for the internal standard, 25 µg of each sample replicate was mixed with 400 pmol of cy2 per sample. after 30 min of on-ice incubation in the dark, the labeling reaction was stopped by adding 1/10 volume of 10 mm l-lysine to the samples. after a short spin, the labeled mixtures were incubated on ice for 10 min in the dark. subsequently, the samples for each replicate gel were mixed in the following manner cy2:cy3:cy5 1:1:1, adjusted with utc solution to the final volume of 225 µl and mixed with an equal volume of the loading/reswelling buffer. ipg strips with non-linear 3-10 ph range were passively rehydrated in 450 µl of the sample mixtures overnight in the dark. the 2d separation protocol was identical to that of the preparative gel with three exceptions -(i) 12% polyacrylamide gel was cast between two low-fluorescence glass plates, not pretreated with bind-silan; (ii) the proteins in the analytical gels were not fixed, but directly scanned (iii) particular emphasis was put on working in the dark. obtained 2d gels were immediately scanned between two low-fluorescence glass plates using the pharosfx molecular imager at 50 µm resolution and at appropriate excitation/emission wavelengths corresponding to cy2, cy3, and cy5 individual channels. quantitative gel analysis was carried out using decyder 7.2 software package (ge healthcare). for those proteins discovered to be different in mock-infected and lcmv-infected samples, analysis of significance was conducted using the student's t-test. only significantly different protein spots (p < 0.05), with an at least 1.5-fold abundance difference (ratio of mock versus infected sample mean normalized spot signals) were regarded as upor down-regulated. these protein spots of interest were chosen for mass spectrometry detection. target spots of interest were excised from the preparative gel using exquest spot cutter (bio-rad) and destained. after reduction and alkylation, the gel plugs were digested overnight with sequencing grade modified trypsin (promega). the digested peptides were extracted with 60 µl of 50% acetonitrile (merck) containing 0.1% trifluoroacetic acid (tfa) (merck). samples with digested peptides were evaporated to 20 µl in concentrator plus (eppendorf) and purified using µ-c18 ziptips (merck millipore). subsequently, 1 µl of peptide mixtures and 1 µl of 0.7 mg/ml chca matrix in 85% acetonitrile, 0.1% tfa, and 1 mm ammonium phosphate were pipetted onto 800 µm anchorchip maldi target (bruker). data were acquired on ultraflextreme tof mass spectrometer (bruker) operated by flexcontrol 3.3 (bruker) in positive reflector mode. for each position on the target 4000 laser shots were summed in 700-3500 m/z range. next, we selected the 25 most intense precursor ions per sample for fragmentation with signalto-noise threshold 15. tandem mass spectra were recorded by accumulation of 3000 shots in lift mode using lid (laserinduced dissociation) mechanism. laser power was boosted by 50% without collision gas, and the detector voltage was increased by 80%. monoisotopic peak lists were generated in flexanalysis 3.3 (bruker) by snap algorithm. precursor spectra with signal-tonoise less than ten were removed, and the remaining masses were externally recalibrated. fragment spectra were smoothed using savitzky-golay algorithm (three cycles with 0.15 m/z width), the baseline was subtracted with tophat algorithm, and filtering was done on signal-to-noise level five. subsequent processing for protein identification was done through proteinscape 2.1 (bruker) interface connected to mascot server 2.3 (matrix science). fixed carbamidomethyl cysteine, variable oxidized methionine, single trypsin miscleavage, and appropriate mass tolerances (40 ppm for precursor ions and 0.4 da for fragments) were specified. queries were done against homo sapiens proteins downloaded from uniprot 1 in april 2014, containing 69,060 sequences. protein identifications were accepted if the ion scores of at least two different matched peptides were higher than the probability of identity threshold (30 at p < 0.05). the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride partner repository (vizcaino et al., 2016) with the dataset identifier pxd005205 and doi: 10.6019/pxd005205. gene ontology annotation was acquired from the gene ontology project using the panther database for biological procedures and molecular function 2 . functional enrichment analysis was performed by fisher's exact test with false discovery (fdr) correction, with fdr p < 0.05 considered significant (mi et al., 2016) . the protein-protein interaction network was analyzed using the string 11.0 database with default settings, while the interaction score was set to medium confidence (0.400) 3 (jensen et al., 2009 ). equal amounts of proteins from untreated mock-infected and lcmv-infected cells or cells treated with 10 mm n-acetyl-l-cysteine (nac) for 24 h were separated in 10% sds-polyacrylamide gel, transferred onto a polyvinylidene difluoride membrane (immobilon-p; millipore), and probed with antibodies specific for lcmv np [in-house generated mab m87 (tomaskova et al., 2011) ], alpha-enolase (abcam #ab85086, 1:1000 dilution), gp96/hsp90-beta 1 (millipore #abf306, 1:5000 dilution), prdx4 (invitrogen #pa3-753, 1:1000 dilution), phospho-akt (ser473) (cell signaling technology #9271, 1:1000 dilution), akt (cell signaling technology #4691, 1:2000 dilution), β-actin (cell signaling technology #3700, 1:5000 dilution), and alpha-tubulin (abcam [ab7291], 1:10,000 dilution). detection was performed with hrp-or irdye-labeled secondary antibodies visualized with enhanced chemiluminescence or odyssey clx imager system, respectively. densitometric analysis of protein abundance was performed using imagej software (schindelin et al., 2015) or image studio lite software, respectively. the generation of reactive oxygen species (ros) was assessed using the fluoroprobe 5-(and-6)-chloromethyl-2 ,7 -dichlorodihydrofluorescein diacetate (cm-h2dcfda). cells (4 × 10 4 cells/well) cultured in 96-well plates were washed with phosphate-buffered saline supplemented with calcium/magnesium (pbs) and incubated with 10 µm cm-h2dcfda (molecular probes) in pbs for 20 min in the dark at 37 • c. after replacement of the reactive agents with pbs, 2 ,7 -dichlorofluorescein (dcf) fluorescence was measured at an excitation wavelength of 492 nm and an emission wavelength of 527 nm using the synergy/h4 microplate reader (biotec instruments). values were corrected for background auto-fluorescence of non-stained cells and normalized to the number of cells assessed by dapi staining. briefly, at the end of the experiment, cells were fixed with ice-cold methanol for 5 min at −20 • c, washed twice with pbs and then incubated with dapi (1 µg/ml) for 5 min in the dark. after a subsequent washing step with pbs, dapi fluorescence was measured in each well using 358 nm excitation and 460 nm emission wavelengths. the genomic dna (gdna) was isolated from hela and hela-mx cells using qiaamp dna mini kit (qiagen) according to manufacturer's instructions. three µg of genomic dna were digested using a mixture of restriction enzymes (hhai, hinf1, mspi, haeiii, rsai, alui) as described by mender and shay (2015) . the dna fragments were separated in 1% agarose gel for 16 h at 1.6 v/cm and stained with 0.5 µg/ml ethidium bromide solution for 20 min (stained gel served as a loading control). the gel was then incubated for 40 min in denaturation solution (1.5 m nacl, 0.5 m naoh), 30 min in neutralization solution (1.5 m nacl, 0.5 m tris, ph 7.4) and 30 min in 20× ssc (3 m nacl, 0.3 m na-citrate, ph 7.0). the dna was then transferred to immobilon ny + membrane (emd millipore) with a vacugene xl blotter (ge healthcare) in 20× ssc and fixed by incubating the membrane at 80 • c for 1 h. the membrane was pre-hybridized for 20 min at 42 • c in hybridization buffer (6× ssc, 5× denhardt solution, 2.5% sds) and hybridized at 42 • c overnight in the same buffer containing radioactively labeled telomere-specific c-rich probe prepared as described by mender and shay (2015) . the membrane was washed once with wash buffer i (2× ssc, 0.1% sds) for 15 min at 37 • c, twice for 15 min in wash buffer ii (0.5× ssc, 0.1% sds) at 37 • c and twice for 15 min at room temperature with wash buffer iii (0.5× ssc, 1% sds). the signal was detected by personal molecular imager fx (biorad). hela and hela-mx cells were seeded into a 6-well plate at 4 × 10 5 cells per well and transfected with 2 µg of the luciferase vector (hre-luc) containing hypoxia-responsive elements the next day, using the turbofect transfection reagent (thermo fisher scientific) according to the manufacturer's instructions. cells were co-transfected with 100 ng of prl-tk renilla vector (promega) to normalize for the transfection efficiency. luciferase reporter construct containing three hypoxia response elements (hre; 24-mers) from the pgk-1 gene was a gift from navdeep chandel (emerling et al., 2008) (addgene plasmid #26731 4 ; rrid:addgene_26731). reporter gene expression was assessed 48 h after transfection using the dual luciferase reporter assay system (promega) and the synergy ht reader with gen5 software (biotec instruments). luciferase activity was normalized against renilla activity. figure 1 | preparative 2d gel. a total of 100 µg proteins from each sample of lcmv-infected and mock-infected hela cells were resolved by 2d page. the protein spots were visualized by sypro ruby protein gel stain. this preparative 2d-gel was used to cut out protein spots that were differentially abundant in analytical gels. arrows indicate the isolated and identified protein spots with at least 1.5-fold up-regulation or down-regulation. spots are numbered according to table 1 . statistical analyses were performed using graphpad prism 8 software for windows, unless stated otherwise. two-tailed unpaired t-test was used to analyze significant differences between two cell groups and one-way anova was used to determine statistically significant differences between three or more independent groups, with p < 0.05 considered significant. to investigate global protein changes in hela cells during persistent infection with the mx strain of lcmv, equal amounts of total proteins prepared from mock-infected and lcmv-infected hela cells were subjected to 2d-dige analysis. we used three independent biological replicates to generate comprehensive and reliable data (supplementary figure s1) . the separation strength was satisfactory; the average yield per gel comprised approximately 2600 protein spots. initial betweengel matching was carried out by manual landmarking, followed by automatic matching and extensive manual evaluation of the matched spots between individual gels in the decyder 2d 7.2 software. only protein spots showing significance (p < 0.05) and at least 1.5-fold difference in abundance were considered as up-or down-regulated. according to our analyses, the abundance of 34 protein spots was significantly altered after lcmv infection. since decyder does not allow identification of proteins present only in one condition and absent in the other, we complemented gel analysis with a manual qualitative study of protein presence between individual samples. in total, 19 protein spots were present only in one condition (mockinfected or lcmv-infected). these 53 protein spots were marked as "proteins of interest (pois)" and matched against the sypro ruby-stained preparative gel. the 30 proteins of interest, which we were able to map on the preparative gel, were excised and analyzed by maldi-tof tandem mass spectrometry. twenty-four protein spots with 21 non-redundant proteins were successfully identified, including 12 up-regulated and 12 down-regulated proteins (figure 1) . detailed information on the identified proteins is provided in table 1 . several proteins were present in more than one spot, namely: heterogeneous nuclear ribonucleoprotein k (hnrnp k; spot 12 and 13, both more abundant upon infection), mitochondrial 60 kda heat shock protein (hsp60; spot 15 and 16, both less abundant in cells affected by persistent virus), and keratin, type ii cytoskeletal 8 (ck-8; spot 19 and 20, which showed opposite changes). these proteins are most likely different post-translational or alternatively spliced variants. proteins present only in one condition were: (a) unique upon lcmv infection -mitochondrial hydroxymethylglutaryl-coa synthase, guanine deaminase, annexin a7, and rab gdp dissociation inhibitor beta; (b) unique in the mock-infected cell line -cellular retinoic acid binding protein 2, prostaglandin e synthase 3, and eukaryotic initiation factor 4a-i. apart from proteins discussed in more detail below, other accumulated proteins upon infection included galectin 1 and macrophagecapping protein, and less abundant endoplasmin, gtp-binding nuclear protein ran, peptidyl-prolyl cis-trans isomerase fkbp4, and adenine phosphoribosyltransferase. to better understand the implications of the cellular response to lcmv infection, the corresponding biological functions of the differentially regulated proteins were categorized according to the gene ontology database, using the panther classification system. most of the proteins with significantly altered abundance were involved in metabolic processes (71.4%; 15 proteins), particularly lipid and glucose metabolism; cellular processes, such as cellular communication and cell cycle (33.3%; 7 proteins); immune system processes (19.0%; 4 proteins); developmental processes (19.0%; 4 proteins); localization (14.3%; 3 proteins); and cellular component organization or biogenesis (14.3%; 3 proteins) (figure 2) . functional enrichment analysis revealed that the most overrepresented were proteins with peroxiredoxin activity (go: 0051920; raw p-value = 1.03e-07; fdr = 4.81e-04) and proteins that play a role in rna binding (go: 0003723; raw p-value = 4.98e-07; fdr = 1.16e-03). furthermore, network analysis was conducted to reveal protein-protein interactions. forty-two associations were observed in the protein network analysis generated by string 11.0 database. the network had significantly more interactions than expected randomly (six expected interactions; ppi enrichment p-value < 1.0e-16). such an enrichment indicates that the proteins are at least partially biologically connected. the confidence view of the protein-protein associations is shown in figure 3 , where the strength of the association is indicated by the thickness of the connecting line (confidence score >0.4). similar to gene ontology analysis, the most connected proteins have important functions in antioxidant activity, protein folding, and rna binding. to validate the changes in protein accumulation patterns in response to lcmv infection, two identified proteins, alphaenolase (eno1; more abundant) and heat shock protein 90 kda beta, member 1 [hsp90b1 (endoplasmin); less abundant] were selected for western blot analysis. the reason to select hsp90b1 was its important functionality, pinpointed by the string network analysis. figure 4a shows a prominent although not statistically significant increase in protein abundance of eno1 and a decrease for hsp90b1 in infected cells. these results are consistent with those observed in the 2d-dige analysis. the densitometric analysis of three biological replicates revealed hela-mx/hela average ratio of 1.8 for eno1 and −2.1 for hsp90b1, while alpha-tubulin was used as a loading control ( figure 4b) . the results were very close to the changes observed in the 2d-dige analysis, which were 1.9 for eno1 and −2.1 for hsp90b1. in this study, we observed a decreased amount of peroxiredoxin 2 (prdx2), peroxiredoxin 4 (prdx4), and peroxiredoxin 6 (prdx6) upon lcmv infection. peroxiredoxins (prxs) are members of an ubiquitous family of thiol-specific peroxidases that reduce mainly hydrogen peroxide (h 2 o 2 ) to water with the use of reducing equivalents provided through the thioredoxin system (wood et al., 2003) . to confirm the alteration in the abundance of peroxiredoxins during lcmv infection, we selected the most affected prdx4 for western blot analysis. figure 5a shows an almost identical decrease in the expression of prdx4 (−2.05) in infected cells as we observed in the 2d-dige analysis (−2.08). since one of the major functions of prxs is cellular protection against oxidative stress, we hypothesized that decreased levels of these antioxidant proteins may lead to an elevation of the ros content in infected cells. the measurement of ros generation in mock-and lcmv-infected cells supported our prediction. we revealed significantly higher production of ros (1.76-fold) in lcmv-infected cells than in mock-infected cells ( figure 5b) . interestingly, another antioxidant enzyme, thioredoxin reductase 1 (trxr1), was more abundant in infected cells (table 1 ), yet this seems to be insufficient to counterbalance the decrease in the levels of peroxiredoxins. in order to determine whether the increased ros level described above is unique to the mx-infected hela cells or can also be seen in other cell lines, we decided to use a549 lung epithelial cells, which are commonly used as an in vitro model system for arenavirus infections. similar to hela cells, we detected a 1. figure s2a) . further, we hypothesized that increased levels of ros may have an effect on dna of the infected cells. telomeric sequences composed of arrays of 5 -ttaggg-3 repeats are particularly vulnerable to oxidative damage due to stretches of g residues that are susceptible to the conversion to 8-oxoguanine (8-oxog). accumulation of 8-oxog in telomeric repeats may affect accessibility of chromosomal ends to telomerase, binding efficiency of telomere-binding proteins, and/or formation of protective secondary structures such as telomeric loops (von zglinicki, 2002; ahmed and lingner, 2018) . as a result, increased levels of ros yield changes in the length of telomeric tracts. to test this hypothesis, we isolated genomic dna from control and infected cells and measured the length of telomere restriction fragments (trfs). we observed that the infected cells contained a more heterogeneous population of trfs and a higher proportion of shorter fragments compared with the control cells (figure 6 ). this indicates that the infected cells have a compromised ability to maintain the standard length of telomeres. it has become apparent in recent decades that less-reactive ros, especially hydrogen peroxide, can function as intracellular signaling molecules regulating multiple physiological and pathological cell processes. it has been shown that ros mediate activation of several signaling pathways and transcription factors, including the pi3k/akt signaling cascade (lee et al., 1998 (lee et al., , 2002 and hif (patten et al., 2010) . to assess biological implications of increased ros in infected cells, we investigated whether lcmv infection affected transcriptional activity of hif-1. using a luciferase reporter containing hre, we detected a modest, but significant increase in hif-1 transactivation in infected hela cells in comparison to control cells ( figure 7a) . further, we evaluated activation of akt by probing cellular protein lysates for phospho-akt (s473), which is a marker for activated akt. figures 7b,c , phosphorylation of akt was induced in infected cells, while overall akt levels were similar in both infected and uninfected cells. since we assumed that activation of akt was triggered by redox signaling, we further investigated the effect of antioxidants on infected cells. we detected decreased levels of phospho-akt in lcmv-infected hela cells treated with n-acetyl cysteine. moreover, inhibition of ros accumulation by the antioxidant also decreased the levels of viral np (figures 7b,d) . in addition, we observed similar impact of antioxidant treatment in lcmvinfected a549 cells, although the reduction in np levels was less pronounced (supplementary figure s2b) . . the signal obtained with anti-alpha-tubulin antibody was used as loading control. presence of np confirms lcmv infection. one representative of three biological replicates is shown. (b) densitometric analysis of protein abundance was performed using imagej software. relative quantity of proteins was calculated as the ratio of the intensity of each signal to the intensity of the related tubulin internal standard. values represent the means of three biological replicates. error bars denote the standard deviations. * * p < 0.01 (hela-mx vs. hela). these data suggest that lcmv maintains its replication in persistently infected cells by regulating redox signaling through modulation of local levels of h 2 o 2 and subsequent activation of cellular processes that it uses for its own benefit. viruses and host cells have established complex, dynamic interactions that either facilitate the efficiency of viral infection or defend the host against invading pathogens. these interactions are crucial for the regulation of metabolism and other processes in the host cell and their elucidation is critical for a detailed description of mechanisms of viral pathogenesis, eventually leading to effective treatment. in this report, we investigated the host response to persistent lcmv infection using 2d-dige-based proteomic approach. as the model system of persistent infection we used human hela cells long-term infected with lcmv mx isolate (over 50 passages). the mx isolate likely represents a lcmv variant that has undergone complex adaptations. it is characterized by reduced accumulation of viral glycoproteins on the surface of . the signal obtained with anti-β-actin antibody was used as loading control. one representative of three biological replicates is shown. densitometric analysis of protein abundance (right) was performed using imagej software. relative quantity of proteins was calculated as the ratio of the intensity of each signal to the intensity of the related β-actin internal standard. values represent the means of three biological replicates. error bars denote standard deviations. * * p < 0.01 (hela-mx vs. hela). (b) ros generation was assessed in a microplate reader using dcf fluorescence and normalized to the number of cells measured by dapi staining. the results represent the mean from three independent biological experiments, each done in eight replicates. error bars denote standard deviations. data are presented as relative increase compared to control (hela cells), which was set to 1. * * * * p < 0.0001 (hela-mx vs. hela). infected cells and by spreading to uninfected cells mainly through cell-cell contact without the release of infectious viral particles, all of which strongly resembles persistent lcmv neuronal infection in its native host (rodriguez et al., 1983) . the use of hela-mx cells has several advantages, such as the feasibility of use of a large number of cells for proteomic analysis, reproducibility, and viral persistence resembling a life-long chronic infection. however, in terms of natural lcmv infection, hela cells possess some limitations and one needs to be cautious about generalizing the results. therefore, we performed some of the experiments also on the a549 cell line, a model of type ii alveolar epithelial cells (supplementary figure s2) . this cell type is the first to be in contact with the virus during primary infection. it has also been shown that a549 cells are capable of antigen presentation (salik et al., 1999) . although the data demonstrating chronic infection in humans are not available, the persisting and figure 6 | lmcv-infected cells exhibit changes in the pattern of telomeric restriction fragments. (a) total dna was isolated from uninfected (hela) and infected (hela-mx) cells, digested by a cocktail of restriction enzymes and the resulting dna fragments were separated by 1% agarose gel electrophoresis. (b) dna was transferred to nylon membrane and hybridized with a radiolabeled telomeric probe. trfs (telomeric restriction fragments), dna ladder (generuler 1 kb dna ladder (thermo scientific). a representative of three independent experiments is shown. reactivated virus may be a source of unexplained complications (often fatal) in immunosuppressed transplant recipients, as well as in developing embryos (peters, 2006; bonthius, 2009 ). thus, a better understanding of cellular processes exploited or subverted by viruses during persistent infection can help develop new strategies to treat mammarenavirus infections in humans. our analysis revealed 53 protein spots differentially accumulated upon persistent lcmv infection in hela cells. 24 proteins were identified with high confidence. remarkably, the abundance of several antioxidant enzymes was shown to be significantly altered. namely, the levels of prdx2, prdx4, and prdx6 were 1.5-2.08 times lower in lcmv-infected cells (table 1 ). in addition, functional enrichment analysis, as well as network analysis, pointed out that the antioxidant system was in the center of virusinduced host proteome response (figure 3) . the key functions of prxs include cellular protection against oxidative stress and modulation of signaling pathways that use hydrogen peroxide as a second messenger (rhee et al., 2005a,b) . a significant amount of published data suggests that prxs are hydrogen peroxide sensors that play a central role in redox signaling (rhee et al., 2012; latimer and veal, 2016; netto and antunes, 2016) . this is implicated in a number of biological processes, including growth factor signaling, cell differentiation, and cytokine production (hampton and o'connor, 2016) . regulation of prxs modifies the concentration of h 2 o 2 and thereby facilitates its signaling functions. we indeed confirmed a slight but significant elevation of the ros content alongside the downregulation of prxs by lcmv both in hela and a549 cells (figure 5b and supplementary figure s2a) . ros, in the form of h 2 o 2 , can modify protein function, and/or structure through the mechanism of cysteine oxidation that influences a number of signaling cascades. for example, ros activate pi3k either directly or by inactivating the phosphatase pten via oxidizing cysteine residues within its active site, resulting in an increased activation of akt and modulation of its downstream targets (lee et al., 2002; connor et al., 2005) . once activated, akt promotes cell survival, growth, metabolism, and proliferation by phosphorylating various effectors (ersahin et al., 2015) . in line with these findings, we observed enhanced levels of active akt in lcmv-infected cells. in addition, inhibition of ros by n-acetyl cysteine led to suppressed phosphorylation of akt in infected cells, suggesting that akt becomes activated in a ros-dependent manner (figures 7b,c) . notably, the treatment with antioxidants also resulted in reduced levels of viral np in hela, as well as in a549 lcmv-infected cells, suggesting a link between ros-dependent signaling and virus replication (figures 7b,d and supplementary figure s2b ). these findings are in accordance with previous studies that have demonstrated that ros generated in response to lcmv play an important role in virus binding and subsequent virus replication (michalek et al., 2008) . moreover, it has been shown that the pi3k/akt pathway plays an important role in different steps of the life cycle of a variety of viruses. for instance, infection with the new world mammarenavirus junin virus has been shown to induce the pi3k/akt pathway, and inhibition of this pathway has resulted in a decreased infectious virus yield because of blocking the recycling of the transferrin receptor engaged in junv cell entry (linero and scolaro, 2009) . furthermore, pi3k/akt pathway inhibition reduced budding and, to a lower degree, lcmv rna synthesis, but not cell entry (urata et al., 2012) . additionally, the sars coronavirus induced weak activation of akt that was crucial for the establishment of persistence (mizutani et al., 2005) . in contrast, pi3k/akt pathway inhibition could not interfere with the establishment of persistent junv infection in vero cells, and this pathway was not crucial for the maintenance of junv persistence (linero and scolaro, 2009 ). data are presented as a relative increase compared to control (hela cells), which was set to 1. * * * * p < 0.0001 (hela-mx vs. hela). (b) immunoblot analysis of p-akt (s473), total akt, and viral np with specific antibodies using whole-cell extracts prepared from untreated (−) mock-infected hela cells (hela) and lcmv-infected hela cells (hela-mx) or cells treated (+) with 10 mm nac for 24 h. the signal obtained with anti-β-actin antibody was used as loading control. one representative of at least four biological replicates is shown. (c) densitometric analysis of protein abundance was performed using imagej software or image studio lite software. relative quantity of p-akt was calculated as the ratio of the intensity of each signal to the intensity of the related signal for total akt. values represent the means of six biological replicates. error bars denote the standard deviations. data are presented as relative change compared to control (untreated hela cells), which was set to 1. * p = 0.0119 (hela vs. hela-mx), * p = 0.0101 (hela-mx vs. hela-mx nac), p = 0.3002 (hela vs. hela nac). (d) densitometric analysis of protein abundance was performed using imagej software or image studio lite software. relative quantity of np was calculated as the ratio of the intensity of each signal to the intensity of the related β-actin or tubulin internal standard, respectively. values represent the means of four biological replicates. error bars denote the standard deviations. data are presented as relative change compared to untreated hela-mx cells, which was set to 1. * * p = 0.0059 (hela-mx nac vs. hela-mx). reactive oxygen species also regulate the activation of several transcription factors, including hifs. hif-1 is the main hypoxiainducible transcription factor responsible for cell and tissue adaptation to low oxygen, by controlling cell metabolism, proliferation and survival, erythropoiesis and angiogenesis (semenza, 2003) . there is increasing evidence demonstrating that non-hypoxic stimuli can also activate hif-1. it has been shown that the pi3k/akt/mtor signaling cascade directly increases the expression of hif-1 encoding gene on the transcriptional and translational levels (dery et al., 2005) . any aberrant stimulation of this pathway leads to activation of hif-1α, even in normoxic conditions. an increased ros production has been reported as essential for increased hif translation through the pi3k pathway (iommarini et al., 2017) and for hif stabilization through the inactivation of prolyl hydroxylases in a nonhypoxic system (diebold and chandel, 2016) . accordingly, we demonstrated enhanced hif-1 transactivation in lcmv-infected cells (figure 7a) , as well as increased expression of eno1 (figure 4) , a typical hif-1 target gene (semenza et al., 1996) . interestingly, hsp90b1 expression was reduced by persistent lcmv (figure 4) , which has also been previously reported in the case of hypoxia in min6 cells (bensellam et al., 2016) . pi3k/akt signaling, as well as the hif transcription factors, have in common that they play an essential role in regulating cellular metabolism (dery et al., 2005; yu and cui, 2016) . in view of that, we observed that a large number of proteins identified in this study, such as the above mentioned alphaenolase or hydroxymethylglutaryl-coa synthase, are involved in metabolism, as indicated by the go analysis (figure 2) . moreover, we have shown previously that the exposure of mx-infected hela cells to chronic hypoxia resulted in a hif-dependent increase of viral replication and enhanced formation of infectious virions (tomaskova et al., 2011) . in addition, there is accumulating evidence that many viruses, through various mechanisms, affect the hif-1 pathway, causing multiple downstream effects, such as modifying host cell metabolism, stimulating inflammation, and facilitating viral replication (morinet et al., 2013; cuninghame et al., 2014) . some viruses, such as the vaccinia virus and hepatitis c virus impair hif-1α prolyl hydroxylation, which leads to its stabilization (nasimuzzaman et al., 2007; mazzon et al., 2013) . hepatitis b virus stabilizes hif-1α by diminishing the interaction between vhl and hif-1α (moon et al., 2004) . influenza virus a h1n1 inhibits the proteasome and decreases fih-1 expression, thereby activating the hif-1 pathway by stabilizing hif-1α (ren et al., 2019) . contrary to downregulated prxs, we observed a 1.6fold increased abundance of cytoplasmic trxr1 (encoded by txnrd1) in lcmv-infected cells ( table 1) . the thioredoxin (trx) system consisting of nadph, trxr, and thioredoxin, is a key antioxidant mechanism in protection against oxidative stress. it regulates protein dithiol/disulfide balance through its disulfide reductase activity (arner, 2009) . as already mentioned, trx provides electrons to peroxiredoxins to remove reactive oxygen and nitrogen species. trx reductase then reduces oxidized trx back using the reducing nadph equivalents. thus, trx reductase plays a crucial role in regeneration of a catalytically active form of prxs (lu and holmgren, 2014) . we can, therefore, speculate that elevated levels of trxr1 can maintain proper accumulation of local h 2 o 2 to ensure that it acts as a signaling molecule and does not cause oxidative damage. based on the abovementioned facts, it is possible that lcmv modulates the delicate interplay inside cells between oxidants and antioxidants, and determines the activity profile for a variety of proteins that maintain persistent infection (such as transcription factors, kinases and phosphatases, cytoskeletal proteins, or metabolic enzymes). on the other hand, elevated ros levels in infected cells can be a double-edged sword, as they can negatively affect genomic stability of host cells. telomeres, the nucleo-protein complexes at the chromosomal ends, are essential parts of the genome providing solutions to both end-protection and endreplication problems (shay and wright, 2019) . the solution to end-protection problem is provided by a specialized protein complex called shelterin (de lange, 2010) , in combination with the formation of a secondary structure called the telomeric loop (griffith et al., 1999) . the end-replication problem is solved by various means. in human germ or stem cells, and most cancer cells (including hela), telomeres are preserved by telomerase, a reverse transcriptase using an rna subunit as a template for extension of an array of 5 -ttaggg-3 repeats (shay and wright, 2019) . guanine residues in telomeric repeats are particularly susceptible to oxidation resulting in their conversion to 8-oxog (oikawa and kawanishi, 1999) . accumulation of this oxidized purine compromises the telomere maintenance system in a quite complex way, including inhibition of binding of the shelterin components trf1 and trf2, the formation of t-loop, and/or the inhibition of telomerase (ahmed and lingner, 2018) . thus, increased intracellular levels of ros often result in telomere shortening (von zglinicki, 2002) , as observed in this study in the case of hela cells infected by lcmv (figure 6 ). furthermore, it was shown previously by the lingner group that telomeres physically interact with prdx1 and prdx2 (aeby et al., 2016) . although the levels of prdx1 were not substantially affected in hela-mx cells (data not shown), prdx2 exhibited a 1.5-fold decrease ( table 1) . as prdx2 was shown to interact with telomeres in the s phase of the cell cycle (aeby et al., 2016) , reduction in its amount in hela cells infected by lcmv may contribute to a detrimental effect of ros on the maintenance of their telomeric repeats. nevertheless, our data suggest that lcmv affects redox signaling and metabolic pathways that support an anabolic program required for efficient virus replication and/or maintenance of persistent infection. the precise mechanism of how lcmv regulates antioxidant-scavenging proteins in order to effectively manage amounts of ros still remains unknown and requires further investigation. although research on lcmv has led to many insights into viral persistence, the answers to several crucial questions about host-lcmv interactions during persistent infection are still lacking. this study analyzed proteomic changes in a human cell line upon persistent infection with lcmv using quantitative gelbased proteomics. we identified 24 modulated host proteins, which play important roles in a number of cellular processes. uncovering their roles in persistent lcmv infection can broaden our understanding of the complex and dynamic virus-host cell interactions and be valuable for antiviral research. we provided experimental evidence that lcmv negatively affects the levels of antioxidant enzymes peroxiredoxins leading to elevated intracellular content of ros. these changes were accompanied by activation of hif-1 and pi3k/akt signaling pathways as well as changes in the profile of telomeric restriction fragments. the treatment with antioxidants resulted in reduced level of viral nucleoprotein, indicating that virus replication and rosdependent signaling are interconnected processes. the datasets generated for this study can be found in the proteomexchange consortium via the pride partner repository, dataset identifier pxd005205. jt and lt conceived and designed the experiments. mb, md, and jt analyzed the data. jt wrote the first draft of the manuscript. mb, md, and lt wrote the sections of the manuscript. all authors performed the experiments, contributed to the manuscript revision, read, and approved the submitted version. this project was supported by grants 2/0053/15, 2/0030/19 to jt, 2/0078/19 to tg, 1/0052/16 to lt from the scientific grant agency of the ministry of education of the slovak republic and the slovak academy of sciences, and apvv-15-0022 to lt from slovak research and development agency. we would like to thank dr. gabriela flores-ramirez from the department of rickettsiology, institute of virology, bmc, for her technical assistance. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.02438/full#supplementary-material figure s1 | 2d-dige images of biological triplicates. a 50 µg of each sample were labeled with 400 pmol of either cy3 or cy5 dye. preferential binding of the dyes was evaluated using dye-swap technique between individual replicates of the analytical gel. in panels (a,c) the hela protein sample was labeled with cy3 dye (green channel) while cy5 labeling (red channel) was used for the hela-mx. panel (b) represents replicate gel no. 2 where the hela sample was labeled with cy5 and the hela-mx was labeled with cy3. intra-and inter-gel variations were normalized according to the internal standard consisting of all analyzed samples in ratio 1:1 labeled with cy2 dye. once normalized, these analytical 2d gels were used to analyze the differential abundance of proteins between hela and hela-mx samples. figure s2 | reactive oxygen species production and antioxidant treatment in lcmv-and mock-infected a549 cells. (a) ros generation was assessed in a microplate reader using dcf fluorescence and normalized to the number of cells measured by dapi staining. the results represent the mean from three independent biological experiments, each done in eight replicates. error bars denote standard deviations. data are presented as relative increase compared to control (a549 cells), which was set to 1. * * * * p < 0.0001 (a549-mx vs. a549). (b) immunoblot analysis of viral np with specific antibodies using whole-cell extracts prepared from untreated (−) mock-infected a549 cells (a549) and lcmv-infected a549 cells (a549-mx) or cells treated (+) with 10 mm nac for 24 h. the signal obtained with anti-β-actin antibody was used as loading control. one of two biological replicates is shown. peroxiredoxin 1 protects telomeres from oxidative damage and preserves telomeric dna for extension by telomerase impact of oxidative stress on telomere biology focus on mammalian thioredoxin reductases-important selenoproteins with versatile functions lymphocytic choriomeningitis virus: pediatric pathogen and fetal teratogen lymphocytic choriomeningitis virus: emerging fetal teratogen hypoxia reduces er-to-golgi protein trafficking and increases cell death by inhibiting the adaptive unfolded protein response in mouse beta cells lymphocytic choriomeningitis virus: a prenatal and postnatal threat differential signaling networks induced by mild and lethal hemorrhagic fever virus infections identification of differentially activated cell-signaling networks associated with pichinde virus pathogenesis by using systems kinomics proteomic analysis of pichinde virus infection identifies differential expression of prothymosin-alpha analysis of the differential host cell nuclear proteome induced by attenuated and virulent hemorrhagic arenavirus infection arenaviridae: the viruses and their replication application of proteomics technology for analyzing the interactions between host cells and intracellular infectious agents mitochondrial h2o2 regulates the angiogenic phenotype via pten oxidation hypoxia-inducible factor 1 and its role in viral carcinogenesis treatment of arenavirus infections: from basic studies to the challenge of antiviral therapy how shelterin solves the telomere end-protection problem hypoxia-inducible factor 1: regulation by hypoxic and non-hypoxic activators mitochondrial ros regulation of proliferating cells. free radic gene expression in primate liver during viral hemorrhagic fever early blood profiles of virus infection in a monkey model for lassa fever pten regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through forkhead transcription factor 3a (foxo3a) the pi3k/akt/mtor interactive pathway transmission of lymphocytic choriomeningitis virus by organ transplantation exotic emerging viral diseases: progress and challenges sequence and characterisation of the z gene encoding ring finger protein of the lymphocytic choriomeningitis virus mx strain mammalian telomeres end in a large duplex loop peroxiredoxins and the regulation of cell death non-canonical mechanisms regulating hypoxia-inducible factor 1 alpha in cancer interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals atpase na+/k+ transporting subunit alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses string 8-a global view on proteins and their functional interactions in 630 organisms characterization of host proteins interacting with the lymphocytic choriomeningitis virus l protein a map of the arenavirus nucleoprotein-host protein interactome reveals that junin virus selectively impairs the antiviral activity of double-stranded rna-activated protein kinase (pkr) a simple method for isolation of cell-associated viral particles from cell culture peroxiredoxins in regulation of mapk signalling pathways; 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shi, hongyan; guo, donghua; chen, jianfei; shi, da; zhu, qinghe; zhang, xin; feng, li title: analysis of protein expression changes of the vero e6 cells infected with classic pedv strain cv777 by using quantitative proteomic technique date: 2015-06-15 journal: j virol methods doi: 10.1016/j.jviromet.2015.03.002 sha: doc_id: 298922 cord_uid: k568hlf4 recent outbreaks of porcine epidemic diarrhea virus (pedv) have caused widespread concern. the identification of proteins associated with pedv infection might provide insight into pedv pathogenesis and facilitate the development of novel antiviral strategies. we analyzed the differential protein profile of pedv-infected vero e6 cells using mass spectrometry and an isobaric tag for relative and absolute quantification. a total of 126 proteins were identified that were differentially expressed between the pedv-infected and mock-infected groups (p < 0.05, quantitative ratio ≥1.2), among which the expression of 58 proteins was up-regulated and that of 68 proteins was down-regulated in the pedv-infected vero e6 cells, involving in integrin β2/β3, cystatin-c. the gene ontology analysis indicated that the molecular function of the differentially expressed proteins (deps) was primarily related to binding and catalytic activity, and that the biological functions in which the deps are involved included metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases. among the disease-related functions, certain anti-viral pathways and proteins, such as the rig-i-like receptor, rap1, autophagy, mitogen-activated protein kinase, pi3k-akt and jak-stat signaling pathways, and integrin β2/β3 and cystatin-c proteins, represented potential factors in pedv infection. our findings provide valuable insight into pedv-vero e6 cell interactions. the porcine epidemic diarrhea virus (pedv) is an enveloped, single-stranded positive-sense rna virus that causes porcine epidemic diarrhea (ped), an acute and highly contagious enteric disease in pigs. ped is characterized by severe diarrhea, vomiting, dehydration, and a mortality rate of up to 90% in suckling piglets (pensaert and debouck, 1978) . ped was first reported in belgium and the united kingdom in 1978, and frequent outbreaks have occurred in various asian countries . since 2007, acute ped outbreaks have continually occurred in thailand, china, and the usa, which have resulted in substantial economic losses (puranaveja et al., 2009; li et al., 2012; chen et al., 2013; huang et al., 2013; marthaler et al., 2013; stevenson et al., 2013; yang et al., 2013; chen et al., 2014) . the continued outbreaks of ped, despite control efforts, have caused widespread concern. the pedv belongs to the genus alphacoronavirus, in the family coronaviridae and order nidovirales (belouzard et al., 2012) . previous studies have investigated various control measures to protect against pedv infection, such as vaccines, diagnostic tools, and therapeutic drugs (sun et al., 2008; ren et al., 2011; sun et al., 2012; zhu et al., 2013; guo et al., 2013; kim and lee, 2013) . various aspects of pedv infection remain unclear, for example, swine testis (st) cells expressing porcine aminopeptidase n of pedv receptor were not susceptible to pedv infection. african green monkey kidney (vero) cells are highly susceptible to pedv infection, and are widely used for the primary isolation and cultivation of pedv guo et al., 2014) . therefore, vero lineages are suitable hosts for understanding the mechanisms of pedv infection. proteomics techniques are effective tools for characterizing protein expression profiles, and have been used widely to investigate disease-associated proteins (hondermarck et al., 2008; boja et al., 2011; he et al., 2012; sun et al., 2013) . among current proteomics methods, quantitative high-throughput proteomics approaches are useful for the analysis of infection-associated proteins of pathogens (linde et al., 2013; papachristou et al., 2013; ye et al., 2013; zeng et al., 2015) . in our current study, we used a quantitative proteomics approach based on an itraq tandem mass spectrometry (ms/ms) technique to identify proteins differentially expressed between pedv-infected and mock-infected vero e6 cells. the functions of the differentially expressed proteins (deps) were analyzed to determine whether they might be associated with pedv infection. our findings provide valuable insight into the changes in cellular processes that occur during pedv infection. the cv777 strain of pedv, kindly provided by maurice pensaert at ghent university (merelbeke, belgium), was used in all of our experiments after being adapted to vero e6 cells, as previously described (hofmann and wyler, 1988) . the vero e6 cell-adapted pedv, the vero e6 cells, and the monoclonal antibody against the nucleocapsid protein (np) of pedv were stored at the diarrhea-related viruses section, division of swine infectious diseases, national key laboratory of veterinary biotechnology, harbin veterinary research institute of the chinese academy of agricultural sciences. the vero e6 cells were cultured in dulbecco's modified eagle's medium containing 10% fetal bovine serum (fbs) in 75-cm flasks at 37 • c in a 5% co 2 atmosphere. when the cells reached 70-80% confluence, they were inoculated with the pedv at a multiplicity of infection of 1 in presence of 5 g/ml trypsin. at 48 h postinoculation, the cells began to exhibit cytopathic effects (cpes) of viral infection, but no cells lysis or shedding had occurred. the cells were washed three times with cold phosphate-buffered saline (pbs, ph 7.4). a 1.5-ml aliquot of lysis buffer containing 4% sds, 1 mm dtt, and 150 mm tris-hcl (ph 8.0) was added to each flask, and the flasks were incubated at 37 • c for 5 min. the cell lysates were collected using a cell scraper, and boiled for 5 min. three cell lysate replicates were prepared for the pedv-infected (v1-v3) and mock-infected (c1-c3) vero e6 cells, and stored at −80 • c. western blotting was performed to confirm pedv infection by detecting the presence of the np of pedv in the vero e6 cells. aliquots of the cell lysates were subjected to sds-page on a 12% acrylamide gel, and the protein bands were transferred to a nitrocellulose membrane using a semi-dry transfer device (bio-rad, hercules, ca, usa). the membrane was blocked using 5% (w/v) nonfat dried milk in pbs at 37 • c for 1 h, before incubation in pbs containing the anti-np monoclonal antibody (1:2000 dilution) at 37 • c for 1 h. after washing three times with 5% tween 20 in pbs (pbst), the membrane was incubated in pbst containing a horseradish peroxidase-conjugated goat anti-mouse igg (1:4000 dilution) at 37 • c for 1 h. after washing three times with pbs, the membrane was incubated with enhanced chemiluminescence detection reagents (biotopped, beijing, china) at room temperature for 3 min, and the peroxidase-mediated luminescence was digitally captured using the molecular imager chemidoc xrs+ system (bio-rad) and the image lab software (bio-rad). to verify the differential expression of the selected deps, equivalent volumes of the cell lysate replicates from the pedv-infected (v1-v3) and mock-infected (c1-c3) vero e6 cells were pooled into the v and c samples, respectively, and western blotting was performed as described above, with the following exceptions: a 1:1000 dilution of the polyclonal antibodies anti-␤ tubulin, anti-integrin-␤3, anti-cystatin-c, anti-protein s100-a2, anti-apolipoprotein e4, and anti-centrin from rabbit (beijing biosynthesis biotechnology, beijing, china) was used as the primary antibody, and a 1:5000 dilution of the hrp-conjugated goat anti-rabbit igg (sigma-aldrich, st. louis, usa) was used as the secondary antibody. protein digestion of the samples was performed according to the fasp procedure described by wiśniewski et al. (2009) . an aliquot of each cell lysate containing 200 g of protein was combined with 30 l of std buffer containing 4% sds, 100 mm dtt, and 150 mm tris-hcl (ph 8.0). the detergent, dtt, and other low-molecular-weight components were removed by dilution in ua buffer containing 8 m urea and 150 mm tris-hcl (ph 8.0) and repeated ultrafiltration using microcon (30 kda) ultrafiltration units. the reduction of cysteine residues was blocked by the addition of 100 l of 0.05 m iodoacetamide to the ua buffer. the samples were incubated for 20 min in darkness before ultrafiltration. the microcon filters were washed three times with 100 l of ua buffer, followed by two washes with 100 l ds buffer containing 50 mm triethyl ammonium bicarbonate (ph 8.5). the final protein suspensions were digested using 2 g of trypsin (promega, madison, wi, usa) in 40 l of ds buffer overnight at 37 • c, and the digested peptides were collected as the filtrate. the peptide content was quantified based on absorbance at 280 nm using an extinction coefficient of 1.1 for a 0.1 mg/ml solution. the digested peptide mixture was labeled using the 8-plex itraq reagent (life technologies, carlsbad, ca, usa), according to the manufacturer's instructions. each itraq reagent was dissolved in 70 l of ethanol, and added to the digested peptide mixture. the samples were labeled as c1-113, c2-114, c3-115, v1-116, v2-117, or v3-118 . the samples were multiplexed, and vacuum dried. the itraq labeled peptides were fractionated by scxc using the akta purifier system (ge healthcare, waukesha, wi, usa). the dried peptide mixture was reconstituted, and acidified by the addition of 2 ml of buffer a containing 10 mm kh 2 po 4 in 25% acetonitrile (ph 2.7). the samples were loaded onto a 4.6 mm × 100 mm column packed with polysulfoethyl (5 m, 200å) chromatography resin (polylc, columbia, maryland, usa). the peptides were eluted at a flow rate of 1 ml/min using a gradient of 0-10% buffer b containing 500 mm kcl and 10 mm kh 2 po 4 in 25% acetonitrile (ph 2.7). the gradient elution consisted of 10-20% buffer b for 25 min, 20-45% buffer b for 5 min, and 50-100% buffer b for 5 min. the absorbance of the eluate was monitored at 214 nm, and fractions were collected at 1-min intervals. thirty fractions were combined into ten pools, and desalted using empore standard density spe c18 cartridges (sigma-aldrich, st. louis, mo, usa) with a bed diameter of 7 mm and a volume 3 ml. each fraction was concentrated by centrifugation in a vacuum, and reconstituted in 40 l of 0.1% (v/v) trifluoroacetic acid. all samples were stored at −80 • c until the ms analysis was performed. the lc-ms/ms experiments were performed using a q exactive mass spectrometer coupled to a proxeon biosystem easy nanolc (thermo fisher scientific, waltham, ma, usa). ten microliters of each fraction was injected for nanolc-ms/ms analysis. the peptide mixture (5 g) was loaded onto a c18-reversed phase column (15 cm × 75 m) packed with rp-c18 (5 m) resin in buffer a containing 0.1% formic acid, and eluted with a linear gradient of buffer b (80% acetonitrile and 0.1% formic acid) at a flow rate of 0.25 l/min for 140 min using the intelliflow technology. the eluate underwent electrospray ionization for the ms/ms analysis. the ms/ms instrument was run in the peptide recognition mode, and the spectra were acquired using a data-dependent top-10 method based on the selection of the most abundant precursor ions from the survey scan (300-1800 m/z) for hcd fragmentation. the determination of the target value was based on the predictive automatic gain control, and the dynamic exclusion duration was 60 s. survey scans were acquired at a resolution of 70, 000 at m/z 200, and the resolution for the hcd spectra was set to 17, 500 at m/z 200. the normalized collision energy was 30 ev, and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%. the ms/ms spectra were compared to the uniprot cercopithecidae database (107 051 sequences, downloaded november 25, 2013) and a decoy database using the mascot search engine, version 2.2 (matrix science, london, uk), embedded in the proteome discoverer 1.4 software (thermo electron, san jose, ca). the following parameters were used for protein identification: a peptide mass tolerance of 20 ppm; an ms/ms tolerance of 0.1 da; trypsin digestion; a missed cleavage value of 2; the fixed modifications included carbamidomethyl, itraq8plex(k), and itraq8plex(n-term); the variable modification was oxidation; and an fdr value ≤0.01. protein quantification was performed using the proteome discoverer 1.4 software based on the centroided reporter ion peak intensity. the average quantitative value of each protein in samples c1, c2, and c3 (mock-infection group) was used as the internal reference. the value of the quantitative ratio for each protein relative to the internal reference was calculated, and averaged to obtain the quantitative ratio (v/c) of the proteins identified in the treatment groups (unwin et al., 2010) . a protein was considered to be differentially expressed between the pedv-infected and mock-infected groups based on the following criteria: the protein had to be present in three replicates of both groups, the difference in the level of the protein between the two groups had to be statistically significant (p < 0.05), and the change ratio for the protein had to be ≥1.2 (yuan et al., 2012) . the expression of a protein with a v/c > 1.0 was considered to be up-regulated, and those with a v/c < 1.0 were considered to be down-regulated. the data were analyzed using a two-tailed, paired student's t test. the statistical analysis was performed using the excel 2007 software (microsoft, redmond, wa, usa). the deps were annotated using the blast2go, version 2.7.0, program (ashburner et al., 2000; quevillon et al., 2005; götz et al., 2008) . the deps were blasted against the kegg genes database (human). the gene ontology categories (gocs) were retrieved, and mapped to pathways in the kegg database (kanehisa et al., 2012) . table 1 the proteins identified from pedv-infected and mock-infected groups. the vero e6 cells inoculated with pedv displayed distinct cpes at 48 h postinoculation, including cell shrinkage, cell fusion, and a rounded cell morphology, but no cells lysis or shedding was observed (fig. 1a) . the immunoblotting analysis confirmed that the vero e6 cells were pedv-infected. the band corresponding to the np of pedv was detected in samples v1, v2, and v3, whereas none was detected in samples c1, c2, and c3 (fig. 1b) . the identified peptides, identified proteins, quantified proteins, known/uncharacterized proteins, and the goc annotations are showed in table 1 . a total of 3178 proteins, including 15 564 peptides, were identified in the pedv-infected and mock-infected groups using the itraq-ms/ms approach, among which 3171 (99.78%) were quantified, 1859 (58.50%) were known proteins, and 1319 (41.50%) were uncharacterized/putative proteins. based on the gocs, 2061 (64.85%) of the proteins were annotated as biological process, 2495 (78.51%) were annotated as molecular function, and 1917 (60.32%) were annotated as cellular components. the quantification and significance of the identified proteins are shown in fig. 2 . the changes in the levels of expression between the two groups were analyzed based on statistical significance. of the 3178 proteins identified, 2496 (78.54%) were not differentially expressed (p > 0.05), and 675 (21.24%) were expressed at statistically different levels between the pedv-infected and mockinfected vero e6 cells (p < 0.05), including 357 proteins (11.23%) with a p-value between 0.01 and 0.05, 227 proteins (7.14%) with a p-value between 0.001 and 0.01, and 91 proteins (2.86%) with a p-value <0.001. the proteins with a p-value <0.05 were also filtered based on whether the v/c or c/v was ≥1.2. based on these criteria, a total of 126 (3.96%) of the 3178 identified proteins were determined to have been differentially expressed between the pedv-infected and mock-infected groups (table 2) . among the 126 deps, 46.03% (58/126) were up-regulated, and 53.97% (68/126) down-regulated. the known proteins and uncharacterized/putative proteins accounted for 69.05% (87/126) and 30.95% (39/126) of the deps, respectively. the dep displaying the greatest increase in expression in the pedv-infected vero e6 cells was isoform 2 of the ovarian cancer immunoreactive antigen domaincontaining 1 protein (1:2.5), and the dep displaying the greatest decrease in expression in the pedv-infected vero e6 cells was cystatin-c (1:2.2). the gene ontology (go) database has been widely used for describing protein function in a standardized format. according to their gocs, the 126 deps were annotated as cellular component, biological process, or molecular function. the go annotations are shown in table 2 , and distributions of the go annotations are shown in fig. 3 . seventy-eight deps were distributed among 16 groups of biological processes (fig. 3a) . the metabolic process (go:0008152), cellular process (go:0009987), single-organism process (go:0044699), and biological regulation (go:0065007) groups contained the highest proportions of the biological process deps. there were more up-regulated proteins in the cellular component organization group (go:0071840) than down-regulated proteins. seventy-four deps were distributed among eight cellular component groups (fig. 3b) , among which the organelle (go:0043226) and cell (go:0005623) groups contained the highest proportion of cellular component deps. there were more down-regulated deps in the membrane group (go:0016020) than up-regulated deps, and there were more up-regulated deps in the macromolecular complex group (go:0032991) than downregulated deps. ninety-seven deps were distributed among eight molecular function groups (fig. 3c) , among which the binding (go:0005488) and catalytic activity (go:0003824) groups contained the greatest proportion of molecular function deps. the kyoto encyclopedia of genes and genomes (kegg) pathway is a collection of pathway maps that represent molecular interactions and reaction networks in cells. seventy-five of the 126 deps identified were annotated, and mapped to a total of six kegg pathway categories, which included the metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases pathway categories (fig. 4) . the annotations in the metabolism, organismal systems, and diseases pathway categories represented 32, 25, and 36 pathway groups, respectively (fig. 4a, b and f). the annotations in metabolism pathways category included the carbohydrate, energy, lipid, nucleotide, amino acid, glycan biosynthesis, cofactors and vitamins, biosynthesis of other secondary metabolites, and xenobiotics pathway groups (fig. 4a ). the annotations in the organismal systems category included the tolllike receptor (tlr) signaling (ko04620), rig-i-like receptor (rlr) signaling (ko04622), and natural killer cell mediated cytotoxicity (ko04650) pathway groups (fig. 4b) , which represent pathways related primarily to the immune response to virus infection. the largest number of deps in the cellular process category were mapped to the lysosome (ko04142) pathway group, all ten of which were down-regulated deps (fig. 4c ). the annotations in the genetic information processing category included pathway groups related to dna replication and repair, transcription, translation, and the folding, sorting, and degradation of proteins (fig. 4d ). the annotations in the environmental information processing proteins and one up-regulated protein. overall, more disease pathway groups were assigned to a single down-regulated dep than those assigned to up-regulated deps. the integrin (␤2 and ␤3 subunits) protein was annotated to the largest number of pathway groups (28), which included the organismal systems, environmental information processing, cellular processes, and diseases categories. the ␤ tubulin as loading control, three down-regulated deps cystatin-c, apolipoprotein e4 and centrin-2, two up-regulated deps integrin-␤3 and protein s100-a2, were selected to verify differential expression between the pedv-infected and mock-infected vero e6 cells. the immunoblotting analysis showed that the ratios of these proteins between the pedv-infected and mock-infected groups were consistent with those obtained using the quantitative proteomics analysis (fig. 5) . in our study, pedv infection significantly alters protein expression in vero e6 cells. the differentially expressed proteins (deps) annotated to virus infection-associated signaling pathways, autophagy, and virus entry-associated proteins were analyzed further to assess their potential roles in pedv infection. in mammals, the first line of defense against virus infection is the innate immune system. early antiviral responses are initiated upon the recognition of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs), resulting in the production of interferons for the innate immune response and the maturation of dendritic cells for establishing acquired immunity (yokota et al., 2010) . the prrs are grouped into the tlrs, rlrs, and nucleotide binding-oligomerization domain-like receptors. our results showed that pedv infection induced the deps that participated in six signaling pathways involved in viral infection, including the rlr, rap1, pi3k-akt, mapk, jak-stat, and tlr signaling pathways. the pedv is an enteric virus that infects the intestinal epithelial cells (iec) of swine, causing severe diarrhea. hirata et al. (2007) reported the rig-i signaling pathway plays an important role in antiviral innate immunity mechanisms in iecs. sheikh et al. (2013) reported the rap1a signaling pathway was associated with secretory diarrhea. the jak-stat signaling pathway regulates the adaptive and innate mechanisms related to mucosal immunity (heneghan et al., 2013; wang et al., 2013) . our results showed that deps induced by pedv infection in vero e6 cells involved in the rlr, rap1, and jak-stat signaling pathways. it has been reported that the tlr, mapk, and pi3k-akt signaling pathways play roles in host cell responses to coronaviruses (mizutani et al., 2004; integrins are cell surface ␣/␤ heterodimeric glycoproteins that contribute to a variety of cellular functions (stewart and nemerow, 2007) . combinations of the various isotypes of the ␣ and ␤ subunits of integrins generate more than 20 different integrin proteins. previous studies have shown that various integrin molecules are used as receptors for virus attachment (stewart and nemerow, 2007; sun et al., 2013) . in our current study, the expression of integrin-␤2 and -␤3 was down-regulated and up-regulated, respectively, in response to pedv infection. the upregulation of integrin-␤3 expression is consistent with that observed in response to dengue virus infection (zhang et al., 2007) . our pathway analysis revealed that both integrin-␤2 and -␤3 are involved in 28 pathways that contribute to organismal systems, environmental information processing, cellular processes, and diseases. the integrin ␣v␤3 protein has been shown to serve as an entry receptor for various viruses (guerrero et al., 2000; neff et al., 2000; chu and ng, 2004; parry et al., 2005; wang et al., 2005) , some of which bind the integrin through an rgd sequence in a viral structural protein to initiate infection (stewart and nemerow, 2007) . the s protein of pedv is a glycoprotein peplomer on the viral surface that plays an important role in receptor-mediated binding and cell membrane fusion. in our study, the integrin recognized sequences of pedv s protein was analyzed based on ruoslahti's (1996) report. the results indicated that four conserved integrin-recognized amino acid motifs (asp-gly-glu, lys-gly-glu, arg-leu-asp, and leu-asp-val) were found in the s proteins of various pedv strains (data not shown). these data suggest that integrin proteins may act as an infection associated protein for the attachment and entry of pedv. autophagy is an essential component of host defenses against viral infection (dong and levine, 2013) . maier and britton (2012) reported that ␤-coronaviruses induced autophagy. in our study, more deps were mapped to the autophagy pathway group than any of the other pathway groups. fifteen deps were mapped to the lysosome and phagosome pathways. of the 15 proteins, 12 (80%) were down-regulated deps. although the autophagy pathway plays an antiviral role in virus-infected cells, the autophagy machinery is exploited by certain viruses for viral evasion and propagation. our results showed that pedv infection induced the downregulation of the expression of many autophagy-associated proteins. therefore, pedv infection might inhibit autophagy in vero e6 cells, thus facilitating virus replication. previous studies have shown that the microtubule-associated protein 1b is a useful biomarker protein for autophagy (dong and levine, 2013) . we found that the expression of map1b was up-regulated 1.37-fold in the pedv-infected vero e6 cells. these results suggest that the pedv induces autophagy. cystatin-c has been shown to reduce the replication of certain viruses, including the poliovirus, rhinovirus, and human coronaviruses oc43 and 229e (korant et al., 1986; collins and grubb, 1991) . the cleavage of s protein has been shown to be essential for the induction of cell-to-cell fusion and coronavirus entry into cells (sturman et al., 1985) . shirato et al. (2011) reported the transmembrane type ii serine protease 2 enhanced infection of pedv in vero cells by increasing virus release. in our study, the reduced expression of cystatin-c might facilitate pedv replication and release through the activation of cysteine-associated proteases in vero e6 cells. apolipoprotein e4, galectin, clusterin, and transferrin receptor 1 have also been shown to be associated with virus infection (hishiki et al., 2010; peng et al., 2011; martin and uprichard, 2013; tripathi et al., 2013) , and may therefore function as infectionassociated proteins in pedv-infected vero e6 cells. additionally, the decreased in vitro expression of the adherens junction protein, such as cadherin, might be associated with a reduced integrity of pedv-infected intestinal epithelial cells in vivo. to the best of our knowledge, our study represents the analysis of the interactions between pedv and vero e6 cells using a quantitative proteomics technique. pedv infection-associated pathways and proteins are described and discussed based on the bioinformatics analysis of the differentially expressed proteins. our analysis of vero e6 cell responses to pedv infection identified relevant targets for subsequent in-depth studies of pedv pathogenesis, expand the current knowledge base regarding the interaction between the pedv and the host cell, and provide useful basic information about other coronaviruses. although the vero e6 cells are highly susceptible to pedv infection and facilitate experimental design and performance for proteomics, the vero e6 cell line is an interferondeficient cell line and not a pig cell line. so, the detailed functions of these pathways and proteins in pedv infection require further verification in the actual host cells of pedv. gene ontology: tool for the unification of biology mechanisms of coronavirus cell entry mediated by the viral spike protein evolution of clinical proteomics and its role in medicine detection and molecular diversity 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mechanisms by signaling through rig-i/ips-1 in intestinal epithelial cells infectivity of hepatitis c virus is influenced by association with apolipoprotein e isoforms propagation of the virus of porcine epidemic diarrhea in cell culture proteomics of breast cancer: the quest for markers and therapeutic targets origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states kegg for integration and interpretation of large-scale molecular data sets ribavirin efficiently suppresses porcine nidovirus replication viral therapy: prospects for protease inhibitors new variants of porcine epidemic diarrhea virus the conserved set of host proteins incorporated into hiv-1 virions suggests a common egress pathway in multiple cell types involvement of autophagy in coronavirus replication complete genome sequence of porcine epidemic diarrhea virus strain usa/colorado/2013 from the united states identification of transferrin receptor 1 as a hepatitis c virus entry factor protective role of toll-like receptor 3-induced type i interferon in murine coronavirus infection of macrophages tyrosine dephosphorylation of stat3 in sars coronavirus-infected vero e6 cells jnk and pi3k/akt signaling pathways are required for establishing persistent sars-cov infection in vero e6 cells high-efficiency utilization of the bovine integrin alpha(v)beta(3) as a receptor for foot-and-mouth disease virus is dependent on the bovine beta(3) subunit isolation and characterization of a variant porcine epidemic diarrhea virus in china the shotgun proteomic study of the human thinprep cervical smear using itraq mass-tagging and 2d lc-ft-orbitrap-ms: the detection of the human papillomavirus at the protein level herpes simplex virus type 1 glycoprotein h binds to alphavbeta3 integrins crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor a new coronavirus-like particle associated with diarrhea in swine chinese-like strain of porcine epidemic diarrhea virus interproscan: protein domains identifier development of a porcine epidemic diarrhea virus m protein-based elisa for virus detection rgd and other recognition sequences for integrins the epac1 signaling pathway regulates cl − secretion via modulation of apical kcnn4c channels in diarrhea role of proteases in the release of porcine epidemic diarrhea virus from infected cells emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences cell integrins: commonly used receptors for diverse viral pathogens proteolytic cleavage of the e2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90 k cleavage fragments generation of a mouse scfv library specific for porcine aminopeptidase n using the t7 phage display system shotgun proteomic analysis of plasma from dairy cattle suffering from footrot: characterization of potential disease-associated factors identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein influenza a virus nucleoprotein induces apoptosis in human airway epithelial cells: implications of a novel interaction between nucleoprotein and host protein clusterin simultaneous analysis of relative protein expression levels across multiple samples using itraq isobaric tags with 2d nano lc-ms/ms porcine reproductive and respiratory syndrome virus nsp1␤ inhibits interferon-activated jak/stat signal transduction by inducing karyopherin-␣1 degradation integrin alphavbeta3 is a coreceptor for human cytomegalovirus universal sample preparation method for proteome analysis genetic variation analysis of reemerging porcine epidemic diarrhea virus prevailing in central china from quantitative proteomics by amino acid labeling in foot-and-mouth disease virus (fmdv)-infected cells the battle between virus and host: modulation of toll-like receptor signaling pathways by virus infection cytotoxicity evaluation of oxidized single-walled carbon nanotubes and graphene oxide on human hepatoma hepg2 cells: an itraq-coupled 2d lc-ms/ms proteome analysis proteome analysis of porcine epidemic diarrhea virus (pedv)-infected vero cells up-regulated expression of beta3 integrin induced by dengue virus serotype 2 infection associated with virus entry into human dermal microvascular endothelial cells expression and purification of the scfv from hybridoma cells secreting a monoclonal antibody against s protein of pedv this work is supported by the national natural science foundation of china (grant no. 31472209), the state national key laboratory of veterinary biotechnology (grant no. sklvbf201506/201302), and the program for new century excellent talents in heilongjiang provincial university (grant no. 1252-ncet-016). key: cord-315570-khm1veuv authors: gonzález-mora, alejandro; hernández-pérez, jesús; iqbal, hafiz m. n.; rito-palomares, marco; benavides, jorge title: bacteriophage-based vaccines: a potent approach for antigen delivery date: 2020-09-04 journal: vaccines (basel) doi: 10.3390/vaccines8030504 sha: doc_id: 315570 cord_uid: khm1veuv vaccines are considered one of the most important bioproducts in medicine. since the development of the smallpox vaccine in 1796, several types of vaccines for many diseases have been created. however, some vaccines have shown limitations as high cost and low immune responses. in that regard, bacteriophages have been proposed as an attractive alternative for the development of more cost-effective vaccines. phage-displayed vaccines consists in the expression of antigens on the phage surface. this approach takes advantage of inherent properties of these particles such as their adjuvant capacity, economic production and high stability, among others. to date, three types of phage-based vaccines have been developed: phage-displayed, phage dna and hybrid phage-dna vaccines. typically, phage display technology has been used for the identification of new and protective epitopes, mimotopes and antigens. in this context, phage particles represent a versatile, effective and promising alternative for the development of more effective vaccine delivery systems which should be highly exploited in the future. this review describes current advances in the development of bacteriophage-based vaccines, with special attention to vaccine delivery strategies. moreover, the immunological aspects of phage-based vaccines, as well as the applications of phage display for vaccine development, are explored. finally, important challenges and the future of phage-bases vaccines are discussed. the development of vaccines represents one of the greatest advances in medical fields which have saved a huge number of human and animal lives [1] . nowadays, several research groups around the world have focused on the development of more effective, better, safer, inexpensive and with long-lasting immune response vaccines for medical applications [2] [3] [4] . currently, different types of vaccines such as live-attenuated, inactivated, synthetic, among others have been successfully developed and tested for preventive purposes. conventional vaccines are made of inactivated or attenuated microorganisms. although significant improvements have been made regarding conventional vaccines, drawbacks as difficulties to culture microorganisms, low efficacies and potential risks related to pathogenic reversion or transmission to immunocompromised patients of these vaccines have been reported [4] . therefore, the administration of specific and contaminant-free antigens has been proposed as a new strategy to overcome the limitations previously mentioned. thus, novel vaccines based on figure 1 . simplified schematic representations of the three types of phage-based antigen delivery systems currently reported. an antigen delivery system based on a hybrid phage-dna vaccine combines the concepts of the other two systems. it is based on a phage displaying on its surface peptides with specific affinity towards antigen presenting cells (apcs) and at the same time, it harbors a dna plasmid encoding the therapeutic antigen in a eukaryotic expression cassette. phages are involved in a wide range of applications such as drug delivery, phage therapy, biosensors development and as vaccine delivery systems [6, 13, 14] . many of these applications are possible as a result of the development of phage display technology, which lies on the manipulation of bacteriophages to present antigens on their surface. until now, phage display vaccines have been used for preventing or treating several diseases including cancer, viral, parasitic and fungal infection as well as their use in immunocontraception and drug abuse, among others [2, [15] [16] [17] [18] . vaccines targeting drug abuse consists on the use of phages particle displaying antibodies that block the effects of different drugs such as the cocaine [15] . bacteriophages are a specific class of viruses that infect and replicate into bacteria and archaea and are incompetent for eukaryotic infection [10, 11] . these viral particles are made by genetic material, dna or rna, packaged in either simple or elaborate protein-made structures known as capsids. phage display is a potent technology that consists on the expression of either peptides or proteins fused to coat proteins of the phage's surface [11, 19, 20] . this technique has been used in biotechnology for several applications such as: directed evolution [21] , identification of ligand binding sites (protein-protein, protein-ligand and protein-dna interactions) [20] , selection of monoclonal antibodies [22] , protein-based drug discovery [23] , development of epitope-based diagnostic tools, identification of b-cell epitopes [24] and for vaccine development [19, 25] . filamentous phages (m13, fd, and f1), lytic phages (t4 and t7) and the temperate phage lambda (λ) have been successfully used as display systems [10, 19, 20, 26] . from all these systems, bacteriophage m13 is the most broadly used in phage display since its purification from an e.coli lysate is easier compared to other phages [10, 27, 28] . filamentous phages are a group of non-lytic viruses with a circular single-stranded dna genome with a length around of 6.4 kb in the case of m13 phage [20, 29] . as a result of the non-lytic nature of filamentous phages, these particles can be obtained in high titers with reduced bacterial figure 1 . simplified schematic representations of the three types of phage-based antigen delivery systems currently reported. an antigen delivery system based on a hybrid phage-dna vaccine combines the concepts of the other two systems. it is based on a phage displaying on its surface peptides with specific affinity towards antigen presenting cells (apcs) and at the same time, it harbors a dna plasmid encoding the therapeutic antigen in a eukaryotic expression cassette. phages are involved in a wide range of applications such as drug delivery, phage therapy, biosensors development and as vaccine delivery systems [6, 13, 14] . many of these applications are possible as a result of the development of phage display technology, which lies on the manipulation of bacteriophages to present antigens on their surface. until now, phage display vaccines have been used for preventing or treating several diseases including cancer, viral, parasitic and fungal infection as well as their use in immunocontraception and drug abuse, among others [2, [15] [16] [17] [18] . vaccines targeting drug abuse consists on the use of phages particle displaying antibodies that block the effects of different drugs such as the cocaine [15] . bacteriophages are a specific class of viruses that infect and replicate into bacteria and archaea and are incompetent for eukaryotic infection [10, 11] . these viral particles are made by genetic material, dna or rna, packaged in either simple or elaborate protein-made structures known as capsids. phage display is a potent technology that consists on the expression of either peptides or proteins fused to coat proteins of the phage's surface [11, 19, 20] . this technique has been used in biotechnology for several applications such as: directed evolution [21] , identification of ligand binding sites (protein-protein, protein-ligand and protein-dna interactions) [20] , selection of monoclonal antibodies [22] , protein-based drug discovery [23] , development of epitope-based diagnostic tools, identification of b-cell epitopes [24] and for vaccine development [19, 25] . filamentous phages (m13, fd, and f1), lytic phages (t4 and t7) and the temperate phage lambda (λ) have been successfully used as display systems [10, 19, 20, 26] . from all these systems, bacteriophage m13 is the most broadly used in phage display since its purification from an e. coli lysate is easier compared to other phages [10, 27, 28] . filamentous phages are a group of non-lytic viruses with a circular single-stranded dna genome with a length around of 6.4 kb in the case of m13 phage [20, 29] . as a result of the non-lytic nature of filamentous phages, these particles can be obtained in high titers with reduced bacterial contamination, making their purification easier and cheaper [30] . due to their inherent viral particle features, filamentous phages are considered vaccine carriers with high immunogenic potential [11, 31] . these particles are flexible protein-based cylinders composed of five coat proteins (piii, pvi, pvii, pviii, and pix). from these, proteins piii, pvi, pvii and pix are the minor coat proteins, meanwhile pviii is the major coat protein which surrounds the whole particle. piii and pviii proteins are the most used for the display of fusion peptides due to their specific location on the phage structure (table 1) [10, 32] . short peptides (8 or fewer amino acids) can be effectively fused to many copies of the protein pviii since the display level have been found to be dependent of the length and sequence of the peptide displayed [33] . since the valency (protein copies per viral particle) has a direct relationship with the immune response to be generated, the proportion of immunogenic peptides displayed on protein pviii has been extensively used for vaccine development [31, 34, 35] . on the other hand, protein piii is preferred for the expression of larger peptides since the phage has fewer copies and steric effects may be negligible [28, 36] . furthermore, important advantages of using filamentous phages for the development of vaccines include that phages are stable under harsh conditions (ph and temperature), the phage size is determined by the length of the dna molecule it harbors and the phage genome capability to be used as cloning vector [10, 28, 37] . although these features surely encourage the use of filamentous phages as antigen display systems, a strategy to address their inefficiency to display peptides above 8 amino acids long on the major coat protein pviii is needed to maximize the display flexibility of the system [38] . lytic phages t4 and t7 have also been employed in phage display for vaccine applications [10] . the capsid proteins of phage t4, hoc and soc [39] , can be used to display larger proteins at high copy numbers more efficiently than filamentous phages (table 1 ) [40] . at the same time, t4 phage capsid proteins have demonstrated to promote an immune response in humans and mice [41] . in addition, oral administration of whole wild type t4 phage to humans demonstrated to be highly safe in clinical trials [39] . some advantages of using t4 phages as vaccine carriers include the high immunogenic activity exerted by their capsid proteins, plasmid compatibility to allow engineering of dual protein expression and the absence of toxic proteins secreted. based on these features, t4 phage has been preferred in some approaches as a display vector over filamentous phages commonly used [42] . on the other hand, the carboxyl-terminus of protein 10 b of phage t7 has been engineered to display heterologous peptides in antigen display strategies [43] . some of the advantages of using the t7 phage for protein display include its high cloning capacity, high stability to harbor foreign gene inserts and its rapid propagation among other features [44] . in addition, humoral and cellular immune responses have been reported to be activated following the administration of recombinant t7 phage particles in animal models [45] . although filamentous phages are the most common vectors used in phage display, the λ phage has also been proposed as an antigen display platform [46] . it has been reported that the λ phage can display properly folded antigenic peptides and 2-3 times larger fusion proteins than filamentous phages, offering a plausible alternative for complex antigens display [10, 47] . for instance β-galactosidase, a protein larger than 400 kda, have been properly displayed on the λ phage surface without affecting phage viability and morphology [35] . moreover, it has been reported that the display of peptides using the λ phage generate particles with higher antigen densities when compared to the density of peptide displayed by filamentous phages [10, 35] . impact vaccine development since the displayed proteins must be properly folded to exhibit the desired activity. on the other hand, in vitro display systems have demonstrated to overcome the limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [29] . desired activity. on the other hand, in vitro display systems have demonstrated to overcome the limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [29] . impact vaccine development since the displayed proteins must be properly folded to exhibit the desired activity. on the other hand, in vitro display systems have demonstrated to overcome the limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [29] . desired activity. on the other hand, in vitro display systems have demonstrated to overcome the limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [29] . desired activity. on the other hand, in vitro display systems have demonstrated to overcome the limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [29] . limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [29] . protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [29] . protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [29] . in vivo and in vitro phage display systems. in an in vivo phage display system, the expression and fusion of the displayed protein occurs at the phage infection stage. although many large proteins have been successfully displayed in phage vectors using in vivo systems, limitations such as variations in intracellular expression, protein aggregation and deficient phage assembly have been described [48] . such drawbacks can negatively impact vaccine development since the displayed proteins must be properly folded to exhibit the desired activity. on the other hand, in vitro display systems have demonstrated to overcome the limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [29] . as mentioned in previous sections, phage display has been proposed as a potent tool for vaccine development [51, 52] . this technology has been used for this purpose in two main strategies: (1) to produce vectors displaying antigens and (2) to identify new protective antigens [6] . the use of phage vectors as antigen display agents is focused on both, the development and production stages of a vaccine. on the other hand, phage display can be employed in early stages of the vaccine design process for the identification of novel antigens through the use of genetic engineering to generate random proteins and peptides aimed to interact specifically with a target, allowing the selection of the best binding antigen. phage display vaccines relies on the successful expression of antigens fused to phage surface proteins to produce viral particles with specific immunogenic activity [25] . table 2 describes the phage display vaccines that have been currently reported. it is worth mentioning that anticancer vaccines successfully developed in recent years have already been used in cancer immunotherapy [53] . administration of these anticancer phage vaccines stimulate the immune system of the host to produce antibodies against cancer antigens to reduce tumor cells proliferation [17] . to identify the best immunogenic antigen for phage-based cancer vaccines, several candidates have been evaluated. for instance, some antigens reported are: epitopes from epidermal growth factor receptor (egfr) [54] , melanoma antigen gene (mage), and fms-like tyrosine kinase 4 (flt4) [55] . since the discovery of the capacity of filamentous phages to exert anti-tumor activity in rabbits and mice [33] , the use of phage particles as immunotherapy to treat melanoma tumors has become a very compelling research field. studies on the administration of m13 and t4 phages to mice tumor models have demonstrated that the mechanism by which anti-tumor phages induce tumor elimination is mediated by the activation of tumor-associated macrophages in a myeloid differentiation primary response 88 (myd88)-dependent way [56] . interestingly, the effect of phage administration was observed with a single dose of 1 × 10 11 plaque forming units (pfu) injected subcutaneously. on the other hand, it has been reported that the administration of anti-tumor phages stimulates the infiltration of neutrophilic granulocytes, leading to the development of metastatic activity that results in damage of tumor tissue and reduction of viable tumor cells [57] . in other study, ericksson et al. reported that the mechanism of action for the tumor degradation caused by bacteriophages is toll-like receptor (tlr) dependent. this study also reported effects on the production of proinflammatory cytokines, on the secretion of molecules necessary for antigen presentation by dendritic cells and co-stimulation of macrophages and t cells [57] . these findings suggest the high capacity of phage particles to induce immunogenicity and prompts the development of phage-based vaccines for cancer immunotherapy. a phage-based vaccine aimed to treat amyloid plaque from alzheimer disease produced an increased igg response in a transgenic mice model, demonstrating the capacity of phage-based vaccines to activate humoral responses [16] . immunocontraceptive vaccines have also gained attention for phage-based therapies. for instance, the gonadotrophin releasing hormone (gnrh) antigen was displayed on the surface of t7 phages for immunocastration in a mice model [58] . antiviral phage-based vaccines have also been successfully developed. epitopes from hiv, hepatitis b, herpes simplex virus 1 (hsv-1), hsv-2 [34] , circovirus 2 (pcv2) [29] , human respiratory syncytial virus [59] among others have been phage displayed. phage display has demonstrated to be an effective, inexpensive and fast technique for the identification of immunogenic proteins and peptides allowing the development of novel and more effective vaccines [19, 36, 64] . this methodology incorporates principles of genetic engineering and combinatorial chemistry [52] . it consists in cloning natural and random cdna sequence variants into the phage genome to be expressed on the phage surface to generate a population of phages carrying different candidate antigens. from these libraries, phages with more affinity to the desired target (antibody for antigen identification) are selected and the dna sequence encoding for the antigen is identified (figure 2 ) [36] . the high efficiency and low costs that have been reported for the design of several vaccines using phage display technology for antigen identification are highly relevant features that must be taken into consideration while choosing the best platform for drug design [20] . phage display has demonstrated to be an effective, inexpensive and fast technique for the identification of immunogenic proteins and peptides allowing the development of novel and more effective vaccines [19, 36, 64] . this methodology incorporates principles of genetic engineering and combinatorial chemistry [52] . it consists in cloning natural and random cdna sequence variants into the phage genome to be expressed on the phage surface to generate a population of phages carrying different candidate antigens. from these libraries, phages with more affinity to the desired target (antibody for antigen identification) are selected and the dna sequence encoding for the antigen is identified (figure 2 ) [36] . the high efficiency and low costs that have been reported for the design of several vaccines using phage display technology for antigen identification are highly relevant features that must be taken into consideration while choosing the best platform for drug design [20] . this technology has allowed the identification of epitopes, mimotopes, bacteria adhesins and vaccine components (table 3 ) [10, 35] . one of the greatest advantages of using this method is the rapid identification and isolation of phages expressing specific targets [20] . at the same time, the protein piii from m13 has been the preferred protein for fusion since high affinity of specific targets towards the displayed antigens have been observed [36] . this technology has allowed the identification of epitopes, mimotopes, bacteria adhesins and vaccine components (table 3) [10, 35] . one of the greatest advantages of using this method is the rapid identification and isolation of phages expressing specific targets [20] . at the same time, the protein piii from m13 has been the preferred protein for fusion since high affinity of specific targets towards the displayed antigens have been observed [36] . table 3 . cases of epitope, mimotope and antigens identification through phage display describing the main findings in regards of the phage display protocol used for selection and the prophylactic and therapeutic results reported. phage display technology has been applied for the identification of new and more specific linear and continuous antigenic determinant epitopes which normally have a length of 4-6 amino acids [20, 69] . for instance, in a recent published paper, chung-tao et al. reported the use of phage display to identify novel enhanced epitopes from the envelope protein of dengue virus which was used later for the development of a dna vaccine [24] . authors concluded that the phage display methodology could be a key element for developing novel and more immunoprotective dengue vaccines [24] . grabowska et al. demonstrated the applicability of phage display technology for the display and discovery of new protective virus hsv-2 epitopes [34] . table 3 summarizes some of the epitopes identified using phage display technology. altogether, this data supports the use of phage display for the development of new vaccines, diagnostic tools and immunotherapies [28] . phage display libraries and biopanning have also been used for the identification of mimotopes of lipid and carbohydrate antigens which often present low immunogenicity [33, 70] . mimotopes are short peptides that structurally mimics epitopes, enabling a conformational interaction with an antibody [28] . these short amino acid sequences are used to produce antibodies against specific antigen epitopes and have several advantages over original antigens or epitopes since they are easy to produce for being short sequences, can mimic non-protein antigens, can be tested against undiscovered antigens and they have demonstrated to possess high bioactivity [17, 70] . thus, several mimotopes displayed on phages have produced effective in vivo responses in murine and swine models (table 3) . nowadays, mimotope-based vaccination is considered an effective immunotherapy approach to induce specific antibody responses [71] . at the same time, use of phage display libraries have allowed the identification of amino acid sequences that mimics specific immunogenic and antigenic regions of toxins. by this approach, epitopes from botulinum neurotoxin a [72] , the beta-mammal toxin cn2 and noxiustoxin (ntx) toxins have been discovered [73, 74] . recently, jahdasani et al. used phage display technology for the identification of immunogenic epitopes from the scorpion venom of hemiscorpius lepturus to develop a preventive strategy against scorpion bites [69] . successful results were obtained demonstrating the applicability of this tool for the development of novel vaccine candidates in the antivenom area. phage display technology has also been used for the identification of new anti-parasite antigens. in this regard, antigens from brugia malayi [75] , plasmodium falciparum [32] , leishmania major [23] have been elicited through this platform. by using phage display to identify peptides with binding capacity to the tegument of schistosoma japonicum, liu et al. discovered the zl4 peptide which showed a potent antiparasitic activity [76] . likewise, phage display for the identification of epitopes for taenia solium paramyosin to develop a sole epitope-based vaccine has been described [77] . nowadays, there are no vaccines against pathogens transmitted by ticks, which have been described as a serious health risk [65] . to overcome this problem, becker et al. used oligopeptide phage display for the identification of antigens present in the saliva of the black-legged tick, ixodes scapularis, which have been found to prevent pathogen transmission. authors identified the metalloprotease 1 (mp1) as a potential candidate for the development of an anti-tick vaccine by testing mp1 immunogenicity in human sera [65] . as can be seen, phage display, a more than 30 years old technique, is an important tool for vaccine development and immunotherapy that had not been harnessed for these purposes until 10 years ago. at the same time, phage display has been applied for the enhancement of antigen-antibody interactions leading to the design of more potent vaccines [20] . the exploitation of this technique for vaccine development may lead to important advances for the prevention of several important infectious diseases [20, 24] . dna vaccination consists in the direct administration of a foreign dna encoding an antigen to induce host immunity. for this approach, use of dendritic cells as transfection targets have demonstrated to increase immunity [35] . dna vaccination has been proposed as a viable alternative when a protein or peptide vaccination is not viable or unsuccessful [3] . several advantages of dna vaccines have been described, for instance: antigen is folded correctly inside the host's cell, downstream processing is not required which significantly decreases overall manufacture costs, null risk of vaccine to become pathogenic, dna can be obtained in large quantities and high purity at relatively low costs and no strict storage conditions are required for dna products [78, 79] . moreover, since this approach have demonstrated to stimulate both cellular and humoral immune responses, dna vaccines have been proposed as promising immunotherapy treatments [80] . unfortunately, standard dna vaccines have shown poor immunogenicity in large primate trials, meanwhile in human trials the use of adjuvants such as dendritic cells have been identified as mandatory [80, 81] . the low stability and the poor distribution of naked dna vaccines diminish hugely the efficacy of the immune response, making imperative the use of a delivery vehicle [8] . to note, currently, there are not commercial dna vaccines for human medical applications. however, novel vaccines based on nucleic acids have been rushed into clinical trials amid the sars-cov-2 pandemic, pointing out their promising efficiency as immune system stimulators [82] . in that regard, since phage particles have shown adjuvant properties, they have been proposed as new and suitable vehicles for dna delivery. in concept, a bacteriophage dna vaccine is made of a eukaryotic expression cassette encoding a specific antigen controlled by target-specific promoters contained within a bacteriophage ( figure 1 ) [3, 25] . the expression cassette must have the necessary regulatory sequences to allow the correct gene expression and protein folding of the antigen. in this strategy, phage particles serve as passive carriers by allowing the transference of the dna encoded antigen to eukaryotic cells where the dna will be expressed [6] . phage-dna vaccines are considered more advantageous than standard dna vaccines since the former are more stable for storage, transport and administration (mainly via oral route), since dna is protected from degradation by the capsid of phage particles. although y phage is the most common phage vector for dna vaccination, filamentous phages have also been tested [37, 83] . an important feature to consider of filamentous phage-dna vaccines is the fact that phagemid vectors can efficiently contain multiple gene copies, which may allow immunization with various epitopes or antigens using a single delivery vector [37] . table 4 describes the phage-dna vaccines currently developed. furthermore, it has been demonstrated that phage-dna vaccines can induce a more effective immunoprotection when compared to naked-dna strategies [10, 79] . for instance, march et al. reported a higher immune response by using a lambda-dna vaccine when compared to the effect of a naked plasmid vaccine even at lower doses [79] . in other work, clark et al. reported a higher immune response when using a dna vaccine compared to the immune effect caused by the commercial vaccine engerix b, based on a recombinant protein-small surface antigen (hbsag) of hepatitis b. the enhanced performance of the dna vaccine was attributed to a better folding of the protein expressed in the host, better compatibility with post-translational modifications on the antigen, the adjuvant effect of the phage particle, the presence of lipopolysaccharides and lipid incorporation when the protein is secreted [3] . notably, phage dna vaccines have exhibited more effectiveness when multiple doses (4 × 10 11 phages per dose at intervals of 0, 5 and 10 weeks) are administered. this effect has been attributed to poor dna expression in the first immunization since the host's immune response focuses on phage coat proteins incapacitating a proportion of the dna-carrying phages. however, the complete mechanism has not been elucidated [3] . based on the favorable characteristics of phage particles and compared to other vaccine systems, production of phage dna vaccines is simple and low-cost, the viral particle do not carry antibiotic resistance genes, the phage genome is suitable for the insertion of large dna antigen sequences (up to 20 kb in lambda vectors) and are quite stable compared to standard dna vaccines [3] . in addition, phage dna vaccines are safer than dna vaccines using adenovirus vectors since the bacteriophage is unable to replicate into eukaryotic cells [79] . hybrid bacteriophage vaccines are produced by the combination of phage display and phage dna vaccines. the most recent advances in phage-based vaccines have focused on improving the interaction and internalization of phage particles by antigen presenting cells (apc) to increase the immune response. this system relies on bacteriophages displaying proteins or peptides with high affinity to antigen-presenting cells or the antigen itself and at the same time they carry on their genome an eukaryotic expression cassette encoding a specific antigen with the final aim of increasing the immune response by combining both effects (figure 1 ) [6] . for such reasons, this strategy has been proposed to be implemented as a directed therapy based on its capacity of delivering antigenic genes to activators of the immune response such as dendritic cells. it has been reported that this kind of vaccines are capable to successfully induce cellular and humoral immune responses [25] . unfortunately, the mechanism for dna delivery used by phages has not been completely elucidated [10] . although the cellular internalization of phage particles seems to be quite efficient, the nuclear uptake efficiency must be improved. in 2008, sartorius et al. developed a double-hybrid filamentous bacteriophage fd co-displaying peptides recognized by the major histocompatibility complex (mhc) class i and mhc class ii cell surface receptors and epitopes from the antigen mage aiming to enhance the anti-tumor immune activity based on ctl responses [62] . by observing an increase in the ctl response through the administration of hybrid phages, sartorius et al. proposed this strategy as a potent tool for the development of more effective anti-cancer vaccines. ideally, a vaccine carrier should be easy to produce in large quantities, safe and capable to effectively present antigens to the immune system to induce a proper immune response. viral particles and more specifically bacteriophages have demonstrated to possess many of the properties previously mentioned. thus, in recent years bacteriophage-based vaccines have gained attention for medical applications considering their low production costs, relative simplicity to prepare and to genetically modify, easiness to produce at large scales, their adjuvant capacity, high stability under a wide range of ph and high temperature, resistance to nucleolytic and proteolytic enzymes and desiccation [25, 29, 47] . the capability of phages to be genetically modified to be used as display or delivery systems represents an important aide in emergency situations, for instance outbreaks of infectious pathogens [35] . in this context, recombinant phages can be produced in large amounts at low cost by basic methods making phage-based vaccines more cost-effective than standard or classical vaccine development strategies [59] . according to munira et al. the cost of producing recombinant vaccines is estimated on average of 2 usd per dose [84] . on the other hand, in, torres-acosta et al. estimated the production cost for a phage-based vaccine was around $0.9 usd per dose (10 12 pfu) [85] . these estimations suggest a 55% reduction on the production costs of phage-based vaccines compared to recombinant-based vaccines, supporting the implementation of phages in vaccine development strategies. the high stability exhibited by filamentous phage preparations and their capacity to protect antigens from degradation makes bacteriophages ideal vaccine vehicles based on their storage and transportation resilience and delivery features. based on the discussion in previous sections of this review, phage-based vaccines are more suitable for immunization than dna naked or recombinant vaccines. in that regard, brigati and petrenko evaluated the stability of landscape phages-multivalent pviii-type filamentous-phage constructs with unique structures and properties-expressing peptides fused to protein pviii at high-temperature (63 • c) and observed that these particles retained stability without affecting their binding sites [86] . this capability of phages is quite remarkable and has to be taken into consideration for the development of vaccines that will be distributed to remote areas where no adequate storage conditions are found or for veterinary applications that requires vaccine administration in the native habitats of animals [11] . the use of phages as antigen carriers has the advantage of increasing the half-life of the antigen in the blood stream facilitating the activation of t-helper cells, enhancing the immune response [87] . to study the immunogenicity efficiency of phage display, wu et al. compared the immune response of recombinant antigens with phage-displayed antigens and reported that the antigens expressed on the phage surface exhibited a better refolding leading to a more efficient activation of the immune system [49] . this results, along with the increase observed on cellular immunity by the administration of hybrid-phages displaying apcs-targeted peptides [11] , demonstrates the remarkable properties of phage-displayed antigens bacteriophages are the most abundant organisms that inhabit the entire planet [88] , suggesting their high capability to coexist with a plethora of micro and macro organisms. for such reason, it is not unusual that these particles have proved to be harmless in mammalian models even in oral administration trials in humans approved by the fda [10, 29, 39, 89] . to reinforce even more the safety of phage administration, as any other viral particle used as vaccine, bacteriophages can be physically or genetically inactivated as well [90] . furthermore, phage particles have also been used in therapeutic applications in humans without side effects being observed [91] . thus, the safe application of phage-based vaccines have been demonstrated as bacteriophages are not capable to infect and replicate into eukaryotic cells and no side effects have been reported contrary to the outcomes observed in mammalian model using other viral vectors [10] . although many proteins and peptides have been successfully displayed in bacteriophages, the correct display of molecules on the phage surface is still considered a potential drawback for the development of these vaccines. the issue relies on two related subjects; proper folding of the polypeptide sequences displayed, and the epitope density displayed. both features, correct folding and presence of enough active epitopes to generate a significative immune response have a direct effect on the overall efficiency of these vaccine [25] . an equally important factor to have in mind while designing any kind of vaccine is the administration route. although phage particles have showed to be safe for animal and human use, phages administered by oral route may infect gut bacteria, potentially leading to a dysbiosis. the infection and further replication of phages could induce the release of endotoxins from infected bacteria, increasing the risk of host damage [92] . this risk can be eliminated by using non-lytic bacteriophages such as the m13 phage. several authors have described that the natural immunogenic properties of phage particles relies mainly on their chemical structure (phage proteins and viral dna) [47] . this capacity makes bacteriophages excellent candidates for their use as antigen delivery vectors. in recent years, although not completely, the immunogenic effects in hosts driven by phage-based vaccines have been described, allowing a more rational design of novel and better phage vaccines. several authors have reported the capacity of filamentous phages to act as natural vaccine adjuvants since these viral particles are capable to achieve an effective antigen presentation to immune cells [47] . this notable feature allows phage-based vaccines to enhance the stimulation of the immune response, obtaining better results than conventional vaccines even at relatively low doses [52, 93] . in addition, it has been reported that phage-based vaccines produce less variability between subjects than conventional vaccines [25] . it has been suggested that filamentous phages evolved to induce low non-specific immune response in eukaryotic organisms [94] . however, filamentous phages still can induce an effective activation of the immune machinery by themselves apart from activation of both cellular and humoral immune responses by being engineered to directly interact with apcs, giving them an advantage over other delivery systems [29] . the immunogenicity exhibited by filamentous bacteriophages have been demonstrated to be caused not only by their repeating coat proteins but also mediated by the phage dna itself [57, 95] . this was demonstrated by the administration of dna from m13 phage to mice in a strategy to induce in vivo heterologous expression of interferon (predominantly the beta type) to confer the mice protection from a vaccinia virus infection. the immune response was in part attributed to the presence of cpg motifs in the phage genome [3, 96] . phage vaccination trials in mice have demonstrated that cpg motifs stimulates t helper type cells (th1) and b cells leading to an enhanced immune response [97] [98] [99] . cpg motifs have been also described to be involved in the stimulation of the toll-like receptor 9 (tlr9) signaling cascade, modifying the production of different immunoglobulin classes [100] . in the case of the t4 phage, the major capsid protein gp23 and the highly immunogenic outer capsid protein (hoc) have shown high antigenicity [55] . hashiguchi et al. investigated the immune response derived from the administration of m13 phages in an in vivo murine model. authors found that the administration of phages induced an equivalent immune response as the typical immunogen sheep red blood cells (srbc), reinforcing the theory that m13 phage may be an important vector for vaccine delivery [95] . in another report, it was found that filamentous phages induce a stronger immune response than the carrier protein ovalbumin [93] , demonstrating that bacteriophages can induce an immune responses by their own, supporting their use as promising vaccines carriers. interestingly, van houten et al. reported that reducing the complexity-quantity of different b-cell epitopes of immunodominant epitopes of filamentous coat proteins in phages enhanced the antibody response against synthetic peptides fused to the phage surface [31] . thus, the adjuvant-like effect exhibited by phages in various vaccination studies along with their capacity to effectively present peptides or proteins to the immune system leading to the activation of cellular and humoral immune responses [40] demonstrates that engineered phage particles are a proper strategy to enhance the efficacy and safety of viral particle-based vaccines. from a metabolic point of view, bacteriophages are considered inert antigen particles whose cell internalization mechanisms are still being studied. it is widely accepted that phages are internalized by endocytosis and processed by apcs [101] . once phage particles are internalized by apcs, antigens are processed and presented through the major histocompatibility complex (mhc) class i and ii pathways (figure 3) , stimulating cellular and humoral immune responses [52] . phage-displayed antigens have been described to induce specific cd4+ and cd8+ t cells lymphocytes (ctls) responses, leading to the production of specific antibodies and activation of t helper cells without the utilization of supplementary adjuvants. also, it has been reported that macrophages are capable to internalize phages via phagocytosis [102] . helper cells without the utilization of supplementary adjuvants. also, it has been reported that macrophages are capable to internalize phages via phagocytosis [102] . antigen presentation via the mhc class i pathway have been demonstrated to activate the ctl through the interaction with cd8+ t cells (figure 3 ). it has been suggested that standard vaccines (soluble exogenous antigen or inactivated pathogens) fail to stimulate the mhc class i pathway, and therefore are incapable to activate t cells, which are important to promote an optimal immune response [103] . in that sense, it has been reported that filamentous phages displaying antigens are efficiently processed and presented by the mhc class i and class ii pathways, demonstrating their high potential as positive stimulants of the immune system [37, 60, 62] . activation of the mhc class i pathway and further triggering of ctl responses have been reported to play a key role in figure 3 . immunological mechanism of phage-based vaccines. phage-based vaccines can stimulate both humoral and cellular responses. phage particles displaying-antigens are taken up by apcs. the antigen is then processed and presented on mhc-i and mhc-ii molecules which leads to the activation of t cells. the direct recognition of the phage particle happens simultaneously, promoting the generation of plasma cells and further secretion of antibodies. antigen presentation via the mhc class i pathway have been demonstrated to activate the ctl through the interaction with cd8+ t cells (figure 3 ). it has been suggested that standard vaccines (soluble exogenous antigen or inactivated pathogens) fail to stimulate the mhc class i pathway, and therefore are incapable to activate t cells, which are important to promote an optimal immune response [103] . in that sense, it has been reported that filamentous phages displaying antigens are efficiently processed and presented by the mhc class i and class ii pathways, demonstrating their high potential as positive stimulants of the immune system [37, 60, 62] . activation of the mhc class i pathway and further triggering of ctl responses have been reported to play a key role in anti-cancer and anti-viral drug approaches [25] . in that regard, wan et al. reported that filamentous phages displaying an exogenous antigen successfully triggered a mhc class i-restricted cd8+ t cell response [61] . in another work, sartorius et al. demonstrated the capacity of hybrid filamentous phages to induce t cell-dependent ctl responses when specific epitopes were displayed on the phage surface [62] . moreover, phages can stimulate apcs for the secretion of costimulatory molecules required for t cell activation [57] . phage display technology allows the design of engineered phages capable to express molecules that specifically targets presenting cells for the induction of a strong cellular response [104] . iwagami et al. reported t cell activation of cd4+ and cd8+ with the co-expression of cd154 and cd137 markers after administration of a y phage-based vaccine displaying the peptidic antigen aspartate-β-hydroxylase (asph) [47] . in the same study, the response also included an increase on the secretion of ifnγ and the asph antigen displayed in the y phage generated specific cd4+ and cd8+ responses in vitro [47] . thus, phages capacity to induce a ctl response supports the use of bacteriophages as vaccine carriers capable to enhance the immune response produced in stand-alone antigen strategies. likewise coat proteins, bacteriophage dna has exhibited to possess an immunostimulatory effect. in that regard, cuesta et al. reported that a dna fragment of the domain i of the coat protein iii of filamentous phages is capable to enhance the t helper 1 humoral and cellular immune responses when fused to a single-chain variable fragment (scfv) [105] . antigen presentation to naïve cd4+ t cells via mhc-ii pathway has demonstrated to leads to the activation of th1 and th2 cells (figure 3 ) [94] . also, in vitro and in vivo studies have confirmed that recombinant filamentous phages are capable to trigger a strong immune response mediated by t helper cells. this response is mediated by phage components such as the coat proteins, phage dna as well as phage-associated lipopolysaccharide (lps) which can direct the activation of th1 and th2 cells [11, 34] . to study the effects of phages in the immune system, iwagami et al. immunized mice using a y phage vaccine at a dose of 10 10 pfu and observed an increased secretion of specific cytokines of th1 (ifn-γ and tnf-α) and th2 (il-4, il-6 and il-10) [47] . in the same study, authors observed that activation of th1 generated a ctl response, suggesting that phage particles may be great candidates to consider for the development of vaccines focused on ctl response activation. on the other hand, a phage-mimotope vaccine against fasciola hepatica showed a mixed th1 and th2 cells responses, advocating the approach of using phage particles in vaccination strategies targeting cellular responses [106] . phage particles have demonstrated their capacity to induce humoral responses through the direct activation of b cells as well as through the activation of th2 cells (figure 3 ) [94] . it has been proposed that for a better b cell response, antigens must be presented in a repetitive and organized configuration [43] . studies focused on the administration of phage display vaccines have also shown the induction of immune response in animal models injected with recombinant vaccines and mimotopes expressed and fused to coat proteins of phages at a dose of 10 11 pfu [95] . also, it has been reported that the antibody response induced by m13 phages is limited to recognition of the first 12 n-terminal residues of protein pviii and to the external domains of protein piii [95] . in 2010, hashiguchi et al. studied the in vivo immune response induced by m13 phage with the aim to characterize this effect for vaccine development [95] . the researchers administered a solution containing highly purified m13 phages (10 11 pfu) to mice and observed the antibody response. they reported a strong primary response based on high production of igg antibodies which was attributed to a complete dependence on the activation of the myd88 pathway involved in innate immune response [95] . other studies have suggested that bacteriophages may play an important role in the innate response activation by stimulation of tumor-associated macrophages (tams), which secretes immunomodulators that assists the recruitment of neutrophils [57] . unfortunately, the complete molecular mechanism of the activation of innate immunity by phages has not been completely elucidated. in 2012, thomas et al. produced a divalent subunit vaccine expressing green fluorescent protein (gfp) as model antigen and the hiv-1 tat protein to enhance cell uptake and thus establish a subunit λ-based vaccine [35] . this platform allows the display of many epitopes at the same time, providing an easy and rapid approach to develop novel vaccines against emerging pathogens. authors reported a strong immune stimulation of splenocytes using the λ-based vaccine demonstrating the effectiveness of phage preparations to induce a strong adaptive immune response. cytokine profile was also evaluated, and the results showed a high ifnγ secretion by splenocytes, suggesting a th1 activation which lead to higher igg2a isotype production. furthermore, it was observed that host's antibodies against the phage-based vaccine recognized more epitopes than those produced with naked dna vaccines. for long time, subcutaneous and intramuscular injections have been acknowledged as the most common routes for vaccine administration. wild-type and recombinant m13 phage particles have proved their capacity to cross the gastrointestinal barrier and remain stable, suggesting the use of phage nanoparticles as antigen delivery systems in oral route administration strategies [107, 108] . nevertheless, based on their native capacity to recognize and infect bacteria, it has been reasonably speculated that oral administration of phages could cause an unbalance of the gut bacteria, compromising the host's health [52] . fortunately, diverse strategies focused on improving the safety of phage-based vaccines administered by of oral route have been developed [109] . a promising strategy could be the utilization of non-lytic phages, as the m13, to diminish the risk of damaging the host's gut microbiome since these phages are not suited to destroy bacterial cells. another strategy is based on the use of viral particles with non-functional tail fibers, required for target recognition and further infection, to make them unable to infect bacterial cells without reducing their capacity to act as vaccine delivery systems [25] . moreover, oral administration of phage-based vaccines has demonstrated strong immunostimulatory effects, reinforcing the idea of using phage particles as oral vaccine carriers [110, 111] . despite the considerable amount of evidence supporting the use of bacteriophages as vaccines, their clinical application still requires further research and support. fortunately, u.s. food and drug administration (fda) has approved recently the first clinical trial for the use of phage therapy in humans. this phage therapy trial consists on the utilization of a bacteriophage combination to treat a resistant staphylococcus aureus infection. this step opens the opportunity to develop more effective clinical trials involving the use of phage particles for vaccine development. the current available knowledge describing the biology, physiology, and purification of phage particles should help to design adequate clinical trials. however, it is important to note that these clinical evaluations are quite expensive which limit their application to phage-based products. moreover, the wide diversity of phage-based vaccines could help in the development of combinatorial vaccination strategies that may surpass issues of current vaccination strategies. based on the wide range of applications that phage-based bioproducts may have and taking into consideration the effectiveness demonstrated of phage-based vaccines currently developed, the design of clinical trials should be taken as a priority for approval of these bioproducts [6] . phage-based vaccines made without the insertion of foreign dna (in vitro display) into the phage genome can be considered as natural bioproducts since no risk of genetic transfer exists. these considerations should be acknowledged to accelerate the commercial use of phage-based vaccines. phage-based vaccines represent a promising approach for vaccine development since this approach offer important advantages over standard vaccine delivery systems. further studies are required for a better understanding of the immunological mechanism of phage vaccines in order to develop more specific antigen delivery systems. furthermore, immunization protocols aimed to test the effect of higher phage vaccine doses as those currently studied (10 10 and 10 11 pfu) should be implemented. future research of phage-based vaccines will focus on optimization the immunogenicity of the antigens displayed. finally, it has been suggested that the expression of mimotopes in a tandem configuration and fused to the pviii protein of bacteriophages could induce an enhanced effect on the immune response compared to the effect observed in other vaccination strategies. therefore, this approach should be thoroughly investigated to improve the efficacy of phage-based vaccine. the contribution of vaccination to global health: past, present and future inexpensive anti-cysticercosis vaccine: s3pvac expressed in heat inactivated m13 filamentous phage proves effective against naturally acquired taenia solium porcine cysticercosis comparison of a bacteriophage-delivered dna vaccine and a commercially available recombinant protein vaccine against hepatitis b the development of veterinary vaccines: a review of traditional methods and modern biotechnology approaches influence of protein fold stability on immunogenicity and its implications for 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peptides on bacteriophage t7 and its immunogenicity in mice model protective immune responses induced by the immunization of mice with a recombinant bacteriophage displaying an epitope of the human respiratory syncytial virus the potential of phage display virions expressing malignant tumor specific antigen mage-a1 epitope in murine model induction of hepatitis b virus-specific cytotoxic t lymphocytes response in vivo by filamentous phage display vaccine de berardinis, p. the use of filamentous bacteriophage fd to deliver hla-a2-restricted peptides and to induce strong antitumor ctl responses mutated and bacteriophage t4 nanoparticle arrayed f1-v immunogens from yersinia pestis as next generation plague vaccines immunodiagnosis of human neurocysticercosis using a synthetic peptide selected by phage-display application of m13 phage display for identifying immunogenic proteins from tick (ixodes scapularis) saliva identification of immunotopes against mycobacterium leprae as immune 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affinity for the tegument of schistosoma japonicum schistosomula synthetic peptide-targeted selection of phage display mimotopes highlights immunogenic features of α-helical vs non-helical epitopes of taenia solium paramyosin: implications for parasite-and host-protective roles of the protein dna vaccines: roles against diseases genetic immunisation against hepatitis b using whole bacteriophage λ particles molecular and cellular mechanisms of dna vaccines the future of human dna vaccines safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial phage displayed biomolecules as preventive and therapeutic agents a cost analysis of producing vaccines in developing countries economic evaluation of m13 bacteriophage production at large-scale for therapeutic applications using aqueous two-phase systems thermostability of landscape phage probes bacteriophage interactions with mammalian tissue: therapeutic applications selection of tumor-binding ligands in cancer patients with phage display libraries the influence of external factors on bacteriophages-review bacteriophages as vehicles for gene delivery into mammalian cells: prospects and problems pros and cons of phage therapy filamentous phage as an immunogenic carrier to elicit focused antibody responses against a synthetic peptide interactions between bacteriophage, bacteria, and the mammalian immune system immunological basis of m13 phage vaccine: regulation under myd88 and tlr9 signaling anti-vaccinia virus effect of m13 bacteriophage dna cpg oligodeoxynucleotides with hepatitis b surface antigen (hbsag) for vaccination in hbsag-transgenic mice cpg motif in phage genome dna enhanced the efficacy of phage therapy by immunostimulation activation of human b cells by phosphorothioate oligodeoxynucleotides targeting toll-like receptor 9 with cpg oligodeoxynucleotides enhances tumor response to fractionated radiotherapy cellular internalization mechanism and intracellular trafficking of filamentous m13 phages displaying a cell-penetrating transbody and tat peptide phage-phagocyte interactions and their implications for phage application as therapeutics a lipid based antigen delivery system efficiently facilitates mhc class-i antigen presentation in dendritic cells to stimulate cd8+ t vaccination with filamentous bacteriophages targeting dec-205 induces dc maturation and potent anti-tumor t-cell responses in the absence of adjuvants enhancement of dna vaccine potency through linkage of antigen to filamentous bacteriophage coat protein iii domain i induction of immunity in sheep tofasciola hepaticawith mimotopes of cathepsin l selected from a phage display library non-specific translocation of peptide-displaying bacteriophage particles across the gastrointestinal barrier identification of peptide sequences that induce the transport of phage across the gastrointestinal mucosal barrier arming filamentous bacteriophage, a nature-made nanoparticle, for new vaccine and immunotherapeutic strategies immunogenicity of filamentous phage displaying peptide mimotopes after oral administration orally delivered foot-and-mouth disease virus capsid protomer vaccine displayed on t4 bacteriophage surface: 100% protection from potency challenge in mice this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors acknowledge the financial support of consejo nacional de ciencia y tecnología (conacyt) for the phd fellowship of alejandro gonzález mora (no.708102) and the support from tecnológico de monterrey (bioprocess research group). the authors declare no conflict of interest. key: cord-314642-oobbdgzh authors: campbell, allan title: the future of bacteriophage biology date: 2003 journal: nat rev genet doi: 10.1038/nrg1089 sha: doc_id: 314642 cord_uid: oobbdgzh after an illustrious history as one of the primary tools that established the foundations of molecular biology, bacteriophage research is now undergoing a renaissance in which the primary focus is on the phages themselves rather than the molecular mechanisms that they explain. studies of the evolution of phages and their role in natural ecosystems are flourishing. practical questions, such as how to use phages to combat human diseases that are caused by bacteria, how to eradicate phage pests in the food industry and what role they have in the causation of human diseases, are receiving increased attention. phages are also useful in the deeper exploration of basic molecular and biophysical questions. bacteriophages (also known as phages) are viruses that infect bacteria. as with all viruses, phages are infectious particles that have at least two components: nucleic acid and protein. if a phage invades a susceptible bacterial cell, the phage (or at least its nucleic acid) enters the cell and triggers a cycle of phage production (fig. 1) . during this cycle, the cell is reprogrammed to become a phage factory in which the components of the biosynthetic apparatus (such as ribosomes and atp generators) are diverted from their normal tasks in bacterial growth. the various pathways for reprogramming are initiated by phagespecified proteins, which are translated from phage mrna that is made after infection. the temporal programme is ordered and regulated. generally, nucleic-acid replication occurs first, followed by the synthesis of structural proteins of the phage particle. new phage particles are then assembled, and are later released from the cell. for most of the classically studied coliphages, release is effected by the disruption of the cell envelope and sudden lysis of the cell, the contents of which (including the new phage particles) spill out into the medium. the whole cycle can last ~40 minutes and produce ~100 new phages (fig. 1) . this type of lytic cycle is the only mode of reproduction for many phages. other one reason that phages have been useful in research is the ease of synchronizing the lytic cycle in a population of cells, so that the progression of molecular events can be measured over the whole culture. synchronization might be achieved either by simultaneous infection or by the induction of phage development in lysogenic cells. a second reason is the ease with which mutations that affect specific stages of the cycle can be isolated and analysed. phages are as varied in size and form as the viruses that infect eukaryotes. several formal classification schemes have been proposed, but none is widely useful or known to reflect phylogeny. table 1 summarizes basic information on common model phages and illustrates some of the diversity in phage morphology and lifestyle. the phylogeny of viruses has eluded generations of virologists. modern methods have redefined the questions rather than achieved definitive answers. in closely related groups, isolates can be arranged by the degree of similarity, but whether all viruses (or even all phages) constitute a monophyletic group remains uncertain. the development of ideas, starting in the pre-genome era, is illustrated by work on phages and their relatives. in the 1950s, numerous temperate phages (lambdoids) were discovered that resembled the phage-λ morphologically and could recombine with it. electron microscopy of heteroduplexes, coupled with the results from genetic crosses, verified that the lambdoid phages have a common genetic map but show diverse after an illustrious history as one of the primary tools that established the foundations of molecular biology, bacteriophage research is now undergoing a renaissance in which the primary focus is on the phages themselves rather than the molecular mechanisms that they explain. studies of the evolution of phages and their role in natural ecosystems are flourishing. practical questions, such as how to use phages to combat human diseases that are caused by bacteria, how to eradicate phage pests in the food industry and what role they have in the causation of human diseases, are receiving increased attention. phages are also useful in the deeper exploration of basic molecular and biophysical questions. approximately 25 years after their discovery in 1915 (ref. 1), the use of phages to answer fundamental questions about the mechanisms of inheritance began, taking advantage of the ease with which they could be manipulated 2 . between the years 1940 and 1970, phage experiments laid much of the groundwork for the science of molecular biology. that science, once dubbed 'modern' as distinct from 'classical' biology, has long since been assimilated into the textbooks. however, recent research indicates that the historical contributions that phage studies have made to molecular biology might be overshadowed by their future influence on biology in general, and on genetics in particular. phages have important roles in global ecology and bacterial pathogenicity. these emerging roles, together with the increasing number of recent publications on phage genomics, highlight the need for a natural selection has preserved some benign combinations. this raises the question of how the parental phages that give rise to a new junction came to be so different from one another. however, before addressing this issue, we leave the myopic consideration of lambdoid phages to look at what genome analysis implies about phages that are essentially unrelated, even those that come from highly diverse hosts. on that level, the message is clear. natural phage genomes are frequently chimeric in origin and contain genes that have been transferred over great evolutionary distances 5 . as with the genesis of new junctions among the lambdoid phages, the potential number of such transfers must greatly exceed what has passed through the sieve of natural selection. among the more marked examples of molecular chimerism is phage n15. whereas most of its phage genes are related to, and in the same order as, the genes of a lambdoid phage, its prophage form persists as a linear plasmid that includes sequences that are related to the f plasmid of escherichia coli as well as to coliphages p1 and p4 (ref. 6 ). this widespread chimerism forces us to believe that phage genomes collectively constitute a pool that is subject to continual mixing. this pool includes groups, such as the lambdoid phages, in which mixing is even more frequent. however, some gene blocks that are found in lambdoid phages might have resided elsewhere for much of their history, and have diverged over long periods during which they were not subject to frequent recombination with other lambdoid phages. it should be noted that molecular clock estimates indicate that some allelic variants of λ-genes diverged long before the existence of e. coli, which is the host of λ, perhaps even as long ago as the branching of bacteria from archaea and eukaryotes 4,7 . there are many reasons to question the accuracy of dating by molecular clocks 8 . however, the antiquity of viral genes is also indicated by an independent line of evidence 9 . there are marked examples (such as adenoviruses and reoviruses) in which the morphology of the viral capsid is similar in a group that includes animal viruses, plant viruses and bacteriophages. these similarities extend from the shapes of individual proteins to the overall morphology of the virus particle (composed of several protein species) and seem unlikely to represent evolutionary convergence. this indicates a common ancestry for reoviruses and phage φ6, for example, although the rna and protein sequences show no significant similarities and, therefore, must have diverged anciently, perhaps before the separation of the main domains of the living world. hendrix 4 offered the most comprehensive interpretation of these results. if different closely related phages, such as lambdoids, encounter one another in nature, they can recombine. the frequency of recombination depends on the degree of similarity of the two partners at the recombination points. so, between any two phages, shared segments of homology can serve as recombinational linkers, and even identities of oligonucleotide length can create preferred exchange points. none of this is new. however, breakage and joining sometimes occur even if there is no homology -which represents new ground -and over evolutionary time this happens often enough to generate the sharp boundaries between homology and non-homology that are observed in phages. most such new junctions should be lethal or deleterious, but specificities for properties such as integration, repression and host range. these studies also showed that any two lambdoid phages are similar in some segments of their genomes and dissimilar in others, with abrupt transitions from similarity to dissimilarity 3 . extensive dna sequencing has confirmed this general picture. it has also shown that segments with altered specificity (such as repressor genes), although differing greatly in sequence, are nonetheless orthologous. comparing all the known lambdoid phages, there is little doubt that they have undergone extensive recombination in nature, mixing and matching heterologous specificity determinants. sequencing also verifies the sharpness of most boundaries between segments of close similarity and those of extreme dissimilarity. at the base of the natural food chain are the photosynthetic microbes: green algae and cyanobacteria. for both of these groups, a substantial fraction of their biomass is liberated as dissolved organic material by viral infection. heterotrophic consumers of dissolved organic matter are also frequently killed by viral infection. no account of the natural food web that omits bacteriophages is even close to being complete 15, 16 . molecular machines. the focus of biochemistry has advanced from chemical catalysis by individual proteins to mechanical processes that are carried out by molecular assemblages. phage systems provide many instructive examples. the forces that drive dna injection during infection by the large-tailed dna phages have long eluded direct identification. for many common phages, such as λ and t4, this remains the case. more progress has been made with phages, such as t7, which inject their dna slowly. the initial step is the injection of t7 proteins from the phage particle to form a channel across the cell envelope that conducts dna into the cell by a process that is apparently enzymatic 17 . next, the dna is pulled into the cell by transcription with an effectively stationary rna polymerase (first host then phage polymerase) that reels dna through itself 18, 19 . the type i restriction enzyme eco ki can replace rna polymerase to reel in dna 20 . in assembling large double-stranded dna (dsdna) phages, viral dna can be pulled into preformed heads by the portal protein that is located at one vertex of the icosahedron. detailed studies of the physical mechanism were made on bacillus phage φ29 (ref. 21 ). the dodecameric portal protein rotates in the protein shell of the phage, in steps of 12 °, which reel in phage dna and are powered by atp hydrolysis (fig. 2) . the forces that act on individual dna molecules that are being packaged into the heads of φ29 have been accurately measured, and show that the φ29 portal protein generates one of the strongest known molecular motors 22 . as well as such motors, phages use numerous molecular machines. the insertion of phage dna into the chromosome during lysogenization is a good example. several molecules of λ-integrase form an assemblage (the intasome) that binds to a specific segment of phage dna, recruits the bacterial insertion sites into the complex, then makes concerted single-strand cuts in the two dnas. the strand cutting proceeds through the covalent joining of a 3′ phosphate to a tyrosine of the integrase protein. the terminal three bases of one characteristic of a phage family such as the lambdoids, is a common genetic map. the main determinants for structural and regulatory elements in the phage genome are present in the same order throughout the family. as discussed previously, these elements have sometimes been replaced by homologues or analogues from other phages. however, at some time in the past these genomes must have come into being, probably by the merging of smaller genomes with more limited functions. so, the original lambdoid phage might have derived its replication genes from one source, its lysis genes from a second source and its structural genes from a third. genes might have been added one at a time or in larger blocks 3, 4 . the exciting point is that such gene addition is not just a long ago fait accompli but is an active continuing process. the hendrix group 4 have identified segments of the genomes of lambdoid phages that seem to have been spliced into the phage genome recently. these segments (dubbed 'morons') typically include a single gene, which is often of unknown function, and generally differ in base composition from the rest of the phage genome. each moron is present in some lambdoid phages and absent from others. the incorporation by phages of toxin genes and longer pathogenicity islands might be an example of the same kind of process (see later). a complete understanding of the natural dynamics that underlie the recombinational origin of new phage types will require more specific information on the role of natural selection -not only the elimination of maladapted types (as mentioned earlier) but also active selection for the few superior variants. further efforts in this direction are desirable, not just for their intrinsic interest but also as models for viruses that infect eukaryotic hosts; for example, it is well documented that members of the coronavirus group, which has attracted attention recently through the emergence of a deadly new human pathogen that causes severe acute respiratory syndrome (sars), undergo frequent recombination in nature 10, 11 . the long-held suspicion that phages are ubiquitous throughout the prokaryotic world has been confirmed as our knowledge has expanded to include more phages and phagerelated sequences in prokaryotic genomes. the numerical abundance of phage-like particles in aquatic ecosystems generally exceeds that of prokaryotic cells and might represent >10% of the biomass of those cells. furthermore, as documented in recent reviews 12, 13, 14 , there is increasing evidence to indicate that a large fraction of these visible particles comprises viable infectious phages. www.nature.com/reviews/genetics p e r s p e c t i v e s predicted by the finding that the repressor assembles to form octamers, as required for cooperative binding 33 . our initial understanding of in vivo switching was incomplete in another respect. the earlier work showed that the or switch, by itself, is bistable. the ci gene, which encodes the repressor, is transcribed leftward from or, whereas the secondary repressor cro and other gene products come from the rightward message. both repressor and cro can bind to three sites in or and shut-off transcription in both directions. however, one or the other will eventually get the upper hand. if repressor synthesis is established, cro synthesis is shut off, and vice versa. the system, therefore, provides an elegant model for how switching events might be controlled in other contexts, such as in cell differentiation in multicellular eukaryotes 28, 34 . as for the phage system, it might seem gratuitous to question whether the crucial events have been identified, as the simple model seems to provide a sufficient explanation. the shut-off of repressor synthesis by cro depends on cro binding to the or3 operator site, which could be the crucial step in activating the lytic cycle. in this view, the establishment of repression depends on a race between cro and the repressor. however, it has been known from early on that another protein, cii, which acts at the promoter site pre, is crucial in repressor establishment 35 and that cro that is bound to the other or sites (or2 and or1) decreases cii production. this makes it equally possible that the decisive race is between cro and cii, with or3 having, at most, an ancillary role. the definitive experiments are yet to be done. a comprehensive modelling of the switch is available 36 , which agrees with the published data on lysogenization frequencies as a function of average phage input 37 . the complex regulation of the switch is shown in fig. 3 . however, in this pioneering work most of the kinetic parameters are estimates rather than direct measurements. this work underscores the potential for modelling whole-cell physiology with the present high-throughput data on transcription rates and protein abundance. still lacking is a complete model that predicts the strong overshoot in repressor concentration that is observed during lysogenization 34 . because the rate of repressor synthesis from cii-stimulated pre is much higher than that observed in a lysogen, it seems unlikely that cro that is bound to or3 can prevent repressor establishment if cii synthesis achieves its maximum rate. it was previously thought that holliday junctions were formed by re-ligation at the cutting site, followed by branch migration to the resolution site (7-bps removed). since then, important progress has been made on the nature of the initial dna-protein interactions 25 gene regulation. in a series of incisive experiments, ptashne and colleagues defined the modus operandi of the lysis/lysogeny decision in phage as a model genetic switch that operates in the right operator sites (or) 29 (fig. 3) . this work has recently been extended by the discovery that the natural switch is strongly influenced by the simultaneous interaction of the repressor with the leftward operator (ol), which is more than 3-kb away [30] [31] [32] ; this was the free 5′ ends then rotate to exchange partners, followed by ligation with removal of the integrase protein from the 3′ ends to form a holliday junction. the assemblage then undergoes a rearrangement (isomerization) and resolves the holliday structure by cutting the two previously uncut strands on the other side of the junction, which allows the exchange of partners by the freed 5′ ends, followed by resealing 23 . the reverse reaction (excision from the chromosome) requires a second phage-specified protein (excisionase) and proceeds by an analogous mechanism, which is, however, not a simple chemical reversal of the integration reaction 24 . probably the most important recent breakthrough was the discovery that both initiation and resolution involve strand swapping of the free 5′ ends, as described earlier 22 . shown by a set of four blue spheres and one red sphere. the dna base that is aligned with the active connector monomer is also shown in red. a | the active prna-atpase i interacts with the adjacent connector monomer (a), which in turn contacts the aligned dna base. b | the narrow end of the connector has moved anti-clockwise by 12 °, to place the narrow end of monomer c opposite atpase ii, the next atpase to be fired; this causes the connector to expand lengthwise by slightly altering the angle of the helices in the central domain (white arrow with asterisk). c | the wide end of the connector has followed the narrow end, as the connector relaxes and contracts (white arrow with two asterisks), thereby causing the dna to be translated into the phage head. for the next cycle, atpase ii is activated, which causes the connector to be rotated by another 12 °, and so on. at present, phage therapy can be described as promising, but despite its long history its ability to deliver on the promise remains to be fully shown. however, there have been clear successes with experimentally infected animals 40, 41 , and mathematical modelling corroborates the expectations of feasibility 42 . much of the earlier work was relatively crude and less controlled, and the whole area was dismissed without a thorough evaluation (largely because of the availability and economy of antibiotic therapy). now, the numerous potential problems, such as the deleterious effects of the phages themselves, the inactivation of phages by neutralizing antibodies and the genesis of phage-resistant mutants, remain to be rigorously addressed. in other areas that require bacterial survival under conditions of incomplete sterility, such as industrial fermentation, phages are the enemy. phage lysis of lactic-acid bacteria has long been a nuisance in cheese factories. extensive studies on phages that infect lactic-acid bacteria are continuing in many countries [43] [44] [45] . a long time 'holy grail' of research that is directed towards the elimination of the problem has been the isolation or engineering of stable bacterial mutants that are resistant to all of the phages that might attack a given culture 46, 47 . there are many occasions on which molecular biologists would like to identify, from a large collection of proteins or peptides, those few that have some special property (such as binding to a low molecular-weight ligand or to a specific site on a protein or dna molecule). it is easy to fractionate the collection on the basis of its binding properties, but if a binding species is rare, it might be impractical to isolate enough material to determine its structure. in such cases, it is preferable to isolate not the protein or peptide, but rather the dna that encodes it and can easily be amplified. the simplest way to do this is in a system in which the fractionation process brings the dna along with the protein it encodes. surface antigens on bacterial cells can be treated in this way. a more powerful method is to display the protein or peptide on the surface of a phage. phage particles are much smaller than bacteria, so larger libraries can be screened. if the library is composed of translational fusions to a protein that is abundant on the surface of a phage particle, the sensitivity is high; under conditions of single infection, the binding properties of a phage particle are determined by the gene fusion in its dna. phage therapy. following their discovery, phages were regarded as possible agents to combat pathogenic bacteria (as discussed by merril et al. 38 ). most early attempts at phage therapy were unsuccessful or inconclusive, apparently for trivial technical reasons such as the absence of placebo controls or the rapid clearance of injected phage from the bloodstream before it had time to reach its intended target 38, 39 . with the advent of antibiotics, the development and use of phage therapy almost disappeared from the western world, although it continued in the soviet union and eastern europe 39 . as the incidence of antibiotic resistance continues to rise, the field has been reactivated 38 . phage therapy has some inherent advantages over antibiotics, in both the specificity and the ability of the agent to propagate in the desired location (advantages that were seldom realized in earlier work with impure phage preparations) 39 . as with antibiotics, the efficiency is limited by bacterial mutation, which will doubtless be a great challenge to future development. (fig. 1) , the phage can either enter the lytic cyle -in which it reproduces and then lyses its host -or the lysogenic cycle -in which the phage dna is incorporated into the host dna and is replicated indefinitely. dashed boxes enclose operator sites that comprise a promoter-control complex. the three operator sites o r1-3 of the 'λ-switch' control the promoters p rm and p r . cro and ci dimers bind to the three sites with different affinities and in the opposite order to control the activation of the p rm and p r promoters 29 . if protein n is available, transcribing rna polymerases (rnaps) can be anti-terminated at the nut r and nut l sites; the termination sites t r1 and t l1 are inoperative for anti-terminated rnaps. the ci dimer acts as either a repressor or activator of the promoter p rm , depending on its concentration. p1 and p2 are proteases that degrade the cii phage protein. ciii is a phage gene product that is also a substrate for p1. by binding to p1, ciii inhibits the degradation of cii. b | λ-decision-circuit dna organization. phage-encoded genetic elements of the decision circuit are located in a 5,000-nucleotide region of the phage dna. genes are separated into leftward and rightward transcribed strands as indicated by the arrows. rightward extensions of the anti-terminated p r transcript transcribe the o and p genes that are essential for phage genome replication, and the q gene that controls the transcription of later genes on the lytic pathway. leftward extension of the anti-terminated p l transcript transcribes xis and int genes, which are essential for phage-chromosome integration and excision both into and out of the host chromosome. the locations of four termination sites are indicated by t r1-2 and t l1-2 . figure modified with permission from ref. 33 . www.nature.com/reviews/genetics p e r s p e c t i v e s direct insertion of toxin genes into phage dna, which might be transposon mediated. the latter possibility is more likely if the toxin gene is not at a terminus of the prophage, but rather in the middle, as in the λ-related coliphage 933w, which encodes a shiga-like toxin 55 . the evolution of bacterial pathogenicity can only be understood by including the role of phages and pathogenicity islands. prophages and pathogenicity islands seem to be closely related; however, assumptions about the nature of the relationship might be premature. pathogenicity islands belong to a broader category of dna known as specialization islands. specialization islands are blocks of contiguous genes that are distinguished by several criteria. first, they are dedicated to a common function, such as pathogenicity, which is not directly needed for simple survival. second, they differ from the surrounding dna in molecular statistics such as the g+c content, codon usage and neighbour relations. third, they are present in certain strains of a bacterial species and absent from others. their widespread occurrence has only come to light through whole genome sequencing, and their molecular statistics indicate that such elements have been derived in the relatively recent past from other bacterial species, to which they might be indigenous 56 . their intercalation in only certain strains of the species in which they are now observed indicates the operation of an insertion mechanism, such as phage integration or transposition; also, some islands are flanked by phage-insertion sites, which technically qualifies the whole unit, whatever its historical origin, as a defective prophage 57, 58 . this raises a host of questions for future research. one scenario (perhaps overly facile) is that the whole unit arose in the species of origin by rearrangements of host and phage dna, that it was introduced into the species in which we now find it by packing into a phage coat, followed by injection and lysogenization, and that, in its present host, it moves from one strain to another by infection and lysogenization. there is, at present, an extreme paucity of evidence to show that the transfer among strains happens by this method rather than by genetic recombination mechanisms that affect all chromosomal genes. at the other extreme, we might imagine that pathogenicity islands, similar to other genes that are scored as alien 59 , could move between species by some other mechanism (perhaps transformation or conjugation) and become inserted into the recipient chromosome by transposition or some other pathogenicity islands that are potentially phage-related. so, a framework for incisive studies on phage-mediated diseases has developed 52, 53 . it is easier to guess the future of the mechanistic studies than that of the evolutionary studies, in which many lines of inquiry converge. even from the vantage point of wholegenome sequencing, the prevalence of lysogeny might be underestimated because the identification of prophages requires homology to known phages. it has also long been known that complete prophages, which are able to generate plaque-forming particles, frequently deteriorate into incomplete (defective) prophages by changes that range from point mutations to extensive deletions and/or insertions. a particular type of defective phage -specialized transducing phages -can result from faulty excision from the chromosome, which replaces some phage dna by host dna adjacent to the insertion site 54 . this is one mechanism whereby phages might have acquired toxin genes. another is recent examples include fusions of domains of surface antigens of the human pathogen neisseria meningitidis onto the surface protein of phage t4, for possible use in vaccine construction, and fusions of random five amino-acid sequences to identify possible binding partners for a nuclease that cuts t4 dna for packaging 48, 49 . it was discovered early on that in some important bacterial diseases, such as diphtheria 50 and botulism 51 , pathogenicity depends on the presence of certain prophages, which generally encode toxins. knowledge has accumulated gradually, but the area has recently blossomed for at least two reasons. first, advances in eukaryotic cell biology and bacterial gene regulation allow a more profound understanding of the interaction between pathogenic bacteria and the human cell. second, whole-genome sequencing of pathogenic bacteria has disclosed the prevalence not only of prophages, but also of glossary analogues genes (or their products) that are not of common ancestry, but which have equivalent functions. the protein-mediated prevention of the termination of rna synthesis. bistable having two steady states that are stable to small fluctuations. coliphage a bacteriophage that infects escherichia coli bacteria. conjugation the intercellular transfer of dna that is mediated by pili, which are surface appendages that are encoded by certain bacterial plasmids and transposons. heteroduplex a dna molecule that is formed by base pairing between strands that are derived from two dna molecules that are not identical in sequence. a point at which the strands of two double-stranded dna molecules exchange partners, which occurs as an intermediate in genetic recombination. homologues genes (or their products) that are descended from a common ancestral gene. intasome an assemblage of integrase molecules that are bound to their dna substrate. lambdoid belonging to a group of phages that are related to λ. ligand a molecule that binds non-covalently to another type of molecule. a bacterium that harbours phages in a latent form, from which it can be activated to produce infectious phage particles. a group that contains all the organisms that are descended from a common evolutionary ancestor. orthologue the form of a gene in one species that corresponds most directly to a similar gene in another species. prophage the latent form of phage dna that is present in lysogenic bacteria. a phage that is able to form lysogenic bacteria. transformation the uptake of exogenous dna that becomes permanently incorporated into the genome of a cell. an artificial construct in which the coding regions of two different proteins are juxtaposed so as to generate a single chimeric protein product if translated. transposition the transposon-mediated movement of a segment of dna. a bacterial enzyme that moves along dna to cleave it far from its specific site of entry. mechanism. in this picture, such foreign dna is frequently found inside prophages or defective prophages simply because, from a bacterial perspective, prophages constitute junk dna the disruption of which does not affect bacterial survival. insertion of island dna into a pre-existing prophage might seem to add an unnecessary step to the mechanism of origin, but that is not the case. the insertion of island dna into the surrounding phage-related dna must have happened somewhere sometime, and it frequently now bears no traces of known insertion processes such as transposition. further work should illuminate two outstanding questions. first, if this and other 'alien' dna came from some distantly related donor species of bacteria, what was the donor? the bacterial genomes that have been sequenced so far provide only a handful of examples of lateral transfer in which both donor and recipient might be inferredperhaps simply because not enough bacteria have yet been sequenced. second, is the potential mobility of the element in its present host a significant factor in the epidemiology of the diseases? a scientific field derives its interest less from what we know than from the unanswered questions that seem approachable by the available methods. i have tried to indicate some such questions here, many of which have been incubating for years and have been explored through more lines of evidence than could be discussed. however, the time is ripe to seek definitive answers with improved methods. future research should include the stochastic kinetics analysis of developmental pathway bifurcation in 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by insertional mutagenesis display of a pora peptide from neisseria meningitidis on the bacteriophage t4 capsid surface a bipartite bacteriophage t4 soc and hoc randomized peptide display library: detection and analysis of phage t4 terminase (gp17) and late σ-factor (gp55) interaction studies of the virulence of bacteriophageinfected strains of corynebacterium diphtheriae conversion of toxigenicity in clostridium botulinum type c a satellite phage-encoded antirepressor induces repressor aggregation and cholera toxin gene transfer bacteriophage control of bacterial virulence transduction and segregation in escherichia coli k-12 sequence of shiga toxin 2 phage 933w from escherichia coli 0157:h7: shiga toxin as a phage late-gene product detecting alien genes in bacterial genomes pathogenicity islands of virulent bacteria: structure, function and impact on bacterial evolution comparative dna analysis across diverse genomes an investigation on the nature of ultramicroscopic viruses the growth of bacteriophage bacteriophages: evolution of the majority evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage the plasmid prophage n15: a linear dna with covalently closed ends ancient phylogenetic relationships nucleotide sequence of coliphage hk620 and the evolution of lambdoid phages evolution of viral structure evidence of natural recombination within the s1 gene of infectious bronchitis virus fundamental virology viroplankton: viruses in aquatic ecosystems viruses and nutrient cycles in the sea dynamics and distribution of cyanophages and their effect on marine synechococcus spp the significance of viruses to mortality in aquatic microbial communities viruses in marine planktonic systems no syringes please, ejection of dna from the virion is enzyme driven rna polymerasedependent mechanism for the stepwise t7 phage transport from the virion into e. coli rate of translocation of bacteriophage t7 dna across the membranes of escherichia coli translocation and specific cleavage of bacteriophage t7 dna in vivo by eco ki structure of the bacteriophage φ29 dna packaging motor the bacteriophage straight φ29 portal motor can package dna against a large internal force swapping dna strands and sensing homology without branch migration in λ-site-specific recombination sitespecific recombination intermediates trapped with suicide substrates architectural flexibility in λ-site-specific recombination: three alternate conformations channel the attl site into three distinct pathways analysis of higher order intermediates and synapsis in the bent-l pathway of bacteriophage λ-sitespecific recombination site-specific photo-cross-linking between λ-integrase and its dna recombination target arm-site binding for λ-integrase: solution structure and functional characterization of its amino-terminal domain phage and higher organisms four dimers of λ-repressor bound to two suitably spaced pairs of λ-operators form octamers and dna loops over long distances octamerization of λ-ci repressor is needed for effective repression of p(rm) and switching from lysogeny the primary self-assembly reaction of bacteriophage ci repressor dimers is to octamer control of λ-repressor synthesis the author thanks d. bamford, b. weisberg and three anonymous reviewers for helpful suggestions. key: cord-292985-w62xaa4f authors: römer, rudolf a.; römer, navodya s.; wallis, a. katrine title: flexibility and mobility of sars-cov-2-related protein structures date: 2020-07-12 journal: biorxiv doi: 10.1101/2020.07.12.199364 sha: doc_id: 292985 cord_uid: w62xaa4f the worldwide covid-19 pandemic has led to an unprecedented push across the whole of the scientific community to develop a potent antiviral drug and vaccine as soon as possible. existing academic, governmental and industrial institutions and companies have engaged in large-scale screening of existing drugs, in vitro, in vivo and in silico. here, we are using in silico modelling of sars-cov-2 drug targets, i.e. sars-cov-2 protein structures as deposited on the protein databank (pdb). we study their flexibility, rigidity and mobility, an important first step in trying to ascertain their dynamics for further drug-related docking studies. we are using a recent protein flexibility modelling approach, combining protein structural rigidity with possible motion consistent with chemical bonds and sterics. for example, for the sars-cov-2 spike protein in the open configuration, our method identifies a possible further opening and closing of the s1 subunit through movement of sb domain. with full structural information of this process available, docking studies with possible drug structures are then possible in silico. in our study, we present full results for the more than 200 thus far published sars-cov-2-related protein structures in the pdb. at the end of 2019 a cluster of pneumonia cases was discovered in wuhan city in china, which turned out to be caused by a novel coronavirus, sars-cov-2. 1 since then the virus has spread around the world and currently has caused over 10 million infections with more than 500,000 deaths worldwide (july 1st). 2 sars-cov-2 is the seventh coronavirus identified that causes human disease. four of these viruses cause infections similar to the common cold and three, sars-cov, mers-cov and sars-cov-2 cause infections with high mortality rates. 3 as well as affecting humans, coronaviruses also cause a number of infections in farm animals and pets, 4 and the risk of future cross-species transmission, especially from bats, has the potential to cause future pandemics. 5 thus, there is an urgent need to develop drugs to treat infections and a vaccine to prevent this disease. some success has already been achieved for sars-cov-2 with dexamethasone reducing mortality in hospitalised patients 6 and a number of vaccine trials are currently ongoing. the viral spike protein is of particular interest from a drug-and vaccine-development perspective due to its involvement in recognition and fusion of the virus with its host cell. the spike protein is a heavily glycosylated homotrimer, anchored in the viral membrane. it projects from the membrane giving the virus its characteristic crown-like shape. 3 the ectodomain of each monomer consists of an n-terminal subunit, s1, comprising two domains, s a and s b , followed by an s2 subunit forming a stalk-like structure. each monomer has a single membrane-spanning segment and a short c-terminal cytoplasmic tail. 7 the s1 is involved in recognition of the human receptor, ace2. this subunit has a closed or down configuration where all the domains pack together with their partners from adjacent polypeptides. 8, 9 however, in order for recognition and binding to ace2 to take place, one of the three s b domains dissociates from its partners and tilts upwards into the open or up configuration. 8, 9 binding of ace2 to the open conformation leads to proteolytic cleavage of the spike polypeptide between s1 and s2. 7 s2 then promotes fusion with the host cell membrane leading to viral entry. 10 drugs that target the spike protein thus have the potential to prevent infection of host cells. the main protease of the virus (m pro ) is another important drug target. m pro is responsible for much of the proteolytic cleavage required to obtain the functional proteins needed for viral replication and transcription. these proteins are synthesised in the form of polyproteins which are cleaved to obtain the mature proteins. 11 m pro is active in its dimeric form but the sars-cov m pro is found as a mixture of monomer and dimers in solution. 12 sars-cov m pro has been crystallised in different, ph-dependent conformations suggesting flexibility, particularly around the active site. md simulations support this flexibility. 13 the protease is highly conserved in all coronaviruses so targeting either dimerization or enzymatic activity may give rise to drugs that can target multiple coronaviruses, known and yet unknown. 14, 15 since the discovery of sars-cov-2, a plethora of structures have been determined including m pro16 and the ectodomain of the spike protein 8, 9 as well as other potential drug and vaccine targets. these structures provide the opportunity for rational drug design using computational biology to identify candidates and optimise lead compounds. however, crystal structures only provide a static picture of proteins, whereas proteins are dynamic and this property is often important in drug development. for example, agonists and antagonists often bind different conformations of g coupled-protein receptors. 17 . flexibility also affects thermodynamic properties of drug binding, 18 , yet the ability to assess flexibility is often hampered by the long computational times needed for md simulations. we use a recent protein flexibility modeling approach, 19 combining methods for deconstructing a protein structure into a network of rigid and flexible units (first) 20 with a method that explores the elastic modes of motion of this network, [21] [22] [23] [24] [25] and a geometric modeling of flexible motion (froda). 26, 27 the usefulness of this approach has recently been shown in a variety of systems. [28] [29] [30] [31] [32] methods similar in spirit, exploring flexible motion using geometric simulations biased along "easy" directions, have also been implemented using frodan 33 and nmsim. 34 we have performed our analysis through multiple conformational steps starting from the crystal structures of sars-cov-2-related proteins as currently deposited in the pdb. this results in a comprehensive overview of rigidity and the possible flexible motion trajectories of each protein. we emphasize that these trajectories do not represent actual stochastic motion in a thermal bath as a function of time, but rather the possibility of motion along a set of most relevant low-frequency elastic modes. each trajectory leads to a gradual shift of the protein from the starting structure and this shift may reach an asymptote, where no further motion is possible along the initial vector, as a result of steric constraints. 31 energies associated with such a trajectory for bonds, angles, electrostatics, and so forth, have be estimated in previous studies of other proteins and shown to be consistent and physically plausible. computing times for the method vary with the size of the proteins, but range from minutes to a few hours and are certainly much faster than thermodynamically equilibrated md simulations. the approach hence offers to possibility of large-scale screening of protein mobilities. we have downloaded protein structure files as deposited on the protein data bank, 35 including all pdb codes that came up when using "sars-cov-2" and "covid-19" as search terms, as well as minor variations in spelling. in our first search of april 18th, 2020, this gave 133 protein structures. a second and third search on may 20th and 29th, 2020, respectively, resulted in a total of 219 structures. in addition we included a few protein structures from links provided in selected publications to give a grand total of 223 protein structure included in this study. many of the structures found, as outlined above, have been deposited on the pdb in dimer or trimer forms. hence one has the choice to study the rigidity and motion of the monomer or the dimer/trimer. clearly, the computational effort for a dimer/trimer is much larger than for a monomer since in addition to the intra-monomer bond network, also inter-monomer bond need to be taken into account. furthermore, it is not necessarily clear whether the possible motion of a monomer or dimer should be computed to have the most biological relevance. we have computed the motion of full dimer and trimer structures only for a few selected and biologically most relevant such structures while the default results concentrated on the monomers. nevertheless, we wish to emphasize that when results for a certain monomer exist, it should nearly always be possible to also obtain their motion in dimer/trimer configuration. for some protein structures, we have found that steric clashes were present in the pdb structures that made a flexibility and sometimes even just the rigidity analysis impossible. usually, this is due to a low crystal resolution. a list of all current protein pdb ids included in this work is given in table s1 . 1 in figs. 1 and 2 we show examples of different rigidity patterns that emerge from the first analysis. in line with previous studies comparing these rigidity patterns across various protein families, 19 we find that they can be classified into about four types. in fig. 1 (a) we see that for the crystal structure of sars-cov-2 nucleocapsid protein n-terminal rna binding domain (pdb:6m3m), the largest rigid cluster in the pristine structure, i.e. at e cut = 0, largely remains rigid through the dilution process of consecutively lowering e cut values. when bonds are opened the rigid cluster shrinks but the newly independent parts are flexible and not part of any new large rigid cluster themselves. only very small parts of the protein chain break to form their own independent rigid structures before at a certain e cut the whole protein is essentially flexible. we shall denote such a behaviour as brick-like. in contrast, in fig. 1 (b) we observe that for chain a of the co-factor complex of nsp7 and the c-terminal domain of nsp8, which forms part of the rna synthesis machinery from sars cov-2 (pdb:6wiq), already the crystal structure has fallen into three independent rigid structures. opening bonds, we find that the largest rigid cluster breaks into 2 rigid structures. gives chain a of the co-factor complex of nsp7 and the c-terminal domain of nsp8 from sars cov-2 (pdb:6wiq). different rigid clusters of the polypeptide chain appear as identically coloured blocks along the protein chain with each c α labelled from its n-terminal at 1 to its c-terminal. when the energy cutoff e cut decreases (left-most column, downward direction towards larger, negative e cut magnitudes), rigid clusters break up and more of the chain becomes flexible. the colour coding shows which atoms belong to which rigid cluster. flexible regions appear as black thin lines. the second column on the left indicates the mean number r of bonded neighbours per atom as e cut changes. we note that the e cut scale is not linear since new rows are only added when the rigidity of a structure has changed. then these now four domains 2 retain their character upon opening more and more bonds until they simply dissolve into full flexibility. while brick-and domain-like behaviour is found only occasionally, more prevalent are two further types of rigidity dissolution. in fig. 2 (a) we see that for, e.g. the monomer of crystal structure of covid-19 main protease (m pro ) in apo form (pdb:6m03), the rigid cluster dominating the crystal structure quickly falls apart upon change in e cut with five newly formed independent rigid clusters emerging towards to n-terminal of the protein chain. these new clusters remain stable to the opening of further bonds, even when the remnants of the original rigid cluster has become fully flexible. such a behaviour relies on a certain critical e cut value to dominate the rigidity dissolution and is reminiscent of so-called firstorder phase transitions in statistical physics. hence this rigidity-type is usually denoted as 1st order. 19 the behaviour seen in 2(b) for the crystal structure of the complex resulting from the reaction between the sars-cov main protease and tert-butyl here, there are many values of e cut where large parts of the original rigid structure break off one after another so that towards the end of the bond-opening process, the original cluster is still present, but no longer dominates the rigidity pattern. this behaviour is called 2nd order. in table s1 , we have indicated the classification for each protein structure into the four classes. obviously, this classification is not perfect and there are also sometimes intermediate rigidity patterns. nevertheless, this rigidity classification already provides a first insight into the possible flexibility and range of motion for each structure. it should be clear, that a brick-like rigidity should show the least flexibility until it dissolves completely. on the other hand, for domain-like structures, one can expect possible intra-domain motion while inter-domain motion might be harder to spot. similarly, for a 1st-order rigidity, one would expect little dynamic mobility until the "transition" value for e cut has been reached, although high levels of flexibility should be possible afterwards. last, a protein with 2nd-order rigidity should have the most complex behaviour in terms of flexibility since new possible mobility can be expected throughout the range of e cut values. for each value of e cut , the first analysis has provided us with a map of rigid and flexible regions in a given crystal structure. we can now translate this into propensity of motion by allowing the flexible parts to move perturbatively, subject to full bonding and steric constraints (see methods section). moving along directions proposed by an elastic normal model analysis of the crystal structure, we can therefore construct possible motion trajectories that are fully consistent with the bond network and steric constraints. each trajectory corresponds to one such normal mode, denoted m 7 up to m 12 for the first low-frequency non-trivial such modes, as well as the chosen e cut value. generally, a larger value of |e cut | implies less rigidity and results in larger scale flexible motion. in fig. 3 we give examples of such motion trajectories. fig. 3(a) shows a monomer of the sars-cov-2 spike glycoprotein (closed state) (pdb code: 6vxx 9 ). we can see that there is a good range of motion from the crystal structure when following the normal mode modes into either positive or negative changes along the mode vector. fig. 3(b) shows the motion for the dimer structure of the sars-cov-2 main protease (pdb code: 6lu7, in complex with inhibitor n3) 16 . again, one can see considerable motion, although due to the complexity of the structure, it is difficult to distinguish individual movement patterns from such a frozen image. much better insight can be gained when watching for full range of motion as a movie. we have therefore included movies for all figures as supplemental materials. we also have made a dedicated web download page 36 where movies for the complete set of pdb codes as given in table s1 are publicly available. the movies are being offered for various modes m 7 to m 12 as well as e cut values, at least containing results for e cut = 1 kcal/mol, 2 kcal/mol and 3 kcal/mol, respectively. in addition, the site includes the rigidity resolutions discussed above and all intermediate structures needed to make the movies. this allows for the calculation of relative distances and other such structural measures along each motion trajectory as desired. for a detailed analysis of the sars-cov-2 main protease with similar methods, we refer the reader to ref. 26 . as discussed above, the trans-membrane spike glycoprotein mediates entry into host cells. 8, 9 as such, it is a "main target for neutralizing antibodies upon infection and the focus of therapeutic and vaccine design". 9 it forms homotrimers protruding from the viral surface. 3 up to the end of may 2020, 3 structures of the trans-membrane spike glycoprotein have been deposited in the pdb. these transmission electron cryomicroscopy (cryo-em) studies have led to structures with pdb codes 6vsb 8 , 6vxx 9 and 6vyb 9 now being available. with rms resolution of 3.5 å, 6vsb has a slightly lower resolution than 6vxx at 2.8 å and 6byv at 3.2 å. in the following, we shall discuss the resulting rigidity and flexibility properties of these three structures in their full trimer form. results for individual monomer rigidities, as given in fig. 3(a) , are also available at ref. 36 . the rigidity pattern of the homotrimer for this structure (pdb:6vsb, 440.69 kda, 22854 atoms, 2905 residues) is dominated by a large rigid cluster encompassing most of the trimer structure except for a region from roughly residue 330 to residue 525. 3 this indeed corresponds to the s b domain of s1 in each monomer. in the trimer configuration, it is known that one of the s b domains can change from the closed to an open configuration 8 at |e cut | = 0.016 kcal/mol, the 6vsb structure of the trimer breaks into many different rigid parts, but the original large cluster remains a dominating feature across the rigidity dilution plot. a motion analysis analysis does not compute but breaks with bad sterics, apparently due to the comparably low resolution of the crystal structure. for the closed state structure (6vxx, 438.53 kda, atom count: 23694 atoms, 2916 residues) of the sars-cov-2 spike glycoprotein, the rigidity pattern is again "brick"-like and the whole of the crystal structure is part of a single cluster. at |e cut | = 0.022 kcal/mol, there is a first order break of the large rigid cluster into dozens of smaller rigid units. nevertheless, the original cluster retains a good presence in each chain of the trimer. in terms of motion, we are now able to produce motion studies for the full set of modes m 7 to m 12 and various e cut 's. in fig. 4 we show motion results, using again the structures for the extreme ranges of the motion as in fig. 3 . in addition, we are showing side, c.p. fig. 4(a) and top, c.p. fig. 4(b) , views similar to ref. 9 . looking at the whole range of modes and e cut 's computed, we find that motion is very reminiscent of the vibrational excitations of a rigid cone or cylinder. there is twist motion around the central axis, bending of the trimer along the central axis, relative twist motion of 2 chains relative to the remaining chain (c.p. fig. 4(b) ), etc. already at m 12 (with |e cut | = 2 kcal/mol), large scale motion has stopped and one only observes smaller scale motion is flexible parts of the trimer chains. overall, this behaviour is very consistent with the elastic behaviour of a "closed" structure 9 similar to a cone or cylinder. we now turn to the last sars-cov-2 spike ecto domain structure (6vyb, 437.65 kda, 22365 atoms, 2875 residues). 9 this structure is in the open configuration similar to the conformation seen in 6vsb. 8 the structure is shown in fig. 5 , again in side and top view. from the rigidity plots we find, in addition to the largest rigid cluster, already for the crystal structure at |e cut | = 0 kcal/mol a second rigid cluster, also roughly spanning from residue 330 to 525. again, this region identifies the s b domain as in the 6vsb structure. compared to the closed state (6vxx), we see that this cluster has more internal structure, i.e. consists of more flexible parts in the s b region from 330 to 525 and also seems to fall apart upon further changing e cut . this suggests that it is more flexible as a whole. upon motion simulation, we observe a very high mobility in that prominent s b subdomain. starting from the crystal structure, we find for |e cut | = 1 kcal/mol a clear further opening towards a negative froda mode, m 7 , while in the other, positive direction of m 7 , the structure can again close the trimer. the distance range of the motion can be expressed as follow: the distance from residue 501 in the middle of the central β -sheet of the s b to the most opened conformation is 57 å while distance to the same residue in the most closed conformation is 28 å. the distance from open to closed is 69 å. hence the motion simulation adds additional insight into the distinction between the open (6vyb) and the closed (6vxx) structures while also showing that a transition from open to closed is indeed possible. as stated in ref. 9 , this interplay of closing and opening is expected to be central to the viral entry into the human cell. sars-cov-2 infectivity is dependent on binding of the spike protein to ace2. this binding is only possible when the spike protein is in the open conformation. structures of both the open and closed conformation have already been determined and the flexibility of the s b domain inferred from these static structures 8, 9 . the spike trimer consists of almost 3000 amino acids and hence is not an easy target for dynamics simulations due to its size. in the open structures (6vsb and 6vyb) the s b domain is clearly identifiable in the rigidity analysis as a separate cluster to the rest of the trimer. this shows that this domain has increased flexibility. we can clearly observe the hinge movement of the s b domain in the open configuration (6vyb) with the s b domain moving back into the closed configuration. the range of movement from the most opened to the most closed conformation can be measured to be quite large and the flexibility within the s b domain itself during the hinge movement is also seen to be considerable. all these findings suggest that the s b domain of the spike protein has the necessary flexibility to attach itself readily to ace2. however, when starting from the closed structure (6vxx), we do not see an opening. this suggests possibly stronger bonds and steric constraints which need to be overcome before the structure is able to open up. nevertheless, to our knowledge this is the first time the hinge motion of the s b domain has been predicted solely based on the dynamics of a structure. in principle, the full structural information provided in our download site 36 we start the rigidity, flexibility and mobility modelling in each case with a given protein crystal structure file in pdb .pdb format. hydrogen atoms absent from the pdb x-ray crystal structures are added using the software reduce 39 . alternate conformations that might be present in the protein structure file are removed if needed and the hydrogen atoms renumbered in pymol. 40 we find that for some protein structures the addition of hydrogen atoms is not possible without steric clashes. consequently, identification of a viable structure and its continued analysis is not possible. these proteins are labelled in table s1 . for the remaining proteins we produce the 'rigidity dilution' or rigid cluster decomposition (rcd) plot using first. 20 the plots show the dependence of the protein rigidity on an energy cutoff parameter, e cut < 0. it parametrizes a bonding range cutoff based on a mayo potential 41, 42 such that larger (negative) values of e cut correspond to more open bonds, i.e. a smaller set of hydrogen bonds to be included in the rigidity analysis. elastic network modes we obtain the normal modes of motion using elastic network modelling (enm) 43 implemented in the elnemo software. 23, 24 this generates a set of elastic eigenmodes and associated eigenfrequencies for each protein. the low-frequency modes are expected to have the largest motion amplitudes and thus be most significant for large conformational changes. the six lowest-frequency modes (modes 1-6) are just trivial combinations of rigid-body translations and rotations of 5/12 the entire protein. here we consider the six lowest-frequency non-trivial modes, that is, modes 7-12 for each protein. we will denote these modes as m 7 , m 8 , . . ., m 12 . the modes are next used as starting direction for a geometric simulation, implemented in the froda module 26 within first. this explores the flexible motion available to a protein within a given pattern of rigidity and flexibility. froda then reapplies bonding and steric constraints to produce an acceptable new conformation. since the displacement from one conformation to the next is typically small, we record only every 50th conformation. the computation continues for typically several thousand conformations. a mode run is considered complete when no further projection along the mode eigenvector is possible (due to steric clashes or bonding constraints). this manifests itself in slow generation of new conformations. we have performed froda mobility simulation for each protein at several selected values of e cut . this allows us to study each protein at different stages of its bond network, roughly corresponding to different environmental conditions, such as different temperatures as well as different solution environments. in a previous publication, we discussed the criteria for a robust selection of e cut . 42 ideally, for each protein structure, a bespoke set of e cut values should be found, with the rcd plots providing good guidance on which e cut values to select. clearly, for a large-scale study as presented here, this is not readily possible due to time constraints. instead, we have chosen e cut = −1 kcal/mol, −2 kcal/mol and −3 kcal/mol for each protein. these values have been used before in a multi-domain protein with 22 kdalton and shown to reproduce well the behaviour of (i) a mostly rigid protein at e cut = −1 kcal/mol, (ii) a protein with large flexible substructures/domains at e cut = −2 kcal/mol and (iii) a protein with mostly flexible parts connecting smaller sized rigid subunits at e cut = −3 kcal/mol. 31, 42 in addition, we have also performed the analysis at other values of e cut when upon inspection of the rcd plots it was seen that the standard values (i) -(iii) would not be sufficient. the exact values used are given in table s1 . we emphasize that these trajectories do not represent actual stochastic motion in a thermal bath as a function of time, but rather the possibility of motion along the most relevant elastic modes. each trajectory leads to a gradual shift of the protein from the starting structure. this shift eventually reaches an asymptote, where no further motion is possible along the initial vector, as a result of steric constraints. energies associated with such a trajectory for bonds, angles, electrostatics, and so forth, can be estimated and shown to be consistent and physically plausible. 31 a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster who coronavirus disease (covid-19) dashboard | who coronavirus disease (covid-19) dashboard viral infections of humans: epidemiology and control vaccination against coronaviruses in domestic animals bat-borne virus diversity, spillover and emergence dexamethasone for covid-19-preliminary report effect of dexamethasone in hospitalized patients with covid-19-preliminary report recovery collaborative structure, function, and evolution of coronavirus spike proteins cryo-em structure of the 2019-ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein unexpected receptor functional mimicry elucidates activation of coronavirus fusion structure of mpro from sars-cov-2 and discovery of its inhibitors coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs ph-dependent conformational flexibility of the sars-cov main proteinase (mpro) dimer: molecular dynamics simulations and multiple x-ray structure analyses structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design targeting the dimerization of the main protease of coronaviruses: a potential broad-spectrum therapeutic strategy structure of m pro from covid-19 virus and discovery of its inhibitors importance of protein dynamics in the structure-based drug discovery of class a g protein-coupled receptors (gpcrs protein conformational flexibility modulates kinetics and thermodynamics of drug binding rapid simulation of protein motion: merging flexibility, rigidity and normal mode analyses protein flexibility and dynamics using constraint theory normal mode analysis of macromolecular motions in a database framework: developing mode concentration as a useful classifying statistic conformational change of proteins arising from normal mode calculations on the potential of normal mode analysis for solving difficult molecular replacement problems elnemo: a normal mode web server for protein movement analysis and the generation of templates for molecular replacement normal mode analysis and applications in biological physics constrained geometric simulation of diffusive motion in proteins docking of photosystem i subunit c using a constrained geometric simulation protein flexibility is key to cisplatin crosslinking in calmodulin inhibition of hiv-1 protease: the rigidity perspective structure and function in homodimeric enzymes: simulations of cooperative and independent functional motions the flexibility and dynamics of protein disulfide isomerase something in the way she moves': the functional significance of flexibility in the multiple roles of protein disulfide isomerase (pdi) generating stereochemically acceptable protein pathways multiscale modeling of macromolecular conformational changes combining concepts from rigidity and elastic network theory the protein data bank flex-covid19 data repository asparagine and glutamine: using hydrogen atom contacts in the choice of side-chain amide orientation the pymol molecular graphics visualization programme automated design of the surface positions of protein helices comparative analysis of rigidity across protein families amplitude elastic motions in proteins from a single-parameter, atomic analysis this work received funding by the cy initiative of excellence (grant "investissements d'avenir" anr-16-idex-0008) and developed during r.a.r.'s stay at the cy advanced studies, whose support is gratefully acknowledged. we thank warwick's scientific computing research technology platform for computing time and support. special thanks to overleaf.com for free premium access during covid-19 lockdown. uk research data statement: data accompanying this publication are available for download. 36 rar conceived the study, nsr and rar assembled table s1 and identified e cut values, rar performed the computations and curated the data, akw identified biologically most relevant structures. all authors wrote and reviewed the manuscript. the authors declare no competing interests. is chain a of complex resulting from the reaction between the sars-cov main protease and a carbamate (pdb:6y7m). different rigid clusters of the polypeptide chain appear as identically coloured blocks along the protein chain with each c α labelled from its n-terminal at 1 to its c-terminal. when the energy cutoff e cut decreases (left-most column, downward direction towards larger, more negative e cut magnitudes), rigid clusters break up and more of the chain becomes flexible. the colour coding shows which atoms belong to which rigid cluster. flexible regions appear as black thin lines. the second column on the left indicates the mean number r of bonded neighbours per atom as e cut changes. 9 . colors are chosen identical to fig. 4 . as in fig. 3 , the arrows in each panel show the range of motion for chain b (blue shades) in (a+b) and also for chain c (reds) in (b). key: cord-298301-p1zj6jg9 authors: dey, lopamudra; chakraborty, sanjay; mukhopadhyay, anirban title: machine learning techniques for sequence-based prediction of viral-host interactions between sars-cov-2 and human proteins date: 2020-09-03 journal: biomed j doi: 10.1016/j.bj.2020.08.003 sha: doc_id: 298301 cord_uid: p1zj6jg9 background: covid-19 (coronavirus disease-19), a disease caused by the sars-cov-2 virus, has been declared as a pandemic by the world health organization on march 11, 2020. over 15 million people have already been affected worldwide by covid-19, resulting in more than 0.6 million deaths. protein-protein interactions (ppis) play a key role in the cellular process of sars-cov-2 virus infection in the human body. recently a study has reported some sars-cov-2 proteins that interact with several human proteins while many potential interactions remain to be identified. method: in this article, various machine learning models are built to predict the ppis between the virus and human proteins that are further validated using biological experiments. the classification models are prepared based on different sequence-based features of human proteins like amino acid composition, pseudo amino acid composition, and conjoint triad. result: we have built an ensemble voting classifier using svm(radial), svm(polynomial), and random forest technique that gives a greater accuracy, precision, specificity, recall, and f1 score compared to all other models used in the work. a total of 1326 potential human target proteins of sars-cov-2 have been predicted by the proposed ensemble model and validated using gene ontology and kegg pathway enrichment analysis. several repurposable drugs targeting the predicted interactions are also reported. conclusion: this study may encourage the identification of potential targets for more effective anti-covid drug discovery. according to the world health organization (who), the coronavirus disease (covid-19) pandemic, caused by a novel strain of coronavirus called severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus infection, is one of the most crucial diseases in the current scenario. it has infected over 15 million people from more than 200 countries while causing death of more than 0.6 million people. the disease has created immense pressure and tension in the worldwide healthcare systems. at the end of the year 2019, wuhan city of china reported the first case of the novel coronavirus infection. now from asia to europe and america, its deadly effect is threatening the whole world [1] . genomic analysis showed that sars-cov-2 is phylogenetically related to sars-like bat viruses. hence, bats could be the possible source of the viral replication [2] . pangolins have also been identified as a potential intermediate host of novel coronavirus [3] . the usual symptoms of covid-19 affected patients are pneumonia, shortness of breath, cough and cold, fever, and multiple organ failure [4] . the genetic characteristics of sars-cov-2 should be well understood to fight against this virus. it is a single-stranded rna virus consisting of approximately 27-32kb with particle size ranging from 65-125nm in diameter [3] . the world healthcare systems are rigorously searching for a vaccine to mitigate the spread of the virus. besides that, they isolate the infected patients along with some general medicine as immediate treatment and care. sars-cov-2 comprises four main structural proteins including spike (s) glycoprotein, small envelope (e) glycoprotein, membrane (m) glycoprotein, and nucleocapsid (n) protein, in addition to many accessory proteins [5] . a comprehension of how these virus proteins interact with the host cells for survival and reproduction is essential for drug exploitation. protein-protein interaction (ppi) is one way the viruses interact with their hosts. identifying ppis between the virus and the host proteins helps explain how the virus proteins work and how they replicate and cause the disease. over the past decades, experimental strategies for recognizing ppis have been established. nevertheless, these experimental high-throughput screens are primarily used to classify intra-species ppis while inter-species interactomes remained largely understudied. in comparison, laboratory identification of ppis is usually time-consuming, laborious, and difficult to achieve complete protein interactomes. therefore, efficient computational methods for ppi prediction are used to bridge the gap by presenting experimentally testable hypotheses and removing protein pairs having a low probability of interaction to reduce the selection of ppi candidates. computational techniques have been popularly used for predicting viral-host interactions previously [6, 7, 8] . few works have already pursued a broad range of applications of artificial intelligence (ai) and machine learning (ml) to cover medical challenges and outbreak prediction of covid-19 pandemic, described in [9, 10] . kassani et al. have used publicly available covid-19 dataset of chest x-ray [3] and barstugan et al. analyzed computerized tomography (ct) images in [11] for automatic covid-19 classification using deep learning method. horry et al. [12] have also followed the same path of x-ray based covid-19 detection using artificial intelligence and pre-trained deep learning models. however, barstugan et al. used only ct images for covid-19 classification using a k-fold support vector machine (svm) classifier [4, 11] . these methods have some drawbacks. there is a possibility to raise the issue of over-fitting in deep learning due to the limited number of trained images [3, 12] . besides that, it also has limitations in classifying more challenging instances with vague, low contrast boundaries, and the presence of artifacts [13] . these approaches are time-consuming, carrying extra cost, space, and overhead. there is a portability issue of collecting sufficient input x-ray and ct images of covid-19 patients for the learning process. ozkaya et al. proposed fusing, and ranking deep features to detect covid-19 [14] . in this work, they generated two (16×16 and 32×32) subdatasets of 150 ct images. after improving the performance by deep feature fusion and ranking method, svm has been applied for classification. apart from image-based analysis and prediction, some works have been done on the optimistic drug discovery of covid-19. edison et al. introduced a vaxign reverse vaccinology tool and the newly developed vaxign-ml machine learning tool to predict covid-19 vaccine candidates [15] . in an article [16] , the authors have used network-based toolset to covid-19 for recovering the primary pulmonary manifestations of the virus in the lung as well as observed comorbidities associated with cardiovascular diseases. they predicted that the virus can manifest to some special tissues such as the reproductive system, and brain regions using network proximity, diffusion, and ai-based metrics techniques. however, similar kind of works on data-driven drug repositioning framework discovery using machine learning and statistical analysis approaches have been proposed in [17, 18] . it helps systematically integrate large-scale knowledge graph, literature, and transcriptome data to discover the potential drug candidates against sars-cov-2 [17] . in [19] , a novel combination of machine learning, bioinformatics, and supercomputing has been used to predict antibody structures capable of targeting the sars-cov-2 receptor binding domain. the potential result of this article [19] suggests that their predicted antibody mutants may bind to the sars-cov-2 rbd and nullify the virus. batra et al. [18] explored the knowledge of small-molecule treatment against covid-19. it also provided a pipeline to perform high-throughput computational modeling and ensemble docking simulations for screening of covid-19 therapeutic agents. in this article, we have tried to predict the target human proteins of the sars-cov-2 virus based on their protein sequences combining amino acid composition, pseudo amino acid composition, and conjoint triad features using machine learning techniques. the problem has been posed as a twoclass classification problem, where the two classes correspond to the interacting and non-interacting proteins of the virus and the host, respectively. this is the first work in this domain as per our knowledge. initially, we have employed the learning vector quantization (lvq) technique for feature subset selection as a preprocessing step. subsequently, after feature reduction, we have used some popular supervised learning algorithms such as support vector machine (svm), naive bayes (nb), random forest (rf) and k-nearest neighbor (knn) along with a deep multi-layer perceptron model and ensemble techniques (voting classifier, xgboost, adaboost) for classification and prediction. we have used 10-fold cross-validation, repeated 10 times strategy for the supervised learning process. in terms of accuracy perspective on the test dataset, the voting classifier ensemble technique performs better than the other algorithms. therefore, we have predicted the 1326 new potential human protein targets of the sars-cov-2 virus using an ensemble algorithm. gene ontology and pathway enrichment for these predicted interactions are investigated. moreover, we have reported a number of repurposable drugs which target the predicted interactions. in this section, we describe the data sets used for our work followed by the methodologies employed. this section describes the different data sets used in this study. in [5] , gordon et al. prepared a sars-cov-2-human protein-protein interactions (ppis) database between human proteins and novel coronavirus proteins using affinity-purification mass spectrometry (ap-ms). the database contains 332 unique interactions between 332 human proteins and four structural and as well as 20 accessory coronavirus proteins. the degree distribution of the sars-cov-2 proteins in the sars-cov-2-human ppi network is given in figure 1 . these experimentally validated 332 human proteins are used to construct positive training and testing datasets in our study. a summary of the ppi network of sars-cov-2-human is shown in figure 2 using cytoscape [20] . the total network is given in supplementary file s5. there is no "gold standard" for planning a negative dataset in the ppi network since noninteracting protein (negative sample) pairs are not experimentally established. therefore, negative samples must be chosen with care, which may adversely influence the accuracy of predicted ppis. there are two main sampling methods, namely random pairing, and subcellular localization. most of the studies have generated non-interacting proteins at random and then eliminates the pairs used in the positive examples [21, 22] . some studies construct the negative samples having different subcellular localization compared to the positive samples [23] . however, both of these methods are not completely reliable. in the case of random sampling, it may incorrectly take a significant number of positive samples as negative samples and produce different accuracy for different random pairing. in [21] ben-hur et al. proved that in ppi prediction, subcellular localization based methods may generate biased accuracy. we have prepared the negative samples in this article by selecting human proteins from the hprd database release 9 [24] that are not present in the positive dataset and that have a low degree in the human ppi network. viral protein pathway-based research showed that they appeared to target higher-degree human proteins rather than lower-degree proteins [25] . with the aid of cytoscape, we determined the degree of each human protein in the hprd database and ordered it in ascending order. then the negative samples are prepared taking into account the minimal degree of human proteins. this degree-based method of negative sample generation achieves better accuracy on the training dataset compared to the random pairing and subcellular localization (table 1 ). in order to prevent statistical differences, the same scale is assumed for the positive and negative sample, i.e., the ratio 1:1. an independent dataset is prepared to predict unknown human proteins that may indulge in protein-protein interaction with corona viral proteins. all the human proteins of the hprd database (around 9155 proteins) except the proteins that are present in the positive and negative datasets are considered as the independent dataset. a set of well-known supervised machine learning algorithms [26] , such as svm, naive bayes, random forest, deep learning, knn, and ensemble techniques are used for predicting ppis. we have implemented all these algorithms in python frameworks. support vector machines (svm) is one of the most powerful supervised learning algorithms which is based on the concept of hyperplane and it is a generalization of a simple and intuitive classifier called the maximal margin classifier. for a given training sample, the algorithm generates an optimal hyperplane that maximizes the margin between data points of different classes. for a two-class dataset, the nearest objects from each class should be well separated from the decision boundary [27, 28] . svm supports the concept of soft margin classifier because it can be violated by some of the training observations [29] . it can be represented as, in equation (1), c is a nonnegative tuning parameter, m is the width of the margin and the optimization problem chooses β 0 , β 1 , ..., β p to maximize m . 0 , 1 , ....., n are slack variables that allow individual observations to be on the wrong side of the margin or the hyperplane. x * are the observations [29] . k nearest neighbor (knn) algorithm is one of the simplest supervised learning algorithm used for both classification and regression problems. it is also called as the lazy learner as it uses all the data for training while doing prediction [27, 28] . we have to choose the value of kfirst (k ≤ √ n), where n is the size of the data). then it calculates the distance between test data and each row of training data with the help of any distance metric function (use hamming distance for categorical naive bayes is a probabilistic classifier, based on bayes' theorem. in simple terms, a naive bayes classifier assumes that the presence of a particular feature in a class is unrelated to the presence of any other feature. so, each feature makes an independent and equal contribution to the outcome. bayes' theorem finds the probability of an event occurring given the probability of another event that has already occurred. bayes' theorem is stated mathematically as the following equation, in equation (2), p (a|b) is the posterior probability, p (b|a) is the maximum likelihood and p (a) is the prior probability. basically, we are trying to find the probability of event a, given the event b is true. event b is also termed as evidence. in naive bayes perspective, p (y|x), y is class variable and x is a dependent feature vector (of size n) where, x = (x 1 , x 2 , x 3 , ......, x n ). therefore, we can rewrite the naive bayes equation for a set of independent features as, now, we need to create a classifier model. for this, we find the probability of a given set of inputs for all possible values of the class variable y and pick up the output with maximum probability. this can be expressed mathematically as, in equation (4), p (y) is also called class probability and p (x i |y) is called conditional probability. naive bayes has been successfully used to predict the protein-protein interactions and binding sites of dna/rna. for ppi, the algorithm generates binary classification output based on the protein sequence vector [27, 29] . ensemble modeling is a powerful way to improve the performance of the model. in order to improve the performance of the model, it combines several base models into an optimal predictive model. there are two kinds of ensemble techniques bagging and boosting. • in bagging (bootstrapping and aggregation) technique, we create multiple bootstrapped subsamples (row sampling with replacement) from the original one and apply the decision tree learning model on each of the bootstrapped subsamples. after each subsample decision tree has been formed, an algorithm is used to aggregate over the decision trees to form the most efficient predictor. in order to choose the most efficient predictor, it follows the majority of the voting classifier technique. this bagging concept is also applicable to various sets of classifier models. in our work, we have applied the majority voting classification technique on combining svm-polynomial, svm-radial and rf models to achieve the best accuracy among all models [29, 30] . a single decision tree classification gives low bias and high variance whereas a random forest classifier gives low bias and low variance. this bagging algorithm is also known as the random forest classifier. • boosting is an ensemble technique where new models are added to correct the errors made by existing models. models are added sequentially until no further improvements can be made. the adaptive boosting (adaboost) algorithm helps us to combine multiple "weak classifiers" into a single "strong classifier". the weak learners in adaboost are decision trees with a single split, called decision stumps. adaboost works by putting more weight on difficult to classify instances and less on those already handled well. it is used for both classification and regression purposes [29, 30] . • the extreme gradient boosting (xgboost) algorithm is an implementation of gradient boosted decision trees designed for speed and performance. it has a wide range of applications, portable, flexible implementation and cloud integration of this model is easy. xgboost is an ensemble tree method that applies the principle of boosting weak learners (carts generally) using the gradient descent architecture [29] . xgboost algorithm provides high bias and low variance [30] . mlp is a multiple feedforward artificial neural network that maps input vectors to output vectors [31] . it can be represented by a directed graph with multiple node layers, where the bottom and top layers are input and output layers respectively and others are hidden layers. dmlp is a fully connected network where every node in the upper level has connections with all the nodes in the lower level. each node represents a neuron (or processing unit) with a nonlinear activation function except for input layer nodes. multiple hidden layers are allowed that makes it deep neural [32] . in our work, we have used an adaptive learning rate optimization algorithm which is designed specifically for training our deep neural network [33] . we have created a five hidden layers mlp network along with the relu (rectified linear unit) activation function and 'sigmoid' function at the output layer. we have fitted our model with varying epochs, varying batch-sizes, and binary-cross-entropy as a loss function. a total of 3 sets of sequence-based features, namely, amino acid composition, conjoint triad, and pseudo amino acid composition of the human proteins are considered to train the machine learning models. the fasta sequences of human proteins are gathered from uniprot and the values of these features are extracted from protr [34] . these 3 feature sets are described below. the composition of amino acids explains the percentage of a type of amino acid found in a protein chain. this is one of the simple and effective predictive functions of ppis. the arrangement of amino acids reflects only the abundance in a sequence of each amino acid [27] . many studies have used conjoint triad to completely explain the essential ppi details to represent the properties of amino acid [35] [36] . in the conjoint triad system, the 20 amino acids are divided into seven classes based on their amounts of dipoles and side chains ( table 2 ). every amino acid of a protein chain is then replaced by the number of clusters. chou first proposed the composition of pseudo-amino acids in 2001 [37] . aac only displays the frequency of each amino acid in a sequence, but information on sequence order is lost. compared to aac, pseaac finds protein sequence order along with amino acid compositions [38, 39] . the order of sequence can be extracted from sequence similarity variables such as hydrophobicity, hydrophilicity, and side-chain mass. the performance of prediction depends on two aspects. they are feature extraction and performance of the classifier. feature selection is one of the important tasks in classification algorithms. the datasets used for classification contain a large number of features. most of them are either partially or completely irrelevant and redundant to the classifier. therefore, feature selection is used to select a number of important features so that it can achieve acceptable classification accuracy compared to considering all features. in this work, we have used a well-known kohonen's learning vector quantization (lvq) technique to identify important features and remove redundant features from the original dataset [40] . lvq is a simple, universal, and efficient learning classifier. it is very similar to the k-nearest neighbors. in an n-dimensional feature space, lvq tries to approximate optimal decision borders between different classes with a number of labeled codebook vectors. the euclidean distance measure helps to label the closest codebook vector of an example vector. a feature selection strategy using lvq is evaluated to optimize the hypothesis margin of lvq classification through minimizing its loss function [41] and it generates importance score of each feature based on the class identification. our proposed methodology is represented in terms of algorithmic steps below. input: collect the human proteins that interact with sars-cov-2 virus [5] (positive dataset). i 2 . collect non-interacting human proteins from hprd using the concept of degree distribution (negative dataset). output: predicted potential human target proteins. procedure: 1. combine i 1 and i 2 to construct the training dataset. the pictorial representation of the ppi prediction methodology has been shown in figure 3 . the performance of each classifier is evaluated with 10-fold cross-validation in 10 repeat runs to acquire average values. different measurements like accuracy, kappa, sensitivity, specificity, precision, and f1 score are calculated considering 1:1 positive and negative datasets. these parameters are defined as per the equations (5) to (10) . kappa = observed accuracy − expected accuracy 1 − expected accuracy , where, p recision = t p t p + f p * 100%, where, t p , f p , f n , and t n respectively denote the numbers of true positives, false positives, false negatives, and true negatives. in this work, we have started with 3 types of features (amino acid composition, conjoint triad, and pseudo amino acid composition) of human proteins which results in a 413-dimensional feature vector. the importance of all these features are calculated using lvq. afterthat, the knee point is evaluated to detect a large change in the importance score and extract the most important features. we have extracted 38 significant features from 413 features of the original dataset by applying the procedure. table 3 shows the comparison of performance between selected best 38 features vs all 413 features using all the used supervised learning algorithms. all these values have been calculated by 10 fold cross-validation repeated 10 times and then the average of them is reported. in case of dmlp classifier, the batch size of 32 or 25 is acceptable, with epochs = 100 unless the dataset is very large. for large datasets, a batch size of 10 with epochs between 50 to 100 can be considered. in our case, our covid-19 ppi dataset is quite large in terms of the number of features (413-dimensional feature vectors) for the training dataset. therefore, we have used epochs = 50 and batch-size = 10. after the feature selection, we kept the same set of epochs and batch-size for showing parity in the result the blind dataset is used to prevent bias of the classifiers. 124 proteins (62 interacting + 62 non-interacting) from the training dataset are treated as test datasets. the rest of the proteins are used as the training dataset. the performance of the classifier models using a blind dataset is given in table 4 . it can be seen from the table that sv m radial , sv m p olynomial , and random forest method achieved better accuracy, specificity, and f1 score over other classifiers. although, dmlp gives better accuracy on the training datasets, on the test dataset it can't do well compared to the other models. therefore, to increase the prediction accuracy and minimize the false positives we have built a majority voting based ensemble model using these three methods sv m radial , sv m p olynomial and random forest) to predict unknown sars-cov-2 target proteins of human. it can be seen that the ensemble method achieved better accuracy (72.33%), recall (71.67%), specificity (74.41%), precision (72.41%), and f1 score (72.03%) than all the other classifier models. on both positive and negative 1:1 dataset we measured aac, conjoint triad, and pseaac features. this dataset is used as the training dataset. all the human proteins of the hprd database, which are not present in the positive and negative dataset, are considered for prediction analysis. we have applied the ensemble method on these large numbers of human proteins (9155) of the hprd dataset and predicted 3603 potential interacting human proteins. the prediction results are listed in supplementary file s1 along with their average probability prediction score. however, standard svm and random forest assume that the probability threshold for both classes is equal, i.e., 0.5 for binary classification problem. in this work, we have changed the probability threshold from 0.5 to 0.7 so that we can predict all high probability human target proteins and avoid possible false positives. a higher threshold indicates higher confidence in predicting the positive class. the average probability of the predicted human proteins varies from 0.926 to 0.461. our motivation is to predict high-throughput target human proteins. if we consider the threshold of the average probability 0.9, then we get only 10 target human proteins. for probability factor 0.8, we obtain 340 proteins. therefore, we have set the probability value to 0.7, so that a reasonable number of target human proteins can be predicted. using this procedure, the potential number of target human proteins comes down to 1326 from 3603 (file s2). we have also calculated the degree of these predicted human proteins with respect to all the proteins of the hprd database using cytoscape and included in the supplementary files. the degree value varies from 1 to 168. the top 10 high-degree proteins with the degree and prediction score are listed in table 5 . go is one of the most used annotation systems to obtain the biological relevance of highthroughput experiments. to examine the functional characteristics of these predicted 1326 highprobability human target proteins, go term analysis is done on the biological process (bp), cellular component (cc), and molecular function (mf) categories. it has been noticed that the proteins that have similar cell locations or involved in some biological process or molecular function, are likely to interact with each other. we have collected go annotations of all predicted human proteins from david 6.8 [42] having corrected p-values less than 0.05 to validate the predictions. we have found that some of the most enriched go-cc terms of the predicted human proteins are cytosol, extracellular exosome, cytoplasm, membrane, and nucleoplasm. the go-cc term describes the different locations of a cell, at the levels of subcellular structures and macromolecular complexes. being an rna-based virus, the novel coronavirus attacks either the nucleus or cytoplasm of the host cell and causes a respiratory block in the lungs. therefore, the proteins involving in these cc terms are likely to act as potential coronavirus targets. antigen processing and presentation of exogenous peptide antigen via mhc class i, tap-dependent, nik/nf-kappab signaling, regulation of the cellular amino acid metabolic process, positive regulation of ubiquitin-protein ligase activity involved in the regulation of mitotic cell cycle transition, negative regulation of ubiquitin-protein ligase activity involved in the mitotic cell cycle, etc. are five most enriched go-bp terms collected from the david server. some of the most enriched go-mf terms are like protein binding, atp binding, gtp binding, cadherin binding involved in cell-cell adhesion, poly(a) rna binding, etc. in [43] , the authors examined that the coronavirus proteins especially the spike proteins bind with target human proteins and increase cell adhesion during infection. all the enriched go terms along with involved human proteins and p-values are listed in supplementary file s3. analysis of the kegg pathway shows potential illnesses that can develop in the human body due to covid-19 infection. all the significant pathways of 1326 predicted target human proteins along with the corrected p-values are listed in table 6 . viruses are well known to be exploiting the machinery of host cells for their own replication. the proteasome pathway is one of these intracellular processes that are hijacked by the viruses [44] . myung et al. showed this proteasome pathway is involved with different viruses like retrovirus, human immunodeficiency virus type 1 (hiv-1), simian immunodeficiency virus (siv), and moloney murine leukemia virus (mo-mulv). therefore, the probability of association of this pathway with novel coronavirus is very high. endocytosis is a biological process of transporting particles, such as large molecules, parts of cells, and even whole cells, to bring into a cell. experiments on the sars-cov-2 virus have shown that the endocytic pathway is the main pathways for regulating the entrance of covs into the host cells and thus the endocytic pathway, as well as the involved human proteins, can be extensively studied for the target of anti-viral therapies [45] . in [46] , wenzhong liu et al. showed that because of the failure to regularly combine carbon dioxide and oxygen during sars-cov-2 virus infection, the lung cells have highly severe toxicity and inflammation, which ultimately results in ground-glass pulmonary images. according to our results, novel coronavirus can alter the synthesis of macromolecules and its growth rate and can cause carbon metabolism. the term biosynthetic pathway suggests manufacturing the antibiotics which can help to combat drug-resistant viruses and diseases. one of the main public health issues of recent times is the exponential growth of antibiotic-resistant pathogens. therefore, the predicted human proteins involving this pathway need to be further studied to develop antibiotics for the sars-cov-2 virus. the amino acid plays an important role in the expression of the different viral functions including synthesis of viral coat proteins and the development of full infectious virions. novel coronavirus may alter this amino acid composition and cause several other diseases in the human body. we can conclude from the above pieces of evidence that the predicted human proteins involving these pathways are strongly involved in coronavirus infection and experimental validation is required to establish direct interactions. in this section, we have tried to find out the relation between our predicted 3603 human proteins with the other viruses. our research included six specific human-pathogenic rna viruses, namely, dengue, hiv-1, hcv, ebola, zika, and h1n1. we have found various pieces of evidence that many predicted proteins interact with different medically important viruses from published literature. table 7 shows the number of overlapping predicted human proteins and experimentally verified 29 aldoa , adpgk, glud1, pgam1, hk2, hk1, agxt, pdhb, got1, idh3g, hk3, idh2, eno2, idh1, eno3, pdha2, cat, rpia, pdha1, eno1, shmt1, pfkl, suclg1, idh3b, pfkm, idh3a, pklr, prps2, prps1 biosynthesis of amino acids (p= 5.6641e-6) 21 aldoa, shmt1, pfkl, pgam1, idh3b, pfkm, asl, idh3a, pycr2, got1, idh3g, pklr, eno2, idh2, eno3, idh1, rpia, prps2, [52] human proteins with other viruses. we found a large number of predicted human proteins interact with more than one virus. for example, human protein ikbke has the highest degree of 4. it interacts with four viruses, namely, dengue virus (denv), hiv-1, hcv, and h1n1. a small ppi network of the predicted target humans that interact with at least three viruses is shown in figure 4 . from no effective drug has yet been discovered targeting sars-cov-2 virus. the traditional mechanism for drug development and its approval is much more expensive and time-consuming. on the other hand, repurposing the existing drugs is an alternative method for identification of effective drugs. it can substantially shorten the time and minimize costs relative to new drug development. the human proteins that interact with the proteins of multiple viruses can be good candidate for as targets for drug repurposing. we have found 30 predicted human proteins that interact with the proteins of at least 3 viruses. the u.s. food and drug administration (fda) approved drugs that interact with these human proteins are queried using dgidb (http://dgidb.org/) and reported in table 8 . most of these drugs are used in cancer-inhibiting microtubules treatment, and other drug classes are used for diseases like atherosclerosis, crohn's disease, antiinflammatory agent, tumor necrosis factor, blood circulation and infection. table 8 : list of drugs associated with the predicted target human proteins that interact with the proteins of at least 3 different viruses. in this study, various sequence-based features and computational approaches are used for the first time in predicting the potential human targets of the sars-cov-2 virus. we have used the lvq algorithm for feature selection. hiv, influenza, and other viruses are well researched in various literature works compared to coronavirus proteins as sars-cov-2 which has emerged recently. hiv, the most well-studied virus, is believed to have around 1000 direct interactions with human proteins and 3000 indirect interactions. both hiv-1 and sars-cov-2 are enveloped rna viruses. the sars-cov-2 virus contains a large number of proteins, even more than hiv-1. therefore, it can be assumed that sars-cov-2 is a virus expected to have a large number of interactions with human proteins. however, due to the lack of experimentally validated sars-cov-2-human ppis, most of the possible interactions are currently unknown. in this paper, we have predicted the 1326 potential target human proteins considering a high probability factor (70%) using ensemble modeling. we have also analyzed the go terms and kegg pathway of these predicted human proteins and some of the pathways are also supported by recent literature.some repurposable drugs that target the predicted interactions have also been reported. we hope that this study will help biologists recognize possible associations between novel coronavirus and human proteins and facilitate the development of anti-viral drugs. the authors declare that they have no conflict of interest. file s1 excel file containing predicted human proteins. columns include predicted human protein's uniprot accession, uniprot name, gene name and prediction score.(xlsx) source: https://sites.google.com/site/supplementcovid19work/files file s2 excel file containing predicted human proteins having 70% average prediction probabilty score. columns include predicted human protein's uniprot accession, uniprot name, gene name and prediction score. source: https://sites.google.com/site/supplementcovid19work/files file s3 excel file containing predicted human proteins go terms. source: https://sites.google.com/site/supplementcovid19work/files excel file containing predicted human proteins that interact with other viruses. 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key: cord-318749-k91oku7h authors: dong, hui-jun; zhang, rui; kuang, yu; wang, xiao-jia title: selective regulation in ribosome biogenesis and protein production for efficient viral translation date: 2020-10-29 journal: arch microbiol doi: 10.1007/s00203-020-02094-5 sha: doc_id: 318749 cord_uid: k91oku7h as intracellular parasites, viruses depend heavily on host cell structures and their functions to complete their life cycle and produce new viral particles. viruses utilize or modulate cellular translational machinery to achieve efficient replication; the role of ribosome biogenesis and protein synthesis in viral replication particularly highlights the importance of the ribosome quantity and/or quality in controlling viral protein synthesis. recently reported studies have demonstrated that ribosome biogenesis factors (rbfs) and ribosomal proteins (rps) act as multifaceted regulators in selective translation of viral transcripts. here we summarize the recent literature on rbfs and rps and their association with subcellular redistribution, post-translational modification, enzyme catalysis, and direct interaction with viral proteins. the advances described in this literature establish a rationale for targeting ribosome production and function in the design of the next generation of antiviral agents. initiation, as the first stage of translation, can be carried out by either cap-dependent or cap-independent mechanism in eukaryotic cells (kapp and lorsch 2004) . the majority of the eukaryotic genome is translated via cap-dependent translation initiation, mainly through the 43s preinitiation complex (pic) binding to the mrna 5′-end cap structure and scanning along the mrna to the aug initiation codon to initiate translation (haimov et al. 2015) . in contrast to the cap-dependent mechanism, the cap-independent mechanism is translated by 43s pic (or a single 40s unit in some specific mrna templates) directly binding to the internal ribosome entry site (iress) in the genome intergenic region (kapp and lorsch 2004) . the number and types of translation initiation factors required for ires-mediated translation initiation can vary significantly among specific mrna species. for example, eif4e is not required for ires-mediated picornavirus genome translation (avanzino et al. 2017) , and ires elements present in the hepatitis c virus (hcv) genome require fewer translation initiation factors to initiate translation (torrecilla et al. 2016; kerr et al. 2016) . ribosomes are apparatuses that catalyze protein synthesis. the eukaryotic 80s ribosome is composed of two subunits: the 40s subunit contains the decoding center, which monitors the complementarity of transfer rna (trna) and messenger rnas (mrna) and is composed of 33 ribosomal proteins (rps), and the 60s subunit, which catalyzes the formation of peptide bonds and is composed of 47 rps (khatter et al. 2015; barna 2013) . ribosome biogenesis is a complex process, which takes place mainly within the nucleoli of eukaryotes, and requires more than 300 ribosomal biogenesis factors (rbfs) (tafforeau et al. 2013) . rps are coordinated with ribosomal rna (rrna) to synthesize, mature, assemble, and export into the cytoplasm, and they are crucial participants in the cellular process of translation from mrna (lafontaine and tollervey 2001; moore 2012) . ribosome-mediated translational regulation can be explained by two different models simsek et al. 2017) . the ribosome-concentration hypothesis communicated by erko stackebrandt. proposes that the changes in cellular ribosome abundance may be a major driver of changes in translation of mrna pools. the specialized ribosomes hypothesis is based on the finding that some rps differ in composition and specifically regulate a subset of specific mrna (such as rpl38 for hox mrna) translation (shi and barna, 2015; xue and barna, 2012; simsek et al. 2017; briggs et al. 2017; genuth and barna, 2018) . rbfs control ribosome biogenesis to participate in the cellular process of mrna translation. several viruses have evolved sophisticated mechanisms that utilize cellular translational machinery to obtain efficient viral protein synthesis and viral replication (hilton et al. 1986; narayanan et al. 2008; li et al. 2018a, b) . the ribosome itself (primarily ribosomal proteins), and the ribosomal biogenic processes that are exploited by viruses to facilitate their own replication, should be considered as targets in the development of antiviral agents, as depicted in table 1 and as summarized in this study. ribosome biogenesis is critical for cell proliferation and stress response. the synthesis of viral proteins depends on the host ribosome, and ribosome biogenesis has implications for viral infection through the effects of rbfs on viral transcription, proliferation, and antiviral immune responses, as well as the effects of viral proteins on ribosomal biosynthesis. ribosomal rna transcription, catalyzed by rna polymerase i (pol i), plays a critical role in ribosome biogenesis. transcriptional activation of rrna is also closely associated with viral infection. however, restricting ribosome biogenesis by interfering with rrna accumulation-triggered ribosomal stress stimulated the viral replication of human cytomegalovirus (hcmv); the process inhibited the expression of innate immune-related genes, such as the high-mobility group box 2 (hmgb2). this reveals that rrna accumulation and/or ribosome biogenesis regulate innate immune responses to restrict virus reproduction and regulate inflammation (bianco and mohr 2019) . similarly, ribosomes are required for the protein synthesis of host cells and viruses, but the biogenesis factor rbfs can also impact the proliferation of virus and cell-intrinsic immune responses. at this intersection of apparently competing processes, it is intriguing to find rbfs that are required specifically for the viral protein biosynthesis, but do not affect global translation. nop53 (gltscr2/pict-1), for example, shares homology with the yeast 60s ribosomal protein nop53p, which acts as an essential ribosome biogenesis factor (sydorskyy et al. 2005; thomson et al. 2005) . a previous study by the present author showed that nop53, expressed as a set of discrete globular structures within the nucleolus, is migrated to the cytoplasm upon infection by herpes simplex virus 1 (hsv-1) (meng et al. 2018) . furthermore, the cytoplasmic translocation of nop53 is required for efficient viral replication, which occurs via depression of the activity of innate immune receptor rig-i and decrease of the phosphorylation level of eif2α to facilitate the production of viral proteins without affecting global protein synthesis (meng et al. 2018 (meng et al. , 2019 . similarly, both nucleolar proteins of ribosomal rna processing 1 homolog b (rrp1b) and nucleolin are involved in ribosomal biogenesis (chamousset et al. 2010) . rrp1b associated with pre-60s ribosomal subunits was translocated to the nucleoplasm upon viral infection of influenza virus, where rrp1b improves viral rna-dependent rna polymerase (rdrp) activity, which is responsible for virus transcription and replication (su et al. 2015) . nucleolin, which is thought to control rna metabolism and ribosome biogenesis (cong, et al. 2012; allain 2000) , is prevented from entering the nucleus in poliovirusinfected cells (waggoner 1998) ; this allows stimulation of ires-mediated translation of viral transcripts (izumi 2001) . upstream binding factor (ubf) plays a major role in the regulation of rrna synthesis and associates with viral replication of adenovirus; the antibody against ubf reduces viral replication (lawrence et al. 2006) . in mammalian cells, there are two sets of ribosome particles, which reside in the cytoplasm and the mitochondria. some viruses manipulate mitochondrial biogenesis to support replication. it has been reported that the protein levels involved in mitochondrial ribosome biogenesis are significantly up-regulated by infection with human cytomegalovirus (hcmv). the inhibition of mitochondrial translation with chloramphenicol or knockdown of ribosome biogenesis factor mrm3 abolished the hcmv-mediated increase in mitochondrial coding proteins and significantly impaired viral growth (karniely et al., 2016) . the main function of the nucleolus is in ribosome biogenesis, regulation of the cell cycle, and the response to cellular stress. many viral proteins, such as the nucleocapsid protein of coronavirus (chen et al. 2002; cawood et al. 2007; dove et al. 2006; emmott et al. 2010; hiscox et al. 2001; you et al. 2005) , the matrix protein of newcastle disease virus (ndv) (peeples et al. 1992) , and the non-structural protein 1 of influenza virus (melén et al. 2002) , transport to the nucleolus to regulate ribosome biosynthesis, change the nucleolar morphology, or affect the function of the nucleolus. during nucleocytoplasmic shuttling, viral proteins can recruit nucleolar rbfs to modulate ribosomal biosynthesis for efficient viral replication. the multifunctional nucleolar phosphoprotein nucleophosmin (npm) plays a lead role in ribosome biogenesis to stimulate rna pol i-dependent transcription (li and hann 2013) and regulates sars-cov, ibv, hiv-1, hcv recruited by viral proteins to facilitate viral replication chen et al. 2009; zhou et al. 2013; goh et al. 2004; bortz et al. 2011 the export of ribosomal proteins and mature ribosomes to the cytoplasm (yu et al. 2006) . the hbx oncoprotein of hepatitis b virus (hbv) directly interacted with the c-terminal domain of npm to stimulate viral transcription (ahuja et al. 2015) . several rna helicases have been demonstrated to facilitate ribosome biogenesis, involving the processing of ribosomal rna (rrna) as well as its assembly into functional ribonucleoprotein complexes (bleichert et al. 2007 ). ddx5 promotes the synthesis and maturation of rrna and ultimately increases ribosome output and proliferation (jalal et al. 2007) . many viral proteins, such as coronavirus nsp13 (chen et al. 2009 ), rev of human immunodeficiency virus 1 (hiv-1) (zhou et al. 2013 ), ns5b of hepatitis c virus (hcv) (goh et al. 2004) , and np protein of influenza virus (bortz et al. 2011 ) have evolved to hijack ddx5 in order to facilitate viral replication. studies on the regulation or manipulation of viral protein in ribosome biosynthesis provides new insights into efficient viral replication. the ribosome was once considered to be a large protein synthesis machine that merely translated mrnas into proteins. in recent years, however, the role of ribosomes in the regulation of initiation and selection of mrnas has been the subject of increased attention. most rps are essential for translation and viral replication, and regulate translation of viral mrnas as constituents of the ribosome, although a few represent the defense signaling of host cells (zhou et al. 2015) . (the role of rps in activating an immune response is beyond the scope of the present paper.) of particular interest are the rps that are non-essential to the function of the ribosome, such as the large ribosomal subunit (rpl40, rpl38) and the small ribosomal subunit (rps27, rps25) (jack et al. 2011; karlas et al. 2010; xue et al. 2015; lee et al. 2013 ). in the process of coevolution with hosts, viruses have evolved different strategies to inhibit host translation or induce host translation shutoff while protecting their own protein synthesis. many viruses globally interfere with host translation by impairing cap-dependent ribosome recruitment to host mrnas. for example, vesicular stomatitis virus (vsv) inhibits the translation of host mrnas in infected cells, but allows the translation of its own capped mrnas (whitlow et al. 2008; wertz and youngner 1970) . rpl40 promotes efficient translation of vsv mrnas in a cap-dependent manner, but is not required for global protein synthesis (lee et al. 2013) . this may represent an endogenous specialized translation function for rpl40. influenza a virus (iav) viral mrnas, on the other hand, are not preferentially translated compared to their host counterparts, and the extensive translation of viral proteins is the result of viral takeover of the mrna pool in the cell (bercovich-kinori et al. 2016) . ribosome footprints and mrna read density analysis reveal that rps are not strongly affected by iav infection but are enriched for genes involved in pathways that the virus may depend on (bercovich-kinori et al. 2016) . moreover, iav encodes c4-type zinc finger peptide (zfp) motif-containing viral protein (such as matrix protein m1) for replication and virus budding (fernandez-pol et al. 2001) ; rps27 also contains a zfp motif, and has recently been reported to facilitate translation and viral replication (fernandez-pol et al. 2001; fernandez-pol 2011) . when rps27 is eliminated, the replication and infectivity of iav is abolished, but it is dispensable for global protein synthesis (karlas et al. 2010) . research on the essential viral and cellular zfp motif-containing proteins has implications for the prevention and therapy of viral diseases, and these proteins should be considered as targets for novel antiviral compounds. in eukaryotes, certain rps control selectivity during ires-driven translation. for example, rpl38 and rpl35 control the selective translation of a specific mrna by promoting translation primarily at the initiation stage (kondrashov et al. 2011; jiang et al. 2015; xue et al. 2015) . several viruses whose genomes lack a 5′-end cap structure use capindependent mechanisms for translation initiation (daijogo and semler 2011; martinez-salas et al. 2015) . these viruses have in fact evolved to hijack specific rps to achieve optimal viral protein synthesis; they facilitate translation of viral transcripts of ires-containing viruses. the rps include rps25 (landry et al. 2009 ), rps5 (fukushi et al. 2001; bhat et al. 2015) , rps6 (huang et al. 2012) , rps9 (fukushi et al. 2001) , and rack1 (majzoub et al. 2014) , as well as rpl10a , rpl22 (wood et al. 2001) , and rplp1/2 (campos et al. 2017) . of these, rack1 (majzoub et al. 2014 ) and rps6 (huang et al. 2012 ) are specifically required for ires activity in various types of viral iress. rpl10a regulates translation from the hcv and cricket paralysis virus (crpv) iress but not from the encephalomyocarditis virus (emcv) ires . in other words, rpl10a is an example of an rp that plays a specific role in promoting translation of particular classes of viral iress. rps25 is essential for initiation from the hcv and crpv ires (landry et al. 2009 ). rps25 deletion in yeast or mammalian cells has minimal effects on cellular protein synthesis, which implies that this ribosomal protein may be selectively required for viral ires-mediated translation (jack et al. 2011; hertz et al. 2013) . similarly, rack1 is not required for cellular iress or global synthesis (majzoub et al. 2014) . overall, rps specifically required for protein synthesis in particular viruses but not for global synthesis may provide an effective target for methods to fight viral infection. some of the related functions of rps are ribosome-dependent, as when rps participate in viral protein biosynthesis, and other functions, typically involved in the regulation of infection in host cells, are independent of the ribosome. alterations in rp characteristics for optional translation are implicated in interaction with viral proteins, post-translational modification, and redistribution, directly or with the assistance of rna helicase. some viruses have developed to hijack specific rps using viral proteins to achieve optimal viral translation. ibdv vp3 is a multifunctional protein playing a key role in virus assembly and pathogenesis. ribosomal protein l4 (rpl4) ) and ribosomal protein l18 were identified as interacting partners of vp3 protein and as being involved in the regulation of ibdv replication. rpl18 interacts with nucleocapsid protein of rice stripe tenuivirus (rsv), and silencing rpl18 significantly reduces viral mrna translation and replication of rsv (li et al. 2018a, b) . rpl7 interacts with the vp51 to participate in viral infection of white spot syndrome virus (wssv), and the addition of anti-rpl7 antibody inhibits wssv infection in litopenaeus vannamei . among flaviviruses, the envelope protein of west-nile virus (wnv), dengue virus (denv), and yellow fever virus (yfv) bind with rpsa, which serves as the viral receptor (zidane et al. 2012) . as regards alphaviruses, rpsa includes different binding sites for venezualian equine encephalitis virus (veev) and sindbis virus (sinv) (jamieson et al. 2008; malygin et al. 2009 ). hiv-1 tat inhibits cell proliferation via an interaction with rps3 and increases the level of rps3 in the nucleus, thereby disrupting mitotic spindle formation during hiv-1 infection (kim and kim 2018) . fmdv vp1 inhibits the antiviral response of rpsa by interacting with rpsa to promote fmdv replication (zhu et al. 2019) . binding of viral proteins to specific rps plays a crucial role in viral infection. effective inhibition of the binding between rps and viral proteins can be a potential target for antiviral design. post-translational modifications such as phosphorylation, ubiquitination, acetylation, methylation, or o-glcnacylation activate, deactivate, or modify rp's functions. over 2,500 modifications of human rps have been reported; these modifications impact rp activity and subsequently modify the global rate of translation by modulating initiation, elongation, and termination rates (emmott et al. 2019) . one wellstudied modification on the ribosome is phosphorylation following activation or inhibition of intra and extracellular signaling cascades in response to stimuli occurring in physiological or pathological conditions (emmott et al. 2019) . phosphorylation of rps6 is perhaps the most widely studied ribosomal protein phosphorylation among the rps involved in virus infection; it is induced by various viral infections decker 1981; banham et al. 1993; beaud et al. 1994 ). in addition, an early study revealed that phosphorylation of rpl30 was induced by herpes simplex virus-1 (hsv-1) (simonin et al. 1995) . rpsa, rps2 and rps13 appear specifically phosphorylated in cells early after infection with vaccinia virus (kaerlin and horak 1978; buendia et al. 1987) . efficient phosphorylation of the ribosomal proteins correlates well with possible translational mechanisms, ensuring efficient expression of early and late genes of vaccinia virus. it has been shown that phosphorylation of the rack1a protein on two residues-ser-122 and thr162-by an atypical serine/ threonine protein kinase wnk8 (with no lysine8) negatively regulates rack1a function in the glucose responsiveness pathway by influencing its protein stability (urano et al. 2015) . rack1a proteins are phosphorylated by tyrosine, depending on diverse environmental stresses (sabila et al. 2016 ). viral infection leads to activation of ribosomal protein phosphorylation, which may be closely related to effective viral translation. the change of phospho-rp level may affect viral translation. however, despite reports on the increasing phosphorylation of rps induced by viral infection (emmott et al. 2019; diaz et al. 2002) , the details of the role of this modification in virus infection remain poorly understood. another extensively studied post-translational modification on the ribosome is ubiquitylation, or covalent bonding of ubiquitin to substrate proteins for disposal by the proteasome (degradative ubiquitylation) or alteration of the function of the protein (regulatory ubiquitylation) (haas et al. 1982; thrower et al. 2000) . degradative ubiquitylation is important for ribosome-independent function. in fact, excess rps that are not incorporated into the ribosome are degraded by the proteasome (lam et al. 2007; genuth and barna, 2018) . several rps have been shown to be dynamically ubiquitylated in response to cellular conditions; rps2, rps3, and rps20, for instance, undergo regulatory ubiquitylation during the unfolded protein response (upr) in yeast, fruit fly, and human cell lines (higgins et al. 2015) . notably, the ubiquitination of rps20 was found to be specifically required for viral replication and synthesis of poxvirus proteins (digiuseppe et al. 2018 ). in addition to ubiquitin, other related proteins have been found to modify the ribosome. rpl26, for instance, is the principal target of ufm1 conjugation, a ubiquitin-related modification (ufmylation), and ufmylated rpl26 is highly enriched on er membranebound ribosomes and polysomes (walczak et al. 2019; wang et al. 2019) . ufm1 is also conjugated to rps3, rps20, and rpl10 (simsek et al. 2017) . the overlap of ufmylation and ubiquitylation on rps3 and rps20 also occurs, and this suggests that certain rps have combinatorial modifications. o-glcnacylation modification was also found to be activated in response to adverse stress to protect cellular proteins from damage hart 2004, 2006; slawson et al. 2006 ). in the stress response, o-glcnac-modified proteins are prominent components of stress granules, including small and large ribosomal subunit proteins (rps3, 9, 11 and 24, and rpl6, 13a, 14, and the ribosomeassociated protein rack1 (ohn et al. 2008) . although the effect of rp o-glcnacylation on viral infection has been as yet little studied, we can to some extent understand rp o-glcnacylation under viral infection through other forms of cellular stimulation, and start to develop an understanding of the possible role of post-translational modification of rps in antiviral strategy. although rps are all synthesized in the cytoplasm, they assemble into functional subunits in different subcellular compartments (kressler et al. 2010; li 2019) . sometimes, ribosomal proteins change their localization under virus infection to exercise functions outside the ribosome. rpl22, for instance, is translocated from the nucleoli to the nucleoplasm and colocalized with icp4 in infection by hsv-1. the location change of rpl22 plays a role in the regulatory functions expressed by icp4 (leopardi and roizman, 1996) . in another example of similar function, after denv infection, rpl18 is redistributed to the perinuclear region for regulation of viral replication (cervantes-salazar et al. 2015) . in eukaryotic cells, acidic ribosomal protein rplp0 binds two p1/p2 protein heterodimers to form a pentameric p-stalk (ballesta et al. 1996; naganuma et al. 2007 ). eukaryotic rplp1 and rplp2 proteins also exist in free form in the cytoplasm, and the exchange between the ribosome-bound rplp1 and rplp2 proteins and the cytoplasmic pools is thought to regulate the activity of the ribosome (lee et al. 2010) . this property may lead to changes in the localization of rplp1/2 in the ribosome and cytoplasmic pools under stress conditions. viral infection is one of numerous possible stress stimuli, so viral infection may cause rplp1/2 to produce this subcellular compartment exchange to affect virushost translation regulation. under nucleolar stress induction, rplp0 accumulates in the cytoplasm of mammalian cells as a free, ribosome-unbound protein (deryło et al. 2018) . a ribosome-free pool of rplp0 has been shown to be a population of proteins released from pre-existing ribosomes. the presence of rplp0 on the ribosome seems to be affected in stressed cells, and it might be considered as a regulatory element responding to environmental fluctuations. in addition, the mitochondrial rps (mrps) are encoded by nuclear genes and then imported into mitochondria for assembly; they are responsible for the translation of 13 mitochondrial mrnas (christian and spremulli 2012) . mitochondrial rpl18 acts as a critical regulator of the stress response and generates a cytosolic isoform in a stress-dependent manner. the cytosolic rpl18 is incorporated into the 80s ribosome and facilitates ribosome engagement in heat-shock protein (hsp) mrna translation to escape from the shutoff of global protein synthesis during stress (zhang et al. 2015) . accumulating evidence has revealed the roles of rps belonging to the large 60s subunit in regulating selective translation of specific mrnas, although they are primarily involved in catalyzing peptide-bond formation (wilson et al. 2012) . a previous study by the present author identified rpl13 as a critical regulator of ires-driven translation during foot-and-mouth disease virus (fmdv) infection, but found that it is not essential for cellular global translation (han et al. 2020) . our results support a model whereby the viral iress recruit helicase ddx3 downstream of the rpl13 to facilitate ires-driven translation and viral replication. specifically, the depletion of ddx3 disrupts binding of rpl13 to the fmdv ires, whereas the reduction in rpl13 expression impairs the ability of ddx3 to promote ires-driven translation directly. this work was the first to identify a connection between ddx3 and rps in modulating viral ires-dependent translation (han et al. 2020) . ddx3 is well known, however, to play roles in various key aspects of rna metabolism, including transcriptional regulation, splicing, mrna export, ribosome biogenesis, and translational regulation (chuang et al. 1997; rocak and linder 2004; guenther et al. 2018) . improved knowledge about these rna helicases and their relation to translation initiation could have important implications for the understanding of selective translation of viral mrna, and thus for the development of effective antivirals. the strategy of targeting a host factor, instead of the virus directly, could circumvent the danger of resistance in mutation-associated variations and the evolution of new viral variants after prolonged use of otherwise-promising antiviral drugs. after 60 years of research on ribosomes, the ribosome has emerged as a major player in translational regulation, and its place in the list of potential antiviral therapeutic targets has been reinforced. targeting the ribosome, either biogenesis or function, may provide an efficient and focused approach for design of antiviral agents. as our understanding of the function of rbfs and rps in viral translation has evolved, the design of therapeutic strategies for rbfs and rps has been explored. recent structural analysis of prokaryotic and eukaryotic ribosomes has demonstrated that certain molecules, including some antibiotics and chemical inhibitors of translation, can bind to the ribosome (svetlov et al. 2017; tereshchenkov et al. 2018) . cx-5461, for example, is a ribosomal biogenesis inhibitor that disrupts rna pol i-mediated transcription (drygin et al. 2011) , and inhibits viral dna synthesis and virus production in the early and late stages of the hcmv infection (westdorp and terhune 2018) . toyocamycin is a small molecule inhibitor of rio1 kinase activity (kiburu et al. 2012) ; rio1 is an essential ribosome biogenesis factor required for maturation of 40 s ribosomal subunit (angermayr et al. 2002) . toyocamycin inhibits ribosome biogenesis to produce antiviral effects (ríman et al. 1969) . notably, translation of ires-containing viruses (such as hcv, htlv-1, crpv, poliovirus, and adenovirus) is dependent on rps25 (landry et al. 2009; hertz et al. 2013; olivares et al. 2014) , and its activity is highly sensitive to the antibiotic edeine (olivares et al. 2014) . picolinic acid (pa) and fusaric acid (fu) disrupt the zfp motifs of rps27 or crucial viral proteins to produce antiviral effects (yu et al. 1995; everett et al. 1993; wakefield and brownlee 1989; love et al. 1996) . ricin is an rplp protein inhibitor (may et al. 2012) ; rplp1/2 is an essential host factor for flavivirus replication (campos et al.2017) . rack1 is essential for the translation of many viruses, including hcv, drosophila c (dcv), cricket paralysis (crpv) (majzoub et al. 2014) , and vaccinia viruses (jha et al. 2017) . thus, rack1 inhibitors may be developed as novel antiviral therapeutics. the drug sd-29 and analogues show high efficacy in inhibition of virus proliferation (ullah et al. 2019) . exploring strategies for targeting rbfs and rps is a natural path for further study in this field. once the function of rbfs and rps in viral translation is understood, targeting inhibitors will need to be identified, designed, and screened. to accelerate the process of drug discovery for both novel targets and existing targets that suffer from drug resistance, basic platforms for drug design and screening are required. in a recent study, structure-based screening of two million commercially available compounds was used to screen for small molecules to target the rack1 y248 phosphorylation site. (as noted, host rack1 protein is an attractive target for developing broad antiviral drugs.) dozens of small compounds were identified that could potentially bind to the experimentally determined functional site of the rack1a protein; the drugs show high efficacy in inhibition of the proliferation of viruses that require rack1 for translation (ullah et al. 2019) . this approach can be used to study the ribosomal proteins that are essential for the translation of many viruses, and eventually to design broad-spectrum antiviral drugs. furthermore, depletion of regulatory rbfs and rps is not essential for basic ribosomal function in experiments, making them potential targets for the design of small molecule antiviral drugs. rna interference (rnai) and strategies based on crispr/cas9 are also promising approaches in targeting of rbf and rp genes. of course, projecting forward to the practical application of antiviral drugs, the adverse cell response caused by targeting of rbf and rp genes must be studied in detail. deciphering the functional details of translation of viral mrna will require the in-depth scrutiny of rps. a number of technological efforts have been made over the last decade to facilitate exploration of rp biogenesis and functions. ribosome profiling is an emerging technique that allows the distribution of ribosome units on each transcript to be obtained, enabling systematic probing of translation and increased sensitivity and efficiency. this method, in conjunction with rna-seq, has been used to probe the complex viral replication of several viruses irigoyen et al. 2016 ). using high-coverage tandem mass tag (tmt) mass spectrometry, the relative monosome and polysome fractions of each rp were compared. a thorough examination of each rp revealed that some rps (such as rps9 and rpl11) are more abundant in monosomes than in polysomes (slavov et al. 2015) . tmt technology has been extended to the investigation of potential heterogeneity of rps. mass spectrometry has also been improved to address directly and more accurately the possibility of variability in rp stoichiometry. this novel technology, called selected reaction monitoring (srm)-based proteomics, uses the spiking of samples with known amounts of labeled peptides derived from rps as a standard for absolute quantification. using this technology, absolute quantification of 15 rps isolated from polysomes was assessed and 4 rps, rpl10a, rpl38, rps7, and rps25, were identified as being substoichiometric in murine esc ribosomes ). based on this technology, the heterogeneity in ribosome composition within a single cell type and a single polysome profile fraction was revealed for the first time. these approaches provide a starting point in the attempt to identify and quantify each rp expression profile in cells challenged with viruses, and at different infectious stages. in a recent study, both the small and large ribosomal subunits in mouse escs were endogenously tagged, and affinity enrichment for each of the tagged ribosomal subunits was performed to define the intersection of the two separate ribosomal subunit datasets. this has led to the identification of ribosome-associated proteins (raps), which fall into unexpected functional categories, such as energy metabolism, cell cycle, and key protein and rna modification enzymes (simsek et al. 2017 ). this method may facilitate identification of a series of proteins (such as raps) that are specifically recruited to ribosomes with the assistance of rps, and act as potential targets for antiviral therapeutic design. technical limitations have thus far impeded the study of selective translation mediated by rps in viral infection, and considerable research will be required before clinical application of viral therapeutics based on the targeting of rps. historically, the ribosome has been viewed as a complex ribozyme, and ribosome activity has been considered to be highly regulated. although the concepts of ribosome heterogeneity and specialization at the level of core rps are still in infancy, solid data have been published in various areas, offering convincing evidence that the ribosome plays a constitutive role as well as a regulatory function in translation. moreover, constitutive components of the ribosome may perform more specialized activities by virtue of their interactions with specific mrna regulatory elements, such as iress. these findings add numerous layers of subtlety to the conventional interpretation of translational regulation. viruses depend on host cell structures and translation systems to complete their life cycle. in order to survive in the host cell and achieve rapid replication and proliferation, viruses have developed various mechanisms to allow the selective translation of viral mrna and repress cellular mrna translation. inducing host translation shutoff is a strategy used by many viruses to optimize their replication and spread by fostering viral protein synthesis and crippling host antiviral responses. in this process, some rps are activated to promote the translation of specific transcripts, and thus the virus can still synthesize its own proteins to optimize their replication and spread when host translation is shut off. in this review, we focus on the important role of rp production and function in viral or host translation processes that support viral replication and infection. understanding the research on the specialized rbfs and rps utilized by a virus can deepen the understanding of selective translation, and expand the depth and breadth of the field of virus-host interactions. this research, data, and theoretical evidence will facilitate the identification of antiviral targets and the eventual design of antiviral drugs, and advance the development of therapeutic strategies to produce optimal antiviral agents for effective control of viral diseases. the hbx oncoprotein of hepatitis b virus engages nucleophosmin to promote rdna transcription and cellular proliferation molecular basis of sequence specific recognition of pre-ribosomal rna by nucleolin yeast rio1p is the founding member of a novel subfamily of protein serine kinases involved in the control of cell cycle progression cellular cap-binding protein, eif4e, promotes picornavirus genome restructuring and translation the large ribosomal subunit stalk as a regulatory element of the eukaryotic translational machinery phosphorylation of ribosomal proteins by the vaccinia virus b1r protein kinase ribosomes take control ribosomal protein s2/sa kinase purified from hela cells infected with vaccinia virus corresponds to the b1r protein kinase and phosphorylates in vitro the viral ssdna-binding protein a systematic view on influenza induced host shutoff nucleolar trafficking of the mouse mammary tumor virus gag protein induced by interaction with ribosomal protein l9 the beta hairpin structure within ribosomal protein s5 mediates interplay between domains ii and iv and regulates hcv ires function ribosome biogenesis restricts innate immune responses to virus infection and dna the long unwinding road of rna helicases host-and strain-specific regulation of influenza virus polymerase activity by interacting cellular proteins subtractional heterogeneity: a crucial step toward defining specialized ribosomes ribosomal protein phosphorylation in vivo and in vitro by vaccinia virus rplp1 and rplp2 are essential flavivirus host factors that promote early viral protein accumulation cell cycle dependent nucleolar localization of the coronavirus nucleocapsid protein dengue virus ns1 protein interacts with the ribosomal protein rpl18: this interaction is required for viral translation and replication in huh-7 cells rrp1b targets pp1 to mammalian cell nucleoli and is associated with pre-60s ribosomal subunits ribosomal protein s6 interacts with the latency-associated nuclear antigen of kaposi's sarcomaassociated herpesvirus interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell interaction between sars-cov helicase and a multifunctional cellular protein (ddx5) revealed by yeast and mammalian cell two-hybrid systems ribosomal protein l4 interacts with viral protein vp3 and regulates the replication of infectious bursal disease virus characterization of the interaction between hantavirus nucleocapsid protein (n) and ribosomal protein s19 (rps19) mechanism of protein biosynthesis in mammalian mitochondria requirement of the dead-box protein ded1p for messenger rna translation interaction of nucleolin with ribosomal rna genes and its role in rna polymerase i transcription mechanistic intersections between picornavirus translation and rna replication phosphorylation of ribosomal protein s6 in avian sarcoma virus-transformed chicken embryo fibroblasts the ul10 protein, a component of the ribosomal p-stalk, is released from the ribosome in nucleolar stress alteration of ribosomal protein maps in herpes simplex virus type 1 infection znf598 plays distinct roles in interferon-stimulated gene expression and poxvirus protein synthesis changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus targeting rna polymerase i with an oral small molecule cx-5461 inhibits ribosomal rna synthesis and solid tumor growth elucidation of the avian nucleolar proteome by quantitative proteomics using silac and changes in cells infected with the coronavirus infectious bronchitis virus ribosome stoichiometry: from form to function a novel arrangement of zinc-binding residues and secondary structure in the c3hc4 motif of an alpha herpes virus protein family conservation of multifunctional ribosomal protein metallopanstimulin-1 (rps27) through complex evolution demonstrates its key role in growth regulation in archaea, eukaryotic cells, dna repair, translation and viral replication essential viral and cellular zinc and iron containing metalloproteins as targets for novel antiviral and anticancer agents: implications for prevention and therapy of viral diseases and cancer ribosomal protein s5 interacts with the internal ribosomal entry site of hepatitis c virus heterogeneity and specialized functions of translation machinery:from genes to organisms cellular rna helicase p68 relocalization and interaction with the hepatitis c virus (hcv) ns5b protein and the potential role of p68 in hcv rna replication the helicase ded1p controls use of near-cognate translation initiation codons in 5' utrs ubiquitin-activating enzyme mechanism and role in protein-ubiquitin conjugation dikstein r (2015) cap-dependent, scanning-free translation initiation mechanisms ribosomal protein l13 promotes ires-driven translation of fmdv in helicase ddx3-dependent manner ribosomal protein s25 dependency reveals a common mechanism for diverse internal ribosome entry sites and ribosome shunting the unfolded protein response triggers site-specific regulatory ubiquitylation of 40s ribosomal proteins translational control in murine hepatitis virus infection the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus rps27a enhances ebv-encoded lmp1-mediated proliferation and invasion by stabilizing of lmp1 attenuation of 40s ribosomal subunit abundance differentially affects host and hcv translation and suppresses hcv replication high-resolution analysis of coronavirus gene expression by rna sequencing and ribosome profiling nucleolin stimulates viral internal ribosome entry site-mediated translation rrna pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells redundant role of dead box proteins p68 (ddx5) and p72/p82 (ddx17) in ribosome biogenesis and cell proliferation crystal structure of the human laminin receptor precursor trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase 60s ribosomal protein l35 regulates beta-casein translational elongation and secretion in bovine mammary epithelial cells identification and characterization of ribosomal proteins phosphorylated in vaccinia-virus-infected hela cells the molecular mechanics of eukaryotic translation genome-wide rnai screen identifies human host factors crucial for influenza virus replication human cytomegalovirus infection upregulates the mitochondrial transcription and translation machineries increased phosphorylation of ribosomal protein s6 in hamster fibroblasts transformed by polyoma virus and simian virus 40 phosphorylation of ribosomal proteins in hamster fibroblasts infected with pseudorabies virus or herpes simplex virus molecular analysis of the factorless internal ribosome entry site in cricket paralysis virus infection structure of the human 80s ribosome interaction of rio1 kinase with toyocamycin reveals a conformational switch that controls oligomeric state and catalytic activity effect of hiv-1 tat on the formation of the mitotic spindle by interaction with ribosomal protein s3 ribosome-mediated specificity in hox mrna translation and vertebrate tissue patterning driving ribosome assembly the function and synthesis of ribosomes analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins rps25 is essential for translation initiation by the dicistroviridae and hepatitis c viral iress nucleolar protein upstream binding factor is sequestered into adenovirus dna replication centres during infection without affecting rna polymerase i location or ablating rrna synthesis solution structure of the dimerization domain of ribosomal protein p2 provides insights for the structural organization of eukaryotic stalk a ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mrnas functional interaction and colocalization of the herpes simplex virus 1 major regulatory protein icp4 with eap, a nucleolar-ribosomal protein regulation of ribosomal proteins on viral infection nucleophosmin is essential for c-myc nucleolar localization and c-myc-mediated rdna transcription ribosomal protein l18 is an essential factor that promote rice stripe virus accumulation in small brown planthopper porcine reproductive and respiratory syndrome virus infection induces both eif2α phosphorylation-dependent and -independent host translation shutoff white spot syndrome virus vp51 interact with ribosomal protein l7 of litopenaeus vannamei the crystal structure of hepatitis c virus ns3 proteinase reveals a trypsin-like fold and a structural zinc binding site rack1 controls ires-mediated translation of viruses c-terminal fragment of human laminin-binding protein contains a receptor domain for venezuelan equine encephalitis and tick-borne encephalitis viruses picornavirus ires elements: rna structure and host protein interactions the p1/p2 proteins of the human ribosomal stalk are required for ribosome binding and depurination by ricin in human cells characterization of the interaction between the hiv-1 gag structural polyprotein and the cellular ribosomal protein l7 and its implication in viral nucleic acid remodeling influenza a h3n2 subtype virus ns1 protein targets into the nucleus and binds primarily via its c-terminal nls2/nols to nucleolin and fibrillarin multifunctional viral proteinγ345 manipulates nucleolar protein nop53 for optimal viral replication of hsv-1 targeting nuclear proteins for control of viral replication how should we think about the ribosome? the n-terminal regions of eukaryotic acidic phosphoproteins p1 and p2 are crucial for heterodimerization and assembly into the ribosomal gtpaseassociated center severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells a functional rnai screen links o-glcnac modification of ribosomal proteins to stress granule and processing body assembly the 5 untranslated region of the human t-cell lymphotropic virus type 1 mrna enables cap-independent translation initiation nuclear entry and nucleolar localization of the newcastle disease virus (ndv) matrix protein occur early in infection and do not require other ndv proteins influence of toyocamycin on rna synthesis in chick embryo cells noninfected and infected with strain mc29 avian leukosis virus dead-box proteins: the driving forces behind rna metabolism tyrosine phosphorylation based homo-dimerization of arabidopsis rack1a proteins regulates oxidative stress signaling pathways in yeast ribosome protein l4 is essential for epstein-barr virus nuclear antigen 1 function translating the genome in time and space: specialized ribosomes, rna regulons, and rna-binding proteins heterogeneous ribosomes preferentially translate distinct subpools of mrnas genome-wide phosphorylation of ribosomal protein l30 after herpes simplex virus type 1 infection the mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity differential stoichiometry among core ribosomal proteins o-glcnac cycling: how a single sugar post-translational modification is changing the way we think about signaling networks a nucleolar protein, ribosomal rna processing 1 homolog b (rrp1b), enhances the recruitment of cellular mrna in influenza virus transcription kinetics of drugribosome interactions defines the cidality of macrolide antibiotics nop53p is a novel nucleolar 60s ribosomal subunit biogenesis protein the complexity of human ribosome biogenesis revealed by systematic nucleolar screening of pre-rrna processing factors binding and action of amino acid analogs of chloramphenicol upon the bacterial ribosome nop53p is required for late 60s ribosome subunit maturation and nuclear export in yeast recognition of the polyubiquitin proteolytic signal silencing of hepatitis c virus replication by a non-viral vector based on solid lipid nanoparticles containing a shrna targeted to the internal ribosome entry site (ires) host targeted antiviral (hta): functional inhibitor compounds of scaffold protein rack1 inhibit herpes simplex virus proliferation arabidopsis receptor of activated c kinase1 phosphorylation by with no lysine8 kinase viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells rna-binding properties of influenza a virus matrix protein m1 ribosomal protein rpl26 is the principal target of ufmylation cloning of mouse genomic ribosomal protein l6 gene and analysis of its promoter the association of ribosomal protein l18 (rpl18) with infectious bursal disease virus viral protein vp3 enhances viral replication ufmylation of rpl26 links translocation-associated quality control to endoplasmic reticulum protein homeostasis interferon production and inhibition of host synthesis in cells infected with vesicular stomatitis virus impact of rna polymerase i inhibitor cx-5461 on viral kinase-dependent and -independent cytomegalovirus replication new mrnas are preferentially translated during vesicular stomatitis virus infection the structure and function of the eukaryotic ribosome hepatitis c virus 3'x region interacts with human ribosomal proteins specialized ribosomes: a new frontier in gene regulation and organismal biology rna regulons in hox 5' utrs confer ribosome specificity to gene regulation deciphering poxvirus gene expression by rna sequencing and ribosome profiling subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein specific disulfide formation in the oxidation of hiv-1 zinc finger protein nucleocapsid p7 nucleophosmin is essential for ribosomal protein l5 nuclear export o-glcnac a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress cell signaling, the essential role of o-glcnac translational control of the cytosolic stress response by mitochondrial ribosomal protein l18 ddx5 facilitates hiv-1 replication as a cellular co-factor of rev ribosomal proteins: functions beyond the ribosome foot-and-mouth disease virus capsid protein vp1 interacts with host ribosomal protein sa to maintain the activation of mapks signal pathway and promote virus replication the folded and disordered domains of human ribosomal protein sa have both idiosyncratic and shared functions as membrane receptors acknowledgments this work was supported by the national natural science foundation of china (31772739) and a cau-grant for the prevention and control of immunosuppressive disease in animals (cau-g-pcida) of the china agricultural university. conflict of interest the authors declare that they have no conflict of interest. key: cord-313988-3xjnpkqp authors: ferraz, rosa maría; vera, andrea; arís, anna; villaverde, antonio title: insertional protein engineering for analytical molecular sensing date: 2006-04-03 journal: microb cell fact doi: 10.1186/1475-2859-5-15 sha: doc_id: 313988 cord_uid: 3xjnpkqp the quantitative detection of low analyte concentrations in complex samples is becoming an urgent need in biomedical, food and environmental fields. biosensors, being hybrid devices composed by a biological receptor and a signal transducer, represent valuable alternatives to non biological analytical instruments because of the high specificity of the biomolecular recognition. the vast range of existing protein ligands enable those macromolecules to be used as efficient receptors to cover a diversity of applications. in addition, appropriate protein engineering approaches enable further improvement of the receptor functioning such as enhancing affinity or specificity in the ligand binding. recently, several protein-only sensors are being developed, in which either both the receptor and signal transducer are parts of the same protein, or that use the whole cell where the protein is produced as transducer. in both cases, as no further chemical coupling is required, the production process is very convenient. however, protein platforms, being rather rigid, restrict the proper signal transduction that necessarily occurs through ligand-induced conformational changes. in this context, insertional protein engineering offers the possibility to develop new devices, efficiently responding to ligand interaction by dramatic conformational changes, in which the specificity and magnitude of the sensing response can be adjusted up to a convenient level for specific analyte species. in this report we will discuss the major engineering approaches taken for the designing of such instruments as well as the relevant examples of resulting protein-only biosensors. conventional biosensors are hybrid elements consisting of a biochemical receptor for a given analyte, physically coupled to a physicochemical transducer that converts such interaction into a macroscopic, analytically useful signal [1] . in the last decades, many types of biosensors have been under continuous development, integrating biological components such as proteins, nucleic acids, membranes cells and even tissues acting as receptors, and different signal transducers devices including microbalances, electrodes, optical components and semiconductors. such instruments have been applied into a diversity of fields but specially for the detection of contaminants in foods and environment [2] . more recently, and pressured by the need of more sensitive and specific detection tools for biomedical applications, in particular diagnosis, new types of protein-only biosensors are being explored [3] , that contain both the receptor and transducer elements in a single polypeptide chain. alternatively, protein-only sensors can specifically act as receptors that exploit the whole living cell where they are synthesised, as a complex signal transducer, enabling the detection of the analyte through intricate global activities such as differential growth or support of viral multiplication among others. both type of protein-only biosensors offer very appealing advantages over classical devices. first, chemical coupling to the signal transducer is not required as straightforward bioproduction results in a ready-to-use final product, either a purified protein of protein-producing cells or cell extracts. also, protein engineering procedures such as sitedirected mutagenesis or directed molecular evolution allow refining the specificity of ligand binding and permit the development of new receptors for new analytes, as demanded from medicine or industry. the mechanics for which a protein responds to a specific ligand in a macroscopically detectable way would generally imply variations in its activity, either enhancement or inhibition, that could be detectable either directly or indirectly through a biological amplification process. in general, natural protein-ligand interactions result in moderate conformational modifications that would be poorly useful in molecular switching, as they have a lim-ited impact on protein's activity. protein engineering allows the modification of the receptor in a way in which the interaction with the analyte promotes profound conformational modifications. there are many examples of useful intracellular indicators of molecular interactions, gene expression or for biological screening [4] that use at different extent end-to-end fusion proteins, including two-hybrid systems [5] , fluorescence resonance energy transfer (fret) [6] , and protein fragment complementation [7] among others. however, insertional protein engineering allows a more versatile combination of functional modules for the construction of highly responsive mosaic proteins exhibiting unusual conformational versatility upon ligand binding [8, 9] . obviously, the protein segment or domain acting as a receptor element must be conveniently displayed on the protein surface to allow a proper interaction with the analyte. although some of the constructs referenced below derive from random insertions and further selection [10, 11] , the previous identification of solvent-exposed permissive sites through different procedures has allowed a more rational designing procedures based on site directed peptide insertion for the construction of biosensors and other type of multifunctional proteins [12] [13] [14] [15] [16] [17] [18] [19] . the principles of protein functionality supporting insertional approaches for biosensor construction are further discussed as exemplified by representative models and specific applications, being most of the resulting protein-only biosensors based on either cleavable ( figure 1a ) or allosteric ( figure 1b table 1 . the most dramatic conformational modification that a given ligand (in this case a protease) might induce on a target protein is hydrolysis, that mostly result in its functional inactivation but being sometimes a requisite for a polypeptide reaching the active form, if existing as an inactive precursor. in fact, targeted proteolysis is a biological principle regulating many complex cellular events [20] [21] [22] . therefore, including a specific protease target site on a protein's surface would made it susceptible to sitelimited digestion resulting in detectable changes in its electrophoretic pattern. the successful implementation of such technology would imply a refined analysis of the protease target site susceptibility, as peptide display in different solvent-exposed sites could result in distinguishable digestion efficiencies, since the protein regions neighbouring the insert seem to have a dramatic influence on the peptide conformation [23] . this has been exemplified by the insertional mutagenesis of the protease resistant, green fluorescent protein (gfp), to make it susceptible to trypsin and other proteolytic enzymes [24] . the detection of specific proteases and proteolytic activities is now of extreme relevance in virology and in particthe biosensing principles of the constructs listed in table 1 are summarized here as split in two groups figure 1 the biosensing principles of the constructs listed in table 1 are summarized here as split in two groups. in a), the sensing principles underlying cleavable platforms are presented in which simple hydrolysis of protease target site-bearing hybrid proteins by an effector protease (p) result in a macroscopic signal. among others, variations of the migration pattern, enzyme activation or inactivation, repressor inactivation, enhanced fluorescence by removal of a quencher or dual fluorescence emission by fret modulation. in b), a ligand (l) promotes conformational modifications in the sensor either multimerization, correct folding or allosteric activation. a few enzyme biosensors are inactivated in presence of the ligand probably by steric hindrance of the active site. http://www.microbialcellfactories.com/content/5/1/15 ular for designing antiviral drugs that inhibit viral protein processing and therefore multiplication. beyond the straightforward electrophoretic analysis of the sensing protein [25] , a rather inconvenient technique from the analytical point of view, monitoring protease-mediated reduction of activity (fluorescence emission or enzymatic activity) would offer a more convenient protease sensing signal [26] . in a step further, it is known that many natural proteins are proteolytically activated by the removal of self inhibitory protein domains [27] . in this context, the convenient insertional engineering of the carboxy terminus of p53, containing such a regulatory element, has resulted in a set of p53 variants that are activated upon its removal mediated by either the lethal factor (lf) or the human immunodeficiency virus (hiv) protease [28] . again, in this case, the sensing signal is detectable by upshift electrophoretic analysis, since the activated p53 gains the ability to interact with specific dna sequences [28] . in an attempt to produce more convenient analytical signals, protease target sites have been introduced in the linker between two end-to-end fused proteins that emit fluorescence at different wavelengths, so the cleavage can be monitored by variations in the fret spectra [29] [30] [31] . although being not a standard insertional approach, the principles governing such engineering processes are similar to those discussed above. in this context, the protein hydrolysis splitting a fluorophore and its quencher has been also a successfully proven biosensing principle [60] . it would be expected that the generation of a signal by a specific proteolytic attack acted as an all-or-nothing switcher rather than as a fine sensing tool. however, a very discriminative monitoring tool for viral proteases activity was implemented as a high-throughput analytical method for antiviral drug testing and evaluation of the enzyme activity. the ci lytic repressor of the e. coli lambda bacteriophage has been engineered to accommodate a selected target site for proteases from either hiv [32] , hepatitis c virus (hcv) [33] and severe acute respiratory syndrome (sars) viruses [34] . the appropriate co-expression of the engineered ci and the protease promotes lytic lambda propagation that is reported by plaque counting. this system serves not only to test protease inhibitors for antiviral drug research but also to quantitatively evaluate the activity of proteases from mutant viruses emerging in patients treated with antiviral, protease-targeted drugs [35] . the cascade events supported by the cell as a network signal transducer permits the quantitative translation of the statistic ci hydrolysis within the cellular pool, what would be probably not possible by using a more straightforward signal transducing system. the regulatable activity of allosteric enzymes lies on a biological principle highly matching with the protein-only biosensing concept [36] . the activity of allosteric enzymes is modulated upon binding of an effector to a receptor site, that being different from the active site, can influence its performance through the conformational impact promoted by the allosteric effector. since most natural effectors are irrelevant for analytical purposes, both allosteric and non allosteric enzymes have been engineered to allosterically respond to new effectors by insertion of appropriate receptor sites, in some cases accompanied by directed or random mutagenesis of the enzyme or directed molecular evolution. this straightforward insertional strategy often requires the identification of permissive sites in which inserted motives do not disturb irreversibly the enzyme activity [12, 37] , and has proven to be efficient in the engineering of β-galactosidase [38, 39] , alkaline phosphatase [40] , β-lactamase [10] and gfp [41, 42] as allosteric biosensors. as the fine mechanics of the conformational signal transduction in allosteric activation is not know, such devices have been constructed by error-and-trial approaches. recently [11] , a random insertional approach has permitted to newly create two allosteric enzymes by domain incorporation, by a strategy, in principle, with general applicability in biosensor design. among enzyme inhibitors and other few ligand species that activate allosteric biosensors, antibodies have been noted to be specially efficient allosteric effectors [36] and the use of antigenic peptides as receptors in only-protein biosensors would offer appealing tools for the fast molecular diagnosis of infectious diseases [39, 43] . allosteric βgalactosidases displaying arginine-glycine-aspartic acid (rgd)-containing antigenic peptides [23] , are activated by anti-peptide antibodies [38] but not by rgd-targeted integrins binding the same receptor [44] . this fact indicates different conformational constraints in the binding of both molecules [45] and suggests that the adaptive antibody binding could be a major force in sensor activation. a main problem of allosteric biosensing is the poor signal-background ratio, that in most of cases does not reach more than 2-fold (table 1) . higher activation factors would be extremely desirable for fine analytical applications where a wide dynamic range is required. in fact, in a few examples, the presence of anti-peptide antibodies (the analyte) even reduces the activity of the enzyme probably by steric hindrance of the active site, as reported by alkaline phosphatase [46] or β-lactamase [10] , when the antigenic peptide was placed in the very close vicinity of the active site. the inhibition of the enzymatic activity is not very desirable as an analytical signal since in highthroughput analysis of complex samples, the presence of enzyme inhibitors might render false positives. by comparing different species of allosteric biosensors sensing anti-peptide antibodies, it was recognized that the activation factor was highly depending on the perturbation that the inserted peptide receptor had promoted on the activity of the enzyme platform after insertion [47] . greater was the reduction in the specific activity of the enzyme, higher the activation mediated by the effector, but reaching only activation factors around 2 that seemed to be a biological upper limit to allosteric activation [47] . however, a deeper exploration of β-galactosidase allosteric sensors has revealed that the signal background ratio can be enhanced up to more than 10-fold, by alternative or combined approaches such as introducing a higher number of receptors per enzyme [43, 48] , optimising the reaction conditions [49] and selecting the appropriate substrate [50] . apart from plain diagnostic utilities, allosteric sensors can intriguingly perform as tools for the analysis of the immune response, as they specially recognize antibodies with a high potential as modifiers of the epitope conformation. in this context, a β-galactosidase sensor displaying an hiv gp41 epitope and responsive to human hivimmune sera is preferentially activated by the igg4 antibody subpopulation [51] . as at least in the case of hiv infection the ability of anti-viral antibodies to modify the epitope's conformation is strongly related to their neutralizing activity [52, 53] and probably to the progression of the infection [54] , allosteric biosensing could eventually offer a valuable instrument for high-throughput sera analysis for prognostic investigation. conformational changes promoted by molecular interactions may generate signals suitable for biosensing other than allosteric responses. the correct folding of a deletion mutant of the human fyn tyrosine kinase (fynsh3), a predominantly β-sheet protein, is induced by the binding of an appropriate proline-rich peptide ligand, and the folding process monitored in real time by tryptophan fluorescence [55] . temperature-sensitive yeast cells lacking dihydropholate reductase (dhfr) are complemented by two mouse dhfr containing foreign different ligand binding domains [56] . culture growth is then enhanced in presence of the respective ligands proving that molecular binding activates the complementing enzyme. although this system can be observed as a generic biosensor [57] , its real potential would lie on the selection of specific or improved ligands by directed molecular-cellular evolution. on the other hand, the presence of bivalent antibodies can promote dimer formation of a mutant p53 in which the tetramerization domain has been removed and antigenic b-cell epitopes of viral origin conveniently inserted [28] . since dimers are much more active than monomers, the presence of antiviral antibodies enables p53 to bind dna in an electrophoretically detectable manner. other conformation-linked effects of molecular interactions might also result in detectable activity changes or phenotypes acting as macroscopic signals for a given analyte. gaining further knowledge about enzyme structure and dynamics would necessarily offer additional possibilities of rational protein engineering [58] for exploitation of such conformational signals. insertion of foreign peptides as receptors of protein-only biosensors confers the resulting protein construct the ability to sense analytes by dramatic conformational changes unusual in the native, non engineered protein. for such a sensor being efficiently responsive, appropriated permissive sites need to be selected permitting proper receptor display and signal transduction, and the whole protein might require further engineering to gain specificity and response range. although most protein-only biosensors derive from trial-and-error engineering approaches, rational and very clever setting-ups are exemplified by combinations of sensing protein segments and conveniently modified acceptor proteins. among the diversity of sensing strategies based on insertional mutagenesis two protein platforms emerge as the most explored, namely cleavable sensors responding to proteases or their inhibitors, and allosteric, among whose most efficient effectors are antibodies. the performance of these two sensor types has been largely proved in the high throughput screening of antiviral drugs and for the molecular diagnosis of infectious diseases respectively. although the potential applications of protein-only biosensors are diverse and still have to be fully exploited, they have arisen as valuable new tools in biomedicine being intriguing alternatives to classical sensing technologies. rosa m ferraz and andrea vera have equally contributed to this review. electrochemical biosensors: recommended definitions and classification survey of the year 2004 commercial optical biosensor literature biosensor profiling of molecular interactions in pharmacology new methodologies for measuring protein interactions in vivo and in vitro using the yeast interaction trap and other two-hybrid-based approaches to study protein-protein interactions analysis of protein interactions using fluorescence technologies 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protein molecular mechanisms for antibody-mediated modulation of peptide-displaying enzyme sensors tailoring molecular sensing for peptide displaying engineered enzymes profiling the allosteric response of an engineered beta-galactosidase to its effector, anti-hiv antibody enhanced molecular recognition signal in allosteric biosensing by proper substrate selection high-throughput, functional screening of the anti-hiv-1 humoral response by an enzymatic nanosensor binding of the 2f5 monoclonal antibody to native and fusion-intermediate forms of human immunodeficiency virus type 1 gp41: implications for fusion-inducing conformational changes hiv-1 evades antibodymediated neutralization through conformational masking of receptor-binding sites antibody neutralization and escape by hiv-1 engineering a signal transduction mechanism for protein-based biosensors a yeast sensor of ligand binding making drug addicts out of yeast enzymes: an integrated view of structure, dynamics and function distinct mechanisms of antibody-mediated enzymatic reactivation in beta-galactosidase molecular sensors conversion of a maltose receptor into a zinc biosensor by computational design the authors appreciate the predoctoral fellowships received by rmf and av (from agaur and uab, respectively) for their work on allosteric biosensing and development of cleavable platforms respectively. the research performed in our laboratory on these topics has been founded through grants bio2004-00700 (from mec, spain) and 2005sgr-00956 (from agaur, catalonia, spain). key: cord-289124-6w2zvvj1 authors: mok, lawrence; wynne, james w.; ford, kris; shiell, brian; bacic, antony; michalski, wojtek p. title: proteomic analysis of pteropus alecto kidney cells in response to the viral mimic, poly i:c date: 2015-11-02 journal: proteome sci doi: 10.1186/s12953-015-0081-6 sha: doc_id: 289124 cord_uid: 6w2zvvj1 background: bats are recognised as an important reservoir for a number of highly pathogenic zoonotic viruses. while many of these viruses cause severe and often fatal disease in humans, bats are able to coexist with these viruses without clinical signs of disease. the mechanism conferring this antiviral response is not fully understood. here, we investigated the differential protein expression of immortalised pteropus alecto kidney cells (pakit03) following transfection with the viral mimic, poly i:c. two complementary proteomic approaches, difference gel electrophoresis (dige) and isobaric tagging for relative and absolute quantitation (itraq) were used to quantify changes in protein expression following poly i:c stimulation at 4, 8 and 20 hr post treatment (hpt). results: the expression of isg54 gene, a known responder to virus infection and poly i:c treatment, was significantly induced in transfected cells compared with mock-transfected cells. through itraq analysis we show that poly i:c up-regulates key glycolytic enzymes at 4 hpt within pakit03 cells. in contrast, at 20 hpt pakit03 cells down-regulated ribosomal subunit proteins. the analysis with dige of poly i:c transfected pakit03 cells showed over 215 individual spots differentially regulated, however only 25 spots could be unambiguously identified by lc-ms/ms. immunoblotting confirmed the up-regulation of eno1 and tpi1 in pakit03 cells following poly i:c transfection. a comparison with human cells (hek293t and hela) and one additional bat cell line (palut02), demonstrated that glycolytic pathways are also induced in these cell types, but at different intensities. conclusion: the two techniques, dige and itraq identified largely overlapping sets of differentially expressed proteins, however dige unambiguously identified significantly less proteins than itraq. poly i:c induced a rapid metabolic shift towards glycolysis within the pakit03 cells at 4 hpt, presumably as a consequence of increased energy requirements. on the other hand ribosomal subunit proteins were seen as down-regulated by itraq, these proteins may be the limiting factors in the translational machinery available for virus replication. this study provides new insight into the antiviral response of bat cells, highlighting the importance of energy metabolism. electronic supplementary material: the online version of this article (doi:10.1186/s12953-015-0081-6) contains supplementary material, which is available to authorized users. bats are the natural reservoir for a number of emerging and re-emerging viruses including severe acute respiratory syndrome (sars)-like and middle east respiratory syndrome (mers) coronaviruses (cov) [1, 2] , hendra and nipah paramyxoviruses [3, 4] , and the filoviruses, ebola and marburg [5, 6] . the spill-over of these viruses from bats to humans, often through an intermediate host, can cause severe and fatal disease in humans. recent high profile examples include the global sars epidemic in 2003, which caused the deaths of over 800 people. investigations led by two independent groups both demonstrated that the natural reservoir for the sars-like cov were bats of the genus rhinolophus [1, 7] . more recent examples of spill over events from bats to humans include the 2014 ebola virus epidemic in west africa that is believed to be of bat origin [8, 9] . while many bat borne pathogens cause severe and often fatal diseases in humans, bats demonstrate no clinical signs of disease when infected with these agents. indeed, experimental infections of bats with highly pathogenic viruses such as hendra and nipah virus yielded no observable clinical signs. however, virus isolation, seroconversion, and the excretion of virus in saliva, urine and faeces were observed [10, 11] . subclinical infections of both fruit and insectivorous bats have also been reported following experimental infection with zaire ebola virus. high titres of ebola virus were successfully obtained from viscera and faecal samples following experimental infection [12] . a multitude of protective responses are invoked following the infection of a cell from both the innate and adaptive immune systems. one of the early innate responses is the induction of interferons (ifns) which exert their effects through the transcription of a large set of interferon stimulated genes (isgs) [13] . the products of these genes have many functions ranging from directly acting on the virus via interfering with virus uncoating to modulating key functions within the host cells such as inhibiting protein translation and apoptosis [14] . beside these known innate processes, there may be others that still await identification and elucidation. previous studies on bats have focused on genome sequencing, transcriptomics and the investigation of specific components of the innate and adaptive immune system, such as pattern recognition receptors, antibody diversity and ifns [15] [16] [17] [18] . important resources generated from these studies include the genome sequences of nine bats species [15, [19] [20] [21] and immortalised cell lines for in vitro studies [22] . the investigation of bat immunoglobulins identified igg and igm in bat serum but iga was only detected in trace quantities and the higher quantities of igg in mucosal secretions is thought to compensate for the lower abundance of iga [23] . all these studies have shown that bats possess genes present in other mammalian species, including components of the innate and adaptive immune system [16] . functional studies of bat ifns show an induction of ifn genes and the subsequent antiviral activity following virus infection [24] . in terms of proteomics research, little has been studied in this area. we have previously identified that hendra virus infection of p. alecto kidney cells sensitises these cells to trail-mediated apoptosis [25] . despite these efforts the exact mechanisms by which bats manage virus infection is yet to be identified. there are a number of different proteomic methodologies that are used for quantitative analyses or proteome expression. fundamentally, these can be grouped as either gel-based or gel-free methods. in gel-based techniques protein separation is achieved by electrophoresis (1-d or 2-d) and separated proteins are stained or labelled and the intensities of protein bands (1-d) or spots (2-d) are quantified prior to protein identification by mass spectrometry (ms). in gel-free techniques the quantitative data and protein identities are obtained from the mass spectra of differentially labelled proteins. both of these approaches have been used to study the host proteome in response to virus infection [26] [27] [28] . here, we undertake a comparative proteomic study utilising two different quantitative proteomic techniques in parallel, namely difference gel electrophoresis (dige) [29] and isobaric tagging for relative and absolute quantitation (itraq) [30] to analyse the proteome of immortalised p. alecto kidney cells (pakit03) [22] challenged with polyinosinic:polycytidylic acid (poly i:c). poly i:c is an analog to dsrna and has been used to stimulate cells and induce a potent antiviral cellular responses [31] . it is recognised by a number of sensory molecules of the cell, termed pathogen recognition receptors [32] resulting in the production of numerous cytokines that in turn induce a number of immune pathways [31] . although the main purpose of this study was to generate a proteomic dataset as a useful resource for future research in bat immunology, we were also interested in assessing the advantages and limitations of two commonly used proteomic methodologies. the viability of pakit03 cells was assessed at 3, 6, 22 and 46 hr following transfection with increasing concentrations of poly i:c (0.5, 1 or 10 μg/ml) delivered with lipofectamine. an initial decrease in viability was observed in all cells following transfection (fig. 1a) . from 6 to 22 hr post transfection (hpt) the viability of cells treated with 0.5 μg/ml, 1 μg/ml of poly i:c or with lipofectamine alone remained stable while the viability of cells treated with 10 μg/ml of poly i:c dropped to below 70 %. at 46 hpt the viability for all cells was found to be at 75-80 % except for those treated with 10 μg/ml of poly i:c, which were still below 70 % viability. based on these findings it was decided that transfection with 1 μg/ ml of poly i:c was the least detrimental to cell viability and thus most appropriate for further studies. to ensure that cells were responsive to poly i:c, the induction of interferon stimulated gene 54 (isg54) was measured using quantitative real-time pcr. up-regulation of isg54 is a known response to virus infection and has been linked to apoptosis [33] . the relative expression of isg54 mrna in pakit03 cells following poly i:c transfection was significantly up-regulated in cells transfected with 1 μg/ml of poly i:c at 4, 8 and 20 hpt compared to the non-transfected control (fig. 1b) . itraq for itraq analysis, mass spectra from samples obtained at the three time points were searched against the translated p. alecto genome using mascot and proteinpilot. three datasets were generated from the three biological replicates. in total, 426 proteins were identified across the three time points with ≥ 2 peptide matches. of the 426 proteins identified 104 were differentially expressed, based on mean fold-change of ≥ 1.5. the number of differentially expressed proteins varied between time points (table 1) . at 4 hpt the majority of differentially expressed proteins were up-regulated ( fig. 2a) . at 8 hpt, only a small number of proteins were differentially expressed, with an almost equal number of proteins up and down-regulated (fig. 2b) . in contrast, at 20 hpt most differentially expressed proteins were down-regulated (fig. 2c ). illustrated by the heatmap (fig. 2d) , proteins that were up-regulated by poly i:c at 4 hpt, did not remain upregulated at 8 and 20 hpt. however, many of the proteins down-regulated by poly i:c transfection at 20 hpt were also down-regulated at 8 hpt. a full list of protein expression profiles obtained from itraq analysis is provided as additional file 1. triplicate cell lysates from pakit03 cells transfected with poly i:c for 4, 8 and 20 hr were also analysed by dige (fig. 3a) . a total of 2,385 protein spots were present in all gels. of the 2,385 spots, 215 (9 %) were differentially expressed (p ≤ 0.05). the majority of differential expression was observed at 20 hpt with 125 spots up-regulated and 90 spots down-regulated ( table 1) . spots of interest were identified using liquid chromatography-mass spectrometry (lc-ms). a total of 42 spots that were differentially expressed were manually excised from gels and processed for lc-ms. of these protein spots, 25 returned a single protein identity, while 17 yielded two or more identities. the 17 spots with ambiguous protein identities were removed from further analysis. the regulation of the 25 unambiguously identified proteins was assessed across the three time points (fig. 3b ) with the majority of these proteins upregulated. in general, proteins followed the same regulation at each time point. proteins that were up-regulated at 4 hpt were also up-regulated at 8 and 20 hpt. this pattern of regulation was also observed for proteins that were down-regulated. a comparison of the itraq and dige datasets revealed significant overlap. of the 25 proteins unambiguously identified by dige, 18 (72 %) were also detected within the itraq analysis. gene ontology (go) enrichment analysis was performed on lists of up and down-regulated proteins. here, we focused on those proteins found to be differentially expressed by itraq, as too few proteins were identified by dige to perform go enrichment analysis. significant enrichment for proteins involved in the glycolytic metabolism pathway was observed in the up-regulated protein list (table 2) . indeed, go terms such as pyruvate metabolic process (go:0006090) and glycolytic process (go:0006096) were significantly enriched within the up-regulated protein list. individual proteins that contributed to this pathway included ldhb, pgk1, gapdh, tpi1, ldha, pgam1, pkm and eno1. in contrast, a strong enrichment for ribosomal processes, including translational termination (go:0006415) was observed in the down-regulated protein list ( table 2 ). contributing to this enrichment was the down-regulation of over 20 different ribosomal proteins at 20 hpt. we further assessed the response of three glycolytic enzymes, α-enolase (eno1), phosphoglycerate mutase 1 (pgam1) and triosephosphate isomerase 1 (tpi1) to poly i:c across two human (hek293t and hela) and two bat cell lines (pakit03, palut02) using immunodetection. polyclonal antibodies specific to eno1, pgam1 and tpi1, successfully detected their protein of interest in both bat (fig. 4a, b) and human cell lines (fig. 4c, d) . using analysis of itraq proteins up-regulated at 4 hpt demonstrated an enrichment of proteins related to the glycolytic metabolic pathway. proteins such as eno1, pgk1, gapdh, tpi1, ldha, pgam1, pkm and ldhb, show an increased expression in the pakit03 at 4 hpt, which suggests an increase in energy requirements. understandably, the initiation of an immune response is energetically taxing and requires the cell to synthesise a wide range of molecules such as isg products and cytokines that serve to defend against the invading pathogen. the induction of eno1, pgk1, gapdh, tpi1, pgam1 and pkm at 4 hpt suggests a rapid shift towards glycolysis early in the anti-viral response in pakit03 cells. glycolysis, the conversion of glucose into pyruvate, consists of multiple steps that require key glycolytic enzymes. tpi1, gapdh, pgk1, pgam1, eno1 and pkm, are the key enzymes that are essential for steps 5, 6, 7, 8, 9 and 10 in the glycolysis pathway [39] . other glycolytic enzymes that were identified, but not differentially expressed, in our itraq data included hk1, pfkp and aldoa. these enzymes control steps 1, 3 and 4 of the glycolysis pathway [39] . immunodetection confirmed the up-regulation of eno1 and tpi1 at 4 hpt within the pakit03 cells, albeit it to a lesser magnitude than quantified by itraq analysis. in contrast, immunodetection demonstrated a down-regulation of pgam1 in pakit03 cells at 4 hpt. in comparison, both human cell lines and the bat lung cell line (palut02) showed up-regulation of pgam1 at 4 hpt. hela cells also showed up-regulation of tpi1 at 4 and 20 hpt. these findings suggest that the induction of glycolytic pathways are not specific to pakit03 cells, however the kinetics of key glycolytic enzymes varies between cell lines. previous study in mice have shown that poly i:c induced type i ifn, rapidly promoting a metabolic shift from oxidative phosphorylation to glycolysis and is required for the maturation of dendritic cells [40] viruses have also been shown to induce metabolic pathways. indeed, hepatitis c virus protein expression has been shown to preferentially promote nonoxidative glucose metabolism over oxidative phosphorylation pathways in human cell lines [41] . similarly herpes simplex virus type 1 was shown to induce key glycolytic enzymes and induced glycolysis in vero cells [42] . in addition to the metabolic proteins that were upregulated, we also identified a large number of ribosomal subunit proteins which were found to be downregulated at 20 hpt. the decrease in levels of these ribosomal proteins by the host cell is a possible defence to virus infection. as not all of the components required for virus replication is encoded by the viral genome, viruses are known to hijack and utilise the host ribosome for the translation of viral proteins. this mechanism has been shown to be utilised by hiv, by binding the 40s ribosomal protein [43, 44] . limiting access to this machinery may be used as a means to slow down replication of virus in infected cells. poly i:c is known to induce isgs, however we were unable to detect any protein expression of isgs within our study. it is likely that the absence of these isgs in our datasets is related to the relative abundance of these proteins in the cell. the dynamic range for cellular proteins spans at least seven orders of magnitude [45] and itraq suffers from low detection sensitivity with high abundance proteins masking low abundance proteins. the enrichment of these proteins by fractionation of the sample may result in the improved detection and the identification of these immune effector proteins. due to technical limitations involved in spot picking we found dige to be unsuitable for whole cell lysate proteomics. in many cases, dige protein spots are simply too close to one another to successfully isolate a single spot. in comparison, itraq does not suffer this technical limitation. we hypothesise that bats may possess a heightened immune response when infected with virus. this allows them to quickly respond to infection and as a result is able to halt or hinder virus replication. the upregulation of proteins involved in glycolysis indicates that there is a rapid shift towards energy production as early as 4 hpt. the down-regulation of ribosomal proteins may serve to limit the machinery available for virus replication. this is the first global proteomic analysis of the p. alecto cell proteome response following transfection with the viral mimic, poly i:c. utilising itraq, we identified proteins related energy metabolism to be up-regulated while proteins associated with the ribosome to be downregulated. the validation of some of these protein's expression by immunodetection demonstrated that there was an induction of proteins related to energy production. subsequent work will involve examining the proteomic response of bat cells using live viruses. immortalised pteropus alecto kidney (pakit03), lung (palut02) cells [22] were maintained in d8437 dulbecco's modified eagle's medium nutrient mixture f-12 ham (dmem) with 15 mm hepes, nahco 3 , pyridoxine and l-glutamine (sigma-aldrich). human embryonic kidney (hek293t) and human cervical cancer (hela) cells were maintained in minimal essential medium (mem) with 10 mm hepes and 2 mm l-glutamine (life technologies). both media used were supplemented with 10 % fetal bovine serum (fbs) (in vitro technologies) at 37°c. cell viability was assessed following transfection with varying concentrations of poly i:c, t24 cm 2 flasks (corning) were seeded with 3.5 × 10 6 pakit03 cells and left at 37°c to incubate overnight. the media were removed the following day and cell monolayers were washed with sterile phosphate buffered saline (pbs). poly i:c (invivogen) solutions at 10 μg/ml, 1 μg/ml, 0.5 μg/ml and mock (control) were prepared with serum free medium according to manufacturer's protocol. poly i:c solutions were added to an equal volume of lipofectamine 2000 (invitrogen) and added to the cells and left to transfect for 6 h. following transfection, supernatant was removed and cells were washed with pbs and fresh growth media added (dmem supplemented with 2 % fbs). at 3, 6, 22 and 46 hpt cells were trypsinised and the cell viability was assessed with trypan blue staining using a hemocytometer. pakit03 cells were seeded into 18 × t75 cm 2 flasks and were incubated at 37°c overnight. for immunodetection all cell types were seeded into individual 6 well plates. cells were stimulated by transfection with 1 μg/ml of poly i:c using 1 μl/ml lipofectamine 2000 and incubated for 6 h at 37°c. the cells were washed with pbs and placed in fresh medium. at the sampling time points 4, 8, 20 hpt the medium was removed and after a wash with sterile pbs the cells were removed with a cell scraper. cells from one flask was resuspended in 5 ml of sterile pbs, 1 ml was removed for rna extraction and the remaining 4 ml centrifuged and pellet resuspended in 5 ml of dige lysis buffer (7 m urea, 2 m thiourea, 4 % chaps, 30 mm tris, ph 8.5). at the sampling time points 4, 8, 20 hpt the medium was removed to a 10 ml tube and centrifuged for 5 min at 1,500 × g. the supernatant was removed and the cells were lysed in 100 μl of 5 % sds. cells in the 6 well plates were lysed with 400 μl of 5 % sds. this was then combined with the centrifuged cells in 100 μl into a fresh microfuge tube. this was boiled for 7 min at 100°c to fragment nucleic acid. real-time pcr of isg54 following poly i:c stimulation the 1 ml aliquot of cells was centrifuged and resuspended in 350 μl of buffer rlt (qiagen). samples were homogenised with a qiashredder (qiagen) and purified rna was extracted using the qiagen rneasy mini kit (qiagen) according to manufacturer's instructions with on column qiagen dnasei digestion (qiagen). extracted purified rna was resuspended in nuclease-free water (promega) and concentration was determined using a nanodrop spectrophotometer (thermo scientific). the purified rna was converted into first-strand cdna using superscript ii reverse transcriptase (invitrogen) according to manufacturer's instructions. the cdna was then used for real-time pcr using the sybr green real-time pcr master mix (life technologies). primer design was completed using primer3 with the following conditions: primer size -20 bp, primer melting point -60°c, primer g-c content -55 % and product size -100 bp minimum, 150 bp optimum, 200 bp maximum. the cycle conditions used were holding stage step 1-95°c for 5 min, cycling stage step 1-95°c for 10 s, step 2-60°c for 20 s, step 3-72°c for 20 s for a total of 40 cycles and a default melt curve stage. proteins were extracted using the 2-d clean-up kit (ge healthcare) according to manufacturer's protocol and the purified protein pellet was solubilised in dige lysis buffer. samples were quantified using the ezq protein quantitation kit (life technologies) with the fluorescence read at 473 nm on a typhoon fla 9000 (ge healthcare). the samples were then divided equally to undergo sample preparation for each proteomic technique. labelling of proteins with cydye fluorophores 50 μg of each protein sample was labelled using the minimal cydye fluors labelling kit (ge healthcare). cydyes were solubilised with dimethylformamide (dmf) to a concentration of 400 pmol/μl and 1 μl of cydye was added to 50 μg of each protein sample. a dye swap was utilised with cy3 and cy5 between groups. an internal standard was prepared by adding equal amounts of protein and labelling with cy2. this resulted in nine individual samples. labelled samples in dige lysis buffer were reduced with 10 mg/ml dithiothreitol (dtt) then applied via cup loading to 24 cm ph 3-10nl ipg strips. iso-electric focusing (ief) was performed on an ipgphor3 instrument (ge healthcare) with the following run conditions: 1) step and hold -150 v for 3 h, 2) step and hold -300 v for 3 h, 3) gradient -1000 v 6 h, 4) gradient -10,000 v 1 h, 5) step and hold 10,000 v 5 h. after focusing for a total of 50,000 vh, strips were incubated in equilibration buffer (6 m urea, 2 % sds, 50 mm tris-hcl ph 8.8, 0.02 % bromophenol blue, 30 % glycerol) with 1 % dtt for 15 min, and then another 15 min in equilibration buffer containing 2.5 % iodoacetamide. strips were then sealed with 0.5 % agarose on top of 12.5 % sds-page gels lab cast in low fluorescence glass plates, and run using the ettan dalt ii system (ge healthcare) under the following conditions: step 1) 1 w/gel for 1 h, step 2) 17 w/gel for 5 h at 25°c. following 2de separation gels were scanned on a typhoon fla 9000 (ge healthcare) using the cy2 (488 nm), cy3 (532 nm) and cy5 (633 nm) excitation wavelengths. scanned gels (triplicates for each time point) were analysed using the decyder software package (ge healthcare v7.0). a spot exclusion filter was applied to spots with a spot volume of <30,000 to remove artefacts from the analysis (fig. 2b) . the remaining spots were then matched to the gel with the highest number of spots detected, designated the master gel. only those spots that were successfully matched to the master gel were used for further analysis. gels were matched using the internal standard with the biological variation analysis (bva) mode. student's t-test was used for statistical analysis. protein spots were assessed as being differentially expressed if they had a p-value of < 0.05. protein spots were manually excised from gels stained with colloidal coomassie blue. spots were subjected to in gel trypsin digestion as described [46] for lc-ms/ms. the resulting ms/ms spectra were searched against the p. alecto genome [15] using mascot version 2.06 [47] with the following parameters: enzyme: trypsin; fixed modifications: carbamidomethyl (c); variable modifications: oxidation (m); ms peptide tolerance: 10 ppm; ms/ms tolerance: 0.1 da; number of missed cleavages: up to 1. itraq extracted proteins from a cell lysate (100 μg) were reduced with tris-(2-carboxyethyl) phosphine (5 mm tcep) for 1 h at 37°c shaking at 1,150 rpm and alkylated with methyl methanthiosulfonate (mmts 10 mm). samples were diluted with triethyl ammonium bicarbonate buffer (teab 100 mm) to reduce the urea concentration to less than 1 m and proteins were digested with 2 μg of trypsin (promega). the final volume of this solution was 133.5 μl and was incubated at 37°c overnight with shaking at 850 rpm. the trypsin digestion was stopped by addition of 10 μl of formic acid (sigma). the samples were then desalted prior to labelling with a sep-pak c18 plus short cartridge (waters, usa). the eluted samples were evaporated in a speed vac at ambient temperature to 10 μl and teab was added to each tube to give a final volume of 30 μl. the ph of each sample was checked with ph 7-10 strips to ensure ph was at least ph 7.5 before addition of the itraq tags. the itraq tags were resuspended in isopropanol and the labelling was performed as recommended by the manufacturer. the itraq labels used were 114, 115, 116, 117, 118 and 119, and the labels were cycled between replicates. following labelling, samples were combined into a new tube and evaporated to approximately 200 μl to remove isopropanol. samples were then desalted on a sep-pak c18 plus short cartridge (waters) and vacuum concentrated to the final volume of 100 μl. samples were then fractionated by strong cation exchange chromatography and fractions analysed by lc/ms/ms as described in [48] . mass spectra obtained were searched against the p. alecto genome using proteinpilot version 4.5with the following parameters: sample type: itraq 8plex (peptide labelled); cys alkylation: mmts; digestion: trypsin; search effort: thorough id. peptide summary report was exported from proteinpilot and filtered by confidence level according to the local 5 % false discovery rate reported by proteinpilot. peak lists generated by proteinpilot were exported and used to search against the same databases using mascot version 2.06. the mascot parameters were: enzyme: trypsin; fixed modifications: itraq4plex (n-term), itraq4plex (k), methylthiol (c); ms peptide tolerance: 10 ppm, ms/ms tolerance: 0.1 da, number of missed cleavages: up to 1. peptide list from mascot was generated with a false discovery rate <1 %, determined using a concatenated reverse sequence decoy database. proteins found using both search algorithms with a minimum of 2 peptides and identified in all three replicates were selected for further analysis as described in [48] . proteins were assessed as being differentially expressed if the foldchange between poly i:c/control was ≥ 1.5. protein samples were analysed by immunodetection in duplicates with commercially available antibodies. antibodies specific to β-tublin (#2128), eno1 (#3810) were purchased from cell signalling while antibodies specific to pgam1 (nbvp1-49532) and tpi1 (nbp1-31470) where from novus biologicals. protein samples in dige lysis buffer were quantified using the ezq protein quantitation kit (life technologies) and separated under reducing conditions on precast 4-12 % bis-tris gels in mops buffer (life technologies). samples were electro-transferred onto polyvinylidene fluoride (pvdf) membrane in 3-(cyclohexylamino)-1-propanesulfonic acid buffer (ph 11) with 10 % v/v methanol. the membrane was blocked over night at 4°c with 5 % skim milk in tris buffered saline with 5 % tween-20 (tbst). the membrane was probed with appropriate species igg conjugated with hrp. visualisation of reactive protein bands was achieved using enhanced chemiluminescence (eclplus, thermo scientific) and fluorescent detection at 473 nm on a typhoon fla 9000. images of blots were imported to imagej (version 1.49v) and densitometry analysis was undertaken. bats are natural reservoirs of sars-like coronaviruses middle east respiratory syndrome coronavirus in bats. saudi arabia emerg infect dis isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus characterization of nipah virus from naturally infected pteropus vampyrus bats fruit bats as reservoirs of ebola virus isolation of 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myd88 toll-like receptor 3 agonist poly(i:c)-induced antiviral response in human corneal epithelial cells freeman: international version direct type i ifn but not mda5/tlr3 activation of dendritic cells is required for maturation and metabolic shift to glycolysis after poly ic stimulation hepatitis c virus-linked mitochondrial dysfunction promotes hypoxia-inducible factor 1 alpha-mediated glycolytic adaptation herpes simplex type 1 activates glycolysis through engagement of the enzyme 6-phosphofructo-1-kinase (pfk-1) viral subversion of the host protein synthesis machinery a conserved structure within the hiv gag open reading frame that controls translation initiation directly recruits the 40s subunit and eif3 the challenge of the proteome dynamic range and its implications for in-depth proteomics mass spectrometric identification and characterisation of the nucleocapsid protein of menangle virus probability-based protein identification by searching sequence databases using mass spectrometry data quantitative proteomic analysis of wheat cultivars with differing drought stress tolerance kf and ab acknowledge the support of a grant from the australian research council to the arc centre of excellence in plant cell walls (ce10001007). the authors would like to acknowledge dr. grant peck and dr. chris cowled for critical review of the manuscript. the authors declare that they have no competing interests.authors' contributions lm performed the experiments and analysed the data. jww assisted in data analysis. kf and bs performed the mass spectrometry and analysed ms data. wpm, jww and ab conceived the study and designed the experiments. all authors were involved in the writing of the manuscript. all authors read and approved the final manuscript. key: cord-315531-2gc2dc46 authors: mcgarvey, peter b.; huang, hongzhan; mazumder, raja; zhang, jian; chen, yongxing; zhang, chengdong; cammer, stephen; will, rebecca; odle, margie; sobral, bruno; moore, margaret; wu, cathy h. title: systems integration of biodefense omics data for analysis of pathogen-host interactions and identification of potential targets date: 2009-09-25 journal: plos one doi: 10.1371/journal.pone.0007162 sha: doc_id: 315531 cord_uid: 2gc2dc46 the niaid (national institute for allergy and infectious diseases) biodefense proteomics program aims to identify targets for potential vaccines, therapeutics, and diagnostics for agents of concern in bioterrorism, including bacterial, parasitic, and viral pathogens. the program includes seven proteomics research centers, generating diverse types of pathogen-host data, including mass spectrometry, microarray transcriptional profiles, protein interactions, protein structures and biological reagents. the biodefense resource center (www.proteomicsresource.org) has developed a bioinformatics framework, employing a protein-centric approach to integrate and support mining and analysis of the large and heterogeneous data. underlying this approach is a data warehouse with comprehensive protein + gene identifier and name mappings and annotations extracted from over 100 molecular databases. value-added annotations are provided for key proteins from experimental findings using controlled vocabulary. the availability of pathogen and host omics data in an integrated framework allows global analysis of the data and comparisons across different experiments and organisms, as illustrated in several case studies presented here. (1) the identification of a hypothetical protein with differential gene and protein expressions in two host systems (mouse macrophage and human hela cells) infected by different bacterial (bacillus anthracis and salmonella typhimurium) and viral (orthopox) pathogens suggesting that this protein can be prioritized for additional analysis and functional characterization. (2) the analysis of a vaccinia-human protein interaction network supplemented with protein accumulation levels led to the identification of human keratin, type ii cytoskeletal 4 protein as a potential therapeutic target. (3) comparison of complete genomes from pathogenic variants coupled with experimental information on complete proteomes allowed the identification and prioritization of ten potential diagnostic targets from bacillus anthracis. the integrative analysis across data sets from multiple centers can reveal potential functional significance and hidden relationships between pathogen and host proteins, thereby providing a systems approach to basic understanding of pathogenicity and target identification. the niaid (national institute of allergy and infectious diseases) biodefense proteomics program, established in 2004, aims to characterize the pathogen and host cell proteome by identifying proteins associated with the biology of microbes, mechanisms of microbial pathogenesis and host responses to infection, thereby facilitating the discovery of target genes or proteins as potential candidates for the next generation of vaccines, therapeutics, and diagnostics [1] . the program includes seven proteomics research centers (prcs) conducting state-of-the-art high-throughput research on pathogens of concern in biodefense and emerging/ reemerging infectious diseases, as well as a biodefense resource center for public dissemination of the pathogen and host data, biological reagents, protocols, and other project deliverables. the prcs work on many different organisms, covering bacterial pathogens (bacillus anthracis, brucella abortus, francisella tularensis, salmonella typhi, s. typhimurium, vibrio cholerae, yersinia pestis), eukaryotic parasites (cryptosporidium parvum, toxoplasma gondii), and viral pathogens (monkeypox, sars-cov, vaccinia). the centers have generated a heterogeneous set of experimental data using various technologies loosely defined as proteomic, but encompassing genomic, structural, immunology and protein interaction technologies, as well as more standard cell and molecular biology techniques used to validate potential targets identified via high-throughput methods. in addition to data, the prcs have provided biological reagents such as clones, antibodies and engineered bacterial strains, other deliverables include standard operating procedures (sops) and new technologies such as instrumental methods and software tools and finally publications related to all of these activities. consequently, there were a number of unique challenges facing the resource center: (i) how to coordinate with the seven prcs with various pathogens, technologies, processes, and data types; (ii) how to provide seamless integration of three institutions that make up the resource center; and (iii) how to provide timely and effective dissemination of newly discovered information to the user community. in particular, due to the breadth of the program, the potential user community is quite broad, from technology or informatics experts who may want to reanalyze the data or develop better algorithms, to a wide group of biomedical scientists who are interested in mining the data for their own studies or just finding new information on a protein or gene of interest quickly and easily. accordingly, we developed a set of functional requirements early in the biodefense resource center development: (i) to implement a center-specific submission protocol and data release plan for timely dissemination, (ii) to promote data interoperability, adopting common standards (such as hupo proteomic standards initiative [2, 3, 4] ), defining a core set of metadata with mapping to controlled vocabularies and ontologies, recommending preferred ids for gene/ protein mapping, and (iii) to provide value-added annotation to capture key findings and integration of the data with related resources for functional interpretation of the data. available online at http://proteomicsresource.org, the architecture, initial content and general features of the biodefense proteomics resource were briefly described elsewhere [5] . a breakdown of the resources content by organism, prc and other criteria can be seen at: http://www. proteomicsresource.org/resources/catalog.aspx. tutorials and help are provided on the website: http://www.proteomicsresource. org/resources/tutorials.aspx , http://proteininformationresource. org/pirwww/support/help.shtml#30 the objective of this study is to provide a systems approach to the study of pathogen-host interactions, connecting the various types of experimental data on genomics, proteomics and host-pathogen interactions with information on pathways, regulatory networks, literature, functional annotation and experimental methods. having most of this information accessible in one place can facilitate knowledge discovery and modeling of biological systems. like many problems in data integration, it is easy to know the general outline of what we want, but often much harder to implement and navigate the information, especially if the original data crosses disciplinary, laboratory and institutional boundaries. here we describe in detail a protein-centric approach for systems integration of such a large and heterogeneous set of data from the niaid biodefense proteomics program, and present scientific case studies to illustrate its application to facilitate the basic understanding of pathogen-host interactions and for the identification of potential candidates for therapeutic or diagnostic targets. several scientific use cases are presented that illustrate how one can search varied experimental data from different laboratories and even ones researching different infectious organism and their hosts to make potentially useful connections that could lead to new hypotheses and discoveries. based on the functional requirements of the resource center, we developed a bioinformatics infrastructure for integration of prc deliverables ( figure 1 ). in our workflow, multiple data types from prcs are submitted to the center using a data submission protocol and standard exchange format, with the metadata using controlled vocabulary whenever possible. for functional interpretation of the data, we then map the gene and protein data based on identifier (id) mapping or if necessary using peptide or sequence mapping to proteins in our data warehouse described below. all of the databases, along with information on the prcs and organisms under study are listed in the proteomics catalog accessible from the web portal. the key design principal in the resource center is protein-centric data integration. here the diverse experimental data are integrated and presented in a protein-centric manner where information is queried and presented via common proteins and connected to experimental data and the network of protein attributes, including information on the encoding genes, protein families, pathways, functions and more. in practice a protein-centric approach works well as proteins occupy a middle ground molecularly between gene and transcript information and higher levels of molecular and cellular structure and organization. proteins are often the functional molecules in biological processes described by pathways, molecular interactions and other networks. protein families, in turn, have proven to be invaluable in studying evolution and for inferring and transferring functional annotation across species. underlying the protein-centric data integration is a data warehouse called the master protein directory (mpd) where key information is extracted from the primary data and combined for rapid search, display and analysis capabilities. the mpd is built on the data and capabilities of iproclass [6] a warehouse of protein information, which in turn is built around uniprotkb [7] but supplemented with additional sequences from gene models in refseq [6] and ensembl [7] and additional annotation and literature from other curated data resources such as model organism databases [8, 9, 10, 11, 12, 13] and generif [14] . the biodefense data are essentially additional data fields added to a subset of iproclass entries to create the mpd. currently the mpd defines and supports information from the following types of data produced by the prcs: mass spectrometry, microarray, clones, protein interaction, and protein structure. more data types or attributes may be added in the future if needed. supplemental table s1 shows the common and unique fields used in the mpd for each data type. an advantage of the data warehouse design is that, if needed, additional fields can be extracted from the primary data and easily added as new attributes without greatly altering the existing database design or query mechanisms. the mpd data is stored in an oracle database along with iproclass data. the mpd including the website and ftp files is updated every 3 weeks in conjunction with iproclass or whenever new prc data is released. the various proteomics research centers all used different sources and identifiers for the nucleotide and protein sequences in their analysis pipelines and occasionally would change sources depending on the experiment. this is a common problem encountered when attempting to combine data across research laboratories unless identical sequence databases, processes, platforms and organism names are used. examples of database identifiers used include genbank/embl/ddbj accessions and locus tags, unigene accessions, refseq accessions, ipi accessions, ncbi gi numbers and ids unique to a sequencing center or organism-specific database. the first step was to map all experimental results to a common representation of a protein. this was achieved by mapping all protein and gene ids and names to iproclass proteins. the majority of the mapping using ids from public resources was done using mapping services and tables provide on the protein information resource (pir) web site (http:// proteininformationresource.org/pirwww/search/idmapping.shtml) and ftp site (ftp://ftp.pir.georgetown.edu/databases/iproclass/). however, some mapping problems needed to be addressed either by automated rules, direct sequence comparisons or manual analysis and annotation. mapping difficulties fell into 4 categories: 1) one-to-many mappings: a common problem, especially when eukaryotic host proteins derived from alternate splicing or viral polyproteins are involved. uniprotkb usually merges information on alternate splice forms or polyproteins, which helped minimize this problem for our purposes, but in cases where multiple mappings exist, we selected as most informative the entry in the manually reviewed uniprotkb/ swissprot section; if no swissprot entry was found, the longer sequence in uniprotkb/trembl section was selected. users could always find the alternate mappings via the iproclass related sequences link on the mpd entry page to precompiled blast results on all iproclass sequences. 2) retired sequences: genomic sequences from databases such as refseq, ipi, or unigene are not static and, as information changes, some gene predictions and translations are retired with each new build. retired sequences often required manual mapping by a curator to match original gene or peptide results to current protein sequences. primarily uniparc (uniprot sequence archive) [15] was used for this purpose. 3) protein sequences not available in iproclass or any public repository: this occurred most often with toxoplasma gondii whose genome sequencing was still in progress and stable builds were not yet available. however, the problem also occurred in some well-characterized organisms like vibrio cholera and bacillus anthracis. several data sets contained information on annotated but not translated pseudogenes. in the case of vibrio cholera, 21 of 48 pseudogenes cloned and sequenced by the harvard institute of proteomics did not contain the annotated point mutation or frame shift and appeared to produce full-length proteins [16] . in the case of bacillus anthracis, microarrays containing probes for 82 of 192 annotated pseudogenes also showed significant changes in rna expression in response to infection or other treatments [17, 18, 19] . in these cases, new database entries were created in the mpd to house the results. 4) alternate species or strain representations: several experimental data sets reported sequence identifiers for strains or variants other than the one used in the experimental sample. this is not an uncommon situation as the genetically most characterized variant is often an attenuated laboratory strain while the more virulent strains are either not yet sequenced or the sequence is of lower quality. often microarray chips or mass spectrometry databases are designed using the best available sequence from the research strain, yet then use rna and protein samples from another similar virulent strain. the question here was what organism strain to map to and represent on our website: the strain the rna or protein the sample came from, or alternatively, the strain that matched the identifier and sequence used in the research to detect the rna or protein. for the mpd we chose to map to the sequence identifier reported in the data files and related publications, with the additional virulent strain information noted in the results summary. data mining design goals. in consultation with niaid and prcs within the project's interoperability working group, we developed 3 goals for data mining. 1) all project data and other deliverables should be available via browsing and simple keyword searches. 2) the data and information provided by the resource should be sufficient to allow a skilled researcher to download and reanalyze or mine the data for additional information. 3) our target user was a biomedical scientist not expert in the technology used to produce the data or in bioinformatics, thus the data, procedures, publications and general results and conclusions of an analysis should be relatively easy to find on the project website for someone not familiar with the details of the particular technologies used to generate it. to allow both simple keyword searches and also boolean searches of the project data, we did the following: 1) we included in the mpd only ''validated'' results that were determined by the research centers to be significant using their methods. to do otherwise would confuse users not familiar with the technology and how results are filtered. results that fell below the significance threshold used by the research center were made available via download of data sets at the ftp site. 2) ''raw'' unprocessed machine specific data would be stored by the prc and available on request. 3) to facilitate and simplify searches across laboratories and data types, we omitted most data type or analysis specific numerical values and statistics from the general mpd search and display. these numerical values are usually platform, laboratory and method dependent and cannot easily be used to compare across datasets so including them might be confusing to users. instead, we focused on providing simple, yet powerful, queries of experimental summaries where a user can query if a gene/protein was presented in the results and, in some cases, if it showed a reported increase or decrease in expression or accumulation based on the prc's criteria. once a set of proteins of interest is identified, a user can then drill down to view the specific experimental values and methods employed to generate the particular dataset. links to details in the publications are provided and full data are available via ftp. since all protein attributes are included as search options, one can query beyond simply protein names, accessions or project data and search pathways, protein families, gene ontology (go) terms, database cross-references and many other attributes, providing many powerful options to the users. to provide a robust text search for the website, we used the pir text indexing system [20] in which over 100 text fields and unique identifiers from the mpd database are indexed using callable personal librarian (cpl) [21] which supports fast exact text search, substring & wildcard text search, range search and boolean searches. entry indexing and retrieval is supported by oracle. i: unstructured keyword search. a simple keyword search was implemented on every page of the resource's website. this searches all fields in the mpd. to further facilitate searches, the protein name field is supplemented by also searching the biothesaurus [10] containing all gene and protein name synonyms and textual variants for each protein from over 35 data sources. in addition, the default option searches all text from the pubmed abstract for all project publications, an abstract of each technology and all text in sops. the text indexed for publications, technologies and sops was annotated with additional standard keywords to facilitate searches. figure 2 shows the results of a simple keyword search with hits for proteins, reagents, publications, technologies and sops. ii: structured text search. the mpd database contains over 100 fields derived from iproclass and proteomics research center's data. currently 75 of these fields are available for individual searches and can be combined with boolean operators as seen in some of the use case examples in this paper. the protein-centric search results are presented in a customizable tabular format where users can add or delete columns. currently 62 fields can be customized. the tabular display has two modes: 1) a default mode which displays fields common to all the supported data types and 2) a data type specific mode which restricts the results to a particular data type and displays fields specific to that data type. see supplemental table s1 for a list of data type specific fields. additional filters for proteomic research center and organism are available as pull down menus to aid browsing and viewing the results of queries. examples of these functions are illustrated in the scientific use cases below and in help pages and tutorials available on the website. http:// www.proteomicsresource.org/resources/tutorials.aspx, http:// proteininformationresource.org/pirwww/support/help.shtml#30. the resource currently contains information on 35,112 proteins from 58 datasets, 35,819 reagents, 75 sops, 31 technologies and 88 manuscripts. table 1 shows statistics on proteins in the mpd. currently ,30% of the proteins are uncharacterized in that they are called either ''uncharacterized'' or ''hypothetical'' and have no other functional annotations or functional domains. of these uncharacterized proteins, ,85% have experimental data, such as mass spectrometry, microarray, or protein interactions, associated with them. the remaining 15% of uncharacterized proteins are available as full length clones for further research. though the program is focused on pathogen proteins, about 32% of the proteins are host proteins from mouse or human, as cell lines or tissue samples from both these organisms were used as infection models for multiple pathogens. simple keyword search. due to the popularity of internet searches, support of unstructured keyword queries, even for structured data, has become critical for any web site. to support this feature, the default protein-centric search returns results for all project deliverables, data, reagents, protocols, technologies and publications. an example is shown in figure 2 where a single keyword search ''bacillus anthracis'' finds 10,988 pathogen and host proteins with mass spec, microarray or protein interaction data and also 13,251 reagents in the master reagent directory (mostly orf and y2h clones and mutant bacterial strains of bacillus anthracis), 2 sops, 16 project publications and 2 technologies. the matched fields column allows users to refine their queries and construct simple boolean searches. example i: integrative analysis. structured fields allow boolean queries across organisms, data types and laboratories. figure 3 shows a query where we make use of the controlled vocabulary in the ''expression condition'' field and searched for common host proteins detected in studies of bacillus anthracis and salmonella typhimurium infection in mouse macrophage cell lines. currently 222 proteins meet these criteria, mostly from mass spectrometry studies by pnnl and the university of michigan; however, if we further restrict the search to include only those also detected in microarray experiments, we find 16 proteins with mass spec data from s. typhimurium infections at one research center, and mass spec and microarray studies done using b. anthracis at another research center. from the customizable results display shown in figure 3 we can view summary information on the proteins detected. full details on the protein and individual experimental results are available via links [5] . some benefits of the protein-centric mapping approach are visible in the default display. 1) minimizing redundancy, the column prc id shows the different identifiers from different databases used by the research centers. in the case of one protein q9wua3/k6pp_mouse 6-phosphofructokinase type c (ec 2.7.1.11), a total of 9 identifiers from 4 different databases (unigene, refseq, ipi, nr) were reported in the research results that all represent either the gene or protein sequence for this single mouse protein. 2) discovery of additional experimental information from other studies. fifteen of the sixteen proteins found in the query also have mass spec data from caprion proteomics, as indicated in the 'experiment' column by caprion_05, 06 and _10. caprion is studying brucella abortus and using a similar mouse macrophage model. currently, only the data for uninfected macrophages is available from caprion. additional data on brucella and mouse proteins from bacterial infected macrophages should be included in future releases. from the results display, one can follow links to view the iproclass protein report with executive summaries of the results from the prcs and information collected from over 100 public resources or drill down to view the specific peptides or expression values seen in these studies, read publications and methods about the experiments or download the data for additional analysis. with a comprehensive protein data warehouse, one can also broaden the search for relevant data and information from related organisms using protein cluster or family information. in figure 4 we illustrate this by selecting one protein, k1033_mouse, an uncharacterized protein seen in three datasets from infected mouse macrophages. using the uniref90 cluster id [22] to query for all proteins with at least 90% identity and no gaps, we find the human homolog k1033_human was detected in hela cells infected with vaccinia and monkypox virus. if we do a batch retrieval using uniref90 ids from all 16 original mouse proteins, we find 12 human homologs were also detected in studies of orthopox infection (not shown). the identification of a hypothetical protein with differential gene and protein expressions in two host systems (mouse macrophage and hela cells) infected by different bacterial (bacillus anthracis and salmonella typhimurium) and viral (orthopox) pathogens suggest that this protein should be prioritized for additional analysis and functional characterization. [23, 24, 25] . although different laboratories use different sample preparation, detection and analysis techniques making some direct comparisons difficult, having the data together in one place allows queries and comparisons between proteins and gene sets to be combined and additional analysis undertaken. in this example, data from different labs and data types are combined for further analysis. a query of the mpd on data type = ''interaction'' and organism name = ''virus'' finds 33 vaccinia virus proteins with interactions with human proteins determined by myriad genetics. browsing the results display shows that 25 of the proteins were also seen in mass spec work published by pnnl [26] . by combining the two experimental data sets experiment = ''myriad_05'' and ''pnnl_ms_09'', we find 83 virus and human proteins with both mass spec and interaction data from each laboratory. further investigation into the experimental details shows that a protein interaction network was determined by myriad genetics using a yeast-two hybrid assay to screen viral bait proteins against a library of human prey proteins cloned from different tissues. the mass spec data from pnnl was obtained from viral preparations isolated from infected human hela cells. the work from pnnl contained quantitative information in the form of spectral counts and accurate mass tag [27] intensities, downloadable from the project ftp site. we combined all the vaccinia plus human interaction data with peptide counts for each protein and visualized the results using cytoscape [28, 29] . the complete network of results is shown in figure 5a . various methods have been tried and compared for filtering large intra-species interaction networks to limit false positives and to select the biologically relevant interactions [30, 31, 32, 33, 34, 35] . relatively little has yet been done for inter-species pathogen-host networks. several common factors that have proved useful in other studies are 1) evaluating network hubs with many interactions and 2) using correlations between interacting pairs with similar gene expression patterns. for this small interactome, we looked for relatively abundant proteins associated with multiple interactions. we identified a single human protein interacting with three viral proteins, three being the largest number of viral interactions seen with a single host protein in this data set. the interactions are shown in figure 5b . the host protein keratin, type ii cytoskeletal 4 (p19013) interacts with three viral proteins (p11258 -14 kda fusion protein, a27l; q805h7 -chemokine-binding protein c23/b29; p17362 -protein c6l). the 14 kda fusion protein a27l is the most abundant protein seen in this data set and participates in virus penetration at during cell fusion [36, 37] . a27l facilitates initial attachment to cells by binding to glycosaminoglycans [38] . a27l is found in all orthopoxviruses and has no cellular or entomopoxvirus homologs. additional viral proteins involved in attachment (d8l, h3l) [39, 40] and fusion (f9l, i2l) [41, 42] were not observed. the chemokine-binding protein c23l belongs to a family of poxvirus chemokine-binding proteins that mimic the chemokine response and prevent activation and chemotaxis of leukocytes [43, 44] . protein c6l belongs to a family of poxvirus paralogs that may function as toll-like receptor inhibitors based on homology to a52r [45, 46, 47] . thus, this protein may modulate toll/il-1r signaling, resulting in a diminished host immune response and enhancing viral survival. p19013 -keratin, type ii cytoskeletal 4, the host protein, has tissue specificity in the suprabasal layer of the stratified epithelium of the esophagus, exocervix, vagina, mouth and lingual mucosa, and in cells and cell clusters in the mucosa and serous gland ducts of the esophageal submucosa [48] . transgenic knockout mice have shown keratin, type ii cytoskeletal 4 to play an important role in maintaining normal epithelial tissue structure [49] . keratin queries on cluster or family information can discover related information across laboratories and host pathogen systems. using the uniref90 cluster id for k1033_mouse, identified in figure 3 , to query for all proteins with at least 90% identity, we find the human homolog k1033_human was detected in hela cells infected with vaccinia and monkypox virus [12] . doi:10.1371/journal.pone.0007162.g004 4 in human saliva has been shown to interact with the protein srr-1 localized on the surface of streptococcus agalactiae and to play a critical role in colonization of this bacterial pathogen [50, 51] . little is known about the mechanisms by which poxviruses attach to and enter host cells. no receptor for virion attachment on the host cell surface has been found. poxvirus infection can occur through interaction with human as well as mice airway epithelia, [52, 53] we propose that the protein interactions outlined above may represent some of the initial interactions between host and pathogen. thus they represent potential therapeutic targets for further investigation. this is the first report describing the interaction of a poxvirus protein with a host keratin, type ii cytoskeletal 4 protein. proteins. unequivocal identification of pathogens is important so that adequate counter measures can be taken. currently over 700 pathogenic and non-pathogenic bacteria have been completely sequenced. the availability of sequence data allows identification of proteins that are unique at different taxonomic levels, thus providing a means to begin to distinguish pathogenic from non-pathogenic species. however, if the initial screening depends on sequence data alone, the list of potential targets for laboratory validation can be relatively long; by supplementing sequence results with experimental data one can prioritize the target list for validation in the laboratory. we used such an approach by computationally screening potential targets using cupid [54] , prc data and other computational means to produce a list of potential targets. identifying species-specific proteins can be done with confidence when multiple species and strains have been sequenced as is the case with bacillus anthracis. the approach relies on the fact that if a gene is conserved over time within multiple strains it gives confidence they will not be lost in the near future and hence are ideal for diagnostic targets. these ''core unique'' proteins have related sequences in all selected organisms (in this case all available strains of bacillus anthracis) but not in other related organisms. an initial total of 327 proteins unique to the bacillus anthracis proteome were identified using the cupid and uniprotkb version 13.0. (bacillus anthracis strain ames isolate porton, bacillus anthracis strain ames ancestor, bacillus anthracis strain sterne were compared to twelve other genomes in the bacillus genus). the two closest relatives of bacillus anthracis as determined by cupid are bacillus thuringiensis and bacillus cereus. the species most closely related to the selected organism is based on the best blast hits of its entire proteome [54] . one needs to be careful in choosing the diagnostic targets that these two non-pathogenic organisms are not being detected. the initial list of 327 was refined to identify ''core unique'' proteins that are 100 amino acids or more in length. the 100 residue cutoff was used to ensure that the target list consisted of proteins that are real (short proteins might not be real) and are unique, as identification of homologs for short proteins is not trivial [55] . this resulted in a list of 21 ''core unique'' proteins in the pathogenic strains. it is possible that the proteins found may have homologs in other organisms which were undetected by cupid because the genes were not annotated as open reading frames to confirm their uniqueness, the 21 proteins were screened for significant regions of similarity at the dna level (either pseudogenes or unannotated genes) using tblastn against the ncbi nr database. using ncbi's nr which is produced independently of similar, but not identical, sources as iproclass also helps assure no sequences were missing from our warehouse. this additional analysis resulted in a total of 10 bacillus anthracis specific proteins proposed as high-quality targets for development of diagnostic probes. we then supplemented this information with data from the prc projects and master protein directory to create a matrix of information ( figure 6 ). six of the ten targets have data from the university of michigan prc showing that they were differentially expressed in published microarray experiments. nine of the ten are available as clones produced by the harvard institute of proteomics. a search of all the microarray data from the university of michigan (using http://proteinbank.vbi.vt.edu/ proteinbank/p/search/searchproteins.dll) showed that the four proteins not differentially expressed (listed as clones only in figure 6 ) were still constitutively expressed well above background in all studies (not shown). either the proteins or the dna coding for these proteins can be used to develop and test pathogen detection systems. all the ''core unique'' proteins detected in this study lacked meaningful functional annotation (i.e., were annotated simply as ''uncharacterized protein''), which is not surprising as such unique proteins are not easy to characterize. one protein identified as a target is a remnant of a prophage protein. such proteins are well known to be related to virulence [56] . another protein is from the pxo2 plasmid. a similar approach was taken for salmonella species using cupid and public prc data and several candidate diagnostic proteins are currently being validated in the laboratory (data not shown). a systems approach to biology or medicine requires the sharing, integration and navigation of large and diverse experimental data sets to develop the models and hypotheses required to make new discoveries and to develop new treatments. to date this has most often been done with selected research data or within an institution or program where common instrumentation and methods make standardization of experimental practices and data management easier to achieve [57, 58, 59] . alternative approaches require a reanalysis of all the data by a common methodology as has been done in some data repositories [60, 61] or assigning some common statistical metric to all data of a certain type to allow functional coupling [62] . these approaches are all potentially useful, but practically difficult to achieve on a large scale with heterogeneous data. the protein-centric approach we employed is a relatively simple, yet powerful and practical, approach to integrate and figure 6 . ten potential diagnostic markers for pathogenic strains of bacillus anthracis. the prioritized protein list was obtained by computationally screening potential targets using cupid [54] , prc data and other computational means described in the text. doi:10.1371/journal.pone.0007162.g006 navigate diverse sets of omics data in a manner useful for systems biology. proteins are often the biologically functional elements in cellular networks; thus, many types of data can be mapped to and through proteins as a common biological object. the lightweight data warehouse approach used for the mpd proved useful in practice, especially with large datasets as its simple design and schema allows greater flexibility to add new data types and to modify search and analysis capabilities. similar lightweight approaches and schemas designed to optimize queries have been shown useful in integration of genomic data [63, 64] . the main drawback of this approach is that the warehouse does not contain all the data. however, this is rarely a problem if the data are available in some other data resource optimized for that particular data type and if some upfront analysis of the user's needs for query and analysis options is performed. for example, our use case analysis suggested that for microarray and mass spectrometry data, individual raw intensities, machine-specific parameters and most calculated numerical values were not required for general queries and analysis across the combined data as these values were only comparable between the particular analysis performed in one lab. as a result, most numerical values were not included in the mpd for the default search but are accessible for display via hyperlinks to our protein data center or ftp site. however, if a new attribute appear or users request searches on a particular value omitted from the warehouse, adding it is a relatively simple matter of adding new data columns. for instance, in example ii our combination and analysis of mass spectrometry and protein interaction data, we could include peptide counts directly in the mpd for immediate download instead of retrieving them from the ftp files. of course, no one approach can be perfect, as in biology and research there always seems to be exceptions and new data and multiple approaches need to be accommodated. efforts to standardize reporting requirements, vocabularies and develop common xml data formats for sharing data are welcome and can greatly ease the transfer and automated processing of a particular data type. however the current standards do not necessarily guarantee integration as the problems of reconciling gene and protein identifiers as well as differences in experimental methodology remain. we investigated and employed a few common data standards and ontologies in developing the biodefense proteomics resource. we provided some data using mzdata [65] and mage-ml [66] but also provided original dataspecific text files for download. we found that several ontologies to describe experimental methods were useful but incomplete and focused on higher eukaryotes and thus did not yet contain terms needed for microbial pathogens. most useful was the gene ontology [67] which has been widely adopted to annotate and classify large scale results and can be used for searching and classification in the mpd. here we have presented some unique examples to illustrate benefits, as well as the difficulties, associated with integration of a very diverse set of omics research data across different data types, laboratories and organisms. we illustrated with three examples how potential therapeutic and diagnostic targets can be identified from integrated data applying relatively simple and established tools and techniques. we continue to focus on data integration to allow biologists to find relevant data sets for further detailed analysis using the approaches and tools of their choice. in general the analysis of diverse omics data is an area of active research and a number of useful tools are under active development including cytoscape [28] , bioconductor [68] and galaxy [69] . in the future a more seamless integration between data repositories and analysis tools such as these would be the most useful approach to add additional analysis options for integrated data. author contributions building integrated approaches for the proteomics of complex, dynamic systems: nih programs in technology and infrastructure development the hupo proteomics standards initiativeeasing communication and minimizing data loss in a changing world the minimum information about a proteomics experiment (miape) the hupo proteomics standards initiative-overcoming the fragmentation of proteomics data an emerging cyberinfrastructure for biodefense pathogen and pathogen-host data the iproclass integrated database for protein functional analysis the universal protein resource (uniprot) ) dictybase, the model organism database for dictyostelium discoideum the arabidopsis information resource (tair): a model organism database providing a centralized, curated gateway to arabidopsis biology, research materials and community the mouse genome database (mgd): the model organism database for the laboratory mouse the yeast proteome database (ypd) and caenorhabditis elegans proteome database (wormpd): comprehensive resources for the organization and comparison of model organism protein information nucleic acids research, database issue the zebrafish information network: the zebrafish model organism database generif quality assurance as summary revision uniprot archive production and sequence validation of a complete full length orf collection for the pathogenic bacterium vibrio cholerae transcriptional profiling of the bacillus anthracis life cycle in vitro and an implied model for regulation of spore formation transcriptional profiling of bacillus anthracis during infection of host macrophages the global transcriptional responses of bacillus anthracis sterne (34f2) and a delta soda1 mutant to paraquat reveal metal ion homeostasis imbalances during endogenous superoxide stress the pir integrated protein databases and data retrieval system callable personal librarian (cpl) (version 6.5). callable personal librarian (cpl) (version 65) uniref: comprehensive and non-redundant uniprot reference clusters a data integration methodology for systems biology: experimental verification from bytes to bedside: data integration and computational biology for translational cancer research integration of metabolomic and proteomic phenotypes: analysis of data covariance dissects starch and rfo metabolism from low and high temperature compensation response in arabidopsis thaliana comparative proteomics of human monkeypox and vaccinia intracellular mature and extracellular enveloped virions the utility of accurate mass and lc elution time information in the analysis of complex proteomes cytoscape: a software environment for integrated models of biomolecular interaction networks exploring biological networks with cytoscape software comparative assessment of large-scale data sets of protein-protein interactions a direct comparison of protein interaction confidence assignment schemes a relationship between gene expression and protein interactions on the proteome scale: analysis of the bacteriophage t7 and the yeast saccharomyces cerevisiae protein interactions: two methods for assessment of the reliability of high throughput observations assessment of the reliability of protein-protein interactions and protein function prediction gaining confidence in high-throughput protein interaction networks vaccinia virus induces cell fusion at acid ph and this activity is mediated by the n-terminus of the 14-kda virus envelope protein a 14k envelope protein of vaccinia virus with an important role in virus-host cell interactions is altered during virus persistence and determines the plaque size phenotype of the virus the oligomeric structure of vaccinia viral envelope protein a27l is essential for binding to heparin and heparan sulfates on cell surfaces: a structural and functional approach using site-specific mutagenesis vaccinia virus envelope h3l protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo vaccinia virus envelope d8l protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells vaccinia virus f9 virion membrane protein is required for entry but not virus assembly, in contrast to the related l1 protein the vaccinia virus gene i2l encodes a membrane protein with an essential role in virion entry blockade of chemokine activity by a soluble chemokine binding protein from vaccinia virus monkeypox virus viral chemokine inhibitor (mpv vcci), a potent inhibitor of rhesus macrophage inflammatory protein-1 identification of a peptide derived from vaccinia virus a52r protein that inhibits cytokine secretion in response to tlr-dependent signaling and reduces in vivo bacterial-induced inflammation a46r and a52r from vaccinia virus are antagonists of host il-1 and toll-like receptor signaling the poxvirus protein a52r targets toll-like receptor signaling complexes to suppress host defense molecular characterization and expression of the stratification-related cytokeratins 4 and 15 mouse keratin 4 is necessary for internal epithelial integrity clumping factor b, a fibrinogen-binding mscramm (microbial surface components recognizing adhesive matrix molecules) adhesin of staphylococcus aureus, also binds to the tail region of type i cytokeratin 10 the surface protein srr-1 of streptococcus agalactiae binds human keratin 4 and promotes adherence to epithelial hep-2 cells vaccinia virus entry, exit, and interaction with differentiated human airway epithelia protective effect of toll-like receptor 4 in pulmonary vaccinia infection computational identification of strain-, species-and genus-specific proteins a search method for homologs of small proteins. ubiquitin-like proteins in prokaryotic cells? the impact of prophages on bacterial chromosomes systems biology at the institute for systems biology a data integration methodology for systems biology integrated genomic and proteomic analyses of gene expression in mammalian cells open source system for analyzing, validating, and storing protein identification data peptideatlas: a resource for target selection for emerging targeted proteomics workflows global networks of functional coupling in eukaryotes from comprehensive data integration ensmart: a generic system for fast and flexible access to biological data biomart central portal-unified access to biological data further steps in standardisation design and implementation of microarray gene expression markup language gene ontology: tool for the unification of biology. the gene ontology consortium bioconductor: open software development for computational biology and bioinformatics galaxy: a platform for interactive large-scale genome analysis key: cord-171099-d0qr84xg authors: buehler, markus j. title: nanomechanical sonification of the 2019-ncov coronavirus spike protein through a materiomusical approach date: 2020-03-30 journal: nan doi: nan sha: doc_id: 171099 cord_uid: d0qr84xg proteins are key building blocks of virtually all life, providing the material foundation of spider silk, cells, and hair, but also offering other functions from enzymes to drugs, and pathogens like viruses. based on a nanomechanical analysis of the structure and motions of atoms and molecules at multiple scales, we report sonified versions of the coronavirus spike protein of the pathogen of covid-19, 2019-ncov. the audio signal, created using a novel nanomechanical sonification method, features an overlay of the vibrational signatures of the protein's primary, secondary and higher-order structures. presenting musical encoding in two versions one in the amino-acid scale and one based on equal temperament tuning the method allows for expressing protein structures in audible space, offering novel avenues to represent, analyze and design architectural features across lengthand time-scales. we further report a hierarchical frequency spectrum analysis of five distinct protein structures, which offer insights into how genetic mutations, and the binding of the virus spike protein to the human ace2 cell receptor directly influence the audio. applications of the approach may include the development of de novo antibodies by designing protein sequences that match, through melodic counterpoints, the binding sites in the spike protein. other applications of audible coding of matter include material design by manipulating sound, detecting mutations, and offering a way to reach out to broader communities to explain the physics of proteins. it also forms a physics-based compositional technique to create new art, referred to as materiomusic, which is akin to finding a new palette of colors for a painter. here, the nanomechanical structure of matter, reflected in an oscillatory framework, presents a new palette for sound generation, and can complement or support human creativity. proteins are the building blocks of virtually all life, providing the material foundation of spider silk, cells, and hair, but also offering other countless functions from enzymes to drugs, and pathogens such as viruses. we cannot see small nanoscopic objects like proteins or other molecules that make up virtually all living matter including our cells, tissues, as well as pathogens such as viruses. here we apply a computational algorithm to a recently reported protein structure, which allows us to make protein material manifestation audible, by rendering molecular structure information such as vibrational spectra, secondary structure, and folding geometry into a complex audio signal [1] [2] [3] [4] [5] . protein molecules consist of 20 naturally occurring amino acid building blocks that are assembled into hierarchical structures across various length-scales [6] [7] [8] . proteins are usually classified by their structural organization, from primary structure (also known as the sequence of amino acids), secondary structure (local folded geometry: e.g., beta-sheet or alpha-helix), as well as tertiary and quaternary structure (overall folded arrangement). more broadly, the folded structure of proteins, encoded by the sequence of dna, determines material function and failure in the context of disease or injury [8] [9] [10] [11] [12] [13] [14] . this biomaterial organization is reminiscent of the way by which musical compositions can be described, starting from basic harmonic waves (for instance a sine wave), different timbres (overlaying harmonic waves modulated in pitch, volume, and other measures, over time), melodies, chords and multiple chords and melodies played together and against each other. the integrated perspective on all aspects of these assemblies, processed simultaneously through the human auditory system, provides an efficient approach for our brains to understand complex data structures in soundwhat humans commonly perceive as "music" [15] . in sonification, we exploit this skill, and use sonification to translate complex data into audible sounds [16, 17] . this article focuses on an analysis of the nanomechanical vibrational spectrum of a virus spike protein that is the pathogen of covid-19, which appears on the surface of the virus as outward facing molecular "spikes", which are critical of the virus' entry into host cells [18] . figure 1 depicts a rendering of this protein structure, revealing its characteristic spike shape, and also outlines the method used to translate the structure in audible signals based on its vibrational patterning. the plan of this article is as follows. we begin with a brief review of materials and methods, introducing to approaches used here, and then present specifics of the sonification examples. we discuss the nanomechanical foundation for the approach, and also review basic elements of the musical choices. sonification is a method to translate data structures into audible signals. here, we use a particular approach of sonification termed materiomusic, to use the actual vibrations and structures of molecules to create audible materials that be used for analysis and design. this method has been used in earlier work to translate from material to music and from music to material, due to its reversible and unique mapping [19, 20] . the main focus of this paper is on the protein structure with protein data bank (pdb) id 6vsb. it features three distinct protein chains that are folded into the virus spike protein, consisting of 1,288 residues each ( figure 2 shows an overview of the structure [21] ). a protein structure is built up from one-dimensional chains of amino acids. the basis to mapping each of the 20 amino acids onto a unique musical tone is the distinct vibrational spectrum of the molecules as explained in these references: [2, 17] . in fact, since each of the amino acids has a unique vibrational spectrum, we may distinguish each of them by its unique sound, or timbre, which is reflected in the overlay of all modal frequencies. since the eigenmodes are orthogonal, energy does not spill from one mode to another, which implies that they are a fundamental way to understand the vibrational spectrum and sounding of a molecule or assembly. since each amino acid features a distinct audible expression, defined by their unique profile of molecular vibrations, we can assume that they can be assigned a particular musical "note" or tone. in this framework, the primary sequence of amino acids defines the note sequence played, which is augmented by the other components [19, 20] the next higher level of organization of a protein is the secondary structure, reflecting the local organization of the 1d chain into helices or sheets (or other structures). we incorporate information about the secondary structure associated with each amino acid in the translation step by affecting the duration and volume of notes. during the translation process from protein to music we use the dssp algorithm to compute the secondary structure from the protein geometry file and sequence [13, 22] . as presented in earlier work [19, 20] the method of transpositional equivalence is used to scale the frequencies to the audible range, however, while maintaining the accurate ratio of frequencies in the signals generated. in addition to mapping primary and secondary structure, we realize the overall vibrational motions of the molecule by incorporating their normal mode frequencies into an audio signal, using the method described in [1] . to obtain the normal modes, we use the anisotropic network model [23] approach, and compute the spectra of frequencies. the data is then imported in the max device [1, 24] , and sounds are rendered using ableton live [25] , forming the basis for the secondary signal. in sound generation, both signalsthe primary and secondary, as well as audio generated by the overall vibrations are overlaid and played together, creating a multi-dimensional image of the protein's structure, true to its accurate reflection of vibrational spectra. sidechain compression is used in some examples to module the volume of the secondary signal based on the level of the primary signal. this renders a dynamic interplay of the two sound sources. in the approach described in section 2.1, all sounds generated are based on the spectrum of the actual molecular vibrations. this, however, poses challenges in matching it with conventional tuning systems. while the sound of amino acids closely resembles that of bells, another approach that can be used is to map each of the 20 amino acids onto a unique tone in a particular musical scale, such as a dorian mode [26, 27] as used in the example described in this article. other possible scale choices could be a major or minor scale, or variations thereof, whereas 20 notes are picked in ascending order to describe each of the 20 amino acids. the note assignments are made in the order tyr, asn, leu, met, glu, pro, trp, arg, gln, his, phe, ser, lys, val, asp, thr, ile, cys, ala, gly, following an ordering based on their lowest vibrational frequency from small to large. as in the other approach described in section 2.1, the next level of organization of a protein is the secondary structure, reflecting the local organization of the 1d chain into helices or sheets, or potentially other structures. we incorporate information about the secondary structure associated with each amino acid in the translation step by affecting the duration and volume of notes, as described in detail in [13, 22] . the format used to represent the distance correlation matrix depicted in figure 3a the concept of rapidly played note insertions, represented musically through strummed notes similar as in a guitar [17] . this approach is further explained in figure 3 , which shows the folding of a protein into a three-dimensional structure. to encode the information that points i and j are neighbors in the folded state, we insert a sequence segment around point j at point i. similarly, a sequence segment around point i is inserted at point j, creating a symmetric representation that reflects the geometric fact that points i and j are neighbors. see figure 3d for the geometry in the folded state, as a visual example of how the coding is implemented in a musical score, in figure 3e . furthermore, additional notes are inserted and played simultaneously, where the volume of the notes is modulated based on the distance of the amino acid inserted from the reference amino acid in the primary sequence, and reflecting the distance correlation matrix shown figure 3 . these reflect the same sequence patterns of the insertions, where a choice can be made about how long the overlaid fragments are. unlike the insertions described in the previous section, the insertions now added are played on top of the primary notes reflecting the primary and secondary structure of the protein. the resulting complex overlapping melodies represent a kind of counterpoint argument in musicthe concept where notes are played against notessuch as used widely by j.s. bach [28, 29] . counterpoint is a powerful means to manifest references of closeness in a time sequence coded manifestation of signals. it is also the basis for pleasantness of music, whereas a balance of repetition and variation is a foundational structural element. this universality, reminiscent of the universality in proteins represented by secondary structure motifs (such as alpha-helices of beta-sheets), or repetitions of sequence patterns within a protein design is common across nature. importantly, the concept a more generalized counterpoint argument represents physical closeness in musical space in an indirect manner. similar concepts of counterpunctual approaches are used in many other fields of communication and art, where referencing conceptsalbeit distortedis a powerful way to represent connections in a coded form. for instance, allegories in art can realize connections to multiple touchpoints and associations [30] . these and other concepts are quite similar to the natural structure of proteins, where one residue may be close to many others, as can be directly confirmed in the distance correlation matrix. moreover, such associations are also reminiscent to other structures, such as the hierarchical organization of neurons in the brain, as visualized in figure 3b [31]. through the transformations described above, a total number of 3,647,770 notes are generated in the raw musical coding, reflecting the hierarchical structure of the protein in musical space. after rendering the sound using max patches [1, 24] and ableton live [25] , spectral analysis is performance using sonic visualizer [32] , and the audio files processed using audacity 2.3.2 [33] . we specifically focus on the melodic spectrogram, which offers a visual representation of how the energy in different frequency bands changes over time. it provides an analytical tool to assess the content of an audio signal, which can complement or validated audible inspection. to show the potential application to distinguish protein structures through sound, we depict a spectral comparison of the overall vibrational spectrum of proteins with protein data bank (pdb) [34] identification code 6vsb and 5i08 in figure 4 . figure 4a shows their normal mode vibrational frequencies, and the figure 4b a spectrogram analysis of the vibrational signal generated of these proteins. representative audio signals are included as supplementary material (comparison_5i08-6vsb.wav), where the distinction is clearly audible, between 5 distinct protein conformations (the order of tones is as depicted in figure 4b to 4d, repeated twice overall, representing a total of 10 tones). this example illustrates how the audible system is capable to quickly discern differences in the spectrum, and hence, the vibrational context of a protein structure. this method to assess vibrational changes can be useful to investigate the impacts of sequence changes, or to screen for effects across a very large number of protein data. figure 4c shows the normal mode vibrational frequencies of three protein complexescomparing scenarios of the virus spike protein attached (6pdb id m17) with proteins without the virus spike protein (pdb ids 6m18 and 6m1d) (for a detailed discussion of these protein confirmations, see [35] ). figure 4d shows the frequency spectrum of the audio signal. it can be clearly recognized how the attachment of the virus spike protein with the human cell receptor changes the frequency spectrum, and it can also be clearly heard. you can hear the transition between the attached state vs. the unattached state between the third and fourth tone, respectively. notably, when looking at the frequency spectrum, the binding of the virus to the ace2 receptor leads to a lowering of the frequency spectrum. coding for the multiple structural levels, the audio file coronavirus_6vsb_aa-scale_excerpt.wav represents a sonification of the novel coronavirus pathogen, with pdb id 6vsb. a spectral analysis of the piece is depicted in figure 5a . the file coronavirus_6vsb_aa-scale-complex_excerpt.wav represents a more complex version of the original score, with added musical ornamentation, expressing creative choices. as can be determined, the raw structure of the original completely algorithmic composition is expanded upon, through added musical experimentation, realizing a more complex exploration of the audible content. the total length of the piece is around 35 minutes. what you hear is a multi-layered algorithmic composition featuring both the vibrational spectrum of the entire protein (expressed in sound and rhythmic elements), the sequence and folding of amino acids that compose the virus spike structure, as well as interwoven melodiesforming counterpoint music -reflecting the complex hierarchical intersecting geometry of the protein. the audio file coronavirus_6vsb_chromatic-scale_excerpt.wav represents a sonification of the novel coronavirus pathogen, with pdb id 6vsb. a spectral analysis of the entire piece is depicted in figure 5b . the techniques used to realize a musical composition include a variety of approaches: â�¢ the primary sequence, all overlaid notes, and all insertions are played as, and rendered as lead sound using physical modeling of a koto (a japanese harp instrument). the use of physical modeling allows us to directly generate sound using a physical basis, accounting for a true reflection of the way by which vibrating strings generate sound. â�¢ overlaid notes, coded through the simultaneous excitation of the sounds of amino acid residues in close vicinity, are played through arpeggios. an arpeggio is a way to play the notes of a chord in succession, rather than sounding them simultaneously. this approach enables to create a flowing expression of the complex chord progressions generated through the protein structure, to offer a contrast to the simultaneously played notes. â�¢ a variety of classical instruments are used in the realization of these scores, and artistic choices are executed to vary the relative loudness of one over other instruments. the realization of the musical score includes a total number of 20 independent musical instruments, including: koto (several), harp (several), strings, strings played pizzicato, celli, solo violin, clarinet (several), oboe, flutes (several), trumpet, fluegelhorn, and percussion. the total length of the piece is around 1:50. this analysis presented two approaches to sonify a large protein structure, to provide access to structural and potentially functional information through sound. it provides not only access to a truthful reflection of protein structures via sound, but also offers intriguing new ideas for musical composition. when applied in this context, human composers may intersect with the compositions generated by alone, forming a new axis of intersection across scales and species. the focus on proteins is motivated since they are the most abundant material component of all living things: especially their motion, structure and failure in the context of both normal physiological function and disease is a foundational question that transcends academic disciplines and can be a footing to explore fundamental nanomechanical questions. we focused on developing a model for the vibrational spectrum of the amino acid building blocks of proteins, an elementary structure from which materials in living systems are built on. this concept is broadly important as at the nanoscale observation, all structures continuously move, reflecting the fact that they are tiny objects excited by thermal energy and set in motion to undergo large deformations as is the case in injury or disease, or due to disruptive mutations. the approach allowed us to extract new musical compositions of the coronavirus spike protein as one way to represent nature's concept of hierarchy as a paradigm to create function from universal building blocks, and exploring the vibrational complexity of a large protein design. this sort of bio-inspired music is an expansion of bio-inspired materials design, and can offer new design ideas for geometric and structural patterning. indeed, the translation from various hierarchical systems into one another poses a paradigm to understand the emergence of properties in materials, sound, and related systems. the use of sonification and comparisons of music generated by proteinsas a core physical material building block of lifewith conventional compositions may shed light into the unique features of the effects of music and sound and interactions with matter and life, including the brain and other non-human forms of life, as demonstrated in earlier work of sonifications of spider webs [36] . the music and its pleasing nature, especially the realization in the dorian mode (coronavirus_6vsb_chromatic-scale_excerpt.wav), is a metaphor for this nature of the virus to deceive the host and exploit it for its own multiplication. a virus' genome hijacks the host cell's protein manufacturing machinery, and forces it to replicate the viral genome and produce viral proteins to make new viruses from it. in light of this nature of viruses, the work explores the fine line between the emergent beauty of life and death as opposite poles, an aspect also critically important for nanomaterials and human health. as one listens to the protein we find that the intricate design results in interesting and actually pleasing, relaxing sounds. the reason for this is that the virus spike protein represents a complex, beautiful design (see also figure 1 ). of course, the intricate protein principles at the basis of the virus do not at all convey the deadly impacts this particularly protein is having on the world. from another vantage point, the pleasing aspect of the music shows the deceiving nature of the virus when it enters the cells by binding to exterior receptors, in order to hijacks our body to replicate. the pleasant sounds represent an analogy for the nature of the virus to deceive the host and exploit it for its own multiplication, emerging as the residue after the transactional infections that diminish or kill the host. a related important aspect of materiomusical coding is the agency of singular eventsin which case, albeit a building block in a system may be small, its impact on the greater context of the overall system behavior is outsized. as in many musical compositions, a single note or sound can be a focal point of a composition (such as a accidentals, or out-of-scale notes or chords in music). in materials science, a pair of atoms may be subject to singular forces near a crack tip [37] . in many systems, a complex structure of a composition of building blocks may solely exist for the purpose of a single, unique, singular note, one of the significant deep foundational questions of many forms of art. specifically in the context of music, when played alone that note would not have particular exposition. it is only in the context of the whole piece that it stands out. in proteins, we can see a similarity to this singular perspective in point mutations, where certain changessuch as point mutationsare particularly relevant. applications of the approach could include the development of antibodies by designing protein sequences that match, through melodic counterpoints, the binding sites in the spike protein, to identify sequences that create strong adhesion for the interruption of disease progression through protein-based drugs. the novel coronavirus is believed to enter human cells via the ace2 receptor on the exterior of cells [18, 35] . ace2 is an enzyme attached to the outer surface of cells found in our lungs, heart, arteries, kidney, and intestines (whereas one of its natural role is blood pressure regulation). one possible engineering task could be to create a protein molecule, through counterpoint composition, that binds more easily to the virus spike, hence shutting down the pathway of infection. an analysis of the mechanism of interaction between ace2 and the spike protein can perhaps provide useful insights, and the intersection of these two proteins could be subject of future experimentation in musical space. not only could this be tackled using human writing, but could also be performed using ai models, as demonstrated in earlier work [2, 20] . the data depicted in figure 4c clearly demonstrated that the binding of the virus spike protein to the human cell leads to an audible change in the sounds generated. other aspects by which vibrations of the virus proteins play an important role include our understanding of the virus stability as temperatures increase, for example. vibrational spectra may also tell us why the sars-cov-2 spike gravitates toward human cells more than other viruses. further applications of materiomusical coding of matter include de novo material design by manipulating sound, detecting mutations, and offering a way to reach out to broader communities to explain the science of nanomaterials such as proteins. it also forms a physicsbased compositional technique to create new art, referred to as materiomusic, which can be explained akin to finding a new palette of colors for a painter. here, the nanomechanical structure of matter, reflected in an oscillatory framework, presents a new palette for sound generation. a summary of the new palette of sounds, specifically the 20 amino acid tones, are summarized graphically in figure 6 . the bio-nano-science of molecular motions can hence offer several avenues of future investigation and applications in the interrelated fields of science, art and engineering. [38] , and visualized using a sequence analysis tool [21] . the analysis shows that the protein features 16% helical and 21% beta sheet content. the image provides a direct visualization of the various secondary structure motifs that make up the protein, which in turn fold into complex shapes. all this information is incorporated into the musical expression of the protein. the plot in panel a shows amino acid residues that are close to each other in brighter red colors, providing an overall image of the protein's 3d folded structure. panel b depicts the connectome as reported in [31] , and rendered via the neuprint server [39] , showing the similarity of folding concepts in the neural network design in the brain. panel c shows a plot with only the closest neighbors highlighted, with distances less than 4.5a. panel d depicts, schematically, how the overall folding geometry is coded in that way. the inlay depicts the folding dynamics of a beta-sheet protein from an initial elongated unstructured protein. panel e shows an example how two sequence patterns are assembled in the overlapped form, creating note-against-note musical content. analysis of the vibrational and sound spectrum of over 100,000 protein structures and application in sonification a self-consistent sonification method to translate amino acid sequences into musical compositions and application in protein design using ai reoccurring patterns in hierarchical protein materials and music: the power of analogies materials by design -a perspective from atoms to structures mater. impuls. natur, acatec ultrathin free-standing bombyx mori silk nanofibril membranes deformation and failure of protein materials in physiologically extreme conditions and disease silk-its mysteries, how it is made, and how it is used materials by design: merging proteins and music materiomics: an -omics approach to biomaterials research nature's hierarchical materials predictive modelling-based design and experiments for synthesis and spinning of bioinspired silk fibres a series of pdb related databases for everyday needs structure and stability of the lamin a tail domain and hgps mutant evaluating hierarchical structure in music annotations sounds interesting: can sonification help us design new proteins? cryo-em structure of the 2019-ncov spike in the prefusion conformation a self-consistent sonification method to translate amino acid sequences into musical compositions and application in protein design using artificial intelligence sonification based de novo protein design using artificial intelligence, structure prediction and analysis using molecular modeling e-msd: an integrated data resource for bioinformatics dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features the anisotropic network model web server at 2015 (anm 2.0) the structure of atonal music formalized music: thought and mathematics in composition, notes a geometry of music : harmony and counterpoint in the extended common practice the craft of tonal counterpoint allegory : the theory of a symbolic mode a connectome of the adult drosophila central brain sonic visualiser audacity multi-track audio editor and recorder the protein data bank structural basis for the recognition of the sars-cov-2 by full-length human ace2 imaging and analysis of a three-dimensional spider web architecture atomistic modeling of materials failure on the nature of the transparent teeth of the deep-sea dragonfish neuprint: analysis tools for em connectomics pre-fusion structure of a human coronavirus spike protein acknowledgements: this work was supported by mit cast via a grant from the mellon foundation, with additional support from onr (grant # n00014-16-1-2333) and nih u01 eb014976. â�¢ comparison_5i08-6vsb.wav: comparison of the overall molecular frequency spectrum of two distinct spike proteins from two different coronaviruses, calculated by a normal mode analysis. the analysis of the spectral content is shown in figure 4 . https://soundcloud.com/user-275864738/comparison-5i08-6vsb â�¢ coronavirus_6vsb_aa-scale_excerpt.wav: sonification of the novel coronavirus pathogen, with pdb id 6vsb (excerpt). key: cord-303555-mwu72q7w authors: dent, paul title: cell signaling and translational developmental therapeutics date: 2020-10-06 journal: reference module in chemistry, molecular sciences and chemical engineering doi: 10.1016/b978-0-12-820472-6.00002-5 sha: doc_id: 303555 cord_uid: mwu72q7w the relationships between drug pharmacodynamics and subsequent changes in cellular signaling processes are complex. many in vitro cell signaling studies often use drug concentrations above physiologically safe drug levels achievable in a patient's plasma. drug companies develop agents to inhibit or modify the activities of specific target enzymes, often without a full consideration that their compounds have additional unknown targets. these two negative sequelae, when published together, become impediments against successful developmental therapeutics and translation because this data distorts our understanding of signaling mechanisms and reduces the probability of successfully translating drug-based concepts from the bench to the bedside. this article will discuss cellular signaling in isolation and as it relates to extant single and combined therapeutic drug interventions. this will lead to a hypothetical series standardized sequential approaches describing a rigorous concept to drug development and clinical translation. the field of cell signaling and signal transduction dates back to the late 19th century. in 1895, epinephrine (adrenaline) was discovered. 1 by the 1920s, insulin and glucagon had been discovered. 2, 3 collectively, these discoveries paved the way for researchers to explore how these hormones acted to regulate glucose metabolism in the liver and skeletal muscle. the laboratory of dr. carl cori played a seminal role in partially unravelling how glycogen could be broken down by glycogen phosphorylase. 4 he, his wife gerty and bernardo houssay received the 1947 nobel prize in physiology or medicine for their work. although the cori laboratory had discovered and described glycogen phosphorylase, it was not until 1959 that leloir discovered the enzyme that made glycogen, glycogen synthase. 5 during the 1950s, fischer, krebs and sutherland not only discovered and characterized the kinase which a dedicated to professor sir philip cohen on the occasion of his 75th birthday. comprehensive pharmacology https://doi.org/10.1016/b978-0-12-820472-6.00002-5 regulated glycogen phosphorylase, phosphorylase kinase, but defined for the first time that the phosphorylation of proteins could regulate enzyme activity. [6] [7] [8] [9] 2 further development of signal transduction up until the late 1950s, however, no-one had been able to elucidate how insulin signaled to make a cell store glucose as glycogen nor how epinephrine and glucagon activated phosphorylase kinase/glycogen phosphorylase to break down glycogen. sutherland and colleagues during their investigations into glycogen phosphorylase discovered a heat-stable factor in liver sections whose levels were regulated by epinephrine and glucagon: cyclic amp, the first second messenger. [10] [11] [12] subsequently, fischer and krebs isolated the kinase regulated by camp, protein kinase a (pka). 13 for these discoveries, sutherland, as well as fischer and krebs, received the nobel prize. sutherland, fischer, and krebs during their studies also discovered an enzyme activity which could remove phosphate from glycogen synthase, i.e. a protein phosphatase. a postdoctoral researcher from the laboratory of fischer in the late 1960s, philip cohen, focused their independent career upon characterizing the many protein phosphatases in cells and above all understanding how phosphatases regulated glycogen metabolism, naming the ser/thr protein phosphatases. 14 over the 20 or so years after the discovery of pka, multiple additional small molecule second messengers were discovered including: calcium ions, diacyl glycerol and ip3; and nitric oxide and cyclic gmp (cgmp). [15] [16] [17] [18] signaling by cgmp in the eye was shown to be essential for the perception of light and cgmp as well as with nitric oxide in the regulation of smooth muscle contractility resulted in the nobel prize being awarded to murad in 1998. 18, 19 during the 1970s and 1980s work by lefkowitz, gilman and johnson led to the discovery of serpentine plasma membrane receptors for hormones, e.g. the beta-adrenergic receptor for epinephrine, as well as receptor-associated large gtp binding protein complexes on the inner leaflet of the plasma membrane which transduced receptor signals to intracellular effectors such as: (1) adenylyl cyclase leading to the generation of camp; (2) activation of phospholipases leading to the generation of diacyl glycerol and inositol 1,4,5-trisphosphate (ip3), with ip3 triggering the release of calcium ions into the cytosol. [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] diacyl glycerol and calcium ion then activated multiple protein kinase c (pkc) isoforms. this resulted in the award of additional nobel prizes. serpentine g-protein coupled receptor (gpcr) signaling can be down-regulated by proteins called arrestins. [31] [32] [33] arrestin proteins prevent both the ga gbg proteins interacting with the gpcr and cause the gpcr to be internalized. internalization can result either in receptor degradation or recycling back to the plasma membrane. thus, by the mid-to late-1980s a large body of literature existed which argued that signal transduction pathways consisted of a receptor linked to a large gtp-binding protein which in turn regulated an enzyme that generated "second messengers;" the second messengers would then diffuse throughout the cytosol activating cellular processes, predominantly for metabolism. in parallel to the study of serpentine receptors, other investigators were focused on the relatively few proteins who became phosphorylated on tyrosine. studies in this field were focused on the insulin receptor (metabolism) and the epidermal growth factor receptor (egfr, erbb1) (growth, cancer). [34] [35] [36] [37] insulin caused the insulin receptor to become tyrosine phosphorylated, and a substrate for the receptor, insulin receptor substrate 1 (irs1), was discovered. 38 for many years prior to the 1990s, diagrams of insulin receptor signaling would include the receptor and irs1, together with downstream insulin targets such as glycogen synthase. in-between the receptor and synthase was drawn a "black box" as the pathway by which insulin regulated glycogen synthase appeared to be intractable to investigation. 39 studies by the laboratory of larner and villar-palasi argued that insulin caused the generation of a "mediator" second messenger which was an inositol phospholipid, that regulated glycogen synthase. [40] [41] [42] although at the time this concept was not widely supported, subsequent studies over the following 10 years demonstrated that insulin activated phosphatidyl inositol 3-kinase whose product, phosphatidylinositol (3,4,5)-trisphosphate (pi(3,4,5)p3), caused activation of the membrane-associated kinase, phosphoinositide-dependent kinase-1 (pdk1). [43] [44] [45] [46] [47] pdk1 was shown to phosphorylate akt t308 causing enzyme activation, and akt to phosphorylate and inactivate glycogen synthase kinase 3 (gsk3). 48 reduced gsk3 activity results in reduced glycogen synthase phosphorylation, leading to activation of synthase activity. one additional component within this process was activation of protein phosphatases to facilitate the dephosphorylation and activation of glycogen synthase. 49 thus, after 60 years of research, by the mid-1990s, the regulation of glycogen metabolism by epinephrine and insulin had largely been elucidated. for the egfr and other subsequently discovered membrane associated tyrosine kinases, e.g. the non-receptor scr family and the fibroblast growth factor receptor (fgfr) family, understanding how these enzymes signaled into the cell again initially rested on studies using traditional biochemical methods. [50] [51] [52] [53] in the mid-1980s, a postdoctoral researcher in the laboratory of dr. ora rosen, thomas sturgill, was given a project in which he was to identify a 42 kilo dalton protein whose tyrosine phosphorylation was increased after exposing cells to insulin. 54 as an independent investigator sturgill continued his studies into the enzyme he called map2-kinase, microtubule associate protein 2 (map2) being the substrate used to measure its kinase activity. 55 it was subsequently renamed to be "mitogen activated protein kinase" (mapk) after it was discovered to not only be regulated by many growth factors, but also that it was an intermediary kinase in the regulation of another insulin-activated kinase p90 ribosomal s6 kinase (p90rsk). 56 this enzyme should not be confused with p70 s6 kinase with is a component of the pi3k pathway. 43, 44 by the end of the 1980s, it had been determined that there was another mapk isoform (p44) and that these kinases were regulated by tyrosine/threonine joint phosphorylation. 57, 58 at that time, kinases were considered to be specific for either serine/threonine or for tyrosine. the discovery of mek1 and mek2 (mitogen/extracellular regulated kinase), kinases that phosphorylated the mapks on both tyrosine and threonine was considered biochemically novel. [59] [60] [61] [62] from work in yeast (cerevisiae, pombe), however, was in parallel demonstrating that they also expressed mapk-like and mek-like enzymes, and that their mek-like enzymes phosphorylated the mapk-like enzymes on tyrosine and threonine. [63] [64] [65] the mammalian mapk/renamed erk1/2 (extracellular regulated kinase) pathway in yeasts regulates the yeast response to pheromones. 66 this understanding facilitated the further characterization of mek1 and mek2. the next question in the development of the "mapk pathway" was to define the kinase(s) upstream of mek1/2. based on data from yeasts, this kinase should have been similar to the mammalian map3k, known as mekk1 (mitogen/extracellular regulated kinase kinase). 67 however, in 1992, two groups linked c-raf-1 and its truncated oncogenic variant v-raf as the kinase activity which enhanced mek1/2 phosphorylation and activity; there are no yeast homologues of the raf family proteins. 68, 69 of note, prior to those studies it was believed that raf-1 was downstream of erk1/2. 70 the function of mekk1 subsequently, and with its family members, was linked in mammalian cells to the regulation of the c-jun nh2-terminal kinase (jnk1/2) and p38 mapk pathways. [71] [72] [73] [74] [75] [76] contemporaneously with these studies, researchers were determining how receptor tyrosine kinases regulated ras family small gtp binding proteins, and other groups determining how ras proteins signaled downstream off the plasma membrane and into the cytosol. [77] [78] [79] [80] [81] it was demonstrated that the proteins grb2 (growth factor receptor-bound protein 2) and sos (son of sevenless homolog 1) linked receptor tyrosine phosphorylation to the exchange of gtp for gdp in ras proteins. within months of these discoveries being published, it was shown that gtp-bound ras would associate with the nh2-terminal domain of raf-1. 82-85 gdp-bound ras proteins did not associate with raf-1. thus, within the period between 1986 and 1994, the first of the "map kinase pathways" had been delineated. because of extant data from yeasts, other parallel mammalian map kinase pathways were rapidly discovered and delineated. for example, as mentioned previously, the p38 mapk pathway in mammalian cells is a stress-induced signaling pathway and was the equivalent of the hog osmo-sensing pathway in yeasts. 86 the jnk pathway has similarities to several yeast and mammalian mapks, but only a 60% best-fit to erk1 and erk2. it was discovered as a uv-activated kinase that bound to the nh2-terminus of the transcription factor c-jun. 87 a parallel mapk pathway, the erk5 "big map kinase pathway" was discovered and inhibitors of mek1/2 also inhibit mek5, demonstrating the close functional alignment of both pathways. 88 hence, by the mid-1990s the basic structures of multiple map kinase as well as the pi3k pathway were in place. broadly, over the past 25 years, signaling by erk1/2 and erk5 were most often linked to tumor cell growth whereas signaling by p38 mapk and jnk were linked to cell death. [88] [89] [90] however, coordinated erk/jnk signaling strongly promoted growth and under prolonged high activity erk1/2 signaling would cause growth arrest via the induction of cyclin dependent kinase inhibitor proteins or tumor cell death. [91] [92] [93] these were also reflected at the level of receptor tyrosine kinases, comparing different ligands for the same receptor with different on-/off-rates, e.g. egf and tgfa, as well as associated with ligand concentration. high ligand levels permanently downregulate the receptor, and ligands such as egf that remain with the receptor in endosomes cycle the receptor for degradation. [94] [95] [96] [97] signaling by p38 mapk regulated chaperone functions but also could cause cell cycle arrest and dna damage repair. 98, 99 what also became readily apparent was that activation of the same pathway to the same extent in different tumor cells could result in different changes in tumor cell biology, with some cells exhibiting growth/growth arrest and other cells becoming moribund either through apoptosis, necrosis or autophagy. [91] [92] [93] some of these behaviors could in part be explained due to the differential expression of driving oncogenes such as mutation of p53, ras proteins, receptor tyrosine kinases or the lipid phosphatase: phosphatase and tensin homologue on chromosome ten (pten). 100-103 the cellular process of autophagy was discovered in the 1960s. 104, 105 the primary purpose of the process is to recycle cellular components into their elemental building blocks during times of metabolic stress, permitting the cell to survive. materials are first encapsulated in a double membrane, called an autophagosome. [106] [107] [108] autophagosomes fuse with lysosomes, the interior acidifies, and they become autolysosomes where materials are digested, ready for recycling. the regulation of autophagy and with it the sensing of nutrient and atp energy levels within a cell are regulated by mammalian target of rapamycin (mtor) and the ampdependent protein kinase (ampk), respectively. [109] [110] [111] [112] [113] the regulation of mtor is complex as it integrates upstream signaling from akt in the pi3k pathway, together with other signals that sense amino acid, lipid and carbohydrate levels. there are two complexes of proteins which associate with mtor, with the kinase being termed mtorc1 or mtorc2 based on the members of the protein complex. [114] [115] [116] the ampk senses amp levels, which are high when the cell is depleted of atp; high amp levels cause allosteric activation of the ampk, and activated ampk then acts to phosphorylate and inactivate mtor. [117] [118] [119] the ampk is itself regulated by phosphorylation, with the most notable regulators being liver kinase b1 (lkb1) and ataxia-telangiectasia mutated (atm). [120] [121] [122] [123] [124] [125] lkb1 is often mutated in tumor cells, leading to dysregulation of energy sensing and autophagy regulation. in the nucleus atm senses dna damage and cytosolic atm senses the levels of reactive oxygen species; atm at both cellular locations phosphorylates and activates the ampk. the key regulatory target for both mtor and the ampk is the kinase unc-51 like autophagy activating kinase (ulk1/2). 126-129 ulk1 is a classic example of a protein whose function is regulated by multi-site phosphorylation. phosphorylation of ulk1 at specific sites by mtor inactivates the kinase. phosphorylation of ulk1 at different specific sites by the ampk activates the kinase. [128] [129] [130] the primary substrate of ulk1 is the gate-keeper protein for autophagosome formation, atg13. phosphorylation of atg13 leads to the formation of multi-protein complexes which act to form a double membrane around the cellular materials that will be digested. autophagic flux occurs where a fully-formed autophagosome fuses with an endosome/lysosome to form an autolysosome. 131, 132 autolysosomes acidify their interior, activating a variety of proteases and other enzymes required to break down the vesicle's contents. many tumor cells exquisitely rely on autophagy to survive, which explains why drugs such as chloroquine, which prevent autophagosome lysosome fusion, have been trialed as cancer therapeutics. 133, 134 alternatively, as tumor cells utilize autophagy for survival, drugs which profoundly stimulate autophagosome formation and autophagic flux cause the over-digestion of cellular proteins and cause the cytosolic release from the autolysosome of active proteases, which collectively leads to a multi-factorial form of tumor cell death. 135 5 using our understanding of autophagy and cell signaling to therapeutically kill tumor cells in all scientific studies, experiments should be performed from an agnostic standpoint. that is, follow the data wherever it may lead, regardless of prior opinions or perceptions. twenty years ago, in collaboration with dr. paul fisher, we began to investigate the molecular mechanisms by which the cytokine il-24 acted to kill tumor cells. [136] [137] [138] at that time, the mechanisms by which tumor cells died were not particularly sophisticated, with death receptor signaling via caspases 8/10 (the extrinsic apoptosis pathway) and mitochondrial dysfunction via caspase 9 (the intrinsic apoptosis pathway) being the two pathways then considered most important in the causation of tumor cell death. because we had observed the cytokine was inactivating mtor, studies were performed to define if "autophagy" played any role in the cytokine's biology. molecular knock down of key autophagy regulatory proteins, atg5 or beclin1, profoundly suppressed il-24 lethality. our studies with autophagy and il-24 resulted in other laboratory projects exploring the role of autophagy in their biology and killing mechanisms. for example, in hepatoma cells, the combination of the multi-kinase inhibitor sorafenib with the histone deacetylase (hdac) inhibitor vorinostat killed cells by activating the death receptor cd95, and in hepatoma cells, knock down of atg5 or beclin1 enhanced drug combination lethality, i.e. autophagy was acting as a protective cellular response. 139 however, in pancreatic cancer cells, knock down of atg5 or beclin1 significantly reduced the ability of this drug combination to kill, i.e. autophagy played a role in the killing process. 140 subsequent studies in the laboratory over the past decade have almost invariably discovered that autophagosome formation was playing an essential role in the tumor cell killing process. one consideration when discussing the role of autophagy in causing cell death is whether the autophagic process caused killing directly, or indirectly by causing, e.g. mitochondrial dysfunction, followed by release of cytochrome c and apoptosis inducing factor (aif) into the cytosol. aif moves to the nucleus to cause dna fragmentation in a fashion similar to necrosis. 141 cytochrome c binds to apoptotic protease activating factor 1 (apaf-1) which together with atp causes the cleavage of pro-caspase 9. activated caspase 9 cleaves and activates caspase 3, which moves to the nucleus to cause apoptotic dna fragmentation, with dna fragments encapsulated in membranes. alongside the apoptotic processes, cathepsin proteases released from autolysosomes can cleave and activate the pro-apoptotic protein bid that is upstream of mitochondria, and which will lead to mitochondrial dysfunction and death. 142 however, it is possible that release of activated proteases by themselves into the cytoplasm can also cause death, without involvement of the mitochondria. we will now illustrate in more detail the role of autophagy in the development of anti-cancer therapeutics and in the development of anti-viral therapeutics. the multi-kinase inhibitor drugs sorafenib and pazopanib are approved for the treatment of liver/kidney cancers and soft tissue sarcoma, respectively. 143, 144 for both drugs, we demonstrated that they synergized with hdac inhibitors to kill liver, kidney, pancreatic and sarcoma tumor cells. [145] [146] [147] [148] contemporaneously with these studies, we were also studying the celecoxib derivative developmental drug, osu-03012. originally osu-03012 was proposed to inhibit pdk1 within the pi3k/akt pathway. 135, 142 osu-03012 has an order of magnitude anti-cancer efficacy than the parent compound. the key, arguably single, mechanism by which we found osu-03012 acted to kill tumor cells was by causing the generation of autophagosomes followed by autophagic flux and the cytotoxic actions of autolysosome localized proteases such as cathepsin b. ultimately, we determined that osu-03012 was an inhibitor of chaperone proteins, in particular grp78. 145 grp78 is an endoplasmic reticulum (er) localized chaperone that plays an essential role in regulating er stress signaling during times of protein overload and protein denaturation. 149 as we compared the chemical structures of osu-03012, pazopanib and sorafenib we realized that had many similarities. compared to osu-03012 which had ic50 values of inhibiting the atpase activities of hsp90 and hsp70 in the 200 and 300 nm range, respectively, the chaperone inhibitory activities of sorafenib were found to be similar, and the inhibitory activity of pazopanib significantly stronger with ic50 values of 50 and 100 nm, respectively. [150] [151] [152] [153] [154] thus, drugs that had been developed and marketed as "multi-kinase inhibitors" for many years also had multiple unknown chaperone targets. hence, just because a drug company states on their packaging that a drug inhibits enzymes a, b and c to cause a therapeutic effect, does not mean that the drug also inhibits unknown enzymes y and z. furthermore, it is probable that without inhibition of y and z, the inhibition of a, b and c together will only have a modest therapeutic effect. in the case of osu-03012, despite a phase i trial in cancer patients (nct00978523), further studies with drug took an unexpected turn away from cancer therapeutics, and towards infectious disease and the development of the drug as an anti-viral agent. 150, 152 all human pathogenic viruses require cells express functional grp78. 155, 156 in a virus-dependent manner, different viruses also recruit other additional chaperone proteins to facilitate their replication and life cycle. 152,153 osu-03012 is not a highaffinity inhibitor of a single chaperone or chaperone family, unlike many chaperone inhibitors developed for use in the cancer therapeutics field. 157, 158 however, because the drug inhibits multiple hsp90 family and hsp70 family chaperones within its clinically relevant safe concentration range, osu-03012 could potentially become a broad spectrum anti-viral drug. osu-03012 prevented the reproduction of viruses including mumps, influenza, measles, coxsackie virus b4, junã­n, rubella, west nile, yellow fever, hiv (wild type and protease resistant), and ebola, effects that were replicated by molecular knock down of multiple chaperone proteins, alone or in combination. 152 very recently we discovered, to some extent not surprisingly, that osu-03012 could also prevent synthesis of the sars-cov-2 spike protein. in three separate animal model systems, rabbit hemorrhagic fever virus, zika and dengue osu-03012 prolonged animal survival and significantly reduced the negative sequelae of virus infection. [159] [160] [161] subsequent studies using the fda approved cancer therapeutic drugs sorafenib and pazopanib also demonstrated that these fda approved drugs also have potent anti-viral properties. 154 thus, a project which began as development of an anticancer drug became a project developing broad spectrum anti-viral drugs. the role of an activating point mutant in the egf receptor was first demonstrated in non-small cell lung cancer (nsclc). [162] [163] [164] [165] subsequently, as patient tumors carrying the activated egfr were treated for prolonged periods with egfr inhibitors such as gefitinib, it became evident that drug resistance, when it eventually evolved, was mediated by the evolution of a second point mutation in the egfr. 163, [166] [167] [168] second and third generation egfr inhibitory drugs such as afatinib and osimertinib potently inhibit double mutant egfr and are in first-line clinical use. [169] [170] [171] at the time of these discoveries we had several research projects determining whether we could combine afatinib with other agents to kill nsclc cells. [172] [173] [174] as part of this work, we generated afatinib-resistant h1975 nsclc cells by treating tumors in mice until the tumor completely regressed and then had begun to regrow. h1975 cells already express a double mutant egfr, so we were expecting to discover novel evolutionary survival signals. initial characterization of the resistant cells demonstrated they had permanently up-regulated signaling by the receptors c-kit, c-met and erbb3 to survive during exposure to afatinib. additional characterization studies then delivered unexpected data; whilst afatinib-resistant h1975 cells were resistant to the irreversible erbb receptor inhibitor afatinib, they were not resistant to the irreversible erbb inhibitor neratinib. 175 furthermore, the ability of neratinib as a single agent or when combined with other drugs, including afatinib, was enhanced in the afatinib-resistant cells. 176 ostensibly, both drugs should mechanistically "do" exactly the same thing to a tumor cell. thus, by implication, in addition to erbb family receptors, neratinib had to have additional "targets" to cause killing in the resistant cells. two molecular modeling manuscripts had stated neratinib, in addition to inhibiting erbb family tyrosine kinases could also inhibit map4k and map3k serine/threonine kinases. 177, 178 in parallel to the studies described above, from our loading control data, we observed that neratinib but not afatinib, could rapidly reduce the protein expression of erbb family receptors in a wide variety of tumor cell types. 175, 179, 180 we also included negative controls in our studies; c-met and c-kit. to our surprise, neratinib also reduced c-met and c-kit levels, albeit in a delayed fashion. to down-regulate the egfr required a ubiquitination step whereas to down-regulate c-met did not. growth factor receptors localize in large quaternary structures in the plasma membrane and we hypothesized that if neratinib was reducing the levels of the egfr, c-met and c-kit, could it also reduce the levels of an important signal transducer on the inner leaflet of the plasma membrane: ras. in pancreatic cancer cells neratinib not only caused internalization and degradation of the egfr, it also caused the degradation of the key oncogenic driver in this disease, mutant k-ras. subsequently, in melanoma cells expressing a mutant n-ras, similar findings with neratinib were obtained. 179, 180 the convergence of the afatinib-resistance studies and the ras down-regulation studies was a project to define the roles of map4k and map3k enzymes in the biological actions of neratinib. 181 from the modeling studies, two potential neratinib targets were mst3 and mst4. this caught our interest because the dose-limiting sequela for neratinib is diarrhea, and mst3 and mst4 play important roles in regulating the integrity of the epithelial brush boarder in the gut. 182,183 because we did not know what effects would be observed, we agnostically examined the activities of multiple map4k enzymes, as well as associated chaperone/docking proteins following neratinib exposure. as map4k/map3k enzymes are expressed in carcinoma cells which express high levels of erbb family receptors as well as in blood cancer cells that express none or very low levels of that receptor family, we performed studies in both tumor cell types. regardless of erbb family receptor expression, neratinib reduced the expression of ras proteins and reduced tumor cell viability. 176 neratinib reduced the phosphorylation of mst1/2, mst3 and mst4 in carcinoma and blood cancer cells; this would a priori predict that phosphorylation of their downstream substrates such as lats1/2 or the cytoskeletal protein ezrin, would be reduced. 176, 181 as was a priori expected, the phosphorylation of ezrin was reduced. however, the phosphorylation of lats1/2 was enhanced, as were the downstream substrates of these enzymes, the co-transcription factors yap and taz. yap and taz are hippo pathway effectors and when phosphorylated leave the nucleus which is followed by degradation in the cytoplasm. 184, 185 as yap and taz cooperate with mutant k-ras to drive pancreatic cancer growth and metastasis, our data suggest that neratinib could be a useful drug to employ in the treatment of this disease. 186, 187 this data also suggests that inhibition of the mst "map4k" kinases probably caused a compensatory activation of another "map4k" kinase(s) which phosphorylated lats1/2. thus, the key take-home messages from this section are that without a full appreciation and understanding of all potential targets of a particular drug, its mechanisms of action cannot be properly understood. because neratinib inhibits map4k/map3k enzymes besides erbb family receptors and particularly her2/erbb2, very few pre-clinical studies were performed in cells that did not over-express her2/erbb2 and none in cells that express mutant ras proteins or in blood cancer cells. these findings emphasize that in developmental drug and therapeutics studies, a broad agnostic approach is essential so as not to miss potential unknown off targets. this is diametrically different to almost all cell biology research projects where intense focus on a particular pathway, or even a component of a pathway is a standard approach. similarly, studying the mechanisms of cell killing by a drug by their nature have to be conceptually broad because very frequently drug-induced killing is not "pure" with only one pathway to tumor cell death being engaged. the drug-induced killing mechanism, for example, could include death receptor signaling, mitochondrial dysfunction and autophagosome formation, all interacting in a contemporaneous fashion. again, this approach is diametrically different to almost all basic science cell biology research projects. developing a compound into a putative drug and eventually into an agent that can be tested in humans is a long process that generally costs in the region of $200-300 million dollars. to some extent, the high cost of all prescription drugs to the consumer is influenced by this math. the screening of millions of compounds may result in the discovery of a new agent with anti-cancer, antiviral or anti-bacterial properties. alternatively, compounds are screened against a specific target until molecules are defined that potently act to inhibit the target's biological activity. optimization of these compounds, either by computer aided design, or by traditional organic chemistry methods, results, hopefully, in a series of compounds all with a low nanomolar ic50 inhibitory activity. drug development companies will then determine which of the drugs has the greatest apparent bioactivity in a range of tumor cell lines, alongside determination of in-animal stability and bioactivity against tumors. these studies collectively will deliver one or two compounds that are considered worthy of further investigation and development. it is at this point where drug companies will often seek outside academic collaborators to assist in their drug development studies. the first thing the independent academic collaborator needs to know is what was the highest safe dose of the compounds used in prior mouse studies? and, ideally, if pharmacodynamic and pharmacokinetic studies were performed, what was the safest peak plasma concentration of the compound, termed the c max and often listed as ng/ml (which requires conversion into a molar value). thus, if the highest safe dose of a compound is 10 mg per kg of animal, with a plasma c max of 1 mm, then all preliminary in vitro cell-based investigative studies must use the compound at concentrations well below 1 mm. to further understand the biology of the compound, preliminary in vitro dose-response studies against tumor cells are most often performed on a log-scale, e.g. 1, 3, 10, 30, 100 and 300 nm. the first question the academic investigator should ask is, in their hands, does the dose-response effect on tumor cell growth/viability correspond to the claimed inhibitory ic50 of the compound against its purified specific target? i.e. if the protein target has an ic50 inhibition of 1 nm and an ic50 for growth inhibition and cell killing of 300 nm, it suggests the compound may be binding tightly to the serum in the culture media, resulting in a very low concentration of free "active" drug. on the other hand, if the target inhibition ic50 is 100 nm but the ic50 for growth arrest/killing is 3 nm, the data implies the compound may have additional unknown higher affinity targets in addition to its primary target which all collectively contribute to the biological efficacy of the agent. in this article we have discussed the fda approved drugs sorafenib and neratinib. sorafenib was originally developed to inhibit raf-1 and b-raf. prior to the discovery that raf-1 phosphorylated mek1/2, it was noted that the catalytic site of the raf-1 serine/ threonine kinase most closely resembled the active sites of src family non-receptor tyrosine kinases. 188 hence, it was no surprise that within a few years sorafenib was also shown to also inhibit class iii receptor tyrosine kinases, and investigators now considered the biology of drug to be as an "anti-angiogenic" agent rather than per se an inhibitor of raf-1. 189, 190 finally, sorafenib was shown to be an inhibitor within its physiological range of hsp90 and hsp70 chaperone proteins. 152, 154 similarly, neratinib was developed solely with the intention of inhibiting the receptor tyrosine kinase her2 (erbb2) as a putative therapeutic for her2 + breast cancer. 191 yet, within several years of neratinib entering the clinic, two groups demonstrated it could inhibit map4k and map3k serine/threonine kinases with low nanomolar ic50 values. 177, 178 so, if the compound under investigation is considered by a drug company to be a "specific" inhibitor of a particular protein kinase, regardless as to whether the agent is also fda approved, the in vitro studies the academic investigator should perform are an agnostic wide-ranging series of assessments, over a clinically-relevant drug dose-response range and over a time course. these studies will define, in your own hands, the changes in phosphorylation of the proposed target but also of multiple other cellular signaling pathways. this involves studying components of each specific pathway, e.g. the regulatory phosphorylation and total expression of erbb1, erbb2, erbb3, erbb4, raf-1, b-raf, mek1/2 and erk1/2, as well as of downstream nuclear transcription factors whose functions are controlled by each pathway, such as camp response element-binding protein (creb). such wide-ranging data-intense screening studies are difficult to perform using traditional sds page and western blotting approaches, and more advanced methods such as dot-blots or staining fixed cells in situ and measuring the intensity fluorescent staining, using validated antibodies, which permit a high-throughput approach, are required. an old phrase in science is: "the data is what it is." thus, if signaling from the primary target in one signaling pathway is only partially inhibited by the drug under examination, but signaling through an unrelated pathway is almost abolished, one would therefore tentatively conclude that the compound has an unknown target in a different signaling pathway. or, if at a low concentration of the drug no inhibition of the primary target is observed, but that this occurs alongside changes in the activities of other pathways, an effect which is also associated with significant levels of growth arrest and tumor cell death, one would conclude that the biological primary target is not the key functional target which regulates tumor cell biology. these examples for drug actions are binary, and in reality, the differential effects upon signaling and tumor cell biology of any drug are more subtle and nuanced. a different set of concepts come into play when rationally combining fda approved drugs to develop a novel anti-cancer therapeutic approach. first, the safe c max values for both agents in patients should be determined alongside their plasma half-lives, c min at 24 h values and serum binding properties. the c max/c min values alongside the drug's half-life should inform the researcher that, for example during a 24 h in vitro time course assay, a drug concentration considerably less than the c max but above the c min should be used to approximate for a physiologic treatment concentration. many clinically relevant drugs are stated to be 99% protein bound in 100% serum; most in vitro studies are performed using 10% (v/v) fetal calf serum. what is self-evident, however from extant data, is that for a drug such as sorafenib, with a safe c max of 13 mm and a stated 99% plasma protein binding, is that a free sorafenib concentration of 130 nm in vitro has a very modest impact on altering tumor cell biology, i.e. the partitioning on-off rate for drug association with plasma proteins and with tumor tissue must also be taken into consideration when deciding the most in vitro physiologic drug concentration. 192 thus, taking all of these parameters into account, studies in the author's laboratory, in vitro with cells in 10% (v/v) serum, and in an attempt to remain within the physiologic range, do not use sorafenib above 2 mm. an additional consideration for drug combination studies is to determine from the literature the doselimiting toxicities of each drug. regardless of excellent laboratory-based data, if the two drugs being combined both have doselimiting toxicities (dlts) in the same tissue, e.g. the gastrointestinal tract (gi), the likelihood that both agents can be safely and successfully combined in a patient is considerably reduced. studies to define hormonal signaling and intracellular signal transduction are almost 100 years old. although much essential biological information was gleaned from work performed in the 1920s to the late 1980s, it was only with the widespread use of more modern molecular biology approaches combined alongside classic biochemical approaches that the signal transduction landscape of the last 25 years evolved. today, essentially all of the building blocks of all signal transduction pathways are known. what is still under investigation are the complex protein-protein interactions which define nuanced signaling during growth, development, and various pathologies. small molecule therapeutic interventions have been and are being developed using ever more sophisticated technologies, of which some have shown considerable clinical utility. however, many of the newly developed "specific" targeted drugs have had little to no testing to fully define off-target effectors of their biology. it is very probable that this on-target/off-target issue for all drugs will never be fully resolved. hence, the step-wise approaches described in this article will still be required to fully understand the use and application of all new drugs. the samuri chemist, and his work on adrenalin observations with insulin on department of soldiers' civil re-establishment diabetics aqueous extracts of pancreas iii. some precipitation reactions of insulin the synthesis of a polysaccharide from glucose-1-phosphate in muscle extract biosynthesis of glycogen from uridine diphosphate glucose conversion of phosphorylase b to phosphorylase a in muscle extracts the interconversion of phosphorylase a and phosphorylase b from dog heart muscle the relationship of epinephrine and glucagon to liver phosphorylase. i. liver phosphorylase; preparation and properties the relationship of epinephrine and glucagon to liver phosphorylase. ii. enzymatic inactivation of liver phosphorylase the role of cyclic adenylic acid in hormone action comparative studies on glycogen phosphorylase. iii. the phosphorylated site in human muscle phosphorylase alpha the role of cyclic-3',5'-amp in responses to catecholamines and other hormones 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protein that regulates beta-adrenergic receptor function manifold roles of b-arrestins in gpcr signaling elucidated with sirna and crispr/cas9 the role of beta-arrestins in the termination and transduction of g-protein-coupled receptor signals insulin-stimulated tyrosine phosphorylation of the insulin receptor in detergent extracts of human placental membranes. comparison to epidermal growth factor-stimulated phosphorylation epidermal growth factor induces rapid tyrosine phosphorylation of proteins in a431 human tumor cells insulin stimulates tyrosine phosphorylation of the insulin receptor in a cell-free system identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in a-431 cell membranes phosphorylation of exogenous substrates by the insulin receptor-associated protein kinase insulin-signaling mechanisms. lessons from the old testament of glycogen metabolism and the new testament of molecular biology d-chiro-inositol glycans in insulin signaling and insulin resistance insulin and a putative insulin metabolic mediator fraction from liver and muscle stimulate p33 messenger ribonucleic acid accumulation by apparently different mechanisms insulin-mediated effect on the activity of udpg-glycogen transglucosylase of muscle molecular basis for the substrate specificity of protein kinase b; comparison with mapkap kinase-1 and p70 s6 kinase mechanism of activation of protein kinase b by insulin and igf-1 characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase b alpha inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase b specific binding of the akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation mechanism of activation and function of protein kinase b the molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle facts and new hopes on selective fgfr inhibitors in solid tumors membrane phosphorylation: a crucial role in the action of insulin, egf, and pp60src? isolation of an additional member of the fibroblast growth factor receptor family, fgfr-3 characterization of sites for tyrosine phosphorylation in the transforming protein of rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src) muscle proteins related to microtubule associated protein-2 are substrates for an insulin-stimulatable kinase rapid stimulation by insulin of a serine/threonine kinase in 3t3-l1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro insulin-stimulated map-2 kinase phosphorylates and activates ribosomal protein s6 kinase ii associated protein 2 kinases, mediate the phosphorylation of tyrosine hydroxylase at serine-31 in situ insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase 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ischemia/reperfusion induction of cyclooxygenase-2 by the activated mekk1 ! sek1/mkk4 ! p38 mitogen-activated protein kinase pathway activation of the ikappab alpha kinase complex by mekk1, a kinase of the jnk pathway identification of c-jun nh2-terminal protein kinase (jnk)-activating kinase 2 as an activator of jnk but not p38 the tao of mekk wiring diagrams of mapk regulation by mekk1, 2, and 3 association of sos ras exchange protein with grb2 is implicated in tyrosine kinase signal transduction and transformation bar-sagi, d. grb2 mediates the egf-dependent activation of guanine nucleotide exchange on ras guanine-nucleotide-releasing factor hsos1 binds to grb2 and links receptor tyrosine kinases to ras signalling the sh2 and sh3 domain-containing protein grb2 links receptor tyrosine kinases to ras signaling association of the shc and grb2/sem5 sh2-containing proteins is implicated in activation of the ras pathway by tyrosine kinases activation of (his)6-raf-1 in vitro by partially purified plasma membranes from v-ras-transformed and serum-stimulated fibroblasts regulation of raf-1 and raf-1 mutants by ras-dependent and ras-independent mechanisms in vitro complexes of ras.gtp with raf-1 and mitogen-activated protein kinase kinase association of mek1 with p21ras the mek kinases mekk4/ssk2p facilitate complexity in the stress signaling responses of diverse systems upstream regulators of the c-jun amino-terminal kinases? isolation of mek5 and differential expression of alternatively spliced forms the role of c-jun n-terminal kinase (jnk) in apoptosis induced by ultraviolet c and gamma radiation. duration of jnk activation may determine cell death and proliferation opposing effects of erk and jnk-p38 map kinases on apoptosis the ras/rac1/cdc42/sek/jnk/c-jun cascade is a key pathway by which agonists stimulate dna synthesis in primary cultures of rat hepatocytes prolonged activation of the mitogen-activated protein kinase pathway promotes dna synthesis in primary hepatocytes from p21cip-1/waf1-null mice, but not in hepatocytes from p16ink4a-null mice bile acid regulation of c/ ebpbeta, creb, and c-jun function, via the extracellular signal-regulated kinase and c-jun nh2-terminal kinase pathways, modulates the apoptotic response of hepatocytes ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo the endocytosis of epidermal growth factor in a431 cells: a ph of microenvironment and the dynamics of receptor complex dissociation recycling of epidermal growth factor-receptor complexes in a431 cells the role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor stress signaling and the shaping of the mammary tissue in development and cancer the role of p38 mapk pathway in p53 compromised state and telomere mediated dna damage response a systematic review of p53 regulation of oxidative stress in skeletal muscle activated forms of h-ras and k-ras differentially regulate membrane association of pi3k, pdk-1, and akt and the effect of therapeutic kinase inhibitors on cell survival role of pi3k/akt pathway in cancer: the framework of malignant behavior egfrviii: an oncogene with ambiguous role functions of lysosomes autophagy wins the 2016 nobel prize in physiology or medicine: breakthroughs in baker's yeast fuel advances in biomedical research aminopeptidase i of saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction isolation and characterization of autophagy-defective mutants of saccharomyces cerevisiae so many roads: the multifaceted regulation of autophagy induction mtor at the nexus of nutrition, growth, ageing and disease two key autophagy-related molecules and their roles in myocardial ischemia/reperfusion injury ampk: regulation of metabolic dynamics in the context of autophagy diverse signaling mechanisms of mtor complexes: mtorc1 and mtorc2 in forming a formidable relationship mtor as a central hub of nutrient signalling and cell growth distinct roles of mtor targets s6k1 and s6k2 in breast cancer allosteric regulation of amp-activated protein kinase by adenylate nucleotides and small-molecule drugs ampk and tor: the yin and yang of cellular nutrient sensing and growth control lkb1/ampk pathway and drug response in cancer: a therapeutic perspective controlling the master-upstream regulation of the tumor suppressor lkb1 multifaceted roles of atm in autophagy: from nonselective autophagy to selective autophagy role of the lkb1/ampk pathway in tumor invasion and metastasis of cancer cells dna-dependent protein kinase regulates lysosomal amp-dependent protein kinase activation and autophagy reactive nitrogen species regulate autophagy through atm-ampk-tsc2-mediated suppression of mtorc1 a review of ulk1-mediated autophagy in drug resistance of cancer recruitment and activation of the ulk1/atg1 kinase complex in selective autophagy canonical and noncanonical functions of ulk/atg1 the mammalian ulk1 complex and autophagy initiation phosphorylation of ulk1 by ampk is essential for mouse embryonic stem cell self-renewal and pluripotency guidelines for the use and interpretation of assays for monitoring autophagy evolution of tools and methods for monitoring autophagic flux in mammalian cells metastatic cells are preferentially vulnerable to lysosomal inhibition regulation of apoptosis by autophagy to enhance cancer therapy osu-03012 promotes caspase-independent but perk-, cathepsin b-, bid-, and aif-dependent killing of transformed cells mda-7/il-24-induced cell killing in malignant renal carcinoma cells occurs by a ceramide/cd95/perk-dependent mechanism caspase-, cathepsin-, and perk-dependent regulation of mda-7/il-24-induced cell killing in primary human glioma cells regulation of gst-mda-7 toxicity in human glioblastoma cells by erbb1, erk1/2, pi3k, and jnk1-3 pathway signaling vorinostat and sorafenib increase er stress, autophagy and apoptosis via ceramide-dependent cd95 and perk activation vorinostat and sorafenib increase cd95 activation in gastrointestinal tumor cells through a ca(2 +)-de novo ceramide-pp2a-reactive oxygen species-dependent signaling pathway apoptosis-inducing factor (aif) in physiology and disease: the tale of a repented natural born killer osu-03012 stimulates pkr-like endoplasmic reticulum-dependent increases in 70-kda heat shock protein expression, attenuating its lethal actions in transformed cells sorafenib: delivering a targeted drug to the right targets therapeutic developments osu-03012 suppresses grp78/bip expression that causes perk-dependent increases in tumor cell killing the role of cell signaling in the crosstalk between autophagy and apoptosis in the regulation of tumor cell survival in response to sorafenib and neratinib not the comfy chair! cancer drugs that act against multiple active sites signaling alterations caused by drugs and autophagy role of the unfolded protein response, grp78 and grp94 in organ homeostasis bip/hspa5/dna k is a universal therapeutic target for human disease osu-03012 and viagra treatment inhibits the activity of multiple chaperone proteins and disrupts the blood-brain barrier: implications for anti-cancer therapies multi-kinase inhibitors can associate with heat shock proteins through their nh 2 -termini by which they suppress chaperone function ar-12 inhibits multiple chaperones concomitant with stimulating autophagosome formation collectively preventing virus replication grp78/dna k is a target for nexavar/stivarga/votrient in the treatment of human malignancies, viral infections and bacterial diseases endoplasmic reticulum stress, and interferon responses master regulator of the unfolded protein response and crucial factor in flavivirus biology he, a. blockade of hsp70 by ver-155008 synergistically enhances bortezomib-induced cytotoxicity in multiple myeloma discovery of novel heat shock protein (hsp90) inhibitors based on luminespib with potent antitumor activity the celecoxib derivative kinase inhibitor ar-12 (osu-03012) inhibits zika virus via down-regulation of the pi3k/akt pathway and protects zika virus-infected a129 mice: a host-targeting treatment strategy ar-12 suppresses dengue virus replication by down-regulation of pi3k/akt and grp78 exploring the in vitro potential of celecoxib derivative ar-12 as an effective antiviral compound against four dengue virus serotypes egfr mutation and resistance of non-small-cell lung cancer to gefitinib irreversible inhibitors of the egf receptor may circumvent acquired resistance to gefitinib activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib gefitinib-sensitizing egfr mutations in lung cancer activate anti-apoptotic pathways egf receptor mutations in lung cancer: from humans to mice and maybe back to humans presence of epidermal growth factor receptor gene t790m mutation as a minor clone in non-small cell lung cancer epidermal growth factor receptor mutations in patients with non-small cell lung cancer third-generation epidermal growth factor receptor tyrosine kinase inhibitors for the treatment of non-small cell lung cancer afatinib in locally advanced/metastatic nsclc harboring common egfr mutations, after chemotherapy: a phase iv study. lung cancer manag observational study of sequential afatinib and osimertinib in egfr mutation-positive nsclc: patients treated with a 40-mg starting dose of afatinib multi-kinase inhibitors interact with sildenafil and erbb1/2/4 inhibitors to kill tumor cells in vitro and in vivo the afatinib resistance of in vivo generated h1975 lung cancer cell clones is mediated by src/erbb3/c-kit/c-met compensatory survival signaling rationally repurposing ruxolitinib (jakafi ( w )) as a solid tumor therapeutic hdac inhibitors enhance neratinib activity and when combined enhance the actions of an anti-pd-1 immunomodulatory antibody in vivo neratinib inhibits hippo/yap signaling, reduces mutant k-ras expression, and kills pancreatic and blood cancer cells comprehensive analysis of kinase inhibitor selectivity the target landscape of clinical kinase drugs exposure permanently reduces erbb1 and ras expression in 4t1 mammary tumors and enhances m1 macrophage infiltration the levels of mutant k-ras and mutant n-ras are rapidly reduced in a beclin1/atg5-dependent fashion by the irreversible erbb1/2/4 inhibitor neratinib neratinib degrades mst4 via autophagy that reduces membrane stiffness and is essential for the inactivation of pi3k, erk1/2, and yap/taz signaling risk of gastrointestinal complications in breast cancer patients treated with neratinib: a meta-analysis targeting neratinib-induced diarrhea with budesonide and colesevelam in a rat model yap/taz functions and their regulation at a glance yap/taz: drivers of tumor growth, metastasis, and resistance to therapy yap1 activation enables bypass of oncogenic kras addiction in pancreatic cancer downstream of mutant kras, the transcription regulator yap is essential for neoplastic progression to pancreatic ductal adenocarcinoma primary structure of v-raf: relatedness to the src family of oncogenes safety and pharmacokinetics of the dual action raf kinase and vascular endothelial growth factor receptor inhibitor, bay 43-9006, in patients with advanced, refractory solid tumors results of phase i pharmacokinetic and pharmacodynamic studies of the raf kinase inhibitor bay 43-9006 in patients with solid tumors neratinib: an oral, irreversible dual egfr/her2 inhibitor for breast and non-small cell lung cancer bay 43-9006: early clinical data in patients with advanced solid malignancies key: cord-332344-upsn0zb4 authors: jeswin, joseph; xie, xiao-lu; ji, qiao-lin; wang, ke-jian; liu, hai-peng title: proteomic analysis by itraq in red claw crayfish, cherax quadricarinatus, hematopoietic tissue cells post white spot syndrome virus infection date: 2016-02-01 journal: fish shellfish immunol doi: 10.1016/j.fsi.2016.01.035 sha: doc_id: 332344 cord_uid: upsn0zb4 to elucidate proteomic changes of hpt cells from red claw crayfish, cherax quadricarinatus, we have carried out isobaric tags for relative and absolute quantitation (itraq) of cellular proteins at both early (1 hpi) and late stage (12 hpi) post white spot syndrome virus (wssv) infection. protein database search revealed 594 protein hits by mascot, in which 17 and 30 proteins were present as differentially expressed proteins at early and late viral infection, respectively. generally, these differentially expressed proteins include: 1) the metabolic process related proteins in glycolysis and glucogenesis, dna replication, nucleotide/amino acid/fatty acid metabolism and protein biosynthesis; 2) the signal transduction related proteins like small gtpases, g-protein-alpha stimulatory subunit, proteins bearing pdzor 14-3-3-domains that help holding together and organize signaling complexes, casein kinase i and proteins of the map-kinase signal transduction pathway; 3) the immune defense related proteins such as α-2 macroglobulin, transglutaminase and trans-activation response rna-binding protein 1. taken together, these protein information shed new light on the host cellular response against wssv infection in a crustacean cell culture. white spot syndrome virus (wssv) is an enveloped dna virus with a wide host range in crustaceans such as shrimp, crab and crayfish [1, 2] . in shrimp aquaculture wssv infection will result in collapse of the whole shrimp stock within 3e10 days [3] . after the emergence of the wssv from 1990s, researchers have been focusing on the virus and the immune response of the host. for example, quantitative determination of the mrna pool in infected cells or animals versa controls has been widely used [4, 5] . with the virus characterized and some knowledge regarding the immune response of the host accumulated [6] , a comprehensive approach to investigate how the host physiological system responds to the viral invasion is the important next step to develop efficient control of the infection. proteomics is a means of comprehensive interpretation used to describe more direct molecular responses than conventional studies assessing the messenger rna [7] . hence, researchers employed proteomic approaches such as twodimensional electrophoresis for studying host protein changes during wssv proliferation [8, 9] . recently, itraq combined with liquid chromatography-tandem mass spectrometry is used to directly quantify and compare the protein expression levels of samples with great efficiency and accuracy [10] . such a global approach helps to correlate different host proteins participating in biological processes, cellular components and molecular function in response to a pathogenic infection. as one of the principle targets by wssv, crayfish hpt cells have been set up as a good model for investigation of wssv infection [11] . intriguingly, the crayfish hpt cells were lately proved by wu et al. [12] to be a better cell model for wssv replication than hemocytes which could not afford the virus for proper replication and viral assemble. previously, we have investigated the transcript alteration of hpt cells from red claw crayfish, cherax quadricarinatus, towards wssv challenge using subtractive library screening. we noticed that numerous genes, with differential expression between wssv infection and mocked infection, were present with annotated functions in immune defense, cytoskeletal organization, signal transduction, stress, metabolism and homeostasis [13] . to further identify proteins or pathways altered during viral infection, here we report proteomic responses of crayfish hpt cells by itraq at both early (1 hpi) and late (12 hpi) stages post wssv infection accordingly. these differentially expressed proteins provide us with better understanding of the host's coordinated effort to defend the wssv infection. adult healthy red claw crayfish were collected from crayfish farms in zhangpu, fujian, china. the crayfish were acclimatized for one week at least in well-aerated tanks and only intermolting animals were sacrificed for hpt cells preparation as described by s€ oderh€ all et al. [14] . the isolated hpt cells were cultured in six well plates with 85e90% confluence using l-15 medium containing 5 mm 2-mercaptoethanol, 1 mm phenylthiourea, 2 mm l-glutamine and a crude astakine preparation [15] . the cells were then inoculated, 1 hpi as early infection stage and 12 hpi as late infection stage accordingly, with 10 5 copies of wssv (chinese isolate, wssv-cnaf332093) kindly provided by prof. xun xu from the third institute of oceanography, state oceanic administration, china. uv-inactivated wssv was used as mocked infection. the cells in duplicates above were harvested by scraping in culture medium with a rubber policeman and collected by centrifugation at 4200 rpm for 5 min at 4 c. the cell pellets were then lysed with ripa lysis buffer (thermo scientific) and the supernatant was collected after centrifugation followed by protein purification using a 2-d clean-up kit (bio-rad), and protein concentration was determined by lowry method using the rc dc protein assay kit (bio-rad). for itraq assays, two independent samples were pooled and each pool was divided into duplicates. total protein (100 mg) of each sample solution as prepared above was digested with trypsin gold (promega) at a ratio of protein:trypsin ¼ 30:1 at 37 c for 16 h. thereafter, peptides were dried by vacuum centrifugation and reconstituted in 0.5 m triethyl ammonium bicarbonate and processed according to the manufacturer's protocol for 8-plex itraq reagent (applied biosystems). two technical replicates were prepared for itraq labeling as suggested by the manufactory instructions. the proteins from the infected (1 hpi labeled with 114/ 118; 12 hpi labeled with 116/121) and mock-infected samples (1 hpi labeled with 113/117; 12 hpi labeled with 115/119) were labeled with itraq tags (fig. s1 ). the tags employed n-hydroxysuccinimide chemistry which consists of three functional groups: an amine-reactive group and an isotopic reporter group (n-methylpiperazine) linked by an isotopic balancer group (carbonyl) for the normalization of the total mass of the tags. the labeled peptide mixtures were pooled and dried by vacuum centrifugation and then reconstituted in buffer a (25 mm nah 2 po 4 in 25% acetonitrile, ph 2.7) followed by separation on a strong cation exchange chromatography column (4.6 â 250 mm, 5 mm, ultremex scx, phenomenex, usa). the peptides were next treated with an isocratic elution at a flow rate of 1 ml/min with buffer a for 10 min followed by a gradient from 5 to 60% of buffer b (25 mm nah 2 po 4 , 1 m kcl in 25% acetonitrile, ph 2.7) for 27 min, and then a gradient elution from 60 to 100% of buffer b for 1 min. the system was then maintained at 100% buffer b for 1 min before equilibrating with buffer a for 10 min prior to the next injection. fractions were collected every 1 min and the eluted peptides were pooled into 20 fractions followed by desalting on a strata x c18 column (phenomenex) and vacuum-dried. each fraction was resuspended in buffer a (5% acetonitrile, 0.1% formic acid) and centrifuged at 20,000 g for 10 min, and the final concentration of peptide was about 0.5 g/l on average. ten microlitre of supernatant was loaded on a lc-20ad nanohplc (shimadzu, japan) by the autosampler onto a 2 cm c18 trap column. then, the peptides were eluted onto a 10 cm analytical c18 column packed in-house. the samples were loaded at 8 ml/min for 4 min, then the 35 min gradient was run at 300 nl/min starting from 2 to 35% buffer d (95% acetonitrile, 0.1% formic acid), followed by 5 min linear gradient to 60% and by 2 min linear gradient to 80%. after that, the samples were maintained at 80% buffer d for 4 min, and finally return to 5% in 1 min. data acquisition was performed with a triple tof 5600 system (ab sciex) fitted with a nanospray iii source and a pulled quartz tip as the emitter (new objectives, ma). data was acquired using an ion spray voltage of 2.5 kv, curtain gas of 30 psi, nebulizer gas of 15 psi, and an interface heater temperature of 150 c. the ms was operated with a rp of greater than or equal to 30,000 fwhm for tof ms scan. for ida, survey scans were acquired in 250 ms and as many as 30 product ion scans were collected if they exceeded a threshold of 120 counts per second. total cycle time was fixed at 3.3 s. the q2 transmission window was 100 da for 100%. four time bins were summed for each scan at a pulser frequency value of 11 khz through monitoring of the 40 ghz multichannel tdc detector with four anode channel detect ion. a sweeping collision energy setting of 35 ± 5 ev, coupled with itraq adjust rolling collision energy, was applied to all precursor ions for collision induced dissociation. dynamic exclusion was set for half of peak width (15 s) and then the precursor was refreshed off the exclusion list. peptide sequence data were processed using mascot software version 2.3.02 to obtain the information of associated proteins. a false discovery rate of less than 1% was used as cut off. the proteins were further classified using gene ontology (go) and pathway enrichment analysis (http://www.geneontology.org). to determine the alteration of protein expression induced by wssv infection, protein expression fold changes were calculated relatively in wssv infected hpt cells compared to those of mock-infected cells. student's t-test was used to identify statistically significant changes in protein expression (p < 0.05). the quantitative proteins ratios of !1.3 were considered as up-regulation and proteins with a label ratio of 0.77 [16, 17] were considered as down-regulated proteins in wssv infected hpt cells versus mock-infected cells. rna isolation was performed by genelute mammalian total rna isolation kit (sigma-aldrich) according to manufacturers protocol. briefly, reverse transcription was done with 1 mg of total rna using superscript ii reverse transcriptase (invitrogen) according to instructions of manufacturer. real-time pcr was performed on abi 7500 using faststart universal sybr master (roche) and the primers used were included in supplementary information (tabel s2). the expression level of mrna was normalized by 16s rrna. the data was presented as relative expression levels (means ± sd, n ¼ 3), and subjected to one-way analysis of variance (anova) followed by fisher's least significant difference test. the significant difference between wssv treated and control (uv treated wssv) samples was represented by one asterisk for p < 0.05. to get insight into the proteins differentially expressed at both early stage (1 hpi) and late stage (12 hpi) of wssv infection as recent publications reported that these two time points have been clearly defined accordingly of wssv replication in crayfish hpt cells [12, 13] , itraq analysis was employed for quantification followed by protein profiling, which resulted in 594 protein hits in mascot. the functional enrichment analysis of go annotations for biological processes showed that the identified proteins were classified into 23 categories (fig. 1) , in which proteins related to cellular process (17.14%) and metabolic process (15.41%) were the most abundant molecules followed by cellular component organization biogenesis (6.43%), biological regulation (6.37%), regulation of biological process (5.85%), developmental process (5.44%), response to stimulus (4.90%), signaling transduction (2.95%), negative regulation of biological process (2.23%) and others. apparently, the protein expression in hpt cells showed variation at both early and late stages of infection if compared to those of mock-infected cells (tables 1e3). among these proteins, alpha aminoadipic semialdehyde synthase, ribonucleoside-diphosphate reductase and thymidylate synthase in metabolism; casein kinase i isoform alpha, camp-dependent protein kinase, pdz domain containing protein gipc1 and rab-1 involved in signal transduction were found for the first time to be involved in the host response to wssv infection. furthermore, the cytoskeletal associated proteins like calponin-3 and beta chain spectrin were also found to be significantly expressed towards wssv infection. here we focus on the brief interpretation of diverse functional classes related to metabolic processes, signal transduction proteins and immune related proteins, which are putatively key molecules involved in metabolism and host defense against cellular stress caused by wssv infection. in addition, numerous proteins were expressed in wssv infected cells but without significant difference in up-or down-regulation when compared to a mock infection (table s1 ). viruses require host cellular metabolism for their energy needs and successful replication. wssv infection perturbs the expression of at least 14 enzymes action in glycolysis, glucogenesis, the pentose phosphate pathway, metabolism of nucleotide, amino acid and fatty acid, dna replication and protein biosynthesis. an increase in activity of metabolic pathways associated with glycolysis, the pentose phosphate pathway, nucleotide biosynthesis, glutaminolysis and amino acid biosynthesis were observed in shrimp hemocytes post wssv challenge [18] . in our present study, phosphoglycerate mutase 2 with a role in glycolysis was downregulated at both 1 hpi and 12 hpi post infection. further studies with glucose starved media should be done to ascertain its role in wssv replication and possible therapeutic applications. the pentose phosphate pathway fulfills two essential functions, the formation of pentose phosphate for synthesis of nucleotides, rna, dna and the generation of nadph for biosynthetic reactions to maintain glutathione in the reduced state and protecting the cell from damaging reactive oxygen intermediates. glucose-6phosphate dehydrogenase, the key enzyme of pentose phosphate pathway showed increased activity in wssv infected litopenaeus vannamei [19] . interestingly, in our study the key enzyme of the oxidative pentose phosphate pathway, 6-phosphogluconate dehydrogenase, was up-regulated at 12 hpi whereas in penaeus monodon it was down-regulated during yhv infection [20] . this difference may reflect alternative energy utilization of these two viruses. viruses use several strategies to ensure adequate supply of nucleotide by expressing virus encoded enzymes that boost nucleotide synthesis and increasing the flux of pentose phosphate pathway [21à23]. wssv-infection perturbs the nucleotide biosynthesis, particularly on nucleoside and nucleotide metabolic enzymes involved in producing monomer intermediates for dna synthesis such as ribonucleoside-diphosphate reductase, thymidylate synthase and gmp synthase. the first two enzymes were down-regulated at 1 hpi whereas gmp synthase, an atp-dependent enzyme involved in purine nucleotide synthesis, and thymidylate synthase was over-expressed at 12 hpi(in comparison to its expression at 1 hpi with live virus infection, table 3 ). a similar increase in nucleotide biosynthesis was observed during wssv infection in l. vannamei [18] . the relative increase of nucleosides at 12 hpi when compared to 1 hpi implies that the dna replication machinery might be sequestered to produce viral dna. recently pharmaceutical compounds are proved to inhibit rna, dna and retroviruses by targeting pyrimidine biosynthesis [24] . broadspectrum antivirals targeting nucleotide biosynthesis could be further tested in vitro such as in the hpt cell cultures. antiviral drugs targeting nucleotide biosynthesis is commonly used against human viruses and this might be extended to wssv. differential expression of enzymes associated with amino acid metabolism was observed in our present study. alpha aminoadipic semialdehyde synthase, involved in the lysine degradation pathway was downregulated at 1 hpi while increased to control levels at 12 hpi. adenosylhomocysteinase, a key enzyme in the regulation of the intracellular concentration of s-adenosylhomocysteine (sah) was up-regulated at 12 hpi (table 3) . sah is an intermediate in the synthesis of cysteine and adenosine. an increased intracellular concentration of sah inhibited vaccinia virus replication in murine cells [25] . aminoacyl-trna synthetases (arss) are highly conserved for efficient and precise translation of genetic codes. evidence is accumulating that several arss form a macromolecular protein complex that would work as a molecular hub linked to the multiple signaling pathways [26] . the up-regulation of bifunctional aminoacyl-trna synthetase at 12 hpi (table 3) implies that protein synthesis might be elevated during later stages of the wssv infection. recent studies in crayfish found that there was a decrease in long chain fatty acids during wssv infection at 12 hpi and later it increased at 24 hpi [27] . the knowledge of virus induced changes in cell metabolome may lead to potential use of inhibitors of metabolic enzymes to treat wssv infection. in total eleven proteins related to signal transduction were differentially expressed in hpt cells upon wssv-infection. three of those are gtp-binding proteins (rab-1, ras opposite, gtp-alpha g s ). gtp binding proteins regulate a wide variety of cell functions acting as biological timers, for instance, that initiate and terminate specific cell functions. in addition they play key roles in determining spatial locations of specific cell functions. rab-1 regulates protein transport from endoplasmic reticulum to golgi apparatus. rab gtpase is essential for the control of intracellular membrane trafficking in all eukaryotic cells and further affects the ability of phagocytic cells to scavenge pathogen. this is in synchrony with the observed upregulation of rab-1 protein during 1 hpi of the current study. in penaeus monodon, rab 7 was found to bind to rvp28 of wssv. the in vivo neutralization assay of wssv infected shrimp in the presence of anti-rab7 antibody revealed less mortality compared to that of wssv alone [28] . depletion of rab-1 significantly decreased the replication of hepatitis c virus in a human hepatoma cell line [29] . the upregulation of rab-1 suggests its role in wssv pathogenesis. g-protein alpha stimulatory subunit (ga s ) is activated by g-protein coupled receptors located in the cell membrane. ga s stimulates adenylate cyclase to produce camp [30] a ubiquitous second messenger that enables cells to detect and respond to extracellular signals [31] . the intracellular camp-level affects the camp-dependent protein kinase (pka) and consequently many metabolic processes of the cell. in our present study wssvinfection caused over expression of ga s at 12 hpi. more in depth investigation is needed to understand its role during a late infection stage of wssv infection. adapter molecules or domains such as gipc1 and 14-3-3 are involved in protein-protein interaction. in the current study the strongest response was the expression of gipc1, a scaffolding protein that regulates cell surface receptor expression and trafficking. viruses usually use carboxyl-terminal pdz domain-binding motif (pbm) that mediates interactions with cellular pdz proteins. gipc1 contains such a pdz domain, a protein-protein interaction module typically found in cytoplasmic and membrane adapter proteins that are involved in a variety of cellular processes of significance to viral infection, for instance maintenance of cell-cell junctions, cellular polarity, and signal transduction pathways [32] . gipc1 was in control level at first hour after viral challenge and later upregulated at 12 hpi, implying that the gipc1 might have modulated the cellular process in favor of wssv replication and dissemination in the cells. to reveal the role of gipc1 during wssv infection, protein-protein interaction studies should be undertaken in the future. 14-3-3 epsilon protein is known for its ability to bind multiple cellular protein ligands. it interacts with a wide range of proteins for functions like cell cycle control, regulation of cell death and apoptosis etc. in addition 14-3-3 epsilon can interact with a diverse array of viral products, and thereby re-direct cellular processes [33à35]. for example, hepatitis c virus core protein can interact with 14-3-3 epsilon protein [33] . also severe acute respiratory syndrome corona virus nucleocapsid protein was phosphorylated and localized in the cytoplasm by 14-3-3-mediated translocation [36] . in our study 14-3-3 epsilon was at control levels at 1 hpi and up-regulated at 12 hpi and therefore indicative of an established viral infection. further studies are therefore needed to elucidate the interaction of 14-3-3 with viral proteins. there was a trend of down regulation observed in proteins that assemble the proteasome (proteasome subunit alpha type, 26s proteasome regulatory subunit rpn2). the main function of proteasome is to degrade unneeded or damaged proteins. there is a high stoichiometric imbalance of host target proteins over those encoded by the virus. to overcome this, many viruses have evolved mechanisms by which their cellular target proteins are directed to the 26s proteasome and subjected to proteolytic degradation [37] . recently a plethora of viral proteins have been shown to direct host-cell proteins for proteolytic degradation. these activities are required for various aspects of viral life cycle like viral entry [38] , replication [39] , enhanced survival [40, 41] , and to viral release [42] . similar to our study, infection of enterovirus 61 in rhabdomyosarcoma cells resulted in down-regulation of proteasome subunit alpha type 2 [43] . the cellular signal transduction is largely controlled by protein phosphorylation. several protein kinases were differentially expressed during wssv infection such as protein casein kinase i (ck1) isoform alpha, a serine/threonine kinase, which was upregulated at 12 hpi. it inhibits hyperphosphorylation of nonstructural protein 5a of hepatitis c virus. the nonstructural (ns) protein 5a is a phosphoprotein and experiments indicated that the phosphorylation state of ns5a is important for the outcome of viral rna replication [44] . similarly, in our study the up-regulation of ck1 might be indicative of its interaction with wssv proteins. wssv envelope proteins like vp28 and vp19 were previously reported to be threonine phosphorylated [45] . further investigations are needed to understand the role of casein kinase i isoform alpha. camp-dependent protein kinase (pka) is involved in the regulation of glycogen, sugars and lipid metabolism. its down-regulation at 12 hpi (table 3 ) might affect the production of essential metabolites during infection. activation of camp can block apoptosis induced by herpes simplex virus 1 associated genes [46] . as the wssv infection is nearly established at 12 hpi, wssv might suppress camp activation for promoting apoptosis in infected cells, spreading viral infection to nearby cells. the mitogen-activated protein kinase (mapk) cascade pathways have global role in the intracellular signaling network pathway. mitogen-activated protein kinase kinase (map2k/mek) phosphorylates mitogen-activated protein kinase (mapk/erk) are part of the protein kinase cascade starting with an extracellular stimulant ending with the expression of transcription factors in the nucleus. it is thought that a viral infection acts as an extracellular stimulant that activates this pathway. rna silencing of p38 mapk homologue ivp38 caused increase in wssv replication on l. vannamei [47] . in our study mapk was in control levels at 1 hpi but down-regulated at 12 hpi while map2k down-regulated at initial period and up-regulated at latter time point (table 3 ). the significant expression of mapk and map2k should be further investigated. among these proteins, nine immune related proteins were differentially expressed during this period of infection within 12 hpi. two of these proteins, a2-macroglobulin and transglutaminase, are well known to play a role in coagulation of hemolymph during pathogen infection. for instance, a2macroglobulin is considered as a broad range proteinase inhibitor involved in phagocytosis [48, 49] . silencing of a2-macroglobulin clearly reduced phagocytosis of chryseobacterium indologenes by hemocytes both in vitro and in vivo in hard tick ixodes ricinus [50] . according to the itraq quantification, no change in protein expression was found for a2-macroglobulin at 1 hpi but its expression was clearly up-regulated at 12 hpi, suggesting that a2macroglobulin might have a role during late infection hours of wssv in crayfish hpt cells. transglutaminase has a regulatory function in antimicrobial peptide production in kuruma shrimp. silencing of transglutaminase caused the down regulation of antimicrobial peptides such as crustin and lysozyme, thus, negatively affecting on the host's defense against pathogen infection [51] . additionally, it has been documented that in a freshwater crayfish transglutaminase prevents hematopoietic stem cells from migrating into the hemolymph [52] . as hemocytes are particularly important for innate immune defense in invertebrates, this prevention on release of matured hpt cells from the hematopietic tissue is likely to have an impact on host immunity. in our present study transglutaminase expression was down-regulated at 1 hpi if compared to those of mock-infection, implying that transglutaminase might act a critical role during virus infection. further functional studies on transglutaminase against wssv infection in red claw crayfish both in vitro and in vivo will benefit the elucidation of an important role of this enzyme. sp€ aetzle is the well-known component of toll signaling pathway, which plays a key role in regulating innate immune responses in invertebrates. gram positive bacteria, fungi and certain viruses can activate toll signaling pathway initiating an intracellular signaling cascade to promote the expression of immunerelated genes such as antimicrobial peptides through the activation of the nf-kb family-dorsal proteins [53] . it has been shown that key components of the toll pathway, like toll [54] and dorsal [55] , are activated during viral infection in shrimp. by recognition of high mannose residues expressed on the surface of viruses or virus surface glycoproteins, toll like receptors are able to mediate virus entry into host cells, in which binding of ligand sp€ aetzle to toll is necessary to initiate this pathway [56] . recently, transcriptomic changes of sp€ aetzle was also reported during wssv and vibrio infection in fenneropenaeus chinensis [57] . protein sp€ aetzle was upregulated in crayfish hpt cells at 12 h post wssv infection (table 3) . recent studies in crayfish hpt shows that by 12 hpi progeny wssv virions will be successfully generated [12] . the presence of progeny virions at this stage might be the reason for up regulation of protein sp€ aetzle. further functional studies are needed to understand the late expression of sp€ aetzle during wssv infection. antioxidant enzymes regulate the concentrations of radical oxygen species, for instance, glutathione peroxidase detoxifies lipids and hydrogen peroxidase while glutathione-s-transferase marks xenobiotics for cellular degradation. this antioxidant activity usually is increased upon pathogenic infection. we found that glutathione peroxidase and glutathione-s-transferase were obviously affected by wssv infection in crayfish hpt cells, in which the former expression was promoted at 1 hpi and the latter was enhanced at 12 hpi. an increased glutathione peroxidase activity at early hours and an up-regulated glutathione-s-transferase activity at late hours were also found in l. vannamei post wssv infection [58] . increased oxidative damage will occur in response to the challenge when rate of free radical formation exceeds its removal by antioxidant system [58] . faulty antioxidant defense causes the failure of sensitive cellular and subcellular components leading to cell death [59] . these findings clearly indicated that antioxidant response was modulated during wssv infection in crustaceans [58] . invertebrates and plants are capable of suppressing viral infection through rna silencing mediated by risc. as a key component of risc, trans-activation response rna-binding protein (trbp) was found to be associated with dicer and argonautes, both of which are involved in rna interference and microrna pathways [60] . combination of trbp with eukaryotic initiation factor 6 leads to an antiviral rna interference pathway in shrimp marsupenaeus japonicus [61] . knockdown of a shrimp tudor staphylococcal nuclease (pmtsn), one of the risc associated protein involved in post transcriptional regulation, resulted in reduced inhibition of yhv infection mediated by rnai of yhv in penaeus monodon [62] . in our studies we found that tar rna-binding protein isoform 1 and tsn, both involved in risc, were down-regulated at 12 hpi. this implied that wssv-infection might negatively affect the immune defense against this viral infection via rnai process, but how this effect is elicited and modulated during wssv replication needs further exploration. to validate the differentially expressed proteins identified by itraq, a randomly selected group of proteins transcriptional level was confirmed by quantitative real-time pcr. as shown in fig. 2 , the gene expression levels were in agreement to our itraq results. among these tested genes, tudor staphylococcal nuclease and thymidylate synthase involved in metabolic process group showed similar gene expression trend to our proteomics data. briefly, tudor staphylococcal nuclease showed a significant decrease at 12 hpi, while thymidylate synthase was significantly down-regulated at 1 hpi but up-regulated at 12 hpi. in regarding to the signal transduction group, the transcriptional level of mapkk, mapk-14, 14-3-3 epsilon and ras opposite were analyzed. mapkk was significantly decreased at 1 hpi but increased at late infection stage, and mapk-14 was clearly down-regulated at late hours of infection. ras opposite showed a decrease on initial hours while 14-3-3 epsilon was obviously enhanced at late hours of infection. in case of immune-related and cytoskeletal group, both transglutaminase and calponin-3 showed significant decline at initials hours post wssv infection, which were in well consistent with our itraq result. the wssv infection in the crayfish hpt cells obviously altered the host cellular expression of proteins related to metabolic processes, signal transduction, immunity and cytoskeletal organization. wssv utilized host metabolic system for its energy needs, replication and dissemination inside the host cells. the virus managed to alter important signal transduction proteins those function as gtp binding, adapter molecules, protein biosynthesis and protein phosphorylation. the protein expression in early hours exposes vulnerability of host defense mechanism to the viral infection. the role of many of the identified proteins in viral infection remains unknown, some of those are first observations and will require systematic studies to show the mechanism of action. by identifying a series of proteins that are affected by viral infection this study opens new opportunities in two directions: 1) to better understand host-virus interactions and 2) to select targets to reduce vulnerability of the host toward the challenge. the proteins identified and host physiological changes elucidated can be further utilized for developing putative drug targets for controlling this virus. protective immunity induced by oral immunization with a rotavirus dna vaccine encapsulated in microparticles white spot syndrome baculovirus (wsbv) detected in cultured and captured shrimp, crabs and other arthropods a handbook of shrimp pathology and diagnostic procedures for disease of cultured penaeid shrimp discovery of immune molecules and their crucial functions in shrimp immunity shrimp molecular responses to viral pathogens antiviral immunity in crustaceans proteome and proteomics: new technologies, new concepts, and new words comparative proteomic profiles of the hepatopancreas in fenneropenaeus chinensis response to white spot syndrome virus protein profiling in the gut of penaeus monodon gavaged with oral wssv-vaccines and live white spot syndrome virus an insight into itraq: where do we stand now? s€ oderh€ all, characterization of white spot syndrome virus replication in in vitro-cultured haematopoietic stem cells of freshwater crayfish, pacifastacus leniusculus crayfish hematopoietic tissue cells but not hemocytes are permissive for white spot syndrome virus replication differential gene expression profile from haematopoietic tissue stem cells of red claw crayfish, cherax quadricarinatus, in response to wssv infection hemocyte production and maturation in an invertebrate animal; proliferation and gene expression in hematopoietic stem cells of pacifastacus leniusculus an ancient role for a prokineticin domain in invertebrate hematopoiesis simultaneous analysis of relative protein expression levels across multiple samples using itraq isobaric tags with 2d nano lc-ms/ms sub-proteomic fractionation, itraq, and offgel-lc-ms/ms approaches to cardiac proteomics an invertebrate warburg effect: a shrimp virus achieves successful replication by altering the host metabolome via the pi3k-akt-mtor pathway white spot syndrome virus induces metabolic changes resembling the warburg effect in shrimp hemocytes in the early stage of infection proteomic analysis of altered proteins in lymphoid organ of yellow head virus infected penaeus monodon systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy metabolic effects of influenza virus infection in cultured animal cells: intra-and extracellular metabolite profiling role of viral ribonucleotide reductase in the increase of dttp pool size in herpes simplex virus-infected vero cells broadspectrum antiviral that interferes with de novo pyrimidine biosynthesis relationship between intracellular concentration of s-adenosylhomocysteine and inhibition of vaccinia virus replication and inhibition of murine l-929 cell growth aminoacyl-trna synthetase-interacting multifunctional proteins (aimps): a triad for cellular homeostasis to complete its replication cycle, a shrimp virus changes the population of long chain fatty acids during infection via q4 the pi3k-akt-mtor-hif1a pathway pmrab7 is a vp28-binding protein involved in white spot syndrome virus infection in shrimp tbc1d20 is a rab1 gtpase-activating protein that mediates hepatitis c virus replication crystal structure of the catalytic domains of adenylyl cyclase in a complex with gs adenylyl cyclase functions downstream of the galpha protein gpa1 and controls mating and pathogenicity of cryptococcus neoformans emerging theme: cellular pdz proteins as common targets of pathogenic viruses hepatitis c virus core protein interacts with 14-3-3 protein and activates the kinase raf-1 rep68 protein of adeno-associated virus type 2 interacts with 14-3-3 proteins depending on phosphorylation at serine 535 parainfluenza virus 5 m protein interaction with host protein 14-3-3 negatively affects virus particle formation the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation viruses and the 26s proteasome: hacking into destruction adenovirus protein involved in virus internalisation recruits ubiquitin-protein ligases herpes simplex virus type 1 immediate-early protein vmw110 induces the proteasome-dependent degradation of the catalytic subunit of dna-dependent protein kinase the role of the e6-p53 interaction in the molecular pathogenesis of hpv a novel class of herpesvirus-encoded membrane-bound e3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction transcriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection hepatitis c virus ns5a is a direct substrate of casein kinase i-alpha, a cellular kinase identified by inhibitor affinity chromatography using specific ns5a hyperphosphorylation inhibitors proteomic analysis of the major envelope and nucleocapsid proteins of white spot syndrome virus herpes simplex virus protein kinase us3 activates and functionally overlaps protein kinase a to block apoptosis the role of litopenaeus vannamei p38 in white spot syndrome virus infection humoral immunity in long-lived arthropods conserved role of a complement-like protein in phagocytosis revealed by dsrna knockout in cultured cells of the mosquito, anopheles gambiae iram-an a2-macroglobulin from the hard tick ixodes ricinus: characterization and function in phagocytosis of a potential pathogen chryseobacterium indologenes transglutaminase regulates immune-related genes in shrimp s€ oderh€ all, i. s€ oderh€ all, transglutaminase activity in the hematopoietic tissue of a crustacean, pacifastacus leniusculus, importance in hemocyte homeostasis the host defense of drosophila melanogaster molecular cloning, characterization and expression analysis of two novel tolls (lvtoll2 and lvtoll3) and three putative spatzle-like toll ligands (lvspz1-3) from litopenaeus vannamei a dorsal homolog (fcdorsal) in the chinese shrimp fenneropenaeus chinensis is responsive to both bacteria and wssv challenge toll-like receptor 4-mediated activation of p38 mitogen-activated protein kinase is a determinant of respiratory virus entry and tropism identification and molecular characterization of a spaetzle-like protein from chinese shrimp (fenneropenaeus chinensis) magall on-barajas, antioxidant enzyme activity in pacific whiteleg shrimp (litopenaeus vannamei) in response to infection with white spot syndrome virus a selenium-dependent glutathione peroxidase (se-gpx) and two glutathione s-transferases (gsts) from chinese shrimp (fenneropenaeus chinensis) trbp, a regulator of cellular pkr and hiv-1 virus expression, interacts with dicer and functions in rna silencing trbp and eif6 homologue in marsupenaeus japonicus play crucial roles in antiviral response tudor staphylococcal nuclease from penaeus monodon: cdna cloning and its involvement in rna interference supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2016.01.035. key: cord-322926-xlwsj3v2 authors: shanmugaraj, balamurugan; i. bulaon, christine joy; phoolcharoen, waranyoo title: plant molecular farming: a viable platform for recombinant biopharmaceutical production date: 2020-07-04 journal: plants (basel) doi: 10.3390/plants9070842 sha: doc_id: 322926 cord_uid: xlwsj3v2 the demand for recombinant proteins in terms of quality, quantity, and diversity is increasing steadily, which is attracting global attention for the development of new recombinant protein production technologies and the engineering of conventional established expression systems based on bacteria or mammalian cell cultures. since the advancements of plant genetic engineering in the 1980s, plants have been used for the production of economically valuable, biologically active non-native proteins or biopharmaceuticals, the concept termed as plant molecular farming (pmf). pmf is considered as a cost-effective technology that has grown and advanced tremendously over the past two decades. the development and improvement of the transient expression system has significantly reduced the protein production timeline and greatly improved the protein yield in plants. the major factors that drive the plant-based platform towards potential competitors for the conventional expression system are cost-effectiveness, scalability, flexibility, versatility, and robustness of the system. many biopharmaceuticals including recombinant vaccine antigens, monoclonal antibodies, and other commercially viable proteins are produced in plants, some of which are in the pre-clinical and clinical pipeline. in this review, we consider the importance of a plantbased production system for recombinant protein production, and its potential to produce biopharmaceuticals is discussed. recombinant proteins are complex exogenous ("foreign") proteins that are produced in expression hosts, and mainly used as medical diagnostic reagents and in human healthcare as vaccines, drugs, or monoclonal antibodies [1] . the prominent role and increasing market demand for high-value recombinant proteins in novel drug discovery creates an opportunity for the development of various protein expression hosts to manufacture proteins by following the existing rigid standards laid down for veterinary and human applications. the industry is focusing mainly on already established production platforms using prokaryotic and eukaryotic expression host systems such as escherichia coli, a selection of yeast, insect, and mammalian cell cultures, due to their well-defined processes in-line with current good manufacturing practice (cgmp) [2] . moreover, industrially established mammalian and other cell cultures have stringent regulatory approval in place, which hinders the industrial acceptance of the new technology or production system. bacterial expression systems offer rapid production with high product yield, whereas saccharomyces cerevisiae and pichia pastoris (yeast) offer post-translational modifications (ptms) which are essential for functional activity of the recombinant proteins [3] . the majority of the table 1 . available expression platforms for recombinant protein production with potential advantages and disadvantages (adapted from shanmugaraj et al., 2020) [25] . the concept of using plants for the production of foreign proteins including pharmaceutical and non-pharmaceutical proteins has been well explored and documented. many reports proved the ability of in vivo and in vitro plant systems to produce vaccine candidates both for veterinary and human applications and showed that plant-produced antigens elicit potential immune responses in animal models and even confer protection in animal challenge experiments. examples of the variety of pharmaceutical and non-pharmaceutical proteins expressed in plant systems are illustrated in tables 2 and 3 . tobacco has been engineered to express a variety of antigens in the nucleus and chloroplast including, but not limited to, chikungunya, dengue, ebola, influenza, and zika. the transformation protocols for recombinant protein production are also established for fruits and vegetables such as tomatoes and potatoes. transgenic potatoes expressing the s1 glycoprotein of the infectious bronchitis virus confers protection to chickens upon virus challenge [26] . leafy crops such as lettuce, alfalfa, and clover have been investigated for molecular farming to obtain the oral delivery of vaccine antigens eliminating purification and injections. the lettuce chloroplast-derived booster vaccine using lyophilized plant cells expressing the poliovirus capsid protein induced neutralization antibodies in mice primed with inactivated poliovirus vaccine (ipv) and conferred protection against all polio serotypes [27] . plant systems have also been evaluated for the expression of virus-like particles (vlp) of many viruses including norovirus, poliovirus, foot-and-mouth disease virus, influenza, [28] [29] [30] [31] , and the potential for plant-derived vlps to be used as candidate vaccines and reagents has been reviewed in detail elsewhere [18, 32, 33] . apart from expressing antigens for human diseases, several antigens for veterinary applications and non-pharmaceutical proteins have also been well tested for expression in plants, and are particularly gaining attention due to the fact that these products can quickly reach the market due to lower regulatory burden [9] . this was clearly evidenced by the commercialization of avidin [34] , β-glucuronidase [35] , and trypsin [36] by the us-based biotechnology company prodigene, inc. the vaccine against newcastle disease virus (ndv) was the first plant-based poultry vaccine (dow agrosciences) that obtained regulatory approval from the united states department of agriculture in 2006, opening a new avenue for the commercialization of plant-derived vaccines. currently, many plant-derived non-pharmaceutical and pharmaceutical proteins are in clinical development. although proof-of-concept and efficacy of many vaccine candidates proved the feasibility and scalability of the robust plant system, it is high time to compete with the established expression systems. now the plant-based good manufacturing practices (gmp) complaint production facilities such as fraunhofer (germany), kentucky bioprocessing (usa), medicago (canada), and protalix biotherapeutics (israel) are available to manufacture gmp materials for human clinical trials. fraunhofer ime received a gmp license for the production of neutralizing anti-hiv antibody 2g12 in tobacco for phase i clinical testing [37] . the plant molecular farming research community continuously thrives to set up a regulatory framework for plant-derived products. the expression methods used for the recombinant protein production in plants can be either stable or transient expression. pmf relies on following approaches for the expression of vaccine candidates, i.e., stable nuclear transformation, stable chloroplast transformation, or transient expression, by using plant viral vectors and stable transformation of hydroponically grown plants in which recombinant proteins are recovered from the medium [119] (figure 1 ). stable nuclear transformation is the traditional strategy of genetic manipulation in plants for recombinant protein production. the transgene in the plant expression vector can be introduced into the in vitro grown plantlets either with agrobacterium tumefaciens-mediated transformation or particle bombardment, and stable transgenic lines can be developed. the best transgenic line for protein production will be subsequently screened from the pool of transgenic lines. by this method, recombinant proteins can be produced in successive generations, as the transgene has been stably integrated into the plant genome. the model plants such as arabidopsis thaliana and tobacco were more commonly used during the early stages of genetic transformation to develop stable transformants [79] . stable transformation in plants requires substantial time and is a labor-intensive process, and the protein expression is insufficient to meet the industrial-level protein production. however, the antigen expression in stable transgenic line could be used for developing oral vaccines that could reduce the cost associated with protein purification [120, 121] . alternatively, transient expression based on agroinfiltration or virus-based vectors have been developed to complement transgenic plants that offer rapid and high-level protein expression within a few days. the drawbacks and challenges associated with stable expression, such as stable nuclear transformation is the traditional strategy of genetic manipulation in plants for recombinant protein production. the transgene in the plant expression vector can be introduced into the in vitro grown plantlets either with agrobacterium tumefaciens-mediated transformation or particle bombardment, and stable transgenic lines can be developed. the best transgenic line for protein production will be subsequently screened from the pool of transgenic lines. by this method, recombinant proteins can be produced in successive generations, as the transgene has been stably integrated into the plant genome. the model plants such as arabidopsis thaliana and tobacco were more commonly used during the early stages of genetic transformation to develop stable transformants [79] . stable transformation in plants requires substantial time and is a labor-intensive process, and the protein expression is insufficient to meet the industrial-level protein production. however, the antigen expression in stable transgenic line could be used for developing oral vaccines that could reduce the cost associated with protein purification [120, 121] . alternatively, transient expression based on agroinfiltration or virus-based vectors have been developed to complement transgenic plants that offer rapid and high-level protein expression within a few days. the drawbacks and challenges associated with stable expression, such as insufficient protein expression, time, and consistency, have been overcome by the development of novel strategies involving deconstructed viral vector systems such as magnicon ® technology, geminiviral, and peaq, which allows rapid accumulation of recombinant proteins in a short time [12] . hence, it is considered as a suitable convenient platform, especially for the production of vaccine antigens or monoclonal antibodies against infectious diseases (figure 2 ). gleba et al. (2007) summarized the application of plant viral vectors for the transient expression of heterologous proteins in plants [122] . plant transient expression holds tremendous potential to produce rapid-response proteins, emergency vaccines, or biologics, which was impressively shown during the ebola outbreak in 2014. mapp biopharmaceutical inc., usa produced an experimental drug zmapp, an anti-ebola antibody cocktail of three chimeric monoclonal antibodies manufactured in tobacco plants (nicotiana benthamiana) to treat humans during the recent ebola outbreak [123] . during a pandemic situation, in order to cope with a rapidly spreading infectious disease, production methods should meet the demand for production targets of strategic vaccines to control the disease. one of the recent examples is the pandemic, corona virus disease (covid-19) . the virus has spread rapidly, and millions of people have been affected across 6 continents in few months, posing a constant threat to global health. this infection has created a massive demand for diagnostic reagents, vaccines, and therapeutic development. given the speed advantages, and proven viability of the plant production platform, the transient expression system in particular could be employed to produce recombinant proteins at high levels to meet the sudden demand for production of viral antigens or antiviral proteins that could be used as research reagents, emergency vaccines (sars-cov-2 subunit and virus-like particle vaccines), or other biopharmaceuticals to fight against covid-19 [25, 124] . the neutralizing monoclonal antibodies against sars-cov-2 could also be produced in plants with minimal investment, which could be used for passive immunotherapy [125] . recently, medicago (quebec, canada), kentucky bioprocessing (owensboro, kt, usa), and ibio (bryan, tx, usa) joined the global race for developing potential plant-based vaccines for covid-19 [126] . by using the transient expression platform, recombinant protein production in plants could be scaled up rapidly, and milligram quantities of proteins could be produced in a timeframe of less than 4 weeks after receiving the corresponding gene construct [5, 59, 127] . plants 2020, 9, x for peer review 12 of 21 has spread rapidly, and millions of people have been affected across 6 continents in few months, posing a constant threat to global health. this infection has created a massive demand for diagnostic reagents, vaccines, and therapeutic development. given the speed advantages, and proven viability of the plant production platform, the transient expression system in particular could be employed to produce recombinant proteins at high levels to meet the sudden demand for production of viral antigens or antiviral proteins that could be used as research reagents, emergency vaccines (sars-cov-2 subunit and virus-like particle vaccines), or other biopharmaceuticals to fight against covid-19 [25, 124] . the neutralizing monoclonal antibodies against sars-cov-2 could also be produced in plants with minimal investment, which could be used for passive immunotherapy [125] . recently, medicago (quebec, canada), kentucky bioprocessing (owensboro, kt, usa), and ibio (bryan, tx, usa) joined the global race for developing potential plant-based vaccines for covid-19 [126] . by using the transient expression platform, recombinant protein production in plants could be scaled up rapidly, and milligram quantities of proteins could be produced in a timeframe of less than 4 weeks after receiving the corresponding gene construct [5, 59, 127] . alternately, chloroplast expression focuses on expressing the transgenes in chloroplast by the precise insertion of foreign dna by homologous recombination into the chloroplast genome. much progress has been made in chloroplast engineering in recent years. the transformation of the chloroplast genome has many advantages over nuclear transformation which includes higher protein production, lack of gene silencing and position effect, polycistronic mrna expression, and prevention of transmission of foreign dna through pollen by uniparental plastid gene inheritance (maternal inheritance) in crop plants [128] [129] [130] [131] . similar to bacterial and mammalian cells, heterologous protein production can be achieved by using individual suspension of plant cells rather than whole plants. the cell suspension derived from undifferentiated callus grown in liquid medium can be scaled up in bioreactors for large-scale protein production under an aseptic environment. the first usda-approved poultry vaccine and the first fda-approved recombinant plant-produced pharmaceutical protein "elelyso" were produced in tobacco and carrot cell suspension cultures, respectively, which proved the importance and competitiveness of plant suspension culture in high-value protein production in the biopharmaceutical industry [121, [132] [133] [134] . hairy root cultures are also being explored as an alternative recombinant protein production system due to their ease in protein recovery and low costs. the recombinant proteins are secreted from the transgenic plant roots into the culture medium viz., rhizosecretion; hence, this allows continuous protein production and recovery from the culture medium without the requirement of cell lysis during extraction. moreover, recombinant proteins produced from root cultures attribute to the improved protein quality and quantity without complex downstream processing that could eventually reduce production costs as well [135] . a recent review on the applications of hairy root cultures for protein production has been extensively discussed by gutierrez-valdes et al. (2020) [136] . although plants are attractive with several unique advantages, they are unable to compete with the existing microbial and mammalian systems, as both are well established and characterized, especially in terms of gmp manufacturing and regulatory approval in an industrial setting. even after many years of research, which has shown the proof-of-concept of expressing many therapeutic proteins in plants, the process of producing therapeutic proteins from the lab bench to commercialization is slow. hence, in order to move forward, the commercial potential and economic sustainability of technology needs to be exploited by developing veterinary vaccines, non-pharmaceutical diagnostic, cosmetic products, and industrial enzymes in plants, as they have a low regulatory burden compared to therapeutic proteins [2, 9] . this technology can also be employed to reproduce rapid response vaccines or diagnostic reagents against emerging infections. for the past few years, extensive research has been carried out to combat the several emerging diseases including zika, chikungunya, nipah, sars-cov, mers-cov, and more recently sars-cov-2. even though several efforts have been made for many years to develop effective vaccine candidates for many of those emerging and zoonotic diseases, still, there are no vaccine candidates or therapeutic measures available commercially. even if a successful vaccine or drug developed against such diseases, it is unlikely that it would have a significant impact on developing and under-developed countries, due to the high cost associated with it, and scalability concern. in such a scenario, a plant-derived vaccine or diagnostic reagent would be a feasible approach to rapidly respond to the demand and need for recombinant proteins. however, harnessing the full potential of this plant molecular farming technology for cost-effective vaccines or drug development will be evident in the upcoming years. plants have both economic and technical advantages over conventional expression systems for the production of pharmaceutical and non-pharmaceutical products. the different pmf technologies such as nuclear, chloroplast expression, viral transfection, and transient expression systems have their unique features, enabling them to address a production of diversified product "targets" with less production constraints in a short time. many scientific and technical challenges associated with the plant platform were met in recent years. however, the regulatory burden associated with therapeutic protein production is a major barrier that hinders the widespread acceptance of the plant system. considering the low costs and greater scalability of plant production systems, the commercialization of non-pharmaceutical proteins is straightforward and faster due to lower regulatory challenges. hence, the universal acceptance of the technology will be strongly influenced by the regulatory framework and restrictions applied to plant-derived 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protalix experience putting the spotlight back on plant suspension cultures. front biologically active recombinant human erythropoietin expressed in hairy root cultures and regenerated plantlets of nicotiana tabacum l hairy root cultures-a versatile tool with multiple applications. front this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-324325-rmlrhyf2 authors: chan, wai s; wu, chun; chow, sammy c s; cheung, to; to, ka-fai; leung, wai-keung; chan, paul k s; lee, kam-cheong; ng, ho-keung; au, deborah m y; lo, anthony w i title: coronaviral hypothetical and structural proteins were found in the intestinal surface enterocytes and pneumocytes of severe acute respiratory syndrome (sars) date: 2005-05-13 journal: mod pathol doi: 10.1038/modpathol.3800439 sha: doc_id: 324325 cord_uid: rmlrhyf2 severe acute respiratory syndrome (sars) is a newly emerging infectious disease that haunted the world from november 2002 to july 2003. little is known about the biology and pathophysiology of the novel coronavirus that causes sars. the tissue and cellular distributions of coronaviral hypothetical and structural proteins in sars were investigated. antibodies against the hypothetical (sars 3a, 3b, 6, 7a and 9b) and structural proteins (envelope, membrane, nucleocapsid and spike) of the coronavirus were generated from predicted antigenic epitopes of each protein. the presence of these proteins were first verified in coronavirus-infected vero e6 tissue culture model. immunohistochemical studies on different human tissues, including a cohort of nine autopsies, two liver biopsies and intestinal biopsies of sars patients, further confirmed the existence of coronaviral hypothetical and structural proteins in the cytoplasm of pneumocytes and small intestinal surface enterocytes in sars patients. with this vast array of antibodies, no signal was observed in other cell types including those organs in which reverse transcriptase-polymerase chain reactions were reported to be positive. structural proteins and the functionally undefined hypothetical proteins were expressed in coronavirus-infected cells with distinct expression pattern in different organs in sars patients. these antipeptide antibodies can be useful for the diagnosis of sars at the tissue level. from november 2002 to july 2003, severe acute respiratory syndrome (sars) had affected more than 8000 people in 28 regions worldwide with mortality upto 9.6%. 1 the clinical picture of this imminent infectious disease was dominated by respiratory system involvement. 2, 3 gastrointestinal symptoms were also common. 4, 5 the primary etiological agent of sars is a novel coronavirus (sars-cov). [6] [7] [8] the genome of sars-cov consists of a single giant positive-strand rna that is approximately 29.7 kb in length. sars-cov genome encompasses two large open reading frames (orfs) encoding nonstructural proteins involved in replication and 12 potential orfs. these potential orfs include four genes encoding structural proteins (envelope, membrane, nucleocapsid and spike proteins). the remaining potential orfs encode hypothetical sars-cov-specific proteins that lack obvious sequence similarity to known proteins. 9, 10 these hypothetical proteins that are purely the prophecies of bioinformatics algorithm need to be studied in infected cells so as to confirm their identities. the functions of these hypothetical proteins and their roles in sars pathogenesis remain obscure. 11, 12 in this study, we have generated specific antipeptide antibodies against each of the putative structural and hypothetical proteins of the sars-cov. using immunohistochemistry, we demonstrated the unique existence of corresponding peptide sequences in sars-cov-infected tissue culture models and clinical specimens. the tissue and cellular distribution pattern of sars-cov hypothetical and structural proteins in sars was studied. clinical specimens were selected retrospectively following the guidelines of the local ethics committee. sars was diagnosed according to the criteria of world health organization. 1 the outbreak in the prince of wales hospital, hong kong, and the sequence of events leading to the world endemic has been reported. 2,3 pathology of case 1 to 8 has been previously described. 13 one probable sars case (negative culture) during the same epoch and two lung sections from autopsies performed in early 2002 (with the diagnosis unrelated to lung diseases) were used as negative controls. the terminal ileum and colon biopsies 5 of one sars patient and liver biopsies 14 were obtained with informed consents. in all, 10 cases of randomly selected autopsies, with causes of death either related or unrelated to pulmonary pathology, in the pre-sars era of 2000-2001 were used as negative control. antigenicities of epitopes on putative orf of the sars-cov (genbank accession no.: nc_004718) were evaluated in silico using clone manager 5 (scientific & educational software), peptide companion (csps pharmaceuticals, inc.) and ds gene (accelrys). these peptides were also compared with other published proteomes of coronavirus (cov) such as human group 1 and group 2 cov, bovine-cov, porcine-cov, feline-cov and canine-cov to select peptides that were sars-cov-specific. antibodies were generated from rabbits immunized with each individual keyhole limpet hemocyanin-conjugated synthetic peptide. all antipeptide antibodies and peptides used in this study were produced by abgent (san diego, ca, usa) and are now commercially available at abgent. the cell line vero e6 (monkey kidney cell line) was obtained from the american type culture collection (manassas, va, usa). the virus cuhk-w1 strain of sars-cov (genbank accession no.: ay278554) was grown and assayed in the cell line as previously described. 15 paraffin blocks were prepared from infected or control vero e6 fixed with 10% formalin as routine clinical cytology specimens. autopsy and biopsy specimens were fixed with 10% formalin and embedded in paraffin blocks. standard avidinbiotin method was used for immunohistochemical studies on 4-mm sections from selected paraffin blocks. antipeptide antibodies (1:100) with strep-tabcomplex/hrp duet reagent set (dako, glostrup, denmark) were used according to the manufacturer's protocols with 3, 3 0 -diaminobenzidine tetrahydrochloride as the chromogen. antigen retrieval was performed by microwave pretreatment twice in 10 mm citrate buffer, ph 6.0 or 0.1 m edta buffer, ph 8.0, with preliminary heating at 780 w for 3 min followed by 480 w for 10 min. the specificity of each antibody for immunohistochemistry was ascertained in formalin-fixed vero e6 cells by suppression of signals when the primary antibody was preincubated with excess corresponding peptides before applying to the cell block sections. intensities of the immunohistochemical signals on each individual cell were judged arbitrarily with the overall assessment as: negative (à), positive ( þ ), moderately positive ( þ þ ) or markedly positive ( þ þ þ ). the coexistence of sars-cov protein and rna in infected cells was detected by immunofluorescence-fluorescence in situ hybridization on formalin-fixed paraffin-embedded tissues as previously described. 16 a 700-bp reverse transcriptase-polymerase chain reaction product corresponding to the m gene of sars-cov strain su10 (genbank accession no.: ay282752) was used as the probe. antipeptide antibodies (1:100) were used for colocalization. fluorescence in situ hybridization signals were detected using anti-dig-rhodamine (roche) and the secondary antibody for immunofluorescence was antibiotin fluorescein thiocyanate (fitc). nuclei were counterstained with 4,6-diamidino-2-phenylindole in antifade mountant (vectorshield, vector laboratories). antigenicity of epitopes of the putative hypothetical and structural proteins was evaluated and anti-peptide antibodies were generated in rabbits (table 1) . putative epitopes were identified in nine out of 14 predicted orfs, representing 64% of the transcriptome, of sars-cov. these antibodies for immunohistochemical analysis were evaluated in vitro on vero e6 infected by sars-cov. uninfected cells were used as negative controls. all epitopes of hypothetical (sars 3a, 3b, 6, 7a and 9b) and structural proteins (envelope, membrane, nucleocapsid and spike) were detected in the cytoplasm of infected vero e6 cells by at least one antibody from each protein ( table 1) . intensities of the signals in individual infected cells were scored arbitrarily. nuclear signals were not detected. a similar cytoplasmic signal was not observed in uninfected vero e6 negative controls. representative results with the antibody sars-abs13a are shown in figure 1 . hence, the peptide sequences of each putative hypothetical and structural protein were expressed in vitro. some of the epitopes, although theoretically antigenic, might be buried in the interior of the proteins. the antipeptide antibodies with strong cytoplasmic signals and minimal background were further applied on formalin-fixed paraffin-embedded sections of clinical specimens. antibodies sars-abs13a, sars-abs-16b, sars-abs-5a and sars-abs21a, against the nucleocapsid protein (n), the membrane protein (m), the spike protein (s) and a putative sars 3a protein, respectively, were selected for further evaluation in 16 different tissues, including the lung, bronchus, small intestine, stomach, colon, pancreas, lymph node, spleen, bone marrow, liver, kidney, adrenal gland, skeletal muscle, heart and skin, from nine fatal cases of sars (table 2) . terminal ileum and colon biopsies from one patient 5 and two liver biopsies 14 from different patients were also included in this study. positive immunohistochemical signals were observed in the lung sections from three fatal cases and the small intestine sections from five autopsy cases. sars-cov proteins were detected only in samples that were positive for viral culture and viral genome. other tissue samples were all immunohistochemically negative. the sars-cov proteins existed either in diffuse signals or in aggregates in the cytoplasm. background in negative controls was minimal. immunohistochemical staining was negative in the lung and intestinal sections from control autopsy and surgical specimens. the histopathology of the lung was dominated by diffuse alveolar damage at various stages of organization. no histological features appeared to predict the positivity of immunohistochemistry. a similar result was obtained previously for in situ hybridization study of the sars-cov genome. 16 in immuno-histochemical-positive lung sections, positive signals were observed in the cytoplasm of pneumocytes lining the alveolar septa (figure 2a, arrows) and in the detached pneumocytes within the alveolar spaces (figure 2a, arrows) . in some of the multinucleated giant cells, a characteristic but rare feature of the lung pathology in sars, the immunohistochemical stainings were strongly positive (figure 2b, arrowhead) . carbon pigments were not seen. these giant cells might represent those originated from syncytial formation of infected pneumocytes. diffuse, weak, granular immunohistochemical signals were seen in carbon pigmentladen macrophages. these signals might represent background or scavengered proteins in these macrophages. no specific histological changes had been identified in the sars-cov-infected intestine. while the virus can be isolated in some of our autopsy cases, no mucosal damage was observed. the lack of tissue damage is particularly striking in the terminal ileum biopsy from an active sars patient with diarrhea. 5 for the small intestine sections, the surface enterocytes showed positive cytoplasmic signals in both autopsy ( figure 3a ) and biopsy (figure 3b ) specimens. crypt cells were rarely positive. macrophages in this organ were negative. the pattern of immunohistochemical-positive cells was consistent with the distribution of viral genome in other reports. 16, 17 the colon biopsy, however, was immunohistochemically negative for all the sars-cov proteins tested. lymphoid follicles within these biopsies were also negative. in liver biopsies, no immunohistochemical signals were detected with the whole battery of antipeptide antibodies in any cell type. in situ hybridization studies using the probe to m-gene in the colon and liver sections were also negative (results not shown). in those tissue sections showing positive signals for immunohistochemical staining, we further performed immunohistochemical studies using all other antibodies tested positive in sars-cov-infected vero e6 cells. these antibodies, including sars-abs18a, 7b, 24a, 20b, 19a, 15b, 9b and 11, all produced similar patterns of immunohistochemical staining in the lung and terminal ileum sections. the number of viral infected cells detected by each antibody was similar, judging semiqualitatively by the pattern of tissue distribution of the positive signals. the intensities of the immunohistochemical signals in each infected cells was correlated with those observed in vero e6 cell culture (table 1 ). these results suggested that, these peptides sars 3a, 3b, 6, 7a and 9b corresponding to envelope, membrane, spike, nucleocapsid, were expressed in sars-cov infected cells in diseased organs. the cellular distribution of sars-cov protein and viral genome in immunohistochemical-positive lung and small intestine sections was further evaluated by immunofluorescence-fluorescence in situ hybridization analysis (figure 4) . sars-cov protein and viral genome were denoted by green (figure 4 -ii) and red fluorescence signals (figure 4-iii) , respectively. in the lung (figure 4a ) and small intestine (figure 4b) sections, colocalization of immunohistochemical signals for sars 3a and fluorescence in situ hybridization signals of sars-cov genome to the same cell was confirmed (figure 4, arrows) . each of these signals was identified in the cytoplasm only. only the cells infected with viral genome expressed the corresponding peptides, and vice versa. in this study, the tissue and cellular distributions of sars-cov hypothetical and structural proteins were evaluated in the autopsy and biopsy samples of sars. we have also demonstrated the use of genome information that is generated from high throughput technologies for the translational research in diagnostic application. a new set of antipeptide antibodies was generated from the predicted orfs of the structural and hypothetical proteins of the sars-cov. immunohistochemical applications of these antibodies were validated on vero e6 infected with sars-cov. vero e6 is the prototype mammalian cell line that is used for sars-cov isolation in routine clinical microbiology investigations. 3 in vero e6 cells, positive cytoplasmic immunohistochemical signals were detected by figure 3 immunohistochemical studies of antipeptide antibody sars-abs13a against the nucleocapsid protein on small intestine sections. sars-cov-infected cells were demonstrated by the strong cytoplasmic signals in surface enterocytes in both biopsy (a, arrows) and autopsy (b, arrows) specimens. the sections were counter-stained with hematoxylin ( â 1 000). at least one antibody from each predicted orfs. the sequences of these peptides were unique and specific for the corresponding sars-cov protein. the specificities of these antipeptide antibodies were further determined experimentally with the suppression of immunohistochemical signals with excess corresponding peptides. in addition, in the study of tissue sections, only those cells with viral genome detected by fluorescence in situ hybridization were positive for immunohistochemical stainings for the antipeptide antibodies. these results demonstrated the existence of sars-cov known structural proteins and provide further evidence for the expression of those hypothetical proteins. the real identities of each of these hypothetical proteins require further investigations. however, our results indicated that at least the genome locations corresponding to the peptides selected for the putative orfs were expressed. further analyses of these hypothetical proteins by western blotting and immunoprecipitation experiments in tissue culture models will be required to confirm the expression and molecular weights of these proteins, to look for possible post-translation modifications and to identify the host interacting partners. the feasibilities of these antibodies to be used in formalin-fixed paraffin-embedded sections of archival clinical specimens were established in the current study. antibodies sars-abs13a, sars-abs-16b, sars-abs-5a and sars-abs21a, against the nucleocapsid protein (n), the membrane protein (m), the spike protein (s) and a putative sars 3a protein, respectively, appeared to provide strong immunohistochemical signals. these antibodies are potentially useful in the histological confirmation of sars-cov infection. the existence of sars-cov proteins was further confirmed in clinical samples. immunohistochemical staining was performed on all available tissues from nine autopsy cases of clinically confirmed sars, two intestine biopsies of sars patients and two liver biopsies. positive cytoplasmic signals were observed in pulmonary pneumocytes and small intestinal surface enterocytes only. the existence of sars-cov in immunohistochemical-positive cells was proven by colocalization of the expressed proteins and viral genome in the same cells by dual immunofluorescence-fluorescence in situ hybridization. both the lung pneumocytes and small intestine enterocytes expressed the full sets of viral proteins. although lung and small intestine were the primary targets of sars-cov infection, the responses of these two tissues were quite different. tissue damage and organization, in the pattern of diffuse alveolar damage, were commonly observed in the lungs of sars. 13, [18] [19] [20] [21] [22] syncytial cell formation is rarely observed although characteristic. little tissue reaction is, however, observed in the small intestine autopsy and biopsy sections. 5, 13 detailed studies of the expression levels and the interactions of viral proteins with host proteome may provide the answer. much interest has been diverted to the figure 4 immunofluorescence-fluorescence in situ hybridization of the hypothetical protein sars 3a and sars-cov genome. sars 3a (ii, green fluorescence) and viral genome (iii, red fluorescence) were detected using the antibody sars abs21a and a dna-probe directed towards the m gene, respectively. the nuclei were counter-stained with 4,6-diamidino-2-phenylindole (iv). pneumocytes (combined, a-i, arrows) and enterocytes (b-i, arrows) infected with the viral genome (iii, arrows) were positive for sars 3a protein (ii, arrows). the background fluorescence of negative controls was limited to red blood cells and some connective tissue components. study of known structural proteins, especially spike and nucleocapsid, in realizing the potentials of developing vaccines. the uniqueness of the hypothetical proteins to sars-cov, as compared phylogenetically to other known coronavirus, might be crucial to the clinical manifestation of sars. at present, the functions of sars-cov hypothetical proteins and their roles in sars pathogenesis are poorly understood. recently, one of the putative proteins, sars 7a was shown to interact with sars 3a and the structural proteins in vero e6. 11, 12 these findings highlighted the potential, undervalued functions of hypothetical proteins in the pathogenesis of sars. the negative immunohistochemical result in liver biopsies is interesting. reverse transcriptase-polymerase chain reaction for the viral genome was positive while electron microscopy failed to demonstrate viral particles in the liver. 14, 22 in situ hybridization aiming at detecting the viral genome were all negative in these same specimens (result not shown). this might be due to the high sensitivity of polymerase chain reaction-based detection method that picked up the small number of viral genome in the scanty infected cells. alternatively, serum contamination in the biopsy might explain the positive reverse transcriptase-polymerase chain reaction. it is, however, important to note that liver dysfunction was observed in sars patients. 14,23-25 a high proliferation index was clearly demonstrated in these liver biopsy specimens. 14 hence, liver metabolism must have been perturbed in these patients. the role of cytokines or other yet unknown paracrine mediators in causing liver dysfunction requires further clarification. a similar scenario is likely happening in the kidneys of sars patients in which 'sensitive' reverse transcriptase-polymerase chain reaction is positive 22 with functional disturbances of the renal function, yet viral particles, proteins and genome were not identified. 26 we are beginning to understand how sars-cov enters its host cells. in various in vitro systems using the spike protein, the angiotensin-converting enzyme 2 (ace2) [27] [28] [29] and the c-type lectins, cd209 30 or cd209l, 31 are identified as receptors of sars-cov. the distribution of ace2, by itself, cannot explain the tissue tropism of the virus. 26 the ace2 protein has a much wilder cellular distribution 32 than even the most extensive, although putative and controversial, list of organs and cell types harboring the virus. 17 in the current study, the dendritic cells were not positive for ihc signals of any of the viral proteins in various different tissues. lymphoid follicles immediately adjacent to infected cells were negative. sars-cov may exist only in low titer in the dendritic cells. parallel to the situations in human immunodeficiency virus-1 (hiv-1) and mycobacterium tuberculosis, 33 the idea of dendritic cells as reservoir and immune mediator is attractive, the actual role of this cell type in sars patients is not clear. we have provided evidence for the presence of known structural proteins, including membrane, envelope, nucleocapsid and spike, as well as hypothetical proteins, sars 3a, 3b, 6, 7a and 9b, in tissue culture model and in clinical specimens. with this full array of antibodies, only limited cell types were shown to harbor the sars-cov. the roles of these proteins and their interaction with the host genome and proteome are currently unknown. this set of antibodies will be useful in tissue diagnosis of sars and in future studies of the in vitro models of sars. severe acute respiratory syndrome (sars) last accessed a major outbreak of severe acute respiratory syndrome in hong kong a cluster of cases of severe acute respiratory syndrome in hong kong digestive system manifestations in patients with severe acute respiratory syndrome enteric involvement of severe acute respiratory syndrome-associated coronavirus infection newly discovered coronavirus as the primary cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome koch's postulates fulfilled for sars virus the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome a novel severe acute respiratory syndrome coronavirus protein, u274, is transported to the cell surface and undergoes endocytosis characterization of a unique group-specific protein (u122) of the severe acute respiratory syndrome coronavirus pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (sars) sars-associated viral hepatitis caused by a novel coronavirus: report of three cases persistent infection of sars coronavirus in colonic cells in vitro tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in situ hybridization study of fatal cases organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways the spectrum of pathological changes in severe acute respiratory syndrome (sars) lung pathology of fatal severe acute respiratory syndrome the clinical pathology of severe acute respiratory syndrome (sars): a report from china lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore pulmonary pathology of severe acute respiratory syndrome in toronto serum hepatic enzyme manifestations in patients with severe acute respiratory syndrome: retrospective analysis retrospective analysis of liver function derangement in severe acute respiratory syndrome temporal patterns of hepatic dysfunction and disease severity in patients with sars exploring the pathogenesis of severe acute respiratory syndrome (sars): the tissue distribution of the coronavirus (sars-cov) and its putative receptor, angiotensin-converting enzyme 2 (ace2) expression cloning of functional receptor used by sars coronavirus angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus susceptibility to sars coronavirus s protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis dc-sign: escape mechanism for pathogens we thank chit chow, cecilia ho, and kw suen for critical comments on the manuscript. the key: cord-312489-ywep0c08 authors: andoh, kiyohiko; suenaga, kiyotaka; sakaguchi, masashi; yamazaki, kenichi; honda, takashi title: decreased neutralizing antigenicity in ibv s1 protein expressed from mammalian cells date: 2015-10-02 journal: virus res doi: 10.1016/j.virusres.2015.06.019 sha: doc_id: 312489 cord_uid: ywep0c08 we evaluated the antigenicity of recombinant infectious bronchitis virus (ibv) s1 protein expressed in mammalian cells. recombinant s1 was expressed as a secreted protein fused with a trimerization motif peptide, then purified using ni sepharose. the purified protein was analyzed by western blotting, mixed with oil adjuvant, and administered to 29-day-old specific-pathogen-free chickens. six weeks after immunization, anti-ibv neutralizing titer and anti-s1 elisa titer were determined; immunized chickens then were inoculated with ibv via the trachea and ciliary activity was observed. results showed that the recombinant s1 protein was highly glycosylated, and the neutralizing antigenicity of recombinant s1 protein was lower than that of inactivated virus. however, anti-s1 elisa indicated that the recombinant s1 protein induced antibodies against s1. these results suggest that the recombinant s1 may retain non-neutralizing epitopes but have unnatural glycosylation pattern and conformation, resulting in lacking neutralizing conformational epitopes. in conclusion, the neutralizing antigenicity of recombinant s1 protein expressed from mammalian cells was decreased, and was not sufficient to induce neutralizing antibodies. infectious bronchitis virus (ibv) belongs to the order nidovirales, family coronaviridae, genus gammacoronavirus, and causes respiratory disease and pathology in the kidney and gonads of chickens and other birds (boltz et al., 2004; cavanagh, 2007) . ibv is an economically important disease in the poultry industry, and several vaccines have been used to prevent the spread of ibv. ibv shows extensive antigenic variation, reflecting mutation of the spike protein gene (cavanagh et al., 1988 (cavanagh et al., , 1997 wang and huang, 2000) . vaccines targeting individual ibv serotypes yield poor cross-protection; therefore, various attenuated and inactivated multivalent vaccines (derived from several different serotypes) are used (deguchi et al., 1998; sjaak de wit et al., 2011) . the spike protein (s) is an envelope glycoprotein that forms a dimer or trimer, and has been shown to play an important role in viral infection (cavanagh et al., 1986; ignjatovic and galli, 1994; wickramasinghe et al., 2011) . s is highly glycosylated, and based on its amino acid sequence, the spike protein is predicted to contain 21 to 35 n-glycosylation sites. s has two main functions: to attach the virus to the host cell receptor, and to activate fusion of the virion membrane with the host cell membrane (casais et al., 2003; wickramasinghe et al., 2011) . the s protein is the most important antigen in inducing neutralizing antibodies against ibv, and the n-terminal s1 region is especially important (cavanagh et al., 1986; ignjatovic and galli, 1994; kant et al., 1992; koch et al., 1990; promkuntod et al., 2014) . the s1 domain forms the bulbous head of the spike protein, and several virus neutralization (vn) epitopes have been reported to reside within the first and third quarter of the s1 sequence (cavanagh et al., 1988; kant et al., 1992; koch et al., 1990; sjaak de wit et al., 2011) . thus, analysis of the antigenicity of the s1 domain is expected to be critical to the development of effective anti-ibv vaccines. to prevent ibv infection, several recombinant subunit vaccine developments have been attempted using the recombinant s1 protein or other proteins. immunization with recombinant s1 protein expressed from baculovirus has been shown to provide in chicken effective protection against ibv infection (song et al., 1998) . other groups also have reported that immunization with recombinant s1 http://dx.doi.org/10.1016/j.virusres.2015.06.019 0168-1702/© 2015 elsevier b.v. all rights reserved. epitope peptide expressed from escherichia coli (e. coli) protected chickens against ibv infection (yang et al., 2009a (yang et al., , 2009b . furthermore, viral vectored vaccines co-expressing the s1 protein and host cytokines have been reported to induce anti-s1 antibodies (chen et al., 2010; shi et al., 2011; tomley et al., 1987; wang et al., 2009; zeshan et al., 2011; zhang et al., 2012) . however, in these reports of protection, recombinant antigen did not provide perfect protection and antibody titers were evaluated only by hemagglutination inhibition (hi) or enzyme-linked immunosorbent assay (elisa); the respective authors did not indicate whether these antigens retained their native neutralizing antigenicity (song et al., 1998; yang et al., 2009a yang et al., , 2009b . therefore, it remains unknown whether recombinant s1 protein, without the s2 domain, completely retains its conformational epitopes and neutralizing antigenicity. to address these issues, we analyzed the recombinant s1 expressed from mammalian and avian cells by western blotting and analyzed the neutralizing antigenicity of recombinant s1 protein and compared titers against those of inactivated virus antigen. for the immunization experiment, recombinant s1 protein was expressed as the secreted protein with a trimerization motif because some researchers have reported that secreted recombinant s1 protein, when expressed fused to the trimerization motif peptide, retains the ability to bind the cell receptors (promkuntod et al., 2013; wickramasinghe et al., 2011) and it seemed to be suited to the vaccine antigen. the present work employed the tm86 ibv strain, an isolate of genotype jp-ii that originally was recovered from a field chicken; this strain subsequently has been used as a vaccine strain (ariyoshi et al., 2010; mase et al., 2004) . tm86 was propagated in specific-pathogen-free (spf) chicken embryonated eggs. ibv tm86 adapted to chicken kidney (ck) cells was used for the vn test and elisa of the antigen. ck cells and chicken embryo fibroblast (cef) cells were incubated in eagle's medium (emem) supplemented with 10% tryptose phosphate broth and 5% heat-inactivated fetal bovine serum (fbs; hyclone), along with 100 units of penicillin and 100 g of streptomycin per ml, and cells were grown at 37 • c in 5% co 2 incubators. 293t cells and 293 cells lacking nacetylglucosaminyltransferase i (293 gnti − , reeves et al., 2002) were propagated in dulbecco's modified eagle's medium (dmem) supplemented with 10% fbs along with 100 units penicillin and 100 g of streptomycin per ml, and cells were grown at 37 • c in 5% co 2 incubators. a segment of the s1-encoding gene, coding for the protein from the n-terminus to the cleavage site between s1 and s2, was amplified from the spike gene of ibv strain tm86 (accession no. ab120655) by reverse transcriptase polymerase chain reaction (rt-pcr). the primer set used for rt-pcr was 5 -caaattattggtcagagatgttgg-3 (s1.1) and 5 -gaatcattaaacagactttttaggtct-3 (s2r1). after amplification, the dna encoding the signal sequence of s1 (mlvkslflvtll-falcs) was replaced with a dna sequence encoding the signal sequence of marek's disease virus (mdv)-glycoprotein a (ga) (mltprvlralgwtglfflllspsnvl). furthermore, dna sequences encoding a 6× his-tag peptide sequence, with or without those encoding a t4 phage fibritin coiled-coil trimerization motif (gsgyi-peaprdgqayvrkdgewvllstflg), were inserted in-phase and downstream of the recombinant s1-encoding gene. the resulting fig. 1 . schematics of the ibv s1 segment expressed as a secreted protein. to construct the secreted s1 expression plasmid, the nucleotide sequence encoding the s1 domain lacking the cleavage sequence (rrfrr) was amplified from the ibv tm86 genome. to increase expression levels of the recombinant s1 protein, the nucleotide sequence encoding the signal sequence was replaced with a nucleotide sequence encoding mdv ga. to facilitate purification and multimerization, the resulting sequence was cloned upstream and in-phase with nucleotide sequences permitting expression as c-terminal fusions to a 6× his-tag peptide sequence with or without a t4 phage fibritin coiled-coil trimerization motif. loci encoded the recombinant s1 protein with c-terminal fusions to the indicated domains ( fig. 1) . the constructed sequence was cloned into the sal i site of the expression plasmid pcaggs, which contains a cag promoter (modified chickenˇ-actin promoter with cytomegalovirus immediate-early (cmv-ie) enhancer), rabbitˇglobin gene sequence including polyadenylation signal, and simian virus 40 (sv40) ori (niwa et al., 1991) . the constructed plasmids were named pcaggs-s1-t4-his and pcaggs-s1-his, respectively. for transfection, the plasmid was purified using a qiaprep spin miniprep kit (qiagen). the mouse mabs against ibv s1 or s2 region were established in our institute. it was confirmed that these mabs recognize nonconformational epitopes by western blotting under denaturing condition. it was also confirmed that these mabs do not show neutralizing activity in vn test. before examination, the mabs were purified using protein g column. 293t, 293 gnti − , and cef cells were transfected with either of the constructed plasmids using polyethylenimine (pei). transfection using pei was performed according to the protocol established by boussif et al. (1995) . briefly, 88 g of the plasmid was mixed with 440 l of pei (2 mg/ml) and then transfected into approximately 2 × 10 7 of 293t cells. transfected cells were incubated in opti-mem (life technologies) and the supernatant of transfected cells was harvested at 72-96 h post-transfection. the recombinant protein was purified using ni sepharose (ge healthcare) according to the manufacturer's protocol. sds-page was carried out under denaturing and nondenaturing conditions. under denaturing conditions, the purified protein was mixed with 2× sample buffer (100 mm tris-hcl (ph 6.8), 4% sds, 20% glycerol, 0.2% bromphenol blue) containing 200 mm dithiothreitol (dtt) and boiled for 5 min at 95 • c. in contrast, non-denaturing sds-page was performed according to the protocol reported by bender et al. (2005) . briefly, the purified protein was mixed with 2× sample buffer containing 0.4% sds (in the absence of a reducing agent) and loaded directly onto a gel (without boiling of the sample). the protein was separated by 5-20% polyacrylamide gradient gel (e-pagel, atto) and transferred to a polyvinylidene difluoride (pvdf) membrane (millipore). the membrane was incubated in 5% skim milk (wako) in t-pbs buffer (phosphate-buffered saline, ph 7.2 (pbs) containing 0.05% tween 20) for 60 min at 37 • c, and then incubated with a mab against s1 protein in 5% skim milk in t-pbs buffer. next, the membrane was incubated with a peroxidase-conjugated second antibody, goat anti-mouse igg (h + l) (jackson). the reacted protein was visualized using a tmb substrate kit (invitrogen). the immunization experiment consisted of 4 groups of 5 or 10 chickens. the spf chickens (layer-type) used for this study were maintained in our institute and 10 chickens were immunized with the purified recombinant s1 protein, which was his-tagged with trimerizaton motif, or inactivated virus, respectively. five chickens were immunized with pbs-mock vaccine (mock group) and 10 chickens were left untreated to serve as an unvaccinated control group. light liquid paraffin, sorbitan monooleate, and polysorbate 80 in a volume ratio of 9:36:4:1 was used as oil adjuvant. recombinant s1 protein was mixed with oil adjuvant to make an s1 suspension at a concentration of 20 g/dose. the ibv tm86 strain was inactivated using formaldehyde and mixed with oil adjuvant to generate a suspension harboring inactivated virus at a virus concentration of 10 7.0 eid 50 /dose. chickens were inoculated intramuscularly. at 6 weeks post-inoculation, blood was collected from each animal, and the resulting sera were used to determine antibody titers. each chicken then was challenged with 10 3.5 eid 50 ibv tm strain administered via the trachea. animals were sacrificed at 4 days post-challenge and ciliostasis was assessed by a slightly modified version of the previously reported protocol (cook et al., 1999) . briefly, tracheas were removed aseptically from euthanized chickens. cilia were observed microscopically and the cessation of ciliary movement was considered to be a sign of symptoms. sera were serially diluted two-fold with emem in a microplate and mixed with 200 tcid 50 of ck-adapted ibv tm strain. after incubation for 1 h at 37 • c, ck cells were inoculated with ibv and incubated at 37 • c in 5% co 2 incubators. vn titer was defined as the reciprocal of the highest dilution showing no cytopathic effect (cpe). the mab against ibv s1 protein was diluted to 2 g/ml with pbs and 50 l was added to each well of a 96-well microplate (maxisorp; nunc, denmark). after incubation overnight at 4 • c, plates were washed three times with t-pbs. next, 300 l of t-pbs containing 5% skim milk was added and incubated at room temperature (rt) for 1 h. after washing three times with t-pbs, 50 l of s1 antigen was added to each well and incubated at rt for 1 h. for the recombinant s1 elisa, supernatant of 293t cells transfected with the expression plasmid pcaggs-s1-his was used. for the native spike elisa, lysate of ibv-infected ck cells was used as the antigen. ck cells were infected with ibv at a moi of 0.2 and extracted 72 h post-infection by treatment with ripa buffer (containing 1% tritonx-100, 1% sodium deoxycholate, 0.05 m tris-hcl (ph 8.0), 0.1 m nacl, and 1 mm edta). antigens were diluted with dilution buffer (t-pbs containing 5% skim milk) and added to wells. the levels of recombinant and native s1 protein used for the elisa test were selected as the respective concentrations that yielded absorbances (following reaction with the positive serum) of 1.0 to 1.5. the contents of the wells were washed with t-pbs; an aliquot (50 l) of sera (diluted 1:100 with dilution buffer) was dispensed to each well, and plates were incubated at rt for 1 h. in the recombinant s1 elisa, primary sera were pre-treated with 1 g/ml of purified his-tagged ibv e protein at 4 • c overnight to remove antibodies against the his-tag peptide. the contents of the wells were washed with t-pbs; an aliquot (50 l) of peroxidaseconjugated anti-chicken immunoglobulin (donkey anti-chicken igy (h + l) (jackson)) was dispensed to each well, and plates were incubated at rt for 30 min. the contents of the wells were washed with t-pbs; an aliquot (100 l) of tmb substrate kit (dako) was dispensed to each well, and plates were incubated at rt for 15 min. after incubation, the enzymatic reaction was stopped by addition of 100 l/well of 1 m sulfuric acid. the absorbance was measured using a spectrophotometer (versamax), with a 450-nm and 650nm filter. a hyperimmune serum against ibv, which was prepared by immunization with attenuated and inactivated vaccines, was used as the positive serum. separate work (data not shown) using chicken antisera against ibv e, m and n proteins and anti-s2 mouse mab (described above) confirmed that native s1 elisa does not react with antibodies raised against ibv component proteins s2, e, m, or n. antisera against ibv e, m and n were prepared from chickens immunized with purified recombinant proteins expressed from e. coli and it was confirmed that these antisera reacted to ibv component by elisa (data not shown). all statistical analyses were performed with ezr (saitama medical center, jichi medical university), which is a graphical user interface for r (the r foundation for statistical computing) (kanda, 2013) . the constructed expression plasmids, pcaggs-s1-t4-his and pcaggs-s1-his, were transfected into cells and the recombinant protein was expressed as secreted protein. the secreted s1 protein was purified using ni sepharose and analyzed by western blotting. the result of western blotting under denaturing condition showed that recombinant s1 protein expressed from 293t cells ran as a broad band of nominal molecular weight ranging from 100 to 150 kda. the size range of this band was increased compared to that of native s1 protein (derived from ibv propagated in chicken embryonated eggs), with the native protein running on the denaturing gel as a single sharp band at a nominal size of approximately 100 kda (fig. 2) . recombinant s1 protein derived from 293 gnti − cells, which lack the ability to synthesize complex n-glycans, ran on the denaturing gel as a sharp band at a nominal molecular weight of 75 to 100 kda, with a size smaller than that observed for recombinant protein expressed from 293t cells (fig. 2) . recombinant s1 protein expressed in cef cells (cells that are derived from the natural host for ibv) also ran under denaturing conditions as a slightly broader and larger band than native s1 obtained from ibv virions; notably, the protein produced in cef cells ran as a sharper band than protein produced in 293t cells (fig. 3) . these results indicated that recombinant s1 protein might have an unnatural glycosylation pattern. using western blotting under non-denaturing conditions, we examined the conformation of recombinant s1 protein produced and secreted from 293t cells. we observed that recombinant s1 protein lacking the fibritin motif existed in monomeric, dimeric, and trimeric conformations. on the other hand, recombinant s1 protein with the fibritin motif existed primarily in the trimeric conformation (fig. 4) . for the immunization experiment, the purified recombinant s1fibritin protein expressed from 293t cells was mixed with adjuvant to generate a s1 inoculum at a concentration of 20 g/dose. vn titer induced by the recombinant s1 protein was lower (p < 0.05) than that induced by the inactivated virus antigen (fig. 5) . to compare the efficacy against ibv infection, ciliostasis was evaluated via ibv challenge in animals previously immunized with s1 or with inactivated ibv virus; as a control, unvaccinated and mock-vaccinated . the asterisk indicates statistical significance (p < 0.05) and the error bar indicates standard deviation. comparison between two groups was performed using t test. protection defined by ciliostasis in the trachea. protection a recombinant s1 4/10 inactivated virus 10/10 unvaccinated control 3/10 pbs-mock 0/5 a birds showing active ciliary movement/total. chickens also were subjected to ibv challenge. following challenge, all (10/10) chickens immunized with inactivated virus antigen retained ciliary movement. in contrast, post-challenge ciliostasis was observed in 60% (6/10) of animals immunized with recombinant s1, 70% (7/10) of unvaccinated animals, and all (5/5) mock-vaccinated chickens (table 1 ). these results indicated that immunization with recombinant s1 protein was not sufficient to provide protection from subsequent ibv infection. to analyze the antigenicity of the recombinant s1 protein, sera obtained from the immunization experiment were analyzed by elisa, using recombinant or native s1 protein as antigen. recombinant s1 elisa showed that sera from chickens immunized with recombinant s1 protein had a slightly high, but not significant (p = 0.14), titer than sera from chickens immunized with inactivated virus (fig. 6a) . native s1 elisa detected similar titers in the sera of animals immunized with recombinant s1 protein compared to those in sera of chickens immunized with inactivated virus (fig. 6b ) (control experiments (data not shown) demonstrated that mock and unvaccinated control groups did not differ from each other in vn and elisa tests of anti-s1 activity.) these results indicated that while recombinant s1 protein retained antigenicity (the ability to induce antibodies against s1 protein), the resulting antibodies was decreased its neutralizing activity, in contrast to those induced by inactivated virus. 6 . indirect sandwich elisa using recombinant s1 protein (a) and the lysate from ibv-infected cells (b). primary sera were collected from immunized chickens and diluted 1:100. in recombinant s1 elisa, primary sera were pre-adsorbed with purified recombinant his-tagged protein to remove the antibodies against the histag peptide sequence. reactivities are shown as the s/p ratio of absorbance at 450 nm and 650 nm. comparisons between three groups were performed using steel-dwass test and n.s. indicated not significant. this study showed that recombinant s1 protein exhibited decreased ability to induce a neutralizing antibody response, although the recombinant s1 induced non-neutralizing antibodies. the elisa using recombinant s1 used sera that already had been adsorbed with his-tag peptides, precluding a role for the purification (his-tag) domain in the induced antibody response. in the elisa using native s1, the native protein may have contained noncleaved spike protein, thus including both s1 and s2 domains as part of the antigen. however, we found (data not shown) that inclusion ("spiking") of s2 protein did not affect the results of the native s1 elisa. this observation is consistent with other reports suggesting that the s2 region contains fewer neutralizing epitopes than does the s1 region; the literature indicates that most epitopes are located in the s1 region (cavanagh et al., 1986; ignjatovic and galli, 1994; kant et al., 1992; koch et al., 1990; promkuntod et al., 2014) . these results excluded the possibility that the other viral components affect the titer in elisa. thus, although recombinant s1 protein induced antibodies, this recombinant protein was decreased the ability to induce neutralizing antibodies, suggesting the recombinant s1 expressed in mammalian cells may change its character. in a separate test, we observed that antigen from inactivated virus that was denatured by boiling (5 min, 95 • c) did not induce neutralizing antibodies (data not shown). this result suggested that the correct conformation is important for s1 protein to induce neutralizing antibodies. in the present study, the recombinant s1 protein used for immunization was expressed in mammalian cells, rather than in avian cells; the expression efficiency of recombinant proteins in avian cells was decreased compared to that in mammalian cells, and expression in avian cells was insufficient to obtain recombinant proteins in the quantities needed for the experiments. the present work also used recombinant s1 expressed as secreted protein because secretion permitted accumulation of recombinant protein to higher levels and made it easy to purify recombinant protein. these modifications, using mammalian cells and expressing recombinant protein as the secreting form, may affect the conformation of the s1. western blotting using denaturing conditions indicated that recombinant s1 protein expressed in 293t cells exhibited increased (and less uniform) sizes compared to the native protein. in contrast, recombinant s1 protein expressed in 293 gnti − cells (which lack n-acetyl-glucosaminyltransferase i activity and so do not generate complex n-glycans) ran as a sharper band of a size similar to that of native s1 protein. together, these data indicated that the glycosylation pattern of recombinant s1 protein might differ from that of native s1, and that this difference reflects the level and nature of n-glycosyl modifications. we hypothesize that the native spike protein is typically decorated with high-mannose or hybridtype n-glycans, and/or that the complex-type glycans decorating the native spike proteins are few in number and are homogeneous. it is known that glycosylation characteristics differ between species, and glycosylation affects the conformation of glycoproteins (helenius, 1994) . therefore, glycosylation pattern differences may affect the conformation and antigenicity of the s1 protein. furthermore, glycans attached on the surface of the envelope protein are known to mask antigenic sites, thereby permitting evasion of the host immune system (sun et al., 2011; zhang et al., 2015) . we infer that recombinant s1 protein antigenic sites also may be masked by glycans. from these hypotheses, there is the possibility that s1 expressed in other expression systems, which have different glycosylation character, also changes its antigenicity. other reports suggest that the s2 region of the ibv spike protein is important for the correct conformation of s (callison et al., 1999; promkuntod et al., 2013) . therefore, the recombinant s1 protein secreted in this study might assume a non-native conformation. we hypothesize that expression of the s1 domain without the s2 region results in the assumption of an unnatural conformation, resulting in unnatural glycosylation patterns, or that unnatural glycosylation of s1 protein causes the recombinant peptide to assume an unnatural conformation. recombinant s1 protein may expose hidden epitope(s) that are normally latent in the protein. however, some researchers have reported that secreted recombinant s1 protein, when expressed fused to the trimerization motif peptide, binds the cell receptors (promkuntod et al., 2013; wickramasinghe et al., 2011) . these reports suggested that the secreted s1 protein retains receptor binding ability. in addition, expression of other viral envelope proteins (specifically, human immunodeficiency virus (hiv) gp120 and influenza virus hemaglutinin (ha)) as secreted proteins have been reported to induce neutralizing antibodies (pancera et al., 2005; wei et al., 2008) . these reports indicated that secreting envelope proteins with a trimerization motif is an effective method for creating vaccine antigens. additionally, cavanagh (2007) reported that ibv s1 protein was sufficient for the induction of neutralizing antibodies (thought s1 was not sufficient for protection against ibv infection). furthermore, cytotoxic t-lymphocyte (ctl) activity induced by immunization of nucleocapsid protein (np) has been shown to be important for virus clearance (collisson et al., 2000; seo and collisson, 1997) . however, in the present study, while immunization with recombinant ibv s1 (expressed as a secreted protein fused to the fibritin motif) induced antibodies, the response mainly consisted of non-neutralizing antibodies. therefore, recombinant s1 protein (expressed as a secreted protein in mammalian cells) may be insufficient for use as a vaccine antigen. recently, virus-like particle (vlp) -based antigens have been tested as new candidates for subunit vaccines against ibv (cavanagh, 2003; lv et al., 2014) . receptor binding domain (rbd) peptides also have been tested as vaccine candidates against severe acute respiratory syndrome (sars) coronavirus (jiang et al., 2012) . these alternative approaches to antigen production may overcome the challenges observed in the present work. in conclusion, we demonstrated that recombinant s1 protein (expressed as a secreted protein in mammalian cells) was decreased the ability to induce neutralizing antibodies in chicken. we infer that the recombinant protein lacked conformational epitopes as a result of changes in glycosylation and changes in conformation compared to the native s1 protein. further research will be required to identify factors necessary to establish a recombinant s1 antigen that retains native neutralizing antigenicity. classification of ibv s1 genotypes by direct reverse transcriptase-polymerase chain reaction (rt-pcr) and relationship between serotypes and genotypes of strains isolated between 1998 and 2008 in japan herpes simplex virus glycoprotein b binds to cell surfaces independently of heparan sulfate and blocks virus entry avian infectious bronchitis virus: a possible cause of reduced fertility in the rooster a versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine infectious bronchitis virus s2 gene sequence variability may affect s1 subunit specific antibody binding recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism coronavirus ibv: virus retaining spike glycopolypeptide s2 but not s1 is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection amino acids within hypervariable region 1 of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes relationship between sequence variation in the s1 spike protein of infectious bronchitis virus and the extent of cross-protection in vivo severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus coronavirus avian infectious bronchitis virus construction and immunogenicity of a recombinant fowlpox vaccine coexpressing s1 glycoprotein of infectious bronchitis virus and chicken il-18 cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry breadth of protection of the respiratory tract provided by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous serotypes influence of inoculation site of combined oil-adjuvanted vaccine on the antibody response in chickens how n-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum the s1 glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens roadmap to developing a recombinant coronavirus s protein receptor-binding domain vaccine for severe acute respiratory syndrome investigation of the freely available easy-to-use software "ezr" for medical statistics location of antigenic sites defined by neutralizing monoclonal antibodies on the s1 avian infectious bronchitis virus glycopolypeptide antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions production and immunogenicity of chimeric virus-like particles (vlps) containing the spike (s1) glycoprotein of infectious bronchitis virus (ibv) phylogenetic analysis of avian infectious bronchitis virus strains isolated in japan efficient selection for high-expression transfectants with a novel eukaryotic vector soluble mimetics of human immunodeficiency virus type 1 viral spikes produced by replacement of the native trimerization domain with a heterologous trimerization motif: characterization and ligand binding analysis contributions of the s2 spike ectodomain to attachment and host range of infectious bronchitis virus mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous n-glycosylation by a tetracycline-inducible n-acetylglucosaminyltransferase i-negative hek293s stable mammalian cell line specific cytotoxic t lymphocytes are involved in in vivo clearance of infectious bronchitis virus evaluation of recombinant fowlpox virus expressing infectious bronchitis virus s1 gene and chicken interferon-␥ gene for immune protection against heterologous strains infectious bronchitis virus variants: a review of the history, current situation and control measures induction of protective immunity in chickens vaccinated with infectious bronchitis virus s1 glycoprotein expressed by a recombinant baculovirus glycosylation site alteration in the evolution of influenza a (h1n1) viruses expression of the infectious bronchitis virus spike protein by recombinant vaccinia virus and induction of neutralizing antibodies in vaccinated mice relationship between serotypes and genotypes based on the hypervariable region of the s1 gene of infectious bronchitis virus protection of chickens against infectious bronchitis by a recombinant fowlpox virus co-expressing ibv-s1 and chicken ifngamma comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian h5n1 influenza virus binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity multivalent dna vaccine enhanced protection efficacy against infectious bronchitis virus in chickens the protective immune response against infectious bronchitis virus induced by multi-epitope based peptide vaccines protective immune responses induced by in ovo immunization with recombinant adenoviruses expressing spike (s1) glycoprotein of infectious bronchitis virus fused/co-administered with granulocyte-macrophage colony stimulating factor protection conferred by a recombinant marek's disease virus that expresses the spike protein from infectious bronchitis virus in specific pathogen-free chicken hemagglutinin glycosylation modulates the pathogenicity and antigenicity of the h5n1 avian influenza virus the authors wish to thank other members of the laboratory for technical assistance, and thank mr. mcculloch for proof reading of the manuscript. the authors declare that they have no conflict of interest. key: cord-323072-4rsgeag7 authors: han, xueqing; bartlam, mark; jin, ying-hua; liu, xiangtao; he, xiaojing; cai, xuepeng; xie, qingqe; rao, zihe title: the expression of sars–cov m gene in p. pastoris and the diagnostic utility of the expression product date: 2004-12-01 journal: j virol methods doi: 10.1016/j.jviromet.2004.08.015 sha: doc_id: 323072 cord_uid: 4rsgeag7 high-level protein expression is an important means of obtaining large amounts of viral proteins to investigate further their biological properties. to express the membrane (m) protein of sars–cov at high-level in vitro, the m gene fragment was amplified and cloned it into the pichia pastoris expression vector ppiczαa. sds–page and western blotting analysis of the induced products of recombinant yeast transformant indicated that successful high-level expression of m protein was achieved, and that the expression product was similar antigenically to the natural protein. purified recombinant m protein was used subsequently as an elisa antigen for detection of eight serum samples screened previously by whole virus elisa and immunofluorescence assay, and consistent results were obtained. these findings suggest that the recombinant m protein may be useful as a diagnostic reagent. severe acute respiratory syndrome (sars), also called atypical pneumonia, is a human severe respiratory infectious disease that emerged recently in asia, north america and europe. the transmission of sars occurs mainly by direct contact, and its incubation period is 2-7 days. infection is usually characterized by high fever, which is followed a few days, later by a dry non-productive cough and shortness of breath, and may progress to generalized interstitial infiltrates in the lung. death from progressive respiratory failure occurs in between 3-10% of cases . at present, no effective therapy or vaccine is available. a novel coronavirus, named as sars-cov, has been identified as the main causative agent of sars. the virions are 80-140 nm in diameter, with 20-40 nm complex surface projections surrounding the periphery. hemagglutinin esterase type glycoprotein projections were not seen (ksiazek et al., 2003) . the genome of sars-cov is a single-stranded, positive-sense polyadenylated rna molecule of approximately 29,727 nucleotides. the genomic organization is typical of coronaviruses, having the characteristic gene order [5 -replicase (rep) , spike (s), envelope (e), membrane (m), and nucleocapsid (n)-3 ] and short untranslated regions at both termini. the sars-cov rep gene is predicted to encode two polyproteins that undergo co-translational proteolytic processing. there are four open reading frames (orf) downstream of rep that are predicted to encode the four structural proteins s, e, m and n, which are common to all known coronavirus. additionally, sars-cov also encodes several non-structural proteins . 0166 the clinical case definition of sars is essentially one of fever and pneumonia, with or without a contact history. there are many causes of pneumonia and in the absence of a definite history of contact with other patients with sars, therefore, it is difficult to differentiate sars from other causes of pneumonia in clinic diagnosis. laboratory tests that can confirm a diagnosis of sars-cov infection early in the course of the illness are, therefore, a critical clinical need (riley et al., 2003) . since the outbreak of sars in 2003, several laboratory diagnostic methods have been established, including real-time rt-pcr assay, whole-virus-based immunofluorescence assay (ifa), recombinant protein-based enzyme-linked immunosorbent assay (elisa) and immunochromatographic tests, antigencapturing enzyme-linked immunosorbent assay, and western blot (wb) assay. these methods were critically important for controlling the transmission of sars; furthermore, many new laboratory diagnostic methods are being developed (poon et al., 2003 (poon et al., , 2004 shi et al., 2003; guan et al., 2004; he et al., 2004) . the m protein is an important structural protein of the coronavirus. in the cases of other known coronaviruses, it is the most abundant among the structural proteins and can induce antibody-dependent complement-mediated virus neutralization (woods et al., 1987) . in addition, the m protein is also involved in the assembly and budding of virions together with the e protein. (rottier, 1995; de haan et al., 2000; molekamp and spaan, 1997; narayanan and makino, 2001; corse and machamaer, 2000; vennema et al., 1996) . the m protein contains highly conserved glycosylated sequences, and its glycosylation may be related to the interaction between virus and host (de haan et al., 2002 (de haan et al., , 1998 . the important role of the m protein in the life cycle of coronaviruses and inducing immunity make it an attractive target for anti-sars drug research, vaccine development and the establishment of a serological detection assay. here, we report the expression of recombinant m protein in p. pastoris and its antigenicity. the genomic cdna of sars-cov strain bj01 was provided by huada gene company. the p. pastoris expression vector ppicz␣a and yeast host strain gs115 were all purchased from invitrogen company. sera from healthy people and sars patients were collected from peking union medical college hospital between april and june 2003. sample # 1-4 were from healthy people, sample # 5-8 were from four sars patients (age 28-40; two males, two females) during the acute phase of infection, which were collected at day 31, 26, 22, 19 post sars onset, respectively. all four sars patients had a history of contact with other patients infected with sars, and exhibited symptoms including persistent fever (>38.0 • c), cough and shortness of breath for several days before they were hospitalized. patients were subsequently confirmed to be infected with sars by clinical diagnosis combined with laboratory diagnostic methods. according to the published genomic sequence of sars-cov strain tor (poutanen et al., 2003) , a pair of primers were designed and synthesized. the sequence of the primers were 5 -gagccgcggccgctcaatgt-ggtcattc-3 (forward primer) and 5 -cgttctaga-tgtactagcaaagcaata-3 (reverse primer), which carried a sacii and xbai restriction site, respectively. the primers were used to amplify the m gene fragment without its transmembrane domain from the genomic cdna of sars-cov strain bj-01. the pcr reaction contained 5 l of cdna, 25 pmol of each of two primers, 5 l of buffer concentrate, 4 l of dntp and 0.5 l of ex taq enzyme (takara, japan), and sterile distilled water up to 50 l. pcr was performed with the following settings: 95 • c for 5 min, followed by 35 cycles at 95 • c for 30 s, 50 • c for 45 s and 72 • c for 1 min, and ending with 72 • c for 10 min. the amplified products were then sent to the takara biotechnology (dalian) co. ltd. for sequencing. the determined sequence was analyzed further by dnaman biological software. the m gene fragment and expression vector ppicz␣a were digested by sacii and xbai, respectively. the larger fragments were purified by qiaquick gel extraction kit (qiagen, germany). the concentrations of purified products were measured by beckman dv-600 (beckman, u.s.a). after mixing in a 3:1 molecular ratio, the purified products were ligated by t4 dna ligase at 16 • c overnight and then transformed into escherichia coli top10 competent cells. transformants selected on low-salt lb plates containing zeocin (25 g/ml) were screened by direct colony pcr, and by restriction digestion of purified plasmids. the sequence of the inserts were verified. the sequence data obtained were compared with the sequence of the m gene of sars-cov strain bj01 (accession number ay278488). the transformation of yeast cells and screening of transformants, as well as expression in p. pastoris, were performed as described previously (david and james, 1998) . the recombinant plasmid was linearized by the restriction enzyme saci, the purified products were transformed into yeast cells gs115 and transformants were selected on low-salt md plates containing zeocin (25 g/ml). the colonies of recombinant yeast cells with high copies of the m gene were screened on lowsalt md plates containing different concentrations of zeocin (500 g/ml, 1 and 2 mg/ml). a screened colony of yeast cells was selected and inoculated into 100 ml of bmmy in the presence of zeocin (25 g/ml). the culture was grown at 28-30 • c until the optical density at 600 nm (od 600 ) reached 4.0. the culture was then centrifuged at 4 • c at 3000 × g for 5 min, the cells were re-suspended in 1/10 prime volume of mmy, and continued to grow at 28-30 • c until the optical density at 600 nm (od 600 ) reached 2-6. methanol was added every 24 h to a final concentration of 0.5% to induce the expression of recombinant m protein, and the incubation was continued for further 3-4 days. control cultures were processed in parallel. the recombinant m protein was secreted into the culture supernatant as soluble form. the supernatant was harvested by centrifugation and used for analysing the expression level of recombinant m protein by sds-page. the highly expressing pichia colony was selected for inoculation into 500 ml of bmmy, and grown at 28-30 • c until the culture reached an od 600 = 4.0. the culture was centrifuged at 4 • c at 3000 × g for 5 min, the cells were resuspended in 1/10 prime volume of mmy, and continued to grow at 28-30 • c until the optical density at 600 nm (od 600 ) reached 2-6. glycerol and methanol were added every 24 h to final concentrations of 0.5 and 0.8%, respectively, to induce the expression of recombinant m protein, and the incubation was continued for a further 3-4 days. the supernatant of culture was harvested by centrifugation at 8000 × g for 30 min at 4 • c, then ammonium sulphate was added to a final saturation of 70% to precipitate proteins. the pellet was collected by centrifugation at 8000 × g for 1 h at 4 • c and dialyzed to 2000 ml of pbs overnight. the dialysis buffer was replaced every 2 h until it was confirmed that the ammonium sulphate was completely removed. the prepared sample was loaded onto an nta column equilibrated with niso 4 . this process was repeated three times. the loaded column was first washed with a 10-fold column volume of mcac0 (20 mm tris, ph 8.0; 500 mm nacl) to remove the contaminant proteins, and then eluted with 10 ml of 200 mm imidazole in mcac0. the different elution parts were analyzed by sds-page, and the part containing recombinant m protein was concentrated by superfiltration. two microgram of the purified recombinant m protein was separated by sds-page and transferred to polyvinylidene difluoride membrane (pvdf) by standard methods. after blocking with pbst (14 mmol/l nacl, 2.7 mmol/l kcl, 10 mmol/l nahpo 4 , 1.8 mmol/l kh 2 po 4 and 0.02% tween-20) containing 5% non-fat milk for 1 h at 4 • c, the membrane was incubated for 3 h at room temperature with sars-covpositive human serum (1:3000 dilution). after washing three times in an appropriate volume of pbst, the membrane was incubated for 1 h with peroxidase labeled goat antihuman igg (1:1000, no: a-0170, sigma). the membrane was washed three times with pbs-t and once with distilled water, for 10 min each time. finally, the washed membrane was transferred into an appropriate volume of ecl solution (amersham) and reacted at room temperature for about 1 min, then put onto a sensitization film for 1-3 min to develop and fix. the purified recombinant m protein was diluted with 50 mmol/l carbonate buffer (15 mmol/l naco 3 , 35 mmol/l nahco 3 , [ph 9.6]), and then used to coat 96-well plates (costar inc.) (1 g/well) overnight at 4 • c. after washing with pbs-t (pbs containing 0.5% tween-20, ph 7.4) for three times, each well of the plate was incubated with blocking solution (3% bsa in carbonate buffer) for 2 h at 37 • c. the wells were washed three times with phosphatebuffered saline (pbs) containing 0.05% tween-20 (pbs-t). hundred microlitres of the primary antibody (sars patient sera), diluted 1:800 in pbs-t, was added in duplicate and the plates were incubated for 1 h at 37 • c. after four washes with pbs-t, plates were then incubated with goat anti-human igg antibody labeled with peroxidase (1:50,000, no.: a-0170, sigma) for 1 h at 37 • c. tmb [3,3 , 5,5 -tetramethylbenzidine] substrate (100 l/well) was added after seven washes with pbs-t and the wells were incubated for 10 min at 37 • c. fifty microlitres of stop buffer (2 mol/l h 2 so 4 ) was added to each well and the optical density (od) was read at 450 nm in bio-rad550. the cut-off value was defined as the mean od plus three standard deviations calculated from the four negative samples used as control. the m gene fragment without transmembrane domain was amplified from the genomic cdna of sars-cov strain bj01. sequence analysis indicated that the nucleotide sequence of that amplified fragment is completely identical to that of sars-cov strain bj01 (accession number ay278488). the deduced amino acid sequence of the m protein of sars-cov strain bj01 has identities of 41.0% or less with the m proteins of other human or animal coronaviruses (fig. 1) . the amplified product of 348 bp was cloned in the ppicz␣a expression vector, and the sequences of inserts were verified by sequencing (data not shown). the nucleotide sequence alignment performed by dnaman software revealed 100% nucleotide identity between the inserted sequence and that of sars-cov bj01. the m gene fragment was cloned in ppicz␣a expression vector and transformed into yeast host strain gs115. the colonies of recombinant yeast cells with high copies of m gene were screened. after inducing with methanol, the supernatant of culture was harvested by centrifugation. the recombinant protein with a polyhistidine tag at the c-terminus was produced in soluble form in the culture supernatant (fig. 2) . twenty microlitres of the supernatant (20 g of protein) was subjected to sds-page and coomassie blue staining, and a protein band corresponding to the expected molecular mass of 14 kda was revealed in supernatant, which was not present either in the culture before the induction or in the control culture before and after the induction. the recombinant m protein was purified by nickel affinity chromatography and the purity was confirmed by single banding in sds-page (fig. 2) . upon elution from the nta column, the protein was >80% pure as estimated from in order to test the reactivity of the recombinant m protein, western blotting was carried out using sars-cov-positive human serum. the protein band of 14 kda showed a strong and specific reaction with the sars-cov-positive serum, whereas the supernatant of the induced culture of recombinant yeast without the insert of the m gene fragment did not react with the sars-cov-positive serum (fig. 3) . these results confirmed the authenticity of the recombinant m protein. to test whether the recombinant m protein is effective as an elisa antigen for detecting sars-cov patient serum, the sera from four healthy people and four sars patients were used. the mean and the standard deviation obtained using four sars-cov-negative sera were 0.106 and 0.003, respectively. therefore, the cut-off value of ods was determined as 0.206. sera from four sars patients were determined to be positive using only the m protein as an elisa antigen. the mean and standard deviation were 0.610 and 0.004, respectively (see fig. 4 for detailed data). the same sera from four sars patients were also positive by whole sars-cov elisa in parallel, using a diagnostic fig. 4 . detection results of eight human sera by elisa using purified recombinant m protein as antigen.# 1-4: sera from four healthy people, respectively, # 5-8: sera from four sars patients, respectively. kit for antibodies to coronavirus (elisa) obtained from beijing bgi-gbi biotech co., ltd., china (unpublished results). coronaviruses are important pathogens in human and animals, where they cause mostly respiratory and enteric diseases. sars-cov is a newly identified human coronavirus that differs from all previously known human coronaviruses (for example, hcov-229e and hcov-oc43) in both the speed of transmission and the severity of disease. both the lack of understanding and effective methods to control the virus means that the threat of sars still exists, and therefore, there is an urgent need to develop effective vaccines and anti-viral drugs. the key role of the m protein in coronavirus assembly, budding and inducing virus neutralization make it an attractive target for the development of vaccines and drugs, as well as for effective diagnostic reagents. sars-cov differs from other coronaviruses in nucleotide sequence, and the exact function of its m protein is unknown. since sars-cov is a pathogen with high infectivity and mortality, special facilities are required when the live virus is used to prepare viral proteins given the high risk of infection. therefore, it is important to obtain large amounts of m protein by expression in vitro. the m protein is a structural glycoprotein, and the glycosylation modification is important for its activity. a previous report indicated that the recombinant m protein, when expressed in inclusion body form in e. coli, reacted weakly with human sars sera . p. pastoris is a eukaryotic expression system with high efficiency, and proteins expressed in this system can get appropriate post-translational modifications, including glycosylation. therefore, the p. pastoris system is more suitable for expression of the m protein in vitro than e. coli (grinner and tschopp, 1989; kukuruzinska et al., 1987; romanos et al., 1992) . however, we cannot rule out the possibility that the glycosylation sites of the sars-cov m gene in p. pastoris, while suitable for detection of antibodies, may be quite different from those present in the virus. we expressed successfully the m protein of sars-cov strain bj01 in p. pastoris, and the expressed m protein was similar to the native protein in its immune response. this point can be demonstrated by the reaction of recombinant m protein with sars-positive sera in western blotting and elisa. the availability of large amounts of recombinant m protein offers a better chance to study its biological characteristics and its role in sars immunity during infection. due to the lack of an effective vaccine or special therapeutic drugs, the control of sars infection relies mainly on early detection of the virus or its antibodies, and rigid quarantine measures. at present, well-established laboratory diagnostic methods for sars include real-time pcr, wholevirus-based indirect immunofluorescence assay and elisa. in these assays, however, the viral nucleic acid and anti-gens needed were commonly prepared from live pathogen, so there is a strong associated risk of individual exposure to this pathogen. compared with these methods, preparing viral antigens by expression in vitro is both easier and safer. among the proteins encoded by the sars coronavirus, the m protein can induce an immunological response and thus may be used for detection of sars-cov antibodies. elisa with the expressed recombinant m protein of canine coronavirus has been reported, and was demonstrated to be a valid and effective detection method (elia et al., 2003) . in order to evaluate whether our expressed recombinant m protein could be applied as a diagnostic antigen for sars, we used it as an elisa antigen to detect eight collected sars-positive and sars-negative human sera. the results were in complete accordance with those of other assays, thus indicating that the recombinant m protein may be useful as an elisa antigen for detecting specific antibodies to sars-cov in human sera. furthermore, the recombinant m protein offers several advantages over the cell-prepared sars-cov antigen. in summary, the p. pichia system is a viable tool to express the m protein at high-levels in vitro. the availability of large amounts of sars-cov recombinant m protein offers an opportunity to better investigate the biological properties of the m protein during sars-cov infection as well as its immunological role, and thus provide a basis for development of sars-cov vaccine and therapeutic drugs. the recombinant m protein expressed in p. pastoris may have better antigenicity than that expressed in e. coli, and is more suitable for use as a diagnostic antigen. infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles pichia protocols coronavirus particle assembly: 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membrane protein of sars coronavirus we are grateful to hai pang from tsinghua university for technical assistance, and to gewei lian and hongkui deng from peking university for providing us with sera and for help with elisa experiments. we also thank hui wang from oxford university for comments and critical reading. this work was supported by project 973 & 863 of the ministry of science and technology of china (grant no. gz236(202/9): "structural proteomics of sars coronavirus"). key: cord-312332-rwmuucsp authors: dicker, kate; järvelin, aino i.; garcia-moreno, manuel; castello, alfredo title: the importance of virion-incorporated cellular rna-binding proteins in viral particle assembly and infectivity date: 2020-09-10 journal: semin cell dev biol doi: 10.1016/j.semcdb.2020.08.002 sha: doc_id: 312332 cord_uid: rwmuucsp rna is a central molecule in rna virus biology due to its dual function as messenger and genome. however, the small number of proteins encoded by viral genomes is insufficient to enable virus infection. hence, viruses hijack cellular rna-binding proteins (rbps) to aid replication and spread. in this review we discuss the ‘knowns’ and ‘unknowns’ regarding the contribution of host rbps to the formation of viral particles and the initial steps of infection in the newly infected cell. through comparison of the virion proteomes of ten different human rna viruses, we confirm that a pool of cellular rbps are typically incorporated into viral particles. we describe here illustrative examples supporting the important functions of these rbps in viral particle formation and infectivity and we propose that the role of host rbps in these steps can be broader than previously anticipated. understanding how cellular rbps regulate virus infection can lead to the discovery of novel therapeutic targets against viruses. viruses are obligate intracellular pathogens that represent a threat to human health and the economy of countries. viral genomes are typically small, encoding only a few proteins. to circumvent this limitation, viruses hijack host resources to complete their infection cycle. rna is a central molecule in rna virus biology since it functions not only as a messenger for protein synthesis (i.e. mrna), but also as a genome. since viruses cannot encode all the proteins necessary for viral (v)rna replication/transcription, translation and packaging, viruses usurp cellular rna-binding proteins (rbps) [1] [2] [3] [4] [5] [6] [7] [8] . on the other hand, cellular rbps are also central to the antiviral defences [9, 10] . the host cell senses viruses through specialised rbps that recognise unusual signatures present in vrnas, known as pathogen-associated molecular patterns (pamps), leading to the stimulation of interferon production and the activation of mechanisms to inhibit protein synthesis [11] . cellular rbps are thus fundamental components of the virus-host cell battlefield, either sustaining or restricting virus infection. understanding how viruses interact with these cellular proteins can thus be instrumental for the development of novel antiviral therapies. rbps interact with rna forming dynamic complexes known as ribonucleoproteins (rnps), which are critical for mediating gene expression [12] . recently, the repertoire of human cellular rbps was increased dramatically to over 1500 proteins by the development of a proteome-wide approach known as rna interactome capture (ric) [13] [14] [15] . ric employs ultraviolet (uv) crosslinking of 'zero' distance (< 2 å) protein-rna interactions, followed by cell lysis under denaturing conditions, isolation of rbps crosslinked to poly(a) rna via oligo(dt) capture and quantitative mass spectrometry. additionally, new methods based on organic-aqueous phase separation to detect also proteins bound to non-poly(a) rnas have contributed to greatly extend the number of rbps in the cell [16] [17] [18] . while a substantial proportion of these proteins interact with rna using a defined set of well-characterised rna-binding domains (rbds), such as rna-recognition motifs (rrms), many rbps lack them, suggesting the existence of unconventional modes of rna binding [13] [14] [15] . indeed, dozens of novel rbds were later discovered using proteomic-based methods. these domains included enzymatic cores, protein-protein interaction surfaces and dna-binding domains [19, 20] , as well as intrinsically disordered regions that emerged as a prominent mode of rna binding [19, 21] . importantly, both classical and unconventional rbps have been linked to virus infection and immunity, as reviewed before [5] . moreover, analysis by comparative ric of the 'rna-binding proteome' of cells infected with the rna virus sindbis (sinv), revealed that the complement of cellular rbps is pervasively remodelled upon infection [7] . strikingly, many rbps activated by sinv either sustain or repress infection, highlighting the critical roles of cellular rbps as regulators of virus infection. because sinv rna is polyadenylated, ric isolates both host and viral rna. indeed, many of the proteins enhanced by sinv infection were shown by orthogonal approaches to accumulate within the viral replication factories, suggesting a direct interplay with vrna. however, ric will also discover rbps with differential rna-binding activity that interact with cellular rna instead of vrna. several approaches have been developed to elucidate the composition of viral ribonucleoproteins (vrnps). initially, studies on influenza a virus (iav) and vesicular stomatitis virus (vsv) focused on isolating viral rna polymerase complexes, as these should reflect the composition of replicating vrnps to some extent [22] [23] [24] . while useful, these datasets were biased towards direct protein-protein interactions. more recently, different groups have employed diverse approaches to capture vrna together with its interacting proteins, which are then detected by mass spectrometry. all these methods start by 'freezing' protein-vrna complexes. this can be achieved by uv protein-rna crosslinking exploiting the excitability of natural bases upon irradiation with uv light at 254 nm [2] . alternatively, photoactivatable nucleotides, such as 4thiouridine (4su), can be incorporated into nascent vrna, and protein-rna crosslinking is achieved by irradiation with 365 nm uv light [1, 3] . uv crosslinking only promotes covalent bonds between proteins and rnas placed at 'zero' distances, thus displaying high specificity with the cost of low efficiency. another approach typically used to immobilise protein-rna interactions is formaldehyde crosslinking [4, 6, 8] . while more efficient than uv, formaldehyde also induces covalent bonds between proteins, which promotes the isolation of indirect binders through protein-protein bridges. once protein-rna complexes are 'frozen', the second step is to isolate the vrna together with its covalently bound proteins. typically, vrna is captured with antisense dna probe(s). this approach substantially enriches for vrna [2, 4, 8] , although can still co-purify rnas in a non-specific fashion through the formation of partial hybrids with non-target sequences. if vrna is labelled with 4su (see above), it can alternatively be isolated by biotinylation of the sulfhydryl group in the 4su base, coupled with streptavidin pull down [6] . since exposed cysteines on protein surfaces can also be biotinylated, it is not surprising that this approach identifies a large proportion of cellular proteins lacking rna-binding activity. collectively, these methods have been applied to several virus models including dengue virus (denv) [2, 3, 8] , zika virus (zikv) [8] , sinv [6] , poliovirus [1] and human immunodeficiency virus 1 (hiv-1) [4] , representing important advances towards understanding vrnp composition. the interactomes between host proteins and incoming viral particles have been recently unravelled using a novel technique named vir-clasp [25] . vir-clasp uses 4su to label the genomic rna in infected cells, which is later incorporated into viral particles. viruses released to the supernatant harbouring 4su-labelled genomes are then used in very high multiplicity to infect new cells. infection is followed by crosslinking with uv light at 365 nm, solid-phase capture of protein-rna complexes and protein identification by mass spectrometry. vir-clasp has been successfully applied to chikungunya virus or iav and have revealed important regulators of the initial steps of the infection cycles of these viruses [25] . despite their strengths and limitations, all these studies agree that both classical and unconventional rbps engage with vrna and play critical roles in infection. in general, infection cycle of rna viruses consist of three main phases ( fig. 1) : (1) attachment of the viral particle to the cell and entry; (2) viral genome replication and expression; and (3) assembly and egress of the virus progeny [26] . the viral cycle begins when a virus binds to specific surface receptors and enters a host cell. once inside the cell, the genomic rna of positive-stranded viruses engages directly with the host protein synthesis machinery to produce the viral proteins required for replication. conversely, negative-stranded rna viruses typically carry the transcription machinery with them into the newly infected cell. retroviral rna must be reverse transcribed into dna, which is then imported into the nucleus and integrated into the host chromosome to be transcribed by the cellular rna polymerase ii. the next stage involves the synthesis of the components (i.e. viral structural proteins and genomes) required to assemble the progeny viral particles that leave the producer cell to infect a new one. virtually all these stages can be regulated by cellular rbps (fig. 1) , although most work so far has focused on viral replication and protein synthesis [27] [28] [29] [30] [31] [32] . however, new discoveries have highlighted the importance of cellular rbps in the assembly of viral particles as well as in the very initial steps of infection, when the rna genome is delivered into a newly infected cell. these steps are the focus of the present review. one of the main open questions is how vrna is recognised specifically by viral proteins with limited, if not non-existent, specificity for vrna during the assembly of viral particles [33, 34] , and whether highspecificity cellular rbps may cooperate with viral proteins to recognise the vrna. moreover, vrna is generally highly structured [35, 36] . thus, it is plausible that cellular rbps with helicase and chaperone activities facilitate the binding of capsid/nucleocapsid proteins across the vrna to enable rna packaging into virions. immediately upon entry into the cell, vrna must either be translated (positive-stranded rna viruses), transcribed and replicated (negative-stranded rna viruses) or reverse transcribed (retroviruses) without alerting the host immune defences, especially in the initial steps of infection where the virus is more vulnerable. how viruses achieve this remains largely unknown; however, recent evidence supports the idea that in certain cases such as hiv-1, some of the lifecycle of the vrna takes place inside the 'protective walls' of the capsid shell [37] [38] [39] . another interesting idea is whether vrnas carry the necessary cellular components from the producer cell to maximise the efficiency of the subsequent initial steps of infection. different proteomic studies have identified hundreds of cellular factors within the particles of several rna viruses [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] , many of which are rbps. here, we discuss the 'knowns' and 'unknowns' of the roles that virion-incorporated cellular rbps could play in the assembly of viral particles and the early steps of infection in the new host cell. in the last two decades, the composition of the particles of several rna viruses has been elucidated by proteomics. however, no work has currently focused on identifying the scope of cellular rbps that are present in virions. to gain insights into this question, we compiled the proteomes of particles from ten different human rna viruses (fig. 2 ) [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] . upon building the superset of host proteins present in virions (table s1a) , we cross-referenced it to the complement of experimentally determined human rbps [15] as well as gene ontology (go) terms and protein domains related to rna binding. the resulting in virion rbps (ivrbps) are listed in table s1b , including the dataset in which each protein was reported. we discover that virions contain many rbps (fig. s1a ), 58 % of which harbour known rbds, while the other 42 % were just recently classified as rbps by ric studies and their modes of rna binding remain unknown [15] . analysis of go biological process terms (fig. s1b ) and proteinprotein interaction networks (fig. s2 ) indicate that ivrbps can potentially participate in a wide variety of processes, e.g. transport, exocytosis, response to stress, immune system pathways, cytoskeleton organization, translation, rna processing, rna stability or protein folding. however, the main limitation of the cited proteomic analyses is that budding of the viral particles results in the uptake of a portion of the cytoplasm and plasma membrane. hence, it is challenging to discriminate between abundant proteins randomly incorporated into virions and with no function in virus biology, from those with an active regulatory role. we reasoned that host proteins with a true function in infection are likely to be identified in multiple datasets and across different virus species and cell lines (fig. s3) . strikingly, we noticed that 14 ivrbps are commonly detected in the particles of different viruses (at least 5 viruses out of 10, table s1b ). for example, cofilin 1 (cfl1) was identified in 8 of the 10 viruses studied here. cfl1 is a regulator of actin cytoskeleton reorganisation and has been shown to play a crucial role in measles virus (mev) rnp formation for vrna synthesis [56] , iav assembly and budding [57] and the production of infectious respiratory syncytial virus (rsv) [58] . however, whether the rna-binding activity of cfl1 plays a role in infection requires further investigation. moreover, the importance of cfl1 incorporation into particles should also be further tested functionally, since it could also be due to passive uptake with actin, which is often present in viral particles. many ivrbps such as annexins, heat shock family proteins (hsp), peptidylprolyl isomerase a (ppia -also cyclophilin a), eukaryotic translation elongation factors (eef), heterogeneous nuclear ribonucleoproteins (hnrnp) or poly(rc) binding protein 1 (pcbp1), have been linked to infection in multiple ways (fig. s2) , and here we show that they are incorporated in the particles of several viruses (table s1b) . whether these proteins are incorporated passively due to high abundance or through convergent, virus-driven active mechanisms such as specific interactions with viral proteins or rna, requires further investigation. however, ppia illustrates well why the second option should not be underestimated, as it interacts specifically with the capsid of hiv and this interaction is critical for capsid core stability [59, 60] . whether ppia rna-binding activity plays a role on this recruitment remains unknown. many ribosomal proteins are also present in the superset of ivrbps, together with rna helicases that aid the unwinding of rna (fig. s2 ). interestingly, several ivrbps are restricted to the virions of a certain superclass of viruses. for example, heat shock protein 90 alpha (hsp90aa1), transgelin 2 (tagln2) and hnrnpd are found exclusively in negative-stranded rna viruses. nevertheless, it is important to note that all these virion proteomes have been generated using different i) virion isolation methods, ii) proteomic approaches and iii) data analyses. these divergences are noticeable when comparing datasets generated in closely related viruses or even the same virus (fig. s3 ). for example, each of the six hiv-1 virion proteomes that we examined identified a distinct number of proteins ( fig. 2 ), reflecting differences in virion isolation stringency, proteomic depth and/or statistical thresholds. the analysis of virion proteomes is complicated by potential contamination with cellular proteins, either within extracellular vesicles that copurify with viral particles or via nonspecific interactions with the virus exterior. most of the viruses used in our metanalysis were purified on sucrose cushions or density gradients, and some lack additional purification steps [41, 43, 47, 48, 50, 52] . few of the latter studies showed that the vast majority of their samples were intact viral particles using transmission electron microscopy. interestingly, several studies incorporated further purification strategies: (1) treatment of viral particles with proteases such as subtilisin [40, 44] or proteinase k [45, 46] to remove non-specific binders and vesicles due to an alteration in their density; (2) cd45 immunoaffinity depletion as cd45 is present on microvesicles but not in hiv particles [42] ; (3) solubilisation of lipid bilayers for isolation of hiv cores by including a detergent layer on the sucrose density gradient [49] ; (4) haemadsorption to and elution from red blood cells to select for iav particles with receptor binding and cleavage activity, provided by haemagglutinin and neuraminidase [51] ; (5) sequential affinity purification with a heparin column to select for hcv particles containing the viral e2 protein, followed by antibody-or tag-mediated capture of envelopecontaining particles [53] . by focusing on proteins identified in multiple datasets, we expect to minimise technical differences between datasets, as well as biological factors, such as the incidence of randomly incorporated proteins (see above). however, passive incorporation due to abundance cannot be excluded a priori, and we recommend interested readers to test the abundance of their candidates in proteomic analyses of whole cell lysates, if available. table s1 highlights many of these robustly identified ivrbps and we discuss below several ivrbps with well-known roles in infection, and others that remain poorly characterised. k. dicker, et al. seminars in cell and developmental biology xxx (xxxx) xxx-xxx 2. do cellular rbps participate in viral particle assembly? the cytoplasm of the cell is a hostile environment for vrna due to the presence of antiviral sensors. these cytoplasmic immune surveillance factors are mostly specialised rbps that detect viral doublestranded (ds)rna, under-methylated cap structures or triphosphate 5′ ends, which are common signatures present in vrna [61] [62] [63] [64] . the recognition of vrna as 'foreign' nucleic acid can trigger the antiviral response and direct the rna decay machinery towards the vrna [65, 66] . hence, it is crucial for vrnas to effectively engage with cellular rbps to increase their stability and translatability [67] . upon accumulation of viral rna and proteins, these components must travel to the virus assembly areas and, in some instances, vrna trafficking has been proposed to be mediated by the cytoskeleton [68] [69] [70] [71] [72] [73] . the role of protein chaperones in the trafficking of vrnps is becoming increasingly evident. many cellular chaperones and peptidylprolyl cis/trans isomerases (immunophilins) are hijacked by viruses to enable viral replication and translation by folding viral proteins [74, 75] . interestingly, new proteome-wide approaches have revealed that both chaperones and immunophilins interact with rna in vivo and exert critical functions in rna metabolism, including regulatory roles within the spliceosome, control of transcription and mrna stability and ribonucleoprotein remodeling [13] [14] [15] . moreover, their rna-binding surfaces have recently been revealed, suggesting that their ability to act on proteins and bind to rna is interconnected [19] . ric analysis of sinv-infected cells revealed that the rna-binding activity of several protein chaperones and immunophilins is enhanced upon infection and that their functional perturbation severely impairs viral fitness [7] . the importance of host chaperones in infection is well illustrated by hiv-1 rnps. viral genomic rna interacts with staufen double-stranded rnabinding protein 1 (stau1) in the cytoplasm forming hiv-1-dependent stau1-rnps [76] [77] [78] . compositional characterisation of these rnp complexes revealed the presence of the chaperones hspd1 (hsp60), hspa1 (hsp70) and hspa8 (hsc70) 78]. it has been proposed that the stress-inducible chaperone hsp70 and its constitutive form, hsc70, interact with nascent hiv-1 gag-vrna complexes and hold them in an assembly-competent conformation during their transport towards the plasma membrane [79] . strikingly, these proteins are also present within hiv-1 virions (fig. s2c) , suggesting that they associate with vrna at different stages of the infection cycle 78]. the participation of hsc70 in viral particle assembly is also illustrated by hepatitis c virus (hcv) infection. hsc70 interacts with hcv rna and colocalises with the hcv capsid and e2 proteins at assembly sites [80] . knockdown of hsc70 [80] or abrogation of protein activity with allosteric inhibitors [81] decreases viral particle production, but has no effect on viral replication. whether direct binding of hsc70 to the hcv rna genome contributes to the formation of infective virions is still unclear. the roles of cellular rna-binding chaperones, therefore, may span replication, translation, trafficking and virion assembly (fig. s2) . cellular rbps are also thought to contribute to capture the vrna, amongst all the cellular rnas in the cytoplasm, to be packaged into the virion. several viruses, such as the bacteriophage ms2, encode capsid proteins with high specificity and affinity for stem loops present within the vrna [82] . however, other viruses, such as hiv-1, do not encode for any protein with high specificity and affinity for the vrna. hence, how these viruses select and package their vrna into virions against cellular counterparts remains poorly understood. for example, it was believed that the hiv-1 nucleocapsid subunit of the viral polyprotein gag recognises vrna from the pool of cellular rnas by a unique binding event at a cis-acting packaging element in the 5′ leader of the vrna [83] . however, a recent study using clip-seq (crosslinking and immunoprecipitation followed by sequencing) discovered that, in addition to the binding site in the 5′ leader, gag interacts in the cytoplasm with other discrete sites across the hiv-1 rna [34] , placed at the rev recognition element (rre) and 3′ utr. however, when the vrna reaches the plasma membrane, this binding pattern changes dramatically, and gag covers virtually the whole vrna. how gag switches from selective to non-selective binding is not understood. since many cellular rbps interact with hiv-1 rna and gag [4, 78, 84] , it is tempting to speculate that they might cooperate with hiv-1 nucleocapsid to recognise hiv-1 genome in the cytoplasm of the infected cell. in agreement, it has been proposed that stau1 assists gag interaction with hiv-1 rna [85] . the resulting rnp is transported through the cytoplasm to the plasma membrane where stau1 facilitates the multimerization of gag on the vrna [86] . this multimerization step enables the formation of immature virus particles, where the hiv-1 genomic rna acts as a nucleation site for assembly [87, 88] . interestingly, stau1 only modulates gag assembly once the tail of gag has anchored into the plasma membrane suggesting that stau1 may play a role in ensuring the rnp is delivered to the membrane [86] . stau2 has also been shown to promote the activity of hiv-1 rev protein in exporting vrna from the nucleus, along with dexh-box helicase 9 (dhx9) and arfgap with fg repeats 1 (agfg1) [89] [90] [91] . together, these studies suggest that staufen proteins play crucial roles in the transport of hiv-1 rnps from the nucleus to the plasma membrane for viral particle formation. additional studies should be carried out to determine if other cellular rbps cooperate with viral capsids and nucleocapsids to recognise the vrna and to enable vrnp trafficking. the cellular endosomal sorting complex required for transport (escrt) is a large multi-component machinery comprised of five complexes, escrt-0, escrt-i, escrt-ii, escrt-iii and vps4, which assemble sequentially in a multi-step manner following ubiquitination of escrt-0 [92] . the escrt machinery is hijacked for viral particle release across most types of viruses including retroviruses, filoviruses, arenaviruses, paramyxoviruses, rhabdoviruses, flaviviruses, reoviruses and picornaviruses [93] . viruses can either hijack escrt for assembly and budding at the plasma membrane or for budding at endosomal membranes into the cytoplasm. escrt is typically involved in the budding of vesicles from the endosomal membrane into the endosomal compartment. however, escrt proteins can also perform a "reverse topology" membrane fission. escrt is the only cellular machinery with such ability and this is perhaps why so many viruses have evolved to exploit it [93] . escrt-i/ii essentially function to recruit the escrt-iii protein chmp4 (charged multivesicular body protein 4) to membranes where the formation of escrt-iii filaments is then promoted. the formation of escrt-iii filaments drives the budding of the membrane, but it is not understood exactly how this is achieved. programmed cell death 6 interacting protein (pdcd6ip or alix) recruits chmp4 and the escrt-iii complex to the plasma membrane. several studies have shown that many viral structural proteins initiate escrt driven budding from the membrane by recruiting alix. examples include gag of hiv-1 [94] , the accessory c protein of human parainfluenza type 1 [95] , ebola virus vp40 [96] , px of hepatitis a virus [97] and the ns3 protein of denv [98] and yellow fever virus [99] . although alix has largely been studied from a proteo-centric perspective, ric studies have revealed that it is endowed with rnabinding activity [15] . in addition, alix has recently been implicated in the recruitment of cellular rnas to extracellular vesicles [100] . importantly, the interaction of hiv-1 gag with alix relies on the presence of vrna. the n-terminal bro1 domain and the central v-shaped domains of alix interact with hiv-1 nucleocapsid and p6 domains of gag respectively [101, 102] . interestingly, the interaction between alix bro1 domain and hiv-1 nucleocapsid is disrupted by rnase treatment, suggesting that the vrna molecule forms a bridge between the two proteins. both alix bro1 and nucleocapsid are highly positively charged and are believed to establish electrostatic interactions with the phosphate backbone of the vrna. the recruitment of alix by vrna raises the question of whether alix is recruited to the hiv-1 rnps in the cytoplasm or in the plasma membrane as the virus assembles 102]. additionally, proteomic-based compositional analysis of the zikv and denv rnps revealed that alix also interacts with these vrnas [8] , although the exact role of these interactions remain unexplored. another class of unconventional rbps implicated in virus release is the annexin protein family. annexins are calcium-dependent membrane binding proteins that carry out a diverse range of functions. they can reversibly associate with components of the cytoskeleton or with regulatory proteins and rnas that mediate stress-induced intra-and inter-cellular signalling [103] . annexin a2 (anxa2) is a multifunctional, ubiquitously expressed protein with roles in membrane domain organisation, membrane fusion, vesicle aggregation and exocytosis [104] . it has been shown to play a role in cell attachment and entry or replication of enterovirus, rsv, hcv and iav, amongst other viruses [104] . however, we still lack a molecular understanding of the relationship between anxa2 rna-binding activity and its regulatory roles in infection. anxa2 was shown to be involved in the formation of the hcv replication complex and can bind both hcv rna, in a sequence-specific manner, and non-structural protein ns5b forming a ternary complex [105] . the silencing of anxa2 has no effect on vrna levels suggesting that it does not influence replication. instead it significantly reduces the number of produced viral particles indicating that anxa2 plays a role in hcv virion formation or release, although the mechanism by which this is achieved remains unknown [106] . anxa2 has also been shown to be involved in virion assembly of mev through recruitment of the viral matrix protein to the plasma membrane [107] . anxa2 and other members of the annexin family have been identified within the viral particles of ebola and marburg virus [48] , hiv-1 [42] , iav [44] , vsv [45] , rift valley fever virus [52] and rsv [47] (table s1b ). together, these data suggest that anxa proteins may be involved in the formation and release of particles of a broad range of viruses. however, the mechanisms of action of anxa proteins and their rna-binding activity in infection remains largely uncharacterised. the compositional analysis of different virions has also revealed the presence of some members of the adp ribosylation factor (arf) gtpase family, suggesting a role in viral particle formation and/or release [55] . this is a ras-related subfamily fundamental for the regulation of vesicle formation, trafficking and docking at target membranes, and has been implicated in the infection cycles of many pathogens [108] . arfs have been involved in the recruitment of hiv-1 rnps to the plasma membrane and the release of hiv-1 particles [109] , and arf1 regulates hiv-1 trafficking to the virological synapse [110] . additionally, arf1 is necessary for hcv rna replication and production of infectious particles [111] . the possibility that the rna-binding activity of arf1 facilitates the localisation of vrnps at the sites of viral particle assembly and egress calls further investigation. after entry into the host cell, most positive-stranded rna viruses release the genomic mrna into the cytoplasm to be immediately translated into the viral replicase complex by the cellular translation machinery (fig. 1) . this is the case for sinv, a representative member of the alphavirus genus [112] . it was recently described that sinv infection of mammalian cells produce two subpopulations of infectious viral particles. one of them, known as 'heavy' viral particles, exhibits an enhanced translation of the vrna once inside the newly infected cell [113] . only one homogeneous population of virions is released from the other natural host of sinv, mosquito, with an infectivity that matches that of 'heavy' viral particles. authors showed that virion-incorporated host-derived factors, including the ribosomal components rps14, rps18 and 18s ribosomal rna, and the cellular rna binding motif protein 3 (rbm3), are responsible for vrna superior translation. rbm3 has been shown to promote translation in different contexts [114] and its incorporation into sinv particles was recently confirmed [54] , together with other ribosomal proteins and many cellular rbps (table s1c) . interestingly, while sinv particles with increased infectivity can be produced in either mammalian or mosquito cells, the enhanced vrna translation only occurs in a newly infected mammalian cell, but not in a mosquito cell [113] . this striking phenomenon is recapitulated in animal models and in other alphaviruses [115] . this suggests that rbps pre-loaded in the viral particle may interact specifically with mammalian proteins controlling translation initiation. the identity of these mammalian proteins remains unknown and uncovering them will be a challenge. however, recent work has shown that the mammalian ribosome establishes interactions with hundreds of cellular rbps, which represent potential regulators of translation [116] . as many of these ribosome interactors are indeed found inside virions (table s1), we anticipate that they may accompany the vrna into the newly infected cell. once the vrna is released into the cytoplasm, these proteins may facilitate translation initiation by interacting with the ribosome. to what extent virion composition determines translation of incoming viral genomes awaits mechanistic characterisation. moreover, whether this phenomenon can be extended to other rna viruses beyond sinv remains to be explored. particles of negative-stranded rna viruses contain a viral rna-dependent rna polymerase and accessory proteins to transcribe the vrna into mrna upon entry into the host cell (fig. 1) . later, this 'transcriptase' complex is modified to form a 'replicase' complex that synthesizes an intermediary positive-stranded rna that serves as a template for producing new copies of the negative vrna genome [117] . thus, viral transcription and replication are two separate processes controlled by distinct vrnp complexes. a representative example is vsv that forms two complexes composed of the viral rna polymerase (l) and different viral and host proteins [22] . the vsv transcriptase complex contains the viral proteins l and p and the host proteins rna guanylyltransferase and 5′phosphatase (rngtt) capping enzyme, eef1a and hsp60 [22, 118] . the last two proteins are important for the rna polymerase activity of l and can be found within virions (table s1b ). in addition, ppia is also bound to the vrnp complex inside purified vsv particles and is used for post-entry primary transcription [119] . interestingly, the mentioned host rbps are absent in the vsv replicase complex, which contains n, l and p viral proteins and synthesizes the plus strand 'antigenome' vrna used as template for copying the viral genome [22] . this suggests that differential association of the rna polymerase complex with host proteins may regulate the switching from transcription to replication. the cellular rbp hnrnpu interacts with the vsv leader rna that is required for vrna replication and inhibition of cellular transcription [120] . this protein is also packaged into vsv particles, but the potential association of virion-incorporated hnrnpu with the transcriptase or replicase complexes and its activity in the early steps of vsv infection is not well understood. in iav, the viral polymerase consists of three subunits: pb1, pb2 and pa. the cellular chaperone hsp90 is involved in the transport of pb1 and pb2 to the nucleus and modulates the interaction of pa with pb1 [121] . after binding of pa to pb1-pb2, hsp90 dissociates from the complex. in this way, authors suggested that hsp90 regulates the assembly of the mature trimeric polymerase complex. on the contrary, hsp90 was found to be associated with the trimeric polymerase complex in a different study [23] . hsp90 relocates to the nucleus after iav infection [121, 122] , and this could reflect its interaction with newly synthesized polymerase subunits. interestingly, hsp90 has been found inside purified iav particles (as well as in other viruses, table s1b), but k. dicker, et al. seminars in cell and developmental biology xxx (xxxx) xxx-xxx the possibility that virion-incorporated hsp90 participates in transport of the incoming vrnp to the nucleus and/or the initial viral transcription has not been explored so far. in vitro, hsp90 stimulates iav rna synthesis by interacting with pb2 122] , and binding of hsp90 to pb2 is increased during rna synthesis [121] . authors suggested that hsp90 may participate in the early steps of transcription elongation by dissociating the polymerase from the vrnp and stabilizing the different subunits during their transfer between rna templates [121] . however, further investigation in the context of iav-infected cells is required to elucidate the role of hsp90 and its recently described rna-binding activity [19] in early iav transcription. it was believed that the capsid shell protecting hiv-1 rna was disassembled upon entry into the host cell, allowing viral rna and proteins to associate with host rbps in the cytoplasm of the newly infected cell to initiate reverse transcription. however, more than a decade of intensive work has challenged this model by discovering that reverse transcription takes place inside the capsid core [37] [38] [39] [123] [124] [125] , protected from the hostile intracellular environment, and uncoating occurs at the nuclear pore complex [126, 127] or even inside the nucleus [128] (fig. 1) . recently, it was shown that the presence of pores in the capsid hexamers allows deoxynucleotides to traverse the capsid shell to feed reverse transcription [38] . however, at only 8 å wide, these pores are too small to allow proteins to pass through. hence, host rbps participating in reverse transcription must be present inside the capsid core prior to cell entry, likely being incorporated in the producer cell during the assembly of viral particles. the idea of bringing up key proteins from the producer cell is well characterised for negative stranded rna viruses, which incorporate viral polymerase complexes including host factors within the viral particles to enable replication upon entry in the host cell (see above). similarly, hiv-1 particles contain reverse transcriptase and integrase, two viral enzymes that are critical in both reverse transcription and proviral dna integration into the host chromosome. recently, integrase was shown to bind not only dna but also specific structures in hiv-1 rna within virions, and these interactions are critical for the correct localization of the vrnp inside mature virions [129] . several studies have revealed that specific cellular ivrbps either promote (e.g. upf1 rna helicase and atpase [upf1] [130] , y-box binding protein 1 [ybx1] [131] , dhx9 [132] , eef1a [133, 134] , ppia [135] and aminoacyl-trna synthetases [136] ) or inhibit (e.g. mov10 risc complex rna helicase [mov10] [137] , pyruvate kinase m1/2 [pkm] [138] , enolase 1 [eno1] [139] ) reverse transcription (table s1e) . nonetheless, the scope of cellular rbps hijacked into hiv-1 particles and whether they play critical roles in these initial steps of infection inside the virion is not well defined yet. as example, upf1 is a cytosolic rna helicase that plays crucial roles in hiv-1 infection and is incorporated into virions (table s1e) . strikingly, reverse transcription fails if virions are generated in cells depleted of upf1 or expressing an atpase-defective upf1 mutant [130] . authors suggested that upf1 could mediate the remodelling the vrnp to facilitate reverse transcription or, alternatively, promote the annealing of trna lys3 primer to the viral genome, as shown in other helicases such as dhx9 [130, 140] . conversely, recent evidence suggested that dhx9 participates in the elongation phase of reverse transcription but not in the annealing of trna lys3 to the vrna [132] . further research is required to elucidate the exact mechanism of action of upf1, dhx9 and other cellular rbps during hiv-1 reverse transcription. interestingly, cellular rbps have also been found in hepatitis b virus (hbv) nucleocapsids. hbv is a 'gapped' dna virus but initially a singlestranded rna pre-genome (pgrna) is packaged into viral particles. the pgrna is reverse transcribed to a circular dsdna within the viral capsid before the virus matures and is secreted from the cell [141] . the pgrna is packaged through recognition of epsilon (ε) stem-loop structure in the 5′ terminus by hbv polymerase (pol). it was shown that eukaryotic translation initiation factor (eif)4e enables this recognition by forming an eif4e-pol-ε rnp complex that is incorporated into the nucleocapsid [142] . reverse transcription of the pgrna then requires hsp90 [143] , which forms a rnp together with other cellular rbps including hsc70 and dnaja1 (hsp40) [144, 145] to enable chaperone-mediated specific binding of hbv reverse transcriptase to pgrna. how the rna-binding activity of these proteins contribute to viral capsid assembly and/or reverse transcription remains poorly understood. finally, other cellular ivrbps such as dead-box helicase 3 x-linked (ddx3x) [145] , apolipoprotein b mrna editing enzyme catalytic subunit 3 g (apobec3 g) [146, 147] and mov10 [148] negatively regulate the very early steps of hbv reverse transcription. ddx3x interacts with hbv polymerase and requires atpase but not helicase activity for minus-strand dna synthesis inhibition [145] ; apobec3 g acts via an unknown deaminationindependent mechanism [147] that may involve direct binding to reverse transcriptase [146] , while mov10 binds directly to hbv rna but not the viral polymerase [148] . however, the precise mechanisms by which these proteins affect hbv dna synthesis still remain unclear. it is an evocative idea that cellular antiviral factors may be also packaged with the vrna into virions in order to interfere with the initial steps of infection. well-known antiviral rbps such as zinc finger ccch-type containing antiviral 1 (zc3hav1), apobec3c and apobec3f are detected in the proteome of purified sinv particles (table s1c) . however, studies exploring their role in the context of the viral particle or the early phase of sinv infection are non-existent to our knowledge. in addition, antiviral rbps might be under-represented in the different virion proteomes analysed here (table s1 ). this is most likely due to 1) the existence of sophisticated mechanisms to avoid the presence of restriction proteins inside virions, or 2) because the viral particles used in these proteomic studies were often produced in highly permissive cell lines with damaged interferon pathway. the first possibility has been widely studied in hiv-1, which excludes antiviral proteins from virions by different mechanisms. for example, serine incorporator 5 (serinc5) and serinc3 are relocalized from cell surface to endosomes by the accessory viral protein nef [149, 150] ; yth n 6 -methyladenosine rna binding protein 3 (ythdf3) is cleaved by hiv-1 protease inside the viral particle [151] , and apobec3 proteins are targeted for degradation by the viral protein vif [152] . ythdf3 has a strong affinity for n 6 -methyladenosine (m 6 a), a posttranscriptional rna modification that has recently emerged as a key regulator of vrna fate [153] . m 6 a can be found in the rna of iav, denv, zikv, hcv, yellow fever virus, west nile virus, enterovirus 71 and hiv-1. in the case of hiv-1, m 6 a modification can influence different steps of the virus infection cycle including reverse transcription. recently, ythdf3 was found to be encapsidated into hiv-1 particles by interacting with nucleocapsid and negatively affected reverse transcription once the capsid core is delivered into the newly infected cell [151] . interestingly, hiv-1 protease cleaves ythdf3 into smaller fragments inside the virion, thus counteracting its antiviral activity. other 'readers' of m 6 a rna can be detected inside the viral particles of different viruses, including eif3, insulin like growth factor 2 mrna binding protein 1 (igf2bp1) and hnrnpa2b1 (table s1b) . further work is required to discover the potential role of m 6 a-modified vrna and ivrbps in the early steps of infection. one of the best studied cases of antiviral rbp acting inside virions is the apobec3 proteins [152] . these are cellular cytidine deaminases that catalyse the irreversible hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine. to exert their antiviral effects in hiv-1, these proteins are recruited into viral particles through binding to the vrna and nucleocapsid. apobec3f, g and h have preference for g-rich and a-rich sequences which resemble the rna-binding pattern of the hiv-1 nucleocapsid domain in gag polypeptide, as determined by clip-seq in cells and purified virions [34, 154] . apobec3 proteins bind to a large proportion of cellular mrnas in infected cells due to their low specificity. however, the g/a-rich sequence bias ensures that a proportion of apobec3 molecules interact with hiv-1 rna [155] as well as with other retroviruses [156] . once inside the viral particle, apobec3 proteins induce cytidine deamination of the reverse-transcribed dna strand. changes from c to u cause complementary g to a conversion during second strand synthesis and this hypermutation inhibits hiv-1 replication [152] . in addition, apobec3 proteins also restrict hiv-1 by a deamination-independent mechanism that consist of altering reverse transcription template switching frequency [157] . apobec3 g has also been shown to hinder replication of mev, mumps virus and rsv [158] . however, it is unclear whether the deaminase activity is responsible for the inhibition of replication of these viruses. apobec3 proteins, including apobec3c, f and h, also restrict replication of the human coronavirus nl63 via two mechanisms: cytidine deamination and binding to nucleocapsid protein [159] . apo-bec3c restricts zikv infection in a non-editing-dependent manner and is partially counteracted by a small subgenomic flavivirus rna that sequesters it [160] . while apobec3s suppress the infection of a wide range of viruses by different, poorly understood mechanisms, it remains unknown if they are incorporated into these viral particles and exert their antiviral activity in the early steps of infection. this possibility deserves further investigation. in summary, many cellular rbps are hijacked by viruses to sustain infection and are involved in virtually all stages of their infection cycle. we have highlighted here many previously known and novel rbps that are found inside virions from different human rna viruses and depicted a clear gap in our understanding of their function in infection. cellular ivrbps may facilitate trafficking and selection of vrna into assembling viral particles, protect the vrna from the hostile cellular environment, or streamline the initial processes of vrna metabolism upon entrance in a new host cell. alternatively, some of these ivrbps may function as part of the immune surveillance system of the cell and are incorporated into viral particles to interfere with infectivity. whilst we have discussed several roles for ivrbps in this review, much of these remain poorly characterised and mechanistic data is still missing. future research must focus on deciphering the roles of ivrbps and their newly described rna-binding activities in virus infection. by broadening our understanding on these proteins, 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spectrometry effectively distinguishes specific incorporated host proteins cellular proteins in influenza virus particles analysis of virion associated host proteins in vesicular stomatitis virus using a proteomics approach quantitative proteomic analysis of lentiviral vectors using 2-de protein analysis of purified respiratory syncytial virus particles reveals an important role for heat shock protein 90 in virus particle assembly identification of essential filovirion-associated host factors by serial proteomic analysis and rnai screen virus-producing cells determine the host protein profiles of hiv-1 virion cores comparative proteomic analysis of hiv-1 particles reveals a role for ezrin and ehd4 in the nef-dependent increase of virus infectivity conserved and host-specific features of influenza virion architecture multi-faceted proteomic characterization of host protein complement of rift valley fever virus virions and identification of specific heat shock proteins, including hsp90, as important viral host factors proteomics of hcv virions reveals an essential role for the nucleoporin nup98 in virus morphogenesis comparative characterization of the sindbis virus proteome from mammalian and invertebrate hosts identifies nsp2 as a component of the virion and sorting nexin 5 as a significant host factor for alphavirus replication host proteins identified in extracellular viral particles as targets for broad-spectrum antiviral inhibitors actin-modulating protein cofilin is involved in the formation of measles virus ribonucleoprotein complex at the perinuclear region cofilin-1 is involved in regulation of actin reorganization during influenza a virus assembly and budding new host factors important for respiratory syncytial virus (rsv) replication revealed by a novel microfluidics screen for interactors of matrix (m) protein the host proteins transportin sr2/tnpo3 and cyclophilin a exert opposing effects on hiv-1 uncoating cyclophilin a stabilizes the hiv-1 capsid through a novel non-canonical binding site the dsrna protein kinase pkr: virus and cell control rig-i detects viral genomic rna during negative-strand rna virus infection ifits: emerging roles as key antiviral proteins cytosolic innate immune sensing and signaling upon infection rna degradation in antiviral immunity and autoimmunity attacked from all sides: rna decay in antiviral defense strategies for viral rna stability: live long and prosper the taking of the cytoskeleton one two three: how viruses utilize the cytoskeleton during egress hiv trafficking in host cells: motors wanted! host cytoskeleton in respiratory syncytial virus assembly and budding microtubule regulation and function during virus infection friend or foe: the role of the cytoskeleton in influenza a virus assembly the role of host cytoskeleton in flavivirus infection role of rna chaperones in virus replication emerging picture of host chaperone and cyclophilin roles in rna virus replication identification of staufen in the human immunodeficiency virus type 1 gag ribonucleoprotein complex and a role in generating infectious viral particles novel staufen1 ribonucleoproteins prevent formation of stress granules but favour encapsidation of hiv-1 genomic rna characterization of staufen1 ribonucleoproteins by mass spectrometry and biochemical analyses reveal the presence of diverse host proteins associated with human immunodeficiency virus type 1 specific incorporation of heat shock protein 70 family members into primate lentiviral virions chaperones in hepatitis c virus infection allosteric heat shock protein 70 inhibitors block hepatitis c virus assembly bacteriophage ms2 genomic rna encodes an assembly instruction manual for its capsid life of psi: how full-length hiv-1 rnas become packaged genomes in the viral particles proteome analysis of the hiv-1 gag interactome the double-stranded rna-binding protein staufen is incorporated in human immunodeficiency virus type 1: evidence for a role in genomic rna encapsidation the host protein staufen1 participates in human immunodeficiency virus type 1 assembly in live cells by influencing pr55gag multimerization dimeric rna recognition regulates hiv-1 genome packaging hiv-1 rna genome dimerizes on the plasma membrane in the presence of gag protein hrip, a cellular cofactor for rev function, promotes release of hiv rnas from the perinuclear region the cellular hiv-1 rev cofactor hrip is required for viral replication human protein staufen-2 promotes hiv-1 proliferation by positively regulating rna export activity of viral protein rev teis, escrt and membrane protein ubiquitination virus budding and the escrt pathway escrt-ii's involvement in hiv-1 genomic rna trafficking and assembly alix serves as an adaptor that allows human parainfluenza virus type 1 to interact with the host cell escrt system alix rescues budding of a double ptap/ppey l-domain deletion mutant of ebola vp40: a role for alix in ebola virus egress hepatitis a virus structural protein px interacts with alix and promotes the secretion of virions and foreign proteins through exosome-like vesicles assembly and release of dengue virus: role of alix protein interaction between the yellow fever virus nonstructural protein ns3 and the host protein alix contributes to the release of infectious particles role of alix in mirna packaging during extracellular vesicle biogenesis human immunodeficiency virus type 1 gag engages the bro1 domain of alix/aip1 through the nucleocapsid identification of the hiv-1 nc binding interface in alix bro1 reveals a role for rna functional association between regulatory rnas and the annexins annexin a2 in virus infection characterization of interactions between hepatitis c virus ns5b polymerase, annexin a2 and rnaeffects on ns5b catalysis and allosteric inhibition role of annexin a2 in the production of infectious hepatitis c virus particles annexin a2 mediates the localization of measles virus matrix protein at the plasma membrane arfs at a glance gga and arf proteins modulate retrovirus assembly and release identification of host trafficking genes required for hiv-1 virological synapse formation in dendritic cells role for adp ribosylation factor 1 in the regulation of hepatitis c virus replication the regulation of translation in alphavirus-infected cells encapsidation of host-derived factors correlates with enhanced infectivity of sindbis virus cold-inducible proteins cirp and rbm3, a unique couple with activities far beyond the cold encapsidated host factors in alphavirus particles influence midgut infection of aedes aegypti the mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity the rna synthesis machinery of negative-stranded rna viruses rna polymerase of vesicular stomatitis virus specifically associates with translation elongation factor-1 alphabetagamma for its activity requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype specific interaction of heterogeneous nuclear ribonucleoprotein particle u with the leader rna sequence of vesicular stomatitis virus involvement of hsp90 in assembly and nuclear import of influenza virus rna polymerase subunits identification of hsp90 as a stimulatory host factor involved in influenza virus rna synthesis hiv-1 dna flap formation promotes uncoating of the pre-integration complex at the nuclear pore isolated hiv-1 core is active for reverse transcription ip6 is an hiv pocket factor that prevents capsid collapse and promotes dna synthesis hiv-1 capsid undergoes coupled binding and isomerization by the nuclear pore protein nup358 single hiv-1 imaging reveals progression of infection through ca-dependent steps of docking at the nuclear pore, uncoating, and nuclear transport hiv-1 uncoats in the nucleus near sites of integration hiv-1 integrase binds the viral rna genome and is essential during virion morphogenesis upf1 is crucial for the infectivity of human immunodeficiency virus type 1 progeny virions y-box-binding protein 1 supports the early and late steps of hiv replication virion-associated, host-derived dhx9/rna helicase a enhances the processivity of hiv-1 reverse transcriptase on genomic rna hiv-1 uncoating and reverse transcription require eef1a binding to surface-exposed acidic residues of the reverse transcriptase thumb domain specific interaction between eef1a and hiv rt is critical for hiv-1 reverse transcription and a potential anti-hiv target cyclophilin a promotes hiv-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cdna role of host trnas and aminoacyl-trna synthetases in retroviral replication p body-associated protein mov10 inhibits hiv-1 replication at multiple stages virion-packaged pyruvate kinase muscle type 2 affects reverse transcription efficiency of human immunodeficiency virus type 1 by blocking virion recruitment of trna(lys3) virion-incorporated alpha-enolase suppresses the early stage of hiv-1 reverse transcription coordinate roles of gag and rna helicase a in promoting the annealing of formula to hiv-1 rna hepatitis b viruses: reverse transcription a different way incorporation of eukaryotic translation initiation k. dicker, et al. seminars in cell and developmental biology xxx (xxxx) xxx-xxx factor eif4e into viral nucleocapsids via interaction with hepatitis b virus polymerase hsp90 is required for the activity of a hepatitis b virus reverse transcriptase hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids ddx3 dead-box rna helicase inhibits hepatitis b virus reverse transcription by incorporation into nucleocapsids reverse transcriptase-and rna packaging signal-dependent incorporation of apobec3g into hepatitis b virus nucleocapsids deamination-independent inhibition of hepatitis b virus reverse transcription by apobec3g the mov10 helicase restricts hepatitis b virus replication by inhibiting viral reverse transcription hiv-1 nef promotes infection by excluding serinc5 from virion incorporation serinc3 and serinc5 restrict hiv-1 infectivity and are counteracted by nef hiv protease cleaves the antiviral m6a reader protein ythdf3 in the viral particle apobecs and virus restriction n(6)-methyladenosine and viral infection the rna binding specificity of human apobec3 proteins resembles that of hiv-1 nucleocapsid promiscuous rna binding ensures effective encapsidation of apobec3 proteins by hiv-1 broad antiretroviral defence by human apobec3g through lethal editing of nascent reverse transcripts apobec3 host restriction factors of hiv-1 can change the template switching frequency of reverse transcriptase the innate antiviral factor apobec3g targets replication of measles, mumps and respiratory syncytial viruses apobec3-mediated restriction of rna virus replication zika virus noncoding sfrnas sequester multiple host-derived rna-binding proteins and modulate mrna decay and splicing during infection key: cord-321013-8pkrg0mx authors: mcbride, ruth; van zyl, marjorie; fielding, burtram c. title: the coronavirus nucleocapsid is a multifunctional protein date: 2014-08-07 journal: viruses doi: 10.3390/v6082991 sha: doc_id: 321013 cord_uid: 8pkrg0mx the coronavirus nucleocapsid (n) is a structural protein that forms complexes with genomic rna, interacts with the viral membrane protein during virion assembly and plays a critical role in enhancing the efficiency of virus transcription and assembly. recent studies have confirmed that n is a multifunctional protein. the aim of this review is to highlight the properties and functions of the n protein, with specific reference to (i) the topology; (ii) the intracellular localization and (iii) the functions of the protein. coronaviruses (covs) have a global distribution and infect a variety of human and animal hosts, causing illnesses that range from mostly upper respiratory tract infections in humans to gastrointestinal tract infections, encephalitis and demyelination in animals; and can be fatal [1, 2] . the international committee for taxonomy of viruses (ictv) reports four coronavirus genera, namely alphacoronaviruses, betacoronaviruses, gammacoronaviruses and deltacoronaviruses [3] . covs are enveloped single-stranded, positive-sense rna viruses with genomes ranging between 26.2-31.7 kb, the largest among known rna viruses [4] . this large, capped and polyadenylated genome contains seven open access common coronavirus genes in the following conserved order: 5'-orf1a-orf1b-s-orf3-e-m-n-3' [5] . orf1a/b encompasses two-thirds of the genome and produces a genome-length mrna (mrna1) that encodes two overlapping viral replicase proteins in the form of polyproteins 1a (pp1a) and pp1ab [6] . these polyproteins are formed as a result of a -1 ribosomal frame shift that involves a complex pseudoknott rna structure [7] and are then proteolytically processed by virally encoded proteases into mature nonstructural proteins (nsp1 to nsp16), which assemble to form a membrane-associated viral replicase-transcriptase complex (rtc) [6, 8, 9] . the last third of the genome produces subgenomic (sg) mrnas that encode the four structural proteins, spike (s), envelope (e), membrane (m), and nucleocapsid (n), as well as a number of accessory proteins [10, 11] . amino acid sequence comparisons have shown that cov n proteins have three distinct and highly conserved domains: two structural and independently folded structural regions, namely the n terminal domain (ntd/domain 1) and c-terminal domain (ctd/domain 3), which are separated by a intrinsically disordered central region (rna-binding domain/domain 2) ( figure 1 ); all three domains have been shown in different covs to bind with viral rna [12] [13] [14] [15] [16] [17] . [16, 18, 19] . the ntd is divergent in both sequence and length. it has been mapped for infectious bronchitis virus (ibv)-n to aa 19-162 [20] , for severe acute respiratory syndrome human coronavirus (sars)-n to aa 45-181 [16] , and for mouse hepatitis virus (mhv)-n to aa [18] . the n-termini of these three covs have been found to associate with the 3' end of the viral rna genome, possibly through electrostatic interactions [21, 22] . there are several common characteristics of cov n protein ntds, including predicted secondary structures such as a central β-sheet platform flanked by α-helices [20] , with a basic rna binding groove along the β-platform and an extended β-hairpin. the ntd is enriched in aromatic and basic residues and the folded shape resembles a hand with basic fingers that extend far beyond the protein core, a hydrophobic palm, and an acidic -wrist‖ [21] . it has been proposed that the flexible, positively charged finger-like β-hairpin extension in the ntd of both ibv and sars-cov n protein is able to grasp rna by neutralizing its phosphate groups, while the base moieties can make contact with exposed aromatic residues from the hydrophobic palm [16, 21] . more precise mapping of the rna-binding site locations has been determined for sars-and ibv-n protein. within the ntd of sars-cov-n, positively charged lysine and arginine residues have been proposed to bind a 32 nucleotide stem-loop structure located at the 3' end of the sars-cov rna genome [16] . site-directed mutagenesis studies on ibv-n have identified two residues that are critical for rna binding; namely tyr-94 and arg-76 [23] . tyr-94 is located in strand β3 of the four-stranded anti-parallel β sheet; arg-76 is located in the immediate vicinity of tyr-94, at the base of the extended flexible hairpin loop [23] . it is however likely that, since no single mutation totally disrupts rna binding, other aromatic/basic residues at the surface of the ntd contribute to nucleic acid binding by creating a broad surface that comes into contact with the viral genomic rna [23] . the ntd possesses some features similar to those of other rna-binding proteins that form a rnp. for example, the u1a spliceosomal protein [24] and the coat protein of ms2 bacteriophage [25] bind viral rna with residues arising from the surface of a four-stranded anti-parallel β sheet. seemingly, strands β2, β3, and the flexible β-hairpin from the ibv n protein could fulfill a comparable role by interacting with phosphate groups on the viral rna [23] . the arg-76 and tyr-94 residues in the ibv n protein are well conserved across the whole cov family, and may structurally correspond to the arg-94 and tyr-122 residues in the sars-cov n protein [23] , meaning that arg-94 and tyr-122 may therefore be critical for sars n-rna binding. the crystal structure of mhv n197 (residues 60-197) adopts a u-shaped β-platform containing five short β-strands (arranged β4-β2-β3-β1-β5) across the platform with an extended β2'-β3' hairpin similar to ntds from other cov n proteins [26] . interestingly, the crystal structure of the mhv ntd shares a similar overall and topology structure with that of sars-cov and ibv but varies in its potential surface, indicating a possible difference in rna-binding module [27] . it has been shown that n219, an mhv-a59 n domain protein fragment that contains the folded ntd and the immediately adjacent intact linker region (lkr; residues 60-219), binds to the trs in the viral genome body (trs-b) and complementary trs (ctrs) with high affinity to form a n219-trs duplex [26] . mhv trs binds across the β-platform of ntd in a defined orientation, with the 5'-end of trs near β4 and the 3'-end of trs near β5; this n219 binding to single-stranded rnas-containing the trs or ctrs-uses base stacking interactions between aromatic side chains on the β-platform with a triple adenosine motif within the trs, 5'-gaaucuaaacu-3' [26] . furthermore, due to its potent helix-destabilizing activity, n219 is able to efficiently melt an rna duplex between the template trs and nascent ctrs strand into component single strands that may be transiently formed during discontinuous transcription of viral sgrna by the coronaviral replicase complex [26] . three residues on the β-platform have been shown to play key roles in trs binding and helix destabilization: arg-125 and tyr-127 on the β3 strand and tyr-190 on the β5 strand, suggesting that the aaa motif in the 3'-end of the trs is anchored here [18] . these three residues are completely invariant in betacoronavirus n proteins and occupy precisely analogous positions on the fold of each ntd, and are therefore likely to define similar rna binding grooves in the closely related sars ntd [18] . the duplex formation and duplex trs unwinding activity exhibited by n219 therefore implicates mhv ntd in template switching during discontinuous sgrna transcription [28, 29] . moreover, the ability of the ntd to melt dsrna may also play a role in rna packaging or other steps in the viral life cycle where rna remodeling is needed [26] . for example, mutations that cripple duplex unwinding are defective in stimulating cov replication in bhk-r cells, and are lethal, providing evidence of a critical role for ntd in viral replication [18, 26] . cov n proteins have also been recognized as rna chaperones [30, 31] , which, as part of their chaperone activities, anneal nucleic acids, and so rna duplex destabilization activity may be important in cov n ntds role in assisting viral rna in reaching its functional three-dimentional structure. viral nucleocapsid and replication accessory proteins from other viruses have also been shown to function as rna chaperones and facilitate helix destabilization, for example hiv-1 ncp7 protein [32] , and adenovirus dna binding protein [33] . the ntd is separated from the ctd by an intrinsically disordered middle region referred to as the linker region (lkr). the charged lkr is also known as the sr-domain because it is rich in serine and arginine residues [34] , and it is involved in cell signaling [15, 35, 36] . the flexible lkr is capable of direct interaction with rna under in vitro conditions [37] . potential phosphorylation sites have been mapped to the ser/arg-rich portion of the lkr of sars-cov n [38] [39] [40] . these lkr phosphorylation sites are thought to function in binding m protein, heteronuclear ribonucleoprotein (hnrnp-a1) and rna to the n protein with high binding affinity [14, [41] [42] [43] . there are conflicting reports regarding the involvement of the lkr in n protein oligomerization. some studies has suggested that the lkr is directly involved [44] and that through electrostatic effects, hyperphosphorylation of the lkr reduces the total positive charge on the sars-cov n protein and leads to enhanced oligomerization of di-domain constructs [45] . other studies have, however, reported that the lkr interferes with oligomerization when the ctd is present [46] or if the lkr is phosphorylated [38] . despite almost no structural information being available for the lkr, possibly due to its high positive charge and flexible nature [47] , there is evidence in support of the functional importance of intrinsically disordered regions in proteins for modulating transcription, translation, post-translational modifications such as phosphorylation, and cell signaling [48] . rna chaperones often have structural flexibility because the rna-protein recognition process often requires conformational changes in the rna, the protein or both [49] . an interaction between n protein and a subunit of the viral replicase-transcriptase complex, namely non-structural protein 3 (nsp3), has been described and key binding determinants localize to the lkr [50,51], highlighting the importance of this unstructured region for a number of potential interactions, such as viral infectivity [52] . it has also been proposed that nsp3 binding induces a conformational change in the lkr, potentially regulating the intracellular localization of n to the site of replication [50] and/or other rna binding functions of n. the ctd, which is a hydrophobic, helix-rich terminal, has been mapped for sars-n to aa 248-365 [17] , and for ibv-n to aa 219-349 [21, 53] . this domain is also referred to as the dimerization domain because it contains residues responsible for self-association to form homodimers, as well as homo-oligomers through a domain-swapping mechanism [16, 17, 42, [53] [54] [55] [56] [57] . oligomerization of n protein is necessary to produce a stable conformation because in its monomeric form, the ctd folds into an extended conformation with a large cavity in its center, making it unstable [47] . sequence comparison shows that the dimerization domain of the n protein is conserved at least among the alpha, beta and gamma groups of covs, suggesting a common structural and functional role for this domain [47] . the monomer of csars-n, a crystalized c-terminal construct of sars-n that contains residues 270-370, comprises five short α-helices, one 3 10 helix, and two β-strands [47] . the general shape of the monomer resembles the letter c, with one edge formed by a β-hairpin extending away from the rest of the molecule [47] . this structure is similar to the crystalline structure of another sars n ctd monomer (np248-365), which consists of eight α-helices and two β-strands [55] . the csars-n dimer interface is formed largely by insertion of the β-hairpin of one subunit into the cavity of the opposite subunit, resulting in the four β-strands of the two subunits forming an anti-parallel β-sheet that is superposed by two long alpha helices [47] . due to the extensive hydrogen bond formation between the two hairpins, together with hydrophobic interactions between the beta-sheet and the alpha helices, this interface is highly stable [17] , and these interactions suggests that the dimeric structure may in fact represent the functional unit of the n protein [47] . the crystal structure of np248-365, a sars-cov ctd spanning residues 248-365, revealed that the n protein dimer has the shape of a rectangular slab in which the four-stranded β-sheet forms one face of the slab and the α-helices form the opposite face [55] . similarly to csars n, the dimerization interface of np248-365 is composed of four β-strands and six α-helices, with each protomer contributing one β-hairpin and helices α5, α6 and α7. the two β-hairpins form a four-stranded intermolecular β-sheet that is stabilized through extensive hydrogen bonding. the other part of the dimerization interface is composed of helices α5 and α6, where strong hydrophobic interactions involving trp302, ile305, pro310, phe315 and phe316 were observed. the dimer is further stabilized by hydrophobic interactions between the longest helix, α7, and the intermolecular β-sheet [55] . similarly to csars-n and np248-365, a nuclear magnetic resonance (nmr) study has reported secondary structural assignments of a sars n protein construct whose dimeric interface also consists of a four-stranded anti-parallel β-sheet and two α-helices [17] . self-association of the n protein has been observed in many viruses, and is required for the formation of the viral capsid which protects the viral genome from extracellular agents [56] . in addition to the ability of n protein to oligomerize, viral capsid formation also requires rna-binding ability [58] . studies revealed that sars-cov n protein fragments containing the dimerization domain (residues 236-384) can bind to a putative packing signal within the viral rna, with the most likely rna binding site being within the basic region between residues 248-280 [59] . nmr studies then showed that the rna-binding site between residues 248-280 formed part of the complete dimerization domain structure [17] . it was not until the crystal structure of sars n ctd was resolved that the molecular basis of rna-binding activity and organization of the ctd octamer was determined. the ctd spanning residues 248-365 (np248-365) revealed that, due to the presence of the eight positively charged lysine and arginine residues, amino acids 248-280 form a positively charged groove, one of the most positively charged regions of the n protein [55] . this groove is similar to that in ibv-n ctd, except that the positively charged surface area is larger in the sars-cov construct than in the ibv [20] , due in part to the presence of additional negatively charged residues in the ibv n protein and in part due to the absence of residues 215-218 from the ibv construct, which contain two lysine residue in the sars-cov construct [55] . the np248-365 construct, which contains both the charge-rich region (residues 248-280) and dimerization core (residues 281-365) of the dimerization domain, is capable of binding to single-stranded rna (ssrna), single-stranded dna (ssdna) and double-stranded dna (dsdna), and np248-365 has stronger nucleic acid-binding activity than the ntd [55, 57] . the strong electrostatic character of residues 248-280 and the fact that both ssrna, ssdna and dddna bind to np248-365, strongly indicates that oligonucleotide binding is based on non-specific charge interactions between the positively charged protein and the negatively charged nucleic acid backbone [55, 60] . by keeping the rna-binding domains in close proximity to the ctd, the formation of a large helical nucleocapsid core is therefore possible [47]. association of the n protein dimers is necessary for further assembly of the core. the full-length dimeric n protein has a tendancy to form tetramers and higher molecular weight oligomers in vitro [54] , and a serine/arginine-rich motif (residues 184-196) has been shown to be important for n protein oligomerization [44] . two dimers arrange themselves into a butterfly-shaped tetramer, while two butterfly-shaped tetramers unite to form an octamer in the asymmetric unit of the ctd crystal [55] . the octamer is held together through hydrophobic interactions and hydrophilic contacts among the four dimers, and networks of inter-dimer hydrogen bonds further help stabilize the octamer [55] . crystallography studies have demonstrated that cov-sars ctd (np248-365) packs as an octamer which stacks to form a helical supercomplex structure with a continuous positively charged surface that could potentially allow viral rna strands to bind and wrap around the helical oligomer structure through electrostatic interactions [55] . the existence of transient self-association between dimers in solution was confirmed using a disulphide trapping technique, and it was shown that by neutralization of excessive charges on the protein, either through environmental charge screening or charge modifications, this transient self-association can be regulated [44] . this proposed biophysical mechanism whereby electrostatic repulsion between n protein molecules acts as an oligomerization switch has implications for understanding how nucleocapsid assembly is then subsequently modulated [44] . in addition, the ctd 45 residues of the mhv n protein have been shown to be the major determinant for interaction with the m protein [61] , and so association of the n protein with the m protein may also play a role in the assembly of the nucleocapsid core into a progeny virion [47] . oligomerization via the ctd has also been reported in human coronavirus 229e (hcov-229e) and a recent study has shown that the c-terminal tail peptide, an intrinsically disordered domain that flanks the ctd, plays an important role in dimer-dimer association [53] . the c-terminal tail interferes with oligomerization of the ctd and has an inhibitory effect on viral titer of hcov-229e; and further understanding this mechanism of oligomerization may provide insight into the viral assembly process and could identify additional targets for drugs to combat covs through the disruption of the n protein self-association [53] . the ctd of sars-n (aa 251-422) is also responsible for stress granule localization that occurs as part of an integrated stress response in arsenite-treated hela cells [38] . once sequestered in these granules, the n protein can induce host translational shutoff. the ntd and the ctd are interspersed by intrinsically disordered regions (idrs) [19, 37] . intrinsically disordered proteins (idps) or idrs lack a tertiary structure and have no fixed 3-dimentional shape in the native form. however, idps and idrs play a role in various biological functions including dna, rna and protein binding with the disordered regions facilitating access to these binding sites [62] [63] [64] . in fact, the three idrs in sars-n (aa 1-44, 182-247 and 366-422) have all been shown to modulate the rnd-binding activity of the ntd and ctd [19, 37] . moreover, both the middle and c-terminal idrs (figure 1 ) have been implicated in the oligomerization of the n protein [44, 65] , with the middle idr also associated with n protein functionality and n-m interaction [19, 39, 40, 66] . it would be interesting to determine whether the presence of three disordered regions in sars n, compared to the one disordered region in hcov-nl63 n for example, would result in sars n having a higher binding affinity to viral, as well as host cellular proteins. if indeed so, could this then indicate a probable basis for the increased pathogenicity of sars-cov compared to hcov-nl63? in order for cov n proteins to package the viral genome with structural proteins to form ribonucleoprotein (rnp) complexes for viral assembly, two key activities are required: the interaction between protein and nucleic acid and the ability of the complex to oligomerize [58] . the n proteins of sars-cov, ibv and mhv have all been shown to perform both these functions. sars-cov-n protein interacts with rna at multiple sites, with all three domains having charged regions [55] . the crystal structures of the ntd and ctd domains of the n protein from sars-cov, ibv and mhv all share a similar overall and topology structure, which corroborates a conserved mechanism of nucleocapsid formation for covs [27] . furthermore, despite a lack of significant sequence similarity, the csars-n had a similar fold to that of the n protein of porcine reproductive and respiratory syndrome virus, a member of arteriviridae family, suggesting an evolutionary link between coronaviridae and arteriviridae in which the n proteins of both viruses have a common origin [47] . in fact, due to their similar genome organization and viral replication mechanisms, the coronaviridae and arteriviridae were united to form the relatively new order nidovirales. in virus-infected cells, cov n proteins can localize to the cytoplasm alone or to the cytoplasm and nucleolus [67] . proteins that are able to localize to the cytoplasm, nucleus and/or nucleolus require multiple signals to determine their subcellular localization [68] . cov n proteins commonly localize in the nucleolus, and although nucleolar localization/retention signals (norss) and pathways are not well characterized, nucleolar localization usually requires regions in the protein that are rich in arg residues and is likely cell-cycle dependent [34, 69, 70] . the n protein of ibv was found to localize in the cytoplasm alone or to co-localize in both the cytoplasm and nucleolus [67, 68, 70] . ibv n protein contains a functional nuclear export signal (nes) to traffic n protein to the cytoplasm [68, 71] , and an 8 amino acid nors motif at its ntd and is necessary and sufficient for nucleolar retention [68] . it is hypothesized that the localization of ibv-n to the nucleolus forms part of a virus strategy to control sgrna synthesis in both the host cell and virus by associating with ribosomal subunits [70] and interacting with nucleolar proteins, nucleolin and fibrillarin [72] . importantly, this interaction is not direct, but mediated through rna and could therefore simply be an artifact of the proteins having rna-binding domains [73] . thus, the nucleolar localization could simply be due to a high density of the host rna attracting a viral rna-binding protein. even so, it has been postulated that the nuclear localization of the n protein may interfere with cellular machinery and thus lead to triggering of apoptosis [39] . the localization of n to nucleoli alone might be cell cycle dependent, because the number and size of nucleoli differ at different stages of the cell cycle: at the beginning of g1 phase, multiple nucleoli can be found, but only single nucleoli can be seen at later g1, s and g2 phases [67, 74] . it was also found that domain 2 of ibv-n predominantly localizes in the nucleus, but when fused with domain 3 (ctd) it localizes to the cytoplasm and thus supports the findings of other studies done on ibv-n localization [35, 68] . the ability for nucleolar localization varies between n proteins of different covs. unlike other cov n proteins, sars-cov n protein is mostly distributed to the cytoplasm [34, 71, 75] . this cytoplasmic localization is somewhat unexpected because there is at least one nors in domain 2 and eight putative nuclear localization signal (nls) motifs within domains i and ii of the sars-cov n protein [35] , of which the short lysine-rich sequence (366-381) near the carboxy-terminus is a putative bipartite nls that is unique to sars-cov n [71, 76] . as a reason for this, it has been suggested that signals for nuclear and nucleolar targeting of sars-cov n protein are poorly accessible to nuclear import machinery due to phosphorylation or conformation restraints [71] . a cytoplasmic nes may be involved in also over-riding the nls, resulting in significantly less n protein (only 10%) being localized to the nucleolus [35] . shuttling of n protein from the nucleus to the cytoplasm occurs through phosphorylated-dependent binding of sars-cov to 14-3-3, with the absence/inhibition of this 14-3-3 molecule resulting in increased nuclear localization of sars-n [38] . also, the deletion of the sr-rich domain contained within the middle region of sars-n can result in dramatic changes in sub-cellular localization of n compared to wild-type n [44]. these results indicate that the localization of n protein to the nucleus or nucleolus is not a conserved property of nidovirales [71] . the primary role of cov n protein is to package the genomic viral genome into long, flexible, helical ribonucleoprotein (rnp) complexes called nucleocapsids or capsids (table 1) . the nucleocapsid protects the genome and ensures its timely replication and reliable transmission. the filamentous nucleocapsids are 10 to 15 nm in diameter and several 100 nm in length, and these macromolecular structures are visible using electron microscopy [77] . within the nucleocapsid there are both n-rna interactions as well as intermolecular association between disulfide-linked n protein multimers [78] . the n-rna interaction is mediated by binding signals contained within the leader rna sequences [79] . during the virus life cycle, multiple copies of the n protein interact with grna and sgrna molecules, indicating a role for n protein in viral transcription and translation [79, 80] . the basic building block for cov nucleocapsid formation is a dimeric assembly of n protein [21] , and it is the ctd of n protein that possesses dimerization function [56] . a structural model of cov proposes that n protein is not only present in the helical nucleocapsid but also in the internal spherical/icosahedral core [81] . the internal core consists of n protein, rna and the ctd of m protein. the m protein is the main core shell component and a 16 amino acid domain (aa 237-252) on the ctd of m protein binds directly to n protein via an ionic interaction, leading to specific genome encapsidation in the budding viral particle [81] [82] [83] .the n protein therefore plays an essential structural role in the cov virion through a network of interactions with (i) the genomic rna; (ii) m protein and (iii) other n proteins. assembly of virus particles is an essential step for a productive viral replication cycle. cov virions contain three envelope proteins, m, e and s, and a viral nucleocapsid, which consists of genomic rna and n protein, within the viral envelope. [87, 88] . it is believed that the interaction of the nucleocapsid with envelope proteins drives the incorporation of the nucleocapsid in enveloped viruses [89] , and such protein-protein interactions are critical for viral assembly, as has been shown for alphaviruses [90, 91] . n and m proteins are the two major structural proteins in cov virions [92] . the m protein is anchored by its three transmembrane domains to the viral envelope and its large carboxy-terminal tail in the virion interior interacts with the nucleocapsid [93] . the nucleocapsid consists of the positive strand genomic rna, mrna 1, helically encapsidated by n protein monomers, and the n protein region that interacts with the c-terminus of the m protein domain seems to be cov specific. the intracellular sites of virus assembly also vary among different viruses [94, 95] . in mhv, the large carboxy-terminal domain of the m protein interacts with the ctd of the n protein [93] . newly synthesized, unglycosylated m protein interacts with n protein at the er membrane, which is a pre-golgi compartment that is also the site of mhv budding [96] , suggesting that the site of interaction overlays with mhv budding sites [93] . the m protein-nucleocapsid interaction is thought to be initiated by a direct binding of m protein to genomic rna that is mediated by a 69 nucleotide (nt) packaging signal (ps) present only on the mrna 1 association [93, 97] . these ps is located about 21kb from the 5' end of mhv mrna 1 [97, 98] , is necessary and sufficient for packaging rna into mhv particles [99] , and it has been suggested that the m protein-ps interaction could lead to the association of m protein with n protein, thereby stabilizing the complex between m protein and the nucleocapsid [93] . although the m protein-nucleocapsid interaction could theoretically also be initiated by direct binding of n protein to genomic rna, this is unlikely because n protein interacts with all mrnas [79, 93] , which makes it difficult to explain how the formation of n protein-mrna 1 rnp complex might lead to specific packaging of genomic rna, and not sgrna, into virus particles [100] . it has since been conclusively demonstrated that m protein selectively interacts with ps-containing rna in the absence of n protein, indicating that the mechanism of m-ps recognition does not require the formation of rnp complex by n protein, and in fact, n protein is not required for rna packaging in that model [100] . mhv m protein was the first example of a viral transmembrane protein that could bind to a specific viral rna element in the absence of any other viral structural proteins [100] , and a proposed model for rna packaging in mhv suggests that once m protein accumulates and oligomerizes in the intermediate compartment between the er and golgi complex, m protein binds to ps-mrna 1, and only thereafter does n protein associated with mrna 1 interact with oligomerized m protein [100] . although n protein appears to be dispensable for mhv rna packaging, n-m interaction might be important in compensating for viral envelope defects that occur due to m protein mutation. the m protein carboxy terminus is extremely sensitive to mutations, and removal of even only the last two amino acid residues from the tail of the m protein appears to be lethal [83] . interestingly, n protein becomes mutated in its ctd, and these changes then compensate for the loss of the two m protein residues, either by increasing the affinity of an adjacent interaction or by providing a new contact point between n and m to stabilize the virion [83] . for the porcine transmissible gastroenteritis coronavirus (tgev), an interaction between the carboxy terminus of m and nucleocapsid has been mapped to residues 233-257 of the tgev m protein [82] . this segment corresponds to residues 201-224 of the mhv m protein [83] , which overlaps with only one of the critical residues identified in the mhv m protein [61] . this region of the two m proteins is, however, poorly conserved and the apparent disagreement between the tgev and mhv results may relate to differences in the respective folds of the m proteins, or differences in how these residues influence those folds [61] . sars-cov is markedly different from other members of the coronaviridae family in the sense that there is only 20%-30% amino acid identity with other known covs, with both the n and m proteins having low sequence homology [76, 101] . one might therefore expect that there could be differences in the mechanism of viral assembly. a mammalian two-hybrid system, which is performed in vivo so that viral proteins will adopt their native state and therefore be more likely to interact in a biologically accurate manner [102] , confirmed that n-m protein interactions occur in vivo [66] . moreover, this study identified a stretch of amino acids in the middle of the n gene that may be critical for n-m protein interaction [66] . this stretch of amino acids spans the lkr and dimerization domain in the ctd, suggesting that this region may be essential in maintaining correct n protein conformation for both self-association and n-m protein interaction [66] . despite sars-cov having the closest genetic resemblance to mhv, the m proteins of these viruses bind to different domains on the n protein. covs assemble and bud intracellularly at the er-golgi complex [96, 103] , and association of the nucleocapsid with this organelle may reflect a role in virus budding. the formation of the virion envelope requires expression of only m-and e-protein, and not n protein, as has been observed for mhv [104] , ibv [105] , tgev [106] , and bcov [107] . it was recently noted however, that these studies all used vaccinia-based expression systems, where overexpression of viral membrane proteins may lead to release in microvesicles, complicating the interpretation of virus-like particle (vlp) experiments [108] . subsequent experiments using transient transfection to express the proteins from plasmids have shown that, at least for mhv [109] , sars-cov [110] and ibv [111] , the presence of n protein can greatly increase vlp yield. therefore, while n protein is not necessarily required for envelope formation, n protein plays an important role in forming a complete virion [108] . n protein binds to both full-length genomic rna (grna) as well as all six sgrnas but displays an increased affinity for grna [112] . the grna functions as a template for the viral rna-dependent rna polymerase as well as a message for translation [52] . during infection, grna is initially transcribed by an early polymerase activity into a genome-sized negative-stranded rna [113] and then a late polymerase activity transcribes the negative-stranded rna into a full length grna that is bound to polysomes [113] and detected in nucleocapsid structures [114] . numerous studies have demonstrated that n protein is required for optimal cov replication [31, [115] [116] [117] [118] [119] [120] [121] . the participation of n protein in an early event in rna synthesis is implied by at least two things: firstly mhv-and sars-cov n protein colocalize intracellularly with replicase components at early stages of infection [122] [123] [124] [125] ; and secondly, stimulation of grna infection is dependent upon n protein translation [52, 126] . a more clearly defined role for n protein in grna synthesis was delineated when an interaction between the sr region of the mhv n protein and a region in the amino terminal segment of the nsp3 subunit of the viral replicase was discovered [51, 52] . the n-nsp3 interaction has been specifically mapped to the ubiquitin-like domain (ubl1) of nsp3, an essential domain to the virus [127] . moreover, it was also shown that this n-nsp3 interaction is required for n protein to promote optimal infectivity of grna [52] . it was proposed that the formation of an initiation complex at the 3' end of the genome requires n protein to tether grna to the newly translated replicase via its interaction with nsp3 [52, 127] . considering that the start of negative-strand synthesis is the first step in both genomic replication and transcription [128, 129] , it is likely that this n-nsp3 interaction is important in both of these rna-synthetic processes [52] . the role of n protein in mhv grna synthesis may not necessarily be limited to early time points of infection as it has also been proposed that n protein sustains ongoing transcription throughout the course of infection [31, 130] . the role of cov n protein in rna synthesis remains controversial as n protein is not essential for tgev rna replication but rather for efficient transcription [31] . rna chaperone proteins assist the proper folding of nucleic acids [131, 132] . chaperone activity has been postulated as a general activity of all cov n proteins, and has been demonstrated for tgevand sars-cov n proteins [30] . it has been proposed that amino acids 117-268 in the central disordered domain (the lkr domain) of the tgev n protein has chaperone activity [31] , and that this region facilitates template switching in vitro by decreasing the energy barrier needed to dissociate the nascent minus rna chain from the grna template during discontinuous rna transcription [30, 31] . this facilitation of template switching is required for efficient transcription and may explain how the presence of n protein therefore increases rna synthesis. deregulation of the cell cycle is a common strategy adopted by many rna (and dna) viruses aimed at exploiting host cell machinery in order to create a more favorable environment for their survival. one of the mhv nonstructural proteins, p28, induces g 0 /g 1 cell cycle arrest by inhibiting hyperphosphorylation of retinoblastoma protein (rb), a step which is necessary for cell cycle progression through late g 1 into the s phase [133] [134] [135] . a model was proposed whereby expressed cytoplasmic p28 induces stabilization of p53, and accumulation of p53 causes transcriptional upregulation of p21, a cyclin-dependent kinase (cdk) inhibitor, leading to suppression of cyclin e/cdk 2 activities, and the resulting reduction in g 1 cyclin-cdk complexes and cdk activities ultimately inhibits rb hyperphosphorylation [133] . certain herpes simplex virus gene products [136, 137] , cytomegalovirus gene products [138, 139] , and zta of epstein-barr virus [140] have similarly also been shown to arrest cell cycle progression at the g 1 phase. ibv infection also perturbs cell cycle progression and arrests cells in the s and g 2 /m phases, and this occurs partly through the interaction of ibv nsp3 and dna polymerase delta [141] . the n protein of sars-cov also modulates the host cell cycle by regulating cyclin-cdk activity such that s phase progression is halted. the n protein bears a signature cyclin box-binding region (rxl motif), can be phosphorylated by cdk and is therefore a substrate for the cyclin-cdk complex [39] . n protein has been shown to have an inhibitory effect on s phase cyclins cdk4 and to a lesser extent cdk6 [39] . the inhibition of kinases means that rb remains hypophosphorylated and cannot release e2f1, and the transcription of s phase genes is halted [135] . similarly to mhv p28, sars-cov n protein also inhibits cdk2 activity, meaning that by blocking the activity of both g 1 and s phase cyclins, n protein doubly ensures the blockage of s phase progression [39] . n protein mediated inhibition of cdk activity; leading to inhibition of rb phosphorylation, is independent of cdk inhibitors (cdkis) such as p21, and so it has been suggested that the n protein mimics the role of cdkis by acting as a competitive inhibitor to cdk4 and cdk2 [39] . the ability of n protein to inhibit cdk4 activity was dependent on the rxl motif, whereas its ability to inhibit cdk2 requires at least two mechanisms: the cdk phosphorylation motif and dependence on host cell signaling pathways [39] . it has been postulated that, p28-mediated (mhv) and n protein-mediated (sars) blocking of the s phase, allows these virus enough time to utilize the cellular raw materials that have been synthesized ahead of the s phase, for replication of its genome, as well as for assembly and budding of progeny particles [142] . these studies provide evidence that even within the cov family, different proteins can deregulate the cell cycle and that those proteins, in the case of sars-n protein, can employ multiple mechanisms to achieve this. in response to environmental stress, eukaryotic cells reprogram their mrna metabolism to adapt to stress-induced damage. translationally stalled mrnas together with a number of translational initiation factors and rna-binding proteins are selectively deposited in cytoplasmic stress granules (sgs) [143] . covs, like many other viruses, induce host translational shutoff, while maintaining synthesis of their own gene products, as a means to interfere with cellular defense mechanisms [144] . mhv-induces a host translational shutoff that is reminiscent of a cellular stress response; sgs appear, numerous mrnas including protein translation-related factors are downregulated and levels of phosphorylated translation initiation factors increase [145] . it is important to remember, though that this translational shutoff could also simply be the host cell's response to the infection. in sars-infected hela cells that are exposed to arsenite treatment, n protein translocates to cytoplasmic sgs where it colocalizes with poly(a)-binding protein 1 (parb) and transiently expressed tia-1 [38] , both of which are components of sgs [143] . this localization and pattern was enhanced when the protein was hypophosphorylated (nδrs), perhaps because nδrs induced larger ribonucleoprotein (rnp) complex formation [38] . the sequestration of n protein in sgs possibly indicates a role in translational suppression. the nucleocapsid protein of both porcine and respiratory syndrome and rubella virus also interact with parb, which promotes protein synthesis by circularizing mrnas, and inhibit host protein synthesis by sequestration of parb through a stoichiometric mechanism [146, 147] . it has been proposed that, at least in the rubella virus, that n-dependent inhibition of translation via parb binding may facilitate the switch from viral translation to packaging rna into nucleocapsids [146] . more specifically, binding of the capsid to parb late in the virus cycle, when the levels of newly synthesized genomes and viral proteins are high, and there is little need for additional replicase components, could prevent recruitment of ribosomes to the nascent 40s rna. this scenario would favour packaging of the 40s genomic rna into nucleocapsids [146] . considering that sars-n and mhv-n protein has been shown to interact with the parb hnrnp-a1 [41, 148] , it is possible that the n-parb interaction also acts as a switch that redirects viral activity from rna synthesis to nucleocapsid formation. the translational suppressive effect of the n protein could also be through its ctd interaction with elongation factor 1α (ef1α), a major translational factor in mammalian cells [75] . n protein of sars-cov binds directly to and induces aggregation of ef1α, resulting in inhibition of protein translation [75] . moreover, ef1α is a multifunctional protein and has unconventional functions related to its association with the cytoskeleton, including interaction with filamentous (f) actin, promotion of f actin bundling and formation of a contractile ring during cytokinesis [149] [150] [151] [152] . n-ef1α therefore blocks ef1α-mediated f actin bundling and the formation of the contractile ring, leading to the formation of multinucleated cells by inhibiting cytokinesis [75] . multinucleated syncytia of macrophages have been observed in late-phase sars-cov but not in other cov-infected patients [153] . the n protein of hcov-229e also binds to ef1α, but with significantly lower affinity than sars-cov n, and induces multinucleation much more slowly in a substantially smaller fraction of transfected cells [75] . in addition, n-protein mediated inhibition of cytokinesis leads to inhibition of cell proliferation in human peripheral blood lymphocytes (pbls) [75] . since actively dividing lymphocytes are a major cell target of sars-cov [154] , sars-cov n may decelerate lymphocyte proliferation and therefore interfere with immune system function [75] . sars-n protein has also been shown to cause cytoskeletal changes in response to a different type of cell stress, namely serum deprivation [155] . n protein induces actin reorganization in cos-1 monkey kidney cells by down regulating focal adhesion kinase and fibronectin via p38 mapk pathway activation [155] . n protein plays an important role in viral pathogenesis since anti-n monoclonal antibodies protect mice from lethal infection of jmhv [156] . coronaviruses seem to have developed mechanisms to overcome the host innate immune response, including the interferon response, a major component of innate immunity. sars-cov infected cells do not allow production of interferon and so it seems likely that sars-cov, like many other viruses, has developed mechanisms to subvert the interferon response [157] . sars-cov n protein is one of three β interferon (ifβ) antagonists, with orf3b and orf6 being the other two, and inhibition of ifβ synthesis by n protein is due to inhibition of irf-3 and nf-kb, two of the transcription factors required for if gene expression [158] . sars-cov n protein inhibits the synthesis of type-1 interferon (1fn) and the ctd of n has been shown to be critical for antagonism of 1fn induction [159] . this inhibition of the interferon response likely contributes to the pathogenesis of sars-cov. cov infection is likely to activate a diversity of host cell signal transduction pathways and kinases, which would lead to phosphorylation of n protein [160] . n protein phosphorylation would therefore only occur at a relatively late stage of the viral cell cycle and may explain why more phosphorylated n protein is incorporated into ibv virions [161] . it has been demonstrated that the sars-cov n protein can bind to dna in vitro [55] . recently, the n protein of hcov-oc43 was shown to interact with the transcription factor nuclear factor-kappa b (nf-kappab) [73] . it was reported that hcov-oc43 n protein potentiates nf-kb activation as a direct result of the ability of the nucleocapsid to bind microrna mir-9, a negative regulator of nf-kb. it was suggested that this previously undescribed interaction between virus and host is a potential mechanism of immune evasion in rna viruses because nf-kb required for if gene expression [73, 158] . 148] , which could act as a switch that redirects viral activity from rna synthesis to nucleocapsid formation.  interaction of n protein ctd with elongation factor 1α (ef1α), a major translational factor in mammalian cells, can suppress translation [75] .  n protein plays an important role in viral pathogenesis. mice infected with jmhv protected by anti-n monoclonal antibodies [156] .  synthesis of type-1 interferon (1fn) inhibited by sars-cov n [158] .  the ctd of n has been shown to be a critical antagonist of 1fn induction [159] 2.5. signal transduction  activation of host cell signal transduction pathways and kinases leads to phosphorylation of n [160] . nf-kb is also involved in sars-cov mediated activation of cyclooxygenase-2 (cox-2), more specifically, n protein binds to nf-kb and ccaat/enhancer binding protein binding sites on the cox-2 gene promoter [162] . activation of cox-2, a proinflammatory factor, by sars-cov n protein is a likely cause of lung inflammation in sars-cov infected patients [162] . sars-cov n protein is also known to activate the activator protein 1 (ap1) signal transduction pathway by increasing the amount of transcription factors binding to promoter sequences of c-fos, atf2, creb-1, and fosb [163] . sars-cov n protein induces apoptosis of cos-1 monkey kidney cells in the absence of growth factor by down-regulating extracellular-signal-regulated kinase (erk) and up-regulating c-jun n-terminal kinase (jnk) and p38 mitogen-activated protein kinase (mapk) pathways, with activation of p38 mapk also causing actin reorganization in serum-deprived cells [155] . the coronavirus n protein is abundantly produced within infected cells. n has multiple functions, including binding to viral rna to form the ribonucleocapsid and has also been proposed to have roles in virus replication, transcription and translation. in host cells, n proteins have been shown to cause deregulation of the cell-cycle [70, 142] , inhibit the production of interferon [158] , up-regulate the production of cox2 [162] , up-regulate the activity of ap1 [163] , and induce apoptosis in serum deprived cells [155] -of all which may have possible pathological consequences [164] . several excellent reviews on the coronavirus n protein have previously been published [165, 166] , including one on the structure and function of the sars-cov n and its interaction with nucleic acids [19] . notwithstanding these reviews, the manner in which coronavirus n proteins carry out its roles during the viral life cycle is still not clearly understood. an important piece of missing information lies in the difficulty in resolving the atomic structure of the rnp complex, which has been hampered by low solubility of the rnp complex and the labile nature of the full-length n protein. in addition, in order to determine whether the rnps from various coronaviruses share a common structural code, the structure of different coronavirus rnps need to be resolved [19] . direct intraviral protein-n interactions identified to date include the interaction between n and m [61, 92, 93] and n and nsp3a, a component of the viral replicase [10, 52] . additionally, in mhv-infected cells, monoclonal anti-n antibody co-immunoprecipitates both m and s proteins; this n-s interaction is not a direct one though. rather, it is due to the interaction between s and m protein [93] , where the s protein forms complexes with m protein in the endoplasmic reticulum (er) [167, 168] . the identification of host proteins targeted by viral proteins during the infection process provides important insights into mechanisms of viral protein function [169] . to date, the interaction of n with numerous host cell proteins have been identified, including hcypa [170] , proteasome subunit p42 [171] , the b23 phosphoprotein [172, 173] , smad3 [174] , nrnp-a1 [148] , the chemokine cxcl16 [175] , translation elongation factor-1 alpha [75] , cellular pyruvate kinase protein [176] , 14-3-3 [39] and nucleolin [73, 177] . more recently, a study using high-throughput mass spectrometry identified a list of cellular proteins that could potentially interact with the ibv n protein [177] . comparative studies between various coronavirus n protein interactions could provide valuable information on host specificity and evolution of the interactions between n and host cell proteins. in turn, this may offer insight into the development of novel antiviral therapeutics that target interactions between host cell proteins and the n protein [177, 178] . sars-cov n protein is extremely antigenic. sars-cov infection causes a highly restricted, immunoglobulin g-dominated antibody response that is directed most frequently and predominantly at the nucleocapsid [179] . dna vaccines encoding sars-cov n protein generate a strong n-specific humoral and t-cell-mediated response and significantly reduce the viral titre of challenging vaccinia virus in c57bl/6-vaccinated mice [180] . importantly though, another study suggests that n does not induce virus neutralizing antibody and as a result, provides no protection to infection in hamsters [181] . in the diagnosis/screening hcov-oc43, rabbit poloyclonal antibodies demonstrated 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and infectivity in cultured cells the virion n protein of infectious bronchitis virus is more phosphorylated than the n protein from infected cell lysates nucleocapsid protein of sars-cov activates the expression of cyclooxygenase-2 by binding directly to regulatory elements for nuclear factor-kappa b and ccaat/enhancer binding protein activation of ap-1 signal transduction pathway by sars coronavirus nucleocapsid protein characterisation of human coronavirus-nl63 nucleocapsid protein the sars-cov nucleocapsid protein: a protein with multifarious activities background paper. functions of the coronavirus nucleocapsid protein protein interactions during coronavirus assembly envelope glycoprotein interactions in coronavirus assembly discovery of host-viral protein complexes during infection nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a interactions of sars coronavirus nucleocapsid protein with the host cell proteasome subunit p42 co-localization analysis between porcine epidemic diarrhea virus nucleocapsid protein and nucleolar phosphoprotein b23.1. wei sheng wu xue bao the nucleocapsid protein of sars-associated coronavirus inhibits b23 phosphorylation severe acute respiratory syndrome-associated coronavirus nucleocapsid protein interacts with smad3 and modulates transforming growth factor-beta signaling cxcl16 interact with sars-cov n protein in and out cell sars-cov nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity the cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology viruses and interactomes in translation antibody response of patients with severe acute respiratory syndrome (sars) targets the viral nucleocapsid generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity immunoreactivity characterisation of the three structural regions of the human coronavirus oc43 nucleocapsid protein by western blot: implications for the diagnosis of coronavirus infection burtram c. fielding receives funding from the senate research fund, university of the western cape and the national research foundation, south africa. any opinion, findings and conclusions or recommendations expressed in this material are those of the author and therefore the nrf does not accept any liability in regard thereto. the authors apologize to any author whose work was inadvertently omitted from this review. the authors declare no conflict of interest. key: cord-304607-td0776wj authors: paszkiewicz, konrad h.; giezen, mark van der title: omics, bioinformatics, and infectious disease research date: 2010-12-24 journal: genetics and evolution of infectious disease doi: 10.1016/b978-0-12-384890-1.00018-2 sha: doc_id: 304607 cord_uid: td0776wj bioinformatics is basically the study of informatic processes in biotic systems. actually what constitutes bioinformatics is not entirely clear and arguably varies depending on who tries to define it. this chapter discusses the considerable progress in infectious diseases research that has been made in recent years using various “omics” case studies. bioinformatics is tasked with making sense of it, mining it, storing it, disseminating it, and ensuring valid biological conclusions can be drawn from it. this chapter discusses the current state of play of bioinformatics related to genomics and transcriptomics, briefs metagenomics that finds use in infectious disease research as well as the random sequencing of genomes from a variety of organisms. this chapter explains the various possibilities of pan-genome, transcriptional reshaping and also enormous progress of proteomics study. bioinformatic algorithms and tools are crucial tools in analyzing the data. the chapter also attempts to provide some details on the various problems and solution in bioinformatics that current-day scientists face while concentrating on second-generation sequencing strategies. although bioinformatics is generally perceived to be a modern science, the term had been put forward over thirty years ago by paulien hogeweg and ben hesper for "the study of informatic processes in biotic systems" (hogeweg, 1978; hogeweg and hesper, 1978) . it is necessarily nebulous-bioinformatics spans many disciplines and can have many shades of meaning. indeed it can be argued that it is the collation and analysis of data from different disciplines that has provided some of the greatest insights. in the field of genomics and transcriptomics, bioinformatics is an incredibly diverse field. evolution, epidemiology, ecology, and the response of an organism to its environment are all fields that require bioinformatics to accurately process and place into context various sources of data. at the heart of genomics and transcriptomics is the generation and analysis of vast quantities of sequence data. dna sequencing took off in the late 1980s when applied biosystems developed the first automated sequencing machine. the subsequent development of more efficient ways to sequence resulted in the phenomenal growth of the number of sequences deposited in genbank (figure 18 .1). obviously, with over 100 million sequences deposited in genbank, it is not feasible to do any serious manual work with such a large dataset. data obtained from modern secondgeneration sequencers is on the order of 1000 times greater than capillary-based sequencers. it is now possible to routinely generate many gigabases of sequence data. bioinformatics is tasked with making sense of it, mining it, storing it, disseminating it, and ensuring valid biological conclusions can be drawn from it. many of the recent high-throughput functional genomics technologies rely on a bioinformatics component, though bioinformatics is just one part of the process. for example, identification of proteins by mass spectroscopy, quantitative analysis of expression data, phylogenetics, and so on all make use of bioinformatics tools, methods, and databases. bioinformatics plays a key role at several steps in genomics, comparative genomics, and functional genomics: sequence alignment, assembly, identification of single nucleotide polymorphisms (snp), gene prediction, quantitative analysis of transcription data, etc. in this chapter, we will discuss the current state of play of bioinformatics related to genomics and transcriptomics and use relevant examples from the field of infectious diseases. the term "metagenomics" was originally used to describe the sequencing of genomes of uncultured microorganisms in order to explore their abilities to produce natural products (handelsman et al., 1998 , rondon et al., 2000 and subsequently resulted in novel insights into the ecology and evolution of microorganisms on a scale not imagined possible before (see cardenas and tiedje, 2008; hugenholtz and tyson, 2008 for an overview). however, metagenomics now finds use in infectious disease research as well as the random sequencing of genomes from a variety of organisms from, for example, patient material that could lead to the identification of the cause of disease. in a quite straightforward metagenomics approach to identify pathogens in sputa from cystic fibrosis patients, standard microbiological culture techniques were compared to molecular methods using 16s rdna pcr (bittar et al., 2008) . the well-known disadvantage of the microbiological methods is that they normally employ "selective" media that are designed to pick up those bacterial pathogens that are thought to be present. emerging pathogens will be missed using traditional culture techniques. indeed, bittar et al. identified 33 bacteria using cultivation while 53 bacterial species were detected using molecular methods (based on blast comparisons; altschul et al., 1990) , interestingly, 30% of the latter were anaerobes, organisms missed in the routine cultivation methods. many bacteria identified using the molecular methods are traditionally not thought to be associated with cystic fibrosis. whether these novel species are associated with the physiopathology of disease remains to be studied. bittar et al. (2008) also noted that the number of bacteria detected increased with increased numbers of clones sequenced, a well-known phenomenon in environmental sequencing that relates to sample depth (huber et al., 2007; huse et al., 2010) . however, with the increased use of next-generation sequencing methods in infectious disease research, the lessons learned from environmental studies relating to diversity and relative abundance of different microbes can be put to effective use. an example of the use of second-generation sequencing in a metagenomics approach of patient material is the study by nakamura et al. (2009) to identify viruses in nasal and fecal material. in this study, rna was isolated from patient material obtained during seasonal influenza infections and norovirus outbreaks. this rna was reverse transcribed into cdna, which was subsequently subjected to large-scale parallel pyrosequencing resulting in 25,000 reads on average per sample. although the influenza samples were mainly (.90%) human in origin, it was nonetheless possible to identify the influenza subtypes in each sample (nakamura et al., 2009) . as the fecal samples were cleared of human and bacterial cells, yields were much better and the complete norovirus gii.4 subtype genome was sequenced with an average cover depth of up to 2583. in addition to being able to identify the influenza and noroviruses, two recently identified human viruses were also identified: wu polyomavirus and human coronavirus hku1 (nakamura et al., 2009) . major bacterial species normally found in the respiratory tract were also identified. although nakamura et al. suggest that the high-throughput sequencing is more sensitive than standard pcr-based analysis and might result in the detection of additional possible pathogens, they also warn that the increased sensitivity might necessitate follow-up work to decide which of the detected pathogens is the actual cause of the disease. important results are expected from the human microbiome project (http:// www.hmpdacc.org/), which will obtain metagenomic information from various human microenvironments such as the gastrointestinal, nasooral, and urogenital cavities as well as the skin. understanding the human microbiome is thought to answer questions such as whether changes in the human microbiome are related to human health. however, large-scale metagenomics projects that include eukaryotic genomes have thus far been quite costly and laborious due to the generally large genomes of eukaryotes. the lowering of sequencing costs may alleviate part of the problem, but sequence data are still accumulating at a faster rate than developments in computational analysis (hugenholtz and tyson, 2008) . organisms that have attracted the attention of genome centers are those that cause disease followed by those from model organisms such as saccharomyces cerevisiae (goffeau et al., 1996) and caenorhabditis elegans (the c. elegans sequencing consortium, 1998), for example. indeed, the first bacterial genomes sequenced were those from pathogens fraser et al., 1995; tomb et al., 1997) , and these were preceded by many bacteriophage genomes such as bacteriophage ms2 (fiers et al., 1976) and ϕx174 (sanger et al., 1977) and viral genomes (fiers et al., 1978) . currently, pathogen genomes represent at least one third of all sequenced genomes. obviously, for comparative genomics two genomes are required, and indeed, when the second bacterial pathogen was sequenced (mycoplasma genitalium by fraser et al., 1995) , it was immediately compared with the first one (haemophilus influenzae by fleischmann et al., 1995) . interestingly, the h. influenzae genome was completed using a "bioinformatics" approach. unlike previous sequencing projects, the used shotgun approach relied on a computational justification that sufficient random sequencing of small fragments would result in a complete coverage of the whole genome. comparing the m. genitalium genome with the haemophilus genome suggested that the percentage of the total genome dedicated to genes is similar albeit that m. genitalium has far fewer genes (fraser et al., 1995) . although the genome of m. genitalium is about three times smaller than that of h. influenzae, its smaller genome has not resulted in an increase in gene density or decrease in gene size. detection of several repeats of components of the mycoplasma adhesin, which elicits a strong immune response in humans, suggests that recombination might underlie its ability to evade the human immune response. that this initial genome study was only the tip of the comparative genomics iceberg was already clear from fleischmann et al. (1995) last sentence: "knowledge of the complete genomes of pathogenic organisms could lead to new vaccines." a whole-genome effort at identifying vaccine candidates appeared some 5 years later when pizza et al. (2000) employed bioinformatics to extract putative surface-exposed antigens by genome analysis. although effective vaccines against neisseria meningitidis, the causative agent of meningococcal meningitis and sepsis, did exist, these vaccines did not cover all pathogenic serogroups. serogroup b had evaded the development of a good vaccine as its capsular polysaccharide (against which the vaccines of the other serogroups were developed) is identical to a human carbohydrate. in order to identify putative candidates for vaccine development, pizza et al. decided to sequence the whole genome of a serogroup b strain. all potential open reading frames (orfs) were analyzed for putative cellular locations using blastx. those orfs likely to be cytosolic were excluded from further analysis. the remaining orfs were analyzed to determine whether they encoded proteins that contained transmembrane domains, leader peptides, and outer membrane anchoring motives using a variety of databases such as pfam (finn et al., 2010) and prodom (servant et al., 2002) . this resulted in 570 orfs encoding putative exposed antigens. these 570 putative genes were cloned in escherichia coli and pizza et al. successfully expressed 350 orfs. these 350 recombinant proteins were used to generate antisera that were tested in enzyme-linked immunosorbent assay (elisa) and fluorescence-activated cell sorter (facs) analyses to test whether they detected proteins on the outer surface of serogroup b meningococcus strains. in addition, the sera were tested for bactericidal activity. of the 350 proteins, 85 reacted positively in at least one assay but only 7 were positive in all three assays. these 7 were subsequently tested on a large variety of strains to analyze their efficacy. a total of 5 seemed able to provide protection against 31 n. meningitidis strains and in addition, those 5 proteins are 95à99% similar to the homologous n. gonorrhoeae proteins, suggesting they might provide successful protection against that pathogen as well (pizza et al., 2000) . arguably the most striking aspect of this study is that in 18 months the authors identified more vaccine candidates than in the preceding 40 years using a novel genomics/bioinformatics approach (seib et al., 2009 ). this study resulted in a vaccine that is currently in phase iii clinical trials (giuliani et al., 2006) . protozoan infections are a major burden on developing nations; they take 8 of the 13 diseases targeted by the world health organization's special program for research and training in tropical diseases (http://www.who.int/tdr). over the last 5 years or so, more than 10 parasitic genomes have been sequenced in the hope that their sequences would reveal weak spots to target these pathogens. the trypanosomatids cause serious disease in africa and south america. trypanosoma brucei causes sleeping sickness in humans and wasting disease in cattle. trypanosoma cruzi is the causative agent of chagas disease and leishmania major leads to skin lesions. the completion of their genomes , el-sayed et al., 2005a , ivens et al., 2005 and the comparative analysis of all three genomes (el-sayed et al., 2005b) may be able to focus efforts toward obtaining vaccines, as current drugs have serious toxicity issues. although their genomes encode a different number of protein-encoding genes (around 8100 in t. brucei; 8300 in l. major; 12,000 in t. cruzi), comparative analysis resulted in the identification of about 6200 genes that entail the trypanosomatid core proteome. all protein coding genes were compared in a three-way manner using blastp (el-sayed et al., 2005b) and the mutual best hits were grouped as clusters of orthologous genes or cogs ( figure 18 .2). trypanosomatid specific proteins from these 6200 might be used in a broadscale vaccine. the remainder of the protein-encoding genes from each parasite (26% of the genes in t. brucei; 12% in l. major; 32% in t. cruzi) consists of species-specific genes. interestingly, a large proportion of these genes encode surface antigens and this might relate to the different mechanisms these parasites employ to evade the host immune system. in addition, it was noted that many genes encoding surface antigens are found at or near telomeres and that many retroelements seem to be present in these regions as well. this might be related to the enormous antigenic variation observed in both trypanosoma species. the presence of novel genes in these areas might suggest that their products play an unknown role in antigenic variation as well which warrants further studies into these uncharacterized genes (el-sayed et al., 2005b) . detailed knowledge of well-studied pathogens might be successfully used to understand the biology of closely related emerging pathogens. this was the driving force for the sequencing of six candida species (butler et al., 2009) . candida species are the most common opportunistic fungal infections in the world and c. albicans is the most common of all candida species causing infection. however, c. albicans incidence is declining while other species are emerging. comparison of eight candida species indicated that although genome size was variable, gene content was nearly identical across all species. as the analysis included pathogenic and nonpathogenic species, butler et al. (2009) specifically studied differences between these two groups. of the over 9000 gene families analyzed, 21 were significantly enriched in pathogenic species. many gene families known to be involved in pathogenesis were present in these 21 families (e.g., lipases, oligopeptide transporters, and adhesins). more interestingly, several poorly characterized gene families were also identified, suggesting these might play an unexpected role in pathogenesis as well. this comparative study revealed a wealth of new avenues to explore, which, combined with the large body of work performed on c. albicans, will aid understanding the newly emerging pathogenic candida species (butler et al., 2009 ). although comparative studies using multiple species can reveal hitherto unknown features as evidenced from the mentioned trypanosomatid and candida studies, they can also reveal something unexpected. because the definition of a bacterial species has been debated for a long time, tettelin et al. (2005) set out to address this question by sequencing multiple strains from streptococcus agalactiae, the most common cause of illness or death among newborns. unexpectedly, despite the presence of a "core-genome" shared between all 8 genomes, mathematical modeling suggested that each additional sequenced genome would add 33 new genes to the "dispensable genome." an additional analysis using s. pyogenes also suggested that sequencing additional genomes would continue to add new genes to the pool resulting in a pan-genome that can be defined as the global gene repertoire of a species . this cannot be extrapolated ad infinitum, as a similar analysis of bacillus anthracis indicated that after the fourth genome, no additional genes were identified in agreement with its known limited genetic diversity (keim and smith, 2002) . subsequent analyses have confirmed the presence of pan-genomes for many bacterial species (hiller et al., 2007; lefébure and stanhope, 2007; rasko et al., 2008; schoen et al., 2008; lefébure and stanhope, 2009) and the ultimate gene repertoire of a bacterial species is much larger than generally perceived. whether this would be the case for eukaryotes remains to be shown. despite the apparently ever-expanding possibilities of the pan-genome, it has also resulted in a universal vaccine candidate for group b streptococcus (gbs). because various gbs serotypes exist, current vaccines only offer protection against a limited set of serotypes. eight genomes from six serotypes were compared resulting in the identification of a core-genome of 1811 genes and a dispensable genome of 765 genes, which were not present in each strain . both genomes were analyzed for the presence of putative surface-associated and secreted proteins. of the 598 identified genes, one third were part of the dispensable genome (193 genes). the authors subsequently produced recombinant tagged proteins in e. coli that were used to immunize mice. ultimately, a combination of four antigens turned out to be highly effective against all major gbs serotypes. three of these antigens were part of the dispensable genome. in addition, this bioinformatics approach highlights the importance of not dismissing unidentified orfs on genomes (generally up to 50% of sequenced genomes) as all four antigens had no assigned function. because of their identification using this method, it became obvious they were part of a pilus-like structure that had never seen before in group b streptococcus (lauer et al., 2005) . the presence of antigens that provide protection on these pilus-like structures suggest that these might play a role in pathogenicity. genomic information is useful as a scaffold. however, in a given environment pathogens and hosts only express a subset of their genes at any one time. the presence of pan-genomes only complicates matters even more. to investigate the response of an organism to an environmental or other stress it is necessary to examine the expression pattern of proteins. at present, this is not possible to accomplish directly on a large scale, but a good approximation can be made by sequencing and counting mrna molecules. at present the process involves converting the rna to cdna, which can introduce biases but nonetheless sequencing has a great many advantages over traditional microarrays (ledford, 2008) . these include high specificity with little or no background noise and one also gains nucleotide level resolution of expression. despite such drawbacks, microarrays are still extremely powerful tools to understand levels of gene expression, and this is obvious from the study by toledo-arana et al., who discovered novel regulatory mechanisms in listeria (toledo-arana et al., 2009) . l. monocytogenes is normally harmless but can lead to serious food-borne infections. environmental change, from the soil through the stomach to the intestinal lumen and ultimately into the bloodstream, is thought to be responsible for the up-and downregulation of a plethora of genes. comparative genomics of the nonpathogenic l. innocua has resulted in the identification of a virulence locus (glaser et al., 2001) . using microarrays, transcripts of one strain grown at 37 c in rich medium were compared to three different conditions: stationary phase, hypoxia, and low temperature (30 c). in addition, knockout mutants in three known regulators of listeria virulence gene expression (prfa, sigb, and hfq) were compared to the control strain as well. rna was also extracted from the intestine of inoculated mice and from blood from healthy human donors that were both infected with three different strains (control and prfa and sigb knockouts). this analysis resulted in the discovery of massive transcriptional reshaping under the control of sigb when listeria enters the intestines. however, in the bloodstream, gene expression is under control of prfa. various noncoding rnas were uncovered, which show the same expression patters as virulence genes suggesting a potential role in virulence (toledo-arana et al., 2009) . because microarray data are based on a comparative difference in hybridization, high-throughput next-generation sequencing is seen as more quantitative as it based on number of hits for each sequenced transcript ( van vliet, 2010) . however, when making cdna for next-generation sequencing transcriptomics in prokaryotes, there are several difficulties not found in eukaryotes, such as high levels of rrna and trna molecules as well as a lack of poly-a tails, making extraction difficult. nontheless, it is possible to overcome these by either reducing the amount of rrna and trna using commercially available kits or by bioinformatic removal of such sequences postsequencing ( van vliet, 2010) . to date, some 20 rna-seq style experiments have been performed on prokaryotes. to give an example of the sort of novel insights that can be gleaned using such technology, passalacqua et al. (2009) sequenced the bacillus anthracis transcriptome using solid and illumina sequencing and clearly showed the polycistronic nature of many transcripts on a whole genome scale. although known for individual operons, this had never been shown on a genome-wide scale. they were also able to test the current genome annotations and discovered that 36 loci that were removed as nongenes showed significant transcriptional activity. in addition, 21 nonannotated regions had clear levels of transcription and should therefore be considered as genes (passalacqua et al., 2009) . as internal methionines could have incidentally been identified as start codons, they also checked whether upstream regions were included in the transcribed region. in 11 cases this proved to be the case suggesting the original start codons were incorrectly annotated. reassuringly, when comparing their data with microarray data, a strong correlation was observed. interestingly, because of the very high resolution of sequence-based transcriptomics studies, it is possible to identify novel regulatory elements. for example, when comparing expression levels under o 2 -and co 2 -rich conditions, the first gene of an eight-gene operon did not show a marked difference in expression level while all the others were significantly upregulated under co 2 (passalacqua et al., 2009 ). indeed, a bioinformatics approach had suggested the presence of a t-box riboswitch between genes 1 and 2 of this operon (griffiths-jones et al., 2005) . a similar approach to study how burkholderia cenocepacia, an opportunistic cystic fibrosis pathogen, responds to environmental changes revealed several new potential virulence factors (yoder-himes et al., 2009). as b. cenocepacia is routinely isolated from soil, two strains (one isolated from a cystic fibrosis patient and one from soil) were analyzed in their response to changes from growth at synthetic human sputum medium and soil medium. although their overall nucleotide identity is 99.8%, 179 and 120 homologous genes showed a significant difference in expression between the two strains when grown in synthetic sputum medium and soil medium, respectively. this suggests that despite the high level of relatedness, differential gene expression plays a large role in adaptation to their ecological niche (yoder-himes et al., 2009) . interestingly, similar to passalacqua et al. (2009) , several expressed noncoding rnas were uncovered with different expression levels depending on environmental condition. the significance of this needs to be investigated but highlights the ability of second-generation sequencing to unearth novel findings. despite the fact that a species' genome could well be larger than the actual genome content of one member of that species due to the pan-genome concept, an organism's proteome is by far much more complex. as discussed earlier, transcriptomics will reveal which subset of the genome is expressed under a given condition. however, posttranslational modifications of proteins make the actual proteome far more complex than the transcriptome. this is also the strength of proteomics, as can be seen in a study of the obligate intracellular parasite chlamydia pneumonia. c. pneumonia is the third-most-common cause of respiratory infections in the world, which, in part, is made possible due to the unique bi-phasic life cycle of this bacterial pathogen. chlamydia spread via a metabolically inert infectious particle called the elementary body. these elementary bodies enter the host cell where they differentiate into reticulate bodies. as the elementary body is the infectious phase, proteins presented on the outer membrane would be ideal candidates for vaccine development, especially as effective vaccines are lacking and treatment is via antibiotic therapy. a large-scale genomics-proteomics study by montigiani et al. (2002) systematically assessed putative exposed antigens for possible use in vaccine development. of the 1073 c. pneumonia genes, 636 have assigned functions, 72 of the latter are predicted to be peripherally located and were therefore selected for follow-up studies. in addition, the remaining 437 orfs were subjected to a series of search algorithms aimed at identifying putative surface-exposed antigens. in total, 141 orfs were identified as being possibly located on the cell surface. these 141 were subsequently used to produce recombinant proteins in e. coli. because both his-tagged as well as gst-tagged versions were made, a total of 173 recombinant proteins were produced and used for immunizations of mice. all antisera were used in facs analysis to test if they could bind to the c. pneumonia cell surface. this resulted in the identification of 53 putative surface-exposed antigens. interestingly, apart from well-known antigens, 14 antigens from unidentified orfs were part of this group of potential vaccine candidates. all 53 candidates were tested on western blots whether they generated a clean band of the expected size or whether they cross-reacted with other proteins; 33 of the 53 were specific. finally, montigiani et al. conducted a proteomic analysis of total protein from the elementary body phase identifying spots using mass spectrometry. protein sequencing using maldi-tof identified 28 putative surface-exposed antigens on the c. pneumonia 2d gels (montigiani et al., 2002) . a follow-up study by thorpe et al. (2007) clearly showed that one of the identified candidates, lcre, induced, amongst others, cd4 1 and cd8 1 t cell activation and completely cleared infection in a murine model. interestingly, lcre is homologous to a protein that is thought be part of the type iii secretion system of yersinia. the exposed nature of lcre on the c. pneumonia cell surface suggests that a type iii secretion system plays a role in chlamydia infection (montigiani et al., 2002) . the importance of exposed outer membrane proteins as potential vaccine candidates has prompted berlanda scorza et al. to assess the complement of outer membrane proteins from an extraintestinal pathogenic e. coli strain (berlanda scorza et al., 2008) . extraintestinal pathogenic e. coli is the leading cause of severe sepsis and current increases in drug resistance warrant the search for novel vaccine targets. in addition, current whole-cell vaccines suffer from undesired cross-reactions to commensal e. coli as well. the novel approach by berland scorza et al. is based on the observation that some gram-negative bacteria release outer membrane vesicles (omv) in the culture media, albeit in minute quantities. a tolr mutant appeared to release much more omvs than wild-type cells and subsequent large-scale mass spectroscopic analysis of its protein content resulted in the identification of 100 proteins. the majority of these were outer membrane and periplasmic proteins. intriguingly, three subunits from the cytolethal distending toxin (cdt) were included. this toxin is unusual in that one of its subunits is targeted to the eukaryotic host cell, where it breaks doublestranded dna resulting in cell death (de rycke and oswald, 2001) . to check whether the presence of cdt in the omv was due to the tolr knockout, wild-type extraintestinal pathogenic e. coli was tested using western blotting. indeed, cdt was detected in wild-type omv as well (berlanda scorza et al., 2008) . this suggests that toxin delivery via vesicles might well be the key event in pathogenesis. interestingly, 18 of the 100 identified proteins were not predicted to be targeted to the periplasm or outer membrane by psortb (gardy et al., 2005) . we see here excellent opportunities to train protein targeting algorithms with new wetbench data as these algorithms generally have been trained on a limited set of model organisms that do not reflect the diversity encountered in real life. despite the enormous progress in genomics of infectious diseases, the discovery of new drugs has not kept equal pace. for example, no candidate drugs have been identified after 70 high-throughput screens using validated bacterial drug targets (payne et al., 2007) . although broad-spectrum drugs might be more desirable, there has been a recent trend in targeting specific proteins from specific pathogens using structural biology. several structural genomics initiatives have been set up to target specific groups of pathogens. for example, the seattle structural genomics center for infectious diseases (http://ssgcid.org) and the center for structural genomics of infectious diseases (http://www.csgid.org) work on category a to c agents listed by the national institute for allergy and infectious diseases (niaid). other centers focus on specific organisms such as mycobacterium tuberculosis. examples are the mycobacterium tuberculosis structural proteomics project (http://xmtb. org) and the mycobacterium tuberculosis structural proteomics consortium (http://www.doe-mbi.ucla.edu/tb). the field of structural genomics aims to solve as many protein structures as possible from human pathogens with the aim to come up with new drug targets or vaccines (van voorhis et al., 2009) . obviously, correct selection of candidates for structural genomics projects is paramount and various criteria have been put forward (anderson, 2009; van voorhis et al., 2009) . if a protein is already a validated drug target obviously aids in selection. the proteins need to be essential for the pathogen and ideally, absent in humans. proteins involved in the uptake of essential nutrients are another target. classically, drug design has been focusing on substrate binding sites. more recently, small molecules interfering with subunit binding have started to attract attention. as eukaryotic and prokaryotic inorganic pyrophosphatases differ in composition (the former are homodimers, while the latter are homohexamers), efforts are aimed at compounds that interfere with the oligomeric state of the enzyme. in contrast, the highly conserved active site of inorganic pyrophosphatase would not have been a good target (van voorhis et al., 2009) . the 2003 sars outbreak that caught the infectious diseases community (if not the whole world) by surprise is one example where structural genomics has made enormous progress. despite knowing that coronaviruses caused serious diseases in animals, the fact that they only caused mild disease in humans meant that there was very little knowledge about coronavirus biology. the subsequent effort to understand viral assembly and replication/transcription, for example, has resulted in the elucidation of 12 sars-cov solved protein structures. interestingly, the novel fold-discovery rate was nearly 50%, while it would normally be more close to 6% (bartlam et al., 2007) . in addition, one key protein, the sars-cov main protease, has since been at the center of structure-based drug discovery. because of the nature of the discipline, structural genomics is dependent on various other disciplines such as biochemistry, microbiology, structural biology, computational biology, and bioinformatics and can only foster in a truly interdisciplinary environment (anderson, 2009 ). it is now possible to sequence the entire genome of a bacterial pathogen, assemble the raw sequence reads, perform automated annotation, and visualize the results within 3 weeks. at the same time (indeed even on the same sequencer) it is also possible to selectively sequence the transcriptome (rna-seq) regions of dna bound to protein (chip-seq) or for relevant species methylated dna to study epigenetic effects as well as small rna molecules. it is also possible to perform the very same sequencing on the host organism at the same time. bioinformatic algorithms and tools are a crucial tool in analyzing such unprecedented volumes of data. these data volumes have emerged as a result of secondgeneration sequencers such as the roche/454, illumina, and abi/solid systems. although useful information can be extracted by single researchers by targeted analysis of the sequencer output, to gain the most information out of such data, it is becoming increasingly common for multiple researchers or research groups with widely differing areas of expertise to collaborate. this collaboration is absolutely crucial if relevant insights are to be gained from large-scale datasets. as a result a vast array of data is generated, which is required to be annotated and curated as well as analyzed for information relevant to any particular experiment. in addition this information needs to be stored, shared, and distributed in a manner that enables reanalysis if and when new hypotheses are generated. platforms as produced by the gmod consortium (http://gmod.org), such as gbrowse, and underlying databases are excellent web-based tools for visualizing and comparing datasets. however, they currently offer limited scope for collaborative annotation or curation of datasets where relevant expertise can be brought to bear from a variety of different research groups. this problem is magnified with the advent of second-generation sequencers since much smaller groups of researchers tend to be involved, meaning that the expertise that large collaborations can muster (such as the influenza research database [fludb], http://www.fludb.org/) is much smaller. thus there is a need for integrated annotation and visualization pipelines to enable individual researchers to perform comparative genomics and transcriptomics. the broad institute offers a number of useful visualization tools to the individual researcher such as argo (http://www.broadinstitute.org/annotation/argo/) and the integrated genome viewer (igv) (http://www.broadinstitute.org/igv/). argo offers the ability to manually annotate and visualize a genome as well as provide a good graphical overview for comparative genomics and transcriptomics. currently, there is no one standard for bioinformatics pipeline development for next-generation sequencing. several efforts are underway or can be adapted from sanger sequencing pipelines. these include the prokaryote annotation pipeline xbase and the isga server (hemmerich et al., 2010) . these enable de novo sequenced prokaryote genomes to be annotated automatically and corrected manually at a later date. alternative sanger adaptations such as maker can also be used once an assembly has been generated. a large array of programs is now available to either align reads to a reference genome or to assemble them de novo (miller et al., 2010; paszkiewicz and studholme, 2010 ). they will not be listed in detail here as there are many considerations, including sequencing platform used, the read length in use, the expected genome size, length of longest repetitive elements, gc content, and whether paired-end reads are in use. the proprietary newbler software from roche is the most popular method of de novo assembly of 454 reads (typically 400à500bp). popular assemblers for short reads (i.e., mostly from illumina or solid platforms) are velvet (http://www.ebi.ac.uk/bzerbino/velvet) for the assembly of genomic dna or oases from the same group dealing with assembly of reads from transcriptomic cdna (http://www.ebi.ac.uk/bzerbino/oases) (zerbino and birney, 2008) . other assemblers such as abyss (simpson et al., 2009) , allpaths (butler et al., 2008) or soapdenovo (http://soap.genomics.org.cn/soapdenovo.html) are also popular. abyss enables assembly to be parallelized, thus speeding up assembly. allpaths has been shown to offer superior performance when multiple pairedend libraries are used. independent of read length, it is crucial that paired-end libraries are used when constructing de novo assemblies of any genome. note that the use of short-read sequences only can lead to significant gaps being left in the final assembly due to repetitive elements. however, for many analyses (especially for prokaryotic organisms) these gaps are generally not considered to be significant. in cases where closure of these gaps is more desirable than the addition of 454, sanger or long-range pcr data can often help. where significant quantities of long-and short-read data are available, then a joint assembly can be attempted. a recommended protocol is to assemble the short and long reads separately using their respective packages and to then merge the two assemblers using programs such as minimus (sommer et al., 2007) . another option is to use a template sequence from a related organism to help guide the assembly (note-this is distinct from remapping as described). the amoscmp package is useful for this purpose (pop et al., 2004) . finally, whatever assembly method is used, it is important to remember that a longer assembly is not necessarily a better one. examining the reads making up a contig (e.g., using the amos package (http://amos.sourceforge.net) or the tablet viewer (http://bioinf.scri.ac.uk/tablet) and alignment to a core-conserved group of genes should be standard practice to ensure that blatant errors are corrected. remapping of short reads to a reference genome is also a valid method of comparison. although software such as blat (kent, 2002) can be used with longer 454 reads, it is not an ideal tool for shorter read technologies where data volumes are much greater. where such a genome is available, software such as maq, its successor, bwa, bowtie, soap, and others offer a wealth of tools to identify indels, snps, and other variants which may be of interest. crucially in these cases it is important to have sufficient depth of coverage to ensure snp calls are valid. paired-end data is also valuable to have to highlight the presence of indels. after remapping it is also common practice to assemble unmapped reads using the de novo assembly software to reveal any novel sequence variants, which may be absent in the reference. in the case where pathogens and hosts are sequenced together, if the sequence of at least one is known, then it is relatively straightforward to separate the two using bioinformatic techniques. to deal with transcriptomic data where a reference sequence is available, softwares, such as erange (http://woldlab.caltech.edu/rnaseq/), tophat (trapnell et al., 2009) , and cufflinks (http://cufflinks.cbcb.umd.edu/), are extremely useful. the cufflinks module in particular offers the ability to predict the most likely exon isoform expression pattern using a combination of bayesian statistics and graphbased algorithms. we are aware that our treatment of the use of "omics" and bioinformatics in infectious disease research is not exhaustive. as mentioned in the introduction, what constitutes bioinformatics is not entirely clear and arguably varies depending on who tries to define it. however, we have attempted to show the considerable progress in infectious diseases research that has been made in recent years using various "omics" case studies. in addition, the last section is an attempt to provide a brief overview of the problems and (bioinformatics) solutions that current-day scientists face who embark on second-generation sequencing strategies. this is a fast-moving field, but the provided references and websites should be a good first approach for those who wish to make further strides toward eradicating infectious diseases from our planet. basic local alignment search tool structural genomics and drug discovery for infectious diseases structural proteomics of the sars coronavirus: a model response to emerging infectious diseases proteomics characterization of outer membrane vesicles from the extraintestinal pathogenic escherichia coli δtolr ihe3034 mutant the genome of the african trypanosome trypanosoma brucei molecular detection of multiple emerging pathogens in sputa from cystic fibrosis patients allpaths: de novo assembly of whole-genome shotgun microreads evolution of pathogenicity and sexual reproduction in eight candida genomes new tools for discovering and characterizing microbial diversity cytolethal distending toxin (cdt): a bacterial weapon to control host cell proliferation? the genome sequence of trypanosoma cruzi, etiologic agent of chagas disease comparative genomics of trypanosomatid parasitic protozoa complete nucleotide sequence of bacteriophage ms2 rna: primary and secondary structure of the replicase gene complete nucleotide sequence of sv40 dna the pfam protein families database whole-genome random sequencing and assembly of haemophilus influenzae rd the minimal gene complement of mycoplasma genitalium psortb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis a universal vaccine for serogroup b meningococcus comparative genomics of listeria species life with 6000 genes rfam: annotating non-coding rnas in complete genomes molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products an ergatisbased prokaryotic genome annotation web server comparative genomic analyses of seventeen streptococcus pneumoniae strains: insights into the pneumococcal supragenome interactive instruction on population interactions microbial population structures in the deep marine biosphere microbiology: metagenomics ironing out the wrinkles in the rare biosphere through improved otu clustering the genome of the kinetoplastid parasite, leishmania major bacillus anthracis evolution and epidemiology blat-the blast-like alignment tool genome analysis reveals pili in group b streptococcus the death of microarrays? evolution of the core and pan-genome of streptococcus: positive selection, recombination, and genome composition pervasive, genome-wide positive selection leading to functional divergence in the bacterial genus campylobacter identification of a universal group b streptococcus vaccine by multiple genome screen the microbial pangenome assembly algorithms for next-generation sequencing data genomic approach for analysis of surface proteins in chlamydia pneumoniae direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach structure and complexity of a bacterial transcriptome de novo assembly of short sequence reads identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing the pangenome structure of escherichia coli: comparative genomic analysis of e. coli commensal and pathogenic isolates cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms nucleotide sequence of bacteriophage phix174 dna whole-genome comparison of disease and carriage strains provides insights into virulence evolution in neisseria meningitidis the key role of genomics in modern vaccine and drug design for emerging infectious diseases prodom: automated clustering of homologous domains abyss: a parallel assembler for short read sequence data minimus: a fast, lightweight genome assembler genome analysis of multiple pathogenic isolates of streptococcus agalactiae: implications for the microbial "pan-genome genome sequence of the nematode c. elegans: a platform for investigating biology discovery of a vaccine antigen that protects mice from chlamydia pneumoniae infection the listeria transcriptional landscape from saprophytism to virulence the complete genome sequence of the gastric pathogen helicobacter pylori tophat: discovering splice junctions with rna-seq next generation sequencing of microbial transcriptomes: challenges and opportunities the role of medical structural genomics in discovering new drugs for infectious diseases mapping the burkholderia cenocepacia niche response via high-throughput sequencing velvet: algorithms for de novo short read assembly using de bruijn graphs we would like to acknowledge our colleague dr. david j. studholme for his suggestions and feedback. key: cord-316983-h4mtpcyc authors: mathé-hubert, hugo; colinet, dominique; deleury, emeline; belghazi, maya; ravallec, marc; poulain, julie; dossat, carole; poirié, marylène; gatti, jean-luc title: comparative venomics of psyttalia lounsburyi and p. concolor, two olive fruit fly parasitoids: a hypothetical role for a gh1 β-glucosidase date: 2016-10-25 journal: sci rep doi: 10.1038/srep35873 sha: doc_id: 316983 cord_uid: h4mtpcyc venom composition of parasitoid wasps attracts increasing interest – notably molecules ensuring parasitism success on arthropod pests – but its variation within and among taxa is not yet understood. we have identified here the main venom proteins of two braconid wasps, psyttalia lounsburyi (two strains from south africa and kenya) and p. concolor, olive fruit fly parasitoids that differ in host range. among the shared abundant proteins, we found a gh1 β-glucosidase and a family of leucine-rich repeat (lrr) proteins. olive is extremely rich in glycoside compounds that are hydrolyzed by β-glucosidases into defensive toxic products in response to phytophagous insect attacks. assuming that psyttalia host larvae sequester ingested glycosides, the injected venom gh1 β-glucosidase could induce the release of toxic compounds, thus participating in parasitism success by weakening the host. venom lrr proteins are similar to truncated toll-like receptors and may possibly scavenge the host immunity. the abundance of one of these lrr proteins in the venom of only one of the two p. lounsburyi strains evidences intraspecific variation in venom composition. altogether, venom intraand inter-specific variation in psyttalia spp. were much lower than previously reported in the leptopilina genus (figitidae), suggesting it might depend upon the parasitoid taxa. strikingly, a high venom diversity was observed between the closely related drosophila parasitoids leptopilina boulardi and l. heterotoma (hymenoptera, figitidae), with none of the abundant venom proteins in common 7 . to assess whether this variation between two figitid species that differ in their host range similarly exists in other parasitoid taxa, we compared here the venom composition of two braconid wasps, psyttalia lounsburyi and p. concolor (hymenoptera, braconidae, opiinae) that belong to the same complex of species 8 . both psyttalia species are used as biological control agents of the olive fruit fly bactrocera oleae 9 and they differ in their host range. p. lounsburyi is specialized on b. oleae 9 whereas p. concolor successfully develops in b. oleae and at least 13 other fruit fly species 10 . comparison of p. lounsburyi and p. concolor venom was performed using a combined transcriptomic and proteomic approach, and it was extended at the intraspecific level using two geographically distant african strains of p. lounsburyi (south africa and kenya). we also compared data with large-scale venomics results from other braconids, either associated with pdvs, as chelonus inanitus 11 and microplitis demolitor 12 , or devoid of pdvs, as aphidius ervi 13 , since using various parasitism strategies could possibly impact venom evolution and composition. this study will contribute to a better picture of the diversification of venom components at a short evolutionary scale, opening the way to the characterization of underlying mechanisms. structure of the psyttalia venom apparatus. as typically observed for braconidae, the venom apparatus of both psyttalia species is composed of venom gland filaments (which secrete venom), a venom reservoir, and a venom duct that extends into the ovipositor (fig. 1a) . as was previously described in opiinae 14, 15 , p. lounsburyi and p. concolor venom gland is multi-lobed, each lobe displaying an external thick layer of tissue and a central lumen filled by a large volume of venom (fig. 1b,c) . the gland lobes are joined together at the base where the ovoid shaped reservoir is connected. the reservoir is composed of a large muscular layer surrounding a small internal volume of venom, suggesting it may serve as a "pump" for injecting venom at the time of oviposition rather than as a storage organ. the reservoir also shows internal structures that form intricate spirals possibly involved in maintaining the shape of the reservoir, like spiral springs, by passively counteracting the muscular contraction (fig. 1b,c) . at the base of the venom apparatus of p. concolor, we also observed a "round gland" filled with large vesicles (fig. 1c) , already described by quicke 14 and called "basal bulb" by wharton 15 . interestingly, the round gland and the intima spirals of the reservoir showed a strong endogenous green fluorescence (fig. 1d) , suggesting the presence of universal cellular auto-fluorophores such as nad(p)h and flavins, pigments, or cuticular compounds 16 . no vlp or pdv in p. lounsburyi and p. concolor. among the few studies on psyttalia species, two had reported the occurrence of unidentified virus-like particles (vlps) within the venom secretory cells (in p. concolor, previously opius concolor 17 , and the close species o. caricivorae fisher 18 ). we thus performed electron microscopy on p. concolor venom glands to search for vlps, as well as on p. lounsburyi and p. concolor ovaries to ensure the absence of pdvs (polydnaviruses). we did not observe vlps or vesicular material resembling vlps in venom gland cells or venom (fig. 2a) , nor viral structures or pdv particles in the ovarian cells and fluid close to the eggs (fig. 2b-d) . accordingly, pl and pc transcriptomes contained no transcript having similarities with genes specific of nudiviruses (from which braconid pdvs derive) or the sister group of baculoviruses. as coronaviruses and cypo-like viruses were also described in p. concolor venom glands 17 , previous observations likely corresponded to small viruses infecting the reproductive tract of the samples, as reported also in other hymenoptera 19 . in the absence of vlps and pdvs, secreted venom proteins are likely the main maternal actors of parasitism success of psyttalia species. comparison of electrophoretic profiles of psyttalia venom proteins. venom samples collected from 50 individuals of p. concolor (pc) and the p. lounsburyi south african (pl sa ) and kenyan (pl k ) strains were the empty space is due to retraction during dehydration. no vesicle is observed in the size range of the vlps described in parasitoids venom (50 to 100 nm). bar = 500 nm. (b) picture of whole mounts p. concolor ovaries. oogenesis occurs in egg tubes (et), late oocytes being located in reservoirs (or) and moving down to the calyx (c) where tubes fuse to form the oviduct. bar = 500 μ m. (c,d) tem micrographs of sections through the calyx region of p. lounsbury (c) and p. concolor (d) ovaries, showing the egg chorion (ec), the ovarian fluid (of) and the calyx cells. no pdv particle is observed inside the cells nor in the fluid surrounding the egg chorion. bar = 500 nm. analyzed by 1d and 2d gel electrophoresis (figs 3 and 4) . on a 6-16% sds-page, the protein content of venom glands was resolved into numerous bands from 10 kda to more than 200 kda (fig. 3) . on the silver-stained 2d gels, 50 to 100 spots were clearly visible, having a 4 to 8.5-9 isoelectric point, and ranging from 10 to more than 120 kda (fig. 4) . some trains of spots were also observed that likely corresponded to post-translational modifications of the same protein. the pl sa and pl k 1d electrophoretic profiles were very similar with presence/absence or intensity variation detected for only a few bands (fig. 3) . the distribution of 2d spots evidenced more differences, in particular the absence of the pl sa spot number 10 (55 kda) in the pl k strain (fig. 4a,b) . we observed a greater variation at the interspecific level with several profile differences in the 25-35 kda range (figs 3 and 4) . comparison of transcriptomic and proteomics data between psyttalia wasps. all the major bands and spots on 1d and 2d electrophoretic profiles of pl and pc venom, and a number of minor spots (70 bands for pl, 46 bands for pc, from at least two 1d gels per species; 117 spots for pl, 57 for pc, from at least three 2d gels per species) were excised, and tryptic peptides were analyzed by lc-ms-ms. in parallel, a transcriptomic analysis of pl and pc venom glands was performed, based on illumina sequencing. de novo assembly of the pl transcriptome was improved using additional 454 (full body) and sanger (venom apparatus) sequence data (supplementary figure s1 ) and it yielded a total of 16,943 and 16,360 unisequences for pl and pc, respectively (supplementary table s1 ). data suggested a similar quality of the transcriptomes, based on general features and similarity searches (supplementary table s1 ), as well as go terms comparison (supplementary figure s2) . the combined transcriptomic and proteomic data resulted in 39 and 40 unisequences for pl and pc, respectively, among which 32 and 36 had a putative function (supplementary tables s2 and s3) . some of these, such as actin-5c or glyceraldehyde-3-phosphate dehydrogenase 2, were typical cellular proteins with no predicted signal peptide (supplementary tables s2 and s3 ). whether their identification in venom is due to cellular damage during collection, or these proteins are actually secreted by non-canonical export mechanisms remains unclear. therefore, we only considered as putative venom proteins the unisequences (i) found in venom proteomics, and (ii) either predicted to be secreted or for which the presence of a signal peptide was not tested due to the incompleteness of the sequence. this resulted in a total of 32 and 30 putative venom proteins for pl and pc respectively (tables 1 and 2), whose relative abundance was compared using (i) the rpkm normalized number of illumina reads from pl and pc venom apparatus, mapped to the assembled transcriptomes and (ii) the number of peptides matches in mascot searches. interestingly, most of the proteins identified in the proteomics of the reservoir (detection of the most abundant putative venom proteins only, data not shown), such as actin or paramyosin, had a predicted muscular function, as expected from microscopy observations (see above; fig. 1 ). comparison of venomics data from the two psyttalia species evidenced that 47% and 43% of the proteins identified in pl and pc were shared with the other species, respectively (tables 1 and 2; fig. 5a ). in comparison, l. boulardi and l. heterotoma shared less than 20% of the identified venom proteins 7 . when considering only the most abundant venom proteins (rpkm > 50 and mascot matches > 10), 9 and 8 out of the 11 pl and 12 pc proteins, respectively, were shared between p. concolor and p. lounsburyi ( fig. 5b ; tables 1 and 2) whereas the two leptopilina species had no protein in common 7 . finally, 20 pl and 21 pc venom proteins (63% and 70%) had already been identified in the venom of another braconid species ( fig. 5c ; tables 1 and 2) . identified venom proteins. putative venom proteins described below are classified based on their abundance in venom (rpkm values and mascot matches; tables 1 and 2 ) and their occurrence in the venom of (i) both psyttalia species, (ii) p. lounsburyi only and (iii) p. concolor only. venom proteins with a putative function are described in table 3 (with the proposed biochemical function, previous identification in parasitoid venom, and demonstrated or proposed role in parasitism success). several proteins with low rpkm values and lacking n-terminal sequence were not considered since they were typical cellular proteins and the number of proteomic matches was low (tables 1 and 2) . proteins identified in the venom of both psyttalia species. proteins of unknown function. several abundant unisequences with no predicted function were characterized by a high number of matches (tables 1 and 2 ) and rather intense protein spots (fig. 4) . among these, a family of five related proteins contains a signal peptide and the duf4803 domain of unknown function (supplementary figure s3 ). they share similarities with venom proteins of the braconids c. inanitus 11 , m. demolitor 12 and m. hyperodae 20 , and of n. vitripennis 21 . leucine-rich repeat protein. two and four unisequences encoding leucine-rich repeat (lrr) proteins were identified in pl and pc, respectively (tables 1 and 2; supplementary tables s2 and s3 ). one of these (pl_009581) was highly abundant (rank 2) in the p. lounsburyi sa strain only, and it corresponded to one of the most intense spot (spot 10, fig. 4a ). the complete unisequences contain a n-terminal signal peptide and 9 to 19 canonical lrr motifs similar to the lrr motif in toll-like receptors (tlrs). yet, the majority of tlrs are multidomain table 2 , locus comp21422_c0). protein not found in the proteomic analysis but with rpkm > 50 and for which a homolog was found in p. lounsburyi. proteins while psyttalia predicted proteins only contain the lrr domain, as already observed for a. ervi venom lrr proteins 13 . as suggested for a. ervi, psyttalia truncated lrr proteins might interfere with the host immune response by targeting the toll pathway. neprilysin-like metalloproteases. three and two unisequences encoded neprilysin-like (nep) zinc-dependent metalloproteases in pl and pc, respectively (tables 1 and 2; supplementary tables s2 and s3) , one of which was in high abundance (rank 8; 37 and 47 peptide matches in pl and pc venom, respectively). nep-like proteins occur in the venom of several parasitoid wasps (table 3) . annexins are a family of ca2 + -dependent lipid binding proteins believed to be engaged in membrane transport processes, although recent work suggests a more complex set of functions. annexins normally lack signal sequences for secretion, but some members of the family have been identified extracellularly where they can act as receptors 44 . annexins had never been described so far in the venom of parasitoids. however, some data suggest that different mammalian parasite clades possess annexins with unique properties that can be secreted and are likely involved in host-parasite interactions and host immune-modulation 45 . moreover, some parasitic nematodes secrete an annexin-like effector into host root cells that may mimic plant annexin function during the parasitic interaction 46 . at last, it has been shown that annexins are also involved in the binding and internalization of toxins in eukaryotic cells 47 . arginine kinase plays a crucial role in the energy metabolism of insects and other invertebrates through the use of atp to catalyze the phosphorylation of arginine in phosphoarginine. this enzyme was detected in the venom of pteromalus puparum 48 and leptomastix dactylopii 49 , but its role in the host-parasitoid interaction is unknown. calreticulin is a calcium (ca2 + )-binding protein with multifunctional properties including chaperone functions 50 . calreticulin was shown to inhibit host cell encapsulation in cotesia rubecula 51 and p. puparum 52 , although the mechanism is still unclear. calreticulin was found in the venom of several phylogenetically distant species 4 and seems thus largely shared among parasitoids. endoplasmin (alternative names: hsp90b1, gp96, grp-94), which belongs to the heat shock protein 90 family, is a molecular chaperone located in the er and involved in the final processing and export of secreted proteins 53 . among parasitoids, endoplasmin has only been detected so far in the venom gland of aphidius ervi 13 . this venom protein was suggested to play a role in the secretion, stabilization, transport and host cell targeting of the different a. ervi venom proteins. enolase is a key enzyme in cell metabolism which is also associated with virulence of several pathogens 54 . an extracellular enolase was recently described in the oviposition injecta from a. ervi 55 and the venom of toxoneuron nigriceps 56 . enolase is also released by teratocytes surrounding the a. ervi embryo 57 . this enzyme was proposed to play an important role in the regulation of the host physiology and the host nutritional exploitation 57, 58 . esterase/lipase-like esterases and lipases belong to a superfamily of hydrolytic enzymes that act on carboxylic esters 59 . proteins belonging to this functional class were previously found in the venom of several phylogenetically distant species 3,4 and appear to be common in parasitoids. the functions of these hydrolase enzymes in host-parasitoid interactions have not been investigated yet. gh1 β -glucosidases gh1 β -glucosidases are a family of enzymes found from bacteria to mammals that hydrolyze glycosidic bonds from glycosides and oligosaccharides, and remove non reducing terminal glucosyl residues 60 . among parasitoids, a β -glucosidase enzymatic activity was detected in the venom of pimpla hypochondriaca 61 . a member of this enzyme family was also recently identified, but not abundant, in the venom of microplitis demolitor 12 and, in a low quantity, in a transcriptomic study of the a. ervi venom apparatus 13 . gh1 β -glucosidases include myrosinases that play a central role in the glucosinolate-myrosinase system, one of the best-studied activated plant defense system 33 . some insects have co-opted this system to defend themselves against enemies, by sequestering plant-derived glucosinolates and producing their own myrosinase-like enzyme [36] [37] [38] . heat shock protein 70 heat shock proteins 70 (hsp70; alternative name: grp-78) are a family of chaperones with distinct sub-cellular localization and function 62 . an hsp70 protein was identified in the venom of the parasitoid p. puparum 48 whose function remains to be elucidated. leucine-rich repeats (lrrs) are motifs involved in protein-protein interactions 63 . lrrs are generally composed of 20-29 amino acid stretches rich in leucine. to our knowledge, lrr domain-containing proteins were only identified in the venom of a. ervi until now 13 . they were suggested to act as scavengers for the pea aphid toll-like receptors, thus impairing the host immune response via the toll pathway. neprilysin-like (nep) proteins are zinc-dependent metalloproteases belonging to the m13 peptidase family. they are involved in the degradation of a number of regulatory peptides in the nervous or immune system of mammals 64 and insects 65 . although they are typically membrane-bound, ectopeptidases such as nep may also be shed from the membrane through a proteolytic process and found in the surrounding fluid 66 . nep-like proteins were detected in the venom of the braconidae a. ervi 13 , microctonus hyperodae 20 , m. demolitor 12 and t. nigriceps 56 , as well as of the figitidae leptopilina boulardi 7 . they were also found associated with the vlps produced in the ovary of venturia canescens 67 . neplike proteins have been hypothesized to modulate the host immune system by degrading immune-specific peptides 67 . secreted phospholipases a2 (pla2s) are a family of relatively stable enzymes found in venoms. pla2 has in vitro and in vivo immunomodulatory effects in bee venom 68 , and neurotoxic and myotoxic effects in snake venoms 69 . this enzyme was recently detected in the venom of m. demolitor 12 and t. nigriceps 56 , but its function in the host-parasitoid interaction is unknown. protein disulfide isomerases (pdis) are enzymes involved in the folding and stabilizing of nascent polypeptides in the endoplasmic reticulum (er) through catalysis of disulfide bond formation and isomerization 70 . although this protein is normally recycled back to the er from the golgi via its c-terminal kdel motif, secreted pdis can escape this turnover mechanism 71 . among parasitoids, pdis have only been detected so far in the venom gland of a. ervi 13 . they have a broad substrate specificity and are involved in the folding of toxin peptides in different venomous organisms 72, 73 . reprolysin-like (rep) proteins are zinc-dependent metalloproteases belonging to the m12 peptidase family, commonly found as constituents of snake venom. they were previously detected in the venom of the parasitoids pimpla hypochondriaca 22 classical serine carboxypeptidases are enzymes that hydrolyze a peptide bond at the c-terminal end of peptides and proteins. a related enzyme (scpep1) that do not show proteolytic activity but is involved in other functions was described in mice 74 . to our knowledge, serine carboxypeptidase has only been identified so far in the venom of m. demolitor 12 and t. nigriceps 56 . interestingly, serine carboxypeptidases have also been described as candidate virulence factors in several pathogens 75 . serpins (serine protease inhibitors) are a large family of functionally diverse protease inhibitors. they share a conserved structural architecture with an exposed reactive center loop (rcl) of about 20 amino acids, which acts as bait for target serine proteases 76 . the l. boulardi venom serpin lbspny was previously shown to be involved in host immune suppression 77 : it interferes with melanization in the drosophila host through inhibition of the phenol oxidase activation. more recently, serpins were described in the venom of a. ervi 13 and m. demolitor 12 but their role in parasitism success is unknown. another zinc-dependent metalloprotease was identified in each psyttalia wasp, with low inter-species sequence similarity. both proteins are weakly related to venom reprolysin-like proteins of p. hypochondriaca 22 and eulophus pennicornis 23 . however, the sequences were incomplete and the number of matches in the venom was rather low (tables 1 and 2; supplementary tables s2 and s3). gh1 β -glucosidases. peptides from major 2d spots at 55-60 kda (spots 6 and 9 in pl k and pl sa , respectively, spots 8, 9, 10 in pc) matched with two unisequences (one for each psyttalia species) that encoded proteins containing a glycosyl hydrolase family 1 (gh1) domain (pfam00232) found in gh1 β -glucosidases (fig. 4) . the high rpkm values and number of peptides matches confirmed that the pl_002819 and pc_001157 unisequences, that share 97% identity (supplementary figure s4) , were among the most abundant in venom (tables 1 and 2; supplementary tables s2 and s3 ). the pc sequence seems full-length, with a predicted signal peptide of 18 residues, while the pl sequence probably lacks the n-terminal part (supplementary figure s4) . the 56.5 kda predicted mw of the mature protein is close to that of the observed spot on 2d gels, suggesting none or few glycosylation, although several n-glycosylation sites are predicted. yet, several spots were observed, having different isoelectric points, suggesting post-translational modification(s) and thus several isoforms (fig. 4) . two pl and one pc additional unisequences, not found in proteomics, shared similarities with gh1 β -glucosidases, suggesting occurrence of a multigene family (supplementary figure s5) . gh1 β -glucosidases were previously identified in venomics data from m. demolitor and a. ervi, but they did not correspond to abundant proteins (tables 1, 2 and 3) . finally, the venom of the ichneumonid pimpla hypochondriaca was reported to display β -glucosidase enzymatic activity (table 3) . gh1 β -glucosidases are found from bacteria to mammals. they play an essential role in the metabolism of glycolipids and exogenous glycosides by hydrolyzing glycosidic bonds and removing non-reducing terminal glucosyl residues from saccharides and glycosides. this enzyme family includes for instance the myrosinases, well-known for their role in the "glucosinolate-myrosinase" plant defense system (table 3) , and also identified in the cabbage aphid brevicoryne brassicae that feed on crucifers 24 . the alignment of pl and pc venom unisequences with that of well-described plant myrosinase (sinapsis alba) and insect β -glucosidases (b. brassicae, phyllotreta striolata, spodoptera frugiperda) shows conservation of all critical enzymatic site residues 25, 26 , suggesting that psyttalia enzymes are indeed functional (fig. 6) . insect β -glucosidases differ from plant myrosinases in that they have two glutamates in the catalytic site instead of one glutamate and one glutamine. the possible role of psyttalia venom gh1 β -glucosidase in host-parasitoid interaction is discussed below. heat shock proteins (hsps). molecular chaperones belonging to the heat shock protein (hsp) class, including hsp70, calreticulin and protein disulfide isomerase (pdi), were found in variable amounts in the two species (tables 1 and 2; supplementary tables s2 and s3 ). endoplasmin, also identified in transcriptomic data from both species (rpkm values of 40.85 for pl and 63.9 for pc) was detected in pl venom only, suggesting a lower abundance in pc venom (tables 1 and 2; supplementary tables s2 and s3 ). in the endoplasmic reticulum (er), all these hsps cooperate to form multi-chaperone complexes involved in the folding and export of secreted proteins. hsp70, pdi and endoplasmin are normally retained in the er through the binding to specific receptors via a k/r/hdel motif. since these proteins are abundant in cells, we cannot totally exclude that their detection in the venom results from tissue contamination during venom collection from the gland. however, one of the venom pdis, identified in both species, contains a substitution that changes the hdel motif into a sdel motif, suggesting a loss of binding and then of retention in the er. moreover, the binding of hsps to er receptors is labile and the retention is not absolute, allowing hsps to be secreted and to play a role in intercellular transport and signaling 27, 28 . endoplasmin, for example, has been associated as a chaperone with the secretion of pancreatic lipases and their further internalization by intestinal cells in rat 29 . venom hsps may thus play a role in stabilization of other venom proteins and/or their transport and targeting of host cells. among these, endoplasmin is of particular interest because it is a master chaperone for tlrs, a family of lrr domain-containing proteins 30 of which members were found in psyttalia venom (see above). in accordance with a possible role of endoplasmin as a chaperone of venom tlrs, endoplasmin was only detected in the venom of pl that contains higher amounts of lrr proteins than the pc venom (tables 1 and 2; supplementary tables s2 and s3 ). interestingly these two proteins are also secreted in the venom of the braconid a. ervi 13 . pdis play a central role in the protection of disulfide bounds of secreted proteins 31 . interestingly, the rapid expansion of a large gene family encoding pdis specifically-expressed in the venom glands of cone snails has been recently suggested to facilitate the folding of the numerous conotoxins produced by these marine predators 32 . here, however, phylogenetic analysis shows that psyttalia venomous pdi sequences group with different pdis from braconid wasps (supplementary figure s6) , suggesting that no specific expansion occurred. proteins of low abundance. proteins with similarities with serpin and enolase (tables 1 and 2; supplementary tables s2 and s3 ) were identified. the pl and pc serpin sequences shared 94% identity but only the pc sequence was complete, the pl serpin lacking the n-and c-termini (supplementary figure s7) . the pc serpin contains a signal peptide as well as the consensus hinge sequence essential for the conformational change involving the rcl and necessary to inhibit the target protease (supplementary figure s7 ). an extracellular enolase was already identified in a. ervi injecta and also produced by specialized extra-embryonic cells (the teratocytes) and suggested to be involved in nutritional host exploitation ( toxin-like peptides were previously identified in a. ervi venom 13 but no toxin-like signature was found in pl venom proteins. proteins of low abundance. all the other proteins found in p. lounsburyi venom only were in low abundance (table 1; supplementary table s2) . among these, an arginine kinase-like protein was identified, and similarities were found with members of the esterase/lipase-like superfamily, detected in many parasitic wasp species (table 3) . proteins detected in p. concolor venom only. proteins of unknown function. two different unisequences (pc_015919 and pc_012023) were detected ( table 2; supplementary table s3 ) but the sequences were not complete and the presence of a signal peptide could not be assessed. pc_012023 (rank 5 based on rpkm values) was one of the most intense 2d spots at less than 15 kda (fig. 4) . pc_015919 was better ranked based on rpkm values, but the corresponding spot was less intense (fig. 4 ) and the number of peptide matches was lower (table 2; supplementary table s3) , suggesting a lower abundance in venom. annexin. one unisequence, corresponding to 16 peptide matches, had similarities with annexins ( table 2; supplementary table s3 ). this protein contains a 20 aa n-terminal signal peptide followed by two annexin domains. based on data from their role in mammalian parasites, p. concolor venom annexin might be involved in binding and cell internalization of other venom proteins ( table 3 ). proteins of low abundance. among these, a secreted phospholipase a2 (pla2s) and a retinoid-inducible serine carboxypeptidase (table 2; supplementary table s3 ) were identified (table 3 ). large scale combined "omics" studies have recently increased knowledge of the nature and diversity of the venom content of parasitoid wasps 3, 4 . yet, very few studies were designed to evaluate how far closely-related parasitoid species differ in venom composition. venom glands of the braconidae m. hyperodae and m. aethiopoides were shown to express a similar set of genes 20 but analyses relied on less than ten genes, and proteomics data were only available for m. hyperodae. more recently, we evidenced striking differences in venom composition of the closely-related figitidae l. boulardi and l. heterotoma 7 , drosophila parasitoids differing in their host range. we observed here a rather similar protein composition of the venom of p. lounsburyi and p. concolor, albeit they also differ in their host specificity level. the host range is determined by both behavioral and physiological traits that include host choice and venom adequacy to the host (i.e. "virulence"). in some taxa, the diversity of venom composition could then be decoupled from the diversity of parasitized hosts in natura if specialization mainly relies on behavioral traits. for instance, although p. lounsburyi is a specialist of the olive fly in the field, it can develop in laboratory conditions on some non-natural hosts such as ceratitis capitata. similarly, although intraspecific variation in p. lounsburyi venom was notably evidenced for a lrr protein, the difference between psyttalia strains from distant geographic origin was much lower than observed between l. boulardi strains 7 . whether the level of diversity of venom differs among taxa (being lower in braconidae than in figitidae), or the extensive venom variation in leptopilina wasps is specific of these genus/species remains an open and stimulating question. unfortunately, a majority of the most abundant unisequences, either common or specific to each psyttalia species, had no predicted function. it is thus difficult to make assumptions about how these wasps counteract the host immune defense and regulate the host physiology, and whether they use similar mechanisms. yet, the identification of a gh1 β -glucosidase as one of the most abundant venom protein in both species suggests a possible role of this protein in parasitoid wasp's success. in plants, glycoside compounds and hydrolytic enzymes form a classic two-component defense system, with glycosides inducing biological effects after being activated by the enzymes. the vast array of secondary metabolites produced is used as a protection against phytophagous organisms and pathogens 33, 34 . hydrolysis-products can indeed be repellent or toxic to insects, nematodes, fungi and bacteria, and they also serve as volatile attractants for specialist herbivores and their parasitoids 35 . plant compounds and endogenous glycosidases are usually stored in separated compartments so that activation only occurs upon tissue damage. to reduce toxic effects, some phytophagous insects were shown to downregulate their gut glycosidases while others have even evolved their own glycosylation system to reglycosylate the produced aglycons. the neo-formed glycosides can then possibly be stored, similarly to the ingested plant glycosides that are indeed sequestered by several insect species. by doing so, these insects prevent the production of toxic compounds [36] [37] [38] , and some even use them for their own defense 39 . the main psyttalia hosts, b. olea and c. capitata, oviposit in developing olives and fruits that contain large quantities of various glycoside compounds. olive is particularly rich in phenol-glucosides such as oleuropein, verbascoside and rutin, which accumulate during its development while b. olea is growing inside 40 . oleuropein was shown to be converted into a toxic compound with a glutaraldehyde-like structure -a potent protein crosslinker -by a defense-related olive β -glucosidase 41 . assuming that fly larvae use a sequestering mechanism to survive within fruits during development, the injection of psyttalia venom β -glucosidase inside their body might result in a burst of toxic compounds that could weaken the host and increase the parasitoid probability of success. the release of sugar moieties from glycosides could also increase the amount of energy available for the developing parasitoid larvae. although this is an attractive hypothesis, the fact remains that the alleged role of psyttalia venom β -glucosidase is based on the sequestration of phenolic glycosides by the olive fly larvae, which has not been tested yet. the importance of a tri-trophic understanding of plant-herbivore and herbivore-predator/parasitoid interactions is increasingly recognized. for instance, the role of glucosides/glucosinolates has started to be evaluated not only in insect-plant interactions but also for their cascading effects on the performance of herbivore enemies through metabolic impacts or emission of volatile products 39 . the report of the large production of a β -glucosidase in a parasitoid venom suggests that this enzyme might participate in parasitoid phenotypes such as counter-defense and parasitism success, in addition to its well-described defensive role in plants and herbivore insects. altogether, this study illustrates that parasitoid venom is a complex mixture of proteins whose relative abundance in a given species or group is still hardly explained. the study of a new species often reveals new types of abundant venom molecules, as exemplified here with the β -glucosidase, and it highlights the role of gene duplication in the rapid evolution of venom and acquisition of new features. deciphering the role of major venom proteins in psyttalia parasitism success, especially those with no predicted function, is a key challenge. this will require further exploration using techniques such as rnai, demonstrated as an efficient approach for impairing the production of parasitoid venom proteins 42 . biological material. the south african (sa) and kenyan (k) strains of p. lounsburyi (pl) were previously described 43 . they were reared on a laboratory strain of the fruit fly c. capitata under a 16:8 h light/dark cycle at 22 °c. the p. concolor (pc) population was collected in 2010 in sicily (italy) and reared for one generation on c. capitata under the same conditions, prior to analysis. c. capitata is a natural host for p. concolor but it is used as a substitute host for p. lounsburyi due to the difficulties of rearing the main natural host, bactrocera oleae (the olive fly). light, fluorescence, and transmission electron microscopy. light and fluorescence microscopies were performed using epifluorescent microscopes fitted with differential interference contrast (dic) optics (imager z1, zeiss) fitted with a black and white camera (axiocam mrm, zeiss). images were pseudo-colorized digitally (adobe photoshop). for transmission electron microscopy (tem), blocks were prepared from 10 ovaries or 10 venom glands per sample. dissected samples were pooled into 100 μ l of ringer's saline (kcl 182 mm; nacl 46 mm; cacl 2 3 mm; tris-hcl 10 mm) in a centrifuge vial on ice. an equivalent volume of fixative (4% glutaraldehyde (sigma) in 0.2 m sodium cacodylate buffer, ph 7.2) was then added and the sample was kept for 24 h at 4 °c. fixed samples were centrifuged (500 × g, 10 min) to pellet tissues and remove the fixative prior to post-fixation in 2% osmium tetroxide in cacodylate buffer. following dehydration in graded series of ethanol solutions, samples were embedded in epon. sample sections were cut with a diamond knife using a lkb ultramicrotome, mounted on copper grids, stained with uranyl acetate and lead citrate, and observed with a zeiss em10cr electron microscope at 80 kv. total rna isolation and cdna library construction. the transcriptomic analysis was performed from samples of 100 pl and 100 pc venom glands using illumina rna-seq. to improve de novo assembly for pl, we also generated sanger sequences from samples of 50 venom glands, and 454 sequences from full insect bodies of 85 males and 85 females obtained from six siblings (supplementary fig. s1 ). pl and pc venom glands were dissected in ringer's saline and stored at −80 °c. total rna was extracted using trizol reagent (invitrogen) according to manufacturer's instructions, and quality was checked using an agilent bioanalyzer. cdna library construction for illumina rna-seq and 454 sequencing was performed by beckman coulter genomics (usa). cdna library used for sanger sequencing was constructed from 1 μ g of total rna using the creator smart cdna library construction kit (clontech). ligation products were transformed into electromax dh10 b escherichia coli competent cells (invitrogen). sequencing and assembly. illumina rna-seq sequencing (hiseq 2000, 2 × 75 pb), 454 sequencing (454 gs-flx titanium platform) and trimming were performed by beckman coulter genomics. quality of illumina raw reads was controlled using fastqc software and reads were cleaned by removing low quality sequences and reads containing n or adaptor sequences. for sanger sequencing, a total of 2,000 clones were analyzed by the genoscope (cea, evry, france) on an abi sequencer using the standard m13 forward primer and bigdye terminator cycle sequencing kit (applied biosystems, foster city, ca, usa). sanger ests were then trimmed using tigr seqclean software. for each species, we performed de novo transcriptome assembly using velvet/oases assembler (https://www. ebi.ac.uk/~zerbino/oases/) after the filtering process of illumina raw reads. the first assembly step used a multiple kmer approach with kmer size ranging from 45 to 65 (k = 45, 55, 65 and coverage = 2). a meta-assembly (kmeta = 51, coverage = 1) was then performed using all previously obtained transcripts (> 100 bases long) ( supplementary fig. s1 ). at both assembly steps, we used cd-hit-est to remove the shorter redundant transcripts entirely covered by other transcripts with more than 99% identity. finally, a clustering of transcripts was performed using tigr-tgicl. to improve the quality of the assembly for pl, we included the cleaned 454 and sanger sequences as long sequences (minimum size of 200 bases) or otherwise as short sequences, in addition to the short illumina reads. sequence annotation and analysis. to identify similarities with known proteins, the unisequences were compared to ncbi non-redundant protein sequence database, uniprotkb/swiss-prot database, insect predicted proteome databases (drosophila melanogaster v5. 46 and nasonia vitripennis v1.2) and all braconid venom proteins found in uniprotkb (96 venom proteins from 9 different braconid species), using blastx with a cut-off e-value of 1e-7. comparisons with previously published venom gland transcriptomes of a. ervi 13 and leptopilina spp 7 were performed using tblastx with a cut-off e-value of 1e-7. search for nudivirus/baculovirus-related specific genes was performed using tblastn with a cut-off e-value of 1e-1. orf prediction and translation were performed with framedp (https://iant.toulouse.inra.fr/framedp/), signal peptide prediction with signalp (http://www.cbs.dtu.dk/services/signalp/), and prediction of n-glycosylation sites with the netnglyc 1.0 server (http://www.cbs.dtu.dk/services/netnglyc/). search for protein domains was achieved using pfamscan (ftp://ftp.sanger.ac.uk/pub/databases/pfam/tools/) and cd-search against conserved domain database (cdd) at ncbi (http://www.ncbi.nlm.nih.gov/structure/ cdd/cdd.shtml). identification of leucine-rich repeats (lrr) in protein sequences was done using lrrfinder (http://www.lrrfinder.com/). search for duf4803 domain-containing, gh1 β -glucosidase and protein disulfide isomerase sequences was performed using blastp at ncbi (http://www.ncbi.nlm.nih.gov/blast/) and hmmsearch from the hmmer package (http://hmmer.org/). pairwise and multiple amino acid sequence alignments were respectively obtained using the needleman-wunsch algorithm and mafft implemented in geneious software (biomatters). phylogenetic analyses were performed using maximum likelihood (ml) with phyml (http://phylogeny.lirmm.fr/). prottest was used to select the best fit model of amino acid substitution for ml phylogeny (https://github.com/ddarriba/ prottest3). gene functions and go terms were automatically assigned to the predicted proteins based on the identification of domains with pfamscan. only the root domain of the hierarchical domain organization available from ebi was conserved. comparison of go terms between pc and pl unisequences and homogenization of the annotation level were performed using go slim terms. for each species, we used bowtie (http://bowtie-bio.sourceforge.net/) to map back all input trimmed illumina raw reads (minimum size of 30 bases) to the assembled transcriptome with up to 3 nucleotides mismatches allowed. to compare the unisequence expression levels, the number of mapped raw reads for each transcript was normalized with the rpkm (reads per kilobase per million reads), using the r package edger (https://bioconductor.org/packages/release/bioc/html/edger.html). analysis was performed independently on pc and pl wasp venom ( supplementary fig. s1 ). venom apparatus were dissected from 50 individuals per sample and glands were collected in 25-50 μ l of ringer's saline supplemented with a protease inhibitor cocktail (sigma). glands were opened to release the venom and centrifuged for 5 min at 500 × g to remove residual tissues. for 1d sds-page, samples were mixed with 4× laemmli buffer containing β -mercaptoethanol (v/v) and boiled for 5 min. proteins were then separated on a 6-16% linear gradient sds-page and the gel was silver stained as previously described 7, 13 . for isoelectric focusing (ief), samples were prepared by boiling the protein solution for 5 min with 4% (v:v) of a denaturing solution (0.15 m dithioerythritol, 10% sds). after cooling, the samples were mixed with an equal volume of a solution containing 9.2 m urea, 0.1 m dithioerythritol, and 2% chaps. ief was performed using slab gel. slab gels were made on glass tubes 14 cm in length (1.5 mm internal diameter) that were filled with 4% acrylamide, 9.2 m urea, 2% ampholytes [1% ph 3-10 (pharmacia) and 1% ph 2-11 (servalytes)], and 2% chaps. isoelectric focusing was run in two steps: a first run at 20 ma, 0.1 w/tube, 700 v for a total of 10,000 v/h, followed by a second run at 20 ma, 0.1 w/tube, 3,000 v for a total of 2,000 v/h. for the second dimension, 6-16% linear gradient sds-page was used. ief gels were incubated with 4x laemmli buffer containing ß-mercaptoethanol and loaded on top of the 1d sds-page. after separation, proteins were silver stained as previously described 7, 13 . identification of proteins by mass spectrometry was performed on 1d bands and 2d spots excised from the gels as previously described 7, 13 . ms/ms data analysis was performed with the mascot software (http://www. matrixscience.com) licensed in house using the combined pc and pl unisequences and non-redundant nr (ncbi). data validation criteria were (i) one peptide with individual ion score above 50 (the mascot significant identity threshold corresponding to p < 0.05 is 45 in our case) or (ii) at least two peptides of individual ion score above 20 (corresponding to 1% probability that a peptide spectrum match is a random event). the mascot score was calculated as −10*log10(p). the calculated fdr (based on an automatic decoy database search) ranged from 0 to 1.4% depending of the individual gel analysis. mascot analysis was performed with a fragment ion mass tolerance of 0.30 da and a parent ion tolerance of 0.30 da. carbamidomethyl of cysteine was specified in mascot as a fixed modification, and oxidation of methionine as a variable modification. the maximum missed cleavage allowed was set to 2. insect immune resistance to parasitoids insights into function and evolution of parasitoid wasp venoms venom proteins from parasitoid wasps and their biological functions diversity of virus-like 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snake venom reprolysin-type metalloproteases a venom metalloproteinase from the parasitic wasp eulophus pennicornis is toxic towards its host, tomato moth (lacanobia oleracae) purification and characterization of myrosinase from the cabbage aphid (brevicoryne brassicae), a brassica herbivore the crystal structures of sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an s-glycosidase crystal structure at 1.1 å resolution of an insect myrosinase from brevicoryne brassicae shows its close relationship to β -glucosidases heavy chain binding protein (bip/grp78) and endoplasmin are exported from the endoplasmic reticulum in rat exocrine pancreatic cells, similar to protein disulfide-isomerase control of pancreatic bile-salt-dependent-lipase secretion by the glucose-regulated protein of 94 kda (grp94) roles of molecular chaperones in pancreatic secretion and their involvement in intestinal 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vasoconstriction via inactivation of endothelin-1 the peptidases of trypanosoma cruzi: digestive enzymes, virulence factors, and mediators of autophagy and programmed cell death serpin structure, function and dysfunction a serpin from the parasitoid wasp leptopilina boulardi targets the drosophila phenoloxidase cascade we are grateful to m. thaon for help in insect rearing, n. ris and t. malausa for fruitful discussions, m.c. bon for providing p. lounsburyi samples, and k. varikou and v. caleca for providing p. concolor samples. we also thank a. schmitz, j. villalba, and the microscopy platform of the sophia agrobiotech institute (isa) for technical advices. we are grateful to the anonymous reviewers for their careful reading of our manuscript. this work received support from the genoscope ( accession codes: p. concolor and p. lounsburyi raw illumina sequencing data are available in the genbank sequence read archive (sra) database under the accessions srr1593901, srr1593902, srr1593906 and srr1593907, and raw 454 sequencing data for p. lounsburyi under the accession srr1593908. all trimmed ests for p. lounsburyi can be found in the genbank dbest repository under the accessions: jz818733 -jz820447. the transcriptome shotgun assembly (tsa) projects were deposited in genbank under the accessions gcdx00000000 (p. concolor) and gceq00000000 (p. lounsburyi). versions in this paper are the first versions gcdx01000000 and gceq01000000. key: cord-296347-fanlvxqs authors: loureiro, joana; ploegh, hidde l. title: antigen presentation and the ubiquitin‐proteasome system in host–pathogen interactions date: 2006-12-02 journal: adv immunol doi: 10.1016/s0065-2776(06)92006-9 sha: doc_id: 296347 cord_uid: fanlvxqs relatively small genomes and high replication rates allow viruses and bacteria to accumulate mutations. this continuously presents the host immune system with new challenges. on the other side of the trenches, an increasingly well‐adjusted host immune response, shaped by coevolutionary history, makes a pathogen's life a rather complicated endeavor. it is, therefore, no surprise that pathogens either escape detection or modulate the host immune response, often by redirecting normal cellular pathways to their advantage. for the purpose of this chapter, we focus mainly on the manipulation of the class i and class ii major histocompatibility complex (mhc) antigen presentation pathways and the ubiquitin (ub)‐proteasome system by both viral and bacterial pathogens. first, we describe the general features of antigen presentation pathways and the ub‐proteasome system and then address how they are manipulated by pathogens. we discuss the many human cytomegalovirus (hcmv)‐encoded immunomodulatory genes that interfere with antigen presentation (immunoevasins) and focus on the hcmv immunoevasins us2 and us11, which induce the degradation of class i mhc heavy chains by the proteasome by catalyzing their export from the endoplasmic reticulum (er)‐membrane into the cytosol, a process termed er dislocation. us2‐ and us11‐mediated subversion of er dislocation ensures proteasomal degradation of class i mhc molecules and presumably allows hcmv to avoid recognition by cytotoxic t cells, whilst providing insight into general aspects of er‐associated degradation (erad) which is used by eukaryotic cells to purge their er of defective proteins. we discuss the similarities and differences between the distinct pathways co‐opted by us2 and us11 for dislocation and degradation of human class i mhc molecules and also a putatively distinct pathway utilized by the murine herpes virus (mhv)‐68 mk3 immunoevasin for er dislocation of murine class i mhc. we speculate on the implications of the three pathogen‐exploited dislocation pathways to cellular er quality control. moreover, we discuss the ubiquitin (ub)‐proteasome system and its position at the core of antigen presentation as proteolysis and intracellular trafficking rely heavily on ub‐dependent processes. we add a few examples of manipulation of the ub‐proteasome system by pathogens in the context of the immune system and such diverse aspects of the host–pathogen relationship as virus budding, bacterial chromosome integration, and programmed cell death, to name a few. finally, we speculate on newly found pathogen‐encoded deubiquitinating enzymes (dubs) and their putative roles in modulation of host–pathogen interactions. relatively small genomes and high replication rates allow viruses and bacteria to accumulate mutations. this continuously presents the host immune system with new challenges. on the other side of the trenches, an increasingly welladjusted host immune response, shaped by coevolutionary history, makes a pathogen's life a rather complicated endeavor. it is, therefore, no surprise that pathogens either escape detection or modulate the host immune response, often by redirecting normal cellular pathways to their advantage. for the purpose of this chapter, we focus mainly on the manipulation of the class i and class ii major histocompatibility complex (mhc) antigen presentation pathways and the ubiquitin (ub)-proteasome system by both viral and bacterial pathogens. first, we describe the general features of antigen presentation pathways and the ub-proteasome system and then address how they are manipulated by pathogens. we discuss the many human cytomegalovirus (hcmv)-encoded immunomodulatory genes that interfere with antigen presentation (immunoevasins) and focus on the hcmv immunoevasins us2 and us11, which induce the degradation of class i mhc heavy chains by the proteasome by catalyzing their export from the endoplasmic reticulum (er)-membrane into the cytosol, a process termed er dislocation. us2-and us11-mediated subversion of er dislocation ensures proteasomal degradation of class i mhc molecules and presumably allows hcmv to avoid recognition by cytotoxic t cells, whilst providing insight into general aspects of er-associated degradation (erad) which is used by eukaryotic cells to purge their er of defective proteins. we discuss the similarities and differences between the distinct pathways coopted by us2 and us11 for dislocation and degradation of human class i mhc molecules and also a putatively distinct pathway utilized by the murine herpes virus (mhv)-68 mk3 immunoevasin for er dislocation of murine class i mhc. we speculate on the implications of the three pathogen-exploited dislocation pathways to cellular er quality control. moreover, we discuss the ubiquitin (ub)-proteasome system and its position at the core of antigen presentation as proteolysis and intracellular trafficking rely heavily on ub-dependent processes. we add a few examples of manipulation of the ub-proteasome system by pathogens in the context of the immune system and such diverse aspects of the host-pathogen relationship as virus budding, bacterial chromosome integration, and programmed cell death, to name a few. finally, we speculate on newly found pathogen-encoded deubiquitinating enzymes (dubs) and their putative roles in modulation of host-pathogen interactions. the vertebrate immune system is equipped to deal with invading pathogens, whether by means of mechanical barriers such as the skin and other epithelial surfaces or by means of innate immunity. innate immunity comprises the phagocytic and inflammatory systems, with phagocytes like macrophages and neutrophils, dendritic cells (dcs), and natural killer (nk) cells, as well as soluble mediators such as cytokines and complement. phagocytes are the immune system's first line of defense: they recognize, engulf, and clear the pathogen and are the main cellular component of the innate antibacterial response. nk cells can directly recognize and kill pathogen-infected cells that fail to express mhc molecules and secrete cytokines that affect the immune response. nk cells are the main cellular effectors of the innate response against viruses. the complement system can lyse infected cells or simply coat the surface of the pathogen or pathogen-derived material, resulting in its neutralization and opsonization. to counteract pathogen infection, host cells also have extracellular and intracellular pathogen recognition receptors to alert the immune system, such as toll-like receptors (tlrs) at the cell surface, and protein kinase r (pkr) and nucleotidebinding oligomerization domain (nod) proteins in the cytosol (akira et al., 2006; inohara et al., 2005) that can detect pathogen-associated molecular patterns (pamps) such as bacterial peptidoglycan or viral dsrna. these ''danger'' signals initiate the synthesis of cytokines like interferons to induce inflammation, a crucial component of the innate defense against pathogens. because innate immunity is not always successful at recognizing or eliminating the infectious agents, a more sophisticated line of defense, adaptive immunity, is also in place. the adaptive immune system includes cells originated in the thymus, the t lymphocytes, and the bone marrow-derived b lymphocytes (b cells), dcs, and macrophages. the two subsets of t lymphocytes, cd8 þ and cd4 þ t cells, possess distinct t cell receptors (tcrs), cd8 and cd4, respectively, that interact with their coreceptors on the surface of the target cell, the polymorphic class i and class ii mhc molecules (ploegh, 1998) . class i mhc molecules are expressed by nearly all nucleated cells, whereas class ii mhc molecules are constitutively expressed only by professional antigen-presenting cells (apcs), such as macrophages, b cells, and dcs. class ii mhc expression however, can be induced in many cells, in particular by ifn-g treatment. apcs can endocytose, process, and display antigen in the context of class ii mhc products at their cell surface to activate cd4 þ t cells. in the presence of antigen displayed by the apc and the appropriate lymphocyte costimulatory molecules, cd4 þ t helper (t h ) cells produce cytokines that ''help'' activate other cells: t h 1 (or inflammatory t cells) activate macrophages to kill the phagocytosed pathogens; t h 2 cells (or helper tcells) trigger tand b cell proliferation and activate the b cell differentiation program into antibody-producing plasma cells. furthermore, activation of cd4 þ t h cells is carefully regulated by a small subset of t cells, the regulatory t cells (tregs). regulatory t cells play an important role in downregulation of the host immune response, limiting the immunopathology resultant from antipathogen reactions, and preventing autoimmune disease (beissert et al., 2006; mills, 2004) . in addition to making antibodies, b cells are a special kind of apcs. unlike dcs and macrophages, b cells are not actively phagocytic. however, stimulation of the membrane immunoglobulin (mig) antigen-recognition component of their b cell receptor (bcr) with cognate antigen triggers the b cell to capture and deliver the antigen to class ii mhc compartments, culminating with antigen presentation for activation of t cells. bone marrow-derived professional apcs include macrophages and dcs. macrophages are phagocytic apcs with a low basal antigen-presenting capacity-owing to low surface expression of class ii mhc and costimulatory molecules-that is induced on macrophage activation, for instance, by ifn-g. macrophages reside in (or are recruited to) peripheral tissues, where they phagocytose and clear pathogens. phagocytosis, in turn, induces release of proinflammatory cytokines like ifn-g that turn macrophages into potent apcs, resulting in initiation of cd4 þ t cell activation. dendritic cells, the consummate professional apcs, travel through the periphery, sampling all tissues for prospective invaders. immature dcs phagocytose pathogens and home to the nearest lymphoid organ to ''educate'' (prime) naïve cd8 þ t cells by cross-presenting antigen in the context of class i mhc molecules-a process described in more detail later. mature dcs can also prime naïve cd4 þ t cells. like resting macrophages, immature dcs have very low antigen-presenting capability, and only on exposure to maturation signals [such as lipopolysaccharide (lps) on bacterial surfaces] does internalized antigen get loaded into class ii mhc products and get displayed at the cell surface to cd4 þ t cells (bryant and ploegh, 2004; stockwin et al., 2000) . ''educated'' (antigen-specific) cd8 þ t lymphocytes survey all cells in the body, ready to destroy any that displays signs of the presence of cellular alterations (such as viral and tumor peptides) within their surface class i mhc molecules (andersen et al., 2006; castelli et al., 2000) . antigen-specific cd4 þ t lymphocytes can coordinate macrophage bactericidal properties, activation of t and b lymphocytes, and antibody production. not only do the cells of the adaptive immune system provide a more elaborate defense, but also an increased level of protection from a subsequent reinfection with the same pathogen, the bedrock principle of vaccine strategies (crotty and ahmed, 2004; pulendran and ahmed, 2006) . adding to the complexity of the immune system is the cross talk between innate and adaptive immunity, which is crucial in eliciting an effective immune response (zingoni et al., 2005) . as mentioned, phagocytes release cytokines that stimulate the adaptive response. conversely, on activation by antigen recognition, t cells synthesize and secrete cytokines that activate macrophages, increasing their ability to kill ingested microbes, an innate immune response (munz et al., 2005; salazar-mather and hokeness, 2003) . the vertebrate immune system, therefore, is the appropriate battleground for microbial pathogens, selecting for those that devise successful strategies to avoid detection and elimination (hilleman, 2004; ploegh, 1998) . intracellular pathogens have evolved sophisticated mechanisms to subvert host processes to ensure their own replication and transmission. the initial hurdle is entry into the host cell, which poses great challenges for avoiding immune detection before establishing infection. to promote entry into host cells without alerting the immune system, bacteria possess capsular surfaces that have evolved to minimize antibody and complement deposition while in circulation through the body. on the other hand, filamentous adhesins (like fimbriae and pili) that protrude through the bacteria's capsule enable binding to host cell receptors, which enables secretion systems to deliver bacterial effectors to modulate uptake and invasion (finlay and mcfadden, 2006; galan and collmer, 1999) . virus particles are very often coated with highly variable capsid (nonenveloped viruses) or envelope (enveloped viruses) proteins to avoid detection and clearance by antibody-mediated responses. these capsids or envelopes can also be studded with immunomodulatory molecules of viral or even host origin and promote attachment to the host cell membrane, fusion and delivery of the virus internal core. alternatively, they may act as signaling devices and induce intracellular cascades required for virus uptake. ultimately, intracellular release of the viral dna or rna occurs (marsh and helenius, 2006; skehel and wiley, 2000) . the establishment of an infection critically depends on bacterial and viral genes dedicated to manipulation of host functions. a number of reviews have covered the bacterial and viral genes involved in manipulation of the host immune system, from control of apoptosis, cytokine signaling, to the antibody response, so the reader is referred to alcami (2003) ; alcami and koszinowski (2000) ; bowie et al. (2004) ; finlay and mcfadden (2006) ; hengel et al . (2005); hilleman (2004) ; and tortorella et al. (2000) . for the purpose of this chapter, we will focus on pathogen manipulation of antigen presentation pathways and the ub-proteasome system. antigen presentation involves the conversion of protein antigens into peptide ligands that can bind to mhc products that are displayed at the cell surface for recognition by t cells. in a simplified view of antigen presentation, the class i and class ii mhc pathways have evolved to sample different sources of antigen to which they have access: the class i mhc pathway usually deals with cytosolic antigens and is crucial for activation of cd8 þ t cells, whereas the class ii mhc pathway deals with exogenous antigens and the activation of cd4 þ t cells (bryant and ploegh, 2004; cresswell et al., 2005; pamer and cresswell, 1998) . antigen presentation is, of course, not as simple and clearcut, as we shall discuss later. there are, however, common principles that apply to the discrete steps of antigen processing and presentation by class i and class ii mhc molecules: antigen must be acquired, it is subjected to proteolysis, delivered to mhc þ compartments, and properly assembled with the mhc product. the complex is then subject to sorting through the secretory pathway and delivered to the cell surface ( fig. 1) . because each of these steps affords a target for interference by pathogens, we shall survey them for each pathway. the class i mhc is a trimeric complex composed of the class i mhc heavy chain (hc), the b 2 -microglobulin (b 2 m) or light chain, and the antigenic peptide. the structure of the fully assembled complex and its interactions with antigen-specific receptors on t cells have been extensively reviewed (alam et al., 1996; rudolph and wilson, 2002; von boehmer, 2006) . the class i mhc hc is inserted into the er membrane and n-glycosylated and binds in its course of synthesis to the membrane-associated chaperone calnexin (cnx), at which point folding and intrachain disulfide bond formation take place. once dissociated from cnx, the hc binds its soluble partner subunit, b 2 m, and enters the peptide-loading complex (plc). the plc is composed of two mhc-encoded components, tap and tapasin, and two ''housekeeping'' er proteins, calreticulin and erp57. the transporter associated with antigen presentation (tap) is an atp-dependent pump with two subunits, tap1 and tap2 that transports peptides into the er. tapasin, a transmembrane glycoprotein, mediates the interaction between the tap transporter and peptide-free hc/b 2 m dimers. the soluble calreticulin and erp57, a chaperone and a thiol oxidoreductase, respectively, normally involved in folding of nascent glycoproteins, promote assembly of the class i mhc complex. the peptide antigen cargo for class i mhc originates from proteasomal proteolysis in the cytosol. the array of proteasome-generated peptides is subject to trimming by cytosolic endopeptidases and delivered to the er lumen by the tap transporter. further trimming by er-resident endopeptidases can also occur to guarantee a custom-fit of the peptide antigens, typically 8-10 amino acids long, into the peptide-binding groove on the hc/b 2 m dimer associated with the plc. empty hc molecules are detained in the er by virtue of interaction with tapasin, until assembly with b 2 m and peptide takes place, at which point the hc/b 2 m/peptide trimeric complex is released from the plc and allowed to exit the er and enter the secretory pathway. once displayed at the cell surface, the antigen-loaded class i mhc complex is ready for inspection by the t cell receptor (tcr) on circulating cytotoxic cd8 þ t cells (cresswell et al., 2005; heemels and ploegh, 1995; rammensee, 2002 rammensee, , 2004 . figure 1 common principles in antigen processing and presentation by class i and class ii mhc molecules. in the class i pathway, endogenous antigens are derived from cytosolic proteolysis and delivered to the er lumen, where loading onto class i mhc products takes place. the assembled complex is then sorted to the cell surface. in the class ii pathway, exogenous material is internalized from the extracellular space and delivered to the lysosome, where processing and loading onto mhc products occur. sorting through the secretory pathway then delivers the class ii complex to the cell surface. class i mhc products on most cells present exclusively ''self'' peptides, derived from the cell's own proteins, the majority of which results from protein synthesis on free ribosomes in the cytoplasm. because of the intrinsic error-prone nature of protein synthesis and folding, a sizable fraction of translation products (estimated at up to 30%) may never result in a finished product. these defective ribosomal products (drips) are destroyed within 30 min of their synthesis by the cytosolic proteasomal pathway and enter the class i mhc antigen presentation pathway (yewdell et al., 2001) . in tumor cells or cells infected by a virus, mutated forms of endogenous proteins or viral proteins will compete with the host's own proteins for presentation by class i mhc products. as ''non-self'' (tumor-or virus-derived) peptides displayed in the context of class i mhc products accumulate at the cell surface, their chance of triggering activation of cd8 þ t cells with a cognate receptor increases. the activated cytotoxic cd8 þ t lymphocytes will then lyse the target cell by releasing perforin and granzymes or by fas ligand engagement. secretion of ifn-g and tumor necrosis factor-a (tnf-a) also aids in elimination of infected and tumor cells by cytotoxic t lymphocytes (ctls) (andersen et al., 2006; castelli et al., 2000) . the selective pressure imposed by immune surveillance has made loss of class i mhc expression a hallmark of some tumors and virus-infected cells, as this allows them to be invisible to ctls. there is, however, a backup system for when lack of class i mhc expression impairs the cd8 þ t cell cytotoxic response: nk cells. nk cells display both activating and inhibitory receptors at their surface, which recognize different ligands at the surface of target cells. nk cell activity is ultimately determined by the integration of signals that are perceived by the nk cell surface receptors (lanier, 2005) . all nk cells express at least one inhibitory receptor, which engages class i mhc molecules on the surface of the target cell, resulting in downregulation of nk cell effector functions. low levels or absence of class i mhc products on the surface of the target cell relieve the inhibitory signals and lead to nk cell cytotoxicity, resulting in clearance of the virus-infected or tumor cells. nk cell recognition has been extensively revised and the reader is referred to backstrom et al. (2004) ; kumar and mcnerne y (2005) ; and lanier (2005) . there is an exception to the rule that the class i mhc pathway is devoted to display of peptide antigens from endogenously generated proteins: the so-called professional apcs, dcs, and macrophages can acquire and process exogenous material and present it at the cell surface in class i mhc products, a process called cross-presentation. cross-presentation allows noninfected professional apcs to prime naïve t cells with pathogen-or tumor-derived peptides acquired through endocytosis of infected cells/cell remnants. this ''cross-priming'' is essential for development of cd8 þ t cell immunity to viruses and tumors in vivo, since only professional apcs can present viral/tumor antigens in the context of class i mhc products without being themselves infected/tumorigenic. there is considerable controversy as to the exact nature of the antigen acquired and modes of antigen acquisition, as well as the intracellular mechanisms leading to cross-presentation and the subsets of apcs endowed with this property (cresswell et al., 2005; groothuis and neefjes, 2005; guermonprez and amigorena, 2005; jutras and desjardins, 2005; shen and rock, 2006) . this controversy is, however, beyond the scope of this discussion. the class ii mhc antigen presentation pathway deals with antigens that reside in extracellular space and are internalized into the endolysosomal pathway. all mammalian cells internalize their own cell surface proteins by constitutive endocytosis. in class ii mhc þ cells, this allows class ii mhc access to self-proteins as a source of peptides. professional apcs, such as b cells, macrophages, and dcs, also engage in receptor-mediated endocytosis to acquire extracellular antigen: the antigen from the extracellular milieu is bound by cell surface receptors, internalized, and delivered to the class ii mhc antigen processing machinery. antibodies, complement system factors, and common bacterial or viral components that coat the surface of pathogens or their toxic products bind receptors on apcs that allow them to recognize and internalize this foreign material. of the many receptors used by professional apcs for this purpose, the mannose receptor, which recognizes mannose residues and glycoproteins on viral and bacterial products, and the scavenger receptor, which recognizes very promiscuously many different classes of macromolecules, are among the most important. professional apcs also have complement receptors and receptors for the fc region of antibodies, the fc receptors, which can assist in the acquisition of opsonized antigen and in its delivery to the proper intracellular destination. b cells can also use their surface immunoglobulin or bcr, to acquire antigen (bryant and ploegh, 2004; cresswell, 1994; kim et al., 2006c) . class ii mhc loading with antigenic peptides takes place mostly in the endocytic vesicles of professional apcs. class ii mhc ab dimers assemble in the er and associate with the chaperone invariant chain (ii), which inserts its class ii mhc-associated ii peptide (clip) portion in the peptide-binding groove of the ab dimer, preventing its premature (prelysosomal) loading. ii is also important for correct assembly and transport of class ii mhc in the endocytic pathway. further class ii mhc maturation and peptide loading takes place in acidified compartments of the endolysosomal pathway of apcs, since low ph favors an ''open'' conformation of the class ii mhc molecule and hence peptide exchange, as well as the action of specific cysteine proteases that displace ii from the class ii mhc-ii complex, and that of the class ii mhclike molecule hla-dm which facilitates peptide loading. the many hydrolase activities present in the endolysosomal compartments of apcs, such as the ifn-g-inducible lysosomal thiol reductase (gilt), numerous cysteine proteases of the cathepsin (cat) family, like catb, cats, catl, and asparaginyl endopeptidase (aep), produce the peptide ligands that are loaded onto class ii mhc products. peptides bound by class ii mhc molecules are usually 13-25 residues long. the end result is a mature class ii mhc-peptide complex at the cell surface, consisting of a class ii mhc ab dimer loaded with peptide, which interacts with the tcr on cd4 þ t cells. the result of this interaction is dependent on class ii mhc-tcr contacts and also on the context provided by lymphocyte costimulatory molecules at the immunological synapse. the cd4 þ t cell response may be cytolytic, but generally these antigen-specific cd4 þ t lymphocytes function as helper cells, releasing cytokines to enhance the overall immune response by inducing macrophage activation, t and b cell proliferation, and b cell differentiation to produce antigen-specific antibodies and different immunoglobulin isotypes with different effector functions (bryant and ploegh, 2004; chapman, 2006; cresswell, 1994; honey and rudensky, 2003; hsing and rudensky, 2005; stern et al., 2006; villadangos et al., 1999) . all cellular proteins, regardless of their half-life, are subject to turnover. the main pathway for degradation of short-lived proteins in the cytoplasm of eukaryotic cells is the ub-proteasome system (hershko and ciechanover, 1992) . since the discovery of ub and ub-dependent proteolysis in the late 1970s, it has become increasingly clear that the ub-proteasome system is pivotal to numerous cellular processes: cell cycle control, transcriptional regulation, signal transduction, antigen presentation and induction of the inflammatory response, degradation from the er, membrane trafficking, receptor endocytosis and downregulation, apoptosis, and development (hershko and ciechanover, 1992; pickart, 2001) . ubiquitin is a small 76-amino acid protein, synthesized as a precursor that is processed by deubiquitinating enzymes (dubs) to expose the glycine-glycine sequence at the ub c-terminus, its site of attachment to target molecules. atp-dependent ub activation is catalyzed by the e1 (ub-activating) enzyme, which adenylates the ub c-terminus, allowing the subsequent formation of a high-energy thioester bond between the glycine residue of ub and the cysteine residue on the e1 active site. ub is then transferred from the e1 cysteinyl side chain to a cysteinyl group on one of several e2 (ub-conjugating) enzymes. finally, one of hundreds of e3 (ub-ligase) enzymes, binds the ub-e2 complex and the substrate, thus facilitating the transfer of ub to a lysine residue in the substrate via an amide (isopeptide) bond (hershko and ciechanover, 1992) . the functions of e3 ligases, in particular, are tightly regulated by signalinduced mechanisms, such as localization, oligomerization, degradation, and posttranslational modifications, which makes e3s the master orchestrators of specificity in the ub conjugation cascade. this multistep mechanism, much like phosphorylation, endows protein ubiquitination with a high degree of specificity and flexibility, which is paramount to its important biological functions (haglund and dikic, 2005; hershko and ciechanover, 1998; pickart, 2001 pickart, , 2004 varshavsky, 2005) (fig. 2a) . overview of the ub-proteasome system. (a) ubiquitin-conjugation cascade and how ub chain linkage type and length influence substrate fate. e1, ub-activating enzyme; e2, ubconjugating enzyme; e3, ub-ligase enzyme; and dub, deubiquitinating enzyme. (b) diversity in e3 ligases. e3s play crucial roles in substrate selection and can be regulated by localization, oligomerization, associated e2s, posttranslational modifications, and degradation. hrd1p and doa10p are yeast e3 ligases that are multispanning membrane proteins of the er and, in the case of doa10p, nuclear envelope. the mammalian scf family of e3 ligases are mainly cytosolic and can recruit substrate adaptor proteins, the f-boxes, with very diverse substrate specificities. we elaborate on ub e3 ligases to some extent, as they are key players in several aspects of the immune system, including immune evasion (liu, 2004) and also in er quality control and degradation kostova and wolf, 2003; romisch, 2005) , that yields to some of the ligands on class i mhc products. e3 ligases can be divided into two broad classes: the homologous to e6-ap carboxyl terminus (hect)-domain ligases or the really interesting new gene (ring)-like domain ligases. the first hect e3 described, e6-associated protein (e6-ap), was shown to be required for ubiquitination and degradation of p53, mediated by the human papillomavirus protein e6 (scheffner et al., 1993) . in hect e3s, ub is transferred from the e2 to a conserved cysteine residue in the hect domain, followed by attack of this thioester by a lysine on the substrate (pickart, 2001) . the ring-ch domain is a ring finger motif with a cysteine residue in the fourth zinc-coordinating position and a histidine residue in the fifth. ring-type e3s are more abundant and do not form an obligatory thioester intermediate with ub; rather they bring the ub-loaded e2s and the substrate into proximity, thus facilitating the ub transfer from the e2 to the substrate (pickart, 2001) . ring-type e3s can be single subunit e3s, which have both a ring-finger domain and substrate recruitment domain(s) on the same protein, like mdm2, a key regulator of p53. multisubunit e3s include the very diverse cullin-ring ligases (crls) (petroski and deshaies, 2005) . crls are composed of a catalytic core that recruits the ub-loaded e2-formed by a nucleating cullin protein and a ring finger protein-as well as a substrate recognition complex. the archetypal crls are the skp-1-cullin-1-f-box protein complexes or scf e3s. the cullin subunit (any one of cullin-1, -2, -3, -4a, -4b, -5, or -7) forms an elongated bent backbone for the multisubunit ligase. the cullin n-terminus binds the s-phase kinase-related protein-1 (skp-1), an adaptor that recruits any one of a number of substrate-specific adaptor subunits called f-box proteins. the f-box protein is the main determinant in substrate specificity, as it binds the substrate through its particular substrate recognition domain (jin et al., 2004a) , although the ring box protein may participate (jin and harper, 2002) . the cullin n-terminus binds the catalytic core composed of the ring-box (rbx) protein with its associated ub-loaded e2 . this arrangement allows the f-box protein to bring its bound substrate close to the ubiquitination machinery of the complex (fig. 2b) . phosphorylation of the substrate very often regulates the f-box protein-substrate interaction, converting the substrate into a form susceptible to e3 activity, adding an extra layer of control to the process (joazeiro and weissman, 2000; schulman et al., 2000; zheng et al., 2002) . crls assemble with numerous substrate receptors. cullins 2 and 5, for example, recruit substrates through suppressor of cytokine signaling/elongin-bc (socs/bc) boxes and form the so-called scf2s and scf5s complexes. in scf2s and scf5s, skp-1 is substituted by the skp-1-like protein elongin c which binds the ub-like elongin b that binds the substrate adaptor subunit (petroski and deshaies, 2005) . many of these e3 complexes have important roles in the immune system (liu, 2004; liu et al., 2005) . the nuclear factor-kb (nf-kb) transcription factor is a master organizer of both innate and adaptive immunity. nf-kb is activated in response to tlr signaling on recognition of pathogen-associated molecules like bacterial peptidoglycan in a process that is crucially dependent on ubiquitination. one of the steps requires the cytosolic scf b-trcp e3 complex. the scf b-trcp substrate adaptor component is the f-box protein b-transducin repeat-containing protein (b-trcp) that possesses wd repeats that bind to phosphorylated inhibitor of nf-kb (ikb), inducing its ubiquitination and degradation. nf-kb is thus released from the ikb-nf-kb dimer and translocates into the nucleus, activating downstream transcription. the elongin-c-elongin-b-cullin-5-socs (ecs) complex uses socs proteins as the substrate adaptors. socs boxes bind janus kinases (jaks), which are recruited and activated in response to ifn and cytokine signaling, promoting ubiquitination and degradation of jaks by the ecs complex. this, in turn, inhibits phosphorylation and activation of the signal transducer and activator of transcription (stat) family of transcription factors that are crucial for the immune response following ifn and cytokine signaling and following viral infections . there are other families of e3s with noncanonical ring-domains, like the k3 homologues and the ufd2homologous box (u-box) e3s (which we discuss in more detail later). for a more comprehensive review of hect and ring e3s and different classifications read (ardley and robinson, 2005; coscoy and ganem, 2003; hatakeyama et al., 2001; petroski and deshaies, 2005; sharrocks, 2006) . originally believed to always deliver a ''kiss of death'' and target the substrate for proteasomal degradation, the much more wide-ranging effects of ub conjugation are beginning to be appreciated. chain length and linkage type also influence the outcome of the ub-conjugated substrate. the multiple ub moieties in a polyub chain (chains of 4 or more ub moieties) are linked to one another by an isopeptide bond between a lysine residue on one ub molecule (usually on lys48) and the c-terminal carboxyl group of the next ub on the chain. at times, extension of a polyub chain on a substrate conjugated with 1-3 ''initiator'' ub moieties requires a special subclass of e3s, the ufd2-homology box (u-box) e3s (once called e4s) (hoppe, 2005) . targeting of proteins for proteasomal proteolysis generally requires polyubiquitination in a lysine (lys) 48-type linkage. by contrast to polyub, substrate monoubiquitination or attachment of noncanonical ub chains-ub chains with non-lys48 linkages such as lys63 and lys29 linkages-usually have nonproteolytic functions in dna repair, endocytosis, signal transduction, transcriptional regulation, and ribosoma l function (d' azzo et al., 2005; pic kart and ed dins, 2004 ) . monoubiquitination can occur on a single lysine residue or on several lysine residues in a substrate (multiubiquitination). monoubiquitination is extremely important as a sorting signal in the endocytic pathway. for example, monoub attachment is sufficient to induce endocytosis of growth hormone receptor and sorting to the lysosome for degradation (hicke, 2001; hicke and dunn, 2003) . direct modification of the cargo (cis-regulation) or modification of the proteintrafficking machinery (trans-regulation) by monoub could have many consequences for antigen presentation, as these processes rely heavily on events that take place in endolysosomal compartments. noncanonical ubiquitin chains play many diverse roles in signaling pathways, in dna replication and postreplication dna repair, and modulating protein-protein interactions. for instance, activation of nf-kb is tightly regulated by a balance between lys48-and lys63-mediated ubiquitination of different components of the nf-kb pathway. triggering of many cell-surface receptors leads to assembly of signaling complexes that recruit tumor necrosis factor receptor-associated factor 6 (traf6), an e3 ligase that binds ubc13, promoting lys63-linked polyubiquitination of the g subunit of the inhibitor of nf-kb kinase (ikk) complex, ikkg. this leads to activation of the ikk complex, which in turn results in ikb phosphorylation. phosho-ikb then recruits the scf complex that catalyzes lys48-linked polyubiquitination of ikb and consequently activates nf-kb (karin and ben-neriah, 2000) . for comprehensive reviews see pickart and eddins, 2004; varshavsky, 2005) . ubiquitin-like molecules or modifiers (ubls) share structural homology with ub and can also be conjugated onto protein substrates, mostly with outcomes other than proteasomal degradation. ubls like the small ubiquitin-like modifier sumo, neuronal precursor cell-expressed developmentally down-regulated 8 (nedd8) or ifn-stimulated gene product of 15 kda (isg15), to name but a few, are implicated in important physiological processes like nuclear transport, maintenance of chromosome integrity, transcriptional regulation, cell cycle control, signaling and regulation of proteolysis (hochstrasser, 2001; schwartz and hochstrasser, 2003) . ubls may regulate ub-mediated proteolysis or signaling through comodification of a substrate, thus modulating the effects of ub conjugation (lamsoul et al., 2005; sobko et al., 2002) or by regulating the activity, specificity, localization, or stability of enzymes in the ub-conjugating cascade, as is the case for nedd8 modification of cullin-ring e3s (kawakami et al., 2001; petroski and deshaies, 2005; wu et al., 2005) . deubiquitinating enzymes can cleave isopeptide bonds to remove ub from the substrate or from polyubiquitin chains. there are about 100 dubs in the human genome, organized in five classes according to their catalytic domain structure: the ubiquitin-specific proteases (usps), the ubiquitin c-terminal hydrolases (uchs), the machado-joseph disease proteases (mjds), the ovarian tumor proteases (otus), and the jab1/mpn/mov34 proteases (jamms). the first four classes comprise cysteine-type proteases, whereas jamms are metalloproteases. for a more detailed inventory of dubs, read nijman et al. (2005) . dubs have very diverse specificity properties, in terms of the ubiquitin or ubl moiety itself (substrate specificity), in terms of the target protein to which the ub or ubl is attached (target specificity), and possibly in terms of the context provided by target and attached modification. dub specificity in vivo can be further regulated by subcellular localization or association with different binding partners (amerik and hochstrasser, 2004; li and hochstrasser, 2003; reyes-turcu et al., 2006; soboleva and baker, 2004) . dub functions are therefore also extremely diverse, ranging from regulation of proteasome function, to regulation of chromatin structure, to membrane protein trafficking, and with obvious implications in processes such as cancer and neurodegeneration (amerik and hochstrasser, 2004; nijman et al., 2005; soboleva and baker, 2004) . the proteasome, very abundant in the cytosol, is a multisubunit protease composed of the 20s and 19s proteasome complexes. the 20s proteasome (or central core particle) has the general architecture of a barrel, formed by four stacked rings of seven subunits each, the outer two rings being composed of a subunits and the innermost two rings of b subunits (groll et al., 1997) . the b subunits, which line the proteasome's inner cavity, carry out the catalytic activity. for mammalian proteasomes, only three of the seven b subunits in each ring are catalytically active. access to this cavity occurs through narrow pores (with a diameter on the order of 10-15 å ) at both ends of the barrel, so it is usually assumed that protein substrates must be unfolded prior to their delivery to the catalytic chamber (groll et al., 1997 (groll et al., , 2000 kohler et al., 2001) . also at both ends of the core particle there is the 19s cap complex, whose functions range from recognition of poly-ub chains on target proteins, to unfolding of the substrate to facilitate entry into the catalytic cavity, to deubiquitination activity (adams, 2003; heinemeyer et al., 2004; rivett et al., 1997; schmidt et al., 2005; seeger et al., 1997) . as mentioned earlier, the proteasome plays an instrumental role in class i mhc antigen presentation and activation of peptide-specific cd8 þ t cell responses. this process requires not only generation of peptides of the right quality-that is, right size and sequence to allow a correct fit into the peptidebinding cleft-but also in the right quantity to trigger a successful response, which is no small endeavor due to many destructive aminopeptidase activities in the cytosol (shastri et al., 2005; strehl et al., 2005) . ifn-g, a crucial component of the innate and adaptive antiviral immune responses, affords the immune system a competitive edge. ifn-g induces expression of auxiliary b subunits, b1i, b5i, and b2i [also known as low molecular weight protein 2 (lmp2), lmp7 and multicatalytic endopeptidase-like complex 1 (mecl1), respectively], as well as synthesis of the proteasome activator pa28, and of the proteasome maturation protein (pomp) (strehl et al., 2005) . the immunosubunits lmp2, lmp7, and mecl1 are incorporated into nascent proteasomes, replacing their endogenous counterparts and constituting the so-called immunoproteasome. the proteasome activator pa28 (or 11s proteasome) binds to the outer rings of the 20s proteasome, thereby opening the central gate and facilitating substrate entry. pomp is important for assembly and maturation of the proteasome (strehl et al., 2005) . this ifn-g-induced proteolytic cascade, mediated by immunoproteasomes and pa28, might be induced to respond to a demand for high proteasome activity when the constitutive cascade is no longer sufficient, altering the proteolytic activity of the proteasome for maximal efficiency in production of the class i mhc peptide repertoire. ifn-g treatment also activates a transcriptional program that increases the synthesis of class i mhc molecules themselves and that of components of the peptideloading complex, thus increasing cell surface presentation (kloetzel, 2004; kloetzel and ossendorp, 2004; kruger et al., 2003; rivett and hearn, 2004; van den eynde and morel, 2001) . therefore, even though class i mhc presentation is constitutive, it can be modulated in the course of an immune response, with a proposed role in the early stages of a cytotoxic response. it is thus not surprising that viruses have targeted the ifn-g signaling cascade so aggressively (alcami and koszinowski, 2000; hengel et al., 2005; salazar-mather and hokeness, 2006) . although tightly controlled, er protein synthesis is not always successful. proteins may sustain damage or fail to complete their synthesis early during biogenesis, or be trapped in an irreversible nonnative conformation, or a mutation may result in a structural alteration that leads to misfolding, as is the case for the cystic fibrosis conductance regulator (cftr) (jensen et al., 1995; ward et al., 1995) , mutant plasma a1-antitrypsin (teckman and perlmutter, 1996) , or tyrosinase (halaban et al., 2000) . they may also be expressed in the absence of their cognate subunits, as is the case for unassembled subunits of tcra (huppa and ploegh, 1997; yang et al., 1998) . er quality control is a homeostatic process that involves an elaborate machinery that recognizes and retains newly synthesized misfolded or misassembled proteins and targets many of them for degradation by the ub-proteasome system (ellgaard and helenius, 2003) . this mode of degradation therefore samples sets of proteins otherwise targeted to extracellular space, where the degradation products are available as peptide ligands for class ii mhc products. in addition to its role in er quality control, this er-associated degradation (erad) can also be employed in the physiological regulated proteolysis of normal er proteins whose degradation is subject to metabolic cues, such as hydroxymethylglutaryl-coenzyme a reductase (hmgr) (hampton, 2002; hampton and bhakta, 1997) . a feature of this er-associated protein degradation is the spatial separation between targeting of substrates and their proteolysis, which requires substrate export from the er lumen or membrane to the cytoplasm by a process termed dislocation (also called retrograde translocation or retrotranslocation) (werner et al., 1996) . dislocation is a complicated multistep process that involves substrate recognition, targeting for dislocation, removal from the er membrane, deglycosylation, ubiquitination, and finally proteolysis (kostova and wolf, 2003; meusser et al., 2005; romisch, 2005) . the proteasome is usually considered to be a nonselective degradation apparatus, with selection of erad substrates being mediated mostly by the ub ligases. however, the er quality control e3 enzymes are mostly cytosolic or membrane-associated and thus are separated from their substrates at least by the er membrane. this invokes the existence of mechanisms, present in e3 ligases themselves or in upstream factors, which facilitate coupling of erad substrate recognition to ubiquitination by e3 ligases in the cytoplasm. owing to the extremely diverse nature of proteins that must be examined by the er quality control machinery, a unifying model for how recognition of erad substrates takes place remains intractable. nonetheless, misfolded proteins cleared from the er enter the class i mhc-processing pathway, and hence this route of degradation is an important aspect of the generation of class i mhc epitopes. instead, erad is likely to be custom-fitted to the client protein in question. for glycoproteins, a possible mechanism is the recognition of terminally misfolded proteins by the calnexin/calreticulin (cnx/crt) lectin-type chaperones, which retain immature glycoproteins in the er until productive folding takes place (ellgaard and helenius, 2003; hammond, 1994; helenius and aebi, 2004) . terminally misfolded proteins-that is, proteins that after extensive cxn/crt cycle folding attempts still fail to acquire their native conformation-are trimmed by er mannosidase i, leading to recognition by er degradation-enhancing alpha-mannosidase-like protein (edem), which presumably targets them for degradation (eriksson et al., 2004; jakob et al., 2001; molinari et al., 2003) . most er lumenal proteins require the lumenal chaperone bip for degradation, while transmembrane proteins with large cytosolic domains usually rely on cytosolic chaperone systems, like the heat shock protein (hsp) complexes hsp70/hsp90 and hsp40/hsp70 (ellgaard and helenius, 2003; romisch, 2005) . yet it appears that a protein's lumenal or er membrane localization matters less than the localization of the folding alteration within the polypeptide itself. cftr whose cytoplasmic domains are recognized first by the hsp70/hsp90 cytoplasmic chaperone system may be targeted for degradation by the cytosolic hsp70/hsp90-interacting chip e3 ligase cochaperone (connell et al., 2001; murata et al., 2001) , even if the protein also has a misfolded lumenal domain (which might also target it to a bip-dependent degradation pathway). if the cytoplasmic domain is properly folded, then the lumenal domains are inspected, and if then the protein is recognized as misfolded, it is degraded in a process that involves bip (connell et al., 2001; meacham et al., 2001; vashist and ng, 2004) . this suggests that er quality control uses sequential checkpoints to select degradation substrates and target them to the appropriate degradation pathway. in the case of nonglycosylated substrates, protein disulfide isomerase (pdi), one of a large number of er-resident oxidoreductases that catalyze disulfide bond formation and isomerization, can play a role in er quality control by unfolding certain substrates prior to degradation. another oxidoreductase, erp57, interacts with cnx and crt to facilitate folding, but in the event of a terminally misfolded protein may aid in transfer of proteins with improper disulfide bonds to the edem pathway . another possibility, at least in yeast, is that misfolded proteins actually escape to the golgi and are then recycled to the er vashist et al., 2001) . this er-golgi shuttling model was proposed because mutations in several secretory pathway genes (like ufe1p, sec23p, and erv29p) compromise degradation of erad substrates vashist et al., 2001) , invoking a functional secretory pathway for efficient degradation of misfolded proteins from the er. the golgi apparatus could presumably endow misfolded proteins with a signal for destruction, but such a modification has not been found. since the ufe1p and sec23p have since been shown to be required for maintenance of proper er structure (prinz et al., 2000) , the effects on protein degradation may simply be pleiotropic consequences of perturbing the normal arquitecture of the er (hammond et al., 1994; romisch, 2005) . ubiquitination at the er membrane is yet another mode of erad substrate selection. most erad e3s are at the er membrane, as is the case for the yeast hrd1p/ der3p and doa10p, or can be brought to the er membrane on demand, as is the case for cytosolic scf complexes and chip, for example (kostova and wolf, 2003; meusser et al., 2005; romisch, 2005 ). an e3 ligase can recruit distinct e2 ub-conjugating enzymes and/or distinct adaptor proteins (as we have seen for the scf and ecs e3 complexes and will discuss later for erad e3s), thus conferring specificity in substrate selection. the yeast hrd1p/der3p e3 ligase was first discovered in a genetic screen for saccharomyces cerevisiae genes involved in hmg-coa reductase degradation (hrp) (hampton et al., 1996) and is an er-resident protein with six predicted transmembrane domains and a c-terminal ring-finger motif facing the cytosol. hrd1p can act in a complex with ubc7p and ubc6p to ubiquitinate substrate proteins ( fig. 2b ). ubc7p is a soluble protein that becomes active only when tethered to the er membrane by the ubc7p cofactor cue1p membrane protein. degradation of transcription factor mata2-10 protein (doa10p) is a transmembrane protein of the er/nuclear envelope, which participates in yeast erad (swanson et al., 2001) . doa10p is predicted to span the membrane 14 times and, like hrd1p, uses cue1p/ubc7p and ubc6p to ubiquitinate its substrates (fig. 2b) . however, the two ligases target different sets of substrates for degradation (bays et al., 2001; swanson et al., 2001) . hrd1p ubiquitination activity can be directed to a specific subset of er degradation substrates, by virtue of its association of hrd1p with hrd3p and der1p. hrd3p is a single-spanning er membrane protein with a large er luminal domain that can recognize misfolded proteins, thus possibly functioning as a substrate recruitment factor for the hrd1p ligase complex (gauss et al., 2006) . the degradation from the er-1 protein (der1p) spans the er membrane four times and is required for degradation of some misfolded glycoproteins (knop et al., 1996) . der1p may function as a substrate adaptor protein or even as a channel for ejection of degradation substrates from the er membrane. hrd3p can associate with der1p, presumably enabling substrate delivery to downstream components. hrd3p can even regulate hrd1p activity, which is necessary for substrate extraction from the membrane and delivery to the proteasome (gauss et al., 2006) (fig. 3) . these multifunctional protein complexes can therefore function in substrate selection in the er lumen or membrane and even facilitate subsequent steps that lead to proteasomal degradation. similarly, doa10p can catalyze ubiquitination of both membrane and soluble proteins, yet the mechanisms of subsequent proteasome targeting differ (ravid et al., 2006) , presumably due to association with other regulatory proteins. the many layers of e3mediated regulation of erad substrate selection are most likely just emerging. in mammals, there are two predicted homologues of hrd1p/der3p, the hrd1 and the gp78 e3 ligases. like its yeast counterpart, human hrd1 is involved in degradation from the er (kikkert et al., 2004) . hrd1 may associate with ubc7 to catalyze ubiquitination of a subset of substrates, like tcra and cd3d. hrd1 is not involved in the regulated degradation of the mammalian hmgr (kikkert et al., 2004) . hrd1 may also associate with sel1l, a homologue of hrd3p, as well as other er membrane and er membrane-associated proteins, including cdc48p(p97)/npl4/ufd1, forming multisubunit complexes that seem to coordinate steps that range from substrate selection to delivery to the proteasome for at least a subset of er degradation substrates (lilley and ploegh, 2005a; ye et al., 2005) . gp78 was identified as the tumor autocrine motility factor receptor (amfr) (nabi et al., 1992) and later as an e3 ligase due to its homology to hrd1p and involvement in degradation of cd3d and apoliprotein b100 (fang et al., 2001; liang et al., 2003) . gp78 is an er-resident protein, predicted to span the membrane five times, with a c-terminal cytosolic ring-domain and an additional ubc7 e2-binding site, the cue domain, arranged in tandem. while hrd1p recruits ubc7 through the transmembrane cue1p protein, it seems that convergent evolution has made the e3-and the e2-docking protein come together in a single human protein. curiously, gp78 is involved in sterol-regulated ub-dependent degradation of hmgr (song et al., 2005) , suggesting it constitutes the true functional homologue of yeast hrd1p. homocysteine-induced endoplasmic reticulum protein (herp) is a singlespanning er membrane protein that is induced by the unfolded protein response (upr) and also required for erad. herp is proposed to improve er protein folding and decrease protein load, protecting cells from er-stress-induced apoptosis (kokame et al., 2000) . furthermore, herp has an n-terminal ubiquitin-like domain (uld) and is required for the degradation of conexin and cd3d (hori et al., 2004; sai et al., 2002) . it forms a complex with hrd1, p97, derlin-1, and vimp (schulze et al., 2005) , a vcp (p97)-interacting membrane protein, that recruits p97 to derlin-1 (ye et al., 2004) . herp may function as another adaptor protein in these multiprotein complexes at the er membrane, influencing substrate selection. the doa10p mammalian homologue, teb4 (or march-vi), is a multipletransmembrane-domain-containing protein of the er membrane that functions as an e3 ligase: it has an n-terminal noncanonical ring-domain in the cytosol that catalyzes ub conjugation and teb4 self-ubiquitination and degradation (hassink et al., 2005) , but its regulation is poorly characterized. parkin is a cytosolic e3 ligase with a c-terminal noncanonical double-ring-finger (ring-ibr-ring), and an n-terminal ub-binding domain, believed to mediate proteasomal degradation of aggregation-prone proteins (imai et al., 2000) , typical of parkinson's disease. both phosphorylation (yamamoto et al., 2005) and er stress-induced association with c-terminus of hsc70-interacting protein (chip), involved in cytosolic chaperone-dependent folding, regulate parkin e3 ligase activity (imai et al., 2002; sahara et al., 2005) , which could be beneficial for reduction of protein aggregates and cellular pathology. how specificity in substrate selection is conferred can be illustrated by e3s involved in glycoprotein turnover. the f-box proteins fbs1 and fbs2 bind high mannose n-linked glycoproteins (winston et al., 1999) . by using fbs1/ fbs2 as its substrate adaptor(s), the cytosolic scf fbs1,fbs2 e3 ligase is rendered specific for glycoproteins that have been dislocated from the er (yoshida et al., 2002 (fig. 4a) . the chip u-box e3 ligase which is involved in the degradation of cftr (meacham et al., 2001) and glucocorticoid hormone receptor (meacham et al., 2001) , can be ''manipulated'' to function in er glycoprotein turnover. chip usually serves as a cochaperone for the cytosolic heat shock protein hsp70/hsp90 chaperone system. the chip n-terminal tpr motif recruits hsp chaperones loaded with misfolded proteins, whereas its c-terminal u-box ring domain recruits e2 enzymes (murata et al., 2003) (fig. 4b) , effectively linking protein folding with ubiquitination. the chip e3 ligase activity can be directed to er glycoprotein turnover by binding to fbs2 (nelson et al., 2006) , through an interaction between its tpr motif and a pest motif in fbs2 (fig. 4c ). n-linked glycans can, therefore, function in er quality control not only to regulate er retention in the folding cycle, but also to function in erad substrate selection and ubiquitination, adding an additional layer of complexity and specificity to glycoprotein quality control (nelson et al., 2006; yoshida, 2003) . export of proteins through the er membrane most likely takes place via an aqueous channel that allows the passage of polypeptides through the highly hydrophobic er membrane environment while maintaining proper ionic balance between the er and the cytoplasm. the sec61 channel, the very same channel responsible for protein import into the er, was initially thought to mediate transport in the reverse direction (hence the name retrograde translocation or retrotranslocation also coined for the dislocation process) (pilon et al., 1997; plemper et al., 1997 plemper et al., , 1998 wiertz et al., 1996b) , with accessory factors regulating directionality and specificity of the channel. the extent to which sec61 is involved in er dislocation or the identity of the ''dislocon'' are not without controversy, and the search for a dislocation channel(s) is a subject of intense research (meusser et al., 2005; romisch, 2005) . mammalian derlin-1, a member of the der1p-like (derlin) family of yeast der1p homologues, is involved in dislocation from the er (lilley and ploegh, 2004; ye et al., 2004) and was proposed to constitute a channel for protein export from the er membrane to the cytosol (lilley and ploegh, 2004; ye et al., 2004) . like their yeast homologue, derlins 1, 2, and 3 are tetraspanning er membrane proteins that can homo-and heteroligomerize and could presumably form higher order structures with channel-like properties (lilley and ploegh, 2004; ye et al., 2004) . conclusive evidence for a role of derlin-1 as a channel is still unavailable, and in any case, derlin-1 is unlikely to be the only channel, as turnover of some erad substrates does not rely on derlin-1 function lilley and ploegh, 2004) . derlins 2 and 3 are obvious candidates that could function in place of derlin-1. alternatively, derlins may act to deliver a particular substrate to a channel/another adaptor in its cognate dislocation pathway. in fact, derlins form a large, multiprotein complex with p97 and the hrd1p and hrd3p mammalian homologues hrd1 and sel1l, respectively (lilley and ploegh, 2005a; ye et al., 2005) , suggesting a very intimate connection between substrate recognition, export through the membrane, ubiquitination, and extraction into the cytoplasm. the existence of such a complex that would integrate all of these different functions, including formation of a channel or dislocon, would offer obvious advantages in terms of control of both specificity and directionality of the dislocation process. we shall return to this substrate ''guidance'' theme. a cytosolic complex containing the aaa atpase cdc48p (yeast) [valosin-containing protein (vcp)/p97 (in mammals)] and its cofactors nuclear protein localization 4 (npl4p) and ubiquitin-fusion degradation 1 (ufd1p), was recently shown to participate in er degradation (lord et al., 2002; romisch, 2005; ye et al., 2001) . cdc48p/p97 is an essential protein of the aaa atpase (atpases associated with various cellular activities) family, conserved from archaea to mammals, whose functions include mitotic spindle disassembly, membrane traffic and fusion, nucleic acid repair and replication, and ubproteasome degradation (woodman, 2003) . cdc48p/p97 is a motor protein that generates energy from atp binding and hydrolysis; it forms a homohexameric barrel structure, with each subunit containing two aaa domains that contain the walker motifs essential for atpase activity, and the 6 subunits arranged in a ring with a pore in the center (zhang et al., 2000) . cdc48p/p97 interacts with many different adaptor proteins, which regulate its function (dreveny et al., 2004) . p97 can recognize denatured proteins nonspecifically (thoms, 2002) and has an affinity for polyubiquitin chains . when complexed with the polyub-binding ufd1p and npl4p, cdc48p/p97 activity is directed to er degradation (ye et al., 2001) . both in yeast and in mammals, the trimeric cdc48p(p97)/npl4/ufd1 complex is proposed to function in a postubiquitination, preproteasomal step (bays and hampton, 2002; jarosch et al., 2002) , in one of two fashions: the atp-hydrolytic activity of the aaa atpase p97 may provide the driving force to extract substrates through the er membrane, or may be required to liberate already dislocated substrates from the cytosolic face of the er membrane (braun et al., 2002; flierman et al., 2003; hirsch et al., 2004; kostova and wolf, 2003; meusser et al., 2005) . the proteasome presumably interacts with the er membrane (hirsch and ploegh, 2000) , either directly or through a receptor that docks the proteasome to the er membrane, perhaps sec61 (kalies et al., 2005) . notwithstanding, the cdc48p(p97)/npl4/ufd1 complex or other accessory factors might aid substrate feeding to the proteasome (hartmann-petersen and gordon, 2004a; richly et al., 2005) . ubiquitin-binding factors, such as rad23p and dsk2p (in yeast), have a ubiquitin-associated (uba) motif that binds polyubiquitin chains and a ubiquitin-like (ubl) motif that binds to the 19s proteasome, and these are required for efficient degradation of a model erad substrate (elsasser et al., 2004; schauber et al., 1998; wilkinson et al., 2001) . the yeast ub regulatory x domain-containing ubx2p/sel1p protein, an integral er membrane protein, was recently shown to recruit the cdc48p/npl4p/ufd1p complex to the er membrane, thereby facilitating the transfer of polyubiquitinated substrates from the e3 ligases hrd1p and doa10p to cdc48/p97 (neuber et al., 2005; schuberth and buchberger, 2005) . these dual function ub-binding factors effectively serve as bridges between the p97/npl4/ufd1 complex and the proteasome. as more of these ub-and proteasome-binding proteins are discovered (buschhorn et al., 2004; decottignies et al., 2004; medicherla et al., 2004; mullally et al., 2006) , a ''guidance'' model, in which erad substrates are escorted from a dislocation channel to the proteasome by a cascade of ub-binding factors, gains strength (hartmann-petersen and gordon, 2004b; hartmann-petersen et al., 2003; hendil and hartmann-petersen, 2004; richly et al., 2005) . these interactions could be responsible for maintaining the substrate in a proteolysis-competent state and protect it from premature deubiquitination, as well as contribute to directionality of dislocation (hendil and hartmann-petersen, 2004; meusser et al., 2005; romisch, 2006) . in fact, it seems cdc48p/p97 may even be capable influencing substrate fate (rumpf and jentsch, 2006) . cdc48p/p97 can simultaneously bind ufd2p, a u-box e3 that catalyzes polyubiquitin chain extension, and one of two factors that can counteract its action: otu1p, a dub, and ufd3p protein, a wd40 repeat protein of unknown function that has been shown to be required for ub-dependent proteolysis (ghislain et al., 1996; johnson et al., 1995) . otu1p can disassemble the polyub chains, whereas ufd3p competes with ufd2p for the same docking site on cdc48p/p97. presumably, cdc48p/p97 can selectively recruit different substrate processing cofactors and thus tip the balance toward substrate degradation or release from the degradation cascade (rumpf and jentsch, 2006) , suggesting a very tight regulation of proteasomal proteolysis. these aspects are important not only to understand how these pathways contribute to class i mhc-peptide epitope presentation, but also how viruses manipulate these routes to avoid detection. pngase is a cytosolic deglycosylating enzyme that presumably removes n-linked glycan chains from misfolded substrates prior to proteasomal degradation (hirsch et al., 2003; suzuki et al., 2000) . both in yeast and mammals, there is generally a tightly knit relationship between pngase and the proteasome: pngase interacts with (at least) the s4 and s5 subunits of the mammalian 19s proteasome and the ub-binding factor rad23p (hr23b in mammals), which seems to recognize only deglycosylated degradation substrates (katiyar et al., 2004) . this suggests that misfolded protein substrates may first be deglycosylated by er-associated or free pngase, then identified by the hr23b adaptor protein, and subsequently targeted to the nearby proteasome (katiyar et al., 2004) . mammalian pngase also associates with the er membrane gp78 e3 ligase and the cytosolic p97 and y33k, a uba/ubx domain protein (li et al., 2006a) . a gp78-y33k-p97-pngase-hr23b complex could therefore be formed that recruits pngase to the cytosolic face of the er membrane that couples the activities of dislocation, ubiquitination, and deglycosylation and escorts misfolded glycoproteins to the proteasome (kim et al., 2006a; li et al., , 2006a . viruses keep evolving and developing sophisticated immune evasion strategies. in particular, they have targeted virtually every step of the class i mhc antigen presentation pathway, inhibiting proteolysis and generation of the antigenic peptide [epstein-barr virus (ebv) nuclear antigen-1 or ebna-1, hcmv e protein pp65, and hiv tat], inhibiting peptide loading and assembly in the er (hsv icp47, hcmv us6, bovine herpes-virus-1 ul49.5), retaining class i mhc molecules in the er (adenovirus e3/19k and hcmv us3), blocking their exit from the er-to-golgi complex (ergic) (mcmv m152), misdirecting mhc complexes to lysosomal compartments (mcmv m06 and hhv-7 u21), internalizing mhc complexes from the cell surface (kshv k3 and k5 and hiv nef ), encoding homologues of class i mhc as decoys for nk cells (hcmv ul18 and ul142 and mcmv m04), and causing degradation of class i mhc products by the ub-proteasome system (hcmv us2 and us11 and mhv-68 mk3) (fig. 5 ). because these topics have been the subject of numerous reviews (alcami and koszinowski, 2000; ambagala et al., 2005; hengel et al., 1998 hengel et al., , 1999 lybarger et al., 2003; mocarski, 2004; yewdell and hill, 2002) , we shall discuss only a few of these mechanisms in more detail, particularly those exploited by hcmv. the b-herpesvirus hcmv is extremely successful in evolutionary terms: it is a ubiquitous, highly species-adapted pathogen that is able to establish a life-long persistent infection with minimal or no disease symptoms in the immunocompetent host. prolonged latency periods (a dormant state with minimal production of viral proteins and absence of viral progeny) and controlled sporadic reactivation ensure transmission to a new host, and thus survival of both host and virus. perturbation of this delicate balance leads to life-threatening infections in immunocompromised patients, transplant recipients and infected newborns and illustrates how the outcome of this host-virus relationship is dependent on viral manipulation of the host immune response (hengel et al., 1998; klenerman and hill, 2005) . the several hcmv-encoded immunoevasins (jones et al., 1995) are presumably aimed primarily, but not solely, at control of the cd8 þ t cell and nk cell responses (falk et al., 2002; mocarski, 2004; pinto and hill, 2005; yewdell and hill, 2002) . here we will discuss the hcmv immunoevasins that interfere with class i mhc antigen presentation, us3, us6, us10, us2, us11, ul16, ul18, ul40, ul141, and ul142. in light of our most recent findings, we will elaborate on the mechanism of er dislocation co-opted by the hcmv us2 and us11 immunoevasins. more specifically, we discuss the similarities and the differences between the two cellular erad pathways that us2 and us11 have allowed us to uncover and the possible implications for er dislocation. we will extend this by comparing the hcmv us2-and us11-mediated dislocation of human class i mhc hc molecules with dislocation of murine hcs by the mhv-68 mk3 immunoevasin. if one goes back to the steps we depicted for antigen presentation (fig. 1) and then examines the immunoevasins encoded by hcmv, we will find that this herpesvirus exploits many aspects of the antigen presentation pathway thus defined. the hcmv phosphoprotein pp65 tegument protein mediates the phosphorylation of the hcmv immediate early antigen-1 (ie-1) during hcmv infection. phosphorylation of ie-1 interferes with the presentation of ie-1-derived antigens (gilbert et al., 1996) . the us3 protein binds to and retains some class i mhc locus products in the er membrane (ahn et al., 1996; jones et al., 1996) . us3 is a type i membrane glycoprotein with an ig-like lumenal domain that is essential for its own retention, albeit transient, in the er (lee et al., 2003) . us3 eventually travels to the lysosome where it is degraded (gruhler et al., 2000) . some evidence suggests that er retention of class i mhc complexes by us3 depends on the er localization signal on us3 and perhaps the ability of the us3 luminal domain to oligomerize (misaghi et al., 2004b) as determinants of retention of both molecules (lee et al., 2003) . another model suggests that association of us3 with tapasin, which inhibits tapasin, is sufficient to mediate er retention (park et al., 2004) . class i mhc alleles that require peptide optimization by tapasin may be retained in the er, whereas tapasin-independent locus mhc products are spared from retention. in fact, there is a perfect correlation between tapasin-dependence and us3 sensitivity (park et al., 2004) . both mechanisms, tapasin inhibition and direct binding, may be in place, perhaps allowing us3 to retain a larger repertoire of class i mhc locus products. us6 inhibits peptide loading of the class i mhc molecules by blocking the tap transporter (ahn et al., 1997; lehner et al., 1997) . us6 is an er-resident type i membrane glycoprotein with a bulky lumenal domain that binds the core transmembrane domains of the tap subunits, tap 1 and tap 2, from within the er lumen, inhibiting atp binding (hewitt et al., 2001; kyritsis et al., 2001) and thus tap-mediated peptide translocation into the er. the us6 luminal domain oligomerizes and may form a bridge between the tap 1 and tap 2 subunits to effectively block tap activity (halenius et al., 2006) . us10 delays trafficking of class i mhc hcs and stalls them in the er; although the block is not absolute and the mechanism is poorly characterized, us10 expression results in downregulation of class i mhc from the cell surface (furman et al., 2002a) . us2 and us11 catalyze destruction of class i mhc hcs from the er membrane by targeting them to the ub-proteasome system (wiertz et al., 1996a,b) , a process we discuss in detail in a later section. expression of each individual immunoevasin results in reduction of cell surface expression of class i mhc peptide-loaded complexes and evasion of cd8 þ t cell-mediated lysis (ahn et al., 1996; jones et al., 1995) . besides cd8 þ t cell recognition, hcmv can frustrate nk cell recognition (orange et al., 2002) . protection of hcmv from nk cell-mediated lysis can be mediated by several immunoevasins, hcmv ul16, ul40, ul18, ul141, ul142, and pp65 (lodoen and lanier, 2005; orange et al., 2002; rajagopalan and long, 2005; reyburn et al., 1997; wills et al., 2005) . the activating receptor nkg2d on the nk cell recognizes divergent families of class i mhc-related ligands, like the mic and ulbp products. hcmv ul16 retains the ulbp 1, ulbp 2, and mic-b nkg2d ligands in the ergic compartment of the target cell, preventing nkg2d recognition and thus nk cell activation . hcmv ul141 and pp65 act at the level of the nk effector cell rather than the apc. the result is, nevertheless, the same: prevention of nk cell activation. ul141 downregulates the nk cell-activating receptors cd226 (dnam-1) and cd96 (tactile) . the pp65 tegument protein engages the activating receptor nkp30 and antagonizes its effects, dampening nk cell-mediated cytotoxicity. how a tegument protein gains access to the receptor on the nk cell is unknown, but pp65 engagement of the nkp30 receptor causes dissociation of a receptor-associated signaling module (orange et al., 2002) , disrupting the activating signaling pathway that would lead to nk cell activation (arnon et al., 2005) . nk cell responses also rely on receptors that recognize class i mhc locus products. the killer cell immunoglobulin-like receptor (kir) genes encode a family of activating and inhibitory receptors that recognize human leukocyte antigen (hla)-a, -b, and -c. the cd94/nkg2 receptors recognize the nonclassical class i mhc molecule hla-e. hla-e presents fragments derived from the signal sequences of classical class i mhc molecules, which delivers an inhibitory signal to cd94/nkg2 receptors on nk cells. ul40 encodes a peptide whose sequence is exactly homologous to the hla-e binding leader peptide from hla-c locus products. ul40 therefore loads hla-e and maintains hla-e on infected cells (tomasec et al., 2000; ulbrecht et al., 2000) even when other class i mhc products are downregulated. ul18 encodes a class i mhc-like molecule that engages the inhibitory cd85j/lir-1/ilt-2 receptor on the nk cell, thus inhibiting nk cell effector functions (cosman et al., 1997; reyburn et al., 1997) . however, ul18 and ul40 expression may be insufficient to confer target cell protection (falk et al., 2002; leong et al., 1998) . ul18 and ul40 may, in fact, be more relevant for control of viral infection by t cells, with hla-e-restricted cd8 þ t cells playing a role in lysis of cells expressing ul40, and non-mhcrestricted cd8 þ t cells playing a role in lysis of ul18-positive cells (pietra et al., 2003; romagnani et al., 2004; saverino et al., 2004) . cd85j is an invariant receptor expressed by many t cells and responsible for transduction of inhibitory signals that downregulate antigen-specific t cell functions (merlo et al., 2001; saverino et al., 2000) . cd85j interaction with ul18 on cd8 þ t cells occurs in a tcr-independent manner and leads to activation (not inhibition) of non-mhcrestricted cd8 þ t cells (saverino et al., 2004) . this expands the repertoire of t cell activation mechanisms and is probably a viral strategy of ensuring survival of the host, by allowing some level of protection from the initial wave of nk cellmediated antiviral response. in vivo hcmv-infected apcs are faced with multiple immunoevasins displaying allelic preferences and expression patterns that are both spatially and temporally regulated. the many immunoevasins expressed by hcmv are thus likely to have both synergistic and antagonistic interactions (ahn et al., 1996; farrell et al., 2000; klenerman and hill, 2005; mocarski, 2004; reddehase, 2002; yewdell and hill, 2002) , as has been experimentally verified for murine cytomegalovirus (wagner et al., 2002) . evidence of er-to-cytosol transport or dislocation, a crucial er quality control step now considered of general importance in dispensing with misfolded or misassembled er proteins, was initially provided by studying the mechanism of action of the hcmv us2 and us11 immunoevasins (wiertz et al., 1996a,b) . the viral proteins appropriate this cellular quality control process to extract (dislocate) class i mhc hcs from the er membrane. on arrival in the cytoplasm, the dislocated hc molecules are destroyed by the proteasome; destruction of the hc component of the class i mhc complex by us2 and us11 abolishes cell-surface expression of class i mhc complexes and, consequently, presentation of viral peptides to cd8 þ t cells, allowing hcmv to remain undetected (wiertz et al., 1996a,b) . us2-and us11-mediated hc dislocation from the er membrane is not only an ingenious viral immune evasion strategy, but also a useful case study in er quality control and degradation. one theme that arises from the characterization of this process over the 10 years that have passed since its discovery is that hc dislocation is unique in many respects: hc molecules do not meet the requirement of being either misfolded or misassembled, yet their dislocation takes place by virtue of the presence of us2 or us11; the speed of hc degradation is unrivaled by that of any other er-associated degradation substrates: hc half-life is reduced from hours to a mere 2-5 min in cells infected by hcmv or in ce lls expressin g either us2 or us11 ( wiertz et al ., 1996a ,b) ; both us2 and us11 have stringent requirements in terms of which hla alleles (barel et al., 2003 (barel et al., , 2006 machold et al., 1997) or assembly, folding and ubiquitination status of the class i mhc complex (blom et al., 2004; furman et al., 2003; gewurz et al., 2001) either viral protein is able to target for dislocation and proteasomal destruction. notwithstanding the unique nature of this virus-mediated process, knowledge from the us11 pathway, and in particular the identification of the derlin proteins, has widened our understanding of the cellular factors involved in er dislocation more generally ploegh, 2004, 2005a) . the us11 transmembrane domain (tmd) is crucial for us11 function: more specifically, mutation of a polar amino acid, glutamine (q) 192, within the us11 tmd to a hydrophobic leucine (l) residue renders this us11 q192l mutant inactive in dislocating hcs from the er membrane. in a screen for proteins that interact specifically with the active version of us11, work from our laboratory showed that the aforementioned derlins, the mammalian der1p homologues, are involved in hc dislocation mediated by us11, but not by us2 (lilley and ploegh, 2004) . the fact that the dislocation mechanism used by us2 is not dependent on the derlins prompted us to investigate what other erad pathw ay is being coopted by the hcmv us2 immu noevasin and allow ed us to unco ver an unexpect ed era d pl ayer. us2 is an er-resident type i membrane glycoprotein of only 199 amino acids, with a noncleavable signal sequence (gewurz et a l., 2002) , a lumenal domain that dictates an allele-specific association with the lumenal domain of hc (gewurz et al ., 2001) , a transmembrane segment, and a short cytosolic tail of only 14 amino acids (residues 185-199), with no obvious sequence homology to known cellular proteins. the us2 tail is essential for dislocation: us2 186 , a cytosolic tail deletion mutant of us2, is dislocation incompetent (furman et a l., 2002b) . by using an affinity purification approach similar to that used for us11 (lilley and ploegh, 2004) , signal peptide peptidase (spp) was found as a specific interacting partner for dislocation-competent (active) us2 (loureiro et a l., 2006) , an interaction that relies solely on the presence of the highly hydrophobic us2 tail. more importantly, reduction of spp levels by rna interference led to inhibition of class i mhc hc dislocation and to the discovery of spp as a necessary factor for the us2-mediated er dislocation pathway. spp is an er -resi dent protein o f ap proximate ly 45 kd a tha t is predic ted to span the er memb rane seven to nine times ( friedmann et al ., 2004 ) , an d a memb er of the presenili n (ps)/sp p-like (sppl ) superfam ily of intrame mbran e-cleaving aspartic protea ses ( weihof en et al ., 2002 ) . these protea ses are charac terized by the ability to cleave substr ate pol ypeptides with in a transm embran e region and by pos sessing two active site aspartat e (d) resid ues (ita licized) within the con served motifs y d and lglg d in adjacent memb ranespan ning regio ns ( martoglio an d golde , 2003; wan g et al., 2006a; weihof en et al., 2002 ) . there are seven related members of the ps/spp l superfam ily in the human genome : ps -1, ps -2, spp an d four spp -like pro teins, spp2 a, spp 2b, and spp2c, an d spp 3 (m artoglio and gold e, 2003 ). presen ilins 1 and 2 are the catalytic compone nts of gsec retase, a tetr americ comp lex containin g ps and three other subunits. pss play a role in processing of the b-amyloid precursor protein (app) into ab40 and ab42, peptides that constitute the principal components of the b-amyloid plaques in alzheimer's disease (ad); pss are also required for development due to processing of the notch receptor by g-secretase (selkoe and kopan, 2003) and might be involved in intracellular traffi cking (si sodia and st george -hyslop , 2002; wang et al ., 2006 c) . the function of the four spp-like proteins is, at this point, unknown, but the role of intramembrane-cleaving proteases is usually to liberate signaling molecules from membrane-bound precursors with consequent activation or repression of signaling cascades (fortini, 2002; kopan and ilagan, 2004; martoglio and golde, 2003; parent et al., 2005; xia and wolfe, 2003) . in humans, spp performs an important immunological function as it generates the peptide ligands for the nonclassical class i mhc molecule hla-e. on insertion of secretory or type ii membrane proteins into the er, their signal sequence is cleaved by the er luminal protein signal peptidase, leaving the signal peptide anchored in the er membrane. the er membrane-anchored signal peptide is subsequently cleaved by the intramembrane-cleaving spp within the transmembrane region. the resulting signal peptide fragments are released into the cytosol (n-terminal portion) or into the er lumen (c-terminal portion). the latter peptides may easily bind to class i mhc molecules in the er lumen. the hla-a2 molecule, for instance, is known to bind signal sequence-derived peptides. the n-terminal signal sequence fragments are tap-transported into the er lumen and bind to hla-e (lemberg et al., 2001) . by presenting fragments derived from the signal sequences of classical class i mhc molecules, hla-e monitors the presence of classical class i mhc molecules. this is, as we discussed earlier, of crucial importance for nk cell recognition. spp is involved in processing of the hepatitis c virus (hcv) core protein (mclauchlan et al., 2002) . hcv is a single-stranded rna virus with a single open reading frame encoding a large polyprotein. the n-terminal portion of the hcv polyprotein encodes the structural components of the hcv virion, the core protein (thought to constitute the virion capsid), and the e1 and e2 envelope glycoproteins. the mature structural components of the hcv virion are produced through a series of cleavage events catalyzed by cellular proteases. the core protein is the most n-terminal portion of the polyprotein and is followed by the signal sequence of the e1 envelope glycoprotein. the e1 signal sequence targets the polyprotein to the er membrane and induces translocation of e1 into the er lumen. cleavage by signal peptidase liberates the n-terminal end of e1, leaving the core protein anchored (by the e1 signal peptide) in the er membrane. spp-mediated intramembrane proteolysis of the e1 signal sequence then results in release of the hcv core protein from the er membrane, thus freeing the mature core protein for incorporation into lipid droplets (martoglio and golde, 2003) . spp-mediated hcv core protein maturation and trafficking to lipid droplets and the outer mitochondrial membrane may be critical for viral assembly and life cycle (ait-goughoulte et al., 2006) and may also affect cellular lipid metabolism and apoptosis, as hcv core proteintransgenic mice display liver pathologies, mitochondrial injury, and enhanced oxidative stress (chou et al., 2005; korenaga et al., 2005; meyer et al., 2005; okuda et al., 2002; omura et al., 2005; schwer et al., 2004; suzuki et al., 2005) . spp-mediated cleavage of the hcv core protein may thus modulate these important cellular functions of hcv. spp may regulate the interaction of signal peptide remnants of hiv gp160 envelope protein and preprolactin (p-prl) with calmodulin. a characteristic feature of a signal sequence is its tripartite structure: a polar n-terminal n-region, a hydrophobic core (h-region) of 7-15 residues, and a polar c-terminal c-region that contains the consensus sequence for signal peptide cleava ge (von he ijne, 1985). the nregion of most signal sequen ces comprise s only a few residues. however, some signal sequences have extended n-regions of up to 150 residues. the function of such long n-regions is not known. both the p-prl and the gp160 signal sequence have an extended basic n-region that can potentially form a basic amphiphilic alpha-helix, a feature of cam-binding domains (o'neil and degrado, 1990) , not found in the majority of signal sequences. spp-mediated cleavage releases this cam-binding domain on the n-terminal fragment of the p-prl and gp160 signal peptides into the cytosol. the functional and physiological significance of an interaction between the p-prl and p-gp160 signal peptide fragments that are released into the cytosol and calmodulin (cam) could be due to a regulatory function of the signal peptide fragments. cam-dependent processes could be enhanced or inhibited depending on the amounts of cam-binding signal peptide fragments generated and released into the cytosol (martoglio et al., 1997) . the spp orthologues in drosophila melanogaster, spp, and in caenorhabditis elegans, imp-2, play an essential role in development: spp protease activity seems to be essential for larval development both in the fly and in the nematode (casso et al., 2005; grigorenko et al., 2004) . although the mechanism by which spp mutations impairs developmental processes in the fly is currently unknown, in c. elegans the molting defect induced by imp-2 deficiency was mimicked by cholesterol depletion and by deficiency in irp-1, a homologue of mammalian lipoprotein receptor-related protein (lrp) receptors suggesting a role in cholesterol and lipid metabolism (grigorenko et al., 2004) . the possibility of a role for spp in er quality control was first advanced by high and colleagues, who reported an association between spp and a truncated version of a polytopic er protein (opsin) in an in vitro system (crawshaw et al., 2004) and proposed spp to be implicated in the recognition of misassembled transmembrane domains during membrane protein quality control at the er. us2-mediated dislocation of class i mhc hc molecules presents the first functional evidence of such a role for the intramembranecleaving protease spp. the us2 tail is necessary and sufficient to recruit spp, and structural predictions suggest that the us2 tail suggests may adopt a 3 10 helical conformation that could form a protein-protein interaction domain (oresic et al., 2006) . spp is crucial for dislocation by us2, as reduction of its levels by rna interference blocks hc degradation. the question remains as to the detailed mechanism of its involvement. an obvious possibility is involvement of the catalytic activity of spp. this would imply a cleavage event during dislocation, such as within the tmd of us2 or hc or another factor, unknown at this point; a postcleavage function of one of these protein fragments could play a regulatory role in the process. sppmediated intramembrane proteolysis requires, among other things, a membrane protein substrate to have access to its catalytic core in a type ii orientation martoglio and golde, 2003) . both us2 and hc are type i membrane glycoproteins, so the topology of their transmembrane and tail segments is opposite to that of predicted spp substrates. spp-mediated intramembrane cleavage within the hc tmd is inconsistent with the observed recovery of full length hc in the dislocation reaction (blom et al., 2004; misaghi et al., 2004a; wiertz et al., 1996a,b) . for us2, while a suggested protein-protein interaction domain in the us2 tail (oresic et al., 2006) may mediate binding to spp, intramembrane cleavage of the us2 tmd by spp in this inverted orientation would presumably not occur, as seen for other proteases (roques et al., 1983; tarasova et al., 2005) . however, the proposed bent-helix conformation on the us2 tail (oresic et al., 2006) might allow us2 to conform to the requirements for spp cleavage. whether spp-mediated us2 cleavage takes place is unknown at this point. one can speculate that this putative processing of us2 by spp, as for the hcv core protein (mclauchlan et al., 2002) , could be necessary for ''maturation'' of us2 into a dislocation-active form. binding of us2 to spp could still modulate spp enzymatic activity and thus affect dislocation. the possibility remains that spp mediates cleavage in trans of an unidentified factor whose function is important. experiments with spp inhibitors and catalytic mutants of spp should allow an assessment of the contribution of the proteolytic properties of spp to dislocation. the involvement of spp need not be related to its catalytic activity. substrate recruitment and subsequent cleavage by spp may be separable events, as shown for the related ps (kornilova et al., 2003; lemberg and martoglio, 2004) . some intracellular cleavage products of g-secretase are proposed to be intermediates that are destined for degradation (kopan and ilagan, 2004; parent et al., 2005) . g-secretase-mediated cleavage of a large number of type i transmembrane proteins releases their c-terminal fragments (ctfs). ps1 deficiency causes delayed turnover and subsequent accumulation of some g-secretase substrates as full-length proteins (esselens et al., 2004; wang et al., 2006c; wilson et al., 2004c) , suggesting cleavage of the ctfs as a prelude to degradation. treatment with g-secretase inhibitors, however, does not phenocopy ps deficiency (wang et al ., 2006 c) , suggestin g tha t this effec t is in dependen t of gsecre tase activ ity. therefore, spp may be crucial for hc dislocation irrespectively of its catalytic properties. binding of us2 to spp could presumably modify the spp structure in a way that affects dislocation. alternatively, recruitment of spp by us2 could perhaps nucleate assembly of a dislocation complex, much like us11 and the derlins (lilley and ploegh, 2005a) . spp may be a component of an erad pathway for a subset of er degradation substrates that includes misfolded transmembrane proteins, such as truncated opsin (crawshaw et al., 2004) , and tha t is recru ited by us2 to dislocat e hc ( fig. 6, a arrow ) . experi ments to address the identity of spp-associated proteins may prove informative. curiously, another class of intramembrane-cleaving proteases, the rhomboid serine proteases, share a homology domain of unknown function with the derlins (lemberg et al., 2005) . it is tempting to speculate that our observations extend the connection from regulated intramembrane proteolysis to a direct involvement in er dislocation. an involvement of spp with the upr is also a possibility. cells deficient for the x-box binding protein-1 transcription factor (xbp-1) show upregulated levels of spp transcripts (shaffer et al., 2004) , but this aspect of the process remains to be explored. although removal of signal peptide remnants from the er membrane, assigned to spp in animals and plants, is not a function exclusive to higher eukaryotes, a gene that encodes an orthologue of this enzyme is absent from the yeast genome (martoglio, 2003; weihofen et al., 2002) . the role of spp in higher eukaryotes might therefore not be limited to signal peptide processing but extend to processes such as protein dislocation from the er. ps and the other spp-like members of the ps/sppl superfamily of intramembrane-cleaving aspartic proteases, so far of unknown function (martoglio and golde, 2003) , and some of which may not reside in the er (krawitz et al., 2005) , may likewise be involved in disposal of different degradation substrates. hcmv might just be exploiting a cellular degradation pathway that involves intramembrane-cleaving proteases for disposal of class i mhc hc. the us2 tmd, although dispensable for interaction with spp, is also required for hc dislocation (loureiro et al., 2006) . hc dislocation is therefore dependent not just on (us2 tail-mediated) recruitment of spp, but also on additional (us2 tmd-mediated) interactions within the plane of the membrane. the us2 tmd may be involved in further engagement of spp (sppmediated cleava ge or o therwise ) ( fig. 6, b arrow ) , o r alternativ ely, in the recruitment or engagement of other protein(s) involved in dislocation (fig. 6 , c arrow ). er m embran e e3s or their adap tor subunits are like ly cand idates. spp is most certainly not the sole host-derived component of the us2 dislocation pathway, and a putative multiprotein complex (cascade of adaptor proteins) analogous to that found for us11 is likely to be found that evokes many (complicated) links between the erad machineries at the level of the er membrane and the cytosol. uncovering the identity of the additional cellular partners of this hcmv us2 immunoevasin will certainly give us insight into the dislocation mechanism. a theme that arises from analysis of the us2-and us11-mediated hc dislocation processes is that these are only superficially similar: although hc dislocation by us2 and us11 has the same outcome (proteasomal degradation) of hc and shares many if not all of the steps that take place after extraction of the hc from the er membrane (such as deglycosylation and degradation kinetics), the differences between the pathways are rather striking in terms of the steps prior to dislocation. as we mentioned earlier, us2 and us11 have different allele and substrate folding, assembly, and ubiquitination requirements. derlin-1 is crucial for us11-but not us2-mediated dislocation of hcs (lilley and ploegh, 2004; ye et al., 2004) , and, conversely, spp is required by us2 but not by us11 (loureiro et al., 2006) . this suggests that the hcmv immunoevasins us2 and us11 are targeting hc molecules to distinct er dislocation pathways, perhaps by serving as adaptor molecules aiding in recruitment and/or assembly of distinct multiprotein complexes at the er membrane ploegh, 2004, 2005b) . the murine g-herpesvirus 68 (mhv-68) mk3 also targets newly synthesized murine class i mhc hc for dislocation from the er and proteasomal degradation (boname and stevenson, 2001; lybarger et al., 2005; wang et al., 2005; yu et al., 2002) . mhv-68 mk3 belongs to a family of structurally related molecules, the k3 homologues, which have e3 ligase activity. k3 homologues are present in several different g-herpesviruses and poxviruses (coscoy and ganem, 2000; ishido et al., 2000; mansouri et al., 2003; stevenson et al., 2000) . all k3 homologues possess a noncanonical ring-finger domain with ubiquitin ligase (e3) activity [also called a plant homeodomain (phd) or leukemia-associated protein (lap) domain], and a conserved integral membrane topology, with the transmembrane domains and cytosolic c-terminal tails mediating interaction with the substrate (coscoy and ganem, 2003) . the mk3 phd/lap-family e3 is a type iii er membrane protein with the phd/lap ring-related domain facing the cytosol (boname and stevenson, 2001; sanchez et al., 2002) . mk3-mediated degradation of murine hcs is absolutely dependent on components of the plc: association of mk3 with tap and tapasin presumably imposes the necessary proximity and/or orientation of the mk3 ring domain that allows mk3 to specifically ubiquitinate class i mhc hc as they enter the plc wang et al., 2004 wang et al., , 2005 . mk3-mediated dislocation of murine hcs is dependent on the atpase activity of p97 and physical association with derlin-1 and vimp (wang et al., 2006 d) . thi s is reminiscen t of the pathway that h cmv us11 is propos ed to co-opt for dislocation of mammalian class i mhc molecules. in a sense, it appears that mk3 may be a more evolved immunoevasin that can couple the ability to recruit other components of the dislocation machinery, like us11 and presumably us2, with e3 ligase activity, in one viral polypeptide. there are several mammalian k3 homologues, the membrane-anchored ring-ch (march) proteins (bartee et al., 2004; goto et al., 2003) , which are likely to be the cellular ancestors of mhv-68 mk3. mk3-mediated ubiquitination of the murine class i mhc hc cytosolic tail, the portion of the hc molecule more likely to come into contact with the cytosolic ring-ch domain of mk3, is not required for dislocation even though ubiquitination and presence of the cytosolic tail are essential for dislocation . in fact, like mk3, hcmv us2 and us11 also induce ubiquitination-dependent degradation of class i mhc molecules in a cytosolic tail lysine-independent fashion (furman et al., 2003; shamu et al., 1999) . how, then, does mk3 access the luminal domain of hc molecules and trigger its ubiquitination? access of the hc lumenal domain to the cytosol would invoke a ''partial dislocation'' model that has been proposed for hcmv us2 and us11 (furman et al., 2003; shamu et al., 1999) : the hc luminal domain must begin to emerge in the cytosolic face of the er so ubiquitination can take place. this would mean that the trigger for dislocation would reside upstream from tail ubiquitination. an alternative explanation would be that the class i mhc tail lysine mutant hc molecules are dislocated as a ''bystander effect'' of dislocation of wild type molecules by mk3, simply because they are in the proximity of the dislocation machinery that has been recruited by the viral protein for the wild type hc clientele. the hcmv us2 and us11 and mhv-68 mk3 immunoevasins all target nascent class i mhc hcs for degradation by inducing their dislocation from the er membrane. the mechanisms used by us2, us11, and mk3, however, when analyzed in detail, are strikingly different. for instance, the stage of class i mhc hc biosynthesis that is targeted by each viral protein is distinct: us11 is the most promiscuous, targeting multiple class i mhc assembly intermediates, whereas us2 targets only properly folded class i mhc complexes and mk3 targets predominantly incompletely assembled hc while in association with the peptide-loading complex. the main difference resides in the fact that mk3 encompasses ubiquitination activity (by means of its ring domain), substrate selection (by means of its association with the peptide-loading complex), and recruitment of er membrane and cytosolic factors necessary for dislocation (like derlin-1 and p97) all in one polypeptide. hcmv us2 and us11 do not possess e3 ligase activity and possess no obvious sequence similarity with known genes/proteins that would hint at their function. however, at least us11, and presumably also us2, are still able to induce assembly of a dislocation complex (fig. 7) that encompasses all the necessary erad activities. the knowledge that the tmd of us11 is essential for its function (lilley et al., 2003) , led to the discovery of derlins (lilley and ploegh, 2004) and of multiprotein complexes at the er membrane that function in us11-mediated dislocation of hcs and us11-independent dislocation of a subset of erad substrates (lilley and ploegh, 2005a; oda et al., 2006; ye et al., 2005) . us2 uses its cytosolic tail to recruit spp and its tmd for an additional step (so far unknown) also critical for hc dislocation (loureiro et al., 2006) , presumably resulting in recruitment of us2-specific-components of the er dislocation machinery. the mechanism by which us2 operates will hopefully be clarified as we continue characterizing the structural and host cofactor requirements for its function in dislocation. the mechanistic details of dislocation catalyzed figure 7 three viral immunoevasins that co-opt distinct erad pathways. the hcmv us11 immunoevasin delivers class i mhc hc molecules to derlins for dislocation from the er membrane and degradation, whereas hcmv us2 uses a pathway that is dependent on signal peptide peptidase. the mhv-68 mk3 immunoevasin is an e3 ligase that uses the plc as a platform to target murine class i mhc molecules for ubiquitination and degradation. although the three pathways are superficially similar, the substrate selection and targeting steps at the er membrane are very distinct. ub, ubiquitin; e1, ub-activating enzyme; e2, ub-conjugating enzyme; e3, ub-ligase enzyme; pngase, peptide-n-glycanase; spp, signal peptide peptidase; tap, transporter associated with antigen presentation; crt, calreticulin. by the hcmv us2 and us11 and the mhv-68 mk3 immunoevasins must be quite diverse and their study will most certainly keep providing new insights into er dislocation. furthermore, it is nothing short of remarkable how sequence and structurally unrelated herpesvirus proteins have converged into (although only superficially) similar mechanisms to dislocate newly synthesized class i mhc molecules from the er membrane. class ii mhc-restricted cd4 þ t cells are crucial for lymphocyte activation, antibody responses, and coordination of the immune response and rely on activation by the professional apcs. immunoevasins aimed at interfering with class ii mhc antigen presentation are not expected to block presentation of exogenous antigens to cd4 þ t cells, unless the apc is infected by the virus (yewdell and hill, 2002) . class ii mhc expression can, however, be modulated by ifn and receptor signaling through the ciita transcription factor (boss and jensen, 2003) . there are relatively few examples of viruses and bacteria that directly infect apcs and affect ifn-induced class ii mhc expression or directly interfere with class ii mhc-restricted antigen presentation (hmama et al., 1998; hussain et al., 1999; miller et al., 1998; rinaldo, 1994; schuller et al., 1998; srisatjaluk et al., 2002; zhong et al., 1999) . it is likely that more examples will be found as this important question is being revisited. for viruses that do not infect professional apcs, the route to avoiding helper t cell and antibodymediated responses is to interfere with activation of cd4 þ t cells by apcs. in this section, we present an overview of some of the mechanisms used mostly by viral pathogens to actively subvert class ii mhc antigen presentation (fig. 8) . the degree to which other pathogens, like bacteria and parasites, actively interfere with class ii mhc antigen presentation, by encoding ''immunoevasins'' rather than passively, due to their residence in endocytic compartments, is difficult to discern. epstein-barr virus is an example of a virus that can infect and establish latency in b lymphocytes and is associated with a number of malignancies. the product of the bzlf2 ebv gene, gp42, is a bifunctional protein. first, it functions as the coreceptor for viral entry into b cells by binding to the hla-dr product. second, gp42 is generated through proteolytic cleavage in the er and matures into a secreted form that binds class ii mhc molecules at the cell surface. gp42 bound to class ii mhc molecules at the cell surface prevents tcr-(peptide-loaded class ii mhc) interactions and cd4 þ t cell activation (li et al., 1997; ressing et al., 2003 ressing et al., , 2005 spriggs et al., 1996) . the hiv-1 nef protein downregulates class ii mhc molecules from the cell surface by restructuring the endocytic pathway such that invariant chain (ii) degradation is impaired and immature class ii mhc complexes (abii) are granted increased access to the cell surface (stumptner-cuvelette et al., 2001) . nef induces a reduction of surface levels of peptide-loaded class ii mhc as well as a strong accumulation of surface-displayed immature class ii mhc complexes, still containing (and thereby blocked by) intact invariant chain (ii). nef expression results in accumulation of both class ii mhc and invariant chain (ii) in multivesicular bodies (mvbs) (stumptner-cuvelette et al., 2003) . mvbs are a specialized type of endosome that constitutes a major pathway of delivery of transmembrane proteins for lysosomal degradation . sequestering in mvbs suggests a reduced capacity of immature class ii mhc complexes to reach lysosomes, either due to a defect in class ii mhc sorting to the lysosomes or due to slower internalization of immature complexes. the mechanism is still unclear (stumptner-cuvelette et al., 2003) . the hcmv us2 immunoevasin was proposed to downregulate class ii mhc from the cell surface, presumably by targeting hla-dra and hla-dma for degradation by the proteasome, thus inhibiting antigen presentation to cd4 þ t cells (chevalier et al., 2002; hegde and johnson, 2003; tomazin et al., 1999) . this effect of us2, however, was only seen in cell lines in which induction of class ii mhc was induced by stable transfection with the class ii mhc trans-activator (ciita), but not in human dcs or several other cell lines, which express class ii mhc endogenously (rehm et al., 2002) . presumably the relative expression levels of class ii mhc and/or us2 could account for the observed differences, and in fact, in ciita-transfected cells, even us3 was seen to downregulate class ii mhc molecules (hegde et al., 2002) . hsv-1 can downregulate surface expression of class ii mhc complexes in b cells and inhibits the ability of b cells to stimulate cd4 þ t cells. hsv-1 inhibits synthesis of ii and also encodes an envelope glycoprotein b (gb) that binds both hla-dr and hla-dm (neumann et al., 2003; sievers et al., 2002) . by binding to hla-dr, gb affects trafficking of the molecule in the secretory pathway and by binding hla-dm it sequesters this peptide editor and thus prevents peptide loading of class ii mhc molecules that may have escaped (neumann et al., 2003) . the cd4 protein serves as the primary cellular receptor for hiv at the surface of cd4 þ cells. however, its presence inhibits virus budding and interferes with incorporation of the gp120 protein into the budding virion, not to mention its crucial role in eliciting of a cd4 þ t cell response (keppler et al., 2006; lama et al., 1999; ross et al., 1999) . not surprisingly, three hiv-1 proteins, nef, vpu, and gp160 dramatically reduce the steady state levels of cd4 on the cell surface piguet et al., 1999a,b) . one of the many mechanisms used by nef involves acceleration of the constitutive endocytosis of cd4. in t cells, cd4 is stabilized at the cell surface by p56lck, a src-family tyrosine kinase, which binds a dileucine motif in the cd4 cytoplasmic tail, preventing cd4 from being recruited into clathrin-coated pits (aiken et al., 1994; jin et al., 2005) . nef can displace lck by directly binding a dileucine sorting motif in the cd4 tail, overcoming the normal lck phosphorylation-dependent route of cd4 downregulation. nef itself contains a c-terminal dileucine motif that recruits a subunit of the tetrameric adaptor protein complex-2 (ap-2), a component of clathrin-coated pits at the cell membrane. thus, nef connects cd4 to ap-2 on clathrin-coated pits, triggering rapid cd4 endocytosis (jin et al., 2004b (jin et al., , 2005 mangasarian et al., 1999; piguet et al., 1998 piguet et al., , 1999a . nef can bind not only the ap-2 components of clathrin-coated pits, but also the regulatory v1h subunit of the vacuolar proton (h þ ) atpase. v1h (also called nbp1 or nef binding protein-1) binds ap-2 in clathrin-coated vesicles. by binding v1h, nef strengthens its weak direct interaction with ap-2 (geyer et al., 2002; mandic et al., 2001) . again, by binding both cd4 (using the aforementioned dileucine motif in the cytoplasmic tail) and v1h, nef directs internalization of cd4 from the cell surface. the endocytosed cd4 accumulates in early endosomes from where it is sorted to lysosomes for degradation. adaptor protein (ap) complexes mediate transport of proteins to numerous compartments within the cell. whereas ap-2 initiates early endocytic vesicle formation at the cell membrane, ap-1 is involved in vesicle formation at the tgn and vesicle targeting to early endosomes and ap-3 participates in vesicle formation at the tgn and targeting to late endocytic/lysosomal compartments (bonifacino and traub, 2003) . the ''coat'' on vesicles in the endocytic pathway is composed of ap complexes and another set of coat proteins, clathrin in clathrin-coated vesicles, and cop proteins, in cop-coated vesicles. the cop proteins are involved in vesicle trafficking early in the secretory pathway between the er and the golgi (mcmahon and mills, 2004) . trafficking in the endocytic pathway can be targeted by hiv nef by direct binding to ap complexes or by interference with the recruitment and release cycles of ap complexes from vesicle membranes. ap complexes cycle from the cytosol to vesicles in a process dependent on the gtpase cycle of adp-ribosylation factor-1 (arf1). nef can bind to and stabilize the small gtpase arf1 on the endosomal membrane, preventing ap complexes from being released, affecting trafficking of host molecules (including cd4) along the endocytic pathway. nef can also mediate the formation of a ternary complex composed of nef, arf1, and a component of the cop-i coat, bcop. cop-i-coated vesicles are mostly subject to retrograde transport within the golgi and between the golgi and the er, but some are involved in transport from early to late endosomes (mcmahon and mills, 2004) , and thus association of nef with arf1 and bcop mediates targeting of cd4 for lysosomal degradation (faure et al., 2004) . nef has a preference for ap-1 and ap-3 complexes in vitro (janvier et al., 2003b) , suggesting that the nef modus operandi is mostly at the level of endosomal membranes. downregulation from the cell surface by binding ap-2 does not seem to be a major route for cd4 downregulation (rose et al., 2005) . which one of these strategies-downregulation from the cell surface or intracellular retention-or what combinations of these strategies and adaptor protein complexes are used may allow the hiv-1 nef protein to downregulate cell surface expression of cd4 perhaps in different cell types (mangasarian and trono, 1997) . vpu targets newly synthesized cd4 molecules in the er for proteasomal degradation (kerkau et al., 1997) by recruiting the cytosolic f-box protein b-trcp, the receptor component of the scf b-trcp e3 ligase, to the er membrane (margottin et al., 1998) . a motif in the vpu c-terminus binds a wd repeat on b-trcp, directing scf b-trcp e3 ligase activity to catalyze the ubiquitination of lysine residues on the cd4 tail, which serves as the trigger for cd4 degradation (margottin et al., 1998; schubert et al., 1998) . gp160 retains newly synthesized cd4 molecules in the er (crise and rose, 1992; kimura et al., 1994) . in the absence of cd4, hiv gp160 is posttranslationally cleaved into its gp120 and gp41 subunits at the level of the er-to-golgi compartment (ergic). in the presence of cd4, gp160 forms a complex with cd4 that mediates er retention of both molecules and downregulation of cd4 from the cell surface. in the context of an hiv-infected cell, coexpression of vpu liberates gp160 from the complex, ensuring gp160 translocation to the ergic and ensuing maturation, and simultaneously accelerating cd4 turnover (crise and rose, 1992; rose et al., 2005) . hiv downregulates class i mhc, class ii mhc, cd4, and cd1d, a class i mhc-like molecule that presents lipid antigens (le gall et al., 1998; piguet et al., 1999b) , from the cell surface by virtue of its nef, vpu, and gp160 proteins, reflecting its ability to manipulate various aspects of the immune response (joseph et al., 2005; piguet et al., 1999b) . furthermore, each of these mechanisms may be more far-reaching than downregulation of each individual receptor. for instance, by directly associating with the proton (h þ ) atpase, which is required for the acidification of lysosomes, nef not only downregulates cd4 but may also interfere with the ph of class ii mhc-positive compartments, affecting antigen processing by lysosomal proteases and class ii mhc antigen presentation. this strategy is used by other pathogens: the e5 protein from both human and bovine papillomavirus (andresson et al., 1995) and the vaca toxin secreted by helicobacter pylori (molinari et al., 1998) actively manipulate acidification in the endocytic pathway, resulting in impaired cd4 þ t cell-mediated responses (brodsky et al., 1999) . by binding to ap complexes or to the small gtpase arf1, nef may interfere with trafficking and alter the fate of numerous host molecules in the endocytic pathway (janvier et al., 2003a) , affecting aspects that range from viral replication to modulation of the immune system (lama and ware, 2000; sol-foulon et al., 2002; swigut et al., 2001) . intracellular pathogens exploit the ubiquitin-proteasome system mostly to destroy or avoid destruction of specific cellular proteins. this serves to create a more hospitable environment for themselves in the host cell, or to prevent destruction of their own proteins, and so to ensure replication or avoid detection by the immune system. many events at the initial stage of infection, entry into the cell, are controlled by signaling pathways-for instance, those involved in cytoskeletal rearrangements or in receptor engagement-that rely heavily on the ub-proteasome system. the same is true for signaling cascades involved in cell survival, differentiation, and proliferation. not surprisingly, pathogens have devoted much energy and coding capacity to interfere with cell signaling events through interference with the ub-proteasome system. an illustrative example is that of tumor viruses and degradation of cellular tumor suppressor proteins, like the retinoblastoma protein or p53, often resulting in malignant transformation (shackelford and pagano, 2005) . there have been a number of recent reviews on viral interference with signaling pathways, so we will not describe this aspect in detail here. for comprehensive reviews, the reader is referred to banks et al. (2003) and pagano (2004, 2005) . in this chapter, we will focus on a few examples of viral and bacterial interference with the ub-proteasome system that illustrate how the latter is crucial for aspects of pathogen life cycles that are as distinct as integration into host chromosomes, exit from the host cell, or rna interference, not to mention control of the immune response. as mentioned in the first section of this chapter, proteasome-mediated degradation of cytoplasmic proteins as well as the proteolytic events in the endolysosomal system are important for class i and class ii mhc presentation and cross-presentation. mounting of an immune response involves complicated signaling cascades like nf-kb and jak/stat signaling, which are heavily dependent on the ub-proteasome system, and that can be manipulated from early events of cell-surface receptor-mediated signaling, for example, to later events of ubiquitination and deubiquitination of downstream targets. certainly, because of the central role of the ub-proteasome system in many different aspects of cellular physiology, interference with this pathway will have pleiotropic effects, and which of the observed effects predominates may be difficult to discern. it is not our intention to make the following a comprehensive list. we rather propose to provide an overview of the possibilities of manipulation of this system that have been described for pathogens (fig. 9) . we discuss in more detail pathogen-encoded modulators of the ubiquitin-proteasome system, ranging from pathogen-encoded proteolysis-resistant peptides, to ubiquitin ligases and dub, with obvious implications for the viral or bacterial life cycle or with consequences for the control of host immune responses. we elaborate on novel pathogen-encoded dubs and their putative functions. the classic example of a viral protein that interferes with proteasomal processing is the ebna-1. the ebna-1 protein contains an internal repeat exclusively composed of glycines and alanines, the gly-ala repeat, which not only interferes with its own proteasomal proteolysis (levitskaya et al., 1995) , but also reduces its rate of translation, blocking viral drips formation and inhibiting the presentation figure 9 pathogen interference with the ub-proteasome system. pathogens interfere with the ub-proteasome system not only to manipulate antigen presentation and other aspects of the immune system, but for processes as distinct as chromosomal integration, virus budding, rna interference, and many others. they may rely on hijacking of host e3 ligase activities or encode their own. pathogens can manipulate host dubs as well as encode dub activities. the function of pathogen-encoded dubs remains a mistery. of ebna-1 class i mhc-restricted t cell epitopes (yin et al., 2003) . a single residue change in the mouse leukemia virus (mulv)-derived ctl epitope (from kspwfttl to rspwfttl) can eliminate the proteolytic cleavage site required for its presentation (ossendorp et al., 1996) . as discussed earlier, phosphorylation of the hcmv ie-1 by the hcmv pp65 tegument protein interferes with production of ie-1-derived peptides (gilbert et al., 1996) . the hiv-1 transcriptional activator (tat) protein manipulates 26s proteasome function by directly interacting with the lmp7 and mecl1 subunits of the proteasome and competing with the 11s proteasome for binding to the 20s proteasome (andre et al., 1998; apcher et al., 2003) , leading to inhibition of proteolytic activity. tat also acts at the transcriptional level by modifying proteasome composition by upregulating the lmp7 and mecl1 subunits and downregulating the lmp2 subunit, leading to an increased presentation of cryptic and subdominant ctl epitopes (gavioli et al., 2004) . by preventing display of viral peptides, these viral proteins interfere with cytotoxic t cell recognition. salmonella enterica serovar typhimurium is an important bacterial pathogen, the causative agent of food poisoning and typhoid fever. s. typhimurium temporally regulates the initial phase of bacterial internalization and host cell recovery after invasion through two type iii secretion system (ttss)-delivered substrates, sptp and sope with different proteasomal half lives (kubori and galan, 2003) . sope is a bacterial guanine nucleotide exchange factor (gef) delivered by a ttss that mimics a host cell gef for the small rho gtpases cdc42 and rac1, involved in actin remodeling and formation of membrane extensions required for bacterial engulfment. sptp, delivered by another ttss, is a rho gtpase activating protein (gap) factor for cdc42 and rac1, which accelerates gtp hydrolysis. spt plays a role once bacterial internalization has taken place, inactivating the rho gtpases, inhibiting actin polimerization, and assuring closure of the plasma membrane and recovery of the normal cellular architecture (pizarro-cerda and cossart, 2006) . salmonella initially delivers equal amounts of sope and sptb to the host cell. however, 15-20 min after infection sope is rapidly degraded, whereas sptb degradation occurs only slowly after 3 h, thereby efficiently timing the actin remodeling events that lead to the initial bacterial engulfment and the later host cell recovery. both processes are proteasome-dependent and catalyzed by the n-terminus of the bacterial proteins, but the mechanism and the host factors involved are currently unknown (kubori and galan, 2003) . the hiv-1 vif protein targets the rna editing protein apobec3g for degradation by a cellular e3 ligase to allow production of infectious viral progeny (yu et al., 2003) . the apolipoprotein b mrna editing enzyme, catalytic polypeptide-like 3g (apobec3g) and related cytidine deaminases are involved in mrna editing and in immunoglobulin gene class switching and hypermutation but are also potent antiretroviral enzymes. the cytosolic apo-bec3g is incorporated into budding virions, where on infection of new target cells, its cytidine deaminase activity induces g to a hypermutation on the minus-strand viral dna, resulting in abortive infection (bishop et al., 2004b; zhang et al., 2003) . vif hijacks the elongin-c-elongin-b-cullin-5-e3 (ecs) complex. the ecs5 e3s recognize substrate receptor proteins containing a bc-box. vif possesses a bc-box motif, through which it binds elongin c . vif hijacks the e3 ligase complex and by bridging apobec3g and elongin c targets apobec3g for ubiquitination and degradation (yu et al., 2003) . this ultimately allows production of infectious virus progeny (bieniasz, 2004; bishop et al., 2004a; harris and liddament, 2004) . the endosomal sorting complexes required for transport (escrt) are highly conserved from yeast to mammals and consist of the escrt i, ii, and iii complexes. escrt complexes are composed of vacuolar protein sorting (vps) proteins (in yeast), which are recruited from the cytoplasm to promote sorting of ubiquitinated proteins to mvbs (katzmann et al., 2002) . mvbs are formed by invagination of the late endosome membrane, which generates internal vesicles into which proteins destined to the lysosomes are sorted; they are critical for receptor downregulation and other normal and pathological cell processes (hierro et al., 2004; hurley and emr, 2006; kostelansky et al., 2006) , as well as for virus budding. the tumor susceptibility gene tsg101, the mammalian homologue of yeast vps23, is essential for sorting of ubiquitinated proteins to mvbs (babst et al., 2000) . tsg101 recruits hepatocyte growth factor-regulated tyrosine kinase substrate (hrs), the mammalian homologue of yeast vps27, to the endosomal membrane. hrs nucleates recruitment of the escrt-1 complex, which in turn recruits escrt 2 and -3, leading to formation of the inner membranes of the mvb. tsg101 is a noncanonical ub e2 variant (uev) protein-it does not possess ub-conjugating activity, only an n-terminal uev domain that binds ubiquitin. binding of the tsg101 uev domain to a p(s/t)ap tetrapeptide motif on hrs recruits hrs to the endosomal membrane, triggering assembly of escrt complexes and genesis of the mvb (clague and urbe, 2003; garrus et al., 2001; pornillos et al., 2002 pornillos et al., , 2003 . many viruses such as hiv-1 and ebola recruit this ub-dependent sorting machinery to the viral release sites at the plasma membrane to promote virus budding from host cells ( li and wild , 2005; liu, 2004 ) , by makin g use of con served p(s/t)ap, ppxy, or fpiv motifs, also called late budding domains ( bieniasz, 2006 ) . retroviruse s like hiv use the p(s/t) ap motif on the ldomain contained in their gag protei n to mimic the tsg10 1-rec ruiting activ ity of the hrs protein ( klinger an d schube rt, 2005; martin -serra no et al., 2003; sor in and kalpana , 2006; stuchell et al., 2004 ) . oth er viruses can also rec ruit neuro nal precurs or cellexpresse d develop mentally dow nregula t ed 4 (nedd 4) and nedd -4-like hect e3 ligases to facilitate viral bud ding ( bieniasz, 2006; harty et al., 20 01; sakura i et al., 20 04; yasuda et al ., 2003 ) . ne dd4 e3s ligase s are a dive rsified group of orthologu es of yeast rsp5p and are in volved in a wid e range of proce sses such as recepto r inter nalizati on and degrad ation (presum ably through the endoly sosomal pa thway), mainten ance of ebv latency, and regulation of cytokine sig naling. ned d4 e3s have a catal ytic cterminal hec t domain , two or more centr al nterminal ww dom ains, an d an n-term inal c2 dom ain. the nterminal c2 domain seems to be responsi ble for me mbrane associ ation and cellu lar localizatio n of the pro tein. the ww dom ain is a protei n-protei n interactio n modul e o f abou t 35 amino acids with two crucia l tryptop han (w) residues spa ced 20-22 amin o acids apart, which appear s to medi ate substr ate selection. it binds mostly pro linerich motifs, such as the ppxy motif present in many cellular proteins , targeting them for degradati on ( ingham et al., 2004 ( ingham et al., , 2005 . viral pro teins with the ppxy mo tif can therefore bind these ww dom ains on nedd 4 e3s, but the na ture of these inter actions and how they facilitate virus budd ing is so far unknown . some viruse s, like ebola, have two different late buddin g motifs, ppxy and p(s/t) ap, and the ebola late domain -con taining vp40 matrix protein can recruit bot h tsg 101 and nedd4 for effec tive budding (lic ata et al., 2003; liu, 2004; yasuda et al ., 2003 ) . the nonen veloped adenov irus pos sesses a ppxy motif on its penton base protein, which is essential for virus internalization that can interact with several nedd4 hect e3 ligases (galinier et al., 2002) . whether or not this interaction or e3 ligase activity is required for adenovirus entry is currently unknown. agrobacterium tumefaciens exploits a host cell scf e3 ligase for integration of its t-dna by encoding an fbox pro tein (tzfira et al., 2004b) . agrob acterium is a common phytopathogenic bacterium that induces ''crown gall'' disease in plants by transfer and integration of a segment of its tumor-inducing (ti) plasmid dna into the plant genome. this process relies also on delivery of several virulence (vir) proteins into the host cell, such as the bacterial vire2 protein, which is thought to package and protect the transported t-dna molecule, and, together with t he host plant vip 1 pro tein, assist its nucl ear import (tzfira et al ., 2004 a) . howeve r, di sassembly of this vire2/t-dna/ vip1 complex mus t occur before in tegration and involves intranu clear pro teolysis of vire2 and vip 1 induced by the virf pro tein, an fboxdomain-co ntain ing protein. t he bacte rial virf f -box hi jacks the scf e3 plant homolog ue. the virf-contain ing scf virf then leads to degrad ation of vip1 and vire2, and integration of the agrobact erium t-dna (tzfira et al., 2004b) . we conside r it unlikely that this pos sibility has been exploited only by pl ant pathogens. the h ost innate response triggered by type i inter feron (ifn-a and -b) innate response is crucial in early immunity against viruses, bac teria, and some parasites, acting t o lim it pathogen i n fection li mi ting replic ation of t he pathogen a nd constraining cellular permissiveness to infection (smith et al., 2005) . type i interferon receptor signaling occurs through the jak/stat pathway and leads to transcription of several ifn-stimulated genes (isgs), of which the gene encoding the ub-like modifier isg15 is one of the most strongly induced (farrell et al., 1979) . isg15 and protein modification by isg15 (isgylation) are induced by viral and bacterial infection or other stresses, suggesting important roles for the isg15 system in innate immune responses ritchie and zhang, 2004) . isg15 is conjugated onto several signaling molecules with immunomodulatory functions, like jak/stat proteins (giannakopoulos et al., 2005; malakhov et al., 2003) . the isgylation cascade is initiated by isg15 activation by an e1-like enzyme, ube1l, transfer to the isg-conjugating ubch8 enzyme , and incorporation into ubch8-compatible ub e3 ligases (dastur et al., 2006; zou and zhang, 2006) . a deisgylating enzyme, ubiquitin-binding protein 43 (ubp43), also called usp18, specifically removes isg15 from isgylated substrates , and is regulated by ubiquitination by the scf skp2 e3 (tokarz et al., 2004) . usp18 is unlikely to be the sole enzyme capable of acting on isg15 conjugates. the role of isg15 in orchestration of the innate antiviral response sets the ground for pathogen interference. although the mechanism is unclear, viral replication in ubp43 knockout mice is impaired, a phenotype that was initially attributed to inhibition of deisgylation and deregulation of stat signaling . there are conflicting views on this (dao and zhang, 2005; kim et al., 2006b; knobeloch et al., 2005) and ubp43 may in fact inhibit type i ifn signaling irrespectively of its isg15 isopeptidase activity, by binding to the ifn receptor and blocking the interaction between jak and the ifn receptor (malakhova et al., 2006) . ifn-mediated inhibition of hiv replication and budding is dependent on isg15 (kunzi and pitha, 1996; pitha, 1994; poli et al., 1989) . expression of isg15 inhibits ubiquitination of the hiv-1 gag protein and tsg101, and disrupts the interaction of the gag late budding domain with tsg101 (okumura et al., 2006) . either of these isg15 effects and/or additional mechanisms of action could lead to failure to recruit escrt complexes and inhibition of hiv budding. isg15 is strongly induced by infection with influenza b virus. the exact cellular function and targets of isg15 are not known, but the ns1 protein of influenza b viruses (ns1b) blocks its conjugation to target proteins: the ns1b n-terminus binds isg15, inhibiting activation of isg15 by its e1 enzyme, ube1l. influenza a viruses also manipulate cellular isgylation processes, through an even less well characterized mechanism: the influenza a virus ns1a protein does not directly bind the isg15 protein, but little or no isg15 protein is produced during infection (yuan and krug, 2001; yuan et al., 2002) . whether this reflects isgylation, deisgylation, and/or ubp43 ubiquitination-mediated control of the ifn innate immune response is so far unknown. suppression of nf-kb signaling is a common theme for many viral as well as bacterial pathogens and can be certainly achieved through modulation of ub-dependent events in the nf-kb cascade (bowie et al., 2004; hiscott et al., 2001; mason et al., 2004) . the human enteric flora may influence intestinal epithelium inflammatory tolerance by inhibiting the nf-kb pathway, which can be achieved by blocking any one of the many ub-dependent steps that control it. shigella flexneri, which causes severe diarrhea in humans, injects ttss-effector proteins into host cells to induce their entry into epithelial cells or trigger apoptosis in macrophages. the ospg effector is a serine/ threonine kinase that binds various e2s, including ubch5, a component of the scf b-trcp e3 complex. ospg binding to ubch5 inhibits the scf b-trcp complex and thereby phospho-ikb degradation, blocking nf-kb signaling in response to the bacterial infection. the cullin subunit of the scf b-trcp complex is itself regulated by nedd8 attachment (pan et al., 2004) . certain enteric bacteria can lead to rapid deneddylation of cullin-1 and consequent repression of the nf-kb pathway (collier-hyams et al., 2005) , but the bacterial activities responsible are still to be determined. in any case, because nf-kb is crucial for both the initial innate inflammatory response and for coordination of the adaptive immune response, dampening of inflammation may endow shigella and other enteric bacteria with the ability to invade and later colonize the gastrointestinal epithelium (kim et al., 2005) . rna interference (or posttranscriptional gene silencing) in plants and invertebrate animals is important for many regulatory processes and a primitive form of antiviral immunity that is nucleic acid based. consequently, plant viruses have evolved proteins, the so-called silencing suppressors, which directly bind to and inactivate the plant micrornas (ding et al., 2004; zamore, 2004) . poleroviruses are small positive-strand rna viruses that cause leafroll phenotypes in many plant species. poleroviruses encode a silencing suppressor p0, which is a viral f-box protein (barry and fruh, 2006) . p0 has a minimal conserved f-box motif that can bind the plant skp-1 homologue and form a functional complex with the plant cullin-1 homologue. the p0 f-box is required for polerovirus infectivity and its silencing suppressor function (pazhouhandeh et al., 2006) . p0 presumably binds the micrornas and targets them for cullin-4a-dependent degradation. this constitutes a remarkable finding, as degradation of rna (and not protein) by an e3 ligase had not been documented before. this primitive form of antiviral immunity, although widely used as a laboratory tool was only recently shown to occur in the context of a natural viral infection in jawed vertebrates (browne et al., 2005) . hiv-1 encodes viral small interfering rna (sirna) precursors that provoke rna silencing in human cells, so not surprisingly, the hiv-1 tat protein also contains a silencing suppressor function (bennasser et al., 2005) , that prevents dicer from processing the precursor double-stranded rnas into sirnas. suppression of rna silencing by other rna viruses is likely to occur. it will be interesting to see whether the mechanism used by the plant poleroviruses is conserved and whether manipulation of the ub-proteasome system for suppressing rna interference is more widely used by mammalian rna viruses. as mentioned earlier, several g-herpesviruses and poxviruses encode phd/ lap e3s, the k3 homologues, that include the kaposi's-sarcoma-associated herpesvirus (kshv) kk3 and kk5 (also called modulator of immune recognition mir-1 and mir-2, respectively), the mhv-68 mk3, and the rabbit myxoma virus m153r (coscoy and ganem, 2003) . we discussed mk3 which catalyzes ubiquitination and proteasomal degradation of nascent murine class i mhc hcs after dislocation from the er membrane wang et al., 2005) . kshv kk3 and kk5 and myxoma virus m153r catalyze ubiquitination of cell surface class i mhc hc, thus providing the trigger for their internalization from the cell membrane, as well as for sorting through mvb formation to lysosomal degradation (duncan et al., 2006) . in addition to downregulation of class i mhc molecules, some of these k3 homologues target the lymphocyte costimulatory molecules cd86 (b7.2) and intercellular adhesion molecule icam-1, cd1d, and cd4 (coscoy and ganem, 2003; coscoy et al., 2001; lehner et al., 2005; mansouri et al., 2003) , as well as the fas/cd95 death receptor (collin et al., 2005; guerin et al., 2002) . by targeting not only class i mhc molecules, the viral k3 homologues are adding an extra layer of protection predicted to be important for viral escape after reactivation from latency (lehner et al., 2005) . the k3 homologues are most likely making use of old ideas: the mechanisms used by the viral ub ligases have probably been ''borrowed'' from their mammalian counterparts, the march proteins, and will certainly continue to yield insights into the ub-proteasome system. a bacterial e3 ligase was recently shown to inactivate another type of immune response in plants (janjusevic et al., 2006) . antipathogen responses in plants, although not as sophisticated as those afforded by the vertebrate immune system, are nevertheless quite efficient. one mechanism, immunity-induced programmed cell death (pcd), is a response that sacrifices a limited portion of the plant to limit spread of the infection. the pseudomonas syringae bacterium, which causes disease in tomato and arabidopsis, delivers its avrptob protein into plant cells through a type iii secretion system. avrptob can inhibit pcd in susceptible hosts, allowing pseudomonas to cause systemic infection and disease. the avrptob c-terminus encodes a u-box e3 ligase activity necessary for the pathogenic role of avrptob, since mutation of the putative e2-recruitment sites abolishes the anti-pcd and virulence activities of the avrptob protein (janjusevic et al., 2006) . determining the host targets of the pseudomonas e3 will be crucial in clarifying its mechanism of action and might even explain plant susceptibility to infection. such experiments may also assist in the identification of similar targets in mammalian species infected with comparable gram-negative microbes. furthermore, it is rather striking that another pathogen uses a mimic of a host e3 ligase to encode an immunomodulatory function. there are bound to be others. mumps virus and other paramyxoviridae family members hijack the cullin-4a-scf b-trcp e3 ligase to suppress ifn-as well as il-6-mediated signaling (ulane et al., 2003) , important for control of inflammation and apoptosis during inflammation (hodge et al., 2005) . ifn and il-6 signaling activates their cognate stat factors and transcription of genes involved in ifn and cytokine signaling. some of these paramyxoviruses use their v protein to bind ddb1, the cullin-4a-scf b-trcp e3 substrate adaptor that recruits stat proteins to the e3 complex, targeting stats for proteasome-mediated degradati on ( li et al., 2006b ; ulane and horva th, 2002; ulane et al., 2005 ) . many other viru ses targe t stat transcri ption factors for protea somal degrad ation (garcin et al., 2002; lin et al., 2005; ramaswamy et al., 2004; zimmermann et al., 2005) . recruitment of cullin e3s is likely to be a more widely used viral strategy of interfering with ifn and cytokine signaling. the interleukin-2 (il-2)-inducible deubiquitinating enzyme dub-2 is induced by il-2 stimulation and may regulate il-2 signaling. the il-2-inducible dub-2 is constitutively expressed in cells transformed by human t cell leukemia virus-1 (htlv-1). like other cytokines, il-2 is an important modulator of apoptosis in t cells that acts through the stat pathway. although the mechanism is not at all clear, dub-2 activity prolongs il-2-stimulated phosphorylation and transcriptional activity of stat5, inhibiting t cell apoptosis in the aftermath of the immune response (migone et al., 2001; shackelford and pagano, 2004) and possibly contributing to cell immortalization. cytokine-inducible dubs could interfere with cytokine signaling, thereby playing an active role in modulation of the immune response. the il-1-inducible dub-1 is specifically induced by interleukin 3 (il-3), gm-csf, and il-5, which suggests a role in responses mediated by these cytokines (d'andrea and pellman, 1998) and might constitute a target for pathogen modulation of the immune response through interference with the ub-proteasome system. although a review of this rapidly expanding field is beyond the scope of this chapter, even this simple example suffices to demonstrate how both ub addition and removal control immune physiology, and consequently are likely targets for interference by pathogens. bacteria of the yersinia genus are the causal agents of plague, septicemia, and gastrointestinal syndromes. enteropathogenic yersinia species are extracellular multiplying gram-negative bacteria that make use of type iii secretion systems to inject virulence factors into host cells. the yersinia yopj virulence factor encodes a protein reported to be a cysteine protease that can cleave ub and sumo. the yopj dub inhibits nf-kb and mitogen-activated protein kinase (mapk) pathways, a function ascribed previously to its somewhat promiscuous deubiquitination of critical cellular proteins, such as traf2, traf6, and ikb. the mapk/jun pathway is involved in transcriptional control of several cytokine genes. the yopj activity induces macrophage death and blocks their ability to activate these inflammatory pathways (orth, 2002; orth et al., 2000; zhou et al., 2005) . however, recent reports show that the true function of yopj is that of a serine/threonine acetyltransferase (mukherjee et al., 2006) . the way by which the functions assigned to yopj have changed as the field advances demonstrates the complexity of assigning a function to proteins that lack obvious mammalian counterparts. both salmonella and the plant pathogen xanthomonas secrete proteins with homology to yopj, but their putative dub activity or function in the host have not been assessed yet (gurlebeck et al., 2006; hardt and galan, 1997) . a novel viral usp or deubiquitinating enzyme, ul36 usp , was recently identified in herpes simplex virus-1 (hsv-1) by labeling with an ub-derived probe . the ul36 usp is located at the n-terminus of the ul36, the large tegument protein of hsv-1, an a-herpesvirus. despite the overall low sequence homology-at the exception of almost only the amino acid residues composing the catalytic triad-the ul36 usp activity is well conserved in all members of the herpesviridae family, as the homologous proteins in murine cytomegalovirus (a b-herpesvirus) and ebv (a g-herpesvirus) also exhibit dub activity in vitro (schlieker et al., 2005) . one of the two severe acute respiratory syndrome (sars) coronavirus proteases responsible for cleavage of the replicase polyprotein, the sars-cov papain-like protease plpro, is also a dub (barretto et al., 2005; lindner et al., 2005) , predicted to be structurally similar to human usp7 (hu et al., 2002) . the sars-cov plpro protease activity is involved in the processing of the viral polyprotein, thereby contributing to replication of the viral rna genome. the function of the sars virus deubiquitinating activity, which extends to isg15-removal activity (lindner et al., 2005) , is unknown at this point. the adenovirus proteinase (avp) and the human cytomegalovirus ul48 protein also encode dub activities, but the viral and/or cellular targets remain unidentified (balakirev et al., 2002; wang et al., 2006b) . chlamydia trachomatis, an obligate intracellular bacterium that causes a variety of diseases in humans has two genes, chladub1 and chladub2, whose products encode deubiquitinating and deneddylating activitie s (misa ghi et al., 2006) . unlike c. pneum oniae who se genome is devoid of chladub genes, c. trachomatis is able to block nf-kb signaling and thus the inflammatory response, as well as host cell apoptosis. both processes could be modulated by the c. trachomatis dubs. these unexpected dub activities encoded by pathogens suggest a novel strategy of modulation of host defense by manipulating the cellular ubiquitination machinery. although the experimental evidence is at best tenuous, one can speculate that many of these other pathogen-encoded dubs may be important for modulation of the ub-proteasome system during the pathogen life cycle and/or in the context of immune evasion. the fact that these dub activities are conserved suggests functional importance. they may be delivered to the host cell at the time of infection (for instance, by a bacterial type iii secretion system or by a viral tegument protein) or may be transcribed early during infection. the pathogen-encoded dub could interfere with the levels of ub-or ubl-conjugated proteins, thereby altering cellular processes ranging from signaling pathways, protein degradation, antigen presentation, vesicular trafficking, and many others. we have elaborated on several examples of how the host immune system can be manipulated by a pathogen-encoded e3 or pathogen hijacking of a host e3. similarly-and although the sample pool is rather small at the moment-there is no reason to believe that encoding their own dubs and/or hijacking of host dubs has not ''occurred'' to pathogens. an attractive possibility is that some of these activities are aimed at manipulating the host immune system. possible targets would include pathways involved in ''housekeeping'' processes like life and death of the cells of the immune system, and extend to, for example, interference with antigen presentation. these dub activities could manipulate membrane trafficking in ways that would prevent mhc molecules from reaching the cell surface, or prevent proteasomal degradation of pathogen-derived proteins. this initial window of deubiquitinating activity could help viruses escape detection. bacterial pathogens could benefit from targeting of transcription factors and cytokine signaling networks that are crucial for coordination of macrophage effector functions. other immune responses that could, in principle, be controlled by dubs are ifn and nf-nf-kb signaling, and the posttranscriptional gene silencing ''immune response'' at least in plants. the possibilities are endless. exploitation of the ub-proteasome system by pathogens to enhance their own survival is beginning to emerge as a central theme. the development of the appropriate genetic and biochemical tools will help place this 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viral modulators of cullin ring ubiquitin ligases: culling the host defense downregulation of major histocompatibility complex class i by human ubiquitin ligases related to viral immune evasion proteins cdc48-ufd1-npl4: stuck in the middle with ub hrd1p/ der3p is a membrane-anchored ubiquitin ligase required for er-associated degradation regulatory t cells evidence that hiv-1 encodes an sirna and a suppressor of rna silencing intrinsic immunity: a front-line defense against viral attack late budding domains and host proteins in enveloped virus release cytidine deamination of retroviral dna by diverse apobec proteins apobec-mediated editing of viral rna a glycosylated type i membrane protein becomes cytosolic when peptide: n-glycanase is compromised mhc class i ubiquitination by a viral phd/lap finger protein signals for sorting of transmembrane proteins to endosomes and lysosomes transcriptional regulation of the mhc class ii antigen presentation pathway viral appropriation of 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neddylation of cullin-1 the poxviral scrapin mv-lap requires a myxoma viral infection context to efficiently downregulate mhc-i molecules the co-chaperone chip regulates protein triage decisions mediated by heat-shock proteins kaposi's sarcoma-associated herpesvirus encodes two proteins that block cell surface display of mhc class i chains by enhancing their endocytosis phd domains and e3 ubiquitin ligases: viruses make the connection a novel class of herpesvirus-encoded membrane-bound e3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition a novel immunoglobulin superfamily receptor for cellular and viral mhc class i molecules a misassembled transmembrane domain of a polytopic protein associates with signal peptide peptidase assembly, transport, and function of mhc class ii molecules mechanisms of mhc class i-restricted antigen processing and cross-presentation human immunodeficiency virus type 1 glycoprotein precursor retains a cd4-p56lck complex in the endoplasmic reticulum immunological memory in humans deubiquitinating enzymes: a new class of biological regulators e3 ubiquitin ligases as regulators of membrane protein trafficking and degradation isg15: a ubiquitin-like enigma herc5, an interferon-induced hect e3 enzyme, is required for conjugation of isg15 in human cells binding of cdc48p to a ubiquitin-related ubx domain from novel yeast proteins involved in intracellular proteolysis and sporulation rna silencing: a conserved antiviral immunity of plants and animals p97 and close encounters of every kind: a brief review lysine-63-linked ubiquitination is required for endolysosomal degradation of class i molecules human cytomegalovirus glycoprotein ul16 causes intracellular sequestration of nkg2d ligands, protecting against natural killer cell cytotoxicity quality control in the endoplasmic reticulum rad23 and rpn10 serve as alternative ubiquitin receptors for the proteasome edem contributes to maintenance of protein folding efficiency and secretory capacity presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway nk cell activity during human cytomegalovirus infection is dominated by us2-11-mediated hla class i down-regulation the tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum cytomegalovirus mhc class i homologues and natural killer cells: an overview accumulation of an mrna and protein in interferon-treated ehrlich ascites tumour cells arf1 regulates nef-induced cd4 degradation anti-immunology: evasion of the host immune system by bacterial and viral pathogens ubiquitin as a central cellular regulator polyubiquitin serves as a recognition signal, rather than a ratcheting molecule, during retrotranslocation of proteins across the endoplasmic reticulum membrane gamma-secretase-mediated proteolysis in cell-surface-receptor signalling consensus analysis of signal peptide peptidase and homologous human aspartic proteases reveals opposite topology of catalytic domains compared with presenilins the human cytomegalovirus us10 gene product delays trafficking of major histocompatibility complex class i molecules membrane-specific, host-derived factors are required for us2-and us11-mediated degradation of major histocompatibility complex class i molecules ubiquitinylation of the cytosolic domain of a type i membrane protein is not required to initiate its dislocation from the endoplasmic reticulum type iii secretion machines: bacterial devices for protein delivery into host cells adenovirus protein involved in virus internalization recruits ubiquitin-protein ligases all four sendai virus c proteins bind stat1, but only the larger forms also induce its mono-ubiquitination and degradation tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding the hrd1p ligase complex forms a linchpin between er-lumenal substrate selection and cdc48p recruitment hiv-1 tat protein modulates the generation of cytotoxic t cell epitopes by modifying proteasome composition and enzymatic activity human cytomegalovirus us2 endoplasmic reticulum-lumenal domain dictates association with major histocompatibility complex class i in a locus-specific manner us2, a human cytomegalovirus-encoded type i membrane protein, contains a non-cleavable amino-terminal signal peptide subunit h of the v-atpase binds to the medium chain of adaptor protein complex 2 and connects nef to the endocytic machinery cdc48p interacts with ufd3p, a wd repeat protein required for ubiquitin-mediated proteolysis in saccharomyces cerevisiae proteomic identification of proteins conjugated to isg15 in mouse and human cells cytomegalovirus selectively blocks antigen processing and presentation of its immediate-early gene product c-mir, a human e3 ubiquitin ligase, is a functional homolog of herpesvirus proteins mir1 and mir2 and has similar activity the caenorhabditis elegans impas gene, imp-2, is essential for development and is functionally distinct from related presenilins structure of 20s proteasome from yeast at 2.4 a resolution a gated channel into the proteasome core particle the many roads to cross-presentation human cytomegalovirus immediate early glycoprotein us3 retains mhc class i molecules by transient association myxoma virus leukemia-associated protein is responsible for major histocompatibility complex class i and fas-cd95 down-regulation and defines scrapins, a new group of surface cellular receptor abductor proteins pathways for antigen cross presentation type iii effector proteins from the plant pathogen xanthomonas and their role in the interaction with the host plant ubiquitylation and cell signaling endoplasmic reticulum retention is a common defect associated with tyrosinase-negative albinism physical and functional interactions of the cytomegalovirus us6 glycoprotein with the transporter associated with antigen processing role of n-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control er-associated degradation in protein quality control and cellular regulation ubiquitin-mediated regulation of 3-hydroxy-3-methylglutaryl-coa reductase role of 26s proteasome and hrd genes in the degradation of 3-hydroxy-3-methylglutaryl-coa reductase, an integral endoplasmic reticulum membrane protein a secreted salmonella protein with homology to an avirulence determinant of plant pathogenic bacteria retroviral restriction by apobec proteins integral ubl domain proteins: a family of proteasome interacting proteins protein degradation: recognition of ubiquitinylated substrates transferring substrates to the 26s proteasome rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction teb4 is a c4hc3 ring finger-containing ubiquitin ligase of the endoplasmic reticulum u box proteins as a new family of ubiquitin-protein ligases generation, translocation, and presentation of mhc class irestricted peptides human cytomegalovirus us2 causes similar effects on both major histocompatibility complex class i and ii proteins in epithelial and glial cells inhibition of hla-dr assembly, transport, and loading by human cytomegalovirus glycoprotein us3: a novel mechanism for evading major histocompatibility complex class ii antigen presentation the ultimate nanoscale mincer: assembly, structure and active sites of the 20s proteasome core roles of n-linked glycans in the endoplasmic reticulum proteasomes: a complex story interference with antigen processing by viruses a viral er-resident glycoprotein inactivates the mhc-encoded peptide transporter immune evasion by cytomegalovirussurvival strategies of a highly adapted opportunist cytomegaloviral control of mhc class i function in the mouse viruses know it all: new insights into ifn networks the ubiquitin system for protein degradation the ubiquitin system the human cytomegalovirus gene product us6 inhibits atp binding by tap a new ticket for entry into budding vesicles-ubiquitin regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins structure of the escrt-ii endosomal trafficking complex strategies and mechanisms for host and pathogen survival in acute and persistent viral infections intracellular targeting of the proteasome a role for n-glycanase in the cytosolic turnover of glycoproteins endoplasmic reticulum-associated protein degradation-one model fits all? hostile takeovers: viral appropriation of the nf-kappab pathway attenuation of hla-dr expression by mononuclear phagocytes infected with mycobacterium tuberculosis is related to intracellular sequestration of immature class ii heterodimers sp-ring for sumo: new functions bloom for a ubiquitin-like protein the role of il-6 and stat3 in inflammation and cancer lysosomal cysteine proteases regulate antigen presentation multiubiquitylation by e4 enzymes: 'one size' doesn't fit all role of herp in the endoplasmic reticulum stress response the lysosomal cysteine proteases in mhc class ii antigen presentation crystal structure of a ubp-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde the alpha chain of the t cell antigen receptor is degraded in the cytosol the escrt complexes: structure and mechanism of a membrane-trafficking network mycobacterium avium infection of mouse macrophages inhibits ifn-gamma janus kinase-stat signaling and gene induction by downregulation of the ifn-gamma receptor parkin suppresses unfolded protein stressinduced cell death through its e3 ubiquitin-protein ligase activity chip is associated with parkin, a gene responsible for familial parkinson's disease, and enhances its ubiquitin ligase activity the nedd4 family of e3 ubiquitin ligases: functional diversity within a common modular architecture the epstein-barr virus protein, latent membrane protein 2a, co-opts tyrosine kinases used by the t cell receptor nod-lrr proteins: role in host-microbial interactions and inflammatory disease downregulation of major histocompatibility complex class i molecules by kaposi's sarcoma-associated herpesvirus k3 and k5 proteins htm1p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast a bacterial inhibitor of host programmed cell death defenses is an e3 ubiquitin ligase hiv-1 nef stabilizes the association of adaptor protein complexes with membranes recognition of dileucine-based sorting signals from hiv-1 nef and limp-ii by the ap-1 gamma-sigma1 and ap-3 delta-sigma3 hemicomplexes protein dislocation from the er requires polyubiquitination and the aaa-atpase cdc48 multiple proteolytic systems, including the proteasome, contribute to cftr processing ring finger specificity in scf-driven protein destruction systematic analysis and nomenclature of mammalian f-box proteins cd4 phosphorylation partially reverses nef down-regulation of cd4 hiv nefmediated cd4 down-regulation is adaptor protein complex 2 dependent ring finger proteins: mediators of ubiquitin ligase activity a proteolytic pathway that recognizes ubiquitin as a degradation signal multiple independent loci within the human cytomegalovirus unique short region downregulate expression of major histocompatibility complex class i heavy chains human cytomegalovirus us3 impairs transport and maturation of major histocompatibility complex class i heavy chains nef: ''necessary and enforcing factor'' in hiv infection phagocytosis: at the crossroads of innate and adaptive immunity the protein translocation channel binds proteasomes to the endoplasmic reticulum membrane phosphorylation meets ubiquitination: the control of nf-[kappa]b activity a complex between peptide: n-glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins a deubiquitinating enzyme encoded by hsv-1 belongs to a family of cysteine proteases that is conserved across the family herpesviridae receptor downregulation and multivesicular-body sorting nedd8 recruits e2-ubiquitin to scf e3 ligase modulation of specific surface receptors and activation sensitization in primary resting cd4þ t lymphocytes by the nef protein of hiv-1 the human immunodeficiency virus type 1 (hiv-1) vpu protein interferes with an early step in the biosynthesis of major histocompatibility complex (mhc) class i molecules human hrd1 is an e3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum the shigella flexneri effector ospg interferes with innate immune responses by targeting ubiquitinconjugating enzymes the png1-rad23 complex regulates glycoprotein turnover ube1l and protein isgylation are not essential for alpha/beta interferon signaling monovalent ligation of the b cell receptor induces receptor activation but fails to promote antigen presentation intracellular membrane traffic of human immunodeficiency virus type 1 envelope glycoproteins: vpu liberates golgi-targeted gp160 from cd4-dependent retention in the endoplasmic reticulum t cells and viral persistence: lessons from diverse infections the ubiquitin-proteasome system in hiv replication: potential targets for antiretroviral therapy the proteasome and mhc class i antigen processing proteasome and peptidase function in mhc-class-imediated antigen presentation reexamination of the role of ubiquitin-like modifier isg15 in the phenotype of ubp43-deficient mice der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast the substrate translocation channel of the proteasome herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress gamma-secretase: proteasome of the membrane? hepatitis c virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ros) production differential effects of inhibitors on the gammasecretase complex. mechanistic implications structural and functional organization of the escrt-i trafficking complex for whom the bell tolls: protein quality control of the endoplasmic reticulum and the ubiquitin-proteasome connection differential localization and identification of a critical aspartate suggest non-redundant proteolytic functions of the presenilin in homologues sppl2b and sppl3 membrane topology of the yeast endoplasmic reticulum-localized ubiquitin ligase doa10 and comparison with its human ortholog teb4 (march-vi) the components of the proteasome system and their role in mhc class i antigen processing temporal regulation of salmonella virulence effector function by proteasome-dependent protein degradation a new self: mhc-class-i-independent natural-killer-cell self-tolerance role of interferon-stimulated gene isg-15 in the interferonomega-mediated inhibition of human immunodeficiency virus replication molecular mechanism and structural aspects of transporter associated with antigen processing inhibition by the cytomegalovirus protein us6 human immunodeficiency virus type 1 nef mediates sustained membrane expression of tumor necrosis factor and the related cytokine light on activated t cells cell-surface expression of cd4 reduces hiv-1 infectivity by blocking env incorporation in a nef-and vpu-inhibitable manner exclusive ubiquitination and sumoylation on overlapping lysine residues mediate nf-kappab activation by the human t-cell leukemia virus tax oncoprotein nk cell recognition nef interacts with the mu subunit of clathrin adaptor complexes and reveals a cryptic sorting signal in mhc i molecules determinant for endoplasmic reticulum retention in the luminal domain of the human cytomegalovirus us3 glycoprotein the human cytomegalovirus us6 glycoprotein inhibits transporter associated with antigen processing-dependent peptide translocation downregulation of cell surface receptors by the k3 family of viral and cellular ubiquitin e3 ligases requirements for signal peptide peptidase-catalyzed intramembrane proteolysis on the mechanism of spp-catalysed intramembrane proteolysis; conformational control of peptide bond hydrolysis in the plane of the membrane intramembrane proteolysis of signal peptides: an essential step in the generation of hla-e epitopes mechanism of intramembrane proteolysis investigated with purified rhomboid proteases modulation of natural killer cell cytotoxicity in human cytomegalovirus infection: the role of endogenous class i major histocompatibility complex and a viral class i homolog inhibition of antigen processing by the internal repeat region of the epstein-barr virus nuclear antigen-1 hiv-1 assembly and budding as targets for drug discovery multiple modes of interaction of the deglycosylation enzyme, mouse peptide n-glycanase, with the proteasome the aaa atpase p97 links peptide n-glycanase to the endoplasmic reticulum-associated e3 ligase autocrine motility factor receptor epstein-barr virus uses hla class ii as a cofactor for infection of b lymphocytes the ulp1 sumo isopeptidase: distinct domains required for viability, nuclear envelope localization, and substrate specificity structure of ddb1 in complex with a paramyxovirus v protein: viral hijack of a propeller cluster in ubiquitin ligase overexpression of the tumor autocrine motility factor receptor gp78, a ubiquitin protein ligase, results in increased ubiquitinylation and decreased secretion of apolipoprotein b100 in hepg2 cells overlapping motifs (ptap and ppey) within the ebola virus vp40 protein function independently as late budding domains: involvement of host proteins tsg101 and vps-4 a membrane protein required for dislocation of misfolded proteins from the er multiprotein complexes that link dislocation, ubiquitination, and extraction of misfolded proteins from the endoplasmic reticulum membrane viral modulation of antigen presentation: manipulation of cellular targets in the er and beyond dislocation of a type i membrane protein requires interactions between membrane-spanning segments within the lipid bilayer hepatitis c virus expression suppresses interferon signaling by degrading stat1 the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme ubiquitin ligases and the immune response immunity by ubiquitylation: a reversible process of modification viral modulation of nk cell immunity er dislocation: cdc48p/p97 gets into the aaact signal peptide peptidase is required for dislocation from the endoplasmic reticulum primate lentiviral virion infectivity factors are substrate receptors that assemble with cullin 5-e3 ligase through a hcch motif to suppress apobec3g viral immune evasion molecules attack the er peptide-loading complex and exploit er-associated degradation pathways virus subversion of the mhc class i peptide-loading complex the hcmv gene products us11 and us2 differ in their ability to attack allelic forms of murine major histocompatibility complex (mhc) class i heavy chains high-throughput immunoblotting. ubiquitiin-like protein isg15 modifies key regulators of signal transduction ubp43 (usp18) specifically removes isg15 from conjugated proteins ubp43 is a novel regulator of interferon signaling independent of its isg15 isopeptidase activity negative factor from siv binds to the catalytic subunit of the v-atpase to internalize cd4 and to increase viral infectivity the multifaceted role of hiv nef nef-induced cd4 and major histocompatibility complex class i (mhc-i) down-regulation are governed by distinct determinants: n-terminal alpha helix and proline repeat of nef selectively regulate mhc-i trafficking the phd/lap-domain protein m153r of myxomavirus is a ubiquitin ligase that induces the rapid internalization and lysosomal destruction of cd4 a novel human wd protein, h-beta trcp vpu connects cd4 to the er degradation pathway through an f-box motif virus entry: open sesame role of escrt-i in retroviral budding intramembrane proteolysis and post-targeting functions of signal peptides intramembrane-cleaving aspartic proteases and disease: presenilins, signal peptide peptidase and their homologs signal peptide fragments of preprolactin and hiv-1 p-gp160 interact with calmodulin new lessons from old pathogens: what parasitic infections have taught us about the role of nuclear factor-kappab in the regulation of immunity intramembrane proteolysis promotes trafficking of hepatitis c virus core protein to lipid droplets cop and clathrin-coated vesicle budding: different pathways, common approaches the hsc70 co-chaperone chip targets immature cftr for proteasomal degradation a genomic screen identifies dsk2p and rad23p as essential components of er-associated degradation cd85/lir-1/ ilt2 and cd152 (cytotoxic t lymphocyte antigen 4) inhibitory molecules down-regulate the cytolytic activity of human cd4þ t-cell clones specific for mycobacterium tuberculosis erad: the long road to destruction inhibition of hepatitis c virus core protein expression in immortalized human hepatocytes induces cytochrome c-independent increase in apaf-1 and caspase-9 activation for cell death the deubiquitinating enzyme dub-2 prolongs cytokine-induced signal transducers and activators of transcription activation and suppresses apoptosis following cytokine withdrawal human cytomegalovirus inhibits major histocompatibility complex class ii expression by disruption of the jak/stat pathway regulatory t cells: friend or foe in immunity to infection? structural and functional analysis of human cytomegalovirus us3 protein using a small molecule inhibitor of peptide: n-glycanase to probe its role in glycoprotein turnover chlamydia trachomatis-derived deubiquitinating enzymes in mammalian cells during infection immune escape and exploitation strategies of cytomegaloviruses: impact on and imitation of the major histocompatibility system role of edem in the release of misfolded glycoproteins from the calnexin cycle selective inhibition of ii-dependent antigen presentation by helicobacter pylori toxin vaca yersinia yopj acetylates and inhibits kinase activation by blocking phosphorylation doa1 is a cdc48 adapter that possesses a novel ubiquitin binding domain dendritic cell maturation by innate lymphocytes: coordinated stimulation of innate and adaptive immunity chip is a chaperonedependent e3 ligase that ubiquitylates unfolded protein chip: a quality-control e3 ligase collaborating with molecular chaperones autocrine motility factor and its receptor: role in cell locomotion and metastasis a novel route for f-box protein mediated ubiquitination links chip to glycoprotein quality control ubx2 links the cdc48 complex to er-associated protein degradation herpes simplex virus type 1 targets the mhc class ii processing pathway for immune evasion a genomic and functional inventory of deubiquitinating enzymes derlin-2 and derlin-3 are regulated by the mammalian unfolded protein response and are required for er-associated degradation mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis c virus core protein innate antiviral response targets hiv-1 release by the induction of ubiquitin-like protein isg15 core protein of hepatitis c virus induces cardiomyopathy how calmodulin binds its targets: sequence independent recognition of amphiphilic alpha-helices viral evasion of natural killer cells a structural determinant of hcmv us2 dictates the down-regulation of class i mhc molecules function of the yersinia effector yop disruption of signaling by yersinia effector yopj, a ubiquitin-like protein protease a single residue exchange within a viral ctl epitope alters proteasome-mediated degradation resulting in lack of antigen presentation mechanisms of mhc class i-restricted antigen processing nedd8 on cullin: building an expressway to protein destruction presenilin attenuates receptor-mediated signaling and synaptic function human cytomegalovirus inhibits tapasin-dependent peptide loading and optimization of the mhc class i peptide cargo for immune evasion f-box-like domain in the polerovirus protein p0 is required for silencing suppressor function function and regulation of cullin-ring ubiquitin ligases mechanisms underlying ubiquitination back to the future with ubiquitin ubiquitin: structures, functions, mechanisms hla-e-restricted recognition of cytomegalovirus-derived peptides by human cd8þ cytolytic t lymphocytes mechanism of nef-induced cd4 endocytosis: nef connects cd4 with the mu chain of adaptor complexes nef-induced cd4 degradation: a diacidic-based motif in nef functions as a lysosomal targeting signal through the binding of beta-cop in endosomes the downregulation of cd4 and mhc-i by primate lentiviruses: a paradigm for the modulation of cell surface receptors sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation viral interference with antigen presentation to cd8þ t cells: lessons from cytomegalovirus multiple effects of interferon on the replication of human immunodeficiency virus type 1 bacterial adhesion and entry into host cells mutant analysis links the translocon and bip to retrograde protein transport for er degradation endoplasmic reticulum degradation of a mutated atp-binding cassette transporter pdr5 proceeds in a concerted action of sec61 and the proteasome viral strategies of immune evasion interferon-alpha but not azt suppresses hiv expression in chronically infected cell lines structure of the tsg101 uev domain in complex with the ptap motif of the hiv-1 p6 protein hiv gag mimics the tsg101-recruiting activity of the human hrs protein mutants affecting the structure of the cortical endoplasmic reticulum in saccharomyces cerevisiae translating innate immunity into immunological memory: implications for vaccine development viral evasion of nk-cell activation specific inhibition of type i interferon signal transduction by respiratory syncytial virus survival of the fitters immunology: protein surgery membrane and soluble substrates of the doa10 ubiquitin ligase are degraded by distinct pathways antigens and immunoevasins: opponents in cytomegalovirus immune surveillance human cytomegalovirus gene products us2 and us11 differ in their ability to attack major histocompatibility class i heavy chains in dendritic cells interference with t cell receptor-hla-dr interactions by epstein-barr virus gp42 results in reduced t helper cell recognition epstein-barr virus gp42 is posttranslationally modified to produce soluble gp42 that mediates hla class ii immune evasion the class i mhc homologue of human cytomegalovirus inhibits attack by natural killer cells the ubiquitin binding domain znf ubp recognizes the c-terminal diglycine motif of unanchored ubiquitin a series of ubiquitin binding factors connects cdc48/p97 to substrate multiubiquitylation and proteasomal targeting modulation of major histocompatibility complex antigen expression by viral infection isg15: the immunological kin of ubiquitin dysregulation of protein modification by isg15 results in brain cell injury proteasome function in antigen presentation: immunoproteasome complexes, peptide production, and interactions with viral proteins regulation of proteasome structure and function hla-e-restricted recognition of human cytomegalovirus by a subset of cytolytic t lymphocytes endoplasmic reticulum-associated degradation cdc48p is ubx-linked to er ubiquitin ligases complete differentiation between enkephalinase and angiotensin-converting enzyme inhibition by retro-thiorphan cd4 down-regulation by hiv-1 and simian immunodeficiency virus (siv) nef proteins involves both internalization and intracellular retention mechanisms inhibition of hiv-1 progeny virion release by cell-surface cd4 is relieved by expression of the viral nef protein the specificity of tcr/pmhc interaction functional division of substrate processing cofactors of the ubiquitin-selective cdc48 chaperone in vivo evidence of chip up-regulation attenuating tau aggregation endoplasmic reticulum stress-inducible protein, herp, enhances presenilin-mediated generation of amyloid beta-protein regulation of human t-cell leukemia virus type 1 (htlv-1) budding by ubiquitin ligase nedd4 calling in the troops: regulation of inflammatory cell trafficking through innate cytokine/chemokine networks cytokine and chemokine networks: pathways to antiviral defense functional organization of mir2, a novel viral regulator of selective endocytosis the cd85/lir-1/ilt2 inhibitory receptor is expressed by all human t lymphocytes and down-regulates their functions specific recognition of the viral protein ul18 by cd85j/lir-1/ilt2 on cd8þ t cells mediates the non-mhc-restricted lysis of human cytomegalovirus-infected cells rad23 links dna repair to the ubiquitin/proteasome pathway the hpv-16 e6 and e6-ap complex functions as a ubiquitin-protein ligase in the ubiquitination of p53 a deubiquitinating activity is conserved in the large tegument protein of the herpesviridae proteasome-associated proteins: regulation of a proteolytic machine cd4 glycoprotein degradation induced by human immunodeficiency virus type 1 vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway membrane-bound ubx2 recruits cdc48 to ubiquitin ligases and their substrates to ensure efficient er-associated protein degradation suppression of major histocompatibility complex class i and class ii gene expression in listeria monocytogenes-infected murine macrophages insights into scf ubiquitin ligases from the structure of the skp1-skp2 complex the ubiquitin-domain protein herp forms a complex with components of the endoplasmic reticulum associated degradation pathway a superfamily of protein tags: ubiquitin, sumo and related modifiers targeting of hepatitis c virus core protein to mitochondria through a novel c-terminal localization motif the 26s proteasome: a dynamic structure notch and presenilin: regulated intramembrane proteolysis links development and degeneration tumor viruses and cell signaling pathways: deubiquitination versus ubiquitination targeting of host-cell ubiquitin pathways by viruses xbp1, downstream of blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation the pathway of us11-dependent degradation of mhc class i heavy chains involves a ubiquitin-conjugated intermediate pias proteins and transcriptional regulation-more than just sumo e3 ligases? all the peptides that fit: the beginning, the middle, and the end of the mhc class i antigen-processing pathway priming of t cells by exogenous antigen cross-presented on mhc class i molecules glycoprotein b from strain 17 of herpes simplex virus type i contains an invariant chain homologous sequence that binds to mhc class ii molecules gamma-secretase, notch, abeta and alzheimer's disease: where do the presenilins fit in? receptor binding and membrane fusion in virus entry: the influenza hemagglutinin type i interferons and the innate immune response-more than just antiviral cytokines regulated sumoylation and ubiquitination of ddmek1 is required for proper chemotaxis deubiquitinating enzymes: their functions and substrate specificity hiv-1 nef-induced upregulation of dc-sign in dendritic cells promotes lymphocyte clustering and viral spread gp78, a membrane-anchored ubiquitin ligase, associates with insig-1 and couples sterol-regulated ubiquitination to degradation of hmg coa reductase dynamics of virus-host interplay in hiv-1 replication the extracellular domain of the epstein-barr virus bzlf2 protein binds the hla-dr beta chain and inhibits antigen presentation modulation of gamma interferon-induced major histocompatibility complex class ii gene expression by porphyromonas gingivalis membrane vesicles mhc class ii compartment subtypes: structure and function inhibition of mhc class irestricted antigen presentation by gamma 2-herpesviruses dendritic cells: immunological sentinels with a central role in health and disease interferon-gamma, the functional plasticity of the ubiquitin-proteasome system, and mhc class i antigen processing the human endosomal sorting complex required for transport (escrt-i) and its role in hiv-1 budding hiv-1 nef impairs mhc class ii antigen presentation and surface expression human immunodeficiency virus-1 nef expression induces intracellular accumulation of multivesicular bodies and major histocompatibility complex class ii complexes: potential role of phosphatidylinositol 3-kinase molecular determinants for subcellular localization of hepatitis c virus core protein png1, a yeast gene encoding a highly conserved peptide: n-glycanase a conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both er-associated and matalpha2 repressor degradation mechanism for down-regulation of cd28 by nef transmembrane inhibitors of p-glycoprotein, an abc transporter er-golgi traffic is a prerequisite for efficient er degradation the endoplasmic reticulum degradation pathway for mutant secretory proteins alpha1-antitrypsin z and s is distinct from that for an unassembled membrane protein cdc48 can distinguish between native and non-native proteins in the absence of cofactors the isg15 isopeptidase ubp43 is regulated by proteolysis via the scfskp2 ubiquitin ligase surface expression of hla-e, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpul40 downregulation of natural killer cell-activating ligand cd155 by human cytomegalovirus cytomegalovirus us2 destroys two components of the mhc class ii pathway, preventing recognition by cd4þ t cells viral subversion of the immune system agrobacterium t-dna integration: molecules and models involvement of targeted proteolysis in plant genetic transformation by agrobacterium paramyxoviruses sv5 and hpiv2 assemble stat protein ubiquitin ligase complexes from cellular components composition and assembly of stat-targeting ubiquitin ligase complexes: paramyxovirus v protein carboxyl terminus is an oligomerization domain stat3 ubiquitylation and degradation by mumps virus suppress cytokine and oncogene signaling cutting edge: the human cytomegalovirus ul40 gene product contains a ligand for hla-e and prevents nk cell-mediated lysis differential processing of class-i-restricted epitopes by the standard proteasome and the immunoproteasome regulated protein degradation misfolded proteins are sorted by a sequential checkpoint mechanism of er quality control distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding proteases involved in mhc class ii antigen presentation shaping the t cell repertoire signal sequences. the limits of variation major histocompatibility complex class i allele-specific cooperative and competitive interactions between immune evasion proteins of cytomegalovirus c-terminal pal motif of presenilin and presenilin homologues required for normal active site conformation highmolecular-weight protein (pul48) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable regulation of tyrosinase trafficking and processing by presenilins: partial loss of function by familial alzheimer's disease mutation model for the interaction of gammaherpesvirus 68 ring-ch finger protein mk3 with major histocompatibility complex class i and the peptide-loading complex requirements for the selective degradation of endoplasmic reticulum-resident major histocompatibility complex class i proteins by the viral immune evasion molecule mk3 the viral e3 ubiquitin ligase mk3 uses the derlin/p97 endoplasmic reticulum-associated degradation pathway to mediate down-regulation of major histocompatibility complex class i proteins degradation of cftr by the ubiquitinproteasome pathway identification of signal peptide peptidase, a presenilin-type aspartic protease proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate the human cytomegalovirus us11 gene product dislocates mhc class i heavy chains from the endoplasmic reticulum to the cytosol sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction proteins containing the uba domain are able to bind to multi-ubiquitin chains human cytomegalovirus encodes an mhc class i-like molecule (ul142) that functions to inhibit nk cell lysis degradative organelles containing mislocalized alpha-and beta-synuclein proliferate in presenilin-1 null neurons a family of mammalian f-box proteins p97, a protein coping with multiple identities neddylation and deneddylation regulate cul1 and cul3 protein accumulation intramembrane proteolysis by presenilin and presenilin-like proteases parkin phosphorylation and modulation of its e3 ubiquitin ligase activity novel aspects of degradation of t cell receptor subunits from the endoplasmic reticulum (er) in t cells: importance of oligosaccharide processing, ubiquitination, and proteasome-dependent removal from er membranes nedd4 regulates egress of ebola viruslike particles from host cells the aaa atpase cdc48/p97 and its partners transport proteins from the er into the cytosol function of the p97-ufd1-npl4 complex in retrotranslocation from the er to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains a membrane protein complex mediates retro-translocation from the er lumen into the cytosol inaugural article: recruitment of the p97 atpase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane viral interference with antigen presentation at the crossroads of cell biology and immunology: drips and other sources of peptide ligands for mhc class i molecules self-inhibition of synthesis and antigen presentation by epstein-barr virus-encoded ebna1 a novel role for n-glycans in the erad system e3 ubiquitin ligase that recognizes sugar chains fbs2 is a new member of the e3 ubiquitin ligase family that recognizes sugar chains induction of apobec3g ubiquitination and degradation by an hiv-1 vif-cul5-scf complex physical association of the k3 protein of gamma-2 herpesvirus 68 with major histocompatibility complex class i molecules with impaired peptide and beta(2)-microglobulin assembly influenza b virus ns1 protein inhibits conjugation of the interferon (ifn)-induced ubiquitin-like isg15 protein structural basis for ubiquitinlike isg 15 protein binding to the ns1 protein of influenza b virus: a protein-protein interaction function that is not shared by the corresponding n-terminal domain of the ns1 protein of influenza a virus plant rnai: how a viral silencing suppressor inactivates sirna the cytidine deaminase cem15 induces hypermutation in newly synthesized hiv-1 dna structure of the aaa atpase p97 the ubch8 ubiquitin e2 enzyme is also the e2 enzyme for isg15, an ifn-alpha/beta-induced ubiquitin-like protein structure of the cul1-rbx1-skp1-f boxskp2 scf ubiquitin ligase complex chlamydia inhibits interferon gamma-inducible major histocompatibility complex class ii expression by degradation of upstream stimulatory factor 1 yersinia virulence factor yopj acts as a deubiquitinase to inhibit nf-kappa b activation a cytomegaloviral protein reveals a dual role for stat2 in ifn-{gamma} signaling and antiviral responses nk cell regulation of t cell-mediated responses the interferon-inducible ubiquitin-protein isopeptide ligase (e3) efp also functions as an isg15 e3 ligase we thank howard c. hang for help with the text and the figures and apologize to all colleagues whose work we may have not included. this work was supported by nih grants to h.l.p. key: cord-306111-wn1gxhk9 authors: dommett, r. m.; klein, n; turner, m. w. title: mannose‐binding lectin in innate immunity: past, present and future date: 2006-09-01 journal: tissue antigens doi: 10.1111/j.1399-0039.2006.00649.x sha: doc_id: 306111 cord_uid: wn1gxhk9 the human collectin, mannose‐binding lectin (mbl), is an important protein of the humoral innate immune system. with multiple carbohydrate‐recognition domains, it is able to bind to sugar groups displayed on the surfaces of a wide range of microorganisms and thereby provide first‐line defence. importantly, it also activates the complement system through a distinctive third pathway, independent of both antibody and the c1 complex. three single point mutations in exon 1 of the expressed human mbl‐2 gene appear to impair the generation of functional oligomers. such deficiencies of functional protein are common in certain populations, e.g. in sub‐saharan africa, but virtually absent in others, e.g. indigenous australians. mbl disease association studies have been a fruitful area of research and implicate a role for mbl in infective, inflammatory and autoimmune disease processes. overall, there appears to be a genetic balance in which individuals generally benefit from high levels of the protein. however, in certain situations, reduced levels of circulating mbl may be beneficial to the host and this may explain the persistence of the deleterious gene polymorphisms in many population groups. it is now 60 years since the australian nobel prize winner sir frank macfarlane burnet, together with john mccrea, identified three inhibitors in serum (called a, b and g), which were able to inactivate influenza virus (1) . we now know that the b inhibitor was, in fact, a protein called mannosebinding lectin (mbl), a component of the innate immune system (2) . during the past 30 years, our understanding of this protein has steadily increased as a result of extensive research activity in three main areas: (a) bio/immunochemistry (including molecular genetics), (b) microbiology and (c) immunodeficiency. work in these areas initially proceeded independently as evidence for both an inexplicable biological function and a clinical deficiency state emerged. the isolation and characterization of the protein were necessary in order to illuminate the observations of the so-called rarf bactericidal activity (3, 4) in the microbiology area and the opsonic deficiency reported in many paediatric populations. some of the main developments are summarized in table 1 . this review briefly addresses issues relating to the early history of mbl, its structure, function, genetics and disease associations. finally, future developments including the potential use of both plasma-derived and recombinant mbl are discussed. the existence of mammalian serum lectins was first predicted in 1975 by robinson et al. (5) , and the protein was first isolated in 1978 from cytosolic fractions of rabbit liver by kawasaki et al. (6) . subsequently, wild et al. (7) were able to isolate mbl from both human and rat liver. more recently, extrahepatic transcription of mbl has been reported and this may have implications regarding its role in localized host defence (8) . mbl belongs to a family of proteins called the collectins, which possess both collagenous regions and lectin domains. the other major human collectins, surfactant protein a and surfactant protein d, possess structural characteristics similar to those of mbl and are found predominantly in the lung and other mucosal sites (9) . plasma-associated phagocytic defect (28) 1975 existence of mammalian serum ôlectin-like proteins specific for mannoseõ predicted (5) 1976 association of opsonic defect with frequent infections in infancy, but deficiency also present in 5% of the general population (29) 1978 mbl isolated from rabbit liver (6) 1980 opsonic deficiency in infants with chronic diarrhoea (30) 1981 association of yeast opsonization defect with suboptimal c3b deposition (31) 1982 description of mouse rarf: a complement-activating bactericidal protein (3) 1983 human mbl isolated from liver (7); human serum mbl isolated (121) prospective study of opsonic deficiency in infancy (122) 1984 rarf activity present in vertebrate classes (4) 1985 bovine serum mbl described (123) opsonic defect linked to absence of an unidentified co-factor of the complement system (124) 1987 mbl activation of classical complement pathway (18) 1988 rat serum mbl a and c described (125) ; description of c-type crd (126) 1989 gene for human mbl cloned (33, 34) ; human mbl has bactericidal activity (127) ; opsonic nature of mbl demonstrated (35) mbl inhibits in vitro infection by hiv (85) correlation of opsonic defect with low serum mbl levels (32) 1990 bovine and mouse serum b inhibitors of influenza a virus identified as mbl (2) correlation of mbl levels with classical complement pathway activation at low serum concentrations (128) 1991 mouse mbl a and c described (129) opsonic deficiency and low mbl levels linked to single point mutation in codon 54 (variant b) (50) 1992 human mbl levels in acute-phase responses (59) ; crystallography of mbl crd (130) ; novel protease (masp-1) and complement activation by mbl (19) human rarf identical to mbl-masp (131) low mbl levels in africans linked to codon 57 (variant c) mutation in the mbl gene (51) 1994 third mbl mutation in codon 52 (variant d) described (52) 1995 polymorphisms found in promoter region of mbl gene (55) 1997 second masp found to activate complement (20) mbl mutations are an important risk factor for infections in children (132) 1998 reconstitution of opsonizing activity by infusion of purified mbl into mbl-deficient humans (112) 1999 truncated form of masp-2 -map19 (21) 2000 complement-activating complex of ficolins and masp (133) mbl shown to bind to clinically relevant organisms (15) structural aspects of mbl the protein structure of mbl has been studied extensively, and aspects are presented in figures 1 and 2 . the protein consists of multimers of an identical polypeptide chain of 32 kda. each chain comprises four distinct regions encoded by different exons of the mbl-2 gene, as will be discussed in more detail later. each chain has a c-terminal, calcium-dependent carbohydrate-recognition domain (crd); a short, a-helical, hydrophobic neck region (in the so-called coiled-coil configuration); a collagenous region containing 19 gly-xaa-xaa triplets and a cysteine-rich n-terminal region. three polypeptide chains form a triple helix within the collagenous region, stabilized by hydrophobic interactions and interchain disulphide bonds within the n-terminal cysteine-rich region. this is the basic building block of all circulating molecular forms of mbl. in serum, mbl consists of oligomers ranging from dimers to hexamers, and x-ray crystallographic studies/electron micrographs have revealed that these oligomers have a sertiform or a bouquetlike structure due to an interruption in the collagenous region, giving rise to a kink/hinge. the ability of the protein to bind effectively to microorganisms and activate complement appears to depend on the presence of higher order oligomers (tetramers and above). work by drickamer and colleagues (10, 11) and also by ezekowitz and colleagues (12) has provided an insight into the structure of the crd. each crd binds a calcium ion, enabling it to form co-ordination bonds with the 3-and 4-hydroxyl groups of specific sugars including mannose, n-acetyl-d-glucosamine, n-acetyl-mannosamine, fucose and glucose. the three crds in each structural subunit are separated by a constant 45-å distance (12) . clustering of the structural subunits provides a flat platform, permitting binding of mbl to the arrays of repeating sugar groups on microbial surfaces. although the binding affinity of each individual crd-sugar interaction is relatively low at 10 23 m (13), the formation of higher order oligomers provides multiple crds, which are able to bind simultaneously with high avidity. mbl is a major pattern-recognition molecule of the innate immune system. it primarily recognizes specific sugar groups (as above) on the surface of microorganisms, enabling it to distinguish self from non-self. it can also bind to phospholipids, nucleic acids (14) and non-glycosylated proteins. mbl has been shown to bind promiscuously to a wide range of bacteria, viruses, fungi and protozoa and some selected examples are listed in table 2 . neth et al. used flow cytometry to demonstrate mbl binding to clinically relevant bacterial isolates from immunocompromised children and noted differences in binding within some species such that one isolate might show strong binding, whereas another was much weaker (15) . the role of specific structural features of microorganisms (e.g. the capsule), which permit or prevent binding to mbl, has been explored in several studies. the earliest work was probably by kawakami et al. on the socalled rarf complex (which was later identified as mbl) and its interaction with salmonella enterica serovar typhimurium (3). this suggested that the structure and composition of lipopolysaccharide play a crucial role in mbl binding and function. other mechanisms that enable microorganisms to avoid recognition and killing by mbl include lipooligosaccharide sialyation (16, 17) . despite much progress in this area, many puzzles remain to be addressed, mostly related to the exact disposition of sugars on microbial surfaces. our understanding of mbl function has grown rapidly over the past three decades. it is now recognized to have a role in processes as diverse as complement activation, promotion of complement-independent opsonophagocytosis, modulation of inflammation, recognition of altered self-structures and apoptotic cell clearance. a role for mbl in host defence was first proposed in 1987 when ikeda et al. observed that the protein was able to activate the classical pathway of complement (18) . however, it is now clear that mbl activates a novel third pathway of complement, often termed the mbl pathway, in an antibody-and c1-independent fashion as illustrated in figure 3 . this functional activity reflects the fact that mbl circulates in association with a group of mbl-associated serine proteases (the so-called masps). in 1992, matsushita and fujita demonstrated the presence of a novel complement enzyme in serum, which was thought to generate the c3 convertase (c4bc2a), associated with classical pathway activation (19) . however, this activity was later found to be mediated by masp-2 (20) , and the original enzyme is now known as masp-1 and may activate c3 directly. subsequently, a small separately synthesized fragment of masp-2 termed smap or map19 was identified (21, 22) and a third masp (masp-3) with no known function was also described (23) . current understanding suggests that on binding to microorganisms, autoactivation of masp-2 occurs, permitting cleavage of c4 and c2 to form a c3 convertase, which is indistinguishable in specificity from the convertases found in the other two activation pathways of complement (24) . it should be noted that the so-called mbl pathway is also activated by another family of proteins called ficolins. the ficolins are structurally similar to collectins, with collagenous domains linked to fibrinogen-like domains having sugar-binding properties. l-and h-ficolins are humoral factors synthesized by hepatocytes, although h-ficolin has also been observed in bronchial/alveolar fluid and in bile (25) . in contrast, m-ficolin is found on peripheral blood mononuclear cells, polymorphonuclear cells and type ii lung epithelial cells (26) . ficolins are also found in complexes with the masps and are considered to have different binding specificities compared with mbl (27) . in 1968, miller et al. reported a plasma-associated defect of phagocytosis in a child with severe recurrent infections, failure to thrive and diarrhoea (28) . in vitro work revealed a failure of the childõs plasma to opsonize heat-killed bakers yeast (saccharomyces cerevisiae). this defect was later detected in the sera of children with recurrent unexplained infections (29) and chronic diarrhoea of infancy (30), but, interestingly, studies in the general population also revealed a relatively high frequency of the defect (5%). in 1981, studies linked this opsonic deficiency to the complement figure 3 complement activation pathway. the lectin pathway of complement is activated by mbl and ficolins. on binding to appropriate targets, mbl-masp-2 complexes cleave c4 and c2 to form c3 convertase (c4bc2a). mbl-masp-1 complexes may activate c3 directly. ficolins also work in combination with the masps. the classical and alternative pathways also generate c3 convertase enzymes, which cleave c3. the lytic pathway (c5-c9) is common to all three routes of c3 cleavage. mbl, mannose-binding lectin; masp, mbl-associated serine proteases; masp-1, mbl-associated serine protease-2; masp-2, mblassociated serine protease-2. system by demonstrating that sera with the deficiency deposited less c3b on yeast surfaces (31) . however, it was not until 1989 that the common opsonic defect was found to be associated with low levels of the mannose-binding protein, which we now refer to as mbl (32) . in that same year, the gene for mbl was cloned (33, 34) (genetics of human mbl). in a study of mbl-coated salmonella montevideo, kuhlman et al. reported that mbl was able to interact directly with cell surface receptors and promote opsonophagocytosis (35) . subsequently, a number of putative mbl-binding proteins/receptors have been proposed including cc1qr/ calreticulin (36), c1qrp (37) and cr1 (38, 39) . however, it is unclear whether mbl is acting as a direct opsonin or is merely enhancing other complement pathways and/or antibody-mediated phagocytosis. the role of mbl as a modulator of inflammation appears to be complex and, accordingly, its mechanism of action remains unexplained. one possible explanation is that mbl is able to trigger proinflammatory cytokine release from monocytes (40, 41) . this concept was addressed in studies by jack et al. using neisseria meningitidis incubated with increasing concentrations of mbl before being added to mbl-deficient whole blood. release of tumour necrosis factor a, interleukin (il)-1b and il-6 from monocytes was enhanced at mbl concentrations below 4 mg/ml but suppressed at higher concentrations (42) . clinical studies in this area are discussed later. the role of mbl in the recognition of altered self and apoptosis a role for mbl in the clearance of apoptotic cells was first proposed by ogden et al. in 2001 (43) . mbl was found to bind directly to apoptotic cells that expose terminal sugars of cytoskeletal proteins, thereby permitting their recognition and directly facilitating their phagocytosis by macrophages. defects in the clearance of apoptotic cells have been implicated in the pathogenesis of certain autoimmune conditions, although the precise role of mbl, if any, remains elusive. for example, in 2005, stuart et al. reported that although mbl-deficient mice displayed defective apoptotic cell clearance, they did not develop autoimmune diseases (44) . in animal studies, mbl has been implicated in the pathophysiology of ischaemia reperfusion injury due to its ability to recognize altered self-structures. stahl and colleagues have proposed the lectin pathway as a mediator of this process in certain organs, and the absence of mbl/masp pathway activation appears to afford protection in these disease models (45, 46) . however, the relevance of these findings to human health needs to be established. changes in cell surface structures during oncogenic transformation appear to promote binding of mbl to cancer cells (47) where the protein can mediate cytotoxic effects including mbl-dependent cell mediated cytotoxicity (48, 49) . the relative importance of such mechanisms in tumour immunology is, at present, unknown. there are two human mbl genes, but mbl-1 is a pseudogene and only mbl-2 encodes a protein product. the functional mbl-2 gene is located on chromosome 10 (q11.2-q21) and comprises four exons as illustrated in figure 1 . exon 1 encodes the signal peptide, a cysteine-rich region and part of the glycine-rich collagenous region. exon 2 encodes the remainder of the collagenous region and exon 3 encodes an a-helical coiled-coil structure, which is known as the ôneckõ region. exon 4 encodes the crd, which adopts a globular configuration. the promoter region of the mbl gene contains a number of regulatory elements, which affect transcription of the protein. in 1991, the complete nucleotide sequence of all four exons of the human mbl-2 gene was determined by sumiya et al. in two british children with recurrent infections and low mbl levels (50) . in both individuals, a point mutation was observed in codon 54, changing the codon sequence from ggc to gac and substituting aspartic acid for glycine in the translated protein. familial studies confirmed that the defect was inherited in an autosomal dominant fashion. in 1992, lipscombe et al. identified a second exon 1 mutation in codon 57 (gly / glu), when studying a sub-saharan african population (51) , and in 1994, madsen et al. reported a mutation in codon 52 (arg / cys) (52) . these point mutations are now commonly referred to as variants b, c and d respectively, with variant a indicating the wild type. the b variant mutation occurs at a gene frequency of approximately 25% in eurasian populations. in contrast, the c variant is rare in eurasians but is commonly seen in sub-saharan african populations, with frequencies of 50%-60%. population studies suggest that the b variant mutation may have arisen between 50,000 and 20,000 years ago (53) since no structural gene mutations have been identified in studies of indigenous australian populations who arrived on the continent approximately 50,000 years ago, whereas the b variant mutation was probably introduced into both north and south america at the time of the last glaciation approximately 20,000 years ago. the effect of these exon 1 mutations on the protein product continues to be the focus of study. they are believed to impair oligomerization and lead to a functional deficiency. the b and c mutations result in the replacement of critical axial glycines in the triple helix by dicarboxylic acids, resulting in distortion of this important part of the protein (50) . in contrast, the d mutation results in the replacement of arginine with cysteine. this extra cysteine has been proposed to cause formation of adventitious disulphide bonds that hinder higher oligomer formation (54) . several polymorphisms have also been reported in the promoter region of the gene. studies by madsen et al. investigating the large interindividual variation in serum mbl levels revealed three polymorphisms, h/l, x/y and p/q at positions 2550, 2221 and 14 of the mbl gene (55, 56) . subsequently, four common haplotypes were identified, namely lxp, lyp, lyq and hyp. of these, hyp, which is associated with medium to high levels of mbl and lxp, which is associated with low levels of the protein, appear to be most important. these promoter haplotypes are in strong linkage disequilibrium with the exon 1 mutations, resulting in seven common extended haplotypes, namely hypa, lypa, lyqa, lxpa, hypd, lypb and lyqc. other rare haplotypes have also been described (57) . figure 4 illustrates the frequency of these various haplotypes in selected populations and highlights the degree of ethnic variation. the combination of structural gene and promoter polymorphisms results in a dramatic variation in mbl concentration in apparently healthy individuals of up to 1000-fold (caucasian: range <20-10,000 ng/ml). in addition, ezekowitz and colleagues presented evidence in 1988 that mbl was an acute-phase reactant (58) . in these investigations, rna was isolated from a ônormalõ liver taken as part of a staging biopsy for hodgkins disease and was compared with rna isolated from a fresh post-mortem liver of a victim with severe trauma. the authors found that mbl messenger rna transcripts were barely detectable in normal liver but that induction was seen in liver exposed to acute stress. subsequent studies have shown that mbl levels can increase between 1.5 and threefold during the acute phase, but this response is variable between individuals (59) . it should also be noted that even during an acutephase response, individuals heterozygous or homozygous for mbl mutations appear unable to achieve the protein levels of those possessing a wild-type genotype. approximately one-third of the caucasian population possess genotypes conferring low levels of mbl, with approximately 5% having very low levels. no absolute level of mbl deficiency has been defined. genotype and phenotype show a relatively strong correlation and studies often use just one measure to infer deficiency. however, there is ôadded valueõ in performing both measures and we would strongly advocate this approach whenever possible. mbl occurs in two distinct forms in rodents and rhesus monkeys (60), but only one form is found in humans and chickens. as discussed previously, there are two human mbl genes, which are most likely due to a gene duplication event (61) . however, mbl-1 is a pseudogene and the potential mechanisms responsible for silencing the mbl-1 (63). such substitutions were also found in other higher primates including chimpanzees and gorillas but not in more distant primates such as the rhesus monkey. the authors concluded that both the mbl-1 and the mbl-2 genes have been selectively silenced by the same molecular mechanisms, but skewed in time resulting in overall downregulation of mbl levels in the present human population. the high frequency of variant alleles observed in certain populations was initially puzzling since it suggests that functional mbl deficiency may well be advantageous. similarities have been proposed between the mbl genetic system and the role of the sickle cell gene in protection against malaria as occurs in carriers of the sickle cell haemoglobin allele (64) . the argument runs as follows: certain intracellular parasites use c3 opsonization and c3 receptors on monocytes/macrophages to enter their host. therefore, any reduction in complement-activating function of the host may reduce the probability of parasitization. in support of this notion is a study on patients with visceral leishmaniasis, which revealed that such patients are more likely to have high mbl levels than uninfected controls (65) . a small study of ethiopian patients with lepromatous or borderline lepromatous leprosy also found that their mbl levels were significantly higher than those of healthy blood donors (66) . an alternative explanation of the unexpectedly high frequency of low mbl phenotype individuals found in many tropical regions is that excessive complement activation can result in immunopathologically mediated host damage; therefore, any mechanism that reduces complement activation may be beneficial (51) . the identification of mbl deficiency as the cause of the so-called common opsonic defect has been followed by a plethora of disease association studies aimed at defining the precise role of this protein. a number of the early studies concentrated on paediatric populations and mbl was suggested to provide substitute ôantibodyõ-like activity during the ôwindow of vulnerabilityõ (approximately 6-24 months), when maternal immunoglobulin g (igg) antibody levels have waned but the infantõs own adaptive immune response is still immature (32) . nevertheless, studies in adults suggested that there might be a role for mbl throughout life (67) . notwithstanding these reports, the majority of individuals possessing a variant mbl allele apparently suffer no ill effects and remain essentially healthy. in a study that apparently confirms this, dahl et al. monitored 9245 adults in a danish caucasian population and found no evidence for significant differences in infectious disease or mortality in mbl-deficient individuals compared with controls (68) . similar findings were reported by tacx et al. in unselected adults admitted to hospital with infections (69) . nevertheless, these studies should not be regarded as proof that mbl levels have no clinical relevance. many groups have undertaken case-control studies, which do indeed suggest that mbl is an important immunological modulator. in some cases, there is evidence that the significance of mbl deficiency is more readily appreciated when there is another co-existing defect (70), as we first proposed in 1991 (71). space does not permit a comprehensive review of all the mbl clinical studies that have been undertaken to date, and the topics covered below have been selected in order to illustrate examples of possible roles for mbl in a variety of clinical situations. most studies have explored the role of mbl in relation to the acquisition of an infectious organism (susceptibility) and the nature of the associated clinical course (severity). in clinical practice, this distinction can be difficult. however, for the purposes of this review, we will highlight examples of infections in which mbl appears to have an influence on one or other of these two aspects of infectious diseases. hamvas et al. have recently shown a role for mbl in mycoplasma infection (72) . they studied cases of infection in patients with primary antibody deficiencies (pad) that are known to be particularly susceptible to such organisms and compared them with a control population. more than two-thirds of pad patients with mycoplasma infections were mbl deficient (in possession of an exon 1 variant allele) compared with one-third of the control group. in the same study, they were able to demonstrate binding of mbl to three strains of mycoplasma using flow cytometry and proposed a role for mbl in prevention of invasive disease. in 2003, severe acute respiratory syndrome (sars) emerged as a highly infectious disease caused by a novel coronavirus (sars-cov). it provided a new challenge to previously unexposed individuals predominantly in asia. specific antibodies to sars-cov could be detected 10 days after the onset of symptoms, making sufferers reliant on innate immune mechanisms during the early phase of infection. since the structure of the virus was rapidly established (73, 74) , it also became clear that this novel infectious agent was rich in the sugars known to be targeted by mbl and it was hypothesized that this lectin might well be involved in first-line defence against this infection. subsequent studies found significant differences in the distribution of mbl-deficient genotypes in patients with sars compared with those in controls (75, 76) . these studies suggested that mbl plays a role in susceptibility to the infection but does not influence subsequent severity. in their investigations, ip et al. were also able to demonstrate binding of mbl to the virus and its ability to inhibit infection (75) . (77). the b variant allele was found more commonly in patients with symptomatic hepatitis b cirrhosis and in those with spontaneous bacterial peritonitis. it was also noted that mbl levels were lower in this patient cohort with chronic infection. screening for mbl mutations in such patients was suggested in order to enable identification of those at increased risk of complications who may benefit from prophylactic antibiotic treatment. in 2005, chong et al. also reported that mbl genotypes correlating with low protein levels were associated with the occurrence of cirrhosis and also hepatocellular carcinoma in hepatitis b carriers (78) . they also demonstrated that mbl is able to bind hepatitis b surface antigen. in the same year, thio et al. published the results of a nested case-control study of 527 patients who had either naturally recovered from hepatitis b (n ¼ 338) or had persistent infection (n ¼ 189). they found that mbl genotypes correlating with high serum levels were associated with recovery from infection, whereas those correlating with lower levels were associated with persistence of the virus (79) . it should be noted that approximately half of the subjects were also infected with human immunodeficiency virus (hiv), but the authors concluded that this did not influence the results obtained. matsushita et al. investigated the influence of mbl mutations in hepatitis c infection and found that sufferers who were homozygous for b variant alleles were less likely to respond to interferon treatment (80) . further work would be warranted in order to define the role of mbl in the pathogenesis of hepatitis infection. secondary immunodeficiencies due to disease or treatment have provided interesting patient populations within which to study the role of mbl. one such group comprises those receiving chemotherapy for malignancy. these patients are rendered neutropenic by their treatment (or underlying disease process) and are subsequently at increased risk of infectious complications. in 2001, two studies were published reporting an effect of mbl deficiency in such patients. neth et al. studied 100 children and measured mbl levels and genotype. children in possession of mbl variant alleles spent twice as many days in hospital with febrile neutropenia during the first 6 months of their treatment compared with wild-type individuals (81) . in the other study, peterslund et al. followed 54 adults undergoing chemotherapy for various haematological malignancies and found that those who developed ôsignificantõ infections (bacteraemia, pneumonia or both) in the 3-week periods post-treatment had significantly lower levels of mbl compared with those without significant infections (82) . subsequent studies have shown differing results, but drawing comparisons between them is inherently difficult. these patients are a highly heterogeneous population, with different underlying disease processes, undergoing treatment regimens of differing intensity, resulting in various degrees of immunosuppression. in one contrasting study, bergmann et al. followed 80 adults undergoing therapy for acute myeloid leukaemia, which involves intense highly myelosuppressive treatment. they found no effect of mbl deficiency on frequency, severity or duration of fever and suggested that the nature of the treatment overwhelmed any potential influence of mbl (83) . further clinical studies in such patients are required in order to delineate the exact role of mbl. an mbl double-knockout mouse model has been used to explore the above clinical conundrum. in 2004, shi et al. demonstrated that mbl null mice were highly susceptible to intravenous inoculation with staphylococcus aureus, all dying within 48 h, compared with 55% survival of mbl wild-type mice. however, when the mice were inoculated via the intraperitoneal route and rendered neutropenic (using cyclophosphamide), neutropenic mbl null mice were found to have higher accumulations of bacteria in the blood and organs compared with neutropenic wild-type mice. by day 8 post-infection, the neutropenic wild-type mice had cleared their blood, but the neutropenic mbl null mice had persistent bacteraemia. the authors were able to reverse the phenotype by treating the mbl null mice with recombinant mbl (84) . to date, nearly 40 million humans have been infected with hiv. the clinical consequences of viral exposure are variable. some individuals can be repeatedly exposed to the virus but remain free from infection. others can be infected but remain free from clinical disease. while numerous viral and host factors will determine the fate of an individual exposed to hiv, there are data to indicate that mbl can influence both susceptibility and severity of hiv infection. the likely target for hiv binding is the heavily glycosylated glycoprotein, gp120. while mbl can be readily demonstrated to bind to purified gp120 (85) , the capacity of mbl to neutralize primary hiv isolates is less convincing. recent data indicate the mbl can opsonize hiv but does not induce neutralization at the levels at which it is normally present in serum. however, binding and opsonization of hiv by mbl may alter virus trafficking and viral antigen presentation during hiv infection. mbl may influence uptake by dendritic cells (dc), which express a cell surface lectin called ôdc-specific intracellular adhesion molecule 3-grabbing non-integrinõ (dc-sign). dc-sign has been shown to mediate a type of infection called ôtransõ-infection, where dc bind hiv and efficiently transfer the virus to t cells. preincubation of hiv strains with mbl prevents dc-sign-mediated trans-infection of t cells and indicates that at least in vitro, mbl may inhibit dc-sign-mediated uptake and spread of hiv (86) . whatever the mechanism of mbl interactions with hiv, a number of clinical studies have suggested that deficiency of mbl is a risk factor for acquiring hiv infection. mbl deficiency appears to increase the acquisition of hiv infection by between three-and eightfold (87) (88) (89) (90) . there is also an increased risk of vertical transmission from infected mothers to their offspring (91) . however, these findings have not been replicated in all populations, with some studies failing to demonstrate a role for mbl in hiv infection (92) (93) (94) . there is even less clarity with regard to the role of mbl in hiv disease progression. garred et al. (87) demonstrated that men with mbl variant alleles had a shorter survival time following the onset of acquired immune deficiency syndrome (aids) than did patients with wild-type mbl alleles. however, in a well-characterized cohort of homosexual men, variant mbl alleles had an insignificant effect on survival following the diagnosis of aids (95) . in this latter study, there appeared to be a protective effect of mbl variant alleles, with a delay in the development of aids from the time of hiv seroconversion. patients with mbl variant alleles had lower cd4 counts at the time of developing aids, indicating that mbl deficiency may influence the onset of aids for any given cd4 count. furthermore, mbl mutations appeared to protect against the development of kaposi sarcoma, a finding that was difficult to explain (95) . in another study, prohaszka et al. (90) found that mbl levels were lower in asymptomatic hiv-positive individuals compared with hiv-negative controls. however, the protective effect of mbl was lost in patients with an aids diagnosis; patients with high mbl levels had significantly lower numbers of cd4 cells. a possible explanation is that enhanced proinflammatory cytokine production in advanced hiv disease acts to increase mbl synthesis (96) , elevating levels in patients with late-stage disease. indeed, a recent study has shown in vitro that mbl can enhance proinflammatory cytokine production and viral replication (97) . in the light of studies indicating a role for mbl in inflammatory modulation, it is tempting to suggest that under some circumstances, mbl may act to promote inflammatory cell activation, thereby accelerating the rate of cd41 t-cell depletion. few studies have assessed the impact of mbl in the context of effective antiviral therapy. however, one study has attempted to relate mbl status and hiv-infected longterm non-progressors (ltnps) (98) . mbl levels were consistent with a wild-type genotype in the six ltnps studied. amoroso and colleagues had also suggested such an effect in a study showing that children with rapidly progressing disease were more likely to have mbl variant alleles (codon 54) than slower progressors (99) . cystic fibrosis provides an example of a clinical condition where mbl appears to be exerting its role as an infection susceptibility gene and inflammatory modulator. garred et al. were the first group to report that patients with mbl variant alleles have significantly impaired lung function and decreased life expectancy in comparison with wild-type individuals (100) . the effect of mbl deficiency on the severity of lung disease was most apparent in patients with chronic pseudomonas aeruginosa infection and it was also found that burkholderia cepacia infection was more common in patients with mbl deficiency. in 2004, davies et al. reported that an effect of mbl was only seen in adults homozygous for mbl mutations. these patients had significantly reduced lung function, more frequent hospital admissions and raised systemic inflammatory markers. however, there was no evidence of increased susceptibility to burkholderia cepacia and pseudomonas aeruginosa (101) . whether mbl has an effect on early colonization with burkholderia cepacia and pseudomonas aeruginosa or subsequent secondary viral infections or whether there is an (anti)inflammatory effect on subsequent lung damage remains unclear. clinical studies of critically ill patients requiring intensive care management have shown that individuals who are mbl deficient are more likely to develop the systemic inflammatory response syndrome (sirs) ( figure 5 ) and progress to septic shock and death (102, 103) , findings which may well relate to the proinflammatory cytokine response. it should also be noted that chronic inflammation is now increasingly accepted to be a risk factor for myocardial infarction (mi), and a recent study by saevarsdottir et al. has found that patients with high mbl levels have a decreased likelihood of suffering a mi -again suggesting a potential role for mbl in modulating the inflammatory response (104). as a component of the complement system with similarities to c1q, but also as a player in infectious and inflammatory processes, the structure and function of mbl have prompted studies exploring a possible role in autoimmune conditions. systemic lupus erythematosus (sle) has been the focus of a number of mbl genotyping studies, but the results have been somewhat inconsistent. nevertheless, a recent meta-analysis has reviewed studies in this area and found that mbl variant alleles are indeed sle risk factors (105) . as with infectious disease, there is some evidence that the risk of pathology increases if there is another co-existing immune defect. for example, in a cohort of spanish patients, the odds ratio for developing sle was 2.4 for individuals with mbl deficiency, but this increased to 3.2 when there was also a co-existing partial c4 deficiency (106) . studies in patients with sle have reported that mbl deficiency also influences their risk of developing certain complications, which include arterial thromboses (107) and respiratory tract infections (108, 109) . a role for mbl in the pathogenesis of rheumatoid arthritis has also been suggested. malhotra et al. reported that changes in igg glycosylation secondary to the underlying disease results in mbl-associated complement activation (110) . such complement activation then contributes to chronic inflammation of the synovial membrane. however, graudal et al. found that patients with lower mbl levels experienced earlier, more severe, symptoms and had more rapid joint destruction as visualized radiologically (111) . several recent research publications suggest the directions in which future work on this collectin and its associated molecules may proceed. these include therapeutic interventions, functional assays and the evaluation of the importance of mbl in disease. these are considered briefly below. therapeutic potential of mbl mbl replacement was first attempted (without any knowledge of the deficiency) when fresh frozen plasma was given to patients and found to correct the opsonic defect (28, 29) . since then, affinity-purified, plasma-derived mbl has been safely given to many patients, resulting in normalization of enzyme-linked immunosorbent assay detectable mbl and complement-mediated opsonic activity (112) . a phase 1 study showed the half-life of the protein to range between 18 and 115 h (113) . the development of recombinant mbl is also at the phase 1 trial stage and such developments provide exciting prospects for the future exploration of the therapeutic potential of mbl. exactly who would benefit from replacement therapy is under debate and the importance of targeting well-defined patient groups will be vital to its success. the discovery of other components of the lectin pathway including the ficolins and the masps indicates that this limb of the immune system is complex and extends beyond mbl and masp-2 alone. this knowledge enables us to question the impact of these molecules either in isolation or in combination. functional assessment of the lectin pathway may be a far more accurate and clinically relevant measurement than mbl level and/or genotype alone. a number of different assays have been reported, which assess activity at different stages of the functional pathway; therefore, the results must be interpreted accordingly (114, 115) . the impact of deficiencies of the various adjunctive components is also the subject of much current research. in 2003, stengaard-pedersen et al. reported the first identified case of masp-2 deficiency (116) . functional analysis of the ability of mbl to activate the lectin pathway, estimating c4b deposition on a mannan surface, was performed on a group of patients with suspected immunodeficiency. one patient was found to have deficient pathway activity despite having sufficient mbl. no masp-2 or map19 was found in the plasma, and genetic analysis indicated that the patient was homozygous for a point mutation in exon 3 of the gene (d105g). clinically, the patient suffered from recurrent infections and autoimmune symptoms. subsequently, the frequency of this mutation has been assessed in a small number of populations and values range from 1.3% to 6.3% (117) . as discussed previously, the contribution of masp-1 and masp-3 in the pathway remains unexplained. the role of ficolins is now beginning to be addressed in clinical studies. like mbl, no absolute levels of deficiency have yet been defined. atkinson et al. studied more than 300 children with recurrent respiratory tract infections and measured l-ficolin levels (118) . an association with mbl deficiency in the same patient cohort had already been reported (119) . in this study, low levels of l-ficolin were more common in patients than in controls and most common in patients with co-existing atopic disorders, suggesting a role for l-ficolin in protection from microorganisms complicating allergic disease. polymorphisms in the ficolins have been identified, although their clinical significance is as yet unknown. mbl is an ancient molecule, which has probably been subject to a large number of evolutionary pressures. the last 50,000 years of human evolution have been associated with major changes as hominids moved from an essentially nomadic lifestyle to increasingly crowded living arrangements in large settled communities. associated with these changes, the spectrum of common infectious diseases would also have changed. more recently, the introduction of antibiotics, the emergence of novel infections and increasing use of immunosuppressive therapies have provided new challenges to our innate host defence system. despite all these changing evolutionary pressures, mbl gene polymorphisms persist at high frequencies, suggesting that they offer potential advantages to the host. thus, there exists a balance in which certain individuals benefit from the expression of high levels of the protein, whereas others (living in differing environments, eg. the tropics) may benefit from reduced levels of circulating mbl ( figure 6 ). mbl status may also be either advantageous or disadvantageous when considered from the viewpoint of the severity of a particular illness. thus, it is known that those with higher levels of mbl are better able to modulate inflammation, probably through an effect on cytokine responses. in contrast, those deficient in mbl appear to be at risk of sepsis and sirs. for these reasons, we believe that analyses of the relevance of mbl (120) should be extended beyond its role in infectious disease and include clinical areas such as autoimmunity and inflammatory disorders. inhibitory and inactivating action of normal ferret sera against an influenza virus strain bovine and mouse serum beta inhibitors of influenza a viruses are mannose-binding lectins properties of a new complement-dependent bactericidal factor specific for ra chemotype salmonella in sera of conventional and germ-free mice a group of bactericidal factors conserved by vertebrates for more than 300 million years affinity chromatography of human liver alpha-d-mannosidase isolation and characterization of a mannan-binding protein from rabbit liver isolation of mannosebinding proteins from human and rat liver extra-hepatic transcription of the human mannose-binding lectin gene (mbl2) and the mbl-associated serine protease 1-3 genes collections and ficolins: humoral lectins of the innate immune defense structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by mad phasing trimeric structure of a c-type mannose-binding protein human mannose-binding protein carbohydrate recognition domain trimerizes through a triple alpha-helical coiled-coil binding of sugar ligands to ca(21)-dependent animal lectins analysis of mannose binding by site-directed mutagenesis and nmr nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins a and d and mannose-binding lectin mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition activation of complement by mannose-binding lectin on isogenic mutants of neisseria meningitidis serogroup b the lipopolysaccharide structures of salmonella enterica serovar typhimurium and neisseria gonorrhoeae determine the attachment of human mannose-binding lectin to intact organisms serum lectin with known structure activates complement through the classical pathway activation of the classical complement pathway by mannose-binding protein in association with a novel c1s-like serine protease a second serine protease associated with mannan-binding lectin that activates complement a truncated form of mannose-binding lectin-associated serine protease (masp)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene masp-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway crystal structure of the cub1-egf-cub2 region of mannose-binding protein associated serine protease-2 hakata antigen, a new member of the ficolin/opsonin p35 family, is a novel human lectin secreted into bronchus/alveolus and bile human m-ficolin is a secretory protein that activates the lectin complement pathway l-ficolin specifically binds to lipoteichoic acid, a cell wall constituent of gram-positive bacteria, and activates the lectin pathway of complement a familial plasma-associated defect of phagocytosis defective opsonization. a common immunity deficiency yeast opsonisation in children with chronic diarrhoeal states a study of c3b deposition on yeast surfaces by sera of known opsonic potential association of low levels of mannan-binding protein with a common defect of opsonisation exon structure reveals its evolutionary relationship to a human pulmonary surfactant gene and localization to chromosome 10 structure and evolutionary origin of the gene encoding a human serum mannose-binding protein the human mannose-binding protein functions as an opsonin human leukocyte c1q receptor binds other soluble proteins with collagen domains mannose binding protein (mbp) enhances mononuclear phagocyte function via a receptor that contains the 126,000 m(r) component of the c1q receptor complement receptor 1/cd35 is a receptor for mannan-binding lectin complement receptor type 1 (cr1, cd35) is a receptor for c1q activation of human monocytes by streptococcal rhamnose glucose polymers is mediated by cd14 antigen, and mannan binding protein inhibits tnf-alpha release induction of tnf-alpha in human peripheral blood mononuclear cells by the mannoprotein of cryptococcus neoformans involves human mannose binding protein mannose-binding lectin regulates the inflammatory response of human professional phagocytes to neisseria meningitidis serogroup b c1q and mannose binding lectin engagement of cell surface calreticulin and cd91 initiates macropinocytosis and uptake of apoptotic cells mannose-binding lectin-deficient mice display defective apoptotic cell clearance but no autoimmune phenotype gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent without involving c1q mannose-binding lectin is a regulator of inflammation that accompanies myocardial ischemia and reperfusion injury tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines antitumor activity of mannan-binding protein in vivo as revealed by a virus expression system: mannan-binding proteindependent cell-mediated cytotoxicity antitumor activity of mannan-binding protein molecular basis of opsonic defect in immunodeficient children high frequencies in african and non-african populations of independent mutations in the mannose binding protein gene a new frequent allele is the missing link in the structural polymorphism of the human mannan-binding protein restricted polymorphism of the mannose-binding lectin gene of indigenous australians molecular determinants of oligomer formation and complement fixation in mannose-binding proteins interplay between promoter and structural gene variants control basal serum level of mannan-binding protein different molecular events result in low protein levels of mannan-binding lectin in populations from southeast africa and south america a new strategy for mannosebinding lectin gene haplotyping a human mannose-binding protein is an acute-phase reactant that shares sequence homology with other vertebrate lectins the concentration of the c-type lectin, mannan-binding protein, in human plasma increases during an acute phase response characterization of two mannose-binding protein cdnas from rhesus monkey (macaca mulatta): structure and evolutionary implications characterization of murine mannose-binding protein genes mbl1 and mbl2 reveals features common to other collectin genes the human ortholog of rhesus mannose-binding protein-a gene is an expressed pseudogene that localizes to chromosome 10 the ôinvolutionõ of mannose-binding lectin protection afforded by sickle-cell trait against subtertian malareal infection mannan-binding lectin enhances susceptibility to visceral leishmaniasis dual role of mannan-binding protein in infections: another case of heterosis? mannose binding protein gene mutations associated with unusual and severe infections in adults a population-based study of morbidity and mortality in mannose-binding lectin deficiency mannan binding lectin in febrile adults: no correlation with microbial infection and complement activation mannan binding lectin deficiency and concomitant immunodefects the molecular basis of a common defect of opsonization role for mannose binding lectin in the prevention of mycoplasma infection characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus mannose-binding lectin in severe acute respiratory syndrome coronavirus infection association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection mannose binding lectin gene mutations are associated with progression of liver disease in chronic hepatitis b infection mannose-binding lectin in chronic hepatitis b virus infection mannose binding lectin genotypes influence recovery from hepatitis b virus infection hepatitis c virus infection and mutations of mannose-binding lectin gene mbl deficiency of mannose-binding lectin and burden of infection in children with malignancy: a prospective study association between deficiency of mannose-binding lectin and severe infections after chemotherapy low levels of mannose-binding lectin do not affect occurrence of severe infections or duration of fever in acute myeloid leukaemia during remission induction therapy mannose-binding lectin-deficient mice are susceptible to infection with staphylococcus aureus a human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus interaction of mannose-binding lectin with hiv type 1 is sufficient for virus opsonization but not neutralization susceptibility to hiv infection and progression of aids in relation to variant alleles of mannose-binding lectin mannan-binding lectin in the sub-saharan hiv and tuberculosis epidemics the level of the serum opsonin, mannan-binding protein in hiv-1 antibody-positive patients mannan-binding lectin serum concentrations in hiv-infected patients are influenced by the stage of disease polymorphisms in the mbl2 promoter correlated with risk of hiv-1 vertical transmission and aids progression absence of association between mannose-binding lectin gene polymorphisms and hiv-1 infection in a colombian population mannose-binding protein in hiv-seropositive patients does not contribute to disease progression or bacterial infections circulating levels of mannose binding protein in human immunodeficiency virus infection presence of the variant mannose-binding lectin alleles associated with slower progression to aids human mannose-binding protein gene is regulated by interleukins, dexamethasone and heat shock modulatory effect of mannose-binding lectin on cytokine responses: possible roles in hiv infection low mannose-binding lectin serum concentrations in hiv long-term nonprogressors? polymorphism at codon 54 of mannose-binding protein gene influences aids progression but not hiv infection in exposed children association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis impaired pulmonary status in cystic fibrosis adults with two mutated mbl-2 alleles association of mannose-binding lectin polymorphisms with sepsis and fatal outcome, in patients with systemic inflammatory response syndrome increased incidence and severity of the systemic inflammatory response syndrome in patients deficient in mannose-binding lectin mannan binding lectin as an adjunct to risk assessment for myocardial infarction in individuals with enhanced risk the mannose-binding lectin gene polymorphisms and systemic lupus erythematosus: two case-control studies and a meta-analysis a dysfunctional allele of the mannose binding protein gene associates with systemic lupus erythematosus in a spanish population mannose-binding lectin variant alleles and the risk of arterial thrombosis in systemic lupus erythematosus association of mannose-binding lectin gene variation with disease severity and infections in a population-based cohort of systemic lupus erythematosus patients association of mannose binding lectin (mbl) gene polymorphism and serum mbl concentration with characteristics and progression of systemic lupus erythematosus glycosylation changes of igg associated with rheumatoid arthritis can activate complement via the mannose-binding protein mannan binding lectin in rheumatoid arthritis. a longitudinal study reconstitution of opsonizing activity by infusion of mannan-binding lectin (mbl) to mbl-deficient humans human plasma-derived mannose-binding lectin: a phase i safety and pharmacokinetic study an assay for the mannan-binding lectin pathway of complement activation functional analysis of the classical, alternative, and mbl pathways of the complement system: standardization and validation of a simple elisa inherited deficiency of mannan-binding lectin-associated serine protease 2 deficiency of the mannan-binding lectin pathway of complement and poor outcome in cystic fibrosis: bacterial colonization may be decisive for a relationship l-ficolin in children with recurrent respiratory infections mannan-binding lectin insufficiency in children with recurrent infections of the respiratory system human mannose-binding lectin in immunity: friend, foe, or both? isolation and characterization of a mannan-binding protein from human serum a common congenital immunodeficiency predisposing to infection and atopy in infancy mannan-binding protein and conglutinin in bovine serum suboptimal c3b/c3bi deposition and defective yeast opsonization. i. evidence for the absence of essential co-factor activity isolation and characterization of two distinct mannan-binding proteins from rat serum two distinct classes of carbohydrate-recognition domains in animal lectins a serum lectin (mannan-binding protein) has complement-dependent bactericidal activity the level of mannan-binding protein regulates the binding of complement-derived opsonins to mannan and zymosan at low serum concentrations molecular characterization of the mouse mannose-binding proteins. the mannose-binding protein a but not c is an acute phase reactant structure of a c-type mannose-binding protein complexed with an oligosaccharide human mannose-binding protein is identical to a component of ra-reactive factor association of mutations in mannose binding protein gene with childhood infection in consecutive hospital series cutting edge: complementactivating complex of ficolin and mannose-binding lectin-associated serine protease mannose-binding lectin accelerates complement activation and increases serum killing of neisseria meningitidis serogroup c activation of the lectin complement pathway by h-ficolin (hakata antigen) differential recognition of obligate anaerobic bacteria by human mannose-binding lectin differential binding of mannose-binding lectin to respiratory pathogens in cystic fibrosis human mannose-binding protein inhibits infection of hela cells by chlamydia trachomatis binding of mannan-binding protein to various bacterial pathogens of meningitis interaction of human mannose-binding protein with mycobacterium avium interaction of mannose-binding lectin with primary isolates of human immunodeficiency virus type 1 high mannose glycans and sialic acid on gp120 regulate binding of mannose-binding lectin (mbl) to hiv type 1 mannose binding lectin (mbl) and hiv mannan-binding protein and bovine conglutinin mediate enhancement of herpes simplex virus type 2 infection in mice mannan-binding lectin modulates the response to hsv-2 infection mannose binding protein is involved in first-line host defence: evidence from transgenic mice binding of host collectins to the pathogenic yeast cryptococcus neoformans: human surfactant protein d acts as an agglutinin for acapsular yeast cells mannose-binding lectin is a component of innate mucosal defense against cryptosporidium parvum in aids recognition of plasmodium falciparum proteins by mannan-binding lectin, a component of the human innate immune system the major surface glycoprotein of trypanosoma cruzi amastigotes are ligands of the human serum mannose-binding protein novel masp2 variants detected among north african and sub-saharan individuals analysis of mannose-binding lectin 2 (mbl2) genotype and the serum protein levels in the korean population association of mannose-binding lectin gene haplotype lxpa and lypb with interferon-resistant hepatitis c virus infection in japanese patients key: cord-324667-wmhdw1qs authors: nishtala, krishnatej; pahuja, natasha; shetty, rohit; nuijts, rudy m. m. a.; ghosh, arkasubhra title: tear biomarkers for keratoconus date: 2016-08-04 journal: eye vis (lond) doi: 10.1186/s40662-016-0051-9 sha: doc_id: 324667 cord_uid: wmhdw1qs keratoconus is a progressive corneal thinning, ectatic condition, which affects vision. recent advances in corneal topography measurements has helped advance proper diagnosis of this condition and increased research and clinical interests in the disease etiopathogenesis. considerable progress has been achieved in understanding the progression of the disease and tear fluid has played a major role in the progress. this review discusses the importance of tear fluid as a source of biomarker for keratoconus and how advances in technology have helped map the complexity of tears and thereby molecular readouts of the disease. expanding knowledge of the tear proteome, lipidome and metabolome opened up new avenues to study keratoconus and to identify probable prognostic or diagnostic biomarkers for the disease. a multidimensional approach of analyzing tear fluid of patients layering on proteomics, lipidomics and metabolomics is necessary in effectively decoding keratoconus and thereby identifying targets for its treatment. keratoconus is a progressive, asymmetric corneal ectasia. it is characterized by the thinning and protrusion of the cornea leading to irregular astigmatism and myopia thereby affecting the visual performance [1] . the disease is clinically diagnosed by corneal thinning and steepening along with the presence of iron oxide hemosiderin deposits in corneal mid periphery (fleischer's ring). around 50 % of subjects exhibit vogt's striae, which are fine stress lines caused by stretching and corneal thinning [2] . although clinical signs are helpful for the diagnosis, keratoconus is typically confirmed by corneal topography or tomography [3] . although keratoconus is bilateral, in certain scenarios, the clinical and topographic signs are present in only one eye (unilateral keratoconus) wherein the other eye is then diagnosed as form fruste keratoconus [4, 5] . the onset of the disease occurs at puberty and progresses up to the third decade of life [6, 7] . hence, a severity grading has been proposed for keratoconus to differentiate a normal eye from a keratoconic eye based on the clinical signs and corneal topography indices [8, 9] . keratoconus has been reported to affect 1 in 500 to 1 in 2000 individuals and has a greater prevalence in asians indicating the role of ethnicity [5, [10] [11] [12] [13] [14] [15] [16] . both genetic and environmental factors contribute to the disease pathology [17] [18] [19] . although atopy, eye rubbing, ocular allergies, down's syndrome and tapetoretinal degeneration have been associated with keratoconus [13] , the etiology and pathogenesis still remains unclear. despite the association of various genetic loci in twin and familial studies, [19] the general consensus remains that the disease is polygenic and is dependent on ocular surface and tear molecular expression changes [20] . tear fluid has been an important source of information in understanding ocular physiology [21] . a large number of proteases and protease inhibitors have been identified in tears [22] . zhou et al., have identified over 1500 proteins in the tear fluid majorly involved in carbohydrate catabolism, proteolysis, protein transport besides immune response and regulation of apoptosis [23] . disease specific molecular signature from tear fluid analysis can help in understanding the etiology of the disease and to help in prognosis. in addition, tear fluid can serve as an optimal source of molecular targets for treating ocular disease conditions [24] . in this review, we attempt to discuss, in a concise manner, the molecular markers for keratoconus that have been recently discovered and the importance of tear film as a source of these biomarkers in understanding the etiopathology as well as in prognosis of the disease. tear film is essential in maintaining ocular homeostasis and monitoring ocular surface conditions [25] . the tear fluid secreted by the lacrimal glands is a complex matrix comprising of an inner mucin layer, middle aqueous layer and the outer lipid layer and is composed of lipids, proteins, peptides, proteases, protease inhibitors and metabolites. the lipid layer provides lubrication, stability to the tear film and plays a protective role by preventing the cornea from drying and shields against pathogens [22, 26, 27] . tear fluid helps in nourishing the corneal epithelium and anterior stroma by delivering nutrients and metabolic products [28] . constitution of the tear film influenced by disease specific changes in the molecular markers can be of diagnostic value. another facet of the tear fluid analysis is the lipidome. analysis of the tear fluid lipidome by rantamaki et al. shows polar lipids, and the majority of tear lipids being phospholipids; the composition of tear lipids differs distinctly from those of the meibomian gland [29] . a more recent and comprehensive analysis of the human tear fluid identified more than 600 lipid species belonging to 17 major lipid classes [30] . besides lipidomics, a metabolome analysis of tears from healthy subjects using mass spectrometry revealed 60 tear metabolites of different classes [31] . tear biomarker discovery tear fluid as a source of biomarkers proteins lipocalin (lcn), lysozyme (lyz), lactoferrin (ltf), serum albumin (albu), proline rich repeat proteins (prr) constitute the most abundant proteins in the tear fluid [32] . inflammatory proteins were observed in tears of patients on long term glaucoma medication [33] and tear fluid proteins were also considered as a means for screening diabetic retinopathy [34, 35] . tear proteins have been reported to be altered in patients with keratoconus with and without contact lens wear when compared to controls [36] . tear composition has been shown to be altered in various inflammatory diseases like dry eye syndrome (des) [37] , keratoconus [38] , primary open angle glaucoma [39] and grave's ophthalmopathy [21, 40] . tear fluid has been studied as a source of biomarkers in the diagnosis of systemic conditions such as breast cancer [41, 42] , diabetes [43, 44] , multiple sclerosis [45] , and severe acute respiratory syndrome (sars) [46] . these reports highlight the importance of tears not only for the study of ocular conditions but also for readouts of systemic conditions. similar to the tear proteome, the tear lipidome and metabolome can also undergo changes in keratoconus as observed in other ocular disease such as des where reduced levels of wax esters and saturated fatty acyl moieties in 93 des patients were observed [47] . besides lipids, the tear metabolome has also been studied to understand disease pathology. using nuclear magnetic resonance (nmr) spectroscopy, levels of about 50 metabolites were found to be varying in the tears of 55 des patients compared with healthy subjects [48] . advances in technologies such as mass spectrometry and nmr have helped in studying and understanding molecular changes in the tear proteome, lipidome and metabolome relating to an ocular disease condition. storage and maintenance of the collected tear fluid under proper temperature conditions is critical for successfully discovering a biomarker. tear fluid from patients can be non-invasively collected either using a glass capillary or schirmer's strip [49] . however, the schirmer's strip method of collecting tears is considered the most effective with minimum discomfort to the patient and is often part of the des ocular surface disease test that patients already undergo in the clinic. since tear composition is complex with various proteolytic enzymes, cytokines, and metabolites, the tear fluid should be stored at sub-zero temperatures i.e., −70 to −80°c. a reduction in total tear protein concentration was observed when stored at room temperature for 4-8 h [50] . genetic variation is one of the factors influencing the incidence of keratoconus [19] . genetic predisposition to keratoconus in familial and monozygotic twins has been well established [51, 52] . single nucleotide polymorphisms (snps) in hepatocyte growth factor rab 3 gtpase activating protein 1 (rab3gap1), interleukin 1β (il1b), cadherin 11 (cdh11), negative regulator of ubiquitin like protein 1 (nub1), collagen type xxvii a1 (col27a1) and lysyl oxidase (lox) were identified by genome wide association studies (gwas) as risk factors for keratoconus [53] . however, the molecular functions associated with these snps are not well understood. gene expression signatures of keratoconus have been reported and offer new insights into the disease process [20] . differential gene expression analysis in debrided corneal epithelia of patients with keratoconus showed dysregulations of lox and collagens -collagen i alpha 1 (colia1) and collagen iv alpha 1 (coliva1) and an elevated expression of matrix metalloproteinase 9 (mmp9) [54] . a positive correlation between the reduced collagen expression with clinical severity of keratoconus was observed indicating their role in structural deformity in keratoconic corneas [54] . these genes were also associated with elevated inflammatory cytokine il-6 in the corneal epithelium and in the tears of keratoconus patients. however, cyclosporine a (cya) treatment of tumor necrosis factor α (tnfα) stimulated corneal epithelial cells and inhibited the expression of il-6, tnfα and mmp9 [55] . cultured human keratocyte cells (hkcs) stimulated with three different isoforms of transforming growth factor β (tgfβ i-iii) showed deregulation of smad6 and smad7, indicating altered tgfβ signaling in the progression of keratoconus [56] . similarly a significant reduction in levels of alcohol dehydrogenase (class 1) betapolypeptide (adh1b) in cultured keratoconic corneal fibroblasts suggested its possible role in keratoconus [57] . reduced levels of beta actin, a protein essential for cell survival and growth, along with human antigen r (hur) was observed in keratoconic and normal corneas both at the transcriptional and translational level suggesting the deregulation of these molecules as a possible trigger for keratoconus [58] . similar reduction in gene expression of beta actin and alpha enolase was also observed in superficial keratoconus epithelial cells compared with normal corneas, suggesting the degradation of these proteins in keratoconus [59] . nielsen et al. performed a proteomic analysis using 2d-gel electrophoresis followed by mass spectrometry from keratoconus patient epithelia. compared to controls, the patient epithelium overexpressed gelsolin (gsn), alpha enolase (enoa), s100a4 and cytokeratin3 (krt3), which suggests a plausible role of these proteins in the pathogenesis of keratoconus [60] while possibly associating the role of structural proteins and enzymes in keratoconus. these molecules can act as prognostic or diagnostic biomarkers and may aid in treatment of the disease. a list of the molecular markers identified in cultured corneal cells and human corneas are summarized in table 1 . the genomic studies thus far have remained inconclusive in terms of a molecular explanation for the phenotypes observed and do not strongly correlate with the molecular expression data from patient corneas. however, the gene and protein expression data from the keratoconic cornea do show deregulation of important factors and pathways such as collagen synthesis, inflammation, extracellular matrix, structural genes, tgfβ pathway and wnt signaling, to name a few. however, in a clinic, it is difficult to obtain corneal tissue for diagnosis and molecular profiling. this makes profiling tears of patients an attractive prospect. keratoconus is associated with tissue degradation that involves extracellular matrix remodeling, collagen deficiency [54] and more recently, increased roles of proinflammatory cytokines, cell adhesion molecules and matrix metalloproteinases [61] . enzyme linked immunosorbent assay (elisa) analysis of capillary collected tears in 28 [61] , 30 [62] and 94 [63] patients with keratoconus in three different studies showed elevated levels of inflammatory markers il-6, tnfα and mmp9. thus, a variety of matrix metalloproteinases (mmp) -1, -3, -7, -13, interleukins (il) -4, -5, -6, -8, and tumor necrosis factor (tnf) -α and -β are elevated in keratoconus tears [64] . studies also demonstrate higher gelatinolytic and collagenolytic activities in keratoconus [65] . hence, patients with elevated mmp9 levels, when treated with 0.05 % cya for 6 months, showed improved corneal topography characterized by reduced disease progression, localized flattening and low tear mmp9 [55] . in another study where tears from 33 keratoconus patients were analyzed by elisa, secreted-frizzled related protein 1 (sfrp1), a protein associated with the wnt signaling pathway, was observed at lower levels compared to age-matched controls, indicating a novel signaling pathway alteration in keratoconus [66] (table 2) . tear cytokine analysis performed to determine the effect of collagen cross-linking on tear fluid composition showed alterations in the tear cytokine levels [67] . these discoveries have significant clinical importance in aiding the [68] . furthermore, pre-existing information based on different experimental methodologies enhances the reliability of pre-selected markers. the high specificity and sensitivity of target specific biochemical assays make them reliable and clinically relevant for detecting molecular changes both in a qualitative and quantitative manner. mass spectrometric analysis for protein biomarkers has a dual advantage of surveying the unknown species thereby discovering novel molecular changes from a repertoire of proteins, and quantifying the changes in a multiplexed manner [69] . using a nano-lc tandem mass spectrometry approach, tears of 44 keratoconus patients were compared with 20 healthy controls that identified the elevation of cytokeratins, matrix metalloproteinase 1 (mmp1) and mammoglobin b (sgb2a1) [70] . additionally, lipocalin (lcn), lysozyme c (lyz), immunoglobulin alpha & kappa (igka & igkc) and precursors to prolactin were deregulated in keratoconus [70] . tear analysis using a 2-de/ms approach, identified zinc-α2glycoprotein (azgp1), immunoglobulin kappa chain, and lactoferrin (ltf) to be reduced in keratoconus [71] . using complimentary proteomic approaches of 2de and lcms e (mass spectral data acquired in a data-independent acquisition mode, ms e ), acera et al. have shown a significant decrease in the levels of cystatins and a higher tear lipocalin-1 in patients with keratoconus [38] . moreover, balasubramanium et al. demonstrated reduction in total tear protein in keratoconus patients [65] . protein levels of gross cystic disease fluid protein-15/ prolactin-inducible protein (pip) and zinc-alpha-2glycoprotein have been found to be elevated in tears of 36 patients by proteomic analysis, suggesting their application as prognostic markers for keratoconus [72] ( table 2 ). one of the major challenges in tear proteomics is the wide dynamic range of tear proteins. as discussed earlier, certain proteins [32] due to their high abundance, are able to mask the identification of low abundant proteins. a comparison of the levels of abundant proteins in tears and serum showed tear fluid to be similar in its protein abundance to serum [23] . though fractionation by 2de helps in resolving the abundance and complexity, it is limited by other factors such as protein isoelectric point and hydrophobicity [73] . a gel-free approach overcomes such limitations and coupled with fractionation methods such as cation exchange chromatography, it aids in resolving sample abundance and complexity [23] . metabolic changes as a consequence of disease can also be diagnosed from tears. tear metabolome analysis by lcms among 45 subjects in 3 clinically defined groups (healthy, keratoconus patients with rgp lens and keratoconus with no correction) identified 296 different metabolites among which more than 40 metabolites associated with glycolysis, gluconeogenesis and the urea cycle showed significant changes in keratoconus tears [74] . thus, the changes in underlying molecular signaling and secretion pathways due to keratoconus could be disease specific, affecting numerous biological processes. metabolite analysis by targeted mass spectrometry performed on 2d and 3d cultured human corneal keratocytes (kcks), human corneal fibroblasts (hcfs) and hkcs detected about 150 metabolites, the majority of which are involved in the oxidative stress pathway, implying its importance in keratoconus [75] . tear proteins lyz, ltf, lcn were also reported to be downregulated in des. besides, tear cytokines il -4, -5 and -6, and tnfα, which were elevated in keratoconus, were also shown to have similar expressions in des [76, 77] , indicating certain similar molecular changes among the two disease conditions. hence, it is imperative to practice caution while considering tear molecular changes as biomarkers for a specific disease. albeit, recent studies have shown that matrix metalloproteinases mmp1 and 9, azgp1, sfrp1, igkc and cell adhesion molecules icam-1 and vcam-1 to be specifically regulated in keratoconus [36, 71] , indicating their probable exclusive role in keratoconus. in addition, the reduced [54] and levels of sfrp1 [66] are specific to the disease and highlight the molecular pathways that can be targeted for disease correction. since collagen cross-linking is the current surgical treatment available for keratoconus [78] , reduction in the natural collagen cross linkers [54] is perhaps expected. the molecular pathways leading to the elevation of collagenolytic factors [55] presents another attractive target for therapy. since these large sample studies [54, 63] indicate a broad range in the expression of such factors, it is imperative to tune future treatment modalities based on specific molecular targets to the correct pathways. this could be possible through tear-based biomarker profiling. it is now understood that inflammation is one of the primary drivers of keratoconus [55, [61] [62] [63] 71] , therefore, it is not surprising that some of the markers between keratoconus and des are similar. however, the amplitude of the deregulation of these markers such as il6 and mmp9, in correlation with disease phenotype can be distinct amongst the different disorders with inflammatory drivers. large sample cohort studies [54, 63] would further validate the role of these proteins as specific and potential biomarkers for keratoconus. therefore, accumulated information about these markers should be considered for application in diagnosis e.g., elevated mmp9 and il6 [55] along with a reduction in markers such as lox, sfrp1 [54, 66] in tears can give a specific diagnosis of keratoconus. many of these factors are deregulated at the corneal tissue (epithelium and stroma) level as well as the tears, further validating their importance as biological processes specific to this disease as established by different groups. with the advent of high throughput technologies such as multiplex cytokine bead array [77] and quantitative mass spectrometry by multiple reaction monitoring (mrm) assays, multiple candidate markers can be validated in a relatively shorter time and over a larger cohort of patients. zhou et al. have demonstrated quantitation of 47 tear proteins in a mrm assay [69] . multiplexed validation also establishes a unique molecular fingerprint of the disease and a more appropriate way of diagnosing and treating keratoconus. tear fluid as a source of biomarkers in ocular and systemic conditions has been well studied and is shown to have translational potential. moreover, the non-invasive way of collecting the tears makes it an optimal biological fluid to study with minimal to no discomfort to the patient. studies performed on tear fluid in patients of keratoconus provided insights into the pathology of the disease and has revealed probable prognostic as well as diagnostic biomarkers for the disease. more importantly, the recent studies and data from tear analysis establishes the definitive role of inflammation as a driver of corneal collagen loss and deformity in keratoconus patients. elevated cytokines move in tandem with elevated matrix degrading enzymes to reduce levels of collagens and collagen crosslinking enzymes resulting in the disease. we term this as "keratoconus inflammaxis", a unique molecular signaling fingerprint identified with this disease and its related pathology. the advances in technology such as high resolution high sensitivity mass spectrometry have enabled mapping of the tear fluid in patients and also the heterogeneity of keratoconus pathology. a multi-omics approach integrating data from proteomics, lipidomics and metabolomics is the need of the hour for studying tear fluid as an important source of biomarkers in keratoconus to lead to effective prognosis and treatment of the disease. the pathogenesis of keratoconus imaging modalities in keratoconus incidence and characteristics of unilateral keratoconus classified on corneal topography keratoconus and related noninflammatory corneal thinning disorders keratoconus in asian eyes at a tertiary eye care facility a new method for grading the severity of keratoconus: the keratoconus severity score (kss) correlation of corneal elevation with severity of keratoconus by means of anterior and posterior topographic analysis estimation of the incidence and factors predictive of corneal scarring in the collaborative longitudinal evaluation of keratoconus (clek) study baseline findings in the collaborative longitudinal evaluation of keratoconus (clek) study a 48-year clinical and epidemiologic study of keratoconus influence of ethnic origin on the incidence of keratoconus and associated atopic disease in asians and white patients incidence and prevalence of keratoconus in denmark prevalence and associations of keratoconus in rural maharashtra in central india: the central india eye and medical study does ethnic origin influence the incidence or severity of keratoconus? genetics of keratoconus: where do we stand? the genetics of keratoconus genetic and genomic perspective to understand the molecular pathogenesis of keratoconus proteomic and gene expression patterns of keratoconus unraveling the molecular repertoire of tears as a source of biomarkers: beyond ocular diseases identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors in-depth analysis of the human tear proteome tears as a source of biomarkers for ocular and systemic diseases complexity of the tear film: importance in homeostasis and dysfunction during disease tear analysis in ocular surface diseases innate defence of the eye by antimicrobial defensin peptides in vivo tear-film thickness determination and implications for tear-film stability human tear fluid lipidome: from composition to function extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles characterization of the human tear metabolome by lc-ms/ms investigation of the human tear film proteome using multiple proteomic approaches proteomic profiling of inflammatory signaling molecules in the tears of patients on chronic glaucoma medication tear fluid proteomics multimarkers for diabetic retinopathy screening combined methods for diabetic retinopathy screening, using retina photographs and tear fluid proteomics biomarkers tear proteomics in keratoconus the pathology of dry eye: the interaction between the ocular surface and lacrimal glands changes in tear protein profile in keratoconus disease level of tear endothelin-1 and plasminogen in patients with glaucoma and proliferative diabetic retinopathy keratoconus: an inflammatory disorder? eye (lond) diagnosis of breast cancer by tear proteomic pattern lacryglobin in human tears, a potential marker for cancer analysis of tear fluid proteins in insulin-dependent diabetes mellitus changes in the tear protein patterns of diabetic patients using two-dimensional electrophoresis exploring the human tear fluid: discovery of new biomarkers in multiple sclerosis the severe acute respiratory syndrome coronavirus in tears lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome a metabolomic approach to dry eye disorders. the role of oral supplements with antioxidants and omega 3 fatty acids schirmer strip vs. capillary tube method: non-invasive methods of obtaining proteins from tear fluid effect of storage on protein concentration of tear samples the genetics of keratoconus the genetics of keratoconus genomewide association studies and assessment of the risk of disease attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity elevated expression of matrix metalloproteinase-9 and inflammatory cytokines in keratoconus patients is inhibited by cyclosporine a keratoconus in vitro and the key players of the tgf-beta pathway marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts downregulation of β-actin gene and human antigen r in human keratoconus molecular changes in selected epithelial proteins in human keratoconus corneas compared to normal corneas proteome profiling of corneal epithelium and identification of marker proteins for keratoconus, a pilot study inflammatory molecules in the tears of patients with keratoconus subclinical keratoconus and inflammatory molecules from tears inflammatory response to contact lenses in patients with keratoconus compared with myopic subjects effects of eye rubbing on the levels of protease, protease activity and cytokines in tears: relevance in keratoconus proteases, proteolysis and inflammatory molecules in the tears of people with keratoconus tear levels of sfrp1 are significantly reduced in keratoconus patients alterations of tear mediators in patients with keratoconus after corneal crosslinking associate with corneal changes sensitivity and specificity of a point-of-care matrix metalloproteinase 9 immunoassay for diagnosing inflammation related to dry eye quantitation of 47 human tear proteins using high resolution multiple reaction monitoring (hr-mrm) based-mass spectrometry urinary matrix metalloproteinase-9 and interleukin-6 and renal manifestations of primary sjogren's syndrome proteomic analysis of the tear film in patients with keratoconus gross cystic disease fluid protein-15/prolactin-inducible protein as a biomarker for keratoconus disease power and limitations of electrophoretic separations in proteomics strategies tear metabolite changes in keratoconus in vitro model suggests oxidative stress involved in keratoconus disease seldi-tof-ms proteinchip array profiling of tears from patients with dry eye analysis of inflammatory cytokines in the tears of dry eye patients corneal collagen cross-linking for keratoconus we thank the narayana nethralaya foundation for funding to ag, kn and np.authors' contributions kn and np wrote the manuscript. rs and rmman helped conceptualize and advised on the manuscript. ag designed and wrote the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-314746-1o0rf0ii authors: bergasa-caceres, fernando; rabitz, herschel a. title: interdiction of protein folding for therapeutic drug development in sars cov-2 date: 2020-08-10 journal: j phys chem b doi: 10.1021/acs.jpcb.0c03716 sha: doc_id: 314746 cord_uid: 1o0rf0ii [image: see text] in this article, we predict the folding initiation events of the ribose phosphatase domain of protein nsp3 and the receptor binding domain of the spike protein from the severe acute respiratory syndrome (sars) coronavirus-2. the calculations employ the sequential collapse model and the crystal structures to identify the segments involved in the initial contact formation events of both viral proteins. the initial contact locations may provide good targets for therapeutic drug development. the proposed strategy is based on a drug binding to the contact location, thereby aiming to prevent protein folding. peptides are suggested as a natural choice for such protein folding interdiction drugs. understanding the biochemistry of the new coronavirus sars-cov-2 has become an issue of prime importance and urgency as the virus spread has triggered an ongoing pandemic that has already cost thousands of lives and large economic disruption. 1−3 many therapeutic strategies are being considered including monoclonal antibodies 4,5 that rely on targeting selected virus proteins based on their native structure. considerable work has been developed against coronaviruses in the past in this direction, and the experience gained is now being deployed against sars-cov-2 based on the crystal structures already available of several of the virus' proteins. 6−9 the purpose of this paper is to introduce a distinct possible new therapy route by (a) presenting predictions of the earliest events along the folding pathway of two of the virus' proteins and (b) building on this foundation to propose an alternative drug development strategy based on reducing the functionality of the virus by interdicting in the folding process of its proteins. in order to fulfill goal (a) of the article, we will focus on the folding initiation events of two of the sars-cov-2 proteins: (1) the adp ribose phosphatase domain of the nonstructural nsp3 protein 10, 11 and (2) the receptor binding domain of the spike protein. 7, 12, 13 the earliest folding initiation event in both cases will be predicted employing the sequential collapse model (scm). 14, 15 in the scm, the multistate folding process of proteins longer than ∼100 amino acids is initiated by formation of specific nonlocal contacts called primary contacts. these primary contacts help constrain the folding process by dividing the protein into several smaller domains. in this way, overall folding becomes vastly more efficient than a purely random statistical search, resulting in what we have referred to as "nature's shortcut" to the native structure. 15 nucleation of the folding process by the primary contacts then constitutes a potential set of emergent natural physical constraints that sidestep levinthal's paradox. 15 in the case of misfolded protein-based diseases, the scm has been applied to investigate some general properties of the folding dynamics of neuropathogenic proteins. 16, 17 in order to unequivocally determine the folding initiation events, we will support the scm predictions with structural information from the crystal structures of both proteins. goal (b) of this article will be addressed utilizing the scm predictions to provide potential target (intraprotein) regions for the development of therapeutic drugs able to interdict the folding initiation event. various possible therapeutic drugs could be considered, but peptides 18 form a natural class for target region binding, ideally preventing subsequent folding, although other molecular categories could also be similarly employed. this therapeutic drug development strategy based on folding interdiction of target regions (fitrs) is similar to an earlier proposal to develop drugs to interfere in protein folding. 19, 20 the novelty in the present work lies in the scm's ability to predict critical target regions for folding initiation from the primary sequence. the broader potential of the fitr strategy and possible development hurdles will also be discussed. in particular, it will be explained that the proposed fitr drug strategy could be extended to other proteins of sars-cov-2 as well as to other diseases in which the presence of specific proteins plays a decisive role. the physical basis of the scm and its most up-to-date formulation have been recently explained in full detail. 15, 17 here, only a brief summary of the main concepts is presented that are relevant to the issues investigated in the present paper. 2.1. scm entropic cost of loop formation. the scm considers early specific nonlocal contacts based on the entropy of formation of the resultant protein loops. the scm has successfully predicted many of the observed features of protein folding pathways. 15 in the scm, two different loop regimes are considered when analyzing early nonlocal contacts: short loops for which the gyration radius, r g (n), is smaller than the average side chain length £(n) [i.e., r g (n) < £(n)], and long loops for which r g (n) > £(n). the loop length at which the transition between the short and long loops takes place [i.e., the length for which r g (n) ≈ £(n)] is called the optimal loop length n op . the optimal loop length has been estimated to be n op ≈ 65 amino acids for typical protein sequences for £(n) ≈ 7.9 å, 17 although some sequence variability exists and n op is expected to be shorter for highly disordered proteins that contain few of the bulky hydrophobic amino acids. 21 this value for n op is consistent with experimental data showing the behavior of poly-alanine, a polypeptide with smaller side chains than the average globular protein, in this case £(n) ≈ 6.7 å, 17 which exhibits deviations from gaussian statistics because of steric hindrance when n < 50 amino acids. 22 the long loop regime is physically equivalent to the classical flory−jacobson−stockmayer (fjs) picture and the entropic cost of forming protein loops is well represented, assuming that the amino acids can be taken to be solid ball-like by a simple logarithmic function of the form 23 this is clearly an approximation, as for example, the side chains would be better represented by solid spheres of different sizes according to the primary sequence. in the scm short loop regime, however, the internal degrees of freedom of the side chains cannot be neglected, and the entropic cost of forming short loops must be higher than when the amino acids are taken to be solid spheres. moreover, because most of the degrees of freedom are in the side chains, we expect the contribution of the side chains to the overall entropic cost to be dominant with respect to that of any constraints imposed by loop formation on the backbone. thus, in the scm it is expected that for a short loop, the entropic cost of loop formation δs loop approximately becomes with δs side-chain-crowding ≪ 0, opposing folding. when r g (n) ≥ £(n), we have δs side-chain-crowding ≈ 0 and the standard fjs regime is recovered. the side chain crowding term δs side-chain-crowding will appear as a correction to the js results for shorter loops. it is extremely difficult to obtain an analytical expression for the side chain crowding term, and in the scm it has been presented in generic boltzmann−gibbs form 15 where f 0 (n,£) is the average configurational freedom per amino acid in the unfolded chain and f loop (n,£) is the average configurational freedom of an amino acid in a loop. consideration of modifying the homogeneous flory-like representation of the protein chain to take into fuller account the microscopic details of the protein−solvent system is not exclusive to the scm and has been employed before to account, for example, for the effects of the solvent. 24 based on the model developed above, in the scm, the folding of proteins with more than ∼100 amino acids likely involves the formation of an early nonlocal contact, called the primary contact within the scm, that defines the earliest folding phase with n ≥ n op ≈ 65 amino acids. as only a few primary contacts can be established at most in proteins of length n ≥ n op , most of the tertiary structure contacts will still be defined by contacts at a shorter range established in later folding phases. 14 formation of the primary contact in the scm defines the primary loop, which subsequently collapses through two-state kinetics. 15 because proteins longer than ∼100 amino acids do not generally undergo two-state collapse 15 but rather fold through multistep pathways, consistent simple physical reasoning implies that there is a limit to the size of the primary loop that can successfully lead to the native scm folding pathway of ∼100 amino acids. the concept of folding nucleated by nonlocal contacts is not exclusive to the scm, having arisen earlier in the context of the diffusion−collision model 25 and in the energy landscape picture. 26 it also has appeared in simulations of the transition state of two-state folding proteins. 27 also, protein topology has been considered an essential element of folding mechanisms in a number of theoretical efforts. 28−32 the particular feature in the scm is that the early nonlocal contacts are highly specific as in the loop hypothesis, 33 and a methodology is developed to derive their location from primary sequence information. 15 2.2. determining the primary contact. based on the model presented in the previous sections, whether there is a nonlocal contact in an otherwise unfolded state is dependent upon the stability of the potential contact candidates at loop lengths of n ≥ n op amino acids. in the scm, the stability of a contact formed by the number n cont of amino acids, δg contact (n cont ,n loop ), can be written as here, δg loop represents the entropic free energy cost of the loop as discussed in section 2.1. the term δg int,h denotes all the enthalpic interactions that help stabilize the contact, possibly including hydrophobic interactions, van der waals interactions, hydrogen bonds, disulfide bonds, and salt bridges, 34 and its value satisfies δg int < 0. the term δg cont,s > 0 represents the entropic cost of constraining the side chains of the amino acids defining the contact such that the contact is stable and it opposes contact formation. a segment-specific determination of the value δg cont,s (n cont ) for a given contact would require detailed molecular dynamics techniques. however, a heuristic estimate can be made from earlier work within the scm, which showed that the average entropic cost the journal of physical chemistry b pubs.acs.org/jpcb article of folding per amino acid for a sample of 13 proteins was δg folding/residue,s ≈ 0.85kt/residue, 35 and the maximum was δg folding/residue,s ≈ 1.09kt/residue. as these are estimates for the entropic cost for folding per residue of complete proteins that include highly buried as well as flexible exposed regions, it is then reasonable to expect that the entropic cost of a contactforming region must be closer to the highest calculated values for δg f o l d i n g / r e s i d u e , s . here, we will assume that δg contact,s (n contact ) for a contact including n cont amino acids is approximately δg folding/residue,s determined by the number of residues defining the contact, such that δg cont,s (n cont ) ≈ 1.09n cont . this result is clearly an approximation, but in section 3 it will be shown to suffice for establishing a cut-off in the number of possible contacts that is consistent with the available structural data. hydrophobic interactions are well understood to constitute the main driving force of the folding process. 34 other interactions such as hydrogen bonds are weaker 34 or like disulfide bonds and salt bridges form later along the folding pathway. 36 thus, for an early contact forming from the unfolded state, we can take δg int (n op ) ≈ δg hyd (n op ), where δg hyd (n op ) represents the stabilizing effect of hydrophobicity in the early contacts, and eq 4 can be written as as the hydrophobic stabilization energy of the contact δg hyd is determined by the hydrophobicity of the segments involved, the hydrophobicity values h k are obtained from the fauchere− pliska scale 37 and assigned to each residue in accord with previous calculations within the scm. because the amino acid side chains are significantly larger than the typical peptide bond length, early contacts between two hydrophobic amino acids will inherently involve segments including several amino acids, adjacent to the initial contact. the stability of this early hydrophobic contact will determine where the folding process is initiated. this picture is not unlike the zapping model of dill and collaborators. 38 here the typical early contact segment size will be taken to be ∼5 amino acids in line with previous calculations within the scm. 14 the 5amino acid window size is based on the geometric considerations underlying the scm: with an average effective fluctuating width of the unfolded protein chain of w ∼ 2£(n) ≈ 15.8 å, and a peptide bond length of 3.5 å, the minimum number n cont of amino acids that can define a contact in the open fluctuating chain should be n cont ∼ int[2£(n)/3.5] = 5 amino acids. the results for the location of the most stable primary contact were seen to be robust to the employment of five and six-amino acid windows, while some deviations were observed when the window was reduced to four amino acids. in practice within the scm, the hydrophobicity h k of each residue is added over a segment contact window of five amino acids centered at residue i, resulting in a segment hydrophobicity h i , 5 (a value of ∼0.45 is equivalent to a change in energy of kt, with the margin of error being ∼0.1kt 35 ) . in order to determine the best contact, the h i,5 values of a segment centered at residue i is added to the h j value of a segment centered at residue j, located at a distance n ij at least n op amino acids apart along the sequence, and no longer than the maximum primary loop length of ∼100 amino acids, to give a contact stability of we have chosen to focus in this paper on two domains of two major functional proteins of sars-cov-2: (a) the nonstructural adp ribose phosphatase domain of protein nsp3; 10 and (b) the structural receptor binding domain of the spike protein. 12 this choice was made based on two distinct considerations: (1) to study both a structural and a nonstructural protein that have a direct involvement in the viral infection mechanism, thus providing options for drug discovery; and (2) to employ the scm within the boundaries of its demonstrated applicability, that is, on proteins long enough that a multi-state folding pathway is expected, but not so long that degeneracies in the results might cloud any definite conclusions. 15 non-structural proteins of coronaviruses have been the object of intense study, concerning both their structures and their functionality. 39, 40 the multi-domain non-structural protein 3 (nsp3) is the largest protein encoded by the coronavirus' genome. 10 it includes up to sixteen domains, of which eight domains and two transmembrane regions are conserved. 41, 42 one of the conserved domains is the macrodomain (also called the x domain), which includes 173 amino acids. 7 the first available crystal structure of a nsp3 domain of any coronavirus was the unliganded x domain of sars-cov, obtained in 2005. 43 the crystal structure for the sars-cov-2 variant of the x domain is known. 11 it has the typical x domain organization, with seven β-strands defining a central β-sheet, surrounded by six α-helices. 11 one of the functions of the x domain is to bind adp-ribose and poly-(adp-ribose). 43−45 the x domains of coronaviruses, also show adp-ribose-1″phosphate phosphatase activity. 43, 44, 46 it has been observed that this property is linked to the ability of the virus to compromise the immune system of the host. 47, 48 our calculations predict that the best possible primary contact for the x domain (i.e., the best folding initiation contact) is established between segments ( 35 ptvvv 39 ) centered at v37, and ( 125 llapl 129 ) centered at a127, with a stability of δg cont ≈ −6.3kt. the predicted primary contact is in clear proximity in the crystal structure of the protein (pdb code 6vxs), and we have represented the two segments in figure 1a . 49 the next-best possible contacts with energies within ∼1kt of the best primary contact (37, 127) are shown in table 1 . none of the possible alternatives to contact (37, 127) is a good native contact on the crystal structure. none of the side chains of the two segments defining these alternative contacts appear within the van der waals interaction distance in the crystal structure. the issue of the multiplicity of possible primary contacts in the scm has been considered in previous work. 50 because no major rearrangements of the protein core are expected post-collapse, it is generally assumed within the model that primary contacts that are non-native on the 3d folded structure are likely not to correspond to native pathways leading to the functional folded structure. 51, 52 in all the proteins studied to date within the scm it has been observed that the most stable primary contact is native-like. 15 thus, our result implies that the majority of the protein molecules are the journal of physical chemistry b pubs.acs.org/jpcb article expected to fold through the initiation event defined by the primary contact predicted here. in this regard, it is a prediction of the model that primary contact (37−127) is the gateway to "nature's shortcut" 15 to the folding of the adp ribose phosphatase domain of sars-cov-2. 3.2. primary contact of the receptor binding domain of the spike protein of sars-cov-2. the receptor recognition mechanisms of coronaviruses have been extensively studied. 53, 54 in particular, for sars-cov-2 7 and its earlier viral variant sars-cov, 53 entry into the host's cells is mediated by a virus-surface spike protein that includes a specific receptor-binding domain (rbd). 54−56 the rbd recognizes angiotensin-converting enzyme 2 (ace2) as its specific receptor. 55 the structure of sars-cov rbd is well known, 56 and the structure of sars-cov-2 rbd is similar, 7 albeit with some specific mutations in the ace2 binding ridge that enhance the ability of sars-cov-2 to bind human ace2. 7 the spike protein of sars-cov-2 is a natural therapeutic target given its critical biological role in facilitating the virus entry in the cell. the search for inhibitors, including peptides, that actively block rbd-ace2 binding of coronavirus has been an active field of investigation for many years. 57−59 the rbd of sars-cov-2 is 223 amino acids long, making it the longest protein investigated within the scm to date. 14, 15, 17 the best possible contacts with energies within ∼1kt of the best primary contact (37, 127) are shown in table 2 . our calculations predict that the best possible primary contact is established between segments ( 114 cviaw 118 ) centered at i116, and ( 193 vvlsf 197 ) centered at l195, 79 residues apart, with a stability of δg cont ≈ −11.5kt. the predicted contact is in clear proximity in the crystal structure of the protein (pdb code 6m0j), and we have represented the two segments defining the contact in figure 1b . 49 the second-best possible contact is defined by segments ( 17 lcpfg 21 ), centered at p19, and ( 114 cviaw 118 ), 97 residues apart, with a stability of δg cont ≈ −10.8kt. contact (19, 116) is not as good a contact on the native structure. thus, our result implies that the majority of the protein molecules will fold through the initiation event defined by the best primary contact (116, 195) predicted here. the identification of the primary contacts along the folding pathway of viral proteins constitutes an important result for at least two reasons: (a) the sequences of the specific segments involved in the primary contacts provide a template to specify candidate peptide drugs of inhibitory effect with the maximum possible contact affinity to compete with the natural folding mechanism; and (b) it provides insight for further investigation into the subsequent folding steps leading to a fully functional viral protein, potentially providing for additional fitrs. the fact that the primary contact is defined by the interaction between two well defined amino acid sequences suggests that a strategy to develop fitr-based therapeutic drugs could be one utilizing trial peptide drugs as suggested above. peptide drugs offer several advantages versus other more classical approaches such as function-blocking monoclonal antibodies. in particular, being much smaller and flexible, peptide drugs can much more easily cross the cellular membrane to reach their intended targets. 60 however, designing therapeutically effective peptide drugs remains an important challenge. 61 there are several reasons why effective peptide drugs are hard to discover: (1) the potential space of peptide candidates is very large; (2) ensuring delivery at the right location on the target molecule is a considerable challenge; and (3) making a peptide drug that is contact-site 49 the color code reflects the location from the nterminus (dark blue) to the c-terminus (yellow). only the side chains corresponding to the segments that define the primary contacts are shown. the journal of physical chemistry b pubs.acs.org/jpcb article specific is not an easy task. as a consequence, no more than ∼60 effective peptide drugs are in use today, 18 although there is active investigation of many more. 18 the scm might prove of assistance in addressing at least difficulty (1) above, and maybe also (2) and (3), through the utilization of the predicted primary contact sequence as a template to search for an effective therapeutic peptide capable of inhibiting primary contact formation. also, other non-peptide molecules have shown potentially therapeutic capabilities by binding to mostly unfolded states of proteins. for example, ceftriaxone binds specifically to the c-terminal region of the intrinsically disordered protein α-synuclein, 62 generally understood to be involved in triggering parkinson's disease, 63 and has shown therapeutic potential. also, recent experimental results suggest that the folding kinetics of proteins with two-state transitions can be modulated by the employment of suitable peptides that mimic specific segments of the protein chain. 64 although the purpose of the experiment was the opposite of the one sought for here, as the goal was to speed up the folding transition, the results provide general support for the contention presented that specific peptide molecules can interfere and alter the folding kinetics of globular proteins. the proposed therapeutic strategy is depicted in figure 2 . to summarize, we have presented a target-specific strategy to develop folding-interdicting drugs. such folding-interdicting drugs would function by specifically inhibiting the earliest folding events. in order to do so, we propose to rely on the a priori capabilities to identify fitrs embedded within the scm. this strategy is generally similar to earlier proposals to develop therapeutic peptide folding inhibitors relying on protein folding information. 65 our expectation is that by developing such scm-based folding-interdicting drugs as figure 2 . proposed inhibitory mechanism of viral functionality based on the employment of specific peptides in order to interdict the initial folding event of the viral proteins. the effect on the viral structure of the employment of such folding inhibitory peptides on the spike proteins of sars-cov-2 is illustrated in generic form as its precise effect on the overall configuration of the virion is not known. additionally, multiple spike proteins would need to be inhibited simultaneously to fully disrupt the functionality of the virion. whether folding interdiction through inhibition of just the dominant folding initiation event suffices to preempt the onset of disease is a matter that calls for experimental assessment. in this article we presented theoretical predictions for the folding initiation events of two functionally relevant proteins of the sars-cov-2 virus. the predicted folding initiation events were shown to map into good contacts on the 3d structure of the proteins. we proposed that knowledge of the protein segments involved in the folding initiation event, opens an attractive route to developing new therapeutic drugs intended to prevent the successful folding of key viral proteins. such reduction of the population of properly folded viral proteins could lead to a decreased level of viral spread, thus reducing the lethality of the infection. on the basis of these findings, we proposed a general therapeutic drug development strategy based on interdicting the folding process through the identification of scmpredicted fitrs. the fitr strategy could be generally used to approach other diseases where specific proteins play an essential role. a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china an interactive web-based dashboard to track covid-19 in real time phase ii trial of 1311-b1 antibody therapy with autologous stem cell and 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(sars)-associated coronavirus using synthetic overlapping peptide library specific asparagine-linked glycosylation sites are critical for dc-sign and l-sign-mediated severe acute respiratory syndrome coronavirus entry getting across the cell membrane: an overview for small molecules, peptides, and proteins reaching for high-hanging fruit in drug discovery at protein-protein interfaces ceftriaxone blocks the polymerization of α-synuclein and exerts neuroprotective effects in vitro lewy bodies rational design of protein-specific folding modifiers design of hiv-1-pr inhibitors that do not create resistance: blocking the folding of single monomers the authors declare no competing financial interest. the authors acknowledge support from nsf grant che-1763198. key: cord-332317-wrztpeb8 authors: zhang, xin; shi, hongyan; chen, jianfei; shi, da; dong, hui; feng, li title: identification of the interaction between vimentin and nucleocapsid protein of transmissible gastroenteritis virus date: 2015-03-16 journal: virus res doi: 10.1016/j.virusres.2014.12.013 sha: doc_id: 332317 cord_uid: wrztpeb8 nucleocapsid (n) protein of transmissible gastroenteritis virus (tgev) packages viral rna genome to form a ribonucleoprotein complex. in addition to its function as a structural protein, n protein is involved in cell apoptosis or cell-cycle regulation. n protein possibly interacts with host factors to modulate cellular functions. to identify cellular proteins that interacted with n protein of tgev, methods of gst pull-down and co-ip were utilized to precipitate cellular proteins of swine testicular (st). bound cellular proteins were resolved by sds-page. analysis of interacting proteins by mass spectrometry allowed identification of 15 cellular protein bands representative of 12 cellular proteins including vimentin that bound to n protein. furthermore, the function of vimentin cytoskeleton in st cells during tgev infection was examined. vimentin cytoskeleton was required for virus replication. the present study thus provides protein-related information about interaction of tgev n protein with host cell that should be useful for understanding host cell response to coronavirus pathogenesis infection and the underlying mechanism of coronavirus replication. coronaviruses (covs), a genus in the coronaviridae family, are pleomorphic, enveloped viruses (perlman and netland, 2009) . covs have been clustered in the cornavirinae subfamily, which includes three approved genera, alpha-, beta-and gammacoronavirus, as well as a tentative new genus, the deltacoronavirus (de groot et al., 2011; reguera et al., 2012) . transmissible gastroenteritis virus (tgev) is a representative cov in the alphacoronavirus genus; severe acute respiratory syndrome-related coronavirus (sars-related cov) is a representative of the betacoronavirus genus; infectious bronchitis virus (ibv) is a representative of the gammacoronavirus genus; and bulbul-cov is a representative of the deltacoronavirus genus (de groot et al., 2011) . the coronaviridae are involved in respiratory, enteric, hepatic and neuronal infectious disease in animals and humans (perlman and netland, 2009) . tgev infection causes severe diarrhea in suckling piglets (about 2 weeks old), which results in enormous economic loss in swine-producing areas in the world (kim and chae, 2001; sestak et al., 1996) . about two-thirds of the tgev genome (28.5 kb) encodes the replicase gene (rep) at the 5 end, and one-third of the genome encodes other viral genes at the 3 end in the order 5 -s-3a-3b-e-m-n-7-3 (penzes et al., 2001) . the genome of the tgev encodes four structural proteins: spike (s), membrane (m), envelope (e), and nucleocapsid (n) . n proteins of covs are highly basic with a molecular mass ranging from 40 to 63 kda, depending on the species and strains. n protein binds to the rna genome, forming a helical nucleocapsid (escors et al., 2001; sturman et al., 1980) . recently, some reports showed that n protein of tgev play an important role in host cell for virus replication. n protein of tgev facilitates template switching and is required for efficient transcription (zuniga et al., 2010) . n protein underwent proteolysis in parallel with the activation of caspases within host cell and n protein of tgev is a substrate for caspases (eleouet et al., 2000) . tgev n protein nucleolus localization was found in transfection experiments and might induced a cell cycle delay or arrest to facilitate virus replication (wurm et al., 2001) . in contrast, tgev n protein was not accumulated in the nucleus in the infection context (calvo et al., 2005) . in addition, the role of tgev n protein in cell cycle arrest has been recently reported (ding et al., 2014) . n protein of tgev may function through direct or indirect interaction with cellular proteins. for better understanding of the mechanisms associated with pleiotropic functions of n protein, cellular proteins of swine testicular (st) cells were pulled down associated with n protein using glutathione (gst)-tagged full-length n proteins immobilized on gst agarose. and cellular proteins of st cells were precipitated using n protein mab. by sds-page coupled with mass spectrometry (ms), a total of 12 cellular proteins interacting with n protein were successfully identified. information on the expanded repertoire of cellular proteins interacting with n protein will provide a framework for future biochemical analyses of these protein functions in tgev infection. st cells were grown in rpmi-1640 medium supplemented with 10% fetal calf serum under standard culture conditions (5% co 2 , 37 • c). tgev infectious strain h (accession no. fj755618) (wang et al., 2010) was propagated on an st cell monolayer. mouse mab to glyceraldehyde-3-phosphate dehydrogenase (gapdh) (ab9484) and rabbit poloclonal antibody vimentin (ab92547) were purchased from abcam. fitc-labeled goat antimouse igg was purchased from kirkegaard and perry laboratories (kpl). tritc-labeled goat anti-rabbit igg was purchased from sigma. the mab to n protein of tgev were donated from mr. wang shao (institute of animal husbandry and veterinary science, fujian academy of agricultural science, china). st cells were plated in six-well plates 1 day prior to infection with tgev infectious strain h at a multiplicity of infection (moi) of 1. tgev not-infected samples were exposed to culture medium alone. after adsorption for 1 h, cells were washed twice and incubated in fresh rpmi-1640. the prokaryotic expression plasmid pgex-tgev-n was constructed previously . escherichia coli bl21 (de3) strain containing pgex-tgev-n plasmid was expressed under induction of 1 mm isopropyl-␤-d-thiogalactopyranoside. gst pulldown assay was performed as previously described . expressed gst protein was used as a control. the lysate of tgev-infected st cells was prepared with ripa lysis buffer (50 mm tris-hcl, ph 7.4, 150 mm nacl, 1% np-40, 0.25% deoxycholate) containing a protease inhibitor phenylmethanesulfonyl fluoride (pmsf) (1 mm). after centrifugation at 12,000 × g for 15 min, lysate supernatant was pretreated with 2 l mouse igg control (beyotime) and protein a/g plus-agarose (santa cruz biotechnology) for 30 min at 4 • c to eliminate non-specific binding to agarose gel. the lysate supernatant (500 g) was incubated with 1 g of mab to n protein of tgev for overnight at 4 • c. then, 20 l resuspended protein a/g plus-agarose was added to this mixture and incubated at 4 • c on a rocker platform for 2 h. after washing four times with lysis buffer, isolated immunoprecipitated proteins (boiling 10 min with page sample loading buffer) were then analyzed by 12% page analysis. the lysate of tgev not-infected st cells was used as a control. 2.6. protein identification by matrix-assisted laser desorption/ionization time of flight (maldi-tof/tof) ms the gels described above stained with phastgel blue r (ge healthcare). protein bands of interest were manually excised from gels. maldi-tof/tof was performed as previously described (zhang et al., 2009 ). data were searched by gps explorer (ver. 3.6) with the search engine mascot (ver. 2.1). the search parameters were as follows: national center for biotechnology information non-redundant (ncbinr) database (release date, july 2011), and the database sus (41,373 sequences; 16,019,616 residues); a trypsin digest was performed with one missing cleavage, ms tolerance was set at 100 ppm and ms/ms tolerance at 0.6 da. known contaminant ions (tryptic autodigest peptides) were excluded. mascot protein scores (based on combined ms and ms/ms spectra) >59 were considered statistically significant (p ≤ 0.05). individual ms/ms spectrum, with a statistically significant (confidence interval ≥ 95%) ion score (based on ms/ms spectra), was accepted. to eliminate redundancy of proteins that appeared in database under different names and accession numbers, single protein member belonging to species sus or with the highest protein score (top rank) was separated from multi-protein family. equivalent amounts of cell lysates were subjected to 12% page and then transferred to 0.22 m nitrocellulose membranes (hybond-c extra, amersham biosciences). after blotting, membranes were incubated with mouse pab to vimentin or with mouse mab to n protein (1:2000) at 37 • c for 1 h. after washing three times with pbst, membranes were inoculated with dylight tm 800-labeled antibody to mouse igg (h+l) (1:10,000, kpl, usa) or dylight tm 800-labeled antibody to rabbit igg (h+l) (1:5000, kpl, usa) at 37 • c for 45 min. images were visualized by odyssey infrared imaging system (li-cor). st cells inoculated with tgev were cultured for 0, 1, 2, 4, 8, and 16 h. cells were washed twice with pbs and fixed with paraformaldehyde (4%) for 30 min at 4 • c, and then allowed to air dry. after blotting with 5% skimmed milk powder, the fixed cells were incubated with mab to tgev n protein (1:200) and rabbit pab to vimentin (1:100, abcam) for 1 h at 37 • c in a humidified chamber. after washing three times with pbst, the fixed cells were incubated with fitc-labeled goat anti-mouse igg (1:100, kpl) and tritc-labeled goat anti-rabbit igg (1:200, sigma). additional nuclear staining with 4 ,6-diamidino-2-phenylindole (dapi, sigma) was performed as described previously (jungmann et al., 2001) . the triple-stained cells were washed three times with pbst and subsequently examined under a leica tcs sp5 laser confocal microscopy. 2.9. transfection of sirna against vimentin sirna against vimentin (genepharma) was used for transfection. sequence of the sirna strands (two rounds of silencing) were as follows: 5 -gcuaacuaccaagacacuatt-3 (sense) and 5 -uagugucuugguaguuagctt-3 (antisense); 5 -ccucugguu-gacacccauutt-3 (sense) and 5 -aaugggugucaaccagaggtt-3 (antisense). negative control sirna strands were as follows: 5 -uucuccgaacgugucacgutt-3 (sense), 5 -acguga-cacguucggagaatt-3 (antisense). transfection with sirna was performed with lipofectamine 2000 reagent (invitrogen) by following the manufacturer's instructions. st cells were cultured overnight in six-well tissue culture plates. the sirna (20 nm) was complexed with lipofectamine 2000 reagent by incubating together at room temperature for 30 min. after removing the cell culture supernatant, the complex was added. after incubation for 36 h, the cells were infected with tgev. total rna was extracted from the st cells transfected with sirna against vimentin and negative control sirna, using the rneasy mini kit (qiagen) according to the manufacturer's protocol. cdna synthesis was performed with total cellular rna using a reverse transcriptase m-mlv kit (takara), according to the manufacturer's protocol. real time rt-pcr was performed using a lightcycler 480 ii (roche) in a total volume of 25 l containing 10 ng of cdna template, 1× sybr ® premix ex taq tm ii (perfect real time, takara), and a 0.4 m concentration of each primer. after initial denaturation at 95 • c for 2 min, the amplification was performed for 40 cycles, each consisting of denaturation at 95 • c for 5 s and primer annealing at 60 • c for 30 s. melting curves were obtained, and quantitative analysis of the data was performed in a relative quantification (2 − ct ) study model. parallel tgev notinfected st cells were used as control (relative expression = 1) and gapdh as an internal reference gene. st cells were re-plated 1 day before infection in 96 well plates for the 50% infectious dose (tcid 50 ) assays. treated samples and their paired controls were thawed as described and immediately serially diluted. cell cultures were then infected for 1 h. after 48 h of incubation, cpe was observed. tcid 50 is calculated using the method of reed and munch. virus titer assay were performed three times for each condition and were performed using the student's t-test. the expressed gst-n protein immobilized on gst-agarose beads was used as a bait to pull down cellular proteins of tgevinfected st cells that form a complex with n protein. gst protein was used as control to eliminate non-specifically binding proteins. after extensive washing the resins with ripa buffer, cellular proteins bound to n protein were analyzed by sds-page and stained with phastgel blue r (ge healthcare). as shown in fig. 1 , 10 cellular protein bands that were not seen in gst control gel. the 10 protein bands of interest were manually excised from gels and plated into 96-well microplates for ms identification. the mab to n protein was used as a bait to precipitate cellular proteins of tgev-infected st cells that form a complex with n protein. after extensive washing resins with ripa buffer, cellular proteins bound to n protein were analyzed by sds-page and stained with phastgel blue r (ge healthcare). as shown in fig. 1 , 5 cellular protein bands that were not seen in tgev not-infected st cells. the 5 protein bands of interest were manually excised from the gels and plated into 96-well microplates for ms identification. tgev n protein-associated cellular proteins were identified using in-gel tryptic digestion, and maldi-tof/tof identification. 15 protein bands binding to n protein were subjected to in-gel trypsin digestion, and peptides were analyzed by ms. database searches with the peptide masses resulted in positive identification for 8 different cellular proteins (table 1, table s1 , and fig. s1 ). two atp-dependent rna helicase proteins were identified as interacting with n protein. protein band 1 was identified as atpdependent rna helicase a (rha) and protein band 4 was identified as atp-dependent rna helicase ddx1 (dbp-rb). four cytoskeleton proteins were identified as interacting with n protein. protein band 3 and band 14 were identified as ␣-actinin-4 (actn4). protein band 5 and band 15 were identified as vimentin (vim). protein band 11 and band 12 were identified as myosin. four ribosome-associated proteins were identified as interacting with n protein: heterogeneous nuclear ribonucleoprotein u (hnrnp u) (band 2 and band 13); 40s ribosomal protein s9 (band 8); 60s ribosomal protein l26 (band 9); and 40s ribosomal protein s13 (band 10). one proteinbiosynthesis-associated protein (elongation factor 1-␣, band 6) and one cell-cycle-arrest protein (vasohibin-1, band 7) were identified as interacting with n protein. maldi-tof ms spectra and tof/tof spectra of vimentin are shown in fig. 2 . three cellular proteins, hnrnp u, actn4, and vimentin, were identified both by gst-n pull down and co-ip in tgev-infected cells, which should have more biological importance in the context of infection. to verify the proteins identified by ms, western blotting was performed. many cytoskeleton associated proteins play important roles in virus infection (dohner and sodeik, 2005) . vimentin protein was identified not only in gst pull-down assay but also in co-ip assay. therefore, the identified protein vimentin was selected for western blot analysis. cellular proteins immobilized on gst-agarose beads in gst pull-down assay were examined with specific antibodies to vimentin (fig. 3) . results validated the maldi-tof/tof identification of cellular proteins. to confirm the interaction between n protein of tgev and cellular vimentin, immunoprecipitation assay was utilized to identify whether tgev n protein interacted with cellular vimentin in tgevinfected st cells. from the immunoprecipitation results (fig. 4) , we can see that the n protein of tgev was precipitated by the pab to cellular vimentin in tgev-infected st cells but not in tgev not-infected st cells. results demonstrate that cellular vimentin interacted with n protein of tgev. subcellular localization of vimentin was investigated in tgevinfected st cells using indirect immunofluorescence confocal microscopy. results indicated that subcellular localization of vimentin was distributed in cytoplasm with enrichment in the perinuclear region after tgev infection (fig. 5) . cellular vimentin labeled with tritc was overlaid with n protein of tgev labeled with fitc, indicating that cellular vimentin was co-localized with n protein of tgev within the st cells during infection. to further investigate the role of vimentin in virus infection, vimentin protein of st cells was inhibited using sirna. the decrease level of vimentin mrna was validated by rt-pcr (fig. 6a) . st cells transfected with vimentin-specific sirna expressed lower level of vimentin by western blot analysis, compared to that transfected with the control sirna (fig. 6b ). after transfection with sirna, st cells were infected with tgev for another 16 h after transfection with sirna at an moi of 1. virus titer assay were performed three times for each condition and were performed using the student's t-test. tcid 50 of virus in control sirna group was 10 5.2 /ml and the vimentin sirna group was 10 3.1 /ml. fig. 5c shows that knock-down of vimentin resulted in significant reduction of cellassociated virus, which reflected viral replication. identifying host cell protein interacting with viral protein is a critical step for understanding protein function and viral replication. recently, affinity purification followed by ms has been widely used to find protein-protein interactions (dunham et al., 2012) . in the present study, using gst pull-down and co-ip coupled with ms identification, 15 cellular protein bands representative of 12 cellular proteins interacting with tgev n protein were identified. several cellular proteins found in this study were identical to those previously described interacting with cov n protein. ef1a, ddx1 and hnrnp u were also observed in infectious bronchitis virus (ibv) n protein using quantitative proteomic approaches (emmott et al., 2013) . interaction between ef1a and n protein of tgev and sars-cov were founded (zhou et al., 2008) . the hnrnp u protein was identified as binding preferentially tgev 3 -end (galan et al., 2009) . a very recent work describes the key role of ibv n protein interaction with ddx1 in viral transcription (wu et al., 2014) . four cytoskeleton-associated proteins were identified that associated with n protein in this study. the cytoskeleton consists of a filament scaffold within cell. filaments are dynamic and divided into three types: microfilaments (actin filaments), microtubules, and intermediate filament (naghavi and goff, 2007) . during infection, viruses interact with components of the cytoskeleton to reach their appropriate intracellular sites of replication (radtke et al., 2006) . in previous study, differentially expressed cytoskeletal proteins were found in tgev infected st cells using two-dimensional difference gel electrophoresis (zhang et al., 2013) . cytoskeletal proteins found in this study provides new insights for understanding the mechanisms of tgev replication. cov replication uses complex mechanisms that involve viral and cellular proteins. similar to other positive-strand rna viruses, cov genome replication takes place in the cytoplasm in a membraneprotected microenvironment that contains all the protein required for viral rna synthesis (enjuanes et al., 2006) . electron microscopy studies of mouse-hepatitis-virus-infected cells have shown that replication complexes of virus consist of double-membrane vesicles (gosert et al., 2002) . cov replication complex formation possibly utilizes components of cellular autophagy (prentice et al., 2004) . although cov replication essentially takes place within the cytoplasm, the virus may control cell machinery by locating some of its proteins in host cell nucleus (enjuanes et al., 2006) . in the present study, vimentin was identified in pull-down assay and co-ip assay, suggesting that it plays an important role in tgev infection. costaining of cellular vimentin and n protein on immunofluorescence microscopy also supported an association between proteins. based on these findings, we speculate that vimentin plays a role in tgev replication. some studies demonstrate that vimentin plays important role in virus infection cycle. vimentin binding is critical for virus entry, such as human cytomegalovirus (cmv) (miller and hertel, 2009 ), japanese encephalitis virus (jev) (liang et al., 2011) , and cowpea mosaic virus (cpmv) (koudelka et al., 2009) . vimentin is required for virus replication, such as bluetongue virus (btv) (bhattacharya et al., 2007) , and dengue virus (denv) (kanlaya et al., 2010) . in this study, vimentin is required for tgev replication. therefore, these may potentially serve as novel therapeutic targets for controlling tgev infection. the efficiency of tgev infection depends on the presence and integrity of vimentin cytoskeleton, which may facilitate virus infection at different steps. vimentin may promote viral replication by interaction with n protein, which may help virions to transport through a functional golgi complex for viral maturation (risco et al., 1998) . additional work will be needed to pinpoint the exact steps during viral infection. overall, this study was the first to identified cellular proteins interaction with tgev n protein using proteomics method. a total of 12 cellular proteins were identified, which provided new information to understand the mechanism of coronavirus replication. the interaction between the cellular vimentin and n protein of tgev was confirmed in tgev-infected st cells. knockdown of vimentin impairs tgev replication in st cells. the present study thus provides insights into interaction of tgev n protein with host cellular proteins that would be useful for further studying viral replication and pathogenesis. interaction between bluetongue virus outer capsid protein vp2 and vimentin is necessary for virus egress phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells coronaviridae tgev nucleocapsid protein induces cell cycle arrest and apoptosis through 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aminopeptidase n and its inhibition by neutralizing antibodies two types of virus-related particles are found during transmissible gastroenteritis virus morphogenesis contribution of passive immunity to porcine respiratory coronavirus to protection against transmissible gastroenteritis virus challenge exposure in suckling pigs isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid molecular characterization of a chinese vaccine strain of transmissible gastroenteritis virus: mutations that may contribute to attenuation nucleocapsid phosphorylation and rna helicase ddx1 recruitment enables coronavirus transition from discontinuous to continuous transcription localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division ef1a interacting with nucleocapsid protein of transmissible gastroenteritis coronavirus and plays a role in virus replication identification of cellular proteome using two-dimensional difference gel electrophoresis in st cells infected with transmissible gastroenteritis coronavirus differential proteome analysis of host cells infected with porcine circovirus type 2 the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription this work was supported by the national natural science foundation of china (grant nos. 31172350, 31101823), heilongjiang provincial natural science foundation (grant no. jc201118) and heilongjiang provincial department of education (2011td001). we thank mr. zhou xin-wen (fudan university, china) for help with maldi-tof/tof mass spectrometry and mr. wang shao (institute of animal husbandry and veterinary science, fujian academy of agricultural science, china) for the donation of mab to n protein of tgev. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.12.013. key: cord-320015-lbr2q4qh authors: chinchar, v. gregory; yu, kwang h.; jancovich, james k. title: the molecular biology of frog virus 3 and other iridoviruses infecting cold-blooded vertebrates date: 2011-10-20 journal: viruses doi: 10.3390/v3101959 sha: doc_id: 320015 cord_uid: lbr2q4qh frog virus 3 (fv3) is the best characterized member of the family iridoviridae. fv3 study has provided insights into the replication of other family members, and has served as a model of viral transcription, genome replication, and virus-mediated host-shutoff. although the broad outlines of fv3 replication have been elucidated, the precise roles of most viral proteins remain unknown. current studies using knock down (kd) mediated by antisense morpholino oligonucleotides (asmo) and small, interfering rnas (sirna), knock out (ko) following replacement of the targeted gene with a selectable marker by homologous recombination, ectopic viral gene expression, and recombinant viral proteins have enabled researchers to systematically ascertain replicativeand virulence-related gene functions. in addition, the application of molecular tools to ecological studies is providing novel ways for field biologists to identify potential pathogens, quantify infections, and trace the evolution of ecologically important viral species. in this review, we summarize current studies using not only fv3, but also other iridoviruses infecting ectotherms. as described below, general principles ascertained using fv3 served as a model for the family, and studies utilizing other ranaviruses and megalocytiviruses have confirmed and extended our understanding of iridovirus replication. collectively, these and future efforts will elucidate molecular events in viral replication, intrinsic and extrinsic factors that contribute to disease outbreaks, and the role of the host immune system in protection from disease. : sizes and coding potentials of iridovirid genomes. iridovirus iiv-9 206,791 191 31 gq918152 iiv-6 aside from differences in virion and genome sizes, the organization of virus particles within the family is generally similar. while transmission electron microscopy (tem) has been conducted using fv3 and other iridovirids, detailed cryo-electron microscopic (cryoem) analyses (discussed below) were performed with iiv-6 and provide the most detailed view of virion structure. virions exist in two forms: non-enveloped particles ~120-180 nm in diameter, and enveloped particles that acquire an envelope by budding from the plasma membrane. in addition to the external envelope, some iridovirids contain a fringe of fine fibers (fibrils) that extend outward from the capsid subunits [15, 16] . non-enveloped particles are composed of three distinct layers: an outer capsid composed of multiple copies of an ~50 kda major capsid protein (mcp), an internal lipid membrane possibly derived from the endoplasmic reticulum (er) or other cellular membranes, and an inner core containing the viral genome and associated virus-encoded proteins [16, 17] . the mcp shows marked sequence conservation within all members of the family [18] . sequence identity within the mcp gene allows primer-based pcr amplification of viral dna and provides an easy way to determine whether a given virus is a member of the family and to which genera it belongs [19] . although the mcp is the primary structural protein and comprises 40% of the total virion protein content, an additional 36 proteins have been identified following gel analysis of purified virions [3] . while many of these proteins likely represent virus-encoded catalytic proteins that play various roles in replication, three minor capsid proteins have been identified by cryoem analysis of iiv-6 and designated as finger, zip, and anchor proteins [17] . finger and zip proteins were suggested to stabilize the virus by acting as intercapsomer cross-links, whereas the anchor proteins appear to be transmembrane proteins that extend into the internal lipid membrane and provide further stabilization. in addition to these proteins, a fifth protein, a putative myristoylated protein (fv3 orf 53r), has been suggested to interact with cellular membrane fragments and play a role in virion assembly [20] . as shown in table 1 , iridovirid genomes range in size from 105-212 kbp. ranavirus genomes occupy the low end of this range and fall into three groups: the genomes of fv3, atv, tfv, and stiv are 105-106 kbp, ehnv is 127 kbp, whereas sgiv and giv, likely isolates of the same viral species, are 140 kbp in size. consistent with their sizes, ranaviruses contain between 92 and 139 close-packed, predominantly non-overlapping orfs of 50 amino acids or greater [3, [11] [12] [13] . repetitive regions are common and may explain the high rate of recombination seen within these viruses. in addition, a small number of putative micrornas of unknown function have been detected [21] . amino acid conservation is marked among iridovirid genes, but gene order, even within members of the same genus, is not conserved suggesting that expression is likely determined by individual promoter elements closely associated with each gene. moreover, although viral genes are expressed in an ordered temporal cascade (consisting of immediate early (ie), delayed early (de), and late (l) genes) nucleotide sequences corresponding to ie, de, and l promoters have not yet been identified. the observation that gene order is not conserved supports the view that genes can be reshuffled through recombination without adversely affecting expression. microarray analysis suggests that fv3 contains 33 ie, 22 de, and 36 l genes [22] . moreover, the classes assigned in that study were generally similar to those assigned to known sgiv genes in two earlier studies [23, 24] . although all ranaviruses contain a core set of 72 genes [11] , unique genes are found within specific viral species. genes held in common (e.g., viral dna polymerase, mcp, the large and small subunits of the viral rna transcriptase, etc.) are thought to be those required for replication in all cell types, whereas those unique to a given viral species may represent specific host adaptations that contribute to virulence, host range, and immune evasion. early studies of iridovirid replication were conducted almost exclusively using fv3, whereas recent studies have included additional ranaviruses such as atv and sgiv and several megalocytivirus isolates that have been linked to massive die-offs in mariculture facilities in japan and south-east asia. below we summarize events in fv3-infected cells and highlight recent work that has appeared since the last comprehensive reviews [1, 2, 15, 25] . fv3 virions, and presumably virions of all other iridoviruses, exist in two forms: non-enveloped (i.e., naked) particles and enveloped virions. although both forms are infectious, enveloped virions were shown to have higher specific infectivity [26, 27] . with regard to entry, enveloped particles likely enter cells by receptor-mediated endocytosis in a ph-dependent manner and require clathrin-coated pits, whereas naked particles enter by fusion at the plasma membrane [26, 28] . recently this model has been questioned by guo and co-workers who argued that tiger frog virus (tfv), a ranavirus nearly identical to fv3, enters cells by an atypical, ph-dependent, caveola-mediated endocytic pathway [29] . their conclusion was based on experiments using chlorpromazine and over-expression of a dominant-negative form of esp15 that inhibited assembly of clathrin-coated pits, but did not affect entry. consistent with a role for caveolae, endocytosis of tfv was dependent on membrane cholesterol and was blocked by caveolin-1 scaffolding domain protein. given that fv3 and tfv are nearly identical in nucleotide sequence [30, 31] , it is surprising that their modes of entry are so different. because the reported differences in entry mechanisms between fv3 and tfv may be due to infection of cells from different non-physiological hosts, e.g., rats and hamsters, resolution of this issue will require direct comparison of tfv and fv3 entry using the above approaches coupled with tem. regardless of whether uncoating takes place at the plasma or nuclear membranes, the viral genome enters the nucleus. unlike herpesviruses, iridovirid genomes are not infectious, indicating that virion-associated proteins are required to initiate viral gene transcription [32] . accordingly, immediate-early (ie) and delayed early (de) viral transcripts are synthesized in reactions mediated by putative virion-associated (for ie transcripts) and virus-encoded (for de mrna) transcriptional trans-activators and, at least for ie transcription, host rna polymerase ii [33] [34] [35] . among the products of early transcription is a viral dna polymerase which catalyzes the synthesis of unit-sized copies of the viral genome within the nucleus [36] . additional ie and de proteins include proteins that may play roles in blocking host immune responses such as a virus-encoded, card (caspase activation and recruitment domain) motif-containing protein (vcard), β-hydroxysteroid dehydrogenase (βhsd), and a rnase iii-like protein, catalytic proteins involved in nucleic acid synthesis (proliferating cell nuclear antigen [pcna], dna methyltransferase [dmtase], the large and small subunits of the viral homolog of cellular rna polymerase ii [vpol-iiα and -iiβ], transcription factor iis), catalytic proteins that may act to increase dttp pool sizes and influence host range (deoxyuridine triphosphatase [dutpase], deoxynucleotide kinase, the large and small subunits of ribonucleotide reductase), and proteins non-essential for replication in vitro, but needed for growth in vivo (the 18k protein) [22] . following its synthesis, unit-sized viral dna is transported into the cytoplasm where it is methylated by a virus-encoded dmtase and, following a second round of dna synthesis, converted into large concatameric molecules that are thought to be the substrate from which viral genomes are derived [36] [37] [38] . virion formation takes place in electron-lucent, morphologically-distinct vas. vas contain viral dna and the structural and non-structural proteins that give rise to virions, but are devoid of ribosomes, mitochondria, and other cellular organelles [39, 40] . although little is known about the precise process of virion morphogenesis, by analogy to african swine fever virus and by study of various iridovirids, it appears that host membranes, perhaps derived from the er, serve as a scaffold to which capsid and shell proteins bind [41, 42] . as progressively larger amounts of viral proteins bind the membrane scaffold, crescent-shaped structures that resemble icosahedral vertices are formed. ultimately both full and empty icosahedral virus-like particles are detected. however, it is unclear precisely how the genome is encapsidated. while packaging via a headful mechanism explains the circularly-permuted terminal redundancy detected within all iridovirids [43] , it is not known whether nearly complete virions bind viral dna and internalize it through a virion portal as seen in some viral systems [44] [45] [46] , or whether developing crescents/icosahedrons engulf the viral genome as they assemble. following their formation, virions remain within vas, accumulate within cytoplasmic paracrystalline arrays, or bud from the plasma membrane. at late times after infection, virions are sometimes seen within the nucleus and elongated tubular structures can be detected in vas, but these are likely artifacts that reflect breakdown of the nuclear membrane and the disruption of the assembly process due to the shortage of key structural proteins and the dysregulation of cellular and viral macromolecular synthesis. a transmission electron micrograph of an fv3-infected fathead minnow cell is shown in figure 1a , and an enlargement of the vas, displaying full and empty virions, is shown in figure 1b . the identities of virus-encoded proteins involved in virion assembly remain to be determined. twelve complementation groups defective in the ability to synthesize infectious virions have been identified through study of temperature-sensitive (ts) mutants (see below), but it is unclear whether the gene products encoded by these 12 complementation groups represent scaffold proteins that are required for virion assembly, but which are not incorporated into mature particles, authentic viral structural proteins, or virion-associated accessory proteins required for viral replication [47] . aside from the mcp, only fv3 orf 53r, which encodes a putative myristoylated membrane protein, has been linked to virion assembly. knock down studies using either asmos or artificial micrornas [20, 48] demonstrated that 53r was required for virion assembly, whereas in vitro studies showed 53r was associated with virus factories and the virion membrane [49] . by analogy to asfv, 53r may play a role in recruiting er-derived membranes into virus factories where they serve as precursors of the inner viral lipid membrane and/or act as a scaffold protein. as with other nuclear and cytoplasmic, large dna viruses (ncldv) late viral gene expression is dependent upon full viral dna synthesis, and is catalyzed by a virus-encoded or virus-modified transcriptase [47] . temperature-sensitive mutants defective in viral dna synthesis show markedly reduced levels of late gene expression as do cells treated with drugs (phosphonoacetic acid [paa], cytosine arabinoside [arac]) that block dna synthesis [50, 51] . fv3 and other iridovirids encode homologs of the two largest subunits of rna polymerase ii and likely use these proteins, and perhaps others, to catalyze late viral gene expression [30] . knock down of the expression of the largest subunit of the viral homolog of rna polymerase ii (i.e., vpol-iiα) results in a marked reduction in the synthesis of late viral proteins and a corresponding reduction in virion formation and supports the notion that vpol-iiα, and other viral proteins, are responsible for late viral gene expression [52] . aside from the mcp, other late proteins tentatively involved in dna packaging and virion assembly include orf 1r, a putative packaging protein, and evr1/alr, a protein involved in the formation of disulfide bonds [53] . to illustrate the above, a schematic diagram of the viral replication cycle is shown in figure 2 . [54] . used with permission. as with other viruses, fv3 infection markedly inhibits host macromolecular synthesis and triggers apoptosis [55] [56] [57] [58] . both phenomena are mediated by heat-or uv-inactivated virus indicating that the inhibitory agent is a virion-associated protein [58, 59] . viral infection appears to trigger activation of pkr, a double-stranded rna-activated protein kinase, which leads to the phosphorylation of the largest subunit of eukaryotic initiation factor 2 (eif-2α) and the subsequent inhibition of protein synthesis [56] . viral translation may be spared due to the synthesis of large amounts of highly efficient viral transcripts and the presence of a virus-encoded protein, e.g., a viral homolog of eif-2α (vif-2α), that acts as a pseudosubstrate and prevents phosphorylation/inactivation of eif-2α [57, [60] [61] [62] . most ranaviruses, with the exception of fv3, contain a full-sized vif-2α gene [63, 64] . however, because the absence of a full-sized vif-2α gene in fv3 does not adversely affect viral protein synthesis, other viral proteins may also play roles in maintaining viral protein synthesis in infected cells. aside from the aforementioned vif-2α protein, fv3 and other ranaviruses encode a number of putative immune evasion gene products including βhsd, vcard, and an rnase iii-like protein. by analogy to a similar protein in vaccinia virus, βhsd is thought to trigger glucocorticoid synthesis in infected animals and enhance virus replication by suppression of the overall immune response [65, 66] . likewise, vcard may inhibit the induction of interferon and/or apoptosis. induction of ifn and/or apoptosis follows interaction of rig-i or mda5, cellular proteins that bind dsrna, with downstream effectors such as mavs and isp-1 [67] [68] [69] [70] [71] [72] [73] . since rig-i/mda5 and mavs/isp-1 interactions are mediated by card motifs, it is possible that a viral card-containing protein might bind either one or both of these proteins and short-circuit the ifn induction pathway [74] . the rnase iii-like protein is found within all iridoviruses and has been postulated to block sirna-mediated interference, or to play a role in the processing of viral mrnas [75] . alternatively, viral rnase iii could act like vaccinia virus e3l and bind and/or degrade dsrna and block the activation of pkr, the induction of interferon, and the inhibition of protein synthesis [62] . as with vaccinia virus [76] [77] [78] , iridoviruses that infect ectothermic animals encode a series of proteins whose function is to ensure an adequate supply of nucleotides for viral dna synthesis. these enzymes include the two subunits of ribonucleotide reductase [79, 80] , thymidine kinase [30] , thymidylate synthase, dutpase [81] , purine nucleoside phosphorylase (pnp), and a dihydrofolate reductase homolog [12, 82] . not all of these putative enzymes are found within each viral species, and it is postulated that specific enzymes might be needed in certain hosts. for example, pnp is found only within giv and it has been suggested that because giv's natural host, the grouper, might not be able to supply the purine nucleotides required for viral replication the virus encodes its own pnp [82] . currently there are seven completely-sequenced ranavirus genomes ranging in size from 105-140 kbp (table 1) . whole genome dot plot analyses show that there are three distinct genomic organizational profiles among ranaviruses that are based on the conservation of gene order [12] : fv3-like viruses (i.e. fv3, tfv and stiv), atv-like viruses (i.e. atv and ehnv) and giv-like viruses (i.e. sgiv and giv). dot plot comparisons of viruses within each group show complete co-linearity. however, comparison of atv-like ranaviruses with fv3-like ranaviruses detected two large inversions, whereas comparing either of these groups to giv-like viruses revealed very little conservation of gene order [11, 83] . in the latter case, gene order is so jumbled between fv3/atv-like viruses and sgiv/giv-like viruses that any trace of the 45 degree diagonal line, indicative of complete sequence alignment between genomes in dot plots, is lost. interestingly, ranavirus genomic organization is markedly different from that of poxviruses. for example, all members of the subfamily chordopoxvirinae share a conserved genomic core structure wherein gene order, with the single exception of avipoxviruses, is conserved [84] . however, despite the conservation of gene order, global sequence alignments show that sequence identity among members of the chordopoxvirinae is only 27-29% [85, 86] . ranaviruses, on the other hand, share a much greater sequence identity (~72 -95% within the mcp) yet have three widely divergent genomic organizations. moreover, as additional ranavirus genomes are sequenced, we may discover novel genomic morphotypes that will elucidate the evolutionary relatedness of ranaviruses. examination of complete ranavirus genomic sequences, including whole genome dot plot and phylogenetic analyses of ranavirus core genes, suggested that ranaviruses have undergone a number of evolutionarily-recent host shifts [12, 87] . phylogenetic analysis using the 26 core iridovirid genes identified by eaton and her co-workers [11] support whole genome dot plot analysis and indicate that fv3-like viruses (fv3, tfv, stiv), atv-like viruses (atv and ehnv), and giv-like viruses (sgiv and giv) cluster on separate branches of the ranavirus tree ( figure 3 ). collectively, these data suggest that for the greater part of their evolutionary history, ranaviruses were restricted to infecting fish. in addition, the shallow branch lengths of fv3-like and atv-like viruses, which infect amphibians and fish, suggest that this group of viruses has recently, at least in evolutionary terms, radiated to infect a wide-range of poikilothermic vertebrates [12] . this hypothesis is supported by studies showing that ranaviruses are multi-host pathogens [88] . as additional ranaviruses are sequenced, particularly those isolates that infect multiple host species, a better understanding of ranavirus evolution, host shifts, and the molecular determinants of ranavirus host range and pathogenesis will be achieved. pathological and immunological aspects of infection by fv3 and various iridovirids will be dealt with by chen and robert [90] and miller et al. [91] in this special issue of viruses. thus, only a brief overview of this topic will be provided here. although iridovirids, in contrast to the chytrid fungus batrachochytrium dendrobatidis (bd), have not been reported to drive species extinction, fv3-like viruses have caused deaths in several amphibian culture facilities and in nature, and megalocytiviruses have triggered wide-spread mortality in mariculture operations in south-east asia [1, 2, [92] [93] [94] [95] . ranavirus infections range from inapparent to fulminant and involve a variety of tissues including the skin, kidney, liver, and intestine [96] . using xenopus laevis as a model host, robert and his colleagues have shown that fv3 infection occurs in both larval and adult animals [96] [97] [98] [99] [100] [101] [102] . however, whereas infections of immunocompetent adult frogs are confined to the kidney and cleared within a few weeks, infection of tadpoles or immunocompromised adults results in widespread infection that spreads to multiple internal organs and often leads to death. protection from fv3 infection appears to involve both humoral and cell-mediated immunity and both antiviral antibodies and cytotoxic t cells have been identified as protective. vaccination may prove useful in protecting susceptible species and vaccines have been developed to protect commercially important fish from megalocytivirus infection [103, 104] . likewise, infection of bullfrog tadpoles (rana catesbeiana) with less pathogenic fv3 protected them against death following infection with the more pathogenic rana catesbeiana virus z [63] . as with the above section, the ecology of ranavirus infections will be more fully discussed by miller and co-workers in their contribution to this issue [91] . suffice it to say iridovirus infections have a marked effect on lower vertebrates. as green and co-workers observed most mortality events seen among amphibians from 1996-2001 were attributable to iridovirus infections [105] . in addition, whereas lymphocystivirus and megalocytivirus infections have, to date, only been detected in fish species, ranavirus infections affect three classes of ectothermic vertebrates: bony fish, amphibians, and reptiles [2] . results from experimental challenges as well as natural infections suggest that ranaviruses isolated from one species can not only infect different host species within the same genus, but also host species from different genera, orders, and classes. for example, amphibian ranaviruses such as bohle iridovirus, fv3, and atv infect a variety of fish and amphibian species, and at least one insect virus appears to also infect reptiles [9,10,88,106-109]. early studies with fv3 attempted to elucidate viral replicative events and the function of viral gene products using a variety of inhibitors, e.g., cycloheximide to confine viral gene expression to ie transcription, α-amanitin to block host rna polymerase ii activity, phosphonacetic acid to block viral dna synthesis, azacytidine to block the methylation of viral dna, flurophenylalanine to block the expression of late viral proteins, elevated temperatures (37 °c) to confine viral gene expression to early transcripts and proteins, etc. (reviewed in [110, 111] ). in addition, panels of ts mutants were generated and subsequently characterized [47, 112, 113] . ts mutants proved useful in confirming and identifying various aspects of the virus life cycle, e.g., the two-stages of viral dna synthesis [114] . ultimately, 28 ts mutants were organized into 19 complementation groups comprising three classes [47] . class i mutants (12 complementation groups) appeared to contain mutants with defects in virus assembly. these mutants synthesized early and late viral proteins and viral dna, but did not generate infectious virions [47] . recently, tem analysis suggested that this class could be further sub-divided into at least two subclasses, those that failed to assemble icosahedral particles and those that formed ostensibly normal, but non-infectious, icosahedral particles at the non-permissive temperature [115] . the remaining complementation groups appeared to contain defects in proteins associated with viral transcription (class ii) and viral dna synthesis (class iii). consistent with an early study suggesting that only early viral proteins were required for assembly site formation [116] , tem analysis suggested that full viral dna synthesis was not required for the formation of viral assembly sites as two ts mutants that synthesized markedly reduced levels of viral dna were able to form large vas, that were devoid of viral dna, most viral proteins, and viral particles [47, 115] . however, although ts mutants have contributed to our understanding of fv3 replication, it has not been possible to associate a given mutant phenotype with a specific viral orf, and thus link a specific viral protein with its function. to address this issue, we have recently developed approaches that target individual viral genes and have used them to determine viral gene function by observing changes in phenotype. to link specific viral proteins with their functions, viral gene expression was knocked down (kd) using antisense morpholino oligonucleotides (asmos) and gene function was inferred by changes in phenotype. asmos are dna-like macromolecules, optimally 25 nucleotides in length, whose backbone contains morpholine rings instead of deoxyribose rings and non-ionic, phosphorodiamidate bonds in place of phosphodiester links [117] . moreover, because phosphorodiamidate bonds are resistant to degradation by cellular nucleases, asmos are remarkably stable in cell culture [117] . furthermore, in contrast to sirnas that sometimes trigger off-targeting effects by activating pattern recognition receptors such as toll-like receptor 3 (tlr-3), asmos are not recognized by cellular dna receptors such as tlr-9 or aim-2 and thus do not induce innate immune responses. asmos bind complimentary sequences within the 5' non-translated region, or the region immediately surrounding the aug initiation codon, of the targeted mrna and are thought to block scanning of the 40s ribosome by steric hindrance. in addition, asmos also block gene expression by inhibiting splicing or preventing interaction of regulatory proteins with their specific target sequence [118] . collectively asmos have been used to block cellular and viral gene expression both in vitro and in vivo [119] [120] [121] . asmos have been used to ascertain the function of several fv3 genes including those encoding the mcp, vpol-iiα, an 18 kda immediate-early viral gene product (18k), a putative myristoylated membrane protein (53r), and two ie proteins of unknown function: a 70 kda protein designated 32r, and 46 kda protein termed 46k [20, 52, 122, 123] . in an initial proof-of-concept study, sample et al. [52] showed that asmo treatment knocked down mcp expression by greater than 80% and resulted in a corresponding drop in the production of infectious virions without any collateral adverse effects on the expression of other viral gene products. moreover, mcp knock down was accompanied by the appearance of atypical elements within vas suggesting that in the absence of full mcp expression aberrant viral structures, perhaps representing altered products of virion assembly, were generated. in the same study, kd of vpol-iiα was shown to result in a marked reduction in the synthesis of late viral proteins. this result provided the first formal evidence that viral homologs of host rna polymerase ii played a role in the synthesis of late viral transcripts. lastly, sample and co-workers observed that kd of the 18k ie protein had no observable effect on viral gene expression or replication in vitro suggesting that, at least in fathead minnow cells, 18k was not required for the production of infectious virions. subsequent kd studies of 53r, a putative virus-encoded myristoylated membrane protein, 32r, and 46k confirmed an essential role for each of these proteins in fv3 biogenesis [20, 122] . kd of 53r resulted in the appearance of putative non-encapsidated viral dna cores within vas and supported the view that 53r plays a major role in capsid formation. kd of 32r and 46k, two ie proteins, did not affect the synthesis of other viral proteins, but resulted in a >80% reduction in virion formation. tem study showed that cells treated with asmos targeting either 32r or 46k formed viral assembly sites, but failed to form virions or recognizable assembly intermediates. studies targeting the virus-encoded rnase iii-like protein (orf 80l), a putative ntpase (orf 9l), the largest subunit of ribonucleotide reductase (orf 38r), a putative viral membrane protein (orf2l), a dna packaging protein (orf 1r), a putative serine/threonine kinase (orf 57r), a putative rad2-like dna repair protein (orf 95r), a 129 kda protein of unknown function (orf 41r), and the viral dmtase (orf 83r) are ongoing and have demonstrated reductions in viral yields ranging from 41-92% [123] . however, unlike the first study in which kd of mcp, vpol-iiα, and 18k was confirmed by 1d sds-polyacrylamide gel electrophoresis, we have been unable to identify unambiguously the targeted protein in these latest studies, and thus do not know if the partial reductions in virus yields reflect the non-essential nature of the gene product or incomplete kd of the targeted protein. to overcome this problem, recombinant viral proteins will be generated and used to produce polyclonal antibodies for western blot and immunofluorescent analyses. lastly, although asmos can be powerful tools for uncovering viral gene function, their use is potentially limited by the nature of some ranavirus mrnas. because many fv3 transcripts possess very short, au-rich 5' non-translated regions, and because sequences surrounding the aug initiation codon may be unfavorable for targeting due to, for example, high au content or secondary structure, asmo-mediated kd approaches may not succeed with all targeted genes. in these cases, sirna-mediated kd or ko approaches, discussed below, provide alternative approaches. nevertheless, despite these limitations, asmos have provided insight into the roles of several fv3 genes and will continue to be a useful tool for elucidating viral gene function in vitro. together with anti-viral antibodies to confirm knock down and identify intracellular locations, these studies will broaden our understanding of the complex story of virus-host interactions. as an alternative to asmos, sirnas have also been used in a limited number of cases to knock down iridovirid gene expression [48, 122, 124, 125] . while knock down has been achieved, we observed that inhibition of fv3 gene expression only took place if cells were infected at low multiplicities of infection, i.e., <0.1 pfu/cell. similar to the situation in some other viral systems, the inverse relationship between multiplicity of infection and sirna knockdown suggests that a virion-associated or virus-encoded protein may block sirna-mediated knockdown by either binding or degrading sirna [126] . to date, expression of fv3 transcripts encoding mcp, dmtase, and vpol-iiα have been knocked down by sirna and resulted in a marked inhibition of replication. a powerful and potentially more useful approach to elucidate viral gene function involves the generation of knock out (ko) mutants since, in contrast to transient kd triggered by asmos or sirnas, the phenotypes of ko mutants can be evaluated both in vitro and in vivo. this feature makes ko mutants especially useful for identifying viral genes that play roles in immune evasion and virulence. ko mutants have been used extensively to elucidate the function of various poxviruses genes [127] [128] [129] , and methodology developed here has been adapted to ranaviruses. however, despite using poxviruses as a guide, ranavirus ko mutants, using homologous recombination to replace the targeted gene with a selectable marker, have only recently been isolated. the reasons for the difficulty in applying this approach to ranaviruses are not clear, but may reflect the presence of a ranavirus-encoded endonuclease that cleaves unmethylated dna [30, 38] . if correct, introduction of unmethylated plasmid dna, bearing the selectable marker, into a virus-infected cell may lead to degradation of the majority of plasmid dna and make recombination unlikely. as was done previously in a recombinant biv vector [130, 131] , positioning the selectable marker, e.g., the neomycin-resistance gene, downstream of the promoter for the 18k immediate early (ie) gene drove high levels of expression of the resistance gene and permitted isolation of rare recombinants. the first ranavirus ko mutant targeted the atv-encoded homolog of eukaryotic translational initiation factor 2α (vif-2α) [132] . as discussed above, following virus infection, host cells activate pkr, an interferon (ifn) inducible double-stranded rna activated protein kinase [133] , that, in turn, phosphorylates the α subunit of eif-2 and, as a consequence, inhibits both host and viral protein synthesis. to prevent translational shut-off, several viruses, including most ranaviruses, encode an eif-2α homolog that has been proposed to function as a pseudosubstrate for pkr [134] . in vaccinia virus this homolog is designated k3l, whereas in ranaviruses it is termed vif-2α. although k3l and vif-2α differ markedly in size, they share a common sequence motif (vdrvdrekgyvdl) that is likely required for activity. in this scenario, pkr binds k3l/vif-2α instead of eif-2α, and as a consequence eif-2α is not phosphorylated and protein synthesis is maintained. to determine if atv vif-2α (orf 57r) is functionally similar to k3l, the ranavirus gene was replaced with a selectable marker, the neomycin resistance (neor) gene. to generate an atv ko mutant, neor was inserted downstream of the atv 18k ie promoter and this construct was bracketed with sequences identical to those in the flanking region of the targeted gene. a pcr product bearing this construct was then transfected into bf-2 cells and infected with wild-type (wt) atv. recombinant virus was isolated by selective growth in the presence of neomycin, and replication of the ko mutant was compared to wt virus in vitro and in vivo. the ko mutant, designated atv∆57r, replicated to titers comparable to that of wt atv in vitro, but had a small plaque phenotype. in addition, atv∆57r was 8-fold more sensitive to ifn than wt atv. the increased ifn sensitivity of atv∆57r was correlated with the increased phosphorylation of eif-2α and the lack of pkr degradation. in contrast, wt atv degraded pkr and inhibited cellular eif-2α phosphorylation. in addition, atv∆57r was less pathogenic than wt atv following infection of tiger salamanders (ambystoma tigrinum) indicating that vif-2α was a likely viral virulence/immune evasion protein. thus the atv vif-2α gene is a putative ifn-resistance gene that inhibits cellular innate immune responses by degrading pkr and maintaining high levels of viral protein synthesis. in an effort to improve the ko methodology, chen et al. [135] recently developed a potentially more powerful dual selection method to generate ko mutants within fv3. in this system, the gene of interest is replaced, via homologous recombination, with a gene encoding the puromycin-resistance gene fused to the gene for enhanced green fluorescent protein (egfp). in initial experiments ko mutants targeting the truncated vif-2α protein (fv3-∆vif-2α) and the 18k ie protein (fv3-∆18k) were generated as well as a control, "knock in" mutant in which the puromycin-resistance/egfp gene was inserted into a non-coding portion of the genome. while all three recombinant viruses grew well in vitro, the vif-2α and 18k ko mutants, but not the control "knock in" mutant, showed a 90% drop in virus levels in x. laevis tadpoles. these results confirmed the previous observation that the 18k protein was not required for replication in vitro [52] , and indicate that 18k plays a role, albeit unknown, in replication in vivo. the finding that the truncated vif-2α protein of fv3 was also required for replication in vivo was surprising because the fv3 homolog of vif-2α is missing the n-terminal two-thirds of this protein, including the region homologous to vaccinia virus k3l and eukaryotic translational initiation factor 2α. inspection of the fv3 nucleotide sequence indicated that fv3 vif-2α is a chimeric protein resulting from deletion and in-frame fusion of a small upstream orf with the larger, downstream vif-2α orf. the resulting product contains 10 amino acids from the n-terminus of the upstream orf and the last 65 amino acids of the full-length vif-2α protein. the reduced replication of fv3-∆vif-2α in vivo suggests that the c-terminal end of the fv3 vif-2α homolog is required for full replication in vivo. in addition to the above ko mutants, a recombinant soft-shelled turtle iridovirus has recently been constructed that expresses egfp. egfp-expressing mutants may provide an alternative, simplified strategy for screening antiviral substrates through the visualization and quantification of fluorescent virus [136] . in contrast to the above studies, a fourth approach for determining viral gene function involves the synthesis of recombinant viral proteins and assessment of their functions in vivo and in vitro. several investigators have used this approach and representative examples are discussed below. a putative homolog of fv3 icp46 was identified in sgiv [137] . icp46 (mol wt 44.1 kda) is an ie gene product that is distributed predominantly within the cytoplasm but is also found within virions. a plasmid expressing icp46 was introduced into grouper embryonic (gp) and fathead minnow cells and stably transfected cells were characterized. over expression of sgiv icp46 resulted in higher cell densities and increased growth of monolayer cultures. sgiv replication was compared in gp cells transfected with an empty vector and in gp cells expressing icp46. sgiv replicated more rapidly and reached titers that were 10-fold higher in icp46 expressing cells than in control cells. although conserved domains suggestive of function were not detected within icp46, the authors speculate, based on the above results, that icp46 might encode a protein involved in cell growth control. the authors suggested that icp46, like ie proteins in other viral systems, might control host cell growth by regulating the cell cycle, preventing growth arrest, or delaying apoptosis and play a critical role in promoting a proliferative environment that enhances virus replication. isknv (genus megalocytivirus) orf 48r encodes a viral protein with marked similarity to vascular endothelial growth factor (vvegf) [138] . microinjection of a plasmid expressing vvegf into zebrafish one-cell embryos resulted in pericardial edema and dilation of the tail region suggesting that vvegf triggers vascular permeability. moreover, vvegf binds and up-regulates the expression of flk-1 and together both proteins likely contribute to the proliferative and highly vascularized nature of isknv lesions in its natural host, the chinese mandarin fish. although these results suggest that isknv orf48r plays a vital role in host infection and viral replication, its precise function remains unclear. following orf virus (family poxviridae) infection, the vvegf homolog aids in scab formation and wound healing and may enhance transmission since scabs contains substantial amounts of infectious virus. a novel histone-binding protein was identified in sgiv by structural analysis of the orf 158l gene product [139] . x-ray diffraction analysis of recombinant 158l determined the crystal structure at a resolution of 2.2 å, and revealed that 158l exhibited partial structural resemblance to the histone-binding region of anti-silencing factor 1 (asf1), a histone h3/h4 chaperon. using recombinant 158l protein, interaction was demonstrated between 158l and histone h3/h4 complexes and h3 by isothermal titration calorimetry. the authors suggested that 158l may be involved in both the regulation and expression of histone h3 and h4 methylation, events which allow the virus to control host cell gene expression and facilitate viral replication. sgiv orf 86r encodes a homolog of the fv3 18k immediate early protein. although kd studies using an asmo targeted against 18k [52] and an 18k knock out mutant [135] indicated that expression of 18k was not required for replication in either fhm or bhk cells, replication of the 18k ko in xenopus laevis tadpoles was reduced about 10-fold compared to wt virus or a "knock in" mutant. to determine the intracellular location of the 18k gene product, xia et al. [140] generated recombinant sgiv 18k protein and used it to produce rabbit anti-18k serum. immunofluorescent assay showed sgiv 18k to be distributed within the cytoplasm adjacent to vas and nuclei, and western blotting using purified and disrupted virions indicated 18k was a non-envelope virion-associated protein. transfection of uninfected grouper cells with a vector expressing 18k enhanced cellular growth rates and led to an increase in the density of monolayer cultures. moreover, sgiv replication in cultures expressing 18k were about 10-fold higher than in cultures transfected with an empty vector. the authors suggest that 18k may be a protein that plays a role in the control of cellular growth and thus indirectly enhances sgiv replication. however, because virus yields were expressed as tcid 50 /ml, it was not clear if the increase in sgiv yield reflected higher virus production per cell or simply equivalent cellular yields in cultures containing varying numbers of cells. rothenburg et al. [61] cloned the vif-2α gene from rana catesbeiana virus z (rcv-z) into an expression vector and examined its function in a yeast-based assay system. yeast expressing human pkr failed to grow due to the toxicity of the pkr protein. however, yeast expressing vif-2α, or the vaccinia virus k3l protein, suppressed the toxic effects of human pkr indicating that vif-2α was functionally equivalent to the vaccinia virus protein. subsequent work showed that whereas k3l was effective only against human pkr, vif-2α suppressed the toxic effects of both human and zebrafish pkr suggesting that pkr antagonists evolved to protect physiologically-relevant/phylogeneticallyrelated targets. in addition, a study using vectors expressing the various domains of vif-2α, i.e., n-terminal, central/helical, and c-terminal, demonstrated that the n-terminal and central/helical domains were sufficient for suppressing pkr toxicity. collectively, these results provide the first formal proof that vif-2α functions as a virus-encoded pkr antagonist. recombinant versions of thymidylate synthase (lcdv-china), erv1 (rana grylio virus, rgv), dutpase (rgv), rnase iii (rbiv), and litaf (sgiv) were generated and evaluated for their ability to influence cellular growth, enhance virus replication, induce apoptosis, and cleave dsrna [51, 75, 141, 142] . recombinant thymidylate synthase from lcdv-china was found to promote cell cycle progression and produced a transformed phenotype [143] . antibodies directed against rana gyrlio virus (rgv) erv1 detected erv1 expression within the cytoplasm and nucleus, but not within vas; transcript analysis indicated that erv1 was a late viral gene product [141] . rgv dutpase was determined to be a de gene product that was localized to the cytoplasm. ectopic expression did not enhance virus replication, however, its effect on cellular proliferation was not examined [51] . recombinant rnase iii from rbiv cleaved dsrna, but the salt optimum for cleavage was inconsistent with a physiological effect [75] . the authors speculated that rnase iii might play a role in the suppression of rna interference, as has been suggested for other viral dsrna-binding proteins, or could be involved in the processing of viral rna. lastly, huang et al. [142] demonstrated that the sgiv homolog of litaf (lipopolysaccharide-induced tnf-α factor) was a de viral gene product. litaf was located within the cytoplasm and was associated with mitochondria. when over-expressed ectopically, it led to the activation of caspase 3 and the induction of apoptosis. collectively, ts mutants, asmo-and sirna-mediated knock down, gene knock out, and recombinant protein studies are slowly elucidating the function of iridovirid genes and providing a clearer and more complete picture of how those genes enhance viral replication and modulate cellular functions. future studies of ranaviruses and other iridoviruses infecting ectothermic animals will focus on the following areas: (1) identifying and elucidating the function of replicative genes and those contributing to virulence, host range, and immune evasion; (2) understanding the correlates of antiviral immunity in an effort to understand the cellular and molecular basis of anti-viral immunity and to protect endangered and commercially important species from infection; (3) determining the impact of iridovirus infections in nature, defining viral host range, identifying susceptible hosts and reservoir species; (4) understanding how intrinsic (e.g., host immune suppression, host mhc repertoire, etc.) and extrinsic (e.g., habitat disruption, environmental stress, introduction of invasive species, etc.) factors influence the clinical outcomes of iridovirus infection. for example, determining whether iridovirids encode viral immune evasion proteins and how these proteins circumvent host immunity will highlight events at the interface of virology and immunology, and may identify possible targets for viral attenuation. successful completion of these studies will involve the interactive efforts of virologists, immunologists, population biologists, ecologists, and veterinary pathologists. the global ranavirus consortium [144] is but one interactive group of scientists interested in the role of 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homolog of icp18 involved in cell growth control and virus replication cloning, expression and subcellular distribution of a rana grylio virus late gene encoding erv1 homologue identification and characterization of a putative lipopolysaccharide-induced tnf-alpha factor (litaf) homolog from singapore grouper iridovirus constitutive expression of thymidylate synthase from lcdv-c induces a transformed phenoptype in fish cells we would like to thank robert sample for the electron micrographs shown in figure 1 and the artwork depicted in figure 2 . the work was partially supported by nsf award no. ios 07-42711. the authors declare no conflict of interest. key: cord-302414-g5onwhg1 authors: tahir ul qamar, muhammad; shahid, farah; aslam, sadia; ashfaq, usman ali; aslam, sidra; fatima, israr; fareed, muhammad mazhar; zohaib, ali; chen, ling-ling title: reverse vaccinology assisted designing of multiepitope-based subunit vaccine against sars-cov-2 date: 2020-09-16 journal: infect dis poverty doi: 10.1186/s40249-020-00752-w sha: doc_id: 302414 cord_uid: g5onwhg1 background: coronavirus disease 2019 (covid-19) linked with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) cause severe illness and life-threatening pneumonia in humans. the current covid-19 pandemic demands an effective vaccine to acquire protection against the infection. therefore, the present study was aimed to design a multiepitope-based subunit vaccine (mesv) against covid-19. methods: structural proteins (surface glycoprotein, envelope protein, and membrane glycoprotein) of sars-cov-2 are responsible for its prime functions. sequences of proteins were downloaded from genbank and several immunoinformatics coupled with computational approaches were employed to forecast band tcell epitopes from the sars-cov-2 highly antigenic structural proteins to design an effective mesv. results: predicted epitopes suggested high antigenicity, conserveness, substantial interactions with the human leukocyte antigen (hla) binding alleles, and collective global population coverage of 88.40%. taken together, 276 amino acids long mesv was designed by connecting 3 cytotoxic t lymphocytes (ctl), 6 helper t lymphocyte (htl) and 4 b-cell epitopes with suitable adjuvant and linkers. the mesv construct was non-allergenic, stable, and highly antigenic. molecular docking showed a stable and high binding affinity of mesv with human pathogenic toll-like receptors-3 (tlr3). furthermore, in silico immune simulation revealed significant immunogenic response of mesv. finally, mev codons were optimized for its in silico cloning into the escherichia coli k-12 system, to ensure its increased expression. conclusion: the mesv developed in this study is capable of generating immune response against covid-19. therefore, if designed mesv further investigated experimentally, it would be an effective vaccine candidate against sars-cov-2 to control and prevent covid-19. viruses have the potential to become dangerous life threat and cause irreparable loss to human beings. hardly the world learns to cope with one strain of virus when another emerges and poses a threat to the future of humanity. a similar situation has emerged when a new strain of novel coronavirus (cov) that has not been previously identified in humans reported in december, 2019 [1, 2] . coronaviruses are the largest among rna viruses belonging to coronaviridae, roniviridae and arteriviridae families. coronaviridae are unsegmented, 3′ polyadenylated and 5′ capped positive sense singlestranded rna viruses cause various respiratory diseases in humans [2, 3] . covs are classified into four classes: alpha, beta, delta, and gamma. amongst them, beta and alpha covs have been reported for infecting humans [4] . recent cov strain has received tremendous attention from researchers, as it causes a global pandemic of coronavirus disease 2019 (covid-19) [5] . severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was identified as the causative agent of this pandemic [6] . the study of genome sequences has cast a shadow that sars-cov-2 is closely related to the sars-cov which is the causative agent of the sars disease in 2002/2003 [7] . initial diagnostic procedures indicated that the sars-cov-2 is primarily spread through respiratory droplets from sneezing/coughing, body contact and to some extent through fecal contact [8] . the sars-cov-2 may show symptoms within 14 days after exposure, or in some cases it takes more than 14 days. symptoms of patients infected with covid-19 include fever, runny nose, cough, and dyspnea [9] . although the entire genome sequence of the virus has been published, the origin and proliferation mechanism of the new coronavirus is still ambiguous as stated by the world health organization [10] . initial reports claimed that bats, snakes, pangolins or civet could be a possible animal source, but the claims are under debate and needs substantial research to prove it [6, [11] [12] [13] . researchers are currently working to sort out the sars-cov-2 source, including possible intermediate animal vectors. the samples taken from a respiratory system-throat swab or lung fluid are helpful in diagnosing its infection in patients [14] . a special clinical diagnostic reverse transcription-pcr based test was developed [15] . over 200 clinical trials are currently underway to test new and repurposed compounds against sars-cov-2 [16, 17] . several medications such as hydroxychloroquine, remedesivir, and dexamethasone are being tested in clinical trials [18] [19] [20] [21] . several vaccines including subunit vaccines [18, 22] , nanoparticle based vaccines, viral vector vaccines (adenovirus vector, ankara vector), inactivated vaccines, fusion-protein based vaccines, recombinant protein, dna vaccines, and live-attenuated vaccines are also being developed and in pre-clinical trials, but these vaccines are long months away from the market [23] [24] [25] [26] [27] . immunoinformatics approaches can be applied to examine viral antigens, prediction of its epitopes and assessment of its immunogenicity [28, 29] . moreover, this approach could be both time and cost-effective [3, 30, 31] . excessive respiratory infection can also resolve with t-cell reactions and antibodies [32] . furthermore, rapid identification, isolation, disease prevention, and control measures are required to hinder its spread of sars-cov-2 at homes, communities and healthcare units [33, 34] . in various studies, therapeutic approaches against the ebola virus, zika virus and middle east respiratory syndrome corona virus (mers-cov) were developed using immunoinformatics approaches [3, 31, 35] . the purpose of this study was to pinpoint the potential t-cell and b-cell epitopes from sars-cov-2 structural proteins which can be further joined through adjuvant and linkers to design a multiepitope-based subunit vaccine (mesv). many in silico approaches were used to validate the structural and physiochemical properties of the mesv. to examine the binding interaction and stability of mesv with human pathogenic receptors, molecular docking analysis has also been carried out. in addition, in silico immune simulation was also performed to validate the immunogenic potential of designed mesv. at the end, the mesv codons were optimized for escherichia coli system and in silico cloning was performed to ensure its expression profiling. flow chart of methodology used in present study is graphically presented in fig. 1 . main structural proteins, surface glycoprotein (s [genbank: qhd43416.1]), envelope protein (e [genbank: qhd43418.1]) and membrane glycoprotein (m [qhd43419.1]) of sars-cov-2 were taken as targets for epitopes screening and vaccine designing against sars-cov-2. their amino acid sequences were collected in fasta format from genbank (https://www.ncbi.nlm.nih.gov/genbank/) [36] . allergenicity and antigenicity (at a threshold of 0.4) of selected proteins were evaluated through aller-top v2.0 (https://www.ddg-pharmfac.net/allertop/) and vaxijen v2.0 (http://www.ddg-pharmfac.net/vaxijen/vaxi-jen/vaxijen.html) respectively [37, 38] . three dimensional (3d) structure of s protein was retrieved from rcsb protein data bank (pdb; https://www.rcsb.org/) [39] . however, 3d structures of other two proteins (e and m) were predicted using homology modeling approach, as their resolved structures are not available yet. raptorx (http:// raptorx.uchicago.edu/) and modeller v.9.12 (https:// salilab.org/modeller/) were employed for homology modeling [40] . predicted models were then visualized by chimera (https://www.cgl.ucsf.edu/chimera/) [41] . galaxy refines server (http://galaxy.seoklab.org/) and modrefiner (https://zhanglab.ccmb.med.umich.edu/modrefiner/) was used to refine the predicted models [42, 43] . besides, the refined structure needs to be validated based on experimentally validated 3d structure of proteins. refined structures were therefore applied in the prosa web (https:// prosa.services.came.sbg.ac.at/prosa.php) providing a quality score for a given structure [44] . the quality score beyond the usual range of native proteins indicates a possible error in protein structure. ramachandran plot was created by rampage server (http://mordred.bioc.cam. ac.uk/~rapper/rampage.php), where the principle of pro-check is applied to validate the protein structure [45] . structural analysis was performed to later investigate the positions of b-cell epitopes on target proteins. the epitopes of b-cells help to detect viral infections in the immune system. abcpred (http://crdd.osdd.net/ raghava/abcpred/) was used to forecast 14-mer b cell epitopes for target proteins at 0.51 threshold [46] . epitopes evident on the outer surface were picked, and other intracellular epitopes were removed. the vaxijen server tested the antigenicity of the selected epitopes at a threshold of 0.5. b-cell epitope identification was based upon antigenicity, flexibility, linear epitope predictions, hydrophilicity, and surface accessibility [47] . parker hydrophilicity prediction algorithms, emini surface accessibility prediction method, kolaskar and tongaonkar antigenicity scale, and karplus and schulz flexibility prediction tool were used to perform hydrophilicity, accessibility of surface, antigenicity and flexibility analysis respectively [48] . as discontinuous epitopes become more evident and have higher dominant properties than linear epitopes, discotop 2.0 server (http://www.cbs.dtu. dk/services/discotope/) was used to forecast discontinuous epitopes from 3d structures of surface glycoprotein, membrane protein and envelope protein [49] . the position of epitopes on 3d structures of proteins was visualized by pymol (https://pymol.org/2/) [50] . in vaccine designing, t-cell epitopes play a crucial role. more specifically, it reduces the cost and time compared with laboratory experiments [51] . iedb consensus method (http://tools.iedb.org/mhcii/) was used to predict 8-11 mer mhc class-i and 11-14 mer mhc class-ii epitopes. the results of this method are very important due to a large number of human leukocyte antigen (hla) alleles used in the calculation. the sequence was given in a fasta format and all the alleles were selected for prediction. epitopes with less than 2 consensus score believed to be good binders and chosen for further research. antigenicity and allergenicity of the selected epitopes were checked by vaxijen v2.0 and allergen fp v1.0 respectively [52] . protein digest server (http://db.systemsbiology.net: 8080/proteomicstoolkit/proteindigest.html) was used to predict epitopes digesting enzymes. toxinpred (http:// crdd.osdd.net/raghava/toxinpred/) was used for nontoxic/toxic properties prediction of epitopes. non-toxic epitopes were selected for further analysis [53] . the degree of conservation of predicted t-cell and b-cell epitopes within the protein sequence was analyzed by iedb conservancy analysis tool (http://tools.iedb.org/conservancy/). epitopes having conservancy among all 3 selected proteins were shortlisted for further analyses [54] . the expression and distribution of hla alleles vary depending on the world's ethnicities and regions, thereby impacting the effective production of mesv [55] . the population coverage was calculated using the iedb population coverage tool (http://tools.iedb.org/population/), and for this purpose mhc class-i and mhc class-ii epitopes and corresponding hla-binding alleles were considered. this tool estimates population coverage for each epitope for various regions of the world based on the distribution of hla binding alleles [56] . epitopes with the following characteristics are generally preferred to design a subunit vaccine: (a) highly antigenic, (b) immunogenic, (c) non-allergenic, (d) non-toxic, and (e) with significant population coverage. therefore, only those epitopes were selected further to construct mesv following the above parameters. an adjuvant was attached with the eaaak linker to the first cytotoxic t lymphocytes (ctl) epitope to improve the immune response. other epitopes were linked using aay, gpgpg, and kk linkers after validation of their interaction compatibility to preserve their independent immunogenic activity. βdefensin has been used as an adjuvant in the present research since it is a simple 45 amino acids long peptide that acts as an immunomodulator and as an antimicrobial agent both [57] . first, blastp analysis was carried out using default parameters to confirm that the designed mesv sequence is non-homologous against the homo sapiens proteome [58] . protein with less than 37% is commonly known to be a non-homologous. physiochemical properties of the designed mesv were accessed by the protparam tool [59] . protparam predicts various physiochemical properties like (half-life, theoretical isoelectric point [pi], instability index, grand average hydropathy, and aliphatic index) based on the amino acid approximations involved in the pk [60] . allertop v.2.0 server was used to analyze the allergenicity of the mesv construct [38] . the secondary structure of the mesv construct was evaluated using a psipred workbench [58] . this test also evaluated various vaccine properties such as alpha helices, extended chain, degree of beta turns, and random coil. the 3d structure of mesv was predicted using the de novo modeling approach of cabs fold server (http://biocomp.chem.uw.edu.pl/cabsfold/), since the designed mesv was a series of epitopes and no appropriate template was available [61] . this server is based on a cabs modeling approach that combines a multi-scale modeling pipeline with an exchange replica monte carlo scheme. predicted mesv 3d structure was modified using a galaxy refine server [62] . the ramachandran plot analysis was carried out using the rampage server (http://mordred. bioc.cam.ac.uk/~rapper/rampage.php) [45] , to confirm the quality of the refined mesv structure, followed by the structural validation analysis using the prosa web server [44] . the errat server (https://servicesn.mbi.ucla.edu/ errat/) was also used to evaluate the calculation of unbounded interactions in the mesv structure [63] . besides, linear b-cell epitopes were predicted from the mesv using the abcpred server [46] . ellipro tool (http:// tools.iedb.org/ellipro/) was used to predict the conformational b-cell epitopes of the designed mesv using default settings (maximum distance: 6 a°; minimum score: 0.5), provided by iedb-ar v.2.22. it predicts epitopes by estimating residual protrusion index (pi), protein shape, and neighbor residue clustering [64] . all together for the appropriate evocation of immune response, the interaction amongst the antigenic molecule and immune receptor molecule is essential. molecular docking was performed to analyze the interaction between mesv construct and human immune receptors. toll-like receptors-3 (tlr3) has been thoroughly studied, and studies found its key role in antiviral immune response generation. gramm-x (http://vakser.compbio. ku.edu/resources/gramm/grammx/) was used for the mesv docking with tlr3 (pdb id: 1ziw) [65] . pymol was utilized for visualization of the docked complexes [50] . moreover, for the achievement of the conventional sketch of interactions among docked proteins, an online server pdbsum (http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=index.html) was utilized. it analyzes the protein-protein interactions among docked molecules [66] . an in silico immune simulation was performed using c-immsim 10.1 server (http://150.146.2.1/c-immsim/ index.php?page=0) to validate the immunological responses of the designed mesv. c-immsim simulates the three main components of the functional mammal system (thymus, lymph node, and bone marrow) [67] . the input parameters for the immune simulations are as follows: volume (10), hla (a0101, a0101, b0702, b0702, drb1_ 0101, drb1_0101), random seed (12345), number of steps (100), number of injection set to 1. the rest of the parameters were considered to be the default. codon optimization is a method to improve the translation effectiveness of foreign genes in the host if the use of codon is different in both organisms. codon optimization was carried out followed by in silico cloning, after the careful evaluation of mesv properties and immune response. to make this method consistent with the commonly used prokaryotic expression system; e. coli k12 [68] , the java codon adaptation tool (http://www.jcat.de/) [69] was used for mesv codon optimization. the other available choices were selected to evade: (i) termination of rho-independent transcription, (ii) binding-site of prokaryote ribosome, and (iii) cleavage-sites of restriction enzymes. codon adaptation index (cai) [70] along with the gc (guanine and cytosine) contents were assessed. sticky ends of the restriction sites of hindiii and bamhi restriction enzymes were added to allow restriction and cloning, in the start/n terminal and end/c terminal of the modified mesv sequence, respectively. the modified nucleotide sequence of mesv was additionally cloned into the e. coli pet30a (+) vector by using snapgene tool (https://www.snapgene.com/ ), to assure its in vitro expression. sequence and structural analysis of the target proteins all target structural proteins were found to be nonallergenic and highly antigenic. e protein was the most antigenic followed by m and s protein with 0.60, 0.51 and 0.46 antigenic values, respectively. the 3d structure of s protein was retrieved from protein-data-bank using id: 6vyb [39] . the 3d structure of e protein was determined using homology modeling. chain-a of envelope small membrane protein of sars-cov (pdb id: 5x29) was found to be the best template (percent identity 88.71%) for e protein of sars-cov-2. however, no suitable template was found for m protein, so its structure was predicted by raptor x [71] . visualization of the models was done by chimera (additional file 1: fig. s1 ). the quality factor (z-score) and ramachandran plot values of refined predicted models are mentioned in additional file 2: table s1 (additional file 1: fig. s2-s3) . total 23 linear epitopes (s-19, e-1, and m-3) were selected. among the chosen linear epitopes, 'ilpvsmtkts vdct' of s protein showed the highest antigenicity (1.6) and predicted score (additional file 3: table s2 ). the positions of epitopes on their respective protein structures were visualized by pymol (additional file 1: fig. s4) . identification of b cell epitope was based on antigenicity, flexibility, linear epitope predictions, hydrophilicity, and surface accessibility. parker hydrophilicity prediction algorithms, emini surface accessibility prediction method, kolaskar and tongaonkar antigenicity scale, and karplus and schulz flexibility prediction tool were used to perform hydrophilicity, accessibility of surface, antigenicity and flexibility analysis respectively (additional file 1: fig. s5-s7) . to further improve the specificity and variety of b-cell epitopes, discotop 2.0 server was used to calculate surface abundance concerning residual contact number and use the novel amino acid score to forecast discontinuous epitopes. 3d structures of the target proteins were used to predict discontinuous epitopes; 90% specificity, − 3.700 thresholds and 22.000 angstroms propensity score radius. fifty-five discontinuous epitopes of s protein, 1 epitope of the e protein and 22 epitopes of m protein were calculated (additional file 5: table s4) . epitopes that are bound to multiple alleles, highly antigenic, non-allergenic and 100% conserved were screened out, and their antigenicity and allergenicity were checked. based on these criteria, 9 mhc class-i (s-3, e-3, and m-3) and 7 mhc class-ii (s-1, e-3 and m-3) were shortlisted (additional file 6: table s5 ). protein digest server was used to estimate epitopes/peptides digesting enzymes. epitopes digestible with many enzymes are not stable. less enzyme digested epitopes, on the other hand, are very stable and favored vaccine candidates (additional file 7: table s6 ). total three ctl epitopes (s-1 and m-2), six htl epitopes (e-3 and m-3), and four b-cell epitopes (s-3 and m-1) were selected to construct mesv ( table 1) . the selected epitopes showed 88.40% of the world population coverage (fig. 2) . results revealed that predicted epitopes are showing promising population coverage of the countries strongly affected by covid-19 including, germany, france, spain, saudi arabia, england, italy, iran, the philippines, the united states, and sweden. a mesv construct was further developed using all selected epitopes. using the eaaak linker, an adjuvant (45 amino acid long b-defensin) was bound at the beginning (to the mev n-terminal). eaaak linker reduces connections to other protein areas with efficient detachment and improves stability [58, 72] . epitopes were merged in a sequential manner with aay, gpgpg, and kk linkers, respectively, based on the compatibility of their interaction. two hundred seventy-six amino acids represented the final mesv construct (fig. 3) . first, blastp analysis was carried out against the homo sapiens proteome, and the results revealed that mesv does not resemble any human protein (higher or equal to 37%). the vaccine structure was then tested for toxicity, allergenicity, and antigenicity. mesv was found to be non-allergenic, highly antigenic (0.6737), and non-toxic. the mean half-life of the construct was calculated as 30 h in vitro, > 20 h in yeast and > 10 h in vivo. molecular weight and theoretical pi of the vaccine were 3157.01 kda and 10.31 respectively. grand average hydropathicity was calculated as 0.395. a positive score of the grand average of hydropathy suggests its hydrophobic nature. the secondary structure analysis show cabs fold server was used to predict the tertiary structure of the mesv (fig. 4) . the structure was refined by the galaxy refine server. ramachandran plot analysis of improved model showed that 89.4% amino acids are in favored region, 6.9% amino acids in the allowed region and 3.6% amino acids in the outlier region. further analysis showed that the qrmsd is 0.544, molprobity is 2.356, poor rotamers are 0.0%, clash score is 17.7 and z-score is − 4.8. in quality check analysis by errat, the refined model score was 82.4561. . aay linkers (blue) used to join the ctl epitopes, gpgpg linkers (green) used to join the htl epitopes and kk linkers (gray) used to join the b-cell epitopes fig. 4 a mesv construct sequence. epitopes sequence is in black. the adjuvant sequence is highlighted in brown color, eaaak linker sequence is highlighted in blue, aay linkers are highlighted with orange, gpgpg linkers are highlighted with green and kk linkers are highlighted with maroon color; b mesv construct refined 3d structure pipes representation (alpha helix: green; beta strands: blue; loops: gray); c ramachandran plot analysis of predicted structure shows 89.4% residues are present in the favored region b-cell epitopes screening from mesv b-lymphocytes also produce antibodies that provide humoral immunity, in addition to the secretion of cytokines. eighteen linear/continuous (additional file 8: table s7 ) and six conformational/ discontinuous epitopes (additional file 9: table s8 ) from the mesv sequence were predicted without altering abcpred 2.0 and ellipro prediction parameters. to start the immune response, an appropriate interaction among the antigenic molecule and immune receptor molecule is needed. to decode the binding potential of mesv to the innate immune receptors, bioinformatic modeling driven molecular docking of the designed mesv to a representative innate immune receptor tlr3 was performed. the docking evaluation forecast that the best complex with a net global energy of − 22.36 kj/mol. visual analysis of the complex leads to the observation of the mesv's deep binding in the center of tlr3 and favors rigorously hydrogen and weak van dar waals interactions with specific tlr3 residues. pdbsum was used to gain insights and pin down possible residues of mesv making stable bonds with tlr3 (fig. 5) . within 3 å, the mesv was observed to form 14 hydrogen bonds with tlr3 potential residues. all secondary and primary immune responses tend to contribute significantly to the pathogen and may be consistent with the actual immune response. the in silico host immune system response to the antigen is shown in fig. 6 . the primary response was characterized by high igg + igg and igm concentration, followed by igm, igg1 + igg2 and igg1 at both the secondary and primary stages with concomitant antigen reduction. additionally, robust interleukin and cytokine response was observed. all of this indicates the mesv's successful immune response and clearance after subsequent encounters. in silico cloning within e. coli system in silico cloning was done to assure the expression of mesv derived from sars-cov-2 in widely used e. coli hosts. first, the codons of mesv were modified according to the use of codons of e. coli expression system (strain k12). the optimized mesv construct contains 828 nucleotides, cai value of 1.0 (0.8-1.0), and an optimal range of gc content of 53.2% (30-70%) demonstrating the strong potential for reliability and positive protein expression. in the following step, both ends of mesv optimized nucleotide sequence were attached to buffer compatible restriction enzymes bamhi and hindiii restriction sites to assist the purification/cloning process. finally, the refined mesv sequence was cloned to the several cloning sites of the pet30a (+) vector between the restriction sites. the clone was 6.23 kb long (fig. 7 ). covs have long been considered as insignificant pathogens causing "colds" in humans. in the twenty-first century, two extremely pathogenic covs named sars-cov and mers-cov emerged from the livestock reservoirs and cause deadly outbreaks. a new strain of cov officially named as sars-cov-2 was identified recently, which started a deadly global pandemic of covid-19. the final dimension and impact of this pandemic are currently uncertain due to the rapidly changing situation [4] . after the recombination of various virus genomes particles, the novel virus infects the host cells rapidly. no reliable medication is currently available for the said infection. covid-19 infection is a severe problem of morbidity and mortality worldwide. unfortunately, the unavailability of the vaccinations against covid-19 has impacted several precious lives, in different regions of the world. the emergence of covid-19 results in a significant global disease burden, for which preventative measures are urgently needed. to successfully eradicate the disease, researchers have been trying to collect data associated with covs to understand its transmission, pathophysiology, and biology [73] . the rapid development of structural and genomic databases combined with computational tools helps in the design and discovery of new vaccine candidates. recent advancements in the immunological bioinformatics area have resulted in a variety of tools and servers that can lessen the time and cost of traditional vaccine advancement. due to the problems in the selection of suitable antigen candidates and immunodominant epitopes, the development of effective multiepitope vaccines remains toilsome. thus, the prediction of appropriate antigenic epitopes of a targeted protein by the immunoinformatics approaches is very essential for designing a mesv [74] . here, we explored the development of epitope-based vaccines targeting the structural proteins (s, m, and e) of the sars-cov-2. these proteins play a crucial in the replication cycle and the virus particle structure. the sprotein plays an important part in binding the virus to the host cell surface receptors and consecutive fusion to promote the viral entrance in the host cell [75] [76] [77] . m and e proteins are important for replication, particle assembly within human cells, and viral entry [78, 79] . tand b-cell epitopes of the target proteins were predicted to support the host's immune response. the research was performed at primary, secondary and tertiary structural levels of proteins. iedb analysis resource and abcpred predicted b-cell conserved epitopes. the position of epitopes on 3d structures of proteins was visualized by pymol. discotop server was used to predict discontinuous epitopes. to further improve specificity and selectivity, allergenicity, toxicity, and physiochemical properties of predicted epitopes were checked. digestion analysis verified that the peptides predicted during the analysis were stable and safe to use. an appropriate mesv should be designed with b-cell, htl, and ctl epitopes and cause effective reactions to a specific virus [80] . few groups developed sars-cov-2 subunit vaccines but only used a single protein for the vaccine design [15, 81, 82] and the use of ctl epitopes only without taking into account the importance of htl or b cell epitopes [83] . however, we have incorporated b-cell epitopes in addition to t-cell epitopes from multiple structural proteins, because of the functions they play in inducing antibody production and mediating its effective features [84] . besides, the humoral response of memory b-cells can be easily overcome by the onset of antigens, while the cell-mediated immunity (t-cell immunity) in many cases leads to long-life immunity [85] . ctl limits pathogen spread through the secretion of unique antiviral cytokines and the identification and destruction of infected cells [86] . therefore, the present vaccine construct has an advantage over already reported constructs. the hla alleles retain their response to t-cell epitopes which are highly polymorphic in different ethnic groups. to gain more population coverage, the t-cell epitopes are paired with more alleles. so we chose the htl and ctl epitopes with their respective hla alleles to predict the worldwide distribution of the alleles. the results showed that the chosen epitopes and their corresponding alleles preferably cover various geographical vaccine candidates were chosen form ctl, htl, and b cell epitopes depending on their antigenicity, toxicity, immunogenicity, population coverage, and allergenicity. the mesv was designed by joining the htl, ctl, and b cell epitopes with gpgpg, aay, and kk linkers respectively. linkers are introduced as an indispensable element in the mesv development to enhance folding, stabilization, and expression. multi-epitope based vaccines are poorly immunogenic when used alone, and need adjuvant coupling [87] . adjuvants are ingredients in a vaccine formulation that protects against infection and affect certain immune responses, growth, stability, and durability of antigens [88] . therefore, 45 amino acids long, an adjuvant β-defensin, was integrated with the eaaak linker whose length is 5, at n-terminal. the eaaak linker is used to integrate the first epitope and adjuvant to facilitate efficient separation of the bifunctional fusion protein domains [89] . the final vaccine stretch with the addition of adjuvant and linkers was discovered to be 276 amino acid long. the analysis of physiochemical characteristics of the mesv construct has shown that it is stable, basic, and hydrophobic. mesv was basic, according to the theoretical pi value, which can ensure stable physiological ph interaction. the calculated aliphatic index and instability index scores showed that the vaccine protein may be stable and thermostable. a positive score of the grand average of hydropathy suggests its hydrophobic nature. mesv has been found to be immunogenic, strongly antigenic, and non-allergenic. this suggests the ability of the epitopic vaccine to elicit a strong immune response without allergic reactions. the 3d structure prediction provides extensive knowledge of the spatial arrangement of essential protein components and provides excellent support for the study of ligand interactions, protein functions, dynamics, and other proteins [90, 91] . after refinement, the desirable characteristics of the mesv construct improved considerably. the ramachandran plot analysis shows that most residues are present in favored and allowed regions with very few residues in the disallowed region, which shows a satisfactory overall quality of the model. the good quality of designed mesv construct is further indicated by rmsd value, poor rotamers, clash score, and mol-probity. various structure validation methods have been used to detect errors in the modeled mesv construct. the errat quality factor (82.4%) and z-score (− 4.8) proved that the overall structure of the refined mesv is of good quality. an adequate interaction between the antigen molecule and the immune receptor molecules is important for triggering an immune response. the refined mesv construct was then docked against tlr3 to examine adequate binding to immediate immune response. stable interactions were observed among the mesv and tlr3 in molecular docking analysis, and less energy was needed for proficient binding. b-and t-cell epitopes consisting multi-epitope vaccine should hypothetically activate both humoral and cellular immune reactions. with substantial il-10 and il-2 activities, our vaccine demonstrated the highest production of ifn-γ. antibodies also provide extracellular sars-cov-2 protection. we have also noticed excess immunoglobulins that are active, i.e., igm, igg, and their isotypes that may be involved in switching isotype. besides, the irrelevant simpson index (d) recommends a diverse immune reaction that is conceivable as a subunit vaccine contains various b-cell and t-cell epitopes. the translation efficiency of foreign genes inside the host system varies because of the incompatibility of mrna codons, which require codon optimization for higher expression [92] . cai value obtained was 1.0 and gc content (53.2%) was also within the optimum limit suggesting possible higher expression in the e. coli k-12 system. the main aim of mesv in silico cloning was to direct genetic engineers and molecular biologists on the expected expression level and the potential cloning sites in a particular expression system i.e., e. coli k12 system. we applied the next-generation vaccine designing approach in this research to create a mesv construct, capable of generating immunological responses against the sars-cov-2. we believe that our vaccine will successfully produce humoral and cell-mediated immune responses. interaction and binding patterns between receptor and vaccine protein were stable and higher. moreover, in immune simulation, effective immune responses were observed in real life. thus, mesv designed carefully using such a methodology could become an important asset in combating viral infections. computational/immunoinformatics approaches rely on experimental methodologies to generate initial raw data for further analyses. the data quality and efficiency of computational algorithms being applied, can limit the accuracy of immunoinformatics predictions. therefore, further in vivo and in vitro investigations are however required to ensure the real potential of designed mesv to combat covid-19. taken together, we characterized sars-cov-2 structural proteins (s, e, and m) for antigenic epitopes and proposed a potential mesv utilizing various immunoinformatics and computational approaches. the findings of this research could save time and related costs for the study of experimental epitope targets. the mesv can activate all host immune system components and has adequate physicochemical and structural properties. it also appears to interact very stably with an innate immune receptor tlr3, making it more likely to be introduced into the host immune system. to reveal its effectiveness 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computational screening of medicinal plant phytochemicals to discover potent pan-serotype inhibitors against dengue virus investigating the molecular mechanism of staphylococcal dna gyrase inhibitors: a combined ligand-based and structure-based resources pipeline novel immunoinformatics approaches to design multi-epitope subunit vaccine for malaria by investigating anopheles salivary protein authors would like to acknowledge guangxi university and government college university faisalabad for providing facilities for this study. supplementary information accompanies this paper at https://doi.org/10. 1186/s40249-020-00752-w.additional file 1: figure s1 . 3d structural representation of sars-cov-2 structural proteins: (a) s protein, (b) e protein and (c) m protein. figure s2 . (a) the e protein contains α-helix (77.33%, 58) and random coil (22.66%, 17) ; (b) the z-score (0.41) of the e protein; (c) the ramachandran plot of refined structure shows 97.3, 2.7 and 0.0% residues in favored, allowed and disallowed region, respectively. figure s3. (a) the m protein contains α-helix (40.54%, 90), β-strand (24.32%, 54) and random coil (35.13%, 78) ; (b) the z-score (− 3.88) of the m protein; (c) the ramachandran plot of refined structure shows 96.8, 2.7 and 0.5% residues in favored, allowed and disallowed region, respectively. figure s4 table s1 . structural details of the sars-cov-2 structural protein predicted models.additional file 3: table s2 . linear b cell epitopes predicted through abcpred 2.0 server (nt: nontoxic).additional file 4: table s3 . emini surface accessibility of sars-cov-2 structural proteins.additional file 5: table s4 . discontinuous epitopes predicted through discotop 2.0 server. additional file 6: table s5 . mhc class-i allele and mhc class-ii binding peptides with their antigenicity scores. additional file 7: table s6 . digestion, allergenicity, toxicity and physiochemical profiling of selected peptides (na: not allergic; nt: nontoxic).additional file 8: table s7 . linear b cell epitopes predicted in vaccine construct.additional file 9: table s8 . conformational epitopes in 3d structure of vaccine. availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. all authors have no competing interests. key: cord-327000-oyg3oyx1 authors: li, shasha; yang, jinping; zhu, zixiang; zheng, haixue title: porcine epidemic diarrhea virus and the host innate immune response date: 2020-05-11 journal: pathogens doi: 10.3390/pathogens9050367 sha: doc_id: 327000 cord_uid: oyg3oyx1 porcine epidemic diarrhea virus (pedv), a swine enteropathogenic coronavirus (cov), is the causative agent of porcine epidemic diarrhea (ped). ped causes lethal watery diarrhea in piglets, which has led to substantial economic losses in many countries and is a great threat to the global swine industry. interferons (ifns) are major cytokines involved in host innate immune defense, which induce the expression of a broad range of antiviral effectors that help host to control and antagonize viral infections. pedv infection does not elicit a robust ifn response, and some of the mechanisms used by the virus to counteract the host innate immune response have been unraveled. pedv evades the host innate immune response by two main strategies including: 1) encoding ifn antagonists to disrupt innate immune pathway, and 2) hiding its viral rna to avoid the exposure of viral rna to immune sensors. this review highlights the immune evasion mechanisms employed by pedv, which provides insights for the better understanding of pedv-host interactions and developing effective vaccines and antivirals against covs. porcine epidemic diarrhea virus (pedv) is the etiological agent of porcine epidemic diarrhea (ped) that causes an acute and highly contagious enteric disease of swine characterized by vomiting, diarrhea, dehydration, and anorexia in pigs of all ages, especially resulting in severe diarrhea and high mortality rate in piglets. in serious cases, outbreak of ped even leads to a mortality rate of 100% in pigs [1] [2] [3] . the causative agent of ped was first described in the 1970s in england [4] . in 1976, an unrecognized enteric disease was reported in several european countries [5] . the viral diarrhea was collectively designated "ped" in 1982 [6] . endemic ped had been described in both developed and developing countries, but with a low impact on the swine industry until 2010. in 2010, outbreaks of ped caused by highly pathogenic variant pedv strains occurred in china, and this was immediately reported in other asian counties, causing up to 100% mortality in suckling piglets [1, [7] [8] [9] . in april 2013, pedv entered the united states (us) for the first time and the virulent strains spread rapidly across the us [10]. apart from almost 100% mortality rate in suckling piglets, pedv infection also damaged the growth performance of finishing pigs [11] . the highly pathogenic strains of pedv spread worldwide, resulting in serious problems to the swine industry and substantial economic losses [7, 10, 12, 13] . vaccination used to be the main strategy to prevent and control the rate of pedv infection [14] , however, the current available pedv vaccines cannot provide complete protection for the pigs affected by the highly pathogenic strains. the optimal vaccines should induce efficient maternal antibodies in sows that could be transmitted to the offspring and protect neonatal suckling piglets from pedv. the vaccination 1 (s1) protein [34] . apart from apn, pedv is able to bind to sialic acids [36] . it remains unknown whether pedv uses sugar coreceptors during viral infection [34, 36] . pedv infects multiple cell lines from different species including bat and primate (human and non-human) in vitro. the ability of pedv to infect the cells of different species indicates that the virus utilizes the evolutionarily conserved cell components as receptors, thereby enhancing the potential for cross-species and potentially, zoonotic transmission [37, 38] . the highly pathogenic variant strains of pedv were identified in 2010 and caused a high morbidity of up to 100% in piglets, and since then, these strains become dominant, leading to most of the acute outbreaks of ped worldwide [1, 7, 8] . the high virulence of these strains is critically associated with the immune evasion mechanisms employed by the virus. pedv has evolved different strategies to delicately manipulate and damage the host innate immune system for their multiplication. clarification of these mechanisms is critical for understanding the host range, tropisms, pathogenesis, and for developing effective vaccines and antiviral drugs to curb the spread of pedv in pigs. in this review, we provide an overview of different mechanisms used by pedv to evade host innate immune responses. pedv is an enveloped, positive-sense, single-stranded rna virus. the pedv genome constitution represents a standard cov arrangement. the viral genome is approximately 28 kb in length, containing a 5 terminal cap, a 3 poly (a) tail, as well as seven open reading frames (orfs), including the orf1a, orf1b, s, orf3, e, m, and n genes ( figure 1 ) [39] [40] [41] . the n terminal orf1a and orf1b encode two large replicase polyprotein precursors (pp1a and pp1ab), which are subsequently processed into 16 nonstructural proteins (nsp1 to 16). orf1a encodes pp1a which is cleaved by viral proteases into 11 nonstructural proteins (nsp1-nsp11). the orf1b generates five additional nonstructural proteins (nsp12-16) that are proteolytically cleaved by the viral proteases from pp1ab [42] . nsp3 contains two papain-like protease domains (plp1 and plp2 or pl pro ) that cleave the nsp1-4 region of the replicase polyprotein at three sites. nsp5, a chymotrypsin-like enzyme also known as 3c-like protease, cleaves the polyprotein at remaining cleavage sites [43] . the c terminus of viral genome contains five orfs, encoding four structure proteins (spike protein (s), small envelope glycoprotein (e), membrane glycoprotein (m), and nucleocapsid protein (n)), as well as a hypothetical accessory protein orf3 [44] [45] [46] . the 16 nsps, together with the n protein, and several host proteins, form a large replication and transcription complex (rtc) that engages in the minus-strand rna synthesis, using viral genomic rna. these nsps play important roles in virion structure modification and the replication and transcription of pedv [47] . the c terminus of viral genome contains five orfs, encoding four structure proteins (spike protein (s), small envelope glycoprotein (e), membrane glycoprotein (m), and nucleocapsid protein (n)), as well as a hypothetical accessory protein orf3 [44] [45] [46] . the 16 nsps, together with the n protein, and several host proteins, form a large replication and transcription complex (rtc) that engages in the minus-strand rna synthesis, using viral genomic rna. these nsps play important roles in virion structure modification and the replication and transcription of pedv [47] . the viral proteins of pedv perform different biological functions during viral entry, replication cycle and propagation (table 1) . pedv s protein, a type i membrane glycoprotein protein located on the envelope of the virus, consists of an n-terminal signal peptide, a large extracellular region, a single transmembrane domain, as well as a short cytoplasmic tail [48, 49] . the ectodomain of s protein comprises s1 and s2 subunits. the n-terminal s1 region, containing n-and c-terminal domains (s1-ntd and s1-ctd), is mainly responsible for receptor binding [50] . the c-terminal membrane-anchored s2 region is mainly involved in triggering the fusion of the viral envelope with host cell membranes [48, 49] . the interaction of the cov s protein with its host cell surface receptor is a key determinant for host tropism. the s1-ctd of most known members of α-cov genus, including pedv, interacts with aminopeptidase n (apn) to entry into the target cell [32, 36, [51] [52] [53] [54] . in addition, s protein contains the epitopes that are the major targets of the neutralizing antibody. n protein is the most abundant viral protein during the early phase of infection in cov-infected cells [55] . similar to the n proteins of other covs, the pedv n protein has multiple functions, such as acting as a structural protein that forms nucleocapsid with viral genomic rna, playing important roles in viral replication, transcription, and assembly [56, 57] . the expression of the n protein in intestinal epithelial cells extends the s-phase of cell cycle, causes endoplasmic reticulum (er) stress, and upregulates interleukin-8 expression [44] . moreover, the n proteins of several α-covs and β-covs, including pedv, pdcov, sars-cov, and mouse hepatitis virus (mhv), have been identified as innate immunity antagonists [58] [59] [60] [61] . however, the involved antagonistic mechanisms are particularly different. pedv m protein participates in virion assembly and virus budding through collaboration with other viral proteins, and engages in the induction of neutralizing antibodies against pedv [62, 63] . pedv m protein is distributed throughout the cytoplasm. it induces the cell growth retardation in intestinal epithelial cells (iec) and arrests the cells in s-phase [64] . in addition, pedv m protein is identified as an interferon (ifn) antagonist with an unrecognized mechanism [65] . pedv e protein is important for the virus packaging and budding [66] . it is predominantly localized in the er, having no effect on cell growth, cell cycle and cyclin a expression in iec. however, it causes er stress and activates the nuclear factor-κb pathogens 2020, 9, 367 5 of 27 (nf-κb) pathway which is responsible for the up-regulation of il-8 and the anti-apoptotic protein bcl-2 expression [67] . orf3 has been predicted to possess multiple transmembrane domains [68] , while it is predominantly distributed in the cytoplasm [69] . orf3 also detains cells at s-phase, facilitating vesicle formation, and thus promoting pedv multiplication [69] . a recent study suggests that orf3 interacts with s protein during pedv assembly and consequently benefits viral replication [70] . cov nsps play multiple roles in the synthesis or processing of viral rna, or in virus-host interactions aiming to create an optimal environment for virus replication, such as facilitating viral entry, viral gene expression, rna synthesis, and virion release. nsp1 is a n-terminal cleavage product of orf1a polyprotein [71] , a 9-kda protein, that exists only in α-covs and β-covs [72] . the nsp1 of α-covs is not very similar to β-covs nsp1 with regard to sequence homology and size [73, 74] . based on the sequence alignment analysis of the genomes of different covs, the viral nsp1 can be regarded as a genus-specific marker [75] . moreover, β-covs nsp1 has been widely reported to inhibit host protein expression. however, the biological functions of α-covs nsp1 remain largely unknown. despite the lack of overall sequence similarity, the nsp1 of different covs shares a similar function to interfere with host protein expression [76] . these studies suggest the importance of nsp1 in the life cycle of different lineages of covs. it is shown that tgev nsp1 inhibits host gene expression and is critical for viral virulence [77] . pedv nsp1 induces the degradation of cbp and nf-κb to abate ifn response [78] , but the detailed mechanisms remain unclear. the sizes and amino acid sequence identity of nsp 2 are variable among different covs [76] . nsp2 of mhv and sars-cov are involved in viral rna synthesis [79] . pedv nsp2 has unknown functions in replication, and may implicate the virus-host interactions and virulence. nsp3 is the largest nsp protein, containing two papain-like protease (plp1 and plp2) domains, of which pedv plp2 acts as a viral deubiquitinase (dub), to negatively regulate type i ifn signaling [80] . covs plps domains exhibit multiple functions, serving as a viral protease, dub, as well as an ifn antagonist [81] . covs, like other positive-stranded rna viruses, induce membranous rearrangements of varying morphologies that are essential for rtcs anchoring [82, 83] . the cov-induced replicative structures consist of double-membrane vesicles (dmvs) and convoluted membranes (cms), which form a large reticulovesicular network that are critical for viral replication and transcription [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] . among the cov nsps, nsp3, nsp4, and nsp6 include the hydrophobic transmembrane domains engaging in anchoring the viral rna synthesis components to the membranes [94] . for mers-cov and sars-cov, co-expression of nsp3 and nsp4 is required to induce dmvs [95] . sars-cov nsp6 has membrane proliferation ability as well, which also contributes to dmvs formation [96] . the structure and functions of α-cov nsp3 are largely unknown [97] . nsp4 is also a marker for cov-induced membrane structures; some results indicate that the nsp4-10 of pp1a act as a large complex through multidomain structure or scaffold during viral rna replication progress, before its cleavage into individual products [98] . covs nsp5 encodes a 3c-like proteinase (3cl pro ). the polyproteins pp1a and pp1ab are processed into individual elements of replicase by 3c-like protease and plps [99] . moreover, pedv nsp5 plays a crucial role in virus replication and also blocks host innate immune responses [100] . crystallographic or nuclear magnetic resonance structures have shown that nsp3, nsp5, nsp7, nsp8, nsp9, and nsp10 have the plprob and the adp-ribose 1 -phosphatase (adrp) activity [101] [102] [103] [104] [105] [106] [107] . the crystal structure of sars-cov nsp9 suggests that nsp9 is dimeric and it is able to bind to single-stranded rna [108] . the crystal structure of sars cov nsp10 protein suggests that nsp10 is a zinc-finger protein, which is existent exclusively in covs so far [107] . moreover, nsp7-10 have rna binding activity and nsp12 encodes a single rna-dependent rna polymerase (rdrp). the biochemical characterization and crystallization of sars cov nsp7 and nsp8 manifests that eight copies of nsp8 and eight copies of nsp7 form a supercomplex [106] . the complex is supportive for nucleic acid binding and may be associated with the processivity of viral rdrp [102, 106] . recently, structural studies have described that the sars-cov nsp12 polymerase binds to the nsp7 and nsp8 complex [109] that may increase the polymerase activity of nsp12 rdrp [110] . cov nsp13, a ntpase/helicase, is also determined to play essential roles in viral replication [111] . cov nsp14 is a multifunctional protein with 3 -5 pathogens 2020, 9, 367 6 of 27 exoribonuclease activity and n-7-methyltransferase [mtase] activity [112, 113] . nsp14 catalyzes the n7-methylation of gppp-rna to form a cap-0 structure. cov nsp15 encodes an endoribonuclease (endou), performing functions through a hexamer in many covs [114] [115] [116] . the endoribonuclease activity of nsp15 is not essential for cov replication [117, 118] . for covs, the 5 end of the viral genomic rna and subgenomic mrna (sgmrna) is supposed to have cap structures: an n-7 methylated guanosine nucleoside (m7gpppn) (cap 0) and a methyl group at the 2 -o-ribose position (cap 1) of the first nucleotide [119] . these cap structures enhance the initiation of translation of viral proteins, protect viral mrnas against cellular 5 -3 -exoribonuclease and limit the recognition of viral rna by host innate system [120, 121] . nsp13 is proposed to catalyze the first step of the 5 -capping reaction of viral rnas [122] . the methylation of the two sites in the 5 cap are catalyzed by three nsps; nsp14 (the n-7-mtase), nsp16 (the 2 -o-methyltransferase), and nsp10 [112, [123] [124] [125] [126] . in addition, the 3 -5 exoribonuclease activity of nsp14 is involved in a replicative mismatch repair system during rna synthesis, which improves the replication fidelity of cov [42] . although these nsps have been demonstrated to play essential roles in viral replication, transcription and/or post-translational polyprotein processing [127] , the nsp12-16 of pedv and other covs are poorly characterized to date, except for sars-cov. nsp7 is an ifn antagonist; nsp8 inhibits type iii ifn response; nsp9 is involved in nucleic acid binding; nsp10 enhances the inhibitory effect of nsp16 on ifn-β production. [65, [131] [132] [133] nsp12 encoding a rdrp; viral replication [134] nsp13 a ntpase/helicase that is essential for viral replication [111] nsp14 n-7-mtase; catalyzing n7-methylation of gppp-rna to form a cap-0 structure; 3 -5 exoribonuclease activity involves in a replicative mismatch repair system during rna synthesis; ifn antagonist [42, 65, 112, 113 host cells generally defend against virus infection by mounting an innate antiviral immune response to prevent the spread of the infection and aid in initiating an adaptive immune response which eventually removes the viruses from host. therefore, the first barrier to restrain viral infections is the host innate immune system, which is related to multiple proteins and mechanisms, including ifns, inflammatory cytokine, apoptosis, autophagy, and so on. the activation of type i ifn responses is composed of three stages: (1) recognition of pathogen-associated molecular pattern (pamp) by prrs; (2) secretion of type i ifns through paracrine or autocrine pathways; and (3) expression of numerous antiviral ifn-stimulated genes (isgs) which bring the host into the antiviral state [136] . at least three important prrs have been identified in recognition of viral nucleic acids, including retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) (detection of viral rna in the cytoplasm) [137] , the membrane-bound toll-like receptors (tlrs) (recognition of viral rna or dna in the endosome) [138] , as well as a structurally unrelated group of viral dna sensors (e.g., cgas (cyclic gmp-amp synthase) and ifi16) localized in the host cytoplasm and/or nucleus [139] . in the cytosol, the formation of specific secondary structure of viral rna is closely related to viral rna delivery and replication. these molecular signatures are detected by rlrs including rig-i (also known as ddx58), melanoma differentiation-associated gene 5 (mda5), and laboratory of genetics and physiology-2 (lgp2) [140, 141] . prrs recognize pamps, inducing an intracellular signaling cascade, thus leading to the activation of transcription factors such as irfs and nf-κb, which in turn induce the production of ifns [142] . both rig-i and mda5 have two n-terminal card domains to interact with the card domains of the downstream adaptor proteins, and a dead/h-box rna helicase domain for rna binding. however, lgp2 does not have the n-terminalcard domains, and the involved functions remain unclear [143] [144] [145] . dsrna, as a specific secondary structure of viral rna, can be sensed by rig-i/mda5 to induce ifn-α/β production through the cascade activation of the rlr pathway [146] [147] [148] . activated rig-i/mda5 forms homo-oligomers and recruits the adaptor mitochondrial antiviral signaling (mavs) to induce the formation of signaling complex of mavs with other proteins on mitochondria. tnf receptor-associated factor 6 (traf6) associates with tnfr1-associated death domain protein (tradd), tripartite motif 14 (trim14), and pyruvate carboxylase (pc), resulting in the activation of iκb kinases (ikks). ikks (ikkα, ikkβ and ikkγ) phosphorylate nf-κb inhibitor (iκb), leading to the ubiquitination of iκb and its subsequent degradation. nf-κb is then activated and translocated into the nucleus to turn on the expression of proinflammatory and type i ifn. mavs also recruits and activates tbk1/ikkε by trafs that are pre-associated with tbk1/ikkε, via direct interaction between the sdd domain of tbk1/ikkε and the coiled-coil domain of trafs [149] . activated tbk1/ikkε phosphorylates ifn regulatory factors (irf3/irf7), that further dimerize and import into the nucleus to promote type i ifn production. on the other hand, mavs interacts with mita (also known as sting), that is located in mitochondria and the endoplasmic reticulum (er) membrane. mita interacts with the trap complex, which may be involved in recruiting tbk1 and ikkε to phosphorylate irf3. ubiquitination and deubiquitination are decisive in the regulation of rlr pathways activation [150] . upon binding to viral dsrna, rig-i and mda5 undergo conformational changes and release the n-terminal tandem card domains [151] [152] [153] . the card domains of rig-i are modified by lysine 63 (k63) polyubiquitin chains through the ubiquitin ligases trim25, rnf135, and riplet. this modification is crucial for rig-i to recruit mavs [154, 155] . in addition to the ubiquitination of rlrs, the polyubiquitylation of traf3 and traf6 also play an important role in the regulation of innate immune signaling by activation of tbk1 and ikks, respectively. the k63 polyubiquitin chains can be removed by dubs such as the tumor suppressor protein cyld, duba and a20, providing a mechanism to downregulate immune responses [156] . ubiquitination and deubiquitination are in a dynamic equilibrium to maintain immune homeostasis. type i ifns are secreted by secretory cells and peripheral cells through self-secretion or paracrine secretion manners. extracellular type i ifns bind to a heterodimeric complex composed of subunits of ifn-α receptors 1 (ifnar1) and 2 (ifnar2) located on the cell surface, which activates the tyrosine kinase 2 (tyk2)/janus kinase 1 (jak1) signal transducer. tyk2/jak1 subsequently induces the phosphorylation of transcription factors stat1 and stat2, which dimerize and in turn recruit ifn-regulatory factor 9 (irf9), to form stat1-stat2-irf9 trimerized complex (isgf3). this complex then translocates to the nucleus, where it binds to the ifn-stimulated response elements (isre motif; conserved sequence is tttcnntttc) [157] . the binding of isgf3 to isre finally triggers expression of ifn-stimulated genes (isgs) that directly or indirectly exert antiviral effects in host cells [157] . three types of ifns (types i, ii and iii) have been identified. type i and type ii ifns have been widely reported. type ii ifns only contain ifn-γ. ifn-γ is produced by natural killer (nk) cells and activated cd4 + and cd8 + t cells in response to the cytokines such as interleukin-12 (il-12) and il-18 [158] . ifn-γ binds to the type ii ifn receptor composed of two subunits, ifngr1 and ifngr2. ifngr1 and ifngr2 induce the formation of stat1-stat1 homodimers. stat1-stat1 homodimers translocate to the nucleus and bind to the promoter of the ifn-γ-activation site (gas) elements, to initiate the transcription of ifn-γ-regulated genes [159] . type iii ifns have been explored in recent years, to unravel the underlying mechanisms that manipulate host innate immune responses. type iii ifns in humans contains ifn-λ1 (interleukin 29 ), ifn-λ2 (il-28a), ifn-λ3 (il-28b), and ifn-λ4 [160] [161] [162] . their expression profiles, signaling pathways, and gene expression programs resemble those of type i ifns. the production of type i and iii ifns are both initiated through the recognition of pamps or damage associated molecular patterns (damps) by prrs [163] . despite the fact that the same transcriptional factors are required for the activation of promoters of type i and iii ifns, the nf-κb pathway is a pivotal regulator in ifn-λ production, whereas the irfs pathway dominates type i ifns expression. the promoter of ifn-λ1 includes more nf-κb binding sites compared with in the ifn-β promoter [164] . the signal transduction of type iii ifns depends on the ifn-λ-specific receptor, il-28ra chain and il-10r2 chain [161, 165] . synthetic ifn-λ binds to il-28rα and induces a conformational change within the receptor subunits, that triggers the activation of the receptor-associated tyrosine kinases (tyk2 and jak1), which then phosphorylate stat1 and stat2. stat1 and stat2 are heterodimerized and interact with irf9 to form the isgf3 transcription complex that binds to isre in the promoters of isgs, to induce the expression of hundreds of proteins with antiviral functions [166] . induction of ifn-α/β is the most rapid and effective mechanism for hosts to initiate antiviral innate immune responses. sars-cov, mhv and many other covs are sensitive to ifns. a great number of viral dsrnas intermediates are generated during covs infection that contribute to ifn production, but these covs remain highly pathogenic. as a matter of fact, covs have developed a set of elaborate mechanisms to evade or inhibit the host antiviral innate immune response during virus evolution [134, 167] . the evasive strategies utilized by pedv are classified into four major types: (1) inhibition of rlrs-mediated ifn production pathways, (2) inhibition of the activation of transcription factors responsible for ifn induction, (3) disruption of the signal cascades induced by ifn, and (4) hiding its viral rna to avoid the exposure of viral rna to immune sensors. in the past decade, accumulating evidence demonstrates that pedv n protein, nsp1, plp2, nsp5, nsp15, and nsp16 antagonize type i ifn or type iii ifns production [58, 65, 78, 80, 100, 132, 135, 168] . this explains why only weak ifns' and cytokines' expression is detected in pedv-infected cells [169, 170] . n protein, as an abundantly produced structural protein within cov-infected cells, has multiple functions, including virus replication, transcription, and assembly [56, 134] . pedv n protein has been identified as an ifn antagonist that blocks the expression of ifn-β and isgs by suppression of the pathogens 2020, 9, 367 9 of 27 irf3 and nf-κb activities [58] . pedv n protein inhibits the activation of the ifn-β promoter induced by tbk1 and its upstream rig-i, mda-5, visa, and traf3, while not affecting the activation of the ifn-β promoter driven by irf3. further experiments confirm that n directly interacts with tbk1 to obstruct the association between tbk1 and irf3, which inhibits tbk1-induced irf3 phosphorylation and ifn-β production [58] . moreover, the effect of pedv n protein on type iii ifn production has also been evaluated [168] . n protein inhibits polyinosinic-polycytidylic acid (poly(i:c))-induced ifn-λ3 production by blocking the nuclear translocation of nf-κb, but does not antagonize the type i or type ii ifn expression induced by poly(i:c) in ipec-j2 cells [168] . recent studies show that sars-cov n protein inhibits type i ifn production through suppressing trim25-mediated rig-i ubiquitination [171] . the mers-cov n protein also blocks ifn production by interacting with trim25 [171] . in addition, both mhv and sars-cov n proteins perturb the function of cellular protein activator of protein kinase r (pact), which can bind to rig-i and mda5 to activate ifn production, and thus antagonize type-i ifn signaling [61] . these results indicate the important function of the covs n protein in modulating host innate immune response. whether pedv n protein targets trim25 or pact should be investigated. although several studies have been performed to understand the pathogenicity of pedv, there remains limited information about the interaction between viral proteins and host cell factors during viral infection. cov n protein is a vital viral protein involved in virus replication. current researches have indicated that n protein interacts with many host proteins, such as hcypa [172] , proteasome subunit p42 [173] , smad3 [174] , hnrnp-a1 [175] , and the chemokine cxcl16 [176] . in the host cells, a large number of host proteins reveal various functions. however, for the virus, the genome only encodes several limited viral proteins. therefore, these viral proteins have to be multifunctional, which is pivotal to virus replication and existence. pedv nsp1 is the n-terminal cleavage product from polyproteins pp1a and pp1a/b processed by nsp3 and nsp5 [177] and is about 110 amino acids in length [74, 178] . nsp1 of many α-cov and β-cov exhibits both functional conservation and mechanistic diversity in suppressing host gene expression and ifn signaling. for sars-cov, nsp1 triggers the decay and cleavage of host mrna and inhibits host protein translation, subsequently inhibiting type i ifn production [179, 180] . sars-cov nsp1 also blocks the expression of ifn-inducible genes, by restraining the signal transduction during virus infection [181, 182] . the tgev nsp1 considerably suppresses host protein expression during viral infection [77] . structural studies show that the core structure of pedv nsp1 is highly similar to those of sars-cov nsp1 and tgev nsp1 [183] . pedv nsp1 inhibits host gene expression and three motifs (amino acids 67 to 71, 78 to 85, and 103 to 110) form a stable functional region for inhibition of host protein synthesis, differing considerably from sars-cov nsp1 [183] . pedv nsp1 has been identified as an ifn antagonist, which constrains poly (i:c)-induced ifn-β promoter activity [65] . nsp1 significantly inhibits the activation of ifn-β promoter triggered by irf3, whereas it does not inhibit irf3 phosphorylation and its nuclear translocation. nsp1 interrupts the association of irf3 with creb-binding protein (cbp), by promoting cbp degradation in the nucleus via the proteasome-dependent pathway. cbp/p300, the transcription co-activator camp responsive element binding protein (creb), forms a complex with the activated irf3 in nucleus. the irf3-cbp/p300 complex binds to the positive regulatory domain (prd) regions of the ifn-β promoter, to assemble the enhanceosome with nf-κb and other factors, which ultimately turn on the transcription of type i ifn genes [184] [185] [186] . therefore, pedv nsp1 blocks type i ifn production in the nucleus. activated nf-κb induces the production of type i ifns and proinflammatory cytokines and is important for inhibiting viral infection. pedv nsp1 has been shown to interfere with the nf-κb activity [78] and is the most potent suppressor of proinflammatory cytokines at early infection. it inhibits the phosphorylation and degradation of iκbα, and blocks p65 nuclear translocation, leading to the suppression of both ifn and the early production of pro-inflammatory cytokines [78] . moreover, pedv inhibits type iii ifn production and nsp1, nsp3, nsp5, nsp8, nsp14, nsp15, nsp16, orf3, e, m, and n are identified as type iii ifn antagonists. among these antagonists, nsp1 is the most potent suppressor [130] . pedv nsp1 blocks the nuclear translocation of irf1 and decreases the amounts of peroxisomes and then suppresses irf1-mediated type iii ifns. the conserved residues of pedv nsp1 protein are crucial for ifn suppression [130] . multiple effects of nsp1 on modulating innate immune response during pedv infection suggest the vital role of nsp1 in the pedv replication cycle. the antiviral innate immune signaling pathways are regulated by several posttranslational modifications (ptms), such as phosphorylation, ubiquitination, glycosylation, neddylation and sumoylation [187] , of which ubiquitination is a critical modification to modulate the stability and activity of prrs and other components of innate immune signaling pathways. during viral infection, a reciprocatory action (occurrence of ubiquitination and deubiquitination) helps maintain the homeostasis of host immune responses. hence, deubiquitinases (dubs) are indispensable in the regulation of virus-induced type i ifn signaling [188] . many host dubs have been reported engaging in the regulation of innate immune signaling pathways [189] [190] [191] . in recent years, a variety of viral dubs have been discovered to target key components of type i ifn pathway during various rna virus infections. for example, foot-and-mouth disease virus leader proteinase (fmdv lb pro ) [192] , and porcine reproductive and respiratory syndrome virus nsp2 (prrsv nsp2) possess ubiquitin-deconjugating activity to deubiquitinate key host components [193, 194] . to counteract host antiviral response, covs likely take advantage of dub activity to break host innate immunity. indeed, the plps of mouse hepatitis virus a59 (mhv-a59) [195] , sars [196] , and human cov nl63 have dub activity and antagonize ifn induction [197] . pedv plp2 has been reported as having a deubiquitinase activity as well, and it can be co-immunoprecipitated by rig-i and sting. as mentioned above, fmdv lb pro , mhv plp2 and sars plps all counteract host innate immune response through blocking the ubiquitination of the components of rlrs pathways. similarly, pedv plp2 removes the ubiquitinated conjugates from rig-i and sting by its dub activity, to negatively regulate type i ifn production. pedv plp2 probably interacts with rig-i and sting, which prevents the activation of rig-i and sting by hindering the recruitment of downstream signaling molecules. as expected, the interference with the ubiquitination of rig-i and sting by plp2 clearly benefits pedv replication [80] . pedv nsp3 contains two core domains of plps (plpl and plp2). it is determined that pedv plp2, but not plp1, inhibits the ifn-β promoter activation in hek293t cells. the dub activity of plp2 is highly dependent on its catalytic activity. three catalytically inactive mutants of pedv plp2 (c1729a, h1888a and d1901a) are defective in the deubiquitination of its targets and fail to impair virus-induced ifn-β production. sars-cov plp2 interacts with mdm2 (mouse double minute 2 homolog) to deubiquitinate and stabilize mdm2, approving the degradation of p53 and the suppression of ifn signaling [198] . pedv infection degrades p53 by upregulation of mdm2 expression [198] . pedv plp2 may be responsible for targeting the p53 pathway and inhibiting p53-dependent apoptosis, leading to immune evasion. a recent study determined that tgev pl1 inhibits the ifn-β expression and interferes with the rig-1and sting-mediated signaling pathway through a viral dub activity [195] . it suggests that different viral proteins are involved in the deubiquitination of host proteins for different covs. however, these studies offer a probability to design a common therapeutic against different viral dubs to reduce the replication and pathogenesis of covs. therefore, further studies are required to understand more about the substrate specificity of these viral dubs and clarify the precise functions of cov protease/dub activity. notably, 3c pro is a critical ifn antagonistic protein identified in multiple different families of viruses. the 3c pro of picornaviruses, including fmdv [199] , hepatitis a virus (hav) [200] , enteroviruses (ev71, ev-d68) and coxsackieviruses (cvb3, cv-a16, cv-a6) [201] [202] [203] [204] , antagonize innate immune signaling by targeting the critical components of the ifn pathways for proteolysis. a newly emerged picornavirus, seneca valley virus (svv), has also evolved an effective mechanism to escape host antiviral innate immune using its 3c pro . moreover, 3c pro cleaves the signaling components (mavs, trif, and tank) of type i ifn pathway and induces the degradation of the transcription factors irf3 and irf7 to constrain host antiviral response [205, 206] . cov nsp5 is called 3c-like protease (3cl pro ), that resembles the 3c pro of other rna viruses. for cov, the polyprotein precursors (pp1a and pp1b) are mainly processed to generate mature nonstructural proteins by 3cl pro . to date, the 3cl pro of covs, including pedv and pdcov, have been confirmed to antagonize type i ifn production by the cleavage of nf-κb essential modulator (nemo) and stat2 [100, 207, 208] . nemo is essential for rna virus-induced activation of nf-κb, irf3, and irf7 [209] . nemo is required for mavs-induced ikkα/β activation and is also crucial for the activation of tbk1/ikkε [149] . to establish successful infections, pedv targets nemo to subvert host innate immune responses. pedv nsp5 significantly inhibits sendai virus (sev)-induced ifn-β synthesis and the process depends on its protease activity [100] . further experiments show that pedv nsp5 inhibits rig-i/mda5 signaling and targets the upstream of tbk1. the cleavage of nemo by nsp5 is identified as responsible for this inhibitive effect. the pedv nsp5-mediated cleavage of nemo efficiently blocks nemo-mediated downstream signaling. the cleavage site within nemo that is grasped by nsp5 has been determined. of these reported immune evasion strategies employed by covs, the cleavage of innate immune adaptors is a particularly effective manner to disrupt antiviral responses. nsp5 is essential for the life cycle of pedv and other covs [210, 211] . it is a potential target for the development of anti-coronaviral therapeutics. although pedv nsp5 does not target stat2 mediated type i ifn signaling pathway, pedv nsp7 has been reported to inhibit the stat1 and stat2 induced activation of isre [212] . nsp7 competes with karyopherin α (kpna1), which is an adaptor mediating nuclear translocation of isgf3, in combination with stat1, to block isgf3 nuclear transport. however, the expression and phosphorylation of stat1 and stat2 are not affected by pedv nsp7. in fact, pedv infection degrades stat1, leading to the inhibition of ifn signaling [170] . therefore, other pedv encoded proteins likely target ifns mediated signaling. covs belong to rna viruses, which produce several rna species, such as dsrna intermediates and rna with a 5 -triphosphate during replication. these rna intermediates are potent stimulators of prrs and are associated with the organelles of viral rna replication, dmvs [213, 214] . dmvs formed from membranous rearrangements seem to sequester the replication intermediates using membrane-bound vesicles or invaginations to keep away from prrs. therefore, the form of dmvs may be a strategy for pedv to escape innate immune recognition in the cytosol (figure 2 ). however, whether dmvs alone are sufficient to shield rna from prrs remains unknown. besides, the replication organelles, the endoribonuclease activity and viral 5 end rna capping/protection mechanisms are also the critical ways of avoiding rna recognition or protecting it from degradation [132, 135] . (1) pedv nsp15 cov nsp15 has endou catalytic activity that was initially thought to play a vital role in virus replication. however, the catalytic-defective endou of mhv shows only a subtle defect in viral replication compared to wt virus in fibroblasts [215] . similar results are found for the nsp15 mutants of sars-cov and hcov-229e [216] . these findings suggest that the endou activity of nsp15 is not required for rna synthesis. recently, nsp15 has been demonstrated to act as a new ifn antagonist of covs [117, 118, 217] . recent reports indicate that covs' endou activity is essential for prevention of rna recognition by mda5, protein kinase r (pkr), and oas/rnase l system [118] . pkr and oas/rnase l recognize and destroy foreign rna in the cytosol to defend viral infections. to counteract the function of pkr and oas/rnase l, the virus hides or modifies its viral rna, to avoid the exposure of viral rna to these molecules. in all covs, the endou catalytic domain in nsp15 is highly conserved. pedv endou activity has been indicated as having an antagonistic effect on ifn signaling [135] . the endou activity of pedv nsp15 not only inhibits the type i ifn response in porcine macrophages, but also antagonizes the type iii ifn response in porcine epithelial cells. the replication of endou-mutant pedv (icpedv-enumt) is considerably impaired in porcine epithelial cells compared to the wild type pedv (icpedv-wt). the icpedv-enumt clearly induces early and robust type i and type iii ifns production, as well as isgs' expression compared with that induced by icpedv-wt. the endou-deficient pedv infected animals also show reduced viral shedding and mortality. these results indicate that the endou activity of pedv nsp15 plays a vital role in evading host antiviral innate immunity (figure 2 ) [135] . (2) pedv nsp16 cov nsp16 is a 2 -o-methyltransferase (2 -o-mtase). to evade recognition by the host immune sensors, many covs encode methyltransferases involved in the capping of viral rna. this modification makes the viral rna indistinguishable with host cell mrna, which is important to avoid the recognition of viral rna by mda5 ( figure 2 ) [121, 218] . methylation of the two sites in the 5 cap is catalyzed by three nsps, including nsp14 (the n-7-mtase), nsp16 (the 2 -o-mtase), and nsp10 [112, [123] [124] [125] [126] . for example, sars-cov nsp16 acts as a 2 -o-mtase to prevent innate immune recognition and promote viral proliferation [113, 121, 123] . pedv nsp14 and nsp16 have been identified as the viral ifn antagonists [65, 132] . the overexpression of nsp14 or nsp16 remarkably inhibits ifn-β production, but nsp16 appears to play a more important role in innate immunity regulation than nsp14 [132] . nsp16 is a highly conserved methyltransferase which contains an invariant kdke motif within the methyltransferase core [219] . this kdke motif is required to mediate its activity. notably, the mutation of any of the kdke active sit has been shown to abolish the 2 -o-mtase activity [123] . pedv nsp16 kdke motif plays a critical role in the inhibition of type i ifn production, suggesting the important role of the 2 -o-mtase in pedv-mediated immune evasion. pedv nsp16 negatively regulates rlr-mediated signal pathway activation, and inhibits the expression of the ifn-stimulated ifit family members (ifit1, ifit2, ifit3), which in turn promotes pedv replication. taken together, these results demonstrate that pedv nsp16 negatively regulates cellular antiviral response to promote viral replication [132] . screening inhibitors targeting the 2 -o-mtase of nsp16 might be a prominent strategy to inhibit cov infections and develop antivirals for treatment of the diseases caused by covs. additionally, covs nsp14 also includes the 3 -to-5 exoribonuclease (exon) activity [113] . a mutation of tgev nsp14 exon generates lower levels of dsrna than wildtype tgev and thus triggers a reduced antiviral response [220] . nsp14 exon activity is also critical for the resistance of host innate immune response in mhv-infected cells [221] . the role of nsp exon activity of pedv in counteracting host antiviral response should be investigated to uncover more functions of pedv nsps. these data suggest that pedv has evolved multiple evasive mechanisms to circumvent viral rna recognition or prevent rna degradation to establish a successful infection in the host. activity is also critical for the resistance of host innate immune response in mhv-infected cells [221] . the role of nsp exon activity of pedv in counteracting host antiviral response should be investigated to uncover more functions of pedv nsps. these data suggest that pedv has evolved multiple evasive mechanisms to circumvent viral rna recognition or prevent rna degradation to establish a successful infection in the host. heat shock protein 27 (hsp27) belongs to the small heat shock proteins family, which has been identified as a multifunctional protein involved in cytoskeletal stability, proinflammatory processes, and the inhibition of apoptosis [222, 223] . several hsps have been reported to be implicated in pedv infection in vitro and in vivo [224, 225] . indeed, the infection of many viruses up-regulates hsp27 expression by different mechanisms to delay cellular apoptosis, then supplies sufficient time for viral replication [226, 227] . however, pedv infection significantly induces the decreased expression of hsp27 in vero cells [225] and marc-145 cells [228] . hsp27 activates the phosphorylation of nf-κb, and thus promotes the mrna expression of ifn-β in marc-145 cells. as hsp27 is an upstream regulator of antiviral immune signaling, overexpression of hsp27 significantly inhibits the pedv replication. pedv has developed a strategy via mediating the suppression of hsp27 production to escape from host innate immune response [228] . hsp70, the most conserved hsp, is also important for the multiplication of several covs. the recruitment of hsp70 is thought to be a viral survival strategy for several viruses in their hosts [229] . the relationship between hsp70 and pedv should be exploited further in future. viral infection triggers host immune response to induce ifns' and inflammatory cytokines' production. released ifns elicit the expression of numerous isgs which limit viral replication in the infected cells. however, the release of excessive amounts of ifns and inflammatory cytokines will lead to autoimmune and auto-inflammatory diseases. the concomitant uncontrolled apoptosis is also one outcome that is harmful to the host. to maintain the reaction in a proper balance, hosts have evolved a series of effective mechanisms to control the antiviral innate immune response [230] . in contrast, viruses often break this balance, causing improper apoptosis reaction, which benefits viral replication. pedv infects various host cells including vero, pk-15 and marc-145 and cause obvious cytopathic effects. pedv-induced apoptosis of the infected cell has been demonstrated both in vitro and in vivo [231] . apoptosis is induced through the activation of apoptotic caspases, including caspase-2, -3, -6, -7, -8, -9, and -10 [232] . pedv infection results in obvious caspase-3 and caspase-8 activation, as well as the cleavage of apoptosis-inducing factor mitochondria-associated 1 (aifm1) and poly adp-ribose polymerase (parp), which leads to apoptotic nuclear fragmentation. pedv spike protein s1 significantly elicits host cell apoptosis, while the nsp1-16 and other structural proteins (m, n, e, s2, and orf3) have none or few effects on cell apoptosis. therefore, s1 protein is probably the critical protein mediating the apoptosis induced by pedv [128] . the multiple stages of cov replication cycle are closely associated with cellular membrane compartments, especially the endoplasmic reticulum (er). the shape and functions of er can be influenced by different physiological states and environmental conditions. when protein synthesis amounts surpass the folding capacity, the accumulation of a large amount of unfolded proteins in the er leads to er stress. consequently, cells manifest a corresponding biological reaction that is widely known as the unfolded protein response (upr) [233] . once the upr is induced, it alleviates the problems by host protein translation inhibition (by the transducer pkr-like er protein kinase (perk)-induced phosphorylation of eif2a), stimulating protein folding. if homeostasis cannot be re-established, apoptosis eventually is triggered. indeed, the activation of upr regulates a wide variety of signaling pathways, such as apoptosis, autophagy, mitogen-activated protein (map) kinase activation, and innate immune response [234] . furthermore, α-cov and β-cov may induce er stress in the infected cells [235] . pedv orf3, as the only accessory protein encoded by pedv, is thought to be related to virus production and virulence of pedv [68] . a series of studies suggest that orf3 plays multiple roles, in addition to acting as an ion channel during pedv replication. recent studies show that pedv orf3 consists of four transmembrane domains (tmds) and localizes in the cytoplasm in the aggregation manner [236] . orf3 is a transmembrane protein, and the confocal microscopy analysis indicates that the aggregated orf3 localizes in the er to induce the er stress associated with either apoptosis or autophagy. however, pedv orf3 induces the autophagy via driving conversion of lc3-i to lc3-ii, but not influencing the apoptosis. orf3-induced autophagy is dependent on er stress response. pedv orf3 triggers er stress response via the up-regulation of grp78 protein expression and the activation of the perk-eif2α signaling pathway. moreover, orf3 protein is identified as an ifn antagonist to block ifn response by an unknown mechanism in pedv-infected cells [65] . the functions of pedv orf3 should be further exploited. pedv has caused epidemic and endemic infections in pig populations in many countries and has become a major economic threat to the swine industry. previous studies have identified viral factors that target key signaling molecules in the rlrs' pathways, as well as viral factors that target the downstream signaling pathways responsible for isgs induction. of the 23 pedv-encoded proteins, at least 10 viral proteins have been identified as type i ifn antagonists [65, 78] . the mechanisms utilized by pedv nsp1, plp2, nsp5, and n protein to antagonize type i ifn production have been clarified (figure 3 , [58, 65, 78, 80, 129] ). however, the specific mechanisms of other viral proteins to inhibit type i ifn production remain largely unknown. at present, 11 pedv proteins have been identified as type iii ifns' antagonists. the suppression of type iii ifn signaling by n protein, nsp1, as well as nsp15, has been reported, while the mechanisms utilized by these viral proteins need to be further investigated. in addition, the covs replication cycle may induce the changes of er stress, cell apoptosis, autophagy pathways, which contain intricate virus-host interactions and cross-talk relationships. thus, more researches for pedv are needed to truly reflect viral evasions from innate immune defenses. the findings of pedv-host interactions will help prevent and control pedv spreading. have been clarified (figure 3 , [58, 65, 78, 80, 129] ). however, the specific mechanisms of other viral proteins to inhibit type i ifn production remain largely unknown. at present, 11 pedv proteins have been identified as type iii ifns' antagonists. the suppression of type iii ifn signaling by n protein, nsp1, as well as nsp15, has been reported, while the mechanisms utilized by these viral proteins need to be further investigated. in addition, the covs replication cycle may induce the changes of er stress, cell apoptosis, autophagy pathways, which contain intricate virus-host interactions and cross-talk relationships. thus, more researches for pedv are needed to truly reflect viral evasions from innate immune defenses. the findings of pedv-host interactions will help prevent and control pedv spreading. removes ubiquitinated conjugates from rig-i; pedv nsp5 induces cleavage of nemo; pedv n protein directly interacts with tbk1 to obstruct the association between tbk1 and irf3; pedv nsp1 causes degradation of cbp and iκbα, as well as inhibition of iκbα phosphorylation and p65 activation. pedv nsp16 inhibits type i ifn production and nsp10 enhances the inhibitory effect of nsp16 on type i ifn production. for type iii ifn, pedv n protein blocks the nuclear translocation of nf-κb; pedv nsp1 blocks the nuclear translocation of irf1 and reduces the amounts of peroxisomes. pedv nsp15 inhibits the type i ifn and type iii ifn responses by unknown mechanisms. pedv nsp7 interacts with stat1 and stat2 to block nuclear translocation of isgf3. removes ubiquitinated conjugates from rig-i; pedv nsp5 induces cleavage of nemo; pedv n protein directly interacts with tbk1 to obstruct the association between tbk1 and irf3; pedv nsp1 causes degradation of cbp and iκbα, as well as inhibition of iκbα phosphorylation and p65 activation. pedv nsp16 inhibits type i ifn production and nsp10 enhances the inhibitory effect of nsp16 on type i ifn production. for type iii ifn, pedv n protein blocks the nuclear translocation of nf-κb; pedv nsp1 blocks the nuclear translocation of irf1 and reduces the amounts of peroxisomes. pedv nsp15 inhibits the type i ifn and type iii ifn responses by unknown mechanisms. pedv nsp7 interacts with stat1 and stat2 to block nuclear translocation of isgf3. author contributions: the manuscript was prepared by s.l., j.y., z.z., and h.z., and reviewed by the co-authors. all authors have read and agreed to the published version of the manuscript. outbreak of porcine 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caspases control antiviral innate immunity porcine epidemic diarrhea virus induces caspase-independent apoptosis through activation of mitochondrial apoptosis-inducing factor caspase function in programmed cell death signal integration in the endoplasmic reticulum unfolded protein response coronavirus infection, er stress, apoptosis and innate immunity regulation of stress responses and translational control by coronavirus porcine epidemic diarrhea virus orf3 protein causes endoplasmic reticulum stress to facilitate autophagy this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we are thankful to everybody who participated in the studies. the authors declare no conflict of interest. key: cord-319609-y0gdjn64 authors: van duyne, rachel; guendel, irene; kehn-hall, kylene; easley, rebecca; klase, zachary; liu, chenglong; young, mary; kashanchi, fatah title: the identification of unique serum proteins of hiv-1 latently infected long-term non-progressor patients date: 2010-07-06 journal: aids res ther doi: 10.1186/1742-6405-7-21 sha: doc_id: 319609 cord_uid: y0gdjn64 background: the search for disease biomarkers within human peripheral fluids has become a favorable approach to preventative therapeutics throughout the past few years. the comparison of normal versus disease states can identify an overexpression or a suppression of critical proteins where illness has directly altered a patient's cellular homeostasis. in particular, the analysis of hiv-1 infected serum is an attractive medium with which to identify altered protein expression due to the ease and non-invasive methods of collecting samples as well as the corresponding insight into the in vivo interaction of the virus with infected cells/tissue. the utilization of proteomic techniques to globally identify differentially expressed serum proteins in response to hiv-1 infection is a significant undertaking that is complicated due to the innate protein profile of human serum. results: here, the depletion of 12 of the most abundant serum proteins, followed by two-dimensional gel electrophoresis coupled with identification of these proteins using matrix-assisted laser desorption/ionization time-of-flight (maldi-tof) mass spectrometry, has allowed for the identification of differentially expressed, low abundant serum proteins. we have analyzed and compared serum samples from hiv-1 infected subjects who are being treated using highly active antiretroviral therapy (haart) to those who are latently infected but have not progressed to aids despite the absence of treatment, i.e. long term non-progressors (ltnps). here we have identified unique serum proteins that are differentially expressed in ltnp hiv-1 patients and may contribute to the ability of these patients to combat hiv-1 infection in the absence of haart. we focused on the cdk4/6 cell cycle inhibitor p16(ink4a )and found that the treatment of hiv-1 latently infected cell lines with p16(ink4a )decreases viral production despite it not being expressed endogenously in these cells. conclusions: identification of these unique proteins may serve as an indication of altered viral states in response to infection as well as a natural phenotypic variability in response to hiv-1 infection in a given population. human serum is derived from the liquid plasma component of the blood with the fibrinogens, or clotting factors, removed and is composed of small molecules such as salts, lipids, amino acids, sugars and approximately 60-80 mg of proteins/ml [1] . serum is a readily obtainable peripheral bodily fluid from which the protein profile directly reflects the normal or disease state of the organism [2] [3] [4] . serum is a complex mixture of "classical" and "non-classical" proteins. classical serum proteins are involved in a number of processes including proteolysis, inhibition, binding, transport, coagulation, and immune response and are often secreted from the liver, through the intestines, and into the bloodstream [5] . "non-classical" proteins are proteins that are not directly tied to any known function within the serum and often originate from cellular leakage or shedding, and may utilize the bloodstream for transportation [5] . it is generally accepted that most of the significant changes in the serum will be found in these low abundant non-classical proteins, due to the hypothesis that the presence of these proteins should reflect changes in the diseased tissue. indeed, serum commonly contains upwards of 10,000 different proteins at any given time that are being actively produced and secreted by all cells and tissues, therefore, the proteomic profile of serum can give insight into the systemic reaction to a disease state and can serve as a pool of differentially expressed proteins [2, [6] [7] [8] [9] [10] [11] [12] [13] . recently, the interest in characterizing the human serum proteome has increased due to the determination of disease biomarkers for early detection, diagnosis, and drug targeting; however, due to the extensive dynamic range of protein concentration within the serum, the identification of low abundance proteins suitable for biomarker determination is often masked. the 22 highly abundant proteins contained within serum constitute approximately 99% of the total serum proteins, including albumin, igg, transferrin, haptoglobin, fibrinogen, etc. and interfere with the identification of low abundance proteins in the ng/ml concentration range. the presence of these highly abundant proteins necessitates the prefractionation of serum samples prior to analysis for low abundant proteins. due to the dynamic insight the analysis of the serum proteome can relate to a disease state, of particular interest is the identification of low abundant proteins that change in expression or abundance in response to a disease state. these low abundant proteins could potentially arise as an early diagnostic for a disease state, or a therapeutic target. serum proteomics has emerged as an integral biomarker identification and diagnostic tool, especially for infectious diseases and oncology. recently, novel serum biomarkers have been identified for liver fibrosis in hepatitis c virus (hcv) infected patients as well as unique protein signatures in sars coronavirus infections, and infant hepatitis syndrome induced by human cytomegalovirus (hcmv) infection [14] [15] [16] . characterization of the serum protein profile of these viral states helps provide insight into the expression changes associated with viral infection. in particular, hiv-1 infection, even at the acute phase, results in dramatic changes in both cellular and viral protein expression levels. as the hiv-1 viral tropism consists primarily of cd4+ t-cells, macrophages, and dendritic cells, the resulting protein changes can be seen systemically as infected cells travel throughout the body. additionally, the nature of this viral infection supports the secretion of altered proteins into the blood and subsequently the serum due to the propensity of the virus to stimulate apoptosis of infected cells, therefore emptying cellular contents into the serum. these characteristics of hiv-1 infection suggest that the analysis of the serum of infected patients is an appropriate reflection of a patients' altered protein expression state. due to innate genetic and phenotypic differences in the human population, significant variability exists in the sus-ceptibility to hiv-1 infection. amongst this diversity includes the well-studied ccr5δ32 inherited mutation, which prevents the binding of r5-tropic hiv-1 strains to the ccr5 chemokine receptor on the surface of cd4+ tcells, therefore preventing entry of the virus [17] . additionally, some individuals can be infected with hiv-1, however will not progress to aids even in the absence of therapy. these long term non-progressors (ltnps) are often characterized as being infected with hiv-1 but are also disease free and sustain a normal cd4 t-cell count and a low viral load. over the past 20 years, multiple studies have been aimed at determining the reason that these individuals are able to resist disease progression. there are studies that suggest that the virus infecting these cells could be deficient in some way, for example, nef deficient viruses and vpr r77q mutations are associated with ltnps [18] [19] [20] [21] [22] . a number of host factors have also been identified that may contribute to the observed resistance. ltnps have a higher prevalence of the ccr5δ32 allele [17, [23] [24] [25] . in addition, the presence of certain hla genes including hla-b27, hla-b*5701, hla-b*5401, and hla-b*1507 have been linked to ltnp [26] [27] [28] however, the identified alterations do not account for all cases of ltnp. therefore, the search for protective host factors is still an area of active investigation in hopes of obtaining information that could be of therapeutic value. here, we describe the detection of unique, low abundant serum proteins in latently infected hiv-1 ltnps as compared to serum from patients undergoing haart treatment, and those not infected with hiv-1. we attempted to characterize the underlying differences in ltnps that contribute to the ability of these patients to combat hiv-1 infections. we have depleted 12 of the most highly abundant serum proteins from three sets of serum samples (uninfected, infected on haart, ltnp) and identified differentially expressed proteins across the samples. in particular, we focus on the identified cellular protein p16 ink4a which is found preferentially in ltnp patient serum samples, but is not present in patients undergoing haart treatment. in vitro viral assays and viability studies confirm the loss of viral replication upon p16 ink4a treatment in latently infected cell lines and the non-toxic effect of the same treatment in corresponding uninfected cell lines. to begin the identification of unique serum proteins, we obtained 18 subject serum samples: six ltnp, six hiv-1 infected subjects receiving haart therapy (haart) and six hiv-uninfected individuals through the washington dc site of the women's interagency hiv study (wihs) georgetown site (table 1) . wihs is an nih multicenter study of the natural history of hiv-1 infection in women [29] . ltnps are defined by wihs as being hiv-1 infected, but disease free for at least five years, having a cd4 count of greater than 500 at all visits and having no history of anti-retroviral therapy. the difficulty associated with analyzing serum is the presence of a high abundance of proteins which mask potential low abundance biomarkers. to overcome this obstacle, we utilized the proteomelab igy serum depletion kit which removes 12 of the most abundant proteins in serum: albumin, igg, transferrin, fibrinogen, iga, α2-macroglobulin, igm, α1antitrypsin, haptoglobin, α1-acid glycoprotein, apolipoprotein a-i, and apolipoprotein a-ii. as can be observed in figure 1a , whole serum (lanes 2, 3) contains many proteins and is too complex to allow for confident identification of specific proteins. however, when the high abundant proteins (figure 1 , lanes 8, 9) are removed, lower abundant proteins that were originally masked ( figure 1 , lanes 4, 5) are able to be analyzed. along these lines, we found the proteomelab igy serum depletion kit to be the most appropriate and reproducible manner in which to fractionate our serum samples into high and low abundant fractions. we applied this depletion strategy to pooled patient samples, combining equal volumes of whole serum from each of the six patients per sample set (ltnp, haart, and negative), which were subsequently depleted into low and high abundance fractions. we began the analysis with pooled samples to assist in the identification of hiv-1 infection specific protein identification as opposed to identifying individual patient and serum variability. these pooled samples were separated based on 1d sds-page ( figure 1b ) and comparisons between ltnp, haart, and negative low abundant samples were carried out via in-gel trypsin digestion, peptide elution and desalting, followed by maldi-tof mass spectrometry as indicated by numbered arrows marking excised bands. the subsequent protein identifications served as a preliminary indication of differentially expressed proteins between the three patient types. these observations, as summarized in table 2 , provide an insight into the relevance of proteins identified in the context of the state of hiv-1 infection. of particular interest in table 2 is the identification of hiv-1 enhancer binding protein 1, (hivep1), ribonuclease iii, and heterochromatin protein 1 binding protein in the low abundance ltnp fraction. hivep1 is a member of the zas family of proteins which bind the promoter and enhancer regions of both cellular genes and infectious viruses, including hiv-1. also known as prdii-bf1 or mbp-1, this transcription factor binds to both the nf-κb and the tar transactivation response dna elements on the hiv-1 ltr in both the presence and absence of hiv-1 tat [30, 31] . it is not surprising that a transcription factor such as hivep1 would be present during hiv-1 infection; however, the identification of this protein is not necessarily a marker for a ltnp phenotype. ribonuclease iii, or drosha, is a cellular enzyme found in the nucleus which serves to cleave double-stranded rna hairpin transcripts as a key step in the production of mirnas in the rna interference pathway. interestingly, heterochromatin protein 1, or hp1 is a member of the chromatin remodeling family of proteins, which can bind histones at methylated lysine residues and can interact with many chromatin-associated nonhistone proteins. the hp1 family of proteins has been associated with promoting a heterochromatic cellular state, where latently hiv-1 infected cells can persist as a transcriptionally silent provirus [32, 33] . it may be of interest that an hp1 binding protein would be present in the serum of an hiv-1 infected patient as hp1, including its subtypes α, β, and γ, could be involved in the control of various stages of infection. it is possible that the association of this hp1 binding protein with varying subtype of hp1 could explain the differences in patient phenotypes, especially those that result in an altered susceptibility to viral infection. following the initial 1d separation and maldi-tof ms assisted identification, the pooled patient samples were subjected to 2d-gel electrophoresis (2dge); isoelectric focusing (ief) using ipg strips with a ph 3.0-10.0 range followed by sds-page using 4-20% tris-glycine criterion gels. the method of 2d-gel electrophoresis is much more sensitive than 1d-gel electrophoresis in that it provides separation of a complex mixture of proteins in two dimensions, therefore removing the complexity associated with overlapping proteins, or masking due to posttranslational modifications. 2dge is a more sensitive front-end purification approach to the isolation and identification of individual protein species by mass spectrometry. figure 2 depicts the ltnp, haart, and negative low abundance fractions in gels "a", "b", and "c", respectively, as well as the ltnp, haart, and negative high abundance fractions in gels "d", "e", and "f", respectively. indicated protein spots from all gels were excised based on a comparison of protein abundance and the presence of unique spots in a given patient set, were subjected to in-gel trypsin digestion, and were identified by maldi-tof mass spectrometry. it is important to note that although gels "d," "e," and "f" contain the majority of the high abundance proteins, unique small protein spots can still be visualized on these gels. this indicates that not only will these high abundant proteins mask proteins of interest; they can also interact with and seclude lower abundance proteins from being identified. peak lists from the collected mass spectra were processed via peptide mass fingerprinting (pmf) analysis using the mascot and profound databases, compared, and compiled into a nonexhaustive list of identified proteins as displayed in table 3 . of particular interest are those proteins identified from gel "a" indicating unique low abundance proteins in the serum of ltnp patients: tropomyosin 3, protein kinase 3, and cdk4/6 binding protein p16. tropomyosin interacts with actin filaments to provide stability and regulates other actin binding proteins. this family of proteins has been shown to be cleaved by hiv-1 protease in vitro, resulting in the dissociation of critical cytoskeletal elements, which may demonstrate the alteration of muscle structure in the presence of an hiv-1 infection [34] . protein kinase 3, or protein kinase c (pkc) is a member of the family of serine/threonine kinases that are integrally involved in key cellular signaling pathways and can phosphorylate a wide variety of substrates. not surprisingly, hiv-1 infection alters the pkc phosphorylation pathway to stimulate tnf-α production by monocytes as well as other cytokines and growth factors such as il-6, il-10, and mcp-1 [35] [36] [37] [38] [39] . pkc has also been shown to be necessary for hiv-1 tat-mediated transactivation as well as directly phosphorylating tat at serine 46 [40, 41] and plays an integral role in the signaling and secretion of cytokines in response to hiv-1 envelope proteins gp120, gp160, and gp41 [42, 43] . of particular interest in the low abundance, ltnp fraction is the presence of the cdk4/ cdk6 binding protein p16, or more specifically, p16 ink4a , a member of the inhibitor of kinase 4/alternative reading frame (ink4/arf) family of endogenous cdk (cyclindependent kinase) inhibitors [44] . dysregulation of the cell cycle, including the manipulation of cdks and their associated cyclins is often a hallmark of cancerous and infectious phenotypes. indeed hiv-1 and its associated proteins have been known to alter the phosphorylation state and activity of these kinases. p16 ink4a inhibits the phosphorylation of rb by competitively inhibiting the association of cdk4/cyclin d therefore inhibiting the release of rb-bound proteins, such as e2f, and the subsequent progression into the s phase of the cell cycle [44, 45] . this small molecular weight protein is an attractive candidate for a secreted, differentially expressed protein in response to hiv-1 infection. in addition to these low abundant protein identifications, the ltnp high abundant samples (gel "d") indicated the presence of the fgfr1 oncogenic partner and pctaire protein kinase 3 as well as an anti-hiv-1 gp120 igg 16 cκ light chain. fgfr1 oncogene partnered with the fibroblast growth factor receptor 1 (fgfr1) is thought to be associated with myeloproliferative disorders and as of yet is not associated with any hiv-1 protein interactions or associated disease phenotypes. pctaire protein kinase 3, however, is a member of the serine/threonine family of protein kinases and more specifically, the cdc2/cdkx subfamily that plays a role in broad signal transduction pathways. this serine/threonine kinase family member has also been associated with the essential regulation of cell cycle progression, as well as transcription and dna repair [46] . this protein identification again demonstrates the role that hiv-1 infection plays in the dysregulation of cellular kinases and specifically, cell cycle progression. the haart responder patient samples (gels "b" and "e") also contained unique protein candidates: serine/ threonine kinase 33, the kelch repeat domain containing protein 11, and the snw1 protein/apaf1 interacting protein in the low abundant fraction as well as the pre-bcell leukemia homeobox interacting protein 1 in the high abundant fraction. of functional interest is the general serine/threonine kinase 33 as we have already identified several cellular kinases of the same family. additionally, the snw1 protein is a transcriptional coactivator that induces the expression of vitamin d, retinoic acid, estrogen, and glucocorticoid associated genes. snw1/skip interacts with hiv-1 tat through the association with p-tefb (cdk9/cyclin t1) at the tar rna complex, stimulating hiv-1 transcription elongation [44] . interestingly, some of the protein spots identified the presence of serum albumin contamination (spots a2, d5, d7, and e2), which both served as an internal positive control for mass spectrometry and also indicated that the depletion columns are not completely efficient at removing contaminating high abundant proteins. in order to further confirm the presence of these proteins in the serum as identified by mass spectrometry, we performed western blots on the same low and high abundant pooled fractions ( figure 3a ). p16 ink4a is present in both the low and high abundant fractions of the pooled ltnps (lanes 3, 4) and is also observed in the high abundance fraction of uninfected patients ( figure 3a , lane 2). interestingly, this protein is not present in haart patient samples at all ( figure 3a , lane 5, 6). the presence of this protein in serum may be specific to individuals that confer resistance to chronic hiv-1 infection. as p16 ink4a is an inhibitor of cell cycle kinases, in particular cdk4 and cdk6, the levels of cdk4 in the serum samples was assayed and was shown to be ubiquitously expressed across all low and high abundance serum samples ( figure 3a , third panel from top). indeed, levels of cdk6 were not detectable in any of the patient serum samples as compared to a 293t whole cell extract positive control (data not shown). this implies that the presence of cdk4 in the serum is not dependent on the presence or absence of p16 ink4a and likewise, p16 ink4a does not affect the expression levels of cdk4 amongst the patient serum samples. the hp1 binding protein was initially identified in the 1d/mass spectrometry analysis in the ltnp low abundance fraction, therefore the serum levels of both hp1α and hp1γ subunits were assayed ( figure 3a ). the family of heterochromatin-associated proteins exist as three distinct isoforms, α, β, and γ and all act as regulators of heterochromatinmediated transcriptional silencing [47] . hp1α has been shown to directly interact with dna methyltransferases and histone methyltransferases to mediate transcriptional silencing [48, 49] and hp1γ, in particular, interacts with the histone methyltransferase suv39h1 to initiate a chromatin-mediated repressive state of the hiv-1 integrated virus [50] . hp1α was shown to be present in the low abundance fractions of all of the patient phenotypes whereas hp1γ was shown to be present in both the low and high abundant fractions across all patient types (fig-ure 3a ). hp1γ is observed in lower amounts in both the negative and haart high abundance fractions and all serum samples indicate the presence of a post-translational modification (i.e. a doublet band) as compared to the 293t whole cell extract positive control. this indicates that the hp1γ found in serum exists in both a modified and unmodified form. interestingly, pctaire was present in the highest abundance in the uninfected (negative), high abundance fraction ( figure 3a , lane 2), however low levels were also seen in both ltnp and haart high abundance fractions ( figure 3a , lane 4, 6). pctaire was identified initially by mass spectrometry in the high abundance ltnp sample and can be seen in the high abundance fractions of all three of the patient types biochemically, however it is present in lower amounts in the hiv-1 infected patients, indicating that this kinase may be differentially expressed upon infection though not necessarily a unique identifier for infection. p16 ink4a is the only protein identified from mass spectrometric analysis and confirmed biochemically that is specific for the 5) were collected as the flowthrough, the column was washed (lanes 6, 7) and the high abundant proteins eluted (lanes 8, 9) . briefly the observed high abundant proteins were compared to the known sizes of the expected proteins as indicated. b) equal volumes of serum from each of the six patients within each category (ltnp, haart, and negative) were pooled together to create a stock of each condition, independent of patient-to-patient variability. twenty microliters of each stock was subjected to depletion and equal concentration of low and high fraction were run on a 1d gel. lanes 2, 3 and 4 are the low abundance fractions of the pooled ltnp, haart, and negative patients, respectively. lanes 6, 8, and 10 are the high abundance fractions of the pooled ltnp, haart, and negative patients, respectively. the indicated arrows represent differentially expressed proteins that were excised, trypsinized, and identified using maldi-tof for preliminary protein screening. low abundance ltnp serum samples; although the protein is also identified in the uninfected and the high abundance ltnp fractions. these results are also interesting due to the involvement of p16 ink4a in alterations of cell cycle control, additionally, mutations in p16 ink4a are found in various cancers including pancreatic, lymphomas, and sarcomas, contributing to cancer progression [45] . these findings also indicate a difference in composition of serum proteins present in hiv-1 infected individuals undergoing haart treatment versus those that are naturally non-progressing. in order to address the concern that the protein signature of the pooled set of samples for each patient type may not be an accurate representation of the individual variability that could be present, we screened the low abundance fractions of the ltnps for the presence of both p16 ink4a and cdk4. results in figure 3b indicate the lack of detection of p16 ink4a in any of the low abundant ltnp samples, especially as compared to the pooled sample "a" (lane 2). in contrast, p16 ink4a was detectable in ltnp patients 1 and 2, as well as in the pooled sample "d" for the corresponding high abundance fractions (lanes 2, 3, 4) . due to the nature of the depletion step based on immuno-affinity, it is not surprising that p16 ink4a is detectable in individual high abundant samples, as it is probably coupled to a larger, more abundant protein and was not efficiently depleted. additionally, post-depletion, the low abundant fractions for each patient were very dilute and the protein levels were undetectable by traditional methods. cdk4 was detectable in the ltnp low abundant individual samples with variable abundance, correlating with the data in figure 3a . interestingly, cdk4 was not detectable in the ltnp high abundant individual samples, indicating that this protein was effectively isolated away from the high abundant proteins. although through a straight western blot, the levels of p16 ink4a were undetectable, figure 3c indicates that p16 ink4a can indeed be immunoprecipitated out of the individual low abundant ltnp samples and subsequently detected by western blot. lanes 1, 2, and 3 represent the pooled "a" sample and patient samples 2 and 3, respectively. the pooled "a" sample was incubated with α-igg, and patient samples 2 and 3 were incubated with α-p16. these three immunoprecipitations were subjected to very stringent wash conditions of tne 600 + 0.1% np-40, tne 300 + 0.1% np-40, and tne 50 + 0.1% np-40 in order to remove any non-specific proteins. as can be seen in figure 3c although we have identified p16 ink4a as differentially present in the serum of hiv-1 infected ltnps as compared to haart treated individuals, this may not directly correlate to viral pathogenesis or functionality of this protein. in order to gain insight into the reason why p16 ink4a may be present preferentially in the serum of ltnp patients, we treated latently infected hiv-1 cell lines (j1.1 and u1) with exogenous purified gst-p16 ink4a figure 4a depicts an rt assay which measures the viral reverse transcriptase activity of infected cells and is an indicator of functional particle production. in the presence of both 0.1 and 0.5 ug of gst-p16 ink4a , j1.1 latently infected t-cells exhibited a decrease in rt activity (cpm) whereas the higher concentration of gst-p16 ink4a was able to elicit a decrease in rt activity in the latently infected monocytes, u1, as compared to the gst treatment alone. this data suggests that the presence of p16 ink4a in serum may result in a decrease in viral replication, which may help to explain why the presence of p16 ink4a in the serum of ltnps could correlate with an overall lack of viral activity. to detect whether p16 ink4a had an effect on normal or uninfected cells, we performed an mtt assay to screen for the percentage of cells viable after p16 ink4a treatment. figure 4b depicts cem, jurkat, and h9 uninfected t-cell lines, as well as the uninfected monocytic u937 cell line treated with gst as well as gst-p16 ink4a . cem, h9, and u937 control cells showed no appreciable decrease in cellular viability upon 48 hours of treatment with any of the four conditions. interestingly, in the presence of 0.5 ug of gst-p16 ink4a , jurkat cells exhibit an almost 40% decrease in cellular viability. we next performed western blots on whole cell extracts from all four of these uninfected cell lines and observed only jurkat cells exhibiting an endogenous expression of p16 ink4a ( figure 4d, lane 2) . this suggests that the decrease in cellular viability seen in jurkat cells ( figure 4b ) treated with p16 ink4a can be correlated with the expression of exogenous p16 ink4a in these cells, resulting in an increase in cdk4,6/cyclin d inhibition and an increase in apoptosis. interestingly, no endogenous levels of p16 ink4a are detected in the infected j1.1 cells. p16 ink4a is a critical member of the rb tumor-suppressor pathway which acts to arrest the cell-cycle at g1/s by inhibiting the binding of cdk4/6 to cyclin d1 and subsequently inhibiting the phosphorylation of rb. in figure 4c , we investigate the levels of rb present in jurkat cells alone (lane 1) compared to jurkat cells treated with an excess of gst or gst-p16 ink4a (2.5 μg, lanes 2 and 3). interestingly, upon treatment of exogenous gst-p16 ink4a , we observed a decrease in cellular levels of rb; indeed there is also a decrease in rb with gst treatment alone the rb antibody used detects total rb levels in the cell, therefore we could not assume a loss of phosphorylation due to the inhibitory effect of p16 ink4a on cdk4/6. a recent paper has addressed the literature-wide discrepancies of rb dephosphorylation vs. degradation in response to drug treatment or cell senescence in various cell types [51] . it is possible that the increased amount of p16 ink4a present in these cells has induced a proteasomal degradation of rb that has not otherwise been characterized in t cells. the cell line panel in figure 4d was also screened for the presence of endogenous levels of rb in these cell lines, and interestingly there is a high degree of variability. the t cell lines cem, jurkat, and the hiv-1 infected j1.1 have the highest endogenous levels of rb. interestingly, the monocytic cell lines u937 and the hiv-1 infected u1 have the lowest amount of rb present, with almost completely undetectable levels in hiv-1 infected u1 cells. the variability supports the discrepancies seen in the literature about hypo-, hyper-phosphorylation of rb, as well as depletion or degradation of rb during cell cycle or cellular responses. in order to confirm that the effects seen by gst and gst-p16 ink4a treatment in figure 4a , b, and c, we checked to ensure that the purified proteins are actually entering the cell. jurkat, j1.1, u937, and u1 cell lines were treated with an excess (2.5 μg) of gst or gst-p16 ( figure 4e ). at 48 hours post treatment, the cells were harvested, washed extensively, lysed, and incubated with glutathione-sepharose beads overnight. the glutathione-sepharose beads were washed extensively to remove any non-specific proteins with buffers containing salts and detergents. the bound proteins were subjected to western blot for the presence of p16 ink4a as shown in figure 4e . jurkat whole cell extract served as the positive control (lanes1 in both blots) and a higher molecular weight band corresponding to gst-p16 ink4a was observed in the gst-p16 ink4a pulldown lanes for each of the cell lines (lanes 4 and 7 in both blots). the lack of detection in the untreated cell lysate incubated with beads alone indicates that the protein detected in lanes 5 and 8 are specifically the gst-bound proteins. these studies confirm that the gst proteins are indeed entering the cells when incubated in the extracellular environment. in order to confirm that the cellular effects shown in figure 4 are specific to the natural biological activity of p16 ink4a as a cdk4/6/cyclin d inhibitor, we attempted to mimic these studies with the small molecule compound inhibitor fascaplysin. fascaplysin (fasc) is a naturally derived molecule isolated from a marine sponge which specifically inhibits the interaction between cdk4/cyclin d at an ic 50 of approximately 0.35 μm, and to a lesser extent cdk6/cyclin d by binding the atp pocket of cdk4, resulting in cell cycle arrest at g1/s [52, 53] . again, we treated latently infected hiv-1 cell lines (j1.1 and u1) with three concentrations of fasc (100 nm, 500 nm, and 1 μm) and collected supernatants at 24, 48, and 72 hours post treatment. the rt activity of both j1.1 and u1 cells in the presence of fasc decreased over time with increasing concentration of the drug. this indicates that the presence of a general cdk4/cyclin d inhibitor is able correlating with the viability assay presented in figure 4b , approximately 50% of jurkat cells were killed due to additional cdk4/cyclin d inhibition by 1 μm of fasc treatment. none of the other cell lines exhibit appreciable cell death which indicates that the drug treatment itself is not toxic to the cells. in figure 5c , we investigate the levels of rb present in jurkat cells alone (lane 1) compared to jurkat cells that have been treated with three concentrations of fasc (lanes 3, 4, and 5). again, correlating with our exogenous p16 ink4a treatment data in figure 4 , we observed a decrease in total cellular rb levels at the highest concentration of fasc. this set of data confirms that the cellular effects we observed with exogenous p16 ink4a may be due to the specific cdk inhibitory activity of this molecule. it is interesting to note that these effects are seen with simple protein treatment of the cells with a purified molecule which may not have efficient entry as compared to transfection or drug treatment. this sug-gests that p16 ink4a in the serum may be able to enter and exit lymphocytes and exhibit its inhibitory effects during an hiv-1 infection. we previously showed that both p16 ink4a and fascaplysin treatment results in a loss of cellular viability in jurkat cells as well as a decrease in viral production in infected j1.1 and u1 cells. we were interested to detect the cell cycle pattern of jurkat, j1.1, u937, and u1 in response to fascaplysin treatment. cells were treated with three concentrations of fasc (100 nm, 500 nm, 1 μm) and were collected after 48 hours. cells were fixed and stained with propidium iodide and cell cycle analyzed using a facscalibur flow cytometer. in figure 6a the presence of an additional cdk4/6/cyclin d inhibitor, we observed the normal g1/s arrest effect. there were no major appreciable differences in cell cycle in the fasc treated u937 and u1, however, looking at the protein profile of endogenously expressed p16 ink4a and rb, it is not surprising that these monocytic cell lines would exhibit a different inhibitory pathway. taken together, the cell cycle data shown in figure 6 , supports the overall notion of cdk4/6/cyclin d inhibitory effect by p16 ink4a and fascaplysin. the global proteomic analysis of serum proteins is not without its challenges; however the presence or absence of proteins in such bodily fluids of patients is often the most accurate reflection of cellular leakage or secretion of proteins in response to a disease state. unfortunately, the classical serum proteome contains a large concentration of high abundance proteins that mask the individual proteins that are unique to a particular phenotype. the identification of such low abundance serum proteins is attractive as a method of early detection of a disease state or a response to infection. here, we demonstrate that the detection of unique proteins in the serum of hiv-1 infected long-term non-progressors may be indicative of a natural "immunity" to the progression of hiv-1 infection. specifically, we have identified p16 ink4a , a cdk4/6 inhibitor, as preferentially present in pooled serum of hiv-1 ltnp patients, as opposed to hiv-1 infected individuals responding to haart treatment. p16 ink4a is a member of the ink4/arf family of endogenous cdki's that serve to regulate cell cycle progression through the inhibition of specific cdk/cyclin interactions. p16 ink4a is a critical member of the rb/p16 tumor-suppressor pathway which inhibits the activation of cdk4/6, preventing the progression through the cell cycle. this tumor-suppressive pathway is mutated in close to 100% of human cancers and specific loss-of-function mutations are found within the rb gene or the cdkn2a gene encoding p16 ink4a [54] . in addition to the direct inactivation of these two proteins, cancer cells also override this regulatory pathway by overexpressing cdk/ cyclins as well as inducing the endogenous loss of expression of other cdk inhibitors. the loss of p16 ink4a results in the constitutive activation of cdk4/6 as well as prb hyperphosphorylation, therefore bypassing the antioncogenic senescence induced by this cdk inhibitor. in addition to necessitating an oncogenic state, the regulation and manipulation of cellular cdks and cyclins is also critical for an active hiv-1 viral infection and occurs mainly through the hiv-1 transactivator tat. tat interacts with the cdk2/cyclin e complex to phosphorylate cdk7 and assist in the phosphorylation of the c-terminal domain (ctd) of rna pol ii at the viral ltr (long-terminal repeat) to promote viral transcription [55] [56] [57] . cdk2 is also involved in the direct phosphorylation of tat at both serine 16 and 46, which are critical for efficient tat activity [56, 57] . hiv-1 transactivation is also heavily dependent on the recruitment of the cdk9/cyclin t1 complex to the viral tar element where it also assists in the phosphorylation of the ctd of rna pol ii as well as an autophosphorylation event which is important for the localization to the nucleus [58] . taking into consideration the recruitment of cellular kinases for hiv-1 transcription, it is not surprising that pharmacological cdk inhibitors have been developed (analogous to endogenous cdki's) as a template to specifically inhibit these kinases. therefore, the presence of the cdk inhibitor p16 ink4a in ltnp serum is suggestive of an existing defense mechanism in these patients that imparts a predisposition to a lack of hiv-1 disease progression. the development of pharmacological cdki's and peptide mimetics is a commonly used approach for both cancer and viral therapeutics. for instance, p16 ink4a peptides have previously been developed, which, when conjugated to localization proteins/peptides, were able to block cell cycle progression in breast cancer and colon cancer cell lines in vitro as well as reduce tumor size in an in vivo mouse model of pancreatic cancer [59] [60] [61] . these peptides have also been used for intracellular delivery to leukemia and lymphoma derived cells [59, 62] . these studies have demonstrated the ability of cdki-derived peptides to be used for both direct cell cycle arrest and the treatment of metastatic cancers. this type of therapy is an appropriate approach to cancerous states where the oncogenic mutation results in a loss of function or expression of the p16 ink4a in this study we also chose to reinforce the p16 ink4ainduced cdki effects seen on cellular viability and viral replication with a known cdk4/6/cyclin d inhibitor, fascaplysin. we show that treatment of jurkat cells with this inhibitor also results in a decrease in cellular viability, consistent with the effect seen by treatment with endogenous p16 ink4a . additionally, the treatment of both stably infected hiv-1 cell lines, j1.1 and u1, with fascaplysin resulted in a decrease in viral replication with a minimal decrease in cellular viability. the correlation of this pharmacological cdki inhibitor data with the functional-cdki p16 ink4a data suggests that the preferential inhibition of the cdk4/6/cyclin d interaction by ltnps may contribute to this phenotype. the high and low abundance fractions analyzed for each of the three patient types were a pooled representation of six individual patient samples that almost certainly have inherent variability. indeed, when assaying for p16 ink4a in high and low abundance serum fractions for each individual ltnp patient, p16 ink4a was present in only a subset of the patients analyzed. this indicates that although p16 ink4a may serve as a unique serum protein indicating a ltnp hiv-1 infected status, individual person-to-person changes will alter the serum proteome profile and needs to be taken into consideration. differences in expression of p16 ink4a can be due to either a host cellular response to the infection or from an influence of the infection itself. two different scenarios need to be finally, many complicating factors can contribute to the altered state of proteins present in the serum, not the least of which is the length of time in which the patient has been infected, age, gender, coinfections, and other preexisting conditions such as cancer or metabolic diseases. therefore, it is important to take into consideration some of these factors when applying global proteomic analyses to hiv-1 patient derived samples. 293t endothelial kidney cell line was harvested for whole cell extract and used as a positive control. 293ts were grown in dulbecco's modified eagle's medium (dmem) containing 10% fbs, 1% l-glutamine, and 1% streptomycin/penicillin (quality biological). latently infected hiv-1 cell lines j1.1 (t-cells) and u1 (monocytes) were used for rt assays and whole cell extracts were obtained for western blots. uninfected cem, jurkat, and h9 cell lines (t-cells) and uninfected u937 cell line (monocytes) were used for viability assays and whole cell extracts were obtained for western blots. uninfected cells were grown in rpmi-1640 media containing 10% fbs, 1% l-glutamine, and 1% streptomycin/penicillin (quality biological). all cells were incubated at 37°c and 5% co2. gst-p16 ink4a was a generous gift from dr. ming-daw tsai, institute of biological chemistry, academia sinica, nankang, taipei, taiwan eighteen subject serum samples (6 ltnp, 6 hiv infected subjects receiving haart therapy, and 6 uninfected individuals) were obtained through washington dc site of the women's interagency hiv study (wihs) ( table 1) . wihs is an nih multicenter study of the natural history of hiv-1 infection in women [29] . ltnps were defined by wihs as being hiv infected, but disease free for at least five years, a cd4 count of greater than 500 at all visits, and no history of anti-retroviral therapy. serum samples were subjected to depletion of the 12 most abundant serum proteins using the proteomelab igy-12 high capacity spin column proteome partitioning kit from this spin column consists of anti-human serum albumin, anti-igg, anti-fibrinogen, anti-transferrin, anti-iga, anti-igm, anti-hdl (anti-apo a-i and anti-apo a-ii) , anti-haptoglobin, anti-α1-antitrypsin, anti-α1-acid glycoprotein and anti-α2-macroglobulin conjugated to polymeric microbeads. twenty microliters of each serum sample was diluted 1:25 in dilution buffer and ran over the spin column. low abundant proteins were collected in the flowthrough and subsequent high abundant, bound proteins were retained and eluted with a low ph stripping buffer. protein concentrations of both low and high abundant fractions were calculated and pooled as indicated. prowl-cgi/profound.exe databases for peptide mass fingerprinting analysis. fifty microliters of pooled low abundance ltnp serum sample "a" and individual low abundance ltnp serum samples (1) (2) (3) (4) (5) (6) were incubated with either α-igg or α-p16 ink4a as indicated, volume was brought up to 500 μl with tne 50 western blots were performed to validate proteins identified from depleted serum samples. whole cell extracts were obtained from cell culture pellets washed twice with 25 ml of phosphate buffered saline (pbs) with ca2+ and mg2+ (quality biological) and centrifuged once more. cell pellets were resuspended in lysis buffer (50 mm tris-hcl, ph 7.5, 120 mm nacl, 5 mm edta, 0.5% np-40, 50 mm naf, 0.2 mm na3vo4, 1 mm dtt, one complete protease cocktail tablet/50 ml) and incubated on ice for 20 min, with a gently vortexing every 5 min. cell lysates were transferred to eppendorf tubes and were centrifuged at 10,000 rpm for 10 min. supernatants were transferred to a fresh tube where protein concentrations were determined using bio-rad protein assay (bio-rad, hercules, ca). antibodies against cdk4 (sc-749), p16 (sc-467), pctaire (sc-174), rb (sc-50), and actin (sc-1615) were purchased from santa cruz biotechnology (santa cruz, ca). antibodies against hp1α and hp1γ were purchased from cell signaling (danvers, ma). j1.1 and u1 cells (2 × 10 6 ) were treated with gst or gst-p16 (0.1 or 0.5 μg) or fascaplysin (100 nm, 500 nm, or 1 μm) and supernatants collected to test for the presence of virus 48 hours post treatment. jurkats were treated with gst or gst-p16 (2.5 μg) for 48 hours prior to hiv-1 infection with the dual tropic strain 89.6. supernatants were collected at various time points post infection to test for the presence of virus post treatment. viral supernatants (10 μl) were incubated in a 96-well plate with reverse transcriptase (rt) reaction mixture containing 1x rt buffer (50 mm tris-hcl, 1 mm dtt, 5 mm mgcl 2 , 20 mm kcl), 0.1% triton, poly(a) (1u/ml), pd(t) (1u/ml), and [3h]ttp. the mixture was incubated overnight at 37°c, and 5 μl of the reaction mix was spotted on a deae filtermat paper, washed four times with 5% na 2 hpo 4 , three times with water, and then dried completely. rt activity was measured in a betaplate counter (wallac, gaithersburg, md). five thousand cells were plated per well in a 96-well plate and the next day cells were treated with gst or gst-p16 (0.1 or 0.5 μg) or fascaplysin (100 nm, 500 nm, or 1 μm). forty-eight hours later, 10 μl mtt reagent (5 mg/ml) was added to each well and plates incubated at 37°c for 3 hours. next, 100 μl of dmso was added to each well to solubilize the violet crystals. the assay was read at 570 nm. cells were washed with pbs and fixed with 70% ethanol. following rehydration in pbs, cells were stained in pbs containing 25 ug/ml propidium iodide (sigma), 10 ug/ml rnase a (sigma) and 0.1% np-40. cells were analyzed on a bd facscalibur flow cytometer. cell cycle analysis and measurement of apoptosis was performed using cell-quest software. aggregates and debris were excluded by gating on the fl2w and fl2a parameters. apoptosis was considered to be the population of cells that were sub-g1. toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry a novel approach and protocol for discovering extremely low-abundance proteins in serum peptidomics-based approach reveals the secretion of the 29-residue cooh-terminal fragment of the putative tumor suppressor protein dmbt1 from pancreatic adenocarcinoma cell lines the role of proteomics in toxicology: identification of biomarkers of toxicity by protein 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progression to aids by a deletion allele of the ckr5 structural gene. hemophilia growth and development study hla-b polymorphism in japanese hiv-1-infected long-term surviving hemophiliacs hla b*5701 is highly associated with restriction of virus replication in a subgroup of hiv-infected long term nonprogressors progression of hiv to aids: a protective role for hla-b27? the women's interagency hiv study transcription factor ap-2 activates gene expression of htlv-i transcription factor prdii-bf1 activates human immunodeficiency virus type 1 gene expression recruitment of chromatin-modifying enzymes by ctip2 promotes hiv-1 transcriptional silencing chromatin remodeling and modification during hiv-1 tat-activated transcription cleavage of human and mouse cytoskeletal and sarcomeric proteins by human immunodeficiency virus type 1 protease. actin, desmin, myosin, and tropomyosin il-16-and other cd4 ligand-induced migration is dependent upon protein kinase c hiv-1 tat protein induces il-10 production in monocytes by classical and alternative nf-kappab pathways human immunodeficiency virus type 1 tat protein induces an intracellular calcium increase in human monocytes that requires dhp receptors: involvement in tnf-alpha production signaling pathways triggered by hiv-1 tat in human monocytes to induce tnf-alpha hiv-1 tat protein induces interleukin-10 in human peripheral blood monocytes: involvement of protein kinase c-betaii and-delta trans-activation of hiv-1 ltrdirected gene expression by tat requires protein kinase c in vitro phosphorylation of human immunodeficiency virus type 1 tat protein by protein kinase c: evidence for the phosphorylation of amino acid residue serine-46 hiv envelope gp120 activates human arterial smooth muscle cells involvement of protein kinase c in hiv-1 gp120-induced apoptosis in primary endothelium mammalian cyclin-dependent kinases cyclin d-dependent kinases, ink4 inhibitors and cancer comparative genomics of cyclin-dependent kinases suggest co-evolution of the rnap ii c-terminal domain and ctddirected cdks heterochromatin formation in mammalian cells: interaction between histones and hp1 proteins functional cooperation between hp1 and dnmt1 mediates gene silencing the hp1alpha-caf1-setdb1-containing complex provides h3k9me1 for suv39-mediated k9me3 in pericentric heterochromatin suv39h1 and hp1gamma are responsible for chromatin-mediated hiv-1 transcriptional silencing and postintegration latency roninson ib: p21(waf1/cip1/sdi1) mediates retinoblastoma protein degradation inhibition of cyclin-dependent kinase 4 (cdk4) by fascaplysin, a marine natural product ca224, a non-planar analogue of fascaplysin, inhibits cdk4 but not cdk2 and arrests cells at g0/g1 inhibiting prb phosphorylation searching for selective cyclin-dependent kinase inhibitors to target the retinoblastoma/p16 cancer gene pathway phosphorylation of hiv-1 tat by cdk2 in hiv-1 transcription hiv-1 tat interaction with rna polymerase ii c-terminal domain (ctd) and a dynamic association with cdk2 induce ctd phosphorylation and transcription from hiv-1 promoter hiv-1 tat-associated rna polymerase c-terminal domain kinase, cdk2, phosphorylates cdk7 and stimulates tat-mediated transcription novel hiv-1 therapeutics through targeting altered host cell pathways therapeutic peptides for cancer therapy. part ii -cell cycle inhibitory peptides and apoptosisinducing peptides inhibition of prb phosphorylation and cell-cycle progression by a 20-residue peptide derived from p16cdkn2/ink4a the p16(ink4a) tumour suppressor protein inhibits alphavbeta3 integrin-mediated cell spreading on vitronectin by blocking pkc-dependent localization of alphavbeta3 to focal contacts highly efficient delivery of p16 antitumor peptide into aggressive leukemia/lymphoma cells using a novel transporter system we would like to thank the members of the kashanchi lab for experiments and assistance with the manuscript. we thank dr. ming-daw tsai (institute of biological chemistry, academia sinica) for the gst-p16 ink4a plasmid constructs. most of the data on the current manuscript was generated using funds from nih grants ai078859, ai074410 and ai043894. data in this manuscript were the authors declare that they have no competing interests. rvd performed the serum depletion, 2d gel analysis, maldi preparation, and mass spectrometry analysis as well as the drafting of the manuscript. ig performed serum depletion, maldi preparation, and confirmatory western blots. kkh performed the rt and mtt assays. re performed the maldi preparation and analysis. zk provided support in drafting the manuscript and maldi analysis. cl established the patient definitions (ltnp, haart responders, etc.) and patient sample identification. my is the wihs contact and provided support and information on patient samples. fk provided overall direction and funding for the project. all authors have read and approved the final manuscript. key: cord-320083-0k15w624 authors: leitão, jorge h. title: microbial virulence factors date: 2020-07-27 journal: int j mol sci doi: 10.3390/ijms21155320 sha: doc_id: 320083 cord_uid: 0k15w624 microbial virulence factors encompass a wide range of molecules produced by pathogenic microorganisms, enhancing their ability to evade their host defenses and cause disease [...]. most represented, followed by enterobacter and citrobacter [2] . the isolates were further characterized concerning the presence in their genomes of genes encoding selected virulence factors, as well as their phenotypes related to biofilm formation and resistance to a selection of antibiotics. results point out that the isolates do not encompass particularly virulent strains and in most of the cases were susceptible to antibiotics [2] . yang et al. investigated the role of the salmonella enterica serovar typhimurium (st) pdxb-usg-trua-deda operon on intracellular survival using deletion mutants constructed with the λ-red recombination technology [3] . the salmonella genus comprises several facultative intracellular pathogens capable of infecting both human and animal hosts. the st deletion mutants was investigated in j774a.1 macrophage cells. the deletion mutants ∆pdxb, ∆usg, and ∆trua exhibited reduced replication abilities compared to st and the deletion mutant ∆deda. the pdxb-usg-trua-deda operon is shown to contribute to st virulence in mice, and to resistance to oxidative stress [3] . aeromonas hydrophila is an aquatic gram-negative bacterium, capable of causing serious and lethal infections to a wide range of hosts, including fish, birds, amphibians, reptiles, and mammals [4] . dong et al. described the identification and functional characterization of the lahs global regulator of a. hydrophila [4] . lahs was identified after the screening of a tn5-derived library of 947 a. hydrophila mutants for reduced hemolytic activity. the lysr family transcriptional regulator family member lahs was found to play a role in biofilm formation, motility, antibacterial activity, resistance to oxidative stress, and proteolytic activity, as well as essential for a. hydrophila virulence to zebrafish [4] . the comparative proteomics analysis performed by the authors confirmed the role of the protein as a global regulator in a. hydrophila [4] . bacteria of the dickeya genus comprise plant pathogens that affect crops such as potatoes. in order to succeed when infecting their hosts, dickeya secrete several proteins with plant cell wall degrading activities, including pectinases, cellulases, and proteases [5] . to investigate the role played by the protease lon on d. solani pathogenicity towards potato, figaj et al. used a λ-red-derived protocol to construct a lon deletion mutant [5] . results presented indicate that the lon protein plays a role in protecting the bacterium to high ionic and temperature stresses, affecting the activity of pectate lyases, the organism motility, and delaying the onset of infection symptoms in the potato host [5] . the plant pathogen candidatus phytoplasma mali is the causal agent of apple proliferation disease, that affects apple production in northern italy [6] . phytoplasma are biotrophic, obligate plant and insect bacterial symbionts, with a biphasic life cycle comprising reproduction in phloem-feeding insects and in plants [6] . the paper of mittelberger et al. focused on the effector protein pme2 (protein in malus expressed 2), expressed by p. mali when infecting apples [6] . the in silico analysis of the pme2 protein sequence performed revealed that the protein has features of effector proteins of gram-positive bacteria, with a predicted final localization at the cytoplasm or nucleus of the host [6] . two main protein variants, pme2st pme2at, were found associated in infected apple trees from italy and germany. using protein variants tagged with gfp, both variants were found to translocate to the nucleus of nicotiana spp. protoplasts. a better understanding of the molecular mechanisms used by p. mali to manipulate its host will rely on genomics analysis, since no genetic manipulation is presently available for these organisms [6] . the necrotrophic fungal pathogen sclerotinia sclerotiorum (lib.) de bary infects a wide range of plants causing devastating agricultural losses. the organism forms a typical structure named sclerotia when vegetative hyphae gather to form a hardened multicellular structure important in its development and pathogenesis, and that under favorable conditions germinate leading to vegetative hyphae or apothecia that will initiate novel disease cycles by producing ascospores [7] . li et al. used a proteomics approach based on 2d gels followed by spot isolation and protein identification by maldi-tof to identify proteins differentially expressed between a wild-type strain and a deletion mutant on the gene ssnsd1 encoding a type ivb gata zinc finger transcription factor [7] . although the gene encoding ssnsd1 was found as expressed at low levels during the hyphae stage, the mutant is unable to form the compound appressoria. the authors were able to identify a total of 40 proteins as differentially expressed, 17 with predicted functions and 23 as hypothetical proteins [7] . the authors emphasize the utility of the approach used to identify important proteins involved in the ssnsd1-mediated formation of appressorium. in addition to other factors, the success of pathogens rely on cell-cell communication. bacterial outer membrane vesicles (omv) are recognized as an efficient means of bacteria-bacteria and bacteria-host communication, not only intra-species, but also interspecies [8] . despite the lack of data on a possible role played by omvs in bacterial-yeast communication, roszkowiak et al. investigated the role played by moraxella catarrhalis omvs on the susceptibility of selected bacterial and fungal pathogens to the cationic peptide polymyxin b, and to the serum complement [9] . using omvs from m. catarrhalis strain 6, the authors found that these omvs conferred protection against the cationic peptide polymyxin b to the non-typeable haemophilus influenzae, pseudomonas aeruginosa, and acinetobacter baumannii. furthermore, omvs also protected serum-sensitive non-typeable h. influenza and promoted the growth of the serum-resistant p. aeruginosa and a. baumannii against the complement [9] . in addition, the results presented also show that omvs facilitate the formation of hyphae by the pathogenic yeast candida albicans, promoting its virulence [9] . as stated by the authors, this work might pave the way to uncover additional roles played by omvs-dependent interactions in multispecies communities [9] . the rna chaperone hfq is a master regulator of gene expression in bacteria, mediating the interaction of small noncoding rnas with their mrna targets, including those related to virulence in gram-negative bacteria [10] . dienstbier et al. performed an integrated omics comparative analysis of the hfq regulon in the bordetella pertussis human pathogen, responsible for respiratory tract infections, in particular of a whooping cough [11] . based on the use of rnaseq, and gene ontology analysis, genes significantly upregulated in the hfq mutant fall into categories including "translation", "regulation of transcription", and "transmembrane transport", while genes downregulated fall in the categories "transmembrane transport", "iron-sulfur cluster assembly", "oxido-reduction process", "pathogenesis", and "protein secretion by the type iii secretion system" [11] . correlations of transcriptome, proteome, and secretome datasets are also presented [11] . results presented corroborate the central role played by hfq on the physiology and pathogenicity of b. pertussis [11] . in their brief report, maisetta et al. performed the ex vivo evaluation of the bactericidal activity of combinations of the semi synthetic antimicrobial peptide lin-sb056-1 in combination with edta (ethylenediaminetetraacetic acid) against endogenous p. aeruginosa present in the sputum from patients suffering from primary ciliary dyskinesia (pcd) [12] . the authors observed that the peptide and edta were almost inactive against pcd sputum endogenous p. aeruginosa when used alone, but exhibited a significant synergistic killing effect with a sputum sample-dependent efficacy [12] . edta, but not lin-sb056-1, was found to inhibit biofilm formation and the production of virulence factors including alginate, pyocyanin, and the metalloprotease lasa [12] . various bacterial species have evolved various strategies to invade, survive, and multiply intracellularly in host cells. the paper of denzer et al. presents an updated review of the mechanisms used by bacteria to invade the host cell, to manipulate their biochemical and gene expression machinery, and to multiply and escape from the host cell [13] . the authors present a thorough review of mechanisms used by intracellular pathogens, including the highjack of host immune defenses to enter into the host cell. central attention is given to the various mechanism used to manipulate gene expression, including histone modification, control of host dna methylation patterns, sabotage of host long non-coding rnas, interfering with the host rna transcription and translation, as well as with host protein stability [13] . the importance of the detailed molecular knowledge of pathogenesis mechanisms to the development of strategies to combat bacterial infections is highlighted [13] . the functions of grimelysin of serratia grimesii and protealysin of serratia proteamaculans that use actin as a substrate and promote bacterial invasion was reviewed by khaitlina et al. [14] . the serratia genus comprises facultative pathogens able to cause nosocomial infections or infections in immunocompromised patients, but nosocomial infections by s. grimesii or s. proteamaculans are low [14] . the paper focused on the discovery, properties and substrate specificity of the two proteases, their high specificity towards actin, and discussed their contribution to the invasiveness of serratia, although further knowledge of the bacterium virulence factors and the cellular response mechanisms is required to fully understand the mechanism of serratia invasion of the host cell [14] . the virulence factors that the bacteria use to cross the blood-brain barrier and cause meningitis is reviewed by herold et al. [15] . meningitis remains a worldwide problem often associated with fatalities and severe sequelae. after reviewing important traits of the central nervous system barriers to bacterial entrance, the authors review the various stages of the virulence processes of bacterial meningitis, including the processes of attachment and invasion, the routes used to enter the central nervous system, and the general mechanisms used to survive intracellularly [15] . the roles played by virulence factors produced by bacteria when crossing the central nervous system is also addressed, followed by the review of the specific traits of bacterial species more commonly associated with meningitis [15] . coagulase-negative staphylococci are a broad group of skin commensals that emerged as major nosocomial pathogens, with the species s. epidermidis, s. haemolyticus, s. saprophyticus, s. capitis, and s. lugdunensis as the most frequent pathogens [16] . in their paper, argemi et al. reviewed the recent progress achieved in the pathogenomics of these species, based on published work supported by whole-genome data deposited in public databases [16] . as stated by the authors, the ever increasing amount of data available at the genomic, molecular, and clinical levels is expected to enhance the development of innovative approaches to characterize the pathogenicity of this bacterial group of pathogens [16] . bacteria of the trueperella pyogenes species are considered as belonging to the microbiota of animals skin and mucous membranes of the upper respiratory and urogenital tracts, but it is also an important opportunistic pathogen to animals, leading to important economic losses [17] . in their paper, rzewuska et al. reviewed the taxonomy of the species, their pathogenicity to animals, and the various diseases associated, as well as their possible involvement in zoonotic infections, as well as the reservoirs and routes of transmission and infections [17] . the authors also present a thorough review of the main virulence factors used by the organism, including pyolysin, fimbriae, extracellular matrix-binding proteins, neuraminidases, and ability to form biofilms [17] . the availability of complete genome sequences and a better knowledge of t. pyogenes virulence factors, transmission routes, and epidemiology of infections is expected to lead to the development of effective vaccines, with particular hope deposited on dna vaccines [17] . candidiasis are on the rise worldwide, with candida albicans and candida glabrata as the more prevalent etiologic agents of these fungal diseases [18] . the paper by galocha et al. thoroughly reviewed the distinct strategies used by the two candida species to successfully cause human infections, starting by the adhesion and ability to form biofilms [18] . while c. albicans is dimorphic, growing as yeast or pseudohyphae, c. glabrata cannot undergo hyphal differentiation. as a consequence, c. albicans relies on the production of proteolytic enzymes and hyphal penetration to invade the host cell, while c. glabrata is thought to invade host cells by inducing endocytosis [18] . the authors extensively review the distinct mechanisms used by the two pathogenic to evade the host immune system, and succeed as pathogens. the detailed knowledge of the virulence mechanisms is critical to develop therapies that specifically target virulence traits of these two pathogenic yeasts [18] . bacterial small non-coding regulatory rnas (srnas) have emerged over the last decade as key regulators of post-transcriptional regulators of gene expression, being involved in a wide range of cellular processes, including bacterial virulence [19] . in their review, pita et al. updated knowledge on srnas from two pathogens associated with respiratory infections and lung function decline of patients suffering from cystic fibrosis, p. aeruginosa and bacteria of the so-called burkholderia cepacia complex (bcc) [20] . as stated by the authors, the knowledge on p. aeruginosa srnas is far more extensive than from bacteria of the bcc. after reviewing the main molecular characteristics of bacterial rnas and their modes of action, including the role played by hfq as a mediator of rna-rna interactions, the authors detail the description of the roles played by p. aeruginosa srnas known for their involvement in virulence traits of the bacterium. despite the shorter information on bcc srnas, the authors make a brief description of known srnas from bcc [19] . the identification and functional characterization of additional srnas from these two pathogens will certainly enlighten our knowledge on their virulence traits. the development of new tools to investigate microbial pathogenesis, at the molecular and cellular level, is of keen importance to comprehend how the microorganism can invade the host and cause infection. the paper from hatlem et al. reviewed the basic molecular traits and applications of the spycatcher-spytag system, originally developed as a method for protein ligation [20] . the system consists of a modified domain of the spycatcher surface protein from streptococcus pyogenes that recognizes the cognate spytag peptidic sequence composed of 13 amino acid residues [20] . upon recognition, a covalent isopeptide bond is formed between a lysine side chain of the spycatcher and an aspartate of the spytag [20] . the authors describe in detail the fundamentals of the system and of related variants, emphasizing their uses in molecular studies of microbial virulence factors, surface proteins, membrane dynamics, as well as in the development of vaccines [20] . microorganisms employ a wide array of virulence factors to successfully thrive and flourish with their hosts, leading this interaction to the development of infections that can often be fatal. the molecular knowledge of the virulence traits, associated with the recent availability of genomics data and bioinformatics tools for the more frequent human pathogens, is expected to lead in the near future of novel molecules and strategies to battle infectious diseases. funding: this research was funded by fundação para a ciência e a tecnologia, through project uidb/04565/2020 from ibb-institute for bioengineering and biosciences. the serine protease autotransporters tagb, tagc, and sha from extraintestinal pathogenic escherichia coli are internalized by human bladder epithelial cells and cause actin cytoskeletal disruption antibiotic resistance, virulence factors, phenotyping, and genotyping of non-escherichia coli enterobacterales from the gut microbiota of healthy subjects relation of the pdxb-usg-trua-deda operon and the trua gene to the intracellular survival of salmonella enterica serovar typhimurium discovery of lahs as a global regulator of environmental adaptation and virulence in aeromonas hydrophila lon protease is important for growth under stressful conditions and pathogenicity of the phytopathogen, bacterium dickeya solani a novel effector protein of apple proliferation phytoplasma disrupts cell integrity of nicotiana spp proteomics analysis of ssnsd1-mediated compound appressoria formation in sclerotinia sclerotiorum types and origins of bacterial membrane vesicles interspecies outer membrane vesicles (omvs) modulate the sensitivity of pathogenic bacteria and pathogenic yeasts to cationic peptides and serum complement hfq: a multifaceted rna chaperone involved in virulence comparative integrated omics analysis of the hfq regulon in bordetella pertussis targeting pseudomonas aeruginosa in the sputum of primary ciliary dyskinesia patients with a combinatorial strategy having antibacterial and anti-virulence potential from gene to protein-how bacterial virulence factors manipulate host gene expression during infection bacterial actin-specific endoproteases grimelysin and protealysin as virulence factors contributing to the invasive activities of serratia virulence factors of meningitis-causing bacteria: enabling brain entry across the blood-brain barrier coagulase-negative staphylococci pathogenomics pathogenicity and virulence of trueperella pyogenes: a review divergent approaches to virulence in c. albicans and c. glabrata: two sides of the same coin small noncoding regulatory rnas from pseudomonas aeruginosa and burkholderia cepacia complex catching a spy: using the spycatcher-spytag and related systems for labeling and localizing bacterial proteins acknowledgments: ibb-institute for bioengineering and biosciences is acknowledged for funding. the author declares no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-300884-rqfxe0x1 authors: zhang, jianqiang; guy, james s.; snijder, eric j.; denniston, doug a.; timoney, peter j.; balasuriya, udeni b.r. title: genomic characterization of equine coronavirus date: 2007-12-05 journal: virology doi: 10.1016/j.virol.2007.06.035 sha: doc_id: 300884 cord_uid: rqfxe0x1 the complete genome sequence of the first equine coronavirus (ecov) isolate, nc99 strain was accomplished by directly sequencing 11 overlapping fragments which were rt–pcr amplified from viral rna. the ecov genome is 30,992 nucleotides in length, excluding the polya tail. analysis of the sequence identified 11 open reading frames which encode two replicase polyproteins, five structural proteins (hemagglutinin esterase, spike, envelope, membrane, and nucleocapsid) and four accessory proteins (ns2, p4.7, p12.7, and i). the two replicase polyproteins are predicted to be proteolytically processed by three virus-encoded proteases into 16 non-structural proteins (nsp1–16). the ecov nsp3 protein had considerable amino acid deletions and insertions compared to the nsp3 proteins of bovine coronavirus, human coronavirus oc43, and porcine hemagglutinating encephalomyelitis virus, three group 2 coronaviruses phylogenetically most closely related to ecov. the structure of subgenomic mrnas was analyzed by northern blot analysis and sequencing of the leader–body junction in each sg mrna. coronaviruses are mainly associated with respiratory and gastrointestinal disease in humans (drosten et al., 2003; holmes, 2001; ksiazek et al., 2003; peiris et al., 2003; van der hoek et al., 2004; woo et al., 2005) and respiratory, enteric, neurological, or hepatic disease in animals (holmes, 2001) . coronaviruses have also been isolated from bats, poultry and other birds (cavanagh, 2005; chu et al., 2006; poon et al., 2005; ren et al., 2006) . on the basis of antigenic and genetic analyses, coronaviruses are divided into three groups (gonzalez et al., 2003; gorbalenya et al., 2004; snijder et al., 2003) . group 1 viruses include human coronaviruses 229e (hcov-229e) and nl63 (hcov-nl63), canine coronavirus (ccov), feline coronavirus (fcov), porcine transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), and bat coronavirus. group 2 viruses are subdivided into group 2a which includes murine hepatitis virus (mhv), human coronaviruses oc43 (hcov-oc43) and hku1 (hcov-hku1), bovine coronavirus (bcov), porcine hemagglutinating encephalomyelitis virus (phev), and rat coronavirus (rcov), and group 2b which includes sars-coronavirus (sars-cov). group 3 viruses include avian viruses, such as avian infectious bronchitis virus (ibv), and turkey coronavirus (tcov). members of the family coronaviridae are enveloped, positive-stranded rna viruses with exceptionally large, polycistronic genomes (27-32 kb). the 5′-proximal two-thirds of the genome comprises two open reading frames (orfs), orf1a and orf1b, which encode the replicase polyproteins (pp) 1a and pp1ab (ziebuhr, 2005) . expression of the pp1ab requires a − 1 ribosomal frameshift during translation of the genomic rna (brierley et al., 1987) . the two replicase polyproteins are processed extensively by two or three viral proteases encoded by orf1a to generate up to 16 end-products termed nonstructural proteins (nsp) 1 to 16 and multiple processing intermediates (ziebuhr, 2005; ziebuhr et al., 2000) . the n-proximal region of the polyproteins is processed by one or two papain-like proteases (pl pro ), whereas the central and c-proximal region is processed available online at www.sciencedirect.com virology 369 (2007) 92 -104 www.elsevier.com/locate/yviro by the viral main protease, 3c-like protease (3cl pro ) (ziebuhr, 2005; ziebuhr et al., 2000) . the 3′-proximal one-third of the genome encodes structural proteins and various accessory proteins. genes encoding the four structural proteins present in all coronaviruses occur in the 5′ to 3′ order as spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins (brian and baric, 2005; lai et al., 2006) . some coronaviruses contain an additional structural protein, the hemagglutinin-esterase (he) protein which is located upstream of the s protein gene (lai et al., 2006) . in contrast to the replicase proteins which are directly translated from the genomic rna, coronavirus structural and accessory proteins are expressed from a nested set of 3′ coterminal subgenomic (sg) mrnas that also possess a common 5′ leader sequence derived from the 5′ end of the genome (pasternak et al., 2006; sawicki et al., 2007) . the common 5′ leader is fused to the 3′ body segments through a mechanism that is presumed to involve discontinuous minus strand rna synthesis to produce subgenome-length templates for subgenomic mrna synthesis, with the transcription regulatory sequence (trs) elements determining the fusion sites of leader and body segments (see recent review of pasternak et al., 2006; sawicki et al., 2007 for details) . equine coronavirus (ecov) was first isolated from feces of a diarrheic foal in 1999 (ecov-nc99) in north carolina, usa (guy et al., 2000) . little is known about ecov and its clinical significance. molecular characterization of ecov and development of diagnostic and prophylactic reagents necessitate sequencing of ecov. in this study, we determined the fulllength nucleotide sequence of the ecov-nc99 strain of equine coronavirus. the viral genome and proteome were analyzed and the predicted features of ecov nonstructural, structural, and accessory proteins were compared to those of other coronaviruses. synthesis of sg mrnas in ecov-infected cells was analyzed by northern blotting. the leader-body junction sequence in each sg mrna was determined and the exact position of trs used for synthesis of each sg mrna was mapped on the genome. the evolutionary relationship between ecov and other phylogenetically closely related group 2a coronaviruses was explored. we report here the full-length genomic sequence of the first ecov isolate, the nc99 strain, and this is also the first reported complete genome sequence of ecov. the nucleotide sequence was determined by directly sequencing 11 overlapping cdna fragments which were rt-pcr amplified from viral rna. the ecov-nc99 genome comprises 30,992 nucleotides (nt), excluding the 3′ poly (a) tail, and has a gc content of 37.2%. the nucleotide sequence data have been deposited in genbank under accession number ef446615. both 5′ and 3′ ends of the ecov genome contain short untranslated regions (utr). the 5′ utr comprises 209 nt (1-209) and includes a potential short internal orf of 8 codons (nt 99-125). four stem-loop structures (i, ii, iii, and iv) were identified in the 5′ utr and a short stretch of nucleotides that are part of the orf1a (see supplementary fig. s1 ). the bulged stem-loop iii (96-115) and iv (189-208) closely resemble the stem-loop iii and iv that have been identified as replication signaling elements in bovine coronavirus and other group 2 coronaviruses (raman and brian, 2005; raman et al., 2003; wu et al., 2003) . the 3′ utr of the ecov genome comprises 289 nt (30, 992) and contains a putative bulged stem-loop structure (nt 30,703-30,770 ) and a putative pseudoknot structure (30,766-30,819) (see supplementary fig. s2 ). similar putative bulged stem-loop structure and pseudoknot structure have been identified in murine hepatitis virus and other group 2 coronaviruses; these have been shown to be essential for viral replication (goebel et al., 2004a,b; hsue and masters, 1997; hsue et al., 2000; williams et al., 1999) . analysis of the ecov-nc99 genome reveals 11 potential orfs (1a, 1b, 2-8, 9a and 9b) as shown in fig. 1 and table 1 . the orfs 1a and 1b encode the replicase polyproteins pp1a and pp1ab. the orfs 2-8, 9a and 9b encode structural and accessory proteins ns2, he, s, p4.7, p12.7, e, m, n, and i, respectively. the replicase orf1a (nt 210-13,499) and replicase orf1b (13,478-21,595) occupy 21.4 kb (69%) of the ecov-nc99 genome. the translation of orf1a generates a precursor pp1a of 4,429 amino acids. similar to other coronaviruses, translation of orf1b involves a − 1 ribosomal frameshift, generating a 7128amino acid pp1ab. the ribosomal frameshift is assumed to be directed by two signals in the orf1a/1b overlapping region: a slippery sequence 5′uuuaaac3′ (nt 13,472-13,478) and a predicted downstream rna pseudoknot structure (nt 13,484-13,559) (see supplementary fig. s3 ). the pp1a and pp1ab proteins are predicted to be proteolytically processed by viralencoded proteases into 16 non-structural proteins (nsp1-16, table 2 ) required for viral replication and transcription. by comparison to other coronaviruses, a number of putative functional domains are predicted in the ecov pp1a and pp1ab and these are summarized in fig. 1 and table 2 (gorbalenya et al., 1991 snijder et al., 2003; ziebuhr, 2005; ziebuhr et al., 2001) . enzymatic activities of nsp3, nsp5, nsp12, nsp13, nsp14 and nsp15 have been experimentally confirmed for some coronaviruses (barretto et al., 2005; cheng et al., 2005; guarino et al., 2005; heusipp et al., 1997; ivanov et al., 2004a,b; ivanov and ziebuhr, 2004; lindner et al., 2005; minskaia et al., 2006; putics et al., 2005 putics et al., , 2006 seybert et al., 2000 seybert et al., , 2005 tanner et al., 2003; ziebuhr, 2005; ziebuhr et al., 2001) . the 3cl pro (catalytic residues his-3333 and cys-3437) is predicted to cleave the c-terminal half of the ecov pp1a and the orf1b-encoded part of pp1ab. the putative pl1 pro (catalytic residues cys-1078 and his-1229) and pl2 pro (catalytic residues cys-1675 and his-1832) are predicted to process the n-proximal regions of the ecov pp1a ( fig. 1 and table 2 ). the most striking differences between the ecov replicase and other group 2 coronaviruses replicases were identified in nsp3. the ecov nsp3 protein has 3 aa deletions and 55 aa insertions compared to the nsp3 proteins of bcov, hcov-oc43, and phev, three viruses phylogenetically most closely related to ecov. these insertions and deletions are clustered at two regions: the ac domain and the region between the pl2 pro and the y domain. the functional significance of these insertions and deletions is unknown as yet; however, the functions of pl1 pro , pl2 pro , and adrp are not anticipated to be affected since insertions and deletions are not located in the functional domains of these enzymes (fig. 1) . orf2 (nt 21,610-22,446) of ecov-nc99 encodes the predicted ns2 protein with 278 amino acids. the ns2 of fig. 1 . schematic diagrams of ecov genome organization. the ecov entire genome organization is depicted (middle). the 5′ leader, orfs 1a and 1b encoding replicase polyproteins are shown, with the ribosomal frameshift site indicated. structural and accessory proteins are also indicated: ns2 protein (encoded by orf2), hemagglutinin esterase (he, orf3), spike protein (s, orf4), p4.7 protein (orf5), p12.7 protein (orf6), envelope protein (e, orf7), membrane protein (m, orf8), nucleocapsid protein (n, orf9a), and i protein (orf9b). predicted cleavage products (nsp1-nsp16) of the replicase polyproteins are depicted (bottom). arrows represent sites in the corresponding replicase polyproteins that are cleaved by papain-like proteases (white arrows) or the 3c-like cysteine protease (black arrows). a number of putative functional domains predicted in the ecov pp1a and pp1ab are indicated. pl1, papain-like proteinase 1 (aa 1059-1275); pl2, papain-like proteinase 2 (aa 1570-1867); x, x-domain which contains adenosine diphosphate-ribose 1ʺ-phosphatase (adrp) (aa 1276-1435); tm, transmembrane domain; 3cl, 3c-like proteinase; rdrp, rna-dependent rna polymerase; z, zinc-binding domain; hel, helicase domain; exon, exonuclease; n, nidoviral uridylatespecific endoribonuclease (nendou); mt, 2′-o-ribose methyltransferase (2′-o-mt). domains ac (aa 846-1058) and y (aa 2310-2796) are described by ziebuhr et al. (2001) . the spike protein (1363 amino acids) of ecov is represented by a black line (top). the n-terminal signal peptide (amino acid residues 1-14 or 17), the heptad repeat 1 (hr1, amino acid residues 991-1902), the heptad repeat 2 (hr2, amino acid residues 1259-1304), the transmembrane domain (amino acid residues 1308-1330), and the cytoplasmic domain (amino acid residues 1331-1363) are depicted. a potential cleavage recognition sequence (rrqrr) at residues 764-768 and the predicted cleavage site between residues 768 and 769 are indicated. the generated cleavage products s1 and s2 subunits are depicted. the positions of the receptor-binding domain on the s1 subunit and the fusion peptide on the s2 subunit are currently unknown. ecov has 67%, 67%, and 45% amino acid identity with the respective ns2 proteins of bcov, hcov-oc43, and phev. the lower amino acid identity with phev may be attributable to the fact that phev has a truncated ns2 protein (vijgen et al., 2006) . sequence analysis revealed that the ecov ns2 protein contains a domain (aa 46-135) with similarity to the putative cyclic phosphodiesterase (cpd, martzen et al., 1999) . the cpd domain has also been identified in the ns2 proteins of other group 2a coronaviruses as well as in the 3′end of the pp1a protein of toroviruses snijder et al., 1991 snijder et al., , 2003 . the ns2 of ecov was predicted to contain 9 potential phosphorylation sites. the ns2 of ecov does not contain a signal peptide and is a non-secretory protein. the function of the ns2 protein in coronaviruses has not been studied in detail. it is known that the ns2 gene is non-essential for mhv replication in transformed cells (schwarz et al., 1990) . however, a recent study showed that a point mutation in the ns2 of mhv led to its attenuation in mice in spite of its wild-type replication in tissue culture (sperry et al., 2005) . orf3 (nt 22,458-23,729) of ecov-nc99 encodes the predicted he protein containing 423 amino acids. nine potential n-glycosylation sites were predicted. signalp analysis revealed a signal peptide probability of 0.802 with a potential cleavage site between residues 17 and 18. it was predicted that the n-terminal 390 amino acids are located outside the cell surface or viral envelope with a transmembrane helix at amino acids 391-413 and an internal domain at amino acids 414-423. the putative active site for esterase activity, fgds (kienzle et al., 1990) , is present at amino acids 36-39 of the he protein in ecov. orf4 (nt 23,744-27,835) of ecov-nc99 encodes the predicted spike (s) protein containing 1363 amino acids. eighteen potential n-glycosylation sites were predicted. an n-terminal signal peptide was identified with a potential cleavage site between amino acids 14 and 15 predicted by signalp-nn or between amino acids 17 and 18 predicted by signalp-hmm. the ecov s protein was predicted to be a typical type i membrane protein with the n-terminal 1307 residues exposed on the outside of the cell surface or virus particle, a transmembrane domain near the c terminus (residues 1308-1330), followed by a cytoplasmic tail (residues 1331-1363). following multiple alignments with the s proteins of other group 2a coronaviruses, a potential cleavage recognition sequence (rrqrr) was identified at residues 764-768 which would predict a cleavage between amino acids 768 and 769, separating the ecov s protein into s1 and s2 subunits (fig. 1) . the ecov s1 subunit is expected to contain a receptor-binding domain whose position has not yet been determined. the s2 subunit is predicted to mediate membrane fusion. two heptad repeat (hr) regions, which are conserved in position and sequence among the three groups of coronaviruses and play important roles in membrane fusion (see reviews of eckert and kim, 2001; hernandez et al., 1996) , were identified in the ecov s2 subunit (hr1: aa 991-1092; hr2: aa 1259-1304) (fig. 1) . the ecov s2 subunit is anticipated to possess a fusion peptide whose position is yet unknown. some coronavirus s proteins have been shown to contain important neutralization epitopes (godet et al., 1994; kubo et al., 1994; yoo et al., 1991) and mutations in the s protein have been associated with altered viral antigenicity and pathogenicity (ballesteros et al., 1997; bernard domains ac and y are described by ziebuhr et al. (2001) . a nucleotide position means the location of the nucleotides encoding corresponding proteins in the entire genome of equine coronavirus-nc99 strain. b pl1 pro , papain-like proteinase 1; pl2 pro , papain-like proteinase 2; adrp, adenosine diphosphate-ribose 1ʺ-phosphatase (formerly known as 'x-domain'); 3cl pro , 3c-like proteinase; tm, transmembrane domain; gfl, growth factor-like domain; rdrp, rna-dependent rna polymerase; zbd, zinc-binding domain; hel, helicase domain; nendou, nidoviral uridylate-specific endoribonuclease; 2′-o-mt, 2′-o-ribose methyltransferase. and laude, 1995; dalziel et al., 1986; gallagher and buchmeier, 2001; leparc-goffart et al., 1997) . whether the s protein of ecov has such properties remains to be determined. orf5 (nt 27,825-27,947) of ecov-nc99 is predicted to encode a hypothetical protein of 40 amino acids with an estimated molecular weight of 4.7 kda (termed p4.7 protein). it was predicted to be a non-secretory protein and did not contain any transmembrane helix. this protein is not closely matched to any known protein based on a search using blastp, psi-blast, or fasta programs. orf6 (nt 28,076-28,405) of ecov-nc99 is predicted to encode a protein of 109 amino acids corresponding to the bcov 12.7 kda non-structural protein (p12.7). this orf overlaps by 15 nucleotides with the orf7 that encodes the e protein. no signal peptide or any transmembrane helix was present. no n-glycosylation site was found. orf7 (nt 28,392-28,646) of ecov-nc99 encodes the predicted e protein containing 84 amino acids. no n-glycosylation site was identified. it was predicted to contain a signal anchor (probability 0.999). one transmembrane domain was predicted at residues 18-36 by tmpred analysis or at residues 15-37 by tmhmm analysis. both programs predicted the n-terminus of the protein to be external to the cell surface or viral envelope. in the case of other coronaviruses, there is increasing evidence that the e protein together with the m protein is instrumental in viral assembly and budding; the cytoplasmic tails of both proteins have an important interactive role in this process (corse and machamer, 2000 , 2003 vennema et al., 1996) . orf8 (nt 28,661-29,353) of ecov-nc99 encodes the predicted m protein containing 230 amino acids. it was predicted to contain a signal anchor (probability 0.947). three transmembrane domains were predicted to be present at positions 25-46, 57-78, and 81-102 by tmpred analysis or at positions 25-44, 49-71, and 81-103 by tmhmm analysis. the n-terminal 24 amino acid residues were predicted to be outside and the c-terminal 127 or 128-amino acid hydrophilic domain was predicted to be inside the virus. one potential nglycosylation site was predicted at position 26 (nfs). the presence of potential o-glycosylation sites was predicted at the extreme n-terminus of the m protein (msstptpapgyt). whether these sites are glycosylated or not needs to be experimentally verified. previous studies have shown that the m protein of group 1 and 3 coronaviruses (e.g. tgev and ibv) are n-glycosylated, whereas the m protein of group 2 coronavirus mhv is only o-glycosylated (de haan et al., 2002; lai et al., 2006) . the m protein is the most abundant envelope component and plays a key role in coronavirus assembly by interacting with the e, s, n and he proteins (bosch et al., 2005; de haan and rottier, 2005 , and references therein). orf9a (nt 29,363-30,703) of ecov-nc99 encodes the predicted n protein containing 446 amino acids. it was predicted to contain 36 potential phosphorylation sites. no signal peptide or any transmembrane helix was present. the n protein of coronaviruses has been shown to be multifunctional, e.g. interaction with the viral rna genome to form a viral nucleocapsid, interaction with the m protein, and the ability for selfassociation (masters, 1992; narayanan et al., 2000 narayanan et al., , 2003 . recently it has also been reported that the n protein may play a role in coronavirus replication (almazan et al., 2004; schelle et al., 2005) . orf9b (nt 29,424-30,044) of ecov-nc99 encodes a hypothetical protein (i) containing 206 amino acids within orf9a which encodes the n protein. it was predicted to contain 10 potential phosphorylation sites. no signal peptide or any transmembrane helix was present. in the case of mhv, expression of the protein i has been detected in virus-infected cells but this protein is nonessential for viral replication and viral production (fischer et al., 1997) . it is generally accepted that the replicase proteins are directly synthesized from the coronavirus genome, whereas the structural and accessory proteins are expressed from a nested set of subgenomic mrnas. however, the number of sg mrnas and the characteristics and expression pattern of the proteins they encode (e.g. a sg mrna may sometimes express multiple proteins) varies for each virus. in order to investigate ecov sg mrna synthesis, northern blot analysis was performed to evaluate the synthesis of genomic and subgenomic rnas in ecov-infected cells. a digoxigenin-labeled rna probe complementary to the 3′ end (nt 30,660-30,946) of the ecov genome was used for a northern blot hybridization analysis. as shown in fig. 2 , nine mrnas were detected in ecov-infected hrt-18g cells at 72 h p.i. absence of such mrnas in mock-infected cells confirms that these mrnas are ecov-specific. according to the estimated sizes of the mrnas, it is reasonable to assume that sg mrnas 2-8 express the ns2, he, s, p4.7, p12.7, e, and m proteins, respectively and that mrna 9 expresses the n protein and probably the i protein as well. there is a general agreement that the trs elements determine the fusion sites of the 5′ leader and the 3′ body segments in coronavirus sg mrnas. in order to determine the precise location of the leader and body trss used for ecov sg mrna synthesis, the leader-body junction and flanking sequences of each ecov sg mrna were determined using sg mrna-specific rt-pcrs (see table 3 and materials and methods for details). the sg mrna sequences were aligned to the leader and corresponding 'body' genomes as shown in fig. 3 . analysis of the leader-body junction sequences revealed that the core sequence of the trs motifs is 5′ucuaaac3′. the leader trs (5′ucuaaac3′) and the body trs (5′ ucuaaac3′) used for synthesizing he mrna, s mrna, and n mrna exactly match each other. there is one mismatch between the leader trs and the body trs (5′ucuaaaa3′) used for generating the mrna of the ns2 protein. there is also one mismatch between the leader trs and the body trs (5′uccaaac3′) used for generating e mrna and m mrna. there are two mismatches between the leader trs and the body trs (5′uuaaaac3′) used for generating the mrna of the p4.7 protein. interestingly, in the case of the mrna of the p12.7 protein, the leader and the body segment is joined at the unusual consensus variant 5′uaaa-cuuuauaa3′. previously it has been shown that the mrna of the p12.7 protein of bcov also utilizes an unusual consensus variant for joining the leader and body segment . from the sequence data, we conclude that the ecov common leader on sg mrnas is the first 64 nucleotides of the ecov genome. phylogenetic analyses of ecovand other coronaviruses were performed based on the amino acid sequences of replicase polyprotein pp1a, the orf1b-encoded part of the pp1ab, s, e, m, and n. phylogenetic analysis clustered coronaviruses into three major groups (g1, g2a, and g3) irrespective of the gene used for analysis (fig. 4) . the sars-cov forms a separate branch and is classified as subgroup 2b (g2b) as suggested previously (gorbalenya et al., 2004; snijder et al., 2003) . phylogenetic analysis clearly demonstrated that ecov falls into the cluster of group 2a coronaviruses and is most closely related to bcov, hcov-oc43, and phev. to further explore the possible evolutionary relationships among ecov, bcov, hcov-oc43, and phev, the genetic distances of ecov, bcov, and phev to hcov-oc43 were determined over the entire genome using the simplot analysis (lole et al., 1999) . as shown in fig. 5 , the bcov strains and hcov-oc43 had lowest genetic distances over the complete genome; the genetic distance between phev and hcov-oc43 was similar to the distance between bcov and hcov-oc43 in most regions of the genome with exception of the spike gene where the genetic distance of phev to hcov-oc43 was significantly greater than the distance of bcov to hcov-oc43; the genetic distance of ecov to hcov-oc43 was significantly greater than the distance of either bcov or phev to hcov-oc43 in the regions of the first half of orf1a, the central part of orf1b, ns2 and he genes; the genetic distance with respect to the spike gene between ecov and hcov-oc43 was similar to the distance between phev and hcov-oc43 but greatly higher than the distance between bcov and hcov-oc43. the genetic distances of bcov and phev to hcov-oc43 observed in this study are consistent with previously reported findings (vijgen et al., 2005 (vijgen et al., , 2006 . vijgen et al. (2006 vijgen et al. ( , 2005 concluded that phev diverged from the common ancestor before bcov and hcov-oc43. our analysis suggested that ecov had diverged earlier than phev from a common ancestor. in summary, ecov had emerged earlier than phev, bcov, and hcov-oc43, notwithstanding the fact that ecov was not isolated until 1999 from a diarrheic foal in usa. in this study, we have determined the first complete genome sequence of ecovand provided the first comprehensive analysis of the ecov genome. completion of the genome sequence of ecov will contribute to our understanding of this virus at the molecular level and also enrich the database of coronaviruses. the sequence data are expected to aid in the development of diagnostic and prophylactic reagents. the sequence data of ecov-nc99 will also help identify and characterize other ecov isolates and enhance our understanding of the molecular epidemiology of coronavirus. neonatal enterocolitis is an economically significant disease for horse breeders. further studies are needed to determine the prevalence of ecov infection in equine populations and the relative role of ecov as a cause of enteric disease in horses. the human rectal tumor cell line hrt-18g (american type culture collection [atcc, crl-11663] ) was grown in dulbecco's modified eagle's medium (dmem) supplemented with 4 mm l-glutamine, 5% fetal bovine serum, and penicillin/streptomycin at 37°c in the presence of 5% co 2 . the equine coronavirus-nc99 (guy et al., 2000) was propagated once in hrt-18g cells to produce the working virus stocks. the complete genome of ecov was determined by sequencing 11 overlapping rt-pcr products encompassing the entire genome (nt 1-3615; nt 3446-5458; nt 4953-6600; nt 5497-9678; nt 9347-13,021; nt 12,451-15,736; nt 15,425-19,307; nt 19,039-22,812; nt 22,566-26,390; nt 26,065-29,662; and nt 29,363-30,992) . viral rna was isolated from ecov stocks using the qiaamp viral rna mini kit (qiagen). viral rna was first reverse transcribed with accuscript reverse transcriptase (stratagene) following the manufacturer's instructions. then, pcr amplification was performed with proof-reading pfuultra highfidelity dna polymerase (stratagene) in a volume of 50 μl: 5 μl pfuultra pcr buffer (10×), 1.0 μl dntp mix (10 mm each), 1 μl of each primer (20 μm), 2 μl cdna template, 1 μl pfuultra dna polymerase, and 39.0 μl nuclease-free water. the reaction mixtures were incubated at 95°c for 2 min, followed by 35 cycles of amplification at 95°c for 45 s, 50-53°c for 45 s, and 72°c for 4.5 min, with a final incubation at 72°c for 10 min. the pcr products were gel-purified using qiaquick gel extraction kit (qiagen). both sense and anti-sense strands were sequenced using the applied biosystems big dye terminator v3.0 sequencing chemistry on abi 3730 dna sequencers (davis sequencing center). partial genomic sequence (9487 nucleotides) of ecov had fig. 5 . genetic distance between ecov, bcov, phev and hcov-oc43. the average genetic distances were calculated over the entire genome using the simplot program with a sliding window size of 400 bp and a step size of 200 bp. each curve represents a comparison of the sequence data of ecov-nc99, the bcov strains, and phev-vw572 to the reference sequence data of the hcov-oc43 atcc strain vr759 (nc_005147). the sequence data of the bcov strains used for comparison are the 50% consensus sequence of six bcov strains: bcov-ent (nc_003045), bcov-alpaca (dq915164), bcov-db2 (dq811784), bcov-mebus (u00735), bcov-quebec (af220295), and bcov-lun (af391542). the linear representation of the ecov-nc99 genome was shown at the top of the diagram. been previously determined by two groups (guy et al., 2000, genbank accession number af251144; wu et al., 2003, af523846 and af523850. h.y. wu, j.s. guy, and d.a. brian, unpublished data, ay316300) . these regions were re-sequenced in this study. to determine the remaining genomic sequence of ecov-nc99, initial rt-pcr and sequencing primers were designed based on multiple alignments of the genomes of bcov (genbank accession number nc_003045), hcov-oc43 (nc_005147), phev (dq011855), and mhv (nc_001846); additional primers were designed based on the results of the first and subsequent rounds of sequencing. all of the primer sequences are attached in the supplementary table s1 . the nucleotide sequences were assembled and manually edited using codoncode aligner version 1.5.2 to produce the complete sequence of the viral genome. orf analysis was performed using vector nti advance 10 (invitrogen). rna secondary structures of 5′ and 3′ utrs and the ribosomal frameshift signals were predicted using the mfold program with the default parameter settings (mathews et al., 1999; zuker, 2003) . potential 3c-like protease cleavage sites were predicted using the netcorona 1.0 server (kiemer et al., 2004) . prediction of signal peptides and their cleavage sites was conducted using signalp 3.0 server (nielsen et al., 1997) . potential n-glycosylation sites, o-glycosylation sites, and phosphorylation sites were predicted using netnglyc, netoglyc, and netphos, respectively (blom et al., 1999; julenius et al., 2005) . prediction of transmembrane domains was performed using tmpred (hofmann and stoffel, 1993) and tmhmm server 2.0 (sonnhammer et al., 1998) . protein similarity searches were performed using blastp version 2.2.16, psi-blast against the protein data bank (pdb) (altschul et al., 1997; schaffer et al., 2001) and fasta version 34.26 against the uniprot protein database with the default parameter settings (pearson and lipman, 1988) . pairwise amino acid comparison was performed using emboss pairwise alignment algorithms with the default parameter settings (http://www.ebi.ac.uk/emboss/align). multiple sequence alignments were performed using clustalx version 1.83 (thompson et al., 1997) . phylogenetic analysis and unrooted neighbor-joining trees were carried out using paup version 4.0b10 with the default parameter settings. bootstrap analysis was carried out on 1000 replicate data sets. the genetic distance between genomes was determined using the simplot version 3.5.1 (lole et al., 1999) . one anti-sense rna probe base pairing to the 3′ end of the ecov genome (nt 30,660-30,946) was developed to evaluate the synthesis of genomic and subgenomic rnas in ecovinfected cells by northern blotting. the ecov rna was amplified using two primer pairs (forward primer 30660p: 5′ agcagatggatgatcccctc3′; reverse primer 30946n: 5′ actgggtggtaacttaacatgctg3′) and the qiagen one-step rt-pcr kit (qiagen). the gel-purified rt-pcr products were cloned into a linearized plasmid vector with overhanging 3′ t residues (pdrive cloning vector, qiagen). the authenticity and orientation of the insert was determined by sequencing both strands of dna with m13 reverse and forward primers. plasmid dna was linearized with bamhi (roche), phenol/chloroform extracted, ethanol precipitated, and resuspended in nuclease-free water. a digoxigenin (dig)-labeled rna probe was prepared using the dig rna labeling kit (roche) according to the manufacturer's instructions. intracellular rna was extracted at 72 h p.i. from ecovinfected hrt-18g cells using the rnaqueous-4pcr kit (ambion). northern hybridization with the dig-labeled rna probe was carried out following the protocols that had been previously described for equine arteritis virus (balasuriya et al., 2004) . the leader-body junction sites of all ecov sg mrnas were rt-pcr amplified and sequenced. briefly, intracellular rna was extracted from ecov-infected hrt-18g cells using the rnaqueous-4pcr kit (ambion). reverse transcription was carried out with an rt primer located downstream to the body trs region in a sg mrna (table 3) using superscriptiii reverse transcriptase (invitrogen) following the manufacturer's instructions. due to the nested nature of sg mrnas, such an rt primer also binds to the corresponding positions in all larger viral mrnas, including the genomic rna. subsequently, cdna was pcr amplified with a forward primer (1p) located in the leader sequence and a reverse primer located just upstream of the rt primer in the body of the mrna (table 3) . amplification was performed in a volume of 50 μl: 5 μl pfuturbo pcr buffer (10×), 0.4 μl dntp mix (25 mm each), 1 μl of each primer (20 μm), 2 μl cdna template, 1 μl pfuturbo® dna polymerase, and 39.6 μl nuclease-free water. the reaction mixtures were incubated at 95°c for 2 min, followed by 35 cycles at 95°c for 45 s, 50-56°c for 45 s, and 72°c for 3 min, with a final incubation at 72°c for 10 min. rt-pcr products corresponding to each mrna species could be distinguished by size differences on agarose gel. pcr products were gel-purified and sequenced to obtain the leader-body junction sequences for each sg mrna. the nucleotide sequence of ecov was deposited in genbank under the accession number ef446615. the nucleoprotein is required for efficient coronavirus genome replication gapped 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two paralogous papain-like proteases that cleave the same peptide bond mfold web server for nucleic acid folding and hybridization prediction this work was partly supported by funds from fort dodge animal health and kentucky agricultural experiment station, college of agriculture, university of kentucky. key: cord-325043-vqjhiv7p authors: gorbalenya, alexander e.; blinov, vladimir m.; donchenko, alexei p.; koonin, eugene v. title: an ntp-binding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive strand rna viral replication date: 1989 journal: j mol evol doi: 10.1007/bf02102483 sha: doc_id: 325043 cord_uid: vqjhiv7p ntp-motif, a consensus sequence previously shown to be characteristic of numerous ntp-utilizing enzymes, was identified in nonstructural proteins of several groups of positive-strand rna viruses. these groups include picorna-, alpha-, and coronaviruses infecting animals and como-, poty-, tobamo-, tricorna-, hordei-, and furoviruses of plants, totalling 21 viruses. it has been demonstrated that the viral ntp-motif-containing proteins constitute three distinct families, the sequences within each family being similar to each other at a statistically highly significant level. a lower, but still valid similarity has also been revealed between the families. an overall alignment has been generated, which includes several highly conserved sequence stretches. the two most prominent of the latter contain the socalled “a” and “b” sites of the ntp-motif, with four of the five invariant amino acid residues observed within these sequences. these observations, taken together with the results of comparative analysis of the positions occupied by respective proteins (domains) in viral multidomain proteins, suggest that all the ntp-motif-containing proteins of positive-strand rna viruses are homologous, constituting a highly diverged monophyletic group. in this group the “a” and “b” sites of the ntp-motif are the most conserved sequences and, by inference, should play the principal role in the functioning of the proteins. a hypothesis is proposed that all these proteins posses ntp-binding capacity and possibly ntpase activity, performing some ntp-dependent function in viral rna replication. the importance of phylogenetic analysis for the assessment of the significance of the occurrence of the ntp-motif (and of sequence motifs of this sort in general) in proteins is emphasized. structural (sequence) motifs thought to be identifiers of certain protein activities are among the main tools used in the functional and evolutionary interpretation of protein sequence data (doolittle 1986a,b; hodgman 1986) . because these motifs are short sequence stretches, and usually include amino acid residues frequent in proteins (i.e., gly, ala, ser, and some others), the presence of such a motif in a protein sequence is, as a rule, in itself not statistically significant. thus, it is important to work out some additional criteria for evaluation of such observations. one of the most widespread sequence motifs is implicated in an activity crucial to the function of a great variety of proteins, namely purine nucleotide binding followed in most cases by hydrolysis of the 8-3' phosphate bond. this motif was first recognized by walker and coworkers in several atp-and gtputilizing enzymes (walker et al. 1982; gay and walker 1983) . it consists of two separate units designated "a" and "b" sites; the "b" site is located in the polypeptide chains c-proximally relative to the "a" site. for the "a" site the following consensus sequence was proposed: gxxxxgk-(t) xxxxxxi/v, and the "b" consensus was r/k-xxxgxxxl***d, where x stands for any amino acid residue, and * for a hydrophobic residue (walker et al. 1982) . results of subsequent analyses of a variety of ntp-utilizing proteins (reviewed by halliday 1984; m611er and amons 1985; doolittle 1986a) suggest much more liberal consensus formulas, namely g/axxxxgkt/s for the "a" site, and an asp residue preceded by five residues, three of which are hydrophobic, for the "b" site. hereafter we accept these loose definitions for the "a" and "b" consensus sequences; taken together, they are designated "ntp-motif." in fact, in recent studies, protein sequences were searched for the "a" consensus alone as the "b" consensus in its loosest form is obviously too degenerate to be unequivocally recognized, except in a family of diverged proteins (see below). for adenylate kinase, escherichia coli tu factor, p2 lras oncoprotein, sv40 t antigen, and some other proteins, there is experimental evidence that the ntp-motif, or at least a larger segment of a protein encompassing it, is involved in ntp binding and/ or cleavage (clertant and seif 1984; jurnak 1985; la cour et al. 1985; fry et al. 1986 ). more specifically, the "a" site has been implicated directly in the binding of the pyrophosphate moiety of ntp, whereas the asp residue of the "b" site appears to interact with the magnesium cation complexed with the same phosphate groups (m611er and amons 1985; bradley et al. 1987) . all these observations make it an attractive idea to search sequences of functionally uncharacterized proteins for the presence of the ntpmotif to the end of prediction of ntpase activity, or at least ntp-binding capacity. following this line, we screened the protein sequences of positive-strand rna viruses (the largest class of viruses, whose single-stranded genomic rna also serves as the mrna for the synthesis of viral proteins) and identified the "a" consensus in nonstructural proteins of several viral families (gorbalenya et al. 1985) . in some of these proteins the presence of this consensus has been independently noticed by other workers too (argos and leberman 1985; doolittle 1986a; dever et al. 1987; domier et al. 1987) . also, the ntp-binding capacity of one of these proteins, p 126 of tobacco mbsaic virus, has been demonstrated experimentally quite independently (evans et al. 1985) . we proposed that such a capacity should be characteristic of all the ntpmotif-containing proteins of positive-strand rna viruses. on the other hand, the validity of such predictions in general has been disputed (argos and leberman 1985; doolittle 1986a) . moreover, doolittle (1986a) identified the "'a'" consensus in several proteins reported to be devoid of ntp-binding properties. in the present study we undertook a more systematic investigation of the primary structures of the proteins of positive-strand rna viruses containing the "a" consensus of the ntp-motif. we demonstrate that the ntp-motif-containing proteins of positive-strand rna viruses (including 8 proteins identified in the previous paper and 13 proteins of viruses whose genomes have been sequenced since then) constitute three monophyletie families that can be brought together into a higher rank taxon. in these homologous proteins the "a" and "b" sites of the ntp-motif are the most strictly conserved sequences and, by inference, should be of principal functional importance, presumably constituting parts of ntpase catalytic centers. protein sequences were extracted from the current literature (for references see table 1 ). the initial screening of the sequences of positive-strand rna viral proteins for the presence of the "a" consensus sequence of the ntp-motif was performed by use of the program srch designed to screen protein sequences for defined amino acid residue strings (motifs). the selected sequences were further analyzed manually for the presence ofcandidate "b" sequences c-proximal to the "a" site. sequences of the viral proteins containing the ntp-motif were compared by use of the programs diagon (staden 1982) and optal (pozdnyakov and pankov 1981) ; the latter program was modified and adopted for multiple sequence alignment as described below. all the programs were written in fortran and run on an es-1060 computer. the program optal, based on the original algorithm of sankoff (1972) , performs optimal alignment of pairs of protein sequences or stepwise alignment of multiple sequences. according to the sankoff algorithm, a series of cumulative similarity matrices for the compared sequences is created. in the present work, for the calculation of the elements of these matrices, weights of amino acid residue pairs were taken from the mutation rate scoring matrix mdm78 (staden 1982) . to accelerate calculations, only those elements of the matrices enclosed within a diagonal window, the width of which was chosen to be equal to v5 of the length of the compared sequences, were computed; it has been shown that this window width is sufficient in most cases for generation of the optimal alignment (pozdnyakov and pankov 1981) . at the first step of the optimal alignment generation, a series of locally optimal alignments with q = 0, 1, 2, . . . ,qmax gaps was obtained. in practice, for sequence lengths of up to 250 residues dealt with in this work, qm~, was chosen to be equal to 15. this exceeds the gap number most frequently observed in related protein sequences (about four gaps per 100 residues; see doolittle 1981) and should guarantee the generation of the optimal alignment. for selection of the best of the alignments with a given q value and concomitant assessment of its statistical significance, the following monte carlo procedure was employed. twenty-five pairs of "random" sequences were generated by scrambling the real compared sequences, and the above alignment procedure was simulated for each pair. the mean score, sq ~d and the standard deviation, aq, were calculated separately for alignments with different gap numbers. for each of the locally optimal real alignments, the deviation ofthe observed score from the mean value for the randomized sequences was calculated in sd units: rezaian et al. 1985 ahlquist et al. 1984 cornelissen et al. 1983 goelet et al. 1982 strauss et al. 1984 takkinen 1986 gustafson and armour 1986 bouzoubaa et al. 1986 bouzoubaa et al. 1987 boursnell et al. 1987 for picorna-and potyviruses, the numbering of complete polyproteins is indicated; for alphaviruses, the numbering of the nonstructural polyproteins is indicated; for cpmv the numbering of the polyprotein encoded by rna b is indicated; and for bnyvv rna 1 the numbering of the entire high-molecular weight product is indicated. the dendrograms were designed to visualize the procedure of multiple sequence alignment in the order of decreasing similarity between proteins; they cannot be automatically regarded as evolutionary trees. for abbreviations of viruses see table 1 . the values refer to the conserved domains, as indicated in table 1 , and, for the proteins of the 1 st family, to the n-terminal subdomains. dq = sq r'l -sq~"d/aq. the alignment with the maximal d value was considered optimal for the selected window width. for multiple sequence alignment, a generalization of this procedure was employed. to align two sets of m and n prealigned sequences, cumulative similarity matrices were created as before, but for the calculation &their elements, values w~j = ~w~, i.e., the combined weights of all possible pairs of residues (m. n total) in the ith position of the set n and the jth position of the set m are used instead of the weights of individual pairs of residues. a weight of 10 was ascribed to a pair of two gaps, and a weight of zero to a pair of gaps with any residue. the procedures of alignment generation and the choice of the optimal alignment were as described, but, for the generation of "random" sequence sets, "columns" of residues occupying each position in the real sets of aligned sequences were jumbled. preliminary comparative analysis of the amino acid sequences of the ntp-motif-eontaining proteins of positive-strand rna viruses by use of the programs diagon and optal (see methods) revealed three distinct families and some additional proteins in whose close relatives the motif was not conserved. within each family, all the proteins contained stretches at least 120 residues long that were similar to each other at a statistically highly significant level. for most pairs, the observed alignment scores exin this table. question marks indicate that the real size of the respective proteins is not known; the large proteins presented in the table may in fact be processed. in those cases where sequences of several serotypes (strains) of a single virus species were available (specifically, for several picornaviruses and tmv), only one sequence was included. an exception is rhinovirus serotypes 14 and 2 with sequences that are substantially different, pr = product the ntp-motif-containing segments displaying statistically significant similarity within each family (see table 1 ) are shown in black; they were aligned by the "a" sites of the ntp-motif (see text). the tricorna-and tobamovirus proteins are multidomain proteins, with the n-terminal domains similar to each other and to alphavirus nspl protein (not indicated; ahlquist et al. 1985) . in the bottom of a and b the respective patterns ofevolutionarily conserved amino acid residues are shown (designated "consensus 1" and "consensus 2"). invariant residues are capitalized. dots stand for variable residues (or gaps) within conserved residue clusters; the lengths of variable regions between these clusters are indicated by bracketed numbers. asterisks denote the amino acid residues constituting the "a" site and the proposed mg2+-binding d residue of the "b" site. ceeded the mean scores for randomized sequences by at least 5 sd (see methods). such a level of sequence similarity between proteins is usually regarded as serious evidence for their monophyletic origin (doolittle 1981 (doolittle , 1986a dayhoffet al. 1983) . to obtain optimal group alignments for each family, the sequences were aligned in order of decreasing similarity (fig. 1) . as is evident from the figure, the significance of the multiple sequence alignments was quite high for each of the families. the families were numbered 1 st, 2nd, and 3rd in order of decreasing sequence divergence between the presently recognized members 9 for part of the proteins constituting the 1st and the 2nd families, analogous sequence comparisons (but with no reference to the ntpmotif) were performed previously by other workers and meaningful similarities have also been observed goldbach 1986) . very recently, domier et al. (1987) compared the sequences of the proteins of the 2rid and the 3rd families and noticed the presence of the "a" consensus of the ntp-motif. the i st family includes the ntp-motif-containing proteins (domains) of alpha-, tobamo-, tricorna-, furo-, and coronaviruses as well as the putative product of the hordeivirus rna ~ open reading frame (orf) 2, totalling 10 proteins (table 1) . these proteins vary greatly in their size ( fig. 2a) , genomic positions of the respective coding sequences, and modes of expression. for the proteins of this family, a statistically significant similarity was observed within a fragment of about 250 amino acid residues ( fig. 2a) . this fragment contains 21 highly conserved residues (of which 14 are invariant) divided between seven clusters of unequal size (or individual residues). the first and third conserved clusters en-compass the "a" and "b" sites of the ntp-motif, respectively. the n-proximal five clusters of conserved amino acid residues in these proteins are separated from the sixth and seventh clusters by a variable region of about 60-90 residues. in fact, the conserved domain appears to be further divided into two subdomains, the n-terminal one containing the ntp-motif, and the c-terminal one of totally unknown function. two specific points are worth noting. first and most remarkably, two segments of the furovirus genome encode two ntp-motif-containing proteins only distantly related (as compared to other members of the family) to each other; this is demonstrated by direct pairwise comparison of their sequences (data not shown). second, the inclusion of the coronavirus ntp-motif-containing domain within the 1 st family is tentative, as the level of its sequence similarity to the other proteins of this family is not much higher than that between different families (see below). also, the distance between the "a" and "b" sites of the ntp-motifis much longer in the coronavirus protein than those in the other proteins of this family. nevertheless, all the amino acid residues invarianl in the latter are conserved in the coronavirus protein also (see below), justifying its inclusion in this family. the 2nd family of ntp-motif-containing proteins includes picornaviral proteins 2c and comoviral protein p58, totalling nine proteins (table 1) . these proteins are much more uniform in their size (fig. 2b) , genomic positions of the respective genes, and mode of expression than those of the 1st family. the region of the most prominent similarity spans the central domain of about 130 amino acid residues; this domain contains 45 conserved residues (23 invariant), more or less evenly distributed (fig. 2b , consensus 2). the "a" and "b" sites of the ntpmotif are located near the n-terminus and in the middle of the conserved domain, respectively. the 3rd family includes ci proteins of two potyviruses (table 1 and fig. 2c ). these proteins are very similar to each other, having more than 50% identical amino acid residues. thus, derivation of a consensus, like those derived for the other two families, made little sense. the "a" and "'b" sites of the ntp-motifare located in the n-terminal parts of ci proteins. comparison of the prealigned sequences of the three families of ntp-motif-containing proteins by the multiple alignment version of optal yielded highly significant alignment scores for all three possible pairs (fig. 1) . however, the final alignment of the sequences of the three families generated by this 261 program (not shown) was not quite satisfactory because the "b" sites of the ntp-motif, as well as some other clusters of residues that seemed good candidates for the conserved regions, did not coincide (although it must be pointed out that the "a" sites did match). presumably, this might be due to different lengths of spacers separating these regions in the proteins of the three families. thus, an overall alignment has been generated by manual fitting of the computer alignments of the three sets of sequences so as to maximize residue coincidence conserved within individual families (fig. 3) . in this alignment five amino acid residues are strictly invariant, four additional residues are common to the 2nd and 3rd families, and three residues are conserved in the 1st and 3rd families. in addition, several positions in all, or nearly all, the sequences are occupied by functionally related residues (fig. 3, consensus) . all in all, a certain degree of conservation was observed in about 40% of the positions of the alignment (highlighted in fig. 3 and further characterized in the legend to this figure) . strikingly, four of the five invariant residues are located within the "a" and "b" sites of the ntpmotif (fig. 3) . these sites and short sequence stretches surrounding them also contain a considerable number of additional coincidences and similar sequence replacements between proteins of different families. thus, the "a" and "b" consensus sequences and short adjacent segments are the most similar portions of the ntp-motif-containing proteins of positive-strand rna viruses. of additional interest is a comparison of the positions of the ntp-motif-containing proteins (domains) in viral multidomain proteins; this approach is illustrated in fig. 4 . only two stretches of similar amino acid sequences are common to all viruses analyzed in this study: (1) the conserved region of the rna polymerase morozov and rupasov 1985; koonin et al. 1987 koonin et al. , 1988 , and (2) the ntp-motif-containing domain (this paper). viruses, with proteins that constitute the 2nd and 3rd families characterized above, possess an additional protein sequence of significant similarity, i.e., the proteases of picorna-, como-, and potyviruses franssen et al. 1984; carrington and dougherty 1987; domier et al. 1987) . in all viruses with nonsegmented genomes (with the probable exception of coronaviruses), in cpmv b polyprotein, and in the furovirus rna 1 product (p237), the proteins (domains) containing similar sequence stretches are positioned in the same order within multidomain proteins, namely n-ntp-motif-containing domain-(protease)-polymerase-c (fig. 4) . in coronaviruses the polymerase has not yet been identified. however, the results of our preliminary analysis indicate that the polymerase doan overall alignment of the evolutionarily conserved segments of the vi~l ntp-moti~containing proteins of the three families. only partial sequences of the conserved regions (table 1 ) were aligned; they encompass the n-terminal subdomains of the proteins of the 1 st family, the sequences of the 2nd family without the five n-terminal amino acid residues, and complete sequences of the 3rd family. the residue numbers shown above the alignment are arbitrary; the numbering begins from the first residues of the aligned stretches and includes gaps. the sets of sequences of the three families aligned by the program optal are separated by blank lines. dots denote conservative positions. these are defined here as positions occupied by similar amino acid residues in at least 50% of the sequences of each of any two of the three families. the upper, middle, and lower rows of dots indicate the conservative positions of the 1st, 2rid, and 3rd families, respectively. thus, if a given position in the alignment contains dots, say, in the upper and lower rows, this indicates the conservation of residues (in the above sense) between the 1st and 3rd families, and so on. similar residues are defined as those belonging to one of the following groups: a, v, i, l, m, and f (hydrophobic); f, y, and w (aromatic); g and a (small); s and t (hydroxy-); k, r, and h (basic); d, e, n, and q (acidic and their derivatives); c and p have no similar residues. the pattern of highly conserved residues is shown under the aligned sequences, designated "cons" for consensus. uppercase letters correspond to invariant residues, and lowercase letters to those conserved in two out of three families; in the latter ease, where a similar residue was conserved in the 3rd family, it was also indicated. * = a hydrophobic residue. the "a'" (positions 6-13 in the alignment) and "b" (positions 93-98) sites of the ntp-motif are denoted by horizontal bars above and below the alignment. for viruses with segmented genomes, the specific designations of the rna segments encoding the ntp-motif-containing proteins are given in parentheses. main also resides in f2, but its position relative to the ntp-motif-containing one is reversed as compared to the "canonical" array described above (unpublished observations). anyway, this single exception certainly does not invalidate the general trend for the specific positioning of these domains in viral rnultidomain proteins. comparative analysis of the amino acid sequences of all positive-strand rna virus rna polymerases provides a strong case for their monophyletic origin . we believe that the sequence similarity between the ntpmotif-containing proteins, together with their similar localization in viral multidomain proteins, indicate that they also constitute a monophyletic group. in the course of the present study we screened all the available protein sequences of positive-stand rna viruses for the presence of the ntp-motif. also, some additional searches have been made: (1) domains occupying positions similar to those of the ntp-motif-containing ones in viral multidomain proteins were searched for the possible presence of degenerate forms of the motif; and (2) partially sequenced proteins were tested for similarity to the ntp-motif-eontaining proteins. the "a" consensus sequence has been found in the c-terminal part of aimv rna polymerase, in the capsid protein of yellow fever virus (a flavivirus), in ns1 proteins of four flaviviruses, and in the f1 polyprotein of ibv; also, a second "a" sequence (besides the one included in our alignment) is present in the furovirus p237. in the first three instances the consensus sequence was not conserved in the relatives of the respective proteins, suggesting that its occurrence was most likely fortuitous. in the last two cases, the absence of other coronavirus and furovirus protein sequences precluded this type of analysis, leaving the significance of these observa. similar sequence stretches are also joined by sloped lines. the ntp-motif-containing protein ("ntpase"), the rna-dependent rna polymerase (polymerase), and the protease (the latter identified in picorna-, como-, and potyviruses) are designated. nominations of specific proteins are given above each rectangle. other designations are: ~, sites of proteolytic processing; ~, leaky termination codons [of two alphaviruses included, nsp4 is expressed only in snbv via a leaky termination codon (takkinen 1986) ]. all the information is given only for the ntp-motif-containing proteins (domains), the polymerases, and the parts of multidomain proteins enclosed between. tions uncertain. however, it should be noted that the segments of f1 and of p237 encompassing the consensus sequence bear no significant sequence similarity to the viral ntp-motif-containing domains described above (unpublished observations). flavivirus protein ns3, which occupies a position similar to that of alphavirus nsp2 in the polyproteins of these viruses, contains an "a" consensus sequence with a single deviation and a "b" sequence strikingly similar to those of the 1 st and 3rd families of viral ntp-motif-containing proteins. comparison of the three available sequences of ns3, those of yellow fever, west nile, and dengue 2 flaviviruses castle et al. 1986; yaegashi et al. 1986) , demonstrated strict conservation of these sequences. a more detailed analysis that we recently performed revealed statistically significant similarity between the putative ntp-binding domains of ns3 and those of potyviral proteins (unpublished observations). it seems quite plausible that ns3 may have some degree of evolutionary and functional relatedness to the group of viral proteins described in this paper. a striking similarity has been detected between the c-terminal sequence of the protein p 120 encoded by bsmv rna ~ [for which only a partial sequence has been reported (rupasov et al. 1986) ] and the c-terminal subdomain of the 1 st family of viral ntp-motif-containing proteins. although the n-terminal part of the p120 sequence is not yet known, in all other proteins of this family, invariably the two subdomains are observed together. thus, the hordeivirus genome, like the furovirus genome, probably encodes two ntp-motif-containing proteins in two genomic segments . in all other complete protein sequences of positive-strand rna viruses reported, namely those of black beetle virus (a nodavirus), carnation mottle virus, and rna bacteriophages, the consensus sequences of the ntp-motif have not been observed. the ntp-motif was first introduced by walker et al. (1982) and was subsequently employed for localization of putative catalytic sites and for prediction of ntp-binding capacity in numerous proteins. however, for reasons mentioned in the introduction, the validity of the whole approach remained rather uncertain. in the present study we demonstrate that in a highly diverged group including similar proteins of positive-strand rna viruses, the consensus sequences of the ntp-motif constitute the most strictly conserved stretches, encompassing four of the five invariant amino acid residues. moreover, the ntp-motif-containing domain is one of the two most conserved domains revealed upon an overall comparison of the sequences of this class of virus proteins. this strongly suggests that this protein domain possesses ntp-binding capacity and possibly ntpase activity, presumably supplying some ntp-dependent function(s) that is of vital importance for viral reproduction. this hypothesis is in agreement with the available experimental data implicating these proteins in viral rna replication and with the reported ntp-binding capacity of tmv p126 (evans et al. 1985) , although direct testing is certainly warranted. in fact, there is experimental evidence that clearly, though indirectly, demonstrates the importance of the ntp-motif in viral rna replication, recently several poliovirus mutants resistant to or dependent on guanidine, a potent inhibitor of rna replication of some picornaviruses, have been thoroughly studied (pincus et al. 1986 (pincus et al. , 1987 . they all mapped to the 2c protein, with the amino acid replacements located in the proximity of the "a" and "b" sites of the ntpmotif, or near the conserved asn residue in the 183rd position of the segments of 2c aligned in this paper (fig. 3) . dever et al. (1987) have recently proposed a consensus for gtp-binding domains that includes, in addition to the "a" and "b" sites of the ntp-motif, a third highly conserved sequence element thought to determine the specificity for guanosine. they identified this sequence in the 2c protein of one serotype of fmdv (but not of the other picornaviruses) and suggested that this protein should possess specific gtp-binding capacity, as opposed to other picornaviral 2c proteins. however, it would be unprecedented for proteins so closely related to have different specificities for nucleotides. in our 265 opinion, it is much more likely that, within groups of highly similar ntp-motif-containing proteins such as picornaviral 2c, the substrate specificities and other principal properties should be identical. on the other hand, when considering more distantly related proteins, such as those belonging to the three distinct families described above, one cannot exclude the possibility that such proteins might differ significantly in their activities and functions in viral reproduction. it seems premature to discuss at length the possible significance of the present observations for understanding the evolution of positive-strand rna viruses. two trends, however are obvious. first, ntp-motif-containing proteins are nearly ubiquitous among eukaryotic positive-strand rna viruses. the existing classification of these viruses (matthews 1982) includes about 30 families (groups), or somewhat more, taking recent developments into consideration. for 13 of these, complete, or nearly complete genomic sequences are available. proteins containing the typical ntp-motif were observed in nine families (table 1 ); in addition, viruses of one family (flaviviridae) probably possess a functionally related protein with a deviant motif. it is tempting to speculate that an ntp-dependent function supported by the amino acid residues constituting the ntp-motif may be indispensable for positivestrand viral rna replication; in some cases this function may be supplied by cellular proteins. in this context it is compelling that the rna replicase of single-stranded rna bacteriophages contains the translation elongation factor tu, an ntp-motifcontaining gtpase, as one of its subunits (reviewed by blumenthal 1979) . second, it appears that the sequence diversity of the ntp-motif-containing proteins as revealed here does not precisely reflect the "phenotypic" diversity of viruses that forms the basis for the existing classification. of the nine virus families (groups) having ntp-motif-containing proteins, six contribute members to the 1 st family of proteins (see above), two to the 2nd, and one to the 3rd family. thus, the 1st family covers a very broad range of viral groups differing greatly in their genomic strategies and biological properties. it is anticipated that sequencing ofgenomes of new viral groups will add new members to this family. the ntp-motif (or the "a'" consensus alone) has been identified in an extremely large class of ntpbinding proteins, mostly ntpases (although it should be noted that the presence of this motif is not an absolute prerequisite for ntp-binding capacity). the ntp-motif-containing proteins include a large group of gtpases, namely the ras family, g proteins, transducins, and some of translation initiation and elongation factors (dever et al. 1987 and references therein). also belonging to this class are numerous proteins involved in bacterial dna synthesis, recombination, and repair, and in membrane transport (doolittle et al. 1986; finch et al, 1986a,b; higgins et al. 1986; husain et al. 1986; yin et al. 1986; gilchrist and denhardt 1987) , proteins implicated in multidrug resistance in mammalian cells (chen et al. 1986; gros et al. 1986) , and several ntp-utilizing enzymes of dna viruses (gorbalenya et al. 1985; anton and lane 1986; doolittle 1986a; astell et al. 1987, and references therein) . from this incomplete list it is obvious that the presence of the ntp-motif brings together numerous proteins with extremely diverse functions. it must be emphasized that many ntp-motif-containing proteins do not bear statistically significant similarity to each other (of. argos and leberman 1985; doolittle 1986a ) and the existence of distinct monophyletic groups of such proteins (excluding very closely related, such as, for example, different ras species) is not obvious a priori. nevertheless, the ntp-motif-containing proteins of positive-strand rna viruses do constitute such a family, whereas the gtpases probably constitute another. although widespread, ntp-motif-containing proteins are not strictly ubiquitous in all biological species. specifically, this motif could not be found upon screening of the protein sequences of two large viral classes, negative-strand rna viruses and retroid viruses (unpublished observations). thus, the presence of proteins of this class in the majority of eukaryotic positive-strand rna viruses appears to be a nontrivial observation, given their small genome size. as for the value of the ntp-motif as a predictor of protein function, we believe that searching amino acid sequences for this motif (and conceivably for other sequence motifs of this kind) may be a very powerful methodology, if accompanied by phylogenetic analysis. during preparation and reviewing of this manuscript, important relevant information became available. genome sequences of viruses of three more groups that encode ntp-motif-containing proteins were determined. these are tobacco rattle virus [a tobravirus (hamilton et al. 1987) ], white clover mosaic virus and potato virus x [two potexviruses (forstcr et al. 1988; krayev et al. 1988 )], and tomato black ring virus [a nepovirus (c. fritsch, personal communication)]. the presumptive ntpbinding domain of the tobravirus is closely related to that of tmv and clearly belongs to the 1 st family of viral ntp-motifocontaining proteins described above. the genomes of potexviruses each encode two ntp-motif-containing proteins. these proteins also beiong to the 1 st family, but their inclusion in the alignment further loosens the consensus. interestingly, in some positions of the potexvirus proteins, residues otherwise invariant in the 1 st family are replaced by those characteristic of the 2rid family. the nepovirus ntp-motif-containing protein belongs to the 2nd family. thus, the new data appear to confirm our prediction that sequencing of genomes of viruses belonging to new groups should add members mainly to the 1st family of ntpmotif-containing proteins. also, the genome sequence of southern bean mosaic virus, a sobemovirus, has been determined (wu et al. 1987) . the authors claimed that it encoded a presumptive ntpbinding domain. however, a more detailed analysis indicates that this domain probably fulfills an entirely different function, namely the protease one, with its sequence being strikingly similar to those ofpicornaviral proteases (gorbalenya et al. 1988a ). thus, sobemoviruses may lack an ntp-motif-conraining protein, which is similar to other positivestrand rna viruses of small genome size (namely nodaviruses, carnation mottle virus, and rna phages). comparison of the sequences of the 1st family of viral ntp-motif-containing proteins with those of several bacterial helicases revealed highly significant similarity, suggesting an rna helicase function for these proteins gorbalenya et al. 1988b,c; hodgman 1988) . nucleotide sequence of the brome mosaic virus genome and its implications for viral replication sindbis virus proteins nspi and nsp2 contain homology to nonstructural proteins from several rna plant viruses thenucleotide sequence of the coding region of tobacco etch virus genomic rna: evidence for the synthesis of a single polyprotein non-structural protein 1 of parvoviruses: homology to purine nucleotide using proteins and early proteins of papovaviruses homologies and anomalies in primary structural 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sequences of the eneephalomyocarditis viral polyprotein coding region analysis of the complete nucleotide sequence of the picornavirus theiler's murine encephalomyelitis virus (tmev) indicates that it is closely related to cardioviruses guanidine-selected mutants of poliovirus: mapping of point mutations to polypeptide 2c guanidine-dependent mutants of poliovirus: identification of three classes with different growth requirements accelerated method for comparing amino acid sequences with allowance for possible gaps. plotting optimum correspondence paths molecular cloning of poliovirus edna and determination of the complete nucleotide sequence of the viral genome nueleotide sequence of cucumber mosaic virus rna 1 nucleotide sequence of yellow fever virus implications for flavivirus gene expression and evolution nucleotide sequence of 3'-terminal regions of barley stripe mosaic virus rnas 1 and 3 matching sequences under deletion/insertion constraints human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region an interactive graphics programme for comparing and aligning nucleic acid and amino acid sequences the complete sequence of a common cold virus: human rhinovirus 14 complete nucleotide sequence of the genomic rna of sindbis virus complete nucleotide sequence of the nonstructural protein genes of semliki forest virus distantly related sequences in the a-and ~-subunits ofatp synthase, myosin, kinases and other atp-requiring enzymes and a common nucleotide binding fold sequence and organization of southern bean mosaic virus genomic rna partial sequence analysis of cloned dengue virus type 2 genome nucleotide sequence of the escherichia coli replication gene dnazx acknowledgments. the authors are deeply grateful to professor v.i. agol for constant interest and encouragement, to dr. k.m. chumakov for help with some of the computer programs, to dr. s.y. morozov for useful discussions, and to drs. c. fritsch and s.y. morozov for communicating their sequence data prior to publication. i f i f i i i f f i i i i i i f f i f i i i i i c key: cord-001835-0s7ok4uw authors: nan title: abstracts of the 29th annual symposium of the protein society date: 2015-10-01 journal: protein science doi: 10.1002/pro.2823 sha: doc_id: 1835 cord_uid: 0s7ok4uw nan c-terminus glutamine-rich sequence deleted) to elucidate the role of metalloprotein hpn-like by fluorescence resonance energy transfer (fret) (figure 2 ) [2] . we found the selective coordination of ni(ii) and zn(ii) to the purified sensors and in e. coli cells. surprisingly, specific interaction between the fret sensors and bi(iii) was observed. our fret analysis confirmed the role of hpnl for ni(ii) storage and revealed the potential association of hpnl with bi-based antiulcer drugs in cells. pb-006 rna fate is controlled by highly-regulated rna binding proteins molecular mechanism that links the mrna-degradation pathway with extracellular signaling networks through the reversible unfolding of a rna binding domain (rbd). rna binding is also controlled by ph conditions. this finding becomes relevant for rbps such as t-cell intracellular antigen 1 (tia-1), which shuttles between two cellular compartments (nucleus and cytoplasm) with slightly different ph values. in fact, rna binding by tia-1 is modulated by slight environmental ph changes due to the protonation/deprotonation of tia-1 histidine residues [3, 4] . the ph dependence of the tia-1/rna interaction provides a new insight into the function of tia-1 in recognizing new rna targets [5] , like the 5' terminal oligopyrimidine tracts (5tops) of translationally-repressed mrnas. along with tia-1, the rbp hu antigen r (hur) is involved in the assembly/disassembly of cytoplasmic stress granules (sg), which arise as a protective mechanism by preventing mrna decay under stress situations. despite wide acceptance that rbps harboring aggregation-promoting prion related domains (prds), such as tia-1, stimulate rapid self-association and formation of sgs, we propose that scaffolding sgs may be driven by rbds, since prd-lacking rbps, like hur, often form oligomers [6, 7, 8] and are included in sgs. under continuous stress, the transition from the physiological to pathological aggregation of rbps in sgs may depend on post-translational modifications of rbds. rna-binding proteinopathies, characterized by the nucleation of irreversible sgs, are often found in neurodegenerative diseases. altogether, resulting insights into rna biology suggest that highly-regulated rbps determine mrna fate from synthesis to decay. a threat to 70 million people in underdeveloped nations around the world, african trypanosomiasis (sleeping sickness) is a neglected tropical disease (ntd) caused by the protozoan parasite trypanosoma brucei (t. brucei). t. brucei is transmitted to humans via the tsetse fly, and replicates in the blood before crossing into the brain, causing death for the infected individual. current treatments that are available for african sleeping sickness are highly toxic and usually difficult to administer past the blood-brain barrier. it is our belief that coupling less toxic compounds with efficient drug delivery systems will contribute to the development of the most effective drug against african sleeping sickness. our goal was to determine a novel and effective chemical inhibitor with the potential to prevent the replication of t. brucei in the human body. the enzyme target for inhibition studied in this research was 6-phosphogluconate dehydrogenase (6pgdh), a cytosolic enzyme in the pentose phosphate pathway (ppp) of t. brucei. 6pgdh is essential in the ppp due to its ability to oxidize 6-phosphogluconate into ribulose-5-phosphate, which is essential for the formation of nucleotides. primer overlap extension polymerase chain reaction (pcr) was used to synthesize the coding dna sequence of the 6pgdh gene, which was then cloned into a pnic-bsa4 inducible expression plasmid with an n-terminal 6 histidine tag, by way of ligation independent cloning. the protein was then expressed in bl21 (de3) escherichia coli (e. coli) cells and purified via nickel column affinity and size exclusion fast protein liquid chromatography (fplc) to perform inhibition assays. through virtual screening, various ligands obtained from the chembridge library and nih clinical collection) were docked into the active site of the crystal structure of tb6pgdh (pubchem identification 1pgj) using gold molecular docking software. the top scoring compounds were selected by utilizing parameters such as hydrophobic interactions, hydrogen bonds, and van der waals forces. the compounds with the best scores that also satisfied lipinski's rule of 5 criteria for druggability were then tested in spectrophotometric enzyme inhibition assays monitoring the absorbance of nadph at 340 nm. compounds that show inhibitory activity in the assays will be taken to higher levels of testing to determine their effect on t. brucei in other organisms. nmr studies of the structural influence of phosphopantetheinylation in nonribosomal peptide synthetase carrier proteins and impact on binding affinities andrew goodrich 1 , dominique frueh 1 1 nonribosomal peptide synthetases (nrpss) are modular enzymatic systems responsible for the production of complex secondary metabolites in bacteria and fungi. each module is comprised of (at least) three core domains whose combined action leads to the selection, activation, and incorporation of a single small molecule into a growing peptide. central to each module is the carrier protein (cp), which is first primed via attachment of a 4'-phosphopantetheine moiety (ppant arm) to a conserved serine to generate the active holo form. an adenylation (a) domain then covalently attaches an amino or aryl abstract acid onto the ppant arm via formation of a thioester. the cp then shuttles activated monomers and growing peptides between the active sites of catalytic domains in both the same and adjacent modules. during cp priming and peptide elongation, a cp thus exists in multiple different post-translational states and interacts with numerous catalytic domains. understanding how nrpss are able to efficiently orchestrate this series of sequential protein-protein interactions between a cp and its partner catalytic domains is key to unraveling the molecular mechanism of nrp synthesis. using a combination of isothermal titration calorimetry and nuclear magnetic resonance (nmr) titrations, we found that converting a cp from the apo to holo form alters its affinity for its partner a domain. this change in binding suggests a means by which directionality in protein-protein interactions is achieved in nrpss. however, we also found that a domain binding affects the same subset of residues in both the apo and holo forms. in order to identify the molecular features underpinning this difference in affinity, we solved the nmr solution structures of the apo and holo forms of the cp. here, we present the solution structures of an apo and holo cp and discuss them in light of their differential binding to an a domain. functional analysis of of conditional analog-sensitive alleles of essential protein kinases in the fission yeast schizosaccharomyces pombe. juraj gregan 1, 2 1 mfpl/imp, 2 the genome of the fission yeast schizosaccharomyces pombe encodes for 17 protein kinases that are essential for viability. studies of the essential kinases often require the use of mutant strains carrying conditional alleles. to inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy. the mutation of a single residue in the atp-binding pocket confers sensitivity to small-molecule inhibitors, allowing for specific inactivation of the modified kinase. using this approach, we constructed conditional analog-sensitive alleles of 13 essential protein kinases in the fission yeast s. pombe. i will present the functional analysis of these mutants during meiosis. peptide conjugates: from self-assembly towards applications in biomedicine ian hamley 1 1 university of reading, dept of chemistry self-assembling peptides and their conjugates offer exceptional potential in nanomedicine. i will present some of our recent work on nanoscale assembled peptides and their conjugates, focussing on lipopeptides [1, 2] and peg-peptide conjugates [3] . pegylation is an important technique in the development of conjugates for applications in therapeutics. it is found to greatly influence self-assembly of peptides and proteins -one example from our own work is a peptide which itself forms twisted fibrils but when peg is attached, self-assembly of the conjugate leads to spherical micelles [4] . the conjugate can be enzymatically degraded using alpha-chymotrypsin, releasing the peptide. this nanocontainer delivery and release system could be useful in therapeutic applications. thermoresponsive telechelic peg/peptides with hydrophobic dipeptide end groups (di-tyrosine or di-phenylalanine) were developed, one of which shows a de-gelation transition near body temperature and which may be useful in bioresponsive delivery systems [5] . examples from our recent work on self-assembling lipopeptides will also be outlined. our focus is to investigate potential relationships between self-assembly and bioactivity, in particular in the fields of regenerative medicine [6] [7] [8] [9] [10] , antimicrobial systems [11, 12] and immune therapies [13] . been shown to become derivatized with argpyrimidine, a prominent nem that occurs on arginine residues [6] , in certain human cancer tissues and cell lines [7, 8] . this nem was linked to the elevated antiapoptotic activity of the protein [7, 8] , whereby modification of arg-188 appeared to be of particular significance [7] . in this work, hsp27 homogeneously modified with argpyrimidine at position 188 is generated for the first time. using expressed protein ligation [9] , the first semisynthesis of the unmodified protein is achieved as well. our approach, which combines organic chemistry, peptide synthesis and protein synthesis, enables complete control over protein composition and thus can provide previously unattainable insight into the properties of this vital chaperone following nonenzymatic modification. the synthesis of argpyrimidine-modified hsp27 and the progress towards structural and functional characterization of the protein will be presented herein. kunitz-type protease inhibitors belong to a widespread protein family present in many plant species and play an important role in plant defense against insect pests and pathogens. members of this family are typically inhibitors of proteases of serine class. interestingly, a few members were identified as inhibitors of proteases of cysteine class, however, they have not been functionally and structurally characterized. our study is focused on kunitz-type inhibitors of cysteine proteases (pcpis) from potato (solanum tuberosum). a series of 20 kda pcpis was purified using a multi-step chromatographical protocol, and two most abundant and effective isoinhibitors named pci 1-5 and pci 3 were characterized in detail. they were screened against a broad panel of model cysteine proteases and digestive cysteine proteases from herbivorous insects. pci 1-5 and pci 3 exhibit different inhibitory specificity pattern and potency up to the nanomolar range. both isoinhibitors were crystallized and their spatial structures were solved and refined at 1.5 å (pci 1-5) and 1.7 å (pci 3) resolutions. a position of reactive sites against cysteine proteases on the conserved b-trefoil fold scaffold was proposed. the work provides the first analysis of pcpis with respect to the structure-function relationships and evolution within the kunitz-type inhibitor family. role of the abcc2 transporter in the mode of action of the bacillus thuringiensis cry1ac toxin in the diamond back moth plutella xylostella 1 protonation pattern influence actively properties of molecules and play an essential role in biochemical mechanisms. for an accurate determination of the protonation equilibria, the absolute proton solvation free energy needs to be known. the determination of this energy represents one of the most challenging problems in physical chemistry. this is particularly difficult for protons solvated in water, where the solvation is dynamically performed by different water clusters and the proton is not attached to a single solvent molecule. the proton solvation is notably important in order to quantify mechanisms of proton transfer and such processes have been investigated for a long time based on different approaches, often leading to contradictory conclusions. a rigorous and accurate protocol for computing proton solvation in solvents of different nature is of prime importance for applied (pharmaceutical and material science) and fundamental sciences. in this study, proton affinities, electrostatic energies of solvation and pka values of a reference set of organic molecules are computed in protic and aprotic solvents. proportional to the free energy of proton dissociation, the pka value calculation is therefore strongly dependent on the free energy of proton solvation. such energy is then determined in acetonitrile (acn), methanol (met), water and dimethyl sulfoxide (dmso) in order to obtain the best possible match between measured and computed pka values. the computation of these values is based on a combination of quantum chemical (qc) and electrostatic approaches by using a thermodynamic cycle connecting gas-phase and solvent-phase of proton dissociation. the computed proton solvation energies in acn, met, water and dmso of the present study are very precise (rmsd much lower than 1 ph value). they will be a basis for better understanding of proton solvation and help to predict pka values of organic compounds in different solvents more precise. biochemical characterization of two evolutionary distant ten-eleven translocation enzymes and their utility in 5-methylcytosine sequencing in the genomes at single-base resolution subtypes leading to an inability to perceive pain and painful neuropathies, respectively. however, as nav ion channels are intimately involved in almost all aspects of physiology, only the most selective inhibitors would be suitable as drug leads. disulfide-rich venom derived mini-proteins from cone snails and spiders are being actively pursued as novel therapeutics for pain, because of their high selectivity and potency at human ion channels, including sodium channels (nav). two main strategies of inhibition have been identified; blocking the pore and interacting with the voltage-sensor domains (vsd) surrounding the pore. the ion-conducting pore is highly conserved between all sodium channel subtypes whereas the voltage-sensor domain binding sites are less conserved. therefore, inhibition of a specific nav isoform is more achievable using inhibitors that modulate vsds than with pore blockers. gating modifier toxins from spider and cone snail venom inhibit nav1.7 and nav1.8 by interacting with the vsd. they appear to reach their target by partitioning into the lipid membrane surrounding the ion channel, thus enabling access to the vsd. toxin pharmacology may therefore not only be driven by the peptide-ion channel interactions, but also including the lipids surrounding the channel protein, a feature that is very much under explored. it is therefore apparent that peptide-lipid interactions in combination with peptide-channel interactions need to be considered when designing potent inhibitors. using a range of biophysical techniques, including surface plasmon resonance and nuclear magnetic resonance, we are studying the interactions underpinning the mechanism of action between toxins and membranes and toxins and ion channels. initial results show that the lipid composition surrounding ion channels play a major role in terms of toxin:lipid interaction and that these interactions can be used in combination with traditional structure-activity relationship studies to design selective and potent nav inhibitors, which will be discussed. we believe that our studies will ultimately delineate what drives toxin pharmacology and nav subtype selectivity and will lead to improve rationally engineering of novel therapeutics for the treatment of pain. micelles promote aß42 assembly into pore-forming oligomers montserrat serra-batiste 1 , mariam bayoumi 2 , margarida gair ı 3 , mart ı ninot-pedrosa 1 , giovanni maglia 2 , nat alia carulla 1 1 institute for research in biomedicine (irb barcelona), 2 biochemistry, molecular and structural biology section, university of leuven, 3 the formation of amyloid-b peptide (ab) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in alzheime rs disease (ad). 1-2 therefore, it is important to understand how oligomers form within a membrane environment. using solution nuclear magnetic resonance (nmr) spectroscopy, combined with size exclusion chromatography (sec), we have studied the two major ab variants-ab40 and ab42, the latter having a more prominent role in ad than the former-under carefully selected micelle conditions intended to mimic a membrane environment. our results indicate that after an incubation period, ab42, but not ab40, assembles into oligomers with specific structural properties, which we have named stabilized micelle oligomers (smos). smo complexes incorporate into lipid bilayers as well-defined pores, a feature linked to neurotoxicity. these results have important implications in the ad field as they provide a new perspective on how ab oligomers cause neurotoxicity. indeed, our findings constitute a first step towards the establishment of a new therapeutic target for ad. dimer formation. it should be noted that this nb peptide contains the autophosphorylatable ser-11 associated with phk activation, and phosphorylated nb; peptide was considerably less effective in promoting b-dimer formation than non-phosphorylated peptide. these results suggest a role for ser-11 autophosphorylation in mediating homodimeric b subunit interactions within the phk complex, and augment previous studies on the activation of phk by phosphorylation in which changes at the nterminus of b are critical in the activation of the catalytic g subunit. summing these results leads to a new model of activation. in this model, in the inactive state, the nonphosphorylated n-terminus of b interacts directly or indirectly with the regulatory c-terminal domain of the g subunit, inhibiting catalytic activity. upon phosphorylation of the n-terminus of b, three important events occur: 1) the interaction between b and g is disrupted, 2) the b subunits of the holoenzyme self-associate, and 3) the catalytic domain is activated. thus, we envision that the n-terminus of b acts as an allosteric switch, with activation triggered by phosphorylation of this region, causing disruption of its previously inhibiting interactions with g and promotion of b b dimerization to stabilize the activated conformation of g . the research was supported financially by the university of kansas medical center biomedical research training program and nih grant dk32953. pb-032 hssb1 is involved in the cellular response to oxidative dna damage christine touma 1 , nicolas paquet 2 , derek j. richard 2 , roland gamsjaeger 1,3 , liza cubeddu 1,3 1 school of science and health, university of western sydney, 2 queensland university of te chnology, 3 school of molecular bioscience, university of sydney cellular dna is subject to oxidative damage in the presence of reactive oxygen species. the 7,8-dihydro-8-oxoguanine (8-oxog) adduct is the most common form of oxidative damage and results in g:c to t:a transversions; these lesions are normally processed by the base excision repair (ber) pathway. singlestranded binding (ssb) proteins of the oligonucleotide binding domain family are heavily involved in dna repair processes, which involve the detection of dna damage and recruitment of repair proteins to the site of damage. using immunofluorescence we demonstrate that hssb1 (a novel human ssb) levels increase in response to oxidative damage (h202). cells depleted of hssb1 are hypersensitive to oxidative damage and are also unable to efficiently remove 8-oxog adducts. we show that hssb1 forms dimers and tetramers under oxidative conditions and that this oligomerisation is likely mediated by inter-domain disulfide bond formation. furthermore, using surface plasmon resonance, we also show that oxidised hssb1 binds to 8-oxo-g damaged ssdna with higher affinity than non-damaged ssdna, indicating a direct role for oxidised hssb1 in the recognition of 8-oxo-g lesions. as oxidative stress is associated with aging, cancer and alzheimer's disease, understanding the molecular mechanisms of how cells repair oxidative dna damage will be crucial in the development of potential therapeutic treatments. epidemic typhus, which is caused by the bacterial pathogen rickettsia prowazekii, is a menacing disease world wide that the nih lists as one of america's greatest biological weapons threats. this research seeks to find novel inhibitors of b-ketoacyl-acp-reductase (fabg), an enzyme that catalyzes one of the reactions in the fatty acid synthesis type ii system in bacteria. this pathway is essential for survival in bacteria. the fabg enzyme uses nadph as a substrate, which facilitates the binding of the second substrate, acetoacetyl-acp into the active site. the acetoacetyl-acp is subsequently reduced into b-hydroxyacyl-acp. the coding dna sequence for the rpfabg protein was cloned into a pnic vector and transformed into e.coli bl21(de3), then the protein was expressed and purified using metal affinity and size exclusion chromatography methods. high throughput molecular docking software (gold) was used to screen a commercial library of ligands against the acetoacetyl-acp region of the active site. the ligands with the best gold scores were selected to be tested in vitro. spectrophotometric enzyme inhibition assays were performed to determine whether the drugs could inhibit rpfabg activity. chlorogenic acid, a previously known inhibitor of homologous fabgs, was tested along with the other potential drugs, and was determined to have moderate inhibitory effects on rpfabg. loop modeling using icm software was performed in order to create a prediction of the complete rpfabg structure, including the disordered loops that are not a part of the 3f9i pdb structure. co-crystallization of rpfabg with both substrates was carried out in order to obtain a structure, but only nondiffracting crystals resulted. further inhibition assays and crystallography trials are being performed in order to continue the search for a novel inhibitor of rpfabg and ultimately a treatment for epidemic typhus. 1 the university of hong kong bioconjugation of proteins has emerged as a useful tool in the study of biological systems. there is an increasing need to develop new synthetic technologies for the bioconjugation reaction of proteins, and metal-catalyzed site-selective modification of proteins has attracted considerable interest in recent years. we have developed a ruthenium glycosylated porphyrin-catalyzed carbenoid transfer reaction for the site-selective modification of proteins. we firstly applied the catalysis to the selective modification of the n-terminus of peptides. by using ruthenium glycosylated porphyrin as catalyst, the n-terminus of a number of peptides can be modified through carbenoid n-h bond insertion in aqueous media with moderate to excellent conversion. the reaction is highly selective, for example, the reaction with ytsssknvvr, which contains various types of oxygenhydrogen and nitrogen-hydrogen bonds possibly available for carbenoid insertion, catalyzed by the ruthenium glycosylated porphyrin gave the n-terminal-modified product with >99% conversion and without the formation of other modified peptides including doubly modified and oxygenhydrogen bond insertion products. we next extended the n-terminal modification method to proteins. eventually success was attained in the modification of rnase a and insulin. the reaction of rnase a with a diazoacetate mediated by ruthenium glycosylated porphyrin gave corresponding n-terminal-modified protein with 65% conversion. we also achieved a bioconjugation to ubiquitin via ruthenium glycosylated porphyrin-catalyzed alkene cyclopropanation in aqueous solution in two steps: (1) incorporation of an alkenic group by the reaction of n-hydroxysuccinimide ester with ubiquitin and (2) cyclopropanation of the alkene-tethered lys6 ubiquitin with the fluorescent labeled diazoacetate in the presence of a catalytic amount of ruthenium glycosylated porphyrin. the corresponding cyclopropanation product was obtained with 55% conversion based on maldi-tof mass spectrometry. in conclusion, we developed a ruthenium porphyrin-catalyzed siteselective modification of peptides and proteins in aqueous media. the method provides an entry to new bioconjugation reactions for protein modifications using metalloporphyrins as catalysts. uridine monophosphate synthase: architecture versatility in the service of late blight control francisco tenjo castaño 1,2 , manuel garavito 1,2 , leonor garc ıa 1,2 , silvia restrepo 2 , barbara zimmermann 1 1 biochemistry and molecular biology research group, universidad de los andes., 2 mycology and plan pathology laboratory, universidad de los andes uridine monophosphate synthase (umpase), a bifunctional enzyme in the de novo pyrimidine biosynthetic pathway, is a protein comprised of orotate phosphoribosyl transferase (oprtase) and orotidine monophosphate decarboxylase (odcase). different fusion orders of the two domains have been documented to exist in nature. in some organisms oprtase and odcase are monofunctional proteins, and act as a complex. here, umpase from solanum tuberosum (potato) and from phytophthora infestans (an oomycete) were examined. p. infestans causes late blight disease in s. tuberosum, destroying crops and increasing production costs. since pyrimidines are fundamental cellular components, we have proposed that umpase could serve as a target to control p. infestans infection. the enzymes from p. infestans and s. tuberosum differ in their fusion order of oprt and odc. the study of these two umpase could facilitate the design of species-specific inhibitors, and might shed light on the effect of fusing umpase domains in one order or the other. to this end we carried out bioinformatic and biochemical characterization of the enzymes. sequence analyses showed 20 residue differences among the p. infestans umpase sequences from three strains: 4084, 1306 and t30-4. strain t30-4 was found to have a duplicated umpase, but neither sequence corresponded to the ones predicted previously from the genome. a recombinant umpase from 4084 strain was expressed in bacteria and purified but it showed low solubility and was inactive in vitro. the recombinant umpase from the 1306 strain complemented both oprtase and odcase deficient e. coli strains. a soluble, active, recombinant protein was expressed and purified in the presence of high salt and the product ump (specific activity 0.2 lmol min-1 mg-1). the sequence skq was found at the c-terminus of the p. infestans umpase sequences and resembles a peroxisome signal peptide (skl). the predicted hydrophobicity of this umpase and its architecture (oprt at the c-terminus and odc at the n-terminus) resembles that of the umpase from leishmania donovani, which has been localized to the peroxisome. we suggest that p. infestans umps could also be located in this organelle. in contrast to the oomycete enzyme, s. tuberosum umpase is highly soluble, and has a higher specific activity (vmax5 8.8 lmol min-1 mg-1). we measured the kinetic parameters km(orotate)5 16.2 lm, km(prpp)5 25.5 lm, and found that it exhibited product inhibition by pyrophosphate. in conclusion, the different architectures of the two umps might be related to distinct biochemical characteristics, further supporting this protein as a good candidate for p. infestans control. we present computer simulation studies of three different antimicrobial peptides we have been studying by md computer simulation in collaboration with experimentalists. the first is daptomycin, a potent lipopeptide currently licensed to treat infections caused by multi-drug-resistent bacteria. the mechanism of action of daptomycin is currently not completely understood. we have solved the nmr structure of this molecule, and attempted to determine the size of its oligomer by small angle neutron scattering (sans) supported by computer simulation. feglymycin is a 13-amino-acid peptide with a high percentage of unusual amino acids such as 4-hydroxyphenylglycine and 3,5-dihydroxyphenylglycine. feglymicin inhibits mura and murc enzymes which are involved in bacterial peptidoglycan synthesis, while also displaying anti-hiv activity by interaction with the viral envelope protein gp120. a previous x-ray structure shows the molecule forming a dimer. here, the molecule was studied by nmr in water and dmso. in water, the molecule is clearly at least a dimer, while in dmso it is a monomer. we have performed noe refinement simulations in order to elucidate a structure, however, due to a lack of long-range noe contacts, a unique structure cannot be determined. labyrinthopeptin a2 is a lantibiotic that contains labionin, a unique carbacyclic posttranslationally modified amino acid that links the protein backbone in three different locations. labyrinthopeptin a2 has shown promising activity as a pain killer. starting from the x-ray structure, we present results from the first md simulation studies of this unique peptide. because of the extensive cross-linking, this peptide is observed to be highly rigid in its native form. simulation results of mutants are also presented. antibiotics with new mechanism of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. here we report the discovery of a new peptidomimetic antibiotic (l27-11), which is active with a minimum inhibitory concentration (mic) in the low nanomolar range, only against pseudomonas sp., and with a non-membrane-lytic mechanism of action. a drug target identified both in a forward genetic screen for resistance determinants and by photoaffinity labeling is the ß-barrel protein lptd, which plays an important role in lps transport and the outer membrane biogenesis. the x-ray structure of lptd in complex with lpte from shigella flexneri shows a 26 stranded b-barrel linked to a periplasmatic n-terminal jelly-roll domain. interestingly the homology model structure for lptd from pseudomonas shows a significant difference: an insertion of around 100 amino acids in the n-terminal domain. the results of our attempts to purify and characterize this large outer membrane protein and to determine the binding site of the peptidomimetic antibiotic will be shown. the theory of how life on earth begun still remains unclear. nevertheless, according to some theories, at the beginning level proteins did not emerge as a complex globular forms as know today. at the times, when solely rna molecules stored both genetic information and catalyzed the chemical reactions in primitive cells, peptides acted as a proteins nowadays [1, 2] . literature postulate that the possible role of primordial short peptides was to catalyze reactions in rna-world, as they possess an excellent ability to self-assemble into well-ordered nanostructures [3, 4] . elementary functional loops (efls) can be considered as a small structures (blocks) having specific signatures and providing functional residues important for binding/activation as well as principal chemical transformation steps of the enzymatic reaction [5] . p-loop efl is a widespread structure across vast majority of protein families such as motor domains, aaa1, reca, pepck and many others. sequential alignment of these protein families reveals existence of a conserved p-loop motif, that is able to bind atp molecule. we investigated the structure and atpase activity of peptides, which sequences possessed strongly conserved gxgk[t/s] motif from ploop. the goal of our work was to check if peptides corresponding to the most conserved p-loop motif fragment are able to bind and hydrolyze atp molecule. all peptides under study were chemically synthesized and their structures was investigated by nmr spectroscopy. the ability to bind atp molecules was analyzed by using hplc chromatography. results of our study show, that peptides with conserved p-loop motif have a suitable structures to promote binding of the molecules with phosphate group, but cannot accelerate pyrophosphate hydrolysis process. conference participation for w. _ z. supported by the fp7 project mobi4health (grant agreement no 316094). computational resources were provided by the informatics center of the metropolitan academic network (ic man task) in gdansk, poland. ck2 is a ubiquitous serine/threonine protein kinase, being one of the most pleiotropic of all protein kinases1. ck2 plays a key role in cell growth, differentiation, cell death and survival, and become the therapeutic target in cancer treatment, since its level is significantly increased in cancer cells2. halogenated ligands have been widely developed as potent inhibitors of protein kinases. among them 4,5,6,7-tetrabromobenzoteriazole (tbbt) is one of the first potent and selective inhibitor of ck2a, directed towards the conserved atp binding site3. to assess contribution of electrostatic interactions to the specificity and strength of binding of multi halogenated inhibitors by a protein kinase, we have studied interaction between ck2a and nine benzotriazole derivatives, representing all possible patterns of halogenation on the benzene ring. herein, we present results that support existence of two alternative regions that are involved in ligand binding. aspartic acid 175 is known for its function in coordination of a mg21 ion, which is required for atp binding 4. asp175 has been identified in crystal structure of ck2:tbbt complex (pdb1j91, fig. 1 ) as the charged residue closest to tbbt. there is also lys68 proximal to tbbt, interaction with which may favor anionic form of ligands5 (pk for tbbt <5), however it is involved in the intramolecular salt bridge, and thus its mutation may significantly change stability of the protein. crystal structure of tbbt complexed with ck2 (pdb:1j91). residues with a distance to tbbt (magenta) shorter than 5a are shown. red residue is negatively charged, blue ones are protonated. abstract comparison of kdiss values determined for ligands at ph 8 and at ph 7 shows that strength of the complex significantly varies upon deprotonation of the triazole ring. this confirms former hypothesis that a negatively charged ligands cluster at the atp binding site region proximal to lys685, which is beneficial both to the specificity and to strength of the binding. we have also observed for the tested ligands variations in their binding to either wild type protein and its d175n mutant (with less negative charge distributed over atp binding site). all ligands displaying higher pka for dissociation of the triazole proton bind to the mutant visibly weaker than to the wild-type protein. altogether reveals the predominance electrostatic intermolecular interactions. although, negatively charged ligands most probably cluster at the atpbinding site proximal to lys68, beneficial for the strength of binding, the less dissociated forms are favored due to unfavorable interactions of the anionic form of ligands with asp175. 1 there are many virulence factors produced by these strains, many of which are encoded on mobile genetic elements. 2 psms are of specific interest because these virulence factors are encoded on the core genome of the bacteria and therefore all strains of staphylococci bacteria produce some variation of psms with a variety of biological functions. 2 the specific mechanism by which psms act as virulence factors has been poorly understood until recently. biological functions of psms include cell lysis, biofilm formation and the ability to kill neutrophils after phagocystosis.1 these toxins are of special interest to our research group due to their genetic similarities to certain bacteriocins, namely leaderless bacteriocins. 3 both groups of peptides are ribosomally synthesized with a n-terminal formyl methionine and secreted from the bacteria by atp-binding cassette (abc) transporters without any leader sequence or signal peptide. abc transporters may also play a role in immunity towards psms and leaderless bacteriocins. these similarities led our group to investigate the solution structure of these peptides through nuclear magnetic resonance (nmr). isolating psms from the producer organisim, s. aureus, typically involves lengthy extractions and low yields. 4 for these reasons, we opted to chemically synthesize the desired peptides using solid phase peptide synthesis (spps). utilizing a variety of spps techniques, psm a1 and psm a3 were successfully synthesized, however, due to the hydrophobic nature of psm b2, an alternate genetic approach was devised to isolate psm b2. formation of a fusion protein between psm b2 and the small ubiquitin like modifier (sumo) protein allowed for heterologous expression. upon cleavage of the fusion protein with sumo protease, and subsequent purification and isolation of the cut peptide, psm b2 was obtained. as previously reported, the psms were found to be alpha-helical in structure inducing solvents. 5 a series of 2 dimensional (2d) nmr experiments were ran to determine chemical shift assignments and to obtain noe data. importing the chemical shift assignments and noe data into the structure calculating software, cyana, we were able to elucidate the solution structure of psm a1 and psm a3 and we are currently working towards the elucidation of psm b2. the synthesis, isolation, characterization and solution structures of the aforementioned psms will be discussed here. 1 transition metals are critical for enzyme function and protein folding, but their excess can mediate neurotoxic oxidative processes [1] . as, energy production involves oxidative phosphorylation, a process requiring a continuous flow of electrons, mitochondria are particularly vulnerable to oxidative damage [2] . as such, mitochondria are the major sites of reactive oxygen species (ros) generation, which are produced as byproducts of the electron transport chain. since free iron and certain ros can engage into potentially deleterious processes such as fenton reaction, mitochondrial iron homeostasis must be tightly controlled, and dysregulation of iron metabolism in this organelle has been associated with various diseases, including friedichs ataxia (fa), alzheimer's, and other neurodegenerative disorders [3] . engineering an efficient mitochondriatargeting, cell-permeable vector is a challenge due to the fact that mitochondrion is impermeable to a wide range of molecules. the development of delivery vectors has been made possible by a greater understanding of mitochondrial structure and chemical features of molecules that selectively localize within this organelle. from these findings, two generalized requirements for mitochondrial localization are delocalized positive charge and lipophilicity [4, 5] . targeting iron in this organelle is proposed as a means to ameliorate fa symptoms. desferrioxamine (dfo) is a bacterial siderophore with high affinity for iron, but low cell penetration. we prepared conjugates of dfo with mitochondria penetrating peptides and studied their iron-binding characteristics in vitro. the lipophilic and charged peptides tat49-57 (h-arg-lys-lys-arg-arg-gln-arg-arg-arg-oh) [6] , 1a (h-cha-arg-cha-lys-cha-arg-cha-lys-nh2) [6] , ss-02 (h-dmt-arg-phe-lys-nh2) [7] and ss-20 (h-phe-arg-phe-lys-nh2) [7] , are known to permeate cytosolic and mitochondrial membranes. they were prepared and conjugated to dfo in solid-phase [8] , an alternative synthetic route. once detached from the resin, fully deprotected, purified and characterized by means of lc/ms and aminoacid analysis, it was observed that the dfo-conjugated peptides displayed iron-binding abilities identical to the free chelator dfo. dfo-conjugated peptides were also able to quench the iron-catalysed oxidation of ascorbate (a model of oxidative stress in plasma of iron-overloaded patients), as probed by a high throughput fluorimetric method [9, 10] . these results indicate that our synthesis and conjugation strategy were successful in preserving the iron-binding moiety and the antioxidant ability of the free chelator dfo. the proteolytic activity and oligomerization status of the human htra3 protease functioning as a tumor suppressor of an n-terminal domain not required for proteolytic activity, a central serine protease domain and a cterminal pdz domain. the latter serves as a substrate or regulator binding domain and may participate in oligomerization. htra3s, its short natural isoform, lacks the pdz domain which is substituted by a stretch of 7 c-terminal amino acid residues, unique for this isoform. down-regulation of htra3 in tumors, shown by other groups and us, suggests htra3s involvement in oncogenesis [1] . htra3 acts as a proapoptotic protein and is suggested to function as a tumor suppressor. it promotes cytotoxicity of etoposide and cisplatin in lung cancer cell lines [2, 3] . to date, htra3 has been poorly characterized from the biochemical point of view, mainly due to the fact that it is difficult to purify recombinant htra3. we were able to express in bacterial system and purify htra3 in quantities sufficient to perform structural studies. the aim of this study was to characterize and compare the proteolytic properties and quaternary structure of the htra3 isoforms. both studied isoforms lacked the n-terminal domain. htra3 with the pdz domain removed (htra3-dpdz) and htra3s (htra3s) were fully active at a wide range of temperatures and their substrate affinity was not impaired. this indicates that the pdz domain is dispensable for htra3 activity. as determined by size exclusion chromatography, htra3 formed stable trimers while both htra3-dpdz and htra3s were monomeric. this suggests that the presence of the pdz domain, unlike in other human htras (htra1 and htra2), influences htra3 trimer formation. the unique c-terminal sequence of dn-htra3s appeared to have little effect on activity and oligomerization [4] . cyclodextrins (cds) are cyclic oligosaccharides that have been recognized as useful pharmaceutical excipients. in aqueous solution cds are capable to form complexes with various ligands, hosting inside their cavity either a whole molecule, or part of a ligand. inclusion complexes with cds offers a variety of physicochemical advantages over the biologically active ligands, including the improved aqueous solubility, solution stability or an increase of bioavailability. ck2 is an ubiquitous, highly pleiotropic and constitutively active ser/thr protein kinase. halogenated benzotriazoles have been developed as potent and selective inhibitors of this enzyme. the interaction of the catalytic domain of human protein kinase ck2 with a series of brominated ligands, which represent all possible patterns of halogen substitutions to the benzene ring of benzotriazole, was previously studied by microscale thermophoresis (mst) [1] . this method alloweddetermination of binding affinities for seven ligands, all of which were found consistent with the values determined independently by isothermal titration calorimetry (itc). however, a very limited aqueous solubility of some brominated benzotriazoles may decrease their bioavability, thus affectingtheir apparent activity [2] . to overcome this limitation, the aqueous solubility of halogenated benzotriazoles in the presence of cyclodextrins has been tested. the formation of inclusion complexes with b-cyclodextrin (b-cd), hydroxypropylb-cyclodextrin (hp-b-cd) and g-cyclodextrin (g-cd) in aqueous solutions, followed by uv-vis spectroscopy, substantially improved the solubility of tbbt and its derivatives. the interaction between protein kinase ck2 and cyclodextrins, and also with their inclusion complexes with halogenated benzotriazoles, was followed with the aid of the microscale thermophoresis. the results obtained clearly demonstrate that the binding of halogenated benzotriazoles by ck2 is only moderately affected by cyclodextrins. oligonucleotide-based molecular circuits offer the exciting possibility to introduce autonomous signal processing in biomedicine, synthetic biology, and molecular diagnostics. here we introduce bivalent peptide-dna conjugates as generic, noncovalent, and easily applicable molecular locks that allow the control of antibody activity using toeholdmediated strand displacement reactions. employing yeast as a cellular model system, reversible control of antibody targeting is demonstrated with low nm concentrations of peptide-dna locks and oligonucleotide displacer strands. introduction of two different toehold strands on the peptide-dna lock allowed signal integration of two different inputs, yielding logic orand and-gates. the range of molecular inputs could be further extended to protein-based triggers by using proteinbinding aptamers. insights of a novel kind of cell wall binding domain that cleaves the peptidoglycan muropeptide: the cw_7 motif noem ı bustamante 1,3 , manuel iglesias, noella silva-mart ın, isabel uson, pedro garc ıa, juan hermoso, marta bruix, margarita men endez 1 institute of physical-chemistry 'rocasolano', csic, 2 institute of physical-chemistry 'rocasolano', csic, 3 ciber of respiratory diseases (ciberes), 4 center of biological research (cib), csic, 5 enzybiotics constitute a hopeful alternative to current treatments to fight against bacterial infections. phage endolysins are consider as enzybiotics due to their capacity to cleave the peptidoglycan (pg) of gram-positive bacteria in a generally species-specific manner and kill bacteria when exogenously added (1, 2) . the cpl-7 endolysin, a lysozyme encoded by the pneumococcal cp-7 bacteriophage, is a remarkable exception among all the pg hydrolases produced by streptococcus pneumoniae and its bacteriophages due to its capacity of degrading pneumococcal cell walls containing either choline or ethanolamine (3, 4) . this fact confers to cpl-7 the advantage of displaying a broader microbicide spectrum comparing to choline binding proteins (5) . this behavior results from the acquisition of a cell wall binding module (cwbm) made of three identical repeats of 48 amino acids each (cw_7 motifs), with unknown specificity and totally unrelated with the choline-binding motives present in pneumococcal hydrolases. interestingly, cw_7 repeats have been identified in many putative proteins potentially involved in cell wall metabolism (pfam entry: pf08230) from different species of gram positive and gram negative bacteria, and some bacteriophages (6) . preliminary studies of thermal stability in presence of a small cell wall structural-analogue (glcnac-murnac-l-ala-d-isogln) point to the muropeptide as the cell wall target recognized by cw_7 motifs (7) . in this communication we have gone in depth in the characterization of cw_7 repeats. we present the first crystal structure of the cw_7 motif, which reveals a three-helical bundle folding. using std_nmr spectroscopy the epitope of binding of the disacharide dipeptide to this repeats has been identified. interestingly, the b anomer of the murnac moiety, the form present in the peptidoglycan, seems to be preferentially recognized with respect to the a anomer. finally, a docking model of the complex cw_7/gmdp compatible with std results was built allowing to identify the major contacts between the protein and the muropeptide and to propose the relevant role of a conserved arginine residue in this interaction. 1 energy-dependent aaa1 proteases carry out regulated proteolysis to ensure protein quality control and post-translational regulation of many cellular processes. control of proteolysis occurs primarily at the level of substrate recognition, which can be modulated by adaptor proteins. the clps adaptor protein enhances and inhibits degradation of different classes of substrates, and thus triggers a specificity switch in clpa. whereas the mechanism for substrate delivery by clps has been described in detail, the inhibition mechanism is poorly understood. we show that clps inhibits ssra substrate recognition and processing, instead of simply preventing substrate binding. we demonstrate that clpa engagement of the clps n-terminal extension (nte) is necessary, and may even be sufficient, for inhibition. in addition, we find that inhibition of substrate processing requires a longer nte, as compared to inhibition of substrate recognition. interestingly, the nte length required for inhibiting substrate processing is also necessary for suppression of the clpa atpase rate. furthermore, preliminary data suggests that clps slows down substrate translocation. these results support a model where there is an ssra•clpa•clps inhibitory complex in which the clpa pore engages the clps nte. this engagement of the nte causes suppression of atpase activity, and therefore slower substrate translocation and processing. this model illustrates how an adaptor protein can inhibit recognition of one type of substrate while efficiently promoting degradation of a different substrate. single-molecule assay development for studying human rna polymerase ii promoter-proximal pausing rna polymerase ii (polii) pausing has been shown to play a significant role in transcription regulation of elongating polii complexes in a large number of metazoan and mammalian genes (1) . the traditional understanding of transcription regulation in mammals involved controlling polii recruitment to promoters and controlling initial steps at the promoter, including pre-initiation complex formation and promoter escape. most works investigating promoter-proximal polii pausing have employed chromatin immunoprecipitation followed by sequencing to determine polii localization or in vitro transcriptional assays using nuclear extracts analyzed with radio-active gel electrophoresis. in order to gain greater mechanistic insight into the regulation of promoter-proximal polii pausing, we have been developing a diffusion-based single-molecule method using alternating laser excitation on the micro-second timescale (msalex). the method detects rna transcripts generated by a reconstituted human polii system in vitro using complementary doubly dye-labeled single-stranded dna (ssdna) probes. the human gene hspa1b for heat shock protein 70 (hsp70) is used as a model system due to its extensive characterization in drosophila. the method would provide a rapid, sensitive and robust avenue to screen for protein factors regulating promoter-proximal polii pausing. controlling of the pic composition using the reconstituted system allows for dissection of the functional roles of different pic components in facilitating regulation of polii pausing. we have demonstrated the hybridization of double dye-labeled ssdna probe to complementary ssdna mimicking rna transcripts and to transcripts generated with bacterial rna polymerase. also, a functional reconstituted human polii system has been verified using radioactive polyacrylamide gel electrophoresis of transcripts from in vitro transcription assays. malaria is a major global health problem. in 2013, there were an estimated 128 million case of malaria and 584 000 deaths, most of them children under 5 years old [1] . among the 5 malaria species that affect humans, plasmodium falciparum is the most deadly form. since no efficient vaccine is available yet, the fight against malaria includes vector control, protection from mosquito bites and artemisinin combined therapy. however, resistances to all known treatments have been observed. therefore, new antimalarial strategies involving novel targets and new mechanisms of action are needed. during its life cycle, in erythrocytic stage, which causes all the malaria symptoms, plasmodium falciparum relies on phospholipids to build the membranes necessary for daughter cell development. approximately 85% of parasite phospholipids consist of phosphatidylcholine (pc) and phosphatidylethanolamine (pe) synthesized by the parasite through the de novo kennedy pathways. in the pathway of phosphatidylcholine biosynthesis, the second step catalyzed by ctp:phosphocholine cytidylyltransferase [ec 2.7.7.15] is rate limiting and appears essential for the parasite survival at its blood stage [2] [3] . we are focused on the structural characterization of this enzyme, the identification of effectors by fragment-based drug design approach (fbdd) and then their optimization to eventually design a lead. the first reported crystal structure of the catalytic domain of the enzyme target (pfcct) has been solved at resolution 2.2 å, 3 enzyme-substrates complexes (cmp-, phosphocholine-and choline-bound forms) at resolutions 1.9-2 å and an enzyme-product (cdp-choline) complex structure at resolution 2.4 å that give detailed images of binding pocket, demonstrate conformational changes between apo-and holo-protein forms and provide the information about the mechanism of the catalytic reaction at atomic level. the fbdd method uses a library of small molecules (fragments) with molecular weight that does not exceed 300 da to explore target binding sites. although fragments often have too low affinities to evoke a biological response, their probability of binding is high because they are small enough to prevent unfavorable interactions with target protein-binding sites. moreover, they represent more attractive and synthetically tractable starting points for medicinal chemistry compared to more complex compounds. as the affinity is low, fragment screening usually depends on detecting binding rather than inhibition. screenings of a fragment library (300 molecules) has been performed by fluorescence-based thermal shift assay and nuclear magnetic resonance saturation transfer difference (nmr std) [4] . this combination of techniques identified so far 4 fragment hits that are currently evaluated for their binding modes and affinities. co-crystallization of the protein-fragments complexes is carrying out to provide accurate information on the molecular interactions. topology of interactions will be used to rationally monitor every iterative round of the optimization process allowing subsequent rational design. [1] world health organization, world malaria report (who press, geneva, switzerland), http://www. who.int/malaria/publications/world_malaria_report_2014/wmr-2014-no-profiles.pdf?ua51 protein scaffolds play a crucial role in signaling pathways by generating signal specificity and increasing signal efficiency and amplitude. engineered protein scaffolds can be used as key regulators for signal transduction in artificial signal transduction cascades where they can regulate in-and output of the network. in this research a 14-3-3 protein scaffold is developed which induces dimerization of proteins mediated by the small molecule stabilizer fusicoccin. as proof of principle caspase 9 is used to constitute proximity induced dimerization. dimerization of caspase 9 leads to its activation and consecutively initiates the caspase cascade involved in the programmed cell death pathway. caspase 9 does not naturally bind to 14-3-3 proteins, therefore the caspase 9 monomer is conjugated to a 14-3-3 binding motif which is known to bind into the binding grooves of a 14-3-3 dimer. this interaction can be stabilized by the small molecule fusicoccin. we showed that upon addition of small molecule fusiccocin caspase dimerization is induced, resulting in caspase activity which is measured using a synthetic caspase substrate. moreover the biphasic effect of the 14-3-3 scaffold could be proven. additionally, the activated caspase 9 is also able to cleave its natural substrate caspase 3, downstream in the caspase cascade. these results indicate that the 14-3-3 platform is a versatile small molecule induced dimerization platform which can be used as tool for engineering of a synthetic signaling network. the g308e variant of the apoptosis inducing factor, responsible of a rare encephalopathy, is hampered in nad1/h binding luca sorrentino 1 , laura rigamonti 1 , mirvan krasniqi 1 , alessandra calogero 1 , vittorio pandini 1 , maria antonietta vanoni 1 , alessandro aliverti 1 1 the apoptosis inducing factor (aif) is a highly conserved mitochondrial flavoprotein known to play two opposite roles in eukaryotic cells: in mitochondria it is required for efficient oxidative phosphorylation (oxphos), while, when released into the cytoplasm, it triggers caspase-independent apoptosis (1) . the mechanism of aif-induced apoptosis was extensively investigated, whereas its mitochondrial role is poorly understood. there are many evidences of aif importance for mitochondrial correct morphology and functions and recently the discovery of its direct interaction with chchd4, a key regulator of respiratory complexes subunits import and folding in mitochondria, was reported (2) . a unique feature of aif, probably pivotal for its vital function, is the ability to form a tight, air-stable charge-transfer (ct) complex with nad1 and undergo dimerization. although some aspects of aif interaction with nad1/ h have been analyzed, its precise mechanism is not fully understood. we investigated the effect of the pathogenic g308e replacement, associated with oxphos defect and neurodegeneration (3) , to understand how it could alter aif properties at the molecular level. to do so, we analysed how the wild type and the g307e forms of murine aif interact with nad1/h and nicotinamide mononucleotide (nmn1/ h), finding that the pathogenic replacement resulted in a dramatic and specific decrease of the rate for ct complex formation and consequent protein dimerization only in the case of the physiological ligand. our results demonstrate that the adenylate moiety of nad1/h is crucial for the ligand binding process and that the g307e replacement causes an alteration of the adenylate-binding site of aif that drastically decreases the affinity for and the association rate of the ligand. in addition, we shed new light on the mechanism of the dimerization process, demonstrating that fad reduction rather than nad1/h binding initiates the conformational rearrangement of aif that leads to quaternary structure transitions. taken together, our results contribute to define how aif works at the molecular level in binding nad1/h and undergoing dimerization and also point out that the g308e replacement, responsible of a rare neurodegenerative disease, has the selective effect of slowing down the formation of aif dimeric ct complex. dipartimento di bioscienze, universit a degli studi di milano, 2 dipartimento di scienze veterinarie e sanit a pubblica, universit a degli studi di mical, from the molecule interacting with casl, indicates a family of conserved cytoplasmic multidomain proteins that catalyze a nadph-dependent f-actin depolymerization activity through their essential n-terminal fad-containing monooxygenase-like domain (mo) in response to semaphorin signaling [1] . this domain is followed by calponin homology (ch) and lim domains, proline-and glutamate-rich regions and a c-terminal coiled-coil motif that mediate the interaction with various proteins (e.g: crmp, casl, plexin, g proteins, ndr) [1] . to contribute to establish the catalytic properties of mical mo and their modulation by the additional domains and by the interacting proteins, we have produced and are characterizing the human mical1 (mical-fl) and forms containing the mo [2] , mo-ch and mo-ch-lim domains. all mical forms contain stoichiometric amounts of fad in the mo domain and 2 zn11 ions in the lim domain. mical-mo catalyzes a nadph oxidase (h2o2-producing) activity. the ch, lim and c-terminal domains lower its catalytic efficiency (kcat/km, nadph) mainly due to an increase of km for nadph. the kcat is similar for all forms excepted for mical-fl where a 7-fold drop is observed, in agreement with the proposed autoinhibitory function of the c-terminal domain [3] . the ph dependence of the kinetic parameters of mo, moch and mochlim is complex suggesting that it does not reflect the ionization state of individual groups, but rather the overall protein charge. mical-mo, -moch and -mochlim catalyze a nadph-dependent f-actin depolymerization with a similar apparent km for actin. f-actin (but not g-actin) stimulates the rate of nadph oxidation by increasing kcat and lowering knadph. the extent of nadph oxidation exceeds total f-actin which is in contrast with the proposal of specific modification of actin met44 or met46 reported for drosophila and mouse moch [4] [5] , but it suggests that f-actin stimulates the nadph oxidase activity or a case of substrate recycling. accordingly, with hmical mo and moch several actin residues are oxidized beside met44 and met46. thus, the ch and lim domains do not seem to be important for the mical-actin interaction and actin modification may be mediated by in situ h2o2 production. in hek293t and cos-7 cells mouse collapsin response mediator protein-1 (mcrmp1) interacts with mical1 inhibiting h2o2 production [3] , suggesting that crmp1 could be a hydroxylatable substrate of mical-mo. we have produced the same mcrmp1 form (8-525 aa) and we have shown that under conditions that limit non specific interactions a mild stimulation (up to 20%) of nadph oxidation is observed. f-actin reversed the effect of mcrmp1 suggesting their competition for mical. these results suggest that crmp1, a major microtubules regulator, is not the substrate of the mo domain, but actin and microtubules cytoskeleton components may be linked through the formation of crmp-mical complex in response to semaphorin-plexin signaling. experiments are in progress to complete the characterization of mochlim and full length mical forms. green fluorescent protein (gfp), owing to its genetically encoded strong fluorescence, has become one of the most important tools in modern biology [1] . enhanced gfp (egfp, f64l/s65t-gfp), frequently used variants of this protein, is thermodynamically more stable and 35-times brighter than gfp [2] . due to the improved fluorescent properties, egfp is commonly used as a fluorescent intracellular marker in bio-imaging in vitro and in vivo. despite sustained interest of the scientific community and numerous practical applications, the actual biological role of gfp remains elusive. recent reports put forward a hypothesis of antioxidant and photo-protective functions of gfp [3] . in this study, we focused on the photo-protective role of egfp against reactive oxygen species (ros) photo-generated by visible light in water suspensions of nano-particular nitrogen-doped titanium oxide (n-doped nano-tio2), that is in the system: 'n-doped nano-tio2)/visible light'. n-doped nano-tio2 (sumitomo tp-s201) was chosen as a photo-catalyst, since it is widely accepted that nitrogen doping enhances visible light photoactivity of tio2. 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl (tempol), a paramagnetic water-soluble compound, belonging to the nitroxide class o superoxide dismutase (sod) mimetics, was used as a target for photo-generated ros. a solar simulator, with the flux output intensity of 1 kw/m2, was used as a visible light source. electron spin resonance (esr) was employed to monitor the changes in the paramagnetic signal of tempol exposed to the action of ros in the absence and presence of egfp. in the absence of egfp and after 50 min of illumination, due to a combined action of superoxide (o2•-) and hydroxyl (oh•) radicals generated by the system 'n-doped nano-tio2)/visible light, the esr signal of 100 um tempol decayed by 20%. moreover, the growth of a new signal, interpreted as 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (tempone), resulting from the attack of oh•radicals on tempol, was also observed. in contrast, in the presence of egfp (7.5 um) , the ros-induced decay of the esr signal of tempol was markedly smaller, not exceeding 5%. concomitantly, the growth of the esr signal of tempone was also partially inhibited (30% smaller amplitude), as compared to the process performed in the absence of egfp. in summary, our results point to a significant inhibition of the photodecomposition of tempol in the presence of egfp and support the hypothesis of the protective role of this fluorescent protein against ros generated by the system 'n-doped nano-tio2)/visible light'. school of chemistry, national university of ireland galway, 2 school of biochemistry and immunology, trinity college dublin by studying a variety of anionic ligands and their interactions with cationic cytochrome c, we are building knowledge of protein recognition geared towards regulating activity. in previous work it was shown that psulfonatocalix [4] arene selectively binds to, and encapsulates, three lysine side chains on cytochrome c 1. here, the binding of two small molecule ligands to cytochrome c was investigated. nmr spectroscopy was used and in one case, a crystal structure of the complex was obtained (fig 1) . the calixarene bound to cytochrome c, reveals a crystal packing assembly that suggests it is a key mediator of crystal formation. nmr data analysis indicates the calixarene's binding site on cytochrome c. the pillararene, a relatively new class of compound, is a symmetrical arrangement with a p-rich cavity 2, related structurally to calixarenes. this suggests good host-guest complexation properties. previously, the carboxylatopillararene showed selective binding to arginine, lysine and histidine 2. with this ligand, an interaction with cytochrome c was observed and a complex formed. additionally, biphasic binding behaviour was observed through analysis of the chemical shift perturbations. this may indicate more than one binding event taking place. the data from these studies indicate that recognition is occurring and again that lysine side chains play an essential role. the enzyme dihydrofolate reductase (dhfr) is necessary for the growth and development of all organisms. 1 the structure and function of escherichia coli dhfr have been characterized in buffer. however, dhfr exists in living cells, where the protein concentration can exceed 300 g/l. 2 we know that weak, non-specific chemical interactions with cytosolic proteins alter protein conformation and dynamics,3,4 both of which are expected to influence dhfr catalysis. investigators have examined steady-state enzyme kinetics under crowded conditions, but conclusions can be conflicting. 5, 6 here, the effects of crowding on e. coli dhfr catalysis are assessed through specific activity measurements in solutions of synthetic polymers. these kinetics studies are complemented by in-cell and in vitro 19f nmr data from fluorinated tryptophan residues. preliminary results suggest that the effects of polymeric crowders on dhfr activity are non-monotonic, which may arise from the polymer's transition from the dilute to semi-dilute regime. the data suggest that synthetic polymers are not a valid representation of the cellular interior. biotechnology department, university of verona calcium (ca21) is one of the most important second messengers in eukaryotes. ca21 binding proteins can be subdivided into two categories: "ca21 buffers" that modulate ca21 ion concentrations in cells, and "ca21 sensors" that decode ca21 signals in a wide array of physiological processes in response to external stimuli. calmodulin (cam) is the prototypical example of ca21 sensor proteins in both animals and plants. in addition to conserved cam, plants possess a unique family of 50 cam-like proteins (cmls). many of these cmls still remain uncharacterized and the investigation of their biochemical and biophysical properties will provide insight into ca21 signalling in plants. herein, a detailed characterization of arabidopsis thaliana cml14 is reported. cml14 is a protein of 148 amino acids with a theoretical molecular weight of 16,579 da and 50% amino acid sequence identity with atcam2. cml14 is predicted to have one functional ca21 binding site despite the presence of three ef-hand motifs (prosite). we overexpressed cml14 in e. coli and analyzed its biochemical and biophysical characteristics, i.e. calcium affinity and stoichiometry and eventual changes in conformation, thermal stability and proteolytic susceptibility upon ca21 binding. isothermal titration calorimetry (itc) and nuclear magnetic resonance (nmr) spectroscopy identified one ca21 binding site in cml14 and showed that ca21 and mg21 compete for the same binding site. the kd values determined by itc established that cml14 has higher affinity for ca21 than for mg21. our data were consistent with the sequence based prediction of one functional calcium binding site. differential scanning calorimetry (dsc) showed that ca21 and mg21 have the same stabilizing effects on protein folding. apo-cml14 undergoes two thermal unfolding transitions, but in the presence of ca21 or mg21 only one unfolding event at an intermediate temperature occurs. limited proteolysis experiments showed that ca21 binding affords protection against cml14 digestion by trypsin. surprisingly, cml14 exhibits very few conformational changes upon calcium binding, which were evaluated by ans fluorescence and stokes radius measurements in the apo-and ca21 bound-forms. these results suggest that cml14 does not show the characteristics of a classical ca21 sensor protein. to better understand the physiological role of cml14 in plants, in vivo analysis will be performed. pb-068 fbp17 controls the hepatocyte morphology through rho signaling jun zhang 1 , mingming ling 1 , qianying zhang 1 , yunhong wang 1 , deqiang wang 2 1 the department of cell biology and genetics, 2 the formin-binding protein 17 (fbp17) widely expressed in eukaryotic cells was previously identified to play a role in morphological maintenance in hepatocyte, but the molecular mechanism keeps still unclear so far. in the present investigation, it was found that rho family proteins cdc42/rac1 signaling was involved in the morphological regulation controlled by fbp17. knockdown of endogenous fbp17 expression with rnai technique or dominant negative mutant of fbp17 could trigger the cell morphological remodeling from the epithelioid to fibroid following the significant down-regulation of cdc42/ rac1 activities and dephosphorylation of paxillin. while the rho protein specific activator could restore the cdc42/rac1 activities, and in turn abrogated the silence effect. overexpression of wild type fbp17 could not result in any of the morphological transition. furthermore, withdrawal of the silence could induce morphological recovery when the fbp17 expression, cdc42/rac1 activities and paxillin phosphorylation were restored to the normal level. the experimental evidences strongly indicated that fbp17 was implicated in morphological control probably via rho signaling pathway in hepatocyte. key words: fbp17; rho signaling; paxillin; morphological control; hepatocyte this work was supported by a grant from national natural science foundation of china (nsfc, no. cytochrome c oxidase (cco) is the final enzyme in the respiratory chain of mitochondria but also an integral part of the metabolism of many types of bacteria. in a complex, stepwise redox-reaction, cco catalyzes the reduction of molecular oxygen to water and utilizes the resulting free energy to pump protons across the membrane thereby creating an electrochemical gradient [1, 2] . to investigate proton pumping spectroscopically it is possible to label the entrance of the proton entrance channel with fluorescein, a ph sensitive dye, which allows determining time resolved local changes in proton concentration at the cytoplasmic cco surface and related properties. it has already been shown that the redox state of copper and heme centers affects such properties at the cytoplasmic surface. [3] this study is a theoretical approach to investigate changes of pka values of the fluorescein label at the entrance of the k-channel for different protonation pattern in both oxidized and reduced cco by performing molecular dynamics (md) simulations. further work is based on calculations of pka values of the fluorescein using software karlsberg1 [4, 5] . methods for genetically and synthetically manipulating protein structure enable a greater flexibility in the study of protein function. we have shown that using inteins as traceless, cleavable purification tags enables the separation of full length unnatural amino acid (uaa) containing proteins from their corresponding truncation products. this method has been used to incorporate uaas in previously unattainable positions in a variety of proteins using a myriad of uaas, inteins, and purification tags. in other applications, we have used e. coli aminoacyl transferase (aat) to selectively modify the n-termini of proteins with uaas in denaturing conditions and conditions that maintain folding. applications of particular interest include overcoming the need for an n-terminal cys residue in expressed protein ligation, transfer of reactive handles for "click" chemistry labeling of proteins, and transfer of fluorogenic molecules for photophysical experiments. we have found that aat can transfer protected cysteine, homocysteine, and selenocysteine to expressed proteins. after ligation, these residues can be converted to met or ala, making the ligation traceless. we continue to develop variants of aat to broaden the substrate scope of both its transferred substrate and n-terminal recognition element. in addition, expressed protein ligation is being used to incorporate backbone modifications, such as the thioamide, into various positions in the protein calmodulin to determine how these modifications can impact the structure and function of an ordered protein. in general, by working at the interface of several protein modification technologies, we have made beneficial discoveries that might be missed by more focused approaches. function and modularity of cw_7 motives in the c-terminal region of the endolysin cpl-7 encoded by the cp7 pneumococcal bacteriophage manuel iglesias-bexiga 1,2 , noelia bernardo-garc ıa 3 , rub en mart ınez-buey 4 , noem ı bustamante 1,2 , guadalupe garc ıa 1,2 , marta bruix 1 , juan hermoso 3 , margarita men endez 1,2 1 dept. of biological physical-chemistry, iqfr-csic, 2 ciber of respiratory diseases (ciberes), 3 department of crystallography and structural biology, iqfr-csic, 4 bacteriophage lytic murein-hydrolases have been proposed as enzybiotics, an efficient way to fight bacterial infections. however, the use of these enzymes is normally restricted to gram-positive bacteria since the outer membrane of the gram-negative bacteria hampers the access of the hydrolases to the peptidoglycan substrates. all the murein hydrolases reported in the pneumococcal system, both from host or phage origin, depend on the aminoalcohol choline to be fully active. there is only a unique exception to this rule, the cpl-7 lysozyme. this hydrolase is encoded by the lytic pneumococcal phage cp-7 and, instead of the common cell wall binding module (cwbm) that recognizes choline, cpl-7 harbors a completely different cell wall binding structure. recent studies have revealed that reducing the net charge of the cwbm, from 214.9 to 13.0, leads to an improvement in the antibacterial activity of cpl-7 (1) . the cwbm of cpl-7 is composed by three identical repeats of 48 amino acids, the cw_7 motives, and it folds both in the presence and in the absence of the n-terminal catalytic module (2) . this module shows the capacity of recognize the glcnac-murnac-l-ala-d-isogln muropeptide (gmdp), structurally related with the peptidoglycan basic unit (3) . here, we report the high resolution structure of the cell wall binding module of the cpl-7 endolysin. each cw_7 repeat is composed of a bundle of three a-helices with a highly negative electrostatic charge at the surface. the strong inter-repeat interactions and the high ionic strength used in the crystallization conditions allow them overcoming the electrostatic repulsions inducing a closed-packed structure with a three-fold symmetry. the module dimensions (49 x 38 x 34 å) and the repeat arrangement in the crystal structure are inconsistent with the gmdp binding characterization, the activity displayed by cpl-7 truncated variants with one or two cw_7 repeats, or the experimental determined hydrodynamic properties. using the small angle x-ray scattering (saxs) technique and the atsas computational platform (4), a different arrangement of the cw_7 repeats is envisaged in solution (fig. 1) , whose rather opened structure (70 x 44 x 46 å) is consistent with the experimental data. additionally, employing the saxs-based structure and the honeycomb structure proposed for the peptidoglycan, a model, where each cw_7 repeat of the cell wall binding module fit in adjacent glycan chains, has been derived. in 2000, the protein structure initiative (psi) was started as to determine three-dimensional structures of proteins within every family. once solved, structures are deposited into the protein data bank (pdb) and termed structural genomics (sg) proteins. as of june 2015, there are over 13,300 sg proteins deposited in the pdb and most of them are of unknown or uncertain biochemical function. in addition, many of these sg proteins have a putative functional assignment based on their sequence and structural similarities with proteins of known function; such comparisons can be made against large databases using programs such as blast or dali. however, these putative functional assignments are often incorrect. this project analyzes members of the crotonase superfamily (cs). the cs consists of five diverse functional subgroups that are well characterized structurally and functionally, representing different types of reactivity, including hydrolase, isomerase, hydratase, and dehalogenase activities. this superfamily also contains at least 70 sg proteins, so it is ideal to test predictions of protein function. our approach is based on local structure matching at the computationally predicted active site. first, partial order optimum likelihood (pool) is used to predict the functionally important residues of each sg protein and of the proteins of known function in the superfamily. next, structurally aligned local sites of activity (salsa) is used to align the predicted catalytic residues of the well-characterized members in the superfamily. from this analysis we generate chemical signatures for each functional subgroup and compare them to the sets of catalytic residues predicted for the sg proteins. we demonstrate based on these computational methods that the majority of the putative annotations in the cs superfamily are likely incorrect. currently, biochemical assays are being used to test these predictions. preliminary biochemical results show that one sg protein, thermus thermophilus q5sls5_thet8, classified as a probable enoyl-coa hydratase, possesses hydrolase activity as predicted by our methods. the outcomes of this project will be to successfully classify the biochemical functions of sg proteins based on their local structure at the predicted active sites and to provide a conceptual framework for the functional classification of the remaining sg proteins within the pdb. this work is supported by nsf-che-1305655. directly observing the synergistic dynamics in f-actin and microtubule assembly jun zhang 1 , deqiang wang 2 1 the department of cell biology and genetics, 2 key laboratory of molecular biology on infectious disease although important in cellular activities, little attention was paid to the synergistic effects of actin and microtubule cytoskeleton assembly. with the time-lapse atomic force microscope (tl-afm), we directly observed the large-scale dynamic structure of actin filaments formed in the presence or absence of microtubulin in solution. in absence of microtubulin, the g-actin could be polymerized into ordered filamentous structures with different diameter from the slimmest filament of single f-actin to giant filament in tree-like branched aggregates. the polymerized actin filaments, to which our most intense attention was attracted, was discretely arranged and showed obvious polymorphism in structures completely distinct from those in the presence of microtubulin. the supra-molecular complex structures of the latter were mainly composed of single f-actin and/or multifilaments clearly consisting of several single f-actin and regularly cross-linked with the assembled microtubular bundles. the experimental results demonstrated that the f-actin dynamics could be coordinated by microtubule assembly. further analyses implied that the interactions between f-actin and microtubule could prevent the emergence of structural polymorphism of f-actin alone, and give rise to organization of specific complex structures instead. it was suggested that dynamic synergy between the f-actin and microtubule would be implicated in living cells. the adaptor protein 14-3-3 is found in a diverse range of pathologically relevant protein-protein interactions (ppis). as 14-3-3 is a hub protein with very diverse interactions, it is able to influence the intracellular localization of their binding partners and they are key regulators of signal transduction processes as well as regulators of cell cycle functions.nevertheless, there are only few examples of 14-3-3 acting extracellularly. one of the extracellular targets for 14-3-3 is aminopeptidase n (apn). apn is an extracellular trans-membrane enzyme that acts as a receptor for 14-3-3. binding to apn, 14-3-3 excreted by keratinocytes can upregulate the excretion of matrix metalloproteinase-1 (mmp1) in fibroblasts. mmp1, by breaking down collagens, is key in the remodeling of the extracellular matrix. modulation of the 14-3-3/apn interaction thereby may play a crucial role in the fundamental understanding and ultimately treatment of wound healing, respiratory diseases and tumor growth. in the eukaryotic cell, the 14-3-3 dimer operates as an adapter platform for binding partners. a wide range of classes of (small) molecules, natural products and peptides has been used to modulate the ppis, providing either stabilization or inhibition of the interactions of 14-3-3 with its binding partner. binding partner fragments or peptides are known to bind to the 14-3-3 binding groove via arecognition motif containing a phosphorylated serine or threonine. making use of the dimeric structure of 14-3-3, novel small-molecule inhibitors may be tethered to exploit the bivalent effect. from a large virtual screening and experimental validation, a scaffold containing a phenyl phosphonic moiety was identified, showing inhibitory properties for 14-3-3 ppis. potent derivatives of this scaffold were bridged by polyethylene glycol (peg) linkers of varying lengths, thereby facilitating the compound to reach both binding sites of the 14-3-3 dimer and concurrently increasing the compound's solubility in aqueous solution. similar bivalent inhibitors have been proven to synergistically increase their efficacy. biophysical evaluation by means of fluorescence polarization (fp) inhibition competition assays, revealed an increase of the half maximal inhibitory concentration (ic50) from approximately 81 lm for the monomeric phenyl phosphonate to approximately 1.8 lm for the bivalent inhibitor with a 60å linker. this demonstrates a 45-fold increase of inhibitory effect towards 14-3-3 and its binding partner peptide mimic. extensive thermodynamic, kinetic and structural analysis of the interaction is in progress.phosphonic moieties have been shown to pass the cell membrane poorly, due to their highly charged character. by being able to specifically inhibit the extracellular interaction between 14-3-3 and apn, these inhibitors are prevented from interfering with the extensive intracellular 14-3-3 interactome. hence, these bivalent phenyl phosphonate inhibitors provide a promising strategy towards extracellular application. the mre11 complex is an oligomeric assembly comprising of dimmers of mre11 and rad50 proteins in archea and additionally nbs1 subunit present in eukaryote. it is the central player in the dna damage response -a functional network comprising dna damage sensing, signal transduction, cell cycle regulation and dna double strand breaks (dsbs) repair [1] . recent structural studies revealed that rad50 hinge domain is rather a short kink in the coiled-coil region and adopts unusual dimerization mode by intermolecular coordination of zn(ii) and formation of so-called zinc hook domain [2] . to date, very limited structural data on the zinc hook domain have been reported, the only known structure was resolved for rad50 homologue from hyperthermophilic archaeon -p. furiosus. unusual zn(ii) coordination mode in zinc hook domain raises question of how zinc hook domain assembles to form interprotein zinc binding site with sufficient stability to function at low intracellular free zn(ii) concentrations [3] . our study on minimal zinc hook domain fragment (14 aa) indicated low femtomolar affinity towards zn(ii) [4] . extended zinc hook domain fragment (45 aa) reveals even zeptomolar affinity. therefore, our main goal was to probe the thermodynamic and structural effects that are hidden in the small interprotein interface and are responsible for the dimerization of the large and critical protein machinery. probing of those effects was achieved by detailed biophysical characterizations (including potentiometry, nmr, hdx ms and cd spectroscopy) of 18 protein fragments of zinc hook domains with a number of point mutations. we showed that extremely high stability of zinc hook domain from p. furiosus is achieved by the formation of hydrogen bond network in b-hairpins and interprotein hydrophobic core. eindhoven university of technology dna-based molecular circuits have become a very attractive tool in molecular imaging, synthetic biology, molecular diagnostics and biomolecular computing. the highly modular and predictable nature of watson-crick base pairing allows the construction of complex circuits using a limited set of logic gates and building blocks. however, the lack of generic approaches to interface dna-based molecular circuits with protein activity limits their application in biomedicine and molecular diagnostics. here we present a new, highly modular approach to control the activity of a reporter enzyme based on the dna-directed assembly and disassembly of a complex between tem1-b-lactamase and its inhibitor protein blip. both proteins are conjugated to a unique oligonucleotide, allowing the assembly of the enzyme-inhibitor pair and inhibition of enzyme activity by the addition of a complementary template strand. addition of an oligonucleotide that is complementary to a loop sequence in the template results in the formation of a rigid dsdna spacer that disrupts the enzyme-inhibitor complex, restoring enzyme activity. using this noncovalent approach allowed easy tuning of the template and target sequences with only a single set of oligonucleotide-functionalized enzyme and inhibitor. to show the modularity of the system, a panel of 8 different template sequences were selected. only in the presence of their complementary viral dna sequences restoration of enzyme activity was observed. in addition to this excellent specificity the system showed to by higly sensitive towards its target, since the presence of as little as 2 fmol of target resulted in an observable increase in enzyme activity. the use of a stable and well-characterized enzyme-inhibitor pair, complemented by the modular design of our reversible dna-directed protein switch make it an attractive system to implement in dna-based molecular circuits. several studies demonstrated important roles of human carbonic anhydrases (hcas) in a variety of physiological and pathological processes. consequently, in recent years the 12 catalytically active hca isoforms have become an interesting target for the design of inhibitors with biomedical applications [1] . derivatized sulfonamides of type r-so2nh2 represent the class of ca inhibitors (cais) mostly used and best characterized. the large number of crystallographic studies so far available on these molecules clarified the main factors responsible for the binding of the sulfonamide moiety to the ca active site. 1 in particular, it has been highlighted that even though these molecules generally behave as very potent cais, they do not show selectivity for the different isoforms. indeed, the sulfonamide moiety plays a predominant role in the interaction with the enzyme, while any change in the nature of the r substituent has generally a rather marginal effect on the enzyme-inhibitor affinity. these characteristics make difficult the design of sulfonamide derivatives selective for the different ca isoforms. consequently, much efforts were dedicated in last years to the development of new inhibitors that, although presenting lower affinity for the ca active site, would be able to be more selective toward the different isoforms. carboxylic acids have been recently investigated as cais, showing that these molecules can adopt different binding modes to the enzyme active site. in particular, they can coordinate directly to the zinc ion or be anchored to the zinc-bound water molecule. however, the structural reasons responsible of this peculiar behavior have not been clarified yet. in a general research project aimed at providing insights into the binding mode of these molecules to cas, we have undertaken the characterization of two carboxylic acids, namely an ortho-substituted benzoic acid [2] and a saccharine derivative, by means of kinetic, crystallographic and theoretical studies. exploring the mechanism of fibril formation using fluorescently labelled human lysozyme variants ana bernardo gancedo 1 1 'exploring the mechanism of fibril formation using fluorescently labelled human lysozyme variants' human lysozyme is a widely characterised protein whose mutational variants misfold into fibrils that are associated with systemic amyloidosis (1) . although the process of aggregation for human lysozyme has been well studied, the details of early events within this process are not fully characterised. single molecule fluorescence microscopy has been used to determine the oligomeric distributions present in the aggregation process of a number of disease-related intrinsic disordered proteins (idps) (2) . recent advances in site-specific labelling of human lysozyme (3) have made this protein amenable to these single molecule fluorescence studies. we have introduced alexa-fluorophores into the i59t variant of human lysozyme and have demonstrated that the process of in vitro fibril formation is not significantly altered. using these fluorophore-labelled proteins we can apply single molecule fluorescence to study the early aggregation events within this system, allowing us to compare protein aggregation in a globular protein and with the aggregation process of idp's. abstract protein structure, folding and function, while specific interactions with lipid molecules can also contribute towards the biological activity of some membrane proteins. improving understanding of the interactions has resulted in the development of artificial lipid systems that allow the bilayer properties to be rationally manipulated in vitro to control protein behaviour. the bacterial transporter lacy is a well known integral membrane protein from the major facilitor superfamily, responsible for the protondriven uptake of d-lactose in e. coli. with a high resolution structure available and considerable understanding of mechanistic detail, and with observed changes to both structure and function in different bilayer environments, lacy is a good model system for examining the behaviour of a major class of membrane proteins in these lipid systems. purified lacy has been reconstituted into liposomes and droplet interface bilayer systems of varying lipid composition and the effect on protein function and bilayer properties examined. targeting abeta oligomers by trehalose-conjugated peptides: a molecular dynamics study alzheimer's disease (ad) is currently one of the most common and devastating forms of dementia correlated with beta-amyloid peptide (abeta) accumulation in human brain tissue [1, 2] . inhibiting abeta selfoligomerization in brain tissue remains one of the main strategies to prevent or treat this disorder. as a consequence, in recent years much efforts have been spent in the understanding of the amyloid fibril growth process and its modulation by putative drug molecules. an interesting class of compounds able to prevent abeta fibrillogenesis, is represented by beta-sheet-breaker (bsb) peptides [3] . although these molecules are thought to recognize in a self-complementary manner the abeta hydrophobic core region, however their precise mechanism of interaction is still unclear. in this context, we have studied the structural basis underlying the inhibitory effect of abeta(1-42) fibrillogenesis explicated by two promising trehaloseconjugated bsb peptides (ac-lpffd-th (thct) and th-succinyl-lpffd-nh2 (thnt)) [4] using an all-atom molecular dynamics (md) approach [5, 6] . the pentameric nmr structure [7] of abeta has been used to model amyloid protofibril, and the two protofibril ends have been investigated as putative binding sites. our simulations suggest that the interaction with the two protofibril ends occurs through different binding modes. in particular, binding on the odd edge (chain a) is guided by a well defined hydrophobic cleft, which is common to both ligands (thct and thnt). moreover, targeting chain a entails a significant structure destabilization leading to a partial loss of b structure and is an energetically favoured process, as assessed by mm/pbsa calculations. a significant contribution of the trehalose moiety to complexes stabilities emerged from our results. the basic structural unit of chromatin is the nucleosome, which is composed of histone proteins forming a scaffold with about 150 base pairs of dna wrapped around. chromatin compacts eukaryotic genomes and regulates gene activity, which is mediated in part by posttranslational modifications (ptms) on the n-terminal tails of the histones. uncovering the detailed relationship between histone tail modifications and gene activity is a major topic of biomedical sciences and general techniques for generating nucleosomes with defined modification patterns in large numbers would greatly facilitate such investigations. to this end we are establishing a chemical toolbox for designer chromatin with defined histone ptm patterns. a protein semysinthesis approach is used that bases on "ligation-ready nucleosomes" with truncated histone h3 that can be ligated with the corresponding synthetic histone tail. we resorted to sortase-mediated ligation as chemoselective ligation method. here we report our recent developments in establishing the envisioned chemical toolbox for designer chromatin. evaluating cation-pi and pi-pi interaction in proteins using various biophysical methods in proteins the aromatic residues phenylalanine (phe), tyrosine (tyr), and tryptophan (trp) can be involved in aromatic interactions known as cation-pi and pi-pi interactions (dougherty 2000). compared to other non covalent interactions in proteins, like h-bonds, dipole-dipole, or van der waals interactions, relatively little is known about the pi-pi and the cation-pi interactions. the strength of both aromatic interactions is dependent on the pi-electron density in the aromatic residues. a lowering of electron density can be created by introducing strong electron-withdrawing substituents like fluorine atoms in the aromatic ring (dougherty 2000). in this way a nearly isosteric change in the aromatic system results in a marked change in electron density. substitution with methyl groups is known to slightly increase the electron density. the response to low cellular oxygen levels in humans and other animals is induced by the hypoxia inducible transcription factors (hifs). these transcription factors are regulated by hypoxia inducible factor prolyl hydroxylases (phds), which act as 'oxygen sensors' by hydroxylating hifs, thus leading to the proteomic degradation of the transcription factors. over the last years, there have been multiple reports that describe additional phd substrates other than hifs. among them are the large subunit of rna pol ii, several transcription factors, and components of signalling pathways. validating these reports is of major medicinal relevance given that phd inhibitors are now in the late stage phase 3 clinical trials. in order to investigate the selectivity of phds, the reported proteins have been tested as substrates for hydroxylation by mass spectrometry, and as binders or competitors of the phds. initial work on peptides that contain the putative hydroxylation sites has indicated that the phds are much more selective for their well-established substrate hif. however, in ongoing work these initial results are going to be validated on protein level by co-expressing phds with the reported substrates. additionally, peptides of reported substrates were screened for their ability to alter the kinetics of hif-hydroxylation by phd2. an inhibitory effect of at least two different peptides on phd2 was observed, suggesting that there is an interaction between the prolyl hydroxylase and these peptides. in order to investigate the mode of binding and inhibition, nmr studies have been carried out and binding of the two inhibitory peptides on phd2 has been shown. altogether, these results indicate that, although phds might be more selective for hif as a substrate as it was initially thought, the enzymatic activity of the prolyl hydroxylases is possibly influenced by a number of other proteins that can directly bind to phds. non-natural aminoacids via the mio-enzyme toolkit alina filip 1 , judith h bartha-v ari 1 , gergely b an oczy 2 , l aszl o poppe 2 , csaba paizs 1 , florin-dan irimie 1 1 biocatalysis and biotransformation research group, department of chemistry, ubb, 2 department of organic chemistry and technology an attractive enzymatic route to enantiomerically pure to the highly valuable a-or b-aromatic amino acids involves the use of aromatic ammonia lyases (als) and aminomutases (ams). all these enzymes have in common an auto-catalically formed 5-methylene-3,5-dihydroimidazole-4-one (mio) electrophilic prosthetic group, and show high structural and sequence similarities. the recent advances in improving the functional properties of these enzymes increased both their biocatalytic and therapeutic applications. we aimed to create a library of recombinant mio-enzymes consisting of the pals and pams with large substrate promiscuity in order to provide access to various non-natural aminoacids through enzymatic ammonia addition and/or ammonia elimination reactions of the substrate library already available in our researchgroup. the developed complementary substrate and enzyme library would provide the mio-enzyme toolkit useful for the synthesis of nonnatural aminoacids. the synthetic gene of the enzymes (pcpal, rtpal, avpal, papam) were cloned into pet19b_j906 expression vector using xhoi and bpu1102i cloning sites. the plasmid dna was transformed to several e.coli host strains (rosetta, bl21, origami 2) in order to optimize the expression yields. the enzymes containing an n-terminal his10-tag were purified with affinity chromatography, followed by ion-exchange or/and size-exclusion chromatography, obtaining pure and homogenous proteins, in their tetrameric, presumably native fold. the enzyme activity and the kinetic parameters of the purified enzymes was determined towards the natural substrate l-phenylalanine, as well as towards novel bulkier aromatic substrates (heteroaryl alanines, styryl alanines, biphenylalanines). furthermore to enhance their biocatalytic applicability we covalently immobilized the enzymes to carboxylated single-walled carbon nanotubes (swcnt cooh) using linkers with different lengths, and tested the activity and recycling of the immobilized enzyme. antibodies that bind protein antigens are indispensable tools in biochemical research and modern medicine. utilizing a phage display selection strategy, we have obtained synthetic antigen binders (sabs), based on a fab fragment of igg, to a wide array of proteins as distinct as membrane proteins, structural proteins, scaffold proteins and nuclear targets. here we demonstrate the applicability of the sabs towards the native, full-length proteins in cells. we show that the generated sabs are able to pull-down endogenous proteins from mammalian cell extracts along with their natural binding partners. we developed a method of utilizing our high affinity and specificity binders as fluorescently labeled tools to visualize target proteins in their native environment in the cells without the need of secondary antibodies or blocking reagents. our system also includes a method of efficient delivery of generated antibodies to living cells, where they can perform their function. the sabs have been successfully used for altering biological processes in a controllable manner. in vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds to target receptors, proteases and trophic factors tai kubo 1 , mohammed naimuddin 1 , seigo ono 1 1 national institute of advanced industrial science and technology (aist) in vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds to target receptors, proteases and trophic factors small molecule natural products are precious resources for drug discovery. during millions of years of evolution, natural products must have been exposed to various selection pressures and have been refined in structure and function to obtain the present features. in some peptide neurotoxins, however, the basic molecular scaffold mainly configured by disulfide (s-s) bridges and/or alpha/beta structures, is strictly conserved within each family even under the evolution pressure. on the other hand the loop regions, which are not heavily involved in scaffold formation, are highly diverged. this mode of molecular evolution named 'accelerated evolution', is reasonable to quickly adapt to the vigorous change of the environment. the evolutionally selected scaffold is compact harboring both rigidity and flexibility in nature, and it may support a topology appropriate for target recognition and selective interaction. inspired by the system, we designed random peptide libraries from the peptide neurotoxins of the accelerated evolution. a three-finger (3f) shaped snake neurotoxin consists of huge family evolved by accelerated gene evolution. we prepared a 3f-peptide library by introducing random sequences in each fingertip. another random peptide library with an ick (inhibitor cystine knot) motif was prepared based on a neurotoxin gtx1-15 from spider; originally identified as a t-type ca21 channel modulator. each library was subjected to in-vitro evolution directed to specific target molecules. for the 3f-peptide library cdna display method was applied to select binders. when interleukin-6 (il-6) receptors were targeted, the selected 3f peptides showed binding affinities (kd 100 nm) comparable to the native ligand il-6. when trypsin was targeted, peptides with serine protease inhibitor activities similar to sti and bpti (ki 30 nm) were isolated. specific binders to a trophic factor vegf were also generated from the 3f library. to target membrane proteins, we developed a unique in-vitro evolution system, and named it as the periss (intra periplasm secretion and selection) method. in the system, target membrane proteins are expressed in inner membrane of e. coli and peptides are secreted to the periplasmic space, in between the inner and outer membranes; and the space is served for interaction and selection. the periss method enabled us to identify a peptide specific to muscarinic receptor m2 subtype from the ick peptide library. in conclusion, it was proved that the library designed from the scaffold of peptide toxin, which evolved in the mode of accelerated gene evolution, has pluripotency in target recognition, interaction and even bioactivity. phenylalanine ammonia lyase from petroselinum cripsum (pcpal) belongs to the class of enzymes containing 4-methylideneimidazole-5-one (mio) as a prostetic group and it is responsible for the conversion of l-phenylalanine into trans-cinnamic acid. this reaction is reversibile under high ammonia concentration. 1 we analyzed several factors that can influence the enantioselective synthesis of nitrophenylalanine mediated by whole cells as well as purified mio-containing and mio-less pcpals. first we investigated the behaviour of the enzymes depending on the ammonia concentration. we also inspected the influence of the ph on the pcpal catalyzed biotransformations. based on our results, we concluded that variation of ammonia concentration and the ph leads to decrease of enantioselectivity, suggesting that pcpal is able to catalyze the formation of both l-and d-enantiomers of electron-deficient structures. all microbial cellulase appears to have a conserved 'sg' amino acid sequence at an identical position in the n-terminal domain. the properties of the n-terminal 15 amino acid sequence were also predicted computationally. this analysis showed that n-terminal sequence of the enzyme is unstable. the nterminal sequence also showed potential cleavage sites by different proteases which may contribute to its instability. the secondary structure analysis showed that the n-terminal sequence has 40% of the 15 a.a. sequence in extended strand and 60% in random coil conformation. the n-terminal sequence was also analyzed for potential phosphorylation sites. while no potential serine and threonine sites were predicted, two tyrosine phosphorylation sites were predicted in the n-terminal sequence. the n-terminal sequence was also examined for the presence of kinase specific phosphorylation sites. the results showed the presence of one potential site which may be phosphorylated by pkc at position 1 of the n-terminal sequence. the analysis for the prediction of the presence of oglcnac sites revealed that two such sites may potentially be present in the sequence. we have also predicted the ligand binding site in the n-terminal sequence of the protein. protein arginine methylation catalyzed by protein arginine methyltransferases (prmts), is a pivotal protein post-translational modification involved in a growing number of physiological and pathological processes including signal transduction, proliferation, differentiation and malignancy. prmt1 accounts for the majority of protein arginine methyltransferase activity in mammalian cells and, in consistence, a large amount of cellular substrates have been identified. several studies have reported that the activity of prmt1 changes upon stimulation in various cellular processes. in mammalian cells, prmt1 exists in a high molecular weight complex. the interacting partners of prmt1, such as antiproliferative proteins btg1 and btg2, protein phosphatase 2a, the orphan receptor tr3, and ccr4-associated factor 1(hcaf1) are shown to play a role in modulating the methyltransferase activity and the substrate selectivity of prmt1. due to the pivotal roles of prmt1 in physiological and pathological conditions, intensive efforts have been put on the search of small synthetic chemical molecules which can efficiently modulate the activity of prmt1 for the potential development of therapeutics. in light of this, the intracellular small molecules that either transmit extracellular stimulation or act as cofactor to dictate the activity of prmts in cells are still poorly understood. our study focused on examining how cellular ions might affect the activity of prmt1 and found that divalent and monovalent ions differentially modulated the catalytic activity of prmt1 toward different substrates. oligomerisation properties of light-dependent protochlorophyllide oxidoreductase prothoracicotropic hormone (ptth) is one of the most important neuropeptide regulators for insect molting and metamorphosis. however, preparation of its recombinant protein has hardly been successful, because it is a homodimer protein with very complicated disulfide-bond structure. for example, silkworm ptth has three intramolecular disulfide bonds in its 109-residue polypeptide chain, and the two chains are further linked by an additional intermolecular disulfide bond to form the homomeric dimer. although the recombinant silkworm ptth was previously expressed in escherichia coli, the product was obtained only in precipitation fractions, and refolding of the precipitated protein provided the active dimer ptth in very poor yield. under such reductive conditions as in cytosol of the e. coli cells, formation of the correct disulfide-bond arrangement must be difficult. alternatively, for the heterologous expression of the silkworm ptth, we employed brevibacillus choshinensis (formally referred to as bacillus brevis), which has achieved good results in expression of various disulfide-bond-containing proteins. in this study, the silkworm ptth was expressed in the brevibacillus cells with an additional his6-tag sequence at the c-terminus, for easier detection and purification. first of all, since the brevibacillus bacteria are equipped with a secretory system of the expressed proteins, a secretory signal sequence to be attached before the silkworm ptth was carefully selected. among four candidates in a commerciallyavailable kit, a signal sequence derived from an intrinsic cell-wall protein mwp gave better results in expression levels of the protein. second, incubation time of the cells was optimized, because an oligomerization state of the secreted ptth in the cell culture medium changed with the time. in the medium, various ptth oligomers including a monomer and a dimer were initially observed, but higher oligomers became a major portion of the secreted product after longer incubation than 48 h. incubation for 24-36 h may be suitable for obtaining the native dimer form of the silkworm ptth. to remove the undesired monomer and higher oligomers, which mostly retained free sulfhydryl groups, the secreted proteins were treated with maleimide-peg2-biotin. in the purification using a ni1-nta column, the dimer of the his6-tagged silkworm ptth was eluted with an imidazole gradient, separately ahead of other biotinylated proteins, probably due to interaction of the peg2 spacer with the ni1-nta groups of the resin. after the reversed-phase hplc purification, the final product showed a single band on the nonreductive sds-page, and it had adequate ecdysone-releasing activity from isolated silkworm prothoracic glands. the brevibacillus bacteria are most promising host cells for the heterologous production of the insect ptth. role of the disulfide bridges in the transmembrane region of the insect prothoracicotropichormone receptor, torso torso is an insect cellular-membrane protein, which was recently identified as a receptor for prothoracicotropic hormone (ptth). although ptth is one of the important regulatory molecules in insect molting and metamorphosis, activation mechanism of torso by the ligand has not been elucidated yet. in this study, an oligomerization manner of the silkworm torso was examined, using heterologous expression in drosophila s2 cultured cells, because torso is a single-polypeptide receptor tyrosine kinase (rtk), and activation of such rtks is often triggered by the ligand-induced receptor dimerization on the cellular membrane. when activated with silkworm ptth, dimerization of the silkworm torso in the s2 cells was observed, using a cross-linking reagent bs3, and the subsequent receptor autophosphorylation and downstream erk phosphorylation were also detected. surprisingly, however, the torso dimerization was revealed to occur even without the ligand stimulation, while the autophosphorylation and the erk phosphorylation were held in response to the stimulation. when fractionated by non-reductive sds-page, the silkworm torso showed an obvious dimer band, in addition to a faint monomer band, both with and without the ptth simulation, even though the receptor was not treated with the cross-linking reagent. this indicates that the torso protein is expressed originally as a disulfide-bond-linked dimer. in addition, by examining oligomerization states of several truncation and substitution mutants, cysteine residues in the transmembrane region were found to participate in the intermolecular disulfide bridges, linking the two receptor molecules in the dimer. when all of the three cysteines in the transmembrane region were replaced by phenylalanines, the disulfide-bond-linked torso dimerization was not observed, but spontaneous, ligand-independent association of the torso molecules was detected using the crosslinker bs3. this spontaneous dimerization caused the apparent torso autophosphorylation, but it could not induce the downstream erk phosphorylation. consequently, without the intermolecular disulfide bridges, torso loses its responsiveness to the ptth stimulation. in conclusion, the disulfide bridges in the transmembrane region may play a role to preserve suitable relative position between the two torso molecules, which could induce ligand-dependent autophosphorylation leading to activation of the downstream signaling pathways in the cells. the yeast enzyme neutral trehalase (nth1, ec 3.2.1.28) from saccharomyces cerevisiae hydrolyses the non-reducing disaccharide trehalose which serves as an energy source and a universal stress protectant in many different organisms. enzymatic activity of nth1 is enhanced by the yeast 14-3-3 protein (bmh1 and bmh2) binding in a phosphorylation-dependent manner. nth1 activity is also regulated by ca21 binding to the ef-hand-like motif containing domain of nth1 [1] .the native tbe page and analytical ultracentrifugation show that nth1 forms very stable complexes with bmh1 and bmh2 [1] . to study the structure of nth1 alone and its complex with the 14-3-3 protein we used circular dichroism, h/d exchange coupled to mass spectrometry, chemical cross-linking [2] and small angle x-ray scattering (saxs) [3] . at the same time protein crystallography of nth1 alone and its complex with bmh1 is performed.the low resolution structure of pnth1:bmh1 protein complex revealed that binding of bmh1 induces a rearrangement of the whole nth1 molecule and that the region containing the ef-hand motif forms a separate domain which interacts with both bmh1 and catalytic domain of nth1. we proved that integrity of the ef-hand motif is crucial for the bmh1 mediated activation of nth1 and ca21 binding. our data suggest that the ef hand-like motif functions as the intermediary through which bmh1 modulates the function of the catalytic domain of nth1. these structural changes probably enable the substrate entry into the enzyme active site [3] . our study of 14-3-3 protein complex with the fully active enzyme nth1 offers a unique structural view of nth1 activation enabling us to better understand the role of the 14-3-3 proteins in regulation of other enzymes. the assembly of self-regulating synthetic biochemical pathways in vitro has great potential as alternative catalysts for the high-yield production of low value/high volume commodity chemicals from biomass. high yields of low-value/high volume compounds that are required for economic viability is particularly difficult via traditional in vivo metabolic engineering of microbes due to competing biochemical pathways and toxicity. we have developed an alternative approach, called synthetic biochemistry, where the glycolysis pathway of central metabolism is reconstituted in vitro with an anabolic pathway that can produce useful compounds at high yield. in the specific synthetic biochemistry system described, reducing equivalents, atp, and carbon from glycolysis are funneled through the anabolic mevalonate pathway to produce the monoterpene limonene from glucose. the successful implementation of the in vitro pathway required development of a molecular purge-valve consisting of an nad1 and nadp1 specific reductase (ie wild-type and mutant pyruvate dehydrogenase), and nadh oxidase, noxe, to maintain proper nadp1/nadph cofactor balance while allowing continuous carbon flux. we find that the purge-valve concept is readily transportable to other nad(p)h generating steps in central metabolism and can be used to convert glucose to limonene at high yield. chitinases (ec 3.2.1.14) are enzymes that randomly hydrolyze b-1,4 glycosidic bonds of chitin and produce n-acetylchitooligosaccharide ((glcnac)n) that has various physiological functions such as immunostimulatory activity. most of fish takes crustacean such as shrimp and crab as food. therefore, the fish has chitinase in the stomach to chemically disrupt the chitinous envelope of crustacean. four chitinase isozymes (42-60 kda), pachia [1] and pachib [2] , and ptchia and ptchib, [3] were purified from the stomach of silver croaker pennahia argentatus and threeline grunt parapristipoma trilineatum, by ammonium sulfate fractionation and column chromatographies, respectively. all the chitinases were stable and showed activity in the acidic ph range (ph3-5). pachia and ptchia preferentially degraded the second glycosidic bond from the non-reducing end of (glcnac)n and pachib and ptchib had a preference for the third glycosidic bond of those. all the chitinases showed different substrate specificity toward insoluble long substrates. moreover, chitinase cdnas (pachi-1 and pachi-2) encoding pachia and pachib, and cdnas (ptchi-1 and ptchi-2) encoding ptchia and ptchib were obtained by cdna cloning using the rt-pcr and race method. the deduced amino acid sequences of all the chitinase cdnas contained n-terminal signal peptide, gh family 18 catalytic domain, linker region, and chitin-binding domain. phylogenetic tree analysis of vertebrate chitinase revealed that fish stomach chitinases form unique chitinase isozyme groups, acidic fish chitinase-1 (afcase-1) including pachia and ptchia, and acidic fish chitinase-2 (afcase-2) including pachib and ptchib, which was different from an acidic mammalian chitinase (amcase) group. [3, 4] the previously reported purified fish stomach chitinases [5] can also be classified into two chitinase isozyme groups, afcase-1 and afcase-2, by the n-terminal amino acid sequence. this study suggested that fish have excellent chitin degrading enzymatic system in which two different chitinases isozyme groups, afcase-1 and afcase-2, with different degradation patterns are expressed in the stomach. recently, the enzymes produced by psychrophilic organisms have gained huge interest especially in the studies of temperature adaptation of the protein. previously, a cold-adapted yeast, glaciozyma antarctica pi12 was isolated from a marine environment in antarctica and the yeast was known to produce lipolytic and proteolytic enzymes. a gene encoding a unique recombinant bifunctional enzyme (lippi12) with cold active lipase with protease activity was successfully expressed, purified and characterized. temperature profile of the bifunctional lippi12 enzyme showed that the lipase functions optimally at 208c whereas the protease was more active at 408c. ph profile showed that both lippi12 lipase and protease were active at near neutral condition. activity of lippi12 lipase and protease were also activated in the presence of cacl2 but its protease counterpart seemed to be more active in the presence of zncl2. effect of surfactants showed lippi12 lipase was activated by tween 80 and sls and in contrast, lippi12 protease was almost deactivated in all surfactants tested. the presence of organic solvents did not affect both the lipase and protease activities. the lipase was more stable at solvents with higher log p value whereas the protease was slightly activated at low log p value particularly with dimethylsulfonyl. inhibitor studies revealed that lippi12 lipase was partially inhibited with edta and pmsf whereby the lippi12 protease was inhibited by pepstatin, edta and pmsf. lippi12 enzyme was successfully crystallized via vapour diffusion method. crystal of lippi12 enzyme was diffracted via synchrotron radiation. the three-dimensional structure of cold-adapted pi12 provided insight into cold adaptation and better understanding of the structural properties of lippi12 enzyme. the bifunctional properties of the enzyme could be potential candidate for low temperature industrial application. conformation-specific antibodies as enhancers and inhibitors of phosphatase activity of dep 1 malgorzata nocula-lugowska 1 , mateusz lugowski 1 , anthony a. kossiakoff 1 1 the university of chicago dep-1 (cd148/ptp-h) is a transmembrane receptor-like protein tyrosine phosphatase (ptp) that has been implicated in the density-dependent regulation of cell growth, differentiation and transformation. it counteracts protein kinases by dephosphorylating a number of their substrates as well as the kinases themselves, thus potentially controlling the specificity of signals. for example egfr, vegfr 2, met, pdgf b receptor have been shown to be dephosphorylated by this phosphatase. dep-1 has been shown to act as a tumor suppressor and it has been proposed as a molecular target in antiangiogenesis therapy. as a result, both enhancers and inhibitors of dep-1 activity have the potential of elucidating pathways responsible for abnormal cell behavior. we generated synthetic antibodies against intracellular catalytic domain of dep-1 that act as modulators of the enzyme's phosphatase activity. by applying a combination of selection pressures an array of antibodies has been raised from phage display libraries of fab fragments which are capable of either enhancing or inhibiting dep-1 activity. in phosphatase assays with catalytic domain of dep-1 the antibodies demonstrate non-competitive or mixed kinetics. the crystal structure of dep-1-inhibitor complex shows that this antibody binds to the part of the protein that is distant from the active site and acts by locking the enzyme in the nonnatural catalytically inactive state by hindering the closure of the wpd loop which is crucial for the reaction to occur. by contrast, as judged from the crystal structure of a complex of dep-1 with the antibody that enhances its phosphatase activity, this antibody seems to act by stabilizing the naturally found active state of dep-1 with wpd loop in the closed conformation. the antibodies are also able to recognize dep-1 in cells, as they stain dep-1 in immunofluorescence experiments. to test the applicability of raised antibodies in cells the activator was additionally used to pull down full-length endogenous dep-1 after being delivered to live cells. inhibition and enhancement of dep-1 activity by locking the enzyme in conformations which are either natural or imposed by allosteric binding of antibodies seems to be a mechanism that can be utilized to modulate activity of other tyrosine phosphatases. investigating acinetobacter baumannii pathogenesis: crystal structure of wbjb epimerase from a polysaccharide biosynthesis cluster oxygen homeostasis is regulated by hypoxia inducible factor, a transcription factor. when the oxygen level becomes too low (hypoxia), hypoxia-inducible-factor 1 (hif-1a) activates the expression of over a hundred genes, associated with angiogenesis, erythropoiesis, vegf (vascular endothelial growth factor), cell migration, and energy metabolism etc. hif-1a cellular level is highly dependent on oxygen concentration and regulated by oxygen sensor enzyme, hif prolyl hydroxylase (phd2 plant sulphite reductase (sir) forms an electron transfer complex with ferredoxin (fd) for the reductive conversion of sulphite to sulphide. although previous studies have highlighted electrostatic interactions between oppositely-charged residues of the two proteins, detailed thermoenergetics of the intermolecular interaction for the complexation remains unknown. we herein carried out isothermal calorimetry of fd:sir complex formation at various nacl concentrations. driving force plot constructed from calorimetry showed that the complex was thermodynamically stabilized by both enthalpy and entropy through favourable electrostatic and non-electrostatic interactions. increasing nacl concentrations weakened interprotein affinity and contribution of the negative enthalpy changes became decreased, while no such significant decrease was found in the contribution of positive entropy changes. furthermore, a negative heat capacity change obtained from the enthalpy changes at distinct temperature indicated a contribution of hydrophobic interactions. these findings suggested that both electrostatic and nonelectrostatic interprotein interactions were energetically important for the complex formation. fddependent sir activity assay revealed a bell shaped activity curve with a maximum under a certain nacl concentration, while the methyl viologen-dependent assay of sir exhibited a profile of saturating curve, suggesting that an optimized interprotein interaction is a crucial factor in control of fd-dependent-sir activity. a residue-based nmr measurement of 15n-labeled fd upon complex formation with sir revealed that charged and non-charged residues were differentially contributed in the complex formation depending on nacl concentrations. we proposed that non-electrostatic forces were also critical for forming the fd:sir complex, and an optimized complex conformation for maximum enzymatic activity was achievable by a delicate balance among non-covalent intermolecular forces. these results may be extended for understanding of complexation between redox proteins containing biased charge clusters. ornithine transcarbamylase has a spatially extended active site as computationally predicted lisa ngu 1 , kevin ramos 1 , nicholas delateur 1 , penny beuning 1 , mary jo ondrechen 1 1 understanding how an enzyme catalyzes a reaction is a fundamental problem in protein science. biochemical experimentation has revealed catalytic mechanisms of many enzymes; however these studies have focused almost exclusively on amino acid residues in direct contact with the reacting substrate molecule(s). here we report on the computational prediction and experimental verification of the importance of distal residues in enzyme catalysis, using e. coli ornithine transcarbamylase as an example. partial order optimum likelihood (pool), developed at northeastern university, is a machine learning technique that only requires the tertiary structure of a protein to predict important catalytic residues, based on computed, residue-specific electrostatic and chemical properties. pool has been shown to predict accurately the catalytic residues and to discern between compact and spatially extended active sites. dynamic conformational changes during catalysis and strong electrostatic interactions give rise to significant coupling between remote residues and the canonical active site residues of an enzyme. this suggests that at least some enzyme active sites are spatially extended, with second-and third-shell residues playing significant roles in catalysis. in this project, we focus on ornithine transcarbamylase (otc), for which dynamic processes are believed to play a role in its catalytic mechanism. otc is reported to undergo induced-fit conformational changes upon binding carbamoyl phosphate, which affects the subsequent binding of ornithine. residues predicted by pool to be catalytically important include five in direct contact with the substrate, r106, h133, d231, c273 and r319. pool also predicted remote residues to form a spatially extended, triple-layer active site. guided by computational predictions and using site-directed mutagenesis and kinetics assays of asp140, his272, glu299 and arg57 variants, we show that these pool-predicted remote residues, located in the second and third layers, are important for catalysis. alternative energy is a major focus of current research efforts. biodiesel, a mixture of fatty acid alkyl esters, is one of the most versatile alternative fuels currently in use. this is due to the fact that it is similar to gasoline and compatible with diesel engines found throughout the existing global infrastructure. biodiesel precursor lipids are abundant in cultivated feedstock organisms such as algae and bacteria. however, the standard process for converting oil to biodiesel is heat-intensive and requires complete removal of water, reducing the overall net energy gained in its production. our work constitutes an attempt to explore enzymatic synthesis of biodiesel from lipids such as those derived from emerging fuel crops. previous literature describes fatty acid alkyl ester formation in human patients with mrsa staphylococcus aureus wound lesions. these esters are formed by partially characterized esterase activity from an unidentified source. we have identified two mrsa enzymes responsible for this activity by using a combination of size exclusion chromatography, gas chromatography-mass spectrometry, and mass spectrometric protein sequencing. these two highly similar enzymes in the glycerol ester hydrolase (geh) family of proteins catalyze the synthesis of fatty acid alkyl esters in aqueous conditions at or near room temperature. we have demonstrated that other non-staphylococcal lipases do not exhibit this behavior. we have expressed these staphylococcal esterases in e. coli, and shown via gas chromatography that the expressed proteins catalyze the formation of fatty acid alkyl esters. based on sequence similarity to homologous proteins that have already been crystallized, we have predicted a structure for these enzymes and have engineered mutant fusions with higher rates of catalysis. our design hypothesis is that increased avidity for substrate molecules will yield a higher substrate concentration in the vicinity to the enzyme. to increase substrate concentration we have designed and expressed one of the enzymes as a chimeric fusion with the drosophila melanogaster alcohol-binding protein lush. gc-ms determination of biodiesel production rate indicates that the chimeric fusion has a lower-order rate constant with respect to ethanol. in other words, the fusion enzyme is less dependent on substrate concentration and is a superior catalyst at low ethanol concentrations. this result indicates that the rationally designed modification of binding avidity constitutes a potential avenue for improving the ability of enzymes to catalyze reactions with low-concentration or low-solubility substrates. functional elements of a human antizyme essential for binding and inhibiting human ornithine decarboxylase proteases are ubiquitous enzymes that catalyze the hydrolysis of peptide bonds within protein substrates; they have served as key model enzymes for studying the molecular basis for catalytic power and specificity. protease substrate specificity is most often defined in terms of linear sequence motifs that flank the cleavage site; however, the natural substrates of proteases are proteins with 3-dimensional shapes and complex conformational dynamics that are not well represented by 1-dimensional sequence alone. these structural and dynamical properties can impact recognition and binding of substrates by proteases, as well as the efficiency of catalysis itself. in this study, we explore the importance of substrate structure and dynamics for proteolysis using as our model the cleavage of the kunitz-bpti family of canonical serine protease inhibitors by mesotrypsin. bovine pancreatic trypsin inhibitor (bpti), an archetypal serine protease inhibitor of the kunitz family, has a high affinity interaction with trypsin, yet its peptide bond hydrolysis is many orders of magnitude slower than other peptide substrates. mesotrypsin, a trypsin variant, has been shown to hydrolyze kunitz family inhibitors at accelerated rates; this is especially true of human kunitz domain inhibitors. amyloid precursor protein inhibitor (appi) and amyloid precursor like protein-2 (aplp2), two human kunitz domain family members, are hydrolyzed by mesotrypsin several hundred times faster than bpti. here, we present a new, unpublished crystal structure of a cleavage intermediate aplp2 bound to mesotrypsin, refined to 1.4å resolution, revealing a dramatic substrate conformational change we hypothesize to be required during cleavage of a kunitz domain. using this structure along with published structures of appi and bpti complexes, we have modeled acyl-enzyme intermediates of mesotrypsin, and we have carried out molecular dynamic simulations that explore the transition of the initially formed native-like acyl-enzyme through the conformational transformation that allows the progression of the hydrolysis reaction. we further identify a specific hydrogen bond, present in bpti but not appi, which forms a stabilizing feature of the bpti scaffold. using site directed mutagenesis, we probe the contribution of this bond to the proteolytic stability of bpti. collectively our data for these highly structured substrates show that proteolysis rates are limited by a necessary conformational change in the substrate as the reaction progresses. rigid substrates possessing stabilizing features that render them highly resistant to this conformational change are proteolyzed more slowly than more flexible substrates of similar structure. lpmos are copper metalloenzymes that carry out the oxidative cleavage of the b-1,4-glycosidic bond, generating new chain ends that can subsequently be processed by cellulases, boosting the cellulose degradation. lpmos have a b-sandwich conformation with a flat binding surface, allowing for the enzyme to bind to crystalline cellulose. the cu21 ion, required for activity, is located in a so-called "histidine brace", in which the n-terminal histidine is highly conserved. regioselectivity according to the carbon atom being oxidized, 3 lpmo types are identified: type 1 and type 2 oxidizing at the c1 and the c4 respectively, type 3 lpmos oxidizing both the c1 and the c4 adjacent to the glycosidic linkage. we were able to express a type-1 lpmo (phanerochaete chrysosporium gh61d) and a type-3 lpmo (trichoderma reesei cel61a) in p. pastoris. this has proven to be very challenging, as lpmo activity requires a perfect cleavage of the signal sequence. after activity assays on pasc, characteristic hpaec-pad traces were obtained which will serve as a reference for engineering experiments. enzyme engineering using the 3dm database, a structure based multiple sequence alignment tool, it is possible to identify residues specifically conserved in subsets of protein sequences. by defining a subset for each lpmo type, we were able to identify residues contributing to regioselectivity. these positions are now being rationally engineered in subsequent rounds of mutagenesis, using trcel61a as a template. the effect of the mutations will be determined by analyzing the hpaec-pad trace released from pasc. the main goal is to investigate the possibility of deleting the c4 specificity in a type 3 lpmo. folding topology determines substrate binding order in the ribokinase superfamily alejandra herrera-morand e 1 , victor castro-fern andez 1 , madrid, españa ribokinase superfamily comprises three enzyme families: the adp-dependent sugar kinases family, the atpdependent coenzyme kinases family and the atp-dependent sugar kinases family. in all these families there is a large domain composed by a rossmann motif but only the atp-dependent enzymes have a b-meander motif in the c-terminal end. interestingly, these enzymes display an ordered kinetic mechanism where the substrate that will be phosphorylated binds first to the enzyme. the adp-dependent enzymes present a topological re-ordering of the secondary structural elements which produces an equivalent tertiary structure, which can be thought as a non-circular permutation (ncp) of the bmeander region. these enzymes also display an ordered kinetic mechanism but with an inversed order being the nucleotide the first substrate to bind to the enzyme. as this b-meander region of the proteins constitutes almost entirely the nucleotide binding site, and given that the permutation is the major structural difference between adp and atp-dependent kinases, it could the responsible for the nucleotide specificity. to test this hypothesis we introduce, by permutation, an atp-dependent topology in the homologous adp-dependent glucokinase from t. litoralis (pergk). size exclusion chromatography and circular dichroism spectra show that both the wild type and the permutated enzyme eluted as monomers with similar hydrodynamic behavior, and have the same secondary structure content. kinetic assays employing atp or adp as substrate demonstrate that even in the presence of 10 mm atp, the pergk enzyme is not able to carry out the phosphoryl transfer. to test if the ncp has an impact in the kinetic constants and substrate binding order we determine the kinetic mechanism through classical protocols, involving initial velocity studies, product inhibition and dead end inhibitors. the results demonstrate that the pergk enzyme presents an altered substrate binding order compared to the wild type enzyme, where glucose was the first substrate to bind to the enzyme and glucose-6-p the last product to be released. also, ligand-induced conformational changes were determined in the crystal structures. the apo, the enzyme-glucose and enzyme-glucose-adpbs structures were determined at 2.14 å, 1.95 å and 2.44 å resolutions, respectively. structure analysis reveals that glucose binding provokes major conformational changes in the pergk enzyme, whereas adp binding does not cause further changes in the conformation of the protein. the results show that although the permutation has no effect on the nucleotide preference it provokes a change in the substrate binding order that correlates well with that those observed in the crystal structures. also, they demonstrate that during the evolutionary history of the ribokinase superfamily folding topology dictates the substrate binding order (fondecyt 1150460). background: human ceruloplasmin (cp) is a circulating copper-containing glycoprotein produced in the liver and first described as a component of alpha2-globulin fraction of human plasma. cp belongs to the multicopper oxidase family and it is nowadays regarded as a "moonlighting" protein, because it changes its function according to substrate, localization and expression. cp plays a key role in copper transport and iron metabolism and it is also a potent inhibitor of leukocyte myeloperoxidase (mpo) (kd5130nm), a major source of oxidants in vivo. the protein is extremely susceptible to proteolysis. in fact, cp is a structural homolog of coagulation factors v and viii, that are physiological substrates of thrombin (fiia). interestingly, thrombin participates in both haemostatic and inflammatory responses: in some focus of inflammation, such as rheumatoid arthritis (ra), the high activity of fiia has been documented. it was demonstrated that fiia can promote the chemotaxis of neutrophils and monocytes and their adhesion to endothelial cells, to increase vascular permeability. all these effect are mediated by par-1 interaction, that are abundantly expressed in inflamed rheumatoid synovial tissues. aims: in this study the interaction of cp with thrombin was investigated to confirm the participation of fiia in "spontaneous" proteolytic degradation of cp. in fact, in vivo the integrity of cp is essential for its role in the transport or metabolism of copper. results: our results indicated that thrombin cleaves cp in vitro at 481arg-ser482 and 887lys-val888 bonds, generating a nicked species that retains the native-like fold and the ferroxidase activity of the intact protein, whereas the mpo inhibitory function of cp is abrogated. analysis of the synovial fluid of 24 ra patients reveals that cp is proteolytically degraded to a variable extent, with a fragmentation pattern similar to that observed with fiia in vitro, and that proteolysis is blocked by hirudin, a highly potent and specific thrombin inhibitor. we demonstrate that fiia has intrinsic affinity for cp (kd 5 60-270 nm), independently of proteolysis, and inhibits cp ferroxidase activity (ki 5 220 6 20 nm). mapping of thrombin binding sites with specific exosite-directed ligands (i.e. hirugen, fibrinogen gamma-peptide) and thrombin analogues having the exosites variably compromised (i.e. prothrombin, prethrombin-2, alpha-thrombin), reveals that the positively charged exosite-ii of thrombin binds to the negative upper region of cp, while the protease active site and exosite-i remain accessible. these results suggest that thrombin can exacerbate inflammation in ra by impairing via proteolysis the mpo inhibitory function of cp and by competitively inhibiting cp ferroxidase activity. an artificial pathway for isobutene production by direct fermentation: combining metabolic engineering and protein engineering benoit villiers 1 , franc¸ois stricher 1 1 the purpose of global bioenergies is to develop innovative metabolic pathways for the production of light olefins from renewable resources, by direct fermentation. light olefins (ethylene, propylene, linear butylene, isobutene and butadiene) are the core of the petrochemical industry. however, microorganisms do not naturally produce light olefins and no bioprocess to convert renewable resources to these molecules has been industrialized so far. global bioenergies has developed an artificial metabolic pathway including all the necessary enzymatic reactions from feedstock to isobutene. the metabolic route leading to isobutene can be divided in three parts, the first one being the use of natural reactions occurring in the host microorganism. second, heterologous natural reactions were introduced into the same host microorganism. finally, in contrast with most former approaches, non-naturally occurring reactions as enzymatic key steps were used, for example the decarboxylation of hydroxyisovaleric acid into isobutene. such non-natural critical steps were made possible by taking advantages of the natural catalytic and substrate promiscuity of exogenous enzymes. candidate enzymes are then evolved using systematic, random and semi-rational approaches in successive rounds in order to reach the desired catalytic efficiency. since all these reactions are enzymatic, isobutene can be obtained by direct fermentation, e.g. a process wherein all the chemical transformations are carried on by the host microorganism. the scale-up of this process began in november 2014 in a pilot plant installed in pomacle-bazancourt, france, with an annual capacity of 10 tons of oxidation-grade isobutene. importantly, production of a volatile compound such as isobutene (and other light olefins) by direct fermentation presents two major advantages: first, the product is spontaneously removed from the culture broth, which alleviates the limitations linked with titer issues. second, the purification process is considerably easier and cheaper since no energy consuming methods such as distillation or phase separation are necessary to purify the end product. for the first time, batches of industrially produced isobutene from renewable resources have been obtained in the first half of 2015. this isobutene has been in turn converted into isooctane, an additive currently used to improve gasoline quality, which could also be used as a standalone fuel. a demonstration plant is planned in leuna, germany, with an annual capacity of 100 tons of polymer-grade isobutene and ibn-one, a joint venture with cristal union (4th european beet processor), has been formed to build and operate the first plant in france converting renewable resources into isobutene. finally, while the isobutene process is progressing towards industrial scale, global bioenergies is also developing new artificial metabolic pathways enabling direct bio-production of butadiene and propylene. the development of a coupled enzyme assay to detect isochorismate pyruvate lyase activity protein folding is typically defined in terms of the spatial arrangement of structural elements, i.e. helices, sheets and loops. we have, however, been developing an alternative and complementary paradigm based on conserved hydropathic interaction networks within proteins. these networks can be viewed as environments comprised of a mixture of polar and hydrophobic interaction fields, and may be the most important factor driving protein folding. this concept applies even to the lowest structural level within a protein: the sidechain conformations (or rotamers). exhaustive statistical analysis of existing crystallographic structures of proteins showed rotameric preferences and led to the creation of rotamer libraries frequently used in multiple aspects of structural biology, e.g., crystallography of relatively low-resolution structures, homology modeling and biomolecular nmr. however, little is actually known about the forces and factors driving the preference or suitability of one rotamer over another. in our study, tyrosine was analyzed since its sidechain has a comprehensive set of hydropathic properties that made it ideal as a proof of concept residue. construction of 3d hydropathic interaction maps of tyrosine residues in our dataset, reveals the environment around each, in terms of hydrophobic (p-p stacking, etc.) and polar (hydrogen bonding, etc.) interactions. after partitioning the tyrosines into backbonedependent bins, a map similarity metric based on the correlation coefficient was applied to each mapmap pair to build matrices suitable for clustering. notably, the first bin representing 631 tyrosines, reduced to 14 unique hydropathic environments with most diversity arising from favorable hydrophobic interactions with many different residue partner types. polar interactions for tyrosine include ubiquitous hydrogen bonding with the phenolic oh and somewhat surprisingly a handful of unique environments for the tyrosine backbone. all but one of the 14 environments are dominated by a single rotamer, the exception being an environment defined by a paucity of interactions with the tyrosine ring and as a consequence its rotamer is indeterminate. this is consistent with it being composed of mostly surface residues. each tyrosine residue attempts to fulfill its hydropathic valences and thus, structural water molecules are seen in a variety of roles throughout these environments. alanine was analyzed using the same protocol as well. having the smallest sidechain (and small hydropathic interaction maps), alanine allowed us to investigate a significantly larger database, permitting us to examine the correlation between hydropathic maps and various structural features. in conclusion, the analysis of hydropathic environments strongly suggests that the orientation of a residue in a three-dimensional structure is a direct consequence of its hydropathic environment, which leads us to propose a new paradigm, interaction homology, as a key factor in protein structure. it is not the surrounding residues that direct sidechain conformations, but rather the hydropathic "field" of the surrounding atoms. folding studies of independent domains of lysine, arginine, ornithine binding protein (lao) protein folding problem has been addressed from the past 50 years until nowadays, however, we still can not explain how proteins acquire their native structure from their amino acid sequence. different approaches has been taken in order to study protein folding, for example, the comparative study of folding mechanism between homologues proteins with high identity of sequence and structure, and the study of independent regions within a single protein. previously in our laboratory, thermodynamic and kinetic folding properties of lysine, ornithine, arginine binding protein (lao), a 238 amino acid periplasmic binding protein (pbp), composed by two rossmann fold domains (one continuous and the other discontinuous) attached by a hinge region, has been studied. even there is a functional research about binding characteristics of histidine binding protei ns (his j) domains of when expressed independently (chu, b. 2013 ); there are no folding studies in these conditions for this or another pbps. it should be noted that his j shares 70% of sequence identity and tertiary structure (rmsd 1å) with lao. in order to know the folding effect of encoding different domains in the same poly peptidic chain, as well as its influence in function, we are studying the thermodynamic and kinetic characteristics of folding of independently expressed lobes of lao, and comparing with those of native protein. by now, we expressed and purified the discontinuous domain. circular dichroism (cd) and fluorescence intensity spectra show that this independent domain has primary and tertiary structure. thermal denaturation has a single cooperative transition, which indicates this domain is folded. thermodynamic analysis of temperature and urea-induced experiments suggest that lao's folding characteristics are not just the addition of those from independent domains. furthermore, folding and refolding kinetics suggest the presence of a burst phase intermediate. a hypothesis to reconcile the physical and chemical unfolding of proteins a comprehensive view of protein folding is crucial for understanding how misfolding can cause neurodegenerative diseases and cancer. when using physical or chemical perturbations, nmr spectroscopy is a powerful tool to reveal a shift in the native conformation toward local intermediates that act as seeds for misfolding. high pressure (hp) or urea is commonly used to disturb folding species. pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the proteinsolvent system. however, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though protein-urea interactions are considered to be crucial. here, we provide nmr spectroscopy and 3d reconstructions from x-ray scattering to develop the "push-and-pull" hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by hp. in studying mpnep2 from moniliophthora perniciosa, we tracked two cooperative units using hp-nmr as mpnep2 moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. at subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by nmr intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle x-ray scattering (saxs)]. this state has a higher susceptibility to pressure denaturation (lower p1/2 and larger dvu); thus, urea and hp share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. these observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes. zinc: a promoter or inhibitor for iapp aggregation? feng ding 1 , praveen nedumpully-govindan 1 1 zinc ions have been found to play an important and yet complex role in human islet amyloid polypeptide (hiapp) aggregation, which is associated with b-cell death in type-ii diabetes (t2d). both concentration-dependent promotion and inhibition of iapp aggregation by zinc ions have been observed in vitro. similarly, at the population level, both positive and negative correlations were reported between the activity of a b-cell specific zinc transporter and t2d risk. zinc ions are able to bind a single histidine in hiapp and coordinate the formation of zinc-bound hiapp oligomers. we hypothesize that the relative zinc/hiapp concentration determines the population of zinc-bound hiapp oligomers with different molecular weights. we have applied molecular dynamics (md) simulations to systematically study the structure and dynamics of a range of zinc-coordinated hiapp oligomers, including monomers, dimers, trimers, tetramers, and hexamers. our computational results suggest that different zinc-bound oligomers have distinct aggregation propensities. high-molecular weight oligomers (2 peptides) have higher aggregation propensity than zinc-free and zinc-bound hiapp monomers at 2 mm concentration in silico. therefore, our results provide a molecular insight into the complex role of direct zinc binding on hiapp aggregation. at low zinc/hiapp stoichiometry, zinc binding promotes aggregation. as the stoichiometry increases and zinc ions bind to single hiapp peptides, the aggregation of hiapp is inhibited due to electrostatic repulsion between the charged zinc ions. our computational study sheds light on the complex role of zinc on hiapp aggregation and t2d development. biomolecules function in the densely crowded and highly heterogeneous cell, which is filled up to a volume of 40% with macromolecules [1] . often, artificial macromolecular crowding agents are used to mimic these conditions in vitro and the excluded volume theory is applied to explain the observed effects [2] . however, recent studies emphasize the role of further contributions aside from a pure volume effect including enthalpic and solvent effects [3, 4] . we study cosolute effects at high molecular and macromolecular concentrations via a thermodynamic analysis of the thermal unfolding of ubiquitin in the presence of different concentrations of cosolutes (glucose, dextran, polyethylene glycol, potassium chloride) [5] . in contrast to the excluded volume theory, we observed enthalpic stabilization and entropic destabilization forces for all tested cosolutes. the enthalpic stabilization mechanism of ubiquitin in macromolecular polysaccharide solutions of dextran was thereby similar to the effects observed in monomeric glucose. further, it remains unclear how such cosolutes reflect the physicochemical properties of the complex cell environment as a characterization of the in-cell crowding effect is lacking. thus, we developed a fret-based macromolecular crowding sensor to study the crowding effect in living cells [6] . the averaged conformation of the sensor is similar to dilute aqueous buffer and cell lysate. we find that the in-cell crowding effect is distributed heterogeneously and can change significantly upon osmotic stress. the presented method allows to systematically study in-cell crowding effects and understand them as a modulator of biomolecular function. the stability of biomolecules under co-solvent conditions is dependent on the nature of the co-solvent [1] . this can alter a protein's properties and structural features through biomolecular interactions between its functional groups and the co-solvent molecules. ionic liquids (ils) represent a rather diverse class of co-solvents. the design flexibility of these molten salts is an attractive feature, allowing the properties of the il to be tuned to meet the requirements of different applications [2] . particularly, the modulation of reaction pathways between folding states, offering possibilities to control irreversibility in non-native protein aggregation [2] . this has led us to investigate the impact of ils as co-solvents with the well-known protein denaturant urea. urea is considered to be a non-ionic chaotrope disturbing considerable the grid of hydrogen bonds with the protein backbone. urea interacts preferentially with the protein surface, mainly apolar residues and that dispersion, rather than electrostatic interactions, is the main energetic contribution to explain the stabilization of the unfolded state of the protein and the irreversibility of the unfolding process in the presence of urea [3] . a large body of multidomain protein folding work has been devoted to study monomeric proteins. how do multidomain multimeric protein fold, avoiding accumulation of stable intermediate is yet to be studied in detail. our present study is focussed on understanding the folding and assembly of the domains of a homodimeric l-aspraginase from a hyperthermophile pyrococcus furiosus (pfa). each monomer of pfa consists of distinct n-and c-terminal domains (npfa and cpfa, respectively), connected by a linker. the folding mechanism of each domain with respect to full length protein was studied by mutating one out of two tryptophans, one in each domain. domains were purified and studied individually to obtain parallel account of the folding of each domain in isolation. subunit assembly was studied by analytical size exclusion chromatography (sec), multiangle light scattering and functional activity. through far uv cd, intrinsic trp fluorescence and sec, we demonstrated that domain folding and subunit association were intimately linked in full length pfa. interestingly, en route to its folding there was complete absence of hydrophobic intermediates as probed by ans fluorescence. folding of npfa was highly cooperative and, it provides interacting surfaces for cpfa to fold and also facilitates subunit assembly. the folding cooperativity of isolated domains was very less compared to the folding cooperativity of their full length counterparts, as indicated by equilibrium m values. to our surprise, during ph induced denaturation, at ph 2 and 13, the dimer dissociates into highly hydrophobic folded monomers which readily underwent amyloidogenesis. we showed that at such extreme conditions, cooperativity in folding process in multidomain multimeric protein is not solely governed by the folding of individual domains, rather by concomitant folding and association of domains directly into a quaternary structure. in other case, where subunit folding occurred prior to association, protein readily underwent extensive aggregation. groel assisted folding of multiple recombinant proteins simultaneously over-expressed in e.coli megha goyal 1 , tapan kumar chaudhuri 1 1 aggregation prone recombinant proteins very often form inclusion bodies and also exhibits poor yield of functional protein during in vitro refolding process from chemically denatured form. bacterial chaperonin groel provides folding assistance to several proteins, when over-expressed with one of the recombinant proteins. there are instances that groel in presence of few other co-expressed chaperones like dnaj, dnak etc provides better yield of folded protein during homologous and heterologous expression. considering the ongoing events in the cells, it is known that molecular chaperone groel assists in the folding of various proteins in the cytoplasm. hence attempt to fold multiple recombinant proteins over-expressing simultaneously with the co-expression of chaperones can be worth trying. this approach may cut down various complexities in the functional recombinant protein preparation, including time and effective cost. keeping this view in mind, folding of two simultaneously expressed aggregation prone proteins, 69 kda e.coli maltodextrin glucosidase (malz) and 82 kda yeast mitochondrial aconitase have been investigated with the co-expression of groel and groes in e.coli cytosol. it has been previously reported that both the chosen proteins undergo co-expressed groel-groes assisted folding in e.coli cytosol, when they over-express alone. in this study we have optimized the overexpression of malz and aconitase simultaneously in e.coli. further optimisation was carried out to coexpress groel along with malz and aconitase. based on the basic philosophy that soluble protein mainly contains folded fraction, the event of groel/es assisted folding of simultaneously overexpressed proteins, malz and aconitase was monitored through the attainment of soluble proteins under various sets of conditions such as temperature. the major outcome of the present study is that, with the groel-groes assistance, the yield of soluble proteins (malz and aconitase) together constitutes higher percentage of folded protein in contrast to the percent yield when a single protein was overexpressed. significance of this type of study relies on the fact that the cells can over-produce higher amount of recombinant proteins, when multiple over-expression takes place. not only pushing up cell's capability of over-expression, co-expression of groel and groes efficiently assists in the folding of multiple proteins simultaneously over-expressed in e.coli. amyloid fibrils associated with serious diseases including alzheimer's, parkinson's, and prion diseases promoted the challenge of studying protein misfolding, leading to the development of amyloid structural biology. amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. here, with b2-microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (itc). the spontaneous fibrillation after a lag phase was accompanied by exothermic heat. the thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. we also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. in order to obtain general thermodynamic properties of protein aggregations, we further investigated aggregation of glucagon and insulin, two of the most famous amyloidogenic peptide hormones, using itc. we also observed characteristic heat of spontaneous amyloid fibrillation of both proteins after a lag time. taken all together, we showed that thermodynamic studies on amyloid fibrillation and amorphous aggregation were indeed possible by means of itc-based qualitative and quantitative calorimetric analyses. itc will become a promising approach for clarifying the thermodynamic properties of protein aggregates. the more case studies are required toward the establishment of thermodynamics of protein misfolding and aggregation when hydrophobic proteins are, for any reason, exposed to the cytosol they are rapidly captured by protective complexes which shield them from the aqueous surroundings and decide their fate (by either targeting them to their correct membrane homes or marking them for degradation by the ubiquitin/proteasome system). the bag6 holdase is a heterotrimeric protein complex, comprising bag6, ubl4a and trc35, which works closely with the cochaperone sgta to triage hydrophobic proteins and pass them along the appropriate pathway. sgta also interacts with viral proteins and hormone receptors and is upregulated in numerous cancer types. these functions require further investigation to determine the scope of sgta as a therapeutic target. our lab has solved the solution structure of the n-terminal dimerization domain of sgta and characterised its interaction with two different ubiquitin-like (ubl) domains in the bag6 holdase (one from ubl4a and the other from bag6 itself) using nmr chemical shift perturbation data and other biophysical techniques including isothermal titration calorimetry and microscale thermophoresis. at this meeting i will report on the progress we have made in structurally characterising further key players that participate in this quality control, with the aim of clarifying the intricate network of molecular interactions that governs these processes in health and disease. ensemble, ribbon and electrostatics spacefill views of the sgta dimerization domain structure. the final panel shows the structure overlaid with its yeast homologue. alpha synuclein is a small protein (14 kda) expressed at high levels in dopaminergic neurons. fibrillar aggregates of a-synuclein inside the dopaminergic neuron are the major components of lewy bodies and lewy neuritis inclusion, which are considered as potential hallmark of parkinson's disease (pd). both in vitro as well as in vivo studies suggest that the soluble, oligomeric forms of a-syn are the more potent neurotoxic species, responsible for neuronal injury and death in pd. therefore, molecules that inhibit the toxicity of oligomers either by reducing their formation or by converting their more toxic oligomeric state to less-toxic fibrillar state would be effective agents for the drug development against pd. curcumin is one of the asian food ingredients which has shown a potential role as therapeutic agent against many neurological disorders including pd. however, the instability and low solubility makes it less attractive for use as potential therapeutic agent. the present work focuses on screening of the compounds similar to curcumin but having better effects on the morphology and toxicity of oligomeric and fibrillar assemblies of a-syn, which could be used as therapeutic agent preferentially over the naturally occurring curcumin. we synthesized and analyzed the effects of nine compounds, which are structurally similar to curcumin, on different stages of a-syn amyloid aggregation. here, we showed that curcumin and its analogs accelerate a-syn aggregation to produce morphologically different amyloid fibrils in vitro. however, there is no significant effect of curcumin and its analogs on the secondary structure of preformed a-syn fibrils. furthermore, these curcumin analogs showed differential binding affinities with the preformed a-syn aggregates, possibly due to difference in their chemical structures. the present data suggest the promising role of curcumin analogs in the treatment of a-synucleinopathy disorders. in vitro folding mechanisms determine the forces applied during co-translational folding there is currently much debate as to whether experiments conducted in vitro describe the folding of proteins in vivo. in particular, it is often suggested that the co-translational folding of nascent protein chains is dominated by the presence of the ribosome and associated chaperones, and that folding mechanisms will be affected by the vectorial nature of translation. here we use an arrest peptide assay to investigate the co-translational folding of a number of all-a spectrin domains that exhibit a range of thermodynamic stabilities and in vitro folding rates. our unexpected finding is that that the force exerted on the ribosome by these domains is not related to either the thermodynamic stability of the domain, or to the folding (loading) rate, but rather to the in vitro folding mechanism. we infer that the in vitro folding mechanisms of these domains are unaffected by the presence of the ribosome -even when part of the nascent chain is retained within the ribosome exit tunnel. there has been much work to date investigating the intermediates present in stalled translation complexes -but now, for the first time, we can begin to directly explore the rate limiting transition state in the co-translational folding of homologous proteins. can the structure of a protein (h3.1) depend on the treatment of a solvent medium (explicit vs effective) in a coarse-grained computer simulation? ras pandey 1 , barry farmer 2 1 university of southern mississippi, 2 air force research laboratory solvent medium plays a critical role in orchestrating the structure and dynamics of a protein. in computer simulation modeling of protein structure in a solvent medium, explicit, implicit, effectivemedium, approaches are often adopted to incorporate the effects of solvation. because of the complexity in incorporating all atomic and molecular details, the multiple components, reaching the large-scale, etc. implicit solvent or effective medium approach is generally more viable than the explicit solvent methods. some of the pertinent characteristics such as excluded volume of the solvent constituents, its concentration, and the underlying fluctuations which may be important in probing some issues are generally ignored in effective medium or implicit solvent approaches. using a coarse-grained approach, we investigate the structure and dynamics of a protein (a histone, h3.1) in the presence of both effective as well as explicit solvent media over a range of temperatures with the monte carlo simulations. the protein is represented by a coarse-grained chain of residues whose interactions are described by knowledge-based residue-residue and hydropathy-index-based residuesolvent interactions. in effective medium approach, each empty lattice site around the protein structure acts as a solvent. only a fraction of lattice sites are occupied by mobile solvent constituents along with the protein chain in explicit solvent medium. large scale simulations are performed to analyze the structure of the protein for a range of residue-solvent interactions and temperature in both explicit and effective solvent media. we study a number of local (e.g. solvation and mobility profiles) and global (radius of gyration and structure factor) physical quantities as a function of temperature. we find that the response of the radius of gyration of the protein in explicit solvent is different from that in effective medium solvent. thus, the presence of fluctuations in explicit solvent approach have considerable effects on the structure and dynamics of protein h3.1. differences due to type of solvent on the response of some of these quantities as a function of temperature as well as general similarities will be presented. single-molecule vectorial folding and unfolding through membrane pores david protein folding and unfolding in vivo is frequently vectorial. for example, proteins are synthesized at the ribosome and emerge n-terminal first. as the polypeptide chain emerges from a 2 nm wide pore is free to fold, interact with partners or misfold1. in another example, proteins are unfolded at the proteasome by pulling from either the n or c terminus against a 1-2 nm wide pore, applying a tension on the residues surrounding the terminus of the protein2. under this conditions, proteins may behave differently than when unfolded/refolded with temperature or urea. this may have important implications, as protein folding and unfolding in vivo is related to both function and disease. we noticed that vectorial folding is inherently linked to nanometer size pores. making use of nanopore technology we developed a method to monitor protein unfolding during membrane translocation at the single-molecule level3. briefly, an oligonucleotide attached at either end of a protein threads a single protein nanopore inserted in a lipid membrane. in response to an applied membrane potential, the oligonucleotide pulls the protein through the pore and as it is forced to translocate it unfolds. analysing the ionic current we obtain the unfolding pathway and information on the polypeptide sequence. this methodology has shown that proteins unfold with different kinetics when pulled from one terminus or the other4. remarkably, it is also possible to say whether the protein has been phosphorylated or not, and where5. we have recently advanced our model system to study protein folding after translocation at the singlemolecule level6. a single-protein molecule was translocated through a pore and forced to translocate back at predetermined times. we measured the stability of the refolded state at different times and we obtained the vectorial folding pathway of the protein. further, we observed that the protein was capable of co-translocational folding and that this premature folding contributed to the complete translocation of the protein. our results show that nanopore technology applied to proteins can be used to describe the vectorial folding and unfolding of proteins, providing insight to how these processes may work in vivo. further, single-molecule protein sequencing is a possibility that could revolutionise our knowledge on biological processes. thermodynamics studies of oligomeric proteins, which are the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. there is not a single report on the reversible equilibrium thermal unfolding of proteins composed by (b/a)8 barrel subunits, albeit this "tim barrel" topology is one of the most abundant and versatile in nature. the eponymous tim barrel, triosephosphate isomerase (tim) is a ubiquitous glycolytic enzyme that catalyzes the isomerization of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. the unfolding of several tims, mainly of eukaryotic organisms, has been extensively studied. regarding thermal unfolding, eighteen tims, mainly from eukaryotes, as diverse as amoebozoa, euglenozoa, ascomycota and chordata, have been studied. even though a full thermodynamic characterization has been hampered by irreversible aggregation and/or the presence of hysteresis in all of them, the activation parameters that describe the kinetic control of five eukaryotic tims have been reported. we characterized the structure, catalytic properties, association state and temperature-induced unfolding of the eponymous tim barrel, triosephosphate isomerase (tim), belonging to five species representative of different bacterial taxa: deinococcus radiodurans (drtim), nostoc punctiforme (nptim), gemmata obscuriglobus (gotim), clostridium perfringens (cptim) and streptomyces coelicolor (sctim). irreversibility and kinetic control were observed in the thermal unfolding of nptim and gotim, while for drtim, sctim and cptim, the thermal unfolding was found to follow a two-state equilibrium reversible process, a behavior not observed previously for others tims. shifts in the global stability curves of these three proteins are related to organismal temperature range of optimal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. reversibility appears to correlate with low isoelectric point, the absence of residual structure in the unfolded state, small cavity volume in the native state structure, low conformational stability and a low melting temperature. furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce the possibility of aggregation and favor reversibility. it appears that there is a delicate balance between several contributions whose concerted interplay is necessary to achieve thermal reversibility in oligomeric enzymes. furthermore, the finding that the three reversible proteins come from organisms from different phyla suggests that unfolding reversibility may be more common than what is currently known supported by a critical step in the late phase of human immunodeficiency virus type 1 (hiv-1) infection is targeting of the virally encoded gag proteins to the plasma membrane (pm) for assembly. prior to assembly, the hiv-1 gag polyprotein adopts a compact "folded over" conformation and exists in the monomeric or low-order oligomeric states. whereas it is established that the nucleocapsid domain of gag specifically recognizes motifs in the viral rna genome for packaging, there is compelling evidence that the myristoylated matrix (ma) domain also binds to cellular rna to prevent premature gag targeting to intracellular membranes. upon transport of gag to the pm, the interaction of ma with rna is exchanged for an interaction of ma with pm components. this molecular switch induces an extended conformation of gag, leading to formation of high-order gag oligomers on the pm. because gag is anchored and therefore captured by its interaction with the available phospholipids, the intracellular targeting of gag is likely to be determined by the relative strength of its interaction with the dominant lipids composing each membrane subcompartment. the key to understanding this essential molecular switch is elucidating at the molecular level the interaction of ma with specific pm components. for over two decades, biochemical, in vivo, in vitro and genetic studies have focused on factors that modulate binding of retroviral gag proteins to membranes but only recently the structural and molecular determinants of gag assembly have begun to emerge. in addition to the electrostatic interactions between a highly conserved basic region of ma and acidic phospholipids, it is now believed that the hydrophobicity of the membrane interior represented by the acyl chains and cholesterol also play important roles. we employ nmr methods to elucidate the molecular determinants of gag binding to the membrane. our structural studies revealed that phosphatidylinositol-4,5-bisphosphate (pi ( the production of functionally antibodies depends on the transition of immature b cells to mature plasma cells and is tightly linked to several "quality control" check points. during b cell development, the pre-b cell receptor (pre-bcr) is the first checkpoint which determines the viability and proliferation of the pre-b cell. the pre-bcr is composed of an immunoglobulin (ig) heavy chain molecule associated with an ig light chain-like molecule called the surrogate light chain (slc). the slc is composed by two proteins k5 and vpreb which possess a unique region at the n-or c-terminus, respectively. vpreb lacks a b-strand which is provided by the k5 protein allowing the non-covalent interaction essential for formation of the slc heterodimer. our understandings of the molecular mechanism of slc function and assembly are still at an early stage. in particular, we do not know how the slc associates and forms the pre-bcr for the selection of all heavy chains (hcs). our study focuses on dissecting the "fab fragment" of the pre-bcr to study the effect of the unexpected structural features of the slc to gain insight in hc selection. the analysis of the assembly of the slc revealed a significant difference between the single domains and the complexes in terms of stability and assembly. the folding behavior of the ch1 domain in the presence of the slc is key for the first quality control mechanism in the endoplasmic reticulum (er) prior to surface expression. our results show that the slc interacts with ch1 domain in a similar manner to the cl domain. thus, the folding of the naturally disordered ch1 domain upon interaction with the slc releases the hc retention in the er by bip. taken together, our study provides new insights into the folding and assembly of the "fab fragment" of the pre-bcr and paves the way for a detailed mechanistic understanding of hcs selection by the unique slc. though the 22-29 (sfgailss) region of human islet amyloid polypeptide (hiapp) has long been known to be crucial for amyloid fiber formation, lack of b-ordering of this region in structures of the final fiber as determined by both nma and x-ray has been puzzling. new evidence now suggests that the fgail region forms ordered b structures only in early intermediates. we present new 2dir studies on the fgail region of hiapp, with uniformly 13c 18o labeled amides, along with spectral and kinetic modelling. evolution of the peak frequency and 2d lineshape of the labeled region clearly present a transition from random coil to a stable b sheet, a conclusion which is substantiated by simulation of the 2d ir spectra. as determined from kinetic modeling, the fgail b-sheet creates a free energy barrier that is the cause of the lag phase during aggregation. these findings help to rationalize a broad range of previous fragment and mutation studies as well as provide a mechanism for fiber formation that has self-consistent kinetics and structures. the temperature dependence of protein stability in living cells studies addressing the consequence of crowding that exist in the interior of cells have reached an interesting stage. experimental data so far, predominantly from, small to medium sized proteins are indicating that, in general, natively folded proteins including, intrinsically disordered, gain structure and stability under conditions mimicking cell interior. however, on the other hand, a few studies on small proteins indicate destabilization of the native state. in very few instances, crowding resulted in compaction and aggregation of the unfolded and partially folded states. experimental data on the consequences of cell-like crowding situation on relatively large proteins with complex folding free energy landscape are absent. alpha subunit of tryptophan synthase, a 29 kda tim barrel protein, provides a unique opportunity to address the consequence of crowding on the structure and stability of the native state and also on a partially folded state stable equilibrium intermediate populated in its (un)folding reactions. in the presence of increasing amounts the most commonly used crowding agent, ficoll-70, a non-monotonous increase in the far uv-cd is observed for the native state. a steady increase up to 250mg/ml ficoll followed by a decrease in far-uv cd region is observed, indicating loss of structure at increased concentrations of the crowding agent. 1h-15n hsqc nmr and fluorescence (fl) spectra confirm the of loss of structure at higher concentrations of ficoll-70. loss of native base line in the urea induced unfolding reaction monitored by cd and fl clearly confirms the destabilization of the native state. similar to the structural changes observed for the native state, for the equilibrium intermediate state maximally populated at 3 m urea also, non-monotonous changes in the far uv cd and fluorescence spectra are observed. the highly populated equilibrium intermediate shows an initial steady increase in the far uv cd signal followed by a sudden decrease. our results suggest that the structure of both native and partially folded states may be affected under crowding conditions. alpha-1 antitrypsin (aat) is a 44-kda serine protein inhibitor (serpin), which acts as an inhibitor of neutrophil elastase within the lungs. during inhibition, the protein undergoes a dramatic conformational change in which its exposed reactive centre loop (rcl) is cleaved and inserts into the central a-sheet as an extra beta-strand. this highly dynamic protein is also susceptible to mutations, resulting in misfolding and the accumulation of ordered polymers as intracellular inclusions within the endoplasmic reticulum of hepatocytes, where aat is synthesized. despite much knowledge of the folding and misfolding properties of aat as an isolated protein, very little is understood of how aat acquires its structure during biosynthesis. like all proteins, the biosynthesis of aat takes place on the ribosome, and protein folding occurs in a co-translational manner as the nascent polypeptide chain emerges from the ribosome's exit tunnel. this study aims to develop the biochemical and nmr structural strategies to characterize the co-translational folding characteristics of aat as it is being synthesized on the ribosome. for these studies, we have designed a series of secm-stalled ribosome nascent chain complexes (rnc) of aat of different lengths, which mimics the "snapshots" of the protein synthesis, capturing the folding process of the nascent chain during its emergence from the ribosome. using this library, we have recently developed a strategy to produce large quantities of the rncs both in vitro and in vivo within e. coli, a prerequisite for detailed biochemical and structural studies. using the aat-rncs, we are developing a suite of biochemical strategies to probe the capacity for aat nascent chains to adopt native structure on the ribosome. we have combined protease inhibition assays, western blot and native-page analysis to demonstrate that aat can fold while bound to the ribosome. in addition, we have employed a cysteine-based modification "pegylation" assay to probe lowresolution structural information of aat-rnc and this will guide our structural studies by nmr spectroscopy to provide a detailed understanding of aat folding on the ribosome at high resolution. thermodynamic properties of proteins vary with the environmental solvent condition (temperature, ions, ph, denaturants, etc.). although the effect of each environmental factor on proteins has been well studied, the complex effect of more than two environmental factors was not studied thoroughly. in this study, we investigate the simultaneous effect of urea denaturation (disruption of non-covalent bonds in proteins) and acid denaturation (titration of protein residues) on the nature of the folding transition for 2cyu protein. we performed the molecular dynamics simulations of bbl (pdb code: 2cyu) protein in various urea concentration at 300k. we calculated ph-dependent free energy landscape using the extended munoz-eaton model and described the phase diagram for the folding transition of bbl at various ph value and urea concentration. we mapped out the phase diagram of the folding transition of 2cyu, which clarifies the condition with which it undergoes the cooperative folding transition or the barrierless folding transition. biophysical analysis of partially folded states of myoglobin in presence of 2,2,2-trifluoroethanol paurnima talele 1 , nand kishore 1 1 the protein folding process involves one or more distinct populated intermediates. one such partially folded structure of particular importance observed during protein folding pathway is molten globule state. the properties of a molten globule state are intermediate between those of native and unfolded protein molecules. the importance of studying equilibrium molten globule is in its greater stability and flexible structure which has been shown to bind a variety of substrates and play a definite role in certain human diseases via aggregation, misfolding or some other mechanism. a protein must assume a stable and precisely ordered conformation to perform its biological function properly. the stability of a protein under specific conditions depends on its interactions with the solvent environment. therefore it is essential to understand protein folding intermediates, protein solvent interactions and protein stabilization. we have made attempts to thoroughly investigate the formation of stable molten globule state of the protein induced by alcohol using combination of calorimetric and spectroscopic techniques. the presentation will cover the topic on biophysical studies on partially folded states of myoglobin in presence of 2,2,2-trifluoroethanol. the thermal denaturation of myoglobin was studied in the presence of 2,2,2-trifluoroethanol (tfe) at various ph values using differential scanning calorimetry and uv-visible spectroscopy. the most obvious effect of tfe was lowering of the transition temperature with increasing concentration of tfe up to 1.5 mol•dm-3, beyond which no thermal transitions were observed. the conformation of the protein was analyzed by a combination of fluorescence and circular dichroism measurements. at ph 5.0 and 11.0, partially folded states of myoglobin were confirmed by cd spectroscopy. quantitative binding of ans to the tfe induced molten globule state of myoglobin was studied by using isothermal titration calorimetry (itc). the results enable quantitative estimation of the binding strength of ans with the molten globule state of myoglobin along with the enthalpic and entropic contributions to the binding process. the results also suggest occurrence of common structural features of the molten globule states of proteins offering two types of binding sites to ans molecules which has been widely used as a fluorescence probe to characterize partially folded states of proteins. modules. each cbr comprises a b-hairpin core followed by a short linker sequence. choline molecules are bound between two consecutive repeats through hydrophobic and cation-p interactions with aromatic side chains. apart from its biotechnological applications as an affinity tag for protein immobilization and purification, clyta is useful as a model for understanding the folding and stability of repeat proteins. in this sense, we proposed to get minimal peptides encompassing the sequence of a single cbr or even only its b-hairpin core able to maintain the native fold and the ability to bind choline. to that end, we first proceeded to analyze the peptide comprising the third b-hairpin core, denoted as clyt3. based on cd and nmr data we demonstrate that the peptide clyt3 conserves its native bhairpin structure in aqueous solution, but forms a stable, amphipathic a-helix in detergent micelles and as well as in small lipid vesicles [1] . considering the great differences in the distribution of hydrophobic and polar side chains shown by clyt3 b-hairpin and a-helix, we propose that amphipathic structures are stabilized in micelles or lipid vesicles. this "dual" behavior is the only up-to-now reported case of a micelle-induced conformational transition between two ordered peptide structures. to check whether other cbr repeats also undertake b-hairpin to a-helix transition in the presence of micelles, so that it represents a general tendency ascribed to all pneumococcal choline-binding modules, we will show new experimental evidences based on cd and nmr structural studies on peptides derived from the bhairpin cores of other clyta repeats, as well as in modified clyt3 peptides. continuing our studies of the effect of like-charged residues on protein-folding mechanisms, in this work, we investigated, by means of nmr spectroscopy and molecular-dynamics simulations, two short fragments of the human pin1 ww domain [hpin1(14-24); hpin1(15-23)] and one single point mutation system derived from hpin1(14-24) in which the original charged residues were replaced with non-polar alanine residues. results, for both original peptide fragments of hpin1 demonstrate the presence of ensembles of structures with a tendency to form a b-chain reversal. understanding the biology of huntington's disease via the pathogenic huntingtin monomer huntington's disease (hd) is caused by an abnormal extension of the polyglutamine (polyq) region within exon 1 of the protein huntingtin from typically 25 glutamines to over 36. disease onset correlates with the huntingtin misfolding and causing the formation of aggregates, however recent studies have postulated that pathogenic huntingtin monomer may form compact structures that are responsible for neuronal toxicity in hd. we sought to examine the conformation of huntingtin monomers, how polyq sequence length affects monomer structure and which protein-binding partners in the cell may exert a gain-of-toxic mechanism in pathology. hydrogen-deuterium exchange mass spectrometry was used to measure the degree of structure in both non-pathogenic (25q) and pathogenic (46q) huntingtin, with results showing that both forms exchanged 79% of potential nh hydrogen bond donors within 30 seconds (n53), with little to no further exchange over the following ten minutes. this result suggested that the pathogenic conformations are not stabilized by slow exchanging hydrogen bonds. binding partners to the monomer were assessed in neuro2a cell culture by immunoprecipitation and quantitative ms/ ms proteomics approaches after depletion of aggregates by pelleting. proteins that more prevalently co-precipitated with pathogenic huntingtin included fused in sarcoma (fus), glycine-trna ligase (gars), peroxiredoxin 6 (prdx6), phosphatidylethanolamine-binding protein 1 (pebp1/rkip), and histone subunit hist1h4a, all of which were significantly enriched by two-fold or greater. rna-seq analysis indicated that none of these proteins had altered expression levels, suggesting that the binding interactions are not due to changes in background abundance. overall we found that the conformational differences are subtle, yet are sufficient to generate several specific proteome interactions that offer clues to a toxic gain-of-function mechanism in pathology. work is ongoing to probe the more subtle changes in conformation and the importance of these interactors to mediating mechanisms of dysfunction. hereditary tyrosinemia type i is an autosomal recesive disorder caused by deficiency of fumarylacetoacetate hydrolase (fah) enzyme. deficiency of fah leads to cellular accumulation of toxic metabolites which include mainly, succinylacetone (sa), maleylacetoacetate (maa) and fumarylacetoacetate (faa) in many body tissues. fah is mainly expressed in hepatocytes and renal proximal tubular epithelium. therefore, liver and kidney are the two primary organs affected by this disorder, and development of hepatocellular carcinoma is the major symptom. missense mutations leads to a loss of enzymatic efficiency which, in a high number of mutations, correlates with loss of kinetic and thermodynamic stability of the enzyme. in our ongoing project, we are trying to elucidate the molecular basis of tyrosinemia by means of biophisical and structural characterization of fah wild type along with its mutations. this knowledge should help us design new therapies based on the identification of pharmacological chaperones that could restore the altered enzymatic stability of the enzyme. human fah wild type and 19 selected mutants were synthesized and inserted in an expression vector for e. coli. the proteins were purified in a fplc and, their thermodynamic and kinetic stability investigated using circular dichroism. our preliminary results confirm the loss of termodinamic stability of different mutants and its variability compared to wild type protein. repulsion between net charges of subunits during ferritin assembly daisuke sato 1 , hideaki ohtomo 1 , atsushi kurobe 1 , satsuki takebe 1 , yoshiteru yamada 2 , kazuo fujiwara 1 , masamichi ikeguchi 1 1 department of bioinformatics, graduate school of engineering, soka university, 2 jasri/spring-8 the organisms have a lot of spherical shell-shaped supermolecules consisting of identical or distinct subunits (e.g., ferritin, virus capsid, lumazine synthase and encapsulin). such multimeric proteins spontaneously assemble into their native structures from the subunits to acquire the specific functions. however, the assembly mechanism of such supermolecules has not been understood in detail. hence, to investigate the assembly mechanism is biologically important. escherichia coli non-heme ferritin (ftn) consists of 24 identical subunits, which are assembled into a spherical shell-shape with 4/3/2 symmetry. ftn is able to store iron inside cavity. the subunit includes a-d helices forming 4-helix bundle, a long bc-loop between b and c-helices and a short e-helix at the c-terminal. ftn dissociates into dimers at acidic ph. the dimer was shown to maintain the native-like secondary and tertiary structures by circular dichroism spectra and small angle x-ray scattering (saxs). the acid-dissociated ftn is able to reassemble into the native structure when ph increases. to clarify ftn assembly mechanism, we performed the stopped-flow time-resolved saxs (tr-saxs) experiments. the saxs profiles could be acquired every 15 ms after the initiation of reassembly. the initial velocity calculated from the forward scattering intensity increment was proportional to the square of the protein concentration, implying that the reaction is second-order. we propose the sequential bimolecular reaction, in which two dimers bind to form tetramer, then another dimer attaches to the tetramer to form a hexamer, and so on. the assembly rate depended on ph and ion strength, indicating that the electrostatic interaction plays an important role in the assembly reaction. the assembly rate decreased with increasing ph in the range from 6.0 to 8.0 and increased with increasing nacl concentration. this indicates that there are repulsive electrostatic interactions between assembly units and that they increases with increasing ph from 6.0 to 8.0. a possible interaction is the repulsion between net charges of dimers since pi of ftn is expected to be 4.6. to test this possibility, we made several mutants with different net charges. as mutational sites, we selected charged residues that are far from the subunit interface. selected sites were glu5, glu8, glu12, glu85 and glu89. we constructed the mutants with one, two, three or four glu -> gln substitutions of selected sites. the structures of those mutants were similar to that of wild-type ftn. if aforementioned hypothesis is correct, the assembly rate is expected to increase with increasing the number of substitution. the result agreed well with this expectation and strongly suggested that the electrostatic repulsion between dimers is an important factor determining the assembly rate of ftn. improved modeling of protein unfolding rates and pathways through solvation and modeling of beta-barrels benjamin walcott 1,2 , lu ıs garreta 3 , christopher bystroff 1,2,4 1 department of biology, rensselaer polytechnic institute, 2 center for biotechnology and interdisciplinary studies, 3 department of computer science, universdad del valle, 4 department of computer science, rensselaer polytechnic intitute an understanding of the folding and unfolding pathways of proteins is integral to improving our ability to associate the structural impact of point mutations and disease etiology. information gained here can also be used for protein structure prediction and design. to model unfolding pathways in proteins we utilize a computational method called geofold. this approach uses recursive hierarchical partitioning of protein structure and finite elements simulation. geofold considers three types of partitioning operations: translational motion (break), single point revolute joints (pivot), and rotation around two points (hinge). from these operations, a directed acyclic graph (dag) is constructed where nodes correspond to the substructures created by these operations and the edges represent the operations. for each operation in the dag, its dissociation and reassociation rates are determined as a function of solventaccessible surface area, hydrogen bonds, voids, and conformational entropy. finite element simulations are carried out to simulate the kinetics of unfolding. this model accurately predicts changes in unfolding pathways due to disulfides in a four-protein case-study, but it fails to produce a realistic pathway for b-barrel proteins such as green fluorescent protein (gfp). to better model these barrel proteins, a new partitioning operation is introduced involving the breaking of all contacts between an adjacent set of b-strands, called a seam. in addition, to improve the accuracy of kinetic modeling, several updates have been made to the energy function, including an improved solvation model and a contact-orderbased estimation of the reassociation rates. the predicted unfolding rates and pathways using this improved geofold are compared with experimentally measured values in kineticdb for proteins with multi-state unfolding kinetics, point mutations, circular permutations, and engineered disulfides. the presence of multiple domains in a protein can result in the formation of partially folded intermediates, leading to increased aggregation propensity. this can be reduced by cooperative, all-or-nothing folding of the multi-domain protein. in good agreement with ensemble folding experiments, a coarsegrained structure-based model of e. coli adenylate kinase (ake) folds cooperatively. ake has three domains, nmp, lid and core. we examine the role of the interfaces between these domains in facilitating folding cooperativity in ake. mutants in which these interfaces are deleted exhibit similar folding cooperativities as wild-type ake. on closer inspection, we observe that unlike a typical multi-domain protein in which one domain is singly-linked to its adjacent domain, nmp and lid are inserted into core, i.e. they are both connected to core by two linkers each. we create circular permutants of ake in which the inserted domains are converted to singly-linked domains, and find that they fold less cooperatively than wild-type ake. domain insertion in wild-type ake facilitates folding cooperativity even when the inserted domains have lower stabilities. the n-and c-termini of nmp and lid are constrained upon the folding of core and this facilitates their folding. thus, nmp and lid which undergo large conformational changes during catalysis can be smaller with fewer stabilizing interactions. in addition, inter-domain interactions need not be optimized for folding, and can be tuned for substrate binding, conformational transition and catalysis. analysis of protein domains using structural bioinformatics suggests several examples of multi-domain proteins in which domain insertion is likely to facilitate folding cooperativity. tuning cooperativity on the free energy landscape of protein folding pooja malhotra 1 , jayant udgaonkar 1 1 national centre for biological sciences, tata institute of fundamental research the mechanism by which a protein explores the free energy landscape during a folding or unfolding reaction is poorly understood. determining whether these reactions are slowed down by a continuum of small ( kbt) free energy barriers or by a few large (> 3 kbt) free energy barriers is a major challenge. in this study the free energy landscape accessible to a small protein monellin is characterized under native-like conditions using hydrogen exchange in conjunction with mass spectrometry. cooperative and noncooperative opening processes could be directly distinguished from the mass distributions obtained in the ex1 limit. under native conditions, where the native state is maximally stable, the unfolded state is transiently sampled in an entirely non-cooperative and gradual manner. under conditions which stabilize the unfolded state or destabilize the native state of the protein, the slowest structure opening event becomes cooperative. the present study provides an understanding of the relationship between stability and folding cooperativity. it suggests that the cooperative transitions observed in unfolding reactions maybe a consequence of the changes in the stabilities of the unfolded state and the transition state. it also provides rare experimental evidence for a gradual unfolding transition on a very slow timescale. role of electrostatic repulsion between unique arginine residues on the assembly of a trimeric autotransporter translocator domain eriko aoki 1 , kazuo fujiwara 1 , masamichi ikeguchi 1 1 haemophilus influenzae adhesin (hia) belongs to the trimeric autotransporter family. the autotransporter consists of an n-terminal signal peptide, an internal passenger domain and a c-terminal translocator domain. the signal peptide directs to export across the inner membrane via the sec system and is cleaved, the passenger domain is a virulence factor, and the translocator domain (hiat) is embedded in the outer membrane. the crystal structure of hia translocator domain (hiat) has shown that hiat forms a transmembrane b-barrel of 12 b-strands, four of which are provided from each subunit. the b-barrel has a pore that is traversed by three a-helices, one of which is provided from each subunit. the protein has a unique arginine residue at 1077. arg1077 side chains from three subunits protrude from the b-strand toward the center of the barrel and are close to each other. these residues seem to have an unfavorable electrostatic effect on the assembly and decrease the trimer stability. to investigate the role of this residue on the trimer assembly and stability of hiat, we replaced this arginine with the neutral amino acid, methionine (r1077m) or the positively charged residue, lysine (r1077k), and properties of these mutants were investigated. hiat and two mutants were dissociated by formic-acid treatment, and they were able to reassemble in the presence of the detergent. to measure the time course of trimer reassembly, amounts of reassembled trimer and monomer were quantified by sds-page at different assembly times. although the neutralized mutation increased the rate of reassembly, the final amount of reassembled trimer decreased, especially at higher protein concentration. these suggest that the neutralized mutation cause the incorrect oligomer formation. the far-uv cd spectrum of reassembled wt hiat was nearly identical with that of the native wt hiat. however, the spectrum of the reassembled r1077m mutant was more intense that of the native r1077m mutant, although the proportion of trimer was much lower than that of the wt hiat. this suggests that the incorrect oligomer has a secondary structure different from the wt hiat. r1077k mutant showed assembly properties similar to those of the wt hiat. therefore, the repulsion between positively charged residues seems to be important for preventing hiat from misassembly. similar proximity of arginine residues is observed for hiv capsid protein, carboxysome shell protein, lumazine synthase and so on. the electrostatic repulsion between arginine residues may be a general mechanism for protein assembly. department of veterinary pathobiology, kagoshima university, 2 institute for food sciences, hirosaki university, 3 faculty of fisheries, kagoshima university, 4 department of veterinary histopathology, kagoshima university, 5 veterinary clinical training center, kagoshima university, 6 department of veterinary anatomy, kagoshima university, 7 sakamoto kurozu inc., 8 the united graduate school of agricultural sciences, kagoshima university kurozu is a traditional japanese rice vinegar. during fermentation and aging of the kurozu liquid in an earthenware jar over 1 year, solid residue called kurozu moromi is produced. in the present study, we evaluated whether concentrated kurozu or kurozu moromi could ameliorate cognitive dysfunction in the senescence accelerated p8 mouse. senescence accelerated p8 mice were fed 0.25% (w/w) concentrated kurozu or 0.5% (w/w) kurozu moromi for 4 or 25 weeks. kurozu suppressed cognitive dysfunction and amyloid accumulation in the brain, while kurozu moromi showed a tendency to ameliorate cognitive dysfunction, but the effect was not significant. we hypothesize the effect is caused by the antioxidant effect of concentrated kurozu, however, the level of lipid peroxidation in the brain did not differ in senescence accelerated p8 mice. dna microarray analysis indicated that concentrated kurozu increased hspa1a mrna expression, a protein that prevents protein misfolding and aggregation. the increase in hspa1a expression by kurozu was confirmed using quantitative real-time pcr and immunoblotting methods. therefore, the suppression of amyloid accumulation by concentrated kurozu may be associated with hspa1a induction. however, concentrated kurozu could not increase hspa1a expression in mouse primary neurons, suggesting it may not directly affect neurons. young-ho lee 1 although amyloid fibrils are associated with a number of pathologies, their conformational stability remains largely unclear. we herein investigated the thermal stability of various amyloid fibrils. a-synuclein fibrils, freshly prepared at 37 c at neutral ph, cold-denatured to monomers at 0-20 c and heat-denatured at 60-110 c. meanwhile, the fibrils of b2-microglobulin, alzheimer's ab1-40/ab1-42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in conformational stability at low temperature in the presence of chemical denaturants. a comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in the fibril cores contributed to the cold denaturation of a-synuclein fibrils. reinforced electrostatic repulsion at low temperatures may promote cold denaturation, leading to a unique thermodynamic property of amyloid fibrils. we propose that although cold-denaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloid structures may explain amyloid-specific cold denaturation due to the unfavorable burial of charged side-chains in fibril cores. key structural differences between tbtim and tctim revealed by thermal unfolding molecular dynamics simulations angel piñeiro 1 , miguel costas 2 , andrea guti errez-quezada 2 1 dept of applied physics, university of santiago de compostela, 2 lab. of biophys. chem., dept of physical chemistry, fac. of chemistry, unam the thermal unfolding pattern obtained by differential scanning calorimetry for trypanosoma cruzi and trypanosoma brucei triosephosphate isomerase (tcim and tctim) are significantly different although the crystal structure of both proteins is almost indistinguishable and the sequences are highly homogolous. in order to explain these differences at molecular level a set of molecular dynamics simulations were performed at different temperatures between 400 and 700 k. the obtained trajectories were analyzed in detail and the residues that showed to be key in the unfolding pathway of each species were identified. a set of residues that behave significantly different between both proteins were selected and proposed for mutations. the general aim is to identify the minimum amount of residue mutations that allow providing tbtim with the behaviour of tctim and vice versa. experimental complementary work is also being performed on the same protein. repositioning som0226 as a potent inhibitor of transthyretin amyloidogenesis and its associated cellular toxicity salvador ventura 1 , ricardo sant'anna 1 , maria ros ario almeida 2 , nat alia reixach 3 , raul insa 4 , adrian velazquez-campoy 5 , david reverter 1 , n uria reig 4 1 universitat aut onoma de barcelona, 2 instituto de biologia molecular e celular, icbas, 3 the scripps research institute, 4 som-biotech, 5 universidad de zaragoza transthyretin (ttr) is a plasma homotetrameric protein implicated in fatal amyloidosis. ttr tetramer dissociation precedes pathological ttr aggregation. despite ttr stabilizers are promising drugs to treat ttr amyloidoses, none of them is approved by the food and drug administration (fda). repositioning existing drugs for new indications is becoming increasingly important in drug development. here, we repurposed som0226, an fda-approved molecule for neurodegenerative diseases, as a very potent ttr aggregation inhibitor. som0226 binds specifically to ttr in human plasma, stabilizes the tetramer in vivo and inhibits ttr cytotoxicity. in contrast to most ttr stabilizers, it exhibits high affinity for both ttr thyroxine -binding sites. the crystal structure of som0226-bound ttr explains why this molecule is a better amyloid inhibitor than tafamidis, so far the only drug in the market to treat the ttr amyloidoses. overall, som0226, already in clinical trials, is a strong candidate for therapeutic intervention in these diseases. neurometals as modulators of protein aggregation in neurodegenerative diseases s onia s. leal 1 , joana s. crist ovão 1 , cl audio m. gomes 1 1 protein misfolding and aggregation is a hallmark across neurodegenerative diseases such as alzheimer's disease and amyotrophic lateral sclerosis (als). since these diseases are mostly sporadic, the formation of protein amyloids in the nervous system depends of chemical and biological triggers within the neuronal environment, such as metal ions [1] . in this communication i will overview the metallobiology of neuronal calcium, zinc and copper, which are key players in brain function and have altered homeostasis in most neurodegenerative conditions. our recent work will illustrate how this allows establishing molecular mechanisms in neurodegenerative diseases [2] [3] [4] [5] [6] . in the pursuit of this goal, in the last years we have been investigating superoxide dismutase 1 (sod1), a cu/zn metalloenzyme that aggregates in the fatal neurodegenerative disorder als, as a model. in sod1-als cases, this ubiquitous protein selectively aggregates in motor neurons, implicating a local biochemical factor in the process: interestingly, zn21 and ca21 levels are upregulated in the spinal and brain stem motor neurons of als patients, and increased ca21 triggers multiple pathophysiological processes which include direct effects on the sod1 aggregation cascade [2, 3] . recently we established that calcium ions promote sod1 aggregation into non-fibrillar amyloid, suggesting a link to toxic effects of calcium overload in als [4] . we showed that under physiological conditions, ca21 induces conformational changes on sod1 that increase sod1 b-sheet content and decrease sod1 critical concentration and nucleation time during aggregation kinetics. we also observed that calcium diverts sod1 aggregation from fibrils towards amorphous aggregates. interestingly, the same heterogeneity of conformations is found in als-derived protein inclusions. we thus hypothesized that transient variations and dysregulation of cellular ca21 and zn21 levels contribute to the formation of sod1 aggregates in als patients [4, 5] . in a follow up study we combined experimental and computational approaches to show that the most frequent ligands for ca21 are negatively-charged gatekeeper residues located in boundary positions with respect to segments highly prone to edge-to-edge aggregation. calcium interactions thus diminish gatekeeping roles by shielding repulsive interactions via stacking between aggregating b-sheets, partly blocking fibril formation and promoting amyloidogenic oligomers such as those found in als inclusions. interestingly, many fals mutations occur at these positions, disclosing how ca21 interactions recreate effects similar to those of genetic defects, a finding with relevance to understand sporadic als pathomechanisms [6] . the amino acid proline is well-known by its disorder promoting and helix breaking properties. prolines can be accommodated within transmembrane (tm) alpha-helices and participate in important biological tasks like signal transduction, ligand binding and helix-helix packing. x-ray crystallography and nmr indicate that proline residues in membrane proteins induce distortions of the helix geometry to different extents ranging from small bends to severe kinks. however, such studies provide essentially a static snapshot of membrane-embedded helices. therefore, the link between proline dynamics and function is not completely understood. in this work we have used singlemolecule f€ orster resonance energy transfer (smfret) and fluorescence correlation spectroscopy (fcs) to probe the structure and dynamics of the tm domain of human glycophorin a (gpa), a widely used model membrane protein for oligomerization studies. a fluorescent dye pair has been attached to both ends of the membrane-spanning region of gpa, which allowed monitoring the average distance and distance fluctuations between the attachment points. site-specifically double-labeled gpa has been reconstituted into two membrane-mimetic systems: sds micelles and phospholipid bilayers assembled into nanodiscs. using proline-scanning mutagenesis we have systematically evaluated the impact of proline residues in different positions along the membrane normal on transmembrane helix length and lateral packing. furthermore, we have investigated the distance distribution in tm helices containing native prolines, namely the insulin receptor and the nesprin protein. our results shed light into the relation between proline dynamics and the folding and function of tm helices. thermodynamic contributions of specific mutations of l30e protein in the rna: protein interface region measured by analytical ultracentrifugation and gel shift assay bashkim kokona 1,2 , sara kim 1 , margaret patchin 1 , britt benner 1 , susan white 1 1 in saccharomyces cerevisiae, ribosomal protein l30e acts as an autoregulator by inhibiting the splicing of its pre-mrna and translation of its mrna. the l30e protein-rna binding site has been previously studied, revealing a rna kink-turn motif, which is characterized by a sharp bend in the phosphodiester backbone due to unpaired nucleotides and internal tertiary interactions. l30e structural flexibility at the rna-binding interface makes such interaction an excellent model to explore the energetics of rna protein binding. we made l30e k28a, f85a, and f85w mutants to quantify the thermodynamic contributions of such interactions to the protein-rna complex. we used analytical ultracentrifugation sedimentation equilibrium (se) and sedimentation velocity (sv) to investigate conformational changes and protein-rna binding free energy changes due to mutations. our computed changes of binding free energy based on the sedimentation equilibrium experiments were consistent with the gel shift assay results. in addition, sedimentation velocity experiments on the l30e wild type indicate that protein-rna interaction is highly dynamic and involves conformational changes of the kink-turn rna induced by l30e protein. our results provide new insights on understanding the binding between ribosomal proteins and their rna molecules counterpart, which can be used to complement the x-ray structure. role of a non-native a-helix in the folding of equine b-lactoglobulin takahiro okabe 1 , toshiaki miyajima 1 , kanako nakagawa 1 , seiichi tsukamoto 1 , kazuo fujiwara 1 , masamichi ikeguchi 1 1 equine b-lactoglobulin is a small globular protein (162 residues). although elg adopts a predominantly b-sheet structure consisting of nine anti-parallel b-strands (a-i) and one major a-helix in the native state, it has been shown that a non-native a-helical intermediate accumulates during the burstphase of folding reaction from the unfolded state in the concentrated denaturant. to ask whether the non-native helix formation is important for acquiring the native b-sheet structure, we determined first where the non-native a-helix is formed. a stable analogue of the burst-phase folding intermediate was observed at acid ph (a state). the amide hydrogen exchange experiment and proline-scanning mutagenesis experiment have shown that the non-native a-helix is formed at the region corresponding to the h strand in the a state. to investigate the role of this non-native a-helix on refolding reaction of elg, we constructed several mutant proteins, which were designed to destabilize the nonnative a-helix in the folding intermediate without perturbation on the native structure. a mutant, a123t, fulfilled this requirement, that is, a123t showed a native structure similar to that of the wildtype protein, and largely reduced cd intensity in the a state. then, the refolding kinetics were investigated by the cd and fluorescence stopped-flow method. a123t mutation resulted in reduction of the burst-phase cd intensity, which confirmed that the non-native a-helix is formed around the h strand region. subsequent to the burst-phase, four kinetic phases were observed for a123t and the wildtype protein. importantly, the folding rate constants of the four kinetic phases were similar between both proteins. furthermore, interrupted refolding experiments demonstrated that the native state was formed in the two parallel pathways in the two slower phases of the four kinetic phases. the relative amplitudes of the two pathways were similar between a123t and the wild-type protein. these results clearly showed that the formation of the non-native helix has little effect on the folding rates and pathways, and suggested that the non-native helix formation may not be a severe kinetic trap for protein folding reaction. impact of the chaperonin cct in a-synuclein(a53t) amyloid fibrils assembly ahudrey leal_quintero 1 , javier martinez-sabando 1 , jose mar ıa valpuesta 1 , begoña sot 1 1 centro nacional de biotecnolog ıa (cnb/csic)., 2 centro nacional de biotecnolog ıa (cnb/csic)., 3 centro nacional de biotecnolog ıa (cnb/csic)., 4 centro nacional de biotecnolog ıa (cnb/csic) and fundaci on imdea-nanociencia cct is a eukaryotic chaperonin that uses atp hydrolysis to encapsulate and fold nascent protein chains. moreover, it has recently been shown that cct is able to inhibit amyloid fibers assembly and toxicity of the polyq extended mutant of huntingtin, the protein responsible of huntington disease. although this opens the possibility of cct being also able to modulate other amyloidopathies, this has not addressed yet. the work presented here intends to determine the effect of cct in the amyloid fibers assembly of a-synuclein(a53t), one of the mutants responsible of parkinson disease. it is demonstrated that cct is able to inhibit a-synuclein(a53t) fibrillation in a nucleotide independent way, suggesting that this effect is based on binding rather than on active folding. furthermore, using deletion mutants and assaying the interaction of cct with monomers, soluble oligomers and fibres, it has been possible to unravel the mechanism of this inhibition: cct interferes with fibers assembly by interacting with a-synuclein(a53t) nac domain once soluble oligomers are formed, thus blocking the reaction before the fibers start to grow. amyloid-like aggregation of nucleophosmin regions associated with acute myeloid leukemia mutations daniela marasco 1 , concetta di natale 1 , valentina punzo 1 , domenico riccardi 1 , pasqualina scognamiglio 1 , roberta cascella 2 , cristina cecchi 2 , fabrizio chiti 2 , marilisa leone 3 , luigi vitagliano 3 1 department of pharmacy, cirpeb: centro interuniversitario di ricerca sui pepti, 2 section of biochemistry, department of biomedical experimental and clinical scie, 3 institute of biostructures and bioimaging nucleophosmin (npm1) is a multifunctional protein involved in a variety of biological processes and implicated in the pathogenesis of several human malignancies. npm1 has been identified as the most frequently mutated gene in acute myeloid leukemia (aml) patients, accounting for approximately 30% of cases (1). the most frequent human npm1 mutations lead to variants with altered c-terminal sequences of the c-terminal domain (ctd) that, in its wild form, folds as a three helix bundle. aml modifications lead to (a) an unfolding of the ctd in the mutated protein and (b) its accumulation in the cytoplasm due to the loss of nuclear localization sequences with mutations of trp290 (mut e) and also of trp288 (mut a) (2) . to gain insights into the role of isolated fragments in npm1 activities we dissected the ctd in its helical fragments. here we describe the unexpected structural behavior of the fragments corresponding to the helices h2 and h3 in both wild-type and aml-mutated variants. h2 region shows a remarkable tendency to form amyloid-like assemblies while only the muta sequence of h3 region is endowed with and b-sheet structure, under physiological conditions, as shown by circular dichroism, thioflavin t and dynamic light scattering. the aggregates of h2, are also toxic to neuroblastoma cells, as determined by using the mtt reduction and ca21 influx assays (3) . furthermore the effects of the local context on the different tendencies to aggregate of h2 and h3 were investigated and appeared to influence for the aggregation propensity of the entire ctd. since in aml mutants the ctd is not properly folded, we hypothesize that the aggregation propensity of npm1 regions may be implicated in aml etiology. these findings have implications to elucidate the pathogenesis of aml caused by npm1 mutants and aggregation phenomena should be seriously considered in studies aimed at unveiling the molecular mechanisms of this pathology. we report a resume of our study regarding the effects of microwaves in the range 900-1800 mhz on a typical protein, myglobin. previous literature have concerned the effects on living and in vitro organic systems induced by high frequencies electromagnetic fields. we have focused our attention on a typical protein, myoglobin, because proteins are the simplest organic systems that are fundamentals in organic functions of livings. myoglobin is a protein found mainly in muscle tissue of vertebrates, consisting of a single protein chain with 153 amino acids and one heme group that stores oxygen in the muscle cells. the physiological importance of myoglobin is mainly related to its ability to bind molecular oxygen. in particular, we focused our attention on the secondary structure of this protein in order to highlight whether exposure to microwaves unfold the protein producing transitions from a-helix component to b-sheet features. to this aim fourier transform infrared (ftir) spectroscopy have been used. the importance of this study is related to previous literature which indicated that transition from a-helix to b-sheet structure in a protein can be responsible for aggregation mechanisms that can lead to neurotoxicity and neurodegenerative disorders that can be considered as the first step to some pathologies [1] [2] [3] . the aggregates consist of fibers containing unfolded proteins with a prevalent b-sheet structure termed amyloid [4] . in our studies myoglobin in deuterium oxide (d2o) solution was exposed for 3 h to mobile phone microwaves at 900 and 1800 mhz at a power density of 1 w/m2. ftir spectra were recorded by a spectrometer vertex 80v from bruker optics, following the protocol accurately described in [5] [6] [7] . ftir spectroscopy analysis evidenced an increase in intensity of b-sheet structures and a significant shift to lower frequencies of about 2.5 cm-1 of the amide i vibration after exposure [8, 9] . these results led to conclude that mobile phone microwaves induce proteins unfolding and formation of aggregates [10, 11] . membrane proteins play a vital role in many biological processes, and yet remain poorly understood as they are frequently unstable in vitro. the goal of this project is to investigate the insertion and folding of membrane proteins into lipid bilayers, using a cell free expression system. we have used both e.colibased cell extracts (s30), and commercial translation systems (purexpress) in combination with synthetic liposomes of defined lipid composition. these studies will aid understanding of cooperative folding, folding intermediates, and the effects of the lipid bilayer on folding and insertion. model e.coli proteins have been investigated, as they can offer important insights into other proteins, and thus facilitate the further study of more biologically relevant proteins. it has been found that the rhomboid protease glpg spontaneously inserts into liposomes without the aid of an insertase such as secyeg. this spontaneously inserted glpg is functional, and is able to cleave bodipy-labeled casein, yielding a fluorescent product. the major facilitator superfamily (mfs) transport proteins lacy, galp and glpt have also been found to insert spontaneously into liposomes. it has been shown that the lipid composition of the liposomes has an effect on the amount of protein inserted into the bilayer, with all proteins tested to date preferring liposomes containing at least 50 mol% dopg. ongoing and future work will involve the use of rare codons to alter the rate of translation, to investigate the effect this has on the final folded structure of the protein. preliminary work is also currently being done into whether the two domains of the mfs family transporters fold cooperatively or independently, thus aiding understanding into the folding and stability of membrane transport proteins. frederic greco 1 , audrey toinon 1 , nadege moreno 1 , marie claire nicola€ ı 1 rabies remains an important worldwide health problem that causes a fatal encephalomyelitis [1] . currently, rabies in humans is under control in europe and north america following the use of efficient vaccines for dogs and wild animals. however, it still kills more than 55,000 people every year mainly in africa and asia [2] . human vaccination prevents infection with very high efficacy. the vaccine contains an inactivated rabv produced on vero cells. rabv is an enveloped, negative single stranded rna virus which encodes five proteins, namely the nucleoprotein (n), the phosphoprotein (p), the matrix protein (m), the glycoprotein (g), and the viral rna polymerase (l) [3] . the viral envelope is covered by trimer spikes of g-glycoprotein which is the most significant surface antigen for generating virus-neutralizing antibodies. here we illustrate the use of dsc (differential scanning calorimetry) to identify structural domains or proteins involved in thermal transitions. the dsc thermogram for intact beta-propiolactone inactivated rabv samples in pbs buffer reveals two major thermal transitions with a tm respectively at 618c and 718c. we have initially focused our investigations on one of the major proteins encode in rabv, glycoprotein g [4] . glycoprotein g contains disulfide bridges on the ectodomain [6] , is sensitive to bromelain cleavage [5] and shows reversible conformation changes at low ph [7] . considering these characteristics, our results provide evidence on the identity of one thermal transition observed by dsc. keywords: rabies virus, differential scanning calorimetry, protein unfolding domain swapping of the dna-binding domain of human foxp1 is facilitated by its low folding stability exequiel medina, sandro l. valenzuela, crist obal c ordova, c esar a. ram ırez-sarmiento and jorge babul departamento de biolog ıa, facultad de ciencias, universidad de chile, santiago, chile protein folding and dimerization (or oligomerization) are biologically relevant processes when reaching the quaternary structure is required for function. proteins that form dimers by exchanging segments or domains of their tertiary structure with another subunit, the so-called domain swapping phenomenon, are examples where folding and dimerization are tightly concerted processes. previous studies on domain swapping proteins, such as p13suc1 and diphtheria toxin, have shown that, in general, a high kinetic barrier separates monomers and domain swapped dimers, and that this barrier can be lowered by promoting protein unfolding and refolding at high protein concentrations, thus favoring the swapped oligomer. recent crystal structures of the dna-binding domain of several human forkhead box (fox) proteins have shown that the p subfamily of these transcription factors (foxp) can form swapped dimers. the human foxp proteins are interesting models of domain swapping, because mutations of the dna-binding domain of these proteins are linked to diverse inherited disorders in humans, such as ipex and language deficits, and some of these mutations are located in the hinge region that connects the exchanged segment with the rest of the protein. moreover, foxp1 and foxp2 have been described to reach monomer-dimer equilibrium in solution after hours of incubation, suggesting that a low kinetic barrier separates both species. using foxp1 as a model of domain swapping, we analyzed the temperature and protein concentration effects on the dimer dissociation, obtaining the free energy change and enthalpy of the process by van't hoff analysis (dh8 of 23.1 kcal•mol-1, ds8 of 0.082 kcal•mol-1•k-1 and dg8 at 258c of 20.95 kcal•mol-1). these results indicate that the monomer-monomer association is an example of an enthalpy-driven process. to understand how foxp1 domains swap without protein unfolding, we performed equilibrium unfolding experiments using gndhcl as denaturant, showing that the wild-type protein has a low stability (dgu 5 6 kcal•mol-1, cm 53.5 m at 258c), in contrast to other domain swapping proteins with high kinetic barriers. we further explore the domain swapping mechanism of foxp1 through biased targeted molecular dynamics simulations, showing that the exchange process can occur by specific local destabilization and unfolding of the hinge region and helix h3. to further corroborate that the low stability of wild-type foxp1 facilitates its domain swapping, we engineered a monomeric version of foxp1 through a single-point mutation in the hinge region, which has been previously described in the literature, and used this protein to visualize the effect of monomer stability in the dimer formation. comparison of the folding stability of the monomeric mutant a39p and wild-type foxp1 shows that ddgu (mutant-wild-type) is 2.5 kcal/mol, concluding that the ability of foxp1 to domain swap rapidly can be explained through its low monomer stability and local unfolding of the exchange region. funding: fondecyt 1130510 and 11140601. determining the coupled interactions that stabilize the structural framework of the ß-propeller fold loretta au 1 , david green 2,3,4 1 department of statistics, the university of chicago, 2 department of applied mathematics and statistics, stony brook university, 3 graduate program in biochemistry and structural biology, stony brook university, 4 laufer center of physical and quantitative biology, stony brook university b-propeller proteins are a highly evolved family of repeat proteins that are involved in several biological pathways, such as signal transduction, cell-cycle modulation and transcription regulation, through interactions with diverse binding partners, despite having a similar fold. as for all repeat protein families, there is a consistent pattern in secondary structure for each repetitive region, in addition to the entire family. typically, four to ten propeller blades (each containing four anti-parallel b-sheets) are arranged in a toroidal shape, thus providing a large binding surface for ligands or other proteins. about 1% of known proteins adopt this distinctive fold, and although the requirements for tertiary structure and protein function are fundamentally encoded in primary structure, this relationship is not fully understood, and addressing it could provide insight on why the b-propeller fold is common. many techniques in comparative sequence analysis can successfully identify amino-acid conservation between closely related proteins, but molecular interactions between amino acids are often neglected, and further experimentation is still needed to determine the reasons underlying conservation. to explore how primary structure can dictate fold and function, we devised a computational approach to perform large-scale mutagenesis, by adapting the dead-end elimination and a* search algorithms (dee/a*), and also leveraged the structural conservation of each repeating region to understand how sequence variation influences protein fitness, defined here as a combination of stabilizing and binding interactions. dee/a* can evaluate low-energy protein sequences and their corresponding three-dimensional structures, and we used the bsubunit of a g-protein heterotrimer (pdb: 1gp2, gia1b1g2) as a model system to demonstrate: (1) how the multiple roles of individual amino acids in protein fitness can be deconvolved, and (2) how epistatic interactions between them can contribute to structural stability. in doing so, we were able to identify important patterns in sequence complementarity between repeating regions that cannot be found using sequencebased methods alone. these results suggest that computational approaches can be used to determine important protein interactions, and help elucidate the prevalence of b-propeller proteins in biology. temperature induced conformational changes of the villin headpiece miniprotein stanislaw oldziej 1 , wioletta _ zmudzi nska 1 , anna hałabis 1 1 the c-terminal subdomain of the actin-binding protein villin called hp35 (villin headpiece) has been used as a model protein in a number of studies of protein folding kinetics and protein folding mechanism [1, 2] . the hp35 is a 35 residue miniprotein with an alpha-helix bundle three-dimensional fold. the goal of our work was to determine conformational ensemble of polypeptide chain of the investigated miniprotein at a wide range of temperatures to get detailed information about how protein structure is influenced by temperature. 2d nmr spectra of the title miniprotein were registered at 278, 293 and 313 k. the three-dimensional structure of the hp35 based on restraints derived from nmr spectra registered at 278 k is almost identical with structure deposited in the pdb database in the record 2f4k [2] . at higher temperatures (293 and 313 k) the general shape of the protein remains unchanged, with well packed hydrophobic core. however, with temperature increase alpha-helices start to melt. at 313 k structure of the protein remains compact and in general shape similar to structure observed at 278k, but none of the alpha-helices could be observed. results obtained for hp35 protein are in agreement with previous observation for the trp-cage miniprotein [3] , that with temperature increase regular secondary structure elements melt first before the break-up of the hydrophobic core of the protein. biological membranes provide a selective and chemically sealed barrier for cells. transport of ions and small molecules across the membrane is mediated by transporter proteins and the breakdown of a cell's ability to produce functionally folded membrane transport proteins can lead to dysfunction and has been implicated in many diseases1. however little is known about the processes that govern the misfolding of a-helical integral membrane proteins, taking into account that these proteins fold and maintain functional structures within membranes of various organelles. the neurotransmitter sodium symporter (nss) protein family is an example of a-helical transporter proteins. the nss family encompasses a wide range of prokaryotic and eukaryotic ion-coupled transporters that regulate the transport of neurotransmitter molecules whose dysfunction has been implicated in multiple diseases and disor-ders2. we have investigated the folding processes of prokaryotic homologue of the nss family leut responsible for the transport of neurotransmitters and amino acids to the sodium electrochemical gradient. previously folding processes of membrane transporters have mainly been characterised within detergent micelles. however, detergent micelles are not an accurate depiction of the environment of the membrane bilayer, with this in mind we have also attempted to investigate folding processes within a bilayer pd-051 nmr investigation of ph-induced unfolding of b domain of an escherichia coli mannitol transporter ii mannitol in the bacterial phosphotransferase system kim gowoon 1 , yu taekyung 1 , suh jeongyong 1 1 the bacterial phosphotransferase system (pts) mediates sugar phosphorylation and translocation across the cytoplasmic membrane. cytoplasmic b domain (iib mtl) of the mannitol transporter enzyme ii mannitol, a pts family protein, delivers a phosphoryl group from a domain to an incoming mannitol that is translocated across the membrane. iib mtl is comprised of a four-stranded ß-sheet and three helices, representing a characteristic rossmann fold. we found that the iib mtl of escherichia coli unfolded at a mildly acidic condition. we made iib mtl mutants to investigate the mechanism of the ph-induced unfolding using nmr spectroscopy. we monitored backbone amide groups and side chain imidazole groups of histidine residues using 2d hsqc nmr, and pointed out a potential histidine residue that might be responsible for the unfolding. histidine residues may be generally important to the folding stability in response to environmental ph changes. can site-directed mutagenesis shed light on the refolding pattern of human glucose 6-phosphate dehydrogenase (g6pd)? nurriza ab latif 1,2 , paul engel 1 1 conway institute, univerversity college dublin, 2 faculty of biosciences and medical engineering, universiti teknologi malaysia human glucose 6-phosphate dehydrogenase (g6pd) is the first enzyme involved in the pentose phosphate pathway (ppp). this oligomeric enzyme catalyses the reaction of glucose 6-phosphate to form 6phosphogluconolactone with concomitant reduction of nadp1 to nadph. in erythrocytes nadph is important mainly for protection against oxidative stress. in connection with its role as the sole source of nadph, g6pd deficiency commonly causes haemolytic disease and is known as the most common human enzyme deficiency globally. protein folding problems and instability are believed to be the major defects in the deficient enzymes. in this study, we employed site directed mutagenesis with hope to give more information on the role of -sh groups in the refolding of human g6pd. two mutants were created: 1) one in which all 8 cys residues were replaced by ser and 2) one in which only c13 and c446 were retained. the refolding of recombinant human g6pd has been studied primarily by measuring the enzyme activity after refolding. we also used a combination of intrinsic protein fluorescence, ans (8-anilino-1-naphthalenesulphonic acid) binding and limited proteolysis to look at the conformational change during the refolding. the results showed that gdnhcl-denatured recombinant human g6pd wild type could be refolded and reactivated by rapid dilution technique. even though, as recombinants in e. coli, the mutants were well expressed and active, they remained inactive after attempts were made to refold them in vitro. the methods we applied may have provided some insights on the refolding pattern of this oligomeric protein, albeit qualitatively rather than quantitatively. a single aromatic core mutation converts a designed 'primitive' protein from halophile to mesophile folding connie tenorio 1 , liam longo 1 , ozan s. kumru 2 , c. russell middaugh 2 , michael blaber 1 1 department of biomedical sciences, florida state university, 2 department of pharmaceutical chemistry, university of kansas experiments in prebiotic protein design suggest that the origin of folded proteins may have favored halophile conditions. these results are consistent with salt induced peptide formation which shows that polymerization of amino acids is also promoted by high salt concentrations. as a result of various origin of life studies, a consensus on which amino acids likely populated early earth has emerged. these residues were synthesized by abiotic chemical and physical processes from molecules present in the surrounding environment. the properties of the consensus set of common prebiotic amino acids (a,d,e,g,i,l,p,s,t,v) are compatible with known features of halophile proteins, meaning these proteins are only stable in the presence of high salt concentrations. the halophile environment, thus, has a number of compelling aspects with regard to the origin of structured polypeptides. consequently, a proposed key step in evolution was, movement out of the halophile regime into a mesophile one commensurate with biosynthesis of "phase 2" amino acids -including the aromatic and basic amino acids. we tested the effects of aromatic residue addition to the core of a "primitive" designed protein enriched for the prebiotic amino acids (a, d, e, g, i, l, p, s, t, v) that required halophilic conditions for folding. the subsequent results show that the inclusion of just a single aromatic residue was sufficient for movement to a mesophile folding environment. thus, the inclusion of aromatic residues into the codon table could have conferred key stability to early proteins enabling adaptive radiation outside of a halophile environment. contact prediction methods that rely on sequence information alone, such as evfold, can be used for de novo 3d structure prediction and identification of functionally important residues in proteins. large multiple sequence alignments of protein families consisting of evolutionarily related and plausibly isostructural members reveal co-variation patterns that can be used to identify interactions between pairs of amino acids. we use a global probability model to disambiguate direct and indirect correlations. specifically, we use a maximum entropy approach called pseudo-likelihood maximization (plm) to distinguish causation (residue interactions) from correlation (correlated mutations) and compute evolutionary couplings (ecs). the inferred set of residue interactions can then be interpreted as physical contacts and used in de novo 3d structure prediction. furthermore, the interactions that are inferred can help guide experiments that measure the phenotypic consequences of protein substitutions, making the method useful for functional studies. the present work can be divided into three areas: (i) methodological improvements related to alignment, folding procedure, structure refinement and ranking; (ii) folding of proteins of known structure for benchmarking and prediction of proteins of unknown structure; and (iii) focused exploration of specific cases of interest. developing shuffle as a platform for expression and engineering of antibodies na ke 1 , alana ali-reynolds 1 , bryce causey 1 , berkmen berkmen 1 1 shuffle is a genetically engineered e.coli strain that allows disulfide bond formationin its cytoplasm with high fidelity. many proteins containing disulfide bonds have been successfully expressed in shuffle. in this study, we expressed, purified and characterized full-length monoclonal antibody igg in shuffle. for the first time, a fulllength igg can be functionally expressed in the cytoplasm compartment of an e.coli strain. in order to improve the folding and assembly of igg, we have investigated the expression of igg in various formats and vectors; we have co-expressed chaperones and other helper proteins with igg. several-fold increase in the yield of fulllength igg was observed. we characterized the shuffle produced igg and found it comparable to hybridoma produced igg. optimization of fermentation conditions for a large-scale production is in progress. we aim to develop shuffle as an easy, fast, robust platform for antibody engineering, screening and expression. experimental and computational studies of the effects of highly concentrated solutes on proteins: insights into the causes and consequences of quinary protein structure and cytoplasmic organization most studies of protein structure and function focus on pure, diluted samples; however, real-world biochemistry and typical biotechnological applications of proteins take place in complex media with very high concentrations of solutes (100-400 g/l) of varied size and chemical nature. on one side, this has recently fostered the study of proteins in vivo, in cell, or at least in media mimicking the native conditions. on the other hand, physical chemistry has for a long time studied the general effects of crowded and viscous conditions on proteins, looking mainly at coarse traits like diffusion and stability. but the general effects on traits relevant at atomic/residue resolutions have been less studied, and one fundamental issue remains unsolved: to what extent are proteins forced into interactions with highly concentrated solutes, and with what direct consequences? i will present here our ongoing efforts to dissect the fine effects of high solute concentrations and macromolecular crowding on proteins, based on nmr experiments and md simulations, two complementary techniques of high spatial and temporal resolutions. our results show that smaller solutes are prone to extensive interactions with proteins when at high concentrations while large solutes act chiefly through excluded-volume effects. overall, we observe location-specific perturbations of a protein's surface, its internal dynamics and internal dielectrics, and its hydration, all very dependently on the solute's size and chemical nature. our results support the growing notion that proteins should be studied in native-like media, adding that not only macromolecular crowders but also small molecules should be considered in these studies. last, the fact that high-concentration conditions affect far more than a protein's diffusion rate and stability suggests critical consequences of quinary protein structure and cytoplasmic organization on the regulation of proteins within cellular biochemistry. aldona jeli nska 1 , anna lewandrowska 1 , robert hołyst 1 1 we developed an analytical technique for the study of interactions of ligands (e.g. cefaclor, etodolac, sulindac) with most abundant blood protein (e.g. bovine serum albumin) using the flow injection method. the experiments were conducted at high flow rates (31 cm/s) in a long (>15m), thin (250mm) and coiled capillaries. the compound of interest (10 ml) was injected into carrier phase, which moved by the poisseule laminar flow. at the detection point we measure the concentration distribution of the analyte. the width of the final profile of the analyte concentration is inversely proportional to the effective diffusion coefficient of the analyte. from the differences between the widths of the concentration distribution of free and bound ligand we can determine value of the association constant. carbohydrate binding modules (cbms), which are defined as contiguous amino acid sequences within a carbohydrate-active enzyme, have been found in both hydrolytic and non-hydrolytic proteins and are classified into 71 families, according to their primary structure similarity. the characterization of cbms by different methods has shown that these modules concentrate enzymes on the surface of polysaccharide substrates. it is thought that maintaining the enzyme in proximity with the substrate leads to more rapid degradation of the polysaccharide. therefore, the study of these kinds of modules or domains is relevant, since they are involved in multiple processes in organisms, like signaling, defense and metabolism; and some of them are involved in allergenic responses. in the present work we studied two different models: the first one is a cbm of the family 26 from lactobacillus amylovorus (lacbm26) that binds starch these domains are present in a a-amylase like a repetitive tandem of five modules that are consecutive and do not present connectors. by means of itc and using a single recombinant lacbm26 domain we determined a ka 5 2.31x104 m-1 for b-cyclodextrin and a ka 5 8.54x104 m-1 for acyclodextrin. when the number of consecutive recombinant modules increased to three or five tandem modules, the ka values increased to 106 m-1; however, these constants did not show an additive or a synergic effect. for these experiments we fitted the isotherms to different models and used different algorithms. additionally, we used circular dichroism in the uv-far region to determine if there existed conformational changes upon binding of the cyclodextrin molecules to the different tandem modules. we could only observe slight changes in a positive band centered around 220-240 nm, which has been explained in terms of p-p; interactions of the aromatic residues at the binding site. these cbms have been used as carriers for in vivo vaccine delivery and affinity tags. the second model is a hevein-like cbm of the family 18 present in a chitinase-like protein from hevea brasiliensis (hbcbm18). hevein is a lectin from h. brasiliensis that shows a 63% identity with hbcbm18. these cmbs are connected to the catalytic domain, in proteins such as chitinases, by a linker of approximately 10 residues. in these experiments we used fluorescence techniques to determine the affinity constants for chitotriose. we previously reported a ka 5 2.08x106 m-1 when using a hbcbm18 that has a met residue at the nterminal region. besides the aromatic residues at the binding site, the met residue also interacts with the ligand, as determined using crystallographic and docking techniques. the mutant hbcbm18-r5w that does not have the met residue showed a ka of 2.8x104 m-1 with chitotriose, similar to the value reported for hevein using itc (ka 1.42x104 m-1). interestingly, there exists an isoform of the hbcbm18 that has a connector between one cbm18 and a half cbm18 (1.5xhbcbm18). this protein has a ka of 6.7x105 m-1 with the same ligand. initiating vesicle formation at the golgi complex: auto-regulation and protein interactions govern the arf-gefs gea1 and gea2 margaret gustafson 1 , j. chris fromme 1 1 molecular decision-makers play critical roles in the effort to maintain efficient and accurate cellular functions. in the case of vesicular traffic at the golgi complex, the decision to initiate vesicle formation is made by a set of guanine nucleotide exchange factors (gefs) that activate the small gtpase arf1, which is the master controller for the recruitment of cargos and coat proteins. saccharomyces cerevisiae possess three golgi arf-gefs, gea1, gea2, and sec7, which work at distinct sub-compartments of the golgi to activate arf1 only when and where appropriate. in the case of sec7 at the trans-golgi network (tgn), this requires a positive feedback loop in which active arf1 relieves autoinhibition of sec7, as well as recruitment to the golgi membrane and catalytic stimulation by signaling rab gtpases. we know far less about the decisionmaking process for gea1 and gea2, which are responsible for retrograde traffic within the golgi and to the endoplasmic reticulum. i have found that both gea1 and gea2 can bind membranes weakly in vitro, an ability which is counteracted by their c-terminal hds3 domains. in addition, i have discovered membrane recruitment in vitro is aided by the rab gtpase ypt1. however, these interactions cannot fully explain the distinct localization patterns of gea1, gea2, and sec7, as all three have been shown to be recruited by ypt1, which is found throughout the golgi. my work has revealed that in addition to the well-established distinct localization from sec7, gea1 also occupies different golgi compartments from gea2, so specific signals must exist which help the gefs decide where to go. my current efforts focus on understanding the roles of the other domains of gea1 and gea2, identifying the signals which send them to different parts of the golgi, and unraveling the different roles they play in vesicle trafficking pathways. sequence variation in archaea through diversity-generating retroelements sumit handa 1 , blair g paul 2 , kharissa l shaw 1 , david l valentine 2 , partho ghosh 1 1 department of chemistry and biochemistry, university of california san diego, 2 marine science institute, university of california protein diversification is an essential tool for the survival and evolution for various species. diversitygenerating retroelements (dgr) in bacteria is known to generate massive variation in dna through an error prone reverse transcriptase and retrohoming, which leads to variation in protein sequence. recent discovery of dgrs in intraterrestrial archaeal systems have opened an opportunity to study this massive sequence variation in third domain of life (paul bg, et al. nat. comm.) here, we present the first crystal structure of variable protein from archaea with ligand-binding pocket is surface exposed. also, it has conserved c-type lectin (clec) fold, as shown by previous work on variable proteins, major tropism determinant (mtd) and treponema variable protein a (tvpa) which bind ligands through the clec fold. despite weak sequence identities (10-15%) among these variable proteins, clec fold was found to be conserved. this variable ligand-binding site for archaea variable proteins can potentially generate 1013 variants. protein synthesis is a dynamic process mediated by a variety of proteins and enzymes. recent studies have shown that hydroxylation is a key post-translational modification involved in translation termination. in particular, the fe(ii)-and 2-oxoglutarate-dependent oxygenase, jumonji domaincontaining 4 (jmjd4), regulates translation termination via the carbon 4 hydroxylation of an invariant lysine residue, k63, of the eukaryotic release factor, erf1. in eukaryotes, translation termination is mediated by a release factor complex that includes erf1. erf1 is comprised of three domains, and it is responsible for recognizing stop codons in mrna transcripts before triggering polypeptide release from the ribosome. the lysine residue hydroxylated by jmjd4 falls within the n-terminal domain and more specifically within the highly conserved niks motif. this motif has been identified by cross-linking and mutagenesis studies to play an essential role in stop codon recognition. while hydroxylation of k63 by jmjd4 has been found to increase translational termination efficiency, the exact molecular mechanism by which hydroxylation influences termination remains unclear. this work aims to understand how hydroxylation of erf1 affects translation termination by exploring the effect of hydroxylation on the structure, dynamics, stability, and binding of the n-terminal domain of erf1 (erf1-n) using mass spectrometry, protein nmr spectroscopy, circular dichroism and differential scanning fluorimetry. in our efforts to understand the effect of hydroxylation, an additional jmjd4-catalyzed modification, characterized by a 130 da mass shift on k63, was identified in vitro. the effect of this modification on erf1 was similarly explored. our findings suggest that hydroxylation has no effect on the in-solution nmr structure of erf1-n, which experiences chemical shift changes localized to the target lysine residue. correspondingly, there are no significant differences in secondary structure content between wild type and hydroxylated erf1-n. hydroxylation was also found to have no effect on protein stability or dynamics. interestingly however, the 130 da modification appears to cause more significant chemical shift changes dispersed beyond the niks motif. this suggests a more global effect on the in-solution nmr structure despite the little differences observed in protein dynamics and secondary structure content. the 130 da modification was also found to have a destabilizing effect on erf1-n. neither hydroxylated nor 130 da modified erf1-n exhibited differences in rrna binding. while hydroxylation of erf1 was found to have little effect on protein structure, dynamics, stability, or binding, the 130 da modification has marked effects on protein structure and stability. such differences suggest that this modification has the potential to play an important role in translation. functional and structural analysis of a gh20 ß-n-acetylglucosaminidase from the marine bacterium vibrio harveyi piyanat meekrathok 1 , arthur t. porfetye 2 , marco b€ urger 2 , ingrid r. vetter 2 , wipa suginta 1 1 biochemistry-electrochemistry research unit, suranaree university of technology, 2 max planck institute of molecular physiology vibrio harveyi b-n-acetylglucosaminidase (so-called vhglcnacase) is a new member of the gh20 glycoside hydrolase family responsible for the complete degradation of chitin fragments, with nacetylglucosamine (glcnac) monomers as the final products. however, the 3d structure of glcnacase is still unknown. in this study, crystal structure and function of glcnacase were investigated based on protein crystallography. size-exclusion chromatography and the native-page were employed to verify the protein state of glcnacase in a native form and the acidic active-site residues were mutated using sitedirected mutagenesis method. the effects of mutations on the binding and hydrolytic activities were studied by enzyme kinetics. to provide a structural basis of glcnacase, the wild-type enzyme was crystalized at 293 k using a solution containing 0.1 m sodium acetate ph 4.6 and 1.3 m sodium malonate and recorded x-ray data. the wild-type enzyme was crystallized within 3 days in the monoclinic crystal form, belonging to space group p21, with unit-cell parameters a 5 90.2, b 5 130.7, c 5 98.5 å. the crystal structures of v. harveyi glcnacase were solved and refined to highest resolution of 2.4 å. structural investigation revealed that glcnacase comprises three distinct domains, designated as the n-terminal carbohydrate-binding domain, the a1b topology domain and the tim-barrel catalytic domain. the substrate binding groove of glcnacase is a small pocket, which is suitable to accommodate a shortchain chitooligosaccharide. kinetic analysis revealed that a group of the adjacent d303-h373-e438 showed a significantly decreased activity as compared with the wild-type enzyme, and these residues might be important for enzyme catalysis. silencing the molecular timekeeper in human cancer alicia michael 1 , stacy harvey 1 , patrick sammons 1 , amanda anderson 2 , hema kopalle 1 , alison banham 2 , carrie partch 1 1 university of california -santa cruz, 2 university of oxford the circadian clock coordinates temporal control of physiology by regulating the expression of at least 40% of the genome on a daily basis.1 disruption of circadian rhythms through environmental stimuli (e.g. light at night) or genetic means can lead to the onset of diseases such as: diabetes, cardiovascular disease, premature aging and cancer.2-5 the circadian clock orchestrates global changes in transcriptional regulation via the bhlh-pas transcription factor clock:bmal1. pathways driven by other bhlh-pas transcription factors have a homologous repressor that modulates activity on a tissue-specific basis, but none have been identified for clock:bmal1. we discovered that the cancer/testis antigen pasd1 fulfills this role to suppress circadian rhythms. pasd1 is evolutionarily related to clock and interacts with the clock:bmal1 complex to repress transcriptional activation. furthermore, deletion of one region, highly conserved with clock exon 19, alleviates repression by pasd1 to suggest that it utilizes molecular mimicry to interfere with clock:bmal1 function. structural and biochemical studies of the direct interaction of pasd1 with the clock:bmal1 complex using recombinant protein expression and biophysical techniques are currently underway. as a cancer/testis antigen, expression of pasd1 is natively restricted to gametogenic tissues but can be upregulated in somatic tissues as a consequence of oncogenic transformation. reducing pasd1 in human cancer cells significantly increases the amplitude of transcriptional oscillations to generate more robust circadian rhythms. our work suggests that mechanisms to suppress circadian cycling can be hard-wired in a tissue-specific manner and our data show that they can be co-opted in cancer cells to attenuate clock function. the scaffolding protein iqgap1 participates in various cellular functions such as cell-cell adhesion, cell polarization and migration, neuronal motility, and tumor cell invasion by binding to target proteins, including rac1 and cdc42, two members of the rho family. to better understand the molecular basis of these interactions, we utilized in this study a novel time-resolved fluorescence spectroscopy to determine individual rate constants for iqgap1 interaction with fourteen different rho proteins. the results indicated that iqgap1 binds among rho proteins selectively to rac-and cdc42-like proteins only in a gtp-dependent manner. moreover, the interaction of rho proteins with the c-terminal half of iqgap1 (grd-c), shorter fragment contains grd-gbd, only the grd and also grd-gbd with single and double phosphomimetic mutations s1441e and s1443d was performed. obtained results showed that, when both grd and gbd are existing, fluorescence changes is detected but for grd alone or in the case of s1443d or s1441e/s1443d no change was observed, suggesting that gbd and specifically, cysteine 1443 is critical for this interaction. furthermore, fluorescence polarization results showed that the grd-c interact with cdc42 and rac1 but not with rhoa, and interestingly the grd domain showed similar behavior, but with 10 to 15 folds lower affinity as compared with the grd-c. consistent with this, a gdp-bound form of cdc42 showed interaction with both grd and the grd-c in quiet comparable affinities. at last, competition experiments utilizing interacting partners of rac1, e.g. tiam1, p50rhogap, plexin-b1, p67phox, pak1 and rhogdia, along with structural analysis, revealed two negative charged areas on the surface of rho-and rnd-like proteins, which might explain their inaccessible interaction with iqgap1. the overlapping binding site of cdc42 and rac1 on the surface of iqgap1 together with the kinetic details of the selective interaction of iqgap1 with rac-and cdc42-like proteins suggests that these interactions are most likely mediated via the same mechanism. ing4 dimerizes through its n-terminal domain, with a symmetric antiparallel coiled-coil structure3, making it a bivalent reader of the h3k4me3 mark. ing5 is highly homologous with ing4, but forms part of a different histone acetyl transferase complex1. here, we show that ing5 is also a dimer and thus a bivalent reader of the h3k4me3 mark. however, the crystal structure of the n-terminal domain of ing5 shows an asymmetric dimer, different from the homologous ing4 domain. our nmr data (backbone assignment and paramagnetic relaxation effects) and saxs data indicate that the structure of the n-terminal domain of ing5 in solution is similar to ing4, suggesting that the crystal structure of ing5 is likely a crystallization artifact. three point mutations in the n-terminal domain of ing5 have been described in oral squamous cell carcinoma: q33r, i68v, and c75r4. we have found that the n-terminal domains of the three mutants are dimeric coiled-coils but with different stability, as measured by thermal denaturation. while the q33r mutant is as stable as the wild type, the i68v and c75r mutants are strongly destabilized, suggesting a role in cancer development at least for these two mutants. efforts so far, to combat alzheimer's disease (ad) have focused predominantly on inhibiting the activity of enzyme(s) that are responsible for the production of the main causative beta amyloid forming peptide. however, the inherent complexity associated with the network of pathways leading to the progress of the disease may involve additional targets for designing effective therapies. recent experimental findings have identified abelson's tyrosine kinase (c-abl), a non-receptor kinase involved in a variety of cellular functions as a new target for ad. in the present study we employed energy optimized multiple pharmacophore modeling strategy from multiple c-abl structures bound with ligands in the inactive atp binding conformation. virtual screening followed by docking of molecules from chembridge_cns database, and maybridge databases resulted in the identification of 15 best scoring molecules. based on docking score and selectivity assessment and druggability parameters, four out of the 15 molecules are predicted to show increased specificity for c-abl in comparison to closely related kinases. given the implied role of c-abl not only in ad but in parkinson's disease, the identified compounds may serve as leads to be developed as effective neurotherapeutics. rafael palomino 1 , glenn millhauser 2 , pietro sanna 2 1 university of california santa cruz, 2 the scripps research institute the central melanocortin system is recognized as a key regulator of energy balance and appetite. the hypothalamic melanocortin receptor, mc4r, is a g-protein coupled receptor that is antagonized by the peptide ligand, agouti-related peptide (agrp), leading to increased feeding and weight gain. while much research has gone into how this ligand exerts its effects at the receptor, less is known regarding nonmelanocortin components of the pathway. syndecan-3, a heparan sulfate proteoglycan, has previously been implicated in potentiating agrp antagonism, however details of this interaction are unclear. this work aims to investigate the role of syndecans at both a molecular level and in vivo. we hypothesize that agrp binds the glycosaminoglycan (gag) components of syndecans, and that this interaction increases the local concentration of the peptide near mc4r. furthermore, we have previously shown that designed positive charge mutations to agrp lead to increased in vivo efficacy that is independent of mc4r activity, and we hypothesize that this is due to greater affinity for the negatively charged gags. using isothermal titration calorimetry we have shown tight binding between agrp and heparan sulfate, the major gag component of syndecan-3, and this affinity is strengthened by additional peptide positive charge. through nmr, we see that both positively charged and polar residues are necessary for binding various heparan sulfate polymers. these data implicate a specific region of agrp that is not required for mc4r binding as being necessary in its role as a heparan sulfate binding protein. expanding on these findings, we are now using a syndecan knockout mouse line to explore the mechanism of differential feeding in our designed mutants. preliminary results indicate a reduction in weight gain in knockouts compared to their wildtype littermates post peptide administration. collectively, these data show that the physiologically relevant form of agrp, previously considered unable to interact with syndecans, is indeed a heparan sulfate binding protein. furthermore, our designed mutants have differential affinities for gags, with increased affinity correlating to increased feeding potency. finally, as the mc4r pathway is thought to be a viable target for wasting disorders such as cachexia, we are interested in leveraging this data to improve the potency and stability of our designed agrp mutants. taken together, this work aims to develop new insights and probe the therapeutic potential of a critical metabolic pathway. evidence of a proteolytic phenomenon in the starch binding domain of the a-amylase from lactobacillus amylovorus zaira esmeralda s anchez cuapio 1 , alejandra hern andez santoyo 2 , sergio s anchez esquivel 1 , romina rodr ıguez sanoja 1 1 instituto de investigaciones biom edicas, universidad nacional aut onoma de m exico, 2 instituto de qu ımica, universidad nacional aut onoma de m exico a-amylases are glycoside-hydrolases that catalyze the hydrolysis of internal a-1,4 glycosidic bonds in starch and glycogen generating smaller oligosaccharides (1). these multidomain proteins contain a catalytic barrel (b/a)8 and, in some cases, one or more non-catalytic domains whose function is generally described as carbohydrate binding module (cbm) and particularly as starch-binding domains (sbd). the sbd can bind granular starch increasing the local concentration of substrate at the active site of the enzyme and may also disrupt the structure of the starch surface (2) . the a-amylase from lactobacillus amylovorus has a structure that consists of a catalytic domain (cd) and an unusual carboxy-terminal starch-binding domain with 5 identical cbms (belonging to family 26) in tandem (3). each repeat acts as an independent fixing module with an additive or synergic effect between the units (4). when we stored pure sbd from l. amylovorus we found multiple forms of low molecular weight with a constant pattern, which does not correspond to random degradation. interestingly, when the protein is stored at ph close to 5 and edta is added, such proteolysis appears to decrease. so far, there is little information about the proteolytic process of amylases and the nature of it. here we show that divalent ions induce a proteolytic cleavage of the sbd, raising the possibility of an autoproteolytic activity. acknowledgments: this work is supported by grants papiit in222113-3 and conacyt 131149. s anchez cuapio z is supported by a personal grant from consejo nacional de ciencia y tecnolog ıa, m exico. unnatural amino acid and related methods provided a special mechanism to implement site-specific spectroscopy active probe incorporation in a specific membrane protein in cells. the site specific incorporation resulted in a single signal during acquisition, resulting in unambiguous signal assignment. the protein specific labeling makes it possible for in situ membrane protein analysis using nmr or fluorescence detection. the 19f containing unnatural amino acid incorporation has been applied for dynamic studies of transporters in native lipid membrane, and the phosphorylation quantification analysis for tyrosine kinase in native lipid membrane with the aid of lipodisc. the fluorescent unnatural amino acid incorporation enabled the site-specific channel responses analysis upon ligand binding in a single cell. heather wiebe 1 , noham weinberg 1,2 1 department of chemistry, simon fraser university, 2 department of chemistry, university of the fraser valley the mechanism by which conformational changes, particularly folding and unfolding, occur in proteins and other biopolymers has been widely discussed in the literature. molecular dynamics (md) simulations of protein folding present a formidable challenge since these conformational changes occur on a time scale much longer than what can be afforded at the current level of computational technology. transition state (ts) theory offers a more economic description of kinetic properties of a reaction system by relating them to the properties of the ts, or for flexible systems, the ts ensemble (tse). the application of ts theory to protein folding is limited by ambiguity in the definition of the tse, although the experimentally observed first-order kinetics for folding of small single-domain proteins lends itself to interpretation by this theory. the pressure dependences of the folding rate constant can be used to obtain activation energies and activation volumes, which are rationalized as the properties of the folding tse. the large amount of activation volume data in the literature has gone largely uninterpreted at the quantitative level. we propose to utilize this data in conjunction with md-calculated volumetric properties to identify the tse for protein folding. the effect of pressure on reaction rates is expressed in terms of logarithmic pressure derivatives, known as activation volumes. according to ts theory, activation volumes can be identified as the difference in volume between the ts and reactant species: activation volumes dv ‡ have been experimentally determined for the folding of several proteins. the concept of activation volume can be extended to that of a volume profile, dv(y), which describes how the volume of a system changes along reaction coordinate y. if the position y ‡ of the ts along the reaction coordinate is unknown, it can be found by locating dv ‡ on the volume profile: such volume profiles can be built using our recently developed md-based displacement volume method.* using this method, volumes of single molecules can be calculated by taking the difference between the volume of pure solvent and solvent containing the desired solute. this method takes into account the strength and type of solvent-solute interactions as well as the geometrical configuration of the solute. in this work, we present the successful application of this method to several conformationally flexible systems. structure of the p15paf/pcna complex and implications for clamp sliding on the dna during replication and repair to the canonical pip-box binding groove on the pcna front face. in contrast to other pcna interacting proteins, however, p15paf also contacts the inside of, and passes through, the pcna ring. the mostly disordered p15paf chain termini thus emerge at opposite faces of the ring, but remain protected from degradation by the 20s core proteasome. we also unveil a novel dna binding activity of p15paf, both free and bound to pcna, which is mainly mediated by its conserved histone-like n-terminal tail. molecular modeling shows that a ternary complex with a duplex dna inside the pcna ring is energetically feasible and our electron micrographs show increased density inside the ring. we propose that p15paf acts as a flexible drag that regulates pcna sliding along the dna, and may facilitate the switch from replicative to translesion synthesis polymerase binding upon dna damage. acknowledgements: this work has been mainly sponsored by mineco grant ctq2011-28680 and juan de la cierva-2010 contract to alfredo de biasio. metabolic syndrome (mets) is one of the leading causes of the death worldwide; however, exact pathophysiological mechanisms of mets remain largely unknown. growing evidence suggests that the increased availability of glucocorticoids at the tissue level play an important in mets development. one of the major determinants of glucocorticoid local action seems to be the enzyme 11b-hydroxysteroid dehydrogenase 1 (11b-hsd1). this enzyme is a well-known member of the short-chain dehydrogenase/reductase (sdr) superfamily. it is an important carbonyl reducing enzyme that, besides its role fine-tuning of glucocorticoids actions, is involved in the biotransformation of drugs and in the development of lung cancer through metabolism of the tobacco specific carcinogen nnk. the phylogenetically closest relative of 11b-hsd1 is dhrs7 enzyme from the same superfamily. unlike 11b-hsd1, dhrs7 is poorly characterized however it can be supposed at least partially overlapping function to 11b-hsd1. moreover its possible association with similar pathological conditions in human as 11b-hsd1 has already been indicated by several studies. the aim of this study is the basic biochemical characterization of dhrs7. the enzyme is a member of cluster 3 of "classical" sdr; such members are considered to be retinoid and steroid metabolizing enzymes, so characterization the enzyme was based on this assumption. dhrs7 was prepared in recombinant form in the sf9 cell line. it was proved that this enzymes is an integral membrane-bound enzyme localized in the endoplasmic reticulum with luminal orientation, similarly to 11b-hsd1. known substrates of 11b-hsd1 and related enzymes were tested also as substrates of dhrs7. it was proved that dhrs7 is nadph-dependent reductase with important substrates as steroid hormones cortisone and androstene-3,14-dione, all-transretinal and also xenobiotics as 1,2-naphtoquinone or carcinogen nnk at least in vitro. for better understanding of the catalytic function of dhrs7 its structural model was prepared and it is used also for the identification of additional substrates by ligand virtual screening. dhrs7 enzyme is expressed in several human tissues as adrenals, liver, prostate, small intestine and kidney. these brand new initial results point to the possible involvement of dhrs7 in important cellular processes that deserve further investigation. these results will lay the foundation for an understanding of dhrs7 role in human physiology resp. pathophysiology. this project was supported by grant agency of charles university (677012/c/2012) and unce 204026/2012). structure-based functional identification of helicobacter pylori hp0268 as a nuclease with both dna nicking and rnase activities bong-jin lee 1 , ki-young lee 1 1 hp0268 is a conserved, uncharacterized protein from helicobacter pylori. here, we determined the solution structure of hp0268 using three-dimensional nuclear magnetic resonance (nmr) spectroscopy, revealing that this protein is structurally most similar to a small muts-related (smr) domain that exhibits nicking endonuclease activity. we also demonstrated for the first time that hp0268 is a nicking endonuclease and a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy. the nuclease activities for dna and rna were maximally increased by mn(21) and mg(21) ions, respectively, and decreased by cu (21) ions. using nmr chemical shift perturbations, the metal and nucleotide binding sites of hp0268 were determined to be spatially divided but close to each other. the lysine residues (lys7, lys11 and lys43) are clustered and form the nucleotide binding site. moreover, site-directed mutagenesis was used to define the catalytic active site of hp0268, revealing that this site contains two acidic residues, asp50 and glu54, in the metal binding site. the nucleotide binding and active sites are not conserved in the structural homologues of hp0268. this study will contribute to improving our understanding of the structure and functionality of a wide spectrum of nucleases. high-fidelity recombinant protein production in a silkworm bioreactor sungjo park 1 , in-wook hwang 1 , tatsuya kato 2 , enoch park 2 , andre terzic 1 1 center for regenerative medicine, mayo clinic, 2 laboratory of biotechnology, shizuoka university the domesticated silkworm, bombyx mori, is an attractive host naturally equipped with a proficient posttranslational modification machinery adequate to fulfill stringent demands of authentic recombinant protein production. silkworm-based protein expression has originally relied on a prototype baculovirus vector system that employs silkworm as a bioreactor in place of more traditional cell lines. recent development of the silkworm trophic b. mori nucleopolyhedrovirus (bmnpv) bacmid launches a second generation of silkworm-based protein production technology. introducing the recombinant bacmid dna into silkworms expedites heterologous protein expression by eliminating prior virus construction and amplification steps. salient examples of heterologous eukaryotic proteins produced in silkworms are acetyl-coa carboxylase 2, malonyl-coa decarboxylase, spot14/mig12 heterodimer and a2,6-sialyltransferase with consistent high levels of protein expression. thus, equipped with a fail-safe post-translational modification machinery, eukaryotic proteins are readily bioengineered using a silkworm-based protein expression platform. studies exploring potential applications of synthetic antifreeze proteins in the frozen food industry ho zee (charles) kong 1 , conrad perera 1 , ivanhoe leung 1 , nazimah hamid 2 , viji sarojini 1 1 school of chemical sciences, the university of auckland., 2 school of applied sciences, auckland university of technology in nature, certain species of plants, insects and fish produce a group of antifreeze glycoproteins and polypeptides which enable them to survive the freezing temperatures of their natural habitat. these naturally occurring antifreeze proteins (afps) were first discovered in polar fishes such as antarctic notothenioids and winter flounder. these afps have the ability to bind to ice crystals and restrict their size and morphology; decrease the freezing point of water and inhibit the ice recrystallization processes. ice crystal formation is of primary concern to the frozen food industry, as ice crystal formation during freezing can be disruptive to and cause damage to the cellular structures in food. the unique properties of afps can be developed into a potential solution to minimize freeze-thaw damage to frozen food. a number of tailor made synthetic analogues based on the naturally occurring afps were successfully designed and synthesized. antifreeze activity studies of the afps were carried out using the clifton nanoliter osmometer attached with a microscope. the afps exhibited thermal hysteresis as well as modification of ice crystal morphology, confirming their antifreeze activity in vitro. the ability of these synthetic afps in preserving the texture and structure of frozen food was evaluated using the techniques of scanning electron microscopy. the afps showed great potential to preserve the cellular structures of frozen food samples during freeze-thaw process. additionally, secondary structure analysis of the afps was carried out using circular dichroism. this presentation will summarize our current results on the design, synthesis and anti-freeze activity analysis of the synthetic afps. invasive fungal infections remain a leading cause of death in immunocompromised patients. current antifungal agents have a host of issues including limited efficacy, host toxicity and an alarming increase in resistance. current research in our laboratories is focused on targeting the calcineurin signaling pathway that has been shown to be required for fungal pathogenesis. calcineurin is a highly conserved serine-threonine-specific ca21-calmodulin-activated phosphatase important in mediating fungal pathogenesis and stress responses. it is a key regulator of a signal transduction network required for survival of the most common pathogenic fungi in humans, making it an ideal target for fungal drug development. calcineurin is a heterodimer of a catalytic (a) and regulatory (b) subunit. phosphatase activity requires association of the two subunits. calcineurin is also the target of the immunosuppressant fk506, which functions as an inhibitor by first complexing with the peptidyl-prolyl cis-trans isomerase immunophilin, fkbp12. the fkbp12-fk506 complex subsequently binds to calcineurin in a groove between the a and b subunits and inhibits its activity. although fungal calcineurins are targeted by fk506, it also targets mammalian calcineurin and is thus immunosuppressive in the host. in order to improve therapeutic efficacy, we have undertaken a unique effort that utilizes both structural biology and molecular mycology in an effort to overcome the fungal versus human specificity barrier. the nmr studies to be presented here have been focused on determining the resonance assignments and solution structures for the fkbp12 proteins from the pathogenic fungi candida albicans, candida glabrata and aspergillus fumigatus. notably, the x-ray crystallography structures of the wild-type candida albicans and aspergillus fumigatus fkbp12 proteins revealed an intriguing intermolecular interaction involving four residues in the 80's loop including pro104 (in c. albicans) and pro90 (in a. fumigatus) which are stabilized in the cis conformation. these data suggest that the protein might use itself as an enzyme substrate. in efforts to establish if this interaction remains in a solution environment, we have determined the nmr structure and measured the t2 relaxation rates for the wild-type a. fumigatus fkbp12 protein and for the p90g mutant variant that adopts a dramatically different orientation of the 80's loop and does not form an intermolecular interaction in the crystal structure. the nmr chemical shift data indicate that, while the remainder of the protein structure remains unchanged, the 80's loops in the two variants are indeed different. in addition, the t2 relaxation rates of the residues in this region are dramatically dissimilar in the two variants, but remain identical throughout the rest of the protein. we have also begun inhibitor binding studies of all of the fkbp12 proteins from each of the pathogens by titrating the fk506 inhibitor into native and mutant fkbp12 proteins in order to examine conformational changes associated in the protein upon complex formation. using this approach we plan to determine the relative kd values for binding of each inhibitor to the fkbp12 protein from each pathogen for comparison of binding proclivities. lupin (lupinus angustifolius l.) b-conglutin proteins: structure functional features, catalytic mechanism modeling and cross-allergenicity identification using protein threading and molecular docking methods lupin is an important pulse, which displays a wide range of benefits in agriculture, particularly these involved in possible plant pathogen suppression. furthermore, lupin seed proteins promote different positive health aspects, preventing cardiovascular disease, and reduction of glucose and cholesterol blood levels. "sweet lupine" seeds seem to be promising as a source of innovative food ingredients due to averaged protein content similar to soybean and an adequate composition of essential amino acids. thus, lupin seeds may be important source of proteins for human and animal consumption. however, and as drawback feature, the number of allergic people to lupin seed proteins is rising, becoming a serious and a growing problem in the western world, because of the rapid introduction of lupin seeds as new ingredients in traditional and novel foods. the goals of this study are the characterization the structure-functional properties of lupinus angustifolius l or narrow leafed lupin (nll) b-conglutin proteins, with a focus in its catalytic mechanism, and its molecular cross-allergenicity with other legumes, i.e. peanut, by extensive analysis using different computer-aided molecular approaches covering (i) physicochemical properties and functional-regulatory motifs, (ii) sequence analysis, 2-d and 3d structural (threading) modeling comparative study and molecular docking, (iii) conservational and evolutionary analysis, (iv) catalytic mechanism modeling, and (v) sequence, structure-docking based b-cell epitopes prediction, while t-cell epitopes were predicted by inhibitory concentration and binding score methods. b-conglutins (vicilin-like or 7s proteins) are seed proteins typically found in reserve tissues (endosperm and cotyledon). they belong to the cupin superfamily of proteins, containing a globular domain constituted by a conserved b-barrel. two barrels were found in all b-conglutin protein isoforms and an additional mobile n-terminal arm constituted bye a-helices. molecular modeling analysis has shown that one of this barrel contain a semi-conserved metal binding motive (hyx. . .r), typically found in oxalate oxidase (oxox) enzymes. interestingly, our results revealed considerable structural differences between b-conglutin isoforms, particularly affecting 2-d elements (loops and coils), and numerous micro-heterogeneities are present in fundamental residues directly involved in epitopes variability, which might be a major contributor to the observed differences in cross-reactivity among legumes. we also identified multiple forms of b-conglutins polypeptides ranging from 15-80kda, with ige-binding characteristics in atopic patients. thus, b-conglutins might be considered as major allergen in different species of lupin, including the "sweet lupin" group, since several of these polypeptides were recognized by human iges, having the potential to trigger an immune response leading to allergy symptoms. 1 influenza virus is one of the most prevalent pathogens causing respiratory illness which often leads to serious post influenza complications such as pneumonia and myocarditis. some viruses, as the avian influenza h5n1, are especially dangerous and draw special attention of who. this highly pathogenic virus spreads quickly among domestic poultry and wild birds resulting in high mortality. what is more distressing, the h5n1 virus may be transmitted to humans. because of antigenic drift it is impossible to deliver an effective vaccine against all subtypes of the h5n1 virus. moreover, traditional egg-based production of influenza vaccines is time-and cost-consuming, what makes it inadequate in case of a pandemic. hence, we have developed an efficient production process of influenza vaccine based on a recombinant hemagglutinin antigen (rha). recombinant vaccines underlay strict regulations and quality requirements. the purpose of this work was to develop a battery of analytical methods that allow to evaluate key quality attributes of rha on each stage of production. at first, we have focused on rha structure as a crucial issue for its activity. the primary structure of rha was confirmed by peptide mapping and tof/tof fragmentation (hplc, maldi tof/tof). furthermore, ftir analysis was used to evaluate the secondary structure of the protein. the disulfide bonds, which stabilize the tertiary structure, were assigned by peptide mapping. additionally, free thiols were measured using ellman's reagent. moreover, we have employed rp-hplc, sec-mals and dls to explore oligomerization of rha. these techniques appeared to be useful not only to confirm existence of native oligomers, but also to find and discard misfolded fraction, aggregates and truncated forms. in addition, two analytical methods (rp-hplc and cge) were developed to assess the purity of rha as required by ich guidelines. we also have determined isoelectric point and heterogeneity of rha by cief. afterward, developed methods were applied in the stability studies that provide a valuable insight into a chemical degradation process and conformational changes of rha during storage. this work was supported by innovative economy operational program, grant no. wnd-poig.01.01.02-00-007/08-00 as a part of project "centre of medicinal product biotechnology. package of innovative biopharmaceuticals for human and animal therapy and prophylactics." muscle cell atrophy via hsp gene silencing was counteracted by celastrol-mediated hsp overexpression molecular chaperone heat shock proteins (hsp) are known to assist protein quality control under various stresses. although overexpression of hsp70 was found to promote muscle mass retention in an unloading state, it is unclear whether muscle atrophy is induced by suppression of hsp expression and is counteracted by active hsp overexpression. in this study, we pre-treated hsp70 sirna to rat l6 cells for the hsp gene-silencing, and determined myotube diameter, hsp72 expression and anabolic and catabolic signaling activities in the absence or presence of triterpene celastrol (cel), the hsp70 inducer. relative to a negative control (nc), muscle cell diameter was reduced by 11% in the sirna-treated group, increased 1.2-fold in the cel-treated group and remained at the size of nc in the sirna1cel group. hsp72 expression was decreased 65% by sirna whereas the level was increased 6-to 8-fold in the cel and sirna1cel groups. expression of foxo3 and atrogin-1 was increased 1.8-to 4.8-fold by sirna, which was abolished by cel treatment. finally, phosphorylation of akt1, s6k and erk1/2 was not affected by sirna, but was elevated 2-to 6-fold in the cel and sirna1cel groups. these results suggest that hsp downregulation by hsp gene-silencing led to muscle cell atrophy principally via elevation of catabolic activities. such anti-atrophic effect was counteracted by cel-mediated hsp overexpression. the centers for disease control and prevention report that at least 2 million people in the united states will become ill due to antibiotic resistant pathogens leading to 23,000 deaths each year. in order to circumvent these resistance mechanisms, it is essential to quantitatively understand how the function of the protein(s) involved relates directly to resistance. integral membrane efflux pumps are known determinants of single-drug and multi-drug resistance in a wide variety of pathogenic organisms. these transporters are proteins whose characterization typically requires reconstitution in an artificial membrane. subsequently, these important proteins are difficult to characterize by traditional in vitro studies. my project aims to determine the physicochemical parameters of the efflux pump tetb utilizing molecular biology and mathematical modeling. tetb is composed of 12 transmembrane (tm) alpha-helices and is found within the inner membrane of gram-negative bacteria. this protein allows for the efflux of tetracycline (tet), doxycycline (dox), and minocycline (mcn) antibiotics from the cytoplasm into the periplasm. these tetracyclines are a bacteriostatic class of antibiotics that inhibit protein synthesis by binding to the 30s ribosomal, therefore, blocking the binding of aminoacyl-trna. for cells grown in tetracyclines, the efflux mechanism of tetb decreases the cytosolic antibiotic concentration allowing for the rate of protein translation to increase. i have inserted a tet(b) expression system into the chromosome of an escherichia coli lab strain and have determined its growth profile under various concentrations of tet, mcn, and dox using a high-throughput 96-well plate format. the growth rate profiles correlate with tetb pumping rates for each drug. tetb more readily pumps out tet compared with dox and mcn and we observe that cells expressing tetb can grow at higher tet concentrations compared with dox and mcn. the shapes of the growth rate profiles produced in the different drugs give insight into the physicochemical mechanism of tetb. we have built a preliminary mathematical model that can simulate these growth profiles and predict efflux pump physicochemical parameters. we are currently working on understanding how efflux expression effects bacterial growth by testing ribosome binding site (rbs) sequences of varying strengths in our tet(b) expression system. future work is geared toward modeling more complex efflux pumps such as the tripartite pumps which traverse both bacterial membranes and cause multi-drug resistance. collectively, this project aims to build an in vivo system which will allow for the characterization of a variety of efflux pumps without the arduous tasks of protein purification and subsequent reconstitution. (2007) identified a small transmembrane region of both kcne1 and kcne3 that are essential for their unique modulation of the kcnq1 channel. by swapping a triplet motif in the transmembrane region of kcne1 and kcne3, we can flip the primary function of these two proteins. while the key for kcne1 and kcne3's unique modulating is believed to lie in this triplet motif, the mechanism and structural changes involved in this modulation is not fully understood. by using nmr spectroscopy, biochemical studies, and computational docking, we aim to look at the structural and conformational differences between kcne1 and the triple mutant kcne1 substituted with the three essential kcne3 residues. we have expressed and purified 15n-labled kcne1 triple-mutant in sufficient quantities for nmr studies in lmpg detergent micelles and other membrane mimetics, and we have collected 2d nmr spectra using a trosy-based pulse sequence. partial backbone assignments of kcne1 triple mutant have been determined by aligning and transfer assignments of the wt kcne1 previous determined in our lab. with the structure of kcne1 triple mutant determined, we aim to computationally dock the triple mutant into a model of the full-length kcnq1 channel in the open and closed state. lastly, we will compare the known structure of kcne1 docked to a model of kcnq1 to that of the kcne1 triple mutant to determine key interactions, significant structural and conformational changes, and how the triple motif region gives rise to its specific structural and functional differences. with this information, we can begin to understand the mechanism of the functional diversity of the kcne family on kcnq1 potassium channel. biochemical characterization of brassica napus diacylglycerol acyltransferase 1 and its regulatory domain .a) expressed in saccharomyces cerevisiae. purified bnadgat1 in n-dodecyl-b-d-maltopyranoside (ddm) micelles behaves as dimers, which can associate further to form tetramers. the acyl donor preference of the major dimeric form with sn-1,2-diolein as acceptor follows the following order: a-linolenoyl-coa > oleoyl-coa 5 palmitoyl-coa > linoleoyl-coa > stearoyl-coa. the first 113 residues of bnac.dg-at1.a corresponding to a soluble regulatory region was expressed in escherichia coli and purified. truncation of this soluble domain reveals that the dimeric interface is located within residues 49-113, while the first 48 residues allow formation of tetramers. this n-terminal region was implicated as an allosteric exosite for acyl-coas as revealed by previous lipidex-1000 binding studies. in the current study, circular dichroism spectroscopy and isothermal titration calorimetry were used to probe the binding kinetics and thermodynamics. dgat1 appears to shift between two oligomerization states, a phenomenon that may be related to regulation of enzyme activity and mediated by the n-terminal domain. alteration of lysine and arginine content as a strategy to modify such an interaction was found to increase the activity of rdrp in vitro. further, deletion of c terminal 43 amino acid residues also resulted in increase in the polymerase activity that was comparable to the full length rdrp-p10 complex. it was proposed that the conserved c terminal disordered domain of rdrp was responsible for interaction with p10 and modulation of the activity. in the present study, role of the c terminal disordered domain was further investigated by determining the oligomeric status of the complex and the c terminal deletion mutants of rdrp and also by quantitating the rdrp-p10 interaction using surface plasmon resonance. size exclusion chromatography revealed that rdrp eluted in the void volume of the column whereas a significant fraction of the rdrp-p10 complex eluted at a position corresponding to the size of the 1:1 complex of rdrp and p10 (77kda). activity measurements indicated that the heterodimeric complex was more active than the aggregate eluting in the void fraction. interestingly, the c terminal deletion mutants of rdrp (c del 43 & c del 72 rdrp) were also found to be less aggregated as compared to full length rdrp and some of the protein eluted at a position corresponding to the respective monomers. these monomers were also more active than the aggregate fractions. these results demonstrate that the increase in activity observed either upon interaction with p10 or deletion of the c terminal domain could be due to the change in the oligomeric state of rdrp. in order to further analyze the interaction of rdrp with p10 surface plasmon resonance was used. rdrp and its deletion mutants were immobilized on biacore sensor surface and p10 protein was used as an analyte. full length rdrp and c del 43 rdrp were shown to interact with p10 with kd values of 0.6 and 1 um respectively. however, c del 72 and c del 85 rdrp did not show any binding with p10. these results suggest that the region 43-72 from the c terminus of rdrp is essential for the interaction with p10. further, the c del 85 rdrp was inactive although c del 72 rdrp continued to be active suggesting that residues 72-85 from the c terminus are crucial for rdrp activity. further studies are in progress to identify the residues within these motifs that may be essential for the activity or interaction with p10. aggregation of androgen receptor in spinal bulbar muscular atrophy is a multistep process spinal bulbar muscular atrophy (sbma) is a member of the polyglutamine (polyq) expansion diseases, like huntington disease, and it is caused by a genetic expansion of the polycag tract in exon 1 of androgen receptor (ar) that codes for the polyq region. sbma is a late onset disease, which involves a progressive degeneration of the motor neurons and consequent muscular atrophy. there is still no treatment available for this disease. ar is a nuclear receptor that responds to testosterone and that regulates the expression of the masculine phenotype. it is composed of an intrinsically disordered nterminal domain (ntd) that bears the polyq tract, a dna binding domain and a ligand binding domain. aggregates of ar protein with an extended polyq are observed in the motor neurons of sbma patients. in vitro studies showed that aggregation of androgen receptor takes place only in presence of testosterone1 and that the cleavage of the protein by caspase 3 is a crucial event for cytotoxicity. however, there is no clear knowledge of the mechanism of aggregation, for this protein. an increasing body of evidence supports the hypothesis that the aggregation of these proteins is controlled by regions flanking the polyq tract, by regulating the rate of aggregation depending on their secondary structure. we have applied nuclear magnetic resonance (nmr) and circular dichroism for generating information on the secondary structure of the n-terminal cleavage product of ar by caspase 3 and we have studied its aggregation with a set of biophysical methods, like dynamic light scattering, an hplc sedimentation assay and transmission electron microscopy. we have found that the polyq tract of ar presents a high degree of helicity. we attribute this conformation to the n-terminal flanking region, characterized by high helicity and we have tested this hypothesis by performing mutations. we have also observed that the rate of the first step of oligomerization is not dependent on the number of glutamine repeats, but instead is due to self interactions of a region n-terminal to and far from the polyq. its progression to fibril is dependent to the number of glutamines in the tract. we have therefore identified two steps in the aggregation process of ar, where a motif far from the polyq at its n-terminal drives the early oligomerization, followed by the interaction of the polyq chains that stabilize it and determine the progression to fibrils. these findings shed a light for possible interventions on the ar oligomerization process, thus suggesting a different strategy to study the onset of the disease in sbma patients. destabilizing the transient helical conformation of islet amyloid polypeptide hastens peptide self-assembly and potentiates cytotoxicity carole anne de carufel 1 , phuong trang nguyen 1 , alexandre arnold 1 , isabelle marcotte 1 , steve bourgault 1 1 amyloidogenic polypeptides can be divided into two different structural classes: those that are intrinsically disordered and those that show a well-defined structure in their monomeric soluble state. natively folded proteins, such as transthyretin, have to unfold (or misfold), at least partially, to form amyloids. in contrast, intrinsically disordered polypeptides, such as the islet amyloid polypeptides (iapp) and abeta peptide, need to undergo conformational rearrangements allowing the formation of locally ordered structure(s) to initiate the amyloidogenic process. studies have shown that iapp and abeta adopt an alphahelix conformation in the initial steps of amyloidogenesis. this intermediate is believed to be on-pathway to fibril formation, although this hypothesis is still the matter of debate. in this study, we designed human iapp (hiapp) derivatives in which alpha-helix destabilizing substitutions were incorporated into the putative helical segment of iapp to probe the initial structural event in amyloid formation. using trifluoroethanol titration, we observed by cd spectroscopy that strategic incorporation of d-amino acids at positions 15 and 16 leads to an iapp derivative (diapp) that cannot fold into a helix. in homogeneous solution, hiapp and diapp show similar kinetics of fibrillization, as measured by thioflavin t fluorescence. although their amyloid fibrils display different characteristics by afm, iapp and diapp are able to self-associate to form amyloids when mixed together and when seeded with one another. studies in heterogeneous environment, notably in presence of glycosaminoglycans and model membranes of dopc/dopg (7:3), showed a helical intermediate for hiapp while only a beta-sheet secondary structure was apparent for diapp. while the rate of amyloid fibril formation was increased for both peptides, diapp was drastically affected by these anionic biomolecules with an absence of lag phase. the incapacity of adopting a transient helical conformation accentuates cell toxicity, supported by the caspase 3/7 activation level and the increase in intracellular calcium level. overall, this study indicates that the helical intermediate is offpathway to iapp amyloid formation and offers novel mechanistic insights for the development of molecular identities modulating peptide self-assembly and iapp-induced cytotoxicity. for an organism to survive, its proteins must adopt complex conformations in a challenging environment where macromolecular crowding can derail even robust biological pathways. the situation is perilous: many diseases arise from improper folding of just a single protein. to cope, cells employ a repertoire of molecular chaperones and remodeling factors that usher unfolded proteins into active conformations, sequester them, or target them for degradation. yet, not all aggregated proteins are the result of mis-folding. yeast prions are self-templating protein-based mechanisms of inheritance that rely upon chaperones for their propagation. the best studied of these is the prion domain (nm) of sup35, which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. using genetic, biochemical, spectroscopic, and solid state nmr techniques, we investigated the structural and dynamic underpinnings of sup35 amyloids and found that prion strains differ in both their atomic structure as well as their dynamic motions. interestingly, these mobility differences correlate with differences in the interaction with molecular chaperones in vivo. limitations on the specificity and sensitivity of biophysical techniques typically restrict structural investigations to purified systems at concentrations that are orders of magnitude above endogenous levels. therefore, i developed an approach to apply a sensitivity-enhancement technique for nmr, dynamic nuclear polarization (dnp), to investigate interactions between sup35 and molecular chaperones at endogenous concentrations in their native environments. critically, i found that the cellular environment induced structural changes in a region of sup35 that is intrinsically disordered in purified samples but known genetically to influence prion propagation from one generation to the next. this approach enables structural and mechanistic investigation of proteins in biologically relevant contexts. genetic instability within regions encoding repetitive proteins as a driver of adaptation stephen fuchs 1 1 more than ten percent of all eukaryotic proteins contain within them a region of repetitive amino acid sequence. these repetitive domains range from short stretches of a single amino acid to multiple copies of longer, heterogeneous amino acid sequences and generally show lack of defined structure. they play diverse roles in cells including acting as structural proteins, promoting cell-cell interactions, and mediating the assembly of molecular machines. tandem repeat proteins are known to be variable in length within cellular populations although the mechanisms dictating this variability have not been elucidated. here we describe work uncovering specific features within the coding sequences of repetitive proteins that contribute to tandem repeat instability in yeast. furthermore, we demonstrate that cells will expand and/or contract repetitive regions in order to adapt to environmental stresses and describe a role for dna repair proteins in this process. lastly, we demonstrate how these mechanisms are likely conserved in higher eukaryotes, including humans. this study uncovers the molecular basis for an important aspect of natural protein evolution and describes a novel mechanism for adaptation in response to environmental changes. a proline-tryptophan turn in the intrinsically disordered domain 2 of ns5a protein is essential for hepatitis hepatitis c virus (hcv) nonstructural protein 5a (ns5a) and its interaction with the human chaperone cyclophilin a (cypa), a peptidyl-prolyl cis-trans isomerase (ppiase), are both targets for highly potent and promising antiviral drugs that are in late stage of clinical development [1, 2] . despite its high interest in the development of drugs to counteract the worldwide hcv burden, ns5a is still an enigmatic multifunctional protein poorly characterized at the molecular level. ns5a is required for hcv rna replication and is involved in viral particles formation and regulation of host pathways. thus far, no enzymatic activity or precise molecular function has been ascribed to ns5a that is composed of a highly structured domain 1 (-d1), as well as two intrinsically disordered domains 2 (-d2) and 3 (-d3). ns5a-d1 structure has been solved by x-ray crystallography and ns5a-d2 and -d3 have been characterized by nmr spectroscopy. these two last domains do not adopt a stable 3d structure but rather exist as an ensemble of highly dynamic conformers. using nmr spectroscopy, hcv ns5a-d2 has been shown to establish a direct interaction with the human cypa and to be a substrate for the enzymatic ppiase activity of cypa [3] . the cypa interaction site in ns5a-d2 is composed of nearly 15 residues that correspond to the most conserved region of the domain, with 3 proline residues being strictly conserved among all hcv genotypes. whereas ns5a-d2 is mainly disordered, some of its nmr resonances, corresponding to residues in the cypa binding site, display unexpected 1h and 15n nmr chemical shifts for an intrinsically disordered domain. thus we have further characterized this region by nmr spectroscopy. a short structural motif in the disordered ns5a-d2 has been identified and we solved its nmr structure. in a cellular assay, we showed that this structural motif, a minimal pro314-trp316 turn, is essential for hcv rna replication. we demonstrated that this pro-trp (pw) turn is required for proper interaction with the host cypa and influenced its enzymatic ppiase activity on residue p314 of ns5a-d2. this work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of hcv ns5a protein. in addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function. [1] [2] . this 27-residue peptide also shows toxicity towards mammalian cells but at higher concentrations, suggesting its possible usefulness as a treatment for trypanosomiasis. here we present the peptide's relative cytotoxicity for bloodstream and procyclic forms of t. brucei and for mammalian cells, the fate of the peptide in t. brucei using fluorescently-labelled bt-6, and its three dimensional structure using nmr spectroscopy.minimum inhibitory assays confirmed the peptide's selective toxicity towards both bloodstream and procyclic forms of t. brucei, demonstrating its potential to serve as a starting point for a trypanocidal drug. fluorescence spectrophotometric experiments, carried out using fluorescein labelled bt-6, show that the peptide is released from the external surface of the parasite into the suspending medium under de-energized conditions but retained in energized cells. heteronuclear and homonuclear biomolecular nmr experiments (tocsy, noesy, 1h-13c-hsqc,1h-15n-hsqc, etc) folowed by structural calculations (chemical-shift based as well as simulated annealing techniques) in the free state indicate that this peptide is mostly unstructured in aqueous solution, suggesting that there is a major conformational change upon binding to t. brucei that is required for uptake. we suggest that the evolutionary pressure that selected for the intrinsically disordered structure of this peptide was the advantage it conferred upon the host to bind to many different surface structures throughout the microbiological world. physikalische biologie, heinrich heine university, 2 structural biochemistry (ics-6), research centre j€ ulich, 3 chemistry and biotechnology, swedish university of agricultural sciences (slu) the misfolding and amyloid formation of proteins featuring intrinsically disordered regions is a pathological hallmark of several neurodegenerative diseases, including alzheimer's disease and parkinson's disease. engineered binding proteins targeting amyloidogenic proteins aid in the elucidation of the aggregation mechanism and suggest therapeutic strategies. we have constructed phage display libraries enriched in binders to amyloidogenic intrinsically disordered proteins, using zab3, a protein with high affinity for the amyloid-beta peptide, as a scaffold. binding proteins selected from these libraries are termed beta-wrapins (beta-wrap proteins). the beta-wrapins as69 and hi18 exhibit nanomolar affinity for monomeric alpha-synuclein or islet amyloid polypeptide, respectively. as69 and hi18 potently inhibit in vitro amyloid formation and toxicity at substoichiometric concentration ratios, indicating that they interfere with the nucleation and/or elongation of amyloid fibrils. the nmr structures of the betawrapin:target complexes reveal beta-hairpin motifs in alpha-synuclein and islet amyloid polypeptide which are stabilized by coupled folding and binding. in the case of alpha-synuclein, the beta-hairpin is formed in the sequence region 35-59 which contains the beta-strand segments b1 and b2 of amyloid fibril models and most disease-related mutations. we show by disulfide engineering, biophysical techniques, and cell viability assays that intramolecular tertiary interactions between the b1 and b2 segments of alpha-synuclein interfere with its aggregation, and moreover inhibit aggregation of amyloid-beta peptide and islet amyloid polypeptide. our results reveal a common preference of different amyloidogenic proteins for formation of beta-hairpin motifs and demonstrate a critical role of hairpin conformers in the control of amyloid formation. interaction profiling through proteomic peptide phage display cecilia blikstad 1 , moon-hyeong seo 2 , norman davey 3 , roland arnold 2 , sachdev s sidhu 2 , philip m kim 2 , ylva ivarsson 1 1 department of chemistry -bmc, 2 donnelly centre a considerable part of the human proteome is intrinsically disordered. the disordered regions are enriched in short motifs serving as docking sites for peptide binding domains. domain-motif interactions are crucial for the wiring of signaling pathways. these interactions are typically transient and difficult to capture through most conventional high-throughput methods. we therefore developed a novel approach for the large-scale profiling of domain-motifs interactions called proteomic peptide phage display (prop-pd) (1). in prop-pd we combine bioinformatics, oligonucleotide arrays, peptide phage display and next-generation sequencing. this allows the interrogation of domain-motif interactions on a proteome-wide scale and the de novo motif discovery.in our pilot experiment we generated two distinct phage libraries, one displaying all human c-terminal sequences and one displaying c-termini of known virus proteins. we used the prop-pd libraries to identify interactions of human postsynaptic density 95/discs large/zonula occludens-1 (pdz) domains. we successfully identified novel pdz domain interactions of potential relevance to cellular signaling pathways and validated a subset of interactions with a high success rate. recently, we created a prop-pd library that displays peptides representing the disordered regions of the human proteome. we validate our disorderome library against a range of peptide binding domains, which provides novel insights into their binding preferences and suggest interactions of potential biological relevance as will be presented here. prop-pd can be used to uncover protein-protein interactions of potential biological relevance in high-throughput experiments and provides information that is complementary to other methods. prop-pd is scalable and can be developed to any target proteome of interest. phosducin is a 30 kda phosphoprotein that regulates visual signal transduction by interacting with the gtbg; subunit of the retinal g-protein transducin. the function of pdc is regulated by phosphorylation at ser54 and ser73 in a process that involves the binding of phosphorylated pdc to the regulatory 14-3-3 protein, but the molecular mechanism of the regulation by 14-3-3 protein is still unknown. pdc was also suggested to be involved in transcriptional control, the regulation of transmission at the photoreceptorto-on-bipolar cell synapse, and the regulation of the sympathetic activity and blood pressure [1] [2] [3] . here, the solution structure of pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, circular dichroism, quenching of tryptophan fluorescence, analytical ultracentrifugation, hydrogen-deuterium exchange coupled to mass spectrometry and nuclear magnetic resonance. we show that the 14-3-3 protein interacts with and sterically occludes both the n-and c-terminal gtbg binding interfaces of phosphorylated pdc, thus providing a mechanistic explanation for the 14-3-3depedent inhibition of pdc function. the 14-3-3 protein dimer interacts with pdc using surfaces both inside and outside its central channel. the n-terminal domain of pdc, where both phosphorylation sites and the 14-3-3 binding motifs are located, is intrinsically disordered protein which remains likely highly flexible when bound to 14-3-3 indicating the fuzzy-like character of this complex. in addition, it has been speculated that the 14-3-3 protein binding decreases the rate of pdc dephosphorylation after a light stimulus through its interaction with phosphorylated ser54 and ser73, thus lengthening the time that pdc remains phosphorylated after a light exposure. pdc is dephosphorylated in vivo by protein phosphatases known to cause neurodegenerative disease in a polyglutamine-length dependent manner. despite intense study, the molecular basis of polyq toxicity in hd or any of the other diseases has only partially been elucidated and potential routes to therapeutic intervention are sparse. the use of genetically tractable model organisms to identify the cellular pathologies caused by mutant huntingtin expression is essential to our understanding of the disease pathology in humans. in eukaryotes, many of the protein folding homeostasis pathways are highly conserved and yeast cells expressing a glutamine-expanded fragment of huntingtin exon 1 exhibit a polyq length-dependent toxicity that recapitulates many of the basic protein folding defects associated with polyq diseases in neurons. taking an unbiased approach, we screened an overexpression library of the entire yeast genome for suppressors and enhancers of polyq toxicity and identified seven proteins with prion-like, q-rich domains that are strong suppressors in yeast. intriguingly, the q-rich domains of these proteins, and several other q-rich domains, suppress toxicity when expressed in isolation. these suppressors are also efficacious in mammalian cells and, strikingly, one suppressor was independently shown to alleviate polyq-expanded ataxin-3 toxicity in a drosophila model. in yeast, the suppressors co-aggregated with an otherwise highly toxic 103glutamine expanded huntingtin exon 1 protein (htt103q), resulting in a non-toxic aggregate and eliminating populations of diffusible oligomeric species. using a transcriptional sensor for protein coaggregation, we determined that yeast and human proteins that normally co-aggregated with htt103q did not co-aggregate with these hetero-aggregates. thus, these q-rich domains may suppress htt103q toxicity by two complementary mechanisms: trapping potentially toxic oligomers in larger aggregates and by limiting the interactome of the larger htt103q aggregates. structuring disorder: the case of the intrinsically disordered unique domain of c-src mariano maffei 1 1 about two thirds of eukaryotic proteins contain large intrinsically disordered regions. they represent a change of paradigm from "structure-function" to "information-function" (uversky, 2011; babu et al., 2011). structured proteins are information rich, but the current challenge is to discover how information is stored in disordered protein. regulation of c-src activity, the first discovered oncoprotein, by its intrinsically disordered n-terminal region has been recently demonstrated (perez et al., 2013). functional studies have revealed that mutations in the ulbr cause strong phenotypes when introduced in fulllength c-src and expressed in xenopus laevis oocytes (perez et al., 2013) or in human sw620 colorectal cancer cells (unpublished). however, the connection with the classical regulatory mechanisms is still missing. c-src domain structure consists of four "src-homology" domains: sh4, sh3, sh2 and sh1, arranged in this order from the n-terminus to the c-terminus, with the intrinsically disordered "unique" domain separating the sh4 and sh3 domains. classically, the sh3 and sh2 domains are involved in regulation and the sh4 domain is the membrane anchoring site. we will present our recent results showing that the unique domain is part of a long loop closed by the interaction of the sh4 and sh3 domains (maffei et al., 2015). the conformational freedom of this disordered region is further restricted through direct contacts between the rt-loop of the sh3 domain and, primarily, residues located within the recently discovered unique lipid binding region (ulbr). the interaction between the unique and sh3 domains is allosterically modulated by a poly-proline ligand binding to the canonical binding site of the sh3 domain (maffei et al., 2015) . these results demonstrate a direct connection between classical c-src regulation involving the sh3 domain and the new regulation mechanisms involving the intrinsically disordered regions and provide new evidence of the functional importance and the underlying mechanism behind regulation of signalling pathways by intrinsically disordered domains. in mammalian cells, the golgi reassembly and stacking proteins (grasp55 and grasp65) are involved in the stacking of golgi apparatus cisternae and in the formation of the golgi ribbon. since grasps have been identified in many organisms, other roles for grasps have already been pointed out, such as chaperoning and transport of other proteins, involvement in cell apoptosis, cell migration, unconventional secretion, and in mitosis. in saccharomyces cerevisiae, it is observed that only 40% of the golgi cisternae are in stacks and do not form ribbon structures. this build yeast contains a single grasp, called grh1, that is analogue to grasp65. the structural differences of the golgi apparatus and the functional repertoire of grasps suggest a structural dynamic of these proteins. here, we used a combination of biophysical/biochemical methods to investigate the behavior of grh1. bioinformatics and circular dichroism (cd) analyses of grh1 indicated a high percentage of either flexible regions or extended loops. the partial unfolded grh1 structure in solution folded into more ordered structures under temperature increasing, dehydration onto a surface and nonaqueous solvents as reported also by cd. hydration of the dehydrated folded protein is a reversible process that is accompanied by unfolding. furthermore, grh1 showed slow migration in sds-page, high susceptibility to proteases and low cooperativity of the chemical-induced unfolding process. fluorescence of trp residues along with cd data showed grh1 preserves a considerable amount of residual secondary structure, and the unfolding transition monitored by trp presented higher cooperativity. another cooperative transition was also reported by the extrinsic hydrophobic fluorescence probe ans upon chemical denaturation. these set of experiments indicate that grh1 behaves as a protein containing intrinsically disordered regions (idrs), characterized by unstructured regions of high polypeptide mobility experiencing many conformations. these findings suggest that an idp-like behavior may be the solution found by nature to account for grh1 functional need for interactions with several different partners in the cell. conformational changes governing dengue virus capsid protein function and its inhibition by pep14 23 andr e f. abstract dengue virus (denv) infection affects millions of people and is becoming a major global disease for which there is no specific treatment available. the interaction of denv capsid (c) protein with host lipid droplets (lds) is essential for viral replication. pep14-23, a peptide designed based on a denv c intrinsically disordered conserved region, inhibits this crucial interaction. combining bioinformatics and biophysics we determined pep14-23 structure and ability to bind different phospholipids, in the context of denv c function. pep14-23 becomes a-helical upon binding to anionic phospholipids. structure prediction of denv c n-terminal intrinsically disordered region reveals orientations that alternatively shield or expose denv c hydrophobic pocket, supporting a novel autoinhibitory role for this region. these findings pave the way for similar studies to understand disordered proteins and improved peptidomimetics drug development strategies against flaviviruses. topics intrinsically disordered proteins protein-lipid interactions pf-015 developing mechanistic insight into modulators of tau aggregation eri nakatani-webster 1 , hannah baughman 1 , shaylin higgins 1 , abhinav nath 1 1 the pathological self-association of microtubule-associated protein tau is implicated in a range of neurodegenerative disorders collectively called tauopathies, perhaps the most prominent of which are alzheimer's disease (ad) and chronic traumatic encephalopathy (cte). tau aggregation in vitro shares many features in common with fibril formation by other amyloid-forming proteins: a nucleationdependent polymerization reaction progressing via oligomeric intermediates into b-sheet-rich fibrillar aggregates, characterized by a distinctive sigmoidal kinetic. over the years, many investigators have advanced our understanding of how these time-courses might best be characterized and interpreted. in particular, elegant analytical and numerical approaches have been developed that supersede the empirical sigmoidal equations typically used to fit fibril formation traces. these modern approaches have enabled more rigorous insight into the mechanism of amyloid formation, and into how small molecules, protein chaperones, and other binding partners can modulate the process. an understanding of a modulator's effects on amyloid formation mechanism is necessary in order for us to predict and engineer its effects on amyloid pathology in a biological context. a given modulator may affect rates of primary or secondary nucleation, elongation, or fibril fragmentation to different extents. each of these perturbations, individually or in combination, can alter the kinetics of aggregation, the final state of the amyloid fibrils, and the sampled ensemble of oligomeric intermediates. unfortunately, fitting of mechanistic models to amyloid formation kinetics is an example of an "ill-posed problem", in that dramatically different combinations of elementary parameters can nevertheless generate very similar sigmoidal kinetic traces. this has typically necessitated global analysis of amyloid kinetic traces collected over a broad range of protein concentrations -a substantial expenditure of time, effort and material that must then be repeated in the presence of a modulator in order to gain insight into its effects. we propose an alternative approach: to fit amyloid formation traces to a large distribution of parameter sets, and determine how various aggregation modulators affect the distribution of parameters. this socalled "parameter distribution analysis" enables the inference of mechanistic effects from measurements at a single protein concentration. parameter distribution analysis based on numerical modeling has been made tractable by advances in computer hardware and software, and can be easily extended to include additional mechanisms or phases relevant to a protein or modulator of interest. here, we illustrate how parameter distribution analysis, complemented by fluorescence correlation spectroscopy (fcs), electron microscopy (em) and other biochemical techniques, can shed light on fundamental aspects of tau amyloidogenesis. we examine the disparate effects that natural products, pharmacotherapies and protein chaperones can have on the mechanism of aggregation, and also discuss the effects of heparin (widely used as an inducer of tau aggregation). these insights demonstrate the value of parameter distribution analysis as applied to amyloid formation and other ill-posed biochemical problems. new insights into amyloidogenesis of tau protein induced by enantiomers of polyglutamic acid amyloidogenesis of tau protein leads to the formation of amyloid fibrils (ordered fibrillar protein aggregates) which are accumulated in neurons of central nervous system during the course of neurodegenerative diseases called tauopathies. studying tau (a typical intrinsically disordered protein) amyloidogenesis has been challenging for many reasons. positive charge on the tau molecule must be compensated (e.g. in the presence of polyanions) in order to initiate the process. heparin (glycosaminoglycan) has been the most intensively studied charge-compensating agent in this context. on the other hand induction of tau aggregation by polyglutamic acid is poorly characterized. mechanisms responsible for the propagation of tau conformations has become an interesting research objective. prion-like features of tau amyloid can be studied in vitro also in the seed-induced regime of aggregation. tau amyloid seeds can act as nuclei for amyloidogenesis. such seeds can be obtained by fragmentation of amyloid fibrils by means of sonication. given that amyloidogenesis can proceed through various assembly pathways resulting in distinct amyloid 'strains' (self-propagating structural variants of amyloid) we have used poly-l-glutamic acid (plga) and poly-d-glutamic acid (pdga) to direct tau onto different amyloidogenic pathways. we have hypothesized that the chirality of the inducers could lead to fibril polymorphism. in our studies, we have used a recombinant human 2n4r tau isoform. we have been using transmission electron microscopy (tem), sedimentation and kinetic measurment. firstly, we have characterized unseeded plga-/pdga-induced tau aggregation to find out that corresponding kinetics were significantly different. secondly, we have used sonicated fibrils to characterize the kinetics of seeded processes. both plga-/pdga-induced amyloid seeds were able to efficiently seed tau aggregation in the presence of plga, whereas in the presence of pdga the aggregation was much less effective. surprisingly, we found that pdgainduced amyloid seeds were able to catalyze fibrillogenesis of tau more clearly in the presence of soluble plga than in the presence of pdga -the primary inducer. we could not induce aggregation of tau in the absence of polyglutamic acids which indicates that positive charge on tau molecules must be unconditionally compensated in order to promote amyloidogenesis. thirdly, using tem we have characterized different morphologies of tau amyloid fibrils generated in unseeded and seeded processes. finally, to further characterize properties of the fibrils we have performed sedimentation experiments. fibrils induced by plga, pdga and heparin revealed different sedimentation properties. heparin-induced fibrils underwent sedimentation more readily than pdga-induced fibrils, whereas plga-induced fibrils remained in the supernatant. these results indicate distinct physicochemical properties of these fibrils. we believe that our findings will contribute to the current understanding of the molecular dynamics of tau amyloidogenesis. self-organizing structures of alpha-synulceins and its aggregates by a coarse-grained monte carlo simulation ras pandey 1 , peter mirau 2 , barry farmer 2 1 alpha-synuclein (asn) consisting of 140 residues, an intrinsically disordered protein, is linked to such neurodegenerative diseases as parkinson's disease (pd) and alzheimer disease via toxic clumping into abstract amyloid fibrils. we investigate the structure and dynamics of an asn chain as a function of temperature by a coarse-grained approach where a residue is represented by a node. in our coarse-grained approach, a residue is represented by a node. the basic idea is borrowed from the 'united atom' approach in polymer chain modeling that has been used extensively where the benefits and pitfalls of the method is explored for decades. such coarse-grained method has also been used protein chain modeling in recent years (e.g. aip advances 5, 092502 (2015)). although the atomic scale structural resolution is sacrificed its specificity is captured via a set of unique knowledge-based residue-residue interactions matrix (e.g. classic miyazawa-jernigan matrix, macromolecules 18, 534 (1985)). a number of local and global physical quantities are analyzed such as contact map, neighborhood and mobility profiles, mean square displacement of protein, its radius of gyration and the structure factor. based on the mobility profile, we are able to identify three distinct segment of asn along its contour, i.e. sluggish nterminal (1-60) and c-terminal (96-140, least mobile) separated by the central region (61-95), the nonamyloid component (nac) with higher mobility. contact profile shows that the probability of intrachain residue aggregation (clumping) is higher in the n-terminal region than the c-terminal with least aggregation in the nac region. we find that the radius of gyration (rg) decays monotonically with the temperature, consistent with the finding of allison et al. (jacs, 131, 18314 (2009) ). from the detail analysis of the structure factor we are able to predict the variation of the spatial mass distribution with the temperature as the residues in asn chain organize and disperse by evaluating its effective dimension d. we find the protein conforms to a globular structure (d3) at the low temperatures and to a random coil (d2) at high temperatures which is consistent with the estimates of uversky et al. (j. biol. chem. 277, 11970 (2002)). in addition, we provide the estimates of d (3 d 2) for the intermediate structures as the protein chain makes a transition from globular to random coil. questions under-investigation includes what are the effects of mutations (e.g. b-and g-synuclein), how does the structure of an isolated asn chain change in presence of many interacting protein chains, and how do they organize over the multiple length scales? attempts will be made to address some of these issues as the data become available. tear down the wall: dismantling the biofilm scaffold of e.coli cesyen cedeno 1 , nani van gerven 1 , wim jonckheere 1 , imke van den broek 1 , han remaut 1 , peter tompa 1 1 csga is the major subunit of the so-called curli fiber system. this is an amyloid structure formed in the outer membrane on e.coli and acts as a scaffold for the biochemical machinery/matrix in the extracellular milieu (biofilms). extracellular matrices of this nature are robust platforms helping bacteria colonization; in this context csga becomes a key target in order to break the architecture within bacterial biofilms. chaperones are molecular machines able to stabilize misfolding prone proteins or even retrieve proteins trapped in non-physiological states. here we show how erd14 acts as a molecular chaperone inhibiting the formation of csga amyloid fibers in vitro. this work illustrates an alternative approach towards biofilm treatment at a molecular level. coupled folding and binding of transcription factors sarah shammas 1 , alexandra travis 1 , jane clarke 1 1 intrinsic protein disorder is ubiquitous in transcription, particularly within transcription factors, which frequently fold into structures upon binding to partner molecules (dna or protein). the coupled folding and binding reactions that take place between individual transcription factors and the key hub co-activator proteins are crucial in determining the expression profile of the cell, and hence its phenotype. these interactions have been well studied by structural and equilibrium methods. here we present mechanistic insights into the process, gained through complementary kinetics experiments, for the binding of five separate transcription factors to a single prototypical co-activator (cbp kix). the transcription factors investigated belong to cellular (cmyb, mll, creb, e2a) and viral (htlv-1 blz) classes. these reactions are remarkably fast; after removing the effect of long-range electrostatic rate enhancement the association rate constant is still approximately 2 x 10 7 m-1s-1, which is just above the typically quoted upper limit for diffusion-limited reactions between pairs of proteins (10 5 -10 6 m-1s-1), and is also the highest such value we have found reported. this, combined with the apparent insensitivity of the association rate to residual structure within the unbound state, indicates that binding preceeds folding (induced fit mechanism). interactions between kix and its transcription factors are additionally modulated by allostery between its two binding sites. we investigate the basis for this, finding it to be mediated by changes in protein flexibility. alternative hit finding strategies for intrinsically disordered proteins, exemplified by forkheadbox transcription factors harm jan (arjan) snijder 1 , maria saline 1 , tomas jacso 1 , frank janssen 1 , mattias rohman 1 , tyrrell norris 1 1 astrazeneca r&d, discovery sciences, se-431 83,pepparedsleden 1 forkhead box o (foxo) proteins are emerging as key transcription factors in insulin and glucose metabolism, regulation of immune responses, and to balance cell proliferation, apoptosis and senescence. foxo proteins are predicted to be intrinsically disordered proteins (idps); idps are largely unstructured and often function as hubs mediating multiple interactions. idps are considered to be largely evasive from classical small molecule interference and lead-generation approaches, as they lack defined binding pockets. the available methods for addressing these targets have been lagging behind and needs to be developed to assess tractability of this target class. here we have evaluated the tractability of fragment screening on various domains of a forkhead box o member. we could confirm the intrinsically disordered character of foxo and used nmr screening to identify fragments that interact with foxo. one of these fragments was subsequently confirmed as a direct foxo binder in 2d hsqc-nmr spectroscopy and this fragment showed an effect in a foxo reporter gene assay. these results demonstrate that fragment screening may be a valuable approach for intrinsically disordered proteins although challenges remain to expand these fragments into more potent hits in the absence of detailed structural data. the characterization of amyloid-beta peptide (abeta) oligomer samples is critical to advance in the field of alzheime rs disease (ad). here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), extensively used in the field, and electrospray ionization ion mobility coupled to mass spectrometry (esi-im-ms), an emerging technique with great potential for oligomer characterization. to evaluate their performance, we first obtained pure cross-linked abeta40 and abeta42 oligomers of specific order. analysis of these samples by sds-page revealed that sds affects the oligomerization state of abeta42 oligomers, thus providing flawed information on their order and distribution. in contrast, esi-im-ms provided accurate information, while also reported on the chemical modifications and on the structure of the oligomers. our findings have important implications as they challenge scientific paradigms in the ad field built upon the sds-page characterization of abeta oligomer samples. coarse-grained simulation of protein association: application to rate prediction and implication for association mechanisms yinghao wu 1 , 1 the kinetics of protein binding is of paramount importance for understanding cellular functions. for instance, the binding kinetics between membrane receptors and their ligands control the speed of signal transduction after cells are exposed to stimulation. the experimentally measured association rates of protein binding span ten orders of magnitude, a range that was divided into two regimes. it was proposed that a fast association regime is limited by protein diffusion, while the other side of the spectrum is controlled by conformational changes. consequently, all previous simulation methods neglected conformational changes when calculating the association rate of a diffusion-limited regime. however, the most updated theory of protein binding suggests that a protein remains in a pre-existing equilibrium of unbound conformations. binding shifts the equilibrium toward its bound state. this highlights the importance of conformational factors for regulating protein binding. enlightened by this conformational selection model, we hypothesize that the conformational flexibility of protein structures regulates association more widely than previously anticipated. we develop a new coarse-grained model to simulate the process of protein association via the kinetic monte carlo (kmc) algorithm. each residue in this model is represented by its ca atom and a side-chain functional site. a simple physically based potential is used to guide the relative diffusion of two interacting proteins. given the size of the simulation box and the length of the simulation, the association rate constant can be derived by counting the frequency of dimerization among a large number of simulation trajectories. we further designed a prediction strategy that accounts for both the conformational and energetic factors of binding. our method is able to predict rates of protein association that are highly correlated with experimentally measured values. due to the coarse-grained feature, our model was further applied to several special cases of protein association. in one example, we studied the binding kinetics of proteins with flexible linkers. the interaction between thrombin and its functional inhibitor, rhodniin, was used as a testing system. we captured the conformational changes of flexible linkers from the all-atom molecular dynamic simulations. we found that the association with full-length flexible rhodniin was faster than its two individual domains and that their dissociation was more difficult, supporting a "flycasting" mechanism in which partial structures of an intrinsic disordered protein (idp) dock to the target first, while the remaining segments undergo conformational searches and sequentially coalesce around the target. in another example, we studied the binding kinetics of membrane receptors from cellular interfaces. the interaction between membrane proteins cd2 and cd58, cell adhesion molecules known to mediate the activation of t cells and natural killer cells, was used as a testing system. the diffusive properties of these proteins on lipid bilayer were captured from all-atom molecular dynamic simulations. we showed that both 3d and 2d association rates could be simulated quantitatively with our method. the calculated values were close to the experimental measurements. we also provided detailed analysis of how molecular diffusions and membrane fluctuations affected 2d association. pf-023 (un)structure-function relationships on the ureg enzyme in the nickel-dependent urease system barbara zambelli 1 , francesco musiani 1 , stefano ciurli 1 1 urease is an essential enzyme for many pathogens and soil microorganisms. its activity relies on the presence of nickel in the active site (1) . the incorporation of this metal ion into the enzyme requires the formation of a supra-molecular chaperone involving four accessory proteins, named ured, uref, ureg and uree. uree is a metallo-chaperone involved in nickel binding and delivery into the enzyme active site. ureg is a gtpase essential for providing energy to the process of nickel site assembly. uref and ured form a complex that regulates the gtpase activity of ureg. the present work focuses on ureg, which exists in solution as an ensemble of inter-converting conformations (2) . this observation made this protein the firstly discovered natural enzyme with an intrinsically disordered behavior, possibly allowing it to interact with different protein partners, such as uree (3, 4) and uref (5) and cofactors, such as metal ions (6), in the urease activation network. ureg folding was studied perturbing protein conformation with temperature and denaturants, and investigating its folding response using circular dichroism, nmr and fluorescence (7). a combination of light scattering, calorimetry, mass spectrometry, and nmr spectroscopy shed light on the effect of metal ion binding onto the conformational equilibrium of ureg ensemble (8) . the results suggest that metal binding and solution conditions modulate affect the protein-protein interactions and enzymatic activity of ureg. nuclear inclusion protein a-protease (nia-pro) is a protease involved in processing of pepper vein banding virus (pvbv) encoded polyprotein to generate various intermediates and mature proteins at different stages of the viral life-cycle. nia-pro has two domains-n-terminal viral protein genome linked (vpg) and the c-terminal protease domain (pro).vpg belongs to the group of proteins that are intrinsically disordered, but attain stable structures upon interaction with other globular proteins. such proteinprotein interactions have a regulatory role on the function of the interacting partners. previously, the influence of vpg domain on the activity of pro was studied and it was shown that there was a substantial increase in the protease activity upon interaction with vpg (both in cis and in trans). in the present investigation, several deletion mutants of vpg and nia were constructed with a view to delineate the domain of vpg involved in interaction with pro. it was observed that deletion of residues from nterminus of vpg resulted in a decrease in the activity of pro in cis and in trans probably because of the abrogation of interaction between the two domains. interaction studies using spr (surface plasmon resonance) and elisa confirmed that the n-terminal 22 residues of vpg are important for interaction with pro. the n-terminal 22 residues of vpg are a part of the disordered region of vpg and their deletion resulted in the change in the secondary structure of the vpg and its oligomeric state. the ser 129 and trp 143 residues of pro domain were shown to be important both for the interaction of the two domains and for the activity of protease by mutational analysis earlier. these residues were identified to be a part of wc loop (w143-c151) which relay the conformational changes to the active site catalytic triad (his 46, asp 81 and cys 151) leading to activation. however, mutations of these residues did not completely abolish the protease activity as well as the interaction with vpg. therefore, in the present study h142 and h167 which are observed to interact with trp143 and c151 (via non-covalent interactions) were mutated to alanine and the h142a and h167a mutants showed a drastic reduction in the activity of protease. molecular dynamics simulations of the wild type pro and the mutants revealed that trp 143 -his 142 -his 167 -cys 151 interaction pathway of the wild type pro was disrupted in the mutants and additional residues were involved in the interaction pathway, such alterations in the network of interactions could be responsible for the loss of activity. however, a change in the oligomeric status of these mutants was also observed as compared to the wild-type pro, suggesting that these residues are important for both the structural and functional integrity of pro and its interaction with vpg. thus, these results provide a molecular insight into the vpg-pro interactions and the modulation of their structure and function upon mutation of residues that are part of the interaction interface. transthyretin (ttr) is one of many proteins that are capable of forming amyloid fibrils in vivo. this protein is associated with two distinct amyloidosis: familial cardiac amyloidosis (fca) that causes a restrictive cardiomyopathy and familial amyloid polyneuropathy (fap) that affect peripheral nerves, they are hereditary and caused by mutations in the ttr gene. the non mutated protein can also aggregate in cardiac tissue in advanced age patients. the diagnosis was established at university hospital since 2008 due to a collaborative between our group and the center of amyloidosis antônio rodrigues de mello (ceparm). the only mutation found in brazil was v30m in 3 patients diagnosed in france. our group discovered 5 new mutation not described in brazil and a novel mutation not described yet a19d. the diagnosed patients are registered in transthyretin amyloidosis outcomes survey (thaos). the novel mutation a19d causes a severe restrictive cardiomyopathy that is certainly related to a higher profile of aggregation observed for this mutant if compared to others amyloidogenic mutants of ttr. structural predictions using a bioinformatics tool called foldx showed that the insertion of the mutation cause a electrostatic clash that facilitates the dissociation and aggregation of protein. this mutant was purified heterologously and biophysical studies revealed that this protein is a dimer and not a tetramer as commonly the ttr structure. the crystallographic structure indicates that this mutant is structurally identical to wild type. biophysical studies revealed that this protein is a dimer and not a tetramer as commonly the ttr structure. the thermodynamic stability of a19d is lower than the wild type ttr. the aggregation profile showed us that this protein can aggregate in a higher manner and with a fast kinetic to that observed for others amyloidogenic mutants of ttr, forming fibers in two hours of aggregation. heterotetramers of a19d and wt are able to aggregate in the same fiber structure. the analysis of interface interaction of this mutant using the pdbsum showed modifications in the profile of hydrogen bonds and non bonded contacts. in addition the oligomers of a19d are toxic for primary culture of cardiomyocytes from murine heart. the amyloidogenic profile displayed by this new mutant can be directly correlated with the aggressiveness observed in the disease developed by the identified patient. furthermore the recent consolidation of ttr diagnosis in our university hospital led to the identification of the rare a19d variant in a brazilian patient, suggesting that other new, uncharacterized mutants could be identified in the coming years. multiple cellular proteins interact with ledgf/p75 through a conserved unstructured consensus motif [1] . the ledgf/p75-mll1-menin complex was structurally characterized, but only partially [2] . using nmr spectroscopy, we identified and mapped a novel mll1-ledgf/ p75 interface. colony forming assays in mll1-af91 leukemic cells expressing mll1 interactiondefective ledgf/p75 mutants revealed that this additional interface is essential for leukemic transformation. interestingly, the newly defined interface overlaps with the binding site of known ledgf/p75 interactor, the hiv integrase [1] . while the pathophysiological interactions of ledgf/p75 are intensively studied, its physiological role remains unclear. since ledgf/p75 contributes to hiv integration and leukemic transformation and has become a new therapeutic target for drug development, it is crucial to study its physiological interactions. in addition to hiv in and mll1-menin, the ledgf/p75 integrase binding domain (ibd) also interacts with several other proteins [3, 4] . our recent data (manuscript accepted in nat. commun.) revealed structural details of ledgf/p75 interactions with physiological binding partners. the interaction with the ledgf/p75 ibd is maintained by an intrinsically disordered ibd-binding motif (ibm) common to all known cellular partners. based on the knowledge of this motif, we identified and validated iws1 as a novel ledgf/p75 interaction partner. naturally occurring single mutants, i56t, f57i, w64r and d67h of lysozyme in human, have been known to form abnormal protein aggregates (amyloid fibrils) and to accumulate in several organs, including liver, spleen and kidney, resulting in familial systemic amyloidosis. these human pathogenic lysozyme variants are considered to raise subtle conformational changes compared to the wild type. here we examined the effects of the aberrant mutant lysozymes i56t, f57i,w64r and d67h, each of which possesses a point mutation in its molecule, on a cultured human cell line, hek293, in which the genes were individually integrated and overexpressed. western blot analyses showed lesser amounts of these variant proteins in the medium compared to the wild type, but they were abundant in the cell pellets, indicating that the modified lysozyme proteins were scarcely secreted into the medium but were retained in the cells. immunocytochemistry revealed that these proteins resided in restricted regions which were stained by an endoplasmic reticulum (er) marker. moreover, the overexpression of the mutant lysozymes were accompanied by marked increases in xbp1s and grp78/bip, which are downstream agents of the ire1_ signaling pathway responding to the unfolded protein response (upr) upon er stress.rnai for the mutant lysozymes' expression greatly suppressed the increases of these agents. next, we addressed the interaction between amyloidogenic lysozyme and grp78/bip as the former proteins were obtained by immunoprecipitation with the latter protein as well as colocalization of both proteins in the er. lysozyme composes of a-domain rich in helices and b-domain rich in sheet. two helices of a1 and a2 in the n-terminal region arrange in parallel and face to face where hydrophobic amino acids at the 3f, l8, l12, l15, l25 and l31 allocate with equal interval there. in the back of dock, there is a core region of amyloid fibril formation, of which the side chain of i56 is exposed on the protruding. probably, these hydrophobic amino acids might be crucial for lysozyme folding. although mutated lysozymes undergo folding by grp78/bip in such environment, the dissociation of the grp from lysozyme by failure of folding is likely inhibited and both proteins remain bound to, resulting in staying to the er. a part of aberrant lysozymes seem to remain bound to grp78/bip during folding and insolubilize with aggregation, thus accumulate in the er accompanied with er stress. lysozyme amyloidosis might be caused by long-term accumulation in the endoplasmic reticulum of the abnormal protein. structural characterization of toxic oligomers that are kinetically trapped during alpha-synuclein fibril formation the accumulation of abnormally aggregated proteins within the body is a common feature of several medical disorders, such as alzheimer's disease, parkinson's disease and diabetes mellitus type 2. while the specific protein found to be the major component of such deposits varies from one disease to another, the formation of the pathological aggregates seems to occur via a common process of misfolding and self-assembly of a normally soluble polypeptide chain into a series of oligomeric intermediates and, ultimately, into insoluble amyloid fibrils that accumulate within specific organs and tissues. increasing evidence indicates that certain oligomeric protein species generated during the self-assembly of specific proteins into ordered fibrillar aggregates can be highly cytotoxic and are likely to be key players in the initiation and spreading of neurodegenerative diseases. however, little detailed structural information is currently available for these oligomeric species due to their often transient nature and, more importantly, because of their variability in terms of size and structure. we report here the isolation and detailed characterization of an ensemble of stable toxic oligomers of alpha-synuclein, the protein whose deposition is the hallmark of parkinson's disease. by defining and minimizing the degree of heterogeneity of these isolated alpha-synuclein oligomers which have accumulated during the process of amyloid formation, we have identified distinct subgroups of oligomers and determined their structural properties and three-dimensional molecular architectures. this characterization has been achieved by the application of a set of complementary biophysical techniques, including a variety of spectroscopic techniques along with analytical ultracentrifugation, atomic force microscopy, and electron microscopy. although these oligomers exist in a range of sizes, with different extents and nature of beta-sheet content and exposed hydrophobicity, all the oligomeric subgroups possess hollow cylindrical architectures with marked similarities to amyloid fibrils. this suggests that these types of oligomers are kinetically trapped during protein self-assembly and that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their beta-sheet structures. our findings reveal the inherent multiplicity of pathways of protein misfolding and the key role the beta-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the rates of structural conversions, and thus the kinetic stabilities and pathological nature of different amyloid oligomers. the results of this study provide the basis for a more complete understanding of the nature of the self-assembly of polypeptides into beta-sheet rich amyloid aggregates, and potentially contributes to efforts to identify specific targets for drug discovery. fish otoliths and mammalian otoconia, biominerals composed of calcium carbonate and organic matrix, are involved in the functioning of the inner ear, the sensory organ that plays an important role in hearing and balance [1] . however, their developmental origins, growth, and the role of the matrix, especially the protein component, are still poorly understood. it has been shown that proteins involved in the formation of biominerals are usually very acidic. they often belong to the group of intrinsically disordered proteins (idps), a class of proteins devoid of a rigid tertiary structure [2, 3] . the shape and polymorph selection of calcium carbonate otolith in danio rerio is controlled by the starmaker (stm) protein [4] . recently, a gene was identified encoding the starmaker-like (stm-l) protein from oryzias latipes, a putative homologue of stm. it has been suggested that stm-l has a similar function as stm, although there is no sequence similarity between stm and stm-l [5] . several methods, such as size exclusion chromatography, cd spectroscopy and analytical ultracentrifugation demonstrated that stm-l is an coil-like idp, with the tendency to form locally ordered structures [6] . because stm-l was suggested to play a crucial role in calcium carbonate mineralization, it is possible that calcium ions may influence its conformation, as was previously shown for stm [7] . however, other ions may also be involved in this process. the aim of this study was to investigate the effect of mono and divalent metal ions on the conformation of stm-l. we used single molecule f€ orster resonance energy transfer (smfret) and fluorescence correlation spectroscopy (fcs), which have shown that calcium ions compacts the proteins most efficiently, followed by magnesium and the monovalent ions. the difference in the effect of monovalent and divalent ions on the protein dimensions is likely to result from the different properties of the ions, like charge density and radius. cd experiments have shown that a high excess of calcium ions caused the formation of ordered secondary structure in stm-l, which may be crucial for the formation of calcium carbonate crystals, when the ratio of building ions to protein is high. it has been demonstrated that dmp1 is proteolytically processed into fragments, including 37k n-terminal region and 57k c-terminal region. as many proteins characterized to be engaged in biomineralization, dmp1 and its fragments belong to the group of intrinsically disordered proteins (idps). it has been suggested that dmp1 and its fragments can take a part in otoconia mineralization, as the protein is present in mouse otoconia, but the role of dmp1 and its fragments in the mineralization of calcium carbonate has not been examined until now. to determine the influence of the dmp1 fragments for otoconia development, 57k dmp1 protein was expressed in bacterial expression system, purified and used in in vitro biomineralization test of calcium carbonate. in particular, immobilized metal anion affinity chromatography (imac) was applied as a first step of purification procedure. because of high content of acidic amino acids, ion exchange chromatography with a mono q column was used as a next step. the development of insects is regulated by the combined action of ecdysteroids and juvenile hormones (jh). pulses of 20-hydroxyecdysone (20e) initiate each step of metamorphosis, while jh modulates its action and prevents precocious differentiation. the biological and molecular mechanism of 20e action is well described. in contrary, the way of the jh activity is still poorly understood. in 1986 wilson and fabian [1] reported that drosophila melanogaster mutants lacking met are resistant to toxic doses of jh and its analogue methoprene. it has been proved, that met binds jh at physiological conditions. therefore met is believed to be a putative jh receptor. met may also be involved in a cross-talk between two hormonal signalling pathways, involving 20e and jh. the detailed structure of met is still unknown. therefore our main aim is to characterize structural properties of met. in silico analysis performed on a full-length met suggested, that n-terminal part of met contains three conserved domains characteristic for bhlh-pas transcription factors, whereas c-terminal part is most probably unstructured. 2010)). capitalizing on self-and cross-amyloid interactions, we designed highly effective, peptide-based inhibitors of amyloid self-assembly of abeta and iapp. due to their favourable properties the designed peptides are promising leads for targeting protein aggregation in ad, t2d or both diseases while the inhibitor design strategy should be applicable to other amyloidogenic polypeptides and proteins as well. apoptosis, the process of programmed cell death, must be carefully regulated in multi-cellular organisms to ensure proper tissue homeostasis, embryonic development and immune system activity. the bcl-2 family of proteins regulates the activation of apoptosis through the mitochondria pathway. dynamic interactions between pro-and anti-apoptotic members of this family keep each other in check until the proper time to commit to apoptosis. the point of no return for this commitment is the permeabilization of the outer-mitochondrial membrane (omm). translocation of the pro apoptotic member, bax, from the cytosol to the mitochondria is the molecular signature of this event. molecular interactions and conformational changes associated with this event have been difficult to obtain due to challenges associated with taking subtle measurements in the complex environment of live cells. to circumvent these challenges, we developed a novel method to reliably detect f€ orster resonance energy transfer (fret) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in bax as this translocation occurs in live cells. in the cytosol, our fret measurements indicated that the c-terminal helix is exposed instead of tucked away in the core of the protein. this coincided with measurements using fluorescence correlation spectroscopy (fcs) that showed that cytosolic bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. we propose that this exposed helix allows for this contact to occur. cross-linking the c-terminal helix (a9) to helix a4 reduced the instances of these interactions while at the same time yielded fret measurements that are consistent with the a9 helix tucked into the core of the protein. after translocation, our fret measurements showed that bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the bh3 domain (helix a2) and the c-terminal helix. these findings provide insight into the molecular architecture that may involve possible contacts with other bcl-2 proteins to permeabilize the omm, which would also be necessary for the regulation of apoptosis. abstract spatial resolution is especially advantageous for bacterial cells because of their small sizes. in the past few years the spatial organization and dynamics of a variety of bacterial cellular structures and protein macromachineries have been revealed with unprecedented details. as the field matures, it is now time to focus on the functional aspect of the observed spatial organizations and dynamics. are they essential in carrying out a specific cellular function? do they play a regulatory role in controlling the on and off of a certain cellular process? in this work i will present a few examples from our laboratory that examine the spatial and functional organization of macromolecules involved in bacterial cell division. transcription factors (tf) exert their function by interacting with other proteins and binding to dna. the nucleus is a compartmentalized space, and the spatial organization of tfs and their partners represents other step of gene expression regulation. we used the glucocorticoid receptor (gr) as a model of tf's mechanism of action. gr is a ligand-activated tf with a relevant role in physiology and a great variety of effects. it can be recruited to specific response elements on dna or interact with other tfs. also, the activity of gr is modulated by different co-regulators, e.g. tif2/grip1. gr and tif2 do not distribute homogeneously within the nucleus but accumulate in distinctive clusters. the functional role of this particular intranuclear organization remains unknown. we used advanced fluorescence microscopy techniques to study the dynamics of gr and tif2 in the nucleus of living cells with high spatial and time resolution. gr and tif2 fused to fluorescent tags were transiently expressed in newborn hamster kidney (bhk) cells and visualized by a confocal microscope. fluorescence correlation spectroscopy (fcs) experiments were carried on to measure the intranuclear mobility of both proteins. the method is based on the analysis of fluorescence intensity fluctuations due to the movement of fluorescent molecules in and out the confocal volume. the data could be fitted with a model that considers a free diffusion of tif2 and gr in the nucleus and their binding to fixed targets. we also studied the dynamics of different gr mutants in the presence of different ligands and our results suggest that the binding depends on dna. both gr and tif2 autocorrelation curves reveal an increase in the bound population upon gr activation by its agonist dexamethasone (dex). a cross-correlation analysis showed that, as expected, dex-stimulus increases the population of gr-tif2 complexes. without hormone, gr shows a homogeneous distribution and tif2 forms large clusters in the nucleus. upon dex-binding, gr accumulates in the nucleus, is rapidly recruited to tif2 foci and there is an important re-distribution of both proteins, that co-localize in the same pattern of small intranuclear clusters. the dynamics of gr and tif2 molecules at these clusters were studied by performing orbital-scanning measurements, tracking the clusters position in silico and analyzing the intensity fluctuations of the clusters along time. a positive cross-correlation between both channels indicates that dex-bound gr and tif2 interact at these foci and dissociate from them forming tif2-gr hetero-complexes. in conclusion, advanced fluorescence microscopy methods allowed obtaining a dynamical map of gr distribution and function in the nucleus of mammalian living cells. assembly of membrane pores as a mechanism for amyloid cytotoxicity by the bacterial prionoid repa-wh1 cristina fern andez 1 , rafael n uñez-ramirez 1 , mercedes jimenez 1 , germ an rivas 1 , rafael giraldo 1 1 amyloid fibril formation is associated with human neurodegenerative diseases. prefibrillar oligomers formed during the fibril assembly process, rather than mature fibrils are known to be central to disease abstract and may be responsible for cell damage. a commonly proposed mechanism for the toxicity of small oligomers is their interaction with the lipid bilayer of cell membranes, leading to loss of membrane integrity [1] . recent studies from our laboratory have shown that repa-wh1, a winged-helix domain from a bacterial plasmid replication protein, can assemble into amyloid fibrils in vitro. when expressed in escherichia coli repa-wh1 functions as a cytotoxic protein that shares features with the mammalian amyloid proteinopathies. these features have proved repa-wh1 to be a suitable synthetic model system to study protein amyloidosis [2, 3, 4] . in this work, using the repa-wh1 bacterial model system, we have studied the interaction between the protein and model membranes (large and giant unilamellar lipid vesicles, luvs, and guvs respectively). repa-wh1 shows association and aggregation to membranes composed of anionic phospholipids. protein association in guvs did not result in lysis of the vesicles, suggesting the assembly of discrete protein pores as the mechanism for repa-wh1 membrane damage. to investigate the formation of pores we analyzed by electron microscopy the aggregation of repa-wh1 in the presence of a pre-formed e. coli lipid monolayer. the em images show the presence of pore-like particles on the monolayer. amyloid pores formation explains the permeabilization effect of repa-wh1 in vesicle models and is in agreement with observations for human amyloidogenic proteins. the approaches presented here provide a deeper insight into amyloid cytotoxicity towards membranes and will make possible the assay of inhibitors and effectors of amyloidosis under controlled conditions. references: b2-adrenergic receptor (b2ar) is a member of g protein-coupled receptors, which represent the single largest family of cell surface receptors involved in signal transduction. b2ar recognizes a variety of ligands and communicates with cytoplasmic g-proteins by transmitting signals through the cellular membrane. thus, investigation of communication pathways for b2ar may give important insights for understanding its allosteric mechanisms and identifying new target sites for more specific and efficient drug molecules to be used in the treatment of pulmonary and cardiovascular disease. in this study, various conformations from 2 ms molecular dynamics (md) simulations and available crystal structures of human b2ar were investigated to reveal alternative signaling pathways between its extra and intracellular regions. specifically, shortest communication paths connecting key residues (more than 35 å apart) at the orthosteric ligand binding site (d113, s203, t286, f289, n312) to either l266 or s329 located near the g-protein binding site were investigated. the conformers from previous md simulations [1] include the intracellular loop 3 (icl3), which especially affects the transmembrane collective dynamics but is lacking in x-ray structures. the protein was described as a graph composed of nodes linked by edges. nodes were placed at the alpha-carbon atoms and the edges were calculated based on the number of atom-atom interactions within a cut-off distance 4.5 å for each residue pair. twenty shortest pathways were revealed using k-shortest path algorithm [2] on the coarse-grained network. our results indicated that distinct signaling paths progressed most frequently on tm6 but alternative paths were also present, which passed partially through tm5, tm7, tm3 or tm2 depending on the conformation. among the critical residues that transmitted the signal between distant sites, f282 and n318 were detected, whose functional roles were reported in previous experimental studies. pathway shifting was observed depending on the open-to-closed transition of icl3 during md simulations. the sulfonylurea receptor 1 (sur1) is an atp binding cassette (abc) protein that forms the regulatory subunit in katp channels found in the pancreas and the brain. mgatp binding and hydrolysis at the two cytosolic nucleotide binding domains (nbd1 and nbd2) in sur1 control gating of the katp channel pore. 1,2 proper regulation of katp channel gating by sur1 is critical. 2 over 100 mutations that lead to diabetes, hyperinsulinism and developmental delay have been identified in different domains of sur1, including the nbds. 3 therefore, molecular-level understanding of the structure and function of the nbds is essential for designing improved treatments for sur-related diseases. here we present biophysical and biochemical studies aimed at understanding the effect of disease-causing mutations on the conformation and nucleotide binding of sur1 nbd1. specifically, we are investigating sur1 nbd1 mutations that cause neonatal diabetes (r826w and h863t) or congenital hyperinsulinism (c717 d, g716v, r824g, r837 d and k890t). 3 our nuclear magnetic resonance (nmr) data shows that the hyperinsulinism mutation k890t causes chemical shift changes throughout the spectrum of nbd1, implying overall changes in protein conformation that may affect mgatp binding and inter-domain interactions with other domains in the sur1 protein. size-exclusion data show that the other hyperinsulinism mutations (c717 d, g716v, r824g, r837 d) produce mostly aggregated protein, likely as a result of misfolding of nbd1. misfolding of nbd1 may be the underlying cause of reduced katp trafficking seen with these mutations and hence decreased katp channel gating observed in hyperinsulinism. in contrast to the k890t mutations, the congenital diabetes-causing mutations (r826w and h863t) cause few nbd1 nmr spectral changes. however, the congenital diabetes mutation r826w decreases the affinity of nbd1 for mgatp, which is unexpected for congenital diabetes mutations. our fluorescence, circular dichroism and microscale thermophoresis data corroborate the results that we have obtained by nmr spectroscopy. our data provide molecular-level details on the effects of disease causing mutations in human sur1. egfr increased stability: rmsf of the ca atoms during the md simulations suggest that glycosylation is associated with dampened motions, suggesting that the glycans stabilize the structure. subdomain iii is the most stabilized while subdomain i is stabilized largely in the proximity of the ligand. both dimer interfaces including the dimerization arm from domain ii and the tip of domain iv fluctuate less upon glycosylation. hydrogen bonding; persistent interactions seen for protein-glycan: in the disaccharide-containing system, we observed three highly occupied hydrogen bonds between the glycans and domain iii and iv of egfr. hydrogen bonds of domain iii involve the residue asp323 in which a sidechain oxygen interacts with oxygen atoms of the n-acetylglucoseamine linked to asn328. in domain iv a hydrogen bond is seen between the cys 515 backbone amide and the oxygen atom of n-acetylglucosamine linked to asn 504. in the oligosaccharide-containing system hydrogen bonds observed between the glycan attached to asn 172 and domain ii. these hydrogen bonds form between the gln193 sidechain oxygen atom and cys 191 backbone oxygen atom and the mannose linked to asn 172. the reduction in the mobility of these amino acids suggests that hydrogen bonds impart stability to both the sugars and to the interacting egfr. insects possess a complement-like immune response utilizing thioester-containing proteins, or teps. the only arthropod tep of known structure is anopheles gambiae tep1, which is a key component in the natural immunity of this mosquito to malaria parasites (genus plasmodium). unlike vertebrate complement factors, agtep1 does not contain an anaphylatoxin domain which acts to regulate a massive conformational change accompanying activation of the protein. the mechanism of agtep1 must therefore involve an alternative mechanism for allosteric regulation of thioester activation. in place of a small internal domain, a large, heterodimeric complex of two leucine-rich repeat (lrr) proteins, lrim1 and apl1c, have been shown to specifically bind and stabilize the active conformation of agtep1. i will present my group's most recent work in this area. we have shown that different alleles of tep1, which are known to influence the vectoral capacity of wild mosquitoes, differ significantly in their susceptibility to thioester hydrolysis. allelic variation is centered on residues at the protein-protein interface within tep1 containing the thioester bond. the lrim1/apl1c heterodimer is shown to form an extended and flexible ensemble in solution. two closely-related genes to apl1c, apl1a and apl1b, can also form a complex with lrim1, and apl1b lrr domain can form a homodimer. we propose that a flexible and heterogeneous group ensemble of lrim1/apl1 dimers interact with the active conformation of tep1, thereby producing an array of immune complexes to protect mosquitoes from a diverse set of pathogens. human flap endonuclease-1 (hfen1) is an essential metallo-nuclease involved in okazaki fragment maturation and long-patch base excision repair. during these processes, bifurcated nucleic acid intermediates with ssdna 5'-flaps are generated by polymerase strand displacement synthesis and then cleaved one nucleotide into the downstream duplex by fen1 to create a nicked-dna that is a suitable substrate for ligase. until recently, how hfen1 achieves tremendous catalytic power (rate enhancements >10exp17) and exquisite selectivity for the scissile phosphate had been understood poorly (1) . in 2011, the grasby and tainer labs solved the structures of hfen1 in complex with product and substrate. this study revealed that scissile phosphate selectivity is largely due to the substrate dna undergoing a novel di-nucleotide unpairing (dnu), which places the scissile phosphate diester in contact with the requisite divalent metal ions. in addition, by comparing the structures of hfen1 alone (2) and in complex with substrate and product dnas (3), grasby and tainer proposed a model, whereby protein conformational changes occur upon binding substrate resulting in placement of key basic residues that position and/or electrophillically catalyse hydrolysis of the scissile phosphate diester. further work using a cd-based assay showed that metals are absolutely required for dnu, whereas the key basic residues in the active site are not. surprisingly, perturbations to the protein structure that are much more distant from the fen1 active site (i.e., helical cap) prevent dna unpairing, implying that the fen1 protein actively participates in the unpairing process (4,5); however, how it does remains a mystery. the maximal multiple turnover rate of hfen1 reaction is rate-limited by enzyme product release, whereas hfen1 kinetics under substrate-limiting conditions ([e]<[s]<km) suggest that the enzyme is approaching catalytic perfection (i.e., almost diffusion controlled -10exp7 m-1s-1) (6) . previous work has shown that a paralogue of hfen1 (i.e., t5 fen1) is not ratelimited by chemistry under single turnover (st) conditions, but rather some physical limitation such as conformational change (6) . to ascertain whether the protein and/or dna conformational changes of hfen1 mentioned above are rate limiting under st conditions, we have initiated kinetic and nmr studies to determine the role of conformational dynamics in the catalytic cycle of hfen1. owing to the latest advance in the computational technologies of microprocessor, high-speed inter-processor connection, and parallelization algorithm, all-atom molecular dynamics (md) simulations of microsecond time scale are getting popular to study the pharmaceutical target proteins. an antibody binds to its antigen with structure changes. a small molecule moves around the target protein and finds an entrance to get into the binding site. these phenomena can be observed in microsecond simulations, but the vital issue is whether the adopted force field is enough accurate to correctly describe the phenomena. we are developing a force field (fuji) based on general amber atom types and amber94 van der waals parameters in order to describe arbitrary organic molecules in a unified manner including proteins and nucleic acids. we use the first principle theoretical method to refine the force field in contrast to common empirical fittings to experimental data. the dihedral torsion parameters of protein backbone were determined to agree with the torsion energy profiles calculated by high-level quantum mechanical theory for the model systems of protein backbone. conformational preferences of dipeptides in water were measured by vibrational spectroscopy. comparing with the experimental distribution of ramachandran angles of dipeptides, how accurately molecular calculations predict the conformation distribution of dipeptides were investigated for various force fields and semiempirical quantum methods. fuji force field gave the best prediction score among the various methods (tzanov et al, 2014) . in this work we examine dynamical structure behaviors of pharmaceutical target proteins by microsecond md simulations with fuji force field. epiregulin (epr) is a transmembrane protein with 140 residues belonging to the epidermal growth factor family. mature epr (46 residues) binds to epidermal growth factor receptor (egfr), which stimulates the proliferative signaling in cancer cells. our epr antibody has a proline at the residue 103 in the third complementarity-determining region (cdr) of the heavy chain, which has cis peptide bond in free state and trans one in the bound state with epr according to x-ray crystallography analysis. in order to clarify the structure changes in binding we performed extensive md simulations of our antibody (fab; 436 residues) and epr. the total simulation time was about 240 microseconds. we also performed md simulations for a protein kinase and a small molecule. we show how the complex system reaches thermal equilibrium states in simulations from the stiff x-ray crystal structure. the calculations are compared with experiments such as x-ray crystal structures and thermodynamic quantities measured by isothermal titration calorimetry (itc) and surface plasmon resonance (spr). this research has been supported by mext spire supercomputational life science (hp130006, hp140228) and first kodama project in japan. structure-based recombination of drug resistance enzymes: structural and functional tolerance to new dynamics in artificially-evolved enzymes our understanding of the contribution of protein dynamics to function is still emergent. in a protein engineering context, do we need to take into account the dynamics in order to maximize the fitness and function of the resulting proteins? using high resolution crystal structures, nmr relaxation dispersion and ms molecular dynamics simulations, we compare two naturally evolved homologous class a b-lactamases, tem-1 and pse-4 which share a high degree of structural and functional conservation. we observed a conservation of dynamics on a catalytically relevant timescale. this is consistent with dynamics being an evolutionarily conserved feature. however, laboratory-engineered chimeric enzymes obtained by recombination of the two homologs exhibit striking dynamic differences, despite the function and structure being conserved. the laboratory-engineered chimeras are thus functionally and structurally tolerant to modified dynamics on the timescale of the catalytic turnover. this tolerance of blactamases to dynamic changes could be linked to the high fitness of the naturally evolved proteins and implies that maintenance of native-like protein dynamics may not be essential when engineering functional proteins. conformations solution. an immense advantage of our recombinant system is the possibility to perturb it by introducing unnatural amino acids for subsequent labelling with fluorescent dyes or to omit subunits from the multisubunit rnap. comparing free, nucleic acid-and factor-bound rnap complexes we found that the position of the clamp is modulated during the transcription cycle in a fashion exceeding the simplified closed-open picture drawn from crystallographic studies. we show that the clamp adopts at least two distinct conformations in both initiation and elongation complexes. moreover, we were able to link conformational states to the catalytic activity of the rnap. transcription factors tfe and spt4/5, which both bind to the rnap clamp domain and stimulate the activity of the enzyme, shift the equilibria towards one of the main conformations suggesting that both prompt an allosteric switch that influences the structure of the rnap. hence, our data provide a mechanistic rationale for the function of these factors and provide new insights into the complex dynamic behaviour of the transcriptional machinery. solvent models for protein simulations -the good, the bad and the applications duy hua 1 , amitava roy 1 , he huang 1 , carol post 1 1 the solvent environment plays a crucial role in determining the structure, dynamics and function of a biomolecule. in order to utilize molecular dynamics (md) simulations to examine the structural changes abstract of biomolecules, it is imperative that the solvent environment be accurately modeled. implicit solvent models (isms), in which water molecules are absent and the solvent effect is estimated using an energy function, offer a low-cost approach to describe the solvent environment in md simulations. isms have been used for a variety of biological studies; yet, the performance of implicit solvation methods in capturing the structural characteristics of proteins has not been rigorously demonstrated. in this work, three isms, namely gbmv ii, facts, and scpism, were evaluated for their abilities to emulate the solvent environment and its effect on the structures, dynamics and electrostatic interacthioredoxins (trx) are ubiquitous proteins that play a key role in redox state regulation of the cell. they interact with multiple targets in the cell. our group solved the structure of s. cerevisiae (ytrx1) and measured its dynamics in different timescales (pico to seconds). it is well established that the asp24 is the proton acceptor in the reduction process of the target protein. we proposed that asp24 also works to couple hydration and motion of the interacting loops. we studied conformational motions of the residues of water cavity and interacting loops. the water molecules in water cavity of the protein play a vital role in the redox activity of thioredoxin. the degree of mutational frustration explains in a great deal the multiple timescales observed in the protein dynamics in ytrx1 and the mutant d24a. the ytrx1 displays a complex equilibrium between the ground state and several excited hydration states. this excited states are more permeable to water and it is key to understand the proton transfer and activation of cys33. in this current study, we examined the mutant d24a, and observed that this small hydrophobic residue induces conformational exchange in residues exposed to the water cavity, such as cys33. along with molecular dynamics simulations and calculation of mutational frustration levels, we were able to describe conformational details of the water cavity. it is formed by three independent contiguous lobes which we called lobes a, b and c. lobe b is the central lobe that contains the catalytic important residues cys33 and asp24. it closed upon mutation d24a. lobe a and c remains open upon mutation, meaning that their hydration are independent. the nmr structures for the oxidized and reduced state of the mutant d24a has been studied. in-vitro and in-silico studies of ligand binding to the nuclear receptor ppargamma using fret and md narutoshi kamiya 1 , gert-jan bekker 1 , takuma shiraki 2 , haruki nakamura 1 1 ipr, osaka university, 2 kinki university the nuclear receptor ppargamma is a ligand-dependent transcription factor which regulates gene expression related to glucose homeostasis and insulin sensitization. it is a potential therapeutic target for metabolic syndrome, cancer, and inflammatory diseases. crystal structures have revealed that its ligands bind deep inside the pocket. however, how the ligand recognizes ppargamma and penetrates inside the pocket remains unclear. we have measured the binding kinetics using the fret method and have calculated potential dissociation paths via molecular dynamics (md) simulations. the binding kinetics were measured using fret between the ligand, c8-bodipy, which is a fluorescent pigment structurally similar to one of its agonists 15-oxo-eicosatetraenoic acid, and cyan fluorescent protein (cfp) which is fused to ppargamma. fluorescence of c8-bodipy is observed when it is close to cfp when cfp is excited by light at 410nm. the ligand was simultaneously added to the ppargamma-cfp solution using the stopped-flow method. time-resolved fluorescence signals of c8-bodipy and cfp were also monitored. two time constants were observed by the fret experiment, which correspond with the landing and docking processes. we used our new md program, psygene-g [1] , which utilizes the gpu for acceleration of the non-bonded interactions, such as electrostatic interaction which uses our original zero-dipole summation method [2, 3] . previously we were able to attain similar thermodynamics with respect to the particle mesh ewald method in membrane proteins and dna-water-ion systems [4, 5] and have applied it to the thermodynamic study of dynein [6] . we have introduced random accelerated md (ramd) into the psygene-g program to predict a ligand's dissociation path from the initial crystal structure (pdb id: 2zk6). in ramd, an external random-directional constant force is added to the ligand. we executed 96-ramd simulations with different random seeds for the initial pull directions. twelve ramd trajectories managed to dissociate within 1 ns, while the remaining simulations did not manage to disassociate within this time-frame. in all twelve cases the ligand went past r288, which is located at the surface of ppargamma. interestingly, a somatic mutant (r288h) is related to colon cancer. we concluded that r288 plays a role of a gate keeper to assist ligand binding. sumoylation is a post-translational modification involved in various cellular processes, such as nuclearcytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle. sumoylation is a conjugation of glysine residue of sumo protein with lysine residue in target(rangap1) protein through the influence of enzyme e2(ubc9) and e3(ranbp2). sumo proteins modify the function of target protein in the cell. but the kinetic mechanism of sumoylation is not well-understand. we traced out the full kinetic process of sumoylation of the sumo1-ubc9 -rangap1-ranbp2 complex in atomic detail via molecular dynamic simulation. we uncovered the kinetics of sumoylation mechanism and verified the important residues which play a key role in the sumoylation process. the comparison of our results with the experimental one are also discussed. human potassium channel kcnq1 is expressed in several tissues including inner ear, heart muscle, lung, intestine, and stomach, each requiring a unique current profile for proper function. to tune its output current, kcnq1 complexes with several accessory proteins from the kcne family, each kcne family member modulating kcnq1 distinctly: kcne1 causes the channel to delay opening and become more conductive in the open state, kcne3 causes the channel to be constitutively conductive, and kcne4 closes the channel. the purpose of this study is to uncover the atomic-scale, mechanical mechanism of how kcne3 modulates kcnq1. to do this we modeled the interaction between kcnq1 and kcne3 with a hybrid experimental-computational approach. our strategy is to determine the nmr structure of kcne3 alone in bilayer-mimicking bicelles, build a homology model of the open-state kcnq1, and dock the structure of kcne3 onto the model of kcnq1 using electrophysiology-based restraints to validate and refine the resulting model complexes. paramagnetic relaxation enhancement, residual dipolar coupling and sequence analysis suggest the single span transmembrane helix of kcne3 is significantly curved. hydrogen-deuterium exchange, nuclear overhauser effect cross peaks, and amber simulation suggest kcne3 carries a significant amount of water in the c-terminal side of the transmembrane helix. the 1h-15n two dimensional nuclear magnetic resonance spectra of a known phenotype-changing mutation in the center of the transmembrane helix, v72t, suggests the helix adopts multiple conformations when the extra side-chain hydroxyl group is present. a docking funnel corroborates a binding pocket suggested by the in vivo electrophysiology data where kcne3 wedges between a helix near the potassium pore and two helices in the voltage sensitive element of the channel. within this pocket, we believe the conformational sampling and structural rigidity-or flexibility, in the context of the v72t mutant-of the accessory protein kcne3 directly influence the modulation profile. watching conformational changes in proteins by molecular dynamics simulations kresten lindorff-larsen 1 1 proteins are dynamical molecules and their ability to adopt alternative conformations is central to their biological function. examples include motions that underlie allosteric regulation or ligand binding, or protein dynamics in enzymes that can modulate the overall catalytic efficiency. protein motions can often be described as an exchange between a dominant, ground state structure and one or more minor states. the structural and biophysical properties of these transiently and sparsely populated states are, however, difficult to study, and an atomic-level description of those states is challenging. in an attempt to determine how well molecular dynamics simulations can capture slow, conformational changes in protein molecules we have studied two different protein systems which are known to undergo conformational exchange on the millisecond timescale, and for which structural information is available for both major and minor states. using enhanced-sampling all-atom, explicit-solvent molecular simulations, guided by structural information from x-ray crystallography and nmr, we show that current force fields and sampling methods allow us to sample experimentally-determined alternative conformations with surprisingly high accuracy. in particular, we find that we can reversible sample both the ground state and minor state, and that the simulations capture the structure of the minor states also. our simulations enable us to calculate the conformational free energy between the two states, and comparison with relaxation dispersion nmr experiments demonstrates a high accuracy. thus, we show for two distinct proteins that we can map the free energy landscapes and that both the structure and energetics are in excellent agreement with nmr experiments. our simulations provide insight into the structural and biophysical properties of transiently populated minor states, and help reinterpret previous experimental measurements. further, our results demonstrate that, at least in the two cases we have studied, modern simulation methods enable us to examine these otherwise "invisible" states of proteins and describe their structural, functional and thermodynamic properties. our results in this way help demonstrate how simulations can now add to our knowledge of transiently formed, "hidden" states of proteins. cell signaling is a fundamental process for all living organisms. g protein-coupled receptors (gpcrs) are a large and diverse group of transmembrane receptors which convert extracellular signals into intracellular responses primarily via coupling to heterotrimeric g proteins. in order to integrate the range of very diverse extracellular signals into a message the cell can recognize and respond to, conformational changes occur that rewire the interactions between the receptor and heterotrimer in a specific and coordinated manner. by interrogating the energetics of these interactions within the individual proteins and across protein-protein interfaces, a communication network between amino acids involved in conformational changes for signaling, is created. to construct this mapping of pairwise interaction energies in silico, we analyzed rhodopsin gpcr coupled to a gai1b1g1 heterotrimer. the structure of this g protein complex was modeled in the receptor-bound and unbound heterotrimeric states as well as the activated, monomeric ga(gtp) state. from these tertiary structural models, we computed the average pairwise residue-residue interactions and interface energies across ten models of each state using the rosetta modeling software suite. here we disseminate a comprehensive analysis of all critical interactions and create intra-protein network communication maps. these networks represent nodes of interaction necessary for g protein activation. structure and dynamics of the polymyxin-resistance-associated response regulator pmra in complex with the promoter dna yuan-chao lou 1 , yi-fen kao 1 , tsi-hsuan weng 2 , yi-chuan li 2 , chwan-deng hsiao 2 , chinpan chen 1 1 institute of biomedical sciences, academia sinica, 2 institute of molecular biology, academia sinica pmra, an ompr/phob family response regulator (rr), takes part in the two-component system that manages genes for polymyxin resistance through a phosphorylation-dependent regulation. phosphorylation of ompr/phob rr induces the formation of a two-fold symmetric dimer in the n-terminal receive domain (rec), promoting 2 c-terminal dna-binding domains (dbds) to recognize tandem repeat dna sequences on the promoter to elicit adaptive responses. recently, narayanan et al. presented the complex structure of active-like kdpe in complex with dna, which reveals a unique asymmetric rec-dbd interface that is necessary to form stable complexes for transcription activation. in this study, we report the 3.2 å resolution crystal structure of bef3-activated pmra in complex with promoter dna as well as its dynamics in solution by nmr. unlike the case of kdpe, pmra-dna complex structure reveals a different asymmetric rec-dbd interaction. interestingly, nmr assignments and dynamics study suggest that rec and dbd do not form stable contacts in solution; instead, 2 domains tumble separately in the absence or presence of dna. relaxation dispersion experiments on methyl groups further show that several rec-dbd interfacial residues exhibit similar slow dynamics in the presence of dna. it is hence highly possible that the slow dynamics observed on these interfacial methyl groups are related to the formation of asymmetric rec-dbd interaction, which reduces the mobility of pmra-dna complex and promotes crystallization. in solution, two domains tumble independently and have diverse orientations, which together with the dbd-dbd interface may facilitate searching best interactions with rna polymerase holoenzyme for transcription activation. time-resolved x-ray observations of nano-scale protein assembly networks yufuku matsushita 1 , hiroshi sekiguchi 2 , noboru ohta 2 , keigo ikezaki 1 , yuji goto 3 , yuji sasaki 1,3 1 the university of tokyo, graduate school of frontier science, advanced materials science, 2 spring-8, 3 osaka university, institute for protein research protein aggregation and network formation in diverse protein species had been studying in various experimental approaches such as absorbance spectrophotometry, dynamical light scattering and x-ray scattering. however, these conventional methods are only able to observe averaged information on bulk conditions and also these techniques have a limitation of time resolved observations. recently, development of single molecular observation techniques are remarkable. diffracted x-ray tracking (dxt) is one of the notable single molecular observation methods to capture detailed intramolecular dynamics of an individual single protein molecule by the labeling x-ray method. in this study, we present an innovative method for observing the protein assembly networks of nano-scale size on the bulk solution using a dxt that possess pico-meter scale positional accuracy and micro-second time resolution. this method is founded by detecting angular rotational displacement of a coexisted and dispersed single gold nanocrystal (approximately 100 nm) on target solution. for target sample, we choose a crystal precursor metastable state of 20 mg/ml lysozyme solution (hen egg white lysozyme: 14331.20 g/ mole on 0.2 m sodium acetate 3 -4 w/v nacl, ph 4.7 buffer) and 10 mg/ml of lysozyme solution such as stable condition. for reference sample, we prepare 20 mg/ml and 10 mg/ml of ribonuclease a solution (ribonuclease a (bovine): 13708.40 g/mol, 0.2 m sodium acetate 3 -4 w/v nacl, ph 4.7 buffer). the experiment was carried out at spring-8 bl40xu. from dxt analysis, we obtained logarithmic rotational velocity distribution of 20 mg/ml, 10 mg/ml lysozyme solution and 20 mg/ml, 10 mg/ml of ribonuclease a solution during 1000 ms and we processed regression analysis of gaussian fitting in each distribution. from this result, 10 mg/ml of lysozyme solution (stable) and 20 mg/ml, 10 mg/ml (reference) have definitely regressed by single peak normal gaussian distribution that are positional peaks at 3.66 mrad and 1.4 mrad and 1.2 mrad, respectively. in contrast, 20 mg/ml of lysozyme solution consisted of double peak logarithmic distribution at 3.42 and 9.54 mrad. this peak separation tendency from dxt are typically observed in a supersaturated condition of the sodium acetate solution which coexisting nano-scale solute networks (dxt and small angle x-ray scattering (saxs) experiment). from this study, dxt measurement results and detailed analysis process for a protein solution such as crystal precursor metastable state of lysozyme solution are concerned that this technique is a powerful tool for observing nano-scale protein network's existence and its local dynamics in bulk solution. at the present time, we will demonstrate the detailed analysis and processing from dxt raw data and the conformity of the results from another experiment confirmation such as saxs and darkfield microscopy. finally, we present a detailed protein network model of lysozyme metastable solution from dxt result. increasing evidence suggests that conformational fluctuations experienced by proteins play a key role in promoting function. however, the experimentally derived dynamic behavior of discrete protein systems has yet to provide clear evidence on the evolutionary conservation of motional events between structural and functional protein homologues. in this work, we used a statistical coupling analysis (sca) approach to analyze the role of co-evolving amino acids in protein function and flexibility. sca facilitates the identification of distinct residue sectors and provides an understanding of the correlation between amino acid co-evolution and biological function. as a model system, we used the ribonuclease a (rnase a) family, which includes canonical members that are structurally similar, but perform diverse activities such as angiogenesis, anti-pathogenicity and neurotoxicity, while preserving the common ribonucleolytic function. analysis of 1922 sequences with sca 6.0 allowed the determination of five independent components (ics) corresponding to amino-acid sectors with distinct functional, structural and/ or dynamical roles within the family. while most ics correspond to residues critical for protein stability and catalytic function, ic4 correlates with residues experimentally determined to be highly dynamic on the catalytic timescale in several rnase a homologues, as we confirmed by nmr 15n-cpmg experiments. additionally, ic5 residues form a hydrogen-bonding network shown to allosterically modulate and coordinate catalytically productive motions. these results provide a direct observation between the role of residue sectors in stability and motions relevant to function in the rnase a family. this study highlights the role of concerted action of multiple residues towards a common function, which can guide experiments for engineering proteins to enhance function such as catalysis. halophilic archea are a type of extremophiles that thrive in hypersaline conditions. in order to avoid the osmotic shock, their cytoplasm is maintained with higher ionic strength up to 4-6m. such a high ionic strength of intracellular environment protects the cells from desiccation or salting out. however, one wonders how such high salt concentrations keep the cell functional and active throughout all the cellular reactions (i.e. enzyme catalysis). the answer lies in the evolution of the halophiles that has left the halophilic proteins with a certain preference for amino acids composition [1] (prefers more acidic residues and overall decrease in hydrophobic side-chains). it's also known that the salt concentration can affect the activity and the underlying mechanism [2] of the halophilic enzymes. in order to further understand the haloadaptation, we have chosen to study a model system, dihydrofolate reductase (dhfr) from haloferax volcanii (hvdhfr). dhfr is a very well understood enzyme and known to bound variety of ligands that can modulate the dynamics and the energy landscapes of the enzyme [3] . we have probed the ls-ms dynamics of hvdhfr bound to the cofactor nadph (holoenzyme) and other substrates (dhf, thf) using relaxation dispersion nmr. in order to understand the salt contribution to the dynamics and catalytic mechanism, the experiments were done in the increasing order of salt concentration, shedding light on the conformational ensembles involved in the catalytic mechanism. the nmr relaxation dispersion analysis of different enzyme complexes in the presence of 3m and 2m kcl has shown more conformational fluctuations than compared to the enzyme with 1m kcl. this result suggests that the enzyme preferably is highly active & samples more flexible conformers in the hypersaline environment. further, quantification of these conformers/invisible-states and their salt-dependence will provide new insights into the mechanism of the enzyme-catalysis. also, it will facilitate designing novel enzymes that can be used in the bioprocess industrial set-up under extreme conditions. 1. tadeo we have identified the extracellular protein hbps in the cellulose degrader streptomyces reticuli and shown that it specifically binds iron ions, heme and aquo-cobalamin. based on 3d crystal structures, structural alignments, sequence comparisons, mutagenesis, and comparative biochemical investigations, we identified the coordination sites for iron, heme and aquo-cobalamin, and binding kinetics were elucidated [1, 2] . hbps interacts with the membrane-embedded sensor kinase sens. while under nonstressed conditions hbps inhibits sens autophosphorylation, under oxidative-stressing conditions activates it. sens in turn phosphorylates the response regulator senr that activates the transcription of anti-oxidative genes [3] . we crystallized hbps and solved its 3d crystal structure that revealed an octomeric assembly which is required for interaction with sens [4] . using mutagenesis, fret, cd spectroscopy, fluorescence spectroscopy and site-directed spin labelling combined with pulse electron paramagnetic resonance spectroscopy, we demonstrated that ironmediated oxidative stress induces both secondary structure and overall intrinsic conformational changes within hbps. we additionally showed that hbps is oxidatively modified, leading to the generation of highly reactive carbonyl groups and tyrosine-tyrosine bonds [5] . we concluded that these molecular events are responsible for the hbps-mediated activation of the sensor kinase sens. this presentation will focus on the hbps structure and dynamics within the protein. indian institute of technology bombay for20 (fop-related protein of 20 kda), a centrosomal protein, is conserved across all ciliated eukaryotes. it has been shown to be involved in ciliogenesis in rpe cells and in the s-phase progression in hela cells. it localizes to the pericentriolar satellite and at the centrosome. the localization of for20 at the centrosome has been found to be throughout the cell cycle. in paramecium, for20 is involved in docking of the basal body to the cell membrane and in the assembly of the transition zone. we found that for20 colocalizes with ciliated microtubules in nih3t3 cells. since, the localization of for20 to the pericentriolar satellite is dependent on microtubules and the cilium itself is a microtubule-based structure; we sought to investigate the role of for20 in microtubule assembly. the over-expression of gfp-for20 in hela cells led to the depolymerization of microtubules while only the gfp expressing hela cells showed typical microtubule network. in addition, the microtubules of gfp-for20 expressing hela cells were found to be more sensitive towards nocodazole, a microtubule-depolymerizing agent, suggesting that for20 destabilizes microtubules. the effect of for20 on the polymerization of tubulin was monitored by light scattering, sedimentation assay and electron microscopy. in vitro, for20 inhibited the rate and extent of polymerization of tubulin. for example, 5, 10, 15 and 20 mm for20 inhibited the polymerization of purified tubulin by 11, 20, 39 and 52%, respectively. further, the sedimentation assay suggested that for20 inhibited the polymerization of tubulin. in addition, electron microscopic analysis of the assembly mixture showed that for20 inhibited microtubule formation in vitro. the binding of for20 to purified tubulin was monitored by several complimentary techniques. using size exclusion chromatography, for20 was found to be co-eluted with tubulin indicating that for20 binds to tubulin. further, the interaction between for20 and tubulin was analyzed by surface plasma resonance technique and the result showed that for20 binds to tubulin with a modest affinity. the data together suggested that for20 regulates microtubule assembly through a direct interaction with tubulin. amide hydrogen exchange (hx) is widely used in protein biophysics even though our ignorance about the hx mechanism makes data interpretation imprecise. notably, the open exchange-competent conformational state has not been identified. based on analysis of an ultra-long molecular dynamics trajectory of the protein bpti, we propose that the open (o) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the n-h group. the hx protection factors computed from the relative o-state populations agree well with experiment. the o states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. the mean residence time of the o state is 100 ps for all examined amides, so the large variation in measured hx rate must be attributed to the opening frequency. a few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. in either case, we argue that an over-coordinated n-h group is necessary for efficient proton transfer by grotthuss-type structural diffusion. differences in redox reactions with nadp1/h between ferredoxin-nadp1 oxidoreductases from bacillus subtilis and rhodopseudomonas palustris daisuke seo 1 , hidehiro sakurai 2 , pierre s etif 3 , takeshi sakurai 1 1 graduate school of natural science and technology, kanazawa univ., 2 research institute for photobiological hydrogen production, kanagawa university, 3 ferredoxin-nad(p)1 oxidoreductase (fnr) is a ubiquitous enzyme that catalyze the redox reaction between soluble small iron-sulfur protein ferredoxin and nadp1/h. fnrs from photosynthetic green sulfur bacterium chlorobaculum tepidum (ctfnr), low-gc content gram-positive bacterium bacillus subtilis (bsfnr) and purple non-sulfur bacterium rhodopseudomonas palustris (rpfnr) are homodimeric proteins containing one fad prosthetic group per subunit. crystal structure analyses of ct-and bsfnrs have revealed their significant structural homology to nadph-thioredoxin reductase from eschelicia coli, which is distinct from the monomeric fnrs from plastids of higher plants, cyanobacteria, g-proteobacteria and putidaredoxin reductase. previous studies on steady-state reaction of bs-and rpfnrs with nadp1/h by diaphorase assay have demonstrated that nadph oxidation rates of bsfnr and rpfnr were comparable to those of the fnrs from plastid and cyanobacteria. but affinities toward nadp1/h and rates for oxidation of nadph differed significantly between bsfnr and rpfnr, despite their high amino acid sequence homology. in this work, we report pre-steady state reactions of bsfnr and rpfnr with nadp1/h by a stopped-flow spectrophotometry. mixing oxidized bsfnr with nadph yielded a rapid formation of charge transfer complexes (ctcs) followed by a reduction of the enzyme. bsfnr was almost fully reduced at equilibrium. mixing photochemically reduced bsfnr with nadp1 also rapidly provided an absorption spectrum of ctc but followed reoxidation of reduced bsfnr was very slow. the amount of oxidized bsfnr after equilibrium depended on nadp1 concentration. kinetic analyses indicated that the rate-determining steps were the hydride-transfer reactions in both directions and the rate for the forward direction was much faster than that for the reverse direction. mixing oxidized rpfnr with nadph exhibited a rapid ctcs formation followed by a slower reduction of the enzyme. increase in nadph concentration reduced the observed rate, suggesting redox potential of rpfnr was similar to that of nadp1/h couple in the presence of excess nadph. mixing photochemically reduced rpfnr with nadp1 rapidly provided ctcs. the decay corresponding to the reoxidation of reduced rpfnr with nadp1 involved two distinctive kinetic phases, which would be due to the similarity in hydride transfer rates of forward and reverse directions, and the presence of excess amount of nadp1. kinetic analyses indicated that the rate-determining steps were the hydride-transfer reactions in both directions and the rate for the forward direction was close to that for the reverse direction. obtained data suggested bsfnr and rpfnr are optimized for different direction of the reaction and may play different physiological roles. stephan k€ ohler 1,2 , friederike schmid 1 , giovanni settanni 1,3 1 institute of physics, johannes gutenberg university mainz, germany, 2 graduate school materials science in mainz, 3 fibrinogen is a serum multi-chain protein which, when activated, aggregates to form fibrin, one of the main components of a blood clot. fibrinolysis controls blood clot dissolution through the action of the enzyme plasmin, which cleaves fibrin at specific locations. although the main biochemical factors involved in fibrin formation and lysis have been identified, a clear mechanistic picture of how these processes take place is not available yet. this picture would be instrumental, for example, for the design of improved thrombolytic or anti-haemorrhagic strategies, as well as, materials with improved biocompatibility. here, we present extensive molecular dynamics simulations of the fibrinogen complex which reveal large bending motions centered at a hinge point in the coiled-coil region of the complex. this feature, likely conserved across vertebrates according to our analysis, suggests an explanation for the mechanism of exposure to lysis of the plasmin cleavage sites on fibrinogen coiled-coil region. it also explains the conformational variability of fibrinogen observed during its adsorption on inorganic surfaces and it is supposed to play a major role in the determination of the hydrodynamic properties of fibrinogen. in addition the simulations suggest how the dynamics of the d region of fibrinogen may contribute to the allosteric regulation of the blood coagulation cascade through a dynamic coupling between the a-and b-holes, important for fibrin polymerization, and the integrin binding site p1. membrane curvature -the assembler of proteins mijo simunovic 1,2 , gregory voth 1 , patricia bassereau 2 1 chemistry department, the university of chicago, 2 physico-chimie curie, institut curie many biological phenomena require the membrane to change its shape. this process is often mediated by curvature-generating proteins, most notably by those containing one of many bar domains. at the same time, membrane curvature controls the way proteins interact with one another and so it acts as a vital signaling mechanism in the cell. we combine theoretical modeling with microscopy imaging techniques to study the driving force underlying the reshaping of biological membranes induced by n-bar proteins. in particular, we employ a combination of coarse-grained molecular dynamics with field-theoretical simulation methods to study the assembly of proteins on the membrane at molecular resolution. this approach allowed us to elucidate the precise mechanism by which cell membranes rapidly and from large distances recruit proteins to membrane-reshaping sites. it also let us identify a surprising role of membrane tension in directing the strength and geometry of protein-protein interactions. by complementing our simulations with fluorescence and atomic force microscopies, we study the way molecular assembly of proteins affects the membrane morphology at a more macroscopic level. we demonstrated how largescale ordering of the proteins on the membrane is mediated by a strikingly long-range interaction that is driven by membrane fluctuations. in sum, our combined theoretical and experimental approach gives vital clues on how the mechanical properties of the membrane may regulate protein dynamics in living cells. adnan sljoka 1 , alexandr bezginov 2 1 kyoto university, 2 university of toronto understanding how a protein functions depends in critical ways on predicting which parts are rigid and which are flexible. using rigidity-theoretical techniques, a number or programs such as first, proflex or abstract kinari can rapidly decompose a protein into flexible and rigid regions. in this study we extend this technique and develop a novel computational approach for detecting protein allosteric interactions. it is widely believed that the binding of a ligand at the allosteric site triggers a conformational change that is transmitted through the protein to cause a rearrangement and alteration of the shape of the active site. allostery was first described more than 50 years ago; however, the underlying allosteric mechanism is still not well understood. we will introduce a rigidity-based allosteric mode of communication together with an algorithm which can detect transmission of rigidity and shape changes between two (or more) distant binding sites in allosteric proteins. our algorithm is also used to predict and identify regions in the protein that are critical for the coupled communication between distant sites (i.e. allosteric pathways). starting with a set of known gpcr 3-dimensional structures, we apply our methods and show how binding of an activating ligand (i.e. agonist) triggers small rigidity changes which propagate to the critical g-protein binding regions. in contrast, in the inactive gpcr structures no such rigidity allosteric communication is observed. detailed predictions and analysis on activated (agonist-bound) and inactive adenosine receptors is discussed and results are compared with experimental evidence. these results show that rigidity-based allosteric model and algorithm is a powerful new tool for detecting allostery in gpcrs. comparing the intrinsic dynamics of multiple proteins using elastic network models reveals global similarities based on their overall shape sandhya tiwari 1,2 , nathalie reuter 1,2 1 with increasing protein structural data, we find ourselves with the opportunity to study the dynamical similarities within large ensembles in order to understand the role of protein dynamics at various levels. is there evolutionary pressure to conserve dynamics? is this necessary to retain functionality, as it has been suggested? we performed strategic comparative analysis using the elastic network model, which has proven to be highly efficient and informative (fuglebakk et al., bmc bioinformatics, 2014) to investigate how the dynamics of related proteins change when their functional or oligomeric state shifts, and if it is conserved within protein families. in our work, we have found that proteins with different ligandbinding states, as in adenylate kinase (tiwari et al., bmc bioinformatics, 2014), or oligomeric states, as in the pyrr family (perica et al., science, 2014), can be classified based on the global similarity of their intrinsic dynamics. moreover, our recent analysis on functionally distinct protein families with the tim barrel fold indicates that the overall similarity in shape dictates similarity in intrinsic dynamics regardless of sequence and functional conservation or evolutionary links. we suggest that since shape dictates a large part of dynamical similarity in proteins, the changes in local flexibility play a strong role in differentiating various functional states. oxygen-affinity and cooperativity of hemoglobin introduction: the widely-held mechanism of allostery of hb has been that the changes from the r-quaternary/tertiary structures of oxy-hb to the t-quaternary/tertiary structures of deoxy-hb exert certain constraints on the coordination structure of the heme group, leading to a lower o2-affinity of the hemes and thus that of hb [1] . however, we found that there is no causal correlation between the static t/r-quaternary structures and the low/high o2-affinities of hb, respectively [2] . we explore an alternative mechanism of the regulation of the ligand-affinity and cooperativity in hb. results and discussion: the o2-affinities of free fe[ii]-protoporphyrin ix-nitrogenous base complexes in organic solvents are very low (p50 > 102 103 torr), whereas the apparent o2-affinities of these metalloporphyrins, which are incorporated in apo-myoglobin, apo-hb, serum albumin, etc., increase substantially to p50 < 10-1 101torr, though their coordination structures are apparently unchanged [3] . such substantial increases in the apparent ligand-affinities of metalloporphyrin-containing proteins are accomplished by preventing/inteferring with the dissociation of the ligand by protein matrix, since the interior of globin is nearly fully packed by protein matrix. in hb, the dissociation process of the ligand proceeds through the "caged" state [4] [5] [6] , which can be produced by cryogenic photolysis of the ligated-states at 4.2k and in which the metal-ligand bond is broken and the un-bonded ligand is trapped near the bonding site within the globin moietiy. this "caged" state has spectral features distinct from those of either deoxyor ligated states of the respective hemoproteins. the apparent ligand-affinities of hb are regulated by heterotropic effectors without detectable changes in either static quaternary/tertiary structures of the globin moiety or the coordination/electronic structures of the metalloporphyrin moiety and thus the ligand-affinity of the metalloporphyrins themselves [7] [8] [9] . the reduction of the apparent ligand-affinities of hb may be caused by increases in the migration rate of ligands through globin matrix from the "caged" state to solvent, resulting from the effector-linked, enhanced high-frequency thermal fluctuations which increase the transparency of the globin matrix toward small diatomic ligands [7] [8] [9] . conclusion: the ligand-affinity of hb is regulated through protein dynamics by heterotropic effectors, rather than static quaternary/tertiary structural changes. thus, the "caged" state of hb acts as a critical transition state in regulation of the affinity for small diatomic ligands in hb [9] . the role of metal ions in the regulation of life processes is extremely important. they act as signal transducers, protein configuration stabilizers, enzymatic cofactors, oxygen transport supporters and many others. for example, subtle perturbations in calcium homeostasis may lead to mental disabilities and are linked to diseases such as autism spectrum disorders (asd). in this study we focus on complex protein systems, mainly those present in the brain. we search for dimers mediated by the presence of metal ions, and determine the impact of the presence or absence of the latter on the structure and energetic properties of the complex in the protein-protein interface. we investigate ions' influence on the interface stability using classic molecular dynamics methods (md), including steered md. moreover, we apply a novel suite of enhanced md-based methods recently developed by our team (rydzewski & nowak) to explore ion diffusion pathways in protein fragments of the synapses. finally, we describe specific inter-protein ion binding motifs with the most important interactions, collating them with various structures deposited in the protein data bank [1] . the binding of integrins to collagen plays a critical role in numerous cellular adhesion processes including platelet activation and aggregation, a key process in clot formation. collagen is an unusually shaped ligand, and its mechanism of recognition and role in selectivity and affinity are unique, and at this stage not well understood. the i-domain of the integrin protein binds to collagen specifically at multiple sites with variable affinities, however the molecular mechanism of integrin i-domain (ai) regulation remains unknown. using nmr, along with isothermal titration calorimetry, mutagenesis, and binding assays we are developing a novel integrated picture of the full recognition process of the integrin a1i binding to collagen. the adhesion of the a1b1 integrin receptors to collagen is cation-dependent with collagen binding a mg(ii) ion that is located at the top of the extracellular integrin a1i-domain (a1i). our results show evidence for a regulatory effect of the mg(ii) ion on a1i affinity, by inducing allosteric ms-ms motions of residues distant from the binding site. we propose a novel model of a1i recognition to collagen, comprising a two-step mechanism: a conformational selection step, induced by mg(ii) coordination, and an induced-fit step caused by collagen binding. hydrogen-deuterium exchange experiments show that the induced-fit step is facilitated by the reduced local stability of the c-terminus. we propose that the conformational selection step is the key factor that allows discrimination between high and low affinity collagen sequences. cytochromes p450 (cyp) are heme containing enzymes involved in the metabolism of endobiotics and xenobiotics, such as drugs or pollutants. [1] in humans, cyps are attached to the biological membranes of endoplasmic reticulum or mitochondria by n-terminal transmembrane anchor and they are partially immersed by their catalytic domain to different level. [2] generally, the composition of lipid membrane may significantly affect behavior of protein embedded in respective membrane e.g. the cholesterol in membrane alters membrane properties such as: thickening of the membrane, changing the stiffness or enhancing ordering of the membrane. furthermore, the increasing amount of cholesterol in membrane may also alter interaction with membrane proteins and affect solute partitioning between membrane and water molecules. [3] cholesterol is also known to noncompetitively inhibit the most typical drugmetabolizing cyp -cyp3a4, [4] however the mechanism was unknown. for this reason, we prepared the set of simulations of cyp3a4 embedded in dopc lipid bilayers with various cholesterol concentrations (0, 3, 6, 20 and 50% wt; figure 1 ) and the 200ns1 long md simulations were carried out. md simulations showed the formation of funnel-like shape of the lipids close to the catalytic domain of cyp. in addition, the cholesterol molecules have tendency to accumulate in the vicinity of membrane-attached f/g loop. the catalytic domain sunk deeper into the membrane with cholesterol and also the number of amino acids in contact with membrane was bigger than in the pure dopc bilayer. in contrast, the presence of higher amount of cholesterol affected the pattern of channel opening effectively blocking the access to the active site from the membrane, which in turn may affect the substrate preferences and catalytic efficiency. [5] finally, we study the effect of different lipid types on membrane-attached cyp3a4. anti-(4-hydroxy-3-nitrophenyl)acetyl (np) antibodies are one of the most widely analyzed type of antibodies, especially with respect to affinity maturation [1] [2] [3] . affinity maturation is a process in which b cells produce antibodies with increased affinity for the antigen during the course of an immune response, and is like "evolution" in term of increasing antigen-binding affinity. during the course of affinity maturation, the structural dynamics of antibodies, which are closely correlated with the binding function, can change. to analyze the structural dynamics at atomic resolution and the single-molecule level, we tried to express and purify single-chain fv (scfv) antibodies against np. using scfv antibodies, we can also analyze the effects of key residues on affinity maturation via site-directed mutagenesis. as the first step, we have succeeded in generating a sufficient quantity and good quality of scfv of affinity-mature anti-np antibody, c6, with a linker composed of four repeats of gggs. the scfv protein was expressed in the insoluble fraction of e. coli, and solubilized using 8 m urea, followed by refolding by step-wise dialysis to decrease the urea concentration. the final step of purification using an antigen column indicated that approximately 2% of the solubilized protein was correctly refolded and possessed antigen-binding ability. the analytical ultracentrifugation (auc) analysis showed that the purified c6 scfv exists in the monomeric state with little oligomeric contamination. the secondary structure and thermal stability of c6 scfv were analyzed using circular dichroism (cd). the far-uv cd spectra of c6 scfv indicated typical b-sheet-rich structures. upon antigen binding, the far-uv cd spectrum remained unchanged, but the thermal stability increased by approximately 20oc. the antigen-binding function of c6 scfv was analyzed using a surface plasmon resonance (spr) biosensor, biacore. the binding affinity and kinetics of c6 scfv for np conjugated to bovine serum albumin immobilized on the sensor chip were similar to those of intact c6. taken together, the results of auc, cd, and spr indicated that c6 scfv could be refolded successfully and would possess its functional structure. next, to analyze the structural dynamics of c6 scfv in the absence or presence of antigen, experiments involving diffracted x-ray tracking (dxt) were performed [4] . c6 scfv with an n-terminal his-tag was immobilized on substrate surfaces using tag chemistry, and au-nanocrystals were labeled on the surface of scfv as tracers. the motions of c6 scfv were analyzed in two rotational directions representing tilting (u) and twisting (v) mean square displacement (msd) analysis from more than 200 trajectories showed that the slope for c6 scfv without antigen, especially in the u direction, was greater than that for c6 scfv with antigen, suggesting that the motion of scfv was suppressed on antigen binding. the antibiotic resistance enzyme aph(2'')-ia confers antimicrobial resistance to aminoglycoside antibiotics in staphylococci and enterococci. this kinase phosphorylates aminoglycosides such as gentamicin and kanamycin, chemically inactivating the compounds. we have determined multiple structures of the enzyme in complex with nucleoside and aminoglycoside substrates and cofactor magnesium. introduction of aminoglycoside to crystals of aph(2'')-ia induce gross conformational changes in crystallo, illustrating several important stages of the catalytic cycle of the enzyme. an interaction between nucleoside triphosphate and an amino acid residue on a conserved loop has also been identified that appears to govern a conformational selectivity and modulates the enzyme activity when no substrate is present. comparisons between multiple protein molecules both within and between crystal structures allow us to infer functional states of the enzyme as it carries out catalysis. these structures collectively highlight an enzymatic flexibility that not only allows the binding of diverse aminoglycosides, but also appears to transition from a stabilized, inactive enzymatic state to a catalytically active enzyme with an active site geometry identical to distantly-related eukaryotic protein kinases. mechanistic insight gained from these studies begin to demystify a widespread staphylococcal resistance factor, and provide a starting point for the development of anti-infectives toward this important antimicrobial resistance machine. ryan godwin 1 , william gmeiner 2 , freddie salsbury 1 1 wake forest university -department of physics, 2 wake forest university health sciences -department of cancer biology the zinc-finger of the nf-jb essential modulator (nemo) is a ubiquitin binding domain, and an important regulator of various physiological processes including immune/inflammatory responses, apoptosis, and oncogenesis. the nominally functioning 28 residue monomer (2jvx) is represented by a bba motif, with a cchc active site coordinating the zinc ion. here, we investigate the effects of a single point mutation that has been linked to the disease states associated with ectodermal dysplasia. the single mutation of the last binding cysteine (residue 26) to a phenylalanine (2jvy) distorts the available conformation and dynamics of the protein, as shown via microsecond, gpuaccelerated molecular dynamics simulations. we examine these two proteins in various states of zinc-binding and coordinating cysteine protonation. in addition to destabilization of the alphahelix induced by the cysteine to phenylalanine mutation, prominent conformations show the bsheets turned perpendicular to the alpha-helix, providing a possible mechanism for the induced disease state. , catalytic (51-220 aa) and c-terminal (220-270 aa)) were expressed in e. coli. several truncated in variants containing amino acids 1-160, 1-220, 51-160 and 51-280 were also prepared. a full-size ku70 with a gst-tag on its n-terminus was purified from e. coli. all the experiments performed showed that neither n-terminal nor c-terminal domains of hiv-1 in are essential for its binding with ku70 despite a weak binding capacity retaining to the c-terminal domain. the catalytic core (51-220 aa) as well as the mutant lacking c-terminal domain (1-220) both demonstrated affinity to ku70 comparable to the affinity of the full-size in, whereas its truncated variant (51-160 aa) bound to ku70 protein only weakly. we also expressed a c-terminal ha-tagged full-length in and its 1-220 variant in hek 293t cells together with a wt ku70-3flag and showed that both in variants are stabilized by co-expression with ku70 by approx. twofold. we hypothesize that the binding surface within in lies in the region from 160 to 230 a.a. that is a long a-helix. we have shown that a homologous integrase from prototype foamy virus that lacks this structural element does not bind to ku70. it is worth noting that ku70 does not affect the interaction of in with its major cellular partner -ledgf/p75 as well as its interaction with the dna substrate. this work was supported by an rfbr grant 14-04-00833 and by an rscf grant 14-14-00489. the nadph-dependent cytochrome p450 oxidoreductase (cypor) is large 677 amino-acid long microsomal multidomain enzyme responsible for electron donation to its redox partner cytochrome p450 (cyp) involved in drug metabolism. electron transfer (et) chain is mediated by two riboflavin-based cofactors -flavin mononucleotide (fmn) and flavin adenine dinucleotide (fad) within their respective domains and nicotinamide adenine dinucleotide phosphate (nadph). during this electron transfer cypor undergoes several structural changes in open and closed state of both domains in different degree of contact. in spite of the fact that cyp-cypor complexes play a key role in drug metabolism, the atomistic mechanism of structural rearrangements during complex electron transfers is still lacking. here, we present the results of our study on structural changes during cypor multidomain complex movement between individual electron transfers using classical molecular dynamics (md) and metadynamics (mtd) simulations with cofactors of nadph, fad and fmn in resting state. homology model of human cypor in both forms (opened and closed) were embedded into pure dioleoylphosphatidylcholine (dopc) bilayer. after system equilibration (figure 1 ), structural changes of protein, anchor and cofactor movement were studied. we were able to select possible cypor-membrane orientation which would allow interaction with cytochrome p450. in addition, spontaneous closing of open cypor was observed. however structural changes between crystal structures and structures obtain from md simulations lead us to the use of metadynamics in order to speed up the process. fmn and fad cofactor remained in close van der waals contact during the 100-ns long simulation stabilized by p stack interaction of fad with trp676, whereas continual movement of nadph continually weakens its p stack interaction with fad. after 100 ns of classical md additional metadynamics simulations were performed in order to investigate internal motion of cofactors during electron transfer. atoms c4n (nadph) and n5 (fad) which are responsible for et were able to move closer to the distance of 3 å after adding biasing potential. this distance is more than sufficient for electron transfer to occur. after switching back to classical md cofactors got into resting positions (8 å) again. our results show that cypor undergo several structural changes and internal motions of cofactors in order to transfer electrons to its redox partner -cyp. research & utilization div., jasri/spring-8, 2 grad. school frontier sci., univ. tokyo, 3 grad. sch. sci., univ. hyogo, japan, 4 national institute of advanced industrial science and technology, japan, 5 pentameric ligand-gated ion channels (plgics) are a major family of membrane receptors that open to allow ions to pass through the membrane upon binding of specific ligands. plgics are made up of five identical (homopentamers) or homologous (heteropentamers) subunits surrounding a central pore. structural information about their multiple allosteric states, carrying either an open or a closed channel, has become available by recent studies by x-ray crystallography. however, dynamic information are needed to understand their mechanism of gating, notably the long-range allosteric coupling between the agonist binding site and the ion channel gate. here we used the diffracted x-ray tracking (dxt) method (1) to detect the motion of the extracellular and transmembrane domain two plgics: the nicotinic acetylcholine receptor (nachr) and a proton-gated bacterial ion channel from gloeobacter called glic. dxt is a powerful technique in biological science for detecting atomic-scale dynamic motion of allosteric proteins at the single molecular level and at tens of micro seconds timescale resolution. the dynamics of a single protein can be monitored through trajectory of a laue spot from a nanocrystal which is attached to the target protein immobilized on the substrate surface (2,3). dxt detects two kinds of rotational motions of nanocrystal, tilting and twisting, based on x-ray incident beam axis. dxt analysis with 0.1ms/f time resolution showed that tilting motion of the transmembrane domain of glic and both tilting and twisting motions of the extracellular domain of glic and nachr were enhanced upon application of agonists (lowering the ph for glic, and binding of acetylcholine for nachr). the detailed dynamic information, including size effect of gold nanocrystal to the motion of them, is discussed. [ proteins possess unique structure-encoded dynamics that underlie their biological functions. here, we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. crystallographic structures were determined for several ancestral gfp-like proteins that were reconstructed based on posterior sequence predictions, using members of the stony coral suborder faviina as a model system. the ancestral proteins belong to the kaede-type class of gfps, a group of proteins that undergoes irreversible green-to-red photoconversion and is therefore frequently employed in superresolution microscopy. surprisingly, we find that the structures of reconstructed common green ancestors and evolved green-to-red photoconvertible proteins are very similar. therefore, we analyzed their chain flexibility using molecular dynamics and perturbation response scanning. we find that the minimal number of residue replacements both necessary and sufficient to support lightinduced color conversion provide for increased fold stiffness at a region remote from the active site. at the same time, the allosterically coupled mutational sites appear to increase active site conformational mobility via epistasis. these data suggest that during evolution, the locations of fold-anchoring and breathing regions have been reversed by allosteric means. therefore, we conclude that the green-tored photoconvertible phenotype has arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the beta-barrel fold. based on titration experiments, we estimate that at ph 6, 0.1% of the protein population harbors neutral side chains for his193 and glu211, residues that form an internal salt bridge near the chromophore. we propose that this reverse-protonated subpopulation constitutes the catalytically competent state. in the electronically excited state, light-induced chromophore twisting may be enhanced, activating internal acid-base chemistry that facilitates backbone cleavage to enlarge the chromophore. in this way, a softer active site appears to be coupled to a mechanism involving concerted carbon acid deprotonation and betaelimination. dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities by tuning motions in the active site. the binding of an agonist to a gpcr causes a conformational change in the receptor that leads to its activated functional state. rhodopsin, the membrane receptor responsible for photoreception in the vertebrate retina, is a prototypical gpcr and has been extensively used in structural, biochemical and biophysical studies of this class of receptors. different small molecules have been described to be capable of binding to rhodopsin. in addition, mutations in rhodopsin have been associated with retinal diseases and efforts have been carried out in order to find potential ligands that can offset the effect of these mutations. cyanidins, a group of flavonoids within the larger family of polyphenols, have been reported to stimulate chromophore regeneration of rhodopsin by means of the formation of regeneration intermediates. the aim of the current study was to evaluate the effect of the flavonoid quercetin on the conformational properties of both native bovine rhodopsin and heterologously expressed recombinant rhodopsin. rhodopsin was purified from bovine retinas by immunoaffinity chromatography, and photobleaching, thermal stability, metarhodopsin ii decay and chromophore regeneration assays were carried out in the absence or in the presence of 1mm quercetin. for recombinant rhodopsin, a plasmid encoding wild-type opsin was transfected into mammalian cos-1 cells, in the absence or in the presence of 1mm quercetin, harvested, regenerated with 11-cis-retinal, or 9-cis-retinal, and subsequently purified in dodecyl maltoside solution. no differences in photobleaching behavior, upon illumination, could be detected in the purified quercetin-containing samples compared to those in the absence of this flavonoid. in the case of rhodopsin, and the recombinant wild-type protein regenerated with 11-cis-retinal, quercetin did not significantly alter the thermal stability and rate of regeneration of the purified proteins under our experimental conditions. however, a two-fold increase in the thermal stability and a 40% increase in chromophore regeneration were observed for the recombinant wild-type protein regenerated with 9-cis-retinal in the presence of quercetin. in contrast, the presence of quercetin did not alter the electrophoretic and basic spectroscopic properties of rhodopsin, or those of the recombinant wild-type protein, suggesting no important structural alterations as a result of quercetin binding to the receptor. the positive effect of quercetin on the stability, and chromophore regeneration of rhodopsin, could be potentially used to counteract the effect of naturally-occurring misfolding mutations in rhodopsin. thus, quercetin could help stabilizing rhodopsin mutants associated with retinal diseases such as retinitis pigmentosa. furthermore, docking of the ligand, carried out on the crystallographic structure of rhodopsin (entry 1gzm), reveals several favorable sites for quercetin binding. one of this would be compatible with 9-cis-retinal suggesting a complementary binding to the receptor of this isomer which would not be compatible with 11-cis-retinal binding. identification of prospective allosteric sites of p38 by computational methods protein function is intrinsically associated with structural flexibility, so that understanding the functional properties of proteins requires going beyond the static picture produced by x-ray diffraction studies. structural flexibility can also be interpreted as a dynamic exchange between different conformational states with low energy barriers at room temperature. allosterism is a mechanism to regulate protein function associated with the plasticity exhibited by proteins. allosteric sites can be considered transient cavities that can be occupied by a small molecule with the subsequent modulation of the protein plasticity. occupation of these sites may modify the affinity of the protein for its native substrate that can be positive when the affinity increases or negative when the affinity decreases. allosterism can be used for the design of non-competitive ligands as new therapeutic agents. this mechanism of activity modulation is particularly interesting for those targets that use a common substrate for activation, like in the case of kinases to search for selective compounds. proteins can be viewed in solution as an ensemble of diverse energy accessible conformations. binding of an allosteric ligand produces a redistribution of the population of the diverse conformational states, which at the end modulate the affinity of the native substrate. allosteric sites can be characterized using computational methods by ensemble docking. it consist of characterize a set of structures that represent the accessible conformations of a protein that can then be used to perform virtual screening. in the present work we have studied prospective allosteric sites of p38 using computational methods. the protein is a member of the mitogen-activated protein kinases (mapks), a highly regulated group of enzymes that control a variety of physiological processes, including mitosis, gene expression, apoptosis and metabolism movement among others. the conformational profile of p38 was assessed using a 4 us trajectory of accelerated molecular dynamics as sampling technique in explicit solvent. we used as starting structure the apoform of p38 in its inactive conformation (entry 1p38). the conformational features of the protein were assessed through the analysis of the variance of the most flexible regions of the protein using principal component analysis. the snapshots of the trajectory were projected onto the two principal components. subsequent cluster analysis permitted us to select a few structures for further studies. specifically, prospective biding sites were identified using a hydrophobic probe as implemented in the sitemap program. the results show previously described regulatory sites and some new prospective ones. hydrogen/deuterium exchange-mass spectrometry provides clues on the mechanism of action of min e maria t. villar 1 , kyung-tae park 2 , joe lutkenhaus 2 , antonio artigues 1 1 cell division in most bacteria is initiated by the formation of the z ring, an essential cytoskeletal element that serves as a scaffold for the cytokinesis machinery, at the mid body of the cell. in e coli the spatial location of the z ring is regulated by the min protein system, comprised by three major proteins: minc, mind and mine. the dynamic interaction between these proteins results in the formation of an oscillating protein gradient between the poles of the cell. this oscillation determines the position of the formation of the z ring. many aspects of this simple mechanism are beginning to be understood. in particular, the conformational changes associated with the interaction of the three min proteins between them and with the cell membrane, are of especial interest. hydrogen/deuterium exchange mass spectrometry (hdx ms) is a sensitive technique for the detection of changes in protein conformation and dynamics. the main advantages of this methodology are the ability to study native proteins in solution, the requirement for low protein concentrations, the potential to discriminate multiple coexisting conformations, and the lack of an upper limit to the size of protein to be analyzed. here we use hdx ms to analyze the dynamics of the wild type mine and of its inactive double mutant d45a d49a. our results show significant differences in the rates of exchange and in the total amount of deuterium exchanged at the end of the reaction between these two forms of mine. the wild type protein exchanges most of the amide hydrogen during the first few seconds of initiation of the exchange reaction. on the other hand, the mutant protein exchanges only 50% of the total amide hydrogen atoms during the first seconds of initiation of the exchange, and the remaining 50% amide hydrogen atoms are exchanged more slowly during the next few minutes of the reaction. our data are consistent with the existence of a highly flexible structure for the wild type protein and the coexistence of at least two rigid conformations for the double mutant that are undergoing a cooperative transition. interestingly, the central b-sheet forming the interface between the two subunits is protected against exchange on both proteins. these results provide insights into the conformational changes that mine undergoes during its interaction with mind. biased signalling and heteromization of the dopamine d2 receptor in schizophrenia and parkinson's disease pablo herrera nieto 1 , james dalton 1 _ , jes us giraldo 1 _ 1 universidad aut onoma de barcelona biased signalling and heteromization of the dopamine d2 receptor in schizophrenia and parkinson's disease as a significant component of dopamine signalling in the brain, the dopamine d2 receptor (d2r), a member of the class a gpcr family, is an important target in the treatment of neurological conditions such as schizophrenia and parkinson's disease. d2r shows a variety of signalling pathways through g proteins, including adenylyl cyclase inhibition, gbgpotentiation of adenylyl cyclase 2, and erk kinase activation, in addition to b-arrestin recruitment,. these pathways are differentially activated by some agonists and it has been suggested that d2r ligands with gai/o antagonist and b-arrestin agonist activity may have anti-psychotic behavioural activity with reduced extra-pyramidal side effects. d2r has also been found to form homodimers or higher-order hetero-oligomers with other gpcrs, which may modulate d2r conformation and activity, thus constituting an additional form of allosteric receptor regulation. based on these findings, we have computationally modelled the full-length structure of d2r, including its long intracellular loop 3 (icl3) that is 1301 residues in length and absent in all homologous gpcr crystal structures. using state-of-the-art tools, such as rosetta for ab initio protein folding and acemd for micro-second1 molecular dynamics (md) simulations we have successfully de novo folded icl3, which primarily consists of extensions to transmembrane helices (tmh) 5 and 6 and an intervening disordered histidine/proline-rich region, which is highly flexible. the latter is observed to interact with other receptor intracellular loops (icl1 and icl2) and appears to restrict access to the g-protein binding-site. in addition, we have docked a structurally diverse collection of 14 ligands (biased agonists, antagonists and allosteric modulators) into our d2r model and observed characteristic binding patterns suggestive of different biased signalling mechanisms. finally, through protein-protein docking with rosettadock, we have generated a complete heterodimer model of d2r with the adenosine a2a receptor (aa2ar), where a mutual interface is formed between their respective tmhs 4 and 5, as well as an association between the c-terminus of aa2ar and icl3 of d2r. this may be a particularly relevant biological complex in the treatment of parkinson's disease where antagonists of aa2ar have been shown to ameliorate disease effects, potentially through direct interaction with d2r. bis-ans as a tool to monitor conformational changes upon assembly of binary and ternary complexes of eif4e, 4e-bp1 inhibitory protein, and the mrna 5'cap specific recognition of the mrna 5' terminal cap structure by the eukaryotic initiation factor eif4e is the first and rate-limiting step in the cap-dependent translation. small 4e-binding proteins, 4e-bp1, 4e-bp2, and 4e-bp3, inhibit the translation initiation by competing with eif4g initiation factor for the same binding site, and by blocking the assembly of the translation machinery [1] . our recent studies revealed intricate cooperativity between the cap and 4e-bp1 binding sites of eif4e [2] . here, we applied a fluorescent dye, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (bis-ans) to investigate conformational changes upon assembly of binary and ternary complexes composed of human eif4e, 4e-bp1, and the mrna 5'cap analogue, m7gtp. the fluorescence quantum yield of bis-ans increases significantly upon binding to hydrophobic sites of proteins, making the probe a convenient tool to determine the accessibility to hydrophobic surfaces, and to monitor structural reorganisation of macromolecules [3] . we characterised the interaction of bis-ans with eif4e and 4e-bp1 by fluorescence titration. the association processes takes up to several hours until the saturation of the fluorescence signal is achieved, reflecting high flexibility of the protein structures. the association constants kas of eif4e/bis-ans complexes are very high for the non-specific interaction. the kas values for eif4e/bis-ans and eif4e/4e-bp1/bis-ans are similar (10 7 m 21 ), whereas the presence of m7gtp results in ca. 5-fold weaker binding of the probe to eif4e. the affinity of bis-ans for 4e-bp1 is 10-fold lower than that for eif4e. we found no effect of either m7gtp or 4e-bp1 on the fluorescence of bis-ans in complex with eif4e, thus indicating lack of conformational changes around the probe on eif4e/m7gtp or eif4e/4e-bp1 complex formation. it also testifies that bis-ans does not bind to the cap-binding site, despite the hydrophobic nature of this eif4e region. on the contrary, addition of m7gtp to the eif4e/4e-bp1/bis-ans complex causes an increase of the probe fluorescence, which indicates differences in the structural reorganisation in the binary, m7gtp/eif4e, compared with the ternary, m7gtp/eif4e/4e-bp1, complexes, and confirms the spatial cooperation between the cap and 4e-bp1 binding sites. we also observed an increase of fluorescence for bis-ans bound to 4e-bp1 in the presence of eif4e, pointing out that 4e-bp1 partially folds upon association with eif4e. in summary, our results provide a deeper insight into the structural aspects of the molecular interaction at early stages of the cap-dependent translation. acknowledgements: this work was supported by the bst 170000/bf project from university of warsaw background: beta2-glycoprotein (b2gpi) is a protein abundantly present in human plasma and highly conserved in all mammals. b2gpi has been identified as the major antigen in the antiphospholipid syndrome (aps), a severe thrombotic autoimmune disease. despite its importance in the pathogenesis of aps, the physiological role of b2gpi is still elusive. in a previous work we have demonstrated that b2gpi significantly prolongs the clotting time in fibrin generation assays, and inhibits aggregation of gel-filtered platelets (ic5050.36um), either isolated or in whole blood, by inhibiting cleavage of par1 on intact platelets (ic5050.32um) and in solution. importantly, b2gpi does not alter the ability of thrombin (fiia) to generate the anticoagulant protein c, with or without thrombomodulin added. hence, we concluded that b2gpi inhibits the key procoagulant properties of fiia, without affecting its unique anticoagulant function. we also proposed that b2gpi, together with other more efficient anticoagulant pathways such as thrombomodulin-fiia -protein c and antithrombin iii-fiia, may function as a mild anticoagulant in vivo especially in those compartments were the efficacy of thrombomodulin is limited, as in the large vessels, or is even absent, as in the brain vasculature. aims: lacking the threedimensional structure of b2gpi-thrombin complex, the aim of this work is to identify the peptide regions either on thrombin and b2gpi involved in complex formation. results: data obtained by fluorescence and surface plasmon resonance (spr) indicated that b2gpi interacts whit fiia whit physiological affinity (kd543 6 4nm). kd values calculated by reverting the interacting systems are very similar to each other (kd598 6 9nm), suggesting that b2gpi in the mobile phase has a conformation which is competent for the binding to immobilized fiia. the affinity of fiia for immobilized b2gpi is markedly decreased by increased ionic strength (i.e. kd increases by 50-fold going from 0.1 m to 0.4 m), suggesting the electrostatic interactions play a key role in fiia -b2gpi recognition. filling/inactivation or perturbation of fiia active site does not alter the affinity of fiia for immobilized b2gpi, confirming that the active site is not involved in the interaction. mapping of thrombin binding sites with specific exosite-directed ligands (i.e. hirugen, gpibalpha, hd1 aptamer) and thrombin analogues having the exosites variably compromised (i.e. prothrombin, prethrombin-2, alpha-thrombin), reveals that the positively charged exosite-ii of fiia plays a key role in b2gpi binding. from the docking model of the bb2gpi-thrombin complex, we identified a highly negatively charged segment 219-232 in domain v of b2gpi interacting with positively charged pathes in thrombin exosite ii. the synthetic peptide b2gpi(219-232) was able to bind to fiia with an affinity (kd538 6 9nm) comparable to that of full-length b2gpi, deduced from fluorescence or spr measurements and to compete in spr measueremnts with the binding of full-length b2gpi to thrombin. hence, combining experimental and theoretical data, we obtained a reliable model of the b2gpi-thrombin complex. metalloproteases are one of the most diverse types of proteases, presenting a wide range of folds and catalytic metal ions. in the case of the merops ma clan, where most of the known metalloproteases are grouped based on the consensus hexxh sequence motif, a single catalytic zinc ion and common fold architecture [1] . despite these common features, members from distinct families present distinct domain composition and topology. given our interest in developing new tailor-made metalloproteases for bioengineering applications, an in-depth understanding of the factors governing their function is required. protein internal dynamics includes the space of functionally-relevant structural changes occurring during an enzymatic reaction, and there is an increasing understanding on how it relates with protein sequence and structure evolution. therefore, we have recently assessed how the structural heterogeneity of metalloproteases relates with the similarity of their dynamical profiles [2] . first, the dynamical profile of the clan ma type protein thermolysin, derived from the anisotropic network model, was evaluated and compared with those obtained from principal component (pc) analysis of a set of 112 crystallographic structures and essential dynamics (ed) analysis of a 20 ns molecular dynamics simulation trajectory [3] . a close correspondence was obtained between normal modes (nm) derived from the coarse-grained model and experimentally-observed conformational changes (rmsip between nm1-nm3 and pc1 of 0.81), corresponding to functionally-relevant hinge bending motions that were shown to be encoded in the internal dynamics of the protein (cumulative overlap of ed1-ed3 and pc1 of 0.85). next, dynamics-based comparison methods that employ a related coarse-grained model (b-gaussian elastic network model) was made for a representative set of 13 ma clan members [4] , allowing for a quantitative description of its structural and dynamical variability. although members are structurally similar (87% pairs with dalilite z-score > 2.0), they nonetheless present distinct dynamical profiles (69% of pairs with aladyn p-value > 0.02), with no identified correlation between structural and dynamical similarity. for cases where high dynamical similarity was observed, the respective modes corresponded to hinge-bending motions encompassing regions close to the active site. further inspection of the produced alignments indicates that for ma clan metalloproteases, conservation of internal dynamics has a functional basis, namely the need for maintaining proper intermolecular interactions between the protein and respective substrate. previously unnoticed dynamical similarity between clan members botulinum neurotoxin type a, leishmanolysin and carboxypeptidase pfu was also found. together, these results suggest that distinct selective pressure mechanisms acted on metalloprotease structure and dynamics through the course of evolution. this work shows how new insights on metalloprotease function and evolution can be assessed with comparison schemes that incorporate additional information of protein dynamics. glucokinase from antarctic psychrotroph pseudoalteromonas sp. as-131 (psgk) has a higher specific activity at low temperatures and a higher thermal stability than its mesophilic counterpart from e. coli (ecgk). in order to elucidate the structural basis for cold-adaptation and thermal stabilization of psgk, we have determined the crystal structure of psgk at 1.69 å and compared it with the ecgk structure. psgk is a homodimer of the subunit of 328 amino acid residues. each subunit consists of two domains, a small a/b domain (residues 7-125 and 314-328) and a large a 1 b domain (residues 126-313). the active site is located in a cleft formed between the two domains. the identity of amino acid sequence between psgk and ecgk was 36%, but three dimensional structures of them are very similar to each other, having the conserved catalytic residues and substrate-binding residues. the analysis of the mainchain temperature factors revealed that the regions of small domain and the hinge region connecting two domains of psgk showed higher temperature factors with a lower number of intramolecular hydrogen bonds and ionic interactions than the corresponding regions of ecgk. however, the large domain regions of psgk showed lower temperature factors with a higher number of intramolecular hydrogen bonds than ecgk. furthermore, the atomic temperature factors of catalytic asp112 on the small domain were higher, but those of glucose-binding glu169, his172, and glu199 on the large domain were lower than ecgk. these results suggest that highly flexible hinge region and the catalytic residue on the small domain of psgk may contribute to its cold-adaptation, namely higher activity at low temperatures, whereas a more rigid structure of the large domain of psgk stabilizes its overall structure more strongly than ecgk. nowadays non-waste technologies in synthetic chemistry become more and more popular. such processes are often carried out using different enzymes. dehydrogenases represent the large group of enzymes, which are widely used in synthesis of chiral compounds and other useful molecules. such enzymes need nadh or nadph as a cofactor and due to high cost of reduced coenzymes a cofactor regeneration system is an obligate part in such kind of processes. it was shown that formate dehydrogenase (fdh, ec 1.2.1.2.) is one of the best enzymes for nad(p)h regeneration. fdh catalyses the reaction of formate oxidation to carbon dioxide coupled with reduction of nad(p)1 to nad(p)h. the main advantages of fdh are the irreversibility of catalyzed reaction, low price of formate ion and wide ph optimum of activity. our laboratory has the largest collection of formate dehydrogenases from different sources. many fdh genes from bacteria, yeasts and plants were cloned and enzymes were expressed in active and soluble forms. mutant formate dehydrogenases from bacterium pseudomonas sp.101 show the highest thermal stability as well as activity in comparison with other reported formate dehydrogenases. now we have focused on eukaryotic genes. the recombinant enzymes from soya glycine max (soyfdh), arabidopsis thaliana (athfdh), moss physcomitrella patens (ppafdh) and yeast ogataea parapolymorpha (opafdh) were obtained by genetic engineering methods. it was revealed, that soyfdh has the best michaelis constants among all known fdhs, but it's less thermally stable compared to other fdhs. new mutant forms of soyfdh with excellent catalytic characteristics and high thermal stability were obtained by protein engineering. other enzymes (athfdh, ppafdh and opafdh) are comparable in their stability with majority of bacterial enzymes (but not with psefdh), so all the new obtained fdhs can be successfully used for cofactor regeneration. marmara university, 2 wellesley college, 3 antibiotics are essential therapeutic drugs widely used in the treatment of bacterial infections. unfortunately, misuse of these drugs resulted in the development of bacterial defense mechanisms. blactamase synthesis is among these mechanisms that renders b-lactam antibiotics ineffective. understanding the dynamic behavior of this enzyme is an important step in controlling its activity. in a former study, the importance of highly conserved w229 in modulating the hinge type h10 motion was reported. in the light of this information, mutant tem-1 b-lactamase enzymes with w229a, w229f and w229y substitutions were constructed. wild-type and mutant tem-1 b-lactamases purified with ni21affinity chromatography were subjected to enzyme assay using centa as the substrate. with w229f and w229y mutations, the remaining activity was approximately 10% of the initial activity. however with the w229a mutation, activity was totally lost. structural studies of the w229a mutant with cd and florescence spectroscopy indicated that there was no major change in the overall structure. however this mutation disrupted the interactions of w229 which resulted in an increase in the flexibility of this region of the protein. this project was supported by t € ub _ itak project no 113m533. light-switchable zn21 binding proteins to study the role of intracellular zn21 signaling stijn aper 1 , maarten merkx 1 1 zn21 plays an important catalytic and structural role in many fundamental cellular processes and its homeostasis is tightly controlled. recently, free zn21 has also been suggested to act as an intracellular signaling molecule. to get increased understanding of the signaling role of zn21 we are developing light-switchable zn21 binding proteins to perturb the intracellular zn21 concentration using light. these protein switches consist of two light-responsive vivid domains and the zn21 binding domains atox1 and wd4, linked together with flexible peptide linkers. in the dark, zn21 is tightly bound in between the two zn21 binding proteins. light-induced dimerization of the vivid proteins disrupts this interaction and thus results in zn21 release. the fluorescent proteins cerulean and citrine were attached to the vivid domains to allow the different conformational states of the protein switch to be monitored using fret. zn21 titrations revealed a 3-fold decrease in zn21 affinity going from dark-to light-state for the initial design, which was further improved to 10-fold by optimizing the linkers between the protein domains. in addition, the zn21 affinities of both states were tuned to be optimal for intracellular applications. switching between the high affinity dark-state and the low affinity lightstate was found to be reversible for at least two light-dark cycles. following the in vitro characterization, we are currently assessing the performance of this genetically encoded 'caged' zn21 in mammalian cells. proteins as supramolecular building blocks: engineering nanoscale structures 5 school of biological sciences, university of auckland, 6 school of biological sciences, victoria university proteins hold great promise in forming complex nanoscale structures which could be used in the development of new nanomaterials, devices, biosensors, electronics and pharmaceuticals. the potential to produce nanomaterials from proteins is well supported by the numerous examples of self-assembling proteins found in nature. we are exploring self-assembling proteins for use as supramolecular building blocks, or tectons, specifically the n-terminal domain of a dna binding protein (nterm-lsr2) and a typical 2-cys peroxiredoxin (hsprx3). non-native forms of these proteins have been designed undergo selfassembly into supramolecular structures in a controllable manner. self-assembly of nterm-lsr2 is initiated via proteolytic cleavage, thereby allowing us to generate supramolecular assemblies in response to a specific trigger. we will show that the degree of oligomerisation can be controlled by variations in environmental conditions such as ph and protein concentration. furthermore, via protein engineering, we have introduced a new "switch" for oligomerisation via enteropeptidase cleavage. the new construct of nterm-lsr2 can be activated and assembled in a controlled fashion and provides some ability to alter the ratio of higher ordered structures formed. hsprx3 has been shown to oligomerise into dimers, toroids, stacks and tubes in response to specific triggers such as ph and redox state. in this work we have utilised the histidine tag to further control the assembly of this versatile protein tecton. we will show that minute variations in ph can induce oligomersation of hsprx3 toroids into stacks and tubes. furthermore, by utilising the histidine tag as a ligand we can bind divalent metals to these supramolecular structures. this not only drives the formation of higher ordered oligomers but also provides a facile route which may facilitate the functionalisation of these protein nanoscale structures after they have been assembled. danielle basore 1,2 , rajesh naz 5 , scott michael 6 , sharon isern 6 , benjamin wright 3 , katie saporita 1 , donna crone 1 , christopher bystroff 1,2,4 1 biological sciences, rensselaer polytechnic institute, 2 cbis, rensselaer polytechnic institute, 3 chemical and biological engineering, rensselaer polytechnic institute, 4 computer science, rensselaer polytechnic institute, 5 obstetrics and gynecology, west virginia university, 6 unintended pregnancy is a worldwide public health concern, with 85 million pregnancies being classed as unintended in 2012 . the magnitude of this number clearly indicates an unmet need in terms of contraception. methods that are currently available are effective, but exhibit many problems. side effects, ease of use, cost, and availability are all concerns. we propose a contraceptive vaccine that would be safe, effective, long-lasting, cheap, and reversible. our vaccine would prevent pregnancy by targeting sperm with antibodies raised in the woman's body. several approaches have been taken to developing a contraceptive vaccine in recent years. the most successful so far has been using human chorionic gonadotropin (hcg), a hormone produced during pregnancy, as an antigen . the hcg vaccine progressed to phase 2 clinical trials, but only displayed an 80% efficacy, which is insufficient for a contraceptive. our lab uses a structure based approach to the design of an anti-sperm antigenic protein. we believe this will raise a more vigorous immune response that will produce a longer lasting titer. the catsper complex is a heterotetrameric calcium channel found in the tail region of sperm . each subunit of the complex contains an exposed loop known as the p-loop. the p-loop is unique on the surface of sperm because it is not glycosylated, allowing antibodies to potentially recognize and bind it. ylp12 is a twelve residue peptide that mimics the glycans in the glycocalyx of sperm . ylp12 is a member of the flitrx library, and in mice, produced protective titers that were reversible both voluntarily and involuntarily. our designs will introduce these two potential antigens into a loop of the l1 protein of human papilloma virus. l1 spontaneously assembles into virus like particles, and will aid in the production of a robust immune response. protein carriers for passage of the blood-brain barrier sinisa bjelic 1 1 medical solutions that help protein therapeutics accumulate into the brain are crucial for future treatment of neurological disorders. biodrugs have a tremendous potential to treat disorders of the nervous system, but their efficiency has been severely restricted. to reach the brain all drugs must traverse the blood-brain barrier (bbb) -a permeable wall that separates blood from the brain -whose main function is to protect the nervous system from environmental influences of bacteria and toxins. unfortunately the bbb is also the culprit that effectively blocks access to therapeutics required for treatment of neurological diseases. a way to boost exposure of therapeutics across the bbb is to piggyback onto the transferrin receptor, a multidomain protein anchored in the membrane, which is involved in the physiological facilitation of iron uptake. here i present research that aims at successfully developing potent protein carriers for transferrin receptor-mediated passage of the bbb by using computational protein design in combination with yeast display methodology for hit validation and optimization. the longterm goal is to couple therapeutics -as for example drugs against alzheimer's -to the designed carriers to increase the brain uptake and cure neurological disorders. medium-throughput multistep purification of coagulation factor viia jais r. bjelke 1 , gorm andersen 1 , henrik østergaard 1 , laust b. johnsen 1 , anette a. pedersen 1 , tina h. glue 1 1 there is a need of medium-to-high throughput purification of low-titre recombinant protein variants for screening to identify the final biopharmaceutical lead. such proteins include coagulation factors to be used for treatment of haemophilia and other bleeding disorders. at novo nordisk we have established a platform for production of recombinant coagulation factor viia variants, which include a spectrum of single-point mutations to large domain insertions. the variants were produced using transiently transfected hek293f, hkb11 or choebnalt85 (qmcf technology) suspension cells. harvest cultivations were typical in the range of 0.3-to 1l. a 3-step continuous, multistep purification method was implemented on € aktaxpress systems (ge healthcare). the interlinked process steps include capture using an immunoaffinity column, polish, concentration and buffer exchange using an anion-exchange column and proteolytic activation of the zymogen variant forms using a coagulation factor xaimmobilized column. buffers were designed such that elution from the capture column was aligned with binding conditions on the polish column to avoid a desalting step in-between. the following and final enzymatic activation was optimized with regards to flow rate to ensure full conversion while minimizing unwanted secondary cleavages in factor viia. the final products were fractionated in sharp chromatographic peaks ready for characterization. hplc and sds-page analyses showed a solid quality of the produced variants and more than 800 variants have been produced in sub mg scale using the outlined method. biomimetic sequestration of co2: reprogramming the b1 domain of protein g through a combined computational and experimental approach esra bozkurt 1 , ruud hovius 1 , thereza a. soares 2 , ursula rothlisberger 1 1 ecole polytechnique f ed erale de lausanne, 2 federal university of pernambuco protein engineering is a powerful tool to generate highly specific enzymes for biomimetic production of chemicals. among many applications, the development of enzymes to accelerate carbon dioxide fixation is a possible route to limit co2 emission. in this project, we are inspired by the ancient enzyme carbonic anhydrase which efficiently catalyzes the reversible hydration of carbon dioxide in the presence of a zinc ion active site.1 to create an efficient biocatalyst, the engineered gb1 domain2 containing a his3cys zn (ii) binding site was used as a starting point.3 in subsequent work, b1 domains comprising of his3wat zn (ii) binding sites have been rationally designed to produce carbonic anhydrase mimics. the re-engineering was accomplished through a series of mutations to orient the zinc bound reactive species to form a hydrogen bond network in the active site while retaining the native secondary structure. we performed classical molecular dynamics (md), quantum mechanics/molecular mechanics (qm/ mm) simulations and metadynamics, with the aim to explore potential catalytic roles of the reengineered b1 domains and to elaborate the reaction mechanism. briefly, we introduced novel zn (ii) binding sites into thermostable b1 domain. in parallel, experiments are underway. wild-type protein was expressed and purified. structural and mutagenesis studies are ongoing. the results emphasize the power of theoretical work to enable the mimicking of nature's enzymes for desired catalytic functions. the roles of entropy and packing efficiency in determining protein-peptide interaction affinities diego caballero 1,2 , corey o'hern 1,2,3,4 , lynne regan 2,5,6 1 physics, yale university, 2 integrated graduate program in physical and engineering biology, yale university, 3 mechanical engineering and materials science, yale university, 4 applied physics, yale university, 5 molecular biophysics and biochemistry, yale university, 6 chemistry, yale university despite many recent improvements in computational methods for protein design, we still lack a quantitative and predictive understanding of the driving forces that control protein stability, for example, we do not know the relative magnitudes of the side-chain entropy, van der waals contact interactions, and other enthalpic contributions to the free energy of folded proteins. in addition, we cannot reliably predict the effects of point mutations on enzyme specificity or sequence tolerance in ligand binding sites. the tetratricopeptide repeat (tpr) motif is a common and versatile protein system that has been used as a model to study protein-protein interactions. for example, recent studies have experimentally measured the binding affinity and specificity for different tpr binding pockets and peptide ligands and generated a ranking of the protein-peptide pairs with the highest affinity. to gain a fundamental understanding of the interplay between atomic close packing and fluctuations of side-chain conformations in protein-peptide binding pairs, we performed all-atom langevin dynamics simulations of key residues near the binding interface of tpr proteins and their cognate peptides. the langevin dynamics simulations enabled us to calculate the entropy and potential energy of side chain conformations in the presence of backbone fluctuations for each protein-peptide pair. we compile rankings of the stability and affinity of mutant tpr-peptide structures to those obtained from experimental studies. this research has enhanced our ability to rationally manipulate protein-peptide interfaces. advances from this research will enable the design of tpr modules that specifically recognize biologically important proteins. monitoring protein-protein interactions using tripartite split-gfp complementation assays protein-fragment complementation assay (or pca) is a powerful strategy for visualizing protein-protein interactions in living cells. previously described split-gfp based sensors suffer from the poor solubility of individual pca fragments in addition to background signal originating from their spontaneous selfassembly (1). we developed a new encoded genetic reporter called "tripartite split-gfp" for visualizing protein-protein interactions in vitro and in living cells. the assay is based on tripartite association between two twenty amino-acids long split-gfp tags, gfp10 and gfp11, fused to interacting protein partners, and the complementary gfp1-9 detector. when proteins interact, gfp10 and gfp11 selfassociate with gfp1-9 to reconstitute a functional gfp (2). using coiled-coils and frb/fkbp12 model systems we characterize the sensor in vitro and in escherichia coli. we extended our studies to mammalian cells and examine the fk-506 inhibition of the rapamycin-induced association of frb/fkbp12. the small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence and for screening modulators of complex formation in cell-based assays. aldehyde dehydrogenases (aldhs) catalyze the oxidation of aldehydes to their corresponding acids using nad(p)1 as coenzyme. these enzymes are responsible for the detoxification of lipid peroxidation products, which have been involved in the etiology and pathogenesis of different diseases involving increments in oxidative stress. recent data from our group, showed that aldh3a1 is resistant to inactivation by lipid peroxidation products, even at concentrations 50-100 times higher than those required to inactivate aldh1a1 and aldh2. the amino acids sequence of the aldehyde-binding site of the three enzymes was analyzed, and it was found that the enzymes susceptible to the effect of lipid peroxidation products (aldh1a1 and aldh2), have cys residues flanking the reactive cys (position 302), based on this criteria and considering that these aldehydes react preferentially with cysteine, a mutant of aldh2 was generated changing the cys residues adjacent to cys302. the mutant aldh2-cys301thr-cys303val, was resistant to the inactivation by acrolein and 4-hne, even at concentrations 1000-fold higher than those required to inactivate aldh2. however, the mutant presented values of km 2, 5 and 50-fold higher for acrolein, propionaldehyde and acetaldehyde, respectively, compared to the wild type enzyme, but showed a catalytic efficiency similar to the parent enzyme. these data revealed that cys residues near to the reactive cys in aldh2 are important in the inactivation process induced by lipid aldehydes, but also participate in determining the specificity for the substrates in this enzyme. small molecule-assisted shutoff: a widely applicable method for tunable and reversible control of protein production h. kay chung 1 , conor jacobs 1 , yunwen huo 2 , jin yang 3 , stefanie krumm 4 , richard plemper 4,5 , roger tsien 0 , michael lin 3 1 department of biology, stanford university, 2 department of pediatrics, stanford university, 3 department of pharmacology, university of california san diego, 4 department of pediatrics, emory university, 5 institute for biomedical sciences, georgia state university, 6 department of chemistry and biochemistry, university of california san diego, 7 howard hughes medical institute, university of california san diego, 8 the ability to quickly control the production of specific proteins would be useful in biomedical research and biotechnology. we describe small molecule-assisted shutoff (smash), a technique in which proteins are fused to a self-excising degron and thereby expressed in a minimally modified form by default. degron removal is performed by a cis-encoded hepatitis c virus (hcv) protease, so that applying clinically available hcv protease inhibitors causes degron retention on subsequently synthesized protein copies and suppresses further protein production. we find that smash allows reversible and dosedependent shutoff of various proteins with high dynamic range in multiple cell types, including yeast. we also successfully use smash to confer drug responsiveness onto a rna virus for which no licensed drug inhibitors exist. as smash does not require permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. furthermore, as smash only uses a single tag and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts. top, a protein of interest is fused to the smash tag via a hcv ns3 protease recognition site. after protein folding, the smash tag is removed by its internal ns3 protease activity, and is degraded due to an internal degron activity. bottom, addition of protease inhibitor induces the rapid degradation of subsequently synthesized copies of the tagged protein, effectively shutting off further protein production. vaccine development has emerged, epitope-focused immunogens, but in the past these have failed to deliver the expected outcome. here, we employed a new computational design methodology (rosetta fold from loops or ffl) to design epitope-focused immunogens. ffl was devised to insert structurally defined functional sites into protein scaffolds. throughout the ffl stages the structure of the scaffold is folded and its sequence designed to stabilize the desired functional conformation of the inserted site. we used ffl to design epitope-focused immunogens for the respiratory syncytial virus (rsv), for which despite the intense research we are still lacking an approved vaccine. we designed three-helix bundles harboring an rsv epitope, that was previously co-crystallized with the neutralizing antibody motavizumab. the designs were thermodynamically stable (tm > 100˚c) and showed extremely high affinities to motavizumab (kd 30 pm). structural characterization through x-ray crystallography of antibodybound and unbound scaffolds showed good agreement to the computational models in the overall structure (rmsd -1.2 å) and exquisite mimicry of the epitope region (rmsd -0.4 å), when compared to the peptide-epitope in complex with motavizumab. the designed immunogens were used to immunize non-human primates (nhp), and approximately 75% of the cohort developed rsv neutralizing activity, in some instances with high potency. to evaluate the therapeutic relevance of the elicited neutralization activity, we compared the nhp neutralization titers to those of human sera after natural rsv infection, which generally yields protective levels of antibodies. the neutralization potency of the best nhp responders was comparable to that of the human sera. to better understand the features of the antibodies elicited, we isolated several rhesus monoclonal antibodies (rhmabs) from the animal that exhibited the most potent neutralization. two of the rhmabs bound to the immunogen with very high affinity (kd 3 pm) and were potent rsv neutralizers. interestingly, these rhmabs were approximately 10 fold more potent than the fda-approved prophylactic antibody palivizumab. our results provide the first proof-of-principle for epitope-focused vaccine design, and demonstrate the power of the ffl figure 1 . schematic of nucleotide binding, exchange and hydrolysis in tubulin, and its coupling to mt assembly. exchange of gdp (orange) for gtp (magenta) at the e-site in b-tubulin (blue) happens in the unpolymerized dimer (left). the active, gtpbound tubulin dimer adds to a growing mt (right). interaction of the incoming a-tubulin (green) with the e-site nucleotide at the plus end of a mt (with b-tubulin exposed) results in gtp hydrolysis. the mt cartoon (bottom right) shows an oversimplified representation of a gtp cap as it first grows by tubulin addition and then shrinks by polymerization-coupled gtp hydrolysis (here b-tubulin that is bound to gtp is shown in red and that bound to gdp is shown in blue). cryo-em density map (emdb-6349) and atomic model (pdb: 3jak) for an eb3-decorated mt bound to gtpgs. a-tubulin, b-tubulin and eb3 are colored green, blue, and orange, respectively. computational methodology. we anticipate that ffl will be useful for a variety of other challenges in the computational design of functional proteins. designed repeat proteins as templates for photoactive molecules and fluorescent nanoclusters sara h. mejias 1,2 , antonio aires 1,2 , javier l opez-andarias 3 , pierre couleaud 1,2 , begoña sot 1,2 , carmen atienza 3 , nazario mart ın 1,3 , aitziber l. cortajarena 1,2 1 imdea nanoscience, c/faraday, 9, ciudad universitaria de cantoblanco 28049, 2 cnb-csic-imdea nanociencia associated unit "unidad de nanobiotecnolog ıa", 3 departamento de qu ımica org anica i, facultad de qu ımica, universidad complutense self-assembly of biological molecules into defined functional structures has a tremendous potential in nanopatterning, and the design of novel bionanomaterials and functional devices. molecular selfassembly is a process by which complex three-dimensional structures with specified functions are constructed from simple molecular building blocks. we present first the study and characterization of the assembly properties of modular repeat proteins, in particular designed consensus tetratricopeptide repeats (ctprs), and their application as building blocks in order to generate functional nanostructures and biomaterials. ctpr proteins can be assembled into self-standing thin films,1 and thin nanometer fibers in solution.2 in this work, we show the use of the designed consensus repeat proteins as scaffolds to template: (1) photoactive organic molecules, and (2) fluorescent nanoclusters. 1.we explore the potential of ctpr proteins to arrange donor-acceptor pairs for electro-active materials. in particular, porphyrin rings arranged by ctprs in a defined distance and orientation for favoring face-to-face orientation which should lead to an improvement in the optoelectronic properties. our results confirm the successful ability of ctpr proteins to be used as scaffold for ordering organic chromophores, while preserving their structure. the unique self assembly properties of ctpr scaffolds have been exploited to generate ordered conductive films of the protein-porphyrin conjugates. these results open the door to fabricate hybrid protein-based solid devices. 2.we show results on the ability of ctpr to encapsulate and stabilize fluorescent gold nanoclusters. we investigated the influence of the protein sequence in the final properties of the nanoclusters. the structural and functional integrity of the protein template is critical for future applications of the protein-cluster complexes. therefore synthetic protocols that retain the protein structure and function have been developed. as a proof of concept, a ctpr module with specific binding capabilities has been successfully used to stabilize nano clusters. biohybrid photoelectrochemical cells have been developed by functionalizing the hematite photoanode with the light-harvesting cyanobacterial protein c-phycocyanin (pc) yielding a substantial enhancement of the photocurrent density. photoelectrochemical cells combining light-harvesting proteins and inorganic semiconductors have potential for the use in artificial photosynthesis. in this work we present processing routes for the functionalization of hematite photoanodes with pc, including in situ co-polymerization of pc with enzymatically-produced melanin and using a recombinantly produced pc 2. moreover, recombinant forms of the light-harvesting protein c-phycocyanin from synechocystis sp. pcc6803 were engineered to carry a peptide with affinity for hematite. similarly, a bacterial laccase was engineered to acquire affinity for hematite. results obtained from the different approaches to hematite functionalization and the advantages offered by protein engineering will be presented. minimizing a suitable free energy expression is arguably the most common approach in (ab initio) protein structure prediction. the achieved accuracy depends crucially on the quality of the free energy expression in use. here, we present corrections to existing free energy expressions which arise from the thermal motion of the protein. we (i) devise a term accounting for the vibrational entropy of the protein, and (ii) correct existing potentials for 'thermal smoothing'. (i) vibrational entropy is almost always neglected in free energy expressions as its consideration is difficult. this practice, however, may lead to incorrect output because distinct conformations of a protein can contain very different amount of vibrational entropy, as we show for the chicken villin headpiece explicitly [1] . for considering vibrational entropy, we suggest a knowledge based approach where typical fluctuation and correlation patterns are extracted from known proteins and then applied to new targets. (ii) at ambient conditions, timeaveraged potentials of proteins are considerably smoothened due to thermal motion where the strength of this effect varies strongly between atoms. distinguishing these inhomogeneities by introducing new atom species regarding their locale environment can therefore increase the precision of time-averaged potentials [2] . extraction of general principles from the continually growing protein data bank (pdb) has been a significant driving force in our understanding of protein structure. atomistic or residue-level statistical potentials, secondary-structural propensities, and geometric preferences for hydrogen bonding are among the classical insights that arose from observations in the pdb. given the magnitude of structural data available today, it is likely that many quantitative generalizations remain to be made. here we hypothesize that the pdb contains valuable quantitative information on the level of local tertiary structural motifs (terms), with term statistics reflecting fundamental relationships between sequence and structure. we define a term to be the structural fragment that captures the local secondary and tertiary environments of a given residue, and put our hypothesis through a series of rigorous tests. first, we show that by breaking a protein structure into its constituent terms, and querying the pdb to characterize the natural ensemble around each, we can estimate the compatibility of the structure with a given amino-acid sequence through a metric we term "structure score." considering submissions from recent critical assessment of structure prediction (casp) experiments, we find a strong correlation (r 5 0.69) between structure score and model accuracy, with poorly predicted regions readily identifiable. this performance exceeds that of leading atomistic statistical energy functions. next, we show that by considering the terms of a structure that are affected by a given mutation, and mining the pdb to characterize sequence statistics associated with each, we are able to predict mutational free energies on par with or better than far more sophisticated atomistic energy functions. finally, we ask whether term statistics are sufficient to enable the design of proteins de-novo. we demonstrate that given a native backbone conformation, term considerations alone with no input from molecular mechanics correctly predict roughly the same fraction of amino acids from the corresponding native sequence as state-ofthe-art computational protein design methods. knowledge-based energy functions have already put pdb statistics to good use by parsing structural environments into geometric descriptors, generally assuming their conditional independence. our results suggest that it may now be possible to instead consider local structural environments in their entirety, asking questions about them directly. if this is the case, then the pdb is an even larger treasure trove of information than it has been generally known to be, and methods of mining it for term-based statistics should present opportunities for advances in structure prediction and protein design. comprehensive understanding of a protein fold is intertwined with successful design. recent advances in designing de novo structures have shown that proteins can be designed for a few globular and helical folds. however, designing all-b structures and barrels remains challenging because loops and intricate long range interactions that are important in these topologies are difficult to control. for designing novel catalysts, the (a/b)8 -barrel (or tim-barrel) fold is one of the most important examples, for it is the most common topology for enzymes. for almost 30 year, attempts in designing de novo tim barrel structures have all resulted in poorly folded proteins. here we describe the successful design of a 4-fold symmetrical (a/b)8 barrel directly from geometrical and chemical principles. 22 designed variants with a wide range of stabilities from being molten globules to cooperatively folded proteins were experimentally characterized, and the results revealed the importance of sidechain-backbone hydrogen bonding for defining the characteristic a/b-barrel. the 184 residue tim barrel structure is among the smallest tim-barrels and has a fully-reversible melting temperature of 888c. the x-ray crystal structure shows atomic-level agreement with the design model. despite this structural similarity, psi-blast searches do not identify sequence similarities to known tim-barrel proteins. more sensitive profile-profile searches suggest that the design is sufficiently distant from other native tim-barrel superfamilies to be in a superfamily of its own, further implying that nature has only sampled a subset of the sequence space available to the tim-barrel fold. the ability to de novo design tim-barrels opens new possibilities for custom-made enzymes. 3 university of texas southwestern medical center, 4 biofrontiers institute, university of colorado creation of new molecular sensors and actuators based on fluorescent proteins relies on methods for identifying complex photophysical phenotypes and subsequently performing separations on cell populations. we developed a microfluidic flow cytometry approach tailored to interrogating the performance of genetically-encoded fluorophores and present the results of studies employing this technology. the system screens cell-based libraries on the basis of multiple photophysical parameters relevant to imaging, including brightness, photostability, and excited-state lifetime (i.e. a proxy for fluorescence quantum yield) at a rate of up to 180 cells/sec. in a first generation of experiments, molecular dynamics-guided design was used to create a library of mcherry mutants that was screened with this system, resulting in the identification of a variant with a higher stability b-barrel and improved photostability but with a decreased brightness due to reduction in the fluorescence quantum yield. to avoid inadvertent decreases in this important performance criterion, subsequent rounds of selection were performed on the basis of both photostability and excited-state lifetime as sorting criteria. in these second generation selections, mutations were designed to target pathways of oxygen access through the bottom of the bbarrel in addition to a position that directly interacts with the chromophore. furthermore, subsequent rounds of screening were used to improve folding and maturation. the multiparameter sort identified multiple clones with up to 8-fold improved photostability and up to double the excited-state lifetime of the parent mcherry fluorescent protein. the best mutant we identified produces one order of magnitude more photons before photobleaching compared to mcherry, at excitation conditions characteristic of confocal fluorescence microscopy. our results demonstrate the utility of combining moleculardynamics-guided library design with technology for photophysics-based selections. we anticipate that the new fluorescent proteins obtained in this work will find use in low-copy-number and long-duration imaging live cell imaging applications in cell-lines created by genomic editing techniques. targeted protein degradation achieved through a combination of degrons from yeast and mammalian ornithine decarboxylase rushikesh joshi 1 , ratna prabha c. 1 1 the maharaja sayajirao university of baroda targeted protein degradation achieved through a combination of degrons from yeast and mammalian ornithine decarboxylase targeting the over accumulated protein in the cell for degradation using specific degrons is an emerging research area. the degradation of the vast majority of cellular proteins is targeted by the ubiquitin-proteasome pathway. but in the case of ubiquitin independent protein degradation, odc/az system is more effective in achieving targeted protein degradation than other types of degradation 1. ornithine decarboxylase (odc) is key regulatory enzyme in the biosynthesis of polyamines. the protein has two domains namely, n terminal a/b barrel domain and c-terminal b-sheet domain. degradation of odc is mediated by polyamine inducible protein, antizyme (az). antizyme interacts with odc on n-terminal region, which results in degradation of odc by proteasomes. in mammalian odc the c-terminal has an unstructured tail of 37 residues, which pulls odc into proteasome for degradation. it was reported earlier by coffino's group that the unstructured tail acts as a degron in chimeric fusion with gfp 2. in yeast, same function is achieved by n-terminal 44 residues 3. present study focuses on accomplishing targeted protein degradation in saccharomyces cerevisiae by adding these two degradation signals or degrons of yeast odc and mammalian odc as tags to a reporter protein. we have selected two degrons namely, n terminal a/b barrel domain of yeast odc and c-terminal 37 residues of mouse odc and grafted them to n and c-terminus of the reporter protein yegfp. degradation of yegfp and yegfp fusion with degrons of odc (degron-yegfp) were monitored by western blot using anti-gfp antibody and fluorescence spectroscopy. initially, the amount of degron-yegfp fusion protein was very low compared to control yegfp. it means that the chimeric protein underwent rapid degradation in the cells. after inhibition of proteasome, increase in the level of degron-yegfp was observed, confirming that the degrons cause rapid degradation of reporter protein through proteasome. earlier, we have also tagged ubiquitin from yeast with last 37 residues of modc and observed enhanced degradation of ubiquitin in saccharomyces cerevisiae. therefore, both the degrons of odc alone and in combination are capable of decreasing stability of reporter protein in the cells. however, the combination of degrons is more effective than either of them in isolation. enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. the requirement that they be stably folded is a likely factor that contributes to their relatively large size (> 10,000 dalton). however, much shorter peptides can achieve well-defined conformations through the formation of amyloid fibrils. to test whether short amyloid-forming peptides might in fact be capable of enzyme-like catalysis, we designed a series of 7-residue peptides that act as zn21dependent esterases. zn21 helps stabilize the fibril formation, while also acting as a cofactor to catalyze acyl ester hydrolysis. the fibril activity is on par with the most active to date zinc-protein complex. such remarkable efficiency is due to the small size of the active unit (likely a dimer of 7-residue peptides), while the protein is at least 15-fold larger in molecular weight. the observed catalytic activity is not limited to ester hydrolysis. we have designed copper binding peptides that are capable oxygen activation. these results indicate that prion-like fibrils are able to not only catalyze their own formation -they also can catalyze chemical reactions. thus, they might have served as intermediates in the evolution of modern-day metalloenzymes. these results also have implications for the design of self-assembling nanostructured catalysts including ones containing a variety of biological and nonbiological metal ions. rational design of the cold active subtilisin-like serine protease vpr with improved catalytic properties and thermal stability abstract proteinase vpr, from a psychrophilic vibrio species and its thermophilic structural homologue, aqualysin i (aqui) from thermus aquaticus, we set out to design a mutant of vpr which would be more thermostable, but would retain the high catalytic activity of the wild type enzyme. our starting protein template was a previously stabilized mutant containing two inserted proline residues close to the nterminus of vpr (n3p/i5p). this vpr_n3p/i5p mutant was shown to have a significantly increased thermal stability but displayed a concomitant tenfold loss of catalytic efficiency. from our previous studies we selected two mutations, one which increased catalytic activity (q142k) of the enzyme significantly and another which stabilized the protein against thermal denaturation (n15d). the n15d mutation had been shown to introduce a salt bridge into the structure of the cold adapted proteinase, yielding higher stability but without negative effects on activity. the q142k exchange had been shown to double the turnover number (kcat) to that of the wild type enzyme. insertions of these selected mutations into the vpr_n3p/i5p mutant were according to predictions; the q142k increased the kcat tenfold, and the n15d mutation increased the thermal stability. in the combination mutant, vpr_n3p/i5p/n15d/q142k, thermal stability was increased by 88c and 108c, in terms of tm and t50%, respectively. furthermore, the catalytic activity of the mutant was somewhat higher than that of the wild type enzyme. critical peptide stretches may not serve as faithful experimental mimics for protein amyloidogenesis bishwajit kundu 1 , dushyant garg 1 1 certain amino acid stretches are considered critical to trigger the amyloidogenesis in a protein. these peptide stretches are often synthetically produced to serve as experimental mimics for studying amyloidogenesis of the parent protein. here we provide evidence that such simple extrapolation may be misleading. we studied the amyloidogenesis of full length bovine carbonic anhydrase ii (bcaii) and compared it with those formed by its critical amyloidogenic peptide stretch 201-227 (pepb). under similar solution conditions and initial monomeric concentrations, we found that while amyloid formation by bcaii followed aggregation kinetics dominated by surface-catalyzed secondary nucleation, pepb followed classical nucleation-dependent pathway. the afm images showed that bcaii forms short, thick and branched fibrils, whereas pepb formed thin, long and unbranched fibrils. atr-ftir revealed parallel arrangement of cross b sheet in bcaii amyloids, while pepb arranged into antiparallel b sheets. amyloids formed by bcaii were unable to seed the fibrillation of pepb and vice versa. even the intermediates formed during lag phase revealed contrasting ftir, far uv cd signature, hydrophobicity and morphology. we propose that for any polypeptide, the sequences flanking a critical region are equally effective in modulating the initial nucleation events, generating prefibrillar and finally fibrillar species with contrasting characteristic. the results have been discussed in light of amyloid polymorphism and its importance in the design of therapeutic strategies targeting such toxic regions. aksana labokha 1 , ralph minter 1 1 all approved biological drugs target extracellular proteins and not the majority of the expressed human genome, which resides within intracellular compartments. included in the latter category are many important, disease-relevant targets which cannot be easily addressed by small molecule approaches, such as the oncology targets c-myc and k-ras. although bacteria and viruses have evolved strategies to deliver biological material to the cell cytoplasm and nucleus, our ability to engineer recombinant proteins to replicate this is somewhat limited by (i) our nascent understanding of protein uptake and trafficking pathways and (ii) the ability to easily quantify cell delivery to the cytoplasm and cellular organelles. the aim of my project is to address these challenges by developing an effective assay for cytoplasmic uptake and then using it to measure the delivery efficiency of recombinant proteins which mimic natural delivery strategies e.g. cell penetrating peptides fusion, exotoxin mimics, and supercharged proteins (proteins with high surface charge which can enter cells). i also intend to explore the influence of the rab superfamily, which are the master regulators of protein trafficking, to influence and control both the kinetics and final subcellular destination of exogenous proteins. protein engineering: what's next? with the growing industrial need for engineering enzymes for the deconstruction and transformation of plant biomass in biorefineries, there is a want for the development of new approaches for designing special purpose biocatalysts. techniques, such as directed evolution, which mimic the natural selection process by evolving proteins towards the improvement of a given property, have unquestionably demonstrated their value and are routinely used in large industrial companies. nevertheless, the brute force employed in these methods, could significantly gain from an all-atom description of the underlying catalytic mechanisms, to center the efforts on more limited areas of the protein. in the last years, we have developed computational tools, which combine the electronic structure description of qm/mm methods with the potential to model long time scale processes of pele,1 to study the details of a variety of reactions. examples, which will be discussed, include rationalizing the selective oxyfunctionalization of steroids using fungal enzymes2 and the study of the effect of point mutations on the oxidation efficiency of laccases.3 these methods have shown their potential not only at the descriptive level but, more importantly, through their high predictive capability that opens many opportunities for their use in biotechnology. in this talk, we will show how recent advances in in silico approaches are setting new grounds for future computer guided directed evolution. several orthogonal bioreactions take place simultaneously within membrane bound organelles in eukaryotes and proteinaceous microcompartments in bacteria. these subcellular structures contain sets of enzymes co-involved in metabolic pathways. towards the goal of creating artificial protein microreactors, we seek to develop an artificial organelle that emulates the metabolic activity of the carbon fixating organelle of autotrophic bacteria, the carboxysome. here, we show that the two key carboxysomal enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and carbonic anhydrase (ca), can be efficiently co-encapsulated using our previously reported encapsulation system which is based on a bacterial capsid formed from the protein lumazine synthase (aals-13). our preliminary results suggest that the enzymes can act in tandem and that the co-encapsulation of ca with rubisco in the capsid is necessary for enhanced rubisco activity in vitro. we attribute this observation to the high local concentrations of the rubisco substrate, co2, produced by ca within the capsid. we are developing a theoretical model of a minimal carboxysome using the kinetic rate constants of our rubisco and ca variants and aals-13 as the shell to complement these experiments. next, we will incorporate our minimal carboxysome within an expression host such as e.coli, opening up the possibility of further optimization through directed evolution. in the past targeting and engineering of chemokines has led to several interesting drug candidates. [1] amongst them, met-rantes, a met-ccl5 with high g protein-coupled receptor (gpcr) affinity but no subsequent signal transduction, as well as mutants addressing the interaction with the so-called glycosaminoglycans (gags) seem to be the most promising candidates. both, gag knockout as well as gag affinity matured chemokine isoforms have been considered as anti-inflammatory drug candidates, out of which an il-8 mutant with 5 modifications reached clinical phase 1 where it was profiled for acute neutrophil-related exacerbation in copd. [2] cxcl10 (ip-10) is a proinflammatory chemokine released by various cells following stimulation by interferon g (ifn-g) . it is therefore considered as a late chemokine being responsible for the attraction of different lymphocytes. [3] any therapeutic indication is consequently related to chronic and multiple applications. we have therefore engineered cxcl10 very conservatively at positions to ultimately generate dominant-negative mutants with a mildly improved gagbinding affinity and an entire knock off gpcr activity. the first steps of our engineering approach were in silico modelling of the mutants and the establishment of a suitable upstream-and downstreamprocessing protocol. next we generated a fluorescently engineered cxcl10 variant for our fluorescence-based affinity studies which was subjected to biocomparability investigations relative to the native, non-fluorescent protein. compared to the wild type, the fluorescently engineered mutant exhibited similar biological, chemotactic and gag-binding properties. next we started to produce sufficient amounts of the members of our nascent mutant library which were tested with respect to their biophysically behavior as well as to their knocked out chemotactic potency on cells. these experiments included gel electrophoresis and western blot analysis to determine identity and purity; circular dichroism (cd) and chaotrope-induced unfolding to approximate structure; isothermal fluorescence titration (ift); surface plasmon resonance (spr) and isothermal titration calorimetry (itc) to quantify gagbinding affinity and boyden chamber experiments to determine the chemotactic activity. our results show that we are able to tune the gag binding strength along with the gpcr activity of human cxcl10 which could lead to therapeutic applications in the future. nanodiscs are composed of a nanometer-sized phospholipid bilayer encircled by two a helical, amphipathic membrane scaffold proteins (msps). these particles provide a unique detergent free lipid bilayer model enabling biochemical and biophysical characterization of membrane proteins in a physiologically relevant medium. previously, the largest diameter reported of a nanodisc assembled using msps was about 16-17 nm. here we present a method to create large nanodiscs (up to 80nm in diameter) assembled with covalently circularized msps (cmsp). we can observe the homogeneity in nanodiscs diameter as a narrow distribution using negative-stain em. using our method, we have created 50 nm nanodiscs and used them to study poliovirus (35 nm diameter) entry and rna translocation. a 50 nm nanodisc is sufficiently large to accommodate multiple copies of the cd155 receptor (also known as the poliovirus receptor), and has enough surface area to act as a surrogate membrane for the rna translocation complex during viral uncoating. the 50 nm nanodiscs functionalized with the his-tagged ectodomain of poliovirus receptor, cd155, were generated by adding lipids derivatized with a nta nickelchelating head group to the lipid mixture during nanodisc assembly. cd155 receptor was added to the already assembled nanodiscs and incubated for 30 minutes at room temperature. the receptordecorated nanodisc complex was purified by size exclusion chromatography. the purified complex was then incubated with poliovirus for 5 minute at 4 c, and then heated to 37 c for 15 minutes to initiate receptor-mediated viral uncoating. virus binding to nanodisc-cd155 complex and subsequent insertion of viral components into and across the membrane were confirmed by negative-stain electron microscopy (figure 1c) . to obtain a high-resolution structure for the rna translocation complex we conducted single-particle cryo-em studies using a polara f30 microscope. unlike liposomes, generating a reconstruction of samples containing nanodiscs is less complicated since the nanodiscs are more homogenous in size, and allow for thinner ice. also, the viral rna can be visualized more easily. the method for making large nanodiscs as well as the negative stain and cryo-em data will be will be presented and discussed. parametric design of alpha-helical barrels and pore-like assemblies with very high thermodynamic stabilities computational design of novel protein structures and enzymes with new functions is a promising tool to create superior biological materials with tailor-made properties, new pharmaceuticals, complex fine chemicals or renewable fuels. it also challenges our understanding of protein folding, protein evolution, molecular recognition and catalysis. here we present a procedure for designing proteins with backbones produced by varying the parameters in the crick coiled-coil generating equations [1] . combinatorial design calculations using the software suite rosetta identify low energy sequences for alternative helix supercoil arrangements. after that, loop modeling is applied to connect the designs with lowest energy. the extent to which the designed sequences encode the designed structures is evaluated using large-scale structure prediction calculations, as well as symmetric and asymmetric protein-protein docking calculations. subsequently, synthetic genes are generated for sequences that converge strongly on the designed structure for experimental characterization. we applied this approach to monomeric three and four helical bundle structures as well as a pentameric five-helix bundle structure using idealized coiled-coil geometries [2] . recently we expanded this approach to higher complexity backbones, which resulted in the de-novo design of monomeric, antiparallel six-helix bundles with untwisted, left-and right-handed geometries. circular dichroism (cd), size-exclusion coupled multi-angle light scattering measurements (sec-mals), negative stain electron micrographs (em) and small angle x-ray scattering (saxs) of these designs suggest that they indeed form the designed structures. in addition, we used rosetta protein-protein interface design functionality to computationally design oligomers out of our previously published three and four helix bundle structures to generate self-assembling pore-like structures with the potential use as channels or transporters. again, experimental validation of these designs by cd, sec-mals, em and saxs show that the designs are correct. we are currently undertaking further structural investigation of all these designs by x-ray crystallography. the designs described above can act as templates for protein or small molecule binding, holding a catalytic machinery or for scaffolding enzymes in reaction cascades. some of these applications are currently under investigation, including a self-sufficient redox system employing two copper-centers, binding of heme-moieties as a prosthetic group and tailoring the pore-like geometries to be used in nanopore sequencing. university of washington, 2 university of california, san francisco, 3 repeat proteins are an example of how evolution proceeds by building on existing structures and functions, but also a source of modular protein scaffolds for molecular recognition and biomaterials. however, it is unclear whether the limited number of folds and families that we know today is the result of the intrinsic limitations of polypeptide chains or the consequence of the path followed by evolution. we explored this hypothesis by computational design of repeat proteins based on modular units formed by two alpha helices and two loops of variable lengths, without relying on information from available repeat protein families. the automated sampling of the conformational space resulted in a large number of architectures from which 83 de novo designs were selected for experimental characterization. 66% of the proteins were stable up to 958c and monodisperse and 42 designs were structurally validated by small angle x-ray scattering. crystal structures were solved for 15 of them, with root mean square deviation from the models between 0.7å and 2.5å. the designs differ from known proteins both at the sequence and structure levels and cover a broader range of geometries than observed in naturally occurring repeat protein families, indicating that existing architectures represent only a small fraction of what can be achieved. our results show that it is possible to expand the range of repeat protein architectures beyond the naturally occurring families, and that computational design can provide new scaffolds and enable the design of proteins tailored for specific applications. the serpin family of proteins consists of over 1500 members, all with a highly conserved native structure that is metastable (1). serpins use this metastability to control the activity of proteases, via a specific inhibitory process. the serpin binds to its target protease through specific residues within the reactive centre loop, the protease cleaves the loop and results in a large conformational change causing the protease to become distorted and catalytically inactive whilst the serpin becomes much more stable (1, 2, 3) . the metastable nature of aat is therefore required to facilitate the rapid and gross conformational changes required for its inhibitory function (2, 3) . several disease-causing mutants of aat have been identified, the most common of them being the z-variant (4). the z-variant has an increased propensity to polymerize in the endoplasmic reticulum of hepatocytes leading to cell death and liver damage (4) . during the past fifteen years, many groups have unsuccessfully screened a number of serpins and a vast range of solution conditions to identify a combination of serpin and conditions that will enable the folding reaction of a serpin to be characterized. we have now taken an alternative approach and designed a synthetic "model" serpin that folds reversibly to its native state. in order to do this, we used a consensus design approach, analysing a sequence alignment of 212 serpin sequences and determining the prevalent amino acid residue at each position, we termed this serpin conserpin (consensus serpin). here we present the structural, biophysical and functional characterisation of conserpin. combined crystallographic and folding studies reveal the characteristics of conserpin that likely dictate its unique stability and folding behaviour, whilst retaining activity as a serine protease inhibitor. the development of enhanced protein binding scaffolds is a key for engineering protein inhibitors and biosensors with advanced characteristics. utilizing the structural variability and designability of repeat proteins offers a means for designing protein binders where the overall shape is customized to optimally match a target molecule. we developed a computational protocol for the design of repeat proteins with a predefined geometry. by combining sequence optimization of existing repeats and de novo design of capping structures, we designed leucine-rich repeat (lrr) proteins where the building blocks assemble into a novel structure. the suggested design procedure was validated by engineering an artificial donut-like ring structure, which is constructed from ten self-compatible repeats. characterization of several designed constructs further suggests that buried cysteines play a central role for stability and folding cooperativity in certain lrr proteins. this effect could provide a means for selectively stabilizing or destabilizing specific parts of an lrr-based protein binder. the computational procedure may now be employed to develop repeat proteins with various geometrical shapes for applications where greater control of the interface geometry is desired. engineering apobec3g enzymes for altered specificity and processivity louis scott 1 , muhammad razif 1 , aleksandra filipovska 1,2 , oliver rackham 1,2 1 harry perkins institute of medical research, 2 school of chemistry and biochemistry, the university of western australia apobec3g (a3g) is a host-encoded protein involved in the defense against hiv-1 and other retroviral infections. a3g is a cytidine deaminase with a 3' to 5' processive nature, causing targeted c to t mutations along a dna strand. the catalytic and processive activity of a3g leads to the hypermutation of nascent retroviral cdna, resulting in premature termination codons and dysfunctional proteins. ultimately, the action of a3g inhibits viral replication. the ability of a3g to jump and slide along a dna strand, deaminating at targeted sequences, makes it an interesting candidate for protein engineering. engineered a3g enzymes for increased activity, altered specificity, and altered processivity are attractive options for expanding the dna modifying enzyme toolbox. mutation of catalytic residues, residues thought to affect its processive nature and those thought to be involved in target recognition, can create novel a3g enzymes. using structure guided selection, residues in key functional sites that are amiable to mutation will be chosen. individuals from the resulting libraries of mutants will be selected by directed evolution for desired characteristics. the resulting a3g enzymes will be examined for the relationship between their structure and function. such engineered a3g enzymes could be targeted to catalyse the reversion of deleterious genetic mutations. furthermore, engineered a3g enzymes could be used in mutational studies that call for targeted deamination along a dna strand, or mutational studies that call for unspecific and high throughput dna deamination. engineering porous protein crystals as scaffolds for programmed assembly thaddaus huber 1 , luke hartje 1 , christopher snow 1 1 a key motivation for nano-biotechnology efforts is the creation of designer materials in which the assembly acts to organize functional domains in three dimensions. crystalline materials are ideal from the validation perspective because x-ray diffraction can elucidate the atomic structure. relatively little work has focused on engineering protein crystals as scaffolds for nanotechnology, due to the technical challenges of coaxing typical proteins into crystallizing, and the likelihood of disrupting the crystallization process if changes are made to the monomers. we have circumvented these limitations by installing guest protein domains within engineered porous crystals (13 nm pore diameter) that have been rendered robust using covalent crosslinks. the retention of the scaffold structure despite changes to the solution conditions and macromolecule uptake can be validated through x-ray diffraction. we have engineered scaffold crystals for the non-covalent and covalent capture of guest macromolecules. by controlling the reversible loading and release, we can prepare "integrated" crystals with spatially segregated guest loading patterns. as assessed using confocal microscopy, such host-guest crystals are highly stable. ultimately, the resulting crystals may serve as a robust alternative to dna assemblies for the programmed placement of macromolecules within materials. engineering ultrasensitive protein probes of voltage dynamics for imaging neural activity in vivo francois st-pierre 1,2 , michael pan 1,2 , helen yang 3 , xiaozhe ding 1,2 , ying yang 1,2 , thomas clandinin 3 , michael lin 1,2 1 department of bioengineering, stanford university, 2 department of pediatrics, stanford university, 3 nervous systems encode information as spatiotemporal patterns of membrane voltage transients, so accurate measurement of electrical activity has been of long-standing interest. recent engineering efforts have improved our ability to monitor membrane voltage dynamics using genetically encoded voltage indicators. in comparison with electrophysiological approaches, such protein-based indicators can monitor many genetically defined neurons simultaneously; they can also more easily measure voltage changes from subcellular compartments such as axons and dendrites. compared with genetically encoded calcium indicators, voltage sensors enable a more direct, accurate, and rapid readout of membrane potential changes. however, several challenges remain for in vivo voltage imaging with genetically encoded indicators. in particular, current voltage sensors are characterized by insufficient sensitivity, kinetics, and/or brightness to be true optical replacements for electrodes in vivo. as a first step towards addressing these challenges, we sought to develop new voltage indicators that further improve upon the performance of the fast voltage sensor accelerated sensor of action potentials 1 (asap1). in asap1, voltage-induced conformational changes in a natural voltage-sensing domain perturb the fluorescence emission of a covalently linked green fluorescent protein (gfp). using a structurebased approach to guide mutagenesis, we discovered several amino acids that tune the kinetics and voltage sensitivity of asap1. these residues are not only located in the voltage-sensing domain, but also in the fluorescent protein and in the linkers bridging sensing domain and gfp. our most improved variant, asap2, exhibits improved sensitivity to voltage transients such as neuronal action potentials and subthreshold depolarizations. we sought to characterize the ability of these new voltage sensors to monitor neural activity in vivo using laser-scanning two-photon microscopy, a technique that allows imaging with lower autofluorescence and deeper tissue penetration. we report that asap sensors were able report stimulus-evoked voltage responses in axonal termini of the fly visual interneuron l2. asap sensors enabled voltage imaging with dramatically improved temporal resolution compared to three recently reported calcium and voltage sensors. overall, our study reports novel voltage indicators with improved performance and highlights how specific amino acids can tune the performance of a proteinbased fluorescent sensor. we anticipate that these results will pave the way for further engineering of voltage sensing proteins, and that our new sensor asap2 will facilitate current and future efforts to understand how neural circuits represent and transform information. assembly of armadillo repeat proteins from complementary fragments erich michel 1 , randall watson 1 , martin christen 1 , fabian bumback 3 , andreas pl€ uckthun 2 , oliver zerbe 1 demonstrated that complementary fragments of a designed consensus armadillo repeat protein (armrp) recognize each other [1] . the two fragments ym2: ma, in which y, m and a denote the n-cap, internal repeats and the c-cap, respectively, form a 1:1 complex with a nanomolar dissociation constant, which is essentially identical to the crystal structure of the continuous ym3a protein. we further demonstrate that structurally intact armadillo repeat protein complexes can be reconstituted from fragments obtained at various split sites -essentially after every repeat but also within repeats. the fragments display variable affinities towards each other, depending on the split site. the low affinity of some complementary pairs can be dramatically increased upon addition of peptide ligands. while a number of proteins are known that can be reconstituted from fragments we believe that the fact that armadillo repeat proteins can be reconstituted from various complementary fragments is novel and opens new interesting perspectives and applications in biochemistry. a reliable method for generating optically controllable proteins would enable researchers to interrogate protein functions with high spatiotemporal specificity. we recently engineered a tetrameric fluorescent protein, dronpa145n, that undergoes light-induced monomerization, then developed a general architecture for lightinducible proteins based on this light-induced transition. we created proteins whose active sites were blocked by fused dronpa145n domains in the dark, but would become unblocked by light. here we present further two extensions to this concept that together enabled the generalization of this method to additional classes of proteins. first, we engineered a photodissociable dimeric dronpa (pddronpa) with tunable affinity, faster photoswitching speed, and decreased level of protein aggregation, enabling better performance of fusion proteins. second, we introduce the concept of caging a protein active site by insertion of dronpa domains into loops rather than strictly at the protein termini. we use the pddronpa system to impose optical control on kinases and the cas9 endonuclease. the resulting light-inducible mek1 kinase, raf1 kinase, and cas9 endonuclease showed high caging efficiency of protein activities in the dark, and robust protein activation upon light illumination. we believe that our efforts on further improving and generalizing this method would bring the power and benefits of light control to a broad community of biologists. exploring the evolution of folds and its application for the design of functional hybrid proteins saacnicteh toledo patiño 1 , birte h€ ocker 1 1 the structural diversity of proteins may appear endless, nevertheless even large protein complexes can be decomposed into protein domains and smaller sub-domain sized fragments. only recently, we could identify such fragments employing sequence-based comparisons of different folds, as the tim-barrel and the flavodoxin-like fold (farias-rico et al., 2014) . as an extension of this work, we compared all a/b proteins and identified several fragments shared by different folds illustrating how nature may have achieved structural and functional diversity from a reduced set of building blocks. inspired by this combinatorial concept, we searched for homologous fragments bearing active sites to engineer a functional fold-chimera. we extracted the vitamin-b12 binding part from methylmalonyl coa mutase, which belongs to the flavodoxin-like fold (fl) and used it to replace the corresponding fragment in uroporphyrinogen iii synthase, which belongs to the hemd-like fold (hdl). the new hybrid resulted in a stable and well-folded protein whose structure was determined by x-ray crystallography. moreover, cobalamin-binding function was successfully transferred to the new protein from the fl parent, which shows the advantage of using this approach for the design of new functional proteins. in addition, profile alignments revealed sequence and structural evidence that suggested an evolutionary path for hdl from fl by gene duplication. to test this hypothesis, we expressed a modified c-terminal half of uroporphyrinogen iii synthase and solved its structure by nmr spectroscopy, thereby confirming the predicted fl architecture. altogether, our approach facilitates the detection of common ancestry among different folds contributing to our understanding of protein development. furthermore, our results show how new complex proteins can be designed using fragments of existing proteins that serve as building blocks in a lego-like manner. we believe that combining fragments containing existing properties will provide a successful method for the design of novel functionalities in the future. [2] . the active site cysteine plays a key role in the reaction mechanism and we investigated this residue in more detail by exchanging this moiety with selenocysteine (sec) and homocysteine (hcy). the sortase mutants were generated by semisynthesis using expressed protein ligation (epl). the resulting cys-, sec-and hcy-sortase enzymes were characterized and showed a moderate 2-3-fold reduction of activity for sec-sortase. the activity of hcysortase was barely detectable with less than 1% of wildtype activity. the alkylation efficiency of the active site nucleophiles correlated with the expected pka values of sec, cys and hcy. analysis of the ph dependency of the transpeptidation reactions showed that the activity optimum of sec-sortase was shifted towards more acidic conditions. these investigations provide further insights into the reaction mechanism of sortase a and the semisynthetic enzymes may provide new tool for further biochemical studies. propanediol oxidoreductase from escherichia coli (fuco) uses nadh/nad1 as cofactors to catalyze the conversion of s-lactaldehyde to s-1,2-propanediol and vice versa. fuco is an attractive enzyme in the search for possible biocatalysts producing a-hydroxy aldehydes, which are important for the synthesis of natural products and synthetic drugs. enzymes catalyzing these types of reactions are unique in catalytic power and stereoselectivity. the usage of fuco in synthetic industry is limited by the restricted substrate scope, which makes fuco inactive with larger phenyl-substituted alcohols. we used reengineering and directed evolution to enable fuco to catalyze the regio-and enantioselective oxidation of arylsubstituted vicinal diols, such as phenylpropanediols, into a-hydroxy aldehyde products. we mutated amino acids considered to restrict the entry into the active site, and modeled the mutants that were most active with the substrates phenylacetaldehyde and s-3-phenyl-1,2-propanediol and performed docking studies with them. as expected, our experimental and in silico results show that the mutations enlarge the active site cavity and enable the mutant enzymes to accommodate the new substrates. we also found specific amino acids in the active site, which need to be conserved to allow the substrates to make stabilizing interactions. interestingly, an asparagine residue makes the mutant enzymes able to discriminate between phenylacetaldehyde and s-3-phenyl-1,2-propanediol. in conclusion, we successfully re-engineered the specialist enzyme fuco to accept also bulkier molecules as substrates, thereby making it more useful for industrial purposes. one way to gain insight into the sequence-structure-function relationship in proteins is to de novo design artificial proteins. despite impressive successes in de novo protein design, designing a folded protein of more than 100 amino acids still remains a challenge. using this approach, an idealized (beta/ alpha)8 fold protein was designed leading to the production of a protein of 216 amino acids (octarellin v). this protein showed a low solubility and stability. through directed evolution we produced a soluble variant, octarellin v.1. the biophysical characterization of octarellin v.1 shows a well folded monomeric and thermostable protein with a tm over 908c. however, after several screenings, we could not find crystallization conditions for this protein. as an alternative, we decided to co-crystallize octarellin v.1 with a protein partner that helps the crystallization process. we used 2 protein partners: alpha-reps and nanobodies. the first one is characterized to interact through a large surface contact, whereas the second is characterized to recognize an specific small epitope. crystallization of both complexes was performed successfully by vapor diffusion and the structures were solved. the experimental structures correspond to the first for an artificial protein of this size and it will allow to criticize the computational design of the octarellin v. generation of synthetic antibodies against membrane proteins in nanodiscs for use in structural biology methods. here, we describe a robust strategy for generating a class of high performance antibodybased affinity reagents that have proven useful in determining the structures of relevant functional states of membrane proteins. these reagents are fab fragments that are generated by phage display from fully synthetic libraries and are called synthetic antibody fragments, or sabs. we have developed phage display sorting strategies that can trap a desired conformational state, making it accessible to structural analysis, or target a particular epitope on the protein surface. however, to maximize this technology for membrane proteins, several limitations of phage display sorting in detergent formats had to be overcome, the greatest being that using detergents can produce non-native conformational biases. we sought to address these limitations by embedding membrane proteins into nanodiscs, soluble lipidfilled discoidal particles, to better mimic the native membrane environment. nanodiscs stabilize the membrane protein and allow it to respond to conformation-inducing stimuli such as ligands, ions and ph during phage display selections. we have established and validated an improved protocol using two membrane protein systems: 1) mj0480, an archaeal membrane protein of unknown function, and 2) cora, a pentameric magnesium ion channel. using mj0480, we compared the nanodisc protocol with the standard method performed in detergent, and as an important byproduct, we characterized the influence of the membrane protein environment on the apparent affinity of sabs to their cognate antigen. using cora, we developed a more sophisticated sorting strategy resulting in a variety of sabs specific to either the open or closed conformation of the channel. finally, using sabs as crystallization chaperones we obtained the structure of mj0480 at 3.5å resolution, and crystallized cora in several new conditions. lipocalin-type prostaglandin d synthase (l-pgds) is a member of the lipocalin superfamily, and binds a large variety of small hydrophobic molecules. using this function of l-pgds, we have already reported the feasibility of l-pgds as a novel drug delivery vehicle for the poorly water-soluble drugs [1] . sn-38, 7-ethyl-10-hydroxy-camptothecin, is a semi-synthetic analogue of anti-cancer alkaloid camptothecin that targets dna topoisomerase i. despite of the potent anti-tumor activity, however, sn-38 was not used directly in a clinical practice due to its poor water solubility. thus, irinotecan hydrochloride (cpt-11), which is the water-soluble prodrug of sn-38, is used for the cancer treatment. however, cpt-11 shows approximately 0.1% cytotoxic activity of sn-38 against the various cancer cell lines in vitro, and its metabolic conversion rate is 10% of the original volume of cpt-11. here, we show the development of the drug delivery system utilizing l-pgds, which enables a direct clinical usage of sn-38. first, we investigated the effect of l-pgds on the solubility of sn-38. in the presence of 2 mm l-pgds, the concentration of sn-38 was 1.7 mm, which was 1,130-fold as compared with that in pbs. then, we carried out isothermal titration calorimetry measurements to investigate the detailed binding mode of sn-38 to l-pgds. as a result, it was revealed that l-pgds binds three molecules of sn-38, and the dissociacontrol over the sensitivity with which artificial biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. such control could likewise be highly useful in artificial biotechnologies in which highly responsive behavior is of value, such as biosensors, genetic logic gates, and "smart" materials and delivery devices. in nature, the control of molecular responsiveness is often achieved using "hill-type" cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. here we use an intrinsic-disorder-based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. to do so we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. the loop spatially separates the two sets of the two halves of the binding sites, preventing a complete binding site that enables target molecule binding without prior closure of the loop. thus, the first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a pre-formed site. using this approach we have rationally introduced cooperativity into three unrelated aptamers, achieving in the best of these a hill coefficient experimentally indistinguishable from the theoretically expected maximum. the extent of cooperativity, and thus the steepness of the binding transition, are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy. essential and non-essential amino acid species for an ancestral protein satoshi akanuma 1 1 the translation system is an essential element for life because it links genetic information embedded in genes to functional molecules, proteins. the modern genetic code, which encodes the standard 20 amino acids (and three terminations) using 64 triplet codons, is shared by most of the extant organisms on the earth. a number of theories have been proposed for the origin and evolution of the genetic code, and these theories suggest that only a fewer amino acids were used in primitive proteins and later the amino acid repertoire gradually increased up to 20 through the course of evolution. if so, one would wonder how many number of and which types of amino acids were involved in the primitive proteins. i have begun to address this issue experimentally. i first resurrected several ancestral proteins and then restricted the amino acid usage of one of the resurrected proteins. i targeted nucleoside diphosphate kinase (ndk) that catalyzes the transfer of a phosphate from a nucleoside triphosphate to a nucleoside diphosphate. ndk may have arisen early because at least one gene that encodes ndk is present in most extant organisms. the first step in the reconstruction of ancestral ndk sequences is to prepare multiple amino acid sequence alignments using homologous sequences of ndk from extant species. then, phylogenetic trees were built. ancestral sequences of ndk that represent the last common ancestors of archaea and of bacteria were reconstructed using the information contained in the predictive phylogenetic trees. the reconstructed ancestral kinases are extremely thermally stable [akanuma et al., 2013] . then, using the most thermally stable ancestral ndk, arc1, as the starting molecule, i restricted its amino acid usage. arc1 does not contain any cysteine residue and therefore consists of 19 amino acid species. i completely replaced one of the 19 amino acid species by other amino acid species and thus created 19 proteins each of which consisted of 18 amino acid species. then, i evaluated the stabilities and activities of the resulting 19 arc1 variants to assess the individual contributions of the 19 amino acid species. as the result, i found that the 19 amino acid species do not equally contribute to the stability and activity of arc1 and that some amino acid species can be easily lacked but others are important or essential for its stability and function. the result clearly shows that the full amino acid species are not necessarily essential and supports the hypothesis that proteins in the early stage of evolution were made from a reduced amino acid set. the protein surface recognition for protein-protein interactions (ppi) is involved in signal transduction, immune reaction, and creation of the nanostructures in living cells. the methods for rational designing of ppi that could provide non-antibody scaffolds and nanostructured materials are required for the therapeutic and nanotechnological applications. although there have been some successful rational designs with computational methods, it is still difficult to design freely the ppi onto arbitrary proteins. the reason for this limitation is decreased solubility in the designed protein due to the additional hydrophobic residues in order to drive ppi. another reason is a limited set of design modes by which proteins can interact, because the target proteins have individual surface structures. therefore, many methods of constructing an interface for numerous target scaffold proteins without loss of their solubility are necessary. surface exposed a-helices are often observed in natural globular proteins. moreover, there are many examples for naturally occurring oligomeric proteins where an a-helix from each subunit interacts to form an intermolecule coiled coil. further, the works related to designing of artificial helical bundle reported by the several other groups have provided information about how to generate and tune the interaction between a-helices. therefore, a surface exposed a-helix would be a good target for designing a de novo interface onto the scaffold protein. here we engineered two different proteins, sulerythrin and cys-larfh, to form the cys-larfh-sulerythrin dimer-cys-larfh heterotetramer via an intermolecular helix-helix interaction. wild-type sulerythrin forms a dimeric eight-helix bundle. cys-larfh is a designed monomeric protein that forms four-helix bundle containing interhelical s-s bonds. both sulerythrin and cys-larfh are extremely thermostable. to design protein-protein interfaces onto the individual proteins, we first introduced six leucines to the two a-helices of sulerythrin and three leucines to a a-helix of cys-larfh. as expected, the introduction of the hydrophobic amino acids reduced their solubilities. to recover the solubility, we then introduced six aspartates or glutamates around the hydrophobic surface of the sulerythrin (hereafter referred to as 6l6d or 6l6e). similarly, three arginines were introduced around the artificial hydrophobic surface of the cys-larfh (hereafter referred as iv-3l3r). the solubilities of the mutants with the hydrophobic interface and additional charged residues were recovered their solubility. in addition, the sulerythrin mutants 6l6d and 6l6e exist mainly as dimer. the cys-larfh mutants iv-3l3r, also exists as monomer. we then examined the interaction between 6l6e or 6l6d and iv-3l3r. a pull-down experiment, in which co21 beads bound to either his-tagged cys-larfh and iv-3l3r were used to pull down wild-type sulerythrin, 6l6d, or 6l6e, demonstrates that 6l6d or 6l6e specifically interacts to iv-3l3r. furthermore, when analysed by size exclusion chromatography, the dominant peaks of the mixture of 6l6d and iv-3l3r appeared at the volume expected for the heterotetrameric complex. thus we successfully created the de novo ppi by using a very simple concept involving hydrophobic interaction in combination with charge interactions. in vitro selection of liposome anchoring peptide by cdna display naoto nemoto 1 , ryoya okawa 1 , yuki yoshikawa 1 , toshiki miyajima 1 , shota kobayashi 1 1 a liposome-anchoring peptide (la peptide) was selected against liposomes composed of dioleoyl-snglycero-3-phosphocholine (dopc) by in vitro selection using cdna display method. the selected peptide la peptide consists of the n-terminal region (hydrophobic) and the c-terminal region (basic) in a characteristic manner. thus, la peptide was synthesized chemically and the interactions between la peptide and particular types of liposomes were investigated and confirmed by confocal laser scanning microscopy. designing of a novel platinum-binding amino acid sequence on a protein surface asumi kaji 1 , hiroya niiro 1 , satoshi akanuma 2 , tetsuya uchida 1 , akihiko yamagishi 1 1 designing of a novel interaction between a metal and a protein is a key to create hybrid materials between organic and inorganic materials. for example, in a glucose biosensor, which is widely used for measuring glucose concentration in blood, glucose oxidoreductase molecules are immobilized on a platinum electrode by polyacrylamide gel. a metal-binding tags that is added to the n-or cterminus of a protein is also used for fix the protein to a metal. however, a technique to create a metal binding site on a desired position of a protein has not been invent. if such a technique would be established, the technique would contribute to developing and improving biosensors and to producing new bionanoelectronic materials. in this study, we created a platinum-binding site on a loop located at a protein surface. we used an artificial protein, larfh, that had been synthesized by connecting four identical alpha helices originated from the c-terminal segment of the escherichia coli lac repressor with three identical loops. we randomized the ser, gly, gln, gly, gly, ser sequence within one of the inter-helical loops and then selected for binding to platinum by a t7 phage display system. most of the selected larfh variants contained the tyr, lys, arg, gly, tyr, lys (ykrgyk) sequence in the randomized segment. we then evaluated the affinity of the larfh variant to platinum by means of quartz crystal microbalance analysis. we found that the variant binds to platinum more strongly than does the original larfh. in the annual symposium, we will also report about the affinity of the isolated ykrgyk sequence to platinum and about the crucial role of the first tyrosine in binding to platinum. engineering of an isolated p110a subunit of pi3ka permits crystallization and provides a platform for structure-based drug design pi3ka remains an attractive target for development of anticancer targeted therapy. a number of p110a crystal structures in complex with the nsh2-ish2 fragment of p85 regulatory subunit have been reported, including a few small molecule co-crystal structures, but the utilization of this crystal form is limited by low diffraction resolution and a crystal packing artifact that partially blocks the atp binding site. taking advantage of recent data on the functional characterization of the lipid binding properties of p110a, we designed a set of novel constructs allowing production of isolated stable p110a subunit missing the adapter binding domain (abd) and lacking or featuring a modified c-terminal lipid binding motif. while this protein is not catalytically competent to phosphorylate its substrate pip2, it retains ligand binding properties as indicated by direct binding studies with a pan-pi3ka inhibitor. additionally, we determined apo and pf-04691502 bound crystal structures of the p110a (105-1048) subunit at 2.65 å and 2.85 å respectively. comparison of isolated p110a (105-1048) with the p110a/p85 complex reveals a high degree of structural similarity, which validates suitability of this catalytically inactive p110a for iterative sbdd. importantly, this crystal form of p110a readily accommodates the binding of non-covalent inhibitor by means of a fully accessible atp site. the strategy presented here can be also applied to structural studies of other members of pi3kia family. identification of structural determinants involved in the differential conformational changes of ef-hand modules emma liliana arevalo salina 1 , joel osuna quintero 1 , humberto flores soto 1 , gloria saab rinc on 1 1 instituto de biotecnolog ıa, universidad nacional aut onoma de m exico identification of structural determinants involved in the differential conformational changes of ef-hand modules calcium signals are regulated by several proteins, most of which belong to the ef-hand superfamily. the ef-hand motif is formed by a helix-loop-helix that binds calcium through its loop1. these motifs occur in adjacent pairs, forming a single globular domain which is the basic structural and functional ca21 binding unit. the proteins in this family can be classified as calcium sensors or modulators, according with their function. the first group undergoes a major conformational change upon calcium binding, while the second one remains practically unchanged1,2. to explain the biophysics behind the different behavior of these proteins upon ca21 binding, we have sought to identify structural determinants that could account for these features, especially for the difference in the conformational change. we examined the primary structure from two ef-hand motifs: a sensor ef-hand from chicken troponin c (sciii) and a modulator ef-hand from bovine calbindin d9k (clbn). the main differences were in the binding ca21 loop and a group of charged residues in the h2 helix of the modulator ef-hand. then, we constructed chimeric clbn motifs containing the loop or the loop and h2 from sciii motif (h1clbnsciii and h1h2clbnsciii). these constructs were analyzed using a reporter system that discriminates ef-hand-sensor motifs from signal-modulators at the single-motif level. this reporter is based on the fusion of genes codifying for the ef-hand and the prephenate dehydrogenase from e. coli (tyra), a protein which is active only as a dimer. isolated ef-hand motifs have the ability to homo-dimerize and in the fusion can stabilize and activate tyra. the sensor motif exhibits a conformational change by binding calcium and in doing so, destabilizes the dimeric conformation of tyra and virtually eliminates its activity. in the modulators, on the other hand, the rather small conformational change only gives rise to a decreased tyra activity. both constructed chimeric ef-hand fusions showed a loss of activity upon ca21 binding, indicating that the 12 residues connector of the sensor ef-hand from sciii is sufficient to confer the conformational change. in addition we used cd and extrinsic fluorescence spectroscopies to analyze any conformational change in the h1h2clbnsciii and h1clbnsciii isolated modules, not finding any difference between the ca21 free and ca21 bound chimeras, suggesting that the change in activity of the reporter protein is due to a change in the orientation of the helices in the ef-hands induced by calcium. the effect of ca21 binding of the chimeras in the context of the entire calbindin d9k protein is under investigation. mapping side chain interactions at the n-and c-termini of protein helices nicholas e newell, independent researcher interactions involving one or more amino acid side chains near the ends of protein helices stabilize helix termini and shape the geometry of the adjacent loops, contributing to supersecondary structure. side chain structures that have been identified at the helical n-terminus include the asx/st n-caps, the capping box, and hydrophobic and electrostatic interactions. at the cterminus, capping is often achieved with main-chain polar groups, (e.g. the schellman loop), but here also particular side chain motifs clearly favor specific loop geometries. key questions that remain concerning side chain interactions at helix termini include: 1) to what extent are helix-terminal motifs that include multiple amino acids likely to represent genuine cooperative interactions between side chains, rather than chance alignments? 2) which particular helix-terminal loop geometries are favored by each side chain interaction? 3) can an exhaustive statistical scan of a large, recent dataset identify new side chain interactions at helix termini? in this work, three analytical tools are applied to answer the above questions for both n-and c-termini. first, a new perturbative least-squares 3d clustering algorithm is applied to partition the helix terminal structures in a large (25,000 example), low-redundancy pdb dataset by loop backbone geometry. the clustering algorithm also generates a set of structural exemplars, one for each cluster, that is used to represent the most important loop geometries at each terminus. next, cascade detection (newell, bioinformatics, 2011), an algorithm that detects multi-amino acid cooperativities by identifying overrepresented sequence motifs, is applied to each cluster separately to determine which motifs are most important in each loop geometry. finally, the results for each motif are displayed in a capmap, a 3d conformational heatmap that depicts the distribution of motif abundance and overrepresentation across all loop geometries by projecting these quantities onto the structural exemplars generated by clustering. the capmap reveals the loop conformations most favored by a motif. actual structures from the clusters corresponding to these favored conformations are then examined in a structure browser to characterize the side chain interaction associated with the motif. this work identifies a 'toolkit' of side chain motifs which are good candidates for use in the design of synthetic helix-terminal loops with specific desired geometries, because they are used in nature to support these geometries. highlights of the analysis include determinations of the favored loop geometries for the asx/st motifs, capping boxes, big boxes, and other previously known and unknown hydrophobic, electrostatic, h-bond, and pi-stacking interactions. a goal of future work is to make these results available in a structurally-addressable database that would enable researchers to immediately retrieve the side chain interactions most compatible with a desired loop geometry. generation of fluorescent protein-tagged gp120 mutants to analyze the intracellular distribution of hiv-1 envelope protein shuhei nakane 1 , zene matsuda 3 1 green earth research center, green earth institute co., ltd., 2 res ctr for asian infect dis, inst of med sci, the univ of tokyo, 3 lab of struct virol and immunol, institute of biophysics, cas hiv-1 is a causative enveloped virus of aids. its envelope protein (env) has two non-covalently associated subunits, gp120 and gp41, which are proteolytically processed from a gp160 precursor. the gp120 subunit is a surface protein and gp41 is a transmembrane protein. the gp120 and gp41 subunits are responsible for the receptor recognition and membrane fusion, respectively. the cytoplasmic tail (ct) of gp41 is about 150 amino acids long and is believed to play a critical role in intracellular trafficking of env. to visualize dynamic trafficking, the c-terminus of gp41 has been tagged with fluorescent proteins such as gfp. however, tagging of ct may cause a concern to affect the interactions between the ct and cellular proteins that are involved in intracellular trafficking. to avoid this problem, here we tried to insert gfpopt, a gfp variant, into five variable regions of gp120. we have analyzed the phenotypes of env mutants, such as the cell surface expression, processing of gp160, membrane fusion activity, and virion incorporation. among 5 variable regions of gp120, the v3 region was most sensitive to insertion. v1/v2 region was less sensitive than v3. consistent with the recently revealed structure, exteriorly located v4 and v5 were highly tolerant to insertion. we used the mutant with the gfp insertion in the v5 region to analyze the intracellular distribution of env with and without ct. we found that deletion of ct increased the presence of vesicles colocalized with late endosome markers. this is consistent with the hypothesis that the ct region contains a motif regulating intracellular trafficking. our results showed that env with gfpopt insertion in its gp120 subunit is a useful tool for the study of intracellular dynamics of hiv-1 env. these mutants would also be useful to trace the fate of virus particles during infection. pi-055 ngs-guided phage panning: comparison to conventional panning strategy buyung santoso 1 , dorain thompson 1 , john nuss 1 , john dwyer 1 1 phage display is a powerful tool for generating binders to a target protein. multiple rounds of panning with conventional phage display strategies typically result in a number of hits, which are then individually screened using in vitro assays. clones screened at this stage are a combination of specific binders, sequences that are selected due to amplification bias, and non-specific binders. if the number of specific clones is low relative to the non-specific sequences, a larger number of clones have to be screened to ensure sufficient diversity of early leads. with the advent of next generation sequencing (ngs) technology, we aim to test whether we can increase the diversity of specific hits and decrease the number of non-specific sequences. in our experiment, four rounds of conventional panning produced ten peptide binders to target protein. ngs analysis after two rounds of panning was done in parallel, yielding more than ten thousand sequences, ranked by abundance. all ten binders from conventional panning were found in the top 150 most abundant ngs hits. more importantly, additional hits were found in ngs analysis but not in conventional panning, highlighting this strategy as a promising alternative for hit discovery with the significant upside of more diverse and higher affinity leads. numerous processes in pharmaceutical development, including construct screening, structural genomics, protein engineering and expression optimization among others, require the use of higher throughput plasmid dna purification. the majority of issues encountered in mini, midi, and maxiprep purification kits involve flocculate removal following alkaline lysis, and there is currently no easy way to produce large amounts of plasmid dna without the addition of complicated and time consuming clarification steps. the existence of a hassle-free automated system that is not restricted by sample size would significantly help in cutting time and costs during the initial processing steps of plasmid purification. the autoplasmid mea instrument provides a fully automated solution to traditional problems faced in plasmid purifications, allowing mini, midi, and maxiprep plasmid purifications to be performed on a single instrument. the data presented here on plasmid yield, purity, and suitability for sequencing and transfection/transformation illustrate a new strategy for automated plasmid preps. by eliminating traditional clarification methods, cell culture volumes between 1 -120 ml can be processed leading to yields ranging from 3 -1000 lg. this flexible system was developed in order to satisfy a wide variety of concentration and yield requirement, while eliminating the time consuming steps previously needed to obtain similar results. the ability to perform fully automated mini, midi, and maxi plasmid preps on one instrument allows for a customized all-in-one purification system that is not restricted by traditional clarification methods, eliminating manual intervention, and streamlining the purification process. the modular nature of protein architectures suggests that proteins have evolved through duplication and fusion to give rise to modular, often symmetric forms, which later diversified under the influence of evolutionary pressure. we have developed a computational protein design method termed reverse engineer evolution (re3volution) to create symmetrically self-assembling protein building blocks. we have used this method to design a perfectly symmetric b-propeller protein called pizza. subsequently, we have engineered a metal binding site into this pizza protein. this new pizza variant carries two nearly identical domains per polypeptide chain, and forms a trimer with three-fold symmetry. the designed single metal ion binding site lies on the symmetry axis, bonding the trimer together. two copies of the trimer associate in the presence of cadmium chloride in solution, and high resolution x-ray crystallographic analysis reveals a nano-crystal of cadmium chloride, sandwiched between two trimers of the protein. this nano-crystal, containing seven cadmium ions lying in a plane and twelve interspersed chloride ions, is the smallest reported to date. our results indicate the feasibility of using rationally-designed symmetrical proteins to biomineralize nano-crystals with applications in bionanotechnology. bacillus licheniformis trehalose-6-phosphate hydrolase structures suggest keys to substrate specificity chwan-deng hsiao 1 , min-guan lin 1 , long-liu lin 2 , yuh-ju sun 3 1 institute of molecular biology, academia sinica, 2 department of applied chemistry, national chiayi university, 3 depaertment of life science, national tsing hua university trehalose-6-phosphate hydrolase (trea) of the glycoside hydrolase family 13 (gh13) catalyzes the hydrolysis of trehalose-6-phosphate (t6p) to yield glucose and glucose-6-phosphate. products of this reaction can be further metabolized by the energy-generating glycolytic pathway. here we present the crystal structures of bacillus licheniformis trea (bltrea) and its r201q mutant complexed with p-nitrophenyl-a-d-glucopyranoside (r201q/ ppng) at 2.0 å and 2.05 å resolution, respectively. the overall structure of bltrea is similar to other gh13 family enzymes. however, detailed structural comparisons revealed that the catalytic groove of bltrea contains a long loop adopting a different conformation from those of gh13 family members. unlike the homologous regions of bacillus cereus oligo-1,6-glucosidase (bcogl) and erwinia rhapontici isomaltulose synthase (nx-5), the active site surface potential of bltrea exhibits a largely positive charge, contributed by the four basic residues his281, his282, lys284 and lys292. mutations at these residues resulted in significant decreases of bltrea enzymatic activity. strikingly, a 281hhlk284 motif and the lys292 residue played critical roles in bltrea substrate discrimination. crystal structure of engineered lrrtm2 synaptic adhesion molecule and a model for neurexin binding anja paatero 1 , katja rosti 1 , alexander shkumatov 2 , cecilia brunello 3 , kai kysenius 3 , prosanta singha 1 , henri huttunen 3 , tommi kajander 1 1 institute of biotechnology, university of helsinki, helsinki, finland, 2 dept of pharmaceutical and pharmacological sciences, ku leuven, leuven, belgium, 3 neuroscience center, university of helsinki synaptic adhesion molecules are key components in the development of the brain, and in the formation of neuronal circuits, as they are central in the assembly and maturation of the chemical synapses. several families of neuronal adhesion molecules have been identified such as ncams, neurexins and neuroligins, and in particular recently several leucine rich repeat protein families, e.g. netrin g-ligands, slitrks and lrrtms. the lrrtms form a family of four proteins. they have been implicated in excitatory glutamatergic synapse function, and were specifically characterized as ligands for neurexins in excitatory synapse formation and maintenance. in addition, lrrtm3 and lrrtm4 have been found to be ligands for heparan sulphate proteoglycans. we report here the crystal structure of a stability-engineered mouse lrrtm2, with a tm 308c higher than the wild type protein, while retaining its function. we localized the neurexin binding site to the concave surface based on protein engineering, sequence conservation and prior information on the ligand interaction with neurexins, allowing us to propose a tentative model for lrrtm:neurexin interaction compex. cell culture studies and binding experiments show that the engineered protein is functional and capable of forming synapse-like contacts. small angle x-ray scattering data suggests that the wild type protein forms transient dimers, which may have importance for the function. the structural and functional data presented here provide the first structure of an lrrtm protein, and a model for molecular mechanism of lrrtm function in adhesion. computational design of phenylalanine binder olga khersonsky 1 , gil benezer 1 , sarel fleishman 1 1 recently, abdesign algorithm was developed in our lab for de novo design of antibodies (1). it is guided by natural conformations and sequences, and exploits the modular nature of antibodies to abstract generate an immense space of conformations, which can be used as scaffolds for design of stable highaffinity binders. we have used abdesign to design a binder of phenylalanine. 30,000 antibody scaffolds were obtained by splicing h3 and l3 fragments into a template (pdb id 2brr), and subsequent optimization of vh and vl orientation. phenylalanine binding site, based on native phenylalanine binders, was introduced into the scaffolds with rosettamatch (2), and the sequences were subsequently optimized by rosetta enzyme design protocol (3) . 30 designs were experimentally tested by yeast display for binding of biotinylated phenylalanine ligand. several designs were found to bind the ligand, and we plan to further characterize this affinity and improve it using directed evolution techniques. in collaboration with the group of prof. johnsonn, the resulting phenylalanine binder will be incorporated in a bio-luminescent (lucid) sensor for phenylalanine (4) . phenylalanine monitoring device would be of primary importance for patients with phenylketonuria, a genetic disease with phenylalanine metabolism problem. cold-adapted enzymes are interesting because of their higher catalytic activity compared to mesophilic and thermophilic homologues. alkaline phosphatase (ap) from a psychrophilic vibrio marine bacteria (vap) has an unusual large surface loop that extends from each of its monomers to stabilize a homodimeric structure (1). in many cold-adapted enzymes, the loop regions are longer compared to proteins of mesophilic organisms and our aim was to study the functional and structural role of this loop. three substitutions (r336l, y346f and f355y) were introduced within the large surface loop as directed by 1microsecond molecular dynamics (md) simulations. with the r336l mutation, two hydrogen bonds were broken that connect the loop to residues on the adjacent subunit, and further two hydrogen bonds broken with the adjacent q334. as a consequence, r336l displayed a 25% higher kcat compared with wild-type and a slight decrease in the km value. overall, the catalytic efficient improved by 45%. the global heat stability (tm) and the active site sensitivity to heat (t50%) were reduced by 68c and 138c, respectively. md simulations showed that hydrogen bonds to arg336 are important for longrange communication to the active site. certain rotamers of two important residues in the catalytic site, ser65 and arg129, were favored, presumably toward states more competent for catalysis upon the replacement of arg336 with leu. in the y346f variant, removal of one hydrogen bond between the loop and the other subunit caused a small drop in stability parameters, whereas both kcat and km were reduced by about half, giving similar kinetic efficiency (kcat/km) to that of wild-type. finally, we changed a residue at the root of the large loop (f355y) such that one new intersubunit hydrogen bond could form. this variant maintained the wild-type characteristics. in conclusion, removing hydrogen bonds connecting the major loop of one subunit to the protein surface of the other subunit in vap produced higher catalytic activity and this shows functional connections between loop mobility and the active site. our study also demonstrates that interactions between residues in the large disordered loop and the opposite subunit in the dimeric vap are determinants of its stability. thus, we managed to show that loosening of interface contacts between the two vap subunits by replacement of crucial residues provides a way to orchestrate structural and kinetic dynamics in a productive way. the de novo design of artificial proteins arises as a stringent test of our understanding of the relationship between sequence, structure, and function. examples include the design of a four a-helix bundle, a new protein topology called top7, and a series of artificial (ba)8-barrels called octarellins. however, de novo design has proven difficult for larger proteins with more than 100 amino acids. here we present two methods to generate the backbone and to perform the de novo design of (ba)8-barrel proteins through the use of the software rosetta; both have different advantages and limitations. the first method for generating the backbone is knowledge-based, with a first analysis of a non-redundant database of natural (ba)8-barrel proteins in order to obtain statistical analysis on preferred secondary structure element length and amino acidic propensities. with this information we use the rosetta cm software to create more than 1000 models which are then ranked in term of rosetta energy. the second method is performed with the parametricdesign package of rosetta, in which only geometrical information are requested (number of strands and helices, radius of the b-and a-barrels, degree of inclination, orientation of the side chains, among others). both methods contain a step of loop refinement and multiple steps of sequence design with the package rosetta design, in order to find low scoring amino acid sequences for each of the starting backbone conformations. thousands of models will be generated by both methods and then analyzed in term of sequence similarity, secondary and tertiary structure prediction, and stability by molecular dynamics simulations. the 30 best candidate sequences will be selected for the experimental verification. in order to identify a putative successfully design, we added a metal binding site during the design step. all the proteins will be expressed in e. coli. the solubility of the designed proteins inside bacteria will be determined thanks to the fusion to green fluorescence protein (gfp). solubility, stability, secondary structure, and cooperativity of folding will be assessed for each protein before determination of their three-dimensional structure. construction of protein capsule possessing drugs controlled release ability shota shimizu 1 , masatoshi nakatsuji 1 , keisuke yamaguchi 1 , yuya sano 1 , yuya miyamoto 1 , takashi inui 1 1 most compounds that exhibit anti-tumor activities are water-insoluble, thus limiting their clinical use. chemical modification of these compounds and the use of solubilizing agents such as organic solvents, surfactants and ph modifiers improve their solubility. however, chemical modification of compounds decreases their potency, and the use of solubilizing agents causes toxicity in many cases. thus, drug delivery systems (dds) for poorly water-soluble anti-tumor drugs which exploit liposomes, cyclodextrins, and lipid nanoparticles have been studied intensely. in these dds, the controlled release of drugs from the delivery vehicle is one of the most important functions. selective release in target cells leads to adequate therapeutic efficacy with few side effects. in our laboratory, we have already demonstrated that lipocalin-type prostaglandin d synthase (l-pgds), an intravital transporter protein, is a novel and valid drug delivery vehicle for sn-38, a poorly water-soluble anti-tumor drug. in this study, we generated l-pgds-based protein capsules with a controlled-release function by introducing a disulfide bond into the upper part of the drug-binding cavity of l-pgds. the intracellular concentration of glutathione (0.510 mm) is known to be substantially higher than the extracellular concentration (2 mm). therefore, it is expected that in the extracellular oxidative environment the disulfide bonds in the protein capsule remain stable, avoiding premature release of the internal drugs during circulation of blood, after reaching the target cells, the disulfide bonds are cleaved in the intracellular redox-environment, and then the internal drugs are released. we generated three kinds of protein capsules which have disulfide bonds in different positions, w54c/w112c, k58c/h111c, k58c/w112c, based on tertiary structure information of human l-pgds (pdb id: 3o2y). firstly, we performed circular dichroism (cd) measurements to confirm the structure of each capsule. the cd spectra of three protein capsules were similar to that of wild-type l-pgds in the far-uv region. therefore, the secondary structures of three protein capsules were not changed from wild-type l-pgds by introducing the mutations. quantitative analysis of the free thiol group in the protein capsule by dtnb assay revealed that the intermolecular disulfide bond was formed by h2o2-induced oxidation and cleaved by dithiothreitol-induced reduction. in addition, to investigate the solubility of sn-38 in the presence of protein capsules, we mixed the protein capsule of reduced-form with sn-38 suspension, and stirred at 37 c for 48 hours. the resulting concentrations of sn-38 in pbs with 1 mm w54c/w112c, k58c/h111c, and k58c/w112c were 374 mm, 194 mm, and 349 mm, respectively. these values were approximately 200-fold higher than without protein capsules. sds-page analysis showed that the bond formation decreased in a time-dependent manner, and that new intermolecular disulfide bond was not formed in the protein capsules after 48 hours' incubation. from the above, we succeeded in generating drug delivery vehicles possessing openable and closable lids that are responsive in an oxidation-reduction environment. takaaki miyamoto 1 , mai kuribayashi 1 , satoshi nagao 1 , yasuhito shomura 2 , yoshiki higuchi 3,4 , shun hirota 1 1 graduate school of materials science, nara institutte of science and technology, 2 graduate school of science and engineering, ibaraki university, 3 department of life science, graduate school of life science, university of hyogo, 4 domain swapping has been of interest as a mechanism of protein oligomerization, where a secondary structural region or a domain of one protein molecule is replaced with the corresponding region or domain of another protein molecule. we have previously shown that c-type cytochromes and myoglobin form oligomers by domain swapping.1,2 in this study, we show that a four-helix bundle protein cyt cb562, in which the heme of cyt b562 is attached to the protein moiety by insertion of two cys residues, forms a domain-swapped dimer. dimeric cyt cb562 was more stable than dimeric cyt b562 at 48c, showing that attachment of the heme to the protein moiety stabilizes the domain-swapped structure. absorption and cd spectra of dimeric cyt cb562 were similar to the corresponding spectra of the monomer, showing that the active site and secondary structures were similar between the dimer and monomer. the redox potential of dimeric cyt cb562 was also similar to that of its monomer. the dissociation temperature of dimeric cyt cb562 was 508c, and its dh on dissociation to monomers was 213.3 kcal/mol (per dimer). according to x-ray crystallographic analysis, dimeric cyt cb562 exhibited a domain-swapped structure, where the two helices in the n-terminal region (helices 1 and 2) in a protomer and the other two helices in the c-terminal region (helices 3 and 4) of the other protomer interacted between each other. the heme coordination structure of the dimer was similar to that of the monomer. we have previously shown that domain-swapped oligomers of horse cyt c form through intermolecular hydrophobic interaction between the n-and c-terminal a-helices at the early stage of folding.3 it has been suggested that helices 2 and 3 form first at the initial stage of folding in wild-type apo cyt b562.4 therefore, we propose that cyt cb562 forms a domain-swapped dimer when helices 2 and 3 interact intermolecularly at the initial stage of folding, whereas the intramolecular interaction of helices 2 and 3 results in formation of a monomer. a highly buried and conserved tryptophan residue close to the dimer interface in a cold-adapted phosphatase is phosphorescent and important for activity. jens g. hj€ orleifsson and bjarni asgeirsson. department of biochemistry, science institute, university of iceland, dunhagi 3, 107 reykjavik, iceland. alkaline phosphatase (ap) from vibrio g15-21 is a cold-adapted dimeric enzyme with one of the highest catalytic efficiency reported for known aps. it contains five intrinsic tryptophan (trp) residues and one additional trp located on the c-terminal streptag used for expression and purification. in this study, we made several single trp-substitutions to determine the role of each of the trp in the fluorescence emission spectrum. we also determined their solvent exposure by acrylamide fluorescence quenching. the results indicate that trp301, trp460 and trp475 are mostly responsible for the fluorescence emission. quenching experiments with acrylamide indicated that all the trp residues were about equally accessible for quenching, except trp460 which was shown to be highly buried in the core of the protein. interestingly, the enzyme was found to be highly phosphorescent at 108c, having two phosphorescence lifetimes. the longer lifetime is due to trp460. trp460 is located close to the dimer interface and points towards a helix in the active site where his277 binds an active-site zinc ion. in other aps, an aromatic amino acid is conserved in the location occupied by the trp460 residue. in most cases for cold-adapted aps it is indeed a trp. interestingly, the mutation of the trp460 to a phenylalanine affected both stability and activity of the enzyme. kcat/km was 10-fold lower than for wild-type. overall, this study reveals that trp460 can be used as a phosphorescent probe of local dynamics and could possibly also serve to study the dimer-monomer equilibrium due to proximity to the dimer interface, an area clearly crucial for enzyme activity and stability. modulating protein-protein interaction with a molecular tether helen farrants 1 , oliver hantschel 1 , kai johnsson 1 1 ecole polytechnique f ed erale de lausanne (epfl) high-affinity scaffolds for protein-protein interactions, such as monobodies and darpins can be engineered in vitro to bind to protein targets. we speculate that the affinity for the target protein can be modulated by incorporating these evolved scaffolds and a synthetic intramolecular tether into protein switches, in a protein construct of composed of snap-tag, a monobody and a circular permutated dihydrofolate reductase. the tether, attached to the construct via snap-tag, was composed of a linker and trimethoprim, which interacts reversibly with the circular permutated dihydrofolate reductase. we have investigated the affinity between the n-sh2 domain of the phosphatase shp2 and an evolved monobody in such a protein construct using a fret assay. when the intramolecular tether was bound the circular permutated dihydrofolate reductase ("closed" conformation), there was an increase in the affinity of the construct to the target n-sh2. in the presence of a small molecule competitor ("open" conformation) the affinity of the monobody construct to its target was reverted to the value reported in the literature. the intramolecular tether in these protein constructs combined with engineered scaffolds for protein-protein interactions may be a general approach towards protein switches. because most proteins are long polymers of amino acids with twenty or more chemically-distinct sidechains, there are an enormous number of potential protein sequences. here, we report the construction of biologically active proteins with minimal chemical diversity. transmembrane domains of proteins can specifically interact with other transmembrane domains to modulate the folding, oligomerization, and function of transmembrane proteins. for example, the bovine papillomavirus e5 protein is a 44-amino acid transmembrane protein that transforms fibroblasts to tumorigenicity by binding directly to the transmembrane domain of the platelet-derived growth factor b receptor (pdgfbr), resulting in ligandindependent receptor activation and cell transformation. these studies showed that a free-standing transmembrane domain could fold properly in cells and act in trans to modulate the activity of a larger transmembrane protein target. because of the relative chemical simplicity of transmembrane domains and this ability to act even when not linked to more complex soluble protein domains, we reasoned that short transmembrane proteins could be used to define the minimal chemical diversity sufficient to construct biologically active proteins. to accomplish this, we infected cultured mouse cells with a retroviral library expressing 26-amino acid proteins consisting of an initiating methionine followed by a randomized sequence of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a single methyl group, and selected rare proteins with transforming activity. we isolated numerous proteins consisting of diverse sequences of leucine and isoleucine that cause morphologic transformation, escape from contact inhibition and focus formation, and growth factor independence. genetic and biochemical analysis of these proteins indicate that like e5 they interact with the transmembrane domain of the pdgfbr to specifically activate the receptor and transform cells. mutational analysis of individual proteins identified specific leucines and isoleucines required for transforming activity, and insertion of a single isoleucine at a particular position in a stretch of leucines is sufficient for activity. these proteins identify the minimal chemical diversity required to generate a biologically active protein and have important implications for biochemistry, protein evolution, protein engineering and synthetic biology. yusuke azuma 1 , donald hilvert 1 1 virus-like particles that are precisely loaded with functional cargo are an important tool to study the effect of spatial confinement and create novel entities with application in biotechnology and medicine. by genetic fusion to a positively supercharged green fluorescent protein (gfp(136)), an enzyme retroaldolase (ra) was efficiently targeted to the negatively charged lumen of an engineered protein cage, aquifex aeolicus lumazine synthase variant 13 (aals-13). the encapsulation is quantitative under mild aqueous condition up to a mixing ratio of 45 guest enzymes per host cages. the chromophoric tag is used for precisely quantifying the enzyme concentration, which allows detailed characterization of the effect of encapsulation on the enzyme activity. the generality of the encapsulation system was examined with 8 structurally different enzymes. introduction and purpose: in the immune system, high affinity antibodies are generated by selection of b cells activated by antigen-stimulation followed by additional optimization through somatic hyper mutation of antibody genes. in the artificial antibody libraries, such as phage libraries, selection of specific antibody clones from the library is performed by in vitro selection process called biopanning and the subsequent binding screening. however, in spite of high efficiency of enrichment in biopanning, there is a possibility that we overlook the minor antigen-specific clones in the screening because of the limitation of the number of clones employed for screening. in recent years, high-throughput analysis of dna sequences by the next-generation sequencer (ngs) has become available not only for genomic analysis of organisms but also repertoire analysis of antibodies. in this presentation, we report the successful isolation of a variety of antigen specific antibodies from patients-derived antibody phage library by a combination method of high throughput sequence analysis on ngs and biopanning. method: we constructed two kinds of human single chain fv (scfv) antibody libraries from pooled mrna of five cancer patients and of a wheat allergy patient, respectively. after biopanning against a cancer antigen or wheat allergy antigen "gluten", the phagemid vector dna prepared from the pooled phages before or after biopanning was used for pcr amplifications of vh genes, adding the index and adapter sequences for ngs analysis. the high throughput sequencing was performed on miseq (illumina) using miseq reagent kits v3. after discarding the short sequences and low quality data, 5'-and 3'-reading sequences were unified by a merge program. the frequencies (%) of all vh sequences were evaluated using a program based on usearch 8.0 clustering software and the changes of the frequency (%) of each sequence between before and after panning were assigned as amplification rate. results and discussion: vh sequences at each round of pooled phages after biopanning against cancer antigen were analyzed on ngs. after three rounds of biopanning, three clusters of antibody sequences were specifically enriched suggesting these are specific binders. to check this, scfv gene were regenerated by pcr using h-cdr3 specific primers and scfv-displaying phages reconstructed were subjected to binding analysis. all three phages showed a clear specific binding to cancer antigen in elisa. subsequently, to test the usefulness of this method, we applied it to identify allergen-specific scfv from allergy patient-derived antibody phage library. the phylogenetic tree analysis of vh sequences which showed the amplification rate higher than 2.5 by a single round of biopanning elucidated total eleven clusters of vh sequences. the vh sequences in the two clusters with the highest amplification factor were selected and the regenerated scfv-displayed phages were tested for binding analysis. the prepared scfvdisplayed phages and also scfv proteins showed a clear binding ability to allergen. thus, it is suggested that the analytical method of vh sequences on ngs before and after biopanning is very useful to isolate a variety of disease related antigen-specific novel antibodies quickly with high degree of certainty. biochemical analysis of the recognition helix of z-dna binding proteins: roles in conformational specificity yang-gyun kim 1 , xu zheng 1 , so-young park 1 1 conversion of right-handed b-dna into left-handed z-dna is one of the dramatic structural transitions in biological processes including gene regulation and chromatin remodeling. z-dna binding motif, zalpha (za), was first discovered from human adar1. subsequently, with sequence and structure similarity to the hzaadar1, families of proteins including viral e3l, interferon-induced protein dai (zbp1) and pkz has been identified to have za domain(s). interestingly, the za domain of the e3l protein from vaccinia virus (vvzae3l) was confirmed to have the ability of z-dna-binding, but it does not have the b-to-z conversion activity. here, we showed that the replacement of the a3-helix of vvzae3l (vvzae3l-a3) with that of hzaadar1 results in acquiring the ability to converting b-dna to z-dna. the detailed biochemical analysis of the a3-helix mutants of vvzae3l further suggested that the contribution of positively charged residues in the c-terminal part of the a3-helix is crucial during the b-to-z transition. in addition, hydrophobic residues of the n-terminal part of the vvzae3l-a3 also influence on the b-to-z conversion activity, possibly through forming a tightly-packed structure. in conclusion, our results revealed the previously-unknown contribution of amino acid residues existed in the a3-helix of the za domains to the b-to-z conversion. moreover, it strongly implies that such residues may play important roles in initiating conformational changes of dna structure during the b-to-z conversion event. the ability of switching the activity of proteins at will is of great interest from an application point of view. one promising approach utilizes a protein modification with an organic photochromic molecule. linking two protein side chains with the photochrome that undergoes a light induced conformational change, protein secondary and tertiary structure can be stabilized or destabilized and thus the structure dependent activity can be switched "on" and "off" by light irradiation. for this the photochrome must fulfil several requirements. foremost, it must possess two states of comparable stability that differ significantly in their geometry. it must further be water soluble and non-toxic, and should not experience fatigue phenomena upon multiple irradiations. there are two classes of molecules that fulfil those requirements: azobenzenes and spiropyrans. we are pursuing two different strategies for the design of photoswitchable proteins. in the first approach we attach an azobenzene compounds to side chains of the alpha-helical antifreeze protein type i. the end to end distance of the photochromic molecule is sterically compatible with the folded helix only in one form, photoisomerization therefore switches the folding state between an active helical state and an inactive unfolded form. in a second, more general approach we use the trp-cage domain as a switching unit. the trp-cage is the smallest known folded protein (20 amino acids). its folding is induced by hydrophobic interactions of a tryptophan side chain in a short helical segment. after modification with a photochromic molecule in appropriate positions, its structure is rendered sensitive to the state of the chromophore. by creating protein chimera of such a trp-cage and biologically active peptides with helical propensity, we aim at conferring the light-dependent fold of the cage to the attached peptide moiety. salt-bridges are electrostatic interactions between groups of opposite charges. net interaction energy (ddgnet) of a salt-bridge is partitioned into bridge (ddgbrd), desolvation (ddgdsolv) and protein (ddgprot) energy-terms of which estimation of ddgdsolv and ddgprot are only possible by computational means. thus, general purpose poisson-boltzmann equation solver: "delphi" (in commercial package of insight-ii) and "apbs" (open-source) are popularly used to determine these energy-terms. nevertheless, the computation-method is highly involved one than other uses of these solvers. moreover, protein-specific saltbridges, grid-points, center, hydrophobic-isosteres-mediated mutation-files of original charge-radius file and others are to be worked out prior to the computation. this might answer as to why only limited numbers of structure files (2% of crystal-structure-database) are worked out till date. at this juncture, an efficient fully automated all-in-one-procedure that could analyze large dataset in a single run would be useful. to the best of our knowledge, such procedure is truly lacking in public domain. at this end, our fully automated all-in-one procedure: adsetmeas (available freely at http://sourceforge.net/projects/adsetmeas/along with detailed documentation) uses "apbs" method to compute component as well as net energy-terms of salt-bridges and redirect compact output in excelformat. further, micro-environments of salt-bridges are also been reported based on the presence of polar, dipolar, acidic, basic and hydrophobic side-chains in their proximity. the procedure provides versatility to users in choosing a] model for computation of energy-terms to-date available in the literature and b] method (default or advanced) for parametric optimization in "apbs" calculations. it works in unix like environment including cygwin. it processes all proteins present in the working directory with any number of salt-bridges in them. a pre-released version of the procedure was successfully applied for energy-terms on 220 salt-bridges from 22 halophilic proteins. overall, our adsetmeas provides intricate details on salt-bridge energetic from crystal structures and find application in the field of computational structural biology. these and other results will be discussed in the conference. next generation analgesics -targeting ion channels with antibody-drug conjugates (adcs) anna wojciechowska-bason 1 , clare jones 2 , chris lloyd 3 1 postdoctoral fellow, adpe, medimmune, cambridge, 2 ria, medimmune, cambridge, 3 adpe ion channels are common targets for chronic pain therapies. small molecule analgesics are widely used therapeutically, but due to poor specificity they often cause a wide range of side effects. as a result, efficacy of existing treatments is very limited. we believe that to achieve the required specificity and efficacy, a novel and innovative approach is required that would combine the potency of the small molecule with the selectivity of an antibody. therefore, we propose to apply antibody-drug conjugates (adcs) to deliver small molecules or peptides to ion channels in order to specifically modulate pain signalling pathways. voltage-gated sodium channel nav1.7 has a well characterised role in the perception of pain. here we present the activity of the peptide huwentoxin-iv (hwtx-iv) and small molecule inhibitors ptc-a, ptc-b and ptc-c on voltage gated sodium channels nav1.7 and nav1.6. in novel findings, we report that these inhibitors show little selectivity between the voltage-gated sodium channel family members, nav1.6 and nav1.7, and that the ic50 values and the impact on channel biophysics (voltage-dependence of activation and fast inactivation) of the inhibitors are largely similar for both channel types. therefore, the use of hwtx-iv and other small molecule inhibitors of nav1.7 for pain therapy could be dose-limited due to side effects mediated by the inhibition of channel nav1.6. in conclusion, we propose that hwtx-iv and the investigated small molecule inhibitors could be used for the treatment of pain as part of a nav1.7 antibody-drug conjugate (nav1.7-adc), establishing nav1.7 specificity and minimising side effects. maria antonietta carillo 1 , daniel varon silva 1 1 malaria is one of the most infectious diseases caused by plasmodium species parasites. the merozoite surface protein 1 (msp1) is the most abundant protein on the surface of the plasmodium species merozoite stage, which plays an important role during the erythrocytes invasion process [1] . msp1 is synthesized as a 200-kda glycosylphosphatidylinositol (gpi) anchored protein precursor which is processed at the end of the schizogony into four different fragments. the primary processing step produces a complex of four fragments that are present on the merozoite surface. the secondary processing step at erythrocytes invasion results in the detaching of the complex from the surface, except for the cterminal 19-kda domain (msp119), which remains anchored to the parasite surface by the gpi moiety. in human malarial infections, the gpi is considered to be a toxin that causes the expression of various host genes and induces a pro-inflammatory immune response, making it a valuable candidate for the development of anti-malarial drugs. in order to study the function of the gpi and evaluate the effects, msp119 fragment has been expressed, purified and anchored to the synthetic gpi molecule using protein trans-splicing strategy based on the split intein method [2] . the role of the gpi moiety will be studied through protein folding experiments and the effect of the anchored protein will be evaluated in vitro in order to understand the function of the gpis. assessment of uch-l3 substrate selectivity using engineered ubiquitin fusions with varying linker lengths peter suon, mario navarro, john love 1 san diego state university, 2 san diego state university, 3 assessment of uch-l3 substrate selectivity using engineered ubiquitin fusions with varying linker lengths peter suon, mario navarro, and john j. love san diego state university the ubiquitin proteasome system (ups) is a complex system composed of multiple structural and functional elements that play key roles in cellular processes such as signal transduction, cell cycle regulation, apoptosis, and protein degradation. proteins destined for degradation are first tagged with the protein, ubiquitin, which is covalently attached to internal lysine residues. once the target has be degraded by the proteasome; the enzyme ubiquitin carboxy hydrolase l3 (uch-l3) is believed to prepare ubiquitin for additional rounds of ubiquitination by cleaving small peptides and chemical adducts from the ubiquitin c-terminus. previously in our laboratory, protein substrates of uch-l3 were engineered and used to characterize uch-l3 substrate selectivity. the engineered substrates consisted of n-terminal monoubiquitinated test variants derived from streptococcal protein g (protein gb1) and staphylococcal protein a (spab). the thermal denaturation temperatures (tm) of the fusion proteins were measured using circular dichroism and span a range of over 608c. more importantly, the rate of hydrolysis for the fusion proteins is demonstrated to be directly correlated to the tm of the test variant fused to the c-terminus of ubiquitin. previously, the engineered substrates were designed to emulate natural ubiquitin fusions and thus did not contain any 'linker' residues between the c-terminus of ubiquitin and the n-terminus of the test protein. to explore the effects of linker length on uch-l3 hydrolysis we are engineering new uch-l3 substrates that contain an unstructured 12 amino acid linker between ubiquitin and the test protein. to further explore the catalytic efficiency of uch-l3 we will revisit diubiquitin (ub-ub), which is not hydrolyzed by uch-l3, and will make mutations in the hopes of generating a hydrolysable substrate. using rational design, the new variants will be engineered to destabilize the c-terminal ubiquitin to determine if this results in hydrolysis of the new ub-ub construct. the thermal stability of these new fusion protein substrates will be measured using circular dichroism spectroscopy (cd) and uch-l3 hydrolysis rates will be characterized using existing assays. our goal is to continue the use of engineered substrates to further explore the catalytic properties of uch-l3 activity and the potential role in protein trafficking and degradation within living cells. we present a biophysical study of a suite of helical proteins that have been modified to contain 12and 17-amino acid additions on their termini that impart increased resistance to degradation in e. coli abstract recombinant expression systems. the b domain of staphylococcal protein a (ab) and the homeobox dna-binding domain from d. melanogaster engrailed (en) are small 3-helix bundles. these domains do not appreciably accumulate in the e. coli bl21 (de3) cytoplasm when expression in a pet vector is chemically induced. this is likely due to host protein degradation/recycling factors that function to efficiently degrade these two proteins. addition of sequences encoding either of two amino-terminal beta-hairpins to either the n-or c-terminus of ab and en results in the accumulation of large amounts of these new chimeric proteins. additionally, destabilization of the ab or en sequence does not abolish the expression enhancement effect of the beta-hairpin addition. we have investigated the biophysical origins and effects of the beta-hairpin additions using circular dichroism (cd) spectroscopy, and have determined that the added sequence does not significantly perturb the secondary structure of ab or en, nor does it significantly influence the unfolding temperature (tm). while investigation into the origin of the accumulation effect is ongoing, we hypothesize that the addition of the sequence is disruptive to recognition events in the native protein degradation machinery in e. coli. thus, this approach represents both a biotechnological tool for expressing helical peptides recalcitrant to expression, as well as a system well-suited to probing mechanisms of protein recycling and homeostasis. a special class of these proteins are lipidated proteins containing a glycosylphosphatidylinositol (gpi) glycolipid moiety at the c-terminus. the lipid chains of the gpi anchor molecule are responsible for the membrane association of the attached protein. a unique feature of gpi-anchored proteins is that after isolation they can be reinserted into the membrane of recipient cells with the retention of the biological function. accordingly, the exogenous introduction of fluorescent gpi-anchored protein analogues into cell membranes is a useful method for visualizing the cellular traffic of membrane associated proteins and for engineering cell surfaces. we have recently shown that cholesterol can be applied for anchoring proteins to the plasma membrane of live cells without perturbing the membrane. in order to introduce proteins containing covalent modifications that are not genetically encoded, an enzymatic method was considered and fused with the c-terminal cholesterylation method. the usefulness of the method is demonstrated via the preparation of multimeric model proteins of 40 kda monomers, that is an appropriate representation of the ligation of domain size proteins. transmembrane domain dimerization drives p75ntr partitioning to lipid rafts irmina garc ıa carpio 1 , marc¸al vilar 1 1 sociedad de biof ısica de españa. sbe p75 neurotrophin receptor (p75ntr), is best known for its role in mediating neuron cell death during development or after injury but it also regulates cell proliferation, axon guidance or survival. the key to understand its signaling could rely in its structure and conformational states. it has been described that p75 forms disulfide-linked dimmers through the cys257 in the transmembrane domain which are essential for its ngf mediated signaling. previous studies have shown that p75 is present in lipid rafts, where it interacts with intracellular adaptors to activate different signaling pathways. we design several p75 mutants in the tm domain that impairs dimerization and study the role of tm domain dimerization in lipid rafts recruitment. our analysis suggests that p75 tm domain dimerization influences lipid raft partitioning. these results could be a key role to understand its signaling and processing pi-079 bioluminescent sensor proteins for therapeutic drug monitoring of the monoclonal antibody cetuximab martijn van rosmalen 1 , remco arts 1 , brian janssen 1 , natalie hendrikse 1 , dave wanders 1 , maarten merkx 1 1 therapeutic drug monitoring (tdm) -adapting the drug dosage scheme to the individual patient's pharmacokinetic and pharmacodynamic characteristics -is still uncommon for therapeutic monoclonal antibodies, despite preliminary studies showing its potential benefits. one of the factors impairing tdm implementation is the lack of equipment and trained personnel to regularly measure drug concentrations in patients receiving treatment. point-of-care diagnostic devices which could be used by patients themselves or by their general practitioners would greatly advance the feasibility of tdm. here we present a biosensor for the therapeutic monoclonal antibody cetuximab. we developed a series of cyclic peptides that specifically recognize cetuximab, covering a fourfold range of affinities, and incorporated these cyclic peptide sequences into a set of luminescent sensor proteins. the sensors translate cetuximab concentrations into a change in emission color that can be read out using a mobile phone camera. together, these sensors can quantify cetuximab levels within the relevant therapeutic concentration range and we propose that they can be used for therapeutic drug monitoring applications. genetically encoded biosensor for cell permeability of inhibitors of the p53-hdm2 interaction silvia scarabelli 1 , thomas vorherr 2 , kai johnsson 1 1 ecole polytechnique f ed erale de lausanne, 2 the evaluation of the permeability across the cellular membrane is a key step in the development of therapeutics, since it affects the distribution and the efficacy of the latters. reliable and versatile techniques for the determination of structural permeability determinants of molecules and information about the entry kinetics are still missing. we introduced in the past a class of semi-synthetic ratiometric sensor proteins (snifits) that has been shown to be suitable for the measurement of intracellular metabolites concentrations. here we describe a totally genetically encoded sensor based on the snifits modular design for the assessment of the cell permeability of small molecules and peptides inhibitors of the protein-protein interaction between p53 and hdm2. we show that our sensor detects the presence of hdm2-binding stapled peptides in vitro, and, when expressed in mammalian cells, it responds to the perfusion of the known small molecule hdm2 inhibitor nutlin-3a. moreover, experiments made with an automated microscope show that the sensor is suitable for measuring and comparing the kinetics of entry of different kinds of inhibitors in the cytosol of living cells. in parallel, we are developing an hcaii-based sensor protein for the sensing of sulfonamides and eventually their peptide derivatives. we show that the sensor responds to the presence of different kinds of hca-inhibitors in vitro and in perfusion experiments. this second sensor would broaden the range of molecules and peptides whose permeability can be studied with our tools beyond the family of the hdm2-binders. our sensors overcome the limitations of the already existing techniques for measurements of permeability while offering a simultaneous measurement of the cell permeability and of the binding efficiency of small molecules and peptides of interest. archer: predicting protein function using local structural features. a helpful tool for protein redesign. jaume bonet 1 , javier garcia-garcia 1 , joan planas-iglesias 2 , narcis fernandez-fuentes 3 , baldo oliva 1 1 structural bioinformatics lab, grib, upf, 2 division of metabolic and vascular health, university of warwick, 3 the advance of high-throughput sequencing methodologies has led to an exponential increase of new protein sequences, a large proportion of which remain unannotated. the gap between the number of known proteins and those with assigned function is increasing. in light of this situation, computational methods to predict the function of proteins have become a valid and necessary strategy. here we present archer, a server that exploits archdb's hierarchy of super-secondary structures to map go and enzyme functions upon protein regions and, thus, infer the function of a protein. the server relies on either the sequence or structure of the protein of interest and returns the mapping of functional subclasses extracted from archdb. moreover, it computes the functional enrichment and significance of each subclass, combines the functional descriptors and predicts the function of the query-protein. combining the functional enrichment analysis of the super-secondary structures with the structural classification of archdb, users can select variants of the target sequence that swap the region of a supersecondary structure by another that putatively fits in the same scaffold minimizing the effect on the global tertiary structure. only variants that modify the predicted function are offered for selection, thus providing a rational, knowledge-based, approach for protein design and functionalization. the archer server is accessible at http://sbi.imim.es/archer. phytochromes are natural photoreceptors known to regulate photosynthesis in plants, fungi and bacteria. phytochromes found in bacteria share common architecture and consist of a pas-gaf-phy photosensory core and a c-terminal output module, responsible for biological function. a bacterial phytochrome, bphp1, from rhodopseudomonas palustris undergoes reversible conversion from the farred absorbing state (pfr) to the red-absorbing state (pr) followed by the conformational change upon 740 nm light irradiation. as most of bacterial phytochromes, bphp1 forms a dimer. it was shown that 740 nm light causes a protomer swapping between the bphp1 dimers; and likely, the output module is involved in this process. however, the mechanism of the light-induced swapping is poorly studied. we tested an ability of the protomer swapping between bphp1 dimers using pull-down biochemical assay. for this, strep-tagged bphp1 was immobilized on strep-tactin sepharose beads in the presence of untagged bphp1 fused to mruby2 at different concentrations. after incubation, the proteins were eluted and visualized in sds-gel using a zinc-induced fluorescence assay. an amount of the bound to beads protein was estimated by densitometry. it was found that more than 75% of heterodimers (streptagged-bphp1 and bphp1-mruby2) form within 2.5 h of incubation under 740 nm light at 8-fold excess of one of the interacting partners. in darkness, the swapping was much slower. in the similar setup we checked the amount of heterodimers after 15, 30 and 120 min of incubation. no difference was observed for different time points, suggesting that the protomer swapping is relatively fast process. next, a role of the c-terminal effector domain of bphp1 in the light-induced interaction was studied. for this, kinetics of the pfr-to-pr transition was analysed by measuring of absorbance at 680 nm and 740 nm for full-length bphp1 and a bphp1 mutant with the deleted c-terminal domain. while full-length bphp1 showed the normal pfr-to-pr transition, absorbance of the mutated bphp1 at 680 nm did not raise. however, 740 nm absorbance changes were similar for both proteins; and surprisingly, the similar dark relaxation kinetics was observed. we propose that the impaired pfr-to-pr transition is caused by restricted pr conformation in the mutant rather than by fast pr-to-pfr relaxation. understanding the mechanisms of the bphp1 light-induced structural changes and the protomer interaction should advance engineering of bacterial phytochromes into fluorescent probes and optogenetic tools. antibody detection is an integral part of many diagnostic strategies, most crucially so when infectious diseases are involved. currently used assays, such as elisa or spr, enable detection of antibodies in the laboratory with high sensitivity, yet a translation of these technologies to an application outside of the laboratory setting is far from trivial. problematically, the burden of disease for many infectious diseases is carried precisely by those countries where access to laboratory facilities is severely limited. we therefore developed a novel, one-step assay that allows the detection of antibodies directly in solution using a luminescent sensor protein. our strategy is based on the use of a bright luciferase, nanoluc, tethered to a green fluorescent protein (mneongreen) via a semi-flexible linker containing two epitope sequences. crucially, two small helper domains were fused to the protein termini. these domains keep nano-luc and mneongreen in close proximity in the absence of antibody, enabling efficient bioluminescence resonance energy transfer (bret). binding of antibody to the epitopes in the sensor proteins linker domain pulls the bret partners apart, effectively changing the color of emission from green to blue. the assay allowed the detection of picomolar amounts of anti hiv1-p17 antibodies directly in solution, both under optimized buffer conditions and in blood plasma. in principle. the modular sensor architecture should allow detection of any antibody with a well-defined epitope of sufficient affinity. to demonstrate this, the hiv-epitopes were substituted for two ha-tag epitopes, yielding a sensor that enabled the detection of picomolar amounts of anti-ha antibodies. the simple optical readout provided by the sensor system allowed us to record the emitted signal with a conventional mobile phone camera. a simple software application that analyzes the image based on rgb values sufficed to interpret the recorded image vis-a-vis the presence of antibody. bearing in mind the eventually envisioned application in a point-of-care diagnostic setting, this combination of sensor recording and interpretation using nothing more than a mobile phone and a software application holds considerable diagnostic potential. beyond point-of-care diagnosis of infectious diseases, a simple assay to detect and quantify antibodies directly in solution could also have a substantial impact in other fields. antibodies are ubiquitous in biotechnology, and this is reflected by the plethora of potential sensor applications, which range from a role in microfluidic circuits or monitoring the biotechnological production of antibodies, including validation of bispecificity, to veterinary applications, diagnosis of autoimmune diseases and monitoring the success of vaccination campaigns. the continually growing protein data bank (pdb) has been a key resource for general principles of protein structure. for example, parsing structural observations in the pdb into simple geometric descriptors has given rise to statistical energy functions. here we present a novel strategy for mining the pdb on the basis of local tertiary structural motifs (term). we define a term to be the structural fragment that captures all local secondary and tertiary structural environments of a given residue, and query the pdb to obtain quantitative information for each terms. first, we show that by breaking a protein structure into its constituent terms, we can describe its sequence-structure relationship via a new metric we call "structure score." using submissions in recent critical assessment of structure prediction (casp) experiments, we find a strong correlation (r 5 0.69) between structure score and model accuracy -a performance that exceeds leading atomistic statistical energy functions. next, we show that querying terms affected by point mutations enables the quantitative prediction of mutational free energies. our simple approach performs on par with state-of-the-art methods fold-x and popmusic on 1300 mutations, and provides superior predictions in certain cases where other methods tend to fail. in all, our results suggest that the data available in the pdb are now sufficient to enable the quantification of much more sophisticated structural observations, such as those associated with entire terms, which should present opportunities for advances in computational structural biology techniques, including structure prediction and design. exploiting natural sequence diversity for protein crystallization sergio mart ınez-rodr ıguez 1 , valeria risso 1 , jos e m sanchez-ruiz 1 , jos e a. gavira 2 , 1 departamento de qu ımica-f ısica, universidad de granada, 2 laboratorio de estudios cristalogr aficos, iact-csic-ugr granada during the last decade, different rational and high-throughput approaches have been successfully applied in the protein crystallography field to widen thejjso-called "protein crystallization bottleneck" [1, 2] . despite the enormous efforts carried out by our community, the statistics presented by structural biology consortiums [3] suggest that so far only the easy-to-pick fruit has been attained; thus, new approaches are necessary to further expand the crystallization limiting step to relevant targets. on the basis of previous hypothesis suggesting that the difficulties found in protein crystallization might be a result of evolutionary negative design [4] , we have used two different protein engineering approaches exploiting natural sequence diversity using beta-lactamase as toolbox: i) ancestral reconstruction and ii) consensus approach [5] . both approaches resulted in hyperstable and promiscuous ancestral derivatives. furthermore, our initial crystallization results also suggest that both approaches increased the crystallizability of the resulting enzymes when compared to the extant tem-1 beta-lactamase. the adipocyte-derived hormone adiponectin has become a key player for the understanding of overweight related diseases like obesity, diabetes, atherosclerosis or the metabolic syndrome. one of its abstract major functions are the insulin sensitizing effects, which are mediated by the activation of ampk, p38-mapk and ppara (1). furthermore adiponectin is involved into glucose regulation and fatty acid oxidation. recently, three adiponectin receptors adipor1, adipor2 and t-cadherin have been described while an unknown fourth receptor is hypothesized (2) . for only two of them (adipor1 and adipor2) the signaling transduction via adiponectin has been confirmed (3). in order to find new binding partners or co-receptors, we cloned and expressed full length adiponectin as a fusion protein with a c-terminal intein and a chitin binding domain (cbd) as well as an n-terminal his10-tag. by using the impactsystem, the fusion protein was cleaved to form the corresponding thioester. to separate the starting materials as well as the cleaved intein chitin binding domain, the purification was performed with chitin beads. furthermore, the product was concentrated by ni-nta-affinity chromatography. accordingly, the obtained adiponectin thioester was reacted with a tamra-or a biotin labeled peptide, respectively, to receive the corresponding ligation product. finally the functionalized adiponectin was purified by size exclusion chromatography. further studies will allow screening for interacting molecules in cell and tissue derived samples. departamento de quimica fisica, facultad de ciencias university of granada, 2 dpto. de quimica fisica biologica. instituto de quimica fisica rocasolano, 3 departamento de quimica organica, facultad de ciencias university of granada, 4 rational design of non-natural enzyme activities has proved challenging. here, we report the introduction of catalysis of the kemp elimination (a model of proton abstraction from carbon) in scaffolds corresponding to precambrian nodes in the evolution of the antibiotic resistance protein b-lactamase. we used a single-mutation, minimalist approach based on chemical intuition, and obtained catalysis levels similar to those reported in the literature for computational kemp-eliminase designs involving multiple mutations. remarkably, the approach was unsuccessful when performed on modern b-lactamases. we provide experimental evidence that enhanced conformational flexibility contributes to the success of the minimalist design in the ancestral scaffolds. this work has implications for the understanding of function emergence in protein evolution and demonstrates the potential of ancestral protein resurrection in enzyme engineering and design. exploring the importance of dimerization for dj-1 function through engineered domain fusions sierra hansen 1 , jiusheng lin 1 , mark wilson 1 1 parkinson's disease is a progressive neurodegenerative disease that affects approximately 6.3 million people worldwide and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. dj-1 (park7) is one of several genes that are mutated in rare forms of familial parkinsonism. dj-1 is a dimeric cytoprotective protein that defends against oxidative stress and preserves mitochondrial function. dimerization of dj-1 is thought to be essential for this function, as some diseaseassociated mutations cause poor folding and disrupt the dj-1 dimer. however, recent reports suggest that dj-1 may be functional as a monomer. to test this, we have engineered a non-dissociable dj-1 dimer that is a fusion of two human dj-1 domains. this construct cannot dissociate into monomers and thus will provide a stringent test of the importance of monomeric dj-1. our engineered construct is modeled on plant dj-1 homologs, which feature naturally occurring duplicate dj-1 domains separated by a small (19 amino acid) linker region. using x-ray crystallography, we confirmed that this engineered non-dissociable human dj-1 dimer has identical structure to the naturally occurring dimeric protein. we have investigated the influence of enforced dimerization of the pathogenic effects of the parkinsonian l166p and l10p mutations. cd spectroscopic analysis reveals that single and double l166p mutations in the non-dissociable dj-1 dimer maintain a higher degree of structure than l166p mutations in the native protein. additional characterization of the protective capacity and subcellular trafficking of this non-dissociable dj-1 dimer is underway. the purification, crystallization and preliminary characterization of sdre from s. aureus the purification, crystallization and preliminary characterization of sdre from s. aureus staphylococcus aureus (s.aureus) is an important human opportunistic pathogen which colonizes about 20% of the human population persistently [1] . surface proteins of s.aureus can excretion a kind of sortase, which represents a surface organelle responsible during the pathogenesis of bacterial infection the host circulation [2] . sdr proteins were a component of cell wall anchored family proteins, including sdrc, sdrd and sdre [3] . sdre could combine with the complement regulatory protein factor h to escape the alternative pathway of complement [4] . to further investigate the functions of sdre, we have expressed and purified the adhesive domain (residues 141-'06:15), and crystallized the recombinant protein. in addition, we also constructed the mutant s.aureus, and the cell experiments confirmed that sdre gene participate in the bacteria invasion. bacterial microcompartments (bmcs) are proteinaceous organelles that sequester key metabolic reactions to increase enzymatic efficiency or to prevent the loss of volatile or toxic intermediates. there is an increasing desire to engineer bmcs for non-native enzymatic processes. it is thought this will increase multi-enzyme pathway efficiency and allow the expression pathways that may produce toxic or volatile intermediates in bacteria. the mechanisms of small molecule transport and retention of toxic intermediates by bmcs remain poorly understood. better understanding of the bmcs pores critical to engineer bmcs for these non-native pathways. in order to better understand the bmc pore we have undertaken structure-guided modifications of the the hexameric pdua shell protein of the 1,2-propanediol utilization microcompartment (pdu mcp). these modifications include pore mutations in an attempt to alter substrate specificity and permutations of pdua to allow more drastic alterations to the structure of the protein. crystal structures of pdua pore mutants, solved to atomic resolution (2-3.3å) provide evidence of the pore residues that confer specificity. further, a pdua permutation (pduap) has resulted in a closed icosahedral cage. this novel pduap cage shows a ph and salt dependent assembly and may serve as a reaction vessel or be utilized for cargo delivery. (1, 2) . anm-mc is used to identify targeted transition pathways and intermediates between open and closed states of proteins. at each step of this iterative technique, the protein is deformed along the collective anm mode showing the best overlap with the target direction and its energy is minimized via short mc run. in this work, optimization of simulation parameters (number of mc moves and their perturbation strength, anm deformation factor in each cycle and force constant for backbone bonds) was performed in order to increase the efficiency of this technique. as a result, this technique can now be applied to much larger systems and conformational changes. the transition pathway between apo and dna-bound conformations of the yeast rna polymerase, which is a hetero-10-mer with more than 3500 residues, will be presented here. moreover, the pathway intermediates for more than 10 diverse proteins were analyzed in terms of changes in local strain energy and backbone torsional angles during apo-to-complex transitions. certain residues interacting with the ligand are detected to exhibit large changes with respect to any of these two parameters for more than half of the proteins in our dataset. department of chemistry and chemical biology, harvard university, 2 howard hughes medical institute, harvard university, 3 transgenic crops have radically reshaped the agricultural landscape. since their introduction in the late 1990s, transgenic crops have affected economic gains greater than us$110 billion globally due to reduced production costs and increased yield gains. crops modified to produce biological insecticides derived from the soil bacterium bacillus thuringiensis (bt) are among the most robust methods of pest control. bt toxins offer many advantages over traditional insecticides, chiefly their inability to affect human biology and exquisite selectivity for defined pest species. however, the evolution of resistance to bacillus thuringiensis oendotoxins (bt toxins) in insects has been widely observed in the field, and greatly threatens the use of this mechanism of pest control in the future. we developed a phage-assisted continuous evolution (pace) platform for the rapid generation of high-affinity protein-protein interactions and validated the system by evolving known high affinity antibody mimetics in <5 days of pace. we applied this system to the evolution of the bt toxin protein cry1ac to recognize a non-cognate cadherin-like receptor from trichoplusia ni, a pest for which bt toxin resistance has been observed in both the laboratory and the field. the resulting evolved cry1ac variants exhibits high affinity for the target receptor, and kill insect cells more potently than wild-type cry1ac. our findings establish that the directed evolution of novel receptor recognition in bt toxins can be used to target resistant pests, and has far-reaching implications for biological reagents and therapeutics. optimization of a designed protein-protein interface brian maniaci 1 , collin lipper 2 , john j. love 1 1 san diego state university, 2 university of california protein-protein interactions play key roles in practically every biological process. protein-protein interactions vary with composition, affinity, and lifetime of the complex. studying designed protein-protein interactions will provide insight into the underlying principles of complex assembly and formation. computational protein docking and amino acid sequence design were used previously to generate protein dimers from monomeric proteins. the normally monomeric b1 domain of streptococcal protein-g (gb1) was computational docked to itself, followed by optimization of the interfacial side chains. two variants, monomera and monomerb, were computationally derived as a result of a designed protein-protein interface. these designed proteins were characterized using analytical ultracentrifugation and heteronuclear nmr techniques. this design resulted in a pair of protein monomers that formed a heterodimer of modest binding affinity. a tetrahedral metal-templated interface design strategy was implemented in an attempt to strengthen the monomera-monomerb complex by introducing cross-monomer metal coordination. another advantage of using the metal-templated interface is the ability to control the protein-protein interaction both temporarily and spatially. a number of newly engineered variants of monomer a and monomer b with metal coordination sites were designed, produced, and tested for increased affinity of the protein-protein complex. while the generation of a metal-templated monomera-monomerb complex was unsuccessful, we were able to obtain monomera variants that form a homodimer assembly only in the presence of zinc (ii) ions. the crystal structures of metal-templated monomera variants in the presence of zinc provide an explanation for the observed dimer formation. the crystal structure indicates that the protein-protein interaction is not driven by the designed protein interface, but rather non-specific association via edge-strand interactions. new variants were designed with the goal of engineering a high affinity homodimer in a helix-to-helix orientation as the originally designed protein-protein interface. current evaluation of monomera variants for self-association via metal coordination are being evaluated using size exclusion chromatography with a multi-angle light scattering detector for oligomerization state quantification. the results of this protein design project should lead to a greater understanding of the biophysical parameters that drive natural protein-protein interactions. continuous evolution of site-specific recombinases with highly reprogrammed dna specificities the ability to precisely modify the genome of human cells has enormous potential as a novel therapy and a powerful research tool. in contrast to reprogrammable nucleases, such as talens or a cas9/ sgrna pair -which specifically cleave dna but then rely on stochastic host cells processes to effect gene insertion -site specific recombinases directly catalyze genomic integration with high efficiency. a major limitation of this approach is that recombinases, such as cre, natively bind with high specificity to long dna target sequences (loxp in the case of cre) that do not exist in the human genome. previous attempts at evolving cre resulted in modest changes to its specificity, or required hundreds of rounds of manual protein evolution. we developed and validated a phage assisted continuous evoluiton (pace) selection for rapidly altering the dna specificity of cre recombinase towards a site present in a human genomic safe harbor locus. the pace experiments resulted in cre variants capable of recombining a substrate with nearly 50% of the nucleotides altered compared to loxp. we successfully used one of these variants to integrate exogenous dna into the genome of unmodified human cells. we are currently using sequencing methods to determine the specificity of the new recombinase clones. aleardo morelli 1 , burckhard seelig 1 1 generation of comprehensive deletion libraries mediated by in vitro transposition analysis of protein enzymes and ribozymes from nature, and from in vitro evolution, revealed that deletions of up to dozens of amino acids (or nucleotides) can be structurally tolerated. furthermore, shortened variants can exhibit better stability and increased catalytic activity. in order to investigate the effects of deletions, we developed a new procedure based on in vitro transposition to build libraries of more than 10,000 deletion mutants in three to four days. we tested our procedure on dna sequences coding for an artificial rna ligase called ligase 10c. we used the generated library for an mrna display selection, and isolated two active mutants containing 18 and 13 amino acids n-terminal deletions. structural characterization of ppsc, a multi-domain polyketide synthase from mycobacterium tuberculosis using a fragment-based approach alexandre faille 1 , nawel slama1 1 , anna grabowska 1 , david ricard 1 , annaik qu emard 1 , lionel mourey 1 , jean-denis pedelacq 1 1 polyketide synthases are of great interest in numerous scientific fields. they are composed by multiple domains, each having a different role to play in the catalysis of sequential reactions including condensation, reduction and esterification. their reaction products, named polyketides, represent a large variety of chemical compounds, from antibiotics to immunosuppressors or even anticancer drugs. ppsc is a 231 kda polyketide synthase, organised into six catalytic domains (ks-at-dh-er-kr-acp) with singular functions. along with other type i polyketide synthases, ppsc is responsible for the biosynthesis of an essential polyketide for the virulence of mycobacterium tuberculosis (mtb) and thus is a target of choice for the design of inhibitors. to date, no structural information of any type i polyketide synthase in its entire form has been described. main reasons are the length of these large size enzymes and the flexibility imposed by the linkers between domains, thus making them very difficult to crystallize. numerous questions about domain-domain interactions, spatial arrangement of this complex machinery, substrate specificity and stereochemistry are still unanswered. addressing the structural and functional characterization of ppsc would then help answering these questions and provide valuable information for drug design. to overcome the length-and flexible-dependent problem originating from the presence of multiple domains and linkers, we decided to study domains expressed alone. for this purpose, we used our domain trapping strategy to identify soluble fragments representing a single domain from ppsc [1] . it has the advantage of not relying on the bioinformatically designed domain boundaries and can even sometimes include parts of linkers to obtain more soluble fragments. using this strategy, we were able to identify relatively small and highly soluble fragments representing each domain of ppsc, thus facilitating the downstream structural and functional characterization. more than 20 fragments have been submitted to crystallization trials. among these, 5 gave crystals and allowed us to determine the x-ray structure of ppsc at, er, in addition to the dh domain in complex with a substrate analog for which activity was confirmed in vitro. the computational design of proteins that bind small molecules remains a difficult challenge in protein engineering. the ability to computationally design native-like interactions with high accuracy and efficiency would be an asset towards therapeutic development, enzyme design, and engineering functional proteins. we have developed a systematic approach to designing interfaces. we first identify ligands with naive binding affinity to our protein scaffold, then use rosettaligand to computationally dock the ligand while designing the interface for a tighter interaction. this way, we are taking a 'shot in dim light' for design as opposed to a 'shot in the dark', allowing us to more thoroughly investigate the successful and not-so-successful designs, and improve the computational methods. of 3500 ligands screened, we identified 28 weakly-binding hits in the range of 340 -1110 mm. thus far, rosettaligand has successfully designed one tighter protein-ligand interface, from 312 mm to 21 mm. in progress experiments include designing and experimentally validating more designed interfaces. structural studies of human acidic fibroblast-growth factor (fgf1) mutants with a probable anticancer activity lectins are carbohydrate-binding proteins ubiquitously present in nature. they play a role in biological recognition phenomena involving cells and proteins. the interaction lectin-carbohydrate is highly specific, and can be exploited for the development of nanoparticles containing on their surface lectins specifically directed to carbohydrate residues present only on malignant cells and absent on healthy ones (1) . lectins have been found to possess anticancer properties and they are proposed as therapeutic agents, binding to cancer cell membranes or their receptors, causing cytotoxicity, apoptosis and inhibition of tumor growth. some lectins are able to prevent the proliferation of malignant tumor cells because they recognize the t-antigen (gal b 1-3galnac) found specifically on the surface of tumor cells (2) . the main problem is that their use as a detection agent for the t-antigen in clinical studies is not possible because the immune system can recognize them as foreign molecules and develop an immune response. previous studies with x-ray crystallography made in our laboratory have characterized a lectin found in mushrooms called bel b-trefoil which has antiproliferative activity on tumor cell lines, because it contains three binding sites for the t-antigen. unlike other lectins with this property, bel b-trefoil shows structural homology with a human protein, acidic fibroblast growth factor (fgf1) (3). superposition of their structures suggests that the human protein could be mutated to contain at least one of the binding sites for the t-antigen. such mutations should create in fgf1 the potential capacity of recognizing tumor cells with less immunogenicity than the fungal protein. fgf1 is mitogenic and chemotactic, and mediates cellular functions by binding to transmembrane receptors, which are activated by ligand-induced dimerization requiring heparin as co-receptor. to reach our purpose, the fgf1 cdna was cloned into a bacterial plasmid and then mutated in five different positions to eliminate its mitogenic activity and to engineer in the protein the t-antigen binding capacity. attempts to crystalize the mutants of fgf1 were made using the hanging drop technique with the final aim to carry out their structural characterization by x-ray diffraction analysis of the crystals. the de novo synthesis of proteins in response to the activation of cellular signaling pathways is a crucial element of many high-level biological processes, including the synaptic plasticity underpinning memory formation in the brain. while of fundamental biological importance, there has been a shortage of tools with which to specifically target pools of newly synthesized proteins of interest for study. thus, we have developed timestamp and smash, methods for drug-dependent tagging, or destruction, respectively, of newly synthesized copies of proteins of interest. both methods rely on protein tags that remove themselves by default via an internal hepatitis c virus (hcv) ns3 protease, but which are retained in the presence of cell-permeable small molecule protease inhibitors. the timestamp tag contains split yfp halves and epitope tags which are reconstituted and preserved, respectively, on proteins of interest following drug application, whereas the smash tag contains a strong degron which remains attached to proteins of interest following drug application, resulting in their clearance. one limitation of time-stamp and smash is that they can only be used to independently manipulate one protein of interest at a time. furthermore, the application of timestamp and smash to study endogenous protein pools in mammals has not yet been explored. here, we report on efforts to extend these techniques by reengineering ns3 proteases which can be inhibited by two different drugs orthogonally to one another. by incorporating different drug resistance mutations into two ns3 protease variants, we engineered ns3 protease domains that are inhibitable either by asunaprevir only, or by telaprevir only. we found that these tags permit simultaneous and independent control over the newly synthesized pools of two proteins of interest within the same population of cells. we also report the development of transgenic knock-in mouse strains incorporating timestamp and smash tags, which allow the interrogation of newly synthesized pools of specific endogenous synaptic proteins in the context of their endogenous regulatory elements, and without relying on overexpression. infectious diseases are often diagnosed by the presence of specific antibodies that are produced in response to the invading pathogen. one example are antibodies that are present in patient blood after infection with the dengue virus serotype 1 and that are directed against an epitope on the virus' nonstructural protein 1 (ns-1). traditional antibody diagnosis relies on time-consuming multi-step assays that require sophisticated equipment in a laboratory environment. a promising alternative are protein switches that are based on bioluminescence resonance energy transfer (bret). these switches comprise a luciferase (nanoluc) and a green fluorescent protein (mneongreen), which are connected via a semiflexible linker. the linker contains two epitope sequences of ns-1 to which the antibodies bind specifically. if no antibodies are present nanoluc and mneongreen are held in close proximity via two helper domains and bret can occur; thus green light originating from mneongreen is visible. if antibodies are present, they bind to the specific epitopes in the linker of the switch and cause stretching of the linker and therewith break the interaction of the helper domains. as a result, nanoluc and mneongreen are separated in such a way that bret cannot occur anymore; thus only blue light originating from nano-luc remains visible. using this principle, monoclonal anti-ns-1 antibodies were detectable in a controlled buffer system and in spiked plasma samples. furthermore, the developed antibody switch was applied to plasma samples of macaques after a primary infection with dengue virus serotype 1. signal readout was possible using a laboratory-based plate reader as well as the camera of a standard smartphone. we demonstrate that this bret-based protein switch can quickly detect antibodies in solution in a single-step assay format using simple equipment for signal readout, such as a standard smartphone. this simplified antibody detection platform has the potential to be carried out outside of a laboratory, thus in areas with limited laboratory infrastructure and a high number of diverse infectious diseases. proteins expressed from more than two-thirds of the human genome reside within intracellular compartments. of these proteins many are important disease-related targets such as kras and c-myc which cannot be easily addressed by conventional small molecule approaches. some of the weaknesses of small molecules can be addressed by biologic drugs, for example high target specificity and inhibition of protein-protein interactions. the challenge for biologics is how to engineer recombinant proteins to access the intracellular space. one strategy is to use systems evolved by bacteria and viruses to deliver material inside the cells. an example of such pathway is used by pseudomonas exotoxin a (pe). the modularity of pe allows the catalytic domain to be replaced with a biologic payload against desired intracellular target. an additional benefit of pe-based delivery is a possibility of targeting the drugs only to relevant cells in the body by modifying the cell-targeting domain of the pe. the aim of this project is to deliver functional payloads against k-ras and c-myc into the cell using a pseudomonas exotoxin a translocation domain. we used phage and ribosome display to select antibody mimetics that bind k-ras and c-myc. here, we present their activity in biochemical assays and the initial results on generation of pe-based constructs. (1) . hemagglutinin is synthesized as ha0 molecule assembled as noncovalently bound homotrimers on the viral surface. this precursor protein is cleaved by trypsin-like proteases to yield two subunits ha1 and ha2 linked by a single disulphide bond (2) . ha0 is also post-translationally modified by n-glycosylation (3). it is well established that the virus hemagglutinin is the main antigen, inducing the neutralizing antibodies. in the attempt towards developing influenza vaccine production (the egg-based manufacturing lasts several months) that would be faster and safer the utilization of recombinant antigen alone is currently being observed. recently we demonstrated that yeast produced influenza h5 protein although cleaved into two subunits induced strong immunological response in mice (4) . in this report, we describe the biochemical and immunological characterization of the h5 antigen, based on hydrolytic domain of the h5n1 gene, with deletion of multibasic cleavage site and expressed in yeast system. the ha encoding gene from h5n1 virus with deletion of 18 nucleotides was cloned into ppiczac vector. rha fusion protein with his6-tag was secreted into the culture medium and was purified to homogeneity in one step using ni-nta agarose. the efficiency of the antigen purification was 200 mg/l. glycosylation sites of rha were determined using lc-ms-ms/ms. analysis of the n-linked glycans revealed that the rha is glycosylated at the same sites as the native ha in the vaccine strain. next we investigated if the hemagglutinin with deletion of the cleavage site oligomerize into higher molecular forms. to determine the oligomeric forms of the recombinant antigen various approaches were applied e.g. native-page, size exclusion chromatography or dynamic light scattering. as a final experiment to measure the size of oligomers in a protein sample a combined technology sec-mals was conducted, using multi angle light scattering (mals) as a detector. the immunological activity of rha was tested in chicken and mice, where antigen elicited high immune response. the data presented here demonstrate that new influenza antigen produced in p. pastoris is highly immunogenic and might be consider as a candidate for subunit vaccine. structural motifs capture redundant patterns that frequently occur in proteins. motifs associated with contiguous fragments of structure (i.e., secondary structural motifs) are well studied and have been successfully used to capture "rules" describing sequence-structure relationships in protein design and structure prediction. we have extended this concept to motifs that capture tertiary information-(i.e., tertiary structural motifs or terms. we have discovered that a relatively small alphabet of terms describes the known structural universe (all secondary, tertiary and quaternary information in the pdb) at sub-angstrom resolution. this alphabet of universal motifs reveals the remarkable degeneracy of the protein structure space, with just a few hundred terms sufficient to accurately capture half of the known structural universe. we have begun to demonstrate the considerable promise this structural alphabet has for applications such as protein design, structure prediction, and docking. we have developed a novel protein design framework that selects amino acid sequences, given a desired structure, using solely information from the universal terms. we show that given a native backbone, this framework recovers the native sequences to a level on par with state-of-the-art atomistic protein design methods, indicating that the motifs capture the salient structural rules governing native proteins. further, predicted sequence distributions agree closely with observed evolutionary variation. given the apparently high degeneracy among even complex features of protein structure, methods based on mining the pdb for tertiary information should provide ample opportunities for advancement in problems of computational structural biology. sortase-mediated synthesis of protein-dna conjugates for sensitive biosensing bedabrata saha 1 , marieke op de beeck 1 , remco arts 1 , maarten merkx 1 1 in recent years, semisynthetic protein-dna conjugates have emerged as attractive biomacromolecules for different applications in bio-nanotechnology, biosensing, diagnostics and therapeutics. in protein-dna conjugates, synthetic oligonucleotides allow the construction of desired molecular architecture with high specificity, while maintaining the original functionality of the protein molecules for desired application. however, the synthesis of site-specific and stoichiometric protein-dna conjugates can be challenging. due to the diversity in composition and physico-chemical properties of the proteins, few generic strategies are available for conjugation of protein molecules to a dna scaffold. a common approach is to use thiol-based covalent conjugation, but the introduction of additional cysteines can lead to the formation of intermolecular disulfides or interfere with the formation of native disulfide bonds. as an alternative, here we have developed a site-directed protein-dna conjugation strategy based on sortase mediated trans-peptidation reaction. the sortase recognizes a 'sorting motif' (i.e. lpxtg, x any amino acid), which is recombinantly introduced by site-directed mutagenesis at the cterminal end of the protein molecule. the sortase cleaves the t-g peptide bond and catalyzed the formation of a new amide bond between the lpxt peptide and the n-terminal amine of any molecule bearing an n-terminal oligoglycine motif. for this purpose, a triglycine motif was introduced at the 5'end of single-stranded dna (ssdna). on-column synthesis of triglycine modified ssdna, protected on a controlled pore glass beads, simplified the purification process and enhanced the yield of triglycinemodified ssdna (> 90%). we used this conjugation strategy in several biosensing applications. for example, we used the method to conjugate ssdna linkers at the c-termini of a range of single-chain antibody fragments (scfv) and applied these constructs to allow oriented display of capture molecules on biosensor surfaces. ssdna-scfv were using an excess of triglycine modified ssdna, we achieved 55% conversion scfv-ssdna conjugate, which can be further purified by in two step purification process consisting of ni-nta affinity column and ion-exchange chromatography. we also extended this sortase-based conjugation strategy to develop a bioluminescence based assay for sensitive target oligonucleotide detection. in this regard, the 5' and 3' end triglycine-modified ssdna molecules were successfully conjugated with a bret protein pairs, nanoluc luciferase and mneongreen fluorescent protein. the introduction of a c-terminal sortase-his5 tag and and n-terminal strep-tag allowed efficient purification of theseprotein-ssdna conjugates from excess oligonucleotides and unreacted protein. mass spectrometry based proteomics to identify the protein differences in human breast milk from breast cancer patients and controls devika channaveerappa 1 , roshanak aslebagh 1 , kathleen f. arcaro 2 , costel c. darie 1 1 breast cancer is the second leading cause of cancer death in women. about 12% women in the us develop breast cancer. death rates due to breast cancer have been declined over the years due to advancements in mammography and treatment. although, mammography helps in the early detection of breast cancer, it has few limitations. dense breast tissue makes mammogram less accurate. breast milk can be assessed to evaluate the risk of one getting breast cancer by comparing the proteomes of breast milk from healthy and breast cancer suffering individual. this study makes use of mass spectrometry based proteomics to identify the differences between the control and cancerous samples which would further help in identifying potential biomarkers for breast cancer. firstly, sds-page was used to separate the proteins from the whole milk sample. the gel bands for each sample was then excised and cut into small pieces. the gel pieces were washed and trypsin digested in order to extract the peptides. peptide mixtures in the solution were cleaned using c18 zip-tipp and then analyzed by liquid chromatography-tandem mass spectrometry (lc-ms/ms). 60 minutes and 120 minutes gradient were used for lc-ms/ms analysis. raw data obtained were converted to pkl files using proteinlynx global served (plgs version 2.4). raw data were then submitted to mascot database search for protein identification. the mascot results were then exported as .dat files and further analyzed using scaffold version 4.1 software. three breast cancer milk samples were investigated against healthy control milk samples. in the sds-page gel, after coomassie staining, the protein patterns did show minor differences. after lc-ms/ms analysis, the proteins identified by mascot database search were imported into the scaffold software and compared for the relative ratio between the proteins from the milk sampled from control donors and the donors with breast cancer. there were significant differences identified in the proteomes of the two sets of samples. some of the proteins were upregulated in the breast cancer samples and some were down regulated when compared with the controls. additional investigation of more breast milk samples is ongoing. this study focuses on identifying biomarkers directly in the milk of donors with breast cancer. leukolike vectors: leukocyte-inspired nanoparticles claudia corbo 1,2 , alessandro parodi 1,2 , roberto palomba 1,2 , roberto molinaro 1 , michael evangelopoulos 1 , francesco salvatore 2,3 , ennio tasciotti 1 1 the houston methodist research institute, 2 fondazione irccs sdn, 3 nanomedicine aims to improve drug efficiency by enhancing targeting and biocompatibility, and reducing side effects. multiple surface modifications have been proposed to provide nanocarriers with these features, based on complex synthesis processes and very often inefficient in contemporary providing biological tolerance and targeting properties [1] . bio-inspired approaches based on surface coatings developed from the purified cell membrane of immune cells represents a new paradigm shift for the development of carrier enable of prolong circulation and proper tumoritropic capabilities. we showed that nanoporous silicon (nps) particles coated with leukocyte cellular membranes -leukolike vectors (llvs) -possess cell-like properties [2] . llvs can escape macrophage uptake, delay sequestration by the reticulo-endothelial system, target tumor inflamed vasculature and accumulate within the cancer parenchyma [2] . llvs were fully characterized for their shape, size, surface charge and coating through dynamic light scattering and scanning electron microscopy. in addition we characterized the content and function of the leukocyte's proteins transferred onto the llvs coating through high-throughput proteomic analysis and the results revealed the presence and the correct orientation of several important markers of leukocytes: cd45, cd47 and mhc-i were identified as key players in determining llvs biocompatibility, while leukocyte associated function-1 (lfa-1) and mac-1 contributed to the llvs targeting ability and bioactivity towards inflamed endothelium [3] . recent investigation showed that the coating induced the formation of a singular protein corona (i.e. the protein adsorption layer) on the surface of the nanoparticles compared to negative control following in vivo injection. in addition, the proteolipid coating favored active extravasation of the llvs in the tumor vasculature by molecular mechanisms similar to those used by tumor infiltrating leukocytes. this work shows that is possible to transfer biologically active leukocyte membrane proteins onto synthetic nanoparticles, thus creating biomimetic carriers retaining cell-like functions that are not affected by the protein corona effect that occurs in vivo. the targeting of the inflamed endothelium can be applied to a broad range of diseases and the approach used to formulate the system could open new avenues for the fabrication of the next generation of personalized treatments by using as cell membrane source the immune cells of patients. references: [1] alessandro parodi, claudia corbo, armando cevenini, roberto molinaro, roberto palomba, laura pandolfi, marco agostini, francesco salvatore, ennio tasciotti. enabling cytoplasmic delivery and organelle targeting by surface modification of nanocarriers. nanomedicine uk. accepted. steroid hormone receptors are intracellular receptors that initiate signal transduction in response to steroid hormones, including oestrogen and androgens. generally, the binding of the steroid to the nuclear receptor induces the protein to form a dimer and relocate onto the chromatin, although the order of these events may vary. the location of receptor binding on the chromatin is defined by specific hormone response elements (hre). once located, the receptor promotes gene activation by the recruitment of other co-factors. it is this process that makes the complex of receptor protein and co-factors play a pivotal role in the regulation and activation of genes. the failure to regulate this process correctly is a key step in the development of several endocrine-driven cancers. for example: oestrogen receptor positive (er1) breast cancer is one of the most common forms of cancer and accounts for 70% of all breast cancer cases. in er1 tumours, the oestrogen receptor (er) drives the tumour growth and cell proliferation. understanding the interactions of the er with other proteins, either directly or indirectly, can provide vital insight to the regulation of the system that drives this cancer. the progesterone receptor (pr) has also been implicated in breast cancer, and the androgen receptor (ar) is a known driver in the majority of prostate cancers. to meet the challenges of elucidating these systems, we have developed methods to purify and analyse cross-linked regulatory complexes bound to dna by mass spectrometry (chip-ms). this allows for the enrichment of proteins involved in gene regulation. chip-ms, combined with tandem mass tags (tmt), makes it possible to realise a quantitative method to investigate the dynamic network of interactions between proteins within complexes that undertake the regulation of biological systems. chip-seq is a well-established method for identifying where these protein complexes are bound to the genome. this work focuses on how to combine these technologies with my previous development of cross-linking coupled mass spectrometry techniques (xcms) to provide a strategy for visualising the dynamic organisation of the proteins on the chromatin. global kinetic analysis of caspase protein substrates in cell lysate reveals selective roles and target specificity olivier julien 1 , min zhuang 1 , arun wiita 1 , james wells 1 1 caspases are cysteine proteases that play important roles in development, cell differentiation and cell death. however, the limited number of known caspase substrates hinders our understanding of caspase function. here we performed a non-biased identification and kinetic analysis of caspase-2 and caspase-6 proteolytic substrates in cell lysate, using an enzymatic n-termini enrichment approach followed by mass spectrometry. we identified 235 and 871 potential substrates for the initiator caspase-2 and putative executioner caspase-6, respectively. our results not only confirm known substrates but also identify many more new substrates with the precise location of proteolysis. given the emerging roles of caspases-2 and 26 in inflammation and neurodegeneration, these new substrates may provide molecular insight into the progression of related diseases. the sequence consensus logo of caspase-2 targets was very similar to a classical executioner caspase motif (devd), while caspase-6 revealed a vevd motif. using selected reaction monitoring (srm), we quantified the kinetics of proteolysis of a large subset of these substrates by measuring the appearance of the caspase cleavage product over time. in the end, we measured 50 and 276 kcat/km values for individual substrates cut by caspase-2 and caspase-6, respectively. by comparing these data with our previous analysis of caspase-3, 27, and 28, we found that substrates that are shared between caspases are often cleaved at rates that differ by orders of magnitude. thus, despite having nearly identical primary sequence motifs, the caspases exhibit remarkable substrate specificity that may reflect their specialized roles within the cell. the rockefeller university, 2 new york university school of medicine, 3 johns hopkins university school of medicine line-1 (l1) retrotransposons are catalysts of evolution and disease whose sequences comprise a significant proportion of the human genome. despite tremendous influence on genome composition, l1 rnas only encode two proteins. consequently, l1 particles include a combination of permissive host factors that are essential to their lifecycle as well as repressive factors that constitute defenses against l1's mutagenic activity. we previously characterized host proteins associated with synthetic and natural human l1 retrotransposons, as expressed in cell culture, using a combination of techniques including metabolic labeling and affinity proteomics. to build on these analyses, we have implemented a series of 2d separations and post-purification treatments to produce a multi-dimensional interactomic characterization of affinity isolated l1s. these studies have revealed the presence of at least two populations of putative transposition intermediates that may exhibit distinctive intracellular localizations. we report a comprehensive, quantitative survey of the proteins partitioning within these distinct l1 populations and their associated in vitro activity. our observations provide a basis for the classification of l1 interactors with respect to their physical and functional links, facilitating hypotheses to direct in vivo experimentation. polyubiquitin recognition by continuous ubiquitin binding domains of rad18 probed by modeling, small-angle x-ray scattering and mutagenesis sangho lee 1 , trung thanh thach 1 , namsoo lee 1 , donghyuk shin 1 , seungsu han 1 , gyuhee kim 1 , hongtae kim 1 1 rad18 is a key protein in double-strand break dna damage response (ddr) pathways by recognizing k63-linked polyubiquitylated chromatin proteins through its bipartite ubiquitin binding domains ubz and lrm with extra residues in between. rad18 binds k63-linked polyubiquitin chains as well as k48linked ones and mono-ubiquitin. however, the detailed molecular basis of polyubiquitin recognition by ubz and lrm remains unclear. here, we examined the interaction of rad18(201-240), including ubz and lrm, with linear polyubiquitin chains that are structurally similar to the k63-linked ones. rad18(201-240) binds linear polyubiquitin chains (ub2, ub3, ub4) with similar affinity to a k63-linked one for diubiquitin. ab initio modeling suggests that lrm and the extra residues at the c-terminus of ubz (residues 227-237) likely form a continuous helix, termed 'extended lr motif' (elrm). we obtained a molecular envelope for rad18 ubz-elrm:linear ub2 by small-angle x-ray scattering and derived a structural model for the complex. the rad18:linear ub2 model indicates that elrm enhances the binding of rad18 with linear polyubiquitin by contacting the proximal ubiquitin moiety. consistent with the structural analysis, mutational studies showed that residues in elrm affect binding with linear ub2, not monoubiquitin. in cell data support that elrm is crucial in rad18 localization to dna damage sites. specifically e227 seems to be the most critical in polyubiquitin binding and localization to nuclear foci. finally, we reveal that the ubiquitin-binding domains of rad18 bind linear ub2 more tightly than those of rap80, providing a quantitative basis for blockage of rap80 at dsb sites. taken together, our data demonstrate that rad18(201-240) forms continuous ubiquitin binding domains, comprising ubz and elrm, and provides a structural framework for polyubiquitin recognition by rad18 in the ddr pathway at a molecular level. optimization of a protein extraction method for the proteomic study of pozol cynthia teresa leyva-arguelles 1 , carmen wacher 2 , rosario vera 3 , romina rodr ıguez-sanoja 1 1 instituto de investigaciones biom edicas, unam., 2 facultad de qu ımica, unam., 3 instituto de biotecnolog ıa, unam key words: proteomics, fermentation, pozol pozol is a mexican traditional no alcoholic beverage elaborated by various ethnic groups in the southeastern of mexico. pozol is obtained from the natural fermentation of nixtamal (heat-and alkali-treated maize) dough. the main carbohydrate in maize dough is starch (72-73%), because others such as sucrose, glucose and fructose are mostly lost during nixtamalization; so, the starch remains as the major carbohydrate available for fermentation [1] . a wide variety of microorganisms have already been isolated from the fermentation of pozol; these microorganisms include fungi, yeasts, lactic acid bacteria, and non-lactic acid bacteria [2] . however, only few bacteria are amylolytic in this fermentation and all of them are weakly amylolytic [1] . in an attempt to explain how a very low content of soluble sugars can support a diverse and abundant microbiota, a proteomic approach was designed to understand the fermentation of pozol [3] . nevertheless, the extraction of proteins from pozol remains a limiting step in proteomic analysis mainly due to the complexity of the sample. on the basis of the aforementioned reasons, the aim of this work was to obtain a suitable extraction method of proteins for proteomic analysis. therefore, the fermentation of pozol was continued for 48 h and samples were taken at 0, 9, 24 and 48 h. for each sample, the total sugar content was determined by the dubois et al. method [4] and protein extraction was performed by two methods: a) direct extraction from the dough [3] and b) initial extraction of microorganisms and soluble proteins (this work). comparison between the two protein methods was performed on two-dimensional gels with silver stain. then, gels underwent to image analysis by the image master 2d platinum software. comparing the 2d-gels, more proteins spots were obtained with method b than that with method a, indicating a more efficient protein extraction with method b. although, using method a higher concentration of total proteins was observed, they were mostly maize proteins, that in turn overlap and reduce the efficiently extraction of the microbial low abundant proteins. then, method b allows a better extraction of those low abundant proteins and removes sample components that may interfere with the determination. these results could help us to find the proteins involved in carbohydrate metabolism of the microbiota and finally elucidate the dynamics of pozol fermentation. proteomics has been applied to the enology field for numerous purposes including fermentation control, improvement of fermentation processes, ensuring wine quality, etc. according to rodriguez et al., (2012), the information provided by wine proteomics is not only useful for these intentions, but also offers excellent prospects for innovation and diversification of winemaking processes in the near future. in this context, our group has focused research on the identification of proteins that might be important for yeast survival under typical wine elaboration conditions (standard fermentation, sherry wine biological aging and sparkling wine second fermentation) as well as proteins that configure the content of metabolites which are ultimately responsible for wine quality. by using novel proteomic (offgel fractionator and ltq orbitrap xl ms) and metabolomic techniques (sbse-td-gc-ms) we have identified a high amount of up-regulated proteins involved in processes like oxidative stress response (in biological aging) or protein biosynthesis (in second fermentation) as well as thirty-three proteins directly involved in the metabolism of glycerol, ethanol and seventeen aroma compounds excreted by the yeast under biological aging conditions. further, in order to validate proteome data; null mutants of genes codifying proteins up-regulated in the biological aging condition were constructed. analyses of correlated phenotypes are in progress. this technique and its combination with metabolomics within the enology context will provide enough knowledge to design or choose yeasts or conditions that satisfy wine production and/or wine characteristics such as color/aroma/texture/flavour profile demands of winemakers and consumers. additional binding sites for cytochrome c on its redox membrane partners facilitate its turnover and sliding mechanisms within respiratory supercomplexes blas moreno-beltr an 1 , antonio d ıaz-quintana 1 , katiuska gonz alez-arzola 1 , alejandra guerra-castellano 1 , adri an vel azquez-campoy 0 , miguel a. de la rosa 1 , irene d ıaz-moreno 1 1 ibvf, ciccartuja, universidad de sevilla -csic, 2 bifi -iqfr (csic), universidad de zaragoza, 3 departamento de bioqu ımica y biolog ıa molecular celular, universidad de zaragoza, 4 gliding mechanisms of cytochrome c (cc) molecules have been proposed to shuttle electrons between respiratory complexes iii and iv within plant and mammalian mitochondrial supercomplexes, instead of carrying electrons by random diffusion across the intermembrane bulk phase [1] [2] . in this work, the binding molecular mechanisms of the plant and human cc with mitochondrial complexes iii and iv have been analyzed by nuclear magnetic resonance and isothermal titration calorimetry. our data reveal that both cc-involving adducts possess a 2:1 stoichiometry -that is, two cc molecules per adduct -. the presence of extra binding sites for cc at the surfaces of complexes iii and iv opens new perspectives on the mitochondrial electron transport chain, where membrane respiratory complexes can be either in independent, free diffusional motion or forming macromolecular assemblies. in the latter context, such new binding sites for cc facilitate the turnover and sliding mechanisms of cc molecules within supercomplexes. indeed, the accommodation of several cc molecules between complexes iii and iv in supercomplexes provide a path for cc diffusion from complex iii to iv. such path could have physiological significance in the electron flow, which is controlled in supercomplexes to optimize the use of available substrates [3] [4] [5] . can bio-functionalities be deciphered from protein sequence information using computational approaches? background: the processes of uncovering bio-functionalities such as pharmacological activities, disease processes, physiological and structural properties by means of clinical approaches are irrational. this is because they are resource and time consuming. sometimes, they involve sophisticated and expensive equipments, reagents and animal tissues. contrarily, sequence information-based computerized approaches are rational and have become relevant in assessing bio-functionalities. they include geno2pheno [coreceptor] [1] , position-specific scoring matrix (pssmsi/nsi and pssmcxcr4/ccr5) [2] , and informational spectrum method (ism)-based phylogenetic analysis (istree) [3] . aim: this presentation demonstrates how bio-functionalities could be deciphered from sequence information using computational approaches. method: ism procedure and peptides, vipmfsals and capagfail are engaged. results: protein sequences of the peptides are converted into bio-functionality (affinity). affinity between the two peptides is demonstrated as significant amplitudes at the point of common interaction also referred to as consensus frequency, signifying remarkable affinity. discussions: bio-functionalities of bio-molecules are known to be expressed in one or two genes, which have been found to provide as much biological information as the bio-molecules. this indicates that biological characteristics, represented in these genes and proteins can now be extracted from their sequence information. for example, multi-drug resistances arising from a variety anti-microbial agent from several classes including alkaloids, flavonoids, etc can be retrieved from the sequence information of their encoding genes (mdr1 and mdr11). similarly, translation of hiv infection to aids disease can be extracted from the protein sequence alterations in the hiv gp120. similarly, effectiveness of anti-retroviral agent, maraviroc on the hiv isolate h2bx2 and ndk can be deciphered from the sequence information of their v3 observed at the predicted sequences. these positions are important as they surround the cleavage site in the three-dimensional structure, and are probably less tolerant to change. moreover in previous studies, cys at p1 position has been shown to be the dominant determinant for cleavage efficiency, while cys, pro and glu at p2 position have also been shown to be correlated with increased cleavage efficiency of ns3/4a protease. for adv2 cysteine protease, on the other hand, bsst produces similar significant results for both type 1 (xgx-g) and type 2 (xgg-x) consensus cleavage sites, where p2 and p1' positions have gly with highest percentage in type 1 (xgx-g) while p2 and p1 positions have gly in type 2 (xgg-x). these indicate that the bsst seems to provide a powerful methodology for predicting the substrate specificity for the hcv ns3/4a serine protease and adv2 cysteine protease, which are targets in drug discovery studies. protein plasticity improves protein-protein binding description chiara pallara 1 , juan fern andez-recio 1 1 an accurate description of protein-protein interactions at atomic level is fundamental to understand cellular processes. however the current structural coverage of protein-protein interactions (i.e. available experimental structures plus potential models based on homologous complex structures) is below 4% of the estimated number of possible complexes formed between human proteins.1,2 for these reasons, computational docking methods aim to become a complementary approach not only to solve the structural interactome but also to elucidate the basis of the protein-protein association mechanism. in spite of the advances in protein-protein binding description by docking, dealing with molecular flexibility is a major bottle-neck, as shown by the recent outcomes of the capri (critical assessment of prediction of interactions) experiment.3 this data clearly confirms that the protein dynamics plays a key role in protein-protein association. the use of conformational ensembles generated from unbound protein structures in combination with computational docking simulations might represent a more realistic description of protein-protein association. here, we present the first systematic study about the use of precomputed unbound ensembles in docking, as performed on a set of 124 cases of the protein-protein docking benchmark 3.0.4 the primary aim of our work is to understand the role of the protein conformational heterogeneity in protein-protein recognition. to do this, small conformational ensembles were automatically generated starting from the unbound docking partners, and then an extensive analysis of their binding properties was performed in the context of pydock docking scheme. 5 the results show that considering conformational heterogeneity of interacting proteins can improve docking description in cases that involve intermediate conformational changes in the unbound-to-bound transition. more interestingly, we found that protein plasticity increases chances of finding conformations with better binding energy, not necessarily related to bound geometries. the relevance for future docking methodology development and for understanding protein association mechanism will be discussed. purpose of the research: there is increasing interest in the development of protein scaffolds that can be used to develop affinity reagents that are alternatives to antibodies. the affimer scaffold is based on the cystatin protein fold. the affimer scaffold is biologically inert, biophysically stable and capable of presenting a range of designed or random binding surfaces defined by peptides inserted at 2 different loops. the result is highly specific, high affinity interactions with a wide range of targets including ones that are inaccessible to antibodies. affimers are designed to work in the same way as the very best antibodies, but with a number of key advantages. affimers are quick to develop (typically 7 weeks) without using animals. they contain no disulphide bonds, are expressed easily in e. coli and have no batch to batch variability. affimers are small molecules (108 aa, 12 kda), robust and stable (resistant to ph range, thermally stable and not sensitive to edta). affimers can be a direct replacement for antibodies -no process or workflow change required -and perform identically to antibodies in assays such as elisa, facs, ihc, western blots, affinity purification, microarray and potentially therapeutics. we describe some applications of the technology in regards of affimer development for custom targets on one hand and for the biomarker discovery workflow using affimer microarrays on the other. main results: by screening of our very large (3 x 1010) library against yeast sumo protein we identified affimers with high affinity allowing their use for elisa. moreover, no cross-reactivity was observed when affimers were used on western blots leading to a unique band specific to yeast sumo when compared to human proteins. a library of 25,000 random affimers, expressed in e. coli, was printed on glass microscope slides and challenged with plasma from children (n5104) with sepsis and from healthy children (n524). unsupervised hierarchical clustering based on the 25,000 affimers allowed differentiation between the control and patient samples. 200 affimers were found to differentially bind proteins between the 2 groups with a > 2 fold change. the affimer arrays identified a strong signature of sepsis and roc curve analysis allowed confident prediction of disease (auroc of 0.9). affinity purification and preliminary mass spectrometry analysis identified known biomarkers of sepsis and also potentially novel biomarkers not previously associated with this disease. major conclusions: this work demonstrates the scope of affimer affinity reagents to develop alternative binders to antibodies, where affimers perform identically in most assays without the disadvantages associated with antibodies. moreover, affimers enable a new protein microarray-based biomarker-discovery workflow and we predict that array-based validation of signatures identified using discovery arrays prior to affinity purification and mass spectrometry will offer a cost-and time-effective methodology compared to purely mass spec-driven workflows. tau pathologies, called 'tauopathies', are related to several neurodegenerative diseases including alzheimer disease (ad). in ad, tau protein is observed hyper-phosphorylated and aggregated as paired helical filament (phf). the neuronal tau protein is an intrinsically disordered proteins (idps). nuclear magnetic resonance spectroscopy (nmr) is here used to study the tau protein phosphorylations and protein-protein interactions (ppis). in in vitro assays, tau phosphorylation by rat brain extract is considered as an hyperphosphorylation model that was furthermore pointed out to enable tau aggregation [1] . in a first step, we have identified all the phosphorylation sites of rat brain extract phosphorylated-tau, using the analytical capacity of nmr. we showed that the protein is modified at 20 ser/thr sites. among the kinases that we have characterized so far using tau as substrate, only the extracellular signal-regulated kinase2 (erk2) shows an ability to modify in vitro tau protein on so many sites. we have indeed identified 14 phosphorylated ser/thr-pro motifs out of 18 potential phosphorylation sites in the sequence of full length 441-residue tau. in addition, we showed using transmission electron microscope (tem) a similar in vitro aggregation capacity of erk-phosphorylated tau protein compared to that of rat brain extract phosphorylated-tau. this shows that phosphorylation by the erk kinase generates an hyperphosphorylated tau. given the high efficiency of erk towards tau, we have next looked into the mechanism of tau recognition. erk kinase possesses two well-characterized docking domains: d recruitment sites (drs) and f recruitment sites (frs), which recruit complementary docking sites and increase the specificity and efficiency of the interaction with both its upstream regulators and downstream substrates [3] . as the interaction between tau protein and erk2 kinase is analyzed by nmr spectroscopy, multiple sites of interaction are observed along the tau sequence, similar to drs docking sites, all located in the so-called microtubule binding domain of tau. these sites are short sequences loosely matching the reported consensus for d sites w1-3uxu (w, u, and x refer to positively charged, hydrophobic, or any intervening residues, respectively) [3] , and also the reverse sequence uxuw 1-3.to confirm the mapping of the interaction, two tau recognition sites were produced as recombinant peptides of about 20 amino-acid in fusion with an n-terminal his-tag sumo. interaction assays using 2d [1h, 15n] hsqc spectra of the peptides confirm their binding to erk kinase. the potential of these peptides to inhibit erk activity with tau as substrate is now being investigated. while rigid-body docking has become quite successful for predicting the correct conformations of binary protein complexes, determining whether two given proteins interact remains a difficult problem. successful docking procedures often give equally good scores for pairs of proteins for which there is no evidence of interaction. studies investigating what we define as the 'pre-docking' problem via in silico approaches have only recently become feasible with the help of supercomputers and gridcomputing systems. in a previous work, on a restricted set of protein complexes, we showed how predictions of interacting partners could be greatly improved if the location of the correct binding interface on each protein was known. experimentally identified complexes are found to be much more likely to bring these two interfaces into contact, at the same time as yielding good interaction energies. we present data from a complete cross-docking (cc-d) study of a database of 168 proteins, including the treatment of more than 14,000 potential binary interactions. the performance of the interaction index we developed to predict binding probability compares well with other methods. by studying the interaction of all potential protein pairs within a dataset, cc-d calculations can also help to identify correct protein interaction interfaces. the present large-scale study also reveals the influence of various protein families (enzyme-inhibitor, antibody-antigen, antigen-bound antibody, etc.) on binding specificity, showing, in particular, the distinctive behavior of antigenic interfaces compared to enzymes, inhibitors or antibodies. the performance of our approach is encouraging. although identifying interaction interfaces significantly helps in the identification of interacting proteins, further refinements will be necessary to make in silico cross-docking a viable alternative to high-throughput experimental methods. whole-protein mass spectrometry reveals global changes to histone modification patterns in hypoxia sarah wilkins 1 , kuo-feng hsu 1 , christopher schofield 1 1 chemistry research laboratory, oxford university cells respond to limiting oxygen availability (hypoxia) by altering the gene expression profile. this primarily involves changes at the level of transcription via the activity of hypoxia-responsive transcription factors, although increasing evidence suggests that changes in chromatin structure (i.e. from a condensed 'silent' state to a more open or 'active' state) are required in order for transcription to take place. in particular, post-translational modifications (ptms) to histones have an important regulatory function in gene expression under hypoxic conditions. the n-terminal tails of histone proteins are accessible to a set of enzymes capable of 'writing' and 'erasing' ptms including acetylation, methylation, ubiquitylation, sumoylation and phosphorylation. to date, studies in hypoxia have employed antibody-based methods to investigate changes in histone modifications, and so have focused on individual marks in isolation. the interplay between coexisting ptms is thought to be much more important than the effect of any single mark. therefore, a global view of the histone modification profile is essential to gain a complete understanding of the function of histone ptms and their roles in gene regulation. in this study, we apply whole protein mass spectrometry to investigate hypoxia-induced changes in histone marks. this 'top-down' approach provides insight into combinational modification patterns that are difficult to establish by antibody-based methods or peptide ms analysis. we investigated changes in the global ptm profiles of histones from a range of human cell-lines and tissues under severe hypoxia (<0.1% o2). we find that hypoxia causes a shift in the overall profile towards a more highly modified state, with significant changes in methylation and phosphorylation. marked changes in histone ptms were also observed following treatment of cells with epigenetic inhibitors and commonly used hypoxia mimetics, including several iron chelators currently in clinical trials for the treatment of anaemia. finally, we show that this method can be used to identify the histone variant h2ax, whose phosphorylation at serine 139 is an indicator of double-stranded dna breaks in cancer. overall, these data provide important insights into the epigenetic changes associated with hypoxia in normal and disease contexts. we hope to further develop this method in combination with different labelling strategies to enable quantitative analysis of histonemodifications in cells. mass spectrometry-based protein biomarker discovery in neurodevelopmental disorders interactions. there is currently no biological diagnosis or known cause of asd. slos is characterized by a cholesterol deficiency due to a mutation on the 7dhcr gene. approximately 1/20,000 babies are born with slos. diagnosis is achieved by measuring cholesterol and 7-dehydrocholesterol (7dhc) levels in the blood, however, there is currently no proven treatment for slos. because of this, research is increasing to determine biomarkers for these disorders. here, samples from people with asd (sera and saliva) and slos (saliva), and matched controls were analyzed using a combination of gel electrophoresis (tricine-page, sds-page and blue native page), in gel digestion or insolution digestion and nanoliquid chromatography-tandem mass spectrometry (nanolc-ms/ms) to investigate differences between the proteomes of people with these neurodevelopmental disorders and matched controls. several alterations in protein expression were identified. these differences may lead to potential biomarkers for diagnosis, possible therapeutic targets and an altogether better understanding of the disorders. understanding protein recognition using structural features protein-protein interactions (ppis) play a crucial role in virtually all cell processes. thus, understanding the molecular mechanism of protein recognition is a critical challenge in molecular biology. previous works in this field show that not only the binding region but also the rest of the protein is involved in the interaction, suggesting a funnel-like recognition model as responsible of facilitating the interacting process. further more, we have previously shown that three-dimensional local structural features (groups of protein loops) define characteristic patterns (interaction signatures) that can be used to predict whether two proteins will interact or not. a notable trait of this prediction system is that interaction signatures can be denoted as favouring or disfavouring depending on their role on the promotion of the molecular binding. here, we use such features in order to determine differences between the binding interface and the rest of the protein surface in known ppis. particularly, we study computationally three different groups of protein-protein interfaces: i) native interfaces (the actual binding patches of the interacting pairs), ii) partial interfaces (the docking between a binding patch and a non-interacting patch), and iii) back-to-back interfaces (the docking between non-interacting patches for both of the interacting proteins). our results show that the interaction signatures in partial interfaces are much less favoured than the ones observed in native and back-to-back interfaces. we hypothesise that this phenomenon is related to the dynamics of the molecular association process. back-to-back interfaces preserve the exposure of the real interacting patches (thus, allowing the formation of a native interface), while in a partial interface one interacting patch is sequestered and becomes unavailable to form a native interaction. structural characterization of the cytoplasmic mrna export platform laboratory of cellular and structural biology, the rockefeller university., 2 laboratory of mass spectrometry and gaseous ion chemistry, the rockefeller univ., 3 university of california, san francisco, 4 the new york structural biology center, 5 department of biochemistry, faculty of medicine, university of montreal mrna biogenesis is an intricate process that begins within the nucleus and culminates with the remodeling and nuclear export of the mrnp particles through the nuclear pore complex (npc). defects in this conserved mechanism have been shown to cause serious human diseases. the protein assembly that performs the last steps in mrnp biogenesis and export is located at the cytoplasmic face of the npc and is formed by 14 different proteins, organized into several subcomplexes whose arrangement and molecular architecture are poorly understood. in this study we applied an integrative approach, combining cross-linking and mass spectrometry (cx-ms), electron microscopy and available high-resolution structures, to describe the molecular architecture of the endogenous npc cytoplasmic mrnp export machinery. we generate a hybrid, close-to-atomic structure of the yeast native nup82 complex, the core of the assembly. our map also reveals how the nup82 complex organizes the entire cytoplasmic mrnp export machinery, and how this in turn docks into the architectural core of the npc. mapping of phenotypic profiles into our structures allows us to generate a first functional map of the ensemble. we expect that our map will serve as a framework to understand the molecular mechanisms underlying this key step of mrnp biogenesis. study of candidate proteins to pore associated with p2x7 receptor in different cell types carla oliveira 1 , anael alberto 1 , mônica freitas 2 , luiz alves 1 1 laborat orio de comunicac¸ão celular -fiocruz, 2 centro nacional de ressonância magn etica nuclear -ufrj aim: the p2x7r is a purinergic receptor, which differs from others subtypes due to its structural and pharmacological characteristics. when exposed for extended time or to high concentrations of its agonist (atp), promotes an increase in membrane permeability, allowing the passage of molecules up to 900da. there is a controversy among several authors that leave in doubt if this receptor needs a second protein for the pore formation and which protein could be. we select five pore-forming proteins: trpv1, trpa1, connexins-43 (cx-43), pannexin-1 (panx-1) and vdac. we believe that different mechanisms and proteins could be associated with p2x7r, depending on the cell type and their microenvironment stimuli. in this context, our main goal is identify possible proteins that could be associated with the p2x7r pore in different cells and species. methods and results: we started with rt-pcr technique of cell lines: j774.g8, n2a, u373, u937, hek-293 and primary cells from wistar mouse and swiss mice. we used different primers and pcr cycle for each target at different species. we observed that the p2x7, panx-1 and cx-43 are the most abundant and are present in all cell types except the absence of p2x7 in u373 cells and panx-1 in mice macrophages and u373 cells (n>3). however, trpv1 was seen at n2a and u937 cells and trpa1 in and primary cells from mouse and mice and in j774.g8 cells (n>3). regarding to the vdac, it is present in mouse macrophages, j774.g8 and hek-293 cells (n>3). the further steps, we verified if those proteins could be physically associated with the p2x7r. we coimmunoprecipitated the p2x7r of j774.g8 (with or without atp), mice macrophages, hek-293 and u937 cells. the samples were applied in two separated 12.5% bis/acrylamide gels: one destined to mass spectrometry (ms) and the other to western blot. at this point, we confirmed the presence of p2x7r, and observed several others proteins associated to p2x7r at different cell conditions, mainly when we exposed, j774.g8 cell, to 5 mm atp (n53). at this condition, we found by ms, hsp70, 75, and 90; alpha and b tubulin; myosin va; alpha, b and g actin; malate and lactate dehydrogenase (n51). although u977 and hek-293 had not received atp treatment, we found several proteins associated to p2x7. the next step was to immunoprecipitated those proteins in j774.g8 (treated or not with atp) and use it to verify if p2x7 are physically associated to them. as result we saw the p2x7 associated to panx-1 in j774.g8 cells. conclusion: we conclude that the p2x7r activated by extracellular atp triggers the recruitment of variety different proteins. at this condition, we can suggest that maybe there is a conformational change, regardless of the numerous recruitment structural proteins. in addition, apparently, the pore-forming protein pannexin-1 is associated with p2x7r, and the others pore forming proteins (vdac, cx-43, trpv1, trpa1) seems not be linked to p2x7r at j774. recently, we developed a series of molecular modeling tools for structure-based studies of protein functions and interactions. these tools are publicly available as web servers that are easily operated even by non-specialists: cabs-fold server for protein structure prediction [1] ; cabs-flex server for modeling of protein structure flexibility [2] ; aggrescan3d server for prediction of protein aggregation propensities and rational design of protein solubility [3] ; and cabs-dock server for prediction of peptide binding sites and peptide docking [4] . the web servers are freely available from the laboratory website: http://biocomp.chem.uw.edu.pl/tools sandy on 1 , pinghui feng 2 1 university of southern california, keck school of medicine, 2 developing a technique to detect deamidated proteins and peptides using rig-i sandy on, pinghui feng university of southern california, norris comprehensive cancer center, department of microbiology, and molecular biology, los angeles ca perhaps the most notable type of post-translational modification of proteins and peptides into a higher order structure is deamidation of asparagine and glutamine. deamidation occurs when an amine group is removed, degrading the molecule for purpose of regulating intracellular levels. previous studies have demonstrated that this notable post translational modification has been uncovered over time for use in dna recombinant technology as well as use as a biological clock to facilitate the rapid turnover of biologically important components of the cell. while the effects of this non-enzymatic chemical reaction have been widely studied, the method to uncover modification sites over a large quantity of proteins remains an issue. one of the most common types of deamidation is of asparagine and glutamine residues. at this time, most researchers will depend on mass spectrometric based proteomic techniques for identification of these post-translational sites. the issue is that mass spectral analysis of deamidated proteins and peptides is complication and can lead to misassigned identification attributed by an overlapping of 13c peak of the amidated form with the deamidated monoisotopic peak; these two peaks are only separated by 19.34 mda. while these issues can be mediated by using a mass spectrometer with a high mass measurement accuracy, and high resolving power, it is essential to establish simpler methods for identifying substrates that have undergone deamidation. if deamidation is present, different protein bands will be exhibited in the western blot, which will be compared to a triple mutant rig-i, which resists deamidation, to observe the location of this modification on the protein. with enough testing, i will determine specific sites of digestion and use this information to make conclusions of unknown proteins. i will make results regarding whether the protein has been modified based on the digestion sites. i will use mass spectrophotometry analysis to compare the proteins on a wider scale and double check my results. i have narrowed it down to a couple of different digestion sites that indicate deamidation. though the analysis work can be tedious, it is crucial to ensure the sites we isolate are accurate in order to establish this technique. from my research, we can apply this method for wider scale use such as in clinical settings. in areas of inflammation of parkinson's' patients, we can review specifically the infected cells versus uninfected and isolate the proteins, usually deamidated, responsible often smaller in size and more specific. in addition, research articles have already shown that suppressing modification of certain cells such as bcl-xl playing a major in leading the regulation of cancer cell death by apoptosis. by leading the discovery of a simpler methods to uncovering deamidation in cells, researchers will more easily and quickly be able to scan through various proteins, some of which discovered eventually may play pivotal roles in cancer research. influenza virus (iv) hemagglutinin (ha) is a homotrimeric integral membrane glycoprotein that mediates receptor-binding and membrane fusion. it constitutes the prominent viral surface antigen and a main target for neutralizing antibodies. bacterial, recombinant ha-based vaccines indicate high potential to confer protection against highly pathogenic (hp) avian iv (aiv) h5n1 and arise as alternative for the traditional egg-or cell culture-based manufacturing. relatively short time of bacterial has production can be of great importance in case of a pandemic. escherichia coli produced protein, based on the ha sequence of a/swan/poland/305-135v08/2006(h5n1) hpaiv*, has been successfully expressed in the form of inclusion bodies at institute of biotechnology and antibiotics. refolded and purified antigen was obtained in a soluble form, isolated by reversed phase hplc and identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry (maldi tof/tof ms). the performed research in a great extent allowed to confirm the amino acid sequence of the recombinant ha (rha) assumed based on the cdna and allowed to establish the location of a total of six disulfide bridges. however, during purification and storage of the rha, apart from desired higher order rosette-like structures of the protein, other non-native species resulting from posttranslational modifications, misfolding, aggregation and degradation may occur what results in reduced vaccine potency. here, besides the properly folded monomers, we indicate non-native aggregates induced by disulfide crosslinking. moreover, several free cysteine residues and unexpected intrachain s-s were identified in rha tryptic peptide maps. cys 43 was found most susceptible to formation of disulfide bridges between the distinct chains of rha. the above findings allow to assume that not all rha particles fold to form the native structure. reduced cys residues exhibit tendency to undergo oxidation and unconnew strategies and approaches to understand how antibodies recognize and neutralize snake toxins represent a challenge to improve the antivenoms. the neurotoxic activity of micrurus venom is carried majority by two distinct proteins families, 3ftx and pla2. the conserved structural folding of these toxins can be appreciated as model to generate inhibitors against them. in this regard, monoclonal antibodies (mabs) can be used as tool to find hot spots for inhibit the toxins and represent the first step in order to develop recombinant neutralizing molecules. in this work our goals were analyse a set of monoclonal antibodies against the most toxic components of m. altirostris venom by proteomics approaches. the venom was fractionated; its major toxic proteins identified by in vivo tests based on murine lethal toxicity analyses (approved by the ethical committee for animal experimentation from center of health and science of the federal university of rio de janeiro -no. 01200.001568/2013-87). the toxic components were used to generate a panel of five monoclonal antibodies. elisa and antivenomics results allowed us identify the specificity of all mab and their neutralizing efficacy was measured by in vitro tests. three mabs showed reactivity towards 3ftx and two against pla2. all monoclonal antibodies against 3ftx lack a broad recognition. however, we identified a pair of monoclonal antibodies able to recognize all pla2 molecules of m. altirostris venom and showed a synergism to inhibit the catalytic activity of them. moreover, we challenge monoclonal antibodies against to micrurus venom for inhibit the pla2 activity of naja naja, specie taxonomically out of micrurus cluster. our results showed that pla2 of m. altirostris venom share a pair of conserved antigenic regions and draw attention to use these epitopes to miming antigen to generate antibodies for antivenom production. moreover, face to the cross reactivity and the pla2 activity inhibition capability by mabs towards the naja naja venom, our results highlight the conservation of neutralizing epitopes across the elapidae family. protein-protein interactions are known to play key roles in the most important cellular and biological processes such as signaling, metabolism, and trafficking. one major goal of structural biology is the structural characterization of all protein complexes in human and other organisms. these efforts can be complemented by computational approaches. in this context, computational docking attempts to predict the structure of complexes from their monomeric constituents. the docking problem presents two main challenges: the generation of structural poses or sampling, and the identification of the correct structures with a scoring function (sf). docking methods can be successful if the interacting partners undergo small conformational changes. however, in a general situation, these algorithms generate a large number of incorrect predictions, and therefore the predictive success strongly depends on the accuracy of the sf used to evaluate the docked conformations. a variety of strategies have been developed to score putative protein-protein docked complexes. they are usually based on atomic level potentials, residue level potentials, or a combination of both. in current work, we have evaluated 73 different sf, taken from cchappi server, on the results of 3 different rigid body docking methods, ftdock, zdock, and sdock, using the docking benchmark 4.0 and a docking set built from capri scorers experiment. our results show 9 sf that showed better or similar success rate than the in-built sf. some of these sf increase the docking success rates especially for flexible or weak-binding cases, which are the most challenging for docking. 6 of them are residue level sf robust enough to detected solutions in cases with large conformation change. in particular we found two sf that shows outstanding robustness, one designed for protein modeling and shared among docking methods, and the other is for protein docking which is also the best success rate in the top100 ranking in the capri scorers set. the other 3 atomic level sf display high success rate to find a solution within weak binding proteins. the 2 most successful sf are shared between the docking methods and display high success rate in the hard cases of the benchmark 4.0 and in the capri scorers set. the difference between them in the resolution level at which they work, one being atomistic the other residue-based. we found that they success rate vary according to the docking method chosen, allowing them to explode different properties of the sampling used. this way to characterize a protein complex can help to develop new combined scoring functions in protein docking or a new ranking strategy to enhance the success rate. multi-ptk antibody: a powerful tool to detect a wide variety of protein tyrosine kinases (ptks) isamu kameshita 1 , noriyuki sueyoshi 1 , yasunori sugiyama 1 1 the eukaryotic protein kinases consist of large families of homologous proteins and play pivotal roles in various cellular functions. these enzymes are classified into two major groups; protein serine/threonine kinases and protein tyrosine kinases (ptks). ptks are believed to be involved in various cellular events such as cell cycle, proliferation, differentiation, apoptosis, and cell adhesion in multicellular eukaryotes. as many as 90 ptk genes have been identified in the human genome and many of these ptks are known to be closely correlated with various diseases such as cancer. therefore, it is important to elucidate the expression profiles of the entire ptk family in cells and tissues. to investigate the expression profiles of the cellular ptks, we produced an antibody that detects a wide variety of ptks. for production of the antibody, antigenic peptides corresponding to amino acid sequences of a highly conserved region (subdomain vib) of ptks were synthesized and immunized to balb/c mice. among various antigens, a peptide with 11 amino acids, cyvhrdlraan, efficiently produced a polyclonal antibody with a broad reactivity to ptks. we established a hybridoma cell line producing a monoclonal antibody, yk34, which appeared to cross-react with various ptks. at least 68 ptks could be detected by yk34 antibody, as evidenced by its reactivity with the recombinant src tyrosine kinases whose subdomain vib had been replaced by those of the other ptks. when differentiated hl-60 cells were analyzed by western blotting after two-dimensional electrophoresis with yk34 antibody, we observed significant changes in the immunoreactive spots in hl-60 cell extracts along with the changes in the morphology of the cells. these results suggest that the multi-ptk antibody, yk34, will be a powerful tool for the analysis of a variety of cellular ptks. analysis of the siglec-9 and hvap-1 interactions leonor carvalho 1 , vimal parkash 1 , heli elovaara 2 , sirpa jalkanen 2 , xiang-guo li 4 , tiina salminen 1 1 structural bionformatics laboratory, department of biosciences, 2 medicity research laboratory, 3 department of pharmacology, drug development and therapeutics, 4 sialic acid-binding immunoglobulin (ig)-like lectins (siglec) are type i transmembrane proteins. siglec-9 has an n-terminal v-set domain followed by two c2-set domains in the extracellular region. it contains an immunoreceptor tyrosinebased inhibitory motif (itims) in its cytoplasmic tail and can function as an inhibitory receptor by dampening the tyrosine kinase-driven signaling pathways. these proteins are expressed primarily on leukocyte subsets and, thus, are thought to be involved in regulation of leukocyte functions during inflammatory and immune responses. recently, phage display screening experiments identified siglec-9 as leukocyte surface ligand for human vascular adhesion protein 21 (hvap-1; aoc3 gene product) and their interaction was confirmed by cell adhesion and enzymatic assays (kivi et al., 2009; aalto et al., 2011) . based on our preliminary data, hvap-1 sugar units with sialic acid (sa) might mediate interactions with the v-set domain in siglec-9. furthermore, it is known that the siglec peptides binding to hvap-1 are located in the ce loop of the second c22-set of domain (siglec-9_c22). based on current hypothesis an arginine in siglec-9_c22 interacts with the tpq residue in the active site of hvap-1. the ce loop of siglec-9_c22 has two arginines (r284 and r290) and, therefore, the interacting arginine is unclear. we will now study the interaction mode of hvap-1 and siglec-9 in silico to predict the role of the arginines in the c22 domain and the role of sa-binding using the 3d model of the full-length ectodomain of siglec-9 and the hvap-1 crystal structure. the in silico analysis will be conducted in parallel with experimental site-specific mutational studies and the result will be combined to elucidate the mechanism of hvap-1-siglec-9 interaction. adam middleton 1 , catherine day 1 1 attachment of ubiquitin to substrate proteins regulates almost all cellular processes, including protein degradation and cell division. ubiquitylation involves a cascade of three families of proteins: ubiquitin activating (e1), ubiquitin conjugating (e2) and ubiquitin ligase (e3) enzymes. the 8.5 kda protein can be attached as a monomeric moiety or as a polyubiquitin chain, and the type of modification spells out the 'ubiquitin code' that directs the fate of the substrate. polyubiquitin chains can be formed via eight different linkage types, and the arrangement of chain formation is typically directed by the e2 enzymes. forming a polyubiquitin chain involves binding of two molecules to the e2: the donor (ubd) and acceptor (uba) ubiquitin. ubd is linked to the e2 via a thioester bond between its c-terminal gly and the active site cys of the e2, and when primed for catalysis it interacts with a particular face of the e2. in contrast, coordination of uba by e2s is transient and cannot be easily measured; however, uba binding defines the linkage type of polyubiquitin chains. the e2, ube2k, directs lys48 chain synthesis, which results in modified proteins being degraded by the proteasome. we generated a stable form of the ube2kub conjugate and crystallized it, and showed that both ube2k and its ubiquitin conjugate are monomeric. using molecular docking, we modelled the position of both ubd and uba and investigated the interfaces with site-directed mutagenesis. these experiments led to a molecular model that revealed how ube2k can synthesise lys48-linked ubiquitin chains. this molecular explanation provides a foundation for understanding how other e2s generate lys48-linked polyubiquitin chains. the two chromophorylated linkers of r-phycoerythrin in gracilaria chilensis marta bunster, francisco lobos-gonz alez, jos e aleikar v asquez, carola bruna, jos e mart ınez-oyanedl 1 fac de cs biol., universidad de concepci on the two chromophorylated linkers of r-phycoerythrin in gracilaria chilensis. francisco lobos-gonz alez, jos e aleikar v asquez, carola bruna, jos e mart ınez-oyanedel, marta bunster. departamento de bioqu ımica y biolog ıa molecular, facultad de ciencias biol ogicas, universidad de concepci on. phycoerythrin is a phycobiliprotein present in phycobilisomes in gracilaria chilensis as a complex with chromophorylated linker proteins. our interest is to discover the role of these linkers in the function of phycobilisomes. phycobilisomes(pbp) are auxiliary light harvesting protein complexes in charge of channeling energy towards photosystem ii in alga, cyanobacteria and cryptophyta. this is possible thanks to fluorescent proteins called phycobiliproteins (pbp) and the chromophores (phycobilins, open-chain tetrapyrrols) attached to specific cysteines. phycobiliproteins share a common general structure; they are organized as (alfab) heterodimers which themselves assemble as trimers(alfab)3 or hexamers (alfab)6; this complexes are organized in high order structures to form the core and the rods. besides pbps, pbs have linker proteins in charge of the assembly and stabilization of the complex, and also it has been proposed that they collaborate in the fine tuning of the energy transfer steps between chromophores. these linkers are located within the rods, the rod-core interface, the core and the core-membrane interface. although most linker proteins are colorless, chromophore bearing linkers have been described, which suggest its participation in the energy transfer process. two of them, g 31 and g 33 are associated to r-phycoerythrin in gracilaria chilensis, nevertheless the information available on these linkers in eukaryots is still limited. to understand how these linkers collaborate with the function of the phycobilisome, we need structural information, especially the coordinates of all the chromophores present in the complex; we have sequenced both linkers from the genomic dna, performed sequence analysis and also we have purified the linkers by anion exchange, molecular sieve and hpl chromatography. the characterization was performed by denaturant electrophoresis, absorption and emission spectroscopy and by mass spectrometry. the results show that they have molecular masses as predicted, with a peptide signal for chloroplasts, an internal sequence repeat; residues 67 -170 with residues 179 -273 for g 31 and residues 107-200 with residues 219-315 for g 33, and the presence of conserved cysteine residues putative sites of chromophorylation. the spectroscopy shows that they have different composition of phycobilins and a very short t1/2. a preliminary model for both linkers shows that they belong to aa structural class and that they share a common fold (heat like motifs) frequently involved in protein-protein interactions. dept. of phys., chuo univ., 2 grad. sch. of inform. sci. and eng., tokyo tech, 3 rigid-body docking algorithms are useful for predicting tertiary structures of near-native protein complexes. however, this algorithms generate many protein complex poses including false positives. then, near-native poses are searched in a post-docking process. there are many computational softwares with rigid-body docking algorithms, for example, zdock. we developed a high-performance protein-protein interaction prediction software, megadock, which is basically used on supercomputing environments for a large scale and network level in this work, we then tried to use these docking softwares and the profile method for understanding mechanisms of protein-protein interactions. we focused on some physicochemical properties, electrostatic and hydrophobicity, of a set of protein complex poses generated by a rigid-body docking process. from these poses, we obtained sets of possible interacting amino acid pairs. a set of interaction profiles has some information of docking spaces. from the view of a network prediction, the docking spaces of a set of protein complex poses are one of the properties for discriminating native protein-protein pairs from non-native pairs. in this work, ensemble docking process is performed by megadock ver. 4.0 and zdock ver. 3.0.1. cluster analysis is used with profiles of physicochemical properties. we used a dataset composed of typical 44 monomer-monomer protein pairs and will discuss mainly differences between native and non-native protein pairs. the structural studies of the two thermostable laccases from the white-rot fungi pycnoporus sanguineus marta orlikowska 1 , grzegorz bujacz 1 1 institute of technical biochemistry, lodz university of technology, poland laccases (ec 1.10.3.2, benzenodiol oxygen oxidoreductases) are enzymes that have the ability to catalyze the oxidation a wide spectrum of phenolic compounds with the four-electron reduction of molecular oxygen to water [1] . it has been found that the active site is well conserved in between laccases from different organisms. it contains four copper atoms: one paramagnetic type 1 cooper (t1) that is responsible for their characteristic blue color and where the oxidation of the reducing substrate occurs, one type 2 cooper (t2) and two type 3 coopers (t3) that conform a trinuclear cluster in which molecular oxygen is reduced to two molecules of water [2] . laccases are present in many different species and they have been isolated from plants, fungi, prokaryotes, and arthropods in most cases laccases are monomeric glycoproteins of around 500 amino acids with molecular weights in the range of 60-85 kda. the various functions carried out by those enzymes include the antagonistic ones such as their involvement in lignin biosynthesis (in plants), lignin degradation, pigment production, fruiting body formation, pathogenesis (in fungi) and spore protection against uv light (in bacteria) [1, 3] . the diversified functions of laccases make them an interesting enzyme for study from the point of view of their structure, function and application. laccases of white-rot fungi (wrf) are of special interest because one of its role is to degrade lignin and most of them are extracellular enzymes helping purification procedures [1] . during the last two decades, there has been an increasing interest in the genus pycnoporus for its ability to overproduce high redox potential laccases as the ligninolytic enzymes. we present the crystal structures of two thermostable lacasses produced by strain pycnoporus sanguineus cs43 (laci and lacii). the molecular weights of laci and lacii, determined by sds-electrophoresis, is 68 and 66 kda, respectively [3] . both isoforms shows high amino acids sequence similarity (91%) between them and high thermal stability, at 508c and 608c. they remained active at high concentration of organic solvent (acetonitrile, ethanol or acetone). the unique properties make them promising candidates for industrial applications in wasterwater treatment. laci exerted a higher thermal and ph stability, tolerance against inhibitors and was a more efficient catalyst for abts and dmp (laccases substrate) then lacii [3] . based on the structures we would like to understand the isoforms differences that confers laci a markedly better performance than lacii in ph and thermal stability as well as better resistance to inhibitors. analysis of liver proteome in cystathionine ß-synthase deficient mice using 2d ief/sds-page gel electrophoresis, maldi-tof mass spectrometry, and label-free based relative quantitative proteomics izabela bieli nska 1 , łukasz marczak 1 , hieronim jakubowski 1,2 1 institute of bioorganic chemistry, polish academy of sciences, 2 rutgers university, new jersey medical school homocysteine (hcy) arises from the metabolism of the essential dietary protein amino acid methionine. levels of hcy are regulated by remethylation to met and transsulfuration to cys. cystathionine bsynthase (cbs) catalyzes the conversion of homocysteine to cystathionine (first step of transsulfuration reaction). human cbs deficiency is a recessive inborn error of homocysteine metabolism that casues severe hyperhomocysteinemia (hhcy) and diverse clinical manifestations, including fatty liver disease [1] . although the causes of fatty liver disease in cbs deficiency have been studied the underlying mechanism is not understood. we hypothesize that cbs deficiency induces changes in gene expression that could impair liver homeostasis. to test this hypothesis and gain insight into hepatic functions of cbs we analyzed the liver proteome of cbs -/-and cbs 1/1 mice [2,3] using 2d ief/sds-page gel electrophoresis and maldi-tof mass spectrometry (n514) we identified twelve liver proteins whose expression was significantly altered as a result of the cbs gene inactivation. expression of three proteins was upregulated and of nine down-regulated by the cbs-/-genotype. two up-regulated liver proteins are involved in iron metabolism (ftl and fth). those proteins are associated with oxidation stress and inflammation. third up-regulated liver protein (cbr3) is related to oxidation-reduction process. the downregulated protein are involved in the hydrolysis of n-acylated or n-acetylated amino acids (acy1), regulation of endopeptidase activity (a1at4), cholesterol biosynthetic process (fpps), amino acid degradation (huth), cellular calcium ion homeostasis and l-ascorbic acid biosynthetic process (rgn). using label-free based relative quantitative proteomics (n58) we identified fourteen liver proteins whose expression was significantly altered as a result of the cbs gene inactivation. expression of four proteins was up-regulated and of ten proteins was down-regulated. the down-regulated liver proteins are linked with regulation of bone mineralization and inflammatory response (ahsg) or regulation of mrna splicing (roa2). the up-regulated liver proteins are involved in tricarboxylic acid cycle (suca), oxidation-reduction process (cy250), cholesterol metabolic process, iron ion homeostasis (fech), fatty acid metabolic process (ssdh; eci1) and response to oxidative stress (lonm). our findings suggests that cbs interacts with diverse cellular processes, including lipid metabolism, that are essential for normal liver homeostasis. deregulation of genes involved in lipid metabolism provides a possible explanation for fatty liver disease associated with cbs deficiency. transcription factors play central roles in coordinating developmental processes, as evidenced by the increasing number of transcription factor-related developmental disorders being uncovered by nextgeneration sequencing and genome-wide studies of copy number variation. the action of a transcription factor in regulating gene expression depends on interactions with other transcription factors, coactivators/co-repressors and chromatin modifying and remodeling complexes. transcription factors are commonly regulated by post-translational modifications. however the study of protein-protein interactions and post-translational modifications of transcription factors by common techniques such as coimmunoprecipitation and mass spectrometry is hampered by the difficulty in preserving interactions and modifications through cell lysis. to circumvent this issue, we developed a bioluminescence resonance energy transfer (bret) assay, which allows protein-protein interactions to be observed in live cells. in this assay, a protein of interest is expressed as a fusion with luciferase from renilla reniformis, and its putative interaction partner as a fusion with yellow fluorescent protein (yfp). upon addition of a cell-permeable substrate, the distance-dependent non-radiative transfer of energy from luciferase to yfp is quantified by measurement of light emission at two wavelengths to assess the interaction between the two fusion proteins. to validate the utility of this assay for investigating transcription factor interactions, we confirmed homodimerization of the foxp2 transcription factor, haploinsufficiency of which causes a rare and severe speech and language disorder, as well as interaction of foxp2 with other members of the foxp family. we also confirmed the interaction between foxp2 and multiple candidate interactors identified through yeast two-hybrid assays, including the autism-related transcription factor tbr1, the co-repressors ctbp1 and ctbp2, and post-translational modification enzymes of the pias family. the role of pias enzymes in sumoylation -the covalent modification of proteins with small ubiquitin-like modifier (sumo) proteins -led us to further explore this process, which is notably difficult to investigate because of the dynamic and labile nature of the modification, which is also typically present on only a minor fraction of molecules of a given protein. combining the bret assay with gel-shift techniques we demonstrated that foxp2 is sumoylated. finally, we used the bret assay to examine the effects of etiological foxp2 variants in speech and language disorder on protein-protein interactions and post-translational modification. in summary, the bret assay is a sensitive, reliable and potentially high-throughput technique for exploring protein biology in the context of live cells. we have demonstrated applications of the assay in validating putative protein-protein interactions, assessing posttranslational modifications, and investigating functional effects of protein variants identified in patient cohorts. these investigations have provided novel insights into the function of the foxp2 transcription factor in neurodevelopment and into the etiology of foxp2-related speech and language disorder. the directly interaction between pres1 of human virus b and human heat shock protein 70 (hsp70) deqiang wang 1 , chen ke 1 , jun zhang 2 1 key laboratory of molecular biology on infectious disease, 2 the department of cell biology and genetics the directly interaction between pres1 of human virus b and human heat shock protein 70 (hsp70). hepatitis b virus (hbv) has infected 2 billion people worldwide, and 350 million of them are chronically infected. the chronic virus infection, a major public health problem worldwide, leads to bout two-thirds of hepatocellular carcinoma (hcc). the hbv envelope consists of the large (l), middle (m) and small (s) envelope proteins, which contain pres1-pres2-s, pres2-s, and s domain alone, respectively [2] . the pres1 domain is believed to mediate virus attachment to the high-affinity receptor. yan et al employed a novel technique to propose sodium taurocholate co-transporting polypeptide (ntcp) as the candidate hbv receptor, and consequently, ntcp is a target for a new family of anti-hbv agents [3] . whereas, it remains a query to clarify that ntcp is the only or major hbv receptor in vivo. to illuminate if other host proteins cooperatively participate the hbv infection, we detect the interaction between pres1 and many candidate host proteins. fortunately, we have found that the human heat shocking protein 70 (hsp70) could directly interact with the pres1 domain of the hbv virus protein. both the pull down and the size exclusion chromatography experiments verify that the grp78 have the ability binding to pres1. whereas, whether the interaction between hsp70 and pres1 relates to the hbv infection need further experiments to clarify. 4 the member sponsorship in the vast world of naturally occurring peptides, where more than 7000 peptides are known and approximately 140 peptide therapeutics are currently being evaluated in clinical trials (fosgerau & hoffmann, 2015), the rapid and accurate determination of their physicochemical properties is key in peptide drug discovery. among these properties, hydrophobicity is crucial for understanding molecular recognition and biomolecular aggregation. hence, there is a great interest in determining hydrophobicity scales for amino acid structures. in this work, octanol/water partition (log p) and octanol/water distribution (log dph, fig. 1 ) of n-acetyl-l-amino-acid methyl amides were determined by means of quantum mechanical ief-mst solvation calculations taking into account the intrinsic conformational preferences of each amino acid according to dunbrack's libraries (dunbrack & karplus, 1993; 1994) . the results reveal log d7.4 differences for a-helical and b-sheet conformations in arg, lys, hid, asn, gln, met, cys, leu and ile. furthermore, by decomposing the octanol/water transfer free energy into electrostatic and non-electrostatic components, we estimated that the non-electrostatic cost of transferring the amino acid side chain amounts to 23.9 6 3.0 cal/mol.å2, in agreement with previous estimates reported in the literature. comparison of our scale with other theoretical and experimental hydrophobicity scales yields satisfactory results, leading to correlation coefficients ranging from 0.61 to 0.94. additionally, the mstderived hydrophobicity scale led to significant correlations with the rp-hplc retention factors measured for eight decapeptides (r 5 0.97) and for 195 influenza virus hemagglutinin 13-mer (ac-ypydvp-dyaslrs-amide) peptides (r 5 0.80). finally, the hydrophobicity scale was able to reproduce the experimental log p for 118 random neutral peptides (r 5 0.92) and log d7.4 for '01:15 random charged peptides (r 5 0.95), fig. 2 . future studies will address the application of this methodology to nonproteogenic amino acids, the prediction of peptide hydrophobicity at global and atomic level in peptides, and the scoring of peptide-protein interactions. docking-based tools for discovery of protein-protein modulators docking-based tools for discovery of protein-protein modulators. protein-protein interactions (ppis) play an essential role in many biological processes, including disease conditions. strategies to modulate ppis with small molecules have therefore attracted increasing interest over the last few years. although protein-protein interfaces (ppifs) are considered difficult to target with small molecules given its lack of well defined cavities. successful ppi inhibitors have been reported into transient cavities from previously flat ppifs. recent studies emphasize on hotspots (those residues contribute for most of the energy of binding) as promising targets for the modulation of ppi. pydock algorithm is one of the few computational methods that use energy of solvation to predict protein-protein interfaces and hotspots residues. we present an approach aimed at identifying hotspots and transient pockets from predicted proteinprotein interfaces in order to find potential small molecules capable of modulating ppis. the method uses pydock to identify ppifs and hotspots and molecular dynamics (md) techniques to propose putative transient cavities. we benchmarked the protocol in a small set of protein-protein complexes for which both structural data and ppi inhibitors are known. the method applies to the unbound proteins of the complexes the fast fourier transform algorithm, followed by the energy-based scoring from pydock to calculate the normalized interface propensity (nip) values derived from rigid-body protein docking simulations to predict the ppifs and hotspots residues without any prior structural knowledge of the complex. then we used md to describe the possible fluctuations of the interacting proteins in order to suggest transient pockets that could be useful as targets of small molecules for the modulation of ppis. finally, we evaluated by ligand docking, the validity of predicted hotspots and pockets for in silico drug design. we found that the nip-based method from pydock protein-protein docking identifies hotspots residues that are located within the binding site of known inhibitors of ppis. predicting ppifs from a three dimensional structure is a key task for the modulation of ppis. the use of the nip-based hotspots prediction method improve the identification of transient cavities from md simulation when compared to known binding cavities. this approach can be extremely useful in a realistic scenario of drug discovery targeting ppifs, when there is no information at all about the protein-protein complex structure. protein complexes are the fundamental molecular organizations that assemble multiple proteins to achieve various biological processes. identification of protein complex membership should provide a genotype-phenotype map to elucidate human gene-disease associations. it has been routinely assumed that network clusters with dense connections inside and sparse connections outside would form functional protein complexes. therefore, searching highly modular subgraphs in protein-protein interaction networks was explicitly or implicitly implemented in the algorithms to find protein complexes. however, to our surprise, we found a large portion of complexes with a medium-to-low modularity from the analysis of 719 experimentally confirmed protein complexes. we also discovered that these complexes have cellular functions enriched in highly time-and space-dependent expression, such as signal transduction or subcellular localization. we further developed an algorithm to find such complexes by weighing network connections to capture transient interactions with intrinsically disordered regions. we confirmed that our method improved the identification of biologically relevant members of protein complexes and covered more complexes with a medium-to-low modularity. furthermore, newly discovered subunits in protein complexes could explain more disease-gene associations, indicating its utility to expand current genotype-phenotype map of human diseases. expanding template-based protein-protein complex prediction using ab-initio docking sergio mares-s amano 1 , luis angel rodr ıguez-lumbreras 1 , juan fern andez-recio 1 1 structural characterization of protein-protein interaction (ppi) networks is crucial for understanding the underlying molecular mechanisms whereby life processes and disease arise. however, due to inherent limitations of experimental techniques, such characterization only covers an extremely reduced fraction of the human ppi network (interactome). recent studies have shown that although available structural templates may suffice to model a significant proportion of the interactome, model accuracy and binding specificity remain unsolved problems. consequently, improving the ability to predict ppis structurally will help to provide a better 3d profile of the known interactome, which may ultimately lead to the development of new therapeutic applications. here we show a novel approach that combines templatebased modeling with protein-protein computational docking to the structure-based prediction of ppis. our approach samples different protein-protein structural models derived from docking simulations. models are subsequently ranked using a function that incorporates an energy-based scoring term and a structural template similarity score. the energy-based scoring function includes electrostatics, van de waals and desolvation calculations, whilst the template similarity score accounts for the degree of structural similarity of models against a high-resolution and diverse dataset of structural templates. our approach highly improved the predictive success rate over individual ab-initio docking and templatebased techniques across a large benchmark dataset, including 176 protein-protein complexes. when compared to the performance of the ab-initio docking algorithm, we found that the approach increased consistently the success rate, by approximately 30%, for the top 1, top 5 and top 10 solutions. the success rate improvement was even more notorious when the comparison was performed against the predictions from the traditional template-based docking. though incorporating ab-initio docking expands considerably the scope of the template-based docking method, challenges remain for interacting proteins in which high conformational changes occur upon binding and also the size and diversity of the repertoire of structural templates needs to be increased. is essential for the development of multicellular organisms. in mammalian cells, early events in pcd involve the release of cytochrome c (cc) from mitochondria to the cytoplasm, so letting cc play a key role in assembling the apoptosome and triggering apoptosis. in plants, pcd is part of a general process -the so-called hypersensitive response -in which mitochondrial cc is likewise released into the cytosol but its further role and cytoplasmic partners remain veiled. such a coincidence in cc release made us think of a common link for pcd in such evolutionarily distant species along evolution. to go deeper in understanding the pcd-dependent role of cc, a proteomic approach based on affinity chromatography with cc as bait was run using human and plant cell extracts. upon combining this approach with bimolecular fluorescence complementation (bifc), a total of eight and nine unknown proteins interacting with cc under pcd conditions were identified in human and plant cells, respectively [1, 2] . such novel cc-partners -which are located in the cytoplasm and even in the nucleus -are involved in protein folding, translational regulation, oxidative stress, dna damage, energetic and mrna metabolism [3] . strikingly, some of the novel human cc-partners are closely related to those for plant cc, so indicating that the evolutionarily well-conserved event of cc release from mitochondria could involve a common signalosome consisting of a wide range of common targets [3] . to also understand such a promiscuity of cc from a structural point of view, the cc surface residues involved in complex formation with each one of its counterparts were mapped by using nmr spectroscopy. the resulting data shows that the heme crevice of cc is at the cc-partner interface in most of the complexes, which is in agreement with the vast majority of known redox adducts of cc. in contrast, however, to the high turnover number of the redox cc adducts inside the mitochondria, the complexes formed by cc under pcd conditions lead to the formation of rather stable nucleo-cytoplasmic ensembles. altogether, these findings suggest that extra-mitochondrial cc interacts with nuclear and/or cytoplasmic pro-survival, anti-apoptotic proteins in both humans and plants so as to lead living cells to dye. keywords: cytochrome c, programmed cell death, signalosome. post-translational phosphorylation often modulates the function of proteins. in particular, they affect the role that cytochrome c (cc) plays in cell life and death [1] . cc is phosphorylated in vivo in tyr48 and tyr97 residues [2, 3] , but recently, two new phosphorylation sites have been described at positions 28 and 47 [4] . hence, we aim at understanding the structural and functional changes induced by thr28 and ser47 phosphorylation cc. for this purpose, we designed two phosphomimetic mutants of cc by replacing either thr28 or ser47 by the canonical amino acid aspartic acid (t28d and s47d). as control, two other mutants at the same two positions (t28a and s47a) were analyzed so as to differentiate the effects due to the presence of a negatively charged residue. remarkably, the s47a mutant is significantly less stable than the wild-type species. we found that phosphorylation at position thr28 diminishes the redox potential and oxygen consumption. in addition, t28d mutation affects the ability of cc to bind the distal site pcc1, thereby suggesting that phosphorylation at this position affects the electron carrier capacity of cc. mass spectrometry (ms) is widely used techniques to gain knowledge about biomolecules [1, 2] . it produces a high amount of data which is often presented as a list containing thousands of proteins. that list usually contains few hits interesting for our research. the pocess to select those proteins may include integrating experimental with annotation data. it requires spending some time in both, performing calculus and searching in databases. in this poster we present msbiodata analysis tool, a web service thought to deal with this tedious work. with this tool, researchers can set rules to select the most interesting hits in his lists using both, experimental data and gene ontology [3] annotation. the data can be upload to the web using an excel spreadsheet or a flat files in a mztab format, and rules are easily constructed by means logical sentences. those sentences are composed by one or more terms linked by logic operators (and and or). each term in the logical sentence indicates to our program the conditions 1 that selected hits must meet. once the alysis is finished, the results are delivered by email. msbiodat analysis tool do not requires any programming knowledge to be used and is freely available at: http://msbiodata.innomol.eu keywords bioinformatics/data analysis/proteomics/data mining/ mass spectrometry. beside the rate of protein synthesis, the regulation of protein degradation plays a crucial role in the white muscle protein accumulation and overall fish growth. intracellular proteolysis in salmonid species, such as atlantic salmon, salmo salar l. and rainbow trout, oncorhynchus mykiss walb., was studied to evaluate the basic mechanisms of protein degradation that could possess a potential target to regulate the body mass accumulation in farmed fish. a number of white muscle proteases such as cathepsins b, l, and d, proteasomes, and calcium-dependent proteases (m-and m-calpains), was studied in the juvenile specimens of different size-and age-groups both wild and farmed salmonids. the correlations between the protease activity and expression levels and morphometric characteristics of fish were found. the size-and age-related differences in intracellular protease activity revealed in fish muscles indicate both general role of proteolysis regulation in salmonid growth and the specific role of the individual proteolytic enzymes as well. the data on negative correlation of cathepsin d and calpain activity in muscles and the rate of weight increase in juvenile salmonids were obtained. a revealed positive correlation of cathepsin b activity and morphometric parameters in fish young presumably indicates its primary contribution to non-myofibrillar protein turnover. ubiquitin-proteasome system seems to contribute to background protein turnover as the proteasome activity was not corresponded with growth rate. summarizing the data obtained the autophagy-lysosomal and calpain-related protein degradation pathways were recognized to be directly involved in body growth and muscle protein retention in salmonid fish. the work was carried out using technical facilities of ib karrc ras equipment centre and financially supported by the russian science foundation, grant no. 14-24-00102 "salmonids of the north-west russia: ecological and biochemical mechanisms of early development". solving the proteomic organization of fitness-related genes in uropathogenic escherichia coli in life threatening sepsis. nowadays, complete genomes for almost all major bacterial pathogens are available, helping researchers to identify virulence factors. however we still ignore how these genes are organized at the proteome level and how this association influences bacteria pathogenicity. we integrated available databases on upec e. coli (strain cft073) to investigate the genomic and proteomic organization of genes related to upec fitness in the host. intriguingly, we found that most fitnessrelated genes have orthologs not only in other pathogenic strains but also in non-pathogenic bacteria such as e. coli k-12. these genes are organized in clusters and operons with similar structure. by integrating protein-protein interaction data we observed that genes with high impact on fitness also display a highly clustered organization when compared to other genes. overall, our results show that proteinprotein interaction clusters associated to upec fitness in the host represent a promising target for the design of new antibiotics. elucidating the molecular mechanisms by which the hnh endonuclease gp74 activates the terminases in bacteriophage hk97 (2) . hnh endonucleases are characterized by two highly conserved his residues and an asn residue(3). gp74 is essential for phage head morphogenesis, likely because gp74 enhances the activity of the hk97 terminase enzymes toward the cos site (4) . notably, enhancement of the terminase-mediated cleavage of the phage cos site requires the presence of an intact hnh motif in gp74. mutation of the canonical metal binding his in the hnh motif abrogates gp74 mediated-terminase activity. although phages are widely studied, there is no definitive structural or mechanistic evidence as to how the hnh endonuclease within gp74 functionally interacts with the adjacent terminase enzymes to facilitate phage morphogenesis. previous work on hnhcontaining bacteriophage proteins does not address explicitly how the requirement for divalent metal binding at the hnh endonuclease site induces interaction with the terminase enzymes that are so crucial for phage dna packaging during morphogenesis (4, 5) . in addition, gp74 possesses no sequence similarity to hnh proteins for which the structure has been determined (3), making structural studies of gp74 necessary. toward these ends, we use nuclear magnetic resonance (nmr) spectroscopy to probe metal and terminase binding of gp74 in the wild type state and bearing metal binding mutations. we also report backbone resonance assignment of gp74. our nmr studies have elucidated residues within gp74 required for metal binding and terminase activity. these data are being used to assess the role of specific gp74 residues in phage morphogenesis. together, this work will identify the enigmatic role describing how metal binding in hnh endonucleases is crucial in the replication and morphogenesis of phages. meat production from pigs for human consumption is a resource heavy process, indeed every part of the animal that is not used constitutes a protein food-chain loss, which is neither economically nor environmentally viable. the goal of this project is to better harness slaughterhouse waste such as the keratin rich pig bristles and nails through microbial conversion. instead of using identified single microorganisms, it is the goal to define microbial consortia where microorganisms synergistically show the ability of efficient keratin degradation/conversion. candidate consortia have been obtained by selecting for microorganisms growing on enriched media that contains milled pig bristles as sole carbon and nitrogen source. by using mass spectrometry and various biochemical analyses to investigate keratinolytic enzymes, methods will be established for identifying and characterizing suitable consortia. protein families likely to be involved are keratinases, which are specialized proteases including serine, cysteine and metallo proteases, as well as systems capable of reducing or otherwise breaking disulfide bonds which are highly abundant in hair and nails. furthermore, interactions and symbiosis of microorganisms in a consortium will be investigated at the meta-proteomics level. the project will lead to development of biotechnological degradation of keratin rich fibers, and provide new insights into functional dynamics and efficacy of microbial consortia. a comprehensive protein domain analysis to map cancer-type-specific somatic mutations interpretation of the genome-wide association studies (gwas) of cancer patients to find cancer-typespecific biomarker is challenging due to the mutational heterogeneity of cancer types. network approaches to find cancer-type-specific variants and biological pathways are increasing since genes tend to act together to display phenotypic or disease outcomes. phenotype similarity has proven to reflect the relationship of functionally related genes. we applied phenotype similarities between various diseases for expanding molecular connections of cancer-type-specific variants to discover cancer-type-specific modules. specifically, cancer-type-specific variants of 7 cancer types from the cancer genome atlas (tcga) were analyzed to find phenotype-inferred relationships among the variants. we find that cancer variants that cause the similar disease phenotypes tend to be linked as a cluster of biological pathways or functions. moreover, cancer-type-specific modules could explain the underlying pathogenicity of specific symptoms which manifest in particular cancer types. cancer-type-specific modules and pathways found from phenotype similarity/dissimilarity based on cancer symptoms improved the discrimination performance to sort cancer-type-specific variants to accurately predict patient groups. our method will be further developed to find genetic biomarkers for the diagnosis or prognosis of specific cancer types pk-009 engineering a stable, symmetric membrane protein scaffold amanda duran 1 , jens meiler 1 1 computational protein engineering has the potential to contribute to various fields including drug design, protein therapeutics, and materials science. protein-ligand interface design and the construction of large, stable proteins rely on stable scaffolds. symmetry is a great tool for protein stability both in protein engineering and nature. several membrane protein structures exhibit pseudo-symmetry and are proposed to be the result of gene duplication, fusion and diversification events originating from a monomeric gene. aquaporins (aqp) are a class of membrane proteins that exhibits a two-fold inverted pseudo-symmetry. the escherichia coli aqp glycerol facilitator protein (glpf) was originally computationally engineered to be perfectly symmetric in sequence and presumably in structure. the symmetric gene was assembled, cloned, and expressed. however, after facing many challenges experimentally, the computational study has been expanded to 13 aqps of known structure for a more extensive symmetric backbone search. mammoth structural alignment was used to align the structures to their inverted counterparts. cutpoints were calculated based on a-carbon distance. finally, the rosetta protein modeling software suite was used to refine and energetically minimize the symmetric backbones. from over 1500 generated symmetric backbones, 20 candidates were chosen for experimental verification. these studies are ongoing.currently, the symmetric backbone models have scored to be more stable than the wild-type proteins. experimental verification of these symmetric backbones will provide valuable information for the current state of membrane protein modeling and design using computational methods. intrinsically disordered proteins drive heritable transformations of biological traits daniel jarosz 1 , james byers 1 , sohini chakrabortee 2 , sandra jones 3 , amelia chang 2 , david garcia 1 1 stanford university, 2 whitehead institute for biomedical research, 3 rockefeller university the transmission of information from one generation to the next generally occurs via nucleic acids. the only known protein-based molecular memories are prions, which drive heritable biological traits based upon self-templating changes in protein conformation. these protein-based genetic elements have previously been identified systematically, but at least three do not share the sequence biases or structural characteristics that have informed such studies. here we employed a comprehensive library of yeast proteins to examine the breadth of protein-based inheritance. transient overexpression of more than forty proteins created new traits that were heritable and beneficial. some shared properties of known prions, but most employed distinct genetic and biochemical mechanisms to act as elements of inheritance. traits with these characteristics were common in wild yeast strains and could also be elicited using orthologous mammalian proteins. the inducing proteins were strikingly enriched in intrinsically disordered sequences that have been widely conserved across evolution. intrinsically disordered proteins are associated with human disease and with dosage sensitivity in yeast, flies and worms. our results suggest another widespread role for such intrinsically disordered sequences: induction of heritable epigenetic switches that transform phenotypic landscapes and drive adaptation to stressful environments. prediction of binding affinity in protein complexes: contacts do matters almost all critical functions in cells rely on specific protein-protein interactions. understanding these is therefore crucial in the investigation of biological systems. despite all past efforts, we still lack a thorough understanding of the energetics of association of proteins. here, we introduce a new and simple approach to predict binding affinity based on functional and structural features of the biological system, namely the network of interfacial contacts. we assess its performance against a protein-protein binding affinity benchmark and show that both experimental methods used for affinity measurements and conformational changes have a strong impact on prediction accuracy. using a subset of complexes with reliable experimental binding affinities and combining our contacts-and contact types-based model with recent observations on the role of the non-interacting surface in protein-protein interactions, we reach a high prediction accuracy for such a diverse dataset outperforming all other tested methods. free radical oxidation -a new method for obtaining stable protein coatings on magnetic nanoparticles magnetically targeted nanosystems (mtnss) are now considered to be applicable in different areas of biology and medicine such as hyperthermia, magnetic resonance imaging, immunoassay, cell and molecular separation, a smart delivery of drugs to target cells. proteins are promising materials for creation of coatings on magnetic nanoparticles (mnps) due to their biocompatibility, an ability to protect magnetic cores from influence of biological liquids and prevent agglomeration of mtnss in dispersion, their possible functional activity as therapeutic products and biovectors. the creation of stable protein coatings with retention of native properties of molecules is still an important biomedical problem because of disadvantages of the commonly used methods such as formation of a polydisperse ensemble of particles, nonselective linking of proteins leading to cross-linking of macromolecules in solution, and desorption of coatings. a novel method in obtaining stable single-layer coatings assembled from protein molecules on the surface of magnetite nanoparticles has been developed. it is based on protein liability to free radical modification, leading to the formation of intermolecular covalent cross links. free radicals are locally generated on the surface of nanoparticles via the fenton reaction thereby proteins adsorbed on the surface are subjected to the cross-linking. o-phenylenediamine was used for detection of free radical generation initiated by nanoparticles. the proteins drastically differing in their structure and properties, namely, serum albumin, thrombin and immunoglobulin g were selected for creating the protein coatings. the properties of the obtained coatings and their stability have been studied with the help of dynamic light scattering (dls), uv/vis spectrophotometry, antibody-antigen test and the method of spectral-fluorescent probes. albumin molecules in mnps coatings have been shown to retain their capability of binding with a dye and be conformationally stable. the dye 3,3'-di-(g-sulfopropyl)25,5'diphenyl-9-ethiloxacarbocyanine-betaine interacting with albumin with a growth of fluorescence and with partial cis-trans conversion of the dye has been used. it has been proven that coatings composed of protein macromolecules are 1) stable, 2) formed around individual nanoparticles and 3) have several nanometers in thickness. the free radical linking of thrombin and immunoglobulin g on the surface of nanoparticles has been shown to almost completely keep native properties of the protein molecules. the free radical linking method reveals new possibilities for design of single-layer multiprotein polyfunctional coatings on the surfaces of all the nano-, micro-and macroobjects containing metals of variable valence (for example, fe, cu, cr). the spectral-fluorescent investigation was supported by the russian foundation for basic research, project nos. 13-03-00863 and 14-03-31196mol_a. regulation of neuronal snares by accessory proteins shrutee jakhanwal 1 , reinhard jahn 1 1 regulation of neuronal snares by accessory proteins 1shrutee jakhanwal and 1reinhard jahn 1department of neurobiology, max planck institute of biophysical chemistry, fassberg, goettingen, germany-37075. synaptic vesicle exocytosis lies at the heart of the process of neurotransmitter release. and, the family of proteins that is central to the process of synaptic vesicle exocytosis is the family of snare proteins. there are three kind of neuronal snare proteins namely syntaxin, snap25 and synaptobrevin. these three snare proteins interact through their snare-motifs to form a highly stable four-helix bundle, which in turn, pulls two membranes together to mediate fusion. years of work in this field have established that the four-helix bundle is critical for the membrane fusion to occur. however, the process of regulation of snare-mediated fusion remains very poorly understood. the major regulatory proteins involved in the process are munc 18, munc 13, synaptotagmin and complexin. the major aim of my project is to obtain a closer look at the regulation process of snare-mediated fusion by focusing on the interaction between the snare proteins and the regulatory proteins. to achieve this objective, i express and purify the different proteins involved in the process of snare-mediated fusion and thereafter subject them to appropriate biochemical characterization. in order to assess the role of the purified proteins in the process of fusion, i reconstitute them into liposomes and perform in-vitro lipidmixing assays. these assays are based on f orster resonance energy transfer (fret). based on the discretion of assessing the protein-protein or protein-lipid interactions, either the proteins or the lipids can be fluorescently labeled. also, the lipid compositions can be varied in order to assess the effect of lipid on the function of the respective protein. fluorescence-based anisotropy measurements can also provide information about the degree of freedom of a protein, indirectly providing information about the kinetics of a reaction. employing these techniques, i observe that munc 18-1 leads to displacement of syntaxin from a complex of syntaxin and snap25. also, a complex of syntaxin and munc 18 is resistant to the action of the aaa-atpase, nsf and its co-factor asnap, implicating this complex as a strong candidate for acting as the starting point for the process of neurotransmitter release. munc 18 also appears to enhance lipid-mixing by interacting with the snare-complex. further investigations on the same lines can provide very useful insights into the process and can help us unravel the secrets that underlie the beauty of the exquisitely regulated process of neurotransmitter release. binding of thymidine nucleotides to a viral thymidine monophosphate kinase aldo a. 3 centro de investigaci on en alimentaci on y desarrollo theme: biochemistry there is great interest in the evolution and activities of fish trypsins, since they appear to have evolved into different families. the cdna for trypsin iii from the monterey sardine (sardinops sagax caerula) was obtained and its deduced amino acid sequence matched its identity with a purified protease from the fish by mass spectrometry analysis. molecular modeling of sardine trypsin iii compared to other homologs showed a typical trypsin fold with all the cognate components for catalysis, and specific amino acid distribution that are possible factors that explain the cold adaptation. from phylogenetic analysis, sardine trypsin iii belongs to the novel y family, which is proposed to have evolved for cold adaptation. the obtained recombinant trypsin iii showed a low catalytic efficiency, but it remained active at cold temperatures, similar to other cold-adapted trypsins. the cold-adaptation of sardine trypsin iii opens a wide range of biotechnological applications for this protease and is also interesting from the serine protease structure-function relationship point of view. fungicidal mechanism of scolopendin 2, a cationic antimicrobial peptide from centipede heejeong lee 1 , dong gun lee 1 drastically (from 6.5 x 10-3 to 2 x 10-3 colonies) upon deletion of this 130 residues domain from the full length trai. we are investigating the structure and function of this very c-terminal end of trai using nmr spectroscopy. for the backbone assignment we used slice-selectively homonuclear broadband decoupled spectra along with standard experiments. three-bond scalar coupling constants were obtained through real-time j-upscaling experiments. with the backbone assignments, we have the first hand evidence which shows that his domain is for the most part intrinsically disordered, but contains short a-helical regions. structural development, interaction studies to find the binding partner and transition of disorder to order orientation of this domain will be further investigated in this project. here we investigated a model system where mab aggregation is induced by increasing the ionic strength (nacl) at low ph. the aggregation depends both on protein and sodium chloride concentration. with nanoparticle tracking analysis (nta) and micro flow imaging (mfi) the aggregation formation was further characterized. aggregation can be partially reverted by lowering the ionic strength as determined by soluble monomer concentration measurement using se-hplc: parts of insoluble aggregates could be solubilized as soluble aggregates, dimers or even monomers. a quasi equilibrium is formed in between the subtypes. the whole aggregation process was examined by ftir and cd-spectroscopy to identify structural changes of the mab. screen of protective additives: the effect of osmolyte additives on aggregation kinetics and final aggregate concentration is investigated, revealing protective effects in both cases. in a screen with more than 200 compounds not only the aggregation propensity was studied but also structural changes. the aggregation index (quantity for colloidal stability) and the melting point (quantity for conformational stability) measured by differential scanning fluorimetry were determined. the used mtp format screen has potential for buffer optimization and formulation development. structural biology and protein dynamics tetraspanin cd81 has a broad range of cellular functions, such as integrin association forming tetraspanin-enriched domains, synapse formation between b and t cells, cell adhesion, motility, invasion and signalling. furthermore, cd81 is one of the four receptors involved in the cell entry of hepatitis c virus (hcv) and therefore infection onset, one of the major causes for chronic liver disease resulting in cirrhosis and hepatocarcinoma. human cd81 large-extracellular-loop (hcd81lel) is composed of a "stalk" and a "head" subdomain; with the latter interacting with hcv-e2 glycoprotein. we present four novel hcd81lel crystal forms. analysis of the fourteen independent observed hcd81lel high-resolution x-ray structures suggests that the dynamism of the hcd81lel head-subdomain is an inherent molecular property, an observation supported also by molecular dynamics (md) studies. we classify the conformations in three distinct clusters (closed, intermediate and open) , which are seen both in the crystal structures and in the molecular dynamics simulations. the md simulations also show that conformational variability is modulated by ph changes, with distinct probability for each cluster at acidic and neutral ph. furthermore, in silico docking of the recent e2core structure with three of the major types of hcd81lel head-subdomain clusters highlights hydrophobic interactions as the major forces in the e2core: hcd81lel recognition mechanism. we propose that the flexibility of the hcd81lel is exploited by hcv at different stages of cell entry from virus attachment to internalization and fusion with the endosomal membrane. our results provide important insights on the basic mechanism governing hcv binding to hcd81, and can help structure-based drug design of entryinhibitors of hcv. allophycocyanin of gracilaria chilensis: from gene to function jorge dagnino-leone 1 , jos e martinez-oyanedel 1 , marta bunster-balocchi 1 1 universidad de concepci on theme: structure-function relationship of proteins the phycobilisomes (pbs) are auxiliary photosynthetic complexes that allow cyanobacteria and red algae to enhance the energy uptake in the range of 490-680 nm. in gracilaria chilensis, an eukaryotic red algae, pbs is composed of phycoerythrin (pe), phycocyanin (pc) and allophycocyanin (apc); these proteins possess chromophores which capture energy and then transfers it to photosytems. pbps are oligomers of a ab heterodimer; it oligomerizes into a trimer (ab)3, this trimer has discoidal shape and it is associated in hexamers (ab)6, several of this hexamers forms cylinder-like structures. pbs has 2 components: antennas and core. the antennas are composed of pe and pc, whose function is to capture energy between 490-570 and 590-625 nm respectively and transfer it to the core. the core is formed by apc, which can absorb energy in the 620-650 nm range. apc emission allows transferring energy to the photosystems with high efficiency. pbs is also composed by linker proteins which allow the correct assembly of pbs and possibly regulate the energy transfer. the main goal in our group is to build an atomic model of the gracilaria chilensis phycobilisome. we have solved the crystal structure of pe and pc and created an antenna model. at present we are working in apc and the chromophorilated linker proteins. the objective of the present work is to create a model of the core of gracilaria chilensis; to achieve these we have used molecular biology, biochemistry and bioinformatics techniques. we designed oligonucleotides primers for the four allophycocyanin subunits genes and for the globular domain of the apce linker. these primers were used in pcr experiments to obtain the genes sequences. the sequences were translated to a aminoacid sequences and used to build a 3d model for apc subunits and trimers using the software modeller. on the other hand we purified and analyzed the spectroscopic properties of apc from gracilaria chilensis using absorption and fluorescence spectroscopy. we also determined apc oligomerization state using gel filtration. molecular docking using the cluspro server was performed to obtain a hexamer and apc cylinder models. based on electron micrographs obtained by our lab a tri-cylindric core model was built. all the models were submitted to a molecular dynamics using gromacs software. finally we determine possible energy transfer pathways in the core model applying the extended forster equation, spectroscopic data from literature and the transition dipole moments of each of the chromophores present in the core. as conclusion of this work we built the first atomic model of gracilaria chilensis phycobilisome core and propose energy transfers pathways inside the core in the context of a phycobilisome. novel practical strategies to access artificial metalloenzymes marco filice 1 , jose miguel palomo 1 1 departamento de biocat alisis, instituto de cat alisis, csic protein chemistry and engineering since the first report, the design of artificial metalloenzymes has rapidly been converted into an important topic in biological and inorganic chemistry due to their potential applications in synthetic chemistry, nanoscience and biotechnology. the combination of a catalytically active organometallic moiety with a macromolecular host has permitted the creation of biohybrids, a new kind of heterogeneous catalytic entities combining the attractive features of both homogeneous and enzymatic systems. presenting our most recent achievements in this research area, here we describe two novel powerful and promising approaches focusing the practical synthesis and large scale production of heterogeneous artificial metalloenzymes showing chimeric activity. the first strategy is based on the in situ synthesis of noble metal nanoparticles and their supramolecular assembly with a microbial lipase from candida antarctica (fraction b) finally creating an ultra-active organometallic-enzyme heterogeneous nanobiohybrid. in the second approach, combining different protein engineering protocols (molecular biology, orienting immobilization, solid-phase bioorganic modification and bioinformatic tools), an orthogonal solid-phase strategy creating novel unnatural catalytic sites was designed and optimized. the application of such a strategy onto the structure of the lipase from geobacillus thermocatelunatus permitted the generation of a heterogeneous artificial metallolipase with chimeric activity. as proof-of-concept, the combinatorial library of generated artificial metalloenzymes obtained by both strategies was successfully assessed in a set of different synthetic reactions (selective c-c bond formation as suzuki, heck or diels-alder reactions) and also combining both activities (metallic and enzymatic) in cascade processes such as dynamic kinetic resolution of amines or production of arylamines. the obtained results were excellent in all cases. extending this strategy to other enzymes, proteins and catalytic metals, we envisage the creation of a combinatorial library of programmable artificial enzymes useful for a wide set of applications (i.e. fine organic and medicinal chemistry, bioremediation or biomedicine). proteomic examination of the yeast nuclear pore complex dynamics protein turnover and exchange nuclear pore complexes (npcs) are proteinaceous assemblies situated in nuclear envelopes of eukaryotic cells. the main function of the npc is the selective transport of macromolecules. npcs also partake in other functions, such as nuclear organization and gene regulation. the core scaffold of the npc is thought to be a stable structure, while the peripheral components exchange at various rates. however, these phenomena have not been elucidated in detail. the recent findings that yeast daughter cells get a higher proportion of the old npcs and the core scaffold hardly turns over raise the possibility that the exchange of the peripheral nucleoporins can be a repair mechanism. yeast provides a useful organism for the interrogation of nucleoporin exchange, as it performs closed mitosis; hence the only mixing of npc constituents is due to exchange. we have developed a panel of genetic tools providing for conditional induction and repression of nucleoporins. by combining these switches with stable isotope metabolic labeling and affinity capture, cross linking coupled to mass spectrometry, we are able to distinguish between pre-existing and newly synthesized proteins and quantify their relative amounts in the npc. our preliminary findings are in agreement with results obtained in other organisms: the core scaffold of the npc (inner ring, outer ring) appears to be stable, however does exchange slowly over time, while peripheral components exchange faster. by looking at the exchange rates of yeast nucleoporins we hope to gain insight into the npc biology of actively dividing eukaryotic cells. active site clustering identifies functional families of the peroxiredoxin superfamily angela harper 1 , janelle leuthaeuser 2 , patricia babbitt 2 , jacquelyn fetrow 3 1 department of physics, wake forest university, 2 department of molecular genetics and genomics,-wake forest university, 3 departments of physics and computer science, wake forest university bioinformatics understanding the relationships between proteins is vital to increasing our knowledge of the protein universe. while there are large databases of sequence information, the massive data influx over the past decade has prevented adequate classification of proteins at the molecular function level. however, it has been previously suggested that a protein's active site information may correlate with these known molecular functional differences; thus, active site profiling was developed to use residues around the active site of a protein to relate proteins. subsequently the deacon active site profiler (dasp) was developed to create these active site profiles and search them in a database, such as gen-bank, in order to find proteins with similar active site environments. by using dasp to computationally cluster proteins based on the similarity of their active site profiles, the peroxiredoxin (prx) superfamily was analyzed through active site similarity methods. the residues from the active site of each prx structure were extracted and clustered, and these profiles were iteratively searched in genbank through a multi-level iterative sequence searching technique (misst). the prx superfamily has been studied by experts, allowing the results of these searches to be compared to a well-annotated group of proteins. while previous sequence based evolutionary methods have been unable to identify functional differences between some subgroups of the prxs, notably the ahpc-prx1 and prx6 subgroups, misst discretely separates these subgroups. classifying prx proteins into functionally relevant groups using computational active site similarity methods lays the foundation for an automated process for identifying protein functional groups beyond the prx superfamily. synthesis and conformational studies of glycoprotein n homolog of bovine herpesvirus 1 (bhv-1) by using cd, nmr and molecular modelling it serves as a chaperone for viral glycoprotein m and, in its gm-unbound form, acts as an inhibitor constraining the transporter associated with antigen processing (tap). the ul49.5/gm complex formation is required for the maturation and proper trafficking of both viral proteins. in the absence of gm, ul49.5 blocks transport of antigenic peptides by tap and their mhc i-restricted presentation. the molecular mechanism of ul49.5 activity still remains elusive. in order to investigate the structural requirements for biological function ul49.5 study was conducted using cd, nmr and molecular dynamics methods. the data obtained with the use of high purity synthetic peptides encompassing ul49.5 confirmed the presence of an alpha-helix structure, formed preferentially in the presence of dodecylphosphocholine (dpc) micelles as a membrane-like environment. in order to determine the three-dimensional structure of ul49.5 protein in the present work its nmr solution structure in the presence of membrane-like environment was performed. the nmr data were used as a set of restraints for a simulated annealing protocol that generated 3dstructures of the colin johnson 1 , sara codding 1 1 membrane proteins resealing of tears in the sarcolemma of myofibers is a necessary step in the repair of muscle tissue. defects in this repair process are responsible for muscular dystrophy and cardiomyopathy. the repair pathway is triggered by the influx of calcium through lesions in the membrane, which result in membrane fusion and patching of the wound. recently dysferlin has been identified as a calcium binding protein essential for sarcolemma repair, as well as other snare mediated exocytotic events including cytokine and acid sphingomyelinase secretion. in this presentation we demonstrate a direct interaction between dysferlin and the snare proteins syntaxin 4 and snap-23. in addition, fret and in vitro reconstituted lipid mixing assays indicate that dysferlin accelerates snare heterodimer formation and snare mediated lipid mixing in a calcium sensitive manner. our results suggest a model whereby dysferlin acts as a calcium sensing snare effector for exocytosis and membrane fusion. exploring the therapeutic potential of a peptide derived from a poxviral immune evasion protein: nmr determination of the solution structure of viper and its inactive mutant toll-like receptors (tlrs) have a role in viral detection leading to cytokine and ifn induction, and as such they are targeted by viruses for immune evasion. the poxviral protein a46 has been identified to inhibit tlr signaling by interacting with tir domain-containing proteins of the receptor complex to collectively inhibit all tlr adaptor proteins that positively regulate transcription-factor activation (1). one 11 aa peptide (kysf-klilaey) termed viper (viral inhibitory peptide of tlr4) was reported to retain the inhibitory properties of full length a46 against tlr4 signaling. a 9r homopolymer delivery sequence at the c-terminus provided delivery of the peptide into cells. structural comparisons are presented between 9r-viper, which is active in preventing tlr4-dependent cytokine induction in cell culture, and a mutant that exhibited loss of function (9r-viper l6a,e10a), through solution nmr spectroscopy. we find that despite a relatively minor sequence difference, the loss of hydrophobicity as well as negative electrostatic interactions result in subtle but potentially significant differences in the region of the peptide proposed to interface with tlr4. reference: wake forest university, 2 wake forest university, 3 university of california san francisco protein function prediction the elucidation of protein molecular function lags far behind the rate of highthroughput sequencing technology; thus, it is essential to develop accurate and efficient computational methods to define functional relationships. protein clustering based on sequence similarity has emerged as a simple, high-throughput method for defining protein relationships, but sequence-based techniques often inaccurately define molecular function details. active site profiling (asp) was previously developed to identify and compare molecular details of protein functional sites. protein similarity networks were created using both active site similarity and sequence similarity for four manually curated superfamilies, and results demonstrate that asp-based clustering identifies detailed functional relationships more accurately than sequence-based clustering. building on this, two iterative pipelines were developed using active site profiling and profile-based searches to cluster protein superfamilies into functional groups. first, the two level iterative clustering process (tulip) utilizes active site profiling and iterative pdb searches to divisively cluster protein structures into groups that share functional site features. across eight superfamilies, tulip clusters exhibit high correlation with expert functional annotations. subsequently, the multi-level iterative sequence searching technique (misst) utilizes iterative profile-based genbank searches to identify protein sequences that belong in each tulip group. the results indicate that these asp-based methods accurately and efficiently identify functionally relevant groups through a process that can be applied systematically and on a large-scale. moreover, the approach can be applied more quickly than detailed manual curation, suggesting its value in guiding annotation efforts. dept. biochemistry and molecular biology. university of valencia, 2 lab of peptide and protein chemistry. centro de investigaci on pr ıncipe felipe membrane proteins changes in the equilibrium between pro-survival and pro-apoptotic members of the b-cell lymphoma-2 (bcl-2) protein family at the mitochondrial outer membrane (mom) induce structural changes that committed cells to apoptosis. bcl-2 homology-3 (bh3)-only proteins participate in this process activating pro-apoptotic effectors and promoting permeabilization of the mom. the membrane association of bh3-only proteins is a controversial issue due to the lack of a canonical carboxyl-terminal (c-terminal) transmembrane (tm) domain. we used an in vitro transcription/translation system to study the insertion capacity of these hydrophobic c-terminal regions of the bh3-members bik, bim, noxa, puma and bmf into microsomal membranes, and an escherichia coli complementation assay to validate our results in bacterial cells. furthermore, we have fused these hydrophobic regions to gfp to investigate the subcellular sorting. these results will allow further refinement in the elaboration of the bcl-2 protein-protein and protein-membrane interactome network. alexis peña 1 , flaviyan jerome irudayanathan 1 , shikha nangia 1 1 syracuse university, dept. of biomedical and chemical engineering computational modeling, biostatistics, biomedical and chemical engineering tight junctions (tj) are vital intracellular barriers that are responsible for regulating paracellular transport. claudins, a family of abstract small transmembrane proteins with approximately 27 members, are an integral part of the tj strands. tight junctions provide molecular-level protection and prevent infection and toxins from entering the body; in the same sense tjs allow nutrients and vital solutes to pass through. claudins are associated with various diseases including metastatic cancer as well as an entry point for many viruses. despite their importance and abundance in all cell membranes and their ubiquitous nature, the exact 3-d structure of claudins has remained elusive to traditional x-ray crystallographic and nmr studies. in this investigation, a computational approach was used to determine the claudin structure of claudin 1-10. homology modeling, molecular dynamic simulations, and reverse mapping were employed to predict the protein structures with relative accuracy. understanding structure of claudin proteins and its interaction at the molecular level can lead to effective drug delivery technology. determination of optimal conditions for an isothermal titration calorimetry essay to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine (sardinops sagax caerulea) idania emedith quintero reyes 1 , francisco javier castillo y añez 1 , enrique fernando vel azquez contreras 1 , roc ıo sugich miranda 1 , david octavio corona mart ınez 1 , aldo alejandro arvizu flores 1 , ivet cervantes dom ınguez 1 1 protein kinetics determination of optimal conditions for an isothermal titration calorimetry essay to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine (sardinops sagax caerulea) trypsin is the most studied alkaline protease and it s very common to found isoforms from this protein as the case for monterey sardine (sardinops sagax caerulea); as it shows an expression of trypsin i and trypsin iii according to the cdna characterization. trypsin i was determine to be a cold adapted enzyme as it shows a higher catalytic efficiency (kcat/km) than the mesophilic counterparts. the kinetic parameters were obtained by spectrophotometric essays, which are not fallible for all the enzymes because native, recombinant or mutant enzyme activity could be below the detection limit of the assay, opaque or turbid solutions interfere with spectrophotometric detection, etc. alternative tools as the isothermal titration calorimetry (itc) can measure enzyme kinetics using thermal power generated by the enzymatic conversion of substrate to product; were the rate of reaction is directly proportional to thermal power. the objective of this study was to stablish the optimum conditions to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine using itc. to reach the objective trypsin i was purified from viscera of monterey sardine using molecular exclusion and affinity chromatography obtaining a yield of 1.1 mg/ml. at 208c kcat and km of tryipsin i form monterey sardine were 14.6 s-1 and 1.4 mm respectively. at 158c were 13.6 s-1 and 4 mm (kcat and km) and at 48c kcat was 0.454 s-1 and km 0.52 mm. the kinetic parameters obtained by spectrophotometric assay at 258c were kcat and km 436 s-1 and 1.8 mm respectively. at 208c the kcat was 409.7 s-1 and km 1.8 mm and at 158c kcat 288 s-1 and km 3mm. comparing the values obtained for kcat with the spectrophotometric essay were higher 29 fold than those obtained by itc and the values in km were similar by both methods. even though the differences in kcat, we can reassert the psychrophilic behavior of trypsin i as the catalytic efficiency is higher by both methodologies. in the understanding that the kinetic behavior of enzymes is important to not only understanding biochemical pathways and catalytic mechanisms but is again a fruitful area for drug discovery and development; so the itc provides a universal approach to determining the kinetic behavior of enzymes and can yield in a single experiment a complete set of kinetic parameters for an enzyme-catalyzed reaction that can be applied for the different alkaline proteases from pyloric caeca of monterey sardine (sardinops sagax caerulea). mysterious world of stress-responding sigma factors in bacillus subtilis olga ramaniuk 1 1 protein-dna interaction bacterial transcription is mediated by the rna polymerase holoenzyme containing sigma factors -essential proteins for the initial step of transcription that recognize and bind to promoter dna. the primary sigma factor is essential in exponential phase of growth while alternative sigma factors are active during transcription under stress conditions. this project has three main aims. the first aim is to explore the binding properties of b. subtilis alternative sigma factors; specifically, whether sigma factors lacking the autoinhibitory domain 1.1 can bind to promoter dna in the absence of rnap. the second aim explores whether rnap associated with alternative sigma factors is regulated by the concentration of the initiation nucleoside triphosphate. the third aim is to define the regulon of sigma i. in order to achieve our aims, 7 out of 17 alternative sigma factors were successfully purified using affinity chromatography and ion exchange chromatography. we set up in vitro transcription system with selected sigma factors and initiated experiments with sigma i regulon determination. results named above and our future findings will help to better understand gene expression regulation on the level of transcription initiation. this work was supported by grant no. p305-12-g034 from the czech science foundation. assessing the costs and benefits of protein aggregation protein aggregation and cell fitness protein aggregation has been associated with numerous diseases but also with important cellular functions such as epigenetic inheritance. here we present a population genetics approach to infer the costs and benefits of protein aggregation on cell fitness. this information is crucial to understand how cellular systems tolerate the formation of protein deposits and which factors modulate this event. using our experimental system, we measured different protein aggregation effects (deleterious, neutral or beneficial) within the same genomic background. single cell analyses, within the same population, showed stochastic variability in the aggregate's size and in its effect on cell fitness. our data indicates that, in certain conditions, protein aggregation can enhance population variability and survival expectancy. overall, these results suggest that the presence and formation of protein aggregates could be almost harmless whereas the associated gain and loss of function are critical for the cell. revealing the key role of negatively charged residues of heme sensor proteins involved in geobacter sulfurreducens' signal transduction pathways marta a. silva 1 , telma c. santos 1 , teresa catarino 2 , carlos a. salgueiro 1 1 ucibio-requimte, departamento de qu ımica, fct-unl., 2 instituto de tecnologia qu ımica e biol ogica, unl signal transduction proteins bacterial chemotaxis systems sense and regulate the microbe mobility in response to environmental conditions. such mechanisms constitute a striking example of cell motility to gain advantages for cell survival and permit the bacteria to fill important niches in a diversity of anaerobic environments [1] . geobacter sulfurreducens (gs) is an anaerobic bacterium with a considerable respiratory versatility whose genome encodes for an unusual family of methyl-accepting chemotaxis proteins (mcp), each containing at least one heme c-binding motif [2] . these sensor proteins, gsu0582 and gsu0935, are involved in signal transduction pathways mediated by chemotaxis-like systems [3] . the thermodynamic and kinetic characterization of the sensors gsu0582 and gsu0935 by visible spectroscopy and stopped-flow techniques, at several ph and ionic strength values revealed that sensor gsu0935 midpoint reduction potentials are lower than those of gsu0582 at all ph and ionic strength values and the same were observed for the reduction rate constants [4] . the origin of the different functional properties of these closely related sensor domains are rationalized in the structural terms showing that gsu0935 has two extra negatively charged residues in the vicinity of the heme group, which have no counterpart in gsu0582: glu89 and asp57. residue asp57 is less exposed compared to glu89 and it was suggested that its carboxylic group might have a role in the modulation of the heme reduction potential of gsu0935. to investigate this, both residues were replaced by a positively charged amino acid (lysine) and by a neutral one (asparagine or glutamine). for the mutants with enough expression, a functional characterization was carry out, using several spectroscopic techniques, including uv-visible and cd, together with kinetics and potentiometric measurements. significant changes on the reduction potential values are observed when a negative charge is replaced by a positive one at position 57 or 89. therefore, the decrease of the reduction potential in asp57 and glu89 mutants reinforces the hypothesis that the higher reduction potential observed for heme sensor domain gsu0582 is related with the less negative electrostatic surface around the heme. this work provides, for the first time, evidence for the co-existence of two similar methyl-accepting chemotaxis proteins functioning in different working potential ranges. these proteins are responsible to allow geobacter sulfurreducens triggering an adequate cellular response in different anoxic subsurface environments. 1 national autonomous university of mexico, faculty of medicine, 2 national autonomous university of mexico, faculty of chemistry, 3 national autonomous university of mexico, institute of chemistry molecular evolution the glycolytic enzyme triosephosphate isomerase (tim) is an oligomeric (b/alpha)8 barrel that catalyses the interconversion of d-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate in a diffusion-limited reaction. although each subunit has its own active site, naturally occurring monomeric tims have not been reported; in fact, monomer association is very tight. tim topology is well conserved among the three domains of life. nevertheless, their folding mechanism and inhibition properties vary across species. comparative studies of proteins have proved to be very useful in understanding the relationship between sequence and physicochemical properties, however, they lack the capacity to give a more integrative and evolutive correlation. in order to elucidate how the catalytic properties, the oligomerization state and the stability of extant tims arose, in this work we examined the molecular history of eukaryotic tim through ancestral protein reconstruction methods (maximum likelihood) and the subsequent physicochemical characterization of the resurrected enzymes. we first characterized in detail the protein corresponding to the last common ancestor of animals and fungi (tim63). the cd and fluorescence spectra of tim63 are similar to those of extant tims. secondary structure is lost in a cooperative transition with tm 5 68.78c. the enzyme loses activity upon dilution suggesting that only the dimer is active. dilution experiments followed by isothermal titration calorimetry indicate that dissociation enthalpy is small; moreover the heat capacity change observed is three times higher than the one predicted for a rigid body dissociation process, suggesting partial unfolding of the monomers. when compared with extant tims, the catalytic efficiency of tim63 is reduced 10-fold, whereas binding of pgh, a transition-state analogue, shows a similar thermodynamic signature. these data indicate that although monomer association may have been less tight in ancestral tims, catalysis has been always linked to oligomerization. analysis of the crystal structure of tim63, obtained at 1.9 å resolution, suggests that the lack of four salt bridges observed in the interface of extant tims is responsible for the low dimer stability. in order to test this hypothesis we also studied the stability of four younger reconstructed ancestors that acquired the salt bridges in two different phylogenetic lineages. we found a correlation between the appearance of stabilizing interactions in the interface, dimer stability and catalysis; suggesting that these salt bridges are partially responsible for extant dimer stability and shed light on the dimeric nature of extant tims. receptor protein-tyrosine phosphatases: dimerization, receptor kinase interaction and allosteric modulation elizabeth dembicer 1 , damien thevenin 1 1 department of chemistry, lehigh university theme: receptor tyrosine kinase and receptor protein phosphatase signaling many cell-signaling events are regulated through reversible tyrosine phosphorylation of proteins, which is controlled by the counterbalanced actions of two key enzyme families: protein tyrosine kinases and protein tyrosine phosphatases. interestingly, both families include transmembrane receptor-like enzymes, namely the receptor tyrosine kinases (rtks) and the receptor-like ptps (rptps). while the regulation and actions of many rtks are well characterized, the mechanisms controlling the enzymatic activity of rptps and how they interact with their substrates remain to be fully explained. thus, understanding how these receptors function and interact will give fundamental insights into how tyrosine phosphorylation is finely tuned in cells, and how it can be modulated. increasing evidence indicates that rptps, like rtks, are regulated by homodimerization. however, it appears that homodimerization inhibits the activity of most rptps. even though the transmembrane (tm) and the juxtamembrane domains have been proposed to be involved in this process, there is no clear structure-based proposal for the role of these regions. moreover, several rptps have been identified as candidate regulators of rtks. in particular, the receptor-type tyrosine-protein phosphatase eta (ptprj; also known as dep1 or cd148) is capable of attenuating egfr tyrosine phosphorylation. physical interactions of egfr with ptprj at the cell surface have been documented, but the basis for these interactions is unknown. here, using a dominant-negative transcriptional activator-based assay (dn-aratm), and mutagenesis analysis, we show that: (1) ptprj has a strong tendency to homodimerize, (2) ptprj heterodimerizes with egfr through tm-tm interactions, (3) these interactions are mediated by specific residues, and can be modulated by the delivery of peptide binders. this work represents the first structure-function study of rptp-rtk interaction, and may not only result in significant progress towards a better understanding of the basic biology of rptps in cancer cells, but also offer new possibilities for targeting protein tyrosine phosphatases for therapeutic modulation of egfr in oncology. inhibiting egfr dimerization and signaling through targeted delivery of juxtamembrane domain peptide mimics using phlip anastasia thevenin 1 , kelly burns 1 , janessa guerre-chaley 1 , damien thevenin 1 1 regulating receptor tyrosine kinase signaling the elevated phosphorylation of key regulatory tyrosines on oncogenic signaling proteins that result from aberrant protein tyrosine kinases activity plays well-abstract established roles in promoting tumorigenesis and in the high frequency with which resistance arises to existing therapeutic treatment. for instance, this is the case for the epidermal growth factor receptor (egfr). thus, there is a clear need for novel specific targeting methods to inhibit the activity of receptor protein tyrosine kinases, such as egfr, in cancer. egfr becomes activated upon ligand binding to the extracellular domain, leading to receptor dimerization. the juxtamembrane (jm) domain of egfr is critical for intrinsic tyrosine kinase activity and receptor dimerization by stabilizing the active conformation of egrr through the formation of a antiparallel helical dimer. therefore, peptides mimicking the jm domain -if specifically delivered to cancer cells -have the potential to prevent egfr dimerization, receptor activation, downstream signaling, and thus to attenuate aberrant egfr activity in cancer cells. here, phlip (ph low insertion peptide), a peptide that can selectively target cancer cells and tumors based solely on their extracellular acidity, is used to selectively translocate the jm domain of egfr in cancer cells to prevent egfr dimerization. at ph above 7, phlip is soluble and unstructured, however, when exposed to lower ph such as observed in tumors, phlip inserts as a transmembrane (tm) alphahelix, allowing the direct translocation of cargo molecules into the cytoplasm. using the dominant negative arac-based transcriptional reported assay (dn-aratm), which assesses jm and tm domain interactions in cells membranes of e. coli, we show that phlip-jm is able to disrupt egfr dimer by 50%. current work is focused on testing the ability of such phlip-jm peptide conjugate to perturb egfr homodimerization and decrease downstream signaling through soluble kinases, such as akt and erk, in cancer cells. the thumb subdomain of yeast mitochondrial rna polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding gilberto velazquez 1 , luis brieba 2 , rui sousa 3 1 universidad de guadalajara, 2 langebio cinvestav, 3 university of texas healthsscience center at san antonio dna protein interaction abstract single subunit rna polymerases have evolved two mechanisms to synthesize long transcripts without falling off a dna template: binding of nascent rna and interactions with an rna:dna hybrid. mitochondrial rna polymerases share a common ancestor with t-odd bacteriophage single subunit rna polymerases. herein we characterized the role of the thumb subdomain of the yeast mtrna polymerase gene (rpo41) in complex stability, processivity, and fidelity. we found that deletion and point mutants of the thumb subdomain of yeast mtrna polymerase increase the synthesis of abortive transcripts and the probability that the polymerase will disengage from the template during the formation of the late initial transcription and elongation complexes. mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric template and, unexpectedly, thumb subdomain deletions decrease the binding affinity for mitochondrial transcription factor (mtf1). the latter suggests that the thumb subdomain is part of an extended bindingsurface area involved in binding mtf1. design principles of membrane protein structures vladimir yarov-yarovoy 1 , diane nguyen 1 1 membrane protein structure membrane proteins play key role in cellular signaling and ion transport. statistical analysis of expanding database of high-resolution membrane protein structures in protein data bank (pdb) provides useful information about membrane protein structure and function. we used rosettamembrane software (yarov-yarovoy v et al (2006) proteins) to analyze 300 unique alpha helical membrane protein structures in pdb and derive knowledge based energy function for membrane protein structure prediction, membrane protein-protein docking, and membrane protein design. the rosettamembrane residue environment energy term is based on amino acid propensities in hydrophobic, interface, and water layers of the membrane and depends on the residue burial state -from being completely buried within a protein environment to being completely exposed either to the lipid or water environments. residue buried state is determined from the number of residue neighbors within 6 and 10 å spheres. the rosettamembrane residue-residue interaction term is based on the propensities of amino acid pairs to be in close proximity to each other within hydrophobic, interface, and water layers. results of our statistical analysis reveal fine details of favorable and unfavorable environments for all amino acids types in all membrane layers and residue burial states. we find that large hydrophobic amino acids are favorable facing the hydrophobic core of the lipid bilayer. small amino acids are favorable facing the protein core within the hydrophobic layer of the membrane. aromatic or positively charged amino acids and favorable facing the lipid head groups. residue-residue interactions are often favored between polar and charged amino acids and also between some of small and large hydrophobic amino acids inside of the protein core within the hydrophobic layer of the membrane. these data will be useful for rational design of novel membrane protein structures and functions. coordinated gripping of substrate by subunits of a aaa1 proteolytic machine ohad yosefson 1 , andrew nager 1 , tania baker 1 , robert sauer 1 1 protein quality control' or 'protein degradation' hexameric aaa1 protein-remodeling machines use conserved loops that line the axial pore to apply force to substrates during the mechanical processes of protein unfolding and translocation. an open question in the aaa1 field is whether pore loops from different subunits of the hexameric ring grip the substrate coordinately (all six subunits involved), independently (one subunit at a time involved), or partially coordinated (two or three subunits at a time). to answer this question, we studied covalently linked hexamers of the e. coli clpx unfoldase bearing different numbers and configurations of wild-type and mutant pore loops and challenged these variants with protein substrates with a broad range of stabilities. we find that successful unfolding of increasingly resistant substrates requires the coordinated action of a greater number of wild-type pore loops. our results support a mechanism in which a power stroke initiated in one subunit of the clpx hexamer results in the simultaneous movement of all six pore loops, which coordinately grip and apply force to the substrate. structure and function of the toc159 m-domain, and its role in targeting the preprotein receptor to the chloroplast outer envelope membrane matthew smith 1 , shiu-cheung lung 2 , prem nichani 1 , nicholas grimberg 1 , j. kyle weston 1 , shane szalai 1 , simon chuong 2 1 deartment of biology, wilfrid laurier university, 2 department of biology, university of waterloo chloroplast biogenesis and function rely on the import of thousands of nucleus-encoded preproteins from the cytosol. preprotein import is supported by the toc and tic (translocon at the outer and inner envelope membranes of chloroplasts) complexes, which work cooperatively to translocate preproteins across the double-membrane envelope to the chloroplast interior. toc159 is one of the preprotein receptors of the toc complex, is also encoded in the nucleus and post-translationally targeted to the chloroplast, and is comprised of 3 distinct domains: 1) the intrinsically disordered n-terminal acidic (a-) domain; 2) the central gtpase (g-) domain; and 3) the c-terminal membrane (m-) domain that anchors the protein to the chloroplast outer membrane (com) through an unknown mechanism. the m-domain has no known homologues and does not contain a predicted trans-membrane domain, but does contain intrinsic chloroplast targeting information at the extreme c-terminus. the m-domain also contains a predicted b-helix motif, which may be important for anchoring the protein to the com. we are interested in characterizing the structure of the m-domain and determining the nature of its association with the com, as part of our larger goal of understanding the role toc159 plays in protein import into chloroplasts. we are also interested in defining the precise nature of the targeting information contained within the extreme c-terminus of toc159, elucidating the targeting pathway that is used, and whether other com proteins use this pathway. we will present our most recent data on the structure, function and targeting of the toc159 m-domain. structural investigation of nlpc/p60 protein acquired by trichomonas vaginalis through a lateral gene transfer event jully pinheiro 1,2 , augusto simoes-barbosa 1 , david goldstone 2 1 microbiology, school of biological sciences, university of auckland, 2 structural biology, school of biological sciences, university of auckland trichomonas vaginalis is an extracellular flagellated protozoan parasite that causes the most common non-viral sexually transmitted disease, with approximately 200 million cases worldwide annually. nevertheless, the biochemical processes behind t. vaginalis infection and its interaction with the vaginal microbiota are still not well defined. in 2007 the draft genome sequence of trichomonas vaginalis strain g3 was described, identifying 60,000 protein-coding genes. of these, nine genes encode nlpc/p60-like members. this superfamily is widely represented in the different kingdoms of life and has diverse enzymatic functions, such as amidases, endopeptidases and acetyltransferases. previous studies have shown that members of this superfamily hydrolyze specific peptide linkages in bacterial cell walls affecting germination, vegetative growth, sporulation and division or cell lysis/invasion. as a typical eukaryote, the protozoan parasite t. vaginalis does not have a cell wall itself. previous studies suggest that the t. vaginalis nlpc/p60 genes were acquired via lateral gene transfer from bacteria and must have an important function, possibly controlling the vaginal microbiota and aiding parasite invasion and infection. to investigate the function of the nlpc/p60 family of proteins in t. vaginalis we have expressed, purified and crystallized a member tvag_119910 and report its three-dimensional structure, determined at 1.5 å resolution, by x-ray diffraction. the structure of the protein reveals a typical papain-like fold resembling peptidoglycan hydrolases from the nlpc/p60 family with a conserved cysteine and histidine; forming the catalytic residues. the protein contains two bacterial sh3 domains at the n-terminus. this domain acts as a general binding domain and is likely to aid the interaction of the nlpc/p60 domain with substrate components. combined with biochemical and enzymatic characterization, the structure of this nlpc/p60 protein will help to elucidate the molecular origin of its hydrolase activity and to decipher their putative role in the parasite infection. novel dna polymerases from red sea brine-pools: new potential polymerases for pcr application masateru takahashi 1 , etsuko kimura 1 , mohamed salem 1 , ulrich stingl 1 , samir hamdan 1 1 protein biotechnology the polymerase chain reaction (pcr) is a key tool in medical and biological research. the most common pcr reaction relies on the thermal cycling method that consists of repeated cycles of heating and cooling steps for dna melting and extension by the dna polymerase, respectively. the introduction of new dna polymerases to the market is a major area of development that tremendously helped in improving the performance and quality of pcr. nonetheless, pcr still requires optimization of salt and metal ion concentrations leaving a room in the market for introducing new dna polymerases that are robuster in their salt and metal ion concentration dependence. in this study, we will present the characterization of a novel archaeal dna polymerase from the red sea brine-pool (termed br3) and demonstrate how its enzymatic activity reflects on every aspects of the environment of the brine-pool -high tolerance to concentrations and types of salts and metal ions including utilization of zn21 ions in its active site. these results suggest that the brine-pool microorganisms are likely to contain novel chemical pathways to deal with its exterior harsh conditions. we will further show the mechanism of br3 polymerase how it was adjusted to be active in harsh condition. structural basis for the identification of the n-terminal domain of coronavirus nucleocapsid protein as an antiviral target ming-hon hou 1 , shing-yen lin 1 , chia-ling liu 1 , yu-ming chang 2 , jincun zhao 3 , stanley perlman 3 1 institute of genomics and bioinformatics, national chung hsing university., 2 institute of biological chemistry, academia sinica., 3 department of microbiology, the university of iowa drug discovery coronaviruses (covs) cause numerous diseases, including middle east respiratory syndrome and severe acute respiratory syndrome, generating significant health-related and economic consequences. covs encode the nucleocapsid (n) protein, a major structural protein that plays multiple roles in the virus replication cycle and forms a ribonucleoprotein complex with the viral rna through the n protein's nterminal domain (n-ntd). using human cov-oc43 (hcov-oc43) as a model for cov, we present the 3d structure of hcov-oc43 n-ntd complexed with ribonucleoside 5'-monophosphates to identify a distinct ribonucleotide-binding pocket. by targeting this pocket, we identified and developed a new coronavirus n protein inhibitor, n-(6-oxo-5,6-dihydrophenanthridin-2-yl)(n,n-dimethylamino)acetamide hydrochloride (pj34), using virtual screening; this inhibitor reduced the n protein's rna-binding affinity and hindered viral replication. we also determined the crystal structure of the n-ntd-pj34 complex. on the basis of these findings, we propose guidelines for developing new n protein-based antiviral agents that target covs. thermal and structural stability of ß-glucosidases gh1 maira artischeff frutuoso 1 1 departamento de bioqu ımica do instituto de qu ımica da universidade de são paulo enzymology we compared the stability of thermophilic b-glucosidases gh1 to mesophilic ones in the presence of denaturants as urea and high temperature by following the transitions between the native and unfolded states by tryptophan fluorescence, enzymatic activity and differential scanning fluorimetry (dsf). the bacterial b-glucosidases (bgla) and (bglb) of the mesophile paenibacillus polimyxa and bglucosidase (bglthm) of the thermophile thermotoga maritima were expressed as recombinant proteins in novablue (de3) and purified by affinity chromatography (ni-nta resin). these recombinant enzymes have very similar folding type structure (b/a)8 barrel, as shown in crystal structures and exhibited a characteristic peak between 330 and 340 nm in the tryptophan fluorescence spectra, indicating that those proteins are folded. circular dichroism analysis in the far-uv region (190 nm to 240 nm) also showed typical spectra of folded proteins with secondary structure composition of 47% of a-helix and 13% of b-sheets for bgla, 61% of a-helix and 2.5% of b-sheets for bglb and 30% of a-helix and 20% of b-sheets for bglthm. the average degree of accessibility to the exposed tryptophan residues in the native enzyme to increasing concentrations of the acrylamide suppressor (stern-volmer constant -ksv) is greater to bgla (9.49), but similar to bglb (3.17) and bglthm (3.84). the thermal stability determined by dsf was higher for bglb (tm 43.8 c) than for bgla (tm 35. 2 c) . the bglthm was stable at 478c and remained stable for up to 4 h at 808c. in addition the thermal inactivation kinetics at 478c evaluated by the relative remaining activity showed that bgla denaturation (kinactivation of 1.9 s-1) is faster than bglb (kinactivation of 31.3 s-1). on the other site, bglthm inactivation at 958c was a two-step process, which exhibited an initial fast step (kinactivation of 2.9 s 21) followed by a slow step (kinactivation of 0.2 s-1). the chemical denaturation by urea followed using tryptophan fluorescence showed a transition pl-039 covalent structure of single-stranded fibrinogen and fibrin oligomers cross-linked by fxiiia. the influence of free radical oxidation anna bychkova 1 , vera leonova 1 , alexander shchegolikhin 1 , marina biryukova 1 , elizaveta kostanova 1 , mark rosenfeld 1 1 n. m. emanuel institute of biochemical physics, russian academy of sciences protein structure and function native fibrinogen is a key blood plasma protein whose main function is to maintain hemostasis by virtue of producing the cross-linked fibrin clots under the effect of thrombin and fibrin-stabilizing factor (fxiiia). fxiiia-mediated isopeptide g-g bonds are known to be produced between g polypeptide chains of adjacent fibrinogen or fibrin molecules. but there are apparently conflicting ideas regarding the orientation of g-g bonds. in this study several peculiarities of self-assembly of fibrin(ogen) and induced oxidation of the proteins have been studied with the aid of elastic and dynamic light scattering, uv-, ftir-and raman spectroscopy methods. in the presence of fxiiia both the non-oxidized and oxidized fibrinogen molecules has been shown to bind to each other in the "endto-end" fashion to form the flexible covalently cross-linked fibrinogen homopolymers. to identify the orientation of g-g bonds in fibrin protofibrils a novel approach based on self-assembly of soluble cross-linked fibrin protofibrils and their dissociation in the urea solution of moderate concentrations has been applied. the results of elastic and dynamic light scattering coupled with analytical ultracentrifugation indicated the protofibrils to exhibit an ability to dissociate under increasing urea concentration to yield single-stranded structures entirely brought about by g-g bonds. the results of this study provide an evidence to support the model of the longitudinal g-g bonds that form between the g chains end-to-end within the same strand of a protofibril. since fibrinogen is known to be sensitive to ros the mechanisms of fibrinogen and fibrin self-assembly under induced oxidation have been investigated. in both cases the polypeptide chains of the oxidized fibrin(ogen) proved to be involved in the enzymatic cross-linking more readily than those of unaffected molecules. the enhancing role of the d:d interaction under oxidation could be considered as an compensatory mechanism in the assembly of fibrin when the d:e interaction is impaired. the experimental data on fibrinogen and fibrin oxidation acquired in the present study, being combined with our earlier findings, make it reasonable to suppose that the spatial structure of fibrinogen could be evolutionarily adapted to some ros actions detrimental to the protein function. the study was supported by the rfbr, research projects 14-04-31897mol_a and 15-04-08188a. structural and thermodynamic analysis of co-stimulation receptor cd28 phosphopeptide interactions with grb2, gads, and pi3-kinese sh2 domains in addition to the signaling produced by the binding of antigen-major histocompatibility complex to tcell receptors, co-stimulatory signals from other receptor-ligand interactions are required for full activation of t-cells. the cd28 receptor on the t-cell surface has been well characterized, and the binding of ligand to cd28 is critical for producing co-stimulatory signals. cd28 has no enzymatic activity and its cytoplasmic region consists of 41 amino acids that contain the sequence ymnm, in which the tyrosine residue is phosphorylated by kinase. the phosphorylated sequence, pymnm, is recognized by src homology 2 (sh2) adaptor proteins, such as growth factor receptor binding protein 2 (grb2), grb2related adaptor downstream (gads), and the phosphatidylinositol 3-kinase (pi3-kinase) regulatory subunit, p85. the consensus sequence for the binding of grb2 sh2 and gads sh2 is pyxnx, and that of p85 n-terminus sh2 (nsh2) and c-terminus sh2 (csh2) is pyxxm. we reported the high-resolution crystal structure of grb2 sh2 in complex with the cd28 phosphopeptide [higo et al., plos one 8, e74482, 2013] , and recently determined those of gads sh2, p85 nsh2, and p85 csh2. these data along with the results of binding thermodynamics analyzed using isothermal titration calorimetry, helped to elucidate the molecular recognition mechanisms of cd28 by adaptor proteins. the sh2 proteins were overexpressed in escherichia coli, and were purified using affinity and gel-filtration chromatography. the cd28 phosphopeptides, 8-residue (octp) and 12-residue (ddcp02), were synthesized using the solidphase supported technique, and were purified using reversed-phase chromatography. the crystals were obtained by the hanging-drop vapor diffusion method. x-ray diffraction data were collected at synchrotron radiation facilities, and the structures were determined by the molecular replacement method. the models of grb2 sh2, gads sh2, p85 nsh2, and p85 csh2 in complex with octp were refined at 1.35, 1.2, 1.0, and 1.1 å resolutions, respectively. the crystal structures showed that the phosphotyrosine phosphate moiety directly interacted with the side-chain of arginine in sh2, which is common in all complex structures. in the grb2 sh2 and gads sh2 complexes, the side-chain of asparagine at the py12 position forms a pair of hydrogen bonds with the main-chain amide and carbonyl groups of lysine in sh2. alternatively, in the p85 nsh2 and csh2 complexes, the side-chain of methionine at the py13 position is located in hydrophobic pockets of nsh2 and csh2, in which the hydrophobic interactions of csh2 would be stronger than those of nsh2. this idea is supported by the observed binding thermodynamics. the binding affinity of csh2 to ddcp02, because of a favorable enthalpy change, is about 10-fold higher than that of nsh2. the binding affinity of grb2 sh2 to ddcp02 is similar to that of gads sh2 to ddcp02, and is about 10-fold lower than that of nsh2 to ddcp02. these results indicate that the contribution of hydrophobic interactions of nsh2 and csh2 at the py13 position are stronger than those of hydrogen bonds of grb2 sh2 and gads sh2 at the py12 position. novel kinetochore protein complex from silkworm holocentric chromosomes takahiro kusakabe 1 , hiroaki mon 1 , jaeman lee 1 1 the kinetochore, which consists of centromere dna and a multilayered protein complex, plays important roles in chromosome organization and segregation. interactions between chromosomes and spindle microtubules allow chromosomes to congress to the middle of the cell, and to segregate the sister chromatids into daughter cells in mitosis, which is followed cytokinesis. in contrast to monocentric chromosomes, in which the centromere is normally present at a single region on each chromosome, the holocentric chromosomes have centromeric activity along the entire length of the chromosome. it has been known that the silkworm, bombyx mori, has holocentric chromosomes since 1970s, none of silkworm kinetochore proteins, however, have been identified so far. here we report the identification of a novel set of genes for outer kinetochore proteins in silkworm by using bioinformatics and rna interference-based screening. under the hypothesis that depletion of essential kinetochore genes causes cell cycle arrest in mitosis, we performed rnai in the silkworm cell line, bmn4-sid1, targeting a set of candidate genes. knockdown of five genes caused significant cell cycle arrest at the g2/m phase. we also found that these five proteins make a complex, and that all of them are localized along the chromosome arms, indicating that the silkworm kinetochore extends along the chromosome. inactivation of bine aldehyde dehydrogenase from spinach by its physiological substrate bine aldehyde to contend with osmotic stress caused by drought, salinity, or low temperatures some plants synthesize the osmoprotectant glycine bine (gb) from bine aldehyde (bal). the last step-the irreversible nad1dependent oxidation of bal-is catalyzed by aldh10 enzymes that exhibit bine aldehyde dehydrogenase (badh) activity. we here report that the spinacia oleracea badh (sobadh) is reversibly inactivated by bal in the absence of nad1 in a time-and concentration-dependent mode to approximately 50% of the original activity. inactivation kinetics are consistent with a partial reversible, two-steps mechanism that involves the formation of an active non-covalent enzyme•bal complex before formation the inactive enzyme-bal complex. crystallographic evidence indicates that in the enzyme previously inactivated by bal the aldehyde forms a thiohemiacetal with the nonessential cys450 (sobadh numbering) located at the aldehyde-entrance tunnel, thus totally blocking the access to the catalytic cysteine. accordingly, bal does not inactivate the c450s sobadh mutant. two crystal structures of the inactivating enzyme-bal complex showed that the trimethylammonium group of bal is inside the active-site aromatic box, as in the productive way of binding. this explains why the inactivation of the a441i mutant-where the binding of the trimethylammonium group is hindered-requires non-physiologically high bal concentrations, while the a441c mutant-where the binding is allowed-is inactivated similarly to the wildtype enzyme. cys-450 is conserved in most plant aldh10 enzymes of known sequence, and in all of them with proven or predicted badh activity. inactivation by bal appears therefore to be a common feature of plants badhs. this short-term regulation may be of great physiological importance since the irreversibility of the badh-catalyzed reaction would unbalance the nad1/nadh ratio if the aldehyde concentrations are high, the nad1 concentrations low and the reaction is not slowed down. plants badhs are prone to this situation since they work under osmotic stress conditions, when high bal concentrations are required for the synthesis of high levels of the osmoprotectant gb. the partial nature of the decamer possesses a donut shaped structure with 10 calcium ions on the surface available for interactions with carbohydrate molecules. binding specificity was evaluated for 20 carbohydrates using differential scanning fluorimetry (dsf) that showed bjcul interacts with galactose and lactose but less with glucose and sacarose. surprisingly, high levels of thermostabilization of bjcul was achieved with the antibiotic aminoglycosides geneticin (g418) and gentamicin in a calcium concentration dependent manner, but not kanamycin. intriguingly, while lactose and galactose inhibited erythrocyte agglutination by bjcul, g418 and gentamicin did not affect hemagglutination implying a second site of binding. dsf analysis also suggested the presence of a second binding site for the antibiotics and crystallization of the complexes are in progress in order to understand fully this new binding mechanism of c-type lectin with antibiotics. ab initio modelling of structurally uncharacterised antimicrobial peptides mara kozic 1 1 institute of integrative biology, university of liverpool ab initio modelling of structurally uncharacterised antimicrobial peptides mara kozic 1* 1 institute of integrative biology, biosciences building, university of liverpool, crown street, liverpool l69 7zb, united kingdom * mara.kozic@liverpool.ac.uk antimicrobial resistance within a wide range of infectious agents is a severe and growing public health threat. antimicrobial peptides (amps) are among the leading alternatives to current antibiotics, exhibiting broad spectrum activity. an understanding of the structure of a protein can lead us to a much improved picture of its molecular function. furthermore, an improved understanding of structure-function relationships facilitates protein design efforts to enhance their activity. currently, the 3d structures of many known amps are unknown. to improve our understanding of the amp structural universe we have carried out large scale ab initio 3d modelling of structurally uncharacterised amps. such ab initio modelling is facilitated by the typical small size of amps as well as their tendency to contain disulphide bonds, these providing valuable additional information to simulations. preliminary results reveal unexpected similarities between the predicted folds of the modelled sequences and structures of well-characterised amps. for example, lacticin q was revealed to contain a helical bundle fold that bears a striking resemblance to enterocin 7a. we also found a remarkable similarity between the predicted structure of silkworm 001 peptide and b-hairpin amps such as tachyplesin i. our results improve the understanding of the structure-function relationship of amps. surface aggregation-propensity as a constraint on globular proteins evolution susanna navarro 1 , marta diaz 2 , pablo gallego 2 , david reverter 2 , salvador ventura 1 1 institut de biotecnologia i biomedicina and departament de bioquimica i biologia, 2 institut de biotecnologia i biomedicina, universitat aut onoma de barcelona in living cells, functional protein-protein interactions compete with a much larger number of nonfunctional interactions. theoretical studies suggest that the three-dimensional structures of present proteins have evolved under selective pressure to avoid the presence of aggregation-prone patches at the surface that may drive the establishment of anomalous protein contacts. however, no experimental evidence for this hypothesis exists so far. the a-spectrin sh3 domain (spc-sh3) has been used as a protein model to decipher the sequential aggregation determinants of proteins. here we use it to address the structural determinants of protein aggregation and their link to protein evolution. to this aim we exploit aggrescan3d (a3d), a novel algorithm developed by our group, which takes into account both protein structure and experimental data to project aggregation propensities on protein surfaces. we used a3d to design a series of spc-sh3 variants with progressively stronger aggregationprone surfaces and characterized their thermodynamic, structural and functional properties. our data support evolution acting to constraint the aggregation propensities of globular protein surfaces in order to decrease their potential cytotoxicity and the protein quality control machinery acting to buffer this negative selective pressure. utilizing 3d structure for the annotation of structural motifs in the conserved domain database narmada thanki-cunningham 1 , noreen gonzales 1 , gabriele marchler 1 , myra derbyshire 1 , james song 1 , roxanne yamashita 1 , christina zheng 1 , stephen bryant 1 , aron marchler-bauer 1 , farideh chitsaz1 1 1 conserved domain database, structure group cbb/ncbi/nlm/nih the conserved domain database (cdd) is a protein classification and annotation resource comprised of multiple sequence alignments representing ancient conserved domains. cdd protein domain models are curated by ncbi and use 3d protein structure explicitly to define domain extent and the location of conserved core structures, and to provide accurate alignments between diverse family members via structure superposition. cdd also imports external collections such as pfam and tigrfam. recently, a novel class of annotation labeled as "structural motifs" has been introduced to supplement current capabilities. these annotations define compositionally-biased and/or short repetitive regions in proteins, which are difficult to model as functional domains conserved in molecular evolution. structural motifs include transmembrane regions, coiled coils, and short repeats with variable copy numbers. for many types of short tandem repeats, a few position-specific score matrices (pssms) suffice to annotate more than 90% of the known instances of that structural motif. unfortunately, a lack of sequence similarity within coiled-coil regions prohibits the development of only a few generic models; therefore, models for coiled-coil regions in the context of specific families have been developed using the spiricoil database as a reference. increased coverage of coiled-coil regions in cdd, specific site annotations of these structural motifs as well as their representation on the webpages will be discussed. specific in vivo ultrasound imaging of e-selectin expression in tumors using a microbubble contrast agent covalently attached to the peptide ligand iellqar, known to bind to e-selectin [2] . however, it was observed that this probe has a limitation in the imaging of cardiovascular diseases where higher shear stresses prevent microbubbles from remaining attached to the target. therefore, peptides with higher eselectin affinity are needed to design probes capable of imaging these diseases. in this context, automated docking and molecular dynamics methodologies were combined and applied to different e-selectin binding peptides. these studies predicted the energetically more favorable binding mode as well as the key interactions between the peptide ligands and the e-selectin receptor. some of these peptides were prepared by solid-phase peptide synthesis and their interactions with e-selectin analyzed by surface plasmon resonance technique. the results showed that these peptides have different affinities for e-selectin. these data were correlated with the computational studies and evaluated to obtain crucial information of the key recognition elements needed for higher e-selectin affinity. these recent results will be presented. burkholderia pseudomallei is the causative agent of melioidosis, a serious invasive disease of animals and humans in tropical and subtropical areas. sedoheptulose-7-phosphate isomerase from b. pseudomallei (bpgmha) is the antibiotics adjuvant target for melioidosis. in general, bpgmha converts dsedoheptulose-7-phosphate to d-glycero-a-d-manno-heptopyranose-7-phosphate (m7p). this is the first step of the biosynthesis pathway of ndp-heptose responsible for a pleiotropic phenotype. therefore, this biosynthesis pathway is the target for searching novel antibiotics increasing the membrane permeability of gram-negative pathogens or adjuvants synergistically working with known antibiotics. the crystal of this enzyme has been solved at 1.9 å resolution. there is an active site pocket where a putative metal binding site is located. to find out inhibitors of bpgmha, in-silico virtual screening with zinc, a free database of commercially-available compounds, has been performed. tens of thousands of chemical compounds were docked into the active site of bpgmha. a number of putative bpgmha binding compounds better than m7p were found using surflex-dock included in the sybyl software package. characteristics of these compounds were surveyed and classified to identify common binding properties with bpgmha. mapping the structure of laminin using cross-linking and mass spectrometry gad armony 1 , toot moran 1 , yishai levin 2 , deborah fass 1 1 weizmann institute of science, department of structural biology, 2 weizmann institute of science, israel center for personalized medicine laminin, a 800 kda heterotrimer, is a major element in the extracellular matrix (ecm). within the ecm, laminin contributes to the adhesion and migration of cells, both in health and disease. the laminin trimer was observed by rotary shadowing electron microscopy to be cross shaped: the three short arms of the cross are formed by the amino-terminal halves of the three subunits, whereas the long arm of the cross holds the three chains together in a long coiled coil. the narrow and flexible arms of the laminin cross complicate studying its structure to high resolution by crystallography or electron microscopy single particle reconstruction. to advance our understanding of this remarkable quaternary structural assembly, we have used cross-linking and mass spectrometry to analyze the organization of the laminin trimer. this technique was validated by known crystal structures of isolated laminin domains. in all cases the crystal structure distances agree with the cross-linker length. the identified cross-links were particularly helpful in assigning the register and the subunit order of the long coiled coil due to the high content of cross-linkable residues in this region. using known x-ray crystal structures, homology modeling, and distance restraints provided by two cross-linker chemistries, a clearer picture of the laminin quaternary structure is obtained. non-sequential protein structure alignment program mican and its applications shintaro minami 1 , george chikenji 2 , motonori ota 1 1 dept. of info. sci., nagoya univ., 2 dept. of comp. schi. & eng., nagoya univ. in some proteins, secondary structure elements are arranged spatially in the same manner, but they are connected in the alternative ways. analysis on such non-sequential structural similarity in proteins is important because it provides a deeper understanding of the structural geometry of protein. this can be also observed even in the homologous proteins, indicating the non-sequential structural similarity is significant in the protein evolution. however, the non-sequential structural similarity in proteins is less investigated. we developed a novel non-sequential structural alignment program mican, which can handle multiple chains, inverse direction of chains, c$�lpha$models, alternative alignments, and non-sequential alignments. we performed comprehensive non-sequential structural comparison among homologous proteins in the same scop superfamily by using the mican program. based on the result, we found that approximately 8% of superfamilies include at least one protein pairs showing non-sequential structural similarity. 85% nonsequential structurally similar pairs are aligned in a simple way, e.g. circular permutation, $�$strand flip/ swap, but 15% are complicated. interestingly, most of such complicated non-sequential similarities can be explicable by combination of 2-4 simple non-sequential relationships. this result indicates that accumulation of simple structural changes in the course of protein evolution produces completely different fold homologs. as early as 1919, ritter surmised that the cell's molecules cooperate to form a "special apparatus and an organised laboratory". 1 despite supporting evidence from srere, mcconkey and others, efforts to understand molecular organisation in vivo are still in their infancy. however, important aspects of the cell interior have already been revealed. for example, weak molecular interactions structure the cytoplasm into time-evolving, functional zones. 2 weak interactions are difficult to capture and can preclude protein detection in cells by many biophysical techniques, including nmr spectroscopy. 3, 4 we explored the effects of cell-like milieus on the cytochrome c (cyt c)-flavodoxin (fld) interaction. these oppositely charged proteins interact weakly with a number of cognate partners. neither cyt c4 nor fld is detectable by nmr in escherichia coli confirming their "sticky" nature ( figure 1a) . the cyt c-fld interaction was assessed in buffer, 8% polyacrylamide gels and in solutions containing 100 g/l of macromolecular crowders ( figure 1b) . 1h, 15n hsqc nmr revealed that the interaction was transient in buffer, proceeding via the known binding site for both proteins. substantial line broadening was effected in crowded and confined solutions suggesting that the cyt c-fld complex is stabilised under native-like conditions. the stabilising effect of macromolecular crowders was also observed by native gel electrophoresis and crystallization. these findings coincide with spitzer and poolman's model for cytoplasmic structuring, emphasising the role of charge-charge interactions and crowding in the formation of macromolecular "clusters". 5 the implications for cytoplasmic structuring will be discussed alongside related investigations of cationic protein interactions in e. coli extracts. 3, 4 detergent:protein ratio. the transmembrane b-barrel of bama is folded in either micelles, bicelles or nanodiscs, however an n-terminally attached single potra5 domain is flexibly unfolded, due to the absence of stabilizing contacts with other protein domains. measurements of backbone dynamics show distinct time scales of dynamic behavior for bama b-barrel and parts of its extracellular loop l6, revealing high local flexibility within the the lid loop. this work presents the first high-resolution 2d solution nmr spectra of the bama barrel and establishes improved biochemical preparation schemes, which will serve as a platform for structural and functional studies of bama and its role within the bam complex. protein arginine methylation is a widespread and important posttranslational modification in eukaryotic cells, shown to be involved in the activation or repression of transcription, modification of the splicing machinery, signaling, and dna repair. mammalian protein arginine methyltransferases include a family of nine sequence-related enzymes that transfer one or two methyl groups onto the terminal guanidino groups on arginine residues, producing monomethylarginine only (mma, type iii), symmetric dimethylarginine (sdma) and mma (type ii), or asymmetric dimethylarginine (adma) and mma (type i). while prmt1, 2, 3, 4, 6, and 8 have been characterized as type i enzymes, and prmt5 as a type ii enzyme, the role and activity types of the two final members of this family of enzymes, prmt7 and prmt9, had been unclear due to conflicting results in the literature, and the substrates for these enzymes had been elusive. both prmt7 and prmt9 are distinct members of the family with two methyltransferase or methyltransferase-like domains and containing acidic residues in otherwise well-conserved substrate double e binding motif, features not seen in the other prmt enzymes. recent work in our laboratory confirmed prmt7 as the only type iii mma-forming enzyme in the group, with a unusual low temperature optimum for activity, and a heretofore not seen preference for a basic stretch of residues in an r-x-r sequence for methylation. mutations of the acidic residues in the substrate-binding motif results in a loss of the specific r-x-r activity and the appearance of a g-r-g specificity typical of many of the other prmts. the physiological substrate of prmt7 has yet to be confirmed, although histone h2b is an effective in vitro substrate. prmt9, on the other hand, had no reported activity, until immunoprecipitation from hela cells showed it pulled down two splicing factors, sf3b2 and sf3b4, in a complex. amino acid analysis showed that prmt9 methylates sf3b2 to produce both mma and sdma, thus making it the second type ii enzyme in mammals. prmt9 knockdown results in modulation of alternative splicing events. this enzyme appears to be relatively specific for the sf3b2 protein; a peptide containing the methylatable arginine residue was not found to be a substrate, and typical substrates of other prmts are not recognized by prmt9. we found that the position of the methylated arginine residue in sf3b2 is important, and the acidic residues in the substrate-binding motif also play an important role in substrate recognition. thus, prmt7 and prmt9 represent unique members of the mammalian prmt family. hydrogen peroxide levels, endogenous hormones (cytokinins, salycilic acid, as well as jasmonic acid and its conjugates), polyphenolics and terpenoids in a model system of a. alba in vitro with inhibition of rootng and stimulation of callusogenesis by means of individual and combined cytokinin and cytokinin/ auxin treatments. results: it was established that inhibition of rooting and stimulation of callusogenesis caused by benzyl adenine (ba) or combinations of ba and indole-3-butiric acid (iba) in vitro were related to elevation of sesquiterpenoids in the essential oils, as well as polyphenolics content, accompanied by a drop of stress hormones, bioactive cytokinins and preservation of oxidative stress and lipid peroxidation levels, as compared with non-treated control. individual treatments with either iba or ba, also increased the sesquiterpenoid content in the essential oil of the plant, in a concentration related manner, this effect being more profound after ba treatment. in addition, ba treated plants exhibited a drop of protein levels of the aerial samples, as well as profound differences of enzymatic activity in the callus tissues, as compared with callus of plants treated with different combinations of ba and iba. conclusion: the results of the present work indicate that alterations of endogenous phytohormonal levels, caused by exogenous plant growth regulators treatment, might be the mediator between primary and secondary metabolism by means of affecting protein levels and activity of key enzymes in vitro. three different additives ((0.1% (v/v); formic acid, acetic acid, ammonium format with formic acid) have been investigated in response to ion intensity of esi-ms for individual hnp 1-4 in saliva. kinetex v r column separation efficiency was evaluated using two different column dimensions (50 x 2.1 mm and 50 x 3 mm.) and two different stationary phases (c18 and c8). kinetex v r column (homogenous porous shell) performance was also compared to new ultra ace v r (encapsulated bonded phase) column. sample optimisation revealed that the spe method removes interference from salivary glycoproteins and consequently yields larger peak area (30-90%) for all hnps. hnps were extracted by spe with a recovery of 80-91%. the meoh: h2o: acetic acid (0.1%) provided enhanced (p>0.05) hnp1-3 ion intensities. the kinetex v r c8 (50 x 3.0 mm, 2.6 mm) column facilitated a better separation efficiency of the four hnps as compared to the ultra core super c18 ace v r (50 x 3.0 mm, 25 mm) column, the kinetex v r c18 (50 x 3.0 mm, 2.6 mm) and the kinetex v r c18 (50 x 3.0 mm, 5 mm) column. the relative levels of the hnps were determined in healthy volunteers before and after a rigorous exercise regime: it is possible that prolonged strenuous exercise will affect oral innate immunity and therefore also the level of salivary defensins. hnp1-3 are traditionally detected in an enzyme-linked immunosorbent assay (elisa) which does not discriminate between the different hnps due to their structural similarities. there has therefore been a need to develop a mass spectrometry method that will discriminate between the defensins. as part of the method validation, the hnp1-3 level was determined by elisa and the data was compared with the lc-ms data. here we present this cross-validation; the data revealed no significance difference between the two methods (r25 0.96) which confirms that the developed lc-ms method is and equal sensitive method for the detection of these potential antimicrobial markers. this method can easily be adopted for similar molecular weight of peptides as hnps and also for any other biological matrix. moonlighting proteins: relevance for biotechnology and biomedicine luis franco serrano 1 , sergio hern andez 1 , alejandra calvo 2 , gabriela ferragut 2 , isaac amela 1 , juan cedano 2 , enrique querol 1 1 institut de biotecnologia i biomedicina. universitat aut onoma de barcelona, 2 laboratorio de inmunolog ıa, universidad de la rep ublica regional norte-salto multitasking or moonlighting is the capability of some proteins to execute two or more biochemical functions. the identification of moonlighting proteins could be useful for researchers in the functional annotation of new genomes. moreover, the interpretation of knockout experiments, in which the result of a gene knocking does not produce the expected results, might be enhanced. the action of a drug can also be facilitated because it might have an off-target or side effect with somewhat hidden phenotypic traits. it would be helpful that bioinformatics could predict this multifunctionality. in the present work, we analyse and describe several approaches that use protein sequences, structures, interactomics and current bioinformatics algorithms and programs to try to overcome this problem. among these approaches there are: a) remote homology searches using psi-blast, b) detection of functional motifs and domains, c) analysis of data obtained of protein-protein interaction databases (ppis), d) matches of the sequence of the query protein to 3d databases (i.e., algorithms like pisite), e) mutation correlation analysis between amino acids using algorithms like mistic. remote homology searches using psi-blast combined with data obtained from interactomics databases (ppis) have the best performance. structural information and mutation correlation analysis can help us to map the functional sites. mutation correlation analysis can only be used in very specific situations because it requires the existence of a multialigned family of protein sequences, but it can suggest how the evolutionary process of second function acquisition took place. we have designed a database of moonlighting proteins, multitaskprotdb (http:// wallace.uab.es/multitask/). from this database we determine the frequencies of canonical and moonlighting coupled functions (being an enzyme and a transcription factor the highest), the percentage of moonlighting proteins involved in human diseases (65% of the human moonlighting proteins in the database) and the percentage of moonlighting proteins acting as a pathogen virulence factor (20% of the moonlighting proteins in the database). correlation between potential human neutrophil antimicrobial peptides (hnp 1-3) and stress hormones in human saliva nadia ashrafi 1 , frank pullen 1 , birthe nielse 1 , cris lapthorn 1 , fernando naclario 2 1 university of greenwich (faculty of engineering and sciene), 2 university of greenwich (centre of sports science and human performance) numerous studies have investigated the effect of exercise on mucosal immunity but the focus has mainly been on salivary immunoglobulins lysozymes and hormones (cortisol, testosterone). this is not surprising given that iga and igg are the predominant immunoglobulins in saliva and there is a relationship between mucosal immunity and upper respiratory illness. it is well known that physical and mental stress provoke the release of cortisol from hypothalamic pituitary adrenal axis, by which stress can modulate various immune responses. in general, cortisol and growth hormones helps to induce the activation of neutrophils. to date, this study represents the first study that investigated the correlation between human neutrophil alpha defensins family against cortisol (stress hormone) and testosterone (growth hormone) in human saliva before and after exercise or training. twelve resistance trained athletes volunteered to participate in the study. participants consumed supplements during exercise and the hnp 1-3, cortisol and testosterone response was investigated pre, post 30 and 60 minutes of the workout. the correlation between salivary antimicrobial peptide (hnp 1-3) and stress hormone (cortisol and testosterone) has been investigated using elisa. cortisol showed no significant (p 5 0.818) difference for (pre to 30 min post) between cho and pl (cho: 483.07 6 912.77 ng/ml; pl5 583.82 6 1134.33 ng/ml) conditions but a strong trend (p 5 0.074) was observed for (pre to 60 min post) post (cho: 1023.19 6 1500.40 ng/ml; pl5 1480.33 6 2214.80 ng/ml) condition. testosterone showed no significant (p 5 0.167; p 5 0.156) difference for (pre to 30 min post) between cho and pl (cho: 23.32 6 44.11 ng/ml; pl5 10.40 6 14.19 ng/ml) and for (pre to 60 min post) post (cho: 26.42 6 19.11 ng/ml; pl5 23.72 6 17.91 ng/ml) condition. hnp 1-3 showed no significant (p 5 0.348) difference for (pre to 30 min post) between cho and pl (cho: 72.26 6 148.82; pl5 125.20 6 70.00) conditions but significant difference (p 5 0.026) was observed for (pre to 60 min post) between cho and pl (cho: 35.18 6 182.69; pl5 228.74 6 151.63) condition. the present findings suggested that there is no correlation between salivary hnp 1-3 and cortisol for (pl: r2 5 0.02 and cho: r2 5 0.01); hnp 1-3 and testosterone (pl: r2 5 0.20 and cho: r2 5 0.10). a worth note from previous study which suggested that using murine skin model (an increase in endogenous glucorticoids (cortisol) by physiological stress reduced mrna levels of antimicrobial peptide (cathelicidin). it is not clear that the correlation between hormones and antimicrobial peptide has been affected by the time interval of the exercise. both cortisol and antimicrobial peptide demonstrated a transient increase after exercise but it is surprising that they are not correlate to each other. one of the hypothesis from the present finding could be cortisol responses slow and it will be interesting to do further research with longer interval. the second hypothesis demands a further investigation to determine the synergism between substances. school of biomolecular and biomedical science, conway institute, ucd., 2 king saud university, sciences, biochemistry department. the crystal structure of a human glucose 6-phosphate dehydrogenase (g6pd) shows that each subunit has two nadp1 sites; in addition to a catalytic site there is a "structural" site which is distant from the catalytic coenzyme site. mutations causing severe deficiency tend to cluster round and close to the dimer interface and the structural nadp1, indicating that the integrity of these areas is important for enzyme stability and therefore for maintenance of activity. in order to understand the molecular basis of g6pd deficiency, and to have a clearer indication about the role of some features of the threedimensional structure, a fuller study of the second, "structural" nadp1 binding site is needed. human g6pd controls the first committed step in the pentose phosphate pathway. it catalyses the oxidation of glucose 6-phosphate to gluconolactone 6-phosphate, generating nadph which is essential, amongst other things, for protection against oxidative stress. the human enzyme can be active in dimer or tetramer forms. human g6pd of "structural" nadp1 per subunit of enzyme. this tightly-bound nadp1 can be reduced by g6p, probably following migration to the catalytic site. the importance of nadp1 for stability is explained by the structural nadp1 site, which is not conserved in prokaryotes. after removing the tightly bound "structural" nadp1 the enzyme is still active but not stable. the effects of different nadp1 fragments on the stability of human recombinant g6pd have been investigated. nadp1 is crucial for the long term stability of human g6pd, and only one of nadp1 analogues which is adenosine diphosphate ribose -2'-phosphate was able to slightly promote the stability of enzyme. . molecular characterization of specific positively selected sites in mammalian visual pigment evolution miguel a. fern andez-sampedro 1 , eva ramon 1 , brandon m. invergo 2 , jaume bertranpetit 2 , pere garriga 1 1 grup de biotecnologia molecular i industrial., 2 visual rhodopsin is a member of the g-protein coupled receptors superfamily. this membrane protein consists of a 11-cis-retinal cromophore bound to a seven transmembrane protein, opsin, by means of a protonated schiff base linkage. it has an important role as a dim light photoreceptor in the retina of the eye. by statistical models, where episodic selection in rhodopsin is tested on one branch of the phylogeny against a background of neutral or purifying selection on the rest of the tree, we have found some significant evidence of specific positively selected sites in early mammalian divergence. we have chosen the three amino acid sites identified with the highest posterior probability of having been targets of positive selection to perform experimental studies, i.e. 13 (positively selected from m to f), 225 (positively selected from r to q) and 346 (positively selected from s to a). we have constructed, expressed, immunopurified and functionally characterized the proposed candidates, f13m, q225r and a346s rhodopsin mutants located at the n-terminus, the transmembrane domain and the c-terminus region of the protein respectively. from the analysis of the molecular features of the f13m mutant, we conclude that position 13 is very important for protein folding and also for proper protein glycosylation, since we only could observe cromophore regeneration after its rescue in the double cysteine (n2c/ d282c) mutant background that stabilizes the n-terminal extracellular domain of the protein. our results also show that mutants q225r and a346s alter the g-protein activation rate, and hydroxylamine susceptibility in the dark-adapted state. in the case of q225r, disrupting critical interactions with the neighbouring y136 of the conserved d/ery motif, critical in gt activation, could cause the lower gt activation ability. the mutant a346s would create a potential additional phosphorylation site in the protein which could affect rhodopsin phosphorylation after photoactivation and, in turn, could affect the binding affinity of arrestin, a regulator of rhodopsin deactivation. this extra phosphorylation site could provide an evolutionary explanation for the enhanced response observed in the case of gt activation. in conclusion, these results highlight the importance of molecular investigations of positive selected sites in rhodopsin evolution and the relevance of structural and functional analysis of these sites in unravelling the molecular basis of visual pigment evolution. natural evolution sheds light on modern drug resistance in protein kinases marc hoemberger 1 , christopher wilson 1 , roman agafonov 1 , dorothee kern 1 1 the anti-cancer drug imatinib exhibits highly specific binding to the human kinase and oncogene abl with a three thousand fold weaker affinity for the structurally and functionally very similar kinase src. it has been shown recently that the major difference in binding of imatinib to abl and src stems from an induced fit after binding of the drug. to further understand the mechanism of imatinib binding to its target we used ancestral sequence reconstruction (asr) and resurrected enzymes along the node from the common ancestor of abl and src up to the extant kinases. we show that imatinib affinity is gained towards the evolution of extant abl while it is lost towards evolving src. the combination of asr and crystallographic data of the ancestors in addition to kinetics data allowed us to identify a subset of residues involved in imatinib specificity sufficient to switch from an intermediate binder to a tight binder. preliminary data shows that a network of hydrogen bonds and packing interactions stabilize the kinked p-loop conformation for tight binders thus allowing for more interactions between the kinase and the drug. strikingly, many of these residues were identified in human cancer patients as "hot spots" for the development of resistance mutations. further investigation into the identified subset of residues in combination with these commonly found imatinib resistance mutations will allow us to understand emerging drug resistances better. an evolutionary view of the cold adapted catalysis of enzymes vy nguyen 1 , christopher wilson 1 , dorothee kern 1 1 the diversity in protein function that we see today arose as a result of life adapting to a cooling earth. how did enzymes, the catalysts of many crucial cellular processes, achieve this cold adaptation? this is a challenging question to answer because ancient sequences of proteins that existed billions of years ago are not available. to address this question we used ancestral sequence reconstruction to create adenylate kinase (adk) enzymes from the divergence of anaerobic and aerobic firmicutes towards modern day thermophilic, mesophilic and psychrophilic organisms. adk is a phosphotransferase that catalyzes the conversion of two adp molecules into atp and amp. we make the following observations. first, all ancestral enzymes are active with optimal catalytic rates linearly corresponding to the temperature of the environments where these proteins would have been found. most strikingly, the catalytic rate of our oldest adk ancestor exhibits a higher enthalpy of activation at low temperatures as compared to the modern thermophilic adk. this suggests a large enthalpic penalty had to be paid for reactions to occur at cold temperatures in an ancestor that existed in a hot environment. second, several high resolution crystal structures of extant proteins that we solved (1.2å -1.6å), show that the oldest ancestors were more rigid than the modern adks due to an intricate salt-bridge network. this work, thus shows for the first time, the molecular and thermodynamic determinants of cold adaptation in an enzyme over a time period that spans billions of years. induced oxidative modification of plasma and cellular fibrin-stabilizing factor anna bychkova 1 , tatiana danilova 1 , alexander shchegolikhin 1 , vera leonova 1 , marina biryukova 1 , elizaveta kostanova 1 , alexey kononikhin 0 , anna bugrova 1 , evgeny nikolaev 0 , mark rosenfeld 1 1 n. m. emanuel institute of biochemical physics, russian academy of sciences, 2 institute for energy problems of chemical physics, russian academy of sciences the main function of plasma fibrin-stabilizing factor pfxiii is to catalyze the formation of the intermolecular covalent cross-links between both gand afibrin polypeptide chains. the crosslinking crucially affects mechanical strength of fibrin and its resistance against fibrinolysis. the precise role of cellular fibrin-stabilizing factor cfxiii remains poorly understood. pfxiii is a heterotetramer (fxiii-a2b2) consisting of two single-stranded catalytic a subunits (fxiii-a2), and two identical single-stranded inhibitory/ carrier b subunits (fxiii-b2). the subunits are held together by weak non-covalent bonds. contrary to plasma fxiii, cfxiii is a dimer (fxiii-a2) devoid of b subunits. as well as many other proteins circulating in the bloodstream, pfxiii is known to be a target for reactive oxygen species (ros) causing processes of protein oxidative modification. since the conversion of pfxiii to the active form of the enzyme (fxiiia) is a multistage process, ozone-induced oxidation of pfxiii has been investigated at different stages of its enzyme activation. the biochemical results point to an inhibition of enzymatic fxiiia activity depending largely on the stage of the pfxiii conversion into fxiiia at which oxidation was carried out. uv-, ftir-and raman spectroscopy demonstrated that chemical transformation of cyclic, nh, sh and s-s groups mainly determines the oxidation of amino acid residues of pfxiii polypeptide chains. conversion of pfxiii to fxiiia proved to increase protein susceptibility to oxidation in the order: pfxiii < pf-xiii activated by thrombin < pfxiii in the presence of calcium ions < fxiiia. with the aid of massspectrometry it has been demonstrated that oxidation leads to decreasing fxiii-a and fxiii-b coverage both in the forms of zymogen and in the presence of calcium ions. a group of amino acid residues involved in oxidation modification of pfxiii is identified in this study. the oxidation of either cfxiii or cfxiiia has revealed an almost complete loss of enzyme activity caused by dramatic changes in the primary and secondary structure of the proteins detected by the ftir data. taking into account these new findings, it seems reasonable to assume that the inhibitory/carrier fxiii-b subunits can serve as scavengers of ros. hypothetically, this mechanism could help to protect the key amino acid residues of the fxiii-a subunits responsible for the enzymatic function of fxiiia. the study was supported by rfbr, research project no. 15-15-04-08188a. mass spectrometry study was supported by the russian scientific foundation grant no. 14-24-00114. performance and quality. making microcalorimetry simple with microcal peaq-itc natalia markova 1 , ronan o'brien 1 , mark arsenault 1 1 microcal, malvern instruments ltd. dynamic interactions involving biomolecules drive and regulate all biological processes. studies of biomolecular interactions are fundamentally important in all areas of life sciences. data provided by isothermal titration calorimetry (itc) enables scientists in academia and industry to directly and quantitatively characterize these interactions in solution. microcal peaq-itc, the latest generation of microcal itc instrumentation, offers a whole range of solutions for addressing current bottlenecks associated with interaction analysis. among the most recognized challenges are the needs to adequately address a broad range of binding affinities and to reliably interpret binding data complicated by the presence of inactive protein fraction or inherent uncertainty in the concentration of a ligand. consistently high performance of microcal peaq-itc enables increased confidence and data resolution when measuring low heats at low or uncertain sample concentrations and complex binding modes. the new microcal peaq-itc analysis software allows for utomated data analysis, minimizing analysis time and user subjectivity in assessing data quality. data quality is determined and advanced fitting performed in a few seconds per experiment allowing for analysis of large data sets of 50 or more experiments in a matter of seconds. glutamine-rich activation domain of transcription factor sp1 -biochemical activity and structure jun kuwahara 1 , chisana uwatoko 1 , emi hibino 2 , katsumi matsuzaki 2 , masaru hoshino 2 1 faculty of pharmaceutical sciences, doshisha women's university, 2 graduate school of pharmaceutical sciences, kyoto university transcription factor sp1 is ubiquitously expressed in a mammalian cell, activates reasonably large subset of mammalian genes, and is involved in the early development of an organism. the protein comprises two glutamine-rich (q-rich) regions (a and b domains) located in its n-terminal half, while three tandem repeats of c2h2 zinc finger motif at its c-terminus binds directly to a gc-rich element (gc box) of dna. in general, q-rich domain is one of the typical motifs found in trans-activation domain of transcription factors together with acidic and proline-rich domains. transcriptional signal of sp1 are transmitted via interaction between q-rich domains of sp1 and different classes of nuclear proteins, such as tata-binding protein (tbp) associated factors (tafs) in components of basic transcription factor complexes (tfii). in addition, self-association of sp1 via q-rich domains is also important for its regulation of transcriptional activity. it has been considered that an sp1! molecule bound to a 'distal' gc-box synergistically interacts with another sp1 molecule at a 'proximal' binding site. although formation of multimers via q-rich domains seems functionally important for sp1, little is known about relevance between biological activity and structural nature of q-rich domains. we analyzed nature of glutaminerich domains of sp1 by biochemical and physicochemical methods. we found that q-rich domains do not have clear secondary structure whereas they can indicate biochemical activity. detailed analysis of nmr spectra indicated interaction between the domains. the q-rich domains of sp1 might be one of the intrinsically disordered proteins (idp). chipping away at the yeast proteome: redesigning an e3 ubiquitin ligase for targeted protein degradation michael hinrichsen 1 , lynne regan 1 1 one of the central goals of synthetic biology is to exploit biological systems in order to produce compounds of therapeutic or industrial value 1. often, these efforts are complicated by the many natural biochemical pathways in cells that can compete for the same small molecule precursors. currently, the most common solution is to simply delete the genes coding for the competing enzymes 2. while such an approach has been successful, it is only applicable to nonessential genes and can produce unintended off-target effects such as decreased cell viability 2. an alternative strategy is to instead target proteins directly for degradation. using this strategy, scientists would first grow cultures of engineered cells to high densities under permissive conditions (i.e. targeted proteins are stably expressed). then, once sufficient cell density has been reached, enzymes of competing pathways would be rapidly degraded, resulting in the rapid production of high concentrations of the compound of interest. we propose to create such a tool by reengineering the c-terminus of hsp70 interacting protein (chip), an e3 ubiquitin ligase. chip recognizes substrate proteins through a short c-terminal peptide tag on target proteins3. we have shown that fusing this tag to non-native substrates is sufficient for ubiquitination in vitro (data not published). cellular assays have also been performed in s. cerevisiae, a model organism commonly used in metabolic engineering applications1. as a number of native yeast proteins possess c-termini similar to that of chip's native substrates (data not published), it was necessary to develop an orthogonal chip-peptide pair. this was achieved by replacing chip's natural tpr ligand-binding domain with a ligand-binding domain engineered previously in the regan lab 4. the altered chip construct has been shown to be active both in vitro and in vivo, and produces an altered growth phenotype when targeted against an enzyme involved in uracil biosynthesis. future work will focus on further kinetic characterization of the engineered enzyme, increasing its activity, and introducing the system into a proof of concept synthetic biology application. advances in modern sequencing techniques have resulted in an explosion of genomic data. correctly classifying this new wealth of information can be daunting not only because of the sheer volume of sequence data, but also because the propagation of erroneous and less-than-ideal names and functional characterizations in the current databases gets in the way of functional classification by mere sequence similarity. we are investigating the extent to which protein domain architecture can be utilized to define groups of proteins with similarities in molecular function, and whether we can derive corresponding functional "labels", starting with some of the most common domain architectures found in bacteria. to this end, we have developed an in-house procedure called sparcle ('specific architecture labeling engine') that lets us track and examine specific or sub-family domain architectures, resulting from annotating protein sequences with domain footprints provided by the conserved domain database (cdd), which includes hierarchical classifications for many common domain families. we will discuss how the proteins are grouped into specific architectures, our successes in assigning functional labels, and the major limitations we have encountered to date. while we will be able to assign functional labels to a large fraction of protein models derived from genome sequences, this effort has the added benefit of pointing out insufficient coverage and resolution of the current protein domain model collections that constitute cdd. we will also discuss alternative procedures that utilize pre-computed domain annotation for clustering protein sequences at a level that is well suited for functional labeling. we hope that this preliminary study will help to identify approaches that facilitate rapid and accurate annotation of genomes with a minimum of manual intervention. pegylated amyloid peptide nanocontainer delivery and release system self-assembly of telechelic peg end-capped with hydrophobic dipeptides collagen stimulating effect of peptide amphiphile c16-kttks on human fibroblasts self-assembly of palmitoyl lipopeptides used in skin care products bioactive films produced from selfassembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions influence of elastase on alanine-rich peptide hydrogels interaction between a cationic surfactant-like peptide and lipid vesicles and its relationship to antimicrobial activity self-assembled arginine-coated peptide nanosheets in water toll-like receptor agonist lipopeptides self-assemble into distinct nanostructures approved drugs containing thiols as inhibitors of metallo-ß-lactamases: a strategy to combat multidrug-resistant bacteria references leukotriene a4 hydrolase -an envolving target. inflammatory diseases -immunopathology, clinical and pharmacological bases the bifunctional enzyme leukotriene-a, hydrolase is an arginine aminopeptidase of high efficiency and specificity lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease a critical role for lta4h in limiting chronic pulmonary this work was supported by the czech science foundation (project p305/11/0708) and czech academy of sciences the tn antigen-structural simplicity and biological complexity bel b-trefoil: a novel lectin with antineoplastic properties in king bolete (boletus edulis) mushrooms acknowledgements: cynthia leyva-arg€ uelles is supported by a personal grant from conacyt, mexico. this work is supported by conacyt grant 131'06:15 and papiit grant in218714 sequence information-based deciphering of biofunctionalities using ism-based techniques has fetched calculation of biological functionalities, designing of biomedical device called computer-aided drug resistance calculator, the understanding of the mechanism of hiv progression to aids [4], and others. they have compared the efficacies of drugs and vaccines, which formed the basis for the innocentive award (id 9933477) for assessing vaccine potency. conclusions: deciphering biological features without engaging reagents, equipments and animal tissues but biological data such as sequence information is one novel, feasible genotypic hiv-coreceptor tropism prediction with geno2pheno [coreceptor]: differences depending on hiv-1 subtype a reliable phenotype predictor for human immunodeficiency virus type 1 subtype c based on envelope v3 sequences available: http:// istree.bioprotection.org signal processing-based bioinformatics methods for characterization and identification of bio-functionalities of proteins an empirical framework for binary interactome mapping estimating the size of the human interactome coming to peace with protein complexes? 5th capri evaluation meeting pj-022 cabs-dock web server for protein-peptide docking with significant conformational changes and without prior knowledge of the binding site while other docking algorithms require pre-defined localization of the binding site, cabs-dock doesn't require such knowledge. given a protein receptor structure and a peptide sequence (and starting from random conformations and positions of the peptide), cabs-dock performs simulation search for the binding site allowing for full flexibility of the peptide and small fluctuations of the receptor backbone cabs-flex: server for fast simulation of protein structure fluctuations cabs-fold: server for the de novo and consensus-based prediction of protein structure cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site mechanism of folding and binding of an intrinsically disordered protein as revealed by ab initio simulations modeling of protein-peptide interactions using the cabs-dock web server for binding site search and flexible docking cabs-fold: server for the de novo and consensus-based prediction of protein structure cabs-flex: server for fast simulation of protein structure fluctuations aggrescan3d (a3d): server for prediction of aggregation properties of protein structures cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site staphylococcal pathogenicity island dna packaging system involving cos-site packaging and phage-encoded hnh endonucleases the etiology of asdis unknown, but it is believed that it involves genetic and environmental components. the purpose of this work is to assess the possible involvement of food contaminants, such as mycotoxins, in the etiology of asd. the hypothesis is that the mycotoxins ingested with the diet could bind to proteins and expose the entire organism,including cns, to the negative effects of xenobiotics, in genetically predisposed patients. in this study some possible protein targets for the mycotoxinswere identified to evaluate if the bond between any protein target and the mycotoxin in exam could play a role in asd. twelve mycotoxins were selected (ochratoxin a, gliotoxin, aflatoxin b1, aflatoxin b2, aflatoxin m1, aflatoxin m2, aflatoxicol, a-zearalanol, b-zeralanol, zearalenone, deoxynivalenol, patulin),which are contaminants of milk and cereals.for each of these molecules,possible protein targets were searched by a reverse docking approach using the idtargetserver[2].from the results given by idtarget, human protein targets expressed in the brain or involved in brain diseaseswere selected. subsequently, a direct docking was made using auto-dock 4.2 [3], in orderto verify the strength of the interaction between selected proteins and each mycotoxin, and to identify the mycotoxins' binding site on each of the selected protein. finally, the bond of some mycotoxins to selected protein targets has been experimentally tested. for each mycotoxin, idtarget returned thousands of possible protein targets,and only those with the best binding energy were selected and evaluated. among them, human protein targets that are expressed in the brain or that are involved in cerebral diseases,have been selected; moreover the protein targets that were not human but that idtargetselected for five or more mycotoxins, were replaced with their human counterparts. at the end of the procedure, nineteen protein targets have been identified for the following direct docking approach. from the docking results, eight proteins have been selected for experimental tests, having a predicted binding energy lower than 27 kcal/mol. finally, the interactions between acetylcholinesterase (ache), b-secretase (bace1) and neuroligin-4, x-linked (nlg4x) with aflatoxin b1, aflatoxin b2, gliotoxin, ochratoxin a and deoxynivalenol, were evaluatedusing fluorescence spectroscopy and microscale thermophoresis. these experiments confirmed the presence of an interaction between bace1 and aflatoxin b1 idtarget: a web server for identifying protein targets of small chemical molecules with robust scoring functions and a divide-and-conquer docking approach the calculation of spatial structure and "assembling" of the whole protein from the obtained peptide structures were performed by using molecular dynamics of the protein in the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (popc) [4]. the obtained structural model may contribute to identification of ul49.5 active sites and elucidation of its mode of action nmr structural studies of membrane proteins acknowledgments: polish national centre for research and development -grant number 178479 pl-017 functional and mechanistic studies of dysferlin, an essential protein in cell membrane repair references moreover the 'm' parameter, which represents the denaturant effect on the protein stability, is 669 cal•mol-1 for bgla and 860 cal•mol-1 for bglb albert einstein college of medicine protein structure modeling, protein-protein interaction computational modeling of ini1/smarcb1 and novel insights into its interaction with hiv-1 integrase savita bhutoria1 epsteain bar virus, nuclear antigen)1. ini1/smarcb1 has no known structural homologues, and its amino-acid sequence yields little insight into its function. a detailed understanding of structure-function relationships is hampered by the lack of structural information for ini1. computational methods that model protein/peptide structures with sufficient accuracy to facilitate functional studies have had notable successes. we carried out combination of sequence analysis ab initio structure modeling and dynamics studies of integrase binding domain of ini1 and found it to be similar to that of phospholipase a2 activating protein, plaa. structural similarity with this distant protein suggests divergent evolution of the two proteins. the modeled structure sheds light on various protein-protein interactions of ini1. by integrating the experimental studies about the binding, we have shown through docking, how a fragment of ini1 binds to the hiv-1 in. molecular docking and experimental studies indicated that two proteins bind tightly through charged/polar residues surrounding a hydrophobic cleft. these studies provide first modeled structure of ini1/smarcb1 or any component of the swi/snf complex, and provide structural basis for in-ini1 interactions. this molecular interpretation of the intermolecular interactions is expected to facilitate design of inhibitors as novel class of anti-hiv-1 therapeutic agents ): e60734. with their catalytic activity towards rna substrates, other biological properties have been reported and evolution studies suggest an ancestral host-defence function in vertebrates. indeed, genetic studies confirmed a rapid molecular evolution within the family, a distinctive trait for host defence proteins exposed to a changing pathogen environment. previous studies from our laboratory characterized the wide spectra antimicrobial activity of two highly cationic human rnases: the eosinophil rnase 3 and the skin derived rnase ribonucleases 6 and 7 have antimicrobial function in the human and murine urinary tract structural determinants of the eosinophil cationic protein antimicrobial activity two human host defense ribonucleases against mycobacteria, the eosinophil cationic protein (rnase 3) and rnase 7 the regulatory mechanism that we are reporting will contribute to prevent both nad1 exhaustion and accumulation of the toxic bal. to the best of our knowledge, this is the first report of a novel reversible covalent modification of an aldh enzyme involving its own substrate anna lewandrowska 1 , aldona jeli nska 1 , agnieszka wi sniewska 1 acknowledgement: this research was supported by the inactivation of the fxr gene reduces aqp2 expression and impairs urine concentrating ability, which leads to a polyuria or urine dilution phenotype. we have previously found that pon1-/-mice exhibit a polyuria phenotype and produce twice as much 24-h urine as their wild type pon11/1 littermates (borowczyk k et al. metabolism and neurotoxicity of homocysteine thiolactone in mice: evidence for a protective role of paraoxonase 1 development and application of novel non-ewald methods for calculating electrostatic interactions in molecular simulations ikuo fukuda 1 , narutoshi kamiya 1 the most time-consuming part of molecular simulation is the calculation of long-range interactions of the particles. in particular, appropriate treatment of the electrostatic interaction is critical, since the simple truncation cannot be used due to the slow decay of the coulombic function. thus, it is highly demanded to calculate the electrostatic interactions with high accuracy and low computational cost. for this purpose we have developed the zero-multipole (zm) summation method [1]. in this method the artificial periodic boundary conditions are not necessary and the fourier part evaluations are not needed, in contrast to the conventional ewald-based methods. instead, a pairwise function that is suitably redefined from the coulombic function is used with a cutoff scheme. the underling physical idea is simple: (a) in a biological system, a particle conformation for which the electrostatic interactions are well cancelled is more stable than other conformations [2]; (b) since such well-cancelled conformations are essentially physical, we should clip a subset of such a conformation out of the conformation within an ad-hoc given cutoff sphere and calculate the interactions only from this subset. this idea is realized by a rigid mathematical consideration that leads to the deformation of the coulombic function. the efficiency of the zm method has been validated in applications to fundamental systems sema 3a) is a protein originally described as an axonal chemorepellent cue involved in many physiological processes ranging from embryonic development to bone homeostasis or immune responses sema3a signal transduction requires the formation of a heteromeric complex with neuropilin-1 (nrp1) and plexina [2]. in addition, sema3a interaction with nrp1 is modulated by the furin protease cleavage at its c-terminal basic domain this c-terminal basic domain has also been suggested to mediate the binding to glycosaminoglycans (gags), an association that locates sema3a to perineuronal nets and enhances its function in restricting neuronal plasticity and inhibiting axonal regeneration in the central nervous system two peptides corresponding to the highly positively charged regions on the domain were shown to bind to immobilized heparin by surface plasmon resonance (spr) and the affinity dramatically increased when the complete domain was assayed. the binding was confirmed by nuclear magnetic resonance (nmr) and circular dichroism (cd) the conserved cysteine within this motif, necessary for the dimerization of sema3a [7], is also critical for the helix formation. in addition, fluorescence spectroscopy studies showed that the n-terminal region also has a contribution in the binding to gags. we acknowledge the financial support from the european union seventh framework programme (fp7/2007-2013) under the project vision semaphorin 3a: a new player in bone remodeling neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex furin processing of semaphorin 3f determines its anti-angiogenic activity by regulating direct binding and competition for neuropilin semaphorin 3a displays a punctate distribution on the surface of neuronal cells and interacts with proteoglycans in the extracellular matrix semaphorin 3a binds to the perineuronal nets via chondroitin sulfate type e motifs in rodent brains mechanistic basis for the potent anti-angiogenic activity of semaphorin 3f. biochemistry collapsin-1 covalently dimerizes, and dimerization is necessary for collapsing activity prior attempts to create functionally relevant groupings of proteins in the crotonase superfamily suggest that this superfamily is difficult to cluster functionally due in part to the functionally diverse nature of the protein superfamily. we have developed two novel procedures to combat this difficulty: tulip (two-level iterative clustering process), a process that utilizes structural information from active sites to cluster protein structures into hypothesized functional groupings, and misst (multi-level iterative sequence searching technique), a process that uses the protein groupings created in tulip as a starting point for iterative genbank searches and further clustering after each search. through these two methods, the total coverage of the crotonase superfamily has increased, and the generated groups contain proteins from subgroups and families that did not have a structural representative. novel hypothesized functional protein groupings have been created, most notably for a large number of proteins that lack annotation data at the subgroup or family level, and for proteins of the enoyl-coa hydratase family fernandes 1 , teresa sorbo 2 , ivan duka 2 , lia christina appold 3 , marianne ilbert 4 , fabian kiessling 3 cnrs, umr 7281, 5 ucibio-requimte, faculdade de ciências e tecnologia e-selectin is a cell-adhesion molecule induced on the surface of endothelial cells in response to cytokines. its upregulation has been reported in many disorders, including inflammatory and cardiovascular diseases, tumor angiogenesis and metastasis [1]. this profile suggests e-selectin as a promising target to develop molecular imaging probes for the detection of these diseases cyt c with 1 equivalent of fld in media mimicking the cytoplasm. these include 8% polyacrylamide gel, 100 g/l bovine serum albumin or polyvinylpyrrolidone 40, and buffer alone for comparison. electrostatic surface representations of the proteins are shown with their in-cell spectrum pl-060 a search for anti-melioidosis drug candidates targeted to d-glycero-d-manno-heptose-1,7-bisphosphate phosphatase from burkholderia pseudomallei bpgmhb converts dglycero-d-manno-heptose-1b,7-bisphosphate to d-glycero-d-manno-heptose-1b-phosphate. this is the third step of the biosynthesis pathway of ndp-heptose responsible for a pleiotropic phenotype. therefore, this biosynthesis pathway is the target for inhibitors increasing the membrane permeability of gram-negative pathogens or adjuvants synergistically working with known antibiotics. to find inhibitors of bpgmhb, we performed homology modeling of bpgmhb and in-silico virtual screening with zinc, a free database of commerciallyavailable compounds. tens of thousands of chemical compounds were docked into the active site of bpgmhb. a number of putative bpgmhb binding compounds better than d-glycero-d-manno-heptose-1b,7-bisphosphate were found using surflex-dock included in the sybyl software package crystal structure of dimeric d-glycero-d-manno-heptose-1,7-bisphosphate phosphatase from burkholderia thailandensis ewha womans university we have solved the crystal structures of d-glycero-d-manno-heptose-1,7-bisphosphate phosphatase from burkholderia thailandensis (btgmhb) catalyzing the removal of the phosphate at the 7 position of d-glycero-d-manno-heptose-1,7-bisphosphate. it belongs to the haloacid dehalogenase (had) superfamily with an a/b rossman fold composed of six parallel b-strands sandwiched between two sets of three a-helices it reveals a conventional rossman-like a-b-a sandwich fold with a novel b-sheet topology. its c-terminus is longer than its closest relatives and forms an additional b-strand whereas the shorter c-terminus is random coils in the relatives. interestingly, its core structure is similar to that of enzyme iib(cellobiose) from e. coli (eciib(cel)) transferring a phosphate moiety. in the active site of the closest eceiib(fruc) homologues, a unique motif cxxgxaht comprising a p-loop like architecture including a histidine residue is found. the conserved cysteine on this loop may be thiolated to act as a nucleophile similar to that of eciib(cel). the conserved histidine residue is presumed to accommodate negatively charged phosphate during enzymatic catalysis leonor morgado 1 , kornelius zeth 1,2,3 , bj€ orn m. burmann 1 , timm maier 1 bama is a b-barrel membrane protein with five periplasmic n-terminal polypeptide transport associated (potra) domains. the bama structure has been determined recently by x-ray crystallography (2,3), however its functional mechanism is not well understood. this mechanism comprises the insertion of substrates from a dynamic, chaperone-bound state into the bacterial outer membrane, and nmr spectroscopy is thus a method of choice for its elucidation we demonstrated that knocked down autophagy by shrna (shatg5, shbecn1, and shatg12) and chloroquine (cq) could enhance high dose of uvb induced cell death in odc overexpressing hela and mcf-7 cells. here, we also observed that knocked down odc in odc overexpressing hela and mcf-7 cells inhibited autophagy and enhanced high dose of uvb radiation. because of atg12 can regulate cell apoptosis and utophagy. site directed mutagenesis was used to mutant the amino acid which can regulate cell apoptosis and autophagy on atg12, respectively in these two odc overexpressing cells. according to the results fish ß-parvalbumin acquires allergenic properties by amyloid assembly using atlantic cod b-parvalbumin (rgad m 1) displaying high ige crossreactivity, we have found that formation of amyloid fibers under simulated gastrointestinal conditions accounts for the resistance to acid and neutral proteases, for the presence of membrane active species at gastrointestinal relevant conditions and for the ige-recognition in allergic patient sera. incorporation of the anti-amyloid compound epigallocathequin gallate prevents rgad m1 fibrillation, facilitates its protease digestion and impairs its recognition by ige. conclusions: rgad m 1 amyloid formation explains its degradation resistance, its facilitated passage across the intestinal epithelial barrier and the epitope architecture as allergen autophagy could degrade the citrullinated and unfolding protein. herein, padi2 could enhance autophagy in jurkat t cells and lead to a degradation of p62 and the accumulation of lc3-ii. autophagy and apoptosis are two critical mechanisms which participate against cellular stress, cell activation, survival and homeostasis. pad2-overexpressed jurkat t cells caused the activation of th17 cells to increase mrna expression of cytokines, such as il-17, il-21, il-22 and tnfa. cytokines provoked caspase expression and led to caspase-mediated cleavage of beclin-1 which was an important factor of apoptotic signaling. knockdown of bcen1 rescued cell survival due to the increase of bcl-xl and the decrease of caspase-3. we suggested that padi2 participated in the activated t cell-induced autonomous death through triggering er stress pathway studies on secondary metabolites production and proteins and enzymes of in vitro cultivated artemisia alba turra and relations with some endogenous phytohormones yuliana raynova 1 , krassimira idakieva 1 , vaclav motyka 2 , petre dobrev 2 , yuliana markovska 3 , milka todorova 1 , antoaneta trendafilova 1 , ljuba evstatieva 4 switzerland aim: artemisia alba turra is an essential oil bearing shrub, characterized with great variability of the essential oil profile of wild grown plants, related to genetic, geographic and environmental factors. it was previously established that inhibition of rooting in vitro caused by cytokinin/auxin treatment affected the essential oil profile of the plant and these changes were also related to bioactive endogenous cytokinin levels in vitro (1, 2) cytokinin and auxin effect on the terpenoid profile of the essential oil and morphological characteristics of shoot cultures of artemisia alba terpenoid profile of artemisia alba is related to endogenous cytokinins in vitro salivary hnp 1-3 are conventionally measured using an enzyme-linked immunosorbent assay (elisa) which does not discriminate between individual hnps due to their structural similarities. considering the biological importance of salivary human neutrophil a-defensin (hnps), there is therefore, a need to develop an analytical method that will discriminate between the defensins. an lc-ms method has been established for the separation and detection of hnp 1-4. the method has been optimised, validated and applied to examine the relative level of hnp 1-4 in participants undertaking a circuit resistance training workout. to date, no studies have systematically investigated the effect of acute (min to hours) and chronic (days to weeks) change in salivary adefensins family before and after exercise by lc-esi-ms systems and models calorimetry showed no difference in dissociation constants at these ph values, while the binding stoichiometry is increased 2.5 fold. furthermore, the binding stoichiometry varied 7 fold among the two alginates corresponding to their difference in average molecular weight and in addition 20 fold higher binding affinity was found with the high as compared to the low molecular weight alginate. in conclusion, the binding stoichiometry of b-lactoglobulin with alginate increases by a factor that correlates to the average molecular weight of the alginate and also a much higher affinity was found for the high molecular weight alginate. acknowledgements: this work is supported by the danish council for the presence of mucins and other high molecular weight glycoproteins in saliva makes the direct analysis of defensins difficult. the lc-ms method was linear for concentrations of hnp-2 between 0.05 and 1 ng/ml (r2 5 0.99) with a lod of 0.05 ng/ml. inter and intra assay precision was 0.94 -15%, respectively. saliva sample were clean up by solid phase extraction (spe) and without-solid phase extraction (wspe) 2.10 mm internal diameter column in relation to the method transfer . during lc-ms optimisation genome-wide docking database (gwidd) provides the most extensive data repository of structures and models of ppi on a genomic scale. currently, we are expanding the gwidd dataset to 800,365 ppi in 1,652 organisms, up from 128,818 ppi in 771 organisms in the previous release. the ppi data were imported from intact and biogrid databases and were subjected to in-house modeling pipeline. gwidd current implementation contains 11,073 experimentally determined complexes, and 12,426 sequence homology and 28,811 structure homology models of complexes. the user-friendly interface offers flexible organism-specific search with advanced functions for a refined search for one or both proteins. the new gwidd version includes also a new interactive visualization screen that allows to view search results in different residue representations with the emphasis on the ppi interface refolding and activation of recombinant trypsin i from sardine fish (sardinops sagax caerulea) amyloid is detectable in human dental plaque and is produced by both clinical and laboratory strains of s. mutans, further supporting a functional role. s. mutans lacking p1 demonstrates residual amyloid forming properties, however, a mutant lacking sortase, the transpeptidase which covalently links p1 and several other proteins to the peptidoglycan cell wall, is defective in cell-associated amyloid-like properties. the objectives of this study were to identify additional amyloid forming proteins of s. mutans and to evaluate the effects of buffering conditions and ph on the ability of the identified proteins to form amyloids. a p1-deficient mutant strain was grown to stationary-phase in defined minimal media, and secreted proteins from spent culture supernatants were fractionated by ion exchange chromatography. partially purified protein fractions were tested for binding of the amyloidophilic dyes congo red (cr) and thioflavin t (tht), and for characteristic birefringent properties following staining with cr and visualization under crossed polarizing filters. proteins from fractions that tested positive for amyloid-like material were separated by sds page, and identified by lc/ms. these included wapa, gbpa, gbpb, smu_2147c and smu_63c. recombinant proteins were expressed in escherichia coli, and purified for confirmation and characterization of individual amyloidogenic properties in vitro. recombinant wapa and smu_63c displayed all the biophysical characteristics of amyloid, including visualization of fibrillar aggregates when viewed by transmission electron microscopy. in contrast, gbpa and smu_2147c produced amorphous aggregates. wapa and smu_63c form amyloid at different ph, smu_63c under acidic conditions and wapa under neutral to basic conditions. this suggests that the prevailing environmental ph may represent different in vivo triggers for amyloid fibrillization of different s like other small gtpases, the activity of rheb is dictated by its guanine nucleotide binding states: it is active in its guanosine 5 0 -triphosphate (gtp) bound form and inactive in the guanosine diphosphate (gdp)-bound form. rheb proteins play critical roles in regulating growth and cell cycle, and this effect is due to its role in regulating the insulin/tor/s6k signaling pathway rheb interacts directly with fkbp38 and prevents its association with mtor in a gtp-dependent manner. moreover, fkbp38 bound to gtp-g-s, a nonhydrolyzable gtp analogon, has a much higher binding affinity for rheb than the gdp-bound form the second study contradicted both studies, since they could not detect any interaction between rheb and fkbp38 [8]. to clarify whether there is an interaction and if it is nucleotide dependent, nmr monitored interaction studies were performed employing a c-terminal truncated construct of human rheb (1-170 5 rhebdct) that cannot be farnesylated and the biochemically defined binding region on fkpb38 (fkbp12-like 5 fkbp38-bd). based on our data rhebdct -gdp does not significantly interact with fkbp38-bp. 15n-fkbp38-bd titrated , we observed a weak interaction between rhebdct bound to a gtpanalogon (gppnhp) and fkbp38-bd. mapping of the observed spectral changes on the structure of rheb-gtp suggests that fkbp38 targets the switch 2 region, loop 109-112 and the neighboring b-sheet region. we further analyzed the backbone dynamics of rhebdct -gdp and -gppnhp using 15n relaxtion data (t1, t2 and heteronuclear noe) .based on these data the phosphorylation loop, the switch regions and the loop around residues 109-112 show increased backbone dynamics that modulated by the nucleotide binding 5 international centre for genetic engineering and biotechnology tdp-43 is an rna processing protein that can form inclusions of debatable nature implicated in neurodegenerative diseases. within the putative aggregation domain, repeats of residues 341-366 can recruit endogenous tdp-43 into aggregates inside cells1. recently, we showed that a coil to b-hairpin transition in a short peptide corresponding to tdp-43 residues 341-357 enables oligomerization2. we have used a broad battery of biophysical experiments, including chromophore and antibody binding, electron microscopy (em), circular dichroism (cd), solution and solid-state nmr, and x-ray to shed light on the nature of these aggregates. based on these findings, structural models for tdp-43(341-357) oligomers have been constructed, refined, verified, and analyzed using computational methods, ranging from docking and molecular dynamics simulations to semiempirical quantum mechanics calculations. interestingly, tdp-43(341-357) b-hairpins assemble into a novel parallel b-turn configuration showing crossb spine, cooperative h-bonding and tight side chain packing3 cellular model of tar dna-binding protein 43(tdp-43) aggregation based on its c-terminal gln/asn-rich region structural characterization of the minimal segment of tdp-43 competent for aggregation structural evidence of amyloid fibril formation in the putative aggregation domain of tdp-43 kyungpook national university scolopendin 2, aglqfpvgrigrllrk, is a 16-mer peptide derived from the centipede scolopendra subspinipes mutilans. to investigate its property against fungal and bacterial pathogens, antimicrobial tests were performed. we observed that this peptide exhibited antimicrobial activity in a salt-dependent manner and showed no hemolysis. the circular dichroism (cd) analysis observed that a-helical structure properties. we determined the mechanism(s) of action using flow cytometry and investigated the release of potassium. the results showed that the microbial membrane in escherichia coli o157 and candida albicans was permeabilized with loss of potassium ions. additionally, the bis-(1,3-dibutylbarbituric acid) trimethine oxonol [dibac4(3)] and 3,3'-dipropylthiacarbocyanine iodide [disc3 (5)] assay showed membrane depolarization. using calcein-encapsulating giant unilamellar vesicles (guvs) and fitc-dextran containing large unilamellar vesicles (luvs), scolopendin 2 disrupted the cell membrane and the damage size is between 4.8 to 5.0 nm against composition of microbial plasma membrane of e. coli and c. albicans. thus, we demonstrated that a cationic antimicrobial peptide, scolopendin 2, possesses broad-spectrum antimicrobial effects that formed pore on the cell membrane. structural and functional investigation of the far c-terminal domain (ctd) of the bifunctional enzyme trai using nmr spectroscopy protein structural biology structural and functional investigation of the far c-terminal domain (ctd) of the bifunctional enzyme trai using nmr spectroscopy b.krishna chaitanya, evelyne schrank and klaus zangger institute of chemistry/organic and bioorganic chemistry university of graz, austria corresponding author email id: krishna.bhattiprolu@uni-graz.at bacterial conjugation is a complex process for the horizontal transfer of single stranded dna from one cell to another. this mechanism also leads, for example, to the spread of antibiotic resistance genes and virulence factors among bacterial species. multi-protein complexes formed at the origin of transfer (orit) region of dna and at the cytoplasmic membrane of the bacterial cell, initiate this process. inside the membrane, the relaxosome identifies the single strand for transfer in a plasmid dna, relaxes and unwinds it, whereas the transferosome is involved in pilus formation (type iv secretion system) and transferring the gene through the cytoplasmic membrane. these events take place in the donor bacterial cell along with several other auxiliary proteins [1] the bifunctional enzyme trai of plasmid r1 plays a crucial role in the relaxosome activity, as it contains both a relaxase and helicase domain. to exert its functions on dna, trai works in close co-ordination with other relaxosome proteins like tray, tram and the integration host factor. trai is a 1756 residual protein and contains 3 major domains: n-terminal relaxase domain, a central helicase domain and a c terminal domain (ctd). the structure of the c-terminal domain until residue 1629 has been solved by crystallography, while the structure and function of the remaining 130 residues remained undetermined [2]. there are saxs models and crystallographic structures for different parts of trai and also for the full length protein. prediction of cleavage specificity in hcv ns3/4a serine protease and adv2 cysteine protease systems by biased sequence search threading gonca ozdemir isik 1 , a.nevra ozer 1 1 department of bioengineering,faculty of engineering,marmara university proteases are enzymes which recognize specific substrate sequences and catalyze the hydrolysis of designated peptide bonds to activate or degrade them. due to the biological importance of proteases, it is particularly important to identify the recognition and binding mechanisms of protease-substrate complex structures in drug development studies. the assessment of substrate specificity in protease systems is crucial, where interpreting the adaptability of substrate residue positions can be useful in understanding how inhibitors might best fit within the substrate binding sites and aid in the design of potent selective inhibitors. substrate specificity is generally determined by the amino acid profile, structural features and distinct molecular interactions. besides experimental methods, computational tools for prediction of natural substrate cleavage sites, such as threading, have emerged as useful alternative approaches to provide valuable insights into complex enzyme-substrate interactions. in this work, the substrate variability and substrate specificity of the hepatitis c virus (hcv) ns3/4a serine protease and the adenovirus 2 (adv2) cysteine protease was investigated by the biased sequence search threading (bsst) methodology. using available crystal structures of the proteases, the template structures for the substrate-bound proteases were created in silico by performing various peptide building and docking procedures followed by energy minimization and molecular dynamics (md) simulations. bsst was performed starting with known binding, nonbinding and some random peptide sequences that were threaded onto the template complex structures, and low energy sequences were searched using lowresolution knowledge-based potentials. then, target sequences of yet unidentified potential substrates were predicted by statistical probability approaches applied on the low energy sequences generated. the results show that the majority of the predicted substrate positions correspond to the natural substrate sequences with conserved amino acid preferences, while some positions exhibit variability. for ns3/4a serine protease cleavage, the significant selection for pro at p2 and cys at p1 positions is zearalenone is a mycotoxin produced by fusarium graminearum and related fusarium species. f. graminearum is a powerful plant pathogen and infects major crop plants around the world. acute toxicity of zearalenone is low, but due to its structural similarity to b-estradiol it has binding affinity to the estrogen receptor, which results in interference with hormonal balance. typical effects seen in animals include symptoms like hyperestrogenism and reproductive disorders (reduced fertility, reduced litter size or swelling of uterus and vulva). to reduce the risk for human and animal health posed by the ingestion of contaminated food or feed different decontamination strategies have been studied, including biotransformation. today many microorganisms are known to degrade zearalenone, but for most of them the degradation pathway and formed metabolites remained unknown, hence it is unknown if this degradation also means detoxification. only for the fungal strains trichosporon mycotoxinivorans and gliocladium roseum zen degradation has been studied in detail and loss of estrogenicity of reaction products has been confirmed. we screened for, and isolated zearalenone degrading bacteria from soil samples. the most promising new bacterial isolate was taxonomically assigned to the species rhodococcus erythropolis and designated pfa d8-1. the zearalenone catabolism pathway of pfa d8-1 was found to be identical as known from g. roseum. the primary reaction product, hydrolysed zearalenone, has so far only been postulated in g. roseum. we prepared hydrolysed zearalenone by preparative hplc and showed loss of estrogenicity in assays with the breast cancer cell line mcf7 and the estrogen reporter yeast strain yzhb817. a genomic library was prepared and screened in zearalenone degradation deficient r. erythropolis pr4. the gene encoding zearalenone hydrolase was found and named zena. the hydrolase was identified as member of the a/b-hydrolase family and named zena. it was cloned, recombinantly expressed in e. coli and purified by 6 x his-tag mediated immobilised metal affinity chromatography. activity of his-tagged and untagged enzyme zena was compared in cleared lysate and zena was purified for enzyme characterisation. the influence of ph and temperature on enzyme activity and stability was evaluated and kinetic parameters were determined. a new biding site for snake venom c-type lectins?maria cristina nonato costa 1 , ricardo augusto pereira de p adua 1 , marco aurelio sartim 1 , suely vilela sampaio 1 1 university of são paulo, fcfrp c-type lectins are proteins that bind different glycan molecules by interactions with a calcium atom present in a carbohydrate recognition domain (crd). many organisms (plants, bacteria, virus and animals) use these proteins in various biological events like lymphocyte adhesion, erythrocyte agglutination and extracellular matrix organization. the c-type lectin fold is plastic and possible for about 1013 different sequences, what promoted its adaptation to diverse functions, similarly to the observed for the immunoglobulin fold (1014-1016 sequences). it is comprised of about 110-130 amino acid residues that folds in two fourstranded b sheets sandwiched by two alpha helices. interestingly, c-type lectins present in snake venoms are possible anti-cancer agents since they are toxic to cancer cells and inhibit the adhesion and proliferation of various cancer cell lines. therefore, we have purified a lactose binding c-type lectin from the venom of bothrops jararacussu (bjcul) to study its structure and binding properties to different sugars. bjcul crystals were obtained by vapor diffusion and the structure solved by x-ray crystallography to 2.9 å resolution. bjcul structure is a decamer formed by a pseudo fivefold axis rotation of a dimer hold by a disulfide bond. each monomer binds a calcium atom and possibly another metal at a second and opposed binding site. key: cord-304616-k92fa15l authors: izes, aaron m.; kimble, benjamin; norris, jacqueline m.; govendir, merran title: assay validation and determination of in vitro binding of mefloquine to plasma proteins from clinically normal and fip-affected cats date: 2020-08-05 journal: plos one doi: 10.1371/journal.pone.0236754 sha: doc_id: 304616 cord_uid: k92fa15l the antimalarial agent mefloquine is currently being investigated for its potential to inhibit feline coronavirus and feline calicivirus infections. a simple, high pressure liquid chromatography assay was developed to detect mefloquine plasma concentrations in feline plasma. the assay’s lower limit of quantification was 250 ng/ml. the mean ± standard deviation intraand inter-day precision expressed as coefficients of variation were 6.83 ± 1.75 and 5.33 ± 1.37%, respectively, whereas intraand inter-day accuracy expressed as a percentage of the bias were 11.40 ± 3.73 and 10.59 ± 3.88%, respectively. accordingly, this validated assay should prove valuable for future in vivo clinical trials of mefloquine as an antiviral agent against feline coronavirus and feline calicivirus. however, the proportion of mefloquine binding to feline plasma proteins has not been reported. the proportion of drug bound to plasma protein binding is an important concept when developing drug dosing regimens. as cats with feline infectious peritonitis (fip) demonstrate altered concentrations of plasma proteins, the proportion of mefloquine binding to plasma proteins in both clinically normal cats and fip-affected cats was also investigated. an in vitro method using rapid equilibrium dialysis demonstrated that mefloquine was highly plasma protein bound in both populations (on average > 99%). successful treatment of feline infectious peritonitis (fip), a fatal, virulent coronavirus infection affecting predominantly younger cats, remains difficult [1] . reviews describing the virus responsible for fip as well as issues with diagnostics and therapeutics are available [1, 2] , providing an explanation as to why re-purposing drugs used in human medicine has been largely unsuccessful in treating fip infected cats [1] . recently, the use of antiviral therapies to alter the replication of virulent forms of feline coronavirus known as feline infectious peritonitis virus (fipv) has shown enormous hope for clinicians [3, 4] . however, veterinary access to these antiviral treatments is currently limited, leading to the global emergence of expensive, unregistered versions of these drugs without the necessary quality control assurances [4, 5] . accordingly, the need for inexpensive, safe, antiviral medications to treat fipv-infected cats remains. plos one | https://doi.org/10.1371/journal.pone.0236754 august 5, 2020 1 / 10 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 mefloquine is currently used for both prevention (as a monotherapy) and treatment (either alone, or in combination with artesunate) of chloroquine-resistant plasmodium falciparum malaria in humans [6] [7] [8] . it also has been shown to reduce the viral load of fipv and feline calicivirus (fcv) at low concentrations in infected crandell rees feline kidney cells without cytotoxic effects [9, 10] . using an in vitro assay, our team demonstrated that while mefloquine undergoes some phase i metabolism in cats, it does not undertake phase ii glucuronidative metabolism in this species [11] . in anticipation of conducting in vivo clinical trials of mefloquine to treat fipv-infected cats, mefloquine's plasma concentrations must be monitored to optimise the dosage regimen. knowledge of the amount of drug bound to plasma proteins is also important when optimising dosage regimens as only the unbound (or free) fraction is therapeutically active [12, 13] . although the plasma protein binding of mefloquine has been determined to be > 98% in humans [14] , its binding percentage in cats has not been reported. this information is particularly germane in any investigation of a potential fip antiviral as fip-affected cats have altered concentrations of the plasma proteins albumin (decreased) and globulins (elevated) compared to clinically normal cats [1, 15] . consequently, the aim of this study was two-fold: first, to develop and validate a high pressure liquid chromatography (hplc) method to detect mefloquine in feline plasma, and second, to determine the in vitro plasma protein binding of mefloquine in both clinically normal and fip-affected cats. mefloquine hydrochloride and verapamil hydrochloride (as the internal standard [is]) were purchased from sigma-aldrich (castle hill, nsw, australia). hplc grade methanol, hplc grade acetonitrile, phosphate buffered saline ph 7.4 (pbs) and triethylamine were purchased from thermo fisher scientific (macquarie park, nsw, australia). mefloquine and verapamil were quantified in feline plasma based on modified hplc method [16] . the hplc system consisted of a shimadzu lc-20at delivery unit, dgu-20a3 ht degassing solvent delivery unit, sil-20a auto injector, spd-20a uv detector and cto-20a column oven. shimadzu lc solution software version 1.24 (kyoto, japan) was used for chromatographic control, data collection and data processing. chromatographic separation was performed with a microsorb-mv c18 column (250 mm × 4.6 mm i.d., 5.0 μm; varian, mulgrave, vic, australia) with a 1.0 mm optic-guard c18 pre-column (choice analytical, thornleigh, nsw, australia) under a pressure of 1,900 psi at 25˚c. the isocratic mobile phase consisted of a mixture of 25 mm sodium phosphate buffer with 1.0% triethylamine adjusted to ph 2.5 and acetonitrile and methanol (50:25:25, v/v/v) at a flow rate of 1.0 ml/min. the injection volume was 10 μl per sample. the diode array detector was set at a wavelength of 220 nm. the retention times of verapamil and mefloquine were approximately 8.5 and 14.5 minutes, respectively. stock solutions of mefloquine and verapamil were prepared in 100% methanol and 100% acetonitrile, respectively, both at a concentration of 1.0 mg/ml. further working solutions of mefloquine were prepared in 100% methanol to achieve final concentrations of 500, 1,000, 2,500, 5,000, 10,000, 25,000 and 50,000 ng/ml. similarly, working solutions of verapamil were prepared in 100% acetonitrile to yield final concentrations of 12.5 μg/ml for the validation study and 5.0 μg/ml for the plasma protein binding study. stock solutions and working solutions were stored at 4.0˚c prior to use. blank, clinically normal, feline plasma was pooled (n > 5) and stored at -20˚c prior to use for preparation of standards and quality control (qc) samples. the origin of the plasma samples is described below. mefloquine standard samples (50, 100, 250, 500, 1,000, 2,500 and 5,000 ng/ml) and three quality control samples (qc) (250, 1,000 and 5,000 ng/ml) were prepared by spiking 10 μl of prepared working solutions of mefloquine into 90 μl of blank feline plasma. the proteins within the plasma samples were extracted through simple protein precipitation. specifically, 100 μl of acetonitrile, containing either 12.5 μg/ml of the is for the validation study or 5.0 μg/ml of the is for the plasma protein binding study, was added to the 100 μl feline plasma samples. the samples were then vortexed and centrifuged at 14,000 × g for ten minutes to remove any particulates. the supernatant was injected into the hplc system. assay selectivity was established by analysing pooled (n > 5) blank, clinically normal, feline plasma to ensure that there were no endogenous interference peaks around the retention times of mefloquine and verapamil. mefloquine concentrations were measured via standard curves performed on three replicates of each concentration of mefloquine (100, 250, 500, 1,000, 2,500 and 5,000 ng/ml) on three consecutive days. a weighting factor (1/x) was used to assign the relative importance of the observations in the regression; in this case, to ensure that larger observations were not over-fitted. the theoretical lower limit of detection (llod, the lowest analyte concentration that can be distinguished from the assay background noise) and the lower limit of quantification (lloq, the lowest concentration at which an analyte can be reliably detected at a specified level of accuracy and precision) were estimated as follows [17] : llod = 3.3 x σ /s, where σ is the standard deviation of the y-intercepts from the regression lines and s is the mean slope of the calibration curves. thereafter, the lloq was calculated using the formula lloq = 3 x llod. acceptance criteria for the lloq was defined as precision with a cv � 15% and accuracy within ± 20% of nominal concentration with repeated analyses [18] . intra-and inter-day precision, expressed as cv (%), were analysed from triplicates of qc samples (250, 1,000 and 5,000 ng/ml), both within a single day and on three consecutive days, respectively. intra-and inter-day accuracy, expressed as bias, were determined by a percentage difference between the estimated value and the nominal value of mefloquine as follows: bias (%) = 100 -(estimated value / nominal value × 100). absolute recovery of mefloquine and is were determined by comparing the peak area of pre-spiked plasma samples (n = 9) at concentrations of 250, 1,000 and 5,000 ng/ml with corresponding concentrations of mefloquine and is in mobile phase. each assay was conducted in triplicate. the linearity of the data was assessed through linear regression analysis with graphpad prism software version 8.1.1 (graphpad software, inc., ca, usa). with owner verbal or written consent, blood was collected from both clinically normal and fip-affected cats. the use of the residual plasma from this patient for this study was approved by the university of sydney animal ethics committee (protocol: 2016/1027). the animals were considered clinically normal based on inclusion criteria modified from that of norris et al. (2007) [19] . specifically, the cats designated for inclusion as clinically normal subjects were systemically well and had their blood collected for purposes other than investigating a current illness. examples of clinically normal cats included those presenting for annual examinations, routine vaccinations or for routine screening tests prior to sedation or general anaesthesia for de-sexing, grooming, routine dental scaling or radiography post-acute trauma [19] . in contrast, a cat's fip status was confirmed by direct immunofluorescence of effusion samples and immunohistochemistry on tissue samples (n = 5), or simply immunohistochemistry (n = 5) using methods outlined by worthing et al. (2012) [20] . additionally, other haematological tests were performed including quantification of albumin: globulin which was consistent with the hyperglobulinaemia observed with cats infected with fipv [15] . all plasma samples were stored at -20˚c prior to analysis. plasma samples from both clinically normal and fip-affected cats were provided by the paddington cat hospital (sydney, nsw) and the veterinary pathology and diagnostic service, sydney school of veterinary science. the in vitro plasma protein binding (ppb) of mefloquine was determined by the rapid equilibrium dialysis (red) method. the red assay was performed according to manufacturer's (thermo fisher scientific, macquarie park) instructions. pooled plasma samples (n > 5) were assigned to one of two experimental groups, depending on health status, i.e., either clinically normal cat plasma or fip-affected plasma. prior to experimentation, the ph of the pooled plasma (approximately ph 8) was adjusted to ph 7.3 [21] , to mimic the physiologic ph of cats. likewise, the total protein (tp), albumin and globulin concentrations of the pooled plasma samples were quantified prior to the assay by the vpds using a thermo fisher scientific konelab prime 30i analyzer (scoresby, vic, australia) employing conventional protocols. for each experimental group, two final concentrations of mefloquine (either 5,000 or 10,000 ng/ml) were tested using three replicates for each concentration. these concentrations were selected as they are comparable to the 10 μm concentration (10 μm = 3.78 μg/ml or 3,780 ng/ml) tested by mcdonagh et al. (2014) [10] in fipv-infected crandell rees feline kidney cells. accordingly, 300 μl of plasma pre-spiked with mefloquine was added to the redringed chamber of the red device whilst 550 μl of pbs was added to the white-ringed chamber. the unit was covered with sealing tape (thermo fisher scientific, scoresby, vic, australia, product no. 15036) and incubated on an orbital shaker at 250 rpm for four hours. the temperature was set at 38˚c to mimic the normal body temperature of cats. following incubation, 100 μl of post-dialysis samples were removed from the red-ringed chambers. these samples were then placed in separate microcentrifuge tubes and precipitated using 100 μl of chilled acetonitrile pre-spiked with the is (5.0 μg/ml verapamil). after vortexing, the mixtures were centrifuged at 14,000 × g for ten minutes. the supernatant was injected into the hplc system. similarly, 200 μl volumes of post-dialysis samples were removed from the whiteringed chambers. these samples also underwent hplc analysis. all samples were prepared and analysed in triplicate. the ppb of mefloquine was determined by the following equation: %free ¼ ðconcentration buffer chamber=concentration plasma chamberþ � 100% %bound ¼ 100 à %free statistical data analysis was performed using graphpad prism software version 8.1.1 (graph pad software, inc., ca, usa). prior to parametric testing, a shapiro-wilk normality test performed on the mefloquine ppb data indicated a normal distribution. likewise, an inspection of a normal quantile-quantile (qq) plot of this data further corroborated its normality. a twoway anova evaluating the effects of health status and drug concentration on mefloquine ppb was performed. results were considered statistically significant at p < 0.05. based on the uv spectra, the greatest area under the mefloquine peak was at a wavelength of 220 nm. the retention times of the is and mefloquine were approximately 8.5 and 14.5 minutes, respectively. no endogenous interference was observed at the retention times of mefloquine and the is. chromatograms of feline plasma pre-spiked with mefloquine and the is as well as blank feline plasma are shown in fig 1. selectivity. pooled blank feline plasma and feline plasma pre-spiked with mefloquine (250, 1,000 and 5,000 ng/ml) and the is (12.5 μg/ml) were used to check the selectivity of this method. due to the limited volume of fip-affected feline plasma available, only blank plasma of clinically normal cats was used. as demonstrated in fig 1, no endogenous plasma components interfered with elution of analytes. linearity, llod, lloq, accuracy and precision. the plasma peak ratio (area of mefloquine divided by the is area) versus the concentration was plotted and determined to be linear for the concentration range used (100 to 5,000 ng/ml). the mean regression standard curves (n = 3) for mefloquine were described as y = 9.374 e-005 x + 0.001417, with a weighting factor of 1/x. the correlation coefficient value (r 2 ) for each curve � 0.97. estimated from standard curves, the llod for mefloquine was 20.5 ng/ml and the lloq was 61.5 ng/ml. however, as mefloquine concentrations < 250 ng/ml were not accurate (>20% accuracy), the lloq was set as 250 ng/ml. intra-and inter-day precision expressed as cvs ranged from 5.30 to 8.74% and 3.77 to 6.32%, respectively. intra-and inter-day accuracy expressed as a percentage of the bias ranged from -12.67 to 13.96% and -15.01 to 9.04%, respectively. these values satisfied the guidelines regarding assay reliability [18] . intra-and inter-day precision and accuracy are summarised in table 1 . drug recovery from plasma. the absolute recovery rates of mefloquine expressed as percentages ± standard deviation (s.d.) from the 250, 1,000, and 5,000 ng/ml qc samples (n = 3) were 107.82 ± 5.93; 99.91 ± 5.58; and 105.21 ± 6.32, respectively. the absolute recovery rate of the is expressed as a percentage ± s.d. was 123.74 ± 3.77 (n = 9). in vitro plasma protein binding of mefloquine in healthy and fip-affected cats. the measured concentrations of total protein, albumin and globulin in the pooled plasma for the respective experimental groups are provided in table 2 . although reference intervals (ri) vary depending on the laboratory and the methodology used for quantification, reference intervals from cornell university's animal health diagnostic center [22] were used as none were provided by the testing laboratory. the results of in vitro plasma protein binding of mefloquine in clinically normal and fipaffected cats are provided in table 3 . a significant difference was found between the plasma protein binding of mefloquine in clinically normal and fip-affected cats (p = 0.0004), even though mefloquine was determined to be highly plasma protein bound (on average > 99%) in both groups. moreover, a significant interaction effect between health status and mefloquine concentration was identified (p = 0.0007). in contrast, no significant difference was discovered between the plasma protein binding of the two concentrations of mefloquine (p = 0.11). there has been recent interest in the suitability of mefloquine as an antiviral treatment for fip [9, 10, 23] . the majority of publications that detect and quantify mefloquine use liquid chromatography. liquid chromatography demonstrates good sensitivity (i.e., it can detect low concentrations of mefloquine), is highly specific and the machinery is affordable for veterinary diagnostic laboratories. although an hplc assay to detect mefloquine in an in vitro matrix has been reported [23] , in contrast, this study describes an assay to quantify mefloquine table 1 . intra-and inter-day precision and accuracy for the qc samples (250, 1000 and 5000 ng/ml of mefloquine) tested in triplicate for each concentration. to the authors' knowledge, this is the first report of mefloquine ppb in a non-human species. this study provides the novel finding that mefloquine is highly plasma protein bound in cats. in humans, mefloquine is also highly plasma protein bound (> 98%) [14] , which may account, in part, for its long half-life of approximately three weeks in healthy human subjects [24] . high plasma protein binding can result in the drug-protein complex acting as a reservoir for the physiologically active free drug concentration and consequently prolonging its duration of action [12, 25] . likewise, it should not be assumed that a drugs' ppb is constant across species [25] . in a comparative species study investigating the plasma protein binding of 574 compounds, drugs tend to be slightly more bound in human plasma proteins in comparison to the plasma proteins of rats, mice and dogs [26] . some of the compounds that showed significant interspecies differences in plasma protein binding included diazepam (98% ppb in humans versus 84.8% in rats), prazosin (94.7% ppb in humans versus 65% in rats versus 63% in dogs) and sildenafil (93.6% ppb in humans versus 74% in dogs) [26] . diseases, such as fip, can alter the concentrations of plasma proteins and in turn impact upon a drug's plasma protein binding and ultimately its therapeutic efficacy [27] . for example, in humans, the proteins albumin and α 1 -acid glycoprotein (aag) provide the largest contribution to the protein binding of drugs [27, 28] . yet, with acute falciparum malaria, serum concentrations of aag in non-immune human patients increase two-fold within 24 hours whereas plasma levels of albumin decrease by 30% [29] . likewise, in fip-affected cats, common serum protein abnormalities may include elevated aag [30] and globulin levels [1, 15] as well as decreased albumin levels and a low a: g ratio [15] . here, as described in the literature, the confirmed fip plasma samples demonstrated elevated globulin and decreased albumin levels as well as a low a: g ratio. mefloquine has a high affinity for aag binding, preferentially binding to aag over albumin [27] . thus, if more aag is present in fip-affected cats, it may be preferentially bound by mefloquine. yet, small changes in the unbound drug fraction of highly protein bound drugs can have a significant therapeutic impact [28] . for example, the reduction of ppb from 99.9% to 99.8% can lead to a doubling of therapeutically active unbound drug concentration in plasma [28] . here, although a significant difference was found between the plasma protein binding of mefloquine in clinically normal and fip-affected cats, due to the unknown biological variability of the assay, it is likely that this difference is equivocal. fundamentally, it is difficult to generalise from two pooled plasma samples to two populations of individuals since the biological variability in each population is unknown. the variability (standard deviation) used to evaluate differences in ppb were derived from the assay method and do not represent biological differences. yet, it is also unlikely that further in vitro mefloquine feline plasma protein binding studies will resolve this issue. on the contrary, observing the clinical response of mefloquine in fip-affected animals ultimately may decide mefloquine's therapeutic efficacy in this patient population. in vitro studies to quantify plasma protein binding have some limitations. whilst the major advantage of the red method include its speed, simplicity, reliability and cost-effectiveness [31] , its drawbacks, which also serve as limitations to this study, involve nonspecific membrane binding of the drug [31] as well as changes in oncotic pressure leading to an overestimation of unbound drug concentration [32] . moreover, in vitro ppb may not mirror in vivo ppb in the live animal as the structure of the plasma proteins and their affinity to bind substrates can vary with age, disease and /or presence of competing endogenous and exogenous compounds such as dietary constituents or other therapeutic drugs [33, 34] . furthermore, the drug-protein dissociation rate, which can also greatly affect highly ppb drugs [35] , was not determined in this study. likewise, an additional limitation to this project was the scarcity of confirmed fipaffected plasma as this impacted upon its availability for use in both the hplc validation and ppb protocols. finally, given mefloquine's purported mechanism of action as a schizonticide, another potential limitation concerns the effects of the intraerythrocytic accumulation of mefloquine on plasma protein binding. however, previous studies have demonstrated that red blood cells do not serve as a significant depot for mefloquine [36, 37] . recently, the broad spectrum coronavirus protease inhibitor, gc376 [38] , and the adenosine nucleoside analogue, gs-441524 [3, 4] have been shown to be safe and efficacious antiviral agents for use against fipv. yet, as neither of these agents has obtained registration for veterinary use, investigations into more readily available drugs with anti-fipv activity, such as mefloquine, are urgently required for this invariably fatal disease. this study has validated an accurate and reliable assay to detect mefloquine in feline plasma and demonstrated that mefloquine is highly plasma protein bound in both clinically normal and fip-affected cats. further studies describing mefloquine's pharmacokinetic profile in the cat should be progressed. an update on feline infectious peritonitis: diagnostics and therapeutics a review of feline infectious peritonitis virus infection: 1963-2008 the nucleoside analog gs-441524 strongly inhibits feline infectious peritonitis (fip) virus in tissue culture and experimental cat infection studies efficacy and safety of the nucleoside analog gs-441524 for treatment of cats with naturally occurring feline infectious peritonitis fifty years' fascination with fip culminates in a promising new antiviral mefloquine for preventing malaria in pregnant women the position of mefloquine as a 21 st century malaria chemoprophylaxis patient age does not affect mefloquine concentrations in erythrocytes and plasma during the acute phase of falciparum malaria antiviral effect of mefloquine on feline calicivirus in vitro identification and characterisation of small molecule inhibitors of feline coronavirus replication in vitro hepatic metabolism of mefloquine using microsomes from cats, dogs and the common brush-tailed possum (trichosurus vulpecula) predicting plasma protein binding of drugs: a new approach drug-protein binding: a critical review of analytical tools clinical pharmacokinetics of mefloquine positive predictive value of albumin: globulin ratio for feline infectious peritonitis in a mid-western referral hospital population a high performance liquid chromatographic assay of mefloquine in saliva after a single oral dose in healthy adult africans defining limit of detection and limit of quantitation as applied to drug of abuse testing: striving for a consensus international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use prevalence of feline immunodeficiency virus infection in domesticated and feral cats in eastern australia risk factors for feline infectious peritonitis in australian cats ph adjustment of human blood plasma prior to bioanalytical sample preparation chemistry (cobas) reference intervals in vitro hepatic metabolism of mefloquine using microsomes from cats, dogs and the common brush-tailed possum clinical application of mefloquine pharmacokinetics in the treatment of p. falciparum malaria in vitro binding of cefovecin to plasma proteins in australian marsupials and plasma concentrations of cefovecin following single subcutaneous administration to koalas (phascolarctos cinereus) species differences in drug plasma protein binding selective plasma protein binding of antimalarial drugs to α1-acid glycoprotein plasma protein binding and blood-free concentrations: which studies are needed to develop a drug? serum protein concentrations in plasmodium falciparum malaria critical assessment of the diagnostic value of feline α1-acid glycoprotein for feline infectious peritonitis using the likelihood ratios approach protein binding of antimicrobials: methods for quantification and for investigation of its impact on bacterial killing errors in estimating the unbound fraction of drugs due to the volume shift in equilibrium dialysis clinical pharmacology: plasma protein binding of drugs what is the true clinical significance of plasma protein binding displacement interactions? drug safety characterization of plasma protein binding dissociation with online spe-hplc characterization of in vivo metabolites of wr319691, a novel compound with activity against plasmodium falciparum studies of the disposition and metabolism of mefloquine hcl (wr 142,490), a quinolinemethanol antimalarial, in the rat reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor professor michael court (washington state university, college of veterinary medicine) provided invaluable insight into the interpretation of the mefloquine plasma protein binding results. we also thank the veterinary pathology and diagnostics services, sydney school of veterinary science and dr. randolph baral (the paddington cat hospital, sydney, nsw), for providing the feline plasma samples. key: cord-319291-6l688krc authors: hung, chun-min; huang, yueh-min; chang, ming-shi title: alignment using genetic programming with causal trees for identification of protein functions date: 2006-09-01 journal: nonlinear anal theory methods appl doi: 10.1016/j.na.2005.09.048 sha: doc_id: 319291 cord_uid: 6l688krc a hybrid evolutionary model is used to propose a hierarchical homology of protein sequences to identify protein functions systematically. the proposed model offers considerable potentials, considering the inconsistency of existing methods for predicting novel proteins. because some novel proteins might align without meaningful conserved domains, maximizing the score of sequence alignment is not the best criterion for predicting protein functions. this work presents a decision model that can minimize the cost of making a decision for predicting protein functions using the hierarchical homologies. particularly, the model has three characteristics: (i) it is a hybrid evolutionary model with multiple fitness functions that uses genetic programming to predict protein functions on a distantly related protein family, (ii) it incorporates modified robust point matching to accurately compare all feature points using the moment invariant and thin-plate spline theorems, and (iii) the hierarchical homologies holding up a novel protein sequence in the form of a causal tree can effectively demonstrate the relationship between proteins. this work describes the comparisons of nucleocapsid proteins from the putative polyprotein sars virus and other coronaviruses in other hosts using the model. identification of protein function is the main theme of the post-genome era. recently, biomedical scientists have been striving to study proteomics and explore the therapeutic potential of genes for curing disease. thus, a powerful and integrated methodology is required for predicting novel protein function. during the past decade, many algorithms applied to computational biology have been used to solve several of the above problems, with the most frequently used methods including dynamic programming [1] , the hidden markov model [2, 3] , the bayesian theorem, and probabilistic modeling [4] [5] [6] . the journal of molecular biology contains an introduction to hidden markov models for biological sequences written by krogh [7] . as a statistical model, the hidden markov model (hmm) is appropriate for many tasks in molecular biology. a profile-like hmm architecture can be used for searching against sequence databases for new family members and also can make multiple alignments of protein families. moreover, the latest review by tsakonas and dounias [8] indicates that artificial intelligence computational methodologies have been widely applied in bioinformatics, for example genetic algorithms [9] , genetic programming [10] , neural networks [11, 12] , fuzzy logic [13, 14] , and some classification and clustering techniques used in data mining [15] . design of automatic models for comparing protein sequences that are homologous to a known family or super-family has become increasingly important. one standard approach to solving this problem uses 'profiles' of position-specific scoring measure estimated using multiple sequence alignment (msa) [16] . in fact, a potential member of a distantly related protein family cannot be identified with the other members in the same family using only the primary protein structures. consequently, a new protein, such as the case of the sars virus, with numerous unknown functions cannot be accurately predicted by modeling a learning model. this study applies a hybrid methodology based on genetic programming with a causal tree [4, 28, 31] model to predicting protein function. the causal tree [28, 31] with a cause-effect form is proposed for locally comparing protein sequences across each pairwise alignment of msa. the clues to this proposal are that comparisons of distantly related sequences may carry more sensitive and accurate messages regarding protein function if given a segmented alignment using the more comprehensive depiction of the protein family. additionally, the probabilistic scoring measures, similar to profile-to-profile comparisons [17] , can be used to achieve one objective of genetic programming, namely aligning the hierarchical homologies based on profile comparisons. the resulting output is capable of function-related representation for further linkages of protein function. furthermore, the proposed model can generate a tree structure output with a special aligned format rather than a traditional local or global alignment format such as clustalw [23] . traditionally, a local alignment based on the smith-waterman algorithm [7] has been used to compare sequences with gaps using various scoring systems. using scoring systems, many methods are used to evaluate the similarity of two profile columns, for example the ffas method [18] , and prof sim [19] . the ffas method can calculate the correlation coefficients and thus score the 'dot-product' scores of the amino acid frequencies in the two columns. meanwhile, the prof sim method utilizes a single similarity score combined with the divergence score and the significance score of the probability distributions in two columns. recently, the method compass [17, 20] generated the occurrence probability of target residue in one profile column based on the other column of given the target residue frequencies to construct the local profile-to-profile alignment. however, the proposed model was designed to detect the potential functions of a novel protein with a hierarchical homology structure. the hybrid model, namely alignment using genetic programming with causal tree (agct), is a heuristic evolutionary method that contains three basic components: (i) genetic programming with innerexchanged individual strategy, (ii) causal trees [4, 28, 31] with probabilistic reasoning, and (iii) construction of hierarchical homologies with local block-to-block alignment using the methods of moment invariant and robust points matching (rpm) [24] . locally, a standard construction of an alignment requires at least a two-step design: the first step is a scoring evaluation for similarity between two given positions, while the second step is an alignment extension that considers the gap penalties and an extension algorithm. the agct model applies two modified steps to an alignment with a standard construction. the first step is to change the compared objects from positions to blocks that initialize extents based on a particular signal wave, while the second step is to iteratively adjust the boundaries using trimming local fragments rather than the extension algorithm. actually, in blast [21] and its successors [22] , the effective local extension is displayed for the cases of sequence-to-sequence and sequence-to-profile comparison. similarly, in agct, the approach to a local block-to-block comparison modified from profile-to-profile alignment described in [17] shifts the leftmost and rightmost boundaries in relation to the parent segments when high local alignment scores are achieved. notably, a detailed and refined procedure of alignment such as the smith-waterman algorithm [7] is also used in a part of the proposed model for confirming a meaningful fragment detected, because a fragment with a short length reduces the computation time and only some individuals in the population have to be compared making the combination of heuristic and exhaustive search models feasible. the proposed agct model is an evolutionary method based on a probabilistic inference model. the proposed model also incorporates a modified rpm [24] method and a local sequence alignment into the probabilistic model. meanwhile, the model uses a moment invariant theorem [32] for pattern recognitions to extract the feature-nodes from some fragmented signal curves. the rpm compares feature points to precisely construct a soft match for two sets of points. moreover, the modification of rpm changes the original transformation between matrices to the current transformation between a matrix and a tree. for achieving this matrix-to-tree transformation, the proposed constraints, namely rooted one-way winner-take-all (ro-wta), resembling the two-way winner-take-all (wta) constraints [27] , are also used. the proposed ro-wta method is a wta method that initially takes a unique root node by applying wta to the slacked matrix column that is involved for the null point correspondences. then wta is applied to all rows to ensure each row only produces a corresponding feature-node in a causal tree, and finally at most three nodes are taken and used as the branches of the parent node using the first three high values in the same column. moreover, the tree-to-matrix transformation simply visits a tree in preorder to derive a set of pairwise points. finally, an equation, eq. (28), is applied to yield a correspondence matrix for optimizing the rpm. a traditional msa method for protein classification compares more than two sequences to provide the aligned sequences for the observation of conserved domains. however, in a distantly related protein family, the data provides neither meaningful knowledge of biochemical function nor a consistent outcome when using different msa algorithms. clearly, the msa comparisons would be worst if it is applied with the distantly related protein family members. in this model, a small protein fragment transformed into a feature-node would be a compared target point with spatially localized features. individual feature-nodes simplified by six moment invariants [32] are matched with each other for the signal registration of 2-d curves [25, 30] . the 2-d curve is depicted using a block-to-block method that can transform a fragmenting sequence of protein residues into a signal curve by iteratively accumulating a block of amino acid properties. the signal curves are plotted by accumulating hydrophobicity against protein sequence here. because the problem has been transformed into the problem of comparing feature-nodes (points), the comparison of spatially localized features for protein function can be considered an optimization problem of point match for the 2-d shape. in fact, the optimization problem is a difficult problem. especially, the mapping problem must also consider the rigid and non-rigid deformations. a hybrid technique, combining genetic programming and the causal tree, is ideal for solving such difficult problems. genetic programming has emerged from the evolutionary based systems [26, [40] [41] [42] , and the causal tree is derived from probabilistic reasoning in intelligent systems [4] . since the genetic programming can avoid local minima of an energy function, the model is appropriate for recognizing a potential pattern which is never found via direct comparisons. this model uses a popular technique for synthesizing a special signal indicating specific protein functions by accumulating the properties of a group of protein residues. comparing these special signals with each other can provide a wider window for viewing the world of the protein. essentially, the signals must be decomposed for comparison using a method that can prevent a suitable waveform from destroying the protein signals. in the implementation, a technique of wavelet reconstruction for signals [29] is used to depict a noiseless signal curve skeleton. next, the reconstructed curve is differentiated with respect to the positions of each protein residue, and let its results equal zero. the technique thus can easily identify some positions with local minimum accumulating properties. subsequently, to preventing the generation of less than 40 residues, the method would discard some of the neighbor positions around the minima. next, all of the fragmented signals are used to provide an initial dataset for the input of evolutionary computation. although the local minimum values can not determine the true boundaries of the signals at the beginning step, the local alignment afterwards for fragmented proteins using the smith-waterman algorithm [7] would iteratively regularize the boundaries until the suboptimal boundaries are found. next, the model must further extract signal features from the fragmented input signals after decomposing the original signals. technically, the vector features can be associated with the signal features. however, the vector value representing a signal must be invariant when the physical linear-transforms deform the signal curve (shape), such as move, rescale, stretching, and rotation. once the problem is transformed into the image and signal processing domains, the rpm method is easily applied to protein function identification. conceptually, this proposed model includes two stages for feature matching. the first stage is designed to obtain the initial signal features for the signal registration [25, 30] . during the second stage, an inner-exchanged genetic programming is used to repeatedly refine the rpm parameters. this work utilizes the theorem of two-dimensional moment invariants [32] for planar geometric figures from the theorem of moment spaces [33] to extract features of the protein fragments. accumulating hydrophobicity of surrounding residues for each position of protein enables the protein fragments to generate signal curves. next, the signal curve further reduces to a fixed length vector, denoting a feature node, using moment invariants for matching the nodes. next, one of the energy functions in the model introduces the energy function of a correspondence algorithm of non-rigid mapping [24] derived from the tps theorem [34] . finally, genetic programming with the inner-exchanged subpopulation strategy is used to refine the comparison results. the theorem of moment invariants, which is extensively used in computer science, was first proposed in [32] for recognizing visual patterns. in the visual field, an essential step must extract patterns for recognizing visual objects from the original objects. the extracted patterns must also be independent of position, size and orientation. likewise, the model uses the moment invariants of the signal curves to substantially reduce the computational complexity. because the flexibility of the moment invariants used for recognizing protein function was such that the model could find more domains of conservation, the domains should be able to attain more biological meaning by this moment invariant based comparison. the following subsections detail how the model obtains a feature vector denoting a feature-node in a causal tree by using the moment invariant theorem for signal recognition. riemann integrals [32] define two-dimensional (k + l)th order moments for a density distribution f (x, y) as follows: where f (x, y) denotes a piecewise continuous bounded function, and has nonzero values if and only if it is on the finite part of the x y plane. vice versa, the f (x, y) may derive the unique double moment sequence {ṁ k,l } in the moment of any order. by directly integrating the double moment sequence, eq. (2) can express a central moment µ k,l derived from the ordinary moments. where x =ṁ 1,0 /ṁ 0,0 and y =ṁ 0,1 /ṁ 0,0 is an expected value of x and y, respectively. the point (x, y) is termed a center of gravity or center of centroid for x and y. for example, the first four orders have µ 0,0 =ṁ 0,0 ≡ µ, µ 1,0 = µ 0,1 = 0, µ 2,0 =ṁ 2,0 − µx 2 , µ 1,1 =ṁ 1,1 − µx y, µ 0,2 =ṁ 0,2 − µy 2 , µ 3,0 =ṁ 3,0 − 3ṁ 2,0 x + 2µx 3 , µ 2,1 =ṁ 2,1 −ṁ 2,0 y − 2ṁ 1,1 x + 2µx 2 y, µ 1,2 =ṁ 1,2 −ṁ 0,2 x − 2ṁ 1,1 y + 2µx y 2 , µ 0,3 =ṁ 0,3 − 3ṁ 0,2 y + 2µy 3 . in a similar mathematical sense, a measure of area for a two-value image b(m, n) with (k+l)th order moment can approximately obtain eq. (4) from eq. (1) . moreover, the corresponding central moment is expressed as eq. (5): m 0,0 ,m andn are the values for the area length and width, respectively. a pattern of the two-value image which must be independent of position, size, and rotation is derived from similitude moment invariants. likewise, the function b(m, n) with respect to a pair of fixed axes can display the signal curve of protein sequences. moreover, the two-dimensional central moments µ k,l expresses image patterns. additionally, numerous properties of the second central moment are equivalent to a covariance matrix in probability fields. therefore, the covariance matrix for the second central moment is expressed as follows: by the diagonalization, eq. (7) is obtained as follows: where e = e 1,1 e 1,2 e 2,1 e 2,2 , a column vector of matrix e is an eigenvector of matrix u , and the eigenvalues of u determine λ = λ 1 0 0 λ 2 . the values of λ max and λ min corresponding to (λ 1 , λ 2 ) and (λ 2 , λ 1 ) are shown in eqs. (8) and (9), respectively. moreover, eq. (10) denotes a direction angle of the area of the image. from the above theoretical deviations, eqs. (1)-(10) can easily formularize some basic properties of the pattern. notably, an added restriction such as µ 2,0 > µ 0,2 can determine the unique angle of θ . clearly, a discrimination property of the patterns increases when the higher order moments are used as the pattern. the higher order moments with respect to the principal axes can also be easily determined using the method of the principal axes described in [32] . because µ 0,0 = m 0,0 can be used to measure the area, a factor √ α of proportionality, a divisor, should be used to further normalize the central moment in eq. (5) by dividing the variables of the function of eq. (2) by the factor. eq. (2) then can adopt the form f x/ √ α, y/ √ α . as a result the normalized central moment ν k,l equals the original central moment of f (x, y) divided by √ α k+l+2 . because of µ 0,0 being able to measure area, α = µ 0,0 . eq. (11) then can be used to define the normalized central moment, as follows: an identification of the pattern independent of position, size and orientation then can be suitably employed to formularize the moment invariants for any signal curve using eq. (11). to improve pattern recognition capability, an absolute moment invariant should further be achieved by using multiple moment invariants. the normalized central ν k,l in the second and third orders can generate the following six absolute and orthogonal moment invariants listed in eq. (12) . while the skewed invariant listed in the original study [32] is useful for distinguishing mirror images, the model discards this invariant since one skewed orthogonal invariant is not necessary for signal recognition here. the six moment invariants in eq. (12) are not only independent of position, size and orientation, but also can be used to present a feature-node for a fragmented protein sequence. finally, a six-valued vector ω = ω 1 ω 2 ω 3 ω 4 ω 5 ω 6 t can denote a feature-node to enable further convenient processing. suppose that a feature-node ω a = (ω a1 , ω a2 , ω a3 , ω a4 , ω a5 , ω a6 ) can represent a signal curve from the fragmented protein corresponding to one point in the 2-d space. assume a set of vectors ψ = { ω a | a = 1, 2, . . . , k } and the other set of x = { x a | a = 1, 2, . . . , n}. since vector ω a with six variables would increase the problem complexity, a simple linear-transform is used to obtain a scalar for solving the point-matching problem using the tps of two variables. one problem of the fragmented sequence-mapping then can simply be transformed into the other new problem. the new problem involving the two feature-node sets of x and ψ fits a mapping function f ( ω a ) using tps, and simultaneously minimizes the tps energy function defined as follows: typically, two types of deformations of a compared object exist, rigid (affine) and non-rigid (non-affine). the rigid deformation includes parallel translation and rotation. meanwhile, the non-rigid deformation includes shrink, dilation, and distortion. by minimizing the first error measurement term of (13), the feature-node set x maps as closely as possible to the other featurenode set ψ . generally, an infinite number of mappings f can minimize the first term since the mapping is non-rigid. the energy function of eq. (13) can uniquely determine a minimizing function f specified by two parameter matricesã andw in eq. (14) if and only if it has a fixed value λ. whereã represents a (d + 1) with c being a constant for any ω [34] , is related to the tps kernel. the kernel comprises the information regarding the internal structural relationships of the feature-node set and a non-rigid warp comes into play when the warping coefficient matrixw is considered in eq. (14) . the second term of eq. (13), which is responsible for regularizing the mapping function between x and ψ , is essentially a smoothness constraint. the lagrange parameter λ in eq. (13) regularizes the warping of the matches of feature-nodes. the precise matches appear if λ approaches zero. when a solution for f in eq. (14) is substituted into the tps energy function in eq. (13) , and using mercer-hilbert-schmidt theorems of riesz and sz-nagy [35] to prove eq. (15), eq. (16) is obtained: whereλ 1 ≥λ 2 ≥ · · ·λ k ≥ 0 denote eigenvalues of the continuous eigenfunction φ 1 , φ 2 , . . . , φ k , andw t is the transposed matrix ofw . where x and ψ are merely concatenated versions of the vector values of the nodes vectors. each row of the matrix derives from the original vectors. in the model, an energy function with the affineã and warpingw parameters, which is derived from eqs. (13)(16), is used as a fitness function with genetic programming. furthermore, the local border between two regions of protein fragments should be considered an important feature for understanding protein functions. therefore, a distinctive characteristic of the tps can always decompose a geometrical transformation into a global affine transformation and a local non-affine warping component. using this characteristic, a functional signal generally can be detected based on the same essential protein family even if it is undergoing various environment parameters. the parameters include the setting of primary forming signal of the sequences, for example the fragment length in relation to signal response. alternatively, the rotation, translation, and global shear of the signals should slightly influence the detection of the functional signals based on the feature geometrical characteristics (pattern). consequently, the smoothness termw in eq. (16) is specifically applied to be the warping components, and this term should be penalized when signal deformation is ineffective to their essential properties. visually, two compared objects with similar patterns should increase the influence of tps to interpolate a fitting spline. correspondingly, the similar protein fragments compared with one another can be identified by tps matching. the proposed model uses tps methods to compare with two feature-node sets for multiple optimization problems. the tps method raises three issues of optimizations. traditionally, the main issues include optimizations of mapping and correspondence. generally, the work of correspondence must permute a one-to-one relationship between two feature-node sets, and the work of mapping must fit the two feature-node sets to an optimal function f . however, this model defines a new issue that the problem solved must simultaneously optimize the hierarchical heaping structure by constructing a hierarchical relationship among feature-nodes. thus, the work of correspondence in the model must achieve the hierarchical relationship of homologies rather than the one-to-one relationship. alternatively, the model must also optimize heaping. heaping optimization should meet the following minimum requirements: (i) only generating the node of a unique root, (ii) a parent node can compare with multiple children nodes, (iii) using the limitation of the number of branches, the model must only choose the first certain children nodes depending on homology likelihood to attach to a parent node, and (iv) the model must place the predicted feature-nodes and the predicting feature-nodes to the external and internal nods of a causal tree, respectively. these requirements describe the one-to-many query strategy in the homology search model. more precisely, heaping also accomplishes a descending order of homology probability for children nodes. nevertheless, the computations have increased difficulty in simultaneously optimizing the hierarchical correspondence, non-rigid mapping, and heaping. the subsection formally defines a correspondence matrix for a causal tree via the strategy of a one-to-many query. assume that a sequence {q 1 } with unknown functions is used as a query sequence to compare with the fragmented member sequences {q x } z x=2 for constructing an optimal hierarchical structure. the optimal structure should comprise the maximum of homology relationships among these protein fragments of both 'query' and 'member'. moreover, z denotes the number of the input sequence classified to the same distantly related protein family. furthermore, letm denote a correspondence matrix representing the correspondence between two node-sets in a tree. assume the matrix contains (n + 1) × (k + 1) elements: where each m ia is an element of the matrix, with a value that is a real number between zero and one. one query sequence q 1 is divided into s 1 fragments. the summation of m ia in eq. (17) for the constraints of each row inm must equal one. the node of the same label thus appears in the tree a maximum of once. the wta method and sinkhorn balancing theorem [36] can be used to normalize m ia to satisfy the row and column constraints in refs. [24, 27, 36] . however, this model only uses the row constraint to modify wta as ro-wta. alternatively, the correspondence matrix for a causal tree must satisfy the constraint n+1 a=s 1 +1,i =a m ia = 1 in eq. (17) to implement a one-to-many relationship. simultaneously, a constraint n+1 a=s 1 +1 m aa = 0 is used to prevent a self-correspondence. the self-correspondence that appears when one feature-node points to itself is invalid. furthermore, a constraint n i=s 1 +1 m i(n+1) = 1 and ∀i ≤ s 1 m i(n+1) can decide the node of the unique root. from the definition of the correspondence matrix in eq. (17), let: where k represents the number of parent nodes with the starting number one and ending at number k . however, the alternative representation is beginning at number s 1 + 1 and ending at number n. in fact, the two numbering representations are identical for the convenience of representation and implementation. from eqs. (18), (19) and (20) define the set of parent and children nodes in a tree, respectively. where j and (l x , l y ) denote different coordinates of the one and two dimensions, respectively. moreover, the variable s p denotes the number of fragments in each protein sequence. next, eqs. (21)-(25) further define a causal tree denoted ct using the correspondence matrix m. a set of causal trees , similarly described in [35] , can be expressed in terms of i as follows: where denotes the set of causal trees i . a causal tree includes the components r i , i i and e i , which denote a root node, internal node-set, and external node-set, respectively. the causal tree is a tree network that is constructed from the binary random variables labeled, and is also illustrated in fig. 1. fig. 2 shows an example of internal and external node assignment. of nine fragments, five corresponding fragments of internal nodes 5, 6, 7, 8, represent a set of fragmenting members from each member sequence {q x } z x=2 . the external nodes numbered 1, 2, 3, and 4 in fig. 1 are for a query sequence {q 1 } in accordance with the query fragments of fig. 2 in the numbering representation. in fact, the internal nodes can be used for inferring the probabilistic reasoning [4] for predicting protein function. the reasoning process infers along a cost path in the causal tree by assessing orderly conditional probabilities combined together from a root-node to an external feature-node. each internal feature-node ω ∈ i indicates a given protein fragment of different properties. likewise, each external feature-node ω ∈ e indicates an unknown protein fragment function. for heaping the causal tree, the constraintsh 0 ,h 1 andh 2 are used to allocate a correct location of feature-nodes in the causal tree. theh 0 in eq. (22) defines a root-node r by applying the wta method to column (n + 1) of correspondence matrixm. alternatively, a feature-node is a root-node if and only if no parent node points to it. additionally, eqs. (23) and (24) define the correct locations of internal and external nodes, respectively. the node-pairs −−→ (a, i ) and − −−→ (a, a ) represent a direction from a parent node to a child node. for clarity, fig. 3 illustrates an example of the correspondence matrix for a causal tree. the proposed ro-wta selects the maximum value from the real numbers within the parenthesis only for each row and the n + 1 column. besides ro-wta, the diagonal elements must all be zero to eliminate self-correspondence. eventually, eq. (25) defines a causal tree with the entities of a correspondence matrix ct as follows: fig. 3 . correspondence matrix for a causal tree. where ∩ and ∪ denote set intersection and union, respectively. because the causal tree ct may heap some feature-nodes to a cause-effect form, a set of given causes can also be inferred to obtain an unknown effect. the model has transformed the tps method of two point-sets correspondence into an optimization of the causal tree. next, the model must control the tree growth by adding an equation in eq. (26) into the resulting energy function. the value n in eq. (26) denotes the total number of all input fragments as the tree size. by adding eq. (26) to the energy function, the model can attain an approximated tree in accordance with the correspondence matrix. where i denotes the labeled identification of a feature-node. in the presence of this feature-node added to a causal tree j , the value n i returns 1, and otherwise it returns 0. finally, eq. (27) combines eqs. (16), (17) and (26) to create the final energy functions, as follows: by minimizing eq. (27), the model can use genetic programming to obtain a globally suboptimal solution with respect to mapping, correspondence, and heaping. the first term in eq. (27) is an error measure term for matching the similarity between two feature-nodes. meanwhile, the second term in eq. (27) can guard against all matches, and a controlling parameter ζ determines a degree of influence for the term. next, the third term of eq. (27) , which refers to a barrier function, is an entropy term with an annealing temperature t in statistical physics. by controlling the entropy barrier function, the distribution of random variables m ia in ct gradually stabilizes. alternatively, the model can generate the minimum number of states required to push the objective minimum away from the discrete points such that some rules emerge from m ia . a gradually reduced temperature t can suitably control thermal fluctuation to yield an optimal solution. regarding related applications, the application of this entropy term to statistical physics is also briefly described in [37] [38] [39] . subsequently, the fourth term in eq. (27) is used to penalize the differences of an affine mapping parameterã and an identity matrix i . the differences stimulate some unphysical reflections that result from flipping through the whole plane. the fifth term in eq. (27) , which is a standard tps regularization term, penalizes the local warping coefficientsw [24] . using an approximated least-squares approach and qr-decomposition technique, the model can solve the parameters (ã,w ) given fixed ct. additionally, two parameters λ 1 and λ 2 are used to control the influence of the fourth and fifth terms, respectively. finally, keeping a matrix consistent with a tree during the evolutionary process of genetic programming is very difficult. thus, the final term in eq. (27) where m ia must conform to ro-wta. currently, a parent node can only connect a maximum of three children nodes for this model. consequently, the summation of all elements in each column must equal three. when the model attains an optimal causal tree, a probability theorem is further used to model a training model for identifying protein function. consideration of a given protein family can be requested and answers obtained to a query with an unknown protein sequence of what the interrelationship among the particular protein fragments is. unfortunately, the calculation of a probability distribution in bayes networks is computationally intractable (np-complete). however, a causal tree of fixed structure may efficiently compute the result of the probabilistic distribution [31] . using the basic probability theorem, then assuming that p(x i , x a ) denotes the joint probability distribution of two random variables x i and x a , and that p(x i | x a ) denotes the conditional probability of x i given x a ; this conditional probability is defined as follows: using the bayes inference described in [4] , eqs. (30) and (31) are easily derived by eq. (29) . where x i and x a are conditionally independent given x a . subsequently, a well defined bayes model can be obtained by further generalizing beyond simple markov models and considering the probability distribution in the form of general trees. by the expansions of eqs. (30) and (31) , a probability distribution can be applied to learning and inferring for biological models when the tree network has a specific predetermined structure. during a learning phase, the variables within internal nodes and external nodes represent a set of member states and query states, respectively. these variables are both observable during the learning phase. instead, these variables are hidden during an inferring phase when the structure and probability distributions of a causal tree are temporally fixed. this process fully corresponds to the operation of hidden states in a hidden markov model. iteratively, the model eventually produces a refined prediction result based on fitness function estimation. recalling the example of a causal tree, fig. 1 defines nine random binary variables on the tree. the case produces a joint probability distribution of the nine variables using a product of the following terms: where each internal node x i separates variables above and below it. for example, using eq. (31) can show that the node x 7 gains the conditional probability p( initially, the root variable x 7 in eq. (32) first generates a value using the result from eq. (28) . given x 7 , the probabilistic model can determine the conditional probabilities p(x 6 | x 7 ), p(x 9 | x 7 ), and p(x 5 | x 7 ). this model then can generate the values of x 6 , x 9 , and x 5 , respectively. this model thus can calculate the remaining variables recursively. finally, this model obtains an estimated value as one of the multiple fitness functions described in the following section. by rearranging and observing the terms in eq. (32) , an ordered product of terms is also very easily implemented by a preorder tree search. besides the fitness function of the probabilistic and rpm models mentioned above, the model needs another fitness function to determine the boundaries of protein fragments. eq. (33) shows the normalized measures of a local alignment score α divided by the sequence length and obtained by the smith-waterman algorithm [7] . where δ ia denotes local alignment score and ρ ia represents sequence length. furthermore, eqs. (34) and (35) are used to determine a value of parameters ζ and λ 2 for adjusting the reasonable parameters in eq. (27) . since temperature t in eq. (27) is gradually decreased during the evolution, these m ia values are rapidly and accordingly decreased. from eq. (28), it is best for m ia to be in the interval of [0.0 1.0] to protect it from an infinite drop in value that cannot be computed due to the reasonable computation capacity limitation. thus, eq. (34) can be easily obtained from the inequality equation and normal distribution. where e 1 is the natural logarithm that provides a rough scope for estimating the resolution of 256 states that sufficiently fit this computing issue. moreover, µ, σ represent the mean and standard deviation of all feature data, respectively. furthermore, hall and titterington [47] proposed a solution for estimating the smoothing parameter λ 2 , by eq. (35) . where f(λ 2 ) denotes an influence matrix, i represents an identity matrix, and σ is a standard deviation of all feature data. a method of qr-decomposition can be used to determine the value of (i − f( the method decomposes the data of featurenodes ψ = (q 1 : q 2 )( r 0 ) into the product of orthonormal matrix and upper triangular matrix. finally, a process of iteratively estimating eq. (35) is used to find the optimal value λ 2 with respect to minimizing the error in eq. (35). this section focuses on an implementation design of genetic programming that evolves three related subpopulations with inner-exchanged individuals. in 1992, koza et al. were early pioneers of the concept of genetic programming [26, [40] [41] [42] which was extended from the genetic algorithm. genetic programming can automatically create programs for solving general problems. genetic programming is based on a grammar-based methodology of an evolutionary theorem using the darwinian survival principle. therefore, genetic programming can describe highly complicated models for any domain problem without sufficient domain knowledge. first, problem solving requires randomly yielding an initial population of individuals. subsequently, a fitness function can be used to evaluate individuals in the population and select excellent individuals for further performing specific genetic operations. the genetic operations typically include crossover, replication, and mutation operations. the operations enable the parent population at a given generation to form a new offspring population at the next generation. iteratively, genetic programming continuously performs the above evolutionary steps until obtaining an optimal individual or satisfying certain conditions. a representation of genetic programming for a domain problem is composed of several functional programs. the programs form a rooted tree structure as a solution for solving the problem. because genetic programming uses a tree form, the model can easily provide a flexible hierarchical presentation to estimate the relationship of homologies for predicting protein function. characteristically, genetic programming provides a mechanism for searching for the fittest individual. the individual is the problem solution provided by using the computer program in the search space of all possible computer programs. the search space comprises terminalnodes (external nodes) and function-nodes (internal nodes) that are appropriate to represent the specific problem [26] . when a causal tree following genetic operations generates an infeasible individual, the model must regularize this infeasible individual by using a tournament pick (tp) method. the tp method using the depth-first search (dfs) strategy is proposed to accelerate the performance of regularizing this infeasible individual to a feasible one. the tp+dfs method recursively cures the infeasible nodes residing in this individual. the method replaces an infeasible node with a feasible node, in a preorder visiting for trees, randomly selected from the tournament that contains numerous feasible nodes. this advantage of the tp+dfs method results from the mutation appearing at a suitable time, and estimates the fitness function simultaneously following the crossover. when all individuals conform to all constraints of a causal tree, a fitness function can be used for individual estimation. to improve prediction speed and accuracy, this model uses a multiple fitness function strategy. this strategy implements an exchanged individual mechanism involving three subpopulations. the model next considers the following parameters for developing a co-evolution with the strategy of inner-exchanged individuals in a population. the model exchanges the individuals in the three subpopulations 0, 1, and 2. there are nn individuals in subpopulation 0, nm individuals in subpopulation 1, and nk individuals in subpopulation 2, respectively. the size of the subpopulations should have nn nm, nk for considering a trade-off between speed and accuracy. the selection method used in the model always uses the tournament selection strategy rather than the fitnessproportionate selection strategy. additionally, the tournament selection (ts) differs from tp in the target level. the former selects the individuals in the population as its targets on the size 7 tournament. meanwhile, the latter selects the programs (nodes) of an individual (tree) to do the same. regarding the probabilities of genetic operations, the genetic operation of crossover and reproduction gives values of 0.9 and 0.1, respectively. to control tree shape, the tree depth must be 20 or less after performing the crossover operation. furthermore, each internal node has between one and three children connected to it. related applications employing genetic programming can be found in [43] [44] [45] . fig. 4 illustrates the inner-exchange strategy of individuals in three subpopulations of a population for achieving the cooperation of the multiple fitness function. initially, a set of initial feature nodes is fed into the subpopulation 0. subsequently, two feature-node sets produced by random orders of all feature-nodes match each other with tps methods to find the best correspondence between them. the subpopulation 0 is responsible for comparing the functional signals via heuristic matching. for effective heuristic match, the model assumes that the identical conserved protein domains demonstrate genetically similar functions given analogous functional signal shapes. consequently, a physical deformation of the signal shapes should be preserved in somewhat ancestral contours. subsequently, in fig. 4 , the n best individuals of subpopulation 0 must emigrate into subpopulation 1 for modifying the correct boundaries of the protein fragments. in subpopulation 1, an exhausted local alignment of two fragments (nodes) is used to adjust the boundaries of two protein fragments. meanwhile, a moment invariant method calculates a new set of feature data for updating the signal data. moreover, the model must realign the protein fragments once the boundaries of the related fragments have been changed. next, the m best individuals of subpopulation 1 must emigrate into subpopulation 2 to confirm the significant protein fragments by training of real cases. moreover, the n best individuals of subpopulation 0 also must emigrate into subpopulation 2 to do the same work. finally, the k best individuals of subpopulation 2 must emigrate into subpopulation 0 to resume the individuals having specific biological evidence. these resumed individuals can improve the quality of the individuals of subpopulation 0 using the guidelines from real cases. simultaneously, subpopulation 2 eliminates the worst individuals from subpopulation 2 to prevent them from backing to subpopulation 0 or 2. when all of the individuals in the three subpopulations have been coevolved by genetic programming, a final solution should select the optimum individual from subpopulation 0. specifically, the solution takes the form of a causal tree. each path from the root node to any external node represents the functional heredity process. this path can provide an auxiliary guide to biologists for comprehensively understanding the unknown function of query sequences. the strategies used by exchanged individuals are feasible and essential for improving the final solution quality. more specifically, the model cooperatively evolves the causal trees via the cooperation of multiple fitness functions. the multiple fitness functions use eq. (27) as the fitness function of subpopulation 0, eq. (33) as the fitness function of subpopulation 1, and the product of eq. (30) for all nodes as the fitness function of subpopulation 2. fig. 5 describes the statements of the whole algorithm constructing this agct model. the protein nucleocapsid (nsp1) of a coronavirus family from various species was tested to clarify the protein function of nsp1 of sars coronavirus. for seven input sequences, one sequence is a query sequence and six are member sequences. the model simultaneously inputs the sequences to analyze its performance. for convenience, table 1 lists the information on sequence number, abbreviated names, accessions, definitions, and sources obtained from the ncbi web site for the proteins, as follows: these coronaviruses are members of a family of enveloped viruses that replicate in the cytoplasm of animal host cells [46] . sars coronavirus tw1 is a severe acute respiratory syndrome associated coronavirus known as the tw1 isolate. for simplicity, the model only inputs the sequence of putative nsp1 protein of tw1 for analysis with the known protein nsp1 in other hosts. however, this analysis is useful for understanding the novel protein based on inference using other proteins with known functions. although the traditional msa can provide accurate knowledge regarding the conserved domains for these proteins, it cannot clearly express homology relationships over these conserved domains. in fact, the proposed model can provide a hierarchical contour of protein fragments. the contour can also help biologists to increase understanding of the functional heredity relationships only depending on the primary structure of proteins. due to the space limitation, this matching causal tree for the problem using this model is not shown. this section analyzes the performance of convergence of energy at different evolutionary generations using the agct model. table 2 lists some of test parameters used for the agct model to obtain a suboptimal solution to the problems. the parameters which are appropriate for the present case are obtained using eqs. (33)(35) . moreover, a reasonable value of λ 2 = 1.8 is used for eq. (27) through analysis using eq. (35) . the analysis attains the reasonable value using a general cross validation. to analyze the agct model, cases (i) to (v) confirm these typically varied situations. first, case (i) designs for illustrating the ideal solution can be found prematurely. next, case (ii) designs a situation involving a sufficiently large subpopulation size but related small migration size for the subpopulations. next, case (iii) designs a situation involving a small population size and small migration size. case (iv) then designs a situation involving sufficient population size and large migration size. finally, case (v) attempts to understand the real circumstances of evolution at subpopulations 0 and 1 when the energy of subpopulation 2 demonstrates convergence. experimentally, the key parameters are the rates of ii 100 10 50 10 50 10 100 iii 20 10 20 10 20 10 100 iv 100 20 50 30 50 30 200 v 1 0 2 1 0 2 8 1 200 table 3 test results of agct model table 3 lists the results of the five cases. the sensitivity column and specificity column of table 3 display the prediction and discrimination capacities, respectively. since the capacity estimation is based on averaging all generations, the ratios of comparison between the heuristic signal match and exhaustive sequence alignment are quite low. in fact, the high prediction capacity can improve the final solution quality. the high discrimination capacity can increase the execution speed. although the model obtains low sensitivity results, the model attains relatively high specificity results. actually, the results of the later generations have higher sensitivity than previous generations (data not shown). thus, the sensitivity results, which decrease the execution performance during the co-evolution in this model, do not influence the prediction accuracy. in fact, the rises and falls depend on a threshold (cut-off) value, because of a trade-off between sensitivity and specificity. thus, eq. (33) specifies a threshold value of 1.5 to determine whether or not two protein fragments are homologous. moreover, eq. (27) specifies a threshold of value of 0.3 to decide whether or not two signals are analogous. from the observation of the data in table 3 , case ii has better sensitivity than the other cases. furthermore, case ii is also competitive with other reported results. although fewer individuals are involved in case iii than case ii, the resulting reports in case iii are the best except for this sensitivity result. the following discussion can further explain the above confusion. although cases ii and iv involve the same numbers of individuals, case iv involves more immigrated or emigrated individuals than case ii. consequently, case iv obtains worse results than case ii. the above demonstrates that, appropriate individual migration is essential for improving solution quality. however, the amount of migration should not be too large to perform the normal evolution by genetic programming. meanwhile, population size is not particularly important in the present cases since the rpm method and smith-waterman algorithm can undoubtedly obtain the local optimal result for each generation of genetic programming. therefore, even through case iii involves fewer individuals than case ii, case iii can still achieve the performance results approaching case ii. finally, the mechanism of premature convergence in cases i and iv attained the worst results. clearly, case v improves the results of case v through a long-term evolution. accordingly, fig. 6 displays the performance of convergence in each subpopulation on fixed temperature. the energy of subpopulations 0 converges at 0.488 and has a different rate of convergence to the other two subpopulations. clearly, subpopulation 2 evolves faster than the other two subpopulations. fig. 7 illustrates the improvement of the energy convergence of subpopulation 0 by using the five temperature rates (tr) for decreasing annealing temperature, including 1, 0.95, 0.90, 0.85, and 0.80. eventually, the suboptimal solution to this present experiment is yielded using the genetic programming via a cooperation of the multiple fitness functions. this cooperation can continuously change the positions appropriate for splitting the protein sequence. due to the space limitation in this paper, the starting and ending positions of each protein fragment are not shown. subsequently, because of case ii containing more individuals than the others, fig. 8 compares the energy convergences based on case ii with each other for obtaining an appropriate tr value. more precisely, the rate of value 0.85 is the best choice for situations involving rapid and steady convergence. additionally, fig. 9 compares four different tr values 0.97, 0.95, 0.9, and 0.85 for analyzing the energy of subpopulation 0. clearly, case iv really is the worst case. next, fig. 10 simultaneously illustrates the energy convergence of three subpopulations based on case v by varying the rates of temperature decrease. clearly, the model proportionally and consistently reduces the energies of each subpopulation. from the observation of generations in fig. 10 , subpopulations 1 and 2 already begin to converge at around the fortieth generation. finally, the model gradually performs the rpm work in subpopulation 0 between the fortieth and eightieth generations. the agct model utilizes a novel representation of tree structure to illustrate the concept of applying functional homologies to predict protein function. this model differs in numerous respects from the other traditional model of msa. nevertheless, this model still includes various traditionally popular methods used for the high level application, predicting protein function. these methods include genetic programming, wavelet transform, profile comparison, local alignment with the smith-waterman algorithm, moment invariant theorem, rpm with tps, bayes inference, and the causal tree model. besides the above methods, this model introduces various strategies, including the inner-exchanged individuals in subpopulations, cooperation of multiple fitness functions, guided evolution involving real cases, and tp+dfs regularization. the prediction accuracy thus can be improved. this hybrid model is developed to exploit global search capabilities in genetic programming for predicting protein functions of a distantly related protein family that have difficulties in the conserved domain identification. moreover, the rpm involved can identify more sequence homologies through softening comparisons. essentially, this work contributed a complex and integrated methodology that enables new advances in predicting protein function in the post-genome era. we believe that the model robustness can be increased in the future if biologists generate more experimental data in laboratories. future works will apply this model to investigate issues related to protein interaction. sequence comparison by dynamic programming a hidden markov model that finds genes in e. coli dna hidden markov models in computational biology: applications to protein modeling probabilistic reasoning in intelligent systems: networks of plausible inference modeling splice sites with bayes networks prediction of the secondary structure of proteins from their amino acid sequence identification of common molecular subsequences hybrid computational intelligence schemes in complex domains: an extended review genetic algorithms in molecular recognition and design searching for discrimination rules in protease proteolytic cleavage activity using genetic programming with a min-max scoring function logistic regression and artificial neural network classification models: a methodology review statistical mechanics beyond the hopfield model: solvable problems in neural network theory applying fuzzy logic to medical decision making in the intensive care unit increasing the efficiency of fuzzy logic-based gene expression data analysis epps: mining the cog database by an extended phylogenetic patterns search biological sequence analysis probabilistic models of proteins and nucleic acids probabilistic scoring measures for profile-profile comparison yield more accurate short seed alignments comparison of sequence profiles. strategies for structural predictions using sequence information within the twilight zone: a sensitive profile-profile comparison tool based on information theory compass: a tool for comparison of multiple protein alignments with assessment of statistical significance basic local alignment search tool gapped blast and psi-blast: a new generation of protein database search programs clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice a new algorithm for non-rigid point matching ant colony system with extremal dynamics for point matching and pose estimation genetic programming on the programming of computers by means of natural selection new algorithms for 2-d and 3-d point matching: pose estimation and correspondence probabilistic prediction of protein secondary structure using causal networks perfect sampling for wavelet reconstruction of signals a computational vision approach to image registration visual pattern recognition by moment invariants moment spaces and inequalities spline models for observational data functional analysis a relationship between arbitrary positive matrices and doubly stochastic matrices a new method for mapping optimization problems onto neural networks statistical physics algorithms that converge a novel optimizing network architecture with applications genetic and evolutionary algorithms come of age genetic programming iii: darwinian invention and problem solving: book review genetic programming and evolutionary generalization discovering knowledge from medical databases using evolutionary algorithms genetic programming for knowledge discovery in chest-pain diagnosis classifying proteins as extracellular using programmatic motifs and genetic programming fields virology common structure of techniques for choosing smoothing parameters in regression problems key: cord-324640-2zhaknbi authors: munday, diane c.; emmott, edward; surtees, rebecca; lardeau, charles-hugues; wu, weining; duprex, w. paul; dove, brian k.; barr, john n.; hiscox, julian a. title: quantitative proteomic analysis of a549 cells infected with human respiratory syncytial virus date: 2010-07-20 journal: mol cell proteomics doi: 10.1074/mcp.m110.001859 sha: doc_id: 324640 cord_uid: 2zhaknbi human respiratory syncytial virus (hrsv) is a major cause of pediatric lower respiratory tract disease to which there is no vaccine or efficacious chemotherapeutic strategy. although rna synthesis and virus assembly occur in the cytoplasm, hrsv is known to induce nuclear responses in the host cell as replication alters global gene expression. quantitative proteomics was used to take an unbiased overview of the protein changes in transformed human alveolar basal epithelial cells infected with hrsv. underpinning this was the use of stable isotope labeling with amino acids in cell culture coupled to lc-ms/ms, which allowed the direct and simultaneous identification and quantification of both cellular and viral proteins. to reduce sample complexity and increase data return on potential protein localization, cells were fractionated into nuclear and cytoplasmic extracts. this resulted in the identification of 1,140 cellular proteins and six viral proteins. the proteomics data were analyzed using ingenuity pathways analysis to identify defined canonical pathways and functional groupings. selected data were validated using western blot, direct and indirect immunofluorescence confocal microscopy, and functional assays. the study served to validate and expand upon known hrsv-host cell interactions, including those associated with the antiviral response and alterations in subnuclear structures such as the nucleolus and nd10 (promyelocytic leukemia bodies). in addition, novel changes were observed in mitochondrial proteins and functions, cell cycle regulatory molecules, nuclear pore complex proteins and nucleocytoplasmic trafficking proteins. these data shed light into how the cell is potentially altered to create conditions more favorable for infection. additionally, the study highlights the application and advantage of stable isotope labeling with amino acids in cell culture coupled to lc-ms/ms for the analysis of virus-host interactions. human respiratory syncytial virus (hrsv) 1 is an enveloped rna virus and a member of the paramyxoviridae family, which includes common respiratory viruses such as those causing mumps and measles. hrsv is a leading cause of serious lower respiratory tract infections in infants and young children (1) and causes repeated infections throughout life. the severity of illness varies from bronchiolitis and pneumonia to common coldlike symptoms. particular individuals at higher risk of disease include preterm infants, the immunocompromised, and elderly patients. one of the pathologies of the disease is an innate inflammatory response to infection in the lung, which could explain possible links between hrsv and asthma (2, 3) . the pathogenesis of hrsv is not well understood, and to date, the development of a vaccine has been unsuccessful (4) . understanding the interaction for hrsv in particular, and for other viruses in general, with the host cell proteome, will aid in the design of effective antivirals and the development of possible vaccine strategies (5) . because of their similar genome organization and gene expression strategy, the paramyxoviruses have been grouped into the mononegavirales order that includes rabies and ebola viruses (6) . hrsv has a cytoplasmic replication strategy, and the genome consists of a single-stranded negative sense rna with the gene order 3ј to 5ј: non-structural protein 1 (ns1), non-structural protein 2 (ns2), nucleo-(n) protein, phospho-(p) protein, matrix (m) protein, small hydrophobic (sh) protein, glyco-(g) protein, fusion (f) protein, m2-1 and m2-2, and the large (l) protein. the viral proteins can be grouped into different functional categories. four proteins are present in the viral envelope: the g, f, m, and sh proteins. the g protein plays a role in virus attachment (7) , and the f protein promotes virus penetration and fusion of infected cells (8) . the m protein is present in the inner viral membrane, is involved in virion morphogenesis, and traffics between the cytoplasm and the nucleus (9) . the sh protein is important to viral infectivity and is a potential viroporin (10) . five proteins are involved in rna synthesis and formation of the ribonucleocapsid structure: n and p proteins, m2-1, m2-2, and the l protein (11) (12) (13) (14) . the l protein is the catalytic component of the replicase-transcriptase complex and possesses rna-dependent rna polymerase activity. ns1 and ns2 are accessory proteins involved in modulating the host response to infection by acting as antagonists of the ␣/␤ interferon (ifn)mediated antiviral state (15) (16) (17) . during infection, the virus has multiple effects on the host cell. these include (but are not limited to) cell cycle arrest through the up-regulation of transforming growth factor ␤1 (18) , alterations in the composition of lipid raft membranes (19) , decreases in members of the ifn pathways such as traf3 (tnf receptor-associated factor 3) and stat2 (signal transducers and activators of transcription protein 2) (20) , activation of the nf-b signal transduction pathway (21, 22) , and the activation of innate immunity through toll-like receptor 2 (23) . many of these processes are regulated by the induction of different gene subsets (24) . the relative level of host cell proteins can have a direct effect on hrsv disease progression where secondary bacterial infections are observed. for example, aberrant expression of a mucosal ␤-defensin leads to bacterial colonization by haemophilus influenzae in hrsv infection (25) . a number of selected cellular pathways have been shown to change in hrsv-infected cells in culture and in vivo. proteomics has been applied in several instances to the study of the interaction between hrsv and the host cell nuclear proteome (26) . in this study, nuclear extracts from uninfected and infected cells were analyzed using two-dimensional gel electrophoresis (2de) coupled to maldi-tof ms. 24 proteins whose abundance altered by ϯ2-fold were identified and included heat shock proteins, oxidant-antioxidant enzymes, and proteins associated with nuclear domain 10 structures (nd10), which are also known as promyelocytic leukemia (pml) bodies (26) . more recently, 2de was used to compare the potential effect of several different negative strand rna viruses, including hrsv, parainfluenza virus, human metap-neumovirus, measles virus, and influenza virus, on the host cell proteome with common changes in proteins involved with apoptosis and endoplasmic reticulum stress being highlighted (27) . to take an unbiased, global approach to investigating changes in the host cell proteome in response to hrsv infection, stable isotope labeling with amino acids in cell culture (silac) was used in conjunction with lc-ms/ms. through this method, cellular and possibly viral proteins were identified and quantified. to reduce sample complexity and to study the interaction of hrsv with different regions of the cell, nuclear and cytoplasmic fractions were purified and analyzed. a549 cells, a human lung carcinoma cell line that has properties similar to those of alveolar cells (which are permissive for hrsv), were used in this study. because of its respiratory origin, this cell line has been used extensively in the characterization of hrsv infection (18, 21, 24, 28 -30) and in the proteomic analysis of cellular and infectious respiratory diseases (26, (31) (32) (33) . the quantitative proteomic analysis was validated using alternative techniques such as western blot, confocal microscopy, and functional assays where appropriate. the data generated in this study indicated that there were cellular proteome alterations in hrsv-infected cells when compared with mock-infected cells and that many alterations were specific to defined protein subsets, pathways, and organelles. these included mitochondrial proteins, cell cycle regulatory proteins, nd10s, components of the nuclear pore complex, and nucleocytoplasmic trafficking proteins. cells and virus-hep2 and a549 cells were obtained from the health protection agency culture collections and grown at 37°c with 5% co 2 in dulbecco's modified eagle's medium (dmem) (invitrogen). all cells were tested to ensure there was no contamination with mycoplasma. the media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. for the silac experiment, a549 cells were cultured in dmem containing either 13 c-labeled arginine and 2 d-labeled lysine (r6k4-medium) or 13 c-and 15 n-labeled arginine and lysine (r10k8-heavy) for a minimum of seven population doublings. labeled media were obtained from dundee cell products ltd. and were supplemented with 10% dialyzed fcs and 1% penicillin-streptomycin. mock-infected supernatant was prepared using r6k4-medium-labeled hep2 cells. the hrsv a2 strain was propagated in r10k8-heavy-labeled hep2 cells. virus was harvested 4 days postinfection and passed through a 0.45-m filter. virus titer was calculated for a549 cells using an antibody-based methylcellulose plaque assay technique based on previous studies (34, 35) . plaques were visualized using a goat anti-hrsv primary antibody (ab20745) and a horseradish peroxidase (hrp)-conjugated secondary antibody (ab6741) that were both obtained from abcam. the latter antibody was detected using the peroxidase substrate 4-chloro-1-naphthol (pierce). a549 cells were then grown in 500-cm 2 dishes until 60% confluent and mock-infected/infected with virus at a multiplicity of infection (m.o.i.) of 1. enrichment of cytoplasmic and nuclear proteins by subcellular fractionation-cell pellets were resuspended in a cold cytoplasmic lysis buffer (20 mm tris-hcl, ph 7.5, 100 mm nacl, 0.5 mm edta, 0.5% nonidet p-40, edta-free complete protease inhibitor mixture (roche applied science)) and incubated for 10 min on ice. the su-pernatant containing predominantly cytoplasmic proteins was collected after a 3-min centrifugation at 2,000 ϫ g at 4°c. the remaining pellet was resuspended in radioimmune precipitation assay buffer (50 mm tris, ph 7.5,150 mm nacl, 1% nonidet p-40, 0.5% sodium deoxycholate, 0.1% sds, edta-free complete protease inhibitor mixture (roche applied science)) and incubated for 30 min at 4°c. the supernatant containing predominantly total nuclear protein was collected after a 2-min centrifugation at 13,000 ϫ g at 4°c. both fractions were incubated for 5 min at 4°c in a sonicating water bath. gel electrophoresis and in-gel digestion-each sample was reduced in sds-page loading buffer containing 10 mm dtt and alkylated in 50 mm iodoacetamide prior to being boiled, then separated by one-dimensional sds-page (4 -12% bis-tris novex minigel, invitrogen), and visualized by colloidal coomassie staining (novex, invitrogen). the entire protein gel lane was excised and cut into 10 gel slices each. gel slices were subjected to in-gel digestion with trypsin (36) . the resulting tryptic peptides were extracted by 1% formic acid, acetonitrile; lyophilized in a speedvac (helena biosciences); and resuspended in 1% formic acid. lc-ms/ms-lc-ms/ms analysis was performed by dundee cell products ltd. as described previously (37) and for completeness is reproduced here. trypsin-digested peptides were separated using an ultimate u3000 (dionex corp.) nanoflow lc system consisting of a solvent degasser, micro-and nanoflow pumps, flow control module, uv detector, and a thermostated autosampler. 10 l of sample (a total of 2 g) was loaded with a constant flow of 20 l/min onto a pepmap c 18 trap column (0.3-mm inner diameter ϫ 5 mm; dionex corp.). after trap enrichment, peptides were eluted off onto a pepmap c 18 nanocolumn (75 m ϫ 15 cm; dionex corp.) with a linear gradient of 5-35% solvent b (90% acetonitrile with 0.1% formic acid) over 65 min with a constant flow of 300 nl/min. the hplc system was coupled to an linear quadrupole trap orbitrap xl (thermo fisher scientific inc.) via a nanoelectrospray ion source (proxeon biosystems). the spray voltage was set to 1.2 kv, and the temperature of the heated capillary was set to 200°c. full-scan ms survey spectra (m/z 335-1800) in profile mode were acquired in the orbitrap with a resolution of 60,000 after accumulation of 500,000 ions. the five most intense peptide ions from the preview scan in the orbitrap were fragmented by collisioninduced dissociation (normalized collision energy, 35%; activation q, 0.250; and activation time, 30 ms) in the ltq after the accumulation of 10,000 ions. maximal filling times were 1,000 ms for the full scans and 150 ms for the ms/ms scans. precursor ion charge state screening was enabled, and all unassigned charge states as well as singly charged species were rejected. the dynamic exclusion list was restricted to a maximum of 500 entries with a maximum retention period of 90 s and a relative mass window of 10 ppm. the lock mass option was enabled for survey scans to improve mass accuracy (38) . the data were acquired using xcalibur software. quantification and bioinformatics analysis-quantification was performed with maxquant version 1.0.7.4 (39) and was based on two-dimensional centroid of the isotope clusters within each silac pair. to minimize the effect of outliers, protein ratios were calculated as the median of all silac pair ratios that belonged to peptides contained in the protein. the percentage of variability of the quantitation was defined as the standard deviation of the natural logarithm of all ratios used for obtaining the protein ratio multiplied by a constant factor of 100. the generation of the peak list, silac-and extracted ion currentbased quantitation, calculated posterior error probability and false discovery rate based on search engine results, peptide to protein group assembly, and data filtration and presentation were carried out using maxquant. the derived peak list was searched with the mascot search engine (version 2.1.04; matrix science, london, uk) against a concatenated database combining 80,412 proteins from the interna-tional protein index human protein database version 3.6 (forward database) and the reversed sequences of all proteins (reverse database). alternatively, database searches were done using mascot (matrix science) as the database search engine, and the results were saved as a peptide summary before quantification using msquant (http://msquant.sourceforge.net/). parameters allowed included up to three missed cleavages and two labeled amino acids (arginine and lysine). initial mass deviation of the precursor and fragment ions were up to 7 ppm and 0.5 da, respectively. the minimum required peptide length was set to six amino acids. to pass statistical evaluation, posterior error probability (pep) for peptide identification (ms/ms spectra) should be below or equal to 0.1. the required false positive rate was set to 5% at the peptide level. false positive rates or peps for peptides were calculated by recording the mascot score and peptide sequence length-dependent histograms of forward and reverse hits separately and then using bayes' theorem in deriving the probability of a false identification for a given top scoring peptide. at the protein level, the false discovery rate was calculated as the product of the pep of the peptides of a protein where only peptides with distinct sequences were taken into account. if a group of identified peptide sequences belongs to multiple proteins and these proteins cannot be distinguished with no unique peptide reported, these proteins are reported as a protein group in maxquant. proteins were quantified if at least one maxquant-quantifiable silac pair was present. identification was set to a false discovery rate of 1% with a minimum of two quantifiable peptides. the set value for false positive rate/pep at the peptide level ensures that the worst identified peptide has a probability of 0.05 of being false, and proteins are sorted by the product of the false positive rates of their peptides where only peptides with distinct sequences are recognized. during the search, proteins are successively included starting with the best identified proteins until a false discovery rate of 1% is reached, an estimation based on the fraction of reverse protein hits. enzyme specificity was set to trypsin allowing for cleavage n-terminal to proline and between aspartic acid and proline. carbamidomethylation of cysteine was searched as a fixed modification; n-acetyl protein and oxidation of methionine were searched as variable modifications. data sets of identified and both identified and quantified cellular and viral proteins in the nuclear and cytoplasmic fractions are presented in supplemental tables 1-7. here, information is included such as the protein identification in the international protein index format, the abundance as a ratio of hrsv/mock, the uniprot identification, number of peptides used to identify the protein, the sequence coverage this represents, predicted protein details such as molecular weight and length, the gel slice in which this protein was present, and the pep score. note that for the ratio of hrsv/mock a score of 0.5 or lower indicates a 2-fold or greater decrease in abundance in hrsv-infected cells and that a score of 2 or more indicates a 2-fold or greater increase in abundance in hrsv-infected cells in the appropriate fraction. protein pathway analysis-data were analyzed through the use of ingenuity pathways analysis (ingenuityா systems, www.ingenuity. com). networks were generated using data sets containing gene identifiers and corresponding expression values that were uploaded into the application. each gene identifier was mapped to its corresponding gene object in the ingenuity pathways knowledge base. a cutoff of 2.0 was set to identify genes whose expression was significantly differentially regulated. these genes, called focus genes, were overlaid onto a global molecular network developed from information contained in the ingenuity pathways knowledge base. networks of these focus genes were then algorithmically generated based on their connectivity. graphical representations of the molecular relationships between genes/gene products were generated. genes or gene products are represented as nodes, and the biological relationship between two nodes is repre-sented as an edge (line). all edges are supported by at least one reference from the literature or from canonical information stored in the ingenuity pathways knowledge base. human, mouse, and rat orthologs of a gene are stored as separate objects in the ingenuity pathways knowledge base but are represented as a single node in the network. the intensity of the node color indicates the increased (red) or decreased (green) abundance. nodes are displayed using various shapes that represent the functional class of the gene product. canonical pathway analysis utilizes well characterized metabolic and cell signaling pathways that are generated prior to data input and on which identified proteins are overlaid. immunofluorescence-coverslip-adhered a549 cell monolayers were infected with hrsv at an m.o.i. of 1 or 2. the plates were rocked periodically at 37°c over a 2-h incubation period before the inoculum was replaced with fresh growth media and the plate was incubated at 37°c for a further 24 h. the cells were fixed with formalin, and the following antibodies were applied. vdac1 (20b12) (ab14734), prohibitin (ii-14-10) (ab1836), tomm20 (ab56783), and tomm22 (ab57523) were obtained from abcam. nadh dehydrogenase 1␤ subcomplex subunit (ndufb) 10 (sab1400181) and flag (m2) (f1804) were obtained from sigma. lamin b (101-b7) (na12) was obtained from merck, and pml (pg-m3) (sc-966) was obtained from santa cruz biotechnology. alexa fluor-conjugated secondary antibodies (a21050, a11004, and a11061) were obtained from molecular probes (invitrogen). hrsv proteins were detected by either a goat anti-hrsv primary antibody (ab20745) recognized by a fitc-conjugated secondary (ab6881) or through the use of a fitc-conjugated anti-hrsv a2-specific primary antibody (ab20391). coverslips supporting the fixed and stained cells were mounted onto glass slides using either vectorshield (vector laboratories) or prolong goldா antifade reagent (invitrogen), both of which contain a dapi counterstain to allow visualization of cell nuclei. confocal microscopy-direct and/or indirect immunofluorescence confocal microscopy images were captured on an lsm510 meta microscope (carl zeiss ltd.) equipped with 40ϫ and 63ϫ, 1.4 numerical aperture, oil immersion lenses. pinholes were set to allow optical sections of 1 m to be acquired. where possible, all fluorescence was measured in the linear range as the detector is a photomultiplier, and the range indicator was utilized to ensure that no saturated pixels were obtained on image capture. however, for certain images depending on the distribution of the protein, some parts of the image were outside of the linear range to clearly show the distribution of the appropriate protein. images were averaged either four or eight times. trafficking analysis of p and m2-1 proteins-the p and the m2-1 genes were amplified by reverse transcription-pcr from total rna extracted from hrsv a2-infected cells. complementary dna served as a template for pcr amplification and was subcloned into topo ta vector pcr2.1 (invitrogen). the p gene was then cloned into the cyan fluorescent vector pecfp-c1 (clontech), and the m2-1 gene was cloned into ptri-exneo vector (novagen). the orientation of the insert was confirmed by sequencing (data not shown). a549 cell monolayers were grown on glass coverslips in 12-well dishes, transfected with 2 g/well plasmid using lipofectamine 2000 in opti-mem (invitrogen) according to the manufacturer's instructions, and added to antibioticfree media. after 24 h at 37°c, the transfection mixture was removed. the plates were then incubated as described above for a further 24 h. control wells of transfection only and media only were included to assess any background reactivity. nuclei were stained with propidium iodide (invitrogen), and coverslips were mounted onto glass slides with glycerol. western blotting-the cellular fractions were obtained as detailed above, and total protein concentration was determined by bca assay (pierce). protein fractions (2 g) were resolved by 12% sds-page and transferred to pvdf membranes (millipore) using a bio-rad semi-dry transfer apparatus. immobilized proteins were detected with the following antibodies. tubulin (yol1/34) (ab6161), lamin b1 (119d5-f1) (ab8982), cell division control protein 2 homolog (cdc2) (a17) (ab18), and nucleolin (4e2) (ab13541) were obtained from abcam. lamin b (101b7) (na12) was obtained from merck, and cyclin a (h-432) (sc-751), cyclin b1 (d-11) (sc-7393), cyclin d2 (m-20) (sc-593), and cyclin e (m-20) (sc-481) were obtained from santa cruz biotechnology. hrsv proteins were detected by a goat anti-hrsv primary antibody (ab20745) from abcam. three hrp-conjugated secondary antibodies (a5795, a4416, and a6154) were obtained from sigma, and one (ab6741) was from abcam; all were detected by enhanced chemiluminescence (ecl). for the pml western blot, 10 g of nuclear protein and 4 g of cytoplasmic protein were resolved by 15% sds-page and transferred to a pvdf membrane as described above. immobilized proteins were detected with pml (pg-m3) (sc-966) obtained from santa cruz biotechnology. the hrp-conjugated secondary antibody (a4416) was detected using supersignal ecl (34095) obtained from pierce. mitochondrial transition pore assay and live cell imaging-glass bottom plate-and coverslip-adhered a549 cell monolayers were infected with hrsv at an m.o.i. of 2 or mock-infected. the plates were rocked periodically at 37°c for 2 h before the inoculum was replaced with fresh growth media and the plates were incubated at 37°c for a further 24 h. the imageit tm live mitochondrial transition pore assay kit (i35103) obtained from molecular probes (invitrogen) was used (according to the manufacturer's instructions) to visualize the mitochondrial transition pore in hrsv-infected, mock-infected, and positive control cells. pml localization-coverslip-adhered a549 cell monolayers in 6-well dishes were transfected with 2 g/well flag-pml-i or flag-pml-ii (40) using lipofectamine 2000 in serum-free media according to the manufacturer's instructions. after the 2-h incubation at 37°c, the transfection mixture was removed, and the cell monolayers were infected and incubated as described above. control wells of transfection only and media only were included to assess any background reactivity. the cells were fixed with formalin and detected with the following antibodies. a fitc-conjugated anti-hrsv-specific primary antibody (ab20391) obtained from abcam was used to detect hrsv proteins. both tagged pml isoforms were detected with an anti-flag m2 (f1804) primary antibody that was obtained from sigma-aldrich. alexa flour-conjugated secondary antibodies (a1106 and a1104) were obtained from molecular probes (invitrogen). the nuclei of the above fixed cells were stained with dapi contained within the vectorshield (vector laboratories) used to mount the coverslips onto glass slides. quantitative proteomic analysis was used to investigate the potential changes in the host cell proteome in response to infection with hrsv. cells were fractionated into nuclear and cytoplasmic extracts to reduce sample complexity for lc-ms/ms but also to provide an excellent way to further probe the interaction of a cytoplasmic replicating virus with the nuclear and cytoplasmic proteomes. to our knowledge, no previous study has used silac coupled with lc-ms/ms to identify and quantify proteome changes in cells infected with hrsv or any other single-stranded negative sense rna viruses. the observed changes were then validated and investigated using a combination of immunofluorescence to study subcellular localization, western blot to study protein abundance, and functional assays applied where appropriate. proteins-labeled a549 cells were infected with hrsv at an m.o.i. of 1 or mock-infected with supernatant that was prepared in the same way as described for the labeled virus stock. labeling, validation, fractionation, protein identification, and quantification were conducted as outlined in fig. 1 . mock-infected cells were grown in media labeled with r6k4-medium, and cells infected with virus were grown in media containing r10k8-heavy. proteins were identified and quantified at 24 h postinfection. indirect immunofluorescence confocal microscopy indicated that 60 -70% of cells treated with hrsv were infected (fig. 1b) . fluorescence was not detected in mock-infected cells (fig. 1b) . cells were enriched into cytoplasmic and nuclear fractions, which were validated by the detection of characteristic cellular (tubulin for the cytoplasm and lamin b1 for the nucleus) (fig. 1c ) and viral (fig. 1d ) marker proteins that were enriched in the respective fractions. the protein concentration was determined by bca assay. note that equal protein concentrations were loaded, and therefore quantitative comparison of the relative abundance of proteins between the cytoplasmic and nuclear fractions should not be made. western blot analysis showed that there was no virus present in mock-infected cells (fig. 1d) . lc-ms/ms analysis identified 606 and 534 proteins in the nuclear (supplemental table 1 ) and cytoplasmic (supplemental table 2 ) fractions, respectively. of these, 561 and 520 proteins were identified and quantified in the nuclear and cytoplasmic fractions, respectively. all proteins were identified by two or more peptides. mitochondrial proteins (74 proteins; supplemental table 3 ) were present in the nuclear fraction, and these were removed from the final list of proteins assigned to the nuclear proteome (supplemental table 4 ). the proteins classified as forming the cytoplasmic proteins are shown in supplemental table 5 . a previous analysis of nuclear fractions obtained from a549 cells (prepared by a different method) also contained mitochondrial proteins, which were shown to be contaminants (33) . five viral proteins were identified in both fractions (ns1, n, p, m, and m2-1 proteins), and one viral protein, f protein, was identified in the cytoplasmic fraction only (see supplemental tables 6 and 7 for viral proteins found in the nuclear and cytoplasmic fractions, respectively). for quantitative analysis, previous investigations using silac and lc-ms/ms have applied ratio cutoffs ranging from near 1.3-to 2.0-fold (41) . a previous study that investigated changes in the nuclear proteins in hrsv-infected cells used a ratio cutoff of 2.0-fold for comparison (26) . in this study, a 2.0-fold cutoff was chosen as a basis for investigating potential proteome changes between data sets through ipa and to provide a basis for comparing the current data set with previous studies. the comparison of mock-infected with hrsv-infected cells indicated that in the nuclear fraction many proteins showed a 2-fold or greater decrease in abundance (supplemental table 1 ), whereas in the cytoplasmic fraction, very few proteins changed in abundance (supplemental table 2 ). bioinformatics analysis of nuclear and cytoplasmic fractions in a549 cells-the nuclear proteome for a549 cells, corresponding to 433 genes, has been resolved previously (33) . in the current study, the nuclear and cytoplasmic proa, a diagrammatic representation of the methodology used in this analysis. stable isotope (medium and heavy)-labeled amino acids were incorporated into newly synthesized cellular proteins. a549 cells were infected with hrsv (heavy) alongside a mock infection (medium), and 24 h postinfection subcellular fractionation was used to enrich the cytoplasmic and nuclear proteins. the infection efficiency and the fraction purity were then validated prior to sample preparation and lc-ms/ms analysis. b, indirect immunofluorescence confocal microscopy validation of hrsv infection efficiency in a549 cells 24 h postinfection. staining with the hrsv antibody combination in mock-infected cells is shown as a control. different magnifications are presented. scale bars, 20 m. c, western blot confirmation of representative proteins enriched from the cytoplasmic and the nuclear fractions of mock-infected (m) and hrsvinfected (i) cells. membrane-immobilized proteins were detected with tubulin (ϳ55 kda) and lamin b1 (ϳ58 kda) antibodies. tubulin and lamin b1 were predominately localized in the cytoplasmic and nuclear fractions, respectively. d, western blot confirmation of hrsv infection (and lack thereof in mock-infected cells). an anti-hrsv-specific polyclonal primary antibody was detected by an hrp conjugate. the locations of molecular mass markers (kda) are indicated on the left, and the tentative assignments of proteins are indicated on the right. rsv, respiratory syncytial virus. teomes were resolved to study the interaction of hrsv with the host cell. ipa was used to assign identified proteins into different molecular and cellular functional classes (see supplemental table 8 for definitions) based upon the underlying biological evidence from the curated ingenuity pathways analysis literature database. the relative proportions of proteins represented in each separate functional class in the nuclear and cytoplasmic proteomes of a549 cells are shown in fig. 2 . the data indicated that the proteomes differed between the nuclear and cytoplasmic fractions. as would be expected, a greater proportion of proteins involved in gene expression, post-transcriptional modification of rna, and rna trafficking were present in the nuclear fraction than in the cytoplasmic fraction, and a greater proportion of proteins involved in protein degradation and lipid metabolism were present in the cytoplasmic fraction than in the nuclear fraction. pathway analysis was used to group proteins into different functional networks to determine whether different cellular activities were altered in hrsv-infected cells. for example, in the nuclear fraction, proteins involved in cell growth and transcription regulation (fig. 3) were less abundant in hrsvinfected cells. such pathways were linked by cell cycle regulatory complexes (e.g. cyclin a) and ifn ␤, which were not identified in the lc-ms/ms analysis. additionally, in the nuclear fraction, proteins involved in molecular transport and protein and rna trafficking could be grouped together, and all were less abundant in the nucleus in hrsv-infected cells (fig. 4) . in the cytoplasmic fraction, proteins involved in cellular assembly and organization, cellular compromise, and protein folding were grouped together. proteins could be linked by nf-b and stat1 signaling for example (fig. 5) . several canonical pathways were highlighted by ingenuity pathways analysis as being disrupted in hrsv-infected cells, including those involved in mitochondrial dysfunction (39 of a possible 172 molecules; p value, 5.89 ϫ 10 ϫ29 ), ubiquinone biosynthesis (15 of 119 molecules; p value, 6.69 ϫ 10 ϫ29 ), and ran signaling (six of a possible 23 molecules; p value, 1.87 ϫ 10 ϫ6 ). other identified cellular proteins altered in hrsv-infected cells included components of subnuclear structures such as the nucleolus (nucleolin and nucleophosmin) and nd10s (e.g. tar dna protein), the latter of which had been previously highlighted as changing in hrsv-infected cells (33) , and cell cycle regulatory molecules. a number of proteins that were ablated could be grouped into specific disease associations, including respiratory disease (22 proteins ; p values, 6.84 ϫ 10 ϫ4 -2.59 ϫ 10 ϫ2 ), the inflammatory response (12 proteins; p values, 5.59 ϫ 10 ϫ4 -4.56 ϫ 10 ϫ2 ), and infectious disease (73 proteins; p values, 5.81 ϫ 10 ϫ10 -2.59 ϫ 10 ϫ2 ). for example, six proteins were associated with pneumonitis, which can be a feature of hrsv infection (42) . selected results of the ipa were validated using alternative techniques. these included western blot, indirect and direct immunofluorescence confocal microscopy, and functional assays from biological replicates. confocal microscopy, unlike western blot, does not rely on subcellular fractionation and purification of proteins from mock-or hrsv-infected cells and thus provides complete, independent verification of the results. this information was combined with an examination of the previously existing literature to form the basis of validation for the quantitative proteomics analysis and to further investigate the findings. validation of ipa analysis for hrsv-infected cells using previously characterized immune signaling-the hrsv-immune response interaction has been well characterized in vitro and in vivo (e.g. ref. 20) . in the quantitative proteomics analysis of a549 cells infected with hrsv, stat1 and its downstream molecule ifn-stimulated gene 15 protein (isg15) were more abundant than in mock-infected cells (fig. 5, left) . this observation reflects previous work in which human diploid fibroblast 2ftgh cells were infected with hrsv, and stat1 protein was shown to be more abundant in hrsvinfected cells when compared with mock-infected cells (43) . the role of stat1 in the immunobiology of hrsv has been investigated in transgenic animal models (44) . the ifn-stimulated gene 15 mrna and protein were shown to be upregulated in a mouse lung epithelial cell line (mle-15) infected with hrsv (45) . in the current data set, these molecules were linked to nf-b-activated transcription, transforming growth factor ␤1, and ifn ␣/␤ (fig. 5) , all of which have been described in hrsv-infected cells (5, 18, 20, 21, 44, 46, 47) . therefore, previously published data were reflected by the bioinformatics analysis of the current quantitative proteomics data. alterations in mitochondrial protein abundance and mitochondrial integrity in hrsv-infected cells-as highlighted by the canonical pathway analysis, mitochondrial proteins formed a group whose abundance differed between hrsvinfected and mock-infected cells. this had not been observed previously. mitochondrial proteins were identified and quantified in both the nuclear and cytoplasmic fractions as being both ablated and enriched proteins in hrsv-infected cells compared with mock-infected cells (supplemental tables 1-3) . this is presented diagrammatically in fig. 6 with the proteins arranged according to their localization in the mitochondria. respiratory complex 1 proteins and other mitochondrial proteins identified and quantified by lc-ms/ms (fig. 6) were decreased in abundance by 2-fold or more in hrsv-infected cells in comparison with mock-infected cells. for example, respiratory complex 1 protein ndufb10 was 60-fold decreased in the nuclear fraction but not represented in the cytoplasmic fraction. the abundance of the translocase of the outer mitochondrial membrane (tom) complex subunits tom20, tom22, tom40, and tom70 were decreased ϳ129-, 26-, 15-, and 12-fold, respectively, in the nuclear fraction enriched from hrsv-infected cells but were not detected in table 8. the cytoplasmic fraction. the abundance of voltage-dependent anion channel (vdac) proteins 1, 2, and 3 were decreased by ϳ31-, 42-, and 26-fold, respectively, in the nuclear fraction in hrsv-infected cells compared with mock-infected cells. vdac1 and vdac2 were also detected in the cytoplasmic fraction with 10-and 9-fold increases, respectively, in hrsv-infected cells when compared with mock-infected cells. prohibitin (phb) subunits phb1 and phb2, which are involved in cell proliferation and the functional integrity of mitochondria (48, 49) , were increased 3-fold in the cytoplasmic fraction from hrsv-infected cells. indirect immunofluorescence confocal microscopy was used to investigate the localization and possible abundance of the mitochondrial proteins in hrsv-infected cells. the ndufb10 localization validation indicated that the subcellular localization of this protein was altered in hrsv-infected cells when compared with mock-infected cells, and increased fluorescence intensity was also observed in the former (fig. 7a) . the fluorescence data for the subcellular localization of rep-resentative tom complex members (tom20 and tom22) indicated that the subcellular localization of these proteins also differed between hrsv-infected cells and mock-infected cells with localization observed in a distinct region of the cytoplasm. there also appeared to be less fluorescence from tom20 and tom22 in hrsv-infected cells (fig. 7 , b and c, respectively). in addition, the data indicated that in hrsvinfected cells tom20 was possibly present in the nucleus or at least closely associated with this structure. this was confirmed in the z-stack image of this cell (supplemental fig. 1) . the relative fluorescence of vdac1 in hrsv-infected cells was greater than in mock-infected cells. the subcellular localization was also altered with increased punctate staining observed (fig. 8a) . the subcellular localization of phb in hrsv-infected cells appeared to be localized with viral complexes and with greater fluorescence intensity in these regions than in mock-infected cells (fig. 8b) . together, the indirect immunofluorescence confocal microscopy images supported the observations from the quantitative proteomic analysis that the abundance of mitochondrial proteins (particularly pore proteins) was altered in hrsv-infected cells. from this, we hypothesized that the mitochondrial permeability transition pores had an altered function in hrsv-infected cells. to test this hypothesis, we made use of a live cell mitochondrial assay in which green fluorescent (calcein am) dye is maintained in healthy mitochondria (which are also stained red with a mitotracker dye). as a positive control, cells were treated with the ca 2ϩ ionophore ionomycin, which results in mitochondrial pore activation from the inner and outer mitochondrial membranes and subsequent loss of green fluorescence. in this live cell assay, it was not possible to use indirect immunofluorescence confocal microscopy to visualize hrsv-infected cells (as the cells could not be made permeable to allow antibody penetration). therefore, cells were infected at an m.o.i. of 2 to ensure that at least 70% of cells were infected at 24 h postinfection, the assay point (after the experiment, cells were fixed, and infection status confirmed using indirect immunofluorescence confocal microscopy (supplemental fig. 2) ). the data illustrated that in mock-infected cells the majority of the cells were stained green and red, indicating functional mitochondria (fig. 9 ). in control cells treated with ionomycin, all of the cells were stained red with no green signal, indicating mitochondrial pore activation (fig. 9) . in hrsv-infected cells, there was a greater population of predominately red cells than predominately green cells, indicating greater mitochondrial pore activation in cell populations infected with hrsv in comparison with the mock-infected cells (fig. 9) . potential disruption of proteins involved in nucleocytoplasmic trafficking-network pathway analysis indicated that the abundance of nuclear pore complex components and proteins involved in the nucleocytoplasmic trafficking of proteins and rna differed between hrsv-infected and mock-infected cells (fig. 4) . the proteins, their position in the nuclear pore complex, and their possible associations can be seen in fig. 10a . assignment of the localization of the localization of these proteins within the nuclear pore complex was based on data from a number of different studies (50 -56) . additionally, nup155 and nup107 identified in the study but not shown in 10a were shown to be involved in nuclear envelope and pore complex formation (57, 58) . the quantitative proteomic analysis indicated that in hrsvinfected cells nucleoporins (nups) were depleted; e.g. nup96 and nup98 were depleted 3.6-fold (and those shown in fig. 10a ). the only exceptions were nup85 and nup160. indirect immunofluorescence confocal microscopy was used to investigate the subcellular localization of the nuclear protein lamin b in mock-infected cells in comparison with hrsv-infected cells (fig. 10b) . the data indicated that in mock-infected cells the protein was localized to the nuclear envelope but also was distributed between the nucleus and the cytoplasm. in contrast, in hrsv-infected cells, lamin b appeared to be more concentrated around the nuclear envelope with less fluorescence observed in the cytoplasm or the nucleus, which is indicative of altered trafficking or loss of nuclear pore complex function. alteration of cell cycle regulatory proteins in hrsv-infected cells-the quantitative proteomics and network pathway analysis (fig. 3) identified several proteins with roles in cell cycle regulation whose abundance differed between mock-infected and hrsv-infected cells. these included cdc2 (also known as cyclin-dependent kinase 1 (cdk1)); histone deacetylase 2 (hdac2); proliferation-associated 2g4, 38 kda (pa2g4); and sin3 homolog a transcription regulator (yeast) (sin3a) (ϳ6-, 4-, 3-, and 3-fold less abundant, respectively, in hrsv-infected cells). network pathway analysis (fig. 3 ) also predicted that cyclin a might be altered in hrsv-infected cells. cdc2 has been shown to bind to cyclins such as a, e, and b types and can regulate cell cycle progression (59, 60) . in addition, in hrsv-infected a549 cells, cell cycle arrest has been observed; however, the abundance of cell cycle regulatory complexes was not elucidated (18) . western blot analysis of nuclear and cytoplasmic fractions from mock-and hrsvinfected cells indicated that cdc2 was less abundant in the nuclear fraction in hrsv-infected cells (confirming the quantitative proteomic analysis) and that cyclins d2, a, e, and b1 were also less abundant (fig. 11) . in such analysis, it is essential to ensure that equal protein loading is used to allow for relative protein abundance observations. lamin b and tubulin were therefore selected as internal controls to confirm protein content. disruption to subnuclear structures, nd10s (pml bodies), and associated proteins-many subnuclear structures such as the nucleolus and nd10s (also known as pml bodies) contain proteins that are involved in the antiviral signaling response that become altered in virus-infected cells (61) (62) (63) . the nucleolus is formed from a complex of protein-protein and protein-nucleic acid interactions centered around nucleolar hub proteins such as nucleolin and nucleophosmin (64) . nd10s are formed around pml protein and are dynamic structures that are in continuous protein exchange with the nucleus, depending on the metabolic state of the cell (65) . pml is a protein with several different isoforms that can localize to nd10s and/or the nucleus or cytoplasm (66) . several components of the subnuclear structures were altered in hrsv-infected cells, including an ablation of nucleolin (decreased 4.5-fold) and nucleophosmin (decreased 6.7-fold) (see supplemental table 1 ; for nucleolin, shown using western blot, see fig. 11 ). the following constituents of nd10s were altered in abundance in hrsv-infected cells: the tar dna-binding protein (ϳϫ3-fold), eukaryotic translation initiation factor 3 (ϳϫ9fold), and dna repair protein rad50 (ϳϫ2.6-fold). nd10 abundance and localization were investigated in hrsv-infected through the use of its major constituent, pml. an antibody that recognized all pml isoforms was applied to both western blot and indirect immunofluorescence analysis. western blot analysis indicated that more pml isoforms were present in the nuclear fraction from hrsv-infected cells when compared with mock-infected cells (fig. 11 ). in the cytoplasmic fraction in hrsv-infected cells, a decreased abundance in the number of pml isoforms was observed when compared with the mock-infected cells (fig. 11) . the indirect immunofluorescence confocal microscopy data indicated that there were a greater number of nd10s present in the hrsv-infected cells when compared with mock infected-cells (fig. 12 ). this observation included not only cells that were positively identified as infected but also the bystander cells (fig. 12 ). this result was in contrast to a previous proteomic analfig. 6 a diagrammatic representation shows the inner and outer mitochondrial membranes with representative proteins identified in the quantitative proteomic analysis shown in their appropriate mitochondrial localizations. in the outer mitochondrial membrane, tom20 and tom22 interact with other toms (some of which were identified in this analysis) to form a pore through which premitochondrial proteins are transported. transmembrane pores such as vdacs may participate in the formation of the permeability transition pore complex, which is responsible for the release of mitochondrial products such as cytochrome c that trigger apoptosis. the inner membrane contains the protein complexes involved in the electron transport chain. respiratory complexes 1, 3, and 4 are proton pumps. those proteins involved in cell proliferation and integrity such as phb subunits phb1 and phb2 are also found in mitochondria. ysis of the nucleus from hrsv-infected a549 cells proposing that pml protein is redistributed from the nucleus to the cytoplasm 24 h postinfection (26) . in the current study, further analysis of hrsv-infected cells at 36 h postinfection indicated that pml remained predominately in the nucleus with no change in the number of nd10s in mock-infected cells (fig. 12 ). to confirm that pml was not redistributed to the cytoplasm in mock-infected and hrsv-infected cells in our experimental system, overexpression analysis of two pml isoforms, recombinant flag-tagged pml-i and pml-ii (40) , was used (fig. 12) . the data indicated that both tagged fusion proteins remained localized to the nucleus at 24 h postinfection, which was the same in mock-infected cells (fig. 12) . fractions-the lc-ms/ms analysis identified several viral proteins in the nuclear and cytoplasmic fractions (supplemental tables 6 and 7, respectively). although the data tables show a quantitative ratio between hrsv proteins and mock-infected cells, in reality this should be infinite and is a result of comparison with cellular peptides of similar sequence. hrsv replication is cytoplasmic, therefore, the potential presence of viral proteins in the nucleus may be considered an unusual observation. however, hrsv m protein has been shown to localize to the nucleus and contains nuclear-cytoplasmic trafficking signals (9, (67) (68) (69) . the presence of viral proteins in the nuclear fraction either may have been an artifact of the fractionation procedure or may reflect the subcellular localization of a protein. to investigate whether the hrsv p and m2-1 proteins were present in the nucleus, overexpression analysis was utilized where these proteins were expressed as c-terminal fluorescent fusion proteins tagged with cyan (ecfp). the direct fluorescence confocal microscopy analysis indicated that both the p and the m2-1 proteins localized predominately to the cytoplasm, but some fluorescent signal was observed in the nucleus but excluded from the nucleolus (fig. 13 ). this correlates with the identification of these proteins in the nuclear fraction. ecfp was localized throughout the cell. note that ecfp and the fusion proteins have been false colored green postimage capture for increased clarity. discussion hrsv infection results in multiple changes in the host cell, and the aim of this study was to provide an unbiased global overview of these effects. the quantitative proteomics and the subsequent bioinformatics analysis using ipa allowed alterations in the host cell proteome to be mapped in hrsvinfected cells. this study serves to reflect the existing in vitro and in vivo data and highlights new interactions. to our knowledge, no such silac-coupled lc-ms/ms study has been applied to hrsv or to any other single-stranded negative sense rna virus. despite the potential of this technique and transferable application to the comparison of treated with non-treated experimental conditions (41, 70) , a few studies have used this methodology to investigate the interactions of viruses with the host cell. these include investigations of the interaction of hepatitis c virus (hcv) with cell lipid rafts (71), pseudorabies virus-infected cells (72) , coronavirus-infected cells (37) , and alterations to the nucleolar proteome in adenovirus(73) and coronavirus (74)-infected cells. identification and quantification of cellular proteins and experimental conditions-potential changes in the host cell cytoplasmic and nuclear proteomes were elucidated for a hrsv subgroup a using optimized conditions to ensure that the majority of cells were infected with hrsv to generate the highest potential differences between the mock-and hrsvinfected cell treatments for lc-ms/ms quantification and data comparison. the laboratory-adapted hrsv a2 strain used in animal-based pathogenesis studies (75) was used in the silac study because it replicated to high efficiency in the a549 model cell line. these cells have been used extensively in hrsv studies because of their ability to retain features of alveolar cells (76) . proteins were identified and quantified at 24 h postinfection as this time point occurs prior to the appearance of a major cytopathic effect; thus, the majority of cells remain viable for downstream processing. in addition, this time point is commonly used in hrsv studies and therefore allows comparison with the previously published data. for example, microarray studies (24) of hrsv-infected cells have demonstrated a high return of data at this time point. in total, 606 cellular proteins were identified in the nuclear fraction, and 534 proteins were identified in the cytoplasmic fraction along with six viral proteins. curiously, of a total of 510 proteins assigned to the nuclear proteome, 431 showed a 2-fold or greater decrease in abundance in hrsv-infected cells. this could not be attributed to a preparation or loading artifact as equal protein concentrations between the mock and infected preparations were validated using independent assays such as a bca assay and staining after separation by one-dimensional sds-page, and large volumes were combined to reduce variation in liquid handling prior to lc-ms/ms analysis. also, nuclear proteins with increased and decreased abundances between mock and infected cells were validated experimentally using alternative techniques on the same and different samples, e.g. the increased abundance of pml versus the decreased abundance of nucleolin and similar levels of marker proteins such as lamin b (e.g. figs. 1 and 11) . a similar study of potential changes in the nuclear proteome in cells infected with an avian coronavirus reflected a general trend in the decreased abundance of nuclear proteins (37) . using ingenuity pathways analysis, these proteins were grouped into functional classes and used to potentially map nuclei are stained blue with hoechst. all mitochondria are stained red with mito-tracker (a), healthy cells are also stained green with calcein am (b), which is concentrated in the mitochondria. merged images are also presented (c). positive control cells were treated with ionomycin to allow the entry of excess calcium into the cells to trigger mitochondrial pore activation, which leads to the release of calcein am and therefore the loss of green florescence. a large proportion of mitochondria in hrsv-infected cells were stained red and only weakly green, indicating that hrsv infection affected mitochondrial transition pore activity. scale bars, 20 m. note that indirect immunofluorescence confocal microscopy was used to demonstrate that cells were infected with hrsv, and these data are presented in supplemental fig. 2. the nuclear and cytoplasmic proteomes of a549 cells (fig. 2) . the nuclear proteome of a549 cells has been investigated previously using 2de and hplc, highlighting the extensive use of a549 cells in respiratory disease research (33) . therefore, our current study complements and expands this analysis and adds further data on cytoplasmic proteins. network pathway analysis-ipa was used to analyze the data sets, investigate potential changes in specific cellular functions, and generate a priority list of proteins and pathways of interest. ipa identified several different biological pathways, some of which may be common to viral infection. for example, in common with the data gathered for hrsvinfected a549 cells, a proteomic analysis of the cd4 ϩ cemx174 cell line infected with hiv-1 highlighted changes in the carrier proteins in nucleocytoplasmic trafficking, cyclindependent kinases, and ubiquitination (77) . the latter process has also been identified in proteomic analysis of cells infected with avian infectious bursal disease virus (78) . highlighted results from the quantitative proteomics study and subsequent network pathway analysis were selected for validation and further investigation using alternative approaches. the results were compared with the previously published literature, and indirect immunofluorescence confocal microscopy, western blot, and functional analysis techniques were also applied in independent experiments separate from the quantitative proteomic analysis. hrsv pathologies and immune signaling-in this quantitative proteomic analysis, several different proteins involved in the pathologies and the immune signaling associated with hrsv infection were altered. for example, ipa highlighted proteins that were previously identified as being associated with pneumonitis, inflammation, and respiratory disease. expansion of a quantitative proteomic analysis to in vivo tissue samples and the use of isobaric tags (e.g. itraq (isobaric tags for relative and absolute quantitation)) may further the investigation of possible links between hrsv infection and diseases such as asthma. stat1 and isg15 were identified as being more abundant in hrsv-infected cells when compared with mock-infected cells (fig. 5) . both of these observations were consistent with previous observations using hrsv-infected cells and in vivo models (20, 45, 79) . in the pathway analysis, stat1 was linked to ifn ␣/␤ (fig. 5) . stat1 activity is also regulated by phosphorylation, and hrsv uses distinct mechanisms to either impair stat1 phosphorylation or increase phosphorylation of stat1␤ (a stat1 variant) to repress ifn expression (80) . hrsv proteins ns1 and ns2 strongly inhibit ifn ␣/␤ by preventing the phosphorylation of the ifn regulatory factor-3 (15, 16) . however, the molecular pathology of hrsv infection and the mechanisms by which it circumvents ifn are still not fully understood. different strains of hrsv can differ in their ability to block ifn type i synthesis, and likewise, cell line choice is important in such pathway studies. therefore, a comparison of different hrsv strains using differential isotopic labeling and different cell lines (e.g. macrophages) may prove useful in further characterizing hrsv infection. integrity-the mitochondria play a role in energy production, membrane potential regulation, proliferation and cellular metabolism, and modulating cell death pathways in response to microbial infection (81) . whether mitochondria are affected by hrsv infection has not been characterized previously. quantitative proteomic analysis coupled to ipa identified changes in mitochondrial proteins in hrsv-infected cells. the localization and abundance of representative mitochondrial proteins (highlighted during data analysis) were then investigated in separate experiments using indirect immunofluorescence confocal microscopy. it is of interest to note that in this analysis mitochondrial proteins were also detected in nuclear fractions. this could have been an artifact of the purification process, or it could have been due to the tubular structures that contain mitochondria and project into the nucleus (82) . certainly, the indirect immunofluorescence confocal analysis of tom20 distribution in hrsv-infected cells reflected the latter hypothesis (supplemental fig. 1) . changes to mitochondrial proteins may be common in various virus-host cell interactions. for example, proteomic analysis of hcv (83) and avian influenza virus h9n2 interactions with the host cell (84) demonstrated an increase in the abundance of prohibitin. the latter study in particular showed an increased abundance value similar to the value observed in the current study. many of the mitochondrial proteins with altered abundance in hrsv-infected cells were associated with mitochondrial membrane pore proteins. validation using a live cell mitochondrial membrane permeability assay demonstrated a loss in the integrity of the pore complexes (fig. 9) . changes in the abundance and localization of mitochondrial proteins such as toms (fig. 7, b and c) , vdacs (fig. 8a) , and phbs (fig. 8b) may be linked to a number of factors relating to the immune response, antiviral state, and viral infection. ipa defines mitochondrial dysfunction as anything (genetic or environmental) that interferes with the regulation of reactive oxygen species (ros) causing the superoxide to overpower the antioxidant system. ros are potent elements in the mitochondrial antimicrobial defense system, and mitochondria may participate in the innate immune response by triggering the production of ros (81) . however, as is the case in the ifn pathway, microbes may have evolved strategies to manipulate such defense mechanisms, and any alterations in mitochondrial function may affect the immune response of cells to virus infection. for example, hcv is able to block the mitochondrial antiviral signaling protein (found on the outer membrane of the mitochondria) downstream signaling pathway, which leads to the expression of ifn ␤, thereby enabling hcv to evade host immunity (85) . furthermore, human cytomegalovirus has been shown to perturb many cellular processes that promote the release of proapoptotic molecules such as cytochrome c (86) . in hrsv infection, activation of retinoic acid-inducible gene-1 (rig-i) induces an antiviral response (87) that involves its association with the mitochondrial antiviral signaling protein, allowing the recruitment of signaling adapters to the mitochondrial surface. subsequent activation of the signaling complex is followed by translocation of nf-b into the nucleus and the activation of associated genes (88) . hrsv has been shown to activate cytoplasmic mitogen-and stressrelated kinase 1 (msk1) via ros, and in turn, msk1 mediates nf-b activity (88) . although the molecular basis for ros-dependent msk1 activation is unknown, the identification of pathways that control nf-b activation in response to hrsv infection may be useful in finding a way to attenuate the proinflammatory effects of hrsv and lung inflammation (88) . bioinformatics analysis of quantitative proteomics data may highlight different pathways that lead to nf-b activation in hrsv-infected cells (e.g. figs. 4 and 5) . potential disruption of nucleocytoplasmic trafficking and nuclear pore complex proteins-in hrsv-infected cells, several proteins associated with nucleocytoplasmic trafficking and the nuclear pore complex were identified as altered, including nup96 and nup98. this could be a common feature of rna virus infection (89) as other negative strand rna viruses that replicate in the cytoplasm, such as vesicular stomatitis virus, have been shown to inhibit ran-dependent trafficking (90) . specifically, the virus-encoded matrix protein can inhibit nuclear import and export (91) . this virus has been shown to target nup96 and nup98 for degradation (92) , potentially to inhibit antiviral activity (93) . nup98 is an ifn-induced nuclear pore complex protein with a major role in the export of mrna. the positive strand rna viruses poliovirus and rhinovirus are also capable of inhibiting nucleocytoplasmic trafficking (94, 95) , and nup98 is degraded in rhinovirusinfected cells (96) . deregulation of cell cycle-the quantitative proteomic analysis, ipa, and subsequent validation with western blotting revealed changes in the abundance of cell cycle regulatory complexes in hrsv-infected cells. most notably obvious was the ablation of cdc2 and the major cyclins responsible for cell cycle progression (fig. 11) . also of interest was the ablation of nucleolin (fig. 11) , which under normal growth conditions is highly expressed in proliferating cells (97) and is responsible for correct mitosis and controlled centrosome duplication (98, 99) . the ablation of cell cycle regulatory complexes may account for the observed cell cycle arrest reported in hrsv-infected primary and a549 cells (18) and in cells infected with bovine respiratory syncytial virus (100) . a number of viruses with rna genomes whose site of rna synthesis is the cytoplasm have been reported to interact with the cell cycle to promote cellular conditions more favorable for viral replication. these include other negative sense rna viruses such as measles virus, which can arrest cells in the g 0 phase to prevent an antiviral response (101) (102) (103) . also, the positive sense rna coronavirus infectious bronchitis virus arrests cells in g 2 /m to increase viral protein translation and progeny virus production (104) . disruption to subnuclear structures-the nucleus contains several subnuclear structures with defined functions. these structures are believed to form around hub proteins and are composed of protein-protein and protein-nucleic acid interactions (64, 105) . in this study, the abundance of proteins associated with the nucleolus and nd10s were altered in hrsv-infected cells. both the nucleolus and nd10s are dynamic structures whose proteins are in constant interchange with the nucleoplasm whose specific composition can depend on the metabolic state of the cell, and nd10s are associated with antiviral defense (63) . both rna and dna viruses target the nucleolus and its structure, and the proteome can change in response to viral infection (61, 106) . in this study, the quantitative proteomic analysis indicated that two of the most common and most studied nucleolar proteins, nucleolin and nucleophosmin, were ablated in hrsv-infected cells. for nucleolin, this was confirmed by western blot analysis (fig. 11) , demonstrating the strength of the silac approach. recently, it has been shown that lamin b1 interacts with nucleophosmin to maintain nucleolar structure for ribosome biogenesis (107) . however, the loss of lamin b1 resulted in the breakdown of the nucleolus (107) . our analysis indicated that lamin b1 was potentially cleaved and depleted in hrsv-infected cells (fig. 1c) . however, the observation of degradation of lamin b was antibody-dependent (e.g. compare fig. 1c with fig. 11 ). nucleolin was also observed to decrease in human metapneumovirus-infected cells using 2de (27) . proteins associated with nd10s were also altered in hrsvinfected cells. nd10 constituents tar and rad50 were significantly decreased in abundance, but the major constituent, the pml protein, was not detected by lc-ms/ms. because pml is an nd10 marker protein, it was used to validate the abundance and localization of nd10s in hrsv-infected cells. western blot analysis indicated that the abundance of pml protein increased in the nucleus of hrsv-infected cells. this was confirmed by indirect immunofluorescence confocal microscopy that showed an increased number of nd10s in the nucleus of hrsv-infected cells. these results are in contrast to a previous report for a549 cells infected with hrsv a2 strain (26) . however, in the current analysis, indirect immunofluorescence confocal microscopy positively identified hrsvinfected cells and showed that tagged pml-i and pml-ii proteins remain in the nucleus. however, this does not preclude that certain isoforms may be more abundant in the cytoplasm of infected cells. other negative sense rna viruses have also been shown to interact with nd10s. for example, in rabies virus-infected cells, nd10s became larger, and the expression of rabies virus p protein led to the sequestration of pml in the cytoplasm, which resulted in an increased viral titer, presumably through the ablation of an antiviral response (108) . along with the observations described above and the contrasting results from previous studies, it is important to note that nd10s are dynamic nuclear structures whose composition is highly dependent on the metabolic state of the cell. fractions-several virus-encoded proteins were identified in the nuclear and cytoplasmic fractions. for the nuclear fraction (supplemental table 6 ), one of these could be predicted, the m protein, as this has well characterized nuclear-cytoplasmic trafficking (69, 109, 110) . overexpression analysis using fluorescently labeled m2-1 and p proteins indicated that these proteins were present in the nucleus but predominately localized to the cytoplasm (fig. 13) , and hence this could explain why they were identified in both fractions. the f protein was detected in the cytoplasmic fraction only (supplemental table 7 ), which may be due to the association of this protein with the cellular membrane. viral proteins could not be quantified in this experimental system as there were no unlabeled virus peptides of known amount in the isolated fractions with which to compare. however, the comparison of viral peptides with mammalian peptides (some of which may have similar sequences) does allow some putative observations to be made regarding the relative amounts of the identified viral proteins. examination of the viral protein ratios in the cytoplasmic fraction (supplemental table 7 ) indicated that the relative protein abundance of the identified proteins reflected their position on the genome and hence the abundance of their corresponding mrna. this correlates with the relationship between gene position on the genome and abundance of mrna in the mononegavirales (111) (112) (113) . this may also explain why no l protein was detected in the lc-ms/ms analysis as this is the least abundant virus protein in hrsvinfected cells. several other hrsv-encoded proteins were not detected, including the ns-2, sh, and g proteins. this may be a function of their post-translational modifications (e.g. glycosylation of g protein) or indicative of protein stability and turnover inside a cell. conclusions-although rna synthesis and virus assembly occur in the cytoplasm, hrsv is known to induce nuclear responses in the cell as replication alters host gene expression. the relative changes in the abundance of cellular proteins in hrsv-infected cells observed in this study confirmed aspects of what is already known about changes in the host cell proteome, validating our novel approach and extending this information as well discovering novel interactions with defined cellular pathways. the application of lc-ms/ms coupled to silac has not been used previously to study negative sense rna viruses and their interactions with the host cell, and 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dysfunction, airway mucus, and augmented il-17 levels reactive oxygen species mediate virus-induced stat activation: role of tyrosine phosphatases the role of mitochondria in cellular defense against microbial infection the nucleus of hela cells contains tubular structures for ca2ϩ signaling with the involvement of mitochondria proteomics analysis of mitochondrial proteins reveals overexpression of a mitochondrial protein chaperon, prohibitin proteomics analysis of differential expression of cellular proteins in response to avian h9n2 virus infection in human cells mitochondrial dysfunction in hepatitis c virus infection complex i binding by virally encoded rna regulates mitochondria-induced cell death retinoic acid-insoluble gene i mediates early antiviral response and toll-like receptor 3 expression in respiratory syncytial virus-infected airway epithelial cells respiratory syncytial virus infection induces a reactive oxygen species-msk1-phospho-ser-276 rela pathway required for cytokine expression inhibition of nucleo-cytoplasmic trafficking by rna viruses: targeting the nuclear pore complex inhibition of ran guanosine triphosphatase-dependent nuclear transport by the matrix protein of vesicular stomatitis virus multiple vesiculoviral matrix proteins inhibit both nuclear export and import vesicular stomatitis virus matrix protein inhibits host cell gene expression by targeting the nucleoporin nup98 role of nucleoporin induction in releasing an mrna nuclear export block effects of poliovirus infection on nucleo-cytoplasmic trafficking and nuclear pore complex composition inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus differential targeting of nuclear pore complex proteins in poliovirus-infected cells nucleolin is regulated both at the level of transcription and translation nucleolin functions in nucleolus formation and chromosome congression molecular dissection of nucleolin's role in growth and cell proliferation: new insights respiratory syncytial virus fusion protein mediates inhibition of mitogen-induced t-cell proliferation by contact cell cycle arrest rather than apoptosis is associated with measles virus contact-mediated immunosuppression in vitro measles virusinduced immunosuppression in cotton rats is associated with cell cycle retardation in uninfected lymphocytes cell cycle arrest during measles virus infection: a g 0 -like block leads to suppression of retinoblastoma protein expression cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication adenovirus type 5 e4 orf3 protein targets promyelocytic leukaemia (pml) protein nuclear domains for disruption via a sequence in pml isoform ii that is predicted as a protein interaction site by bioinformatic analysis involvement of the nucleolus in replication of human viruses lamin b1 maintains the functional plasticity of nucleoli rabies virus p and small p products interact directly with pml and reorganize pml nuclear bodies the matrix protein of vesicular stomatitis virus inhibits nucleocytoplasmic transport when it is in the nucleus and associated with nuclear pore complexes complex nuclear localization signals in the matrix protein of vesicular stomatitis virus unravelling the complexities of respiratory syncytial virus rna synthesis recombinant respiratory syncytial virus with the g and f genes shifted to the promoterproximal positions gene rearrangement attenuates expression and lethality of a nonsegmented negative strand rna virus key: cord-329844-w969lczb authors: robson, b. title: bioinformatics studies on a function of the sars-cov-2 spike glycoprotein as the binding of host sialic acid glycans date: 2020-06-08 journal: comput biol med doi: 10.1016/j.compbiomed.2020.103849 sha: doc_id: 329844 cord_uid: w969lczb sars-cov and sars-cov-2 do not appear to have functions of a hemagglutinin and neuraminidase. this is a mystery, because sugar binding activities appear essential to many other viruses including influenza and even most other coronaviruses in order to bind to and escape from the glycans (sugars, oligosaccharides or polysaccharides) characteristic of cell surfaces and saliva and mucin. the s1 n terminal domains (s1-ntd) of the spike protein, largely responsible for the bulk of the characteristic knobs at the end of the spikes of sars-cov and sars-cov-2, are here predicted to be “hiding” sites for recognizing and binding glycans containing sialic acid. this may be important for infection and the ability of the virus to locate ace2 as its known main host cell surface receptor, and if so it becomes a pharmaceutical target. it might even open up the possibility of an alternative receptor to ace2. the prediction method developed, which uses amino acid residue sequence alone to predict domains or proteins that bind to sialic acids, is naïve, and will be advanced in future work. nonetheless, it was surprising that such a very simple approach was so useful, and it can easily be reproduced in a very few lines of computer program to help make quick comparisons between sars-cov-2 sequences and to consider the effects of viral mutations. as far as is known to history, no coronavirus [1] has been as disturbing to humanity as the human pandemic of the 2019 novel coronavirus [2] now known as sars-cov-2. in quick response to determination of the final version of the rna sequence of the wuhan seafood market isolate, the present author examined functional sites of sars-cov-2 that are highly conserved across the coronaviruses [3, 4] , and which thus likely to exhibit escape mutation that can quickly undo the good work of the developers of vaccines and therapeutic agents. so far, the published papers have concerned the spike glycoprotein [3] [4] [5] . exploration of known and newly found proteolytic cleavage sites in the spike glycoprotein of sars-cov-2 that are well conserved is a popular area of inquiry for sars researchers because such sites can interact with human host airway proteases that could be the target for protease inhibitors as potential drugs (e.g. ref [6] ). the difficulty is that inhibiting the action of human proteins could have undesirable effects on the host [6] , so parallel work on other kinds of functional sites in the coronavirus proteins is of great importance. the present paper explores another potential functional site in the spike protein, but this one is a different because the site is not a proteolytic cleavage site, and it is not well conserved, except, it is argued, for a characteristic composition of particular amino acid residues. expressed another way, there can exist certain subsequences of a protein sequence that are well conserved, but only in respect to some pattern or property that is less obvious than the order of amino acids. finding them (or as is more correctly stated, predicting them) may therefore require a more subtle and, in the present case, novel bioinformatics tool, compared with the standard bioinformatics tools which were essential in the preceding papers [3] [4] [5] . comparisons with other proteins as described below suggest that the subsequence of interest in this paper could have a crucial function, and a high degree of conservation is, even by itself, also a clue as having a role important to the virus [5] . hence such a site may represent a potential therapeutic target, perhaps as well as representing a synthetic vaccine target. however, until very recently, that crucial function did not even seem to be possessed by sars-cov and sars-cov-2, and the details have yet to be elucidated. the particular virus function that is considered in the present paper is noncovalent binding to the sialic acid glycans, i.e. oligosaccharides or polysaccharides that contain sialic acid residues. they are sometimes called sialylated glycans. interest in this binding arose as follows. it seems unlikely (although of course possible) that functions important for many different kinds of virus are of little importance to others, especially if they have a common lifestyle such as infection of the respiratory system or alimentary tract, typically reflected by common symptoms. if such functions are absent, it begs the question of how the virus copes. though glycan binding of sars-cov and sars-cov-2 seems absent, diminished, or relatively neglected in the literature (see section 1.5), many coronaviruses such as human coronavirus oc43 and bovine coronavirus appear to recognize sialic acid as a receptor. however, most biology students are more familiar with the hemagglutinin and neuraminidase of influenza, the h and n in, for example h1n1 (the numbers such as 1 being based on immunological typing of these proteins), that bind to glycans, (sugar chains, oligosaccharides or polysaccharides) at cell surfaces notably those chemically bound to membrane proteins, hence called glycoproteins, of host cells. the surfaces of many animal and all by host enzymes and so would appear, again at first consideration, to be more specific. enveloped viruses such as sars-cov-2 also have their own bound sialic acid glycans (the spike protein is usually referred to as the spike glycoprotein), but these are of less direct interest here although they can clearly influence binding of a virus to various receptors. despite their diversity, and perhaps because of it (i.e. because that diversity implies more information content) sialic acid glycans of host cells are key molecular recognition features not only for entry of viruses such as influenza, but also in embryonic development, neurodevelopment, reprogramming, and oncogenesis. correctly speaking, even sialic acid itself is diverse. it is a generic term for a family of derivatives of the nine-carbon sugar neuraminic acid. the sialic acid family includes some 43 derivatives of neuraminic acid, but these acids rarely appear free in nature. members include n-acetylneuraminic acid, 2-keto-3-deoxy-d-glycero-d-galactononulosonic acid, 5,7-diamino-3,5,7,9-tetra-deoxy-d-glycero-d-galacto-nonulosonic acid, and 5,7-diamino-3,5,7,9-tetra-deoxy-l-glycero-l-manno-nonulosonic acid. if the term "sialic acid" is used unqualified, it usually refers to the representative member of this group, n-acetylneuraminic acid. the variability of glycans is not random but reflects their modes of synthesis. in eukaryotes generally, a typical n-linked glycan has an initial core that consists of 14 residues (3 glucose, 9 mannose, and 2 n-acetylglucosamine). this preassembled glycan is usually transferred by a glycosyltransferase oligosaccharyltransferase to a nascent peptide chain within the reticular lumen. this initial core 14-sugar unit is assembled in the cytoplasm and endoplasmic reticulum and other sugars may be added later. in contrast, o-linked glycans are assembled one sugar at a time at the outset on proteins in the golgi apparatus. there are some specific features of medical interest as relevant to the human host of viruses (but by no means unique to humans). the lung epithelial glycans are typical by having sialic acids as the distal residues, and it is these that the influenza neuraminidase cleaves away. most soluble secreted proteins are also similarly decorated with such glycans. that includes the proteins that make up saliva and mucus in the airway, and are in general important for viral infection. both n-and o-and glycosphingolipid-glycans are found in human lungs, and they include large and complex-type n-glycans with linear poly-n-acetyllactosamine [-3galβ1-4glcnacβ1-] n extensions, which are predominantly terminated in α2,3-linked sialic acid. in contrast, the smaller n-glycans lack poly-n-acetyllactosamine but are enriched in α2,6-linked sialic acids. there are also large glycosphingolipid glycans, which also consists of poly-n-acetyllactosamine, usually terminating in α2,3-linked sialic acid. while it is commonly maintained that viruses such as influenza virus bind to the sialylated glycans, and this is assumed in the present paper, some care is required, because there are also nonsialylated glycans in human lungs on which viral binding could occur. while it is binding of viruses to sialic acid glycan that is of interest here, it should be kept in mind that it is often associated with a catalytic activity in which the sialic acid glycan is the substrate. in influenza these two aspects are particularly distinct by being on separate surface proteins, the hemagglutinin and neuraminidase respectively, but that separation is not true of all viruses. not surprisingly, as noted above, most coronaviruses of the coronaviridae family also have capabilities to bind to sialic acid glycans, but they also have the ability to cleave the glycans, which is often described by authors as a "receptor destroying activity". like influenza c viruses, purified bovine coronavirus preparations have an esterase activity which inactivates o-acetylsialic acidcontaining receptors on erythrocytes; diisopropyl fluorophosphate completely inhibited this receptor-destroying activity suggesting that the viral enzyme is in this case a serine esterase [8] . this is believed to facilitate the spread of virus infection by removing receptor determinants from the surface of infected cells (see discussion below) and prevent the formation of virus aggregates. another coronavirus, porcine transmissible gastroenteritis virus (tgev) recognizes n-glycolylneuraminic acid. nor does it depend on the sialic acid binding activity for infection of cultured cells, but interaction with sialoglycoconjugates appears to help the virus to pass through the sialic acid-rich mucus layer in the epithelium of the small intestine. hemagglutinin-esterases are a family of viral envelope glycoproteins that mediate reversible attachment to oacetylated sialic acids. these too are said to be receptor-destroying, but the enzymic activity reaction is in this case not a cleavage in the sense used concerning neuraminidase, but rather a change in molecular recognition by removal from of the acetyl group from the c9 position of the above acetylated neuraminic acid residues. the other and probably major reason for researchers thinking of these actions as "receptor destroying" is because the picture is a little more complex than just allowing entry and exit from the host cell. because many viruses attain host cell specificity by being selective for particular types of sialic acid, these may occur as decoys to the virus on off-target host cells and on free molecules in the extracellular environment. to prevent irreversible binding to these decoys, many viruses including many coronaviruses have receptor-destroying enzymes that are therefore interesting targets for antiviral intervention, exemplified by the influenza a virus neuraminidase [9] . even though such glycan binding domains and enzymes as neuraminidases are found in many coronaviruses, there seems to be no such enzymes in sars-cov and sars-cov-2. viruses of the lower respiratory tract, such as influenza virus, respiratory syncytial virus, and sars-related coronaviruses, are generally considered as having key differences that require different therapeutics [10] even though relatively little is lost in considering already approved drugs for one of such viruses against the other (e.g. ref [11] ). typically, the apparent absence of glycan binding and enzymic sites in sars-cov and sars-cov-2 has been dismissed as due to the fact that the virus enters on ace2, i.e. angiotensin converting enzyme type 2 (e.g. see ref [5] for discussion), not on a glycoprotein. this does not, however, escape from the intuitively important need for preliminary binding, cell entry and exit through the glycan layer, and probably the decoy-related function discussed above. there appears to be growing evidence of significant lectin-binding capability. lectins are the carbohydrate-binding proteins that are highly specific for sugar groups of other molecules. activation of ctype lectin receptor and other similar receptors contributes to pro-inflammatory response to many coronavirus infections. there also are studies over several years that locate glycan binding and even related catalytic activities in the spike glycoprotein. it has been noted that e3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity [8] , and the 3d structure of coronavirus hemagglutinin-esterase offered insight into coronavirus and influenza virus evolution, with implications for drug and antibody discovery [9] . the location of any sialic acid glycan binding region of sars-cov-2 is, a priori unclear, although intuitively (a) it would likely be associated with the cap or knob at the outer end of the spike protein, or (b) at least not involve exactly the same domain as is required for other important functions. although throughout the coronaviruses various external proteins and domains can recognize either protein or sugar receptors or both, the majority of such studies like those above implicate the s1 region in their spike glycoproteins, but as discussed in the present paper, there are other potential sugar binding sites that are still within the spike protein. overall, the sars-cov-2 spike glycoprotein has 1273 amino acid residues and until early 2020 understanding of structure was heavily based on sars-cov spike glycoprotein (1255 amino acids) with 20-27% amino acid residues similarity among non-sars coronaviruses. most of the spike protein appears to be involved in the specific stages of cell entry. the spike glycoprotein of sars-cov and sars-cov-2 is translated as a large polypeptide that is later cleaved to s1 and s2 sites. after binding to the main receptor that that is held to be primarily ace2, the host proteases activate the virus by cleaving first at the s1/s2 boundary (i.e. s1/s2 site) and then within s2, i.e. at the s2' site. the spike of similar coronavirus have long been considered as being in two main states (i) the pre-fusion form (the form of the mature virion) and (ii) post-fusion form, the form after membrane fusion has been completed). more detailed studies have split the latter into a prehairpin intermediate state, and post-fusion hairpin state. somewhat like in all virus class i fusion proteins, the s2 protein contains two heptad repeat regions (hrs) of which one (hr2) is located close to the transmembrane anchor. membrane fusion occurs when there is a conformational change in the hrs to form a fusion core. the hrs of the protein fold into a coiled-coil structure, known as the "fusogenic state". as virus and target cell membranes fuse, the coiled coil regions (called heptad repeats) become a trimer-of-hairpins structure. the s2' cleavage site appears particularly important by being well conserved [3] [4] [5] and proteolysis by cathepsin appears sufficient to expose the fusion peptide of s2 and activate fusion within the host cell endosome. in general, s2' is now considered as the key viral fusion peptide which is unmasked following s2 cleavage. subsequently, s1 dissociates from s2, allowing s2 to transition to the post-fusion structure. the following locations in the sequences of amino acid residues apply specifically to sars-cov-2. these vary somewhat with author, and the following are used here. the signal peptide (sp) comprises residues 1-19. on the inside of the lipid membrane, the carboxyl terminus (c-terminus) is comprised of the transmembrane region (tm) comprising residues 1214-1236 and the cytoplasmic tail (ct) residues1237-1273. the extracellular domain of the spike glycoprotein is comprised of n-terminal domain (s1-ntd) comprising residues 20-286, and is of particular interest here. the host cell receptor binding domain (rbd) comprising residues 319-541. in summary the key regions are as follows. sp see text in regard to the significance of the other colors. fig. 1 shows the external part 20-1213 of the spike glycoprotein of sars-cov-2 in the closed state prior to ace2 binding, with s1-ntd domain (the "ears", dark blue) of interest here, rbd (at tip, subdomains light blue, blue-green), s2 (subdomains, orange, green, yellow). the orange-white, green-white and yellow-white helical structures are the α-helices of the trimer that form the neck associated with s2, and the red-white helical structures are the start of the transmembrane α-helices tm. in at least some coronaviruses, s1-ntd is known to be involved in binding host proteins or glycans, but coronaviruses show great diversity in their binding which presumably underlies their ability to jump between very different host species. while the role of s1-ntd compared with the current reasonably detailed knowledge of the remarkable mechanism of cell entry involving ace2 and changes to the spike protein on cleavage, the specific function of s1-ntd of sars-cov-2 has not been elucidated (at least, not by the time of writing in april 2020). as noted above, s1 in sars-cov-2 is now well known to have a region which is the receptor binding domain to human ace2 but also, significantly for what follows in the text below, sars-like coronaviruses can bind clec4m/dc-signr c-type lectin domains on host cells. see ref [12] for review of the diverse receptor recognition mechanisms of coronaviruses up to 2015, which represented the body of understanding until the cov-19 pandemic. bovine coronavirus is an example of a coronavirus for which it seems clear that s1-ntd has an established glycan-binding function. although the structure of a sugar-bound bovine cov s1-ntd was not available, some conclusions could be reached by researchers using structureguided mutagenesis and comparisons with different coronaviruses. as well as evidence in 2008 linking hemagglutinin-esterase to the s1 domain of at least some coronaviruses [9] , zhang and yap [13] had reported in 2004 a rational 3d model for s1 domain of sars-cov spike protein by fold recognition and molecular modeling techniques, and there they noted a suggestive structure similarity between s1 protein and influenza virus neuraminidase [14] . this opened up the possibility for those authors that existing antiinfluenza virus inhibitors and anti-neuraminidase antibody could be used as a starting point for designing anti-sars drugs, vaccines and antibodies [14] . based on such observations and discussion so far, it is therefore reasonable to propose that s1-ntd could be important in the binding of certain alternative host cell surface receptors, or perhaps which aid in targeting the virus to ace2, and so might provide a helpful therapeutic target (as well as candidate antigenic site for synthetic vaccine design). nonetheless, such functions if present in sars-cov-2 could, a priori, reside in other domains at the virus surface. the challenge for research here is that the substantial knowledge concerning such matters in well-studied coronaviruses is not readily transferable. variously throughout the coronaviruses, s1-ntd, ctd and s2 regions can recognize either protein or sugar receptors or both in various cases, and very similar coronavirus spike protein domains within the same genus may recognize different host cell receptors, while many very different coronaviruses may recognize the same host cell receptor. the studies mentioned above also suggested that at least some coronavirus s1-ntds are evolutionarily related to human galectins, the term typically used for the lectins as carbohydrate-binding proteins that are specifically involved in inflammation, immune responses, cell migration, autophagy and signaling; however the viral domains derived from them have diverged with specificities for different sugar receptors [12] . further review is given throughout results section 4 where appropriate. another challenge discussed in results section 4 is that key regions for which an experimental 3d structure would help resolve the matter are disordered, and hence invisible, in current available spike protein structures. less familiar theoretical principles do arise in the knowledge gathering, inference, and prediction methods developed by the author and colleagues are used in the present cov-19 project as described in ref [4] . they also include approaches to facilitate interaction with the standard bioinformatics web tools which are stated below in methods section 3. however, while tools of these various kinds do speed and facilitate a project of this nature, the usual methods of investigating literature and accessing bioinformatics data bases and tool are sufficient for reproducibility of the work described in this paper, at least by researchers reasonably familiar with bioinformatics and protein structure. an algorithm for predicting the domains and proteins involved in sialic acid glycan binding is developed in the course of the project described in results section 4, but this is primarily of a highly empirical nature. future work to advance this algorithm on a sounder theoretical basis is underway. see brief discussion on future work in discussion section 5.1, where some of the above-mentioned theoretical approaches of the author and colleagues, also to be used in the development of the algorithm, are cited. the overall approach comprised the following steps. i. automated gathering of information from the world wide web regarding hemagglutinins, neuraminidases, and sugar binding proteins, particularly but not solely of viruses, using "autosurfing", natural language processing, and knowledge extraction techniques [4] . note that it is in particular non-covalent sialic acid glycan binding sites that are being explored, not for example asparagine or serine or threonine sites to which glycans are covalently linked. this knowledge gathering approach, as described in ref [4] , is not essential for reproducing the present work or for carrying out comparable studies, but it does greatly accelerate research and preparation of the scientific paper, allowing fast responses to a new epidemic [3] [4] [5] . ii. attempted discovery of continuous short sequences of amino acid residues (potential "sequence motifs") with patterns and amino acid content common to sars-cov-2 spike protein amino acid sequences and hemagglutinins and neuraminidases, particularly those of influenza viruses. also, more generally, in preparative work, comparison of the spike protein sequence of the spike protein with sialic acid glycan binding proteins and other sugar binding proteins, or domains of them. protein sequences or parts of them used as input for any part of the study were obtained from genbank https://www.ncbi.nlm.nih.gov/genbank/. the standard method of bioinformatics used for detecting in large protein sequence databases any amino acid residue sequences similar to those of an input sequence was primarily blastp at https://blast.ncbi.nlm.nih.gov/ blast.cgi. the standard tool for a more formal and typically multi-sequence alignment was clustal omega at https://www.ebi.ac.uk/ tools/msa/clustalo/. these tools can be automatically accessed by the present author's methods [4] but again that is not essential for reproducibility of the present work. iii. examination of patterns in potential or known short subsequences in small proteins or domains known to have a function involving non-covalent sialic acid binding and, in absence of any clear patterns, study of the amino acid content of the subsequences. this established a preliminary sialic acid glycan binding score (sabs) for the twenty naturally occurring amino acid residues. however, the short subsequences identified were to be considered as "signals" or "fingerprints" for sialic acid glycan binding domains as a whole. that is, direct contact with sialic acid was not necessarily at (or solely at) the specific short subsequence, for reasons discussed in results section 4. this, plus a sequence rather than three dimensional structure perspective, and a specific focus on binding sialic acid glycans rather than sugars in general, resulted in a substantial difference in scores from another major method of predicting sugar binding regions of proteins also discussed later below. iv. development of the above as an algorithm sabr-p for identifying potential small proteins or domains of proteins that non-covalently bind sialic acid glycans, by predictions on a test data set of protein sequences. as noted above the subsequences predicted are taken to indicate the glycan binding domain as a whole, not necessarily the sialic acid sites per se, but they may be. this approach involved noting true positive and negative predictions and false positives and negative predictions so as to optimize sensitivity and specificity. this was done specifically in regard to non-covalent binding of sialic acid glycans. in other words, it was done so as to distinguished sialic acid glycan binding domains not only from those domains known not to bind sugars but also from those that bind sugars and glycans that do not contain sialic acid. v. examination of the three dimensional structures of regions of the regions of the sars-cov-2 spike protein predicted as binding sialic acid glycans to propose and locate a sialic acid binding function of sars-cov-2 (possibly but not necessarily associated with some kind of enzymic activity). results and discussion in the present paper used the same amino acid codes as the above tools and data bank use, i.e. the iupac (international union of pure and applied chemistry) one letter amino acid codes, given in table 1 below. for completeness, conservative replacements in column 3 of table 1 are given. they relate largely to substitutions that can usefully be made in the design of synthetic peptides [4, 5] . this is an application which is not specifically discussed in the present paper but which could be a basis for design of synthetic vaccines and preventative or therapeutic agents [4, 5] , in this case targeted at sialic acid glycan binding site of a virus. as discussed in the sequence of steps for the methodology above, the sialic acid glycan binding motifs are taken to be indicators of the sialic acid binding domain and not necessarily of the target sites per se, but they may be, and often are, potential target sites. the list of conservative replacements also remains useful for considering substitutions that are conservative in maintaining similar amino acid properties when detecting and comparing related sequences. since for both reasons they be useful in deeper consideration of many results in the present paper, some comment may be useful to researchers less familiar with bioinformatics. see ref [4] for a further account. note that the work of considering what is a conservative replacement is done automatically by the standard bioinformatics tools used. the replacements in table 1 are consistent with the conservative replacement rules implied by the tables of weights implemented automatically in blastp and clustal omega mentioned above, which are discussed at those sites. however, the original intent as an application to peptide design means that in table 1 there is a degree of asymmetry based on the author's experience in peptide design [4] because one is going from a natural protein state to less natural one without evolution making compensatory changes in the rest of the protein or system. for example, empirical studies show that serine (s) can be replaced by alanine (a) or threonine (t) but it is frequently important that a replacement to threonine should be isoleucine (i) in order to retain stability of a βpleated sheet in which they occur. strictly speaking, these are just fairly crude rules-of-thumb: the best replacements are dependent on more specific circumstances and detailed conformational and binding calculations. the assignment in table 1 are not seen as controversial because apart from the asymmetry they relate to the "interchangability" or "alternative rule" of amino acid residues by many authors that are intended as universal, i.e. intended to apply to all proteins. this is because they relate to similarity of amino acid residues in terms of physicochemical, conformational, as well as biological properties of many sequences that are at least universal to, say, vertebrates. however, they are historically more directly empirically based on wellknown studies probabilities of amino acid differences found by comparing amino acid residue sequences amongst fairly related proteins from a wide range of sets of different proteins, such that one is comparing sequences of hemoglobins, or of lysozymes, or of cytochromes c, and so on. as is to some extent customary in the field, three letter codes (such as gly for glycine) are used for the amino acids in the molecular graphics figures; these codes are fairly self-evident at least in the direction of deducing the full name of the amino acid being represented. there was also use of data and the associated graphics tools in the protein data bank (pdb) at https://www.rcsb.org/ and in japan https://pdbj.org/ which was used for fig. 1 . energy calculations by the author's own krunch and by a commercial sculpt protein modeling package were used in ref [4] , but were not required for the present study, with the exception that some calculations by these tools were used to obtain earlier unpublished results on sugar binding to amino acid residue sidechains. this provided a check on the preliminary sialic acid binding capability shown in column 4 of table 1 , used initially in the present paper. krunch is a molecular mechanics modeling package that essentially functions like many standard molecular modeling packages. there is the arguable exception that it gives much more attention than usual to novel algorithms for navigating through multiple energy minima and discovering new conformers, but that capability did not appear to be too important in the present study. for the much greater part, however, these binding assessments were based on the amino acid residues observed by the author in sequences involved in sugar binding sites in proteins (found by visual examination of binding sites of entries in the pdb) and similar qualitative observations by other authors. that is, they also reflect rather general opinion of what amino acids are involved in sugar binding and in its most general formulation this intuitively comprises aromatic residues, and hydrogen bonding residues to interact with the sugar hydroxyl groups. at the outset, as a starting point only, column 4 of table 1 of these preliminary sialic acid binding amino acid scores (sabs) are really assignments that are qualitative, using 0 for not often present in sialic acid glycan binding sites and 1 for often present,. however, tryptophan was assigned a double score of 2 reflecting its larger size and double ring. how reliable these assignments are in regard to sialic acid glycan binding is what is assessed on a more objective basis by the prediction method developed in this paper, including a degree of recalibration. the marginally modified parameters are also shown in the last column table 1 for convenience of comparison. while as a methodological strategy it was tempting to start from an alternative more objective and established approach discussed in results section 4, or at least to use it as a starting point or as an important "gold standard" for comparison, it has substantially different aims. as a first step in an investigation making use of bioinformatics, a common strategy is the use of protein sequence alignments so that a functional part that is known in one protein might suggest an analogous functional part in another. as discussed here, this approach has proven somewhat less successful for sialic acid glycan binding sites than for other functions considered elsewhere (e.g. refs [3] [4] [5] ), and necessitated development of alternative techniques of which discussion occupies the major part of this paper, but the results are consistent and to some degree suportive. as the basis of discussion and starting point for the present study, the following is a sars-cov-2 spike protein sequence in which the s1-ntd is shown in italics. the short section of sequence underline emerges below as of particular, but by no means sole, interest. more subtle computational techniques based on protein conformation have in this project suggested relationships between sars-cov and hemagglutinins and neuraminidases of other viruses, but they essentially support prior work. recall that zhang and yap [13] noted a suggestive structure similarity between sars-cov s1 and influenza virus neuraminidase. using various computational techniques they compared the 3d structure of sars-cov s1 protein data bank 3d structures 1iny (neuraminidase from influenza a virus complexed with a sialic acid phosphonate analogue inhibitor) and 1b9t (neuraminidase from influenza b virus with novel aromatic inhibitors). this observation does not necessarily suggest by itself a common function, because many protein folding patterns are used by nature for diverse purposes, but armed with that information a more careful use of sequence alignment is helpful. the following part of a clustalw sequence alignment done in the present study also includes the hemagglutinin esterase sites in e3 of the bovine coronavirus [8] (e3-cov-q14eb1.1 below) and that studied by zeng et al. [9] (hem-est-cov-3cl5 below). substrate binding residues in deduced for sars-cov s1 by zhan and yap [13] are shown by #. : :. . *: . ::.: *. ---qiifyegvnfnphhrf------kcffngsndvwifnkvrfyralysnmalfryltfv 140 . : *:*:. .: : :.. * . :: the situation is of course not clear when the degree of amino acid residue match between sequences is poor because similarities can arise by chance, but aligning more sequences can be insightful, and in this case including influenza a and b sequences seemed particularly helpful. although still barely conserved compared with the motifs in the current project (published [4, 5] , submitted, or under investigation) the most plausible match for further study is at sssgwtagaaayy of sarscov2-s-mn908947.3, because others would include at least one extensive deletion areas from one protein. including influenza a and b sequences interferes with much of the alignment but it does preserve the above match. this preserved match region also places the notable tryptophan (w) within short distance of alignment in the second subsequence corresponding to the zhan-yap binding site, but in procedures of this kind it is important to establish that such features are not simply artifacts of the particular sequences included. the essential features of the above alignments at the binding site are not perturbed by removing the influenza neuraminidases which provided a basis for the zhan-yap catalytic regions, as follows. : :. . *: . ::.: *. ---qiifyegvnfnphhrf------kcffngsndvwifnkvrfyralysnmalfryltfv 140 . : *:*:. .: : :.. * . :: . . #### alignment studies like those above are helpful in forming initial hypotheses, but they are clearly not convincing by themselves when the degrees of match between aligned residues is weak; however, it is possible that the actual composition in terms of amino acids in the subsequence and potential motif of interest is more important than their actual order in that subsequence. based on many studies exemplified by the above, it was noted that tryptophan is a recurrent, albeit not absolutely essential, feature of sugar binding including sialic acids. references to that also recur throughout the biochemical literature. for example, see e.g. ref [14] , and also fig. 2 shows the tryptophan interaction with and sialic acid in the influenza virus b neuraminidase (pdb entry 2bat). as might be expected by their similar aromatic character, the alternative amino acid residues as sugar binders, and residues frequently supporting tryptophan in the binding site, tend to be aromatic sidechains, notably tyrosine (y), sometimes phenylalanine (f) and histidine (h). a preliminary survey of sequence motif patterns in sugar binding proteins suggests that invariant amino acid residues across a family of proteins tend be one or more of the above aromatic residues supported by negatively charged aspartate (d), asparagine (n), serine (s), threonine (t) glycine (g) and sometimes alanine (a) that provide the hydrogen bonding. however, particularly in regard to the non-aromatic residues, the binding of acidic and non-acidic sugars should probably be distinguished. as discussed later below, charged amino acid residues glutamate (e), arginine (r), and lysine (k) also frequently make intimate contact with sialic acids but that is in three dimensions, not together in a subsequence. a likely relevant observation was that the first set of amino acid residues (the set containing aspartate) and binding sialic acids tended to occur in a subsequence that adopted a local loop conformation, while the second set (that containing glutamate) were frequently associated with α-helices and particularly their termini. however, this was an empirical and qualitative observation regarding a tendency, and a more objective quantification of the importance of the aspartate set is the purpose of the prediction algorithm developed below. three dimensional considerations, however, give insight and sometimes explain why influences can be somewhat indirect. hydrogen bonding that occurs between the hydroxyl groups of carbohydrate ligands and polar amino acid residues at the binding site is typically supported by water-mediated hydrogen bonding networks in which serine and threonine are fairly commonly involved. nonetheless, the most outstanding feature of carbohydrate binding sites from a three dimensional perspective would appear to be the position and orientation of tryptophan (w), tyrosine (y), and/or phenylalanine (f), which usually provide a hydrophobic plate for close interaction with the planar face of sugar rings, an interaction resembling hydrophobic stacking interactions, as in fig. 2 . the importance of these and to some extent of histidine (h) in a sequence motif seems reasonable. along with the occasional appearance of cysteine (c), sometimes as a serine (s) and particularly a threonine (t) substitution, the residues mentioned above can be used as the basis of a preliminary and essentially qualitative model for assessment of sugar binding as given column 4 of table 1 . often a valine (v) substitution was seen in potential glycan binding sites, although this was not significantly supported by the optimization of the predictive technique described below. however, glycans containing sialic acids appear to bind somewhat differently to other sugars. taking this as a hypothesis and focusing on these, protein sites for binding them may have a variety of affinities for different subtypes. for example, all influenza a virus strains critically depend on sialic acid to bind to host cells and the different forms of sialic acids all show different affinities that change with influenza a virus variety, important because it determines which species can be infected. there has also been very relevant work that can complicate the details of what can be meant by, for example, "sugar binding motif". indeed, zhang and yap [13] themselves noted that tryptophan was frequently involved in strong protein fold interactions that stabilized the sugar binding domain fold, yet lacked any direct interactions with sugars. in the influenza neuraminidase they comprised three pairs of main chain-side chain interactions: tryptophan (w) 171 (donor) and phenylalanine (f) 179 (acceptor), alanine (a) 210 (donor) and phenylalanine (f) 29 (acceptor), leucine(l) 209 (donor) and tryptophan (w) 171 (acceptor). for example, tryptophan accepted one n-h⋯π bond from leucine (l) 209 and donates one n-h⋯π bond to phenylalanine 179. it is possible that the aromatic residues could be induced to be more exposed in certain types of binding, but the above interactions appeared to stabilize the structure of s1 fold motif while reducing the active site cavity for ligand binding [13] . features of sialic acid glycan binding regions represented by a run of amino acid residues have diverse sequence patterns, but evidently the simple presence of the above residues in a section of sequence is itself a strong signal feature; this seems particularly so for tryptophan. to examine this further, many sequence alignments were examined that relate to role of the aromatic amino acids and the contribution of tryptophan to known or suspected sialic acid glycan binding, and these were were investigated in a more quantitative way. for example, the following represent a summary of influenza subsequences that contain tryptophan. with it is associated the preliminary, essentially qualitative sialic acid binding residue score (sab-s) discussed above and based on observation of multiple sugar binding sites and literature survey, to each of the amino acids mentioned above. at this stage, there is no significant algorithm except that the sum of scores over the residues in the short sequence is divided by the number of residues (mostly 16 or 17) so that it expressed on an averaged, per residue, basis for that sequence. recall that it is qualitative that the amino acid residues are given a score of 1 (and 0 otherwise), except that tryptophan is given an extra weight of 2. in many cases the sidechain is involved, especially for the aromatic residues, but this not obligatory: it could be a backbone interaction (as is necessary for glycine (g) that lacks a sidechain). this is the "qualitative" model that was shown in table 1 , which will be extended to the sabr-p method later below. there is in the following an attempt at local alignment to highlight similar features because there is often some indication that a motif is reused even within a protein, although that assertion is not required for present purposes. the sum of score is divided by the sequence length that excludes relative deletions '-'. many hemaglutanin and neuraminidase matches were found with sssgwtagaaayy using blastp, the top 100 varying from 64% match and 63% identities that preserve the tryptophan, such as hemagglutinin influenza a virus, genbank axb35920.1 ssgw--gavn, and neuraminidase, influenza a virus afk13818.1 sgw----aay, and neuraminidase, influenza a virus anz90284.1, ssawsasa. looking at the larger sequence context, 95% of these scored over 0.75 on the above system. however, nucleocapsid proteins of influenza a virus such as sequence qiq4588 with sg-tagaa and aaz08011.1 as stsg-aagaa also appear in the top 100 matches along with the above and with comparable scores, but with one obvious and significant difference, that the tryptophan (w) is missing. the possible importance of tryptophan in binding in sars-cov-2 is exemplified in the following sequence of the sars-cov-2 s1-ntd section of the spike glycoprotein (including the signal peptide, in order to conserve standard numbering). the sequence is from the s1-ntd of sars-cov-2 genbank entry mn908947.3, along with a brief description of accessibility in pdb entry 6vvx, which is for the spike protein in the closed state. trtqlppaytnsftrgvyypdkvfrssvlhstqdlflpffsnvtwfhaihv 60-70 w exposed sgtngtkrfdnpvlpfndgvyfasteksniirgwifgttldsktqsllivnnatnvvikvcefqfcndpf 112-124 cleft lgvyyhknnkswmesefrvyssannctfeyvsqpflmdlegkqgnfknlrefvfknidgyfkiyskhtpi 147-160 disord. nlvrdlpqgfsaleplvdlpiginitrfqtllalhrsyltpgdsssgwtagaaayyvgylqprtfllkyn 253-265 disord. engtit here, "disord." means a disordered (flexible) loop: the tryptophan (w) and adjacent residues are not seen in the experimental 3d structure determination 6vvx. this is also true of pdb entry 6vsb the spike protein in the open state, 6vyb and other sars-cov-2 accessible to the author at the time of writing. see fig. 3 . the fact that disordered suggest an open loop like structure that be may be capable of binding to, and perhaps adjusting to, disordered targets, and certainly does not prohibit functional significance. the following summary table 2 has comments on the status of exposure of the tryptophan and surrounding residues, and on the involvement in binding based on alignments with the zhang-yap analysis. evidently the above is not perfect as a basis for a prediction method, and the next step in this study required more detailed considerations. it also required a more comprehensive analysis of predictive capability that allows for false positives and false negatives. the method developed here takes account of the above results in the light of several pieces of experimental and theoretical evidence, which is usefully briefly reviewed at this point in the narrative. several kinds of evidence were taken into account in development of sabr-p, the sialic acid binding region prediction. the emerging principles used, not all of which are immediately intuitive, are argued as follows so that they may help researchers develop improved versions. the method specifically concerns predicting regions that interact with glycans containing sialic acid residues. the development of the method started with the qualitative sialic acid binding region sabs score in table 1 and used in preliminary studies above. other prediction methods such as that discussed below also address sugars or oligoscharrides in general. recall, however, that even the sialic acid components comprise a fairly diverse family of sugar types (see introduction section 1.3). also, while described as a sialic acid binding region prediction, and this is believed to be the naturally emerging emphasis, it is formally the binding to glycans that contain sialic acids that matters. (ii) the emphasis is also on binding, not catalysis. the method is not specifically concerned with catalytic amino acid residues involved in neurominidase action (nor any other activity of cleaving modifying the glycan to reduce virus binding, e.g. esterases, deacetylases). while evidence of neuraminidase activity in sars-cov-2 would be very important, the current evidence is that focus should be on sialic acid binding. this is because of lack of any strong evidence for such enzymic activities at the time of writing, and also because neuraminidase inhibitors approved as dugs, oseltamivir (tamiflu), and zanamivir (relenza) have been tested on sars-cov-2 in vitro and were not found effective [15] . it is nonetheless the binding of the appropriate glycans containing sialic acid that may still be important for sars-cov and sars-cov-2 infection in vivo, as it is for other viruses [16] . the fact that other viruses, including other coronaviruses, require that function, followed by any demonstration that sars-cov and sars-cov-2 still possess that function, would suggest that it could still be a target for pharmaceutical drug development, at least as a preventative. this argument is not unique in its general form. for example, fantani and colleagues believe that the sars-cov-2 also uses sialic acids linked to host cell surface gangliosides to somehow facilitate host cell entry and so could represent a therepeutic target [17] . (iii) the method seeks to predict whole domains and proteins that bind sialic acid, not specific short sequence motifs. that is the case even though the predictions assign a sialic acid binding score to each residue in the domain or protein, which can of course still be inspected as of potential involvement in direct binding. the threshold and scale of the sabr-p prediction method is set such that any residue (in practice it is almost always a continuous run of residues) with a sialic acid binding score of more than 100 signals that the whole domain or protein, whichever was provided as the whole sequence in input, is a binding module for glycans containing sialic acid. there are several reasons for using this as a marker of a domain or protein of interest. one is that the binding is to the whole glycan as an oligosaccharide or polysaccharide, not necessarily the sialic acid components per se, and therefore the ligand is a large structure that could involve several binding sites. recall that tryptophan was found to be frequently involved in strong protein fold interactions that stabilized the sugar binding domain fold, yet were lacking any direct interactions with sugars and even buried [13] . there are at least somewhere between 10 and 100 amino acid residue sequence motifs known to be associated with sugar binding in general, and they fall into some 7 fold motifs [18] . also, as the function of sialic acid recognition may be to guide the virus to and across the host cell surface [17] , sites on the virus that enable this motility and hunt out points for catalysis or simply the strong binding appear to be as important as key strong binding and catalytic sites themselves (e.g. ref [18] ). in addition, predictions of localized regions of proteins as sugar binding sites are naturally not as good as those based on predicting that a whole domain or protein is involved in sugar binding, but such a prediction remains valuable and indeed well suited to the present study. in a particularly detailed investigation by taroni et al., analysis of the characteristic properties of sugar binding sites was performed on a set of 19 sugar binding proteins [19] . their prediction was optimized on a training set of 19 non-homologous carbohydrate binding structures and tested on a test set of 40 protein-carbohydrate complexes. the thoroughness of that study and the inclusion of many kinds of information would make it a reasonable choice of "gold standard" for comparison, except that the aims were substantially different, and the overall accuracy of prediction achieved was only 65%. it is true nonetheless that results were very good for carbohydrate-binding enzymes as opposed to lectins, with a rate of success of 87%. this emphasis on enzyme catalytic sites again argues for avoiding the use of this kind of approach in the present paper, as covered by point (i) above. in summary, it may be that a large domain or protein that has strong signals for sialic acid binding may be indicative of a sialic acid binding and/or catalytic function even if a specific short subsection of the sequence predicted as binding sialic acids is not in the position for which a strong binding or catalysis has been observed. subsequences. only the sequence is considered in the method, not residues brought together by the folding of the protein in space. most prediction methods for predicting sugar binding such as the example of taroni et al. [19] discussed above are not based on sequence and the sugar-binding propensity of amino acids in short segments alone, but on regions on a protein surface that require account of 3d structural information, analogous to "discontinuous" epitopes or "discontinuous determinants" of a pathogen protein in the study of immune response and in synthetic vaccine design. this kind of approach was abandoned in the present study not just because it took away the emphasis on the binding functions of whole domains or proteins but most importantly because many of the sites of particular interest are in disordered regions, or otherwise invisible in the available experimental 3d structure, as discussed later below. it might even be that a degree of disorder is important for binding some sugars. also and not least, 3d structural information is not always available. (v) glycophilicty parameters for amino acid residues or sequence patterns as obtained by other workers were not used. this is particularly because, the aims, i.e. what things are wanted to be known, are usually very different in these methods. although taroni et al. showed that out of 6 partially parameters partially dependent on 3d information the sugar binding propensities of certain amino acids was prominent in having discriminatory power, this was in regard to the tendency for being in putative a sugar binding patch compared with protein surfaces in general (requiring ordered conformation and experimental information about it). at the same time, it still concerns a specific region of a kind rather than predictions of domain or proteins as sugar binding as a whole. perhaps most importantly these studies were concerned with binding sugars in general (not specifically sialic acids). not surprisingly, therefore, the amino acid residue propensities have no significant overall correlation with the propensities in table 1 used and developed in the present paper. notably, alanine (a), serine (s), threonine (t) and cysteine (c) have a propensity against sugar binding in their approach. there is nonetheless the significant exception that tryptophan (w) is the strongest in both that and the present study. (vi) a simple predictive model was developed based on optimized parameters. if a simpler method works as well as a more rigorous approach, it can sometimes provide insight and help build even better rigorous approaches. the method used in the present case was initially based on the gor method [20] for protein secondary structure prediction in which sialic acid glycan binding state of residues replaced α-helix, β-sheet, and coil (or loop) states of residues. however, it was found that the results were essentially reproduced by a simpler model. this is primarily because the directional effects (in terms of n-terminal direction or c-terminal direction along the amino acid residue sequence) was found to be equivalent (i.e., symmetrical) and to persist for some 8 residues in both directions, very like the influence of alanine on α-helix formation in the gor method [20] . consequently the final method used in the present study consisted of just two changes to that used in previous results section 4.3 above, the second being described in point (vii) below. the qualitative scores as parameters to types of amino acid residue as shown in table 1 were based on visual observations by the present author and a survey of sites in the literature, and this was improved by optimization on essentially the same set of proteins examined. the 20 amino acids were initially assigned the qualitative parameters of table 1 , then these 20 parameters were optimized to optimize sialic acid glycan binding predictions as positive for 20 sialic acid glycan binding proteins, and negative for 10 proteins not considered as binding sugars, and 10 proteins such as lectins that bind other sugars that do not contain sialic acids. this gave as before glycine (g), alanine (a) aspartate (d), asparagine (n), serine (s), threonine (t), cystine/cysteine (c) each with a score of 1, and the rest assigned 0, except for tyrosine (y phenylalanine (f) and histidine (h) that were now assigned larger parameter values of 2 and tryptophan (w) that was assigned a larger parameter value of 4. (vii) parameters describing directional influences of amino acid residues were modeled in a simple way. in part this is a response to the above point that the directional effects (in terms of n-terminal direction or c-terminal direction along the amino acid residue sequence) are symmetrical, but more specifically the essential features of the interactions between neighboring residues can be deduced from parameters like those in table 1 in much the same way as the shape of an equilateral triangle standing on its base can be deduced from its height. as well as using new parameters, the initial score for a particular residue was computed in the same way as that for the whole segment of 17 residues in section 4.3, but was specifically considered as associated with the central residue, and the sum was retained as that sum rather than the overall score being averaged by the number of residues in the segment. deletions to consider alignments of segments were not applied in the sabr-p method. the above sum associated with each central, i.e. 9 th , residue, was then averaged over the corresponding sums from the residues up to 8 away on the n-terminal direction up to 8 residues in the c-terminal direction including the central residue being addressed. this was done for every residue in the input sequence. it formally models parameters describing the directional characteristics of different types of amino acid residue, albeit in a highly empirical way. however, it is simply analogous to a smoothing of the propensity plot for residues along the overall input sequence. (viii) plot scaling was applied to produce a convenient decision threshold. an appropriate height scale of the plot, in which a residue with a score of over 100 would be considered as the whole input sequence indicative of a sialic acid residue binding domain or protein, was obtained by an empirical pseudo-normalization consisting of dividing each residue score by 300. whenever the final results by sabr-p are expressed as a percentage-like score by multiplying all residue scores on the plot by 100, this is of course equivalent to dividing by 3. on that percentage basis, there seems no benefit it describing scores more accurately than to the nearest integer. in summary, the essential features of the simple resulting algorithm are as follows. (1) residue binding parameter assignment. examine every residue in the sequence and assign it the parameter 0,1,2,3, or 4 of the sabr-p prediction method refined parameter (last column of table 1 ). (2) basic motif score. for each residue number i, obtain a score by summing over the run of 17 residues of which i is the central (9 th ) residue and assign the resulting sum (the score) to each residue i (providing that a residue numbers i-8, i-7 etc. up to i+8 lies within the sequence). [20] by repeating step (2) with the resulting scores, but do not add the basic motif score for each residue i to itself. more specifically stated, examine the above basic motif score for each i th residue from i=1 to end of sequence in turn, and add it to the score of the residue at i-8, i-7, etc up to i-1, and to the score of the residue i+1, i+2, etc. up to i+8 (providing that the residue numbers i-8 etc. lie within the sequence). (4) normalized score as sabr-p propensity. examine each smoothed score resulting from the above for each residue i, and divide by 300. this is the current value of the optimized normalization parameter that sets the threshold as 100 above which sialic acid glycan binding is predicted. (5) reporting. the normalized score (sabr-p propensity) is plotted as a function of residue number from 1 to end of the sequence, i.e. as a "sabr-p propensity plot". the actual predictions are also written out separately as text and these report only the amino acid residue (as g, a, v, etc.) along with the score, for those residues for which the normalized scores exceed 100. particularly because of step (3), these will tend to occur conveniently in runs of approximately 8-18 contiguous residues, but sometimes shorter. in the present study the normalized score was formalized as a simple measure by rounding to the nearest integer prior to reporting, though this made no significant difference in the present paper. this simple approach will be better developed as a gor-like method, but for present purposes it suffices to give an estimate of the likely sialic acid glycan binding domains on the spike protein and proceed with further investigations and results described below. at the above optimized threshold, this the example sabr-p propensity plots shown in fig. 4 . the initial studies using a random selection (more correctly stated, an arbitrary selection) of proteins except that a predominance of sialic acid or sialic acid glycan binding proteins were sought, the method initially gave 18 true positives out of 20 proteins representing group a, i.e. those that are known to bind sialic acids or glycan molecules containing them, 10 true negatives for prediction of the above same kind of sialic acid binding out of group b, i.e. 10 proteins believed not to bind any kinds of sugars significantly, and 6 true negatives for the 10 proteins comprising group c, i.e. proteins that bind sugars but not those containing sialic acids, and so representing the biggest challenge. this initial result represented 85% accuracy, 82% sensitivity, and 80% specificity, a reasonable preliminary result for such a simple model. note that there were initially no false negatives for those proteins not believed to bind any kinds of sugars, so a specific search for at least one case of a false negative (discussed below), which is strictly speaking a bias, dropped the prediction quality to 83% accuracy, sensitivity 82%, and 76% specificity. a recent number of further studies exemplified in the discussion below were also, strictly speaking, a bias including comparisons between weakly homologous proteins, extended the sample to 80 proteins and reproduced the original quality to within the nearest percentage: 85% accuracy, 82% sensitivity, and 80% specificity. fig. 4 . examples of prediction of sialic acid binding sites by sabr-p. in each case, the abscissa (x axis) is the distance along the sequence (residue number) and the ordinate (y axis) is the predicted sialic acid glycan binding propensity. scores above a threshold of 100 (red line) for any residues are taken as a prediction that the domain or protein binds sialic acid glycans. at this stage of the present project the predictive method was only required to give approximate results as guidelines as to the regions the spike protein that might be further examine for sialic acid glycan binding capability, but the method appears promising and justifies some further comments and some further exploratory investigation, as follows. in group a, proteins being tested are believed to be able to accommodate sialic acid, sialic acid glycan, and related compounds by non-covalent binding. predicted correctly, these would represent the true positives, but predicted incorrectly, they would represent false negatives. such proteins include various hemagglutinins such as that of influenza a at genbank caa24291. generally arbitrarily selected neuraminidases and hemagglutinins are true positives but the above kind of pattern, a segment of approximately 9-23 residues with a score exceeding 100 and centered on tryptophan with the peak score, is not universal. sometimes it is another hydrophobic residue. in a "haemaglutinin repeat-containing protein" of a yet to be fully classified micororganism candidatus kentron a tryptophan (w) with a score of 118 is slightly displaced from the central peak of 119 associated with a run of three glycines (g). it is two glutamate residues (e), negatively charged that are in the vicinity in the sequence that appear to perturb the usual central role of tryptophan. similarly, a rat neuraminidase is of some interest in that the predicted sequence sldhghtw surrounds the glycine (g) with peak score 106 and terminates at tryptophan (w) with a score of 102. the tryptophan is, however, immediately followed by a glutamate (e). the significance of the above comments is as follows. of the amino acids with charged sidechains, only aspartate (d) and histidine (h) appear to favor such binding in the present author's analysis, while glutamate (e) arginine (r), lysine (k) have zero valued parameters and so are contraindications of sialic acid or sialic acid glycan binding. however, the latter three charged amino acid residue can sometimes be found in scialic acid binding sites and in sites predicted as such by the present method. indeed, they are often dominant features of residues directly interacting with sialic acid. in the sialic acid binding site of the globular head region of the newcastle disease virus haemagluttinin a glutamate (e), three arginine residues (r) and a lysine (k) are intimate contact with sialic acid ligand. a difference is that these residues make intimate contact in space and are not together in a one or very few subsequences. that is, they do not necessarily constitute a sequence motif. the actual extent of any counter-predictive impact of the above charged residues other than aspartate (d) and histidine (h), when they together in a subsequence of amino acids examined by the present algorithm, can be seen in false negatives. the neuraminidase of the plant striga asiatica (asian witchweed), was one of the false negatives: the analogous region to many of the above, at least in the sense of surrounding the only tryptophan (w) and with the peak value, is egavdrwrgeanf where the tryptophan (w) has only a peak value of 88, and has an arginine (r), positively charged, on both sides. the other false negative in the original study was a bacterial (chryseobacterium) haemagluttinin with the peak value of 93 at a tryptophan, but with two lysine residues (k), also positively charged sidechains, in the vicinity. later studies also noted that n-acetyl neuraminic acid synthetase neub of e. coli, which has score of any residue to more than 100. for example, in the case of the witchweed neuraminidase, changing rwr to sws raised the peak value at the tryptophan to 99, a significant increase but still not exceeding 100. in some cases a value exceeding 100 can of course attained. a false negative also found in later studies investigating these issues was the ox neuraminidase, with the subsequence ddhgvswrygggvs containing the tryptophan (w) with peak value 96. changing to an serine (s) the arginine (r) adjacent to the tryptophan (w) did have an effect, albeit that the only change was having the tryptophan as the only residue predicted, with a marginal score of 101. two kinds of potential true negative or false negative were distinguished in the study. the group that appeared to do particularly well at predicting those proteins that do not binding sialic acid or sialic acid glycan was, perhaps not surprisingly, group b, i.e. those proteins not expected to bind any kind of sugars. out of the original sample of 10 proteins not expected to bind any kinds of sugars significantly, and that also confirmed that expectation, i.e. true negatives as far as predicting sialic acid binding is concerned, two (i) hemoglobin and (ii) trypsin precursor for which the prediction plots for which are shown in fig. 4 . the others not shown are mostly quite large proteins containing more than 5 tryptophan residues (w) sites, for which the method still correctly predicted as non-sialic-acid binders, and so worthy of some comments. they included (iii) human ubiquitin c of 685 residues and no tryptophans (w) and no residue score exceeding 56, in contrast to (iv) human progesterone receptor of 933 residues of which 6 were tryptophan but none of which exceeded 100 (one had the highest score of 95). the remaining true negative cases are (v) fatty acid oxidation complex subunit alpha fadb of acinetobacter calcoaceticus of a substantial 717 residues, (vi) the mitochondrial nadh-ubiquinone oxidoreductase 75 kda subunit of the camel, which comprised a substantial 733 residues but did notably not exceed a score of 77 for any residue, (vii) human cytochrome c with a maximum score of 87 for the second tyrosine (y) in tgqapgysytataankn, and (viii) alcohol dehydrogenase (human, 1a) that did not exceed a score of 81 for any residue despite the "concern" that ethanol having basic sugar-like features and so could, a priori, be marginal. perhaps unfairly included as rather small, (ix) proinsulin nonetheless does contain a tryptophan (w) which correctly did not exceed 100 and indeed only had a score of 72 in llallalwgpdpaaa; the phenylalanine (f) in gpdpaaafvnqhlcg had a highest score of 81 in the sequence. in the initial study, the case most closely approaching a false positive in this group was (x) human prothrombin with a substantial number of 622 residues, there was only one residue, glycine (g), that reached a score of 100, and a residue score should exceed 100 to classify the whole domain or protein as sialic acid binding. it is possibly best declared as an example of a marginal case. although at the outset false positives were expected to appear in this non-sugar-binding group groups as the sample is increased, not least because of the preliminary nature of the method. human angiotensin converting enzyme type 2 (ace2) was the first exception found and it is a significant exception in that it had two substantial regions 198-276 and 599-610 both exceeding scores of 100 throughout and peaking at substantial scores of 112. this group is interesting in that persistently predicting these as false positives might incline a researcher to abandon specifically predicting sialic acid glycan binding and instead consider their method as better positioned to predict sugar binding in general, but this turned out not to be the case. for group c, i.e. those sugar-binding proteins that are believed to bind sugars but not to bind sialic acids or glycans containing them, the prediction plots for the 6 true negatives are shown to the lower right in fig. 4 . human α-amylase, not shown, was an interesting false positive with subsequence of residues all only marginally exceeding 100 ysgwdfwgegw but containing three tryptophans (w) of which the first had the highest score of 103. the human lysozyme precursor was also a false positive, albeit only marginally so, with one sequence kwesgyntra exceeding 100 and with the peak at tyrosine (y) with a score of 103. in later studies examining the effect of considering weakly homologous sequences, the significantly different hen lysozyme precursor is interesting as still being a false positive but representing and even more marginal case, having one short subsequence with residues exceeding 100, namely wv, with tryptophan (w) with final score 102 followed by valine (v) with final score 101. while there are significant amounts of sialic acids in hen egg white, the author is not aware of any extract of hen egg white lysozyme that has these bound to the protein. evidently comparisons of homologous proteins might be helpful to improve prediction power. another false positive is the human atp-dependent translocase abcb1 isoform 2 which has a ribose binding site has a considerable size of 1,280 residues yet with just one subsequence, syalafwygmmyfsyagcf, exceeding 100, and has the peak of 107 at the tryptophan (w). for such reasons, one may suspect that a fairer set of criteria for assessing predictive performance would be an average per residue basis. one of the more recent studies included the sars-cov-2 polyprotein which contains proteins with known rna and ribose binding functions. out of 7095 residues, 184 residues exceeded scores of 100. the proteins and domains there, however, include several of which all possible functions are not yet known. evidently, those regions with scores over 100, by virtue of being in proteins of sars-cov-2, are certainly worthy of future further examination in this project. however, this was considered beyond present scope for the present paper, as the focus is on the spike glycoprotein. the more recent studies of sugar binding proteins that did not include suspected sialic acid or sialic acid glycan binding regions included sugar isomerases, such as the l-rhamnose isomerase of rubinisphaera brasiliensis, which with 423 residues containing 9 tryptophans (w) was a true negative. they also included a comparison between sugar transporter proteins, such as the udp-galactose transporter of drosophila melanogaster had 357 residues including 4 tryptophans (w), the pts fructose transporter subunit eiic of aeromonas hydrophila with 589 residues containing 7 tryptophans, and a facilitated glucose transporter member 1, which despite having 492 residues containing 6 tryptophans (w), all of which were true positives by not predicting any sialic acid or sialic acid glycan ability. however, some sugar transporters were false positives. for example, the arabinose transporter of e. coli had the subsequence fwlyta which exceeded 100 with the tryptophan scoring 104. armed with the above predictions and insights, one may make better informed judgements as to whether a domain for non-covalently binding host sialic acid glcycans may exist in the sars-cov-2 spike protein. the above results suggesting the ability to distinguish between three classes of protein in relation to sialic acid glycan binding is perhaps surprising, not least because non-sialic sugars such as fucose and mannose can occur in cell surface glycans (including sialic acid glycans) as indicated in introduction section 1.3. this is discussed in discussion section 5.2. at this stage, only the ability to show propensities in different regions of the sars-cov-2 (genbank entry mn908947.3) spike glycoprotein is required. in this stage of the study the predicted regions of the sars-cov spike protein were examined in more detail. while as discussed above tryptophan can be involved in the fundamental structure of a sialic acid binding domain, an exposed tryptophan like that binding a sialic acid glycan in fig. 2 would be particularly persuasive. the first predicted sialic acid binding motif is segment ffsnvtwfhaihv 58-70 with sabr-p score ranging from 101 for valine (v) to 113 for tryptophan (w) and for phenylalanine ( as shown in fig. 6 , the tryptophan sidechain in ffsnvtwfhaihv as residues 58-70 of the sars-cov-3 spike glycoprotein is exposed in a site that has all the appearance of a sialic acid glycan binding site, comparable with the influenza virus b neuraminidase (pdb entry 2bat) tryptophan site known to have interaction with and sialic acid, that was shown in fig. 2 . the tryptophan sidechain in ffsnvtwfhaihv 58-70 of spike glycoprotein is exposed in a site that has all the appearance of a sialic acid glycan binding site. it is useful to see how recurrent a potential more universal motif may be, to give insight, to avoid cross reactions of vaccine in human and veterinary patients as hosts, to detect an underlying common function that one might not wish to inhibit in the host with an anti-viral therapeutic, and so on. a blast search on non-viruses picks this subsequence up as ffsnvtniawihai of parasteatoda tepidariorum, the common house spider, and related sequences, a zinc figure domain because it contains fyve the zinc finger motif, but more abundantly it picks up sugar-binding proteins such as glycosyl transferases such as ffspvwartpnvtwfh-hv of actinobacteria, ribulosephosphate 3-epimerase of animals, a-amylase of the chitinophagia ("chitin eating") bacteria, c-type lectin 37db-like of drosophila hydei, and related sugar binding proteins, all varying around 100% cover and 55% match, 92% cover 71% match, 76% cover 70% match, and so on. the second predicted sialic acid binding subsequence sssgwtagaaa has been of interest in the preceding sections 4.1-4.4, where it seemed a likely sugar binding site based on circumstantial evidence such as homologies to neurmaindiases and glycan esterases. unfortunately, it lies in the range that generally disordered in experimental 3d structure, e.g. residues 246-262 in vvx (spike closed state) and 243-262 in vyb (spike open state). it also has modest maximum scores of 109, but the above considerations do not prohibit its potential importance. again, as for the previous subsequence, it is useful to see how recurrent a potential more universal motif may be. blastp searches of non-viruses find subsequences in sugar binding proteins such as ssagwtagaa of microbacteria (90% cover 90% match), but compared with ffsnvtwfhaihv discussed above, searche generate a lot of closer matches (many 100% matches with the top 99 at 100% cover 82% match or better) which, however, involve an even more diverse set of proteins. at first examination most appear less directly relevant to sugar, but many of these merit more examination. details of each case are beyond present scope, but for example a particularly recurrent match example is sssgwtaga or similar sequences of proteins that contain the twin-arginine translocation pathway signal of most bacteria and archaea. for example such as ruegeria marisrubri, of the rhodobacteraceae has the above matching subsequence. the twin-arginine translocation pathway transports folded proteins across the cytoplasmic membrane of these microorganisms. the proteins are targeted by signal peptides containing a conserved twin-arginine motif, and the literature does not always mention any n-glycosylation and indeed there may not in every case be any directly relevant evidence of such. however, there is certainly known involvement in the production of secretory and extracellular n-linked glycoproteins in bacteria such as escherichia coli. the third predicted subsequence is hwkwpwyiwl 1102-1218 with a peak score of 108 relates to ikwpwyiwl in the original wuhan seafood market isolate (genbank mn908947.3) and lies at the boundary between the c-terminal end of s2 and the transmembrane part 1214-1273. its 3d structure is not, to the present author's knowledge, available, as it lies in residues 1147 onward are generally excluded from experimental structure (e.g. vvx and vyb). again, matches with non-virus or host proteins may be of interest as biologically and medically important. blastp searches on non-viruses with the sequence as query inappropriately pick up a lot of coronaviruses by accidentally relating to the host name, but this is indicative of a strong recurrence of the motif across coronaviruses. otherwise, there is a diverse set of matches especially but not solely with bacterial proteins, and unfortunately most are described as hypothetical proteins for which the function is typically unclear. of those that are named, there are some indications of involvements with sugar binding in many cases. many are ribosome proteins or phosphatases relevant directly or indirectly to rna or ribose binding. the pap2 superfamily is characterized by a core consisting of a 5-helical bundle and includes functions involving glycerol phosphates but also sugars such as in the case of glucose-6-phosphatase. this subsequence hwkwpwyiwl also matches sequences in proteins with an spfh domain which is implicated in regulating targeted protein turnover in stomatins and other membrane-associated proteins. hwkwpwyiwl has of course a notable tryptophan (w) repeat that hints a special role of its own, certainly worthy of analysis but outside present scope. in general, however, it does appear that across many proteins it has other functions than sugar binding (perhaps diverse or multiple functions), although sugar binding is not excluded. the significance and innovation of the present work is that it proposes a sialic acid glycan binding function for the sars-cov-2 spike protein that has been largely neglected by other workers, apparently on the rationale that ace-2 binding is the important first step in cell entry. sites involved in the characteristic cap or knob of the spike protein appear partially persuasive in the light of their role as binding to host cells. there is a further possible site towards the base of the external part of the spike protein, which seems less likely by virtue of its position and weaker prediction. interaction with sialic acid glycan with or without associated catalytic activity would be consistent with such functions observed in many respiratory and alimentary tract viruses, and not least in many or most other coronaviruses, and so such a function must be important to these viruses. on these grounds, it may be a target for therapeutic agents against sars-cov-2, particularly perhaps preventatives as well as means of impeding spread from lung cell to lung cell, and an exposed target for antibodies raised by synthetic vaccines. although other authors have recently touched on such a glycan binding ability in sars (as discussed in this paper above and particularly below), it has not been to the present author's knowledge analyzed in comparable detail and do not appear to relate to the same site. nor do they propose a general prediction method for sialic acid glycan binding as described in the present paper. of course, in the present paper this is still a prediction and not an experimental result, but it will hopefully encourage experimental researchers to investigate the glycan binding properties of sars-cov-2 more extensively. a further innovative feature is that predictive method, which is expected to be worthy of investigation for the proteins of other viruses and even of other organisms. like many predictive methods in bioinformatics it is not perfect, i.e. there are false positives and false negatives in prediction, so it is actually conceivable that the method is useful even if it is not correct in the particular case of sars-cov-2. in that sense, it may emerge as the more important contribution. work. the current sabr-p predictive algorithm is naïve and it is not expected that it will resemble closely the final refined form of the algorithm, which will based on more rigorously on principles closer to those of the gor method [20] , the hyperbolic dirac net [21] [22] [23] , the association q-uel language, and the bioningine implementation including its new algorithms [25] [26] [27] [28] . the impression of good performance for the current sabr-p method largely arises from the fact that it is only required to predict the sialic acid glycan binding properties of whole domains or proteins, not highly localized subsequences or surface patches. in essence, the method is really doing little more than capture and quantify in an algorithm the visual inspection of sugar binding domains and proteins and the observations of other workers as discussed above. however, the method was only required to help explore potential non-covalent sialic acid glycan binding sites in the spike glycoprotein, and in that regard it has proven adequate and valuable for present purposes. it also suggests a more refined approach may perform well because false positives and false negatives were mainly just over the boundary and just under it respectively. the current predictions also indicate a research direction in which to explore. the parameters for the general sugar binding capacity of amino acids residues are very different to those used by other workers and here the focus has been on sialic glycans versus other saccharide-based molecules. in this, perhaps the most surprising finding of all is the apparent ability of the method to distinguish between sialic acid containing glycans and other sugars in the case of lectins. this is because non-sialic sugars such as mannose and fucose can occur in in sialic acid glycans, and prediction results hint that there is likely to be some distinguishing feature for a majority of cases that makes a specific recognition. in this respect it would seem initially of concern for the sensibleness of the predictions that, for example, mannose-binding lectin binds to a range of sugars that also include n-acetyl-d-glucosamine, n-acetyl-mannosamine, fucose and glucose. it is therefore possible that research in this direction will not be so profitable because the above distinguishing behavior of the algorithm might be to some extent coincidental. be that as it may, the predictions are remarkably much better than expected, and should certainly be challenged by researchers in order to improve such methods. specifically, a larger sample may require a threshold adjustment or corresponding rescaling, perhaps resulting in a deterioration of performance particularly in regard to distinguishing lectins. nonetheless, it is noteworthy that ultimately human glycan binding proteins have to overcome the same problem as the above kind of prediction algorithm. while this broad range of sugar recognition by the mannose-binding lection permits that lectin to interact with a wide selection of pathogens (viruses, bacteria, yeasts, fungi and protozoa) decorated with such sugars, there must be some kind of distinguishing aspect such that is not decoyed by the sialic acid glycans of the human host. a more mundane problem in extending the study is that the correct state as sialic acid glycan binding, other sugar binding, or not binding any kind of sugar, may be uncertain or a matter of degree. further studies at time of writing suggested only about 70% for each of accuracy, sensitivity, and specificity, but this larger set is, as yet, of dubious quality for the purpose. some proteins were believed, rather than known, to bind sialic acid glycans, binding might be weak or less specific or of multiple types, or the domain or approximate location of the binding site can be unclear. related to that is a difficulty that the performance of any prediction method of this kind is defensible, and possibly unfairly defensible, in regard to false positives: it may be that experiment shows that a particular virus predicted to bind sialic acid glycans does not specifically do so, but perhaps it once did, in evolutionary terms. this is particularly relevant in regard to studying coronaviruses because, as discussed above, many coronaviruses certainly do bind sialic acid glycans. of course, the prediction method would then still be subject to the criticism that it insufficiently sophisticated to manage the impact of small changes. for purely theoretical methods, that may be an issue for some time: in the present author's experience even simulations of binding of sugars to proteins in atomic detail tend to be difficult in view of the complex role of water molecules. for example, water molecules commonly represent protein-to-sugar bridges as discussed in this paper. potential biological implications arguably support the above prediction for sars-cov-2 spike protein. that is, the story "makes sense". although the involvement of sars-cov and sars-cov-2 with sialic acid glycans has been rather neglected in the literature (but see below), such involvement represents a prominent and well known feature in the life history of influenza and other viruses, and appears no less important in the life of many other coronaviruses. admittedly, hiv and many other enveloped viruses do not encode hemagglutinin for sialic acid binding. instead, they interact using n-terminal sialic acid bound to envelope-associated proteins, like gp120 on hiv-1. however, the mode of infection is different. the sars-cov-2 coronavirus cannot jump in a magical way from contaminated surfaces to the lung, and it is doubtful that infective small loads of virus rely on chance to travel from the infecting person to the lung cell ace2 receptor of the next human host. it is as yet unclear how many virus particles of sars-cov-2 are needed for infection, but the virus is clearly very contagious, and this may be because rather few particles are needed for infection. in any event, the virus has to survive, and ideally even benefit for its survival, stages in a complex journey in mucus of a sneeze or on hands, face, eye, nose, or mouth, and in the various stages of the airway. initial cell entry points are unlikely to be only the lung epithelium. sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes [20] . viral mechanisms relating to these various surfaces could be fairly sophisticated. in biology in general, flexibility in carbohydrate recognition contributes to the targeting efficiency of carbohydrate-active enzymes in environments where there is diverse range of saccharides [18] . in a virus, more than one saccharide-binding site or multiple sugar binding sites in a protein could act to increase or decrease the overall affinity and increase or decrease virus mobility at different locations, while conformational changes could make available some sites and not others could regulate the extent of movement of the virus. some binding sites have evolved to distinguish not just the sugar residue components but several types of monosaccharide or glycosidic bond linkage. once having reached the vicinity of a cell with an ace2 receptor, the virus still needs to recognize the cell surface and raft across the cell surface to reach the ace2 receptor. fantani et. al [17] argued that a new type of ganglioside-binding domain exists at the tip of the n-terminal domain of the sars-cov-2 s protein, and that the subsequence 111-158, conserved among clinical isolates, may improve attachment of the virus to lipid rafts and facilitate contact with the ace-2 receptor. this study also showed that, in the presence of clq or its more active derivative, hydroxychloroquine, the spike protein is no longer able to bind gangliosides [17] . the present study does not support (nor necessarily refute) their conclusions in terms of such specific details, but the general argument concerning guidance to the ace-2 receptor is compatible. very recently milanetti and colleagues have made available a preprint [30] that is tune with such ideas, and specifically states that binding silvic acids provides a second means of entry, other than ace2. rather like the approach of thornton et al. [19] , this is based more on interactions between surfaces of molecules in three dimensional space. the results and conclusions of this study are speculative in the sense that they are applications of computers (using the techniques of bioinformatics and a new predictive method), and hence they are essentially theoretical. their role has been to highlight the likelihood that the sars-cov-2 spike has a biological function of binding host cell sialic acid glycans (and probably across cells surfaces by that means, as discussed below). in particular, a domain in the cap or knob of the sars-cov-2 spike, which has so far been somewhat neglected, is involved in the non-covalent binding of host sialic acid glycans. it is perhaps curious that subsequences found as conserved by use of bioinformatics tools such blastp and clustal omega (also used here as described in methods section 4.1), or detected as a known or new functional motif, often seem in the literature to be considered as having the status of experiment or observation, while consideration of more complex patterns with more sequence options tend to be treated as theory and prediction. this caution is justified in the present study because further study and confirmation is required along the lines discussed in discussion section 5 above. above. to the extent that it is a prediction, it is a prediction for sars-cov-2 made in advance of experiment in order to provide an objective and fair test of the methodology and it is hoped that it will stimulate experimental study in this area whether the experiments confirm or refute that prediction. either result would likely be of ultimate medical importance. this is essentially typical of the more interesting roles of computers in biomedical research, although the general infrastructure and support that they provide for more routine tasks is of course of great importance. the present paper possibly still stands as the first reported attempt to establish means of making use of sequence motifs that could be recognized between strains, albeit that the order and to some extent precise nature of the amino acid residues of amino acid residues appears less important, or perhaps more subtle, than has been considered in previous papers in this series [4, 5] , which is why it required the development of the predictive technique (sabr-p). this will be advanced in future work, but the present method already helps to make quick comparisons between sars-cov-2 sequences and to consider the effects of viral mutations. however, it was surprising that a very simple approach was so useful, and it can easily be reproduced in a very few lines of computer program. the important consequence of the present study, however, is that there are already a variety of inhibitors of sialic acid binding that may serve as anti-viral agents, and this will be examined elsewhere. • this paper extends the studies of the author's previous sars-cov-2 papers. • designing vaccine and drugs must seek to avoid escape mutations. • strangely, sars-cov and sars-cov-2 appear to lack sialic acid binding functions. • sequence motifs are found, but they require a simple prediction method. the molecular biology of coronaviruses genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding, www.thelancet preliminary bioinformatics studies on the design of synthetic vaccines and preventative peptidomimetic antagonists against the wuhan seafood market coronavirus. possible importance of the krsfiedllfnkv motif preliminary bioinformatics studies on the design of a synthetic vaccine and a preventative peptidomimetic antagonist against the sars-cov-2 (2019-ncov, covid-19) coronavirus covid-19 coronavirus spike protein analysis for synthetic vaccines, a peptidomimetic antagonist, and therapeutic drugs, and analysis of a proposed achilles' heel conserved region to minimize probability of escape mutations and drug resistance the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade neuraminidase is important for the initiation of influenza virus infection in human airway epithelium the e3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution overview of current therapeutics and novel candidates against influenza, respiratory syncytial virus, and middle east respiratory syndrome coronavirus infections drug treatment options for the 2019-new coronavirus (2019-ncov) receptor recognition mechanisms of coronaviruses: a decade of structural studies journal of virology the 3d structure analysis of sars-cov s1 protein reveals a link to influenza virus neuraminidase and implications for drug and antibody discovery tryptophan residues and the sugar binding site of potato lectin inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs sialic acids as receptor determinants for coronaviruses structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov-2 infection carbohydrate-binding modules analysis and prediction of carbohydrate binding sites gor method for predicting protein secondary structure from amino acid sequence hyperbolic dirac nets for medical decision support. theory, methods, and comparison with bayes nets split-complex numbers and dirac bra-kets bidirectional general graphs for inference. principles and implications for medicine suggestions for a web based universal exchange and inference language for medicine implementation of a web based universal exchange and inference language for medicine. sparse data, probabilities and inference in data mining of clinical data repositories studies in using a universal exchange and inference language for evidence based medicine. semi-automated learning and reasoning for pico methodology, systematic review, and environmental epidemiology studies in the extensively automatic construction of large odds-based inference networks from structured data. examples from medical, bioinformatics, and health insurance claims data extension of the quantum universal exchange language to precision medicine and drug lead discovery. preliminary example studies using the mitochondrial genome sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes in-silico evidence for two receptors based strategy of sars-cov-2 barry was a pioneer in bioinformatics, protein modelling, and computer-aided drug design, interests that he actively continues today. he is the recipient of several honours including the asklepios award for outstanding vision in science and technology at the future of health technology congress at m.i.t. in 2002. he has helped start up several other companies or divisions in the uk and usa. barry continues as ceo of the dirac foundation in the uk, and distinguished scientist prior to that, he was cso of gryphon sciences (later gryphon pharmaceuticals) in south san francisco, california, a bio-nanotechnology ultrastructural chemistry start-up largely held and then acquired by smithkline beecham. before moving to the us, barry was the scientific founder of proteus international plc in the uk, designing and leading the development of the prometheus expert system and its underlying global expert system, bioinformatics and simulation language for drug, vaccine, and diagnostic discovery. it sold for the equivalent of $9.4 million to the pharmaceutical industry in the mid-1990s. at proteus, he also led the team that used the above expert system to invent and patent several diagnostics and vaccines including the mad cow disease diagnostic subsequently marketed worldwide by abbott as a whitepaper to the president of the united states. he was also an advisor in relation to a major scientific computer-aided drug design collaboration and network for peter feinstein consultants between work between us scientists and the russian science city arzamas. for five years, barry was a nature "news and views" correspondent on biomolecules. he was visiting scholar stanford university school of medicine 1997-1998, professorial lecturer mount sinai nyc during part of his period at ibm corporation, and held visiting positions and professorships in inra and u quantum universal exchange language and hyperbolic dirac nets for precision medicine and drug design. proposals with examples from mitochondrial studies studies in the use of data mining, prediction algorithms, and a universal exchange and inference language in the analysis of socioeconomic health data bidirectional general graphs for inference. principles and implications for medicine studies in the extensively automatic construction of large odds-based inference networks from structured data. examples from medical, bioinformatics, and health insurance claims data studies in using a universal exchange and inference language for evidence based medicine. semi-automated learning and reasoning for pico methodology, systematic review, and environmental epidemiology studies of the role of a smart web for precision medicine supported by biobanking data-mining to build a knowledge representation store for clinical decision support. studies on curation and validation based on machine performance in multiple choice medical licensing examinations interesting things for computer systems to do: keeping and data mining millions of patient records, guiding patients and physicians, and passing medical licensing exams implementation of a web based universal exchange and inference language for medicine. sparse data, probabilities and inference in data mining of clinical data repositories split-complex numbers and dirac bra-kets suggestions for a web based universal exchange and inference language for medicine. continuity of patient care with pcast disaggregation popper, a simple programming language for probabilistic semantic inference in medicine hyperbolic dirac nets for medical decision support. theory, methods, and comparison with bayes nets suggestions for a web based universal exchange and inference language for medicine the concept of novel compositions of matter. a theoretical analysis towards new tools for pharmacoepidemiology the role of information drug discovery using very large numbers of patents. general strategy with extensive use of match and edit operations considerations , for a universal exchange language for healthcare quantum mechanics in artificial intelligence and decision support. a review this project used some of the knowledge gathering methods described in preceding papers although the work is described entirely in terms of standard bioinformatics tools. these knowledge gathering methods are used, amongst many others in an integrated way, in the algorithms and internal architectural features of the bioingine.com, a distributed system developed by ingine inc. cleveland, ohio, for the mining of, and inference from, very big data for commercial purposes. key: cord-310909-nc82a70n authors: qiu, maofeng; shi, yuling; guo, zhaobiao; chen, zeliang; he, rongqiao; chen, runsheng; zhou, dongsheng; dai, erhei; wang, xiaoyi; si, bingyin; song, yajun; li, jingxiang; yang, ling; wang, jin; wang, hongxia; pang, xin; zhai, junhui; du, zongmin; liu, ying; zhang, yong; li, linhai; wang, jian; sun, bing; yang, ruifu title: antibody responses to individual proteins of sars coronavirus and their neutralization activities date: 2005-04-13 journal: microbes infect doi: 10.1016/j.micinf.2005.02.006 sha: doc_id: 310909 cord_uid: nc82a70n a novel coronavirus, the severe acute respiratory syndrome (sars) coronavirus (sars-cov), was identified as the causative agent of sars. the profile of specific antibodies to individual proteins of the virus is critical to the development of vaccine and diagnostic tools. in this study, 13 recombinant proteins associated with four structural proteins (s, e, m and n) and five putative uncharacterized proteins (3a, 3b, 6, 7a and 9b) of the sars-cov were prepared and used for screening and monitoring their specific igg antibodies in sars patient sera by protein microarray. antibodies to proteins s, 3a, n and 9b were detected in the sera from convalescent-phase sars patients, whereas those to proteins e, m, 3b, 6 and 7a were undetected. in the detectable specific antibodies, anti-s and anti-n were dominant and could persist in the sera of sars patients until week 30. among the rabbit antisera to recombinant proteins s3, n, 3a and 9b, only anti-s3 serum showed significant neutralizing activity to the sars-cov infection in vero e6 cells. the results suggest (1) that anti-s and anti-n antibodies are diagnostic markers and in particular that s3 is immunogenic and therefore is a good candidate as a subunit vaccine antigen; and (2) that, from a virus structure viewpoint, the presence in some human sera of antibodies reacting with two recombinant polypeptides, 3a and 9b, supports the hypothesis that they are synthesized during the virus cycle. an outbreak of atypical pneumonia, first emerged in guangdong province of china and termed severe acute res-piratory syndrome (sars) by the world health organization, rapidly spread to many regions and countries from late 2002 to early 2003. thousands of people got infected and hundreds of them were claimed. a novel coronavirus, the sars coronavirus (sars-cov), was identified as the causative agent of sars [1] [2] [3] . since this virus is a novel pathogen for humans and its many aspects remain unknown, accurate diagnostic tools and protective vaccine will play an indispensable role in its control. the genome of the sars-cov was predicted to encode replicase 1a and 1b,4 structural proteins, including spike glycoprotein (s protein), envelope glycoprotein (e protein), membrane protein (m protein) and nucleocapsid protein (n protein), and some putative uncharacterized proteins [1, 4, 5] . although these putative uncharacterized proteins have not been found in other known coronaviruses, it is possible for them to be present and play certain function in the sars-cov. for example, a unique novel protein 3a of the sars-cov was identified and characterized recently [6, 7] . humoral immunity is one of the immune responses for defense against viral infection. hence, investigation of human immune responses to the pathogens will provide us important information about the mechanism of how the pathogens interact with their hosts. to understand the humoral immunity to the sars-cov, li et al. [8] studied the profile of specific igg and igm antibodies in sars patient sera by indirect elisa. they found that specific igg antibodies were present in all tested convalescent patient sera and persisted for a long time (until week 12), but specific igm antibodies remained measurable for a much shorter period, suggesting that igg antibodies to the sars-cov may represent the primary humoral immune response protecting patients against sars. although several articles have been published on the antibody response against the structural proteins of sars-cov and their contribution to protective immunity [9] [10] [11] [12] [13] , more extensive study on the profile of specific antibodies to individual proteins of the sars-cov remains to be uncovered, and it could help us not only to understand the humoral immunity but also to develop specific diagnostic tools and vaccine. protein microarrays with the ability to detect antibodies in parallel have a wide range of potential applications in epidemiological research, vaccine development, diagnosis of allergies, autoimmune and infectious diseases [14] [15] [16] . mezzasoma et al. [17] generated protein microarrays by printing five microbial antigens to simultaneously determine the specific antibodies in human sera. human igg and igm bound to the printed antigens were detected by confocal scanning microscopy and quantified with internal calibration curves. the determination results were well concordant with those by elisa. this suggested the feasibility of utilizing antigen microarrays to determine specific antibodies in human sera. in our previous study [18] , to investigate the antigenicity of n protein of the sars-cov by protein microarray containing different recombinant truncated n proteins, human igg at serial concentrations were printed onto slides to act as internal calibration standards. the fluorescence intensity (fi) values gave good linear correlation with human igg concentrations, indicating that fi values could be used to assess the relative levels of specific antibodies captured by respective antigens on a microarray. in the present study, to understand the profile of antibodies to individual proteins of the sars-cov, 13 recombinant proteins associated with four structural proteins (s, e, m and n) and five putative uncharacterized proteins (3a, 3b, 6, 7a and 9b) of this virus were prepared and used to screen and monitor their specific igg antibodies in sars patient sera using protein microarray, and the rabbit antisera to recombi-nant proteins s3 (aa 241-591), n (full length), 3a (aa 125-274) and 9b (full length) were prepared and used to investigate their neutralizing activity to the sars-cov infection in vero e6 cells. the sars-cov strain used for this study was isolated by another group in our institute (isolate bj01; genbank accession number ay278488) [5] , and it was passaged in vero e6 cells to generate a virus stock with a titer of 10 7 50% tissue culture infective doses (tcid 50 ) per ml. all work with infectious virus was performed inside a biosafety cabinet, in a biosafety level 3 (bsl3) laboratory. a total of 58 sera were collected from sars patients at convalescent-phase in beijing in 2003, and they were confirmed for the presence of anti-sars-cov igg antibodies by an elisa kit with whole virus lysate as coating antigen (beijing gbi biotechnology). a group of serial sera were also collected from 13 recovered patients from weeks 2 to 30 after their onset of sars in guangdong province. the normal human sera were collected from healthy people more than 1 year before 2003 sars outbreak. all human sera were inactivated at 58°c for 30 min. the sars-cov was cultured in vero e6 cells at 37°c with 5% co 2 for 48 h. the supernatant medium of the infected cells was collected as crude sars-cov virus, and the viral rna was extracted and purified by qiaamp viral rna mini kit (qiagen). cdna synthesis was performed with super-script ii system (invitrogen) by using gene specific primer. amplification of the full length or truncated fragments of the four structural proteins and five putative uncharacterized proteins (table 1) was performed by pcr. the sequences of primers were referred to by their positions within the corresponding genes, with the note that 5′ ends of the forward and reverse primers contained bamh i, ecor i or nco i sites and sal i, xho i or bamh i sites, respectively, to facilitate cloning. the amplified products were purified with montage pcr kit (millipore), digested with specific restriction enzymes (promega), ligated into the plasmid pet32a vector (novagen) using t4 dna ligase (promega) and finally transformed into escherichia coli (e. coli) strain bl21 (de3) (novagen). the positive recombinant clones were first identified by pcr and further confirmed by enzymatic cutting and sequencing. the recombinant bacteria were grown at 37°c to an optical density of 0.5-0.6 at 600 nm in luria-bertani medium with ampicillin (final concentration, 100 µg/ml) and were induced with 1 mmol/l isopropyl-b-d-thiogalactoside (iptg) for 6 h at 30°c. the expressed recombinant fusion proteins were purified using ni-nta agarose (qiagen) following the manufacturer's instructions. the purified proteins were dialyzed against pbs (ph 7.5) at 4°c overnight, and then quantified by spectrophotometer (beckman du640). however, the insoluble proteins, which were dissolved in 8 mol/l of urea during purification, were renatured in a refolding solution (50 mmol/l tris cl, ph 8.0, 200 mmol/l l-arginine, 1 mmol/l edta, 100 mmol/l urea, 0.25 mmol/l glutathione oxidized, 1 mmol/l glutathione reduced) at 4°c for at least 24 h before dialysis and quantification. the purified fusion proteins were dissolved in 40% glycerol-pbs solution at a concentration of 200 µg/ml, and then printed approximately 40 pl in triplicate onto aldehyded glass slides (cel associates) using gsi flexys arrayer (genomic solutions inc.). human igg and the lysate of e. coli bl21 (de3) were printed onto the slides as positive and negative controls, respectively. printed slides were stored at 4°c overnight and used within 2 weeks after being printed. the serum samples were absorbed with lysate of e. coli bl21 (de3) carrying pet32a to eliminate the corresponding antibodies. for serum reaction with the proteins on the slide, the slides were first blocked with 3% nonfat milk in pbs for 1 h and then 40 µl of 1:10 diluted absorbed sera were added onto the slides and covered with coverslips. following a 1 h incubation at room temperature, the slides were rinsed three times for 3 min each with pbst (0.1% tween-20 in pbs) and then pbs twice. to probe the captured human igg, 40 µl of 1:250 diluted cy5-labeled goat-anti-human igg (goatanti-human igg was labeled with cy5 dye using cy5 antibody labeling kit, amersham biosciences) was applied to the slides, using the same incubation condition and rinsing protocol as described above. the slides were spun dry prior to scanning at 635 nm using a genepix personal 4100a microarray scanner (axon instruments). the fi value of each spot was taken by subtracting the median intensity of the local background from that of the spot, utilizing the genepix pro 4.0 software provided by the same company. the triplicate spot signals for each protein were averaged and ready for further analysis [18, 19] . each sample was analyzed three times. one new zealand white rabbit was immunized with each of the recombinant fusion proteins s3 (aa 241-591), n (full length), 3a (aa 125-274) and 9b (full length), respectively. 0.2-0.8 mg of these proteins in 1 ml of pbs were emulsified with an equal volume of freund's complete adjuvant and 3-month-old rabbits were immunized by subcutaneous injection. after 3 weeks, boosters of each protein emulsified in freund's incomplete adjuvant were given by subcutaneous injection; this was followed by another intravenous injection of protein alone in another 3 weeks. antisera were collected 12 days after the last boost and the immunoreactivity titers were monitored by double agar diffusion precipitation performed in 0.8% agarose in pbs. twofold serial dilutions of heat-inactivated antiserum from each immunized rabbit were tested by a microneutralization assay for the presence of antibodies that neutralized the infectivity of 100 tcid 50 of the sars-cov in vero e6 cell monolayers, with four wells per dilution on a 96-well plate. the presence of viral cytopathic effect was read on days 3 and 4. the dilution of serum that completely prevented cytopathic effect in 50% of the wells was calculated by the reed-muench formula [20, 21] . besides encoding replicase 1a and 1b, the genome of the sars-cov was predicted to encode four structural proteins and some putative uncharacterized proteins. the number of predicted open reading frames (orfs) encoding putative uncharacterized proteins is not the same in different publications, depending on the analysis method used [1, 4, 5] . in this study, we cloned and expressed the five putative uncharacterized proteins larger than 50 amino acids (3a, 3b, 6, 7a and 9b) according to the prediction of qin et al. [5] and all the four structural proteins ( table 1 ). all the positive recombinant clones were confirmed by dna sequencing. among the expressed proteins, s protein was expressed as five truncated fragments including s1 (aa 1-668), s2 (aa 669-1255), s3 (aa 241-591), s4 (aa 419-591) and s5 (aa 647-935); proteins 7a, n and 9b were expressed as full length; proteins 3a, 3b, e, m and 6 were expressed as truncated fragments because their full length could not be expressed with pet32a vector in e. coli, or the expression level was very low. all these recombinant proteins were expressed with trx/6_his-tag at nh 2terminus and their molecular weights were same as predicted (fig. 1, s1 and s2 were not shown). the 13 expressed proteins were in two forms, soluble (3a, 3b, e, m, 6, 7a, n and 9b) or insoluble (s1, s2, s3, s4 and s5). the soluble proteins were purified under native conditions using ni-nta agarose, while the insoluble ones were purified under denaturing conditions, and then renatured before dialysis and quantification. thirteen recombinant proteins were printed in triplicate onto glass slide as protein microarray for screening their specific igg antibodies in 58 sera from convalescent-phase sars patients, with 23 sera from healthy people as control. the fi values for proteins s1, s2, s3, s4, s5, 3a, n and 9b to sera from sars patients were statistically significantly more than those from healthy people (p < 0.01, using spss 10.0 software) (fig. 2) , but the fi values for proteins e, m, 3b, 6 and 7a to sera from sars patients had no significant difference with those from healthy people (p > 0.05). to establish the baseline for the assays, the cutoff value for each protein was calculated using the fi values for this protein to the normal human sera (cutoff = mean + 3s.d.). with these cutoff values, the specificities of the protein microarrays for antibody detections to proteins s1, s2, s3, s4, s5, 3a, n and 9b were all 100%, and the corresponding sensitivities were 44/58 (75.9%), 17 five truncated recombinant s proteins showed different immunoreactivities to the patient sera, among them s3 could react with almost all the samples, indicating that it is the most sensitive antigen for detecting anti-s antibodies. in the one sample (no. 496) missing anti-s3 antibody, both anti-s2 and anti-s5 were detected (fig. 3) . combining these results, anti-s antibodies were present in all the patient sera. in order to investigate the kinetics of the igg antibodies to recombinant proteins s3, 3a, n and 9b in humans after onset of sars, a group of serial sera collected from 13 people from weeks 2 to 30 were analyzed. both anti-n and anti-s3 antibodies were positive in all the serum samples, whereas anti-3a and anti-9b antibodies were only positive in part of these samples. as fig. 4 shows, anti-n and anti-s3 antibodies persisted until week 30; their levels rapidly increased from weeks 2 to 4, and decreased from weeks 20 to 30. at week 30, the average fi values were about half of those at week 4. in addition, anti-3a and anti-9b antibodies remained at low levels all the while. although it is regretable that we could not obtain the patient sera from weeks 5 to 19, the available data provided us valuable information, which is helpful in screening diagnostic markers and the development of vaccine and immunity protocol. the antisera were generated by immunizing new zealand white rabbits with recombinant fusion proteins s3, n, 3a and 9b, respectively. their immunoreactivity titers to respective recombinant proteins were not less than 16 using double agar diffusion precipitation (table 2 ). these antisera were heatinactivated and diluted serially twofold for testing neutralizing activity to the infectivity of 100 tcid 50 of the sars-cov in vero e6 cells. anti-s3 serum showed significant neutralizing activity to the sars-cov infection, with a neutralizing antibody titer of 708, whereas the other antisera and control serum from non-immunized rabbit lacked neutralizing antibodies ( table 2) . compared with elisa-based assays, the protein microarrays have the advantage of parallelism, miniaturization and thirteen recombinant proteins were applied to analyze human sera. the control and patient sera bands indicate the average fi values for each protein to the sera from 23 healthy people and 58 convalescent-phase sars patients, respectively, and the cutoff values were calculated from the data from healthy people, cutoff = mean + 3s.d. * indicates statistically significant difference between patient sera and control. high throughput to detect antibodies [19, 22] . in this study, the recombinant proteins associated with four structural proteins (s, e, m and n) and five putative uncharacterized proteins (3a, 3b, 6, 7a and 9b) of the sars-cov were expressed in e. coli and were used to screen their specific igg antibodies in 58 convalescent-phase sars patient sera by protein microarray, and the antibodies to proteins s, n, 3a and 9b were detected with different positive rates. s and n are structural proteins present in the sars-cov and other known coronavirues [23] , and a number of research groups have reported that the specific antibodies to s and n could be detected in sars patient sera [13, [23] [24] [25] [26] [27] [28] . 3a and 9b are predicted proteins of the sars-cov that have not been found in other known coronavirues, and our serological evidence implies that these two unique novel proteins may be actually present in the sars-cov. during completion of this manuscript, 3a protein has been identified and characterized by other laboratory and our group [6, 7] , respectively; and the antibody to 3a (also known as u274) has also been detected in sars patient sera [13] . however, the possible presence of novel protein 9b, consisting of 98 amino acids residues, in the sars-cov needs to be further proved. on the other hand, no significant antibodies against proteins e, m, 3b, 6 and 7a were detected in the tested sera. proteins e and m are structural proteins of the sars-cov. our results indicate that they may not induce antibody production in humans or the antibody concentrations may be too low to be detected. another possible reasons that could not be ruled out are that the bacteria expressing proteins were not folded or post-translationally modified correctly to recognize the corresponding antibodies. similar results for proteins e, m and 7a (also known as u122) could be seen in a newly published paper [13] . tan et al. expressed full length proteins e, m and 7a in mammalian cells and the total protein lysates were analyzed by western blotting with sera from three convalescentphase sars patients, but no antibodies to these proteins were detected. so far, serological diagnosis for sars has been the most reliable laboratory method. in contrast to the whole virusbased assays, recombinant protein-based assays have the advantages of safety and economy. several research groups have evaluated the possibility of using recombinant proteins n, s and/or 3a for serological diagnosis in the past year [13, 18, [24] [25] [26] [27] [28] . anti-n has been proved to be the most frequent antibody in sars patients and bacteria expressing n protein or even some truncated n proteins are highly immunogenic [13, 18, [24] [25] [26] [27] . although anti-s antibody was detected in 100% (74/74) of convalescent-phase sera by immunofluorescence with mammalian cells expressing s protein [13] , it was only detected in 47.5% (19/40) of sars patient sera by western blotting with bacteria expressing s protein [24] . anti-3a was detected in 73% (59/81) of convalescentphase sera by western blotting with bacteria expressing 3a protein (aa 134-274) [13] . in the present study, we used a protein microarray to qualitatively and quantitatively screen the serological diagnostic markers. among the detectable antibodies to proteins s3, 3a, n and 9b, anti-n and anti-s3 were the dominant, with 100% and 98.3%, respectively, of positive rates and high-level of fi values (9848 and 3581, respectively) in the convalescent-phase sars patient sera, and even in the sample missing anti-s3, both anti-s2 and anti-s5 were detected. this indicates that anti-n and anti-s antibodies are sensitive diagnostic markers for the sars-cov infection, and bacterial expressed recombinant proteins n and s3 (aa 241-591) could be used for antibody detection. however, anti-3a and anti-9b were only detected in 60.3% and 41.4%, respectively, of the sera, with low-level of fi values (1122 and 721, respectively), suggesting that there is limitation of using these two antibodies as diagnostic markers for the sars-cov infection. immunization with one or more sars-cov subunit antigens, either administered as purified protein or expressed from viral or dna vaccine vectors, is one approach to designing a vaccine against sars. this approach would be facilitated by knowledge of the relative importance of the various viral structural proteins in inducing protective immunity. in the present study, the rabbit antisera to recombinant fusion proteins s3 (aa 241-591), n (full length), 3a (aa 125-274) and 9b (full length) were tested for their neutralizing activity to the infectivity of 100 tcid 50 of the sars-cov in vero e6 cells, but only anti-s3 serum showed significant neutralizing activity to the sars-cov infection, with a neutralizing antibody titer of 708. this suggests that s protein is a neutralization antigen for the sars-cov, and recombinant protein s3 (aa 241-591) may be used as a candidate subunit vaccine. although anti-n antibody is also present in sars patient sera at a highlevel and persisted for a long time (until week 30), recombinant n protein could not induce a detectable neutralizing antibody in rabbit to the sars-cov infection. in other papers about the sars-cov vaccine studies, only s among the structural proteins could induce protective immunity in experimental animals [9, 10] . for example, buchholz et al. [9] investigated the contributions of the structural proteins (s, e, m and n) of the sars-cov to protective immunity by expressing them individually and in combinations from a recombinant parainfluenza virus (piv) type 3 vector called bhpiv3, table 2 the immunoreactivity and neutralizing activity of rabbit antisera to recombinant proteins of sars-cov immunoreactivity titer a neutralizing antibody titer b s3 32 708 3a 32 <4 n 1 6 < 4 9b 32 <4 non-immunized control serum <2 <4 a titer of antibody that reacted with its respective recombinant protein, using double agar diffusion precipitation. a titre is the reciprocal value of the last dilution when the property was observed. b titer of antibody that neutralized infectivity of 100 tcid 50 of the sars-cov, using a microneutralization assay. a titre is the reciprocal value of the last dilution of serum that completely prevented cytopathic effect in 50% of the wells, calculated by the reed-muench formula [21] . then evaluating bhpiv3/sars recombinants for immunogenicity and protective efficacy in hamsters. the results identified protein s among the structural proteins as the only significant neutralization antigen and showed that a single mucosal immunization is highly protective in an experimental animal that supported efficient replication of the sars-cov. s protein is very promising in the development of a diagnostic antigen and vaccine. the antigenicity of five truncated recombinant fusion s proteins was preliminarily analyzed in this study, and anti-s antibodies were detected in all the convalescent-phase sars patient sera using s3 and either s2 or s5. the sequence of individually expressed s1 (aa 1-668) and s2 (aa 669-1255) formed the whole sequence of s protein, but these two recombinant proteins only reacted with 75.9% and 29.3% of the patient sera, respectively. although the sequence of s1 covered that of s3 (aa 241-591), s3 was much more sensitive to detect the patient sera, suggesting that some important epitopes appeared in the conformation of s3 but not in that of s1. in addition, s2 (aa 669-1255) and s5 (aa 647-935) could react with the sample missing anti-s3 antibody, suggesting one or more important epitopes may be present in the overlapping sequence of s2 and s5, aa 669-935. further work should be done for full antigenicity analysis of protein s to elucidate its epitopes for diagnostic antigen and vaccine development. characterization of a novel coronavirus associated with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a complete sequence and comparative analysis of a sarsassociated virus (isolate bj01) identification of a novel protein 3a from severe acute respiratory syndrome coronavirus characterization of the 3a protein of sars-associated coronavirus in infected vero e6 cells and sars patients profile of specific antibodies to the sarsassociated coronavirus contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice protective humoral responses to severe acute respiratory syndrome-associated coronavirus: implications for the design of an effective protein-based vaccine an exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers protein and antibody arrays and their medical applications recent developments in protein microarray technology high-throughput protein arrays: prospects for molecular diagnostics antigen microarrays for serodiagnosis of infectious diseases antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein printing proteins as microarrays for high-throughput function determination prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice a simple method of estimating fifty percent endpoints protein arrays for gene expression and molecular interaction screening mass spectrometric characterization of proteins from the sars virus: a preliminary report evaluation of antibody responses against sars coronaviral nucleocapsid or spike proteins by immunoblotting or elisa antibody response of patients with severe acute respiratory syndrome (sars) targets the viral nucleocapsid detection of specific antibodies to severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein for serodiagnosis of sars coronavirus pneumonia recombinant protein-based enzyme-linked immunosorbent assay and immunochromatographic tests for detection of immunoglobulin g antibodies to severe acute respiratory syndrome (sars) coronavirus in sars patients cleavage and serum reactivity of the severe acute respiratory syndrome coronavirus spike protein we are grateful to dr. david bastin, tianjin biochip co., tianjin, china, for careful reading of the manuscript and valuable suggestions. we also thank dr.yang liu, lei lan, mingnan liu and wen li, lab of visual information processing, institute of biophysics, chinese academy of sciences, for participating in gene cloning and protein expression. key: cord-006229-7yoilsho authors: nan title: abstracts of the 82(nd) annual meeting of the german society for experimental and clinical pharmacology and toxicology (dgpt) and the 18(th) annual meeting of the network clinical pharmacology germany (vklipha) in cooperation with the arbeitsgemeinschaft für angewandte humanpharmakologie e.v. (agah) date: 2016-02-06 journal: naunyn schmiedebergs arch pharmacol doi: 10.1007/s00210-016-1213-y sha: doc_id: 6229 cord_uid: 7yoilsho nan in vitro systems and mechanistic investigations i the demand of alternative test systems which closely mirror the in vivo situation is one of the main challenges in modern toxicity testing. the major goal is the development of in vitro systems that partly display the complexity of an organism and thus may mimick in vivo conditions. despite great efforts in the past no adequate in vitro systems are available yet. on the other hand, cell cultures from almost every organ are easily accessible and therefore may help to roughly assess the toxic potential of substances at target structures. nonetheless, the complex interactions which take place in vivo cannot be addressed in single cell cultures. in the liver, hepatocytes comprise 80% of the total liver volume while non-parenchymal cells -endothelial cells, stellate cells and kupffer cells (that is, liver resident macrophages) -contribute only 6.5% of the volume, but 40% of the total cell number (kmiec 2001) . it has been increasingly recognized that in the liver neighboring non-parenchymal cells release molecules which contribute to the inflammatory damage and even aggravate it (adams et al. 2010) . in our project a human in vitro co-culture system was established by combining a hepatic and a monocytic cell line, the latter of which can be differentiated to a macrophage-like phenotype. in this system the hepatotoxicty of substances has been analyzed, and the results were compared to single cultures and to published data from in vivo studies. using ketoconazole, an antifungal, as a known hepatotoxic substance, inflammatory markers were studied and proved to be significantly upregulated only in coculture. conversely, cultures of hepatic cells only did not display this increase in inflammatory markers. at the same time, a negative substance, caffeine, failed to show any hepatotoxic potential in the co-culture system. our results demonstrate that this novel in vitro co-culture model represents a promising tool to evaluate the hepatotoxic potential of substances. in drug research, it might help to reduce animal testing as drugs with a high dili potential can be dropped early in the development phase. question: raman spectroscopy (rs) is a highly sensitive analytical method for markerfree and non-invasive identification and characterization of cells. here, we present rs as a novel tool for gentle yet precise cell analysis in three independent experiments, focusing on monitoring cellular reactions upon treatment. we could provide evidence that rs is a suitable tool to monitor cell differentiation, analyze cell modification and study cell apoptosis after drug application. methods: in a first experiment, mesenchymal stem cells (mscs) were treated with erythropoetin (epo) for certain time points and subsequently fixed with paraformaldehyde (pfa) for raman analysis. in addition, skbr3 breast cancer cells were exposed to the anti-cancer drug herceptin (20μg/ml). cells were then fixed in pfa for rs. in a last experiment, molm-13 cells were separately cultivated in microwells and treated with thymidine for different time points prior to raman analysis. results: raman spectroscopy was able to monitor differentiation of epo treated mscs and found that around 35% of treated cells showed fibroblast like raman profiles. in case of herceptin treated skbr3 cells, rs found internal changes of the cell´s metabolism as reaction on drug application. analyzing the most prominent differences in raman spectra revealed discrimination of cells to be mainly due to changes in amid i, lipid and protein content. in the last experiment, rs was able to follow apoptosis of molm-13 cells after thymidine application and discriminate early from late apoptotic states. discussion: rs is a photonic marker for gentle yet highly specific cell analysis, which allows monitoring of single cell reaction after drug treatment. thereby, rs provides information about changes within the entire metabolome on a single cell level. raman spectra are as characteristic as a "fingerprint". rs works label-free and non-invasive and thus does not impare cell viability. this allows to gain new insights in pharmacological development and toxicological survey. acknowledgement: this project received funding from the eu 7th health program grant agreement no 279288. deutsches zentrum für herzinsuffizienz, würzburg, germany extracellular signal-regulated kinases 1 and 2 (erk1/2) are essential for the regulation of cell growth and cell survival and their kinase activity is up-regulated for example in different types of cancers and pathological cardiac hypertrophy. while inhibition of erk1/2 activity by kinase inhibitors prevents tumor growth, it can also lead to exacerbated cardiomyocyte death and impaired heart function. interestingly, we have previously identified an erk autophosphorylation at threonine 188 as a prerequisite for nuclear erk1/2 signaling and erk-mediated cardiac hypertrophy. here, we investigated an alternative strategy to interfere with erk1/2 signaling: since activation of erk1/2 triggers erk dimerization, a prerequisite for erk t188autophosphorylation, we chose the erk dimer interface as a possible target to selectively interfere with erk t188 -phosphorylation. first, we investigated the impact of monomeric erk2 on cardiac function. to address this issue, we generated mice with cardiac overexpression of monomeric erk2 ∆174-177 and performed transverse aortic constriction (tac) to induce cardiac hypertrophy. compared to wild-type mice, erk2 ∆174-177 overexpression attenuated tac-induced cardiac hypertrophy, interstitial fibrosis and mrna expression levels of collagen and brain natriuretic peptide (bnp), while cardiomyocyte survival and cardiac function were largely preserved. because of the positive effects of monomeric erk ∆174-177 in the heart, we designed a peptide to interfere with endogenous erk dimerization. cross-linking and coimmunoprecipitation experiments showed that the peptide binds to erk2 and prevents its dimerization. moreover, the peptide effectively inhibited erk t188 -phosphorylation and nuclear translocation of yfp-tagged wild-type erk2 after phenylephrine stimulation. further, adenoviral or adeno-associated virus serotype 9 (aav9)-induced overexpression of the peptide in neonatal rat cardiomyocytes (nrcm) or mouse hearts resulted in a significantly reduced hypertrophic response to phenylephrine or tac, background: g i -proteins have been proposed to be cardioprotective. it's matter of debate whether this depends on the particular g i isoform and/or on the particular conditions (e.g. cardiac "stress") only. in our study we investigated effects of a gα i2knockout on cardiac function and survival in a murine heart-failure model of cardiac β 1adrenoceptor overexpression. methods and results: β 1 -adrenoceptor overexpressing mice lacking gα i2 (β 1 -tg/gα i2 -/-) were compared to wild-type (c57bl/6) mice and littermates either overexpressing cardiac β 1 -adrenoceptors (β 1 -tg) or lacking gα i2 (gα i2 -/-). at 300 days of age mortality of mice only lacking gα i2 was higher compared to wild-type or β 1 -tg, but similar to β 1tg/gα i2 -/mice. beyond 300 days mortality of β 1 -tg/gα i2 -/mice was enhanced compared to all other genotypes (mean survival time: 363±21 days). echocardiography revealed similar cardiac function of wild-type, β 1 -tg and gα i2 -/mice, but significant impairment for β 1 -tg/gα i2 -/mice (e.g. ejection fraction 14±2% versus 40±4% in wild-type mice). significantly increased ventricle-to body-weight ratio (0.71±0.06% versus 0.48±0.02% in wild types), left-ventricular size (length 0.82±0.04 cm versus 0.66±0.03 cm in wild types) and anp and bnp expression (mrna: 2819% and 495% of wild type, respectively) clearly indicated hypertrophy. gα i3 was significantly upregulated in gα i2 -knockouts (protein compared to wild-type mice: 340±90% in gα i2 -/and 394±80% in β 1 -tg/gα i2 -/-, respectively). radioligand binding experiments confirmed cardiac overexpression of β 1adrenoceptors in β 1 -tg mice. of note, overexpression levels differed depending on the particular wild-type background. on an fvb/n background we found the overexpression level to be more than 2-fold higher (b max : 1425 ± 68 fmol/mg) than on the otherwise used c57bl/6 background. accordingly, fvb/n-based β 1 -tg mice showed a significantly impaired cardiac function at an age of 300d while c57bl/6-based β 1 -tg mice did not. conclusions: gα i2 -deficiency combined with cardiac β 1 -adrenoceptor overexpression strongly impaired survival and cardiac function. on a c57bl/6 background β 1adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction at day 300 while there was overt cardiomyopathy in mice additionally lacking gα i2 . we propose an enhanced effect of increased β 1 -adrenergic drive by lack of protection via gα i2 . the observed gα i3 upregulation was not sufficient to compensate for gα i2 deficiency suggesting an isoform-specific and/or a concentration-dependent mechanism. the role of gα i3 is currently addressed in a subsequent study using β 1 -tg and gα i3deficient mice. heart failure is accompanied by morphological and functional alterations (e.g. hypertrophy, decreased contractility) which are summarized by the term "cardiac remodeling". while the β-adrenergic signaling pathway is essential for short-term modulation of cardiac performance, its chronic stimulation by elevated plasma catecholamines and the subsequent activation of camp-dependent signal transduction pathways is regarded as a fundamental factor in the pathogenesis of cardiac remodeling. however, the mechanisms mediating the transition of physiological conditions of short-term to detrimental remodeling under long-term β-adrenergic stimulation are not understood in detail so far. in this context, icer, an isoform of the camp dependent transcription factor crem (camp responsive element modulator), acts as an early response gene strongly induced by beta-adrenergic stimulation via camp responsive elements (cre) in its promoter. contrary to its cre-mediated induction, icer is a strong inhibitor of cre-mediated transcription by itself. here we study the role of icer induction in the catecholamine-induced cardiac remodeling in a time dependent manner by the use of icer deficient mice (iko) and wild type (wt) controls, which were treated with isoproterenol (iso; 10 mg/kg per d) for 6 and 24 hours and 7 days. overall 7 days of iso stimulation resulted in an elevation of cardiomyocyte length in iko (in µm; 7d iso 156±1) vs. wt cardiomyocytes (7d iso 143±2). at this time point a 29 % decrease of cardiac output and a 16 % decrease of the maximal rate of rise of left ventricular pressure (dp/dt max ) in iko vs. wt animals was detectable. the maximum increase of icer mrna in wt cardiomyocytes already occurred after 6 h (75-fold), and declined after 24 h (29-fold) to 2.5 fold increase after 7 days, while icer mrna was not detectable in iko mice. this raised the hypothesis, that the early induction of icer modulates transcriptional processes after beta-adrenergicstimulation, involved in cardiac remodeling of the heart. profiling of mrna expression levels between iko vs. wt cardiomyocytes at the different time points revealed: 55 regulated genes (up-regulated: 45 %) in untreated; 103 altered genes (up 35 %) after 6h; 1437 changed genes (up 97%) after 24 h and 131 altered genes (up 19%) after 7 days of iso treatment. in summary, the absence of icer induction in myocytes resulted in an increase of cardiomyocytes length and a decrease of heart performance after 7 days of betaadrenergic stimulation. this is preceded by upregulated mrna levels of several hundred genes at 24 h, which is going along with the induction of transcriptional inhibitor icer after a few hours of beta-adrenergic stimulation. this suggested a protective role of icer by inhibiting the progression of cardiac remodeling after betaadrenergic stimulation in an early responsive manner. (supported by the dfg) the performance of the adult heart is tightly regulated by g protein-coupled receptors. adrenergic and angiotensin receptors efficiently control heart rate and contractility. muscarinic receptors, on the other hand serve as master regulators of the conduction system, which is often lost upon myocardial infarction. this function of muscarinic receptors has been well described in the adult or late embryonic heart. here we provide evidence that muscarinic receptors are crucial to constrain pacemaker cell identity. we applied subtype-specific inhibitors of muscarinic receptors to zebrafish embryos of different stages. we observed that both, early cardiac function as well as specification are specifically regulated by muscarinic m3 receptors, while m2 receptors appear to exert a heart-specific function only at later stages. continuous m3 blockage renders zebrafish with greatly altered cardiac morphology, particularly of the conduction system. furthermore, embryos with m3 inhibition display impaired ventricular function most likely due to an av-block and substantial arrhythmia in the atrium. importantly, to observe these phenotypes it was sufficient to block m3 receptors during stages of cardiac differentiation, which is long before a heart tube has formed. we corroborated our findings regarding these morphological changes using marker gene analysis. furthermore, we obtained evidence for m3 receptors preventing a transcriptional program towards the induction of pacemaker cells at the expense of av canal cells. importantly, this is not only true during heart development. a pacemaker program is also induced in adult hearts upon m3 inhibition. taken together, we postulate that muscarinic m3 receptors confine a pacemaker lineage during early steps of heart development as well as in the adult heart. our data suggests m3 receptors as potential new therapeutic targets for the regeneration of hearts with an injured sinoatrial node. systemic inhibition of mir-21 has proved effective against fibrosis of the myocardium and in other organs. mir-21 has been reported to exert detrimental effects in cardiac fibroblasts and protective roles in cardiac myocytes and other myocardial cell types. a better definition of the cell types that contribute to the beneficial effects of inhibiting mir-21 in vivo may aid the development of strategies with enhanced therapeutic efficacy. thus far, no approach to selectively manipulate micrornas in the non-myocyte population of cardiac cells in vivo has been available. in this study, we developed an icre-encoding mml virus for application in mir-21 fl/fl mice. delivery of this vector to neonates achieved targeted genetic ablation of mir-21 in non-myocyte cardiac cells. immunohistochemistry and flow cytometry confirmed that mmlv was highly selective and effective for cardiac fibroblasts and endothelial cells. in parallel, an aav9-icre vector allowed for specific and almost complete deletion of mir-21 in cardiac myocytes. when tested in a model for chronic left ventricular pressure overload, mmlv-icremediated deletion of mir-21 in cardiac fibroblasts and endothelial cells significantly reduced cardiac fibrosis and hypertrophy and improved cardiac function. the benefit of this cell-type-specific inhibition exceeded that observed upon global genetic deletion of the mir-21 gene in mice. aav9-mediated deletion of mir-21, albeit lowering cardiac hypertrophy, had no effect on fibrosis or cardiac function. taken together, neonatal delivery of engineered icre-encoding viruses enabled for the first time a differential gene targeting in non-myocyte and myocyte cells in myocardium. non-myocyte deletion of mir-21 demonstrated that mir-21 exerts its cardiac profibrotic activity directly in cardiac fibroblasts and in endothelial cells. this novel finding should encourage tailoring of antimir-21 therapy towards cellular tropism. chronic inflammatory diseases, such as psoriasis or rheumatoid arthritis, are characterized by constant leukocyte infiltration and ongoing angiogenesis in the inflamed tissue. as current anti-inflammatory pharmacotherapy is not always satisfying, there is a great demand for the discovery of new drug leads as well as novel drug targets. the synthetic carbazole alkaloid derivative c81 acts as a multikinase inhibitor. results of a thermal shift assay revealed that c81 shows by far the highest binding affinity to the bmp-2-inducible kinase (bmp2k/bike). bmp2k represents an as yet largely uncharacterized protein, which is not regulated by bmp-2 in endothelial cells. therefore, we aimed to analyze (i) the pharmacological potential of c81 and (ii) the role of bmp2k in angiogenic and inflammatory processes in the vascular endothelium. initial experiments show that only high concentrations of c81 affected the viability of human umbilical vein endothelial cells (huvecs). both c81 and the knock-down of bm2k (rnai) reduced the migratory capacity of a human microvascular endothelial cell line (hmec-1). also the proliferation of hmec-1 was reduced by c81 treatment (ic 50 : 7 µm). a tube formation assay on matrigel demonstrated that c81 significantly impaired the formation of capillary-like structures in a dose-dependent manner. interestingly, the analysis (western blot) of signaling molecules in huvecs that play a crucial role in cell proliferation (e.g. erk, akt) revealed that these pathways are not influenced, neither by c81 treatment nor by bmp2k gene silencing. in regard to inflammatory processes, c81 treatment or bmp2k silencing of huvecs decreased the adhesion of thp-1 cells, a monocytic cell line, onto the activated endothelial cells. as the interaction of leukocytes is mainly mediated by cell adhesion molecules (cams), the effect of c81 or bmp2k silencing on their expression was analyzed (flow cytometry, qpcr). while the expression of cams was strongly decreased after c81 treatment, the knock-down of bmp2k did not markedly affect their expression. furthermore, both approaches did not lead to the reduction of tnf-induced iκbα degradation (western blot) or p65 translocation into the nucleus (microscopy). our study provides first insights into the anti-inflammatory and anti-angiogenic potential of the carbazole alkaloid derivative c81 in vitro. the precise role of bmp2k in angiogenic and inflammatory endothelial processes as well as the involved pathways during bmp2k silencing and c81 treatment will be further elucidated. moreover, since the inhibition of bmp2k seems not to be responsible for all actions of c81, we will investigate the role of other kinases affected by the compound in these processes. the chemokine receptor cxcr4 is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine 12 (cxcl12). cxcr4 seems to be part of the lipopolysaccharide sensing complex, suggesting that an intervention with cxcr4 agonists or antagonists could result in reduced tlr4 signaling. however, the role of cxcr4 and the influence of different cxcr4 ligands in acute as well as chronic inflammatory diseases are still contradictious. therefore, we aimed to characterize the systemic effects of cxcr4 activation in severe systemic inflammation and to evaluate its impact on endotoxin induced organ damages by applying a sublethal lps dose (5 mg/body weight) in mice. the plasma stable cxcl12 analog ctce-0214d was synthesized and administered subcutaneously shortly before lps treatment to ensure a delayed release and thereby a prolonged effect of the drug. 24 hours following lps administration, mice were sacrificed and blood was obtained for tnf alpha, ifn gamma and blood glucose evaluation. additionally, histopathological changes and oxidative stress in the liver and spleen were assessed and liver biotransformation capacity was determined. finally, cxcr4, cxcl12 and tlr4 expression patterns in liver, spleen and thymus tissue as well as the presence of different markers for oxidative stress and apoptosis were evaluated by means of immunohistochemistry. ctce-0214d improved the health status and distinctly reduced the lps mediated effects on tnf alpha, ifn gamma and blood glucose levels by approximately 35%, 50% or 70%, respectively. it attenuated oxidative stress in the liver and spleen tissue and enhanced liver biotransformation capacity unambiguously. ctce-0214d diminished the lps induced expression of cxcr4, cxcl12, tlr4, nf-κb, cleaved caspase-3 and gp91 phox, whereas heme oxygenase 1 expression and activity were induced above average. furthermore, tunel staining revealed anti-apoptotic effects of ctce-0214d in all organs. the cxcr4 is undoubtedly involved in inflammation. its activation was accompanied by anti-inflammatory, anti-oxidative and cytoprotective effects as ctce-0214d attenuated tlr4 signaling, induced heme oxygenase 1 activity and mitigated apoptosis. thus, the administration of cxcl12 analogs seems to be a promising treatment option to control acute systemic inflammation, especially when accompanied by a hepatic dysfunction and an excessive production of free radicals. the neurodegenerative disease friedreich ataxia (frda) is caused by a gaa triplet repeat expansion in the first intron of the frataxin gene, which results in a reduction of the corresponding mitochondrial protein. despite several cellular and animal models the exact function of frataxin is still a matter of debate, but the role of frataxin in iron sulfur cluster biosynthesis is generally accepted. however, we still don't know which primary metabolic events are caused by a frataxin deficit and until now, there is no therapeutic option available. we developed a new cellular model for frda by using the cre/loxp recombination system in mouse embryonic fibroblasts (mef). c57bl/6j mouse strains with a loxp flanked exon 4 of the frataxin gene and an tamoxifen-inducible cre-recombinase (creer t2 ) were crossed and several mef cell lines isolated. after selection by genotype and growth manner the fx-mef 2-1 (fxn -/-) and fx-mef 2-8 (fxn +/-) cell lines were finally choosed. the generation of the homozygous or heterozygous frataxin knockout was successfully tested on rna and protein level. long maintenance of the frataxin depleted fibroblasts revealed a strong growth inhibition consistent to earlier observations in other cell systems. therefore, we established a pattern of treatment over 12 days, with medium and substance changes at day 4 and 8, which allows us to get a fully functional knockout and overcome the growth inhibition problem. endpoint measurements of known metabolic phenomena from mammalian and non-mammalian models were studied at day 12 of our novel cell system. the induced total disruption of frataxin leads to a clearly reduced aconitase activity, cell division and oxygen consumption as well as an increase in ros production. in the heterozygous knockout with residual frataxin activity no such changes were observed. in addition, our pattern of treatment enables us to monitor the full and partial frataxin knockout in the course of time, to detect early and late metabolic events after frataxin disruption. therefore we analysed the mentioned parameters (with additional atp and iron content) in parallel at day 3, 5, 7 and 10 and could identify an initial event followed by secondary consequences and parameters, which seem to play only a minor role in the frda pathogenesis. on the contrary, a partial deficit of frataxin didn't result in any differences over time and suggests that there are only cellular alterations below a critical threshold. in conclusion, our new established mammalian cellular frda model mimics typical metabolic consequences of the human disease and seems to be qualified for frda research. the model shows for the first time six different metabolic events in the course of time in parallel and reveals insights into primary and secondary events of frda pathogenesis. these observations can be used to better understand the function of frataxin and can help to develop new therapeutic strategies to address the consequences of frataxin deficiency. moreover, the transfer of this cell model into 96well plates offers the possibility for a high-throughput screening of potential therapeutic substances. the disease diphtheria is caused by the diphtheria toxin (dt) which belongs to the group of single-chain ab-type bacterial protein toxins. receptor-binding of the b-domain on the target cell surface is followed by receptor-mediated endocytosis and internalization into early endosomal vesicles. endosomal acidification triggers membrane insertion and pore formation of the transmembrane (t) domain together with translocation of the (partially) unfolded catalytic (c) domain into the cytosol. herein, dta catalyzes adp-ribosylation of elongation factor 2 which leads to disruption of protein synthesis and finally causes cell death [1] . in hela cells, these events are related to cell-rounding functioning as a specific endpoint to monitor the uptake of dta into the cytosol of the host cell. as for other adp-ribosylating toxins such as c. botulinum c2 toxin, c. perfringens iota toxin and c. difficile cdt, also in case of native dt we demonstrated the involvement of several host cell factors during the translocation step of the catalytic domain across the endosomal membrane [2, 3] . in detail, we confirmed the involvement of the host cell chaperone hsp90 and the thioredoxin reductase (trxr), the latter presumably responsible for the reduction of the interchain disulfide bond between the dta and dtb moieties [4, 5, 6] . furthermore, we identified another group of protein folding helpers, the family of peptidyl-prolyl cis/trans isomerases (ppiases) including cyclophilin a (cypa), cyp40 and fk506-binding protein (fkbp)51 as required cytosolic factors for dta translocation. to characterize the role of the protein folding helpers in more detail, we investigated their interaction with purified dta in vitro by performing dot blot analysis with immobilized recombinant host cell factors, co-precipitation of cellular factors with dta from hela lysate and isothermal titration calorimetry with purified proteins therewith determining the thermodynamic parameters of the individual binding events. thereby, we detected binding of dta to hsp90, cypa, cyp40, fkbp51 and fkbp52. the data increase the knowledge of the molecular mechanisms underlying dt uptake and especially dta translocation which can be medically used to develop novel therapeutic strategies against the disease diphtheria. [1] murphy (2011) toxins 3, 294-308. [2] barth (2011) naunyn-schmied arch pharmacol 383, 237-245. [3] kaiser et al. (2012) cell. microbiol. 14, 1193-1205. [4] dmochewitz et al. (2011) cell. microbiol. 13 , 359-373. [5] ratts et al. (2003) j. cell biol. 160, 1139-1150. [6] schnell et al. (2015) novel afflictions as for example clostridium (c.) difficile associated diseases (cdad) caused by clostridium difficile are on the increase and challenging to treat. cdad most frequent occur in hospitalized patients after prolonged treatment with antibiotics. cdad includes among others diarrhea and the severe form of pseudomembranous colitis. not only the treatment of the infection, but also the treatment of the toxins has a high clinical significance. c. difficile secretes the exotoxins a (tcda) and b (tcdb), which glycosylate and thereby inactivate rho-gtpases in mammalian cells. tcda and tcdb are considered as the causative agents of cdad. in the last few years, more and more hypervirulent strains of c. difficile were described. in these hypervirulent strains, the adp-ribosyltransferase cdt was found as a third toxin in addition to tcda and tcdb. given the lack of agents effective against antibiotic-resistant bacterial strains and bacterial exotoxins, the development of novel pharmacological strategies is needed. the antimicrobial activity of naturally occurring substances is already known for a long time. one important mechanism of the innate immune system is the production of natural peptides showing antibiotic features. in recent years, it was shown that human antimicrobial peptides as important part of the native innate immune system plays a crucial role not only in inactivation of bacteria but also in inhibition of bacterial toxins (1). prompted by these result, we found that only human α-defensin-1 (hnp-1) but not human β-defensin-1 (hbd-1), both important effectors of the innate immune system, protected cultured epithelial cells from intoxication with tcda and cdt when applied prior to the toxins to the cells. moreover, α-defensin-1 prevented also the cytotoxic effects of all three c. difficile toxins tcda, tcdb and cdt combined in the medium. the combined investigation of all three toxins might be even more suitable to mimic the situation after an infection with hypervirulent c. difficile. the inhibition of the toxins was monitored by cell rounding caused by each of the toxins. currently, the molecular mechanisms underlying the inhibitory effects are still unknown and will be investigated in different cell lines. in conclusion, our results demonstrate that hnp-1 causes a loss of cytotoxicity of the c. difficile toxins and may act as novel drugs to cure c. difficile infections that contribute to cdad. neurodegenerative diseases like parkinson´s disease (pd) are accompanied by altered gene expression levels in the brain. recent studies support a role of regulatory noncoding rnas, such as micrornas (mirnas), which silence a specific set of mrnas at the post-transcriptional level. upon their aberrant expression, they are likely involved in the pathophysiology of specific neuronal loss. manipulation of neuronal gene expression is pivotal for understanding the function of proteins and the development of new therapeutic strategies. rna interference (rnai) strategies can be employed through the administration of small interfering rnas (sirna), which mediate the specific knockdown of a selected target gene. however, the main challenge is the delivery of these rnas into the neurons of interest. in this pilot study, we present a method for delivering sirnas in polymeric nanoparticles based on low molecular weight polyethylenimines (peis). their intracerebroventricular (icv) injection leads to in vivo silencing of neuronal gene expression in the brain of mice overexpressing α-synuclein (thy1-asyn mice). in a first step, pei-complexed sirna tagged with afluorescencedye were injected to track the localization and distribution after icv administration. five days later, fluorescent cells were visible throughout the brain, with the highest fluorescence intensity around the ventricles. fluorescence was also observed in large cells of the lumbar spinal cord. moreover, preliminaryresultsdemonstrate a 42.6% knockdown (p<0.05 student's t-test, n=6) of human α-synuclein (snca) in thetargetstructurestriatum upon a single icv injection of pei-complexed specific sirna compared to the control injection group (n=9). hence, our first results support the usability and efficacy of pei nanoparticle-mediated delivery of short rnas, namely sirnas, for rapidly and efficiently reducing the expression of a neuronal target gene of interest in the brain in vivo. this may allow the development of gene therapy strategies for the treatment of neurodegenerative diseases. is a propenylic alkenylbenzene found in several plants, e.g. acorus calamus. bacontaining plant materials are used to flavor foods, and are active ingredients in traditional plant medicines. thus, human exposure results primarily from the intake of bitters and teas, as well as from calamus-containing medicines and plant food supplements. although many (positive) pharmacological properties/effects of asarone isomers are described in the literature, ba was found to be carcinogenic in rodents (liver, duodenum) when given daily or in a single dosage. early experiments indicated that ba is not activated via hydroxylation and sulfonation as it is the case for known hepatocarcinogenic allylic alkenylbenzenes such as estragole or methyleugenol. because the mechanism of metabolic activation of ba in not known, we investigated the metabolism of ba in liver microsomes and human cytochrome p450 (cyp) enzymes, the mutagenicity of ba and its metabolites in the ames fluctuation assay and the dna adduct formation in primary rat hepatocytes. we found that side-chain epoxidation (leading to diols and a ketone) was by far the most dominating metabolic route of ba in liver microsomes and human cyp enzymes. ba was mutagenic in the ames test (+s9 mix), as was the synthesized ba-epoxide (-s9 mix). furthermore, we were able to synthesize and characterize a ba epoxide-derived dna adduct with deoxyguanosine. this dna adduct was formed in a concentration-dependent manner in rat hepatocytes incubated with ba. our results strongly indicate that ba is genotoxic with the side-chain epoxide being its ultimate carcinogen. morbid obesity is an independent risk factor for cardiovascular disease, type 2 diabetes mellitus and certain types of cancer. bariatric surgery -with the roux-en-y gastric bypass (rygb) being the gold standard -has become the therapeutic option of choice as a sustained weight loss and improvement of associated morbidity is achieved in the majority of patients. there is, however, a lack of evidence focusing on bariatric surgery induced sustained weight loss and its possible impact on cancer risk. we investigated the association between obesity, oxidative stress and genomic damage after roux-en-y gastric bypass surgery (rygb) or caloric restriction induced weight loss in the obese zucker rat. obese male zucker fa/fa rats were divided into three groups: sham surgery (sham), rygb and caloric restriction (cr) and were compared with lean controls (lean; zucker fa/+ rats). shams showed impaired glucose tolerance and elevated plasma insulin levels, which were less severe in rygb and cr. oxidative stress was elevated in kidney, liver and colon tissue of sham and reduced again after weight loss induced by either rygb or bwm. urine-derived oxidization products of lipids, dna and rna increased in shams and decreased after weight loss (rygb and cr). dna double strand breaks were more frequent in shams than in the weight loss groups or lean. dna damage in zucker fa/fa rats correlated with their basal plasma insulin values. obese rats showed elevated oxidative stress and genomic damage in comparison to lean rats. after body weight loss, achieved by either rygb or caloric restriction alone, oxidative stress level and genomic damage were decreased. this may indicate a reduction of the elevated cancer risk in obesity. ) mice were treated with the nocrelated compound azoxymethane (aom) followed by the administration of dextran sodium sulfate to trigger crc. tumors were quantified by non-invasive mini-endoscopy, which revealed a non-linear increase in crc formation in wt and aag -/mice. in contrast, a linear dose-dependent increase in tumor frequency was observed in mgmt -/and mgmt -/-/aag -/mice. the data was corroborated by hockey stick modeling, which yielded similar carcinogenic threshold doses for wt and aag -/mice. o 6 -meg levels and depletion of mgmt activity correlated well with the observed dose-response in crc formation. aom dose-dependently induced double strand breaks (dsbs) in colon crypts including in lgr5-positive colon stem cells, which coincided with atr-chk1-p53 signaling. intriguingly, mgmt-deficient mice displayed significantly enhanced levels of γ-h2ax, suggesting the usefulness of γ-h2ax as an early genotoxicity marker in the colorectum. this study demonstrates for the first time a non-linear dose-response for alkylation-induced carcinogenesis and reveals dna repair by mgmt, but not aag, as a key node in determining a carcinogenic threshold at low alkylation dose levels [2] . obesity is characterized as a status where the excessive accumulation of fat in adipocytes leads to local inflammation and hypoxia; both contributing to severe obesity associated co-morbidities such as cardiovascular disease and type 2 diabetes mellitus. local inflammation is mediated by macrophages, stromal vascular cells, preadipocytes, and adipocytes as well as by a number of proinflammatory cytokines and chemokines (1). in particular, the chemokines monocyte chemoattractant protein (mcp-1, ccl2), interleukin-8 (il-8/cxcl8) and stromal cell-derived factor 1 (sdf-1a/cxcl12) secreted by stromal vascular cells, preadipocytes, and adipocytes exert paracrine effects by recruiting neutrophils, monocytes/macrophages, and t-and b-cells. interestingly, deficiency in cxcl14 was shown to attenuate obesity in mice (2) . the cc chemokine ccl2 is known to stimulate ccr2 receptors, and the cxc chemokines cxcl8 and cxcl12 to activate cxcr1 and cxcr2 or cxcr4 and cxcr7, respectively. the receptor(s) activated by cxcl14 is currently unknown. a role of cxcl14 in modulating cxcr4 signaling has been proposed. we initiated our studies to determine the presence and functional significance of chemotaxin receptors in human adipocytes and their precursor cells. to this end, the mrna expression pattern of cc chemokine receptors, cxc chemokine receptors formylated peptide receptor fpr1 and the related receptor fprl1, and cc and cxc chemokines was analyzed during in vitro adipose differentiation of human simpson-golabi-behmel-syndrome (sgbs) preadipocytes, and under conditions mimicking an inflammatory response. in particular, we focused on the expression pattern of human ccr2 receptors, since previous reports indicated a role in adipogenic differentiation. however, our comprehensive analysis using different sources of adipocytes and their precursors indicated that ccr2 receptors were absent (3) . yet, the analysis revealed appreciable levels of mrna encoding ccl2, cxcl12, and cxcl14, and ccr1, cxcr2, cxcr7, fpr1, and fprl1, and cxcr4. of interest, cxcr4-and cxcr7-mrna were found to be up-regulated under the proinflammatory conditions. to analyze the responses of adipocytes and their precursors to chemokine receptor agonists, we used chemokine-mcherry fusion proteins purified from baculovirus-infected insect cells, e.g ccl2, cxcl12, cxcl14. while sgbspreadipocytes and adipocytes did not accumulate ccl2-mcherry upon stimulation, they showed a small accumulation of cxcl12-mcherry, and a strong accumulation of cxcl14-mcherry in the endosomal compartment. similar results were obtained in murine 3t3l-1 preadipocytes. using mass spectrometry analysis, we set out to identify the cxcl14-binding putative receptor protein(s) in murine 3t3l-1 preadipocytes. (1) makki, k. et al., (2013) in personalized medicine tumors are screened for several mutations in oncogenes or tumor suppressors. however, the cellular protein content not exclusively depends on the dna. we identified new rac variants generated on the mrna level in androgenindependent prostate cancer cells. all variants represent active forms of the gtpase. they are capable to suppress rhoa-induced apoptosis and additionally, mediate the synthesis of genes which are under the control of the androgen receptor. importantly, expression of the rac variants is sufficient to support tumor growth in mice. we prove the existence of the variants and verify their clinical appearance and relevance in tissue samples of a prostate cancer patient. dna analysis, however, revealed the wildtype sequence of rac. therefore, routine analysis of patient tumor tissue would miss the detection of active rac which precludes the success of therapy. the existence of active rac variants in prostate cancer tissue that promote resistance towards androgen deprivation suggest rac inhibition as an effective add on therapeutic strategy against prostate cancer. the bacterial effector protein exotoxin y (exoy) of pseudomonas aeruginosa is delivered into host cells via the bacterial type iii secretion system. once arrived in the host cell nucleotidyl cyclase activity of exoy is activated by a yet unknown cofactor and thus has a profound effect on concentrations of cyclic nucleotides: in addition to production of cyclic amp (camp) and cyclic gmp (cgmp) there is a massive synthesis of cyclic 3', and to some extent of the corresponding cytidylyl analogue ccmp 1,2 . currently, the role of cump and ccmp during the pathogenesis of p. aeruginosa infection remains unknown 3 . one of our hypotheses is that these cyclic nucleotides fulfil a role as first messengers, e.g. in the communication between individual bacteria or bacterial populations during establishment of acute or chronic infections. to test this hypothesis, the intra-and extrabacterial concentrations of cyclic nucleotides were measured via hplc-ms/ms at different time-points in liquid cultures of p. aeruginosa, either in a complete (lb medium) or a starving medium (vogel-bonner medium). additionally, we tested if supplementation of the media with extrinsic cump or ccmp had an effect on these measured concentrations. the influence of extrabacterial cyclic nucleotides on the bacterial metabolism and homeostasis was evaluated with a microarray of bacterial total cdna extracted at different time points of p. aeruginosa liquid culture with or without extrinsic cump/ ccmp. furthermore we investigated a potential function of the cyclic nucleotides in biofilm formation. cyclic ump and cyclic cmp have differential roles in bacterial metabolism and communication. for example, whereas ccmp is synthesized by p. aeruginosa when the bacteria are in a nutrient-rich environment, we could not detect bacterial cump under any tested circumstance. in our biofilm formation assays, only ccmp had a biofilmpromoting effect, but only in very high concentrations. the currently ongoing analysis of gene expression data in the presence or absence of cump may reveal a role of this cyclic nucleotide as first messenger, too. in further studies we will elucidate the signal transduction processes underlying the observed cump / ccmp effects, for example by identifying cump and ccmp binding proteins and their coupling mechanisms to intracellular signalling cascades. synapses are complex computational platforms that transmit information encoded in action potentials but also transform their functionality through synaptic plasticity. g protein-coupled receptors (gpcrs) play a major role in modulating the strength of the synapses via the second messenger camp 1 . however the spatio-temporal dynamics of the mode of action of camp underlying synaptic plasticity are still controversial. the role of this study was to investigate the dynamics of camp signaling at the drosophila neuromuscular junction, where octopamine binding to its receptors has been shown to cause camp-dependent synaptic plasticity 2 . for this purpose, we generated a transgenic drosophila expressing the camp sensor epac1-camps 3 in motor neuron. this allowed us to directly follow the octopamine-induced camp signals in real time by fluorescence resonance energy transfer (fret) in different compartments of the motor neuron (i.e. cell body, axon, boutons). we found that octopamine induces a steep camp gradient from the synaptic bouton (high camp) to the cell body (low camp), which was due by higher pde activity in the cell body. high octopamine concentrations evoked a response also in the soma. notably, these signals were independent and isolated form each other. moreover, application of octopamine by iontophoresis to single synaptic boutons induced bouton-confined camp signals. these data reveal that a motor neuron can posses multiple and largely independent camp signaling compartments, and provide new basis to explain how camp could control neurotransmission at a level of a single synapse. 1 kandel, e.r., dudai, y. & mayford, m.r. the molecular and systems biology of memory. cell 157, 163-186 (2014) . 2 koon, a.c., et al., autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling. nat neurosci. 14 (2): p. 190-9 (2010) . 3 nikolaev vo, bünemann m, hein l, hannawacker a, lohse mj novel single chain camp sensors for receptor-induced signal propagation. j. biol. chem.279, 37215-37218 (2004) cardiovascular pharmacology 039 hyaluronic acid deposition determines engineered heart muscle characteristics and can be pharmacologically targeted to enhance function s. schlick background: engineered human myocardium (ehm) can be generated from psc derived cardiomyocytes (cms) and primary fibroblasts suspended in a collagen i hydrogel (70%:30%:0.4 mg/ml). ehm development encompasses an early consolidation phase followed by functional maturation. the presence of fibroblasts is essential for consolidation into a force-generating ehm. here we assessed the hypothesis that fibroblasts of different origin support ehm formation differentially as a function of hyaluronic acid deposition. methods and results: oscillatory rheology (1% strain, 1 hz) on cell-free and cell containing collagen i hydrogels directly after casting revealed enhanced consolidation in the presence of human foreskin fibroblasts (ffbs) compared to primary adult cardiac fibroblasts (cfbs) -change in storage modulus over time (pa/min): collagen 0.03; collagen + cms 0.03; collagen + cms + cfbs 0.09, collagen + cms + ffbs 0.2. we next generated ehm with cms and ffbs or cfbs. after 4 weeks of culture under serum-free conditions, we assessed ehm function by contraction measurements. ffb-ehms developed a significantly (p<0.01) higher force of contraction (foc) per cross sectional area (csa) than cfb-ehms (maximal foc/ csa are in mn/mm 2 : 1.8±0.1, n=29 vs. 0.3±0.1, n=20). cross sectional area (csa) of tissues was greatly increased (p<0.01) in cfb-ehms (csa in mm 2 : 1.6±0.1, n=20 vs. 0.6±0.03, n=30) and nonmyocyte content was higher in cfb-ehms (5.6±0.7, n=9 vs. 3.1±0.4, n=15; x10 5 cells/ml). histological analysis revealed that cardiomyocytes were only poorly matured in cfb-ehms compared to ffb-ehms. extending ehm functional data, principal component analysis of rnaseq data revealed distinct expression patterns for ffbs and cfbs, in which hyaluronic acid synthase 2 (has2) enzyme was significantly (p<0.01) upregulated. based on these findings, we pharmacologically intervened with has2 mediated hyaluronic acid (ha) deposition by treating cfb-ehms with hyaluronidase during all 4 weeks of culture. interestingly, ecm manipulation with low concentrations of enzyme significantly (p<0.01) reduced csa (csa in mm 2 : control 1.8±0.4, n=8; hyaluronidase of concentrations from 0.15u to 150u 0.9±0.2, n=4) with a concurrent, statistically significant (p<0.01), increase in contractile function and improved cardiomyocyte morphology on a histological level (maximal foc/csa in mn/mm 2 : control 0.2±0.05, n=8; hyaluronidase of concentrations from 0.15u to 150u 0.4±0.08, n=4). summary and conclusions: our data suggest that ehm consolidation is influenced differentially by fibroblasts of different tissue origin with hff-ehm being functionally superior to cfb-ehm. cfb-ehm could be rescued by hyaluronidase leading to reduced ha deposition. the latter demonstrates that extracellular matrix composition is centrally involved in ehm development. angiogenesis is the process of formation of new blood vessels from the pre-existing ones. vascular endothelial growth factor (vegf) is the most studied regulator of this process. by binding to its type 2 receptor (vegfr2), it has been shown to activate a variety of different signaling-pathways leading to enhanced angiogenesis. camp, on the other hand, is a versatile second messenger which regulates various endothelial functions including barrier function. it directly activates protein kinase a (pka) or the exchange protein directly activated by camp (epac) which is a guanine exchange factor (gef) for the small monomeric gtpase rap. as human umbilical vein endothelial cells (huvec) express both camp effectors (epac1 and pka), we investigated the role of camp-signaling using a spheroid based sprouting assay as an in vitro model for angiogenesis. interestingly, the activation of β-adrenergic receptors with 5 µm of isoproterenol significantly increased the cumulative sprout length. similarly, the selective activation of epac with 30 µm of the camp analog 8-pcpt-2'-o-camp (007) significantly increased the basal and the vegf-induced cumulative sprout length. in accordance, sirna-mediated depletion of epac1 in huvec decreased the basal and vegf-induced sprouting. surprisingly, 10 µm of forskolin increased basal and vegf-induced cumulative sprout length stronger than 007, indicating an additional role of pka. in accordance, 1 µm of myristoylated pki, a membrane-permeable specific pka inhibitor, significantly attenuated the forskolin-induced increase in sprouting. in all conditions tested, 50 ng/ml of vegf always showed an additive effect to the same extent on cumulative sprout length. therefore, our data indicate that the vegf-pathway is acting independently of the camp-pathway in the regulation of the sprouting angiogenesis. the β-adrenergic receptor-mediated activation of camp signaling in huvec induces angiogenic sprouting by activation of epac1 and pka. introduction: hypertension is one major risk factor for the development of chronic heart and kidney disease. mineralocorticoid receptor (mr) antagonists are a cornerstone in the therapy of heart failure and there is first evidence for a beneficial effect on the kidney as well. inflammation plays an important role in hypertensive organ injury. thus, this study was designed to evaluate and directly compare the effect of mr deletion in endothelial cells on blood pressure and cardiac vs. renal injury in a mouse model of deoxycorticosterone acetate-induced hypertension. methods and results: mice lacking the mineralocorticoid receptor in endothelial cells (mr cdh5cre ) were created using the cre/loxp system. mr cdh5cre and cre-negative littermates (mr wildtype ) underwent unilateral nephrectomy and received 1 % nacl with drinking water for 6 weeks. the mineralocorticoid deoxycorticosterone acetate (doca, 2.5 mg/d) was delivered by subcutaneous pellets. untreated mice served as controls (ctrl). ambulatory blood pressure was determined by implantable telemetry in awake mice. doca/salt treatment increased mean blood pressure in mr wildtype (141.7 ± 8.0 vs. ctrl 97.8 ± 3.2 mmhg, p<0.001) and mr cdh5cre (150.0 ± 3.0 vs. ctrl 104.1 ± 4.7 mmhg, p<0.001) without differences between genotypes. cardiac hypertrophy after doca/salt treatment was ameliorated in mr cdh5cre mice (ventricle weight 162.4 ± 4.2 mg vs. mr wildtype 189.0 ± 10.9 mg, p<0.05). doca/salt significantly increased cardiac fibrosis and the expression of fibrotic marker genes in mr wildtype but not in mr cdh5cre mice. this was accompanied by an increased expression of the vascular cellular adhesion molecule (vcam1) in mr wildtype cardiac endothelial cells. renal function was not altered by mr deletion in endothelial cells at baseline. doca/salt treatment lead to marked interstitial fibrosis in the kidneys of mr wildtype (sirius red fibrosis score: 2.4 ± 0.2 vs. ctrl 0.1 ± 0.1, p<0.001) and mr cdh5cre (2.3 ± 0.2 vs. ctrl 0.1 ± 0.1, p<0.001) mice. mrna expression of the fibrosis marker gene col3a1 (mr wildtype 4.3 ± 0.9-fold; mr cdh5cre 3.9 ± 1.1-fold vs. ctrl) was similarly increased. periodic acid-schiff staining revealed glomerular injury in both genotypes. this was associated with a marked rise in urinary albumin / creatinine ratio (mr wildtype 20.4 ± 5.7fold; mr cdh5cre 34.3 ± 12.5-fold vs. ctrl). in the kidney vcam1 mrna expression and the number of macrophages was increased by doca/salt treatment independently from endothelial mr deletion. conclusion: in conclusion, mr deletion from endothelial cells ameliorated doca/saltinduced cardiac but not renal inflammation and remodeling independently from blood pressure. these findings suggest different mechanisms for the beneficial effect of mr antagonists in hypertensive heart vs. kidney disease. platelets are relevant cells implicated in morbidity and mortality provoked by cardiovascular thrombosis. even with the actual antiplatelet therapy there is still a substantial incidence of arterial thrombosis. therefore, a better understanding of the mechanisms involved in platelet activation and aggregation is required to develop improved antiplatelet therapies. the increase in the intracellular ca 2+ concentration due to ca 2+ entry from the extracellular space is critical for platelet activation and aggregation. ca 2+ entry follows activation of plasma membrane receptors including gqcoupled receptors for adp, thromboxane a 2 (txa 2 ) or thrombin, as well as the collagen receptor glycoprotein vi (gpvi). the cellular signalling pathways downstream these receptors involve activation of phospholipase c and second messengers that are known to mediate activation of trpc channels. trpc proteins form receptor-operated cation channels, but their regulation and permeability differ depending on the cell type. it has been proposed that trpc proteins might contribute to platelet function as constituents of agonist-activated ca 2+ entry channels; however, the experimental approaches used so far and the lack of specific agonists or antagonists have not allowed to determine the individual contribution of trpc proteins for agonist-induced ca 2+ entry in platelets, aggregation and thrombosis formation. we detected the expression of trpc3 and trpc6 in human and mouse platelets. we identified that those proteins together are essential components of a system of coincidence detection in cellular ca 2+ signalling. this coincidence detection triggered by simultaneous stimulation of both thrombin and collagen receptors is required for the phosphatidylserine exposure in human and murine platelets, indicating the role of trpc3/c6 proteins for procoagulant activity. in addition, we detected the expression of s12 trpc1 transcripts in mouse platelets. therefore, we tested trpc1/c3/c6-deficient mice in an in vivo model of arterial thrombosis where they showed reduced thrombus formation. regardless of the protective effect of trpc1/c3/c6 inactivation observed in the thrombosis model, no differences were detected in tail bleeding. to evaluate the relevance of these trpc proteins in platelet aggregation we measured in vitro platelet aggregation in platelets from trpc1/c3/c6-deficient mice and we observed that the aggregation was reduced after adp (3µm) or txa 2 -analogue (1µm) stimulation, but not after collagen stimulation (10µg/ml). we are currently analyzing the in vitro aggregation and the agonist-evoked ca 2+ response in platelets from different trpc-compound and single deficient mouse lines to understand the mechanisms behind this phenotype with regard to its complementarity to actual antiplatelet therapy. background: thrombin signaling initiates inflammatory events directly and through activation of platelets. endogenous and pharmacologic inhibitors of thrombin are therefore of relevance during atheroprogression and for therapeutic intervention. the small leucine-rich proteoglycan biglycan (bgn) is such an endogenous thrombin inhibitor that acts through activation of heparin cofactor ii (hcii). here, the effect of genetic deletion of bgn on thrombin activity, inflammation and atherosclerosis was addressed. methods and results: bgn concentrations were elevated in the plasma of patients with acute coronary syndrome. in apoe -/mice, bgn was detected in the plasma as well as in the glykokalyx of capillaries. additionally, bgn expression occurred in the subendothelial matrix of arterioles as well as in atherosclerotic plaques. in line with a role of bgn in balancing thrombin activity, apoe -/-/bgn -/0 mice exhibited higher activity of circulating thrombin and increased numbers of activated platelets than did apoe -/mice. furthermore, higher concentrations of circulating cytokines in apoe -/-/bgn -/0 mice suggested a pro-inflammatory phenotype. likewise, immunohistochemistry and facs analysis of the aorta demonstrated increased macrophage content in atherosclerotic lesions of these mice. in addition, apoe -/-/bgn -/0 mice exhibited higher aortic plaque burden and larger atherosclerotic lesions at the aortic root. of note, apoe -/-/bgn -/0 mice showed progressive dilatation of the aortic arch corresponding to a decrease in collagen fibril density suggestive of an outward remodelling in the absence of bgn. no differences were evident with respect to lipid content of the aortic root plaques or circulating plasma lipids. treatment with the thrombin inhibitor argatroban reversed platelet activation and aortic macrophage accumulation in apoe -/-/bgn -/0 mice. conclusions: the present results strongly suggest a protective role of bgn during the progression of atherosclerosis by inhibiting thrombin activity and platelet activation, and ultimately macrophage-mediated plaque inflammation. the exposure to environmental or human-made xenobiotics including drugs induces the hepato-intestinal transcription of metabolizing enzymes and transporters. the time-span of induction is thought not to exceed xenobiotic exposure, in order to minimize disturbances of endobiotic metabolism. in contrast, we find cross-generational transmission of the induction of the phase i enzyme cyp2b10 (1200-fold in females, 700-fold in males) in 6-day old offspring of adult female mice exposed one week prior mating to tcpobop (3 mg/kg i.p.) , the model ligand of the xenosensing nuclear receptor car. such cross-generational effects of xenobiotics are of great clinical interest as they could have profound consequences on the health status of the offspring, including interferences with drug therapies. the multigenerational transmission of tcpobop-driven induction could be mediated by pre-uterine/pre-conceptional epigenetic changes of oocytes. alternatively, they could be brought about by direct intrauterine/post-conceptional contact with tcpobop released from long-term depots. to discriminate between these mechanisms we conducted embryo transfer experiments. both donor mothers and foster mothers were injected with tcpobop (3 mg/kg) prior to mating. the analysis of hepatic cyp2b10 expression in 6-day-old offspring is clearly consistent with a post-conceptional onset of tcpobop effects. thus, offspring of solvent-injected donor mothers transferred to tcpobopexposed foster mothers display a 700-fold induction while offspring from the reciprocal experiment show no changes. cesarean sections on day e18.5 followed by crossfostering proved transmission to be mediated predominantly via lactation (f1 hepatic cyp2b10 induction 3000-fold) and only to a minor part via intra-uterine exposure (300fold). this mechanism is consistent with the absence of induction transmission via the male germline. to analyze if tcpobop leads to functional consequences in drug metabolism of f0 and f1 generation, we conducted in vivo zoxazolamine paralysis assays taken as a functional test for cyp2b10 catalytic activity. in both tcpobop-pretreated f0 and in their f1 descendants, the induction reduced the duration of paralysis evoked by zoxazolamine by >50%. the characterization of cross-generational tcpobop-mediated effects on other processes controlled by car such as energy and bone metabolism is in progress. first tests indicate a transmission of anabolic effects on bone, as evidenced by the induction of serum osteocalcin expression by 45% in 12-weeks-old offspring. in summary, the car-mediated cyp2b10 induction by tcpobop is transmitted to the offspring mainly via lactation, resulting in lasting phenotypic consequences in drug and bone metabolism. the effects of similarly lipophilic drugs and anthropogenic environmental pollutants are currently being investigated. such compounds could affect offspring despite discontinuation of intake or exposure well ahead of pregnancy. age-related cognitive decline can eventually lead to dementia, the most common mental illness in elderly people and an immense challenge for patients, their families and caregivers. cholinesterase inhibitors constitute the most commonly used antidementia prescription medication. the standardized ginkgo biloba leaf extract egb 761 ® is approved for treating age-associated cognitive impairment and has been shown to improve the quality of life in patients suffering from mild dementia. a clinical trial with 96 alzheimer´s disease patients indicated that the combined treatment with donepezil and egb 761 ® had less side effects than donepezil alone (yancheva et al., 2009) . in an animal model of cognitive aging, we compared the effect of combined treatment with egb 761 ® or donepezil monotherapy and vehicle. we compared the effect of chronic treatment (15 days of pretreatment) with donepezil (1,5 mg/kg p. o.), egb 761 ® (100 mg/kg p. o.), or the combination of the two drugs, or vehicle in 18 -20 month old male ofa rats. learning and memory performance were assessed by morris water maze testing, motor behavior in an open field paradigm. in addition to chronic treatment, the substances were administered orally 30 minutes before testing. compared to the first day and to the control group, only the combination group showed a significant reduction in latency to reach the hidden platform on the second day of testing. moreover, from the second day of testing onwards, the donepezil, the egb 761 ® and the combination group required less time to reach the hidden platform compared to the first day. the control group did not reach the same latency reduction until day three. there were no effects on motor behavior. these results suggest a superiority of the combined treatment of donepezil with egb 761 ® compared to monotherapy. literature: yancheva, s., ihl, r., nikolova, g., panayotov, p., schlaefke, s., & hoerr, r. (2009) . ginkgo biloba extract egb 761(r), donepezil or both combined in the treatment of alzheimer's disease with neuropsychiatric features: a randomised, double-blind, exploratory trial. aging ment health, 13 (2) , [183] [184] [185] [186] [187] [188] [189] [190] institut für vegetative physiologie und pathophysiologie, göttingen, germany values are well below the plasma concentrations (3-28 µm; just et al. expert opin emerging drugs 20: 161-164, 2015) observed in patients treated with dantrolene. although not yet proven directly, oat3 may be involved in renal secretion of dantrolene and 5-oh dantrolene by mediating the first step, i.e. the uptake across the basolateral membrane of proximal tubule cells. the second step, the release of these compounds into the urine across the luminal membrane, is possibly mediated by mrp4. since oat3 was also detected in the cytoplasmic membrane of skeletal muscle cells (takeda et al. europ. j. pharmacol. 483: 133-138, 2004) , dantrolene may reach its target, the intracellular ryanodine receptor, ryr1, by influx through oat3, where it inhibits calcium efflux by ryr1 thereby preventing severe muscle contraction and malignant hyperthermia. this assumption, however, awaits a direct demonstration of dantrolene transport by oat3. in addition, we identified, besides dantrolene and 5-oh dantrolene, several further fdaapproved drugs such as tyrphostin ag 1478, ceefourin 1, glafenine, nalidixic acid, and prazosine, as inhibitors of es uptake by oat3. controls 1 with other defects and controls 2 with genetic disorders. all drugs used in the first trimester were identified from the database, and were cross-referenced against previously compiled lists of drugs with reactive intermediates and drugs with faa. drugs with reactive intermediates, with systemic absorption and with a daily dose ≥50mg were considered os-inducing drugs. when there was an association between os-inducing drugs and a group of birth defects, we further investigated two different faa exposure categories: concurrent exposure to both os-inducing drugs and faa drugs (os+/faa+) and exposure to os-inducing drugs only (os+/faa-). when the number of subjects allowed (at least five cases/controls were exposed), we examined the role of folic. odds ratios (ors) with 95% confidence intervals were adjusted for maternal smoking and alcohol use in the first trimester in controls 1 and additionally adjusted for maternal age in controls 2. results: a total of nine groups of birth defects were investigated. only nervous system defects were associated with os-inducing drugs. exposure rates were 65/464 (14.0%) for cases, 512/6033 (8.5%) for controls 1 and 130/1564 (8.3%) for controls 2 and adjusted ors (95%cis) were 1.71 (1.29-2.26) and 1.77 (1.27-2.46), respectively. this association was unchanged when we examined os+/faa+ and os+/faa-separately. the os+/faa+ category, however, had slightly higher or values than the os+/faa-(2.41 vs. 1.61 for controls 1, and 2.55 vs. 1.67 for controls 2). because of the low number of exposed subjects, we could only examine folic in relation to os+/faa-. using os-/faa-/folic+ as reference, we found the highest risk with os+/faa-/folicand a lesser magnitude with os+/faa-/folic+ (ors being 2 and 1.6 times respectively for both controls). conclusion: our study suggests an increased risk of having a child with nervous system defects in mothers who were exposed to os-inducing drugs during pregnancy, and a potential risk reduction with folic. background: inhibition of rho-gtpases with statins as well as specific inhibition of the small gtpase rac1 protects non-transformed cells from topoisomerase ii-(top2)-poisoninduced cleavable complex formation and thereof derived dna double-strand breaks. this effect rests at least partially on rac1-mediated regulation of topoisomerase ii activity. however, the link between rac1 and top2-poisoning is only poorly understood. furthermore, it is unclear whether mitochondrial or nuclear type ii topoisomerases are the most relevant target for top2-poison-induced cytotoxicity. here, we investigated the relevance of rac1-regulated actin cytoskeleton integrity as well as mitochondrial integrity in top2-poison-induced dna damage responses as well as cytotoxicity under situation of rac1 inhibition. methods: since endothelial cells are the first barrier for any kind of systemically administered chemicals and cardiomyocytes are particular sensitive to anthracyclines, endothelial cells (h5v) as well as cardiomyocytes (h9c2) were chosen as in vitro model systems for top2-poisoning. the cells were pre-treated with rac1 inhibitors, statins or actin cytoskeleton disruptors and were subsequently treated with the topoisomerase ii poisons doxorubicin or etoposide. to compare the levels of induced dna damage, γh2ax foci quantifications as well as the comet assay were employed. actin disruption was visualized by phalloidin-fitc staining. to be able to detect relevant changes in mitochondrial mass or integrity, high doses of top2-poisons had to be used in both cell lines. changes in mitochondrial homeostasis as well as integrity were detected by the jc1-assay, mitotracker assay as well as atp-assay. additionally, pcr-and gelelectrophoresis-based methods were used for detecting mitochondrial dna damages. selected components of the dna damage response machinery as well as factors of mitochondrial homeostasis were detected by western blot. results: disruption of the integrity of the actin cytoskeleton attenuated the dna damage response to a similar extent as seen by rac1 inhibition, pointing to a role of actin filaments in the dna damage response after genotoxic insults. the actin cytoskeleton seems to participate in genotoxin-induced dna damage, -repair or in the dna damage response as reflected by reduced numbers of nuclear h2ax-foci as well as the comet assay after treatment with doxorubicin. this was not related to nuclear import or export of doxorubicin. disturbance of mitochondrial homeostasis or integrity was only detectable at high doses of topoisomerase ii poisons. this was largely unaffected by pre-treatment with statins or rac1-inhibitor. top2-poison-induced raise in mitochondrial mass was slightly enhanced by the rac1-inhibitor and statins. interestingly, inhibition of rac1 counteracted doxorubicin-induced phosphorylation of the amp-kinase in endothelial cells but not in cardiomyocytes. conclusion: mitochondrial toxicity seems to play only a minor role in top2-poisoninduced cytotoxicity in h9c2 and h5v cells. the data point to a role of rac1-regulated filamentous (nuclear?) actin in the dna repair and/or dna damage response after treatment with top2-poisons. poly(adp-ribose) polymerase 1 (parp1) and the recq helicase werner syndrome protein (wrn) are important caretakers of the genome. they physically interact with each other and are both localized in the nucleus and in particular in the nucleoli. both participate in various overlapping mechanisms of dna metabolism, in particular genotoxic stress response and dna repair [1] . previously, we and others have shown in biochemical studies that enzymatic functions of wrn are regulated by parp1 as well as by non-covalent poly(adp-ribose)-wrn interaction [2] [3] [4] . furthermore, pharmacological parp inhibition as well as a genetic parp1 ablation in hela cells alters the recruitment kinetics of wrn to sites of laser-induced dna damage [5] . here we report a novel role for parp1 and poly(adp-ribosyl)ation in the regulation of wrn's subnuclear spatial distribution upon induction of oxidative stress. we could verify previous reports that wrn is transiently released from nucleoli upon induction of oxidative stress, camptothecin (cpt) treatment, and laser-induced dna damage in a time-dependent manner. while, cpt-induced translocation appears to be a parpindependent process, our results reveal that upon h 2 o 2 -induced oxidative stress, parp1 is essential for the translocation of wrn from the nucleoli to the nucleoplasm. parp1 activity only partially contributes to wrn release from nucleoli, underlining the importance of a direct wrn-parp1 interaction for subnuclear wrn redistribution. furthermore, we identified a novel par-binding motif within the wrn sequence that is located in its rqc domain, which also harbors the binding site for parp1 and is necessary for wrn's nucleolar localization under non-stress conditions. currently, we are testing corresponding wrn mutants to analyze if this region is responsible for the parp1-dependent release of wrn from nucleoli to sites of dna damage. in conclusion, we provide novel insight into the role of parp1 in wrn's spatio-temporal regulation in the nucleus during the oxidative stress response. host-cell reactivation (hcr) is an assay used to determine dna repair capacity of cells. in its canonical layout, the test utilised a virus or a plasmid with a marker gene, inactivated by uv-damage [1, 2] . among the infected or transfected host cells types, only those with functional dna repair pathway would re-activate the damaged dna, thus providing a rationale for identification of dna repair genes in the mutant screens. an obvious advantage of hcr is that repair can be measured in cells that have not been exposed to a damaging agent. however, because of multiple variables of the damage generation, transfection and interpretation of results, the assay has been hard to harmonise and develop into a widely accepted quantitative dna repair assay. over the last years, my team has developed and validated several major improvements of the mammalian hcr assay. exploiting sequence-specific nicking endonucleases and customised design of the reporter vectors, we proposed an innovative and very efficient technique for incorporation of synthetic oligonucleotides, containing single structurally defined dna base and backbone modifications, into desired gene elements [3] . this ad hoc approach allows examination of the repair in a stand-specific manner and at single nucleotide resolution. we efficiently applied hcr in its new layout for measurement of the nucleotide excision repair of various dna adducts. moreover, we demonstrated that the enhanced hcr assay can differentiate between the transcription-coupled (tc-ner) and global genome (gg-ner) subpathways of ner [4] . we further obtained new significant insights into the lesion-specific mechanisms of base excision repair of several endogenously occurring aberrant dna bases [5 -7] and plan to adapt the assay to the detection of mismatch repair and translesion dna synthesis. in addition to the applications in the dna repair field, the enhanced hcr assay provides a tool for investigation of the dynamics and transcriptional impact of the regulatory dna bases 5methylcytosine and 5-hydroxymethylcytosine as well as their derivatives (5formylcytosine and 5-carboxycytosine a coordinated and faithful dna damage response is of central importance for maintaining genomic integrity and cell survival. transcriptional activation of dna repair genes is an important regulatory mechanism contributing to the adaptation of cells to genotoxic stress and protection against genotoxin-mediated cell death. here we show that exposure to a low dose of benzo(a)pyrene 9, , the active metabolite of benzo(a)pyrene (b[a]p), which represents the most important carcinogen formed by incomplete combustion during food preparation and smoking, causes upregulation of several dna repair genes. combined induction of the nucleotide excision repair (ner) genes ddb2, xpc, xpf and xpg enhanced repair activity and protected cells against a subsequent bpde exposure. furthermore induction of the translesion polymerase polh was also involved in protection against bpde-induced apoptosis, however led to an enhanced mutation frequency in the surviving cells. activation of these dna repair pathways was also observed upon exposure to b[a]p and in vivo in buccal cells of male individuals upon smoking, indicating that this mechanism may be involved in the formation of smoking-related cancers. altogether, we could show that low-dose bpde exposure activates a complex network of transcriptional alterations, leading to protection against cell death, at the cost of increased mutation frequency, highlighting the danger of occasional smoking. poly(adp-ribosyl)ation (parylation) is an essential posttranslational modification with the biopolymer poly(adp-ribose) (par). the reaction is catalyzed by poly(adp-ribose) polymerases (parps) and plays key roles in cellular physiology and stress response by regulating physico-chemical properties of target proteins. of the 17 members of the human parp gene family, at least four have been shown to exhibit par-forming capacity. upon dna damage parp1 is catalytically activated and is thought to contribute to the bulk of the cellular par formation. parp inhibitors are currently being tested in clinical cancer treatment, in combination therapy, or as monotherapeutic agents by inducing synthetic lethality (mangerich and bürkle 2011, mangerich and bürkle 2015) . here we generated a genetic knock out of parp1 in one of the most widely used human cell systems, i.e. hela parp1 ko cells, via talen-mediated gene targeting and characterized these cells with regards to parylation metabolism and genotoxic stress response. furthermore, by reconstituting hela parp1 ko cells with a series of artificial and natural parp1 variants, we analyzed structure-function relationships of parp1 in a cellular environment without interfering with endogenously expressed wt-parp1. we confirmed that the parp1e988k mutant exhibits mono-adp-ribosylation activity and extended previous reports by demonstrating that the parp1l713f mutant is constitutively active in a cellular environment, leading to high cellular par levels even in unchallenged cells. additionally, both mutants exhibited significantly altered recruitment and dissociation kinetics at sites of laser-induced dna-damage, which can partially be attributed to non-covalent parp1-par interaction via at least one specific par binding motif located in zinc finger 2 of parp1. expression of both artificial mutants led to distinct cellular consequences, caused by the altered cellular biochemistry. while the expression of parp1l713f itself triggered apoptosis, parp1e988k expression led to a strong g2-arrest during cell cycle and sensitized cells to camptothecin treatment. interestingly, pharmacological parp inhibition with abt888 mitigated effects of the e988k mutant, suggesting distinct functions of mono-adp-ribosylation. finally, by reconstituting parp1 ko cells with a natural cancer-associated parp1 snp variant (v762a), as well as a newly identified parp1 mutant present in a patient of pediatric colorectal carcinoma (f304l-v762a), we demonstrate, that these variants exhibit altered biochemical and cellular properties, potentially supporting carcinogenesis. together, this study establishes a novel model to study parp1-dependent parylation during genotoxic stress response and reveals new insight into the structure-function relationships of artificial as well as natural parp1 variants in a cellular environment, with implications for parp research in general. mangerich, a. and a. bürkle (2011) . "how to kill tumor cells with inhibitors of poly(adpribosyl)ation." int j cancer128 (2): 251-265. mangerich, a. and a. bürkle (2015) . multitasking roles for poly (adp-ribosyl) ation in aging and longevity. parp inhibitors for cancer therapy, springer: 125-179. leukemic cells frequently overexpress the transcription factor wilms tumor 1 (wt1) and the persistence of wt1 expression after chemotherapy indicates remaining leukemic stem cells. hydroxyurea induces replicative stress by its ability to inhibit ribonucleotide reductase, an enzyme that catalyzes the synthesis of dntps from ntps. we demonstrate that the expression levels of wt1 determines the extent of dna damage and apoptosis in a panel of leukemic cells treated with hydroxyurea. accordingly, inhibiting apoptosis through chemical inhibition of caspases or by overexpression of mitochondrial anti-apoptotic bcl proteins prevents the hydroxyurea-induced depletion of wt1 and cell death. in addition, we show that an rna interference-mediated elimination of wt1 sensitizes leukemic cells to the pro-apoptotic and dna damaging effects of hydroxyurea. furthermore, such a loss of wt1 suppresses hydroxyurea-induced erythroid differentiation. pharmacological approaches that diminish wt1 also sensitize cells to hydroxyurea. these include the tyrosine kinase inhibitor (tki) imatinib or epigenetic modifiers belonging to the histone deacetylase inhibitor (hdaci) group. thus, an inhibition of wt1 is therapeutically exploitable for a targeting approach against leukemic cells undergoing replicative stress. our novel findings reveal that wt1 is a novel biological target of hydroxyurea and they suggest that wt1 has a previously unrecognized ability to prevent dna damage when replication forks halt and eventually collapse. fret (fluorescence resonance energy transfer)-based cell assays were developed to directly monitor receptor activation and receptor-stimulated camp response. mutant ß 1 ar were generated by insertion of cyan and yellow fluorescent proteins (cfp and yfp) into the third intracellular loop and the c-terminus, respectively (bornholz et al., cardiovasc res 97:472, 2013) and stably transfected to hek 293 cells (hekß 1 -fret). to monitor the camp response the epac1-based fret sensor of camp, was stably transfected alone (hekwt-e1) and together with a moderate level of native ß 1 ar to hek293 cells (hekß 1 -e1; nikolaev et al. jacc 50: 423, 2007) . fret-activity was measured with recently developed fluorescence detectors (12 channels) equipped with fast semiconductor technology, avoiding any movable optical and mechanical parts, using 438 nm for excitation and 483/540 nm for the emmission ratio. cells were cultivated in 96-format 12-well strips, incubated in physiological hepes-buffered salt solution and treated with ßar agonists of different selectivity and affinity to determine their ßar-subtype preference. catecholamines tested in hekß 1 -fret cells exhibited ec 50 -values (-log, m) which matched k d -values (-log, m) known from native heart receptor membranes (isoprenaline, iso: 6.9±0.1, adrenaline 5.7±0.1, noradrenaline 6.2±0.1). ßar-expression levels were controlled by radioligand binding with [ 3 h]-(-)-cgp12,177 resulting in different densities of ~4x10 6 and ~1.4x10 4 receptors/cell in hekß 1 -fret and hekß 1 -e1, respectively, whereas in hekwt-e1 cells only ~1000 ß 2 ar were found. surprisingly, the low level of ßar in hekwt-e1 cells allowed the measurement of the action of ß 2 -sympathomimetics (ß 2 sym), e.g. fenoterol, thereby amplifying receptor binding (pk d~6 .7) to an effective regulation of fret activity in the presence of 0.06 mm ibmx (pec 50~8 .3±0.1), nearly matching the ~100-fold amplification of iso (pk d~6 .9; pec 50~8 .9). in order to determine ß 1 ar-mediated side effects of ß 2 sym, hekß 1 -e1 cells characterized by a 14-fold higher level of ß 1 ar over ß 2 ar were assayed for fret activity. fenoterol maximally inhibited fret activity with a pec 50~8 .4 whereas the high affinity ß 2 sym salmeterol acted a partial agonist (~75% of iso maximum, pec 50~8 .6), both compounds being rather insensitive against the highly effective ß 1 ar-blockade with 1 µm cgp 20,712 a. for that reason hekß 1 -fret cells were used characterizing fenoterol as partial agonist (~25%) whereas salmeterol activated less than 10% of maximum receptor activation by iso. thus, it has to be concluded that the low level of effectively coupled wt-ß 2 ar present in hekß 1 -e1 cells precludes the exact determination of ß 1 ar-mediated side effects of ß 2 sym, and that cfp/yfp labelled receptors have to be used for the determination of the subtype specific intrinsic activity of an agonist. they account for about one third of all drug targets. their regulation from desensitization to internalization and alternative signal transduction is largely dependent on phosphorylation of intracellular serine and threonine residues of the activated receptor. even though the β 1 -adrenoceptor is of tremendous importance in a number of diseases its phosphorylation remains poorly understood. we addressed this question in a qualitative and quantitative way. by using radioactive phosphorylation assays and mass spectrometry, we were able to elucidate the phosphorylation pattern of the human β 1 -adrenoceptor in vitro. we identified ten previously unknown phosphorylation sites in the third intracellular loop and the receptor's c-terminus. labeling hek293 cells with stable heavy isotopes (silac) lead to the discovery of a stimulation-dependent regulation of several of these phosphorylation sites. furthermore, mutagenesis studies in stably transfected hek293 cells revealed the impact of phosphorylation for arrestin binding and internalization of the receptor. fluorescence resonance energy transfer experiments with β 1 -adrenoceptor variants carrying point mutations of putative phosphorylation sites identified two c-terminal phosphosites that determine arrestin recruitment. our current goal is to further investigate the functional implications of these newly identified phosphorylation sites on downstream signal transduction, with an emphasis on the map kinase pathway. a moderate increase in arrestin affinity to the β 2 -adrenergic receptor is sufficient to induce arrestin internalization. the homologous desensitization of g-protein-coupled receptors is a two-step process. initially, g-protein-coupled receptor kinases phosphorylate agonist-occupied receptors which are subsequently bound by arrestins. in many cases, the resulting receptorarrestin complex is then internalized via clathrin-coated pits. dependent on the identity of the receptor and the ligand, the complex between receptor and arrestin may exist only in the proximity of the plasma membrane or internalize into the cell interior. we constructed mutants of the β 2 -adrenergic receptor carrying three additional serine residues in various positions at the c-terminal tail. one of these mutants which carried the serine residues in close proximity to the endogenous grk phosphorylation sites (β 2 ar-sss) showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. the affinity of arrestin-3 to the receptor was measured by fluorescence resonance energy transfer (fret) between the receptor and arrestin-3 and by two-color fluorescence recovery after photobleaching (frap). in the fret assay, arrestin-3 dissociation from the β 2 ar-sss receptor upon agonist washout was prolonged approximately two-fold compared to the wild-type receptor. frap was performed with an n-terminally tagged receptor immobilized with an antibody against the n-terminal tag either in solution or on a micropatterned surface. in these assays, the recovery of arrestin-3 into the bleached region was prolonged between two-and fourfold for the β 2 ar-sss receptor compared to the wild-type. even though this two-to fourfold increase in affinity seemed rather modest, it resulted in the trafficking of receptorarrestin complexes to the early endosome whereas the wild-type receptor interacted only transiently with arrestin at the plasma membrane. furthermore, the increased affinity of arrestin led to more efficient internalization of the β 2 ar-sss compared to the wild-type receptor. however, recycling to the plasma membrane after agonist washout was very similar for both receptors. we conclude that even a modest change in affinity between a g-protein-coupled receptor and arrestin can lead to substantial alterations in arrestin trafficking which in turn may have effects on cellular signaling. despite recent structural research allow for better understanding of gpcr structure, the crucial aspects of the selectivity mechanism of receptor -g protein subtype coupling remain unresolved. based on the hypothesis that the affinity of the ternary complex (agonist/gpcr/g-protein) in the nucleotide-free state determines the selectivity of gpcr-g protein coupling, we set out to measure gpcr-g protein interaction in membranes of single cells. in order to quantify the affinity of gα-subunit towards gpcrs in single cell, we determined the lifetime of the receptor-g protein complex in living cells upon agonist withdrawal under conditions of gtp-depletion. therefore, we utilized förster resonance energy transfer (fret) based assays to study interactions between fluorescent muscarinic receptors and heterotrimeric g proteins in single permeabilized hek293t cells transfected with the appropriate cdnas. here we focused on muscarinic m1-, m2-, and m3-receptors and characterized the kinetics of agonist-induced binding of go/i -and gq/11-proteins to muscarinic receptors and their subsequent dissociation in the absence of nucleotides. as a measure of affinity we calculated the rate constant of g protein dissociation from the receptor after agonist withdrawal. the dissociation kinetics of go protein from m3-and m1-achrs was found to be 10-fold faster in comparison to gq. similarly, we observed a 15-fold right shift of the concentration-response curves of go proteins binding to m3-achr in comparison to gq. in order to ensure, that the affinity of the ternary complex correlates with the efficiency of g protein activation, we performed experiments on the g protein activity in intact cells expressing non-fluorescent m3-achr by using a fret-based assay. our results showed that gq activation required 10-fold lower agonist concentration compared to go activation, suggesting that indeed the stability of the ternary complex in the absence of nucleotides determines the selectivity of gpcr-g protein coupling. we further explored the subtype selectivity of m2-achr for gi family members by comparison of dissociation kinetics of gi1-, gi2, gi3-, and go-proteins from m2-achr under nucleotide depleted conditions. k off of gi1 and gi3 were found to be two-fold higher in comparison to gi2 and go proteins, indicating the higher affinity of the latter ones to m2-achr. our fret-based assay to study receptor-g-protein interactions in membranes of single cells has been proven to be a fast and reliable method to quantify the affinity of the ternary complex. the g protein subtype dependent differences in the affinity towards activated receptors correlate with the g protein coupling efficiency of this receptor. despite their tremendous pharmacological relevance and potential for the development of new drugs, our understanding of g protein-coupled receptor (gpcr) architecture and signaling mechanisms are still limited. major reasons for this are the low abundance and poor biophysical properties of gpcrs, which makes them one of the most challenging class of proteins for structural and biophysical studies. among the superfamily of gpcrs, the class b receptors comprising 15 receptors are structurally least understood because to date it has not been possible to obtain a crystal structure of this receptor class. to overcome these limitations, we have developed a method for improving functional expression and simultaneous thermo-stabilization of gpcrs by directed evolution which is based on expression of receptors in saccharomyces cerevisiae and subsequent selection of highly expressing variants by flow cytometry with fluorescent ligands. by this strategy, key residues within a receptor sequence can be rapidly identified that are responsible for improved biophysical properties without greatly affecting the pharmacological features of the receptor. we have now applied this method to the human parathyroid hormone 1 receptor, a member of the class b of gpcrs which is a major regulator of calcium homeostasis in the body and a key target for the treatment of osteoporosis. from two rounds of directed evolution in yeast we obtained several mutants of parathyroid hormone 1 receptor that exhibit strongly improved expression levels and that remain stable after solubilization in detergents. these receptor variants are ideal candidates for subsequent structural and biophysical analysis. opioid drugs exert nearly all of their clinically relevant actions through stimulation of mors (μ-opioid receptors). the molecular biology of endogenous opioid peptides and their cognate receptors has been studied extensively in vitro. for mor, signaling efficiency is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. morphine induces a selective phosphorylation of serine 375 that is predominantly catalyzed by g protein-coupled receptor kinase 5. as a consequence, the selective morphine-induced s375 phosphorylation does not lead to a robust beta-arrestin mobilization and receptor internalization. by contrast, high-efficacy opioid agonists such as fentanyl or etonitazene not only induce phosphorylation of s375 but also drive higher order phosphorylation on the flanking residues threonine 370, threonine 376, and threonine 379 in a hierarchical phosphorylation cascade that specifically requires grk2 and grk3 isoforms. as a consequence, multisite phosphorylation induced by potent agonist promotes both betaarrestin mobilization and a robust receptor internalization. however, little is known about agonist-selective phosphorylation patterns in vivo after acute and chronic drug administration. to learn more about mor regulation in vivo we have generated a new μopioid receptor knock in mouse with an n-terminal ha-tag. using these mice, we were able to study in vivo phosphorylation of an endogenous g protein-coupled receptor using both mass spectrometry and phosphosite-specific antibodies. we were also able to address the question which of the many putative mor splice variants detected on the mrna level are indeed expressed as functional receptors in mouse brain. ion channels 061 hcn4 in thalamic relay neurons is necessary for oscillatory activity in the thalamocortical system institut für physiologie i, westfälische wilhelms-universität, münster, germany hcn channels underlie the i h current and are involved, among other functions, in the genesis of epilepsy. the significance of hcn1 and hcn2 isoforms for brain function and epilepsy has been demonstrated, however the role of hcn4, the third major neuronal hcn subunit, is not known. here we show an unexpected role of hcn4 in controlling oscillations in the thalamocortical network. hcn4 is predominantly expressed in several thalamic relay nuclei, but not in the thalamic reticular nucleus and the cerebral cortex. hcn4-deficient thalamocortical relay neurons showed a massive reduction of i h and strongly reduced intrinsic burst firing. evoked thalamic oscillations in a slice preparation were completely abolished. in vivo, brain-specific hcn4 null mutants were protected against induced spike-and-wave discharges (swd), the hallmark of absence seizures. our findings indicate that hcn4 is necessary for rhythmic intrathalamic oscillations and that the channels constitutes an important component of swd generation. ludwig-maximilians-universität, walther-straub-institut für pharmakologie und toxikologie, münchen, germany trpc4 and 5 channels are members of the classical transient receptor potential (trpc) family whose activation mechanism downstream of phospholipase c (plc) largely remained elusive until now. while trpc3/6/7 channels are directly activated by diacylglycerol (dag), trpc4 and 5 channels are commonly regarded as daginsensitive. in contrast to trpc3/6/7 channels, they contain a c-terminal pdz-binding motif allowing for binding of na + /h + exchanger regulatory factor (nherf) 1 and 2. interestingly, performing electrophysiological measurements, co-immunoprecipitations and intermolecular dynamic fret experiments, we found that dissociation of nherf proteins from the c-terminus of trpc5 confers dag-sensitivity on trpc5 channels. trpc5 channels were dag-sensitive under the following experimental conditions: inhibition of protein kinase c, amino acid exchange in the c-terminal pdz-binding motif, pip 2 depletion with and without involvement of plc, over-expression of g-protein coupled receptors, down-regulation of endogenous nherf1 and 2 proteins and overexpression of a nherf1 mutant incapable of trpc5 binding. these findings strongly argue for nherf proteins as molecular determinants for channel activation. interestingly, pip 2 depletion itself caused slight trpc5 current increases while during pip 2 depletion, the membrane permeable dag analogue oag evoked even higher trpc5 currents suggesting that pip 2 depletion induces an active and dag-sensitive channel conformation. receptor mediated pip 2 depletion also resulted in dissociation of nherf1 and 2 from the c-terminus of trpc5 thereby eliciting a dag-sensitive trpc5 channel conformation. thus, our findings suggest that dag-sensitivity of trpc5 is the result of an activation cascade starting with pip 2 depletion and subsequent dynamic dissociation of nherf1 and 2 from the c-terminus of trpc5. altogether, dagsensitivity is a unifying functional hallmark of all trpc channels. the melastatin-related transient receptor potential channel trpm3 is a heat-activated nonselective cation channel expressed in sensory neurons of dorsal root ganglia. since trpm3-deficient mice show impaired inflammatory thermal hyperalgesia, the pharmacological inhibition of trpm3 may exert antinociceptive properties. fluorometric ca 2+ assays and a compound library containing approved drugs were used to identify trpm3 inhibitors and to characterize their potency and selectivity. biophysical properties of the block were assessed using electrophysiological patch-clamp methods. microfluorometry in fura-2-loaded single cells was applied to monitor [ca 2+ ] i signals in isolated dorsal root ganglion (drg) neurons. analgesic effects were assessed applying pregnenolone sulfate (pregs)-induced chemical pain and heat stimuli at mice. in the screening approach using stably transfected hek trpm3 cells we identified the nonsteroidal anti-inflammatory drug (nsaid) diclofenac, the tetracyclic antidepressant maprotiline and the anticonvulsant primidone as highly efficient trpm3 inhibitors. the compounds exhibited half-maximal inhibitory concentrations of 0.6-6 µm. the selectivity profiles of maprotiline and primidone for trpm3 were promising with no inhibitory effects on trpm2, trpm8, trpa1, trpv1, trpc5, trpc6 and p2x7 receptor channels. primidone inhibited pregs-induced [ca 2+ ] i signals in rat drg neurones, indicating a block of native trpm3 channels. consistently, primidone attenuated nocifensive responses of mice to paw-injected pregs. furthermore, intraplantar primidone reduced nociception in healthy and hyperalgesic cfa-inflamed paws in the hot plate test. the finding that an approved drug can inhibit trpm3 at concentrations that may be therapeutically relevant and thereby can act as an analgesic, provides a method to study trpm3-related effects by acutely challenging the channel´s function. pharmacological interference with trpm3 applying an approved drug or optimised successor compounds may pave the way to better understanding of physiological functions of trpm3 in humans and may represent a novel concept for analgesic treatment. excitotoxicity, calcium deregulation, mitochondrial dysfunction and neuroinflammation contribute to progressive cell death in many neurodegenerative diseases. therefore, proteins that prevent deregulation of these pathways are considered as drug targets. potential therapeutic approaches may benefit from modulation of small-conductance calcium-activated potassium (sk) channels, since recent data supports the hypothesis that sk channel activity promotes neuronal survival against cellular stress via a dual mechanism of action: i) by controlling neuronal excitability and ii) by preventing mitochondrial dysfunction and inflammation. our previous studies showed that activation of sk channels in neurons exerted protective effects through inhibition of nmdarmediated excitotoxicity. further, we revealed recently that in a model of glutamate oxytosis, activation of sk channels attenuated mitochondrial fission, prevented the release of pro-apoptotic mitochondrial proteins, and reduced cell death. however, little is known about the function of sk channels in cell metabolism and neuroinflammatory processes in non-neuronal cells, such as microglial cells. in this study, we addressed the question whether sk channel activation affected primary mouse microglia activation upon lps and α-synuclein challenge. we found that activation of sk channels significantly reduced activation of microglia in a concentration-dependent manner, as detected by real-time xcelligence cell impedance measurements. further data on cytokine (tnf-alpha and il-6) analysis revealed that activation of sk channels attenuated α-synuclein-induced cytokine release. inhibition of glycolysis prevented microglial activation and cytokine release. although sk channel activation slightly reduced atp levels, it attenuated α-synuclein-induced no release. furthermore, glycolytic products and ampk signaling were evaluated. overall, our findings show that activation of sk channels attenuates microglial cell activation. thus, sk channels are promising therapeutic targets for neurodegenerative disorders, where neuroinflammation and cell metabolic deregulation are associated with progression of the disease. mutant of the residue pair e103/k308 can be crosslinked efficiently in both states, the closed-and open state of the p2x2r. interestingly, oxidative crosslinking of cysteine substitution mutants of each individual residue pair significantly reduced the atpinduced current amplitudes. charge reversal or swapping mutagenesis and cysteine modification by charged mts-reagents indicated the electrostatic nature of the pairwise interactions in these four residue pairs. furthermore, preliminary data from triple, tetra and penta mutant cycle analysis indicated energetic coupling between the residue pairs e84/r290, e103/k308, e167/r290 and e167/k308 and thus indicates the cooperative interaction in a larger salt bridge network. together with the markedly reduced current amplitudes following disulfide crosslinking, our data suggest that the salt bridge network serves to stabilize the closed-state conformation of the p2x2r. the comparison of the closed-state and open-state model of the rat p2x2r showed that atp promotes a marked rearrangement of the side chains of the residues r290 and k308 to enable the strong ionic coordination of the γ-phosphate oxygen of atp. in summary, our data are in line with the concept that the electrostatic interaction of r290 and k308 with atp competitively releases e84, e103 and e167 from their strong electrostatic coupling and thus initiates a destabilization of the closed-state, which favors channel opening. fig. 1 in a similarity search using sequence motifs conserved amongst various members of the trp protein family we identified three non-annotated putative membrane proteins that we initially termed tmem1, tmem2 and tmem4. expression analysis using the nanostring ncounter system, northern blotting and rt-pcr showed that murine tmem2 is expressed in various tissues including heart, brain, lung, endothelium, colon, cardiac myocytes, cardiac fibroblasts, embryonic fibroblasts, mast cells and pancreatic acinar cells. hydropathy analysis predicts that tmem2 proteins exhibit 6 to 10 plasma-membrane spanning domains, but fluorescently labeled tmem2 fusion constructs expressed in mouse embryonic fibroblasts revealed a vesicular subcellular localization pattern. in contrast to the prediction by the psort ii algorithm, tmem2-eyfp could not be identified in the plasma membrane of fibroblasts, cardiac myocytes, mast cells or pancreatic acinar cells but showed a significant colocalization with markers and fusion constructs specific for acidic compartments including lysosomes. in tmem2 -/mice, a marked elevation of amylase and lipase plasma levels was observed. we found that constitutive but not stimulated amylase secretion from tmem2-deficient acinar cells is elevated indicating a cell autonomous defect. calcium (ca ++ ) is an important signaling molecule regulating stimulated as well as constitutive secretion from pancreatic acinar cells. microfluorimetric measurements using fura-2 or indo-1 indicate higher resting ca ++ concentrations in tmem2-/-pancreatic acinar cells correlating with elevated basal enzyme secretion. in tmem2-yfp-knock-add-on mice we identified tmem2 in organelles of the apical acinar cell pole and a partial colocalisation with lamp2 proteins. furthermore, largely increased elevations in cytoplasmic ca ++ concentration were observed upon osmotic lysis of lysosomes triggered by gly-phe β-naphthylamide (gpn) or by nh 4 cl application. the role of tmem2 for ca ++ release was evaluated by stimulation with low concentration of cholecystokinin 2pm) in the absence of extracellular ca ++ using both microfluorimetric recording of cytosolic ca ++ transients as well as electrophysiological recordings of ca ++ -activated chloride currents. these measurements revealed a higher frequency of intracellular ca ++ oscillations and a larger area under the curve of ca ++ activated chloride currents upon cck-8 stimulation indicating that tmem2 inactivation leads to an enhancement of the globalization of cck-8 evoked ca2+ release from intracellular organelles. taken together, our study identifies tmem2 as a novel regulator of ca ++ release from intracellular organelles including endo-lysosomes and as a critical determinant of constitutive protein secretion in pancreatic acinar cells. while generally highlighting toxicology as a translational science that requires academic anchoring, gundert-remy and co-workers [1] have called for efforts to improve the relevance of in vitro methodologies in predicting in vivo effects. against this background, the german society of toxicology working group on alternative approaches to animal testing proposes specific quality criteria (qc) for in vitro methods and for research work using in vitro methods. these qc may serve to evaluate in vitro methods that are developed or applied in-house or that are described in work plans, peer reviewed articles, etc. for the time being, the qc focus on in vitro cell or tissue culture methods that address human health endpoints in the context of substance-related regulatory toxicity testing. nevertheless, these qc are also generally applicable to in vitro research conducted for other toxicological purposes. relevant work from, e.g., the organisation for the economic co-operation and development has been taken into account in specifying the qc that cover the following aspects:  the 3rs impact of an in vitro method in replacing, reducing (and refining) a specific animal test for a specific toxicological endpoint. this aspect also includes scientific hurdles that, in the past, had impeded the successful development of in vitro methods for the given toxicological endpoint.  scientific relevance and reliability, i.e. which fundamental requirements should an in vitro method meet to ensure that its results are relevant and reliable.  practicability and applicability, i.e. what is the expected expenditure for the in vitro method, and have relevant authorities and industrial sectors been involved in the development of the in vitro method. qc related to the scientific relevance of research work using a specific in vitro method provide a tool to justify, e.g., the suitability of the selected test system and in vitro endpoint(s) for the given purpose; the selection of test substances, positive and negative controls; the setting and control of test concentrations; and the definition of acceptance criteria to determine the relevance of test results. the proposed qc may serve as a framework to assess the relevance of in vitro methods and in vitro research work. thereby, they aim at improving in vitro predictivity of in vivo toxicological effects, which in return contributes to reducing and replacing the need for animal testing. [1] gundert-remy, u. et al. (2015) . toxicology: a discipline in need of academic anchoring -the point of view of the german society of toxicology. arch toxicol 89: 1881-93. during the past decades, considerable progress has been made in implementing 3r approaches in routine safety assessment. in spite of these achievements, animal tests still need to be conducted if legal requirements prescribe in vivo tests or if no reliable, accepted alternative method exists. efforts to foster 3r approaches and make them 'ready for use' focus on three levels: development and validation of scientific methods/strategies, regulatory acceptance of acknowledged approaches and global harmonization of standards. strong cooperation between toxicological experts from scientific bodies, national and international authorities and industry is needed to advance on all three levels for the benefit of animal welfare. 3r approaches that have already been implemented in routine safety assessment of consumer goods do not focus solely on replacement of animal testing by use of accepted alternative methods. in cases where reliable alternative approaches are not yet available, reduction in animal numbers and refinement of testing procedures can be achieved on a case-by-case basis. a tailor-made, tiered testing strategy is usually pursued that involves knowledge on specific characteristics of the test item and makes use of all available data, including details on exposure and results obtained with structural homologues. hurdles to apply alternative approaches can even occur for established methods. as legislations give different priority to alternative approaches, it remains challenging to fulfil conflicting legal requirements in different regions of the world, or even to address horizontal legislations of the same region. furthermore, successfully validated and legally implemented alternative approaches might not always provide the safety assessor with meaningful test results. with gaining experience, limitations of test systems can become evident that affect for example the applicability domain of the method, as has been the case both for some in vitro and in vivo methods. in these cases, the new information needs to be shared not only among safety assessors, but also with method developers and regulators to facilitate refinement of scientific approaches and/or amendments of regulations. leibniz-institut für arbeitsforschung (ifado), vistox, dortmund, germany two-photon microscopy facilitates imaging of biological processes in vivo. establishing this recent technique in mouse liver allowed us to record in a real-time the sequence of events during acetaminophen (apap) induced-liver damage. although apap is intensively studied and described in vivo imaging revealed so far unknown scenarios of cell death. the hepatocytes close to the central vein of a liver lobule went within hours into cell death as commonly described due to the toxic metabolite napqi. surprisingly, we observed a distinct way of cell killing at the outer border of the dead cell area which is accompanied by bile acid decompartmentalization. there, within an hour after apap administration dilatation of bile canaliculi was observed. subsequently, bile acids containing invaginations arouse from the apical side of a hepatocyte into the cytosol. these invaginations ballooned until the bile leaked into the hepatocyte volume and subsequently the plasma membrane of the affected hepatocytes lost its integrity leading to cell death. this mechanism emerged in an environment for hepatocytes where moderate napqi levels meet intracellular high bile salt concentrations of the midzonal region. in conclusion, establishing in vivo imaging in mouse liver enabled us to identify new cellular mechanisms which cannot be discovered by conventional methods. universitätsklinikum düsseldorf, institut für toxikologie, düsseldorf, germany introduction: lung inflammation and fibrosis are considered as major toxicities after thoracic cancer radiotherapy. up to now effective pharmacological interventions for normal tissue protection are largely missing. hmg-coa reductase inhibitors (statins), which are used in the clinic for lipid-lowering purpose, are reported to have multiple inhibitory effects on genotoxic stress responses. for this reason we aim to investigate the usefulness of statins to protect normal lung cells in vitro and lung tissue in vivo from damage provoked by ionizing radiation (ir). methods: according to clinically relevant anticancer radiation regimens, we used fractionated irradiation schemes (4 x 4 gy) for both in vitro as well as in vivo experiments. we analyzed the effect of lovastatin on ir-induced dna damage formation and repair, dna damage response (ddr) and cell death in non-proliferating human lung fibroblasts, epithelial as well as endothelial cells. furthermore, we established an irradiation device that is useful to selectively irradiate the right lung of mice and investigated the influence of lovastatin on lung damage following fractionated and selective irradiation of the lung in vivo (balb/c mice). results: compared to lung fibroblasts and epithelial cells, endothelial cells exhibited the highest radiosensitivity and underwent ir-induced apoptosis which was partly prevented by lovastatin. by contrast fibroblasts and epithelial cells did not undergo apoptosis upon irradiation. lovastatin did not affect initial dna damage formation in any of these cells. in all three lung cell types lovastatin enhanced the repair of dna double-strand breaks as analyzed 24 h after the last irradiation by γh2ax nuclear foci formation. depending on the cell type lovastatin affected various components of the ddr machinery in vitro. in vivo, lovastatin prevented ir-mediated increase in breathing frequency as determined two and four weeks after fractionated irradiation. moreover, statin treatment attenuated the level of residual dna damage and ir-induced apoptosis as analyzed four weeks after irradiation. these results were mimicked when eht1864, a small molecule inhibitor of the small rho-gtpase rac1, was applied in vivo, pointing to an involvement of rac1 in statin-mediated radioprotective effects. conclusion: bearing in mind that statins are well tolerated in humans, we suggest the application of statins as a promising pharmacological strategy for the prevention of irradiation-induced damage of the lung. targeted genome engineering by crispr/cas9 is an evolving tool for generating specific knockout cell lines. co-expression of crispr/cas9 allows for efficient dna cleavage and introduction of so called indel mutations (insertion/deletion point mutations) that lead to either misfolded non-functional proteins or complete knockout. we exploited this tool to generate a bid (bh3-interacting domain death agonist) knockout cell line in neuronal ht-22 cells. bid has been shown to be involved in regulated cell death pathways like oxytosis where its activation mediates mitochondrial demise, subsequent release of apoptosis inducing factor (aif) and cell death. in the cell death model of oxytosis the cystine/glutamate antiporter (x c -) is inhibited by high extracellular glutamate concentrations. following events such as increasing lipid peroxidation and ros production resemble major characteristics of another emerging cell death pathway, called ferroptosis. in this study we generated a bid crispr/cas9-knockout cell line to elucidate the role of bid as a potential link of oxytosis and ferroptosis in the ht-22 cell line. in order to investigate the potential mechanistic overlap at the level of mitochondrial death pathways, we induced oxytosis with glutamate or ferroptosis with erastin in wild-type cells and analyzed the respective effects of the well-established inhibitors ferrostatin-1 and the bid inhibitor bi-6c9 on cell death and mitochondrial paradigms. these results were then compared to the effects of glutamate or erastin in crispr/cas9-bid-knockout cells. bi-6c9 inhibited glutamate-induced morphological changes of ht-22 cells and also prevented cell death as assessed using the mtt assay and annexin v/pi staining. similar results were observed with ferrostatin-1 in the model of erastin-induced ferroptosis. subsequent facs analysis of lipid peroxidation by bodipy staining demonstrated that bi-6c9 abolishes lipid peroxide formation in the erastin model and ferrostatin-1 in the model of oxytosis. facs analysis was further employed for the detection of mitochondrial ros formation. mitosox staining revealed a significantly decreased production of mitochondrial ros by bi-6c9 and ferrostatin-1 in the respective model systems. investigating the crispr/cas9-bid-knockout ht-22 cell line revealed that bid knockout prevented cell death, lipid peroxidation and mitochondrial toxicity in both model systems of cell death, oxytosis and ferroptosis. in conclusion, the present study exposes bid as a pivotal molecular link between the previously separated cell death pathways oxytosis and ferroptosis at the level of mitochondria. parkinson's disease is a common neurodegenerative movement disorder characterized by midbrain dopaminergic neuronal loss in the substantia nigra that has been linked to alpha-synuclein toxicity. however, the molecular mechanisms underlying alphasynuclein-mediated toxicity in human dopaminergic neuronal loss are not well defined. the goal of this study was to investigate the deleterious effects of alpha synuclein in particular mitochondrial toxicity in human dopaminergic cells. therefore, we have generated neuron specific, adeno associated virus type 2 (aav2) expressing cytosolic as well as mitochondrial targeted alpha synuclein and egfp expressing viruses used as respective controls. overexpression of both, the cytosolic and the mitochondrial variants of alpha synuclein severely disrupted the dendritic network, induced loss of cellular atp, enhanced mitochondrial ros production, and was associated with activation of caspases and dopaminergic cell death in a time-dependent manner. in addition, real-time analysis of mitochondrial bioenergetics using the seahorse bioscience system following aav infection elicited a complete damage to mitochondrial respiration capacity in the dopaminergic neurons. our results suggested that mitochondrial targeted expression of alpha synuclein appeared to be more toxic than the cytosolic form of alpha synuclein. in addition, ultrastructural mitochondrial morphological analysis by transmission electron microscopy illustrated a number of deformed cristae in cells expressing the cytosolic alpha synuclein and a complete loss of cristae structure and massively swollen mitochondria following the expression of mitochondrial targeted alpha synuclein in the human dopaminergic neurons. in addition, we found that inhibition of caspases by the broad spectrum caspase inhibitor qvd significantly ameliorated alpha synuclein-induced dopaminergic neuronal death. interestingly, inhibition of caspases preserved neuronal network integrity, atp levels and mitochondrial respiration capacity in both paradigms of cytosolic and mitochondrial alpha synuclein overexpression. overall, our findings show that cytosolic as well as mitochondrial targeted expression of alpha synuclein is detrimental to human dopaminergic neurons, while inhibition of caspases amend alpha synuclein toxicity at the level of mitochondria. thus, caspase inhibitors provide promising therapeutic potential to prevent dopaminergic neuronal death in parkinson's syndromes that are associated with alpha synuclein toxicity. degradation of and adverse effects caused by tattoo and permanent make-up pigments upon sunlight exposure and laser removal have been occasionally reported in the last decades. until now, only the ban of certain azo-pigments has been addressed in the national legislation. the regulation was based on a number of studies showing the cleavage of azo-bonds by ultra violet light and laser-irradiation leading to the formation of carcinogenic aromatic amines. as a result, especially german tattoo ink manufactures switched to the use of more light-fast polycyclic pigments assuming these would be safer for this kind of application when compared to azo-pigments. to assess the potential risks of polycyclic pigments in terms of decomposition in the skin, we compared the photochemical cleavage of the widely used azo-pigment orange 13 and the polycyclic pigment copper phthalocyanine blue. main decomposition products are qualitatively and quantitatively analyzed after q-switched laser irradiation of 1 mg/ml aqueous suspensions and tattooed pig skin. irradiated specimen were extracted with ethyl acetate and analyzed with gas chromatography coupled to mass spectrometric detection (gc/ms) using liquid injection and head-space sampling techniques. we were able to confirm the cleavage of pigment orange 13 at the azo-and other weak bonds in our experimental set-up (fig.1a) . amongst other substances, the carcinogens aniline (max. conc. 1.01 ± 0.12 µg/ml) and 3,3-dichlorobenzidine (max. conc. 0.88 ± 0.18 µg/ml) are formed. despite the lack of such weak bonds, the highly stable porphyrin-like structure of copper phthalocyanine blue is as well decomposed upon laser-irradiation (fig. 1b) . here, 1,2-benzenedicarbonitrile (max. conc. 1.11 ± 0.12 µg/ml) were found as the main decomposition product in all experimental setups. concentrations of cleavage products were generally higher in aqueous suspensions compared to pig skin extracts with both pigments. additionally, the highly toxic gas hydrogen cyanide (max. conc. 35.8 ± 4.32 µg/ml) and the human carcinogen benzene (max. conc. 0.19 ± 0.06 µg/ml) were formed from both pigments, dependent on the laser wavelengths used. cyanide levels of ≥50 µg/ml evolving upon ruby laser irradiation of >1.5 mg/ml aqueous suspensions of phthalocyanine blue were proven to significantly reduce cell viability in human skin cells in vitro. reference 1 schreiver, i., hutzler, c., laux, p., berlien, h. p. & luch, a. formation of highly toxic hydrogen cyanide upon ruby laser irradiation of the tattoo pigment phthalocyanine blue. sci rep 5, 12915 (2015) . understanding the interactions between nanoscaled objects and living cells is of great importance for risk assessment, due to rising application of nanomaterials in foodrelated products. several studies show that silver nanoparticles can reach the intestinal epithelia in nanoform in a human in vitro digestion model. nevertheless, only sparse data concerning the direct quantification of cellular uptake of silver nanoparticles are available. therefore, this study was focused on a systematical quantitative comparison of the cellular uptake of differently coated silver nanoparticles of comparable size. intracellular uptake was determined quantitatively via a transwell tm -system with subsequent elemental analysis (aas) and ion beam microscopy (ibm). silver nanoparticles were coated with poly (acrylic acid) and polyvinylpyrrolidone and characterized extensively by tem, dls, saxs, zetasizer and nanosight. agpure tm as a widely used reference nanoparticle coated with tween 20 and tagat to v was also used for comparison. different intestinal cell models were applied to get closer to the complex in vivo situation: beside the widely used caco-2 model we also investigated particle uptake in a model which considered the enterocyte-covering mucus layer, as well as in a model specialized on particle uptake, the so-called m-cell model. our findings suggest that silver uptake is clearly a particle-and not an ion-related effect. the internalization of silver nanoparticles was enhanced in uptake-specialized m-cells, although no enhanced transport through the cells was observable. furthermore, the mucus did not providing a substantial additional barrier for nanoparticle internalization. rutherford backscattering spectrometry (rbs) via ibm allowed distinguishing between adsorbed an internalized material and the results were in accordance with the transwell tm -data. additionally, ibm investigations via particle-induced x-ray emission (pixe) showed intracellular association of silver with sulfur. the quantification of silver nanoparticle internalization revealed a clear particle-specific and a coating-related uptake. furthermore, a high amount of silver nanoparticles is taken up in cell models of higher complexity. thus, an underestimation of particle effects in vitro might be prevented by considering cell models with greater proximity to the in vivo situation. analyzing iron oxide nanoparticles for drug delivery -innovative investigation tools for nanotoxicology nanoparticles offer promising new possibilities for medical applications including therapy and diagnosis of various diseases. especially nanoparticle systems with magnetic cores provide a broad application spectrum as contrast agents, magnetic transporters, or heat carriers in hyperthermia treatment. for bench to bedside translation of superparamagnetic iron oxide nanoparticles (spions) for medical applications, safety issues have to be clarified. for that, reliable standards must be established on the basis of comprehensively validated physicochemical and biological characterization methods. spions consisting of maghemite and magnetite are usually of brown or black color. due to these special properties, spions and other metal oxide nanoparticles are prone to interfere with classical toxicological assays relying on optical detection of colorimetric, fluorescence or luminescence signals. particularly, nanoparticle concentration and cellular uptake are further influencing factors. consequently, for reliable analysis of nanoparticle mediated effects, alternative robust and interference-free readouts have to be established. based on long lasting experience working with spions, we suggest a combination of complementary methods to analyse nanoparticle-mediated effects: multiparameter analyses in flow cytometry deliver statistically relevant data and link uptake of nanoparticles (side scatter increase) with cellular effects in a high-content style. combination of noninvasive, label-free impedance measurements (xcelligence system) with real-time (fluorescence) microscopy enables us to monitor cellular proliferation and morphology over several days without interference by nanoparticles. additional experiments in multicellular tumor spheroids provide information about tissue infiltration and thus, more closely resemble the in vivo situation. using those complementary methods, several drug-loaded spion systems dedicated for medical applications have been successfully characterized previously. in sum, nanotoxicology is a complex and interdisciplinary challenge, where physicochemical parameters, as well as in vitro and in vivo behavior of nanoparticles have to be considered. to address these basic requirements, we are working on a stringent standardized road of characterization for iron oxide nanoparticles synthesized for medical applications. reference: lyer s, tietze r, unterweger h, zaloga j, singh r, matuszak j, poettler m, friedrich rp, duerr s, cicha i, janko c, alexiou c. nanomedical innovation: the seon concept for an improved cancer therapy with magnetic nanoparticles. nanomedicine (lond). 2015; 10 (21) acrylamide (aa) is an α,β-unsaturated compound, which is categorized as probably carcinogenic to humans [1, 2] . aa is known to arise in foods by heat treatment in the course of the maillard reaction between reducing sugars and amino acids at processing temperatures > 120 °c [3] . dietary aa exposure has mainly been estimated on the basis of dietary recall, assessing consumption of foods with known aa contents. the use of human biomarkers of aa exposure, primarily haemoglobin adducts of aa and its genotoxic metabolite, glycidamide ( ga) in red blood cells, as well as mercapturic acids excreted in the urine, is a promising alternative. such biomarkers are to be validated by exact measurement of aa uptake in duplicates of food as consumed (duplicate diet studies) [4] . we here present results of a nine-day human intervention study with 14 healthy male volunteers. aa contents were determined in duplicates of servings as consumed and kinetics of aa-associated mercapturic acids (aama and gama) monitored in total urine [5] . the study design included washout periods with an aa-minimized diet (21 -41 ng /kg bw), a low aa intake day (0.6 -0.8 µg /kg bw) as well as a high aa intake day (1.3 -1.8 µg /kg bw). after a three-day washout period an aama baseline level of 93 ± 31 nmol/d was determined. low aa intake led to an aama excretion within 24 h of 225 ± 37 nmol/d, high intake to 404 ± 78 nmol/d corresponding to an aama excretion rate of about 30% of the ingested aa dose within 24 h, whereas aama output within 72 h corresponded to 58% of the respective aa intake, the aama baseline after 3 days washout corresponds to a net exposure level of 0.2 -0.3 μg aa/kg bw/d. whether this represents a true baseline level is to be clarified in a follow-up study. in summary, this study provides important quantitative information on kinetics of urinary short-term exposure biomarkers validated by analytically verified dietary aa intake at present day food contamination levels. [1] deutsche forschungsgemeinschaft (dfg), mak-und bat-werte-liste 2013 , 2013 doi: 10.1002 iarc, iarc monographs on the evaluations of carcinogenic risks to humans 1994, 60. [3] tareke et al., j. agric. food chem. 2002, 50, 4998-5006. [4] efsa panel on contaminants in the food chain (contam), efsa journal 2015; 13(6):4104 [321 pp.] . [5] ruenz et al., arch. toxicol. 2015 doi: 10.1007 /s00204-015-1494 heinrich-heine-universität, institut für toxikologie, düsseldorf, germany objective: flavonoids are known to modulate distinct signaling pathways thereby causing different physiological effects. effects of the flavonoids baicalein and myricetin as well as several methylated derivatives were analyzed in the nematode caenorhabditis elegans and in in hct116 colon carcinoma cells and to get insights in molecular mechanisms modulated by these compounds. methods: radical-scavenging activity (teac, dcf), stress resistance (sytox, sodium arsenite), modulation of signaling pathways (nrf2/skn-1, daf16), life span. results: baicalein enhances the resistance of c. elegans against lethal thermal and sodium arsenite stress and dose-dependently prolongs the life span of the nematode (median life span: + 57%). using rna interference we were able to show that the induction of longevity and the enhanced stress-resistance were dependent on skn-1 (homolog to mammalian nrf2), but not daf-16 (homolog to mammalian foxo), another pivotal transcription factor. negletein was the only methylated derivative which was able to enhance the life span of the nematode. in hct116 cells, baicalein activates nrf2; the methylated derivatives oroxylin a and negletein showed a comparable redox-active potential in these cells, but only negletein was able to activate nrf2. the dietary flavonoid myricetin as well as the methylated derivatives laricitrin, syringetin and myricetintrimethylether strongly enhance life span of c. elegans, decreased oxidative stress (dcf) and accumulation of lipofuscin. in contrast to myricetin, the methylated compounds strongly enhanced the resistance against thermal stress. furthermore, treatment with the derivatives induced a much stronger nuclear localization of the daf-16 transcription factor. conclusion: baicalein increases stress-resistance and life span in c. elegans via skn-1 but not daf-16. experiments with methylated baicalein derivatives suggest that the redox-active potential has a minor impact on the nrf2/skn-1 activation since only distinct derivates activate this pathway. in case of myricetin, the methylation increases the stress resistance of the flavonoid. methylation seems to enhance the biofunctionality of the flavonoids. our results may be useful to understand molecular mechanisms of flavonoids and methylated derivatives used as food supplements or pharmacological extracts. the loss of progesterone during menopause is linked to common sleep complaints of the affected women. consequently, a previous study of our laboratory demonstrated sleep promoting effects of oral progesterone replacement in postmenopausal women [1] . the oral administration of progesterone, however, is compromised by individual differences in bioavailability and metabolism of the steroid. we therefore investigated the sleep-eeg effects after intranasal application of progesterone in 12 healthy postmenopausal women (50-70 yrs).in a randomized doubleblind protocol each subject received four treatments, 2 doses of intranasal progesterone (4.5mg mpp22; 9.0 mg mpp22), 10mg of zolpidem and placebo. the 4 conditions consisted of 2 experimental nights (adaptation + examination) separated by at least one week. during each examination sleep eeg was recorded from 23:00 to 07:00. simultaneously blood was collected every 20 min between 22:00 and 07:00 by long catheter for later analysis of the hormones growth hormone (gh), cortisol, melatonin and progesterone. conventional sleep-eeg was statistically evaluated by multivariate analyses of variances (manovas) with repeated measures designs after removal of two outliers, which showed a low sleep efficiency index (sei) after 4.5 and 9.0mg mpp22. univariate f-tests in the manovas pointed to the following results (significant p-values at α=0.05). sei was higher after zolpidem than after the other three treatments. after 9.0mg mpp22 sei was elevated significantly in comparison to placebo. subjects spent more time in nonrem sleep and less time in intermittent wakefulness after 9.0mg mpp22 and after zolpidem than after placebo. total sleep time was elevated and wake after sleep onset (waso) was reduced after 9.0mg mpp22 and after zolpidem. after all active treatments with mpp22 and zolpidem the time spent in sleep stage 2 was higher than after placebo. the amount of slow-wave sleep was higher after zolpidem than after placebo. in addition, the higher dose of mpp22 resulted in an increase of spindle and β frequencies combined with a decrease of δ oscillations during nonrem sleep. in comparison, administration of zolpidem resulted in strong increase of δ, spindle and high β frequencies as well as strong decrease in θ and α frequencies. nocturnal progesterone levels increased after 9.0 mg mpp22. no other changes of hormone secretion were found. our study show sleep promoting effects of 9.0 mg mpp. as expected the sleep promoting effect of zolpidem was confirmed. the spectral signature of intranasal progesterone partly resembled the well-known sleep-eeg alterations induced by gaba active compounds. progestereone levels were elevated after 9.0 mg mpp22. no other endocrine effects were observed. introduction: anticholinergic drugs or drugs with anticholinergic side effects are commonly used for the treatment of various diseases in the elderly population. elderly patients are particularly vulnerable to anticholinergic-related cognitive effects. moreover, there is a relationship between anticholinergic exposure and cognitive impairment. however, there is currently a lack of data on the anticholinergic burden in geriatric patients in germany. it was therefore the aim of this study to evaluate the anticholinergic burden in a large representative cohort of geriatric patients. materials and methods: in this retrospective cohort study, (co)-prescriptions of anticholinergic drugs as well as anti-dementia drugs were evaluated using the discharge medication of geriatric patients between january 2013 and june 2015 from the geriatrics in bavaria-database (gib-dat). anticholinergic drugs were classified according to the anticholinergic cognitive burden (acb) scale in three groups (definite anticholinergics with a score of 2 or 3 and possible anticholinergics with a score of 1). the acb scale was modified by omitting trospium and by adding the three drugs biperiden, metixen and maprotilin, which are used in germany, with a score of 3. a patient's individual score of 3 or higher is considered to be clinically relevant. in total, 130,186 geriatric patients (median age 82 years, 66.3% female, median no. of drugs 9) were evaluated. of these, 41,456 (31.8%) patients took at least one drug with anticholinergic properties. two or more anticholinergic drugs were coprescribed in 10,941 (26.4% of the patients taking anticholinergic drugs) patients. 11,241 (27.1% of the patients taking anticholinergic drugs) patients had a score of 3 or higher. the most common anticholinergic drug combinations involving two definite anticholinergic drugs were amantadine/quetiapine (58), amitriptyline/quetiapine (41) and amitriptyline/carbamazepine (35). 2,885 (7.0%) patients received anticholinergic drugs in combination with anti-dementia drugs. conclusions: one third of patients in a large geriatric population were prescribed at least one anticholinergic drug. one quarter received a co-prescription of anticholinergic drugs. caution is advised prescribing anticholinergic drugs to elderly patients especially with dementia. the antiglaucoma agents brimonidine and timolol are novel substrates of the organic cation transporters oct2 and mate1 expressed in human eye c. neul purpose: glaucoma is a leading cause of visual loss in the world population. lowering intraocular pressure by topical administration of antiglaucoma agents is still the mainstay for glaucoma treatment. 1,2 although many effective drugs exist, the major challenge is their efficient intraocular delivery, which is estimated to amount to 3 the involvement of membrane drug transporters in the intraocular delivery of the widely prescribed antiglaucoma prostanoid latanoprost has been described. 4 however, it is currently unknown whether the cationic drugs brimonidine and timolol, which are also commonly used antiglaucoma agents, are similarly transported by drug transporters and whether these transporters are expressed in human eye. brimonidine is an α 2 -adrenergic agonist, which inhibits the activity of the adenylate cyclase subsequently leading to a reduced production of aqueous humor. timolol is a β-adrenergic receptor antagonist, which blocks β-receptors on the ciliary epithelium also resulting in a reduced aqueous humor production. the aim of the present study was to determine whether brimonidine and timolol are substrates of the organic cation drug transporters oct1 (encoded by slc22a1), oct2 (slc22a2), oct3 (slc22a3) and mate1 (slc47a1). a further aim was to investigate whether these transporters are localized in different human eye substructures. experimental design: transport of brimonidine and timolol was studied using the mammalian cell line hek293 stably expressing the organic cation transporters oct1, oct2, oct3 or mate1. 5 intracellular accumulation of brimonidine and timolol was analyzed by mass spectrometry. immunohistochemistry and immunofluorescence experiments were performed to study the localization of these transporters in different substructures from glaucomatous and non-glaucomatous human eyes. results: uptake experiments revealed that brimonidine is transported by oct2 and mate1 in a time-and concentration-dependent manner, but not by oct1 or oct3. timolol is only transported by mate1, but not by the octs. as shown by immunolocalization studies, the oct2 and mate1 transporter proteins were expressed in all anterior eye substructures of non-glaucomatous and glaucomatous eyes, i.e. the cornea, the conjunctiva and the ciliary body. conclusion: our data demonstrate that oct2 and mate1 may play a role in the ocular disposition of the antiglaucoma drugs brimonidine and timolol and may contribute to interindividual variability of drug concentrations and effects. references: 1. zhang et al., nat rev drug discov. 2012 jun 15; 11(7) :541-59. 2. lavik et al., eye (lond). 2011 may; 25(5):578-86. 3. gaudana et al., pharm res. 2009 may; 26(5):1197 -216. 4. kraft et al., invest ophthalmol vis sci. 2010 51(5):2504 -11. 5. nies et al., plos one. 2011 6(7) :e22163. supported by the robert bosch foundation, stuttgart, germany. immature platelet count or immature platelet fraction as optimal predictor of antiplatelet response to thienopyridine therapy c. stratz 1 , t. nuehrenberg background: previous data suggest that reticulated platelets impact significantly on antiplatelet response to thienopyridines. it is unknown which of the parameters describing reticulated platelets is the optimal predictor of antiplatelet response to thienopyridine therapy. methods: this study is a prespecified subanalysis of the excelsiorload trial that randomized elective patients undergoing coronary stenting to loading with clopidogrel 600mg, prasugrel 30mg or prasugrel 60mg (n=300). adp-induced platelet reactivity was assessed by impedance aggregometry before loading (=intrinsic platelet reactivity) and on day 1 after loading. multiple parameters of reticulated platelets were assessed by an automated whole blood flow cytometer: immature platelet fraction (ipf, proportion of reticulated platelet of the whole platelet pool), highly immature platelet fraction (hipf), absolute immature platelet count (ipc). results: each parameter of reticulated platelets correlated significantly with adpinduced platelet reactivity: ipf (r s =0.18; p=0.002), hipf (r s =0.18; p=0.002), ipc r s =0.26; p<0.001). in a multivariable model including all three parameters, only ipc remained as significant predictor of platelet reactivity (p<0.001). after adjustment to known predictors of on-clopidogrel platelet reactivity including cytochrome p450 2c19 polymorphisms (*2 and *17), age, body mass index, diabetes, smoking and intrinsic platelet reactivity, ipc s21 was the strongest predictor of on-treatment platelet reactivity (partial η 2 =0.045; p<0.001) followed by intrinsic platelet reactivity (partial η 2 =0.034; p < 0.002). these findings prevailed when analyzing subgroups of patients on clopidogrel or on prasugrel. conclusion: immature platelet count is the strongest platelet count derived predictor of antiplatelet response to thienopyridine treatment. given its easy availability together with its even stronger association with on-treatment platelet reactivity when compared to known predictors including the cyp 2c19*2 polymorphism, immature platelet count might become the preferable predictor of antiplatelet response to thienopyridine treatment. cutaneous squamous cell carcinoma (cscc) is the second most common human cancer with continuously rising incidences worldwide. primarily caused by cumulative uvb exposure, cscc accounts for considerable costs for health care systems and poses a deadly risk especially to organ transplant recipients [1] . current chemotherapy needs to be improved, because even the topical treatment for cscc's carcinoma in situ bears limited efficacy and painful adverse effects [2] . however, animal-based approaches in preclinical development contribute to the frequent failure of investigational new drugs in clinical trials [3] . herein, we characterized a human cell-based cscc model, normal reconstructed human skin (rhs) served as control. whereas rhs exhibited low proliferation, the co-culture with cscc increased ki-67 index 23-fold in the cscc model (p£0.001). while the presence of claudin-4 and occludin were distinctly reduced, zonula occludens protein-1 was more wide-spread, and claudin-1 was heterogeneously distributed within the cscc model compared with rhs. this is in accordance to the in vivo situation [4] and likely contributes to the impaired barrier function of the cscc model, as demonstrated for 2.6-fold increased caffeine permeation. finally, the ingenol mebutate effects in the cscc model and rhs closely mimic the anti-tumor effect and the adverse reactions in patients [2] , both linked to the drug's inherent cytotoxicity. in conclusion, the thoroughly characterization of disease models fosters both advanced preclinical drug development and improved cscc treatment. funded by the german government, the berlin-brandenburg research platform bb3r with integrated graduate education was launched in 2014. the aim of this research platform, along with the associated graduate school, is to close substantial knowledge gaps in the fields of the 3rs and to find alternatives to animal experimentation within the next years. a panel of 3r experts has been set up to provide advice and assistance and to raise awareness in society for 3r-related issues. research in bb3r investigates physiological functions on different levels to establish alternative methods for preclinical drug development and basic research. the principal investigators aim at facilitating research collaborations and sustainable research activities in the region berlin-brandenburg and abroad. an integrated bb3r graduate program has been developed to offer structured training to graduate students in a specific, mandatory course program on 3r including modules on ethics and legislation. currently, 14 phd students are qualified for management positions in professional areas related to the 3rs, and three junior research groups are now ready to expand their regional research activities nationwide. furthermore, the concept of a novel lecture series for master students and undergraduates has been designed and awarded the animal welfare research award for berlin-brandenburg in teaching and education. the state government of berlin supports the research platform bb3r and will be funding an additional professorship at the fu berlin to further promote research on alternative testing. finally, co-operations with national and international partners are being built to facilitate the project-based exchange of scientists and joint research. currently, the identification and evaluation of skin sensitizers is mainly restricted to animal testing using the guinea pig maximization test, buehler test or the murine local lymph node assay. recently, an adverse outcome pathway of skin sensitization has been released by the oecd, identifying the key events leading to allergic contact dermatitis. in vitro tests address these key events and two assays are now regulatory adopted (oecd 442c and 442d). the use of the current in chemico and in vitro models is, however, limited since they do not reflect dermal penetration, complete biotransformation and cell cross-talk in an organotypic environment. in this study, we aimed to overcome these limitations by establishing reconstructed skin tissues containing langerhans cells (lcs). in vitro generated immature monocyte-derived (molcs) or mutz-3-derived cells (mutz-lcs) cultivated with keratinocytes on a dermal compartment with fibroblasts form a stratified epidermis after 14 days as indicated by the expression of epidermal differentiation markers. molcs or mutz-lcs were mainly localized in suprabasal layers of the epidermis and distributed homogeneously in accordance with native human skin. topical application of the extreme contact sensitizer 2,4-dinitrochlorobenzene (dncb) induced il-6 and il-8 secretion in skin models with lc-like cells, whereas no change was observed in control rhs lacking immune cells. increased gene expression of cd83 and pd-l1 in the dermal compartment indicated lc maturation. we confirmed the enhanced mobility from epidermal to dermal compartments for mutz-lcs and molcs in the presence of dncb. in summary, we successfully integrated immature and functional lc-like cells into reconstructed human skin. this fosters the development of animal-free test systems for advanced and potentially individualized hazard assessment of skin sensitization. computational methods for prediction of in vitro activity of new chemical structures. background: with a constant increase in the number of new chemicals synthesized every year, it becomes highly important to employ the most reliable and fast in silico screening methods to predict their safety and activity profiles. in recent years, in silico prediction methods received great attention as alternatives to animal experiments for evaluation of various toxicological endpoints, complementing the theme of replace, reduce and refine (3rs). various computational approaches have been proposed for prediction of toxicity of chemicals ranging from quantitative structure activity relationship modeling to molecular similarity based methods and machine-learning methods. within the "toxicology in the 21st century" screening initiative, a crowdsourced platform was established for development and validation of computational models to predict the interference of chemical compounds in nuclear and stress receptor pathways based on a training set containing more than 10,000 compounds tested in high-throughput screening assays. methods: here we present the results of various molecular similarity-based and machine-learning-based methods over an independent evaluation set containing 647 compounds. further, we compare the performance of these methods when applied individually and together. in retrospect we also discuss the reasons behind the superior performance of an ensemble approach, that combines a molecular similarity approach and a naïve bayes machine learning algorithm in achieving best prediction rates in comparison with other individual methods, explaining their intrinsic limitations. results and conclusions: our results suggest that, although prediction methods were optimized individually for each modeled target, an ensemble of similarity and machinelearning approaches provides promising performance indicating its broad applicability in toxicity prediction. charité -universitätsmedizin berlin, structural bioinformatics group, berlin, germany a multitude of drug candidates (approx. 20 %) fail in the late drug development due to toxicity and adverse effects [1] . immunotoxicity can be divided into four types of immune-mediated adverse effects: immunosuppression, immunostimulation, hypersensitivity and autoimmunity. current safety evaluations of drug candidates with respect to immunotoxicity are comprised of in vivo and in vitro assays. here, we present an in silico approach for predicting immunotoxic substances based on immune suppressive and not toxic compounds using the laplacian-modified naϊve baysian model as a supervised machine learning method. therefore, we examined the relations between about 51,000 compounds and 115 cancer cell lines from the national cancer institute's (nci) nci-60 growth inhibition data [2] with focus on five immune cell lines (rpmi-8226, ccrf-cem, . different fingerprints encoding the chemical structures have been evaluated for their predictive power (e. g. extended-connectivity fingerprints, substructure fingerprints) and showed good prediction rates in a retrospective analysis. acting in phase ii metabolism, sulfotransferases (sult) transform endo-and exogenous molecules such as drugs into more hydrophilic entities that are easily excreted from the human body [1] . although serving detoxification, sult-mediated transformation of molecules has also been associated with the formation of chemically reactive metabolites that could promote adverse reactions [2] . the development of a computerbased model that allows prediction of molecules susceptible to metabolism would improve drug development and drug safety [3] , and encourage the reduction of in vivo testing according to the principles of the 3rs (replacement, reduction and refinement). in our study, we developed an in silico model to predict human sult subtype 1e1 activity acting in phase ii metabolism. structure-based molecular modelling of sult activity is challenging due to the broad and overlapping substrate spectrum of sult subtypes. this low substrate specificity can be attributed to the high degree of conformational flexibility of the enzyme, particularly in the active site. therefore, molecular dynamics simulations were performed to address enzyme flexibility and the broad substrate spectrum of sult ( figure 1 ). based on a collection of selected enzyme conformations from molecular dynamics simulations and a dataset of active sult1e1 ligands, ensemble docking was utilized to generate ligand-protein complexes that served as a basis for 3d pharmacophore development. eight specific 3d pharmacophores were created that allow identification of sult1e1 substrates and inhibitors. for further refinement of the computer-based prediction, machine learning models and post-filtering steps were generated that allow classification of predicted hits. the final prediction model was used to screen the drugbank (a database comprising over 6,500 experimental and approved drugs). a major part of the predicted hits could be confirmed from literature. from the remaining hits, a representative selection of molecules was experimentally tested for sult1e1 inhibition or biotransformation. experimental results were in agreement with our computer-based models and revealed previously-unknown biotransformation by or inhibition of sult1e1 for compounds listed in the drugbank. introduction: vascularization of the dermal equivalent of full-thickness skin constructs by endothelial cells is highly desirable, because such constructs closely mimic the architecture of real skin. unfortunately, the realization of a capillary network in skin constructs is still difficult. in our study of full-thickness skin constructs, following the methodologies of küchler et al. (2011) , there were alterations in the epidermal differentiation after endothelialization of the dermal equivalent. the aim of this study was to characterize these changes on a morphological level. material and methods: non-endothelialized constructs (keratinocytes, fibroblasts) were prepared according to küchler et al. (2011) . to obtain endothelialized constructs, the dermal equivalent of the non-endothelialized constructs was enriched with endothelial cells. after two weeks of in vitro culture, the skin constructs were processed for quantitative as well as qualitative assessment by light and electron microscopy. results: both types of skin construct developed all strata, i.e., stratum basale, spinosum, granulosum, corneum of a stratified soft-cornified epidermis, although the two constructs displayed differences in every stratum: significantly more mitoses occurred in the epithelial germ layers of the endothelialized constructs (p=0.013). in addition, significantly more keratohyalin granules were counted within their stratum granulosum (p=0.010). 50% of the shapes of the spinous and the granulosum cells were irregular and these cells were separated by wide intercellular spaces. the typical epidermal lamellar bodies appeared in the endothelialized constructs more often than in the nonendothelialized ones. at the stratum granulosum -stratum corneum interface, no cohesion between the strata was present. background and novelty: during the last decade organ-on-a-chip technologies received increasing attention in the scientific community. the idea of combining different tissue types on a physiological-like system creates completely new options on how substances can be characterized without the use of animal experiments. animal models were used for the investigation of skin sensitization as a standard method until animal testing for cosmetic substances was banned in the e.u. in 2013. by combining skin models with an immunological counterpart, new data will be presented to see if the multi-organ-chip add value to the current need of alternative methods regarding skin sensitization and immunotoxicity testing. experimental approach: the multi-organ-chip platform is designed to combine different human cell and tissue types like 3d-spheroids, barrier models or biopsies in one microfluidic system. a peristaltic on-chip micropump enables circulation of medium, allowing for a constant perfusion between the compartments. first experiments were performed using a dendritic-cell-only approach in the 2-organ-chip. in subsequent cocultivation experiments ex vivo human epidermis and dendritic cells were cultivated each in one culture compartment connected by the microfluidic channels. for analysis we measured the typical activation marker cd86 and the vitality of the dendritic cells by flow cytometry. functionally different sensitizers were selected to investigate their effects in our model. finally a more complex 3d matrices including different immunological cell types to emulate in vivo-like reactions like in the human lymph node were cultivated in the 2-organ-chip. results and discussion: our data show a strong influence of pump pressure and pumping frequency on the activation of dendritic cells. hence, we established an adequate set up by cultivating the dendritic cells in cell culture inserts, preventing cell activation due to shear stress. compared to existing sensitization assays, the main advantage of the perfused 2-organ-chip sensitization assay is the presence of an epidermis equivalent, partially integrating important parameters such as metabolism and skin barrier function. we compared our data with reference cd86-values from the pbmdc (peripheral blood monocyte derived dendritic cells) skin sensitization assay. for identical substances, we observed differences in dendritic cell activation between the pbmdc assay and the 2-organ-chip perfused assay. here we present the first-time cultivation of primary derived immune cells cultivated on our microfluidic system which is a promising enhancement to integrate immunological reactions on further multi-organ combinations. due to growing social and political pressure, the interest in alternatives to animal testing has constantly increased during the past 10 years stimulating the development and validation of new in vitro test systems including reconstructed skin models. additional to toxicological studies and permeability assays, skin models are of high interest for fundamental research to elucidate basic physiological and pathophysiological processes in human skin [1, 2] . as for today, most of the in vitro skin models are grown from primary keratinocytes and fibroblasts that were either isolated from excised human skin or from juvenile foreskin following circumcision. in this project, we aimed for the generation of in vitro skin models using hair folliclederived cells. therefore, different methods to optimize cell isolation and expansion of outer root sheath (ors) cells from human hair follicles were systematically investigated. the best procedure for isolation of ors cells was the direct cell outgrowth on a cell culture insert which was co-cultured with a feeder layer of postmitotic human dermal fibroblasts. following outgrowth, the cells were either further cultivated with feeder cells in specific serum-enriched cell culture medium to obtain hair follicle-derived keratinocytes or using the same culture medium without feeder cells to obtain fibroblasts. afterwards, the generation of hair follicle-derived fibroblasts and keratinocytes was verified via the fibroblast-specific markers vimentin and desmin and the keratinocyte marker cytokeratin (ck) 14 clearly showing that vimentin and desmin are expressed in hair follicle-derived fibroblasts and in dermal fibroblasts. as expected, these cells were negative for ck14, which was abundantly expressed in hair folliclederived keratinocytes. moreover, the expression of collagen type i, iv, tgf-beta, alpha sma and il-1 alpha in hair follicle-derived fibroblasts and dermal fibroblasts showed no significant differences. ultimately, hair follicle-derived keratinocytes and fibroblasts were used to grow full-thickness skin models which were subsequently characterized with regard to epidermal differentiation, skin permeability and skin surface ph. again, no significant differences compared with skin models grown from skin-derived cells were detected showing the potential of using hair follicle-derived cells for generating in vitro skin models. [1] vávrová, k., henkes, d., strüver, k., sochorová, m., školová, b. et al. filaggrin deficiency leads to impaired lipid profile and altered acidification pathways in a 3d skin construct. the journal of investigative dermatology. 134, 746-753 (2014 bundesinstitut für risikobewertung, experimentelle toxikologie und zebet, berlin, germany background: the eu directive 2010/63 has been drawn up with the aim of ultimately replacing animal testing. wherever animal experimentation is necessary, the 3-rprinciple of russel and burch (replace, reduce, refine) has to be observed. the primary goal of the 3-r-principle is to replace animal testing with alternative methods. if no alternative method can be applied, the total number of animals is supposed be reduced. consequently, some animals are used multiple times in the course of an experiment. for example, in imaging studies, rodents are exposed to anesthesia several times in order to control the progress of a disease. however, the directive claims that "the benefit of reusing animals should be balanced against any adverse effects on their welfare, taking into account the lifetime experience of the individual animal". objective: we are looking into whether multiple exposures to anesthesia cause more stress than a single exposure. methods: the most common mouse strain c57/bl6 j and anesthetics isoflurane and the combination of ketamine/xylazine are used. with regard to recent studies, the animals are anesthetized six times for 45 minutes over a period of three weeks. all parameters observed are compared between controls, animals with a single and repeated anesthesia. the interval between the administration of the anesthesias is three to four days. when the animals are under anesthesia, their vital parameters are continuously monitored and afterwards their general condition is examined. the grimace scale is scored 30 and 150 minutes after anesthesia. besides pain, the grimace scale can also assess anxiety, stress and malaise. the display of so-called luxury behaviors like nest building and burrowing behavior serves as an indicator of wellbeing. furthermore, activity, food and water intake are monitored for 24 hours. a behavioral test battery including the free exploratory paradigm, open field, balance beam and rota rod test is performed one, seven and ten days after the last anesthesia. motor coordination and balance are assessed by the balance beam and rota rod. the open field is a test to investigate anxiety-related and exploratory behavior, the free exploratory paradigm estimates trait anxiety. moreover, corticosterone metabolites are measured in feces and fur in order to prove evidence of cumulative stress. results: the first results of our study will be presented at the 82 nd meeting of dgpt. conclusion: we are confident that the results of our study will contribute to the assessment of the severity level caused by multiple exposures to anesthesia and in this way be a benefit for the welfare of laboratory rodents. bb3r is funded by bmbf. in 1959 the 3r-principle was defined by the british scientists william russel and rex burch in their book 'the principles of human experimental techniques'. the 3r refer to replace, reduce, and refine. they set the goals to use alternative non-animal methods (replace), to reduce the number of laboratory animals (reduce) and to minimize the distress of laboratory animals and to refine their welfare (refine). the implementation of the 3r-concept is the overall goal of scientific animal welfare. article 4 of the 'directive 2010/63/eu on the protection of animals used for scientific purposes' state that the research on refinement is as important as on replacement and reduction [1] . according to the current statistics on laboratory animals, the mouse is the most commonly used animal in experiments with approximately 70 % [2] [3] . effective pain treatment is crucial not only for ethical and legal considerations but also to achieve highquality results [4] . pain in mice is only obvious if it is severe or acute pain. however, it is difficult to identify slight or moderate pain. the determination of pain levels and effective dosages of analgesics is therefore challenging. the most commonly used analgesics for postsurgical pain treatment in mice are opioids. however, the recommendations for their use show vast dosage ranges. for example, the recommended dose of buprenorphine ranges from 0.05 to 2.5 mg/kg per bodyweight [5] [6] [7] depending on the pain model used. additionally, putative pharmacogenetic strain differences have to be considered. for example, the analgesic treatment with morphine shows mouse strain specific differences in pain sensitivity [8] . the goal of the project is the refinement of the recommendations for effective dosage of opioid analgesics in laboratory animals for mouse inbred strains by incorporating strain specific differences. regarding the phenotype, we want to identify a putative inbred strain dependent pain threshold and measure the drug level in plasma and brain. additionally the metabolic capacity and mrna expression level will be investigated. genotype identification is based on a data bank analysis used for correlation with phenotypical parameters. [1] rl 2010/63/eu. [2] bmel (2014) . anzahl der für versuche und andere wissenschaftliche zwecke verwendeten wirbeltiere. [3] eu commission (2013) . seventh report on the statistics on the number of animals used for experimental and other scientific purposes in the member states of the european union. [4] carbone l (2011) pain in laboratory animals: the ethical and regulatory imperatives. plos one 6, e21578. [5] gv-solas, a.f. a.d. (2013) . empfehlung zur schmerztherapie bei versuchstieren. [6] carpenter, j.w., t.y. mashima, and d.j. rupiper (2001) . exotic animal formulary, 2nd edition, w.b. saunders co., phila. [7] flecknell, p. (1996) . laboratory animal anaesthesia, 2nd edition, academic press, san diego, ca. introduction: göttingen minipigs™ are frequently used large animal models for orofacial research, for example dental implant surgery. requests from experimental surgeons for detailed anatomical information can not be answered because there is no existing data, especially not age-related. because of unavailable data and the false choice of animal age, surgical interventions fail or lead to enormous post-operative suffering. therefore the aim of this study is the acquisition of detailed anatomical data of the mandibula and other organs and structures without sacrificing pigs for this reason. animals, materials and methods: ct scans of a 64-slice scanner were collected from 18 female minipigs, consisting of 6 animals aged 12 months (group 1, n=6) and 12 animals (group 2; n=12) examined at the age of 17 and 21 months. these minipigs were involved in experiments, approved by the regional office for health and social affairs, berlin. image analysis was performed using vitrea advanced® (vital images). more than 50 parameters concerning teeth, the mandibular body, frame and canal, coronoid process and mandibular condyle were defined and measured. for now, we focused on the development of the mandibular canal and the distance between the dorsal borders of the mandibular canal to the alveolar ridge at the most posterior mental foramen, parameters immensely important for planning interventions when testing new dental implants. results: the measurements by computed tomography showed variations of several parameters between left and right ramus mandibulae and within the different age groups. the volume of the canalis mandibulae increases in time. animals of the same age show significant differences in volume, with a range of up to 65% where the largest volume was 13,4 ml and the lowest 4,7 ml. the distance between the dorsal borders of the mandibular canal to the alveolar ridge decreases between 12 and 17 months of age. comparing 17 and 21 months old minipigs, no significant difference in distance could be observed. from the age of 17 months the position of the mandibular canal in relation to the alveolar ridge remains constant. conclusion: the decrease of the distance between the mandibular canal and the alveolar ridge between 12 and 17 months of age indicates ongoing anatomical changes of this parameter until the age of 17 months. therefore animals should be older than 17 months if included in long-term studies after orofacial experiments, like implant surgery of the mandibula. because of the described individual differences, the authors strongly suggest to support the planning of orofacial interventions by ct imaging or other radiographic techniques. background: laboratory housing conditions are standardized to a high level. under these conditions, the occurrence of stereotypic behaviour (sb) can be observed. stereotypies are commonly known as deviations from normal behaviour that are repetitive, invariant and without any obvious function or aim for the animal [1]. worldwide it is estimated that over 85 million animals perform sb, with the highest prevalence in laboratory animals and the agricultural sector. fvb/n is a typical inbred mouse-strain that shows different types of stereotyped movements. it is known that environmental enrichment decreases the incidence of sb, yet they still occur [2] . since animal's behaviour highly influences its metabolism and immune system, differences in handling, caring and keeping can lead to varying results, even with an identical experimental setup [3] . aim: of the study aim of the study is to observe different life stages of fvb/n-mice and immediately detect the development of sb. observations are linked to various behavioural tests and the characterisation of the metabolic and immunological phenotype. the results will lead to a better understanding of the mechanisms driving the development of sb and clarify its implication to animal welfare or to what extent the performance of stereotypies even reflect emotional suffering. the strain-related behaviour and sb are recorded with computer-assisted programmes. observational periods already begin with the parental generation. as possible indicators for later developing sb, data on reproductive success and maternal care are collected, such as different behavioural tests. the animals are characterized by a specific protocol for detecting the metabolic and immunological phenotype. finally, histological and molecular biological analyses follow. outlook: it is of paramount importance to take good care for the welfare of laboratory animals (3r-refinement). though, the knowledge about the ethological particularities of animals and the motivational base of animals performing sb are not enough to generally avoid stereotypies. therefore the meaning according to the character of sb has to be analysed more intensively to understand the needs of laboratory animals and evolve recommendations for optimizing the breeding and keeping such as for the assessment of possible distress in animals performing sb. objective: thermoregulation is a vital function in both humans and animals with the serotonin (5-ht) system, in particular the 5-ht 1a receptor, playing a major role. activating 5-ht 1a receptors by the 5-ht 1a receptor agonist 8-hydroxy-2-(dipropylamino)tetralin (8-oh-dpat) leads to reduced body temperature. while there is consensus that hypothermia is induced by the stimulation of postsynaptic 5-ht 1a receptors in rats and humans, the regulatory mechanisms in mice are less clear. in our group, within phenotyping a transgenic mouse line permanently overexpressing the 5-ht 1a receptor in serotonergic projection areas, bert et al. (2008, pmid: 18396339) revealed exaggerated 8-oh-dpat-provoked hypothermic response. thus, the objective of the present study was to substantiate the contribution of postsynaptic 5-ht 1a receptors to thermoregulation, more precisely to the hypothermic effect of 8-oh-dpat, in mice. methods: we used radio telemetry technique to monitor the basal body temperature and the hypothermic effect of different doses of 8-oh-dpat (0.1 mg/kg -4 mg/kg i. p.) in male transgenic mice in comparison to nmri wild-type males. additionally, we investigated whether reduction of serotonergic activity by pretreatment with the 5-ht synthesis inhibitor parachlorophenylalanine (pcpa; 100 mg/kg, i. p. on four consecutive days) would alter the effects of 8-oh-dpat on body temperature in transgenic mice postsynaptically overexpressing the 5-ht 1a receptor. results: 5-ht 1a overexpressing mice revealed lower levels of basal body temperature than wild types (transgenic mice: 36.0 °c; nmri wild-type mice: 37.4 °c). in both genotypes, systemic administration of 8-oh-dpat dose-dependently decreased body temperature, being significantly more pronounced in mutant mice (-2.8 °c compared to -1.5 °c in nmri wild types). dose response curves of 8-oh-dpat revealed an ed 50 = 0.4 mg/kg in transgenic and an ed 50 = 0.57 mg/kg in nmri wild-type mice. pcpa pretreatment did not alter the hypothermic response to 8-oh-dpat in mice. the dose-response curves indicate a higher potency of 8-oh-dpat in transgenic mice. the exaggerated hypothermic response to 8-oh-dpat in mutant mice implies that postsynaptic 5-ht 1a receptors could be involved in thermoregulatory function in mice. this assumption is further confirmed by the fact that 8-oh-dpatevoked thermal responses were not influenced by pretreatment with pcpa, most notably in transgenic mice overexpressing 5-ht 1a receptors postsynaptically. genetic variation within g protein-coupled receptors compromises the therapeutic application of drugs targeting these receptors. one of the most intensely studied variation is p.arg389gly in the human beta1-adrenoceptor (adrb1). the adrb1 carrying arginine at position 389 in helix 8 in the proximal carboxy terminus is more frequent among caucasians and is hyperfunctional. yet, the molecular basis for the differences between the beta1-adrenoceptor variants arg389-adrb1 and gly389-adrb1 is poorly understood. despite its hyperfunctionality, we found the arg389-variant of the adrb1 to be hyperphosphorylated upon continuous stimulation with norepinephrine when compared to the gly389-variant. using adrb1 sensors to monitor activation kinetics by fluorescence resonance energy transfer (fret), the arg389-adrb1 exerted faster activation speed and arrestin recruitment than the gly389-variant. both depended on phosphorylation of the receptor as shown by knockdown of g protein-coupled receptor kinases and by fret experiments using phosphorylation-deficient adrb1 mutants. point mutation of single serines and threonines in the carboxy terminus of the adrb1 finally revealed a variant-specific phosphorylation pattern that determines arrestin recruitment. taken together, these findings suggest that differences in receptor phosphorylation determine the differences in activation speed, efficacy and arrestin recruitment of adrb1 variants. opioid drugs are the most potent analgesics, which are used in the clinic; however, by activating the μ-opioid receptor (mor) they also produce several adverse side effects including constipation, antinociceptive tolerance, and physical dependence. there is substantial evidence suggesting that g protein-coupled receptor kinases (grks) and βarrestins play key roles in regulating mor signaling and responsiveness. following phosphorylation by grks, β-arrestins bind to phosphorylated mors, which prevents further interactions between the receptor and g proteins even in the continued presence of agonist resulting in diminished g protein-mediated signaling. we have previously shown that agonist-induced phosphorylation of mor occurs at a conserved 10-residue sequence, 370 trehpstant 379 , in the carboxyl-terminal cytoplasmic tail. morphine induces a selective phosphorylation of serine 375 (s375) in the middle of this sequence that is predominantly catalyzed by g protein-coupled receptor kinase 5 (grk5). by contrast, high-efficacy opioids not only induce phosphorylation of s375 but also drive higher-order phosphorylation on the flanking residues threonine 370 (t370), threonine 376 (t376), and threonine 379 (t379) in a hierarchical phosphorylation cascade that specifically requires grk2/3 isoforms. to investigate this mechanism further, we have developed novel β-galactosidase complementation assays to monitor agonist-dependent recruitment of grk2 and grk3 to activated mors. using this assay, we were able to show that activation of mor by high-efficacy agonists such as damgo results in a robust translocation of grk2/3. in contrast, activation by low-efficacy agonists such as morphine results in a much less pronounced recruitment of grk2/3 isoforms. interestingly, damgo-induced β-arrestin recruitment was strongly inhibited by sirna knock down of grk2 or grk3. conversely, the morphine-induced β-arrestin recruitment was strongly enhanced by overexpression of grk2 or grk3. mutation of s375 to alanine strongly inhibited both grk and β-arrestin recruitment. however, mutation of all 11 carboxyl-terminal serine and threonine residues of mor was required to completely abolish its interaction with arrestins and grks resulting in a complete loss of mor internalization and desensitization. heterotrimeric g proteins are located at the inner leaflet of plasma membranes and are a major primary transducer of cell signaling initiated by g protein-coupled receptors (gpcrs). based on sequence similarity, heterotrimeric g proteins can be subdivided into four main classes, i.e. gi/o, gs, gq/11, and g12/13, which interact with distinct cellular effectors to shape the final cellular response 1 . identification of new selective and cell-permeable g protein inhibitors is of great interest as these may be beneficial in complex pathologies that involve signaling of multiple gpcrs 2 . mechanistically, small molecule g protein inhibitors may, for instance, block nucleotide exchange by inhibiting gdp release (i.e. guanyl nucleotide dissociation inhibitors, gdis) or allow gdp release but block gtp entry by stabilizing the g protein in an empty pocket conformation 3 . here, we set out a new approach to classify g protein inhibitors regarding their mechanism of action in radioligand binding experiments. in particular, we investigated the influence of the specific gα q/11/14 inhibitor fr900359 4 on agonist-radioantagonist binding experiments performed with membranes of cho cells stably expressing the muscarinic m1 acetylcholine receptor (cho-m1). agonistradioantagonist competition under these conditions is biphasic because agonists bind with higher affinity in the ternary complex consisting of agonist, receptor and nucleotidefree g protein compared with a g protein-free receptor 1,5 . we show that fr900359 can be classified as a gdi as it significantly reduced high affinity binding of carbachol in cho-m1 membranes. in contrast to this, bim-46187, a pan g protein inhibitor with a cell-type-dependent preference for gq, did not influence high affinity agonist binding and thus stabilized gq in an empty pocket conformation 3 . interestingly, inhibition of high affinity agonist binding by fr900359 was incomplete in agonist-radioantagonist displacement studies and also when a radioagonist was applied. to fully prevent high affinity agonist binding by fr900359, combined uncoupling of both gi and gs proteins from m1 was required by pre-treatment with pertussis toxin and cholera toxin, respectively. these data demonstrate that not only gq, but also gi, and gs, contribute to the high affinity fraction of m1 receptors. taken together, our findings show that radioligand binding experiments are an attractive approach to classify new g protein inhibitors according to their mechanism of action. universität, würzburg, germany g protein-coupled receptors (gpcrs) are cell surface receptors which, upon a conformational change in the receptor protein induced by an extracellular stimulus, can transduce the signal onto intracellular adaptor proteins such as heterotrimeric g proteins [1] . gpcr-induced cell signaling can be rather complex as several gpcrs may activate multiple different adaptor proteins and can additionally be activated via distinct binding sites, i.e. the orthosteric transmitter binding site and other "allosteric" binding sites [2] . in the present work, we wanted to investigate the influence of an allosteric binding site on receptor activation of muscarinic acetylcholine receptors (machrs). to this end, we employed the orthosteric full agonists acetylcholine and iperoxo as well as several dualsteric compounds consisting of iperoxo linked to an allosteric phthalimide (phth) or naphthalimide (naph) moiety through alkyl chains of different length or through a diamide linker (fri). binding of the allosteric part to the receptor protein may restrict the conformational flexibility of the receptor protein and thus interfere with receptor activation [2] . therefore, application of different linker length may control the signaling outcome. here, we applied the human m1 machr which preferentially activates g proteins of the g q/11 type but can also promiscuously stimulate g s proteins. g q/11 -and g sdependent signaling pathways were analyzed by application of cho cells stably transfected with the human m1 machr in ip1 and camp accumulation assays, respectively. in comparison to the orthosteric building block iperoxo, all dualsteric compounds under investigation showed a decrease in potency for both g q -mediated and g s -mediated signaling. our findings show that the bulkier allosteric naph residue impaired both signaling pathways to a greater extent than the smaller substituent phth. particularly, the compound iper-6-naph completely lost intrinsic activity for both g q/11 and g s activation at the m1 machr. moreover, g s -mediated pathway activation is more sensitive to spatial restriction in the allosteric vestibule than g q -signaling. interestingly, longer linker length led to improved signaling for both pathways (g q and g s ) in both hybrid series. iper-7-phth seems to be an exception as it had a higher intrinsic efficacy for g s -dependent signaling than the other phth hybrids with longer linker chains. strikingly, only iper-fri-phth, which corresponds to iper-8-phth in linker length, but is able to engage increased hydrogen bonding with the receptor protein, acted as a full agonist on m1 machr for both signaling pathways under investigation. taken together, these data strongly suggest that, in comparison to g q/11 -mediated signaling, activation of the g s protein in m1 machr is more sensitive to spatial restriction in the allosteric vestibule. thus, it appears to be possible to control signaling of the m1 machr by allosteric constraint of the receptor's conformational flexibility. for more than three decades 3-(1h-imidazol-4-yl)propylguanidine (sk&f-91,486 (1) [1] ) is known as the prototypic pharmacophore of highly potent histamine h 2 -receptor (h 2 r) agonists of the guanidine class of compounds including, e.g., impromidine and arpromidine. [2] in order to get more insight into the structure-activity relationships of alkylated analogues of sk&f-91,486, we characterized 78 newly synthesized derivatives including several bivalent compounds (e.g., 2) by using different pharmacological in-vitro methods. [3] the potential h 2 r agonists were subjected to a broad screening procedure utilizing radioligand binding assays with membranes of sf9 cells [4] (hh 1,2,3,4 r). compounds were also functionally characterized in the [ 35 s]gtpgs assay (hh 2 r, sf9 cell membranes). [5] selectivity vs. hh 1,3,4 r was determined for selected derivatives also using this technique. organ bath studies (gph 1 r (ileum), gph 2 r (right atrium)) yielded functional data in a more physiological environment. the major part of the new sk&f-91,486 analogues displayed partial or full agonism via hh 2 r and gph 2 r, respectively. the most potent analogue, bivalent thiazole-type bisguanidine 2, was a partial agonist (e max = 88%) and 250-times as potent as histamine vis-à-vis the gph 2 r. attempts to antagonize the positive chronotropic effect of (partial) agonists by preincubation with cimetidine, or by adding a cimetidine bolus at the end of the concentration-response curve, respectively, were successful and furnished pa 2 values for the antagonist (5.87 -6.38) which are in accordance with literature data. however, in the functional in-vitro assay on gph 2 r, the positive chronotropic response evoked by sk&f-91,486 was surprisingly resistant to antagonism by cimetidine and other typical h 2 r antagonists (ranitidine, famotidine), although the compound so far has been unanimously classified by others as a weak partial h 2 r agonist. this behaviour is unique within the large series of sk&f-91,486 analogues studied so far under similar conditions. probably the positive chronotropic effect of the lead compound is -at least in part -the result of a second molecular interaction which has been overlooked so far. [1] parsons, m.e. et al.; agents actions 1975, 5, 464. [2] buschauer, a.; j. med. chem. 1989 , 32, 1963 -1970 [3] pockes, s.; dissertation, univ. regensburg 2015. [4] seifert, r. ; j. pharmacol. exp. ther. 2003, 305 (3) , 1104-1115. the nociceptin/orphanin fq (n/ofq) peptide (nop) receptor is the fourth most recently discovered and least characterized member of the opioid receptor family (mor, kor and dor). nop receptor is widely distributed and modulates several physiological processes by its endogenous ligand nociceptin. the nop receptor is a potential target for the development of ligands with therapeutic use in several pathophysiological states such as chronic and neuropathic pain. consequently, there is increasing interest in understanding the molecular regulation of nop receptor. recently, we generated two phosphosite-specific antibodies directed against the carboxyl-terminal residues serine 351 (s351) and threonine 362 /serine 363 (t362/s363), which enabled us to selectively detect either the s351-or the t362/s363-phosphorylated forms of the receptor. our results show that nociceptin, mcoppb, sch221510 and ro64-6198 induce a stably phosphorylation at s351 and t362/s363 followed by a profound internalization of the receptor. the nociceptin-induced s351 and t362/s363 phosphorylation can be blocked by selective nop receptor antagonists such as j113397 or sb612,111. nnc63-0532, buprenorphine and norbuprenophine failed to induce a phosphorylation at these sites. in the presence of nociceptin, s351 phosphorylation occurred at a faster rate than phosphorylation at t362/s363 indicating that s351 is the primary site of agonistdependent phosphorylation. activation of pkc by phorbol 12-myristate 13-acetate facilitated receptor phosphorylation only at s351 but not at t362/s363, indicating that s351 can also undergo heterologous phosphorylation. using nop-gfp knock in mice, we detected nop receptors in brain, spinal cord and dorsal root ganglia (drg). we were also able to demonstrate a dose-dependent nop receptor phosphorylation at t362/s363 in mouse brain in vivo using western blot and mass spectrometry. in contrast, mcoppb and sch221510 failed to induce phosphorylation in vivo. together, these data provide new insights into the molecular regulation of the nop receptor in vitro and in vivo. several findings indicate that inflammatory diseases are initiated or maintained by an imbalance of receptor baised signaling; the latter referring to the ability of distinct ligands to endue individual receptors with qualitatively different g-protein-and/or b-arrestindependent signaling (1). chemokines and their receptors regulate a wide array of leukocyte functions, including chemotaxis, adhesion, and transendothelial migration, and thus play important roles in regulating inflammation (2) . in man, two cc chemokine receptors ccr2a and ccr2b are present that only differ in their carboxyl terminal portions; the latter known to interact with multi-protein complexes made up of heterotrimeric g proteins (pertussis toxin sensitive and -insensitive) and non-g-protein components, including b-arrestin (2) . interested in differential signaling of ccr2a and ccr2b we comparatively analyzed ligand-induced g-protein-regulated signaling pathways (e.g. activation of phospholipase c isoenzymes and rhogtpase-induced serum response factor [srf] activity) and b-arrestin-regulated pathways (e.g. internalization of receptors and phosphorylation of erk1/2) in the presence of ccl2, ccl8, ccl7, and ccl13. all these chemokines have been shown to interact with human ccr2 receptors. in addition, the structural requirements of the carboxyl terminal portions involved in determining specificity in g-protein-dependent signaling was addressed by using ccr2 receptor mutants. the comparative analysis revealed that differences in ligand-induced activation of g-protein-dependent (pertussis-toxin-sensitive versus pertussis-toxin-insensitive) and/or b-arrestin-dependent signaling exist. for example, activation of ccr2b receptors by ccl2 induced both rho-dependent srf activation and receptor internalization, while ccl8-stimulation resulted in srf but little if any receptor internalization. in contrast, ccr2a-expressing cells showed ccl2dependent srf-activation but any receptor/ligand internalization. analysis of the structural requirements of ccr2 receptors for coupling to g proteins revealed that arginine 313 within the putative 'eighth helix' of the carboxyl-terminal portions of ccr2a and ccr2b is not involved in ga i -mediated induction of erk1/2 and plays a minor role in ccr2b receptor internalization, but is specifically required for the ccr2a/ and ccr2b/ga q -mediated activation of srf. serotonin 5-ht 2c receptors (5-ht 2c r) are functional engaged with gq proteins and expressed in the central nervous system (cns). 5-ht 2c r significantly regulate emotion, feeding, reward or cognition and thus might serve as promising targets for drugs against psychiatric disorders or obesity. due to the technical difficulties in isolating cells from the cns and the hitherto lack of suitable cell lines that would endogenously express 5-ht 2c r, our knowledge about this receptor subtype is rather limited. recently established hypothalamic mhypoa2-10 cells show some characteristics of appetite-regulating hypothalamic neurons of the paraventricular nucleus, where 5-ht 2c r in vivo expression has been detected. thus, we tested mhypoa2-10 cells for putative 5-ht 2c r expression by performing single cell calcium imaging. we observed that serotonin and the specific 5-ht 2c r agonist, way161,503 induced robust calcium transients, which were strongly inhibited by two unrelated 5-ht 2c r-selective antagonist (sb206,553, rs102,221). serotonin and way161,503 also activated a camp response element-dependent reporter gene construct and induced significant phosphorylation of extracellularregulated kinases-1/2 in a sb206,553 and rs102,221 sensitive manner, providing further evidence for functional 5-ht 2c r expression in mhypoa-2/10 cells. intrinsic activity of way161,503 ranged between 0.3 and 0.5 compared to serotonin in all assays, defining way161,503 as a partial 5-ht 2c r agonist. in conclusion, we provide convincing data that hypothalamic mhypoa-2/10 cells endogenously express 5-ht 2c r and thus represent the first cell line to analyse 5-ht 2c r pharmacology, signaling and regulation in its natural environment. optical and electrophysiological methods allow detection and characterization of g i/o -protein coupled receptors j. straub 1 1 walther-straub-institut für pharmakologie und toxikologie, münchen, germany g-protein mediated signaling pathways are essential components of basic cellular functions. of note, g-protein coupled receptors (gpcrs) constitute one of the major drug targets in modern medicine. however, despite their clinical importance, fundamental properties of these receptors remain unknown. in particular, regulation of the major second messenger camp by g s -and g i/o -protein coupled receptors is of special interest. the classical biochemical method to detect receptor-mediated camp level changes uses pre-labeling with 3 h-adenine and calculation of the conversion rate to 3 h-camp. although, this multi-cellular method is highly sensitive and reproducible, it lacks time resolved and spacial assessment of camp formation in single living cells. to measure g s -protein induced increases of intracellular camp levels in single living cells in a time resolved manner, the fret-based camp-sensor epac is commonly used. however, it was unknown whether this sensor might be suitable to detect g i/o -protein mediated decreases of intracellular camp levels as well. in this study, we show that fret-based camp sensors can be deployed to reliably monitor g i/o -protein mediated camp level decreases. fret experiments with adrenergic α 2a or µ 1 opioid receptors in combination with different fret-based camp sensors showed a notable reduction of intracellular camp levels upon receptor activation which could be significantly reduced by selective receptor antagonists. of note, hek293 cells had to be pre-incubated with forskolin in submaximal concentration to increase basal camp levels. our findings suggest that fret based epac sensors are suitable to detect g i/o -protein activation similar to electrophysiological whole-cell measurements with g i/o -protein coupled receptors and trpc5 or heteromeric kir3.1/kir3.2 or kir3.1/kir3.4 channels coexpressing cells. hereby, agonist stimulations caused current increases with characteristic current-voltage relationships. altogether, our findings indicate that both optical and electrophysiological approaches allow time resolved detection and characterization of g i/o -protein coupled receptor activation in single living cells. histamine can exert positive inotropic and chronotropic effects in humans via histamine h 2 -receptors. we have generated and partially characterized transgenic mice (tg) which overexpress the human histamine h 2 -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), histamine increased heart rate and ejection fraction (ef) measured in vivo by echocardiography under isoflurane anesthesia. to investigate some aspects of the signaling pathway in these mice, we crossed the tg mice with pp2a mice leading to double transgenic mice (h 2 xpp2a = dt). pp2a mice (j biol chem 2004; 279:40827) overexpress the catalytic subunit of protein phosphatase 2a (pp2a) in cardiac myocytes and develop a cardiac hypertrophy. at an age of about 240 days we noted reduced ef in pp2a (33.1 ± 3.5 %, n=16) compared to wt (54.4 ± 4.5 %, n=10) and tg (59.8 ± 2.9 %, n=10). interestingly, in dt the ef (43.9 ± 4.9 %, n=11) was higher than in pp2a at similar heart rates. e´/a´ was elevated in dt compared to wt. relative heart weights were unchanged between these groups. in summary, we demonstrated that pp2a is involved in h 2 -receptor signaling and we tentatively conclude that the h 2 -receptor is able to ameliorate systolic but not diastolic cardiac function of pp2a mice. serotonin (5-ht) can exert positive inotropic and chronotropic effects in humans via 5-ht 4 -receptors. we have generated transgenic mice (tg) which overexpress the human 5-ht 4 -receptor selectively in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), serotonin induced increases in heart rate and ejection fraction. we treated the mice with 30 µg lps (lipopolysaccharide, i.p.; a standard model of sepsis) per g body weight or isotonic sodium chloride solution (as solvent control). echocardiography with isoflurane anesthesia was performed before and 3, 7 and 24 hours after lps treatment. lps led to a time-dependent deterioration of cardiac function in both tg and wt. the deterioration included systolic function (left ventricular ejection fraction=ef) as well as diastolic function (height of a and e waves through the mitral valve: e/a). for instance, ef amounted to 58.8 ± 20.7% seven hours after lps in wt and to 61. [4] [5] [6] [7] [8] [9] [10] [11] [12] p<0 .05 vs pre-lps value). however, 24 hours after lps, diastolic function, measured as e/a, amounted to 1.86 ± 0.43 in p<0 .05 tg vs. wt). moreover, after 24 hours a less pronounced decline in body temperature (probably due to superficial abdominal hyperemia) occurred in tg versus wt. in contrast, while all flow parameters declined after 3 and 7 hours of lps, they were not different between wt and tg. for instance, maximum flow (in mm/s) through the ascending aorta declined from 1158.3 ± 212.2 to 782.5 ± 154.3 in wt and from 1208.4 ± 235.1 to 798.8 ± 170.1 in tg (tg vs. wt, p>0.05, n=10-12; after 7 hours). we tentatively conclude: 5-ht 4 -receptors overexpression protects the heart against sepsis, putatively by interference with the intracellular biochemical pathway of lps in cardiomyocytes. histamine can exert positive inotropic and chronotropic effects in humans via histamine h 2 -receptors. we have generated transgenic mice (tg) which overexpress the human h 2 -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in tg, but not in wild type mice (wt), histamine (ec 50 = 34 nm) or amthamine (ec 50 = 10 nm), a more selective and potent h 2 -receptor agonist, induced positive inotropic effects (pie) and positive chronotropic effects (pce) in isolated left and right atrial preparations, respectively. in order to investigate the signal transduction for the pie in atrium, contractile studies using partially depolarized preparations were performed. therefore, left atrial preparations were equilibrated in the organ bath to 44 mm potassium chloride. thereafter, histamine (100 µm) induced a pie (31.1 ± 10.4 % of control, n=7) in tg but not in wt preparations whereas isoprenaline (10 µm) increased force in both wt and tg. the pie of histamine in potassium treated tg atrium could be blocked by cimetidine. compound 48/80, a releasing agent of endogenous histamine, increased force of contraction in tg left atria to a higher extent than in wt. furthermore, we tested whether analgetics known to release histamine were inotropically active in tg. however, morphine (10 µm) was unable to affect contractility in wt or tg, whereas ketamine and fentanyl increased left atrial contractility in both tg and wt. in summary, we demonstrated an involvement of the l-type calcium channel current in the h 2 -receptor mediated pie in tg atria. we failed to release inotropically active histamine using classical analgetics, arguing that a direct effect also in the human heart is unlikely to occur. the initial step in the homologous desensitization of g-protein-coupled receptors is their phosphorylation by one of the g-protein-coupled receptor kinases (grks). we demonstrate here measurement of the interaction of grk2, a ubiquitously expressed grk, with the μ-opioid receptor (µor) by fret and its dependence on agonist efficacy. hek293t cells transfected with yfp-tagged µor and mturquoise-tagged grk2 as well as non-fluorescent gα i1 , gβ and gγ subunits showed a robust increase in fret upon superfusion with 10 µm [d-ala 2 , n-mephe 4 , gly-ol]-enkephalin (damgo) which was reversible upon agonist washout. the partial agonist morphine (30 µm) also caused a fret increase but the amplitude of the fret signal was reduced to approximately 15-20% of that of the corresponding damgo signal. grk2 binds g-protein βγ (gβγ) subunits, and therefore we aimed to find out how cotransfection of grk2 affected the interaction of gβγ with the µor. however, we could not measure any damgo-induced fret changes between yfp-tagged µor and mturquoise-tagged gβ in the presence of non-fluorescent gα i1 and gγ. this was unexpected because we had previously successfully determined interactions between gβγ and the α 2a -adrenergic receptor or the m 3 muscarinic acetylcholine receptor. this lack of fret was not due to an inability of the tagged gβ to interact with the µor as we could measure damgo-induced fret changes between yfp-tagged gα i1 and gβγ (gβ tagged with mturquoise) in the presence of non-fluorescent µor. moreover, when we attempted to establish an effect of grk2 on the interaction between the µor and gβγ, we could pick up fret between the µor and gβγ. comparison of the on-and off-kinetics of the µor-grk2 interaction with that of the µor-gβγ interaction in the presence of grk2 revealed similar time constants both for the on-and off-kinetics (grk2: k on 0.16 s ). this suggests that, in the absence of grk2, the orientation of the two fluorophores on the µor and gβγ may be unfavorable or the interaction may be too short-lived to produce an appreciable fret signal. in the presence of grk2, however, gβγ changes its position relative to the µor in a way that allows the interaction of the grk2-gβγ complex with the µor to be detected by fret. similarly, we measured fret between gβγ and the α 2a -adrenergic receptor or the m 3 muscarinic acetylcholine receptor in the absence and presence of grk2 and compared the kinetics with the kinetics of grk2 binding and unbinding to these receptors. in both cases, we found that grk2 and gβγ in the presence of grk2 associate and dissociate from these receptors with comparable kinetics. our results suggest that ligand efficacy for µors is already apparent on the level of receptor-grk interaction. institute of pharmacology, university medical center göttingen, ag lutz, göttingen, germany introduction: the monomeric gtpase rhoa is dysregulated in heart disease and in vivo models provide evidence of rhoa signaling being involved in the progression of cardiac fibrosis and hypertrophy. how rhoa is regulated within this context on a cellular level is not defined. objective: the goal of this study was to analyze rhoa activation in adult cardiomyocytes (amcm) from normal and diseased mouse hearts in response to g protein-coupled receptor activation. this project also aimed at providing new insight into the dependence of rhoa localization and activation on the signaling organizing caveolae in neonatal as well as adult cardiomyocytes and engineered heart muscles. methods: cardiomyocytes from sham-operated c57bl/6 mice, from mice subjected to transverse aortic constriction (tac) or from neonatal rats were either directly fixed after isolation or cultured in the presence or absence of methyl-b-cyclodextrin (mβcd). for analyses of rhoa activation cells were treated with endothelin-1 (et-1) for 90 sec. cells were prepared for immunofluorescence analysis or lyzed for immunoblotting. imaging was performed using confocal microscopy. effects of mβcd were further studied in 3d engineered heart muscles (ehm) made from total neonatal rat cardiac cells. the contractile function of ehm was assessed in the organ bath and cells were studied in sections by immunofluorescence analysis. results: in amcm from sham mice active rhoa mainly localizes at the sarcolemma and is augmented in response to et-1 treatment. in tac-amcm the basal level of active rhoa is increased and surprisingly et-1 had no further effect. immunoblot analysis demonstrated that in tac-amcm rhoa expression was per se higher and the major caveolae protein caveolin-3 was reduced. to test the influence of caveolae on rhoa activation and expression, we treated nrcm with mβcd and found the expression of rhoa as well as of rhoa target genes ccn1 and ccn2 to be moderately up-regulated after 24h. in addition, an intensified longitudinal alignment of sarcomeric actin fibers was detectable, which could also be seen in mβcd-treated ehm. however, mβcd had no effect on ehm twitch tension but increased the resting tension compared to control. we further treated amcm with mβcd and found rhoa expression to be increased and its activity less sensitive to et-1 treatment. finally, we could show that the perinuclear localization of cav3 and rhoa was strongly reduced after mβcd treatment. whereas g-protein coupled receptors (gpcrs) have been long believed to signal through cyclic amp only at cell surface, our group has previously shown that gpcrs not only signal at the cell surface but can also continue doing so once internalized together with their ligands, leading to persistent camp production (1). this phenomenon, which we originally described for the thyroid stimulating hormone receptor (tshr) in thyroid cells, has been observed also for other gpcrs (2) (3) (4) . however, the intracellular compartment responsible for such persistent signaling was insufficiently characterized. the aim of this study was to follow by live-cell imaging the internalization and trafficking of tshr, tsh and effector proteins in thyroid cells. mouse primary thyroid cells were transfected with fluorescent-protein tagged tshr, g-proteins, nanobody specific for active g-proteins and/or subcellular markers by electroporation, stimulated with fluorescently labeled tsh and visualized using highly inclined thin illumination (hilo) microscopy. our results suggest that tsh is internalized in complex with its receptor and they traffic retrogradely via the trans golgi network (tgn). while we could not find any evidence of internalized tsh/tshr complexes activating g-proteins in early endosomes, we show that tsh/tshr complexes meet the intracellular pool of gαs in the tgn and activate it, as visualized in real-time by a nanobody specific for active gαs. upon acute brefeldin a-induced golgi collapse, the retrograde trafficking of tsh/tshr via tgn is hindered. bulk tsh stimulations in primary mouse thyroid cells isolated from transgenic mice expressing the camp sensor, epac1-camps, also show a significantly reduced camp production in the presence of brefeldin-a. these data provide evidence that internalized tsh/tshr complexes meet and activate g-proteins at the tgn, which might serve as the main platform of persistent camp signaling after receptor internalization. objective: sphingosine 1-phosphate (s1p) is generated by sphingosine kinase (sk)-1 and -2 and acts mainly as an extracellular ligand at five specific g protein-coupled receptors, denoted s1p 1-5 . after activation, s1p receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration methods and results: here we demonstrate that dexamethasone treatment lowered s1p 1 mrna and protein expression levels in rat mesangial cells measured by taqman® and western blot analyses. this effect was abolished in the presence of the glucocorticoid receptor antagonist ru-486. in addition, in vivo studies showed that dexamethasone downregulated s1p 1 expression in glomeruli isolated from c57bl/6 mice treated with dexamethasone (10 mg/kg body weight). functionally, we identified s1p 1 as a key player mediating s1p-induced mesangial cell migration. using boyden chamber assays, we could show that dexamethasone treatment significantly lowered s1p-induced migration of mesangial cells. this effect was again reversed in the presence of ru-486. conclusion: we suggest that dexamethasone inhibits s1p-induced mesangial cell migration via downregulation of s1p 1 . overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells (koch et al., biol. chem. 2015; 396: 803-12) . the g protein-coupled receptor mrgd is a receptor for angiotensin-(1-7) involving g alphas , camp, and phosphokinase a rationale: angiotensin (ang)-(1-7) has cardiovascular protective effects and is the opponent of the often detrimental ang ii within the renin-angiotensin system. although it is well-accepted that the g-protein coupled receptor mas is a receptor for the heptapeptide, the lack in knowing initial signalling molecules stimulated by ang-(1-7) prevented final verification of ligand/receptor interaction as well as the identification of further hypothesized receptors for the heptapeptide. objective: the study aimed to identify a second messenger stimulated by ang-(1-7) allowing confirmation as well as discovery of the heptapeptide's receptors. we identified camp as the second messenger for ang-(1-7). the heptapeptide elevates camp concentration in primary cells such as endothelial or mesangial cells. using camp as readout in receptor-transfected hek293 cells, we provided final pharmacological proof for mas to be a receptor for ang-(1-7). more important, we identified the g-protein coupled receptor mrgd as a second receptor for ang-(1-7). consequently, the heptapeptide failed to increase camp concentration in primary mesangial cells with genetic deficiency in both mas and mrgd. furthermore, we excluded the ang ii type 2 receptor at2 as a receptor for the heptapeptide, but discovered that the at2 blocker pd123119 can also block the mas and mrgd receptors. conclusions: our results lead to an expansion and partial revision of the reninangiotensin system, by identifying a second receptor for the protective ang-(1-7) but excluding the at2 receptor, and by enforcing the revisit of such publications which concluded at2 function by using pd123119 as a specific at2 blocker. members of the g protein-coupled receptor (gpcr) superfamily are integral membrane proteins that are activated by extracellular ligands and induce cell signaling via g proteins and other adaptor proteins. rhodopsin, the prototypical gpcr that mediates vision, is activated by photons that isomerize its covalent ligand. spectroscopic analyses of the cognate agonist retinal allow a detailed description of rhodopsin dynamics at submillisecond resolution. using rhodopsin as a model, it has been demonstrated that receptor activation, i.e. a switch from the fully inactive to the fully active state, occurs within 1 ms 1 . activation kinetics of other receptors have been studied mainly using fluorescence resonance energy transfer (fret) which allows kinetic studies with high resolution in living cells 2 . in contrast to rhodopsin, activation constants of several gpcrs using agonist superfusion have been determined to range between 30-80 ms 3 . however, it is unknown if all gpcrs with diffusible ligands are really activated on a longer timescale or if ligand diffusion to the binding site is rate limiting. in this study, we intend to overcome ligand diffusion by using photodestruction of caged ligands. we monitor activation-related conformational changes of homodimeric metabotropic glutamate receptor 1 (mglur1) sensors by fret after uncaging of an inert glutamate derivative. 4-methoxy-7-nitroindolinyl-l-glutamate (mni-glutamate) is a caged derivative of glutamate that does not activate mglur1s. upon pre-incubation of hek-tsa cells expressing both cfp-and yfp-tagged mglur1 protomers with mniglutamate, a short uv laser pulse releases active l-glutamate close to the receptor binding site. we demonstrate very rapid mglur1 activation kinetics and this allows us to study the process of signal transduction of this homodimeric gpcrs opioids are still the mainstay of modern pain treatment. most of the clinically established substances primarily exert their effects via the µ-opioid receptor (mor). however, many side effects such as tolerance, constipation and respiratory depression limit their therapeutic use. the efficacy of mor agonists in the treatment of chronic pain is unsatisfactory. in general analgesic effects can be mediated by all four members of the opioid receptor family. the nociception receptor (nop) is the latest member of the opioid receptor family. there is a rapidly growing interest for the development of novel nop and combined mor/nop agonists. the aim of this developement is novel therapeutic agents with improved analgesic characteristics and less classical mor-mediated side effects. here we used buprenorphine as a clinically established opioid which exerts its effect on multiple opioid receptor subtypes. recently, nalfurafine, a potent kappa-opioid receptor (kor) agonist was granted by japanese authorities for the treatment of uremic pruritus. even though kor agonists are known to mediate dysphoria and hallucinations this has not been reported for nalfurafine. rudolf-virchow-zentrum für experimentelle biomedizin, würzburg, germany g protein-coupled receptors (gpcrs) belong to a superfamily of cell surface signaling proteins that mediate many physiological responses to hormones and neurotransmitters. they represent the prime targets for therapeutic drugs in healthcare. however, due to the limited knowledge about the pharmacology of the majority of gpcrs, only few of them are employed as therapeutic target. in our lab, the activation kinetics of the α 2aadrenergic receptor, among others receptors, has been extensively studied in single cell assays (1) (2) . the activation kinetics of the labeled α 2a -adrenergic were monitored by förster resonance energy transfer (fret). the goal of our study is to design a sensor to monitor receptor activation kinetics in high throughput screening assays. for the proof of concept, we used the α 2a -adrenergic receptor as a prototype. to optimize the fret efficiency we exchanged the previous acceptor (yfp) with the halotag technology (3) . additionally, we used halotag in combination with the nanoluc (4) to explore the possibility of using bret as a high-throughput approach to monitor receptor activation kinetics. the fret-based sensor α 2a -halo/cfp showed an increase in fret upon application of the full endogenous agonist norepinephrine with an ec50 value in accordance with the previously published data. this suggests the functionality of the fret-based α 2a -halo/cfp sensor. similar results were obtained with the α 2a -adrenergic receptor bret-based sensor. in contrast to the full agonist norepinephrine, the inverse agonist, yohimbine, decreased the ratio in both, fret as well as bret-based α 2aadrenergic receptor sensors. this suggests the sensitivity of the methods to discriminate among agonist (increased ratio) and antagonist (decreased ratio) induced receptor kinetics. our results show the feasibility of using halotag to monitor receptor activation via fret in a single cells format and halotag, in combination with nanoluc, can be used to monitor receptor activation in high-throughput format. , c. hoffmann the homologous visual arrestins) that has recently been solved by x-ray crystallography. here we investigated both the interaction with gpcrs and β-arrestin conformational changes in real time and in living cells with a series of fret-based β-arrestin2 biosensors. upon stimulation, β2-adrenergic receptors bound β-arrestin2 with a time constant τ = 1.3 ± 0.17 s, indicating that β-arrestin2 binding rapidly terminates their gprotein signaling. we observed a subsequent receptor-mediated conformational change in β-arrestin2 with a τ = 2.2 ± 0.22 s. stimulation of β2-adrenergic vs. m2 muscarinic or ffa4 receptors resulted in different patterns of conformational changes in the various β-arrestin2 sensors and also in downstream kinase signaling, revealing receptor-specificity in β-arrestin2 activation. upon agonist removal, first the interaction (delay = 1.9 ± 0.51 s) and only then the active state of β-arrestin2 (delay = 4.2 ± 0.85 s) were reversed. accordingly, β-arrestin localization at the cell membrane lasted much longer than the direct interaction with β2-adrenergic receptors. our data indicate a rapid, receptorspecific, two step binding and activation process between gpcrs and β-arrestins; they further suggest that β-arrestins remain active following dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. thus, gpcrs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signaling. patghogenic clostridium difficile produce two large glucosyltransferases, tcda and tcdb, which are the main pathogenicity factors. the cytotoxin tcdb is about 1,000 fold more potent than tcda. tcda and tcdb are a/b structure toxins exhibiting an enzymatically active (a) domain and a binding/translocation domain (b) to deliver the active glucosyltransferase domain into the cytosol of host cells. beside its glucosyltransferase activity by which the substrate proteins mainly of the family of rho gtpases are inhibited, tcdb has additional cytotoxic effects that are independent of rho glucosylation. to investigate the mechanism by which tcdb induces early cell death, we applied chimeras of tcdb from different toxinotypes where different glucosyltransferase domains were combined with different translocation domains. to this end we cloned tcdb from strain vpi10463 (historical strain), strain 1479 (serotype f, variant tcdbf), strain r20291 (hypervirulent strain, ribotype o27), and strain r9385 (hypervirulent strain with tcdbf characteristics). we were able to investigate the impact of the glucosyltransferase domains with different substrate specificity when translocated into the host cell by identical translocation domains. furthermore, we tested different translocation domains to deliver the same glucosyltransferase domain into host cells. we found that the glucosyltransferase domain of tcdbf (strain 1470) is less cytotoxic with respect to early cell death mediated by reactive oxygen species than that from reference strain vpi10463. in addition, the translocation domain also showed significant impact on cytotoxicity, probably by faster intracellular delivery of the gtd. by using glucosyltransferase deficient mutants where the highly conserved dxd-motif was changed to nxn, we were able to show that glucosylation of rho gtpases counteracts the cytotoxic effect, since the mutants were more cytotoxic than wildtype toxins. in conclusion, the cytotoxicity of tcdb mainly depends on the translocation efficiency into the host cell and on the kinetic of glucosylation of their substrate gtpases. thus, sensitivity of target cells towards cytotoxic effect also depends on receptor abundancy and intracellular status of rho gtpases, whereas the cytopathic effect, i.e. cell rounding, is predominatly determined by the substrate specificity. introduction: p63rhogef activates the g protein-coupled receptor (gpcr)-mediated induction of rhoa in different cells. however, its role in cardiac fibroblasts (cf) is not defined yet. thus we studied its localization and function in cf in 2d and 3d culture experiments. methods: neonatal rat cardiac fibroblasts (nrcf) and adult ventricular fibroblasts (amcf) from wild type mice and p63rhogef-knockout mice were adenovirally transduced for 48 to 72 h with recombinant adenoviruses or directly used. for 2d studies the cells were treated with angiotensin ii (ang ii). the location of the involved signaling components, rhoa activation and down-stream effects were studied by confocal microscopy and biochemical analyses. in addition, cf were used to prepare cf-containing engineered connective (ect) or muscle (ehm) tissues. results: we could show that p63rhogef locates at the plasma membrane, adjacent to the golgi apparatus and at the base of primary cilia. in accordance, p63rhogef regulates in response to ang ii the expression and secretion of the connective tissue growth factor (ctgf) in nrcf involving the serum response factor. in ect, p63rhogef increases the stiffness of these tissues and in ehm containing cf expressing gain-and-loss-of-function p63rhogef variants it influences the contractility. interestingly, the increase in ect stiffness was independent of p63rhogef's regulatory function of ctgf, as overexpression of ctgf in cf had no impact on ect properties arguing for a more general role of p63rhogef in auto-and paracrine signaling. moreover, our data on amcf with a genetic deletion of p63rhogef implies that p63rhogef is not only a transducer of gpcr-dependent rhoa activation as its loss led to an increase in rhoa expression accompanied by an increase in rhoa-dependent gene expression suggesting a role of p63rhogef in the feedback regulation of this signaling cascade. conclusion: in summary, our data show that p63rhogef regulates auto-and paracrine signaling in cardiac fibroblasts. the atrophic rhinitis is characterized by a drastic destruction of nasal turbinate bones in different animals. it leads to shortening and twisting of the snout and a growth retardation of young pigs. this bone degradation is induced by pasteuralla multocida toxin (pmt), a toxin produced by p. multocida serogroups a and d. this destructive effect indicates an interaction of pmt with bone cells like osteoclasts and osteoblasts. we demonstrated that pmt stimulates the differentiation of osteoclasts and inhibits the differentiation of osteoblasts in a gq-dependent mechanism. the underlying molecular mechanism of the toxin is the deamidation of an essential glutamine residue in the αsubunits of heterotrimeric g proteins, which results in the constitutive activation of the g protein. until now only the function and the pmt-dependent effects on osteoblasts and osteoclasts were studied in detail, but there is also a third important cell type in bone, the osteocytes. osteocytes are discussed as the regulator of the bone turn-over by interacting with osteoclasts and osteoblasts e.g. via secretion of several osteoclastogenic and osteoblastogenic cytokines. therefore, we studied the effects of pmt on the function of osteocytes in more detail. we utilized an osteocyte like cell line and primary osteocytes isolated from tibiae and femurs from mice. the susceptibility of primary osteocytes and the osteocyte like cell line towards pmt was demonstrated by detection of toxin-induced deamidation of g proteins. we also observed a pmt-induced secretion of different cytokines, like rankl, il-6 and tnf-α, which are known to induce osteoclastogenesis or inhibit osteoblastogenesis. furthermore, we studied the underlying signal transduction pathways and other pmtinduced effects on osteocytes, like morphological changes. in summary, we show that pmt acts on osteocytes by stimulating heterotrimeric g proteins. this might have impact on overall bone metabolism due to modulation of osteoblast and osteoclast activity. pasteurella multocida is an opportunistic pathogen often residing in the nasal pharyngeal space of animals. one virulence factor of p. multocida serogroups a and d is the protein toxin pmt (p. multocida toxin), which is the causative agent of atrophic rhinitis characterized by degradation of nasal turbinate bones in pigs and other animals. on the molecular level, pmt activates distinct members (g q/11 , g 12/13 and g i ,) of heterotrimeric g proteins leading to a modulation of bone metabolism: the toxin stimulates osteoclastogenesis but blocks osteoblastogenesis which results in bone loss. this mechanism of action of pmt might be exploited to counteract the human disease fibrodysplasia ossificans progressiva (fop), a rare and highly disabling disorder of extensive heterotopic bone growth. the underlying cause of fop is a point mutation in the activation domain of acvr1 (r206h), a bmp (bone morphogenic protein) type 1 receptor. this mutation leads to an inflated bmp-signaling and heterotopic osteoblastogenesis. here, we report that c2c12 cells, a mouse myoblast cell line often used as a fop model, are susceptible to pmt intoxication. pmt induces deamidation of g proteins in these cells. furthermore, pmt very efficiently inhibits bmp4-induced osteoblast differentiation in c2c12 cells. this has been shown by measuring alkaline phosphatase expression which is an early marker of osteoblast differentiation. additionally, the impact of pmt on acvr1 r206h induced osteoblastogenesis will be investigated and the involved cellular signaling pathways will be characterized in detail. the data indicate that activation of heterotrimeric g-proteins might be a rationale for pharmacological therapy of fop. p63rhogef is an activator of the monomeric gtpase rhoa and was shown to be expressed in the heart. in cardiac fibroblasts and smooth muscle cells, p63rhogef regulates rhoa in response to angiotensin ii and controls the actin cytoskeleton as well as protein expression and secretion. its role in cardiomyocytes, however, has not been elucidated so far. cardiomyocytes were isolated from neonatal rat hearts (nrcm), wild type mouse hearts (wt-amcm) and homozygous (ko-amcm) p63rhogef knockout mouse hearts. the cells were either directly fixed or adenovirally transduced for 48 h in culture. for activation of the g q/11 -signaling the cells were treated with endothelin-1 (et-1), angiotensin ii (ang ii) or phenylephrine (pe) for 90 s. rhoa activation was assessed by affinity binding or with a specific active-rhoa antibody. other proteins were detected by immunoblot or immunofluorescence analysis with subsequent confocal imaging. in nrcm p63rhogef is involved in the regulation of the et-1-induced rhoa activity and thus increases the expression and secretion of the rhoa target gene ctgf. in accordance, p63rhogef was found to be localized at the sarcolemma as well as in intracellular membrane compartments. the strongest co-localization was detected with the kdel-receptor 3 (kdelr3) which resides in the endoplasmatic reticulum membrane. next, we analyzed rhoa activation in wt and ko-amcm and could show that a loss of p63rhogef led to an increase in basal rhoa activity and an uncoupling from the gpcrs. interestingly, in the ko-amcm caveolin-3 the major component of caveolae, in which several gpcrs are clustered, was reduced in its expression and a shift in localization from transverse to longitudinal membrane tubules was found, arguing for a role of p63rhogef in intracellular protein transport. in accordance, golgi apparatus particles, which were demonstrated to play role in caveolae formation, were reduced in size in ko-amcm. to further address the role of p63rhogef in the transport of membrane proteins, we overexpressed p63rhogef in wt-amcm and could show that this led to an increase in the expression of the kdelr3 and its co-localization with p63rhogef in the perinuclear region and at the sarcolemma. no sarcolemmal localization of kdelr3 was found in control-transduced cells. further, p63rhogef was localized adjacent to golgi apparatus particles which were similar reduced in size as detected in the ko-amcm. finally, we expressed the dominant negative construct p63dn and detected similar changes with respect to kdelr3 localization and golgi structure suggesting that this regulatory function of p63rhogef is not dependent on its activity. conclusion: p63rhogef mediates the activation of rhoa from gpcrs coupled to g q/11 proteins. moreover, it has a function in intracellular transport and distribution of membrane proteins independent of its activity. universität bonn, pharmacology and toxicology section, bonn, germany g protein signaling is a means allowing cells to quickly respond and adapt to environmental changes. four major g protein classes (gs, gi/o, gq/11, g12/13) exist in mammals and these must suffice to convey signals from about 800 g protein-coupled receptors to the cell interior. as such, g proteins receive, interpret, and finally route the gpcr signals to diverse sets of downstream target proteins and thereby permit cells to respond to their ever changing environment. 1 understanding contribution of individual g protein classes or even isoforms to complex signaling networks in living cells requires capacity to activate or inactivate proteins with great precision and selectivity. one approach towards inactivation of g protein function is via chemical inhibition. however, "true specificity" of chemical inhibitors for their associated targets may often be debated. in this study we posit that fr900359, a cyclic depsipeptide isolated from the leaves of ardisia crenata, may clearly be designated as "truly specific" for inhibition of gq signaling. using a broad set of complementary methods based on label-free holistic cell sensing, classical endpoint assays, and bioluminescence resonance energy transferbased g protein biosensors we assign exceptional selectivity to fr for inhibition of gq/11/14 over all other mammalian isoforms ("on-target effects"). in holistic label-free recordings using hek293 cells that lack functional gq/11 alleles by crispr-cas9 genome editing, bona-fide gq stimuli were undetectable. however, reintroduction of gq into the knockout background was required and sufficient to fully restore both, agonist responses and their inhibition by fr. moreover, fr was completely ineffective in cells lacking gq/11 using phenotypic assays that examine basic cellular functions such as cell growth, viability, morphology and expression of housekeeping genes ("off-target effects") 2 . from these results we conclude that fr is of outstanding value as molecular probe to unravel contribution of gq signaling in complex biological processes in vitro, ex vivo and in vivo. just as pertussis toxin, applied world-wide by numerous laboratories to diagnose signaling of gi/o proteins, we anticipate fr to stand out at least equally for investigations into the biological relevance of gq. binary actin adp-ribosylating toxins like c. perfringens iota toxin and c. difficile transferase cdt cause depolymerisation of the cortical actin cytoskeleton, induce the formation of microtubule-based cell membrane protrusions and redirect rab-dependent intracellular traffic (schwan et al. 2009 ). here, we employed the model of toxin-induced protrusions to study the formation of cilia. we found that toxin-induced microtubule-based protrusion formation at the cell membrane depends on recruitment of septins, which are highly conserved, small gtpbinding proteins. similar to toxin-caused protrusions, septins are also recruited to the site of ciliogenesis. inhibition of septins by shrna-based knockdown inhibits ciliogenesis as well toxin-induced protrusion formation. septins are suggested to be involved in exocytotic processes, which are important for ciliogenesis and also for toxin-induced protrusion formation. accordingly, translocation of septins is accompanied by a recruitment of rab proteins and proteins of the exocytotic machinery. the data indicate that septins function as a scaffold at the base of cellular processes like cilia and toxin-induced protrusions, organizing the cross-talk between the actin cytoskeleton and microtubules to regulate the vesicle traffic-and exocytotic machinery. hypervirulent clostridium difficile strains are associated with increased morbidity and mortality. these strains produce the actin-adp-ribosylating clostridium difficile toxin cdt. cdt depolymerizes the actin cytoskeleton, causes formation of microtubule-based protrusions and increases pathogen adherence. septins are essential for cdt-induced protrusion formation. sept2, 6, and 7 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. the septins are a prerequisite for protrusion formation. the inhibitor forchlorfenuron or knock-down of septins inhibit protrusion formation. septins colocalize with active cdc42 and its effector borg which act as up-stream regulators of septin polymerization. microtubules interact with septin structures. precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to microtubule plus end tracking protein eb1 thereby guiding incoming microtubules. the data indicate that cdt hijacks conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, cdc42, borgs and restructuring of the actin cytoskeleton. the zebrafish danio rerio has become an important vertebrate model organism for a wide range of scientific questions [1] . current studies are mainly focused on development, genetics and disease for which the zebrafish is particularly well suited due to its small size, rapid development, short generation time, optical transparency of embryos and larvae as well as conservation in functional domains [1] . hitherto, nothing is known about the composition and endogenous level of different cnmps in various developmental stages and organs of danio rerio. therefore, we used the zebrafish in our study as a vertebrate model to characterize systematically the temporal-and organspecific occurrence(s) of all cnmps including cump in vivo. cyclic nucleotides were quantified by high performance liquid chromatography quadrupole tandem mass spectrometry. we observed specific cnmp patterns in developmental stages and different organs from adult zebrafish, which is in support of the hypothesis of a distinct cnmp signaling code [2] . camp, cgmp and cump were present in tissue samples of both developmental stages (embryos at 24 hours post fertilization, larvae at 5 days post fertilization) and within all harvested organs. remarkably, these three cnmps were the only ones detected in the brain. camp concentration of entrails as well as camp and cgmp concentrations in the brain were similar to those previously described in mouse tissues [3] . ccmp was detected throughout development and was present in all organs except the brain. the identity of ccmp and cump in the zebrafish was confirmed by high performance liquid chromatography quadrupole time-of-flight mass spectrometry (hplc-ms/tof). thus, we unequivocally show for the first time that cump occurs in vertebrates. furthermore, we detected cimp in several developmental stages of the zebrafish, and observed the highest concentrations in testes and heart, but we were unable to unequivocally identify cimp via hplc-ms/tof. in the zebrafish, sac is evolutionarily not conserved and absent, since a search in the ncbi gene data base revealed no entry for sac (also referred to as ac10). therefore, sac can be excluded as cump-and ccmp generator in this system and sgc remains as the only bona fide cump-and ccmp generator in the zebrafish. to test this hypothesis, the effects of no donors, sgc stimulators and sgc activators on cump levels in zebrafish should be examined in future studies. recently, induction of apoptosis in mouse lymphoma cells by ccmp-am has been described [4] . thus, it would be interesting to examine the effect of ccmp-am on zebrafish embryos in future studies as well. [1] seifert, r.: ccmp and cump: emerging second messengers, trends in biochemical sciences 40, 8-15 (2015) . ccmp, 3',5'-cump and 3',5'-cimp were ineffective. to further characterize the action of 3',5'-cgmp on hut-78 cells, we determined apoptosis (propidium iodide/annexin v staining) and proliferation (carboxyfluorescein succinimidylester staining). 3',5'-cgmp significantly increased apoptosis (ec 50 = 75 µm) and inhibited proliferation (ec 50 = 63 µm) of okt3-activated hut-78 cells. interestingly, also 2',3'-cgmp exhibited comparable effects on apoptosis and proliferation with ec 50 values of 92 µm and 75 µm, respectively, while 2',3'-camp, 2',3'-ccmp and 2',3'-cump were ineffective. this indicates that the pro-apoptotic and antiproliferative action of cgmp does not depend on the position of the phosphodiester bond. we also tested 3',5'-cgmp degradation products under the same experimental conditions and found that both 5'-gmp and guanosine increased apoptosis and inhibited proliferation with ec 50 -values between 50 and 100 µm. by contrast, adenosine did not influence cell growth and viability, suggesting that adenosine receptors are not involved in the observed effects. our results suggest that the guanosine moiety is responsible for the pro-apoptotic and antiproliferative effects of 3',5'-cgmp, 2',3'-cgmp, 5'-gmp. it has been reported earlier that guanosine is toxic to jurkat cells, another t cell lymphoma cell line [1]. 3',5'-cgmp may be hydrolyzed by an ekto-phosphodiesterase on the cell surface of hut-78 cells (e.g. enpp1), yielding 5'-gmp, which could be further degraded to guanosine by the 5'-ekto-nukleotidase cd73. a similar pathway may lead from 2',3'-cgmp to guanosine. a previous analysis of phosphodiesterases (pdes) revealed that the dual-specific pde isoforms 3a and 3b as well as the cgmp-selective pde9a also degrade the emerging second messenger cump [1, 2] . we analyzed the enzyme kinetics of pde3b-mediated cump-hydrolysis using recombinant gst-tagged pde3b and a highly sensitive and specific hplc-ms/ms method. our data show that pde3b is a low-affinity enzyme for cump with a cump k m -value of >100 µm. the pde3-selective competitive inhibitor milrinone inhibited pde3b-mediated cump degradation, suggesting that cump binds to the catalytic center. pde3b is highly expressed in adipose tissue [3, 4] . thus, we differentiated murine 3t3-l1-mbx-fibroblasts into adipocytes and analyzed differentiation-dependent alterations of pde3b expression and basal cnmp-concentrations. in both differentiated and undifferentiated 3t3-l1 cells cump and ccmp were detected in addition to the established second messengers camp and cgmp. differentiation to adipocytes reduced camp and cgmp by 66 % and 60 %, respectively, while ccmp was reduced by 78 % and cump even by 85 %. these findings suggest that cump plays a distinct role in adipocyte differentiation. the cump-hydrolyzing pde3b was upregulated ~1000-fold on mrna level after adipocyte differentiation, which may contribute to the observed reduction of basal cump concentrations. we currently investigate the potential biological role of cump in differentiation and lipolysis experiments, analyzing the effects of the membrane-permeant cumpacetoxymethyl ester cump-am. in future experiments, we will also analyze the enzyme kinetics of pde9a-mediated cump hydrolysis. pde9a is the first example of a cgmp-"specific" cump-hydrolyzing pde. background: cgmp and camp are cyclic nucleotide messengers relevant to many physiological and pathophysiological conditions. live-cell imaging with fret-based biosensors is a powerful method to study the spatiotemporal dynamics of cgmp and camp under close-to-native conditions. however, with the existing biosensors it is difficult to resolve potential membrane-associated cgmp microdomains and to monitor cgmp and camp signals in parallel in the same cell. we have generated novel versions of the "green" cfp/yfp-based cytosolic cgmp biosensor, cgi500. they comprise a "green" membrane-targeted version (mcgi500) and a "red" variant (red cgi500) that contains the fluorophores tsapphire and dimer2. methods: the sensors were expressed and characterised in primary vascular smooth muscle cells (vsmcs). intracellular cgmp was elevated in intact vsmcs by application of a nitric oxide donor or natriuretic peptides, and the sensor's sensitivity to each stimulation and its signal-to-noise ratio were determined. to test each sensor's sensitivity and specificity for cgmp versus camp, sensor-expressing cells were permeabilised with β-escin and exposed to defined concentrations of cyclic nucleotides. results: the original cgi500 sensor showed a good signal-to-noise ratio, an ec 50 value of ≈1.1 µm for cgmp, and a high selectivity for cgmp over camp (>100-fold). flincg3, a non-fret-based cgmp sensor, showed similar properties as cgi500. the new membrane-targeted mcgi500 and the new red cgi500 displayed ec 50 values of ≈0.4 µm cgmp and a high selectivity for cgmp over camp (>300-fold). in vsmcs, the red cgi500 showed a better signal-to-noise ratio than the previously described "red" cgmp sensor, red cges-de5. the "green" fret-based camp sensor, epac1-camps, showed a signal-to-noise ratio comparable to that of cgi500, an ec 50 value of ≈3 µm for camp and a selectivity for camp over cgmp of ≈6-fold. finally, imaging of cells expressing both the epac1-camps and the red cgi500 demonstrated the feasibility of combined visualisation of camp and cgmp signals in the same cell. the new cgmp biosensors should be useful for a broad spectrum of applications requiring real-time monitoring of cgmp signals. for example, mcgi500 would be useful to investigate membrane-associated cgmp compartments and red cgi500 to study the crosstalk between cgmp and camp signalling in living cells and tissues. the cgmp-system is a major regulator of blood pressure. cgmp-dependent protein kinases (cgks), located in the smooth muscle layer of vessels, enable cells to dilate and therefore cause a decrease in blood pressure (bp). to the contrary, the reninangiotensin-aldosteron-system (raas) acts as opponent and causes an increase in bp; furthermore, it influences fluid-electrolyte balance. renin, which is secreted from reninproducing cells located in the juxtaglomerular apparatus (jga), is the key regulator enzyme in this system. pharmacological inhibition of the raas, e.g. via ace-inhibitors or at1-receptor-antagonists, is a powerful tool to treat hypertension, but chronically challenges this endocrine system, resulting in an enhancement of renin expression. this is caused by an increased number of renin-expressing cells (the so-called reninrecruitment), which derive from a reversible metaplastic retransformation of extraglomerular and smooth muscle cells of afferent arterioles. next to regulation of renin-function via camp/pka, it has been shown that enos-derived no supports this recruitment via activation of sgc and subsequent generation of cgmp [1] . whether this causes an activation of cgks is not known. these enzymes exist in 3 different isoforms, cgkiα, cgkiβ und cgkii. in contrast to the β-isoform, cgkiα (as well as cgkii) is highly expressed in the jga [2] , [3] . therefore, we analyzed, whether cgkiα also plays a role regarding renin synthesis, secretion or recruitment. to characterize the function of cgkiα in jga-cells, we generated renin-cell specific cgkiα-knockout mice (ren cre-cgki fl/fl) and stimulated renin recruitment via administration of a low salt diet (0.02% na + ) and enalapril (10mg/kg/d) for 3 weeks. we analyzed blood pressure, mrna and renal protein content of renin and cgkiα, plasma renin activity and renin recruitment. furthermore, we activated the cgmp-system in these mice using bay41-8543, a sgcstimulator, and re-analyzed the above-mentioned parameters. our results indicate that cgkiα could be an additional system supporting renin recruitment but is not a crucial pre-requisite. in contrast, the basal renin concentration and activity appears to be downregulated in ren cre-cgki fl/fl-mice, thus, cgki could be an important regulator of renin synthesis. universität regensburg, pharmakologie und toxikologie, regensburg, germany jaw1/lrmp (lymphoid-restricted membrane protein) is a type 2 membrane protein, localised to the cytoplasmic face of the endoplasmic reticulum. it encodes a 539 amino acid protein with a highly conserved coiled-coil domain in the middle third of the protein and a cooh-terminal transmembrane domain [1] , [2] . jaw1 and irag have a limited homology throughout the length of the protein. the coiled-coil domain and the putative transmembrane anchor at the c-terminus of jaw1 and irag share the highest homology [3] , [4] . the coiled coil domains of irag and jaw1 are important for the interaction with ip 3 rs. as already known, irag forms a trimeric complex with cgkiβ and ip 3 r1 and gets phosphorylated by cgkiβ [5] . hence we examined if jaw1 is a new target protein of cgkiβ. the recognition site, where a substrate can be phosphorylated by cgki, is composed of the following amino acids: (k/r)(k/r)x(s/t). in the amino acid sequence of jaw1 possible phosphorylation sites can be found. our in vitro studies with jaw1 and cgki showed that jaw1 gets phosphorylated in a cgmp-dependent manner by cgkiβ. in contrast, jaw1 was not phosphorylated by cgkiα. furthermore, no stable interaction between jaw1 and cgkiβ was detected. to examine the importance of jaw1 in vivo, we generated a conditional knockout mouse. mating with a cmv cre mouse, resulted in an ubiquitous deletion of jaw1. mrna analysis and western blot analysis approved the deletion. the expression pattern revealed high expression in the thymus and weaker expression in the lung, spleen, colon, pancreas and the tongue. as already published by shindo et al., jaw1 was found in sweet, bitter, and umami taste receptor-expressing cells of mouse circumvallate, foliate, and fungiform papillae. we confirmed these results by x-gal staining and mrna analysis. therefore, we decided to analyse if jaw1 influences taste perception. two bottle preference tests did not result in significant differences between wildtype and knockout mice, indicating that taste perception is not altered by jaw1. hence, the function of jaw1 in taste receptor expressing cells has to be further examined in future studies. the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (camp) and guanosine 3',5' cyclic monophosphate (cgmp) are well-characterized second messengers. both are generated by nucleotidyl cyclases and degraded by phosphodiesterases. several binding partners of camp and cgmp were already identified and functionally analyzed, e.g. camp-dependent protein kinase (pka) and cgmp-dependent protein kinase (pkg) as well as exchange protein activated by camp 1 and 2, hyperpolarization-activated cyclic nucleotide gated channels and phosphodiesterases. recent data indicate that the cyclic pyrimidine nucleotides cytosine 3',5'-cyclic monophosphate (ccmp) and uridine 3',5'-cyclic monophosphate (cump) also fulfill the criteria of second messengers [1, 2] . the interaction of ccmp with the regulatory subunits of pka (pkariα and pkariiα) has already been shown by using ccmpagarose [3] . additional ccmp-and cump-binding proteins such as calnexin (chaperone), myomegalin (phosphodiesterase-interacting protein) and akap9 (a-kinase anchoring protein) were identified by mass spectrometry analysis. to verify the interaction of ccmp and cump with these potential target proteins, ccmp and cump linked to biotin were used as another approach. the biotin-constructs exhibit lower steric interference than the ccmp-and cump-agarose matrices, which were previously used to confirm the binding of pkariα to ccmp and cump [4] . flag-tagged calnexin, flag-tagged myomegalin and myc-tagged yotiao (smallest splice-variant of akap9) were examined as potential ccmp and cump target proteins. hek293 cells were transiently transfected with the cdna of the respective proteins. the lysates of the protein-overexpressing cells were then incubated with ccmp-and cumpbiotin matrices and bound proteins were purified using strep-tactin® beads (iba). afterwards, the interaction of ccmp and cump with the potential binding partners was analyzed by western-blotting. a pkariα antibody was used as a positive control. analogous experiments were also performed using ccmp-and cump-agaroses. once the interaction between the cyclic pyrimidine nucleotides and the potential binding partners has been confirmed, deletion mutants will be cloned to localize the ccmp-and cump-binding area of the target proteins in further studies. axonal branching is essential for the correct formation of neuronal circuits and enables the simultaneous transmission of information throughout the body. in mice, the bifurcation of axons of sensory neurons at the dorsal root entry zone of the developing spinal cord depends on a cgmp signaling cascade that includes c-type natriuretic peptide (cnp), natriuretic peptide receptor 2 (npr2, also termed gc-b), and cgmpdependent protein kinase iα. in this study it was investigated, if a disturbance of cgmp signaling induced by manipulation of cgmp breakdown or cnp scavenging affects axon bifurcation of murine embryonic dorsal root ganglion (drg) neurons. rt-pcr screens, in situ hybridization, and fret-based cgmp imaging in living neurons revealed phosphodiesterase 2a (pde2a) as the major enzyme for degradation of cnp-induced cgmp in embryonic drg neurons. interestingly, cgmp measurements and dii labeling of pde2a knockout embryos indicated that a strongly elevated concentration of cgmp does not impair sensory axon bifurcation of drg neurons in vivo. the natriuretic peptide receptor 3 (npr3) was found to be expressed in the roof and floor plate of the spinal cord as well as in the dorsal roots of e12.5 embryos. because npr3 binds natriuretic peptides, but does not generate cgmp, it is thought to act as a natriuretic peptide clearance receptor. by scavenging cnp, npr3 could lower the activity of the cnp-npr2-cgmp signaling cascade in drg neurons. in the absence of npr3, the majority of sensory axons showed normal bifurcation, but »13 % of the axons turned only in rostral or caudal direction. this study shows (1) that pde2a is important for the degradation of cgmp in embryonic drg neurons, (2) that the bifurcation of sensory axons in the spinal cord can tolerate high levels of intracellular cgmp in the absence of pde2a, and (3) in the central nervous system, no-dependent cgmp signalling is associated with many different developmental processes and brain functions, and plays an important role in memory consolidation and cognition. to analyse cgmp signals in primary cells, a knock-in mouse was generated which stably and ubiquitously expresses a fret-based cgmp indicator (cgi500). cultured cortical and hippocampal neurons were found to respond to exogenously applied no (gsno). in these cell types, endogenous no is mainly generated by the neuronal no synthase (nnos) isoform which requires a rise in intracellular calcium for activation of the no/cgmp-signalling cascade. here, we show that ampa-type ionotropic glutamate receptors were capable to induce cgmp response in cultured cortical and hippocampal neurons. surprisingly, amparinduced cgmp signals were independent of nmdar activation, as inhibition of nmdars with the nmdar antagonist d-apv (d-2-amino-5-phosphonopentanoic acid) did not block ampar-induced cgmp response. however, cgmp accumulation depends on no synthase activation as the nos inhibitor l-nna (ng-nitro-l-arginine) completely abolished cgmp accumulation. whether ampar-induced nos activation depends on calcium influx via calcium permeable ampars, vgccs (voltage gated calcium channels) or calcium release from intracellular stores will further be investigated in detail. cyclic adenosine monophosphate (camp) is an important and ubiquitous cellular second messenger. a dogma in signaling is that camp is distributed homogenously in the cell and that its concentration changes equally upon stimulation. in contrast, a large body of evidence suggests the existence of concentration gradients (so-called microdomains) of camp. in this regard, phosphodiesterases (pdes), the only enzymes which can degrade camp, have been suspected to be responsible for maintaining those gradients. however, how pdes establish camp gradients is entirely unknown. here, we measure local camp levels in hek cells and cytosolic fractions thereof using the camp fretsensor epac1-camps fused to a phosphodiesterase (pde4a1). we demonstrate the existence of low camp concentrations in close proximity to pdes and show that this gradient is maintained by pde hydrolytic activity. further we establish that camp gradients cannot be maintained solely on the basis of pde activity as the camp turnover is very slow. we provide evidence that pdes are structurally organized in yet unspecified 'microstructures' in which camp diffusion must be considerably slowed down. taken together, we suggest that camp gradients are established by pde hydrolytic activity in cellular regions with slow diffusion of camp. our study sheds light on the organization and maintenance of signaling compartments in cells. the influence of pde hydrolytic activity on camp gradients universität würzburg, pharmakologie und toxikologie, würzburg, germany phosphodiesterases (pdes) are a family of enzymes that degrade cyclic amp (camp) and cyclic gmp (cgmp) to their respective monophosphates. although several pdes have been shown to play an important role in a wide variety of physiological and pathological processes, their complexity and function in cell signalling is only beginning to be understood. it is especially astonishing that eukaryotes express more than 100 different pde isoforms while their single function is to degrade camp, cgmp or both. in recent years, a large body of evidence has suggested that pdes (especially isoforms of the pde families 3, 4 and 5) are key players in establishing signalling compartments. these so-called microdomains are yet unspecified regions in cells where the concentrations of camp and cgmp are higher or lower than in the bulk cytosol. in a companion abstract (bock & lohse) we provide evidence that camp nanodomains, i.e. regions of low camp concentrations, exist in cells in the direct vicinity of pde4. however, the mechanisms by which pdes establish and maintain camp gradients are largely unknown. here we study if establishing camp gradients is a general role of pdes and, moreover, if the size of camp nanodomains mainly depends on pde hydrolytic activity. by fusing an ultra-fast pde2a3 (v max = 120 µmol/min/mg) to a camp fret sensor (epac1-camps) we monitor camp concentrations in direct vicinity of pde2a3. in comparison to pde4, we show that pde2a3, albeit displaying a high camp turnover, only establishes a small camp gradient in both cytosol preparations of transfected hek cells and in living cells. interestingly, this gradient can be increased by deleting the nterminal regulatory domains while maintaining fast camp turnover. biochemical mapping of the camp gradient gives an estimate of the size of nanodomains. taken together, our data suggest that establishing camp gradients does not exclusively depend on pde hydrolytic activity. arglabin is a plant-derived sesquiterpene lactone used for cancer therapy in kazakhstan and russia. signaling pathways targeted by arglabin are poorly understood. we have isolated arglabin by using high performance liquid chromatography from a methanolic extract of artemisia glabella, a plant endemic in kazakhstan. mass spectrometric analyses confirmed the chemical structure and the purity of the isolated arglabin. in j774 macrophages, arglabin strongly induced accumulation of lc3 type ii protein in the absence of inflammasome activators, and also in cells activated with lps and cholesterol crystals. in addition, arglabin induced clustering of lc3-ii at autophagosomal membranes, as evidenced from its punctuated pattern in confocal microscopic images of arglabin-treated macrophages, which is a characteristic sign for autophagy. since autophagy activation leads to increased degradation of nlrp3 and pro-interleukin (il)-1β, we further analyzed whether arglabin inhibits the nlrp3 inflammasome. arglabin reduced expression of nlrp3 and pro-il-1β, inhibited activation of caspase-1, and release of mature il-1β by lps-and cholesterol-crystal-stimulated macrophages consistent with inhibition of the nlrp3 inflammasome. intraperitoneal injection of arglabin into female apoe2.ki mice fed a high fat diet resulted in significantly decreased plasma levels of proinflammatory il-1β. moreover, arglabin markedly reduced mean lesion areas in the sinus and whole aorta in mice. thus, arglabin may represent a new promising drug to treat diseases associated with inflammasome activation, e.g. atherosclerosis. this work was supported in part by a grant from the nouvelle société francophone d'athérosclérose (nsfa). recently, we found that activation of the proteinase-activated receptor (par) 2 stimulates renin release in the isolated perfused kidney model. therefore, we determined in the current experiments the response of plasma renin concentration (prc) to acute intraperitoneal administration of the par2 activating peptide sligrl (100µg/kg), hydralazine (2 mg/kg), isoproterenol (10 mg/kg), losartan (3 mg/kg) and furosemide (40 mg/kg) in conscious wild-type (wt) and par2-deficient mice. prc was measured in plasma obtained by tail vein puncture. renal renin expression was determined by quantitative rt-pcr. renal protein expression was measured by immunohistochemistry. on a control diet (0.6% nacl), plasma renin concentration (in ng angiotensin i per ml per hour) was significantly lower in par2-deficient mice than in wild-type mice (190 ± 35 versus 380 ± 91). renin mrna expression was 50 ± 9% of wt. renin-expressing cells were located at the juxtaglomerular position and renal renin protein expression was lower in par2-deficient mice. as measured by tail-cuff method, systolic blood pressure was not different between par2 (-/-) and wt mice. administration of sligrl increased renin secretion about 6-fold (p<0.01). acute stimulation of renin release by furosemide, isoproterenol, losartan and hydralazine caused significant increases of plasma renin concentration in both par2 (-/-) and wt mice. the absolute changes (delta prc) were similar (3780 ± 770, 2230 ± 400, 2780 ± 70, 1390 ± 460 in wt, and 2320 ± 270, 2530 ± 250, 3010 ± 210, 1230 ± 470 in par2 (-/-)). in conclusion, chronic absence of par2 reduces basal renin expression and renin release. however, par2-deficiency does not alter renin release in response to typical stimuli for renin secretion. therefore, par2 does not appear to be a mandatory and specific requirement for acute regulatory responsiveness. increased myofilament ca 2+ sensitivity could be the underlying cause of diastolic dysfunction. we evaluated acute effects of epigallocatechin-3-gallate (egcg), which has been shown to decrease myofilament ca 2+ sensitivity, on cardiac myocyte contractility and force-ca 2+ relationship of skinned cardiac muscle strips in an hcm mouse model with left ventricular hypertrophy and both systolic and diastolic dysfunction. methods: the hcm mouse model used in this study carries a point mutation in the cardiac myosin-binding protein c gene at the homozygous state (mybpc3-targeted knock-in; ki). we isolated ventricular myocytes from adult ki and wt mice and analyzed sarcomere shortening and ca 2+ transients at 37 °c under 1 hz pacing using the ionoptix system in the absence or presence of egcg (1.8 µm). furthermore, force-ca 2+ relationships of skinned cardiac muscle strips of ki and wt mice were obtained ±egcg (30 µm). results: in baseline settings and absence of fura-2, ki cardiomyocytes displayed higher sarcomere shortening ( type-1 serine/threonine protein phosphatase (pp1) comprises a family of enzymes that dephosphorylate cardiac regulatory proteins, thereby modulating ca 2+ handling and contractility. all pp1 heterodimers possess a catalytic subunit, which is selectively inhibited by inhibitor 2 (i-2). it has been shown by our group that the heart-directed overexpression of a truncated, constitutively active form of i-2 resulted in an improved basal ca 2+ handling and contractility. in contrast, chronic pressure overload by transverse aortic constriction exacerbated the progression of cardiac remodeling and heart failure in transgenic mice. in the present study, we tested whether the overexpression of a full-length form of i-2, regulated by gsk3-dependent phosphorylation at thr 72 , resulted in comparable functional alterations using a model of induced heart failure. for this purpose transgenic (tg) and wild-type (wt) mice were subjected to chronic application of isoprenaline (iso, 30 mg/kg/d) via osmotic minipumps. iso-stimulated mice were compared to mice treated with 0.9% nacl (n=5). after one week of iso administration, cardiac hypertrophy was comparable in tg and wt. ca 2+ transients were measured in isolated, indo-1-loaded myocytes. the peak amplitude of [ca] i was reduced by 39% in tg nacl compared to wt nacl (p<0.05), whereas chronic iso application was associated with comparable effects in tg and wt. [ca] i decay kinetics were comparable in nacl-treated groups but hastened by 25% in tg iso compared to wt iso (p<0.05). consistently, sr ca 2+ load was diminished by 28% in tg nacl compared to wt nacl (p<0.05). chronic iso stimulation led to an unchanged sr ca 2+ content in tg and wt myocytes. biochemical analyses revealed that chronic βadrenergic stimulation was accompanied by a more than 4-fold higher phospholamban phosphorylation at ser 16 in tg (p<0.05). thus, these findings suggest that overexpression of i-2 is able to reduce the progression of heart failure by an improvement of myocyte ca 2+ handling. the ligand-activated farnesoid x receptor (fxr) is a nuclear receptor highly expressed in gastrointestinal and metabolic tissues, such as the duodenum, jejunum, ileum, colon and the liver, but also in lower amounts for instance in macrophages. the endogenous agonists for this receptor are bile acids with the primary bile acid chenodeoxycholic acid as the most active one. activation of fxr regulates the transcription of target genes relevant in bile acid homeostasis, glucose and lipid metabolism, liver protection, inflammation, and cancerogenesis. agonists for fxr have been discussed as possible therapeutic options for the treatment of obesity and the metabolic syndrome. atherosclerosis is the main pathology underlying cardiovasular diseases and often occurs side-by-side with the metabolic syndrome. cholesterol deposition and the formation of cholesterol-loaded foam cells from macrophages lead to the formation of atherosclerotic plaques. this can be prevented by stimulation of cholesterol efflux from macrophages. based on leoligin, a lignan-type secondary plant metabolite, naturally occuring in leontopodium alpinum cass., 168 derivatives were synthesized and subjected to a fxr pharmacophore-based in silico screening. testing of 56 virtual hits in a luciferase-based fxr transactivation assay yielded one compound with promising activity on fxr. moreover, the heterodimer partner of fxr, rxrα, was not activated by this leoligin derivative in a luciferase-based rxrα assay. in addition, this compound was able to increase cholesterol efflux in thp-1 macrophages without affecting cell viability. western blot experiments revealed an increase in atp-binding cassette transporter a1 (abca1) expression in human thp-1 macrophages by this leoligin derivative. the transporters abcg1 and scavenger receptor class b (sr-bi), which also play a key role in macrophage cholesterol efflux, will be investigated. moreover, the effect of the leoligin derivative on liver x receptor activation, the nuclear receptor responsible for upregulation of these transporters, has to be studied. based on this data, further characterization of the molecular mechanism underlying the described effects will provide valuable insights in a possible crosstalk between macrophage cholesterol efflux and fxr activation. background: rho-associated kinases rock1 and rock2 are serine/threonine kinases that are downstream targets of the small gtpases rhoa, rhob, and rhoc. rock1 and rock2 are known to play a pivotal role in the pathogenesis of myocardial fibrosis. however, their specific function in cardiac fibroblasts (cf) remains unclear. remodelling of the diseased heart results in the transition of fibroblasts to a myofibroblast phenotype exemplified by an increased proliferation, migration rate and synthesis of extracellular matrix (ecm) proteins. therefore, we sought to investigate whether rock protein signalling intermediates have an impact on cellular characteristics, intracellular protein expression and mechanical properties in cf and engineered tissues. methods: neonatal cardiac fibroblasts were isolated from wild type rats and downregulation of rock1 and rock2 by 75% was achieved by lentiviral transduction or transfection. wild type fibroblasts were treated with 10 μm fasudil or 3 µm h1152p for general rock inhibition and 3 µm slx-2119 for inhibition of rock2. protein expression and modification was determined by immunoblot analysis, gene expression by qpcr analysis, cf morphology and the localisation of cytoskeletal proteins by immunofluorescence analysis, cell proliferation by automated nuclei counting, cell migration on a planar surface by life cell imaging, and rigidity of engineered tissues by rheological measurement. results: our results show that both rock1 and rock2 influence cf morphology, gene expression, proliferation and migration. the knockdown and inhibition of rocks was associated with changes in cf morphology accompanied by a disorganization of higher-order actin structures including stress fibers and geodesic domes. moreover, the knockdown of rock1 and rock2 in cf increased adhesion velocity, whereas proliferation was attenuated. interestingly, downregulation of rock2, but not of rock1 led to a significantly decreased migration velocity and distance suggesting an isolated principle role for rock2 in cardiac fibroblast migratory behavior. analysis of a three dimensional engineered tissue model composed of cardiac fibroblasts (engineered connective tissue, ect) suggested that rocks are involved in the regulation and turnover of the extracellular matrix (ecm) and thus influence viscoelastic properties of engineered tissues. destructive tensile strength measurement in ect treated with rock inhibitors showed that rigidity was significantly reduced when compared to control tissues. rna sequencing of ect treated with the rock inhibitor h1152p and qpcr analysis of cf with a downregulation of rock1 and rock2 showed that both rocks are involved in the regulation of ecm proteins, such as collagens 4a2, 6a, and 8a1, biglycan, decorin, elastin and its respective degrading enzyme mmp12. conclusion: this study demonstrates that rock signalling controls myofibroblast characteristics of cf via remodelling of the cytoskeleton and the ecm. background: regulation and fine-tuning of gene transcription in cardiomyocytes (cms) is a centerpiece of cardiac development, function, and disease. in order to obtain authentic data, cell type-specific analyses are indispensable. recently, high-purity isolation protocols for cm nuclei were established [bergmann, exp cell res, 2011] and employed for detailed genetic and epigenetic studies on cardiac gene transcription [gilsbach, nat commun, 2014] . however, corresponding protein analyses, which bridge from transcriptional control to cm function are still lacking. therefore, we aimed to map the landscape of nuclear protein expression in newborn and adult mice in order to complement and extend our epigenetic studies. methods and results: cardiac nuclei were isolated from homogenized adult and p1 mouse frozen hearts by sucrose gradient centrifugation. magnetic-assisted cell sorting (macs) with pcm-1 as a nucleus-specific marker was used to enrich cm nuclei to >95%. proteins were extracted from nuclear lysate with 2% sds. quantitative protein data were obtained from silac-based liquid chromatographytandem mass spectrometry (lc-ms/ms) experiments with an ltq orbitrap xl mass spectrometer after in-gel digestion with trypsin. nuclear protein extracts from 3 murine cell lines served as silac (lys8/arg10)-labeled internal standard. finally, protein data are correlated with corresponding mrna data obtained by rna-sequencing. we identified 1041 proteins, 688 of which are annotated to the nucleus. 21%/23% (adult / p1) of nuclear proteins are annotated as dna-binding. 1.5%/1.4% belong to transcription factor complexes; 7.3%/7.5% are able to bind transcription factors. 7.9%/8.3% have chromatin modification functions; 4.3%/4.4% modify histones. nuclearenriched go terms include mrna processing and transport, transcription, nucleosome assembly and protein degradation. 71% of proteins are shared between p1 and adult nuclei. 142 proteins are exclusive to p1, 34 are only found in adult. 93 proteins are enriched (1.3-fold increase in abundance) in p1 hearts, 89 proteins in adult proteins related to heart development, gene silencing, and dna replication are more prominent in or exclusive to newborn mice. adult nuclei strongly express proteins related to regulation of actin fibers and cm function, proteins involved in protein degradation, and chaperones. although high mrna expression increases the chance of protein identification, a significant correlation between mrna and protein level could not be observed on a genome-wide scale. conclusions: we present a comprehensive and specific protein landscape of newborn and adult cm nuclei. young cm nuclei appear as a developing tissue, show the ability for proliferation, and indicate ongoing alterations in gene expression. adult cm nuclei prominently display a focus on regulation of contractile fibers and cm function, as well as chaperones and proteasomal proteins indicative of its arduous function. background: fibrosis is a hallmark of many myocardial pathologies and contributes to distorted organ architecture and function. recent studies have identified premature senescence as regulatory mechanism of tissue fibrosis. however, its relevance in the heart remains to be established. objective: to investigate the role of premature senescence in myocardial fibrosis. methods: murine models of cardiac disease and human heart biopsies were analyzed for characteristics of premature senescence and fibrosis. results: senescence markers p21 cip1/waf1 , senescence-associated ß-galactosidase (sa-ß-gal) and p16 ink4a were increased 2-, 8-and 20-fold (n=5-7; p < 0.01), respectively, in perivascular fibrotic areas after transverse aortic constriction (tac) when compared to sham-treated controls. similar results were observed with cardiomyocytespecific β1-adrenoceptor transgenic mice and human heart biopsies. senescent cells were positive for vimentin (92 ± 0.9%), platelet derived growth factor receptor α (94 ± 0.9%) and α-smooth muscle actin (71 ± 2.3%), specifying myofibroblasts as the predominant cell population undergoing premature senescence in the heart. conclusion: our data provide first evidence for an essential role of premature senescence of myofibroblasts in myocardial fibrosis. it is tempting to speculate that pharmacologic modulation of premature senescence might provide a novel therapeutic target for anti-fibrotic therapies in the heart. introduction: the endocannabinoid system is increasingly studied in cardiac research due to its role in fibrosis, inflammation and cell fate modulation. the deregulation of this system has been implicated in myocardial infarction (mi) and consequent heart failure development. a recent study suggests cannabinoid receptor 1 (cb1) inhibition to improve cardiac function and to reduce adverse remodeling after cardiac stress, but the exact underlying molecular mechanisms of these beneficial effects are still unknown. micrornas (mirnas, mirs) provide a complex layer of post-transcriptional regulation modulating key biological processes such as tissue remodelling in heart failure. the aim of the present study was to explore microrna pathways in the chronic effect of cb1 receptor inhibition after angiotensin-fibrosis induction and left ventricular remodeling. methods and results: adverse cardiac remodelling was induced in mice by chronic administration of angiotensin ii (angii, 1,5 mg/kg/day) with osmotic minipumps for 14 days. treatment with cb1 antagonist, or vehicle was performed every second day during the angii administration period. hemodynamic parameters were measured by echocardiography and cardiac pressure volume catheter and tissue samples were taken for molecular and histological analysis. after two weeks of angii infusion, left ventricular dysfunction was prevented by cb1 antagonist treatment. this was shown by significantly improvements of the myocardial performance index and end-diastolic pressure values. at the tissue level, anti-fibrotic effects of cb1 antagonist treatment was confirmed histologically and by expression analysis of pro-fibrotic genes. these beneficial effects were also observed in cb1 ko mice and in an aging mice model. the particular role of tissue fibroblast in aii-induced cardiac fibrosis was further explored. primary cardiac fibroblasts (cf) from each experimental group were isolated and analysed by next generation deep rna sequencing to identify differentially regulated micrornas. microrna-181a/b family was downregulated by in vivo angii delivery and vice versa upregulated after cb1 antagonist treatment and foxb1 (a direct target of mir-181a family) was differentially regulated, suggesting a possible mechanism of action for the benefits of cb1 receptor inhibition. conclusion: we found that in angii-induced cardiac remodelling, lv function is preserved by chronic cb1 antagonist treatment and that cardiac fibrosis is reduced with concomitant downregulation of fibrogenic genes. also, cf-enriched mir-181a/b family seems to be sensitive to cb1 antagonist treatment, thereby affecting cardiac fibrosis. the current study employs a novel concept regarding chronic cb1 inhibitor treatment and may provide important details and novel targets for anti-fibrotic approaches in heart failure. background: low homoarginine (harg) was recently identified as an emerging biomarker for stroke, myocardial infarction, and heart failure in clinical and epidemiological studies. harg competes with arginine as a substrate for nitric oxide (no) synthase and weakly inhibits arginase. both mechanisms might lead to increased no formation in vivo. the aim of this study was to investigate whether harg effects the development of atherosclerosis as a potential underlying mechanism of cardiovascular diseases. methods: harg-deficient agat-knockout (agat -/-) and wildtype (wt) mice were crossed with apolipoprotein e (apoe) deficient mice and fed with high fat diet (hfd) for three months to induce atherosclerosis. harg plasma concentrations were determined using mass spectrometry. en face preparation of aortae followed by red oil staining of atherosclerotic plaques and quantitative evaluation of plaque areas was performed for female mice. endothelial function of male mice was tested with acetylcholine (ach) and nitroglycerin (ntg) after contraction with prostaglandin f2α. background: the direct oral thrombin inhibitor dabigatran etexilate (dabigatran) is used for the prevention and treatment of venous thromboembolism. obese patients as well as patients with type 2 diabetes mellitus (t2dm) have an increased risk for thrombotic disease and show enhanced thrombin generation. besides its role in blood coagulation, thrombin is known to be involved in many pro-inflammatory processes. in obesity, adipose tissue (at) inflammation plays a crucial role in the development of insulin resistance and t2dm and contributes to atherosclerosis development. the aim of the present study was to analyse the effects of dabigatran on at inflammation in a mouse model of diet-induced obesity in the context of accelerated atherosclerosis. methods: 10-week-old female low-density lipoprotein receptor-deficient (lldr -/-) mice were fed a high-fat diet containing 5 mg/g dabigatran or respective placebo for 20 weeks. results: analysis of visceral at revealed a significant increase in adipocyte size in dabigatran-treated mice, although body weight, fat mass, glucose tolerance, and insulin resistance were unchanged between groups. this effects seemed to be directly mediated by thrombin, as treatment with another thrombin inhibitor (argatroban) also resulted in the development of adipocyte hypertrophy. accordingly, in vitro studies in 3t3-l1 cells revealed an inhibitory effect of thrombin on lipid accumulation in adipocytes. the amount of pro-inflammatory cd11c-positive macrophages (atms) in visceral adipose tissue was significantly reduced, and the secretion of pro-inflammatory il-6 from visceral at was significantly lower in dabigatran-treated animals. in vitro studies using 3t3 l1 cells and primary bone marrow-derived macrophages revealed that the changes in macrophage polarization were not directly mediated by thrombin, but indirectly by a change in the secretion profile of adipocytes. a similar reduction in proinflammatory macrophages as detected in at could also be observed in the aortic wall of dabigatran-treated mice. conclusions: the direct thrombin inhibitor dabigatran inhibits at inflammation and the accumulation of pro-inflammatory macrophages in vat but also the aortic wall of ldlr -/mice. these anti-inflammatory effects of dabigatran might contribute to the known atheroprotective effects of dabigatran. background: sarco/endoplasmic reticulum ca 2+ -atpase (serca2a) and its inhibitor phospholamban (pln) are critical determinants of cardiomyocyte calcium cycling and hence, cardiac contractility. pln exists in an equilibrium between mono-and pentamers. while monomeric pln has been implicated in direct serca2a inhibition, a functional role for the pentamers remains ambiguous. recently it has been shown that pln pentamers modulate pka-dependent phosphorylation of pln monomers in vitro. 1 using transgenic mouse models we now investigated the effects of pln pentamers on pln phosphorylation, myocyte ca 2+ cycling and contractility in cardiac myocytes. methods: phosphorylation patterns of pln were analyzed by western blot using phospho-specific antibodies as well as phosphate affinity sds-page. to assess the phosphorylation at baseline, pln knockout (pln-ko) mice expressing either wild type pln (tgpln) or the solely monomeric pln afa mutant (tgafa) transgene were deeply anesthetized, whereas pln phosphorylation by pka was induced using the betaadrenergic agonist isoproterenol. the consequences on myocyte ca 2+ kinetics were measured in isolated, fura2-loaded and electrically paced (0.5hz) cardiomyocytes as the time to 50% decay of the ca 2+ signal (t 50% ). the time to 50% baseline of sarcomere length (t 50% baseline) characterized the speed of myocyte relaxation. results: under basal conditions, we found stronger phosphorylation of pln pentamers than monomers, pointing at pentamers as the preferred pka target. pln afa monomers showed 3.3-fold stronger phosphorylation signals if pentamers were absent (p<0.0001). consistent with a higher basal phosphorylation of pln afa monomers, measurements of calcium kinetics revealed a faster decay of calcium signals in tgafa compared with tgpln cardiomyocytes (t 50% [ms]: 92±8 and 122±8, respectively, p<0.05). notably, t 50% of pln-ko myocytes was 79±5ms (p= not significant versus tgafa), indicating that the strong basal phosphorylation of monomers leads to near complete inactivation of pln in tgafa. upon stimulation of pka, pln monomer phosphorylation and calcium kinetics of tgpln and tgafa mouse myocytes were indistinguishable, because monomer phosphorylation and the speed of cytosolic ca 2+ clearance strongly increased only in tgpln. acceleration of sarcomere relaxation upon pka stimulation was also more pronounced in tgpln than in tgafa and pln-ko myocytes (increase of t 50% baseline [ms]: 32±8 in tgpln versus 7±4 in tgafa-pln and 5±7 in pln-ko, p<0.05). even high-dose isoproterenol induced phosphorylation of only about half of all protomers of pln pentamers suggesting a high capacity of pentamers to attenuate monomer phosphorylation by acting as a phosphate scavenger. conclusions: our data demonstrate that pln pentamers reduce basal phosphorylation of pln monomers in myocytes. nevertheless, pentamers allow strong phosphorylation of monomers during beta-adrenergic stimulation, thereby extending the range within which pln can modify diastolic ca 2+ kinetics and myocyte relaxation. therapeutic inhibition of micrornas is a promising field in cardiovascular research. vector-based overexpression of an inhibitor construct (e.g. microrna sponge) is one approach to achieve sustained inhibition with potential applicability in humans. yet the strength of expression achieved by the currently available gene therapy vectors (e.g. aavs) in humans remains a limiting factor, therefore inhibitory constructs with increased potency would provide an improvement of this approach and bring it closer to therapeutic application. micrornas are believed to discriminate between potential binding sites, based on additional factors provided by the endogenous untranslated regions at the 3' end of mrnas (3' utrs) and the proteins that are bound to them. aim of this project was to investigate whether selected endogenous 3' utrs can likewise increase the potency of microrna inhibitory constructs. to this end several known targets for a cardiac microrna were selected and their relative potencies of microrna inhibition was compared. to accurately assess changes in the activity of the respective micrornas we constructed dual-fluorescent reporter plasmids and established an automated fluorescent microscopy acquisition and analysis pipeline. among several tested utr constructs we found one which strongly increased the inhibitory potency of the microrna binding site in primary rat cardiac myocytes (nrcms). furthermore a similar effect was obtained when the binding site was exchanged for that of a different microrna and analyzed in the nih-3t3 fibroblast cell line. we therefore conclude that endogenous utr contexts can indeed be successfully applied to increase the potency of vector-based microrna inhibitors. the g-protein-coupled protease-activated receptor-2 (par2) regulates inflammatory responses including monocyte migration and cytokine release. par2 is activated by the coagulation factor-xa or by the tissue-factor (tf)/factor viia complex. the immunomodulatory lipid sphingosine-1-phosphate (s1p) is released from activated platelets and interlinks blood coagulation and inflammation. this study investigates the impact of s1p on the expression of par2, tf and of the anticoagulant protein thrombomodulin (tm) in human monocytes and after pma-induced differentiation into macrophage-like cells. monocytic thp1 and u937 cell lines were used as human monocyte models. primary monocytes were isolated from healthy volunteers using a magnetic bead-based monocyte isolation kit. expression of par2, tf and tm was measured by quantitative real-time pcr and western blotting. differentiation of monocytes into macrophage-like cells was induced by incubation with 50 ng/ml pma (phorbol 12-myristate 13-acetate) over 72 h. calibrated automated thrombin (cat) generation was determined in plateletrich plasma from healthy volunteers. in thp1 and u937 cells s1p induced a time-(1 to 24 h) and concentration-dependent (0.1 to 10 µm) significant upregulation of par2 mrna and total protein expression. par2 total protein was upregulated maximally (about 1.5-fold, n=7) with 1 µm s1p after 16 h incubation. comparable effects were seen in human primary monocytes. in comparison, tf mrna and protein were only marginally elevated in non-differentiated thp1 monocytes and tm was not regulated by s1p. after differentiation of cultured monocytic cells with pma into adhesive macrophage-like cells, incubation with s1p resulted in a time-(1 to 24 h) and concentration-dependent (0.1 to 10 µm) significant upregulation of tf expression within 3 to 6 h of incubation. conversely, par2 total protein expression was reduced by about 50% after 24 h s1p incubation. the expression of tm was again not affected. the generation of thrombin in platelet-rich plasma was determined using pma-differentiated thp1 cells as tf source. timedependent incubation with s1p (1 µm) in differentiated monocytes shortened the time to the onset (lag time) of thrombin generation in plasma from 8.0±1.6 to 6.5±1.4 min and elevated total the thrombin generation capacity from 814±130 to 1286±129 nm. peak thrombin formation was elevated from 66±31 to 130±30 nm/min (control versus s1p for 6h, mean±sd, n=5, respectively). these data suggest that s1p induces an enhanced expression of par2 in undifferentiated human monocytes while tf and tm are not regulated. in differentiated monocytes/macrophages, s1p does upregulate tf expression but attenuates par2 levels. since par2 is involved in regulation cell migration, s1p may stimulate a phenotypic switching from a migratory to a procoagulant phenotype during differentiation of monocytes into macrophages. the pro-inflammatory cytokine interleukin-6 (il-6) plays an important role in vascular inflammation. coagulation factors such as the activated factor-x (fxa) may regulate local inflammatory responses of the vessel wall. in this study we investigated whether fxa regulates il-6 expression and secretion in human vascular smooth muscle cells (smc) as well as in failed thrombosed vein grafts. also, we analysed its possible prothrombotic impact on monocytes. il-6 mrna expression was determined in primary human saphenous vein smc by taqman® real-time pcr. secretion to the cell culture media was measured by elisa. tissue factor (tf) expression in monocytic thp-1 cells was determined by western blot. immunostainings for il-6 and the smc marker smoothelin were performed on paraffin embedded tissue sections from failed thrombosed vein grafts and control veins. incubation of cultured human venous smc with fxa (30 nm) induced a time-dependent (3 -16 h) increase in il-6 mrna expression. maximum expression was observed within 6 h to a 7.2±3.3 fold increase (mean±sd, n=5, p<0.05). incubation with an inhibitor of p38 map kinase (sb203580, 10 µm) or pi3k (ly294002, 10 µm) significantly attenuated fxa-induced il-6 mrna expression (n=5). inhibition of p42/44 mapk, rho kinase or nf-kb had no significant effect. stimulation with fxa for 24h resulted in a markedly increased il-6 secretion into the smc culture media from 0.6±0.2 to 1.1±0.3 ng/ml (p<0.5, n=4). stimulation of thp-1 cells with il-6 (1 ng/ml) induced a time dependent (6 -24 h) increase of up to 2.1±0.6 fold (p<0.05, n=5) in tf protein expression. immunostainings of tissue sample of failed vein grafts revealed enhanced il-6 expression in smc-rich regions in vessel walls compared to non-thrombosed control veins suggesting an elevated il-6 regulation in thrombosed vein grafts in vivo. in conclusion, fxa induced il-6 expression and secretion in venous smc which may be regulated via p38 and pi3k signaling. il-6 enhanced tf expression in thp-1 monocytes and was found in smc-rich regions in failed thrombosed vein grafts. fxa-stimulated il-6 release may be involved in regulating local pro-thrombotic processes during vascular inflammation and possibly vein graft failure. rationale: the transcription factors camp-response element binding protein (creb) and camp-responsive element modulator (crem) bind to camp response elements (cres) and mediate a camp dependent gene regulation. suppression of cre mediated transcription is linked to atrial remodeling in genetic mouse models. inhibition of creb target genes is associated with atrial fibrillation (af) susceptibility in patients. creb and crem affect histone acetylation recruiting the creb-binding protein (cbp/p300). the histone acetyltransferase (hat) activity of cbp facilitates gene transcription by loosening chromatin structure. histone deacetylases (hdacs) catalyze the inverse reaction: histone deacetylation with consecutive gene silencing. mice with heart directed expression of the human cardiac isoform crem-ib∆c-x (tg) show atrial dilatation, morphological and physiological alterations in atria preceding spontaneous-onset af. the hdac inhibitor (hdac i ) valproic acid (vpa) reduced atrial weight and af incidence in tg mice. here we tested the hypothesis, that vpa attenuates the structural remodeling in tg atria by reversing changes in atrial gene regulation due to the transgene. methods and results: tg and wt mice were treated from week 5-16 with vpa (0.71% in drinking water, ad libitum) or vehicle (veh). atrial ultrastructure was studied by electron microscopy (em) (week 7 and 16). veh-treated tg atria showed a progressive dysorganisation of sarcomeres (sm) with less mitochondria and more collagen fibers between cardiomyocytes as compared to veh-treated wt atria. the fraction of sm structure in veh-treated tg atria was significantly reduced as compared to veh-treated wt atria (week 7: tg veh : 33±2%, wt veh : 47±1%; week 16: tg veh : 19±2%, wt veh : 44±1%, p<0.05). vpa led to a more organized ultrastructure and restored, at least partially, the degradation of the sm in the tg atria (tg vpa at week 7: 40±2%, tg vpa at week 16: 34±1%, p<0.05 vs. tg veh ). the structure of wt atria was not affected by vpa. we further analyzed the protein abundance profiles in the groups of all animals (wt veh , wt vpa, tg veh , tg vpa) by using lc-ms/ms. between veh-treated genotypes (tg veh vs. wt veh , p<0.05) 998 proteins were significantly changed while 854 proteins were differentially abundant between vpa-treated groups (tg vpa vs. wt vpa, p<0.05). 109 proteins were regulated by vpa in wt atria (wt vpa vs. wt veh , p<0.05), whereas vpa affected 525 proteins in tg atria (tg vpa vs. tg veh , p<0.05), out of which 43 proteins were common. 295 prominent changed proteins between veh-treated tg and wt atria were significantly regulated by vpa in tg atria in the opposite direction. a functional pathway analysis showed that pathways activated in tg atria such as cardiac fibrosis, mitochondrial dysfunction were inhibited by vpa treatment. conclusion: similar to human af, crem-tg mice present atrial dilatation, ultrastructural changes and impaired conduction and spontaneous af. while vpa had little to no effect in wt mice, valproate improved the tg phenotype by interfering with pathways involved in structural remodeling. this supports the idea that hdac inhibition by vpa antagonizes effects of crem expression in atria. in isolated mouse cardiac preparations, histamine is ineffective regarding inotropic or chronotropic effects, presumably because of lack of receptor protein expression. on the other hand, histamine can exert positive inotropic and chronotropic effects in humans via cardiac histamine h 2 -receptors. hence, we have generated transgenic mice (tg) which overexpress the human h 2 -receptor specifically in cardiomyocytes. in isolated left and right atrial preparations of these mice, we investigated the histamine metabolism on a functional level. preparations of wild type mice (wt) served as control. histamine induced positive inotropic effects (pie) and positive chronotropic effects (pce) in left and right atria of tg mice, respectively, but not in wt. interestingly, the inhibitor of histamine oxidation, aminoguanidine (1 mm), shifted the concentration response curves for the pie of histamine from ec 50 = 110 nm to 37 nm (p<0.05). furthermore, the unspecific inhibitor of mono amine oxidase, tranylcypromine (10 µm), shifted the pie of histamine from ec 50 = 70 nm to 38 nm and increased the efficacy of histamine for the pie (p<0.05). these data indicate that exogenously applied histamine is subject to degradation in the mouse heart by two different pathways namely via diaminoxidase and mono amine oxidase. drugs that inhibit theses enzymes could conceivably alter cardiac function also in the human heart. protein phosphorylation by kinases and dephosphorylation by protein phosphatases has a crucial function in cell signal cascades. it has been shown that cardiomyocyte specific overexpression of serine /threonine protein phosphatases pp1, pp2a, pp2b (calcineurin) and pp5 in mice leads to cardiac hypertrophy and alters cardiac function. to examine the function of another important protein phosphatase in the heart we established a mouse model overexpressing protein phosphatase 2cβ (pp2cβ) under control of the α-myosin heavy chain promoter. cardiac overexpression was demonstrated by western blotting. like other serine/threonine phosphatases, pp2cβ can lead to cardiac hypertrophy. in transgenic mice (tg), relative ventricular weight was increased (4.24 ± 0.14 mg/g) compared to wild type (wt) littermates (3.78 ± 0.20 mg/g; p<0.05) whereas weights of right and left atria were unchanged. therefore, relative heart weight was increased in tg (4.78 ± 0.17 mg/g) vs. wt (4.13 ± 0.22 mg/g; n=8-12; 10-11 months of age; p<0.05). left ventricular function, measured in vivo by echocardiography under isoflurane anesthesia was diminished in tg compared to wt (ejection fraction: 58.33 ± 3.16 % (tg) versus 76.94 ± 0.92 % (wt); n=12-16; 8-11 months; p<0.05 and fractional shortening: 30.84 ± 2.02 % (tg) versus 44.94 ± 0.87 % (wt); n=12-16; 8-11 months; p<0.05). the left ventricle was dilated (systolic diameter: 2.78 ± 0.21 mm (tg) versus 1.84 ± 0.06 mm (wt); n=12-16; 8-11 months; p<0.05; diastolic diameter: 3.97± 0.19 mm (tg) versus 3.33 ± 0.07 mm (wt); n=12-16; 8-11 months; p<0.05). in contrast, atrial function measured as response to β-adrenergic stimulation in isolated left and right atrial preparations was unchanged in tg vs. wt. in summary, our results indicate that pp2cβ overexpression can lead to ventricular dysfunction and hypertrophy. the underlying signal transduction pathways need to be elucidated. the insulin-like growth factor binding protein 5 (igfbp5) -a potential developmental gene is regulated upon cardiac stress m. wölfer background: cardiac remodeling is a complex biological adaptation process of the failing heart accompanied by a re-activation of embryonic gene expression, which so far has unclear pathophysiological relevance. we and others showed that insulin-like growth factor binding protein 5 (igfbp5) is expressed in the early pre-cardiac region in mouse embryos and its up-regulation impairs cardiac progenitor differentiation. igfbp5 functions as an extracellular growth factor binding protein for igf and also has igfindependent activities. the role of this factor in the context of cardiac remodeling is still unknown. the aim of this study was to investigate the relevance of igfbp5 in cardiogenesis and cardiac remodeling and its role as a potential target for ameliorating stress-induced cardiac remodeling methods and results: we investigated the expression of igfbp5 in murine cardiac tissue at different developmental stages by qpcr normalized to tpt1 (tumor protein, translationally-controlled 1). this analysis showed temporal changes of cardiac igfbp5 expression from developing to postnatal hearts, where a high expression was detected in early heart stages, which decreased during cardiac development and became low in the postnatal heart. the analysis of igfbp5 expression in different heart cells showed a very low igfbp5 in adult cardiomyocytes in contrast to a high expression in undifferentiated sca-1 positive cells. in a mouse model with cardiac specific wnt/βcatenin activation, which led to cardiac dysfunction, igfbp5 was found up-regulated (p<0.05). further we found an increased igfbp5 expression after pressure induced cardiac hypertrophy using mice with transverse aortic constriction (tac) (p<0.01). in line with this data, an in vitro model of human heart muscle hypertrophy using engineered cardiac heart muscle (ehm) showed an up-regulation of igfbp5 upon adrenergic activation via norepinephrine stimulation accompanied by a functional deterioration in comparison to untreated controls (p<0.05). all these findings were further supported by rna-sequencing analysis from human aortic stenosis patient samples, where igfbp5 expression was found increased in patients with compensatory hypertrophy and in a higher extent in patients with heart failure in comparison to non-failing heart samples. interestingly, the expression of igfbp5 in angiotensin 2 or norepinephrine stimulated neonatal murine cardiomyocytes, as well as in hearts of mice treated with angiotensin 2, showed the opposite results, namely a reduction in its expression (p<0.01; p<0.05, respectively). summary and conclusion: our results show active igfbp5 transcription in the early developing heart but a low expression in the postnatal heart. a re-activation of expression was found in the process of pathological heart remodeling in mouse and human, in vivo as well as in vitro, indicate the participation of igfbp5 in a conserved manner. we hypothesize that igfbp5 may participate in the developmental gene program becoming activated in the diseased adult heart again. the functional role and regulation of igfbp5 is under investigation. we have recently shown that perivascular adipose tissue (pvat) plays a crucial role in obesity-induced vascular dysfunction. in pvat-free aortas isolated from male c57bl/6j mice fed a high-fat diet (hfd) for 22 weeks, the endothelium-dependent nitric oxide (no)-mediated vasodilator response to acetylcholine remained normal. in contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when pvat was left in place. these results suggest that the reason for vascular dysfunction in diet-induced obese mice is a pvat dysfunction rather than an endothelial dysfunction. treatment of hfd mice during the last 4 weeks with crataegus extract ws® 1442 (150 mg/kg/day) completely normalized vascular function in pvatcontaining aorta. the expression of endothelial no synthase (enos) was not changed by ws® 1442, neither in pvat nor in aorta. phosphorylation at serine 1177 is the most important positive modulation of enos activity. hfd-induced obesity was associated with a reduction in enos phosphorylation at serine 1177 in pvat, but not in aorta. ws® 1442 treatment significantly improved enos serine 1177 phosphorylation selectively in pvat but had no effect in aorta. a major upstream kinase for enos serine 1177 phosphorylation is akt. the activity of this kinase is inhibited in the pvat of hfd mice, which was largely reversed by ws® 1442 treatment. in addition, ws® 1442 treatment enhanced the mrna expression of the nad-dependent deacetylase sirtuin-1 (sirt1), known also as a longevity gene. the activity of sirt1 depends, among others, on the intracellular content of its cofactor nad. ws® 1442 treatment led to an upregulation of nicotinamide phosphoribosyltransferase (nampt), a rate-limiting enzyme in the salvage pathway of nad biosynthesis. one of the non-histone substrates of sirt1 is enos. deacetylation of enos at lysine residues 497 and 507 by sirt1 enhances the activity of the enos enzyme. currently, we are studying the effect of ws® 1442 on nad synthesis, sirt1 activity, and enos (de)acetylation. in conclusion, crataegus extract ws® 1442 reverses obesity-induced vascular dysfunction by improving pvat function. the molecular mechanisms may involve enos phosphorylation at serine 1177 and upregulation of sirt1. the raf kinase inhibitor protein (rkip) inhibits g-protein-coupled receptor kinase (grk2) and the raf-erk1/2 pathway. these two functions of rkip could counteract each other. while grk2 inhibition is cardio-protective, inhibition of the pro-survival erk1/2 axis promotes signs of heart failure in patients and experimental models. in view of this ambivalent nature, the function of rkip in vivo is not clear. furthermore, rkip could have a pathophysiological role because heart specimens from patients with heart failure showed rkip up-regulation (ref. 1). to investigate the impact of cardiac rkip upregulation in vivo, we generated transgenic mice with myocardium-specific expression of the human rkip gene (pebp1) under control of the alpha-mhc promoter. two different rkip-transgenic lines with 2.7-fold and 3.4-fold increased cardiac rkip protein level were generated (ref. 2, and jax id number 911819). we investigated the cardiac phenotype and found that tg-rkip mice developed cardiac hypertrophy with a significantly increased heart weight to body weight ratio and a decreased left ventricular ejection fraction relative to non-transgenic fvb controls, as early as 10 weeks of age. histology analysis revealed progressive atrial and ventricular enlargement of tg-rkip hearts. ecg abnormalities, a lower maximum rate of left ventricular pressure rise, and a strongly decreased left ventricular ejection fraction of 32.9±2.3 % (n=6; ±s.d.) were documented at an age of 8 months. down-regulation of the transgenic rkip by lentiviral transduction of an rkip-targeting mirna retarded the cardiac phenotype of tg-rkip mice. thus, dual-specific inhibition of the grk2 and raf-erk1/2 axis by the human rkip gene (pebp1) triggers signs of heart failure in vivo, and the documented upregulation of the cardiac rkip in heart failure patients could aggravate disease pathogenesis. these findings are in contrast to rodent rkip (pebp1), which does not seem to inhibit the raf-erk1/2 axis in vivo but instead confers grk2 inhibitionheterodimerization between the at1 receptor (at1r) for the vasopressor peptide, angiotensin ii, and the b2 receptor (b2r) for the vasodepressor peptide, bradykinin, enhances angiotensin ii-stimulated signalling in cells. in addition, at1r--b2r heterodimerization has a major pathophysiological role and contributes to the angiotensin ii hypersensitivity in women with preeclampsia hypertension. to analyse the vascular function of the at1r--b2r heterodimer in vivo, we generated a transgenic model of at1r--b2r heterodimerization (tg-b2r+) by transgenic expression of the b2r gene (bdkrb2) in the b2r-deficient tg-b2r-/-strain. fluorescence resonance energy transfer (fret) imaging was applied to analyse the interaction between different gprotein-coupled receptors in the aorta of transgenic mice. we report here that fret imaging detected the close interaction between the aortic at1r and b2r at a distance of less than 9 nm in tg-b2r+ mice whereas the at1r--b2r heterodimer was absent in tg-b2r-/-mice. in contrast, fret was not detectable between the endothelin eta receptor (etar) and the b2r in the aorta of tg-b2r+ mice, although immunofluorescence and immunohistology confirmed the aortic (co-)-localization of both, etar and b2r. the efficient at1r--b2r heterodimerization in tg-b2r+ mice was accompanied by an enhanced angiotensin ii at1r-stimulated vasopressor response relative to that of tg-b2-/-mice, which lack the at1r--b2r heterodimer. as a control, the endothelin-1-stimulated vasopressor response mediated by the etar, which did not dimerize with b2r, was not significantly different between tg-b2r+ and tg-b2r-/-mice. together these findings provide strong evidence that at1r--b2r heterodimerization occurs in vivo and enhances the angiotensin ii at1r-stimulated vasopressor response. dysfunction of the cardiac energy substrate metabolism is a characteristic feature of late-stage heart failure. the dysfunctional cardiac substrate metabolism contributes to insufficient energy generation and has limited treatment options. in search for a treatment approach, we investigated whether inhibition of g-protein-coupled receptor kinase 2 (grk2) could confer cardioprotection by targeting the dysfunctional cardiac substrate use. the impaired substrate metabolism of late-stage heart failure was reproduced in a transgenic model with myocardium-specific expression of fatty acid synthase (fasn), which is the major palmitate-synthesizing enzyme. experiments with a seahorse xf24 extracellular flux analyzer revealed that in an adult-like lipogenic milieu, fasn-transgenic cardiomyocytes reproduced the overall depressed substrate use of late-stage heart failure with a switch from fatty acid to predominant glucose utilization. the impaired substrate use was largely retarded by co-expression of a small peptide inhibitor of grk2, grkinh. the grkinh-mediated protection against cardiometabolic remodelling required an intact raf-erk1/2 axis and involved the erk1/2-dependent inactivation of the heart failure-promoting peroxisome proliferatoractivated receptor gamma (pparg) by phosphorylation of serine-273. as a consequence of erk-dependent phosphorylation of pparg on serine-273, the expression of heart failure-related pparg targets such as fatty acid synthase, resistin and adiponectin was decreased. the importance of pparg serine-273 phosphorylation was further shown in transgenic mice with myocardium-specific expression of the phosphorylation-deficient pparg serine-273a mutant, which was resistant to the cardioprotective activity of grkinh. taken together our experiments show that grk2 inhibition could target cardiometabolic remodelling by inhibition of the heart failure-promoting transcription factor pparg. the effect of sodium valproate on the action potential of atrial myocytes of crem ib∆c-x transgenic mice introduction: in mouse, cardiomyocyte directed over-expression of transcription factor crem (camp response element modulator) causes an atrial phenotype characterized by hypertrophy, reduced contractility and increased duration of the monophasic action potential (map). moreover, this animal model (crem ib∆c-x) showed spontaneous atrial fibrillation (af) episodes as early as 5 weeks of age in homozygous mice and 10-12 weeks of age in heterozygous mice (phenotype delayed towards adult stage). previous studies in heterozygous mice targeted hdac2 inhibition by sodium valproate (vpa, an anticonvulsant drug, acting also as inhibitor of hdac class i>ii). vpa treatment delayed significantly the development of atrial hypertrophy and the incidence of af episodes, without affecting cellular hypertrophy. our aim was to investigate the effect of chronic vpa treatment on the electrical activity of atrial myocytes isolated from crem ib∆c-x and wild type (wt) littermate mice. methods and results: atrial myocytes were isolated from 12 weeks old wild type mice (wt) and heterozygous crem ib∆c-x transgenic mice with enlarged atria (tg), treated for 7 weeks with vpa (0.4 mm in the drinking water) vs. water (vehicle control). action potentials (ap) were measured at room temperature using the patch-clamp technique. atrial myocytes of water treated tg mice had the ap amplitude significantly reduced by 7 mv compared to water treated wt, and, in line with previous results for the map, the tg cells depolarized with a slower slope of 90.2±8 v/s (tg: n=33 cells) vs. 109.8±5.1 v/s (wt: n=30, p=0.05). moreover, ap of atrial myocytes isolated from water treated tg mice had longer duration (apd) at 50% (tg: 11.6±1.4 ms, n=35 vs. wt: 6.9±0.5 ms, n=30, p<0.01), at 70% (in ms: 21.2±2.5 vs. 13.6±1, p<0.05) and at 90% (in ms: 46.8±4.5 vs. 34±2.5, p<0.05) repolarization. vpa treatment reduced ap amplitude in wt mice by 6 mv (n=22, p<0.05) vs. water treated wt, without altering the slope of depolarization or the apd. in vpa treated tg mice the apd was reduced (50%: 7.3±0.8 ms, 70%: 13.8±1.5 ms, 90%: 30.9±3.2 ms, n=21, p<0.05 vs. untreated tg), the amplitude was increased by 5 mv (n.s.) and the slope of depolarization was increased by 21% (p=0.16, n.s.). membrane capacitance evaluation, as an estimation of atrial myocyte size, showed that in untreated tg mice the cells were larger than in wt (tg: 104±7.2 pf, n=27 vs. wt: 55±5.4 pf, n=30, p<0.001), in line with the occurrence of cellular hypertrophy in tg atria. chronic vpa treatment did not change the cell size in either genotype (wt-vpa: 64.8±7 pf, n=17 n.s. vs. wt; tg-vpa: 88±7 pf, n=14, p<0.05 vs. wt-vpa; p<0.01 vs. wt; n.s. vs. tg). conclusions: in hypertrophied atrial myocytes of crem-ib∆c-x, ap were characterized by smaller amplitude, slower onset of depolarization and increased duration compared to wt cells. despite having no effect on atrial myocytes size, vpa treatment reduced the duration and showed a tendency to increase the amplitude and the slope of depolarization of the action potential in tg mice to values similar to wt. these data suggest that chronic treatment with vpa restored partially the electrical activity of atrial myocytes and may reverse the electrical remodeling via hdac2 inhibition. (supported by the dfg) of the transcription factor crem (camp response modulator) icer, smicer and crem-ib∆c-x are inducible by β-adrenergic stimulation and code for similar or even identical proteins. thus, these isoforms are able to repress expression of respective target genes in response to camp and might play a role in an arrhythmogenic remodeling during the development of chronic heart diseases. here we test this hypothesis in a mouse model with transgenic expression of crem-ib∆c-x (tg). these mice develop not only spontaneous onset atrial fibrillation but likewise arrhythmogenic alterations in the ventricle. patch clamp experiments revealed an increased na + -ca 2+ exchanger current (i ncx ) and decreased transient outward current (i to ) in tg ventricular cardiomyocytes (vcms) vs. wild-type controls (ctl). these alterations were associated with an increased arrhythmogenicity in tg vcms. action potentials were prolonged in tg vcms vs. ctls leading to an increased proportion of vcms displaying early afterdepolarizations. ca 2+ imaging revealed that the transduction rate of spontaneous sub-threshold ca 2+ -waves into supra-threshold transient-like ca 2+ -events which is mediated by the ncx was increased in tg vcms. at the same time the serca mediated ca 2+ transport rate (r serca ) was enhanced in tg vcms potentially limiting ca 2+ extrusion by the ncx. underlining the in-vivo relevance of our findings ventricular extrasystoles (ves) were augmented in ecgs of tg mice (ves/mouse during 10 -6 m isoproterenol challenge, tg: 3.65*, ctl: 0.4; n=20/condition). the increase in i ncx and r serca and the decrease in i to went along with an increase of ncx1, serca2a and decrease of kchip2 protein levels. however, the respective mrna levels (slc8a1, atp2a2 and kcnip2) were unaltered between groups pointing to a post-transcriptional regulation of these genes. in a mrnasequencing approach we identified the downregulation of precursor mirnas inter alia for mir-369 (fold change in tg: 0.04*) and mir-1 (fold change in tg: 0.13*) (n=10/condition). atp2a2 is a predicted target of mir-369 and mir-1 has recently been shown to regulate ncx1 and i to related potassium channel subunits. (*p<0.05 vs. ctl) our results demonstrate that transgenic expression of crem-ib∆c-x in mouse vcms leads to distinct arrhythmogenic alterations. they further indicate that the repression of micro rnas by short crem repressor isoforms may lead to the upregulation of genes in the context of an arrhythmogenic remodeling. since crem-repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of credependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease. ( chronic overstimulation of cardiac β-adrenergic receptors (β-ar) is a major trigger for the development and maintenance of cardiac hypertrophy and heart failure. although the camp activated proteinkinase a (pka) is known as a prominent downstream effector of β-ar signaling, its functional contribution to pathological cardiac remodeling is neither well understood nor directly studied so far. to address this issue we used mice carrying a point mutation in the regulatory pka subunit riα (pkariαb), which prevents binding of camp and consequently diminished kinase activity. this dominant negative mutation was controlled by a tamoxifen (tam) inducible αmhc promotor driven cre transgene which allows a selective expression in the ventricular myocardium. the inducible and tissue specific gene expression was analyzed and confirmed by pcr, rt-pcr and immunohistochemistry. furthermore diminished phosphorylation of several pka targets verifies impaired pka activity in tam treated double transgenic animals. hypertrophic response in ventricular pka mutants was studied in genetic, pharmacological and surgical mouse models of heart disease as well. genetically induced heart failure was observed following tam treatment in mice expressing an inducible myocardial-specific cre transgene. this deleterious cardiac phenotype develops independently of the presence of the floxed transgene. 8 days after tam treatment, controls displayed elevated heart weight to bodyweight ratio (hbr) and heart weight to tibia length (htr). hbr shifted from 6.2(mg/g) in untreated control animals to 10.9(mg/g) in tam injected mice. in contrast pka inhibited mutants displayed a minor increased hbr of 7.7 (mg/g). for the pharmacological induction of cardiac hypertrophy we implanted osmotic mini pumps, delivering a combination of isoproterenol and phenylephrine. control animals showed a significantly increased hbr (8.4 mg/g) compared to saline treated animals (6.8mg/g) and pka mutants (7.1 mg/g). paradoxically, all pka inhibited animals displayed a consistent elevation in important hypertrophic markers like anp. surgical constriction of the aortic arch (transverse aortic constriction tac) led to a pressure induced hypertrophic response (hbr: 6.5 vs 7.9 mg/g) followed by a pronounced elevation in several hypertrophic factors such as anp, myh6/7 ratio and myocytes size. in contrast pka mutants displayed an irregular progression of cardiac hypertrophy presented by two groups with either an unchanged (6.9 mg/g) or a strongly elevated hbr (13.5 mg/g). however, additional hypertrophic factors including anp, myh6/7 ratio and myocyte size were significantly increased in both groups. to our knowledge this is the first report, which directly studies the role of ventricular pka activity in cardiac hypertrophy in a genetically altered mouse model. our results suggest that in an early stage of cardiac remodeling pka inhibition alleviates cardiac weight gain but provokes a detrimental shift during further progression, which implicates a protective role of ventricular pka activity in cardiac disease. acetyl-coa carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacterial and eukaryotic cells, i.e. the conversion (carboxylation) of acetyl-coa into malonyl-coa. acc-generated malonyl-coa functions as a substrate for de novo lipogenesis and acts as an inhibitor of mitochondrial β-oxidation of fatty acids. because of its role in lipid metabolism this enzyme has become an interesting target in drug discovery in the field of metabolic diseases and cancer. despite this interest in acc, no attention has as yet been given to the role of acc in endothelial cells. we aimed to investigate the role of acc in two functional key aspects of angiogenesis: endothelial cell proliferation and migration. we used the acc inhibitor soraphen a, a polyketidic natural compound isolated from the myxobacterium sorangium cellulosum, as well as an rnai-based approach to inhibit the function of acc. primary human umbilical vein endothelial cells (huvecs) were used as in vitro model. first, we analyzed the action of soraphen a on cell viability. the compound did neither lower the metabolic activity of huvecs up to a concentration of 100 µm after 24 and 48 h (ctb assay) nor increase in the apoptosis rate after 24, 48, or 72 h up to 100 µm. measuring adenosine triphosphate (atp) levels revealed that 30 µm soraphen a does not alter the atp levels in huvecs after 24 h treatment. in contrast, a 48 h treatment significantly lowered the atp levels by 12 %. also gene silencing of acc1 in huvecs attenuated the atp levels by 11 %. mitochondrial membrane potential (mmp) assays showed decreased mmp levels (10 %) in soraphen a-treated cells after 24 h. interestingly, the compound inhibited the proliferation of endothelial cells with an ic 50 value of 34 µm. cell cycle analysis showed that soraphen a decreases the amount of cells in the g 0 /g 1 phase by 26 % and increases the number of cells in the g 2 /m phase by 50 %. the compound also inhibited the activation of akt (western blot analysis). in a wound healing/scratch assay, 30 µm soraphen a lowered the migration of endothelial cells by 65 %. gene silencing of acc1 in huvecs strongly decreased endothelial migration, whereas a knockdown of acc2 had no influence. furthermore, boyden chamber assays revealed that soraphen a can also lower chemotactic migration by 34 %. since actin rearrangement is necessary for migratory processes, we analyzed the factin cytoskeleton (microscopy) and found that soraphen a decreases the number of filopodia by 60 % but did not influence stress fiber formation. surprisingly, soraphen atreated cells did not exhibit significant alterations in their capacity to form tube-like structures on matrigel. in summary, we could gather first hints that inhibiting acc has an immense impact on the proliferation and migration of primary endothelial cells. the mechanistic basis of this phenomenon will be investigated in future studies by analyzing the lipid profile and the transcriptome of endothelial cells. acknowledgement: this work was supported by the german research foundation (dfg, for 1406, fu 691/9-2) . introduction: statins are among the best examined drugs with excellent efficacy and safety profiles. lowered low-density lipoprotein (ldl) cholesterol goals, new indications for treatment and new knowledge about their pleiotropic effect have promoted a considerable increase in statin use. but as statin use becomes more widespread, awareness of their adverse effects as well as the recognition of statin intolerance problems increase. statin intolerance is a significant problem in the treatment of dyslipidemia, understood as the inability to tolerate a dose of statin required to reduce individual cardiovascular risk sufficiently and could result from different statin-related side effects. muscle-related adverse events, elevation of liver enzymes, cognitive problems and new onset diabetes mellitus have all been described, especially at higher doses. although muscle symptoms are the common side effects observed, excluding other adverse events might underestimate the number of patients with true statin intolerance. these patients represent a target population for the newest lipid lowering drug category i.e. the proprotein convertase subtilisin/kexin type 9 (pcsk9) inhibitors. this work aimed to give an overview of published definitions derived from clinical studies, associations as well as major drug regulatory agencies. we discussed overlaps, differences and limitations in the current definitions. methods: literature based search included pubmed and uptodate publications in english and german language until october 2015. we performed hand searches of the references retrieved and performed an overview. results: a definition of statin intolerance of the european medicines agency (ema) or the us food and drug administration (fda) is not available. in clinical studies, different definitions are chosen and the results are not comparable. also different associations, such as the american heart association (aha), the european atherosclerosis society (eas), the canadian working group or the national lipid association (nla) are not able to agree on one common definition. statin intolerance definitions included different types of muscle symptoms, integration of ck levels and minimal requirements of statin doses. there are currently no validated questionnaires or specific laboratory parameters available. in addition, the term 'myopathy' is often considered as a synonym to statin intolerance. overall, only a few major studies have been conducted with statin intolerant patients so far using inconsistent definitions. discussion and conclusion: there is an unmet need to find a robust and clear definition of statin intolerance as overemphasizing it might hinder appropriate clinical use of this important drug class. thus, further work is required to develop a consensus definition on statin intolerance or a more focused definition regarding statin-associated muscle symptoms only. subsequently, these definitions could be implemented in patient care and their relevance being analyzed and tested in future studies. background: development of cardiac hypertrophy is characterized by reactivation of genes involved in cardiac development. wnt/β-catenin signaling is essential for embryonic cardiac development and is known to be dysregulated in pathological heart remodeling. our previous work suggested a cardiac specific protein complex regulating wnt/b-catenin/tcf transcription in the adult heart. we aim to identify and to characterize this complex in order to find potentially interesting targets for pharmacological therapy preventing maladaptive cardiac remodeling and the onset of heart failure. results: we previously demonstrated that the krüppel-like-factor 15 (klf15) is a βcatenin interaction partner and a cardiac specific nuclear inhibitor of the wnt/β-catenindependent transcription. because klf15 and β-catenin are ubiquitously expressed, we suggest the existence of cardiac specific co-factors responsible for cardiac specificity in this complex. we identified the basic leucine zipper and w2 domain containing protein 2 (bzw2), a phylogenetically conserved protein, as a β-catenin and klf15 interaction partner using yeast-two-hybrid screen. in vitro overexpression experiments and coimmunoprecipitation validated these interactions, which were also confirmed by mass spectrometry. in the developing mouse embryo bzw2 mrna expression is detectable in the heart, neuronal tissue, somites, limbs and branchial arches as shown by whole mount in situ hybridization. in the adulthood expression of bzw2 is confined to the heart, predominantly in cardiomyocytes and in cardiac progenitor cells compared to cardiac fibroblasts (*p<0.05, cm n=4, cfb n=4, cpc n=2), and skeletal muscle. bzw2 was localized in both the cytosol and in the nucleus. mutation analysis showed the importance of the n-terminus of bzw2, containing a putative bzip dna interaction domain, for the nuclear placement of the protein. bzw2 protein expression was significantly increased under cardiac wnt/β-catenin signaling activation in vivo in two mouse models (klf15 knockout (ko) mice **p<0.01 n=3, and in a cardiac specific β-catenin stabilized mouse model, **p<0.01 n=6). a mouse model with constitutively bzw2 loss of function (bzw2 ko) showed cardiac specific upregulation of β-catenin on rna level (***p<0.001, p<0.05, ctrl n=4, bzw2 ko n=5) and on protein level (**p<0.05, ctrl n=7, bzw2 ko n=9). echocardiography analysis in eight-weeks-old bzw2 ko mice showed increased left ventricle wall thickness indicating a hypertrophic phenotype at baseline. we also observed increased levels of bzw2 expression in angiotensin ii treated mice as a model for cardiac hypertrophy (*p<0.05 ctrl n=4, angii n=3) as well as in human samples derived from patients with dilated cardiomyopathy and ischemic cardiomyopathy (*p<0.05 ctrl n=3, dcm n=11, icm n=10). conclusion: these data demonstrated that bzw2 is associated with components of the canonical wnt cascade and suggest its relevance in the constitutive regulation of the wnt/β-catenin components specific in the heart. this study further contribute to the elucidation of the tuning of the wnt-off/-on states aiming to establish a proof-of-concept model for wnt-modulation as a therapeutic strategy in hypertrophic-induced heart failure. objective: sphingosine-1-phosphate (s1p) is involved in the regulation of cell growth, survival, migration and adhesion. it is formed by sphingosine kinases and degraded by phosphatases and s1p lyase [1] . mice that lack s1p lyase are characterized by the accumulation of s1p and sphingosine in their cells and tissues, and by lymphopenia, generalized inflammation, multiple organ damage, and a strongly reduced life span [2] [3] [4] . on the other hand, embryonic fibroblasts from s1p lyase-deficient mice (sgpl1 -/--mefs) are resistant to chemotherapy-induced apoptosis [5] , in part due to an upregulation of multidrug transporters of the atp-binding cassette (abc) transporter family [6] . interestingly, s1p lyase-deficient mice have elevated plasma levels of cholesterol and triglycerides, while suffering from strongly reduced body fat [7] . the aim of the present study was to analyze the link between s1p lyase deficiency and altered cholesterol homeostasis using sgpl1 background: cardiac gene expression changes during cardiac development and under pathophysiological conditions. these alterations in gene expression are regulated by several processes and the exact regulation of gene expression is essential for the proper development and function of the heart. crucial steps in transcription regulation are rna polymerase ii (pol ii) recruitment and changes in pol ii activity. pol ii activity is tightly linked with phosphorylation at serine-2 (p-ser2) of the carboxyterminal domain of pol ii. thus, the aim of the present study was to identify cardiomyocyte-specific genomewide pol ii and p-ser2-pol ii enrichments to get insight into pol ii activity and recruitment in development and disease. methods and results: to get insight into rna polymerase ii dynamics, genome-wide maps of rna polymerase ii occupancy were generated by chromatinimmunoprecipitation in cardiomyocyte nuclei purified from normal neonatal and adult mouse hearts. in addition, cardiomyocyte nuclei were obtained from adult hearts after 4 weeks of pressure overload induced by transverse aortic constriction (tac). cardiomyocyte nuclei were isolated by magnetic beads with an anti-pcm1 antibody. nuclei were used for pol ii chromatin-immunoprecipitation followed by deep sequencing (chip-seq). to test if pol ii marks correlate with nuclear mrna expression in cardiomyocyte nuclei all coding genes were ranked according to their expression level. genes expressed in cardiomyocyte nuclei (> 0.062 fpkm, gene expression rank < 12,653) showed high pol ii enrichment at promoters as well as in genomic regions. many of the gene promoters showed high levels of pol ii accumulation at the transcription start site, as compared to genic regions, which have been associated with pol ii pausing. in contrast, p-ser2-pol ii showed enrichment downstream of the transcription start site. the genomic region of troponin i type 1 (tnni1) which is expressed in cardiac muscle only during development but not in adult cardiomyocytes, was enriched for pol ii in neonatal cardiomyocytes. at the tnni1 gene, pol ii was absent in adult or pressure-overloaded cardiomyocytes. in contrast, pol ii enrichment at the alpha actin 1 (acta1) locus was only present in pressure-overloaded cardiomyocytes. these data are consistent with cellular rna-seq data showing an induction of acta1 after tac. furthermore no pol ii enrichment could be detected in the genomic region of biglycan (bgn), a matrix proteoglycan that is not expressed in cardiomyocytes. this confirms a high purity of cardiomyocyte chromatin. conclusions: this study provides, for the first time, cardiomyocyte-specific landscapes of rna polymerase ii occupancy in heart development and disease. cardiac myocyte maintenance dna methyltransferase 1 is essential for embryonic heart development but is dispensable for cardiac function and remodeling postnatally t. nührenberg background: recent studies have identified dynamic changes in dna methylation in cardiac myocytes during development, postnatal maturation and in disease. however, the enzymes involved in shaping the cardiac myocyte dna methylome are only partially known. here, we explored the role of maintenance dna methyltransferase dnmt1 in cardiac development and in remodeling after chronic left ventricular pressure overload. methods: in mice, deletion of the dnmt1 gene was accomplished by use of two different cre recombinases. crosses of homozygous dnmt1 fl/fl mice with heterozygous dnmt1 fl/+ mice expressing a cre recombinase under control of the atrial myosin light chain gene promoter (myl7-cre) resulted in embryonic deletion of dnmt1 (ko). embryos without myl7-cre served as controls (ctl). embryos were dissected and genotyped at e11.5, e12.5, e13.5 and e14.5. rna-seq and pyrosequencing of genomic dna was performed on e11.5 hearts, histology on e12.5 hearts and electron microscopic imaging on e13.5 hearts. for deletion of dnmt1 in adult mice, homozygous dnmt1 fl/fl mice expressing an inducible cre recombinase (myh6-mcm) were given tamoxifen i.p. over 4 days. homozygous dnmt1 fl/fl mice not carrying myh6-mcm as well as myh6-mcm carrying mice without dnmt1 fl alleles were also injected with tamoxifen and served as controls. cardiac phenotyping including histology, echocardiography and qpcr was carried out without (sham) or with left ventricular pressure overload induced by transverse aortic constriction (tac). results: myl7-cre mediated loss of dnmt1 resulted in progressive embryonic lethality with absence of living ko embryos after e14.5. ko embryos displayed loss of cardiomyocyte gene expression patterns, decreased promotor cpg methylation of aberrantly expressed genes and ultrastructural features of wide-spread cardiac myocyte cell death. in contrast, tamoxifen-induced ablation of dnmt1 in adult mice did not affect survival of ko mice. cardiac phenotyping of adult mice revealed no significant differences between ko and ctl mice under sham and tac conditions. conclusion: dna methyltransferase dnmt1 in embryonic cardiac myocytes is essential for proper heart development. in adult cardiomyocytes, dnmt1 is dispensable for normal cardiac function and for adaptation to chronic cardiac pressure overload. background: recent studies showed that mice with general deletion of the oxidoreductase tet3 involved in dna demethylation are embryonic lethal, the underlying cause remaining unknown. this prompted us to investigate wether embryonic lethality is caused by cardiomyocyte-specific loss of tet3. methods: female mice homozygous for tet3 flox and male mice heterozygous for tet3 flox and heterozygous for myl7-cre were mated and offspring were genotyped after weaning. mice homozygous for tet3 flox and heterozygous for myl7-cre (ko) or homozygous for tet3 flox without myl7-cre (controls) were sacrificed at 12 weeks of age and ventricles were harvested. mrna of the ventricles was isolated and expression of cardiomyocyte-specific genes was evaluated by quantitative real-time pcr. cardiomyocyte-specific genomic dna from ko and control mice was obtained from facs-sorted cardiomyocyte nuclei and bisulfite-converted for analysis of dna methylation by pyrosequencing. results: ko mice showed embryonic lethality of nearly 50 %. born ko mice developed without phenotypic abnormalities (normal heart weight/tibia length ratio) and displayed compensatory upregulation of tet1 and tet2. cardiomyocyte genomic dna of ko mice showed significantly higher methylation levels in the body of the atp2a2 gene and at the binding site of the transcription factor gata4 but not near the promotor and the binding site of the transcription factor tbx5. higher methylation levels were not accompanied by changes in atp2a2 expression. however, both myh7 and nppb were upregulated in ko mice compared to control mice. conclusions: our findings suggest that tet3 is involved in dna demethylation in cardiomyocytes. loss of tet3 expression resulted in embryonic lethality. compensatory upregulation of tet1 and tet2 isoenzymes may contribute to the incomplete penetrance of this phenotype. further studies are ongoing to investigate the functional relevance of tet3 in cardiomyocytes. to identify novel proteins secreted by the myocardium, we have previously conducted a genetic screen, which led to the identification of protease inhibitor 16 (pi16). a recent gwas analysis showed an association of a genetic variant in the pi16 genomic locus (rs1405069) with chemerin plasma levels. here we tested the hypothesis that pi16 determines chemerin plasma levels through regulation of chemerin processing. we generated mice deficient for pi16, which did not display an overt phenotype under basal conditions. plasma levels of chemerin were found significantly lower in pi16-deficient animals compared to littermate controls. to investigate whether pi16 and chemerin interact, we performed co-immunoprecipitation experiments. indeed, we found pi16 to co-precipitate with chemerin from both murine plasma and cell culture supernatants. as chemerin is proteolytically processed and activated, we next asked whether the presence of pi16 would affect the processing of pro-chemerin to its processed forms in native tissue. western blot analysis on cardiac and adipose tissue lysates that detected both the unprocessed precursor and the processed forms of chemerin showed a significant shift towards the processed forms upon genetic deletion of pi16. when we assayed for the activity of the chemerincleaving protease cathepsin k, we found recombinant pi16 to potently inhibit cathepsin k activity. taken together, we propose pi16 to act as a regulator of chemerin processing. the transient receptor potential canonical 6 (trpc6) is a second messenger-gated cation channel, which mediates depolarization and ca 2+ entry. it is known to be activated by diacylglycerol derivatives (dag, 1-oleoyl-1-acetyl-sn-glycerol oag) [1] in a pkc-independent manner and plays important roles in lung and kidney physiology. gain-of-function mutations in the trpc6 gene can cause focal segmental glomerulosclerosis (fsgs), a kidney dysfunction leading to end stage renal disease. [2] thus, the discovery of potent inhibitors of trpc6 may help to develop new therapeutic strategies. urban et al. discovered that larixol, a natural product with a labdane skeleton found in the oleoresin of the european larch (larix decidua), blocks the oag-dependent activation of trpc6. [3] larixyl acetate, another component of the resin showed an even higher potency in trpc6 inhibition (ic 50 = 0.26 µm) and a 12-fold selectivity compared to trpc3. these findings led to the idea that further modifications of the larixol lead structure may reveal even more potent inhibitors. furthermore, changes in selectivity and efficacy of such compounds may also provide a deeper insight about relevant structural elements for channel binding. as larixyl carbamate was assumed to exhibit a higher metabolic stability as larixyl acetate, this compound was already investigated in previous studies. it showed a potent and subtype-selective inhibition of trpc6. hence, the development of further carbamates was a priority objective. as an alternative to the use of different isocyanates for the introduction of a carbamate function at the c1 position of the molecule we found an elegant way via formation of an active ester with carbonyldiimidazole. this precursor allowed the design of several isosteric compounds like larixyl methylcarbamate, larixyl hydrazide and larixyl methylcarbonate, which were all able to block trpc6 with similarly low ic 50 values. the introduction of more bulky side chains appeared to diminish the bioactivity of the compounds, the stereochemistry at the c1 position, however, seems to play no important role for the inhibition of trpc6 currents. larixyl methylcarbamate lead to trpc6 inhibition with an ic 50 value of 0.15 ± 0.06 µm. compared to larixyl carbamate and larixyl methylcarbonate, which are also very potent blockers of trpc6, this compound bears the benefit of high subtype selectivity towards trpc3. even with concentrations up to 50 µm of larixyl methylcarbamate, no complete inhibition of the ca 2+ influx via trpc3 channels could be achieved. this fact distinguishes this larixol derivative as a very promising compound for further studies of trpc6 in health and disease. poisoning by organophosphorus compounds (opc) including pesticides and highly toxic nerve agents is based on irreversible inhibition of acetylcholinesterase (ache) resulting in an excess of acetylcholine causing accumulation. the subsequent overstimulation at nicotinic and muscarinic receptors finally leads to respiratory arrest due to paralysis of the respiratory muscles. therapy focuses on competitive antagonism at muscarinic acetylcholine receptors and reactivation of inhibited ache by bisquarternary pyridinium oximes. thereby, nicotinic malfunction is not directly approached. for that reason, an alternative strategy appears rational using nicotinic acetylcholine receptor (nachr) active substances to counteract the effects of accumulated acetylcholine and thus to restore the loss of function of nachrs. different bispyridinium-non-oxime-compounds (bps) have been demonstrated to be able to serve as target structures for the identification of new positive allosteric modulators of nicotinic receptors. unlike nicotinic agonists, positive allosteric modulators can reinforce the endogenous cholinergic neurotransmission despite of acetylcholine accumulation in the synaptic cleft. to this end, the following electrophysiological in vitro study investigated the effect of twelve diversely substituted bps on human α7 nachr using whole-cell patch clamping under voltage-clamping conditions (-70 mv) performed with planar electrodes in an automatic system (nanion technologies gmbh, munich). cholinergic currents of hα7 nachr that have been expressed in stably transfected cho cells were activated by the agonist nicotine. measurements of the effect of various bps concentrations in the presence of nicotine were performed to establish concentration-response relations. cholinergic inward currents were generated by human α7 nachrs in response to low nicotine concentrations. at high concentrations of the drug the currents were decayed reflecting both, desensitization of the receptors and presumably block of the open channel by high agonist concentrations. four out of twelve bps co-applicated with nicotine showed a concentration-dependent enhancement of peak agonist-evoked currents and, most pronounced, 4-tert-butyl-substitued-bp, also demonstrated a marked elongation of the evoked response. this suggests a positive allosteric effect of these compounds on the nicotinic receptor. however, at high bp concentrations in the presence of agonist, responses were decayed significantly, presumably resulting from an open channel block induced by bps. hence, further compounds have to be synthesized to identify promising candidate compounds for improvement of effective therapy against nerve agent poisoning. the transient receptor potential channels (trp) are a family of tetrameric nonselective cation channels, which are involved in a variety of physiological and pathological processes (1). among the 28 mammalian trp channels, the canonical channel 5 (trpc5) is a ca 2+ -permeable ion channel, which is predominantly expressed in the brain (2) . many aspects of trpc5 function are still elusive although behavioral experiments with trpc5-knock-out mice suggest a role in innate fear-response (3) and some studies indicate a trpc5-mediated down regulation of neurite outgrowth in nerve cells (4, 5) . to elucidate trpc5 function on a cellular level, selective and potent compounds are required to acutely control channel activity. despite extensive research, trpc5 modulators often lack selectivity or exhibit toxicity, limiting their applicability in vivo (6, 7) . thus, there is still a need for identifying novel and efficient trpc5 modulators. we therefore screened a compound library (chembionet) and identified a benzothiadiazine derivative (btd) as a novel, potent, and selective trpc5 activator. hek293 cells heterologously expressing trpc5 upon tetracycline induction (hek trpc5 ) show a btd-induced concentration-dependent activation in both ca 2+ assays (ec 50 = 0.71 µm) and in electrophysiological whole cell patch clamp recordings (ec 50 = 1.1 µm). btd elicits currents with an n-shaped i/v curve, typical for trpc5. the resulting activation is long lasting, reversible and sensitive to clemizole, a recently established trpc5 inhibitor (8) . mtt assays revealed that incubating hek trpc5 cells for 24h with btd concentrations above 1 µm results in a concentration-dependent decrease in viability and cell proliferation, indicating a ca 2+ -mediated cytotoxic effect in consequence of sustained channel activity. non-induced control cells remain unaffected by btd at concentrations up to 10 µm. ca 2+ assays showed no influence of btd on closely related trpc4 channels, as well as trpc3/6/7 at concentrations up to 10 µm. the same applied to more distantly related trpv and trpm channels. besides a homotetrameric organization, trpc5 subunits can also assemble to heteromeric channel complexes with their closest relatives trpc1 and trpc4 (9) . trpc1/5 and trpc4/5 heteromers can also be activated by btd as evident from their typical i/v curves in patch clamp experiments, suggesting a high selectivity of btd for channel complexes bearing at least one trpc5 subunit. transient receptor potential canonical channels 3/6 and 7 are controlled by membrane lipids and highly expressed in neuronal and cardiac tissues. the involvement of these channels in development and (patho)physiology of these tissues is well documented, while our understanding of structure-function relations, specifically in terms of the lipid sensing machinery, in these channel proteins is still incomplete. using a homology model of trpc3, based on the recently available structural information on trpv1, we performed structure-guided mutagenesis and identified a single residue in transmembrane domain 6 (g652), which is conserved within the canonical family of trp channels. single point mutations at position 652 in trpc3 largely eliminated lipid sensitivity. trpc3 g652a expressed in hek293 cells was found resistant not only to activation via the phospholipase c pathway but also to direct administration of diacylglycerols. on the contrary, a synthetic agonist of trpc3/6/7 channels (gsk1702934a) activated wild-type trpc3 and trpc6 channels as well as the respective lipid insensitive mutants (trpc3 g652a, trpc6 g709a ). interestingly, the synthetic activator was found to generate substantially enhanced trpc conductances in cells expressing the lipid-insensitive mutants as compared to wild-type proteins. closer inspection of sensitivity of the wt and mutant proteins to various gsk derivatives argue against a contribution of g652 to gsk recognition by trpc3. our results demonstrate the existence of two different mechanisms of trpc3/6 activation supposedly involving distinct gating movements in the channel complex. we suggest that lipid gating of trpc3/6 involves a hinge-point and/or requires a certain level of flexibility within transmembrane segment s6 provided by g652. lipids and synthetic activators of trpc3/6 may be capable of initiating markedly different structural rearrangement in these channels. objective: organophosphorus compounds (opcs), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (ache). inhibited ache results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nachr) in the postsynaptic membrane is provoked. as the therapeutic efficacy of oximes is limited, e.g. poisoning by soman or tabun, the direct targeting of nachr may be an alternative therapeutic approach. studies with the non-oxime bispyridinium compound (bp) mb327 (1,1'-(propane-1,3-diyl) bis (4-tert-butylpyridinium) di(iodide)) demonstrated a therapeutic effect against soman in vitro and in vivo. consequently, studying the affinity of bps at muscle-type nachrs and functional effects are topics of interest. to identify potential candidates, homologous series of substituted and non-substituted analogues (linker c 1 -c 10 ) of mb327 were investigated by using binding and functional assays. experimental procedures: crude membranes from frozen electric organ of torpedo californica were purified by sucrose-gradient density centrifugation and used in both affinity and functional assays. in competition radio-ligand binding assays, the influence on [³h]epibatidine binding sites of torpedo muscle-type nachr was determined. functional assessments were carried out with a bilayer method to investigate the effect on the cholinergic signal induced by 100 µm carbamoylcholine. results: bispyridinium compounds bearing unsubstituted pyridinium rings and long alkyl linkers (> c 7 ) inhibited the binding of [³h]epibatidine and decreased the cholinergic signal of 100 µm carbamoylcholine in the functional assay. mb327 and several bispyridinium structure analogues (mainly c 2 -c 4 linker) exhibited no regular displacement curves at [ 3 h]epibatidine binding sites and enhanced the carbamoylcholine-induced signal. the results demonstrate that the described affinity and functional screening methods detected some structure-activity-relationships (sar). depending on linker length and substitution pattern, the investigated bispyridinium compounds seemed to interact as positive allosteric modulators. further research is necessary to verify this hypothesis. non oxime bispyridinium compounds with an effect on soman-blocked respiratory muscle function have no effect on normal muscle function the life threatening toxicity of organophosphorus compounds (op), like nerve agents or pesticides, lies in the inhibition of acetylcholinesterase (ache) which causes cholinergic crisis. the accumulated acetylcholine in neuromuscular synapses results in the desensitization of nicotinic acetylcholine receptors (nachr) and paralysis of respiratoric muscles. the 4-tert-butyl-substituted bispyridinium compound mb327 showed therapeutic efficacy in soman and tabun poisoned guinea pigs in vivo. partial restauration of neuromuscular transmission by bispyridinium compounds (bp), e.g. mb327 or mb420, could also observed in soman paralysed respiratory muscles in vitro and was partly attributed to an interaction of bp with nachrs. however, it is unknown, whether these bp might affect normal respiratory muscle function in the absence of cholinergic crisis. therefore this study investigated the effect of bp on physiological rat diaphragm muscle function. force generation of rat diaphragm hemispheres was determined after incubation with increasing bp concentrations (1-300 µm) and compare to sham treatment. the diaphragm hemispheres were stimulated every ten minutes by an indirect electrical field (20, 50, 100 hz). muscle force was analyzed as time-force integral and is expressed as percentage of the individual control values, measured at the outset of the experiment. the muscle force dropped during the experiment. the application of the bispyridinium compounds mb327 (1,1′-(propan-1,3-diyl) bis (4-tertbutylpyridinium) di(iodide)) and mb420 (1,1′-(propan-1,3-diyl) bis (2-ethylpyridinium) di(iodide)) in the tested concentration range (1-300 µm) did not change muscle force production compared to the sham treated muscle. this was equally true for low (20 hz) and high (50 and 100 hz) stimulation frequencies. this study showed that bispyridinium compounds which can partially reverse somaninduced neuromuscular block in rat diaphragms show no effect on respiratory muscle function in absence of the op-induced neuromuscular block. these results suggest that the bp tested in this study interacted with desensitised nachrs only, but do not affect physiological neuromuscular transmission. this effect needs to be investigated with further, promising bp compounds. interaction of recombinant pain-relevant atp-and proton-gated ion channels in an expression system; potentiation of the p2x3 receptor-induced current by the opening of asic3 channels g. stephan 1 , p. illes 1 1 universität leipzig, rudolf-boehm-institut für pharmakologie und toxikologie, leipzig, germany the p2x3 receptor (r) is a ligand-gated cationic channel, which is activated by extracellular atp. the acid sensing ion channel 3 (asic3) belongs to the enac/degenerin family and is gated by extracellular protons. despite their different amino acid sequences both ion channels share the same structure and pore architecture, by i.e. consisting of three identical subunits. besides, they are both located at partially overlapping subpopulations of dorsal root ganglia neurons and are implicated in acidic pain signaling. consequently, their physical interaction in the cell membrane or even the formation of heteromeric receptor channels from p2x3 and asic3 subunits has to be taken into consideration. we transfected rat (r)p2x3r and rasic3 constructs individually or together in a ratio of 1:1 into cho cells. we further used the whole cell patch clamp technique to analyze the current responses either elicited by the application of α,β-methylene-atp (α,β-meatp) or by a decrease in the extracellular ph value. the functionality of the individually transfected p2x3r-and asic3-constructs was verified by recording concentration-response curves for the agonists α,β-meatp and protons, respectively. after co-transfection of both ion channels, a ph-shift from 7.4 to 6.7 caused a rapidly desensitizing current response and a subsequent strong potentiation of the α,β-meatp-induced current. an even larger potentiation was achieved after a decrease of the ph value to 6.5. the opening of asic3 channels failed to facilitate the p2x3r current when 2-guanidine-4-methylquinazoline was used to stimulate a non-proton ligand-sensor of asic3. then, we substituted ca 2+ in the extracellular medium by ba 2+ or decreased the intrapipette concentration of egta, to modify the free intracellular ca 2+ concentration. in cells individually transfected with the receptor-channels, external ba 2+ increased the effect of α,β-meatp but decreased the effect of protons. the lowering of intrapipette egta modified p2x3r-and asic3-specific currents in a similar manner as external ba 2+ . in cells co-transfected with p2x3r/asic3, the ionic manipulations mentioned above abolished the potentiation of the α,β-meatp currents by asic3 activation. taken together, our results suggest that p2x3r and asic3 interact with each other, since the activation of asic3 had a marked impact on the p2x3r specific current response. further experiments are required to clarify the mechanism of this interaction, although it has been shown that extra-and intracellular ca 2+ and the proton sensor of asic3 appear to critically participate in this process. university of duisburg-essen, institute for anatomy, essen, germany background: the sperm acrosome reaction is an all-or-none secretion process, mainly following the conserved principles of calcium-regulated exocytosis in neurons and neurosecretory cells. however, the relationship between the formation of hundreds of fusion pores and the required mobilization of calcium from the lysosome-related acrosomal vesicle has only been partially defined. hence, the second messenger, nicotinic acid adenine dinucleotide phosphate (naadp), known to promote efflux of calcium from lysosome-like acidic compartments, was analyzed for its ability to trigger acrosome reaction in mouse sperm. in addition, the expression of two-pore channel (tpc) proteins, which are primarily localized in lysosome-related acidic organelles and which present potential molecular targets of naadp were examined in mammalian spermatozoa. methodology/ principal findings: our results show that treatment of spermatozoa with naadp resulted in a loss of the acrosomal vesicle, which shows typical properties, described for tpcs: (i) registered responses were not detectable for its chemical analogue nadp, and (ii) where blocked by the naadp antagonist trans-ned-19. in addition, (iii) two narrow bell-shaped dose-response-curves were found, with maxima either in the nanomolar or low micromolar naadp concentration range. performing immungold-electron microscopy with a tpc1 specific antibody, a co-localization with naadp-binding at the acrosomal region was detectable. moreover, quantifying loss of the acrosomal vesicle in tpc1 null sperm upon application of different naadp concentrations, responsiveness to low micromolar naadp concentrations was completely abolished. conclusions/significance: our finding that two convergent naadp-dependent pathways are operative in driving acrosomal exocytosis and that zona pellucida induced acrosomal exocytosis is prevented by trans-ned-19 support the concept that both naadp-gated cascades match local naadp concentrations with the efflux of acrosomal calcium, thereby ensuring reliable and complete fusion of the large acrosomal vesicle. since the acrosome reaction shares the same basic sequence of events typical for the conserved process of calcium regulated exocytosis, such as tethering, docking, priming and final vesicle fusion, the sperm model system may also be useful to comparatively examine whether the same convergence of naadp-dependent pathways is also operative in cellular systems with many secretory vesicles. walther-straub-institut, münchen, germany trpv4 channels are members of the vanilloid family of trp proteins. the channel is nearly ubiquitously expressed and can be found in brain, kidney, skin, heart, blood vessels as well as in the lung. pulmonary expression of trpv4 has been identified in endothelial cells (1), epithelial cells (2) and arterial smooth muscle cells (3) . most interestingly, the channel is known to be involved in the development of several lung diseases such as cough, asthma and pulmonary edema formation, due to its activation by heat, changes in osmolarity and shear stress (reviewed in 4). it is a matter of debate however if trpv4 activation in pulmonary endothelial as well as epithelial cells induces disruption of the barrier and an increased fluid leak into the alveolus as described for trpc6 (5) which is also expressed in both cell types. to analyze the potential role of trpv4 on ischemia-reperfusion-injury(iri)-induced pulmonary edema formation we utilized the isolated perfused mouse lung model. much to our surprise, we detected a significantly enlarged edema formation after 90 minutes of ischemia in trpv4-deficient mice in comparison to wild-type (wt) mice. this effect was observed by constant weight measurements as well as wet-to-dry ratio gain and was not dependent on the initial perfusion rate. most interestingly, edema formation of trpv4/trpc6 double deficient lungs was indistinguishable from wt lungs, indicating antagonizing effects of both channels, because trpc6 deficiency protected lungs from iri-induced edema (5) . moreover, we identified reduced expression levels of aquaporin 5 in trpv4-deficient lung lysates compared to wt lungs. these findings raise the intriguing possibility that trpv4 might be involved in the regulation of aquaporin expression in lung endothelial cells. endosomes and lysosomes are cell organelles involved in transport, breakdown and secretion of proteins, lipids, and other macromolecules. endolysosomal dysfunction can cause storage disorders such as mucolipidoses, sphingolipidoses, or neuronal ceroid lipofuscinoses, but is also implicated in the development of metabolic and neurodegenerative diseases, retinal and pigmentation disorders, trace metal dishomeostasis, infectious diseases, and cancer. endolysosomal ion channels and transporters are highly critical for the tight regulation of the multiple endolysosomal fusion and fission processes including endo-and exocytotic events as well as the regulation of proton and other ionic concentrations in the lumen of endolysosomal vesicles. methods to patch-clamp endolysosomal organelles are continuously improving. yet until now it has not been possible to selectively enlarge endosomal or lysosomal organelles with pharmacological tools for patch-clamp experimentation. we show here by using a combination of two small molecules that we can selectively enlarge early endosomes to a degree sufficient for patch-clamp experimentation. the ability to more selectively patch-clamp intracellular organelles will substantially improve the functional investigation of endolysosomal ion channels under physiological and pathophysiological conditions. the transient receptor potential (trp) channels are a superfamily of non-selective ion channels involved in a variety of physiological processes and in the pathgenesis of many disorders. in the kidney, trp channels have been implicated to be involved in diabetic nephropathy, focal, segmental glomerulosclerosis, polycystic kidney disease, hypomagnesemia with secondary hypercalcemia and idiopathic hypercalcuria. the melastatin-like trp channel subfamily 3 (trpm3) has been shown to be expressed in human kidney 1, 2 . using newly developed anti-trpm3 antibodies, we are able to visualize trpm3 protein in epithelial cells of proximal tubule as well as collecting ducts in the mouse kidneys. therefore, we compared renal function of male, five month old mice lacking trpm3 (trpm3 the atp-gated p2x7 receptor (p2x7r) is a non-selective cation channel widely expressed in epithelia, endothelia, and cells of hematopoietic origin. it plays a central role in cytokine release and studies in p2x7 -/animals indicate its involvement in inflammatory and neurodegenerative diseases. in addition, accumulating data suggests a functional role in neurons and its involvement in neurotransmitter release in the brain. however, despite its importance as a drug target, its precise localization and its molecular and physiological functions remain poorly understood. in particular, the location and function of p2x7r in neurons remain a matter of ongoing debate. to clarify the cellular and subcellular distribution of the p2x7r and to investigate its physiological and pathophysiological role in the brain we generated bac transgenic mouse models in which murine polymorphic variants of egfp-tagged p2x7r are overexpressed under the control of the endogenous p2x7 promoter. the egfp-tagged p2x7rs are efficiently overexpressed in the plasma membrane and can be directly visualized by green fluorescence or indirectly by anti-egfp antibodies. the obtained mouse lines show different expression levels but identical expression patterns with predominant expression in the cerebellum, hippocampus, and thalamus. using cell type-specific markers, p2x7-egfp was identified in almost all microglia and subpopulations of oligodendrocytes and astrocytes in the brain. in the spinal cord, numerous astrocytes in the white matter showed egfp immunoreactivity. so far, no egfp immunostaining was found on map2-and neun-positive cells indicating that under non-pathological conditions, the p2x7 receptor is not expressed in neurons of the cns. interestingly, a higher expression level of cd68 protein was observed in the p2x7r-overexpressing mice. these results suggests that overexpression of p2x7rs alone is sufficient to induce microglia activation, even under non-pathological conditions. since cd68 primarily localizes to lysosomes and endosomes, this further supports a role of the p2x7r in the regulation of phagocytosis. to validate these data, conditional knockout mice are generated. the current status of the project will be presented. the two-pore channels (tpcs) -tpc1 and tpc2 -are located in membranes of intracellular organelles of the endo-lysosomal system. the tpc-protein-monomer contains two homologous domains with six transmembrane α-helices each. a functional tpc probably consists of a dimer of two tpc-proteins resembling an ion channel architecture with the typical four domain organization like voltage gated na + or ca 2+ channels or such as trp channels. due to their biophysical properties, tpc1 and tpc2 are assumed to be involved in the efflux of ca 2+ from intracellular organelles and thereby contribute to fusion/fission processes of endosomes and lysosomes. thus, tpcs are supposed to be important regulators for vesicle trafficking, sorting and degradation/recycling processes. recently, it was shown that virus entry and replication of certain strains of filoviridae -such as ebola -depends on functional tpcs and that either block or genetic inactivation of tpcs reduces virus infectivity. a large family of bacterial protein toxins elicit their effects by modification of intracellular target proteins of host cells. these toxins are taken up by receptor mediated endocytosis and follow different endosomal routes to reach their final cytosolic destination. these toxins principally use two different intracellular routes: the first group uses an entry route via early or late endosomes (short-trip toxins), the second group takes a retrograde route via endosomes, golgi network and the endoplasmic reticulum (long-trip toxins) to get access to the cytosol. translocation of short-trip toxins -such as diphtheria toxin (dt), pasteurella multocida toxin (pmt) and bacillus anthracis lethal factor (pa/lf) -from early and late endosomes into the cytosol is driven by ongoing acidification. long-trip toxins -including cholera toxin (ct) -are retrogradely transported after endocytosis via the golgi apparatus to the endoplasmic reticulum (er). within the er a specific peptide-motif allows the translocation into the cytosol. due to the role of tpc1 for vesicle fusion & fission processes we investigated a potential impact of tpc1 on the uptake of bacterial toxins. first we determined the precise localization of tpc1 in intracellular compartments. to deduce its role for trafficking processes we performed co-localization and correlation studies with a whole set of established markers such as rab-gtpases and pips. second we intoxicated wild-type and tpc1 deficient cell lines with different bacterial protein toxins such as cholera (ct), diphtheria (dt) or pasteurella multocida (pmt) toxin. using cell viability and other intoxication assays we investigated the consequences of tpc1-deletion on bacterial toxin uptake, translocation and cytotoxicity. universität des saarlandes, institut für experimentelle und klinische pharmakologie und toxikologie, homburg/saar, germany trpm3 ion channels are considered to be involved in hormone release from pancreatic islets and the pituitary gland 1,2 . the trpm3 gene encodes a number of different splice variants that differ in their permeation properties and their activity in response to agonists 3, 4 . variations include the presence or absence of five stretches of 10 to 25 amino acid residues within the aminoterminus, long or short pore loops and long or truncated carboxytermini 5 . furthermore, three different aminotermini of human and mouse proteins have been described 5 , but presumably these differences are caused by the activity of different promoters. screening of a mouse pituitary gland cdna library identified 12 variants that differ in exons 8, 13, 15, 17 and 20 . however, only variants bearing the short, ca 2+ permeable pore loop were detected. 3´ rapid amplification of cdna ends (3´race) revealed that trpm3 proteins of the pituitary gland carry truncated c-termini exclusively. 5´race identified five independent regions of transcription initiation within the trpm3 gene implying the presence of five independent promotors. the different transcripts encode four trpm3 amino termini α, β, γ and δ of 1-155 amino acid residues including those described in humans (β,γ). however, the activity of these variants after stimulation with pregnenolone sulfate varied largely just as their frequency in the pituitary with shortened γ-variants being most abundant (~76 %). two-pore channels (tpcs) are a small family of ion-channels found throughout the endolysosomal system of eukaryotic cells. phylogenetically tpcs belong to the voltagegated ion channel superfamily sharing common traits with ca v / na v and trp channels. tpcs show a duplicated architecture with two homologous trans-membrane domains. each domain is build up by six membrane spanning alpha helices linked by short loops. it is very likely that tpcs form dimers maintaining the four-fold symmetry found in other members of the voltage-gated ion channel superfamily. due to their localization in the endolysosomal system tpcs are not accessible to conventional patch clamping. to investigate tpcs electrophysiological properties we use black lipid bilayer measurements. purified channels are integrated into an artificial phospholipid bilayer that separates two chambers enabling us to apply different buffers. upon activation by naadp ions flow through the channel and can thereby pass the diffusion barrier. the movement of charged molecules through tpcs results in currents which can be amplified and recorded. the controlled environment of the lipid bilayer setup allows testing of different ions as well as putative activating and inhibitory substances. one of the major drawbacks of conventional lipid bilayer setups are long preparation times between each measurement. stability of the phospholipid bilayer can pose another issue. often several membranes have to be established before a measurement can be performed making it very time consuming to achieve adequate experiment numbers. new multichannel systems resolve this issue supporting fast formation of bilayers while allowing measurement of up to 16 different bilayers at a time. here we utilize the "orbit" multichannel system with a meca16 chip by ionera to measure tpc1 channel activity. we will present data generated by the "orbit" system and a conventional bilayer setup using different channel constructs and charge carriers. cardiac action potentials are generated and propagated through the coordinated activity of multiple ion channels, including voltage-gated sodium channels (nav1) and potassium channels (like kv1, kv4). the voltage-gated na + channel nav1.5 initiates the cardiac action potential (ap), is essential for rapid depolarization, and is also known to control the ap duration in cardiomyocytes. the voltage-gated k + channel kv4.3 is responsible for the early repolarization of action potentials in human heart. similar to many membrane proteins, nav1.5 and kv4.3 have been found to be regulated by several interacting proteins. the transmembrane β subunit dipeptidyl aminopepidase-like protein (dpp) 10 is known to interact with the kv4.3 channel complex, modulating kinetics and voltage dependence. the overexpression of dpp10 in ventricular cardiomyocytes of rats revealed strong reduction of ap amplitude and significant slowing of ap upstroke velocity and ap duration, which could not be explained by the effects on cardiac kv4 channels. to study the potential influence of dpp10 on nav1.5 channels, we performed whole-cell patch-clamp analysis of transiently transfected cho-k1 cells, expressing scn5a alone or with dpp10. surprisingly, we observed significant effects of dpp10 on nav1.5 channel voltage dependence and kinetics. thus, the co-expression of dpp10 significantly shifted the half-maximal voltage of steady-state activation and steady-stateinactivation to more positive potentials compared to nav1.5 channels alone. in addition we analysed the effects of dpp10 on the kinetics nav1.5 currents. while time to current peak was not affected in cells co-expressing nav1.5 and dpp10 compared to nav1.5 alone, dpp10 slightly accelerated the inactivation. in addition, the time course of recovery from inactivation was clearly accelerated in cells expressing both nav1.5 and dpp10 compared to nav1.5 alone. in summary, we provide first evidence that dpp10 not only interacts with kv4 channels, but also influences nav1.5 channels modulating the depolarization as well as the early repolarization phase of the cardiac ap. therefore, it becomes likely that these ion channels are part of large, multi-protein complexes, and that the pore-forming subunits kv4.3 and nav1.5 behave very differently depending on the expression of its associated proteins like dpp10. cardiac fibroblasts (cf) comprise the most abundant cell type of the mammalian heart and it is known that they contribute to maladaptive cardiac remodeling processes. in response to pressure or volume overload, ischemia-reperfusion injury or myocardial infarction, cardiac fibroblasts proliferate and transdifferentiate into myofibroblasts which produce collagen and pro-hypertrophic cytokines influencing cardiomyocyte function and size. it was shown that β-adrenergic stimulation of cfs with isoproterenol leads to angiotensin ii (at-ii) production and autocrine stimulation of these cells (jaffre et al, 2009 ). activation of phoypholipase c triggered by at-ii leads to formation of inositol trisphosphate (ip 3 ) and subsequent release of calcium from intracellular stores as well as calcium entry across the cell membrane. the focus of our research is the identification of the plasmalemmal channel proteins such as trpc channels mediating this calcium entry, and whether these calcium entry pathways in cfs contribute to pathological remodeling. to date the precise role of calcium entry for these pathological processes is largely unknown. trpc channels are candidates for the analysis of calcium homeostasis in cf. recently, we showed that trpc1/c4-deficient mice are protected from maladaptive cardiac remodeling after neurohumoral stimulation or pressure overload, respectively, which can be explained by a significant reduction of a background ca 2+ -entry (bcge) pathway in cardiomyocytes; this bgce is enhanced by stimulation with agonists such as isoproterenol or angiotensin ii and it critically depends on trpc1 and trpc4 (camacho londoño et al., 2015) . nevertheless, the role of trpc1/c4 for calcium homeostasis in cfs has not been analyzed so far. we established an in vitro model that allows the analysis of calcium release and entry triggered by several (patho)physiological agonists in cultured primary adult cfs from mice. cfs were isolated using langendorff-perfusion and were cultured for maximal 6 days. our results show that there is no difference in 100 nm at-ii induced ca 2+ -release or ca 2+ -entry in trpc1/c4-deficient cf compared to wt. to evaluate whether the lack of trpc1/c4 can be compensated by other trpc channels we currently analyze cfs from trpc hepta ko mice lacking all seven trpc channel proteins concerning at-ii induced ca 2+ -release and ca 2+ -entry and we will also analyze the influence of other agonists on cfs which are known to evoke a longer lasting rise in the [ca 2+ ] i like isoproterenol, 5-ht, thrombin and endothelin-1. cardiovascular and metabolic diseases are currently the primary cause of morbidity and mortality in the western world and are spreading to the rest of the world following globalization. adipose tissue, in particular perivascular adipose tissue (pvat) is recognized as an important player in the development of these diseases. the release of relaxing factor(s) from the pvat has been a matter of interesting and highly spirited debates about its nature, the channels that govern its activities and its role in vascular dysfunction. data from our laboratory indicate that adipose-derived relaxing factor (adrf) is an important player, however the potential channels necessary for its downstream activities are still under study. our recent research primarily focuses on kv7.1 channels, which are known to be expressed in vascular smooth muscle cells. k v 7.1 voltage-gated potassium channels are expressed in vascular smooth muscle cells (vsmc) of diverse arteries, including mesenteric arteries. based on pharmacological evidence using r-l3 (k v 7.1 opener), hmr1556, chromanol-293b (k v 7.1 inhibitors), these channels have been suggested to be involved in the regulation of vascular tone. however, the specificity of these drugs in vivo is uncertain. we used kcnq1-/-mice to determine whether k v 7.1 plays a role in the regulation of arterial tone. we found that r-l3 produces similar concentration-dependent relaxations (ec 50 ~1,4 µm) of wild-type (kcnq1+/+) and kcnq1-/-arteries pre-contracted with either phenylephrine or 60 mm kcl. this relaxation was not affected by 10 µm chromanol-293b, 10 µm hmr1556 or 30 µm xe991 (pan-k v 7 blocker). the anti-contractile effects of pvat were normal in kcnq1-/-arteries. chromanol-293b and hmr1556 did not affect the anti-contractile effects of perivascular adipose tissue (pvat). isolated vsmcs from kcnq1-/-mice exhibited normal peak k v currents. the k v 7.2-5 opener retigabine caused similar relaxations in kcnq1-/-and wild-type vessels. we conclude that k v 7.1 channels are apparently not involved in the control of arterial tone by alpha 1 adrenergic vasoconstrictors and pvat. in addition, r-l3 is an inappropriate pharmacological tool for studying the function of native vascular k v 7.1 channels in mice. introduction: according to international guidelines [1] [2] [3] [4] [5] [6] [7] , both human and animal skin in vitro models have been used and validated to predict percutaneous penetration in humans. excellent correlations have been found for domestic pig as surrogate for human skin [8] [9] . material and methods: tissue the skin samples are descended from female pigs of german landrace (50 kg weight, approx. 4 month). process approved under german welfare law. after narcotization and euthanization the animals were shaved with an electric shaver, washed and dried. microbiological investigation swabs from 10 different skin area (fig. 1) were taken before next preparation step. skin areas were cut, stretched and subcutaneous fatty tissue was carefully removed. the skin was harvested at thickness of 1.000 µm by dermatome. after dermatomization, samples were taken for histological examination. skin disks of 30 mm were punched out from the frozen skin stripes and stored at -20°c. hplc and skin absorption waters corporation hplc containing 2767 sample manager, 2545 binary gradient pump, 2998 pda detector (optional: 3100 electron spray mass spectrometer); column: nucleodur® 100-5 c18 ec 50mm x 4.0 mm id. hanson microette™ vision® diffusion test system (hanson vision® autoplus™ autosampler/autofill™ collector, 6-cell drive system with vertical diffusion cell "standard". the permeation experiment was performed over a period of 48 h at 32°c. the dosage compartments of each cell were filled with approximately 300 µl of the caffeine solution (10 mg/ml). samples of 1 ml are taken after 0, 12, 24, 36 and 48 hours from receptor medium of each cell. aliquots from each vdc are analyzed by hplc in duplicate. microbiology staphylococcus spp. were found explicitly on pig skin surface. bacteria are facultative anaerobe, gram-positive bacteria that are physiologically colonizing the skin, oropharynx and the gastrointestinal tract. histology and skin thickness he staining and mechanical skin thickness determinations confirmed intact dermatomizing process of skin (fig. 2) . thickness was in the order of magnitude between 897 to 1.230 µm and intra variations were less than 10 %. caffeine skin absorption permeability coefficients and lag-phases recorded are in the same order of magnitude of previous work [10], demonstrating intact barrier properties of the membranes after 3 month storage process. until today, intra-assay variations are superior or equal to interregional and inter-animal variations. discussion: well characterized dps1000 provides ready to use research tool for locally and systemic skin investigations. ongoing experiments will cover skin structure analysis by raman spectroscopy, biophotonics and storage impact on skin barrier function. respirable biopersistent granular dusts (gbs) should fulfill the criteria of i.) a negligible solubility in physiological lung fluid that ii.) do not exhibit a specific surface chemistryrelated toxicity at volumetric non-overload conditions in lungs. in 2012, the mak commission derived a new threshold value of 0.3 mg/m 3 for gbs with a density of 1, recognizing that at this concentration a chronic inflammation and increase of the lung cancer risk will not occur. -the objectives of the project were i.) to determine an experimental dissolution value for 'low soluble' gbs using six candidate dusts; ii.) in addition, to measure the inflammatory response in lung lavage fluid and to decide on the criterion 'inert dust'; iii.) to investigate whether nanoscaled dusts could possibly fulfill the criteria to be included in the gbs class. -six micro-and nanoscaled dusts (one of them a well-characterised inert tio 2 dust (microscaled; rutile modification) were compared analysing the solubility in the lung fluid (day 3, 28 and 90) and the lung toxicity after intratracheal instillation in rats (day 3 and 28): tio 2 (rutile, micro), tio 2 (anatase, nano), eu 2 o 3 (micro-nano mixed), baso 4 (micro), zro 2 (micro) and amorphous sio 2 (nano). two doses of 0.5 and 1.5 µl per rat were administered to wistar rats; these volume doses resulted in a non-overload and moderate overload of lungs, respectively. -the differential cell count showed only slight inflammatory cell levels after treatment with tio 2 (rutile) and baso 4 (pmn < 5% after 3 days in the low dose group; < 15% in the high dose group; full recovery after 28 days). in contrast, the tio 2 (anatase) showed a stronger response (pmn > 30% after 3 and 28 days). the rare earth eu 2 o 3 (micro-nano) dust showed the strongest effect (approx. 40% pmn after 3 and 28 days) including a red-coloured lung lavage fluid. µ-zro 2 and amorphous sio 2 showed a strong acute response after 3 days, however, mostly complete recovery after 28 days. the low solubility criterion was met by the following dusts: tio 2 (both) and zro 2 . -similar volumetric lung burdens were deposited in a parallel validation experiment (14-day subacute inhalations). overall the physiological inhalation route confirmed the results obtained in the instillation study, thus suggesting the applicability of the latter as a tool for identification for gbs dusts. however, for nanoscaled dusts an individual toxicological characterization seems to be adequate. polycyclic aromatic hydrocarbons (pah) represent a complex mixture of compounds and occur in considerable amounts as contaminants in the environment and food. some pah have been demonstrated to be carcinogenic and mutagenic. benzo[a]pyrene (bp), the most known and studied member of the potent carcinogenic pah, is classified by iarc as group 1 carcinogen and is present in a wide variety of food items. however, other non-carcinogenic pah such as pyrene (pyr) and fluoranthene (fa) are also found in substantial amounts in the diet and are strongly suspicious to cause interactive effects. reporter gene assays were used for analyzing interactive effects of a ternary mixture including bp, pyr and fa in relative proportions occurring in food on the nuclear receptors aryl hydrocarbon receptor (ahr) and constitutive androstane receptor (car). the observed activations were verified at the gene expression level in the human hepatoma cell line heparg. beside the well characterized ligand bp, 25 µm of pyr and 20 µm fa also activated the ahr, even though to a much lesser extent. no significant higher activation over the level of bp alone was reached when testing different pah mixtures. however, in heparg cells the analysis of cyp1a1 gene expression as a model target gene of ahr showed synergistic effects after pah co-exposure. in addition, the activation of human car was analyzed. pyr and fa are each strong agonists, whereas bp was less potent. for the ternary pah mixture with bp a strong decrease of the induction was observed. this inhibiting effect was verified at the mrna level using the model car target gene cyp2b6 in heparg cells. in conclusion, really occurring mixtures with non-carcinogenic pah can modulate the effects of carcinogenic pah. such effects warrant to be investigated in more detail to enhance our knowledge of interaction of pah mixtures at the molecular level. cytochrome p450 enzymes and transporters are important for the turnover of pharmaceutical compounds. their expression levels and activity influence bioavailability and convey drug-drug interactions. moreover, transporters mediate barrier maintenance of several organs such as blood-brain-barrier and placenta-barrier. overexpression of export transporters in tumors can lead to multiple drug resistance. however, membrane associated proteins are difficult to quantify by conventional bioanalytical methods such as sandwich immunoassays because of their hydrophobicity. antibody -based analysis of cytochrome p450 enzymes and transporters is challenging due to their sequence homology. therefore, we developed a test system for protein quantification which combines the sensitivity of immunoprecipitation and the specificity of mass spectrometry: this method is especially convenient for hydrophobic proteins because denatured samples are analyzed on peptide level. one peptide from each protein, which can be assigned unambiguously, is identified via tandem ms and quantified by means of an isotope labeled reference. prior to ms-read-out, the peptides are enriched by antibodies which recognize a very short c-terminal epitope. these epitopes are selected in such way that they are common in peptides derived from target proteins and therefore allow the analysis of protein groups with few antibodies. the major advantage of this method is that whole cell or tissue lysates -without preparation of microsomal fractions -can be used for quantification by lc-ms. also, samples from different model organisms can be analyzed with the same assay which enhances the comparability of experiments. physiologically based toxicokinetic modeling (pbtk) is an in silico tool to predict compound kinetics based on test substance related properties and physiological parameters of the organism. pbtk is a key element for inverse dosimetry to relate effect concentrations in vitro to external, e.g. oral doses. in our investigations, we use 8 compartment models for the rat including adrenals and testes or ovaries. test substance specific properties taken for pbtk modeling are molecular weight and logp o/w as well as ivis based tissue specific partition coefficients, hepatic clearance, intestinal permeability and plasma protein binding. berkeley madonna software was applied to solve consequent differential equations. here we present the above described model for the 3 test substances bisphenol a (bpa), fenarimol (fen) and ketoconazole (keto). using the lowest effect concentrations (loecs) of bpa, fen and keto from 1) an in vitro yeast based assays with human estrogen and androgen receptor combined with a reporter gene and 2) the interaction of steroidogenesis model calculations were made to relate in vitro concentrations to oral doses in the rat. model calculations, based on in vitro loecs of 10 µm (bpa), 3 µm (fen) and 0.01 µm (keto), for concentrations in target organs resulted in estimated oral loels of 16, 4 and 0.04 mg/kg. when calculations were made for plasma levels oral loels were estimated to be 608, 77 and 16 mg/kg for bpa, fen and keto, respectively when compared to existing in vivo data with endocrine related loels of 375 mg/kg bw day for bpa (1), 50 mg/kg day for fen (2) and 6 mg/kg day for keto (3) , it can be concluded that for the exemplary test substances addressed, ivis related risk assessment approaches based on target tissues seems overpredictive, whereas plasma related loels were closer to the in vivo situation, to study the activation of the nuclear receptor pxr a gal4/uas-based pxr transactivation assay was used. the pxr-mediated induction of cyp3a4 promotor activity was investigated using a pxr-dependent cyp3a4 reporter gene assay. cyp3a4 induction was analyzed at the mrna and protein levels in hepg2 cells using qpcr and western blot. to cover the most frequently occurring pa structures (retronecine, heliotridine and otonecine type as well as monoester, open-chain diester and cyclic diester) the four pa senecionine, heliotrine, echimidine and senkirkine were selected as representative pa for initial analyses. out of the four investigated pa only echimidine activated pxr. accordingly, pxrmediated induction of cyp3a4 promotor activity could only be detected for echimidine. cyp3a4 induction by echimidine was verified at the mrna and protein level in hepg2 wildtype and hepg2 pxr-overexpressing cells. taking heinrich-heine universität düsseldorf, institut für toxikologie, düsseldorf, germany introduction: in higher concentrations, the blood pressure regulating hormone angiotensin ii (ang ii), leads to vasoconstriction, hypertension and oxidative stress by activation of the renin angiotensin system (ras). here we investigate if nadphoxidases are responsible for ras-mediated oxidative stress in kidney and heart. nadph-oxidases (7 isoforms are known, nox 1-5, duox 1 & 2) are membrane-bound enzymes that produce reactive oxygen species (ros) for example during the immune response and cell signaling. material and methods: to clarify the role of nadph-oxidases, wildtype mice and nox 1-, nox 2-and nox 4-deficient mice were equipped with osmotic minipumps, delivering ang ii in a concentration of 600 ng/kg during 28 days to stimulate high blood pressure. kidney and heart were investigated for steady-state ros levels and dna damage (dna single and double strand breaks). results: in wildtype mice, ang ii leads to hypertension, a declined renal function, formation of ros in kidney and heart and to dna single and double strand breaks in the kidney. all nox-knockout mice exhibited ang ii-mediated hypertension and albuminuria. the lack of nox 2 and nox 4 could neither protect from the formation of oxidative stress in the kindey nor from dna double strand breaks in the kidney. initial findings from the nox 1-knockout mouse do not show an increase in dna double strand breaks in the kidney. discussion: contrary to published results of nox 1-knockout mice, we observed a constant rise in blood pressure over the treatment period compared to the control. this can possibly be due to different ang ii doses. in nox 4-knockout mice we observed increased oxidative stress and increased renal dna damage already in untreated control animals, which is in line with reports suggesting a protective effect of nox4. conclusion: separate eliminations of 3 nadph-isoforms did not allow the identification of the enzyme which is responsible for ang ii-induced oxidative stress. a possible explanation is that oxidative stress is caused by more than one nox-isoform or other enzymes like xanthine oxidase or nitrogen synthase take major part in the formation of ros. of mice (rats, cats, dogs, monkey) and men -how to measure kidney biomarkers across species introduction and objectives: there is a need for reliable biomarker assays to detect organ toxicity induced by drug candidates. in the last 50 years about 60 drugs were withdrawn from the market due to liver and/or kidney damage. for the detection of druginduced organ injury, safety-tox studies in rodents, dogs, non-human primates and humans are mandatory in the drug development process. currently, several protein biomarkers for kidney, liver and cardiovascular organ toxicitity are being clinically validated by international consortia like the safer and faster evidence-based translation (safe-t) or the predictive safety consortium (pstc). we are developing mass-spectrometry-based immuoassays suitable for the detection of these markers in animal models to support these efforts method: urinary proteins are proteolytically digested to peptides using trypsin. subsequently synthetic isotope-labelled peptide standards are spiked in at known concentrations. we employ multi-specific antibodies (txp-antibodies) targeting cterminal amino acid motifs for the enrichment of peptides derived from the protein biomarkers. finally, the protein biomarkers are quantified using nanolc-parallel reaction monitoring-ms. the use of our group-specific txp-antibodies allows protein analysis of samples from different species using the same antibody. results and discussion: we established an ms-based immunoassay platform for the analysis of kidney (diki) injury protein biomarkers in urine across 5 species; human, cynomolgus, mouse, rat and dog. we analyzed the potential diki biomarkers aquaporin 2, podocin, synaptopodin, retinol-binding protein 4, clusterin and osteopontin in urine samples from toxicity studies in cynomolgus monkeys, rodents and humans. conclusion: the application of group-specific txp-antibodies and mass spectrometry allows the quantification of biomarkers in urine of all relevant model organisms. the results strongly support the validation of translational drug-induced organ injury protein biomarkers. although effective anticancer-therapeutic regimen are available, they are accompanied by severe adverse effects on normal tissue, for instance chemotherapy induced peripheral neuropathy (cipn) caused by platinum compounds. the pathophysiology of this clinically highly relevant side-effect is still unknown and neither prophylaxis nor specific treatment is available. therefore, further research elucidating the underlying molecular mechanisms of platinating anti-tumor drugs leading to cipn is required as basis for future development of preventive or therapeutic strategies. in general, platinum compounds lead to cell death mainly via dna damage induction (mostly intrastrandcrosslinks) and through interference with the redox homeostasis of cells. here, we introduce and suggest the well-known nematode model organism c. elegans to elucidate mechanisms of neurotoxicity triggered by platinating agents. so far, we determined doses for cis-and oxaliplatin, which have only moderate effects on development, reproduction and body movement (muscular read-out). however, these doses are sufficient to trigger apoptosis in c. elegans and to induce a considerable amount of 1,2-intrastrand crosslinks in dna (measured by south-western blotting). even more important they lead to strong neurotoxicity in a functional read-out (pharyngeal pumping). with regard to redox homeostasis, we determined the oxidative stress resistance showing that e.g. cisplatin sensitizes c. elegans to reactive oxygen species (ros), which could be prevented if worms were co-or pretreated with n-acetylcysteine. furthermore we determined the level of ros in living c. elegans after treatment with platinating agents and also in combination with protective compounds. using the advantages of c. elegans as a genetic model system, we will further clarify the relevance of different defense mechanisms, including dna repair (nucleotide excision repair, base excision repair), detoxification systems (antioxidative stress factors, metallothioneins) as well as drug transporters and signaling proteins. this will be achieved by using rna interference approaches that allow targeting either the whole animal or specific tissues (i.e. neurons) only. first results of this approach will be presented. finally we aim to use this setup to identify neuroprotective compounds that prevent chemotherapy induced peripheral neuropathy induced by platinating anti-tumor drugs. clostridium botulinum c3 exoenyzme (c3) exclusively adp-ribosylates rhoa, b and c leading to reorganization of the actin cytoskeleton and morphological changes. in addition to the enzyme-based inhibition of rho-gtpases, c3 promotes in an enzymeindependent manner axonal and dendritic growth in neurons. as c3 lacks the canonical binding and translocation domains of bacterial protein toxins, cell entry is currently not well understood. based on overlay assays and mass spec analyses the intermediate filament vimentin was identified as the putative membrane receptor for c3. knock down of vimentin by sirna and application of the selective vimentin disruptor acrylamide led to a significantly delayed uptake of c3. moreover, addition of extracellular vimentin to cells induced an enhanced uptake of c3. proof of principle experiments in astrocytes and neurons from vimentin knock out mice showed c3-induced morphological changes (astrocyte stellation and axon growth) to a reduced extent and a significantly delayed uptake of c3 compared to wild type cells. as vimentin knock out did not completely inhibit c3 uptake into cells, an additional uptake mechanism or additional receptor for c3 is likely. nevertheless, our data reveals that c3 employs a specific endocytosis mechanism with involvement of the intermediate filament vimentin to gain access to host cells. the primary target organ of organic hg species-mediated toxicity is the central nervous system (cns). humans are exposed to organic hg mainly in the form of methylmercury (mehg) via the consumption of contaminated fish and other seafood products. in terrestrial food sources hg is mostly found as inorganic hg. thiomersal is a further organic hg compound which is used as a preservative in medical preparations. exposure to organic hg promotes primarily neurological effects. the understanding of transfer mechanisms regarding the cns is an important precondition for an evaluation of hg species-induced neurotoxicity. thus, primary porcine in vitro models of the bloodbrain barrier and the blood-cerebrospinal fluid (csf) barrier were used to investigate effects of mehgcl, thiomersal and hgcl 2 on the barriers as well as transfer properties into and out of the cns in vitro. the results show significant transfer differences of the various incubated species as well as in the different barrier systems. whereas the bloodbrain barrier seems to account for the transfer of organic hg species from the blood side to the brain side, these species are transferred in the contrary direction by the blood-csf barrier. inorganic hgcl 2 was not transferred across both brain barriers towards the brain side but was able to leave the brain side across the blood-brain barrier. additionally, cytotoxic effects of the hg species by themselves as well as the combination of organic and inorganic hg species have been investigated in human astrocytes and human differentiated neurons. differentiated neurons were much more sensitive towards all hg species. organic species exerted stronger cytotoxic effects in both cell types as compared to hgcl 2 . interestingly, a coincubation of organic and inorganic hg species led to an increased cytotoxicity in the astrocytes. this cocytotoxic effect is currently investigated in differentiated neurons. the species-specific differences with respect to both, effects on and transfer across the blood-brain and the blood-csf barrier in vitro as well as toxic effects in brain target cells, clearly emphasizes the necessity for comparative analyses. introduction: the neural crest is a multipotent stem cell population that arises at the neural plate border during early fetal development. neural crest cells (nccs) migrate to target sites in the periphery, where they differentiate into multiple cell types, including melanocytes, cranial bones and peripheral neurons. failure of ncc migration can lead to severe disorders, such as hirschsprung's disease. aim: to test whether toxicants interfere with human ncc migration, a high-throughput migration assay was established. this test system was used to screen an 80 compound library of potential developmental toxicants. methods: nccs were derived from human embryonic stem cells. the cells were allowed to migrate for 24 h before toxicants were added to the cells. migration and viability of the cells were then measured after another 24 h by high-content image analysis and a custom-developed software package. results: the screening library was assembled by the us national toxicology program (ntp) and consisted of different substance classes, e.g. organophosphates, organochlorines, drug-like compounds, pesticides and polycyclic aromatic hydrocarbons (pahs). out of the tested potential developmental toxicants, 26 compounds reduced ncc migration at non-cytotoxic concentrations. hit-confirmation testing confirmed 23 of the compounds as concentration-dependent inhibitors of ncc migration. among the potential developmental toxicants identified here, there were several organophosphates (e.g. chlorpyriphos) and drug-like compounds as well as polybrominated diphenyl ethers (pbdes) and organochlorine pesticides (e.g. ddt and dieldrin), while none of the tested pahs inhibited ncc migration. the negative controls in the screening library, like acetylsalicylic acid, acetaminophen and saccharin, proved to be non-toxic. conclusion/outlook: the newly established test system allows screening of potential developmental toxicants in a high throughput manner for interference with human ncc migration. confirmation in other types of migration assays is ongoing, and selected compounds from amongst the screen hits are undergoing mechanistic evaluation. oxidative stress is regarded as a major trigger for neuronal dysfunction and death in the ageing brain and in multiple neurodegenerative disorders. how oxidative stress mediates neuronal death and whether the associated mechanisms are accessible for therapeutic intervention strategies is not clarified. increasing evidence suggests, however, that oxidative stress triggers molecular mechanisms of regulated necrosis that involve the activation of receptor interacting protein 1 (rip1) independently of death receptor activation. here, we show that erastin-induced ferroptosis which involves inhibition of the glutamate-cystein transporter (xc -), glutathione depletion and lethal formation of reactive oxygen species (ros) 1 , triggers mechanisms of regulated necrosis independent of tnfα-signaling. in hippocampal ht-22 cells erastin promotes activation of rip1 and subsequent rip1-rip3 necrosome formation which has been investigated as a hallmark of regulated necrosis 2 . in fact, silencing of rip1 by sirna or by the rip1 inhibitor necrostatin-1 prevents ferroptosis-induced cell death whereas the ferroptosis inhibitor ferrostatin-1 fails to protect cells against tnfα-induced classical necroptosis, a form of programmed cell death that is mediated by receptor interacting protein-1 (rip1) and rip3 kinases downstream of death receptor activation (e.g. tumor necrosis factor receptor tnfr) 2, 3 . recently, a genome-wide sirna screen linked cylindromatosis (cyld) to rip1/rip3-dependent necroptosis 4 and also in the present paradigm of ferroptosis, cyld depletion promotes neuronal survival and decreases rip1-rip3 complex formation, suggesting a role of cyld in intrinsic pathways of regulated necrosis triggered by oxidative stress. the ns5a inhibitor daclatasvir is used in combination with other antivirals such as the polymerase inhibitor sofosbuvir for treatment of chronic infection with the hepatitis c virus. daclatasvir is embryotoxic and teratogenic in rats and rabbits at exposures at or above the clinical exposure. in contrast, no teratogenic effects were observed in rat and rabbit developmental toxicity studies with ledipasvir, another ns5a inhibitor. we studied these compounds in the embryonic stem cell test (est) alone and in combination with sofosbuvir. the ns5a inhibitors were obtained from selleckchem, the main metabolite of sofosbuvir, psi-6202, was from medchem express. murine embryonic stem cells (es-d3) were obtained from atcc. they were kept in iscove's modified dulbecco's medium (imdm). substances were dissolved in dmso at a final dmso-concentration of 0.1% in the culture medium. a cytotoxicity assay as well as a differentiation assay were performed. after 10 days in culture the cells were evaluated. cytotoxicity was measured by an mtt test. differentiation into contracting myocardial cells was determined using direct phase contrast microscopy. the substances were tested at concentrations between 0.1 and 30 mg/l, which is a broad coverage of the therapeutically relevant concentrations reached in patients. at a concentration of 10 mg daclatasvir / l medium and higher the substance inhibited differentiation of cells. we observed contracting myocytes in 23, 22 and 2 wells out of 24 wells in total at concentrations of 1, 3 and 10 mg/l. at 30 mg/l no differentiation was observed. effects on cell viability were observed at 30 mg/l. unexpectedly, we found a higher potency with ledipasvir. at the low, therapeutically relevant concentration of 1 mg/l this nsa5-inibitor showed a clear impact on differentiation with 6 out of 24 wells affected and no differentiation at higher concentrations. addition of sofosbuvir or its main metabolite psi-6206 at concentrations up to 30 mg/l had no influence on the concentration effect curves established for daclatasvir or ledipasvir. this is the first indication of an embryotoxic potential of ledipasvir. the difference to the results from the routinely performed animal experiments is unknown. possibly, metabolic activity in the maternal organism is responsible for this discrepancy. dimoxystrobin is a european-registered pesticidal active ingredient. biologically it is acting as an inhibitor of the fungal respiratory chain. for the purpose of european registration a full set of toxicological studies has been conducted with dimoxystrobin, including reproduction toxicity studies (according to the most recent oecd tg 416) and developmental toxicity studies (oecd tg 414) in rats and rabbits. dimoxystrobin interferes with the iron transport in rats and mice. this leads to lower serum iron levels and anemia in rats after repeated exposure. this holds true for treated dams and offspring in reproduction toxicity studies. furthermore, offspring effects seen at the high dose of the 2-generation toxicity study were a hypochromic microcytic anemia, impaired body weight development, which only developed postnatally, and reversible cardiomegalies in some 21-days old pups. for all effects clear noaels were determined. in the 2-generation toxicity study no dose adjustment during pregnancy and lactation was performed, which resulted in considerably higher food and compound intakes in dams and offspring during these lifestages. as a result, it seemed, that pups were more severely affected by body weight effects compared to the parental generation. by performing a life-stage specific comparison of body weight and substance intakes, as well as benchmark dose calculations (bmd) for these parameters, it could be demonstrated that the point of departures (pods) and the loaels for direct dimoxystrobin-related effects were comparable for offspring and parents. the heart effects (cardiomegaly), which were reversible, occurred only after direct dimoxystrobinexposure and are considered to be secondary to the detected offspring anemia. both effects (lower body weights and offspring cardiomegalies) only occur postnatally and are not the consequence of in-utero exposure, as no respective effects at higher doses in rat prenatal toxicity studies were seen. two new mechanistic studies (1-generation toxicity study and a 3-week study in young and adult rats, additionally investigating serum iron levels and anemia) confirmed, that pups and young rats were not more sensitive than adult animals to develop anemia or decreased serum iron levels. in 2006, dimoxystrobin was classified with r63 (possible risk of harm to the unborn child) by the ecb, which was the european authority responsible for classification and labeling, before echa in helsinki was formed. the r63 (which has been translated into the ghs classification repr. 2, h361d) was based on offspring body weight and heart effects seen in the 2-generation toxicity study. based on a comprehensive re-evaluation of existing and on new data of dimoxystrobin, the conclusion can be drawn, that a classification for reproduction toxicity is scientifically not justified and should be reconsidered. perfluorooctanesulfonic acid (pfos) and perfluorooctanoic acid (pfoa) are perfluorinated substances (pfas) which are used for the fabrication of surfaces with water-and dirt-repellent properties. due to their reprotoxic properties and their persistence in the environment, the use of pfos was restricted in 2009 and a restriction program for pfoa was initiated in 2013. therefore, industry switches to pfoa and pfos substitutes, which are predominantly pfas with a shorter carbon chain length, or structure-related compounds. in contrast to pfoa and pfos only few toxicological data are available for their substitutes. aim of this study was to examine endocrine effects of the substitutes perfluorohexanesulfonic acid (pfhxs), perfluorobutanesulfonic acid (pfbs), perfluorohexanoic acid (pfhxa), perfluorobutanoic acid (pfba) and 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propionic acid (genx) in comparison to pfoa and pfos. a hek-293t cell-based dual-luciferase reporter gene assay was used to investigate the potential of these compounds to affect the activity of the human estrogen receptors herα and herβ. the reporter gene assay revealed no activation of herα or herβ by the pfas tested in this study. to investigate the potential inhibition of herα and herβ by pfas, a coincubation with the estrogen receptor agonist 17β-estradiol was performed. none of the tested pfas inhibited herα or herβ activity. however, in the case of herβ an enhancement of 17β-estradiol-stimulated activity was observed. thus, pfas do not directly activate or inhibit the human estrogen receptors but have an impact on herβ activity as they amplify the activation mediated by 17β-estradiol. further studies will be conducted to examine this synergistic effect in more detail. the xeer-reporter cell line: a novel dual-color luciferase reporter assay for simultaneous detection of estrogen and arylhydrocarbon receptor activation p. tarnow consumers are exposed to a multitude of anthropogenic and natural substances capable of activating or inhibiting ligand activated transcription factors, respectively. this in turn can lead to adverse health effects, particularly for substances acting on signalling pathways that are subject to regulatory crosstalk such as xenoestrogens and polycyclic aromatic hydrocarbons (pahs). xenoestrogens are known to activate human estrogen receptors (ers), whereas pahs or dioxins act on the arylhydrocarbon receptor (ahr). importantly, both receptor signalling pathways are interconnected by a complex crosstalk on multiple levels. this ranges from direct protein-protein interactions to competition for common co-factors. however, although this cross-talk has been long known we still lack a deeper understanding of its molecular mechanisms and physiological implications. one reason for this is a lack of tools to visualise and investigate receptor interaction in vivo. based on the breast cancer cell line t47d we thus developed a dualcolour reporter assay which allows time-resolved simultaneous monitoring of the activation of er and ahr in living cells. the assay uses two beetle luciferases emitting luminescence in the red (slr) and the green (eluc) spectrum, respectively. while eluc is expressed under the control of a 6-fold repeated xenobiotic response element (xre) slr is subject to transcription regulation by a 6-fold repeated estrogen response element (ere). both constructs were stably transfected into t47d human breast cancer cells, which endogenously express erα and ahr and are thus ideally suited for monitoring interactions with both receptors. the respective "xeer"-cell line has been successfully subjected to proof of principle studies, using prototypical er-and ahrligands as well as various phytochemicals and xenobiotics. besides e2 and tcdd ligands included various pahs, polychlorinated biphenyls, alpha-and betanaphthoflavone, cosmetic ingredients (butylparaben, benzophenone-2 and 4-mbc), bisphenol a, genistein, resveratrol, diindoylmethane as well as pharmacological antagonists of both receptors. asian women consuming soy rich food throughout life possess lower levels of 17betaestradiol (e2) in plasma (pl) than western women, whose diet is characterized by less soy consumption during early life and possible intake of soy based dietary supplements during adulthood. however, the impact of these soy exposure scenarios on estrogen (biotrans)formation and the consequence thereof in female mammary glands (mg) has not been investigated yet. thus, female august copenhagen irish rats were fed either isoflavone (if) depleted diet (idd, western exposure scenario without if supplement) or if rich diet (ird, asian exposure scenario) until the end of the study at postnatal day (pnd) 81. furthermore, rats fed idd until pnd74 were fed ird for 7 days (idd+ird, western dietary exposure scenario with if supplement). estrous was determined histologically. levels of transcripts were determined by qpcr and e2 and estrone (e1) in pl and mg were quantified by gc-ms/ms. statistical analyses of estrogens were performed by kruskal wallis and unpaired wilcoxon tests and of transcript levels by linear regression models considering the explanatory variables tissue levels of e2 and diet (idd vs ird and idd vs idd+ird). e2 levels in pl and mg did not coincide with those predicted by estrous. furthermore, median levels of e1 and ratios e2/e1 in mg and ratio of e2 levels in pl/mg were not affected by diet. in contrast, diet tended to affect e2 concentrations in pl (p=0.1211) due to an increase in the ird group (p=0.0056) whereas e2 levels in the idd+ird group only tended to be elevated (p=0.0788). in mg, ird and idd+ird increased e2 levels only weakly (p=0.0788 each). likewise, besides significant changes in transcript levels of cyp1a1 and 1a2, putatively decreasing oxidation of e2 to catechols, in the idd+ird group and (not significantly) also in the ird group, no changes in transcript levels putatively affecting e2 levels were observed. moreover, no decrease in levels of transcripts indicative for cellular (oxidative) stress (gclc, tp53, mt1a) was observed in the idd+ird group. e2 mg levels were significantly associated with an increase in transcript levels of areg and pgr, indicating activation of estrogen receptor (er). in contrast, ird was associated with a significant and idd+ird with a not significant decrease in pgr transcript levels. e2 levels but not diet were significantly associated with gata3 transcript levels, indicating tissue differentiation. furthermore, levels of transcripts involved in intercellular communication (egfr, wnt4) were significantly decreased by idd+ird and not significantly by ird and differed from that affected by e2 (increase in gdf15, hgf, igf1r, wnt5a). bmf, a marker transcript for apoptosis was increased by ird, but not affected by e2 and even decreased not significantly by idd+ird. taken together, despite an increase in e2 levels in pl, less er activation was observed after dietary exposure to if. whereas e2 and transcript levels of enzymes involved in e2 (biotrans)formation as well as er activation and cellular communication were affected similarly but to a different extend in both asian and western if exposure scenarios, differences in apoptosis were observed between ird and idd+ird groups. supported by dfg le 1329/10-1. august copenhagen irish (aci) rats with 17β-estradiol (e2)-releasing implants are an accepted model to study the etiology of breast cancer, but neither e2 (biotrans)formation in mammary gland tissues (mg) during tumorigenesis, nor the impact of isoflavones (if) shown to affect tumorigenesis in aci rats, has been investigated, yet. therefore at postnatal day (pnd) 75 and 175, placebo (-e2) or silastic implants containing 4 mg e2 were implanted in female aci rats exposed to either if depleted diet (idd) or if rich diet (ird) since conception until the end of the study at pnd 285. palpable mg tumors (pt) and 1-2 mg per animal without pt were characterized histologically and categorized into normal (-e2 group, n=12), hyperplasia and non-pt and pt with and without solid tumors (+e2 group, n=32). e2, estrone (e1), their hydroxylation products and methylation (meo-) products thereof, as well as conjugates of e1 and e2 in plasmas and mg were analyzed by gc-and uhplc-ms/ms, respectively. levels of 49 transcripts involved in (biotrans)formation of e2 and estrogen receptor (er) activation were determined by taqman®-pcr. without exogenous e2, plasma e2 as well as e1 and (borderline) e2 levels in mg were higher in ird. plasma e2 as well as e1 and e2 levels in mg were lower in the -e2 group than that in the +e2 group. e2 levels as well as e2/e1 and e2 mg/plasma ratios were elevated in pt, accompanied by a significant increase in transcript levels indicative for estrogen receptor activation (areg, pgr) and proliferation (mki67). ird increased e2/e1 ratio in pt and, although ird did not affect er activation (areg, pgr), ird increased differentiation (gata3) in normal and hyperplastic tissues and tended to decrease proliferation in hyperplastic (ccnd1) tissues. levels of e1 and 2-meo-e1 were highest in hyperplastic tissues, accompanied by an increase in transcript levels of hsd17b2 (conversion e2 to e1) and cyp1a1. transcript levels of gstm1 and gstm2 were decreased in the whole +e2 group and of gstt1 and gstt3 in hyperplastic tissues, possibly decreasing inactivation of electrophilic metabolites. accordingly, maximum transcript levels of tp53 and mt1a indicating cellular (oxidative) stress were observed in hyperplastic tissues. ird did neither affect levels of 2-meo-e1 nor cellular stress (gclc, mt1a, tp53). of note, neither 4-meo-e1, nor e1 catechols, nor e2 catechols nor methylation products of the latter were observed in any sample. furthermore, no conjugates of e1 or e2 were detected in plasmas and mammary gland tissues. thus, changes in transcript levels of conjugating enzymes induced by tumorigenesis and by ird were not related with detectable conjugate levels of e1 or e2. taken together, whereas hyperplastic tissues were characterized by maximum oxidative metabolism of e1 and cellular (oxidative) stress, pt exhibited highest e2 levels and er activation. ird increased differentiation and decreased proliferation in normal and hyperplastic tissues but increased e2/e1 ratio in pt. supported by dfg le-1329/10-1. level of 17beta-estradiol (e2) in human breast tissue is considered to affect breast cancer initiation, promotion and progression. although putatively beneficial and adverse effects of soy isoflavones (if) on the human mammary gland, in particular in western women, have been discussed extensively, the influence of if levels on estrogen formation in human mammary gland tissue has not been investigated yet. thus, glandular tissues were dissected from 37 mammoplasty specimen obtained from women (age 18-66 years old) not taking estrogen active drugs. 14 of these women had been exposed to if by their usual diet or by intake of a soy-based dietary supplement for 7 days prior to mammoplasty. information on soy consumption and lifestyle were collected by questionnaire and tissues were characterized histologically. genistein, daidzein their conjugates (n=12) and bacterial metabolites (n=7) as well as the estrogens estrone (e1)-sulfate, e1, e2 and 2-methoxy-e1 were determined by uhplcand gc-ms/ms, respectively and transcript levels of 19 enzymes involved in e2 (biotrans)formation were quantified by taqman®-pcr in glandular tissues. isoflavonoids were categorized into the if parameters aglycones (agl) and conjugates (con) of either genistein, daidzein or sum of both and were further statistically analyzed by spearman`s rank correlation analysis. a positive correlation of e2/e1 ratio with agl(+con) was observed in glandular tissues (r=0.49, p=0.002), accompanied by a significant negative correlation of e1 levels with agl (r=-0.35/p=0.032), possibly due to reduction of 17beta-hydroxysteroid dehydrogenase 2 (conversion of e2 to e1) expression as indicated by a weak negative correlation of transcript levels of 17beta-hydroxysteroid dehydrogenase 2 with agl+con (r=-0.25, p=0.080). further statistical analysis taking into account multiple variables using linear regression models will provide more insights into variables affecting e1/e2 ratio. taken together, estrogen profile in human glandular breast tissue seems to be affected by if levels. supported by dfg le-1329/10-1. allergic contact dermatitis (acd) is a widespread disease often caused by substances in consumables. the eu prohibits the testing of cosmetic ingredients in vivo. this urges the development of reliable in vitro testing strategies. activation of dendritic cells (dcs) represents a key step during sensitization as they are essential for selection and priming of allergen specific effector t cells. in an integrated omics approach we aimed to further elucidate the molecular mechanisms of dc activation using quantitative metabolomics and proteomics. monocytic thp-1 cells were used as a model system and treated with the sensitizer 2,4dinitrochlorobenzene (dncb; 5, 10 and 20 µm) and the irritant sodium dodecyl sulfate (sds; 100 µm). samples were taken after 4, 8 and 24 hours. thp-1 activation was analyzed by measuring the established activation markers cd86 and cd54 after 24 hours. a targeted lc-ms/ms approach was used to analyze 188 metabolites including amino acids and lipids. protein levels were quantified by nano-lc-maldi-ms/ms after stable isotope labeling by amino acids in cell culture (silac). data sets were examined by multivariate analyses for identification of biomarker candidates. regulated metabolites and proteins were subjected to pathway analysis. the data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization. drug induced liver injury (dili) is one of the most frequent causes of acute liver injury and a main cause for drug withdrawals. currently there are no reliable models to test the dili potential of new compounds available. kupffer cells (kc) play an important role in hepatic cell stress mediated through chemokines and release of endogenous proteins. kc activation by damaged or stressed hepatocytes can lead to activation of the nf-κb signaling pathway transmitted by reactive oxygen intermediates (roi). we have recently established a liver model composed of primary human hepatocytes (phh) and kc which enables investigation of immune reactions after induction of hepatocyte stress (kegel et al., 2015) . aim of the present study was the kinetic investigation of hepatic cell stress induction and macrophage activation after treatment with subtoxic concentrations of hepatotoxic drugs. primary human hepatocytes (phh) and kc were isolated from human liver resectates using a two-step collagenase perfusion technique. initial kc activation was characterized by the dcf assay and immunofluorescence staining. phh were incubated with different concentrations of acetaminophen (apap) and diclofenac (dic) for different time intervals. cell stress was evaluated by measurement of oxidative stress (dcfassay) and viability (xtt-assay). in order to simulate macrophage activation following hepatocyte damage, kc and macrophages derived from the monocytic cell line thp-1 were incubated with supernatants of phh treated with hepatotoxic compounds. kc and thp-1-macrophage activation were investigated by measuring intracellular formation of roi using the dcf-assay and cell activity using the xtt-assay. the characterization of kc activation revealed a donor and disease dependent kc activation resulting in kc differentiation to pro-and anti-inflammatory macrophages. therefore, kc were substituted by macrophages derived from thp-1 cells. evaluation of hepatic cell stress showed the strongest effect on thp-1-macrophages when phh were incubated with apap or dic for 4 h.treatment of kc and thp-1-macrophages with supernatants of phh challenged for 4 h with hepatotoxic compounds indicates that thp-1-derived macrophages react similar to kc when treated with phh supernatants in terms of cell activity and roi-production. in conclusion, thp-1 derived macrophages might be a suitable alternative to kc concerning macrophage activation. the evaluated kinetic window of 4 h covering hepatic stress induction and immune reaction allows to perform these measurements in a since the use of cerium dioxide nanoparticles is known to be beneficial e.g. in terms of reducing fuel consumption when added to diesel fuel it has become a frequently used nanomaterial. to compensate the concurrent lack of information on its toxicology a 90day nose-only inhalation study was initiated. by comparing the results to a combined chronic inhalation toxicity and carcinogenicity study using the same test items and experimental conditions (basf, ludwigshafen, germany) early indicators for genotoxic and carcinogenic effects should be determined. rats were exposed to 0, 0.1, 0.3, 1 and 3 mg/cm³ ceo 2 as well as 50 mg/m³ baso 4 nanoparticles (6 h/day, 5 days/week, 13 weeks). animal dissections were conducted at five time points (exposure day 1 and 28; recovery day 1, 28 and 90) aiming for endpoints mandatory according to oecd guideline 413. additionally, gene expression analyses in isolated pneumocytes type ii were performed using pathway arrays for inflammation, oxidative stress, genotoxicity, apoptosis and lung cancer. the given results intend the identification of marker genes displaying modulated expression in response to nanoparticle exposure. investigations on ceo 2 and baso 4 retention in the lung are also included in this project. in bronchoalveolar lavage fluid (balf) a time and dose-dependent increase of inflammatory cells has been detected up to the end of exposure. the amount of inflammatory cells decreased during post-exposure; however, in the high dose group a persistent inflammation up to 90 days was detected by balf and histopathology examination. based on our current results effects of ceo 2 nanoparticles on the respiratory system are suggested. its relevance in the context of long term effects such as tumor development needs to be estimated considering all investigations included in this study. the inhalt-90 project is funded by the german federal ministry of education and research (bmbf) -03x0149a. core or coating material? what dictates the uptake and translocation of nanoparticles in vitro? nanoparticles are becoming increasingly important role in consumer-related products. understanding the interactions between nanoscaled objects and living cells is therefore of great importance for risk assessment. in this context, it is generally accepted that nanoparticle size and shape are crucial parameters regarding the potential of nanoparticles to penetrate cell membranes and epithelial barriers. current research in this field additionally focuses on the particle coating material. in order to distinguish between core-and coating-related effects in nanoparticle uptake and translocation behavior, this study investigated two nanoparticles equal in size, coating and charge but different in core material. silver and iron oxide were chosen as core materials to ensure similar nanoparticles characteristics after particle synthesis. nanoparticles were coated with poly (acrylic acid) (pas) and extensively characterized by tem (transmission electron microscopy), saxs (small-angle x-ray scattering), zetasizer tm and nanosight tm . for uptake and transport studies the widely used human intestinal caco-2 model in a transwell tm -system with subsequent elemental analysis (aas) was used. for evaluation and particle visualization transmission electron microscopy (tem) and ion beam microscopy (ibm) were conducted. although similar in size, charge and coating material, the behavior of particles in caco-2 cells was quite different. the internalized amount was comparable, but pas-coated iron oxide nanoparticles were additionally transported through the cells. by contrast, pascoated silver nanoparticles remained in the cells. our findings suggest that the coating material influenced only the uptake of the nanoparticles whereas the translocation was determined by the core material. in summary, a core-dependent effect on nanoparticle translocation was revealed. both the uptake and transport of nanoparticles in and through cells should be considered when discussing nanoparticle fate and safety. nanotechnology is having a great impact not only on basic research but also on many sectors of industry opening the market for numerous new applications ranging from electronics to the health care system. besides their great innovative potential, the large variety of existing synthetic nanomaterials used in the last decade represents a major challenge for scientists and regulators in terms of measuring and assessing the potential hazard caused by the materials or the products themselves. equally, consumers often miss reliable and easy-to-understand information on nanomaterials and nanotechnology and do not know where to get such information. therefore, the international dana 2.0 expert team brings together its expertise and knowledge from different research areas dealing with all aspects of nanosafety research in order to create and provide easy-to-understand, up-to-date and quality-approved nanomaterials' knowledge base on www.nanopartikel.info. this information platform covers the 25 market-relevant nanomaterials focusing on their effects on the safety of humans and the environment.in order to manage and asses the rapidly increasing number of publications related to nanosafety issues, the dana 2.0 project developed a customised methodology «literature criteria checklist», which includes mandatory and desirable assessment criteria covering physico-chemical characterisation, sample preparation and (biological) testing parameters. this checklist facilitates the discrimination between high-and low quality publications and all positively evaluated literature is then fed into the dana knowledge base. accounting for the need to harmonise experimental practices, the dana team also developed a template for standard operation procedures (sop) to support careful scientific practice. validated protocols generated within the german bmbf-funded nanosafety research projects are presented together with results from the swiss ccmx project v.i.g.o. and available for download. another unique feature of the dana knowledge base is the integrated application-based database that provides a unique link between nanomaterials in real applications (e.g. environmental remediation or medical products) and their potential impacts/ toxicological effect(s) that can be easily accessed by the interested visitor. additionally, dana2.0 provides a list of faqs, a link platform with contact data to other information portals and the opportunity to directly pose questions to our experts via e-mail. dana 2.0 is also present on twitter, follow us @nano_info. background: particulate matter of combustion processes enhances cardio-vascular diseases and increases associated mortality rates. around 13% of total pm10 emissions are emitted by wood burners (uba 2006) . how wood combustion aerosols (particles and gasses) can affect human lung cells and how such cellular responses depend on the usage of different wood types and burners is widely unknown. methods: in an exposure chamber imitating the human respiratory tract human alveolar cells (a549) were exposed at an air-liquid-interface (ali) to gasses and particles of wood combustion aerosols. log wood of beech, birch and spruce was burnt in a conventional oven and compared to the combustion of wood pellets in a modern pellet burner. the combustion aerosols were diluted 1:40 and directly delivered to the exposure chamber. after 4h exposure the lung cells were lysed and rna was isolated. in an array based transcription analysis of the whole genome the effects of the aerosol exposures on lung cells was assessed. in parallel, physical and chemical parameters of the combustion aerosols were analyzed. results: the combustion aerosol of wood pellets contained less organic substances than the log wood aerosols, but was higher in its zinc content. genome-wide we found a higher number of regulated genes with combustion of pellets compared to combustion of log wood. the gas phase alone (filtered aerosol) showed comparable gene regulatory activity as the particle-containing total aerosol. aerosol from log wood burning induced mainly genes of the xenobiotic metabolism and cellular signaling. pellet aerosols additionally regulated apoptosis and dna repair processes. conclusions: modern pellet burners reach better combustion efficiencies than conventional log wood ovens, but their emissions seem to stress human lung cells stronger. one reason might be the higher zinc content of wood pellet aerosols. multiwalled carbon nanotubes (mwcnts) may pose as a risk similar to asbestos in causing cancer, notably mesothelioma, which is a malignant tumor originating from mesothelial cells. to identify molecular cues leading to mesothelioma development, we performed genome-wide transcriptome analysis using microarrays in primary human peritoneal mesothelial lp9 cells treated with two different tumor-inducing tailor-made mwcnts (rat model; rittinghausen et al. 2014 part fibre toxicol 11:59), or amosite asbestos at 3 µg/cm2 for 24 h. specifically, we determined how the transcriptomic changes of the highly tumorigenic mwcnt a would differ from another tumor-inducing mwcnt with albeit lesser potency (mwcnt d), long amosite asbestos as positive control, and milled mwcnt a as material control. initial analysis using bioinformatic tools, revealed 3788 significantly differentially regulated genes for mwcnt a, 1680 for mwcnt d, 145 for amosite, and 4 for milled mwcnt. further analyses with ingenuity pathway analysis comparing the two different mwcnt types and amosite, found common as well as exclusive biomarkers. interestingly, we identified many differentially regulated genes implicated in cellular senescence, a growth arrest in response to different stressors including dna damage, disrupted chromatin, and strong mitogenic signals. paradoxically, cellular senescence can represent both tumor suppression and tumor promotion mechanisms. more important, we found differential expression of genes associated with senescence-associated secretory phenotype (sasp) such as inflammatory cytokines, chemokines, proteases, and growth factors, which were manyfolds up-and down-regulated in mwcnt a, compared to mwcnt d and amosite. the mechanisms leading to mesothelioma induction by mwcnts are from far clear, but the key information emerging from the present transcriptomic data, together with our previously identified senescence markers, indicate that cellular senescence has a likely role. nanotechnology offers great advantages for the food industry despite its partly unknown risks, whose enlightenment is the main target of nanotoxicology. due to variability in terms of size, material, shape, surface texture and several endogenous influences, the toxicity of most frequently used and ingested nanomaterials is difficult to estimate. therefore, the aim of this study was the in vitro investigation of toxicological endpoints such as cell viability, dna integrity and the induction of apoptotic processes in human colon carcinoma cells (ht29). for this purpose ht29 cells were exposed for 24 hours with metal nanoparticles (gold, silver) and metal oxide nanoparticles (copper oxide, titanium dioxide, zinc oxide) in concentrations of 2-10 µg/ml. at first the cellular uptake of the nanoparticles by means of an icpms was determined. the influence of cell viability was demonstrated by the trypan blue staining and the mtt assay. the alkaline comet assay gave information about possible dna damages and the use of the repair enzyme formamidopyrimidine dna glycosylase (fpg) additionally allowed the detection of oxidized bases. the induction of programmed cell death was examined using by annexin v fitc assay. the icp-ms data showed a maximum particle content of 1.39 pg per cell for the used concentration range. the metal oxide nanoparticles resulted in a significant reduction of cell viability with a decrease up to 40 % after copper oxide and zinc oxide treatment. for metal particles, only for silver a reduced cell metabolism of about 50 % was detectable by the mtt assay. low genotoxic effects could be determined for silver nanoparticles (tail intensity about 12%; control about 6%), while for titanium dioxide the amount of oxidized bases was additionally increased (tail intensity about 20%; control about 6%) for concentrations above 8 µg/ml. induction of apoptosis was determined for silver particles (up to 24% early apoptotic and 20% late apoptotic cells) as well as for titanium dioxide and zinc oxide (10% each early apoptotic cells), whereby the most significant increase in late apoptotic cells was detected for zinc oxide (up to 90%). the results obtained in our studies indicate a clear particle-dependent influence on cell viability and apoptosis-triggering processes, depending on the used material or the concentration deployed, while only minor changes of dna integrity were detected. the evolution in the field of nanotechnology led to a variety of novel materials at the nanoscale. among them are different carbon materials like buckyballs, graphene nanoplates and carbon nanotubes (cnts). cnts are hollow carbon fibres with either one (sw) or multiple sidewalls (mw). mwcnts usually show a diameter of up to 100nm and can be several micrometres long. because of their nanoscale diameter cnt-uptake can take place directly through the plasma membrane of cells by the so called nanoneedle effect [1] . additionally cnts, like most nanomaterials, show a high surface to volume ratio and, because of their micro scale length, a potentially high loading capacity. these properties make cnts interesting for the potential use as drug delivery carriers (ddcs). mwcnts, produced via chemical vapour deposition with a diameter of 45nm and 16µm length, were used in three different forms, unmodified, acid oxidized (ox_cnt) and ground. cytotoxicity testing was performed in human umbilical vein endothelial cells (huvec). the cells were seeded in 48-well plates and exposed to doses of 1, 5, 10 and 25µg/cm² growth area of the respective cnt type for 24h. the wst-8 assay was applied for testing cell viability and the ldh cytotoxicity assay to identify potential damage to the plasma membrane and to calculate overall cytotoxicity. the results show that an increased oxidation time for the ox_cnts, in a h 2 so 4 /hno 3 mixture, leads to decreased cytotoxicity in huvec, compared to unmodified mwcnts. during the oxidation reactive oxygen groups are formed on the cnt surface [2] . these groups lead to a reduced hydrophobicity of the cnt surface which could be responsible for the decline in cytotoxicity. future investigations will include the toxicological analysis of mwcnts functionalized with polyethylene glycol (cnt-peg). the hydrophilic polymer peg will be covalently bound to the cnt surface and is expected to further reduce the cytotoxic effect. for these investigations different analytical methods will be used. among others, cell cycle analysis, the brdu assay, pathway arrays and qrt-pcr for the investigation of gene expression and cytokines will be measured. these methods will involve a co-culture model of huvec and human umbilical vein smooth muscle cells (huvsmcs) for a better approximation to the cellular in vivo situation. additionally the peg modified mwcnts will be tested for their loading capacity and efficacy with the anticancer drug doxorubicin for a potential use as an intravenous drug delivery carrier in vivo. although aluminium is one of the most common elements in the biosphere, up to now little is known about its impact on human health. aluminium and its chemical derivatives are highly abundant in food, food contact materials and consumer products what leads to an exposition via the gastrointestinal tract (gi tract), the lung and via skin contact. recently, aluminium is hypothized to cohere with cancer and neurodegenerative disorders. lately, due to an increasing attentiveness on this topic, limiting values for food additives have been tightened by the eu commission. however, cellular effects of aluminium and especially aluminium-containing nanomaterials, are in the focus of our research activities, for example in the international solnanotox project. we established an in vitro simulation system of the gi tract, where nanomaterials undergo the different physiological, chemical and proteinbiochemical conditions of saliva, gastric juice and the intestine. the artificially digested nanomaterials, as well as soluble aluminiumchloride as ionic control substance, were subjected to several analytical and biochemical methods to characterize their change of appearance and their cytotoxic effects on intestinal cellular models. we observed the fate of the nanomaterials during typical ph-values of saliva, gastric and intestinal juice with dynamic light scattering measurements and icp-ms in the single particle mode. after observable disappearance at ph 2 the particles recovered in the simulated intestinal fluid. the simulation of the gi tract, mainly the change of ph settings, may lead to a certain chemical activation of aluminium that can increase bioavailability in the intestine after oral uptake of aluminium-containing food products. in vitro assays like ctb, mtt and cellular impedance measurements showed that there were no acute cytotoxic effects measurable after a period up to 48h after incubation, comparable to undigested particles. in contrast, high amounts of aluminium ions showed additional effects on cell viability compared to non-digested aluminium ions. although toxicological potential of al ions to healthy tissue appears to be low, increased hazardous potential cannot be ruled out to pre-damaged tissue and can have a relevance for special consumer groups with for example chronical intestinal inflammation or dietary eating behavior combined with high exposure to al-containing food products. in the eu, there is a strong need for solutions to substitute halogenated flame retardant (hfr) additives, employed in the fabrication of fr thermoplastic and thermoset materials. these materials are used in diverse commercial products, applications and markets, such as electrical/electronic devices, low-voltage wires or household appliances. the phoenix project, funded by the european union 7 th framework program (grant agreement no. 310187), therefore investigates e.g. several tailor-made, nano-layered hybrid particles as alternatives to hfr additives. considering "safer-bydesign" strategies, potential fr nanomaterials (nm) were physico-chemically characterized (e.g. particle size distribution, zeta potential) and screened early in development for their (geno)toxic and pro-inflammatory potential to timely reject nm with high health hazard. as inhalation is the most important exposure route for nm, lungrelevant cells were used as in vitro screening models. to better enable detection and differentiation of the biologic effects of the most promising nm, screening was started with primary rat lung alveolar macrophages (am; cells of first contact for inhaled nm), at a high concentration of 50µg/cm 2 (24h of incubation), using membrane damage (lactate dehydrogenase release assay), direct dna-damage (alkaline comet assay), and il-8 liberation (elisa) as primary endpoints, and quartz dq12 and al 2 o 3 as particulate positive and negative controls, respectively. in this screening system, biologically inert nm could be differentiated from more active ones. thereby, mg(oh) 2 nanoplatelets (mg1; mean lateral size: 1,5-2 µm; mean thickness: 15-20 nm) represented the least, and pristine few layers graphene nanoplatelets (gr1; mean lateral size: 2 µm; mean thickness: 3 nm; graphene layers: 8 ± 0.5) the most biologically active nm. clear concentration dependencies were detected for gr1 in follow-up experiments. mg1 and gr1 were further tested in other lung-relevant cell types, i.e. mrc-5 primary human lung fibroblasts and a549 lung adenocarcinoma epithelial cells. interestingly, mrc-5 cells were less sensitive towards biological effects of gr1, compared to am, whereas a549 cells showed nearly no effect, keeping in mind that lung epithelial cells are the target cells of lung tumor development. to test the hypothesis that the observed cellspecific differences in sensitivity might in part be based on cellular uptake, cells were exposed for 24 h to 12.5 or 25 µg/cm 2 of gr1 on chamber slides. slides were finally stained with dapi and analyzed by dark field microscopy. cells indeed demonstrated differences in uptake capacity and also showed unique pattern of cellular localization of gr1, i.e. with tight perinuclear agglomeration in am, a more scattered cytoplasmic distribution in mrc-5 cells and limited uptake in a549 cells. additionally, biological activity of the diverse nm seemed also to correlate with cellular uptake, as determined by light and dark field microscopy in am and mrc-5 cells. in conclusion, an am-based screening system was able to differentiate biological activity of diverse nm, with morphology, physico-chemical characteristics, and related cellular uptake most likely to be key for nm-and cell type-specific hazard. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. 453 with several protocol extensions. female rats (100/group) were exposed to cerium dioxide (nm-212, 0.1; 0.3; 1; 3 mg/m³) for two years; a control group was exposed to clean air. after one year exposure, 42 µg/lung was found in animals exposed to 0.1 mg/m³ and 2.6 mg/lung in animals exposed to 3 mg/m³. histological examination of lungs revealed several adverse and non-adverse effects in the lung. the non-adverse effects comprised accumulation of particle-laden macrophages in alveolar/interstitial areas and in the balt, particle-laden syncytial giant cells in the balt and bronchiolo-alveolar hyperplasia (alveolar bronchiolization). the adverse effects included (mixed) alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, interstitial fibrosis and alveolar lipoproteinosis. the incidence and severity of the effects were concentration-related. alveolar lipoproteinosis was not observed at low concentrations of 0.1 and 0.3 mg/m³ ceo 2 . neither pre-neoplastic nor neoplastic changes were observed after 12-months exposure. a no observed adverse effect concentration could not be established in this study. the comprehensive histopathological examinations of lungs and other tissues will be finalized in 2017. this project is part of the eu project nanoreg. moreover, german federal ministry for the environment, nature conservation, building and nuclear safety, german federal institute for occupational safety and health, german federal environment agency funded this project. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. 453 with several protocol extensions. female rats (100/group) were exposed to cerium dioxide (nm-212, 0.1; 0.3; 1; 3 mg/m³) and barium sulfate (nm-220; 50 mg/m³) for two years; a control group was exposed to clean air. lung burdens and burdens in extrahepatic tissues were measured at various time-point. the two year exposure period was successfully terminated and 50 animals per dose group were examined for organ burden and histopathology. the remaining animals currently are kept exposure-free for maximally 6 additional months. up to two years exposure to both nanoparticles did not lead to body weight reduction compared to control animals. the mortality rates were in an acceptable range. macroscopically evident tumors were not detected after two years. the ceo 2 lung burdens were maximally 3.5 mg/g lung tissue at the highest exposure concentration of 3 mg/m³. in comparison, highest ceo 2 burdens in organs remote to exposure were liver and spleen with maximally roughly 1 x 10 -3 g/g tissue. in brain, maximum ceo 2 levels were 7x10 -6 mg/g lung tissue. baso 4 lung burdens were comparatively low (1 mg/g) within the first 13 weeks of exposure and steeply increased to 6 mg/g lung tissue after one year. the comprehensive histopathological examinations of lungs and other tissues will be finalized in 2017. the european centre for ecotoxicology and toxicology of chemicals (ecetoc) 'nano task force' proposes decision-making framework for the grouping and testing of nanomaterials (df4nano) that consists of 3 tiers to assign nanomaterials to 4 main groups, to perform sub-grouping within the main groups and to determine and refine specific information needs. the df4nanogrouping covers all relevant aspects of a nanomaterial's life cycle and biological pathways, i.e. intrinsic material and systemdependent properties, biopersistence, uptake and biodistribution, cellular and apical toxic effects. use (including manufacture), release and route of exposure are applied as 'qualifiers' within the df4nano to determine if, e.g. nanomaterials cannot be released from a product matrix, which may justify the waiving of testing. the four main groups encompass (1) soluble nanomaterials, (2) biopersistent high aspect ratio nanomaterials, (3) passive nanomaterials, and (4) active nanomaterials. the df4nano aims to group nanomaterials by their specific mode-of-action that results in an apical toxic effect. this is eventually directed by a nanomaterial's intrinsic properties. however, since the exact correlation of intrinsic material properties and apical toxic effect is not yet established, the df4nano uses the 'functionality' of nanomaterials for grouping rather than relying on intrinsic material properties alone. such functionalities include system-dependent material properties (such as dissolution rate in biologically relevant media), bio-physical interactions, in vitro effects and release and exposure. the df4nano is a hazard and risk assessment tool that applies modern toxicology and contributes to the sustainable development of nano-technological products. it ensures that no studies are performed that do not provide crucial data and therefore saves animals and resources. the the grouping decisions of df4nano for 24 nanomaterials were validated against grouping by results of existing in vivo data and demonstrated 23 concordant grouping decisions. serum concentrations in cell culture medium influence the effect of cerium oxide nanoparticles on human lung a549 cells: cytotoxicity, proinflammation, cellular uptake, and particle properties k. burchardt the wide use of cerium oxide (ceo 2 ) nanoparticles (np), e.g. as fuel additive and for industrial and biomedical applications, evoked intense studies to understand the effects those particles might have on living organisms. contradictory results of ceo 2 nanoparticle toxicity have been published as they depend on many variables like shape, size, diluent and others. in our work we used an in vitro model of ceo 2 nanoparticles and lung carcinoma cells to investigate the role of serum content in the cell culture medium on cellular toxicity, particle uptake, proinflammation and particle characteristics. proinflammatory ceo 2 np with an average diameter of 15-30nm and different concentrations were diluted in cell culture medium with different fetal calf serum (fcs) concentrations (0.01, 0.1, 1 and 10%) and were used to expose human lung adenocarcinoma cells (a549) for up to 24 hours. at 100µg/ml ceo 2 np showed little to no toxic effecton growth arrested a549 cells at fcs concentrations of 1% or below, but cell viability was decreased to about 80% in proliferating cells in cultures with 10% fcs. the proinflammatory effect of ceo 2 np was investigated through measurement of il-8 mrna expression after 3 hours and cellular il-8 secretion after 24 hours. the qrt-pcr showed that the expression of il-8 mrna in cells treated with 100 µg/ml ceo 2 np in 10% fcs medium was three times higher than in cells treated with lower fcs concentrations. this finding correlated with the cellular il-8 secretion, which showed a stronger increase by cells treated at 10% fcs. differences in cellular uptake of ceo 2 np was determined by fluorescence-activated cell sorting (facs) after 2 and 24 hours of exposition. after 2 hours, the cells treated with ceo 2 np in 10% fcs medium showed a lower mean granularity (∆gmean) as measure for cellular particle uptake than those with less fcs in medium. after 24 hours all probes showed about the same granularity. to examine the effect the fcs concentrations on ceo 2 np characteristics in cell cultures we used dynamic light scattering (dls) and phase contrast microscopy (pcm). dls measurements revealed an increasing hydrodynamic diameter of the particles with decreasing fcs concentrations (about 1030nm (10% fcs) to 4090nm (0.01% fcs)), which was correlated by an increasing particle agglomeration shown by pcm. our results show that the fcs concentration in cell culture medium has a direct or indirect impact on the cytotoxicity, the proinflammatory effect, the facs parameter for cellular particle uptake as well as on particle properties, which should be taken into account when designing, performing and interpreting in vitro experiments to investigate the toxicity of nanoparticles. toxic and inflammatory effects of shape-engineered titanium dioxide nanoparticles in nr8383 rat alveolar macrophages. knowledge about the contrasting toxicity of nanoparticles (np) of different chemical composition has steadily increased over the past decade. however, available literature often reveals considerable differences in effects within a specific type of nanomaterial. these contrasts have been contributed to different handling and testing protocols as well as to sample-specific differences in physico-chemical properties of np that could affect their mode of interaction with cells. within the nanometrology project setnanometro, the highly controlled generation and characterisation of a large set of shape-engineered tio 2 np allows us to investigate the potential role of subtle shape-and surface structure changes on np toxicity. as inhalation represents the most relevant uptake route of np, the nr8383 rat alveolar macrophage cell line was selected for in vitro toxicological testing. since oxidative stress and inflammation are considered as key biological pathways in nanotoxicity, we evaluated the expression of the oxidative stress marker genes heme oxygenase-1 (ho-1) and g-glutamylcysteine synthetase (g-gcs) as well as the pro-inflammatory genes interleukin (il)-1β, il-6, il-18 and inducible nitric oxide synthase (inos) by qrt-pcr. protein levels of il-1β and tumour necrosis factor-α (tnf-α) were measured by elisa. cytotoxicity testing of the tio 2 np by wst-1 assay overall revealed only minimal toxicity in comparison to sio 2 np which were used as reference material. ho-1 and g-gcs mrna analyses indicated that specific tio 2 np triggered a moderate induction of oxidative stress. il-6 was only induced after sio 2 treatment, whereas il-18 was not affected by any of the tested np. in contrast, various tio 2 np caused a significant induction of il-1β mrna expression. however, no significant induction of il-1β and tnf-α protein secretion was observed for any of the tio 2 np. the results obtained from these and ongoing investigations will be linked to the physico-chemical database as being developed for all tio 2 np within the setnanometro project, with the overall aim to build and model nano-structure activity relationships (nsar) for this widely applied type of nanomaterial. acknowledgements: the setnanometro project is supported by the eu-fp7 programme. specific types of tio 2 particles were obtained by solaronix (switzerland), evonik and cristal. potential of silver and silver nanoparticles to reduce n-acetyltransferase 1 (nat1) activity j. lichter 1 , b. blömeke 1 1 trier university, department of environmental toxicology, trier, germany humans are exposed to various kinds of engineered nanoparticles including silver, which is frequently used in consumer and biomedical product due to its bactericide properties. despite their widespread usage, knowledge about influences on cellular functions is still incomplete. n-acetyltransferase 1 (nat1), an enzyme which is ubiquitously expressed in human tissues, catalyzes the transfer of an acetyl group to its substrates and although its endogenous function is not clear yet, it is well known to be involved in the n-acetylation of arylamines. in addition nat1 enzyme activity is known to modulated by non-substrates including metals and certain nanoparticles, however, the influence of silver on nat1 has not been analyzed yet. to address whether human nat1 is a target of silver nanoparticles and released irons on protein, purified nat1 was exposed to silver ions (agno 3 ) and silver nanoparticles (ag10-cooh, average size 10 nm, carboxyl functionalized), and nat1 enzyme activity was analyzing the n-acetylation of the nat1 substrate para-aminobenzoic acid (paba). therefore, purified nat1 (1 ng/µl) was co-exposed for 20 min to paba (1 mm) and agno 3 or ag10-cooh (0.01, 0.1, 1, 10 and 100 µg/ml each), resulting in a nat1:silver relation of 1:0.01-100 (w/w). both, agno 3 and ag10-cooh inhibited the n-acetylation of paba in a concentration dependent manner. using equal amounts of silver and nat1 (w/w, 1 µg/ml) enzyme activity was reduced about 98±0.2% (agno 3 ) and 82±0.6% (ag10-cooh). the lowest concentration analyzed (0.01 µg/ml) reduced nat1 activity about 24±5% (agno 3 ) and 17±5% (ag10-cooh). fifty percent activity reduction was caused by 0.11 ± 0.01 µg/ml of agno 3 and 0.21 ± 0.04 µg/ml of ag10-cooh, which is 10-fold lower compared to the published ic50 values for other metal oxide nanoparticles (3-15 µg/ml). these data indicate that both chemical silver species are able to modulate paba acetylation. further studies will be performed to clarify whether silver ions and/or silver nanoparticles could affect the specific n-acetylation of arylamines in human cells. colloidal silver has been used in medicine for centuries and nanosilver is present in many consumer-related products. however, despite of intense research in the past few years, the potential of nanosilver to induce effects different from ionic silver in vivo and in vitro, is still under debate. in this study, we compared proteomic effects of nanosilver (agpure™) and ionic silver (silver acetate) in the kidney of male rats after repeated oral delivery in a rat 28-days toxicity study. in order to avoid overt signs of toxicity, silver was dosed moderately in amounts of 60 and 6 mg/kg body weight for nanosilver and corresponding amounts of silver acetate (9.3 and 0.93 mg/kg). accordingly, no pathologic effects, including results from clinical chemistry and hematology, were reported. kidney tissue protein crude extract was separated by 2-d gel electrophoresis and differentially expressed spots were identified by maldi-ms. 374 unique proteins, showing a log 2 ratio of ≤ -0.3 for downregulation and ≥ 0.3 for upregulation were identified in all treatment groups. protein lists were analyzed with ingenuity pathway analysis (ipa). when comparing effects of particulate and ionic treatments, similar alterations were indicated for canonical pathways associated with glycolysis, gluconeogenesis and tricarboxylic acid cycle. regarding inflammatory responses, stronger effects were derived for ionic treatments. for both types of silver exposures, changes of protein expressions were linked to changes of fatty acid metabolism and nrf2-mediated stress. mitochondrial dysfunction was highlighted for both nanosilver treatments only, as well as activation of the insulin receptor. in the top-scored network of the higher dose nanosilver treatment, upregulated 14-3-3 protein zeta (ywhaz) displays a central position. ywhaz, an important regulator of cell cycle and apoptosis, interacts with the insulin receptor and is well known to be envolved in many types of cancer. overall, both forms of silver treatment revealed similar patterns of affected cellular and molecular functions in rat kidney, supporting common and overlapping mechanisms of particulate compared to ionic silver. because of the widespread application of nanomaterials and the fact that for some nanomaterials effects on different organisms where shown, nanomaterials are still in focus of interest. moreover the fate of nps is only partiallyassessed over the lifecycle of products containing nanomaterials. while general toxicological properties of nps are well described in diverse in-vitro and in-vivo experiments, the distribution of these particles during the whole and complex process of waste incineration shows big knowledge gaps. in the "nanoemission"-project the entire route from the residual material via incineration, filtering of the exhaust gas up to a possible release into the environment are considered together with the toxicological evaluation of effects on humans and the environment. in these experiments the influence of thermal waste treatment on the toxicological profile of nanoparticles, contained in the waste, will be described. after a complete characterization of the two types of baso 4 -nps from two different manufacturers by scanning electron microscopy/ energy dispersive x-ray analysis (sem/edx), measurement of the specific surface area (bet) and dynamic light scattering (dls) we investigated the impact of the pure baso 4 -nps on primary cells (normal human bronchial epithelial cells (nhbec) and peripherial lung cells (plc)). both materials show statistically significant cytotoxic effects in the resazurin-assay (decreased viability below 40 % for 1 mg/ml after 72 h). in general the effects of both nps were almost similar. additionally the effects of the baso 4-nps were compared more in detail. uptake of the baso 4 was quantified by icp-ms after 24 h and 72 h as well as the release of ba-ions into the cell culture medium after centrifugal separation. the incubation with baso 4 of plc and nhbec to 0.1 mg/ml over 24 h and 72 h leads to ~200 µg baso 4 /1 mio cells. the uptake is dose dependent but not time dependent. the impact on the secretion of inflammatory cytokines was determined by bead-based multiplex-elisa flow cytometry. tnf-α, il-8 and il-33 could be detected in nhbec and plc after np incubation. the possible induction of apoptosis was measured by flow cytometry as well. first investigations showed no induction of apoptosis for both materials. the impact of both nps on the intracellular glutathione level was measured by hplc and showed a decrease of gsh after 72 h. summing up baso 4 -nps showed toxic effects in primary human lung cell cultures after 72 h for concentrations under 1 mg/ml. c3 exoenzyme from c. botulinum is an adp-ribosyltransferase that inactivates selectively rhoa, b and c by coupling an adp-ribose moiety. rho-gtpases represent a molecular switch integrating different receptor signalling to downstream transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton, cell proliferation and apoptosis. previous studies with the murine hippocampal cell line ht22 revealed a c3-mediated inhibition of cell proliferation and a prevention of serum-starved cells from apoptosis (rohrbeck et al., 2012) . former results of studies on map kinase signalling indicated c3-induced modulations of downstream signalling modules. therefore, ht22 cells treated with 500 nm c3 for 48 h were applied for screening of the activity of 48 various transcription factors followed by luciferase reporter assays. five transcription factors namely sp1, atf2, e2f-1, cbf, stat6 were identified as significantly regulated in their activity. for validation of identified transcription factors, studies on the protein level of certain target genes were performed. western blot analyses exhibited an enhanced abundance of sp1 target genes p21 and cox-2 as well as a raise in phosphorylation of c-jun. in contrast, the level of apoptosisinducing gadd153, a target gene of atf2, was decreased. our results suggest that c3 is able to modulate the activity of transcription factors whose target genes are involved in the regulation of cell proliferation and apoptosis. via covalent binding of chemical compounds to dna, adducts can be formed. as a consequence, mutations may occur, which represent stages of chemical mutagenesis and carcinogenesis. the isolation of leucocytes represents an essential field of work within dna-adductomics of blood cells, in which adducts serve as markers of exposure for biological monitoring. peripheral mononuclear blood cells (pbmc) comprising monocytes and lymphocytes can be separated from other blood cells like granulocytes, erythrocytes (and dead cells) by isopycnic density gradient centrifugation of buffy coats (bc´s). bc´s are blood concentrates rich in leucocytes and thrombocytes, prepared from whole blood samples thorough removal of plasma and erythrocytes. for the experiments only bc´s from healthy donors were used. while erythrocytes contain no dna, lymphocytes, which constitute a subcategory of leucocytes, carry genetic information. a variety of commercially available fluids with a density of 1.077g/cm 3 , based on different polymers, exists. these materials form the gradient required for the centrifugation. furthermore, there are several methods available, which differ in the ratio blood vs. fluid, duration of the different work up steps, centrifugation parameters and others. the aim of this work was to compare the effectivity of the different fluids and optimize the workflow. for isolation of pbmc the fluid was put in a tube and then covered by a thin layer of blood. after centrifugation, five layers were obtained (figure 1 ). the interphase was removed carefully, washed and the cells were counted. the yield is expressed as percentage of isolated vs. total leucocytes in the bc, which was based on the leucocyte count of the whole blood sample. as separation fluids, ficoll® paque plus (ge healthcare), histopaque® (sigma aldrich) and lsm® (ge healthcare) were tested. no significant differences between the different fluids were observed. although dilution is often recommended in literature, it was found that dilution had no effect on the yield. similarly, using different fluids (rpmi or pbs) for the washing steps did not reveal any differences. increasing centrifugation speed from 400 to 500 xg resulted in higher yields. in general, large variations in yield (46.8% ± 17.1%) among the various bc´s arose. this result is due to the different parameters such as age, storage, leucocyte and erythrocyte counts of the different bc´s used. therefore, the test conditions were optimized using the same batch of bc. the results show that the low priced separation fluids are comparable in performance tothe more expensive ones. by the direct lamination with bc (without dilution) the wastage could be minimized, the yield increased and thus the isolation made more efficient. the franz cell is a well-established model in lots of skin research fields. we adapted the diffusion cell system to additives and contaminants of consumer products which are designated for skin contacts. we were aimed to simulate real exposure as realistic as possible. to meet requirements like longevity, haptic properties and factory costs, different polymers are used as the raw material of choice and modified by a variable number of additives in the majority of commodities. besides additives with a defined function such as plasticizers, stabilizers, colorants and vulcanization accelerators, contaminants as well as decomposition products or so-called non-intentionally added substances (nias) can be part of the material, among them potentially harmful substances. in a first step, polymers of consumer products like flashlights or tools have been characterized concerning additive composition. possible breakdown products were identified by means of gc-ms/ms or pyrolysis-gc-ms. we focused on analytes of toxicological relevance including antioxidants such as n-phenyl-2-naphthylamine (neozon d), which is suspected of causing cancer, and on degradation products like cresol and its derivatives (e.g., mesitol or 2-tert-butyl-4-methylphenol). subsequently, analytes of interest were brought into direct skin contact using porcine, human and artificial skin models in the franz cell chamber assay. the analytes were either followed up layer by layer using tape stripping or examined utilizing cryo sections. for visualization purposes, analytical evaluation has been completed by use of imaging techniques like he staining or atr-ftir (attenuated total reflectance fourier transform infrared) microscopy. the latter was used with intrinsic markers for tissue specific distribution. our project provides evidence for the potential of polymer components to overcome the natural epidermal barrier and, in part, to enter the viable layers of the epidermis. during skin contact with consumer products several substances migrate out of the matrix and penetrate the skin, among them substances which are hazardous to health such as neozon d. depending on its dose, the blister agent sulfur mustard (sm) may lead to painful erythema, blistering with complicated wound healing, pulmonary edema, pulmonary bleeding and temporary blindness. sm is listed as a schedule 1 chemical in the chemical weapons convention and thus its production, stockpiling and use is prohibited. sm still represents a serious threat for civilians and military forces, especially in asymmetric, terroristic and accidental scenarios. after exposure, the highly reactive molecule alkylates nucleophilic sites in endogenous biomacromolecules forming the characteristic and stable hydroxyethylthioehtyl (hete) residue. hence, bioanalytical methods targeting theses adducts in forensic post-exposure verification analysis are of high interest. herein, we present an optimized, accurate and comparably simple method to detect adducts of sm and human serum albumin (hsa) alkylated at its cysteine 34 -residue. since albumin extraction from human plasma is a time-consuming and expensive step of an established procedure, an alternative method for direct proteolysis of human plasma was developed. plasma samples were cleaved directly with pronase resulting in the alkylated dipeptide hete-cysteine-proline (hete-cp) which is detected by micro-liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µlc-esi hr ms/ms). in order to optimize reproducibility and yield of proteolysis, kinetics were investigated for different kinds of plasma (edta-, citrate-and heparin-) as well as serum. two different mass spectrometers, a triple quadrupole system (4000 qtrap) and a hybrid quadrupole time-of-flight instrument (tt5600 + ), were compared. the latter one proved to be the more selective and sensitive system. the method was successfully applied to in vitro and in vivo samples of real cases of sm poisoning. organophosphorus compounds (op), which were originally intended to be used as pesticides to increase agricultural yields at the onset of the 20 th century, still represent a considerable threat to human health. by irreversible inhibition of acetylcholinesterase op lead to cholinergic crisis due to uncontrolled increase of acetylcholine in the synaptic cleft. finally, sudden death by respiratory failure may result if medical countermeasures are lacking. therefore exceptionally toxic compounds were designed und synthesized as chemical warfare agents (cwa), among which v-type nerve agents, i.e. vx, chinese vx and russian vx, belong to the most toxic artificial substances. recent events like the first gulf war, terrorist attacks in tokyo and the conflict in syria underline the need for ongoing and strict surveillance of cwa prohibition by the chemical weapons convention. unambiguous evidence of such substances (verification analysis) plays an important role with great political and legal impact. a variety of such bioanalytical methods have been established at the bundeswehr institute of pharmacology and toxicology in munich, which is accounted for medical chemical defence in germany. exhibiting quite short half-lives in vivo nerve agents can hardly be detected days or even weeks after exposure. accordingly, there is a great need for additional long-term biomarkers like specific protein adducts. consequently, the current work focuses on examination of adducts between nerve agents and human serum albumin (hsa) as its high abundance and stability in vivo provide relative ease of sampling. after incubation of hsa with v-type nerve agents in vitro the protein was subjected to proteolysis. subsequently resulting peptides were separated using microbore high-performance liquid chromatography (µlc) and detected on-line by modern high-resolution tandemmass spectrometry (hr ms/ms). this allowed unambiguous identification of already known phosphylated tyrosines as well as novel adducts between cysteine-proline dipeptides and the thiol-containing leaving group of v-type nerve agents. simultaneous detection of both biomarkers was realized by a new method, which was applicable even at the very low toxicologically relevant concentrations of v-type agents. therefore, this method represents a valuable and novel supplement of existing methods for verification. fatty acid esters of glycidol (glycidyl esters) are processing contaminants formed as byproducts of industrial deodorizing of plant fats or during other heating processes. following oral intake, glycidyl esters are mainly cleaved to release the reactive glycidol in the gastrointestinal tract. according to the national toxicology program (ntp), glycidol is carcinogenic, genotoxic and teratogenic in rodents. it is classified as probably carcinogenic to humans (iarc group 2a). the exposure assessment of the oral intake of glycidyl esters in humans is difficult, because the current data set for glycidyl ester contents in food is incomplete. we developed a method for the determination of the internal exposure to glycidol by mass spectrometric quantification of a hemoglobin adduct reflecting the total glycidol burden over approximately three months. a modified edman degradation was adapted for the cleavage of the valine residues from the n-termini of hemoglobin by fluoresceinisothiocyanat (fitc) (von stedingk et al. (2011 ) chem res toxicol 24, 1957 resulting in the formation of dihydroxypropyl-valine-fluorescein thiohydantoin (dhp-val-fth). the target analyte is purified with mixed-mode anion-exchange solid-phase extraction and analyzed by lc-ms/ms. a major advantage of the technique is the application to whole blood samples, which renders the time-consuming isolation of erythrocytes unnecessary. we synthesized dhp-d 7 -val-fth as an internal standard for the quantification of the glycidol adduct by lc-ms/ms multiple reaction monitoring. a limit of detection of 5 fmol per injection (5 pmol adduct/g hemoglobin) was achieved. the application of this method will possibly allow future monitoring of the internal exposure of glycidol in human studies. glycidol and 3-monochloropropane-1,2-diol (3-mcpd) are carcinogenic food contaminants, which are present in heat-processed oils and fats mainly in form of fatty acid esters. the risk assessment concerning human consumption of these substances is complicated by various reasons. for example, the data on the occurrence in food stuffs is incomplete. also, the amounts of the proximate carcinogens released from ester hydrolysis in the gastrointestinal tract in humans are not known. monitoring of the internal exposure would be an alternative strategy to support the assessment of possible health risks related to the intake of glycidol and 3-mcpd and their fatty acid esters. for short-term monitoring of the internal exposure, urinary metabolites are suitable biomarkers. we study the potential use of two different substances as descriptors of the oral intake of glycidol and 3-mcpd. the metabolite 2,3-dihydroxy mercapturic acid (dhpma) is generated following glutathione conjugation of both compounds. the second target analyte is 3-mcpd itself, which may also be formed from glycidol in the reaction with hydrochloric acid in the stomach. a method for the quantification of urinary 3-mcpd by gc-ms is currently developed. an lc-ms/ms multiple reaction monitoring technique was devised for the quantification of dhpma in urine samples with the isotope-labeled reference compound 13 c 2 -dhpma. the limit of quantification is 10 µg dhpma/l. related to creatinine, the analyte was detected to be in a relatively small concentration range in urine samples from humans. the average concentration in urine samples (n = 45) of one male volunteer collected over ten days was 154 ± 21 µg dhpma/g creatinine. a meal of a highly contaminated, commercially available frying fat (containing 1.1 mg of glycidol equivalents) did not lead to a visible increase of the urinary concentrations. the considerable background levels of dhpma in urine of humans and also in urine samples of other mammals support the hypothesis that dhpma may be also be formed from an endogenous c 3 -metabolite, as already reported by eckert et al. furfuryl alcohol is a common food contaminant formed by acid-and heat-induced dehydration from pentoses. it induced renal tubule neoplasms in male b6c3f1 mice and nasal neoplasms in male f344/n rats in a study of the national toxicology program (ntp). the neoplastic effects may originate from sulfotransferase (sult)-catalyzed conversion of furfuryl alcohol into the dna reactive and mutagenic 2-sulfoxymethylfuran. the incomplete data set of furfuryl alcohol contents in food does not allow estimating the human exposure. thus, we sought a method for the determination of the internal exposure. recently, the dna adduct of furfuryl alcohol n 2 -[(furan-2-yl)methyl]-2′deoxyguanosine was detected in specimen of human lung tissue. however, human biomonitoring of dna adducts has various disadvantages. for example, dna adducts are removed by various repair systems and human dna samples are usually not accessible in sufficient quantities. we decided to develop a biomarker for the internal exposure to furfuryl alcohol using blood proteins as dosimetric targets. bladder cancer (bc) is a smoking and occupation related disease showing a substantial genetic component. though the prognosis is generally good, a major problem is the frequent relapses affecting about half of the patients. n-acetyltransferase 2 (nat2) is well-known to modulate bc risk in persons heavily exposed to carcinogenic aromatic amines. we aim to investigate the impact of nat2 genotypes, in particular, the ultraslow genotype, on relapse-free time after first diagnosis in 772 bladder cancer cases. we used follow-ups of three case-control studies from lutherstadt wittenberg (n=207), dortmund (n=167) and neuss (n=398). nat2 was genotyped using seven characteristic polymorphisms (rs1801279, rs1041983, rs1801280, rs1799929, rs1799930, rs1208, rs1799931). haplotypes were reconstructed using phase v.2.1.1. we compared slow to rapid acetylators. additionally, we differentiated between the most frequent slow nat2*5b/*5b and *5b/other slow haplotypes as well as between the ultra-slow *6a/*6a and *6a/other slow haplotypes compared to rapid acetylators. chi-square tests used to check the frequency of relapses in ultra-slow, slow and rapid acetylators. genotype differences in relapse-free time up to 5 yr after first diagnosis of bc were analysed using cox proportional hazards models adjusted for age, gender, smoking habits, invasiveness and study group. a total of 371 (49%) patients showed a relapse within the first 5 yr after bc diagnosis. slow acetylators show a higher frequency of relapses than rapid acetylators (51% vs. 46%, p=0.154). this frequency is even higher in ultra-slow acetylators (61%, or=1.81, p=0.019) but not in slow *5b/*5b genotypes (49%, p=0.609). ultra-slow acetylators had a significantly shorter relapse free time within 5 yr after bc diagnosis than rapid acetylators (median 0.66 vs. 0.94 yr, hr=1.57, p=0.009). this trend was not that pronounced in all slow acetylators combined (0.78 yr, hr=1.19, p=0.127) nor in the subgroup of nat2*5b/*5b genotypes (0.79 yr, hr=1.20, p=0.255). the effect of ultraslow nat2 is even more pronounced in smokers (hr=1.78, p=0.003) but absent nonsmokers (hr=0.89, p=0.781). ultra-slow nat2 seems to be associated with a higher recurrence risk and a shorter relapse-free time, especially in smokers. slow nat2 in general seems to have less impact on recurrence. aalborg university, department of biotechnology, chemistry and environmental engineering, aalborg, denmark xenoestrogens with the potential for endocrine disruption like bisphenol a (bpa) may bind to the estrogen receptors (ers) and modulate expression of er target genes mimicking the natural ligand 17β-estradiol (e2). the potential for endocrine adversity is still predominantly assessed in vivo as existing in vitro tests have only limited value for an exposure-based risk assessment. thus, the development of reliable bioassays for the detection of endocrine disruptors is one of the paramount challenges faced by modern toxicology. a targeted metabolomics approach in mcf-7 cells treated with e2 or bpa revealed potential biomarkers for the estrogenic potency of the studied compounds. among them were several phosphatidylcholines and amino acids. we further addressed proline levels that were found to be strongly increased. investigations of proline levels over time showed a clear proliferation correlated concentration dependency after both e2 and bpa stimulation. furthermore, sirna knockdown experiments suggested an influence of the oncogenic transcription factor myc and the dependency of erα activation on the estrogen-mediated proline increase. our study demonstrates metabolomics as a powerful tool for biomarker identification and hypothesis generation. the results could be used further to develop bioassays for the detection of endocrine disruptive chemicals. children are considered to be more sensitive to most chemicals than the general population due to a variety of factors, including dynamic growth and developmental processes as well as physiological, metabolic and behavioral differences [1]. however, only a few data are available on the magnitude of preschool children's exposure to most chemicals present in many consumer products. several of these chemicals are linked to endocrine disrupting effects in animal studies and are suspected to have also adverse effects e.g. on development and function of the reproductive organs as well as on neurological and behavioral development in humans. among of the chemicals that have been a major focus of discussion in the last years are phthalates, dinch, parabens, bisphenol a, and triclosan due to their suspected health effects. therefore we aimed to investigate exposure levels to metabolites of different phthalates and parabens as well as to bisphenol a and triclosan in urine samples collected from preschool children in german day-care centers from north rhine-westphalia (lupe iii; 2011/12). urine specimens from children aged from 20 to 80 months from 23 different day-care centers were analyzed. in total, 253 preschool children were recruited with mean age of 54 months. our study results show that nearly all children (>95 %) of the study population had urine concentrations equal to or above the limit of quantification for five most common phthalates metabolites (mnbp, mibp, 5oh-mehp, 5oxo-mehp, 7oxo-minp), for bisphenol a and methylparaben. triclosan was detected in 16 % of the study population. in general, the median urinary concentrations of the above mentioned phthalate metabolites were about 5-50 µg/l in spot urine samples. the highest amount among the phthalate metabolites was observed for mibp with maximal values of about 1000 µg/l. median urinary concentration for methylparaben and bisphenol a were about 48 µg/l and 2 µg/l respectively. the maximum methylparaben, bisphenol a and triclosan level found were 1770 µg/l, 72.4 µg/l and 55,6 µg/l respectively. in conclusion, our study shows a widespread exposure of young children to various phthalates, parabens and bisphenol a in north rhine-westphalia, germany. a follow-up human biomonitoring study (2014/2015) has finished recruitment and is in the process of analyzing data. polycyclic aromatic hydrocarbons (pahs) represent a large group of organic compounds that are common environmental contaminants. they are formed by incomplete combustion of organic matter such as coal or crude oil and are often known to be carcinogenic, mutagenic and teratogenic. the acute toxicity of pahs is rather low, but because of their stability and lipophilic character those compounds can accumulate in the human body and cause severe chronic effects. additionally pahs may enter the food chain when preserving meat or fish by exposure to smoke. in the european union maximum levels of 2 µg/kg benzo[a]pyrene and 12 µg/kg as the sum of benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene and chrysene in the meat of smoked fish and smoked fishery products are set, respectively. smoked fish is often handmade in small fishery stores in schleswig-holstein, where self caught fish is prepared in smoke houses. this technique implies the danger of pahs to accumulate in smoked fishery products above allowed maximum levels. here, we report our findings of pahs in smoked fishery products bought in local convenience and fishery stores in schleswig-holstein and give a brief overview about actual contaminant levels. hplc with fluorescence detection was used to determine the quality and quantity of several toxic pahs in smoked fishery products made locally. pahs may constitute risks for human health when exposed to hazardous levels and therefore it is important to have knowledge about given contaminant levels. colorectal cancer (crc) is one of the most frequent cancers worldwide and is tightly linked to dietary habits. epidemiological studies provided evidence that the intake of red meat is associated with an increased risk to develop crc [1]. red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. the underlying molecular mechanism, however, remains elusive and may involve increased cell proliferation and dna damage induction by heme-iron. in this study, we set out to analyze the genotoxic and cytotoxic effects of heme-iron in human colonic epithelial cell lines. we used hemin (fe iii ) as commercially available heme source, which was compared to inorganic iron chloride (fecl 3 ). first, the time-dependent internalization of hemin and fecl 3 into hct116 cells was determined using icp-ms/ms analysis. treatment of cells with inorganic iron resulted in a maximum of intracellular iron content after 1 h at all doses tested, while hemin particularly at high doses caused an iron accumulation up to 24 h. hemin catalyzed the formation of reactive oxygen species (ros) in a dose-dependent manner in caco-2 and hct116-cells as shown by flow cytometry. consistent with this finding, hemin dose-dependently induced the oxidative dna lesion 8-oxoguanine (8-oxog) as revealed by slot blot analysis and fpg-modified alkaline comet assay. using a pharmacological inhibitor of mutt homologue 1 (mth1), which protects the nucleotide pool by hydrolysis of 8-oxogtp, 8-oxog dna adduct levels in hemin-treated cells were further enhanced. in contrast, inorganic iron hardly affected the cellular ros level and only slightly increased oxidative dna damage. subsequently, a time-and dose-dependent activation of the dna damage response (ddr) by hemin was shown in hct116 and caco-2 cells using western blot analysis, which was followed by a reduction in cell viability at high doses after 72 h. finally, the cytotoxic effects of hemin and inorganic iron were tested using an ex vivo model of intestinal crypt organoids. preliminary results indicate that hemin is highly cytotoxic in organoids, whereas inorganic iron does not impair their viability. taken together, this study demonstrated that hemin induces oxidative stress and dna damage, resulting in the activation of the ddr and subsequent cytotoxicity. in contrast, inorganic iron displayed only modest effects. further in vivo studies using dna-repair deficient and proficient animals will shed light on the contribution of specific dna lesions to hemeassociated colorectal carcinogenesis. red and processed meat consumption is known to be a crucial risk factor in the development of colon cancer, which is one of the most common cancers worldwide. a diet rich in red and processed meat increases n-nitrosation within the colon leading to an increase in endogenously formed nitroso compounds. these compounds are able to alkylate dna of gastrointestinal tract cells, resulting in the formation of dna adducts such as -meg is repaired by mgmt, the potential of this enzyme to repair o 6 -cmg in cells is not well characterized. additionally, adenomas containing a k-ras gc-at transition mutation have lower mgmt levels than adenomas without this mutation. therefore, the aim of this study is to determine the role of mgmt in protecting colorectal cells from genotoxicity by repairing o 6 -cmg adducts. for this purpose, an mgmt-deficient non-transformed human colon epithelial cell line was established by stable transfection via rna interference to inhibit the expression and therefore the activity of mgmt. the transfected cell line was analyzed for complete mgmt gene silencing by activity and expression analyses and cell characterization. results confirmed that there was neither expression nor activity for mgmt in the transfected cells, and the cell characterization data showed that mgmt deficiency did not lead to differences in growth behavior or cell morphology or malignant cell transformation. cytotoxicity experiments performed in the transfected and nontransfected cell lines by treatment with dna alkylating agents suggest that the mgmtdeficient cell line is more sensitive to dna alkylating agents than the non-transfected cell line. these results were also supported by dna damage analysis via comet assay. asarones are secondary plant constituents mainly occurring in acorus calamus l. and guatteria gaumeri. the essential oil of the rhizome of acorus calamus l. is used for flavoring of food and alcoholic beverages. the concentration of β-asarone (ba) in these oils varies between 0.3% and 95%. the bark of guatteria gaumeri, which is rich in αasarone (aa), is used as cholesterol-lowering drug and to treat gallstones in traditional mexican medicine. both, aa and ba are carcinogenic in rodents and mutagenic in the ames test after metabolic activation and thus classified as genotoxic carcinogens. [1, 2] previously, the major metabolites in microsomal incubations with aa and ba were identified and synthesized in our laboratory. side-chain epoxides, the corresponding diols, side-chain alcohols and aldehydes were identified as the major metabolites of aa and ba. [3] the investigation of the mutagenic potency in the ames fluctuation test showed positive results in the salmonella strain ta 100 for aa and ba with metabolic activation and for aa-and ba-epoxide without metabolic activation. while it is known that epoxides are reactive against nucleophiles we set up the hypothesis of dna-adduct formation by epoxides to explain the mutagenic effect. this adducts are currently isolated, characterized by nmr-spectroscopy and used to quantify adducts in cells. at the same time primary rat hepatocytes were incubated with non-cytotoxic concentrations of aa and ba for 24 h. after harvest and lysis of the cells, the dna was isolated by chloroform/phenol extraction and enzymatically hydrolyzed. [4] the residual hydrolysates will be used to identify the dna-adducts formed in cells and to determine the adduct formation rate. preliminary experiments indicate that both, aa and ba also form dna-adducts in intact cells in a concentration-dependent manner. [1] göggmann, schimmer (1983) . mutagenicity testing of α-asarone and commercial calamus drugs with salmonella typhimurium. mutation research. 121, [191] [192] [193] [194] wiseman et al. fatty acid esters of 3-chloro-1, and of 2-chloro-1, are food process contaminants that are formed, e.g., during refinement of vegetable oils. after ingestion, the esters are hydrolyzed in the gut, thereby releasing free 3-mcpd and 2-mcpd. 3-mcpd has been identified by the international agency for research on cancer (iarc) to be possibly carcinogenic to humans (class 2b) and is therefore in the focus of food safety authorities. to elucidate the toxicological properties of these compounds at the molecular level (mode of action) a proteomic study was conducted in which 10 mg/kg b.w./day of either 3-mcpd or 2-mcpd were orally administered to rats over a period of 28 days. total protein was extracted from different tissues of the animals and separated via twodimensional gel electrophoresis (2de). among others, the redox sensor protein dj-1 was identified to be deregulated in liver, kidney, testis, and heart of rats treated either with 3-mcpd or 2-mcpd. up to six different isoforms of dj-1 were identified by 2de-western blot analysis, all of them having the same molecular weight but different pi values, indicating protein modifications of low molecular weight but with a strong impact on the protein charge. treatment of the animals with either 3-mcpd or 2-mcpd predominantly led to a shift of the abundance between two dj-1 isoforms in the rat tissues. this effect was more pronounced with 3-mcpd compared to 2-mcpd. mass spectrometric analysis of these two isoforms identified an oxidation of a conserved cysteine residue (cys106) of dj-1 to a cysteine sulfonic acid to be the protein modification induced by treatment of the rats with either 3-mcpd or 2-mcpd. dj-1 is discussed to participate in a number of biological processes such as proteolysis, protein folding, or redox regulation. oxidation of cys106 was shown to be crucial for the activity of dj-1, and the irreversible oxidation of cys106 to a cysteine sulfonic acid as observed in the present study was shown to result in a loss of protein function and was correlated with diseases related to oxidative stress such as parkinson's disease. thus, the potential impact of 3-mcpd and 2-mcpd on cellular oxidative stress and on associated neurodegenerative diseases has to be considered in the ongoing risk assessment of these food contaminants. pyrrolizidine alkaloids (pa) are secondary plant compounds and widely spread in plant kingdom. humans can therefore be regularly exposed via direct or indirect contamination of food, like herbal teas, honey, wheat or salad. 1,2-unsaturated pa are known for their potentially harmful effects. an acute intoxication may cause venoocclusive disease leading to hepatomegaly, ascites as well as liver hardening resulting in high mortality. on the other hand, chronic pa intoxications due to regular consumption of small amounts of pa are characterized by hepatic necrosis, fibrosis and cirrhosis. an initial whole genome transcriptome study in hepatocytes revealed that molecular pathways related to cancer development, cell cycle regulation and cell death are regulated by the four structurally different pa echimidine, heliotrine, senecionine and senkirkine in short-term exposure (24h). additionally, lipid and bile acid metabolism was affected. however, the uptake of pa by food is more likely to be continuous than singular. therefore, the aim of this study was to investigate molecular effects of longterm exposure (14d) comparatively to short-term exposure (24h) in the hepatoma cell line heparg with the four structurally different pa. in this context we analyzed selected cellular parameters like cytotoxicity and morphology. in contrast to short-term exposure, structure-and concentration-dependent cytotoxicity was found for the long-term exposure (sn>he>em/ski). furthermore, obvious morphological changes such as destructuration and perforation of the heparg cell monolayer were seen after 14d of incubation. based on these findings, a possible induction of apoptosis or necrosis by pa was examined. effects of long-term exposure to pa on gene expression were investigated for a set of genes found to be regulated in the short-term whole genome transcriptome study. the identified regulation of gene expression was confirmed for both terms, with the strongest regulation for cyp7a1 (down-regulation), a gene involved in cholesterol metabolism. therefore, the effects of pa on various parameters related to cholesterol metabolism were analyzed, showing pa effects on cholesterol levels and bile acid secretion. short-term exposure to pa did not affect cell viability. however, repeated doses of pa resulted in severe effects on hepatic cells, concerning viability and morphology. at the mrna level, short-and long-term incubation with pa seem to affect a wide range of signaling pathways. in conclusion, we show for the first time that heparg cells can serve as an in vitro model for hepatotoxicity following chronic intake of pa. shiga toxin-producing e. coli (stec) strains cause a diversity of enteric symptoms in humans, ranging from mild diarrhea to severe diseases such as the hemolytic uremic syndrome (hus). hus is a life threatening disease with microvascular endothelial damage in the gastrointestinal tract and the kidneys, which often leads to haemolytic anemia, thrombocytopenia and acute renal failure. shiga toxin plays a major role for the pathogenesis of hus but the subtilase cytotoxin, which was identified during an hus outbreak in 1998 in stec strains, might contribute as an additional potent enterotoxin. like shiga toxin, subab is an ab 5 protein toxin. the pentameric binding/transport subunit (subb) delivers the enzymatic active subunit (suba), a protease, into the endoplasmic reticulum (er) of human target cells. in the er, suba cleaves the chaperone bip/grp78, which results in cell stress and caspase 3/7dependent cell death. recently, we discovered that higher concentrations of suba (10 mg/ml) causes cytotoxic effects in human epithelial cells (hela, in the absence of subb. the cytotoxic effects were investigated in hela cells in more detail. here, suba binds in a concentration dependent manner to the cell surface and induces dramatic morphological changes as well as caspase-dependent cell death [1] . in contrast to hela and caco-2 cells, cho fibroblasts did not respond to suba. currently, further cell types including macrophages are tested for suba effects and the molecular mechanisms underlying the cytotoxic effects caused by suba and the cell type selectivity are investigated. although there are strong cytotoxic effects caused by suba on some human epithelial cells in vitro, the situation in vivo and the pathogenic relevance of the newly observed suba effect are not known. thermal treatment of fat-containing foodstuff in the presence of salt leads to formation of 3-mcpd and its esters. high amounts of 3-mcpd esters detected in food raised toxicological concern. recent studies revealed that food may also contain significant amounts of structurally related 2-mcpd and its fatty acid esters. toxicological studies indicate genotoxicity in vitro and a carcinogenic potential of 3-mcpd, pointing to kidney and testes as main target organs. 3-mcpd esters were shown to cause similar, but milder effects. few unpublished data exist for 2-mcpd, showing similar organ toxicity compared to 3-mcpd and identifying heart as additional target organ. no such data exist for 2-mcpd diesters. in consequence, further toxicological data were required in order to complete risk assessment for 3-mcpd, 2-mcpd and their esters. hence, an oral 28-days study was performed in male rats in order to fill data gaps and provide essential information for risk assessment. a proteomic analysis was included in order to compare molecular effects induced by 3-mcpd and 2-mcpd and its dipalmitic ester in rat liver, kidney, testes and heart. in order to avoid overt toxicity, moderate doses of 3-mcpd + 2-mcpd (10 mg/kg body weight), and 2-mcpd-dipalmitate (53 + 13.3 mg/kg body weight) were applied. accordingly, no pathologic effects were reported. here, we present proteomic results obtained after analysis of heart tissue. after separation of heart tissue protein crude extract by 2-d gel electrophoresis , differentially expressed spots were identified by maldi-ms. a total of 270 unique proteins deregulated at a log 2 ratio of ≤ -0.5 for downregulation and ≥ 0.5 for upregulation at p ≤ 0.05 were identified. comparing deregulated spots induced by different treatments revealed that a higher number of spots was deregulated by 2-mcpd versus 3-mcpd. dipalmitate treatment even caused more deregulation than 2-mcpd. upregulated cytochrome b-c1 complex subunit rieske (ucri) and downregulated acetyl-coa acetyltransferase (thil) were among the top deregulated proteins after 2-mcpd and 2-mcpd dipalmitate treatment. pronounced upregulation of respiratory chain protein ucri, not deregulated after 3-mcpd treatment, indicates an effect on energy metabolism. downregulation of thil, involved in ketone body metabolism, was only weakly affected after 3-mcpd treatment. protein dj-1 (park7), a multifunctional redoxsensitive chaperone and protease protecting the cell against oxidative stress, was significantly downregulated after treatment with 3-mcpd and the higher dose of the 2-mcpd diester. tropomyosin beta chain (tpm2) was commonly upregulated after 3-mcpd and 2-mcpd treatments, possibly indicating a tgf-beta induction of actin stress fibers. for rat heart, data show that similarities but also some significant differences of 2-mcpd-and 2-mcpd dipalmitate-induced proteomic changes exist compared to 3-mcpd, indicating different mechanisms of toxicity for this structural analogues. oxidation products (oxy) of cholesterol (chol) such as 7α-hydroxy-chol (7α-ho-chol), 7β-hydroxy-chol (7β-ho-chol), 7-keto-chol (7-o-chol), 5,6α-epoxy-chol (α-epoxy-chol) and 5,6β-epoxy-chol (β-epoxy-chol) are formed by autoxidation of chol and are discussed as biomarkers for inflammation and oxidative stress in human tissues to be used in the identification of risk factors for disease. however, oxy-chols are also present in processed foodstuff where 7β-ho-chol (milk) and 7-o-chol (fish, meat, and egg) tend to represent the main oxychols, whereas epoxy-chols, are generally minor constituents. thus, levels of oxychols in tissues may result from both endogenous formation as well as dietary exposure. since quantitative profiles of oxy-chols have not been determined in human adipose tissues yet, levels of oxychols and chol were quantified using gc-ms/ms (internal standards: deuterated oxychols) and gc-fid (internal standard: 5α-cholestan-3β-ol), respectively in breast adipose tissues of 48 healthy women undergoing mammoplasty. furthermore, tissue levels of 25 fatty acids in adipose tissues were determined by gc-fid (internal standard: undecanoic acid) to assess relative levels of pentadecanoic acid, docosahexaenoic acid and elaidic acid, indicative for consumption of dairy products, fish, and processed fats, respectively. all oxychols were detected in all breast adipose tissues. the most abundant oxychol was β-epoxy-chol (median: 0.36 nmol/g tissue, range: 0.06-1.55 nmol/g), followed by α-epoxy-chol > 7-o-chol > 7α-ho-chol> 7β-ho-chol (0.009 nmol/g, range: 0.002-0.11 nmol/g). tissue levels of chol (2.8 micro mol/g, range: 1.88-3.87 micro mol/g) did not correlate (spearman's rank analysis) with that of epoxy-chols and correlated even negatively with that of 7α-ho-, 7β-ho-, and 7-o-chol (r = -0.29, p=0.024-0.044) median oxychol/chol ratios ranged from 0.0119 (5, to 0.0003 (7β-ho-chol). 7-o-chol and 7-ho-chols correlated strongly with each other (r=0.81-0.91, p oxy-chol levels did not correlate with relative amounts of pentadecanoic acid and docosahexaenoic acid, whereas total oxychol, 7β-ho-chol, and β-epoxy-chol levels correlated with relative amounts of elaidic acid (r=0.30, 0.30, and 0.31, respectively, p=0.037, 0.034, 0.028, respectively) . no correlations of oxychol levels, individual or total oxychol/chol ratios with age or bmi were observed. taken together, oxychol profiles in breast adipose tissues were different from that usually present in food but could be affected by dietary habits. classification and labelling of hazardous substances and hazardous consumer products (1) has proven to be a very efficient tool for risk communication. consumer products, such as glue, varnish, or washing and cleansing products need to be classified and labelled if they contain dangerous ingredients that render the mixture hazardous. personal care products, however, need not be classified and labelled if they contain dangerous substances above the thresholds for classification. they are excluded in the clp regulation. what would happen without this exception? when i applied the criteria for classification and labelling to a selection of cosmetic product formulas in a conservative approach, most products would have to be labelled and classified, mainly due to hazardous effects to the eye and to the skin (2) . benefits of personal care products can go along with unwanted properties such as the hazards for the human health, and consumers should be informed about them (3) . risk communication for every day products like personal care products should be clear, easily and quickly understandable. according to the cosmetic regulation (4) the ingredients must be listed on the containers. it must be questioned whether the listing of the ingredients without hazard pictograms on the products could be considered as a clear, easily and quickly understandable risk communication instrument (3). the results show that it is urgent to inform consumers better about the potential dangers of personal care products, because cosmetics need to be applied even with more care than any other consumer product. it is strongly recommended to delete the exception provision in the clp regulation for personal care products. the infochemical effect describes that anthropogenic substances can interfere with the chemical communication and influence organisms so that they perceive their chemical environments differently (1,2). infochemicals play a role in life history, habitat search, food related aspects and survival which shows that disturbed communication (infodisruption) could affect population vulnerability at various decisive points (3). the classical ecotoxicological standard tests do not allow to observe the infochemical effect. systematic analyses are needed to find out more about the relevance of this new chapter in ecotoxicology for natural ecosystems. the first crucial step is to select suitable test substances. repellents (substances used to keep away target organisms, e.g. invertebrates such as midges or fleas via olfaction) enter surface waters mainly indirectly via wastewater discharges from sewage treatment plants or directly by being washed off from the skin and clothes of bathers. there are various indications that repellents which are not toxic for aquatic animals could induce effects like organismic downstream drift of non-target species (downstream dislocation of e.g. crustacean and insect larvae in streams). in a literature study, three repellents were identified to be suitable test compounds for proof of concept of the infochemical effect. deet (cas 134-62-3), icaridine (cas 119515-38-7) and ebaap (cas 52304-36-6) (4). these substances are investigated in new test designs in a subsequent experimental part of the project which are designed to detect and quantify the infochemical effect. persistent, bioaccumulative and toxic (pbt) substances or very persistent and very bioaccumulative (vpvb) substances are compounds with hazardous properties. the non-biodegradability (persistence) and high accumulation in organisms (bioaccumulation) may elicit long-term adverse effects in the environment. persistent and bioaccumulative substances concentrate in the environmental compartments (water, sediment, soil, air) and can be distributed in the food chains. ecotoxicological effects are strengthened by bioaccumulation and appear often in remote areas like marine and polar regions. in the framework of pbt assessment, contrary to the risk assessment, the substances are evaluated regardless of the emission into the environment. an evaluation of medicinal active ingredients under assessment in the german federal environment agency (uba) identified less than 10% as potential pbt candidates. due to data lacks in many cases a definite pbt classification is not possible. the poster presents results of an extensive literature research on the global occurrence of pharmaceuticals in the environment. data on measured environmental occurrences from more than 1,000 international publications have been transferred to a database, with more than 120,000 entries. according to the database, pharmaceuticals have been found in the environment of 71 countries worldwide in all five un regions. most published data are for the compartments surface water and sewage effluent; less information is available for groundwater, manure, soil, and other environmental matrices. more than 600 active pharmaceutical substances (or their metabolites and transformation products) have been detected in the environment. most findings have been published for industrialized countries. monitoring campaigns are increasingly being conducted in developing and emerging countries. these have revealed the global scale of the occurrence of pharmaceuticals in the environment. for example, diclofenac, a non-steroidal inflammatory drug, has been detected in the aquatic environment in 50 countries worldwide. a number of globally marketed pharmaceuticals have been found in both developing and industrialized countries. the available ecotoxicological information indicates that certain pharmaceuticals pose a risk to the environment at measured concentrations. options for cooperative action to address the risk of are also presented. the aim of the research presented was to support the discussion of the proposed emerging policy issue pharmaceuticals in the environment under the strategic approach to international chemicals management (saicm), which is a global initiative of united nation environment programme (unep). the european chemicals' legislation reach aims to protect man and the environment from substances of very high concern (svhc). chemicals with (very) persistent, (very) bioaccumulative and toxic properties (pbt and vpvb compounds), substances that are carcinogenic, mutagenic and toxic to reproduction (cmr compounds), as well as chemicals of comparable concern like endocrine disruptors (eds) may be subject to authorization. the identification of eds is limited as yet, because specific experimental assessments are not required under reach. evidence is currently based on a combination of few experiments, expert judgement and structural analogy with known eds. structural alerts for the identification of potential eds: predictions of properties and effects from chemical structures are based on similarities with either active or inactive substances. structural alerts for the identification of potential estrogen/androgen-ed activities are relevant parts of the structures of compounds that are known to interact with estrogen and androgen receptors as either agonists or antagonists. in addition to the backbones of the chemical structures (pharmacophore) for steric fit to the receptors, modulating factors may be small substructures for local interactions with receptor binding sites and physicochemical properties related to the strength of binding to the receptor. comparison of evidence from in silico, in vitro and in vivo assays for potential eds: identification of potential eds based on findings from mammalian long-term reproduction studies, fish life-cycle tests, receptor assays, and chemical alerts were compared and differences analysed. agreement is limited because in vivo, in vitro and in silico methods address different aspects of potential effects on endocrine systems regarding toxicological targets, modes of action and functional similarity of chemical structures. a combination of toxicological and chemical assays can provide comprehensive and complementary information to support evidence-based identification of potential eds among the chemicals released into the environment. application of structural alerts for the identification of potential eds to the einecs inventory: more than 33000 discrete organic einecs compounds are within the applicability domain of the structural alerts for potential estrogen/androgen-ed activities. among them, 3585 chemicals (ca. 11%) are indicated as potential candidates for endocrine effects based on structural alerts. due to the possibility that these chemicals may interact with estrogen/androgen receptors they should be subject to further investigations regarding their potential for endocrine effects, eventually leading to regulatory actions. within the imi (innovative medicine initiative) project "intelligence-led assessment of pharmaceuticals of the environment " (ipie;http://i-pie.org/), a more intelligent environmental testing and tools for prioritisation of legacy compounds shall be developed. regarding the evaluation of fish toxicity, screening approaches in fish embryos are pursued. while the standard fish embryo toxicity (fet) test is restricted to lethal parameters more relevant as substitutes for acute effects, additional sublethal endpoints may provide expanded applicability of the fet assay for chronic effect assessment in fish. in this respect, the analysis of the metabolome could provide additional insights into biochemical processes elicited by pharmaceutical compounds and could potentially support the extrapolation from fish embryo to chronic fish toxicity as displayed in the standard early life stage (els) test. in the context of ipie a pilot study was performed with the aim to quantify and comparatively assess changes in the metabolic signatures of fish and fish embryos induced by the reference compound amikacin, an aminoglycoside antibiotic, in order to identify metabolic patterns applicable as biomarker profiles that can be linked to apical endpoints in terms of an integrated approach. therefore, toxic effects in fathead minnow embryos and els fish were investigated following a 4 and 32 days exposure, examining conventional endpoints and additionally using a combined direct injection and lc-ms/ms assay for metabolite identification and quantification. metabolic endpoints were found to be at least as sensitive as standard apical endpoints such as growth and mortality as detected in the longer-term fish study. furthermore, multivariate data analysis (pca-x, opls-da) revealed substance induced metabolic perturbations specific for fish and fish embryos, respectively. beyond that, the statistical approach of shared and unique structure (sus) identified some metabolites from the classes of amino acids, biogenic amines and lipids to be similarly changed in both developmental stages related to amikacin treatment, representing shared biomarker candidates. in this first pilot study, the integrated metabolomics approach yielded insights into the molecular consequences of amikacin exposure and provided indications for biomarkers for common effects in fish embryos and fish. due to the different exposure levels in the fet (1 -100 mg/l) and els study (0.0003 -0.03 mg/l), more definitive conclusions regarding the predictivity of metabolic responses in fish embryos for apical endpoints in chronic fish tests are yet not possible. further studies are ongoing with a range of pharmaceutical compounds of different chemical classes which will reveal more substantial information on the applicability of this technology in the prediction of longterm effects in fish. the human cationic amino acid transporter hcat-1 (slc7a1) represents the major uptake route for arginine and other cationic amino acids (such as the essential amino acid lysine) in most mammalian cells. it thus provides these amino acids for protein synthesis as well as for essential metabolic pathways. in endothelial cells, special attention has been given to the role of hcat-1 for supplying arginine to nitric oxide synthase. in spite of its wide distribution, hcat-1 expression is highly regulated both, on the transcriptional and post-transcriptional level. however, the genetic elements involved in transcriptional regulation a largely unknown. here we studied the expressional regulation of hcat-1 in human ea.hy926 endothelial cells. starvation of these cells from cationic amino acids led to a pronounced induction of both, hcat-1 mrna and protein. the mrna induction was almost completely abolished by the transcription elongation inhibitor drb (5,6-dichloro-1-β-dribofuranosylbenzimidazole), suggesting the involvement of transcriptional regulation. we thus aimed at identifying the promoter elements in the hcat-1 gene responsible for this regulation. to our surprise and in contrast to data published by others 1 the chromosomal region up to 5 kb upstream of the first hcat-1 exon exhibited no promoter activity in either endothelial or dld-1 colon carcinoma cells that exhibit a very strong endogenous hcat-1 expression. we could however identify a promoter element within the first intron of the hcat-1 gene. transcriptional activity of this element increased upon amino acid starvation in a similar way as endogenous hcat-1 expression. our results indicate starvation-induced transcriptional regulation of hcat-1 through newly identified promoter regions distinct from those published previously. the transport of a multitude of drug molecules into the cell is mediated by multispecific organic cation transporters (octs), belonging to the solute carrier group (slc). one of these families within this group of membrane transport proteins is the slc47-family consisting of the two multidrug and toxin extrusion transporters mate-1 (slc47a1) and mate2-k (slc47a2). while mate-1 is highly expressed in several different tissues like kidney, liver, skeletal muscle, adrenal glands, testes and heart, mate2-k exclusively occurs in the apical membrane of proximal tubular epithelial cells within the kidney. both transport proteins translocate organic cations in exchange of protons into as well as out of the cell. to define the affinity of both transporters for the anti-diabetic drug metformin and to investigate their interactions with 26 different anti-neoplastic agents comparative transport experiments with the model substrate 1-methyl-4-phenylpyridinium (mpp) have been carried out. therefore stably transfected hek293-cells expressing mate-1 or mate2-k transport proteins generated by portacelltec biosciences gmbh and vector transfected hek293-cells were used. the interaction analyses were carried out by determining the uptake of [ 14 c]-metformin and the inhibition of [ university of basel, basel, switzerland drug transporters play a pivotal role in pharmacokinetics by modulating the cellular entry or efflux of compounds. one transporter facilitating the transport of bile acids, steroid hormones, and statins is the organic anion transporting polypeptide (oatp) 2b1 that is highly expressed in placenta, liver, and small intestine. especially its activity in small intestine and liver is assumed to be basis for specific drug-drug interactions. understanding mechanisms involved in pharmacokinetics is a prerequisite in drug development. to test whether there are species differences in transport activity we compared the expression and function of the human and rat orthologue. first, we determined the transport activity of the known oatp2b1 substrate estrone 3sulfate (e 1 s), and observed a significantly lower k m for mdck-hoatp2b1 compared to .00 ± 10.23 µm, *p<0.05 student´s t-test) whereas there was no difference in v max (mdck-hoatp2b1 119.4 ± 11.3 fmol/min/µg protein; mdck-roatp2b1 102.3 ± 12.8 fmol/min/µg protein). the human oatp2b1 exhibits multiple binding sites for its substrates that may explain specific drug-drug interactions [1] . to identify whether rat oatp2b1 owns the same characteristics, the cellular accumulation of 50 µm e 1 s (low affinity site) or 0.005 µm e 1 s (high affinity site) was determined in presence of specific inhibitors/substrates of oatp2b1. as observed for atorvastatin, a known inhibitor of both affinity sites, the rat transporter failed to exhibit the low affinity site. in detail, while atorvastatin reduced the accumulation of e 1 s in mdck-hoatp2b1 cells (110.0 ± 14.6 fmol/min/µg protein vs. 60.7 ± 7.5 fmol/min/µg protein, *p<0.05 student´s t-test), there was no inhibition of e 1 s accumulation in mdck-roatp2b1 cells (53.2 ± 13.2 fmol/min/µg protein vs. 49.0 ± 17.4 fmol/min/µg protein). additionally, we observed a different membrane localization of both transporters as assessed by immunofluorescent staining showing an apical localization for rat oatp2b1 while human oatp2b1 is localized at the basolateral pole of the cellular model. absolute quantification of mrna (copy number per 1000 ng of total rna) in different tissues of rat revealed a high expression of oatp2b1 in liver (159.6 ± 45.5), a moderate expression in small intestine (14.9 ± 4.0) and colon (57.0 ± 12.3), and a low expression level in kidney (2.3 ± 0.8) . the latter is in accordance with previous findings showing that oatp2b1 is abundant in rat kidney as quantified by absolute proteomics technics [2] . our data demonstrated species differences in localization and activity of the drug transporter. further studies are warranted to proof whether this knowledge will help in future drug development and which molecular cause is responsible for the observed data. background: intestinal drug absorption depends on various factors including aqueous volume, site-specific ph and intestinal motility. moreover, the expression of efflux-and uptake-transporters vary in dependence of the anatomical localization making the gut a complex barrier for drug transfer into the body. in a recent study, site-specific protein and mrna expression levels of 10 drug transporters were determined along the entire length of the human gut. interestingly, discrepancies between mrna expression and protein levels were observed. moreover, there were quantitative differences between small intestine and colon. as a consequence the question arose if this observation could be explained by small non-coding rnas acting as highly tissue-specific posttranscriptional regulators of gene expression. hence, in our current study, we aimed to investigate the impact of mirnas on site-specific transporter expression along the human intestine. methods: total rna was isolated from biopsies obtained from six disease-free organ donors. tissue samples were acquired from the duodenum, the upper and lower jejunum, the upper and lower ileum, and the transversal or descending colon. the expression of 754 mirnas was measured using rt-pcr based low density arrays. expression of all detected mirnas was correlated with transporter protein data recently determined by lc-ms/ms-based targeted proteomics. mirnas and transporter genes showing an inverse pearson's correlation between mirna and protein expression underwent an in-silico search (microcosm targets v.5, targetscan7.0) for putative mirna/mrna interaction. to investigate those interactions in more detail, reporter gene assays and site directed mutagenesis were conducted. results: out of 754 mirnas, 248 were detected in all tissue types. out of ten transporters five showed significant inverse correlations with 10 putatively targeting mirnas (e.g. pept1 and hsa-mir-27a, r= -0.498, p=0.002). reporter gene assays indicated interactions of mir-193a/b with pept1 3'-utr (p = 0.0025 and p = 0.0012) as well as of mir-27a with abcb1 3'-utr (p = 0.0006). the site-specific abundance of intestinal drug transporters is significantly affected by microrna-mediated post-transcriptional regulation under physiological conditions as exemplified for pept1 and abcb1. background: the human uptake transporter nact [gene symbol slc13a5; also known as mindy, the human orthologue of the drosophila indy (i´m not dead yet) transporter] is expressed in liver and brain. nact is important for energy metabolism and brain development and mediates the uptake of tricarboxylic acid (tca) intermediate such as citrate and succinate. reduced expression of this transporter, as studied in knock-out mice, mimics aspects of dietary restriction, promotes longevity and protects against insulin resistance and adiposity. furthermore, mutations in the human slc13a5 gene are associated with epileptic encephalopathy with seizure onset in the first days of life, possibly due to the reduced uptake of tca intermediates into neurons. to gain more insights into the role of nact in drug transport and into structure-function relationships, we studied the inhibition of nact-mediated citrate transport by various drugs and analyzed the effect of known mutations in the slc13a5 gene on nact-mediated transport. methods: drug inhibition studies were performed using hek cells stably expressing human nact with citrate as prototypic substrate. twenty-four drugs were used as potential inhibitors of nact-mediated uptake. the effects of eight mutations, three of them (nactp.g219r, -p.t227m and -p.l488p) associated with epileptic encephalopathy, were analyzed using a transient transfection approach. furthermore, the first computational-based structural model of the nact transporter was established and the impact of all mutations on substrate transport was modeled. results: inhibitions studies demonstrated that only a few drugs (three out of 24 tested) inhibited nact-mediated citrate transport at the tested drug concentration of 100 µm. from these, benzbromarone shows the strongest inhibition with an ic 50 value of 83.2 µm. furthermore, citrate transport was also slightly inhibited by pioglitazone and rosiglitazone. citrate transport of the mutated proteins nactp.g219r, -p.g219e,p.t227m, -p.l420p and -p.l488p was totally abolished and the effect of these mutations could be explained on the basis of the structural model. conclusion: inhibition studies demonstrated that simultaneously administered drugs can inhibit nact-mediated uptake of the prototypic substrate citrate. nact-mediated uptake is abolished by mutations in the slc13a5 gene associated with epileptic encephalopathy. the effect of these mutations can be explained on the basis of the first structural model of this uptake transporter. the atp-binding cassette subfamily b member 1 (abcb1) is a drug efflux pump responsible for the classic multi-drug resistance phenotype in cancer cells. increased activity of abcb1 in cancer cells contributes to protection against a wide range of chemotherapeutic agents. this dramatically decreases therapeutic options and the chance of patient survival. knowledge of the underlying mechanisms for abcb1 deregulation is a critical step for the reversal of this unfavorable condition. of note, phosphatidylinositol-4,5-bisphosphate (pip 2 ) is a known regulator of abcb1. the protein "myristoylated alanine-rich c-kinase substrate" (marcks) is known for its ability to bind and sequester the phospholipid pip 2 . in various forms of colorectal cancer the deregulation of marcks protein goes hand in hand with an increase in malignancy and therapeutic resistance. in this study, we characterized the enigmatic marcks as a modulator of abcb1 activity. we focused on a subgroup of colon cancers, where marcks resides in a hyperphosphorylated state for which the established cell line ht-29 served as a model. we employed various in-vitro methods for the measurement of abcb1 activity, in combination with imaging experiments, assays for cellular viability and classical methods of molecular biology. we combined these approaches with pharmacological inhibition of marcks phosphorylation state or rnai-mediated depletion of marcks. with these interventions we could modulate endogenous abcb1 activity and re-sensitize our cell model against chemotherapeutic agents like 5-fluorouracil which are known to be substrates of abcb1. taken together, our findings suggest a new way how a cancer cell can gain a state of therapeutic resistance. the exploitation of marcks as modulator of abcb1 might be a new approach to target resistant tumors without interfering with the natural function of abcb1 in non-malignant tissue. background: in several large clinical studies low blood concentrations of homoarginine were identified as independent risk marker for stroke, cardiovascular events and mortality. experimental data suggest an active role of homoarginine deficiency in disease development. interference with l-arginine-dependent pathways and signaling has been implicated as a possible mechanism. it was the aim of the present study to identify transport mechanisms involved in the cellular uptake and tissue distribution of homoarginine, which is poorly understood, so far. the experiments focused on cationic amino acid transporters (cats) and possible interactions with known cat substrates. methods: uptake assays were performed using [ 3 h]-labeled homoarginine as substrate and human embryonic kidney (hek293) cells stably overexpressing human cat1 [gene: slc7a1 (solute carrier family 7)], cat2a (slc7a2a) or cat2b (slc7a2b). cells transfected with an empty vector were used as control. unlabeled known catsubstrates l-arginine and asymmetric dimethylarginine (adma) were investigated as inhibitors. results: compared to the uptake into control cells, uptake of homoarginine was significantly higher in hek cells overexpressing cat1 (7-fold), cat2a (1.6-fold) or cat2b (2.2-fold) after 2.5 min using 100 µm substrate (each p<0.001). apparent k m values for cellular net uptake of homoarginine were 174.6 µm for cat1, 3640 µm for cat2a and 522.8 µm for cat2b. homoarginine uptake by the three cats could be inhibited by addition of l-arginine and adma. conclusion: the protective biomarker homoarginine is a substrate of the human cationic amino acid transporters cat1, cat2a and cat2b. compared to other cat substrates, the cat1-and cat2b-mediated homoarginine transport is characterized by relatively high affinity. the uptake of homoarginine by all three cats can be inhibited by l-arginine and adma. taken together these findings make cat-mediated transport of homoarginine a possible target for interactions and pharmacological interventions aimed at homoarginine homeostasis. this project is supported by the doktor robert pfleger-stiftung. background and aim: organic cation transporter 1 (oct1, alternative name slcc22a1) is a polyspecific membrane transporter, which has been suggested to play a role in absorption, distribution and elimination of drugs and toxins. besides endogenous compounds like thiamine (vitamin b1), known oct1 substrates are toxins like mpp + as well as drugs like metformin, o-desmethyltramadol, ranitidine, and sumatriptan. tissue distribution of oct1 has been shown to have strong inter-species differences. in rodents oct1 is strongly expressed in both the sinusoidal membrane of hepatocytes and the basolateral membrane of kidney epithelial cells. human oct1 is strongly expressed on the sinusoidal membrane of hepatocytes, but not in the kidney. furthermore, substrate specific differences have been observed between the human and rodent oct1 orthologs. the aim of this study is to characterize the mechanisms causing substrate specificity between rodent and human oct1 orthologs. methods: overexpression of oct1 orthologs in mouse, rat and human cells was performed by targeted chromosomal integration of t-rex™ 293. the cells were characterized in detail for their ability to transport the model substrates tea + , mpp + , and asp + , the drugs ranitidine, sumatriptan and fenoterol. results: mouse and rat oct1 orthologs showed similar transport activity for all the model substrates and drugs tested. however, significant differences were observed when rodent orthologs were compared to the human oct1. human oct1 showed a 5fold higher v max for the uptake of asp + and 2-fold increase of v max for sumatriptan in comparison to the rodent oct1 orthologs. conversely, human oct1 showed a 4-fold lower v max for the uptake of fenoterol compared to rodent oct1s. there was no difference between rodent and human oct1 in the uptake of ranitidine. these differences were further characterized in detail using chimeric mouse-human oct1 constructs and by comparing the effects of key point mutations in mouse and human oct1 orthologs. conclusions: these data demonstrate strong differences in the substrate specificity between rodent and human oct1 orthologs. therefore oct1 pharmacokinetic data obtained in mouse or rat models could not be directly extrapolated for use in human. furthermore, comparative functional analyses of orthologs may help reveal the mechanisms underlying polyspecificity of oct1. ranitidine is a histamine h 2 -receptor antagonist which is commonly used without prescription for the treatment of pyrosis and gastric ulcers. approximately 30% of the systemic clearance of ranitidine is via hepatic metabolism. ranitidine is known to be a substrate of the hepatic organic cation transporter oct1 [1]. oct1 is expressed on the sinusoidal membrane of human hepatocytes and is highly genetically variable. sixteen major oct1 alleles are known [2] . thereof 12 alleles confer partial or complete loss of oct1 activity. the observed loss of activity was highly substrate specific and should be analyzed substrate by substrate. in this study we analyzed the effects of genetic polymorphisms in oct1 on the uptake of ranitidine. we used hek293 cells stably transfected to overexpress wild type or variant oct1 isoforms. the variant oct1 alleles oct1*1a (met408val), oct1*1c (phe160leu), oct1*1d (pro341leu/met408val), oct1*2 (met420del), oct1*3 (arg61cys), oct1*4 (gly401ser), oct1*5 (gly465arg/met420del), oct1*6 (cys88arg/met420del), oct1*7 (ser14phe), oct1*8 (arg488met), oct1*9 (pro117leu), oct1*10 (ser189leu), oct1*11 (ile449thr), oct1*12 (ser29leu) and oct1*13 (thr245met) were analyzed. we characterized in ranitidine uptake determined k m and v max for the different polymorphic oct1 isoforms. wild type oct1 showed a time and concentration dependent uptake of ranitidine with a k m of 62.91 ± 4.32 µm and a v max of 1125.41 ± 86.12 pmol/min/mg protein. variants oct1*5, oct1*6, oct1*12 and oct1*13 completely lacked ranitidine transport activity. variants oct1*2, oct1*3, oct1*4 and oct1*10 showed v max values decreased by 64, 77, 91 and 50 %, respectively. oct1*8 variant showed an increase of v max by 25 %. there was no significant changes in ranitidine uptake by variants oct1*1a, oct1*1c, oct1*1d, oct1*7, oct1*9 and oct1*11 compared to the wild type. there were no significant differences in the k m values between the wild-type and variants. in conclusion, we confirmed ranitidine as an oct1 substrate and demonstrated that genetic polymorphisms in oct1 can strongly affect ranitidine uptake. the effects of oct1 polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed. otto-von-guericke university, institute for pharmacology and toxicology, magdeburg, germany serotonergic hallucinogens ( s hgs), such as lysergic acid diethylamide (lsd), induce profound alterations of human consciousness, which are thought to be mediated by activation of 5-ht 2a receptors. with repeated application, the mind-altering effects of most s hgs rapidly are undermined by tolerance. the only exception seems to be dimethyltryptamine (dmt), whose mind-altering effects for reasons unknown even with repeated application do not decrease. assuming that dmt differs from other s hgs in its capacity to regulate 5-ht 2a receptors, we here compare lsd and dmt with regard to processes of 5-ht 2a downregulation. in heat-exposed rats, lsd and dmt induce a marked increase in body core temperature (hyperthermia) accompanied by "defensive hypersalivation". both effects are mimicked by the 5-ht 2 selective agonist dimethoxybromoamphetamine (dob) and can be blocked by the selective 5-ht 2a antagonists ketanserin and mdl100907. after repeated application, the temperatureregulatory effects of lsd are subject to tolerance, whereas the ones of dmt are not. tolerance to lsd (as measured by dob induced [ 35 s]gtpуs binding) is paralleled by a desensitisation of frontocortical 5-ht 2 receptors; for dmt, there is no such decrease. applying techniques of immunocytochemistry, transphosphatidylation, and quantitative real-time pcr to (ha-5-ht 2a transfected) hek293 and (endogenously 5-ht 2a expressing) c6 glioma cells, respectively, we furthermore demonstrate that dmt in contrast to lsd fails to internalise 5-ht 2a receptors, fails to activate the phospholipase d (which is needed for 5-ht 2a internalisation), and fails to inhibit the synthesis of 5-ht 2a receptors. given that dmt unlike lsd turns out to be inactive as to all processes of 5-ht 2a downregulation investigated, our data suggest that the differential tolerance development noted for dmt and lsd indeed might be accounted for by differential regulation of 5-ht 2a receptors. lsd and dmt both have recently regained scientific attention as potential therapeutics in the treatment of depression and/or anxiety disorders. providing mechanistic insights into their action, thus, is of timely clinical relevance. increased gaba release in human neocortex at high intracellular sodium and low extracellular calcium -an anti-seizure mechanism? ] e , this reduction might induce an anti-seizure mechanism by augmenting gat-mediated gaba release, a mechanism absent in rats. aging is complex on the systems as well as on the molecular level. the process of aging is characterized by a progressive loss of physiological functions and accumulation of cellular damage. one hallmark of aging is an impaired protein homeostasis. the imbalance of the quality control of both de novo protein synthesis and protein degradation, therefore, is likely to contribute to the phenotype of aging. we investigated protein turnover rates with the state-of-the art techniques funcat (dieterich et al., 2010) and sunset (schmidt et al., 2009) in aging neuronal cell cultures . using these techniques we show a prominent decrease in protein synthesis and degradation that progressed gradually in aging neuronal cells cultured up to div 60. in order to rejuvenate the protein turnover in aged neuronal cells we applied the selective eukaryotic elongation factor-2 kinase inhibitor a 484954 and the polyamine spermidine and observed protein translation utilizing funcat and sunset. whereas both a 484954 and spermidine had no effect on de novo protein synthesis in juvenile neurons (div 20) , both substances increased the de novo protein synthesis to a juvenile level in aged neuronal cultures (div60). this effect is seen in neuronal somata and dendritic spines. the molecular function of spermidine as an "anti-aging agent" is not defined yet. thus, additional pharmacological interventions are used for further examination of specific molecular spermidine targets. in conclusion, the described experimental setup is used to investigate impaired protein homeostasis as one hallmark of aging. agents with a presumed "anti-aging" effect can be tested for a potential rejuvenating effect on the level of protein homeostasis. a screening approach to test tolerability of multitargeted drug combinations for antiepileptogenesis in mice a large variety of brain insults can induce the development of symptomatic epilepsies, particularly temporal lobe epilepsy. in the latent period after the initial insult multiple molecular, structural, and functional changes proceed in the brain and finally lead to spontaneous recurrent seizures. prevention of these developments, called antiepileptogenesis, in patients at risk is a major unmet clinical need. several drugs underwent clinical trials for epilepsy prevention, but none of the drugs tested was effective. similarly, most previous preclinical attempts to develop antiepileptogenic strategies failed. in the majority of studies, drugs were given as monotherapy. however, epilepsy is a complex network phenomenon, so that it is unlikely that a single drug can halt epileptogenesis. recently, multitargeted approaches were proposed ("network pharmacology") to interfere with epileptogenesis. developing novel combinations of clinically used drugs with diverse mechanisms that are potentially relevant for antiepileptogenesis is a strategy, which would allow a relatively rapid translation into the clinic. we developed an algorithm for testing such drug combinations in a screening approach, modelled after the drug development phases in humans. tolerability of four repeatedly administered drug combinations was evaluated by a behavioral test battery: a, levetiracetam and phenobarbital; b, valproate, losartan, and memantine; c, levetiracetam and topiramate; and d, levetiracetam, parecoxib, and anakinra. as in clinical trials, tolerability was separately evaluated before starting efficacy experiments to identify any adverse effects of the combinations that may critically limit the successful use in preclinical studies on antiepileptogenesis and translation of these preclinical findings to the clinic. based on previous studies, we expected that tolerability would be lower in epileptic mice than in nonepileptic mice. therefore nonepileptic mice were used as a first step, followed by epileptic mice and mice during the latent period shortly after status epilepticus. except combination b, all drug cocktails were relatively well tolerated. in contrast to our expectations, except combination c, no significant differences were determined between nonepileptic and post-status epilepticus animals. as a next step, the rationally chosen drug combinations will be evaluated for antiepileptogenic activity in mouse and rat models of symptomatic epilepsy. major depression is one of the most common mental disorders worldwide, with serious social and economic consequences. there are many different hypotheses concerning the pathophysiology of this disease. the complex brain serotonin system and particularly the serotonin 1a receptors (5-ht 1a r) apparently play a pivotal role in the development of depression. the involvement of an altered, 5-ht 1a r-mediated signalling in adult neurogenesis is also discussed. however, in this context the effects of pre-and postsynaptically located 5-ht 1a rs have not been clarified yet. mice with a permanent overexpression of postsynaptic 5-ht 1a rs (oe mice) represent a unique tool to elucidate the effects of postsynaptic 5-ht 1a rs in adult neurogenesis and depressive-like behaviour. previous studies demonstrated an increased proliferation and survival of newborn cells in the adult dentate gyrus of female oe mice in comparison to controls. in the present study, we investigate the proliferation and survival of adult born cells after chronic treatment (15 days) with the selective 5-ht 1a r agonist 8-oh-dpat (0,5 mg/kg/day) in young adult oe and wt mice. on the last three days of treatment, newly generated cells of oe and wt mice are labelled by injections with bromodeoxyuridine (brdu; 50 mg/kg/day). mice are sacrificed either one day (proliferation) or 21 days (survival) after the last injection. we hypothesise that the data we will present will confirm our previous results, with possibly more pronounced proneurogenic effects and differences in male mice. further immunohistochemical studies post-exercise and behavioural analyses are in progress to identify the relation between chronic postsynaptic 5-ht 1a r stimulation, depressive-like behaviour and hippocampusdependent learning. dystonia is a common movement disorder characterized by intermittent and prolonged muscle contractions resulting in involuntary movements and/or abnormal postures. the lack of knowledge of the pathophysiology of dystonia hampers the development of effective therapeutics. although benzodiazepines can improve dystonic symptoms, tolerance and side effects limit their use. there is evidence for striatal dysfunctions in human dystonia. gabaergic striatal interneurons (in) are important for the regulation of striatal signaling. in the dt sz mutant hamster, a model of paroxysmal dystonia, immunoreactive in were reduced at the age of maximum severity of dystonia (33 days), but not after spontaneous remission (age 90 days). as indicated by unaltered homeoboxprotein nkx 2.1 (cell density, mrna), the age-dependent deficit seems not to be related to a disturbed migration, but to a retarded maturation of in in mutant hamsters. here we further determined the maturation of striatal gabaergic neurons in the dt sz hamster compared to healthy controls. kcc2 and cavii mrna, used as markers for the gaba-switch, were unchanged in 18 and 33 day old mutant hamsters, indicating that there is no general delay in gabaergic maturation. as a retarded maturation seems to be specific for in, we used another marker for gabaergic maturation: the expression of specific gaba a receptor (gaba a r) subunits (mature striatal in express the alpha1 subunit). by stereological determination, we found a 46% decrease in alpha1 subunit expressing neurons. a lower immunoreactive intensity was restricted to the somata of dorsomedial striatal in (32%) of dt sz hamsters, indicating both a reduced density as well as a delayed maturation. these findings prompted us to examine the effects of the αlpha1 gaba a r preferring compound zolpidem in comparison with the benzodiazepine clonazepam. zolpidem (2.0 and 10.0 mg/kg i.p.) only exerted moderate antidystonic effects compared to the benzodiazepine clonazepam (0.5 and 1.0 mg/kg i.p.) in the dt sz hamster. examinations of αlpha2 gaba a r preferring compounds are ongoing. in summary our studies indicate that there is no general defect in striatal gabaergic maturation in the dt sz mutant but a specific alteration of striatal gabaergic interneurons which express αlpha1 gaba a r subunits. changes in αlpha1 gaba a r subunit expression and differences in the antidystonic efficacy of zolpidem and clonazepam indicate that further investigations on the role of gaba a r subunits could lead to new therapeutic approaches for the treatment of dystonia. 2005) . therefore, the hypothesis of the present study was that pregnenolone attenuates the inhibition of synaptic transmission elicited by cannabinoids. methods: 250 µm-thick slices containing the cerebellum and the nucleus accumbens were prepared from the brains of mice and rats. spontaneous and electrically evoked gabaergic inhibitory postsynaptic currents (sipscs and eipscs) and evoked glutamatergic excitatory postsynaptic currents (eepscs) were analyzed in superfused brain slices with patch-clamp electrophysiological techniques. results: a) the synthetic cannabinoids jwh-018 (5 x 10 -6 m) and jwh-210 (5 x 10 -6 m) inhibited the spontaneous gabaergic synaptic input (sipscs) to purkinje cells in mouse cerebellar slices. the inhibition by jwh-018 was not affected by pregnenolone (10 -7 m), the inhibition by jwh-210 was only marginally attenuated. b) the depolarization of the purkinje cells induced suppression of the gabaergic input to purkinje cells (dsi); pregnenolone (10 -7 m) did not affect this endocannabinoid-mediated form of synaptic suppression. c) in rat nucleus accumbens slices, gabaergic and glutamatergic synaptic input to medium spiny neurons was activated by electrical stimulation of axons. ∆ 9 -tetrahydrocannabinol (2 x 10 -5 m) suppressed the gabaergic and glutamatergic synaptic transmission in the nucleus accumbens. these suppressive effects of ∆ 9tetrahydrocannabinol were not changed by pregnenolone (10 -7 m). d) finally, we tested whether we can observe neurosteroid-mediated effects in our brain slice preparations. tetrahydro-deoxycorticosterone (thdoc, 10 -6 m) markedly prolonged the decay time constant (τ) of spontaneous gabaergic postsynaptic currents (sipscs), similarly as in previous experiments (ej cooper et al., j physiol 521: 437-449, 1999) . the results show that inhibition of gabaergic and glutamatergic synaptic transmission by synthetic-, endogenous,-and phyto-cannabinoids is not changed by pregnenolone. therefore, it is unlikely that interference with cannabinoid-induced inhibition of synaptic transmission is the mechanism by which pregnenolone attenuates behavioural and somatic effects of ∆ 9 -tetrahydrocannabinol in vivo. the hypothalamus is one of the key players in the regulation of the energy homeostasis. cold stress leads to an activation of neurons in the paraventricular hypothalamic nucleus (pvn) and increases thermogenesis. the thyrotropin-releasing-hormone (trh) neurons have an important function in this effect. however it is hardly understood which role the trh neurons exactly play and how they are connected to other regions of the brain. we have transduced neurons in the pvn of mice with a recombinant adeno associated virus which contains an activating "designer receptors exclusively activated by designer drugs" (dreadd) system under the control of a shortened trh promotor. two weeks after transduction the animals were injected with clozapine-n-oxide (cno). to analyse the physiological function of this neurons we performed indirect calorimetry, measured rectal temperature and thermogenesis in the brown adipose tissue (bat), analysed drinking feeding behaviour and the home cage activity. after stimulation we measured the expression of genes in bat as well plasma hormone levels of pituitary hormones. propranolol and the specific β3-antagonist sr59230a were used to analyse the relevance of the sympathetic system. to further characterise the transduced neurons and their projections we used immunohistochemistry methods. after stimulation with cno the energy expenditure and body temperature were increased. these effects were mostly driven through an activation of the brown adipose tissue (bat). in dreadd transduced trh-receptor 1 (trh-r1) knockout mice this effects were abolished. in parallel the plasma levels of tsh, the ucp1 mrna level in the bat, the home cage activity as well the food and water intake were increased. after the treatment with propranolol and sr59230a the effects on the thermogenesis were reduced, but the home cage activity was not affected. sr59230a treatment normalised the food intake and increased in parallel the plasma leptin concentrations after cno stimulation. transduced neurons project into the raphe nucleus, the medial part of the thalamus and the spinal cord. with our experiments we could provide strong evidence for a sympathetic connection of the transduced neurons in the pvn to the bat and for the involvement of thr neurons in these effects. therefore, this system is a suitable tool to investigate the metabolic relevance of trh neurons in detail. background & objective: during obesity development, tissue factor signalling contributes coagulation-independently to inflammatory and metabolic dysfunction of adipose tissue. adipogenesis involves proliferation and differentiation of preadipocytes, apoptosis and hypertrophic growth of differentiated adipocytes, angiogenesis and extracellular matrix reorganisation. the coagulant protease thrombin promotes similar processes in various cell types, through activation of protease-activated receptors par-1, par-3 and par-4. in human adipose tissue, par-1 is found in vascular stromal cells and par-4 in preadipocytes and differentiated adipocytes. thrombin stimulates mitogenic kinase signalling and induces inflammatory cytokine and angiogenic growth factor secretion in adipocytes. we have examined the contribution of thrombin receptor activation to adipogenesis processes in 3t3l1 cells. results: differentiation of 3t3l1 preadipocytes with insulin, dexamethasone and isobutylmethylxanthine increases leptin and pparg gene expression and accumulation of triglycerides and oil red o-stained lipids. par-4 is time-dependently upregulated in maturing cells while par-1 expression is detectable but not altered. in preadipocytes, thrombin (3 u/ml) activates the mitogenic kinase erk1/2, promotes cell proliferation and induces gene expression of the maturation markers leptin and pparg and the inflammatory marker tumor necrosis factor alpha (tnfa). repeated stimulation of differentiating adipocytes with thrombin suppresses induction of leptin and pparg and attenuates lipid accumulation, while expression levels of the proliferation marker ki67 and the inflammatory cytokine interleukin (il)-6 are increased compared to differentiated control cells. similar proliferative and anti-adipogenic effects are seen with the selective par4-activating peptide (gypgkf, 100 µm) and cathepsin g, a proteolytic par-4 activator released from neutrophils and mast cells. repeated exposure of maturing 3t3l1 cells to conditioned medium from degranulating mouse peritoneal mast cells (mccm) augments lipid accumulation and il-6 expression. pretreatment of mccm with a par-4 inhibitor further drives lipid accumulation, the induction of il-6 by contrast is suppressed. conclusion: par-4 activation by thrombin or inflammatory cell-derived cathepsin g appears to suppress adipogenesis, possibly by maintaining proliferative capacity and preventing the growth arrest essential for initiating matuation. increased par-4 expression in maturing adipocytes may instead support inflammatory changes, thereby promoting the onset of insulin resistance. the chemokine receptor cxcr4 antagonist amd3100 exerts deleterious effects in endotoxemia in vivo s. seemann 1 , a. lupp the chemokine receptor cxcr4 is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine 12 (cxcl12). although a blockade of the cxcr4/cxcl12 axis revealed beneficial outcomes in chronic inflammatory diseases, its importance in acute inflammatory diseases remains contradictious and not well characterized. as cxcr4 seems to be part of the lipopolysaccharide sensing complex, cxcr4 agonists or antagonists may have a positive impact on tlr4 signaling. additionally, cxcr4 is involved in the production of pro-inflammatory cytokines, suggesting the receptor to be a promising target in terms of mitigating the cytokine storm. therefore, we aimed to investigate the impact of a cxcr4 blockade on endotoxemia by applying a sublethal lps dose (5 mg/body weight) in mice. the selective cxcr4 inhibitor amd3100 was administered intraperitoneally shortly after lps treatment to ensure an immediate effect after endotoxemia onset. 24 hours after lps administration, the clinical severity score, the body temperature and the body weight of the animals were determined. afterwards, the mice were sacrificed and serum tnf alpha as well as ifn gamma levels were measured. furthermore, the oxidative stress in the brain, liver, lung and kidney tissue was assessed. in addition, the biotransformation capacity of the liver was evaluated and finally, the expression of gp91 phox as well as of heme oxygenase 1 in the spleen and liver were determined by means of immunohistochemistry. the mice of the amd3100 plus lps treatment group displayed a significantly impaired general condition, a reduced body temperature and a decreased body weight in comparison to the control and to the lps treated animals, respectively. tnf alpha levels were significantly increased by more than 200% or 35% when compared to the control or to the lps group, respectively, whereas ifn gamma levels were elevated by about 11% in comparison to mice which had received lps only. in all investigated organs, but especially in the liver and in the kidney, co-administration of amd3100 and lps caused massive oxidative stress. furthermore, the protein contents and the activities of several cyp enzymes in the liver were significantly reduced. immunohistochemistry revealed gp91 phox to occur above average, whereas heme oxygenase 1 expression was distinctly decreased. our results indicate that a blockade of the cxcr4 in endotoxemia is disadvantageous and even worsens the disease. co-administration of amd3100 and lps impaired the health status of the animals, caused massive oxidative stress and diminished the biotransformation capacity. thus, handling acute systemic inflammation with a cxcr4 antagonist cannot be recommended, hence indicating the activation of cxcr4 to be an attractive treatment option. toll-like receptors (tlrs)recognizemicrobial pathogens and trigger inflammatory immune responses to control infections. in acne vulgaris, activation of tlr2 by propionibacterium acnes contributes to inflammation. although glucocorticoids have immunosuppressive and anti-inflammatory effects, acne can be provoked by systemic or topical treatment. enhanced tlr2 expression by glucocorticoids has been reported in undifferentiated keratinocytes, however, human skin cells of different epidermal and dermal layers have not been investigated. in this study, the modulation of tlr expression by dexamethasone was assessed in monolayer cultures of primary human keratinocytes and dermal fibroblasts, as well as the immortalized keratinocyte cell line hacat. constitutive tlr2, tlr1 and tlr6 mrna and protein expression was confirmed in basal keratinocytes, calcium-induced differentiated keratinocytes, hacat cells and fibroblasts by qpcr and western blotting. dexamethasone induced tlr2 expression in a time-dependent and concentration-dependent manner and reduced tlr1/6 expression in keratinocytes but not in hacat cells or fibroblasts. stimulation with dexamethasone in the presence of the pro-inflammatory cytokines tnfα or il-1β further increased tlr2 mrna levels. gene expression of mapk phosphatase-1 (mkp-1) was also upregulated by dexamethasone. glucocorticoid-induced tlr2 expression was negatively regulated by p38 mapk signalling under inflammatory conditions through mkp-1 induction which functions to deactivate mapks. as expected, dexamethasone inhibited the immune responses linked to tlr2 signalling as demonstrated by reduced il-8, il-1β, mmp-1 and mcp-1 levels. however, the expression of traf6, a critical cytosolic regulator of tlr-and tnf family-mediated signalling, was further upregulated by the tlr2 agonist hklm (heat-killed lysteria monocytogenes) in dexamethasonepretreated basal keratinocytes.conclusively, our results provide novel insights intothe molecular mechanismsof glucocorticoid-mediatedtlr expressionand function in human skin cells. psoriasis is a cutaneous chronic inflammatory disease characterized by increased amounts of il-1 cytokines and t helper 17 (th17) related cytokines in lesional psoriatic skin. treatment with beta-adrenoceptor antagonists is associated with induction or aggravation of psoriasis, however, the underlying mechanism is poorly understood. previously, we could demonstrate a pivotal role for langerhans cells and dermal dendritic cells in antimalarial-provoked psoriasis by maintaining a potent th17 activity. in the present study, we investigated the effect of propranolol on human monocyte-derived langerhans-like cells (molc) and dendritic cells (modc) under inflammatory conditions. in the presence of il-1β, propranolol induced the th17 priming cytokines il-23 and il-6 in a concentration-dependent manner. the increased cytokine release was not mediated by camp suggesting gpcr-independent pathways. in contrast, il-36γ and lps failed to increase il-23 release in molc and modc in the presence of propranolol but further induced secretion of il-1β. autophagy has been linked with the secretion of il-1 family cytokines that are upregulated in chronic inflammatory disorders such as psoriasis. propranolol upregulated the expression levels of the autophagy marker p62 and lc3-i to lc3-ii conversion, induced accumulation of lc3 positive vesicles, as well as expression of il-1 signalling downstream adapter molecule traf6, indicating a late-stage block in autophagy. in summary, our results suggest a prominent role of cutaneous dendritic cell subtypes in psoriasis-like skin inflammation mediated by propranolol and possibly other beta blockers. langerhans cells (lcs) represent a highly specialized subset of epidermal dendritic cells (dcs), yet not fully understood in their function of balancing skin immunity. in the present study, we investigated in vitro generated langerhans-like cells obtained from the human acute myeloid leukaemia cell line mutz-3 (mutz-lcs) to study tlr-and cytokine-dependent activation of epidermal dcs. mutz-lcs revealed high tlr2 expression and responded robustly to tlr2 engagement, confirmed by increased cd83 and cd86 expression, upregulated il-6, il-12p40 and il-23p19 mrna levels and il-8 release. tlr2 activation reduced ccr6 and elevated ccr7 mrna expression and induced migration of mutz-lcs towards ccl21. similar results were obtained by stimulation with pro-inflammatory cytokines tnf-α and il 1β whereas ligands of tlr3 and tlr4 failed to induce a fully mature phenotype. despite limited cytokine gene expression and production for tlr2-activated mutz-lcs, co culture with naive cd4+ t cells led to significantly increased ifn-γ and il-22 levels indicating th1 differentiation independent of il-12. tlr2-mediated effects were blocked by the putative tlr2/1 antagonist cu-cpt22, however, no selectivity for either tlr2/1 or tlr2/6 was observed. computer-aided docking studies confirmed non-selective binding of the tlr2 antagonist. taken together, our results indicate a critical role for tlr2 signalling in mutz-lcs considering the leukemic origin of the generated langerhans-like cells. the stagnation in the development of new antibiotics during the last decades and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that pep19-2.5, a synthetic antimicrobial and lps-neutralizing peptide (salp), efficiently neutralizes pathogenicity factors of gram-negative and gram-positive bacteria and protects against sepsis. in the present study, we investigated the potential of pep19-2.5 and the structurally related compound pep19-4lf for their therapeutic application in bacterial skin infections. primary human keratinocytes responded to tlr2 (fsl-1) but not tlr4 (lps) activation by increased il-8 production, as determined by elisa. western blot analysis showed that both salps inhibited fsl-1-induced phosphorylation of nf-κb p65 and p38 mapk. furthermore, the peptides significantly reduced il-8 release and gene expression of il-1β, ccl2 (mcp-1) and hbd-2 as assessed by qpcr. no cytotoxicity (mtt test) was observed at salp concentrations below 10 µg/ml. in lps-stimulated monocyte-derived dendritic cells, the peptides blocked il-6 secretion, downregulated expression of the maturation markers cd83 and cd86, as analysed by flow cytometry, and inhibited ccr7-dependent migration capacity. similarly, monocyte-derived langerhans-like cells activated with lps and pro-inflammatory cytokines showed reduced il-6 levels and cd83/cd86 expression in the presence of salps. in addition to acute inflammation, bacterial infections often result in impaired wound healing. since re-epithelialization is a critical step in wound repair, we tested whether pep 19-2.5 affects keratinocyte migration using the scratch wound assay. the peptide markedly promoted cell migration and accelerated artificial wound closure at concentrations as low as 1 ng/ml and was equipotent to tgf-β. conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections. recently, we and others have shown that the transcription factor nuclear factor erythroid 2-related factor 2 (nrf2), a major regulator of the cellular antioxidant defence system, is activated by mechanical ventilation. during ventilator-induced lung injury, nrf2 exerts a protective role by interaction with the stretch-induced growth factor amphiregulin. in the current study, we aimed to investigate the role of nrf2 in acid-induced lung injury, a model for aspiration-induced ards. methods: nrf2-deficient (nrf2 -/-) mice and wild type (wt) littermates were tracheotomised and ventilated for 30min (v t =16ml/kg, f=90min -1 , peep=2cmh 2 o, fio 2 =0.3), before 50 µl hydrochloric acid (hcl) with ph=1.5 or ph=1.3 were instilled intratracheally, controls received nacl. mice were then ventilated for further 6h under monitoring of lung mechanics and vital parameters. blood gases as well as proinflammatory mediators, neutrophil recruitment and microvascular permeability were examined to assess lung injury. results: instillation of hcl ph=1.5 induced mild lung injury, indicated by hypoxemia (po 2 /fio 2 ~300mmhg) and continuously increasing lung tissue elastance (stiffness), from which nrf2 -/mice were protected. pulmonary inflammation, characterized by liberation of cytokines, chemokines and oedema formation, was attenuated in nrf2 -/mice. in contrast, hcl ph=1.3 caused more severe lung injury (po 2 /fio 2 ~200mmhg) with a steeper incline in elastance and more severe inflammation in both wt and nrf2 -/mice. conclusion: we conclude that the presence of nrf2 augments mild acid-induced lung injury, but plays no role in more severe injury. these discrepant results will be elucidated in future investigations. uniklinik rwth aachen, institut für pharmakologie und toxikologie, aachen, germany 2 fakultät für maschinenwesen der rwth aachen, werkzeugmaschinenlabor, aachen, germany rationale: reproducibility is key to science. in recent times, the reproducibility of biomedical research has been questioned increasingly. this reproducibility crisis also affects complex animal experiments, which -if not reproducible -might also be regarded as unethical and lose public acceptance. part of the problem is frequently that the provided documentation is not sufficient for reproduction. therefore, in this study we analyzed the potential of conventional quality management tools -used as standard in machine production -as an approach to improve the documentation and ascertain the quality of complex animal experiments. methods: quality management tools were transferred to an experimental animal set up -the mouse intensive care unit (micu) -which we use for lung injury studies. the tools included visualization of the experimental set-up, transfer of the experimental procedures to an event-driven process chain (epc) and statistical process control (spc) of all crucial pulmonary and cardiovascular parameters. data from ventilator-and acidinduced lung injury studies acquired in the micu were analyzed retrospectively. results: schematic visualization of the micu resulted in a chart comprising medical components, hardware, software and generated data types. the customized epc included all important activities and the resulting events for preparation of the mouse and the workplace, the actual animal ventilation experiment and sample-taking. in addition, checklists were provided for these activities and events, to ensure standardization of every work step. lung impedance and cardiac functions from ventilator-and acid-induced lung injury models were analyzed by spc and correlated with events in the epc. the spc proved to be suitable to identify outliers, predict processes and thereby validate the lung injury models. conclusions: conventional quality management tools were successfully adapted to analyze the quality of lung injury experiments in the micu. we suggest that this new approach is suitable to standardize animal testing procedures and increase the reproducibility of animal studies. background: a dysfunctional endothelial l-arginine-nitric oxide (no) pathway is a key pathomechanism of idiopathic pulmonary arterial hypertension (ipah) that can be provoked by hypoxia in cell culture models [1] [2] [3] [4] . the small peptide apelin is involved in the maintenance of pulmonary vascular homeostasis and angiogenesis although its precise mechanism of action is still unclear [5] . asymmetric dimethylarginine (adma) is known to be an endogenous inhibitor of endothelial no synthase and is associated with several cardiovascular diseases [6] . adma is degraded by dimethylarginine dimethylaminohydrolase 1 and 2 (ddah) enzymes [7] . objective: to determine the effect of apelin on the l-arginine/no pathway in human pulmonary microvascular endothelial cells (hpmecs). methods: hpmecs were cultured under normoxic and ph-related hypoxic conditions and treated with apelin. the expression of regulators of the l-arginine/no pathway were analysed using real-time pcr. the effect of apelin on the phosphoinositide-3 kinase (pi3k)/akt signalling pathway was determined using immunoassays and specific inhibitors[lh1] . apelin and adma concentrations were measured in cell culture supernatants using an enzyme-linked immunosorbent assay and a liquid chromatography-tandem mass spectrometry assay. results: treatment with apelin resulted in a reduced expression of the apelin receptor (aplnr) on hpmecs suggesting a negative feedback mechanism. apelin directly influenced the l-arginine/no pathway by increasing the expression of ddah1 and ddah2 enzymes. thus, the concentration of adma was decreased in hpmecs supernatant following treatment with apelin. the effect of apelin could be abrogated by modulation of the pi3k/akt pathway. conclusion: apelin modulates the l-arginine/no pathway and mediates enhanced degradation of adma via an upregulated expression of ddah 1 and 2 enzymes. the pi3k/akt pathway might play a decisive role in regulation of the effect of aplein. an apelin receptor agonist could be a novel and promising therapeutic option for ipah treatment. background and purpose: there is presently no proven pharmacological therapy for the acute respiratory distress syndrome (ards). recently, we and others discovered that the heptapeptide angiotensin (ang)-(1-7) shows significant beneficial effects in preclinical models of acute lung injury (ali). here, we aimed to identify the best time window for ang-(1-7) administration to protect rats from oleic acid (oa) induced ali. experimental approach: the effects of intravenously infused ang-(1-7) were examined over four different time windows before or after induction of ali in male sprague-dawley rats. hemodynamic effects were continuously monitored, and loss of barrier function, inflammation, and lung peptidase activities were measured as experimental endpoints. key results: ang-(1-7) infusion provided best protection from experimental ali when administered by continuous infusion starting 30min after oa infusion till the end of the experiment (30-240min). both pretreatment (-60-0min before oa) and short-term therapy (30-90 min after oa) also had beneficial effects although less pronounced than the effects achieved with the optimal therapy window. starting infusion of ang-(1-7) 90min after oa (late-term infusion) achieved no protective effects on barrier function or hemodynamic alterations, but still reduced myeloperoxidase and angiotensin converting enzyme activity, respectively. conclusions and implications. our findings indicate that early initiation of therapy after ali and continuous drug delivery are most beneficial for optimal therapeutic efficiency of ang-(1-7) treatment in experimental ali, and presumably accordingly, in clinical ards. airway epithelium functions as a physicochemical barrier against dust, air pollutants and other pathogens and plays a critical role in physiological and pathological processes including modulation of the inflammatory response, innate immunity and airway remodeling such as in human asthma, copd and equine recurrent airway obstruction (rao). models of the airway epithelia are, indeed, missing for the horse; thus, we established long-term equine bronchial epithelial cell cultures using the rock inhibitor y-27632 and cell growth and differentiation was characterized. bronchial epithelial cells (ebec) from adult horses were cultured in the presence and absence of 10 µm y-27632 under conventional and air-liquid-interface (ali) culture conditions. cell proliferation and differentiation were analyzed. formation of a functional epithelial barrier was investigated by transepithelial electric resistance (teer) measurement and immunocytochemical staining of the tight-junction-protein zonula occludens-1 (zo-1). under conventional culture, y-27632 induced higher growth rate of primary ebec and increased the passage number up to 5 passages with retained epithelial cell behavior. in the presence of y-27632, ebecs under ali showed higher teer values. expression of zo-1 correlated with the increase in teer, but in y-27632-treated ebec tight-junctionformation was more rapid, indicating accelerated differentiation, as well h/e-staining and scanning electron microscopic imaging showed a higher amount of cilia and microvilli and pas-positive cells. in conclusion, the data suggest that the rock inhibitor y-27632 facilitates long-term culture of equine bronchial epithelial cells which can be used to study airway disease mechanisms and to identify pharmacological targets. leishmaniasis is a neglected disease of tropical and subtropical regions with millions of people at risk of infection with severe consequences including death. current antileishmanial drugs exhibit serious side effects and also development of resistances is rising. this disease is caused by protozoal organisms from the genus leishmania. in their insect vector they exist in the promastigote form, while in the mammalian host they survive as amastigotes inside the phagolysosomes of macrophages. this makes a specific pharmacotherapy complicated. due to the success of artemisinin in malaria therapy, it was of interest whether endoperoxides are also useful to treat leishmaniasis. in a previous study we demonstrated that ascaridole, an endoperoxide from chenopodium ambrosioides, can cure cutaneous leishmaniasis in a mouse model and exhibited ic 50 values for the viability in the low micromolar range [1] . even though in chemical model systems some basic ideas about the mechanism of activation of these endoperoxides exist, in biological systems including leishmania parasites this activation step has never been demonstrated. therefore, we set up experiments to identify primary drug intermediates formed from ascaridole by activation in leishmania tarentolae promastigotes using electron spin resonance spectroscopy in combination with spin trapping methods. ascaridole was activated in a cell-free system by fe 2+ . the radicals were trapped by 2-methyl-2-nitrosopropane (mnp). the resulting esr spectra consisted of the triplet of duplets. spectral simulations revealed coupling parameters of a n = 16.8 g, and a h = 1.8 g. these coupling constants are compatible with iso-propyl radicals as primary intermediates. in the cellular system, consisting of leishmania tarentolae promastigotes, instead of mnp the less cytotoxic 5,5-dimethyl-1pyrroline-n-oxide (dmpo) was used for spin trapping. without addition of fe 2+ a six line esr signal was observed. spectral simulations of the dmpo spin adduct revealed coupling constants of a n = 16.1 and a h = 24.6 g. according to previously published data [2] from other spin trapping experiments, this corresponds to the formation of carboncentered radicals from ascaridole by leishmania parasites. additional experiments using iron chelators and antioxidants as well as a comparison with the endoperoxide artemisinin were performed. in summary, this study for the first time demonstrated the activation of the endoperoxide ascaridole by a protozoal organism to its active intermediate as a prerequisite to understand its mechanism of action. [1] l. monzote, j. pastor, r. scull, and l. gille. antileishmanial activity of essential oil from chenopodium ambrosioides and its main components against experimental cutaneous leishmaniasis in balb/c mice. phytomedicine 21:1048-1052, 2014. nitric oxide (no), produced by the inducible nitric oxide synthase (inos) has many functions in physiological and pathophysiological pathways. after induction of inos expression by cytokines and other agents the enzyme produces high amounts of no in a ca 2+ -independent way. this high no production can have beneficial microbicidal, antiparasital, antiviral and antitumoral effects. in contrast, aberrant inos induction may have detrimental consequences and seems to be part of many diseases such asasthma, arthritis, multiple sclerosis, colitis, psoriasis, neurodegenerative diseases, tumor development, transplant rejection or septic shock. analysis of the human inos-mrna structure revealed the existence of an upstream open reading frame (µorf) and a putative internal ribosome entry site (ires) in the 5' untranslated region (5'utr) in front of the start codon of the inos coding sequence (cds). to analyze the function of the µorf and the putative ires we cloned different egfp and luciferase reporter constructs and transfected them into the human colon carcinoma cell line dld1. using a plasmid construct with the µorf fused with the egfp cds, we could show that the µorf can be translated. however, compared to the positive control plasmid less egfp was produced, which can be explained by a weak kozak sequence of the µorf. blocking the mrna cap-dependent translation by cloning a stem loop structure in front of the inos 5'utr within a luciferase reporter plasmid led to a remarkable loss of luciferase production. thus, the expression of inos seems to be cap-dependent. furthermore, transfection experiments with dld1 cells using constructs coding for a bicistronic renilla-firefly luciferase mrna showed that there is no ires in front of the inos cds. taken together, the inos expression seems to be cap-dependent and without influence of an ires, while the µorf is translatable. therefore we speculate that inos expression is only possible due to a leaky scanning mechanism depending on the weak kozak sequence of the µorf. objectives: vascular oxidative stress is considered a pathophysiologic factor promoting cardiovascular diseases such as coronary artery disease, heart failure, diabetes and hypertension. there are several sources of superoxide in vascular smooth muscle and endothelial cells but whether an impairment of the catalytic function of enos and thus generation of oxidative stress is involved in blood pressure (bp) regulation and/or the development of hypertensive disease states is unknown. methods: we generated a mutant enos in which one of the two essential cysteines required for the coordination with the central zn-ion, correct dimer formation and normal activity is replaced by alanine (c101a-enos). normal enos (enos-tg) or a novel dimer-destabilized c101a-enos described previously (antioxid redox signal. 2015 sep 20; 23(9) :711-23) were introduced in c57bl/6 in an endothelial-specific manner. mice were monitored for enos expression and localization, aortic relaxation, systolic blood pressure, levels of superoxide and several post-translational modifications indicating activity and/or increased vascular oxidative stress. some groups of mice underwent voluntary exercise training for 4 weeks or treatment with sod mimetic tempol. results: c101a-enos-tg showed significantly increased superoxide generation, protein-and enos-tyrosine-nitration, enos-s-glutathionylation, enos 1176/79 phosphorylation and amp kinase (ampkα) phosphorylation at thr172 in aorta, skeletal muscle, left ventricular myocardium and lung as compared to enos-tg and wild type (wt) controls. the localization of enos-c101a-tg was restricted to endothelium as evidenced by immunohistochemically staining for enos and an endothelial-specific marker cd31. exercise training increased phosphorylation of enos at ser 1176/79 and of ampkα at thr 172 in wt but not in c101a-enos-tg. aortic endothelium-dependent and endothelium-independent relaxations were similar in all strains. in striking contrast, c101a-enos-tg displayed normal blood pressure despite higher levels of enos, while enos-tg showed significant hypotension. tempol completely reversed the occurring protein modifications and significantly reduced bp in c101a-enos-tg but not in wt controls. conclusions: by means of a novel transgenic mouse model we demonstrated that vascular oxidative stress generated by endothelial-specific expression of a dimerdestabilized variant of enos selectively prevents bp reducing activity of vascular enos, while having no effect on aortic endothelial-dependent relaxation. these data suggest that oxidative stress in microvascular endothelium may play a role in the development of essential hypertension. the herbal medicinal product myrrhinil-intest ® consists of myrrh, chamomile flower dry extract and coffee charcoal. clinical data prove the effectiveness of this herbal preparation for inflammatory intestinal disorders. to further investigate the anti-inflammatory potential of the single components as part of a multi-target principle, an ethanolic (my) and aqueous (mya) myrrh extract, ethanolic chamomile flower extract (ka) and aqueous coffee charcoal extract (cc), were examined in an in vitro tnbs inflammation model using rat small intestinal preparations. the effect of the plant extracts on tnbs induced inflammatory damage was characterised based on tnfα-gene expression analysis, isometric contraction measurement and histological analysis. furthermore, tnfα-release from lpsstimulated thp-1 cells was determined. budesonide was used as positive control. additionally, microarray gene expression analysis was performed in lps/ifnγ stimulated native human macrophages to determine potential underlying mechanisms. the tnbs-induced overexpression of tnfα-mrna was reduced after ka (0.1 mg/ml) and mya (1 mg/ml) treatment down to 24% and 16% resp.; tnbs-induced loss of contractility and reduction of mucosal layer thickness was inhibited after ka (3 mg/ml) treatment by 26% and 25% resp.; after mya (0.1-1mg/ml) treatment by 17% and 44% resp. lps-induced tnfα release from thp-1 cells was inhibited concentrationdependently by my (ic50 = 60.65 μg/ml; 97% inhibition), ka (ic50 = 439 μg/ml; 71% inhibition) and cc (ic50 = 1886 μg/ml; 44% inhibition). furthermore, ka (200 µg/ml) and cc (500 µg/ml) inhibited the lps/ifnγ-induced expression of genes associated with chemokine signalling up to 100-fold (for cxcl13). the presented study demonstrates further evidence for anti-inflammatory properties of the herbal components which contribute to the reported clinical effectiveness. introduction: the purine nucleoside adenosine, which is involved in a variety of physiological functions, regulates immune and inflammatory responses and acts as a modulator of gut functions. although it is present at low concentrations in the extracellular space, stressful conditions, such as inflammation, can markedly increase its extracellular level up to micromolar range. by activation of different receptor subtypes adenosine is able to induce anti-inflammatory or pro-inflammatory impacts. aim: the current study examined the impact of adenosine a2a receptors (a2ar) and adenosine a2b receptors (a2br) to regulate contractility in untreated and inflamed rat colon preparations using a specific a2ar agonist (cgs 21680) and an a2br antagonist (psb-1115) on acute inflammation in rat colon preparations. further it focused on interactions of the multi-herbal drug stw 5 with a2ar as a possible mechanism of the protective effect of stw 5 in gastrointestinal disorders. methods: inflammation was induced by intraluminal instillation of 2,4,6-trinitrobenzene sulfonic acid (tnbs). contractions were measured isometrically in an organ bath set up. gene expression was determined using rt-pcr. radio ligand binding assays (competition experiments) were carried out with rat brain homogenates. morphological changes were estimated after van gieson staining. results: all four adenosine receptor subtypes were expressed in untreated colon preparations. activation of a1, a2b, and a3 receptor with specific agonists reduced the acetylcholine (ach, 10µm)-induced contractions, while activation of a2br enhanced it. after incubation with tnbs morphological damages in colonic mucosa and muscle walls were detectable followed by reduced ach-contractions. the tnbs-mediated decrease of ach-contractions as well as the morphological damages were partially normalized by co-incubation of tnbs with cgs 21680 (10µm) or with psb 1115 (100µm). the same effects with smaller intensity were found for stw 5 (512 µg/ml) in female but not in male colon preparations. these results are in accordance with ligand binding studies indicating that stw 5 interact with the a2ar. conclusion: anti-inflammatory mechanisms and cell protective actions of stw 5 are partly due to the interaction with adenosine receptors. the results give a clear-cut correlation with symptom improvements in clinical trials and thereby highlight the relevance of stw 5 as a therapeutic approach in ibs. (allescher 2006) . therefore, a multi-target approach is a promising therapeutic strategy, as is exemplified by stw 5(ottillinger et al. 2013) . stw 5 (iberogast®) is a fixed combination of nine plant extracts with iberis amara (stw 6) as one of its components. it is successfully used for treatment of functional dyspepsia and irritable bowel syndrome (ibs). to allow an overview of targets addressed by stw 5 and the role of its components in relation to the different forms and causes of functional gi diseases, an evaluation of the data, which have been gained from more than 150 pharmacological tests, is needed. all data from studies including stw 5 alone, or stw 5 and its components, were retrieved and sorted according to types of study models (human and animal systems, animal disease models, gi-preparations, cell cultures, in vitro-systems) and respective etiologic mechanisms related to fgds and then visualized in the form of 2d histograms (lorkowski et al. 2015) . results: more than 150 pharmacological tests indicated anti-oxidative activity, electrophysiological effects, ulcer protection, anti-inflammatory actions, pro-kinetic and spasmolytic effects as well as reflux and acid reduction. moreover, the analysis indicated that the components of stw 5 contribute differently to the overall effect of stw 5. altogether, the evaluation of the data shows that stw 5 is active in response to multiple etiologic factors involved in fgds, especially functional dyspepsia and irritable bowel syndrome, and to which extent the herbal extract components of the combination are relevant for the different mechanisms of action and their translation to clinical efficacy. conclusion: multi step clustering allows the transformation of complex data sets. it makes the allocation of specific actions to the different components of stw 5 manageable, so also giving support to its clinical use in patients with different symptoms. introduction: stw 5 ii has been recently developed in an effort to reduce the number of active extracts in the mother multi-component herbal preparation, stw 5 (iberogast ® , steigerwald arzneimittelwerk gmbh, darmstadt, germany) without affecting the overall therapeutic efficiency. stw 5 consists of a mixture of 9 standardized extracts: bitter candytuft (iberis amara), lemon balm (melissa officinalis), chamomile (matricaria recutita), caraway fruit (carum carvi), peppermint leaf (mentha piperita), liquorice root (glycyrrhiza glabra), angelica root (angelica archangelica), milk thistle (silybum marianum) and celandine herb (chelidonium majus), whereas stw 5 ii lacks the last 3 components. stw 5 was shown to be effective clinically to treat functional dyspepsia (1) and irritable bowel syndrome (2) and was shown experimentally to be effective to guard against the development of radiation induced intestinal mucositis (3) and in the management of ulcerative colitis (4) . the present study was initiated to show whether stw 5 ii with the reduced component extracts would also be as effective in the latter condition. this was induced in wistar rats by feeding them with 5% dextran sodium sulfate in drinking water for 1 week when lesions were observed in the colon evidenced by histological examination as well as colon shortening and reduction of colon mass index. this was associated with a rise in myeloperoxidase and a fall in reduced glutathione, glutathione peroxidase, and superoxide dismutase in colon homogenates as well as a rise in tnfα in serum. oral administration of stw 5 in doses of 2 and 5 ml/kg or stw 5 ii in a dose of 2 ml/kg for 1 week before and continued during dss feeding tended to normalize all the changes in a fashion comparable to sulfasalazine, used as a reference drug in a dose of 300 mg/kg. conclusions: the modified preparation, stw 5 ii thus proved to be as effective as stw 5, thereby reflecting its potential usefulness in ulcerative colitis possibly by virtue of its anti-inflammatory and anti-oxidant properties. (1) schmulson mj (2008) the emetic pathways include the action of neurotransmitters dopamine, serotonin and substance p in the emetic centers localized in the brainstem, area postrema and vagal nerve afferents. previous in vivo studies in beagle dogs revealed that the plant alkaloid lycorine potentially induce nausea and emesis. though antagonists of the tachykinin receptor 1 (maropitant) and serotonin receptor 3 (ondansetron) prevented lycorinemediated emesis, the molecular mechanism of nausea and vomiting remain still unknown. to study the mechanism of action of the emetic agents, we analyzed the effect of lycorine (direct activation of nk1) and channel opening (activation of 5ht3) on the intracellular calcium homeostasis (using fluorometric ca 2+ analysis) and cell proliferation rates in endogenously nk1 and 5ht3 receptor expressing cell lines as well as in cho and hek cells stably expressing the receptors. neither endogenously receptor expressing nk1 or 5ht3 cells nor receptor overexpressing cells showed calciumflux or calcium mobilization after stimulation with lycorine. furthermore, we are measuring the receptor number and subtypes using radioligand binding studies. it is planned, moreover, to obtain fluorescent labeled constructs of the nk1 receptor to gain insights into the involvement of receptor internalization which might mediate emesis. by characterizing these molecular principles of the nk1 and 5ht3 receptors, we are attempting to obtain more information in predicting drug-induced side effects such as nausea and emesis. the intestinal epithelium is completely renewed every 4-5 days. this process is driven by stem cells, which reside within specialized niches in the intestinal crypts and give rise to several differentiated cell types, including enterocytes, paneth, enteroendocrine, goblet and tuft cells. however, the molecular mechanisms that establish and maintain differentiated cell numbers and proportions remain largely unknown. here, we systematically analyzed the intestinal expression of semaphorins and plexins, which constitute a ligand-receptor system that plays central roles in cell-cell communication in various biological contexts. we identified plexin-b2 and its semaphorin ligands to be highly expressed in intestinal epithelial cells. genetic inactivation of plexin-b2 in intestinal organoids strongly reduced the number of enteroendocrine cells. our data suggest that semaphorin-plexin-b2 signaling promotes differentiation of intestinal epithelial cells towards the enteroendocrine lineage. the gastric epithelium contains several types of differentiated cells, including foveolar cells that produce mucus, parietal cells that secrete gastric acid and intrinsic factor, chief cells that synthesize pepsinogen and gastric lipase, and enteroendocrine cells that release different hormones. these differentiated cell types all originate from multipotent stem cells, yet little is known about how this differentiation process is regulated on a molecular level. the gap protein rasal1 controls the activity of small gtpases of the ras family, and its expression levels have been shown to inversely correlate with progression of stomach cancers. however, functional studies on the physiological role of rasal1 in the gastric epithelium are lacking. here, we established and characterized a mouse line with inactivation of the rasal1 gene. we observed that these mice showed increased numbers of enteroendocrine cells in the gastric mucosa. conditional inactivation of rasal1 in enteroendocrine cells, using a mouse line in which cre expression is driven by the atoh1 promoter, further corroborated that rasal1 expression in enteroendocrine cells determines enteroendocrine cell numbers. these findings identify rasal1 as a regulator of gastric epithelial cell differentiation. ). the present study investigates the impact of two faah inhibitors (arachidonoyl serotonin [aa-5ht], urb597) on a549 lung cancer cell metastasis and invasion. lc-ms analyses revealed increased levels of faah substrates (aea, 2-ag, oea, pea) in cells incubated with either faah inhibitor. in athymic nude mice faah inhibitors were shown to elicit a dosedependent antimetastatic action. in vitro, a concentration-dependent anti-invasive action of either faah inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (timp-1). using sirna approaches, a causal link between the timp-1-upregulating and anti-invasive action of faah inhibitors was confirmed. moreover, knockdown of faah by sirna was shown to confer decreased cancer cell invasiveness and increased timp-1 expression. inhibitor experiments point toward a decisive role of cb 2 and transient receptor potential vanilloid 1 in conferring the anti-invasive effects of faah inhibitors and faah sirna. finally, antimetastatic and anti-invasive effects were confirmed for all faah substrates. collectively, the present study provides first-time proof for a pronounced antimetastatic action of the faah inhibitors aa-5ht and urb597. as underlying mechanism of its anti-invasive properties an upregulation of timp-1 was identified. regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (mscs). the present study focused on inhibitors of the enzyme fatty acid amide hydrolase (faah), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (n-oleoylethanolamine, n-palmitoylethanolamine). in boyden chamber assays, the faah inhibitors, urb597 and arachidonoyl serotonin (aa-5ht), were found to increase the migration of human adipose-derived mscs. lc-ms analyses revealed increased levels of all four aforementioned faah substrates in mscs incubated with either faah inhibitor. following addition to mscs, all faah substrates mimicked the promigratory action of faah inhibitors. promigratory effects of faah inhibitors and substrates were causally linked to activation of p42/44 mitogen-activated protein kinase (mapk), as well as to cytosol-to-nucleus translocation of the transcription factor, peroxisome proliferator-activated receptor α (pparα). whereas pparα activation by faah inhibitors and substrates became reversed upon inhibition of p42/44 mapk activation, a blockade of pparα left p42/44 mapk phosphorylation unaltered. collectively, these data demonstrate faah inhibitors and substrates to cause p42/44 mapk phosphorylation, which subsequently activates pparα to confer increased migration of mscs. this novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by faah inhibitors. background: the hematopoietic disorder chronic myeloid leukemia (cml) is one of the most extensively studied neoplasms. it is caused by translocation between chromosomes 9 and 22 leading to the formation of the philadelphia chromosome and the bcr-abl fusiongene. first-line targeted therapy is still the tyrosine-kinase inhibitor imatinib (im), which led to tremendous success in treatment. however, the amount of therapeutic resistances is increasing, caused either by bcr-abl-dependent mechanisms (e.g. bcr-abl amplification/overexpression, point mutations) or bcr-ablindependent mechanisms. these might be linked to alterations in drug transporter expression or particularly, microrna-expression levels. in our previous study, we analyzed the changes of microrna expression profiles during the development of imresistances in the leukemic cell line k562. an inverse correlation of mir-212 expression and protein levels of the efflux transporter atp-binding cassette transporter g2 (abcg2) was observed in cells resistant to different im-concentrations, pointing to a relation of mir-212 to im-resistance. hence, we investigate in current studies, how the influence of mir-212 on im-sensitivity could be explained. methods: we transfected k562 cells, sensitive treatment-naïve cells and cells resistant to various im-concentrations, either with mir-mimic pre-mir-212 or inhibitory anti-mir-212, challenged them with im and analyzed effects on cell viability, activation of apoptosis and cell death using wst-1-, caspase glo 9-assay and cell counting. in addition, we analyzed changes in abcg2 expression using flow cytometry and qrt-pcr and investigated alterations in im-efflux using hplc and hoechst efflux assay. results: under im-treatment, sensitive k562 showed an effect of mir-212-inhibition using anti-mir-212. this led to a significant promotion of cell survival apparent on the level of respiratory chain function (p<0.01) and cell membrane integrity and reduced capase-9 activity (p<0.05). furthermore, these mirna-effects are dose-dependent as confirmed in concentration row-experiments. regarding transport and abcg2 expression, we found that 2 µm im-resistant k562 do not express higher amounts of abcg2, but showed higher transport rates of im or the abcg2-substrate hoechst 33342. conclusions: overall, these experiments indicate that mir-212 does not only affect abcg2-expression, but also influences cell sensitivity to im in a more direct manner. further analysis will now be performed to reveal the underlying mechanism, how cell sensitivity to im is altered and if these effects occur due to a direct regulation of abcg2. in summary, these findings could be relevant in cml-therapy, overcoming imresistances with a better understanding of mirna-and drug transporter alteration in cml. acknowledgments: we would like to thank all the authors for their contribution to this project. this work was funded by the university hospital schleswig-holstein. oxidized silicon nanoparticles and iron oxide nanoparticles for radiation therapy s. klein radiation therapy often combined with surgery and/or chemotherapy is applied to more than 50 % of patients at some point of their treatment. the cytotoxic effects of ionizing radiation occur from their ability to produce dna double-strand breaks through the formation of free radicals within cells. however, the curative potential of radiotherapy is often limited by intrinsic radio resistance of cancer cells and normal tissue toxicity. to overcome this resistance and enhance the effectiveness of ionizing radiation, radio sensitizers are used in combination with radiotherapy. in our studies we used amino functionalized, oxidized silicon nanoparticles (sinp), superparamagnetic iron oxide nanoparticles (spion) and iron doped silicon nanoparticles (fe(1%)-sinp) to increase the formation of reactive oxygen species (ros) in cells. cancer and tissue cells loaded with the various nanoparticles were irradiated with a single dose of 1-3 gy using a 120 kv x-ray tube. after irradiation, the formation of the different ros species including superoxide, hydroxyl radicals and singlet oxygen was investigated. sinps with sizes around 1 nm can easily cross the cell and nuclear membrane. the positively charged amino functionalized sinps stick in all membranes as well in those of the mitochondria. irradiation of the mitochondria may cause the depolarization of the mitochondrial membrane, which enables the release of cytochrome c and simultaneously, an inhibition of the respiratory chain, which leads to an increased generation of superoxide. amino functionalized sinps, as being embedded in the outer mitochondrial membrane, evidently enhance the depolarizing effect of the x-ray radiation on the mitochondria and therefore increase the concentration of superoxide. [1] oxidized sinps with larger sizes accumulate in the cytoplasm and generate mainly singlet oxygen after irradiation. spions enter the cells via endocytosis, whereas the uncoated spions remain in the vesicles and the citrate coated spions accumulate in the cytoplasm. cells loaded with citrate coated spions show no higher ros concentration than in media-cultured cells. but after irradiation, the ros formation increased drastically. this enhancing effect is explained with the impact of x-rays onto the surface of spions which is due to the destruction of surface structures. the freed spion surface contains easier accessible iron ions. this ions can participate in the fenton and haber-weiss chemistry and thus, catalyze the hydroxyl radical formation. [2] 1 to 5 % iron doped sinp increase the formation of hydroxyl radicals as well as the generation of singlet oxygen after irradiation. chronic pain in response to tissue damage (inflammatory pain) or nerve injury (neuropathic pain) is a major clinical health problem, affecting up to 30% of adults worldwide. currently available treatments are only partially susceptible and are accompanied with therapy limiting side effects. thus it is important to elucidate molecular mechanisms of pain signaling in detail to obtain new insights in potential future therapies. recent data indicate that hydrogen sulfide (h 2 s) contributes to the processing of chronic pain, however pro-as well as antinociceptive effects have been described so far. moreover the sources of h 2 s production in the nociceptive system are only poorly understood. here we investigated the expression of the h 2 s releasing enzyme cystathionine g-lyase (cse) in the nociceptive system and characterized its role in chronic pain signaling using cse deficient mice. paw inflammation and peripheral nerve injury led to upregulation of cse expression in dorsal root ganglia. however, conditional knockout mice lacking cse in sensory neurons as well as global cse knockout mice demonstrated normal pain behaviors in inflammatory and neuropathic pain models as compared to wt littermates. thus, our results suggest that cse is not critically involved in chronic pain signaling in mice and that sources different from cse mediate the pain relevant effects of h 2 s. this work was supported by the deutsche forschungsgemeinschaft (sfb815-a14) and in part by loewe-schwerpunkt "anwendungsorientierte arzneimittelforschung". heinrich-heine-universität, institut für toxikologie, düsseldorf, germany 2 heinrich-heine-universität, urologie, düsseldorf, germany background: cisplatin (cispt) is frequently used in the therapy of advanced stage urothelial cell carcinoma (ucc). yet, inherent and acquired drug resistance limits the clinical use of cispt. here, we comparatively investigated the response of epithelial-like (rt-112) and mesenchymal-like (j-82) uc cells following cispt treatment. methods: upon selection with equitoxic doses of cispt for months, we obtained cispt resistant variants (rt-112 r , j-82 r ). cell viability was measured using the alamar blue assay. cell cycle distribution was analysed by flow cytometry. immunocytochemistry was used to quantify the number of nuclear γh2ax and 53bp1 foci representing dna double strand breaks (dsbs), while western blot was used to unravel the role of dna damage response (ddr) to acquired cispt resistance. qrt-pcr was performed to analyse the mrna expression of genes associated with cispt resistance. j-82 and j-82 r cells were treated with different concentrations of lovastatin and selected ddr inhibitors to elucidate their influence on cell viability. results: untreated rt-112 cells showed an about 2-3-fold higher resistance to cispt than j-82 cells. both cell lines differed in the expression pattern of genes that are associated with cispt resistance. rt-112 r and j-82 r revealed a 2-3-fold increased cispt resistance as compared to the parental cells. during the selection procedure, we observed that acquired cispt resistance goes along with morphological alterations that resemble epithelial mesenchymal transition (emt). cell cycle analysis of rt-112 r cells disclosed a reduced apoptosis and enhanced g2/m arrest following cispt exposure as compared to rt-112 wild-type cells. by contrast, induction of cell death was similar in j-82 and j-82 r cells. notably, j-82 r cells showed a reduced formation of cispt-induced dsbs. correspondingly, the related ddr was diminished in j-82 r as compared to their parental cells. this was not found when ddr was comparatively analysed between rt-112 r and rt-112 cells. data obtained from qrt-pcr analysis indicate that different mechanisms contribute to acquired drug resistance of j-82 r and rt-112 r . unexpectedly, j-82 r and rt-112 r shared the upregulation of xaf-1. treatment of j-82 r cells with statins and protein kinase inhibitors revealed an enhanced sensitivity to pharmacological inhibition of chk-1 and, moreover, re-sensitization to cispt by chk-1 inhibitor. based on the data we suggest that mechanisms of acquired cispt resistance of epithelial and mesenchymal uc cell lines are different with apoptosisrelated mechanisms appear to be more relevant for epithelial-like rt-112 cells and ddr-related mechanisms dominating cispt susceptibility in mesenchymal-like j-82 cells. furthermore, our findings indicate that chk-1 might be an appropriate target to deal with acquired cispt resistance in ucc. in many patients, gastric cancer treatment with conventional cytostatic agents shows only limited clinical response. novel therapeutics, which inhibit rtk signaling by targeting c-met or her family receptors, have demonstrated some efficacy; however, primary resistance of gastric cancer cells against these inhibitors is still a major problem. in the present study we investigated the mechanism of heregulin (hrg)-promoted survival of gastric cancer cells after treatment with c-met inhbitors or sirna-mediated downregulation of c-met. we found that hrg treatment of gastric cancer cells with a c-met amplification partially rescued the cells from the antiproliferative effects of pharmacological c-met inhibition or sirna-mediated downregulation of c-met. moreover, c-met inhibition or downregulation led to an induction of her3 expression on mrna and protein level, whereas other her family receptors were unaffected. downregulation of her3 impaired the hrg-mediated rescue of cell survival upon c-met inhibition. in other tumor entities the chromatin organizer special at-rich sequence-binding protein 1 (satb1) has been described as a regulator of her family receptor expression involved in adaptive responses of tumor cells. thus, we investigated the contribution of satb1 in the upregulation of her3 after c-met inhibition. of note, c-met inhibitors as well as c-met-specific sirnas markedly induced satb1 expression in gastric cancer cells, and the downregulation of satb1 by sirnas completely prevented the induction of her3 upon c-met inhibition. in contrast, her1 or her2 expression levels were not affected by satb1-specific sirnas. the function of satb1 as transcriptional regulator is controlled by its phosphorylation status, which in turn is modulated by pkc activity. thus, we also tested the effect of pkc inhibitors on her3 expression after c-met inhibition. interestingly, the upregulation of her3 in gastric cancer cells was significantly reduced by pkc inhibitors. to summarize, satb1 and pkc are critically involved in the regulation of her3 expression in gastric cancer cells after treatment with c-met inhibitors and the oncogene her3 plays a crucial role for tumor cell survival in this context. thus, inhibition of pkc or satb1 may help to overcome resistance against c-met inhibition in this tumor entitiy. in the rising field of nanomedicine, development of new approaches in diagnosis and treatment of cancer is a challenging task. typically, a nanocarrier is synthesized and linked to functional compounds displaying either diagnostic or therapeutic effects in cancer models. recently, nanomaterials combining both diagnostic and therapeutic properties, so-called 'theranostics', became of primary interest. here we used a human albumin-polyethylene glycol (peg) copolymer (hsa) as a theranostic platform for molecular integration of the chemotherapeutic drug doxorubicin (dox) and the magnet resonance imaging (mri) contrast agent gadolinium (gd) yielding gd-hsa-dox nanoparticles. besides in vitro testing, which demonstrated cytotoxic efficacy of gd-hsa-dox, we used the chorioallantoic membrane (cam) of fertilized chick eggs as a preclinical xenotransplantation model. the cam assay, which in legal terms does not represent an animal experiment, allows testing of compounds in an in vivo setting. this model is particularly helpful to narrow the gap between in vitro and in vivo applications in rodents, because it can help to reduce number of elaborate experiments with typically nude mice, and it reduces or even avoids exposure of those animals to adverse effects and distress. treatment-resistant mda-mb-231 breast cancer cells stably transfected with luciferase were xenotransplanted onto the chorioallantoic membrane. after formation of solid breast cancer xenografts, gd-hsa-dox was injected intravenously and its antiproliferative effect was evaluated by ivis imaging of luciferase activity and by immunohistochemical analysis of the tumor xenografts for the ki-67 proliferation antigen. in comparison to conventional dox, gd-hsa-dox showed increased antiproliferative efficacy and reduced general toxicity in the cam assay. on the basis of these findings, a rodent model was established, where the mda-mb-231 breast cancer cells were orthotopically xenotransplanted into the mammary fat pads of female nmri nu/nu mice. in this model, we further investigated biocompatibility, as well as diagnostic and therapeutic properties of the engineered nanomaterial. after repeated administration of gd-hsa-dox into the tail vein of the animals, biocompatibility of gd-hsa-dox was confirmed by uncompromised liver, kidney and hematopoietic parameters. to warrant diagnostic properties, accumulation of the nanomaterial in tumor tissue is indispensable. by small animal mri of gd, kinetics of intravenously applied gd-hsa-dox in tumor tissue was monitored. an enhancement of the engineered nanomaterial in tumor tissue was detected for up to 47 h after injection indicating successful enrichment of gd-hsa-dox within the tumor tissue, which can be ascribed to the enhanced permeability and retention (epr) effect observed in the microenvironment of many solid tumor tissues. we are currently investigating the antitumor efficacy of gd-has-dox in this mouse model and preliminary data seem to indicate a dose-dependent anticancer effect. supported by the volkswagenstiftung. tubulin-binding agents are the most important anti-tumoral drugs. due to the side effects and the development of resistances, the discovery of new agents is still of importance. recently, pretubulysin (pt), a naturally occurring precursor of the myxobacterial compound tubulysin, was identified as a novel tubulin-binding compound. in the dfg research group for 1406, pt was characterized as anti-tumoral, antiangiogenic and vascular-disrupting compound. moreover, pt was also found to inhibit the formation of metastases in vivo. aim of the present study was to gain first insights into the mechanisms underlying this anti-metastatic effect by investigating the influence of pt on the interaction of endothelial and tumor cells in vitro. pt treatment of primary human endothelial cells (huvecs) strongly increased the adhesion of breast cancer cells (mda-mb-231) on huvecs, but limited their transmigration through the endothelium (transwell assay). based on this data, the gene expression of presumably involved adhesion molecules was determined by qrt-pcr: icam-1, vcam-1, e-selectin, n-cadherin, and galectin-3. moreover, the chemokine system cxcl12/cxcr4 was analyzed. it could be demonstrated that the mrna level of endothelial n-cadherin is upregulated by pt. while total protein expression of ncadherin was enhanced in pt treated huvec, its surface expression was not largely influenced by pt (western blot, flow cytometry). in line with this, blocking endothelial ncadherin by a neutralizing antibody revealed that this protein is not involved in ptevoked tumor cell adhesion. interestingly, pt strongly augmented the mrna and protein expression of cxcl12 in huvecs (qrt-pcr, western blot), whereas its endothelial secretion was not affected by pt (elisa). an autocrine action of cxcl12 could be excluded, since blocking the cxcl12 receptor cxcr4 on endothelial cells with plerixafor did not influence cancer cell adhesion. by microscopic analyses, we observed that pt treatment causes transient gaps in the huvec monolayer, where tumor cells prefer to adhere. since β1-integrins on the tumor cells could mediate interactions between cancer cells and extracellular matrix proteins in the gaps (e.g. collagen), their influence in cell adhesion and transmigration assays was examined. both the pt-evoked increase in cell adhesion and decrease in transmigration was completely abolished when β1-integrins were blocked on mdas by a neutralizing antibody. these results indicate that the anti-metastatic action of pretubulysin might be based on the trapping of tumor cells on the endothelium. whether this effect is also relevant in vivo, will be analyzed in future studies using intravital microscopy. this work was supported by the german research foundation (dfg, for 1406, fu 691/9-2). introduction: tyrosine kinase inhibitors (tkis) for the treatment of non-small cell lung cancer (nsclc) patients harboring activating mutations in the epidermal growth factor receptor have shown prominent success. nevertheless, patients treated with tkis eventually acquire resistance and relapse (1). based on an evolutionary cancer model (2) , weekly high dose-pulsed tki regimens were proposed to delay resistance. using data from nsclc bearing mice treated with erlotinib at different dosing regimens, we developed a semi-mechanistic pharmacokinetic/pharmacodynamic model for erlotinib effects on tumor killing and resistance development. methods: data was available from experiments in xenograft mice bearing nsclc tumors (pc9 and hcc827 cell lines; both erlotinib sensitive) (3). plasma concentrations from two single-dose groups, 30mg/kg and 200mg/kg, were used for pharmacokinetic modeling. relative tumor volume changes in mice randomized to five dosing regimens (15mg/kg daily, 30mg/kg daily, 200mg/kg every 2 days, 200mg/kg every 4 days, or vehicle) was the pharmacodynamic endpoint. a tumor growth inhibition model was developed by testing linear, exponential and logistic models to account for the tumor growth kinetics, as well as fitting an emax model to explain the effect of exposure on killing the sensitive tumor cells, and resistance development. analysis was performed using nonmem 7.3. results: absorption was dose dependent, and a precipitate compartment accounted for dissolution limited absorption for the 200mg/kg dose. a 1-compartment model with first order elimination kinetics described distribution and elimination. to describe tumor volume changes, a tumor was assumed to be a mixture of sensitive and resistant cells (represented by distinct compartments and ordinary differential equations). exponential kinetics best described natural growth (doubling times: 13 and 52 days, for sensitive and fully resistant cells, respectively). a tumor was found to transit through a less sensitive phase before acquiring full resistance. an e max model (less than linear) best described effect on the sensitive cells (ec 50 =0.53μm for both cell lines), and on the partially sensitive transit phase (ec 50 =1.24μm and 3.00μm, for hcc827 and pc9 cell lines, respectively), urging to provide adequate trough erlotinib concentrations for optimal effects. conclusions & future perspectives: an exposure-driven tumor growth inhibition model accounting for the kinetics of resistance development was developed. the model emphasizes the need for establishing an adequate trough erlotinib concentrations to delay disease progression. extracts of the stem bark of ficus platyphylla (fp) have been used in traditional nigerian medicine to treat psychoses, depression, epilepsy, pain and inflammation. previous studies have revealed the analgesic and anti-inflammatory effects of fp in different assays including acetic acid-induced writhing, formalin-induced nociception, and albumin-induced oedema. in this study, we assessed the effects of the standardised extract of fp on hot plate nociceptive threshold and vocalisation threshold in response to electrical stimulation of the tail root in order to confirm its acclaimed analgesic properties. we also investigated the molecular mechanisms underlying these effects, with the focus on opiate receptor binding and the key enzymes of eicosanoid biosynthesis, namely cyclooxygenase (cox) and 5-lipoxygenase (5-lo). fp (i) increased the hot plate nociceptive threshold and vocalisation threshold. the increase in hot plate nociceptive threshold was detectable over a period of 30 min whereas the increase in vocalisation threshold persisted over a period of 90 min. (ii) fp showed an affinity for µ opiate receptors but not for δ or κ opiate receptors, and (iii) fp inhibited the activities of cox-2 and 5-lo but not of cox-1. we provided evidence supporting the use of fp in nigerian folk medicine for the treatment of different types of pain, and identified opioid and non-opioid targets. it is interesting to note that the dual inhibition of cox-2 and 5-lo appears favourable in terms of both efficacy and side effect profile. despite the fact, that the enormous economic burden and individual suffering caused by gastrointestinal infections permanently persists in developing and newly industrialized countries, healthcare systems in first world countries underestimated its significance for a long time. the alarming prevalence of multidrug-resistant gram-negative bacteria, combined with a high epidemic potential of gastrointestinal pathogens, however, demonstrates the urgent need for new antibiotics and antiinfectives worldwide. 2,5 million deaths per year were actually caused by acute diarrheal infections. the most common causative agents of acute diarrheal infections, amongst others, are yersinia enterocolitica, campylobacter jejuni, salmonella spp., shigella spp., escherichia coli, vibrio cholerae, and clostridium difficile. the established treatment based on antibiotics is mostly ineffective or may even have adverse side effects and result in prolonged shedding. in either way, antibiotic treatment also eradicates at least parts of the intestinal microbiome, and thereby disrupts colonization resistance, fosters overgrowth of pathogens and prolongs shedding times. therefore, the development of future drugs should be focused on highly specific antiinfectives, which enable a direct pathogenspecific treatment. one very promising strategy is the inhibition of the biogenesis of outer membrane virulence factors. due to the fact that many decisive virulenceassociated outer membrane proteins (omps) of gram-negative enteropathogens are substrates of the periplasmic chaperone sura exclusively, we developed a new assay format to determine sura in vitro chaperone activity. previous publications by behrens et al., 2001 and buchner et al., 1998 documented an assay to determine sura in vitro chaperone activity with extremely limited sensitivity and minimal detectable concentration, which was not suitable for high throughput screening (hts). we now developed a luciferase-based screening assay. this highly sensitive and robust test system has been validated extensively and now gives reliable output with an appreciable z-factor of > 0,6. in cooperation with the hzi braunschweig (germany) and the hzi saarbrücken (germany), we were able to screen over 7000 purified compounds and over 500 extracts of myxobacteria. during the ongoing screening period, the assay generated four validated primary actives, which corresponds to a positive hit rate of 0,05 %. additionally, we developed an elaborate follow-up strategy to validate positive hits, which includes a well-established mouse infection model. we are looking forward to escalate our screening efforts and would like to use this abstract to invite all scientist who are interested in testing compound/natural extract libraries for an activity against the target structure sura. the potential atypical antipsychotic and dopamine d 2 receptor partial agonist 2bromoterguride antagonizes phencyclidine-and apomorphine-induced prepulse inhibition and novel object recognition deficits in rats e. tarland objectives: schizophrenia is a disabling mental disorder affecting more than 21 million people worldwide. available medical therapies are effective in the treatment of psychosis and other positive symptoms, however come with considerable side effects and often fail to ameliorate cognitive deficits and negative symptoms of the disorder. the dopamine d 2 receptor partial agonist 2-bromoterguride (2-bt) has recently been shown to exhibit antipsychotic effects in rats without causing adverse side effects common to antipsychotic drugs [1]. to determine its atypical character in vivo, the ability of 2-bt to antagonize the disruptive effects of phencyclidine (pcp) and apomorphine on sensory motor gating was determined in the prepulse inhibition paradigm. the effect of 2-bt on cognitive deficits was assessed in the novel object recognition (nor) test after object recognition memory deficits were induced by pcp treatment. method: 10 week old male sprague-dawley rats were injected with 2-bt (0.1 or 0.3mg/kg; i.p.) followed by pcp (1.5mg/kg; s.c.) or apomorphine (0.5mg/kg; s.c.). prepulse inhibition was measured in two sound-proof startle chambers. the attenuating effect of 2-bt (0.1 or 0.3mg/kg; i.p.) on visual learning and memory deficits following subchronic administration of pcp (5.0mg/kg; i.p. twice daily for 7 days) was assessed in the nor task consisting of a 3min acquisition trial and a 3min retention trial separated by a 1h inter-trial interval. clozapine (5.0mg/kg; i.p) or haloperidol (0.1mg/kg; i.p) were used as positive controls. results: the dopamine d 2 receptor partial agonist 2-bt (0.3mg/kg) and the typical antipsychotic haloperidol successfully antagonized apomorphine-induced ppi-deficits. interestingly 2-bt also ameliorated the pcp-induced ppi-deficits to the same extent as the atypical antipsychotic clozapine. preliminary data from the nor test indicate that 2-btreduces subchronic pcp-induced cognitive deficits in novel object recognition analogous to clozapine. the disrupting effects of pcp on ppi are mediated by non-competitive antagonism at nmda sites indirectly influencing a series of neurotransmitter systems. our results indicate that 2-bt mediates actions at multiple neurotransmitter receptors as it successfully ameliorated both the pcp-and apomorphine-induced ppi disruptions in rats, showing an atypical antipsychotic character. furthermore, our preliminary results support the potential atypical antipsychotic effect of 2-bt as it restored performance in the nor test, a test with good predictive validity. due to the previously shown properties and antipsychotic-like effects of 2-bromoterguride [1], this substance may be a promising candidate for treatment of schizophrenic patients. ongoing experiments investigate the potency of 2-bt to improve social deficits following a sub-chronic pcp regime in rats. background and objectives: cannabinoid-1 receptor signaling increases the rewarding effects of food intake and promotes the growth of adipocytes, whereas cb2 possibly opposes these pro-obesity effects by silencing the activated immune cells that are key drivers of the metabolic syndrome. pro-and anti-orexigenic cannabimimetic signaling may become unbalanced with age because of alterations of the immune and endocannabinoid system. methods: to specifically address the role of cb2 for age-associated obesity we analyzed metabolic, cardiovascular, immune and neuronal functions in 1. 2-1.8 year old cb2 -/and control mice, fed with a standard diet and assessed effects of the cb2 agonist, hu308 during high fat diet in 12-16 week old mice. results: the cb2 -/mice were obese with hypertrophy of visceral fat, immune cell polarization towards pro-inflammatory sub-populations in fat and liver and hypertension, as well as increased mortality despite normal blood glucose. they also developed stronger paw inflammation and a premature loss of transient receptor potential responsiveness in primary sensory neurons, a phenomenon typical for small fiber disease. the cb2 agonist hu308 prevented hfd-evoked hypertension, reduced hfdevoked polarization of adipose tissue macrophages towards the m1-like proinflammatory type and reduced hfd-evoked nociceptive hypersensitivity but had no effect on weight gain. conclusion: cb2 agonists may fortify cb2-mediated anti-obesity signaling without the risk of anti-cb1 mediated depression that caused the failure of rimonabant. leishmaniasis is a neglected tropical disease caused by leishmania, eukaryotic protozoal organisms, which infect humans and other mammals. this disease is transmitted by sandflies of the genus phlebotomus. due to global warming the endemic region of these vectors expands further to northwards and threatens south european countries as well. the treatment of leishmaniasis is difficult due to toxicity and resistance development for current drugs. the so far unexplored inhibition of mitochondrial functions in leishmania by natural products or even food ingredients seems to be an interesting alternative. two food ingredients, resveratrol (res) and xanthohumol (xan), were widely studied in mammalian cells but little is known about their actions on protozoal parasites. therefore, we compared the influence of res and xan on the function of leishmanial and mammalian mitochondria. anti-leishmanial activities of xenobiotics were assessed in cell culture of leishmania tarentolae promastigotes (ltp), leishmania amazonensis amastigotes (laa) and compared to peritoneal macrophages from mouse (pmm) using viability assays. furthermore, mechanistic studies regarding mitochondrial functions were conducted in ltp, mitochondrial fractions isolated from ltp and bovine heart submitochondrial particles using oxygen consumption measurements, assays of individual mitochondrial complex activities, membrane potential and superoxide radical formation by photometry, fluorimetry and electron spin resonance spectroscopy. in ltp, xan inhibited the viability more effective than res (ic 50 : xan 23 µm, res 161 µm). likewise, xan and res demonstrated anti-leishmanial activity in laa (ic 50 : xan 7 µm, res 14 µm) while had less influence on the viability of pmm (ic 50 : xan 68 µm, res > 438 µm). in contrast to res, xan strongly inhibited oxygen consumption in leishmania. further studies demonstrated that this is based on the inhibition of the mitochondrial electron transfer complex ii/iii by xan which was less pronounced with res. however, xan also demonstrated inhibitory activity on mammalian mitochondrial complex iii. in addition, xan caused no decrease of the membrane potential in leishmanial mitochondria, while res resulted in mitochondrial uncoupling. neither xan nor res increased mitochondrial superoxide release in ltp. these data show that res, a major polyphenol from red wine, and xan, an ingredient of hop-containing beer, may have selective anti-leishmanial activity. tryptophan hydroxylase (tph) is the rate-limiting enzyme in serotonin (5-ht) biosynthesis. its two existing isoforms are exclusively expressed in the periphery (tph1), or the raphe nuclei of the brainstem (tph2) and the respective 5-ht populations are distinctly separated by the blood-brain barrier, offering the possibility to pharmacologically modulate central and peripheral functions in an independent manner. peripheral 5-ht is mainly produced by tph1-expressing enterochromaffin cells of the gut and taken up into platelets and transported in the blood stream. upon platelet activation, 5-ht is rapidly released and locally induces multiple effects, such as vasoconstriction, cell proliferation or fibrosis and is furthermore involved in the regulation of e.g. vascular tone, gut motility, primary hemostasis, insulin secretion and t-cell-mediated immune response. following the classical early drug development pathway, we developed a fluorescencebased tph activity assay and performed a high-throughput screening of about 37000 small chemical compounds. we discovered a novel class of tph inhibitors, which was thoroughly validated in a variety of in vitro assay setups. combining medicinal chemistry and x-ray crystallography, we further aimed to develop these inhibitors into preclinical drug candidates. to date we were able to generate and patent a series of novel tph inhibitors with optimized affinity and an in vitro ic 50 in the low nanomolar range. this novel class of tph inhibitors could potentially be used to treat a variety of disorders with aberrant peripheral 5-ht signaling, such as gastrointestinal disorders (e.g. irritable bowel syndrome, crohn's disease, various forms of diarrhea), cardiac valve diseases, pulmonary hypertension, chronic respiratory diseases and some neuroendocrine (carcinoid) tumors. primary hepatocellular carcinoma (hcc) is the most frequent type of liver cancer. therapeutic options are rare. beside sorafenib, a tyrosinkinase inhibitor, which is only used in end stage liver cancer, the surgical intervention is the only successful clinical treatment option. hence there is an urgent need to develop new therapeutic strategies and to identify new drugs for therapy of hcc. hcc often arises in fibrotic or cirrhotic liver, which is accompanied by a change of the extracellular matrix (ecm) composition. in addition it was shown that hepatoma cells express different integrins, which interact with ecm and intracellular cell signaling, compared to hepatocytes. snake venoms have gained increased attention, as it was shown that some of their enzymes and peptides directly act on tumor cells and their multicellular arrangement or indirectly by influencing the stroma environment of the tumor. aim of the present study was to investigate the effect of snake venoms on liver cancer related cell lines as well as their specific action on the ecm-integrin axis. the effects of the snake venoms vipera palestinae (vp), calloselasma rhodostoma (cr) and echis sochureki (es) on a cellular level (mtt, ldh release), on cell-cellconnections (caco2 permeability assay) and on cell-matrix-interactions (adherence test) were investigated. cell-matrix interactions were tested with an adhesion assay using collagen i (c-i), collagen iv (c-iv), fibronectin (fn) and laminin (lm) as ecm compounds. in our in vitro models we used hepg2 as a hcc tumor cell line and the fibroblast cell line fi301 as stroma simulation. additionally caco2 cells were used, a colon carcinoma cell line representing colorectal liver metastasis. the toxicity of snake venom on liver cancer related cell lines was determined in the range of 0.01 -100 µg/ml and plotted into dose response curves. the noaels were calculated from these dose response curves: vp: 0.5 µg/ml -cr: 1 µg/ml -es: 5 µg/ml. performance of the caco2-transwell permeability assay revealed no influence of the tested venom concentrations on the integrity of the cellular arrangement. investigations for integrin inhibition revealed that the venom from vp reduced adherence on lm coated plates and the venom of ec reduced adherence on lm and fn coated plates compared to untreated cells. there was no effect on the adherence on any matrix from the venom of cr observable. co-incubation of the snake venoms of vp and es (below or near noael concentrations) with 5-fluorouracil (5fu), which is used as a chemotherapeutic agent, caused a reduction of its ic50 values. the results indicate that components of vp and ec inhibit the formation of cell-matrixinteractions possibly acting as disintegrins. the co-incubation experiments demonstrated a synergistic effect of 5fu and snake venoms. further experiments should enable the isolation of therapeutic active venom compounds, identification of disintegrins and their role in synergistic mechanisms in liver cancer therapy. modulation of the blood-brain barrier with peptidomimetics to improve drug delivery s. dithmer after decades of research, the blood-brain barrier (bbb) still remains a major problem for successful delivery to the brain for the vast majority of drugs. the main component forming the bbb is the brain microvascular endothelium. the paracellular permeation is limited by tight junctions (tjs), a multiprotein complex composed of the members of the claudin family claudin-1, -3, -5, -12. claudin-5 is known to be the key tj protein tightening the bbb. therefore, claudin-5 has been selected as target to modulate the bbb. for this reason, drug enhancer peptides (peptidomimetics) were designed to modulate transiently claudin-5 and, thereby, permeabilize the bbb. by combining biochemical protein/peptide interaction and tissue culture methods, we identified, validated and optimized peptide sequences modulating claudin-5 containing barriers. the claudin-5 targeting peptides decreased the transcellular electrical resistance and increased the permeability through mdck-ii cell monolayers stably expressing yfpclaudin-5 and immortalized brain endothelial cells (bend.3). the peptides decreased the amount of claudin-5 and zo-1 at cell-cell contacts and changed the cell morphology from spindle-shaped to more round-shaped. all tested peptides showed no signs of toxicity on cell cultures and in vivo (intravenous injection). permeability measurements in mice proved enhanced permeation of na-fluorescein (376 da) through the bbb, which was confirmed by magnet resonance imaging of contrast agents (gd-dtpa, 547 da). in summary, we identified new peptides with the potential to enhance cerebral delivery of small molecules through the bbb. treatment of cerebral diseases is limited by the capability of pharmacologically active agents to penetrate the blood-brain barrier (bbb). this paracellularly tight diffusion barrier is formed by brain capillary endothelial cells. the paraendothelial cleft is sealed by tight junctions (tjs), a multiprotein complex. cerebral tjs predominantly consist of claudin-5 (cldn5) which tightens the bbb for molecules <800 da. consequently, cldn5 is a potential target for transient and size-specific modulation of the bbb to improve cns penetration for pharmaceutically active agents. in high throughput screening using a cldn5 assay, the barrier opener 1 (bo1) was identified as a cldn5 modulator. initially, a significant removal of cldn5 from the plasma membrane was shown by confocal microscopy using epithelial and endothelial cell lines. measurement of transcellular electrical resistance and of paracellular permeability using lucifer yellow (mw 521 da) demonstrated the effect of bo1. concentration dependent treatment (50-150 µm) of cell monolayers with bo1 reduced tightness of the tjs between some hours and 24 h. applying 2-hydroxypropyl-ß-cyclodextrin as a solubilizer, opening activityof bo1 became detectable in mice. due to short stability (< 2 h) of bo1 in the bloodplasma repeated administration (1.5 mg/kg i.v.) was required to induce significantly increased permeability of the bbb for na-fluorescein(mw 376 da). the small molecule bo1 is a promising new approach for transient opening of the bbb in vivo. further modification of the stability and solubility of bo1 is necessary to optimize its applicability. the complex of tight junction (tj) proteins is located between opposing epithelial or endothelial cells. tjs restrict the paracellular permeation of ions and other solutes. tricellulin (tric) tightens tricellular tjs (ttjs) and regulates bicellular tj (btj) proteins like claudins and occludin (occl). current data suggest an important role of ttjs at the blood-brain barrier (bbb). a main pharmacological problem is modulation of the bbb to improve drug delivery to the cns. therefore, tricsi has been developed as a peptide taken from tric to open tissue barriers specifically and transiently.initially, a recombinant protein was generated based on a sequence of an extracellular loop of tric, tagged with maltose binding protein. the fusion protein caused down-regulation of tric, internalization of both tric and occl (confocal laser scanning microscopy), and a significant decrease in transcellular electrical resistance (ter) of a human epithelial colorectal adenocarcinoma cell line. then, studies with the synthetic peptide tricsi indicated its capacity of cell barrier openingafter about 16 h of incubation with concentrations varying from 100 to 150 µmaffecting the membrane localization of tricand occl. barrier opening was proven by decreasing ter, increasing permeability coefficient of lucifer yellow (457 da) and fitc-dextran (10 kda); the localization of tric elongated from ttjs towards btjs and cldn1 was weakened at btjs.physiochemical properties of tricsiexamined by circular dichroism spectroscopy suggested ß-strand structure and no helical propensity. taken together, a tric-derived peptide has been identified increasing the paracellular permeability of tissue barriers and redistributing the cellular localization of tj proteins. tricsi is a novel, promising tool to overcome cerebral barriers with the potential to improve drug delivery to the cns. further experiments are needed to better understand the role of tric in tissue barriers as well as to clarify the mode of action of tricsi. introduction: lung transplantation has become an established treatment option for a variety of end-stage lung diseases, but the long-term survival is often disappointing. the leading cause of death is generally chronic rejection which is characterized by inflammation and fibrous obliteration of the small airways, progressively leading to a reduction of the airflow. the mouse heterotopic tracheal transplantation model is widely used as an experimental model to study the development of obliterative airway disease. despite its widespread application, the heterotopic transplantation model does have a number of limitations, as for example the lack of airflow. the present study provides a description of the orthotopic tracheal transplantation mouse model, which shares more similarities with transplant situation in humans, and provides the analysis of airway obliteration via micro ct and histological evaluation. methods: a seven-ring donor trachea from balb/c mice was implanted into the recipient c57bl/6 mice. c57bl/6 mice without transplantation were used as normal controls. donor c57bl/6 mice to recipient c57bl/6 mice were served as the isograft group. 42 days after transplantation, mice were scanned using an in vivo small animal µct (skyscan 1176). tracheal tissue was harvested and fixed in formalin, embedded in paraffin, cut and stained with hematoxylin and eosin (h&e) as well as sirius-red/fast-green. results and conclusions: histologic evaluation showed luminal narrowing with subepithelial inflammatory cell infiltrates and fibrosis, as well as partially damaged and flattened epithelium. the aerated volume of the allogeneic grafts, analyzed by micro ct was significantly reduced compared to the isogenic control grafts and normal controls. non-invasive imaging via micro ct may offer an option for longitudinal monitoring of the progression of obliterative airway disease as well as response to treatment. c. elegans is a well-established model organism to study the aging process as well as effects of various substances in vivo. its lifespan is regulated by multiple signaling pathways (e.g. insulin or mtor signaling), which are well conserved up to humans. the insulin/igf-1 pathway was the first pathway shown to effect ageing in animals. mutations that decrease the activity of daf-2 (igf1r) lead to a significant increase of lifespan accompanied by a decrease of age pigment accumulation in c. elegans. the relevant effector of the insulin/igf-1 pathway is the transcription factor daf-16 (hfoxo3a). inhibition of hmg-coa reductase (enzyme of mevalonate pathway) by statins, which are frequently used as cholesterol-lowering agents in the clinic, has been shown to attenuate protein prenylation and glycosylation. notably, prenylated-, membrane-bound small gtp-binding proteins are important for the regulation of the afore mentioned age-related signaling pathways like the insulin/igf-1 pathway. recently, a cohort study showed that a decreased mortality rate in humans between age 78 -90 correlates with statin treatment, but is independent of total cholesterol levels. as c. elegans harbors the mevalonate pathway, but the branch leading to cholesterol synthesis is missing, it is a well-suited model to study cholesterol -independent effects of statins on aging-associated phenotypes and the underlying molecular mechanisms. here, we show that exposure of c. elegans to statins substantially decelerated the accumulation of age pigments. while the level of age pigments roughly doubled in control animals, there was only a slight increase in the lovastatin group. the use of atorvastatin gave comparable results indicating a more general effect of the inhibition of the hmg-coa reductase. the retarded accumulation of age pigments could be partly phenocopied using an inhibitor of the small gtpase rac1 or using rnai against the hmg-coa reductase. a reduced level of age pigments is prognostic for an elevated mean lifespan (about 20%) in c. elegans. a post reproductive treatment with lovastatin, mimicking the use of statins in patients of advanced age increased the mean lifespan in c. elegans even further. in addition, we could show a mild reduction of fertility and a developmental delay as well as a marked increase in acute thermal stress resistance mediated by lovastatin. besides the reduced accumulation of age pigments and the increased lifespan these are phenotypes which are usually observed under accumulation of daf-16 overactivity. consequently we found an increased nuclear localization of daf-16 in the presence of lovastatin and lovastatin completely failed to reduce age pigments in a daf-16-ko mutant background. rt-qpcr brought jnk-1, a known activator of daf-16, into play as a possible effector induced by statins. this is currently under investigation. in summary, statin exposure induces a longevity phenotype in c. elegans, which might be daf-16 dependent. this findings indicates that a product of the mevalonate pathway might influence the insulin/igf-1 pathway and particularly the transcription factor daf-16. the high-fat diet (hfd)-fed, streptozotocin (stz)-treated rat model is one of the experimentally-induced animal models of diabetes. this model is often used to evaluate the antidiabetic activity of several agents. according to srinivasan et al. (2005) , prolonged exposure of high-fat diet leads to insulin resistance, and the development of diabetes occurs only in insulin-resistant hfd-fed rats following low dose stz, because the hfd-fed rats are already mildly hyperglycemic due to insulin resistance (1). in hfd/stz model, the rats are fed with high-fat diet for 2-4 weeks or for a relatively long time (≥ 3 months) in order to simulate the insulin resistance and/or glucose intolerance. after induction of diabetes with multiple or single low-dose of stz (30-35 mg/kg), some of the diabetic rats receive treatment (2) . in this way, the impact of treatment can be determined by comparing the differences between groups. despite the lack of methodological information concerning the feeding time in some studies, all rats should be allowed to continue to feed on their respective diets until the end of the study. but what would happen if the hfd was switched to normal pellet diet in these diabetic rats? in our experience, the feeding of npd for 4 weeks significantly decreased fbg in diabetic rats compared to hfd-fed diabetic rats (234.40 ± 42.71 mg/dl vs. 464.00 ± 23.88 mg/dl, p < 0.05). although diet regulation could not restore normal blood glucose, such a decrease was unexpected. in addition, the body weights of the npd-fed diabetic rats were significantly lower than the body weights of the hfd-fed diabetic rats (249 ±6.00 gvs. 288.00 ±4.41 g, p < 0.05). there was no significant difference in body weight between nondiabetic control rats and diabetic rats fed npd for 4 weeks. further details can be found in table 1 . diet regulation and weight loss may prevent, control and reverse diabetes. however, at later stages of the disease, it is difficult to improve blood glucose control without medication, because the disease progresses from insulin resistance to insulin deficiency (3) . according to some diabetes researchers, the amount of residual functional betacells mass is an important issue, and another important question is whether hfd/stz rat mimics an early or late stage of type 2 diabetes (4). these preliminary findings suggest the possibility that hfd/stz rat model may simulate the characteristics of early stage more than the final stage of type 2 diabetes, and hyperglicemia in the experimental model can partially reverse with diet regulation. references: 1. srinivasan, k., viswanad, b., asrat, l., kaul, c. l., ramarao, p. (2005) . combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. pharmacol res 52 (4): 313-320. 2. oztürk z, gurpinar t, vural k, boyacıoglu s, korkmaz m, var a. (2015) . effects of selenium on endothelial dysfunction and metabolic profile in low dose streptozotocin induced diabetic rats fed a high fat diet. biotech histochem 90 (7): 506-515. 3. franz, m. j. (2007) . the dilemma of weight loss in diabetes. diabetes spectr 20 (3) animal models are pivotal for studies of pathogenesis and treatment of movement disorders. dystonia, characterized by sustained or intermittent muscle contractions causing twisting movements/postures, is regarded as a basal ganglia disorder. the pathophysiology is however poorly understood. in mouse models of dyt1 dystonia, which is caused by a gag deletion in tor1a that encodes for the protein torsin a, ex vivo electrophysiological studies have shown an abnormal d2 receptor mediated release of acetylcholine from striatal interneurons. in these models, which do not exhibit a dystonic phenotype, the functional relevance of the increased d2 receptor mediated acetylcholine release has not been examined yet. the aim of present study was to (1) generate more powerful tests to detect behavioural alterations in the dyt1 knock-in mouse and to (2) examine the behavioral effects of the d2 receptor agonist quinpirole. for this purpose, a sequence of cognitive, motoric and sensorimotor tests were performed in this mouse model. only the adhesive removal test that explores sensorimotor connectivity revealed significant impairments in the dyt1 knock-in mice compared to controls. to induce a more characteristic and stronger phenotype, the "rotating beam test" was developed. this motoric test measures motor coordination and balance. interestingly, dyt1 knock-in mice showed significant motor deficits in the rotating beam test. based on these results, the acute effects of quinpirole (0.25 -1 mg/kg i.p.) were tested in dyt1 knock-in and wildtype mice. subsequent to the injections, mice were tested in the open field, the rotating beam test and the adhesive removal test, respectively. in the open field test, dyt1 knock-in mice showed increased thigmotaxis at a dose of 0.5 mg/kg quinpirole. in the rotating beam test, both groups showed a dose-dependently reduced performance. in the adhesive removal test, quinpirole improved the reaction time in dyt1 mice independently of dosage, while no effects were observed in the wildtype littermates. however, in vehicle follow-up (post-drug control), this effect remained consistent in the dyt1 model, suggesting a habituation effect. in conclusion, we generated a new test, i.e., the rotating beam test which improves the detection of mild motor impairments in dyt1 knock-in mice. furthermore, the adhesive removal test revealed sensorimotor dysfunctions in this animal model. these results represent an important step for our ongoing optogenetic examinations on the role of abnormal neuronal plasticity in dyt1 dystonia and for pharmacological studies. the first data on the effects of quinpirole do not indicate a critical role of d2 dysregulated acetylcholine release, but this has to be clarified by ongoing local striatal injections of quinpirole and by pharmacological manipulations of the cholinergic system. renal fibrosis is characterized by decreased nitric oxide (no) bioavailability and pronounced transforming growth factor β (tgfβ) signalling subsequently excessive extracellular matrix (ecm) deposition. here, the effects of the soluble guanylate cyclase (sgc) stimulator bay 41-8543 after unilateral ureter obstruction (uuo) have been studied. kidney fibrosis was induced by unilateral ureter obstruction (uuo) in wild type (wt) and cgki knock-out (cgki ko) mice. starting one day after uuo, the sgc stimulator bay 41-8543 was (4mg/kg/daily) i. p. injected for seven days. biomarkers indicating remodelling processes in the kidney were analysed via mrna expression and protein expression. bay 41-8543 administration influenced the activity of the ecm degrading matrix metalloproteases (mmp2 and mmp9) and their inhibitor timp-1, the expression pattern of extracellular matrix proteins (e.g. collagen and fibronectin) of profibrotic mediators (e.g. connective tissue growth factors (ctgf) and plasminogen-activator inhibitor-1 (pai-1)) and the secretion of cytokines, e.g. il-6. thereby, bay 41-8543 increments the cgmp pool among others via modulation of endothelial no synthase (enos) expression. agents, which enhance no and cyclic guanosine monophosphate (cgmp) ameliorate the progression of fibrotic tissue. however, the molecular mechanism by which cgmp via cgki affects the development of kidney fibrosis has not fully been elucidated. accordingly, the present study investigates the functional role of sgc stimulation in regulating the fibrotic process, the signalling pathway and the underlying mechanisms involved. we hypothesize that the antifibrotic potential of bay 41-8543 might be related to the increased cgmp pool and the inhibition of the mapk and smad signalling pathway. the elucidation of the signalling allows the development of new therapeutic options. infection of mice with listeria monocytogenes (lm) results in a strong t-cell response that is critical for an efficient defense. here, we demonstrate that the adapter protein sly1 (sh3-domain protein expressed inlymphocytes 1) is essential for the generation of a fully functional t-cell response. the lack of sly1 leads to reduced survival rates of infected mice. the increased susceptibility of sly1 ko mice was caused by reduced proliferation of differentiated t cells. ex vivo analyses of isolated sly1 ko t cells displayed a dysregulation of forkhead box protein o1 (foxo1) shuttling after tcr signaling, which resulted in an increased expression of cell cycle inhibiting genes, and therefore, reduced expansion of the t-cell population. foxo1 shuttles to the cytoplasm after phosphorylation in a protein complex including 14-3-3 proteins. interestingly, we observed a similar regulation for the adapter protein sly1, where tcr stimulation results in sly1 phosphorylation and sly1 export to the cytoplasm. moreover, immunoprecipitation analyses revealed a binding of sly1 to 14-3-3 proteins. altogether, this study describes sly1 as an immunoregulatory protein, which is involved in the generation of adaptive immune responses during lm infection, and provides a model of how sly1 regulates t-cell proliferation (schäll et al., eur j immunol. 2015) . the catalytical isoforms p110γ and p110δ of phosphatidylinositol-4,5-bisphosphate 3kinase γ (pi3kγ) and pi3kδ play an important role in the pathogenesis of asthma. two key elements in allergic asthma are increased eosinophil and ige levels. whereas dual pharmacological inhibition of the catalytical subunits p110γ and p110δ reduces asthmaassociated eosinophilic lung infiltration and ameliorates disease symptoms, it has been shown that dual genetic deficiency in pi3kγ and pi3kδ in p110γ ko δ d910a mice increases serum ige and basal eosinophil counts in mucosal tissues and blood. this suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. here we analysed p110γ/δ -/mice and determined ige and eosinophil counts in a basal state and the immune response to ovalbumin (ova)-induced allergic asthma. we found that serum concentrations of ige, il-5 and eosinophil numbers in blood, spleen and bone marrow were significantly increased in p110γ/δ -/mice in comparison to single knock-out (ko) and wildtype (wt) mice. nevertheless, p110γ/δ -/mice were protected against ovainduced infiltration of eosinophils, neutrophils, b cells and t cells into the lung tissue and the bronchoalveolar space. moreover, p110γ/δ -/mice, but not single ko mice, showed a reduced bronchial hyperresponsiveness as measured with the isolated and perfused lung. we conclude that although the dual deficiency of p110γ and p110δ causes eosinophilia and ige hyperproduction, p110γ/δ -/mice are not prone to develop ovainduced allergic asthma. an increase of plasma extravasation induced by activation of constitutively expressed endothelial bradykinin type 2 receptors (b2) has been shown to contribute to the development of angioedema occurring as a sometimes life-threatening side effect of angiotensin-converting enzyme inhibitors such as enalapril (new engl j med 2015:372; 418-425) . these drugs inhibit the degradation of bradykinin and increase its vascular steady-state concentration. hence, it is reasonable to assume that bradykinin may destabilise the endothelial barrier, i.e. may increase physiologic extravasation. while the commonly used miles assay provides a useful and relatively easy tool to study the effect of permeabilizing mediators in-vivo, it does not distinguish between intravascular and interstitial evan's blue dye. likewise, extravasation can only be quantified at one particular time point per animal, usually 20-30 min. furthermore, evaluation of physiologic extravasation is not possible. in contrast, non-invasive twophoton laser microscopy may allow separating the intravascular from the interstitial compartment and thereby investigations of changes of the physiologic endothelial barrier induced by drugs or transgenes. therefore, we have evaluated this methodology for its suitability to study endothelial permeability in mice in vivo. to establish this, we used two different fluorescent dyes of different molecular weight. a 200,000 kda dextran equipped with a green fluorescent chromophore which cannot leave vascular lumen was injected intravenously to visualize small dermal blood vessels of the mouse ear located approximately 200 µm below the surface. after stabilization of the green fluorescent signal, a 10,000 kda dextran equipped with a red fluorescent chromophore which easily traverses the endothelial barrier was applied by intravenous injection. the red fluorescence permeates into the interstitium during physiologic extravasation and accumulates in the interstitial space. this process can be followed by measuring the decrease of intravascular red fluorescence over various time periods. using this methodology we have studied whether endothelial-specific overexpression of b2 changes physiologic endothelial permeability. this newly developed transgenic mouse line (b2 tg ) was established using a plasmid consisting of pbluescript ii sk+ -vector, the tie-2-promotor, the human b2 cdna, the sv40 poly-a-signal and a tie-2 intron fragment. we observed that b2tg showed a significantly stronger extravasation than their transgene negative littermates as evidenced by the more rapid extravasation of the 10,000 kda dextran at each time point (fig. 1) .we conclude that two-photon laser microscopy is suitable to study endothelial permeability non-invasively in-vivo and that this methodology allows to study the effects of drugs and transgenes on the endothelial barrier under non-inflammatory conditions. furthermore, our results suggest that endothelial-specific overexpression of b2 increases physiologic extravasation. non-allergic angioedema such as angioedema induced by angiotensin converting enzyme inhibitors (acei) develops as a consequence of increased activation of bradykinin receptor type 2 (b2). using a plasmid consisting of pbluescript ii sk+ -vector, the tie-2-promotor, the human b2 cdna, the sv40 poly-a-signal and a tie-2 intron fragment a transgenic mouse line harbouring an endothelial-specific overexpression of b2 was generated and backcrossed to c57bl/6 for more than 10 times (b2 tg ). lung mrna using primers specific for the human or the mouse b2 cdna revealed a 12.5-fold stronger expression of human b2 in b2 tg (n=6), while the expression of murine b2 mrna was unchanged and similar to transgene negative littermates (b2 n ). we have evaluated the specificity of several antibodies directed against b2 and found that a rabbit monoclonal anti b2 antibody appears to be reliable, i.e. there was just a faint staining in lung tissue of b2 -/mice. however, this antibody primarily stains rodent b2 and has only little cross-reactivity to human b2. hence, we were not able to detect a significant increase of b2 protein in tissues of b2 tg . previous experiments have shown that bradykinin induced concentration-dependent constrictions of aortic rings with a maximal effect at 1 µm of bradykinin. the contraction due to bradykinin was completely inhibited by icatibant or diclofenac indicating that it is mediated by endothelial b2 activation and dependent on cyclooxygenase activity. in striking contrast to their transgene negative littermates b2 n , we found a significant icatibant sensitive aortic dilation in b2 tg following preincubation with diclofenac which indicates functional overexpression of b2 in conductance vessels of b2 tg . to evaluate whether this applies to dermal micro vessels we used the miles assay to quantify dermal extravasation of the albumin-bound dye evans blue following intradermal injection of 30 µl of either vehicle, bradykinin, labradimil and histamine (control). increasing concentrations of bradykinin caused a significant increase of extravasation reaching 4.41±0.11 fold at 18.9 nmol bradykinin in c57bl/6 (n=6 each, p<0.0001 vs. vehicle). a similar increase was found in b2 n (4.45±0.25 fold, n=7, p<0.0001 vs. vehicle), while there was a stronger response in b2 tg (5.50±0.16 fold, n=7, p<0.0001 vs. vehicle) which was significantly different to b2 n (p<0.01) and c57bl/6 (p<0.01). in another set of experiments the specific and ace-resistant b2 agonist labradimil (1.89 nmol) was used instead of bradykinin. labradimil increased extravasation by 3.736±0.121 fold in c57bl/6 (n=6 each, p<0.0001 vs. vehicle), by 4.51±0.11 fold in br2 n (n=7 each, p<0.0001 vs. vehicle) and 4.88±0,21 fold in b2 tg (n=6, p<0.0001 vs. vehicle) which was significantly different to c57bl/6 (p<0.01) but not to b2 n (p>0.05). the effects of bradykinin and labradimil were largely blocked by 10 nmol icatibant (i.v.) in c57bl/6, b2 n and b2 tg mice (p<0.0001) and hence mediated by activation of b2. these data suggest that overexpression of b2 in b2 tg is functionally active in endothelial cells of large conductance and small dermal vessels. therefore, b2 tg represents a new animal model suitable for cardiovascular and non-allergic angioedema research. pharmacokinetic pharmacodynamic modeling of irreversible effects: the rituximab example f. keller 1 1 universitätsklinikum, innere 1, nephrologie, ulm, germany background: for pharmacokinetic-pharmacodynamic modeling usually the sigmoid emax model is used as described by the hill equation. however, treatment regimens exist where the effect is only exerted as long as the drug concentration increases whereas decreasing concentrations produce no longer an effect. examples are the pulse anti-cancer therapy such as originally proposed by the devita protocols. methods: here, the new model for irreversible drug action is derived from the time dependent change of the concentration that must be larger than the time-dependent growth of the number of target cells (tumor or bacteria). the irreversible effect can be assumed if the is no further growth of the target cells occurs. de/dt = + dc/dt -dn/dt dn/dt = 0 a solution for the above differential equation can be obtained by use of the integral exponential function iec based on the euler-mascheroni constant (gamma = 0.5772 …). this model of an irreversible effect was applied.to the example of rituximab where the initial effect on cd19+ and cd20+ b-cells completely persists for 6 month. to obtain a numerical solution, the following parameters are needed to be determined: the target concentration ctarget = 100 mcg/ml and the infusion time t = 2 hours. results: it can be shown that a plausible result for rituximab can be estimated only under the condition that a short distribution half-life is assumed of t1/2 = 2 hours (not shorter than the time t of infusion). with the terminal half-life of 460 hours no plausible solution is obtained. under these conditions two observations are made: there is a negative effect both, initially for low concentrations and after cessation of the infusion when concentrations decrease (in reality this means no effect in both cases). the irreversible effect is proportional to the target concentration. the shorter the half-life comes out relative to the infusion time (t1/2 < t) the stronger is the effect (figure) . occasionally there are specific questions occurring on the ruminant xenobiotic metabolism: 1) are the observed metabolites ruminant specific and formed directly in the rumen? 2) are ruminants able to cleave plant specific metabolites like glycosides to the respective aglycon? in the past new additional in vivo goat metabolism studies with at least one animal were performed. the aim of the project was to elucidate an alternative in-vitro method to replace the existing in vivo method in order to address robustly specific questions on xenobiotic metabolism in ruminants for registration of ppp. fresh sheep rumen fluid was incubated in-vitro >7 days by using rumen simulation technology (rusitec). the conservation of the physiological conditions were proven by measurement of ph (~ph 6.6) and redox potential (~-300 mv). the microflora composition and their viability (bacteria, protozoa and fungi) of the rumen were monitored by microscopy, incubation on agar plates and performing several viability tests (e.g. glycosidase-test, nh3 and short fatty acids). all the tests showed that the rusitec is a successful tool to maintain sheep rumen fluid for at least 7 days in -vitro. the metabolic behavior and performance of the rumen fluid was tested by e.g. incubating 14c-triazol derivative metabolites (tdm) like triazol-alanin (ta); triazol-acetic-acid (taa) and triazol-lactic-acid (tla), which are usually formed in plants after application of triazol-containing fungicides. it was shown that ta was cleaved within 72 h to 1.2.4.-triazol, while taa and tla were stable under these conditions. these data are in a good accordance with available in vivo data in cows. moreover glycosides (12c-polydatin, octyl-14c-β-d-glucopyranosid) were cleaved within 1 hour completely. all these data show, that the rumen fluid maintained its metabolic performance by using rusitec. basf identified the rusitec method, which is usually used in different areas (e.g. investigation of methane production in-vitro) as suitable and adapted this method for the purpose of investigation of ruminant xenobiotic metabolism. it was shown that rusitec is a robust method to analyze rumen xenobiotic metabolism and therefore can clearly substitute in vivo animal studies on ruminant metabolism studies beyond oecd503 and contribute significantly to animal welfare (3r: replacement). results: by using the training set, physicochemical (e.g. lipophilicity) and pharmacokinetic characteristics of mtx (e.g. v max for active tubular secretion) were slightly adjusted. using the gfr formula of morris et al. (1982) and including an empiric correction factor, mrd for the training set was 2.49 whereas bias was 2.80 µmol/l. by applying the developed pbpk model to the test set the respective values were mrd=3.92 and bias=1.43 µmol/l. for the covariates "at least one potentially interacting co-medication" and "trimethoprime/sulfamethoxazole" a significant impact on the prediction quality was found. conclusions: using the developed pbpk-model, a good prediction of the pharmacokinetics of hd mtx in severely ill children was found. by including additional factors influencing the prediction of mtx characteristics (e.g. co-medications) an improved prediction of mtx-sl might be reached. in prospective clinical trials, those more complex models should be evaluated and might be helpful to predict hd mtx pharmacokinetics and reduce unwanted side effects. hypericin is a natural polycyclic quinone found in hypericum perforatum. although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound, presumably, because hypericin is a highly lipophilic compound which is poorly soluble in physiological medium. recently we have shown that quercitrin and isoquercitrin, but not hyerosid, quercetin or rutin increased the uptake of hypericin in caco-2 cells. the major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the caco-2 cell system under different experimental conditions. the permeation characteristics of hypericin (5 µm) in absence or presence of hypericum extract 145, 62.5 µg/ml) were studied in the absorptive direction. following application of hypericin to the apical side of the monolayer only negligible amounts of the compound were found in the basolateral compartment. the amount of hypericin in the basolateral compartment increased concentration-dependently in the presence of the extract (from 0 to 7.5 %). the majority of hypericin was found after cell extraction (44% in absence and 76% in presence of the extract). the recovery was in the range of 90 %, and significant amounts of hypericin found after cell extraction. fluorescence microscopy and imaging analysis revealed that hypericin is mainly accumulated in the cell membrane. the precise mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated. however, our experiments demonstrated that the permeation characteristics of hypericin significantly improved in presence of the extract. background: the combination of gamithromycin (gam), a novel drug with the big advantage of a once weekly administration, and rifampicin (rif) is used in the treatment of lower airway disease in foals. both are effective in the therapy of infections with rhodococcus equi, a gram-positive coccobacillus bacterium, which is known to survive and reproduce within alveolar macrophages. macrolides are combined with rif to prevent resistance developing with single agent therapy. both drug classes reach high concentrations in the lung, penetrate into phagocytes and kill intracellular pathogens. methods: a controlled, single-and multiple dose study with four-periods was conducted in 10 healthy foals (5 ♂ and 5 ♀, age: 42-63 days, body weight: 100-177 kg) which were treated once with rif alone (10 mg/kg s.i.d., p.o., a) followed by the administration of gam (6 mg/kg once weekly, i.v., b) for 3 weeks. study period 3 ("rif-gam acute", c) includes the administration of gam and rif for 7 days with an administration interruption after the first rif dose for blood sampling. for the last study period ("rif-gam chronic", d) both gam and rif were coadministered for 2 weeks. all periods were completed with blood sampling for pharmacokinetic analysis for 48 ( . rif is also accumulated in the lung, but to a much lower degree than gam (elf/c 24 h : 1.2 ± 0.5; balc/c 24 h : 2.01 ± 1.24). conclusion: pharmacokinetic data of the present study provides surprising results. in previous studies coadministration of clarithromycin and rif show a dramatic decreased plasma exposure of the macrolide, whereas the balc/c 24 h -ratio was unaffected (peters et al. 2011) . in contrast, systemic exposure of gam increase significantly in case of the combined therapy and the balc/c 24 h -ratio was nearly halved. both macrolides have in common, that they are intensively accumulated in the lung (elf << balc). at the moment there is further research required (e.g. in vitro studies) for a better understanding of the very interesting in vivo data. 1 1 institut für klinische pharmakologie, göttingen, germany background and aim: desvenlafaxine is a selective serotonin and norepinephrin reuptake inhibitor, which is approved in the usa (but not in europe) for treatment of major depressive disorder. desvenlafaxine is the major active metabolite of the antidepressant venlafaxine. desvenlafaxin is produced by o-desmethylation via cyp2d6. direct administration of desvenlafaxine should bypass the variability in venlafaxine pharmacokinetics caused by the highly polymorphic cyp2d6. however, desvenlafaxine is less lipophilic than venlafaxine and may require carrier-mediated transport to penetrate cell membranes. based on our in vitro data, desvenlafaxine is a substrate of the hepatic organic cation transporter oct1 and common genetic polymorphisms abolished desvenlafaxine cellular uptake. about 9% of caucasians are compound heterozygous carriers of loss-of-function oct1 polymorphisms. therefore, oct1 polymorphisms may cause substantial inter-individual variability in the hepatic uptake and plasma concentrations of desvenlafaxine. in this study we evaluated the influence of genetically-determined loss of oct1 function on the pharmacokinetics and pharmacodynamics of desvenlafaxine. primary aim was dependence of desvenlafaxine plasma concentrations (represented by auc as primary endpoint) on the number of active oct1 alleles. methods: 50 mg desvenlafaxine (pristiq®) was orally administered to 41 healthy subjects preselected according to their oct1 genotypes. oct1*1 allele was regarded full active, oct1*2 to *6 alleles were regarded loss of function. plasma concentrations of desvenlafaxine and its main metabolite didesmethylvenlafaxine were quantified in plasma sampled up to 60 hours after administration using lc-ms/ms. pupillographic measurements were performed as possible surrogate markers for desvenlafaxine pharmacodynamics. results out of the 41 subjects 14 carried two active, 13 one active, and 14 zero active oct1 alleles. age, height and weight were 26.9 ± 6.4 years (mean ± standard deviation), 1.75 ± 0.11 m and 70.9 ± 12.1 kg with no significant differences among the oct1 genotypes. there were strong variations in the pharmacokinetics of desvenlafaxine and its metabolite didesmethylvenlafaxine. the auc 0-infinity of desvenlafaxine varied between 52.8 and 282.2 min*mg/l and auc 0-infinity of didesmethylvenlafaxine between 3.5 and 30.7 mg*min/l. however, neither desvenlafaxine nor didesmethylvenlafaxine pharmacokinetics significantly differed among the three oct1 genotypes. concerning pharmacodynamics of desvenlafaxine, pupil diameters at maximal constriction after a standardized light exposure were on average 14% greater around the time of t max than before administration. in line with the pharmacokinetic results there were no significant differences in maximal constriction or other pupillographic parameters among the oct1 genotype groups. conclusions: our results suggest that oct1 genotype does not affect the pharmacokinetics of desvenlafaxine and therefore no dose adjustment in respect to oct1 genotype should be considered. other factors like renal transporters or polymorphic glucuronidation may explain the great variability in desvenlafaxine pharmacokinetics. background: cardiovascular disorders and medication are highly prevalent in elderly (1). due to age related changes in the body, the elderly are particularly vulnerable to side effects and adverse drug reactions. some psychotropic drugs are linked with reports of cardiac side effects. additionally, some cardiac drugs may also cause psychiatric symptoms. of these, angiotensin converting enzyme inhibitors, beta blockers, methyldopa and calcium channel antagonists can induce or exacerbate symptoms of depression (2) . the aim of this study was to provide information on the concomitant use of cardiovascular drugs among elderly patients who took psychotropic medication. methods: we conducted a single-center, retrospective study between september 2013 and december 2013 using the medical records of elderly patients (≥65 years of age) admitted to basin sitesi polyclinic, izmir ataturk research hospital, turkey. demographic characteristics of patients, diagnoses, prescription drugs were evaluated, and spss 16.0 statistical software was used for data analysis. number, percent, mean and standard deviation were used as descriptive statistics. results: a total of 541 elderly patients with psychiatric disorders were identified. one in four patients receiving psychotropic medication took at least one cardiovascular agent concomitantly (n=135). median age was 72 (min:65, max:98), 84 patients were female (62.2%). according to medical records of 135 patients, the most commonly used drugs were escitalopram, sertraline, mirtazapine, quetiapine, mianserin and risperidone. the proportion of the concomitantly use of cardiovascular drugs was higher among the patients who took more than one psychotropic drug (69.6% vs. %30.4) compared to patients taking psychotropic monotherapy. a higher percentage of women used diuretics (65.4% vs. 33.3%) and angiotensin receptor blockers (36.9% vs. 23.5%) concomitantly with psychotropic drugs when compared to men. the proportion of men using angiotensin-converting enzyme inhibitors and lipid-modifying agents was higher than women (58.8% vs. 38%, 68.6% vs. 20.2%, respectively). conclusions: the world's population is ageing rapidly. according to world health organization, over 20% of elderly suffer from a psychiatric or neurological disorder. our data showed that use of cardiovascular drugs among elderly patients with psychiatric disorders was extensive. the effects and interactions of these drugs should be discussed and carefully evaluated before starting treatment in the elderly. further studies focusing on drug use in elderly will increase the success in geriatric pharmacotherapy. since the adoption of the ich e14 guideline [1], the thorough qt (tqt) study has become a standard element of clinical drug development. however, with the iq/csrc study [2] the ability to detect qtc-prolongations of about 10 ms in a phase i setting has been demonstrated. as a consequence, regulatory agencies have begun to grant waivers for a tqt study based on negative qt findings obtained from first-in-man studies. a concentration-response model is the key tool that gives sufficient power to an analysis based on data from single or multiple ascending dose studies. this power has been investigated in subsampling studies that simulate situations comparable to those encountered in first-in-man studies [3, 4] . other topics to be addressed are the assurance of sufficient quality of the ecgs obtained, in particular in doses that cause adverse reactions, and a replacement for the active control that is part of a tqt study. if a model based statistical analysis is used for confirmatory inference, it must be specified in advance. this pre-specification includes tests to ascertain that the model assumptions are met and alternative methods to be used in case they are violated. in particular linearity and the absence of a hysteresis, i.e. a delay between the drug concentration and the observed qt effect need to be tested. this is an area of active research. in this contribution, i will share current experience from a statistical perspective, both based on real data and on simulated studies. i will also discuss critical points in the design of first-in-man studies that are intended to be used to obtain a waiver for a tqt study. human platelets express the g-protein-coupled angiotensin receptor-like-1 (apj) receptor. apj is activated by apelin, which is produced as pre-apelin and cleaved into several bioactive peptides such as apelin-12, -13 and -17. apelin and apj are expressed in a variety of tissues such as the heart, the vessel wall, several tumor types, and in platelets. to date, there is no description or a suggested function of the apelin/apj system in platelets to date. here, we investigate apj expression and function in human platelets. apelin and apj expression were determined in platelet-rich plasma from healthy donors by immunofluorescence, western blotting and flow cytometry. in a pilot study apelin and platelet apj expression were analyzed in 23 patients with nstemi, 4 stemi patients and 14 controls. here, platelet aggregation was analyzed by light transmission aggregometry (lta); platelet cd62 and apj expression by flow cytometry and circulating plasma apelin by elisa. in resting human platelets, apj receptor expression was observed predominantly in the outer cell membrane, as determined by immunofluorescence staining and flow cytometry. activation with a selective thrombin receptor-activating peptide (ap1) resulted in decreased apj protein levels determined by western blotting in platelet lysates compared to untreated controls. preincubation of platelets with different apelin isoforms for 30 sec to 5 min reduced platelet aggregation in lta studies by up to 20 % for apelin-17. this effect was inhibited by preincubation of the platelets with the enos inhibitor l-name (300 µm), suggesting the involvement of a no-dependent mechanism. in patients with myocardial infarction the expression of platelet apj was significantly reduced compared to the control group (56,84% ± 9,28 % in mi versus 100 % ± 19,35 %in controls; p = 0.029). this reduction in apj expression on platelets was accompanied by decreased plasma levels of apelin-17 in patients with mi (14.95 ± 0.6 pg/ml versus 16.98 ± 0.6 pg/ml; p = 0.035). interestingly, the decreased apj expression on platelets in mi patients significantly correlated inversely with the troponin t plasma levels (r = -0.46; p = 0.03). this may suggest an association of apj expression with lower plasma levels of troponin t and possibly tissue damage. in conclusion, our study shows for the first time the expression of apj and a possible function in human platelets. apj may act as an endogenous inhibitor of platelet aggregation in response to certain apelin isoforms, predominantly apelin-17. upon platelet activation, apj is internalized and surface expression is reduced by about 50 %. in mi patients, plasma levels of apelin-17 and platelet apj expression were reduced. this correlated inversely with troponin t levels. reduced circulating apelin-17 levels and platelet apj expression may be associated or partly account for platelet hyperactivity in mi patients. anticholinergic drug use, m 1 receptor affinity and dementia risk-a pharmacoepidemiological analysis using claims data f. thome background: dementia is characterized by cumulative cognitive decline and progressive inability for independent living. the lack of suitable therapies for terminating the progression of this disease underlines the importance of the detection of risk factors. anticholinergic drugs have been shown to enhance cognitive decline in the elderly. the classification of anticholinergic drugs according to their anticholinergic burden, however, is inconsistent. since cholinergic transmission is mainly mediated by the m 1 muscarinic acetylcholine receptor in the brain, we classified anticholinergic drugs from anticholinergic risk lists according to their affinity for the m 1 receptor subtype and calculated the risk for the onset of incident dementia. methods: our analyses are based on claims data of the public health insurance fund aok. data include information of inpatient and outpatient diagnoses and treatment from 2004 to 2011. inclusion criteria comprised the initial absence of dementia and age of 75 years or older in 2004. anticholinergic drugs were taken from three anticholinergic risk lists. the pdsp-database and literature search were used to define k i -values for the substances. hazard ratios were calculated using time-dependent cox regression including covariates like age, gender, and several comorbidities. results and conclusion: anticholinergic drug exposure increases the risk for dementia. we found that anticholinergics with small k i -values are at a higher risk than those with greater k i -values. furthermore, conventional risk factors for dementia (e.g. age, depression, stroke) could be confirmed. in conclusion, the intake of anticholinergic drugs increases the risk for incident dementia in the elderly. taking into account the m 1 receptor affinity may be a contribution in determining the anticholinergic load in view of the risk for incident dementia. safety signal detection in a large german statutory health insurance databasefirst results of a feasibility assessment f. andersohn 1,2 , s. schmiedl 3, 4 , k. janhsen 3, 5 , p. thuermann 3, 4 , j. walker background: during the last years, approaches to routinely screen health care databases based on electronic medical records or claims to identify drug safety signals were proposed. to evaluate the performance of such methods, reference sets of index drugs have been compiled consisting of (1) drugs with a known association to a certain adverse event (=positive controls) and (2) drugs without any evidence to cause this adverse event (=negative controls). the best possible signal detection method would identify a safety signal for 100% of the positive control drugs, and for none of the negative controls. ryan et al. 2013 developed drug reference sets for four adverse events of interest (acute myocardial infarction=ami, acute kidney injury =aki, acute liver injury=ali, and upper gastrointestinal bleeding=ugib) and have shown feasibility of using these reference sets in us health care databases. if the use of these us specific drug lists for evaluation of signal detection methods is also feasible within german health care databases, is unknown. aims: to evaluate if the drug reference sets developed by ryan et al. (drug saf. 2013 ;36 suppl 1:s33-47) could be used for testing signal detection methods in a large german statutory health insurance database. methods: data source was the health risk institute (hri) database, an anonymized healthcare database with longitudinal health insurance data from approximately six million germans. new users (initiators) of index drugs in 2010 to 2013 were identified and followed-up for one year from their first prescription. exposed person-time to the respective index drug was assessed to estimate for which of these drugs an increase in risk of 50% (relative risk 1.5) compared to the background incidence of the respective adverse event (ami, aki, ali or ugib) could be identified with 80% statistical power. results: from a total of 182 index drugs in the reference sets of ryan et al., 142 (78.0 %) were also available on the german drug market and were used by at least one insurant in the hri database during the study period. a total of 5,485,722 index drug initiators were included in the analysis. for a total of 16 index drugs, a relative risk of 1.5 could be detected with 80% power. the numbers of index drugs for each of the outcomes of interest were: ami (3 positive controls; 6 negative controls); aki (3 positive controls; 1 negative control); ugib (2 positive controls; 1 negative control). as the background incidence of ali was low, no positive or negative control with sufficient power was identified for this outcome. conclusions: using the set of reference drugs proposed by ryan et al., the number of drug-event pairs with 80% power to detect a relative risk of 1.5 was low, despite the magnitude of the database used. this may be attributable to differences of drug exposure in germany and the us. hence, an adaptation of the drug list to the german drug market and consumption data might be relevant for future evaluations of signal detection methods using german databases. correlation of sativex™ doses to steady state concentrations of 11-nor-9-carboxyadministration of the oromucosal spray sativex™ represents a therapy option for treatment of spasticity in patients with multiple sclerosis. sativex™ is an extract containing equal amounts of the cannabis-derived cannabinoids ∆ 9tetrahydrocannabinol (thc) and cannabidiol (cbd). in cases of cannabis abuse a long elimination half life of some thc metabolites is known. therefore, in patients receiving sativex™, the long elimination half life of these metabolites should allow a drug monitoring under conditions of steady state. due to the fact that immunologically based methods for thc determination are very common in medical chemistry, a monitoring might be simply performed even in patients under sativex™ therapy. in a preliminary observational study 11-nor-9-carboxy-∆ 9 -thc (thc-cooh) concentrations were measured with a commercial immunoassay in urine samples of 16 patients with multiple sclerosis obtaining sativex™. in addition, thc-cooh, thc, cbd as well as the hydroxy metabolites of thc and cbd were measured by gc/ms in urine and blood samples. using this analytical technique, only an excessive dosing (as compared to the declaration by the patient) can be detected. as a result of this approach, thc-cooh concentrations determined by the immunoassay were found not to correlate to the daily applicated amount of sativex™ as indicated by the patients (spearman rang order test: p > 0.05). two patients mentioned not to have taken sativex™ on the day the samples had been taken, and one patient predicated an additional cannabis abuse. in three patients the immunological thc-cooh determination was negative or nearly negative. interpretation of the data is hampered by the fact that an incorrect declaration of sativex™ applications by the patients cannot be excluded. introduction: learning analytics seeks to enhance the learning process through systematic measurement and analysis of learning related data to provide informative feedback for students and lecturers. however, which parameters have the best predictive power for academic performance remains to be elucidated. objective: to analyze the potential of different learning analytics parameters to predict exam performance in undergraduate medical education of pharmacology. methods and results: hypertext preprocessor (php) as server-side scripting language was used to develop a learning analytics platform linked to a my structured query language (mysql) database for storage and analysis of data (www.tumanalytics.de). the database consisted of 440 lecturer-authored multiple choice questions that were made available to a cohort of undergraduate medical students enrolled in a pharmacology course (winter term 2014/15) at technische universität münchen (tum). the course consisted of a 28-day teaching period, followed by a 12-day self-study period and a final written exam. students' assessment data of tumanalytics was collected during the self-study period and correlated to the individual exam results in a pseudonymized manner. a total of 224 out of 393 (57%) students participated in the study. the coefficient of multiple correlation (r) was calculated for different parameters in relation to exam results as a measure of predictive power. of different parameters investigated, the total score and the score of the first attempt in tumanalytics had the highest positive correlation with exam performance (abb. 1). no sex-specific differences were observed. summary and conclusion: in this study we systematically investigated the potential of different learning analytics parameters to predict learning outcome and exam performance. total score and score of the first attempt were identified as parameters with the highest predictive power. in conclusion, our study underscores the potential of learning analytics as valuable feedback source in undergraduate medical education of pharmacology. in educational settings, tests (e.g., written or oral exams) are usually considered devices of assessment. however, a recent and intriguing line of evidence from basic cognitive psychological research suggests that tests may not only help to assess what students know, but may also help to improve the learning and long-term retention of information. the goal of the present study was to apply such test-enhanced learning to pharmacological teaching. after the last lecture of a pharmacology class (n=194 3 rd -year medical students, n=213 4 th -year medical students: basic or clinical pharmacology, respectively), one week prior to the final exam, students were given the opportunity to voluntarily participate in online exam. because pilot work from previous semesters had revealed relatively low levels of participation in such formative exams, students were offered bonus points for (successful) participation as an incentive. the online exam consisted of 60 items (i.e., selected pieces of information from the lectures and seminars) and was provided on the e-learning platform ilias. twenty of the 60 items were presented as statements for restudy, 20 items were tested using single-choice questions, and 20 items were tested using short-answer questions. randomly a third of the students were assigned to different sets of questions. the summative final written exam for each group consisted of 30 single-choice questions, 15 questions of which had not been used before (as a standard of comparison). the remaining 15 questions of the final exam were taken from the previous online exam, but were slightly reworded to avoid ceiling effects. each of 5 of these reworded 15 questions from the final exam corresponded to restudy items, single-choice items, and short-answer items from the online exam, respectively. the main points of interest were (i) whether the re-processing (rewording and asking for transfer of knowledge) of information in the online exam affected participants' performance on the final exam, and (ii) whether any effect depended on the specific type of re-processing (restudy vs. single-choice test vs. shortanswer test). if previous findings from basic cognitive psychological research on testenhanced learning can be generalized to more applied settings and educationally more relevant materials such as pharmacological information, students' performance in the final exam should be better for questions corresponding to previously tested items than for questions corresponding to previously restudied items. moreover, if more difficult tests lead to more test-enhanced learning than less difficult tests, as is suggested by recent findings from cognitive psychological research, performance for questions corresponding to (supposedly more difficult) short-answer items should even exceed that for questions corresponding to (supposedly less difficult) single-choice items. the present findings bear direct implications for educational practice. safe and rational prescribing is one of future physicians' key skills [1] . in order to address the persisting prescribing deficiencies [2] , we set out to develop a learning tool for pharmacotherapies of the most important diseases worldwide. the format, scope, information architecture, and functionalities of the app were identified through assessment of existing apps, literature analysis, app simulation-based student surveys, and expert advice. a fully functional offline app format for smartphones was selected based on the trends in using digital technologies for educational purposes [3] and on the unreliable internet and power availability in many learning settings. a relational database based on semantic relationships was chosen to minimize information redundancy and to enable the retrieval of drug-related information in the context of mechanisms, contra-and indications, adverse drug reactions, interactions, and common prescribing situations. the usability was optimized using a simulation of the app evaluated by medical students from germany and tanzania, and by experts. a list of 67 indications was assembled beginning with disease burden data for the seven who world bank regions. each disease accounts for at least 0.3% of life years lost due to premature death or lived with ill-health or disability (daly) in at least one region. the list was further complemented according to expert recommendations. therapeutic recommendations are based on current guidelines, considering cheaper treatment alternatives provided in the who list of essential medicines. a novel dual-scale classification system lists drug mechanisms according to the affected physiological process and to the resulting therapeutic effect [4] . contraindications, adverse drug reactions, and interactions were compiled using drug monographs of the european medical agency, the us food and drug administration, and health canada. unexpectedly, we found significant differences among these sources in respect of adverse drug reactions. this necessitated the ongoing verification through surveying general practitioners and specialists in internal medicine. during the dgpt meeting we will present the results of testing of the cardiovascular section comprising 8 indications, 28 drugs representing 25 mechanisms, and up to 120 adverse drug reactions. the european certified pharmacologists (eucp) programme was lauched in july 2014 by the federation of european pharmacological societies with the intention to identify experts in the field of pharmacology whose competency profile, in addition to their personal specialised scientific expertise, covers expert knowledge in all major fields of the discipline. seventeen ephar member societies have declared their active participation in the eucp programme so far (austria, croatia, czech republic, finland, france, germany, greece, hungary, italy, the netherlands, norway, poland, portugal, serbia, slovenia, spain, turkey) . eacpt, the european association of clinical pharmacology and therapeutics, has also recently decided to participate in the eucp programme. national programmes must meet all requirements of the eucp guidelines including a clear catalogue of criteria with respect to knowledge, practical awareness and skills, as well as general rules including rules for final assessment of candidates. such programmes may be based on existing diplomas or training schemes or may consist of a set of rules how applicants may submit credentials for their expertise with respect to the eucp criteria. so far, three ephar member societies have submitted a national eucp program: austria, italy and the netherlands. the programmes differ in structure and reflect the flexibility of the eucp programme with respect to the respective national conditions. while the italian programme is based on a catalogue of criteria where applicants have to certify and document their expertise on the basis of this catalogue and the dutch program is based on a structured phd training course, the eucp scheme submitted by the austrian pharmacological society aphar is based on the legally regulated diploma medical specialist (facharzt) in pharmacology and toxicology (aphar also plans to submit separate regulations for specialists in clinical pharmacology and for nonmedically qualified pharmacologists). the aphar eucp scheme has been approved by the eucp committee in november 2015 and its regulations are available on the eucp website (www.eucp-certification.org). the differently structured programs of the italian and dutch pharmacological societies will also be available at this website, once approved by the eucp committee and may thus serve as 'case studies' for other ephar and eacpt member societies wishing to take part in the eucp programme. introduction: the outstanding importance of pharmacovigilance (pv) for the safe use of medicines has increasingly been recognised during recent years. the multidisciplinary character of pv requires know-how in topics as different as pharmacology, clinical medicine, pharmacoepidemiology, information technology, pharmaceutical manufacturing, legal aspects, public health policies, and medical traditions in different regions of the world. in this complex situation there is a growing need for pv capacity building, in particular by professional training through high quality pv courses with different focuses and different levels of detailing. against this background, the world health organization (who) and the international society of pharmacovigilance (isop) have co-operated to create a curriculum for teaching pv which can be used for a wide range of audiences and in very different settings and situations. the purpose was to provide an inventory and systematically structured overview of pv including recent developments of topics like pharmacogenomics, consumer reporting of adrs, risk management and who-led international projects, and to propose a range of tasks for practical training. we made use of several relevant already existing packages of pv topics and concepts of pv teaching from national and international institutions. we also drew from extensive printed material as well as comprehensive reviews, textbooks and guidelines developed by international organisations which are often available online. results: the curriculum includes a main component consisting of a major part for theoretical lecture-based training and a minor component with suggestions for hands-on exercises. the theoretical part has a three-level hierarchical and modular structure with evenly divided tiers. there are 15 chapters. each of them is divided into four sections and each section into four to six sub-sections. the practical part consists of twelve times three or four proposals for practical tasks which are related to the theoretical lectures. since its launch in 2014 1 it has successfully been used in several international courses. currently a pilot project is under way to explore its use for 'crowd sourcing': it is placed on the isop homepage with a programme allowing for institutions or persons experienced in pv teaching to upload any relevant presentations they may have with a link to related chapters, sections or subsections. these presentations will be offered for downloading by interested users. the curriculum provides a comprehensive coverage of almost all areas of pv. the structure and content allows almost every kind of focusing on specific issues and going into depth, while maintaining the overall context. it offers opportunities of tailoring courses specifically to the needs of different audiences and can be applied to various forms of training, such as broad, comprehensive and intensive courses, short overviews or focuses on specific narrow topics in perspective. according to the reach regulation (ec) no. 1907/2006 chemicals produced, marketed or used within the european union have to undergo a registration process, wherein the registrants have to provide information on hazard and potential risks presented by the substances. however, the standard information requirements defined in annexes vii to x of the regulation might be waived or adapted by the registrants if adequate documentation and justification according to criteria specified in annexes vii to xi are provided. to evaluate the data availability in registration dossiers of high tonnage substances (above 1000 tpa) and their compliance with the reach regulation, the federal institute for risk assessment (bfr) in cooperation with the federal environment agency (uba) developed a systematic web-based scheme. in total, 1932 dossiers were checked for selected human health and environmental endpoints such as repeated dose toxicity, genetic toxicity and ecotoxicity. a remarkable high rate, 43% to 82 % depending on the endpoint, of the evaluated dossiers included waiving or adaptations from the standard information requirements. therefore, those dossiers were not concluded, but categorised as 'complex' (springer et al., 2015) . the use of waiving and adaptations in 'complex' endpoints were part of a follow-up project. herein, it was evaluated whether the given justifications were in accordance with the criteria set out in the respective reach annexes. the results will show the frequency and pattern of waiving/adaptation approaches for the human health as well as the environmental endpoints. besides this general overview, specific problems regarding the application of the reach regulation were identified and their significance with regard to remaining data gaps will be discussed. the german commission for the investigation of health hazards of chemical compounds in the work area has re-evaluated dimethylformamide, and classified it in the carcinogen category 4. this category is for chemicals with carcinogenic potential for which a non-genotoxic mode of action is of prime importance and genotoxic effects play no or at most a minor part provided the mak and bat values are observed. under these conditions no contribution to human cancer risk is expected. dmf was identified as a substance of very high concern by european commission. the amount of dmf manufactured and/or imported into the eu is, in the range of 10000-100 000 t/y. n,n-dimethylformamide is a hepatotoxin in humans and rats. the carcinogenicity studies in both mouse and rat were conducted with test material of an acceptable purity and physical form. the critical study involved administration of dmf via inhalation, which is relevant to human exposure. there is conclusive evidence that dmf induces significant increases of hepatocellular carcinomas in rats after exposure to 800 ml/m³ and in mice in response to 200 ml/m³ and higher. several in vitro and in vivo studies have indicated that dmf is not genotoxic. the results of the long-term studies reveal that the tumors develop in the liver only after chronic toxic inflammatory and degenerative changes have developed in this organ. the commission concluded that the tumors are a result of chronic liver damage, occurring at high exposure concentrations. the available evidence therefore suggests that there is a threshold dose for the carcinogenic effects of dmf. accordingly, dmf was classified in carcinogen category 4 with a mak-value of 5 ml/m³, an exposure concentration which does not induces liver toxicity and as a consequence is not associated with an increased cancer risk. today, a large majority of people is constantly exposed to electromagnetic radiation. many studies have been performed to investigate whether this type of radiation has a potential to affect biological systems at low intensity levels. even though no complete consensus has been reached so far in this issue, most of the investigations do not indicate a harmful potential of this radiation. two questions remain open until today, i. e. long-term effects and specific effects on children. it has been demonstrated that in comparison to adults, children absorb far higher doses of mobile phone radiation in the skull, particularly in the bone marrow, where hematopoiesis takes place. these absorptions occasionally exceed the recommended safety limits. the aim of this study was to elucidate, whether cells of the hematopoietic system can be affected by different forms of mobile phone radiation. as biological systems, two cell types were investigated, hl-60 cells as an established cell line, and human hematopoietic stem cells. the radiation was modulated according to the two major technologies, gsm (900 mhz) and umts (1950 mhz). additionally, lte (2.535 mhz) modulation was applied because this technology is used worldwide already but has not been studied sufficiently. cells were exposed for a short and a long period and with different intensities ranging from 0 to 4 w/kg. studied endpoints included oxidative stress, differentiation, dna repair, cell cycle, dna damage, histone acetylation, and apoptosis. appropriate negative and positive controls were included and three independent replicate experiments were performed. exposure to radiofrequency radiation did not induce any alterations of cell functions, measured as oxidative stress and cell cycle. cell death in the form of apoptosis was not observed. primary dna damage was not induced and dna repair capacity for nucleotide excision repair was not changed. epigenetic effects (measured as histone acetylation) were not observed. finally, differentiation was not affected. the effect of treatment with various chemicals as positive controls was different in the two cell types. all in all, mobile phone radiation did not induce effects on human hematopoietic cells. in 2014 who published guidance on evaluating and expressing uncertainties in human health hazard characterisation (hc). in this approach, the outcome of hc is expressed as an interval or distribution rather than a "traditional" deterministic point estimate, such as a reference dose (rfd), thereby communicating potential uncertainties more clearly. risk management protection goals, such as the acceptable magnitude of effect (m) and incidence (i) in the population, are made explicit quantitatively along with the confidence with which they are achieved, e.g. by an rfd. specifically, the goal of this approach to hc is to estimate the "hd m i" , i.e. the "true" human dose associated with m and i (e.g. body weight decreased by ≥ 10% (m) in 5% (i) of the population). if uncertainties are expressed by providing estimates of the hd m i as confidence intervals or uncertainty distributions, both the "degree of uncertainty" (ratio of upper and lower limit of the interval/relevant distribution segment) and the "coverage" (statistical confidence) associated with a given rfd value can be characterised. alternatively, one may start from a chosen coverage and calculate the associated "probabilistic rfd". uncertainty in each hc aspect, e.g. inter-/intraspecies or time extrapolation, can be characterised by a "generic default uncertainty distribution" which has been derived from historical data, but may be replaced by a substance-or effect-specific distribution (analogous to a chemical-specific adjustment factor in the "traditional" deterministic approach), where available. the uncertainty distributions for the individual hc aspects can then be combined into an overall uncertainty interval/distribution in (1) a simple non-probabilistic way, (2) a more refined "approximate probabilistic analysis" (aproba, a free spreadsheet tool for easy implementation also by non-statisticians is available from the who website), or (3) a fully probabilistic monte carlo analysis. the hc aspects contributing most to overall uncertainty are also identified and may be prioritised for refinement in a next assessment tier. the who approach uses a tiered strategy which may start with evaluating the uncertainties in the outcome of a "traditional" deterministic hc. in this way it represents a unified framework integrating deterministic and probabilistic hc methodologies. moreover, the concept can be easily combined with exposure uncertainty assessment. risk managers may use the additional information in better weighing potential health effects against other interests. when they consider the overall uncertainty larger than desirable in view of the problem formulation, they may decide to ask for a more refined (higher tier) assessment. if all possibilities for refinement are exhausted, the new approach can also aid in the selection of new data which might need to be generated. due to a constant improvement in analytical methods an increasing number of substances are found in drinking water. the joint project toxbox aimed for the development of a reliable test battery, allowing for a rapid evaluation of single substances in water. eleven partners either from the research (nine) or the business sector (2) formed the project. the attention was focused on genotoxic, neurotoxic and endocrine effects, which are considered to be of most concern to the consumer. by the end of the project a set of guidelines is published that describes the analytical methods in detail. the project was based on a theoretical concept, called "health-related indicator value" (hriv), which was developed by the german federal environment agency (uba) for the assessment of substances with incomplete toxicological data. depending on the type of effect a hriv between 0.1 and 3.0 µg/laws derived for the substance which had to be evaluated. during the years an increasing amount of substances as well as an increase in finds was observed in drinking water. this called for the creation of an evaluation scheme that offers rapid and at the same time reliable evaluations of chemicals for which there are no data available. the concept is in accordance with tox21, which envisages the trustworthy evaluation of relevant endpoints by two or three in vitro assays. in the context of toxbox this was provided for the endpoints genotoxicity, neurotoxicity and endocrine effects. in all cases a hierarchic strategy is applied that enables a first assessment via relatively simple test assays and only when these test give a hint towards an effective more elaborate techniques are applied for a final assessment. the ames test and micronucleus assay in combination with the umu test will form the panel for genotoxicity testing. neurotoxicity will be assessed by comparing necrotic and apoptotic effects as well as the development of reactive oxygen species in human nervous cell with human liver cells. additionally neuron specific assay like the neurite outgrowth test are performed. this is complemented by measuring the activity of acetyl choline esterase activity and the development of the side line organ in zebra fish (danio rerio). the test battery for endocrine effects consists of hormone specific reporter gene s81 assays in addition to the h295r assay. when necessary a reproduction assay in the mud snail potamopyrgus antipodarum is carried out. during the project some 60 substances were evaluated. this allowed for the development of a reliable test strategy. currently the guidelines for performing the required tests are in the making. metabolomics has gained increasing interest over the last years with numerous possible applications ranging from strain optimization for industrial production over drug discovery to improved toxicity testing. however, regulatory acceptance of this promising approach is still not reached, mostly because standardization and evaluation of reproducibility are still mostly lacking. the metamap®tox database has been developed by basf over the last ten years containing toxicity and metabolome profiles of more than 750 different compounds. to ensure maximum reliability, data was gained from plasma samples of highly standardized 4 week rat studies. animal maintenance and treatment, sampling and work-up of plasma, measurement of the metabolome as well as data interpretation and storage were standardized including thorough documentation, the compliance with sops and safe data storage. data from more than 80 control groups with each 10 males and females were analyzed to assess variability. an in depth analysis of this showed a high stability and robustness of the metabolome over a period of ten years. after artificially splitting the groups of 10 control groups into groups of five animals and comparing the number of statistically significantly regulated (false positive) metabolites, the peak of the distribution curve was located to the left of the exact (gaussian) center, but tailed off to the right more than expected under the normal distribution. from this analysis we were able to calculate density distributions (relative ratio and standard deviation) for the control values of each metabolite, which can serve as a historical control displaying the range of changes which can be expected as normal. during the course of our project we have used more than ten exact repeats to show reproducibility and reliability of the metabolome analysis (kamp et al., 2012) . comparing these exact repeats at different levels of statistical significance, we noted that at a level of statistical significance of approximately p = 0.1, the best balance between matches (metabolites regulated in the same direction) and mismatches (metabolites regulated in opposite directions) was obtained. the high quality standards applied as well as the examination of control data increase the robustness of this approach, going also hand in hand with improved data quality. this significantly facilitates decision making based on the gained data. due to these improvements a new level of transparency is reached, which might allow inclusion of metabolome data in a regulatory environment. hydroxycitric acid (hca) is a fruit acid naturally occurring in fruits of the tropical plant garcinia cambogia. a number of dietary supplements intended for weight loss contain hca, which is added in form of g. cambogia extracts. the composition of these extracts is often not clearly specified. health concerns about safety of the hca-containing supplements have been raised, based on results from animal studies, which observed toxic effects on the testis and on spermatogenesis after administration of preparations containing high hca doses. in the current risk assessment, the possible health risks associated with consumption of hca-containing dietary supplements (hca doses of approximately 1000 to 3000 mg per day) were evaluated based on relevant animal and human studies with the focus on testicular toxicity as a critical endpoint. in several published animal studies, repeated (short-term or subchronic) ingestion of certain hca-preparations (g. cambogia-extract or ca 2+ -hca salt) induced testicular atrophy (i. e. atrophy of seminiferous tubules, degenerative changes of sertoli cells at histological examination) and impaired spermatogenesis (i. e. decreased sperm counts) in male rats at high doses (noael and loael of 389 and 778 mg hca/kg body weight & day, respectively). animal studies with other hca-preparations (ca 2+/ k + -hca salt) found no such effects at the highest hca-doses tested (noael: 610,8 mg hca/kg bwt & day). human intervention studies which addressed the safety of hca in healthy test persons reported no substancespecific adverse effects after ingestion of hca doses up to 3000 mg per day over the period of up to 12 weeks. however, the question of possible adverse effects of hca on the human testes was not adequately addressed in studies with human volunteers. in a single clinical study with 24 male test subjects, no significant changes in endocrinologically relevant parameters such as serum inhibin b or fsh were observed after consumption of 3000 mg of hca for 12 weeks. however, no investigations of direct parameters that might inform on potential effects on spermatogenesis, such as sperm quality and sperm count, were conducted in this study. considering the serious adverse effects on the testes observed in several animal studies as well as in view of lack of the adequate human data on the safety of the long-term use of hca-preparations, it is concluded that knowledge gaps and substantial uncertainties exist regarding the safety with respect to human health of high amounts of hca found in commercially available food supplements, particularly with regard to the human male reproductive system. a critical look on the passing-bablok-regression b. mayer 1 1 universität ulm, institut für epidemiologie und medizinische biometrie, ulm, germany background: the passing-bablok (pb)-regression is a commonly used approach to prove the equality of different analytical methods when studying quantitative laboratory data. it is based on the assumption that the measurements of two methods are linearly related. if then one method is regressed onto the other and the respective confidence intervals of the intercept and the slope include 0 and 1, respectively, it is assumed to have a proof of methods equality. however, this conclusion is problematic in respect of an essential principle of statistical hypothesis testing. methods: in this talk the general idea behind the pb-regression is discussed critically. although the method makes use of confidence intervals a decision is made, which is why it is important to discuss how the results of a statistical hypothesis test have to be interpreted. moreover, alternative statistical approaches to investigate agreement in biometrical practice are pointed out by means of a practical example and their advantages and limitations are addressed. results: all approaches applied to a sample data set led to the same conclusions. demonstrating methods equality though necessitates an a-priori definition of an appropriate equivalence margin. conclusion: the pb-regression may give useful advice when comparing two measurement methods towards equality. however, its results are statistically inconclusive, since the pb-method does not follow the principle of equivalence testing. alternative measures of agreement should be applied instead to ensure results which are not attackable and serve as a statistical proof. insulin is an important parameter both in toxicology (toxicity to the endocrine pancreas) and pharmacology (models of diabetes and metabolic syndrome). currently available elisa and ria methodologies for insulin often require up to 100 µl plasma or serum for a single measurement. in order to meet the general trend to include more relevant parameters in animal studies and restrictions through animal welfare requirements to limit the volume of interim blood draws we explored the rat/mouse insulin singleplex assay of meso scale discovery (msd) as an alternate assay consuming only 10 µl serum or plasma or less for a single measurement. the assay is a sandwich immunoassay, whereby insulin in the sample binds to the capture antibody immobilized on the working electrode surface at the bottom of each well and recruitment of the labeled detection antibody (anti-insulin labeled with electrochemiluminescent compound, msd sulfo-tag™ label) by bound analyte completes the sandwich. voltage applied to the plate electrodes then causes the label bound to the electrode surface to emit light the intensity of which is a quantitative measure of insulin. the msd insulin assay was characterized by a robust calibration and only small variations within repeated measurements. the assay presented a broad dynamic range and differences in insulin levels of normal and rats suffering from metabolic syndrome could readily be demonstrated. furthermore, the high sensitivity may even allow the use of smaller sample volumes. these features render this assay an attractive alternative for the measurement of insulin. the lack of corresponding quality control samples for internal quality control may be considered as a relative drawback. however, the cross reactivity of the assay with human insulin provides the opportunity to use qcs designed for human assays and to possibly participate in ring trials for human insulin for external quality control if needed. surfactants are main constituents of different consumer products, e.g. detergents or cosmetic cleansing products. since surfactants show an intrinsic skin irritation potential, dilutions are used in the final products to avoid adverse effects like irritant contact dermatitis from product use. in addition, mixtures of different surfactants are typically formulated, as it is a long-standing experience that those mixtures exhibit much lower acute irritation potential than expected from the mere summation of their individual irritation potential, an effect coined surfactant antagonism. only few studies were performed to gain a more fundamental understanding of the effect, and it's mechanistic basis remains unclear. however, a thorough understanding of the surfactant antagonism is not only of value for the formulation of products that are considered 'mild to the skin'. it is also important for the classification of products according to the clp regulation in cases when data of the mixtures is missing, because summation of the ingredients' irritating effects usually results in over-classification as skin irritant. due to the progress in the development of alternatives to animal testing, different in vitro methods have become available to determine skin irritating properties of substances. methods like the oecd tg 431 and 439 especially aim at deriving a classification for skin irritation/corrosion effects according to the clp regulation. however, even though these methods became the preferred test methods for skin irritation testing, to our knowledge hitherto isolated investigations on the surfactant antagonism were only performed either by human patch test studies or by non-standard in vitro assays. in this study, the irritation potential of binary mixtures of sodium dodecylsulfate (sds), linear alkylbezene sulfate (las), cocamidopropyl betaine (cabp) and alkylpolyglucosid (apg) compared to the single compounds was investigated using open source reconstructed epidermis (os-rep) models. combinations of sds or las with cabp and apg, respectively, resulted in a clear decrease of the irritation potential compared to the irritation exerted by the single surfactants, even though the total surfactant concentration was higher in the mixtures. in addition, the effect of surfactant antagonism was also observed in a mixture of cabp and apg. the reduced irritation potential of mixed surfactants came along with both reduced skin penetration of fluorescein and reduced release of ldh. since no surfactant antagonism is observed in monolayer cultures of keratinocytes that were exposed to mixtures of surfactants, it is assumed that keratinocytes in the viable parts of the reconstructed epidermis are promptly damaged by the surfactants once the model's barrier is destroyed. hence, surfactant antagonism appears to be primarily driven by the mixture's lower ability to damage the skin model's barrier. the micronucleus (mn) test is a reliable method for the detection of cytogenetic damage in proliferating cells. in recent years, substantial progress has been made on automated, thus faster and more objective scoring of mn test samples, i.e., methods based on flow cytometry. the aim of the present study was to use the adherently growing human bladder cancer cell line rt4 to carry out a comparison between traditional (fluorescence microscopy) and automated (flow cytometry) mn scoring. for this purpose, different substances which are known to be positive controls were used. rt4 cells were either continuously incubated for 72 h (approximately 1.5 cell cycles duration) with methyl methanesulfonate (mms; 50-200 µm), benzo[a]pyrene (b[a]p; 0.1-1 µm), vincristine (3-20 nm) , and colcemid (10-20 nm) or cells were irradiated with xrays (0.5-2 sv) and then cultured for 72 h. for standard mn scoring, cells were harvested, subjected to hypotonic treatment, fixed with methanol/acetic acid, placed on glass slides, stained with acridine orange and observed by fluorescence microscopy. for the flow cytometric method, harvested cells were stained in two sequential steps. intact cells were subjected to ethidium monazide bromide followed by photoactivation (75 w, 20 min) to label dead or dying cells. then, cells were lysed and stained with sytox green for a pan-dna labelling and analyzed on a flow cytometer. both, chemically-and radiation-induced treatment led to a dose-dependent induction of mn when evaluated by fluorescence microscopy. when the flow cytometry-based method was applied, clearly positive results including a dose-dependent induction of mn, however, were obtained only for 3 out of the 5 treatments (vincristine, colcemid and x-rays); whereas, treatment with mms and b[a]p led to only minor increases in relative mn frequencies (≤2-fold), even at the maximum concentrations. in summary, flow cytometry-based mn scoring has been successfully applied in rt4 cells. however, our initial results suggest that flow cytometry-based mn scoring is less sensitive than microscopic scoring when rt4 cells are used. so far, only few adherently growing cell lines have been applied to flow cytometry-based mn scoring. further substances (positive and negative controls) and possibly other adherent cell lines need to be tested to expand our knowledge on the effectiveness of automated mn scoring in vitro and compared to traditional approaches. background: the platinating agent cisplatin is commonly used in the therapy of various types of solid tumors, especially urogenital cancers. its anticancer efficacy largely depends on the formation of bivalent dna intrastrand crosslinks, which impair dna replication and transcription. these crosslinks stimulate mechanisms of the dna damage response (ddr), thereby triggering checkpoint activation, gene expression and cell death. the clinically most relevant adverse effect associated with cisplatin treatment is nephrotoxicity, which mainly results from damaged tubular cells. here, we analyzed the influence of the hmg-coa-reductase inhibitor lovastatin on the cisplatin-induced geno-and cytotoxicity in the rat renal proximal tubular epithelial cell line nrk-52e. methods: cell viability was determined by using the alamar blue assay, as well as by electrical impedance measurements via the icelligence system. alterations in cell cycle progression were assayed by flow cytometric analysis. the formation of pt-(gpg) intrastrand crosslinks was determined via southwestern blot. the amount of dna double-strand breaks (dsbs) was quantified by measuring the level of s139 phosphorylated h2ax (γh2ax) via immunocytochemistry as well as by western blot. additionally, neutral and alkaline comet assays were performed to determine the amount of dna single-and dna double-strand breaks. mechanisms of the ddr were analyzed by western blot as well as by quantitative real-time pcr. results: the data show that pretreatment of nrk-52e cells with a subtoxic dose of lovastatin reduced the cytotoxicity evoked by high doses of cisplatin by protection from cisplatin-stimulated apoptotic cell death. moreover, lovastatin had extensive inhibitory effects on cisplatin-induced ddr, as reflected on the level of p-atm, p-p53, p-chk1, p-chk2 and p-kap1. furthermore, activation of mitogen-activated kinases (mapks) was also reduced. the lovastatin-mediated mitigation of cisplatin-induced ddr was independent of the initial formation of dsbs as well as of pt-(gpg) intrastrand crosslinks. lovastatin protects nrk-52e cells from cisplatin-induced cytotoxicity by interfering with proapoptotic mechanisms of the ddr independently from initial dna damage formation. with respect to the clinic, the data indicate that lovastatin might be useful to mitigate cisplatin-induced nephrotoxicity. the influence of oxidant tert-butylhydrochinone (tbhq) on endothelial cell migration in wrn-deficient cells k. laarmann 1 , g. fritz 1 1 institut für toxikologie, düsseldorf, germany introduction: wrn is a dna helicase and possesses a 3´-5´exonuclease and atpase activity as well as a single strand annealing activity. it is involved in dna repair, by interacting with proteins of base excision repair (ber) and nucleotide excision repair (ner). defects of wrn are marked by genome instability which, in turn, is caused by defects in dna damage repair. patients with a mutation in the wrn gene show premature aging and early mortality. the latter is mainly caused by arteriosclerosis. furthermore, wrn participates in the regulation of genotoxic stress responses stimulated by reactive oxygen species (ros) and alkylating agents. the aim of this study was (i) to investigate whether endothelial cell migration and adhesion were effected by sub-toxic (ic 10 ) and moderate toxic (ic 40 ) concentrations of the oxidant tertbutylhydrochinone (tbhq) and (ii) whether wrn influences migration and adhesion in the presence or absence of tbhq. methods: endothelial-like ea.hy926 cells were treated with different concentrations of the redox cycling and thus ros producing oxidant tbhq. viability was measured by the alamar blue assay. ic 10 and ic 40 were determined after 24h permanent treatment. to investigate the influence of wrn on endothelial cell migration and adhesion, a wrn knock-down was performed in ea.hy926 cells using rna interference. to measure migration, a confluent cell monolayer was scratched using a pipet tip, 24h after permanent tbhq treatment. pictures were taken at the time points 0h, 4h and 8h after performing the scratch. the non-closed area was measured. in a second part, adhesion of the calcein-labeled colon adenocarcinoma ht-29 cell line on the ea.hy926 monolayer was investigated. wrn-deficient or non-deficient cells were treated with 100 µm and 160 µm tbhq or with tnfα. results: for ea.hy926 cells, 100 µm and 160 µm tbhq were determined as ic 10 and ic 40 , respectively. performing the migration assay, ea.hy926 cells showed 75% gap closure, whereas wrn-deficient cells showed a closure of only 35% after 8h. the gap was closed of 65% and 55% after 100 µm and 160 µm tbhq treatment. in wrndeficient cells no remarkable effect on migration was observed after 100 µm tbhq treatment, whereas the treatment with 160 µm tbhq showed a slight decrease in migration of about 10% compared to wrn-deficient cells. no effect on adhesion was observed after tnfα treatment. after 160 µm tbhq treatment a slight increase of adhesion was detected in ea.hy926 cells. the influence of moderate tbhq concentration on adhesion was reduced in the absence of wrn. conclusion: wrn influences endothelial cell migration. in contrast to wild-type ea.hy926 cells, no significant effect of tbhq was observed on migration of wrndeficient cells. furthermore, the moderate toxic concentration of tbhq showed slightly increased ht-29 adhesion to ea.hy926, which was not found in wrn-deficient cells. outlook: in forthcoming studies we analyse the effect of alkylating agents on migration and adhesion. data will be presented and discussed. the aim of the present work was to compare the sensitivity of leukemia cell lines (hl60, jurkat and tk6) and hematopoietic stem cells with regard to the response to genotoxic agents. chromosomal damage was analyzed by evaluation of the micronucleus frequency. furthermore, changes in the proliferation index and the frequencies of apoptotic and mitotic cells were assessed. several cytostatic drugs with different mechanisms of action were used as genotoxic agents. doxorubicin was used as an intercalator, radical producer and topoisomerase ii inhibitor. also, the effects of vinblastine, a mitosis-inhibiting drug and of methyl methanesulfonate, which forms dna-adducts and stalls replication forks, were analyzed. in general, a difference in sensitivity between the different substances was observed. with regard to the formation of micronuclei after treatment with doxorubicin, jurkat and tk6 cells showed similar increasing trends, whereas hl60 cells showed a much higher increase in micronucleus frequency. a clear decrease in proliferation and the frequency of mitotic cells was observed at the highest concentration (100 nm doxorubicin) investigated, and only a slight increase in the number of apoptotic cells could be shown. the biggest differences in formation of micronuclei could be detected after treatment with vinblastine. hl60 cells showed only a slight increase of micronuclei, but the effect on jurkat cells was stronger. the highest micronucleus frequency after vinblastine treatment was detectable for the tk6 cells. the results for the highest investigated concentrations (31.6 nm and 100 nm vinblastine) showed a significant reduction of the proliferation index. this effect is reflected by the increasing numbers of apoptotic cells in all cell lines. the results for methyl methanesulfonate demonstrated only a small increase in micronucleus formation for the jurkat cells, but higher values for the tk6 cells. in contrast the hl60 cells did not lead to a concentration-dependent effect with methyl methanesulfonate. these results are complemented by preliminary findings in hematopoietic stem cells at selected compound concentrations. the different results between the leukemia cell lines and the stem cells might possibly originate from the different p53 status of hl60 (null), jurkat (multiple mutations), tk6 (wild type) and hematopoietic stem cells (wild type). this difference might also cause differences in cell cycle control or repair mechanisms, and needs further investigations. hyperinsulinemia is thought to enhance cancer risk. a possible mechanism is induction of oxidative stress and dna damage by insulin, here, the effect of a combination of metformin with insulin was investigated in vitro and in vivo. the rational for this were reported antioxidative properties of metformin and the aim to gain further insights into mechanisms responsible for protecting the genome from insulin mediated oxidative stress and damage. comet assay, micronucleus frequency test and a mammalian gene mutation assay were used to evaluate the dna damage produced by insulin alone or in combination with metformin. for analysis of antioxidant activity, oxidative stress and mitochondrial disturbances, the cell-free frap assay, the superoxide-sensitive dye dihydroethidium and the mitochondrial membrane potential-sensitive dye jc-1 were applied. accumulation of p53 and pakt were analysed. as an in vivo model, hyperinsulinemic zucker diabetic fatty rats, additionally exposed to insulin during a hyperinsulinemic euglycemic clamp, were treated with metformin. in the rat kidney samples, dhe staining, p53 and pakt analysis, and quantification of the oxidized dna base 8-oxodg was performed. metformin did not show intrinsic antioxidant activity in the cell free assay, but protected cultured cells from insulin mediated oxidative stress, dna damage and mutation. treatment of the rats with metformin protected their kidneys from oxidative stress and genomic damage induced by hyperinsulinemia. metformin may protect patients from genomic damage induced by elevated insulin levels. this may support efforts to reduce the elevated cancer risk that is associated with hyperinsulinemia. the human skin is the primary barrier against environmental and chemical impacts. as such it shields us against a plethora of xenobiotics such as potentially carcinogenic polycyclic aromatic hydrocarbons (pahs). at the same time it is the second most densely populated organ, harbouring more than 1000 bacterial species and population densities of up to 10 7 cfu per cm 2 . yet little is known about this microbiome's potential to metabolise and toxify pahs such as benzo[a]pyrene (b[a]p). previous work at the bfr showed that degradation of b[a]p and other pahs is a universal feature of the skin's microbiome (sowada et al., 2014) . the corresponding metabolites only partly overlap with those known from eukaryotic metabolism and possess cytotoxic as well as genotoxic properties. excretion of these metabolites will lead to exposure times of 20-30 hours or longer for full and partial metabolisers, respectively. while in vitro studies show the corresponding substances to exert their effects synergistically, an assessment of their potential impact on human carcinogenesis is pending. one obvious mode of action would be direct genotoxicity. however, another option is interference with uv-damage repair. ultraviolet radiation (uvr) from sunlight is regarded the main causative factor for the induction of skin cancer. it induces two of the most abundant mutagenic and cytotoxic dna lesions, that is cyclobutane-pyrimidine dimers (cpds) and 6-4 photoproducts (6-4pps). these lesions are repaired primarily by nucleotide excision repair (ner), a system that is also responsible for the removal of pah-derived dna adducts. we therefore wanted to know whether and to what extent bacterial b[a]p metabolites have the capacity to interfere with ner, potentially contributing to uv-induced dna-damage. to investigate this selected genotoxic metabolites were examined for their potential to affect the dna repair capacity of skin cells (hacat). following treatment with uva/b and bacterial b[a]p-metabolites the skin's repair capacity was assessed using a modified comet-assay. ionizing radiation (ir) is a well-established model to induce dna double-strand breaks (dna-dsbs), but it also generates a broad range of other dna lesions including dna single-strand breaks as well as oxidative dna base modifications. furthermore, ir is able to modify membrane components and triggers the activation of epidermal growth factor receptor. a more specific dsb-inducer is cytolethal distending toxin (cdt), which is produced by a variety of gram-negative bacteria and harbours an intrinsic dnase-like endonuclease activity [1] . dsbs are potent cytotoxic lesions and promote genomic instability, e.g. by formation of chromosomal aberrations. a cellular mechanism to prevent genomic instability and maintain cell homeostasis could be autophagy. this process is highly regulated involving the lysosomal degradation of damaged organelles and proteins. here, we study autophagy induction following dsb generation in human colorectal cancer cells as well as in primary human colonic epithelial cells (hcec) and analyzed regulatory mechanisms. first, the autophagy-specific marker lc3b was shown to increase in a dose-and time-dependent manner after treatment with both cdt and ir as assessed by confocal immunofluorescence microscopy and western blot analysis in hct116. similar results were obtained in sw48 and hcec cells via western blot. these findings are in agreement with the enhanced formation of autophagosomes and the dose-dependent decrease of the autophagy substrate p62 as observed by flow cytometry and western blot analysis in hct116, sw48 and hcec. cdt-and irinduced autophagy rates in hct116 increased over time correlating well with the dsb induction. importantly, a dnasei-defective mutant of cdt did neither cause dsbs nor induce autophagy. additionally, the time-dependent accumulation of the lysosomal associated membrane protein 1 (lamp-1) was observed by confocal immunofluorescence microscopy. dsb-induced autophagy was blocked by chemical inhibitors. next, we showed that both ir and, to a lesser degree, cdt induce the phosphorylation of akt at ser473. pharmacological inhibition of akt in hct116 cells enhanced the cdt-and ir-induced autophagy shown by accumulation of lc3b and lamp1 after 48 h and increased autophagosome formation. upregulation of dsbinduced autophagy by akt inhibition resulted in a decreased cytotoxicity after 72 h and significantly lower apoptosis/necrosis rates after 48 h, which were determined by mts cell viability assay and annexin-v/pi staining. ongoing studies will evaluate the impact of other dna damage response pathways and the potential protective role of autophagy against genomic instability. mustard agents are potent dna alkylating agents. among them, the bi-functional agent sulfur mustard (sm) was used as a chemical warfare agent due to its vesicant properties. although the use of sm in warfare has been banned in most countries of the world, its use in terroristic attacks or asymmetrical conflicts, such as the syrian civil war, still represents a realistic and significant threat. on the other hand, especially nitrogen mustards, such as cyclophosphamide or melphalan, have been used as chemotherapeutic agents due to their cytostatic properties. thus, mustard-induced dna damage, in particular dna crosslinks, can trigger complex pathological states, as it is observed in sulfur mustard exposed victims, but on the other hand also lead to the chemotherapeutic effects of clinically-used nitrogen mustards. mass spectrometric monitoring and quantitation of mustard-induced dna adducts can help to unambiguously identify and verify sm-exposed victims and to monitor the efficiency, as well as potential side-effects of mustard-based chemotherapy. up to now, the verification of mustard-induced nucleic acid damage is mainly based on immunohistochemical methods, which have several drawbacks such as limited specificity, sensitivity, and low dynamic range of quantitation. with this project, we aim to develop a (hplc/uplc)-ms/ms-based platform for the quantitation of the most common mustard-induced dna adducts including bis(n7-guanine-ethyl) sulfide dna crosslinks. up to date, we established methods for the quantitation of the several common dna adducts induced by the mono-functional sulfur-mustard derivative 2chloroethyl ethyl sulfide ("half mustard", cees). for that reason purification protocols, chromatographic conditions and mass spectrometric settings were developed to detect n7-ethylthioethyl-2´desoxyguanosine (n7-ete-dg) and n3-ethylthioethyl-2´desoxyadenosine (n3-ete-da) and their thermal hydrolysis products n7ethylthioethyl-guanine (n7-ete-gua) and n3-ethylthioethyl-adenine (n3-ete-ade), respectively, and the sensitivity was compared to immunohistochemical methods. additional non-radioactive isotope-labelled standards are being synthesized, which will be spiked into samples to account for technical variability during sample work-up and to improve ms-based quantitation. this procedure requires minimal cellular material and therefore should be transferred to quantitation of dna adducts in human blood samples. this will allow to monitor dna adducts as biomarkers of exposure in potential smexposed victims as well as in mustard-based chemotherapy. this method also sets a basis to investigate specific mustard-induced dna repair mechanisms and their cellular consequences. the γh2ax assay vs. comet assay for genotoxicity testing universitätsmedizin mainz, institut für toxikologie, mainz, germany dna damage leads to activation of the cellular dna damage response (ddr). this signalling network results in activation of various dna repair proteins and chromatin structure modulators. a frequent manifestation of ddr is the phosphorylation of histone 2ax (gh2ax), which can be visualised as gh2ax foci by immunocytochemistry. in the present study, we tried to assess if gh2ax is a reliable biomarker for detecting the cellular response to dna damage. we selected 14 well-characterised genotoxic compounds and compared them with 10 non-genotoxic chemicals in the wellcharacterised cho cell system. we measured quantitatively γh2ax by manual and automatic scoring of γh2ax foci, and by flow cytometry counting of γh2ax positive cells. the cytotoxicity dose-response was determined by the mtt cell proliferation/viability assay. we show that a) all genotoxic agents were able to induce dose-dependently γh2ax in the cytotoxic range whereas no induction was observed after treatment with non-genotoxicants; b) manual scoring of γh2ax foci and automated scoring gave similar results, with the automated scoring being faster and more reproducible; c) data obtained by foci counting and facs analysis of γh2ax positive cells showed a significant correlation. further we compared dna damage induced by 4 selected genotoxins at the time-points using the alkaline and neutral comet assay. significant correlation with the alkaline and neutral comet assay was observed for some but not all genotoxins and, predominantly, at earlier time points. we suggest that comet assays detect mainly primary dna damage, whereas γh2ax assay detects a specific response to dna damage which can persist longer. the γh2ax foci and flow-cytometry assays allow for a rapid and reliable determination of genetic damage in mammalian cells and can be used as additional genotoxicity assays. available in vitro methods to investigate the genotoxic potential of drugs fall short of throughput, specificity and mode of action information. a set of mechanistic biomarkers for clastogenic, aneugenic or apoptotic effects may help to overcome these limitations. thus, a staining assay amenable to flow cytometric analysis is being developed by litron laboratories, rochester, ny, supported by international collaborators. the experimental design of this assay consists of 3 stages. the objective of this work is the evaluation of this assay in the laboratories of bayer pharma ag. the biomarkers covered by the assay are associated with dna damage response pathways that have potential for class discrimination (clastogen/aneugen/cytotoxicant) of in vitro genotoxicants: dna double strand breaks (γh2ax), nuclear division (phospho-h3, dna content), apoptosis (cleaved parp). based on the pilot work at litron laboratories, tk6 cells were introduced to the genetic toxicology of bayer pharma ag. cells were exposed for 4 and 24 hrs in triplicates on a 96 microwell plate to one reference clastogen (etoposide, eto), aneugen (vinblastine, vb) and cytotoxicant (carbonyl cyanide 3-chlorophenylhydrazone, cccp). after staining, the samples were analyzed with the flow cytometer bd accuri c6 (bd biosciences, heidelberg, germany). the reference substances yielded the responses expected from the pilot study at litron laboratories: vb showed distinct increases of phospho-h3 events at 4 and 24 hrs and polyploidy at 24 hrs time point. eto induced a clear increase of yh2ax with a simultaneous reduction of phospho-h3 at 4 and 24 hrs. finally, cccp caused a reduction of phospho-h3 events, increased cleaved parp events and did not influence γh2ax. moreover, benchmarking experiments under pilot work conditions were performed with high content imaging analysis. we compared yh2ax and phsopho-h3 pilot study results as well as cleaved parp with caspase 3/7. in addition, the tunel assay (click-it ® tunel alexa fluor, thermofisher) was executed to benchmark cleaved parp. the benchmarking results support the selected biomarkers of the multiplexed assay. in stage 2, additional reference compounds (three aneugens/clastogens/cytotoxicants) were investigated. so far, the chosen biomarkers of dna damage response appear useful for class discrimination and provide additional information to existing genotoxicity tests. cell-cell contacts are involved in keeping a physiological balance between proliferation, differentiation and apoptosis. far less is known about the role of cell-cell-contacts in regulating necrosis, for instance in response to oxidative stress. previous findings of our group demonstrated that, in contrast to semi-confluent proliferating cultures, confluent murine fibroblasts (nih3t3, mef) and human keratinocytes (hacat) are protected against necrosis induced by tert-butyl hydroperoxide (t-booh). comparison of confluent cells (g0/g1 = ~70 %) and semi-confluent cultures, similarly arrested in the g0/g1 phase by serum-starvation or the mek inhibitor u0126, ascertained that the resistance against t-booh is mediated by cell-cell contacts and not by cell cycle arrest. we further revealed that confluent cultures are protected against t-booh-induced dna double strand breaks as assessed by the neutral comet assay and against mitochondrial damage detected by flow cytometric analysis of dioc6 staining. to better understand the protective role of cell-cell-contacts in ros-mediated necrosis, we started characterizing the signaling cascade induced by t-booh in semi-confluent proliferating cultures. in accordance with the observed formation of dna double strand breaks in response to t-booh, we detected phosphorylation of the checkpoint kinase chk2. however, inhibition of atm, the kinase responsible for chk2 activation, did not influence t-booh-induced cell death. interestingly, first experiments gave a hint for the participation of rip1, since the chemical rip1 kinase inhibitor necrostatin-1 (nec-1) blocked cell death up to averagely 50 %, what is described as a specific marker for regulated necrosis. in line with this observation, t-booh-induced cell death could not be blocked by the pan-caspase inhibitor z-vad-fmk strongly indicating that caspase activity is not required. moreover, parp-1 and p53 are probably not involved. deeper analyses could give evidence that nec-1 did not block formation of dna double strand breaks nor mitochondrial damage indicating that the kinase blocked by nec-1, possibly rip1, acts downstream of dna double strand breaks and / or mitochondrial damage. in the end, we could identify a crucial role of ca 2+ signaling for t-booh-mediated toxicity. as the calcium chelator bapta-am was able to completely block not only cell death, but also mitochondrial damage and dna double-strand break formation, there is a strong need for further investigations of the possible interplay between regulated necrosis and calcium, regulated by cell-cell contacts among oxidative stress. the work was supported by the hoffmann-klose-stiftung, the promotionsförderung rheinland-pfalz, the johannes gutenberg-university and the university medical center of the johannes gutenberg-university. the mammalian target of rapamycin (mtor) forms two multiprotein complexes (mtorc1 and mtorc2) and influences cell growth, proliferation, survival and metabolism. constitutively activated mtor was found to be deregulated in several cancer types, which makes it an interesting target for therapeutic cancer strategies. rapamycin is able to inhibit mtor and its downstream targets and is currently studied for its anticancer properties in clinical trials. despite previous evidence, there are studies that show an adverse effect in cancer treatment causing tumour growth, evolving the question of the effectiveness of the drug in cancer treatment. therefore, we examined the transformational potential of rapamycin in a balb/c cell transformation assay (cta) as well as markers of proliferation and protein synthesis. the balb/c 3t3 cell transformation assay mimics different stages of in vivo carcinogenicity (initiation, promotion, post-promotion phase) and is a promising alternative to rodent bioassays. balb/c fibroblasts are treated for 3 days with the tumour initiator mca (3-methylcholanthrene) followed by 13 days with the promotor tpa (12-o-tetradecanoylphorbol-13-acetate). upon treatment with these chemicals cells are transformed into morphologically aberrant foci and can be visualized after six weeks by giemsa staining. it is possible to apply additional substances during the whole assay or in several phases of transformation and evaluate the colony formation. furthermore, our improved protocol allows additional westernblot or immunofluorescence analysis. the influence on cell proliferation of different concentrations of rapamycin was investigated by cell counting (living and dead) to choose a suitable concentration for the cta. performances of balb/c ctas with 10 nm rapamycin showed, contrary to expectations, an increase in cell transformation. by administration of rapamycin only in the promotion phase we could detect an increase in colony formation, whereas a treatment with rapamycin in the post-promotion phase with already established foci, seemed to reveal its therapeutic properties. to better understand the role of mtor in our cell transformation system we used another mtor inhibitor called osi-027. surprisingly, an incubation with 3 µm osi-027 led to a decrease in colony formation. we are now able to investigate the underlying mechanism with westernblot and immunofluorescence analysis and can compare regulations of downstream targets like the marker of protein synthesis p-s6. our investigations revealed different cell transformation outcomes by comparing the two known mtor inhibitors rapamycin and osi-027, which need to be further evaluated. in the ongoing project we want to detect differences between rapamycin and osi-027 by protein analysis and identify key proteins, which are involved in this opposed colony formation of the balb/c cells. these results can be helpful to better understand mtor inhibition in matters of tumour therapy. introduction: over the past 50 years, the biguanide compound metformin has been widely prescribed as an insulin sensitizer in type 2 diabetes mellitus. interestingly, recent meta-analyses of epidemiological studies have shown that metformin might be involved in risk reduction of carcinogenesis. in vitro studies have described amp-activated protein kinase (ampk)-dependent, by inhibition of the respiratory chain complex i, as well as ampk-independent actions of metformin. however, the detailed molecular mechanisms by which metformin affects cell proliferation and carcinogenesis have not been well identified up until now. method: to evaluate the protective potential of metformin, balb/c 3t3 cell transformation assays were performed. this valid toxicological method is an alternative to in vivo carcinogenic testing and mimics the different stages of cell transformation during carcinogenesis. in detail, mouse fibroblasts are treated with metformin and/or the tumour initiator 3-methylcholanthrene (mca) and the tumour promotor 12-otetradecanoylphorbol-13-acetate (tpa). in the first experiment several metformin concentrations (0.1-1 mm metformin) were applied answering the question of an effective metformin concentration. next, metformin treatment during the different phases of carcinogenesis (initiation, promotion, post-promotion phase) was done determining the most effective phase for an intervention, i.e. chemopreventive or chemotherapeutical properties of metformin. additionally, the effect of metformin on the energy metabolism of the cells was analysed using various methods like immunoblot and oxygen measurement by clark electrode. results/discussion: analysis of different metformin concentrations revealed a concentration-dependent effect of metformin. in detail, decreased colony forming potential of balb/c cells was most prominent using 1 mm metformin. this effect was not caused by growth inhibition of metformin itself since 1 mm metformin showed no growth inhibitory properties in a cellular growth pretrial. interestingly, the 2 phase cell transformation assay showed that the metformin effect is more pronounce in the postpromotion phase than in the initiation and promotion phase pointing to a chemotherapeutical potential. investigating several energy metabolism parameters, the results indicate that metformin may affect cell respiration as well as energy-dependent mechanistic markers like ampk. the presented results support rather the idea of the chemotherapeutic potential of metformin than a chemopreventive, using 1 mm metformin. the initial analysis of energy metabolism markers discovered interesting starting points for further investigations. johannes gutenberg university, institute of toxicology, 55131 mainz, germany nvp, widely used e. g. as a monomer for polyvinylpyrrolidones (pvp) with applications in food technology or cosmetics is a known hepatocarcinogen in rats after inhalative exposure to 5, 10, and 20 ppm for 2 years. nvp is tested in a battery of genotoxicity assays (e.g. ames, hprt, mouse lymphoma, uds, chromosome aberration, cell transformation, micronucleus test (mnt) in mice bone marrow) [1]) that all yielded negative results. however, nvp induces cell proliferation in liver (loaec: 0.5 ppm) after whole body exposure to vapor [2] . to confirm the absence of genotoxicity in the context of a potentially non-genotoxic mode of action, a five day whole body inhalation study to nvp vapor with concentrations of 0, 5, 10, 20 ppm was conducted in wistar rats (six animals per gender and group, ethyl methanesulfonate 200 mg/kg bw p.o. as positive control). genotoxicity was investigated by the mnt in bone marrow and the comet assay (± fpg) in liver and lung. further investigated endpoints related to possible non-hepatogenotoxic moa were: enzyme induction (erod, prod, brod), oxidative stress (gsh-, gssg-, non-protein sulfhydryl group level), and peroxisome proliferation (cyp4a, cyanide-insensitive palmitoyl-coa-oxidase). at carcinogenic inhalative doses, the results of this study proved the absence of genotoxicity in lung, liver and bone marrow as neither the tail intensity in the comet assay nor the number of micronuclei in the mnt was increased compared to the controls. however, also the non-genotoxic parameters (cyp-enzyme activity, glutathione levels, cyanide-insensitive-palmitoyl-coa-oxidase) were not affected by nvp-treatment. as potential metabolic activation cannot be excluded and may essentially contribute to the understanding of the carcinogenic mechanism, in vitro investigations in rat liver systems (subcellular fractions, hepatocytes, precision cut liver slices (pcls)) were performed additionally. up to now, 2-pyrrolidone is the only identified in vitro metabolite. as these results cannot mimic the in vivo situation of two described, ring-and vinylmajority containing unidentified metabolites [3] detailed investigations on metabolism may be a future perspective to approach the overall understanding of the carcinogenic mechanism of nvp. introduction: in ischemic conditions such as wound healing and myocardial infarction, new vessels are generated by vasculogenesis and angiogenesis. these processes are stimulated by the signalling peptide vascular endothelial growth factor which therefore has been proposed as a promising compound for the treatment of ischemic conditions. however, results of respective clinical studies have not been fully convincing yet. here, we investigated principles underlying the selforganization of newly formed vessels to functionally adequate microvascular networks indispensable for proper tissue substrate supply. intravital microscopy of the chick chorioallantoic membrane (cam), a non animal model as defined by the american national institutes of health's office for protection from research risks, was used to study peripheral expansion of existing arteriolar and venular trees by recruiting segments of the dense polygonal capillary mesh. this process we call "emerging angiogenesis". methods: white leghorn chicken eggs were put into incubators on embryonic day 0 (e0) at 37.5°c and 82% humidity. on e3, the eggs were cracked open and transferred into petri dishes. on e10, cam microcirculation was recorded using time-lapse intravital videomicroscopy at discrete time points for up to 48 hours. to improve the visibility of the capillary mesh, videorecordings were processed offline by generating coefficient of variation images of pixel grey values over time. changes of network topology during the observation time were investigated. results: in the cam, a sequence of specific events leading to extension of existing vessel trees was observed: in a capillary mesh region near terminal branches of existing vessel trees, homogeneous flow distribution is transferred to inhomogeneous flow distribution: preferred flow pathways through the mesh evolve carrying most of the blood. over time, these flow pathways exhibit diameter increase, straighten and connect the mesh to arteriolar and venular trees. in contrast, less perfused parallel mesh flow pathways and transversal mesh segments exhibit progressive decrease of flow and diameter resulting in vessel regression. as a result, hierarchical vessel tree structures are extended into the mesh region. while newly generated tree extensions are located above the mesh at the beginning, they sink to a lower level at later stages until they are finally covered by a reconstituted mesh network. the cam ex ovo model is well suited for studying emerging angiogenesis. vessel tree extension occurs via parallel processes of vessel maturation and capillary mesh segment regression. at later stages, newly formed vessel tree branches sink and the capillary mesh is reconstituted above. in the next step of our project, we will implement these phenomena in a computer simulation and use theoretical modeling to further investigate and better understand principles underlying microvascular network maturation. this will allow us to derive effective therapeutic strategies which could be tested in the cam model. chemicals are able to induce cancer in a wide range of organs. therefore, it is very important to investigate the toxic properties of chemical substances, especially their carcinogenic potential. in this context the number of animal experiments will drastically increase in the future. in order to avoid the use of expensive and time consuming animal experiments for long-term carcinogenic studies, the development of an in vitro system to test the carcinogenic potential of a high number of chemicals in a highly reproducible manner within a short period of time is imperative. by combination of the well-established balb/c cell transformation method with the soft agar colony formation assay, we developed a high-throughput in vitro system to identify effects of chemicals on cell transformation for the first time. balb/c mouse fibroblasts are treated with 3-methylcholanthrene as a tumour initiator and 12-o-tetradecanoylphorbol-13-acetate as promotor for several days, whereby foci of transformed cells are developed. after the promotion phase of the common balb/c cell transformation assay, cells are transferred into soft agar to further monitor the anchorage independent growth of transformed cells only. the established soft agar transformation assay reproduces the foci growth of previous experiments and is performed in 96-well plate format. hence, we can analyse the carcinogenic potential of several chemical substances in parallel and are also searching for alternative endpoint analysis, e.g. the usage of fluorescing cells stably expressing irfp, instead of the former time-consuming microscopic assessment. the here presented new technique is a high-throughput and low priced alternative for the evaluation of the carcinogenic potential of chemical substances in a short period of time without animal testing. the effort to develop new or refine established in vitro test systems rises due to animal welfare, scientific and/or regulatory reasons (e.g. the animal testing ban concerning the risk assessment of cosmetic product ingredients in march 2013). this progress, among others, leads to an increased performance of cell-based assays. the majority of model cell lines are routinely cultured using medium supplemented with fetal bovine serum (fbs) in amounts between 5-10%. the application of serum-substitutes will provide a reduction of the animal number needed, which corresponds to the guiding principles of the three r's (3r), described by russel and burch in 1959. in addition, chemically defined serum-substitutes have the potential to reduce the inter-experimental variability of test conditions caused by the inherent differences in chemical composition across fbs batches 1 , resulting in a refinement of in vitro testing. in this study, human tk6 cells were gradually adapted to serum-free conditions, where they show comparable growth gradients at the exponential phase. for cells under serum-free conditions a mean doubling time of 19.3 (± 1.7) h was observed while fbs supplemented cells showed a doubling time of 13.5 (± 0.8) several non-animal test methods addressing key events in the sensitization process have passed formal validation and oecd (draft) test guidelines are available. one of these methods is the direct peptide reactivity assay (dpra) assessing the ability of a chemical to bind to proteins to form a complete antigen (oecd tg 442c). the test is used to obtain a yes/no answer on whether the substance has a protein-binding potential. for a complete risk assessment, however, an estimation of a chemical's potency is also needed. in this study we examined if an assessment of potency could be achieved by 1) determining reactivity class cut offs based on published data on 199 substances for the dpra performed according to oecd 442c to predict un ghs sensitizer classes, 2) a variant of the dpra assessing reaction kinetics (time and concentration) for 12 substances or 3) an extended protocol testing several test substance concentrations for 50 reference substances and estimating the concentration of a test substance that is needed to cause a peptide depletion of 6.38% (ec6.38%). results of the first approach indicated that cut offs to differentiate the un ghs sensitizer classes 1a and 1b could indeed be defined. secondly, evaluating the reaction time based assay in which several time points between 5 min and 24 hours were assessed, it was found that not all reactions followed ideal kinetics. hence further investigations are needed to eventually derive a reaction time based prediction model. the results of the 3 rd approach (the standard protocol of the dpra was amended by testing three concentrations i.e. 1, 10, and 100 mm) indicated that potency classes could be assigned using the ec6.38% value to assess potency. in summary, using quantitative information derived from the dpra in particular using ec6.38% value may support the assessment of the skin sensitizing potency. identification of pre-and pro-haptens with non-animal test methods for skin sensitization since pro-haptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (xme) activities in cell lines used for testing of sensitization in vitro is of special interest. metabolic activity of e.g. n-acetyltransferase 1 (nat1) and esterase in the keratinocyte (keratinosens tm and lusens) and dendritic cell-like cells (u937 and thp-1) was previously demonstrated. aldehyde dehydrogenase (aldh) activities were found in keratinosens tm and lusens cells. activities of the investigated cytochrome p450-dependent alkylresorufin o-dealkylases, flavin-containing monooxygenase, alcohol dehydrogenase as well as udp glucuronosyl transferase activities were below detection in all investigated cell lines. a set of 27 putative pre-and pro-haptens (no obvious structural alert for peptide reactivity but positive in vivo) was routinely tested using the above mentioned cell lines as well as in the direct peptide reactivity assay (dpra). 18 of the compounds were unexpectedly positive in the dpra und further analyzed by lc/ms techniques to clarify the reaction mechanism leading to true positive results in this assay. oxidation products like dipeptide formations or the oxidation of the peptide-based sulfhydryl group led to positive results for benzo[a]pyren or 5-amino-2-methylphenol, respectively. in contrast, covalent peptide adducts were identified for 12 putative pre-haptens, indicating the dpra to be suitable for compounds requiring abiotic oxidation to get activated. for some dpra negatives, the keratinocyte and dendritic cell based assays provided true positive results. a combination of dpra, keratinosens tm and h-clat within a '2 out of 3' prediction model provided a high sensitivity of 81% for the set of the pre-/pro-haptens. the sensitivity of this combination of non-animal test methods in the '2 out of 3' prediction model in a set of 95 direct haptens was comparable (sensitivity = 87% when compared to llna skin sensitization testing is mandatory for all substances produced or marketed in volumes larger than 1 tonne per year under the european reach legislation. with reach supporting in vivo testing only "as a last resort" and the marketing ban for finished cosmetic products with ingredients tested in animals, attention has been given to developing integrated testing strategies combining in vitro, in silico and in chemico methods. key challenges are which tests to select and how to combine non-animal methods into testing strategies. this study suggests a bayesian value of information (voi) approach for developing non-animal testing strategies, which consider information gains from testing, but also expected payoffs from adopting regulatory decisions on the use of a substance, and testing costs. the 'value' of testing is defined as the expected social net benefit from decision-making on the use of chemicals with additional, but uncertain information from testing. the voi is calculated for a set of individual nonanimal methods including dpra, oecd qsar toolbox, are-nrf2 luciferase method covered by keratinosens and lusens, and hclat, seven battery combinations of these methods, and 86 two-test and 360 three-tests sequential strategies consisting of nonanimal methods. their voi is compared to the voi of the local lymph node assay (llna) as the animal test. we find that battery and sequential combinations of nonanimal methods reveal a higher voi than the llna. in particular, for small prior beliefs (i.e. a chemicals is, prior to testing, assumed to be a non-sensitiser), a battery of dpra + lusens reveals the highest voi. if there are strong beliefs that a chemical is a sensitizer, a sequential combination of the battery dpra + lusens, followed by keratinosens + hclat at the second stage and by the oecd qsar toolbox at the third stage performs best. for given specifications of expected payoffs the voi of the nonanimal strategy significantly outperformed the voi of the llna, for the entire range of prior beliefs. this underlines strong economic potential of non-animal methods for skin sensitization assessment. a chemical series to predict the proarrhythmic potential of drugs with low solubility for which no reliable purkinje fiber results could be obtained. these validation results showed that this cardiosafety in silico model can successfully be applied in r&d to predict the proarrhythmic potential of drug candidates within the model ad. introduction: the use of p-phenylenediamine (ppd) and derivatives (tab. 1) in oxidative consumer hair dye products is considered as key in hair dye allergic contact dermatitis [1] [2] [3] [4] . in recent supplement, 2-methoxymethyl-ppd (me+) shows significantly reduced sensitizing properties [5, 6] . since overcoming the skin barrier is a prerequisite for sensitization, numerous in vitro an in vivo studies on skin penetration of ppd and derivatives have been performed. the aim of the present study is the in silico prediction of the penetration of ppds, because such computations may help in understanding the processes involved in sensitization. for the first time, software dskin [7] is challenged to simulate this class of compounds. in silico results are retrospectively compared to previously published experimental data and may assist in future tailoring of in vitro experiments. material and methods: the permeabilities, lag-times and the time-dependent accumulated amounts of ppds were computed using dskin. input parameters for the latter were a concentration of 1 mg/ml (1%), finite dosing and 30 min in use incubation periods. molecular structures were optimized ab initio and the condensed fukui functions (ff) were estimated from mulliken population analyses [8] and electrostatic potentials using gamess [9] . results: initial results agree with experimental results using ppd in white petrolatum, demonstrating the applicability of dskin to ppds. the four ppds exhibit only small differences in permeabilities in silico (tab. 1). toluene-2,5-diamine shows a higher accumulated mass due to increased lipophilicity (fig. 1) . in general, the ff were very similar for all ppds and indicated that the n atoms would be the preferred targets for radical and electrophilic attack. discussion and outlook: in silico methods may be used to model the permeation of ppds despite their low molecular weight and low lipophilicity. the low amounts of ppds under in use conditions result from oxidative conditions. computed me+ permeation was not different to other ppds, therefore other properties account for the reduced sensitization potential. the very similar ff values hint at similar reaction pathways. furthermore, ppd and its derivatives are prone to n-acetylation in living skin resulting in metabolites exhibiting higher molecular weight and greater lipophilicity than the parent compounds. the effects of n-acetylation and reactions of ppd and its derivatives with histidine and cysteine residues are subject of upcoming computations. dermal absorption is an important factor in regulatory science regarding the registration of chemicals, agrochemicals and cosmetics. the issue has gained importance since it has been realized that the skin is not completely impenetrable for chemical substances. [1] the different ways to assess dermal absorption range from qsar models to complex in vivo studies including a complete toxicokinetic examination. the choice of method depends on the question that has to be answered as different systems give different results: absorption as % of applied dose in in vivo studies or permeability coefficient and lag time in infinite dose in vitro studies [1] . ideally both data would be available. since the oecd has adopted a guideline for assessing dermal penetration in vitro in 2004 the number of in vitro studies is rising continuously. depending on the chosen method results may vary in reliability and in acceptance by regulatory authorities. the skinab database [ba1] contains data for about 600 substances on dermal absorption which has been found through the echemportal [3] and extended with data from the edetox database. for selected substances with a broad spectrum of data available further analysis has now been started. 165 chemicals have been investigated in a comparable test system; from these 79 were shown to have a low dermal absorption of less than 1% and 51 compounds showed a high absorption rate of more than 50%. for the assessment of dermal exposure either the absorbed dose in percent or the flux can be measured. data analysis showed that only for 15 substances both is available: flux data from in vitro studies and absorption data from in vivo studies. this data could be used to clarify which parameter would be most useful for exposure assessment regarding dermal exposure. seven substances in the dataset were conspicuous for their range of absorption rates in different studies: less than 1% to more than 50%. an in depth analysis revealed the complex influence that different exposure parameters have on the results of dermal absorption studies. for some chemicals the influence of exposure time on increasing absorption values could be clearly demonstrated. beside other factors such as the chosen vehicle, and the (non-)occlusion of the site of exposure especially the choice species introduced a high variability; this holds even for the most common laboratory animals t. a review published by jung in 2015 [5] which comes to the conclusion that i.e. hairless species are usually not a good model to predict dermal absorption in humans. [1]who (2006) ehc 235 dermal absorption [2] scholz et al (2014) naunyn-schmiedeberg´s arch pharmacol 3 (suppl 1):s86 [3] www.echemportal.org [4] http://edetox.ncl.ac.uk [5] jung et al (2015) in-silico methods have evolved to indispensable tools in various areas of life sciences. several stages in drug development including hit identification and lead optimization, for instance, highly benefit from an accurate estimation of binding free energies associated with biological host-guest systems. as a consequence, the need for laboratory experiments including in-vivo experiments and animal testing is considerably reduced. another field profiting from free energy calculations is human as well as ecotoxicology. upon the development and risk assessment of new chemicals, transformation products arising from biotic or abiotic degradation of the parent substance have usually been neglected. however, since few years, the risk assessment of new chemicals often includes transformation products probably causing more harm than the parent substance itself. such studies as well are mostly carried out on the basis of in-vitro and in-vivo tests. moreover, many metabolites can be detected but neither enriched nor synthesized in amount sufficient for toxicological evaluations. at this stage, computational methods come into play. using classical molecular dynamics simulations in combination with an empirical linear prediction model, we have investigated several metabolites of the drugs sulfamethoxazole and carbamazepine and prioritized them according to their estimated binding affinities to potential biological target proteins. consequently, a couple of metabolites were identified that bind to one or more human cytochrome p450 variants and the bacterial enzyme dihydropteroate synthase, respectively, which are known to be sensitive to the two drugs. the investigations were carried out in the framework of the bb3r poject funded by the german government through bmbf. instituto superiore di sanità, environment and primary prevention dept., rome, italien introduction: in vitro methods have been increasingly used to characterize pharmacological and toxicological properties of substances. to address the problem of nominal versus actual concentrations, in vitro biokinetic studies were recently undertaken (truisi et al., toxicol lett 233:172-86, 2015) . we use those data as input into a physiologically based human kinetic model (pbhkm) to model the in vivo doses leading to the in vitro measured concentrations. methods: a pbhkm was used to simulate the concentration time profile of ibuprofen in the hepatic vein after oral administration. the details of the model and the physiological parameters used have been described elsewhere (abraham et al., arch. toxicology 79: 63-73, 2004) . we modelled the concentration time profile exploring the dose which would lead to a concentration at 1 hour and at 24 hour as similar as possible to the concentration measured in the supernatant of human freshly prepared cell cultures after dosing the culture with ibuprofen. we parametrized the pbhkm with the parameters which have been estimated from the in vitro kinetic studies (clearance between 3 and 15 µm 3 /sec (truisi et al., 2015) and an absorption of 100% and an absorption rate of 1/h (cristofoletti and dressman, j pharm sci 103: 3263-75, 2014). results: the data of the in vitro study with 100 µm ibuprofen could well be modelled. when assuming a clearance of 15 µm 3 /sec the dose of 1480 mg resulted in an 1 hour concentration of 66.5 µm in the hepatic vein of pbhkm equal to 133.0 nmol/well (volume of the well = 2 ml) in the in vitro study in which the measured concentration was 138.75 nmol/well. the concentration at 24 hours of 8.7 µm (equal to 17.4 nmol/well) corresponded with the in vitro concentration (16.5 nmol/well). the modelling approach was less successful with in vitro dosing of 1000 µm. the 10 fold higher dose of 14800 mg lead to nearly double the concentration at 1 hour than measured in vitro. with a dose of 8500 mg/kg an approximation was feasible resulting in 396.7 µm in the hepatic vein at 1 hour which is equal to 793.4 nmol/well whereas the measured concentration in vitro was 782.85 nmol/well. even with a clearance value as low as the 2.5 percentile (3 µm 3 /sec), the concentration at 24 hours was modelled to be lower than the in vitro measured value (in vivo model: 98.7 µm which corresponds to 197.4 nmol/well; measured in vitro concentration: 979.2 nmol/well). discussion: this is the first attempt to use kinetic data obtained in vitro to feed in in a pbhkm for reverse dosimetry finding the dose which corresponds in vivo to the in vitro situation. in the case presented here, the in vitro dose assumed to be low in vitro (100 µm) corresponds to a dose of 1480 mg (note: the highest approved daily dose is 2,400 mg). for the high in vitro dose modelling was successful only for the concentration 1 hour after dosing and a dose of 8,500 mg. conclusions: in vitro kinetic parameters, such as clearance, can successfully be used for parametrizing a pbhkm. it is of utmost importance for the relevance of in vitro finding to assure that the concentrations used in vitro can be obtained with relevant in vivo doses. in this case, the in vitro concentrations were within (low dose) and 3.5 fold above (high dose) the in vivo relevant therapeutic concentration range. introduction: a variety of drug residues have been detected in sewage plant run-offs, rivers and lakes, but also in groundwater and tap water samples. studies have yet to identify a risk for human health from these contaminants, but adverse health effects have been reported for various species, including fish and birds. it has recently been suggested that for a comprehensive risk assessment toxicologists should also consider transformation products (tps) of such water contaminants that may arise from abiotic and biotic (metabolic) reactions. with aciclovir (acv), a well-known antiviral drug, as the parent drug we tried an in-silico approach to identify tps that might be of interest due to some mutagenic or carcinogenic toxicophores. methods: from a literature and database search we picked up 12 acv-tps. predicted acute toxicities and mutagenic / carcinogenic properties for these tps were derived from an expert system analysis using the lazar portal (http://lazar.in-silico.ch/) as front end. results: two of the identified acv-tps could not be handled by lazar because of insufficient training data in one out of eight queried categories. the highest score (4 positive out of 8 possible genotoxicity categories) was assigned to 9 of the 12 tps, including acv itself. this is a rather low score when compared to other water-borne drug residues, e.g. carbamazepine. cofa, an imidazole derivative of acv seen in advanced oxidation processes, had shown antiproliferative effects in several ecotoxicologic screening assays, e.g. [1], but was unremarkable in our tests. additionally, a computer-based simulation of the respective tps interacting with human cyp isozymes did not support concerns that these tps may pose a risk for human health. conclusions: our in-silico analyses of 12 acv-tps did not provide evidence for any adverse health effects in the micromolar concentration range. further studies are needed to clarify if the biological activity of some acv-tps in ecotoxicological assays may eventually affect yet unidentified biological targets in the human body. sulfur mustard (sm) is a chemical warfare agent which was first used in world war i, but has found use in several conflicts afterwards. although sm is prohibited by geneva protocol, terroristic attacks cannot be ruled out. latest news give rise to concern that is may be in the possession of sm and is willing to deploy it. even 200 years after the initial synthesis of sm its mode of action is not fully unraveled. thus, no antidote does exist. however, chemosensing ion channels have been shown to be activated by highly toxic chemicals and might represent a specific therapeutic target. previous studies have shown that the sm-surrogate cees (mono-functional alkylating agent) is able to activate transient receptor potential ankyrin 1 (trpa1) channels that are known to affect mapk cell signaling. mapk-pathways, especially perk1/2, are known to increase protein biosynthesis through activation of transcription factors binding to the serum response element (sre). it is unknown whether alkylating agents have also impact on mapk signaling mediated through trpa1 activation. our results demonstrate that aitc resulted in phosphorylation of the mapk perk1/2 and increased protein biosynthesis of sre-regulated genes in hek293 cells overexpressing htrpa1. cees increased perk1/2 levels already after 2.5 min which could be prevented by the trpa1 blocker ap18. activation of target genes through perk1/2 signaling was also evident, but less pronounced compared to aitc. our results demonstrate that alkylating agents have impact on cell signaling through trpa1 channel activation. thus, trpa1 might represent a promising target for counteracting sm toxicity. sulfur mustard (sm) is a chemical warfare agent that provokes severe inflammation and blistering upon exposure to the skin accompanied by disturbed wound healing. the potential use of sm in terroristic assaults amplified the interest in understanding the underlying cellular and molecular pathomechanisms in order to improve therapeutical intervention. autophagy is a highly conserved catabolic pathway in eukaryotes that ensures the degradation and recycling of cellular components through the lysosomal machinery. autophagy is important for cell survival in physiological and pathological stress situations. emerging knowledge indicates that imbalanced regulation of autophagy disturbs basal cell functions including proliferation, differentiation and migration, thus contributing to the pathophysiology of various diseases. after penetration into skin cells, sm alkylates and thereby modifies nucleic acids and proteins thus forming aggregates of dysfunctional proteins destined for autophagic disposal. in our studies, we analyzed the influence of sm on protein expression (western blotting) of autophagy-related (atg) genes as well as proliferation (wst-1) of primary normal human keratinocytes (nhek) and primary normal human dermal fibroblasts (nhdf). preliminary results demonstrate that sm strongly dysregulates the biosynthesis of atg proteins that may contribute to the diminished cell migration and proliferation under these conditions. our findings suggest that sm affects autophagy in correlation with an impairment of physiological functions in keratinocytes and fibroblasts that are essentially required for normal tissue regeneration. thus, application of pharmacological modulators of autophagy might be useful in the treatment of the delayed wound healing in skin upon exposure to sm. exposure of the respiratory tract to airborne particles is a major risk to human health. due to the ubiquitous application of these particles in the field of pharmacy, industry and in daily life, there is a strong necessity to investigate the toxic properties and the underlying pathomechanisms of these inhalable substances. in addition, the eu chemicals regulation requires not only that all substances placed on the market have to undergo a toxicological characterization, including the identification of potential toxic inhalation hazards (reach), but also that animal testing shall be undertaken only as a last resort ("3rs" principle) and the promotion of the development of alternative methods. thus, the development, establishment and validation of alternative in vitrobased test systems for the assessment of pulmonary toxicity are in the focus of current research. until now, most of the available in vitro cell culture models are limited to some extent as in those studies the exposure is either done under submerged conditions, not resembling the exposure conditions in vivo, or a homogeneous particle distribution is not guaranteed. the cultex ® radial flow system (rfs) is a specially designed in vitro modular exposure system that overcomes these limitations. it enables the homogenous exposure of human lung epithelial cells at the air-liquid interface (ali), thereby mimicking the physiological conditions of the alveolae. however, further optimizations are needed for the enhancement of the cultex® methodology. aim of this study was first the optimization of the test methodology in general (i.e. focus on clean air controls of the human lung epithelial cell line a549), and second the improvement of cultivation conditions. parameters such as handling of the cultex® device (proper closing and opening operation of the cultex® rfs module; improved washing conditions and media supply), treatment of the incubator controls, adjustment of clean air pressure and flow rates, and integration of two additional filters were sequentially adjusted in order to enhance the methodical setup. our results show that the test parameters for clean air exposure of the a549 cells were successfully optimized resulting in more accurate and robust data. cultivation conditions were improved by changing from closed-wall cell culture inserts to open-wall cell culture inserts. the openwall inserts turned out to be more suitable for exposure experiments as they provided a better medium supply and preserved humidity. deductive, the change of the cell culture inserts was identified as the deciding factor for the improvement of cell morphology. hence, we have successfully optimized the cultex ® rfs methodology for clean air exposure of a549 cells. human primary hepatocytes represent the gold standard in in vitro liver research. due to their low availability and high costs, alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. the human hepatocarcinoma cell line hepg2 is often used as a model for liver toxicity studies. however, under two-dimensional (2d) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers is very low. cultivation for 21 days in a three-dimensional (3d) matrigel culture system has been reported to strongly increase the metabolic competency of hepg2 cells. in our present study we extended previous studies and compared hepg2 cell cultivation in three different 3d culture systems: collagen, matrigel and alvetex culture system. cell morphology, albumin secretion, cytochrome p450 monooxygenase (cyp) enzyme activities, as well as expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. our results show that the previously reported increase of metabolic competency of hepg2 cells is not primarily the result of 3d culture but a consequence of the duration of cultivation. hepg2 cells grown for 21 days in 2d monolayer exhibit comparable biochemical characteristics, cyp activities and gene expression patterns as all 3d culture systems used in our study. however, cyp activities did not reach the level of heparg cells. in conclusion, the increase of metabolic competence of the hepatocarcinoma cell line hepg2 is not due to 3d cultivation but rather a result of prolonged cultivation time. in vitro assessment of the neurotoxic potential of arsenolipids arsenolipids are organic, lipid-soluble arsenic compounds, which occur mainly in marine organisms. major human exposure routes are fatty fish including herring or fish oil-based food supplements. about 55 different arsenolipids have been identified so far. thereby, arsenic-containing hydrocarbons (ashc) and arsenic-containing fatty acids (asfa) represent two subgroups of the arsenolipids [1]. our in vitro studies have demonstrated high cellular bioavailability and a high cytotoxic potential of ashcs in human liver and bladder cells [2] , whereas asfas were less toxic [3] . a substantial transfer across an intestinal barrier model (caco-2) indicated that ashcs are highly intestinal available. in comparison, asfas showed lower intestinal bioavailability and underwent a presystemic metabolism [4] . moreover, in drosophila melanogaster ashcs exerted late developmental toxicity and accumulated in the fruit fly's brain. these results suggest that ashcs might pass the blood-brain-barrier due to their amphiphilic structure [5] . in order to assess the neurotoxic potential we currently investigate the toxicity of several arsenolipids in differentiated, human neurons (luhmes). after 48 h incubation with ashcs or asfas, cell number (hoechst) as well as cellular dehydrogenase activity (resazurin) were measured, with the latter endpoint turning out to be more sensitive. ashcs showed substantial cytotoxic effects (ic 50 ~ 7-12.5 µm) in a concentration range comparable to that of arsenite (ic 50 ~ 7.5 µm), whereas asfas were less cytotoxic (ic 50 > 100 µm). after incubation with ashcs the cellular arsenic concentrations increased 10-20fold as compared to incubation with arsenite. further studies indicated that one possible toxic mode of action of arsenolipids could be a disruption of the cellular energy level. therefore, the mitochondrial membrane potential was investigated after incubation with the arsenic compounds in differentiated neurons. whereas arsenite did not exert an impact, ashcs reduced the mitochondrial membrane potential significantly. this might be due to interactions of the amphiphilic ashcs with mitochondrial membranes. currently we investigate the impact of the arsenolipids on neurite outgrowth as a developmental toxicity endpoint. standard treatment of poisoning by organophosphorus compounds (op; e.g. nerve agents and pesticides) consists of co-administration of atropine and an oxime-based reactivator of inhibited cholinesterases. due to lack of efficacy of clinically used oximes against various op-inhibited human acetylcholinesterase (ache) (e.g. soman) research started focusing on new therapeutic approaches. several research groups conducted in silico screenings [1, 2] in order to identify new non-oxime reactivators, presenting amodiaquine as a promising candidate for paraoxon-inhibited hache. for decades, antimalarial drugs like amodiaquine and chloroquine have been closely investigated regarding their side effects, thereby discovering interaction with cholinesterases, which could pose a new potential therapeutic benefit for inhibited cholinesterases. therefore, in this study interactions between antimalarial agents in presence or absence of ops were examined spectrophotometrically by a modified ellman assay. reversible inhibition of cholinesterases was observed with both antimalarial agents. amodiaquine had higher inhibitory potency for hache than human butyrylcholinesterase (bche), being confirmed by ic 50 values of 0.67 ± 0.02 µm for hache and 81.28 ± 0.04 µm for hbche. ic 50 values with chloroquine were 28.37 ± 0.02 µm for hache and 55.62 ± 0.02 µm for hbche, thus representing a weaker inhibition of hache than amodiaquine. furthermore, reactivation of paraoxon-(pxe), sarin-(gb), cyclosarin-(gf), and vxinhibited hache and hbche by amodiaquine and chloroquine was determined. after 60 minutes, only paraoxon-inhibited hache (50%) and cyclosarin-inhibited hbche (10%) were reactivated by 500 µm chloroquine. on the contrary, 10 µm amodiaquine reactivated all tested ops after 60 minutes in the following order: pxe > vx > gf > gb. in contrast, with hbche the highest reactivation was generated with 100 µm amodiaquine in the following order: vx > gb > gf > pxe. due to the high reversible inhibitory potency of amodiaquine, an increased concentration does not result in a higher reactivation of op-inhibited hache. in summary, our results show that amodiaquine is a reactivator of op-inhibited cholinesterases. in the future, non-oxime reactivators that are structurally-related to amodiaquine should be further investigated. [1] bhattacharjee, a.k., marek, e., le, h.t., gordon, r.k., eur. j. med. chem., 2012, 49, 229-238. [2] katz, f.s., pecic, s., tran, t.h., trakht, i., schneider, l., zhu, z., ton-that, l., luzac, m., zlatanic, v., damera, s., macdonald, j., landry, d.w., tong, l., stojanovic, m.n., chembiochem., 2015 , 16, 2205 -2215 bundesinstitut für risikobewertung, lebensmittelsicherheit, berlin, germany development of mammary gland tumors is connected to a deregulation of breast epithelial cell differentiation, a complex process which cannot be reproduced in vitro under standard cell culture conditions. however, cultivation of cells in a tissue-like environment in an in vitro three dimensional (3d) model can mimic general architecture, function and differentiation of mammary bulks. in this project, a 3d model was used consisting of the permanent breast epithelial cell lines mcf10a (er -, estrogen receptor negative) and mcf12a (er + , estrogen receptor negative) grown in matrigel tm , which mimics the complex extracellular matrix in vivo. the 3d culture of mcf10a and mcf12a cells in matrigel tm results in the formation of growth-arrested, polarized spheroids with a lumen (acini-like organoids). in order to perform a semi-quantitative estimation on the influence of substances on the differentiation of the breast cells for the identification of non-genotoxic carcinogens a scoring method was developed. this scoring method provides information about substance-induced morphological changes of the spheroids during differentiation based on the following parameters: size of the spheroids, the formation of the lumen, and the degree of polarization. furthermore, the model allows distinguishing between erdependent (mcf12a) and er-independent (mcf10a and mcf12a) effects. the 3d in vitro model is a useful tool for toxicologists to study substance effects on differentiation processes. the system will be used to examine the potential of e.g. food contaminants such as phthalates or perfluorinated substances (pfas) to disrupt the differentiation process of breast epithelial cells and will therefore serve as a valuable in vitro tool to assess their carcinogenic potential. inflammatory episodes occur erratically throughout life and are likely to play a critical role in the alteration of the individual susceptibility of a person to idiosyncratic druginduced liver injury (idili), a particular severe form of drug-induced liver injury (dili). in concordance with the inflammatory stress hypothesis, modest inflammatory stress can lower the threshold for hepatotoxicity and make an individual susceptible to develop liver injury during exposure to therapeutic doses of a drug. in order to evaluate the role of immune cells and its secreted factors during drug therapy, we established an in vitro test battery consisting of two cell culture systems in presence or absence of proinflammatory factors (lps, tnfα): (a) the monoculture of human hepatoma (hepg2) cells and (b) co-culture systems of human monocytic or macrophage-like (thp-1) and hepg2 cells. with these different test settings we aimed to identify whether the introduction of inflammatory immune cells and/or pro-inflammatory factors could increase the sensitivity of liver cells towards idili compounds. three reference substance pairs were tested, namely troglitazone -rosiglitazone, trovafloxacin -levofloxacin, and diclofenac -acetylsalicylic acid, each of them being composed of a compound that is known to induce idili and a partner compound of the same substance class that does not induce idili. first, all compounds were tested for cytotoxicity towards the single cell systems using the wst-assay. co-culture experiments with hepg2 and thp-1 monocytes or macrophages as well as co-exposure experiments with lps or tnfα were then done at about 20% cytotoxicity of the respective substance in the most sensitive cell type. subsequently the results were compared to the experiments in the monoculture of hepg2. we observed that every idili compound showed a significant increase in cytotoxicity in a minimum of one exposure combination while this effect was not observed with the corresponding non-dili partner compound. in conclusion, a combination of different culture systems and co-exposures with proinflammatory factors is needed for a valid differentiation between non-dili and idili compounds. this test battery could provide a useful tool for the prediction of inflammation-associated idiosyncratic drug-induced hepatotoxicity. furthermore, our results support the inflammatory stress hypothesis and points to an involvement of proinflammatory factors in the development of idili. extensive animal models of carcinogenicity ensure a safe usage of chemicals. to elucidate fundamental molecular mechanisms of carcinogenicity these methods are expensive, time consuming and above all too complex. in contrast, most in vitro methods are rather simple and detect only selected endpoints, like dna damage, mutations or changes in proliferation. the balb/c cell transformation assay is a validated toxicological method to identify potential tumour initiators and promotors. first, balb/c mouse fibroblasts form a monolayer culture and get contact-inhibited after reaching confluence. upon treatment with a tumour initiator (3-methylcholanthrene) and promotor (12-o-tetradecanoylphorbol-13-acetate) transformed cells do not stop proliferation and grow as morphologically aberrant foci over the monolayer of normal cells. after fixation with methanol at day 42, morphological aberrant foci can be visualized with giemsa staining. because the balb/c assay mimics different stages of the malignant cell transformation process (initiation, promotion and post-promotion phase) and detects with the colony formation a late endpoint of carcinogenicity we improved this method for mechanistic cancer research. using the example of insulin-signalling pathway we can show that (1) several substances have a different impact on the transformation process, (2) it is possible to identify for each substance the phase with the greatest effectiveness and (3) we can detect additional endpoints to elucidate the mechanistic mode of action. therefore we used several compounds (linsitinib, metformin, rapamycin, …) to manipulate the insulin-signalling pathway on different levels (insr, ampk, mtor, …) and analysed a number of characteristic endpoints of carcinogenesis. changes on protein level and signalling (westernblot, immunofluorescence, flow cytometry) or parameters of energy metabolism (oxygen consumption, glucose or atp measurement) are measurable and enable new insights into the process of cancer origin. summing up, the balb/c 3t3 assay proves to be a cheap and short-time alternative to rodent bioassays. although this method does not mimic the whole in vivo neoplastic process, it can be used to provide essential information regarding key proteins and their signalling, during the different stages of transformation. is there hope to correctly classify severe ocular irritant agrochemical formulations using in vitro methods: a proof of concept using the isolated chicken eye test, two modified bcop protocols and an epiocular™ et50 protocol while some in vitro methods addressing ocular irritancy have gained regulatory acceptance, to date the draize rabbit eye test (oecd tg 405) is the only world-wide regulatory accepted test for the determination of the full range of eye irritation potential. further although several in vitro methods for the severe eye irritation have gained regulatory acceptance, agrochemical formulations are nor explicitly included nor excluded from the applicability domain to predict severe ocular irritant formulations. systematic analyses are only available for e.g. the hen's egg test-chorioallantoic membrane (het-cam), and bovine corneal opacity and permeability (bcop, oecd tg 437) assays both showing that the used protocols do not provide sufficient sensitivity to reliably predict severe ocular irritating formulations. the purpose of this study was to evaluate whether the regulatory accepted isolated chicken eye (ice, oecd tg 438) test including corneal histopathology (as suggested for evaluation of the depth of injury), as well two modified protocols of the bcop and/or an et50 (exposure time reducing viability of treated tissue to 50%) protocol using the reconstructed cornea model epiocular™ are useful to predict severe ocular irritant agrochemical formulations. a proof of concept comprising the testing of ten to twelve agrochemical formulations with available in vivo data in each assay was conducted. in summary, based on the ice evaluation described in oecd tg 438, one of the five severe ocular irritant formulations (un ghs cat 1) was predicted correctly. using both modified protocol versions of the bcop the result for one of the four tested un ghs cat 1 formulations was just above the un ghs cat 1 classification border for using one of the modified protocols. lastly and most promising, the epiocular™ et50 predicted four of five tested un ghs formulations correctly with the fifths being close to the classification border. additional agrochemical formulations will be tested to further evaluated the epiocular™ et50 protocol to identify severe ocular irritant agrochemical formulations. drug-induced pancreatic toxicity comprises effects on the exocrine and/or the endocrine pancreas, which both can have serious clinical implications, e.g. acute pancreatitis or diabetes mellitus. adverse effects on the pancreas are occasionally observed during drug discovery and development and often prohibit further development. hence, there is a need for reliable in vitro models to early on identify the pancreas-toxic potential of drug candidates. permanent cell lines and primary cells have many shortcomings, e.g. loss of cell-to-cell and cell-to-matrix relationships or changes in cell physiology due to the isolation procedure. pancreas tissue slices are a potential alternative, circumventing most of these limitations. their preparation is rather elaborate which may explain its rare use. so far, pancreas tissue slices have predominantly been used to address physiological or pharmacological questions, although they might also serve as valuable in vitro model for toxicological applications. therefore, this work aimed to establish and characterize rat pancreas tissue slices as in vitro model for studying drug-induced pancreatic toxicity. results will be compared to the responses of the permanent endocrine (ins-1e) and exocrine (ar42j) pancreatic cell lines to evaluate a potential added value. rat pancreas tissue slices were prepared by a protocol adapted from marciniak et al. (nat protoc, 2014 . 9(12): p. 2809 . briefly, pancreas was infused and embedded with agarose. tissue sections of app. 200 µm were prepared using a vibratome and maintained in cell culture medium for up to 6 days. cell viability was determined by daily measurement of lactate dehydrogenase (ldh) in medium supernatants and by microscopic evaluation following fixation in 10 % formalin and h&e staining. functional integrity of acinar and beta cells were assessed by cell-type specific secretory responses (i.e. insulin, amylase, lipase) to physiological stimuli. moreover, the effects of the pancreas toxins streptozotocin (stz), alloxan (all), and the cholecystokinin (cck) analogue cerulein on the viability and functional integrity of tissue slices were compared to the respective responses of the cell lines. we were able to establish an optimized isolation and cultivation procedure for rat pancreas tissue slices applying minor modifications to the original protocol. cell viability declined over the cultivation period. stimulation of the cell lines with glucose or cerulein increased secretion of insulin (ins-1e cells) or amylase/lipase (ar42j cells), respectively. the pancreas slices responded to both stimuli, demonstrating functional integrity of endocrine and exocrine cells. treatment of ins-1e islet cells with the betacell toxicants all or stz only slightly affected islet cell viability, whereas treatment of ar42j acinar cells with cerulein at supraphysiological concentrations had no effect. this set of experiments is currently completed by investigating the effects of all, stz and cerulein on the viability of acinar and islet cells in pancreas slices. our preliminary data demonstrate feasibility to prepare and cultivate rat pancreas tissue slices over a period of 6 days thereby maintaining functional integrity to some extent. coculture of human monocytes with the keratinocyte cell line hacat in serumcontaining medium leads to higher sensitivity to weak contact allergens: an improvement for the loose-fit coculture-based sensitization assay (lcsa) a. sonnenburg 1 , j. the loose-fit coculture-based sensitization assay (lcsa) has proved reliable for the in vitro detection of contact sensitizers in the past. however, the use of primary human keratinocytes has some disadvantages. to facilitate high throughput screening of chemicals, we replaced primary keratinocytes from the original assay setup (setup a) by the human keratinocyte cell line hacat. these cells were cocultured with monocytederived dendritic cells in serum-free medium (setup b) or fetal calf serum (fcs)containing medium (setup c). upregulation of the dendritic cell maturation marker cd86 assessed by flow cytometry served as endpoint. we have tested four substances known as sensitizers and four non-sensitizers in both new setups as well as in the original setup with primary cells. three out of four sensitizers (2,4-dinitrochlorobenzene, 2-mercaptobenzothiazole, and coumarin) , and three out of four non-sensitizers (glycerol, monochlorobenzene, and salicylic acid) were correctly assessed under all culture conditions. the weak sensitizing potency of resorcinol was only detected by setup b with fcs supplemented medium. a false positive reaction to caprylic (octanoic) acid in all three setups confirms earlier results from our laboratory that some fatty acids are able to induce cd86 on dendritic cells in vitro. culture in fcs supplemented medium led to generation of dendritic cells showing a more pronounced upregulation of cd86 after application of substances with rather high sensitization potency compared to dendritic cells which are formed under serum-free conditions. therefore, we characterized dendritic cells from setups b and c by flow cytometric measurement of additional dendritic cell surface markers. dendritic cells from the original setup a had been characterized extensively before (schreiner et al., toxicology 2008; 249:146-1529) . dendritic cells generated in fcs supplemented medium were cd1a+/cd1c+, whereas dendritic cells from serum free culture conditions were cd1a−/cd1c− regardless whether cocultured with primary human keratinocytes or hacat. populations with cd1a+/cd1c+ dendritic cells in coculture seem to show a higher sensitivity to weak sensitizers, which proved beneficial for the identification of resorcinol. in conclusion, modification of the lcsa protocol led to an increased sensitivity of the assay. due to ethical and social reasons, in vitro assays are being developed to replace animal tests for addressing e.g. toxicological questions. for the induction of skin sensitization by chemicals, resulting in tolerance or allergic contact dermatitis after repeated exposure, prerequisites are the induction of inflammatory responses in keratinocytes supporting maturation of dendritic cells (dc), which is needed for the t cell response. although related in vitro assays consisting of one single cell type have good hazard prediction capacities, they have limitations in predicting sensitization potency. one drawback could be the lack of communication between keratinocytes and dc. with respect to the activation of keratinocytes and maturation of dc, intercellular communication between these two cells may include the release of danger molecules such as cytokines, damage-associated molecules such as atp, and metabolized chemicals. beside this, microrna (mirna), among them those that can regulate dc activation or maturation, can be differentially expressed upon stimulation but can also be transferred between cells. for skin sensitizers, we reported already that cross talk between hacat keratinocytes and thp-1 cells, as model for dc, enhanced cyp1 enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol, belonging to a subgroup of chemicals (prohaptens) whose sensitizing potential depend on prior metabolic activation e.g. via cytochrome p450 (cyp) enzymes. furthermore, coculture clearly increased the upregulation of the cell surface molecule cd86 on thp-1 cells after incubation with these prohaptens and also several other skin sensitizers. in this study we further elucidate the cross talk between thp-1 cells and hacat cells by analyzing the impact of hacat cells on the expression of mirnas in thp-1 cells by microarray technology. we identified 6 differentially expressed mirnas in cocultured thp-1 cells compared to monocultured thp-1 cells irrespective of the treatment (medium, 0.2% dmso as solvent control, b[a]p). in the presence of dmso and b[a]p (after 48h) 8 additional mirnas are differentially expressed. up to now it is not clear whether the cross talk between hacat and thp-1 cells comprises the exchange of mirna between the cocultured cells or whether it influences the expression of these mirna in thp-1 cells, or both. given that one mirna has several gene targets these results illustrate that the cross talk between thp-1 and hacat cells also impacts on the mirnome. walther-straub-institut der lmu-münchen, münchen, germany transient receptor potential (trp) proteins represent a large superfamily of nonselective cation channels sensing toxic stimuli in the human body. trpa1 expresses a high number of aminoterminal ankyrin repeats and is the only member of the trpa family. channel monomers form homotetramers in the plasma membrane with six transmembrane segments (tm) and a pore forming loop between tm5 and 6. trpa1 has been extensively described in sensory nerve endings as an important cellular detector for toxic stimuli and as an oxygen sensor (reviewed in 1). although recently two reports identified trpa1 in pulmonary epithelial and endothelial cells (2, 3) , its expression in non-neuronal tissues is still a matter of debate. after isolation and identification of different murine lung cells we were able to identify murine trpa1 protein in primary endothelial cells, pneumocytes type ii (atii) and fibroblasts by using specific antibodies in a western blot analysis, but not in cells from trpa1-deficient mice. atii cells were identified by specific cell markers such as surfactant protein c and were further differentiated to ati cells characterized by their specific expression of podoplanin. quantitative trp expression patterns will now be evaluated by quantitative reverse transcription (rt)-pcr as well as utilizing nanostring ® technology in different lung cells. to characterize trpa1 on a cellular level we cultured a hek293 cell line stably expressing trpa1 (4) . allylisothiocyanate (aitc) a specific activator as well as hypoxia and hyperoxia was able to induce ca 2+ -influx in this cell line, which was blocked by the specific inhibitor a96079. in the future, we will utilize the isolated perfused lung model (5) to quantify toxin-induced edema formation in ex vivo lungs from wt and trpa1-deficient mice after exposure to potential toxic inhalation hazards (tih see 6) to challenge the hypothesis of trpa1 as an important toxin sensor in the lung. by this strategy we hope to understand trpa1 function in lung cells and to evaluate trpa1 proteins as potential pharmacological targets for a specific therapeutic intervention during toxin-induced edema formation. metabolism by the intestinal microbiota is likely to contribute essentially to the plasma metabolite profile of the mammalian host organism and it requires adequate identification of effects of the microbiome on the endogenous plasma metabolite patterns. the current investigations present insights in the mammalian-microbiome cometabolism of endogenous metabolites. antibiotics have a profound effect on the micro-organism composition of the microbiome and hence on the mammalian-microbiome co-metabolism. the consequences, however, on the functionality of the microbiome (defined as the production of metabolites absorbed by the host) and which of these changes are related to the microbiome are not well understood. to identify plasma metabolites related to microbiome changes due to antibiotic treatment, we have employed a metabolomics approach. to this purpose broadspectrum antibiotics belonging to the class of aminoglycosides (streptomycin, neomycin, gentamicin), fluoroquinolones (moxifloxacin, levofloxacin) and tetracyclines (doxycycline, tetracycline) were administered orally for 28 days to male rats including blood sampling for metabolic profiling after 7, 14 and 28 days. fluoroquinolones and tetracyclines can be absorbed from the gut whereas aminoglycosides cannot. to distinguish between metabolite changes caused by systemic toxicity of the antibiotics and microbiome related changes, the metabolites identified in the metabolome pattern were compared to a list of metabolites known to be produced by the gastro-intestinal micro-organisms. beside changes mainly concerning amino acids and carbohydrates, hippuric acid and indole-3-acetic acid were identified as key metabolites being affected by antibiotic treatment. for each class the following gut metabolites were found to be unique: indole-3-propionic acid for aminoglycosides, taurine for fluoroquinolones, 3indoxylsulfate, uracil and allantoin for tetracyclines. for each class of antibiotics specific and selective metabolome patterns could be established. the results suggest that plasma based metabolic profiling (metabolomics) could be a suitable tool to investigate the effect of antibiotics on the functionality of the microbiome and to obtain insight in the mammalian-microbiome co-metabolism of endogenous metabolites. drug-induced liver injury (dili) is still a major reason for termination of clinical trials and thus is an important concern in drug development. identification and prediction of dili in the clinic and in preclinical safety testing still relies on the classical clinical chemistry panel and histopathology with known limitations in sensitivity and specificity. in the last years bile acids (bas) have been studied as potential biomarkers to better characterize drug-induced liver injury with promising results (ellinger-ziegelbauer et al., 2011; luo, schomaker, houle, aubrecht, & colangelo, 2014; yamazaki et al., 2013) . to evaluate whether a targeted bile acid profiling via lc-ms/ms in plasma and liver tissue can improve assessment of liver injury, methapyrilene (mpy) a known hepatotoxin, or corresponding vehicle, was administered daily to male wistar rats at a low (30 mg/kg) and a high (80 mg/kg) dose. rats were sacrificed following 3, 7, or 14 consecutive daily doses, or after recovery 10 days following 14 consecutive administrations of mpy or vehicle. in addition to bile acids which were determined both in plasma and tissue, conventional preclinical safety endpoints (histopathology and clinical chemistry) assessment and gene expression profiling was performed in liver to obtain mechanistic information about potential changes in regulation of bile acid levels. conventional findings included periportal necrosis, inflammation and biliary hyperplasia, and increased liver enzyme activity and bilirubin levels during the treatment phase. the bile acid pattern showed increased levels of conjugated and unconjugated bile acids in low dose and high dose groups compared to the controls after administration of methapyrilene. furthermore, although liver enzyme activity and bilirubin levels in serum were decreased again in the recovery groups, suggesting recovering liver injury, bile acid concentrations remained elevated with no signs of recovery. analysis of transcriptomics data revealed decreased levels of mrna encoding α-methylacyl-coa racemase (amacr) 4 and 15 days after dosing, a gene responsible for bile acid synthesis. membrane transport systems for bile acids like sodium/taurocholate co-transporting polypeptide (ntcp) and organic anion transporting polypeptide 1 (oatp1) expression were down regulated as well, indicating that the increased bile acid concentrations in plasma and tissue could be attributable to reduced uptake by the hepatocyte. in summary the data suggest that targeted bile acid profiling could be used as potential biomarkers to enhance assessment of drug-induced liver injury. photorhabdus asymbiotica is an entomopathogen and emerging human pathogen causing soft tissue infections in humans. photorhabdus asymbiotica produces the bacterial protein toxin patox, which is cytotoxic for various cell lines and kills insect larvae. previous studies have established that patox harbors two enzymatic active domains, a glycosyltransferase and a deamidase domain. the glycosyltransferase domain inactivates host gtpases of the rho family by glcnacylation of a tyrosine residue in the effector binding loop, which results in the disassembly of the actin cytoskeleton. the deamidase domain deamidates a crucial glutamine residue in heterotrimeric gα i and gα q/11 proteins, which renders the g proteins constitutive active. sequence and structural homology analyses of patox revealed a third domain (patox p ) resembling peptidases of the c58 protease family. patox p contains the conserved catalytic triade (c/h/d) of papain-like cysteine proteases and shares sequence similarity with effectors from yersinia pestis (yersinia outer protein yopt) and pseudomonas syringae (avirulence protein avrpphb). transient expression of patox p in hela cells induces cell rounding and indicates a cytotoxic potential of patox p . incubation of patox p with linearized bovine serum albumin (bsa) results in cleavage products of bsa assuming proteolytic activity of patox p . mutation of the catalytic cysteine in patox p prevents cleavage of bsa and blocks cytotoxicity. we were not able to observe autocatalytic cleavage of patox constructs under various conditions. the intracellular activity of the protease domain is most likely involved in the pathogenicity of patox. vitamin d metabolism -involved in triazole fungicide toxicity? a. lehmann 1 1 background: in a 28-day rat feeding study with the azole fungicides cyproconazole (c), epoxiconazole (e), propiconazole (p), tebuconazole (t), prochloraz (pz) as well as combinations c+e and c+e+pz, a reduction of vitamin d (vitd) receptor mrna levels was reproducibly observed in adrenals for c, e and p. transcription of various enzymes related to vitd homeostasis (including cyp2r1, gc, cyp3a, ugt1a) in liver was also affected, while initial indications for modulation of renal cyp24a1 and renal and hepatic cyp27b1 could not be confirmed. a possible induction of parathormone (pth) was noted for the high dose of c, but statistical significance could not be shown. we have now performed supporting analyses for serum vitd levels, measured additional transcript levels and will provide a framework for the interpretation of the findings. methods: male wistar rats (n=5 for single substances, n=10 for combinations) were treated for 28 days at dose levels tested based on noaels from 90-day subchronic feeding studies and ranged from noael/100 to noaelx10. quantitative rt-pcr analyses were performed on organ samples obtained at sacrifice. serum vitd levels were determined using the total (25-oh) vitamin d elisa (drg instruments gmbh, marburg, germany). results: the elisa established for diagnostic analysis of human serum and plasma samples could be applied to rat serum. vitd levels in control animals (n=30) were 64.7±8.3 ng/ml (min/max: 48/78 ng/ml), i.e. in the range of values reported previously for rats. for the high dose of c (1000 ppm in food, n=5), there was a statistically nonsignificant reduction of vitd levels to 71.3±17.6% of the concurrent control (n=5). however, for 4 of 5 animals of this group, measured vitd level were below the range observed in pooled controls (n=30). an according follow-up is ongoing. qrt-pcr analysis of adrenal tissue showed deregulation of apoptosis related genes (p21 for c, e and pz; cdk1 and gadd45a for e; cdkn1c for c), which is in agreement with an involvement of vitd in the autocrine/paracrine regulation of cell proliferation. conclusion: reduction of circulating vitd levels would be plausible as a result of induction of hepatic cyp3a1/2 and ugt1a. however, this could not be confirmed by elisa as a general mechanism for all azole fungicides under investigation. only for rats fed with 1000ppm cyproconazole, there were indications for a moderate reduction of 25-oh vitamin d, which would correlate with the previously reported moderate increase in serum pth for this group. hansen's disease during pregnancy and lactation: two babies born to a mother using antileprosy drugs z. ozturk hansen's disease, also known as leprosy, during pregnancy has been rarely reported in europe and united states. early diagnosis is important, and medication can decrease the risk of those living with leprosy patients from acquiring the disease. this report presents a case of multidrug antileprosy therapy during pregnancy and lactation. a 26-year-old multiparous woman with a known case of multibacillary leprosy presented with unplanned pregnancy. her pregnancy was discovered in the 9th week, and she has been taking a multidrug therapy (dapsone 100 mg/day, rifampicin 600 mg/month, clofazimine 50 mg /day and clofazimine 300 mg/month) for the past 8 months. diagnosis of leprosy was established in her previous pregnancy. the patient was informed about the risks of drugs used in pregnancy. the treatment was continued unchanged during pregnancy. a detailed fetal ultrasonography was offered to scan the development of the fetus at about 20 weeks. in the 8th, 22nd, 28th weeks of pregnancy, prenatal sonographic examinations revealed normal fetal growth and amniotic fluid volume. at 28 weeks pregnant, she was diagnosed with gestational diabetes. diabetes did not cause any symptoms during pregnancy, and it was controlled with a reduced-calorie diet in a week. the patient delivered a healthy baby girl by vaginal birth in the 39th week of gestation without perinatal complications. the baby was also healthy (apgar 8-9,3300 g,51 cm), and its growth and development were normal during a 6-month follow-up period. the patient decided to breastfeed while taking medication. she had a previous experience with use of anti-leprosy drugs while breastfeeding, her other child was 15 months old and healthy. as well as in the first child, skin discoloration was observed in newborn due to clofazimine during lactation. after 3 months, she stopped breastfeeding, and the infant's skin changes were reversed. for pregnant women and practitioners, treatment of leprosy in pregnancy can be complicated. physical and neurological damage may be irreversible even if cured. multidrug therapy consisting dapsone, rifampicin and clofazimine is highly effective for people with leprosy and considered safe, both for the mother and the child. antileprosy drugs are excreted into human milk but there is no report of adverse effects except for skin discolouration of the infant due to clofazimine. therefore, multidrug therapy for leprosy patients should be continued unchanged during pregnancy and lactation. methods: individuals included in the analysis were participants of the berlin initiative study (bis). bis is a population-based prospective cohort study initiated in 2009 in berlin, germany, to evaluate kidney function in people ≥ 70 years. medication was assessed through personal interviews and coded using the anatomical therapeutic chemical classification system. for estimation of glomerular filtration rate (egfr) we used the ckd-epicr equation. predictor analysis was conducted via logistic regression. results: figure 1 illustrates the percentage of drug use for the three noacs and phenprocoumon, the most common vitamin k antagonist in germany, over the course of four years. table 1 shows the characteristics of patients for each oral anticoagulant group during the four-year follow-up visit (from january 2014 until april 2015). the probability of dabigatran use rose with increasing age (+12%), and the probability of phenprocoumon use rose in case of egfr < 60 ml/min/1.73m 2 (+54%) or male sex (+82%). discussion: our data show that also in the elderly noac use increased over the past years. characteristics such as age, sex or kidney function had an impact on the choice of oral anticoagulation. objective: orthostatic hypotension (oh) is an important factor in determining cardiovascular mortality especially in older age. different factors were discussed to influence oh. arterial stiffness, medication and frailty were demonstrated as modifying factors of oh. the aim of this study was to assess prevalence of and influencing factors on oh in nursing home residents (nhr) in germany. methods: systolic (sbp) and diastolic (dbp) blood pressure as well as pulse pressure (pp) and pulse wave velocity (pwv) as markers of arterial stiffness were measured in nhr aged ≥ 65 years in 12 nursing homes in berlin, germany. measurements were first performed in the sitting position and then repeated after standing up. oh was defined as a sbp decrease of > 20 mmhg and/or dbp decrease of > 10 mmhg within 3 min after standing up. hypertension was defined as the presence of diagnosis arterial hypertension, the prescription of at least one antihypertensive drug, or mean sbp values > 139 mmhg and/or mean dbp >89 mmhg. information about antihypertensive medication was received from interviews and medical records. frailty was determined by geriatric assessments, e.g. "timed up and go test" (tug) or barthel scale. results: oh testing could be performed with 96 nhr (mean age = 84.5 ± 7.3 years). in total, 15 subjects (15.6%) had oh. the mean change in sbp from sitting to standing was 19.2 ± 15 mmhg (range +8.5 to -52.5 mmhg) in patients with oh and 1.5 ±10.9 mmhg (range +44.5 to -17 mmhg) in patients without oh. mean sbp was significantly higher (143.6 ± 17.1 mmhg) in people with oh than in those without (131.5 ± 20.1mmhg). all of the nhr with oh were hypertensive compared to 89% of the nhr without oh. sex, mean age, pwv and pp was not significantly different between individuals with or without oh (p>0.05). medication data was available for 89 patients. all individuals with oh and 60 nhr without oh (80%) had antihypertensive medication. more than 2 different antihypertensive drugs were present in 11 patients with oh (78.5%) and in 43 patients without oh (57.3%). the intake of beta-blockers had no impact on oh development. geriatric assessments did not differ significantly between the oh group and the non-oh group. more than 75% of patients in both groups reached 80 points as maximum in barthel scale defining a need for assistance and tug analyses demonstrated that around 50% of patients with oh as well as patients without oh needed more than 19 sec showing a motor slowing. conclusion: we found a relatively low prevalence of oh in our very old patient cohort and the overall bp control was good. similar to earlier publications mean sbp was significantly higher in nhr with oh. all of the other investigated factors were not associated with the occurrence of oh. the small cohort size might have limited the detection of cardiovascular, epidemiological or geriatric associations. in addition, important confounding factors such as the inability to stand of some nhr and the lack of standardized fraility assessments must be addressed. impact of reticulated platelets on the initial antiplatelet response to thienopyridine loading in patients undergoing elective coronary intervention c. stratz 1 , t. nuehrenberg are known to be involved in cell metabolism pathways and therefore ccrcc is supposed to be a metabolic disease. in order to facilitate a better understanding of cancer metabolism and to support tumor classification on the metabolite level we have developed a novel analytical approach for comprehensive metabolomic profiling of small molecules and lipids in kidney tissue. the method was established and validated based on porcine tissue and, as proof of concept, applied to a small cohort of human normal and ccrcc tissue samples for molecular tissue differentiation. methods: five fresh frozen ccrcc samples and corresponding normal tissue were used for cancer-specific metabolomic profiling and were derived from patients who underwent partial or radical nephrectomy. metabolites and lipids were recovered from tissue samples by a two-step extraction protocol. tissue homogenization and extraction of polar metabolites was performed in methanol/water (aqueous extract) by a beadbeating approach. lipids were recovered by consecutive extraction of the pellet with methanol/methyl tert-butyl ether (organic extract). metabolites in aqueous extracts were separated by hydrophilic liquid interaction chromatography whereas compounds in organic extracts were separated by reversed phase chromatography prior high resolution mass spectrometry. results: reproducibility of tissue extraction and metabolite analysis was assessed by the analysis of multiple individually prepared porcine kidney samples. more than 1000 metabolic features including amino acids, nucleotides, small organic acids, phospholipids, sphingolipids, glycerolipids and fatty acids could be reproducible (cv ≤ 30 %) analyzed with the novel non-targeted metabolomics approach. the validated protocol was applied for metabolomic profiling of kidney tissue derived from ccrcc patients. based on unsupervised multivariate statistics, a clear differentiation between cancerous and normal tissue for the small metabolites profile as well as for the lipid profile could be observed. a first subset of differentially regulated metabolites responsible for tissue differentiation could be tentatively identified. conclusion: metabolomic profiling of kidney tissue extracts enables differentiation between ccrcc and normal kidney tissue samples based on the lipid and small molecule metabolomic profiles. further studies on larger and independent sample groups are necessary to confirm and validate our preliminary findings. in summary, the presented approach provides a first basis for comprehensive metabolomics studies in human kidney tissue and thus offers great potential for the metabolic characterization of ccrcc with important prognostic and therapeutic implications in the future. introduction: clomiphene (clom) citrate as mixture of trans-and cis-isomer (60:40) is the first line therapy for the treatment of infertility caused by the polycystic ovary syndrome. treatment schedule includes dose escalation from 50 mg/d clom citrate to up to 150 mg/d in case of non-ovulation. however, therapy outcome is variable and approximately 10 -30% of patients do not benefit from clom treatment. the pro-drug clom is bioactivated via 4-hydroxylation of trans-clom by the highly polymorphic cytochrome p450 (cyp) 2d6 leading to the major active metabolite trans-4hydroxyclomiphene (trans-4-oh-clom) [1] . recently, we identified a less active trans-3-oh-clom which is also formed by cyp2d6. besides the formation of the active metabolites, their plasma concentrations are influenced by their clearance e.g. via glucuronidation and sulfation. here we investigated the glucuronidation and sulfation of both hydroxyl-metabolites. methods: isoforms of udp-glucuronosyl-transferase (ugt) and sulfotransferase (sult) responsible for conjugation of oh-clom were identified using commercially available supersomes. glucuronidation and sulfation kinetics were determined in pooled human liver microsomes. conjugated clom metabolites were quantified in plasma and urine samples obtained from healthy female volunteers who received a single dose of 100 mg clom citrate. results: incubations with human liver microsomes revealed an almost 60-fold higher glucuronidation rate for trans-3-oh-clom, which is exclusively catalyzed by ugt2b7, compared to the more potent trans-4-oh-clom. for the latter a pattern of multiple ugts was identified. in contrast, the intrinsic clearance of trans-4-oh-clom to its sulfate is 16-fold higher compared to 3-oh-clom. for both metabolites a participation of sult1a1 and sult1e1 was identified. these results were in line with previous studies, which identified the same sults [2] and ugts [3] responsible for the conjugation of the structurally related trans-4-hydroxytamoxifen. in addition, in vivo data from plasma and urine samples confirmed the reverse regioselective glucuronidation and sulfation of trans-3-oh-clom and trans-4-oh-clom. overall, concentrations of clomglucuronides were significantly higher than those of sulfates. highest concentrations in plasma and urine samples were measured for trans-clom-3-o-glucuronide. conclusion: our results suggest a new metabolic route via trans-3-oh-clom which appears to be a potential inactivation pathway of clom. 1 1 institut für pharmakologie und toxikologie der bundeswehr, münchen, germany for decades the biological effect of sm has been investigated. it is well known how sm interacts and destroys cells. unfortunately, it is still unknown if and how a cell can become resistant against sm. within the here described experiments we investigated a new approach adapting cells to the presence of sm. over a time period of nearly three years the cells were cultivated in presence of sm with increasing concentrations. before starting the initial sm sensitivity was investigated. at the beginning cells were cultivated with a concentration of 0.07 µm sm (ic 10 ). today the cells are able to tolerate a concentration of 7.2 µm sm (ic 90 ), which reflects to a concentration of which 90% of the original cells would have died. to determine cellular characteristics, the resistant cells were compared with wildtype cells. the following cell characteristics were investigated: proliferation, apoptosis, clonogenicity, size of nuclei and cytoplasm, cell-cell contacts, dna adducts formation, secretome, screening of mirna expression, next generation sequencing, vital observation and scratch assay, nad(p) + /nad(p)h, h 2 o 2 , glutathione, ca 2+ -influx, mdrchannels, resistance to other alkylating agents and the reversibility of the resistance. the resistant cells demonstrate smaller nuclei and cytoplasm, less dna adducts, a higher clonogenicity as well as proliferation and less apoptosis. the secretome analysis showed an up-regulation of anti-apoptotic acting cytokines timp and ang and the proproliferative acting cytokines timp and pdgf-aa. in contrast, immunologically active cytokines were down-regulated. concerning cell-cell contacts no differences were seen. in the mirna screening 49 significant up-regulated and 20 significant down-regulated mirnas have been observed. noteworthy was the regulation of various members of 11 different families. during vital observation and in a scratch-assay the resistant cells were show to have disadvantages. the observed resistance was not unique for sm but also towards other alkylating agents and cytostatic drugs. by analyzing the reversibility cells stayed resistant over more than 35 weeks. in conclusion, many aspects investigated in this study have an influence on the sm resistance, pointing out that it is a combination of various effects that are involved to switch on resistance. more likely, there are many aspects working together. the present results are an important step in the characterization of the sm-resistant cell line and further studies may be able to directly use these as a start for target identification in antidote or prophylactic agent discovery. the arylhydrocarbon receptor (ahr) is localized in a cytosolic complex that contains several co-chaperones and associated factors. the protein is shifted into the nucleus in response to endogenous and xenobiotic ligands. however, a transient nuclear transport does also occur in the absence of any ligands, while the predominant cytoplasmic compartmentalization is maintained by parallel export. we have analyzed the interplay between this basal nucleo-cytoplasmic shuttling and ligand induced transport in hepg2 cells, using a yfp-tagged fusion protein that is capable to respond to ligands and to trigger the induction of cyp1a1 expression. basal import was assessed in cells that had been treated with leptomycin b (lmb), an inhibitor of crm1-mediated nuclear export. interestingly, the apparent ahr import rate in lmb-treated cells was comparable with nuclear import as trigged by xenobiotic (b-naphthoflavone) or endogenous (kynurenine) ligands. this observation was confirmed for endogenous ahr in hepg2 cells, since both ligands and lmb showed comparable effects on nuclear compartmentalization. however, the basal nuclear import rate in lmb-treated cells was strongly increased by ahr ligands. ligand-induced nuclear transport was therefore confirmed as an import step in receptor activation. interestingly, lmb did also accelerate nuclear import of ahr after pretreatment of cells with ahr ligands. these data suggest that nuclear export of the ahr is maintained in the presence of ligands. receptor activation might therefore comprise several rounds of shuttling, thereby involving both accelerated import and continued export of the ahr protein fraction that has not already undergone interactions with arnt or dna. we suggest that nuclear export provides an additional kinetic control of ahr activation and function. mitochondrial toxicology: rescuing mitochondria in wilson disease avoids acute liver failure h. zischka 1 1 institut für molekulare toxikologie und pharmakologie, ag zischka, neuherberg, germany in wilson disease (wd) functional loss mutations in the hepatocyte atp7b gene cause dramatic copper overload leading to acute liver failure, posing an unmet therapeutic issue. we find that the pathology of severe wd cases is mirrored in lpp (-/-) rats carrying a functional loss atp7b mutation. this is especially apparent in the hepatocyte mitochondrial compartment. a progressive copper deposition increasingly harms the lifesustaining mitochondrial membrane integrity. thus, depleting this devastating mitochondrial copper burden is a core requirement for a treatment strategy against acute liver failure in this wd animal model. preparation for the master degree program in toxicology started in 2006 as a cooperation of charité universitätsmedizin berlin with the university of potsdam and other institutions of the region. first enrollment of students was done in 2008. the program was accredited in 2011 by the central evaluation and accreditation agency. it offers a modern curriculum encompassing a wide variety of scientific aspects with an interdisciplinary character. this training program in toxicology is organized in modules and ends with the degree "master of science" (m.sc.). the goal of this program in toxicology is to teach the basis of the interactions between substances at toxic concentrations and living organisms, as well as the molecular mechanism of the adverse effects of chemicals. the understanding of the mechanism of a toxic action is an important prerequisite for the scientifically based evaluation of a hazard associated with a substance. furthermore, only with the knowledge of the mechanism of action and a deduction of structure activity relationships it is possible to predict toxic effects of new substances. this knowledge should enable students to perform a risk evaluation of chemicals or to predict the adverse effects of chemicals with the aim that human beings and the environment can be protected from harmful consequences of chemical exposure. the program allocates 30 places per year to an average of 60 applicants. most applicants have a basic training in the fields biology, chemistry, pharmacy, veterinary medicine and nutritional sciences. about 75% of the students are female. the majority of them have a bachelor's degree before starting the master program, other degrees are diploma and state examination as pharmacists or physicians. ninety percent of the students pass the final examination consisting of the master's thesis and disputation at the end of the four semesters. afterwards, most of the graduates aim to obtain a phd degree. the program is well established in the education of toxicologists in germany. respiratory injury due to chlorine developed from consumer products. still an issue in germany u. stedtler 1 , m. hermanns-clausen 1 1 uniklinikum freiburg, vergiftungs-informations-zentrale, freiburg, germany objective: in the last decades strong effords have been took to improve product safety, especially in products intended for domestic use. hypochlorite-containing cleaners may develop chlorine gas when acidified e.g. by adding an acid sanitary cleaner. usually these cleaners contain sodium hydroxide or other strong alkalines to avoid this reaction. we analysed reports to our poisons center concerning inhalation exposure to chlorine developed from hypochlorite-containing mixtures. method: retrospective search in the case database of the poisons center. human inhalative exposures to chlorine released from mixing hypochlorite as well as human inhalative hypochlorite exposure alone were analysed. frequency and symptoms were compared. results: from 2010 to 2015 in total 85 cases of human exposures to chlorine developed from mixtures of hypochlorite and acids (0.8 of 1000 cases) were registered. in 55 cases the exposure was due to mixtures of products intended for domestic use. 94 % of the exposed patients reported symptoms. only in two cases the symptoms were not considered to be caused by the inhalation accident. most frequent symptoms reported were (percent of symptomatic patients): cough (45 %), dyspnea ( 33 %), irritated upper airway (26 %), abdominal discomfort (pain, nausea, vomiting) (21 %), thoracic pain (20 %), irritated eyes (11 %), dizziness (8%), and bronchospasm (6 %). further symptoms were malaise, headache, irritated nose, sweating, muscle pain, and others. in 12 patients (14 %) the symptoms were graded as moderate severe. main symptoms in this group were dyspnoea ( 83% ), cough, and irritated airway. one third of the patients experienced bronchial obstruction. all symptomatic patients developed symptoms while exposed or shortly after exposure. there were no severe or fatal cases (especially no lung edema) and all symptoms were expected to resolve completely. because hypochlorite containing procucts sontanously release "chlorine-like" smelling gases, we additionally analysed inhalation exposures to hypochlorite solutions alone in the same period. there were 42 patients in the same period exposed to hypochlorite evaporation alone. 36 of them (86 %) had symptoms of which in 30 cases these were considered to be caused or possibly be caused by the hypochlorite. most frequent symptoms were irritated upper airway (33 %), nausea or vomiting (30 %), cough (23 %), irritated eyes (20 %). dyspneoa was less fequent than in the mixture group (10 %). all symptoms were considered mild. there was no bronchospasm or thoracic discomfort. conclusion: respiratory injuries by chlorine from hypochlorite-containing solutions still occur despite clear warning on the label. the majority of cases was due to products for domestic use. symptoms develop shortly after exposure. the γh2ax assay for genotoxic and nongenotoxic agents: comparison of h2ax phosphorylation with cell death response perturbation of mitosis through inhibition of histone acetyltransferases: the key to ochratoxin a toxicity and carcinogenicity? regulation of chromatin by histone modifications transcriptomic alterations induced by ochratoxin a in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model in vitro gene expression data supporting a dna non-reactive genotoxic mechanism for ochratoxin a fragment ion patchwork quantification for measuring site-specific acetylation degrees combinatorial patterns of histone acetylations and methylations in the human genome inroads to predict in vivo toxicology -an introduction to the etox project value of shared preclinical safety studies -the etox database acknowledgements: support of the bfr through grant 1322-530 is gratefully acknowledged. acknowledgement: supported by the robert bosch foundation, stuttgart, germany. [1] mürdter t, et al. hum mol genet, 2011, 21:1145-54 [2] nishiyama t. et al., biochemical pharmacology, 2002, 63:1817 -1830 [3] sun d. et al., drug metabolism and disposition, 2007, 35:2006 background: infections are a major problem in patients with burn diseases (bd). due to severe injuries of their total body surface area (tbsa), burn patients have altered pharmacokinetic characteristics. therefore, insufficient plasma concentrations may be achieved, when standard dosing schedules are applied for antibiotics such as piperacillin. for time-dependent antibiotics, the duration how long drug concentration exceeds the minimal inhibition concentration (mic) is crucial for their antibacterial effects. since pseudomonas spp. is the main problematic pathophysiological bacterium for bd patients. the aim of the present study was to monitor the plasma concentrations of piperacillin during piperacillin/tazobactam treatment in bd patients. patients from intensive care units (icu) served as controls. methods: 10 bd patients (5/5 m/f, 43.4±5.3y, tbsa 40 .9±5.9%) and 5 patients (74.4±3.9y) from the icu were included in this observational study. blood samples were taken within the 3 rd interval of the 8h-lasting dosing period of piperacillin/tazobactam (4/0.5g within 0.5h) at 1, 4 and 7.5h after the end of infusion. total and free piperacillin concentrations were determined in plasma using hplc-uv after deproteinisation with acetonitrile and by ultrafiltration, respectively. pharmacokinetic parameters and dosing simulations were calculated by tdmx (www.tdmx.eu). free plasma concentrations of piperacillin exceeding at least 1xmic but preferably 4xmic over the whole dosing interval were considered to be sufficient for antibiotic efficacy (mic 16 mg/l for pseudomonas spp.,www.eucast.org). results: the pharmacokinetic parameters of total piperacillin, calculated for each bd or icu patient using the concentrations at 1, 4, and 7.5h, were as follows: c max 69.6±7.9 vs.116.3±10.4 mg/l, p<0.05, half-life 1.8±0.3 vs. 2.3±0.3h, p>0.05, clearance 12.6±1.9 vs. 6.5±0.4 l/h, p<0.05, volume of distribution 27.8±2.0 vs. 20.9±1.3l, p<0.05. free concentrations (which were included in tdmx calculations) were 87±2 vs. 81±2% (p<0.05) of total concentrations. duration per day while concentrations exceeded 1xmic (15.6±1.9 vs. 22.0±1.1h, p<0.05) or 4xmic (5.4±1.3 vs. 9 .4±0.7h, p<0.05) were lower in bd than in icu patients. moreover, tdmx simulations predicted that the duration per day for 4xmic could be enhanced to 16.2±1.5h if the piperacillin amount will be increased to 4x8 g/d and the infusion duration to 3h. pharmacokinetic parameters have, however, to be determined in a pilot study with bd patients to ensure predicted values. conclusions: standard dosage regimens for piperacillin/tazobactam could result in suboptimal plasma concentrations of piperacillin in bd patients as well as in icu patients. drug monitoring and tdmx simulation of kinetic parameters may easily help to improve piperacillin treatment in bd patients. background: high dose methotrexate (hd mtx), defined as >1000mg mtx/m 2 bodysurface-area (bsa) is used in children to treat a variety of malignant diseases since the 1950s. clinicians observe relevant rates of severe unwanted side effects. identifying patients having an increased risk for toxicity due to altered mtx pharmacokinetics is urgently needed. we aim to develop and evaluate a physiology-based pharmacokinetic (pbpk) model for hd mtx in children using pk-sim® (bayer technology services gmbh, leverkusen, germany) with a special emphasize on relevant covariates. methods: in this non-interventional observational study, children receiving hd mtx intravenously at two major german pediatric oncology departments during the years 2004-2009 were included if at least one mtx serum level (mtx-sl) was determined during clinical routine. 29 patients aged 2-18 years (male = 19, female = 10) with following diagnoses were included: acute lymphoblastic leukemia, non-hodgkin lymphoma, burkitt lymphoma, brain stem glioma and glioblastoma multiforme. in total, 103 mtx treatment cycles corresponding to 300 mtx-sl were used in this study. patients were randomized into two patient sets (training set and test set). based on literature data, mtx pbpk-models were developed and slightly adapted taking into account mean relative deviation (mrd) and bias of predicted versus observed mtx-sl of the training set. the pbpk model with the lowest mrd and bias was chosen and finally evaluated using the test set. the impact of the covariates urine ph <6.5, trimethoprime/sulfamethoxazole, proton-pump-inhibitors, non-steroidal anti-inflammatory drugs and ß-lactam antibiotics on the prediction quality was assessed using the mann-whitney u test. ochratoxin a (ota) is a wide-spread food contaminant and one of the most potent renal carcinogens [1] . recent data by our group demonstrate that ota inhibits histone acetyltransferases (hats), thereby causing a global reduction of lysine acetylation of histones and non-histone proteins [2] . based on these findings and the importance of specific histone acetylation marks in regulating gene transcription [3] , we speculated that repression of gene expression as the predominant transcriptional response to ota [4, 5] may be linked to loss of histone acetylation. in this study we therefore used a novel mass spectrometry approach, which is based on chemical acetylation of unmodified lysine residues of histones using 13 c-labeled acetic anhydride and subsequent calculation of the degree of acetylation based on the measured intensities of heavy and light acetylated isotopologues [6] , to identify and quantify site-specific alterations in histone acetylation in human kidney epithelial (hk-2) cells treated with ota. our results demonstrate ota-mediated loss of acetylation at almost all important lysine residues at histones h2a, h2b, h3 and h4. we further selected acetylation at histone h3 lysine 9 (h3k9), a well-known euchromatic hallmark that is elevated at promoter regions of transcriptionally active genes [7] and which was reduced from ~ 3% in controls to < 0.1% in response to ota, to establish a link between loss of h3k9 acetylation and expression of genes consistently shown to be down-regulated in response to ota [4, 5] . using chromatin immunoprecipitation followed by quantitative real-time pcr (chip-qpcr), we observed ota-mediated loss of h3k9 acetylation at promoter regions of the selected genes (% of controls: amigo2: 45%, clasp2: 60%, ctnnd1: 54%). overall, these data provide first evidence for a mechanistic link between h3k9 hypoacetylation as a consequence of ota-mediated inhibition of hats and repression of gene expression by ota. a new paradigm to assess the proarrhythmic potential of drugs is proposed by the cipa (comprehensive in vitro pro-arrhythmia assay) initiative combining a suite of a priori in vitro assays (7 most important ion channels for cardiac activity) coupled to in silico reconstructions of cellular cardiac action potential (ap). the etox consortium has developed a multiscale simulation in silico model based on o'hara/rudy incorporating the principles of this new paradigm. the core model simulates the effects of drugs on a virtual cardiac tissue composed by different types of cardiomyocytes. the input of this model, the blockade of a set of 3 ion channels (ikr/herg, iks, ical), can be obtained experimentally or predicted using advanced 3d-qsar models. the system predicts the % change of the qt interval at different drug concentrations in order to facilitate risk assessment. this in silico model was validated using purkinje fiber assay results (input: ap prolongation and arrhythmogenic risk assessed by early after-depolarisation occurrence) from 500 in-house drug candidates. the validation showed that predictivity is highly dependent on the model's applicability domain (ad): for some chemical series the proarrhythmic potential could not be identified, for others, however, most of the positive drugs were correctly predicted with sensitivities up to 80-90% (average prediction accuracy was 70%). retraining of this model with additional internal data should help to improve the model ad and predictivity. it is important to note that ap prolongation was correctly predicted for many proarrhythmic drugs with only low (> 30 µm) in vitro herg inhibition. furthermore, the model showed high additional benefit for read-across within bayer pharma ag, investigational toxicology, berlin, germany etox [1] [2] started in 2010 and is a public-private partnership project within the european innovative medicines initiative (imi) [3] . the etox project is building a toxicology database relevant to pharmaceutical development and to elaborate innovative strategies and software tools. the overall goal is to better predict the toxicological profiles of new chemical entities in early stages of the drug development pipeline based on existing in vivo study results contributed by the participating efpia * companies in the consortium. the etox database is a relational database with a specifically designed schema to store complex and comprehensive preclinical safety data like the study design, toxicokinetics, adme data, clinical chemistry, hematology, gross necropsy, histopathological findings and general toxic effects. in addition relevant data from public sources has been included into the database. the primary focus for data collection are systemic toxicity (up to 4 week) repeated dose studies, mostly in rodent. overall more than 7000 study reports for approximately 1400 investigated compounds. in order to optimize the usage and mapping of data from different sources the development of common ontologies was a key task within the project. this timeconsuming step was necessary to make a high quality read-across analysis possible and valuable. therefore the ontobrowser [4] tool was developed to curate and harmonize the verbatim terms to standardize terms which are used within the etox database. until now more than 13 million verbatim terms were curated. additionally to the toxicology database, a web-based user interface called etoxsys was developed to allow the retrieving of toxicity information, as well as the prediction of toxic endpoints for chemical compounds. due to the complex search capabilities, the database can be queried for structural similarity, similar target classes and specific toxicological endpoints. approximately 150 prediction models based on public data are available and first models based on in vivo data are in development. the etox database therefore represents a valuable tool for early animal-free assessment of drug candidates [5] . * european federation of pharmaceutical industries and associations cell lines background: consumers are constantly exposed to chemical mixtures e. g. to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. since substances are tested for regulatory purposes on an individual basis at generally high dose levels, there is only limited data available on potential mixture effects especially in the low dose range. with more than 400 active substances approved for being used in pesticides and over 100000 chemicals registered under reach there are more possible combinations than one could test with classical animal experiments. the development of in vitro tools for assessment of mixture effects consequently is of tremendous importance. methods: as a first step in the development of such in vitro tools we used a group of fungicides, (tri-)azoles, as model substances in a set of different cell lines from known target tissues, basically liver (human: hepg2, heparg, rat: h4iie) and adrenal gland (human: h295r). concentrations were taken from measured tissue concentrations in vivo to ensure that used concentrations of the (tri-) azoles reflect realistic effect levels. the cell lines were exposed with the triazoles cyproconazole and epoxiconazole as well as with the azole prochloraz as individual substances and in binary or ternary combinations of these substances at three dose levels and three different time periods. the effects of the substances were subsequently analysed by transcriptomics and metabolomics. a support vector machine will be utilized to integrate the data from the different sources to gain a complete picture of affected adverse outcome pathways and mechanistic information about the applied fungicides. first results indicate combination effects of the substances also at the omics level depending on the specific endpoint and the concentration used. some of these are comparable to effects found with similar methods in a standard toxicity test, a 28-day feeding study in the rat, thus raising hope for the development of in vitro methods suitable to detect combination effects. background: plant protection and biocide products are chemical mixtures, which contain one or more active substances as well as several co-formulants (e.g. solvents, wetting agents, thickener or preservatives). nevertheless, to this day extensive toxicological testing is performed only with the individual active substances, while the plant protection products are only evaluated for acute toxicity, ie, a single dose group experiment with rats is performed as well as testing for skin-and eye-irritation. current pesticides regulation foresees testing of potential harmful mixture effects but only when adequate methods are available making the development of such methods a high priority. several published studies both in vitro and in vivo have shown fortified toxic effects of plant protection products compared to individual active substances. methods: here we present effects of plant protection products as a whole as compared to the individual active substances or co-formulants in a set of human cell lines of hepatic and renal origin (hepg2, heparg, hek293). cytoxicity has been analysed by wst-1 and nru assay as well as gene expression of several marker genes involved in xenobiotic metabolism. additionally reporter gene assays have been conducted for nuclear receptors such as ahr and car. results: while some active substances showed lower toxicity as compared to the respective products, this cannot be confirmed as a general rule for all endpoints for all of the analysed fungicides or herbicides containing active substances such as epoxiconazole, cyproconazole, azoxystrobin or glyphosate. chemical compounds may induce skin sensitization in humans, resulting in tolerance or allergic contact dermatitis after repeated exposure. mechanistically, the activation of dendritic cells is one of the prerequisites for the induction of skin sensitization. a subgroup of sensitizing chemicals, prohaptens, need metabolic activation, e.g. via cytochrome p450 (cyp) enzymes. thus, xenobiotic metabolism may crucially impact on a chemical's potential for the induction of skin sensitization by activation, but also deactivation of reactive molecules via conjugation, which determines the concentration and the chemical species available for protein haptenation and cell activation. we established a coculture model consisting of hacat keratinocytes and thp-1 as surrogate dendritic cells for the detection of sensitizing chemicals and found enhanced cyp1 enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol as well as clearly increased expression of cell surface molecule cd86 on thp-1 cells after incubation with these prohaptens (hennen et al., 2011) . here, we studied the impact of intercellular cross talk on activation and conjugation capacities in more detail. treatment of thp-1 with b[a]p and eugenol in coculture with hacat cells augmented cyp1a1 and/or cyp1b1 mrna levels, while this was not found for thp-1 monoculture. augmentation of cyp1a1 mrna needed continuous presence of hacat cells. in coculture, levels of 3-oh-b[a]p as exemplary cyp-dependent metabolite were increased compared to single cultures. in contrast to this, total glutathione contents as well as n-acetyltransferase 1 enzyme activities in both cell types were not modulated in coculture, furthermore the capacity for sulfation/glucuronidation of 3-oh-b[a]p was maintained in coculture. additionally, the decrease of the total glutathione content in thp-1 cells by 2,4-dinitrochlorobenzene (dncb) was much less pronounced when exposed in coculture with hacat cells, showing that hacat cells provide additional targets for cysteine-reactive chemicals such as dncb, diminishing the total amount of chemicals available for thp-1 cells.overall, results indicate that the cross talk between keratinocytes and antigenpresenting cells enhances their capacities for metabolic activation of chemicals, while hacat cells also provide supplementary capacities for phase ii reactions. references: hennen j et al. cross talk between keratinocytes and dendritic cells: impact on the prediction of sen-sitization. toxicol sci 2011 123:501-510.toxicology -toxic pathway analysis/aop 395 background: reticulated platelets are associated with impaired antiplatelet response to thienopyridine treatment. this interaction might be caused by intrinsic properties of reticulated platelets or a decreased drug exposure due to high platelet turnover reflected by reticulated platelets as surrogate. we investigated the impact of reticulated platelets on antiplatelet response to thienopyridines and if this effect is linked to platelet turnover. methods: this study randomized elective patients to loading with clopidogrel 600mg or prasugrel 60mg (n=200). adp-induced platelet reactivity was assessed by impedance aggregometry 30 to 120 minutes and day 1after loading but before intake of the next dose of thienopyridines. immature platelet count (ipc) was assessed as marker of reticulated platelets by whole blood flow cytometry. results: platelet reactivity increased with rising tertiles of ipc (figure) . this effect was more pronounced in patients on clopidogrel as compared to patients on prasugrel. overall, ipc correlated well with on-treatment platelet reactivity at 120min (r=0.21; p<0.001). this correlation did not change over time indicating an effect independent of platelet turnover (comparison of correlations 120min/day 1: p=0.57 for clopidogrel, p=0.76 for prasugrel). conclusion: a high immature platelet count is associated with impaired response to thienopyridine loading. this effect is independent of platelet turnover indicating a relation to intrinsic properties of reticulated platelets. introduction: one of the biggest drawbacks of protein-based therapeutics with intracellular targets is their inability to enter the cytosol. targeted toxins are known to be used in drug delivery. aim of the study was to target epidermal growth factor (egf) receptor overexpressed on pancreatic carcinoma using a novel well-defined targeted toxin consisting of egf fused to the toxic plant ribosome-inactivating protein dianthin and a glycosidic triterpenoid (so1861) as efficacy enhancer. methods: the enzymatic activity of dianthin-egf was verified by an adenine release assay. the kinetics of cytotoxicity were evaluated in pancreatic adenocarcinoma bxpc-3 and miapaca-2 cells in comparison to the non-target cell line nih3t3 with an impedance-based real time cell analyzer (xcelligence) and final cytotoxicity analyses with conventional end-point mtt assays. acute toxic of dianthin-egf was studied in male balb/c mice. a xenograft solid tumor model was developed in male nude mice by injecting bxpc-3 cells into the dorsal part subcutaneously. dianthin-egf was administered at the vicinity of the tumor and so1861 by subcutaneous injection at the neck. after the tumor reached a diameter of 2 to 3 mm in size 6 treatments were given in total. tumor volumes and body weight shifts were observed twice weekly to determine the potency of dianthin-egf when given alone and in combination with so1861 in comparison to placebo. immunohistochemical detection of egf receptor was performed according to the manufacturers's advice (dako, glostrup, denmark, k1492). complete blood count analysis was done by labor 28 gmbh, berlin. results: the adenine release mediated by dianthin-egf was 47.8 pmol adenine/pmol toxin/h. the in vitro efficacy of the targeted toxin was proven by an ic50 value of approximately 1 nm for egf receptor expressing miapaca-2 and bxpc-3 cells as compared to 100 nm for non-target nih3t3 cells. real time measurement of the cytotoxicity showed a dose-dependent decrease in cell viability from 10 pm to 1 µm. toxicity studies in balb/c mice revealed 0.4 µg/mouse to be non-toxic and maximum tolerated dose (mtd) whereas 40 µg caused moribundity accompanied with white ocular discharge. efficacy studies were performed for a period of 28 days. the combination therapy showed that the average tumor volume measured by a digital vernier caliper was found to be 80% less than for placebo whereas single therapy using dianthin-egf alone caused a further increase in tumor volume which was although yet 50% less when compared to placebo. immunohistochemistry slides showed egf receptor expression in each of all untreated xenograft tumors, which further confirms the presence of egf receptor overexpression in the target bxpc-3 cell line. enlarged spleen was only observed in untreated xenografts. no significant change in various blood parameters (rbc counts, wbc counts, hgb, hct, mcv, mch and mchc) were observed on hematological analysis except for the platelet (plt) counts in comparison to healthy male nude mice. conclusion: combination therapy with so1861 proves to be a promising approach for the targeted delivery of toxins instead of single therapy administering targeted toxin alone. the strategy is specific for egf receptor overexpressing tumors such as pancreatic cancer. introduction: moringa oleifera (mo) is a popular herbal supplement used for treatment and management of diverse diseases in sub-saharan africa. its intake among individuals infected with hiv/aids has increased recently due to the purported immune boosting property. limited information, however, is available regarding its potential to cause interactions with commonly prescribed medications that are substrates of cyp3a4 and p-glycoprotein. methods: the methanol extract and four fractions of mo were tested on recombinant cyp3a4 at different concentrations with and without nadph to determine the ic 50 shift reduction. the crude methanol extract of mo was incubated with testosterone (tst) and cryopreserved hepatocytes to evaluate its influence on clearance of tst. effect of mo on the efflux transporter, p-glycoprotein was investigated by incubating the methanol extract with mdr1 -mdckii cells. virtual screening was conducted to predict physicochemical properties, bioavailability and interaction potential of phytochemical compounds unique to mo using combination of molinspiration version 2014.11 and admetsar. results: fractions (f1-f3) indicated ic 50 shift reduction ≥5 post-incubation with and without nadph. mo showed moderate interaction (auc i /auc = 2.46) with tst in cryopreserved hepatocytes. also, mo mildly inhibited the transport of digoxin (ic 50 = 35.45 µg/ml) across mdr1 -mdckii cells. niaziminin indicated 85.57% bioavailablity via the human intestinal membrane with 61% chance of inhibiting cyp3a4. βsitostenone showed strong p-gp inhibition (83.27%) with 100% absorption via the intestine. conclusions: mo has the potential to inhibit the metabolism or excretion of other medications that are eliminated by cyp3a4 or p-glycoprotein, respectively, if adequate amounts of the active constituents such as niaziminin and β-sitostenone enter the circulation. background: herb-induced liver injury (hili) has attracted attention in the past years due to an increasing number of publications reporting cases of hepatotoxicity associated with use of phytotherapeutics. here, we present data on hili from the berlin case-control surveillance study fakos. methods: fakos was initiated in 2000 to study serious toxicity of drugs including hepatotoxicity. potential cases of liver injury were ascertained in more than 180 departments of all 51 berlin hospitals from october 2002 until december 2011. through a standardised face-to-face interview and review of medical charts information on all previous intakes of drugs or herbals, on co-morbidities, and demographic data was ascertained. inclusion criteria were an elevation of alanine aminotransferase or aspartate aminotransferase threefold above the upper limit of normal or an elevation of total bilirubin higher than 2 mg/dl. excluded were patients with underlying liver disease (e.g., alcoholic fatty liver disease). drug or herbal aetiology was assessed based on the updated council for international organizations of medical sciences (cioms) scale. results: of all 198 cases of hepatotoxicity included into the fakos study, herbs were involved in ten cases (5.1%). demographic, clinical, and laboratory characteristics of these ten cases are illustrated in table 1 . among the six patients with available liver biopsy results, five patients showed signs of necrosis, either disseminated or predominantly near the central vein. portal inflammation was more common than lobular inflammation, and the infiltrates contained mostly lymphocytes, neutrophil or eosinophil granulocytes. herbal aetiology was judged two times as probable (ayurvedic herb in patient 1, pelargonium sidoides in patient 6), and eight times as possible (valeriana in patients 3, 4, 8, 9, 10 , mentha piperita in patient 5, hypericum perforatum in patient 2, eucalyptus globulus in patient 7). in nine cases other non-herbal drugs were also suspected as potentially hepatotoxic (exception: patient 6). seven cases occurred in the ambulatory setting requiring hospitalisation, three cases occurred during hospital stay. discussion: this case series provides further information on laboratory and clinical aspects of hili. it corroborates known risks for valeriana and ayurveda treatment, and suggests that further herbals rarely or never associated with liver injury before such as pelargonium sidoides, hypericum perforatum or mentha piperita could also exhibit a hepatotoxic potential. clinical routine often requires to evaluate the cause of a newly occurring adverse event. if this event is regarded to be iatrogen, further information of the association between the drugs in the current medication list and the adverse event is needed. this information should ideally reflect the true risk and allow ranking of the drugs according to this risk to identify which drug to discontinue first. we discuss the summary of product characteristics (spc), the sider side effect resource and openvigil 2 as possible sources of information. spcs are becoming more and more a vindicative charter for pharmaceutical companies that contain misleading information which is not based on evidence (ref. 1). since it relies on the spcs, sider inherits these shortcomings and flags warnings that result from confounding factors (ref. 1, fig. 1 ). furthermore, if any rates are given, they are not easily comparable since they stem from different studies. pharmacovigilance data are biased by the very nature of the data and the collection method. however, once confounders are eliminated, pharmacovigilance offers better information om how to rank the drugs than spcs/sider. we present decision-guiding information obtained by sider and by openvigil 2 for one of our patients ( fig. 2 & 3 ) and discuss how this information was used to modify the therapy. institut für naturheilkunde und klinische pharmakologie, universität ulm, ulm, germany background: differences (polymorphisms) in target genes or genes encoding drug transport proteins or drug metabolizing enzymes may be responsible, among other factors, for observed variation in patients' response to medications. pharmacogenetics aims at identification of patients at higher, genetically determined, risk of adverse drug effects or ineffective medication, to modify dosage or switch to alternative therapy. there is, however, a lack of awareness of pharmacogenetic-based clinical practise guidelines. methods: a systematic literature review was conducted which focused on published guidelines on genotype-based (germ-line genetic variants) dosage modification or selection of drugs. we serched the medline and the pharmacogenomics knowledgebase (pharmgkb) databases. prescribing information was also screened for pharmacogenetic guidance. results: the systematic review revealed recommendations for 61 drugs (table) that enable the translation of genetic test results into actionable prescribing decisions. for 20% of these drugs the respective german drug labels recommend or even require pharmacogenetic testing (table, 3rd column). although pharmacogenetic testing is recommended, the prescribing information not always provides guidance on how to adjust the drug dosage based on the pharmacogenetic test result. compared with the german or european drug labels, the fda drug labels povide more detailed information on pharmacogenetic dose modifications. conclusions: academic working groups have a front-runner role in the development of prescribing recommendations based on genetic markers. to date, drug labels rarely contain detailed guidelines how available genetic test results should be used to adjust drug dosage. because pharmacogenetics has a growing role during drug development and pre-prescription genotyping will become more widespread, it is expected that specific pharmacogenetic guidance for the treating physicians will become increasingly important. bisphenol a (bpa) is a high production volume compound mainly used as a monomer to make polymers for various applications, including food-contact applications. people are exposed to low levels of bpa because very small amounts of bpa may migrate from the food packaging into foods or beverages. however, other potential sources of exposure, such as dermal contact have also been identified (efsa, 2015) . a substance evaluation process (corap) was initiated for bpa by the european chemicals agency (echa). as part of the safety evaluation of bpa, a study was required by echa to assess absorption and metabolism of bpa following dermal exposure to human skin. an in vitro study with human skin was requested according to oecd tg 428 under consideration of the scientific committee on consumer safety (sccs) criteria for the in vitro assessment of dermal absorption. to investigate potential dermal bpa metabolism fresh human skin was used. abdominal skin was obtained fresh from surgery from 4 different donors. split-thickness human skin membranes were mounted into flow-through diffusion cells (n=4 per dose and donor) and the receptor fluid was pumped underneath the skin at a constant flow rate. the skin surface temperature was maintained at 32°c throughout the experiment and electrical resistance barrier integrity testing was performed at the start (0 h) and end of the experiment (24 h). four test preparations at final bpa concentrations of 2.4, 12, 60, and 300mg/l were investigated. the highest concentration was chosen based on the maximum solubility of bpa in water and the lowest concenration was chosen based upon the specific activity of the radiolabelled [ 14 c]-bpa that could be used for mass balance. percutaneous absorption was assessed by collecting receptor fluid (tissue culture medium (dmem), containing ethanol (ca 1%, v/v), uridine 5'-diphosphoglucuronic acid (udpga, 2 mm) and 3'-phosphoadenosine-5'-phosphosulfate (paps, 40 µm)), at multiple time points througout the experiment. at termination the skin was removed from the cells and the stratum corneum was removed with 20 successive tape strips. the exposed epidermis was separated from the dermis using a scalpel. metabolism was investigated for the highest concentration (300 mg bpa/l) only, using a hplc with in-line radiodetection and confirmed bpa-glucuronide (bpa-g) and bpa-sulfate (bpa-s) standards for comparison. no metabolism was observed in any of the epidermis samples, however some metabolism is observed in dermis and receptor fluid samples. metabolites were identified with retention consistent with bpa-g and bpa-s, and also some more polar components. the mean total absorbed dose (receptor fluid + receptor chamber wash + receptor rinse) was between 1.7 and 3.6% of the applied dose and the mean dermal delivery (epidermis + dermis + total absorbed dose) was between 16 and 20% of the applied dose, with the majority of the radioactivity associated with epidermis samples compared to dermis and receptor fluid samples. a linear dose-response relationship is observed over the whole concentration range. anastrozole is a well-known non-steroidal aromatase-inhibiting drug approved for the second-line treatment of breast cancer after surgery and for treating postmenopausal women. treatment with the only available dosage form, anastrozole film-coated tablets for oral administration, is frequently associated with concentration-dependent unwanted side effects like hot flashes, fatigue, joint pain, joint stiffness, vaginal dryness, hair loss, skin rash, nausea, diarrhea and headache. in order to minimize the local gastrointestinal as well as systemic side effects, a system for transdermal anastrozole delivery has recently been developed. in this study, we describe the first experimental in vivo application of a transdermal therapeutic system (tts) to beagle dogs and, as a necessary prerequisite for the analysis of the time course of anastrozole release and uptake, a simple, sensitive and accurate lc-ms method for quantifying anastrozole in plasma. the detection of fragment ions at m/z 225 and 237 instead of the molecule ions (m/z 294 and 306) generated from the elevated collision energy, and the use of a deuterated internal standard resulted in increased relative abundances and improved signal-to-noise ratios.the lower limit of quantification and the limit of detection were 1.4 ng/ml and 0.5 ng/ml, respectively. the developed method was successfully applied in a pharmacokinetic study of anastrozole plasma levels in beagle dogs, measuring percutaneous drug absorption from an experimental, newly designed glycerol-based patch / tts. a distinct time course was observed, with an initial linear increase over 24 hours and a plateau thereafter. this offers promising strategies for the transdermal application of anastrozole with improved pharmacokinetics. background: the monocarboxylate transporter 4 (mct4), encoded by the slc16a3 gene, mediates h + -coupled transport of lactate across the plasma membrane. for cells with high glycolytic activity lactate export is of major importance for the maintenance of the glycolytic metabolism and for the prevention of intracellular acidification. in glycolytic tumor cells, the acidic extracellular environment resulting from export of lactate and h + , furthermore promotes anti-apoptotic effects and metastasis. clear cell renal cell carcinoma (ccrcc) is the most common subtype of renal cell carcinoma (rcc) and is characterized by a metabolic shift towards enhanced aerobic glycolysis and hence, increased lactate production. mct4 and its epigenetic regulation by slc16a3 promoter methylation has previously been identified as prognostic marker for ccrcc outcome and as target for ccrcc treatment. since metastatic ccrcc is associated with poor overall survival and represents a major challenge for treatment, mct4/slc16a3 might represent a promising prognostic marker and a target for therapeutic intervention also for metastatic disease. methods: mct4 protein expression was analysed in 130 paraffin embedded tissue samples of distant metastases derived from different organs by immunohistochemical staining of tissue microarrays. protein expression was evaluated semi-quantitatively using tissue studio v.3.6 (definiens ag). dna methylation in the slc16a3 promoter, specifically at the previously identified cpg site with prognostic potential in primary ccrcc, was analysed in 82 paraffin embedded metastasis samples by maldi tof-ms. mct4 protein expression data and dna methylation at the specific cpg site in the slc16a3 promoter were correlated with clinicopathological parameters and outcome data. results: distant metastases of primary ccrcc showed high mct4 protein expression irrespective of the affected organ. the most frequently affected organs like lung or bone, with approximately 28% and 14% in our cohort respectively, showed similar expression levels as less frequent metastatic sites such as thyroid gland or spleen. accordingly, dna methylation at the identified cpg site in the slc16a3 promoter was low in metastatic tissue in all investigated organ sites. an association of low promoter dna methylation level at the previously identified prognostic cpg site in metastases with poor tumor-specific survival of the patients was observed. conclusion: from these results we hypothesize that dna methylation at specific cpg sites in the 5'-regulatory region of mct4 may not only serve as a predictor for patient outcome and as potential novel target for therapeutic intervention in primary, but also for metastatic disease. tamoxifen is used to treat pre-and postmenopausal women with estrogen-receptor (er) positive breast cancer. as a prodrug, tamoxifen undergoes extensive hepatic metabolism resulting in a complex mixture of metabolites with estrogenic and antiestrogenic effects. while endoxifen and (z) 4-hydroxytamoxifen are the most potent antiestrogenic metabolites, bisphenol and both isomers (e) and (z) of metabolite e are the most potent compounds with estrogenic properties at the er. the mixed antagonist/agonist pharmacodynamic effects of the selective estrogen receptor modulator tamoxifen at the er have been mainly attributed to tissue specific action of er coregulators, yet little is known about agonistic metabolites contributing to its estrogenic actions. the aim of the present study was to clarify whether there is a genetic component for interindividual differences in the formation and clinical effect of agonistic tamoxifen metabolites. a genome-wide association study (gwas) was conducted on steady-state agonist plasma levels in 390 postmenopausal breast cancer patients of european origin who were treated with 20 mg/day of tamoxifen for at least 6 months. plasma concentrations of estrogenic metabolites bisphenol, (e), and (z) metabolite e were quantified using a recently established lc-ms/ms method 1 . promising snps for an association between genotype and either plasma metabolite concentration or clinical outcome were confirmed for their relevance in an independent patient cohort of 313 premenopausal breast cancer patients mainly of european descent 2, 3 . twelve snps close to or above genome-wide significance (p <5e-08) were found to be associated with allele-dependent variable (e) or (z) metabolite e plasma levels, while no genomic hit was found for the tamoxifen metabolite bisphenol. here, positive intergenic or genic regions mapped to chromosomes 1, 2 and 16 for (e) metabolite e and to chromosomes 15 and 18 for (z) metabolite e. upon genotyping of the validation cohort, two genetic loci with minor allele frequencies < 5% were confirmed as putative candidates: rs662106 was associated with a 21-39% variant allele-dependent increase of (e) and (z) metabolite e isomers (p< 0.05), and rs3731872, mapping to a gene encoding zinc finger protein znf124, was associated with increased risk of reccurrence or death (hr carriers 2.6, 95% ci: 1.3 -3.4; p < 0.005). these findings suggest the existence of genetic loci that may contribute to the formation and clinical effect of estrogenic tamoxifen metabolites and therefore could explain therapeutic failure of tamoxifen and/or the occurrence of adverse events during treatment. introduction: metabolomic monitoring of endogenous biomarkers is of increasing importance for the assessment of drug safety and efficacy during clinical drug development. myrcludex b, a novel lipopetide-based entry inhibitor for the therapy of hepatitis b and d, exerts its function through inhibition of the hepatic bile acid transporter na + -taurocholate cotransporting polypeptide (ntcp). in order to assess a myrcludex binduced metabolomic response in humans, lc/ms-based monitoring of endogenous metabolites was performed in blood and urine samples from healthy individuals before and during treatment with myrcludex b. methods: plasma and urine samples were collected from healthy volunteers participating in clinical phase i trials to evaluate safety, tolerability, and pharmacokinetics of single doses of the ntcp inhibitor myrcludex b. using quadrupole time-of-flight mass spectrometry coupled to reversed-phase chromatography (lc-qtof-ms) a set of 15 known ntcp substrates (bile acids) was quantified by targeted metabolomics. protein precipitation was performed in the presence of deuterium-labeled internal standards (istds) which allowed absolute bile acid (ba) quantification in low amounts of plasma. ba profiling in urine was performed after dilution with methanol/water (1:1) in the presence of istds. both methods were validated according to fda guidance and applied to monitor the effect of myrcludex b treatment on human bile acid homeostasis. results: dynamic quantification in plasma and urine was achieved in the range from 7.8 nm to 10000 nm depending on the ba species analyzed. intraday-and interday accuracy and precision were in the 15% tolerance range for all analytes in all matrices. matrix effects were between 39-104% (plasma) and 31-95% (urine), apparent recoveries in plasma were above 97%. basal plasma ba level (mean ± sd) in fasting healthy subjects were 667 ± 574 nm (unconjugated bas), 935 ± 629 nm (glycine-conjugated bas) and 104 ± 62 nm (taurine-conjugated bas). urinary ba level (nmol/g creatinine) were 193 ± 225 nm (unconjugated bas), 89 ± 29 nm (glycine-conjugated bas) and 6 ± 2 nm (taurine-conjugated bas). myrcludex-induced ntcp inhibition resulted in significantly elevated amounts of conjugated ba species demonstrating a spillover of ntcp substrates into the systemic circulation. furthermore, higher urinary ba level were observed during treatment indicating accelerated elimination of excessive bas from the body. conclusion: lc/ms-based monitoring of endogenous biomarkers has been successfully established and applied to study the effect of myrcludex b treatment on human ba metabolism. the results obtained by our assay demonstrate that a myrcludex-induced ntcp inhibition drastically affects human ba homeostasis. this observation provides valuable insights into the drug´s mode of action and will be indispensable for the assessment of side effects and dose-finding processes during future clinical trials. further studies are required to assess a possible role of ba modification (e.g. sulfation) in the process of ba detoxification during myrcludex treatment. key: cord-307731-a2fqmaly authors: vázquez, javier; lópez, manel; gibert, enric; herrero, enric; luque, f. javier title: merging ligand-based and structure-based methods in drug discovery: an overview of combined virtual screening approaches date: 2020-10-15 journal: molecules doi: 10.3390/molecules25204723 sha: doc_id: 307731 cord_uid: a2fqmaly virtual screening (vs) is an outstanding cornerstone in the drug discovery pipeline. a variety of computational approaches, which are generally classified as ligand-based (lb) and structure-based (sb) techniques, exploit key structural and physicochemical properties of ligands and targets to enable the screening of virtual libraries in the search of active compounds. though lb and sb methods have found widespread application in the discovery of novel drug-like candidates, their complementary natures have stimulated continued efforts toward the development of hybrid strategies that combine lb and sb techniques, integrating them in a holistic computational framework that exploits the available information of both ligand and target to enhance the success of drug discovery projects. in this review, we analyze the main strategies and concepts that have emerged in the last years for defining hybrid lb + sb computational schemes in vs studies. particularly, attention is focused on the combination of molecular similarity and docking, illustrating them with selected applications taken from the literature. predicting with chemical accuracy the biological activity that a small drug-like compound can attain against its target is a major challenge in drug discovery. in the late stages of lead optimization, this task can be accomplished by resorting to enhanced sampling techniques, such as free energy calculations [1] [2] [3] , which can estimate the binding affinity between a ligand and its macromolecular target. remarkably, the effort spent in developing robust algorithms in conjunction with efficient configurational sampling methods permit to estimate the binding affinity with a chemical accuracy close to the 1 kcal/mol limit [4] [5] [6] [7] , though at the expense of a significant computational cost that prevents their application in large datasets. nevertheless, attempts have been made to alleviate this limitation through the development of automated workflows for the in silico prediction of binding affinities [8, 9] , which will facilitate the usage of these sophisticated techniques to nonexpert researchers in computational chemistry. several strategies have been proposed to combine lbvs and sbvs in order to reinforce the mutual complementarity of these approaches and palliate their individual weaknesses [39] [40] [41] . the major shortcoming in lbvs is the bias toward the reference template, which may result in overfitting to the input structures. when a pharmacophore is used to guide the screening of the compounds, the chemical features of the ligands present in the training set may affect the optimal choice of the pharmacophoric restraints. moreover, the available activity data may turn out to be inadequate for selecting a structural and functional pool of compounds, often limited by the absence of data relative to poorly active or inactive compounds, which may be valuable to calibrate the merits of the pharmacophore model in distinguishing between actives and inactives. on the other hand, accounting for protein flexibility is a major drawback for docking methods. the binding site of a protein is flexible and can adopt diverse conformational states, generally at the level of side chain residues but often also involving structural changes in loops and the remodeling of secondary structural elements induced upon ligand binding [42] [43] [44] [45] . furthermore, the outcome of docking studies may be largely affected by the identification of water molecules that mediate the interactions of the ligand in the binding pocket, making it necessary to explore the potential role of bridging waters or networks of ordered waters in docking calculations [46] [47] [48] [49] [50] . on the other hand, providing an accurate score and even estimating the binding affinity at a reasonable cost compatible with the screening of large chemical libraries is still challenging for docking methods [51] [52] [53] [54] . finally, the outcome of lbvs and sbvs also appear to exhibit a strong target dependency [55, 56] . for the sake of brevity, a detailed discussion of these weaknesses is omitted here, and the reader is addressed to previous studies in the literature [57] [58] [59] [60] . in this context, searching for computational strategies that can mitigate the limitations of lb and sb methods has been actively pursued in the last years. one alternative is to focus the screening effort on targeted chemical libraries that would facilitate the task of hit identification [61] [62] [63] [64] [65] . this can be achieved via automated algorithms of molecular generation or de novo design, often assisted by artificial intelligence techniques, which aim to create sets of compounds endowed with properties similar to the structural and chemical features found in real cases, including a bias toward specific ranges of physicochemical properties or toward compounds active against a given target. alternatively, a balanced combination of lb and sb methods may be devised to exploit synergistically the merits of these vs techniques, while counterbalancing their limitations, in order to increase the success rate in the screening of large chemical libraries. several strategies have been proposed to combine lbvs and sbvs in order to reinforce the mutual complementarity of these approaches and palliate their individual weaknesses [39] [40] [41] . the major shortcoming in lbvs is the bias toward the reference template, which may result in overfitting to the input structures. when a pharmacophore is used to guide the screening of the compounds, the chemical features of the ligands present in the training set may affect the optimal choice of the pharmacophoric restraints. moreover, the available activity data may turn out to be inadequate for selecting a structural and functional pool of compounds, often limited by the absence of data relative to poorly active or inactive compounds, which may be valuable to calibrate the merits of the pharmacophore model in distinguishing between actives and inactives. on the other hand, accounting for protein flexibility is a major drawback for docking methods. the binding site of a protein is flexible and can adopt diverse conformational states, generally at the level of side chain residues but often also involving structural changes in loops and the remodeling of secondary structural elements induced upon ligand binding [42] [43] [44] [45] . furthermore, the outcome of docking studies may be largely affected by the identification of water molecules that mediate the interactions of the ligand in the binding pocket, making it necessary to explore the potential role of bridging waters or networks of ordered waters in docking calculations [46] [47] [48] [49] [50] . on the other hand, providing an accurate score and even estimating the binding affinity at a reasonable cost compatible with the screening of large chemical libraries is still challenging for docking methods [51] [52] [53] [54] . finally, the outcome of lbvs and sbvs also appear to exhibit a strong target dependency [55, 56] . for the sake of brevity, a detailed discussion of these weaknesses is omitted here, and the reader is addressed to previous studies in the literature [57] [58] [59] [60] . in this context, searching for computational strategies that can mitigate the limitations of lb and sb methods has been actively pursued in the last years. one alternative is to focus the screening effort on targeted chemical libraries that would facilitate the task of hit identification [61] [62] [63] [64] [65] . this can be achieved via automated algorithms of molecular generation or de novo design, often assisted by artificial intelligence techniques, which aim to create sets of compounds endowed with properties similar to the structural and chemical features found in real cases, including a bias toward specific ranges of physicochemical properties or toward compounds active against a given target. alternatively, a balanced combination of lb and sb methods may be devised to exploit synergistically the merits of these vs techniques, while counterbalancing their limitations, in order to increase the success rate in the screening of large chemical libraries. here, our attention is focused on the methodologies and computational approaches undertaken to enrich the outcome of vs by combining lb and sb techniques. in particular, we review the main strategies that have been proposed combining molecular similarity and docking. the strengths and weaknesses of the combined approaches are illustrated by selecting representative studies reported in the literature, primarily dealing with the efforts reported in the last five years. overall, the aim of this review is to provide useful guidelines for the application of combined lb and sb methods in drug discovery. different schemes can be adopted to combine lb and sb methods. the classification proposed by drwal and griffith will be adopted in this review [40] . accordingly, the discussion of the combined lb and sb strategies can be completed following three main categories: sequential, parallel, and hybrid, which are summarized in figure 2 . here, our attention is focused on the methodologies and computational approaches undertaken to enrich the outcome of vs by combining lb and sb techniques. in particular, we review the main strategies that have been proposed combining molecular similarity and docking. the strengths and weaknesses of the combined approaches are illustrated by selecting representative studies reported in the literature, primarily dealing with the efforts reported in the last five years. overall, the aim of this review is to provide useful guidelines for the application of combined lb and sb methods in drug discovery. different schemes can be adopted to combine lb and sb methods. the classification proposed by drwal and griffith will be adopted in this review [40] . accordingly, the discussion of the combined lb and sb strategies can be completed following three main categories: sequential, parallel, and hybrid, which are summarized in figure 2 . (i) sequential approaches divide the vs pipeline in consecutive steps with the aim to perform a progressive filtering in the library of chemical compounds toward the most promising candidates, which will be selected for biological testing at the end of this multi-step process. generally, prefiltering is performed at the beginning of the vs process using lb techniques due to their reduced computational cost, whereas the most computationally demanding sb methods are exploited in the final stages of the selection process. thus, this strategy attempts to optimize the tradeoff between the computational expensiveness and the complexity of the formalism that underlies the filtering technique along the vs process. however, they do not exploit all the available information at once and maintain the limitations of the individual methods. (ii) in the parallel approach, both lb and sb methods are run independently, and the best candidates identified from each separate method are selected for biological testing. swann et al. reported a prospective application of this approach in 2011 [66] , and subsequent studies have examined distinct functional forms for combining the ranks obtained from lb and sb methods (see below). in particular, the compounds obtained in the final rank order lead to meaningful increases in both performance and robustness over the single-modality approaches, but the results also demonstrate the sensitivity of the performance to the target structural details (i.e., the nature of the template ligand in measurements of molecular similarity and the reference protein pocket in docking studies) [67, 68] . (iii) finally, the hybrid strategies comprise approaches that represent a true combination of lb and sb techniques into a standalone method. two main combinations have been followed to (i) sequential approaches divide the vs pipeline in consecutive steps with the aim to perform a progressive filtering in the library of chemical compounds toward the most promising candidates, which will be selected for biological testing at the end of this multi-step process. generally, prefiltering is performed at the beginning of the vs process using lb techniques due to their reduced computational cost, whereas the most computationally demanding sb methods are exploited in the final stages of the selection process. thus, this strategy attempts to optimize the tradeoff between the computational expensiveness and the complexity of the formalism that underlies the filtering technique along the vs process. however, they do not exploit all the available information at once and maintain the limitations of the individual methods. (ii) in the parallel approach, both lb and sb methods are run independently, and the best candidates identified from each separate method are selected for biological testing. swann et al. reported a prospective application of this approach in 2011 [66] , and subsequent studies have examined distinct functional forms for combining the ranks obtained from lb and sb methods (see below). in particular, the compounds obtained in the final rank order lead to meaningful increases in both performance and robustness over the single-modality approaches, but the results also demonstrate the sensitivity of the performance to the target structural details (i.e., the nature of the template ligand in measurements of molecular similarity and the reference protein pocket in docking studies) [67, 68] . (iii) finally, the hybrid strategies comprise approaches that represent a true combination of lb and sb techniques into a standalone method. two main combinations have been followed to achieve this goal: (i) interaction-based methods and (ii) similarity-docking methods. the former translates the observed protein-ligand interactions into pharmacophoric features and quantitative structure-activity relationship (qsar) models [39, 69, 70] , which have been used for several applications, such as vs, the profiling of ligands, the analysis of pseudo-receptors, and de novo designs [71] [72] [73] [74] . on the other hand, the combination of molecular similarity and docking techniques has been examined in the last years as an alternative procedure to assess the reliability of predicted poses of ligands by measuring the overlay against suitable templates [75] [76] [77] [78] [79] [80] . high-throughput vs may be computationally demanding when large sets of compounds have to be evaluated. in this scenario, decomposing the vs pipeline into a multi-step process can be valuable to reduce progressively the number of compounds and enrich the chemical library toward the most promising scaffolds before screening with more expensive methods. lb techniques are generally used in the prefiltering step, as illustrated in different works that have exploited 2d fingerprints [81, 82] , 3d molecular similarity [83] [84] [85] and pharmacophore models [86] [87] [88] . to enhance the drug-likeness of the compounds, knowledge-based in silico admet or pan-assay interference compounds (pains; [89] ) filters can also be applied. for cases with a reduced number of compounds, the results obtained from the sbvs can be further refined, resorting to the structural stability observed in md simulations [90] [91] [92] [93] . as an example that illustrates the sequential application of lb and sb techniques, khan et al. [84] performed multi-step lbvs and sbvs to identify g protein-coupled estrogen receptor-1 (gper-1) modulators ( figure 3a) . lbvs was performed based on a gper-1 selective agonist (1-((3ar,4s,9bs)-4-(6-bromobenzo[d] [1, 3] dioxol-5-yl)-3a,4,5,9b-tetrahydro-3h-cyclopenta(c)quinolin-8yl)ethan-1-one) as a query model for screening of the emolecules library (about 7.2 million compounds used in [84] ) using rapid overlay of chemical structures (rocs; [32] ) and electrostatic potential screening (eon; [94] ). then, after generation of a gper-1 homology model, fred [95, 96] was used to screen the top-scored hits from lbvs. next, the top-ranked hits retrieved by molecular docking were clustered based on the similarity between their scaffolds. finally, the prospective validation in sk-br-3 and mcf-7 cell lines resulted in two compounds with an ec 50 (i.e., effective drug concentration that gives half-maximal response) antiproliferative activity in the micromolar range. alternative lb and sb sequential protocols have also been adopted, as in the study by dawood et al. [97] , where the sbvs was followed by a lbvs ( figure 3b ). an in-house database of 1720 phytochemicals used in traditional egyptian medicine was screened to search for inhibitors of the human aromatase enzyme. the initial size of the library allowed the direct use of molecular docking using glide [98] [99] [100] . subsequently, a lb pharmacophore was used to filter the ranked compounds with phase [101, 102] . in vitro testing revealed that the methylene chloride extract of artemisia annua showed the most significant aromatase inhibitory activity with an ic 50 of 2.2 µg/ml, thus opening a path for the use of secondary metabolites in the search for new therapeutic leads. potential screening (eon; [94] ). then, after generation of a gper-1 homology model, fred [95, 96] was used to screen the top-scored hits from lbvs. next, the top-ranked hits retrieved by molecular docking were clustered based on the similarity between their scaffolds. finally, the prospective validation in sk-br-3 and mcf-7 cell lines resulted in two compounds with an ec50 (i.e., effective drug concentration that gives half-maximal response) antiproliferative activity in the micromolar range. the application of different lb and sb methods generates distinct sets of ranked compounds for the same target. given that there is no single method that consistently ranks a database of compounds in the best decreasing order, a combination of the ranks obtained from multiple lb and sb searches into a single ranking could lead to a better overall enrichment and a wider diversity of hit structures [103] . in this context, lbvs approaches have been combined under the framework of data fusion [104, 105] , targeting the search for new entities, drug repurposing, polypharmacology, and safety profile analysis [106] [107] [108] [109] . with regard to sbvs, distinct methods have been examined to yield a "consensus scoring" [110] [111] [112] [113] , relying on three main strategies: (i) the same docked poses have been evaluated with different scoring functions to build the final ranking, (ii) the results obtained for an ensemble of different protein structures of the same target have been combined to obtain a final score, and (iii) multiple docking methods have been used against a single-protein structure [54, 114, 115] . efforts have also addressed the development of parallel protocols for combining lb and sb methods [66, 67, 103] . table 1 summarizes different fusion strategies that have been adopted to combine the results of lb and sb techniques in benchmarking studies. the parallel selection seems to perform better than other rank fusion metrics, although the quality of the structural information and the specific physicochemical features of the target system may influence the overall performance. for instance, tan et al. [116] evaluated the performance of a parallel protocol that combined docking and similarity search calculations using 2d fingerprints on nine target enzymes. the results were combined through rank fusion, where the ranks from docking and similarity searching were added to generate the final ranking, or the parallel selection method, where compounds are alternately selected according to the ranks obtained separately for lb and sb screenings. these combinations yielded an overall improvement in compound recall in 25% of the calculations. furthermore, parallel selection was found to be more effective than rank fusion. table 1 . description of selected fusion algorithms implemented in parallel ligand-based (lb) and structure-based (sb) strategies. vs: virtual screening. adds together the ranks from the different vs methods rank lists. standard statistical measures, weighted or not, are used (i.e., sum, average, and median or max. value) to combine rank positions. [66, 67, 103, 116] pareto ranking ranks a compound based on how many other compounds are better in all screening methods. ties could be broken using the sum rank, as example. [103] parallel selection compounds are alternatively selected among the top-ranked compounds obtained from each screening method until the desired number of compounds is reached. [81, 103] swann et al. examined the combination of lbvs (2d graph-based extended connectivity fingerprint (ecfp6) [117] and rocs) and sbvs (chemical gaussian overlay, cgo [95] ) within a probabilistic framework that returns a quantitative likelihood (or probability) of observing bioactivity for the selected compounds [66] . the analysis of the results obtained for a set of 18 targets showed that the retrieval rates for the cumulative probability (obtained from the fusion of the individual lb and sb values) are equal to or better than the highest retrieval rate achieved with any single method. similar trends were observed for an additional external validation set of six targets, and, importantly, the method was successful in the identification of novel hit compounds in a prospective study performed against four targets not included in the training and validation sets. a number of studies dealing with the application of the parallel strategy in the search of novel hits have been reported in the last years [118] [119] [120] [121] . an illustrative example is the work by vucicevic et al. [119] , who reported the identification of compounds with anticancer potential effects through a parallel lb and sb screening protocol ( figure 4a ). starting from a large virtual library with more than 9 × 10 6 compounds, those molecules that showed good ranking in both approaches were selected for biological testing. the most active compound exhibited a cytotoxic profile similar to the positive control and enhanced the apoptotic response to doxorubicin, thus representing an adjuvant chemotherapeutic strategy for doxorubicin-insensitive cancers. [117] and rocs) and sbvs (chemical gaussian overlay, cgo [95] ) within a probabilistic framework that returns a quantitative likelihood (or probability) of observing bioactivity for the selected compounds [66] . the analysis of the results obtained for a set of 18 targets showed that the retrieval rates for the cumulative probability (obtained from the fusion of the individual lb and sb values) are equal to or better than the highest retrieval rate achieved with any single method. similar trends were observed for an additional external validation set of six targets, and, importantly, the method was successful in the identification of novel hit compounds in a prospective study performed against four targets not included in the training and validation sets. a number of studies dealing with the application of the parallel strategy in the search of novel hits have been reported in the last years [118] [119] [120] [121] . an illustrative example is the work by vucicevic et al. [119] , who reported the identification of compounds with anticancer potential effects through a parallel lb and sb screening protocol ( figure 4a ). starting from a large virtual library with more than 9 × 10 6 compounds, those molecules that showed good ranking in both approaches were selected for biological testing. the most active compound exhibited a cytotoxic profile similar to the positive control and enhanced the apoptotic response to doxorubicin, thus representing an adjuvant chemotherapeutic strategy for doxorubicin-insensitive cancers. finally, a more recent example is the work by costa et al. [121] , where they performed a parallel vs application followed by md simulations in the search of a novel compound able to inhibit human immunodeficiency virus type 1 (hiv-1) reverse transcriptase (rt) rna-dependent dna polymerase activity ( figure 4b ). more than 143,000 natural compounds commercially available in the zinc database were screened. as a result, 20 hit molecules were chosen and tested in biochemical assays. however, instead of merging the output rankings, compounds shared in both lb and sb vss were selected. among them, three compounds were identified as novel nonnucleoside rt inhibitors in the low micromolar range. as a final remark, let us note that, compared to sequential methods, parallel lb and sb screenings imply a larger computational cost, as several vs techniques have to be simultaneously finally, a more recent example is the work by costa et al. [121] , where they performed a parallel vs application followed by md simulations in the search of a novel compound able to inhibit human immunodeficiency virus type 1 (hiv-1) reverse transcriptase (rt) rna-dependent dna polymerase activity ( figure 4b ). more than 143,000 natural compounds commercially available in the zinc database were screened. as a result, 20 hit molecules were chosen and tested in biochemical assays. however, instead of merging the output rankings, compounds shared in both lb and sb vss were selected. among them, three compounds were identified as novel non-nucleoside rt inhibitors in the low micromolar range. as a final remark, let us note that, compared to sequential methods, parallel lb and sb screenings imply a larger computational cost, as several vs techniques have to be simultaneously run in order to derive their respective rankings, which will subsequently be used to generate the final selection. as noted above, hybrid lb and sb strategies can be grouped into two major categories, which are denoted as (i) interaction-based approaches and (ii) similarity-docking methods. these methods rely on the identification of patterns of protein-ligand interactions, which are subsequently used in the screening of compounds through the usage of pseudo-receptor and pseudoquery methods. since sb information is not effectively incorporated in pseudo-receptor models, we limit ourselves to giving a brief description for the sake of completeness but omit a detailed discussion, which can be found elsewhere [73] . pseudo-receptor methods rely on the mapping of the potential interactions that may be formed by a set of reference ligands suitably aligned in their bioactive conformation to mimic their overlaid arrangement in the binding pocket [122] [123] [124] . this process leads to a rough definition of the overall shape and key anchoring points of the binding pocket, which can be exploited for the screening of chemical libraries. the performance of these models is strongly affected by the chemical space of the ligand dataset and the overlay of the ligands. the model can only account for those features present in the starting set of ligands, and the superposition of ligands is sensitive to minor modifications in the chemical scaffold, especially for highly flexible ligands. in contrast with the preceding approaches, pseudoquery methods exploit the experimental structures of protein-ligand complexes in order to extract a profile of the interaction pattern established by the ligands bound to the protein target. this pattern is generally translated into fingerprints that encode ligand-target interactions or, alternatively, into pharmacophoric features and then used in similarity searches to find ligands that match the interaction pattern [123] [124] [125] [126] [127] [128] [129] [130] [131] (see table 2 for a brief description of several formalisms). in addition, the search of novel hits can be performed, imposing constraints related to the shape and volume of the binding site. table 2 . selection of pseudoquery methods categorized by the underlying protein-interaction model. interaction fingerprint-based sift [132] fingerprint encoding seven predefined types of target-ligand interactions. plip [133] a web service for the detection and visualization of seven protein-ligand interaction types considering a 3d space. flip [134] for each residue, 7 different interactions are represented in 10 bits. padif [135] fingerprints with the inclusion of information relative to the strength of interactions and unfavorable ones. ligandscout [125] pharmacophores derived from six types of nonbonded protein-ligand interactions and volume constraints. flap [126] four-point pharmacophore fingerprints with a shape component. ichem [136] converts the protein-ligand interaction pattern in fingerprints and graphs. tifp [137] encodes a string of unique triplets (two interacting atoms and an interaction pseudo-atom). the pioneering methods included key elements of the protein-ligand complex, such as the formation of hydrogen bonds, hydrophobic or aromatic interactions, or contacts with acidic and basic groups, often supplemented by isocontours of the binding site. as an example, salentin et al. [138] resorted to plip to perform a pharmacophoric search of over more than 170,000 complexes using protein-ligand interaction profiles, leading to the disclosure of the fda-approved malaria drug amodiaquine as the top-ranking hit, which was subsequently validated as a potential anticancer agent showing inhibitory activity on the target protein hsp27. this demonstrates the potential of pseudoquery methods for drug repurposing. recent methods have evolved to include solvation and entropy effects. for instance, tran-nguyen et al. [131] included in their pseudoquery pharmacophoric tool the desolvation component of the protein-ligand interaction energy using a poisson−boltzmann treatment. furthermore, the analysis was decomposed in three consecutive steps: (i) the detection of druggable cavities at the surface of the protein target and the identification of pharmacophoric features, (ii) the generation of cavity-based pharmacophore queries in the 3d space, and (iii) molecular alignment exploiting the cavity-based feature. the proposed pharmacophoric model was benchmarked using dud-e [139] . unique chemotypes were retrieved from high-throughput vs, being as efficient as state-of-the-art docking [140] and shape-matching [32] methods in both pose prediction and ranking power. as noted above, pseudoquery methods have also exploited interaction fingerprint patterns (ifp) containing information about the contacts of the ligand with the protein, thus condensing the 3d structural binding information into a 1d binary string, leading to a drastic reduction in the cost of vs. this is exemplified by the structural interaction fingerprint (sift; [132] ) method, where crystallographic ligands are divided into two groups of fragments: (i) atoms involved in protein-ligand interactions (interaction fragments, ifs) and (ii) fragments generated by the random deletion of ligand atoms used as a control. then, for each ligand and the corresponding fragments, maccs (molecular access system) structural keys [141] were calculated and used as a fingerprint for similarity searching. the results of their validation work suggested that ifs used as templates can increase the similarity search performance of conventional structural fingerprints. sift is included in arpeggio [142] , a web server for the analysis of protein interactions with small-molecule ligands, proteins, and dna. the concept of if has been adopted in alternatives models to outperform conventional scoring functions in predicting the correct poses for drug-like compounds [143] , similarity-based screening [137, [144] [145] [146] [147] [148] , binding/unbinding kinetics [146] , and drug resistance [147] . an important challenge in vs is to create accurate scoring and ranking functions to identify hit compounds active against specific targets. in this context, similarity measurements between compounds can be used to assist molecular docking to score sampled poses [148] [149] [150] [151] [152] [153] [154] and to discriminate between active and inactive molecules [155, 156] . exploiting the synergy between molecular similarity and docking has received increasing interest in the last years, leading to hybridized tools, such as homdock [157] and hybrid [96] . a direct application of merging molecular similarity and docking is related to improving the prediction of the ligand pose in the binding pocket. at this point, exploiting the experimental information on the binding mode of active compounds has been shown to enhance the performance of predicting the pose of drug-like compounds [158] [159] [160] . for instance, the participants of the drug design data resource (d3r) grand challenge 3 were challenged in predicting the binding poses of 24 cathepsin s ligands. kumar and zhang [161] tested the performance of three methods (popss [150] , cdvs [162] , and popss-lite) based on the concept of ligand 3d shape similarity. popss evaluates the shape similarity with existing crystallographic compounds bound to the target protein for predicting the poses of query ligands with unknown binding modes. the ligand with the highest shape similarity score was selected and placed into the binding pocket. after ligand placement, side-chain residues of the binding pocket were repacked based on the query ligand conformation, followed by monte carlo energy minimization of the protein-ligand complex. finally, ligand-bound structures were scored using the rosetta energy function [163] . for cdvs and popss-lite, 3d shape similarity calculations were also used to identify the ligand pose. however, once the suitable ligand-receptor pair was identified, cdvs performed a standard docking using glide, and popss-lite refined the pose with an energy minimization. popss-lite exhibited an excellent performance in this challenge, leading to the lowest mean root mean square deviation (rmsd) values between the native and predicted poses. moreover, cdvs and popss were located among the best 15 methods tested for both metrics. shape similarity between the ligand conformation and the crystallographic ligand is the most common scheme adopted for guiding the pose prediction. however, other similarity measurements and methodological refinements have been explored. as an example jacquemard et al. defined a benchmark constituted by 2376 high-quality structures representing 64 proteins and compared the performance of three rescoring schemes applying the similarity of ifp, graph matching of interaction patterns (grim [137] ), and rocs [78] (figure 5 ). grim and rocs were more efficient than ifp rescoring based on 2d fingerprints, even when the comparison involved structurally dissimilar molecules. in addition, the speed of calculation for all the methods was improved, facilitating the processing of a large number of poses. the search for methodological innovations is also exemplified by kumar and zhang [151] , as they modified popss to account for water-mediated protein-ligand interactions using a continuum benchmark constituted by 2376 high-quality structures representing 64 proteins and compared the performance of three rescoring schemes applying the similarity of ifp, graph matching of interaction patterns (grim [137] ), and rocs [78] (figure 5 ). grim and rocs were more efficient than ifp rescoring based on 2d fingerprints, even when the comparison involved structurally dissimilar molecules. in addition, the speed of calculation for all the methods was improved, facilitating the processing of a large number of poses. the search for methodological innovations is also exemplified by kumar and zhang [151] , as they modified popss to account for water-mediated protein-ligand interactions using a continuum poisson-boltzmann (pb) solvation model, leading to the popss-pb model. popss-pb demonstrated an excellent performance in d3r gc4, with mean and median rmsds of 1.20 (ranked 10th out of 74) and 1.13 (ranked 9th out of 74) å, improving the performance obtained for popss and popss-lite. finally, varela-rial et al. [164] also evaluated in the d3r grand challenge 4 an algorithm named skeledock to define the binding mode based on the structure of a protein−ligand complex. finally, varela-rial et al. [164] also evaluated in the d3r grand challenge 4 an algorithm named skeledock to define the binding mode based on the structure of a protein−ligand complex. the algorithm defines graphs for the query and the template molecules, and then, these graphs are compared to extract a common subgraph, which describes a continuous set of atoms whose element (node) and bonds (edges) are equivalent in the two molecules ( figure 6 ). thus, a mapping that links atoms in query and template compounds can be identified, facilitating the conformational adjustment of the atoms in the query ligand onto those in the template molecule, whereas atoms in the query molecule with no equivalent counterpart in the template are positioned by using a tethered template docking protocol. the algorithm was ranked 15th out of 74 according to the mean rmsd (1.33 å) and 9th according to the median rmsd (1.02 å). (node) and bonds (edges) are equivalent in the two molecules ( figure 6 ). thus, a mapping that links atoms in query and template compounds can be identified, facilitating the conformational adjustment of the atoms in the query ligand onto those in the template molecule, whereas atoms in the query molecule with no equivalent counterpart in the template are positioned by using a tethered template docking protocol. the algorithm was ranked 15th out of 74 according to the mean rmsd (1.33 å) and 9th according to the median rmsd (1.02 å). in addition, to assist the prediction of the ligand pose in the binding cavity, similarity measurements can also be used as a weighting factor in the reranking of the docked compounds. in fact, considering that lb 3d-shape matching algorithms often produced better enrichments than docking, assessing the overlay of docked poses relative to known crystallographic ligands could be valuable to retrieve active compounds in screening studies. to this end, the scoring function in docking calculations could be supplemented with 3d molecular similarity measurements to build the final ranking in the vs process, taking into account that positive candidates accommodated in the active site are expected to share similar structural and physicochemical features that resemble those of known actives in cocrystal structures and improve the ranking of the screened ligands. the first implementations of this lb and sb scheme in docking programs were performed by marialke et al. [157] and mcgann [96] in the development of homdock and hybrid, respectively. homdock is a combination of optimization methods with graph-based molecular alignment (gma; [165] ) that superposes a query molecule on a rigid template. gma places candidate ligands over the template and optimizes their placement in the field of the protein. then, the ligands are ranked according to their interaction with the protein and/or their structural similarity with the ligand. on in addition, to assist the prediction of the ligand pose in the binding cavity, similarity measurements can also be used as a weighting factor in the reranking of the docked compounds. in fact, considering that lb 3d-shape matching algorithms often produced better enrichments than docking, assessing the overlay of docked poses relative to known crystallographic ligands could be valuable to retrieve active compounds in screening studies. to this end, the scoring function in docking calculations could be supplemented with 3d molecular similarity measurements to build the final ranking in the vs process, taking into account that positive candidates accommodated in the active site are expected to share similar structural and physicochemical features that resemble those of known actives in cocrystal structures and improve the ranking of the screened ligands. the first implementations of this lb and sb scheme in docking programs were performed by marialke et al. [157] and mcgann [96] in the development of homdock and hybrid, respectively. homdock is a combination of optimization methods with graph-based molecular alignment (gma; [165] ) that superposes a query molecule on a rigid template. gma places candidate ligands over the template and optimizes their placement in the field of the protein. then, the ligands are ranked according to their interaction with the protein and/or their structural similarity with the ligand. on the other hand, hybrid uses an exhaustive search algorithm, treating ligand and protein structures as rigid bodies. both the protein and ligand flexibility are addressed through multiple conformers. subsequently, the cgo ligand-based scoring function is applied. cgo scores based on how well the docked molecule matches the shape and 3d arrangement of the chemical features of the crystallographic ligand bound to the active site. in another vein, anighoro and bajorath published a series of comparative studies where the best poses of commercial docking software are directly scored using 3d similarity methods, such as the whole-ligand 3d shape similarity and protein-ligand ifp similarity [76, 155, 156] . the protocol was validated by performing retrospective vs calculations for different targets, including dihydrofolate reductase, glucocorticoid receptor, hiv-1 protease, vascular endothelial growth factor receptor-2, adenosine a2a receptor, and β2 adrenergic receptor. as noted in figure 7 , the hybridized approach yielded better performance in retrieving active compounds against the targets included in the validation set. thus, the results showed that ranking by whole-ligand 3d similarity calculations outperformed the force field-based ranking tested for both global performance and early enrichments. it was also shown that a ligand was less suitable as a reference for 3d similarity calculations if it contained large solvent-exposed groups not directly interacting with the target. finally, they highlighted the importance that reference ligands should be engaged in interactions within the binding site as much as possible. as rigid bodies. both the protein and ligand flexibility are addressed through multiple conformers. subsequently, the cgo ligand-based scoring function is applied. cgo scores based on how well the docked molecule matches the shape and 3d arrangement of the chemical features of the crystallographic ligand bound to the active site. in another vein, anighoro and bajorath published a series of comparative studies where the best poses of commercial docking software are directly scored using 3d similarity methods, such as the whole-ligand 3d shape similarity and protein-ligand ifp similarity [76, 155, 156] . the protocol was validated by performing retrospective vs calculations for different targets, including dihydrofolate reductase, glucocorticoid receptor, hiv-1 protease, vascular endothelial growth factor receptor-2, adenosine a2a receptor, and β2 adrenergic receptor. as noted in figure 7 , the hybridized approach yielded better performance in retrieving active compounds against the targets included in the validation set. thus, the results showed that ranking by whole-ligand 3d similarity calculations outperformed the force field-based ranking tested for both global performance and early enrichments. it was also shown that a ligand was less suitable as a reference for 3d similarity calculations if it contained large solvent-exposed groups not directly interacting with the target. finally, they highlighted the importance that reference ligands should be engaged in interactions within the binding site as much as possible. figure 7 . roc plots obtained for four validation targets (dhfr: dihydrofolate reductase, gr: glucocorticoid receptor, hiv1pr: hiv-1 protease, and vegfr2: vascular endothelial growth factor receptor-2) using shape-based similarity (rocs; green), docking (autodock [166] and moe [167] ; cyan), and the hybridized method (yellow). reprinted with permission from the american chemical society [76] . under the same framework, our group has recently presented a 3d similarity scheme to enrich the docking performance based on the usage of lipophilic descriptors [168] determined from quantum mechanical-based continuum solvation models [80] . the 3d similarity was determined by comparing the 3d distribution of atomic lipophilicity computed by pharmscreen [31, 169] (figure 8) , and the similarity measurements were exploited in conjunction with the poses obtained by using three docking programs: glide, rdock [170] , and gold [171] . two hybrid algorithms were examined over 44 sets: (i) rescoring ranking (rr), where the final ranking was determined according to the score obtained from the 3d lipophilic similarity of the best pose generated by the docking method and the co-crystallized ligand, and (ii) consensus ranking (cr), where the final score of the compounds was obtained by merging the rankings provided directly from the docking method and from the rr. the results obtained support the synergy of the hybrid lb and sb approaches, as the cr consistently showed better performance than using only either the lb or sb methods. in addition, the results suggest that cr may overcome the existence of multiple binding modes differing from the experimental pose of the co-crystallized ligand. receptor-2) using shape-based similarity (rocs; green), docking (autodock [166] and moe [167] ; cyan), and the hybridized method (yellow). reprinted with permission from the american chemical society [76] . under the same framework, our group has recently presented a 3d similarity scheme to enrich the docking performance based on the usage of lipophilic descriptors [168] determined from quantum mechanical-based continuum solvation models [80] . the 3d similarity was determined by comparing the 3d distribution of atomic lipophilicity computed by pharmscreen [31, 169] (figure 8) , and the similarity measurements were exploited in conjunction with the poses obtained by using three docking programs: glide, rdock [170] , and gold [171] . two hybrid algorithms were examined over 44 sets: (i) rescoring ranking (rr), where the final ranking was determined according to the score obtained from the 3d lipophilic similarity of the best pose generated by the docking method and the co-crystallized ligand, and (ii) consensus ranking (cr), where the final score of the compounds was obtained by merging the rankings provided directly from the docking method and from the rr. the results obtained support the synergy of the hybrid lb and sb approaches, as the cr consistently showed better performance than using only either the lb or sb methods. in addition, the results suggest that cr may overcome the existence of multiple binding modes differing from the experimental pose of the co-crystallized ligand. while selecting different lb and sb strategies may provide alternative approaches to enrich the results of vs, the identification of a novel lead compound may be conditioned by the structural diversity of the chemical space encoded in compound libraries. therefore, the choice of a representative dataset well-suited to the specific structural, physicochemical, and biological features of the macromolecular target may be crucial for the successful outcome of a vs campaign, especially keeping in mind that the available chemical libraries comprise only a small portion of the synthesizable chemical universe of compounds. to facilitate this task, there has been continued progress in the availability of experimental data in the public domain during the last two decades [172] , which is exemplified in the consolidation of curated databases of bioactive molecules with drug-like properties, as exemplified with chembl [173] , pubchem [174] , and drugbank [175, 176] , where the user may find a comprehensive compilation of diverse information, such as the chemical structure of the compounds, physicochemical properties, biological assays data, related targets, pharmacokinetics and pharmacodynamics properties, metabolic and signaling pathways, and patents. currently, selection of the compounds can be performed through a variety of chemical databases, which can be categorized into three main groups: public, commercial (provided by vendors), and proprietary (table 3 ; see references 177 and 178 for a detailed discussion). table 3 . examples of public and commercial databases (data taken from [177, 178] in this context, rather than focusing the computational effort on the massive screening of larger databases, one might consider the possibility to enhance the success of lb and sb strategies in the search of novel hit compounds by resorting to the screening of targeted chemical libraries. an example is the work by miyao et al., who reported an algorithm for the exhaustive generation of chemical structures based on inverse quantitative structure-property (qspr)/activity (qsar) relationships to build datasets of compounds endowed with a suitable range of desired properties [179] . the synthetic feasibility of the compounds may also be accounted from the availability of information about known chemical reactions [180] [181] [182] . more recently, the de novo design algorithm for exploring chemical space (daecs) exploits the combination of a two-dimensional distribution of the chemical properties with the projection of the biological activity for a set of training compounds in order to generate structures in a specific target area of the chemical space [61, 182] . a library of novel designed structures is constructed through an iterative process that involves the selection of seed structures characterized with selected chemical features and the generation of novel compounds by means of introducing slight structural changes from the seed dataset. the design of target chemical libraries is actually an undertaking of increasing interest, as illustrated by a number of recent studies that have reported the implementation of artificial intelligence-based algorithms [63] [64] [65] [183] [184] [185] [186] . one of them is the development of release (reinforcement learning for structural evolution), which integrates a generative deep neural network with a predictive one into a joint framework for the design of novel compounds satisfying certain chemical requirements, as illustrated with the biased selection of compounds fulfilling a specific range of physical properties (i.e., melting temperature and lipophilicity) or inhibitory activity against the desired target protein (janus protein kinase 2) [62] . another example is the transfer-learning-based generation algorithm proposed by amabilino et al. [186] , where recurrent neural networks are used as smiles (simplified molecular-input line-entry system) generators and trained on a smaller set of molecules with the biological activity of interest for the design of focused libraries. on the other hand, the reinvent code proposed by olivecrona et al. [187] also relies on a recurrent neural network model that operates on a smiles representation of molecules for the automated creation of molecules with predicted biological activity. very recently, this algorithm was adapted to enable the pair-based multi-objective optimization of several molecular features based on pareto dominance [188] and applied to the de novo design of datasets of inhibitors targeting neuraminidase, acetylcholinesterase, and the main protease of the severe acute respiratory syndrome coronavirus 2. another issue that deserves a brief discussion concerns the discovery of ligands able to modulate protein-protein interactions (ppis) in the early stages of drug discovery. given the estimated 650,000 ppis that comprise the human interactome, the stabilization and inhibition of ppis may represent a valuable strategy to alter the oligomerization equilibria of supramolecular protein complexes, thus altering their physiological functions in the cell [189] [190] [191] , which may thus be exploited in the search of novel therapeutic approaches [192, 193] . nevertheless, the success of these studies may be affected by the availability of chemical libraries with ligands suitable to interact with druggable pockets at the interface of protein-protein complexes. in this context, it is worth noting the efforts made toward the design of specific databases enriched in protein-protein modulators, such as ppi-hitprofiler, which was developed to provide for any drug-like compound collection a focused chemical library enriched in putative ppi inhibitors [194] , 2p2i hunter , which is a learning machine tool for filtering potential ppi modulators [195] , and fr-ppichem, which reflects the collective effort of a french consortium to provide a unique chemical library for ppi inhibition [196] . a comparative analysis of different ppi-focused libraries was reported in the study by zhang et al. [197] , where they noticed that ppi inhibitors tend to be larger and more hydrophobic than standard drugs and that ppi-focused libraries, although designed using different strategies, tend to share common chemical subspaces. efforts have also been conducted for the application of combined strategies in the identification of ppi modulators. as an example, singh et al. [198] proposed a vs protocol using a hybrid sb and lb method, highlighting the benefits of 3d topological descriptors to assess the post-docking output. as a validation set, 11 ppi targets with known active and inactive compounds were considered. in a first step, the compounds were docked using surflex, and the docked poses were post-processed to calculate the shape similarity and the structural interaction fingerprint similarity to the co-crystallized ppi inhibitor. notably, the hybrid protocol showed an improved performance for numerous targets, supporting the application of these combined techniques for prospective studies of ppi modulators. in a different context, it is also worth mentioning here the systemic chemogenomics/qsar procedure introduced by cruz-monteagudo et al. [199] , which aimed to generate a disease-relevant pool of ligands by combining phenotypic data with lb and sb information in a sequential process that ended up with a phenotypic vs performed with qsar models (figure 9 ). the first step is the selection of a representative set of ligands targeting a disease with a measurable therapeutic phenotype, such as compounds successfully evaluated in clinical trials. then, information about the ligand-target interaction, key genes, or protein targets involved in the molecular interactions and reaction networks are compiled to build a disease-relevant chemogenomics space, which should encompass potential targets directly or indirectly (i.e., cascading effects) implicated in the physiological response. gene ontology is subsequently used to encode the systemic effect of each ligand in fingerprints containing both chemical descriptors and biological information. the codified compounds are split into two classes: ligands that significantly interact with at least one target (phenotype-positive class) and compounds with no significant interaction with any of the targets associated with the desired phenotype (phenotype-negative class). finally, a qsar-based vs methodology is performed. this protocol was utilized in a retrospective study aimed at prioritizing ligands acting as neuroprotective agents in parkinson's disease, and a significant fraction of the drug candidates used as starting points could be recovered at early fractions of the screened data. compounds are split into two classes: ligands that significantly interact with at least one target (phenotype-positive class) and compounds with no significant interaction with any of the targets associated with the desired phenotype (phenotype-negative class). finally, a qsar-based vs methodology is performed. this protocol was utilized in a retrospective study aimed at prioritizing ligands acting as neuroprotective agents in parkinson's disease, and a significant fraction of the drug candidates used as starting points could be recovered at early fractions of the screened data. as a final remark, it is worth emphasizing the relevance of curating the activity data in the analysis of the compounds and the preparation of targeted chemical libraries, minimizing the bias introduced by spurious hits, which can arise from a number of unexpected factors, such as covalent modification of specific protein residues or changes in the redox state of a range of ligands. in particular, a large number of cases have been attributed to self-aggregation of the ligand in an aqueous solution [200] [201] [202] . the tendency of small organic molecules to spontaneously form colloidal self-aggregates can lead to undesired artifacts in the screening of drug-like compounds, resulting in the identification of false positives [203, 204] . the colloidal aggregates formed by these types of compounds exhibit common trends, such as the lack of robust structure-activity relationships, as well as the identification of time-dependent noncompetitive-like inhibition [200, 205] , likely reflecting the nonspecific inhibitory mechanisms related to adsorption of the target protein onto the aggregates or the induction of conformational alterations that affect the protein's activity [206] . in these cases, a critical parameter to be considered is the critical aggregation concentration of the compound, which turns out to be in the micromolar range for a significant number of aggregating drug-like compounds [202, 207] . overall, this discussion suffices to emphasize the need to perform a detailed curation of the biological data and of the conditions used in experimental assays, as this information may have an unexpected influence on the efficacy of the bioactive compounds. as a final remark, it is worth emphasizing the relevance of curating the activity data in the analysis of the compounds and the preparation of targeted chemical libraries, minimizing the bias introduced by spurious hits, which can arise from a number of unexpected factors, such as covalent modification of specific protein residues or changes in the redox state of a range of ligands. in particular, a large number of cases have been attributed to self-aggregation of the ligand in an aqueous solution [200] [201] [202] . the tendency of small organic molecules to spontaneously form colloidal self-aggregates can lead to undesired artifacts in the screening of drug-like compounds, resulting in the identification of false positives [203, 204] . the colloidal aggregates formed by these types of compounds exhibit common trends, such as the lack of robust structure-activity relationships, as well as the identification of time-dependent noncompetitive-like inhibition [200, 205] , likely reflecting the nonspecific inhibitory mechanisms related to adsorption of the target protein onto the aggregates or the induction of conformational alterations that affect the protein's activity [206] . in these cases, a critical parameter to be considered is the critical aggregation concentration of the compound, which turns out to be in the micromolar range for a significant number of aggregating drug-like compounds [202, 207] . overall, this discussion suffices to emphasize the need to perform a detailed curation of the biological data and of the conditions used in experimental assays, as this information may have an unexpected influence on the efficacy of the bioactive compounds. in the past decades, vs has been a powerful alternative to high-throughput screening assays due to the reduced expensiveness, the continued progress in computer resources, and the refinement in lb and sb techniques, often leading to hit rate enrichments that outperform the results obtained with experimental screenings. being the most widely used strategy for disclosing novel hit compounds in the early stages of drug discovery, the success of vs campaigns is also severely affected by the intrinsic shortcomings of both lb and sb methods, which makes it necessary to search for novel computational strategies that exploit the merits of the individual techniques synergistically. in the last years, we have witnessed a flourishment of different combined lb and sb approaches, ranging from the hierarchical application of techniques in multi-step filtering process to novel methods that integrate lb and sb techniques into a standalone framework. the progress is encouraging, but it can be anticipated that the adoption of these integrated strategies will depend on two main factors. first, an extensive benchmarking of the distinct combination strategies, including a diverse sets of targets covering distinctive structural and physicochemical features, the calibration of different descriptors for similarity measurements, and docking algorithms in retrospective studies should be necessary, eventually complemented with the prospective application to drug discovery projects. these studies should be valuable to judge not only the improvement obtained with the usage of integrated methods relative to either pure lb or sb techniques but, also, to identify the optimal combination strategy in light of the druggability characteristics of the target protein. second, the ability to implement the combined lb and sb strategies in automated modeling platforms should provide user-friendly access to the screening of targeted-oriented chemical libraries, guidelines for an appropriate design of the combination strategy, and graphical display facilities to analyze the results. the implementation in modern software will be necessary to facilitate the adoption of the combined strategies by the drug discovery community. the authors declare no conflict of interest. advancing drug discovery through enhanced free energy calculations free energy methods in drug design: prospects of "alchemical perturbation" in medicinal chemistry accuracy assessment and automation of free energy calculations for drug design exploring the 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self-associations into nano-entities case studies of minimizing nonspecific inhibitors in hts campaigns that use assay-ready plates how do small molecule aggregates inhibit enzyme activity? a molecular dynamics study stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors key: cord-029747-8f463wz0 authors: viedma-poyatos, álvaro; pajares, maría a.; pérez-sala, dolores title: type iii intermediate filaments as targets and effectors of electrophiles and oxidants date: 2020-07-17 journal: redox biol doi: 10.1016/j.redox.2020.101582 sha: doc_id: 29747 cord_uid: 8f463wz0 intermediate filaments (ifs) play key roles in cell mechanics, signaling and homeostasis. their assembly and dynamics are finely regulated by posttranslational modifications. the type iii ifs, vimentin, desmin, peripherin and glial fibrillary acidic protein (gfap), are targets for diverse modifications by oxidants and electrophiles, for which their conserved cysteine residue emerges as a hot spot. pathophysiological examples of these modifications include lipoxidation in cell senescence and rheumatoid arthritis, disulfide formation in cataracts and nitrosation in endothelial shear stress, although some oxidative modifications can also be detected under basal conditions. we previously proposed that cysteine residues of vimentin and gfap act as sensors for oxidative and electrophilic stress, and as hinges influencing filament assembly. accumulating evidence indicates that the structurally diverse cysteine modifications, either per se or in combination with other posttranslational modifications, elicit specific functional outcomes inducing distinct assemblies or network rearrangements, including filament stabilization, bundling or fragmentation. cysteine-deficient mutants are protected from these alterations but show compromised cellular performance in network assembly and expansion, organelle positioning and aggresome formation, revealing the importance of this residue. therefore, the high susceptibility to modification of the conserved cysteine of type iii ifs and its cornerstone position in filament architecture sustains their role in redox sensing and integration of cellular responses. this has deep pathophysiological implications and supports the potential of this residue as a drug target. intermediate filaments (ifs) are cytoskeletal structures critical for cell mechanics, which maintain a close interplay with the other cytoskeletal systems, namely, actin microfilaments and microtubules [1, 2] . ifs act as integrators of cytoskeletal responses and cell behavior. they are constituted by homo-or heterooligomers of proteins, which are classified into six different families depending on their structure. keratins (types i and ii), vimentin (type iii) and nuclear lamins (type v) are examples of the different protein families and illustrate the critical roles of ifs in epithelial biology, cytoplasmic and nuclear mechanics and stress sensing, and nuclear support and function, respectively [3] . mutations in many if proteins lead to devastating diseases, ranging from epidermolysis bullosa (keratin) [4] to premature aging syndromes (lamins) [5] , which highlight their key function in cell and organism homeostasis. the type iii if protein family comprises vimentin, glial fibrillary acidic protein (gfap), desmin and peripherin, which are important components of the cytoplasmic cytoskeleton and are expressed in specific cell types. vimentin is present mainly in cells of mesenchymal origin and is the most widely expressed type iii if protein [6, 7] , whereas the expression of desmin, gfap and peripherin is mainly restricted to the muscle and glial cells and neurons of the peripheral system, respectively [6] . vimentin is also the most thoroughly studied member and is frequently considered as a prototype for the assembly, regulation and implications of the constituents of this family [7] . therefore, many of the features outlined below derive from research on vimentin. type iii if proteins form a dynamic network that typically extends from the periphery of the nucleus to the cell membrane, interacting with and seemingly modulating the behavior of the actin and tubulin cytoskeletons [7] [8] [9] [10] . vimentin influences many critical cellular processes including cell plasticity and mechanics, deformability, cell cortex properties in interphase and mitosis, migration and adhesion, cell division, organelle positioning, and nucleus and dna integrity under stress [6] [7] [8] [11] [12] [13] . in turn, gfap has been involved in astrocyte https://doi.org/10.1016/j.redox.2020.101582 received 11 february 2020; received in revised form 5 may 2020; accepted 13 may 2020 only cysteine residue. moreover, this residue is conserved in all species, from zebra fish to humans. the conserved cysteine residue is located in the last coil segment of the rod, facing outwards from the vimentin dimer [25] . notably, whereas in the a11 tetramer cysteine residues from different dimers will be far apart, in the a22 tetramer conformation, the cysteine residues would be closer. nevertheless, information on the three-dimensional organization of tetramers in the body of the filament would be necessary in order to ascertain the relative positions of cysteine residues from different tetramers. from the existing models of vimentin [19, 24] , it could be hypothesized that the cysteine residue would occupy a position close to the space needed for the interdigitation of adjacent ulfs during elongation (fig. 1 ). therefore, bulky modifications of this residue could lead to alterations in assembly. in the context of oxidative modifications, other nucleophilic residues prone to oxidation or adduction of electrophiles, including several histidine, lysine and arginine residues, as well as the single tryptophan (w290 in vimentin), are also conserved among members of the type iii if family, as well as between species [26, 27] . in cells, type iii ifs are under constant remodeling through the exchange between the assembled polymers and the pool of soluble subunits, and their dynamics is tightly controlled by posttranslational modifications (ptms), mainly phosphorylation [28] . regulation of vimentin by phosphorylation is critical in mitosis, where the spatiotemporally concerted action of several kinases allows its reorganization, which is important for completion of cytokinesis [29] . in addition, the extent of phosphorylation together with protein-protein interactions influences whether vimentin is disassembled in mitosis or remains in filaments [30] that intertwine with and modulate the actin cortex, allowing proper mitosis progression [8] . type iii ifs can suffer diverse enzymatic ptms, including ubiquitination and sumoylation [31] , glycosylation [32] , proteolysis [33] , and acetylation [34] . one important modification with significant pathophysiological consequences is citrullination [35] . citrullinated vimentin is an autoantigen involved in the pathogenesis of rheumatoid arthritis and in antitumor immunity [36, 37] . in turn, citrullinated gfap has been detected in several diseases, including alzheimer's disease (ad) and multiple sclerosis, and it has been reported to constitute an early response to retinal injury [38, 39] . enzymatic ptms of type iii if proteins have been considered in several important reviews [31, 40] . nevertheless, type iii ifs are also subjected to numerous non-enzymatic modifications, including those induced by oxidants and electrophiles, which are arising as important determinants in filament structure and function and will be considered in more detail below. all cells are exposed to reactive oxygen and/or nitrogen species (ros/rns) and possess defense mechanisms to avoid excessive oxidation. oxidative stress arises when oxidant levels, either from endogenous or exogenous sources, surpass the cellular antioxidant defenses. protein oxidation can occur upon exposure to diverse oxidant species and induce multiple structural alterations. several excellent alzheimer's disease axd alexander's disease 15d-pgj 2 [41, 42] , and of cysteine residues in particular [43, 44] , and provided detailed methodological information for their assessment. in addition, oxidative stress can increase the production of electrophilic species eliciting additional ptms. depending on their type and/or extent, oxidative and electrophileinduced ptms can contribute to cell homeostasis, signaling or stimulation of antioxidant defenses, or result in protein dysfunction, (caption on next page) á. viedma-poyatos, et al. redox biology 36 (2020) 101582 misfolding or aggregation, and to the impairment of protein interactions and functions, in the cell or in the extracellular medium [45] [46] [47] . importantly, these ptms can lead to structure-specific outcomes. thus, depending on the protein, certain modifications can have virtually no consequences on protein function, whereas others can lead to activation or inhibition, or to the modulation of protein-protein interactions or subcellular localization [45, 48, 49] . cysteines are the most nucleophilic residues and readily attack and form adducts with electrophiles of very diverse structure. therefore, the moieties bound to a protein through cysteine residues can be very varied, and range from the addition of one oxygen atom or one oxygen plus one nitrogen atom, in the case of sulfenylation (sometimes called sulfenation) and nitrosation (frequently referred to as nitrosylation) [50] , respectively, to bulky substitutions by endogenous reactive species or drugs (fig. 2) . formation of disulfide bonds can result in covalent binding of the protein to small molecules, like cysteine or glutathione (gsh) (cysteinylation and glutathionylation, respectively), or to other proteins. importantly, cysteine modifications can sometimes interconvert. disulfide exchanges between proteins and small molecules can occur. moreover, the nitroso moiety can be transferred between proteins or lead to no release. interestingly, nitrosoglutathione (gsno) can induce transnitrosation or glutathionylation of target cysteine residues depending on their nucleophilicity. thus, highly nucleophilic thiols break the s-no bond leading to glutathionylation, whereas moderately nucleophilic thiols are more likely to become transnitrosated [51] . in turn, oxidized and electrophile adducted moieties can suffer further transformations leading to derived species and/ or protein crosslinks [41] . an important aspect of cysteine oxidative modifications is stability. while some modifications are readily reversible (e.g. sulfenylation, disulfide formation and nitrosation), others are considered irreversible due to their high stability under biological conditions (e.g. sulfonylation). some reversible modifications of cysteine residues, including glutathionylation or nitrosation have been considered as protective mechanisms preventing the occurrence of "irreversible" modifications, thus preserving protein function [52] . cysteine residues can also undergo a plethora of enzymatic ptms, including some catalyzed oxidative modifications, acylations, prenylation and methylation (for cterminal isoprenylated cysteine residues), etc. [53] [54] [55] . therefore, in certain cases there can be interplay between these ptms leading to structurally and functionally diverse proteoforms [49] . the term lipoxidation refers to the modification of proteins by reactive products of lipid peroxidation [56] . these electrophilic lipids are generated under physiological conditions at moderate levels and they participate in signaling pathways and adaptive and cytoprotective responses that have been recently reviewed [57] . however, under pathological situations commonly associated with oxidative stress, the levels of these agents increase, favoring their reaction with other biomolecules including other lipids, dna and proteins. the structures and concentrations of electrophilic lipids can be very varied (fig. 3) , and can range from small aldehydes, like acrolein or malondialdehyde (mda) (56 and 72 da, respectively), to cyclopentenone prostaglandins (cypg) or isoprostanes (300-400 da), or even oxidized or nitrated phospholipids (700-900 da). indeed, the number of reactive lipid species expands to several hundred [56] . adducts with protein residues occur mainly through schiff base formation with amino groups or , rod and tail domains is shown for the four members of the type iii class of if (upper panel) . dots indicate the position of the conserved cysteine residue (black) and that of the additional cysteine of peripherin (red). amino acid numbering for the various domains corresponds to the following entries from uniprot: vimentin, p08670; gfapα, p14136; desmin, p17661; peripherin, p41219. the lower panel depicts a cartoon view interpretation of the assembly process of type iii ifs, as inferred from numerous in vitro works (see the text for details). please note that in unit length filaments (ulfs), individual tetramers are deliberately not perfectly aligned. the 2-ulf "filament" shown at the bottom illustrates the potentially critical position of the cysteine residue, which could lie close to the region required for the "plugging" of two ulfs. modifications of this residue with bulky moieties may have important consequences for filament assembly or elongation. although the a11 tetramer assembly is considered the starting unit, once assembled in filaments, vimentin dimers can fall in different relative positions, giving rise to several tetrameric configurations, including those outlined in the figure by color shadowing: a11 tetramer (blue), acn tetramer (yellow) and a22 tetramer (green). these tetrameric configurations have been schematically extracted at the right for easier visualization. depictions of if monomer and ulf are modified from ref. [13] . (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) fig. 2 . schematic representation of several modifications of vimentin c328. the figure shows a cartoon of a stretch of the alpha helical structure of a segment of vimentin and the c328 lateral chain prepared using pymol and data from the pdb entry 3klt [202] . structures of nitrosocysteine, acrolein and 15d-pgj 2 have been obtained from pubmed compound. this schematic representation illustrates the diverse size of the moieties that can bind to c328, potentially imposing steric hindrances or altering interactions required for filament assembly. through michael addition with various residues bearing nucleophilic moieties, like the thiol group of cysteine, imidazole of histidine and amino of lysine residues [48] . the stability of adducts formed will be influenced by several factors including the nature of the adducted residue and the occurrence of additional intramolecular reactions [58] . moreover, both, non-enzymatic and enzymatic reversal of certain electrophilic lipid-protein adducts has been reported in vitro as well as in cellular models [59] [60] [61] . therefore, lipoxidation can constitute a dynamic modification contributing to signaling mechanisms [60, 61] . given the structural variety of adducts their functional consequences (structure-activity relationships) can be diverse. there are several examples of differential effects of lipoxidation of cysteine residues, depending on the adducted moiety. adduction of distinct electrophilic lipids to ras proteins can differentially influence activation and subcellular localization [49, 62] . similarly, the aldo-keto reductase enzyme akr1b1 is activated by addition of small moieties but inhibited by larger ones [63] . cypg are electrophilic eicosanoids derived from prostaglandins by non-enzymatic dehydration [64] . they are characterized by the presence of an α,β-unsaturated carbonyl group in the cyclopentane ring that allows michael adduct formation, preferentially with cysteine residues. one example of these eicosanoids is 15-deoxy-δ 12,14 [13] ; network fragmentation alone or accompanied by solubilization, characterized by the diffuse background, correspond to treatment with diamide or diamide plus calyculin a, respectively, as in ref. [91] ; filament bundling is exemplified by treatment with 15d-pgj 2 as in ref. [91] , and peripheral condensation, by treatment with no 2 -popc as in ref. [115] . scale bar, 20 μm. please note that the effects observed in cells can be the consequence of direct and/or indirect modifications of if proteins, as well as of ptm interplay. prostaglandin j 2 (15d-pgj 2 ) (fig. 2) , which has been broadly studied both in cell culture systems and in vivo. the effects of cypg, as those of many lipoxidation agents and oxidants, can have a dual or hormetic nature, depending on time or concentration. low concentrations can activate the cellular antioxidant defenses and potentiate the initial inflammatory response, whereas high levels can elicit anti-inflammatory and antiproliferative effects and induce cytotoxicity [65, 66] . intracellular gsh is an important determinant of cypg distribution and effects through various mechanisms, including the formation of cypg-gsh adducts that can be involved in cypg transport or sequestering, leading to a differential modification of proteins depending on the subcellular compartment [49] . numerous studies on protein oxidative damage use the term carbonylation to refer to the formation of carbonyl groups on proteins. chemically, carbonylation refers to the addition of carbon monoxide to a compound leading to the appearance of a carbonyl group. appearance of carbonyl groups on proteins can occur by several mechanisms, including oxidation of amino acid lateral chains, oxidative deamination or formation of certain adducts with compounds that retain a free carbonyl group, as in some types of lipoxidation, like michael addition of cypg or of hne, or after glycation or glycoxidation. as none of these processes involves carbon monoxide addition the term "formation of protein carbonyls", should be preferred instead of protein carbonylation, which, as stated above is not selective for a particular type of modification, but can arise by multiple mechanisms, as detailed in ref. [67] . early functional studies in cells lacking vimentin showed altered nuclear morphology and impaired distribution of certain organelles [68] . however, the finding that vimentin knockout mice were fertile and apparently developed normally [69] dimmed the interest in this protein. nevertheless, subsequent works have established the role of vimentin as a key player in cellular organization and as a modulator and effector of numerous biological and pathophysiological processes. indeed, vimentin knockout animals do not respond normally to many kinds of stress and show alterations in immune function [70] , autophagy, cell migration and division, wound healing, function of the central nervous system, and arterial remodeling [71] , among others. in addition, vimentin is an important marker and player of epithelial mesenchymal transition and its expression in certain types of cancer can be considered a sign of malignancy and invasiveness [16] . it is also necessary for cellular entry and/or toxic cellular actions of diverse bacterial and viral pathogens [72] [73] [74] . in contrast to other if proteins, only a few mutations in the vimentin sequence have been identified. the e151k mutation [75] , the heterozygous frameshift from v6c [76] and the missense (q208r) [77] mutation have been associated with a hypothetical model to conciliate the differential effects of certain oxidants on the assembly of soluble if proteins and preformed filaments is shown. a very simplified representation of if dimers (rectangles) and if tetramers is depicted, showing the approximate positions of the cysteine residues (black dots). on the left, disulfide formation (red ellipses) or modification of cysteine residues by bulky moieties (yellow shapes) in soluble if tetramers may interfere with their subsequent assembly in ulfs or with ulf elongation upon induction of polymerization, or may impede conformational rearrangements needed for proper assembly. conversely, on the right, preformed filaments could undergo a certain degree of modification on accessible residues, or of disulfide formation between close cysteine residues, without suffering substantial disruption of their assembly. nevertheless, modification above a certain threshold could lead to filament disruption. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) cataract development. recently, a dominant vimentin variant (l387p) causing a rare syndrome with premature aging has been reported [78] . vimentin is the target of numerous enzymatic and non-enzymatic ptms, some of which have been outlined above. accumulating evidence indicates that vimentin behaves as a sensor for several kinds of stress, including oxidative stress and generation of lipid-or sugar-derived electrophiles that target several residues leading to versatile network remodeling [56, 79] (fig. 3) . the single cysteine residue of vimentin (c328) appears to be highly susceptible to oxidation or addition of diverse species, both in vitro and in cells [25, [80] [81] [82] [83] [84] , suggesting that it is a preferred site of modification. moreover, modifications of c328 play a key role in redox-related reorganizations of vimentin filaments, with diverse consequences on cell behavior. disulfide formation and other cysteine oxidations. early studies using cytoskeleton preparations from several cell types subjected to oxidative in vitro crosslinking with cu 2+ -phenanthroline showed the formation of homo-and heterodimers of either vimentin and desmin or vimentin and gfap, which led to propose that both proteins were present in hybrid filaments [85, 86] . these studies indicated that cysteine residues in the "crosslinkable" units were exposed to the protein surface at a distance sufficiently close to allow interaction. oxidized oligomeric species of vimentin were also detected in fibers isolated from eye lenses of several species, when extracted in the absence of beta-mercaptoethanol [87] . later studies putatively identified vimentin as one of the proteins undergoing disulfide bonding in fibroblast-like synoviocytes subjected to oxidative stress by treatment with h 2 o 2 [88] , and soon after, traub et al., showed that oxidized vimentin and desmin species, including homo-and heterooligomers of both proteins, formed abnormal filaments, which could be restored by reducing agents [89] . the involvement of the cysteine residue in disulfide formation and its impact on assembly was confirmed by using a c328a vimentin mutant that showed resistance to crosslinking by oxidation with h 2 o 2 and cucl 2 and formed apparently normal filaments in vitro [25] . it was then postulated that crosslinking of vimentin wild type could occur between neighboring dimers arranged in the a22 conformation, in which the cysteine residues fall in a closer position (fig. 1 ). however, it was noted that this arrangement was still not ideal for disulfide bond formation unless additional flexibility or molecular motion within the tetramer occurred to allow a closer contact between the cysteine lateral chains [25] . dimeric species of vimentin have also been observed in sds-page gels upon treatment with bifunctional cysteine crosslinkers, like dibromobimane (spacer arm 4.66 å), both in vitro and in cells [13] , indicating that in intact cells cysteine residues within filaments can also be present at a close distance. moreover, the use of this reagent allowed obtaining vimentin-gfap heterodimers from intact astrocytoma cells [90] , thus suggesting the presence of both proteins in the same filaments as proposed in earlier in vitro works [85, 86] . recently, the electrophilic reagent diamide has been used to induce vimentin disulfide bonding [91] . this reagent reacts sequentially with two thiol groups facilitating disulfide bond formation. diamide-treated purified vimentin appears as monomer and dimer species in non-reducing sds-page gels in approximately a 1:1 proportion. notably, disulfide formation profoundly impaired subsequent polymerization into filaments of vimentin wild type, but not of a cysteine-deficient mutant. however, preformed filaments underwent disulfide formation to a similar extent than the soluble protein but were not disrupted by short diamide treatments. therefore, these results suggest that promoting disulfide bond formation in soluble tetramers may lead to their association in conformations that are not productive for subsequent assembly, whereas once filaments are formed and the subunits are in place, a certain number of disulfide bonds can form between cysteines that fall within close distance without disrupting the whole filament (fig. 4) . remarkably, although resistant to oxidation by diamide, filaments formed in vitro by a c328s mutant showed subtle, but significant, differences with vimentin wild type, calling attention on the importance of this residue for filament assembly [91, 92] . interestingly, diamide induces a major disruption of vimentin filaments in cells in such a way that filaments rapidly reorganize into discrete dots [13, 91] . in cells, both soluble and polymerized vimentin coexist and exchange. therefore, oxidation of soluble vimentin could hamper its incorporation into filaments leading to network disruption. moreover occurrence of additional ptms as a consequence of oxidative stress, could also contribute to diamide-induced filament fragmentation. nevertheless, the fact that the c328s vimentin mutant is completely resistant to this effect, confirms the critical role of the modification of the cysteine residue in vimentin reorganization [91] . formation of mixed disulfides can also occur between vimentin and small thiol-containing molecules, like gsh (glutathionylation) or cysteine (cysteinylation). vimentin glutathionylation has been observed in several experimental models. stimulation of isolated aortic rings with acetylcholine led to vimentin glutathionylation through an enos-dependent mechanism [93] . notably, in this model system, glutathionylation can occur directly or after denitrosation. as enos possesses basal activity in endothelial cells, these observations suggest that glutathionylation could occur under basal conditions and constitute a physiological regulatory mechanism. indeed, vimentin glutathionylation has been detected in control mouse embryonic fibroblasts [94] . nevertheless, this modification increases under conditions of oxidative stress. in particular, loss of function of the krit1 gene, which is associated with the pathogenesis of cerebral cavernous malformations, induces a decrease in the gsh/gssg ratio and increased glutathionylation of several proteins, including vimentin, in cellular models of the disease [94] . glutathionylation of vimentin has also been observed in oxidatively stressed t-lymphocytes or cos7 cells exposed to no donors [93, 95] , and in human senescent fibroblasts, where it has been connected with cell growth arrest and cell death [96] . moreover, data mining of the oxidized thiol proteome, has found a high proportion of vimentin c328 involved in disulfide bonding in aging and cataractous lenses [82] . from the functional perspective, the vimentin cysteine residue has been compared to cryptic cysteines in other proteins, in which glutathionylation could contribute to protein elasticity [97] . in addition, recent studies indicate that quantitative vimentin glutathionylation impairs vimentin elongation and provokes pre-formed filament severing [98] , supporting the view that modification of c328 with bulky moieties could have important consequences for filament assembly. nevertheless, the functional implications of vimentin glutathionylation in cells have not been fully elucidated and could depend on the proportion of the protein modified, and/or represent a protective mechanism against irreversible modifications. the oxidation of vimentin in the extracellular space deserves special attention. vimentin can be exposed at the cell surface or secreted by several cell types [99, 100] . cell surface vimentin can be labeled by cellimpermeable cysteine reagents. the observation that labeling can be increased by pretreatment with the reducing agent n-acetyl-cysteine suggests that part of the protein exposed is reversibly oxidized [101] . interestingly, oxidative stress increases extracellular exposure of vimentin, frequently in an oxidized form [100] , and it has been proposed that this phenomenon could constitute an early response of cells to oxidants from the extracellular space, and at the same time, generate oxidized and secreted species of vimentin that could be involved in pathogenesis of autoimmune diseases or act as a vehicle amplifying oxidative stress and lipid oxidation [74, 102] . nitrosation. the above observations show that vimentin cysteine can be modified under (patho)physiological conditions. shear flow is a physiological stimulus that activates enos in endothelial cells leading to increased no levels that can induce protein s-nitrosation. indeed, vimentin was identified among the s-nitrosated proteins detected in endothelial cells after treatment with an exogenous no donor or upon stimulation of endogenous no production [103] . moreover, c328 has been identified as the nitrosated residue in endothelial cells after shear stress, depending on enos activity [104] , and after no-donor exposure [105] . vimentin nitrosation can also occur under basal conditions and increase as a consequence of drug treatment. statins, drugs widely used to control cholesterol levels, modulate enos expression in endothelial cells [106] , and lead to increased enos activity and nitrosation of endothelial proteins, including vimentin [107] . nitrosation has been proposed to occur in hydrophobic environments, and indeed, hydrophobicity plots predict c328 to be located in a hydrophobic motif, qsltcevda [104] . interestingly, the sequence -[i/l]-x-c-x 2 -[d/e]-, has been reported to constitute a consensus motif for inos-s100 catalyzed transnitrosation [108] , that can take place for instance in activated peripheral blood monocytes. vimentin c328 is located in one such motif and has been validated as a target for nitrosation through this mechanism. the functional significance of vimentin nitrosation is not fully understood, although in endothelial cells it has been proposed to contribute to endothelial remodeling under flow. a recent study indicates that s-nitrosation of vimentin at c328 does not hinder, but slows down, vimentin elongation in vitro. these results shed light on the different functional outcomes of structurally different c328 modifications [98] . in addition, being a readily reversible modification, snitrosation could also act as a defense mechanism protecting proteins from deleterious oxidative modifications. lipoxidation. the stability of some adducts formed between vimentin and electrophilic lipids has allowed obtaining structural and functional information on the role of the lipoxidation of vimentin cysteine, both in vitro and in cells. modification of vimentin by cypg in vitro occurs selectively at c328 [80, 91] , and disrupts subsequent naclelicited polymerization, inducing filament widening and bundling and formation of aggregates, in wild type but not c328s vimentin. notably, these differential effects occurred in conditions under which the extent of vimentin lipoxidation was estimated to be approximately 10% [80] , which is in the range of that estimated after treatment of cells with biotinylated cypg analogs [109] . nevertheless, at high 15d-pgj 2 concentrations this selectivity is lost, since these compounds can form adducts with other nucleophilic residues, namely, histidine. interestingly, as observed with oxidants, preformed filaments appear to be protected from disruption in vitro, even when modified to the same extent, potentially due to a different topology of the modification (fig. 4) . the amenability of cypg to labeling with biotin or other moieties has allowed their use as probes for lipoxidation. using biotinylated 15d-pgj 2 or pga 1 , vimentin was first identified as one of the major targets for these eicosanoids in mesangial cells, in which they induce a marked cytoskeletal rearrangement leading to perinuclear condensation of vimentin filaments into thick bundles [109] . besides filament condensation, cypg provoke the dislodgement of vimentin from the actin cell cortex in mitosis, which could have deleterious consequences for cell division [8] . the use of a vimentin c238s mutant has allowed identifying the cysteine residue as the key site for adduct formation and/or for the induction of vimentin filament disruption by cypg in several cell types [13, 80] . as other electrophilic lipids, cypg covalently bind to multiple cellular targets and induce oxidative stress on their own, thus the possibility exists that some of their effects on the vimentin network may be indirect. indeed, in addition to lipoxidation, 15d-pgj 2 has been shown to induce reversible cysteine oxidation (cysteinylation) of certain proteins [110] . nevertheless, the fact that the effects of cypg are attenuated in the c328s mutant, both in vitro and in cells, indicate that this residue is a critical site for the action of these eicosanoids. hne is one of the most studied electrophilic lipids. the levels of hne, and of hne-protein adducts, increase under many pathophysiological conditions, including inflammatory, neurodegenerative diseases and aging [56, 111] , in some of which hne can reach micromolar concentrations [112] . lipoxidation of vimentin by hne has been detected in human monocytic thp1 cells treated with this lipid by means of immunological and derivatization methods, and c328 was identified as the modification site using lc-ms/ms [81] . hne-vimentin adducts have also been identified in senescent fibroblasts, mainly in soluble vimentin and vimentin fragments [113] . notably, hne can form several types of adducts with proteins, both through michael addition and schiff base formation [114] . in addition, hne adducts can give rise to protein crosslinks. consistent with this broad reactivity, hne can modify both vimentin wild type and c328s in vitro [91] . however, the functional consequences of the modification are different; whereas hne-pretreated vimentin wild type formed thicker and shorter filaments with a tendency to laterally associate, c328s vimentin was protected from these effects [91] . these results suggest that even though hne can bind to other residues, the modification of c328 is functionally the most important. this is also supported by observations in cells showing that hne treatment induces a juxtanuclear accumulation of vimentin wild type in thick bundles or aggregates, whereas in cells expressing vimentin c328s, the network is preserved and filament retraction is attenuated [13, 56] . among other lipoxidation agents, vimentin can be modified by mda. interestingly, immunization of mice with senescent cells led to the isolation of a polyreactive antibody that recognized vimentin at the surface of senescent primary human fibroblasts. in addition, an increase in vimentin modified by mda at c328 was observed at the cell surface of senescent cells and in plasma of aged senescence-accelerated mice [83] . based on these findings it was proposed that mda-modified vimentin could serve as a senescence marker and potentially be recognized by the innate immune system to eliminate senescent cells. other electrophilic lipids potentially interacting with vimentin include nitrated phospholipids (fig. 3) , which alter the cellular vimentin network in a manner dependent on the presence of c328, and shield this residue from alkylation in vitro [115] . importantly, although the c328s mutant is protected from oxidative insults in cells, it shows a lower efficiency than the wild type at forming extended networks, supporting aggresome formation and promoting organelle position [13] . therefore, even conservative substitutions of the cysteine residue have a measurable impact on vimentin function. in spite of the importance of c328 in vimentin lipoxidation, modification of k235, but not of c328, by nonealdehyde has been detected in hepatic tissue from patients with end stage alcoholic liver disease or non-alcoholic steatohepatitis [116] . this modification eliminates the positive charge of k235, disrupting its electrostatic interaction with e230 [117] . this brings about the potential of lipoxidation to alter protein-protein interactions, with consequences for cell activation and/ or cytotoxicity. indeed, lipoxidized proteins are ligands for the receptor for advanced glycated end products (rage), which has been involved in the pathogenic consequences of hyperglycemia or inflammation. by abolishing the positive charges of lysine residues, lipoxidation disrupts the ion pairs with negative residues in their vicinity, enabling the latter to interact with positive residues in rage, favoring the activation of this receptor [118] . through this mechanism, lipoxidized proteins could contribute to propagate the damage. protein carbonyl formation (pcf). formation of protein carbonyls on vimentin has been reported in numerous studies using derivatization approaches, although, in many instances, the sites of modification have not been identified. among evidence from cellular models, pcf on vimentin has been detected in rat aortic smooth muscle cells treated with homocysteine or bone morphogenic protein-4 (bmp-4) [119, 120] , huvec treated with sodium arsenite [121] , and lung epithelial cancer cells exposed to acrolein or smoke extracts [122, 123] , these latter cells showing also vimentin crosslinked products [122] . vimentin bearing protein carbonyls has been identified in non-tumor lung specimens of patients with lung cancer with or without chronic obstructive pulmonary disease [124] . increased vimentin pcf has also been detected in cultured fibroblasts from ad patients with respect to those of control subjects, after exposure to aβ (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) . these observations suggest a dysfunction of antioxidant responses in ad [125] , and raise the importance of vimentin as a target for oxidation in cns diseases [126] , together with gfap, as it will be detailed below. pcf can occur also as a consequence of glycation. in primary cultured human dermal fibroblasts vimentin was glycated predominantly at lysine residues located in linker regions likely exposed to the cytosol. this caused vimentin redistribution into perinuclear aggregates, which was accompanied by loss of fibroblast contractile capacity. glycated aggregated vimentin was also detected in facial skin biopsies. therefore, the accumulation of life-long vimentin glycation was proposed to contribute to loss of skin contractile properties during aging [79] . other modifications. vimentin tyrosine nitration has been detected by immunoprecipitation with an anti-3-nitrotyrosine antibody in mitochondrial diseases [127] . a similar approach was used to identify tyrosine nitrated vimentin in lung cancer [128] . however, in these studies, identification of the nitration sites was not achieved. tryptamine-4,5-dione is a product of the oxidation of serotonin that is highly reactive with thiols. vimentin has been identified as a target for this putative neurotoxin in sh-sy5y neuroblastoma cells, suggesting the modification of the thiol group, although this was not confirmed [129] . in summary, a plethora of vimentin modifications has been identified to date in many pathophysiological situations. oxidative and nonoxidative modifications can coexist in many instances allowing their interplay, but an understanding of their structural and functional consequences requires further investigation. gfap is the main if expressed in mature astrocytes, which also express vimentin [130, 131] . gfap can be expressed in other cell types outside the nervous system, including fibroblasts and hepatic stellate cells [80, 132] . a particular feature of gfap among type iii if proteins is the existence of multiple isoforms that arise by alternative splicing and differ mainly in the tail domain, some of which are not able to form filaments on their own [14] . here, we will focus on the most abundant isoform that is gfapα (fig. 1) . this protein plays complex functions in astrocyte homeostasis which, to date, have not been totally unraveled. studies in knockout animals have yielded diverse results pointing to the complexity of gfap functions and their dependence on the experimental system. roles in astrocyte migration, organelle distribution and mitosis have been defined. the presence of gfap also influences neuronal functions, axonal and neuronal regeneration and several aspects of neurotransmission [14] . gfap plays an important role in the response to damage, including inflammation, physical or ischemic trauma. in these processes, astrocytes become activated, overexpress gfap and resume the expression of other ifs, like nestin and synemin [130] . gfap appears to play a dual role in trauma, favoring the formation of a glial scar that limits the damage at the early stages, but slowing down regeneration at later stages [133] . the importance of gfap in brain homeostasis is highlighted by the devastating consequences of its mutations, which lead to alexander disease (axd), a rare neurodegenerative disease that causes leukodystrophy, epilepsy, loss of brain white matter and ultimately death [17] . in axd, gfap forms aggregates known as rosenthal fibers that accumulate other proteins as well, and show oxidative damage [134] . interestingly, axd associated mutations often involve substitutions of the wild type residues by cysteines [135] , which raises the possibility that they undergo additional oxidative modifications. gfap is also a hub for ptms and it has been reported to undergo phosphorylation at multiple residues. phosphorylation plays an important role in the regulation of filament assembly and in the progression of mitosis and cytokinesis [136, 137] . moreover, increased phosphorylation of certain residues, in particular s13, has been associated with pathological conditions including frontotemporal lobar degeneration, axd and hypoxia [138] [139] [140] . other ptms have also been associated with disease, including glycosylation and citrullination, the latter, rendering gfap an autoantigen for autoimmune disease or appearing in conditions such as ad or ischemia reperfusion injury [38, 39] . moreover, gfap degradation products have been observed in neurodegenerative diseases [141] as well as in models of viral infection [33] . gfap can be targeted by numerous oxidative modifications and appears frequently in proteomic studies of pathophysiological conditions. nevertheless, most studies focus on the variations of its levels as a marker of nervous system injury and less attention has been paid to its role as a target of damage. as specified above, disulfide formation between gfap monomers, likely belonging to different tetramers, has been observed in vitro upon oxidative crosslinking [86] . moreover, bifunctional cysteine reagents also lead to homo-and heterodimers of gfap and vimentin in vitro and in cells [90] . these studies have substantiated the complex nature of these filaments in astrocytes and in astrocyte models. moreover, results from cellular models have shown the requirement for the presence of the cysteine residue in gfap for proper filament assembly, which highlights the functional importance of this residue. interestingly, in primary astrocytes from mice deficient in both gfap and vimentin, the requirement for the cysteine residue was more marked for gfap than for vimentin, and this latter protein seemed to rescue the defect in c294s gfap assembly to some extent [90] . gfap has been found both in reduced and in reversibly oxidized forms in brain tissue from a subject with mild ad, by employing a differential thiol labeling protocol followed by 2d electrophoresis and ms identification [142] . finally, the appearance of high molecular weight oligomers containing gfap has been noted in several models of disease, including axd [143] , although the nature of these oligomers has not been characterized. gfap appears s-nitrosated in several conditions. a marked increase in gfap nitrosation has been observed in synaptosomes of transgenic mice overexpressing mutated human amyloid precursor protein compared to wild type, suggesting its involvement in pathology [144] . however, s-nitrosation of gfap has also been detected under control conditions and found not to increase in experimental models of murine autoimmune encephalomyelitis [145] . therefore, the role of this modification in pathogenesis or as a defense mechanism requires further investigation. lipoxidation. evidence on gfap lipoxidation is gathering in recent years. pick's disease is a type of fronto-temporal dementia characterized by severe atrophy of frontal and temporal lobes, neuronal loss and astrogliosis [146] . gfap has been shown to be a major target of lipoxidation by hne and glycoxidative damage in brain samples from pick's disease patients compared to control subjects [146] . down's syndrome patients develop symptoms of premature aging, including neuropathological features of ad. therefore, observations in this syndrome have been considered clues for the understanding of ad. a 3.3-fold increase in hne-modified gfap has been observed in brain tissue from down's syndrome patients compared to controls by means of immunological detection with an anti-hne antibody [147] . hne also targets elements of the protein degradation machinery and quality control, the impairment of which could contribute to accumulation of damaged or misfolded proteins. gfap has also been recognized as a major target for hne-protein adduct formation in frontotemporal lobar degeneration [148] , although the site(s) of modification have not been identified. notably, an 8-fold increase in mda-modified gfap has also been reported in brain samples from ad patients compared to agedmatched controls [149] , whereas non-significant differences were found for vimentin modification in this study. nevertheless, it should be taken into account that many of these reports use whole tissue samples for the proteomic studies. although the evidence for lipoxidation is solid, novel approaches able to assess oxidative damage in specific regions and/or cell types or subpopulations in the brain may shed valuable information on pathogenic mechanisms [150] . using cellular models such as fibroblasts and astrocytes, we have recently identified gfap as a target for modification by cypg [80, 90] . in astrocytoma cells, cypg produced an intense disruption of the gfap network with retraction of filaments from the cell periphery and formation of curly bundles or aggregates in the proximity of the nucleus, whereas in mouse primary astrocytes the most obvious effect was filament retraction and perinuclear condensation [90] . biotinylated analogs of cypg bind to gfap in a manner dependent on the presence of its single cysteine, c294. moreover, a cysteine deficient gfap mutant, although showing altered filament assembly per se, is protected against network disruption induced by these electrophilic lipids. similarly, treatment of cells in the presence of dtt attenuated network disruption, pointing to the importance of thiol modification in cypg effect [90] . protein carbonyl formation. most of the evidence on protein carbonyl formation on gfap has been obtained through the use of derivatization techniques combined with immunological detection after immunoprecipitation or 2d analysis, or by using adduct-specific antibodies (reviewed in ref. [151] ). these approaches have demonstrated pcf in ad, tautopathies including progressive supranuclear palsy and pick's disease, and in huntington disease [146, [151] [152] [153] ]. an 8-fold increase in gfap protein carbonyl levels has been found in samples from the frontal cortex of down's syndrome patients compared to controls [154] . moreover gfap has been identified as the major carbonyl containing protein in the cerebral cortex of a patient with aceruloplasminemia, the hallmark of which is intracellular iron overload [155] . from all this evidence, it seems clear that gfap constitutes a highly susceptible target for oxidative modifications in various neurodegenerative diseases. nevertheless, whether the modifications lead to significant network rearrangements and whether they represent a toxic or a defense mechanism requires further study. nitration. nitrated gfap has been identified in normal rat cortex [156, 157] , and marked increases in nitration have been observed in a model of hypobaric hypoxia and reoxygenation [157] . in this model, intense nitration of other cytoskeletal components, including tubulin and γ-actin has been observed, for which a cooperation of these proteins in cytoskeletal disruption has been proposed. although in this review we have not considered the modifications of gfap isoforms, the study of their specific modifications could yield valuable pathogenic information. indeed, in proteomic studies, up to 46 forms of soluble gfap could be separated by 2d immunoblotting from control and ad brain samples, which differed in size, phosphorylation, glycosylation and oxidation state [158] . desmin is the main if protein of mature striated muscle, where it is located in the sarcolemma, z-lines, neuromuscular and myotendinous junctions. this protein provides strength to the muscle fiber during contraction and facilitates mechanochemical signaling [15, 159] . desmin and its protein binding targets form a scaffold that links the contractile system to the nucleus, mitochondria and other organelles through the z-discs, allowing their crosstalk. this interconnecting role also enables transport between the extracellular and nuclear matrix [15, 159] . more than 70 mutations in the desmin gene (des) have been identified, which lead to different types of myopathy (e.g. myotilinopathies and desminopathies), including cardiomyopathy or isolated myopathies. mutations have been identified in all the protein domains. skeletal and cardiac muscle phenotypes involve mainly mutations affecting rod 2b, abnormal phosphorylation levels, and some oxidative modifications that correlate with desmin related myopathies (drms) and cardiac disease [15, 159] . desmin is a target for a variety of ptms including phosphorylation, adp-ribosylation, ubiquitination, glycation, oxidation and nitration. most of them cause disassembly of the desmin network [160] . also in this case, phosphorylation is by far the most studied ptm. although alterations in desmin levels or distribution have been frequently encountered in pathophysiological situations associated with oxidative stress or in cellular models of electrophilic stress [161] , the precise characterization of oxidative modifications and their impact on the function of the protein is not always available. indeed, muscle activity induces thermal, mechanical and redox stress, which in cellular models of desminopathies elicit aggregation of desmin mutants [162] that can be attenuated by treatment with antioxidants, like, n-acetyl-cysteine or tocopherol [163] . as described above, oxidative crosslinking-induced dimers of desmin or of desmin and vimentin have been generated in vitro and showed impaired assembly [85] . moreover, purified desmin subjected to in vitro oxidation with h 2 o 2 and fe 2+ increases its content of random coil and β-sheet, in turn, decreasing its α-helix content according to far-uv cd spectra [164] . also, oxidation can alter its susceptibility to proteolytic cleavage [164] . in myocytes, several redox modifications of desmin have been reported including disulfide linked and sulfenylated forms of the protein, as has been identified in studies using diamide, dimedone analogs or peroxide [165, 166] . desmin oxidation has also been confirmed in muscle biopsies from patients with desminopathies or myotilinopathies [159] . desmin is also a target for lipoxidation. functional derangement by hne treatment has been reported [161] , and acrolein-modified desmin has been identified in myocytes exposed to this toxic agent [167] . among other modifications, desmin has also been identified as a target for nitration [127, 159] , and increased formation of protein carbonyls in skeletal muscle has been detected in sepsis, ischemia-reperfusion, diabetes and chronic obstructive pulmonary disease (reviewed in ref. [168] ). however, the functional consequences of these modifications are not yet clear. peripherin expression is mainly restricted to the peripheral nervous system, but also occurs in neurons of the cns. there are several forms of peripherin generated by alternative splicing, some of which increase in response to traumatic neuronal injury [169] . the role of this protein is unclear, but it seems to contribute to if cytoskeleton organization and axonal elongation. the protein is a major autoantigen in type 1 diabetes [170] and appears in compact inclusions in several diseases, including amyotrophic lateral sclerosis (als). screening for sequence variants in the peripherin gene (prph) has been performed in patients with als and several snps have been identified [171] . these include an insertion in intron 8 (ivs8-36insa), a 1 bp deletion in exon 1 (228delc) leading to a truncated form of the protein, and a homozygous mutation in the first linker generating the d141y mutation. peripherin overexpression induces motor neuron degeneration in transgenic mice, whereas the knockout mice seem to compensate lack of prph expression by inducing that of vimentin and α-internexin, apparently leading to decreased myelination of only a subset of sensory fibers [172] . peripherin is by far the less studied of the type iii if class, and is the only one that possesses two cysteine residues. nevertheless, several oxidative modifications have been reported. a disulfide-linked peripherin dimer of 130 kda has been detected in rat sciatic nerve and dorsal root ganglia [173] . non-differentiated n2a neuroblastoma cells treated with hydrogen peroxide showed concentration-dependent changes in the levels of peripherin, together with alterations in the proportion of the peripherin forms, per-58 and per-45. moreover, peroxide (100-500 μm) induced loss of filament structure and inclusions in nondifferentiated cells, whereas retraction of neurites was detected at 10 μm. however, peripherin filaments and aggregates did not colocalize with carbonyl markers, despite increased total protein carbonyl formation [174] . the best characterized oxidative modification of peripherin is nitration, which has been described upon ngf-induced differentiation in pc12 cells, and occurs at the head (y17) and tail (y376) domains [175] . apparently, the ratio between nitration levels and peripherin expression before and after ngf addition increases during differentiation. remarkably, nitrated peripherin is only present in the insoluble cytoskeletal fraction. heavy tyrosine nitration and phosphorylation of peripherin have been observed in als, in association with disruption of filament association and appearance of inclusions or aggregates [169] . in any given situation, proteins are subjected to multiple ptms that can occur on the same or on different protein molecules in different combinations, giving rise to structurally and functionally different proteoforms. type iii ifs and, in particular, their conserved cysteine residues, are emerging as hot spots for modification by oxidants and electrophiles. indeed, the studies summarized above highlight the susceptibility of these cysteine residues to a plethora of modifications including nitrosation, disulfide formation, sulfenylation and addition of various electrophilic lipids including hne, mda and cypg. interestingly, a complex interplay between modifications can take place both by direct and indirect mechanisms (fig. 5) . reversible cysteine modifications, for instance, glutathionylation, could prevent irreversible oxidations or adduct formation; however, some oxidative modifications can increase or decrease the reactivity of the affected residues [176] . phosphorylation is probably the most studied modification regulating protein function. many oxidant species act on signaling proteins, for instance, kinases and phosphatases, triggering additional modifications of ifs. vimentin is a target for phosphorylation by numerous kinases, including pka and akt, which are directly and/or indirectly regulated by redox mechanisms [177] . the impact of oxidative modifications on kinase activity can lead to activation or inhibition. in turn, tyrosine phosphatases are generally inhibited by oxidative stress, whereas oxidation of serine/threonine protein phosphatases can result in activation or inhibition. interplay of nitric oxide with vimentin phosphorylation has been reported through modulation of pp2a activity [178] . phosphatase inhibition would result in vimentin hyperphosphorylation, which could contribute to network disruption by oxidative modifications. in this context, we have recently observed that the phosphatase inhibitor calyculin a, an inhibitor of pp2a and pp1 phosphatases, potentiated the fragmentation of vimentin network induced by diamide, whereas a phosphorylation-deficient vimentin mutant "sa mutant", in which 11 phoshorylatable residues of the n-terminal domain had been substituted by alanines, was partially protected from the diamide-disruptive effect [91] . a complex interplay can also occur with the protein degradation machinery. certain oxidative insults or other types of stress can lead to the degradation of vimentin and gfap by activating proteases [179] , although some ubiquitin ligases and subunits of the proteasome can be inhibited by lipoxidation [180] , which, as stated above, would result in under oxidative stress, several oxidants are generated that can react with unsaturated lipids in membranes giving rise to the formation of electrophilic lipid species. these can directly form adducts with proteins in a process called lipoxidation. oxidative stress also diminishes the ratio gsh/gssg and favors the formation of disulfide bonds between glutathione and thiol groups in proteins (glutathionylation). this modification can also be mediated by nitrosoglutathione, which can also induce s-nitrosation. oxidative modifications of cysteine residues can also lead to the formation of sulfenic, sulfinic and sulfonic acids. other oxidative modifications include protein carbonyl formation, nitration or formation of protein crosslinks (see ref. [41] for review). other ptms that are under redox control, such as phosphorylation and protein degradation will be modulated in situations of oxidative stress, thus contributing to the diversity and complexity of proteoforms arising under these conditions. the function of type iii ifs will be also affected by oxidative modifications of interacting proteins, such as those described for plectin, actin, and several chaperones. importantly, interplay can also take place with other ptms, including those listed in the lower panel, which can occur on the same or nearby residues, affecting their availability or accessibility to modification. accumulation of damaged proteins. interestingly, due to their polyelectrolyte properties, ifs interact with cations such as ca 2+ , mg 2+ or zn 2+ which, depending on their size and concentration, influence if assembly [13, 92, 181] . importantly, zn 2+ availability can influence the occurrence of certain ptms on vimentin [13] , which could occur through direct interaction or through its capacity to elicit antioxidant or pro-oxidant effects [182, 183] . although zn 2+ is a redox-inert ion, it can influence cellular redox status because it is necessary for the activity of enzymes involved in the antioxidant defense, whereas its deficiency or excess cause oxidative stress [183] . in addition, modification of interacting proteins can have an important impact on the regulation of ifs localization and function. the linker protein plectin has been reported to be nitrosated, affecting vimentin distribution [184] , whereas actin and tubulin are targets for various oxidative and lipoxidative modifications [56] . moreover, certain modifications can compete for the same site, for instance, carbamoylation, resulting from modification by isocyanic acid [185] , can occur at citrullination sites, and tyrosine nitration could preclude phosphorylation. modification of a given residue can also affect that of nearby amino acids, either by altering their reactivity or their accessibility. therefore, bulky moieties can shield nearby residues, but certain modifications can cause unfolding and exposure of additional amino acids for modification. additionally, many conditions associated with oxidative stress and oxidative modifications trigger important changes in gene expression, including the antioxidant stress response [57] , contributing to the complexity of the effects. given their implication in pathological processes, ifs in general, and those belonging to the type iii family in particular, are becoming important drug targets. indeed, hypothesis-driven, as well as virtual and pharmacological screening approaches are being used to find small molecule modulators of these proteins. high expression levels of type iii if proteins have been considered as pathogenic factors in several diseases. therefore, efforts have been devoted to find strategies to modulate their levels or organization in cells [186, 187] . nevertheless, this section will focus on approaches related to ptms and more precisely on drugs that directly bind to these proteins and preferentially interact with their conserved cysteine residue. as vimentin c328 and the equivalent cysteine residues in the other members of the family seem to be highly susceptible to modification by oxidant and electrophilic species, their modification by cysteine-reactive drugs is worth exploring. among the first compounds reported to target vimentin c328 is the withanolide natural product withaferin a (wfa). this molecule was studied based on its antitumor and antiangiogenic properties and was reported to covalently bind to c328 and induce vimentin aggregation in vitro and filament disruption in cells [188] . in addition, wfa caused vimentin downregulation in cells and showed in vivo antiangiogenic effects, reportedly mediated by vimentin [188] . it was later described that this molecule could bind to the single gfap cysteine residue, c294 [189] . moreover, it was shown that wfa reduced the levels of both vimentin and gfap in primary astrocytes as well as in mouse retina in vivo [189] . therefore, these wfa actions could be beneficial in situations associated with increased expression of these proteins, such as reactive gliosis. thus, wfa has become a popular tool employed to target and disrupt the vimentin network in several experimental models [190] . however, being a cysteine reactive molecule, wfa could interact with other cellular targets. in fact, several works have called attention to its lack of specificity [191] . indeed, wfa has recently been reported to covalently bind the heterogeneous nuclear ribonucleoprotein k (hnrnp-k) [192] , and tubulin, and induce the degradation of the latter [193] . moreover a vimentin mutant lacking c328 appears to respond to wfa as well [191] . recent molecular dynamics simulations indicate that wfa binds in the vicinity of c328, but not directly to this residue [192] . therefore, although wfa may be useful to disrupt vimentin and other type iii if proteins, its use as a selective target should be performed cautiously. interestingly, the compound arylquin 1 also binds to vimentin at a region close to the cysteine residue. in this case, interaction of arylquins with vimentin displaced the binding of par4, an apoptosis-inducing protein, with consequences for cell viability [194] . additional evidence of the importance of c328 as a drug target has risen from the garlic compound ajoene [195] , which has been shown to form a disulfide bond with this residue. this modification is associated with condensation of vimentin filaments and with an impairment of several vimentin-related processes, including cell migration and invasion of cancer cells. these effects were attenuated in cells in which vimentin expression had been transiently knocked down, thus pointing to vimentin importance as mediator of ajoene actions. notably, another garlic defense substance, allicin, has been shown to induce s-thioallylation of several cytoskeletal proteins, including vimentin, in jurkat cells [196] . in addition, phenyl vinyl sulfones, mechanism-based inhibitors of protein tyrosine phosphatase, covalently bind vimentin c328 in cells, although the significance of this finding is still not known [197, 198] . a number of proteomic studies searching for protein targets of drugs or natural products have found vimentin [199] , although the precise site of interaction or the functional consequences have not always been elucidated. additional compounds found to bind vimentin include two quinone methide metabolites of the phenolic antioxidant butylated hydroxytoluene. these metabolites are believed to be responsible for promoting lung tumor formation in mice [200] . in contrast, the green tea polyphenol epigallocatechin gallate binds to vimentin and inhibits its phosphorylation in cells, which is associated with an antiproliferative effect [201] . type iii if proteins are arising as integrators of basic cellular processes and as sensors of stress. these proteins, and in particular their conserved cysteine residue, have been proposed as hot spots for ptms. modification of this cysteine residue leads to marked reorganization of type iii ifs in cells, which is supported by the lack of response of cysteine-deficient mutants. remarkably, the nature of the reorganization appears to depend on the extent and the structure of the modification, illustrating an exceptional versatility of type iii ifs remodeling. moreover, this suggests that the conserved cysteine residue of type iii ifs functions as a hub that can accept different types of modifying moieties and transduces them into distinct cytoskeletal responses, either per se or in cooperation with other ptms, thus acting as a sensor for various signals. the position of the conserved cysteine residue in the structure of the protein could be critical for the functional outcome of the modifications, affecting its organization in filaments. nevertheless, many aspects of structure and function of type iii ifs still need investigation. elucidation of the structure of filaments and of their various arrangements in cells will be key to understand the basis of their response to stress. moreover, the downstream consequences of the diverse reorganizations are still poorly understood. the modification of type iii ifs by oxidant and electrophilic species could impact on many cellular processes, but it also could represent a defensive mechanism by which these proteins would act as decoys or scavengers of reactive species. therefore, further identification and characterization of the ptms occurring in vivo, both structurally and quantitatively, will be necessary to elucidate their role in physiological if regulation and their potentially protective or pathogenic effects under pathophysiological conditions. finally, certain oxidative ptms of type iii ifs can be considered as disease biomarkers, whereas their modulation 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vimentin binding and involvement in if collapse protein carbamylation: chemistry, pathophysiological involvement, and biomarkers antisense suppression of glial fibrillary acidic protein as a treatment for alexander disease an image-based small-molecule screen identifies vimentin as a pharmacologically relevant target of simvastatin in cancer cells the tumor inhibitor and antiangiogenic agent withaferin a targets the intermediate filament protein vimentin withaferin a targets intermediate filaments glial fibrillary acidic protein and vimentin in a model of retinal gliosis the use of withaferin a to study intermediate filaments withaferin a alters intermediate filament organization, cell shape and behavior 3-dihydro-3beta-methoxy withaferin-a lacks anti-metastasis potency: bioinformatics and experimental evidences the natural compound withaferin a covalently binds to cys239 of beta-tubulin to promote tubulin degradation arylquins target vimentin to trigger par-4 secretion for tumor cell apoptosis the garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells the human allicin-proteome: s-thioallylation of proteins by the garlic defence substance allicin and its biological effects, free radic aryl vinyl sulfonates and sulfones as active site-directed and mechanism-based probes for protein tyrosine phosphatases target identification reveals protein arginine methyltransferase 1 is a potential target of phenyl vinyl sulfone and its derivatives a vimentin binding small molecule leads to mitotic disruption in mesenchymal cancers immunochemical and proteomic analysis of covalent adducts formed by quinone methide tumor promoters in mouse lung epithelial cell lines the intermediate filament protein vimentin is a new target for epigallocatechin gallate atomic structure of vimentin coil 2 this work was supported by grants from agencia estatal de investigación, ministerio de ciencia e innovación (mci, spain) and european regional development fund, rti2018-097624-b-i00, instituto de salud carlos iii (spain) retic aradyal rd16/0006/0021, and csic pti global health (pie202020e223/csic-cov19-100). av-p is supported by the fpi program from mci, reference bes-2016-076965. feedback from cost action eurocellnet ca15214 is gratefully acknowledged. supplementary data to this article can be found online at https:// doi.org/10.1016/j.redox.2020.101582. key: cord-330475-mameyzih authors: shi, da; lv, maojie; chen, jianfei; shi, hongyan; zhang, sha; zhang, xin; feng, li title: molecular characterizations of subcellular localization signals in the nucleocapsid protein of porcine epidemic diarrhea virus date: 2014-03-13 journal: viruses doi: 10.3390/v6031253 sha: doc_id: 330475 cord_uid: mameyzih the nucleolus is a dynamic subnuclear structure, which is crucial to the normal operation of the eukaryotic cell. the porcine epidemic diarrhea virus (pedv), coronavirus nucleocapsid (n) protein, plays important roles in the process of virus replication and cellular infection. virus infection and transfection showed that n protein was predominately localized in the cytoplasm, but also found in the nucleolus in vero e6 cells. furthermore, by utilizing fusion proteins with green fluorescent protein (gfp), deletion mutations or site-directed mutagenesis of pedv n protein, coupled with live cell imaging and confocal microscopy, it was revealed that, a region spanning amino acids (aa), 71–90 in region 1 of the n protein was sufficient for nucleolar localization and r87 and r89 were critical for its function. we also identified two nuclear export signals (nes, aa221–236, and 325–364), however, only the nuclear export signal (aa325–364) was found to be functional in the context of the full-length n protein. finally, the activity of this nuclear export signal (nes) was inhibited by the antibiotic lepomycin b, suggesting that n is exported by a chromosome region maintenance 1-related export pathway. porcine epidemic diarrhea (ped) was first recognized as a devastating enteric disease in feeder and fattening pig, resembling transmissible gastroenteritis (tge) in pigs in the united kingdom, in 1971. it is a member of coronavirinae, which are single-stranded, positive-sense rna viruses with the largest genome that are known to infect humans, other mammals, and birds, usually causing subclinical or respiratory and gastrointestinal diseases. the porcine epidemic diarrhea virus (pedv) subgenomic mrnas, which are transcribed from the genome, produce viral proteins, such as the spike (s, 180-220 kda), envelope (e, ~8.8 kda), membrane (m, 27-32 kda), nucleoprotein (n, [55] [56] [57] [58] , and several other proteins of unknown function [1] [2] [3] . among the proteins, n, as the rna-binding protein, play an important role in both virus rna synthesis and modulating host cell processes, and phosphorylation may regulate these processes by exposing various functional motifs [4, 5] . several other functions have been postulated for the coronavirus n protein throughout the virus life cycle, including encapsidation, packaging, correct folding of the rna molecule, the deregulation of the host cell cycle [6] [7] [8] , inhibition of interferon production [9, 10] , up-regulation of cox2 production [11, 12] , up-regulation of ap1 activity [13] , induction of apoptosis [14] [15] [16] , association with host cell proteins [17] , and rna chaperone activity [18] . therefore, it is clear that n is a multifunctional protein involved in biological processes related to the survival of pedv. the nucleolus was one of the first subcellular structures to be identified by early users of the light microscope, appearing as a highly refractive black dot(s) in the nucleus of the cell [19] . the nucleolus is a highly specialized structure that participates in regulation of several host cell processes, including ribosome subunit biogenesis, rna processing, control of cell growth and response [20] . interestingly, a cytoplasmic-nucleolar distribution pattern has been reported for the n proteins of several coronaviruses, including representative members of alphacoronavirus (transmissible gastroenteritis virus, tgev), betacoronavirus (mouse hepatitis virus, mhv; and severe acute respiratory syndrome coronavirus, sars-cov), and gammacoronavirus (infectious bronchitis virus, ibv) [2, 21, 22] . further study indicated that n protein co-localize with major nucleolar proteins, including nucleolin, fibrillarin, and nucleophosmin [23] [24] [25] . how viral and cellular proteins traffic to the nucleolus and what determines their sub-nucleolar localization is not clearly understood, but proteins that localize to the cytoplasm and nucleus or nucleolus contain multiple signals that determine their subcellular localization [26] , such as nucleolar localization signal (nols). active nuclear import of proteins is mediated by nuclear localization signals (nlss), which are then recognized by proteins of the importin super-family (importin α and β) that mediate the transport across the nuclear envelope using ran-gtp [27] . similar to nuclear import, export of a protein from nucleus depends on the presence of a specific nuclear export signal (nes) [28] . the chromosome region maintenance 1 (crm1; also known as exportin 1 or xpo1) has been identified as an export receptor that interacts with the predominant nes, the so-called leucine-rich nes, which is found in a large variety of nucleocytoplasmic shuttling proteins [29, 30] . in fact, some of these ness are not necessarily leucine rich but rather characterized by several hydrophobic residues. the pharmacological compound leptomycin b (lmb) directly interacts with crm1 and blocks nes-mediated protein export [31] . therefore, the proteins can shuttle between the nucleus and the cytoplasm with their subcellular localization signals. it was reported previously that n protein nucleolar localization is a common feature in coronaviruses, however, there are different results regarding n subcellular localization in a strain of sars-cov [4] . within the alphacoronavirus coronaviruses, the precise nols and nes of pedv n and its traffic mechanism are still elusive. therefore, we have attempted to characterize these signals, and the molecular mechanism responsible for its subcellular localization. in this study, we examined the intracellular localization of the pedv n protein in pedv-infected and transfected cell lines using mouse polyclonal antisera and confocal microscopy. by generating a series of deletion and mutagenesis constructions, we found that amino acids 71-90 in region 1 were sufficient for nucleolar retention and we also identified two ness (aa221-236 and 325-364), but only the nes (aa325-364) was found to be functional in the context of the full-length n protein. the nucleocytoplasmic shuttling of n and the nuclear export of gfp-nes could be blocked by lmb, an inhibitor of the crm1, which is the receptor for exportin-1-dependent nuclear export. a polyclonal antibody specifically against the n protein was produced to determine its intracellular localization. we generated anti-n mouse antisera using an e coli-produced fusion protein as the antigen. the antigenicity of the recombinant n protein was confirmed by immunoreactivity with pedv pigs sera using elisa assay, which showed high sensitivity and specificity (data not show). to examine the reactivity and specificity of the mouse antiserum, blot results demonstrated that mouse antisera notably reacted with n protein from cv777 strain pedv, and the cell lysate from vero e6 cells transfected with pcdna3.1-n showed a band with the same molecular mass to n protein, whereas no band was detected from samples of cells uninfected pedv and transfected with an empty vector alone ( figure 1a ,b). our results showed that n protein was localized predominantly in the cytoplasm in pedv cv777-infected cells, while in a few cells fluorescence was also observed in the nucleus (or nucleolus) ( figure 2a ). no significant fluorescence was observed in uninfected cells (data not shown). a similar observation was also found in vero e6 cells transfected with plasmid expressing full length n protein ( figure 2b ). the n protein was observed to localize mainly in the cytoplasm with some protein in the nucleus (or nucleolus). the results suggested that the n protein localized to a subnuclear structure and may contain functional signals. to identify predicted nuclear (or nucleolar) localization signals, and whether they participate in this process or not, we conducted further experiments. after 24 h, infected or transfected, cells were fixed and analyzed by indirect immunofluorescence using mouse anti-n polyclonal antibody (green) and stained with pi (red) to visualize the nuclear dna. differentially fluorescing images were gathered separately using confocal microscopy. images were obtained with a 63× oil objective. to identify whether there were subcellular localization signals in pedv n protein, we first conducted a bioinformatics analysis on the protein using existing motif prediction algorithms. predict nls [32] and psort ii [33] were used to identify potential nlss, and the nes predictor (net nes) [29] was used to identify potential ness. predict nls found no nlss, whereas psort ii indicated that pedv n protein contained a pat7 motif (261pkknksr267). net nes found no nes. in other studies, coronaviruses, such as tgev, mhv, ibv, and sars-cov showed a common characteristic, that of nls-rich in c-terminal. so through amino acid sequence comparison among groups we also found a basic amino acid-rich short peptide (383rkkekknkre393) in c-terminal. we presumed it might play a role in n protein localization as a new localization signal, and named it patx. although n protein contains putative nuclear (or nucleolar) localization signals (n (or no) ls), it is not known whether they are functional or not. to investigate whether these and other unknown signals operated to determine the subcellular trafficking of n protein, we utilized acgfp as a fusion marker to observe the localization characteristics of n and truncated n protein in live cells. the b23.1-dsred fusion protein was used to tag the nucleolus, so we could analyze nucleolar localization properties and colocalization in cotransfected cells by live cell imaging (direct fluorescence) or confocal microscopy. at 24 h post-cotransfection, live cell imaging indicated that as previously shown, acgfp evenly distributed throughout the cytoplasm and the nucleoplasm but not the nucleolar, on the contrary acgfp-n protein localized to both the cytoplasm and nucleolus but not the nucleus in vero e6 cells ( figure 3 ). from the above results we hypothesize that there is a nols in n, which can guide exogenous protein to the nucleolus. to determine the exact nols, the acgfp-tagged truncated different regions of the n protein were transfected into vero e6 cells, which did not destroy the integrity of the signals. in our observation, acgfp-nr1 mainly localized to nucleolar structures, acgfp-nr2 localized predominantly to the cytoplasm and appeared also to accumulate in the nucleolus to the low level as the cytoplasm, whereas acgfp-nr3 predominantly localized to the cytoplasm; acgfp-nr1+2 protein same to acgfp-n and localized to the cytoplasm and nucleolus, acgfp-nr2+3 protein localized predominantly in cytoplasmic ( figure 3 ). this evidence demonstrated that region 2 targeted to the nucleolus with weaker enrichment, while region 1 localized to the nucleolus with stronger enrichment. thus, we speculated that region 1 have an effect on the nucleolar localization of n. this data also suggested that region 2 and 3 may contain ness because region 2 and 3 was mainly directed to the cytoplasm. interestingly, although region 2 and 3 contained predicted pat7 and patx motifs, respectively, they could either have been submissive to the nes or not functional. none of these fusion proteins had a distribution similar to acgfp only. to further identify the sequence for nucleolar localization in detail, a series of expression constructs containing fragments of region 1 were constructed. vero e6 cells were cotransfected with, either pacgfp-nr1 1-50 , pacgfp-nr1 51-100 , or pacgfp-nr1 101-147 , and pdsred-b23.1, analyzed using live cell imaging and confocal microscopy at 24 h post-transfection. the data indicated that acgfp-nr1 51-100 colocalized with b23.1, whereas the other two fusion proteins did not ( figure 4 ). to further refine the amino acids involved in nucleolar localization, 20 amino acid overlapping motifs encompassing amino acids 51-100 were cloned into downstream of acgfp, creating plasmids pacgfp-nr1 51-70 , pacgfp-nr1 61-80 , pacgfp-nr1 71-90 , and pacgfp-nr1 81-100 for the expression of recombinant fusion proteins. vero e6 cells were cotransfected with these constructs and pdsred-b23.1, at 24 h post-cotransfection analyzed using live cell imaging and confocal microscopy ( figure 5 ). the data indicated that acgfp-nr1 71-90 localized to the nucleolus and colocalized with b23.1. therefore, the amino acids at positions 71-90 in pedv n protein were capable to localize in the nucleolus. comparison of the pedv n protein nols with known cellular and viral nolss showed that the r87 and r89 of the pedv n protein nols might be conserved, although some cellular and viral nolss in this site did not contain basic amino acids ( figure 6 ). current work is directed at further resolving pedv nols sequence, including the contribution of individual amino acid residues. red squares indicate the amino acids of conservation. the cellular and viral nolss are described in nols ibv n protein [34] , nols prrsv n [20] , nols htlv-1 rex [35] , nols hsv gamma1 34.5 [36] , nols mdm2 [37] , nols mdv meq [38] , nols nf-kappa [39] , nols nuclear vcp-like protein (nvl2) [40] , nols p 120 [41] , nols surviving-deltaex3 [42] , nols (ggnnv) protein alpha [43] , bhv-1 bicp27 [44] , earning-associated protein 1-19 [45] , hsv-1 icp27 [46] , nols angiogen [47] , nols fibroblast growth factor-2 [48] , nols herpes-mareks meq [38] , nols hic p40 [49] , nols hiv-1 rev [50] , nols hiv-1 tat [51] . to map the amino acid sequence of region 2 responsible for its nuclear export, similar to the approach used to identify the nols in region 1, region 2 was subdivided into two distinct components. amino acids 148-220 and 221-294 were placed downstream of acgfp, creating expression vectors pacgfp-nr2 148-220 and pacgfp-nr2 221-294 . these expression plasmids were transfected into vero e6 cells, the nuclear was stained with dapi at 24 h post-transfection indicated that amino acids 148-220 directed acgfp to the cytoplasm and nucleus and had a subcellular localization similar to acgfp. in contrast, amino acids 221-294 directed acgfp to the cytoplasm; further investigation revealed that amino acids 241-260 and 261-294, when fused to acgfp, directed this protein to the cytoplasm and nucleus, whereas amino acids 221-240 directed acgfp to the cytoplasm (figure 7) . to further define the amino acids involved in cytoplasm trafficking, we conducted a tetra-alanine substitution mutagenesis of amino acids 221-240. these were placed down stream of acgfp, creating expression plasmids, pacgfp-nr2 221-224dlva-aaaa , pacgfp-nr2 225-228avkd-aaaa , pacgfp-nr2 229-232alks-aaaa , pacgfp-nr2 233-236lgig-aaaa and pacgfp-nr2 237-240enpd-aaaa . therefore, in some cases, the wild-type alanine was not substituted. the data indicated that substituting 221-224dlva-aaaa, 229-232alks-aaaa, and 233-236lgig-aaaa abolished cytoplasm trafficking. the remaining tetra-alanine substitutions had no effect on cytoplasm trafficking (figure 8 ), indicating that amino acids 221 dlvaavkdalkslgig 236 were involved in cytoplasm trafficking. to test whether this amino acid sequence was involved in directing the cytoplasm trafficking of n protein, this motif was deleted in the context of full-length n protein tagged to acgfp (plasmid pacgfp-n ∆221-236 ). this plasmid was transfected into vero e6 cells and the subcellular localization of the resulting fusion protein acgfp-n ∆221-236 investigated using confocal microscopy. there was no difference at 24 h post-transfection ( figure 9 ) compared with cells expressing acgfp-n protein. this data also indicated that the nes identified in region 2 was not necessary for cytoplasm trafficking in pedv n protein. as region 3 of pedv n protein localized to the cytoplasm, thus, the similar approach was used to identify the nes in region 3. the data indicated that amino acids 295-394 directed acgfp to the cytoplasm; further investigation revealed that amino acids 325-364 directed acgfp to the cytoplasm ( figure 10 ). taken together, we proposed that amino acids 325-364 are necessary and sufficient to direct n protein to the cytoplasm, and no other signals are involved. to determine if the pedv n protein was exported via the crm1-mediated pathway, the subcellular localization of the pacgfp-n, pacgfp-nr3 or pacgfp-nr3 325-364 , was compared between vero e6 cells left untreated or treated with 2.5 ng/ml lmb. as shown in figure 11 , the nucleocytoplasmic shuttling of pedv n, nr3 and nr3 325-364 was completely inhibited by lmb. the finding indicates that pedv n shuttling activity was affected by lmb and, thus, suggests that pedv n protein was transported via the classical crm1-dependent pathway. pedv is an important pathogen causing viral diarrhea in the swine industry. although much research has been carried out on the general characteristics of pedv, few reports have been reported on the functions of the pedv structural proteins, especially n protein. n protein plays an important role in virus replication and modulation of host cellular machinery, which should be a result of its self-interaction and interaction with other viral and cellular proteins and with virus and host cell nucleic acids. therefore, it is important to understand the subcellular localization properties of the pedv n protein. figure 11 . the nuclear export mechanism of pedv n. vero e6 cells were transiently transfected with plasmids encoding pacgfp-n, pacgfp-nr3, pacgfpnr3 365-394 , with or without treatment with lmb, and examined live 24 h after transfection by confocal microscopy. each image is representative of the majority of the cells observed in the same cells. the nucleolus (no) is arrowed where appropriate. to enter and export from the nucleus, all molecules or cargoes must traverse a large macromolecular structure called the nuclear pore complex (npc), which is located in the nuclear envelope. small molecules up to 40-60 kda or less than 10 nm in diameter can passively diffuse through the npc, but if proteins are larger than this size-exclusion limit and/or are required to move against a concentration gradient, then transport requires energy-driven mechanisms [52] . in this case, most proteins should contain the appropriate trafficking motifs, such as nls. the rules and signals that govern the nuclear localization of proteins are well defined. nlss can be classified into several categories, including the pat4 and pat7 motifs and bipartite nlss, are composed of basic amino acids of a given sequence length. the pat4 motif consists of a continuous stretch of (usually) four basic amino acids, and the pat7 motif starts with a proline residue and is followed by six amino acids [32, 53] . by contrast, the signals that govern nucleolar localization and retention are not well defined [26] . the motifs involved are usually rich in arginine and lysine residues; however there is no immediately obvious consensus sequence or structure. proteins that localize to the nucleolus can also have nuclear-import motifs. the nolss that have been identified so far can be grouped into those that contain single motif and those that contain multiple motifs [54] . in this study, the living cells fluorescence microscopy and confocal microscopy were employed for investigating the subcellular localization and nuclear import and export mechanisms of pedv n protein. it is well known that living cells fluorescence microscopy and confocal microscopy has an advantage over conventional in vitro nuclear transport assays in that cells are not physically damaged by microinjection, detergent, or mechanical perforation, this means that cellular components important for trafficking, such as nuclear import and export receptors, the mt network, intact [55, 56] . our results indicated that pedv n protein mainly distributes throughout the cytoplasm with localization to a substructure within the nucleolus in infected cells. similar results were also obtained in transfected cells. to eliminate the influence of charged protein migration to the nucleolus post-fixation, and to investigate the nuclear (or nucleolar) localization of pedv n protein and the functions of these motifs in more detail, we generated constructs that express the protein (or parts of the protein) as a fusion with enhanced green fluorescent protein. the protein could then be detected by direct fluorescence using both live-cell and confocal microscopy. no difference in the localization of either protein was observed between different cell lines, and the presence of a fluorescent tag at either the n terminus or c terminus of n protein did not affect the localization of the fusion protein compared with native protein [2, 4] . furthermore, the b23.1 gene was amplified from the vero e6 cells and then fused with dsred in order to visualize the nucleolus. transfection assay results indicated that acgfp localized predominately to the cytoplasm and the nucleus, but not the nucleolus. the characteristics of acgfp-n protein localized to either the cytoplasm alone or the cytoplasm and nucleolus, with a maximum of 60% of transfected cells exhibiting this phenotype at 24 h. our studies indicated that fusing acgfp with pedv n protein increased the molecular weight of this protein (82 kda) above the size exclusion limit of the nuclear pore complex, and it could not diffuse passively through the npc. this result suggested that there must be some signals in n protein that determined the nucleolar localization. a number of viruses and viral proteins can disrupt nucleolar architecture [57] , and the n protein of coronavirus can also localize to the nucleolus in a cell cycle dependent manner and this may be related to dynamic trafficking [58] . a previous study of sars-cov indicated the n protein inhibited b23 phosphorylation and might influence ribosome biogenesis to suppress host gene expression and create a more favorable milieu for virus survival [25] , so the n protein of pedv might take part in some cellular process. to investigate whether pedv n protein contained a nols, the protein was expressed as a series of single and overlapping regions. this preliminary analysis indicated that pedv n protein contained a nols in region 1. deletion mutagenesis delineated amino acid pedv n 71-90, a 20 aa motif that modulated nucleolar localization. furthermore, comparison with cellular and viral nolss, the data indicated that r87 and r89 may be critical for the nucleolar localization. for the site of replication and virus assembly is the cytoplasm, if the subcellular localization of the nucleocapsid protein is in the nucleolar, then the protein is not available for rna synthesis, encapsidation and assembly, and therefore progeny virus production might be less efficient. however, if such proteins do target the nucleolar as part of a virus replication strategy then they will contain appropriate targeting signals. thus, these will include not only nlss and nolss, but perhaps more importantly nes. by using a series of n deletion mutants fused to the acgfp and tetra-alanine substitution mutagenesis, we identified two nes site in region 2 (aa221-236) and region 3 (aa325-364), but nes in region 2 was deleted in the context of full length n protein did not affect cytoplasm trafficking, suggesting that the nes presented in region 2 submissive to the nes in region 3 or not function. furthermore, pedv n protein nucleocytoplasmic shuttling was specifically blocked by lmb treatment suggested that pedv n protein is transported via the classical crm1-dependent pathway. vero e6 was grown and maintained in dulbecco's modified eagle's medium supplemented with 10% heat-inactivated fetal calf serum and penicillin-streptomycin, and incubated at 37 °c in 5% co 2 . vero e6 cells were infected with pedv strain cv777 kindly provided by pensaert m b at moi = 1, and were seeded and after 24 h for both indirect immunofluorescence assay and western blot analysis. all enzymes used for cloning procedures were purchased from takara (dalian, china) except t4 dna ligase from new england biolabs (ipswich, ma, usa). pdsred-b23.1 encodes a fusion of dsred protein to the amino terminus of b23.1 and pacgfp-n encodes a c-terminally gfp-epitope-tagged version of pedv n and has been previously described [59] . the regions and subregions were amplified by pcr using pacgfp-n as a template. primers incorporated 5' bamh i site and 3' xho1 used for cloning into pet30a; primers incorporated 5' kpn i site and 3' xho i site used for cloning into pcdna3.1 and the other primers incorporated 5' xhol i site and 3' kpn i site used for cloning into pacgfp-c1. the cloning procedures for constructing most of recombinant plasmids were similar and all primers are listed in table 1 . at all times, numbers used in primer or construct names denoting amino acid numbers refer to their position on the full length n protein. for the tetra-alanine substitution, each pair of oligonucleotide strands was annealed, and the resultant double-stranded dna fragments were cloned into the vector pacgfp-c1. additionally, the nes of nr2 was deleted in the context of full-length n protein by overlapping pcr using forward primer 1-u and reverse primer 1-l (table 1) to generate one pcr product and forward primer 2-u and reverse primer 2-l (table 1) to generate the second pcr product. a second round of pcr was performed using both pcr products as templates and 1-u and 2-l as the forward and reverse primer, respectively. the resulting product was subcloned into pacgfp-c1. all plasmid were confirmed by sequencing analysis. the recombinant 6× his-tagged n protein was expressed in e. coli bl21 (de3) cells after induction with 1 mm iptg for 5 h in lb-medium at 37 °c . the recombinant protein was purified by a gravitraptm affinity column (ge healthcare, bio-sciences, piscataway, nj, usa) according to the manufacturer's instructions. balb/c mice were immunized with 0.1~0.2 mg of purified recombinant n protein injected subcutaneously at multiple sites on the back. booster injections were given two and four weeks later. blood was drown from the mouse at the fifth week following the immunization, and the blood was allowed to clot at 4 °c and the antiserum was recovered by centrifugation at 5,000× g for 10 min at 4 °c . a control serum was made by injecting normal saline under the same conditions. the animal experiment was approved by harbin veterinary research institute and performed in accordance with animal ethics guidelines and approved protocols. the animal ethics committee approval number is heilongjiang-syxk-2006-032. on the day before transfection, 0.5~2 × 10 5 cells grown on 35 mm culture dishes so that 60%-80% confluent cell monolayers were transfected with plasmid dna with lipofectamine™ 2000 (invitrogen, groningen, netherlands) according to the instructions of the manufacturer. protein samples were collected 48 h after transfection by direct lysis of the cells in 5× sds protein sample buffer with 5% 2-mercaptoethanol. the samples were boiled for 5 min, resolved with 12% sds-page, and blotted with the following antibodies. for the infection and transfection protein samples: the n protein polyclonal antiserum was the first antibody and irdyetm700dx conjugated affinity purified anti-mouse igg (h&l)(mouse) (li-cor biosciences, lincoln, ne, usa) was the second antibody; the images were acquired by the odysseytm infrared imaging system (li-cor). vero e6 cells were grown on coverslips and fixed 24 h post-infection (or post-transfection) with 50% methanol-50% acetone for analysis by indirect immunofluorescence using mouse anti-pedv n polyclonal sera (1:50 dilution) followed by fluorescein isothiocyanate (fitc)-labeled goat anti-mouse antibody (sigma, st louis, mo, usa). the cells were washed twice with phosphate-buffered saline (pbs) and subjected to perforation using 0.2% triton x-100. then the cells were stained with propidium iodide (pi) (50 μg/ml) (sigma) to visualize nuclear dna. samples were analyzed using fluorescence microscopy. for acgfp and dsred fusion expression constructs, co-transfection analyses were carried out. at 24 h post-transfection, subcellular localization properties in living cells were analyzed by laser confocal scanning microscope (leica laser technik, heidelberg, germany) with appropriate filters. in order to better display the results at the time of identify the nes in the regions of nr2 and nr3, at 24 h after transfection, cells on glass cover slips were rinsed with pbs and subjected to fixation using 50% methanol-50% acetone for 30 min and permeabilized with 0.2% triton x-100. then the nuclear was stained with 4',6-diamidino-2-phenylindole (dapi) (0.05μg /ml) (sigma) and analyzed by confocal microscope. cells were transfected with pacgfp-n, pacgfp-nr3, or pacgfp-nr3 325-364 . at 18 h post-transfection, cells were washed with pbs followed by either treated with 2.5 ng/ml lmb diluted with medium or left in unsupplemented cell culture medium for a further 6 h, then analyzed by used confocal microscope. a nols (aa71-90) and a functional nes (aa325-364) of pedv n protein 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signal of human angiogenin nuclear and nucleolar localization of 18-kda fibroblast growth factor-2 is controlled by c-terminal signals sequence requirement for the nucleolar localization of human i-mfa domain-containing protein (hic p40) identification of sequences important in the nucleolar localization of human immunodeficiency virus rev: relevance of nucleolar localization to function effects of a highly basic region of human immunodeficiency virus tat protein on nucleolar localization classical nuclear localization signals: definition, function, and interaction with importin alpha transport into and out of the nucleus. microbiol ppar γ mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance function of dynein and dynactin in herpessimplex virus capsid transport p53 is associated with cellular microtubules and is transported to the nucleus by dynein rna viruses: hijacking the dynamic nucleolus cell cycle dependent localization of the coronavirus nucleocapsid protein co-localization analysis between porcine epidemic diarrhea virus nucleocapsid protein and nucleolar phosphoprotein b23.1 this work was supported by grants from the national natural science foundation of china (31172350), the natural science foundation for distinguished young scholars of heilongjiang province (jc201118), the agricultural scientific and technological transformative project (2011gb23260003), and higher school science and technology innovation team project of heilongjiang province (2011td001). the sponsors had no role in study design, collection, analysis, and interpretation of the data, writing the report, and in the decision to publish the results of the study. the authors declare no conflicts of interest. key: cord-319884-d8n0aokl authors: natesan, mohan; ulrich, robert g. title: protein microarrays and biomarkers of infectious disease date: 2010-12-16 journal: int j mol sci doi: 10.3390/ijms11125165 sha: doc_id: 319884 cord_uid: d8n0aokl protein microarrays are powerful tools that are widely used in systems biology research. for infectious diseases, proteome microarrays assembled from proteins of pathogens will play an increasingly important role in discovery of diagnostic markers, vaccines, and therapeutics. distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods. depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods. new developments, challenges, and future demands in infectious disease research will impact the application of protein microarrays for discovery and validation of biomarkers. large-scale genome sequencing projects first advanced knowledge of the theoretical composition of proteomes and led to the development of dna microarrays for studying gene transcription at the organism-scale. using genome sequence data to guide the direct examination of proteomes then enabled the study of host-pathogen interactions occurring beyond the level of gene transcription. it soon became evident that direct correlations between gene expression and protein abundance were rare [1, 2] , driving the development of new approaches to study complex proteomes. protein microarrays are ideally suited to serve this purpose and have enormous potential applications in biomarker discovery, diagnosis, vaccine development, and drug discovery for infectious diseases. fluorescencebased detection of protein interactions is similar to gene array methods, and data analysis often employs approaches previously developed for genome and transcription studies. the number of proteins that can be printed on a single microarray surface also approaches the same upper limits as nucleic-acid based systems. multiplexing of fluorescent probes is limited by the ability to separate signals of overlapping emission spectra. generally, pair-wise comparisons of 2-3 differentially labeled, experimental and control samples can be analyzed. for example, serum igg and igm binding to arrayed proteins can be independently probed by fluorescently labeled secondary antibodies, and detected by a confocal laser scanner (such as genepix, molecular devices, sunnyvale, ca, usa). a complete high-throughput screening of thousands of interactions can be performed accurately and rapidly ( figure 1 ). direct labeling of probes may also be used, though structure and function may be adversely affected. most label-free techniques [3] provide real-time measurements and in some cases yield kinetics (figure 1 ), providing further insight into molecular interactions. surface plasmon resonance (spr) [4] , nanowire surfaces [5] , and mass spectrometry [6] are examples of label-free methods that are applied in the field of protein microarrays. of these, only a limited number of spr-based instruments are currently available for analysis of complex protein microarrays. spr imaging (spri) is a more recent and promising development [3, 7] . while planar arrays that use slides or chips for immobilizing capture molecules are most common, suspension arrays based on microbeads have many important applications. the xmap technology developed by luminex corporation (austin, tx, usa) uses 5.6 micrometer polymer beads infused with two fluorescent dyes at different ratios to yield up to 100 distinct bead sets. the binding measurements are performed by flow cytometry with two lasers, one for the identification of the bead and the other for the sample. bead-based arrays are very useful for standardizing assays, with an upper limit of 80 independent immobilized probes. protein microarrays are increasingly used in infectious disease research to identify new biomarkers that are involved in the disease process or that are targets of immune responses [8] [9] [10] [11] [12] [13] . in contrast with other proteomic techniques such as 2-d gel electrophoresis and mass spectrometry, the arrayed probes are known, conforming easily to high-throughput applications that require examination of the entire proteome of an infectious agent. a variety of formats have been developed for the study of pathogen proteomes. capture and reverse-phase are examples of abundance-based microarrays produced by spotting either antibodies [10] or antigens [14] on a substrate. the arrays can be probed with serum, plasma, and other biological samples to detect binding interactions. microarrays based on wellcharacterized antibodies are specifically useful for identifying biomarkers and quantification of proteins. aptamers [15] , affibodies [16] , or engineered antibody fragments [17] are alternative capture molecules. antigen arrays find applications in serodiagnosis of disease, antibody-response profiling, and evaluation of immunity after vaccination. in reverse-phase protein microarrays (rppms), small amounts of biological samples from cells, tissues, sera, or plasmas are directly printed on the slide [18, 19] . these arrays are probed with a single protein of interest that is detected by labeled antibodies. rppms were successfully used for the screening of cell-signaling pathways and posttranslational modifications of proteins [20] [21] [22] . the biochemical activities of arrayed proteins have also been analyzed [23] by enzyme activity or substrate activity, and protein interaction with other proteins, nucleic acids, or drugs [24] [25] [26] . the host antibody response provides a signature of pathogen proteins displayed by infectious diseases that can be captured and detected by microarrays. our studies with vaccinia virus [12] and the bacterium yersinia pestis [11] exemplify the application of antigen microarrays for the analysis of immune responses ( figure 2 ). vaccinia is a large dna virus (orthopoxviridae) that replicates in the cytoplasm of host cells from genomes encoding 150-300 proteins. the virion is comprised of approximately 100 proteins. most phenotypic variability occurs in proteins encoded in the terminal regions of the genome that are associated with host virulence or immune evasion. some of these terminal-region proteins are secreted during cell infection and interfere with host immunity by binding complement factors, cytokines, and chemokines, while others interfere with signaling pathways regulating host gene expression and apoptosis. to construct a proteome microarray, genomic dna of the copenhagen (nc_001559.1) vaccine strain (dryvax) was used as the template for pcr amplification of the 273 open reading frames (orf). gateway recombination cloning (invitrogen, carlsbad, ca, usa) was employed to facilitate high-throughput production of proteins from all orf clones. all dna clones were sequence-verified through the entire length of their inserts. baculovirusbased expression was used to produce the recombinant viral proteins as gst-tagged fusions to ensure high yield of properly folded proteins with posttranslational modifications that are similar to those encountered in the human host. the gst-tagged proteins from cell lysates were affinity purified to 90% homogeneity in a single step by using glutathione-agarose in 96-well plates and analyzed for correct size and abundance by western blots. virus and control proteins were printed onto glass slides coated with a thin layer of nitrocellulose. ultimately, 95% of the proteome was successfully expressed, purified, and arrayed. a standard assay for measuring antibody interactions with proteins of the vaccinia proteome microarray was first developed with a pool of therapeutic human sera collected from vaccinia-immune individuals (vig) and this data was compared to results obtained from individuals vaccinated against smallpox using dryvax. the assay consisted of incubating a dilution of serum on the microarray surface, washes, and finally detection with a fluorescently labeled anti-ig antibody. fluorescent images were captured by scanning with a confocal laser (genepix 4000b; molecular devices). because only a very small sample volume (1-2 l) is required, this approach is ideal for limiting the amount of sample consumed, often necessary with clinical material. a high level of reproducibility with a very low background was apparent in repetitive assays that confirmed previously reported antigens and identified new proteins that may be important for neutralizing viral infection. incubation of the microarray with vig identified nine proteins that consistently bound antibody, while antibody interactions with all other proteins were insignificant, requiring no further treatment to suppress non-specific signals. these antigens were also recognized by antibodies from individual subjects following primary smallpox vaccination and were diverse in function, consisting of regulatory, surface, core, and secreted proteins. the identical vaccinia proteins clustered into proteins recognized by primary and secondary vaccinated subjects based solely on signal intensities. the vaccinia proteins c3l and i1l were not previously reported as antibody-recognized antigens. the nine antigenic proteins we identified did not bind antibody from non-vaccinated sera, confirming the specificity of these antibody-antigen interactions. however, o2l and h7r were reactive with antibodies from both vig and non-vaccinated control sera, suggesting that these were cross-reactive or non-specific interactions. these results indicated that only a small subset of proteins present within the complex orthopoxvirus proteome was associated with antibody responses. in addition, the human response to individual proteins was variable as sera from more than half of the subjects contained igg that recognized >4 vaccinia proteins, while the remaining samples recognized one to three proteins. further, antibodies from most individuals recognized a greater number of viral proteins after a boost vaccination compared to a single vaccination, suggesting that repeated infection expands the total number of proteins recognized by igg. a previous report [27] similar to our study on vaccinia [12] , analyzed the serological response to vaccinia and smallpox, observing that a significant amount of the antibody response to vaccinia was not involved in the virus neutralization, but could be used as potential markers for diagnostic and vaccine development. a later report [28] evaluated an attenuated smallpox modified vaccinia virus ankara (mva) an alternative to dryvax vaccine by comparing antibody profiles in the sera of humans and monkeys. the overall immunogenicity of mva was reported to be comparable to that of the dryvax vaccine and the study concluded that mva may be a useful alternative to dryvax. our vaccinia virus microarray was expanded to allow analysis of additional poxviruses. monkeypox is a zoonotic viral disease that occurs primarily in central and west africa. immunity to monkeypox is provided by vaccination against smallpox caused by the closely related variola virus. to differentiate antibody responses to monkeypox virus infection from human smallpox vaccination, we developed a protein microarray covering 92-95% (166-192 proteins) of representative proteomes from monkeypox viral clades of central and west africa, including 92% coverage (250 proteins) of the vaccinia virus proteome as a reference orthopox vaccine. serum igg of cynomolgus macaques that recovered from monkeypox recognized at least 23 separate proteins within the orthopox meta-proteome, while only 14 of these proteins were recognized by igg from vaccinated humans. there were 12 of 14 antigens detected by sera of human vaccinees that were also recognized by igg from convalescent macaques. the greatest level of igg binding for macaques occurred with the structural proteins f13l and a33r, and the membrane scaffold protein d13l. significant igm responses directed towards b16r, f13l, and a33r of monkeypox virus were detected before onset of clinical symptoms in macaques. results from this study suggested that antibodies from vaccination recognized a small number of proteins shared with pathogenic viral strains, while recovery from infection also involved humoral immunity to antigens uniquely recognized within the monkeypox virus proteome (unpublished data). to identify antibody biomarkers that could distinguish plague from infections caused by other bacterial pathogens [11] , we developed a microarray comprised of the proteome from the bacterium yersinia pestis. the chromosome of y. pestis co92 encodes approximately 3885 proteins, while an additional 181 are episomally expressed by pcd1, pmt1, and ppcp1. for comparison, the proteome of y. pestis kim contains 4202 individual proteins, 87% in common with co92, and the closely related enteric pathogen y. pseudotuberculosis contains approximately 4038 proteins (chromosome plus plasmids). the microarray was comprised of proteins representative of over 75% of the 4066 orfs present within the y. pestis genome. in a manner similar to the vaccinia microarray, the y. pestis proteins were produced as full-length polypeptides fused to gst as an affinity isolation tag. an in vitro translation method, based on e. coli lysates, was used to express the proteins in a gram-negative background. the orf clones were fully sequenced to confirm quality and identity. the affinity-purified proteins were characterized by sds gels and western blots probed with an anti-gst antibody. different approaches for studying the antibody repertoire for plague in rabbits and non-human primates were investigated. based on results from experiments using the y. pestis proteome microarray, we identified new candidates for antibody biomarkers of bacterial infections and patterns of cross-reactivity with a panel of other gram-negative pathogens [11] . we were able to cluster the proteins recognized by the antibodies into three groups: common proteins of all gram-negative bacteria, combinations of proteins unique to one pathogen, and proteins unique to one pathogen. this example demonstrates the power of a high-density proteome microarray to dissect the complex immunological relationships among a group of related human pathogens. convalescence sera from vaccinated non-human primates that survived an otherwise lethal aerosol challenge with y. pestis co92 (plague) or bacillus anthracis ames spores (anthrax) were also examined. in addition to the caf1 and lcrv proteins comprising the vaccine components, a subset of proteins from the y. pestis proteome were recognized by antibodies from plague survivors and none of these were detected with antibodies from anthrax survivors or non-challenged controls. signature patterns of antibody recognition were identified that reflected the orthologous relationships among proteomes of these bacteria. a high degree of cross-reactivity in the antibody response to burkholderia and related bacteria was also previously reported [29] . further, a protein microarray consisting of 4% of y. pestis proteins was used by li et al. to profile antibody responses to live plague vaccine in rabbits [30] , sentinel animals [31] , and plague patients [32] . predominant responses were observed for 11 proteins in addition to f1 and v antigens in rabbits. in plague patients, they identified 14 proteins that were potential serodiagnostic biomarker candidates and found antibody responses from different plague foci responsible for different clinical symptoms. additional microarray studies of antibody responses to bacterial pathogens were reported. sundaresh et al. [33] developed a protein microarray with 244 of the most reactive francisella tularensis proteins derived from a larger chip. in this study, they identified a set of immunodominant antigens from f. tularensis patients suitable for diagnosis development. in another study [34] , a similar protein microarray approach was employed to find immunodominant antigens in mice vaccinated with killed f. tularensis vaccine and challenged with a virulent f. tularensis strain. the study found 31 antigens in addition to 12 known immunodominant antigens of f. tularensis discovered by other methods. this was also the first published report to use protein microarrays for studying protective immune responses to an infectious agent. immunoreactive antigens of coxiella burnetti, the causative agent of q fever, were identified using a protein microarray consisting of 75% of the c. burnetti proteome assembled by using a cell free protein expression system [35] . serum from q fever patients and individuals vaccinated with q-vax vaccine strongly reacted to 50 antigens of c. burnetti that were confirmed by elisa including antigens isolated from an e. coli expression system. in yet another study, human and goat antibody responses to brucella melitensis infection were analyzed [36] , finding that there was differential recognition of antigens by the two host species. the serodiagnostic proteins were of potential value for diagnostic purposes as brucellosis is diagnosed by measuring the antibodies to lipopolysaccharide (lps), which cannot distinguish between past and present infection in endemic areas. antibody response to lyme borreliosis disease in humans and mice was studied by barbour et al. [37] , using a protein microarray comprising 80% of the proteins from borrelia burgdorferi. a comprehensive analysis of the sera from b. burgdorferi patients and rodents revealed that only a small set of antigens elicited antibody responses in both hosts, while the majority of b. burgdorferi proteins did not induce antibodies. it is also possible to focus on specific subsets of antigens identified by bioinformatics. for example, a protein array produced from the outer membrane proteins of pseudomonas aeruginosa was constructed [38] to study the immune response in patients, and several antibody-binding antigens were identified by this group as potential diagnostic markers. in another example, a recombinant niesseria meningitidis protein array was used for screening serum from meningitis patients [39] for immune responses to phase-variable expressed proteins. the development of new proteomics methods has been driven in part by the need to identify new types of infection biomarkers. though studies of antibody responses dominate the field, protein microarrays should be considered as an alternative approach to examine other catagories of host-pathogen interactions. research areas with well-established cell proteomics data are prime targets. for example, because many of the host factors associated with phagosomes have been identified [40] , protein microarrays may provide a convenient model system to examine interactions with pathogen proteomes associated with this portal of entry. in a similar manner, targeted microarrays comprised of pathogen proteins may be used to examine host interactions. the few published examples from this developing field of study should be mentioned. in one report, rppm along with other techniques [41] were used to examine phosphoprotein signaling pathway in primary human small airway epithelial cells infected with bacillus anthracis, identifying reduced akt (protein kinase b) phosphorylation as a contributor to increased bacterial survival and pathogenicity. in another study [42] , proteins from group a and b streptococci were arrayed on a chip and probed with human fibronectin, fibrinogen, and c4 binding protein, all well-known targets of gram-positive bacteria. the use of purified and well-characterized proteins to assemble the microarray will insure the highest quality and most reproducible results. however, the high level of quality control required to sequence, purify, and characterize elements of a proteome microarray in the examples we described may not be as important for provisional screening purposes. a coupled transcription/translation reaction was previously reported as a rapid method to develop protein microarrays against a number of infectious organisms [43, 44] , bypassing sequencing and purification steps. in this technique, both transcription and translation occur simultaneously to synthesize proteins in parallel from pcr products, directly on the chip. presumably, results obtained by these methods will require follow-up studies with more rigorous controls for confirmation. the protein in situ array (pisa) developed by he and taussig [45] first produces dna constructs from a pcr of the protein of interest and a t7 promoter for translational initiation followed by immobilization using a n or c terminal tag sequence. another approach [25, 46] called nucleic acid programmable protein array (nappa) included slides coated with avidin to capture biotinylated plasmid dna for synthesizing proteins. proteins with fused tags were synthesized and immobilized on the chips coated with molecules to bind the tags [25] . he et al. [47] described a technique called dna array to protein array (dapa) where they utilized a single dna array to make multiple copies of the same protein array. in one example, a self-assembled nappa-based on a cell-free expression system was used for constructing a protein microarray of all 69 proteins produced by varicella zoster virus (vzv), the cause of chickenpox [48] . three sets of antigens in the vzv proteome were identified from human sera that may be useful in defining the clinical status of the infection and diagnostics. another vzv protein array produced from an e. coli expression system was later used to detect antibodies in human sera reactive to vzv viral proteins [49] . the orf68 (ge) antigen identified in the study showed high-confidence for determination of the serological response to vzv. although these alternative in situ synthesis methods are rapid, they are not without significant shortcomings. it is difficult to characterize proteins expressed on the chip in contrast to direct spotting of purified proteins. two studies investigated the antibody response to vzv by protein arrays made from either nappa cell-free expression system [48] or e. coli expression system [49] . antibodies from the sera of patients recognized three microarrayed vzv proteins produced recombinantly with e. coli, whereas these identical proteins were not recognized on a nappa protein expression-based protein microarray. interestingly, these proteins were abundantly expressed in the nappa protein microarrays, suggesting that they did not fold correctly to expose their linear epitopes for binding. despite these limitations, microarrays based on in situ synthesis offer simplicity, efficiency, and highthroughput capabilities. the relatively small proteomes of most viruses increase in diversity by considering variations in clinical isolates. for example, while human papilloma virus produces only a few proteins, sequences for these may diverge in the more than 100 strains of the virus. a protein microarray developed with 13 human papilloma virus types [50] was used to examine patient sera compared to asymptomatic subjects. the study identified e7 proteins as the most reactive antigens in the patient group. however, the e7 proteins were not useful as a diagnostic biomarker because the differences between patients and asymptomatic subjects were not significant. in another report, a bead suspension multiplex array (luminex xmap) was used to study serological responses to 27 antigens of human papilloma virus [51] . the data correlated well with elisa results and in addition the bead array was able to measure weak antibody responses. a protein array constructed from six coronaviruses including severe acute respiratory syndrome (sars) was used for screening serum samples for virus-specific antibodies [52] . the microarray data predicted sars infection in 90% of the patients correctly with a specificity of 93%. the high efficiency, low cost, and high-throughput nature of chemically synthesized peptides are significant advantages compared to production of recombinant polypeptides. therefore, peptide microarrays have been explored as another alternative to recombinant proteins, with a limited degree of success. a chemically synthesized peptide microarray representing the major antigens of hepatitis b and c viruses, human immunodeficiency virus, epstein-barr virus, and syphilis was constructed on a glass slide and antibody responses were simultaneously detected [53] . the assay showed very high sensitivity and specificity for the diagnostic identification of these viruses. another proof of principle study [54] was reported to identify diagnostically relevant peptides using a peptide array deduced from bioinformatics data for echinococcus granulosa (tapeworm) infection, achieving only 57% sensitivity and 94% specificity compared to elisa. as an alternative to full-length polypeptides, expression and purification of protein domains may improve yield of stable products for use in microarrays. in one reported study, a total of 212 protein domains (sh3, sh2, pdz etc.) were immobilized on nitrocellulose and screened with peptides [55] . the results proved that the immobilized domains were stable and could be used for binding studies. kaushansky et al. [56] constructed an array based on protein interaction domains such as src homology, phosphotyrosine binding domains, and mouse pdz domains produced from recombinant e. coli. these arrays required low sample consumption and may reduce false positive rates. advanced knowledge of the specific recognition events will be required to substitute peptides or single protein domains for full-length proteins to ensure that important epitopes are not missed for the purpose of studying antibody interactions. protein stability is an important and particularly challenging factor in using protein microarrays. the high-throughput nature of construction tends to sacrifice detailed knowledge about individual proteins. there are few methods available to assess folding and functionality of all arrayed proteins immediately before use. recombinant proteins constructed with fusion tags to facilitate folding during translation offer a partial solution, especially if proper folding of the fusion protein can be readily assessed. for example, unfolded glutathione-s-transferase (gst) will not bind to glutathione, the tripeptide ligand of gst, hence purification schemes employing gst fusions will favor proteins with stable structures. fusion tags also provide a convenient marker for monitoring quality control of protein spotting (figure 2) . however, the fusion tag may hinder protein accessibility or alter function in other ways. because proteins may denature during printing and storage, enzymes or other denaturing-sensitive proteins that can be assessed after printing should be included in the microarray. antibodies are a natural choice to be used as capture molecules due to their specificity, affinity, ease of production, and potential for engineering. for probe development, the analysis of antibody specificity and cross-reactivity [57] [58] [59] can be simplified by screening against target or off-target proteins printed as microarrays. antibody arrays are routinely used in the field of biomarker discovery and validation. the detection of binding molecules to an antibody array is usually done by direct labeling of proteins in the sample (serum, cell, or tissue) or by a sandwich format similar to elisa. for example, protein abundance in cells was determined by haab et al. [60] , using 115 antigen and antibody reactions. this was the first published report to use a two-color differential labeling of samples in a protein microarray. the microarray format is also convenient for standardizing the routine analysis of analytes present in screening assays, such as cytokines released by cell cultures [61] . an antibody-based microarray for the multiplexed detection of cholera, diphtheria, staphylococcal enterotoxin b, tetanus toxins, anthrax protective antigen, and lethal factor was reported [62] . a competition assay between labeled and unlabeled toxins in serum was used to simultaneously detect all the toxins in the antibody microarray. in another report, a sandwich fluorescence immunoassay based on an antibody microarray format was developed for the capture and detection of e. coli o157.h7 [63] , and later to screen large number of food samples in a high-throughput manner to simultaneously detect e coli o157:h7 and salmonella typhimurium [64] . the major drawback of capture microarrays is that few well-defined and high-quality antibodies against microorganisms are available. engineering antibody fragments is a potential alternate approach to accelerate the development of antigen-capture molecules. antibody fragments such as the fab portion of igg [65] , phage display libraries or recombinant single chain variable fragment (scfv) containing the antigen-binding motif can be used in place of antibodies in protein microarray development [66, 67] . a decreased cross-reactivity was also observed with fragments as compared to full-length antibodies. thin-layered nitrocellulose or chemically modified 2-d surfaces (glass, silicon, gold, and polymer) are generally used for immobilization of microarrayed proteins. buffer additives like glycerol, polyethylene glycol, or sugars that prevent drying of the proteins and choice of buffers should be carefully selected to match the type of surfaces used for arraying. alternatively, gel-based 3-d surfaces made from polyacrylamide or agarose provide a hydrophilic environment [68] . regardless of the printing substrate selected, microarrayed proteins must retain functionality and stability during preparation and use. though it may not be possible to directly examine all elements of an array, it is important to include printing controls of interacting proteins that can be tested for functionality before use. there are several methods by which proteins are spotted onto substrates. the choice of technology used impacts spotting consistency, speed, spot diameter, and ease of use. in turn, the final spot quality required for printed proteins will depend on the method of detection. control of the laboratory environment to maintain constant temperature, humidity, and clean-room conditions will vastly improve printing consistency. printing with solid pins (for example stealth pin, arrayit corportion, sunnyvale, ca, usa) relies on capillary forces to release spots on contact with the surface, resulting in 60-600 m diameter spots depending on buffer composition and pin diameter. dip-pen lithography (dpn) uses atomic force microscopy microcantilevers to deposit spots in the range of 1-60 m diameter (nano enabler, bioforce nanosciences, ames, ia, usa) and inkjet printers (for example arrayjet, roslin, scotland) use piezoelectric elements to transfer the protein solution in the form of droplets to the target surface, resulting in spot diameters of 80-150 m. both pin and inkjet spotting methods deposit a very small amount of protein, requiring highly concentrated samples. unfortunately, highly concentrated samples may fail to adsorb completely on the surface and tend to spread during hydration. a new continuous flow microspotting method (wasatch microfluidics, taylorsville, ut, usa) uses microfluidic channel networks to continuously circulate protein samples over a spot to achieve uniform and maximum protein adsorption [69] . this technique may be especially useful for dilute and crude protein sample arraying. fluorescence-based labeling of probes is most commonly used to detect binding events, due primarily to simplicity and sensitivity. examples are amine-reactive dyes such as cyanines (cy3 and cy5) or labeling probes with biotin followed by detection with an avidin-fluorescent dye conjugate. ccd cameras or fluorescent slide scanners are used for detection of the labeled probes within defined grid areas. sandwich immunoassay methods provide increased sensitivity and at the same time reduce non-specific binding, but their use is limited by the availability of paired antibodies. using a rolling circle amplification (rca) method can dramatically increase fluorescent signal intensity [70] , thus increasing sensitivity in protein microarray detection methods. in this scheme, proteins are labeled with biotin and recognized by an antibody conjugated with a primer to which a circular dna is hybridized. fluorescently labeled oligonucleotides are used for elongation of dna and detection. a universal rca signal amplification scheme was used [71] for measuring cytokines in a multiplexed format. a total of 75 cytokines were simultaneously measured on an antibody microarray, demonstrating that even larger number of analytes can be measured by this technology. thus, rca is a powerful tool for improving the detection limits of protein microarrays by signal amplification. current fluorescence-based detection in protein microarrays is limited to two to three independent probes because broad emission profiles exhibited by the dyes used for detection often overlap with each other and with the background fluorescence emitted by substrates, such as the commonly used nitrocellulose. also, multiple excitation and emission channels are required to detect combinations of fluorescent markers. quantum dot (qds) nanoparticles are one alternative to fluorescent dyes. the qds are semiconducting fluorophores that are extremely bright, resist photobleaching, and have multiplexing capability. each contains an inorganic fluorescent core, commonly cdse, coated with a shell of another semiconductor such as cds or zns. the most important property of qds with reference to protein microarray detection is that they absorb light at broad wavelengths, from uv to visible range and emit light at a very narrow bandwidth, in contrast to fluorescent dyes. additionally, the qd surfaces may be modified with suitable chemistries for immobilizing biomolecules. streptavidin-coated qds in a protein microarray format were used to detect six different cytokines at picomolar concentrations [72] , currently the practical limit for multiplexing with qds. additional multiplexing strategies have been previously described in detail [23] . bead-based microarrays offer one solution to multiplexing. most commercially available methods were developed by luminex (xmap platform). the xmap technology is based on polymer beads incorporated with fluorescent dyes that produce unique signatures. these beads can be conjugated to different molecules to perform multiplexed assays. up to 100 discrete interactions can be monitored simultaneously using this technology [73] , perhaps extended to 500 bead sets in the near future. antigens or peptides have been attached to the luminex beads and antibody responses to papillomavirus [74] , epstein-barr virus [75, 76] , and mycobacterium tuberculosis [77] were reported. becton dickinson (franklin lakes, nj, usa) markets a cytometric bead array (cba) system, which is another multiplexed bead-based assay that includes flow cytometry for cytokine, chemokine, mouse isotyping, and signaling molecule analysis. label-free detection methods are used for real-time monitoring of binding events and to minimize artifacts caused by using labeled probes. spr imaging technology is widely used in a microarray format for many applications. spr is label-free, allows real-time monitoring, and has the additional ability to measure kinetics of the molecular interactions ( figure 1 ). spr imaging was developed to simultaneously measure spr dip changes in an array format, recorded with a ccd camera. grating-coupled (flexchip, ge/biacore, piscataway, nj, usa) and prism-coupled (spri-plex, horiba scientific, edison, nj, usa, proteon xpr36, bio-rad, hercules, ca, usa) spri systems are some examples of commercially available spri microarray systems. a model protein-protein interaction system was reported [78] , consisting of the e6 protein of human papillomavirus complexing with the proteins e6ap and p53 using a spri system. in another example, an array-based spectral spr system was used for measuring antibody response to mumps virus infection [79] . in this system the reflected light from the array was collected into a fiber optic spectrometer for analysis. our laboratory routinely uses spri microarrays to validate interactions that were detected during primary microarray screens from a fluorescent probe read-out. because spot sizes must be large enough for resolution by the commonly used ccd imaging, practical array densities are currently less than 1000 independent immobilized probes. additional label-free detection methods have been described. surface-enhanced laser desorption/ionization time of flight mass spectrometry (seldi-tof-ms) has been used as a low throughput method for on-chip purification of proteins, ionization, and detection [80] . however, this approach was not very successful in identifying biomarkers in parasitic diseases, and most markers found in the study were intact host proteins [81, 82] . matrix-assisted laser desorption/ionization (maldi)-ms in combination with 3-d gel surfaces [83] and silicon nanoporous nanovials [6] were also used for biomarker discovery. the ms-based detection systems are important for specific applications, but there have been only limited advancements in the development of microarray formats, currently limiting use as a primary screening tool. nanowire-based systems detect changes in conductivity as a result of molecular binding [84, 85] . it is possible to make an array of nanowires such that each nanowire is coated with a specific capture molecule. gantelius et al. [86] reported a magnetic bead-based multiplexing format for the detection of auto-antibodies to 12 antigens, comparing the assay results to fluorescence-based detection system. although the magnetic bead-based system was rapid, it was not sufficiently sensitive for routine use. despite the rapid progress in protein microarray development standardized methods for data handling and analysis are not universally accepted. fluorescence scanners made by different commercial vendors use stand-alone software to analyze data, while assay protocols used by investigators may vary widely. hence, it is often difficult to compare studies reported by different groups, and there is a need to formulate standardized methods for protein microarray data handling and analysis. a number of reports have outlined ways to normalize protein microarray data, mainly for different approaches to antibody microarrays. these normalization methods include concentrationdependant analysis [87, 88] , spiked internal standards [89] , and algorithm-based [90] , and all have significant pros and cons. invitrogen/life technologies (carlsbad, ca, usa) developed a data analysis package (protoarray prospector) specifically designed for protein antigen microarrays, including a suite of statistical methods. bioarray software environment (base), a software package for microarray data management and analysis (http://base.thep.lu.se) developed by lund university, sweden, also addresses some of the issues we have outlined. however, it remains to be seen how rapidly standards will be adopted by the research community. antibodies are primary biomarkers of infection that are commonly used for diagnosis, measuring vaccine efficacy, and discovery of disease interventions. protein microarrays are important tools for measuring these complex antigen-antibody interactions and other host-pathogen interactions at different stages of the disease. the major impediments to development of protein microarrays are the inherent complexity of proteins themselves, availability of well-characterized antibodies to infectious agents, and standardized statistical methods for analysis of data. however, we expect many of these issues to be resolved in the coming years as these tools are used more frequently in infectious disease research. quantifying e. coli proteome and transcriptome with single-molecule sensitivity in single cells correlation between protein and mrna abundance in yeast label-free detection methods for protein microarrays present and future of surface plasmon resonance biosensors multiplexed electrical detection of cancer markers with nanowire sensor arrays high-speed biomarker identification utilizing porous silicon nanovial arrays and maldi-tof mass spectrometry protein and antibody microarrays: clues towards biomarker discovery. front. drug des protein-protein interactions and selection: array-based techniques for screening disease-associated biomarkers in predictive/early diagnosis defining the 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imaging mutation detection and single-molecule counting using isothermal rolling-circle amplification multiplexed protein profiling on microarrays by rolling-circle amplification protein microarrays and quantum dot probes for early cancer detection antibody suspension bead arrays within serum proteomics serologic response to oncogenic human papillomavirus types in male and female university students in evaluation of three different assays for the assessment of epstein barr-virus immunological status evaluation of a multiplexed bead assay for assessment of epstein-barr virus immunologic status profiling antibodies to mycobacterium tuberculosis by multiplex microbead suspension arrays for serodiagnosis of tuberculosis surface plasmon resonance imaging protein arrays for analysis of triple protein interactions of hpv, e6, e6ap, and p53 array-based spectral spr biosensor: analysis of mumps virus infection is seldi-tof a valid tool for diagnostic biomarkers? identification of novel diagnostic serum biomarkers for chagas' disease in asymptomatic subjects by mass spectrometric profiling use of proteinchip technology for identifying biomarkers of parasitic diseases: the example of porcine cysticercosis (taenia solium) analysis of protein interaction and function with a 3-dimensional maldi-ms protein array nanowire nanosensors for highly sensitive and selective detection of biological and chemical species protein recognition via surface molecularly imprinted polymer nanowires magnetic bead-based detection of autoimmune responses using protein microarrays a concentration-dependent analysis method for high density protein microarrays development of an internally controlled antibody microarray optimized normalization for antibody microarrays and application to serum-protein profiling determination of relative protein abundance by internally normalized ratio algorithm with antibody arrays the authors appreciate the many contributions of sarah keasey, christine pugh, emily cisney, stefan fernandez, and other members of the riid protein interactions team, the contributions of barry schweitzer and our other collaborations at life technologies for pioneering studies in protein microarray technology, and justin rodrigo for critical reading of the manuscript. opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the united states army. key: cord-333262-xvfl7ycj authors: robson, b. title: covid-19 coronavirus spike protein analysis for synthetic vaccines, a peptidomimetic antagonist, and therapeutic drugs, and analysis of a proposed achilles’ heel conserved region to minimize probability of escape mutations and drug resistance date: 2020-04-11 journal: comput biol med doi: 10.1016/j.compbiomed.2020.103749 sha: doc_id: 333262 cord_uid: xvfl7ycj abstract this paper continues a recent study of the spike protein sequence of the covid-19 virus (sars-cov-2). it is also in part an introductory review to relevant computational techniques for tackling viral threats, using covid-19 as an example. q-uel tools for facilitating access to knowledge and bioinformatics tools were again used for efficiency, but the focus in this paper is even more on the virus. subsequence krsfiedllfnkv of the s2′ spike glycoprotein proteolytic cleavage site continues to appear important. here it is shown to be recognizable in the common cold coronaviruses, avian coronaviruses and possibly as traces in the nidoviruses of reptiles and fish. its function or functions thus seem important to the coronaviruses. it might represent sars-cov-2 achilles’ heel, less likely to acquire resistance by mutation, as has happened in some early sars vaccine studies discussed in the previous paper. preliminary conformational analysis of the receptor (ace2) binding site of the spike protein is carried suggesting that while it is somewhat conserved, it appears to be more variable than krsfiedllfnkv. however compounds like emodin that inhibit sars entry, apparently by binding ace2, might also have functions at several different human protein binding studies. the enzyme 11β-hydroxysteroid dehydrogenase type 1 is again argued to be a convenient model pharmacophore perhaps representing an ensemble of targets, and it is noted that it occurs both in lung and alimentary tract. perhaps it benefits the virus to block an inflammatory response by inhibiting the dehydrogenase, but a fairly complex web involves several possible targets. coronaviruses have been known to medicine for some time [1] , but it is of course only very recently that the coronavirus sars-cov-2, the covid-19 virus new and dangerous to humans, was identified. it is believed to be related to an initial cluster of pneumonia cases associated with a seafood and fresh meat market in wuhan, china, [2] . current case rates at the time of writing are close to one million with close to 60,000 deaths. the genomic relationships to other coronaviruses were quickly examined by lu et al. to shed light on the origins, epidemiology, and receptor binding of the virus [2] . on january 17th , 2019, the wuhan isolate genbank entry mn908947.3 replaced mn908947.2, and mn908947.3 probably represents an adequate stable description of the sequence for research into that strain isolate, and was immediately investigated by the present author [3, 4] . originally, it was seen by authorities as a coronavirus outbreak but not as sars (severe acute respiratory syndrome). however, its genomic relationships examined in refs [3, 4] also showed many fairly close correlations with the genomes of sars-cov in the previous human (but not pandemic) outbreaks and in pigs, bats and civets, and the emphasis was on finding subsequences that are well conserved across coronavirus strains and species. the earliest patients suffering from what is now called covid-19 had overall 99·98% genome sequence identity to the above wuhan isolate, so that one may reasonably say that it is the origin of covid-19, and its virus sars-cov-2 [2] . the earlier wuhan isolates also related (with 88% identity) to two bat-derived severe acute respiratory syndrome (sars)-like coronaviruses collected in 2018 in zhoushan, china, but differed more from sars-cov (at about 79%) and mers-cov (at about 50%) [2] . the wuhan and related isolates revealed a coronavirus that resides in the subgenus sarbecovirus of the genus betacoronavirus [2] , and although genetically distinct from its predecessor sars-cov it appeared to have similar external binding proteins, meaning here the spike glycoprotein discussed extensively in the present paper. see section 1.3 below for introduction to this protein, which also discusses some further early identified genomic correlations. in addition, the rest of this present paper discusses many other genomic relationships relevant to the design of synthetic vaccines and therapeutic antagonists againstcovid-19. one problem is that covid-19 is a new pathogen posing a global threat and so presents new challenges both in primary prevention, where a vaccine is required, and in secondary prevention, where a therapeutic compound (ideally, "in a pill") is required to treat patients who are infected. it might also present challenges for tertiary prevention, which seeks to remedy a persistent level of infection, or to prevent recurrence even to essentially the same strain, as discussed in section 1.2. a main problem of concern, and a point of the present paper, is the likely appearance of new strains with resistance to vaccines and therapeutic agents. at the time of writing, confirmed cases double globally every 6 days, and undetected cases are expected to be much higher (the current plateauing of reported cases in china offers a glimpse that this this should attenuate soon, but estimates of how and when are varied). with a significant portion of humanity already infected, there is enhanced probability of successful "escape mutations" in the genome of the virus. development of vaccines and perhaps particularly therapeutics that could, but do not, take account of this by targeting less variable protein regions could be a huge waste of resource and a dangerous delay. covid-19 is, of course, by far the most serious, but not the first sars outbreak of concern to humans, and coronaviruses have for decades been of veterinary concern [1] . however, it still remains true that zoonotic coronaviruses have only rather recently seriously impacted humans, as far as is known. they include sars-cov (2002, betacoronavirus, subgenus sarbecovirus), and mers-cov (2012, betacoronavirus, subgenus merbecovirus). although the idea that sars-cov-2 was distinct from sars-cov was originally discouraged, distinction is here a matter of degree. by usual criteria they are fairly closely related, genetically clustering within betacoronavirus subgenus sarbecovirus. until very recently, sars-cov, effectively sars-cov-1, was the primary reference point and model regarding molecular and functional details, and it remains important. shortly after the appearance in genbank of the apparently final version of the wuhan seafood market isolate mn908947.3 [2] , the present author compared a variety of coronavirus genomes [3, 4] . the krsfiedllfnkv protein subsequence was seen as a potential achilles' heel because it is exposed or potentially exposable, being required for proteolytic activation cleavage, and importantly is also a well-conserved feature on the surface of the virus [3, 4] . being well conserved suggests that mutations are much less easily "accepted", meaning that the virus is less likely to survive more than one or two generations. as discussed below, the conservation is in a region of protein on the virus surface concerned with at least one step of lung cell entry, interesting because coronaviruses seem to be able readily adjust to alternative means of entry, possibly hinting at additional roles for the subsequence. whether or not that is the case, the above motif seems a likely primary target for synthetic vaccines and a basis for drug discovery, and was proposed as such [3, 4] . it is a motif that was found to be quite well conserved even in more distantly related coronaviruses [3, 4] , and the present paper also explores how far that seems to extend. it includes the common cold coronaviruses. another potential subsequence of interest popular with researchers is also examined (the ace2 binding domain discussed below), but the above remains popular with the present author because of its relatively high degree of conservation. present authors' opinion, however, it relates to a specter that recently haunted covid-19 vaccine research, and which might still cause some concerns. this is the question of why there is no significant immunity acquired by the body to prevent recurrence of common cold, of which up to roughly 30% of cases are believed to be due to coronaviruses. fortunately, at time of final writing of this paper, news reports indicate that neutralizing antibodies can be found in patients who have had covid-19. however, with the risks of escape mutations of the virus in mind, it remains worthwhile considering whether the subsequence krsfiedllfnkv, again, found to be well conserved [3, 4] across many coronaviruses [3, 4] , is still present in common cold coronavirus. this is in order to force better immune response by targeting using synthetic or cloned vaccines with this epitope. most common cold strains fall into one of two coronavirus serotypes: oc43-like and 229e-like, which are the main examples discussed below. while the common cold is generally considered as mainly an upper respiratory tract infection and a mundane inconvenience, common human coronaviruses betacoronavirus hcov-oc43 and hcov-hku1, as well as alphacoronavirus hcov-229e, also cause severe lower respiratory tract infections in children and the elderly. some discussion is also given to hcov-hku1 in this paper. the above motif krsfiedllfnkv occurs in the spike glycoprotein [4] responsible for initial binding of previous sars coronaviruses to lung cells and their activation of the spike protein by a proteolytic cleavage [5] [6] [7] . the spike glycoprotein (or just "spike protein") is the familiar spike that studs the surface of the coronavirus, giving it the appearance of a crown to electron microscopy, hence "corona" (latin: crown). after the completion of the first version of the previous paper [3] , a bat virus with 97.41% identity of the amino acid sequence of the spike protein discussed extensively in the present paper, was entered into genbank as entry qhr63300.1. as of the time of final writing this on april 2 nd 2020, there is 100% match of this protein with entry yp_009724390.1 that appears to be a same or similar to the above wuhan isolate. the top hundred nonredundant matching entries found using mn908947.3 by blastp at https://blast.ncbi.nlm.nih.gov/blast.cgi (see below) for mn908947.3 spike protein used here vary from the above 100% match down to 75 .80%, such as aau04646.1, which is a civet isolate. in viruses, proteins of a similar protruding nature, e.g. the hemagglutinin of influenza a, are primary targets for vaccine development, and important targets for development of therapeutic drugs that seek to block the virus from infecting host cells. at the time of the current project, only the three dimensional structures of the sars-cov spike proteins of the earlier sars outbreak was known (e.g. ref [8] ), which has only 75%-81% sequence match to sars-cov-2 [3] . note that it is customary to write sars-cov rather than sars-cov-1. rna viruses mutate with high frequency but so far the differences in spike proteins in emergent sars-cov-2 variants are much less. at the time of the study in late february and early march 2020, the amino acid residue sequences of the spike proteins of covid-19 isolates from different states and countries, such as california, brazil, taiwan, and india, remain identical or almost so. for example, with respect to the original wuhan isolate [2] , phenylalanine (f) is replaced by cysteine (c) as residue 797 in a swedish isolate, and alanine (a) is replaced by valine (v) as residue 990 in an indian isolate. as of 21 st march 2020, largest variants in the sars-cov-2 genome as a whole show 99.9% nucleotide sequence match, which for a genome of 29,858 rna bases, suggests approximately 30 base changes, and of the order of 5 in the spike glycoprotein gene of 3821 nucleotides. that then suggests roughly 1 to 3 amino acid differences in the spike protein of current (march 2020) sars-cov-2 variants, consistent with the above more specific observations of isolates from california etc. a single amino acid change can, of course, sometimes have significant effect, e.g. on the aggressive character of a coronavirus, and so be considered as creating a new strain. some new strains are being reported at the time of writing, but to the author's knowledge none of them are spike protein variations, and more specifically none are as yet in the krsfiedllfnkv subsequence. the left hand side of fig. 1 shows the sars-cov (previous sars) s1 spike glycoprotein within the trimer that makes up the spike. the right hand side shows sars-cov-2, the sars of current concern. all human sars coronaviruses (and indeed the spike proteins of many other related coronaviruses) appear similar in overall conformation, and the variations seen in experimental structures are probably more to do with crystallization or other preparation methods, particularly regarding solvent details and ligands. sars-cov, on the left, has been well studied and still serves as the reference model. in order to fuse with and infect cells, the spike protein needs to be in an open state; presumably the closed state makes it less vulnerable to antibodies. on the left, fig. 1 also shows the approximate positions of the cleavage points superimposed on the protein data bank (pdb) entry 5xlr for sars-cov. reading from the n-terminus of s1, the important functional elements of sars coronaviruses deduced from sars-cov studies [5, 6] and applicable to sars-cov-2 are the s1 nterminal domain (s1-ntd), the s1 c-terminal domain (s1-ctd), the s1/s2 site as the first protease cleavage site as a loop between a pleated sheet and a-helix, the fusion peptide (fp) associated with a highly disordered loop between two a-helices which contains the second cleavage site s2', and a heptad repeat (hr). the arginine (r) in the conserved motif krsfiedllfnkv that was of interest in the previous study [2] is the cleavage point in s2'. recall that the krsfiedllfnkv subsequence associated with s2' is potentially important, not least because it must be exposed or exposable (because it permits proteolytic cleavage) and therefore the site cannot be well shielded. the experimental three dimensional structures of coronavirus spike proteins do not for the most part reveal the large amount of glycosylation that protects most of the spike protein surface. possibly the major problem, however, is not so much in the selection of accessible surface regions as a basis for design entry inhibitor and vaccine design [8, 9] but that the coronavirus readily escapes from such agents by mutation, including in the spike protein [10, 11] . this is the further importance of being a highly conserved motif, i.e. a subsequence that does not readily change from strain to strain except for a conservative sidechain replacement in more remote strains. of course, as one carries the study forward to more distantly related viruses, one expects the motif to differ at some stage, and this is investigated later below. in contrast, nonetheless, the pigag motif "associate with the s1/s2 cleavage site disappears in coronaviruses that are not too distantly related [3, 4] . as noted above, a high degree of conservation of krsfiedllfnkv in the face of genetic indicates that it is in some way important to the virus, presumably for the proteolytic activation cleavage, and/or initial binding to lung cells, but there could be other interactions with other proteins, i.e. to reduce an inflammatory response, as discussed later below. agents. modern computer-driven strategies, and the kind of chemical products that they help produce, differ substantially from the earlier and more familiar approaches in which the computer played little if any role. in large part this is due to the invention of automatable peptide synthesis by merrifield in 1963, who used solid phase peptide synthesis based on crosslinked polystyrene beads [12] . traditional vaccines are purely biological, being composed of dead or attenuated strains of pathogen (meaning mainly, viruses and bacteria). in contrast, a synthetic vaccine is a vaccine consisting mainly of synthetic peptides but also sometimes carbohydrates, often linked to a carrier protein to render it immunogenic. such vaccines produced via chemical synthesis are safer because they do not involve cell-derived material or biological processes for production. their purity can be controlled as in the case of classical drugs. the world's first synthetic vaccine was created in 1982 from diphtheria toxin by louis chedid (scientist) from the pasteur institute and michael sela from the weizmann institute. in 1986, manuel elkin patarroyo created spf66, the first version of a synthetic vaccine for malaria. primarily applications so far have been veterinary. many early vaccines used dead samples of foot and mouth disease virus to inoculate animals, but they caused real outbreaks. scientists discovered that a vaccine could be made using only a single key protein from the virus, and later also found that loops from the surface proteins could be cloned or used in cloned or synthetic constructs. novartis vaccine and diagnostics, among other companies, developed a synthetic approach that very rapidly generates vaccine viruses from amino acid sequence data in order to be able to administer vaccinations early in a pandemic outbreak. traditional vaccines have so far remained the popular choice, but during the h1n1 outbreak in 2009, they only became available in large quantities after the peak of human infections. this was a learning experience for vaccine companies. creating vaccines synthetically would be currently more expensive but has the ability to increase the speed of production and to retune and fine tune the solution to combat new variations in pathogens. this is all especially important in the event of a pandemic. synthetic vaccines are also considered to be safer by researchers than vaccines grown from e.g. eggs or from bacterial cultures (in the latter case there may even be other viruses present). cloned proteins can however reflect the same desirable principles; regions of pathogen amino acid sequence acting as epitopes (see below) can be presented as loops at the surface of a cloned protein. the general idea is that synthetic vaccines are freer of contaminants and focus on the essential features of the required immune response. they can also be developed in a more logical step by step approach. for example, sometimes diagnostics are considered as a useful early step on the way to a vaccine, since they are only required to raise antibodies in animals such as sheep for diagnostic kit production, not to be safe in humans and also raise immune system memory and a cellular as well as antibody response. synthetic vaccines also have the advantage that they can be seen as cartridge vaccines, meaning that they contain bits and pieces that can readily be replaced by others to update the vaccine in order to combat new strains of pathogen. a synthetic vaccine thus has several functional components, looking somewhat like a swiss army knife under the electron microscope. the key component reproduces the essential features of a pathogen protein that the immune system sees. it is an epitope that typically means a patch of some 5 to 20 amino acid residues. reproduced as a short peptide, epitopes can be considered as haptens. haptens are substances with a low molecular weight such as peptides, small proteins and drug molecules that are generally not immunogenic and require the aid of a carrier protein to stimulate a response from the immune system in the form of antibody production. there are two main types of epitope, b and t, discussed in theory section 2. a synthetic vaccine consists of t-epitopes as haptens (for cell response and immune system memory), molecular adjuvant (e.g. muramyl dipeptide), and possibly excitatory or anti-inhibitory peptides. b-epitopes are good for raising antibodies in e.g. sheep to use in diagnostics/biosensors, all attached to, or cloned into, a carrier protein. the latter must be safe but at the same time sufficiently different from any human protein to avoid autoimmune disease. used extensively as a carrier protein in the production of antibodies for research, biotechnology and therapeutic applications, keyhole limpet hemocyanin (klh) is the most widely employed carrier protein, and least for studies using laboratory animals. for humans the food and drug authorities may have other preferences for carrier protein, but klh illustrates the desired features. its large size and numerous epitopes generate a substantial immune response, and abundance of lysine residues for coupling haptens allows a high hapten:carrier protein ratio, increasing the likelihood of generating hapten-specific antibodies. because klh is derived from the limpet, a gastropod, it is phylogenetically distant from mammalian proteins, thus reducing false positives in immunologically-based research techniques in mammalian model organisms, and clinically avoiding autoimmune effects. so far, the food and drug authorities do not seem to have favored synthetic vaccines for human use, but this may be more to do with the peptides themselves than the carrier proteins available. the earlier methods of peptide synthesis did not achieve high levels of purity. however, this has changed and quite elaborate peptides as well as proteins can be made, facilitated by making peptide synthesizers run fast to avoid the slower side reactions, and by methods that join shorter synthetic peptides into longer chains [43] [44] [45] [46] . one of the original motivations for the present study was to capture experience and design strategies from vaccine, diagnostic and antagonist design [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] . methods by the author and colleagues ranged from the expert system approach to automated bioinformatics and protein modeling [26] [27] [28] and automated drug design (e.g. refs [29] [30] [31] [32] ). see also ref [33] . more recently there has been an automated approach based on the proposed q-uel language [34] [35] [36] [37] . the more fine-grained principles for the design of synthetic peptide vaccines, and antagonist peptides made of d-amino acids, are discussed in some detail in the previous paper [3] . a variety of bioinformatics techniques are available to help in development of these solutions (e.g. ref [38] [39] [40] [41] [42] ), as well as computational (e.g. refs [33] ) and synthetic techniques (e.g. ref [43] ). the q-uel language [33] [34] [35] [36] [37] used in the preceding work [3] is also a means of gathering relevant information from the world wide web efficiently when encountering a new problem such as an epidemic caused by a new virus, or at least a problem new to the researcher [3, 4, 38] . it also enables more automated interaction with websites for publically available bioinformatics tools. the motivation for this was all the stronger because the popular highly integrated approach to bioinformatics' called the biology workbench at the university of san diego supercomputer center has been no longer available for some time [39] . however, the standard bioinformatics tools (e.g. refs [40] [41] [42] ) used in the present study can of course be used readily by researchers reasonably experienced in bioinformatics. although peptidomimetics (containing amino acids that would not occur in normal ribosome-based biosynthesis) have been considered by authors as a basis for haptens in synthetic vaccines, they are in the author's opinion probably best considered as potential therapeutic antagonists. in the present study, the specific aims include design of molecules to impede binding and activation of the spike glycoprotein at the surface of lung cells [5] [6] [7] . synthetic peptides copied from subsequences in the spike protein could be used directly for such clinical purposes, but then an important design step would be to render them resistant to biodegradation by human proteases. this is typically by inclusion of d-amino acid residues [44] [45] [46] [47] . previously, in the author's personal opinion, peptides and peptidiomimetics have been currently best considered as first steps in the research and development of small organic "in a pill molecules" of the traditional kind favored the by the pharmaceutical industry. their role there nonetheless is a powerful one, linking amino acid sequences seen in nature, conveniently already "designed" by millions of years of evolution, to (typically) smaller novel organic molecules designed to have van-der-waal's and electrostatic features in the binding site. however, the author's reticence has been largely based on cost, including cost in changing traditional production strategy, and in the reservations of food and drug authorities, but fairly recently all that appears to be changing. thpdb (http://crdd.osdd.net/raghava/thpdb/) includes an example of a manually curated repository of peptides and related molecules approved by the us food and drug administration (fda). over some 70 peptide drugs are approved in the us and other major markets, and those in pipelines and in or approaching clinical trials may now be exceeding 200 entries. as natural compounds, peptide drugs are typically less toxic than more traditional chemical candidates. although d-amino acids are not natural features of ribosomal production of peptides and proteins, human metabolism can handle them. they occur in gut microbes and ingested material and in human proteins they form spontaneously in a kind of aging process from some amino acids in situ in protein sequences (e.g. l to d-aspartate). diverse d-amino acids such as d-serine, d-aspartate, d-alanine, and d-cysteine are found as free amino acids and small peptides as well as in some proteins, and quite commonly in mammals. they are often found having playing important roles in the nervous system. for example, n-methyl-daspartate (nmda) receptors are associated with learning and memory and d-serine, daspartate, and d-alanine bind to those receptors. hydrogen sulfide generated from dcysteine reduces disulfide bonds in receptors and potentiates their activity. peptides made of d-amino acids resist not only normal proteolytic degradation but also resist an immune response (unless attached to a carry protein) [44, 46] . they persist some 4-6 days in the body, which is an ideal time period for pharmaceutical action, and are ultimately degraded to safe products (probably mainly in the peroxisomes and by enzymes in the kidneys) [44, 46] . the negative aspect is that they do have higher entropy to overcome than many drugs of more traditional form, but in practice this appears to be more a barrier to computer simulation of binding than to the real molecule, as extensively discussed below. studying the binding of synthetic peptides or small organic molecules to human proteins benefits from computer simulations of the solute-solvent system, and it was early found that these should ideally include water molecules in a detailed way because there are hydrogen bonding options between water molecules and amino acid residues which are not particularly intuitive [48, 49] . in most cases, the spatial locations of hydrogen atoms are deduced rather than seen in experimental protein and peptide three dimensional structures. this is likely to impact considerations of docking ligands to protein targets. in the present author's opinion, this provides a beneficial possibility for retroinverso designs [3] made by reversing the sequence and using d-amino acids that has the unfortunate or complicating effect of interchanging the c=o and n-h groups in the backbone of the synthetic peptide [3] . the beneficial possibility is that, for example, a repulsive c=o…o=c electrostatic interaction between a synthetic peptide and the spike protein could be ameliorated in the manner c=o…h…o=c where the h is a water, serine or threonine hydrogen atom, or by c=o…h-o-h…o=c, albeit that in practice the water molecule likely lies more to the side of the o..o interaction vector. somewhat similar considerations apply to any n-h…h-n interactions that can ameliorated by the lone pair orbitals of an oxygen atom. both could also involve tautomerization and/or rearrangement of internal hydrogen bonding networks (e.g. in the manner …o-h…o-h…. to …h-o..h-o…). today, to take care of such matters, researchers consider docking of ligand to protein and high grade molecular dynamics simulations of the overall solute-solvent system by molecular dynamics, at least as the final refinement step [50] , but even the awareness that the above compensations and others can take place can make it worthwhile to synthesize and test a proposal. somewhat similarly, design of peptide synthetic vaccines and diagnostics can make direct use of peptides duplicating sequence motifs in the pathogen protein found by bioinformatics and relatively simple computational tools. after that, researchers often go straight to synthesis and experimental immunological testing of the constructs rather than using complex simulations [51] [52] [53] . epitope predictions for sars-cov-2 (simply meaning the choice of amino acid residue subsequences to synthesize for synthetic vaccines, but also for peptidomimetic antagonists) have been made by several authors (e.g. ref [54] ). they have typically made use of extensive historical experimental data about the amino acid residue sequences of epitopes such as the epitope database and analysis resource (iedb) and the virus pathogen resource (vipr) (e.g. ref [54] ). the immune system by its nature can make its own adjustments to recognize pathogens and vaccines, but designing some kind of therapeutic antagonist against virus binding to the lung cells requires rather more consideration about what human protein the spike protein is binding. bioinformatics as the study of biosequences is a powerful tool, but it is well known that having the detailed three dimensional structure of the human protein target for a potential new pharmaceutical agent, or to which a virus attaches, is a great benefit to rational computer-aided design. studies specifically investigating human protein binding and activation of previously known sars viruses have for some years been carried out by several groups (e.g. [54] [55] [56] [57] ). it seems reasonably well agreed that angiotensin converting enzyme type 2 (ace2) is responsible for binding the sars associated with the 2002 outbreak, combined with a proteolytic cleavage to activate the spike protein, for which type ii transmembrane serine protease (tmprss2) is the current popular candidate [3] . several three dimensional structures are known for ace2 complexed with sars spike protein e.g. protein data bank (pdb) entry (6acg) and of variants of the latter (e.g. tmprss2 protein data bank entry 2oq5). however, the full story involving human cell surface proteins (with which sars-cov-2 interacts in order to infect and replicate) is possibly not quite as firmly established at the time of this present study as some summaries would suggest. the origin of the general problem for a more detailed conformational chemistry approach is that diversity of genome and means of infecting cells are readily generated in nature in the case of different virus hosts, virus strains, and species jumps, and it is long established that the binding shows variation in the receptors used that correspond to viral groups. there have been alternative proposed candidates for initial binding receptors, e.g. carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1), and various dipeptidyl peptidases. highly virulent coronaviruses that form syncytia between cells can even spread in a receptor-independent fashion. even when an initial binding receptor such as ace2 is identified for a coronavirus, initial uncertainty or enduring complexity for the rest of the entry process may be the norm. many other human proteases present in the lung seem capable of cleaving various sites on the spike protein and which could cause its activation. for example, a variety of proteases such as trypsin, tryptase clara, mini-plasmin, human airway trypsin-like protease (hat), and tmprss2 (transmembrane protease, serine 2) are known to cleave the glycoprotein hemagglutinin (ha) of influenza a viruses as prerequisite for the fusion between viral and host cell membranes and viral cell entry. human airway trypsin like protease (hat), tmprss3, tmprss4, tmprss6 have also all been considered by sars researchers at various stages. other human proteins that might have similar involvement to the above in the sars-cov-2 case, and that are also affected by the same antagonists against the sars-cov-2 targets in the preceding paragraph, have also attracted the attention of researchers. the trypsin-like serine protease hepsin which has a fairly broad action and which is significantly inhibited by a diverse set of ligands, a particular example of one such binding is represented by protein data bank entry 5ce1. even intracellular proteases could be released on cell damage resulting from the first wave of lung infection or from other disease or tissue trauma. some variants and strains may use other, as yet unknown, proteins, or sugars, to assist entry. it is also plausible the spike protein might be activated by other proteases on exit from the epithelial lung cells, so allowing it efficiently to infect other cells. the spike glycoprotein of sars-cov-2 also has the so-called furin cleavage sequence (prrars or prrars), which is an extension to the so-called pigag motif of ref [3] . consistent with the present author's preferred choice of krsfiedllfnkv motif, coronaviruses with high sequence homology (such as that isolated from a bat in yunnan in 2013), lack the furin cleavage sequence. nonetheless, because furin proteases are abundant in the respiratory tract, sars-cov-2 spike glycoprotein might be cleaved on exit from cells. even if the means of binding, activation and entry is well established for a viral strain, recall that a single rna base difference resulting in a single amino acid residue difference could alter all that, and there also appear to be several other possibilities that the virus can exploit in parallel. indeed, somewhat similarly, potential inhibitors of sars entry and/or activation proposed by researchers (e.g. refs [55] [56] [57] [58] [59] [60] [61] ) may work by several routes in parallel, and significantly at least three mechanisms were reported in one relevant study [61] . once a target protein and its relevant binding site are clearly understood, methods are available for screening available ligands (binding molecules) to bind to those sites as potential antagonists, or even for "growing" or evolving antagonist molecules in those sites, whether smaller organic molecules [29] [30] [31] or peptides [32] . pharmaceutical chemists have long used evidence and hunches to deduce a pharmacophore, i.e. an abstract description of recurrent molecular features that are necessary for molecular recognition of the ligand by the protein [3] . a pharmacophore ultimately implies at least a schematic model of the interfacial surface between ligand and protein, but in practice, a pharmacophore tends to be either considered from the perception of the ligand (one compares similar inhibitors etc.) or from the perception of the binding site (one compares positions of key residues in the binding site). the choice depends on the quality of each kind of data, but could involve both. historically, drug design was frequently based only on indirect deduction of binding site features using the chemical features of the ligands which successfully inhibit (or in a few instances excite) a response. this is essentially the use of quantitative structure activity relationships (qsar). in effect the perception of the binding site was indirect and typically based on the chemist's expertise and hunches, and so often extremely "fuzzy". subsequent elucidation of many protein structures with clear pictures of their binding sites led to a crisper physical perspective, exemplified by a study [50] that included many ligand molecules in the present investigation, and so faced some similar issues. in the approach which may now be considered traditional, docking calculations are fast, using grid maps that consist of a three dimensional lattice of regularly spaced points, centered on some region of interest of the protein target under study. as discussed above, ace2 and tmprss2 are very likely correct targets, but again they are not necessarily the only targets even for cell entry of current sars-cov-2, and the mechanisms used by each new coronavirus strain can differ, as the result of even a single amino acid residue change. in such circumstances, the conservation of the krsfiedllfnkv motif might be considered suspicious. the activation cleavage is at the arginine (r) and workers tend to conclude that this site is more essential for action than s1/s2, and mutation of the arginine (r) specifically inhibits trypsindependent fusion in both cell-cell fusion and laboratory assays. but also, with the arginine retained, many other proteases can active the spike protein as above, and others can do so in laboratory conditions. because of the conservation, one might therefore hold the seemingly reasonable hypothesis that this site is not also susceptible to cleavage and activation by other extracellular proteolytic enzymes, but also doing something else. whether or not this is so, all this complexity makes detailed interaction models of spike protein binding and activation difficult, and while the "best bet" for ranking the choices of target protein may currently seem obvious, making a reasonable, currently conventional, choice which is actually an incorrect assumption can delay productive research into therapeutic agents. in the case of the hunt for prevention and cure of virus diseases, and particularly covid-19, there seems to be increased justification for a "fuzzier" set-theoretic picture of a pharmacophore as an ensemble of different binding sites, or of ligands in a ligand-oriented perspective, as follows. many of these, and perhaps all, suggest that even if one is using an incorrect picture of the mechanisms of entry and replication, even using the "wrong" or less important protein target, one might achieve some success. in brief summary, the justifications for the ensemble pharmacophore in the coronavirus case, i.e. the contributions to "fuzziness", include parsimony, that proteins and parts of proteins sometimes have more than one function [12] encouraged by limited numbers of accessible sites (due to e.g. glycosylation) and exemplified by parallel alternative mechanisms of cell entry, multiple methods of drug action, escape from scientific defense measures by virus mutation, polymorphism of human proteins involved, different expression levels of human proteins involved, and the potential problem of the "specter of vaccine development" (concerns about missing the appropriate region of the virus that allows common cold viruses to escape the appropriate immune response). to the above may be of course added the fact that even if an experimental researcher is convinced of the value a specific protein as appropriate target, the picture for the computational chemist is a fuzzy one. the system itself, real and simulated, is to be seen as a statistical mechanical ensemble of multiple states, sampled over the population of molecules and across their conformational behavior in time. not least, protein binding sites are often partially disordered before binding, and in any case there may be several binding modes. picking the right one can be difficult because there is a fine balance between solvent and conformational entropy, and entropy is notoriously hard to compute [12] . given this argued uncertainty as to the nature of the target protein and its binding site, a broader initial net as an ensemble pharmacophore can help. docking approaches are continually being improved by researchers, and recently include ways of combining features that could ultimately relate to different protein binding sites. while many authors of these studies include the word "ensemble" in their discussion of pharmacophores, they appeared to be significantly different to the particular means of combining multiple pharmacophores that was explored here. however, the present author has had his attention drawn to some that are rather similar and the approach of kumar [33] appears to particularly akin, especially in regard to distributions of expected values and use of weighting. kumar's description [33] thus suffices, and briefly stated, it explores the ability of an ensemble of selected protein-ligand complexes to populate pharmacophore space in the ligand binding site, assesses the importance of pharmacophore features using poisson statistical and information-theoretic entropy calculations, and generates the pharmacophore models with high probabilities. a scoring function then combines all the resultant high-scoring pharmacophore models. there is one significant operational difference between kumar's approach and that used here. recall that in the more traditional docking approach, it is the ligand as candidate drug that is typically seen as the variable and constantly changing and in many studies "evolving" the ligand chemistry with the pharmacophore is the basis of drug design [29] [30] [31] [32] . ref [50] , related to the present study, has aspects of that applied in a different way. kumar's approach can, however, combine the perspective of both pharmacophore and ligand as conceptual variables. despite that, the present author's approach, as used in the present overall project, considers one candidate ligand at a time. this seems less efficient from the point of view of designing candidates, and not even as smart as the older single, non-probabilistic pharmacophore approaches [29] [30] [31] [32] . nonetheless, a single, simpler one-ligand-at-a-time strategy is both adequate and appropriate in the present case. this is because there is already a data collection of candidate antagonists to build on [50] , as discussed in section 1.7. approaches of the ensemble pharmacophore kind are currently highly recommended for investigation of sars-cov-2 and for the spike protein in particular, again because of some uncertainties and the likely multiple functions of some spike protein features. however, it has not as yet had significant impact in the present study. the approach actually taken remains consistent in the sense that inclusion of one particular source for a pharmacophore, an enzyme considered by the author, was evidently going to dominate the ensemble because of certain similarities in the antagonists of sars virus entry and inhibitors of the enzyme [3] , given the knowledge available at this time. that choice is not, however, obvious, as follows. pharmacophore. what may be more controversial is the case when there is a representative choice and it is a protein that is not obviously relevant to the target protein, or simply not "on the radar" of coronavirus researchers. what makes it a candidate is not necessarily that it is relevant to viral infection and not necessarily that it has an evolutionary relationship to proteins that are considered relevant, although this is a question addressed briefly in this paper. rather, it may simply be based on the pragmatic notion that there may be ligands, potential binding molecules as antagonists, which are common to both more popular choices human target proteins and a less obvious candidate. the small organic molecule emodin has been found to inhibit sars coronavirus entry [59, 60] , as also so have other compounds some of which have emodin-like features [3, 61] . similar molecules, and importantly emodin itself, are also inhibitors of 11β-hydroxysteroid dehydrogenase type 1, an example of a steroid binding enzyme [62] . it is normally anchored within the endoplasmic reticulum through an nterminal transmembrane domain. its involvement as a protein target is here based on a chemical justification. a biological justification might be that this enzyme is involved in the inflammation response which a coronavirus might also benefit by inhibiting. if so, the goal is not, of course, to help the virus by inhibiting at the same target which it would also gain by inhibiting, but rather to inhibit protein targets more crucial to it, i.e. for cell entry and possibly replication which are even more crucial to the virus. some inhibition of 11β-hydroxysteroid dehydrogenase type 1 might even be a desirable thing because excessive or prolonged inflammation (including in response to pathogens) is well known to be potentially damaging to the host. an excessive inappropriate immune response may also include the basis of allergic reactions and even of autoimmune diseases. a pragmatic reason for this choice of protein as pharmacophore is that was also one of those protein-ligand interaction systems that have been well studied by the present author and collaborators [50] . such studies pursued the idea of using a more rigid molecular framework, including the steroid framework and fragments of it, as a more rigid scaffold for active drug groups [50] . importantly, that study and subsequent work has already established data base of compounds that bind to 11β-hydroxysteroid dehydrogenase type 1, and it includes many molecules including some discussed in this paper that again have some of the features of emodin. it also includes many weak binders that could also be much stronger binders at what turns out to be a more obviously relevant protein target. these issues can be addressed quickly in the laboratory and certainly seem worthy of investigation before addressing the more popular targets. there was a further implication in the previous paper [3] , though not a requirement for its main arguments, that the peptides designed on the basis of the krsfiedllfnkv motif bind the same krsfiedllfnkv site as do emodin-like molecules. that seems currently to be an even less reliable assumption than the assumption that the above steroid dehydrogenase enzyme is relevant to coronavirus biology, and it is not of course an assumption that even matters if either a peptidomimetic and/or small organic molecule is found effective. however, again keeping in mind that there are a limited number of accessible, conserved sites in the spike protein, and that these may be involved in multiple mechanisms as discussed above, common targets for action of both peptides and smaller organics like emodin seems plausible. partially the problem is extensive glycosylation. it is well known that glycosylation plays an important role in receptor-ligand recognition but also have structural influence in receptor-ligand recognition because of its bulky shape caused by branched side chains. for that and other reasons it may be that the krsfiedllfnkv site is, with just a little variation, almost the only site on the spike protein that is persistently recognizable in coronavirus strains, and so presumably carrying out an important function and accessible, as also discussed in this paper. angiotensin converting enzyme 2 (ace2) binding is however also considered in this paper. a number of ideas and principles, borrowed in established and recent design of synthetic vaccines and petidomimetics, were used (see ref [3] for discussion and e.g. refs [63] [64] [65] [66] [67] [68] [69] ), as well as some of the ideas that lie behind the popular zinc data base [70] . as discussed in refs [3, 4] , the present investigation started as a use case for the hyperbolic dirac net (hdn) and particularly the associated q-uel language for automated inference [34] [35] [36] [37] . the theory has been discussed elsewhere, e.g. in refs [34] [35] [36] [37] , which relate more to the practical and general uses of q-uel. these considerations are less important here because present studies can be reproduced by standard bioinformatics and molecular modeling means. nonetheless, it is doubtful that the research for refs [3, 4] could have been done and written up so rapidly without the aid of q-uel to interact with websites of the world wide web, gather knowledge, and facilitate use of the publically available bioinformatics tools [3] . the challenge is ultimately one of molecular recognition but in practice many key principles for hapten design relate to distinguishing types of naturally occurring epitope. by the term "epitope" in this paper is meant "continuous epitope", though several smaller epitopes may be joined to represent a discontinuous epitope in which conformation and relative position in space can sometimes be important. while a synthetic construct implies the use of synthetic chemistry typically combined with a judicious carrier protein to which the peptide is linked chemically, constructs can also be obtained by cloning, using protein engineering principles [12] . the terms b-epitope and t-epitope relate to the traditional picture of a bone marrow b or thymus t response. b cell epitopes occur at the surface of the protein against which an immune system response is required. they are recognized by b cell receptors or antibodies in their native structure, and are concerned with the bone marrow response and antibody production. t epitopes may be buried inside protein structures and released by proteolysis, and are traditionally considered as concerned with a cellular response and immune system memory, i.e. active immunity. continuous b cell epitope prediction is very similar to t cell epitope prediction. the focus is on b-epitopes here, though a bepitope can also be (or overlap with) a t-epitope especially if it has a significant content of hydrophobic residues. prediction of these has traditionally been based has mainly been based on the amino acid properties such as hydrophilicity, charge, exposed surface area and secondary structure. there are many predictive algorithms available, but the present author prefers a more "expert system" kind of approach that incudes experimental data, though the above biophysical considerations certainly still play a strong role (see below). infection. the previous paper [3] focused primarily on design of synthetic peptides as infection antagonists. however, partly for the reason of greater conformational flexibility discussed below, smaller less flexible organic molecules (i.e. with fewer rotatable bonds) are the traditional province of the synthetic chemist rather than use of an automated peptide synthesizer, are preferred for pharmaceutical application. consideration of peptides is more often considered as merely a useful intermediate step in more traditional pharmaceutical compound design. biodegradability per se of peptides is not the main concern, since including d-amino acids in the design prevents proteolysis. in preliminary docking and simulation studies, the peptides do bind to 11βhydroxysteroid dehydrogenase type 1, but less strongly and with several binding modes [3] . this weaker binding is not in itself a contraindication of the idea that these peptides bind at the same site as the more rigid non-peptide molecules, because it is an expected consequence of the much greater flexibility of peptides compared with molecules with, for example, multiple aromatic ring scaffolds. conventional wisdom (e.g. ref [12] ) frequently uses the rule-of-thumb that the total change in intramolecular (bond rotational) entropy of a peptide ligand is roughly t∆s total = 1.5 kcal⋅mol −1 per residue at 300 k, corresponding approximately to a 12-fold reduction in conformational freedom per residue on binding. because van der wall's and hydrogen bonding tend to be very roughly equivalent for peptides in water and in well bound forms, the water entropy effects known as hydrophobic effects (along with electrostatic forces) play an important role in determining the balance of energies and final outcome. krsfiedllfnkv would thus cost about +19.5 kcal/mole entropic contribution to bind rigidly, primarily compensated by hydrophobic contacts at up to about -1.7 kcal/mole in going from an aqueous to a non-polar environment, i.e. -22.1 kcal/mole for a 13 residue peptide or analogue of krsfiedllfnkv. that example would not favor binding, but the proper calculation is in the details which of should show balance that favors good binding if that is found to be the case experimentally. despite the above comments, the flexibility of peptides does provide more opportunities to fit a specific binding site, i.e. they can show some accommodation and they are more tolerant to imperfections in the design process. however, this is also an argument for their importance as an intermediate step in the design of more conventional pharmaceutical agents. the main methods are essentially standard bioinformatics approaches as used in refs [3, 4] . some methods, e.g. rules for epitope prediction, are best discussed in context in results section 4. the q-uel methods specifically for bioinformatics are discussed in [38] , and those for computational chemistry and docking of compounds are those using krunch as described in ref [3] and the appendix to ref [50] . they are somewhat unorthodox by focusing on heuristics to handle the multiple energy minimum problem, but the end effect is probably similar to that of long runs using high grade molecular dynamics calculations, given opportunities for calibration [50] . epitope predictions lie in more traditional "one dimensional" bioinformatics, and in this paper and the previous paper depended on predictions using a gor4 secondary structure prediction of α-helix (h), extended chain or β-sheet (e), and coil or loop (c). the reason for this and the particular use of gor4 is discussed in ref [3] , but briefly, it is in part because sections predicted by runs of c tend to be immunogenic even if they are incorrect as structure predictions [3] . however, charged residues in α-helices and βsheets are believed to be occasionally b-epitopes, and short sections extended chain can effectively imply loops. the core and initial rules for b-epitope prediction used in the present study consider (i) surface exposure when a three dimension structure is known, but allowing for conformational adjustment to expose residue when in a likely disordered or flexible loop, scores +2, (ii) known exposure based on other kind experiment, which also recognizes the possibility that a partially buried site by the above criteria can be brought to the surface on binding, notably for proteolytic cleavage [3] , (iii) runs of amino acid from the set [stnqy] score +1, from the set [dekhr] they score +2, and from the set [livfcm] they score -1, (iv) runs of secondary structure prediction as coil or loop c, though runs of three or less e and the first and last three of helix h can be considered as c for this purpose, score +1, and (v) the motif nx(s/t)x of asparagine (n) serine (s) or threonine (t), where x means "not a proline" (p) scores +2. however, this will not permit a corresponding peptidomimetic or vaccine without considering glycopeptide synthesis technology. see discussion below, which would justify a negative score, depending on the technology available. in addition, these may be combined with predictions based on significant homology with proven epitopes in data bases, which has already been done by several groups for sars-cov-19 (e.g. ref [54] ). for sources of data concerning covid-19 virus spike proteins, genbank and the protein data bank were the main sources. there was some use of in-house collections of data, e.g. of typical b-epitopes and t-epitopes, although publically available collections would probably serve the same function. there was also use also of a data base of non-peptide ligand molecules of potential interest already generated during and since the work described in ref [50] that was used where appropriate. many of these molecules (including emodin) are also found on the public zinc data base [70] as indicated in results section 4 below, but several, including derivatives of carbenoxelone, are not, and these derivatives are of interest as potential coronavirus antagonists. to look up an entry on the zinc data base by the codes used in this and other papers, one can go to http://zinc15.docking.org/ substances/ and enter zinc00011032. in an automated approach such as that favored by q-uel, a variable (such as a perl variable $mol) to zinc00011032 and is set an the q-uel application goes to http://zinc15.docking.org/substances/search/?q=$mol. any references to experimental binding results concern data from cited papers, and see for example ref [69] for typical methods used for natural herbal compounds. as discussed in ref [3] , q-uel helped gather these in the form of q-uel knowledge representation tags, so they become part of the growing knowledge representation store. in regard to peptides and proteins, table 1 used in ref [3] shows the standard iupac one-letter codes used for amino acid residues in sequences throughout this paper. table 1 . one letter amino acid codes used in the text. conservative replacements are those common substitutions from a peptide design perspective, but for example phenylalanine (f), isoleucine (i), and alanine (a) are seen as natural substitutions that appear in discussion of spike protein sequence motifs later below. these amino acid residues have hydrophobic sidechains but they are not conservative replacements but rather substantially different size. a reasonable explanation is of course that sidechain size conservation matters less when the sidechains are at exposed at the surface of the protein. similar notions underlie the idea that what can readily replace what is not always an equal probability in each direction. in that respect, table 1 tends to reflect the changes that are used in the present project for design, when starting from epitopes. the previous paper [3] should not give the impression that the specific motifs discussed (and particularly krsfiedllfnkv) are the only sections likely of the sars-cov-2 spike protein to be of interest in the above respect. the preference for one choice was based on (a) conservation across many strains, suggesting that the site has an important function and is likely at the spike surface, and (b) avoiding the shielding of the spike protein by extensive glycosylation. the dramatic effect of relaxing these restrictions is a major point of this section, in which a large number of candidates are found. over-prediction is not necessarily a bad thing, because once a laboratory has a peptide synthesizer and other tools for constructing and testing designs, it is relatively easy and cheap to test and reject ideas, and more problematic to miss opportunities. the intention here is also to cover most possibilities, to enable index numbers to be assigned to them according to their order in the sequence (putative epitope 1 etc). consequently, in future one may then readily refer to the index number, or speak of a new proposal or experimental epitope extending, overlapping, or even lying between two of these epitopes. they are primarily to be seen as b-epitope predictions, though they are favored if some t-epitopic character is also expected. an initial step is based on adding up weights as described in methods section 3.2. in practice, there was also some judicious use of expertise and an epitope data base in an attempt to refine assignments. recall that the trimeric sars coronavirus (sars-cov) spike glycoprotein consists of three s1-s2 heterodimers. some of these will be shielded by that configuration during most of the life cycle of the virus, but not necessarily in every s protein monomer, and also shielded by glycosylation. the higher scoring predicted epitopes in the sequence below are underlined and in bold, and are primarily to be considered as b-epitopes but with some extension to include t-epitope character where possible. also included in these predictions are those using the immune epitope database and analysis resource (iedb) and the virus pathogen resource (vipr) which have already been made [54] (see later below). these are shown in underlined, bold, and in italics in the following, and since some are contiguous sections that look like a single long representation in the following, they are also stated separately below. it is apparent that while focus was on just krsfiedllfnkv, if strain variation and glycosylation are ignored then much of the spike protein sequence contains epitope candidates. 10 20 many of the epitopes predicted in the present study overlap with prediction made using the immune epitope database and analysis resource (iedb) and the virus pathogen resource (vipr) [54] , and these comprised the following. recall that one of the reasons for the original single preferred candidate krsfiedllfnkv was that many of predicted epitopes contain evidence of glycosylation, reflecting the last of the "rules" (v) in methods section 3.1 above. that rule has, however, a special status, and the present author has tended to consider them undesirable for synthetic vaccine or diagnostic development. it indicates likely glycosylation of the protein. the bulky oligosaccharides so attached can be immunogenic, but they are rather difficult to work with synthetically, traditionally expected to make bulk production expensive, and may be variable in structure which cannot typically be seen in detail in experimental three dimensional protein structures (typically as obtained by x-ray crystallography or high grade electron microscopy). antibodies that are raised against the glycosylated surface patch of the protein or corresponding synthetic glycopeptides may be specific for their carbohydrate units. these can be recognized irrespective of the peptides, or in the context of the adjacent amino acid residues. conformation and exposure of b-peptide epitopes of glycoproteins may be modulated by glycosylation because of intramolecular carbohydrate-protein interactions. the beneficial versus undesirable effects of glycosylation in synthetic vaccines is also a complex matter. glycosylation may be essential for reactivity with the antibody, but conversely it may in effect inactivate the capabilities of a section of amino acid sequence to function as a b-epitope, which seems to be a very good reason for giving the glycosylation motif a strong negative rather than positive score. unfortunately this will depend on the structure of the antigenic site and antibody fine specificity, and the recognition mechanisms involved are not fully clear. there is a (usually) positive aspect, however, in the current view that similar effects of glycosylation apply to t-celldependent cellular immune and igg antibody responses, and that glycosylated peptides can elicit glycopeptide-specific t cell clones after being bound and presented by mhc class i or ii molecules. it is of course only a positive aspect if the intended effect is obtained by the synthetic construct. the overall spike glycoprotein protein sequence shown above changes across the coronaviruses, but the krsfiedllfnkv subsequence is most notable amongst the exceptions. it extends to the common cold coronaviruses with minor variation, and may imply a better targeted approach to stimulate immunity. for common colds caused by the rhinovirus, recent research suggests misdirection of antibody responses against a non-protective epitope as a mechanism how the virus escapes immunity and so permits recurrent infections [71] . a clearer understanding of conserved subsequences in coronaviruses may also help tune the action of toll-like receptors to initiate the appropriate response. these are a class of proteins that play a key role in the innate immune system. they are single-pass membrane-spanning receptors usually expressed on sentinel cells (e.g. macrophages and dendritic cells) that recognize structurally conserved molecular features of pathogens [72] . despite concerns about two or more strains of covid-19 virus appearing, these are not big changes for present purposes. it is sufficient to consider the sequence of the original wuhan isolate as reference in comparisons for present purposes, i.e. for comparing the spike protein sequences of other coronaviruses. recall that as discussed in introduction section 1.3, at the time of the study in late february and early march 2020, the sequences of the spike proteins of covid-19 isolates from different states and countries, such as california, brazil, taiwan, and india, remain identical or almost so. for example, with respect to the original wuhan isolate [2] , phenylalanine (f) is replaced by cysteine (c) as residue 797 in a swedish isolate, and alanine (a) is replaced by valine (v) as residue 990 in an indian isolate. neither of these relate to the sequence motif krsfiedllfnkv of particular interest here. in the initial studies [3, 4] , the genome of the common cold coronavirus, and particularly the sequence of the spike protein, was considered sufficiently far from that of the covid-19 virus so as to be less relevant to that problem. while looking at differing sequences is essential for detection of conserved motifs, very different and less relevant pathogens are unlikely to preserve them, except perhaps as pattern matches involving quite complex substitution rules. however, the appearance of the covid-19 krsfiedllfnkv motif does appear in common cold coronaviruses and with typically at most two relatively conservative substitutions. that is in the sense of preserving hydrophobic sidechain as discussed above in methods section 3. the conservative aspartate (d) and asparagine (n) replacement is also fairly common in the motif in the sequences examined. an example shown below is a clustal omega alignment of the covid-19 virus spike protein original wuhan seafood market isolate (genbank entry mn908947.3) with spike proteins representatives members of the two major common cold coronaviruses strains 229e and oc43 (genbank entries np_073551.1 and aiv41987.1). despite radical sequence differences for the spike protein sequences overall (only 12.8% identity, well within the range for a random match), the underlined sequence motif krsfiedllfnkv of covid-19 virus is essentially retained as that sequence, except that alanine (a) replaces phenylalanine (f) in the common cold coronavirus (which is moderately conservative at the surface of a protein) and a conservative leucine for valine substation in one case. in the sequence (not shown) of hcov-hku1 which is often associated with more serious cases of cold-like diseases the above motif is still noticeable as rsffedllfdkv in which the isoleucine (i) is replaced by phenylalanine (f). the "a for f" modified motif rsaiedllfdkv is also found in the coronaviruses of dogs, cats, rodents, pigs, rabbits, camels, ferret badgers, raccoon dogs, amongst others. all of these might be eaten by humans in certain countries and notably they are, for the most part, species that live in close proximity to humans. the "pigag" motif does not show up in the above alignment, as is also the case in many other distantly related coronaviruses [3, 4] . however there is a subsequence pigtnyrscestt in the hcov-hku1 spike protein that appears to relate to pigagicasyqtq in the covid-19 virus (recall that hcov-hku1 is a common cold virus, albeit usually associated with more severe, lower respiratory tract cases). in contrast, not only does the krsfiedllfnkv motif stand out as potentially important to the covid-9 virus by virtue of such comparisons, but also a match with that motif is almost the only continuous stretch of amino acid residues in most alignments like that above. the subsequence kwpwyiwl is an exception that is of interest and a characteristic feature of many sars coronaviruses. it is not, however, considered further in the present paper, except to note that it does not appear to be associated with a covid-19 virus spike protein proteolytic cleavage site. these sites are most prominently trypsin: s1/s2 htvsllrstsqksivaytmsl, s2' lpdplkptkrsfiedllfnkv; cathepsin: s1/s2 htvsllrstsqksivaytmsl; elastase: s2' lpdplkptkrsfiedllfnkv, plasmin: s1/s2 htvsllrstsqksivaytmsl, s2' lpdplkptkrsfiedllfnkv, tmprss1: s1/s2 htvsllrstsqksivaytmsl; tmprss2: multiple sites; tmprss11a: s1/s2 htvsllrstsqksivaytmsl, s2' lpdplkptkrsfiedllfnkv. as one looks out to more distant relatives, there are a number of variations in the krsfiedllfnkv motif which, despite large variations in spike protein sequence as a whole, are still recognizable in the spike proteins of coronaviruses of diverse various host species, as shown for some examples in table 2 . the most noticeable variation is the occasional substitution of the cleavage point arginine (r) by a g. rather than disrupt the possibility of cleavage, however, it is seemingly displacing that role to a arginine (r) or lysine (k) that lies to the n-terminal (left) side of the motif. it is interesting that this commonly retains firmly the iedllf core of the motif. the notion that the krsfiedllfnkv motif overall plays an important role, and presumably a common or similar function across at least a very large number of known coronaviruses, still seems a reasonable one. most important of course is that it is at least the case for the sars-cov-19 virus and its near relatives. at this time, no match with a coronavirus in genebank has been detected by the author by blast-p using queries with no phenylalanine (f), e.g. rsaiedllldkv, rsaiedllidkv, rsaiedlladkv, rsaiedllmdkv, rsaiedllwdkv, and rsaiedllydkv as queries, but the search has not been exhaustive because it would not be too contradictory to any of the current hypotheses if some were found. in the group with the inserted glycine (g) replacement of initial argine (r) by the similar positively charged lysine (k) is common. however, as long as the motif is significantly recognizable, no histidine (h) as opposed to initial arginine (r) has been found. the motif cannot extend to other strains indefinitely as recognizable because at some point the evolutionary tree will bring up virus and hosts subject to quite different selective pressures, and the motif is not the definition of coronaviruses. however, it still persists as recognizable in birds such as duck (e.g genbank kx266757, kc119407 white-eye bird cov hku (nc016991), magpie-robin (shama) cov hku18 (nc016993)) strains, a selction which spans a large range of coronavirus genome sizes. see the alignments below compared with the wuhan seafood market isolate genbank mn908947.3, showing the motif underlined and in bold. :.: .: : : eeyihklnatlvdldwlnrvetyikwpwwvwllitlaivafvvilvtiflctgccggcfg 1188 : . ** ::::*: *. : *:****::** : .: : : : *.**. some indication of the limit of the survival of the motif rsfiedllfnkv as the researcher departs from sars-cov-19 might be given by the nidoviruses other than coronaviruses. somewhat coronavirus-like nidoviruses are common as e.g. reptile viruses. the order nidovirales contains enveloped, positive-strand rna viruses with the largest known rna genomes. nidoviruses have been identified in snakes. they appear to be most closely related to coronavirus subfamily torovirinae, and might be best represented as a genus in this subfamily. sequences suggestive of rsfiedllfnkv, e.g. knfidlllagf do occur in genomes such as the ball python genome, but these really lie beyond the limit of serious detection. for example, clustal omega gives 18% exact match between the wuhan isolate and spike protein nidovirus 1 of the reptile shingleback, but the motif is barely recognizable. including fish nidovirus of the pacific salmon (genbank qeg08239.1) is notable here because it supports the above alignment because it is preserved, but gtlywldy of the salmon nidovirus is far from krsfiedl and the nearest preceding plausible cleavage point is an arginine (r) 10 residues in the n-terminal direction (to the left). however, a similar occurs in some mammalian coronaviruses and so that residue may still play a similar role as an activation cleavage. looking for similar motifs in human proteins has a somewhat different motive. it makes sense in that, if there is significant match with subsequences, they might represent features of proteins to which both the spike protein and other human proteins may bind, irrespective of any other justification for commonality. even if coincidental, as epitopes similar to those in a proposed synthetic vaccine they are always of possible interest in assessing the risk of cross-reaction and inducing autoimmunity in synthetic vaccine designs, and on certain occasion with peptidomimetics that induce an immune response, perhaps by a binding strongly to a human protein that the designer did not intend. as discussed in ref [3] , there is a motif match at 56% identity with 77% coverage is with tumor protein d55 isoform 2 [homo sapiens], id: np_001001874.2, and similarly with tumor protein d52-like 3 [homo sapiens] id: aah33792.1. next match is in regard to neprilsyn entries at only 56% match and 55% coverage. none of these are sufficient close of concern regarding induction of an autoimmune response. some fairly close matches of krsfiedllfnkv and of the "a for f" modified motif rsaiedllfdkv have come to light that might plausibly have a biological significance if supported by biological relevance, but are more likely to be random matches. selecting only for human proteins, hits vary from 100% cover with 50% identity to 62% cover with 92% identity. these hits cannot be considered significant for peptides of this length in isolation from other evidence. however, a few seem worth recording for future reference in regard a potential biological function for the virus. as already noted [3] , rrsfidelafgrg a section of a human semaphorin (genbank np_001243276.1) produced in response to lung disorders. running rsaiedllfdkv itself in blastp generates 100 coronavirus hits. rnareellfd is found in human mhc class ii antigen, genbank axn55588.1. rnareellfd is found in human immunoglobulin heavy chain junction region genbank mcg49633.1. dllfekv is found in human tubulin, gamma complex associated protein 6, isoform cra_d genbank eaw73510.1. e3 is of interest with 75% identity 87% matches for sfleellfin khksfleellf in ubiquitin. the cellular e3 ubiquitin ligase ring-finger and chy zinc-finger domain-containing 1 (rchy1) have been identified as interacting partners of the viral sars-unique domain (sud) and papain-like protease (pl pro ), with the involvement of cellular p53 as antagonist of coronaviral replication. down-regulation of p53 is a major player in antiviral innate immunity [72] . again, however, these matches remain tenuous. genbank has of the order of 0.2 billion nucleic acid sequences but a 13 residue peptide can have 81,920,000 billion sequences. while the krsfiedllfnkv motif remains favored by the author as a target at this time, identifying the amino acid residues in ace2 and the spike protein is important. it may for example involve conserved residues that are not together in a continuous sequence. while a conserved run of amino acid residues is sufficient to be on the list of candidates for an important site, important sites are not necessarily conserved runs of amino acid residues. here is shown that there is some conservation, but significant variation compared with rsfiedllfnkv. subsequences rsfiedllfnkv and pigagicasy…r discussed in ref [3] as motifs associated with activation cleavage sites do not lie in the receptor (ace2) binding domain of the sars-cov-2 spike glycoprotein. the relationships between the whole spike protein and the receptor binding domains in pdb entries 6m17 and pdb 6vw1 are shown in the alignment below. note that the above receptor binding domain precedes the above motifs in the sequence. a three dimensional perspective is required for an appreciation of the important sequence features. in fig. 2 , the pdb 6vw1 binding domain is on the right, bound to ace2 on the left. fig. 2 of course, not all the receptor binding domain is interacting intimately with ace2. the sections of the receptor binding domain that do interact with ace2 are also shown (underlined). to facilitate deeper analysis, the loops on the spike protein receptor binding domain were initially classified as loops a,b,c,d,e, and f in order of visual perspective, then joined into three subsequences 1, 2, 3 that contain these loops. the part of the spike glycoprotein sequence that represents the receptor (ace2) binding domain can be shown by considering the proteins used in the two structural determinations 6m17 and 6vw1 in the protein data bank, shown below in an alignment made using clustal omega alignment. note that the above receptor binding domain precedes the above motifs in the sequence. the amino acids residues in bold and underlined font are the subsequences of ace2 that interact with the above spike protein ace2 binding domain loops, which are indicated above each subsequence. these include some longer range electrostatic interactions and potential solvent effects. those also in italics dkfnheaedlfy, dkfnheaedlfy and kgdfr have particularly strong interactions. as a reference perspective, the full sequence for ace2 as angiotensin-converting enzyme 2 isoform x1 [homo sapiens] genbank entry xp_011543851.1, is as follows. the part in the three dimensional structure above is in bold underlined font. note that at least in these particular experimental structures there is an involvement of "glycosylation-like" molecules. for example, in 6vw1 there are well localized n-acetyl-d-glucosamine, β-d-mannose, and 1,2-ethanediol molecules that make significant interactions in a glue-like manner, and essentially "glue around the edges". however there no obvious indication of involvement of glycosylation in the main interior interaction face of the complex. the intimate interactions are proteinprotein, i.e. between amino acid residues. primarily, but not solely, ace2 and spike glycoprotein association involves interaction between the bent α-helix residues 19-54 (stiee…nyntn) of ace2 and an extended chain configuration, effectively a stretched loop, that runs from residue 485-500 (gfncy….ygqpt) of the spike glycoprotein and involves or ends in loops 3a, 3b, and 3f. in the case of ace2 interacting with the ace2 binding domain of the spike glycoprotein, one could in principle imagine blocking the ace2 as receptor with a mimic of the spike protein surface, or blocking the receptor binding site of the spike glycoprotein protein with a mimic of the ace2 receptor surface. in other kinds of infection the former is usually considered more plausible, but the latter would not interfere with normal function of ace2 and it is of course essentially the way in which immune system, and notably antibodies, work. possibly the main argument against this second choice it is that it is essentially equivalent to using antibodies raised against the spike protein, i.e. in effect, passive immunization. at the time of final writing, various news articles are drawing attention to potential use of the upper sequence or parts of it, which is that of the α-helix of ace2 (for example https://scitechdaily.com/mit-chemists-have-developed-a-peptide-that-could-block-covid-19/). in the above "alignment", the helix contains 35 residues and the extended chain below contains 16. they have similar length as is as expected for such structures. an αhelix has a rise of 1.5 å per residue along its axis and there are in typical protein helices with turn variations that imply up to 2.0 å. the β-strand or a similar extended chain in the spike glycoprotein that interacts with it has a rise of circa 3.5 å per residue. this general geometry naturally puts the two sequences above in roughly the one-to-one spatial correspondence shown. note that this is not intended to be a sequence match representation; these chains have to interact. in that respect, there is a lack of charged residues (acidic and basic sidechains) in the extended chain of the spike glycoprotein in structure 6vw1, although an aspartic acid (d) replaces the serine (s) in some strains, arginine (r) replaces asparagine (n) in others, and so on (e.g. see blastp alignments later below). a detailed backbone view is confusingly cluttered, but one may identify residues that interact at the boundary between ace2 and spike protein. all sidechains in the above spike protein subsequence gfncyfplqsygfqpt either make close contact or are likely to have some influence at the interface. it is perhaps useful to have the initial mental picture that, very roughly speaking, the planes of the peptide groups are tangential to the above α-helix surface, rather than constituting an extended chain that makes an edge-on approach. as even fig. 2 suffice to makes clear, however, the extended chain, like any so-called extended chain in proteins in practice, is essentially a visible helix of larger pitch, resembling a very stretched-out α-helix, and is itself slightly supercoiled to wrap around the ace2 α-helix. in this case, this tends to follow the elbow or bend in the α-helix, staying roughly parallel to the local axis of the α-helix, so as to make intimate contact overall. if gfncyfplqsygfqpt is to be use as an epitope analogue, the cysteine (c) may be tested as a convenient linker to a carrier protein, otherwise replaced by serine (s) as a close analogue. as far as peptide antagonists are concerned, the difficulty with using the above sequences stiee…. and gfncy…. is that they are readily degraded by host proteases. this would not occur if the peptide is made entirely of d-amino-acid restudies. a retro-inverso peptide [3, 46] is made up of d-amino acids in a reversed sequence to the subsequence which is seeks to mimic, and in the extended conformation assumes a side chain topology similar to that of the original native peptide, but with backbone n-h and carbonyl c=o groups interchanged. these are peptidomimetics of the subsequences sequences stiee…. in ace2 and gfncy….. in the spike glycoprotein respectively. the cysteine (c) in ….cnfg in the second molecule may be a convenient linker for an epitope for a vaccine but should be replaced by serine (s) in an antagonist. recall that the problem of having the backbone amide n-h and carbonyl c=o groups interchanged is that if, in the original section of backbone being mimicked, any n-h and c=o groups form a hydrogen bond with recipient and donor groups in the protein, those hydrogen bonds are now disrupted in the intended competitive antagonsist, e.g. they would be unstable n-h…h-n or c=o…o=c interactions. it would thus seem a significant advantage in using the ace2 mimic, because that is essentially an α-helix which uses up its backbone amide and carbonyl groups. however, retro-inverso α-helices are not typically found in the areas that have shown some degree of success [47] , such as antigenic mimicry. it would nonetheless seem to be of value to test both of the retro-inverso peptides in laboratory studies. as to developing the above further both as the basis of a synthetic vaccine, or as a peptidomimetic, and as to the worth of extending the studies to small organic drug molecules, everything in the above depends on the extent to which gfncyfplqsygfqpt can produce escape mutations which might soon rend such solutions useless. as in the previous paper, we can relate this to variations of the above sequence bother in closer and much more distant relatives. as the following shows, using blastp we do not have to go very far from sars-cov-2 to find matches with only part of this sequence (coverage) and differences within that area of partial match: in the original wuhan seafood market pneumonia virus isolate wuhan-hu-1, genbank id mn908947.3, the subsequence in this regions is fncyfplqsygf, and the following are examples of coverage as found by blastp. the last of the above blastp at https://blast.ncbi.nlm.nih.gov/blast.cgi match results differs in total alignment by clustal omega at https://www.ebi.ac.uk/tools/msa/clustalo/, as follows, but of course this illustrates the high degree of variation that occurs as one proceeds on to coronaviruses less related to the wuhan seafood market isolate that is believed to be associated with the origin of covid-19. phenylalanine (f) commonly immediately precedes many of these matching subsequences ncyfp… ncywp… etc., and the conservative substitution tryptophan (w) substitution for the second phenylalanine (f) is also very common, so it may be worth noting that fnctwp is a subsequence in the mammalian vomeronasal type-2 receptor 1 on sensory cells within the main nasal chamber that detects heavy moistureborne odor particles, and fnctwp is also found in in dynein. many viruses require the minus-end-directed dynein motor complex transport on microtubules from cell surface toward the nucleus, and dynein in addition to kinesins for the transport toward the plasma membrane. however a direct connection to viral infection, while tempting, is far from obvious as to any mechanistic or evolutionary explanation. also, dynein nuclear shuttle transport may be less relevant to the coronavirus (an rna virus), but certainly rna viruses can rely on the dynein system (e.g. hanta virus uses it for endoplasmic reticulum-golgi intermediate compartment). at very least, the above illustrates the kinds of further, perhaps immediately less obvious, functions that the above ace2 binding domain of the spike glycoprotein, and the above motif, might have. within the coronaviruses, there is some degree of conservation that suggests that ncywplndygf is a segment for the virus to conserve, and a hint that fnctwpgf is the key part, but there are soon very clearly significant variations across coronaviruses of different hosts as we depart from the wuhan seafood market isolate compared with the rsfiedllfnkv motif in the s2' cleavage regions [3] . small organic drugs design to mimic this section, or simply designed to designed to antagonize ace2 binding, are thus potentially susceptible to escape mutations, i.e. rapid appearance of drug resistance. binding studies with 11β-hydroxysteroid dehydrogenase type 1 as model pharmacaphore. 11β-hydroxysteroid dehydrogenase type 1, which is inhibited by emodin, was an interim model pharmacophore of choice [3] . at this point in the development of the argument for optimal targets for vaccines and therapeutic antagonists, the above target fits in as follows. while the above regarding ace2 binding must be kept in mind for antagonist development, as noted above the motif is not well conserved, and so could be prone to development of escape mutations, i.e. acquired resistance to vaccines and therapeutic antagonists. because of the dominant theme of an ace2 α-helix interacting with an extended chain loop of the spike glycoprotein, the structure of the interaction region is fairly easy to deduce for various sars strains, and there was as yet no obvious strongly recurrent theme of significant conserved residues that are discontinuous (i.e. not together in the same subsequence) that could be interacting closely with ace2. at the same time, while emodin appears to act at the ace2 binding site [59] , it remains of interest because there are complexities [60, 61] as discussed in introduction section 1.6. notably, the ace2 binding domain of the spike protein and the binding sequence discussed above might bind other human proteins and might have other functions that emodin and related compounds, related in the sense that they are at least consistent with pharmacophore features, might inhibit. a priori, the binding properties of emodin and the choice of 11β-hydroxysteroid dehydrogenase type 1 as model pharmacophore could equally relate to the rsfiedllfnkv site, or some other site, or a mix of several. the case for interaction vomeronasal type-2 receptor 1 and dynein discussed above was at best marginal, but these examples illustrated the diversity other kinds of functions, important to the virus, that might apply. in any event, any relations between emodin and similar and potentially related molecules remains of interest to impeding sars-cov-2 entry and the worse casualty would be the continuity of the story developed above, which is intended to illustrate a flow of reasoning in using the standard tools of bioinformatics. 11β-hydroxysteroid dehydrogenase type 1 is interesting as accommodating a great variety of ligands at the steroid binding site, but not without a degree of specificity as to general features of the ligands, and so far these resemble those of potential sars-cov-2 therapeutics. keeping in mind the refutation principle [3] that a pharmacophore (or contribution to a pharmacophore ensemble) the dehydrogenase is worthy of use until a new ligand or other information proves otherwise. so far pharmacophore validation here, i.e. a demonstration that it is a suitable pharmacophore model until proven otherwise, has been based circumstantially on emodin and compounds looking chemically similar to it, that are known in practice or argued theoretically to interact with sars virus entry in some way and bind at least weakly, experimentally or computationally, to 11β-hydroxysteroid dehydrogenase type 1 [3] . a review of compounds that are known experimentally to inhibit the dehydrogenase and known experimentally inhibit coronavirus entry, replication and maturation is being prepared. however, validation is also extensively based on a weaker but larger body of preliminary binding studies involving a variety of antagonists of coronavirus infection and very often other kinds of virus infection, that also bind at least very weakly to the dehydrogenase (see discussion on "very weakly" below). most of these, emodin-like and otherwise, were first found by q-uel knowledge gathering tools as used in the initial coronavirus study [3] combined with "very early candidate selection rules" based on estimates of the mean binding strength of groups when binding well. note that a hydrogen bond worth about -4 kcal/mole is nonetheless effectively zero when binding well, because it is relative to binding to water. in contrast aromatic and large aliphatic and are worth circa -3 kcal/mole due to hydrophobic interactions which depend on being considered relative to water. there are more complex electrostatic and intramolecular entropic considerations beyond present scope, noting that at least preliminary study of the interaction with 11β-hydroxysteroid dehydrogenase type 1 is the arbitrator. weak and very weak candidates are also considered because there may be multiple binding modes that will take a great deal of computer time to explore but which could yield lower binding fre energies. this produces a fairly "mixed bag" of compounds, based on the argument that viruses and coronavirus in particular may use each of its limited number of exposed or exposable sites for several purposes, and the coronavirus seems to be able to readily adjust to new mechanisms under the selective pressure of drugs and vaccines. the details of these molecules and studies are the subject of a further paper that will also discuss some interesting unifying themes. briefly, they include many names as hopedfor drugs against the coronavirus that appear in the news and internet discussion. it is convenient to see them as dividing into three classes (i) quinone-like. a "quinone" is any of a class of aromatic compounds having two carbonyl or ketone c=o functional groups in the same six-membered ring, though in "quinone-like" the author includes include many compounds resembling steroid fragments that may have many or just one carbonyl groups and several rings. this group includes 9,10anthraquinone and derivatives that relate to many important drugs some with suggestive laxative and antiinflammatory functions, collectively called anthracenediones. they include ubiquinone as coenzyme q, and various shorter aliphatic chain forms hydroxyl-decylubiquinone and shorter aliphatic chain forms, laxatives such as dantron, emodin, and aloe emodin, and some of the senna glycosides, antimalarials such as rufigallol, antineoplastics used in the treatment of cancer, such as mitoxantrone, pixantrone, and the anthracyclines. caution is reuired in reading this list as a list of potential therapeutics, because anthraquinone derivatives rhein, aloe emodin or anthrone that lacks the methyl group, parietin (physcion), to some extent emodin itself, and chrysophanol extracted from cassia occidentalis are toxic and known to cause hepatomyoencephalopathy in children. it is a medical term effectively defined to cover lethargy, jaundice, and altered senses of children in india after consumption of cassia seeds. (ii) steroid-like. this group includes some plant steroid-like compounds such as carbenoxolone itself from liquorish (licorice) and others found in soy and sprouts.17βestradiol (the endogenous ligand responsible for the growth and development of many tissues) diethylstilbestrol (a synthetic estrogen); 7-methyl-benz[a]anthracene-3,9-diol (a possible natural product from a common polyaromatic hydrocarbon) is also of interest. this group resembles group (i), but the concern for this group (ii) is that molecules like emodin that are known to antagonize viral or other infections are generally smaller, so it possible that a more relevant pharmacophore would sterically exclude a large steroid-like ring. (iii) quinine-like. these should not be confused with "quinone-like". quinine is an alkaloid derived from cinchona bark, used to treat malaria and as an ingredient of tonic water. a common feature is, nonetheless the abundance of aromatic and other rings that in the quinine-like case include nitrogen, so variously resembling pyrimidines, purines, histidine and tryptophan. this group is of current considerable interest as potential thereputics for covid-19. of particular interest are chloroquine, theophylline, tavipiravir, baloxavir marboxil. some ace and ace2 inhibitors can be classified in this group. they are weak but not very weak binders as discussed later below. camostat, a serine protease inhibitor that has been considered as a potential therapeutic for covi-19 is convenient to place in this class because of its analogues but it does not itself include a nitrogen atom within a ring. there are several possible intriguing biological connections that will be discussed elsewhere. one might be briefly mentioned when considering combined therapeutic use of a member of each set. ubiquinone-like compounds can inhibit ubiquinone sites that work in concert with nadh and nadph cofactor sites. the latter in turn are often inhibited by the quinine-like members. many other above compounds generally bind "very weakly", though steroid-like compounds are strong binders and many quinine-like compounds are medium binders: these are discussed below. binding strength is of course a matter of degree. rt (where r is the gas constant and t the absolute temperature) is 0.593 at 298 o k, i.e. circa 0.6 at biological temperatures, so 1 kcal/mole is not significant above thermal noise. free energies of 2, 3, 4, and 5 correspond to binding association constants of 5, 148, 786, and 4160. the above free energies are usually expressed as negative, for the perspective from the associated system. considering that absolute values are much less reliable than relative values in this field, one might conservatively consider a binding energy of -3.5 kcal/mole as worthy justification for keeping a compound on a list, if one does not wish to reject prematurely, and this seems reasonable if one still has in mind the refutation principle. this includes the mental picture that a model pharmacophore such as 11β-hydroxysteroid dehydrogenase type 1 has a fairly large cavity which does not provide strong steric inhibition to the candidate ligands, but new evidence might show that a large ligand such as a steroid might be too big to fit the real target which the experimental data is describing. in other words, deficiencies in the pharmacophore model will start to show up when considering larger potential drugs. there is also the benefit of using 11β-hydroxysteroid dehydrogenase type 1 as model pharmacophore that the present author has a data base of experimental and computational studies on compounds that bind to it. it should be stated, nonetheless, that any case for any common evolutionary relationship between this dehydrogenase and the spike protein binding receptor ace2 would be, at best, marginal. 11βhydroxysteroid dehydrogenase type 1 has 292 residues and ace2 has 613. there is a 24% identity match of amino acid residues in the region of best possible match of the dehydrogenase. there is also further 19% conservative substitution (clustal ':', i.e. conservation between groups of strongly similar properties with a score greater than .5 on the pam 250 matrix). if taken alone, this would provide some basis for further exploring a relationship. admittedly, the conventional rule of thumb is that any two sequences are considered homologous if they are more than 30% exact amino acid residue matches, and strictly speaking this should apply over their entire lengths (this is discussed in chapter 8 of ref [12] and a brief review of standard tools is given in refs [2] and [38] ). nonetheless, caution is required because the 30% exact match criterion is well known to miss many easily detected homologs and 15-20% is sometime found supported by evidence of evolutionary and functional relationship. for example, alignments between common cold and sars-cov-2 spike proteins already discussed above are in this range, but there is every good reason to believe a common ancestry, there is an overall conformational similarity, and essential features of some sequence motifs are preserved. there is some sense of comparable fold motifs with ace2 comprising two 11β-hydroxysteroid dehydrogenase type 1 folds. the dehydrogenase is a bundle of some 12 well defined, roughly parallel and antiparallel α-helices of up to about 30 residues, interspersed by 7 short β-pleated sheet strands. ace2 has some 20 well defined, predominantly and very roughly parallel and antiparallel α-helices of up to about 30 residues, interspersed by 6 short β-pleated sheet strands. if there is a common evolutionary origin of 11β-hydroxysteroid dehydrogenase type 1 and ace2 domains, it is distant, but it remains marginally possible and more extensive conformational analysis is underway. there is even less evidence of homology between 11β-hydroxysteroid dehydrogenase type 1 and tmprss2, although a serine residue is highly conserved in the catalytic site in both cases, which arguably makes it worthy of some initial exploration. tmprss2 comprises distinct cystine rich scavenger domain (residues 150-242) and a serine protease domain (residues 255-484). clustal o(1.2.4) multiple sequence alignment gives an exact match of amino acid of only 17.5%. there are some grounds for further investigation in the future. there is also further 17.5% conservative substitution (clustal ':', see above). for tmprss2 there are some suggestive short section matches in same order of appearance, e.g. aqyyys with ayyyys, vvshc with vvshc, lyhsd with lfhdd, and gilrqs with galrqe, which by some arguments slightly increase statistical significance. no significant conformational homology is apparent, so it is even more likely to be a chance match, and any argument for similarity between the proteins would be on the basis of some kind of convergent evolution based on certain common ligands, recalling again that the coronavirus might benefit from inhibiting an inflammatory response. preliminary studies on the panel of ligands discussed below suggest some degree of binding (-4.5 kcal/mole and better, i.e. more negative) to both the above and 11β-hydroxysteroid dehydrogenase type 1, but these studies are still not fully complete and low energies may yet be obtained. the most substantial data base of results that can reasonably be considered final is in large part from the original studies [50] . there carbenoxolone was automatically evolved (by automatic editing of its chemical structure) under the combined selective pressure of improve binding to 11βhydroxysteroid dehydrogenase type 1 while avoiding significant match with compounds covered by all us patents [50] , and subsequent docking and high grade molecular dynamic simulations were carried out on ibm's blue gene [50] . many subsequent studies have, however been carried out on using krunch on a personal computer, because in the initial study it predicted well the blue gene results providing that the krunch binding energies obtained were corrected (or refined) to fit the blue gene results by a linear regression formula [50] . recall again that it is on the basis of similarities between some compounds that antagonize sars virus entry and bind the steroid dehydrogenase, plus a notable commonality in the case of emodin (i.e. it binds both), that this model pharmacophore was chosen. since emodin and many other compounds of interest contain two or three or more aromatic rings, it is reasonable, at least as an initial tactic, that one may regard them as pieces of the steroid ring system and start them in the steroid binding cavity in the same "plane" as the steroid ring. in such a case involving minor variations as sidechains on the original steroid core, the way to make initial fit to using carbenoxolone as guide is obvious. however, the flat view of a steroid is misleading. the steroid ring system can "buckle" in various cis-trans combinations of bonds in the rings, and the longer sidechain conformations preferred on the basis of intramolecular energy are perhaps not obvious. although the rotation barriers for most of the transitions are clearly above the thermal energy (kt) energy conformations (0.6 kcal/mole), the associated energy demands for buckling of parts of the steroid ring system of variously and roughly 2.5 to 5.0 kcal/mole are less that the ligand-receptor binding the associated energy demands are below the gain in energy from ligand-receptor binding to the protein target. this is shown in the high grade quantum mechanical hartree-fock gamess calculations on blue gene in the original study [50] but which have not been described in the literature. minimized energy conformers of steroid-like compounds considered are shown in fig. 3 . calculations. such calculations in vacuo are less reliable for the charged species, but one may obtain a qualitative assessment from relative values and comparative uncharged species. these compounds are also shown more clearly from the chemist's perspective in two dimensional formula format, later below. fig. 4 shows one of early analogues of carbenoxolone (the thioketone derivative cbos1 discussed later below) in the 11βhydroxysteroid dehydrogenase type 1 steroid binding site. the particular interest in this compound is as follows. since in the original study [50] krunch judged this as the strongest binder at -16.8 kcal/mole, this compound was frequently used as a starting template for initial docking configurations when using krunch. this is even though (a) it is probably an unlikely choice for a chemist to use in practice because of likely oligimerization of the thioketone groups, and even though (b) corphos (also known as cortisol 21-phosphate, cortisol, phosphate, hydrocortisone-21-phosphate or 21-hydrocortisonephosphoric acid) was the strongest at -16.8 kcal mole when using instead the amber force field for molecular dynamics on ibm's blue gene [50] . the thioketone still retained a reasonable binding energy of -16.3 kcal/mole in the latter study, however, i.e. effectively the same binding strength within the state of the art. fig. 4 does not of itself give details of any ligand-protein interactions (but see discussion in refs [3, 49] ), although it does illustrate the tightest of fit. that is, except to the lower right of the thioketone ring of the ligand, which does appear to relate to genuine opportunities for additional groups to be added to carbenoxolone at that position. carbenoxolone and initial closely related derivatives derived in that study [50] are shown in fig. 4 , binding in the range, -17 to -14 kcal/mole. accuracy and limited realism of such methods does not really justify more precise statements on binding energy, and the classification of binding below is as strong, medium, and weak, but see ref [50] for more detail on some of the compounds. authors variously consider binding energies -5 to -9 as a safe requirement for significant binding, but again this is subject to considerations of accuracy and almost all agree that it is only the relative values that are significant. note that while they are often interpreteted as estimates of binding free energy, the entropy component, particularly of the aqueous solvent and solute-solvent intercations, is difficult to estimate. experimental binding values of ligands in general in biological systems typically range from -4 to -16 kcal/mole, though over 95% lie in the range -7 to -13 kcal/mole. the thiioketone derivatives are more of theoretical interest in binding studies because in practice they may cause oligmerization organic compounds binding the pharmacophore. strong binders. from the original study [50] . the estimated binding energy is in the range -17 to -14 kcal/mole. these were deigned from carbenoxolone with the intent to have a stronger or comparable strong binding (-16 kcal/mole). corphos, cbonring, and cbos2 bind at -17 kcal/mole. recall that the two peptide analogues of features of the spike protein of interest [3] are as follows. so far, simulations only show these to be binding relatively weakly at -10 and -8 kcal/mole respectively, but these compounds are highly flexible with a theoretical internal in vacuo conformational entropy corresponding to about -19.5 kcal/mole as discussed in theory section 2, show multiple binding modes and conformers on binding, and may not yet be complete. a high performance computer like ibm's blue gene used in the earlier drug design study [50] would certainly help. in fig. 6 is shown a set of compounds from the zinc data base [69] , and most were identified from the original 11β-hydroxysteroid dehydrogenase type 1 study [50] and subsequent studies. these bind significantly by usual criteria, but more weakly. they are in the range found for the synthetic peptides of interest, but have much less conformational freedom. a small few not shown here did appear in the original higher grade studies, but the reasonable binding energies could not be reproduced for reasons that are not as yet clear. organic compounds binding the pharmacophore. medium binders. from the zinc data base. the estimated binding energy is in the range -9 to -11 kcal/mole. fig. 7 shows some weaker binding results using krunch [50] . great caution is required in drawing conclusions from the compounds in fig. 3 . camostat is definitely of interest as an inhibitor of the ace2 protein to which the spike protein initially binds for cell entry and does seem effective in blocking entry [74] , and similarly hepsin results are of related interest, e.g. as it is a potential alternative entry point. the ace inhibitors also looked initially interesting by virtue of certain similarities to the other potential ligands, and of course because of their binding in this theoretical study, but most of the traditional ace inhibitors are commonly viewed as not inhibiting ace 2. taking a drug such as valsartan that acts on ace might up-regulate ace2, so facilitating virus entry, [75] but emerging information is revealing a complicated picture: see discussion and conclusions. there are possible explanations that would still allow for competing with spike protein binding, but these seem somewhat unlikely. most probably, the binding is sufficiently weak that the normal substrate, and also the spike protein, displace it. aromatic group interactions may be important here [76] . organic compounds binding the pharmacophore. weak binders. selected known drugs or proposals (caution: use of ace inhibitors might be counterproductive, see text). the estimated binding energy is in the range -5 to -7 kcal/mole. some consideration has been given to prediction of ligand binding site motifs, but so far these have proven essentially negative as regards interesting results that might shed any further light on the above, although some clues may well have been missed. binding sites are often comprised of conserved residues that are not contiguous (continuous in a sequence), which will require further and more detailed study, although subsequences of 2 to 6 amino acid residues in length are worthy of a quick preliminary study because they are commonly involved in ligand interactions. the matches involved in here as judged by blastp and clustal omega are not statistically significant, but one might think of weak matches as ligand binding site predictions in much the same way that one thinks of epitope predictions. in much of the present paper, the structure of emodin, carbenoxolone and related compounds have involved discussion of aromatic rings and hence phenylananine (f), tyrosine (y) and tryptophan (w) and more generally amino acid residues with hydrophobic character. very polar subsequences are also strong binders of charged ligands, or have a role for charged molecules or inorganic ions in some way. as far as such subsequences in the coronavirus spike protein are concerned, very polar charge-pattern motifs such drets and dreds are common in ligand binding some of the molecules that may be of interest as antagonizing sars entry, activation or replication in some way, in the present author's experience. specifically, motifs like this were of initial interest in the present project because sars-cov) nonstructural proteins have zinc finger motifs, and ret and especially red are common in prosite motifs at https://prosite.expasy.org/cgibin/prosite/prosite_search_full.pl, including zinc-finger motifs. this is not considered directly relevant to the spike protein but, for example respectively in genbank entry aia62240.1 and dreds and drets align with srldkv in three-way clustal omega alignment with srldk of the original wuhan spike protein sequence mn908947.3 and drldt of np_073551.1 spike protein. however, these and many similar alignments also illustrate considerable sequence variation, and the weak matches are not close in the sequence to the subsequences of interest neither for coronavirus spike protein nor human proteins of potential discussed above. as far as ace2 is concerned, the closes match with drets and dregs is drkkps, but this weak match again lays well away from regions of current interest, e.g. in the sequence from the region that interacts with the spike glycoprotein. dteta and drfin do occur in the c-terminal half of human 11β-hydroxysteroid dehydrogenase type 1, but again these are expected to be coincidental matches. like the first vaccines [77] therapeutics too have, of course, been drawn directly or almost directly from nature, until the late 19 th century when chemical synthesis became a science, and only in the 1970s did use begin to made of computers for rational drug design. the advantages of still seriously considering herbal remedies is that they tend to be tolerated by cells because they are produced in cells, they are already subjected to hundreds of years of human trial, are often economic solutions for bulk production, and are leads for further drug development and discovery. the principal non-peptide compounds considered above as possible therapeutics have such convenient and herbal sources. as reviewed previously [3] , the herbal extract emodin is a convenient product extracted from rhubarb, buckthorn, and japanese knotweed, and several fungi. the previous paper [3] also noted that emodin had certain molecular similarities with anti-inflammatory drugs such as carbenoxolone, derived from an extract, glycyrrhizic acid, from liquorish (licorice), that variously inhibit or are believed to inhibit human11β-hydroxysteroid dehydrogenase type 1. the above does not guarantee efficacy of emodin carbenoxolone against sars-cov-2, not least because even the emodin studies concerned sars-cov not sars-cov-2, and the case for the dehydrogenase is circumstantial, but these and related substances are worthy of investigation. indeed, this paper has described a number of compounds that bind the dehydrogenase. importantly, however, recall that the weak binders that are also ace inhibitors may be more dangerous and promote infection because they upregulate ace2 [75] . nonetheless, that situation is not resolved, as follows. the above considerations as to the action and possible usefulness are empirical observations that are largely independent of bioinformatics and molecular computation and they are even independent of whether the correct human protein targets discussed here are correct and relevant; however, it would be valuable to know what might relate ace2 and 11β-hydroxysteroid dehydrogenase type 1, and ideally also understand why both enzymes might "benefit" the coronavirus by interaction with them. also, this paper could not provide any evidence of an evolutionary relationship between these proteins, despite certain similarities, or with tmprss2. so far, there is no obvious relationship with the dehydrogenase, and some other studies by the author on other transmembrane serine proteases do not, as yet, suggest any relationship. without such connections, the dehydrogenase can only be considered as a rather arbitrary model pharmacophore. as such, it is possibly meritorious as correctly representing an ensemble of multiple targets, but that may be fortuitous, and hence only to be used until refuted by evidence. nonetheless, possible clues as to mutual relevance of these human protein targets might come noting their tissue distribution and considering how this may relate to their biological role. as regards ace2, its mrna is known to be present in virtually all organs. studying sars entry into human cells, hamming et al. [78] considered their most remarkable finding to be the substantial surface expression of ace2 protein not only on lung alveolar epithelial cells but also enterocytes of the small intestine, as in arterial and venous endothelial cells and arterial smooth muscle cells in all organ studied (oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain). there is the attractive prospect that several many herbal remedies considered as laxatives interact with ace2 and inhibit sars-covid-2 entry. recall that emodin is an antagonist of both ace2 [59] [60] [61] and 11β-hydroxysteroid dehydrogenase type 1 [62] . in the past, 11βhydroxysteroid dehydrogenase type 1 has been considered to be distributed mainly in the human liver, with no detectable levels in the intestine or kidney, mostly membranebound and retained in the liver microsomal fraction [79] . this was not however the finding of bruley et al. 80] . they found it to be highly expressed in glucocorticoid target tissues including liver and notably the lung, and modest levels in the brain. it was also found in modest levels in adipose tissue where it is of medical interest that selective increase expression occurs in obese humans and rodents and is likely to be of pathogenic importance in the metabolic syndrome [80] . lung expression appears to be managed differently: a new promotor that the authors discovered and called p1 predominated in lung while the previously known promotor predominated in liver, adipose tissue, and brain [79] . researchers therefore need to sort out an intriguing web of information. it is possible that a complex web of laxative and anti-inflammatory effect may provide clues by somehow relating to the body's attempts to reject and eject viruses of this kind, and the virus's attempts to resist. it is well known that some covid-19 patients complain of stomach upsets and diarrhea. to recapitulate the essential themes in terms of action in the alimentary tract, recall again that emodin had earlier been shown by several groups of researchers (e.g. ref [79] ) to inhibit sars-cov entry into cells (apparently initially by binding ace2), and emodin is taken as a herbal laxative. licorice has, conversely, been sometimes taken as a soother for alimentary disorders, and carbenoxolone has been used commercially in the past specifically to treat peptic ulcers. intriguingly, carbenoxolone is also known to influence the renin-angiotensin system involving ace2, so at least there appears to be a connection in terms of networks of physiological control. as noted above, while traditionally 11β-hydroxysteroid dehydrogenase type 1 has been thought of as a liver enzyme, many researchers have indicated that both ace2 and the dehydrogenase are available in both lung and intestinal tract (e.g. refs [78] [79] [80] ). this all hints also that some of the other laxatives that work in a similar stimulatory way might block viral entry on ace2 and perhaps other targets, and should be explored. of course, absolutely nothing should be done by patients without physician direction, because dosages are difficult matters (not least in herbal products) and there are potentially serious side effects on such as salt balance and blood pressure, and some might cause birth defects, all potentially worse than covid-19 would be, for most people. but more worryingly still, the situation is not settled, and physicians and patients could take action in the wrong direction. gurwitz [81] has emphasized that the picture is even more complex. he examined reports from china suggesting that a mechanism of production of lung injury during the viral infection may be due to excess free angiotensin-ii, which might be displaced from ace2 by the sars virus particles. if so, then increasing the amount of ace-2 could be desirable and administering angiotensin receptor antagonists could beneficially upregulate the production of ace-2. it now becomes important to examine medical records of patients who have, and who have not, been infected by sars-cov-2, with a particular eye on who is, and who is not, taking ace inhibitors. as noted in section 1.4, merrifield developed first solid phase peptide synthesis on crosslinked polystyrene beads in 1963 [12] . somewhat like natural compounds discussed above, peptides and petidomimetics are potentially important first steps in more detailed rational design of small organic molecules convenient as traditional "in a pill" drugs. however, as in the present paper, the ability to propose specific peptides and peptidomimetics does depend on bioinformatics, and benefits from some computational chemistry. note that in this case, one is thinking largely not of screening natural products, but now considering truly novel molecules using theoretical methods because they do not yet exist. the variations in the krsfiedllfnkv motif that might be appropriate to synthetic vaccine and peptidomimetic antagonist design suggest the following where the amino acid residues in square brackets [ ] represent alternatives. (g?) means an optional glycine insertion. the above is also valid as a regular expression, i.e. a match query in operating systems and software. more generally and colloquially, (positive charge)-(optional glycine)-serine-hydrophobic-hydrophobic-glutamate-aspartatehydrophobic-leucine-phenylalanine-(hydrophilic or alanine)-lysine-valine considerations at the n-terminus and c-terminus to design a synthetic vaccine, and the retro-inverso approach for a peptidomimetic agonist, are described in ref [3] . other variations appear as the strain becomes more distant; there is not a universal clear indication of any sharp point of departure, although the above glycine (g) insertion is evidence that a significant jump can happen. one may therefore ask what variations should be included. with the emphasis on sars-cov-2, only closely and medium distance relatives are of interest, with the purpose of prevent mutations that escape from vaccines and antagonists, and elude diagnostics. as far as sars-cov-2 is concerned, krsfiedllfnkv is a satisfactory basis because a large number of coronaviruses significantly different from sars-cov-2 preserve it, or in a few cases have very conservative substitutions. it may well be that the fact that residues are, for example, hydrophobic or positively charged is sufficient to for the approach to be applicable to other mammalian coronavirus diseases, if successful for the above basic motif form. attempting to tackle the common cold is not a priority. in other words, it may well be that an immune response against krsfiedllfnkv will also illicit a response against the motif variants, providing of course that krsfiedllfnkv elicits a response itself. it remains that this motif is one of very few subsequences that still recognizable when moving on to rather distantly related coronaviruses such as those of the common cold. one feature of both figs. 5 to 7 is of course the constant appearance of aromatic rings, and this is also noticeable in many of the studies of antagonist's against sars virus binding and activation. of course, the aromatic (i.e. benzene) ring makes copious appearance in many pharmaceutical agents in any event, because they provide rigid scaffolds for added groups supported by many long-established recipes for synthesis. the compounds in figs. 5 to 7 should be distinguished from those such as lopinavar, ritonar, promazine and particularly niclosamide that have been explored for sars viruses in the past, because these are targeted by drug designers against the sars virus own protease required for maturation of the assembling virus. nonetheless, some of these do have a visual similarity to the compounds in fig. 3 , particularly niclosamide (which is normally a medication used to treat tapeworm infestation). also, in the present case, prevalence of aromatic rings in figs 1 to 3 is hardly surprising, since carbenoxolone and derivatives shown in fig. 1 were the starting point for their evolution or selection from the zinc data base. nonetheless, there is, in principle, nothing to constrain the evolution to aromatic chemistry [50] and later unpublished studies did produce molecules departing from aromatic chemistry. however, these bound relatively weakly. with the possible importance of aromatic rings and avoidance of escape mutations by the coronavirus in mind, a question is whether occasional loss of phenylalanine (f) from the krsfiedllfnkv motif discussed above contests the tentative hypothesis that the peptidomimetic candidates derived from krsfiedllfnkv bind to a similar site as the smaller organic ligands considered here, because of two phenylalanine residues (f) in the original motif and a tendency to several benzene rings in the case of organic ligands. the answer is: perhaps. there seems to be a need to have one aromatic ring present in the motif, and no match with a coronavirus in genebank was detected by the author by blast-p using queries with no phenylalanine (f), e.g. rsaiedllldkv, rsaiedllidkv, rsaiedlladkv, rsaiedllmdkv, rsaiedllwdkv, and rsaiedllydkv as queries, though as also noted above, the search has not been exhaustive. it would not be too contradictory to any of the current main hypotheses if some examples were found. the fact that tyrosine (y) does not seem to readily substitute here for phenylalanine (f) (from which it differs only by a hydroxyl -oh, i.e. phenolic group) suggests an important hydrophobic feature of the pharmacophore at that point. of course, many or most drug-like molecules contain at least one aromatic ring and this is almost certainly because they can form especially strong stacking interactions in the binding site. one very relevant report in the same month of writing the present paper emphasizes that the use of protein and other fragments to characterize binding pocket and determine the strengths of ligand-protein interactions is common in both a computational and experimental approach, and that aromatic interactions are both strong and need special attention [76] . because of resonance and the special nature of the π orbitals, the strength of stacking is best calculated using high level quantum mechanical approaches, not empirical force fields [76] . however, as these calculations are performed in vacuum, solvation properties are neglected, and this led to the proposal of a grid inhomogeneous solvation theory (gist) to describe the properties of individual heteroaromatics and complexes; this gave good correlation for the estimated desolvation penalty and the experimental binding free energy, and prediction of binding sites [76] . a main conclusion is that peptide krsfiedllfnkv remains of special interest as well conserved across coronaviruses. other sites and other proteins of the virus may, of course, emerge as the solutions to this formidable problem. all aspects of the virus must be considered. however, even the ace2 binding domain is significantly more prone to accepted mutations. the recurrence of the 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explanation for recurrent rv infections cooperative molecular and cellular networks regulate toll-like receptor-dependent inflammatory responses p53 down-regulates sars coronavirus replication and is targeted by the sars-unique domain and pl pro via e3 ubiquitin ligase rchy1 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor rapid response (comment): sars-cov-2, hypertension and ace inhibitors stacked -solvation theory of aromatic complexes as key for estimating drug binding the jenneration of disease: vaccination, romanticism, and revolution tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis 11β-hydroxysteroid dehydrogenase 1 human tissue distribution, selective inhibitor, and role in doxorubicin drug metabolism and disposition a novel promoter for the 11β-hydroxysteroid dehydrogenase type 1 gene is active in lung and is c/ebpα independent angiotensin receptor blockers as tentative sars-cov-2 therapeutics drug discovery using very large numbers of patents. general strategy with extensive use of match and edit operations towards automated reasoning for drug discovery and pharmaceutical business intelligence towards new tools for pharmacoepidemiology the concept of novel compositions of matter. a theoretical analysis suggestions for a web based universal exchange and inference language for medicine hyperbolic dirac nets for medical decision support. theory, methods, and comparison with bayes nets popper, a simple programming language for probabilistic semantic inference in medicine suggestions for a web based universal exchange and inference language for medicine. continuity of patient care with pcast disaggregation split-complex numbers and dirac bra-kets implementation of a web based universal exchange and inference language for medicine. sparse data, probabilities and inference in data mining of clinical data repositories interesting things for computer systems to do: keeping and data mining millions of patient records, guiding patients and physicians, and passing medical licensing exams data-mining to build a knowledge representation store for clinical decision support. studies on curation and validation based on machine performance in multiple choice medical licensing examinations studies of the role of a smart web for precision medicine supported by biobanking studies in using a universal exchange and inference language for evidence based medicine. semi-automated learning and reasoning for pico methodology, systematic review, and environmental epidemiology studies in the extensively automatic construction of large odds-based inference networks from structured data. examples from medical, bioinformatics, and health insurance claims data • this paper "drills down" into the studies of the author's previous covid-19 paper.• designing vaccine and drugs must seek to avoid escape mutations.• subsequence krsfiedllfnkv seems recognizable across many coronaviruses.• the ace2 binding domain is a target, but shows variation.• a steroid dehydrogenase is argued to remain an interesting model pharmacophore.this paper is provided to the community to promote the more general applications of the thinking of professor paul a. m. dirac in human and animal medicine in accordance with the charter of the dirac foundation , to emphasize the advantages and simplicity of the basic form of the hyperbolic dirac net, to encourage its use, and to propose at least some of the principles of the associated q-uel, a universal exchange language for medicine, as a basis for a standard for interoperability. these mathematical and engineering principles are used, amongst many others in an integrated way, in the algorithms and internal architectural features of the bioingine.com, a distributed system developed by ingine inc. cleveland, ohio, for the mining of, and inference from, very big data for commercial purposes. immediately prior to joining ibm in 1998 he was hired as principal scientist at mdl information systems in california to help put together the technology for the multimillion sale of a bioinformatics system to the holding company forming craig venter's celera genomics that produced the first draft of the human genome. prior to that, he was cso of gryphon sciences (later gryphon pharmaceuticals) in south san francisco, california, a bio-nanotechnology ultrastructural chemistry start-up largely held and then acquired by smithkline beecham. before moving to the us, barry was the scientific founder of proteus international plc in the uk, designing and leading the development of the prometheus expert system and its underlying global expert system, bioinformatics and simulation language for drug, vaccine, and diagnostic discovery. it sold for the equivalent of $9.4 million to the pharmaceutical industry in the mid-1990s. at proteus, he also led the team that used the above expert system to invent and patent several diagnostics and vaccines including the mad cow disease diagnostic subsequently marketed worldwide by abbott. he has some 300 scientific publications including some 50 patents and two books: "the engines of hippocrates. from the dawn of medicine to medical and pharmaceutical informatics" robson and baek, 2009, wiley, 600 pages)" and "introduction to proteins and protein engineering" (b. robson and j. garnier, 1984 garnier, , 1988 , elsevier, 700 pages). he has contributed to several reports to governments including panels of the national innovation initiative including "innovate america" published by the council on competitiveness, washington d.c. (2004) as a whitepaper to the president of the united states. for five years, barry was a nature "news and views" correspondent on biomolecules. key: cord-328046-5us4se5o authors: xu, h. y.; lim, k. p.; shen, s.; liu, d. x. title: further identification and characterization of novel intermediate and mature cleavage products released from the orf 1b region of the avian coronavirus infectious bronchitis virus 1a/1b polyprotein date: 2001-09-30 journal: virology doi: 10.1006/viro.2001.1098 sha: doc_id: 328046 cord_uid: 5us4se5o abstract the coronavirus 3c-like proteinase is one of the viral proteinases responsible for processing of the 1a and 1a/1b polyproteins to multiple mature products. in cells infected with avian coronavirus infectious bronchitis virus (ibv), three proteins of 100, 39, and 35 kda, respectively, were previously identified as mature cleavage products released from the 1b region of the 1a/1b polyprotein by the 3c-like proteinase. in this report, we show the identification of two more cleavage products of 68 and 58 kda released from the same region of the polyprotein. in addition, two stable intermediate cleavage products with molecular masses of 160 and 132 kda, respectively, were identified in ibv-infected cells. the 160-kda protein was shown to be an intermediate cleavage product covering the 100and 68-kda proteins, and the 132-kda protein to be an intermediate cleavage product covering the 58-, 39-, and 35-kda proteins. immunofluorescent staining of ibv-infected cells and cells expressing individual cleavage products showed that the 100-, 68-, and 58-kda proteins were associated with the membranes of the endoplasmic reticulum, and the 39and 35-kda proteins displayed diffuse distribution patterns. coronavirus gene expression involves the expression of six to seven mrna species. in cells infected with the prototype species of the coronaviridae, avian coronavirus infectious bronchitis virus (ibv), six mrna species are detected. these include the genome-length mrna (mrna1) of 27.6 kilobases (kb) and five subgenomic mrna species (mrnas 2-6) with sizes ranging from 2 to 7 kb. the 5ј-terminal unique region of mrna 1 contains two large orfs, 1a and 1b, encoding the 441-kda 1a and 741-kda 1a/1b polyproteins (boursnell et al., 1987) (fig. 1) . the two polyproteins are cleaved by two viral proteinases to produce functional products associated with viral replication (ziebuhr et al., 2000) (fig. 1) . the first proteinase was identified to be included in a 195-kda cleavage product, which contains a papain-like proteinase domain encoded by orf 1a from nucleotides 4242 to 5553 (lim et al., 2000) . this proteinase was shown to be involved in cleavage of the n-terminal region of the 1a and 1a/1b polyproteins at two g-g dipeptide bonds (g 673 -g 674 and g 2265 -g 2266 ) to release two mature products of 87 and 195 kda lim and liu, 1998; lim et al., 2000) . the second proteinase was identified as a 33-kda protein in ibv-infected cells lim et al., 2000; ng and liu, 2000) . this serine proteinase belongs to the picornavirus 3c proteinase group (3c-like proteinase) and was shown to mediate cleavage of the 1a and 1a/1b polyproteins at more than 10 q-s(g, n) dipeptide bonds to release mature cleavage products (liu et al., 1994 (liu et al., , 1997 liu, 1998, 2000) . in addition to the viral proteinases, understanding of the functions of other cleavage products from the 1a and 1a/1b polyproteins is emerging. for example, the 71-kda protein released from the human coronavirus 1a/1b polyprotein was shown to possess atpase and rna duplex-unwinding activities, confirming the previous predication that the protein may be the viral helicase (heusipp et al., 1997a; seybert et al., 2000a,b) . immunofluorescence and biochemical studies demonstrated that several cleavage products are membrane-associated and colocalize with the viral rna replication-transcription machinery (bost et al., 2000; denison et al., 1999; ng and liu, 2000; schiller et al., 1998; shi et al., 1999; sims et al., 2000; ziebuhr and siddell, 1999; , suggesting the involvement of these products in viral rna replication. more recent studies showed that the first cleavage product of mouse hepatitis virus-a59 (mhv-a59) 1a and 1a/1b polyproteins, p28, might play a direct role in viral rna synthesis together with polymerase and helicase (bost et al., 2000; sims et al., 2000) . other cleavage products, such as the mhv 22-kda protein, may segregate into different but tightly associated membrane populations which may serve independent functions during viral replication (bost et al., 2000; sims et al., 2000) . in previous reports, we demonstrated that four previously predicted q-s(g) dipeptide bonds located in the 1b region of the 1a/1b polyprotein are genuine cleavage sites of the 3c-like proteinase (liu et al., 1994 . taken together with the n-terminal cleavage site identified for releasing the 100-kda protein, cleavage at these positions would result in the release of five mature products with molecular masses of approximately 100, 68, 58, 39, and 35 kda, respectively (fig. 1 ). among them, the 100-, 39-, and 35-kda proteins were specifically identified in ibv-infected cells. in this communication, we report the identification of the two previously unidentified products, the 68-and 58-kda proteins, in ibvinfected cells with newly raised region-specific antisera. meanwhile, two relatively stable intermediate cleavage products with molecular masses of approximately 160 and 132 kda were identified. analysis of the expression and processing kinetics showed that both the 160-and 132-kda intermediate cleavage products coexist with their final cleavage products during the viral replication cycle. furthermore, immunofluorescence analysis showed that the 100-, 68-, and 58-kda proteins were associated with the membranes of the endoplasmic reticulum (er), while the 39-and 35-kda proteins displayed diffuse distribution patterns. in our previous reports, four q-s(g) dipeptide bonds, q 891(1b) -s 892(1b) , q 1492(1b) -g 1493(1b) , q 2012(1b) -s 2013(1b) , and q 2350(1b) -s 2351(1b) , located in the orf 1b region and encoded by nucleotides 15129-15134, 16929-16934, 18492-18497, and 19506-19511, respectively , were demonstrated to be the cleavage sites of the 3c-like proteinase (liu et al., 1994 (fig. 1 ). taken together with the q 3928 -s 2929 dipeptide bond (encoded by nucleotides 12310-12315) identified as the n-terminal cleavage site of the 100-kda protein, cleavage at these positions would result in the release of five mature products with molecular masses of approximately 100, 68, 58, 39, and 35 kda. among them, the 100-, 39-, and 35-kda proteins were specifically identified in ibv-infected cells (liu et al., 1994 . to further identify and characterize the cleavage products, four new region-specific antisera, anti-100, anti-68, anti-58, and anti-35, were raised in rabbits against bacterially expressed viral proteins. the ibv proteins used to raise these antisera were encoded by nucleotides 12447-15131, 15536-16787, 16932-18494, and 19509-20414, respectively (fig. 1) . anti-100 was raised to replace v-58, which was raised against the ibv sequence encoded by nucleotides 14492-15520, and was used to identify the 100-kda protein in ibvinfected cells (liu et al., 1994) . the specificities of these antisera were tested by immunoprecipitation assay, showing that they could specifically precipitate their target proteins synthesized in both the in vitro system and intact cells (data not shown). to identify the cleavage products in ibv-infected cells, confluent monolayers of vero cells were infected with ibv at a m.o.i. of approximately 3 pfu per cell. to reduce the background, 5 g/ml of actinomycin d was added to the culture medium at 2 h postinfection and cells were labeled with [ 35 s]methionine and cysteine at 6 h postinfection. cell lysates were prepared from cells harvested at 8 h postinfection and subjected to immunoprecipitation with anti-100, anti-68, anti-58, v17, and anti-35. v17 was raised against the ibv polypeptides encoded by nucleotides 19154-20414 and used to detect the 39-and 35-kda proteins in ibv-infected cells in a previous report . immunoprecipitation with anti-100 resulted in the detection of the 100-kda protein and a protein with an apparent molecular mass of 160 kda (fig. 2a, lane 10) . the 160-kda protein was also detected by anti-68 (fig. 2a, lane 9) . no product corresponding to the 68-kda putative helicase domain-containing protein was detected by anti-68 (fig. 2a, lane 9) . the detection of the 160-kda protein with the two n-terminally specific antisera and the apparent molecular mass of the protein suggest that it may be an intermediate cleavage product containing the 100-kda and the putative 68-kda proteins. immunoprecipitation of the same lysates with anti-58 resulted in the detection of two products with apparent molecular masses of 58 and 132 kda (fig. 2a, lane 8) . the 132-kda protein was also immunoprecipitated by v17 and anti-35 (fig. 2a, lanes 6 and 7) . in addition, v17 also precipitated specifically the 39-and 35-kda proteins (fig. 2a, lane 7) . once again, very weak immunoprecipitation of the 35-kda protein was observed (fig. 2a, lane 7) . the 35-kda protein was also weakly immunoprecipitated by anti-35 (fig. 2a, lane 6 ). the detection of the 132-kda protein by the three c-terminally specific antisera and the apparent molecular mass of the protein suggest that it may be an intermediate cleavage product derived from the c-terminal region of the polyprotein. interestingly, the three c-terminally specific antisera also weakly immunoprecipitated the 160-kda protein (fig. 2a , lanes 6-8). the reason for this result is currently unclear, but it may reflect the interaction among the cleavage products. as immunoprecipitation with anti-68 failed to detect the putative 68-kda protein in ibv-infected vero cells, we then tried to detect the protein by western blot with the same antiserum. for this purpose, cell lysates were prepared from ibv-infected vero cells harvested at 8, 24, and 32 h postinfection, respectively, and subjected to western blotting analysis. as shown in fig. 2b , a polypeptide with an apparent molecular mass of 68 kda was specifically detected in lysates prepared from cells harvested at 24 h postinfection (lane 2), and the expression of the protein was dramatically increased at 32 h postinfection (lane 3). expression and processing kinetics of the orf 1b region of the 1a/1b polyprotein in ibv-infected vero cells as the 160-and 132-kda proteins may represent stable intermediate cleavage products, time-course experiments were carried out to investigate the expression and processing kinetics of the two products in ibv-infected cells. for this purpose, confluent monolayers of vero cells were infected with ibv at a m.o.i. of approximately 3 pfu per cell and were labeled for 2 h with [ 35 s]methionine at 6 h postinfection. the cells were then washed with complete medium and chased with a 10-fold excess of cold methionine until they were harvested at appropriate times. immunoprecipitation of cell lysates with anti-100 showed the detection of both the 100-and 160-kda proteins throughout the time course (fig. 3a) . the 100-kda protein appeared at the beginning of the time course, peaked after chase for 1.5 h, and remained stable at the end of the time course (fig. 3a, lane 2) . the 160-kda protein peaked at the beginning of the time course and remained detectable after chase for 7.5 h (fig. 3a, lanes 1-6) . slight and gradual reduction of the 160-kda protein over time was observed (fig. 3a) . these results demonstrate that the 160-kda protein is a relatively stable intermediate cleavage product coexisting with the 100-kda mature cleavage product during the ibv infection cycle. similarly, immunoprecipitation of cell lysates with anti58 showed the detection of both the 132-and 58-kda proteins (fig. 3b) . the 132-kda protein appeared at the beginning of the time course, peaked after chased for 3 h, and remained detectable at the end of the time course (fig. 3b, lanes 9-12) . the 58-kda protein was first seen after chased for 3 h and gradually increased over time (fig. 3b, lanes 9-12) . interestingly, a product with an apparent molecular mass of 97 kda, representing an intermediate cleavage product containing the 58-and 39-kda proteins , was weakly detected and briefly observed during the time course (fig. 3b , lanes 9 and 10), indicating that it is not a stable intermediate cleavage product. the coexistence of the 132-and 58-kda proteins over time suggests that the 132-kda protein is a stable intermediate cleavage product. further characterization and definition of the coding sequences of products processed from the cterminal 200-kda region of the 1a/1b polyprotein two dicistronic constructs, p3c-cite-ibv20 and p3c-cite-ibv8, were made and expressed to characterize the expression and processing patterns of the c-terminal 200-kda region of the 1a/1b polyprotein. in these two constructs, the region coding for the 3c-like proteinase was placed between the t7 promoter and the internal ribosome entry site (ires) of encephomyolitis virus (emcv), and the ibv sequences from nucleotides 15132-20506 and 15132-18495, respectively, were cloned downstream of ires (fig. 1) . expression of both constructs in cos-7 cells resulted in the detection of the 33-kda 3c-like proteinase, which comigrates on sds-page with the 33-kda protein detected in ibv-infected cells (fig. 4, lanes 2-4) . immunoprecipitation of cell lysates prepared from p3c-cite-ibv20-transfected cells with anti-58 led to the detection of three protein species with apparent molecular masses of 200, 132, and 58 kda, respectively (fig. 4, lane 7) . the 200-kda protein represents the full-length product encoded by the ibv 1b sequence present in this plasmid, and the 132-and 58-kda proteins comigrated with the 132-and 58-kda proteins detected in ibv-infected cells (fig. 4, lanes 6 and 7) . immunoprecipitation of cell lysates prepared from p3c-cite-ibv8-transfected cells with anti-58 led to the detection of the 58-kda protein and a product with an apparent molecular mass of 125 kda (fig. 4, lane 8) . the 125-kda protein may represent the full-length product encoded by the 1b sequence present in this construct. immunoprecipitation of cell lysates prepared from p3c-cite-ibv20-transfected cells with antiserum v17 led to the detection of the 200-, 132-, 39-, and 35-kda proteins (fig. 4, lane 11) . the 132-, 39-, and 35-kda proteins comigrated with the three equivalent fig. 4 . comparative analysis of the 132-and 58-kda proteins expressed and processed in cells transfected with p3c-cite-ibv20, p3c-cite-ibv8, and pibv1b4 and in ibv-infected cells. plasmid dnas were expressed in cos-7 cells using the vaccinia virus-t7 expression system (fuerst et al., 1986) . semiconfluent monolayers of cos-7 cells were infected with a recombinant vaccinia virus (vtf7-3), transfected with plasmid dna using dotap according to the instructions of the manufacturer (roche), and labeled with [ 35 s]methionine and cysteine, and lysates were prepared. cell lysates prepared from mock-infected (lanes 1, 5, and 9) and ibv-infected (lanes 2, 6, and 10) vero cells and transfected cells (lanes 3, 4, 7, 8, 11-13) were immunoprecipitated with anti-3c (lanes 1-4) , anti-58 (lanes 5-8, 12, and 13), and v17 (lanes 9-11). the radiolabeled polypeptides were separated on an sds-12.5% polyacrylamide gel and detected by fluorography. numbers indicate molecular mass in kilodaltons. products detected in ibv-infected cells (fig. 4, lanes 9-11) . as mentioned earlier, the apparent molecular mass and processing pattern of the 132-kda protein suggested that it may be derived from the c-terminal region of the 1a/1b polyprotein covering the 58-, 39-, and 35-kda proteins. to further confirm this possibility, plasmid pibv1b4, which covers nucleotides 16932-20490 and therefore encodes the 132-kda product , was expressed in cos-7 cells. as expected, immunoprecipitation of cell lysates prepared from cells transfected with pibv1b4 with anti-58 resulted in the detection of the full-length 132-kda protein, which comigrated on sds-page with the 132-kda intermediate cleavage detected from cells transfected with p3c-cite-ibv20 (fig. 4, lanes 12 and 13) . to gain clues of the functions of the five cleavage products in the viral replication cycle, indirect immunofluorescence analysis of cells expressing individual cleavage products was carried out and representative confocal microscopy images are present in the proteins were labeled with the fitc-conjugated secondary antibodies. panels b, e, h, k, and n refer to cells stained with r6, a dye for the er. the green images represent fitc-derived green fluorescence, and red images represent rhodamine and texas red-derived red fluorescence. colocalization of viral proteins with the organelle markers is represented by the yellow region within each cell in the merged images (c, f, i, l, and o). panels a-o show cos-7 cells transfected with the empty vector pkt0 and stained with antisera or r6 as indicated. the fluorescence was viewed using a confocal scanning zeiss microscope. nofluorescent pattern of anti-pdi (data not shown). these results suggest that the three proteins may be associated with the er membranes. in cells expressing the 39and 35-kda proteins, a diffuse staining pattern was observed for each protein (figs. 5j and 5m), which does not coalign with the staining pattern of r6 (figs. 5j-5o). this diffuse distribution pattern of the 39-and 35-kda proteins was unexpected, as the majority of cleavage products were shown to be membrane-associated. the same antisera were used to stain cells transfected with the empty vector pkt0 (liu et al., 1994) , showing weak background staining (figs. 5a-5o) . the subcellular localization patterns of the five proteins were then analyzed in ibv-infected cells (fig. 6) . immunofluorescent staining of ibv-infected vero cells with anti-100, anti-68, and anti-58, respectively, showed similar er localization profiles (figs. 6a-6i), and a diffuse staining pattern was observed in cells stained with antiserum v17, which reacts with both the 39-and 35-kda proteins (figs. 6j-6l) . similarly, a diffuse distribution pattern was observed in cells stained with anti-35 (figs. 6m-6o). the same antisera were used to stain mockinfected cells, showing weak background staining (figs. 6a-6o). in our previous reports, we showed the identification of three mature cleavage products of 100, 39, and 35 kda, processed from the 1b region of the 1a/1b polyprotein (liu et al., 1994 . however, we were unable to detect the two other cleavage products of 68 and 58 kda. in this study, we report the identification of the 68-and 58-kda proteins in ibv-infected cells. in addition, two stable intermediate cleavage products of 160 and 132 kda, respectively, were identified, which coexist with the mature cleavage products in virus-infected cells. immunofluorescence analysis showed that the polymerase domain-containing 100-kda protein and the helicase domain-containing 68-kda protein as well as the 58-kda protein may be associated with the er and ic membranes, the viral replication and assembly sites. however, the 39-and 35-kda proteins display diffuse distribution patterns in both ibv-infected cells and cells expressing each of the proteins. it is intriguing that immunoprecipitation and western blot of ibv-infected cells harvested at 8 h postinfection failed to detect the 68-kda protein. the protein was detected by western blot in ibv-infected cells harvested at 24 and 32 h postinfection. as the equivalent human coronavirus 71-kda protein was shown to be an rna helicase (heusipp et al., 1997a; seybert et al., 2000a,b) , this protein is likely the ibv helicase. it is expected that such a functional product directly involved in viral rna replication would be expressed at early stages of the viral replication cycle. in fact, the equivalent 71-kda protein of human coronavirus was first seen in virus-infected cells at 5 h postinfection (heusipp et al., 1997a) . the reason for the failure to detect the 68-kda protein in ibv-infected cells at earlier time point is uncertain, but it might reflect the folding property, as discussed later, of the protein. the dramatic increase in the detection of the 68-kda protein at 24 and 32 h postinfection may partially be due to the secondary infection of cells that remain uninfected during the primary infection. as significantly more cells (over 95% of cells) were infected at 24-32 h postinfection, it is understandable that more protein would be detected. a slight, but gradual increase of the accumulation of the human coronavirus 71-kda protein over a time course of 15 h was also observed (heusipp et al., 1997a) . alternatively, the increase in the detection of the 68-kda protein at 24-32 h postinfection may reflect the genuine accumulation pattern of the protein in virusinfected cells at late stages of the viral replication cycle. if this were the case, it may indicate that the protein might also be involved in processes other than viral rna replication. the identification of two stable intermediate cleavage products of 160 and 132 kda coexisting with the mature cleavage products raised two interesting questions. first, the two products may have certain functions during the viral replication cycle. the 160-kda product is particularly interesting in this aspect, considering the fact that it contains both the polymerase and helicase domains and the 68-kda helicase protein is undetectable at early stages of infection. it is plausible that the 160-kda protein might have both polymerase and helicase functions in the replication of viral rna at early stages of the infection. in fact, some smaller positive-stranded rna viruses encode single proteins containing both the polymerase and helicase domains (buck, 1996) . the second interesting question is why only these two intermediate cleavage products were detectable in ibv-infected cells. as shown in fig. 4 as well as in our previous report , other intermediate cleavage products were also observed when this region was expressed in intact cells. one obvious distinct feature of the cleavage site between these two products is that it is a q-g dipeptide bond, while all the other cleavage sites in this region of the polyprotein are q-s dipeptide bonds (fig. 1) . however, no experimental data indicate that cleavage at the q-g site is more efficient than at the q-s sites. the 68-kda protein migrated on sds-page as a multiprotein species, heterogeneous smear, probably due to the formation of protein aggregates, and deletion analysis showed that a stretch of 30 amino acid residues in the c-terminal region was responsible for the aberrant migration property of the protein (data not shown). it suggests that the 68-kda protein may misfold when expressed in intact cells in the absence of other viral components. in recent years, it was found that the proper folding of certain proteins requires the assistance of molecular chaperones (ellis and van der vies, 1991; gething and sambrook, 1992) . as the 68-kda protein may be a component of the viral rna replication complex, the protein is expected to interact with viral rna and other viral proteins. those viral rna/proteins may act as a chaperone for the correct folding of the 68-kda protein in ibv-infected cells. it would be of interest to define if the region of the 30 amino acid residues that were shown to be responsible for the aberrant migration of the 68-kda protein on sds-page contain either rna binding or protein interacting domains. however, no such domains were found in this or the neighboring regions by computer analysis using relevant programs. the 58-kda protein was recently shown to be able to induce programmed cell death when expressed alone in intact cells . this is the first ibv product that was demonstrated to be a proapoptotic protein. in our previous report, we were unable to identify the 58-kda protein in ibv-infected cells due to the cross-reactivity of the antiserum used with a cellular protein . a newly raised antiserum was used in this study, leading to the successful identification of the protein in virus-infected cells. understanding of the expression, processing, and subcellular distribution pattern of the 58-kda protein would help us to further characterize the proapoptotic property of the protein and to study the functions of the protein in the pathogenesis of ibv-induced infection in chicken, the natural host of ibv. currently, no functions have been assigned to the 39and 35-kda proteins. a counterpart of the ibv 39-kda protein, the 41-kda protein of human coronavirus, was shown to exhibit a punctate, perinuclear distribution pat-tern in virus-infected cells (heusipp et al., 1997b) . interestingly, the 39-and 35-kda proteins display a diffuse distribution pattern in both ibv-infected cells and in cells overexpressing the proteins. as the majority of the cleavage products from the 1a and 1a/1b polyproteins were shown to be associated with cellular membranes at or near the viral replication and assembly sites, the diffuse distribution pattern may exclude the direct involvement of the two proteins in the formation of viral replication complexes. the egg-adapted beaudette strain of ibv (atcc vr-22) was obtained from the american type culture collection (atcc) and was adapted to vero cells as described by alonso-caplen et al. (1984) . briefly, the virus was passaged three times in 11-day-old chicken embryos and then adapted to vero cells (atcc ccl-81) by a series of passages at 24-48 h intervals. the cytopathic effects, including syncytium formation and rounding up of cells, were initially observed after three passages in vero cells. virus stocks were prepared after the 36th passage by infecting monolayers of vero cells at a m.o.i. of approximately 0.1 pfu/cell. the virus was harvested at 24 h postinfection and the titer of the virus preparation was determined by plaque assay on vero cells. vero cells were grown at 37°c in 5% co 2 and maintained in glasgow's modified minimal essential medium (gmem) supplemented with 10% newborn calf serum. confluent monolayers of vero cells were infected with ibv at a m.o.i. of approximately 3 pfu/cell. prior to being labeled, the cells were incubated in methionine-free medium for 30 min. after 4 h of labeling with 25 ci of [ 35 s]methionine, the cells were scraped off the dishes in phosphate-buffered saline (pbs), recovered by centrifugation, and stored at ϫ80°c. open reading frames placed under control of the t7 promoter were expressed transiently in eukaryotic cells as described previously (liu et al., 1994) . briefly, semiconfluent monolayers of vero cells were infected with 10 pfu/cell of a recombinant vaccinia virus (vtf7-3) which expresses the bacteriophage t7 rna polymerase and then transfected with appropriate plasmid dna using the dotap transfection reagent according to the instructions of the manufacturer (roche). after incubation of the cells at 37°c for 4 h, 25 ci/ml of [ 35 s]methionine was added directly to the medium. the radiolabeled cells were harvested at 18 h posttransfection. appropriate primers and template dnas were used in amplification reactions with pfu dna polymerase (stratagene) under standard buffer conditions with 2 mm mgcl 2 . the pcr conditions were 30 cycles of 95°c for 45 s, x°c for 45 s, and 72°c for x min. the annealing temperature (x°c) and the extension time (x min) were adjusted according to the melting temperature of the primers used and the length of the pcr fragments synthesized. plasmid dna-transfected vero cells were lysed with ripa buffer (50 mm tris-hcl, ph 7.5, 150 mm nacl, 1% sodium deoxycholate, 0.1% sds, 1% np-40) and precleared by centrifugation at 12,000 rpm for 5 min at 4°c in a microfuge. immunoprecipitation was carried out as described previously (liu et al., 1994) . sds-polyacrylamide gel electrophoresis (sds-page) of virus polypeptides was carried out using 12.5% polyacrylamide gels (laemmli, 1970) . labeled polypeptides were detected by autoradiography or fluorography of dried gels. cells were grown on four-well chamber slides (iwaki) and infected with ibv or transfected with appropriate plasmid dnas. after washing with pbs, the cells were fixed with 4% paraformaldehyde (in pbs) for 15 min at room temperature and permeabilized with 0.2% triton x-100 (in pbs), followed by incubation with specific antiserum at room temperature for 2 h. antibodies were diluted in fluorescence dilution buffer (pbs with 5% normal goat serum). the cells were then washed with pbs and incubated with anti-rabbit igg conjugated to fluorescein isothiocynate (fitc) (sigma) in the fluorescence dilution buffer at 4°c for 1 h before mounting. confocal microscopy was performed on a zeiss axioplan microscope connected to a bio-rad mrc 1024 laser scanner equipped with an argon laser with appropriate filters. fluorescent images were superimposed to allow fine comparison and colocalization of green (fitc) and red (tritc) signals in a single pixel produces yellow, while separated signals are green or red. plasmid pibv1b4, which contains nucleotides 16932-20490, was previously described . plasmid pibv1b3 contains nucleotides 15132-16931 and codes for the 68-kda protein, pibv1b6 contains nucleotides 16930-18495 and codes for the 58-kda protein, pibv1b9 contains nucleotides 18496-19508 and codes for the 39-kda protein, and pibv1b10 contains nucleotides 19509-20506 and codes for the 35-kda protein. these constructs were made by cloning an ncoi-and bamhi-digested pcr fragment into ncoi-and bamhidigested pkt0 (liu et al., 1994) . the sequences of the two primers used to construct pibv1b3 were 5ј-cgact-tccatggcttgtggcgttј-3 and 5ј-ccaaaggatccta-ttgcagacttg-3ј. the sequences of the two primers used to construct pibv1b6 were 5ј-acaagtcccatggg-tacaggtt-3ј and 5ј-tattggatcctacggagagctg-3ј. the sequences of the two primers used to construct pibv1b9 were 5ј-gtttttcagctcccatggctatcgac-aat-3ј and 5ј-aaccacacgtcggatcctattgaagctg-tg-3ј. the sequence of the two primers used to construct pibv1b10 was 5ј-ccacagcttcccatggcatg-gacgtg-3ј. plasmid pibvpol, which contains nucleotides 12451-15131 and codes for the 100-kda protein with a 37-aminoacid truncation at the n-terminus, was constructed by cloning a bamhi-and xhoi-digested pcr fragment into bamhi-and xhoi-digested pet22b(ϩ) (novagen). the sequences of the primers used were 5ј-gtaataag-gatccagctggtatg-3ј and 5ј-aaggcctcgagttgta-aagtcgtaggagc-3ј. the two dicistronic constructs, p3c-cite-ibv20 and p3c-cite-ibv8, were constructed as follows. the ires sequence was obtained by digestion of pcite-1 (novagen) with ecori, end-repair with klenow, and redigestion with ncoi. this 592-bp fragment was then cloned into pvuii-and ncoi-digested pkt0, giving rise to pkt-cite. the ibv sequence that codes for the 3c-like proteinase was obtained by digestion of pibv3c (liu et al., 1997) with bglii and bamhi and was cloned into bglii-digested pkt-cite, giving rise to p3c-cite. digestion of p3c-cite with ncoi produced a 1510-bp fragment containing both the ibv 3c-like proteinase and the ires sequences. this fragment was then cloned into ncoi-digested pibv1b8 and pibv20, respectively, creating the two dicistronic constructs. plasmids pibv1b8 and pibv20, which cover the ibv sequences from nucleotides 15132 to 18495 and 15132 to 20506, respectively, were constructed by cloning ncoi-and bamhi-digested pcr fragments covering the relevant regions into ncoi-and bamhi-digested pkt0. replication and morphogenesis of avian coronavirus in vero cells and their inhibition by monensin impaired integrinmediated adhesion and signaling in fibroblasts expressing a dominant-negative mutant ptp1b subcellular distribution of normal and mutant vitamin d receptors in living cells four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus comparison of the replication of positive-stranded rna viruses of plants and animals the putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral rna synthesis molecular chaperones eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase protein folding in the cell identification of an atpase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229e identification and subcellular localization of a 41 kda, polyprotein 1ab processing product in human coronavirus 229e-infected cells cleavage of structural proteins during the assembly of the head of bacteriophage t4 characterization of the two overlapping papain-like proteinase domains encoded in gene 1 of the coronavirus infectious bronchitis virus and determination of the c-terminal cleavage site of an 87 kda protein identification of a novel cleavage activity of the first papain-like proteinase domain encoded by orf 1a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products the missing link in coronavirus assembly: retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-golgi compartments and physical interaction between the envelope and membrane proteins induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus characterisation and mutational analysis of an orf 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein a 100-kilodalton polypeptide encoded by open reading frame (orf) 1b of the coronavirus infectious bronchitis virus is processed by orf 1a products identification, expression, and processing of an 87-kda polypeptide encoded by orf 1a of the coronavirus infectious bronchitis virus proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10 kda polypeptide and determination of its cleavage sites proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3c-like proteinase and identification of two novel cleavage products identification of a 24 kda polypeptide processed from the coronavirus infectious bronchitis virus 1a polyprotein by the 3c-like proteinase and determination of its cleavage sites further characterization of the coronavirus infectious bronchitis virus 3c-like proteinase and determination of a new cleavage site processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning 400 kilodaltons of orf1a the human coronavirus 229e superfamily 1 helicase has rna and dna duplexunwinding activities with 5ј-3ј polarity biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases colocalization and membrane association of murine hepatitis virus gene 1 products and de novo-synthesized viral rna in infected cells mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes characterization of endoplasmic reticulum by colocalization of b-p and dicarbocyanine dyes localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication integral membrane proteins of the nuclear envelope are dispersed throughout the endoplasmic reticulum during mitosis processing of the human coronavirus 229e replicase polyproteins by the virus-encoded 3c-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab virus encoded proteinases and proteolytic processing in the nidovirales acknowledgment this work was supported by the national science and technology board of singapore. key: cord-304343-m7tbdfri authors: khandia, rekha; dadar, maryam; munjal, ashok; dhama, kuldeep; karthik, kumaragurubaran; tiwari, ruchi; yatoo, mohd. iqbal; iqbal, hafiz m.n.; singh, karam pal; joshi, sunil k.; chaicumpa, wanpen title: a comprehensive review of autophagy and its various roles in infectious, non-infectious, and lifestyle diseases: current knowledge and prospects for disease prevention, novel drug design, and therapy date: 2019-07-03 journal: cells doi: 10.3390/cells8070674 sha: doc_id: 304343 cord_uid: m7tbdfri autophagy (self-eating) is a conserved cellular degradation process that plays important roles in maintaining homeostasis and preventing nutritional, metabolic, and infection-mediated stresses. autophagy dysfunction can have various pathological consequences, including tumor progression, pathogen hyper-virulence, and neurodegeneration. this review describes the mechanisms of autophagy and its associations with other cell death mechanisms, including apoptosis, necrosis, necroptosis, and autosis. autophagy has both positive and negative roles in infection, cancer, neural development, metabolism, cardiovascular health, immunity, and iron homeostasis. genetic defects in autophagy can have pathological consequences, such as static childhood encephalopathy with neurodegeneration in adulthood, crohn’s disease, hereditary spastic paraparesis, danon disease, x-linked myopathy with excessive autophagy, and sporadic inclusion body myositis. further studies on the process of autophagy in different microbial infections could help to design and develop novel therapeutic strategies against important pathogenic microbes. this review on the progress and prospects of autophagy research describes various activators and suppressors, which could be used to design novel intervention strategies against numerous diseases and develop therapeutic drugs to protect human and animal health. phosphatidylinositol-3-kinase (pi3k) inhibitors and beclin 1 inhibits the starvation-induced mitochondrial autophagy, but not the neurotoxin (1-methyl-4-phenylpyridinium)-mediated autophagy [107] [108] [109] . although autophagy was discovered over 50 years ago [54] , its molecular mechanisms were only understood in the late 1990s following a genetic screening in yeast, which revealed mutations in autophagy-related genes. at least 30 yeast autophagy genes (atgs) have been identified, many of which have mammalian cell homologs [85] . many molecular mechanisms have been explored to reveal the basic processes underlying autophagy. multiple signaling pathways focus on two protein complexes to initiate autophagy, the ulk1 (unc51-like autophagy activating kinase 1) protein kinase complex and the pi3kc3-c1 (class iii phosphatidylinositol 3-kinase complex i) lipid kinase complex [110] . novel autophagy regulators with rna-related activities have also been shown to be involved in this process [111] . furthermore, upstream signaling pathways common to both autophagy and apoptosis are known to be induced by er stress via signaling molecules such as perk/atf4, ire1α, atf6, and ca 2+ [112] . the details of these mechanisms will shed light on the different forms of autophagy and the numerous intermediates involved. three types of autophagy [macroautophagy, microautophagy, and chaperone-mediated autophagy (cma)] are depicted in figure 1 . autophagy refers to the process of delivering cytoplasmic or extracellular components to the lysosomes of an animal cell or the vacuoles of plant or yeast cells [113] . the production and maturation of autophagosomes are directly regulated by location, timing, and intensity [114] . the phosphoinositide-binding protein, hs1bp3, is a negative regulator of autophagosome biogenesis that regulates the lipid composition and phosphatidic acid (pa) levels of autophagosome precursor membranes [114] . increased levels of systemic autophagy have been reported in caenorhabditis macroautophagy is initiated when a portion of cytoplasm containing a cellular organelle is sequestered to form the autophagosome [83] . the autophagosome fuses with the lysosome or late endosomal multivesicular bodies (mvbs) to degrade the materials within it. atg8 (microtubule-associated protein 1a/1b-light chain 3, lc3, is an atg8 homolog in humans) was the first autophagosomal protein to be characterized [119] . macroautophagy can be classified as cargo-specific or non-selective [83, 119, 123] . mitophagy has been observed in yeasts when a shift occurs between non-fermentable and fermentable carbon sources, such as glucose, following which the surplus mitochondrial population undergoes mitophagy [120, 124] . the first protein identified to cause mitophagy in yeast was uth1p, a member of the sun family, which is present in the outer mitochondrial membrane and allows excessive mitochondria to be removed during starvation [125] . the mitochondrial outer membrane protein, atg32, is a receptor for selective autophagy [126] that is not conserved in mammalian species; instead, fundc1 and bnip3, bnip3l/nix, and sqstm1/p62 act as mitophagy receptors, and are dependent upon hypoxia, erythrocyte maturation, and damage-induced mitophagy, respectively [123, 127, 128] . pexophagy is also induced in saccharomyces cerevisiae and pichia pastoris via the atg36 and ppatg30 receptors, respectively, when the fungal medium is switched from an oleic acid or methanol to a glucose or nitrogen starvation medium [129, 130] . starvation has also been shown to induce non-selective macroautophagy [9] , whereas mitochondrial phospholipids have been demonstrated to be required for autophagy [17] . the machinery required for selective autophagy has been studied extensively using yeast cells, revealing that the cytoplasm-to-vacuole targeting (cvt) pathway is used to specifically transport vacuolar hydrolases into the vacuole of budding yeast cells [131] . a high degree of curvature in the initiating membranes (phagophores or isolation membranes) is a prominent feature of cvt vesicles during mammalian autophagy [132] . after the lysosome has formed vesicles by invaginating and engulfing small sections of the cytoplasm, lysosomal proteases degrade the contents of these vesicles [119] . microautophagy occurs during the biogenesis of multi-vesicular bodies (mvbs), which deliver soluble proteins to the late endosomes, and relies on electrostatic interactions between endosomal sorting complexes required for transport (escrt) i and iii and the heat-shock cognate protein 70 (hsc70). hence, microautophagy involves both endocytic and autophagic components [133, 134] . only proteins with a c-terminal pentapeptide kferq motif undergo cma; the hsc70 cochaperone identifies cytosolic proteins containing this sequence and delivers them to the lysosome [135, 136] . chaperones bound to the substrate are transported to the lysosomal surface, where they interact with the monomeric lamp-2a [137, 138] . lamp-2a must form a multiprotein complex to translocate the substrate [139] ; lamp-2a complex assembly is a dynamic process that occurs when the substrate binds to the receptor. the unfolded substrate protein (chaperon-mediated) is then translocated into the lysosome by lamp-2a for degradation, following which lamp-2a disassembles and its monomers are degraded in lipid microdomains. the levels of lamp-2a tightly regulate the rate of cma at the lysosomal membrane [15, 140] . in the mammalian anti-viral defense system, a cell-autonomous autophagy mechanism has been identified wherein cellular p62 adaptor-mediated autophagic viral protein clearance induces cell survival [141] . some positive-strand rna viruses, including picornaviruses and influenza virus, promote autophagic membrane formation, and inhibit their final maturation (lysosomal fusion) [142] [143] [144] . consequently, studying the interactions between autophagy and adenoviruses could improve adenoviral-based oncolytic virotherapies [145] . the process of cma is depicted in figure 2 . after the lysosome has formed vesicles by invaginating and engulfing small sections of the cytoplasm, lysosomal proteases degrade the contents of these vesicles [119] . microautophagy occurs during the biogenesis of multi-vesicular bodies (mvbs), which deliver soluble proteins to the late endosomes, and relies on electrostatic interactions between endosomal sorting complexes required for transport (escrt) i and iii and the heat-shock cognate protein 70 (hsc70). hence, microautophagy involves both endocytic and autophagic components [133, 134] . only proteins with a c-terminal pentapeptide kferq motif undergo cma; the hsc70 cochaperone identifies cytosolic proteins containing this sequence and delivers them to the lysosome [135, 136] . chaperones bound to the substrate are transported to the lysosomal surface, where they interact with the monomeric lamp-2a [137, 138] . lamp-2a must form a multiprotein complex to translocate the substrate [139] ; lamp-2a complex assembly is a dynamic process that occurs when the substrate binds to the receptor. the unfolded substrate protein (chaperon-mediated) is then translocated into the lysosome by lamp-2a for degradation, following which lamp-2a disassembles and its monomers are degraded in lipid microdomains. the levels of lamp-2a tightly regulate the rate of cma at the lysosomal membrane [15, 140] . in the mammalian anti-viral defense system, a cell-autonomous autophagy mechanism has been identified wherein cellular p62 adaptormediated autophagic viral protein clearance induces cell survival [141] . some positive-strand rna viruses, including picornaviruses and influenza virus, promote autophagic membrane formation, and inhibit their final maturation (lysosomal fusion) [142] [143] [144] . consequently, studying the interactions between autophagy and adenoviruses could improve adenoviral-based oncolytic virotherapies [145] . the process of cma is depicted in figure 2 . autophagy is an evolutionarily conserved process induced via multiple signaling pathways by numerous stimuli including nutrient starvation [16, 105] , hypoxia [82, 146] , oxidative stress [109, 147] , pathogen infection [39, 148] , and er stress [149] . in the presence of nutritional substances and cytokines, mechanistic/mammalian target of rapamycin (mtor) can prevent apoptosis and stimulate cell growth [150] , whereas stress and nutrient starvation inhibit mtor to initiate autophagy via at least four molecular complexes, including the unc-51-like kinase (ulk) complex, consisting of ulk-1, atg13, atg101, and fak-family interacting protein (fip200); the pi3k complex, consisting of atg15, vacuolar protein sorting (vps)15, vps34, beclin 1, and beclin 1-regulated autophagy protein 1 (ambra1) [151] [152] [153] ; transmembrane protein complexes, including atg9 and wipi; and two ubiquitin-like protein conjugation systems (atg12 and lc3) [154, 155] . autophagy is initiated by the assembly of the ulk complex, which phosphorylates ambra1 and leads to activation of the pi3k complex [155, 156] . class iii pi3k is known to participate in various membrane trafficking events, whilst pi3k and beclin 1 mediate membrane nucleation. the atg5-atg12-atg16 complex is recruited to the pre-autophagosomal structure (pas) where it associates with the outer membrane of the phagophore, essentially preventing the premature fusion of vesicles and lysosomes [157] . the second ubiquitin-like system stimulates the binding of phosphatidylethanolamine (pe) and atg8/microtubule-associated protein 1 light chain 3 (lc3). lc3 has a high affinity for the lysosome when bound to the phagosome (laposome); thus, any engulfed pathogens will be killed and degraded at a higher rate [158] . atg4, atg7, and atg3 process lc3 into lc3-ii, a molecular marker for autophagosomes [86] that is present on both its inner and outer surfaces and is essential for the expansion and completion of the autophagic membrane. following autophagosomal closure, the atg5-atg12-atg16 complex dissociates from the autophagosome. atg9 is required for the formation of intraluminal vesicles and is localized within the autolysosome for acidification [159] ; atg9 is also translocated to the site of autophagosome formation where it provides a membrane to elongate the limiting membrane, known as the phagophore [160] . the autophagosome then fuses to the lysosome to form the autolysosome, which is regulated by lysosomal membrane proteins and cytoskeletal proteins [2] . the lamp-1/2 protein controls autophagosomal maturation. genetic mutations in lamp-2 are known to cause danon disease, a glycogen storage disorder linked to hypertrophic cardiomyopathy, skeletal muscle weakness, and intellectual disability [161] . within the autolysosome, hydrolytic enzymes digest the internalized cargo and the internal autophagosome membrane, then the digested products such as amino acids are released into the cytosol to be recycled. autophagosomes are also directly related to cell trafficking pathways. recently, holland et al. identified that the phosphoinositide-binding protein hs1bp3 negatively regulates autophagosome production [162] . hs1bp3 is thought to reduce phospholipase d1 (pld1) activity and its localization to atg16l1 and transferrin receptor (tfrc)-positive vesicles. it is also known to regulate the levels of pa and the lipid content of autophagosome precursor membranes [114] . two large families of e3 ubiquitin ligases, trim and cullin, have been recognized as important autophagy regulators which promote or inhibit the process, respectively [81] . the gtpase ras-related protein in brain 7 (rab7) also plays a key role in autophagy regulation, particularly in modulating its flux [163] . knockdown of the small gtpase rab13 has been shown to inhibit pterostilbene-induced autophagy in vascular endothelial cells (vecs), whilst its upregulation stimulates autophagy in vecs [164] . under basal autophagy conditions in humans, proteomic analysis of the autophagy interaction network (ain) revealed a network of 751 interactions between 409 candidate proteins [165] . in order to identify the proteins modulating starvation-induced autophagy, genome-wide screening of sirna in a gfp-lc3-expressing human cell line was carried out [166] , shortlisting nine proteins. one of these, short coiled-coil protein (scoc), forms an essential starvation-sensitive trimeric complex with uv radiation resistance-associated gene (uvrag) and wac (ww domain-containing adapter protein with coiled-coil), which is a negative regulator of the ubiquitin-proteasome system. genome-wide studies in c. elegans identified 139 genes that promote autophagy when inactivated [167] . long ncrnas (lncrnas), which are longer than 200 nucleotides and do not encode proteins, often possess regulatory functions; for example, mir188-3 has been found to regulate atg7 expression. rna-linked strategies have revealed several autophagy regulators such as rna-binding proteins (rbps), which are post-transcriptional and co-translational regulators with rna-related functions. surprisingly, various key autophagy proteins, including lc3b and lamp-2c, have been found to bind rna [111] . a considerable amount of autophagy research is being carried out worldwide; however, these innovative findings have raised numerous additional questions. to some extent, autophagy research has been protein-centric, and innovative new approaches have been developed to strengthen this focus in recent years. among these, genome-wide screens and proteomics-based strategies have revealed substantial interlinking between autophagy and rnas; however, the precise mechanisms underlying this association require further investigation. future studies must develop and evaluate novel agents that specifically target the autophagy pathway. a pictorial representation of the process of autophagosome formation is presented in figure 3 . starvation-sensitive trimeric complex with uv radiation resistance-associated gene (uvrag) and wac (ww domain-containing adapter protein with coiled-coil), which is a negative regulator of the ubiquitin-proteasome system. genome-wide studies in c. elegans identified 139 genes that promote autophagy when inactivated [167] . long ncrnas (lncrnas), which are longer than 200 nucleotides and do not encode proteins, often possess regulatory functions; for example, mir188-3 has been found to regulate atg7 expression. rna-linked strategies have revealed several autophagy regulators such as rna-binding proteins (rbps), which are post-transcriptional and co-translational regulators with rna-related functions. surprisingly, various key autophagy proteins, including lc3b and lamp-2c, have been found to bind rna [111] . a considerable amount of autophagy research is being carried out worldwide; however, these innovative findings have raised numerous additional questions. to some extent, autophagy research has been protein-centric, and innovative new approaches have been developed to strengthen this focus in recent years. among these, genomewide screens and proteomics-based strategies have revealed substantial interlinking between autophagy and rnas; however, the precise mechanisms underlying this association require further investigation. future studies must develop and evaluate novel agents that specifically target the autophagy pathway. a pictorial representation of the process of autophagosome formation is presented in figure 3 . liu and levine [168] described a novel form of non-apoptotic autophagic gene-dependent cell death, termed autosis, which is mediated by the na + /k + -atpase pump. autosis involves enhanced cell-substrate adhesion, focal ballooning of the perinuclear space, and dilation and fragmentation of the endoplasmic reticulum. the tat-beclin 1 peptide complex may initiate autosis, with the fusion of the evolutionarily conserved, 18-amino acid-long beclin 1 domain with 11 amino acids from the hiv tat protein transduction domain aiding the cellular entry of the fusion peptide [169] . the tat-beclin 1 fusion peptide has been shown to inhibit the replication of hiv, chikv, sindbis (sinv), and wnv, as well as intracellular bacteria such as listeria monocytogenes [23] . tat-beclin 1 treatment also reduced mortality in neonatal mice infected with chikv and wnv, demonstrated using a tunel assay, and cleared mutant huntingtin protein aggregates [170] . autosis can be partially rescued by knocking down atg13 or atg14 or using 3-methyladenine. under serum/amino acid starvation, approximately 1% of dying cells exhibit a morphology similar to that of cells treated with tat-beclin 1 and autosis is selectively blocked when the na + /k + -atpase pump is inhibited [171] . during cerebral hypoxia or ischemia, the neonatal brain releases cardiac glycosides (ouabain or endobain), which inhibit the na + /k + -atpase pump and reduce autosis [172] . autosis has also been observed in patients with severe liver anorexia nervosa who display focal ballooning of the perinuclear space, convoluted nucleus, dilated and fragmented er, empty vacuoles, and several autolysosomes in their hepatocytes [173] . ischemic injury can also lead to autosis in other organs, including the kidney and heart, which is attenuated in beclin 1 +/− mice [168, 174] . autophagy can promote or inhibit cell death depending on the cellular context; many other death mechanisms are intricately involved in the processes, with several mechanistic links elucidated between autophagy and other death mechanisms. autophagy and apoptosis regulation overlap when the bh3 domain of the beclin 1 autophagy protein interacts with anti-apoptotic proteins of the bcl-2 family, including bcl-2, bcl-xl, and mcl-1 [175] [176] [177] [178] . the bh3 domain has a critical role in the interaction between these proteins and has been shown to interact with the receptor domain of the bcl-2 family in nutrient-rich cells [178] . beclin 1-mediated autophagy is inhibited by er-localized bcl-2 [179] ; the transgenic expression of bcl-2 was shown to inhibit autophagy in mouse heart muscles. beclin 1 mutants, which are unable to bind to bcl-2, induce higher levels of autophagy than their wild-type counterparts [179, 180] ; hence the physical beclin 1-bcl-xl/bcl-2 interaction regulates beclin 1-mediated autophagy [179] . abt737, a compound which mimics the bh3 domain and thus inhibits this interaction, increases the aggregation of lc3, an autophagy marker which is present on autophagosomes [181] , in both nutrient-rich and nutrient-deprived media. furthermore, the knockdown of beclin 1 and other essential atg proteins using sirna heteroduplexes was shown to reduce abt737-stimulated lc3 aggregation [168] . atg12 is a dual-functioning protein that participates in both autophagy and apoptosis [108] ; non-conjugated atg12 can bind and inhibit mcl-1 and bcl-2 via a bh3-like domain to positively regulate mitochondrial apoptosis. atg12 knockout inhibits the release of cytochrome c from the mitochondria and apoptosis, whilst abnormal atg12 expression represses the anti-apoptotic activity of mcl-1 [182] . autophagy promotes apoptosis by degrading a negative regulator of the fas ligand [183] ; however, it can also protect cells against apoptosis induced by tumor necrosis factor (tnf)-related apoptosis-inducing ligand (trail) by altering the concentrations of bcl family members [184] . similarly, components were found to be degraded by autophagy during developmental apoptosis [185] , whilst it was recently shown that inhibiting autophagy increased apoptosis and accelerated mortality in murine sepsis models with inadequate autophagy pathways in cd4 + t cells, indicating that autophagy has a functional role against apoptosis and immunosuppression in t cells in sepsis [186] . furthermore, trail combined with a novel chalcone derivative, chal-24, was found to remarkably increase lung cancer cell cytotoxicity via autophagy-mediated apoptosis [187] . necroptosis is often associated with inflammation [63] . the relationship between autophagy and necroptosis is complex, elusive, and slightly controversial since reports have indicated that necroptosis may promote [188] , inhibit [189, 190] , or do not affect autophagy [191] . in several cell lines, including l929 cells, lymphocytes, and cancer cells, autophagy is activated in the presence of tnfα and under starvation to suppress necroptosis [190] . the apoptosis-inhibiting peptide, carbobenzoxy-val-ala-asp (zvad), prevents autophagy and induces necroptosis in response to tnfα by regulating lysosomal cathepsins, highlighting the pro-survival function of autophagy against necroptosis [192, 193] . similarly, inhibiting the mtor signaling pathway can prevent apoptosis and even enhance necroptosis, whereas starvation, which induces autophagy, protects cells from zvad-mediated necroptotic death [194] . sirtuins (sirt) are nad + -dependent protein deacetylases which are actively involved in both autophagy and necroptosis, as well as transcription, stress resistance, and aging. sirt-1 deacetylates various components of the autophagy pathway, including atg5, atg7, and atg8 [195] , thus promoting autophagy. in cancer cells, dissociation of the foxo1 transcription factor from sirt-2 during oxidative stress or starvation results in the acetylation and binding of foxo1 to atg7, which subsequently induces autophagy [196] . the binding of sirt-2 to receptor-interacting protein (rip) 3 mediates rip1 deacetylation in response to tnfα; rip1 and rip3 then form a complex, which triggers necroptosis [197] . the switch between necroptosis and apoptosis is achieved by recruiting necrosome components to autophagy machinery. atg5 knockdown reduced the association between ripk1 and mlkl, suggesting that atg5 is important in trail-induced necrosome activation. furthermore, atg5 knockout in the atg5 -/-df-1 cell line inhibited autophagy but promoted apoptosis [198] . autophagy machinery also affects the mechanism of cell death by promoting efficient necrosomal activation and mlkl phosphorylation, thus inducing necroptosis [199] . several anti-cancer agents, including sorafenib, cause deficient autophagosome formation and facilitate the interaction between p62 and ripk, resulting in cell death by necroptosis [200] ; however, there is still much to be elucidated about the interplay between these two processes. necrosis refers to the increase in cell volume caused by organelle swelling, which results in plasma membrane rupture and the loss of intracellular contents. when atp is depleted, the cell is unable to undergo apoptosis and undergoes necrosis instead [201] . poly adp ribose polymerase (parp1) is an enzyme with roles in dna repair, transcriptional regulation, and chromatin modification [202] . parp1 over-activation decreases the atp reservoir and induces necrotic cell death by bypassing energy-dependent apoptotic cell death [203] . atp depletion also activates amp-activated kinases (ampk) [204] , which induce autophagy by activating the ulk1 complex or inhibiting mtor signaling [205] . thus, dna damage-induced parp1 activation leads to a decline in atp levels, ampk activation, mtor inhibition, and autophagy induction [206] . parp1 plays a dual role in autophagy and necrosis since autophagy is a pro-survival mechanism, whilst necrosis is a pro-death mechanism. the fate of the cell depends on the balance between autophagy and necrosis, where autophagy represents the final attempt of the cell to survive before necrosis. autophagy plays a beneficial role against infectious diseases by simultaneously degrading pathogens and activating the host immune system [91] . this enables infections to be countered directly, by killing infectious agents, and indirectly, by inducing host immunity against pathogens. autophagy provides an excellent intracellular defense system against bacterial pathogens, including salmonella enterica serovar typhimurium [51, 207] , listeria monocytogenes [50, 208] , and shigella flexneri [209] . anti-bacterial autophagy is termed xenophagy [21, 53, 210] . numerous cellular, membrane-associated, or cytoplasmic moieties modulate xenophagy; and those cells unable to carry out xenophagy, exhibit higher rates of infection. bcl-xl regulates xenophagy, and bcl-xl knockout cells are more susceptible to streptococcus pyogenes infection [53] . the infection of non-phagocytic cells by shigella flexneri is dependent upon type-iii secretion system (t3ss) effector proteins [52] which reorganize the host cell cytoskeleton, ruffle the cell membrane, and cause bacterial uptake. following internalization, bacterial peptidoglycans are detected by nucleotide-binding oligomerization domain (nod)-like receptors (nlrs) which trigger a pro-inflammatory immune response [211] (figure 4 ). the bacteria-sensing nod proteins interact with atg16l1 to initiate anti-bacterial autophagosome biogenesis in response to bacterial invasion [212] . intracellular bacterial sensing either by nlrs or sequestosome-1-like receptors (slrs) recruits autophagy proteins, including unc-51-like kinase (ulk) 1/2 and lipid kinase complexed with beclin 1 and atg16l1, to initiate phagophore membrane nucleation and engulf invading bacteria [213] . mutant c. elegans with defective autophagy genes exhibit increased susceptibility to bacterial infection [214] . in addition, it has been reported that hlh-30/tfeb-mediated autophagy and autophagy pathways can regulate the tolerance of c. elegans to bacillus thuringiensis infection by protecting against its pore-forming toxins [215] , suggesting a novel association between intrinsic epithelial defenses and hlh-30-mediated autophagy against in vivo bacterial attacks. liang et al. [176] reported that beclin 1 overexpression inhibits sindbis virus replication, indicating that autophagy protects against infectious pathogens. autophagy may activate innate immunity against mycobacteria via pattern recognition receptors (prrs) or non-receptor-mediated processes [49] . infection with group a streptococcus species (gas; streptococcus pyogenes) induces anti-apoptotic bcl-xl expression which inhibits autophagy directly by suppressing autophagosome-lysosome fusion, and indirectly by interacting with beclin 1-uvrag to suppress gas internalization [53] . in addition, mycobacterium tuberculosis is known to induce mir144 expression in human macrophages and monocytes and adversely affect their antimicrobial activities and innate host immune responses against the bacterial infection by targeting dram2 (dna damage-regulated autophagy modulator 2), which is a critical element of the autophagy response [216] . the ubiquitin ligase, smurf1, has also been shown to control m. tuberculosis replication in human macrophages by associating with bacteria in the lungs of patients with pulmonary tuberculosis. the murine macrophage cell line, raw264.7, has been used to study bacillus amyloliquefaciens sc06-induced autophagy and its anti-bacterial response against escherichia coli; b. amyloliquefaciens stimulated autophagy by increasing the expression of beclin 1 and the atg5-atg12-atg16 complex, but not activating the akt/mtor signaling pathway [217] . several autophagy-inducing drugs have been used to treat microbial infections; for example, ar-12 [2-amino-n-[4-[5-(2 phenanthrenyl)-3-(trifluoromethyl)-1h-pyrazol-1-yl] phenyl]-acetamide] inhibits phosphoinositide-dependent kinase-1 and eliminates salmonella typhimurium in murine macrophages and francisella tularensis in human leukemic thp-1 macrophages [218, 219] . furthermore, 1α,25-dihydroxycholecalciferol, a form of vitamin d, can enhance autophagy and inhibit human immunodeficiency virus (hiv) replication in macrophages [220] . the bacteria-sensing nod proteins interact with atg16l1 to initiate anti-bacterial autophagosome biogenesis in response to bacterial invasion [212] . intracellular bacterial sensing either by nlrs or sequestosome-1-like receptors (slrs) recruits autophagy proteins, including unc-51-like kinase (ulk) 1/2 and lipid kinase complexed with beclin 1 and atg16l1, to initiate phagophore membrane nucleation and engulf invading bacteria [213] . many bacteria have evolved mechanisms to overcome autophagy and allow them to replicate within infected cells or even within autophagosomes. these bacteria may express receptors to prevent or enhance phagosome formation, capture nutrient containing phagosomes, subvert autophagy machinery, prevent fusion, or resist autophagy. certain bacteria hijack autophagosomes and use the by-products of autophagic degradation for microbial replication [221] . anaplasma (formerly ehrlichia) phagocytophilum, the causative agent of human anaplasmosis, uses the effector anaplasma translocated substrate 1 (ats-1) to enhance autophagosome formation and acquire nutrients from inside the autophagosome [222] . after entering the cell, a. phagocytophilum replicates inside a double-lipid bilayer membrane associated with lc3 (atg8), beclin 1, and atg6 but lacking lysosomal markers. inhibiting autophagy with 3-methyladenine did not prevent bacterial internalization but arrested its growth [223] , indicating that the autophagic machinery had been subverted to facilitate bacterial proliferation. another bacterium, yersinia pseudotuberculosis, replicates intracellularly inside specific compartments called yersinia-containing vacuoles (ycvs), which contain autophagy markers; however, ycvs are not acidified and sustain bacterial replication [224] . during y. pestis infection, lc3-i is conjugated with pe to recruit lc3-ii, a marker of autophagy progression, to the phagosomal membrane [225] . a similar mechanism is used by coxiella burnetii, the causative organism of q fever. coxiella-replicative vacuoles contain lc3, beclin 1, and rab24; overexpression of these proteins increases the number of coxiella-replicative vacuoles [226] . brucella abortus replicates within brucella-containing vacuoles (bcvs) which traffic from the endocytic compartment to the endoplasmic reticulum, where the bacteria proliferate. bacterial proliferation requires the autophagy-initiation proteins ulk1, beclin 1, and atg14l; however, atg5, atg16l1, atg4b, atg7, and lc3b are not required [227] . pathogens such as brucella spp. and porphyromonas gingivalis have evolved to survive inside autophagosomes by preventing its fusion with the lysosome, thus escaping host innate immunity mechanisms [228, 229] . salmonella typhimurium studies have revealed that autophagy targets invading intracellular bacterial pathogens for degradation [230] ; s. typhimurium regulates the sirt1/lkb1/ampk complex of the mtor pathway by targeting sirt1, lkb1, and ampk to lysosomes for rapid degradation, restricting autophagy and disrupting ampk-mediated mtor regulation [231] . autophagy is differentially regulated in tuberculoid and lepromatous leprosy [232] ; in tuberculoid skin lesion cells, autophagy controls mycobacterium leprae, whereas in lepromatous cells, the blocking of bcl-2-mediated autophagy promotes bacterial persistence. ifn-γ may counteract the m. leprae-mediated inhibition of autophagy in lepromatous macrophages as autophagy levels were restored in lepromatous patients who developed the reversal reaction, an inflammatory state associated with augmented ifn-γ and rapamycin treatment, indicating that autophagy is an important innate mechanism associated with m. leprae control in skin macrophages [232] . autophagy has a beneficial role in cellular defense against invasion by viruses; therefore, it has been used for antiviral immunity [141, 233, 234] . autophagy helps to clear viral pathogens during infection via various molecular mechanisms, regulates immune responses, and prevents harmful overactivation and inflammation [235] . for example, autophagy increases the presentation of endogenous viral antigens in the peptide grooves of major histocompatibility complex (mhc) class i molecules on the cell surface during herpes simplex virus type 1 (hsv-1) infection. studies of viral peptides have suggested a complex interaction between vacuoles and mhc class i presentation pathways in autophagosomes [236] . in contrast, mhc class ii molecules continuously accept input from autophagosomes, which facilitates antigen presentation by mhc class ii molecules [237, 238] . autophagy is a major component of drosophila immunity against vesicular stomatitis virus (vsv) [239] as it can deliver viral antigens to tlrs for presentation. during anti-viral signaling, pattern recognition receptors (prrs) at the plasma membrane (i.e., toll-7) that are engaged by vsv stimulate an autophagy-dependent innate immune response mediated by pi3k-akt-signaling [239, 240] . furthermore, toll/tlr signaling has been shown to regulate the rift valley fever virus (rvfv) replication in both flies and mammals [241] , whilst sirt1, an nad(+)-dependent deacetylase, modulates the activation of dendritic cells and autophagy during induced immune responses against respiratory syncytial virus (rsv) [242] , thereby directing an effective anti-viral immune response. furthermore, autophagy is stimulated by the salicylamide derivatives against cytopathic bovine viral diarrhea virus (cp-bvdv), a flaviviridae pestivirus [243] . foot-and-mouth disease virus (fmdv) infection suppresses autophagy and nf-κb anti-viral activities by degrading atg5-atg12 using the viral protein, 3c pro , suggesting that atg5-atg12 positively modulates anti-viral nf-κb and irf3 pathways during fmdv infection to limit fmdv proliferation [244] . however, autophagy is often hijacked by viral pathogens and can be modulated to their own benefit. subverting the autophagic pathway can have adverse consequences by giving pathogens access to nutrients for growth and reproduction [245] . autophagy plays an important role in viral replication and pathogenesis [246] , with coronaviruses [247] , coxsackievirus b3 [248] , poliovirus [249] , hepatitis c virus (hcv) [142, [250] [251] [252] , and denv [143] all known to stimulate and require autophagy for accelerated replication. for instance, autophagy has been demonstrated to be actively involved in the replication of influenza a virus (iav), which induces autophagosome formation during the early phase of infection and later inhibits autophagosomal maturation by preventing autophagosomal-lysosomal fusion and promoting autophagosomes to accumulate in virus-infected cells [253] . autophagy-deficient cells are more susceptible to apoptosis upon influenza infection [253, 254] , while using pharmacological reagents or rna interference to alter cellular autophagy can impair viral protein accumulation [255] . human single-chain antibody variable fragments (scfvs) which bind to the influenza a virus ion channel protein (m2) and inhibit viral replication [256] were found to restore autophagosome maturation suppressed by the infecting virus (personal communication). it has also been reported that hcv can trigger autophagy via immunity-related gtpase m, which promotes hcv replication [257] . paramyxoviruses such as newcastle disease virus (ndv) have been shown to trigger autophagy in u251 glioma cells to enhance viral replication [258] . in addition, modulating ndv-induced autophagy using rapamycin, chloroquine, or small interfering rnas which target genes critical for autophagosome formation (atg5 and beclin 1) affects virus production, suggesting that ndv may utilize autophagy to promote its replication [259] . human immunodeficiency virus (hiv) uses multiple methods to regulate autophagy and enhance its replication [260, 261] . hiv induces the early stages of autophagy but inhibits the later stages which would suppress the production of new virions. the hiv-1 accessory protein, nef, inhibits autophagosomal maturation by interacting with beclin 1 [262] , whilst the hiv protein vpr can trigger autophagy in transfected thp-1 macrophages, indicating that autophagy may be involved in maintaining hiv reservoirs in macrophages [263] . hsv-1 [264] , kaposi's sarcoma-associated herpesvirus (kshv) [265] , and mouse herpesvirus 68 (mhv-68) encode proteins that bind beclin 1 to prevent autophagy initiation [266] . during poliovirus (pv) infection, vesicle acidification, which can mature autophagosomes, has been shown to induce the maturation of virions into infectious particles [267] . one of the most important characteristics of high-risk human papillomavirus (hrhpv) etiopathogenesis is that inhibiting host autophagy could cause cervical cancer via hrhpv [268] . in epithelial cells, flavivirus ns4a-induced autophagy protects infected cells and induces viral replication [269] . autophagy also plays a critical role in the replication of coronaviruses and the generation of their replicative structures [270] . coronavirus nonstructural proteins (nsp6) induce the formation of omegasomes and autophagosomes from the er via an omegasome intermediate [271] . in addition, autophagy has been shown to induce the replication of infectious spleen and kidney necrosis virus (isknv) in the chinese perch brain (cpb) cell line, suggesting complex interactions between isknv and host cells during viral pathogenesis and for anti-viral treatment strategies [246] . treating fmdv-infected cells with rapamycin, an autophagy inducer, was shown to increase viral replication, whilst inhibiting the autophagosomal pathway using 3-methyladenine or small-interfering rnas decreased viral replication [272] . furthermore, disrupting autophagy using the knockdown approach in hepatitis c virus (hcv)-infected hepatocytes stimulated the interferon signaling pathway and induced apoptosis, indicating that hcv-induced autophagy can impair the innate immune response [251] . suppressing hcv-induced autophagy could be a promising approach for inhibiting exosome-mediated viral transmission [273] , besides autophagy has been shown to reduce hcv clearance following ifn-α/ribavirin (rbv)-based anti-viral therapy [274] . a denv study revealed that autophagy inhibitors are better candidate targets than conventional anti-viral therapies using interferons (ifns) [275] ; upregulating cellular autophagy was reported to inhibit rlr-mediated type-i ifn-independent signaling and cause the antibody-dependent enhancement (ade) of denv [274] . suppressing autophagic vacuoles has been demonstrated to stimulate the maturation of infectious bursal disease virus [276] . adenoviral infection may be favored by autophagy via an increase in atp, essential to increase anabolism of the infected cells and amino acid pools for the synthesis of viral proteins. in the later stages of adenoviral infection, atg12-atg5 complex is significantly upregulated as an evidence of enhanced autophagy [277] ; therefore, autophagy may improve the virulence of some viruses. autophagy genes are involved in the regulation and execution of autophagy [278] . beclin 1 was the first mammalian gene identified to stimulate autophagy [279] . some viruses, such as α-, βand γ-herpesviruses, encode the neurovirulence protein, icp34.5, which associates with atg6/beclin 1 and inhibits autophagy by preventing the formation of the pi3 kinase complex [264] . the autophagy genes fip200, beclin 1, atg14, atg16l1, atg7, atg3, and atg5 have been found to promote the reactivation of latent murine gamma-herpesvirus 68 by inhibiting virus-induced systemic inflammation molecules, such as ifn-γ [280] . in contrast, autophagy inhibition has been reported as a new molecular mechanism by which hsv-1 escapes innate immunity, resulting in fatal disease [281] . the autophagic cell death of alveolar epithelial cells has been observed to play a major role in the high mortality rate caused by h5n1 influenza virus infection; hence autophagy-blocking agents could have preventative and therapeutic effects against this virus [282] . activating the pi3k /akt/mtor pathway and inhibiting autophagy have been shown to promote the cellular entry of hpv type 16 [283] , contrarily autophagy has been shown to be essential for the replication of coronavirus and mouse hepatitis virus (mhv) [284] . hsv-1 mutants, those are unable to inhibit autophagy grows to low virus titer and are less pathogenic [264] . autophagy evokes antiviral adaptive immunity via the endogenous presentation of viral antigens through the mhc class ii pathway [278] . the proviral and anti-viral actions of autophagy are illustrated in figure 5 . [280] . in contrast, autophagy inhibition has been reported as a new molecular mechanism by which hsv-1 escapes innate immunity, resulting in fatal disease [281] . the autophagic cell death of alveolar epithelial cells has been observed to play a major role in the high mortality rate caused by h5n1 influenza virus infection; hence autophagy-blocking agents could have preventative and therapeutic effects against this virus [282] . activating the pi3k /akt/mtor pathway and inhibiting autophagy have been shown to promote the cellular entry of hpv type 16 [283] , contrarily autophagy has been shown to be essential for the replication of coronavirus and mouse hepatitis virus (mhv) [284] . hsv-1 mutants, those are unable to inhibit autophagy grows to low virus titer and are less pathogenic [264] . autophagy evokes antiviral adaptive immunity via the endogenous presentation of viral antigens through the mhc class ii pathway [278] . the proviral and anti-viral actions of autophagy are illustrated in figure 5 . initially, autophagy was thought to be involved in tumor suppression by stimulating gene expression, inhibiting proinflammatory mediators, inhibiting inflammation or inflammatory products, and stimulating signaling pathways. the essential atg6/beclin 1 gene was found to be lost monoallelically in 40-75 % of human prostate, breast, and ovarian cancers [285] , whereas excessive autophagy stimulation by beclin 1 overexpression has been reported to inhibit tumor progression [286, 287] . autophagy causes necrosis and chronic inflammation by inhibiting the release of proinflammatory hmgb1, which is involved in tumorigenesis [288] . in cell-based assays, inhibiting autophagy was shown to enhance cancer cell growth [289] . p62 (a signaling adaptor/scaffold protein) is involved in the formation of intracellular ubiquitin-related protein aggregates because of autophagy deficiency. atg7-deficient mice exhibit enhanced accumulation of p62 and ubiquitinated protein aggregates in hepatocytes and neuron [290] . autophagy has also been implicated in benign hepatomas [291] , and the inactivation of beclin 1 and atg5 was shown to increase the incidence of initially, autophagy was thought to be involved in tumor suppression by stimulating gene expression, inhibiting proinflammatory mediators, inhibiting inflammation or inflammatory products, and stimulating signaling pathways. the essential atg6/beclin 1 gene was found to be lost monoallelically in 40-75 % of human prostate, breast, and ovarian cancers [285] , whereas excessive autophagy stimulation by beclin 1 overexpression has been reported to inhibit tumor progression [286, 287] . autophagy causes necrosis and chronic inflammation by inhibiting the release of pro-inflammatory hmgb1, which is involved in tumorigenesis [288] . in cell-based assays, inhibiting autophagy was shown to enhance cancer cell growth [289] . p62 (a signaling adaptor/scaffold protein) is involved in the formation of intracellular ubiquitin-related protein aggregates because of autophagy deficiency. atg7-deficient mice exhibit enhanced accumulation of p62 and ubiquitinated protein aggregates in hepatocytes and neuron [290] . autophagy has also been implicated in benign hepatomas [291] , and the inactivation of beclin 1 and atg5 was shown to increase the incidence of cancer in mice [292] . atg5-and atg7-deficient mice exhibited liver tumors, indicating that defective autophagy can affect the suppression of tumorigenesis [292] . heterozygous beclin 1 (beclin 1+/-mutant) was shown to have a high incidence of spontaneous tumors [289, 293] , whilst beclin 1 inhibited tumor growth in cell lines such as the breast cancer cell line, mcf-7, in which beclin 1 expression was lower than in normal epithelial breast cells [294] . the uvrag protein was found to suppress the tumorigenicity and proliferation of human colonic cancer cells [295] (figure 6 ). cancer in mice [292] . atg5-and atg7-deficient mice exhibited liver tumors, indicating that defective autophagy can affect the suppression of tumorigenesis [292] . heterozygous beclin 1 (beclin 1+/mutant) was shown to have a high incidence of spontaneous tumors [289, 293] , whilst beclin 1 inhibited tumor growth in cell lines such as the breast cancer cell line, mcf-7, in which beclin 1 expression was lower than in normal epithelial breast cells [294] . the uvrag protein was found to suppress the tumorigenicity and proliferation of human colonic cancer cells [295] (figure 6 ). (4) mtor is implicated in cancer and its substrates include the eukaryotic initiation factor 4e (eif4e)-binding proteins (4e-bps) and the ribosomal s6 kinases (s6ks) 1 and 2, which promote cell cycle progression. the mtor, which is inhibited by rapamycin, induces autophagy. (5) a novel anti-cancer molecule, ha15, which targets hspa5/bip was shown to induce endoplasmic reticulum stress and increase the unfolded protein response, resulting in cancer cell death through autophagy and apoptosis. autophagy suppresses tumor formation by preventing inflammation, the accumulation of proteins and organelles damaged by necrosis, and cellular transformation caused by gene instability [296] [297] [298] [299] . the conserved protein kinase, mtor, has been implicated in cancer since its substrates (eukaryotic initiation factor 4e (eif4e)-binding proteins (4e-bps) and ribosomal s6 kinases (s6ks) 1 and 2) promote cell cycle progression [299] . mtor, which is inhibited by rapamycin [300] , and molecules such as phosphatase and tensin homolog (pten) and tuberous sclerosis (tsc) (products of tumor suppressor genes) can induce autophagy [301, 302] . pogostone, a medicinal herb widely used to treat gastrointestinal diseases, was shown to possess anti-colorectal tumor activities by stimulating autophagy and apoptosis via the pi3k/akt/mtor axis [303] . in addition, the novel anti-cancer 4) mtor is implicated in cancer and its substrates include the eukaryotic initiation factor 4e (eif4e)-binding proteins (4e-bps) and the ribosomal s6 kinases (s6ks) 1 and 2, which promote cell cycle progression. the mtor, which is inhibited by rapamycin, induces autophagy. (5) a novel anti-cancer molecule, ha15, which targets hspa5/bip was shown to induce endoplasmic reticulum stress and increase the unfolded protein response, resulting in cancer cell death through autophagy and apoptosis. autophagy suppresses tumor formation by preventing inflammation, the accumulation of proteins and organelles damaged by necrosis, and cellular transformation caused by gene instability [296] [297] [298] [299] . the conserved protein kinase, mtor, has been implicated in cancer since its substrates (eukaryotic initiation factor 4e (eif4e)-binding proteins (4e-bps) and ribosomal s6 kinases (s6ks) 1 and 2) promote cell cycle progression [299] . mtor, which is inhibited by rapamycin [300] , and molecules such as phosphatase and tensin homolog (pten) and tuberous sclerosis (tsc) (products of tumor suppressor genes) can induce autophagy [301, 302] . pogostone, a medicinal herb widely used to treat gastrointestinal diseases, was shown to possess anti-colorectal tumor activities by stimulating autophagy and apoptosis via the pi3k/akt/mtor axis [303] . in addition, the novel anti-cancer molecule ha15, which targets hspa5/bip, was shown to induce er stress and increase the unfolded protein response, resulting in cancer cell death via autophagy and apoptosis [304] . trichosanthin (tcs), a 27 kda protein from the bioactive component of the root tuber of trichosanthes kirilowii (chinese cucumber plant; gua lou in mandarin), also exhibited anti-cancer properties against different human ovarian cancer cells via a pathway common to both autophagy and apoptosis [305] . various factors and mechanisms are involved in autophagy-mediated tumor progression. tumor-induced nutrient shortage, cell debris (degraded proteins), inflammation, and oxidative cascades are all molecular mechanisms affecting tumor progression. during nutrient starvation, autophagy induction promotes the survival of normal cells and may also promote tumor cell survival; however, hypoxia, metabolic stress, energy shortage, oxidative stress-damaged mitochondria, and organelles can be caused by cancer-causing genes or cancer treatments [306] . the undifferentiated colon cancer cell line, ht-29, and other transformed cells have shown an increased tendency to degrade autophagic proteins [287, 307, 308] . the elevated expression of the autophagy signature protein, bnip3, a pro-apoptotic bcl-2 member, has been demonstrated in colorectal and gastric epithelial carcinomas, suggesting that bnip3 expression may be important for the development of these cancers [309] . the activation of autophagy and peroxisome proliferator-activated receptor gamma (pparγ) was shown to protect colon cancer cells against apoptosis induced by the interaction between butyrate and docosahexaenoic acid (dha) in a cell type-dependent manner [310] . additionally, an atg8/lc3 family member implicated in autophagy and tumor suppression was associated with alterations to cell death and cytokine secretion in mice lacking gamma-aminobutyric acid receptor-associated protein (gabarap) [311] . it has been well documented that inhibiting autophagy in cancer cells increases their death, with this strategy proving most useful in tumors that behave like ras-activated tumors [312] . inhibiting autophagy is expected to cause ubiquitinated proteins to accumulate and p62 levels to increase; in hepatic tumors, autophagy suppresses spontaneous tumorigenesis via cell-intrinsic pathways whilst p62 accumulation promotes tumor formation [291] . the elimination of damaged organelles via autophagy may allow cancer cells to survive despite the stress caused by chemotherapeutic agents [313] . it has also been reported that upregulating autophagy after chemotherapy causes cancer cells to enter a dormant state, which may then propagate at a later stage [314, 315] . the state of cell cycle arrest, termed senescence, has been postulated to underlie autophagy-induced tumor cell dormancy [316] . ras-induced senescence is mediated by autophagy, with autophagy inhibition delaying senescence [317] . moreover, it has been reported that psmd10/gankyrin stimulates autophagy in hepatocellular carcinoma (hcc) in response to starvation or stress. a physical association occurs between psmd10 and atg7, and is translocated to the nucleus to bind to atg7 promote and upregulates atg7 expression [318] . there is growing evidence that autophagy performs both physiological and pathological functions in the nervous system, and that autophagic stimulation plays critical roles in neuronal survival and activity. autophagy is the only method by which neurons degrade and excrete expired organelles, and is responsible for clearing abnormal intracytoplasmic contents from normal cells which would otherwise cause protein accumulation and damage neuronal activity, inducing severe functional impairment. autophagy also clears protein aggregates from old neurons; thus, inhibiting autophagy can lead to neuronal degeneration and intraneuronal protein accumulation. mutations in atg5 confined to neural tissues can cause impaired growth, progressive motor and behavioral deficits, prominent neurodegeneration, and axonal swelling in regions of the brain with increased levels of ubiquitinylated proteins, indicating that autophagy has a neuroprotective role [319] . in mammals, the absence of atg59 and atg710 can cause severe neurodegeneration, further supporting the neuroprotective role of autophagy [100, 320] . furthermore, neuronal death can be attributed to the loss of basal autophagy or an imbalance in autophagic flux. in some neurodegenerative diseases, such as alzheimer's, parkinson's, and huntington's, as well as in the brain or spinal cord trauma, the damaged neurons exhibit abnormally high numbers of autophagosomes. therefore, understanding the interaction between pathophysiological mechanisms and autophagy could be a promising approach for therapies against neurological disorders [321] . prenatal alcohol exposure has been shown to increase the number of autophagic vacuoles in the cortical micro-vessels of human fetal and mouse neonatal brains, impairing autophagy [322] . furthermore, autophagy can modulate notch degradation, stem cell development, and neurogenesis [323] . several reviews have evaluated the relationship between autophagy and neurodegenerative diseases [324, 325] . autophagy is vital for neuronal homeostasis [326] , and its deregulation is highly associated with numerous neurodegenerative effects, such as the accumulation of damaged and toxic molecules with pathological consequences in neurodegenerative disorders such as alzheimer's, parkinson's, and huntington's diseases [327, 328] . lysosomal system inactivation is responsible for the accumulation of autophagosomes observed in alzheimer's disease [329] , whilst the disease is thought to be due to either excessive or impaired autophagosomal degradation, or the activation of autophagy genes in response to temporary injury/stress in neuronal tissues. alzheimer's [330] , parkinson's [331, 332] , and huntington's diseases [333] are key examples where autophagosomal accumulation and anomalies in the endosomal-lysosomal pathway have been observed in post-mortem human brain tissues via electron microscopy. autophagy deficiency resulted in neuronal loss in the cerebral and cerebellar cortices in a mouse model [334] . dysfunction or abnormalities in autophagy, including mutations in autophagy-regulating genes, are accompanied by neurodegenerative diseases across the age spectrum with exceptional frequency. atg7-deficient mice exhibited ubiquitin accumulation in their cns, causing nervous symptoms, neurodegeneration, and ultimately death [31, 334] , whilst atg5-deficient mice developed cytoplasmic inclusions and exhibited motor dysfunctions [319] , and ambra 1-deficient mouse embryos displayed neuronal tube defects [335] . autophagy is involved in the cytoplasmic clearance of α-synuclein (α-syn), which is observed in parkinson's disease [336] . in a mouse model, beclin 1 overexpression was found to reduce the clearance of α-syn, leading to pathological neuron abnormalities [337] . pharmacological and genetic pathways are involved in the degradation of α-syn by polo-like kinase 2 via autophagy, suggesting that these two proteins are concomitantly co-degraded [338] . the pink1 and parkin genes regulate mitophagy [339] , indicating that mutations in these genes can cause defects in mitophagy which have been correlated with parkinson's disease [340, 341] . beclin 1 expression is lower in the brains of patients with alzheimer's disease and not only affects autophagy but also increases the deposition of β-amyloid proteins causing neurodegeneration [330] . huntington's disease is caused by the extension of the polyglutamine (polyq) proteins aggregate intracellularly, which causes neuronal death. atg-knockout c. elegans exhibit increased polyq toxicity [342] , whilst in drosophila the autophagy-enhancing small molecule 2-(4-phenylphenyl)-5,6-dihydroimidazo[2,1-b] [1, 3] thiazole, also known as autophagy enhancer-99 (auten-99), has been shown to prevent the symptoms of neurodegenerative diseases [343] . the dual role observed for autophagy may be due to our poor understanding of this ubiquitous cellular recycling system. the differences between physiological and pathological autophagy may help design therapeutic strategies specifically targeting pathological autophagy without hindering its physiological roles [344] . for example, the apoptosis-stimulating protein p53-2 (aspp2/53bp2l) was reported to have different effects on autophagy in neurons stimulated with different levels of gp120, a soluble envelope glycoprotein of hiv-1 that interacts with chemokine receptors such as cxcr4 and ccr5. thus, regulating autophagy in the cns could be a potential therapeutic approach against hiv-associated neurocognitive disorders [77] . 3.6. autophagy in the immune system and autoimmune diseases 3.6.1. autophagy in the immune system the roles of cellular autophagy in immunological processes and autoimmune diseases have been reviewed extensively [82, 345, 346] . autophagy plays important roles in both innate and adaptive immunity [347] , modulates cellular and humoral immune responses [348] [349] [350] , and has roles in the non-metabolic and metabolic functions of immune cells [349] . furthermore, autophagy is involved in innate immune cell differentiation, degranulation, phagocytosis and extracellular trap formation involving neutrophils, eosinophils, mast cells, and natural killer cells, and plays an essential role in the renewal, differentiation, and homeostasis of immune cells [351] . autophagy also regulates the functional responses of immune cells, such as phagocytosis, antigen presentation, cytokine production, control of inflammasome activation, tolerance, and their consequences on overall host defense via monocytes, macrophages, dendritic cells, and antigen presentation [350] . additionally, autophagy plays important roles in b cell development, activation, and differentiation, which enables b cells to adapt to various events, and determines their fate, survival, and function [352] . since b cells produce antibodies, autophagy can determine humoral immune responses. in one study, the b cells of atg5-deficient mice had defective antibody responses, indicating that autophagy has a role in antibody production [353] . several studies have documented interplay between autophagy and the nf-κb signaling pathway. members of the nf-κb family of transcription factors regulate the transcription of genes involved in cell proliferation, survival, differentiation, and development, whilst activation of the inhibitor of nf-κb (iκbα) kinase complex is essential for autophagy induction. t-cell receptor-mediated nf-κb activation in b-cell lymphoma/leukemia is linked with the autophagy adaptor p62/sqstm1 [354] , which modulates the nlrp3-inflammasome activation and il-1β production in macrophages [355] . il-1α secretion is enhanced in atg5-deficient macrophages, whilst inhibiting autophagy results in il-1β overexpression [356] . the anti-inflammatory cytokine, il-10, inhibits autophagy by activating mtor complex 1 [357] and inhibits starvation-and ifn-γ-induced autophagy via bcl-2 and beclin 1 in various autoimmune and inflammatory disorders [358] . autophagy has predisposing, pathogenic, and therapeutic roles in autoimmunity. defects in autophagy pathways and/or autophagy-related genes have been implicated in numerous autoimmune and autoinflammatory diseases, including multiple sclerosis, systemic lupus erythematosus (sle), rheumatoid arthritis, psoriasis, psoriatic arthritis, inflammatory bowel disease, diabetes mellitus, crohn's disease, and vitiligo [82, [358] [359] [360] . abnormalities in the maintenance of homeostasis via autophagy result in the accumulation of dysfunctional or defective cellular organelles, abnormal proteins, infectious agents, and metabolite accumulation. this predisposes cells to the generation of autoantibodies and proinflammatory mediators and exposes vital and susceptible cellular structures to deleterious agents that can cause disease [358] [359] [360] [361] . a recent study revealed a correlation between the expression pattern of autophagy-related genes and the type of lupus nephritis (ln); thus, autophagy could indicate of the type of ln when formulating a treatment regimen [362] . several atgs are known to be involved in autoimmune disorders, including atg5, pr domain zinc finger protein 1 (prdm1; also known as blimp-1), and dna-damage regulated autophagy modulator 1 (dram1) in sle patients and atg16l1 and immunity-related gtpase m (irgm) in crohn's disease and ulcerative colitis. autophagy defects have been observed in t cells, b cells, and macrophages [363] . mhc class ii antigen presentation by macrophages occurs via cma; lysosomal proteins have a central role in antigen processing, which is essential for a correct immune system function. studies in mrl/lpr mice which develop a full panel of lupus autoantibodies revealed that increased lysosomal ph might be an important lysosomal malfunction involved in autoimmunity, and that perturbed lysosomal turnover may lead to hyperactive antigen presentation by antigen presenting cells (apc) in autoimmune disorders [363] . in innate immunity, reduced atg5 and mtor expression result in defective autophagy, affecting the clearance of dead cells, increasing levels of nucleic acid remnants and self-antigens, increasing type 1 ifn by dcs, and inducing b cell hyper-differentiation and autoantibody production [82, 346, 358] . it has also been reported that autophagy-related gene knockdown can have therapeutic effects on autoimmune diseases [357] . modulating autophagy can manage immunity-related and inflammatory diseases [82, 345, 347, 364] by regulating cytokine and antibody production against immunogenic insults to prevent autoimmune diseases. therefore, regulating autophagy has clinical potential in cancer immunotherapy [365] , whilst autophagy and adenoviral combinations are proving beneficial in adenoviral-based oncolytic virotherapy [145] . under normal conditions, the myocardium exhibits low levels of autophagy, whilst stressful conditions can increase the level of autophagy to increase cell survival [366, 367] . patients with congestive heart failure, coronary artery disease, hypertension, and aortic valvular disease display increased autophagosomal accumulation in their myocardial biopsies [368] . autophagy levels vary in normal and affected or stressed hearts, with constitutive autophagy maintaining normal structure and function, and upregulated autophagy occurring during cardiac disease or stress [369] . in atg5-deficient mice, contractile dysfunction and hypertrophy have been observed during cardiomyopathy [370] , whilst cell culture studies have revealed that autophagic gene deficiency can cause the accumulation of unwanted proteins and contribute to myocardial disease [371] . similarly, lamp-2-deficient mice displayed increased autophagic vacuole accumulation and could not degrade proteins, thereby promoting cardiomyopathy [371, 372] . it has been postulated that during early life, between birth and suckling, autophagy provides the energy required for cardiac cells [373] , whilst mitophagy protects cardiac muscles under ischemic stress [374] . increased autophagy can cause heart failure [375] , with autophagy-induced degeneration resulting in the death of cardiomyocytes. this knowledge has helped our understanding of the pathogenic role of autophagy in cardiac failure models and helped devise therapeutic targets [376, 377] . autophagy can cause myocardial cell damage via parp1, which promotes autophagy in cardiomyocytes by modulating foxo3a transcription [378] . increased autophagy causes pathological remodeling of the heart, whilst decreased autophagy reduces remodeling [88] . thus, it has both protective and destructive roles in the cardiovascular system. iron homeostasis involves a form of macroautophagy known as ferritinophagy, wherein ferritin, an iron storage protein, is degraded in the lysosome [379] . iron levels are tightly regulated in cells; nutrient deficiency induces autophagy, during which cellular proteins and organelles are engulfed by the autophagosome, which then fuses with the lysosome. the degradation of these contents provides essential resources that either promote cell survival or lead to cell death. iron (fe), copper (cu), zinc (zn), and aluminum (al) react with molecular oxygen to produce reactive oxygen species (ros) and reactive nitrogen species (rns). besides acting as a cofactor for metalloprotein enzymes involved in redox reactions, iron also plays a major role in mitochondrial atp metabolism and other cellular processes. the fenton and haber-weiss redox reaction is highly involved in ros production and alzheimer's progression [380] . to maintain iron homeostasis, storage and recycling are critical. when engulfed by macrophages, the iron within erythrocytes is either stored as a ferritin complex or exported from the cell by the ferroportin iron-exporter [381] . hsp70 and ferritin bind iron in the cytosol and autophagocytosis of these proteins can sequester redox-active iron in the lysosomes [382] . cells rich in these proteins exhibit increased resistance to oxidative stress; therefore, autophagy plays a major role in maintaining cellular redox status [383, 384] . the autophagy inhibitor ncoa4, which is a substrate of pi3k, has been shown to physically bind to the ferritin protein complex and direct it to autolysosomes for degradation [385] . ncoa4 knockdown prevents the localization of ferritin in the lysosomes and increases the levels of iron-responsive element-binding protein 2 (irp2), which is a free intracellular fe antagonist that prevents cell death via exogenous ros [386] . ncoa4 also acts as an autophagy receptor for ferritin and delivers it to the lysosome to maintain iron homeostasis. experimentally simulating low-iron conditions by chelating iron revealed that ferritin is degraded to release the stored iron [387] . autophagy is involved in obesity [388] and diabetes mellitus [389] . improper lipid and glycogen processing can affect the liver activity and thus, insulin synthesis, resulting in diabetes. studies have shown that hepatocytes from mouse models of obesity display reduced autophagy [390] , with decreased atg7 expression causing er stress and affecting insulin signaling [390] . mutant atg7 mice also exhibit reduced β cell mass, reduced insulin circulation, and glucose intolerance, indicating that autophagy defects can reduce insulin levels and cause hyperglycemia [391, 392] . a genetic mosaic screen for mutations that increase lysosomal and/or autophagic activity in d. melanogaster larva revealed that autophagy-lysosome pathways underlie novel cytoprotective features in drosophila [393] . obesity impairs autophagy in the liver via s-nitrosylation, a process induced by nitric oxide (no). s-nitrosylation of the lysosomal enzymes cathepsin b (ctsb) and hexosaminidase subunit β (hexb) impairs normal lysosomal functioning and is carried out by denitrosylation enzymes, particularly s-nitrosoglutathione reductase (gsnor) and thioredoxin [394] . obesity inhibits the denitrosylation ability of the liver, impairing hepatic autophagy and insulin resistance [395] . in obese animals, hepatic insulin signaling is impaired by no-induced hepatic autophagy repression, which ultimately causes the progression of type 2 diabetes [396] . the potential roles of autophagy in ameliorating diseases while maintaining homeostasis have been enumerated in table 1 . details of applied/granted patents for treating autophagy-related ailments and dysfunctions are presented in table 2 . human nuclear ribonucleoprotein k (hnrnp-k) and ubiquilin 4 (ubqln4) help in viral replication. ndp52 human autophagy receptor interacts with chikv nsp2 and acts as proviral factor wong and chu, 2018 [37] increased autophagy-associated protein lc3 and bnip3-linked to colorectal and gastric cancers; elevated expression of nip3 (a pro-apoptotic member of the bcl-2 family of cell death factor) in gastric carcinomas lee et al., 2007a [309] autophagy inhibition leads to cell death in tumors acting like an ras-activated tumor guo et al., 2011 [312] in the absence of autophagy -accumulation of ubiquitinylated protein aggregates and higher p62 level-responsible for liver tumor takamura et al., 2011 [291] activation of autophagy and peroxisome proliferator-activated receptor gamma (pparγ) protect colon cancer cells against apoptosis tylichová et al., 2017 [310] in ras-activated tumors, inhibition of autophagy leads to increased cancer cell death guo maintaining homeostasis is the most important role of autophagy, which is the final survival mechanism used to escape cell death. mutations or genetic dysfunctions in autophagy-associated genes can have a variety of pathological consequences, as described below: 3.10.1. static encephalopathy of childhood with neurodegeneration in adulthood (senda) senda is a recently discovered type of neurodegeneration associated with iron accumulation in the brain [422] . it begins with early-onset spastic paraplegia and mental retardation during childhood, followed by symptoms of parkinsonism and dystonia during adulthood along with eye movement abnormalities, dysautonomia, and sleep disorders. whole-exome next generation sequencing has revealed that senda is associated with a mutation in the wipi4 gene (also known as wdr45), located at xp11.23 [399, 423, 424] . the disease is characterized by iron accumulation in the globus pallidus [425] . wipi4, which is the mammalian homolog of yeast atg18, is involved in autophagosome formation [426] by binding phosphatidylinositol 3-phosphate, which is recruited to the autophagosome formation site [427] . a severe decline in wipi4 expression has been reported in lymphoblastoid cell lines derived from patients with senda [424] . as the wipi4 gene is found on the x chromosome, one of which undergoes inactivation in females, the loss of wipi4 function is expressed in a mosaic pattern in females. crohn's disease is a major inflammatory bowel disease whose symptoms include abdominal pain, diarrhea, vomiting, and weight loss. atg16l1 mutations have been found to be linked with crohn's disease [428] . atg16l1 forms a complex with atg12-atg5 which initiates autophagosome formation by inducing the conjugation of lc3 with pe [429] . a study on the 300t > a atg16l1 mutation revealed that the mutation is differentially involved in crohn's disease and canonical autophagy [430] . in mice, the mutations that eliminated or reduced atg16l1 expression indicated a relationship between atg16l1 mutations and crohn's disease [429] . other autophagy-associated proteins, including irgm-101, 102, and 103 and nod2-104, 105, and 106 have also been linked to crohn's disease in humans [431] . autophagy is thought to be involved in the pathogenesis of crohn's disease, but it can also control disease severity by reducing the levels of inflammatory mediators [401] . hsp is a neurodegenerative disorder that causes axonal degeneration in the corticospinal or pyramidal motor and sensory tracts that control distal organs and is caused by a recessive mutation in the tecpr2 gene. tecpr2 interacts with six human atg8 orthologs to positively regulate autophagy, with tecpr2 knockdown in hela cells reducing autophagic activity and indicating its role in autophagy [432] . the zinc-finger protein spastizin interacts with the beclin 1-uvrag-rubicon complex and is involved in autophagosome maturation. in fibroblasts derived from patients with hsp, knockout of the spastizin gene reduced autophagy and caused autophagosome accumulation by impairing lysosomal fusion [433] . the zfyve26/spastizin and spg11/spatacsin mutations were recently associated with hsp [434] ; however, there are currently no treatments available to reverse nerve degeneration in hsp, with efforts instead directed towards reducing symptoms by physiotherapy and improving spasticity using medication. danon disease is a rare cardiomyopathic disease caused by lamp-2 deficiency and characterized by the accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscles, cardiomyopathy, and intellectual dysfunction [435] . the lamp-2 gene is alternatively spliced into three isoforms (lamp-2a, -2b, and -2c) which have different functions in autophagy. lamp-2a acts as a receptor for cma, in which proteins carrying the kferq motif are selectively trafficked into the lysosome and degraded. lamp-2b has been suggested to play a prominent role in macroautophagy and is responsible for the danon phenotype. lamp-2b-deficient mice exhibit higher mortality and autophagic vacuole accumulation in the liver, kidney, pancreas, and cardiac and skeletal muscles [372] . whole genome sequencing identified a nonsense mutation (codon 520c>t in exon 4) in lamp2 in a family with danon disease [436] . lamp-2 knockout resulted in failed autophagic progression, as evidenced by inappropriate cathepsin d processing in the autophagic vacuoles and abnormally high numbers of mannose-6-phosphate receptors that limited the degradation of long-lived proteins during starvation [437] . 3.10.5. x-linked myopathy with excessive autophagy (xmea) xmea is a rare x-linked recessive skeletal myopathy caused by a single-nucleotide substitution (c. 164-7t > g) in vma21, whose protein product assembles proton pumps in the lysosome which generate and maintain the acidity required for the activity of various lysosomal hydrolases [438] . impaired hydrolase activity can block the final step of autophagy or induce autophagy by inhibiting mtor. if autophagy is impaired, a feed-forward pathogenic loop is activated, which results in the accumulation of autophagolysosomes containing incompletely-digested products [439] . xmea is characterized by progressive muscle weakness, particularly in the proximal muscles of the legs, and muscle degeneration (atrophy) in adulthood. recently, two vma21 non-coding microdeletions [440] , one intronic (c.54-16_54-8del) and the other in the 3 utr (c.*13_*104del), were reported to result in more severe clinical manifestations with extra-ocular and upper extremity involvement and earlier disease onset. besides long-lived proteins and membranes, xmea also affects membrane repair, interrupts sarcolemmal membrane homeostasis, and increases serum creatine phosphokinase (cpk) levels [440] . sibm is an age-related progressive muscle disorder which presents in the elderly and is characterized by the presence of autophagic vacuoles and ubiquitinylated misfolded multiprotein aggregates. two major lysosomal proteases, cathepsins d and b, exhibit decreased activation in sibm muscle fibers [441] whilst lc3-ii is increased, indicating that autophagosome formation is increased due to reduced cathepsin d and b activity during autophagosome maturation [442] . t cells may have a role in sibm [443] , whilst allelic variants of the hla-dr3 locus, other genetic factors, and the hla genotype are also thought to play major roles in the progression and severity of the disease [444] . dysphagia (difficulty in swallowing) may occur due to weakness in the neck muscles of patients with sibm, whilst myalgia (muscle pain) and difficulty in manipulating objects may occur due to weakness in the fingers. an overview of diseases caused by genetic defects in autophagy genes is presented in table 3 . to alleviate autophagy-associated diseases, a variety of drugs, biomolecules, chemicals, and epigenetic strategies have been developed to either promote or inhibit autophagy. autophagy can be suppressed during any stage of autophagic flux. the pro-apoptotic role of autophagy may lead to hyperstimulation-induced excessive activation, which results in detrimental self-cannibalism that can go beyond the limit of cellular recovery. numerous chemical inhibitors have been identified and tested in various cell and animal models. most autophagy inhibitors are poorly selective, limiting their wider applications. 3-methyladenine (3-ma), a class-iii pi3k inhibitor, is commonly used to inhibit autophagy [460] . class iii pi3k and beclin 1 are essential during the first step of autophagy induction [461] ; thus, inhibiting pi3k reduces autophagosome formation. bafilomycin a1 is a vacuolar h + -atpase inhibitor, which functions at concentrations as low as 1 nm to inhibit both the early and late stages of autophagy by activating the mtor pathway [462] . in early 1998, bafilomycin a1 was reported to inhibit autophagic vacuoles in rat hepatoma [463] by dissociating the beclin 1-vps34 complex and preventing autolysosome formation, thus attenuating functional autophagy. bafilomycin a1 has been tested in a mouse model and found to be safe [464] . furthermore, disruption of the vacuolar-type h + -atpase complex in mouse liver cells has been shown to induce the rapamycin complex 1 (mtorc1)-independent aggravation of autophagic vacuoles and lysosomes [465] . concanamycin a belongs to the same class of vacuolar h + -atpase inhibitors as bafilomycin a1 and in the presence of autophagosomes causes autophagic bodies to accumulate in the central vacuole and cytoplasm of tobacco by-2 cells [466] . therefore, vacuolar-type h + -atpases have been proposed as potential therapeutic agents. cycloheximide is a protein biosynthesis inhibitor that is frequently used in biomedical research. in mouse pancreatic acinar cells, cadmium chloride-or hyperosmotic sucrose-stimulated autophagy was found to be inhibited by cycloheximide [467] , which likely inhibits autophagy at the segregation step. cycloheximide is used to prevent the autophagy-lysosome pathway [468] and its effects can be rapidly reversed by its removal [469] . chloroquine, hydroxychloroquine, nh 4 cl, and neutral red are chemicals that can rapidly neutralize the acidic environment of the lysosome; therefore, they are used to block autophagosome maturation. chloroquine and hydroxychloroquine are repurposed drugs that have been used to treat malaria, sle, and rheumatoid arthritis [470] ; however, higher hydroxychloroquine concentrations are required to induce active autophagy as a cancer treatment, which are usually not achievable in cancer patients. chloroquine-mediated lysosomal dysfunction is thought to have increased anti-cancer functions when combined with nutrient deprivation [471] . lys01, a dimeric form of chloroquine in which each molecule is separated by the spacer molecule n-bis(2-aminoethyl)-methylamine, has been reported to inhibit autophagy at a level 10-fold higher than chloroquine lys05, a water-soluble salt of lys01, effectively accumulates within the lysosome to concurrently deacidify and inhibit autophagy [472] . lysosomal proteases are active at an acidic ph. the protease inhibitor leupeptin inhibits cysteine, serine, and threonine peptidases to block protein degradation and thus inhibits the final step of autophagy, as evidenced by the accumulation of vacuolar autolysosomes [473] . cathepsins are aspartic, cysteine, and serine proteases that are transported to lysosomes via mannose-6-phosphate receptors [474] and can be inhibited by the lysosomal protease inhibitors e64d and pepstatin a. cathepsins b, h, and l are inhibited by e64d, whilst cathepsins d and e are inhibited by pepstatin a. cells treated with e64d and pepstatin a exhibit increased lc3-ii levels [475] . bafilomycin and chloroquine, which inhibit autophagy by targeting lysosomes, have been reported to increase levels of the autophagosome marker lc3-ii, block key aspects of autophagy, decrease mitochondrial quality, and increase mitochondrial dna damage in primary neurons [476] . the atg genes are crucial in autophagy; therefore, the inactivation or knockdown of these genes may be a useful way to manipulate the process [477] . mir-30a has been reported to downregulate beclin 1 and atg5 expression, whilst mir-101 can inhibit basal and rapamycin-induced autophagy. autophagy is induced by the activation of the ulk complex, which consists of ulk1/2, atg13, fip200, and atg101. in melanoma cells, mir-290-295 clusters can inhibit ulk1 and atg7 expression to suppress autophagic cell death [478] . mir-885-3p targets ulk2 to regulate autophagy [479] , whereas mirna-30a/b, mirna-376b, mir-216a, mir-519a, and mir-17-5p inhibit beclin 1 expression to suppress vesicle nucleation [480] [481] [482] . recently, the leucine-rich repeat kinase 2 gene (lrrk2), which is associated with crohn's disease, parkinson's disease, and leprosy, was shown to inhibit the non-canonical autophagy cascade, indicating that the negative regulation of this cascade could directly induce disease [483] . in addition, the atg16l1 300 t > a polymorphism has been demonstrated to disrupt unconventional tmem59.110-mediated autophagy in mice [484] . rapamycin, also known as sirolimus, is a natural mtor inhibitor [485] that stimulates autophagy both in vitro and in vivo; however, its long-term use is often complicated as it suppresses ribosome biogenesis and protein translation [486] . the rapamycin ester cci-779/temsilorimus, which is also an mtor inhibitor, prevents the development of intracellular tau protein inclusions (abundant protein that stabilizes microtubules in cns neurons) which are known to accumulate in neurons in alzheimer's disease and other tauopathies and can also reduce the density of neurofibrillary tangles by stimulating mtor-dependent autophagy [487] . rad001 and ap23573 are rapamycin derivatives with comparatively high safety and low toxicity [488, 489] . during the early stages of carotid atherosclerosis, which is an inflammatory step in the primary pathogenesis of cerebrovascular diseases, mirna-155 plays an important role in the activation of autophagy by rapamycin [490] . rapamycin has also been shown to increase autophagy, decrease cyclin d1 expression, and attenuate aggressive iga nephropathy progression in a rat model [491] . the immunosuppressive activities of rapamycin limit its frequent use; therefore, safer molecules are required. three smers were identified from 50,729 compounds that can induce autophagy [492] ; smers 10, 18, and 28 can reduce the pathogenesis associated with polyglutamine aggregation and a53t α-synuclein, which are linked to huntington's disease and familial parkinson's disease, respectively [493] , and induce autophagy in an mtor-independent manner. in addition, the small molecule auten-99 activates autophagy in cell cultures and animal models and inhibits the progression of neurodegenerative symptoms [343] . trehalose is a natural disaccharide known to stimulate autophagy via ampk [494] . in cultured cells from an animal model of huntington's disease, trehalose has been shown to stimulate autophagy and reduce huntingtin protein aggregation during starvation by inhibiting a family of glucose transporters known as the solute carrier 2 or the glucose transporter family [495] . as well as being an autophagy activator and a non-reducing disaccharide, trehalose can also reduce the levels of aggregate-prone proteins and ameliorate cytotoxicity in neurodegenerative disease models [496] . trehalose treatment has been shown to increase the conversion of lc3-i to lc3-ii via an mtor-independent pathway, whilst sucrose and raffinose are also known to induce autophagy [497] . it has been shown that trehalose can be used to safely induce autophagy in neurodegenerative disorders such as parkinson's disease [498] , alzheimer's disease [499] , and prion disease [500] . the bioavailability of trehalose in the body for autophagy induction is low due to the hydrolytic enzyme, trehalase, which is expressed in the intestine and kidney; therefore, the enzyme-stable trehalose analogs, lentztrehaloses a, b, and c, were synthesized and found to be as effective as trehalose [501] . lithium chloride (licl) can induce autophagy by inhibiting inositol monophosphatase (impase), which reduces inositol and inositol-1,4,5-triphosphate (ip3) levels [502] . since lithium has already been approved by the fda for patients with bipolar disorder, it can easily be adapted for other diseases [503] . long-term oral lithium administration enhanced autophagy in a tauopathy mouse model by inhibiting glycogen synthase kinase-3. l-690,330, a bisphosphonate inhibitor of impase, exhibits a similar function to lithium by clearing mutant synucleins and egfp-hdq74 [504] . valproic acid is an angio-suppressive compound which suppresses the akt/mtor signaling pathway by acting as a histone deacetylase inhibitor and promotes autophagy, as evidenced by the increased concentrations of lc3-ii and beclin 1 after its administration in prostate cancer cell lines [505] . carbamazepine and valproic acid can both reduce intracellular inositol-1,4,5 trisphosphate levels [506] . therefore, other impase inhibitors could also be incorporated into therapeutic strategies. anacardic acid, curcumin, garcinol, and spermidine increase autophagy by reducing the level of acetylation in cultured human cells, as evidenced by depleted sequestosome-1 levels and mtorc1 inhibition [507] . spermidine also inhibits ep300 and the major autophagy proteins, atg5, atg7, atg12, and lc3 to repress autophagy [508] . supplementing the diet of mice with coffee beans rich in polyphenols concomitantly increased autophagy and decreased acetylation levels [509] , whilst natural polyamines can inhibit acetyltransferases, and their dietary intake can improve the life span of short-living mouse strains and the health of long-living ones [510] . fluoxetine, which selectively improves the secretion of the pro-inflammatory cytokine tnf-α, and gefitinib, which inhibits the epidermal growth factor receptor (egfr), can also enhance autophagy to impart neuroprotection and restrict m. tuberculosis growth [511] . the antiepileptic compound, carbamazepine, has been found to protect cells against m. tuberculosis infection, likely by inducing autophagy [512] . elevated intracytosolic ca 2+ levels can inhibit autophagy; penitrem a, which inhibits ca 2+ -dependent k + channels, is known to induce autophagy and has been used to treat neurodegenerative disorders [513, 514] . furthermore, bacterial pore-forming toxins can induce the expression of the transcription factor hlh-30 (tfeb) in c. elegans, which stimulates autophagy genes and thus inhibits bacterial infection [215] . metformin, an ampk activator, has been shown to induce autophagy and reduce the risk of hepatocellular carcinoma (hcc) in diabetic patients [515] . a patent studied by gorski and qadir [402] revealed the potential of a thioxanthone-based autophagy inhibitor for suppressing atg gene expression by using sirna-directed therapy against cancer cells receiving anti-cancer therapies, and for treating cancer in combination with other therapies. the use of dimeric quinacrine derivatives to inhibit lysosomes and autophagy in cancer cells where autophagy allows them to survive metabolic and therapeutic stresses [403] is currently at the patent filing phase. the herbal product onjisaponin b, from radix polygalae (polygala tenuifolia)-a traditional chinese herb, is known to enhance autophagy. upon administration, it prevents, treats, or delays the onset of neurodegenerative diseases, thus a patent for this product has been granted to law et al. [406] . imidazo [4-5f] [1, 10] phenanthroline derivatives (1-6) have been evaluated for their anti-cancer effects, with their use of upregulating lc3-ii and beclin 1 expression and thus inducing autophagy. of the different compounds evaluated, the phenanthroimidazole 6 derivative was found to induce autophagy and apoptosis; thus, it could prove to be a novel anti-cancer drug [516] . in nanosilica stimulated raw264.7 cells, melatonin was found to increase lc3 expression and decrease bax expression, suggesting that autophagy was promoted, and apoptosis was inhibited [517] . sasanquasaponin iii obtained from schima crenata korth was found to upregulate lc3-ii expression in melanoma cells and induce autophagy; hence, it can be used as an anti-cancer drug to treat melanoma [518] . epigallocatechin gallate (egcg) was found to promote autophagosome formation and increase lysosome acidification in müller cells, thus increasing autophagy. a gliosis experimental model revealed that egcg reduced reactive gliosis and retinal damage; hence, egcg should be explored as a treatment of diabetic retinopathy [519] . compounds that inhibit and activate autophagy are depicted in figure 7 . therapies. the use of dimeric quinacrine derivatives to inhibit lysosomes and autophagy in cancer cells where autophagy allows them to survive metabolic and therapeutic stresses [403] is currently at the patent filing phase. the herbal product onjisaponin b, from radix polygalae (polygala tenuifolia)a traditional chinese herb, is known to enhance autophagy. upon administration, it prevents, treats, or delays the onset of neurodegenerative diseases, thus a patent for this product has been granted to law et al. [406] . imidazo [4-5f] [1,10] phenanthroline derivatives (1-6) have been evaluated for their anti-cancer effects, with their use of upregulating lc3-ii and beclin 1 expression and thus inducing autophagy. of the different compounds evaluated, the phenanthroimidazole 6 derivative was found to induce autophagy and apoptosis; thus, it could prove to be a novel anti-cancer drug [516] . in nanosilica stimulated raw264.7 cells, melatonin was found to increase lc3 expression and decrease bax expression, suggesting that autophagy was promoted, and apoptosis was inhibited [517] . sasanquasaponin ιιι obtained from schima crenata korth was found to upregulate lc3-ii expression in melanoma cells and induce autophagy; hence, it can be used as an anti-cancer drug to treat melanoma [518] . epigallocatechin gallate (egcg) was found to promote autophagosome formation and increase lysosome acidification in müller cells, thus increasing autophagy. a gliosis experimental model revealed that egcg reduced reactive gliosis and retinal damage; hence, egcg should be explored as a treatment of diabetic retinopathy [519] . compounds that inhibit and activate autophagy are depicted in figure 7 . compounds that inhibit and activate autophagy. autophagy inhibitors: 3-methyladenine inhibits pi3k. bafilomycin a1 causes dissociation of the beclin 1-vps34 complex and prevents the formation of autolysosome. chloroquine/hydroxychloroquine, nh4cl, and leupeptin rapidly neutralizing the acidic environment of the lysosome and are used to block lysosomal degradation of substrates. leupeptin inhibits cysteine, serine and threonine peptidases, and hence blocking protein degradation at the last step of autophagy. autophagy activators: rapamycin inhibits the mtor. rad001 and ap23573 are rapamycin derivatives having comparatively higher safely with minimum dose toxicities. trehalose causes lc3-i to lc3-ii conversion in an mtor-independent pathway. valproic acid increases lc3-ii and beclin 1 concentrations. compounds that inhibit and activate autophagy. autophagy inhibitors: 3-methyladenine inhibits pi3k. bafilomycin a1 causes dissociation of the beclin 1-vps34 complex and prevents the formation of autolysosome. chloroquine/hydroxychloroquine, nh 4 cl, and leupeptin rapidly neutralizing the acidic environment of the lysosome and are used to block lysosomal degradation of substrates. leupeptin inhibits cysteine, serine and threonine peptidases, and hence blocking protein degradation at the last step of autophagy. autophagy activators: rapamycin inhibits the mtor. rad001 and ap23573 are rapamycin derivatives having comparatively higher safely with minimum dose toxicities. trehalose causes lc3-i to lc3-ii conversion in an mtor-independent pathway. valproic acid increases lc3-ii and beclin 1 concentrations. autophagy is a fast-moving area of science which has had an excellent positive impact on animal and human health-related issues and threatening diseases and has considerable future potential. autophagy is a highly complex process, and understanding the complexity of its mechanisms and internal regulations will be highly beneficial for developing novel methods such as simple ameba-based experimental models. emerging studies have established many interesting connections in the field of autophagy research. since autophagy is one of the most conserved evolutionary processes, present in lower organisms and all highly evolved mammals, much work has been carried out to understand its physiological and pathological features. pathogenesis-associated autophagy is involved in degenerative, inflammatory, infectious, and neoplastic diseases, whereas physiological autophagy is associated with maintaining liver iron homeostasis, cns development, endothelial cell alignment, atheroprotection, and preventing infection. numerous mechanisms of action involving various signaling pathways have been linked to autophagy, with novel mechanisms currently being investigated. mtor acts as a nutrient sensor in autophagy; however, the process may also be regulated via mtor-independent pathways. intracellular ca 2+ regulates autophagy, with intracellular ca 2+ mobilization via the ip3 receptor stimulating calmodulin and erk pathways to inhibit autophagy. notably, ca 2+ also inhibits autophagy by increasing mitochondrial atp levels, which inhibits the camkkβ/ampk pathway, yet promotes autophagy by promoting the ampk-dependent inhibition of mtor. autophagy is linked with other cellular mechanisms such as apoptosis, necrosis, autosis, and necroptosis, and can take the form of microautophagy, macroautophagy, and cma. autophagy can be selective, neutralizing specific substances, or non-selective, degrading different materials irrespective of their nature. it can play physiological and pathological roles by maintaining homeostasis and causing disease, thereby affecting both health and disease. autophagy encounters infectious and non-infectious diseases, either mediating protection or aggravating the disease. autophagy has dual roles in an anti-bacterial, anti-viral, and tumor suppressing capacity and by favoring bacterial infections, viral infections, and tumor progression. among its dual physio-pathological roles, autophagy can promote brain development, immunity, and cardiovascular and endocrine development, or cause neurodegeneration, autoimmune diseases, cardiovascular diseases, obesity, and diabetes. autophagy has a genetic basis and many diseases are caused by genetic defects in autophagy genes, including senda, crohn's disease, hsp, danon disease, xmea, and sibm. efforts are being made to explore the positive aspects of autophagy to overcome various health and disease problems. novel therapeutics and interventions are being investigated to counter autophagy-associated diseases, including apoptosis inhibitors and activators. excessive autophagy stimulation may cause self-destruction which could be controlled with drugs such as vacuolar-type h (+)-atpase inhibitors, cycloheximide, lysosome alkalizers (chloroquine, hydroxychloroquine, nh 4 cl, and neutral red), and acidic protease inhibitors (e64d and pepstatin a), whilst knocking down beclin 1 and atg5 using mir-30a could be used to inhibit the excessive cannibalism caused by autophagy. upregulating autophagy could therapeutically benefit a range of diseases caused by reduced neuronal apoptosis, including the neurodegeneration-associated impairment of learning/memory capabilities, motor dysfunction, seizures, adult stroke, neonatal asphyxia, cardioskeletal myopathy, and cancers. autophagy could also prevent various bacterial and viral diseases, inflammatory and autoimmune conditions, and also increase lifespan. rapamycin, small-molecular enhancers of rapamycin (auten-9), trehalose, impase inhibitors (carbamazepine and valproic acid), epigenetic modulators (anacardic acid, curcumin, garcinol, and spermidine), and chemicals (fluoxetine, penitrem a, and metformin) are all autophagy stimulators which help to ameliorate disorders associated with reduced autophagy. the apparent dual role of autophagy may be due to our poor understanding of this ubiquitous cellular recycling system. understanding the differences between physiological and pathological autophagy may help us design therapeutic strategies to target pathological autophagy without hindering its physiological effects. studies on mouse models combined with human genetic studies provide an important insight into the role of autophagy in neurological diseases like parkinson's and inflammatory disorders like crohn's disease. some critical issues have yet to be addressed regarding the role of autophagy in therapeutics and diagnostics. there are few efficient markers for studying autophagy modulation and those that exist have limitations. these markers are important for determining the effect of autophagy on disease initiation and progression. furthermore, autophagy modulating drugs are imprecise and nonspecific; hence, more specific drugs are required. similarly, autophagy upregulation appears to be beneficial for removing misfolded and aggregated proteins, intracellular pathogens, damaged mitochondria, and other organelles; however, it is not yet clear how selective autophagy could be upregulated in other situations. these are just some of the questions that need to be addressed in order to use autophagy as a therapeutic molecular process. nonetheless, autophagy modulation-based therapies will soon become a reality and will help safeguard human health against various pathological conditions. author contributions: all the authors substantially contributed to the conception, design, analysis, and interpretation of data, checking and approving the final version of the manuscript, and agree to be accountable for its contents. funding: this compilation is a review article written, analyzed, and designed by its authors and required no substantial funding to be stated. till death do us part: the 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associated with lysosomal dysfunction and autophagic stress in npc1 −/− mouse brain niemann-pick type c disease and intracellular cholesterol trafficking lipid imbalance in the neurological disorder, niemann-pick c disease amino acid starvation sensing dampens il-1β production by activating riboclustering and autophagy sex differences in autophagy contribute to female vulnerability in alzheimer's disease basal and starvation-induced autophagy mediates parasite survival during intraerythrocytic stages of plasmodium falciparum intracellular cholesterol trafficking and impact in neurodegeneration regulation of autophagy by extracellular signal-regulated protein kinases during 1-methyl-4-phenylpyridinium-induced cell death crosstalk between autophagy and apoptosis, potential and emerging therapeutic targets for cardiac diseases wogonin induces beclin-1/pi3k and reactive oxygen species-mediated autophagy in human pancreatic cancer cells mechanisms of autophagy initiation emerging connections between rna and autophagy crosstalk of autophagy and apoptosis: involvement of the dual role of autophagy under er stress lysosomal degradation of intracellular nucleic acids-multiple autophagic pathways hs1bp3 provides a novel mechanism of negative autophagy regulation through membrane lipids hormetic heat shock and hsf-1 overexpression improve c. elegans survival and proteostasis by inducing autophagy epigenetic and transcriptional regulation of autophagy vitamin d receptor regulates autophagy in the normal mammary gland and in luminal breast cancer cells lysosomal signaling in control of degradation pathways monitoring and measuring autophagy mechanisms and physiological roles of mitophagy in yeast the role of alfy in selective autophagy regulation of starvation-and virus-induced autophagy by the eif2α kinase signaling pathway mechanisms of mitophagy mitochondria-anchored receptor atg32 mediates degradation of mitochondria via selective autophagy mitophagy: mechanisms, pathophysiological roles, and analysis atg32 is a mitochondrial protein that confers selectivity during mitophagy nix is a selective autophagy receptor for mitochondrial clearance autophagy receptors in developmental clearance of mitochondria peroxisome degradation in saccharomyces cerevisiae is dependent on machinery of macroautophagy and the cvt pathway peroxisome size provides insights into the function of autophagy-related proteins structural biology of the cvt pathway the journey of the autophagosome through mammalian cell organelles and membranes microautophagy of cytosolic proteins by late endosomes chemotherapy-induced tissue injury, an insight into the role of extracellular vesicles-mediated oxidative stress responses peptide sequences that target cytosolic proteins for lysosomal proteolysis role of chaperone-mediated autophagy in metabolism a receptor for the selective uptake and degradation of proteins by lysosomes the roles of endo-lysosomes in unconventional protein secretion the chaperone-mediated autophagy receptor organizes in dynamic protein complexes at the lysosomal membrane methods to monitor chaperone-mediated autophagy autophagy protects against sindbis virus infection of the central nervous system expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex autophagic machinery activated by dengue virus enhances virus replication subversion of cellular autophagosomal machinery by rna viruses adenovirus, autophagy and lysis: ecstasies and agonies hypoxia induces microglia autophagy and neural inflammation injury in focal cerebral ischemia model icariin promotes angiogenic differentiation and prevents oxidative stress-induced autophagy in endothelial progenitor cells bovine viral diarrhea virus infection induces autophagy in mdbk cells a pathogenic role for er stress-induced autophagy and er chaperone grp78/bip in t lymphocyte systemic lupus erythematosus tor directly controls the atg1 kinase complex to regulate autophagy role of ulk-fip200 complex in mammalian autophagy: fip200, a counterpart of yeast atg17? the role of the atg1/ulk1 complex in autophagy regulation structure and function of the ulk1 complex in autophagy autophagy, renovation of cells and tissues a molecular perspective of mammalian autophagosome biogenesis termination of autophagy and reformation of lysosomes regulated by mtor autophagy at the crossroads of catabolism and anabolism lc3-associated phagocytosis-the highway to hell for phagocytosed microbes atg9 is required for intraluminal vesicles in amphisomes and autolysosomes an atg9-containing compartment that functions in the early steps of autophagosome biogenesis clinical outcome and phenotypic expression in lamp2 cardiomyopathy hs1bp3 negatively regulates autophagy by modulation of phosphatidic acid levels rab7 may be a novel therapeutic target for neurologic diseases as a key regulator in autophagy up-regulation of the active form of small gtpase rab13 promotes macroautophagy in vascular endothelial cells network organization of the human autophagy system genome-wide sirna screen reveals amino acid starvation-induced autophagy requires scoc and wac genome-wide screen identifies signalling pathways that regulate autophagy during caenorhabditis elegans development autosis and autophagic cell death: the dark side of autophagy autosis is a na + , k + -atpase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia identification of a candidate therapeutic autophagy-inducing peptide autosis, a new addition to the cell death tower of babel multiple interacting cell death mechanisms in the mediation of excitotoxicity and ischemic brain damage, a challenge for neuroprotection autosis occurs in the liver of patients with severe anorexia nervosa distinct roles of autophagy in the heart during ischemia and reperfusion: roles of amp-activated protein kinase and beclin 1 in mediating autophagy recent insights into cell death and autophagy protection against fatal sindbis virus encephalitis by beclin, a novel bcl-2-interacting protein the bcl-2 family in host-virus interactions beclin 1 phosphorylation-at the center of autophagy regulation bcl-2 antiapoptotic proteins inhibit beclin 1-dependent autophagy p53-mediated molecular control of autophagy in tumor cells methods in mammalian autophagy research the autophagy protein atg12 associates with antiapoptotic bcl-2 family members to promote mitochondrial apoptosis sorting cells for basal and induced autophagic flux by quantitative ratiometric flow cytometry autophagy controls the kinetics and extent of mitochondrial apoptosis by regulating puma levels autophagy as a trigger for cell death: autophagic degradation of inhibitor of apoptosis dbruce controls dna fragmentation during late oogenesis in drosophila suppression of t cell autophagy results in decreased viability and function of t cells through accelerated apoptosis in a murine sepsis model combination of trail and chal-24 synergistically induces autophagy-mediated apoptosis in lung cancer cells induction of autophagy-dependent necroptosis is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resistance fadd and caspase-8 control the outcome of autophagic signaling in proliferating t cells identification of small molecule inhibitors of phosphatidylinositol 3-kinase and autophagy fas-associated death domain (fadd) is a negative regulator of t-cell receptor-mediated necroptosis caspase inhibition prevents tumor necrosis factor-α-induced apoptosis and promotes necrotic cell death in mouse hepatocytes in vivo and in vitro an ursolic acid derived small molecule triggers cancer cell death through hyperstimulation of macropinocytosis l929 cells depends on autocrine production of tnfα mediated by the pkc-mapks-ap-1 pathway the sirtuin family, therapeutic targets to treat diseases of aging antineoplastic activity of the cytosolic foxo1 results from autophagic cell death rip3, a molecular switch for necrosis and inflammation knockout of atg5 inhibits proliferation and promotes apoptosis of df-1 cells the autophagy machinery controls cell death switching between apoptosis and necroptosis sorafenib-induced defective autophagy promotes cell death by necroptosis nad+ depletion triggers macrophage necroptosis, a cell death pathway exploited by mycobacterium tuberculosis adp-ribose) polymerase 1, parp1, modifies ezh2 and inhibits ezh2 histone methyltransferase activity after dna damage receptor interacting protein kinase-3 determines cellular necrotic response to tnf-alpha amp-activated protein kinase, an emerging drug target to regulate imbalances in lipid and carbohydrate metabolism to treat cardio-metabolic diseases role of ampk-mtor-ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks atm signals to tsc2 in the cytoplasm to regulate mtorc1 in response to ros autophagy recognizes intracellular salmonella enterica serovar typhimurium in damaged vacuoles listeria monocytogenes acta is a key player in evading autophagic recognition interactions between shigella flexneri and the autophagy machinery nod proteins: regulators of inflammation in health and disease the protein atg16l1 suppresses inflammatory cytokines induced by the intracellular sensors nod1 and nod2 in an autophagy-independent manner autophagy in infection, inflammation and immunity autophagy genes protect against salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance hlh-30/tfeb-mediated autophagy functions in a cell-autonomous manner for epithelium intrinsic cellular defense against bacterial pore-forming toxin in c. elegans mir144* inhibits antimicrobial responses against mycobacterium tuberculosis in human monocytes and macrophages by targeting the autophagy protein dram2 probiotic bacillus amyloliquefaciens sc06 induces autophagy to protect against pathogens in macrophages eradication of intracellular salmonella enterica serovar typhimurium with a small-molecule, host cell-directed agent eradication of intracellular francisella tularensis in thp-1 human macrophages with a novel autophagy inducing agent vitamin d inhibits human immunodeficiency virus type 1 and mycobacterium tuberculosis infection in macrophages through the induction of autophagy the role of autophagy in intracellular pathogen nutrient acquisition autophagosomes induced by a bacterial beclin 1 binding protein facilitate obligatory intracellular infection subversion of cellular autophagy by anaplasma phagocytophilum autophagosomes can support yersinia pseudotuberculosis replication in macrophages host response during yersinia pestis infection of human bronchial epithelial cells involves negative regulation of autophagy and suggests a modulation of survival-related and cellular growth pathways coxiella burnetii modulates beclin 1 and bcl-2, 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compartments for major histocompatibility complex class ii molecules continuously receive input from autophagosomes autophagy is an essential component of drosophila immunity against vesicular stomatitis virus virus recognition by toll-7 activates antiviral autophagy in drosophila antiviral autophagy restricts rift valley fever virus infection and is conserved from flies to mammals sirtuin 1 regulates dendritic cell activation and autophagy during respiratory syncytial virus-induced immune responses stimulation of autophagy by salicylamide derivatives-implications for viral infection foot-and-mouth disease virus infection suppresses autophagy and nf-κb antiviral responses via degradation of atg5-atg12 by 3c pro autophagy in health and disease: a double-edged sword autophagy promoted infectious kidney and spleen necrosis virus replication and decreased infectious virus yields in cpb cell line coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication autophagosome supports coxsackievirus b3 replication in host cells modification of cellular autophagy protein lc3 by poliovirus induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro knockdown of autophagy enhances the innate immune response in hepatitis c virus-infected hepatocytes matrix protein 2 of influenza a virus blocks autophagosome fusion with lysosomes autophagy, apoptosis, and the influenza virus m2 protein autophagy is involved in influenza a virus replication human monoclonal scfv that bind to different functional domains of m2 and inhibit h5n1 influenza virus replication hepatitis c virus triggers golgi fragmentation and autophagy through the immunity-related gtpase m. proc. natl. acad. sci newcastle disease virus triggers autophagy in u251 glioma cells to enhance virus replication autophagy benefits the replication of newcastle disease virus in chicken cells and tissues human immunodeficiency virus type-1 infection inhibits autophagy autophagy pathway intersects with hiv-1 biosynthesis and regulates viral yields in macrophages dysregulation of autophagy by hiv-1 nef in human astrocytes the role of autophagy in thp-1 macrophages resistance to hiv-vpr-induced apoptosis autophagy in herpesvirus immune control and immune escape kaposi's sarcoma-associated herpesvirus k7 modulates rubicon-mediated inhibition of autophagosome maturation targeting γ-herpesvirus 68 bcl-2-ediated down-regulation of autophagy intracellular vesicle acidification promotes maturation of infectious poliovirus particles autophagy knocked down by high-risk hpv infection and uterine cervical carcinogenesis flavivirus ns4a-induced autophagy protects cells against death and enhances virus replication involvement of autophagy in coronavirus replication coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate foot-and-mouth disease virus utilizes an autophagic pathway during viral replication knockdown of autophagy inhibits infectious hepatitis c virus release by the exosomal pathway hepatitis c virus infection induces autophagy as a prosurvival mechanism to alleviate hepatic er-stress response antibody-dependent enhancement of dengue virus infection inhibits rlr-mediated type-1 ifn-independent signalling through upregulation of cellular autophagy infectious bursal disease virus subverts autophagic vacuoles to promote viral maturation and release adenovirus's last trick: you say lysis, we say autophagy unveiling the roles of autophagy in innate and adaptive immunity hepatitis b virus x protein sensitizes cells to starvation-induced autophagy via up-regulation of beclin 1 expression autophagy genes enhance murine gamma herpesvirus 68 reactivation from latency by preventing virus-induced systemic inflammation hsv-1 icp34.5 confers neurovirulence by targeting the beclin 1 autophagy protein. cell host microbe inhibition of autophagy ameliorates acute lung injury caused by avian influenza a h5n1 infection cellular entry of human papillomavirus type 16 involves activation of the phosphatidylinositol 3-kinase/akt/mtor pathway and inhibition of autophagy coronavirus replication complex formation utilizes components of cellular autophagy autophagy in human health and disease induction of autophagy and inhibition of tumorigenesis by beclin 1 autophagy modulation in cancer: current knowledge on action and therapy endogenous hmgb1 regulates autophagy promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice autophagy-deficient mice develop multiple liver tumors cell biology, autophagy and cancer beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor lower beclin 1 downregulates her2 expression to enhance tamoxifen sensitivity and predicts a favorable outcome for er positive breast cancer autophagic and tumour suppressor activity of a novel beclin1-binding protein uvrag autophagy, mitochondrial quality control, and oncogenesis autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis autophagy suppresses tumor progression by limiting chromosomal instability the expanding role of mtor in cancer cell growth and proliferation inhibition of mtor induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of huntington disease the coordinate regulation of the p53 and mtor pathways in cells autophagy and cancer, dynamism of the metabolism of tumor cells and tissues pogostone induces autophagy and apoptosis involving pi3k/akt/mtor axis in human colorectal carcinoma hct116 cells new anti-cancer molecules targeting hspa5/bip to induce endoplasmic reticulum stress, autophagy and apoptosis trichosanthin inhibits human ovarian cancer cells growth due to apoptosis and autophagy autophagy is activated in colorectal cancer cells and contributes to the tolerance to nutrient deprivation guanine nucleotide exchange on heterotrimeric gi3 protein controls autophagic sequestration in ht-29 cells autophagy inhibition by chloroquine sensitizes ht-29 colorectal cancer cells to concurrent chemoradiation mutational and expressional analysis of bnip3, a pro-apoptotic bcl-2 member, in gastric carcinomas activation of autophagy and pparγ protect colon cancer cells against apoptosis induced by interactive effects of butyrate and dha in a cell type-dependent manner: the role of cell differentiation tumor suppression in mice lacking gabarap, an atg8/lc3 family member implicated in autophagy, is associated with alterations in cytokine secretion and cell death activated ras requires autophagy to maintain oxidative metabolism and tumorigenesis autophagy as a target for anticancer therapy the tumor suppressor gene arhi regulates autophagy and tumor dormancy in human ovarian cancer cells role of autophagy in suppression of inflammation and cancer autophagy, senescence and tumor dormancy in cancer therapy autophagy mediates the mitotic senescence transition psmd10/gankyrin induces autophagy to promote tumor progression through cytoplasmic interaction with atg7 and nuclear transactivation of atg7 expression suppression of basal autophagy in neural cells causes neurodegenerative disease in mice promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult drosophila autophagy mechanisms for brain recovery. keep it clean, keep it alive. in neurobiological and psychological aspects of brain recovery prenatal alcohol exposure impairs autophagy in neonatal brain cortical microvessels autophagy regulates notch degradation and modulates stem cell development and neurogenesis neurodegenerative lysosomal disorders: a continuum from development to late age liaisons dangereuses, autophagy, neuronal survival and neurodegeneration autophagy gone awry in neurodegenerative diseases gene of the month: becn1 brain metabolism as a modulator of autophagy in neurodegeneration lysosomal system pathways, genes to neurodegeneration in alzheimer's disease the autophagy-related protein beclin 1 shows reduced expression in early alzheimer disease and regulates amyloid beta accumulation in mice wild type alpha-synuclein is degraded by chaperone-mediated autophagy and macroautophagy in neuronal cells regulation of neuronal survival factor mef2d by chaperone-mediated autophagy trehalose, a novel mtor-independent autophagy enhancer accelerates the clearance of mutant huntingtin and alphasynuclein loss of autophagy in the central nervous system causes neurodegeneration in mice ambra1 regulates autophagy and development of the nervous system alpha synuclein is degraded by both autophagy and the proteasome beclin 1 gene transfer activates autophagy and ameliorates the neurodegenerative pathology in alpha-synuclein models of parkinson's and lewy body diseases dissecting the molecular pathway involved in plk2 kinase-mediated α-synuclein-selective autophagic degradation pink1/parkin mitophagy and neurodegeneration-what do we really know in vivo? a gene implicated in autosomal recessive juvenile parkinsonism, is a candidate tumor suppressor gene on chromosome 6q25-q27 pink1-dependent recruitment of parkin to mitochondria in mitophagy autophagy genes protect against disease caused by polyglutamine expansion proteins in caenorhabditis elegans the small molecule auten-99 (autophagy enhancer-99) prevents the progression of neurodegenerative symptoms autophagic-lysosomal dysfunction and neurodegeneration in niemann-pick type c mice: lipid starvation or indigestion? autophagy in immunity, implications in etiology of autoimmunity/autoinflammatory 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targeting of ferritin. international and national patent collections microrna that regulate autophagy use of hepcidin as a regulator of iron homeostasis. u.s. patent 7169758b2 erythroferrone and erfe polypeptides and methods of regulating iron metabolism. patent ca 2890040a1 syndromes of neurodegeneration with brain iron accumulation exome sequencing reveals de novo wdr45 mutations causing a phenotypically distinct, x-linked dominant form of nbia de novo mutations in the autophagy gene wdr45 cause static encephalopathy of childhood with neurodegeneration in adulthood pathophysiology and treatment of neurodegeneration with brain iron accumulation in the pediatric population the wd40 repeat ptdins(3)p-binding protein epg-6 regulates progression of omegasomes to autophagosomes mammalian atg18 (wipi2) localizes to omegasome-anchored phagophores and positively regulates lc3 lipidation crohn's disease loss of the autophagy protein atg16l1 enhances endotoxin-induced il-1beta production 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conditions this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: all the authors acknowledge and thank their respective institutes and universities. all authors declare that there exist no commercial or financial relationships that could in any way lead to a potential conflict of interest. key: cord-321386-u1imic5l authors: li, chun; zhao, jialing; wang, changzhong; yao, yuhua title: protein sequence comparison and dna-binding protein identification with generalized pseaac and graphical representation date: 2018-02-17 journal: comb chem high throughput screen doi: 10.2174/1386207321666180130100838 sha: doc_id: 321386 cord_uid: u1imic5l aim and objective: the rapid increase in the amount of protein sequence data available leads to an urgent need for novel computational algorithms to analyze and compare these sequences. this study is undertaken to develop an efficient computational approach for timely encoding protein sequences and extracting the hidden information. methods: based on two physicochemical properties of amino acids, a protein primary sequence was converted into a three-letter sequence, and then a graph without loops and multiple edges and its geometric line adjacency matrix were obtained. a generalized pseaac (pseudo amino acid composition) model was thus constructed to characterize a protein sequence numerically. results: by using the proposed mathematical descriptor of a protein sequence, similarity comparisons among β-globin proteins of 17 species and 72 spike proteins of coronaviruses were made, respectively. the resulting clusters agreed well with the established taxonomic groups. in addition, a generalized pseaac based svm (support vector machine) model was developed to identify dna-binding proteins. experiment results showed that our method performed better than dnabinder, dna-prot, idna-prot and endna-prot by 3.29-10.44% in terms of acc, 0.056-0.206 in terms of mcc, and 1.45-15.76% in terms of f1m. when the benchmark dataset was expanded with negative samples, the presented approach outperformed the four previous methods with improvement in the range of 2.49-19.12% in terms of acc, 0.05-0.32 in terms of mcc, and 3.82-33.85% in terms of f1m. conclusion: these results suggested that the generalized pseaac model was very efficient for comparison and analysis of protein sequences, and very competitive in identifying dna-binding proteins. dna-binding proteins (dna-bps) are very important functional proteins in a cell. these proteins play vital roles in various cellular processes, including dna replication, transcription, regulation of gene expression, packaging, and other activities associated with dna [1] [2] [3] [4] [5] . it is therefore substantially important to distinguish dna-bps from non-dna-binding proteins (nbps). in the past, many experimental and computational techniques have been developed for identifying dna-bps. experimental techniques can provide a clear-cut answer to a query protein. however, the experimental methods are cost-intensive and time-consuming, and thus impractical for large datasets [3] [4] [5] [6] [7] . computational methods can be broadly divided into two categories: structure-based method and sequence-based *address correspondence to this author at the school of mathematics and statistics, hainan normal university, haikou 571158, china; tel: +86-898-65883210; e-mail: lichwun@163.com method. the former can discriminate dna-binding and nonbinding proteins with high accuracy, but these methods can't be employed in high throughput annotation, as they require the structure information of a query protein [1] . though tremendous progress has been achieved in experimental determination of protein structures in the past five decades, it can't keep pace with the explosive growth of sequence information resulting from modern sequencing technology [8] . yet as suggested by anfinsen [9] , proteins contain within their amino acid sequences enough information to determine their native conformation. therefore, it is more promising to use sequence-based methods to identify dna-bps. one of the core issues to the sequence-based methods is how to characterize protein sequences and harvest the fruits hidden in them. the most typical approach is using the amino acid composition (aac) to formulate a protein sequence. owing to its simplicity, the aac model was widely applied in a number of earlier statistic-based methods. however, as pointed out in ref [6] , if we denote by the counts of 20 standard amino acids in a protein sequence, then we can see that there are a total of different sequences/strings possessing the same aac. the reason is that aac model neglects the order relation among elements of a sequence. to overcome this drawback, the concept of pseudo amino acid composition (pseaac, or chou's pseaac) was proposed [10] [11] [12] [13] [14] [15] [16] [17] [18] . the essence of pseaac is that it not only covers aac, but also contains additional order-correlated factors along a protein sequence. another popular way for sequence analysis is to convert the protein primary sequence over 20 amino acids into a reduced one. the earliest and simplest reduction was the well-known hp model, in which 20 standard amino acids are divided into two types, hydrophobic (h) (or non-polar) and polar (p) (or hydrophilic). on the basis of the classic model, a detailed hp model was introduced by dividing the polar class into three subclasses: positive polar, uncharged polar and negative polar [19] . in addition, a few five-group classifications of amino acids were presented for practical purposes [20] [21] [22] [23] . by considering property-based triples, li et al. [6] put forward a six-letter model of amino acids. also based on three physical-chemical properties of amino acids, yao et al. [24] mapped the 20 standard amino acids to eight vertices of a cube with the center of origin, and thus an eightgroup model of amino acids is obtained. motivated by the work mentioned above, we propose a generalized pseaac which is grounded on a three-letter model and 2-d graphical representation of a protein sequence. we summarize the main work of this paper as follows: in section 2, we briefly introduce five datasets used in this study. in section 3, on the basis of two important physicochemical properties of amino acids, we cluster the 20 standard amino acids into three groups. by assigning to each group a representative symbol, we transform a protein sequence into a three-letter sequence. then a 2-d graph without loops and multiple edges and its geometric line adjacency matrix are obtained. a sequence-derived feature vector of dimension (25+ ) is thus constructed to characterize a protein sequence. our scheme is similar to, but obviously different from that of pseaac. in section 4, we apply the presented feature vector to compare -globin proteins of 17 species and 72 spike proteins of coronaviruses respectively. also, we develop a svm (support vector machine) model using the generalized pseaac to identify dna-binding and non-binding proteins on three datasets. experiment results show that the presented method outperforms the existing methods including dnabinder [1] , dna-prot [2] , idna-prot [3] and endna-prot [4] . finally, conclusions are given in section 5. in this study, the following five datasets are used. for convenience, they are denoted by betaset, covset, dnaset, dnaeset and dnaiset, respectively. the dataset called betaset is composed of -globin protein of 17 species: human (alu64020), gorilla (p02024), chimpanzee (p68873), cattle (caa25111), banteng (baj05126), goat (aaa30913), sheep (abc86525), european hare (caa68429), rabbit (caa24251), house mouse (add52660), western wild mouse (acy03394), spiny mouse (acy03377), norway rat (caa29887), opossum (aaa30976), guttata (ach46399), gallus (caa23700), muscovy duck (caa33756). this dataset is used to determine the adjustable parameters in a feature vector. this dataset consists of 72 spike proteins of coronaviruses (covs), 23 of which are mers-covs, and 30 are sars-covs. covs can be divided into three groups according to serotypes. group alpha (formerly known as cov-1) and group beta (formerly cov-2) contain mammalian viruses, while group gamma (formerly cov-3) contains only avian viruses. the name, accession number, and abbreviation of the 72 sequences are listed in table 1 . according to the existing taxonomic groups, sequences 1-5 belong to the first group, sequences 6-8 belong to the third group, and the remainings belong to the second group. this is a benchmark dataset created in 2007 by kumar et al. [1] . it contains 396 sequences, 146 of which are dna-bps (positive samples), and 250 nbps (negative samples). in both the positive and the negative sets, the sequence similarity between any two proteins is not more than 25%. this dataset was also generated by kumar et al. [1] which is based on the work of wang and brown [25] . it originally contains 92 dna-bps and 100 nbps. in order to avoid overestimating a given method, those sequences having sequence similarity with dnaset were removed by xu et al. [4] , and the final dataset is composed of 82 dna-bps and 100 nbps. as an expanded benchmark dataset, dnaeset was constructed in 2014 by xu et al. [4] . according to a sequence filter criteria which is identical to dnaset, they added a number of nbps to dnaset, and the total number of nbps is 2125. by removing the sequence which has sequence identity with dnaiset, the current version of dnaeset has 146 dna-bps and 1710 nbps. isoelectric point (pi) and relative distance (rd) are two important physicochemical properties of the 20 standard amino acids [26] [27] [28] . their original numerical values are listed in table 2 (relative distance) varies between 1469 and 3355. therefore, the normalization of these values is needed. here, we scale them into the interval [0,1] by the formulary below: the corresponding values are listed in table 3 . the last row in this table gives the average values. for the i-th amino acid , if , then we label it by "+", otherwise we will label it by "-". similarly, if property is considered, the second label for amino acid can be obtained. in this way, each of the 20 standard amino acids has a label pair. in table 3 , the corresponding labels are also listed. amino acids with a same label pair are viewed as members of a same group. thus, the 20 standard amino acids are distributed to the following groups: for each group, the first amino acid is used to stand for the group. thus the three groups have three representative letters, they are a, c and h, respectively. the value for the property of a group is defined as the average value for the property of all members in the group. in the left-hand side of table 4 , we list the corresponding values of the three groups. obviously, each group can be viewed as a 2-d vector. in order to make the vectors of the three groups have unit length, we further normalize them to be unit vectors, and list the normalized values ( ) in the right-hand side of table 4 . in fig. (1) , we show the 2-d map of the 20 standard amino acids according to the classification above. by substituting each amino acid with its representative letter, a protein primary sequence is reduced into a threeletter sequence. for example, the three-letter sequence of the sequence segment ekaavtgfwgkvkvdevgaea is ahaaahcccchahacaacaaa. to obtain the graphical representation of a reduced sequence, we start from the origin (0,0) and move in xoy-plane in the direction dictated by fig. (1) . in mathematics, one can let be a given three-letter sequence. and then one has a map , which maps s into a plot set. explicitly, , and is given by where, t represents the transpose of a matrix, (j=1,2) represents the j-th component of the unit vector corresponding to (cf. fig. 1 and table 4 ). connecting all points of the plot set in turn, a 2-d curve is drawn. in fig. (2) , we show the 2-d graphical representation of sequence ahaaahcccchahacaacaaa. it is not difficult to find that the 2-d graphical representation has no degeneracy, and thus is a simple graph, that is, a graph without loops and multiple edges. in this section, we give a numerical characterization of a protein sequence that will facilitate quantitative comparisons of protein sequences. as is known, once a graphical representation is given, it can be transformed into some structural matrices, such as the matrices ed, gd, m/m, and l/l [6, 24, [29] [30] [31] [32] [33] [34] [35] [36] [37] . here we employ the l/l matrix. l/l is a nonnegative symmetric matrix whose off-diagonal entries are defined as a quotient of the euclidean distance between two vertices of the graph and the sum of geometrical lengths of edges between the two vertices. by definition all diagonal elements are zero. obviously, the entries in a l/l matrix are less than or equal to one. the higher order k l/ k l matrix is the matrix whose (i,j)-entry is . as the exponent k approaches positive infinity, k l/ k l converges to a (0,1) matrix (denoted by b l/ b l). with respect to the proposed 2-d graph, [ b l/ b l] ij =1 if and only if the two corresponding vertices lie on a straight line in the curve, including the cases of adjacency and non-adjacency. in this sense, we call such a matrix a geometric line adjacency matrix (glam), or simply a generalized adjacency matrix (gam), generated by a graph, and denote it by . the first zagreb index is a well-known vertex-degreebased molecular structure descriptor. this index was first time considered by gutman and trinajstic about 45 years ago, and since then discussed and used in numerous studies (see [38] [39] [40] and the references cited therein). the first zagreb index is defined as (2) where du denotes the degree (=number of first neighbors) of the vertex u in graph g. if g is a simple graph (i.e. without loops and multiple edges), z g1 can be also obtained directly from its adjacency matrix since the row-sums of this matrix are equal to degrees of the corresponding vertices. it should be mentioned that the zagreb index gives greater weights to inner vertices and edges than to outer vertices and edges of a graph [38] . one way to amend it is to insert inverse values of the vertex-degree into eq(2), and thus the modified zagreb index has been proposed [38] : clearly, m z g1 gives greater weights to outer vertices/edges than to inner ones in a graph. at the same time, on the basis of our geometric line adjacency matrix, we can count the vertex-pair with generalized adjacency relationship. it should be noted that, in our case, the 'neighbors' include not only the conventional neighbors, i.e. the first neighbors, but also the second neighbors, the third neighbors, and so on. we call the corresponding number of graph g a line-adjacency index, and denote it by la(g). then we have a graph-based index: for a symmetric matrix, eigenvalue-based indices, such as the leading eigenvalue [29] [30] [31] [32] [33] 35] and the graph energy [17] , are often used as the matrix invariants. moreover, in our previous paper [41] , an alternative invariant called 'ale-index' was proposed. the ale-index is defined by the following formula: (4) where l is the order of the matrix, and are the m1-and f-norms of a matrix respectively. in order to reduce variations caused by comparison of matrices with different sizes, we consider a normalized ale-index instead of . for convenience, we denote this matrixbased index by . in addition, with respect to three-letter sequence , we define a coupling mode function by , (n=1, 2) where p 1 and p 2 are values for properties of the corresponding representative letter (group), integer k represents the counted rank (or tier) of the coupling mode. then, following the similar procedures in [10, 11] , we can extract global sequence-order information of the three-letter sequence s by , , . where is called the k-th tier correlation factor. clearly, reflects the coupling mode between the most contiguous elements along three-letter sequence s, is the coupling mode between the second most contiguous, the third most contiguous, and so forth. furthermore, if the respective counts of the three representative letters (a, c and h) in sequence s are , respectively, then we can obtain a so-called group composition (gc): where, denotes the size of a group (set). consequently, elements are derived, which reflect the information about the reduced sequence and, particularly, the 2-d graphical representation. by combining these elements with the conventional amino acid composition (aac), a dimensional feature vector can be constructed to numerically characterize a protein sequence: , where (8) here, are frequencies of occurrence of the 20 standard amino acids in a protein sequence, and are weight factors. as will be described later in detail, the four adjustable parameters in eqs (7) and (8) can be determined by a set of known samples. roughly speaking, the vector contains the feature of aac, and the information beyond aac as well, which is similar to chou's pseaac in form. therefore, we call such a vector formulated by eqs (7) and (8) the generalized pseaac of a protein sequence. in this section, we will discuss the use of the generalized pseaac. as can be seen from eqs (7) and (8), the present mathematical descriptor contains four uncertain parameters: , w 1 , w 2 and w 3 . here represents the total number of correlation ranks counted (cf. eq(6)), which is an integer. generally speaking, the greater the value of , the more sequence-order effects will be incorporated. however, if the value is too large, it might cause the overfitting problem or 'high dimension disaster' [15] , therefore, we endeavour to limit the value of to a small integer. in this study, the five datasets (betaset, covset, dnaset, dnaeset and dnaiset) are arranged into two groups: one contains betaset, the other includes the rest. the first group is used for determining the four adjustable parameters, and the second group for testing purpose. according to the method mentioned above, we first associate each of 17 protein sequences in betaset with a dimensional vector (cf. eqs (7) and (8)), and then calculate the pair-wise euclidean distance between any two of the 17 protein sequences via their m-d vectors. thus a real symmetric matrix is obtained. on the basis of the achieved distance matrix , a upgma tree is constructed using mega4 package. the result will depend on values of the rank and the three weight factors. it is found that when , , and , the three non-mammals (muscovy duck, gallus and guttata) form a separate branch and stay outside of the mammals. moreover, in the subtree of mammals, primate species (human, chimpanzee, gorilla) are grouped closely. also, rodent species (norway rat, spiny mouse, house mouse, western wild mouse) and lagomorph species (rabbit, european hare) are situated at independent branches, respectively. while goat, sheep, cattle and banteng appear to cluster together (fig. 3) . this result is analogous to that reported in the literature [6, 29, 30, 35, 36] . accordingly, the four numerical values are respectively used for the four uncertain parameters, and a 31-d feature vector is thus obtained. fig. (3) . the relationship tree of 17 species. in order to evaluate the effectiveness of our method, we test it by phylogenetic analysis on the covset dataset. coronaviruses (covs) belong to the genus coronavirus of family coronaviridae [42] . the first coronavirus (hcov-229e) was isolated from humans in 1965. until 2003, coronaviruses attracted little interest beyond causing mild upper respiratory tract infections. however, this phenomenon changed dramatically with the emergence of sars-cov and mers-cov. as of july 2017, 2040 laboratory-confirmed cases of mers-cov infection were reported in over 27 countries, and at least 710 individuals have died (crude cfr 34.8%) [43] . using the above-determined values for parameters , w 1 , w 2 , and w 3 , we calculate the 31-d feature vectors of 72 coronavirus spike proteins and their euclidean distance matrix; then the corresponding phylogenetic tree (fig. 4) is constructed. observing fig. (4) , we find that the 72 coronavirus spike proteins are clustered into three groups: one contains the five alpha coronaviruses (pedvc, pedv, tgevg, tgev, and hcov-229e), the second includes the three gamma coronaviruses (ibv, ibvbj, ibvc), and the third corresponds to the group beta. a closer look at the subtree of beta coronaviruses shows that mers-covs are clearly clustered together, so it is with sars-covs, while mhv, mhva, mhvm, mhvp, mhvjhm, bcov, bcove, bcovl, bcovm, bcovq and hcov-oc43 are situated at an independent branch. the resulting cluster agrees well with the established taxonomic groups. to further assess the effectiveness of the porposed method, we conduct a series of experiments of identification of dna-binding proteins on three datasets: dnaset, dnaeset and dnaiset. among them, dnaset and dnaeset serve as training datasets, while dnaiset serves as an independent testing dataset. support vector machine (svm) is employed as the classifier, and r package 'e1071' v1.6-8 [44] is used to implement svm. for a given set of binary-labeled training examples, svm maps the input space into a higherdimensional space and seeks a hyperplane to separate the positive samples from the negative ones [25] . the optimal hyperplane maximizes the separation margin between the two classes of training data. the distance measurement between the data points in the high-dimensional space is defined by the kernel function. in this study, we use the radial basis function (rbf) kernel . this model involves two tunable parameters: the kernel width and the penalty parameter c. prediction performance can be assessed using some quality indices including accuracy (acc), sensitivity (se), specificity (sp), fmeasure (f1m) and matthews correlation coefficient (mcc) [2, 4, 5, 25, 37, 45] : , , , . where tp, tn, fp, and fn are defined as the numbers of true positive, true negative, false positive, and false negative samples obtained from the prediction respectively, while p and r denote precision value and recall value, respectively. one can also use the alternative definition by a series of studies published recently [15, [46] [47] [48] . the higher the values of these measurements, the better the quality of prediction. this experiment is made on dnaset itself. to obtain a reliable result with few error, the svm model on dnaset is established by 5-fold cross-validation (5cv) with 3 runs. here the 31-d feature vector of a protein sequence serves as the input for svm. in a 5cv, the positive and negative samples are randomly distributed into five subsets or the socalled folds, and the test is repeated five times. in each of the five iterations, one subset is used as the testing set, while the remaining four subsets are combined together and used to build a classifier (training). the predictions made for the test data instances in all the five iterations yield the final result. the sensitivity, specificity, acc, mcc and f1m are calculated for each run, and the corresponding results and their average values are listed in table 5 . as can be seen fig. (4) . the relationship tree of 72 coronavirus spike proteins. t a lw a n t c 2 t a lw a n t c 1 t a lw a n t c 3 tw 1 tw 2 tw h tw j urbani from this table, we achieve the accuracy (acc) of 89.65%, with mcc of 0.776 and f1m of 84.91%. this result shows that our svm model performs well on the benchmark dataset dnaset. it is important to examine the performance of the newly developed method on an independent dataset. in this experiment, we establish the classifier with the benchmark dataset dnaset and then test it on the independent dataset dnaiset. to decide the parameter pair (γ, c), we utilize a systematic grid search for and , where integers i and j are in ranges [-3, 3] and [0, 3], respectively. it is find that and are the optimal values for dnaset. with the best pair (γ, c), dnaiset is fed to the svm. as a result, our model correctly predicts 68 out of 82 dna-bps and 92 out of 100 nbps. the acc arrives at 87.91%, with the mcc, sensitivity, specificity, and f1m of 0.756, 82.93%, 92.00% and 86.07%, respectively (see table 6 ). this demonstrates that our svm model performs equally well on independent dataset. for convenience of comparison, results of some existing methods including dnabinder [1] , dna-prot [2] , idna-prot [3] and endna-prot [4] are also listed in table 6 . dnabinder developed by kumar et al. [1] can extract evolutionary information in form of position specific scoring matrix (pssm) from the corresponding protein sequence. pssm-21 and pssm-400 are two feature vectors generated by means of pssm, whose dimensions are 21 and 400, respectively. in [1] , pssm-400 based svm model was mainly used for predicting dna-bps. dna-prot [2] is a random forest based method, in which the feature vector includes sequence information and structure information, such as the composition of 20 standard amino acids, composition of 10 amino acid groups, and secondary structure information predicted from a protein sequence. idna-prot [3] constructs the feature vector via the grey model, and random forest is also used as the operation engine. endna-prot [4] is a predictor which encodes a protein sequence into a feature vector with dimension of 188 and adopts an ensemble classifier constructed with four types of machine learning classifiers. all these methods are tested on the same datasets to make an unbiased comparison with our method. observing table 6 , we can see that the current approach outperforms other methods by 3.29-10.44% in terms of acc, 0.056-0.206 in terms of mcc, and 1. .76% in terms of f1m. this result indicates that our method achieves highly comparable performance. when the size of positive samples is comparable to that of negative samples, many machine learning algorithms should have better performance. however, in real life, the number of non-binding proteins is much greater than that of dna-bps, i.e., . in this case, the frequency of nbps is generally much greater than that of the binding ones in the predictions, that is, . eqs (10) and (11) lead to that the value of acc defined by eq (9) tends towards 1. to solve this problem, instead of using the definition of acc in eq (9), here we use the alternative definition [49, 50] : . (12) in order to analyze the influence of the number of negative samples in a benchmark dataset on the predictive performance of the current method, we construct a series of subsets of dnaeset and use them as training set in turn, while dnaiset is always used as the testing set. each subset contains all the 146 dna-bps and a part of nbps in dnaeset. in detail, if the set of nbps in is denoted by , k=1, 2, ..., then consists of 250 nbps randomly selected from dnaeset. and is obtained by adding 50 nbps to , until 1700 nbps are contained in it. for each subset , k=1, 2, ..., 30, we develop the svm model by 5cv with 3 runs. the results averaging over the three runs are given in fig. (5) . from fig. (5) we can see that the curves of acc and acc visibly split with each other when n, the size of , is larger. with increasing of n, acc increases rapidly, while acc tends to be steady. the value of acc seems higher and higher on the surface, but it cannot correctly reflect the performance because it is nothing but a false appearance. in order to show the advantage of their method, xu et al. [4] created a dataset called expanded benchmark dataset1100 with all the 146 positive samples and 1100 negative samples in dnaeset, which is employed as another training dataset to evaluate the predictive performance on the independent dataset dnaiset. for convenience of comparison, we also select the expanded benchmark dataset to establish the classifier and test it on dnaiset. repeating this procedure five times, the average results are given in table 7 (the first row). results obtained by the other four methods (dnabinder, dna-prot, idna-prot and endna-prot) trained on the expanded benchmark dataset with n=1100 are also listed in table 7 . from this table we see that the overall accuracy of our method is about 92%, with mcc of 0.84 and f1m of 91.24%, which outperforms other methods with improvement in the range of 2.49-19.12% in terms of acc, 0.05-0.32 in terms of mcc, and 3.82-33.85% in terms of f1m. this suggests that our method performs well on unbalanced datasets. based on two important physicochemical properties, 20 standard amino acids were distributed into three groups, and to each of which a representative symbol was assigned. by replacing each amino acid with its representative letter, a protein primary sequence was converted into a three-letter sequence, which can be viewed as a coarse-grained description of the protein primary sequence. on the basis of the three-letter sequence, a graph without loops and multiple edges was obtained. by taking the advantage of the 2-d graph, we constructed a geometric line adjacency matrix (glam) and then the corresponding ale-index, the lineadjacency index, the first zagreb index and its modification were calculated. in addition, order-correlated factors were extracted via the reduced sequence. by combining these elements with the frequencies of occurrence of 20 standard amino acids and their three representative letters, a generalized pseaac model of a protein sequence was constructed. on five popular datasets, the 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genomes irna-pseu: identifying rna pseudouridine sites recognition of protein coding genes in the yeast genome at better than 95% accuracy based on the z curve using a euclid distance discriminant method to find protein coding genes in the yeast genome the authors' greatest gratitude goes to the anonymous referees for their insightful suggestions and generous support.the authors are also indebted to the previous programs: the natural science foundation of liaoning province (201602005), the program for liaoning innovative research team in university (lt2014024), and the national natural science foundation of china (61762035). not applicable. the authors declare no conflict of interest, financial or otherwise. key: cord-302490-em1tiz7s authors: cañadas, olga; olmeda, bárbara; alonso, alejandro; pérez-gil, jesús title: lipid–protein and protein–protein interactions in the pulmonary surfactant system and their role in lung homeostasis date: 2020-05-25 journal: int j mol sci doi: 10.3390/ijms21103708 sha: doc_id: 302490 cord_uid: em1tiz7s pulmonary surfactant is a lipid/protein complex synthesized by the alveolar epithelium and secreted into the airspaces, where it coats and protects the large respiratory air–liquid interface. surfactant, assembled as a complex network of membranous structures, integrates elements in charge of reducing surface tension to a minimum along the breathing cycle, thus maintaining a large surface open to gas exchange and also protecting the lung and the body from the entrance of a myriad of potentially pathogenic entities. different molecules in the surfactant establish a multivalent crosstalk with the epithelium, the immune system and the lung microbiota, constituting a crucial platform to sustain homeostasis, under health and disease. this review summarizes some of the most important molecules and interactions within lung surfactant and how multiple lipid–protein and protein–protein interactions contribute to the proper maintenance of an operative respiratory surface. in the lung, the essential function of respiration takes place through gas exchange between air and blood. to allow this, a perfect architecture has been developed in which alveoli constitute the functional units of the pulmonary system. there, a thin exchange surface structure is needed to ensure an effective diffusion of oxygen to the capillaries, which is achieved by the existence of flattened alveolar epithelial type i cells (ae1c) that constitute 95% of the total alveolar area [1] . on the other hand, the presence of an air-water interface physically requires a reduction of the surface tension generated by the cohesive forces between liquid molecules that, otherwise, would lead to shrinkage of alveoli [2] . to fulfill this crucial role, alveolar epithelial type ii cells (ae2c) secrete pulmonary surfactant, a lipid-protein complex that forms a surface active film at the respiratory interface, allowing its stability and avoiding alveolar collapse during expiration [3, 4] . moreover, beyond this main function, the huge respiratory area exposed to external potentially harmful particles and microorganisms, entails an important role of the lung in protection against pathogens, for which alveolar macrophages (am) are critical, together with the first line of defense constituted by surfactant [5] . pulmonary surfactant is mainly composed of lipids (approximately 90% by mass), with phospholipids constituting the principal class, with phosphatidylcholine (pc) as the predominant species (70-80%) . among these, the saturated dipalmitoylphosphatidylcholine (dppc) accounts for 40% in average (although it can be as much as 50-55% of surfactant mass in humans, pigs, rats or mice), whereas unsaturated [20, 21] . upon secretion to the alveolar fluid, surfactant is efficiently adsorbed into the air-liquid interface, in a process highly dependent on the presence of proteins sp-b and sp-c [22, 23] . multilayered membranous surfactant structures associate to the interfacial film forming a reservoir, which provides mechanical stability and a source of material upon respiratory dynamics [19] . extracellular surfactant structures include a membrane network promoted by sp-a and sp-b (tubular myelin) [24] and membrane arrangements of different complexity [25] . after exposure to air, surfactant "used" membranes are converted into small vesicles [26] in a sp-d-mediated process [27] that can be at least partly re-uptaken by ae2c, a process promoted by sp-a [28] . sp-a also acts as a secretion inhibitory signal [29] , in an opposing role to that of sp-b as secretion inducer [30] . components captured by ae2c are routed to the recycling pathway or degraded [14] . besides, alveolar macrophages account for a 20% of surfactant clearance [31] , for which the presence of granulocyte-macrophage colony stimulating factor (gm-csf) is required [32] . alveolar homeostasis highly depends on the effective cross-talk between alveolar macrophages (am) and ae2c and the maintenance of lipid homeostasis in both cells, involving a proper reverse cholesterol transport (rct) mediated by abca1 and abcg1 lipid transporters [33] [34] [35] . sp-a is represented as its most abundant octadecameric form; sp-b as double rings, each formed by six dimers; sp-c as monomers; sp-d as dodecamers. (lb) . proper formation of these highly packed lipid organelles requires the presence of sp-b and abca3 [20, 21] . upon secretion to the alveolar fluid, surfactant is efficiently adsorbed into the air-liquid interface, in a process highly dependent on the presence of proteins sp-b and sp-c [22, 23] . multilayered membranous surfactant structures associate to the interfacial film forming a reservoir, which provides mechanical stability and a source of material upon respiratory dynamics [19] . extracellular surfactant structures include a membrane network promoted by sp-a and sp-b (tubular myelin) [24] and membrane arrangements of different complexity [25] . after exposure to air, surfactant "used" membranes are converted into small vesicles [26] in a sp-d-mediated process [27] that can be at least partly re-uptaken by ae2c, a process promoted by sp-a [28] . sp-a also acts as a secretion inhibitory signal [29] , in an opposing role to that of sp-b as secretion inducer [30] . components captured by ae2c are routed to the recycling pathway or degraded [14] . besides, alveolar macrophages account for a 20% of surfactant clearance [31] , for which the presence of granulocyte-macrophage colony stimulating factor (gm-csf) is required [32] . alveolar homeostasis highly depends on the effective cross-talk between alveolar macrophages (am) and ae2c and the maintenance of lipid homeostasis in both cells, involving a proper reverse cholesterol transport (rct) mediated by abca1 and abcg1 lipid transporters [33] [34] [35] . sp-a is represented as its most abundant octadecameric form; sp-b as double rings, each formed by six dimers; sp-c as monomers; sp-d as dodecamers. hydrophobic proteins sp-b and sp-c are required for efficient surfactant unpacking upon secretion, and for the formation of the surface film [22] . sp-b in particular plays a vital function in surfactant biophysics. this protein, which belongs to the saposin family, shows lipid membrane fusion and lysis activities that have been ascribed to the different segments of the sequence [49] [50] [51] . the lipid-protein and protein-protein interactions behind these sp-b properties are responsible for its role in promoting efficient surfactant adsorption and re-extension as well as in maintaining film stability at low surface tensions [17, 23] . sp-b is known to be able to generate contacts among membranes [52] , and we have shown that sp-b is able to oligomerize into ring structures formed by association of dimers, with a hydrophobic cavity inside [53] . docking of sp-b oligomers would allow the connection of contiguous surfactant structures and the generation of a hydrophobic tunnel through which lipids could rapidly be transferred across the alveolar fluid phase, adsorbing towards the interfacial film [45, 46] . the cohesivity among surfactant membranes provided by sp-b protein-protein interactions would also be important for supporting film stability [54] . besides offering a hydrophobic pocket, the orientation of the amphipatic α-helices of sp-b at the surface of surfactant layers allows electrostatic interaction of positively-charged residues at the protein with the polar heads of surfactant anionic phospholipids, mainly pg. within the literature, there is some evidence suggesting a preferential interaction of sp-b and sp-c with anionic phospholipids [45, [55] [56] [57] . this pg/sp-b interaction could be important for the orientation of the protein in the membrane. as a matter of fact, the effect of pg to facilitate surfactant adsorption and in maintaining the stability of the film, was early related to its interaction with cationic hydrophobic proteins, and in particular, with sp-b [58] . the possibility that sp-b oligomer could form a proteolipidic pore implies also the involvement of phospholipids, especially pg, as essential determinants of lipid transfer/adsorption properties of surfactant [45, 53, 59] . apart from pg, unsaturated phospholipids play also a role in favoring surfactant adsorption, facilitating a dynamic insertion of the material into the interfacial film [60] . on the other hand, molecular dynamics simulations have very recently proposed potential selective interactions of sp-b with cholesterol, an interesting possibility whose functional implications have still to be clarified [45] . protein/protein and lipid/protein interactions involved in the formation, stability and dynamics of the surfactant film are summarized in table 1 . regarding sp-c, beyond its implication in promoting surfactant adsorption [22, 61] , it has been ascribed a role in maintaining attachment of the surfactant reservoir to the interfacial monolayer, thus providing stability [62] [63] [64] [65] . a potential cooperation between sp-b and sp-c in biophysical functions in surfactant could be inferred from several studies, including their synergic action in permeability and lipid transfer properties of surfactant [22, 23, 59, 66] . as a matter of fact, a surfactant containing both proteins, sp-b and sp-c, exhibits superior functional properties to facilitate breathing, as tested in vivo, than surfactant preparations lacking one of the hydrophobic proteins [64, 67] . thus, the existence of direct interactions between both proteins has been considered as an interesting possibility, which finally was recently demonstrated by advanced fluorescence spectroscopy techniques [68] . a model has been proposed in which sp-c could also regulate the interaction between sp-b oligomers. the specific and opposing roles of both hydrophobic proteins is revealed by the destabilizing effect of sp-c upon lipid vesicles versus the fusogenic activity of sp-b, and might have functional implications along the surfactant cycle [66, 69] . concerning the preferential interactions of sp-c with lipids, besides pg the protein has been proposed to specifically interact with cholesterol [70] . this neutral lipid is important for maintaining a proper fluidity and viscosity in surfactant, thus facilitating the dynamical properties of the membranes [71] [72] [73] . however, cholesterol proportion in surfactant must be tightly controlled, so that its increase has been related to functional inhibition [74] [75] [76] . the presence of sp-c seems to be critical to ensure the proper function of surfactant in the presence of cholesterol [47, 77, 78] , and the combined action of sp-b and sp-c could be important for the removal of cholesterol during film refining at expiration [69] . despite hydrophobic proteins are the principal players in the biophysical activities of surfactant, the hydrophilic sp-a is also important for an optimal function. this protein, which forms oligomers consisting of six trimers, is able to bind dppc and cholesterol through its carbohydrate recognition domain (crd) [79] . sp-a induces calcium-dependent vesicle aggregation and enhanced surface adsorption [80, 81] , requiring for it a supratrimeric assembly [82] . the interaction of the protein with cholesterol could be involved in the protecting role of sp-a against the surfactant dysfunction produced as a consequence of supraphysiological concentrations of this neutral lipid [83] . interestingly, slightly different biophysical activity has been found in the two proteins encoded by the two sp-a-related human genes, sp-a1 and sp-a2, which differ in several features of its structure [84, 85] . in particular, sp-a1 has been shown to be important for the efficient adsorption of surfactant phospholipids and for the reorganization of the surface film during breathing-like compression-expansion cycles. this role could be related to the high level of oligomerization of sp-a1, which could assist in the formation of the highly cohesive multilayer film, for which sp-b is essential [43] . the existence of recently found protein-protein interactions between sp-a and sp-b [86] would also support the concerted action of both proteins in surfactant processes, and some evidences even suggest the possibility of different modes of interaction of sp-a1 and sp-a2 with sp-b [87] , although precise information is still lacking. thus, protein-protein cooperation could be crucial for developing sp-a physiological roles in surfactant at different levels, including enhancing surfactant adsorption, optimizing film stability and allowing the formation of specific surfactant structures at the alveolar spaces, as tubular myelin [10] . finally, sp-d, which is not involved in the biophysical activity of surfactant, is not lipid-associated although preferential binding to pi has been proposed, with potential implications for the regulation of surfactant homeostasis, as will be discussed later [88] . interfacial adsorption [58] cholesterol cholesterol removal from interfacial film (refining) cholesterol removal from alveolar spaces * crosstalk ae2c-am [47, 69, 77] [69] [78] sp-d pi regulation of re-uptake to ae2c [27, 93, 94] gpr116 * regulation of secretion and re-uptake * [95] * suggested potential interactions or roles. as previously described, the surfactant film is essential for reducing surface tension avoiding collapse of alveoli. the presence of surfactant itself is therefore the first indispensable requirement for maintaining lung homeostasis, stabilizing alveoli and preventing edema formation. besides this, surfactant precludes interfacial stress forces that could deform and cause dysfunction of ae2c as a consequence of their contact with the air-liquid interface [96, 97] . this role of surfactant in alveolar micromechanics entails that its deficiency or dysfunction, either by primary (e.g., absence of sp-b, ae2c injury) or secondary (as lung inflammation) causes, triggers lung injury [98, 99] . surfactant deficiency or alteration has been associated with the leading causes of acute respiratory distress syndrome (ards), which refers to inflammatory processes frequently associated with lung injury. inflammation itself mediates ae2c injury, but also liberation of pro-inflammatory mediators and cell and tissue debris, all contributing to an altered composition of surfactant. a complex group of alterations are found in alveolar spaces at ards: reduced amount of surfactant lipids and proteins, altered profiles of surfactant protein intermediates and byproducts, increased levels of plasma proteins (because of the increased capillary permeability) that produce surfactant inhibition, increased surfactant hydrolysis of surfactant by secretory phospholipase a2 (pla2) and release of reactive oxygen species [100] [101] [102] [103] [104] . absence of sp-c and decreased levels of sp-a and sp-b are associated with familial interstitial lung disease [105] . decreased levels of sp-a and/or sp-d are also observed in neonates with respiratory distress syndrome (rds) and patients with idiopathic pulmonary fibrosis, bacterial pneumonia, chronic obstructive pulmonary disease, and asthma (reviewed in [106] [107] [108] ). reduced amounts of lung collectins in the lung increase the susceptibility to respiratory infections, promote inflammation and hamper the elimination of apoptotic cells leading to abnormal lung tissue regeneration as described in the following sections. the finding that elderly people show reduced levels of sp-a in the lungs [109, 110] suggests that ageing may aggravate the clinical consequences of surfactant deficiency, particularly in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis [111] . on the other hand, since lung collectins levels are usually reduced in the lung because of protein leakage towards the vascular compartment, serum levels of these proteins are considered biomarkers of disease severity and predictive factors of unfavorable disease outcome [112] [113] [114] [115] . genetic deficiency of sp-b is rare, and occurs as a consequence of mutations leading to the absence of sp-b or prosp-b proteins. the disease is characterized by the lack of properly packed lamellar bodies, absence of mature sp-c and appearance of unprocessed prosp-c forms [102, [116] [117] [118] . unless partial deficiency in sp-b production still allows a certain production of sp-b [119, 120] , this genetic deficiency gives rise to a lethal rds upon birth, requiring early lung transplantation to survive [121] . gene therapy would be thus an essential tool to reverse the usually lethal sp-b deficiency, and promising progress has been recently made in the production of human lung organoids [122] . in the case of adults carrying mutations in heterocygosis, susceptibility to lung diseases in the adulthood has been described, particularly among smokers [123] . unlike sp-b, genetic sp-c deficiency is not usually a cause of neonatal rds, but interstitial lung disease, including pulmonary fibrosis, can appear at long-term [124] [125] [126] . pathophysiology of the disease caused by sp-c mutations is mainly related to ae2c cell damage, produced by endoplasmic reticulum (er) stress due to prosp-c misfolding, or to its altered traffic to lamellar bodies [102] . on the other hand, different studies have shown that mutations in the genes encoding sp-a1 and sp-a2 are also involved in lung diseases. for example, mutations that affect the crd domain of sp-a1 (w211r) or sp-a2 (g231v and f198s) are associated with idiopathic interstitial pneumonia [127] and pulmonary fibrosis [128, 129] , respectively. these mutations impair protein secretion and promote protein aggregation within the er, increasing er stress at resident ae2c [129] . in addition, genetic polymorphisms of lung collectins are known to be associated with susceptibility to acute lung injury [130] , rds [131] and infections [132, 133] . in the case of acute lung injury, a polymorphism that causes the insertion or deletion of three amino acids within the collagen-like domain of sp-a1 has been related to the reduced water levels in the lung characteristic of this disease, probably due to alterations in the structure and binding properties of the protein [130] . complex interactions of single nucleotide polymorphisms of the genes that encode all surfactant proteins seem to contribute to the pulmonary disease in cystic fibrosis patients [134] . similarly, interactions between sp-a1 and sp-b polymorphisms are involved in genetic susceptibility to rds [135] . to ensure the presence of a functional surfactant film at the respiratory interface, synthesis, secretion, reuptake and degradation of surfactant components must be perfectly regulated and balanced. this includes achieving both a precise composition of lipids and proteins and their proper organization in defined surfactant structures at the alveolar spaces, together with an efficient cross-talk established between epithelial cells and alveolar macrophages. protein and lipid interactions involved in surfactant homeostasis are also summarized in table 1 . de novo synthesis of surfactant phospholipids takes place at the endoplasmic reticulum in ae2c. pc is synthesized from choline by the kennedy pathway, which includes several enzymatic steps, with the cytidine triphosphate-phosphocoline cytidyltransferase (cct) controlling the synthesis rate. a parallel formation of diacylglycerols occurs coupled to this pathway (for a recent review on surfactant metabolism see [13] ). direct interaction of acyl-coa:lysophosphatidylcholine acyltransferase (lpcat1) with the star10 (steroidogenic acute regulatory-related lipid transfer protein 10) protein has been found to be important for trafficking of newly synthesized dppc from endoplasmic reticulum to lb [136] , which seems to occur by direct non vesicular transport between both organelles, apparently skipping golgi and multivesicular bodies [137] . in fetal mouse lung, glycogen storages present in ae2c, in which cct is also found, seem to provide substrate for pc biosynthesis [138, 139] . besides this, triglycerides have been also shown to be directed from alveolar lipofibroblasts to ae2c and contribute to the fetal surfactant phospholipid pool, pointing to a cross-talk between these two cell types to ensure surfactant homeostasis [140, 141] . apart from this de novo synthesis, in the lands cycle dppc is also obtained by remodeling from unsaturated pc by pla2 and lpcat1, being the latter also involved in the synthesis of pg. these two enzymes, together with cct, have been shown to be key players for the proper regulation of surfactant synthesis, so that their alteration is cause of respiratory distress [142] [143] [144] . in the case of lpcat1, it has been ascribed a role in coordinating the two pathways of dppc synthesis according to the physiological demands of surfactant at the alveolar spaces [145] . besides this, the finding that peroxiredoxin 6, a multifunctional enzyme with pla2 and lpcat activities, is located in lamellar bodies, pointed to these organelles as a sufficient location for dppc remodeling [146] . regarding to pla2, not only this enzyme has a key role in regulating the turnover of surfactant phospholipids because of its implication in their synthesis and degradation, but it is also involved in antioxidant and cell signaling roles [147] . finally, the interaction of sp-a with peroxiredoxin leads to inhibition of its pla2 activity, pointing to a potential role of sp-a in regulating surfactant phospholipids turnover [91, 148] . with regard to cholesterol, apart from the principal plasmatic source, it can be also synthesized in peroxisomes in ae2c, whereas cholesterol-binding proteins have been found in lamellar bodies, suggesting a potential implication of the niemann-pick c pathway in the regulation of surfactant cholesterol and homeostasis [149, 150] . finally, alveolar lipofibroblasts could also participate in cholesterol supply to surfactant under certain conditions [141] . the synthesis and complex processing of proteins sp-b and sp-c initiate in the endoplasmic reticulum of ae2c, and occur coupled to surfactant lipid synthesis and intracellular trafficking. both proteins are produced as large soluble precursors where the mature hydrophobic modules are protected by flanking n-and c-terminal domains [13] . processing of precursors takes place in transit to lbs via multivesicular bodies, and consists of several proteolytic cleavages and post-translational modifications, in a route coupled to sequential acidification. thus, maturation of both proteins is conducted in a concerted way, being sp-b required for proper sp-c processing [117] by mechanisms that are still unknown, although they could well involve the previously described sp-b/sp-c interactions [68] , or even potential interactions between its precursors. furthermore, the fusogenic and lytic properties behind membrane remodeling abilities of sp-b, together with its role in generating membrane contacts, turn sp-b into an essential requirement for the formation of correctly assembled lbs [20, 151] . these organelles are formed by packed lipid membranes in a well-defined structure that, in the case of humans, is arranged as concentric lamellae [152] . surfactant lipids, at least pc, pg and cholesterol, are imported and accumulated into lbs by their limiting membrane transporter atp binding cassette subfamily a member 3 (abca3), in an atp-depending process [21, 153, 154] . the coupling between lipid and protein synthesis, processing and assembly in lbs places all these actors as essential providers of surfactant homeostasis, so that their disruption leads to alteration affecting the rest of components of the system and inadequate surfactant production. thus, absence of cct is cause of changes in surfactant protein levels and aberrant lbs [142] , mutations in abca3, or in its interacting protein emc3 (endoplasmic reticulum membrane protein complex subunit 3), which is required for abca3 stabilization, produce absence of lamellar bodies and misprocessed surfactant proteins [155, 156] , and decrease of sp-b levels affects sp-c processing and lb formation, as previously stated. on the other hand, mutations in the sp-c gene, as previously described, are related to long-term lung diseases, including fibrosis, and injury to ae2c by endoplasmic reticulum stress due to protein misfolding (mutations affecting sp-c precursor) [102] . finally, absence of abca3 has been also related to ae2c apoptosis by endoplasmic reticulum stress due to the accumulation of phospholipids and cholesterol, which are then rerouted to the endoplasmic reticulum from lb [154] . hydrophilic proteins sp-a and sp-d are synthesized in the endoplasmic reticulum, where post-translational modifications and oligomerization also occurs. the main route of secretion of these proteins is via constitutive non-regulated vesicular transport, bypassing lbs, although a certain proportion of synthesized sp-a seems to be directed to these organelles, together with the recycled protein recovered from alveolar spaces [13] . due to the role of hydrophilic proteins in lung immunomodulation, their absence produces increased inflammation [157] , although alterations in surfactant homeostasis are also found, related to their involvement in surfactant catabolism [90, 158] , as it will be discussed later. lbs are secreted to the alveolar fluid in response mainly to alveolar stretching produced during inspiration, through stimulation by purinergic agonists such as atp. the stimulation of ae2c by atp leads to an increase in cytoplasmic calcium, both by entry from the alveolar medium and by release from intracellular stores, mainly endoplasmic reticulum. calcium triggers lb secretion by regulating the direct fusion of lbs with the plasma membrane and by cytoskeleton activation. generation of the fusion pore, involving soluble snare (n-ethylmaleimide-sensitive factor attachment receptor) proteins at both the lb and the cell membrane, triggers a local entry of extracellular calcium (fusion-activated calcium entry) that expands the pore and allows surfactant release, which requires compression of the vesicle by an actin coat [13] . regulation of surfactant secretion is essential to maintain alveolar homeostasis. extracellular sp-a was described as an inhibitor of secretion by interaction with its receptor tumor protein 63 (p63) at the ae2c membrane [28, 29] , although little is known about the mechanisms involved in sensing the availability of surfactant at the alveolar spaces and the possible need for secretion of material. stretching sensing has been traditionally ascribed to ae1c, which would as a result induce lb secretion through the increase of extracellular atp [159] . however, an implication of the air-liquid interface itself has been reported, so that ae2c could be able to sense the mechanical forces produced by the thinning of the aqueous phase and the increase in surface tension, responding with intracellular calcium increase, thus promoting the secretion of a surplus of new surfactant material. besides this, ae2c could be also capable of activating cellular responses involved in cell repair, to prevent lung injury [96, 97] . on the other hand, extracellular sp-b has been recently found to stimulate surfactant secretion by the purinergic pathway, so that the protein could ensure the availability of enough density of surfactant and a proper membrane network at the alveolar spaces, and thus, surfactant homeostasis [30] . the molecular mechanism of this regulation needs still to be investigated, as well as the potential existence of a "sp-b receptor" at the ae2c plasma membrane. the possibility that sp-b/sp-a interactions [86] , as a secretion activator/inhibitor couple, might be involved in a concerted regulation of surfactant secretion will require further investigation. finally, a potential role as sensor of the surfactant pool size or its composition has been postulated for g-protein coupled receptor 116 (gpr116), a transmembrane receptor of ae2c that has been identified as a negative regulator of surfactant secretion. the sensing mechanism of this protein could rely on its interaction with some surfactant component(s) which, up to now, has not been yet identified [160, 161] . however, suggested possibilities for gpr116 sensing include interaction with sp-d or its direct modulation by mechanical stretching upon inspiration [162] . once surfactant is secreted into the alveolar fluid, fast adsorption of material takes place and new components are transferred into the air-liquid film. the specific mechanisms converting the secreted highly packed membrane structures into part of the interfacial film are not still completely understood. sp-b/sp-c common machineries have been shown to be essential for unpacking of secreted surfactant lb-like particles and formation of the surface film, pointing to the possibility that, upon contact with the air-water interface, sp-b nanorings, regulated by sp-c, would trigger the fast transfer of lipids into the film [22] . apart from these freshly secreted structures, another surfactant assembly that could act as an intermediate between lb and the interfacial film, is tubular myelin (figure 1 ). tubular myelin is a complex three-dimensional membranous tubular structure, whose formation requires the presence of sp-a (in humans, both sp-a1 and sp-a2), sp-b, and anionic lipids, highlighting once again the essential role of the interactions among these surfactant components [24, 163, 164] . although the particular function played by tubular myelin is still unknown, as its presence is not essential for respiration [165] , potential roles could be related with the involvement of sp-a in antimicrobial defense. the study of the extracellular surfactant recovered from alveolar spaces led to the finding that two different pools of surfactant are present, in terms of size, composition and structural complexity. the more dense surfactant material known as large aggregates (la), which contains secreted lbs, tubular myelin and sp-a, sp-b and sp-c, is thought to be somehow metabolically converted into another form rich in small vesicles (small aggregates or sa) with decreased content in proteins and a lower adsorption activity [25, 26] . of note, an increased sa/la ratio is found in patients suffering ards [103] . the conversion of la to sa can be reproduced in vitro by compression-expansion cycling of the interfacial film, so that it is generally assumed that the smaller structures are generated by the "used" surfactant components once surfactant has been exposed to a dynamic air-water interface ( figure 1 ). the physiological role of these structural changes of surfactant seems to be encompassed in an important mechanism of surfactant homeostasis regulation, as it will be discussed below. the proper regulation of surfactant pool size includes the clearance of the used surfactant from the alveolar spaces, thus avoiding the excessive accumulation of a spent, substantially altered (including extensive oxidation) material that can lead to lung injury and inflammation. surfactant is removed from the alveolar fluid by re-uptake to ae2c for recycling or degradation, and to alveolar macrophages for its catabolism. surfactant pool size itself has been shown to induce the increase of catabolic rate, thus maintaining alveolar homeostasis, although the molecular pathways involved in such response are not clear [166] . apart from inhibiting surfactant secretion, sp-a contributes to alveolar homeostasis by stimulating the phospholipid uptake by ae2c via clathrin-mediated endocytosis upon interaction with its receptor p63 [28] . a similar case is gpr116, which acts both as secretion inhibitor and as stimulator of pneumocyte re-uptake, for which potential interactions with sp-d may be involved [95, 162] . as a matter of fact, sp-d seems to play an essential role in the regulation of surfactant pool size. this protein is involved in the structural remodeling of surfactant structure into sa, which is the form taken up by ae2c to proceed with surfactant recycling or degradation ( figure 1 ). to achieve this role, for which sp-d oligomeric structure is required [167] , the protein seems to induce fragmentation of surfactant membranes, for which its interaction with pi has been suggested to be a determinant factor [27, 94, 168] . on the other hand, the structure of surfactant aggregates does not influence its uptake by ams [169] , and although sp-d is able to bind to the surface of these cells to modulate inflammation, the homeostasis/immune roles of the protein seem to be independently regulated [94] . the regulation of surfactant homeostasis carried out by sp-d is particularly important upon birth. newborn mice show a much larger pool size and an ultrastructure of sa and la pools different to that observed in adults. the postnatal change leading to a normalization of the pools has been suggested to be initiated by the increased amount of pi of newly secreted surfactant (both in newborns and adults) that promotes the association of sp-d to surfactant lipids, triggering the structural conversion to small vesicles [93, 94] . all these findings highlight how the particular composition and structure of surfactant at the different levels of its functional cycle regulate alveolar homeostasis in detail, and how the interactions between surfactant proteins and lipids are particularly essential for this task. maintaining lipid homeostasis inside the cell is crucial both for ae2c and am, mainly through the atp-binding cassette transporters abca1 and abcg1. these proteins are involved in the reverse cholesterol transport by mediating efflux of excess cholesterol and phospholipids towards the plasma (figure 1 ). in ae2c, alteration of these proteins produces lipid accumulation and affects lb morphology, secretion and recycling, suggesting an important role in surfactant homeostasis [35, 170] . alveolar macrophages are essential for surfactant lipids catabolism, accounting for around 20% of its clearance [31] . alterations affecting lipid degradation by am entails accumulation of lipids in these cells (the so-called foamy macrophages) that causes inflammation, and reduced clearance of surfactant, leading to accumulation of impaired surfactant at the alveolar spaces. factors involved in these pathways, such as cluster of differentiation cd44, are thus regulators of lung homeostasis and inflammation [171] . the main regulator of lipid catabolism in am has been shown to be granulocyte-macrophage colony stimulating factor (gm-csf) (figure 1 ). this factor does not affect surfactant uptake by am directly but promotes the maturation of these cells and activates proteins involved in phospholipid and cholesterol clearance, as abca1 and abcg1 [32, 34, 172] . on the other hand, the administration of gm-csf to mice with impaired am development has been recently shown to produce a decrease in the amount of sp-d, pointing to a cross-regulation of ae2c re-uptake and am catabolic pathways [33] . surfactant would then mediate somehow the communication between both cells, as it seems to occur also in relation to cholesterol metabolism, so that the increase of cholesterol in surfactant has been described to trigger alteration of cholesterol metabolism in am [34] . on the other hand, an involvement of sp-b has been suggested in facilitating cholesterol efflux from am to surfactant, which would act as acceptor with the aim of reducing inflammation-induced cholesterol accumulation in these cells [173] . a potential role of sp-b/cholesterol interactions in this activity has still to be demonstrated. regarding sp-c, its reported membrane fragmentation ability, together with its tendency to partition into cholesterol-rich environments have been also suggested to be potentially involved in regulatory mechanisms of surfactant homeostasis [69] . intrinsic absence of sp-c in am with cholesterol accumulation in sp-c-ko mice leads to an impaired metabolism and the generation of cholesterol crystals in am cytoplasm, a phenotype that has been suggested to be the consequence of the impairment of ae2c/am cross-talk, and that could be mediated by the alveolar instability generated by the absence of sp-c [78] . an altered lung surfactant homeostasis as a consequence of unbalanced anabolism/catabolism leads to the generation of pulmonary alveolar proteinosis (pap). pap is a syndrome characterized by the accumulation of alveolar surfactant and dysfunction of am, thus producing respiratory deficiency and alteration of the lung immune defense. the existence of such numerous proteins involved in the regulation of lung lipid homeostasis entails a broad etiology for pap, including alterations of re-uptake/catabolism by ae2c or am (as autoantibodies to gm-csf, absence or alteration in gm-csf receptors colony stimulating factor 2 receptors alpha and beta, abca1, abcg1, sp-d and gpr116) or of surfactant secretion (gpr116). besides this, mutations in genes required for the proper production of surfactant (abca3, sp-b or sp-c) also leads to congenital pap [16] . regarding to the nature of the accumulated lipids, the analysis of the lipidome of pap samples from different etiologies revealed similar results, including drastic increase of sphingolipids (mainly ceramide), free and sterified cholesterol, pc and lysophosphatidylcholine [174] . alteration of surfactant homeostasis can also occur upon exposure to external toxicity. electronic cigarettes have been found to alter lipid homeostasis in ae2c and am, with effects in abca1, abca3, lb structure and immune functions [175] . finally, chronic alcohol consumption also disrupts lipid homeostasis and immune responses in am, increasing susceptibility to pulmonary infections [176] . in conclusion, the importance of a proper lipid homeostasis in the lung goes beyond the necessity of maintaining sufficient but proportionate lipid levels to ensure surfactant function and alveolar stability. lipids play essential roles in lung physiology, and constitute a link between metabolism and host defense, sensing and translating extracellular information to immune cells. as will be detailed later, anionic phospholipids influence am response to inflammatory stimuli and oxidized lipids and cholesterol mediate and expand inflammatory responses [177] . of note is the role of pla2, involved in surfactant lipid remodeling and degradation, in connecting surfactant turnover and oxidative stress responses in am [147] . the constant exposure of the lung to microorganisms from inhaled air at the upper respiratory tract activates the immune response, which leads to transcriptional expression of inflammatory mediators that coordinate the elimination of infected cells and pathogens. resolution of inflammation and return to homeostasis is induced by activation and secretion of endogenous anti-inflammatory factors [178] . however, aberrant activation of the inflammatory response leads to immunodeficiency, septic shock, or induction of autoimmunity [179] . therefore, the activation and resolution of inflammation must be tightly regulated by immune mechanisms that decrease microbial burden by facilitating clearance of inhaled pathogens, and control tissue damage caused by pathogens or by the host immune response by modulating inflammatory responses [180] . surfactant proteins and lipids have been shown to provide immune protection against respiratory pathogens, both directly by limiting inflammation and promoting pathogen clearance, and indirectly by activating molecular and cellular mechanisms that contribute to restore lung homeostasis [15, [181] [182] [183] . this part of the review summarizes the role of surfactant proteins and lipids on the immune response against bacterial lipopolysaccharide (lps), the most potent pathogen-associated molecular pattern among bacterial cell wall components. bacterial lipopolysaccharide, the major component of the outer membrane of gram-negative bacteria, strongly stimulates the innate or natural immunity in eukaryotic species [184] . lps is composed of a poly-or oligo-saccharide part linked to a membrane-anchoring lipophilic domain known as lipid a or endotoxin (figure 3 ). the polysaccharide part is commonly subdivided into the terminal o-specific chain, whose composition varies within bacterial species, and the rather invariable core region, most proximal to lipid a. bacteria express either smooth lps, which contains the o-antigen and complete core oligosaccharides, or rough lps, which lack o-antigen and possess progressively shorter core oligosaccharides. rough lps is designated as ra, rb, rc, rd, and re in order of decreasing core length [185] . strains with rough type lps are more common among pathogens colonizing pulmonary compartments [184, 186, 187] . lipid a, which represents the conserved molecular pattern of lps, consists of a β(1→6)-linked diglucosamine backbone carrying two phosphate groups at positions 1 and 4' of the glucosamine residues and six or seven ester-and amide-linked saturated acyl chains of 12 and mostly 14 carbon atoms in length [184] (figure 3 ). both phosphate groups and the myristoyl and lauroyl acyl chains have been shown to be major determinants for specific recognition and activation of innate immunity. lps recognition by cell receptors requires a specific three-dimensional non-lamellar supramolecular structure of lps aggregates [185, [188] [189] [190] (figure 3) . host responses to lps are mainly mediated by the pattern recognition receptor toll-like receptor 4 (tlr4) [191] . the activation of tlr4 by lps requires the interaction of lps with several receptor-proximal proteins (figure 3 ). the lipopolysaccharide binding protein (lbp) binds to the aggregated structures of lps and transfers lps monomers to the soluble or membrane-bound forms of cluster of differentiation 14 (cd14) via specific recognition of the lipid a domain. cd14 subsequently transfers lps to the myeloid differentiation factor 2 (md2), which forms a complex with tlr4. lps binding to md2 induces a conformational change in tlr4 that promotes tlr4/md2 dimerization and the subsequent cd14-controlled internalization of tlr4 into endosomes [192] , initiating the signalling cascade [193] . using different lps analogs, triantafilou and co-workers [194] showed that the recruitment of these receptors is triggered by the 1-phosphoryl lipid a moiety of lps. and an inner core region proximal to lipid a that contains 3-deoxy-d-manno-octulosonic acid [184] . the o-antigen, also termed o-specific chain, contains up to 50 highly variable oligosaccharide units. both the acyl chains and the negative charges of the phosphate groups bound to the diglucosamine backbone of lipid a are required for the recognition of lps by cell receptors and the subsequent activation of the host immune system. lps molecules associate forming non-lamellar supramolecular structures that are recognized by lbp, which transfers lps monomers to cd14. lps is subsequently transferred to the tlr4/md2 complex, triggering complex dimerization and initiating the signaling cascade. interaction of surfactant protein sp-a (functional form, octadecamer) with lps modifies the aggregated lps structure, inducing formation of lamellar phases, and therefore preventing the recognition of lps by lbp. [195] [196] [197] , contributing to the clearance and inactivation of lps released by gram-negative bacteria. these proteins interact with different lpss [198] [199] [200] [201] [202] [203] . while sp-d binds to the acyl chains of lipid a [204] as well as to the heptoses and mannoses of the core [205, 206] and o-antigen [207] , respectively, sp-a only binds to lipid a [208, 209] . as a result, sp-a only binds to rough lps since the bulky headgroup of smooth lps hinders the access of sp-a to the lipid a moiety. although the interaction of sp-a and sp-d with lps was firstly described as being mediated by calcium [208] [209] [210] , it has been shown that both collectins bind to re-lps in the absence of this cation [202, 203, 211] . on the other hand, sp-c binds to the lipid a moiety of lps by its n-terminal segment, probably through electrostatic interactions between the polar and basic residues of the protein and the negatively charged terminal 1-phosphate group at the reducing end of the lipid a disaccharide [199] . similarly, sp-a binding to the 1-phosphate of lipid a [212] may involve electrostatic interactions with a cluster of basic residues located in the crd of the protein [203, 213] . since the negative charges of the phosphate groups are required for lps binding to lbp [214] and the activation of the tlr4/md2 complex [215] , the interaction of sp-c and sp-a with 1-phosphate may shield this functional group, blocking some of the physiological responses to lps in the lungs. this hypothesis is supported by the finding that sp-a prevents the interaction of re-lps with lbp [216] . sp-a also agglomerates rough lps aggregates in the presence of calcium [203] and rearranges the aggregated structure of lps, figure 3 . host response to lipopolysaccharide (lps). lps is composed of lipid a, the hydrophobic membrane-anchoring region, and a hydrophilic part formed by the core oligosaccharide region and the o-antigen. the core oligosaccharide region includes an outer core region, enriched in hexoses, and an inner core region proximal to lipid a that contains 3-deoxy-d-manno-octulosonic acid [184] . the o-antigen, also termed o-specific chain, contains up to 50 highly variable oligosaccharide units. both the acyl chains and the negative charges of the phosphate groups bound to the diglucosamine backbone of lipid a are required for the recognition of lps by cell receptors and the subsequent activation of the host immune system. lps molecules associate forming non-lamellar supramolecular structures that are recognized by lbp, which transfers lps monomers to cd14. lps is subsequently transferred to the tlr4/md2 complex, triggering complex dimerization and initiating the signaling cascade. interaction of surfactant protein sp-a (functional form, octadecamer) with lps modifies the aggregated lps structure, inducing formation of lamellar phases, and therefore preventing the recognition of lps by lbp. [195] [196] [197] , contributing to the clearance and inactivation of lps released by gram-negative bacteria. these proteins interact with different lpss [198] [199] [200] [201] [202] [203] . while sp-d binds to the acyl chains of lipid a [204] as well as to the heptoses and mannoses of the core [205, 206] and o-antigen [207] , respectively, sp-a only binds to lipid a [208, 209] . as a result, sp-a only binds to rough lps since the bulky headgroup of smooth lps hinders the access of sp-a to the lipid a moiety. although the interaction of sp-a and sp-d with lps was firstly described as being mediated by calcium [208] [209] [210] , it has been shown that both collectins bind to re-lps in the absence of this cation [202, 203, 211] . on the other hand, sp-c binds to the lipid a moiety of lps by its n-terminal segment, probably through electrostatic interactions between the polar and basic residues of the protein and the negatively charged terminal 1-phosphate group at the reducing end of the lipid a disaccharide [199] . similarly, sp-a binding to the 1-phosphate of lipid a [212] may involve electrostatic interactions with a cluster of basic residues located in the crd of the protein [203, 213] . since the negative charges of the phosphate groups are required for lps binding to lbp [214] and the activation of the tlr4/md2 complex [215] , the interaction of sp-c and sp-a with 1-phosphate may shield this functional group, blocking some of the physiological responses to lps in the lungs. this hypothesis is supported by the finding that sp-a prevents the interaction of re-lps with lbp [216] . sp-a also agglomerates rough lps aggregates in the presence of calcium [203] and rearranges the aggregated structure of lps, which changes from inverted to lamellar phases [212] (figure 3 ). both the clustering of lps aggregates and their lamellar structure would reduce lps toxicity by preventing the recognition of lps by lbp, and the subsequent initiation of the lbp/cd14 pathway towards inflammatory reactions. surfactant lipids can facilitate the scavenger action of surfactant proteins. in this regard, it has been shown that lps incorporates in vesicles of a modified porcine pulmonary surfactant [217] and films of surfactant lipids [196, 211] . repulsive interactions between smooth lps and surfactant lipids facilitate the interaction of sp-a with the lipid a moiety of smooth lps, which results in the extraction of lps molecules [196] . additionally, incorporation of lps into surfactant membranes would decrease the endotoxicity of lps by abrogating the interaction of lipid a with its cellular receptors. this hypothesis is supported by earlier studies reporting that the biological activity of lps is reduced when lps is incorporated into liposomes [218] . alternatively, surfactant lipids may incorporate into lps aggregates, changing the aggregated structure of lps, and hence, reducing its ability to stimulate immune cells [190] . in addition to the direct interaction with lps, surfactant proteins also modulate the cellular inflammatory response via interaction with pattern-recognition receptors and associated molecules. for instance, sp-a modulates the cellular response to smooth lps, although this lps is not a ligand for sp-a, by binding to cd14 [210] and the tlr4/md2 complex [219] . thus, the interaction between sp-a and lps receptors significantly decreases the binding of smooth lps and consequently prevents the smooth lps-elicited cellular response [210, 219] . in contrast, sp-a significantly enhances the binding of rough lps to cd14 [210] and does not attenuate rough lps binding to tlr4/md2 [219] . this effect has been related to the formation of a sp-a/cd14/rough lps complex in which the amphipathic neck domain of sp-a would bind to the leucine-rich region of cd14, whereas the crd would bind to the lipid a moiety of rough lps, allowing the transfer of lps molecules to the tlr4/md2 complex and initiating physiological responses against pathogens [208] . formation of a ternary complex with cd14 and lps has also been proposed for sp-c, which, like sp-a, binds to cd14 increasing the binding of rough lps [220] . sp-c-containing phospholipid vesicles, but not lipid vesicles alone, block lps-induced cytokine production by a tlr4-dependent mechanism in human embryonic kidney hek293t cells [197] . whether this effect is due to the direct interaction of sp-c with tlr4 and/or md2 needs to be clarified. on the other hand, sp-d binds to cd14 and tlr4/md2 through its crd [208, 221, 222] , decreasing the binding of both smooth and rough lps to cd14 [208] and tlr4/md2 [223] . this suggests that sp-d may act as a negative regulator to protect against a chronic inflammatory state induced by the release of numerous inflammatory mediators as a consequence of the interaction of sp-a and sp-c with rough lps and its receptors. on the other hand, anionic surfactant lipids pg and pi inhibit lps-induced cell activation by competitive binding to lps receptors [224, 225] . while pi interferes with the interactions between lps and cd14 [225] [226] [227] , pg inhibits lps binding to lbp, cd14 and md2 [190, 224, 225, 228] . alternatively, surfactant lipids can prevent activation of tlr4 by lps by incorporating it into the plasma membrane. thus, dppc has been shown to inhibit the translocation of tlr4 to raft domains [229] , precluding the co-localization of cd14 and tlr4 in lipid rafts that is required for lps-induced activation of tlr4 [230] . voelker and co-workers [231] have suggested that inhibition of toll-like receptor function by surfactant lipids would set a threshold for the engagement of inflammatory cascades in the lung, preventing inflammation by casual environmental stimuli like environmental levels of particulated lps while allowing a robust inflammatory response during established bacterial infections. surfactant proteins and lipids also contribute to lung homeostasis by exerting antimicrobial effects (figure 4 ). animal models of sp-c and collectin deficiencies show a significant defect in host defence since: (i) sp-c, sp-a, and sp-d knock-out mice are more susceptible to bacterial, fungal, and viral infections [157, [232] [233] [234] [235] , (ii) sp-a −/− and sp-d −/− mice show reduced uptake of these microbes by alveolar macrophages (reviewed in [236] ), and (iii) bacterial clearance is restored by intratracheal administration of sp-a [237] . lung collectins sp-a and sp-d prevent microbial dissemination by direct binding to a broad range of microorganisms, including viruses, fungi and gram-positive and gram-negative bacteria (reviewed in [236, 238, 239] ). these proteins recognize different pathogen-associated molecular patterns on the surface of microorganisms: lps in gram-negative bacteria, lipoteichoic acid and peptidoglycan in gram-positive bacteria, lipoarabinomanan in mycobacteria, phospholipids in mycoplasma, glucan and mannose in fungi, and glycoproteins in fungi and viruses [236, [240] [241] [242] [243] [244] . the association of sp-a and -d with pathogens may result in microbial aggregation, which facilitates mucociliary clearance by the respiratory tract, prevents attachment of pathogens to cell surfaces, inhibits microbial colonization and invasion, and may favor microbial phagocytosis [245] . sp-a and sp-d also act as opsonins, increasing the association, uptake and killing of the microorganisms [246] . this requires specific interactions with different receptors on phagocytic cells, such as surfactant protein receptor 210 (sp-r210) [247] , a specific receptor for sp-a [248] , the putative opsonin receptor glycoprotein 340 (gp340), a macrophage scavenger receptor family member, to which both sp-a and sp-d bind [249, 250] , or the immunoglobulin g [251] . interaction of the globular heads of lung collectins with pathogen-associated molecular patterns promotes the presentation of the collagenous tails to calreticulin/cluster of differentiation 91 (cd91) on the surface of phagocytic cells, stimulating phagocytosis and pro-inflammatory responses [252] . lung collectins can also stimulate phagocytosis through upregulation of the expression of cell-surface receptors involved in microbial recognition in phagocytic cells [253] , such as the mannose receptor [254, 255] or the class a scavenger receptor [256] . the precise receptor(s) involved in the activation of the signal transduction pathways that lead to an increased trafficking of pathogen recognition receptors to the plasma membrane remains elusive. alternatively, lung collectins may promote microbial clearance by regulating the complement system. thus, sp-d enhances the activity of complement component 1q (c1q) by forming additional binding sites for c4b and c3b [257] , two complement binding protein complexes involved in opsonization [258] . on the contrary, association of sp-a with c1q may down-regulate complement activity, preventing c1q-mediated complement activation and inflammation [259] . lung collectins are also involved in pathogen clearance by neutrophil extracellular traps (nets), dna-based extracellular traps that capture and kill microbes. simultaneous binding of sp-d to bacteria and nets promotes bacterial trapping by the nets [260] . on the other hand, since lps efficiently induces the formation of nets [261] , binding of sp-d to lps has been recently shown to attenuate nets formation [262] . this suggests that sp-d may regulate the balance between the antimicrobial activity of nets and the exacerbated immune response and tissue injury as well as surfactant inhibition induced by an excessive presence of nets in the lung. sp-a and sp-d directly affect the growth and viability of microorganisms [263] . lung collectins have been found to inhibit the growth of different bacteria like escherichia coli, klebsiella pneumoniae, enterobacter aerogenes, legionella pneumophila, mycobacterium avium, and mycoplasma pneumoniae [236, 240] . this bacteriostatic effect has been related to the ability of sp-a and sp-d to increase the permeability of the microbial cell membrane [264] . gram-negative bacterial strains decorated with rough lps are permeabilized more effectively by sp-a and sp-d than strains containing smooth lps [264] . although the mechanism of bacterial permeabilization by lung collectins is unclear, it seems to require a direct interaction between sp-a and lps. in this regard, kuzmenko and co-workers [265] suggested that sp-a distorts or perturbs membrane structure, creating defects that allow small hydrophilic molecules to enter and translocate through the bilayer. such defects seem to be related to the formation by sp-a of extensive lattice-like structures on the surface of lps-containing layers [211, 266] . pulmonary collectins also decrease the viability of histoplasma capsulatum by increasing the permeability of the fungal cell wall in a calcium-dependent manner [267] . although the precise mechanism involved in membrane permeabilization of this fungi is unknown, mccormack and co-workers [267] have suggested that membrane disruption could be due to the formation of calcium bridges between the collectins and fungal phospholipids. growth of microorganism is also affected by the agglutinating activity of lung collectins, i.e., sp-d agglutinates candida albicans [195, 238] decreasing the availability of substrate to agglutinated yeasts [195] . sp-a and sp-d directly affect the growth and viability of microorganisms [263] . lung collectins have been found to inhibit the growth of different bacteria like escherichia coli, klebsiella pneumoniae, enterobacter aerogenes, legionella pneumophila, mycobacterium avium, and mycoplasma pneumoniae [236, 240] . this bacteriostatic effect has been related to the ability of sp-a and sp-d to increase the permeability of the microbial cell membrane [264] . gram-negative bacterial strains decorated with rough lps are permeabilized more effectively by sp-a and sp-d than strains containing smooth lps on the other hand, a surfactant lipid mixture composed of dppc, palmitoyloleoylphosphatidylcholine and palmitic acid has been shown to interfere with the interaction of non typable haemophilus influenzae with pneumocytes by binding to the bacteria and preventing bacterial adhesion and internalization in alveolar epithelial cells [268] . in addition, these lipids can be endocytosed by pneumocytes by binding to the scavenger receptor class b type i, blocking bacterial uptake [268] . in vivo studies show that administration of the hydrophobic fraction of native surfactant, containing surfactant lipids and proteins sp-b and sp-c, significantly diminishes bacterial load in bronchoalveolar lavage and lung tissue of mice infected with this pathogen [268] . although this antimicrobial activity has been ascribed to surfactant lipids, it is plausible that surfactant proteins sp-c and sp-b may play a bactericidal role against other pathogens. in this regard, bovine lung surfactant extract, a mixture of dppc, palmitoyloleoylphosphatidylglycerol (popg) and sp-b/-c, and a synthetic model lung surfactant, but not the lipids alone, all show antimicrobial activity against e. coli and staphylococcus aureus [269] . sp-b and sp-c have a strong affinity for iron-regulated surface determinant proteins a and c [269] , cell-wall receptors of s. aureus involved in the process of heme-iron acquisition. binding to these receptors would disturb the bacterial surface, enhancing bacterial killing [269] . sp-b and sp-c also bind to β-barrel assembly machinery protein a and lipopolysaccharide transport protein d, two β-barrell proteins at the outer membrane of e. coli, lethally disrupting the bacterial outer membrane [269] . additionally, incubation of e. coli with sp-c incorporated into liposomes of dppc and popg, but not the lipids alone, decreases bacterial growth by altering the membrane [269] . although the mechanism by which sp-c disturbs the outer membrane is not clear, sp-c binding to lps could be a key feature to disrupt the outer membrane and induce the release of lps, rendering the cells permeable. this would facilitate the access of sp-c to the plasma membrane, where this protein would exert an antimicrobial activity [269] . the antimicrobial action of sp-b, which has been shown to aggregate and kill clinical isolates of k. pneumoniae, p aeruginosa, s. aureus and group b streptococcus by increasing membrane permeability [270] , is inhibited by surfactant lipids, mainly popg [270] . this suggests that endogenous sp-b may not play by itself a significant role in alveolar host defense. however, prosp-b, the sp-b precursor may be proteolitically cleaved into smaller peptide fragments that could retain its antimicrobial activity in the presence of surfactant lipids. in this regard, sp-b n , the n-terminal propeptide of sp-b (residues , which encodes a saposin-like domain, promotes the uptake of bacteria by macrophages and exerts a bactericidal effect at acidic ph, consistent with a lysosomal, antimicrobial function [271] . binding of surfactant lipids and proteins to viruses results in enhanced phagocytosis and viral neutralization at the initial stages of infection [243, 272] . regarding lung collectins, the viral neutralizing activity of sp-d is enhanced compared to sp-a, playing an important role in the innate immune response to different viruses such as vaccinia or influenza a virus. the antiviral activity of lung collectins is related to their ability to bind to different proteins on the viral surface [243] . in general, interactions of lung collectins with viral proteins are calcium-dependent, involve the crd and are facilitated by the multimerization of the full-length protein. the collagen domain of sp-a may also play a role in the antiviral action of the protein, since a recombinant variant of human sp-a composed of the neck and carbohydrate recognition domains can promote the replication of influenza a virus [273] . while sp-a prevents the recognition of influenza a virus by host cell receptors through binding of the sialylated asparagine 187 residue of sp-a to hemagglutinin [274, 275] , binding of sp-d to mannose-rich oligosaccharides close to the sialic acid-binding sites of hemagglutinin reduces viral uptake into epithelial cells [276, 277] , and sterically blocks the access of neuraminidase to surface-bound substrates [278, 279] thereby preventing viral dissemination. this inhibitory effect has been related to the bulky collagen domain in the extended structure of dodecameric sp-d [279] . sp-a and sp-d also bind to the human immunodeficiency virus envelope glycoprotein 120 [280] [281] [282] and to the fusion and attachment glycoproteins of respiratory syncytial virus, f and g, respectively [283] [284] [285] [286] , neutralizing viral infectivity and inhibiting viral replication [281] . sp-a can mediate phagocytosis of herpes simplex virus type 1 by alveolar macrophages via its n-linked oligosaccharides [287] . to which viral surface protein(s) sp-a binds is not known. however, since sp-a binding to herpes simplex virus is inhibited by heparin, and heparin is known to bind to viral glycoproteins b and c, these proteins are likely candidates [287] . other viral surface proteins recognized by lung collectins are the spike glycoprotein of the severe acute respiratory syndrome (sars) coronavirus [288] , the outer capsid glycoprotein 7 of non-enveloped rotavirus [289] , and the a27 protein of vaccinia virus [290] . the potential interaction of surfactant proteins with proteins at the surface of the sars-cov-2 virus responsible of the emergent covid19 pandemic is currently under intensive scrutiny. interactions of lung collectins with viral proteins can also result in deleterious effects during viral infections. in this regard, binding of sp-d to the glycoprotein of ebola virus enhances infection in mammalian cells, facilitating the attachment of sp-d-bound virus to host cell co-receptors and probably affecting the inflammatory response [291] . on the other hand, the finding that constitutive expression of misfolded sp-c increases susceptibility to viral-induced cell death [292] suggests an antiviral activity for sp-c. this hypothesis is supported by the increased susceptibility of sp-c deficient mice to respiratory virus infection [235] , which is reduced upon transgenic restoration of sp-c [197] . surfactant lipids also play antiviral roles. different molecular species of pg and pi have been shown to bind to respiratory syncytial virus and influenza a virus, inhibiting their interaction with the epithelial cell surface [231, [293] [294] [295] [296] . neither the structural basis for lipid antagonism nor the inhibition mechanisms are known. it has been proposed that popg and pi could act as decoy ligands for toll-like receptors, given the structural similarities between the lipid a moiety of lps and the anionic surfactant lipids [183] . likewise, the structural similarities between anionic surfactant lipids and sialic acid containing lipid receptors for the hemagglutinin of h1n1 influenza a virus suggest that these lipids could also act as decoy receptors for viral attachment proteins [183] . the antimicrobial function of surfactant components may be modulated by interaction with other antimicrobial components of the alveolar fluid. cooperative interactions of sp-a and sp-d with antimicrobial peptides have been suggested. for example, binding of sp-a to sp-b n results in synergistic activity against sp-a-and sp-b n -resistant capsulated k. pneumoniae both in vitro and in vivo [253] . on the other hand, binding of sp-d to human neutrophil defensins results in antagonistic or cooperative effects on the hemagglutinin inhibitory and neutralizing activity of sp-d [297] . these effects depend on the viral strain and the multimeric forms of both proteins, with higher weight multimers precipitating sp-d in bronchoalveolar lavage fluid and thereby, reducing its hemagglutinin inhibitory activity [297] . on the contrary, combinations of sp-d with human β-defensins 5 and 6 show additive activity against influenza a virus [298] . sp-a, sp-d and the scavenger receptor-rich gp340 act cooperatively against influenza a virus, inducing viral aggregation and inhibiting hemagglutination [299] . although both collectins bind to gp340 [249, 250] , this cooperative effect does not appear to require a direct interaction with gp340, at least for sp-d [299] . whether this is also true for sp-a, needs to be clarified. additionally, lung collectins can modulate the activity and toxicity of some antimicrobial peptides. by binding to β-defensin 3, sp-a negatively regulates the cytotoxicity of this peptide on epithelial cells without affecting its antimicrobial activity, protecting the lung epithelium from injury caused by an excess amount of β-defensin 3 liberated during inflammation [300] . surfactant proteins also contribute to the homeostasis of the alveolar epithelium by modulating apoptosis, promoting clearance of apoptotic cells, and enhancing tissue repair [15] . alveolar epithelial apoptosis is an important contributor to the pathophysiology of lung diseases since severe alveolar epithelial apoptosis increases alveolar capillary permeability, and causes leakage of serum into alveoli and loss of compartmentalization with unrestricted mechanical transduction and signaling from the airways [301] . excessive apoptosis may therefore affect the outcome of lung diseases, including adult respiratory distress syndrome [302] , chronic obstructive pulmonary disease [303] , and pulmonary fibrosis [304] . therefore, a well-balanced interplay of cell apoptosis and proliferation is required to maintain the integrity and function of the lung. lung collectins have been shown to play a key role regulating apoptosis of alveolar epithelial cells apoptosis. in this regard, sp-d has been shown to decrease apoptosis both in vitro and in vivo. a recent study shows that sp-d-deficient mice have higher level of apoptotic cells in their alveolar spaces than wild type mice and intratracheal administration of recombinant sp-d reduces levels of apoptotic cells [263] . this reduction in apoptosis is due to a delayed activation of caspase 8 and 3 and externalization of phosphatidylserine [305] , the aminophospholipid that in normal conditions is located in the inner leaflet of the plasma membrane, but flips to the outer leaflet in dying cells. janssen et al. [306] have proposed that sp-a and sp-d suppress the phagocytic function of alveolar macrophages through tonic interaction with the transmembrane receptor signal inhibitory regulatory protein alfa (sirpα) in the resting, noninflamed lung. binding of sp-a or sp-d to sirpα may facilitate the interaction of this receptor with cluster of differentiation cd47, and the subsequent negative control of phagocytosis by innate immune cells [307] . in type ii cells, binding of sp-a to its receptor initiates a signaling pathway that regulates apoptosis through tyrosine phosphorylation and activation of phosphatidylinositol 3-kinase [308] . on the other hand, it has been recently demonstrated that the interaction of interleukin 8, a chemokine produced by macrophages and epithelial cells, with sp-a exacerbates cell damage in acute lung injury through inhibition of sp-a, promoting cell viability and suppressing apoptosis [309] . interleukin 8 also binds to sp-b [309] , inhibiting the protective effect of this protein on calcium ion transport. this would result in an increase of intracellular calcium ions that may induce passive depolymerization of cytoskeleton and destroy the endothelial barrier [310] . regarding surfactant lipids, little is known about their role in apoptosis. supplementation of poractant alfa (curosurf), an exogenous porcine-derived surfactant composed of surfactant lipids, sp-b, and sp-c, with popg or dioleoylphosphatidylglycerol reduces alveolar epithelial cell apoptosis [311, 312] . how these phospholipids regulate apoptosis is unknown, but they are supposed to act directly on alveolar epithelial cells [311] . surfactant proteins sp-b and sp-c are not involved in principle in regulation of apoptosis. however, it has been shown that mutations in the sp-c gene produce aberrant forms of sp-c that can generate accumulation of aggregated forms of prosp-c, causing misfolded protein stress responses, inhibiting proteasome function, and activating caspase-3-mediated apoptosis [313] . non-inflammatory removal of dying cells is important for maintenance of lung tissue homeostasis since apoptotic cells can undergo necrosis, releasing potentially damaging pro-inflammatory molecules and antigens that cause autoimmunity [314] and the eventual destruction of alveolar walls [315] . lung collectins enhance the ingestion of apoptotic cells by alveolar macrophages [316, 317] , being sp-d a more potent promoter of apoptotic cell clearance than sp-a [316] . removal of apoptotic cells by sp-a and sp-d occurs through simultaneous interaction with apoptotic cells and surface receptors on alveolar macrophages. the interaction of sp-a and sp-d with dying cells involves recognition of intracellular molecules that become exposed on the outside of the cell upon apoptosis. in this regard, both collectins have been shown to bind to dna [318, 319] and myeloperoxidase [320] , a specific intracellular defence molecule of neutrophils that has been proposed to act as a bridging molecule between phosphatidylserine epitopes on apoptotic cells and sp-a, facilitating the internalization of dying cells by macrophages. on the other hand, the macrophage receptors calreticulin/cd91 and myosin 18a are involved in collectin-mediated clearance of dead cells [15, 321] . interestingly, the interactions of sp-a and sp-d with these receptors, as well as the binding of sp-d to dna, occur through their collagenous domains [15, 319, 322, 323] . therefore, the potency of sp-d as promoter of apoptotic cell clearance may be linked to its large and extended collagen steams, which would facilitate the interaction between the macrophage receptors and apoptotic cells. this would allow the binding of calreticulin to dying cells and their subsequent targeting for removal by phagocytosis [324] . in vivo administration of poractant alfa increases the clearance of apoptotic neutrophils [325] . whether this effect is due to surfactant lipids or to surfactant proteins sp-b and/or -c is not clear. although the mechanisms involved in this effect remain elusive, willems and co-workers [325] have proposed that surfactant lipids could compete with sirpα for binding sp-a and sp-d, thereby preventing their suppressive effects on phagocyte function. pathogens, damaged cells or toxic compounds may trigger an exacerbated or chronic inflammatory response that damages lung tissue, playing a critical role in the pathology and progression of different lung diseases such as emphysema [326] and lung fibrosis [327] . resolution of inflammation and the subsequent regeneration of lung tissue requires replacement of injured cells by cells of the same type and the subsequent fibroplasia or fibrotic scarring in which connective tissue replaces normal parenchymal tissue, associated with an alternative, anti-inflammatory m2 polarization of macrophages [328] [329] [330] . willems and co-workers demonstrated that pulmonary surfactant plays a role in the regeneration and remodeling of lung parenchyma [325] . sp-a has been shown to participate in the processes that control lung epithelial cell proliferation and apoptosis by binding to transforming growth factor β (tgf-β) [325, 331] , a potent inhibitor of epithelial cell proliferation. formation of the sp-a/tgf-β complex protects tgf-β from inactivation by the latency-associated peptide [331] and facilitates tgf-β binding to its receptors, inducing signaling cascades involved in cell cycle progression and proliferation [127] . sp-a and sp-d also contribute to tissue repair by activating the polarization of alveolar macrophages to a m2 phenotype [323, 332] . this effect has been related to the binding of the collagen domain of sp-a to myosin 18 [323] . this receptor, also known as sp-r210, forms a pro-inflammatory complex with cd14 and class a scavenger receptor (sr-a) that promotes internalization of lps and subsequent signaling [333] . therefore, sp-a may inhibit the sp-r210/cd14/sr-a complex, contributing to regulation and resolution of the inflammatory response [333] . in addition, through binding to interferon gamma (ifn-γ), sp-a suppresses ifn-γ interaction with its receptor, inhibiting the polarization of alveolar macrophages to a pro-inflammatory m1 phenotype [334] that has been shown to hinder fibroplasias [328] . on the other hand, sp-c [78] also plays a key role in maintaining alveolar integrity and repair since sp-c-null patients and animal models of sp-c deficiency develop fibrosis [105, 335] . moreover, mutations or decreased expression of the gene encoding sp-c, causes alveolar type ii cell injury and aberrant repair of lung tissue to develop pulmonary fibrosis [335] [336] [337] . in this regard, glasser and co-workers [234] showed that sp-c-deficient mice express markers of macrophage alternative activation associated with advancing pulmonary fibrosis. this suggests that sp-c negatively regulates the alternative activation of macrophages, preventing the development of fibrosis. although the role of sp-c deficiency in fibrosis development has been documented by numerous lines of evidence, little is known about the interactions of sp-c with macrophage receptors involved in regulation of macrophage polarization. the extensive network of lipid-protein and protein-protein interactions described by this review to occur between lung surfactant molecules and host and pathogen cells at the alveolar spaces put surfactant structures at the center of lung homeostasis. through these interactions, the surfactant network likely integrates signals from epithelial cells and the cells responsible for the innate and induced immune response, leading to a coordinated modulation of the respiratory scenario. this includes a proper control over an environment that needs to be at the same time protective against mechanical and biophysical demands and against infection, but relatively tolerant with the occasional entrance of exogenous particles and microorganisms inhaled 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conformation of bacterial deep-rough lps lead to reduced activity on human macrophages elucidation of lipid binding sites on lung surfactant protein a using x-ray crystallography, mutagenesis, and molecular dynamics simulations reconstruction of lps transfer cascade reveals structural determinants within lbp, cd14, and tlr4-md2 for efficient lps recognition and transfer the structural basis of lipopolysaccharide recognition by the tlr4-md-2 complex surfactant protein a inhibits lipopolysaccharide-induced immune cell activation by preventing the interaction of lipopolysaccharide with lipopolysaccharide-binding protein the perturbation of pulmonary surfactant by bacterial lipopolysaccharide and its reversal by polymyxin b: function and structure altered in vivo activity of liposome-incorporated lipopolysaccharide and lipid a surfactant protein a directly interacts with tlr4 and md-2 and regulates inflammatory cellular response. importance of supratrimeric oligomerization interaction of 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phosphatidylinositol in the signaling via toll-like receptor 4-md-2 structural analogs of pulmonary surfactant phosphatidylglycerol inhibit toll-like receptor 2 and 4 signaling surfactant lipids regulate lps-induced interleukin-8 production in a549 lung epithelial cells by inhibiting translocation of tlr4 into lipid raft domains lps-induced clustering of cd14 triggers generation of pi(4,5)p2 anionic pulmonary surfactant lipid regulation of innate immunity host defense against fungal infections and allergies surfactant protein (sp)-a and sp-d as antimicrobial and immunotherapeutic agents. recent patents anti-infective drug discov macrophage dysfunction and susceptibility to pulmonary pseudomonas aeruginosa infection in surfactant protein c-deficient mice surfactant protein c-deficient mice are susceptible to respiratory syncytial virus infection diverse functions of pulmonary collectins in host defense of the lung pulmonary collectins and innate host defense of the lung role of soluble innate effector molecules in pulmonary defense against fungal pathogens an insight into the diverse roles of surfactant proteins, sp-a and sp-d in innate and adaptive immunity. front surfactant protein a binds mycoplasma pneumoniae with high affinity and attenuates its growth by recognition of disaturated phosphatidylglycerols characteristics of surfactant protein a and d binding to lipoteichoic acid and peptidoglycan, 2 major cell wall components of gram-positive bacteria respiratory fungal infections: their role in the inflammatory response surfactant proteins a and d: trimerized innate immunity proteins with an affinity for viral fusion proteins collectins and fungal pathogens: roles of surfactant proteins and mannose binding lectin in host resistance surfactant protein d binding to terminal alpha1-3-linked fucose residues and to schistosoma mansoni opsonic activities of surfactant proteins a and d in phagocytosis of gram-negative bacteria by alveolar macrophages surfactant protein a (sp-a)-mediated clearance of staphylococcus aureus involves binding of sp-a to the staphylococcal adhesin eap and the macrophage receptors sp-a receptor 210 and scavenger receptor class a purification of a cell-surface receptor for surfactant protein a lung surfactant proteins (sp-a and sp-d) in non-adaptive host responses to infection glycoprotein-340 binds surfactant protein-a (sp-a) and stimulates alveolar macrophage migration in an sp-a-independent manner collectin surfactant protein d binds antibodies and interlinks innate and adaptive immune systems by binding sirpalpha or calreticulin/cd91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation natural anti-infective pulmonary proteins: in vivo cooperative action of surfactant protein sp-a and the lung antimicrobial peptide sp-bn pulmonary surfactant protein a up-regulates activity of the mannose receptor, a pattern recognition receptor expressed on human macrophages pulmonary collectins enhance phagocytosis of mycobacterium avium through increased activity of mannose receptor pulmonary surfactant protein a augments the phagocytosis of streptococcus pneumoniae by alveolar macrophages through a casein kinase 2-dependent increase of cell surface localization of scavenger receptor a ten haken, b.; et al. pulmonary surfactant protein sp-d opsonises carbon nanotubes and augments their phagocytosis and subsequent pro-inflammatory immune response overview of complement activation and regulation surfactant protein a regulates complement activation innate immune collectin surfactant protein d simultaneously binds both neutrophil extracellular traps and carbohydrate ligands and promotes bacterial trapping hemoglobin induction in mouse macrophages sp-d attenuates lps-induced formation of human neutrophil extracellular traps (nets), protecting pulmonary surfactant inactivation by nets surfactant protein d attenuates acute lung and kidney injuries in pneumonia-induced sepsis through modulating apoptosis, inflammation and nf-kappab signaling surfactant proteins a and d inhibit the growth of gram-negative bacteria by increasing membrane permeability pulmonary collectins selectively permeabilize model bacterial membranes containing rough lipopolysaccharide sp-a permeabilizes lipopolysaccharide membranes by forming protein aggregates that extract lipids from the membrane macrophage-independent fungicidal action of the pulmonary collectins paradoxical bactericidal effects of hydrophobic lung surfactant proteins and their peptide mimics using liposome molecular trojan antimicrobial activity of native and synthetic surfactant protein b peptides surfactant protein b propeptide contains a saposin-like protein domain with antimicrobial activity at low ph the role and molecular mechanism of action of surfactant protein d in innate host defense against influenza a virus full-length human surfactant protein a inhibits influenza a virus infection of a549 lung epithelial cells: a recombinant form containing neck and lectin domains promotes infectivity interactions of surfactant protein a with influenza a viruses: binding and neutralization impact of ozone exposure on the phagocytic activity of human surfactant protein a (sp-a) and sp-a variants evidence for a protective role of pulmonary surfactant protein d (sp-d) against influenza a viruses mechanisms of anti-influenza activity of surfactant proteins a and d: comparison with serum collectins collectin-mediated antiviral host defense of the lung: evidence from influenza virus infection of mice inhibition of influenza viral neuraminidase activity by collectins surfactant protein a binds to hiv and inhibits direct infection of cd4+ cells, but enhances dendritic cell-mediated viral transfer surfactant protein d binds to human immunodeficiency virus (hiv) envelope protein gp120 and inhibits hiv replication surfactant protein d modulates hiv infection of both t-cells and dendritic cells 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adaptation and increased susceptibility to infection associated with constitutive expression of misfolded sp-c pulmonary surfactant phosphatidylglycerol inhibits respiratory syncytial virus-induced inflammation and infection phosphatidylinositol inhibits respiratory syncytial virus infection pulmonary surfactant lipids inhibit infections with the pandemic h1n1 influenza virus in several animal models phosphatidylglycerol provides short-term prophylaxis against respiratory syncytial virus infection innate defense against influenza a virus: activity of human neutrophil defensins and interactions of defensins with surfactant protein d interactions of alpha-, beta-, and theta-defensins with influenza a virus and surfactant protein d respiratory innate immune proteins differentially modulate the neutrophil respiratory burst response to influenza a virus pulmonary surfactant protein a protects lung epithelium from cytotoxicity of human beta-defensin 3 role of apoptosis in amplifying inflammatory responses in lung diseases the role of apoptosis in the pathophysiology of acute respiratory distress syndrome (ards): an up-to-date cell-specific review role of apoptosis in the pathogenesis of copd and pulmonary emphysema apoptosis in lung injury and fibrosis surfactant protein d regulates caspase-8-mediated cascade of the intrinsic pathway of apoptosis while promoting bleb formation surfactant proteins a and d suppress alveolar macrophage phagocytosis via interaction with sirp alpha functional cd47/signal regulatory protein alpha (sirp(alpha)) interaction is required for optimal human t-and natural killer-(nk) cell homeostasis in vivo elevated expression of surfactant proteins in newborn rats during adaptation to hyperoxia mechanism of il-8-induced acute lung injury through pulmonary surfactant proteins a and b endothelial mitochondria determine rapid barrier failure in chemical lung injury novel therapeutic roles for surfactant-inositols and -phosphatidylglycerols in a neonatal piglet ards model: a translational study 18:1/18:1-dioleoyl-phosphatidylglycerol prevents alveolar epithelial apoptosis and profibrotic stimulus in a neonatal piglet model of acute respiratory distress syndrome misfolded brichos sp-c mutant proteins induce apoptosis via caspase-4-and cytochrome c-related mechanisms efferocytosis and lung disease functional significance of apoptosis in chronic obstructive pulmonary disease role of surfactant proteins a, d, and c1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and cd91 as a common collectin receptor complex surfactant protein a enhances alveolar macrophage phagocytosis of apoptotic neutrophils surfactant protein d binds genomic dna and apoptotic cells, and enhances their clearance, in vivo nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins a and d and mannose-binding lectin surfactant protein a (sp-a) binds to phosphatidylserine and competes with annexin v binding on late apoptotic cells cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of lrp on the phagocyte surfactant protein a inhibits t cell proliferation via its collagen-like tail and a 210-kda receptor local amplifiers of il-4ralpha-mediated macrophage activation promote repair in lung and liver programmed cell removal by calreticulin in tissue homeostasis and cancer poractant alfa (curosurf(r)) increases phagocytosis of apoptotic neutrophils by alveolar macrophages in vivo immune-mediated inflammation in the pathogenesis of emphysema: insights from mouse models inflammation in cystic fibrosis: an update p-selectin suppresses hepatic inflammation and fibrosis in mice by regulating interferon gamma and the il-13 decoy receptor macrophage plasticity and polarization in tissue repair and remodelling dual polarization of human alveolar macrophages progressively increases with smoking and copd severity surfactant protein a binds tgf-beta1 with high affinity and stimulates the tgf-beta pathway surfactant protein-d is essential for immunity to helminth infection sp-r210 (myo18a) isoforms as intrinsic modulators of macrophage priming and activation surfactant protein a prevents ifn-gamma/ifn-gamma receptor interaction and attenuates classical activation of human alveolar macrophages histone deacetylase inhibitor restores surfactant protein-c expression in alveolar-epithelial type ii cells and attenuates bleomycin-induced pulmonary fibrosis in vivo expression of mutant sftpc in murine alveolar epithelia drives spontaneous lung fibrosis genetics of fibrosing lung diseases key: cord-288673-ku3tmjd3 authors: sabotič, jerica; kos, janko title: microbial and fungal protease inhibitors—current and potential applications date: 2012-01-05 journal: appl microbiol biotechnol doi: 10.1007/s00253-011-3834-x sha: doc_id: 288673 cord_uid: ku3tmjd3 proteolytic enzymes play essential metabolic and regulatory functions in many biological processes and also offer a wide range of biotechnological applications. because of their essential roles, their proteolytic activity needs to be tightly regulated. therefore, small molecules and proteins that inhibit proteases can be versatile tools in the fields of medicine, agriculture and biotechnology. in medicine, protease inhibitors can be used as diagnostic or therapeutic agents for viral, bacterial, fungal and parasitic diseases as well as for treating cancer and immunological, neurodegenerative and cardiovascular diseases. they can be involved in crop protection against plant pathogens and herbivorous pests as well as against abiotic stress such as drought. furthermore, protease inhibitors are indispensable in protein purification procedures to prevent undesired proteolysis during heterologous expression or protein extraction. they are also valuable tools for simple and effective purification of proteases, using affinity chromatography. because there are such a large number and diversity of proteases in prokaryotes, yeasts, filamentous fungi and mushrooms, we can expect them to be a rich source of protease inhibitors as well. applications of protease inhibitors are intimately connected to the proteases they inhibit. so, as a preface to protease inhibitors, an overview of proteases with the modes of regulation of their proteolytic activity is provided. then, known microbial and fungal protease inhibitors are reviewed, with the emphasis on protein (tables 1 and 2 ) rather than small-molecule protease inhibitors (table 3) . finally, their potential applications in the fields of medicine, crop protection and biotechnology are described, based on their target proteases. microorganisms (prokaryotes, yeasts and filamentous fungi) and higher fungi or mushrooms have been selected for review since protease inhibitors of microbial origin have already proven useful in many different applications. higher fungi have emerged as a valuable source of new protease inhibitors with unique characteristics only in the last decade and therefore offer great potential for future applications. proteases, also called peptidases or proteolytic enzymes, constitute a large group of enzymes that catalyse the hydrolysis of peptide bonds. cleavage of peptide bonds can be general, leading to complete degradation of protein substrates into their constituent amino acids, or it can be specific, leading to selective protein cleavage for post-translational modification and processing. peptidases that cleave peptide bonds at the termini of polypeptide chains are called exopeptidases, while endopeptidases cleave peptide bonds within the polypeptide chain. peptidases are classified according to their catalytic type into aspartic, cysteine, glutamic, serine and threonine peptidases, according to the main, functional amino acid residue at the active site. metallopeptidases, on the other hand, are those whose catalytic activity depends on the presence of a divalent metal ion bound within the active site. in the mer-ops database (http://merops.sanger.ac.uk/), peptidases are classified further into families, according to their sequence similarity, and into clans, according to their structural similarity. there are 226 peptidase families assigned in the merops database (release 9.5, july 2011) and 57 clans, based on structural data (barrett 2001; rawlings et al. 2010) . peptidases are present in all living organisms, including viruses, bacteria, archaea, protists, fungi, plants and animals. serine peptidases form the most abundant class, followed by metallo-, cysteine, aspartic and threonine peptidases. there has been an explosive growth of the number of peptidase families observed in eukaryotic organisms, there being 100 peptidases in bacterial genomes and half as many in archaeal genomes and from 400 to 700 peptidase genes in plant and mammal genomes. furthermore, there is a striking difference between the compositions of eubacterial and eukaryotic degradomes, (i.e. the complete set of proteases present in an organism). sixteen peptidase families constitute the core of the nearly ubiquitous peptidase families present in all living forms. additional 34 peptidase families are widely distributed in eukaryotic organisms, while another ten are unique to higher metazoan organisms, performing mainly limited proteolysis in extracellular environments (page and di cera 2008; rawlings et al. 2010) . in addition to the merops database, information on proteases can be found in several other online databases, including the degradome database (http:// degradome.uniovi.es/) (quesada et al. 2009 ) and the proteolysis map (pmap) (http://www.proteolysis.org/) that comprises five α 2 m a1a, a2a, c1a, c2a, c11, m4, m10a, m10b, m12a, m12b, s1a, s1b, s8a a underlined families include protease inhibitors exclusively of microbial and/or fungal origin b × denotes the number of sequence homologues found in each group of organisms: × less than 10, ×× 11-200, ××× 201-1000 and ×××× more than 1000 (armstrong and quigley 1999; sottrup-jensen 1989) serine protease inhibitors ovomucoid (kazal-type) chymotrypsin (s1) and subtilisin (s8) families tight-binding, laskowski described in stramenopiles oomycetes (fungus-like microorganisms distantly related to fungi); e.g. involved in pathogenicity of phytophtora infestans (tian et al. 2004; rawlings and barrett 2011) aprotinin i2 chymotrypsin family (s1) broad inhibitory specificity (ascenzi et al. 2003; rawlings and barrett 2011) peptidase b inhibitor i9 subtilisin family (s8) bacterial inhibitors are propeptides of subtilisin-like proteases. fungal inhibitors are separate polypeptides, e.g. pleurotus ostreatus proteinase a inhibitor 1 poia1 and yeast proteinase inhibitor 2 yib2 they are potent but unstable inhibitors, gradually degraded by subtilisin (ascenzi et al. 2003; dohmae et al. 1995; kojima et al. 1999; kojima et al. 2005; kojima et al. 1997; rawlings and barrett 2011) marinostatin i10 proteases of family s8 (subtilisin) and certain proteases of family s1 (chymotrypsin) tight-binding, laskowski. structure stabilized by two internal ester bonds that are essential for their inhibitory activity exclusive to marine bacteria (kanaori et al. 2005; rawlings and barrett 2011) ecotin i11 chymotrypsin family (s1) tight-binding, laskowski for primary binding site. active as dimers, each monomer binds the protease at two binding sites ecotins from enterobacteria and parasites perform a protective role against host digestive proteases and target host proteases to facilitate colonization. structure enables inhibition of multiple proteases with the chymotrypsin fold (eggers et al. 2001; eschenlauer et al. 2009; rawlings and barrett 2011) streptomyces subtilisin inhibitor (ssi) family s8 (subtilisin, kexin), family s1 (trypsin, plasmin) and the metalloprotease griselysin (family m4) tight-binding, laskowski exclusive to bacterial actinomycetales order. they probably control endogenous proteases involved in proteolytic activation of transglutaminase (kantyka et al. 2010; taguchi et al. 1997; tsuyuki et al. 1991; rawlings and barrett 2011) carboxypeptidase y inhibitor i51 serine carboxypeptidase y (family s10) a defensive role against predatory insects has been shown, but a regulatory endogenous role is also possible (avanzo et al. 2009; odani et al. 1999; sabotič et al. 2012; rawlings and barrett 2011) human elastase-2 and endogenous aspergillus elastases (family s1) unknown homologues have been found in a few other ascomycete species and in proteobacteria, but none was characterized biochemically (okumura et al. 2008; okumura et al. 2006; rawlings and barrett 2011) serpin i4 chymotrypsin (s1) and subtilisin (s8) families. some members inhibit also cysteine proteases of papain (c1) and caspase (c14) families trapping. suicide inhibitors in which a rapid conformational change traps the cognate protease in a covalent complex the physiological role of microbial serpins has been proposed to be to protect the cellulosedegrading apparatus (cellulosome) against proteolytic degradation. the only fungal serpin (celpin), was characterized from the anaerobic fungus piromyces sp. e2 (kantyka et al. 2010; law et al. 2006; roberts et al. 2004; steenbakkers et al. 2008) cysteine protease inhibitors thyropin i31 papain-like proteases (family c1), and equistatin inhibitor unit 2 inhibits an aspartic protease cathepsin d (family a1) tight-binding present in animals and in one bacterial pathogen (coxiella burnetii) that has presumably acquired the gene by lateral transfer (kantyka et al. 2010; rawlings and barrett 2011) survivin i32 caspases-aspartate-specific cysteine proteases (family c14) tight-binding, several mechanisms survivin plays a dual role as a mitotic regulator of cell division and as an inhibitor of caspase activation in the process of apoptosis. fungal homologues have been identified in ascomycete and a few basidiomycete genomes. the fission yeast homologue is a conserved chromosomal passenger protein (huang et al. 2005; luthringer et al. 2010; rawlings 2010) chagasin i42 protozoan and mammalian papain-like cysteine proteases (family c1) tight-binding parasitic chagasins are involved in regulating endogenous cysteine proteases essential for their life cycle. in bacteria and archaea, chagasins serve as endogenous regulators, and in some pathogenic species, they also serve a protective role against host proteases (kantyka et al. 2010; santos et al. 2006) clitocypin (i48) and macrocypin (i85); together named mycocypins i48, i85 papain-like cysteine proteases (family c1) and legumain (family c13) and serine protease trypsin (family s1) tight-binding. mycocypins are small and exceptionally stable proteins. they have a β-trefoil fold formed by the core six-stranded β-barrel that supports 11 loops which provide a versatile surface for the inhibition of several types of proteases unique to basidiomycetes. they probably have an endogenous regulatory role or a role in defence against pathogen infection and/or predation by pests. a defensive role for mycocypins is further supported by their high genetic variability and conformational stability as well as a broad inhibitory profile (brzin et al. 2000; kidrič et al. 2002; renko et al. 2010; sabotič et al. 2007a; sabotič et al. 2006; sabotič et al. 2009b the high specificity is the result of the structural stabilization of the ia3 inhibitor in complex with saccharopepsin since the unstructured inhibitor in solution forms an alpha helix upon interaction with the enzyme active site (green et al. 2004; phylip et al. 2001) different databases (cutdb, pathwaydb, proteasedb, substratedb and profiledb) (igarashi et al. 2009 ). the occurrence of proteases in all living organisms indicates their critical role in essential metabolic and regulatory functions in many biological processes. proteases are important in the production of nutrients for growth and proliferation. extracellular proteases catalyse the hydrolysis of proteins into smaller peptides and amino acids for subsequent absorption into cells, constituting a very important step in nitrogen metabolism. proteases perform critical regulatory functions in numerous physiological processes since they regulate the fate, localization and activity of many proteins, modulate protein-protein interactions and contribute to the generation, transduction and amplification of molecular signals. proteases are involved in a wide span of cellular and metabolic processes, including regulation of gene expression, dna replication, transport of proteins, cell growth and differentiation, cell cycle, heat shock response, sos response to dna damage, misfolded protein response, oxidative stress response and programmed cell death (lopez-otin and bond 2008; rao et al. 1998 ). furthermore, in plants, proteases are important in the build-up and breakdown of seed storage proteins during seed germination, protein remobilization upon organ senescence and in many developmental processes such as embryogenesis, chloroplast biogenesis, photomorphogenesis, hormone signalling, flower development, pollen-pistil interaction and local and systemic defence responses against pathogens and herbivores (simoes and faro 2004; salas et al. 2008; schaller 2004; van der hoorn 2008) . moreover, in animals, proteases are involved in tissue morphogenesis and remodelling, angiogenesis, neurogenesis, ovulation, fertilization, wound repair, stem cell mobilization, haemostasis, blood coagulation, inflammation, immunity, autophagy and senescence (lopez-otin and bond 2008). proteases of microbial and fungal origin offer a wide range of biotechnological applications. alkaline proteases have been used in the detergent industry for over 50 years. proteases with elastolytic and keratinolytic activities have been used in the leather industry for de-hairing and baiting of skins and hides. the food industry uses proteases in cheese making, baking, preparation of various protein hydrolysates, meat tenderization and manufacturing protein-rich diets. in the pharmaceutical industry, proteases have found uses as therapeutic agents as well as additives in preparations of slow-release dosage forms. bioprocessing of used x-ray films for silver recovery involves the use of alkaline proteases. proteases allow potential applications for the management of wastes from various food processing industries and from household activities. in addition to industrial and medical applications, proteases are used in basic research; for example, proteases with very selective peptide bond cleavage are used in protein sequencing, unselective proteinase k is used in nucleic acid isolation, and trypsin is widely used in maintaining animal cell cultures (kumar and takagi 1999; rao et al. 1998) . there is also the downside to proteases as some are important virulence factors of many pathogenic bacteria, parasites and viruses. these proteases are involved in acquiring nutrients for growth and proliferation through host tissue degradation and evasion of host immune defences. in addition to colonizing and facilitating dissemination functions, they are also involved in evading the host immune system by interrupting the cascade pathways, disrupting the cytokine network, excising cell surface receptors and inactivating host protease inhibitors (maeda 1996; travis and potempa 2000; supuran et al. 2002) . because proteases play essential roles in life and death processes in all living organisms and because peptide bond hydrolysis is irreversible, anomalies in proteolytic activities lead to numerous pathological conditions, including cancer, neurodegenerative disorders and inflammatory and cardiovascular diseases, as well as bacterial, viral and parasitic diseases (lopez-otin and bond 2008; turk 2006) . activity of proteases is regulated on several levels, including regulation of gene expression at transcriptional and post-transcriptional levels, synthesis as inactive zymogens, blockade by endogenous inhibitory proteins, spatial and temporal compartmentalization, post-translational modification (glycosylation, phosphorylation, co-factor binding), proteolysis and degradation (lopez-otin and bond 2008). protein protease inhibitors constitute a very important mechanism for regulating proteolytic activity. they can be classified approximately according to the class of proteases they inhibit (for example, serine or cysteine protease inhibitors). however, those composed of multiple inhibitor units and pan-inhibitors (such as α 2 -macroglobulin of family i39) that target proteases of different catalytic classes prevent unambiguous classification. a more detailed classification is included in the merops database (http://merops.sanger. ac.uk/), which follows a hierarchy similar to that for proteases. protease inhibitors are grouped into families based on sequence homology and into clans based on protein tertiary structure. in release 9.5 (4 july 2011) of the mer-ops database, there are 71 families of protease inhibitors, and those with available three-dimensional structural data have been assigned to 39 different clans (rawlings 2010) . of the 71 families, 27 include members of microbial and fungal origin (tables 1 and 2 ). of these, seven families include members of exclusively bacterial origin (i10, i16, i36, i38, i57, i58, i69), and five families include members of exclusively fungal origin (i34, i48, i66, i79, i85). in addition to protein protease inhibitors, the merops database includes a list of small-molecule inhibitors that are well known and widely used. many of them have been synthesized in the laboratory; however, those that occur naturally (table 3 ) have been isolated from bacteria and fungi (rawlings 2010) . there are two general mechanisms of protease inhibition, namely, irreversible "trapping" reactions and reversible tight-binding reactions. trapping reactions work only on endopeptidases and are the result of a conformational change of the inhibitor triggered by cleavage of an internal peptide bond by the host protease ( fig. 1a ). only three families utilize a trapping mechanism: i4 (serpins), i39 (α 2 -macroglobulin) and i50 (viral caspase inhibitors). reversible tight-binding inhibition is widespread, the best known being the "standard canonical" or "laskowski mechanism", in which the inhibitor has a peptide bond that is cleaved by the peptidase active site in a substrate-like manner. the inhibitor is only slowly released due to the conformational stability of the stabilized loop that can mimic a substrate. this mechanism has been conclusively demonstrated only for inhibitors of serine proteases. other reversible tight-binding protease inhibitors physically block the protease active site by high-affinity binding to sites on either side of the active site (fig. 1b, c) . binding of an inhibitor to the active site can also be irreversible, when an electrophilic reactive group of the inhibitor forms a covalent bond with an amino acid residue in the enzyme active site. there are also some inhibitors that block the exosites, to which substrate binding occurs in addition to the active site in some proteases (christeller 2005; rawlings 2010; ). the families of protein protease inhibitors that include members of microbial and fungal origin are described in table 2 , and information of their distribution in taxonomic groups is given in table 1 . protease inhibitors are described in groups according to the catalytic class of protease they inhibit and following the merops inhibitor classification (rawlings 2010; rawlings and barrett 2011) . since prokaryote-derived protease inhibitors have been reviewed recently (kantyka et al. 2010 ), more information on protease inhibitors of fungal origin, including yeasts, filamentous fungi and mushrooms, is provided here. among the small-molecule protease inhibitors isolated from bacteria and fungi (table 3) , there are several that show broad inhibitory specificity and inhibit proteases of different catalytic classes. several inhibit both serine and cysteine proteases (antipain, chymostatin, leupeptin), serine and metalloproteases (bestatin, puromycin), metallo-and cysteine proteases (amastatin) or metallo-, cysteine and serine proteases (bacitracin a). of the small-molecule cysteine protease inhibitors, the best known is e-64 (1-[l-n-(trans-epoxysuccinyl) leucyl] amino-4-guanidinobutane), an irreversible inhibitor originally isolated from aspergillus japonicus (hanada et al. 1978) and routinely used as a class-specific cysteine protease inhibitor. a number of e-64 analogues have been synthesized in order to improve selectivity for a particular cysteine protease. several inhibitors are specific for metalloproteases and inhibit more than one protease family (e.g. phosphoramidon). the only natural small-molecule inhibitor of aspartic proteases is pepstatin, originally isolated from various species of actinomycetes (umezawa et al. 1970) , which inhibits several families of aspartic proteases. it is a hexa-peptide containing the unusual amino acid statine (rawlings 2010) . proteases play an important part in almost every biological process; therefore, unregulated activity often leads to disease. in this review, only excessive proteolysis will be addressed as it is the one that can be reversed by protease inhibitors. excessive proteolysis plays an important role in cancer and in cardiovascular, inflammatory, neurodegenerative, bacterial, viral and parasitic diseases. due to the obvious relevance of protease inhibitors, they have been studied extensively with the intent to develop therapeutic drugs (drag and salvesen 2010; turk 2006; haq et al. 2010) . proteases that have a potential as therapeutic targets are reviewed, according to their catalytic type, for each group of disease-causing organisms and for other human diseases. information on the availability of protease inhibitors for each protease described is provided, with the emphasis on those for which specific inhibitors have not yet been identified. proteolytic processing of virus polyprotein into structural and non-structural proteins is an essential part of the viral replication cycle, making the proteases an important antiviral drug target. several viral proteases have been studied as therapeutic targets. although proteases of any virus could be potential antiviral targets, viruses that cause chronic diseases (e.g. hiv, herpes virus) and those that could cause large-scale epidemics (e.g. sars coronavirus, dengue virus) have received most attention. several protease inhibitors acting against the human immunodeficiency virus 1 (hiv-1) protease, a homodimeric aspartic protease, have been used in treating hiv-1 infection. fig. 1 examples of protease inhibitors utilizing irreversible "trapping" reaction (a) and reversible tight-binding reactions (b and c). proteases are shown in light grey, their active site residues in black and inhibitors in dark grey. a serine protease trypsin in complex with serpin (family i39) (pdb id 1k9o). the protease cleaves the reactive centre loop of serpin, which triggers a conformational change in the inhibitor and trapping of the protease in an inactive covalent complex. b cysteine protease cathepsin v in complex with clitocypin (family i48) (pdb id 3h6s). the inhibitor binds to the protease active site cleft and obstructs access of substrate. c aspartic protease plasmepsin iv in complex with the small-molecule inhibitor pepstatin a (pdb id 1ls5). the inhibitor binds in the active site of the protease these are all low molecular weight peptidomimetic inhibitors whose design has been based on the structures of the compounds binding to the protease active site. in therapy, they are usually used in combination with inhibitors of reverse transcriptase (abbenante and fairlie 2005; anderson et al. 2009 ). in addition to a number of designed synthetic inhibitors, a potent peptidic inhibitor of hiv-1 protease of bacterial origin (atbi) has been found in an extremophilic bacillus sp. (dash and rao 2001; vathipadiekal et al. 2010) . other targeted viral proteases belong to the serine catalytic type. human cytomegalovirus (hcmv) is one of eight human herpes viruses and is widespread in populations worldwide, with infection rates of 80-100%. it causes asymptomatic infections in healthy individuals but high morbidity and mortality in immunocompromised individuals. a few inhibitors of the cytomegalovirus protease have been described from bacterial (streptomyces) and fungal (cytonaema) origins (stoeva and efferth 2008; anderson et al. 2009 ). an additional target in antiviral therapy against cytomegalovirus is the proteasome, where the aim is to hinder the recruitment of host proteins by the virus for its replication. several synthetic and a few natural proteasome inhibitors (e.g. lactacystin from streptomyces lactacystinaeus) are known and have been reported to obstruct replication of several viruses, including influenza virus, herpes simplex virus type 1, paramyxovirus and rhabdoviruses, as well as cytomegalovirus (kaspari and bogner 2009 ). the serine proteases ns3 and ns2 of flaviviruses are targets for antiviral drug development against hepatitis c virus and dengue virus, with the former blood-transmitted virus causing various liver diseases (including cirrhosis and liver cancer) and the latter being a mosquitotransmitted disease causing dengue hemorrhagic fever. several structure-based designed low molecular weight inhibitors are in different phases of clinical evaluation (anderson et al. 2009; lange et al. 2010; tomlinson et al. 2009 ). the coronavirus associated with severe acute respiratory syndrome, sars, encodes a chymotrypsin-like cysteine protease m pro that is similar to picornavirus 3c protease. since the 2003 sars global outbreak, several strategies of structure-based design of low molecular weight protease inhibitors have been applied in the search for antiviral drugs against sars (anderson et al. 2009; sirois et al. 2007 ). the first to be considered were the protease inhibitors targeting the picornavirus 3c protease. the picornaviruses, which encode a 3c protease, are important human and animal pathogens such as poliovirus, hepatitis a virus, coxsackievirus, human rhinovirus and foot-and-mouth disease virus. inhibitors targeting the 3c protease of human rhinoviruses that cause common cold, as well as 3c proteases of other picornaviruses and coronaviruses, have been developed based on structural data, but none has yet successfully passed all the phases of clinical evaluation (neubauer et al. 2009; wang and liang 2010) . due to the rapid development of resistance in viruses, the search for novel strategies for developing inhibitors targeting different sites on proteases is encouraged, including the search for novel lead compounds from natural sources and structure-based drug development. bacterial pathogens employ an array of virulence factors that enable their colonization, evasion of host defences and dissemination. proteases are important virulence factors of many pathogenic bacteria, which play roles in acquiring nutrients by direct degradation of host tissue components. the even more important aim of disrupting host immune response and signalling cascades has been reviewed by potempa and pike (2009) . most currently available antibiotics target bacterial cell wall synthesis or protein synthesis. in the light of rapidly spreading antibiotic resistance, bacterial proteases are promising targets for the design of novel antibiotics. metalloproteases are most abundantly represented in primary and opportunistic pathogens, although all catalytic classes are found. these proteases are often associated with mobile genetic elements (plasmids, pathogenicity islands, integrated phages), and their expression is not constitutive but regulated through environmental or cellular signals (travis and potempa 2000; wladyka and pustelny 2008) . omptins (outer membrane proteins t) are aspartic proteases (family a26) found in several gram-negative bacteria, including the pathogenic species escherichia coli (ompt), yersinia pestis (pla), shigella flexneri, shigella dysenteriae (sopa), salmonella enterica (pgte), legionella pneumophila (lpa) and plant pathogens agrobacterium tumefaciens and erwinia pyrifoliae (plaa). omptins are bacterial virulence factors and, in addition to their proteolytic activity, possess adhesive and invasive activities. they modulate the coagulation system since they act as plasminogen activators (ompt, pla, pgte, lpa), inactivate tissue factor pathway inhibitor (ompt, pla, pgte), degrade thrombin-activatable fibrinolysis inhibitor (pla, pgte), degrade the complement system proteins (pla, pgte) and antimicrobial peptides (ompt, pla, pgte), and process autotransporters (ompt, sopa). lipopolysaccharide (lps) is required for their enzymatic activity. omptins have a unique catalytic mechanism that combines the elements of both serine and aspartic proteases, and partial inhibition by serine protease inhibitors has been reported (hritonenko and stathopoulos 2007; valls seron et al. 2010; yun et al. 2009 ). other than a weak substrate-based peptide inhibitor with a d-arg, shown to inhibit ompt (dekker et al. 2001) , and a colicin immunity protein shown to protect colicin e2 from degradation by ompt in escherichia coli (duche et al. 2009) , no specific inhibitors have been reported. no specific inhibitor has so far been found for the exfoliative toxin a, a glutamate-specific serine protease (family s1) produced by staphylococcus aureus, which is the causative agent in staphylococcal scalded skin syndrome. the target of the toxin is a transmembrane glycoprotein desmoglein-1 of the cadherin superfamily, which plays an important role in keratinocyte cell-cell adhesion (ladhani 2003) . immunoglobulin a1 proteases (iga1 proteases) are serine proteases (family s6) produced by several pathogenic bacteria, including species causing bacterial meningitis, haemophilus influenzae, neisseria meningitidis and streptococcus pneumoniae. the iga1 proteases enable colonization of human mucosal surfaces by cleaving the secretory iga antibodies, thus disrupting the specific immunity response. except for an early report on substrate analogue inhibitors (burton et al. 1988 ) and small synthetic peptide inhibitors (bachovchin et al. 1990 ) of neisseria gonorrhoaeae iga1 protease, there has been no further development of iga1 protease inhibitors (mistry and stockley 2006) . other immunomodulating serine proteases are c5a peptidases from streptococci (family s8)-those of group a (streptococcus pyogenes scpa) and group b (streptococcus agalaciae scpb) streptococci have been described in more detail. streptococcus pyogenes is the causative agent of pharyngitis and also causes rheumatic fever and skin infections, which can develop severe complications, including toxic shock syndrome. c5a peptidases are important for streptococcal pathogenesis as they specifically cleave complement c5a and therefore prevent the recruitment of phagocytic cells to the infection site (cheng et al. 2002; collin and olsen 2003) . antibodies raised against c5a peptidase were used to inhibit c5a peptidase in vivo (park and cleary 2005) , but no peptidase inhibitors have been described. serine proteases are important pathogenesis factors in bacteria involved in dental diseases. treponema denticola is a spirochete implicated in the progression of periodontal diseases. a serine protease, trepolisin (also called dentilisin), of family s8 is an important pathogenesis factor, a mediator of cytopathic effects by degrading host proteins, including extracellular matrix components and host protease inhibitors (sela 2001) . the broad-range inhibitor of serine proteases (families s1 and s8), chymostatin, inhibits trepolisin; however, no specific inhibitors have been described. another important oral cavity pathogen involved in periodontal disease, porphyromonas gingivalis, in addition to a few cysteine proteases (discussed further in the following), produces a serine protease, a prolyl tripeptidyl peptidase ptpa (family s9), which is involved in degrading host connective tissue, providing nutrients for bacterial growth (banbula et al. 1999) . a substrate-based specific inhibitor of ptpa has been developed (xu et al. 2008 ). bacterial type i signal peptidases spase (family s26) are serine proteases widespread among gram-negative and gram-positive bacteria and are membrane proteases required for processing newly synthesized secreted proteins. in addition to their essential role in bacterial viability, they are important antimicrobial drug targets as they are involved in the secretion of many virulence factors (paetzel et al. 2000) . synthetic penem inhibitors have been developed for inhibiting both gram-negative (escherichia coli) and grampositive (staphylococcus aureus, staphylococcus epidermidis) spases. the various penem derivatives display different degrees of activity against these pathogenic bacteria spases (allsop et al. 1995; harris et al. 2009 ). recently, substratebased peptide aldehydes have been shown to be promising inhibitors of escherichia coli and staphylococcus aureus spases (buzder-lantos et al. 2009 ). however, the in vitro inhibitory activity and in vivo antimicrobial activities of these inhibitors did not correlate well, so further optimization of and search for novel spase i inhibitors are expected. no specific inhibitors have been described for the streptococcal pyrogenic exotoxin b (speb, streptopain), a cysteine protease (family c10) produced by all strains of streptococcus pyogenes. it is a multifunctional protease and an important pathogenesis factor with several immunomodulating activities, including release of proinflammatory molecules, degradation of extracellular matrix, cleavage of igg in the hinge region and degradation of other immunoglobulins. in addition to class-specific cysteine protease inhibitors, a peptide derivative was shown to inhibit speb, as well as α 2 -macroglobulin and an s-nitrosylated form of α 1 -protease inhibitor (collin and olsen 2003) . ides (family c66) is another cysteine protease from streptococcus pyogenes that specifically cleaves igg, its only known substrate (vincents et al. 2004 ). in addition to specific, inhibitory igg antibodies (akesson et al. 2006) , synthetic reversible inhibitors were designed, with aldehyde compounds being the most promising; however, no specificity data are yet available for these inhibitors (berggren et al. 2009 ). staphylococcus aureus causes a range of diseases, from mild skin infections to life-threatening disorders, including septicaemia, endocarditis, toxic shock syndrome and pneumonia (lowy 1998) . it expresses several extracellular proteases with proposed roles in pathogenicity, including a serine protease v8 (sspa), cysteine proteases staphopains a and b (scpa, sspb) and a metalloprotease aureolysin (aur) (shaw et al. 2004) . staphopains a and b (family c47) are papain-like cysteine proteases that are co-expressed with their respective specific inhibitors staphostatins a and b (dubin 2003) . their role in pathogenicity has not been determined. staphopain b (sspb) is activated by the glutamyl peptidase sspa (v8 protease, family s1), which is expressed from the same operon (shaw et al. 2004 ). v8 protease modulates the surface protein profile and so influences the binding of fibronectin by staphylococcus aureus (mcgavin et al. 1997) . sortases are cysteine proteases (family c60) of grampositive bacteria that catalyse the covalent attachment of proteins to the cell wall peptidoglycan. they have been shown to contribute to the virulence of several important pathogens, including staphylococcus aureus, bacillus anthracis, listeria monocytogenes and streptococcus pneumoniae. therefore, they have been considered to be important targets for the development of novel antiinfective agents (suree et al. 2007; clancy et al. 2010) . staphylococcus aureus sortase (srta) has been at the focus of sortase inhibitor development due to the emergence of antibiotic-resistant strains and the need for novel antimicrobial strategies. several types of srta inhibitors have been investigated, including nonspecific sulfhydryl modifiers, peptide analogues of the sorting signal, compounds from plants and marine organisms, and synthetic small-molecule inhibitors. several inhibitors of srta have been described with varying strength and specificity; however, good in vitro inhibitory activity has not yet led to an effective in vivo sortase inhibitor (maresso and schneewind 2008; suree et al. 2007; clancy et al. 2010) . the cysteine protease clostripain (family c11) is a secreted protease of the anaerobic bacterium clostridium histolyticum, a causative agent of gas gangrene (clostridial myonecrosis). clostripain selectively hydrolyses arginyl bonds and constitutes an important clostridial virulence factor (jozwiak et al. 2005; manabe et al. 2010 ). in addition to oxidizing agents, thiol-blocking agents and heavy-metal ions that all inhibit clostripain, good reversible inhibitors have been described, namely, aziridine peptide esters, which are thus promising lead compounds for the development of specific clostripain inhibitors (schirmeister and peric 2000; barrett et al. 2004) . gingipains are extracellular cysteine proteases (family c25) produced by the oral pathogenic bacterium porphyromonas gingivalis, a major etiological bacterium of chronic periodontal disease. gingipains comprise two argininespecific cysteine proteases (rgpa and rgpb) and a lysinespecific cysteine protease (kgp). they constitute the major virulence factor of this periodontopathogenic bacterium as they are involved in multiple facets of its virulence and survival, including the destruction of periodontal tissues, disruption of the host immune system by inactivation of host proteinase inhibitors and deregulation of several proteinase cascades, as well as acquisition of nutrients required for bacterial growth and survival in the periodontal pocket (fitzpatrick et al. 2009; travis and potempa 2000) . due to their importance in pathogenesis, considerable efforts have been put into discovering or designing specific inhibitors of gingipains. tetracyclines, inhibitors of prokaryotic protein synthesis, have been shown to have, in addition to their antibiotic activity, inhibitory activity against cysteine proteases through binding to the proteinase outside the substrate binding site (imamura et al. 2001 ). peptidyl chloromethanes have been used as specific rgp and kgp inhibitors for their characterization (potempa et al. 1997) , and compound a71561 was shown to attenuate porphyromonas gingivalis virulence through specific kgp inhibition (curtis et al. 2002) . based on histatin cleavage specificity, small peptide analogues were designed, which specifically inhibit rgp and kgp (kyt-1 and kyt-36, respectively), which display attenuation of several virulence traits of porphyromonas gingivalis (kadowaki et al. 2004) . chlorhexidine, which has been used in oral healthcare preparations on account of its antimicrobial effects, also inhibits proteolytic activities, including those of gingipains. moreover, chlorhexidine inhibitory activity against r-gingipains was enhanced by the addition of zn(ii), which has also been used in human oral health care (cronan et al. 2006) . metalloproteases are important virulence factors of many primary and opportunistic pathogenic bacteria and cause major infectious diseases such as cholera, salmonellosis, legionnaires' disease, cystic fibrosis, botulism, tetanus and anthrax (miyoshi and shinoda 2000) . they have either direct roles in host interaction or indirect roles in processing other important virulence factors. therefore, much has been invested in the search for an effective protease inhibitor for use in treatment (jacobsen et al. 2007 ), but none has yet been developed, which would be used in clinic. metal chelators, including edta (ethylenediaminetetraacetic acid), egta (ethylene glycol-bis(2-aminoethylether)-n, n,n′,n′-tetraacetic acid) and 1,10-phenanthroline, inhibit metalloproteases in general. the ubiquitous presence of metalloproteases prevents the use of broad-spectrum inhibitors, and the search for potent and specific inhibitors of individual metalloproteases that could find clinical applications is important. the most studied bacterial metalloproteases are those of the thermolysin family (m4), including mpi protease of listeria monocytogenes, coccolysin of enterococcus faecalis, hemagglutinin/proteinase of vibrio cholerae and helicobycter pylori, pseudolysin of pseudomonas aeruginosa, aureolysin of staphylococcus aureus, legionella pneumophila protease and λ-toxin of clostridium perfringens. inhibitors of thermolysin family proteases are of bacterial origin, including those isolated from streptomyces, the small-molecule inhibitor phosphoramidon and protein inhibitor smpi (streptomyces metalloproteinase inhibitor) of family i36. another family of inhibitors targeting bacterial thermolysins is family i8 of animal origin (adekoya and sylte 2009; supuran et al. 2002; rawlings and barrett 2011) . of the bacterial metalloproteases, the light chain domains that are zinc metalloproteases (family m27) of tetanus and botulinum neurotoxins (tent and bonts) from clostridium tetani and clostridium botulinum, respectively, have also been studied extensively. various β-aminothiols have been considered as selective bont and tent inhibitors and have been further developed into strong and selective pseudotripeptide inhibitors of bont/b (blommaert et al. 2004; supuran et al. 2002) . clostridium histolyticum collagenases and their homologues from vibrio (family m9) are very effective in connective tissue degradation and hydrolyse triple helical regions of collagen under physiological conditions. they are targeted for both therapy and diagnosis of clostridial infections, and several types of compounds have been found to inhibit them. however, in addition to bacterial collagenases, they also inhibit vertebrate collagenases (supuran et al. 2002; barla et al. 2009) . no potent and selective inhibitor has yet been found for metalloproteases of family m10 from pathogenic bacteria, including serralysin from serratia, pseudomonas and erwinia, aeruginolysin from pseudomonas aeruginosa, mirabilysin from proteus mirabilis and fragilysin from bacteroides fragilis. they are, however, inhibited by protein inhibitors of bacterial origin belonging to family i38 and hydroxamate inhibitors, including batimastat (supuran et al. 2002; rawlings and barrett 2011) . another group of medicinally important bacterial proteases for which specific inhibitors have not yet been described comprises the metalloexopeptidases, which belong to several merops families (m1, m2, m14, m15, m17, m18, m19, m20, m24, m28, m29, m32, m42, m54, m55 and m61). of the metallocarboxypeptidases (belonging to families m14, m15, m20, m32) the zinc-containing d-ala-d-ala dipeptidase vanx (family m15) has been studied in view of its ability to mediate antibiotic resistance against vancomycin (crowder 2006) . similarly, the family m19 of membrane dipeptidases includes members that degrade β-lactam antibiotics. other carboxypeptidases, such as glutamate carboxypeptidases (family m20), have been studied with a view to clinical use in treating different types of cancer (holz et al. 2003) . bacterial metalloaminopeptidases, which perform essential cellular functions in protein synthesis and maintenance, have been studied as targets for novel antibiotics. a few reviews on inhibitors designed to inhibit bacterial aminopeptidases of families m17 (e.g. leucyl aminopeptidases or laps), m1 (alanyl aminopeptidase) and m24 (methionine aminopeptidases) cover the natural and designed compounds that could serve as lead compounds for inhibitors aimed at this group of proteases (mucha et al. 2010; holz et al. 2003; supuran et al. 2002; rawlings and barrett 2011) . bacterial metalloproteases that cleave immunoglobulin a (iga proteinases, families m26 and m64) constitute important colonization factors for several pathogenic bacteria (e.g. streptococcus, neisseria, haemophilus, clostridium, prevotella, capnocytophaga, bacteroides). no strong and specific inhibitor is available for these enzymes. the same is true also for another family of pathogenesis-related metalloproteases (family m23), including staphylolysin from pseudomonas aeruginosa and lysostaphin from staphylococcus simulans. in addition to their function in increasing virulence, staphylolysin and lysostaphin show bactericidal activity against staphylococcus aureus and have been studied with a view to their use in countering drug-resistant staphylococci (e.g. methicillin-and vancomycin-resistant staphylococcus aureus) (barequet et al. 2009; desbois and coote 2011) . the metalloprotease anthrax lethal factor lf (family m34) is a component of the anthrax toxin responsible for the major symptoms and death associated with bacillus anthracis infection (kim and yoon 2006 ). an increased interest in anthrax vaccination and treatment methods has been provoked by the use of bacillus anthracis spores as a bioweapon. lethal factor (lf) inhibitors would provide two-fold protection, namely, in preventing early spore protection in macrophages and, later, inhibiting lf disruption of signalling pathways through inactivation of mitogen-activated protein (map) kinase kinases. the search for an lf inhibitor to use in combination with antibiotic treatment is aimed at finding a selective and potent lf inhibitor (shoop et al. 2005; turk 2008 ), which would be non-(cyto) toxic and have good biological stability and bioavailability (johnson et al. 2009; kim et al. 2011; li et al. 2011 ). the predominant fungal diseases afflict immunocompromised patients and are caused by opportunistic pathogens candida sp. (e.g. candida albicans, candida glabrata, candida parapsilosis) and aspergillus sp. (e.g. aspergillus fumigatus, aspergillus niger, aspergillus nidulans, aspergillus calidoustus), followed by other ascomycetes of genus fusarium, basidiomycete genera malassezia, cryptococcus and trichosporon, and zygomycete genera rhizopus and mucor (boekhout et al. 2009 ). the importance of proteases in the pathogenicity of these opportunists is controversial; however aspartic, serine and metalloendopeptidases, as well as aminopeptidases, carboxypeptidases and dipeptidylpeptidases that are secreted by these species, have been proposed as the virulence factors that facilitate colonization and invasion by hydrolysis of host proteins or damage cells and molecules of the host defence system (segal 2006; yike 2011). the most studied are the secreted aspartic proteinases of candida albicans (saps) (naglik et al. 2003) . interestingly, the protease inhibitors targeted against the viral aspartic protease used in treating hiv infection also inhibited candida saps and reduced occurrence of candidiasis in these patients. secreted aspartic proteases are thus an important target for the development of new protease inhibitor based compounds for treating candidiasis (braga-silva and santos 2011; naglik et al. 2004; dash et al. 2003) . fungal proteases are also important fungal allergens, and most belong to the serine catalytic type. addition of protease inhibitor during aspergillus fumigatus and aspergillus niger protease and antigen sensitization attenuated allergic inflammation and hyper-responsiveness in an animal model (yike 2011) . secreted alkaline serine protease of aspergillus fumigatus was shown to help evade the host immune response by degrading human complement proteins, and it is therefore a good target for drug development (behnsen et al. 2010) . a network of proteolytic enzymes is very important for the survival of dermatophytes such as microsporum canis and trichophyton rubrum, the specialized pathogenic fungi that infect stratum corneum, nails or hair of healthy individuals. it includes the metalloendopeptidases, fungalysins (family m36), serine endopeptidases, subtilisins (family s8a) and several exopeptidases (dipeptidyl peptidases (family s9), aminopeptidases (family m28) and carboxypeptidases (family s10)) (monod 2008) . although proteases are only one of the several groups of virulence factors of pathogenic fungi, they aid in the invasion of tissues and evasion of immune responses. therefore, specific protease inhibitors aimed at them would constitute a valuable addition to the presently used antifungal drugs that target fungal cell wall and cell membrane integrity or dna replication-and to which many pathogenic strains have acquired resistance (marie and white 2009) . the classspecific (broad-spectrum) aspartic protease inhibitor pepstatin has been shown to inhibit adhesion of candida albicans and prevent invasion or mucosal tissue damage by inhibiting the saps (naglik et al. 2004) . recently, it has been shown that ergosterol production transcriptional regulator (sre1), which is activated by a metalloprotease stp1, is essential for the survival of cryptococcus neoformans in the presence of antifungal drugs that inhibit sterol biosynthesis. therefore, regulators of the ergosterol pathway, including the metalloprotease stp1, constitute promising targets for novel antifungal therapeutics to be used in combination with a sterol synthesis inhibitor for treating cryptococcosis in immunocompromised individuals (bien et al. 2009 ). protozoan parasitic diseases, including malaria, leishmaniasis and african and american trypanosomiasis, are some of the most important infectious diseases in the world, with high mortality and morbidity rates in developing countries. reasons for their persistence, despite prolonged use of antiparasitic drugs, include their toxic side effects and the increasing emergence of drug resistance. therefore, research over the past 15 years has been focused on identifying new targets for antiparasite treatment and on developing substances suitable for human therapy. parasitic proteases constitute one of the very important druggable targets since they are key virulence factors due to their essential roles in cell metabolism and interaction with the host (zucca and savoia 2011; renslo and mckerrow 2006; mckerrow et al. 2008) . aspartic proteases plasmepsins (family a1), which are important for haemoglobin catabolism in parasites causing malaria (plasmodium falciparum, plasmodium vivax, plasmodium ovale, plasmodium malariae), have been targeted for the design of novel antimalarial drugs, i.e. selective protease inhibitors. although plasmepsin inhibitors (including the broad-spectrum pepstatin) have been shown to have antimalarial effects, the main challenge still remains to design an inhibitor that would be active against different plasmepsins i, ii and iv, and the histoaspartic protease hap involved in haemoglobin degradation, but also inactive against the homologous human aspartic proteases (cathepsins d and e) (zucca and savoia 2011; rosenthal 2003; ersmark et al. 2006; gil et al. 2011 ). in addition to plasmepsins, the cysteine proteases falcipains (family c1), metalloprotease falcilysin (family m16) and aminopeptidases have been targeted for development of new antimalarial protease inhibitors. a synergistic effect has been observed for their combined use (e.g. a cocktail of aspartic and cysteine protease inhibitor) (mckerrow et al. 2008; rosenthal 2003; zucca and savoia 2011; trenholme et al. 2010) . cysteine proteases similar to falcipain (family c1) have been associated with the pathogenesis of trypanosomiasescruzipain of trypanosoma cruzi (chagas disease) and brucipain or rhodesain of trypanosoma brucei (sleeping sickness). cruzipain (or cruzain) has been extensively studied and is considered to be a promising target for chemotherapy since it is critical for parasite viability in all stages of infection, especially for nutrition acquisition, tissue invasion and host immune response evasion (duschak and couto 2009 ). an irreversible inhibitor, k777, was effective in pre-clinical models of chagas disease; however, a safer treatment would be achieved by utilizing a reversible and highly specific cruzipain inhibitor (beaulieu et al. 2010; mckerrow et al. 2009; brak et al. 2010 ). cysteine proteases have been implicated in the pathogenesis of leishmania species and are major virulence factors as they substantially modify the immune response. a surface metalloprotease gp63 or leishmanolysin (family m8) has been shown to be essential for establishing and maintaining the infection and would make a promising target for development of a selective protease inhibitor for antiparasitic chemotherapy (olivier and hassani 2010; yao 2010) . in addition to the computational design, development and optimization of a suitable protease inhibitor, based on the 3d structure of the target protease, the search for novel types of inhibitors from natural sources (pereira et al. 2011) , such as fungi and microbes, is important for identifying new lead compounds. serine proteases are also a neglected group of potentially targetable protozoan proteases involved in their pathogenicity, although several subtilisins (family s8) have been described from plasmodium falciparum and toxoplasma gondii, and two oligopeptidases (family s9) from trypanosoma cruzi (mckerrow et al. 2008) . in addition to protozoan parasites, helminths, which include parasitic roundworms (nematodes) and flatworms (trematodes and cestodes), also cause important parasitic infections. cysteine cathepsins (family c1), which are important for many aspects of the helminth-host relationship, are a potential target for developing antihelminthic drugs. cysteine protease inhibitors have been shown to impair fecundity of the liver fluke (fasciola hepatica) and blood fluke (schistosoma mansoni) in animal models. however, the similarity of host cathepsins, together with their importance, calls for the design of a very specific and selective protease inhibitor. alternatively, drug design could exploit bioavailability and pharmaco-kinetic and -dynamic properties to target parasitic proteases preferentially (robinson et al. 2008 ). in addition to proteases, parasite-derived protease inhibitors play important roles in manipulating the host immune system and establishing a niche for the successful feeding and reproduction of helminth parasites. therefore, protease inhibitors are also important targets for immunological control of helminth parasitic infections and make proteases that are insensitive to parasite-derived protease inhibitors valuable candidates for new types of antihelminthic therapy (knox 2007; stepek et al. 2006) . the ability of tumour cells to invade extracellular barriers and to metastasize to distant sites is associated with the activity of proteases (kos and lah 1998) . the major molecular mechanism, which involves the active role of various intra-and extracellular proteases, is the dissolution and remodelling of connective tissue and the basement membrane. it includes matrix metalloproteases (mmp), serine proteinases such as urokinase, tissue types of plasminogen activator (upa, tpa) and plasmin, aspartic proteinase cathepsin d and cysteine proteinases cathepsins b, h, s and l (schmitt et al. 1992 ). in addition to extracellular matrix remodelling, proteases regulate several other processes, leading to the progression of malignant disease, such as cell adhesion, migration, differentiation, proliferation and signalling of tumour cells. it is well accepted that tumourassociated proteolytic activity escapes the control of endogenous protease inhibitors and that treatment of patients with exogenous protease inhibitors may suppress the progression of the disease and improve the outcome of cancer patients. however, the treatment should specifically target the tumour-associated proteases that cause harmful actions and not those involved in the numerous physiological processes in cells and tissues. several protease inhibitors failed in clinical trials due to lack of specificity, resulting in severe side effects and/or lack of clinical benefit in treated patients (turk 2006; coussens et al. 2002) . new approaches to developing protease inhibitors applicable in therapeutic interventions include structure-based medicinal chemistry and development of molecular systems to deliver inhibitors to the site of action. among the small-molecule inhibitors of bacterial and fungal origin, peptidyl aldehydes such as leupeptin and antipain, hexapeptide pepstatin and epoxysuccinyl peptide e-64 and their analogues have been studied as anticancer agents. the thiol-protease specific inhibitor, e-64, originally isolated from aspergillus japonicus (hanada et al. 1978) , has been studied extensively as a potential antitumour agent in cell culture and animal models. derivatives of e-64, displaying selectivity between different cysteine proteases (frlan and gobec 2006) , represent the next step towards their application in treating cancer and other diseases. they were designed on the basis of the x-ray crystal structures of individual cathepsins, and the most studied were cathepsin b specific inhibitors ca-074 and ca-030, cathepsin l specific inhibitors clik-148 and clik-195, and cathepsin x specific inhibitor ams-36. cathepsin s specific inhibitor clik-060 was designed on the basis of the structure of leupeptin and antipain (katunuma 2011) . antitumour activity was exhibited particularly by ca-074, a specific inhibitor of the cysteine protease cathepsin b (johansson et al. 2000) , which appears to be crucial for tumour cell invasion (lah et al. 2006) . in animal models, it was shown that ca-074 reduces tumour growth, invasion and angiogenesis of many cancer types, including pancreatic cancer, melanoma and breast tumours, all tested on animal models. the cell permeability of epoxysuccinyl inhibitors was improved by esterification. the esters are less active than free acids; however, in cells, they are rapidly hydrolysed to their active form. better cell permeability was demonstrated for ethyl ester e-64d and the methyl ester of ca-074, which are also highly soluble and effective for prolonged periods (frlan and gobec 2006) . metalloproteases are an important group of proteases that have been considered extensively as targets for cancer therapy due to the many roles they play in carcinogenesis and tumour invasion, growth and dispersion. however, the diversity of endogenous metalloproteases and their numerous and versatile physiological roles have prevented the use of broad-spectrum metalloprotease inhibitors. much effort is invested in determining which metalloproteases to target and in designing highly selective and potent protease inhibitors based on the structural characteristics of individual target metalloproteases (coussens et al. 2002; bialas and kafarski 2009; dorman et al. 2010; overall and kleifeld 2006) . several proteases of the serine catalytic type have also been targeted for the design of specific protease inhibitors for use in cancer treatment, including the urokinase plasminogen activator and matriptase (abbenante and fairlie 2005; bialas and kafarski 2009; ulisse et al. 2009 ). the broad-range microbial inhibitors of serine, cysteine and threonine proteases, leupeptin and antipain, were also shown to inhibit malignant transformation (vaccari et al. 1999) or tumourigenesis (hozumi et al. 1972) . threonine catalytic type proteolysis is present in proteasomes, and several chemical classes of natural and synthetic proteasome inhibitors have been considered as anticancer agents because of their preferential antiproliferative and proapoptotic activity on cancer cells (kisselev and goldberg 2001; abbenante and fairlie 2005; bialas and kafarski 2009; cecarini et al. 2011; chen et al. 2011) . the aspartic protease inhibitor pepstatin has also been frequently tested as an antitumour agent since cathepsin d was identified as an important tumour promoting factor (greenbaum and sutherland 1983; benes et al. 2008 ). irregular function of proteases is associated with a variety of other diseases, representing targets for therapeutic application of all catalytic types of protease inhibitors. smallmolecule inhibitors of bacterial and fungal origin, described already in the previous section, particularly epoxysuccinyl inhibitors, have been reported as potential protective agents in autoimmune, neurodegenerative, antiinflammatory and cardiovascular diseases; osteoporosis; muscular dystrophy; diabetes and others. ca-074 and clik-148 were demonstrated to cause a switch between th1 and th2 t cell response due to the different roles of cysteine cathepsins b and l in antigen processing and presentation (katunuma 1997) . cathepsin s inhibitor clik-060 has been shown to suppress sj gren syndrome, an autoimmune disease associated with the processing of α-foldin by cathepsin s (saegusa et al. 2002) . the cathepsin x inhibitor ams-36 reduces the activation of integrin receptor lfa-1 (lymphocyte functionassociated antigen 1), a molecule involved in t cell adhesion, proliferation and migration (jevnikar et al. 2008) . lfa-1 overexpression and activation by cathepsin x is typical of autoimmune diseases, particularly psoriasis. the same inhibitor significantly enhanced the proliferation of neuronal cells and neuritogenesis, preventing the processing of neurotrophic factor γ-enolase by cathepsin x (obermajer et al. 2009 ). epoxysuccinyl inhibitors have been tested in several animal models of neurodegenerative diseases. e-64 was shown to restore normal synaptic function in the app/ps1 mouse model of alzheimer's disease with overexpressed amyloid precursor protein (app) and presenilin 1 (ps1) (trinchese et al. 2008 ). e-64d and ca-074me also reduced the accumulation of neurotoxic beta-amyloid peptides in brains, presumably inhibiting cathepsin b involved in processing the amyloid precursor protein (hook et al. 2007 ). aspartic proteases β-secretase (also known as memapsin-2 or bace1 and belonging to merops family a1) and γ-secretase (composed of two presenilins belonging to merops family a22) are important therapeutic targets for treating alzheimer's disease, and several compounds designed to reduce their activity are in clinical trials (de strooper et al. 2010) . another important target of aspartic proteases is renin (family a1), which is part of the complex renin-angiotensin-aldosterone system that regulates blood pressure and electrolyte balance. several inhibitors have been designed based on the renin structure and activity, and one of them, aliskiren, is the first non-peptide, orally administered, direct renin inhibitor available on the market for management of hypertension (nguyen et al. 2008; barrios and escobar 2010) . the very first protease inhibitor used in humans as a therapeutic agent, namely, an inhibitor of metalloprotease angiotensin-converting enzyme (ace), was also hypertension related. it is an important regulator of the reninangiotensin system, and ace inhibitors are used for treating hypertension, but are also implicated in other cardiovascular and renal diseases (abbenante and fairlie 2005; ondetti et al. 1977) . the inhibitors of collagenolytic enzymes, such as cathepsins l and k, prevent bone remodelling and can be useful in treatment of osteoporosis. several synthetic cathepsin k inhibitors are in different stages of clinical trials. in addition to osteoporosis, they are also considered for application in arthritis, atherosclerosis, obesity and cancer (bromme and lecaille 2009) . administration of cathepsin l inhibitor clik-148 significantly reduced invasion and metastasis formation in bones (katunuma 2011) . e64d, a methyl ester of e64, was tested for treating muscular dystrophy; however, its further development was stopped in phase iii due to insufficient effectiveness and hepatic injury in rats (fukushima et al. 1990 ). antipain, leupeptin and pepstatin have also been tested for treatment of muscular dystrophy; however, persuasive benefit was not demonstrated in animal models. specific inhibitors designed to target the serine aminopeptidase dipeptidyl peptidase iv are in clinical trials for management of type 2 diabetes (abbenante and fairlie 2005; tahrani et al. 2011) . endogenous plant protease inhibitors constitute one of the plant defence strategies against pathogenic, parasitic and herbivorous organisms. they target the important proteolytic virulence factors of phytopathogenic bacteria, fungi, parasites and viruses, preventing their roles in nutrient acquisition and evasion of host defence. furthermore, they target digestive proteases of herbivorous pests (e.g. insects, mites, slugs), preventing the utilization of food-derived organic nitrogen for their growth and development (ryan 1990; haq et al. 2004) . since pests and pathogens depend on utilization of these proteases, there is a strong selection pressure operating to develop resistance to plant endogenous defensive protease inhibitors (haq et al. 2004; jongsma and beekwilder 2008) . therefore, the search for novel protease inhibitors with potential protective function is very important for the development of environmentally friendly pest and pathogen management strategies. in addition to investigations aimed at augmenting crop endogenous resistance by conventional breeding, there are several protease inhibitors of plant origin that have been used in the preparation of genetically modified crop plants with superior ability for biotic stress resistance (ferry and gatehouse 2010) . the use of protease inhibitors for insect pest management has gained most attention. insect pests cause major economic losses annually, of which lepidopteran (butterflies' and moths') larvae are considered the most destructive. agricultural pests causing significant economic impact also belong to orders coleoptera (beetles), diptera (true flies), hemiptera (e.g. aphids), orthoptera (e.g. locust) and thysanoptera (thrips). they cause either direct damage to crops by feeding or indirect damage by transmitting viral diseases or secondary microbial infections. the most notable for their destructive capacity are the migratory locust (locusta migratoria), several beetles, including colorado potato beetle (leptinotarsa decemlineata), boll weevil (anthonomus grandis), japanese beetle (popillia japonica), the western corn rootworm (diabrotica virgifera virgifera) and many species of aphids belonging to all families of the superfamily aphidoidea. different catalytic types of proteases provide the predominant proteolytic activity in different groups of insect pests. while serine proteases are predominant in digestive proteolysis in most insect species (e.g. lepidoptera, diptera), cysteine proteases predominate in hemiptera, coleoptera and thysanoptera. in addition, aspartic and metalloproteases complement protein digestion to different degrees in most insect orders (terra and ferreira 1994) . therefore, protease inhibitors targeting different groups of proteases have shown variable antinutritional effects when fed to different insect pests. the catalytic, classspecific, small-molecule protease inhibitors of microbial origin have often been used for proof-of-principle feeding experiments, but protein protease inhibitors were then employed to generate insect-resistant transgenic plants. these were predominantly of plant origin, with a few exceptions of animal-derived protease inhibitor genes (haq et al. 2004; dunaevsky et al. 2005; malone et al. 2008) . other important proteins that have been used for constructing pest-resistant transgenic crops are insecticidal proteins derived from bacillus thuringiensis (bt), and genetically modified maize and cotton varieties that express bt toxins have become an important component in agriculture worldwide and have reduced the use of pesticides and lowered production costs (ferry and gatehouse 2010; kumar et al. 2008) . the involvement of endogenous protease inhibitors in natural plant resistance against herbivores is probably the basis of the adaptation of lepidopteran and coleopteran species to ingestion of protease inhibitors that has been observed for several species (jongsma and beekwilder 2008; wu et al. 1997; bonade-bottino et al. 1999; lara et al. 2000) . they circumvent the antinutritional effect of the ingested protease inhibitors either by overexpression of native gut proteases or by production of insensitive proteases; some of which can degrade the ingested protease inhibitors (ferry and gatehouse 2010; jongsma and beekwilder 2008) . therefore, the pyramiding or stacking of different families of inhibitors to increase the spectrum of inhibitory activity has been shown to have synergistic effects, as well as combining protease inhibitor genes with genes of other insecticidal proteins, namely, lectins, bt or other bacterial toxins and α-amylase inhibitors (ferry and gatehouse 2010; schluter et al. 2010; christou et al. 2006; malone et al. 2008) . furthermore, the use of protease inhibitors of microbial and fungal origin could offer superior characteristics, such as stability, resistance to proteolytic degradation and diverse inhibitory patterns, for a more potent antinutritional or insecticidal effect. only a few examples of utilization of microbial small-molecule inhibitors as antinutritional agents are available, e.g. aminopeptidase inhibitors of actinomycetes amastatin and bestatin against the red flour beetle (tribolium castaneum) (oppert et al. 2011) , aspartic protease inhibitor pepstatin a from actinomycetes against the cowpea bruchid (callosobruchus maculatus) (amirhusin et al. 2007) , the serine and cysteine protease inhibitor leupeptin from actinomycetes against western corn rootworm (diabrotica virgifera) (kim and mullin 2003) and cysteine protease inhibitor e-64 from aspergillus japonicus against colorado potato beetle (leptinotarsa decemlineata) (bolter and latoszekgreen 1997) . the use of protein protease inhibitors is advantageous since transgenic plants expressing a pest resistance gene in a controlled manner represent a stable and cheap propagation source that would lower the amount of pesticides needed, making plant protection environment friendly. to our knowledge, the only examples of a protein protease inhibitor of microbial or fungal origin as an effective antinutritional agent are the cysteine protease inhibitors macrocypins (family i85) from the edible parasol mushroom (macrolepiota procera), which have been shown to be detrimental to the growth and development of colorado potato beetle larvae (istinič et al. 2011) . the high capacity for development of resistance to insecticidal proteins in major insect pests drives the search for effective protease inhibitors. novel protein protease inhibitors aimed at serine proteases would be useful for management of major agricultural pests such as beet armyworm (spodoptera exigua), cotton bollworm (helicoverpa armigera and helicoverpa zea) and tobacco hornworm (manduca sexta). a combination of serine and cysteine protease inhibitors would be useful against, e.g. boll weevil (anthonomus grandis), cowpea weevil (callosobruchus maculatus) and red flour beetle (tribolium castaneum), and cysteine protease inhibitors would, for example, be useful against the colorado potato beetle (leptinotarsa decemlineata), western corn rootworm (diabrotica virgifera), banana weevil (cosmopolites sordidus) and pea aphid (acyrtosiphon pisum). microorganisms that are generally recognized as safe and edible mushrooms offer a valuable source of such novel protease inhibitors that would also be acceptable for use in crops for human consumption. in addition to insect pests, other herbivores causing significant crop losses worldwide include mites and slugs. in both cases, cysteine proteases constitute the predominant digestive proteolytic activity. plant cystatins have been shown to be detrimental to mite development and reproductive performance in feeding trials on one of the major mite pests on agricultural crops, the two-spotted spider mite tetranychus urticae (acari: tetranychidae) (carrillo et al. 2011) . similarly, the growth of juvenile slugs deroceras reticulatum, the important agricultural and horticultural pest, was significantly reduced when fed with leaf tissue overexpressing a plant cysteine protease inhibitor (walker et al. 1999) . therefore, protease inhibitors of microbial and fungal origin have great potential for protecting plants from important mite pests and suppressing the growth rates of slug populations, in addition to their antinutritional characteristics for different insect pests. another advantage of the application of protease inhibitors in crop protection is that in addition to protection against herbivorous pests, they offer cumulative protection against nematodes and bacterial, fungal and viral pathogens (haq et al. 2004 ). the use of protease inhibitors is one of many strategies to protect plants from parasitic nematodes. their targets are intestinal proteases, mostly of the cysteine catalytic class. therefore, heterologously expressed plant cysteine protease inhibitorsphytocystatins (family i25)-have been effective to different degrees against beet-cyst nematode (heterodera schachtii), root-knot nematode (meloidogyne incognita), potato cyst nematode (globodera pallida) and burrowing nematode (radopholus similis) (mccarter 2009; haq et al. 2004) . a plant-derived serine protease inhibitor offered satisfactory nematode resistance in transgenic wheat against the cereal cyst nematode (heterodera avenae) (vishnudasan et al. 2005; mccarter 2009 ). the antifungal effect of serine protease inhibitors has been determined with plant-derived protease inhibitors that target secreted proteases (mainly families s1 and s8) of phytopathogenic fungi involved in plant cell wall penetration by hyphae (wong et al. 2010 ). in addition, aspartic and cysteine proteases that are also fairly widespread (valueva and mosolov 2004) represent potential targets for protective protease inhibitors. similarly, phytopathogenic bacteria secrete proteases involved in pathogenesis, mainly aiding plant cell wall degradation. together with pectinases, cellulases and hemicellulases, they contribute to massive degeneration of plant tissue, for example, in wilt (e.g. ralstonia solanacearum) and soft-rot diseases (e.g. erwinia chrysanthemi, erwinia carotovora) (kunkel and chen 2006) . cysteine proteases belonging to families c48, c55, c58, c70 and c72, which have been implicated in the pathogenicity of many phytopathogens belonging to genera pseudomonas, xantomonas, ralstonia, erwinia and pantoea as effectors of the type iii secretion system, constitute potential targets for protective protease inhibitors (kunkel and chen 2006; mosolov and valueva 2006) . the importance of polyprotein processing in replication of viruses, especially those of families potyviridae and comoviridae, indicates that protease inhibitors could mediate resistance to plant viruses by inhibiting target viral proteases. indeed, a rice cystatin (oryzacystatin) expressed in tobacco plants conferred resistance to potato virus y (pvy) and to tobacco etch virus (tev) which correlated to increased inhibition of the target papain-like protease sudarshana et al. 2007 ). in addition to their protective role against biotic stress, endogenous plant protease inhibitors have been implicated in the control of proteolytic systems in plants under abiotic stresses with a dehydration component such as drought, increased salt concentration and freezing (vaseva et al. 2012) . it has been shown that the changes in metabolism triggered by water deficit involve active involvement of regulated proteolysis that assists the protective proteins (dehydrins and chaperones) in the cellular response to the increased levels of denatured, aggregated or oxidatively damaged proteins that accumulate during dehydration stress (brzin and kidrič 1995; benchabane et al. 2010; bray 1993; hoekstra et al. 2001; feller 2004) . overexpression of endogenous protease inhibitors increased resistance to drought stress, as shown for overexpression of the cysteine protease inhibitors cystatins atcys1 and atcys2 in the model plant arabidopsis thaliana ) and for overexpression of a serine protease inhibitor ocpi1 (oryza sativa chymotrypsin inhibitor 1) in rice, with the latter showing improved drought resistance in terms of yield loss in the field (huang et al. 2007) . although protease inhibitors have great potential in protecting crops under abiotic stress conditions, detailed knowledge of the specific roles of different proteases in response to abiotic stress is needed before exogenous protease inhibitors can be used to manipulate and improve drought resistance in plants (vaseva et al. 2012) . small-molecule protease inhibitors are routinely used as buffer additives when preparing protein extracts, in order to prevent proteolytic degradation during protein purification procedures. broad-spectrum inhibitors that cover all the different catalytic classes are generally used, including pepstatin a for aspartic proteases, e-64 for cysteine proteases, chymostatin for serine proteases, antipain for cysteine and serine proteases, and leupeptin for cysteine, serine and threonine proteases; all of which were originally isolated from actinomycetes. for inhibition of serine proteases, synthetic protease inhibitors such as aebsf (pefabloc; 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride), the physiologically more stable derivative of pmsf (phenylmethanesulfonyl fluoride), are used. chelating agents such as edta and 1,10-phenanthroline are generally used to inhibit metalloproteases and, for certain applications, inhibitors of actinomycete origin-phosphoramidon (m4 and m13 metallopeptidases inhibited) or bestatin (aminopeptidases inhibited). unwanted proteolytic degradation can cause severe reduction in yield of heterologously expressed proteins in various expression systems. therefore, natural or engineered protease-deficient strains are often used as hosts for bacterial (e.g. escherichia coli bl21), yeast (e.g. pichia pastoris smd 1163, 1165 or 1168) and filamentous fungal (e.g. aspergillus niger prt pep) expression systems. however, these strains may not exhibit optimal bioprocessing characteristics due to lower fitness traits. alternative strategies are therefore used for preventing proteolytic degradation of recombinant proteins, including modification of the recombinant protein sequence to remove protease cleavage sites without affecting protein function, expression with a stabilizing fusion partner, optimization of cultivation conditions (ph, temperature, medium composition, bioprocess strategy) and use of protease inhibitors. for secreted recombinant proteins, small-molecule inhibitors can be added to the culture medium to inhibit the predominant secreted proteolytic activity of the host organism that is often of the serine and aspartic catalytic type. another strategy is coexpression of an appropriate protein protease inhibitor with the recombinant protein, which may, however, influence the yield or complicate the downstream purification procedures (potvin et al. 2010; sharma et al. 2009; rozkov and enfors 2004) . in addition to protection against proteolytic degradation of recombinant protein during the expression process, protease inhibitors as fusion partners have other advantages. one example is the use of the bacterial periplasmic serine protease inhibitor ecotin (family i11) as a vehicle for secretion to the periplasmic space of escherichia coli (paal et al. 2009 ). another example is the serine protease inhibitor from oyster mushroom, the pleurotus ostreatus proteinase a inhibitor 1 (poia1) (family i9) that serves as an intramolecular chaperone enabling proper refolding of the fused subtilisin protease from inclusion bodies expressed in a heterologous bacterial expression system (kojima et al. 2005) . the strategy of using co-expression of protein protease inhibitors for reducing proteolytic degradation of heterologously expressed proteins has been successfully implemented in plant expression systems. preparation of a protease-deficient host is, in this case, not applicable because of the essential roles of proteases in growth and development. plant expression systems offer many advantages over bacterial and yeast expression systems (e.g. posttranslational modification capability) and over animal cell lines (e.g. lower cost and contamination risks) for largescale recombinant protein production, but have not yet been commercialized due to low levels of protein expression and of heterologous protein accumulation. the latter can be influenced by targeting the expression to specific organelles (e.g. endoplasmic reticulum) or tissues (e.g. seeds and tubers) or by co-expression with stabilizing fusion partners or protease inhibitors. co-expression of protective protease inhibitors does not have any adverse effects on plant growth and development, as described previously for examples of protease inhibitor expressing, insect-resistant transgenic plants. furthermore, the heterologously expressed protease inhibitors offer the added advantage of protection against proteolysis also ex planta, especially in the early steps of purification of crude protein extracts, thus minimizing the need for addition of protease inhibitors to the extraction buffers (rivard et al. 2006; desai et al. 2010; doran 2006; benchabane et al. 2008) . so far, only protease inhibitors of plant and animal origin have been used as co-expression partners; use of microbial or fungal protease inhibitors could offer superior protective characteristics. however, the properties of the protein of interest and of the selected host plant species influence the selection of an appropriate protective protease inhibitor, and the choice still has to be made on a case-by-case basis. another important biotechnological application of protease inhibitors is in protein purification, where they can be used as ligands in affinity chromatography. affinity chromatography is a simple one-step purification method of a molecule from a complex mixture based on specific and high affinity binding to a ligand immobilized on a solid support. reversible protease inhibitors of microbial origin are excellent ligands for purification of their cognate proteases by affinity chromatography. depending on the target protease, inhibitors with broad range or very specific inhibitory spectrum can be selected for a ligand. attention must be paid to the strength of the inhibitor as purification is not possible when the binding is too weak (no binding) or too strong (ineffective elution) (ge 2007) . the advantage of using microbial and fungal protease inhibitors is that many of them display unique inhibitory profiles and resistance to proteolytic cleavage, as well as high thermal and broad ph range stability, with the latter being very convenient since harsh conditions may be used for immobilization to the matrix as well as for the several cycles of elution steps, usually involving extreme change in ph and/or ionic strength. the broad-range inhibitor of pepsin-like aspartic proteases, pepstatin a, has been used for purification of aspartic proteases from several different sources, including higher fungi (sabotič et al. 2009a) , plants (payie et al. 2000) and insect recombinant enzyme expressed in a bacterial expression system (volkov et al. 2004) . several different types of inhibitor have been used for purification of serine proteases, including the synthetic inhibitor benzamidine and plant-and animal-derived protease inhibitors of different families (polanowski et al. 2003) . differences in the inhibitory spectra of immobilized protease inhibitors can be used in serial affinity chromatography, where, first, a broad-range protease inhibitor can be used to purify proteases from a crude protein extract, followed by the use of a highly specific inhibitor for isolation of a single protease. the microorganisms of prokaryotic domains archaea and bacteria and of the kingdom of fungi, including higher fungi or mushrooms, constitute important sources of protease inhibitors. microbial protease inhibitors are versatile in their structures and mechanisms of inhibition in ways that differ from those of other sources. they have therefore found countless applications in the fields of medicine, agriculture and biotechnology. the diversity of processes in which proteases are key players, together with their multiplicity, drives the search for further novel protease inhibitors that could find applications, directly or as leads in 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discovery of dengue protease inhibitors bacterial proteinases as targets for the development of second-generation antibiotics aminopeptidases of malaria parasites: new targets for chemotherapy inhibition of calpains improves memory and synaptic transmission in a mouse model of alzheimer disease purification and characterization of streptomyces griseus metalloendopeptidases i and ii targeting proteases: successes, failures and future prospects discovery and development of anthrax lethal factor metalloproteinase inhibitors the urokinase plasminogen activator system: a target for anti-cancer therapy pepstatin, a new pepsin inhibitor produced by actinomycetes effects of the protease inhibitor antipain on cell malignant transformation thrombin-activatable fibrinolysis inhibitor is degraded by salmonella enterica and yersinia pestis role of inhibitors of proteolytic enzymes in plant defense against phytopathogenic microorganisms plant proteases: from phenotypes to molecular mechanisms the cladosporium fulvum virulence protein avr2 inhibits host proteases required for basal defense the response of plants to drought stress-the role of dehydrins, chaperones, proteases and protease inhibitors in maintaining cellular protein function molecular cloning, over expression, and activity studies of a peptidic hiv-1 protease inhibitor: designed synthetic gene to functional recombinant peptide enzymatic characterization of the streptococcal endopeptidase, ides, reveals that it is a cysteine protease with strict specificity for igg cleavage due to exosite binding assessment of nematode resistance in wheat transgenic plants expressing potato proteinase inhibitor (pin2) gene new aspartic proteinase of ulysses retrotransposon from drosophila virilis transgenic arabidopsis leaf tissue expressing a modified oryzacystatin shows resistance to the field slug deroceras reticulatum (muller) picornaviral 3c protease inhibitors and the dual 3c protease/coronaviral 3c-like protease inhibitors regulation of bacterial protease activity proteins with antifungal properties and other medicinal applications from plants and mushrooms adaptation of helicoverpa armigera (lepidoptera: noctuidae) to a proteinase inhibitor expressed in transgenic tobacco novel inhibitor for prolyl tripeptidyl aminopeptidase from porphyromonas gingivalis and details of substrate-recognition mechanism major surface protease of trypanosomatids: one size fits all? fungal proteases and their pathophysiological effects proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins two cysteine proteinase inhibitors from arabidopsis thaliana, atcysa and atcysb, increasing the salt, drought, oxidation and cold tolerance current developments in the therapy of protozoan infections acknowledgements we are grateful to dr. jože brzin for numerous discussions and suggestions for the improvement of the review and to professor roger h. pain for critical reading of the manuscript and language editing. we would like to thank dr. miha renko for the assistance with fig. 1. key: cord-310947-aqau2n7q authors: pan, ji'an; peng, xiaoxue; gao, yajing; li, zhilin; lu, xiaolu; chen, yingzhao; ishaq, musarat; liu, dan; dediego, marta l.; enjuanes, luis; guo, deyin title: genome-wide analysis of protein-protein interactions and involvement of viral proteins in sars-cov replication date: 2008-10-01 journal: plos one doi: 10.1371/journal.pone.0003299 sha: doc_id: 310947 cord_uid: aqau2n7q analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. in this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of sars coronavirus (sars-cov) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. three pairs of the interactions identified were detected in both directions: non-structural protein (nsp) 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. the interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of nidovirales with large rna genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. using a sars-cov replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (n, x and sud domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins. interactions between viral proteins play pivotal roles in many processes during the viral infection cycle. this is the case in the formation of virus replication complexes, coordinated functions between different viral proteins, assembly of virions, and counterdefense of host immune responses. analysis of protein-protein interactions is essential to understand protein functions and the molecular mechanisms underlying biological processes. as the viral genomes are of limited sizes, they are particularly well suited for genome-wide analysis of all possible protein-protein interactions. however, the viral protein interaction maps have been generated until now only for a limited number of viruses, including t7 bacteriophage [1] , vaccinia virus [2] , potato virus a [3] , pea seed-borne mosaic virus [3] , wheat steak mosaic virus [4] , hepatitis c virus [5, 6] , porcine teschovirus [7] , kaposi sarcoma-associated herpesvirus [8] , and very recently severe acute respiratory syndrome coronavirus (sars-cov) [9, 10] . although a large variety of methods have been developed to detect protein-protein interactions, only a few of them are suited for largescale and high throughput protein interaction analysis. until now, all the genome-wide analysis of protein interaction networks for viruses and cells have been carried out mainly with the yeast two-hybrid systems, in combination with glutathione-s-transferase (gst) pulldown assays to verify major interactions [8, 9] . considering that protein modifications that are likely to influence protein interactions may be different for certain proteins in the context of yeast and mammalian cells, the mammalian two-hybrid system may better reflect genuine protein interactions for human viruses. accordingly, we adopted the mammalian two-hybrid system for detecting genomewide protein-protein interactions of sars-cov. the coronaviruses are classified into the family coronaviridae in the order nidovirales and possess the largest rna genomes known. the genome of sars-cov contains a single-stranded, plus-sense rna of approximately 29.7 kb in length. fourteen open reading frames (orfs) have been identified, of which 12 are located in the 39 end of the genome [11, 12] . the two large orfs (1a and 1b) in the 59proximal two-third of the genome encode the viral replicase and are translated directly from the genomic rna, while orf 1b is expressed by 21 ribosomal frameshifting. the large polypeptides encoded by 1a and 1b are considered to be cleaved into 16 functional replicase proteins by two proteinases, a papain-like proteinase 2 encoded by nsp3 and a 3c-like proteinase (or main proteinase) encoded by nsp5 [12] . a number of functions or characteristics have been identified for the 16 non-structural proteins (nsps). nsp1 was proved to be able to suppress host gene expression by promoting host mrna degradation and was involved in cellular chemokine deregulation [13, 14] . nsp2 seems not to play a crucial role in the generation of infectious viruses in cell culture [15] . nsp3 is involved in many activities including papain-like proteinase activity, deubiquitinating activity, and adp-ribose-10-phosphatase activity [16] [17] [18] 60] , which are essential for viral replication and transcription. nsp5 encodes a 3c-like proteinase which is considered to be an important target for antiviral drug design [19] . coronavirus nsp4 and nsp6 are transmembrane proteins that could anchor the replication complexes to double membrane vesicles [20] . based on the structural analysis, hexadecamer of nsp7 and nsp8 may possess dsrna-binding activity [21] . nsp8 was shown to have rna-dependent rna polymerase (rdrp) activity that could be involved in producing primers utilized by nsp12 which is normally accepted to be the rdrp for sars-cov [22, 23] . nsp9 is a single-stranded rnabinding protein [24] . though the structure of nsp10 is resolved, its function is still poorly understood, except that of nsp10 of mhv, homologous to that of sars-cov, which regulates viral rna synthesis [25] [26] [27] . nsp13 is shown to be rna helicase and 59-triphosphatase that may play a crucial role in the viral rna capping [28, 29] . nsp14 of coronaviruses possesses a 39-.59 exoribonuclease activity which may be involved in the proof-reading ability during the viral rna replication and transcription [30] [31] [32] . besides the exoribonuclease activity, sars-cov also possesses the endoribonuclease activity that is rendered by nsp15 [33] . according to the bioinformatic prediction, nsp16 and sud domain of nsp3 of sars-cov may function as 29-o methyltransferase and guanine n7 methyltransferase, respectively [34, 35] . very recently, the 29-o methyltransferase activity was confirmed experimentally for nsp16 of feline coronavirus [36] . nsp14, 15 and 16 were shown to be essential for efficient replication of coronavirus [30, 37, 38] . the 39 one-third of genome encodes 12 viral proteins including the structural and accessory proteins, which are translated from about 10 subgenomic rnas [39, 40] . proteins s, e, m, and n are four well described structural proteins, and 3a, 6, 7a, and 7b were also reported to be virion-associated proteins or viral structural proteins [41] [42] [43] [44] . multiple functions and activities have been identified for the structural and accessory proteins, including apoptosis induction, interference with the innate immunity response, and regulating the cellular protein expression [45] . however, these proteins are not essential for viral replication and transcription at least in cell culture and tested animal models, except nucleocapsid (n) protein [46] [47] [48] [49] . previous studies on sars-cov focused mostly on the molecular characterization and functional analysis of individual proteins encoded by sars-cov and limited information is available on viral protein-protein networks of coronaviruses [50] . to provide more insights into the functions of individual proteins, we analyzed interactions between all sars-cov-encoded proteins. by using mammalian two-hybrid assays, 40 different interactions between viral proteins have been identified, and six novel interactions could be confirmed in vitro by biochemical assays. moreover, a sensitive replication and transcription reporter system of sars-cov was established in this study, and, based on this system, we examined the impacts of all the individual viral proteins on the viral replication and transcription and found that the n protein played an important role at the early stage of sars-cov genome replication. in this study, a mammalian two-hybrid system was adopted to analyze the protein-protein interactions of sars-cov as it was assumed that the viral proteins expressed in mammalian cells were prone to be in their native conformations and therefore the interactions detected were more likely to be biologically relevant in comparison with other in vitro biochemical methods and assays performed in yeast cells [51] . for analysis of genome-wide protein interactions of sars-cov, all known orfs were amplified by pcr from viral cdnas of sars-cov isolate whu [39] and cloned into pgem-t vector ( table 1 ). the large polyprotein encoded by orf 1a/b was split into 18 domains according to the proteinase cleavage sites, except for nsp3 which was divided into 3 parts based on predicted functional domains. for structural and accessory proteins, the intact orfs including start and stop codons were amplified, except for s protein which was divided into s1 and s2 as these domains were proved to be two separate domains with distinct functions [52] . all the primers used for amplification were designed by inserting appropriate restriction sites which could be used for subcloning all the fragments from pgem-t cloning vectors into mammalian two-hybrid vectors pm (bait) and pvp16 (prey) and other protein expression vectors (see below) in correct reading frames. in total, 1024 interaction combinations between all sars-cov proteins were examined in a pairwise matrix. as a result, 40 different interactions were detected using the mammalian twohybrid assays (fig. 1a) . all the interactions shown ( fig. 1) were most likely specific for the viral protein domain of the fusion proteins as the activities of the reporter genes were reduced to background levels when one interacting partner in any of the combinations was replaced with non-relevant fusion protein (negative control) (data not shown). to further show the specificity of the interactions (fig. 1) , a quantitative assay for a typical positive interaction exemplified by nsp10-nsp14 and various controls were shown (fig. 1b) . to test the specificity of individual interactions, competitive assays were performed by co-expression of viral proteins using a different vector within the cells transformed with the bait and prey constructs. the co-expressed partner proteins interfered with the nsp10-nsp14 interaction, leading to reduced reporter activity (fig. 1c) , indicating that the same proteins with different fusion domains competed with each other and impaired the specific interactions needed for activation of the reporter genes. most interactions showed directionality, indicating the influence of fusion domains on the interacting sites. nevertheless, three pairs of interactions were detected in both directions: nsp10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. nsp11 is a small polypeptide containing only 13 amino acids and no interaction was detected with it in various assays but nsp11 in the fusion with nsp10 (nsp10/ 11) could significantly enhance the binding capability of nsp10 with either nsp14 or nsp16, indicating that the small nsp11 may also play important roles in viral protein interactions and replication. self-interactions of nsp3, nsp5, nsp8, nsp15 and n were observed, suggesting that these proteins could form dimeric or multimeric complexes by interacting with themselves. furthermore, 8 interactions between non-structural proteins and accessory proteins (3b, 7b, 8b, 9b) and one interaction between nonstructural protein nsp16 and structural protein n were detected, indicating that some of the accessory proteins might be involved in viral replication and transcription processes. nevertheless, that being the case, these interactions could have a minor effect on modulating virus replication as the virus-specific proteins are not strictly essential for viral replication at least in cell culture and mouse models [48, 49] . to confirm the interactions which were newly detected by mammalian two-hybrid assays, pull-down assays were performed. proteins that were related to the interactions not reported previously were expressed in different bacterial systems including pet30a (his-tagged), pgex-6p-1 (gst fusion) and pmal-c2x (mbp fusion). using pull-down assays that were described in materials and methods section, six protein-protein interactions were confirmed, including those between: nsp10 and nsp14, nsp10 and nsp16, nsp13 and 3b, nsp8 and 3b, nsp16 and n, and 8b and n (fig. 2 ). due to difficulties in bacteria expression, the interactions involving nsp12, nsp3.2 and nsp3.3 could not be confirmed. although a large number of protein-protein interactions were detected for sars-cov in virtue of the large-scale screening analysis in mammalian two-hybrid system, the roles of these interactions in the viral replication and transcription were still not clarified. to obtain more clues to the general roles of individual proteins, we constructed a sars-cov replicon (rep-scv-luc/ neo) that expresses the firefly luciferase gene, a sensitive reporter, [19, 68, 69] , nsp5-nsp7 and nsp8 [9] , nsp7-nsp7 [21, 70] , nsp7-nsp8 [21] , nsp7-nsp9 [9] , nsp8-9b [9] , nsp10-nsp14 and nsp10-nsp16 [10] , nsp15-nsp15 [54, 56, 71, 72] , nsp7-e and 7a-m [73] , n-n [55, 57] and n-n [55, 57] . (b) a typical result for a positive interaction with the example of nsp10-nsp14. the combination of pm-53 and pvp16-t represents a positive control. (c) a typical interaction inhibition assay performed to confirm that the interaction was not resulted from self-activation. error bars represent standard deviations from three independent experiments. doi:10.1371/journal.pone.0003299.g001 under the control of m gene transcription regulatory sequence (trs) as described in the materials and methods section (fig. 3a) . the effects of individual proteins or protein domains provided in trans on the replication/transcription of the sars-cov replicon were evaluated. any effect of the protein provided in trans on the luciferase expression levels could in principle be due either to changes in the extent of the replication, the transcription, or a combination of both. to evaluate the reporter gene expression of the replicon generated in this work, the bac plasmid encoding rep-neo/luc (pbac-rep-scv-neo/luc) was transfected into bhk21 cells. a significant increase in luciferase activity, in comparison with that of the parental replicon plasmid pbac-sars-cov-rep was observed ( fig. 3b ). as the luciferase gene is located in the 39-end of the genome and under the control of a viral trs, it can only be expressed from subgenomic rnas but not from the viral genome rna. therefore, expression of luciferase was expected to reflect the genome replication and transcription of sars-cov replicon. to further clarify whether the luciferase expression of rep-scv-luc/ neo derived from the viral replication and transcription processes but not from nuclear splicing products of the large viral replicon rna during the cmv promoter-driven transcription in nucleus, the positive and negative subgenomic rnas were examined according to the strategy described in our previous work [39] and the existence of correct subgenomic rnas was confirmed (data no shown). moreover, when the gene segment between two mlui restriction sites in viral genome, which encodes nsp4-nsp11 and part of nsp12, was deleted, the construct pbac-rep-scv-dmluluc/neo produced much lower levels of luciferase expression although it still retained detectable level of luciferase activity (fig. 3b) . to analyze the luciferase expression from the mlui deletion mutant, the identity of the luciferase mrna was sequenced. the results showed that a low level of luciferase mrna was generated either by cryptic promoters or by splicing of cryptic introns within the replicon transcripts in nucleus but not in the mrnas derived from the cytoplasmic replication and transcription of the replicon (data not shown). taken together, these results showed that the reporter-containing sars-cov replicon could be used as a model system for analyzing the functions of viral proteins. to study the effect of sars-cov proteins on replication and transcription, individual viral proteins were co-expressed in trans with the sars-cov replicon. renilla luciferase was employed as an internal control reporter to normalize the transfections among different wells (fig. 4) . though most of the viral proteins did not exert significant impact on the replication and transcription of the replicon, the nsp3.1 containing x and sud domains, and the rna polymerase nsp12 (fig. 4a) , and nucleocapsid n (fig. 4b ) significantly increased the luciferase activity, whereas nsp3.2 containing the papain-like (pl) proteinase domain reduced the viral replication and transcription. other proteins, such as nsp10/11, also showed a minor but significant increased expression. these results confirmed that the function of n gene fragment was not resulted from protein 9b that is internally nested in n sequence, as 9b showed no obvious impact on the replication and transcription of replicon when it was co-expressed (fig. 4b ). in addition, these results indicate that nucleocapsid protein, nsp3 and rna polymerase may possess a dominant function in trans, while the pl proteinase is involved in important cis functions like cisprocessing of the large viral polyprotein. trans-activation activity of n protein at the early stage of genome replication or transcription of sars-cov to make further analysis on the enhancement effect of n protein on the viral replication and transcription, we examined the dynamic changes of the luciferase activity expressed from the reporter replicon rep-scv-luc/neo. when the replicon plasmid pbac-rep-scv-luc/neo was transfected alone, the luciferase activity could be detected 13 h post transfection (more than 10 times compared with background levels) while the highest value of activity was reached around 32 h post transfection (fig. 5a) . the luciferase activity became stable 72 h post transfection and the activity level was as low as that of 14 h post transfection (fig. 5a) . to study the role of n protein provided in trans, we next transfected cells with equal amount of the reporter replicon together with either n protein expression construct (pcdna3.0-n) or the empty vector pcdna3.0. luciferase activity became detectable at 7 h post transfection when n protein was provided in trans (fig. 5b) , which was 9 h earlier than similar transfection without additional n protein (fig. 5b) . it was also observed that the luciferase activity was significantly enhanced in the first 40 h post transfection (fig. 5b) . the ratios of luciferase activities with and without co-expression of n protein at different time points post transfection were shown in fig. 5c . the ratios became lower step by step from over 50 times at 16 h post transfection to around 4 times at 38 h post transfection (fig. 5c) . the corresponding ratios for nsp3.2, nsp3.1 and nsp12 were also investigated and no similar phenomena were observed (data not shown). collectively, these data showed that the nucleocapsid n protein provided in trans could enhance the efficiency of sars-cov genome replication and transcription at early stages. coronaviruses have the largest rna genome known, which encodes a large number of proteins that are involved in viral replication, assembly, and other important functions that are essential to viral amplification in host cells. except for some viral proteins that might perform their activities individually, most of the viral proteins could associate with other proteins or themselves to carry out their functions, indicating that the interactions between these proteins may play a crucial role during the viral life cycle. for sars-cov, at least 17 proteins, including 16 nonstructural proteins and the structural nucleocapsid protein, are most likely involved in the replication process [12, 53] . in this study, a mammalian two-hybrid system was used to determine the genome-wide matrix-based protein-protein interactions of sars-cov. in total, 40 different interactions for 28 predicted mature proteins were observed, and most of them have not been previously reported. interestingly, 32 percent of the interactions tested could be confirmed by pull down assays. to our knowledge, this is the first genome-wide protein-protein analyses for an intact viral orfeome by using mammalian two-hybrid system. in our screen, 1.4 protein interactions per viral protein on the average was detected, and this result is in the upper range of the detection rates of viral protein interactions obtained by yeast two-hybrid systems [58] . therefore, this study indicates that mammalian two-hybrid system can also serve as a convenient system to detect viral protein interactions of the whole orfeomes. in addition, mammalian two-hybrid system may have some advantages over yeast two-hybrid system for detecting genuine viral protein interactions due to the native posttranslational modifications and folding of the proteins. very recently, two large-scale analyses of protein interactions of sars-cov were carried out by using yeast two-hybrid systems [9, 10] . in the report by von brunn et al, 70 pairs of interactions were revealed, among which 30% could be verified by coimmunoprecipitation [9] . in the work by imbert et al, 17 pairs of interactions were observed for non-structural proteins, about half of which were related to nsp3 [10] . surprisingly, none of the interactions revealed in the two studies was overlapping although they both employed the yeast two-hybrid system for the screening. in contrast, overlapping interactions could be identified between that detected by mammalian two-hybrid system in current work and that by different yeast two-hybrid systems. there are 9 pairs of interactions found in our work that are overlapping with that of von brunn et al, and they are nsp5 with nsp5, nsp7 and nsp8, nsp7 with nsp7, nsp8 and nsp9, nsp8 with nsp8, nsp9 and 9b. there are 4 pairs of interactions found in this study that are identical to that of imbert et al. and they are nsp10 with nsp14 and nsp16 in both directions. interestingly, all the overlapping interactions represented strong interactions in the mammalian two-hybrid assays and could be verified by biochemical assays. in the two previous studies with yeast two-hybrid system, four proteins (nsp2, nsp3, nsp8 and 9b) were shown to have a wide range of interactions with other viral proteins, but these phenomena could not be observed in mammalian two-hybrid assays. in contrast, a number of interactions including n-n and nsp15-nsp15 were detected in current study and proved previously by other studies [54] [55] [56] [57] but they could not be revealed by the yeast two-hybrid systems [9, 10] . intriguingly, the two strong interactions nsp10-nsp14 and nsp10-nsp16 identified in mammalian two-hybrid system was also revealed in a yeast two-hybrid system by imbert et al. [10] but not by von brunn et al [9] . although it is difficult to judge which system offers more biologically relevant results, great caution should be taken when interpreting the interaction data as both two-hybrid screenings as well as biochemical methods may generate false positive and false negative interactions. nevertheless, the above comparative analysis of overlapping interactions of different systems may suggest that mammalian two-hybrid system could provide more reliable assays for detecting human viral protein interactions. in other scenario, the profiles of sars-cov protein interactions revealed by mammalian two-hybrid system (in this work) and yeast systems [9, 10] , respectively, may be complementary to each other and jointly serve as a framework for further characterization of protein-protein interactions and their biological functions on coronavirus replication cycle. similar to that of yeast two-hybrid system, the detectable protein interactions all take place in the nucleus whereas, on the contrary, all positive-stranded rna viruses replicate in cytoplasm, the natural location for viral proteins. thus, the two-hybrid systems may have obvious limitation on detecting proteins that contain transmembrane domains or change to abnormal conformations in acidic conditions, and this may represent one reason for generating false negative and false positive interactions. in current screen, only few interactions were detected for the sars-cov membrane proteins such as nsp4, m, e and 3a proteins, and no interactions could be detected for nsp1, nsp6, spike and the small transmembrane protein 6, possibly reflecting the limitation of the two-hybrid system. therefore, using shorter or random cdna fragments by removing the transmembrane and other inhibitory domains may help detect more interactions in this system. in this study, we have detected several interactions between accessory proteins and replicase proteins, for example, 3b with nsp8, nsp12, nsp13 or nsp14, 7b with nsp9 or nsp16, 8b with nsp9, and 9b with nsp8. interestingly, among these interactions those between 3b with nsp8 and nsp13 were confirmed by pull-down assays. however, previous studies showed that deletions of orfs 3b, 7b, 8b and 9b alone or in combination with other virus-specific proteins did not significantly influence the level of viral rna and replication efficiency in cell culture and mouse models [48, 49] , suggesting either that these interactions may not play significant roles in viral replication or that these systems are not sensitive enough to detect minor differences. in any case, it would be reasonable to speculate that such interactions may contribute to the virus-host interplay and hence to the viral pathogenicity. the interactions between nsp10 with nsp14 and nsp16 showed bi-directionality in the mammalian two-hybrid analyses, which could be confirmed in pull-down assays and were also revealed in one of the two yeast-hybrid analyses [10] . recently, nsp10 was shown to form a dodecameric rna-binding protein complex [26, 27] involved in regulation of rna synthesis and polyprotein processing [25, 59] . nsp14 and nsp16 were proved to play an essential role in viral replication and transcription as shown by mutational analysis [37] . considering that nsp14 has exoribonuclease activity and could play an essential role in rna proofreading activity [30] [31] [32] , and that nsp16 is involved in the viral methyltransferase activity and rna capping [34, 36] , the interactions of nsp10 with nsp14 and nsp16 may indicate that nsp10 could play an important role in the formation of replication complex bound to rna (i.e., nsp10 could mediate the binding of nsp14 and nsp16 to the rna) and in the replication/transcription processes. although no interactions were detected for nsp11, which was predicted to be small polypeptide with 13 amino acids, fusion of nsp11 with nsp10 strengthened the respective interactions of nsp10 with nsp14 and nsp16. these observations may imply that nsp11 could act as a cofactor for nsp10 and the predicted cleavage site between nsp10 and nsp11 may be inefficient in vivo, thus resulting in the production of nsp10/nsp11 fusion protein in cells. indeed, no evidence for existence of the fully processed nsp11 has been presented in published researches. however, such speculation needs to be confirmed by further investigations. several interactions detected in current work have also been observed in previous studies. for example, nsp7 and nsp8 could associate with each other and thus form a hexadecameric supercomplex with a central channel that has dimensions and positive electrostatic properties favorable for nucleic acid binding [21] . consistent with our results, self-interactions of n protein and nsp15 have also been revealed and the results indicate that these proteins have the propensity to oligomerize [56, 57, 61] . in this study, a sars-cov replicon containing a sensitive luciferase reporter was constructed, with its expression under the control of m gene trs. in this situation, the reporter activity would indicate the efficiency of both genome replication and transcription. when individual viral proteins were provided in trans to the reporter replicon, most proteins did not exert significant influence on the reporter activity, with exception of nsp3, nsp12 (polymerase), and nucleocapsid protein n. as protein nsp3 is a large multidomain protein, different domains were tested separately in this system. while nsp3.1 that covers domains predicted for adenosine diphosphate-ribose 10phosphatase [12] and methyltransferase [35] could enhance the reporter activity, the nsp3.2 had a negative effect, indicating the domains in nsp3.2 may play structural roles in the formation of replication/transcription complex and could behave in a dominantnegative manner when separated from other domains. in accordance with this hypothesis, nsp3.2 was found to associate with itself, nsp4 and nsp12 in this work. n protein of coronaviruses increases the rescue efficiency of coronaviruses from infectious rna transcripts [37, 49, 62] and is required for efficient genome replication [53, 63] . in this study, we showed that n protein was not absolutely essential for starting the replication but may play important roles at the early stages of genome replication and/or transcription because the reporter replicon in absence of additional n protein provided in trans also resulted in obvious replication and transcription but in lower efficiency and delayed manner. the enhancement effects of n protein phased out at late stage probably when the n protein expressed from the genome had accumulated to certain level. although the exact mechanisms for n-mediated stimulation activity at early stages are not known, several scenarios could be envisaged, such as protecting viral genome rna, increasing the translation efficiency of viral rna, stabilizing replication/ transcription protein complex and inhibition of cellular innate immune responses, as is supported by the recent reports on diversified functions of n protein [64, 65] . in summary, current work constructed an interaction network of sars-cov proteins and established a sensitive replicon for studying the functions of individual viral proteins. the intraviral protein interactions identified in this study, in combination with the data obtained by using other systems, could serve as a basis for further studies of viral protein functions and molecular mechanisms of the genome replication/transcription processes of coronaviruses. african green monkey kidney (vero e6) cells, baby hamster kidney (bhk21) cells and 293t cells were grown and maintained in dulbecco's modified eagle medium and modified eagle medium (gibco invitrogen), respectively, supplemented with 10% heat-inactivated fetal bovine serum and 100 u/ml of penicillin and 100 mg/ml of streptomycin (gibco invitrogen). viral cdnas encoding individual sars-cov orfs were generated and described in our previous work [39, 66] . the primers for individual orfs were designed according to the genome sequence of sars coronavirus strain whu (genbank accession number: ay394850) with restriction sites which were compatible for the downstream subclonings. the reagents for pcr were 0.2 mm dntps, 0.4 mm forward and reverse primers, ,10 ng template dna and 1 u of kod dna polymerase (toyobo) in 50 ml reaction system. the amplification conditions were 94uc for 2 min and 30 cycles of 94uc for 15 sec, 52uc for 15 sec and 68uc for 3 min, followed by 68uc for 10 min and 4uc for 10 min. after the separation by agarose gel electrophoresis, pcr fragments were recovered from the gels by dna extraction kit (omega bio-tech) and cloned into pgem-t vector (promega) after a-tailing reaction according to standard protocols. the sequences of positive clones examined by restriction and pcr analyses were confirmed by dna sequencing on abi 3730 dna analyzer (applied biosystems) and ceq tm 8000 sequencer (beckman coulter). subsequently, the coding sequences were cut from pgem-t constructs with appropriate restriction enzymes and cloned into the mammalian two-hybrid vectors pm and pvp16 (clontech), escherichia coli protein expression vectors pet30a (novagen), pgex-6p-1 (amersham biosciences) and pmal-c2x (new england biolabs), and mammalian expression vectors pcdna3.0 (invitrogen corporation), pcmv-tag2b (stratagene corporation) and pcih that was generated by adding an atg-ha-tag oligo at the xhoi-ecori sites of vector pci-neo (promega). details of the cloning processes and various constructs can be provided upon request. the reporter construct pg5-luc was reconstructed from pg5cat (promega) by replacing chloramphenicol acetyl transferase (cat) gene with luciferase gene sequence. for protein interaction analysis, 0.3 mg of dna-binding domain fusion constructs in plasmid pm, 0.3 mg of transcriptional activation domain fusion constructs in plasmid pvp16, 0.15 mg of reporter construct pg5-luc and 0.1 mg of vector prl-tk (promega) as internal control were transfected into 293t cells by calcium phosphate transfection method. one day before transfection, 293t cells were seeded in 24-well plate with ,1610 5 cells per well. on the following day, the dna of specified amount and 2.5 ml of 2.5 m cacl 2 were diluted with deionized h 2 o to reach a final volume of 25 ml. after an incubation for 5 minutes, 25 ml of 26hbs solution (50 mm hepes, 10 mm kcl, 12 mm dextrose, 280 mm nacl, 1.5 mm na 2 hpo 4 , ph 7.05) was added and mixed. the calcium phosphate-dna solution was added dropwise to the cell culture medium while swirling the plate gently. after incubation for 6 h at 37uc with 5% co 2 , the medium was replaced with fresh one. 24 h later, the luciferase assays were performed by dual-luciferase reporter assay system (promega corporation) according to the provider's instructions. several control constructs were adopted in the analysis, including pm-53 which encodes the p53 protein, pvp16-t which encodes the sv40 large t antigen, and the empty vectors pm and pvp16. the known interaction between p53 and t antigen was used as positive control, and the combinations of test construct and one control plasmid were assigned as negative controls. the assays on each pair of plasmid combinations were repeated at least 4 times and 3 wells were examined per assay. recombinant glutathione s-transferase (gst)-fusion proteins and maltose-binding protein (mbp)-fusion proteins were expressed in e. coli bl21 and dh5a, respectively. bacterial cells harvested were resuspended in phosphate-buffered saline (pbs, 10 mm sodium phosphate, 150 mm nacl, ph 7.5) and lysed by sonication in ice. the lysate was subsequently clarified by centrifugation at 12,000 g for 30 min at 4uc. gst fusion proteins and gst protein immobilized on glutathione-sepharose were coated firstly with mbp protein and were subsequently incubated with the lysate for mbp-fusion proteins in pbs containing protease inhibitor cocktail. after incubation with rotation for 2 h, the resin of glutathione-sepharose was precipitated by brief spin and washed by pbs at least for 5 times. the resin was re-suspended in sds-page loading buffer and heated at 100uc for 5 min. the samples were separated in 10% sds-page and transferred to pvdf membrane following the standard procedures of westernblotting. anti-mbp rabbit serum (new england biolabs, dilution 1:5000) was used in the immunoblot analysis to identify mbpfusion proteins. construction of a replication and transcription report system for sars-cov the reporter system was based on the primary sars-cov replicon (pbac-sars-cov-rep) [37] . to construct a reportercontaining replicon including a selection gene for mammalian cells, the luciferase and neomycin fusion gene cassette was amplified by pcr from a hepatitis c virus replicon provided by dr. ralf bartenschlager [67] . the luciferase-neomycin gene sequence was fused with and thus 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infectious cdna of severe acute respiratory syndrome coronavirus selective replication of coronavirus genomes that express nucleocapsid protein ribonucleocapsid formation of severe acute respiratory syndrome coronavirus through molecular action of the n-terminal domain of n protein severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists isolation of virus from a sars patient and genome-wide analysis of genetic mutations related to pathogenesis and epidemiology from 47 sars-cov isolates enhancement of hepatitis c virus rna replication by cell culture-adaptive mutations dissection study on the severe acute respiratory syndrome 3c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor structural genomics of the severe acute respiratory syndrome coronavirus: nuclear magnetic resonance structure of the protein nsp7 the structure of the endoribonuclease xendou: from small nucleolar rna processing to severe acute respiratory syndrome coronavirus replication crystal structure and mechanistic determinants of sars coronavirus nonstructural protein 15 define an endoribonuclease family severe acute respiratory syndrome coronavirus protein 7a interacts with hsgt we thank yu chen for valuable discussion and comment on the manuscript and nian xiang for excellent technical assistance. key: cord-317675-s1ac5vcx authors: de marco, ario title: strategies for successful recombinant expression of disulfide bond-dependent proteins in escherichia coli date: 2009-05-14 journal: microb cell fact doi: 10.1186/1475-2859-8-26 sha: doc_id: 317675 cord_uid: s1ac5vcx bacteria are simple and cost effective hosts for producing recombinant proteins. however, their physiological features may limit their use for obtaining in native form proteins of some specific structural classes, such as for instance polypeptides that undergo extensive post-translational modifications. to some extent, also the production of proteins that depending on disulfide bridges for their stability has been considered difficult in e. coli. both eukaryotic and prokaryotic organisms keep their cytoplasm reduced and, consequently, disulfide bond formation is impaired in this subcellular compartment. disulfide bridges can stabilize protein structure and are often present in high abundance in secreted proteins. in eukaryotic cells such bonds are formed in the oxidizing environment of endoplasmic reticulum during the export process. bacteria do not possess a similar specialized subcellular compartment, but they have both export systems and enzymatic activities aimed at the formation and at the quality control of disulfide bonds in the oxidizing periplasm. this article reviews the available strategies for exploiting the physiological mechanisms of bactera to produce properly folded disulfide-bonded proteins. the success of recombinant protein expression in e. coli depends mainly on the capability of avoiding unproductive interactions of newly expressed polypeptides. such interactions lead to aggregation of folding intermediates instead of yielding native proteins. the efficiency of the process can be increased by favoring conditions that stabilize folding intermediates and promote the formation of mature structure. several strategies may help in preventing protein aggregation by masking hydrophobic patches on their external surfaces. these include the introduction of chaperone molecules, adding detergents, or co-expressing interacting sub-units of larger complexes. once the conditions have been optimized for keeping the folding intermediates monodispersed, it becomes crucial to speed up the folding process to reach stable native structures and avoid the accumulation of metastable configurations that remain potentially prone to aggregation. foldases and isomerases may strongly enhance the folding (fig 1) . the attention of this review will be focused on the technically available solutions to improve the bacterial expression of proteins that rely on disulfide bond formation to reach their native state. such cys-cys bridges block folding units into stable conformations by linking residues in a covalent manner and their formation is necessary for a protein to achieve its stable tertiary structure. the equilibrium between reduced and oxidized cysteines is regulated by the redox conditions of each cell compart-ment. in eukaryotic cells, the oxidative environment in which disulfide bonds are preferentially formed is the endoplasmic reticulum (er). therefore, polypeptides expressed in the reducing cytoplasm need to be directed to er to complete their folding. the correct targeting to the subcellular compartment is mediated by signal peptides fused to the protein amino terminus that are removed after the import into the organelle. prokaryotes share with eukaryotic cells the reducing cytoplasm, but do not have structures resembling the er. instead of it, they possess an oxidizing periplasm to which pro-peptides with an extra n-term export peptide can be translocated. therefore, eukaryotic protein expression in bacteria periplasm is possible following the substitution of the er with a bacterial signal sequence for periplasm translocation. alternative strategies consider promoting the formation of disulfide bonds by targeting the nascent polypeptides to the external medium or by modifying the redox state of cytoplasm to reach a mild oxidative environment ( figure 1 ). both overexpression and direct fusion to chaperones, foldases, and stabilizing carriers has been tested for improving the yields of functional target proteins. finally, protein aggregates can be first dissolved in chaotropic solutions to reach monodispersity and later be used as a starting material for oxidative refolding processes. a flowchart of the different alternatives is reported in figure 2 . the most intuitive method to exploit e. coli for recovering folded disulfide-bond dependent recombinant proteins is to direct the translated polypeptides to the bacterial periplasm. there are clear physiological reasons for such an approach: the periplasm, in contrast to the cytoplasm, is an oxidizing compartment and it hosts enzymes catalyzing disulfide bond formation and their isomerization, as well as specific chaperones and foldases [1] [2] [3] . however, the necessity of translocating nascent polypeptides through the inner membrane introduces a delicate step since there is only a limited number of available gates for schematic representation of folding pathways and cellular localization of proteins that depend on oxidative environ-ment to reach their native structure figure 1 schematic representation of folding pathways and cellular localization of proteins that depend on oxidative environment to reach their native structure. unfolded proteins can be translocated into the periplasm post-translationally (sec mechanism) or co-translationally (spr mechanism), according to their specific leader sequences. secb is the physiological chaperone involved in stabilizing unfolded proteins bound to sec translocation machinery, but the activity of other cytoplasmic chaperones like dnak can be beneficial. once in the periplasm proteins folding is mediated by the dsb oxidases/isomerases, by chaperones such as skp, degp and fkpa, and by peptidyl-prolyl isomerases such as sura, ppia, and ppib. double mutant strains, in which the thioredoxin/glutaredoxin reductase (trxb -, gor -) pathway is silenced, are characterized by the presence of an oxidizing cytoplasm, compatible with the folding of proteins that need disulfide bridges for stabilizing their structure. cytoplasmic accumulation of correctly folded disulfide-dependent proteins is improved by disulfide isomerase dsbc co-expression and cytoplasmic chaperone (dnakje, groels, clpb). finally, proteins provided with a tat export leader sequence first fold in the cytoplasm and then are tanslocated into the periplasm. chaperones like dnakje, can stabilize the precursor in the cytoplasm and chaperones such as slyd and tord support the pathway efficiency probably binding to the export leader peptide. flow-chart summarizing the different possibilities for produc-ing disulfide-dependent proteins in bacteria figure 2 flow-chart summarizing the different possibilities for producing disulfide-dependent proteins in bacteria. expression is optimized and protein folding directed either in the cytoplasm or in the periplasm. once folded in the cytoplasm, proteins can accumulate in the same cell compartment, or be exported to the periplasm by the tat-pathway, or be secreted to the medium by the type i secretion system. unfolded proteins can be translocated post-translationaly (sec) or co-translationaly (spr) into the periplasm and accumulate there, leak to the medium, or be exposed on the outer membrane. precipitated proteins can be recovered into native structure by means of oxidative refolding. considering the potential danger of having an overcrowded cytoplasm, some authors proposed that protein secretion could be effectively modulated at the translational level by modifying the shine-dalgarno sequence [4] . other authors found that the crucial tuning region starts upstream of the shine-dalgarno region and spans approximately twenty nucleotides downstream of the initiation codon (translational initiation region) [5] . coherently, also other parameters influencing the expression rate have been evaluated in relationship with the production of disulfide-bond dependent proteins, such as growth medium, plasmid origin of replication, and expression promoters. for instance, the expression features of tac, uspa, uspb, t7, trc, lacuv5, malk, pm/xyls have been investigated to achieve high product yields by preventing protein precipitation and cell lysis [6] [7] [8] [9] [10] [11] [12] . however, data have been collected under different experimental conditions and, therefore, they are not comparable, preventing the identification of surely preferable systems. in the absence of an n-terminal signal peptide for periplasmic secretion, recombinant polypeptides expressed in bacteria accumulate in the cytoplasm. the fusion to suitable leader peptides allows for the translocation of unfolded precursors into the periplasm by either the sec (relatively slow, post-translational translocation) or the srp (fast, co-translational translocation) system [13, 14] , even in the case of aggregation prone proteins such as pngase and large molecules like full-length immunoglobulins [15, 16] . the search for optimal leader peptides to use in combination with recombinant proteins has been initially undertaken by comparing the efficiency of natural signal sequences identified in the precursors of bacterial periplasmic proteins, including the leader peptides from spa, phoa, ribose binding protein, pelb, ompa, ompt, dsba, tora, tort, and tolt. furthermore, both synthetic sequences and the phage piii leader peptide were used [6, 9, [17] [18] [19] [20] [21] [22] . initially, the approach was not systematic and no clear preference for any among them was apparent, although ompt resulted preferable when coupled to overexpression of chaperones involved in the stabilization of intermediates translocated through the sec export machinery [23] . however, a wide survey performed by beckwith and coworkers identified a strong correlation between hydrophobicity of the leader peptide and export mechanism [24] . apparently, cotranslational translocation by srp needs the presence of highly hydrophobic leader sequences, even though further unknown biophysical features may be critical. the physiological necessity of the srp pathway as an alternative to the post-translational secretion meditated by the sec route is required to avoid premature folding of the proteins in the cytoplasm. the biotechnological implication of these conclusions is that poor periplasmic accumulation of rapidly folding recombinant proteins may be the consequence of their non-productive cytoplasmic (mis)folding that prevents efficient translocation and correct periplasmic folding. therefore, the choice of the leader peptide may make the difference in terms of secretion efficiency, as demonstrated for thermodynamic stable proteins [25, 26] . spontaneous protein oxidation in extracytoplasmic compartments is extremely slow and is incompatible with cell activity. therefore, it is necessary that the disulfide bond formation is enzymatically catalyzed. periplasmic protein oxidation is regulated by the five members of the dsb protein system (dsba, b, c, d, g) [1, 2] . with the exception of dsbb, these proteins belong to the thioredoxin protein superfamily and are involved in both disulfide-bond formation and rearrangement. specifically, the soluble monomer protein dsba donates its disulfide bond to newly synthesized polypeptides ( figure 3 ) that are folding intermediates and need disulfide bonds to reach their native structure. interestingly, dsba binds its substrates by hydrophobic interactions using a chaperone-like recognition mechanism specific for partially unfolded proteins [27] . such substrate-independent chaperone-like activity dsb-dependent protein oxidation and isomerization in the bacterial periplasm figure 3 dsb-dependent protein oxidation and isomerization in the bacterial periplasm. dsba couples consecutive cysteines providing the disulfide bond and is re-charged by the inner membrane dsbb. incorrect disulfides of oxidized proteins are scrambled by dsbc and dsbg. these isomerases are kept reduced by the inner membrane dsbd that, in return, is reduced by cytoplasmic thioredoxin. periplasm cytoplasm seems to have a general stabilizing effect on passenger proteins fused to dsba mutant deprived of its oxidoreductase activity and, therefore, it has been successfully used as fusion partner even for the production of cytoplasmic proteins [28] . dsba active site is regenerated by the transfer of electrons to the dsbb integral membrane protein, whose activity is crucial in maintaining dsba in its active state ( figure 3 ) [27, [29] [30] [31] [32] . the dsba/dsbb mediated oxidative process is very efficient, but may result in incorrect cysteine pairing and in trapping the target protein in non-native conformations. therefore, a quality control mechanism for the rearrangement of the disulfide bonds was initially postulated and later identified. dsbc and dsbg are the isomerases that scramble incorrect cystin bridges and, therefore, play a key-role in the periplasmic protein folding process ( figure 3 ) [33] [34] [35] [36] . the active cysteines of dsbc and dsbg must remain reduced in an oxidizing environment to be functional. this condition is achieved by the action of the integral inner membrane dsbd. such protein constantly reduces the isomerases by transferring to them the electrons made available by the cytoplasmic thioredoxin [37] [38] [39] [40] [41] . similarly to dsba, both dsbc and dsbg have chaperone activity. since misfolding could be a direct consequence of incorrect disulfides, dsbc/dsbg chaperone activity favors the recognition and interaction with substrates necessiting disulfide isomerization [1, 42] . such homodimer isomerases are v-shaped and use the structure flexibility of the cleft embodied by the two arms to bind unfolded structures of various sizes. their adaptability allows the recognition of largely heterogeneous substrates and explains the elevated efficiency of bacterial dsbc/ dsbg in isomerasing also heterologous proteins [1] . although the two routes of keeping dsba oxidized and dsbc/dsbg reduced have been always considered strictly separated [1] , recent data indicate a possible direct cooperation between dsba and dsbc [43] , and chimeras of dsba and dsbc proved to be able to serve both as oxidase and as isomerase, thus reconciling the competing pathways leading to dsba oxidation and dsbc reduction [44] . furthermore, it has been demonstrated that dsba can be mutated to become an isomerase by adding a linker with peptide binding capacity and that provides a dimerisation domain. these characteristics prevent its immediate oxidation by dsbb and allow substrate recognition and binding [45] . such results will probably simplify the biotechnological applications of dsb proteins in vitro. the relevance of the dsb protein family for the correct folding of disulfide-bond dependent proteins in the e. coli suggested that boosting such molecular machinery could result in increased yields of correctly folded recombinant constructs. the accumulation of a functional scfv was positively affected by co-expression of dsba, b, c, d [46] , both dsbab and dsbcd overexpression improved the production of active horseradish peroxidase [47] , whilst co-expression of dsba and dsbc increased the yield of functional human plasma retinol-binding protein [48] . fusion to dsba increased the accumulation of few mg/l culture medium of active bovine enterokinase and human pro-insulin, a polypeptide stabilized by three disulfide bonds [49, 50] . however, in the case of ragi bifunctional inhibitor, a protein with five overlapping disulfide bonds, dsba activity resulted in the accumulation of non-native intermediates, whilst the availability of dsbc significantly increased the yield of the native protein both in vitro and in vivo [51] . again, the overexpressoion of dsba, b, c, d and dsbcd, but not dsbab, increased the accumulation of active nerve growth factor beta [52] and dsbc was the key foldase also for enhancing the production of horseradish peroxidase and brain-derived neurotrophic factor, although its synergistic effect with dsba, b, and d was detectable [53, 54] . these results were confirmed by experiments performed with plasminogen activator and a scfv [55, 56] and seem to indicate that the isomerase dsbc activity could be the limiting factor for the correct folding of at least part of the recombinant proteins expressed in the e. coli periplasm. the different responses observed when using eukaryotic substrates and dsba or dsbc overexpression might be explained by examining the structure of each single substrate protein. dsba couples cysteines as soon as they are available and, therefore, only disulfides between consecutive residues are formed. as a consequence, dsbc activity is not necessary for proteins in which only disulfide bonds between consecutive cysteines are present in the native structure [57] . in contrast, bond scrambling is dsbcdependent. the correlation between the presence of nonconsecutive disulfides and dsbc-dependence for achieving the correct folding has been demonstrated for eukaryotic as well as for bacterial proteins [51, 58] . however, although the bacterial rnasei is a natural dsbc substrate with three consecutive and one nonconsecutive disulfide bonds [59] , it is possible to artificially tune the periplasm redox potential to achieve conditions that allow dsba to correctly catalyze the bond formation between the nonconsecutive residues [60] . the pivotal role of thiol-disulfide oxidoreductases in supporting the correct folding of disulfide-bond dependent proteins is conserved in gram-negative bacterial periplasm [61] . interestingly, functionality seems to be retained also by phylogenetically distant proteins, such as endoplasmic reticulum disulfide isomerase from mammalian and yeast since, despite the low sequence homology, a marked structural similarity exists [62] [63] [64] . this observation led to successful engineering of human disulfide isomerase in the e. coli periplasm and even in the gram-positive bacillus brevis, with consequent functional restoration in dsba mutants and yield improvement of functional proteins [65, 66] . the process leading to correct protein folding in the e. coli periplasm is mediated by the activity of proteins belonging to several classes and that often have partially overlapping functions (fig 1) . there are peptidyl-prolyl isomerases and chaperones such as sura, fkpa, ppia, ppid, and skp, and chaperone/proteases such as degp [67] [68] [69] [70] [71] [72] [73] . fkpa and sura overexpression contributed to an increase in the accumulation of functional human plasma retinol-binding protein [48] , fkpa, degp, dsba, and dsbc rescued the activity of lipase b [74] , and both the chaperone and protease activities of degp were necessary to enhance the yields of penicillin acylase [71, 75] . the prolyl-cis/trans isomerase activity of ppi apparently catalyzes the rate-limiting step of the recombinant antibody folding represented by the isomerization of proline 95 (kabat numbering) [76] . its coexpression increased the solubility of scfvs and fab fragments [77] [78] [79] , but this enzyme was also successfully used as a fusion partner for recombinant antibody expression [4, 80] . similarly, fusions to fkpa resulted in increased yields of functional recombinant antibodies [81, 82] . the mechanisms by which cytoplasmic chaperones can help periplasmic folding are less clear. in a few cases such molecules were directly secreted to the periplasmic space, as was the case of dnaj and hsp25 that increased the yields of native plasminogen activator [83] . probably such molecular chaperones had an unspecific stabilizing effect on folding intermediates, but no sound rationale supports the strategy of boosting the periplasm with cytoplasmic chaperones, instead of using periplasmic ones. in contrast, the idea of overexpressing cytoplasmic chaperones in the cytoplasm might be justified by the consideration that nascent polypeptides are translocated as unfolded intermediates into the periplasm by the sec/srp systems and, therefore, their potential aggregation must be prevented ( figure 1 ). secb is the natural chaperone that binds polypeptides to be secreted post-translationaly. apparently, secb possesses specificity for 9-residue sequence motifs enriched in aromatic and basic residues [84] . the presence of these motifs within recombinant proteins may considerably influence their chances to be correctly delivered to the periplasm by the sec system. however, the overexpression of other chaperones could compensate for poor secb binding to non-specific substrates. they might unspecifically recognize and stabilize unfolded polypeptides in the cytoplasm, as suggested by the observation that chaperone overexpression in the cytoplasm was beneficial for the accumulation of some periplasmic proteins [85] . different combinations have been tested, such as dnakje (dnak, dnaj, grpe) that increased the accumulation of functional scfvs against domoic acid and the mycotoxin deoxynivalenol [56, 86] . dnakj co-expression prevented the formation of inclusion bodies of cora and improved the periplasmic accumulation of granulocyte-colony stimulating factor [87, 88] . in contrast, cytoplasmic chaperone overexpression failed to improve the yields of horseradish peroxidase [47] . the accumulation rate of secb, the physiological chaperone for the sec system, is apparently efficiently tuned by the effective amount of potential substrates and, consequently, its exogenous overexpression was not considered necessary [13, 89, 90] . however, in at least one case higher periplasmic accumulation of heterologous proteins was detected as a consequence of cytoplasmic secb overexpression [91] . other authors found that the overexpression of secb and dnakj was beneficial in promoting recombinant accumulation of human proteins in the periplasm, but only in combination with signal sequence optimization [92] . the problem of stabilizing the folding intermediates has been addressed also by fusing the target polypeptide to protein carriers. periplasmic eeti-ii, erythropoietin, and scfv production was increased by fusions with maltosebinding protein and immunoglobulin-constant domain, whilst human proinsulin and pepsinogen yields were enhanced by fusions with ecotin, a trypsin inhibitor that could favor target protein accumulation by reducing its degradation [93] [94] [95] [96] . a double ecotin-ubiquitin tag has been recently engineered. it should allow both the stabilization of the passenger proteins and their recovery with their authentic amino terminus [12] . it must be underlined that protein fusion always carries an inherent risk of resulting in soluble but incorrectly folded target protein. for instance, fusions of maltose binding protein and some scfvs resulted in higher periplasmic yields, but the antibodies were not functional, whilst the fusion to alkaline phosphatase stabilized both production and activity [97] . alkaline phosphatase has been often used as a fusion partner for recombinant antibodies. it is not only useful for enhancing the production but, dimerizing, it increases the avidity of the immunoproteins for their substrate and allows their direct detection by enzymatic assay [97] [98] [99] [100] . protein a or single and tandem repeats of its b-and zdomains were successfully used for expressing and purifying a scfv and human proteins such as proinsulin and coagulation factor vii [9, [101] [102] [103] [104] [105] . fusion to barnase resulted in correct folding of ick and mcoeeti, but the construct was directed to the culture medium [106, 107] . other fusion partners, especially in combination with recombinant antibodies, were considered not for facilitating folding and increasing yields, but to obtain reagents more suitable for the final applications. a biotinylationconsensus sequence has been fused to fab fragments to obtain streptavidin-affine immunoconstructs [108] , gfp has been fused to scfvs for recovering immunuofluorescent reagents [109] , and fusion to nuclear import/export sequences have been used to direct scfvs to specific subcellular localizations [110] . a positive effect of low molecular weight additives (chemical chaperones) supplemented in the culture medium was sometimes observed in terms of yields of periplasmic expressed proteins. sorbitol addition to the culture medium resulted in higher accumulation of a functional scfv [46] , glycine betaine and sucrose were beneficial for the folding of immunotoxin and cytochrome c550 [111, 112] , whilst l-arginine and ethanol increased the yields of human pro-insulin, plasminogen activator, and a scfv [50, 83] . also the supply of reduced glutathione, alone or in combination with dsbc overexpression, increased the accumulation of disulfide-dependent proteins [51, 83] . although the sec and srp systems are responsible for translocating most of the proteins into the periplasm, bacteria possess another mechanism -the twin-arginine translocase (tat) pathway-that enables the transport of folded proteins across the inner plasma membrane (figure 1 ) [113] . e. coli genome encodes some tens of proteins with a signal-peptide capable of interacting with tat machinery [114] and such export route has been exploited for recombinant expression as well [115, 116] . the overexpression of all the tatabc genes expressing the proteins involved in the transport increased the translocation of a fluorescent reporter [117] . also the overexpression of the cytoplasmic chaperone dnak seems particularly useful in increasing the efficiency of the tat pathway at both physiological and protein overexpression-induced stress conditions [118, 119] . such effect could be a consequence of the dnak role in facilitating the substrate folding into native structures. another explanation considers that virtually all tat leader peptides contain recognition sequences for dnak [120, 121] . therefore, santini et al. [122] proposed that dnak sequesters the protein intermediates in the cytoplasm by masking their leader peptide until the folding is complete. other chaperone-like proteins as slyd and tord have been reported improving tat efficiency [118, 123, 124] . interestingly, scfvs correctly folded in the cytoplasm (intrabody mutants or wild type constructs expressed in trxbgorcells) were efficiently transported by the tat system [125, 126] . protein accumulation varies from cell to cell inside a population and, therefore, strategies for selecting the most productive clones are relevant. similarly, it may be necessary to identify, within large bacteria cultures expressing clone libraries, the few cells producing proteins/antibodies with desired features. flow cytometry represents a powerful methodology since it enables a quick and thorough screen of large numbers of constructs and it has been successfully applied for selecting proteins, recombinant and full-length antibodies accumulated in the periplasm [127] [128] [129] [130] [131] [132] . several approaches have been described, all referring to the same principle of labeling proteins accumulated in the periplasm to enable the isolation of the productive bacteria. in the simplest protocol, proteins accumulate directly in the the periplasm, otherwise recombinant antibodies are anchored to the periplasmic side of the inner membrane through a lipoprotein domain, or full-length antibodies, secreted and folded in the periplasm, are captured through their fc domain by an anchoring fusion construct consisting of the z-domain of the protein a and an inner membrane lipoprotein [129, 132] . the outer membrane is successively permeabilized to form spheroplasts and the recombinant proteins/ antibodies are labeled by binding to fluorescent antibodies/antigens. finally, the cultured bacteria are sorted by flow cytometry according to the expression and affinity of the produced constructs. such approach may become a paradigm for efficiently screening tagged proteins and antibodies expressed in the periplasmic space. despite the periplasmic quality control and the observation that the fkpa chaperone activity seems to be effective in suppressing inclusion body formation [133] , proteins can aggregate also in this cellular compartment. the simple overexpression is already sufficient to induce the accumulation of the bacterial beta-lactamase protein inclusion bodies into the periplasm [134, 135] . similarly to what was observed in cytoplasmic aggregates, the proteins trapped in periplasmic inclusion bodies may partially preserve their physiological activity [135] and induce stress response [136] . there are relatively few papers reporting the accumulation of heterologous protein inclusion bodies in bacterial periplasm [8, 75, [137] [138] [139] . however, the number might be underestimated because cell fractionation is rarely undertaken and negative results are often not communicated. from the available information it seems that construct instability is often present in engineered polypeptides. for instance, both the addition of unpaired cysteines and sub-optimal length or composition of the linkers connecting the variable domains may be strongly detrimental for the scfv yields [140, 141] . jeong and lee systematically tried to alleviate heterologous protein precipitation in the periplasm by analyzing the effect of bacterial strain, growth temperature, expression vector features (signal peptide, promoter, codon usage optimization, linker compositions), and dsba co-expression, but general conclusions require further studies [8, 142] . the recovery of periplasmic proteins is usually achieved by disruption of the outer membrane by osmotic shock. such purification protocol has the advantage of limiting the contamination with cytoplasmic material. however, as an alternative it has been also proposed to induce the lysis of the outer membrane to recover the target protein directly from the culture medium avoiding a cumbersome purification step (figure 2 ). the most investigated solution is based on the expression of kil protein, a bacteriocin that induces periplasmic protein release through induced membrane solubilization [143, 144] . lysis can be induced by regulating the kil protein expression with a stationary-phase or a temperature inducible promoter [145, 146] and the strategy was successfully used to recover from the medium interleukin-2, beta-glucanase, and streptavidin [144, 145, 147] . also the co-expression of the tola protein has been proposed for inducing the external membrane permeabilization [17] . the accumulation of proteins in the culture medium, and specifically of recombinant antibodies, expressed for being secreted into the periplasm has been often observed [148, 149] . the reasons remain unknown and cell lysis does not seem to be always involved in the protein leakage. however, it has been shown that several different factors such as growth conditions, inducer concentration, and aminoacid substitutions can tune the leakage of scfvs and have been proposed as means to regulate scfv partition between periplasm and medium [149, 150] . the influence of the expression level on protein leakage from the periplasm to the growth medium has been directly demonstrated comparing different promoters [10] . early observations indicated that the disulfide bridges in both î²-lactamase and alkaline phosphatase precursors expressed in e. coli were formed only after their translocation and processing in the periplasm and that it was pos-sible to prevent their oxidative folding by trapping the precursors in the cytoplasm [151, 152] . these results were explained by the presence of a reducing cytoplasm and an oxidizing periplasm in bacteria. furthermore, they indicated that no protein requiring the formation of disulfide bonds for reaching its native structure can be produced in a functional form in the cytoplasm. the correlation between presence of disulfide bonds and native, functional structures was exploited by beckwith and co-workers in their pioneering attempt to identify mutations that enabled the formation of active alkaline phosphatase in bacteria cytoplasm. it turned out that alkaline phosphatase folded correctly when expressed in bacteria hosting mutations that blocked the reduction of cysteines in the cytoplasm by silencing the activity of the thioredoxin reductase [153] . the preliminary model indicated that nadph was the source of reducing potential used by thioredoxin reductase to reduce oxidized thioredoxin. however, the data suggested that another thioredoxin-like protein could be involved in a parallel reducing route. the search for complementary reducing mechanism(s) led to the identification of a second thioredoxin and of glutathione oxidoreductases, all involved in the mechanism aimed at reducing cytoplasmic cysteines [154, 155] . the apparent redundancy of the reducing machinery is probably due to the necessity of obtaining the maximal electron transfer efficiency between protein pairs with varying redox potentials [156] . under physiological conditions the directionality of the electron flux is maintained. however, it was shown that thioredoxin acts as a reductase only when it remains constantly reduced by the thioredoxin reductase activity. in contrast, it can catalyze substrate oxidation when exported to periplasm and oxidized by dsbb [157, 158] . also glutaredoxin 3 can catalyze disulfide bond formation in the periplasm, but its activity depends on oxidized glutathione availability rather than dsbb [159] . a similar process has been already demonstrated in eukaryotic cells [160] and its existence may also be hypothesed in the bacterial cytoplasm wherethe environment becomes oxidized as a consequence of impaired reducing activities. summarizing, it is possible to obtain an oxidizing cytoplasm in a cellular system in which glutaredoxin activity is abrogated by gormutation, and both thioredoxin 1 and 2 are kept oxidized as a consequence of thioredoxin reductase mutations (figure 1 ). reduced glutathione, necessary to preserve cell viability, is produced by the disulfide reductase activity of mutated peroxiredoxin ahpc [161, 162] . interestingly, ahpc reductase activity does not significantly influence the oxidizing condition of the cytoplasm in trxb -, gorcells [162] . such observation prompted to investigate the biotechnological potential of both single (trxb -) and double (trxb -, gor -) mutant strains -now commercialized with the names of ad494 and origami (novagen), respectively-for the cytoplasmic expression of recombinant proteins with multiple disulfide bonds in their native structure. the encouraging data obtained in the first attempts [163] were further improved by combining cytoplasmic oxidation conditions and cytoplasmic accumulation of dsbc isomerase (figure 1 ) [164] . recently, a new expression strain has become available. shuffle (new england biolab) is trxb -, goras the commercial origami (novagen), but overexpresses cytoplasmic dsbc and is spectinomycin selectable, allowing the transformation with the majority of the currently used vectors. although no literature is available so far, it is expected that the shuffle combination of oxidizing conditions and isomerization capability would strongly improve the correct folding of disulfide bond-dependent proteins in the e. coli cytoplasm. the method of expressing disulfide-bond-dependent proteins in oxidized cytoplasm was validated by several groups and progressively optimized. the collagen prolyl 4-hydrolases yield was higher in the cytoplasm of trxb -, gorbacteria than in the periplasm of the corresponding bl21 wild type strain [165] . the oxidized cytoplasm allowed the accumulation of functional igg-like extracellular domain receptor [166] , ig2 domain of neurolin [167] , lipase b [168, 169] as a consequence of the constantly increasing importance of recombinant antibody expression, a significant effort has been dedicated for optimizing their production in bacteria. although few antibodies are stable in the absence of disulfide bonds and can be directly expressed as intrabodies in the reducing cytoplasm of host cells [175] [176] [177] [178] [179] , the structure and the functionality of most of them strictly depends on correct cys-cys bridges. recombinant antibodies in scfv format have one intrachain disulfide bond for each variable region, those in fab format have a further inter chain bond. a scfv fragment fused to a consensus peptide for obtaining in vivo site-specific biotinylation, a bivalent fv antibody fragment fused to molybdopterin synthase to induce its oligomerization, and some fab fragments were successfully produced in the cytoplasm of trxb -, gorcells [180] [181] [182] . camelidae recombinant antibodies in vhh format possess one intrachain disulfide bond between framework residues and may have a further one to constrain the cdr3 loop. also these antibodies and have been successfully produced in the cytoplasm of trxb -, gorbacteria [183] . the cytoplasmic accumulation of thioredoxin as a consequence of its recombinant overexpression in wild type bacteria was proposed for increasing the yields of coexpressed eukaryotic proteins without disulfide bonds in their native structure [191] . similarly, the precipitation of eukaryotic disulfide-bond independent proteins in the bacteria cytoplasm was prevented after their fusion to thioredoxin [192] . cytoplasmic expression of thioredoxin fusions rescued also polypeptides that failed to correctly fold in the periplasm [187], plant viscotoxins, and porcine pepsinogen a, although these proteins contain multiple disulfide bridges [193, 194] . the reasons as to why thioredoxin can stabilize its passenger proteins in wild type bacteria is not well understood, although its contribution to scfv folding might be due to its chaperone prop-erties [195] , since a catalytic cysteine thioredoxin mutant was still effective in supporting both disulfide bond formation and functionality of the target protein [196] . in contrast, there is a logical reason for overexpressing cytoplasmic thioredoxin in double mutant trxb -, gorbacteria since such enzyme can act as an oxidant when it operates in an oxidized milieu [157] . therefore, it can actively contribute to maintain oxidizing conditions in the cytoplasm. fusions between recombinant scfvs and thioredoxin 1 expressed in trxb -, gorbacteria resulted in increased cytoplasmic yields [196] , correct folding of a scfv against the c-met receptor [188] , and of the first domain of the multiple kazal-type inhibitor lekti, a polypeptide that contains two disulfide bridges in its native structure [197] . both the his-and gst-fusions of bsph1, a protein containing 2 fibronectin type-ii domains each consisting of 2 disulfide bonds, aggregated when expressed in the cytoplasm of trxb -, gorbacteria, but their fusions with thioredoxin resulted insoluble and active proteins [198] . other proteins have been used as fusion partners and showed positive effects on folding and functionality of recombinant proteines accumulated in bacteria cytoplasm. carboxyterminus fusions to maltose binding protein stabilized several scfvs independently on the cytoplasm redox conditions [199] , although less efficiently than thioredoxin [196] , whilst fusion to nusa was strictly necessary to yield another functional scfv and april in the cytoplasm of trxb -, gorcells [200, 201] . sumo has also been proposed as solubilizing carrier for disulfide-bonded proteins, but the structural quality of the recovered proteins has not been investigated [202, 203] . however, in a recent report it has been shown that a scfv against vegf-165 was soluble and active when expressed fused to sumo [204] . although the rational background for the expression of disulfide-bond dependent proteins in double mutant strain trxb -, goris generally accepted and such strain has been successfully exploited, alone or in combination with the expression of foldases/chaperones, the usefulness of the single trxbmutation remains unclear. the glutathione oxidoreductase is still functional in such bacteria and its activity should be sufficient to keep the cytoplasm partially reduced [156] . consequently, disulfide bond formation should be at least partially impaired. in practice, only minimal amounts of all expressed scfvs remained soluble and functional in the cytoplasm of trxb -(ad494) cells [109] and the advantage over controls was limited also in the presence of thioredoxin co-expression [205] . a direct comparison between ad494 and trxb -, gorbacteria clearly indicated an advantage in using the double mutant [196] . in contrast, the same cells resulted significantly more efficient than conventional xl-blue cells in producing an anti-progesterone scfv [206] . however, data concerning the oxidation state of this antibody are not reported. information concerning folding features or functionality is limited also in other cases in which proteins were successfully produced in the cytoplasm of ad494 cells. however, at least in the case of the scorpion neurotoxin lqq-v and fibronectin ii-2 domain from mmp-2, the proteins were both functional and correctly folded [207, 208] . furthermore, the trxbstrain, in combination with dnakj coexpression, allowed the production of biological active human sparc in the cytoplasm [209] . in the case of a serpin domain, ad494 cells did not improve the total amount of soluble recombinant protein accumulated in the cytoplasm with respect to wild type bacteria, but it was correctly folded and active, whilst the protein expressed in the control cells did not form the essential disulfide bonds [210] . groels co-expression further increased the serpin domain solubility. in some cases, the combination of ad494 cells and thioredoxin fusions significantly improved the expression of functional target proteins. leishmania chitinase, the extracellular domain of human thyrotropin receptor, and the disintegrin domain of jararhagin venom were purified, in large amounts, as soluble and active proteins [211] [212] [213] . in contrast, it has been reported that a pro-urokinase fusion with thioredoxin expressed in ad494 (de3) strain accumulated in inclusion bodies, even when the system was supplemented with disulfide isomerase and chaperonines [214] . however, there is scarce access to negative results that would help in understanding the factors regulating disulfide bond-dependent protein expression in the cytoplasm of wild type and mutant strains. a time-consuming but definitive approach for avoiding trial-and-error attempts in identifying the optimal cytoplasmic expression conditions to yield functional proteins with disulfide bonds is the generation of mutants that maintain a stable structure even in the absence of cys-cys bridges [215] . such protein engineering efforts were particularly successful in the case of recombinant antibodies. various strategies of mutation and molecular evolution have been used to generate disulfide-independent stable scfvs starting from a reduction-sensitive precursor [175, [216] [217] [218] . intrabodies were recovered from libraries in which cdr variability was introduced by hypermutation of a naturally disulfide bond-independent vhh or scfv backbone [219, 220] , and by selecting natural intra-bodies by biopanning [186, [220] [221] [222] . such molecules are valuable tools with great application potentiality for intracellular immunization [179, 223, 224] . the substitution of critical residues in both the framework and in the cdrs has also been used to improve the periplasmic accumulation of recombinant antibodies [150, [225] [226] [227] . expressing secreted proteins anchored on the external side of bacteria membranes [228] has been initially considered to render them accessible for external binders rather than to produce material for purification purpose. avirulent bacteria displaying peptides corresponding to pathologyrelated antigen determinants on their surface were designed as vaccine tools [229] [230] [231] . they succeeded in stimulating immunological reactions [229] [230] [231] [232] [233] [234] , but the externally anchored peptides were not thoroughly characterized at structural level and, therefore, it cannot be ruled out that immunoresponse was promoted by partially unfolded antigen domains. however, more recent data show that correctly folded fusions of outer membrane transporters and both scfv and vhh recombinant antibodies were efficiently translocated on the outer bacteria surface [235, 236] . once displayed, anchored proteins can be automatically released into the medium by cleavage of a specific sequence recognized by a membrane protease like ompt [233, 237] . several bacteria transporter proteins have been used to develop vectors in which they are fused to the proteins of interest [238, 239] , such as flic [240] , pullulanase [241] , oprf [242] , opri [243] , phoe [229] , misl [237, 244, 245] , and cytolysin [234] . exposing the protein to the outer bacteria surface is a particularly valuable approach when the proteins can be directly used in such format. for instance, enteric coronaviruses were successfully neutralized in cultured epithelial cells treated with e. coli expressing fusions of iga protease beta domain from neisseria gonnorrhoeae with specific scfvs [246] . an adhesion domain of intimin has been used as an anchoring partner for exposing both proteins and peptide libraries on the surface of the bacteria outer membrane. the resulting bacteria were suitable for cell sorting and used to identify epitope-specific antibodies [247, 248] . the type i secretion pathway is used by gram-negative bacteria to transfer toxins and exoenzymes provided of a carboxyterminus export signal directly from the cytoplasm to the external medium [249] [250] [251] . these proteins are devoid of disulfide bridges but the work performed by fernandez and co-workers demonstrated that fusions of the secreted protein alfa-haemolysin c-domain and antibody fragments devoid of periplasm export signal peptide were efficiently secreted in oxidized and functional form [252] [253] [254] . functional alfa-haemolysin fusions of both scfvs and vhhs were recovered from the culture medium [252] [253] [254] [255] and hemolysin-fusion of shiga toxin b subunit [245] was able to induce host immunoreaction in inoculated rabbits. despite the great effort made by several groups for obtaining native proteins with correctly folded disulfide bridges in bacteria, many attempts failed and target proteins accumulated as precipitates. this observation prompted other groups to turn the disappointing results into an opportunity of pulling together large amounts of proteins and developing oxidative refolding strategies. the methodologies available for protein refolding have been thoroughly reviewed [256] [257] [258] and here only parameters that are of specific interest for the disulfide bond-dependent proteins will be briefly presented. two common requisites for a refolding buffer are the presence of a redox couple and of alkaline ph. the redox couple (gsh/gssg, mesna/dimesna, heteroaromatic thiols) is necessary to activate the cys residues [259] [260] [261] , whilst the alkaline ph facilitates the nucleophilic attackdependent disulfide bond formation [262] . low temperature generally prevents non-productive contacts of metastable folding intermediates and can improve the final yields [263] . non-productive interactions have been also successfully limited by modifying the reduced thiol groups before starting the refolding process. the cysteines of denatured proteins are first s-sulfonated or transformed into mixed disulfides and then the disulfide bonds are formed in the presence of a suitable redox system [259, 264] . surfactants and polyethylene glycol also showed stabilizing properties [265, 266] , whilst a dynamic redox environment, in which the conditions pass progressively from reductive to oxidative, has been proposed to maximize disulfide bond shuffling [267] . however, also simple dilution was effective for refolding heavy chain single domains [268] . an interesting recent paper shows the contradictory effect that stabilizing molecules may have on the refolding of disulfide-bond dependent proteins [269] . l-arginine is known to slow down the refolding process while suppressing hydrophobic interactions. both these factors have been usually interpreted as an advantage for efficient refolding. however, although hydrophobic aggregation is prevented, the accumulation of intermediates with free cysteines leads to the formation of intermolecular disulfide bonds and the formation of progressively larger oligomers in an arginine-concentration dependent manner. productive oxidative refolding can be improved by different means. chromatography allows separating the target protein from contaminants that can interfere during folding and has been successfully used to achieve the refolding of thioredoxin-scfv fusion proteins [270] . furthermore, binding unfolded protein to a substrate impairs the physical interaction amongst single molecules and their refolding can be performed in a sort of "independent microenvironment" in which aggregation-driving contacts are prevented. optimization is necessary with respect to salt concentrations to avoid protein-matrix interactions. oncolumn refolding was successfully performed by using metal affinity chromatography, as in the case of ribonuclease a [260] , chemokines [271] , and î±-glucosidase [272] , ion-exchange substrates, as for bovine serum albumin [273] and alfa-lactalbumin [274] , or by using zeolite [275, 276] . different classes of proteins can contribute to an increase in the refolding yields of native proteins (figure 1 ). thioredoxin supported the productive refold of both reduced denatured and oxidized but incorrectly paired disulfides of pancreatic rnase, citrate synthase and alphaglucosidase [195, 277] . the peptidyl-prolyl isomerase was beneficial for the refolding of fab fragments [278] , whilst the disulfide isomerase, alone or in combination with quiescin-sulphydryl oxidase and glutaredoxin, facilitated the oxidative folding of ribonuclease a and riboflavin binding protein [279] [280] [281] . the fusion to the chaperone slyd had a positive effect on the refolding of the ectodomain e1 of rubella virus [282] . dsba, peptidyl-prolyl-isomerase, and groel minichaperone were immobilized on an agarose gel to refold the scorpion toxin cn5 [283] , immobilized dsba, dsbc and groel minichaperone were effective in refolding single-chain fragments [284] , disulfide isomerase was beneficial for ribonuclease and lysozyme refolding [285] , and dnak in combination with trigger factor or disulfide isomerase helped the functional folding of scfv alone and fused to a toxin domain [286, 287] . the availability of clpb and dnak/dnaj/grpe improved the refolding efficiency of the cysteine-rich protein gloshedobin [288] . finally, high-pressure has also been used to prevent the formation of nonnative disulfide bonds [289] . often, when very heterogeneous molecules as the proteins are handled, it seems that the only possible approach towards optimization of the production process is the trial-and-error strategy. however, the comprehension of the physiological mechanisms involved in the protein folding and aggregation allowed for more rational solutions that simplified the selection of the conditions to be used in the trial panel ( figure 2 ). robust tools, such as oxidizing mutant strains or plasmids for the overexpression of chaperones and foldases, are now available and several successful expression alternatives have been described. there is still no certainty that a specific disulfide-bond dependent protein will be expressed in a functional form in bacteria, but a series of rational approaches can easily be compared. once optimized the construct dna sequence and the expression conditions, the main choice is between cytoplasmic and periplasmic folding and accumulation of the target protein. it will guide the selection of opportune leader sequences and suitable bacteria strains. finally, the purification strategy will request further modification if the proteins are to be recovered from inclusion bodies, total lysate, periplasmic fractions, or culture medium. at the end of this review, it becomes apparent that there is a large amount of information missing concerning the accurate description of the molecular mechanisms involved in protein folding, translocation and molecular quality control. there is also a huge gap between the theoretical knowledge and the results of protein production, as these are only sometimes in agreement with the hypothetical expectations and the apparent irrationality of some successful conditions may seem as if it were a "magical" activity. in contrast, what we are missing is a coherent system for the comparison of experimental data that remain anecdotal unless precisely annotated. our understanding of why some conditions work in one case, but not in another, will substantially increase when a sufficient amount of homogeneous and comparable data will be available for bioinformatic analyses [290] . data organization and their direct accessibility would help in extrapolating the information necessary to improve the identification of optimal strategies for each specific class of polypeptides. therefore, the future collective endeavor should be addressed at rationalizing rather than accumulating data. one example to follow for future attempts might be the public repository refold database http:// refold.med.monash.edu.au/ that lists the successful methods for refolding of proteins [291] . the author declares that they have no competing interests. protein disulfide bond formation in prokaryotes pathways of disulfide bond formation in escherichia coli thioredoxins and glutaredoxins as facilitators of protein folding increased yield and activity of soluble singlechain antibody fragments by combining high-level expression and the skp peripasmic chaperonin translational level is a critical factor for the secretion of the heterologous proteins in escherichia coli secretion of active kringle-2-serine protease in escherichia coli fed-batch production of recombinant beta-galactosidase using the universal stress promoters uspa and uspb in high cell density cultivations secretory production of human leptin in escherichia coli evaluation of inducible promoters on the secretion of a zz-proinsulin fusion protein in escherichia coli medium and copy number effects on the secretion of human proinsulin in escherichia coli using the universal stress promoters uspa and uspb broad-host-range plasmid pjb658 can be used for industrial-level production of a secreted hosttoxic single-chain antibody fragment in escherichia coli a novel ecotin-ubiquitin-tag (ecut) for efficient, soluble peptide production in the periplasm of escherichia coli sec-dependent preprotein translocation in bacteria srp-mediated protein targeting: structure and function revisited using secretion to solve a solubility problem: high-yield expression in escherichia coli and purification of the bacterial glycoamidase pngase f expression of full-length immunoglobulins in escherichia coli: rapid and efficient production of aglycosylated antibodies tolaiii co-overexpression facilitates the recovery of periplasmic recombinant proteins into the growth medium of escherichia coli identification of factors impeding the production of a singlechain antibody fragment in escherichia coli by comparing in vivo and in vitro expression a novel fusion protein system for the production of native human pepsinogen in the bacterial periplasm the presence of n-terminal secretion signal sequences leads to strong stimulation of the total expression levels of three tested medically important proteins during high-density cultivations of escherichia coli srp and sec pathway leader peptides for antibody phage display and antibody fragment production in e. coli the dsba signal sequence directs efficient, cotranslational export of passenger proteins to the escherichia coli periplasm via the signal recognition particle pathway improvement of post-translational bottlenecks in the production of penicillin amidase in recombinant escherichia coli strains use of thioredoxin as a reporter to identify a subset of escherichia coli signal sequences that promote signal recognition particle-dependent translocation signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display efficient selection of darpins with sub-nanomolar affinities using srp phage display investigation of the dsba mechanism through the synthesis and analysis of an irreversible enzyme-ligand complex expression of eukaryotic proteins in soluble form in escherichia coli identification of a protein required for disulfide bond formation in vivo a pathway for disulfide bond formation in vivo identification and characterization of the escherichia coli gene dsbb, whose product is involved in the formation of disulfide bonds in vivo two cysteines in each periplasmic domain of the membrane protein dsbb are required for its function in protein disulfide bond formation characterization of dsbc, a periplasmic protein of erwinia chrysantemi and escherichia coli with disulfide isomerase activity chaperone activity of dsbc a new escherichia coli gene, dsbg, encodes a periplasmic protein involved in disulphide bond formation, required for recycling dsba/dsbb and dsbc redox proteins in vivo and in vitro function of the escherichia coli periplasmic cysteine oxidoreductase dsbg identification and characterization of a new disulfide isomerase-like protein (dsbd) in escherichia coli six conserved cysteines of the membrane protein dsbd are required for the transfer of electrons from the cytoplasm to the periplasm of escherichia coli transmembrane electron transfer by the membrane protein dsbd occurs via a disulfide bond cascade beckwith : reduction of the periplasmic disulfide bond isomerase, dsbc, occurs by passage of electrons from cytoplasmic thioredoxin ndsbd: a redox interaction hub in the escherichia coli periplasm dsbg, a protein disulfide isomerase with chaperone activity the disulphide isomerase dsbc cooperates with the oxidase dsba in a dsbd-independent manner engineered dsbc chimeras catalyze both protein oxidation and disulfide-bond isomerization in escherichia coli: reconciling two competing pathways de novo design and evolution of artificial disulfide isomerase enzymes analogous to the bacterial dsbc combination of dsb co-expression and an addition of sorbitol markedly enhanced soluble expression of single-chain fv in escherichia coli improvement of productivity of active horseradish peroxidase in escherichia coli by coexpression of dsb proteins a system for concomitant overexpression of four periplasmic folding catalists to improve secretory protein production in escherichia coli production of recombinant bovine enterokinase catalytic subunit in escherichia coli using the novel secretory fusion partner dsba increased production of human proinsulin in the periplasmic space of escherichia coli by fusion to dsba dsba and dsbccatalyzed oxidative folding of proteins with complex disulfide bridge patterns in vitro and in vivo overproduction of bacterial protein disulfide isomerase (dsbc) and its modulator (dsbd) markedly inhances periplasmic production of human nerve growth factor in escherichia coli overexpression of protein disulfide isomerase dsbc stabilizes multiple-disulfidebonded recombinant protein produced and transported to the periplasm in escherichia coli production of brainderived neurotrophic in escherichia coli by co-expression of dsb proteins expression of active human tissuetype plasminogen activator in escherichia coli optimisation of production of domoic acid-binding scfv antibody fragment in escherichia coli using molecular chaperones and functional immobilization on a mesoporous silicate support in vitro and in vivo redox states of the escherichia coli periplasmic oxidoreductases dsba and dsbc the nonconsecutive disulfide bond of escherichia coli phytase (appa) renders it dependent on the protein-disulfide isomerase, dsbc in vivo substrate specificity of periplasmic disulfide oxidoreductases the oxidase dsba folds a protein with a nonconsecutive disulfide dsbc activation by the n-terminal domain of dsbd thioredoxin -a fold for all reasons the protein disulphide-isomerase family: unraveling a string of folds protein disulfide isomerase: the structure of oxidative folding human protein disulfide isomerase functionally complements a dsba mutation and enhances the yield of pectate lyase c in escherichia coli a protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of bacillus brevis new components of protein folding in extracytoplasmic compartments of escherichia coli sura, fkpa, and skp/omph a new heat-shock gene, ppid, encodes a peptidyl-prolyl isomerase required for folding of outer membrane proteins in escherichia coli skp, a molecular chaperone of gram-negative bacteria, is required for the formation of soluble periplasmic intermediates of outer membrane proteins defining the roles of the periplasmic chaperones sura, skp, and degp in escherichia coli degp-coexpression minimizes inclusion-body formation upon overproduction of recombinant penicillin acylase in escherichia coli protein quality control in the bacterial periplasm physiological improvement to enhance escherichia coli cell-surface display via reducing extracytoplasmic stress heterologous expression of lipase in escherichia coli is limited by folding and disulfide bond formation roles of degp in prevention of protein misfolding in the periplasm upon overexpression of penicillin acylase in escherichia coli the rate-limiting steps for the folding of an antibody scfv fragment selection for a periplasmic factor improving phage display and functional periplasmic expression escherichia coli skp chaperone coexpression improves solubility and phage display of single-chain antibody fragments. prot expr purif a step-wise approach significantly enhances protein yield of a rationally-designed agonist antibody fragment in e. coli isolation and expression of recombinant antibody fragments to the biological warfare pathogen brucella melitensis production of soluble and functional engineered antibodies in escherichia coli improved by fkpa expression of a functional scfv fragment of an antiidiotypic antibody with a beta-lactam hydrolytic activity cosecretion of chaperones and low-molecular-size medium additives increases the yield of recombinant disulfide-bridged proteins substrate specificity of the secb chaperone chaperone-based procedure to increase yields of soluble recombinant proteins produced in e. coli cloning, expression, and characterization of singlechain variable fragment antibody against mycotoxin deoxynialenol in recombinant escherichia coli dnak and dnaj facilitated the folding process and reduced inclusion body formation of magnesium transporter cora overexpressed in escherichia coli dnak/dnaj supplementation improves the periplasmic production of human granulocyte-colony stimulating factor in escherichia coli effect of secb chaperone on production of periplasmic penicillin acylase in escherichia coli effects of pre-protein overexpression on secb synthesis in escherichia coli engineering and overexpression of periplasmic forms of the penicillin-binding protein 3 of escherichia coli combined effects of the signal sequence and the major chaperone proteins on the export of human cytokines in escherichia coli improved expression characteristics of singlechain fv fragments when fused downstream of the escherichia coli maltose-binding protein or upstream of a single immunoglobulin-constant domain the cysteine knot of a squash-type protease inhibitor as a structural scaffold for escherichia coli cell surface display of conformationally constrained peptides the extracellular domain of the human erythropoietin receptor: expression as a fusion protein in escherichia coli, purification, and biological properties periplasmic production of native human proinsulin as a fusion to e. coli ecotin. prot expr purif a simple vector system to improve performance and utilization of recombinant antibodies construction, bacterial expression, and characterization of hapten-specific singlechain fv and alkaline phosphatase fusion protein schots a: pskap/s: an expression vector for the production of single-chain fv alkaline phosphatase fusion proteins single-chain fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for elisa effect of modification of connecting peptide of proinsulin on its export immobilization and purification of enzymes with staphylococcal protein a gene fusion vectors expression of the second epidermal growth factor-like domain of human factor vii in escherichia coli tandem affinity tags for the purification of bivalent anti-dna single-chain fv expressed in escherichia coli evaluation of bottlenecks in proinsulin secretion by escherichia coli a fusion protein system for the recombinant production of short disulfide bond rich cysteine knot peptides using barnase as a purification handle barnase fusion as a tool to determine the crystal structure of the small disulfide-rich protein mcoeeti in vivo biotinylated recombinant antibodies: high efficiency of labeling and application to the cloning of active anti-human igg1 fab fragments production of fluorescent single-chain antibody fragments in escherichia coli nucleocytoplasmic shuttling of antigen in mammalian cells conferred by a soluble versus insoluble single chain antibody fragment equipped with import/export signals compatible-solute-supported peroplasmic expression of functional recombinant proteins under stress conditions expression, mutagenesis, and characterization of recombinant lowpotential cytochrome c550 of photosystem ii the bacterial twinarginine translocation pathway export pathway selectivity of escherichia coli twin arginine translocation signal peptide export of active green fluorescent protein to the periplasm by the twinarginine translocase (tat) pathway in escherichia coli genetic analysis of the twin arginine translocator secretion pathway in bacteria quantitative export of a reporter protein, gfp, by the twin-arginine translocation pathway in escherichia coli dnak plays a pivotal role in tat targeting of cueo and functions beside slyd as a general tat signal binding chaperones an essential role for the dnak molecular chaperone in stabilizing over-expressed substrate proteins of the bacterial twin-arginine translocation pathway identification of a twin-arginine leader binding protein a little help from my friends: quality control of presecretory proteins in bacteria a novel sec-independent periplasmic protein translocation pathway in escherichia coli coexpression of tord enhances the transport of gfp via the tat pathway solubilization of aggregation-prone heterologous proteins by covalent fusion of stress-responsive escherichia coli protein a scfv antibody mutant isolated in a genetic screen for improved export via the twin arginine transporter pathway exhibits faster folding efficient isolation of soluble intracellular single-chain antibodies using the twin-arginine translocation machinery display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (pecs) anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from escherichia coli-expressed libraries engineering of recombinant antibody fragments methamphetamine by anchored periplasmic expression binding and enrichment of escherichia coli speroplasts expressing inner membrane tethered scfv antibodies on surface immobilized antigens isolation of engineered, full-length antibodies from libraries expressed in escherichia coli chaperone function of fkpa, a heat shock prolyl isomerase, in the periplasm of escherichia coli localization of inclusion bodies in escherichia coli overproducing beta-lactamase or alkaline phosphatase formation of active inclusion bodies in the periplasm of escherichia coli temperature effect on inclusion body formation and stress response in the periplasm of escherichia coli too hp: parameters influencing the expression of mature glial-cell-line-derived neurotrophic factor in escherichia coli a bacterial single-chain fv antibody fragment that inhibits binding of its parental anti-e-selectin monoclonal antibody to activated human endothelial cells improving production of penicillin acylase in escherichia coli via efficient degpmediated processing of precursors in periplasm optimizing the stability of single-chain proteins by linker length and composition mutagenesis effects of unpaired cysteines on yield, solubility, and activity of different recombinant antibody constructs expressed in e. coli secretory production of human colony-stimulating factor in escherichia coli structure-function relationships of the non-lanthionine-containing peptide (classii) bacteriocins produced by gram-positive bacteria factors that influence the extracellular expression of streptavidin in escerichia coli using a bacteriocin release protein increasing the secretion ability of the kil gene for recombinant proteins in escherichia coli by using a strong stationary-phase promoter efficient specific release of periplasmic proteins from escherichia coli using temperature induction of cloned kil gene of pmb9 production of soluble and active recombinant murine interleukin-2 in escherichia coli: high level expression, kil-induced release, and purification bacterial expression of anti-dna antibody domains high level production of soluble single chain antibodies in small-scale escherichia coli cultures identification of framework residues in a secreted recombinant antibody fragment that control production level and localization in escherichia coli role of primary structure and disulfide bind formation in î²-lactamase secretion escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm mutations that allow disulfide bond formation in the cytoplasm of escherichia coli the role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the escherichia coli cytoplasm disufide bond formation in the escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins the thioredoxin superfamily: redundancy, specificity, and gray-area genomics the reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the escherichia coli periplasm complementation of dsba deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm disulfide bond formation by exported glutaredoxin indicates glutathione's presence in the e. coli periplasm monitoring disulfide bond formation in the eukaryotic cytosol mutant ahpc peroxiredoxins suppress thiol-disulfide redox deficiencies and acquire deglutathionylating activity functional plasticity of a peroxidase allows evolution of diverse disulfide-reducing pathways functional antibody single-chain fragments from the cytoplasm of escherichia coli: influence of thioredoxin reductase (trxb) efficient folding of proteins with multiple disulfide bonds in the escherichia coli cytoplasm high-level production of human collagen prolyl 4-hydroxylase in escherichia coli expression and characterization of recombinant c-terminal biotynilated extracellular domain of human receptor for the advanced glycation end products (hsrage) in escherichia coli expression and purification of neurolin immunoglobulin domain 2 from carrassius auratus (goldfish) in escherichia coli effect of folding factors in rescuing unstable heterologous lipase b to enhance its overexpres expression of active human c1 inhibitor serpin domain in escherichia coli leishmania donovani: expression and characterization of escherichia coliexpressed recombinant chitinase ldcht1 characterization of soluble, disulfide-bond stabilized, prokaryotically expressed human thyrotropin receptor ectodomain jararhagin ecd-containing disintegrin domain: expression in escherichia coli and inhibition of the platelet-collagen interaction fusion expression of human pro-urokinase with e. coli thioredoxin evolution rescues folding of human immunodeficiency virus-1 envelope glycoprotein gp120 lacking a conserved disulfide bond antibody scfv fragments without disulfide bonds made by molecular evolution in vitro folding and thermodynamic stability of an antibody fragment selected in vivo for high expression levels in escherichia coli cytoplasm a semi-synthetic repertoire of intrinsically stable antibody fragments derived from a singleframework scaffold identification of a universal vhh framework to graft non-canonical antigen-binding loops of camel single domain antibodies a focused antibody library for selecting scfvs expressed at high levels in the cytoplasm immunomodulation of enzyme function in plants by singledomain antibody fragments targeting and tracing antigens in live cells with fluorescent nanobodies intracellular immunization with cytosolic recombinant antibodies intrabodies: turning the humoral immune system outside in for intracellular immunization engineered turns of a recombinant antibody improve its in vivo folding expression studies of catalytic antibodies engineering antibodies for stability and efficient folding biotechnological applications for surfaceengineered bacteria protection of guinea-pigs against foot-and-mouth disease virus by immunization with phoe-fmdv hybrid protein surface expression of maliaria antigens in salmonella typhimurium: induction of serum antibody response upon oral vaccination of mice oral vaccination with attenuated salmonella typhimurium strain expressing borrelia burgdorferi ospa prevents murine lyme borreliosis outer membrane phoe protein of escherichia coli as a carrier for foreign antigenic determinants: immunogenicity of epitopes of foot-and-mouth disease virus a fusogenic peptide expressed on the surface of salmonella enterica elicits ctl responses to a dengue virus epitope enhanced immunity to plasmodium falciparum circumsporozoite protein (pfcsp) by using salmonella enterica serovar thypi expressing pfcsp and a pfcsp-encoding dna vaccine in a heterologous primeboost strategy development of an optimized expression system for the screening of antibody libraries displayed on the escherichia coli surface structural tolerance of bacterial autotransporters for folded passenger protein domains display and release of the plasmodium falciparum circumsporozoite protein using the autotransporter misl of salmonella enterica versatility of a vector for expressing foreigner polypeptides at the surface of gram-negative bacteria transport and anchoring of î²-lactamase to the external surface of escherichia coli peptide display on bacterial flagella: principles and applications the normally periplasmic enzyme î²-lactamase is specifically and efficiently translocated through the escherichia coli outer membrane when it is fused to the cell-surface enzyme pullulanase pseudomonas aeruginosa outer membrane protein oprf as an expression vector for foreign epitopes: the effects of positioning and length on the antigenicity of the epitope a new membrane-bound opri lipoprotein expression vector. high production of heterologous fusion proteins in gram (-) bacteria and the implications for oral vaccination expression of the plasmodium falciparum immunodominant epitope (nanp4) on the surface of salmonella enterica using the autotransporter misl delivery of heterologous protein antigens via hemolysin or autotransporter systems by an attenuated ier mutant of rabbit enteropathogenic escherichia coli neutralization of enteric coronaviruses with escherichia coli cells expressing singlechain fv-autotransporter fusions display of passenger proteins on the surface of escherichia coli k-12 by the enterohemorragic e. coli intimin eaea epitope mapping and affinity purification of monospecific antibodies by escherichia coli cell surface display of gene-derived random peptide libraries escherichia coli haemolysin: characteristic and probable role in pathogenicity isolation and analysis of the c-terminal signal directing export of escherichia coli haemolysin protein across both bacterial membranes the e. coli alpha-haemolysin secretion system and its use in vaccine development specific secretion of active single-chain fv antibodies into the supernatants of escherichia coli cultures by use of the hemolysin system formation of disulfide bonds during secretion of proteins through the periplasmic-independent type i pathway secretion of proteins with dimerization capacity by haemolysin type i transport system of escherichia coli improved secretory production of recombinant proteins by random mutagenesis of hlyb, an alphahemolysin transporter from escherichia coli advances in refolding of proteins produced in e. coli practical considerations in refolding proteins from inclusion bodies strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins in vitro folding of inclusion body proteins one-step refolding and purification of disulfide-containing proteins with a c-terminal mesna thioester in vitro-refolding of a single-chain fv fragment in the presence of heteroaromatic thiols identification of in vitro folding conditions for procathepsin s and cathepsin s using fractional factorial screens inhibition of aggregation side reactions during in vitro protein folding reversible protection of disulfide bonds followed by oxidative folding renders recombinant hcgî² highly immunogenic polyethylene glycol enhanced protein refolding control of aggregation in protein refolding: a variety of surfactants promote renaturation of carbonic anhydrase ii dynamic redox environment-intensified disulfide bond shuffling for protein refolding in vitro: molecular simulation and experimental validation an antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain vh molecules with a unique interface different effects of larginine on protein refolding: suppressing aggregates of hydrophobic interaction, not covalent binding chromatographic methods for the isolation of, and refolding of proteins from, escherichia coli inclusion bodies on-column refolding of recombinant chemokines for nmr studies and biological assays improved refolding of an immobilized fusion protein controlled oxidative protein refolding using an ion-exchange column continuous matrix assisted refolding of alpha-lactalbumin by ion exchange chromatography with recycling of aggregates combined with ultradiafiltration sakaguchi k: a novel protein refolding method using a zeolite use of zeolite to refold a disulfide-bonded protein thioredoxin-catalyzed refolding of disulfide-containing proteins prolyl isomerases catalyze antibody folding in vitro catalysis of the oxidative folding of bovine pancreatic ribonuclease a by protein disulfide isomerase effect of glutaredoxin and protein disulfide isomerase on the glutathione-dependent folding of ribonuclease a oxidative protein folding in vitro: a study of the cooperation between quiescin-sulphydryl oxidase and protein disulfide isomerase chaperone-aided in vitro renaturation of an engineered e1 envelope protein for detection of anti-rubella virus igg antibodies oxidative refolding chromatography: folding of the scorpion toxin cn5 immobilized oxidoreductase as an additive for refolding inclusion bodies: application to antibody fragment catalysis of protein folding by agaroseimmobilized protein disulfide isomerase chaperone-assisted folding of a single-chain antibody in a reconstituted translation system renaturation of a single-chain immunotoxin facilitated by chaperones and protein disulfide isomerase chaperone-assisted column refolding of gloshedobin with the use of refolding cocktail high-pressure refolding of bikunin: efficacy and thermodynamics minimal information: an urgent need to assess the functional reliability of recombinant proteins used in biological experiments the matrix refolded the author wishes to thank alicja gruszka for her critical review of the manuscript. key: cord-304953-ntg8w5k4 authors: modis, yorgo title: relating structure to evolution in class ii viral membrane fusion proteins date: 2014-02-11 journal: curr opin virol doi: 10.1016/j.coviro.2014.01.009 sha: doc_id: 304953 cord_uid: ntg8w5k4 enveloped viruses must fuse their lipid membrane to a cellular membrane to deliver the viral genome into the cytoplasm for replication. viral envelope proteins catalyze this critical membrane fusion event. they fall into at least three distinct structural classes. class ii fusion proteins have a conserved three-domain architecture and are found in many important viral pathogens. until 2013, class ii proteins had only been found in flaviviruses and alphaviruses. however, in 2013 a class ii fusion protein was discovered in the unrelated phlebovirus genus, and two unexpectedly divergent envelope proteins were identified in families that also contain prototypical class ii proteins. the structural relationships of newly identified class ii proteins, reviewed herein, shift the paradigm for how these proteins evolved. viral envelope proteins are the principal effectors of virus assembly and cell entry. enveloped viruses must fuse their lipid membrane with a host-cell membrane in order to deliver their genome into the cytoplasm for replication. this membrane fusion event is catalyzed by viral envelope proteins. viruses also rely on their envelope proteins to recognize host cells by binding cellular receptors. envelope proteins shield viruses from the immune system and bear most of the neutralizing antibody epitopes against any given virus. the envelope proteins of many viruses form a rigid outer structural shell, which usually takes the form of a quasi-spherical icosahedral assembly. viral membrane fusion proteins fall into at least three distinct structural classes. the influenza virus hemagglutinin (ha) is the prototype of ''class i'' fusion proteins [1] , which encompass those of other orthomyxoviruses and paramyxoviruses, retroviruses, filoviruses, and coronaviruses [2] . the unifying structural feature of class i fusion proteins is a core consisting of three bundled ahelices [3, 4] . class ii fusion proteins are a structurally unrelated class found in flaviviruses, alphaviruses, and most recently in rubella virus (sole member of the rubivirus genus) and rift valley fever virus (from the phlebovirus genus) [4,5 ,6 ]. class ii proteins share a threedomain architecture consisting almost entirely of bstrands, with tightly folded ''fusion loops'' in the central domain serving as the anchor in the cellular membrane targeted for fusion ( figure 1 ). class iii fusion proteins, found in herpesviruses, rhabdoviruses and baculoviruses, possess structural features from both class i proteins (a core three-helix bundle) and from class ii proteins (a central b-stranded fusion domain) [7] . until recently, class ii proteins had only been found in flaviviruses and alphaviruses (in the flaviviridae and togaviridae families, respectively), which share many key characteristics. indeed viruses from these two genera all have positive-stranded rna genomes of 11-12 kilobases with similar gene organizations, icosahedral outer protein shells with a diameter of approximately 50 nm, and lifecycles that alternate between vertebrates and arthropod vectors [8] . the most plausible evolutionary model had thus been one in which flaviviruses and alphaviruses evolved from a common ancestor virus. however, a class ii fusion protein was recently discovered in the unrelated bunyaviridae family [5 ]. conversely, divergent fusion protein architectures have emerged within the flaviviridae and togaviridae families in which the prototypical class ii proteins were first identified [6 ,9 ,10 ]. together, these recent discoveries shift the evolutionary paradigm from a divergent model (common ancestor virus), to a model in which class ii fusion proteins evolved independently by borrowing from a common (or related) ancestral class ii cellular membrane fusion protein. unifying structural features of class ii envelope proteins envelope proteins from flaviviruses and alphaviruses assemble into icosahedral outer shells, but the mode of assembly differs in the two families, with alphaviruses forming canonical (t = 4) quasi-equivalent assemblies [19,22 ,23 ] and flaviviruses forming unusual non-equivalent icosahedral assemblies [24, 25, 26 ] . class ii proteins are anchored in the viral membrane via a c-terminal transmembrane anchor, which is linked by a flexible ''stem'' region to the ectodomain (figure 2 ). the ectodomain consists of three domains: a b-barrel (domain i); an elongated, mostly b-stranded domain bearing a tightly folded ''fusion loop'' that inserts into the target cellular membrane (domain ii); and an igc-like module that bears the epitopes responsible for cellular tropism and efficient antibody neutralization (domain iii) [11, [27] [28] [29] . remarkably, despite evidence that domain iii is directly involved in cellular attachment of flaviviruses [30, 31] , no receptors that bind to class ii proteins in flaviviruses or alphaviruses have yet been identified. however, protein-glycan interactions involving class ii glycoproteins have been shown to contribute to attachment (but not endocytosis [32, 33] ) of certain flaviviruses in a subset of host-cell types. these interactions involve the c-type lectins dc-sign and l-sign [15, [34] [35] [36] , mannose receptor [37] , and cell-surface heparan sulfate [38] . in alphaviruses, it is the non-fusogenic e2 spike protein that mediates receptor binding but interestingly e2 also recognizes dc-sign, l-sign and heparan sulfate [39, 40] . crystal structures of various class ii envelope proteins before and after the conformational change that catalyzes membrane fusion provide a molecular outline of the fusion mechanism (figure 1 ) [11] [12] [13] [14] [15] [19] [20] [21] [41] [42] [43] [44] [45] . complementing these prefusion and postfusion structures, structures thought to represent fusion intermediates provide invaluable insights on the steps required for fusion [5 ,20,45,46] . in the mechanism that is emerging until 2013, class ii proteins had only been found in flaviviruses and alphaviruses, but recent studies suggest that the class ii fold is more widely prevalent than previously anticipated. indeed, dessau and modis showed that glycoprotein c (gc) from rift valley fever virus (rvfv) is a class ii fusion protein [5 ] . rvfv belongs to the phlebovirus genus in the bunyaviridae family, which is unrelated to flaviviruses or alphaviruses. moreover, rubella virus e1 was shown to have a class ii fold, albeit with a more divergent structure than expected for a virus in the same togaviridae family as alphaviruses [6 ] . however, despite the presence of some novel structural features in both rvfv gc and rubella e1, the two proteins still possess each of the core structural features (described earlier in this section) that unify class ii fusion proteins (figures 1 and 2) . these parallels even extend to receptor binding in the case of phleboviruses, since rvfv and uukuniemi virus were recently shown to utilize dc-sign as a receptor [49] . in the case of rubella, myelin oligodendrocyte glycoprotein (mog) was recently identified as a putative receptor for e1 [50] , making mog the first receptor reported to bind to a class ii protein via protein-protein interactions. the identification in 2013 of a class ii fusion protein in rvfv [5 ], although it had been predicted by amino acid analysis [51] , was nevertheless unexpected because phleboviruses such as rvfv do not have any of the key characteristics shared by flaviviruses and alphaviruses. phleboviruses have segmented negative-sense and ambisense rna genomes, undergo membrane fusion much later in late endosomes [52] , and their envelope proteins form much larger (103 nm diameter) t = 12 icosahedral lattices [53, 54] with a novel mode of assembly [5 ] . the structure of rvfv gc is strikingly similar to flavivirus e structures (especially dengue e), more similar in fact than flavivirus and alphavirus envelope proteins are to each other. the most notable similarity is that gc forms dimers that have the same head-to-tail configuration as flavivirus e dimers, with the fusion loop buried at the dimer interface ( figure 3 ). this is particularly surprising given that the e dimer is the building block of the flavivirus non-equivalent ''herringbone'' assembly, which is very distinct from the t = 12 phlebovirus assembly, although interestingly a nonequivalent configuration has been proposed for the latter [5 ] . another noteworthy similarity between gc and e is the fusion loop, which has the same tightly folded glycinerich structure in the two proteins ( figure 3) . together, the structural similarities of gc and e are strongly suggestive of some sort of evolutionary link between the bunyaviridae and flaviviridae families. divergence of the class ii fold within the togaviridae family in another recent advance, the e1 protein of rubella virus was found to have the most divergent class ii fold identified so far. this was unexpected given that rubella virus belongs to the same togaviridae family as alphaviruses. the most notable differences of the rubella e1 structure, which was crystallized in the trimeric postfusion conformation, are in domain ii (figure 1 ). domain ii is larger due to three insertions. instead of a single 10-15-amino acid fusion loop, rubella e1 has two fusion loops that project a total of 15 aromatic side chains (mainly tyrosines) for interaction the cellular membrane ( figure 3) [6 ]. a metal ion (na + or ca 2+ ) is coordinated between the two fusion loops and bound ca 2+ allows rubella e1 to bind lipid membranes a neutral ph [6 ] . there are no metal sites in the other class ii fusion proteins, or in the fusion motifs of any other viral fusion protein reported to date. another distinctive feature of the rubella e1 structure is that domain iii is swapped in the e1 trimer, occupying the position of domain iii from the neighboring subunit in the flavivirus and alphavirus postfusion e trimers. additionally, the rubella e1 structure includes the stem (figure 1) , which connects domain iii to the transmembrane anchor and is either absent or mostly disordered in the structures of other class ii proteins. lastly, rubella virus particles exhibit a large degree of pleomorphy [55 ] , making rubella e1 the only class ii fusion protein known not to form an icosahedral assembly. a new envelope protein fold in the flaviviridae family . the gc dimers are strikingly similar to the flavivirus e dimers (dengue type 2 e shown here [12] ). e dimers are the building block of the icosahedral outer protein shell in flaviviruses [26] . structures were available only from the flavivirus genus. envelope proteins from pestiviruses and hepaciviruses had been predicted to have class ii folds based on the disulfide bonding pattern [56] and on amino acid sequence analyses of the e1 and e2 envelope proteins [57] . it was therefore surprising when two groups discovered in 2013 that the larger envelope protein, e2, from the pestivirus bvdv (bovine viral diarrhea virus) is not a class ii fusion protein. instead bvdv e2 has a novel fold, suggesting that pestiviruses have a non-class ii fusion machinery. since e1, with its 174-amino acid ectodomain, is too small to be a class ii fusogen, the e2 structure appears to define a new structural class of fusion proteins (figure 4) [9 ,10 ]. the structure of bvdv e2 provides an even more striking example than rubella e1 of how structurally divergent viral envelope proteins can be within a single virus family. the discovery of a class ii fusion protein in a phlebovirus [5 ], in a virus family otherwise unrelated to flaviviruses and alphaviruses, reveals that the class ii fold is more prevalent and more widely distributed across virus families than was previously anticipated. the striking structural similarity between the flavivirus e proteins and rvfv g -which extends to the mode of dimerization even though e and gc dimers form different types of icosahedral lattices -is strongly suggestive of a common evolutionary origin for certain envelope proteins within the bunyaviridae and flaviviridae families. but what is the nature of this link? the two virus families clearly differ in their genomic organization, coding strategies and outer protein shell assemblies (figure 4 ). in the light of these differences it is tempting to speculate that, rather than diverging from a common ancestor virus, class ii fusion proteins may instead have evolved independently from a common (or related) and as yet unidentified ancestral cellular class ii membrane fusion protein. the concept of independent transmission of class ii fusion proteins from hosts to viruses is supported by the discovery that certain viruses within the same family with similar genomic organizations can have distinct fusion machineries. indeed, pestiviruses have a non-class ii fusion machinery distinct from that of flaviviruses even though the two genera are adjacent to each other in phylogenetic tree of the flaviviridae family [9 ]. thus, although pestiviruses and flaviviruses may have evolved from a common ancestor virus, they evidently borrowed their fusion machineries from different sources. these could presumably be structural relationships of viruses that contain class ii fusion proteins. the class ii fold is highly conserved in flaviviruses, alphaviruses and phleboviruses, even though these viruses differ in their genomic organization, coding strategies and outer protein shell assemblies. these three genera have in common that they have lifecycles that alternate between vertebrate and arthropod hosts. rubella virus (rv) e1 has the most divergent class ii fold even though rubella belongs to the same family as alphaviruses (togaviridae). glycoprotein e2 from the pestivirus bovine viral diarrhea virus has a novel fold even though pestiviruses belong to the same family as flaviviruses (flaviviridae) [9 ,10 ]. rubella virus and pestiviruses, and their close relatives the hepaciviruses, have in common that they infect strictly vertebrate hosts, and also that they do not form rigid icosahedral outer protein shells. thus, structural conservation in viral fusion proteins does not correlate with overall phylogenetic relatedness. the virus particles shown here are, clockwise from top right, dengue virus, semliki forest virus, rv, rift valley fever virus and hepatitis c virus (hcv). the electron micrographs of rv [55 ] and hcv [61] are not drawn to scale with the particles in color. the phylogenetic tree is based on qualitative structural and genetic relationships between envelope proteins and is not based on a quantitative phylogenetic analysis. different host fusion proteins, but alternatively different virus species could conceivably have borrowed fusion proteins from each other during co-infections with multiple viruses. the conservation of an a-helical coiled coil architecture in class i viral proteins and in the snare family of intracellular vesicle fusion proteins provides a compelling precedent for the evolutionary transfer of a structural membrane fusion fold between host and virus during evolution. although similarities between class i fusion proteins and snares have long been recognized [58] , the link was further strengthened by a recent study demonstrating that a paramyxovirus class i fusion protein resembles snares in that it has ahelical transmembrane anchors in both membranes before fusion, with subsequent zippering of the coiled coils during fusion resulting in a bundle of helical hairpins that extends across the fused membrane [59 ,60] . alphaviruses and flaviviruses seem to have undergone a more conservative evolution, despite belonging to different families. the discovery of a divergent class ii fold in rubella virus within the same family as alphaviruses (togaviridae) was therefore unexpected [6 ] . notably, the more canonical class ii folds have all been found in viruses alternating between arthropod and vertebrate hosts, whereas rubella virus infects only humans. the structural conservation of class ii proteins in viruses with vertebrate-arthropod lifecycles may reflect more stringent evolutionary restraints exerted on these viruses. rubella virus, along with pestiviruses and hepaciviruses, each have a single vertebrate host with which they seem to have co-evolved more rapidly. together, the structural relationships that have emerged between envelope proteins across different virus families are consistent with an evolutionary model in which class ii fusion proteins originate from an as yet unidentified set of ancestral class ii membrane fusion proteins in the host. moreover, fusion proteins appear to have been transferred as independent modules, implying that the class ii membrane fusion fold may have been hijacked by different viruses at different times throughout evolution. . this study showed that the gc envelope protein from rift valley fever virus (from the bunyaviridae family) has a class ii fold with striking resemblances to that of e from dengue and other flaviviruses, including a propensity to form head-to-tail dimers with a hydrophobic membrane anchor, or fusion loop buried at the dimer interface. rvfv gc was the first class ii protein identified in a virus family otherwise unrelated to flaviviruses and alphaviruses, suggesting that class ii proteins may have been transferred as independent modules during evolution from a host or another virus. this study showed that the e1 envelope protein from rubella virus has the most structurally divergent class ii fold identified so far. this was unexpected because rubella virus is in the same togaviridae family as alphaviruses, which have canonical class ii proteins. rubella e1 is the first class ii fusion protein to be identified in a virus that that does not alternate between vertebrate and arthropod hosts -rubella virus only infects humans. this suggests that the envelope proteins of viruses with both insect and vertebrate hosts may be subject to more stringent evolutionary restraints. a ligand-binding pocket in the dengue virus envelope glycoprotein crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes crystal structure of the japanese encephalitis virus envelope protein variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein structural insights into the neutralization mechanism of a higher primate antibody against dengue virus the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph structural changes of envelope proteins during alphavirus fusion glycoprotein organization of chikungunya virus particles revealed by x-ray crystallography chiu w: 4.4 a cryo-em structure of an enveloped alphavirus venezuelan equine encephalitis virus 23 ] reveal the structure of an alphavirus particle in unprecendented detail. the electron microscopy structure clearly resolves the transmembrane helices and cytoplasmic tails of the class ii fusion protein e1, and of the receptor binding envelope protein e2 the structure of barmah forest virus as revealed by cryo-electron microscopy at a 6-angstrom resolution has detailed transmembrane protein architecture and interactions structure of west nile virus structure of dengue virus: implications for flavivirus organization, maturation, and fusion cryo-em structure of the mature dengue virus at 3.5-a resolution this study reveals the structure of a flavivirus particle in unprecendented detail, at near-atomic resolution. the electron microscopy structure reveals features that were missing in previously determined crystal structures, including the unusual truncated helical hairpin transmembrane anchor of the class ii fusogen, e, and a segment of envelope protein m that contains three ph-sensing histidines monoclonal antibodies that bind to domain iii of dengue virus e glycoprotein are the most efficient blockers of virus adsorption to vero cells development of a humanized monoclonal antibody with therapeutic potential against west nile virus structural basis of west nile virus neutralization by a therapeutic antibody an external loop region of domain iii of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells evolutionary reversals during viral adaptation to alternating hosts dermal-type macrophages expressing cd209/ dc-sign show inherent resistance to dengue virus growth dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals west nile virus discriminates between dc-sign and dc-signr for cellular attachment and infection dendritic-cellspecific icam3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquitocell-derived dengue viruses dc-sign (cd209) mediates dengue virus infection of human dendritic cells the mannose receptor mediates dengue virus infection of macrophages dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate dc-sign and l-sign can act as attachment receptors for alphaviruses and distinguish between mosquito cell-and mammalian cell-derived viruses adaptation of sindbis virus to bhk cells selects for use of heparan sulfate as an attachment receptor structure of the dengue virus envelope protein after membrane fusion structure of a flavivirus envelope glycoprotein in its low-ph-induced membrane fusion conformation conformational change and protein-protein interactions of the fusion protein of semliki forest virus structure of the st. louis encephalitis virus postfusion envelope trimer structure of a dengue virus envelope protein late-stage fusion intermediate a stable prefusion intermediate of the alphavirus fusion protein reveals critical features of class ii membrane fusion characterization of a structural intermediate of flavivirus membrane fusion viral membrane fusion and nucleocapsid delivery into the cytoplasm are distinct events in some flaviviruses dc-sign as a receptor for phleboviruses identification of the myelin oligodendrocyte glycoprotein as a cellular receptor for rubella virus proteomics computational analyses suggest that the carboxyl terminal glycoproteins of bunyaviruses are class ii viral fusion protein (betapenetrenes) entry of bunyaviruses into mammalian cells insights into bunyavirus architecture from electron cryotomography of uukuniemi virus electron cryomicroscopy and single-particle averaging of rift valley fever virus: evidence for gn-gc glycoprotein heterodimers cryo-electron tomography of rubella virus the authors use electron cryomicroscopy to examine the structure of rubella virions. the rubella virus envelope proteins, e1 and e2, are shown to be organized into extended rows on the viral surface the disulfide bonds in glycoprotein e2 of hepatitis c virus reveal the tertiary organization of the molecule proteomics computational analyses suggest that hepatitis c virus e1 and pestivirus e2 envelope glycoproteins are truncated class ii fusion proteins coiled coils in both intracellular vesicle and viral membrane fusion transmembrane orientation and possible role of the fusogenic peptide from parainfluenza virus 5 (piv5) in promoting fusion the authors show that the n-terminal fusion peptide of a paramyxovirus class i fusion protein forms a transmembrane a-helix after membrane insertion. this helix extends the helical coiled coil core of the fusion protein and contributes to the zippering of the coiled coils during fusion. in the trimeric postfusion conformation, the n-terminal and c-terminal helices form a bundle that extends across the fused membrane. snares also have n-terminal and c-terminal transmembrane helices that contribute to the fusogenic zippering of helices. the additional parallels between class i proteins and snares strengthen the case for an evolutionary link between these two protein families helical extension of the neuronal snare complex into the membrane ultrastructural and biophysical characterization of hepatitis c virus particles produced in cell culture this work was supported by a burroughs wellcome investigator in the pathogenesis of infectious disease award, and nih grant r01 gm102869 to y.m. key: cord-323358-05bk91lm authors: bhaskar, sathyamoorthy; lim, sierin title: engineering protein nanocages as carriers for biomedical applications date: 2017-04-07 journal: npg asia mater doi: 10.1038/am.2016.128 sha: doc_id: 323358 cord_uid: 05bk91lm protein nanocages have been explored as potential carriers in biomedicine. formed by the self-assembly of protein subunits, the caged structure has three surfaces that can be engineered: the interior, the exterior and the intersubunit. therapeutic and diagnostic molecules have been loaded in the interior of nanocages, while their external surfaces have been engineered to enhance their biocompatibility and targeting abilities. modifications of the intersubunit interactions have been shown to modulate the self-assembly profile with implications for tuning the molecular release. we review natural and synthetic protein nanocages that have been modified using chemical and genetic engineering techniques to impart non-natural functions that are responsive to the complex cellular microenvironment of malignant cells while delivering molecular cargos with improved efficiencies and minimal toxicity. smart nanosized materials with high stability, suitable pharmacokinetics and efficient cell permeability for delivering cargo molecules to target cells have been the requirements for an ideal drug delivery system. 1 advances in the design and fabrication of synthetic carriers such as cationic liposomes, micelles, block copolymers, carbon nanotubes, dendrimers and inorganic nanoparticles are restricted by severe toxicity and low delivery efficiency. 2 nature-derived nanocarriers are potential alternatives to synthetic ones as they satisfy most of the key features, such as biocompatibility, water solubility and high cellular uptake efficiency with minimal toxicity. 1, 3 examples of naturederived nanocarriers include protein nanocages such as viruses, ferritin and many others that are formed by the self-assembly of protein subunits, resulting in a cage-like structure. the monodispersed subunits are modifiable through chemical and genetic methods. 3 the key biomedical applications involving the use of protein nanocages that we focus on in this review are therapeutics and diagnostics. among myriad nature-derived nanocarriers, protein-based biological systems such as viruses have been a subject of intense study owing to their innate ability to penetrate cell membranes. the structure of viruses best represents the principles of protein assembly in nature. structural analyses of viruses show that they consist of a protein shell comprising a definite number of subunits that surrounds and protects its genome. 4 viral capsids are naturally programmed for host-cell targeting and cell entry. they have evolved to mediate the exchange of nucleic acids between different chemical environments. 5 viruses are stable structures that have the ability to withstand environmental pressures but are sensitive enough to detect signals or the change in signals in cellular environment, thereby releasing the nucleic acids they carry in the target microenvironment. viruses have thus been an inspiration in developing diverse self-assembling protein nanocages from natural sources. important aspects of protein nanocages such as biocompatibility, functional diversity, biological fabrication and flexibility of design by protein engineering make them powerful materials for various applications. 5 viruses have been engineered to perform specific functions. for example, bacteriophages have been used in peptide display, filamentous phages have been used as templates for nanofabrication and virus-like particles (vlps) have been used as immunogens. with the exception of peptide-displaying filamentous phages commonly used for nucleic acid and conjugated drug delivery in vitro and in vivo, these nanoparticles are hardly suitable for celltargeted drug delivery. 6 specific functional polypeptides can be modified to self-assemble into nanoparticles with or without caged structures with desirable nanoscale properties in terms of size and geometry. the assembly of the functional building blocks to form protein nanoparticles, as observed in natural viruses, can seldom be mimicked by general nanofabrication techniques. 6 the intricacy stems from selecting protein sequences that promote protein-protein interactions without nonspecific aggregation. these protein sequences can be derived from both natural and non-natural amino-acid sequences that lead to selfassembly in various patterns. 6 natural protein scaffolds are structures that already exist in nature with intrinsic self-assembling properties, including viruses, ferritin and eukaryotic vaults. in contrast, synthetic protein scaffolds are designed de novo to mimic the properties of the natural scaffolds and carry out specific functions. the advantage of both natural and synthetic protein scaffolds is that they can be exploited for the design of novel functional architectures. 5 specific applications of protein nanocages for drug delivery have been discussed in a review by molino et al. 7 and schoonen et al. 8 in this review, we focus on natural and synthetic protein scaffolds engineered with specific functional groups to impart non-native functions, including aiding the delivery of active molecules through targeting of malignant cells and overcoming cellular barriers. natural scaffolds protein scaffolds have been found to facilitate the formation of various inorganic structures in nature. for example, the single-celled algae emiliania huxleyi form fine calcium carbonate structures called coccoliths. proteins control the formation of these structures by guiding the assembly of crystallites into three-dimensional structures. 4 an examination at the molecular level shows that complex systems such as cells use proteins, in addition to phospholipids, as physical boundaries that define and separate the inside of the cell into subcellular compartments. here, we present five classes of structures that are distinct in size and morphology: viruses, ferritin, e2 protein, vaults and other protein nanocages. viruses and virus-based particles. years of studies to deduce information about viral assembly, replication and infection pathways provide a clear idea on the stability and functionality of viruses. 3 viral transduction has been observed to be facilitated by self-assembling protein structures called capsids, which interact with host cells and deliver their genome into the target cell compartment. 1 the outer surface of the viral capsid opens up several opportunities for diverse chemical functionalization and site-specific modifications to synthesize organic/inorganic materials and to link targeting moieties. 9 viruses have an innate mechanism for protecting their genome, transferring it to the extracellular environment, escaping the immune system upon reaching target cells by interacting with their receptors, and delivering the packaged nucleic acids into the desired cell compartment. therefore, viruses are used as functional carriers for targeted delivery with built-in advantages over synthetic delivery vehicles. 1 viruses are the dominant class of the bionanoparticle family, with sizes ranging widely from 10 to 100 nm ( figure 1 ). 3 in addition to cell targeting ability and gene delivery efficiency, these protein-based multifaceted systems have highly ordered spatial configurations, and the stability and functionality of these materials have already been established through intensive research with advances in understanding virus infection, replication and assembly pathways. 3 the cowpea mosaic virus (cpmv) (pdb id 2bfu) was the first nanocage among viruses shown to be conjugated to a variety of functional molecules. this nanocage is a well-characterized plant virus that can be produced in high amounts. the composition of the amino-acid residues in the virus's structure is well understood for bioconjugation with functional molecules. the virus is stable over a wide range of ph levels, temperatures and organic solvents. it could therefore be used for assembling other inorganic nanoparticles. 10 genetically engineered cpmv possessing thiol groups are produced and covalently attached to gold nanospheres. the nanospheres self-assemble into icosahedral plasmonic nanoclusters following the locations of the thiol groups on the cpmv and exhibit a 10-fold increase in local electromagnetic fields. these plasmonic nanoclusters have unique optical properties that can be used for spectroscopy and cancer treatment. 10 multiple orthogonal reactive sites on viral nanocages can be established by genetic engineering for bioconjugation with signalling moieties and biological recognition motifs. 11 two different amino-acid residues of turnip yellow mosaic virus (pdb id 1auy) are used for conjugation to luminescent terbium complexes and biotin molecules. the modified nanocages can be used as a scaffold for the development of sensor by time-resolved fluoroimmunoassay. 11 recent advances in selective chemical modifications of viruses and viral-based particles have broadened the scope of modulating bionanoparticles beyond genetic mutations. 3 ferritin. in 1937, ferritin was discovered as a novel protein structure for storing and transporting iron molecules. ferritin isolated from horse spleen contained 20% iron in its native structure. 9 subsequently, it has been found to exist almost ubiquitously in biological systems, s bhaskar and s lim regulating the storage and release of iron to maintain homeostasis. these findings have shed light on the idea that proteins can be used to encapsulate and deliver active molecules in vivo. the first x-ray diffraction data of horse spleen ferritin were reported in 1943 12 and the structure in 1984, 13 but it was not until 1991 that the crystal structure of human ferritin (1fha) was first reported and the cagelike architecture was clearly elucidated. 14 since then, numerous ferritins and ferritin-like proteins have been developed for exogenous applications other than iron storage. 9 the globular, multisubunit caged ferritin stores iron in an insoluble non-toxic state while keeping it bioavailable within cells by readily converting it to its soluble form. the iron in the central cavity is maintained as small crystalline particles in an organic ferric oxyhydroxide form. ferritin can withstand high temperature and a wide range of ph levels for limited periods without significant disruption to its caged quaternary structure, which is formed by 24 subunits. these subunits self-assemble into a hollow spherical structure with an outer diameter of 12 nm and an internal cavity with a diameter of 8 nm 5 ( figure 2 ). ferritin's iron core is approximately the size of its internal diameter. this critical diameter and several molecular characteristics, including electrophoretic mobility, solubility and antigenicity, are the most significant properties of the protein shell. 13 the self-assembly and disassembly properties of the ferritin nanocage can be controlled by metal ions, thereby aiding the uptake and release of active molecules from the protein structure. 17 similarly, the structural and biochemical properties of ferritin protein are used for tailoring it to a wide range of applications, from the synthesis of nanoparticles to the design of vaccines in biomedicine. 18 e2 protein. the e2 protein is derived from the e2 core domain (dihydrolipoamide acetyltransferase) of the pyruvate dehydrogenase multienzyme complex. the protein is formed by 24 subunits in e. coli or 60 subunits in geobacillus stearothermophilus that self-assemble into a hollow structure with a cubic core or an icosahedron with twelve 5-nm openings (pdb id 1b5s), respectively. 19 pyruvate dehydrogenase enzyme complex is composed of three enzymes: pyruvate decarboxylase (e1), dihydrolipoamide acetyltransferase (e2) and dihydrolipoamide dehydrogenase (e3) (figure 3 ). it has been shown that the complex can be produced separately and the component enzymes can be reassembled in vitro to form the native structure. 19 because e1 and e3 do not dissociate from each other in the presence of the enzyme e2, it is evident that the latter forms the structural core of the assembled complex. g. stearothermophilus is a thermophilic organism. hence, the pyruvate dehydrogenase complex derived from it has innate stability that allows it to survive under extreme conditions. unlike other nanocages such as viral capsids, the modification of the external surface with functional ligands does not hinder the assembly of the e2 core domain. this property is identical to that in its natural state, in which the e2 domain is linked to two distinct folding protein domains at its surface. 19 similar to other natural protein nanocages, e2 protein nanocages can be subjected to genetic or chemical modifications to enable several functions such as drug loading and specific targeting protein attachment. ren et al. showed that the e2 protein can be modified both on the internal and external surfaces for loading of drug molecules inside and for displaying functional epitopes on the outer surface of the nanocage simultaneously. 21 thus, the e2 protein cage offers promising avenues for tailored engineering of the exterior, the subunit-subunit interfaces and the interior to produce the desired modifications. 22 vaults. first reported in 1986, vaults are ribonucleoparticles with a mass of 13 mda found in most eukaryotic cells. 23 vaults have a barrellike structure measuring 41 × 41 × 72.5 nm 3 with extending caps at their extremities and a hinged waist region. 24 the nanostructure consists of several highly conserved proteins, including the major vault protein (mvp: pdb ids 2qzv, 4v60), which constitutes over 70% of the overall mass of the nanoparticle, vault poly(adp-ribose) polymerase (vparp), telomerase-associated protein 1 (tep1) and some untranslated rna molecules. 25 the basic vault structure consists of 78-96 mvps arranged from the c to n terminus from the cap to the waist, forming a thin protein coat covering an internal cavity of 5 × 10 7 å 3 . the n terminus is tucked towards the interior of the vault. the mvps are non-covalently associated. despite the strong binding between mvps, the vault has a dynamic structure that opens and closes transiently, a phenomenon referred to as breathing, to incorporate small molecules, proteins and other macromolecules within the inner core. 24 . peptide extensions at the n termini (shown in red) and c termini (shown in blue) from the waist region (light red) and the extreme ends of the caps, respectively (aa and b). by fusing an exogenous protein at int binding site (ac) and additional peptide at the n terminus (ad), a composite assembly with multifunctional units could be formed (ae) 26 recombinant production of mvps using a baculovirus expression system in insect cells shows that the protein structure can sufficiently aid in directing the formation of recombinant vault particle with a structure analogous to the wild-type particles. the naturally occurring vault nanocages can thus be engineered for a wide range of novel applications. 28 other natural scaffolds. bacterial species have been found to host self-assembling nano-and microsized structures called bacterial microcompartments (bmcs) containing protein complexes formed by 60-20,000 copies of one or more self-assembled protein species. bmcs are part of specific metabolic pathways of the cell, encapsulating unstable or toxic intermediate metabolites. 1 carboxysomes were the first bacterial organelles identified to perform such functions. as described in 1973, carboxysomes have icosahedral structures with a cross-section measuring 100-150 nm and a protein coat containing 6-10 distinct proteins; they are involved in autotrophic co 2 fixation in bacteria via rubisco and carbonic anhydrase enzymes. 1 similar to viral capsids, the formation of carboxysomes' icosahedral structure requires a combination of hexameric and pentameric self-assembling units of bmc proteins. interestingly, the self-assembly property of the carboxysomes was retained in vivo even after the deletion of the rubisco enzyme. 29 the flat sheet of the icosahedron is formed by the hexameric units that assemble side by side, whereas the vertices are occupied by the pentameric units ( figure 5 ). a common bmc subunit consists of 90 amino acids with an α-/β-fold structure. 1 in 1994, a protein-based organelle from the hyperthermophilic bacterium thermotoga maritima, referred to as 'linocins', was described as the smallest bmc, measuring 20-24 nm in diameter. 31 later, these protein structures were renamed 'encapsulins'. 1 constant efforts are being made to engineer natural protein sequences to impart new or additional functions. recent studies have taken the efforts to a higher level by creating self-assembling protein structures through de novo design. the assembly of small peptides occurs through various non-covalent interactions, van der waals, hydrophobic, π-π staking, electrostatic interactions, hydrogen bonds and disulfide bond formation. 1 on the smallest possible scale, small proteins that self-assemble to form helical bundles consisting of two to four helices are designed by focusing on simple folding patterns, particularly the α-helices or coiled-coil arrangements. 32 the phenomenon of biological self-assembly observed in amyloidogenesis, in which a soluble protein is transformed into insoluble aggregates (amyloid fibrils) by a chemically specific molecular assembly process involving domain swapping and symmetric oligomerization, could be considered the inspiration in designing artificial supramolecular protein structures. 32 stringing multiple protein domains to construct modular proteins allows for the incorporation of multiple functions into a single polypeptide chain. the modular protein has been used for in vitro condensation and targeted cell delivery of nucleic acids. 1 this novel approach has paved the way to designing a variety of protein/dna complexes to deliver nucleic acid therapeutics both in vitro and in vivo. self-assembling polypeptides are created by leveraging natural protein oligomerization domains to form higher-order structures. the interfacing surfaces of these domains can be tuned for assembly into tetrahedral and octahedral structures. 1 yeates group leverages on symmetry in designing novel protein cages. 33, 34 two or more subunits are fused in a geometrically predefined manner which subsequently self-assemble into highly symmetrical structures ( figure 6 ). 35 the design principles have led to the production of megadalton protein nanocages with icosahedral symmetry. 36 gradišar et al. 37 proposed a different strategy inspired by dna origami approach. polyhedral structures consisting of individual self-assembling polypeptide nanostructures based on modularized orthogonal dimerizing segments. the formation of these dimerizing segments is controlled by electrostatic and hydrophobic interactions that serve as a platform for designing new artificial polypeptide folds (figure 7 ). 37 the interlinked coiled-coil segment-based design can lead to asymmetric structures that are s bhaskar and s lim challenging to construct by the assembly of organic and inorganic subunits. the enormous size of existing protein structural databases could aid the design of a diverse class of self-assembling structures. 35 hybrid protein scaffolds to expand their applications in diagnostics/therapeutics and to modulate the immune response, protein nanocages have been conjugated with other moieties (i.e. sugar, lipid, nucleotides and polymer) that result in hybrid protein scaffolds. synthetic polymer conjugates of viruses, such as tobacco mosaic virus, adenovirus, and cowpea mosaic virus, are the most widely reported hybrid conjugates of protein scaffolds. 38 lucon et al. 39 synthesized poly(2-aminoethyl methacrylate) from the interior of p22 viral capsids through atom-transfer radical polymerization, producing particles with increased loading capacity for magnetic resonance imaging (mri) contrast agents, gd-dtpa. 39 protein nanocages could be enclosed within a polymeric shell attached covalently to the external surface of the protein core. 2 polymerizable vinyl groups are grafted to the protein, and further polymerization with crosslinkers and monomers favors the formation of a thin polymer coat around the protein. such strategies are designed to produce protein nanocages with a non-degradable or degradable polymeric shell for designated purposes ( figure 8 ). 2 similarly, introducing a certain class of 'smart polymers' could impart stimuliresponsive properties to protein nanocages. 28 smart polymers are designed to be sensitive to external factors such as heat, ph, magnetic field and enzymatic degradation, as well as other microenvironmental changes. for instance, pnipaam (poly(n-isopropylacrylamide)) is a well-known temperature-sensitive polymer that undergoes a reversible phase change, losing its water solubility above the lower critical solution temperature. 28 matsumoto et al. 28 produced novel biohybrid nanoparticles through covalent linkage of pnipaam to recombinant cp-mvp (cysteine-rich 12-amino-acid peptide to the n-terminus of the mvp) vaults ( figure 9 ). 28 the resulting polymerized particles were thermally responsive as expected. although synthetic polymer-protein nanocage hybrids have been widely explored, reports on sugar, polysaccharide, nucleotide or lipid modifications are limited. the properties of carriers for delivery of drugs, contrast agents or other active molecules should be considered depending on the target and specific therapeutic index to be achieved. certain essential factors include drug efficacy, the target physiological environment, stability, safety, loading capacity and drug release kinetics of the carrier. 40 carriers measuring 25-50 nm favor receptor-mediated endocytosis and target cell membrane encapsulation. 8 an advantage of nanocagebased carriers is the ability to decorate these nanocarriers with multiple functional elements precisely and uniformly. 8 these functional elements aid the carriers because of their ability to modulate the immune response, their penetration efficiency, and their targeting capability for delivery of active molecules. most importantly, the carrier should be non-toxic for clinical applications. 40 three main properties underlying the design of a drug carrier are discussed in the following subsections. an ideal drug carrier should have significant biocompatibility. the definition of biocompatibility pertaining to drug delivery systems has been concisely reviewed by kohane and langer. 41 a more comprehensive review by naahidi et al. 42 emphasized that the ideal drug carrier should be devoid of intolerable toxic, thrombogenic, carcinogenic and immunogenic effects. the carrier should be sustained in the host long enough to carry out its intended function; therefore, the half-life of the carrier is an essential factor for ensuring its effectiveness. the delivery vehicle should be cleared from the biological system after performing its function without settling in any organs, which can lead to long-term side effects. the biocompatibility of a carrier nanoparticle is thus a relative property that depends on the risk-benefit ratio regarding its overall role without causing considerable damage to the body of the host. the suitability of the type of carrier used might vary from one context to another, but the end result should be an appropriate host response. 42 one of the major concerns associated with protein nanocarriers, which have repeated virus-like structural motifs, is their immunogenicity. elevated engineered protein cages in biomedical applications s bhaskar and s lim immunoglobulin g levels and b-cell numbers have been noted following a single dose of heat-shock protein and cpmv. 8 multiple administrations could thus enhance immune responses and neutralize the carriers. nanocarriers are cleared when they start to bind to proteins called opsonins, which mediate phagocytosis by the reticuloendothelial system of the host. 42 a common modality for reducing the immunogenic response would be to modify the carrier surface with poly(ethyleneglycol) (peg), polymethylmethacrylate, poly (lactic-co-glycolic acid) or polyamidoamine. 3, 42 alternative methods include coating with polyketals or glycan shielding. 8 biodegradable carriers that are digested and cleaved by the body are often chosen over non-biodegradable ones, for they might cause toxic side effects. such preferred carriers can be prepared from polysaccharides (e.g. chitosan), proteins, gelatin or biodegradable synthetic polymers such as poly(lactic-co-glycolic acid) and polylactic acid. 42 as they are made of proteins, protein nanocages are naturally biodegradable. one of the key requirements of drug delivery systems is the effective loading and release of drugs from the nanoparticle carriers. the strategies used to choose a delivery system rely on the system's structure and functions, the nature of the drug carried and the microenvironment the drug encounters. the delivery of drugs/active molecules can be facilitated by chemical immobilization, non-covalent interaction and environment-dependent conformational changes of the carrier. protein-based carriers and drugs could be chemically conjugated by post-translational attachment of drug molecules to reactive side chains of amino acids such as amines, carboxyls, sulfhydryls and hydroxyls, as well as non-side chains through click chemistry. covalent conjugation allows for adequate control over release kinetics. the mode of drug release is chosen based on the conjugation chemistry between the drugs, the carrier and the cellular microenvironment encountered. the interior cavity of the protein nanocages often contains natural reactive sites for attachment of nucleic acids, drugs or metals. drug molecules may also be loaded by nonspecific interactions with secondary carriers that have a strong affinity for the internal surface of the carrier. the nonspecific interactions may modulate drug release kinetics under physiological conditions. diffusion through the native pores in the carrier facilitates entry into the central cavity. gated pores in certain vlps could swell open at low salt concentrations, high ph or osmotic shock, helping load the drug. when the conditions are reversed, the drug is retained, preventing outward diffusion. this mechanism can be applied to deliver drugs to acidic cancer microenvironments. the disassembly and reassembly of the protein cages based on environmental conditions could also aid drug loading and release. the release mechanism of loaded molecules could be tuned by introducing repulsive forces at the intersubunit interfaces to facilitate environment (e.g. ph)-triggered dissociation of protein subunits of the nanocarrier. 8, 43 targeting nanoparticles accumulate in the cancer microenvironment by enhanced permeation and retention (epr) effect due to the leaky vasculature of the tissue. nonspecific accumulation through the enhanced permeation and retention effect is referred to as passive targeting and may not be very effective. to enhance the affinity for target cells, the carriers can be decorated with targeting ligands to impart active targeting. targeted delivery of drugs by carrier systems can reduce the amount of drug-carrier complex needed for therapy. an example of active targeting ligand is the peptide rgd, which is present in adenoviruses and has a natural affinity for upregulated integrin receptors in endothelial cells of tumor vessels. attaching this peptide to the external surface of a carrier aids tumor-targeted delivery. certain tumor delivery platforms focus on targeting the surface receptors using natural ligands that facilitate endocytosis, 44 engineered protein cages in biomedical applications s bhaskar and s lim such as folate receptors in cancer cells, transferrin receptors and epidermal growth factor receptors. 40 although there are many diseases that delivery systems target, research has focused on solid tumors, and cardiovascular diseases. one of the emerging areas of interest is the modulation of the immune response using protein carriers for application in tumor immunotherapy and the treatment of autoimmune diseases. 8 other ligands used for biological targeting include antibodies, engineered antibodies (e.g. single-chain variable fragment) and non-natural peptide ligands. non-natural peptide ligands are often identified using phage-display libraries that provide the opportunity to screen multiple ligands with different affinities towards a specific cell receptor. aptamers are nucleotide-based molecules that have been garnering interest as targeting ligands. the targeting ability of a carrier depends mainly on its surface ligand density, which can be tuned on protein nanocages to suit a given application. 8, 45 engineering of protein scaffolds for specific biomedical applications similar to other nanoparticles, the application of protein nanocages in biomedicine involves several challenges: (1) lack of a natural capability to carry drugs, (2) lack of specificity, (3) low cell uptake efficiency, (4) absence of endosomal escape mechanism, (5) limited circulation time, (6) potential to trigger an immunological response and (7) lack of tuneable release properties. furthermore, clinical applications of nanocages are limited because of the structures' poor stability and cell permeability. 2 to overcome some of these challenges, the engineering of protein nanocages is required to impart non-natural functions. for example, the translocation of these carriers into cells through endocytosis is not always advantageous as these materials are digested in the lysosome rather than shuttled into the target cell organelle. hence, incorporating an endosomal escape mechanism into protein nanocages is beneficial. what distinguishes nanocage-based delivery systems from other inorganic nanoparticles is the spatial control of functional groups and the ligands attached to the protein structure. multifunctional properties can be achieved by combining the desired modifications for loading, targeting and chimeric assembly, leading to the development of smart nanocarriers with tremendous potential in nanomedicine. 4 selective covalent chemical modifications in protein nanocages are made by leveraging native amino acids such as lysine, glutamic acid, aspartic acid and cysteine. 7 table 1 provides a library of natural protein cage structures that have been chemically or genetically engineered to impart functional groups to different cage surfaces, such as the interior, exterior or intersubunit interface. the structure of protein nanocages offers three distinct surfaces for engineering: the interior, exterior and intersubunit interface ( figure 10 ). 64 these interfaces can be exploited to induce chemical and genetic modifications for various purposes. 64 these modifications can also be made under the same reaction conditions, providing the opportunity to control the size, position and orientation of the loaded external active molecules. interior modification of protein nanocages. the interior cavity of the protein nanocages provides an ideal containment for molecular cargos. tailoring the cage interior increases the encapsulation efficiency, binding affinity and modulated release profile. genetic or chemical alterations can be made at precise locations to manipulate the nucleation and attachment of molecules. molecular cargos such as small molecules, peptides, protein drugs, rna/dna drugs, imaging agents and polymers have been encapsulated and released from the interior of protein nanocages. antibodies are potential cargo for larger protein nanocages, such as vaults. modification of the nanocage interior by introducing cysteine residues has been a widely used approach. the modification facilitates covalent attachment of dye molecules, drugs and other active molecules through disulfide bonds. 5 other modifications include the introduction of phenylalanine and the attachment of lipid substances and polymers inside the cage architecture. 70 the attachment of polymer chains to the interior surface offers spatial control of reactive sites, which has been shown to stabilize cages. abedin et al. 71 created a polymer network inside a protein cage by introducing cysteine reactive residues by genetic manipulation. the polymer network was created by the sequential conjugation of multifunctional monomeric units by click chemistry, allowing the free amines to be incorporated into the polymer for internal functionalization (figure 11 ). the polymer network covalently crosslinks the subunits of the protein cage, increasing its thermal stability to at least 120°c. 71 exterior modification of protein nanocages. the exterior surface of protein nanocages is engineered to impart increased circulatory halflife, local accumulation, cellular penetration and triggering of specific cellular responses. the unique geometry of protein nanocages allows for multiple ligands and functional molecules to be displayed on their surfaces. modifying molecules include small molecules (e.g. folate), peptides (e.g. rgd, transactivator of transcription 47-57 peptide (cell penetrating peptide), immunoglobulin g binding peptide (z domain)), antibodies (e.g. anti-epidermal growth factor receptor and anti-cd4), nucleotides (e.g. dna, rna, aptamers) and polymers (e.g. peg, pga, poly(lactic-co-glycolic acid), polymethacrylate, polyamidoamine). the exterior surfaces of cpmv, cowpea chlorotic mottle virus (ccmv) and bacteriophages m13, qb and ms2 have been modified with antibodies, peptides, transferrin and cell transduction domains or cell-penetrating peptides. 58 the adenovirus-based peptide rgd has a natural affinity for integrin receptors that are upregulated in tumor vessels. modification of protein carriers with this peptide has been proven to aid tumor targeting. 8 short, singlestranded oligonucleotides called aptamers with secondary structures capable of recognizing specific molecules can be incorporated onto the nanoparticles to enhance their specificity. these aptamers can bind to both extracellular (membrane) and intracellular proteins, thus facilitating aptamer-nanoparticle conjugate therapy. 72 other functionalities can be imparted to the nanocages by attaching functional ligands to extend their half-life (e.g. peg) and enhance penetration into cells (e.g. cell penetrating peptides). folate-peg displayed on adenoviral nanoparticles has been delivered to folate receptoroverexpressing cells. these nanocarriers were proven to decrease the interleukin-6 levels of macrophages, showing that the engineered viral scaffolds counteract the innate immune response. 1 peg acts as a stealth layer shielding the immunogenic epitopes on the surface of the nanocage structure. 73 this layer prevents the protein opsonin from adsorbing onto the surface, thereby shunting the recognition of the nanocage by phagocytic cells (reticuloendothelial system). 42 the peg modification also benefits the tuneable solubility and structural integrity of the protein nanocage. 73 the spatial control of multifunctional groups on protein nanocages equips them for both hierarchical self-assembly and display of distinct functional ligands. display of multiple types of ligands in a precise arrangement on nanoparticles is challenging. a distinct advantage of protein nanocages over other nanoparticles is that the position of each amino acid is spatially defined, allowing for precise spatial control of the displayed ligands. the n or c termini of the nanocage subunits 22, 43, 68 engineered protein cages in biomedical applications s bhaskar and s lim that face the external surface are a natural choice for displaying functional ligands. fusion of short peptides to these termini has been achieved through genetic engineering. modification of recombinant vault by fusing a cysteine-rich 12-amino-acid peptide to the n terminus of the mvp (cp-mvp) leads to increased particle stability. 67 engineering vault mvp c-terminal regions with tags such as epitopes, 33-amino-acid immunoglobulin g-binding peptide (z domain) or 55-amino-acid epidermal growth factors, which are displayed at the caps of the vault, were shown to facilitate cell-specific targeting. 27 in another approach, domingo et al. 74 denatured, mixed and reassembled protein subunits carrying different chimeric peptides to display multiple types of ligands (i.e. green fluorescent protein and two types of malaria epitopes) on the surface of a single e2 protein nanocage. the display of two types of hiv-derived antigenic epitopes (pep23 and rt2) provokes specific antibodies and t-cell responses. 74 despite the natural spatial control in protein nanocages, achieving the desired symmetry is still a great challenge. 64 production of protein nanocages with a dual architecture was achieved by toposelectively modifying the exterior surfaces of dna-binding protein from starved cell cages by a masking/unmasking method based on solid supports. the distribution of the two functional domains, fluorophore and affinity tag, was manipulated by using different materials for the solid supports ( figure 12 ). 75 this toposelective modification provides more sophisticated designs of multifunctional nanoplatforms. by combining genetic fusion with toposelective attachment of streptavidin, the universal coupling protein to which a biotinylated molecule could be attached, suci et al. 76 achieved asymmetrical placement of streptavidin. this asymmetrical placement provides further spatial control over the display, providing tools with which to explore the effects of the polarized orientation of conjugated functional molecules on interactions with specific cell surface receptors. intersubunit modifications. the self-assembly of nanostructures in cells depends on specific protein-protein interactions. understanding these interactions is essential in the design of novel nanostructures for various applications. for example, controlling the assembly and disassembly process to respond to ph variations has implications for the release of molecular cargos from the protein nanocage. other triggers include salt concentration and nucleic acid content, as well as metal ions. 5 the display of ligands can also be modulated by modifying how the subunits interact. the interface between subunits of viruses such as ccmv was studied to determine the mechanism of supramolecular assembly of protein nanocages. 77 in-depth study of the interactions between protein subunits at the interface has paved the way to optimizing the conditions for the induction of self-assembly. genetic modification of specific locations on the subunits, especially at the n and c termini, yields vlps with different sizes and total number of subunits. 77 the ccmv viral cage was also tested in terms of low symmetrical assembly. two different strategies were developed to break the symmetry of ligand presentation on the virus. 5 ccmv nanoparticles were labeled differently with either biotin or digoxigenin and then were disassembled into individual subunits ( figure 13 ). these differentially labeled subunits were purified, mixed and reassembled in optimal stoichiometry to form dual-function nanocages with controlled display of ligands. the maximum benefit of this strategy can be reaped when a functional nanocage structure is assembled based on a library of differentially engineered subunits, each having its own ligand or epitope. 5 protein-protein interactions often involve large areas of buried surface that can be interrupted by small molecules that target engineered protein cages in biomedical applications s bhaskar and s lim 'hot spots' where the energy for binding is concentrated. 78 zhang et al. 78 identified these hot spots in dna-binding protein from starved cell by the alanine shaving method. this method works by conceptually shaving the individual side chains to methyl residues; the stabilities of the resulting mutants are deduced by comparing them with the wild type. this work could aid the basic understanding of the assembly of miniferritin and the control of ferritin's oligomerization state, which can be applied as templates for nanomaterial synthesis and drug delivery. by modifying the interactions at the subunit-subunit interface, the assembly profile of e2 protein nanocages can be modified such that they are stable at ph 7.4 but disintegrate at acidic ph. 43, 79 in other words, the ph transition point at which this change occurs can be controlled. using this strategy, the release of the encapsulated drug/active molecules can be achieved by modulating the interactions between the subunits to respond to ph changes in the target cell microenvironment. 79 for example, the tumor microenvironment is slightly more acidic than healthy tissues. ph changes are also experienced by molecules entering cells by endocytosis. the molecules will experience a ph level of 7 near the cells and then a ph level of 5 upon entering the lysosome later in the pathway. thus, ph-based assembly and disassembly responds could be used to trigger the release of molecules encapsulated within protein nanocages. engineering and production of protein nanocages. protein nanocages are plastic and powerful materials because of their low toxicity, functional diversity and flexibility in being suitably engineered for specific purposes. 6 the construction of protein nanocages from their building blocks could be achieved by selecting protein domains that facilitate protein-protein interactions without nonspecific aggregation. environmental parameters such as temperature and ph have been used to control peptide self-assembly. 6 additional functional ligands can be incorporated into naturally existing protein nanocages by genetic engineering and chemical modification techniques. genetic engineering of the protein nanocages is achieved by recombinant dna technologies. they are produced in small and large scales using both microbial and non-microbial systems as the cell factories. bacterial cells, in particular, are popular hosts for producing a variety nanomaterials, including those for medical applications. 6 chemical modifications can be used to prevent the interbatch variability of protein production observed in recombinant dna technologies. 1 the francis group has developed new methods for chemically modifying proteins that may be applicable to protein nanocages. 80 application of protein nanocages as drug/gene carrier by leveraging on facile genetic or chemical modifications to promote molecular attachment and encapsulation, protein nanocages can be engineered to carry drugs, genes, imaging contrast agents or other active molecules. to date, small molecules such as doxorubicin, bleomycin, cisplatin, carboplatin, nucleic acids dna/rnas, peptides and proteins such as cre recombinase, capase-8, interferon-γ and interleukin-2 have been delivered in vitro, whereas paclitaxel, doxorubicin, interleukin-2, cd40l, small interfering rna, microrna and cpg oligos have been applied in vivo as therapeutics using protein nanocages. 8 covalent attachment of small molecules to the interior of protein nanocages prevents leakage, protects the drugs by preventing degradation and prevents nonspecific interaction. for instance, the low endosomal ph of the cell could be used as a trigger to release the encapsulated drug. the heat-shock protein cage interior and e2 protein are modified by genetically inserting cysteine residues at the interior surface for selective attachment of antitumor drugs with a ph-sensitive linker. 68, 81 the attachment of 6-maleimidocaprol, a hydrazone derivative of doxorubicin, to the inner surface of cysteine-modified heat-shock protein and e2 protein nanocages allows for the selective release of the drug under acidic conditions through the hydrolysis of the hydrazone linkage. ferritin has been used to encapsulate the platinum-based anticancer drugs cisplatin and carboplatin. 82 drugs encapsulated within a protein nanocage have been reported to retain their efficacy and exert cytotoxic effects on cancer cells. however, efficacy comparisons between the encapsulated drugs and free drugs and their effects on healthy cells are still under investigations. vlps such as the brome mosaic virus, ccmv, papillomavirus and polyomavirus have been used to package and deliver exogenous dnas. 83 pan et al. 1 used bacteriophage ms2 vlps to deliver human pre-mirna. the nanocages were simultaneously decorated with cellpenetrating peptides and transactivator of transcription 47-57 to enhance cellular penetration. the designed delivery vehicle promotes subcellular localization of nucleic acid mir-146a, which suppresses the target gene effectively. 1 small interfering rna encapsulated in hbv capsid nanocarriers was shown to systemically deliver small interfering rna to cancer sites in mice and effectively silences the target gene. 84 protein nanocages have also been explored as carriers for protein-based therapeutics. delivery of protein therapeutics requires protein nanocages with larger cavities. model therapeutic molecules, such as prodrugs and enzymes, have been packaged in vlps by fusing them to gag proteins. 85 viral vectors are naturally taken up by cells through the interaction of the envelope protein on their outer surface with cell receptors. 1 vsv-g envelope 86 or other localizing targeting ligands further increase the efficiency of delivery of the cargos to particular cells. vaults with a volume of~122,000 nm 3 are good non-viral protein nanocages candidates for packaging of protein-based therapeutics 26 . loading can be facilitated by fusing the protein of interest with int, which serves as a shuttle protein to package the molecule inside the vault. 27 modifications of the vault interior and exterior to accommodate other therapeutics or other ligands are reviewed in other sections. the basic requirements for in vitro and in vivo imaging is to achieve high local concentrations of imaging agents and suppression of the quenching of fluorescent probes. various contrast agents for enhanced mri, positron electron tomography and near infrared fluorescence imaging have been encapsulated in protein nanocages. single or coencapsulation of the contrast agents will allow for the development of protein nanocages with multimodal imaging ability. of all protein nanocages, ferritin has dominated bioimaging applications. the inherent ability of ferritins to store iron as ferric oxyhydroxide particles makes them attractive as an mri contrast agent. the ferric oxyhydroxide nanoparticles are superparamagnetic; therefore, endogenous ferritin loaded with these particles can act as a natural t2 mri contrast agent. 87 at clinically significant magnetic field strengths (i.e. 1.5 and 3 t), ferritin has 10-100-fold less relaxivity per iron than commercially produced iron oxide nanoparticles. loading of superparamagnetic iron oxide nanoparticles in ferritin nanocages shows higher r 2 relaxivity than endogenous ferritin. 64 protein nanocages can be engineered in a chemoselective manner to increase the loading capacity of fluorophores (e.g. alexa fluor or fluorescein dyes on cpmv) with precisely defined positions on the exterior and interior of the cage. the imaging agents are attached to the rigid and ordered protein structure at distinct positions. the spatial confinement of dye molecules prevents them from reacting or aggregating with each other and therefore reduces the quenching possibility. the inherent ability of some near-infrared fluorescence dyes to penetrate tissues with reduced background noise makes them attractive for in vivo imaging. 3 attachment of these dyes on the protein nanocages increases the local concentration resulting in reduced dosing. a notable example is luminescent semiconductor nanocrystals (quantum dots), which were displayed on engineered cpmv nanocages. 3 bacteriophage ms2 nanocages were tested for their ability to carry the positron electron tomography imaging agent [ 18 f] fluorobenzaldehyde. the radiolabel was attached to the interior of the cage by bioconjugation. positron electron tomography imaging analysis in rats showed that conjugation with protein nanocages increases the blood circulation time of the imaging agent without affecting biodistribution. 88 protein nanocages can also be used as contrast agents in ultrasound imaging. gas-filled protein based nanostructures derived from cyanobacterium anabaena flos-aquae has been genetically engineered for enhanced harmonic properties that is optimal for in vitro and in vivo ultrasound imaging. 87 it has been shown that these protein nanocages can also be engineered for multimodal and targeted ultrasound imaging. as each imaging modality has unique advantages such as tissue penetration depth, spatial and temporal resolution and cell specificity, the combination of multiple molecular probes of different imaging modalities will allow for more accurate diagnosis of a disease condition. to date, only near-infrared fluorescence-positron electron tomography multimodal imaging using protein nanocages has been reported, in which ferritin is used as a carrier to deliver the two imaging probes cy5.5 and 64 cu and a targeting ligand for imaging tumors. 65 application of protein nanocages as vaccine/immune modulators protein nanocages have shown promising potential as a display platform for pathogenic epitopes to elicit the production of neutralizing antibodies. hbv, hpv, influenza, hiv, hepatitis c virus, rsv, chlamydia infections and cancers such as cervical cancer and p815 tumors are a few diseases that have been targeted for vaccine development using the protein nanocage platform. [90] [91] [92] [93] current vaccines are mostly based on whole viruses, both live attenuated and inactivated. in 1976, edward jenner presented the first virus-based vaccine against small pox. since then, other vaccines have entered the market, including vaccines for hepatitis a, rubella, measles, mumps and influenza. despite their initial success, the live attenuated viral vaccines are relatively less stable and difficult to administer. there is also the risk that these attenuated viruses will revert to their pathogenic form, which can be detrimental to the host. neutralized virus vaccines do not carry the risk of reversion, but they are weaker, expensive and exhibit reduced vaccine coverage in a given population. to overcome this problem, subunit vaccines have been considered because they prime the immune response by the administration of a preload of viral proteins, which are often taken from the viral capsid engineered protein cages in biomedical applications s bhaskar and s lim portion. because there is no viral replication, these vaccines are safer but are less immunogenic when produced and purified without any other viral counterparts. however, the innate ability of viral capsid subunits to assemble into vlps could be used to mimic the whole virus to improve vaccine efficacy. vlps could also be used as a platform for displaying foreign ligands of viral or non-viral origins. 8, 92 both enveloped and non-enveloped vlp-based vaccines have been developed. two of the most successful non-enveloped vlp-based vaccines, namely hbv and hpv vaccines, are licensed and commercialized for clinical applications. viruses such as influenza, hepatitis c virus and hiv can give rise to enveloped vlps, which lack the ability to replicate. the immunogen is composed of assembled particles consisting of some or all of the surface subunits of the plasma membrane. 92 current research is focused on the conserved epitopes of the envelope protein of hiv-1 gp-41. developing successful hiv vaccine requires engineering different vlps. the development of vlp-based vaccines against hepatitis c virus, filoviruses ebola virus, marburg virus, severe acute respiratory syndrome, coronavirus and chikungunya virus is rapidly progressing. 92, 94 plant viruses are also used as a platform for displaying antigenic epitopes. cpmv is an extensively studied plant virus for vaccine applications. other plant viruses include cmv and papmv. bacteriophage and insect virus platforms are also explored for heterologous epitope display. 92 a variety of cancer vaccines could be developed by using toll-like receptor agonist cpg peptide-loaded protein nanocages. 8 vaccines against respiratory infections, such as influenza and respiratory syncytial virus, have been explored. h1n1-virus specific hemagglutinin was fused genetically to ferritin subunit such that eight trimeric viral spikes were presented on the outer surface after the self-assembly of the nanocage (figure 14 ). 91 respiratory syncytial viral epitopes have been displayed on vlps such as baculovirus and newcastle disease virus. 95, 96 chlamydia trachomatis immunogenic epitope pmpg-1-loaded vault nanocages, vaginally injected in mice, stimulated specific t-cell responses against the pathogen. 97 the engineered protein nanocage-based vaccines have been shown to induce protective immunity. protein nanocages are a versatile platform for biomedical applications. the main advantage of protein nanocages is the spatial control of functional groups displayed at well-defined locations through genetic or chemical modifications. to date, only a few nanoplatforms provide for the ability to simultaneously tune size, shape and biocompatibility. protein nanocages have been shown to be amenable to engineering to cater to specific functions. despite the multitude of functionalities, the detailed mechanisms for the uptake of the protein nanocages and their intracellular fates are not fully understood. although cells internalize some of the carrier molecules, the efficiency of delivery to the intended intracellular compartment remains a challenge. decorating protein nanocages with ligands, such as cell-penetrating peptides, appears to improve the delivery of drug cargos into the cells via nonspecific cellular uptake. to improve the selectivity and enhance local accumulation on target cells, targeting agents have been displayed on the external surface of protein scaffolds. the advantage of the spatial control of ligand attachment on protein nanocages, however, is limited with respect to the display of different types of ligands on the same nanocage. engineering peptides and proteins could have a crucial role in creating multifunctional materials by combining two or more distinct ligands on a single protein nanocage. selective display of peptides, proteins or nucleotides with such spatial control may open a new avenue for the design and development of novel self-assembling functional hierarchical supramolecular structures. whereas a similar approach is being pursued in the general field of nanotechnology and still remains a challenge, protein nanocages may present new opportunities. although the majority of efforts have been focused on using protein nanocages to deliver anticancer drugs, exploiting protein nanocages to modulate the immune response is an emerging area of research and potentially the most attractive application. protein nanocages are good candidates for delivering immune modulating agents for application in cancer immunotherapies or autoimmune disease treatments. in nature, viruses have the innate ability to condense and deliver nucleic acids through cell receptor interactions. by studying the structure and functions of the protein subunits in viruses that are responsible for packaging and releasing nucleic acids, novel gene delivery systems can be constructed de novo. other limited explorations include the use of protein nanocages for the treatment of skin conditions such as pigmentation disorders and ocular delivery of therapeutics. further development of the vast applications of protein nanocages requires a deeper understanding of fundamental concepts that s bhaskar and s lim remain understudied. for example, the self-assembly mechanism of many protein nanocages remains elusive. a detailed mechanistic understanding is important to better design drug loading and release properties. the mechanism of internalization and intracellular processing of these nanoparticles is yet to be determined. although cell-specific delivery of protein nanocages is possible, the targeting efficiency of these carriers presents a major challenge. in addition, penetration through other physiological barriers such as the epidermal skin layer and corneal layer has yet to be addressed. the advent of synthetic biomaterials such as artificial peptides, modular proteins and polymers widens the scope for additional properties such as reversible aggregation, which can complement natural scaffolds. the design of hybrid nanoscaffolds de novo via nature's bottom-up approach using smart synthetic materials could provide critical advantages, such as biocompatibility, site-specific modification, control of self-assembly with respect to environmental stimulus, stability and drug/nucleic acid loading. these novel bioinspired materials, which are formed by the self-assembly of distinctly engineered protein subunits for various functions, are of immense value in nanomedicine and associated medical fields. drug delivery and gene therapy a novel intracellular protein delivery platform based on single-protein nanocapsules adaptations of nanoscale viruses and other protein cages for medical applications a library of protein cage architectures as nanomaterials biological containers: protein cages as multifunctional nanoplatforms engineering building blocks for self-assembling protein nanoparticles functionalization of protein-based nanocages for drug delivery applications caged 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cryoelectron microscopy thermostability and molecular encapsulation within an engineered caged protein scaffold biomimetic design of protein nanomaterials for hydrophobic molecular transport multiple display of peptides and proteins on a macromolecular scaffold derived from a multienzyme complex isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small rna the vault exterior shell is a dynamic structure that allows incorporation of vault-associated proteins into its interior draft crystal structure of the vault shell at 9-a resolution targeted vault nanoparticles engineered with an endosomolytic peptide deliver biomolecules to the cytoplasm targeting vault nanoparticles to specific cell surface receptors smart vaults: thermally-responsive protein nanocapsules halothiobacillus neapolitanus carboxysomes sequester heterologous and chimeric rubisco species bacterial microcompartment organelles: protein shell structure and evolution isolation and characterization of linocin m18, a bacteriocin produced by brevibacterium linens designing supramolecular protein assemblies nanohedra: using symmetry to design selfassembling protein cages, layers, crystals, and filaments o principles for designing ordered protein assemblies structure and flexibility of nanoscale protein cages designed by symmetric self-assembly accurate design of megadalton-scale twocomponent icosahedral protein complexes design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments hybrid virus-polymer materials. 1. synthesis and properties of peg-decorated cowpea mosaic virus use of the interior cavity of the p22 capsid for site-specific initiation of atom-transfer radical polymerization with high-density cargo loading protein-based nanomedicine platforms for drug delivery biocompatibility and drug delivery systems biocompatibility of engineered nanoparticles for drug delivery trimer-based design of ph-responsive protein cage results in soluble disassembled structures polymer conjugates as anticancer nanomedicines cell-specific delivery of diverse cargos by bacteriophage ms2 virus-like particles enhanced adenovirus infection of melanoma cells by fiber-modification: incorporation of rgd peptide or ad5/3 chimerism human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain chemical modification of a viral cage for multivalent presentation display of peptides on the surface of tobacco mosaic virus particles dual-surface modification of the tobacco mosaic virus dual-surface-modified bacteriophage ms2 as an ideal scaffold for a viral capsid-based drug delivery system viral capsids as self-assembling templates for new materials design and validation of a bifunctional ligand display system for receptor targeting modification of turnip yellow mosaic virus coat protein and its effect on virion assembly chemoselective modification of turnip yellow mosaic virus by cu(i) catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction and its application in cell binding melanoma and lymphocyte cell-specific targeting incorporated into a heat shock protein cage architecture the small heat shock protein cage from methanococcus jannaschii is a versatile nanoscale platform for genetic and chemical modification development of a microrna delivery system based on bacteriophage ms2 virus-like particles self-assembled virus-like particles from rotavirus structural protein vp6 for targeted drug delivery virus-based nanocarriers for drug delivery binding of external ligands onto an engineered virus capsid a simple tagging system for protein encapsulation short n-terminal sequences package proteins into bacterial microcompartments the ferritin superfamily: supramolecular templates for materials synthesis chimeric ferritin nanocages for multiple function loading and multimodal imaging utilization of a protein 'shuttle' to load vault nanocapsules with gold probes and proteins engineering of vault nanocapsules with enzymatic and fluorescent properties protein nanocapsules containing doxorubicin as a ph-responsive delivery system active protein aggregates induced by terminally attached self-assembling peptide elk16 in escherichia coli reactions inside nanoscale protein cages synthesis of a cross-linked branched polymer network in the interior of a protein cage nanocarriers as an emerging platform for cancer therapy protein-polymer therapeutics: a macromolecular perspective induction of specific t-helper and cytolytic responses to epitopes displayed on a virus-like protein scaffold derived from the pyruvate dehydrogenase multienzyme complex janus-like protein cages. spatially controlled dual-functional surface modifications of protein cages a streptavidin-protein cage janus particle for polarized targeting and modular functionalization the role of subunit hinges and molecular 'switches' in the control of viral capsid polymorphism rational disruption of the oligomerization of the mini-ferritin e. coli dps through protein-protein interface mutation design of a ph-dependent molecular switch in a caged protein platform one-step site-specific modification of native proteins with 2-pyridinecarboxyaldehydes selective attachment and release of a chemotherapeutic agent from the interior of a protein cage architecture encapsulation of platinum anticancer drugs by apoferritin analysis of the size of dna packaged by the human jc virus-like particle systemic delivery of sirna by chimeric capsid protein: tumor targeting and rnai activity in vivo protein delivery using engineered virus-like particles efficient gene transfer of vsv-g pseudotyped retroviral vector to human brain tumor molecular engineering of acoustic protein nanostructures genome-free viral capsids as carriers for positron emission tomography radiolabels the ferritins: molecular properties, iron storage function and cellular regulation a vault nanoparticle vaccine induces protective mucosal immunity self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h1n1 antibodies viral nanoparticles and virus-like particles: platforms for contemporary vaccine design vaccination against chlamydia genital infection utilizing the murine c. muridarum model a virus-like particle vaccine for epidemic chikungunya virus protects nonhuman primates against infection virus like particle vaccine induces protection against respiratory syncytial virus infection in mice newcastle disease virus-like particles containing respiratory syncytial virus g protein induced protection in balb/c mice, with no evidence of immunopathology activation of the nlrp3 inflammasome by vault nanoparticles expressing a chlamydial epitope key: cord-328003-yovp8squ authors: duan, liangwei; zheng, qianqian; zhang, hongxia; niu, yuna; lou, yunwei; wang, hui title: the sars-cov-2 spike glycoprotein biosynthesis, structure, function, and antigenicity: implications for the design of spike-based vaccine immunogens date: 2020-10-07 journal: front immunol doi: 10.3389/fimmu.2020.576622 sha: doc_id: 328003 cord_uid: yovp8squ the ongoing pandemic of coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), poses a grave threat to global public health and imposes a severe burden on the entire human society. like other coronaviruses, the sars-cov-2 genome encodes spike (s) glycoproteins, which protrude from the surface of mature virions. the s glycoprotein plays essential roles in virus attachment, fusion and entry into the host cell. surface location of the s glycoprotein renders it a direct target for host immune responses, making it the main target of neutralizing antibodies. in the light of its crucial roles in viral infection and adaptive immunity, the s protein is the focus of most vaccine strategies as well as therapeutic interventions. in this review, we highlight and describe the recent progress that has been made in the biosynthesis, structure, function, and antigenicity of the sars-cov-2 s glycoprotein, aiming to provide valuable insights into the design and development of the s protein-based vaccines as well as therapeutics. the coronavirus disease 2019 (covid-19) global pandemic represents an unprecedented public health, social and economic challenge (1, 2) . the etiological agent of covid-19 is a new member of the coronaviridae family that is closely related to severe acute respiratory syndrome coronavirus (sars-cov) and was recently referred to as sars-cov-2 by the coronavirus study group of the international committee on taxonomy of viruses (3) . the virus has spread rapidly and sustainably around the global resulting in over twenty-one million cases and more than 750,000 deaths as of august 15, 2020 (4) . coronaviruses (covs) are enveloped positive-sense rna viruses (5) . enveloped covs entering host cells and initiating infection is achieved through the fusion of viral and cellular membranes (6, 7) . membrane fusion is mediated by the large type i transmembrane s glycoprotein on the viral envelope and the cognate receptor on the surface of host cells (8) (9) (10) . the surfaceexposed location of the s glycoprotein not only allows it to carry out membrane fusion but also renders it a direct target for host immune responses, making it the major target of neutralizing antibodies (11) . because of its central roles in viral infection and eliciting protective humoral and cell-mediated immune responses in hosts during infection (10) , the s protein is the primary target for vaccine design as well as antiviral therapeutics (12) . here, we provide a comprehensive overview of the wealth of research related to the sars-cov-2 s glycoprotein biosynthesis, structure, function, and antigenicity, aiming to provide useful insights into the design and development of the s protein-based vaccines as well as therapeutics to prevent or treat the ongoing global spread of sars-cov-2/covid-19. the sars-cov-2 s glycoprotein is synthesized as a 1273-amino acid polyprotein precursor on the rough endoplasmic reticulum (rer) (figure 1 ) (13) . the unprocessed precursor harbors an endoplasmic reticulum (er) signal sequence located at the n terminus, which targets the s glycoprotein to the rer membrane and is removed by cellular signal peptidases in the lumen of the er (14, 15) . a single stop-transfer, membrane-spanning sequence located at the c terminus of the s protein prevents it from being fully released into the lumen of the er and subsequent secretion from the infected cell (16, 17) . co-translationally, n-linked, highmannose oligosaccharide side chains are added during synthesis (18, 19) . shortly after synthesis, the s glycoprotein monomers trimerize, which might be thought to facilitate the transport from the er to the golgi complex. once in the golgi complex, most of the high-mannose oligosaccharide side chains are modified to more complex forms (20, 21) , and o-linked oligosaccharide side chains are also added (22, 23) . in the trans-golgi network, the sars-cov-2 s glycoprotein is proteolytically cleaved by cellular furin or furin-like proteases at the s1/s2 cleavage site, comprising multiple arginine residues that are not found in the closely related sars-cov (24, 25) . cleavage at the s1/s2 site yields a surface subunit s1, which attaches the virus to the host cell surface receptor, and a transmembrane subunit s2, which mediates the fusion of viral and host cell membranes (10) . the s1 and s2 subunits remain associated through noncovalent interactions in a metastable prefusion state (11) . furin-like cleavage is essential for the sprotein mediated cell-cell fusion and viral infectivity, and is required for efficient sars-cov-2 infection of human lung cells (24) and airway epithelial cells (26) . following cleavage, an er retrieval signal (errs) consisting of a conserved kxhxx motif (27) located at the extreme c terminus ensures that the mature sars-cov-2 s protein accumulates near the er-golgi intermediate compartment (ergic) (27, 28) , where driven by interactions with another structural protein, the membrane (m) protein, the s protein participates in virus particle assembly and is incorporated into virus envelope ( figure 1 ) (29, 30) . besides, a fraction of mature sars-cov-2 s proteins travel through the secretory pathway to the plasma membrane, where they can mediate fusion of infected with uninfected cells to form multinucleated giant cells (syncytia) (24, 31) . this may allow direct spreading of the virus between cells and potentially alter the virulence of sars-cov-2 (24) . notably, a deletion of~20 amino acid containing the errs from the cytoplasmic tail of the sars-cov-2 s protein has been shown to increase the infectivity of single-cycle vesicular stomatitis virus (vsv)-s pseudotypes (9) and replicationcompetent recombinant vsvs bearing the s glycoprotein (32, 33) , which likely could be translated to single-cycle human immunodeficiency virus (hiv)-s or other retrovirus-s pseudotypes straightforward (33) . presumably, this deletion may enhance the cell surface expression of the sars-cov-2 s glycoprotein (32) , thereby facilitating the s protein incorporation into pseudovirions and replication-competent virions. as mentioned above, the sars-cov-2 s glycoprotein plays pivotal roles in viral infection and pathogenesis. mature s glycoprotein on the viral surface is a heavily glycosylated trimer, each protomer of which is composed of 1260 amino acids (residues 14-1273) ( figure 2a) . the surface subunit s1 is composed of 672 amino acids (residues 14-685) and organized into four domains: an n-terminal domain (ntd), a c-terminal domain (ctd, also known as the receptor-binding domain, rbd), and two subdomains (sd1 and sd2) ( figure 2a ) (34) . the transmembrane s2 subunit is composed of 588 amino acids (residues 686-1273) and contains an n-terminal hydrophobic fusion peptide (fp), two heptad repeats (hr1 and hr2), a transmembrane domain (tm), and a cytoplasmic tail (ct), arranged as fp-hr1-hr2-tm-ct ( figure 2a ) (34) . as a typical class i viral fusion protein (35) , the sars-cov-2 s glycoprotein shares common structural, topological and mechanistic features with other class i fusion proteins, including hiv envelope (env) glycoprotein and influenza virus haemagglutinin (ha) (36) (37) (38) . like other class i viral fusion proteins, the sars-cov-2 s glycoprotein is also a conformational machine that mediates viral entry by rearranging from a metastable unliganded state, through a prehairpin intermediate state, to a stable postfusion state (38, 39) . since the first genome sequence of sars-cov-2 became publicly available (40) , a number of structures have been determined for the sars-cov-2 s glycoprotein trimer fragments in both the prefusion and postfusion states ( figures 2b-d) (11, 34, 41) . the overall architecture of the prefusion sars-cov-2 s ectodomain stabilized by two consecutive proline mutations in two conformations determined by single particle cryo-electron microscopy (cryo-em) is a~160 å long trimer with a triangular cross-section, with the s1 subunit adopting a "v" shape contributing to the overall triangular appearance and the s2 subunit forming the stalk ( figures 2b, c) (11, 34) . the structural difference between these two conformations only lies in the position of one of the three s1 rbds ( figures 2b, c) (11) . when all three rbds are in the "down" position, the resulting s ectodomain trimer assumes a closed conformation, in which the receptor-binding surface of the s1 rbd is buried at the interface between protomers and cannot be accessible by its receptor ( figure 2b ) (11) . the s ectodomain trimer with one single rbd in the "up" position assumes a partially open conformation and represents the functional state, as the receptorbinding surface of the "up" rbd can be fully exposed ( figure 2c ) (11, 34) . the structural information provides a blueprint for structure-based design of vaccine immunogens and entry inhibitors of sars-cov-2. in the closed sars-cov-2 s ectodomain trimer, interprotomer interactions occur through the s1 ctd packed against the other two s1 ctds and one ntd from an adjacent protomer because of domain swapping and through s2, primarily between helical interactions formed by the upstream and central helices from each subunit around the trimer axis ( figure 2b ) (11) . the s1 subunits rest above the s2 trimer, the life cycle of sars-cov-2 begins with membrane fusion occurring at the plasma membrane or within acidified endosomes after endocytosis, which is mediated by conformational changes in the s glycoprotein triggered by angiotensin-converting enzyme 2 (ace2) binding. following viral entry, sars-cov-2 releases its genomic rna into the host cell cytoplasm. genome rna is first translated into viral replicase polyproteins (pp1a and 1ab), which are further cleaved by viral proteases into a total of 16 nonstructural proteins. a replication-transcription complex (rtc) is formed based on many of these nonstructural proteins. in the process of genome replication and transcription mediated by rtc, the negative-sense (− sense) genomic rna is synthesized and used as a template to produce positive-sense (+ sense) genomic rna and subgenomic rnas. the nucleocapsid (n) structural protein and viral rna are replicated, transcribed, and synthesized in the cytoplasm, whereas other viral structural proteins, including the s protein, membrane (m) protein and envelope (e) protein, are transcribed and then translated in the rough endoplasmic reticulum (rer) and transported to the golgi complex. in the rer and golgi complex, the sars-cov-2 glycoprotein is subjected to co-translational and post-translational processing, including signal peptide removal, trimerization, extensive glycosylation and subunit cleavage. the n protein is subsequently associated with the positive sense genomic rna to become a nucleoprotein complex (nucleocapsid), which together with s, m, and e proteins as well as other viral proteins, is further assembled and followed by budding into the lumen of the er-golgi intermediate compartment (ergic) to form mature virions. finally, the mature virions are released from the host cell, waiting for a new life cycle to start. this figure is adapted from the template in biorender (https://biorender.com/). stabilizing the later in the prefusion conformation ( figure 2b ) (11) . when the s ectodomain trimer adopts a partially open conformation, the rbd in the "up" position will abolish the contacts with the s2 subunit of an adjacent protomer, destabilizing the partially open conformation ( figure 2c ) (11, 34) . this will be beneficial to the dissociation of the s1 subunit and facilitate conformational rearrangements that the s2 trimer undergoes to mediate viral entry. prefusion structures of human coronavirus hku1 (hcov-hku1) and mouse hepatitis virus s protein ectodomains without two consecutive proline mutations reveal only fully closed conformation (37, 42) , similar to that observed for a full-length, wild-type prefusion form of the sars-cov-2 s glycoprotein (41) . notably, it is well established that trimeric prefusion hiv-1 env primarily resides in a closed configuration that is conformationally masked to evade antibody-mediated neutralization (43, 44) and can spontaneously sample a transient, functional configuration (45) . it can thus be speculated that native cov s glycoproteins on mature and infectious virions share a similar conformational masking feature (46) , concealing the receptor-binding surface (for those utilizing ctds as rbds) ( figure 2c ), which is further discussed below. several lines of research have established that angiotensinconverting enzyme 2 (ace2) is an entry receptor for sars-cov-2 (47) (48) (49) . detailed interactions between the sars-cov-2 rbd and its receptor ace have been revealed by several structures of ace2 in complex with rbd (50) (51) (52) (53) . structurally, rbd consists of two subdomains: a core and an external subdomain (51, 52) . an extended loop (residues 438-506), which lies on one edge of the core subdomain, presents a gently concave surface to cradle the n-terminal helix (a1) of ace2. analysis of the interface between the sars-cov-2 rbd and ace2 reveals that a total of 17 residues in rbd are in contact with 20 amino acids in ace2, forming a network of hydrophilic interactions that are suggested to predominate the virus-receptor engagement (51) . outside this extended loop, residue lys417 located in helix a3 of the core subdomain, was shown to form ionic interactions with asp30 of ace2. as the extended loop contains almost all the amino acids of the sars-cov-2 rbd that contact ace2, it is referred to as the receptor-binding motif (rbm) (51) . it has been proposed that inhibiting the interaction between rbd and ace2 might be useful in treating sars-cov-2 infection. recombinant soluble ace2 (54) and ace2-fc (55, 56) have been shown to have potential applications in the prevention and treatment of sars-cov-2 infection in vitro. as the interaction between the rbd and ace2 is extensive, small molecules probably cannot be used as entry inhibitors to effectively block the virus entry by targeting the interaction interface. however, peptides would be able to engage most of the residues belonging to rbm (57) . a pioneering study demonstrated that a 23-amino acid peptide (residues 21-43), derived from the n-terminal helix (a1) of ace2, specifically associates with the sars-cov-2 rbd with low nanomolar affinity and disables receptor interactions (57), representing a promising strategy for preventing the virus from invading human cells. in another study, a 65-amino acid peptide (residues 19-83), derived from the n-terminal back-to-back helices (a1 and 2) and composed of most of the residues of ace2 that mediate interactions with the s protein, shows a similar but probably more potent inhibitory effect (58) . the formation of a trimer-of-hairpins structure (also known as six-helix bundle) comprising hr1 and hr2 in the postfusion conformation is a unifying feature of class i viral fusion proteins (37) . the crystal structure of a protein construct in which sars-cov-2 hr1 and hr2 were connected by a six-residue hydrophilic flexible linker was determined to be a canonical six-helix bundle structure with a rod-like shape ∼115 å in length and ∼25 å in diameter (59) . three hr1 helices form a parallel central coiled-coil with three hr2 helices packing in an oblique, antiparallel manner against deep hydrophobic grooves on the surface of the central coiled-coil (59) . notably, when a full-length s protein construct bearing the native furin-like cleavage site was transiently expressed by expi293f cells, the purified s proteins contained the dissociated s2 trimer in the postfusion conformation (41) . the cryo-em structure of this trimeric postfusion s2 shows that the central helix (ch) extended regular helices from the central coiled-coil, oriented toward target cells ( figure 2d ) (41) , which forms the longest central triple helical coiled-coil (~180å) among all known class i transmembrane subunit structures. the sars-cov-2 s trimer in the pre-hairpin intermediate state is very unstable and is just transiently present in vivo after triggering by ace2 engagement, stymieing structural characterization of the s protein in this state (60) . however, although this fusion-intermediate phase is very short, it is enough for inhibitory peptides to associate with the prehairpin intermediate and block the six-helix bundle formation (39) . furthermore, it has already been shown that the hr1 regions in various human covs are highly conserved (61) , and therefore could serve as an attractive target for the design and development of potent and broad-spectrum inhibitors of pan-covs, including sars-cov-2. a highly potent pan-coronavirus fusion inhibitor, ek1c4, has been reported to have good prophylactic and therapeutic potential against sars-cov-2 infection (59). as mentioned earlier, the sars-cov-2 s proteins are heavily decorated by heterogeneous n-linked glycans projecting from the s trimer surface. the sars-cov-2 s sequence encodes up to 22 n-linked glycan sequons per protomer, which likely plays an important role in protein folding (19) and host immune evasion as a glycan shield (62) . of the 22 potential n-linked glycosylation sites on the s protein, 14 were identified to be predominantly occupied by processed, complex-type glycans (63) . the remaining eight sites were found to be dominated by oligomannose-type glycans, which are divergent from those founded on host glycoproteins (63) . although glycosylation sites (n165, n234, n343) proximal to the receptor-binding sites on the sars-cov-2 s protein can be observed, ace2 bound to the glycosylated and deglycosylated s ectodomains with nearly identical affinity (1.7 nm vs 1.5 nm) determined by a biolayer interferometry binding assay (64) . this observation suggests that the high binding affinity between the sars-cov-2 s protein and ace2 does not depend on the s protein glycosylation. when the site-specific n-linked glycans are mapped onto the prefusion structure of the sars-cov-2 s ectodomain (63), the resulting model exhibited substantially higher levels of glycanfree surface than that revealed by structures of fully glycosylated, trimeric hiv-1 env ectodomains (65, 66) . this suggests that the sars-cov-2 s protein is covered by a less dense and less effective glycan shield compared to viral glycoproteins from hiv-1 (36, 66) and lassa virus (67) , which may be beneficial for the induction of humoral immunity and could be good news for a sars-cov-2 vaccine (68) . notably, it has been shown that multiple major viral surface antigens have neutralizing epitopes that are partly or even exclusively composed of carbohydrate moieties (69, 70) , exemplified by the hiv-1 env spike, which could be recognized by a large number of carbohydrate-binding antibodies, including 2g12, pg9, pg16, ch04, pgt121, pgt128, pgt135, and pgt145 (70, 71) . in the case of sars-cov-2, more recently a potent neutralizing antibody against both sars-cov and sars-cov-2, s309, has been shown to recognize a highly conserved glycan-containing rbd epitope (72) . these observations suggest that carbohydrate moieties could be immunogenic and highlight the need for immunogens to display the glycans important for the recognition of neutralizing antibodies (73) ; in support of this, specific n-linked glycans on hemagglutinin has been shown to be essential for the elicitation of broadly neutralizing antibodies against influenza (74) . accordingly, there has been mounting interest in exploring the potential of immunogenic glycan moieties as vaccine candidates against multiple viruses, including sars-cov-2 (75, 76) . membrane fusion and viral entry of sars-cov-2 is initiated by binding of rbd in the viral s glycoprotein transiently sampling the functional conformation to ace2 on the surface of target cells (figure 1 ) (10). after receptor engagement at the plasma membrane or ensuing virus endocytosis by the host cell (8), a second cleavage (s2′ cleavage site) is generated, which is mediated by a cellular serine protease tmprss2 (48) or endosomal cysteine proteases cathepsins b and l (10) (figure 1) . protease cleavage at s2′ site frees the fusion peptide from the new s2 n-terminal region, further destabilizes the sars-cov-2 s glycoprotein and may initiate s2-mediated membrane fusion cascade. following the second cleavage, the fusion peptide at the n terminus of the s2 trimer is inserted into the host membrane (8) , forming the pre-hairpin intermediate state (39) . since the pre-hairpin intermediate state is extremely unstable, the s2 fusion protein is refolded quickly and irreversibly into the stable postfusion state (39, 77) . these large conformational rearrangements pull the viral and host cell membrane into close proximity, leading ultimately to the membrane fusion (8, 39) . since sars-cov-2 was identified as the causative agent of covid-19, and its first genome sequence was released immediately and freely by a chinese research group (40), sars-cov-2 vaccine candidates based on various vaccine platforms, such as inactivated or live attenuated vaccines, dna and mrna vaccines, viral vector-based vaccines, and recombinant protein-based vaccines, have been developed (12, 78) . most of these vaccine strategies are based on the full-length s glycoprotein, the major viral surface antigen (12) . when a vaccine strategy requires that the sars-cov-2 s protein be recombinantly expressed in the human body, the errs should be omitted to enhance the cell surface expression level of the resulting protein. theoretically, the native hiv-1 env trimer present on the surface of intact virions is thought to be a most ideal immunogen (60) , as most of the neutralizing antibodies thus far described could recognize and bind to the prefusion form of trimeric hiv-1 env, although it is with great difficulty that such neutralizing antibodies against this glycan-covered, sequence-variable native form are induced (36) . for sars-cov-2, different lines of research have shown that convalescent sera from sars-cov and sars-cov-2 patients showed no or limited crossneutralization activity against these two viruses by pseudotyped and authentic viral infection assays, despite significant crossreactivity in binding to the s glycoproteins of both viruses (9, (79) (80) (81) . similar results were also observed in infected or immunized animals (48, 79, 81) . together with the finding that although the sars-cov-2 s protein shares a high degree of amino acid sequence identity with that of sars-cov (~76% overall), the rbm is less conserved (~47% identity) than any other functional region or domain (82) , it can thus been surmised that the rbm has the most immunodominant neutralizing epitope(s) of the whole s protein, capable of readily eliciting strong neutralizing antibody responses. however, the native trimeric sars-cov-2 s protein could conceal each of its immunodominant rbms by adopting the closed conformation (41, 83) . therefore, sars-cov-2 evades immune surveillance also through conformational masking, which is well-documented for hiv-1 (43, 44) ; while at the same time, the s protein could transiently sample the functional state to engage ace2, consistent with the notion that the fusion glycoprotein of highly pathogenic viruses have evolved to perform its functions while evading host neutralizing antibody responses. another concern for vaccine candidates based on the fulllength s glycoprotein of sars-cov-2 is raised by the observation that the s1 subunit could spontaneously dissociate from the s glycoprotein probably as a trimer that still assumes the rbd closed conformation, leaving only the postfusion s2 trimer (41) . the resulting s1 and s2 subunits might expose immunodominant, nonneutralizing epitopes that are utilized by sars-cov-2 to serve as decoys to distract the host immune system, inducing a large proportion of ineffective antibody responses, as documented for hiv-1 (60) and respiratory syncytial virus (rsv) (84) . it should be noted that although vaccine candidates based on the full-length s protein of the closely related sars-cov could elicit neutralizing antibody responses against infection of sars-cov, they may also induce harmful immune responses, including liver damage of the vaccinated animals, infection of human immune cells by sars-cov, and antibody-dependent enhancement of sars-cov infection (85) (86) (87) (88) (89) . therefore, although the s proteins of both sars-cov and sars-cov-2 are thought to be promising vaccine immunogens for generating protective immunity, optimizing antigen design is critical to ensure an optimal immune response through exposing more neutralizing epitopes and displaying fewer potentially weakly or non-neutralizing epitopes (90) . vaccines containing or expressing the full-length s protein or its soluble ectodomain form should thus be engineered to sample a rbd(s) "up" conformation while the rest is still kept in the prefusion state (91, 92) . apart from recombinant, soluble, stabilized ectodomains that are engineered to expose the immunodominant rbd by adapting the rbd(s) "up" conformation, rbd proteins of sars-cov and sars-cov-2 have also been widely used as recombinant protein-based vaccines (85, (93) (94) (95) . the rbd of sars-cov is highly immunogenic (96, 97) and is targeted by most of the neutralizing monoclonal antibodies that have been characterized (98) . based on the observation that a 193-amino acid fragment (residues 318-510) was previously identified to be the minimal rbd region of sars-cov (99), a corresponding 194-amino acid fragment (residues 331-524) can be readily selected as the minimal rbd region of sars-cov-2 and has already been characterized (100) . this minimal form of rbds of both viruses could serve as a vaccine candidate (100) . however, a conserved cysteine residue is located immediately upstream of the minimal rbd fragments of both viruses and always forms a disulfide bond in nearly all published structures containing this residue (101, 102) ; this is also the case for middle east respiratory syndrome coronavirus (mers-cov) (103, 104) and hcov-hku1 (37), consistent with the observation that all rbds of these viruses share a conserved structural core. the disulfide bond contributes to stabilization of the rbd structure and likely modulates the protein immunogenicity. this notion is consistent with the observation that mice immunized with a longer form of the sars-cov rbd (residues 318-536) produced a higher titer of neutralizing antibodies compared with mice immunized with the minimal rbd region (residues 318-510) (105) . therefore, when each of the minimal rbd fragments of sars-cov and sars-cov-2 is used as vaccine candidates, the critical cysteine residue should not be ignored and thus should be included (106) . besides the rbd, which has been shown to a major target for human neutralizing antibody responses (107), the ntd was recently identified to be a new vulnerable site of the sars-cov-2 s protein for antibody neutralizing and therefore could also serve as a recombinant protein-based vaccine (108) (109) (110) . as expected, ntd-specific neutralizing antibodies could target the s protein in both closed and open conformations (108) . in addition, the apparent accessibility of the fusion peptide and hr1 region in published structures of the sars-cov-2 s ectodomain trimer as well as their high sequence conservation among covs suggests that they would be good immunogen candidates for epitope-focused vaccine design aimed at raising broadly cov neutralizing antibodies (46) . the epitope-focused vaccine design has proven to be successful in generating neutralizing antibodies against rsv fusion glycoprotein (111). however, neutralizing antibodies targeted against these two regions still need to be isolated in infected individuals to support this notion. unlike wild-type full-length s protein of sars-cov-2, the above monomeric fragments do not induce any infection-enhancing antibodies or harmful immune or inflammatory responses (106, 112) , all of which could be potentially avoided through structure-based immunogen design to improve immunogenicity (113, 114) . however, wide-type full-length or soluble ectodomain form of the sars-cov-2 s protein could trigger stronger cellular immune responses (115) , which have been demonstrated to play an important role in controlling diseases caused by covs (116, 117) , including sars-cov-2 (118) , and are probably also an important determinant of effective vaccines against sars-cov-2 (115, 119) . additionally, when more than one rbd of the s protein trimer is engineered to be locked in the "up" conformation (120, 121) , the antigenicity and immunogenicity of the resulting rbds would be significantly enhanced compared to monomeric rbd form (97, 122) . moreover, improved protection is likely to be achieved when vaccinated with full-length or soluble ectodomain form of the sars-cov-2 s protein in that both forms can elicit neutralizing antibodies directed against non-rbd sites, as observed for mers-cov (123) . genetic variation has been used by many viruses that have rna genomes (124) , including hiv and influenza, as a mechanism to avoid antibody-mediated immunity, and is partially responsible for the great difficulty in developing effective and durable vaccines against these viruses (36) . as an rna virus, however, sars-cov-2 has a very low mutation rate overall (125) likely because covs have a genetic proofreading mechanism (126) . all reported variations occurred in the sars-cov-2 s glycoprotein have a prevalence of no more than 1% (127) , with an exception of d614g, which has become the most prevalent genotype in the global covid-19 pandemic (127) . fortunately, although the d614g mutation of the sars-cov-2 s protein has been shown to enhance viral infectivity (128) (129) (130) , until now there is no evidence that infection with sars-cov-2 carrying the g614 mutant will be associated with disease severity (127, 131) . furthermore, assays using both monoclonal and polyclonal antibodies generated from individuals naturally infected with d614-or g614-carrying viruses demonstrated that the d614g mutation retains or even increases viral susceptibility to neutralization (127, 130, 132, 133) . this suggests that the d614g mutant maintains or favors an open, functional conformational state (134) . although at an extremely low frequency, natural variations, including l452r a475v, v483a, and f490l that render the s glycoprotein resistant to certain neutralizing antibodies targeting the rbd, emerged under no selection pressure exerted by approved vaccines or neutralizing antibodies or entry inhibitors (127, 132) . however, it has been shown that sars-cov-2 escape mutants could be easily selected and quickly amplified under the selection pressure of single antibody treatment (135) . these observations suggest that a combination of at least two neutralizing antibodies that recognize and bind to distinct and non-overlapping epitopes on the sars-cov-2 s glycoprotein (e.g., rbd and ntd, as well as hr and glycan) is required to restrict the possible occurrence of viral escape mutants and potential subsequent loss of single antibody-mediated neutralization (135) (136) (137) (138) . when these observations are taken into consideration for vaccine design and development, an ideal sars-cov-2 immunogen should contain as many exposed neutralizing epitopes as possible, although the rbd also possesses extra epitope(s) besides the epitope in the rbm region (72, (139) (140) (141) . sars-cov-2 is a highly contagious pathogen that continues to spread quickly around the globe, causing covid-19 to be one of the worst pandemics in recorded history. a safe and efficacious vaccine represents one of the best ways to reduce or eliminate the covid-19 pandemic (142) . unfortunately, no vaccines for any of the known human covs have been licensed (143, 144) , although several potential sars-cov and mers-cov vaccines have advanced into human clinical trials for years (117, 145) , suggesting the development of effective vaccines against human covs has always been challenging. however, it has been shown that both sars-cov and sars-cov-2 could readily induce neutralizing antibodies following natural infection or immunization (146) (147) (148) (149) . moreover, a growing number of neutralizing monoclonal antibodies targeting the sars-cov-2 s glycoprotein with high potency have been isolated from plenty of convalescent donors (33) as well as humanized mice (136, 141) , some of which have been shown to afford protection against sars-cov-2 challenge in animal models. it thus seems that vaccine candidates designed to elicit such neutralizing antibodies are feasible. it is widely accepted that the s protein of sars-cov-2 is a most promising immunogen for producing protective immunity (150) . however, it is likely that the s protein has evolved to perform its functions while evading host neutralizing antibody responses and thus should be engineered to ensure an optimal immune response (151, 152) . the immunogen design strategies described in this review based on the wealth of the sars-cov-2 s glycoprotein research related to its biosynthesis, structure, function, antigenicity as well as immunogenicity will likely contribute to the ultimate success of safe and efficacious vaccines against sars-cov-2/covid-19. covid-19: emergence, spread, possible treatments, and global burden highlight of immune pathogenic response and hematopathologic effect in sars-cov, mers-cov, and sars-cov-2 infection 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recovered from covid-19. medrxiv immunogenicity of a dna vaccine candidate for covid-19 dna vaccine protection against sars-cov-2 in rhesus macaques antibody signature induced by sars-cov-2 spike protein immunogens in rabbits progress and prospects on vaccine development against sars-cov-2. vaccines (basel) (2020) 8:153 what are the most powerful immunogen design vaccine strategies? reverse vaccinology 2.0 shows great promise structure-based vaccine antigen design all authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. we would like to thank prof. xinqi liu for critical reading of the manuscript; and drs. yanbin feng, mengyuan xu, jing ma and jianrong feng for helpful comments and discussions on the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 duan, zheng, zhang, niu, lou and wang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-314321-klb8oe9q authors: chen, serena h.; young, m. todd; gounley, john; stanley, christopher; bhowmik, debsindhu title: distinct structural flexibility within sars-cov-2 spike protein reveals potential therapeutic targets date: 2020-04-18 journal: biorxiv doi: 10.1101/2020.04.17.047548 sha: doc_id: 314321 cord_uid: klb8oe9q the emergence and rapid worldwide spread of the novel coronavirus disease, covid-19, has prompted concerted efforts to find successful treatments. the causative virus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), uses its spike (s) protein to gain entry into host cells. therefore, the s protein presents a viable target to develop a directed therapy. here, we deployed an integrated artificial intelligence with molecular dynamics simulation approach to provide new details of the s protein structure. based on a comprehensive structural analysis of s proteins from sars-cov-2 and previous human coronaviruses, we found that the protomer state of s proteins is structurally flexible. without the presence of a stabilizing beta sheet from another protomer chain, two regions in the s2 domain and the hinge connecting the s1 and s2 subunits lose their secondary structures. interestingly, the region in the s2 domain was previously identified as an immunodominant site in the sars-cov-1 s protein. we anticipate that the molecular details elucidated here will assist in effective therapeutic development for covid-19. a new coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), causes respiratory illness and has now reached a pandemic scale. denoted as coronavirus disease 2019 (covid19) , its global spread is currently ongoing with symptoms that can range from mild flu-like to severe pneumonia, leading to death in certain cases. early investigations in china showed that sars-cov-2 has a high genomic sequence similarity to the previous sars-cov-1, along with a bat coronavirus [1] , [2] . similar to sars-cov-1, sars-cov-2 is a positive-sense, single-stranded rna virus of the betacoronavirus genus. given the health crisis caused by the mounting number of covid-19 cases worldwide, there is an urgent need to develop effective therapeutics and eventual vaccines. a critical step in developing targeted treatments is obtaining a detailed notice: this manuscript has been authored by ut-battelle, llc under contract no. de-ac05-00or22725 with the u.s. department of energy. the united states government retains and the publisher, by accepting the article for publication, acknowledges that the united states government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for united states government purposes. the department of energy will provide public access to these results of federally sponsored research in accordance with the doe public access plan (http://energy.gov/downloads/doe-public-accessplan). understanding of the molecular pathways of sars-cov-2 and constituent structures. the structural biology community has made rapid progress towards this goal by experimentally determining several sars-cov-2 proteins, including the spike (s) [3] [6] , nucleocapsid (n) [7] , and main protease (m pro ) [8] . the s protein is particularly important since it resides on the viral envelope and is responsible for host cell entry by engaging angiotensin-converting enzyme 2 (ace2) receptors [2] , [9] , [10] . recent experimental structures of the sars-cov-2 s protein receptor binding domain (rbd) in complex with ace2 provide detailed interface information [4] , [6] ; targeting this interface represents an active area of research for therapeutic development [11] . however, there may exist potential targets on the s protein besides the rbd domain. further motivating our work is the need to understand sars-cov-2 structure in the context of structures from previous coronaviruses. prior to the emergence of sars-cov-2, comparisons of the trimeric s protein from different viruses showed they possess overall similar structures but with some local differences [12] , [13] . with the appearance of sars-cov-2, it is necessary to investigate the s protein structure [3] , and compare it to previous human coronaviruses. here, to obtain deeper insights into s protein structure for biological understanding and therapeutic targeting, we employ a combined molecular dynamics (md) simulation and artificial intelligence (ai) methodology on a series of coronavirus s proteins. specifically, we investigate the s proteins from the current sars-cov-2, sars-cov-1, middle east respiratory syndrome coronavirus (mers-cov), and human coronavirus hku1 (hcov-hku1). from structural analysis of extensive md simulations, we find substantial flexibility between the subunits of the s proteins. we find that reduced distance matrix representations, interpreted by unsupervised deep learning (dl), reveal important regions for s protein trimerization. these regions present potential targets for therapeutic development. human coronavirus spike (s) proteins are molecular complexes each formed by three protein chains [3] , [14] [16] . to better understand s protein structure [3] , we started by studying the protomer, the structural unit of the trimeric complex, and compared the protomer structure of the current sars-cov-2 with protomers of previously identified human coronaviruses, including sars-cov-1 [15] , mers-cov [16] , and hcov-hku1 [14] . we modeled each protomer system from the corresponding cryo-em s protein structure (see methods for more details). there are three major structural domains which constitute the protomer: the amino-terminal domain (ntd), receptor binding domain (rbd), and the s2 domain. the ntd and rbd are within the s1 subunit and are responsible for binding to host receptors, while the s2 domain is in the s2 subunit which mediates fusion of the viral and host membranes [14] , [15] . fig. 1 a-d shows the initial protomer structures of the four s proteins, each highlighted by the three domains. to determine the dynamic change of the domain organization in the protomer structures, we measured the distribution of the interdomain distances from 5,000 structures of each system taken from 25 independent 200 ns md simulations (fig. 1 e-g) . given the limited timescale, we made no attempt to reproduce the thermodynamics but focused on computing the structural features of the systems [17] . the overall distributions of the three interdomain distances are broad, ranging from 40å to 110å between s2 and ntd/rbd domains and 30å to 100å between ntd and rbd domains. these wide distributions indicate enhanced structural flexibility in the domain arrangement of the four protomer systems compared to the cryo-em structures. to gain further insight into the 20,000 protomer structures, we applied convolutional variational autoencoders (cvaes) to encode the high dimensional protein structures from the md simulations into 3-d latent spaces for visualization. compared to commonly applied structural analysis methods, which are mostly limited to specific regions of the protein of interest, our dl approach allows us to evaluate a protein structure as a whole yet still capture detailed local structural features. we found individual clusters corresponding to each of the four protomer systems, suggesting that the structural features of the four systems embedded in the latent spaces are distinct from each other ( fig. 2 a-c). representative structures selected from the clusters also show high structural flexibility in their domain arrangement ( fig. 2 d-f). the hinge region connecting the s1 and s2 subunits opens up, causing broad distributions of the interdomain distances observed in fig. 1 e-g. the exposed surface in the protomer may provide new targets for therapeutic action. therefore, we further explored the structural flexibility of the sars-cov-2 s protein. we compared the structures between the protomer and trimer to investigate whether the large structural arrangement of the domains in the protomer is affected by the trimeric state. we found that domain organization is stabilized by trimerization. unlike the protomer, the three domains are closely arranged in the trimer (figs. 3 a-b and s4). in addition to their interdomain distances, the differences in structural flexibility between the protomer and trimer is captured in solventaccessible surface area (sasa) values (fig. 3 c) . the sasa values of the protomer and one chain of the trimer are 532 ± 10 nm 2 and 450 ± 8 nm 2 , respectively. the difference in these values largely comes from their s2 domains, which have the values of 228 ± 7 nm 2 and 170 ± 5 nm 2 , respectively. again, we applied a cvae to encode the 10,000 protomer and trimer structures into 3-d latent spaces. we found two individual clusters, one corresponding to the protomer and the other to the trimer, suggesting that the structural features of the two oligomeric states embedded in the latent spaces are different (fig. 3 d-g) . to locate the different structural features between the protomer and trimer in their 3-d structures, we compared the difference between the distance matrices of the representative protomer and trimer structures selected from the clusters in the latent dimensions (fig. 4 a) . most differences between the distance matrices result from interdomain arrangement. however, there are some disparities within the s2 domain (fig. 4 b) . one region of difference corresponds to residue numbers 784 to 810 in the pdb. in the 3-d structure, this region forms a beta sheet in the trimer while the structure is lost in the protomer (fig. 4 c) . further investigation into this region reveals that the beta sheet in the trimer is stabilized by another beta sheet constituted by residues 700 to 710 of another chain (fig. 4 d) . without the presence of other chains, this stability is lost; not only do residues 784 to 810 in the s2 domain lose the beta sheet structure, but residues 700 to 710 in the hinge region connecting the two subunits become an extended loop (fig. 4 e) . oligomeric proteins are often stabilized by oligomerization and hold highly flexible regions in the protomer state [18] . the transition between the structured and unstructured form of these flexible regions is sometime reversible, but due to the size of the s protein protomer and the timescale applied in this study, we did not observe reversible behavior of the two loop regions in the protomer state. interestingly, a fragment between residues 784 and 810 was identified previously as an immunodominant site in sars-cov-1 s protein [19] . complementary antibodies acting on this site provided the dominant immune response for patients who recovered from the sars-cov 1 infection. our results provide structural comprehension on the previous experimental observation and posit that targeting these residues might not only interfere with s protein trimerization and the subsequent viral activity but also aid antibody immune response. our findings provide insights into therapeutic design targeting the s protein beyond the oft-targeted rbd domain. here, we used unsupervised deep learning routines based on cvaes to systematically compare s protein ensembles from md simulations across lower-dimensional, latent spaces. by first comparing the s protein protomer structure of sars-cov-2 to those from previous human coronaviruses, we identified distinct clusters for each virus in the 3-d latent space, where representative structures from these clusters highlight their differences in domain flexibility. next, we compared the sars-cov-2 s protein protomer and trimer structures, which also displayed a clear separation of clusters in the latent space. while the main distinctions between these two states arise from the general gain in structural stability as the protomer self-assembles into the trimer state, we pinpointed structural transitions in specific flexible regions of the protomer that warrant consideration as potential therapeutic targets. these regions are promising as natural targets of immune recognition, but more importantly, they are involved in s protein oligomerization, suggesting they are susceptible to therapeutic action for protein destabilization. overall, our study provides a more complete molecular view of the sars-cov-2 s protein that may assist in accelerating both vaccine and drug design efforts. to generate initial systems for the structural study of human coronavirus s proteins, we built protomer structures from chain a of the cryo-em s protein structures of sars-cov-2 (pdb 6vsb [3] ), sars-cov-1 (pdb 6crz [15] ), mers-cov (pdb 6q05 [16] ), and hcov-hku1 (pdb 5i08 [14] ). the trimeric state of the sars-cov-2 s protein consists of all three chains in pdb 6vsb. each structure was solvated in the center of a water box with a minimum distance of 15å from the edge of the box to the nearest protein atom, neutralized with counter ions and ionized with 150 mm nacl. table 1 summarizes the details of the five molecular systems studied. following a similar protocol to our previous studies [20] , [21] , the resulting systems were each subjected to 20,000 steps of energy minimization, followed by 1 ns equilibration with harmonic restraints placed on the heavy atoms of the protein. the force constant was 1 kcal/mol/å [15] . after equilibration, the restraints were released, and a 200 ns trajectory was generated in a production run. for each system, 25 independent 200ns trajectories were performed. structures were taken every 1 ns for analysis, yielding a total of 5,000 structures for each system. all md simulations were performed with namd [22] in npt ensemble at 1 atm and 310 k with a time step of 2 fs. the charmm36m force field [23] and tip3p water model [24] were used. the nonbonding interactions were calculated with a typical cutoff distance of 12å, while the long-range electrostatic interactions were enumerated with the particle mesh ewald algorithm [25] . to further understand the molecular structures of different human coronavirus s proteins and the oligomeric state of sars-cov-2 s protein, we deployed a custom-built deep learning architecture, a convolutional variational autoencoder (cvae), to encode the high dimensional protein structures from the md simulations into lower dimensional latent spaces. the goal of our ai method is to reduce the high dimensionality of the molecular system while preserving the inherent characteristics of the system and learning novel behavior in a latent space that is normally distributed. the direct comparison between the decoded and original input data ensures the accuracy of the latent space representation. this customized cvae approach has been successfully applied to study the folding pathways of small proteins and structural clustering of biomolecules [26] [28] . 1) data preparation: we used translation and rotation invariant input data for the dl networks. we represented each md structure by a distance matrix using the c ↵ atoms of the protein and generated two input datasets for the cvaes. input 1 included the matrices of the protomer structures of sars-cov-2, sars-cov-1, mers-cov, and hcov-hku1. input 2 included the matrices of the protomer structure and the chain a of the trimer structure of sars-cov-2. for input 1, as the proteins are different in length, we first performed a multiple sequence alignment of the protomer structures of sars-cov-2, sars-cov-1, mers-cov, and hcov-hku1 by clustal omega [29] . based on the alignment, we inserted gaps into the corresponding aligned residue location in the distance matrix and set the distance between gaps to be 0å. the size of each distance matrix after the alignment was 1,342 ⇥ 1,342. to reduce the matrix size, we applied a convolution layer with padding of 2 and a 14 ⇥ 14 filter with strides of size 7 in both the x and y directions. the size of each resulting matrix became 191 ⇥ 191. finally, after alignment and size reduction we merged a total of 20,000 distance matrices of the four s proteins. an example of a distance matrix following the alignment and size reduction is represented in fig. s1 . for input 2, as the proteins are of the same length, no alignment was required. the size of each matrix was 959 ⇥ 959. we again applied a convolution layer with padding of 1 and a 10 ⇥ 10 filter with strides of size 5 in both the x and y directions to reduce the matrix size. the size of each resulting matrix was also 191 ⇥ 191, and we merged a total of 10,000 distance matrices of the protomer and trimer of sars-cov-2 s protein. 2) cvae implementation: for each of the two input datasets, we randomly split the aligned matrices into training and validation datasets using the 80/20 ratio and applied a cvae to capture the important structural features and projected them in the three-dimensional (3-d) latent space for visualization. the encoder network of each cvae consisted of three convolutional layers and a fully connected layer. we used a 3 ⇥ 3 convolution kernel and a stride of 1, 2, and 1 at the three convolutional layers, respectively. we trained each cvae until the training and validation loss converged. fig. s2 shows the loss curves along the 150 epochs. the difference between decoded and original images is minimal, suggesting the models were trained successfully (fig. s3) . we then selected representative structures from the clusters in the latent space and visualized them using vmd [30] . all md simulations and dl analysis were performed on the summit supercomputer at the oak ridge leadership computing facility. the distances calculated from the cryo-em structure (pdb 6vsb) are marked by a pentagon. a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin cryo-em structure of the 2019-ncov spike in the prefusion conformation structural basis for the recognition of sars-cov-2 by full-length human ace2 structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor crystal structure of nsp15 endoribonuclease nendou from sars-cov-2 crystal structure of sars-cov-2 main protease provides a basis for design of improved -ketoamide inhibitors functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target structure, function, and evolution of coronavirus spike proteins cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains pre-fusion structure of a human coronavirus spike protein stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis structures of mers-cov spike glycoprotein in complex with sialoside attachment receptors the energy landscape of a protein switch in silico design and validation of high-affinity rna aptamers targeting epithelial cellular adhesion molecule dimers b-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the s2 domain of the surface spike glycoprotein graphene-extracted membrane lipids facilitate the activation of integrin ↵ v 8 stability of ligands on nanoparticles regulating the integrity of biological membranes at the nano-lipid interface scalable molecular dynamics with namd charmm36m: an improved force field for folded and intrinsically disordered proteins comparison of simple potential functions for simulating liquid water particle mesh ewald: an n log (n) method for ewald sums in large systems deep clustering of protein folding simulations mechanism of glucocerebrosidase activation and dysfunction in gaucher disease unraveled by molecular dynamics and deep learning visual analytics for deep embeddings of large scale molecular dynamics simulations the embl-ebi search and sequence analysis tools apis in 2019 vmd: visual molecular dynamics key: cord-048360-n9sih438 authors: villard, viviane; agak, george w.; frank, géraldine; jafarshad, ali; servis, catherine; nébié, issa; sirima, sodiomon b.; felger, ingrid; arevalo-herrera, myriam; herrera, socrates; heitz, frederic; bäcker, volker; druilhe, pierre; kajava, andrey v.; corradin, giampietro title: rapid identification of malaria vaccine candidates based on α-helical coiled coil protein motif date: 2007-07-25 journal: plos one doi: 10.1371/journal.pone.0000645 sha: doc_id: 48360 cord_uid: n9sih438 to identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. the corresponding synthetic peptides are expected to mimic structurally “native” epitopes. indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. these antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. this strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens. human plasmodium falciparum (pf) infection is a dramatic public health problem. today approximately forty percent of the world's population is at risk of malaria. malaria causes more than 300 million acute clinical cases, and at least one million deaths annually. ninety percent of malaria deaths occur in sub-saharan african countries mostly among young children and pregnant women (http://rbm.who.int). thus, there is an urgent need of a malaria vaccine. however, vaccine discovery, in general and particularly in malaria, is still a very empirical process. in fact, protective antigens do not bear any structural, physico-chemical or sequence-related characteristics that would allow their identification. for protective humoral responses, the only recognized characteristics of antigens are their antigenicity/immunogenicity and accessibility. in addition, the lack of surrogate markers of protection renders the vaccine discovery process difficult and time consuming. hence, in spite of a constant and impressive progress in molecular biology techniques and antigen identification and expression [1, 2] , vaccine discovery is still labor-intensive, making the approach fastidious, costly and poorly adapted to highthroughput screening. proper protein folding and solubility remain a limitation in numerous cases. thus, overcoming the bottlenecks of manufacturing and identification of fragments/proteins, as possible targets of a protective immune response still constitute a scientific and technical challenge. we addressed this challenge by combining bioinformatics, chemical peptide synthesis and functional protection assays. we focused on the search for a-helical coiled coil motifs that, in general, do not exhibit a folding problem, and are a target of effective antibodies for several current malaria vaccine candidates (eg. lsa-1 [3] , lsa-3 [4] , msp-3 [5] , and msp-6 [6] and other pathogens [7] . msp-1, another leading malaria vaccine [8] , also contains predicted a-helical coiled coil regions (unpublished results). our choice was based on the following considerations. first, the a-helical coiled coil motif bears a characteristic seven amino acid residue repeat (abcdefg) n with hydrophobic residues located in a and d positions and hydrophilic residues generally elsewhere. this motif can be easily identified by bioinformatic analysis. secondly, an important known characteristic of the ahelical coiled coil domains is that, taken separately from the whole protein, they frequently and readily fold into the same stable oligomeric structure [9] . thirdly, for this reason, the a-helical coiled coil fragments are frequently recognized by conformational dependent antibodies, and can similarly elicit antibodies reactive with structurally ''native'' epitopes. in addition, these domains are short (about 40 residues) and can be rapidly produced by chemical synthesis. furthermore, when the antibodies have an anti-parasite biological activity, this designates the corresponding proteins/ fragments as potential, novel vaccine candidates to be further developed and assessed. here, we focus on the pf parasite erythrocytic stage, a target of protective antibodies and describe a straightforward, rapid procedure based on bioinformatic analysis of a-helical coiled-coil motifs and peptide synthesis. the screening of the pf genome [10] using generalized sequence profiles [11] identified several hundred proteins containing putative a-helical coiled coil motifs. through proteome and transcriptome data [12] [13] [14] we assessed which of these molecules are expressed in the pf parasite erythrocytic stage. the combined analysis/assessment identified over 100 segments associated with this stage and displaying the putative a-helical coiled coil motifs with high probability score (table s1) . out of these a-helical coiled coil fragments, in general 30-40 amino acids long, present either in the same protein or in different ones, 95 were chemically synthesized and hplc purified. among them, longer peptides (up to 70 amino acids), which contained one or more a-helical coiled coil domains, were also synthesized (antigens 1, 12 and 83; table s1 ). the selected antigens were then tested in elisa assays for reactivity with three panels of sera obtained from adult donors from burkina faso, tanzania and colombia, respectively. to our surprise, all of the a-helical coiled coil fragments were antigenic, though the prevalence of responders varied greatly (tables 1 and s1 ). in this manner, 71 proteins were identified whose lengths varied from 200 to 10,000 amino acids. twenty-one peptides with the highest prevalence of responders and elisa mean od value were selected for further studies. variation in recognition among the three panels of sera may be due to differences in the genetic background of the hosts, of the parasites and, most likely, to distinct malaria transmission conditions in the three regions. the high level of recognition of the a-helical coiled coil motifs may be explained by the fact that taken separately from the whole protein these fragments readily fold into the same stable structure in aqueous solution. indeed, circular dichroism (cd) studies of selected peptides associated with biological activities (tables 1 and 2) indicate that they predominantly assume an a-helical conformation in water. peptides 14, 27 and 45 ( figure s1a ) exhibit a cd pattern characteristic of a high a-helical content, whereas the remaining peptides show cd profiles similar to that shown for peptide 12 ( figure s1b ) or intermediate between those shown in figures s1a and s1b characteristic of a partial a-helical organization. when analyzed by size exclusion chromatography on fplc columns, peptides presented elution profiles between those exhibited by chymotrypsin and ribonuclease (mw 24 and 13kda, respectively). the cd and size exclusion chromatography results suggest that peptides adopt an a-helical coiled-coil structure, which need to be unambiguously ascertained by nmr and ultra-centrifugation studies. to test the biological activity of peptide-specific antibodies, the latter were purified by affinity chromatography using three serum pools obtained from papua new guinean adults. the 3 serum pools were first tested in elisa assays against 21 peptides that were the most antigenic (table 1) ; from these, 18 peptide-specific antibodies were purified from the most positive serum pool and tested again in elisa. these 18 antibodies all reacted with parasite native proteins in infected red blood cells as shown by ifat ( figure 1a ; table 2 ). reactivity was restricted to blood stages, since the antibodies did not react with sporozoites stages (data not shown), and this reactivity was also peptide-specific as shown by ifat competition assays with the corresponding peptide ( figure 1a ). the specificity of the antibodies obtained was investigated in detail, particularly since several peptides contain glutamic acid (glu)-rich sequences which are known to generate cross reactivity among several malarial glu-rich proteins [15] . cross-reactions were systematically investigated using each of the 18 affinitypurified antibodies on each of the 18 peptides. results show thatwith few exceptions-each antibody preferentially recognizes the peptide against which antibodies were affinity-purified, i.e. they are specific for the corresponding peptide (table s2) . to determine if non-specific antibody binding to solid phase-adsorbed antigens could be responsible for the rare cross-reactivities detected, elisa competition assays were performed. to this end, binding of antibodies to the solid phase-adsorbed antigen was competed against increasing concentrations of the homologous or cross-reacting peptides. only homologous peptides competed best whereas peptides having sequence similarity did not (figures 2a, 2b , and s2b), or at a much higher concentration ( figure s2a ) including the shorter glu-rich peptides derived from the cterminus of peptide 27 and 45 (figures 2a and 2b) . finally, the pattern of recognition of peptides by the various sera tested, which differ markedly from one to the other (data not shown), confirms the above results i.e., specificity of antibodies to the corresponding peptide. antibodies corresponding to the 18 selected peptides were tested for direct and cell-mediated anti-parasite activity. clinical experiments have shown that the antibody-dependent cellmediated inhibition (adci) of p. falciparum malaria represents one of the mechanisms controlling parasitemia and thereby clinical manifestations in humans [16] . twelve peptide-specific antibodies proved able to induce a strong (more than 40%) and intermediate (lower than 40%) monocyte-dependent parasite killing (table 1) , whereas, in the absence of monocytes, no direct effect of antibodies on parasite growth was observed. the effects were in the range observed with antibodies from african adults who have the highest natural protection known against malaria. therefore peptidespecific, human affinity-purified antibodies were functionally effective as shown by their ability to react with parasite proteins and to inhibit parasite growth. thus, in vitro functional assays show that peptide-specific antibodies elicited by natural exposure to the parasite can induce protective mechanisms effective against malaria. sixteen peptides -twelve targeted by adci positive antibodies and four controls-were used to immunize cb6f1 mice ( table 2) . eleven of them elicited an intermediate or high antibody response, four of which also recognized the parasite protein in infected erythrocytes as determined by ifat ( figure 1b ; table 2 ). as seen before for human antibodies, recognition was restricted to blood stages since sporozoites were negative in ifat assays (data not shown) and by ifat competition assays with the corresponding peptide ( figure 1b ). anti-peptide 27 mouse antibodies, which are positive in ifat, are also specific for the homologous peptide 27 but not for the sequence related peptides 9, 12 and 45 (table s2) . thus, peptides, which were chosen for their propensity to form ahelical coiled coil, can induce the production of antibodies that recognize epitopes present in the native protein. improvement of the immunogenicity and structural specificity of the remaining peptides might be achieved in the future by a) a short elongation at the n-and c-terminal ends, b) stabilizing the a-helix as suggested by cooper et al. and lu and hodges [17, 18] and/or c) use of other adjuvants. genetic polymorphism in current vaccine candidates is a major limitation to vaccine development. however available genotyping studies of our peptide sequences in parasite isolates of worldwide origin indicate very limited polymorphism (plasmodb 5.2, and unpublished sequencing data). a few peptide dna sequences show deletion of one entire heptad repeat so that the shorter region still preserves its potential for the a-helical coiled coil formation. with regard to the structural features and cellular location prediction of the proteins corresponding to the peptides selected for adci assays ( table 1) , 15 of the proteins contain a pentapeptide conforming to the pexel consensus [19, 20; 21, 22] , but that none of these have a position within the amino acid sequence that conforms to the location of known active pexel motifs (see materials and methods and membrane segments, and none of them has a gpi anchor. only one protein contains a signal sequence. fourteen proteins are predicted to be in the cytoplasm, one in the nucleus, one in the mitochondria, and one in the peroxysomes (table s3 ). the prediction of the sub-cellular localization of these proteins should be taken with caution because gene annotation is being constantly updated and/or protein trafficking of the parasite is complex and not fully elucidated [23] . further investigations will be required to determine the actual localization of the corresponding antigens. the predicted localization is a priori surprising for molecules able to trigger an adci activity. however, recent studies have shown that in addition to merozoite surface proteins, soluble proteins released at the time of schizont rupture were equally effective at triggering adci provided they defined at least two epitopes [24] , which is the case for a-helical coiled coil heptad repeats. therefore, molecules expressing a trans-membrane domain that can be exported to the parasite or host cell membrane, as well as molecules present in the cytoplasm of maturing schizonts and released by bursting schizonts can trigger antibodies to cross-link fc-c receptors on monocytes to achieve pf parasite killing. in conclusion, an approach combining a genome-wide search by bioinformatics of a-helical coiled coil protein motifs and chemical synthesis can lead to the rapid identification and development of new malaria vaccine candidates. in fact, this approach is straightforward and easy to scale up; vaccine formulations may comprise mixtures of peptides or single constructs made up of several epitopes. in principle, this strategy can be extended to the discovery of proteins and vaccine candidates in other complex pathogens. the pf 3d7 genome [10] was used for the bioinformatics analysis. the generalized sequence profile method and the pftools package [11] were used to search for the short a-helical coiled coil domains. the coiled coil profiles were constructed using an alignment of several amino acid sequences corresponding to the known coiled coil domain. two profiles containing four and five heptad repeats were used for the analysis. the cut-off levels of the profiles were chosen by tests performed against sequence database of proteins with the known 3d structures. subsequently, the coiled coil figure 1 . immunofluorescence microscopy analysis of pf 3d7 parasites with peptide specific antibodies. acetone/methanol-fixed schizonts and merozoites were reacted with a: human peptide specific, affinity purified antibodies obtained with peptides 12 and 14 (table 1) and b: sera from mice immunized with peptide 27 (table 1) . grey: bright field images; blue staining: indicates dapi nuclear staining of schizont stage parasites; red staining shows labeling of peptide specific antibodies by cy3-conjugated anti-human or anti-mouse igg specific antibody. merge picture is an overlay of the blue and red fluorescence channel. doi:10.1371/journal.pone.0000645.g001 regions selected by this approach were tested manually for the presence of the characteristic heptad repeats. these proteins were also analyzed by the coils program [25] . the selected a-helical coiled coil containing proteins were further tested on their possible surface location and gpi anchoring by using the following programs: identification of potential signal peptides, secretomep and signalp (http://www. cbs.dtu.dk/services/) [26] ; transmembrane spanning regions (tmpred http://www.ch.embnet.org/software/tmpred_ form.html and tmhmm http://www.cbs.dtu.dk/services/ tmhmm; [27, 28] ), and gpi-anchored proteins (http://mendel. imp.univie.ac.at/sat/gpi/gpi_server.html; [29] ) and prediction of sub-cellular localization (ptarget http://bioinformatics. albany.edu/,ptarget; [30] ). to identify the pexel-like motifs in sequences of the selected proteins we used the following pattern [ [deq] that represents a combination of the pexel patterns indicated in recent papers [21, 22] . the presence of the identified proteins in the asexual erythrocytic stages was also checked using the published data on the transcriptome and proteome of this stage of development of p. falciparum (www.plasmodb.org; [31] ). peptides were synthesized on the advanced chemtech (hatley st george, uk) ac t348 omega multi channel synthesizer and the applied biosystem synthesizer 431a and 433a (foster city, ca) using solid-phase fmoc chemistry. crude peptides were purified by rp-hplc (c18 preparative column) and analyzed by mass spectrometry (maldi-tof; applied biosystem, foster city, ca). chemicals and solvents used for peptide synthesis were purchased from fluka (buchs, switzerland) and novabiochem (laufelfinger, switzerland). circular dichroism (cd) spectra of peptides were recorded on a jasco j-810 spectrometer (jasco corporation, tokyo, japan) equipped with a temperature controller and a 0.1 cm path length cuvette. the measurements were made in water at ph 7.3 and 22uc and at a peptide concentration of 0.2 mg/ml. the sera from burkina faso were collected in the village of goundry located in the central mossi plateau, between 15 and 50 km north of the capital ouagadougou, in the province of oubritenga. the climate is characteristic of areas of sudanese savannah, with a dry season from november to may and a rainy season from june to october. malaria transmission is very high during the rainy season and markedly seasonal. ethical clearance was obtained from the ministry of health, burkina faso. after obtaining informed consent from parents and caretakers, heparinized venous blood samples were collected during a crosssectional survey during the malaria low transmission season 1998. the tanzanian sera came from a large-scale community based study undertaken in kikwalila village, kilombero district, morogoro region from 1982 to 1984. blood samples from adults (.15 years) were taken by finger prick and the serum was kept at -70uc until use. research and ethical clearance for the study was obtained by the tanzanian commission for science & technology. the colombian sera were collected in buenaventura the main port on the colombian pacific coast after human informed consent, during a cross sectional survey carried out from february to may 2002 within the framework of a project supported by the colombian research council, colciencias. the area has unstable transmission of both p. falciparum and p. vivax malaria. ethical clearance to draw blood from human volunteers was human purified antibodies were used at 5 mg/ml; ifat was not performed on ring stages. valle. blood was taken by venipuncture into tubes containing edta and sera fractionated and stored frozen until use. the sera from adults from papua new guinea (png) pooled for affinity purification were collected in the maprik district of the east sepik province, during a cross sectional survey in july 1992 within the framework of the malaria vaccine epidemiology and evaluation project (mveep) supported by the united states agency for international development [32] . the area is highly endemic for malaria. ethical clearance for mveep was obtained from the png medical research advisory committee. blood was taken by venipuncture into tubes containing edta. the pool of immune african globulins (piag) used for adci was prepared from immune individuals living in endemic areas and negative control igg (n-igg) was obtained from a pool of more than 1000 french adult donors with no history of malaria. briefly, the igg fractions from both positive and negative controls were purified using a size exclusion trisacrylh gf05m (pall bioseprah; pall life sciences, ny) column followed by an ionic exchange deae ceramic hyperdh f column (pall bioseprah). purified igg were then extensively dialyzed against rpmi and kept at 4uc until use. cb6f1 mice were injected 3 times with 20 mg of the indicated peptide in montanide isa 720 at the base of the tail on day 1, 22 and 78. bleeding was performed 10 days after the second and third immunization. elisa was performed according to lopez et al. [33] and anti human igg-or anti mouse igg conjugated to alkaline phosphatase was used (sigma, st louis, mo) as second antibody. individual human sera from 37, 42 and 39 adults donors from burkina faso, tanzania and colombia respectively were used at 1:200 dilution. serum was considered positive if the optical density (od) reading was higher than the mean od value+3 standard deviation (sd) of the negative controls (individual serum samples from 8 to 11 naïve swiss donors) or if the od ratio of the mean of duplicate experimental values to the mean od of the negative control was higher than 2. for mouse sera, the end point value was determined as the last dilution of the mean od value+3 standard deviation (sd) of the negative control (non immune sera). elisa competition assays were performed by incubating either each of the 18 selected human affinity-purified antibodies, or antibodies elicited in mice, together with each of the 18 antigens over the indicated range of concentrations for 30 minutes at room temperature prior to addition to the elisa peptide-coated plate wells. antigen-sepharose conjugate preparation: 5 mg of antigen was dissolved in 1 ml of coupling buffer (0.1 m nahco 3 containing 0.5 m nacl, ph 8.0). the cnbr-sepharose 4b (amersham bioscience ab, uppsala, sweden) was activated by swelling in 1 mm hcl and then washed with coupling buffer. the antigen solution was added to the gel and the mixture was stirred for 1h at rt. after the coupling reaction, excess antigen was washed away with coupling buffer. the unreacted activated groups were blocked by treatment with ethanolamine (0.25 m; ph 8.0) for 30 min at rt. the gel was then washed with sodium acetate buffer (0.1 m; ph 4.0), followed by coupling buffer. the antigensepharose beads were either used or stored at 4uc in pbs (1x) containing 1 mm azide. isolation of specific antibody: pooled human serum was diluted five times with pbs (1x) containing 0.5 m sodium chloride and mixed with antigen-sepharose conjugate. this mixture was then stirred gently on a wheel o/n at 4uc. after centrifugation the supernatant was collected and stored at 220uc for further use. the antigen-sepharose beads were then washed with 5 ml of trizma base tris (20 mm containing 0.5 m nacl, ph 8.0) then with 5 ml of tris (20 mm, ph 8.0). the elution of bound antibody was achieved with glycine (0.1 m, ph 2.5). the fractions obtained were instantly neutralized with tris (1 m, ph 8.0), dialyzed against phosphate buffer (0.1m, ph 7.0) and the antibody concentration was determined by the absorbance of the solution at 280 nm. slides coated with pf sporozoites were dried at rt for 30 minutes, fixed with 100% acetone at 4uc for 10 minutes, washed 2 times in pbs-0.05% tween 20, dried carefully and blocked with 20 ml/ well of pbs-3% bovine serum albumin (bsa) for 30 minutes at rt. slides coated with pf merozoites were fixed with 100% acetone at 220uc for 15 minutes and dried o/n at rt. the appropriate antibody or serum dilutions prepared in pbs-3% bsa were distributed (10 ml/well) and incubated for 1h at rt in a humid chamber. after washing with pbs-0.05% tween-20, goat anti-human or goat anti-mouse polyvalent immunoglobulins conjugated to cy3 (molecular probes) diluted 1/500 in pbs-3% bsa or anti-human igg (fc specific) fitc conjugate (sigma) diluted 1/50 in evans blue solution (1/50000) was added (400 ml/slide) and incubated for 1h at rt in a humid chamber in the dark. slides were washed as above, covered with 50 % glycerol, sealed and read using a fluorescence microscope (leica dmirb dc200). the uganda palo alto strain (fup/c) was cultured in rpmi-1640 supplemented with 0.5% albumax i (gibcobrl-invitrogen, san diego, ca). for adci assays, blood stage parasite cultures were synchronized by at least two successive sorbitol treatments followed, after maturation over 24 h, by floatation on 1% porcine skin gelatin type a (sigma). blood monocytes (mn) were prepared from cytapheresis samples obtained from healthy blood donors with no previous history of malaria (lecourbe blood bank, paris, france). peripheral blood mononuclear cells (pbmc) were separated on ficoll density gradients j prep (techgen, les ulis, france) and washed in ca 2+ and mg 2+ free hbss buffered with 10 mm hepes (both from gibcobrl-invitrogen). cells were then distributed on polystyrene 96-well flat-bottomed culture plates (tpp, trasadingen, switzerland) and adherent mn were selected by incubation for 2 h at 37uc, in a humidified 5% co 2 atmosphere. more than 90% of the adherent cells obtained in this manner were mn as estimated by the non-specific esterase test (a-naphtyl acetate esterase; sigma). mn from each donor were tested prior to adci assays and only those without direct inhibitory effect were used in assays. to wells containing 2610 5 mn purified as described above, 50 ml of an asynchronous parasite culture at 0.5 % parasitemia and 4 % hematocrit were added. wells were then supplemented with test or control antibodies (ab) and the total volume adjusted to 100 ml with culture medium. after 48 h and 72 h, 50 ml of culture medium were added to each well and after 96 h the adci assay was stopped and the final parasitemia was determined by light microscopy on giemsa-stained smears by counting $50,000 red blood cells. for each ab tested, duplicate wells included the following controls 1) non-specific monocytic inhibition, both mn+parasite, and mn+n-igg+parasites and 2) direct inhibition by control or test igg, both n-igg+parasites, and test abs+parasites. piag and n-igg were used at a final concentration of 1 mg/ml as positive and negative controls respectively. immunopurified tests abs were used at 15 mg/ml. the specific growth inhibitory index (sgi) which considers the parasite growth inhibition due to the effect of test abs cooperating with mn was calculated as follows: sgi = 1006[12(% parasitemia with mn and test abs/% parasitemia test abs)/(% parasitemia with mn and n-igg/% parasitemia n-igg)]. figure s1 cd spectra of the peptides 45 (s1a) and 12 (s1b) found at: doi:10.1371/journal.pone.0000645.s001 (12.32 mb tif) figure s2 elisa inhibition assay using anti-human peptide specific antibodies. binding of peptide specific antibodies to peptides 76 (s2a) and 9 (s2b) absorbed on elisa plates was inhibited by incubating specific antibodies (1-2 mg/ml) with peptides 14, 76 and 81 (s2a) and peptides 8 and 9 (s2b), respectively (see material and methods). peptides 14, 76 and 79 share nnm or mnn as sequence similarity while peptides 8 and 9 do not exhibit any apparent sequence similarity. table s3 structural feature and cellular location prediction of the proteins containing the peptides whose specific antibodies were tested in adci (table 1) . found at: doi:10.1371/journal.pone.0000645.s005 (0.05 mb doc) identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing reverse vaccinology and genomics plasmodium falciparum liver stage antigen-1 is well conserved and contains potent b and t cell determinants protection against plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen 3 phase i malaria vaccine trial with a long synthetic peptide derived from the merozoite surface protein 3 antigen plasmodium falciparum merozoite surface protein 6 displays multiple targets for naturally occurring antibodies that mediate monocyte-dependent parasite killing template-based coiled-coil antigens elicit neutralizing antibodies to the sars-coronavirus phase 1 randomized double-blind safety and immunogenicity trial of plasmodium falciparum malaria merozoite surface protein fmp1 vaccine de novo design of alpha-helical proteins: basic research to medical applications genome sequence of the human malaria parasite plasmodium falciparum a flexible motif search technique based on generalized profiles transcriptomics and proteomics: tools for the identification of novel drug targets and vaccine candidates for tuberculosis a proteomic view of the plasmodium falciparum life cycle the transcriptome of the intraerythrocytic developmental cycle of plasmodium falciparum crossreactive antigens between life cycle stages of plasmodium falciparum antibodies that protect humans against plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes mapping of conformational b cell epitopes within alpha-helical coiled coil proteins a de novo designed template for generating conformation-specific antibodies that recognize alpha-helices in proteins targeting malaria virulence and remodeling proteins to the host erythrocyte a host-targeting signal in virulence proteins reveals a secretome in malarial infection proteomic analysis identifies novel proteins of the maurer's clefts, a secretory compartment delivering plasmodium falciparum proteins to the surface of its host cell multi-character population study of the vir subtelomeric multigene superfamily of plasmodium vivax, a major human malaria parasite a maurer's cleft-associated protein is essential for expression of the major malaria virulence antigen on the surface of infected red blood cells a novel antibody-dependent cellular cytotoxicity mechanism involved in defense against malaria requires costimulation of monocytes fcgammarii and fcgammariii predicting coiled coils from protein sequences improved prediction of signal peptides: signalp 3.0 tmbase-a database of membrane spanning proteins segments predicting transmembrane protein topology with a hidden markov model: application to complete genomes prediction of potential gpimodification sites in proprotein sequences ptarget [corrected] a new method for predicting protein subcellular localization in eukaryotes plasmodb: the plasmodium genome resource. a database integrating experimental and computational data the malaria vaccine epidemiology and evaluation project of papua new guinea: rationale and baseline studies a synthetic malaria vaccine elicits a potent cd8(+) and cd4(+) t lymphocyte immune response in humans. implications for vaccination strategies the authors wish to thank luis rodrigues and florela penea for the synthesis and purification of peptides and thomas smith for discussion and all of the blood donors. key: cord-320092-0qnvydux authors: ehsani, sepehr title: covid-19 and iron dysregulation: distant sequence similarity between hepcidin and the novel coronavirus spike glycoprotein date: 2020-10-16 journal: biol direct doi: 10.1186/s13062-020-00275-2 sha: doc_id: 320092 cord_uid: 0qnvydux the spike glycoprotein of the sars-cov-2 virus, which causes covid-19, has attracted attention for its vaccine potential and binding capacity to host cell surface receptors. much of this research focus has centered on the ectodomain of the spike protein. the ectodomain is anchored to a transmembrane region, followed by a cytoplasmic tail. here we report a distant sequence similarity between the cysteine-rich cytoplasmic tail of the coronavirus spike protein and the hepcidin protein that is found in humans and other vertebrates. hepcidin is thought to be the key regulator of iron metabolism in humans through its inhibition of the iron-exporting protein ferroportin. an implication of this preliminary observation is to suggest a potential route of investigation in the coronavirus research field making use of an already-established literature on the interplay of local and systemic iron regulation, cytokine-mediated inflammatory processes, respiratory infections and the hepcidin protein. the question of possible homology and an evolutionary connection between the viral spike protein and hepcidin is not assessed in this report, but some scenarios for its study are discussed. (ace2) is thought to be its (main, but perhaps not exclusive) receptor on human host cells [132, 136] . the spike protein is formed of a receptor-binding subunit (s1), a membrane-fusion subunit (s2), a single-pass transmembrane (tm) domain, and a cytoplasmic/intracellular tail (ct) [75, 115] . of note, the s1 domain has a similar fold as human galectins (galactose-binding lectins) [97] . briefly, in terms of the putative primary function of the spike protein, li comments that "because coronaviruses must enter cells for replication, membrane fusion is the central function of coronavirus spikes" [75] . a basic question that might arise is: what exactly makes the pathobiology and disease course of these particular viruses unique? and, could it be that, in addition to viral replication inside the particular types of human host cells, other intracellular processes specific to these viruses are involved? (for a thematically related inquiry, see e.g., ref. [58] .) having this in mind, we wondered if there might be any sequence similarity (and thereby potential structural similarity) between the sars-cov-2 spike protein (which has 1273 amino acids [133, 134] ) and any vertebrate protein(s). a simple blast search does not reveal any similarities with human proteins. however, based on previous experience with the pufferfish takifugu rubripes proteome [41, 42, 111] and its unique evolutionary history, we initially restricted the search to this species. briefly, teleost ancestor species underwent an ancient wholegenome duplication event [125] , and teleost species with well-annotated genomes such as the pufferfish can provide invaluable insights into the sequence evolution of genes with a clear phylogenetic linkage to mammalian genes (for a related commentary, see ref. [76] ). interestingly, a query using the full-length sars-cov-2 spike protein (accession no. yp_009724390.1) revealed a sole hit with the pufferfish hepcidin protein (xp_ 003965681.1; score: 32.7, e-value: 0.54). given that sars-like coronaviruses can be found in bats [133, 134] , we also used a full-length bat coronavirus sequence (ana96027.1) as the query, which showed a closer match with pufferfish hepcidin (score: 38.5, e-value: 0.005). conversely, a blast search in the coronaviridae family of viruses using the pufferfish hepcidin (xp_ 003965681.1) revealed the bat coronavirus spike protein (ana96027.1) as the top hit (score: 38.5, e-value: 5e-04). the scores and e-values here are not meant to indicate any claims of statistical significance but are rather provided for the purpose of comparison. this similarity between the spike protein and hepcidin is at the cytoplasmic tail [80] of the spike protein, or, depending on the exact domain delineation, perhaps at the junction between the tm and ct domains. a multiple sequence alignment of this sequence region (generated using the alignx feature of vector nti advance 11.0, invitrogen, carlsbad, ca, usa), using three coronavirus spike proteins and four hepcidin proteins (from pufferfish, bat and human) is illustrated in fig. 1a . the alignment depicts a number of conserved motifs, particularly between the first pufferfish hepcidin sequence (various pufferfish species have at least two hepcidin-like genes [55] ) and the coronavirus spike proteins. in a sense, the pufferfish sequence seems to act as a 'bridge' between the coronavirus motifs and those in the human hepcidin sequence. this may not be surprising particularly given the evolutionary context of teleost species described earlier. the similar cysteine-rich motif takes the following form: 'lxxxtxccxcckgxxxcgxcc(r/k)f'. of note, the eight cysteines of the mature human hepcidin in the similarity motif, and the aligned cysteines of the sars-cov-2 spike protein, are not all specifically coded by one of the two cysteine-coding codons (tgc and tgt). both codons are present in the respective gene segments. also, for comparison purposes, as the coronavirus envelope protein [112] contains a related 'lcayccn' motif [135] , this sequence was added as the last line of the alignment. although this is a 'distant' and limited sequence similarity, it cannot be attributed to 'chance'. the search that found hepcidin did not reveal a range of similarities with other teleost proteins. moreover, there are many cysteine-rich protein sequences in teleosts and vertebrates in general, yet this similarity to the hepcidin gene family (not merely a one-off sequence) was unique and specific. how or why this similarity arose is then a separate, and potential follow-up, question. on this point, rather than framing the question as one having to do with a chance/random occurrence, it could more aptly be framed under concepts related to convergent/adaptive evolution versus common ancestry. clearly, however, no conclusive claims about homology and sequence conservation can be made at present without a concerted investigation focused on this topic. nevertheless, the similarity reported here raises a potential and intriguing question of whether there could be mimicry of human hepcidin (structural, functional or otherwise), perhaps inside the host cell, by the tm-ct junction of the spike protein. these possibilities should not be dismissed based on the mere phylogenetic gap between coronaviruses and vertebrate species. furthermore, the phylogenetic gap, and the absence of clear evolutionary homologies between genes that may otherwise have unexplored functional linkages, should not dissuade one from pursuing such connections. as a case in point, gene network and protein structure/function linkages between certain yeast (unicellular eukaryotes) and mammalian genes proved to be quite beneficial in investigating neurotoxicity in human cells [123] . the question now is if the linkage between the spike protein and hepcidin could be expanded, and what reasonable scenarios for its experimental validation could be envisaged. hepcidin is a small peptide hormone that was discovered in 2000/2001 [7, 63, 69, 96, 102] , and initially named leap-1 (liver-expressed antimicrobial peptide). it has an antiparallel beta-sheet fold and contains four disulfide bonds, and is involved in iron trafficking and the host's response to infection [34] . in fact, it has been remarked by a number of commentators that "hepcidin is to iron, what insulin is to glucose" [57] . clear hepcidin orthologs appear to be missing in birds and invertebrates [105] . the human hepcidin (encoded by the hamp gene on chromosome 19q13) is an 84-amino-acid prepropeptide, leading to a mature 25-amino-acid peptide that is detectable in blood and urine [65] . the proprotein convertase furin has been demonstrated to cleave prohepcidin at a polybasic site [73, 124] . of note, a furin-like cleavage site ('rrar') has been reported to exist in the ectodomain of the sars-cov-2 spike protein, which is fig. 1 comparison of select hepcidin and coronavirus spike protein sequences. a a multiple sequence alignment of the c-terminal region of a number of coronavirus spike proteins (encompassing portions of the putative transmembrane and cytoplasmic tail segments), four hepcidins and the sars-cov-2 envelope protein is presented. the envelope sequence is provided only to demonstrate the cysteine residues with which the spike protein is proposed to form disulfide bridges [135] . the residue numbers are shown on the sides of each protein segment, and for proteins whose c-terminal sequences continue beyond the alignment, the full residue length is provided to the right. as per a color scheme used previously [111] , dark green, grey and black highlights depict conserved, similar and identical residues, respectively. 'tr' stands for takifugu rubripes (japanese pufferfish), 'rf' for rhinolophus ferrumequinum (greater horseshoe bat) and 'hs' for homo sapiens (human). the protein accession numbers of the sequences shown are, in order: (1) awh65954.1, (2) yp_009724390.1, (3) ana96027.1, (4) xp_003965681.1, (5) xp_029694670.1, (6) ensrfet00010014064.1, (7) np_066998.1 and (8) qhd43418.1. the domain illustration of the spike protein is based on ref. [132] . b a solved nmr structure of human hepcidin [65] (pdb: 2kef), adopting an antiparallel beta-sheet fold, is visualized with its putative four disulfide bonds formed between eight cysteine residues. c the position of the disulfide bonds in the sequence of the mature human hepcidin is illustrated along with the potential palmitoylation residues (ten cysteines) of the cytoplasmic tail of the sars-cov-2 spike protein. the palmitate visual is as per ref. [77] with permission from the publisher (springer nature) absent in coronaviruses of the same clade [5, 28] . the protein-coding part of the hamp gene is split over three exons, with the 25-amino-acid mature peptide occurring on the last exon. in terms of its putative function, prentice notes that "although the hepcidin molecule does itself possess some antimicrobial activity, this is rather weak compared to peptides such as defensins, and its primary contribution to innate immunity is via regulation of iron" [105] . in fish species, one of the two hepcidin paralogs has been shown to potentially possess antimicrobial effects in innate immunity [78, 79] . finally, hepcidin binds to and mediates the degradation of ferroportin (encoded by the slc40a1 / fpn1 gene), the only known cellular iron exporter. the structural details of this interaction are being mapped and studied in ever more detail [18, 88, 108] . there are a number of solved structures of hepcidin [65, 72] . an nmr structure of human hepcidin is depicted in fig. 1b (visualized using 3-d molecule viewer, vector nti advance 11.0, invitrogen) including the locations of the four putative disulfide bonds. of note, in addition to various hepcidin orthologs containing eight cysteines, four-cysteine variants have been described in notothenioid fish species [137] . available solved structures of the coronavirus spike glycoprotein, as far as our search could reveal, mostly utilize expression constructs that stop just short of the tm domain. as noted, this is partly because the protein's ectodomain is the main focus of studies on viral binding to host surface receptors [129, 131, 132] . for example, wrapp, wang and colleagues have reported the cryo-electron microscopy structure of ectodomain residues 1-1208 of the spike protein (trimer in the prefusion conformation) [132] , but this excludes the tm and ct domains. it goes without saying that the inclusion of transmembrane domains would require complicated structural elucidation protocols, and even then, one may still not be able to solve the structure of the protein in its entirety. moreover, we would like to note that using the pfam-a (ver. 32) structural/domain database [46] in the hhpred remote homology and structure prediction toolkit [145] , the coronavirus spike protein regions analyzed here show some predicted structural similarity to lipolysis-stimulated receptor (lsr) lipoprotein receptor family (pf05624) [139] , and hepcidin sequences show some predicted structural similarity to the sar8.2 protein family found in solanaceae plants (pf03058) [2] . what, if any, significance these findings may hold is unclear at present. of more importance right now would be the theoretical and/or actual elucidation of the structure of the spike protein tm-ct junction region and a comparison with the available hepcidin structures (the pfam hepcidin entry, pf06446, currently references six pdb structures). of note, a recent in silico study has reported on a predicted structural similarity and compatibility between hepcidin and an allosteric site in the sars-cov-2 spike protein [32] . given the prominence of cysteines in the aligned motif ( fig. 1a) , how are they utilized in the respective similar domains? at first pass, the usages appear to be different: as noted earlier, in hepcidin, the cysteines may give rise to a compact disulfide-bridged peptide [65, 89] (fig. 1b) , whereas in coronavirus spike glycoproteins, the cysteines in the tm-ct junction serve as palmitoylation acceptor residues [117] (fig. 1c ) that facilitate membrane fusion [24] . it should be mentioned, however, that 'mini-hepcidins' conjugated to palmitoyl groups have been synthesized and studied previously [106] , but these do not occur naturally. as for the sars-cov, at least a portion of the palmitoylation of the spike protein has been reported to occur in a pre-medial golgi compartment [81] . however, there is also the possibility of crossdisulfide bond formation with a non-homologous small cluster of cysteines within the envelope protein [135] . moreover, if s-palmitoylation is a reversible and dynamic process [118] , it is to be determined if the spike protein junction cysteines might in fact have a different post-translational modification in the host cytoplasmic environment (although there is no evidence of this at the moment, and the reversibility of palmitoylation in viral spike proteins has not been reported [47] ). that being said, in the cited paper by mcbride and machamer, the authors conclude that the palmitoylation of the sars-cov spike protein "was not necessary for s protein stability, trafficking or subcellular localization" nor "for efficient interaction with m protein" [81] . to what extent the post-translational modifications of the spike protein and hepcidin, be it furin cleavage, disulfide bonds or palmitoylation, are in any way similar in an intracellular context, remains to be examined. as alluded to earlier, in terms of the possibility of an evolutionary connection between the spike protein and hepcidin, one could imagine a scenario whereby an ancestral spike protein acquired a hepcidin-like sequence from a host organism, and the new sequence was palmitoylated to aid with membrane association. li points out that "the primordial form of coronavirus spikes might contain s2 only" [75] , and the cytoplasmic motif features highlighted in the current report do not appear to be present in other class i viral membrane fusion proteins (which include the influenza virus hemagglutinin [20, 66] ), although we have not performed an exhaustive search. however, a number of questions might then arise under this scenario, such as the difference between the primary localizations of hepcidin (considering its putative interaction with extracellular and transmembrane regions of ferroportin [12, 18, 108] ) versus the ct domain of the spike protein. alternatively, it might be argued that perhaps a case of convergent sequence evolution is at play. for example, the influenza hemagglutinin glycoprotein appears to have a conserved 'cxici' motif in its cytoplasmic tail domain [93, 128] , and perhaps an ancestral spike protein with similar features convergently acquired hepcidin-like sequence motifs. these are of course speculations and remain open questions. investigations pursuing these topics could also make use of studies that attempt to trace the evolutionary history of hepcidin itself [67, 137] . one of the first questions that may arise from this potential sequence similarity is whether the comparison is equal for all seven known human coronavirus strains, or if it is more limited to the severe disease-causing viruses. figure 2 depicts a sequence alignment to start to answer this question. the alignment is restricted to the length of the mature human hepcidin protein, which is depicted at the bottom row. the first four spike protein sequences are comprised of the mild/asymptomatic strains, i.e., 229e and nl63 (alpha coronaviruses) and hku1 and oc43 (beta coronaviruses). these are followed by the spike proteins of mers-cov, sars-cov-1 and sars-cov-2. the bat coronavirus sequence and the four hepcidins are depicted in the same order as in fig. 1a . paying particular attention to the region between the two conserved cysteines (marked by two black arrows), there appears to be less similarity in the motif between the first four spike proteins than the rest of the sequences. specifically, for example, a 'conserved' glycine in the sixth position from the first overall-conserved cysteine (indicated with a red arrow) appears to be an important residue that groups together the three disease-causing spike proteins with the bat sequence and the two teleost hepcidin proteins. how, if at all, these differences play out in the cell might be an intriguing experimental direction. what can also be noted from fig. 2 is that although there are appreciable differences in the spike protein domains visualized in the figure between the mers-cov and the two sars-cov sequences, the sars-cov-1 and sars-cov-2 spike proteins are almost identical in this region (the tabulated amino acid identity and conservation values shown were calculated for the aligned region using the alignx program). therefore, based on the hypothetical link presented in this report, it might be reasonable to assume that any pathobiological differences between the two sars strains would not be as a result of any differing biology attributable to the hepcidin similarity domain. for reference, the sars-cov-2 genome has been reported to be 96% identical to bat cov (nucleotide identity), 80% to sars-cov-1, 55% to mers-cov and 50% to common cold cov (e.g., the 229e and oc43 strains) [13] . also of note in fig. 2 is the percentage residue identity and conservation between takifugu hepcidin and the corresponding bat coronavirus spike protein region (54% identity, 62% conservation), which are the same identity and conservation values between the two takifugu hepcidin paralogs. more broadly, the potential sequence link reported here points to the need to build on previous research on the cytoplasmic tail of the spike protein (e.g., refs. [80, 100, 101] ) to better understand its distinct roles from the time of viral attachment to possible protein-protein interactions inside the host cell. of the rare mutations currently reported in the sars-cov-2 spike protein (using patient-isolated genomic data in the gisaid repository), only one (p1263l) appears to be in the cytoplasmic tail domain [68] , placing it outside of the similarity region reported here. moving back to the biology of the hepcidin protein itself, as noted previously, hepcidin binds to and mediates the degradation of the iron exporter ferroportin. if the sequence similarity reported here is actually playing a significant role at the cellular level, could it be that, although the cellular localizations appear to be different based on current knowledge, the sars-cov-2 spike protein cytoplasmic tail can partly mimic the structure of hepcidin and interact with ferroportin? could the cytoplasmic tail even coordinate and bind iron? these remain to be investigated experimentally, but of note, rishi and colleagues recently reported on the intracellular localization of ferroportin dimers, and concluded that both the carboxy-and amino-termini of the protein are intracellular [109] . as cited earlier, the details of hepcidin's own interaction sites with ferroportin remain the subject of different structural determination projects [12, 18, 108] . relatedly, and of interest, neves and colleagues, using experiments on iron overload in european bass (dicentrarchus labrax), have discussed the functional partnership between hepcidin and ferroportin from an evolutionary perspective and suggested that this may "open new possibilities for the pharmaceutical use of selected fish [â�¦] hepcidins during infections, with no impact on iron homeostasis" [90, 91] . notwithstanding the specificities of the hepcidinferroportin interaction, the sequence similarity reported here also points to a possible broader focus on iron biology. following calcium, oxygen and lead, iron has historically been one of the most studied elements in cell biology [40] , and as such there is a vast body of (at times conflicting) literature to draw upon relating to iron in coronavirus infections. other investigators have reviewed broad themes from iron biology in the context of covid-19 [39, 78, 79] , but here i will only touch upon salient features of iron biology that revolve around hepcidin. in the three subsections that follow, i will briefly discuss relevant and recent research with fig. 2 comparison of the core hepcidin-like motif among the seven known coronavirus human infection-causing strains. following the alignment presented in fig. 1a , the core similarity motif, corresponding to the length of the mature human hepcidin sequence, is shown in an alignment containing the spike protein cytoplasmic domain of the four mild/asymptomatic human coronavirus strains and the three severedisease-associated strains. hepcidin sequences from the previous figure appear on the last four lines. the alignment color scheme is the same as in fig. 1 . focusing on the region between the two black arrows, fewer similar residues could be observed between the group of mild/ asymptomatic coronavirus strains and the mers/sars-cov strains. one such residue that can act to distinguish the groups is indicated with a red arrow. the accession numbers of the sequences shown, in order of appearance, are: perspectives on inflammation, hypoxia and diagnostics. moreover, the main themes discussed in these segments have been summarized pictorially in fig. 3 . foremost among the potential physiological connections related to the present discussion is the so-called 'cytokine storm' mediated by interleukin-6 (il-6) reported in some covid-19 patients [70, 84, 87, 138] . this is not, however, restricted to il-6 and in fact elevated levels of a bundle of pro-inflammatory cytokines has been reported in severe covid-19 cases [25, 26] . indeed, researchers have proposed that "reduced innate antiviral defenses coupled with exuberant inflammatory cytokine production are the defining and driving features of covid-19" [19] . in terms of the chronology of events, we could consider an initial "immune defense-based protective phase" and a subsequent "inflammation-driven damaging phase" in the disease [116] . how could these relate to hepcidin biology? first and foremost, an early set of findings on hepcidin was that it is regulated by anemia, hypoxia and inflammation [92] . a general understanding of this regulatory network is that inflammation brought about by infections increases hepcidin production, which in turn can lead to the anemia of inflammation [50, 52] . hepcidin production in the liver is induced by il-6 [51, 94] , and it has been reported that hepatic heparan sulfate affects and regulates il-6-stimulated hepcidin expression [103] . furthermore, heparin, the anticoagulant glycosaminoglycan that is a highly sulfated form of heparan sulfate, has been shown to be a potent inhibitor of hepcidin expression [104] . of interest, anticoagulant treatment has been reported to be effective in a subset of severe covid-19 patients [120] [121] [122] , and researchers have determined that the procoagulant transferrin [120] [121] [122] is upregulated in sars-cov-2 infections [83] . related to such concerns with inflammation and coagulation in covid-19, there have also recently been proposals centered on ace2 and the vasopressor system protein bradykinin [53, 110] . these are various leads requiring further investigation. it is pertinent to note that dysregulated hepcidin is a defining feature of hemochromatosis, a condition characterized by hepcidin deficiency, increased plasma iron levels and transferrin saturation [21] . in addition, a notyet-fully-established link of relevance here is the observations of a kawasaki-disease-like systemic vasculitis syndrome in children infected with the novel fig. 3 summary of salient facets of coronavirus spike protein and human hepcidin biology. on the top left of the figure, three trimeric spike proteins are depicted on a segment of the viral membrane, two in a figurative style and the middle trimer in a more detailed domain-specific format. one palmitate is shown attached to the cytoplasmic tail of the spike proteins, as per fig. 1c . following the binding step to ace2, a stylized viral fusion step is depicted based on refs. [120] [121] [122] , where an early fusion scenario (as opposed to endocytosis) is envisioned. the assembly of new viral spike proteins is shown to be taking place in an er-golgi intermediate compartment. on the right half of the figure, various elements of hepcidin and iron biology are depicted, which could result in potential outcomes such as coagulopathy, ferroptosis and hyperferritinemia. in terms of hepcidin's binding to ferroportin, two possible scenarios are depicted, whereby hepcidin could bind to the extracellular face of ferroportin or deeper inside the transmembrane cavity. lastly, the geometric shapes for the spike and ace2 proteins were adapted from ref. [119] , for ferroportin from ref. [30] , and for ferritin from ref. [29] , all with permission from the publisher (springer nature) ehsani biology direct (2020) 15:19 coronavirus [64, 127] , which is also being called multisystem inflammatory syndrome in children (mis-c) [37, 44] . incidentally, an association between a novel human coronavirus and kawasaki disease was reported in 2005 [43] , although other investigators were apparently not successful in confirming the link [38] . nevertheless, if the association with the kawasaki-disease-like syndrome is real, then it is noteworthy that increased hepcidin levels have been suggested as a biomarker for kawasaki disease [61] . as opposed to the inflammation-based upregulation of hepcidin, anemia and hypoxia are usually taken to have the opposite effect on hepcidin's expression [92] . further to cytokine storms and characteristic immune reactions, hypoxemic respiratory failure could be another major warning sign in covid-19 patients [31] . less established, but still important to mention, is also the debate surrounding the issue of hypoxia/hypoxemia and certain symptoms resembling, but differentiable from, altitude illness [8, 9, 15, 54] . congruously, hepcidin expression levels have also been investigated in the context of highaltitude acclimatization [59] (see also ref. [27] ). moreover, hepcidin levels are known to increase in patients with acute respiratory distress syndrome (ards) [48, 86] . it may also be important to point to a number of circumstantial but perhaps important findings in the literature pertaining to lung disease. these include (i) a link between sars and liver function abnormalities [74] , (ii) the association of pulmonary iron overload and restrictive lung disease [49, 90, 91] , (iii) the role of iron in pulmonary fibrosis [3] , (iv) hepcidin's modulation of the proliferation of pulmonary artery smooth muscle cells [107] , and (v) the vital role of hepcidin in alveolar macrophage function [98] . furthermore, (vi) hepcidin upregulation along with serum iron reduction has been reported in influenza infections [10, 45] . importantly, however, iron dysregulation changes may only be at a local cellular/tissue level and not reach a systemic response [71] , and may demonstrate selective tissue tropisms during different viral infections [11] . presently, dexamethasone and other corticosteroids are among the very few widely-used treatments for covid-19 [130] , and it would be of great significance to have robust diagnostic correlates of the course of the disease. could systemic changes in serum iron levels [60, 114, 126, 141] (with a possible view on the degree of abovenormal serum ferritin in patients [25, 26] ) or levels of hepcidin itself be detected in patients with varying covid-19 severities? first, on the latter point, researchers have recently reported that increased serum levels of hepcidin and ferritin are indeed associated with the severity of covid-19 [143, 144] . this combined hepcidin/ferritin diagnostic might potentially be promising. serum ferritin levels alone are usually considered a general indicator of inflammation and infection, and as pointed out by baron and colleagues, "the use of ferritin to diagnose iron deficiency may be problematic in patients with covid-19 disease, who may have normal or high ferritin levels despite very low iron stores" [14, 143, 144] . in fact, covid-19 has been proposed to be a part of the hyperferritinemic spectrum of conditions [56, 99] . as for correlations with plasma iron levels, shah and colleagues have reported that "compared with patients with non-severe hypoxemia, patients with severe hypoxemia had significantly lower levels of serum iron", and that "the association of serum iron with lymphocyte counts could reflect the requirement of the adaptive immune response for iron and may contribute to possible t cell dysfunction reported in covid-19" [114] . the authors did not find significant differences in transferrin saturation or serum ferritin levels between the nonsevere and severe hypoxemia patient groups. congruently, a retrospective study of covid-19 patients and serum iron deficiency has found that "the severity and mortality of the disease was closely correlated with serum iron levels" [142] . beyond the immediate issue of diagnostic markers, two broader questions that could follow from the present work can be raised: first, does the spike protein, similar to hepcidin, potentially promote iron sequestration in (alveolar) macrophages [85] and hence impede the host's initial immunological response? second, could reports of the common presence of digestive symptoms in covid-19 patients (e.g., ref. [95] ) be explainable in part by a link to hepcidin? with respect to the biology surrounding these gastrointestinal symptoms, it is important to point out that a recent study concluded that in gut epithelial cells, the "expression of two mucosaspecific serine proteases, tmprss2 and tmprss4, facilitated sars-cov-2 spike fusogenic activity and promoted virus entry into host cells" [140] . it is also known that the liver-specific serine protease tmprss6 (matriptase-2) negatively modulates hepcidin [16, 22, 36] . again, what if any significance these connections might hold remains to be determined. overall, the observations in this report suggest that, as a starting point, serum iron status would be a critical data category to be systematically collected from patients at various stages of the disease's progression. furthermore, iron dyshomeostasis in the case of covid-19 may be a more specific pathobiological feature of this infection, and we can only know the answer to this scenario if more data related to iron biology is collected during the current pandemic. could, for instance, clinical evidence be gathered to clarify if disorders of iron homeostasis might exacerbate symptoms in covid-19 patients? as a final note, given the many known and yetto-be-discovered intricacies of iron homeostasis in the body, research on therapeutic strategies that, for example, propose to utilize iron chelation or hepcidin antagonists should proceed cautiously [1, 52] . theoretical analyses in new areas may necessarily entail reasonable speculations based on limited or disparate data. this is expected, but one should remain cognizant of overinterpretation, and pursue a rational course of theoretical inquiry to hopefully inform subsequent experimental investigations. here, a purposeful and restricted protein sequence search revealed a potential sequence similarity between the relatively less-studied cysteine-rich cytoplasmic domain of coronavirus spike proteins and the vertebrate hepcidin protein. this is quite unlikely to be a spurious and random similarity. there are many cysteine-rich protein sequences in vertebrates, but the motif identified here is unique and specific, and also appears to tentatively set apart the disease-causing strains from the milder coronavirus strains. following from this link, a number of emerging clinical strands of evidence (summarized in table 1) were discussed which further link a biology surrounding hepcidin with coronavirus-caused pathobiology. while each piece of clinical evidence discussed does not by itself provide overwhelming corroboration for the hypothesis of the paper, the totality of evidence presented we believe make a strong case that if sequence and/or structural mimicry to hepcidin is taking place upon viral attachment to and entry in the host cell, then perhaps a local disease condition resembling iron dysregulation (e.g., iron overload) might ensue in the infected tissue(s). this hypothesis can be immediately tested in one of three ways. first, in the clinic, levels of various serum markers for iron biology could be more systematically and comprehensively measured and analyzed. this strand of investigation has already produced corroborating evidence in the form of increased serum levels of hepcidin and significantly lower levels of serum iron in covid-19 patients. second, computational investigations could examine potential structural mimicry between the two proteins and explore the effect of differing post-translational modifications. and third, the potential link to hepcidin could be relied upon in cellbased assays to determine the possibility of the involvement of the spike protein in iron biology. the first draft of this manuscript was posted on 27 march 2020 on the arxiv preprint server (arxiv.org/abs/2003.12191v1). thanks are due to gerold schmitt-ulms (university of toronto) and farnaz fahimi hanzaee (university college london) for very helpful discussions. author's contributions s.e. performed the analysis and wrote the paper. the author(s) read and approved the final manuscript. author's information s.e. holds a ph.d. in laboratory medicine and pathobiology from the university of toronto, and was a postdoctoral fellow at the whitehead institute for biomedical research and the computer science and artificial intelligence laboratory at the massachusetts institute of technology. he is currently a ph.d. student at the department of philosophy at university college london. the author is supported by a university college london overseas research scholarship. availability of data and materials all sequences utilized are from publicly available repositories. accession numbers are provided in the relevant sections. table 1 points of similarity and divergence between hepcidin and the coronavirus spike protein cytoplasmic domain based on current knowledge of the viral protein and its pathobiology (see main text for references) potential similarities potential differences 1. protein sequence: a unique but restricted sequence similarity exists between mature hepcidin and the cysteine-rich cytoplasmic tail of coronavirus spike proteins. 1. protein length and domains: coronavirus spike proteins are much larger, multi-domain, proteins compared to the relatively short-length hepcidin proteins. post-translational processing: the proprotein convertase furin cleaves prohepcidin, and has been reported to also activate (the ectodomain of) the sars-cov-2 spike protein. 2. post-translational modification: cysteines in the cytoplasmic tail of coronavirus spike proteins are thought to be palmitoylation acceptor residues, and this modification has thus far not been reported to be reversible. cysteines in hepcidin are used to form disulfide bonds. 3. cytokine storm: il-6-mediated inflammatory responses have been reported in covid-19 patients. hepcidin production in the liver is induced by il-6 and is well-studied in the context of the anemia of inflammation. in addition, covid-19 may be associated with a kawasaki-disease-like systemic vasculitis manifestation in children, and hepcidin levels have also been suggested as a biomarker for kawasaki disease. 3. localization: hepcidin is thought to interact with its main (iron-bound) interactor, ferroportin, extracellularly, whereas the spike protein cytoplasmic tail does not face the environment outside the viral membrane. 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neutral with regard to jurisdictional claims in published maps and institutional affiliations ethics approval and consent to participate not applicable. not applicable. the author declares no competing interests.received: 3 july 2020 accepted: 8 october 2020 key: cord-321762-7kiahjyy authors: nandy, ashesh title: chapter 5 the granch techniques for analysis of dna, rna and protein sequences date: 2015-12-31 journal: advances in mathematical chemistry and applications doi: 10.1016/b978-1-68108-053-6.50005-3 sha: doc_id: 321762 cord_uid: 7kiahjyy abstract: the very rapid growth in molecular sequence data from the daily accretion of large gene and protein sequencing projects have led to issues regarding viewing and analyzing the massive amounts of data. graphical representation and numerical characterization of dna, rna and protein sequences have exhibited great potential to address these concerns. we review here in brief several different formulations of these representations and examples of applications to diverse problems based on what this author had presented at the second mathematical chemistry workshop of the americas in bogota, colombia in 2010. in particular, we note several insights that were gained from such representations, and the applications to the bio-medicinal field. my first brush with a dna sequence, in around 1990, left me totally puzzled: i could not "see" nor get a "feel" of anything noteworthy in the apparent jumble of characters that symbolized a dna, not the least because i had never studied biology myself. my background was physics, and i began a search for, to me, a more meaningful exposition of the sequence of characters that represented the dna sequence. my studies led me to appreciate and anticipate the immense potential opening up with the sequencing of genome length sequences and the concomitant need for rapidly scanning and analyzing dna sequences for matters of interest [1] , and to get excited at the new insights being gained from a global perspective of the dna sequences: jeffreys [2] had shown through his chaos generator representation that such sequences had an inherent fractal nature; peng et al. [3] speculated that dna sequences had long-range correlations, an observation that raised a storm of papers in very short order; and voss [4] showed that long range fractal correlations existed in dna sequences with the degree of correlation varying with evolutionary divergence. but a close up look at a dna sequence and how the bases were distributed along it still lacked an appealing representation. experimenting with various formats i determined that a 2d graphical representation, as explained later in this chapter, was what i could relate to on a purely personal basis. after many graphs of various sequences and discussions with some eminent persons to ensure that such a simple stratagem was not already familiar to cutting edge biologists, i published a paper on it in current science (bangalore) in 1994 [5] . imagine my consternation when i was informed soon after that gates had already anticipated such a device, albeit with different axes assignments, way back in 1986 [6] , but which seemed to have been in limbo since! a short note had to be published soon after informing of this oversight and explaining the differences although both used cartesian co-ordinate system to plot the graphs [7] . however, a physics background demands some quantitative appraisal of whatever nature has to offer. i had observed certain similarities and changes in plots of conserved gene sequences of various species, but coming up with some way to measure the changes posed difficulties with these plots of discrete numbers. i had done some number crunching with individual gene segments like introns and exons [8, 9] , but now the need was for whole sequences for which we came up with a geometrical interpretation to describe in general a macro-molecular sequence and measure sequence differences. we presented our scheme at the first indo-us workshop on mathematical chemistry in shantiniketan, west bengal, india in 1998 [10] where we reported, as stated in the abstract, that "geometrisation of macromolecular sequences in the form of a graphical representation provides one … technique where the nucleotides in a gene sequence can be viewed as objects in a 4-dimensional space; the method can be extended, in principle, to include, say proteins, in a 20-dimensional space. we have found a reduced 2-dimensional representation of dna sequences very useful in studies of nucleotide distribution and composition. …. we here propose a new measure of the dispersion of dna graphs that can be used to quantify the differences between two or more graphs of genes of various organisms …. lt also appears that once standardized the proposed scheme may help study molecular phylogeny in evolutionary time scale." although the participants in the shantiniketan workshop included stalwarts in the field like prof. milan randić, prof. haruo hosoya, prof. paul mezey and others, our scheme did not seem to evoke any response, not surprising since they apparently did not know about dna issues. but prof. subhash basak of the university of minnesota, usa and co-organiser of the workshop was intrigued enough by our work and its potential to describe dna sequences through graph invariants to meet me in kolkata after the workshop to discuss the possibility of using invariants for dna sequences as descriptors. subsequently prof. basak invited me the following summer to duluth to carry out further research on dna mathematical descriptors in his group funded by the natural resources research institute (nrri). prof. milan randić and some other distinguished scientists were also invited there to begin to work on dna descriptors in a project funded by nrri. it began with a talk i gave at the university of minnesota, duluth about my work on mathematical descriptors of dnas arising from my graphical representation method. among the attendees was prof. milan randić who, with prof. basak, immediately saw the potential for converting a dna sequence graph to a matrix and thereby extract numerical invariants which could be a more meaningful way to characterize dna sequences. we collaborated then on a proposal for a 3d graphical representation and a matrix method for extracting graph invariants for the first exons of beta globin sequences of several species. this was published in 2000 in the journal of chemical information and computer science [12] and led very soon to a whole host of papers on the dna graphical representations and numerical characterisations and applications of them that continues still as more and more areas keep opening up and a new field of research seems to have begun. this review is a brief introduction to the readers of this new and exciting field of research on graphical representation and numerical characterization (granch) of bio-molecular sequences, based on the talk i presented at the second mathematical chemistry workshop of the americas in bogota, colombia, in july 2010 [13] . some of the various applications made to date using these techniques are also covered briefly, with special emphasis on our recent work that provides a possible approach to anti-viral vaccine design that could be expected to be less susceptible to invalidation through mutational changes in the viral proteins. more details can be found in the several reviews [14] [15] [16] [17] [18] and book chapters [19] [20] [21] that have appeared on the subject, and of course there are always the original papers. (note added in proof: see also bibliography in ref [82] .) as sequence data on long stretches of dna began to become available in the late 1980's, there arose a problem on how to view them and a curiosity to know whether any systematics lay hidden in the apparently random arrangement of characters representing the bases in the sequence. h j jeffrey [2] came up with the idea of plotting them in a square grid where the four corners were identified with the four bases a, c, g, t. the algorithm was to start from the center of the square and for the first base plot a point midway between the origin and the home corner of the base. for the second base he started from the point representing the first base and plotted a point midway between it and the home corner of the second base. continuing in this way filled up the square with a series of points until the entire sequence was plotted. this diagram he called the chaos generator representation (cgr) of the dna sequence. he noticed that different animal kingdoms showed different patterns -double scoop depletion regions for vertebrates, striped patterns for plant sequences, an apparently random distribution for bacterial genomes. overall, each sub-square section of the cgr pattern seemed a replica of the whole, i.e. dna sequences had properties of selfsimilarity or fractal nature. the cgr diagrams of various sequences were investigated by several researchers to find different properties of biological interest. burma et al. [22] showed the structures observed in cgr diagrams arises from skews in base composition and presence of repetitive sequences or specific motifs. dutta and das [23] reported that a cgr plot can be reproduced by suitable algorithms by manipulating different combinations of strings of bases with appropriate frequencies. thus, the double scoop depletion patterns seen in vertebrate cgrs arises from scarcity of cg dinucleotides, and so on. baranidharan et al. [24] developed quantitative methods to generate similarity/dissimilarity maps of genomic sequences and showed that for certain mitochondrial genomes species wise characteristic features could be seen when nucleotide stretches of 7 or more bases at a time were analysed. in a slightly different vein, peng et al. [3] considered the structure of the bases in a dna sequence and analysed them on the basis of their being pyrimidines (c,t) or purines (a,g) only. on an x-y graph where the x-axis counted the nucleotide number, they plotted the appearance of the bases in a sequence by taking a step diagonally upwards if it was a purine or downwards if it was a pyrimidine to the next nucleotide number. plotting the whole sequence step by step in this manner they generated a graph with an irregular up-down structure which they called a "dna landscape". by taking subsections of the graph they found that the subsections also looked similar to the up-down structure of the whole, and the same was true of sub-sub-sections and so on showing that the purine-pyrimidine structure of a dna sequence had self-similarity, which was what jeffrey had remarked two years ago. peng et al. then searched for possible correlations by estimating frequencies of different lengths of nucleotide stretches and found that all gene sequences with the mosaic structure of introns and exons had long-range correlations whereas intronless genes did not show this feature. the implications of such an observation, on the face of it, are huge: the beginning of the dna sequence should, theoretically, be knowing what the end would be like! such an observation quite naturally led to a storm of papers on the subject until it quitened down after the observation that since dna sequences are known to elongate by duplicating long stretches of subsequences, it was possible that such sequences showed apparent long range correlations. around the same time voss [4] conducted a rigorous analysis of 25000 dna sequences with over 50 million bases covering organisms of all classes to search for long range correlations. using a spectral density function analysis he concluded that "(a) long range fractal correlations exist in dna sequences, (b) the degree of correlation as measured by a spectral exponent varies systematically with evolutionary category and (c) short range periodicities of period 3 are prominent while other periods, e.g. 9, are also present. the fractal correlations have been seen to extend over long ranges of nucleotide positions, with the smallest for phage and bacteria and extending to over 100,000 bases for the higher classes" [14] . to get a feel for the actual distribution of bases along a dna sequences you need a more direct graphical depiction than what the abstract representations of jeffrey [2] or peng et al., [3] can offer. this problem was addressed many years ago by hamori and ruskin [25] with their proposal for a 3-dimensional graphical representation of a dna sequence. they proposed a hypothetical square on the xy plane with four corners (nw, ne, se, sw) identified with the four bases a, c, g, t and the nucleotide number to be counted along the z-axis. thus for a dna sequence like acggt, one would plot a point on the a-corner at z-coordinate 1, then draw a line to the next base, c in this case, in its corner with z now equal to 2, and so on. for a sequence like acgtacgtacgt this would generate a spiral around the z-axis; in case there was a preponderance of one base or the other the curve would flow along those corners. these curves the authors called h-curves. visualizing such a 3d image on the 2d plane of the paper is admittedly difficult. however, the authors suggested that drawing two such curves at slightly different angles would allow stereoscopic vision so that the dna could be seen in 3d. taking the bacteriophage m13 as an example they showed that in their representation they could easily identify regions of sharp changes in base composition through visualization that would be difficult to determine from the normal character representation. this author's search for a meaningful display of dna sequence information led him to propose a 2-dimensional graphical representation where the four cardinal directions are associated with the four bases [5] . the method is to take a walk in the negative x-direction if there is an adenosine in the sequence, in the positive ydirection for a cytosine, positive x-direction for a guanine and the negative ydirection for thymine. proceeding to walk in succession in the appropriate direction in the order of the bases making up the particular dna sequence generates a path that visually depicts the arrangement of bases in the sequence. these dna plots were found to be characteristic of the types of gene sequences and that the same genes from different species showed almost the same pattern. since we know that specific genes from different species have significant homology, and in fact that is how often new genes are recognized, it is not surprising that their graphical plots will show basically the same shape. it was found later that gates [6] had already proposed a similar scheme to depicting gene sequences, although his assignment of bases were different from nandy's scheme. a year later leong and morgenthaler [26] independently proposed another 2d scheme, where the base assignments were again different from the two just mentioned. on a 2d cartesian co-ordinate system the assignments of the bases with the cardinal directions in the three schemes are, starting from the negative x-direction and going clockwise, gtca (gates), acgt (nandy) and ctag (leong and morgenthaler). it is interesting to note that these three axes representations exhaust all possible 2d schemes of this type, and these can be seen to be like 2d projections of the hamori-ruskin h-curves. the 2d plots can be scaled to accommodate from the largest to the smallest dna sequences depending on the level of detail one wishes to observe. in reference 27 (the illustration (3)) depicts the 73326 thousand base long human beta globin sequence that contains the beta, eta, delta and the gamma globins of less than 2000 bases each, which can individually be plotted on a smaller scale. plots such as these provide a quick estimation of base composition and distribution along a dna sequence. an inspection of the human beta globin sequence graph shows that it has two sections that are mainly a-t rich with one part in between that is t-dominated. a plot of a sequence like the chicken myosin heavy chain gene is represented in illustration 2 (loc. cit.,) shows that it also is at-rich; from the angle it makes with the axes, it is evident that the sequence is dominated by larger percentage of t's than a's, and likewise one can determine preponderance of structures like amtn from inspections of such plots. further applications of these graphs are taken up later in this chapter. the 2d representations, however, suffer from degeneracy in that nucleotide pairs, like ag or ct in the nandy scheme, will result in only one step instead of two. bielińska-wąż et al. [28] have shown that this can be accounted for by a mathematical method of using a weight parameter for each visit to the same location, but a number of researchers has been to propose different ways to represent dna sequences graphically that reduces or removes this degeneracy. an extensive coverage of these methods can be found in nandy, harle and basak [16] , but we may mention here that one of the first proposals to reduce the degeneracy was the scheme of guo, randić and basak [29] where the unit vectors for the four bases were aligned at a small angle to the cardinal directions. yau et al. [30] used a two-quadrant representation in 2d space where a,g were inclined to the x-axis in the 4 th quadrant and t,c were inclined to the x-axis but in the first quadrant, and the nucleotide count was recorded along the x-axis; this generated a dna graph extending in the positive x-direction and had no degeneracy. he et al., [31] proposed to characterize a dna sequence by their chemical (amino, keto), structural (purine, pyrimidine) and bond strengths (weak, strong) and plotted this set of three reduced sequences as characteristic curves that extended along the x-axis with nucleotide number thus avoiding degeneracy altogether. randić proposed several constructs, among them a 4 horizontal line scheme [32] where the four bases were plotted in order of the sequence along four lines parallel to the x-axis and placed unit distance apart wile the nucleotide number was again counted along the x-axis, a compact "worm curve" representation [33] , four-color maps [34] , "spectrum-like" curves [35] among others which reduced or eliminated the degeneracy inherent in the classical 2d approach. 3d and higher dimensional representations have been proposed to more faithfully reproduce the features of a dna sequence or enable more accurate calculations. hamori and ruskin [25] had originally proposed a 5d model where the four bases were plotted in four dimensions and the fifth was for nucleotide count, but since this was difficult to visualize he had moved to the 3d h-curve representation. 3d representations and variations were also proposed by randić, vracko, nandy and basak [36] , and li and wang [37] to name a few. a 4d method was proposed by chi and ding [38] , a 6d method by liao and wang [39] and an 8d method by liu and wang [40] . the interested reader can refer to the reviews [e.g. ref 16] and the literature for details of these interesting developments. thus, the study of dna sequences is facilitated in many ways by graphical representation, but making intra-and inter-sequence comparison becomes meaningful when the similarities and differences can be quantified in some manner. the difficulty is that since the graphical plots are composed of a set of discrete points, one has to apply either novel geometrical methods or use graph theory where the points are considered as nodes and the connections between the nodes as edges. we describe below first the geometrical methods and then the graph theoretic methods. two techniques were devised, one for intra-sequence comparison and another for inter-sequence comparison. for the variations within a sequence arising from the base distribution, we had observed [27] that coding regions of mammalian gene sequences appeared as a dense cluster of points in the 2d graphical representations implying high degree of mixing of the four bases in almost equal proportions, whereas the non-coding regions that were a-t or g-c rich usually appeared as long filaments. we therefore devised a cluster density measurement by enclosing such regions in a square grid and dividing the number of points in the grid by the area of the square. this was complemented by an inverse displacement method and a fractal coefficient method to numerically assess the differences between these two types of regions. analysis of 386 introns (noncoding regions of a gene) and exons (coding regions) of 35 genes from various species by these measures showed [27] that (a) cluster density of non-coding regions are very small and fall off exponentially rapidly, (b) the cluster density of coding regions grows to about 0.8 per unit area and falls off gradually, (c) exons of evolutionarily later genes have higher cluster densities, (d) cluster densities of intronless genes like the phage m13 genome or the bacteriophage lambda are very low, closely paralleling intron densities and (e) more recent genes show greater fragmentation and smaller lengths of the exons. the cluster density measure also enabled us to propose a way of predicting protein coding regions in new dna sequences [41] and was used to analyse the human chromosome 3 contig 7 and predict existence of several genes [42] . gates [6] had proposed a manhattan distance computation to compare two or more sequences, but this method is suitable for equal length sequences, whereas gene sequences are not generally of equal lengths. to study similarities and dissimilarities of genes from various species we devised a new and different methodology, which was reported for the first time in the first indo-us workshop 1998 [10] and published the following year [11] , as mentioned earlier. since we have in the 2d graphical representation a set of discrete points comprising each gene sequence, we defined a function to describe the sequence as where s 0 is the zeroth-order term representing the coordinates x f , y f of the end points, s 1 is the linear term representing the first-order moments about the two axes, s 2 the second-order term representing the variance about the mean, s 3 the third order term representing the skewness, etc., all of which taken together became a descriptor for the sequence. for the initial presentation we computed the first order moments as weighted center of mass only and defined a graph radius, g r , the distance of the weighted center of mass from the origin, for each sequence and a g r to estimate the difference between two sequences plotted on the same scale; this scheme gave a reasonable fit to the dispersion of the beta globin genes from various species [11] . because of the cumulative nature of the sequence plots, differences in base distributions will lead to progressively increasing differences in the plots. closely related sequences with less mutational changes between them will have smaller g r while unrelated sequences can be expected to lead to larger values of the g r . as remarked by the authors, this method could clearly be generalized to apply to the case of protein and other sequences where one may represent the sequences in a multidimensional hyperspace with a view to eventually develop phylogenetic trees. these techniques have been used by several authors (e.g., [43, 48] ). bielińska-wąż et al. [28] have computed the moments to various higher orders in a 2d dynamic graph with statistical moments of mass-density distributions as new descriptors. computing the moments for a set of histone genes, they showed that the larger number of descriptors improved the characterization of the object and different aspects of the dna could be compared separately while retaining the simplicity of the 2d graphs. nandy and nandy [44] showed that the g r s were quite sensitive measures where base composition or base arrangement differences caused the g r to change and that two or more sequences will not have the same g r value except in some pathological cases. the graph theoretic method arose out of deliberations after the first presentation of the 2d graphical representations in duluth in 1999. the method described in the paper by randić, vracko nandy and basak [36] was to first represent a dna sequence graphically in a 3d cartesian grid and then convert the points to elements of a matrix by computing the ratio of euclidean distance to graph theoretic distance between all possible pairs of points taken systematically. matrix methods are well studied and have well recognized properties. the d/d matrix generated by the distance measures was analysed to yield a set of eigenvalues with the leading eigenvalue being taken as invariant of the matrix and therefore of the sequence. differences between the leading eigenvalues of various gene sequences could then be taken as indicative of their evolutionary distances, although this seminal paper limited itself to computation on the basis of the first exons of 11 beta globin genes only. the interesting point to note is that this paper led to generation of intense interest among researchers and many different ways of representing dna sequences and computation of evolutionary distances subsequently ensued (see review nandy et al. [16] ). authors such as randić et al. [33] , randić [35] , he and wang [45] , song and tan [46] and many others proposed different ways to graphically represent dna sequences and convert the plots to mathematical objects, and derive leading eigenvalues as invariants of the sequences. for example, he and wang [45] reduced the dna sequences to a set of three sequences based on their structural, chemical and bonding nature and devised a vector of the three leading eigenvalues of the matrices associated with each of the reduced sequences which they proposed as being characteristic sequences of the original dna sequence. distances between two sequences then were computed by determining the distances between the end points of the two vectors. song and tan [46] similarly devised a 24-component vector characterizing a sequence, others came up with other ways of computing the intersequence distances based on vectors devised out of the matrix eigenvalues. such matrix invariants from their own representations were used by liao et al. [47] , wang et al. [43] , liao et al. [48] and others to draw phylogenetic trees for mitochondrial genes, sars coronavirus genomes, etc. the graph theoretic method, however, does not seem to have been applied so far to determine specific features within a sequence. developments in the graphical representation and numerical characterization of dna sequences raised the possibilities of using similar analysis of protein sequences, albeit with difficulty arising from the fact that now we have to contend with 20 amino acids making up a protein chain whereas dna sequences were made up of only four nucleotides. although meeta rani [49] had shown as early as 1998 the presence of statistical self-affinity, a kind of self-similarity, in protein sequences that implies a fractal nature, graphical representation methods for proteins drew attention with the paper of randić [50] . the basic idea here was to start with the cgr method of jeffrey to plot a rna sequence drawing triangles for every triplets of bases, i.e., the genetic codes, and taking the centers of each such triangle as corresponding to the residue the triplet would code for. thus starting with the mrna, this method generates a cgr-equivalent 2d graphical representation for the protein sequence. randić et al. [51] carried the method further to construct a zigzag curve for the a-chain of human insulin which allows a direct conversion of a protein sequence into a numerical sequence of (x,y) coordinates that can be used subsequently for construction of the graph-theoretic matrices and sequence invariants. the technique was refined to remove some arbitrariness that were inherent in the 2d scheme by converting the 2d graph to a 3d graphical representation where the triplets were assigned to the corners of a tetrahedron structure; although visual inspection of the graphical patterns had to be discarded in this scheme, the authors claimed that construction of graph invariants in this manner was more accurate and unique. randić et al. [52] proposed a magic circle representation where the protein sequence graph starts from the centre following the sequence by moving half way towards the corresponding amino acids which are positioned equally spaced on the circumference of a unit circle. the result of the complete execution of the protein sequence within the circle produces a typical graph for a particular protein, except for large protein sequences which are often found to have lesser visual benefits. bai and wang [53] considered the triplet codon concept and using a complex coordinate scheme constructed a purine-pyrimidine graph on the left half of the complex plane, with purines (a and g) in the first quadrant and pyrimidines (t and c) in the fourth quadrant. a protein sequence can then be drawn from the triplet codons extending along the x-axis allowing visual inspection of the trends and also from the co-ordinates generate graph-theoretic matrices and their leading eigenvalues as descriptors of the sequences. bai and wang [54] next proposed a 3d graphical representation for protein sequences where the 20 amino acids are represented as end points in a dodecahedron embedded in the 3d space, i.e. each amino acid is represented at one of the vertices of the dodecahedron. this allows construction of a sequence graph following the amino acids in the sequence where each point in the plot can be considered as a node of the graph, from which one can again generate matrices and sequence invariants. liao et al. [48] used a 2d graphical representation method to compare 24 coronavirus sequences where the four cardinal directions were associated with particular properties of the amino acids. they classified the 20 amino acids of a protein sequence into four separate groups according to the chemistry of their r groups: amino acids a,v,f,p,m,i,l to the hydrophobic chemical group; amino acids d,e,k,r to charged chemical group; amino acids s,t,y,h,c,n,q,w to polar chemical group; and the g amino acid to glycine chemical group. starting with the nucleotide sequence, this enabled them to construct three 2d graphs (one for each reading frame) for each gene sequence and compute a distance matrix. in a similar construction, aguero-chapin et al. [55] grouped the 20 amino-acids into four categories: acidic, basic, polar and non-polar and assigned the four groups to the four cardinal directions of a cartesian frame to compute numeric descriptors of 108 sequences of polygalacturonases. in recent years the field has progressed rapidly to numerically characterize protein sequences for application to different issues. gonzález-díaz and collaborators have extended these representations to the study of protein sequences [56] and applied to mass spectral data of proteins and protein serum profiles in parasites [57] . gonzalez-diaz has found that using different type of numerical indices derived from the protein 2d molecular graphics to perform qsar studies is simpler than having to work with the protein 3d structures [58] . integrated qsars [59] developed using chemodescriptors for ligands and biodescriptors of a molecular entity connect structural information of drug molecules, dna and rna sequences or rna secondary and protein tertiary structures. basak et al. [60] using a new differential qsar approach for study of dihydrofolate reductases (dhfr) from multiple strains of plasmodium falciparum showed that dhfr from the wild strain is substantially different from four mutant strains of their study; this indicated that the protocols indicated in the paper can be used for the development of drugs to combat drug-resistant pathogens arising continuously in nature due to mutations. nandy et al. [61] showed that their 20d graphical representation of protein sequences (explained later) was useful in generating phylogenetic relationships between sequences without necessity of multiple alignments and for determining conserved surface exposed stretches on viral proteins that could be useful in drug and vaccine designs [62] . we mention in passing that randić [63] , basak and gute [64] had developed mathematical techniques for analysis of proteomics data drawing parallels with dna granch techniques, but we do not go into any details about this topic in this review. a detailed review of graphical representations of proteins including of proteomics has been made by randić et al. [65] . any new technique needs to be tested through applications to real problems and these methods of graphical representation and numerical characterization of biomolecular sequences are no exception. the intense interest which these granch techniques have evoked amongst researchers have led to many and varied applications which shows the wide applicability and great potential of the methods. we cover some of these applications in brief here, with a novel application to anti-virus drug targeting in slightly more detail. as a natural application of the graphical representation of dna sequences, consider the visualization of patterns in base arrangements that are otherwise difficult to see in the normal character representation. as already mentioned, gates [6] had noticed large scale repeats that were revealed by his 2d graphical plots and nandy [5] showed that conserved genes have shapes on the 2d maps that are similar across species. from a detailed analysis of the graphical plots of families of conserved gene sequences that these altered with evolution such that the constituent bases appear to tend to greater homogeneity in base composition and higher complexity in base composition in the protein coding sequences [66] . also, visual inspection of the graphical plots can enable new insights into similarities of different stretches of dna sequences. larionov et al. [67] had thus found long range palindromes in the mouse and human chromosomes. nandy, gute and basak [68] reported on a stretch of the h5n1 avian flu neuraminidase gene that appeared to be well conserved among the various strains of the avian flu and reported on the possibility of using this site as drug or vaccine target so that these can be effective over many mutational changes (see below). further observations and numerical computations on over 600 h5n1 neuraminidase sequences showed the wide dispersion and mutations of the gene sequences and especially the possible exchange of structural parts of the genes, which was a new observation for this type of virus [69] . based on the observations of the plots of several conserved gene sequences, nandy [70] showed that the base arrangements of these sequences could be conceived as bound by a characteristic function of the instantaneous population of the four bases as one moves along the sequence. based on spot mutations, nandy proposed an equation connecting the instantaneous values of the purine and pyrimidine population asymmetries. it was hypothesized that this may have important consequences for genetic engineering since it implied that stability of engineered gene sequences required these constraints to be followed. an important issue in molecular biology is identification of protein coding regions in dna sequences. nandy showed from the 2d graphical representations that exon and intron regions of mammalian genes showed distinctly different patterns and how these could be used to discriminate between the exons and introns [41] . this method was used by ghosh et al. [42] to analyse a newly sequenced human chromosome iii contig 7 dna to identify coding regions and predict, using webbased tools, possible genes in the sequence. he, li and wang [31] used the numerical characterization of characteristic sequence representation of he and wang [45] to suggest a protein coding gene finding algorithm specific for the yeast genome and found that the total number of protein coding genes in the yeast genome was 5897, which matches very well with estimates from other methods of 5800-6000. discrimination between protein coding and non-coding regions was also proposed on an entropy-based approach [71] differentiating the dna sequence into three subsequences and using shannon's formula. wiesner and wiesnerova [72] did an interesting application of granch techniques to study plant germplasm identificators. for their study of multiallelic marker loci from 18 begonia × tuberhybridas, they used a 2d random walk digitization of the dna sequences by three transform classes according to the prescription of bai et al. [73] and derived invariants from the respective matrices to compute sequence similarities and dissimilarities. principal component analysis done to compare the 18 marker loci to the dna invariants found statistical correlations between the genetic diversity of the marker loci and the random walk invariants. based on their results, the authors concluded that "dna walk representation may function as an efficient pre-scanning procedure, which can predict allele-rich genomic loci as highly informative dna markers solely using the information from their primary sequence." one of the early observations was that these graphical and numerical techniques allowed comparison of dna and protein sequences without having to do multiple sequence alignment since here we are dealing with numbers derived from the method rather than having to compare base by base or residue by residue. almost all proposals of schemes for graphical representations have computed distances between dna sequences to determine similarities and dissimilarities without multiple sequence alignment and obtained fairly good, though not uniform, results. for example, liao et al. [47] used a 2d graphical representation proposed by liao [74] to derive a phylogenetic tree from the elements of a similarity matrix for eleven mitochondrial gene sequences without having to go through any multiple alignment procedure. they constructed a 2x2 covariance matrix of the weighted centers of masses from the co-ordinates of each base of a sequence and computed the euclidean distance between pairs of sequences to obtain their similarity/dissimilarity matrix. liao et al. [48] also investigated the phylogeny of 24 sars coronavirus genomes by their 2d graphical representations for protein sequences where they could draw three plots for each sequence by considering the three reading frames. these generate three eigenvalues for each sequence which are then used to compute a distance matrix from which they could diagrammatically show the relationships of various strains of the virus. in another exercise, bai and wang [75] compared nine different neurocan nerve protein sequences in their 3d dodecahedron representation scheme. a direct comparison of these protein sequences through alignments is difficult since these protein sequences have different lengths. using 10-and 35-component vectors from their model, they compared the distances between end-points of the vectors corresponding to each of the nine genes and built phylogenetic trees. nandy et al. [61] used their 20d representation of protein sequences to compute distances between sequences of the families of globin, the rat and human voltage gated sodium channel alpha subunit and their phylogenetic relationships. it is to be noted that deriving phylogenetic trees from protein sequences is usually a difficult matter when the sequences are of different lengths; but with the granch techniques where d/d and other matrices can be computed for any length sequence and only the eigenvalues compared, the sequence length differences become irrelevant. jayalakshmi et al. [76] generalized these methods to compute alignment free sequence comparison using n-dimensional similarity space. h gonzalez-diaz and his group have used 2d graphical methods for extensive work in the bio-medicine field. based on pseudo-folding lattice network (ln) and star-graphs (sg) topological indices they proposed two dna promoter qsar models to predict promoter sequences in the function regulation of several mycobacterial pathogens [57] . aguero-chapin et al. [55] using their reduced four groups of amino acids on a 2d cartesian co-ordinate framework computed numerical descriptors for 108 polygalacturonases through a markov model and were able to discriminate between these and other proteins and predict polygalacturonase activity of a new protein. comparison of rna secondary structures are important to understand their catalytic properties. bai et al. [77] considered a 3d graphical representation of rna characteristic sequences taken 2 bases at a time to compare similarities and dissimilarities in viral rnas of nine species. they computed three modular lengths and three phases for each sequence from which they constructed a 6component vector characteristic for each viral sequence. two sequences were considered to be similar if their vectors pointed in the same direction and difference between sequences could be quantified by computing the euclidean distance between the end points of the two vectors: the bigger the distance the less similar the sequence. the resultant difference table showed how methods such as these could be used to do cluster analysis without having to use alignment tools which are time consuming and requires several assumptions. in another instance, gonzalez-diaz et al. [78] has computed 2d-rna coupling numbers by adapting the 2d graphical representation method for dna sequences. a novel application of granch techniques was proposed by ghosh et al. [62] to determine targets on viral proteins for drug and vaccine design. viruses are known to mutate very fast and therefore become resistant to drugs and vaccine sin short time scales; the virulence of the avian flu led to an apprehension that it might mutate to a form that would enable human to human transmission of the disease and thus cause widespread infection and possible death as had happened in the case of the spanish flu outbreak in 1918 when millions died. new drugs and vaccines, especially ones that could be readily moved from table to dispensaries were badly needed. we had already noticed in early 2006 that certain parts of the neuraminidase gene appeared to be fairly well conserved [68] . the neuraminidase, along with hemagglutinin, are surface proteins that enable the viral particles to enter and leave the human cells where they proliferate, and of these the neruaminidase is the preferred target of the currently available drug, tamiflu. we therefore determined to search the neuraminidase protein for surface residues that were well conserved. our procedure was to scan a small stretch of the neuraminidase protein sequences of 600+strains of the h5n1 virus and then slide the window by one base and scan again to calculate the protein graph radius in our 20d representation system. we know that these radii are very sensitive to any changes in the sequence, so equal values of the radii in one stretch over all the strains implied that this stretch was conserved. by covering the entire sequence for all strains we could get a good profile of regions of least variability. the next step was to determine which parts of the sequences were surface exposed. there are several on-line engines available to scan a sequence and assign parameters to predict the degree of probability that certain portions were surface exposed. matching these predictions with the hard facts we had on low variability we were able to identify six regions in the neuraminidase protein that were surface exposed and largely stable to mutational changes. these included the peptide we had identified earlier as being exceptionally stable. however, in a recent report on influenza virus rna structure [79] , it has been noted that the structures seen in the crystalline form may be one of several structural forms in vivo and confirmation will need to be experimentally determined. the results of the analysis on the h5n1 neuraminidase protein sequence was published in 2010 [62] . subsequently we have done a similar study on the vp7 protein of the rotavirus, a mainly tropical disease responsible for causing deaths to over half a million children every year. we identified four regions on the vp7 which appeared to be surface situated and quite stable. our findings were reported at the 2 nd mathematical chemistry workshop at bogota, colombia in 2010 [13] and the indian biophysics conference, delhi 2011 [80] . while a number of applications have shown the usefulness of the granch approach to analyzing dna, rna and protein sequences, this remains as yet a nascent field where many issues need to be looked into and problems resolved for the potential to be well realized. an early indication of some of these areas was outlined some years ago [81] , but they are worth recapitulating along with some more issues that may bear scrutiny. the intense interest in this field of graphical representation and numerical characterization of bio-molecular sequences have led to proposals for a vast array of models for depicting the sequences, some real and some virtual, more for dna sequences, less for protein and rna sequences. this has almost become an intellectual sport, with new ideas being propounded on regular basis, generally without a proper rationale for yet another method or critical comparison with earlier proposals. what appears to be lost in the process is the target: how useful are these representations to the practicing biologist? critical to this issue is the problem of determining the domains of applicability of the various representations if different, i.e., which model is best suited to address which classes of problems. as of now, the vast majority of proposals have addressed themselves to comparisons of similarity and dissimilarity, but as we have seen in the previous section, the issues that we can address and which biologists need answers for are more varied. from the applications made to date, the 2d graphical representations where the sequence data are easily viewed have generated the most interest. even aside for the global characteristics revealed by the investigations of jeffreys [3] and peng et al. [3] , the particular patterns of intron and exon segments [9] or characteristic curves of he et al. [31] have led to models to predict protein coding regions, determination of long-range palindromes [67] , identification of target segments for vaccine development for viral proteins [62] and determination of allele-rich genomic loci for plants [72] among other applications have been based on 2d representation schemes. hamori had identified regions of sharp changes in base compositions from his 3d h-curves [25] , but for almost all other 3d, 4d and higher dimensionality representations applications have been restricted to sequence similarities and generation of phylogenetic trees. the mathematical technique involved in generating the descriptors and characterizers for dna sequences are still at a preliminary level. while the first moments in the geometrical method for generating descriptors have generally yielded reasonable results in comparing intra-and inter-genic sequences, attempts to calculate higher moments to increase the accuracy and effectiveness of these descriptors have only lately begun [28, 82] . the leading eigenvalues from the euclidean and graph theoretic distance ratios matrix have so far been used mainly to compute inter-sequence distances; given the rigorous mathematics of matrix mechanics, it may be worthwhile to try and extend the applications to other areas. for the benefit of users of these methods, it would be useful to have a comparison of the geometrical and graph theoretic models to determine at what level the two could give comparable results. in the case of 2d graphical representations using cartesian co-ordinates, we had seen that gene sequences take characteristic shapes [5] . this raises the possibility that some day we could create an atlas of gene sequences where samples of each gene would be depicted and the descriptor parameters listed for easy reference and rapid visual identification. we have described quantitatively the gross features of the graphical plots in the 2d representations by using the first moments in a geometrical method [10, 11] ; better descriptors can be determined through higher order moments [28] to quantify the curvature, skewness and other properties. these, and the leading eigenvalues from the graph theoretic approach, could be considered as a list of parameters describing the sequence, akin to the quantum numbers that are used to describe elementary particles. such a scheme then provides a method to electronically store, retrieve and compare data between various sequences more efficiently, especially with a view to quickly scan newly sequenced dna, rna and proteins to determine the genes and functions. we have considered the moments calculated from the geometrical approach to 2d graphical representations as numerical "descriptors" of the dna sequence and taken tentative steps to enhance the number of descriptors of a sequence by computing higher order moments to more completely describe the sequences. in the matrix method applied to different varieties of graphical representation, leading eigenvalues arising from the matrices have been taken as "invariants" of the sequence in the strict mathematical sense. however, the concept of invariants derived from these matrix methods of numerically characterizing dna sequences may require some modification to account for the fact that dna sequences constantly change due to mutations in the bases. the vast majority of these changes do not affect the functioning of the protein or the enzyme coded by the gene due to synonymous mutations in the coding segments or in the non-coding part; e.g. in the case of intronless gene like the neuraminidase of the avian flu h5n1 we had found [69] 447 out of total 682 sequences prevalent over the period 1997 to 2008 had undergone mutations in one or more bases in the gene, but even then, all of these variants coded for a functioning flu neuraminidase protein. for a beta globin gene, the common standard example of most graphical representation schemes, a sample from one person may differ by a base or more from the next person due to mutational changes. determining an "invariant" from one sample sequence of these genes, while being mathematically precise, may not adequately express or characterize a gene sequence from a practical point of view. perhaps a biologically more relevant measure would be a sampling of several such sequences and from them to compute an average eigenvalue with a standard deviation and derive a numerical to characterize the gene. in fact, in the absence of a sensitivity analysis or a standard deviation, it would be difficult to accept that the computations through leading eigenvalues of distances between several sequences that are only a few percentage points apart could be statistically meaningful. the descriptors are no exception either. once these basic issues are attended to, the granch techniques can become a useful tool in the medicare field. since the computations of the numerical descriptors/characteristics are quite simply done, they can be incorporated into the dna sequencing schemes so that there will be automatic computations of, e.g., g r and p r values which would enable the physician to immediately ascertain the presence of any harmful genetic disorders; huntington's potential to degenerate into a disease for the patient, or some similar genetic problem areas could be easily read out as the genome is sequenced provided we know the characteristic locus and have a standard genome, for example the readout for a normal person from the family, available for comparison. the viral application already discussed in detail in the previous section could be automated and extended to other viruses and bacterial genomes to promote new generation of drugs and vaccines. the researches of gonzalez-diaz [55, 58, 78] and basak [59, 60] are already pointers to new directions. many potential application areas remain to be explored. since the numerical descriptors mentioned previously are seen to be quite sensitive to changes in base composition and distribution, the potential exists to devise schemes to index various aspects related to the bio-molecular sequences. initial attempts have been made to index toxic chemicals that have damaging effects on dna sequences [83] , and to index snp gene sequences measured against some standard sequences [84] . however, these need to be refined and made more useful for the confidence to be generated for their use in laboratory situations. one area that requires in depth study is how to address non-contiguous sequence segments. for example, in the case of epitopes, it is found that there could be continuous epitopes and discontinuous epitopes; in the latter case the folded protein brings residues from different parts of the amino acid sequence close together, which then become sites for the antibodies to act upon. the methods delineated so far for g r and p r or leading eigenvalue evaluation require contiguous span of the bases or residues for the numbers to be calculated. one way to circumvent this difficulty is to work on small segments of the sequence at a time as had been done in ref. [62] . however, this is time consuming and inefficient, and more improved methods to be able to focus on regions of interest and calculate a minimum number of the parameters could offer better rewards. in summary it is apparent that graphical representation and numerical characterization of molecular sequences hold far-reaching potential of rapidly analyzing the sequences to extract numerous information. it opens up new ways to look at these sequences, and to gain new insights such as long range palindromes, fractal properties and intra-purine intra-pyrimidine relationships not seen by any other means. it allows one to compute many aspects of biological and medicinal interest and provide novel methods of tackling old problems; we have seen examples of gene identification, analysis of evolutionary trends and generation of phylogenetic trees, identification of conserved sites on viral proteins for drug and vaccine targeting, predict promoter sequences and new properties of polygalacturose proteins, among others and many possibilities remain unexplored, or barely scratched. still, from plants to viruses, from mammalian genes to mitochondrial genomes, a varied series of applications have been formulated. although many issues doubtless remain yet such as handling non-contiguous stretches of bases and residues like discontinuous epitopes, it is apparent that the granch techniques hold a lot of promise for a new direction in molecular analysis. recent investigations into characteristics of long dna sequences. ind chaos game representation of gene structure long range correlation in nucleotide sequences evolution of long-range fractal correlations and 1/f noise in dna base sequences a new graphical representation and analysis of dna sequence structure: i. methodology and application to globin genes simple way to look a dna graphical representation of long dna sequences graphical analysis of dna sequence structure: iii. indications of evolutionary distinctions 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representation and numerical characterization of h5n1 avian flu neuraminidase gene sequence computational study of dispersion and extent of mutated and duplicated sequences of the h5n1 influenza neuraminidase over the period 1997-2008 empirical relationship between intra-purine and intra-pyrimidine differences in conserved gene sequences relative entropy of dna and its application 2d random walk representation of begonia × tuberhybrida multiallelic loci used for germplasm identification a representation of dna primary sequences by random walk a 2d graphical representation of dna sequence on graphical and numerical representation of protein sequences alignment-free sequence comparison using n-dimensional similarity space analysis of similarity between rna secondary structures 2d-rna-coupling numbers: a new computational chemistry approach to link secondary structure topology with biological function influenza virus rna structure: unique and common features characterization of conserved regions in rotaviral vp7 proteins: a graphical representation approach towards epitope prediction theory and computation: old problems and new challenges, g. maroulis and t. simos graphical and numerical representations of dna sequences: statistical aspects of similarity simple numerical descriptor for quantifying effect of toxic substances on dna sequences quantitative descriptor for snp related gene sequences the author confirms that this chapter contents have no conflict of interest. key: cord-319780-rfj9t99r authors: alexander, s.p.h.; armstrong, j.; davenport, a.p.; davies, j.; faccenda, e.; harding, s.d.; levi‐schaffer, f.; maguire, j.j.; pawson, a.j.; southan, c.; spedding, m.j. title: a rational roadmap for sars‐cov‐2/covid‐19 pharmacotherapeutic research and development. iuphar review 29 date: 2020-05-01 journal: br j pharmacol doi: 10.1111/bph.15094 sha: doc_id: 319780 cord_uid: rfj9t99r in this review, we identify opportunities for drug discovery in the treatment of covid‐19 and in so doing, provide a rational roadmap whereby pharmacology and pharmacologists can mitigate against the global pandemic. we assess the scope for targetting key host and viral targets in the mid‐term, by first screening these targets against drugs already licensed; an agenda for drug re‐purposing, which should allow rapid translation to clinical trials. a simultaneous, multi‐pronged approach using conventional drug discovery methodologies aimed at discovering novel chemical and biological means targetting a short‐list of host and viral entities should extend the arsenal of anti‐sars‐cov‐2 agents. this longer‐term strategy would provide a deeper pool of drug choices for future‐proofing against acquired drug resistance. second, there will be further viral threats, which will inevitably evade existing vaccines. this will require a coherent therapeutic strategy which pharmacology and pharmacologists are best placed to provide. pubmed has already accumulated a vast repository of information on sars-cov-2/covid-19, which increases on a daily basis (on 2020-03-23, there were 1369 hits for covid-19; this number more than doubled in the space of two weeks, so that by 2020-04-06 there were 2780 hits in pubmed for . clearly, there is a need to summarise this information critically and prioritise the elements which are constructive and useful for each individual sector. this document suggests priorities for how drug discovery and development might be rationally focussed for the rapid identification and successful translation of therapeutic agents to treat covid-19. given the urgency of the current situation, clearly initial drug discovery should focus on repurposing licensed drugs, as dosage and safety information are largely to hand. unfortunately, there is controversy over proof of efficacy for essentially all the potential repurposed agents for which preliminary, and, in many cases, non-peer reviewed data have surfaced. some of this controversy is addressed below but efforts are underway from both who and nih to coordinate larger, higher powered and better controlled studies in an attempt to demonstrate efficacy unequivocally. as a 'second wave', de novo discovery focussing on novel agents may allow future refinement and capacity to treat patients who are unable to be treated by, or are unresponsive to, the repurposed agents, but it would be very unlikely to have these new drugs available to treat the current crisis. the iuphar/bps guide to pharmacology (gtopdb) is an open-access database, developed by the international union of basic and clinical pharmacology (iuphar) and the british pharmacological society (bps). it provides expert-curated descriptions of almost 3,000 human proteins and over 10,000 ligands, including more than 1400 approved drugs. management of the new resource is the responsibility of the nomenclature and standards committee of iuphar (nc-iuphar), which acts as the scientific advisory and editorial board. the committee has an international network of over 700 expert volunteers organized into ∼60 subcommittees dealing with individual target families. the database is notably enhanced through the continued linking of relevant pharmacology with key immunological data types as part of the iuphar guide to immunopharmacology (supported by the wellcome trust) and by a major new extension, the iuphar/mmv guide to malaria pharmacology (in partnership with the medicines for malaria venture). the gtopdb team centred at the university of edinburgh have constructed a resource (faccenda et al.) , which provides a precis of the current understanding about the virus and potential associated drug targets and drugs. as with the other databases, the emphasis of the curation process is on stringent provenancing of the information provided, although inevitably the current situation limits the capacity for triangulation of data. sequencing analysis of the novel virus has identified a high level of similarity with the virus identified to cause the severe acute respiratory syndrome (sars) outbreak in china in 2002/03/04, which was known as the sars coronavirus or sars-cov. provisionally named as 2019-ncov, the virus has been renamed sarscov-2 (viruses, 2020) . for the purposes of this document, the virus is described as sars-cov-2, while the infectious disease is named as covid-19 (world health organization, 2020) . one of the positive aspects of the emergence of sars-cov-2 and covid-19 is the rapidity at which aspects like genome sequencing (for example, wu et al., 2020) and 3d structures (for example, yan et al., 2020) have been described. protein targets and drugs in the current review follow nomenclature as presented on the guidetopharmacology.org website (alexander, ball & tsoleridis. sars-cov-2 proteins, accessed on 2020-04-24) and the concise guide to pharmacology 2019/20 (alexander et al., 2019) . for general reviews of the coronaviruses, see masters, 2006; fehr and perlman, 2015; de wit et al., 2016; zumla et al., 2016; cui et al., 2019; desforges et al., 2019; song et al., 2019 . sars-cov-2 is a betacoronavirus; a lipid-enveloped, single-stranded, positive sense rna virus. other human coronaviruses include alphacoronaviruses, such as human coronavirus-229e (hcov-229e), and betacoronaviruses, such as sars-cov and mers-cov (responsible for the middle east respiratory syndrome) (for review, see zumla et al., 2016; corman et al., 2018; pillaiyar et al., 2020) . more than 200 viral types have been associated with the common cold, of which 50% of infections are rhinovirus, but also include respiratory syncytial virus, influenza and coronaviruses, particularly hcov-229e. although hcov-229e is regarded as 'relatively benign' since monocytes are much more resistant to infection, it does rapidly kill dendritic cells (mesel-lemoine et al., 2012) . classically, the viral lifecycle can be divided into six elements: cell attachment; cell entry; viral uncoating; nucleotide replication; viral assembly, and release (see figure 1 ). positive-stranded rna viruses replicate in the cytoplasm of infected cells, in close contact with intracellular membranes. this organization allows a concentration of viral and host factors to enable virus production and to evade innate immune responses (reviewed by yager and konan, 2019) . the sars-cov-2 coronavirus 30 kb genome encodes 29 proteins has 15 open reading frames, two of which encode viral polyproteins that generate 16 non-structural proteins (see below) (wu et al., 2020) . historically, therapeutic benefit has been gained through exploitation of the differences between viral and host proteins that subserve superficially similar functions (proteases and nucleotide polymerases, for example). the rapidity with which structural elements of the sars-cov-2 proteome have been identified provides hope that drug discovery approaches will soon provide agents to target the virus selectively, with minimal impact on the host. based on the evidence from orthologous proteins from other betacoronaviruses and the information currently available on sars-cov-2 (some of it not yet from peer-reviewed sources), we propose here the priority targets for pharmacological investigation. that should not be taken to mean that research should be limited to these targets, since there are undoubtedly a number of functions of the viral proteins still to be ascertained. it would be remiss not to conduct a thorough examination of all the viral proteome, both in isolation and in combination. the strategies we learn from investigation of the host:viral interaction from sars-cov-2 will stand us in good stead for future viral threats. coronavirus binds to cell surface proteins on target cells and, following proteinase priming of spike proteins on the virus surface, the virus is internalized into endosomal fractions that are subsequently acidified, or accumulates through a non-endosomal route (fehr and perlman, 2015) (figure 1 ). the endosomal route appears to involve clathrin (inoue et al., 2007) , but there are contradictory reports of the importance of the intracellular c-terminus of ace2 in this mechanism (inoue et al., 2007; haga et al., 2008) . a fusion domain permits insertion of a key protein (s, see below), which then allows mixing of the viral and cellular membranes and subsequent release of the coronaviral genome into the cytoplasm. following entry into the host cell cytoplasm and viral uncoating, the replicase gene of the viral rna is translated. the genome of coronaviruses consists of a single, continuous, linear, ssrna, capped at the 5' end and with a 3'-polya tail (fehr and perlman, 2015) . translation occurs from open reading frame (orf) 1a and 1b at the 5' terminus, with a ribosomal frameshifting mechanism allowing the overlap between orf1a and orf1b to generate the two polyproteins pp1a and pp1ab (fehr and perlman, 2015; perlman and netland, 2009; snijder et al., 2003; thiel et al., 2003) . in sars-cov-2, the polyproteins are long, 4405 and 7096 aa, respectively. encoded within the polyproteins of betacoronaviruses are two proteinases: papain-like proteinase, plpro, and chymotrypsin-like proteinase, 3clpro. in sars-cov, plpro, derived from the polyproteins, has three endoproteinase target sites, which release nsp1-3 . 3clpro has 11 cleavage sites to release the remaining non-structural proteins. in the coronavirus family, these proteinases process the polyproteins to generate 16 functional non-structural proteins identified as nsp1-16 (anand et al., 2003; thiel et al., 2003; ziebuhr et al., 2007; kindler et al., 2016; cui et al., 2019) . downstream of the orf1a and 1b are genes encoding four structural proteins (spike, envelope, membrane and nucleocapsid) (figure 2 ) and a short series (described as at least 13 in total, srinivasan et al., 2020) of other proteins (see below). once sufficient protein and rna accumulate, coronavirus assembly takes place, centred on the structural proteins. the release of coronavirus particles involves the secretory pathway of the endoplasmic reticulum and golgi apparatus and vesicular exocytosis (for review, see de haan and rottier, 2005; fehr and perlman, 2015) , and it is likely, but as yet unconfirmed, that sars-cov-2 adopts this mechanism also. to date, there is more evidence about the molecular detail involved in (and the possibilities to modify) viral recognition, entry and replication compared to uncoating, assembly and release, hence the attention paid here to the former three mechanisms. the cell-surface anchor -ace2 among the coronaviruses, the spike protein interacts with proteinases to anchor on host cell surfaces. the cell-surface anchoring point for the alphacoronavirus hcov-229e is aminopeptidase n (also known as cd13, yeager et al., 1992) . for the betacoronavirus mers-cov, dipeptidylpeptidase 4 (also known as cd26, raj et al., 2013) is an anchor. analysis of the co-crystal structure suggested that the sars spike protein binds to the active site of angiotensin converting enzyme 2 (ace2, li et al., 2005) . binding of sars-cov spike to ace2 seems to require cholesterol-rich rafts in the host cells (glende et al., 2008) 00. recent evidence points to the spike protein of sars-cov-2 also binding to ace2. both sars-cov (li et al., 2003) and sars-cov-2 (hoffmann et al., 2020; letko et al., 2020) have been described to require ace2 to enter cells ( figure 1) . a particular domain of the spike protein of sars-cov-2, a so-called receptor-binding domain (rbd), has been shown to facilitate binding to ace2 (hoffmann et al., 2020) . the ace2 peptidase active site is located remotely from the cell membrane (li et al., 2005; wrapp et al., 2020; yan et al., 2020) , into which the spike protein binds. the rbd of the spike protein is located in the s1 ectodomain, approximately a third of the way along the protein. ace2 is a carboxypeptidase, which means it removes the terminal amino acid from oligopeptides, and so it seems unlikely that the spike protein is a substrate for ace2. in sars-cov-infected mouse lung, ace2 protein expression was downregulated compared to uninfected mice . following sars-cov spike protein administration to mice, angiotensin ii was increased in the lungs . these observations led to the suggestion that this was the molecular mechanism for the frequent development of acute respiratory distress syndrome (ards) during sars-cov infections kuba et al., 2005) . ace2 activity has been reported to be released from plasma membranes by proteolysis, thought to be through the action of tnfα convertase (adam17, a disintegrin and metalloproteinase domain containing protein 17, lambert et al., 2005) (figure 1) . the activity of adam17 can be increased by g protein-coupled receptor activation, including the at1 angiotensin receptor (schafer et al., 2004) . ace2, and ace, activity can be measured in human plasma (ocaranza et al., 2006; herath et al., 2007; lew et al., 2008) . human plasma ace2 activity is reported to be 'masked' by the presence of endogenous inhibitors (lew et al., 2008) , which don't yet appear to have been precisely defined. blood ace2 activity can be altered in pathology; for example, serum ace2 was found to be decreased in patients following acute ischemic stroke (bennion et al., 2016) . the expression of ace2 mrna and enzyme activity in cardiac tissues were increased following repeated oral administration of the at1 angiotensin ii receptor antagonist losartan, while oral administration of an ace inhibitor lisinopril only increased cardiac mrna expression, but not enzyme activity (ferrario et al., 2005) . studies using disruption of the ace2 gene in mice indicated an increase in circulating angiotensin ii levels and a severe cardiac contractility defect, which could be 'rescued' with simultaneous genetic disruption of ace (crackower et al., 2002) . an early investigation of ace2 polymorphisms in man failed to show an association with hypertension (benjafield et al., 2004) . a study of sars victims and ace2 polymorphisms failed to find a correlation with patient outcomes (chiu et al., 2004) . the spike protein is the largest viral structural protein (~1200-1400 aa) and is heavily glycosylated, forming extended trimeric structures providing the characteristic 'crown' feature of coronaviruses (belouzard et al., 2012 ) (see figure 2 ). the ectodomain is divided into the s1 domain responsible for binding to ace2, whereas the s2 domain is responsible for the fusion machinery. following binding of the s1 domain to ace2, a deformation of the pre-fusion trimer results (wrapp et al., 2020) . surface plasmon resonance of the binding of human ace2 to the immobilized sars-cov-2 indicated an affinity (kd value) of 15 nm, an order of magnitude larger than sars-cov binding to ace2 (wrapp et al., 2020) . using a related label-free technique, biolayer interferometry, affinities of 5 and 1.2 nm for binding of sars-cov and sars-cov-2 spike protein, respectively, to human ace2 has been reported (walls et al., 2020) . although a proteolytic cleavage site at the s1/s2 boundary of the sars-cov spike protein is the best characterised, a second site upstream of the fusion peptide in the s2 domain, called s2' has also been described (belouzard et al., 2009) . this raises the possibility that multiple other proteases might be targetted to influence coronavirus activation (millet and whittaker, 2015) . a key difference between the spike proteins in sars-cov and sars-cov-2 is the presence in the latter of a site at the s1/s2 boundary predicted to be sensitive to the proteinase furin, and which may be targetted during viral assembly and maturation (walls et al., 2020) . the sars-cov s2 domain has a pair of α-helices, which may participate in coiled:coil structures during membrane fusion (petit et al., 2005) . the host complex of zdhhc9 (link to uniprot) with golga7 (link to uniprot), a palmitoyltransferase, which modifies the low molecular weight g proteins nras and hras (swarthout et al., 2005) , also palmitoylates the cysteine-rich s2 endodomain of the sars-cov to facilitate membrane fusion (petit et al., 2007) . very recently, in a comparison of the s2 domains of sars-cov and sars-cov-2, an enhanced capacity of the novel virus' s2 domain for membrane fusion was observed and suggested to result from eight differing amino acids . using a series of oligopeptides conjugated to lipid entities, high affinity (ic50 values in the nanomolar range) inhibitors of cell fusion were identified. given that the spike protein binds to the active site of ace2 (li et al., 2005) , in theory, any alteration in the availability of the active site should influence the binding of the spike protein and, hence, interfere with sars-cov-2 infection. one option would be to provide an excess of an endogenous peptide substrate, or more conventionally to apply a selective enzyme inhibitor. endogenous substrates of ace2 ace2, discovered in 2000 (donoghue et al., 2000) , shares 40% sequence similarity to ace within the n-terminal domain and is a type i transmembrane metallopeptidase. unlike ace, it functions as a zinc carboxypeptidase to cleave single c-terminal amino acids from peptides, particularly hydrolysing pro-phe residues in angiotensin-(1-8) to angiotensin-(1-7), [pyr 1 ]-apelin 13 to [pyr 1 ]-apelin-(1-12) and [des-arg 9 ]-bradykinin to bradykinin-(1-8) with high efficiency. it may also cleave other peptides less effectively (vickers et al., 2002) , shown below: angiotensin i  angiotensin-(1-9) + leu angiotensin ii  angiotensin-(1-7) + phe apelin-(1-13)  qrprlshkgpmp + phe apelin-(1-36)  ….qrprlshkgpmp + phe [des-arg 9 ]-bradykinin  rppgfsp + phe dynorphin a-(1-13)  yggflrrirpkl + lys 115 other peptides were not hydrolysed by ace2 including adrenocorticotrophic hormone, calcitonin, cholecystokinin, met-enkephalin, glucagon, glucagon-like peptide-1, melaninconcentrating hormone, pituitary adenylyl cyclase-activating polypeptide, somatastatin-14, urocortin or vasoactive polypeptide (vickers et al., 2002) . in humans, levels of mrna encoding ace2, together with immunoreactive peptide, are highest in the gastrointestinal tract, followed by heart, kidney, testes and gall bladder and other tissues (uhlen et al., 2015) . within organs, ace2 immunoreactivity was predominantly localised to epithelial (for example, in the lungs) and endothelial cells from all vascular beds examined (yang et al., 2017) . importantly, the ace2 antisera used in this study for immunocytochemistry was the same as that employed in the study described in the section below "using biopharmaceutical/antibody approaches to target ace2:spike interactions" (hoffmann et al., 2020) , to block entry of the virus in cell culture. the epitope of this antisera would be a rational starting point for the development of selective therapeutic antibodies. the presence of ace2 on airway epithelial cells is consistent with the isolation of sars-cov-2 from broncho-alveolar lavage of patients with covid19 and the infection of cultured airway epithelial cells . in humans, levels of ace2 immunoreactivity tend to be low. however, in addition to being upregulated by ace inhibitors and angiotensin receptor antagonists (see above), ace2 expression has been reported to be increased in human cardiovascular disease, for example, in the cardiomyopathic heart (zisman et al., 2003) . since ace2 is critical for viral entry, it may be one explanation for the high incidence of co-morbidity of covid-19 patients with cardiovascular disease. manipulation of ace2 activity by synthetic agents assays employing fluorogenic surrogate substrates to screen for inhibitors of ace2 activity are wellestablished, for example using methoxycoumarin-rppgfsafk(dnp)-oh (ocaranza et al., 2006; bennion et al., 2016) , or methoxycoumarin-apk(dnp)oh (herath et al., 2007; lew et al., 2008; mores et al., 2008) . detailed protocols for the use of methoxycoumarin-apk(dnp)oh have been described for fret-based high throughput screening xiao and burns, 2017) . this style of assay identified that ace2 was not inhibited in the presence of 10 µm lisinopril, enalaprilat, or captopril, inhibitors of angiotensin-converting enzyme (tipnis et al., 2000) . there are no licensed drugs described to inhibit ace2 activity. however, dx600 is a peptide-based ace2 inhibitor (huang et al., 2003) , while mln4760 and compound 28 are described as sub-nanomolar potency ace2 inhibitors (mores et al., 2008) . there is evidence for allosteric regulation of ace2 activity, in that a xanthenone derivative (xnt) was observed to enhance ace2, but not ace, activity in vitro with a potency of 20 µm (hernandez prada et al., 2008 ). an in silico study later identified a binding site in an allosteric hinge region of ace2, distinct from the proteinase active site, against which 1217 fda-approved drugs were screened (kulemina and ostrov, 2011) . a subsequent kinetic assay with the recombinant enzyme and a fluorigenic substrate identified labetalol and diminazene as agents able to double the maximal velocity of ace2 enzyme activity. whether any of these compounds alter the binding of the spike protein from either sars-cov or sars-cov-2 or viral infection in general does not appear to have been examined yet. a speculative area that should be explored further is the concept of enhancing the activity of the serine proteinase adam17 to increase cleavage and release of membrane bound ace2. peptides such as angiotensin ii are reported in animal models to cause release ('shedding') following binding to at1 receptors (xu et al., 2017) . although angiotensin ii is licensed by the federal drug administration to treat sepsis (known as giapreza, davenport et al., 2020) , it would be inadvisable as a treatment for covid-19 given the detrimental action of angiotensin ii on the lungs. in contrast, the investigational agent [pyr 1 ]-apelin-13 is currently used in clinical studies (davenport et al., 2020) and may also interact with its cognate receptor to downregulate membrane-expressed ace2. this peptide also has beneficial effects on the heart, including an increase in cardiac output (japp et al., 2010) . using biopharmaceutical/antibody approaches to target ace2:spike interactions an alternative approach to the small molecule manipulation of the ace2 enzyme would be to target the spike or ace2 proteins with selective antibodies. antibodies directed against ace2 led to a reduction in sars-cov-2 virus entry into target cells (hoffmann et al., 2020) , although this is likely to be some distance away from a therapeutic application. a truncated version of human recombinant ace2, lacking the transmembrane domain, mitigated against sars-cov infection of cells (li et al., 2003) and has been used in animal models to reduce symptoms of severe acute lung failure , diabetic nephropathy (oudit et al., 2010) and cardiac hypertrophy and fibrosis . treating sars-cov-2 victims with a soluble form of ace2 (batlle et al., 2020) or a fusion protein of the spike-binding portion of ace2 combined with the fc portion of human igg (lei et al., 2020) has been suggested. apeiron biologics has approval to conduct a phase ii clinical trial of apn01 (human recombinant ace2) for the treatment of covid-19 in three european countries (austria, germany and denmark) (nct04335136). this recombinant version of ace2 was originally licensed to glaxosmithkline and previously tested as gsk2586881 in a phase 2 multicentre trial (nct01597635) in patients with lung injury or ards, both features of sars and mers (and now covid-19). the study tested the hypothesis that cleavage of angiotensin ii (which causes lung injury -vasoconstriction, inflammation, fibrosis, vascular leak, and sodium absorption) to angiotensin-(1-7), would have counter regulatory beneficial action and reduce long term injury. gsk2586881 was well-tolerated in patients with ards, and the rapid modulation of peptides of the renin-angiotensin system demonstrated target engagement, in that levels of angiotensin ii decreased rapidly whereas angiotensin-(1-7) levels increased and remained elevated for 48 h, although the study was not powered to detect changes in acute physiology or clinical outcomes (khan et al., 2017) . sera from convalescent sars-cov patients prevented the cell entry of sars-cov-2 (hoffmann et al., 2020) and this approach has been used with some success in the sars, mers and covid-19 outbreaks (for review, see bloch et al., 2020) . the difficulty in identifying the precise molecular mechanism/s of convalescent sera action and issues with collection, standardization and scaling-up will be a challenge (bloch et al., 2020) . a bacterial equivalent of ace2 (based on 3d structure rather than primary sequence) termed b38-cap has been described, which is reported to reduce hypertension and limit cardiac dysfunction in an animal model (minato et al., 2020) . whether this agent might provide a decoy anchor to 'chelate' viral particles prior to cell entry has not been investigated. in a preliminary (as yet, not peer reviewed) study, a conformational change in the s1 rbd of the sars-cov-2 spike protein in the presence of heparin was noted (mycroft-west et al., 2020) . cellsurface heparan sulphate glycosaminoglycans have previously been suggested to be a lactoferrinsensitive alternative attachment point for the sars-cov virus (lang et al., 2011) . these observations suggest further routes for pharmacological targetting of viral infection and propagation. the cell-surface priming mechanism -tmprss2 tmprss2 is a single transmembrane domain protein with an extracellular serine protease domain, which appears to cleave substrates preferentially at basic residues (arg/lys), with a calcium-binding ldl receptor class a domain (paoloni-giacobino et al., 1997) . the tmprss2 gene encodes a cell-surface proteinase (transmembrane serine protease 2, tmprss2) and is located at chromosomal locus 21q22.3 in close proximity to erg, a gene encoding an ets transcription factor (link to uniprot, paoloni-giacobino et al., 1997) . (erg fusion with ews leads to ewing's sarcoma) fusion of the tmprss2 and erg (or the related etv1) genes has been reported to occur in the majority of prostate cancers and is suggested to lead to an androgen-dependent amplification of ets-regulated genes (tomlins et al., 2005) . tmprss2 expression is androgen-regulated (lin et al., 1999; chen et al., 2019) ; it is expressed highly in prostate cancer (lin et al., 1999; lucas et al., 2008) (for review, see tanabe and list, 2017) and loss of tmprss2 in the prostate is associated with reduced metastatic potential (lucas et al., 2014) . in aggressive versions of prostate cancer, tmprss2 undergoes autocatalytic proteolysis at arg 255 -ile 256 (afar et al., 2001) , where the two chains may remain in combination due to interchain disulphide bridges (chen et al., 2010) or the catalytic moiety may be secreted (chen et al., 2010) . in lncap human prostate cancer cells, the pparα agonist fenofibrate was able to mitigate against the androgen receptor agonist-evoked increase in tmprss2 expression (zhao et al., 2013) . following binding of the s protein to ace2, tmprss2 'primes' the spike protein to facilitate entry of the virus into the target cell (hoffmann et al., 2020; matsuyama et al., 2020) . pathogenesis of two strains of influenza virus has been reported to be markedly diminished by gene disruption of tmprss2 in mice (hatesuer et al., 2013; tarnow et al., 2014) , inferring that targeting this enzyme may have antiviral potential. interfering with the tmprss2:spike interaction using immunohistochemical analysis (bertram et al., 2012) and, very recently, using single nuclei and single cell rna sequencing (lukassen et al., 2020) , as yet not peer reviewed) of lung samples from otherwise healthy subjects, ace2 and tmprss2 were shown to be co-expressed in human bronchial epithelial cells, which could be a nexus for primary infection. a similar approach identified coexpression of ace2 and tmprss2 in nasal goblet cells, lung type ii pneumocytes and small intestine absorptive epithelia (ziegler et al., 2020) . in the same study, human primary nasal epithelial cells showed an upregulation in ace2 expression following 12 h incubation with interferon-α2 and interferon-γ, which suggests the potential for a feed-forward mechanism whereby the virus interacts preferentially with 'activated' cells to suppress the innate immune response (see below) (ziegler et al., 2020) . by analogy with the previous consideration of ace2 (above), alternatives to manipulate tmprss2 activity would be to provide endogenous substrates or synthetic inhibitors. however, the potential to make use of endogenous substrates seems limited. thus, although tmprss2 has been described to hydrolyse and activate the cell-surface g protein-coupled receptor proteinase-activated receptor 2 (wilson et al., 2005) , mice lacking tmprss2 failed to display an overt phenotype (kim et al., 2006) . as with ace2, there are no reports of licensed drugs which inhibit tmprss2 activity. cbz-ggraminomethylcoumarin has been described as a surrogate fluorogenic substrate suitable for highthroughput screening (paszti-gere et al., 2016) , although it is also a substrate for other proteinases, such as chymotrypsin. i432, a 3-amidinophenylalanine, has been described as a high affinity selective inhibitor (compound 92, ki of 0.9 nm) of tmprss2 (meyer et al., 2013) . in ipec-j2 pig jejunal epithelial cells, 10-50 µm i432 reduced tmprss2-derived product in cell media (paszti-gere et al., 2016) . in an investigation of sars-cov entry into hela cells expressing recombinant ace2 and tmprss2, a number of serine proteinase inhibitors (benzamidine, aprotinin, gabexate, tosyl lysyl chloromethyl ketone and camostat) were tested (mostly) at 10 µm for 30 min before exposure to pseudotyped viruses. only camostat was effective at reducing viral entry (kawase et al., 2012) , and further experiment suggested that 1 µm camostat was also effective, but only when tmprss2 was expressed. at 10 and 50 µm, camostat inhibited cell entry of the sars-cov and sars-cov-2 spike protein (hoffmann et al., 2020) . a direct inhibition of tmprss2 activity appears not to have been reported for camostat. potential ancillary proteins for virus entry -b 0 at1/slc6a19 and b 0 at3/slc6a18 the slc6 family of transporters includes the well-characterised net, sert and dat monoamine transporters, as well as the less well-exploited neutral amino acid transporter subfamily. b 0 at1/slc6a19 and b 0 at3/slc6a18 allow sodium-and chloride-dependent accumulation of neutral, aliphatic amino acids at the apical membranes of epithelial cells in the small intestine (b 0 at1/slc6a19) and kidney (b 0 at1/slc6a19 and b 0 at3/slc6a18) (for review, see broer and gether, 2012) . b 0 at3/slc6a18 is also highly expressed in the gi tract and gall bladder (protein atlas) and may play a role in the faecal:oral transmission of coronavirus (yeo et al., 2020) . the cell-surface expression of these neutral amino acid transporters is dependent on co-expression of ace2 (kowalczuk et al., 2008; fairweather et al., 2012) , aminopeptidase n (fairweather et al., 2012) or collectrin (an adaptor protein, which has high homology to the transmembrane region of ace2, camargo et al., 2009, link to uniprot) , in an apparently tissue-dependent manner (kuba et al., 2010) . a recent cryo-em structure suggested that ace2 and b 0 at1/slc6a19 form a heterodimer which pairs up through interfaces between the two ace2 partners (figure 1) , with the rbd of sars-cov-2 spike protein binding to the peptidase active site of ace2 suggesting that b 0 at1/slc6a19 may facilitate entry of the novel coronavirus. in the small intestine, absorptive epithelial cells were identified to co-express mrnas encoding for ace2 and tmprss2 (ziegler et al., 2020) . although it is not yet tested, it would be attractive to speculate that the colocalized expression of these targets may play a role in the faecal:oral transmission of coronavirus (yeo et al., 2020) . assays for b 0 at1/slc6a19 and b 0 at3/slc6a18 tend to be traditional accumulation of amino acids labelled with ionising or stable isotopes. recently, a primary screen using a membrane potentialsensitive fluorescence-based assay was used and followed up with a stable isotope accumulation assay to identify a novel inhibitor, cinromide, which exhibited modest potency (0.3-0.4 µm) for inhibiting b 0 at1/slc6a19 in cell-based assays (danthi et al., 2019) . once inside the cell, the endosomal cysteine proteases cathepsin b and cathepsin l have been described to process sars-cov (simmons et al., 2005 ) and this appears to be maintained for sars-cov-2 (hoffmann et al., 2020) although the significance of such intracellular proteinase activity is unclear. potent inhibitors for these two proteinases have been reported as pharmacological probes, but there are no licensed drugs identified to target them. following entry into the cell, many viruses accumulate in acidified lysosome-like vesicles, and so weak bases (including ammonium chloride and chloroquine) which target the lysosome have been used in vitro to target this mechanism. ammonium chloride (at 20 mm) has been described as a nonspecific inhibitor of viral replication in vitro, targeting viral uncoating (mizzen et al., 1985) and, at 50 mm, ammonium chloride inhibited cell entry of both sars-cov and ssars-cov-2 (hoffmann et al., 2020) . chloroquine was also observed to reduce infection of l cells by mouse hepatitis virus 3 (krzystyniak and dupuy, 1984) . following entry into the cell, the virus subverts nucleotide, protein, lipid and carbohydrate turnover of the host cell to produce multiple copies of itself. the viral rna is translated into multiple proteins to produce the replication machinery. as protein translation from the viral genome occurs, the two polyproteins are the first to be synthesised, with the two intrinsic proteases able to cleave the polyproteins into their constituent products. targetting the viral proteinases the low sequence similarities between mammalian and viral proteases has allowed successful drug targetting of viral diseases, including both hiv/aids and hcv/hepatitis c. the genome of sars-cov-2 contains two proteinases intrinsic to the polyproteins, plpro and 3clpro. the more n-terminally-located plpro is the larger (~2000 aa) of the two proteins (for review, see baez-santos et al., 2015; lei et al., 2018) , and, in sars-cov, is a membrane-associated, polyfunctional entity (harcourt et al., 2004) . sequence modelling of sars-cov-2 plpro suggested the presence of 6tm domains towards the c terminus, with the majority of the protein extending into the cell cytoplasm (angeletti et al., 2020) . in other coronaviruses, the enzyme is also capable of hydrolysing ubiquitin from protein substrates (barretto et al., 2005; ratia et al., 2006) , as well as removing the ubiquitin-like protein interferon-stimulated gene 15 (isg, link to uniprot) from isgconjugated proteins (yang et al., 2014) . using the orthologous proteinase from the mouse hepatitis coronavirus, analysis of three distinct structural domains suggested that the papain-like proteinase domain coincided with the deubiquitinylating and deisgylating functions (chen et al., 2015) . in sars-cov, the plpro also contains an adrp functional phosphatase domain directed at adp-ribose-1''-phosphates, although the functional significance of the hydrolase activity may be less impactful than the capacity to bind adp-ribose, at least for the enzyme from hcov-229e (putics et al., 2005) . this domain is thought to contribute to processing of the viral subgenomic rnas and the suppression of the innate immune system through reducing interferon production (lei et al., 2018) . investigating the peptidase activity of sars-cov plpro suggested a preference for larger proteins (ubiquitinated or isgylated) rather than simpler fluorescent-tagged oligopeptide substrates (lindner et al., 2005; lindner et al., 2007; baez-santos et al., 2014; ratia et al., 2014) making screening more complicated. the chymotrypsin-like proteinase, 3clpro the smaller proteinase from sars-cov-2 is 3clpro (sometimes called the main prote(in)ase, mpro). in silico docking models of sars-cov-2 3clpro has led to suggestions that particular existing antiviral agents, including velpatasvir and ledipasvir (licensed agents for treating hepatitis c when combined with sofosbuvir in the uk), should be screened for functional activity . a recent screen of ~10 000 compounds including approved drugs, candidate drugs and natural products used a substrate derived from the n-terminal autocleavage site of the sars-cov-2 3clpro which was modified (methylcoumarinylacetyl-avlqsgfr-lys(dnp)-lys-nh2) to allow a fret-based assay . the same substrate was used in a screen of the equivalent enzyme from the another coronavirus, hcov-hku1, which transferred to humans (zhao et al., 2008) . a number of inhibitors of the sars-cov 3clpro proteinase have been described (lu et al., 2006; yang et al., 2006; goetz et al., 2007) , without progressing into the clinic. recently, an in silico approach using orthologues of the sars-cov 3clpro from other coronaviruses and enteroviruses allowed production and testing in vitro of a series of α-ketoamides . one compound (11r) exhibited submicromolar potency against sars-cov 3clpro in a cell-free fret-based assay, and micromolar potency in a cell infection assay with sars-cov . in a preliminary (not yet peer-reviewed) report, the sars-cov-2 3clpro expressed in hek293 cells was found to interact with histone deacetylase 2 (hdac2) by affinity purification/mass spectrometry (gordon et al., 2020b) . a number of approved drugs target hdac2 in the treatment of various t cell lymphomas, including romidepsin, belinostat, and vorinostat with nanomolar potency (bradner et al., 2010) . targetting nucleotide turnover a relatively large proportion of the viral genome is inevitably devoted to nucleotide turnover. for sars-cov-2, this includes nsp7/nsp8/nsp12 as an rna-dependent rna polymerase; nsp13 as a helicase; nsp10/nsp14 as an 3'-to-5' exonuclease complex; nsp15 as an endoribonuclease and nsp16 as a 2'-o-ribose methyltransferase. remdesivir (currently in clinical trials to treat covid-19), is described as a non-selective inhibitor of multiple rna viruses, and has shown some efficacy in mers-cov and sars-cov infection of monkeys (de wit et al., 2020) . in in vitro investigations, the triphosphate analogue of remdesivir inhibited rna synthesis of mers-cov rna-dependent rna polymerase (primarily nsp8/nsp12 complexes derived from co-expression in insect cells of a construct containing nsp5, nsp7, nsp8 and nsp12) with an ic50 value of 32 nm when nucleotide levels were low, increasing to 690 nm at higher nucleotide concentrations (gordon et al., 2020a) . in silico modelling identified that remdesivir, as well as the approved antiviral drugs ribavirin, sofosbuvir and tenofovir could bind tightly to the active site of nsp12 from sars-cov-2, based on the crystal structure of sars-cov (elfiky, 2020) . however, ribavirin alone had no significant effect in a clinical trial with sars patients, although a combination of ribavirin with lopinavir-ritonavir and corticosterone had lower rating of ards and death (for review, see zumla et al., 2016) . in-depth analysis has not been completed with mers patients, although an ongoing phase 2 clinical trial for mers uses a combination therapy of lopinavir/ritonavir and interferon β1b (arabi et al., 2020) . nsp13 is a helicase, which enables unwinding of duplex rna. the exoribonuclease activity of nsp14 sets the coronaviruses apart (snijder et al., 2003) , as the enzyme is suggested to remove damaging mutations from the genome (eckerle et al., 2010; sevajol et al., 2014) . in other coronaviruses, the endoribonuclease nsp15 has some selectivity for hydrolysing polyu sequences (hackbart et al., 2020) . this enables the virus to delay or minimise initiation of the innate immune system by hydrolysing negative sense polyu nucleotides, which activate the mda5 system to evoke interferon production (discussed further below). nsp16 is a methyltransferase, which uses s-adenosyl-lmethionine as a co-substrate to assist in cap formation (decroly et al., 2008) . protein: protein interactions in recombinant expression in a preliminary (not yet peer reviewed) report, a series of tagged recombinant proteins from sars-cov-2 were expressed in hek293 cells and then protein partners were identified by affinity purification/mass spectrometry (gordon et al., 2020b) . for nsp12 (rna-dependent rna polymerase) and nsp14 (3'-5'-exonuclease) of sars-cov-2, interactions with receptor interacting protein kinase 1 (ripk1) and inosine monophosphate dehydrogenase 2 (impdh2), respectively, were identified. for these two targets, there are established approved drugs. thus, ponatinib, which is used to treat acute myelogenous leukemia or chronic myelogenous leukemia (philadelphia chromosome), targets multiple protein kinases, inhibiting ripk1 with an ic50 value of 12 nm (najjar et al., 2015) . mycophenolic acid and ribavirin are impdh2 inhibitors with ic50 values of 20 nm (nelson et al., 1990) and 1-3 µm (wittine et al., 2012) ranges, respectively, with clinical uses in organ transplantation and antiviral therapy, respectively. reservations about the use of ribavirin have already been noted above. mycophenolic acid as a monotherapy was examined in a mer-cov-infected non-human primate model, where the authors concluded it actually worsened the condition . nsp13 (helicase) and nsp15 (endoribonuclease) have been described to bind to centrosomeassociated protein 250 (cep250) and rnf41 (also known as nrdp1, link to uniprot), respectively, in a preliminary report of recombinant expression (gordon et al., 2020b) . cep250 is suggested to influence centrosome cohesion during interphase (de castro-miro et al., 2016) and to be elevated in peripheral t cell lymphomas (cooper et al., 2011) . the functional relevance of nsp13 interaction with cep250 is not yet clear. rnf41 is an e3 ubiquitin ligase, which polyubiquitinates myeloid differentiating primary response gene 88 (myd88, link to uniprot), an adaptor protein for toll-like receptors, which allows activation of tbk1 and irf3 (see below) and thereby increases type i interferon production (wang et al., 2009) . the lipid profile of viruses appears to be important in terms of viral entry into the cell, either as sites for anchoring or for endocytosis (for review, see heaton and randall, 2011; mazzon and mercer, 2014) . replication of sars-cov is reported to take place associated with the endoplasmic reticulum in 'replicative organelles' incorporating convoluted membranes and interconnected doublemembrane vesicles, inferring a capacity for the virus to induce extensive reorganization of intracellular phospholipid membranes (knoops et al., 2008) . three non-structural proteins from sars-cov with transmembrane domains, nsp3 plpro (see above), nsp4 and nsp6 when co-expressed in model cells prompted the formation of these double-membrane vesicles (angelini et al., 2013) , although it is unclear whether specific catalytic activities are necessary for this action. the lipidome of influenza virus (also a positive strand rna virus) consists of glycerophospholipids, sterols (mainly cholesterol) and sphingolipids, with sphingolipids and cholesterol enriched compared to the host cell membrane (gerl et al., 2012) , but there does not yet appear to be a parallel investigation of sars-cov. cytosolic phospholipase a2α, cpla2α, hydrolyses phospholipid to produce lysophospholipids and free fatty acids. using alphacoronavirus hcov-229e-infected huh-7 cells, inhibition of cpla2α using pyrrolidine-2 at higher concentrations (20 µm) evoked an inhibition of viral titre (muller et al., 2018) . arachidonoyl trifluoromethylketone, a non-selective inhibitor of multiple eicosanoid-metabolising enzymes including pla2 isoforms, also inhibited viral titres at higher concentrations (muller et al., 2018) . transmission electron microscopy suggested that cpla2α inhibition reduced the density of double-membrane vesicles (muller et al., 2018) . analysis of lipid metabolites indicated that hcov-229e-infected huh-7 cells showed increases in levels of ceramides, lysophospholipids and phosphatidylglycerols, with decreases in phosphatidic acids (muller et al., 2018) . 20 µm pyrrolidine-2 inhibited the elevations in lysophospholipids and phosphatidylglycerols, but not the ceramides. intriguingly, some selectivity of the involvement of pla2α was suggested as pyrrolidine-2 also displayed antiviral activities against other members of the coronaviridae (and togaviridae) families, while members of the picornaviridae family were not affected. although speculative, there is the possibility that some of the benefits of glucocorticoid administration in the clinic might be the up-regulation of annexins, and the subsequent binding and concealment of membrane phospholipid from further metabolism (for review, see lemmon, 2008) . while clearly some distance from a validated target, since phospholipids are an essential component of enveloped viral proliferation, targeting the host availability of key structural lipids, particularly sphingolipids, has been proposed to be a useful strategy in preventing propagation of enveloped human rna viruses, including influenza, hiv and hepatitis c (yager and konan, 2019) . currently, however, assays to screen inhibitors of cpla2α are relatively limited. targetting carbohydrate turnover given that a number of the viral proteins, including the two structural proteins spike and membrane, are glycoproteins, there is clearly a diversion of sugars from the host. it is unclear as yet, whether specific sugars are involved and whether specific host glycosyltransferases are involved in the processing of coronavirus glycoproteins and might, therefore, form further tractable targets for drug discovery. notably, in studies using site-directed mutagenesis of the spike protein from sars-cov, glycosylation was identified at three glutamine residues within the s1 region, with no loss of binding to ace2-expressing cell of mutated (non-glycosylated) fragments (chakraborti et al., 2005) . the e envelope protein the envelope proteins of sars-cov, hcov229e and mers are small (<100 aa) single transmembrane domain proteins (see figure 2 ) and constitute ion channels with selectivity for monovalent cations over monovalent anions (wilson et al., 2004; zhang et al., 2014) apparently forming homopentamers in model membranes (pervushin et al., 2009; surya et al., 2015) . infecting or transfecting the coronavirus e message into cells results in accumulation of protein in the golgi region (ruch and machamer, 2012) . conserved cys residues proximal to the transmembrane domain internally within the virus are palmitoylated (lopez et al., 2008) , a post-translational modification suggested to allow an internal inflexion point in the protein (ruch and machamer, 2012) . hexamethylene-amiloride has been described as an inhibitor of the hiv-1 virus vpu ion channel (ewart et al., 2002) and to reduce virus proliferation in human macrophages in culture . hexamethylene-amiloride, but not the clinically-used amiloride, inhibited the sars-cov envelope protein-associated ion channel activity when expressed in hek293 cells (pervushin et al., 2009) . amantadine has had multiple uses clinically, including in the therapy of parkinson's disease (for review, see vanle et al., 2018) . it has been used to treat influenza a infection through targeting the m2 ion channel (pinto et al., 1992; wang et al., 1993; holsinger et al., 1994) , although it is no longer recommended in the uk or us because of drug resistance (for review, see li et al., 2015) . amantadine at higher concentrations (100 µm) was found to inhibit the sars-cov e protein expressed in model membranes (torres et al., 2007) . sars-cov e protein was identified as being pro-apoptotic upon transfection into vero e6 monkey epithelial cells, where it localized to both plasma membrane and punctate cytoplasmic locations (chan et al., 2009) . indeed, the sars-cov e protein's ion channel function has been linked to calcium entry into endoplasmic reticulum/golgi membrane complexes and the subsequent activation of the nlrp3 inflammasome, leading to interleukin-β (il-1β) production (nieto-torres et al., 2015) . sirna targeting of the envelope protein of sars-cov reduced virus release in culture media, without altering n and p gene expression in frhk-4 monkey kidney epithelial cells (lu et al., 2006) . a similar observation was reported for the orf4a protein (derived from the orf4a gene) of hcov229e (zhang et al., 2014) . infecting mice with sars-cov in which the e protein ion channel function was disrupted showed unchanged viral proliferation but reduced il-1β and oedema levels in the lungs and better survival over 10 days post-infection (nieto-torres et al., 2014) . in a preliminary (as yet, unreviewed) report, the e protein of sars-cov-2 has been reported to interact with brd2/brd4 bet family bromodomain kinases when expressed in hek293 cells (gordon et al., 2020b) . jq1 and rvx208 are brd2/4 inhibitors with ic50 values with 40-120 and 50-1800 nm ranges, respectively. the membrane protein is usually regarded as the most abundant protein in the coronavirus envelope (see figure 2 ) and is of intermediate size in sars-cov-2 (222 aa). it is thought to assist in viral assembly by collating the other surface structural proteins (ruch and machamer, 2012) . the n protein is of moderate size in sars-cov-2 (419 aa), highly basic and binds the viral rna as a dimeric entity (fan et al., 2005) into nucleocapsids (see figure 2) , which afford protection for the viral genome, while also providing access for replication at appropriate times (for review, see mcbride et al., 2014) . in a preliminary (not yet peer reviewed) report, the n protein of sars-cov-2 was tagged and expressed in hek293 cells and then protein partners were identified by affinity purification/mass spectrometry (gordon et al., 2020b) . the n protein was suggested to interact with casein kinase 2 (ck2), la-related protein 1 (larp1, link to uniprot) and stress granule protein ras gtpase-activating protein-binding protein 1 (g3bp1, link to uniprot). ck2 phosphorylates a broad range of cellular targets, mostly in the nucleus, to regulate development and differentiation (for review, see gotz and montenarh, 2017) . although not in use clinically, two inhibitors are described to target ck2 with high affinity. silmitasertib is a ck2 inhibitor with an ic50 value of 1 nm (pierre et al., 2011) , while tmcb has a ki value of 21 nm (janeczko et al., 2012) . larp1 is an rna-binding protein, which releases rna when phosphorylated by mtorc1 (fonseca et al., 2015; hong et al., 2017) . larp1 seems to preferentially bind 5'-terminal oligopyrimidines with an as-yet unclear cellular role (philippe et al., 2020) . of the three targets suggested to associate with sars-cov-2 n phosphoprotein, g3bp1 seems a relevant focus for therapy against covid-19. g3bp1 regulates the innate immune response liu et al., 2019; wiser et al., 2019; yang et al., 2019) and stress granules reduce the replication of mers-cov (nakagawa et al., 2018) , so there is a potential for targetted drug discovery. interactions with the host innate immune system sars-cov produces proteins that interfere with interferon pathways (nsp1, nsp3, nsp16, orf3b, orf6, orf9b, m and n proteins, wong et al., 2016) and nlrp3 inflammasome activators (e, orf3a, orf8b) which are closely related to orthologues found in sars-cov-2. fung et al (2020) have recently reviewed the molecular aspects whereby sars-cov and, by inference, sars-cov-2, evades immune surveillance, activates the inflammasome and causes pyroptosis. other coronaviruses may give an indication as to how this is happening. hcov-229e rapidly kills dendritic cells, while monocytes are much more resistant. the rapid invasion of, and replication in, dendritic cells kills them within a few hours of infection (mesel-lemoine et al., 2012) . dendritic cells are sentinel cells in the respiratory tract, and plasmacytoid dendritic cells are a crucial antiviral defence via interferon production, and by modifying antibody production. thus, these viruses can impair control of viral dissemination and the formation of long-lasting immune memory. penetration of sars-cov-2 infection deep into the lungs, and eventually the alveolae, results in the 'cytokine storm' which accompanies pneumonia and lung fibrosis and is probably a major determinant of the necessity for intubation, and also mortality (shi et al., 2020b) . it is currently not known what specific factor/s differentiate the patients who develop this; although mortality among younger health workers may indicate that initial viral load may play a role. immunological agents which can prevent or control the 'cytokine storm' could therefore have a major effect on necessity to intubate and mortality. tocilizumab is a monoclonal antibody targeting interleukin-6 receptors, as a means to interfere with the effects of chronic autoimmune disorders such as rheumatoid arthritis. the chinese clinical trials registry has two studies that are designed to evaluate tocilizumab efficacy in patients with severe covid-19 pneumonia (registration numbers chictr2000029765 and chictr2000030442). similarly, anakinra, which is a slightly modified version of an endogenous antagonist of interleukin-1 receptors, is being investigated in clinical trials in multiple locations in patients with covid-19 infection (nct04324021, nct04330638 and nct02735707). it has been reported that in stage iii of covid-19, a critical point with a high viral load and severe respiratory involvement, lungs of patients appear with 'ground-glass' patches in ct scans, while autopsy reports indicate that the lungs are filled with a 'clear liquid jelly' (shi et al., 2020c; xu et al., 2020) , similar to an observation in drowning victims. on the hypothesis that inflammation-driven hyaluronan production (via hyaluronan synthase 2, has2, link to uniprot), and associated water retention may be critical; a recent study proposed therapy via administration of recombinant hyaluronidase or inhibitors of has2 (shi et al., 2020c) . the interaction between the virus and the innate immune system is complex and multifactorial, with temporal intricacies. it is beyond the scope of this review to identify all the multiple components and so we discuss here those pathways we consider most tractable. the positive sense rna of coronaviruses is translated to produce the replication machinery, which allows complementary negative sense rna to be synthesised, which itself is the template for the synthesis of positive strand rna. as a consequence, double-stranded rna is produced, which act as a pathogen-associated molecular pattern (pamp) targetting mda5 (interferon induced with helicase c domain i, also known as melanoma differentiation antigen 5, kato et al., 2006) from the rig-1-like receptor family of cytoplasmic pattern recognition receptors (for reviews, see schlee, 2013; bryant et al., 2015) . mda5 differs from rig-1 (dexd/h-box helicase 58, also known as retinoic acid-inducible gene 1) in recognising longer dsrna (kato et al., 2006; goubau et al., 2014) , and it has been proposed this differentiates the sensing of positive-stranded viruses by mda5 compared to negative strand virus sensing by rig-i (kato et al., 2006; goubau et al., 2013) . rig-1-like receptors have an nterminal caspase activation and recruitment domain (card), which shows ligand-dependent interaction with cards from other proteins, such as mitochondrial antiviral signalling protein (mavs, link to uniprot). mavs activates ikk family kinases, such as tank binding kinase (tbk1) and ikk-ε, leading to the phosphorylation of interferon regulatory factors, such as irf3 (link to uniprot) and irf7 (link to uniprot). this induces the transcription of type i interferon genes, such as interferon-β and ccl5 (also known as rantes) (doyle et al., 2002; fitzgerald et al., 2003; sharma et al., 2003) . mavs present in peroxisomes is also able to recruit short-acting, interferon-independent defense factors (dixit et al., 2010) . the orf9b protein from sars-cov has also been reported to target mitochondrial mavs to limit the interferon response, as well as triggering proteolysis of dynamin-like protein 1 (link to uniprot) thereby prompting the formation of mitochondria-associate autophagosomes claimed to create 'havoc' in energy production in infected cells (shi et al., 2014) . in a preliminary (as yet, unreviewed) report, orf9b of sars-cov-2 has been reported to interact with translocases of outer membrane 70 (tom70, link to uniprot) when expressed in hek293 cells (gordon et al., 2020b) tom70 activates mitochondrial irf3 and so this is a potential locus for pharmacological intervention, but as yet with no inhibitors described in the literature. a number of other coronavirus proteins have been identified to influence the irf3 pathway to restrict interferon production. this includes the mers-cov plpro proteinase (yang et al., 2014) , as well as the orf6 and nucleocapsid proteins from sars-cov (kopecky-bromberg et al., 2007) . the orf6 protein of sars-cov has also been described to reduce the activity of a series of karyopherindependent host transcription factors (sims et al., 2013) . karyopherin is an importin, which traffics proteins between the cytoplasm and the nucleus (for review, see kosyna and depping, 2018; . translocases of outer membrane 70 (tom70, link to uniprot) activates mitochondrial irf3 . the orf9b protein of sars-cov-2 has been reported to interact with tom70 when expressed in hek293 cells (gordon et al., 2020b) . clearly, the induction and suppression of interferon production are central to numerous human diseases and have been extensively studied; the 'trick' to treat covid-19 will be to identify a novel angle for therapeutic exploitation. working with sars-cov (not sars-cov-2), pfefferle and colleagues used yeast two-hybrid screens to identify interactions between the viral and human proteomes (pfefferle et al., 2011) . they identified an interesting interaction between viral nsp1 and a group of host peptidyl-prolyl cis-transisomerases (ppia, ppig, ppih and fkbp1a, fkbp1b), all of which modulate the calcineurin/nfat pathway important in immune activation (reviewed by hogan et al., 2003) . the nsp1 protein acts on these to activate nfat signalling and immune activation. cyclosporine a, an inhibitor of this pathway, has used for several decades to control transplant rejection and some autoimmune diseases and, in a simple in vitro assay, cyclosporine inhibited sars-cov transcription/replication in (non-immune-system) cells (pfefferle et al., 2011) . sars-cov-2 has an nsp1 protein closely related to that of sars-cov (dong et al., 2020; srinivasan et al., 2020) , though its effect on the nfat pathway seems not to have been reported. nevertheless, cyclosporine has been shown to inhibit sars-cov2 in an in vitro vero cell-based assay in a preliminary report (as yet not peer-reviewed, jeon et al., 2020) . it has therefore been suggested as a drug target (see, for example, li and de clercq, 2020) . it may seem paradoxical to suggest an inhibitor of immune activation as a treatment for viral disease, but for the subgroup of patients that might suffer cytokine storms (mehta et al., 2020) , the doubleaction might be useful. the orf3a protein of sars-cov appears to bind calcium in a cytoplasmic domain (minakshi et al., 2014) and to elicit a response from the innate immune system by enhancing the ubiquitination of apoptosis-associated speck-like protein containing a card (asc, link to uniprot), which in turn activates the nlrp3 inflammasome and caspase 1 (siu et al., 2019) . the potential for targetting asc and the nlrp3 inflammasome for therapeutic benefit in inflammatory conditions has recently been reviewed (mangan et al., 2018) , although there are no inhibitors in the clinic as yet. in sars-cov, the orf8a and orf8b genes became separated as the disease progressed by a 29nucleotide deletion (chinese sars molecular epidemiology consortium, 2004; oostra et al., 2007) . the orf8a gene of sars-cov encodes a short (31 aa, 1 tm, link to uniprot) protein, which forms a cation channel of predicted pentameric structure (chen et al., 2011) . in sars-cov-2 and a batderived coronavirus, in contrast to the sars-cov-2 genome, orf8 encodes a continuous 121 aa orf8 protein (cagliani et al., 2020) . given that sequence analysis of different strains of sars-cov-2 suggests that the orf8 locus displays only limited evidence of positive selection (cagliani et al., 2020) , it seems germane to investigate the profile of orf8 protein in more depth. sequence comparisons led to prediction of secondary structure composed of an α-helix and a β-sheet containing six strands , but there appears not to be any literature as to whether this entity is a functional ion channel. in a preliminary (as yet, unreviewed) report, the orf14 protein (link to uniprot) of sars-cov-2 has been reported to interact with nod-like receptor x1 (nlrx1), proteinase-activated receptor 2 (par2/f2rl1) and nedd4 family-interacting protein 2 (ndfip2, impdh2 link to uniprot), among other proteins of the iκb/nfκb pathway, when expressed in hek293 cells (gordon et al., 2020b) . at the moment, there are no approved drugs targeting par2, although az3451 (link to gtop) acts as a negative allosteric modulator with pic50 values of 5-23 nm (cheng et al., 2017) . there is a limited insight into the roles or potential exploitability of the remaining range of other viral proteins (nsp2; nsp9; nsp11, proteins orf3b; orf6; orf7a; orf7b; orf10). the spike glycoproteins in sars-cov and mers-cov are crucial for host specificity and jumping between species, e.g. from bats to humans (lu et al., 2015) , and from dromedary camels to humans (mers-cov) and also the recent cross-over of a hku2-related coronavirus to pigs as a swine acute diarrhoea syndrome (sads-cov) (zhou et al., 2018) . sads-cov appears to influence the innate immune system by reducing interferon-β production evoked through ips-1 and rig-i pathways, but not through irf3, tbk1 and ikkε (zhou et al., 2020) . ace2, as an anchoring point for the spike glycoprotein, is present throughout the animal kingdom, but small structural differences are critical for interaction with the spike protein (li et al., 2020b; luan et al., 2020) . key sequences of the spike protein from sars-cov and sars-cov-2 are responsible for binding to ace2. luan et al. (2020) found that the key residues in s protein, from sars-cov and sars-cov-2, recognised in ace2 from dog, cat, pangolin and circetidae mammals (simulated through homology modelling) were broadly similar. mouse ace2 is inefficient in prompting entry of both sars-cov and sars-cov-2 (fung et al., 2020) . cats and dogs suffer from their own specific coronavirus infections (e.g. canine respiratory coronavirus, feline coronavirus) without significant cross-over to humans. a preliminary (as yet lacking peer review) very recent report has suggested that cats and ferrets are sensitive to sars-cov-2, but dogs, pigs, chickens and ducks are much less sensitive (shi et al., 2020a) . ferrets, which have previously been used as models for respiratory tract infections,and retained the sars-cov-2 virus in the respiratory tract, while. shi et al. (2020) showed that the infection was transmitted between cats by aerosol (which may have implications for confinement); infected cats subsequently produced antibodies (shi et al., 2020a) . the syrian hamster has been used as a model for sars-cov (roberts et al., 2005; roberts et al., 2006; de wit et al., 2013) and studies with mice and syrian hamsters are ongoing with sars-cov-2. a preliminary report (as yet not peer-reviewed) suggests that monkeys can be infected and show signs of sickness similar to covid-19, producing antibodies which minimize the signs of subsequent infection (). in a small study where three juvenile (3-5 years old) and two mature (15 years old) rhesus macaques were infected intratracheally with the sars-cov-2 virus, all the monkeys showed symptoms of inflammation and interstitial pneumonia, with a greater apparent severity in the older animals . thus, while there is intensive research in animal models, a clearly validated model is still not apparent. given the similarities in the viruses and their symptoms, there is clearly a value to comparing the profiles of sufferers from the original sars and subsequent mers outbreaks with covid-19 to evaluate the risk factors associated with each event individually and collectively. a detailed consideration is beyond the scope of this review, but there are some obvious questions to ask (not in an order of priority). 1. what factor/s determine resistance to infection? it is apparent that many individuals who test positive for sars-cov-2 infection only experience 'mild' symptoms, others suffer a level of debilitation requiring hospitalization with limited supervision, and a third group require assisted breathing. there is preliminary evidence (as yet, not peer-reviewed) suggesting that people with type a blood might be more at risk of covid-19 than those with other blood types one potential explanation for the relatively high proportion of male victims has been suggested to be previous smoking history (cai, 2020; olds and kabbani, 2020; vardavas and nikitara, 2020) , clearly a general risk factor for many diseases. is there evidence from the sars and mers outbreaks to suggest a commonality of susceptibility? 6. what is the impact of contracting the virus on individuals with other underlying conditions? for example, what are the mechanism/s underlying why some sufferers of hypertension and/or diabetes might be at higher risk (https://www.immunopaedia.org.za/breakingnews/why-are-hypertension-and-diabetes-patients-at-high-risk-of-severe-covid-19/)? is there evidence that patients on ace inhibitors or angiotensin receptor blockers were at higher risk with sars-cov and mers-cov infections and, currently, for sars-cov-2 infection? 7. how will the evolution of the virus alter rates of infection and the severity of symptoms? some level of mutation is to be expected, and indeed has been noted for the sars-cov-2. at the moment, it is too early to identify the significance of any influence of these mutations on the course of covid-19. some of these questions are more tractable since the sars and mers outbreaks because of the strides being made in sophisticated molecular biological techniques (e.g. nextgen sequencing). an additional distinction compared to the previous outbreaks is the major increase in patient numbers associated with covid-19, allowing greater comparisons to be made in many more geographical locations. inevitably other questions will form as greater detail becomes available. this review has concentrated on the prevailing hypothesis that an essential first step in infection is sars-cov-2 binding to ace2 and for tmprss2 to prime the viral spike protein. we further hypothesise that both proteins must be expressed on a target cell for the virus to gain entry. tmpess2 has an extensive cellular expression profile, whereas ace2 is more limited and is usually at low levels, unless increased by risk factors such being sex, age, and smoking history, so is likely to be rate-limiting. other potential target proteins such as cathepsin l or b 0 at1 may also prove important. currently, although there are no drugs approved for the treatment of patients with covid-19, the pandemic has triggered a stampede into clinical trials with both approved and investigational agents. the pharmacological rationale for these trials is sometimes obscure, but there is a logic to focus on viral entry and replication, as well as limiting the host immune response. for the immediate term, the highest priority would be to investigate known antivirals to mitigate effects of covid-19. for the longer term, a vaccine (for review, see amanat and krammer, 2020) seems to hold the most promise to reduce covid-19 damage. there is also a role in the mid-term, however, for drug discovery conducted in mainstream pharmacology labs. the goal here would be an international co-ordinated approach to drug re-purposing; examining the spectrum of licensed drugs (likely to be less than 2000, varying dependent on jurisdictions). these would ideally be screened in a co-ordinated, blinded fashion in multiple labs simultaneously to account for any minor methodological differences. this requires the re-opening of screening and protein biosynthesis labs closed at the start of the pandemic, while ensuring that workers are kept safe. if one were to write a target product profile for a drug to treat covid-19, several parallel profiles can be identified. there are clear considerations, which may be identified as desirable pharmacodynamic, screening methodologies, drug metabolism and pharmacokinetic and formulation profiles. from a pharmacodynamic perspective, a priority would be to screen the proteinases identified in this review (ace2, tmprss2, adam17, cathepsin l, cathepsin b, plpro and 3clpro). a second parallel stream would assess inhibitors of the viral rna polymerase and endoribonuclease complexes, as well as the ion channel functions of the viral envelope (and potentially the orf8 protein). clearly there are multiple other targets, which might bear fruit, and so further studies should assess the tractability of b 0 at1/slc6a19, b 0 at3/slc6a18, impdh2 and has2. further, the molecular mechanism of action of ivermectin should be assessed, since it has recently been shown to inhibit in vitro sars-cov-2 replication (caly et al., 2020) . this agent is used clinically as an anthelmintic, probably through blocking invertebrate glutamate receptors although it also inhibits mammalian glycine receptors and acts as a positive allosteric modulator of other mammalian ligand-gated ion channels. from a screening aspect, biophysical and biochemical screens would probably take a matter of daysto-weeks. following mass availability of the recombinant proteins involved, the capacity for inhibition should be assessed using a library of already approved drugs. biophysical methods can be applied, such as surface plasmon resonance or biolayer interferometry, to monitor the affinity of interaction between host ace2 and viral spike glycoprotein in the presence of these agents, as well as the relevant proteins where multimerization is critical, such as the trimeric spike glycoprotein. assessing the remainder of the targets would likely adopt standard, fluorescent-based pharmacological methodologies. if the assay involves the use of viral proteins, the constructs should acknowledge the inevitable mutations which the viral genome has/will undergo. an overarching priority for the in vitro screening would be to recognise and replicate, as much as possible, relevant features of the virus and its lifecycle. this would include post-translational modifications of the viral proteins, such as glycosylation of the spike and membrane proteins. additionally, while the high throughput screens described above for identifying inhibitors associated with components of the viral entry system, such as ace2, should be confirmed in more translational assays, such as have been described for hiv cell entry in an automatable format (bradley et al., 2004) . a desirable element would also be to minimise adverse effects on the cardiovascular and respiratory system, given the high incidence of damage described associated with those systems (esler and esler, 2020; li et al., 2020a; lippi et al., 2020) . candidate drugs should also not increase activity of the il-6 (or any other pro-inflammatory cytokine) pathway to avoid provoking a cytokine storm. if a similar approach were taken to the ways in which targetted therapy is applied for certain types of cancer, there would be an increased benefit in a multimodal strategy. thus, in cancers where egf/egf receptors are involved, it is possible to target the ligand using chelating antibodies, to antagonise the receptor using blocking antibodies, to use specific antibodies to prevent dimerization of the receptor and to inhibit the catalytic activity of the receptor with small molecular inhibitors. it should be possible to reproduce this approach by simultaneously targetting several steps in the viral cycle (while naturally being cognisant of the potential for phenomena of drug:drug interactions, for instance in terms of convergent pathways of drug metabolism). this approach, enacted for the treatment of hepatitis c and human immunodeficiency viruses, for example, should also show benefit in reducing the capacity for drug-driven mutation in the enzyme. from a dmpk perspective, a beneficial profile for any agent would avoid drug:drug interactions by not converging on key metabolic enzymes and/or transporters. ideally, a once-daily treatment regimen would be optimal, but if more frequent administration were needed, there is likely to be good patient adherence, given the public response to 'spatial distancing'. from a formulation perspective, prophylactic use or for treatment of mild symptoms, an orally-administered or inhaled formulation would be appropriate. for more severe cases, where breathing is significantly impaired, an inhaled aerosolised version may be difficult to administer effectively; in this circumstance, a soluble version to be applied intravenously is likely to be useful. micro-organisms, such as viruses and bacteria, continue to evolve to evade our immune systems and previous pandemics contributed to the decline and fall of civilizations. there is a widespread hope that the current pandemic will be controlled by the rapid development of a safe and efficacious vaccine. clearly, there are major successes with vaccines targetting viral disease, but, to date no vaccine has been successfully produced to protect against human betacoronaviruses such as those causing sars and mers. on the contrary, multiple viral diseases have been successfully controlled by pharmacological agents. hiv-aids became more widespread in the last century and was associated with high morbidity and mortality. as a result of the discovery of novel pharmacological treatments, including specific antivirals, it is now a chronic condition and a cure has been effected in at least two individuals. similarly, the highly variable hepatitis c virus has resisted vaccines, but can be treated with direct antiviral agents allowing elimination of the virus in a very high proportion of those treated. this gives us hope that the roadmap outlined in this review may provide some relief from covid-19 (and indeed for viral threats yet to come). the novel virus uses angiotensin converting enzyme 2 (ace2) to attach to target cells, including epithelial and endothelial cells, particularly in the lungs. sars-cov-2 requires the camostat-sensitive serine proteinase tmprss2 to prime the spike protein for fusion and internalization. thereafter, host cellular processes are exploited for viral replication and release from the cell. ace2 is also expressed in high levels in the gi tract where it is associated with b 0 at1/slc6a19 that actively transports neutral amino acids across the apical membrane of 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recombinant ace2-ig nsp3 of coronaviruses: structures and functions of a large multi-domain protein membrane recognition by phospholipid-binding domains functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses angiotensin-converting enzyme 2 catalytic activity in human plasma is masked by an endogenous inhibitor prevalence and impact of cardiovascular metabolic diseases on covid-19 in china structure of sars coronavirus spike receptor-binding domain complexed with receptor therapeutic options for the 2019 novel coronavirus (2019-ncov) analysis of angiotensin-converting enzyme 2 (ace2) from different species sheds some light on cross-species receptor usage of a novel coronavirus 2019-ncov clinical implications of antiviral resistance in influenza angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus prostate-localized and androgenregulated expression of the membrane-bound serine protease tmprss2 the 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coronavirus tropism and pathogenesis the sars coronavirus 3a protein binds calcium in its cytoplasmic domain b38-cap is a bacteria-derived ace2-like enzyme that suppresses hypertension and cardiac dysfunction attenuation of murine coronavirus infection by ammonium chloride development of potent and selective phosphinic peptide inhibitors of angiotensin-converting enzyme 2 inhibition of cytosolic phospholipase a2alpha impairs an early step of coronavirus replication in cell culture the 2019 coronavirus (sars-cov-2) surface protein (spike) s1 receptor binding domain undergoes conformational change upon heparin binding structure guided design of potent and selective ponatinib-based hybrid inhibitors for ripk1 the endonucleolytic rna cleavage function of nsp1 of middle east respiratory syndrome coronavirus promotes the production of infectious virus particles in specific human cell lines synthesis and immunosuppressive activity of some side-chain variants of mycophenolic acid severe acute 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signalosome susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 susceptibility of ferrets, cats, dogs, and different domestic animals to sars-coronavirus-2 covid-19 infection: the perspectives on immune responses inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry release of severe acute respiratory syndrome coronavirus nuclear import block enhances host transcription in human lung cells severe acute respiratory syndrome coronavirus orf3a protein activates the nlrp3 inflammasome by promoting traf3-dependent ubiquitination of asc unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage from sars to mers, thrusting coronaviruses into the spotlight structural genomics of sars-cov-2 indicates evolutionary conserved functional regions of viral proteins determining the enzymatic activity of angiotensin-converting enzyme 2 (ace2) in brain tissue and cerebrospinal fluid using a quenched fluorescent substrate mers coronavirus envelope protein has a single transmembrane domain that forms pentameric ion channels dhhc9 and gcp16 constitute a human protein fatty acyltransferase with specificity for h-and n-ras the role of type ii transmembrane serine protease-mediated signaling in cancer tmprss2 is a host factor that is essential for pneumotropism and pathogenicity of h7n9 influenza a virus in mice mechanisms and enzymes involved in sars coronavirus genome expression a human homolog of angiotensinconverting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase recurrent fusion of tmprss2 and ets transcription factor genes in prostate cancer conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of sars coronavirus envelope protein proteomics. tissue-based map of the human proteome nmda antagonists for treating the non-motor symptoms in parkinson's disease covid-19 and smoking: a systematic review of the evidence hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 structure, function, and antigenicity of the sars-cov-2 spike glycoprotein ion channel activity of influenza a virus m2 protein: characterization of the amantadine block the e3 ubiquitin ligase nrdp1 'preferentially' promotes tlr-mediated production of type i interferon sars coronavirus e protein forms cation-selective ion channels the membrane-anchored serine protease, tmprss2, activates par-2 in prostate cancer cells g3bp1 enhances cytoplasmic dna pattern recognition novel 1,2,4-triazole and imidazole derivatives of l-ascorbic and imino-ascorbic acid: synthesis, anti-hcv and antitumor activity evaluations a molecular arms race between host innate antiviral response and emerging human coronaviruses cryo-em structure of the 2019-ncov spike in the prefusion conformation a new coronavirus associated with human respiratory disease in china inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion this article is protected by copyright. all rights reserved measurement of angiotensin converting enzyme 2 activity in biological fluid (ace2) clinical relevance and role of neuronal at1 receptors in adam17-mediated ace2 shedding in neurogenic hypertension pathological findings of covid-19 associated with acute respiratory distress syndrome sphingolipids as potential therapeutic targets against enveloped human rna viruses structural basis for the recognition of sars-cov-2 by full-length human ace2 synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent sars coronavirus 3cl protease inhibitor g3bp1 inhibits rna virus replication by positively regulating rig-i-mediated cellular antiviral response proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease human aminopeptidase n is a receptor for human coronavirus 229e enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov-2 possible? age-related rhesus macaque models of covid-19 -ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication: structure-based design, synthesis, and activity assessment the orf4a protein of human coronavirus 229e functions as a viroporin that regulates viral production fenofibrate down-regulates the expressions of androgen receptor (ar) and ar target genes and induces oxidative stress in the prostate cancer cell line lncap relationship between the abo blood group and the covid-19 susceptibility structure of the main protease from a global infectious human coronavirus, hcov-hku1 angiotensin-converting enzyme 2 suppresses pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin swine acute diarrhea syndrome coronavirus (sads-cov) antagonizes interferon-beta production via blocking ips-1 and rig-i a novel coronavirus from patients with pneumonia in china human coronavirus 229e papain-like proteases have overlapping specificities but distinct functions in viral replication sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues increased angiotensin-(1-7)-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensinconverting enzyme homologue ace2 coronaviruses -drug discovery and therapeutic options this article is protected by copyright. all rights reserved.we would like to thank our colleagues in the universities of cambridge, edinburgh and nottingham for helpful discussions and wellcome trust 107715/z/15/z (to a.p.d., j.j.m.). this article is protected by copyright. all rights reserved. key: cord-318551-c1qr27lg authors: boguszewska‐chachulska, anna m.; haenni, anne‐lise title: rna viruses redirect host factors to better amplify their genome date: 2005-12-29 journal: adv virus res doi: 10.1016/s0065-3527(05)65002-6 sha: doc_id: 318551 cord_uid: c1qr27lg this chapter provides an updated view of the host factors that are, at present, believed to participate in replication/transcription of rna viruses. one of the major hurdles faced when attempting to identify host factors specifically involved in viral rna replication/transcription is how to discriminate these factors from those involved in translation. several of the host factors shown to affect viral rna synthesis are factors known to be involved in protein synthesis, for example, translation factors. in addition, some of the factors identified to date appear to influence viral rna amplification as well as viral protein synthesis, and translation and replication are frequently tightly associated. several specific host factors actively participating in viral rna transcription/replication have been identified and the regions of host protein/replicase or host protein/viral rna interaction have been determined. the chapter centers exclusively on those factors that appear functionally important for viral amplification. it presents a list of the viruses for which a specific host factor associates with the polymerase, affecting viral genome amplification. it also indicates the usually accepted cell function of the factor and the viral polymerase or polymerase subunit to which the host factor binds. as obligatory intracellular parasites, viruses depend on their host for all steps of their reproduction, starting from uptake by the cell to the final encapsidation of progeny virions and exit from the cell, as well as their spread within the host. thus, host factors can be expected to participate at most-if not all-levels of virus reproduction. the present review aims at providing an updated view of the host factors presently believed to participate in replication/transcription of rna viruses. however, it does not deal with viruses that resort to reverse transcription, nor does it discuss the many protein-modifying enzymes such as phosphorylating enzymes whose indirect participation in viral genome amplification is undeniable. the reader may wish to turn to various other reviews dealing with certain aspects of genome amplification of rna viruses for complementary information buck, 1996; de and banerjee, 1997; lai, 1998) . determining what host factors are required for viral protein synthesis has been rather straightforward since viruses rely entirely on the host protein synthesizing machinery to produce their own proteins (ehrenfeld, 1996) , although in certain cases they recruit additional cell proteins not normally involved in translation regulation. on the other hand, searching for host factors involved in transcription and replication of rna viruses has proven to be much trickier. originally, the prevailing seemingly simple view of virus genome replication was modeled on the replication of rna phages such as q (klovins and van duin, 1999; schuppli et al., 2000) . yet it soon became apparent that the situation is far more complex for eukaryotic rna viruses than for rna phages. multiplication of the genome of most rna viruses takes place in the cytoplasm. however, influenza virus (orthomyxoviridae) (wang et al., 1997) and borna disease virus (paramyxoviridae) (pyper et al., 1998) replicate in the nucleus. an interesting consequence of this nuclear localization is that transcription in these viruses can be accompanied by splicing, a process requiring the host's splicing machinery (lamb and horvath, 1991; tomonaga et al., 2002) . in addition, an increasing number of rna viruses require the nucleus in certain steps of their life cycle (hiscox, 2003) . arenaviruses cannot complete their replication cycle in cells enucleated soon after infection (borden et al., 1998) . on the other hand, certain (þ) as well as (à) strand rna viruses grow in enucleate cells, virus yield being related to the length of the virus growth cycle (follett et al., 1975) . several viruses target some of their proteins to the nucleus (hiscox, 2003; urcuqui-inchima et al., 2001) , whereas others such as hepatitis c virus (hcv; flaviviridae) code for proteins that contain nuclear localization signals but do not seem to enter the nucleus (song et al., 2000) . finally, infection by members of the picornaviridae, such as poliovirus and coxsackievirus, and by vesicular stomatitis virus (vsv; rhabdoviridae) (belov et al., 2000; gustin and sarnow, 2001 ) triggers relocation of certain nuclear proteins to the cytoplasm, suggesting that these proteins probably participate in the life cycle of the virus (hiscox, 2003) . for viruses that produce subgenomic (sg) rnas, a distinction must be made between two mechanisms, replication of the entire genome and transcription, which usually entails amplification of only certain regions of the genome to yield the sg mrnas. since sgrnas are generally 3 0 coterminal, an internally located open reading frame (orf) in the genomic (g) rna can become 5 0 proximal in the sgrna, a more favorable situation for the eukaryotic translation apparatus. to date, most of the information available concerning the role of host factors in viral rna amplification deals with replication, while information concerning host factors and transcription is less abundant except for (à) strand rna viruses and for viruses of the coronaviridae, arteriviridae, and closteroviridae families (miller and koev, 2000) . all rna viruses contain within their genome the information for the synthesis of an rna-dependent rna polymerase that shall be referred to as the rdrp or polymerase. the viral polymerase and other viral proteins as well as host factors participate in rna transcription and replication. yet, how the switch occurs between transcription and replication remains largely enigmatic. during replication, the rna strand complementary to the genome strand is produced in far lower amounts than the newly synthesized genome strands. how this imbalance is regulated and what factors may be involved in this bias is also far from clear. nevertheless, some results have appeared suggesting that certain host factors participate in synthesis of one but not of the other strand. the sequence of events that leads to virus amplification depends on whether the viral rna entering the cell is of (þ) or of (à) polarity. the incoming genome of many (þ) strand rna viruses ( fig. 1 ) serves as mrna for virus protein synthesis including the rdrp, and is then replicated yielding the complementary (à) rna strand. in many virus groups, the (à) strand serves as a template to transcribe the sgrnas fig 1. replication/transcription of (þ) rna viruses that produce sgrnas. that will serve as mrnas (miller and koev, 2000) . the (à) strand is also replicated, producing nascent full-length (þ) strands; these are used as mrna for protein synthesis, as a template for further (à) strand synthesis, and ultimately as a genome to be encapsidated, yielding progeny virus particles. exceptions to this general scheme exist. red clover necrotic mosaic tombusvirus (sit et al., 1998) and citrus tristeza closterovirus (gowda et al., 2001) generate (à) strand sgrnas that might function as templates for sg mrna synthesis. production of sgrnas in viruses of the coronaviridae and arteriviridae families (both of the order nidovirales) involves a mechanism similar to splicing known as discontinuous sgrna synthesis: the transcripts are both 3 0 and 5 0 coterminal (fig. 2) . in this strategy, synthesis of a sgrna proceeds on a template. the initiated sgrna, together with the polymerase, then switches from its position to another complementary region called transcriptionregulating sequence (trs) on the template to which it can base-pair and resume synthesis. the step at which the nascent rna chain is transferred from one region of the template to another remains controversial-it could occur during (þ) or (à) strand synthesis. the recent demonstration of the presence of (à) sg strands favors the latter possibility (pasternak et al., 2001; sawicki et al., 2001) . trss present in the 3 0 end of the leader and at the 5 0 end of the sgrna body regions in the grna could allow the nascent rna chain to be transferred to the leader trs by trs-trs (sense-antisense) basepairing, or conversely could allow the rna chain in the leader to be transferred to the sgrna. the scheme for (à) strand rna viruses is different from that of (þ) strand rna viruses because the incoming rna cannot serve as a template for protein synthesis. consequently, all viruses with a (à) strand rna genome encapsidate their polymerase complex, including host factors. the genome tightly bound to the nucleocapsid protein as a ribouncleoprotein (rnp) with its associated polymerase (de and banerjee, 1997) is either replicated into complementary (þ) strands, or transcribed to produce sg mrnas. the mechanism of transcription depends on whether the virus contains a nonsegmented or a segmented genome. in nonsegmented (i.e., monopartite) (à) rna viruses (order: mononegavirales), such as the paramyxoviridae and rhabdoviridae, the 3 0 and the 5 0 termini of the (à) sense grna are known as the 3 0 leader promoter and 5 0 trailer region, respectively (fig. 3) . the 3 0 region of the complementary (þ) sense rna or antigenome, known as the trailer, directs synthesis of the grna. in transcription, synthesis of complementary (þ) sgrnas begins at the 3 0 end (3 0 leader) of the incoming (à) strand genome producing a short leader rna, followed by monocistronic capped and poly(a)-tailed mrnas by sequential transcription. the 5 0 trailer region of the genome and the intercistronic regions separating the orfs are not transcribed (kolakofsky et al., 2004 ). fig 2. two possible models for the synthesis of sgrnas by the discontinuous transcription strategy used by the nidovirales (coronaviridae and arteriviridae). model i, during (þ) strand synthesis (leader-primed transcription); model ii, during (à) strand synthesis (recombination during (à) rna strand synthesis). thin vertical grey bars, transcription-regulating sequences (trss) which determine the stop and start points during discontinuous transcription. the leader is composed of the 5 0 -positioned nucleotides (stippled box) and the trs in the (þ) rna strand. (adapted with permission from pasternak et al., 2001.) transcription of segmented (à) strand rna viruses such as the orthomyxoviridae, arenaviridae, bunyaviridae, and tenuiviruses requires a primer to initiate synthesis of the mrnas. this is achieved by cap-snatching in which the replicase complex, or a protein thereof, binds to the 5 0 region of cell mrnas, cleaves off the cap together with generally 7-15 nucleotides from the 5 0 end of the cell mrna, and uses this fragment as a primer to initiate synthesis of the viral mrnas (bouloy et al., 1978; nguyen and haenni, 2003) . hence, the viral mrnas are capped and have cell-derived nucleotides at their 5 0 end; they also contain a poly(a) tail at their 3 0 end. replication, on the other hand, is not primer-dependent, and the rnas complementary to the genome are devoid of cap and of poly(a) tail. host factors participate in viral genome amplification based on their specific binding to or copurification with replicase subunits, their binding to specific regions of the viral rna, their influence on replication/ transcription, the occurrence of host mutations that influence virus multiplication, and the reduced effect on replication/transcription observed in the presence of small interfering rnas (sirnas) directed against specific candidate host proteins. the unambiguous identification of host proteins that persistently copurify with viral polymerases during multiple purification steps has been fraught with difficulties, in spite of the fact that certain cell proteins bind with high affinity to the purified polymerase, or that cell proteins are often encapsidated together with the polymerase as in the case of the (à) strand rna viruses. similar difficulties have been encountered in searching for specific host factors that interact with the viral genome. these difficulties are exacerbated by the fact that it is often difficult to distinguish between factors that directly affect rna replication/transcription from those that have an indirect effect on viral rna synthesis, such as by interacting with host factors that in turn are directly involved in rna amplification. gel-shift mobility assays, uv-crosslinking, coimmunoprecipitation, and the yeast two-or three-hybrid systems have served to search for interactions between host factors and the viral polymerase and/or the viral rna. yet the relevance of such interactions remains equivocal in the absence of functional rna amplification assays. in many cases, particular caution must therefore surround published data, and further experiments are needed to determine whether copurification and/or interaction with viral proteins or rna is fortuitous or has a functional significance. technologies such as the systematic search for host genes became involved in virus rna amplification using an ordered genomewide set of deletion mutants and rna interference (ahlquist, 2002; isken et al., 2003; lan et al., 2003; li et al., 2002a) , are beginning to provide promising results that nevertheless still require further verification. one of the major hurdles faced when attempting to identify host factors specifically involved in viral rna replication/transcription is how to discriminate these factors from those involved in translation. as has become apparent, several of the host factors shown to affect viral rna synthesis are factors known to be involved in protein synthesis such as, for example, translation factors. in addition, some of the factors identified to date appear to influence viral rna amplification as well as viral protein synthesis, and translation and replication are frequently tightly associated. this situation adds a further level of complication to the identification of host factors involved in a specific viral function. it will hopefully become possible to overcome this problem by examining the effect of a given host factor on the replicase and its activity, and in parallel its effect on translation in vitro and in vivo. such experiments have been undertaken (choi et al., 2002) and have already provided interesting information concerning transcription in a coronavirus. on the other hand, to identify host factors that specifically favor synthesis of rna strands complementary to the genome but not synthesis of further genome strands, one can take advantage of the observation that a few additional nontemplated nucleotides positioned at the 5 0 end of a viral genomic rna can still allow synthesis of the complementary strand, but can hinder subsequent synthesis of new genome strands. experiments based on this approach have been used to distinguish certain aspects of (à) from (þ) strand synthesis of poliovirus (murray and barton, 2003) . in spite of all the pitfalls outlined previously, several specific host factors actively participating in viral rna transcription/replication have been identified and the regions of host protein/replicase or host protein/viral rna interaction determined. the present review centers exclusively on those factors that appear functionally important for viral amplification. table i presents a list of the viruses for which a specific host factor associates with the polymerase, affecting viral genome amplification. it also indicates the usually accepted cell function of the factor and the viral polymerase or polymerase subunit to which the host factor binds. the replication complex of rna phages, such as q is composed of four subunits: the virus-encoded rna polymerase, and three host factors, the protein elongation factors tu (ef-tu) and ts (ef-ts), and the ribosomal protein s1 (table i) . another host factor, designated hf, is also required for (à) strand synthesis from the (þ) strand rna template. in the generally accepted scenario, the polymerase provides the polymerizing activity, whereas s1 allows the enzyme complex to bind to two (s and m) internal sites in the viral genome (table ii ; fig. 4 ). the long-standing observation that the polymerase subunit binds to internal sites on the viral genome remained a paradox for several decades. indeed, if the enzyme is to faithfully replicate the template, it must somehow also lie close to the 3 0 end of the template. it has now been established that this is achieved at least in part by elaborate secondary structures in the viral rna ( fig. 4 ) that bring the 3 0 end of the viral rna in close proximity to the internal sites where the polymerase is positioned (klovins and van duin, 1999) , facilitating this interaction. (1997) tev hpiv-3 paramyxoviridae (à) leader rna (à), leader rna (þ) gapdh glycolysis de et al. (1996) pv, poliovirus; mhv, mouse hepatitis virus; wnv, west nile virus; bvdv, bovine viral diarrhea virus; hpiv-3, human parainfluenza virus-3; ig (à), intergenic region in (à) rna; utr, untranslated region; leader rna (à), 3' end of (à) rna; leader rna (þ), 5' end of (þ) rna; hf, host factor; pcbp, poly(c)-binding protein; pabp, poly(a)-binding protein; hnrnp a1, heterogeneous nuclear ribonucleoprotein a1; ptb, polypyrimidine tract-binding protein; tia-1, t-cell-activated intracellular antigen; tiar, tia-1-related; rha, rna helicase a; gapdh, glyceraldehyde-3-phosphate dehydrogenase. the eukaryotic initiation factor 3 (eif3) stimulates binding of the mrna to the 40s ribosomal subunit and stabilizes the interaction between the 40s and initiator met-trna. it has been shown that the 2a polymerase-like protein involved in brome mosaic bromovirus (bmv) amplification interacts with a 41-kda protein, one of the 10 subunits of eif3 or of a closely related protein (quadt et al., 1993) thereby greatly stimulating (à) strand rna synthesis in vitro. similarly, the membrane-bound replication complex of tobacco mosaic tobamovirus (tmv) contains a subunit of eif3, the 56 kda component which is the rna-binding subunit of the wheat germ eif3. hence, the host components of the two viral polymerases correspond to different subunits of eif3. antibodies against the 56 kda subunit inhibit tmv rna synthesis in vitro (osman and buck, 1997) . nia forms part of the replication complex of potyviruses, such as tobacco etch potyvirus (tev; schaad et al., 2000) , by providing the viral genome-linked protein (vpg) and proteolytic functions. it interacts with the protein initiation factor eif4e, a factor that binds to the klovins and van duin, 1999.) cap structure and facilitates recruitment of the mrna as well as other factors on the 40s ribosomal subunit. to investigate this interaction further, full-length tev transcripts were produced harboring different mutated versions of the eif4e coding sequence. after transfection of protoplasts with these in vitro-transcribed constructs, cells containing the mutated eif4e transcripts accumulated far lower amounts of virus than those harboring the wild-type eif4e transcript. the tumor suppressor retinoblastoma protein (rb) is a nuclear phosphoprotein that binds to many proteins and plays a fundamental role in suppression of various neoplasms. its capacity to suppress cell proliferation is caused mainly by binding and inhibiting the transcription factor e2f (taya, 1997) . the nsp90 protein, the putative rdrp of rubella virus (rv; togaviridae), binds to the rb in vitro and in vivo. this interaction requires the presence of at least some rb in the cytoplasm where amplification of rv is believed to be confined (forng and atreya, 1999) . rb facilitates rv replication, since in null-mutant (rbà/à) mouse embryonic fibroblasts, the level of rv replication is lower than in wild-type (rbþ/þ) cells (atreya et al., 1998) . nsp90 contains the rb-binding motif lxcxe. in mutant viruses containing a c to r mutation in this motif, replication is reduced to less than 0.5% of the wild-type virus, and deletion of the motif is lethal (forng and atreya, 1999) , clearly demonstrating the requirement of the rb protein for efficient rv replication. interaction of the hcv rna polymerase (ns5b) with -actinin, a member of a superfamily of actin cross-linking proteins, was demonstrated using various in vitro techniques. it was confirmed by reducing the expression of -actinin by rna interference in the hcv replicon system, resulting in decrease of hcv rna levels (lan et al., 2003) . likewise, a ubiquitin-like protein hplic1 (the human homolog 1 of the protein linking integrin-associated protein and cytoskeleton) also interacts with ns5b, and its overexpression in the replicon system reduces the levels of ns5b and of replicon rna probably by promoting ns5b degradation via a ubiquitin-dependent pathway (gao et al., 2003) . using the two-hybrid system to screen a human cdna library with ns5b as bait, it was demonstrated that the cellular rna helicase p68 is another protein that interacts with the hcv polymerase (goh et al., 2004) . the c-terminal part of ns5b is responsible for this interaction. the p68 helicase is redistributed from the nucleus to the cytoplasm in ns5b expressing cells. the use of two different cell systems to study the effect of p68 on replication led to opposite results when the level of p68 was reduced using an sirna: in an hcv replicon-bearing cell line, no effect on hcv replication was observed, while in cells transiently transfected with a full-length hcv construct, synthesis of the negative strand was inhibited. overexpression of ns5b and its c-terminally truncated mutant in the transiently transfected cell line also inhibited negative strand synthesis, probably due to sequestration of p68. the helicase activity of p68 does not seem to be required for replication, suggesting that it acts as a transcription factor rather than as a helicase (goh et al., 2004) . the rdrp or l protein of vsv has been overexpressed in recombinant baculovirus-infected cells. the purified, transcriptionally active fraction contains the subunits , , and of ef-1. the subunit binds to the rdrp with the highest affinity yielding a partially active enzyme, and addition of the and subunits significantly enhances enzyme activity. all three subunits are packaged with the l protein in the virions (das et al., 1998) . tubulin binds to the rdrp (l protein) of measles virus (mv; paramyxoviridae) and may be part of the polymerase complex of this virus, as suggested by its coimmunoprecipitation with the rdrp from extracts of mv-infected cells (moyer et al., 1990) . it is required for efficient viral rna replication and transcription, since anti-tubulin antibodies inhibit viral rna synthesis and addition of purified tubulin stimulates mv rna synthesis in vitro. experiments performed with canine distemper virus (cdv; paramyxoviridae) (de and banerjee, 1997; lai, 1998) strongly suggest that heat shock proteins may also be required for optimal transcription when closely associated with the nucleocapsids (ncs) of this virus. indeed, when the cdv ncs from non-heat-shocked and heat-shocked cells are compared, an increase in-and/or accumulation of-viral transcripts is observed in the heat-shocked cells. the heat-shock protein hsp72 seems to directly enhance viral rna transcription in vitro (oglesbee et al., 1996) . three proteins, pb1, pb2, and pa compose the polymerase of influenza a virus. pb2 cleaves 5 0 oligonucleotides containing the cap structure from cellular mrnas in the cap-snatching scenario. pb1 is the catalytic subunit for viral rna synthesis and uses the capped oligonucleotides as primers for transcription. the function of the phosphoprotein pa remains unclear. in addition, the nucleoprotein np is required for synthesis of full-length rna (momose et al., 2001) , and as such it forms part of the replicase complex. various proteins have been shown to interact with the np. among them is the nucleoprotein interactor 5 (npi-5, also known as raf-2p48, bat1 or uap56), a cellular splicing factor (momose et al., 2001) . it interacts with free np, but not with np-rna complexes, and stimulates synthesis of the viral rna. its interaction with np is disrupted by the addition of large amounts of viral rna that bind to np, suggesting that npi-5 may facilitate loading of np on the rna, thus acting as a chaperone. in influenza virus-infected cells, npi-5 disappears from spliceosomes, where it is concentrated in uninfected cells. its relocalization is similar to that of another splicing-related host protein, the ns1-binding protein (ns1-bp), which depends on the presence and distribution of the viral nonstructural protein ns1 (wolff et al., 1998) . the same distribution pattern of the three proteins (npi-5, ns1-bp, and ns1) in infected cells suggests that npi-5 may indirectly contribute to shutoff of host protein synthesis in these cells, due to inhibition of splicing and 3 0 -terminal processing of pre-mrnas by the ns1 protein (momose et al., 2001; wolff et al., 1998) . the search for host factors involved in viral rna amplification has also focused on cellular proteins that specifically bind to the 3 0 or 5 0 untranslated regions (utrs) of either the (à) or (þ) rna strand, as well as to intergenic regions in the nidovirales and mononegavirales. several host factors that bind to specific regions of viral rnas have been identified. all those whose implication in viral rna replication has also been clearly demonstrated are presented in table ii . the poly(c)-binding protein (designated pcbp, hnrnp e or cp) isoforms 1 and 2 regulate the stability and expression of several cellular mrnas (herold and andino, 2001; walter et al., 2002) . they are involved in poliovirus rna translation and replication (gamarnik and andino, 1998) . when bound to the internal ribosome entry site (ires) of poliovirus rna, pcbp probably enhances translation , but it also regulates rna synthesis since its binding to the disrupted stem-loop iv of the ires negatively affects replication (gamarnik and andino, 2000) . it also binds to the 5 0 cloverleaf structure of the poliovirus rna upstream of the ires andino, 1997, 1998; parsley et al., 1997) to which the poliovirus 3cd polymerase also binds, and where it is essential for initiation of (à) rna synthesis. studies have aimed at investigating the functions of the different pcbp isoforms as well as identifying the domains of these factors involved in poliovirus translation and replication using pcbpdepleted cell extracts. using this approach, it was shown that pcbp2 rescues both rna replication and translation initiation, whereas pcbp1 rescues only replication (walter et al., 2002) . pcbp thus seems to be involved in the switch from viral rna translation to replication that may result from a change in the relative homo-and hetero-dimer concentrations of both isoforms. the ternary ribonucleoprotein (rnp) complex formed between the 5 0 cloverleaf structure, 3cd, and pcbp can interact in vitro with the poly(a)-binding protein pabp bound to the poly(a) tail, thus circularizing the rna (herold and andino, 2001) . such a circularized rnp complex consisting of an rna-protein-protein-rna bridge is required to initiate (à) strand rna synthesis. nucleolin is an abundant nucleolar protein. it is highly modified by phosphorylation and methylation, and at times also by adp-ribosylation. one of the multiple functions of this rna-binding protein is to promote processing of ribosomal rna in the presence of u3 snornp (hiscox, 2002) . upon poliovirus infection, it relocalizes to the cytoplasm and interacts strongly with the poliovirus 3 0 utr (waggoner and sarnow, 1998) . cell-free extracts depleted of nucleolin produce fewer infectious particles than control extracts at early times after infection. this is overcome at later times for reasons that remain undefined. ongoing viral gene expression is necessary for relocalization of nucleolin to the cytoplasm and is not the result of inhibition of host protein synthesis, since addition of cycloheximide to uninfected cells does not trigger relocalization of nucleolin. an interesting example of seemingly contradictory results obtained using different approaches to identify host factors required for viral genome amplification, is the case of the possible role of the heterogeneous nuclear ribonucleoprotein (hnrnp) a1 in coronavirus sg mrna synthesis. hnrnp a1 binds to the pyrimidine tract-binding protein (ptb) and is believed to be part of a splicing complex involved in alternative rna splicing (lai, 1998; miller and koev, 2000) , although new data indicate that it may act as a splicing silencer as demonstrated for human immunodeficiency virus-1 (retroviridae) (tange et al., 2001) . on one hand, by uv-cross-linking and immunoprecipitation, it was reported that hnrnp a1 binds to the common trss in the mouse hepatitis virus (mhv; coronaviridae) rna of the complementary (à) leader and (à) intergenic regions, which conform to the consensus ptb-binding motif. hence, hnrnp a1 was proposed to bring together the two elements of the viral rna involved in transcription (huang and lai, 2001; li et al., 1997) . moreover, mhv rna transcription and replication were stimulated by overexpression of hnrnp a1, and inhibited by dominant-negative mutants of hnrnp a1 (shi et al., 2000) . on the other hand, however, the hnrnp a1-binding motif found in mhv transcription-regulating sequences is not conserved in other nidovirales, and mhv replication and transcription were not affected when a cell line not expressing hnrnp a1 was used (shen and masters, 2001) . the protein is thus dispensable for mhv replication and transcription. furthermore, recent results indicate that hnrnp a1-related proteins can replace hnrnp a1 for mhv rna synthesis in hnrnp a1-depleted cells . therefore, similar functions may be exerted by closely related proteins, making it difficult to assign conclusive roles to certain cellular proteins in viral genome amplification. ptb is a member of the hnrnp family of proteins and shuttles between the nucleus and the cytoplasm. it binds to the polypyrimidine tract of the adenovirus major-late pre-mrna, and regulates alternative splicing of certain pre-mrnas. its role in stimulating internal initiation of translation of the genome of certain rna viruses containing an ires has attracted considerable attention (anwar et al., 2000; hunt and jackson, 1999; kaminski and jackson, 1998) . in the cases where it favors internal initiation, this appears to be achieved by maintaining the ires in the appropriate configuration for efficient translation (kaminski and jackson, 1998) . in mhv rna, ptb binds to a stretch of four ucuaa repeats in the 5 0 (þ) leader and to the 5 0 utr of the complementary (à) strand. binding to these sites is required for transcription (huang and lai, 1999) . to further elucidate the role of ptb in mhv rna synthesis and discriminate between an effect on transcription and translation, defective interfering (di) rnas harboring different reporter genes were transfected into stable cell lines overexpressing full-length or truncated ptb (choi et al., 2002) . when these overexpressing cell lines were infected with a di rna-coding for chloramphenicol acetyl transferase (cat) preceded by the intergenic region-that had to be transcribed during mhv infection to undergo translation, cat activity was much lower than in control cell lines, suggesting that ptb overexpression had a dominant-negative effect on translation and/or transcription. to determine whether ptb was affecting translation or transcription, cell lines overexpressing ptb were transfected with an in vitro transcribed di rna containing part of the mhv orf1a fused to the luciferase gene and flanked by the mhv 5 0 and 3 0 utrs. the same level of luciferase activity was obtained in cells overexpressing ptb as in control cells. likewise, synthesis of a reporter protein in a reticulocyte lysate previously depleted of ptb was the same as in a control undepleted lysate. these last two series of experiments demonstrate that ptb is directly involved in rna synthesis, not in translation. moreover, ptb interacts in vivo and in vitro with the viral n protein, an element of the replication complex, supporting the idea that ptb is part of the viral transcription/replication machinery (choi et al., 2002) . ptb interacts specifically with the iress of several picornaviruses enhancing translation. however, as opposed to its proposed function in directly enhancing mhv rna transcription, ptb probably indirectly participates in poliovirus rna synthesis. it has been suggested that early in infection, ptb enhances poliovirus translation. however, because the poliovirus translation products accumulate, and because ptb is cleaved by the viral protease 3c pro and/or 3cd pro , during late stages of infection the concentration of ptb would no longer be sufficient to favor translation, leading to a switch from translation to replication (back et al., 2002) . mitochondrial aconitase is involved in the citric acid cycle; it is a posttranscriptional regulator and possesses iron-regulatory functions (nanda and leibowitz, 2001) . it is one of several proteins that bind specifically to the mhv 3 0 (þ) rna region harboring the poly(a) tail. early in infection such binding increases production of infectious virus particles as determined by western blots of the infected murine cell extracts, in particular when the cell cultures are supplemented with iron. binding of mitochondrial aconitase to the 3 0 utr of the viral genome might increase the stability of the mrna, thereby favoring translation and the production of virus particles at early times after infection. the multifunctional tia-1 (t-cell-activated intracellular antigen) and tiar (tia-1-related) proteins belong to a family of rna-binding proteins and are involved in translation regulation and apoptosis; they have also been identified in mammalian stress granules, where they promote recruitment of untranslated mrnas during environmental stress (dember et al., 1996; kedersha et al., 1999; li et al., 2002b) . one of the host proteins that bind to the west nile virus (wnv; flaviviridae) 3 0 hairpin in the (à) rna strand has recently been identified as tiar (li et al., 2002b) . tia-1 also binds to the same region in the viral rna. moreover, virus growth is inefficient in tiar knockout cells, and when such cells are complemented with a vector expressing tiar, wnv growth is partially restored. thus, tiar and possibly also tia-1 are functionally important for wnv replication. this has not been shown for sendai virus (sev; paramyxoviridae), although its trailer rna binds tiar, and the protein is involved in virus-induced apoptosis (iseni et al., 2002) . a group of cellular proteins belonging to a family with diverse functions and containing a common double-strand (ds) rna, binding motif (dsrbm) was identified when searching for proteins interacting with the 3 0 utr of the bovine viral diarrhea virus (bvdv; flaviviridae) genome. interaction of the three proteins nf90/nfar-1, nf45, and rna helicase a (rha), known as the "nfar" group to both the 3 0 and 5 0 utrs, was unambiguously demonstrated (isken et al., 2003) . mutations of the proteins' interaction sites in the 3 0 utr decreased protein binding and rna replication (measured as progeny (þ) rna synthesis). similarly, transfection of cell lines supporting bvdv replication with sirnas directed against rha led to 60-85% reduction of viral replication. the data suggest that the nfar proteins may mediate circularization of the viral rna leading to a switch from translation to replication. interestingly, this group of proteins is also believed to be part of the antiviral response of the cell that seems to be subverted by the virus for the purpose of its propagation. the direct involvement of the nfar proteins in viral replication and not in translation will require confirmation by further experiments. glyceraldehyde-3-phosphate dehydrogenase (gapdh) is a key enzyme of glycolysis. it appears as multiple isoforms consistent with its multifunctional nature (takagi et al., 1996) . it interacts with various proteins and with rnas including the leader rna (à) and leader rna (þ) of human parainfluenza virus type 3 (hpiv-3; paramyxoviridae) (de et al., 1996) and has helix-destabilizing activity. specific phosphorylated forms of the protein associate with the viral rna and inhibit transcription in vitro (choudhary et al., 2000) . thus, gapdh is a negative regulator of viral gene expression in this case. specific, highly phosphorylated forms of gapdh are encapsidated in the hpiv-3 virions (choudhary et al., 2000) . the la autoantigen is a ubiquitous phosphoprotein and an rnabinding protein. although it interacts with the utrs of several rna viruses (de nova-ocampo et al., 2002; duncan and nakhasi, 1997; gutiérrez-escolano et al., 2000 , 2003 lai, 1998; wolin and cedervall, 2002) , to date, it has not been shown to have a clear functional effect on viral genome amplification and therefore is not discussed further here. tubulin and actin activate rna transcription of members of the rhabdoviridae and paramyxoviridae families (table iii) . these cytoskeletal proteins might stabilize the various components of the transcription/replication complexes, or could transport or orient the viral rnas and the complexes to specific cellular compartments in the appropriate topology. synthesis in vitro of vsv and sev mrnas requires tubulin (moyer et al., 1986) . in sev, tubulin and at least two other cellular proteins, phosphoglycerate kinase (pgk) and enolase, both glycolytic enzymes, are required for transcription. once associated with the transcription complex, tubulin might recruit pgk and enolase into the complex that would become active for elongation of viral mrna chains (ogino et al., 2001) . in mv-infected cell extracts that support transcription and replication, actin stimulates both these reactions in vitro (moyer et al., 1990) . actin microfilaments are involved in hpiv-3 replication and transcription in vivo, and in transcription in vitro (de and banerjee, 1999; gupta et al., 1998) as judged by the action of cytochalasin d that depolymerizes actin microfilaments and results in inhibition of viral rna synthesis. actin is detected in purified human respiratory syncytial virus (hrsv; paramyxoviridae) (ulloa et al., 1998) ; transcription in vitro is stimulated by monomeric actin, as well as by actin microfilaments (burke et al., 1998) . in addition to actin, profilin (an actin-modulatory protein) is required for optimal transcription of hrsv (burke et al., 2000) . among (þ) strand rna viruses, interesting clues have been gathered using bmv, taking advantage of the fact that yeast cells support bmv amplification. using this approach, several yeast factors have family ( been characterized that stimulate virus amplification . mutations in the small protein lsm1p involved in mrna decapping preceding mrna degradation modulate bmv rna replication and sg mrna synthesis in yeast, leading to a selective reduction of rna3 replication, and hence of sgrna synthesis (diez et al., 2000; . mutations in ydj1p, a chaperone thought to participate in proper polymerase folding to produce an active replication complex, inhibit the formation of such a complex required for (à) rna strand synthesis (tomita et al., 2003) . finally, mutations in the ded1p required for translation initiation (noueiry et al., 2000) appear to also indirectly decrease bmv rna replication. ded1p was also shown to directly influence replication of the yeast dsrna l-a virus (totiviridae) because its addition to extracts of virusinfected yeast cells led to stimulation of (à) but not of (þ) strand rna synthesis. however, the mechanism of action of ded1p remains only partially elucidated, as only interaction of ded1p with the capsid protein gag could be clearly demonstrated (chong et al., 2004) . a systematic search for host factors involved in bmv replication has been performed using 4500 single-gene yeast deletion mutants transformed with constructs expressing replicase proteins as well as luciferase from a replication template. this led to the identification of 58 and 38 genes whose deletions respectively inhibited and enhanced luciferase expression at least threefold . some of these genes, such as the lsm and ski genes, had been previously identified as being implicated in viral replication and/or rna function and turnover. for a selected group of genes, a more detailed analysis was carried out that further supports the direct effect of the gene products on rna synthesis. further studies are needed to elucidate more precisely how these newly implicated host factors influence viral replication, and to uncover possible functions of essential yeast genes in bmv rna replication. the replication complexes of all (þ) strand eukaryotic rna viruses are associated with intracellular membranes (lee et al., 2001) , and virus infection frequently leads to membrane proliferation or vesiculation. in addition, proliferation of membrane proteins is linked to lipid biosynthesis-both phenomena appear to be important for the amplification of most viral genomes. thus, it is to be expected that membrane association is important for viral rna synthesis. the polymerase of members of the togaviridae family is active in modified endosomes or lysosomes designated cytoplasmic vacuoles or double-stranded vesicles (kää riä inen and ahola, 2002) . in equine arteritis virus (arteriviridae) (pedersen et al., 1999; snijder et al., 2001) , the rna polymerase is found in presumably endoplasmic reticulum-(er-) derived double-membrane structures. rna synthesis of members of the flaviviridae family, such as that of wnv, probably occurs in trans-golgi membranes (mackenzie et al., 1999) . rna synthesis of hcv occurs on membranes of the er or on a membranous matrix designated membranous web (moradpour et al., 2003) or lipid raft (detergent-resistant membrane) (gao et al., 2004) . poliovirus infection leads to the appearance of greatly rearranged vesiculated membranes (suhy et al., 2000) , and poliovirus rna replication occurs on membranes that contain markers of lysosomes, the golgi apparatus, and the er (egger et al., 2000; schlegel et al., 1996) . flock house virus (fhv; nodaviridae) is an insect virus that multiplies in diverse host cells such as plant, animal, insect, and yeast cells. using anti-rdrp antibodies, it was demonstrated that the rdrp is tightly associated with the outer mitochondrial membranes of drosophila cells, implying that fhv replication occurs on these membranes (miller et al., 2001) . this interaction results from the presence of an n-proximal sequence in the rdrp that contains a transmembrane domain. when this domain was replaced by a yeast or hcv ertargeting sequence, the rdrp chimeras were redirected to the er membranes in yeast cells, and viral genomic and subgenomic rna synthesis was enhanced considerably (miller et al., 2003) . rna synthesis of plant rna viruses occurs on cell membranes whose nature depends on the virus such as er-derived membranes (carette et al., 2000; den boon et al., 2001; dunoyer et al., 2002; má s and beachy, 1999; ritzenthaler et al., 2002; schaad et al., 1997) , cytoplasmic inclusions (dohi et al., 2001) , vacuolar membranes (hagiwara et al., 2003; van der heijden et al., 2001) , invaginations of chloroplast membranes (prod'homme et al., 2001) , or multivesicular bodies derived from peroxisomes, vacuoles, or mitochondria . cowpea mosaic comovirus (cpmv) replication is linked to small membranous vesicles that proliferate upon virus infection. cpmv but neither alfalfa mosaic bromovirus nor tmv replication requires ongoing lipid biosynthesis, and replication of cpmv is inhibited by the antibiotic cerulenin, an inhibitor of lipid biosynthesis (carette et al., 2000; van der heijden and bol, 2002) . a yeast mutant defective in bmv rna replication was characterized; it is defective in the ole1 gene (lee et al., 2001) that encodes a á9 fatty acid desaturase, essential for the conversion of saturated to unsaturated fatty acids (ufas) ( table iv) . this desaturase is an integral protein of the er, the site of bmv rna replication. as such, it is not required for bmv rna replication, but high levels of ufas are required at one of the steps leading to rna replication. ufas could participate in the assembly of a functional complex for replication. isolated membrane complexes can, in certain cases, support the production of replicative intermediates in vitro and the elongation of rna chains to produce genomic-length viral rnas. early work demonstrated that nuclease digestion of the membrane-bound fhv rna yielded a template-dependent rna polymerase. however, upon addition of the template rna, only dsrna intermediates were produced due to synthesis of only (à) rna strands. yet, upon addition of certain phosphoglycerolipids (pgls), both (à) and (þ) strands were produced, and complete replication of the genomic rna was restored (buck, 1996; wu and kaesberg, 1991; wu et al., 1992) . it was postulated that either interaction of pgls with the polymerase was required to initiate (þ) strand synthesis, or that the pgls modified the configuration of the membrane to mimic the modifications occurring in virus-infected cells. in cells infected with viruses such as semliki forest virus (sfv; togaviridae) (perez et al., 1991) or poliovirus , the level of lipid synthesis increases, and inoculation with cerulenin prevents replication of these viruses. cerulenin also inhibits poliovirus replication in vitro. continuous lipid synthesis is required for synthesis of sfv rna (perez et al., 1991) . the sfv replicase is composed of the four nonstructural proteins 1 to 4 (nsp1-nsp4). nsp4 is the catalytic subunit of the replicase, and nsp1 contains the capping enzyme activities. nsp1 directly associates with several cell membranes such as the plasma membrane, endosomes, and lysosomes, and its attachment to membranes is required for its activity. solubilization of nsp1 with various detergents strongly inhibits these activities, but they can be restored by the addition of anionic phospholipids, in particular phosphatidylserine (ahola et al., 1999) . as of yet, the elements in the membrane that are affected by the detergents remain undefined, as does how the lipids affect the membrane-associated replication system. tom1 and tom2a are arabidopsis thaliana transmembrane proteins normally located with vacuolar membranes and other uncharacterized cytoplasmic membranes. tom1 interacts with tom2a and with the helicase domain of the tmv polymerase (tsujimoto et al., 2003; yamanaka et al., 2000) . inactivation of either of the corresponding genes significantly reduces tmv replication, implying that in vivo both proteins are involved in the formation of the replication complex (ohshima et al., 1998; yamanaka et al., 2000) . mutations in either the tom1 or the tom2a gene decrease tmv multiplication but do not affect replication of three unrelated plant rna viruses. it was recently shown (gao et al., 2004 ) that binding of the hcv polymerase ns5b as well as of ns5a to the cellular vesicle membrane transport protein hvap-33 is necessary for the formation of the hcv replication complex on lipid raft and for viral rna replication. experiments were carried out using a subgenomic hcv replicon in a hepatocyte cell line transfected with sirna against hvap-33 or overexpressing truncated versions of hvap-33. in both cases, the association of the viral nonstructural proteins with the lipid raft was inhibited, and the overall level of viral proteins was lower than in control cells. in the hvap-33 mutant-expressing cells, the level of hcv (þ) rna synthesis was correlated with disruption of this association, supporting the significance of interaction of hvap-33 with ns5b and ns5a for hcv replication. virus multiplication negatively affects its host as reflected by the appearance of symptoms and diminished performance of the host that are the basis of disease. the organism retorts with a series of programmed responses to counteract the development of virus infection. this situation triggers reiterated counteroffensives between the host and the virus. whereas the host is indispensable for the virus, the reverse is rarely true. it is only in very few cases that the presence of a virus is beneficial for the host as in the case of yeast strains infected with dsrna viruses that secrete peptide toxins lethal to virus-free yeast cells (sesti et al., 2001) . thus, it can appear surprising that so many host factors normally engaged in vital cell functions can be subverted by the virus for its own benefit. the participation of an everincreasing number of host factors in virus replication/transcription is indeed somewhat unexpected, as is the variety of these factors. moreover, the borrowed factors do not necessarily serve the same function in virus development such as in rna replication/transcription as they serve in the host, making their identification more laborious. indeed, some of them are usually associated with functions distantly related to nucleic acid multiplication. in many cases, it still cannot be dismissed that these factors act by stimulating other factors that are more directly involved in viral rna genome amplification. basically, the viral rdrp constitutes the core of the polymerizing enzyme complex, essentially providing the machinery that will produce complementary rna copies of the template. it has been shown that relatively pure preparations of several rdrps can replicate certain natural or synthetic rnas. specificity is provided in large part by ancillary viral factors and by a cohort of satellite host factors (klovins and van duin, 1999; lai, 1998) . intracellular virus-host interactions are both intimate and varied. certain viruses are tissue specific, restricted to certain cell types, and this must be ascribed to the presence or absence of adequate host factors anywhere along the path of virus development. other viruses replicate in diverse hosts, indicating that critical cell proteins required by these viruses must be highly conserved between host species. an interesting example of this situation is provided by experiments showing that the insect virus fhv not only multiplies but also systemically spreads through a transgenic plant expressing the viral movement protein of a plant virus (dasgupta et al., 2001) . as more is learned about viral genomes, it is becoming clear that interactions of proteins with secondary and tertiary rna structures allow the rna to adopt various folding patterns and constitute major elements modulating gene expression. for example, the 5 0 and 3 0 utrs as well as rna structures located within the coding regions are vital for (à) rna strand synthesis of rhinoviruses, enteroviruses, and cardioviruses (murray and barton, 2003) . there is increasing evidence that circularization of mrnas is an important step in protein synthesis, presumably because it allows ribosomes to be redirected from the 3 0 to the 5 0 end of the mrna for a new round of translation and probably because it ensures that only intact mrna is translated. circularization involves the participation of cap-and poly(a)-binding proteins. it is also important for translation and replication of rna viruses. for viral genomes containing a cap structure and a poly(a) tail, circularization could be brought about by a mechanism similar to the one used by cellular mrnas. for viral genomes devoid of cap and/or poly(a) tail, other mechanisms must be invoked. host factors participate in bringing close to one another distantly located regions of the rna in poliovirus (gamarnik and andino, 1998; sadowy et al., 2001; wimmer, 1982) and q phage (klovins and van duin, 1999; schuppli et al., 2000) . there are numerous other cases of viral rna circularization involving essentially rna-rna interactions, and the functional importance of these interactions for viral gene expression has been demonstrated (khromykh et al., 2001; shen and miller, 2004; yang et al., 2004) . it is, however, not unlikely that as for q, viral and/or host factors could be implicated in regulating circularization and triggering a switch between translation and replication. some of the same host factors could be involved in both mechanisms. none of the strategies used so far to study the involvement of host factors in viral rna amplification has been without pitfalls, and frequently the data reported attempt to answer certain but not other questions. what then might be the most appropriate strategy to unambiguously distinguish between factors involved in replication and/or transcription but not in translation, between (à) and (þ) strand rna synthesis, and between those directly involved in a given mechanism and those that affect this mechanism indirectly? it would, for instance, be instructive to investigate by appropriate methods, not only synthesis of the viral genome rna and of its complementary strand, but at the same time also translation. such questions must ultimately be addressed both in vitro and in vivo. having identified potential factors by a screening process such as the one involving the yeast system , a reductionist approach might best be adopted to pinpoint the exact step at which a given factor is required, using various constructs, each allowing only one specific step to be examined. following these experiments, more intricate models could be envisaged. this strategy demands huge investments in time and effort. however, it can be hoped that the availability of eukaryotic genome sequences and of an ever increasing number of infectious viral clones and replicons, the development of more reliable replication systems, and finally the advent of nanotechnology will facilitate such endeavors. moreover, the rapidly developing rna interference methodology and its application to genome-wide screening of gene functions and protein interactions, including in mammals, now enables large-scale identification of host genes whose down regulation (knock-down) will have a severe impact on viral replication (fraser, 2004; paddison et al., 2004 ). an hcv replicon-bearing human hepatocarcinoma cell line that has become one of the favorite models to study host factor influence on viral genome amplification would be a good target for such an experiment. in conclusion, over the past few years, much has been done to establish which host factors participate in viral genome amplification. although the exquisite function of many of these protein-protein and rna-protein interactions must still be established, their identification is a first step towards further understanding of the function of these interactions in the virus life cycle. rna-dependent rna polymerases, viruses and rna silencing host factors in positive-strand rna virus genome replication semliki forest virus mrna capping enzyme requires association with anionic membrane phospholipids for activity demonstration of functional requirement of polypyrimidine tract-binding protein by selex rna during hepatitis c virus internal ribosome entry site-mediated translation initiation the rubella virus putative replicase interacts with the retinoblastoma tumor suppressor protein translation of polioviral mrna is inhibited by cleavage of polypyrimidine tract-binding proteins executed by polioviral 3c pro early alteration of nucleocytoplasmic traffic induced by some rna viruses rna replication: function and structure of q -replicase requirement of poly (rc) binding protein 2 for translation of poliovirus rna two ring finger proteins, the oncoprotein pml and the arenavirus z protein, colocalize with the nuclear fraction of the ribosomal p proteins globin mrna are primers for the transcription of influenza viral rna in vitro comparison of the replication of positive-stranded rna viruses of plants and animals role of cellular actin in the gene expression and morphogenesis of human respiratory syncytial virus profilin is required for optimal actin-dependent transcription of respiratory syncytial virus genome rna cowpea mosaic virus infection induces a massive proliferation of endoplasmic reticulum but not golgi membranes and is dependent on de novo membrane synthesis polypyrimidine-tract-binding protein affects transcription but not translation of mouse hepatitis virus rna ded1p, a conserved dexd/h-box translation factor, can promote yeast l-a virus negative-strand rna synthesis in vitro specific phosphorylated forms of glyceraldehyde 3-phosphate dehydrogenase associate with human parainfluenza virus type 3 and inhibit viral transcription in vitro rna polymerase of vesicular stomatitis virus specifically associates with translation elongation factor-1 for its activity systemic spread of an rna insect virus in plants expressing plant viral movement protein genes specific interaction in vitro and in vivo of glyceraldehyde-3-phosphate dehydrogenase and la protein with cis-acting rnas of human parainfluenza virus type 3 role of host proteins in gene expression of nonsegmented negative strand rna viruses involvement of actin microfilaments in the transcription/replication of human parainfluenza virus type 3: possible role of actin in other viruses individual rna recognition motifs of tia-1 and tiar have different rna binding specificities identification of sequences in brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes translation elongation factor-1, la and ptb interact with the 3 0 untranslated region of dengue 4 virus rna identification and characterization of a host protein required for efficient template selection in viral rna replication brome mosaic virus replicase proteins localize with the movement protein at infection-specific cytoplasmic inclusions in infected barley leaf cells la autoantigen binding to a 5 0 cis-element of rubella virus rna correlates with element function in vivo intracellular localization of the peanut clump virus replication complex in tobacco by-2 protoplasts containing green fluorescent protein-labeled endoplasmic reticulum or golgi apparatus formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral rna synthesis initiation of translation by picornavirus rnas virus development in enucleate cells: echovirus, poliovirus, pseudorabies virus, reovirus, respiratory syncytial virus and semliki forest virus mutations in the retinoblastoma protein-binding lxcxe motif of rubella virus putative replicase affect virus replication human genes hit the big screen two functional complexes formed by kh domain containing proteins with the 5 0 noncoding region of poliovirus rna switch from translation to rna replication in a positive-stranded rna virus interactions of viral protein 3cd and poly(rc) binding protein with the 5 0 untranslated region of the poliovirus genome interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis c virus rna-dependent rna polymerase interactions between viral nonstructural proteins and host protein hvap-33 mediate the formation of hepatitis c virus rna replication complex on lipid raft cellular rna helicase p68 relocalization and interaction with the hepatitis c virus (hcv) ns5b protein and the potential role of p68 in hcv rna replication characterization of the cis-acting elements controlling subgenomic mrnas of citrus tristeza virus: production of positive-and negative-stranded 3 0 -terminal and positive-stranded 5 0 -terminal rnas effects of fatty acids on lipid synthesis and viral rna replication in poliovirus-infected cells involvement of actin microfilaments in the replication of human parainfluenza virus type 3 effects of poliovirus infection on nucleo-cytoplasmic trafficking and nuclear pore complex composition interaction of cellular proteins with the 5 0 end of norwalk virus genomic rna la, ptb, and pab proteins bind to the 3 0 untranslated region of norwalk virus genomic rna subcellular localization of host and viral proteins associated with tobamovirus rna replication poliovirus rna replication requires genome circularization through a protein-protein bridge the nucleolus-a gateway to viral infection? the interaction of animal cytoplasmic rna viruses with the nucleus to facilitate replication polypyrimidine tract-binding protein binds to the complementary strand of the mouse hepatitis virus 3 0 untranslated region, thereby altering rna conformation heterogeneous nuclear ribonucleoprotein a1 binds to the 3 0 -untranslated region and mediates potential 5 0 -3 0 end cross talks of mouse hepatitis virus rna polypyrimidine-tract binding protein (ptb) is necessary, but not sufficient, for efficient internal initiation of translation of human rhinovirus-2 rna sendai virus trailer rna binds tiar, a cellular protein involved in virus-induced apoptosis members of the nf90/nfar protein group are involved in the life cycle of a positive-strand rna virus functions of alphavirus nonstructural proteins in rna replication the polypyrimidine tract binding protein (ptb) requirement for internal initiation of translation of cardiovirus rnas is conditional rather than absolute rna-binding proteins tia-1 and tiar link the phosphorylation of eif-2 to the assembly of mammalian stress granules essential role of cyclization sequences in flavivirus rna replication a long-range pseudoknot in q rna is essential for replication viral rna polymerase scanning and the gymnastics of sendai virus rna synthesis systematic, genome-wide identification of host genes affecting replication of a positive-strand rna virus cellular factors in the transcription and replication of viral rna genomes: a parallel to dna-dependent rna transcription diversity of coding strategies in influenza viruses direct interaction between -actinin and hepatitis c virus ns5b mutation of host á9 fatty acid desaturase inhibits brome mosaic virus rna replication between template recognition and rna synthesis induction and suppression of rna silencing by an animal virus heterogeneous nuclear ribonucleoprotein a1 binds to the transcription-regulatory region of mouse hepatitis virus rna cell proteins tia-1 and tiar interact with the 3 0 stem-loop of the west nile virus complementary minus-strand rna and facilitate virus replication markers for trans-golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral rna flock house virus rna replicates on outer mitochondrial membranes in drosophila cells engineered retargeting of viral rna replication complexes to an alternative intracellular membrane synthesis of subgenomic rnas by positive-strand rna viruses cellular splicing factor raf-2p48/npi-5/bat1/uap56 interacts with the influenza virus nucleoprotein and enhances viral rna synthesis membrane association of hepatitis c virus nonstructural poteins and identification of the membrane alteration that harbors the vial replication complex tubulin: a factor necessary for the synthesis of both sendai virus and vesicular stomatitis virus rnas host cell proteins required for measles virus reproduction poliovirus cre-dependent vpg uridylylation is required for positive-strand rna synthesis but not for negative-strand rna synthesis mitochondrial aconitase binds to the 3 0 untranslated region of the mouse hepatitis virus genome expression strategies of ambisense viruses a mutant allele of essential, general translation initiation factor ded1 selectively inhibits translation of a viral mrna brome mosaic virus rna replication: revealing the role of the host in rna virus replication yeast lsm1p-7p/ pat1p deadenylation-dependent mrna-decapping factors are required for brome mosaic virus genomic rna translation enolase, a cellular glycolytic enzyme, is required for efficient transcription of sendai virus genome the highly inducible member of the 70 kda family of heat shock proteins increases canine distemper virus polymerase activity isolation of a mutant arabidopsis thaliana carrying two simultaneous mutations affecting tobacco mosaic virus multiplication within a single cell the tobacco mosaic virus rna polymerase complex contains a plant protein related to the rna-binding subunit of yeast eif-3 a resource for large-scale rna-interference-based screens in mammals poly(rc) binding protein 2 forms a ternary complex with the 5 0 -terminal sequences of poliovirus rna and the viral 3cd proteinase sequence requirements for rna strand transfer during nidovirus discontinuous subgenomic rna synthesis open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex synthesis of semliki forest virus rna requires continuous lipid synthesis detection and subcellular localization of the turnip yellow mosaic virus 66k replication protein in infected cells the nucleolus is the site of borna disease virus rna transcription and replication characterization of a host protein associated with brome mosaic virus rna-dependent rna polymerase grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes proteins attached to viral genomes are multifunctional the rna structures engaged in replication and transcription of the a59 strain of mouse hepatitis virus formation of plant rna virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein strain-specific interaction of the tobacco etch virus nia protein with the translation initiation factor eif4e in the yeast two-hybrid system cellular origin and ultrastructure of membranes induced during poliovirus infection synergism of mutations in bacteriophage q rna affecting host factor dependence of q replicase immunity to k1 killer toxin: internal tok1 blockade the 3 0 untranslated region of tobacco necrosis virus rna contains a barley yellow dwarf virus-like cap-independent translation element evaluation of the role of heterogeneous nuclear ribonucleoprotein a1 as a host factor in murine coronavirus discontinous transcription and genome replication heterogeneous nuclear ribonucleoprotein a1 regulates rna synthesis of a cytoplasmic virus multiple type a/b heterogeneous nuclear ribonucleoproteins (hnrnps) can replace hnrnp a1 in mouse hepatitis virus rna synthesis rna-mediated trans-activation of transcription from a viral rna nonstructural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex nuclear localization and intramolecular cleavage of n-terminally deleted ns5a protein of hepatitis c virus remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles positive and negative host factors for sendai virus transcription and their organ distribution in rats the hnrnp a1 protein regulates hiv-1 tat splicing via a novel intron silencer element rb kinases and rb-binding proteins: new points of view mutation of host dnaj homolog inhibits brome mosaic virus negative-strand rna synthesis molecular and cellular biology of borna disease virus infection arabidopsis tobamovirus multiplication (tom) 2 locus encodes a transmembrane protein that interacts with tom1 interactions between cellular actin and human respiratory syncytial virus (hrsv) potyvirus proteins: a wealth of functions alfalfa mosaic virus replicase proteins p1 and p2 interact and colocalize at the vacuolar membrane composition of alphavirus-like replication complexes: involvement of virus and host encoded proteins viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells distinct poly(rc) binding protein kh domain determinants for poliovirus translation initiation and viral rna replication the npi-1/npi-3 (karyopherin ) binding site on the influenza a virus nucleoprotein np is a nonconventional nuclear localization signal genome-linked proteins of viruses ns1-binding protein (ns1-bp): a novel human protein that interacts with the influenza a virus nonstructural ns1 protein is relocalized in the nuclei of infected cells the la protein synthesis of template-sense, single-strand flockhouse virus rna in a cell-free replication system active complete in vitro replication of nodavirus rna requires glycerophospholipid tom1, an arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein translation enhancer in the 3 0 -untranslated region of rotavirus gene 6 mrna promotes expression of the major capsid protein vp6 we are very grateful to karla kirkegaard, leevi kääriäinen, masayuki ishikawa, andrzej palucha, peter sarnow, and eric snijder for careful reading of the manuscript, fruitful discussions, and constructive comments. this work was partly supported by the centre national de la recherche scientifique (cnrs, france), the center of excellence of molecular biotechnology (poland), the french-polish center of plant biotechnology (cnrs and polish academy of science), and the university of antioquia and colciencias (colombia). key: cord-329625-hx2rsi91 authors: you, jae-hwan; howell, gareth; pattnaik, asit k.; osorio, fernando a.; hiscox, julian a. title: a model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (prrsv) nucleocapsid protein based on live cell imaging date: 2008-08-15 journal: virology doi: 10.1016/j.virol.2008.04.037 sha: doc_id: 329625 cord_uid: hx2rsi91 porcine reproductive and respiratory syndrome virus (prrsv), an arterivirus, in common with many other positive strand rna viruses, encodes a nucleocapsid (n) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing n protein. the dynamic trafficking of positive strand rna virus nucleocapsid proteins and prrsv n protein in particular between the cytoplasm and nucleolus is unknown. in this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (frap and flip), was used to investigate the trafficking of fluorescent labeled (egfp) prrsv-n protein. the data indicated that egfp-prrsv-n protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. further the nuclear import of n protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of n protein between the cytoplasm and the nucleolus. a model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (prrsv) nucleocapsid protein based on live cell imaging introduction porcine reproductive and respiratory syndrome virus (prrsv) is a spherical, enveloped virus, which causes highly contagious, severe disease in the natural host, the swine, with a spectrum of disease states ranging from respiratory failure in neonates to abortions in sows and persistence (wills et al., 2003) . prrsv is endemic to most swine producing countries and imposes a heavy economic burden due to the high mortality associated with the disease. prrsv is a positive strand rna virus belonging to the arteriviridae family, order nidovirales. although the primary site of replication is the cytoplasm, the viral encoded nucleocapsid (n) protein (an rna binding protein) localises predominately in the cytoplasm and nucleolus during virus infection and when over-expressed in cell culture (rowland et al., 1999) . this property is common with related viruses including the equine arterivirus (eav) n protein (tijms et al., 2002) and the coronavirus n proteins wurm et al., 2001) with the possible exception of the severe acute respiratory syndrome coronavirus (sars-cov) n protein (li et al., 2005; rowland et al., 2005; timani et al., 2005; you et al., 2005; you et al., 2007) . localisation and trafficking of the avian coronavirus infectious bronchitis virus (ibv) n protein to the nucleolus (cawood et al., 2007) correlates with the stage in the cell cycle (g2) when virus protein synthesis and progeny virus production is most efficient harrison et al., 2007; li et al., 2007) . the nucleolus is a dynamic sub-nuclear compartment found principally during interphase in eukaryotic cells (andersen et al., 2005; boisvert et al., 2007; hernandez-verdun, 2006; matthews and olson, 2006) . solution of the nucleolar proteome (andersen et al., 2002) has indicated that the nucleolus has a number of functions ranging from ribosome subunit biogenesis to non-classical functions such as the stress response (mayer and grummt, 2005; rubbi and milner, 2003) and regulation of cell growth (hernandez-verdun and roussel, 2003) . virology 378 (2008) there are over 700 proteins associated with the nucleolar proteome (leung et al., 2006) and these include nucleolin, fibrillarin and b23.1. localisation of positive strand rna virus capsid proteins and replication proteins (and in one case partial rna synthesis) in the nucleus and/or nucleolus appears to be an emerging feature of positive strand rna viruses. the reason why positive strand rna virus capsid proteins localise to the nucleolus is unknown, and several hypotheses have been proposed including; as part of a cellular defense mechanism to sequester viral proteins away from sites of virus replication and assembly, to recruit nucleolar proteins to facilitate virus replication, to usurp cellular processes by disrupting the nucleolar proteome or because the viral protein contains motifs which mimic nucleolar localisation signals (nolss) (hiscox, 2002 (hiscox, , 2003 (hiscox, , 2007 uchil et al., 2006; weidman et al., 2003) . however, the majority of rna synthesis in positive strand rna viruses (ahlquist, 2006; de haan and reggiori, 2008; taylor and kirkegaard, 2008) including arteriviruses (posthuma et al., 2008; van hemert et al., in press ) is thought to occur on cytoplasmic membrane bound replication complexes. assembly also occurs in the cytoplasm; therefore capsid proteins are required in this cellular compartment to facilitate these processes. this would infer the need for appropriate trafficking signals to ensure import and export from the nucleus and several such motifs have been identified. mutational analysis and abrogation of nuclear/nucleolar localisation of viral proteins for the prrsv n protein (lee et al., 2006; pei et al., 2008) and the positive strand rna plant rna virus groundnut rosette virus orf3 (kim et al., 2007a,b) have elegantly demonstrated the importance of nucleolar localisation in the biology of these viruses. for prrsv n protein, the motifs which direct nucleolar localisation are composed of a nuclear localisation signal (nls) acting in concert with a nols (lee et al., 2006; . whereas for ibv n protein an eight amino acid motif was shown to be necessary and sufficient to act as an nols (reed et al., 2006) . for nuclear export of the arterivirus and coronavirus n proteins, nuclear export signals (ness) have either been identified or inferred from molecular genetic and/or bioinformatic analysis, structural modeling or use of specific metabolic inhibitors of nuclear export tijms et al., 2002; you et al., 2007) . the dynamic trafficking of positive strand rna virus capsid proteins between the nucleolus and the cytoplasm or the mobility in the nucleolus is not clearly understood. previous studies which have been mainly based on preparations of fixed cells could not distinguish whether there were for example two populations of capsid protein; one which sequestered to the cytoplasm and one which sequestered to the nucleolus or whether protein trafficked between the two compartments. in order to distinguish between these possibilities and derive a model for prrsv n protein trafficking, this study used live cell confocal microscopy to investigate the trafficking of fluorescent labeled n protein in porcine and non-porcine cell lines which were permissive and non-permissive for prrsv. to investigate whether the localisation of n protein within live cells was equivalent to that previously observed in fixed cells and for subsequent trafficking analysis, the prrsv n gene was pcr cloned downstream of the enhanced green fluorescent protein (egfp) in the expression vector pegfpc2. this created plasmid pegfp-prrsv-n, which when transfected into cells led to the expression of prrsv n protein fused to egfp. although several cell lines were used in this study, primarily, the continuous porcine monocytic cell line, 3d4/31, was used in the majority of experiments. although not permissive for prrsv infection, these cells were derived from porcine alveolar macrophages (which are permissive) following expressing of the sv40 large t antigen (weingartl et al., 2002) . to investigate the localisation of egfp-prrsv-n protein, 3d4/31 cells were co-transfected with pegfp-prrsv-n and a vector which directed the expression of the nucleolar protein b23.1 fused to dsred (dsred-b23.1) (reed et al., 2006; you et al., 2005) to act as a marker for the nucleolus (fig. 1a) . two different localisation patterns of egfp-prrsv-n protein were observed: in 3d4/31 cells which expressed lower amounts of egfp-prrsv-n protein, localisation was predominately nucleolar with some nuclear signal (fig. 1a, panel a) . in 3d4/31 cells which expressed higher amounts of protein localisation was predominately either cytoplasmic and nucleolar (fig. 1a , panel c) or predominately nucleolar with some cytoplasmic and nuclear signal (fig. 1a, panel d) . this difference in expression was evident in that the fluorescent signal from egfp-prrsv-n protein in fig. 1a , panel a was captured in the linear range. however, using the same setting led to over-saturated pixels in cells expressing higher amounts of egfp-prrsv-n protein e.g. fig. 1a , panel b (red indicates that the image range is not linear). gain adjustment to ensure pixels were captured in the linear range restored image clarity (fig. 1a , panel c). nucleolar localisation of egfp-prrsv-n protein was confirmed due to colocalisation with the nucleolar marker protein dsred-b23.1 (yellow signal). previously, n protein has been shown to interact with the nucleolar protein fibrillarin . there are several different mechanisms which may account for the concentration dependent localisation pattern of n protein. n protein could diffuse and/or be actively transported into the nucleus through the nuclear pore complex (npc), diffuse through the nucleoplasm and be permanently sequestered in the nucleolus. as the nucleolar concentration of n protein becomes saturating so excess protein could either diffuse or be actively transported through the npc to the cytoplasm. alternatively, n protein could be continuously exchanged between the nucleolus and the nucleus and the cytoplasm, with localisation being driven by possible differential relative trafficking rates between nuclear import and export. to investigate and discriminate between these possibilities dynamic live cell imaging using fluorescence loss in photobleaching (flip) and fluorescence recovery after photo-bleaching (frap) was used to study movement of egfp-prrsv-n protein. relative trafficking within the nucleolus and trafficking from the cytoplasm to the nucleolus was investigated using flip to continuously photo-bleach a defined portion of the nucleolus in 3d4/31 cells expressing egfp-prrsv-n protein. this was compared to the trafficking of three nucleolar marker proteins (reed et al., 2006; you et al., 2005) ; egfp-nucleolin, egfp-b23.1 and efp-fibrillarin (fig. 1b) . loss of fluorescence is then a result of the egfp-tagged fusion protein trafficking into and out of the photo-bleached area with time. the data indicated that by 180s, loss of fluorescence from cells expressing egfp-nucleolin and egfp-fibrillarin was greater than egfp-b23.1. in cells expressing lower amounts of egfp-prrsv-n protein, fluorescence was completely photo-bleached by 120s and in higher expressing cells egfp-prrsv-n protein fluorescence was completely photo-bleached in the nucleus and reduced in the cytoplasm by 180s (fig. 1b) . this indicated that prrsv n protein was potentially as mobile as the cellular nucleolar proteins examined. to compare the relative nucleolar mobility between egfp-prrsv-n protein and egfp-nucleolin, egfp-b23.1 and efp-fibrillarin in 3d4/ 31 cells, frap was used to photo-bleach a defined portion of the nucleolus ( fig. 2a ) and the time taken to refill the photo-bleached area was measured (fig. 2b ) and t 1/2 values were compared (fig. 2c ). the data indicated that egfp-prrsv-n protein had the lowest t 1/2 value and therefore had a slightly faster mobility than the three cellular nucleolar marker proteins used in this study. cellular nucleolar proteins can have different mobility rates and nucleolar localisation patterns depending on their functions (andersen et al., 2005) and can show a continuous rapid turn over between the nucleus and the nucleolusyet the overall structure of the nucleolus is maintained (misteli, 2001) . the diffusion coefficient (d) value of egfp-prrsv-n protein and egfp-nucleolin, egfp-b23.1 and efp-fibrillarin was calculated from the recovery curves (phair and misteli, 2000) . this value is the rate at which a protein repopulates a photo-bleached area. this confirmed that egfp-prrsv-n protein had an equivalent (or slightly higher) mobility (d = 0.839 μm 2 /s) than egfp-nucleolin (d = 0.156μm 2 /s), egfp-fibrillarin (d = 0.278μm 2 /s) or egfp-b23.1 (d = 0.166μm 2 /s). in a previous study the half time of egfp-fibrillarin in bhk cells was reported to be approximately 3s with a d value of 0.53μm 2 /s (phair and misteli, 2000) , which is in similar agreement to this study. phair and misteli (2000) noted that the d values of egfp-fibrillarin and other fluorescent tagged nuclear/nucleolar proteins were 100 times lower than that reported for free solutes in the nucleus and suggested that this was due to interactions with other nuclear components. therefore, similar to the cellular nucleolar proteins the mobility of egfp-prrsv-n protein indicated that n protein probably localised to the nucleolus by diffusing through the nucleoplasm until it located an appropriate binding site, possibly a nucleolar protein or ribosomal rna, and thus appeared to accumulate in this compartment. prrsv n protein has been shown to interact with fibrillarin . the precise mechanism of trafficking of n protein within a cell and to the nucleolus is unknown, although n protein has been shown to interact with and pull down fibrillarin and thus may potentially localise to the nucleolus via this interaction . for example, the nucleolin binding activity of hepatitis delta antigen is associated with nucleolar localisation (ning and shih, 2004) . prrsv n protein has also been shown to interact with importin-α and importin-β and is sensitive to leptomycin b (lmb), an inhibitor of crm1-dependent export . several different nuclear-cytoplasmic trafficking pathways are utilized by viruses (whittaker et al., 2000) and have been described for a number of different virus nucleocapsids and nucleocapsid proteins. for example, in vesicular stomatitis virus nucleocapsids are trafficked via a microtubule mediated process (das et al., 2006) , as are the hantaan virus nucleocapsid protein (ramanathan et al., 2007) and the west nile virus capsid protein (chu and ng, 2002) . to investigate whether the trafficking of n protein between the cytoplasm and the nucleus/nucleolus was associated with microtubules or energy dependent, the mobility of egfp-prrsv-n protein was compared in 3d4/31 cells incubated at 37°c in the presence and absence of nocodazole (60ng/ml nocodazole (sigma) for 16h), a microtubule inhibitor, or incubated at 10°c. in preparations of fixed cells, in the absence of nocodazole (fig. 3a ) microtubules were visualized (observed as filaments) using an anti-tubulin antibody. as before egfp-prrsv-n protein localised predominately to the nucleolus. in preparations of fixed cells in the presence of nocodazole (fig. 3b ) microtubule polymerization was inhibited (diffuse staining of tubulin), although similar to untreated cells, egfp-prrsv-n protein remained predominately localised in the nucleolus. the activity of nocodazole was also confirmed by analyzing the cell cycle stage of treated cells (using flow cytometry as described previously harrison et al., 2007) ), which demonstrated enrichment in the g2/m stage (fig. 3b ) compared to untreated cells (fig. 3a) , and is a result of the inhibition of spindle formation in metaphase. to investigate the trafficking of egfp-prrsv-n protein in live cells, a nucleolus in 3d4/31 cells expressing egfp-prrsv-n protein was continuously photo-bleached (flip) in the presence of nocodazole at 37°c (fig. 3c ) and in the absence of nocodazole in cells that had been pre-cooled to 10°c (fig. 3d) . in both cases, photo-bleaching resulted in the loss of fluorescent signal in both the cytoplasm (where appropriate) and nucleus and the non photo-bleached nucleolus, indicating that cytoplasmic to nuclear trafficking occurred, and was not inhibited by either the presence of nocodazole or cooling to 10°c. this suggested that the trafficking of n protein from the cytoplasm to the nucleus was not microtubule or energy dependent. to investigate trafficking within the nucleolus a defined portion of the nucleolus was photobleached (frap) in cells incubated at either 37°c, 10°c or 37°c in the presence of nocodazole (fig. 4a ) and the time taken to refill the photobleached area was measured (fig. 4b) . the data indicated that egfp-prrsv-n protein at 37°c had a t 1/2 value of 0.74 ± 0.07 which was similar to the t 1/2 value (0.75 ± 0.18) at 4°c and at 37°c in the presence of nocodazole (t 1/2 value of 1.37 ± 0.55). the mobility of egfp-prrsv-n protein between the nucleolus and the cytoplasm and vice versa was compared to egfp using frap and flip on the cytoplasm or the nucleus/nucleolus in 3d4/31 cells. egfp is approximately 27kda and contains no known nls, nols or nes motifs and therefore the relative localisation of egfp between the nucleus and the cytoplasm would reflect the relative trafficking of the protein across the npc, most likely due to diffusion and concentration gradients, such that equilibrium would be reached depending on the relative balance between the rate of nuclear import versus nuclear export and the relative volume of the cytoplasm versus the nucleus. prrsv n protein is approximately 15kda (and with the addition of egfp approximately 42kda) and although the fusion protein has the potential to freely diffuse through the npc (which has a size exclusion limit for passive transport of approximately 50kda (macara, 2001; terry et al., 2007) ), clearly egfp-prrsv-n protein has a discrete localisation pattern being cytoplasmic and nucleolar (with some nuclear signal) (rowland et al., 1999) . without the presence of trafficking signals n protein would be predicted to display similar trafficking and localisation to egfp. the trafficking of egfp compared to egfp-prrsv-n protein between the nucleus and the cytoplasm and vice versa was analyzed by frap (fig. 5 ) and the relative level of fluorescence between the nucleus and the cytoplasm was measured using imagej software (nih) as described for the 3d4/31 cell line. this takes into account the reduction in fluorescence during photo-bleaching due to ongoing trafficking into and out of the bleach area. in egfp-expressing 3d4/31 cells the pre-bleach level of egfp in the nucleus to that of the cytoplasm was 0.56 ± 0.05 to 0.44 ± 0.03, respectively. to examine trafficking into the nucleus, this area was photo-bleached and subsequent recovery of fluorescence in the nucleus (due to import of egfp from the cytoplasm) indicated that after 180s the relative level of egfp between the nucleus and the cytoplasm was 0.60± 0.04 compared to 0.41 ± 0.05, respectively, which was not significantly different from the pre-bleach levels (fig. 5a) . analysis by 180s was then used as a base line for comparison of trafficking of egfp and egfp-prrsv-n protein between the nucleus and the cytoplasm in frap analysis. in 3d4/31 cells expressing egfp-pprsv-n protein with nucleolar/nuclear and cytoplasmic distribution, the prebleach level of egfp-prrsv-n protein between the total nuclear content and the cytoplasm was 0.78 ± 0.07 and 0.22 ± 0.03, respectively. 180s after photo-bleaching, the nuclear to cytoplasm level was 0.81 ± 0.08 to 0.24 ± 0.04, respectively (fig. 5b) , which was not significantly different from the pre-bleach level. to examine trafficking of egfp into the cytoplasm, this area was photo-bleached in 3d4/31 cells and subsequent recovery of fluorescence in the cytoplasm (due to import of non photo-bleached egfp from the nucleus) was compared to levels before photo-bleaching (nucleus to cytoplasm level of 0.58 ± 0.04 to 0.41 ± 0.05, respectively) to levels 180s after photo-bleach when the nuclear to cytoplasm ratio was 0.72 ± 0.05 compared to 0.28± 0.04, respectively (fig. 5c ). this indicated that egfp had not reached pre-bleach levels, which was different when considering cytoplasm to nucleus trafficking. to examine trafficking of egfp-prrsv-n protein into the cytoplasm, this area was photo-bleached and subsequent recovery of fluorescence in the cytoplasm (due to import of egfp-prrsv-n protein from the nucleus) was compared before photo-bleach (nucleus to cytoplasm relative level of 0.71 ± 0.06 to 0.29 ± 0.03, respectively) to 180s after photo-bleach, when the nuclear to cytoplasm ratio was 0.92 ± 0.08 compared to 0.08 ± 0.05, respectively (fig. 5d ). (note that this experiment was performed on cells in which egfp-prrsv-n protein localised also in the cytoplasm). this data indicated that the ratio of egfp-prrsv-n protein had not reached pre-bleach levels, which was different when considering cytoplasm to nucleus trafficking. this indicated that the export of egfp-prrsv-n protein from the nucleus was potentially slower than import and given the difference in relative ratios, slower than the export of egfp from the nucleus to the cytoplasm. the relative difference between nuclear import and nuclear export observed with egfp-prrsv-n protein was investigated using flip in which either a defined area of the nucleus (not the nucleolus) was photo-bleached for either egfp (fig. 6a ) or egfp-prrsv-n protein (fig. 6b ) expressing 3d4/31 cells or a defined area of the cytoplasm was photo-bleached for either egfp (fig. 6c ) or egfp-prrsv-n protein (fig. 6d ) expressing 3d4/31 cells. again, comparison of the data from the two proteins indicated that more fluorescent signal of egfp-prrsv-n protein was lost during import than export, suggesting that the former process operates faster than the latter. reflecting the frap analysis, the relative trafficking of egfp-prrsv-n protein between the nucleus and the cytoplasm was in favor of nuclear import. the observed live cell trafficking patterns of egfp-prrsv-n protein may have been specific to 3d4/31 cells. therefore, to investigate this possibility, the localisation and dynamic trafficking of this protein was compared to egfp in marc-145 cells (an african green monkey kidney cell line permissive for prrsv infection (kim et al., 1993) ) and hela cells (derived from a human cervical cell line, and non-permissive for infection). the data indicated that in both marc-145 and hela cell types egfp-prrsv-n protein localised predominately to the nucleus/ nucleolus compared to egfp which localised throughout the cell (figs. 7a and 8a, respectively). very few cells (less than 5%) exhibited more than 10% of the total fluorescence from egfp-prrsv-n protein in the cytoplasm. note that for egfp, the transmission phase image is presented to highlight the differentiation between the cytoplasm, nucleus and nucleolus. (such images were used in subsequent analysis for placement of photo-bleaching in the egfp fluorescent cell, where distinction between the nucleus/nucleolus/cytoplasm was not possible). the sub-cellular localisation patterns for both fluorescent proteins were similar to that observed in 3d4/31 cells. investigation of the trafficking of egfp-prrsv-n protein compared to egfp between the cytoplasm and the nucleus and vice versa by continuous photo-bleaching (flip) a defined portion of the nucleus or the cytoplasm in both marc-145 cells (fig. 7) and hela cells (fig. 8) , indicated that photo-bleaching in the nucleus in cells expressing egfp resulted in apparent simultaneous loss of fluorescence from the nucleus and the cytoplasm (figs. 7b and 8b), indicating rapid trafficking of egfp between the two compartments. in comparison, little fluorescence (approximately~5% of total fluorescence) of egfp-prrsv-n protein was observed in the cytoplasm in both marc-145 and hela cells and photobleaching in the nucleus resulted in loss of fluorescence from the nucleoplasm and then the nucleolus (figs. 7c and 8c upper panels, respectively) . in approximately 10% of cells expressing egfp-prrsv-n protein fluorescence was observed in the cytoplasm, but only if imaging was adjusted so that the fluorescent signal in the nucleus/nucleolus was above the linear range (for example fig. 8c, lower panels) . in this case photo-bleaching in the nucleus resulted in loss of fluorescence from the cytoplasm which could be accounted by faster trafficking into the nucleus of non-photo-bleached protein which was then photo-bleached than the export of photo-bleached protein into the cytoplasm. photobleaching in the cytoplasm of either marc-145 cells or hela cells expressing egfp resulted in loss of signal from both the nucleus and the cytoplasm (figs. 7d and 8d, respectively) . in comparison, photobleaching in the cytoplasm of either marc-145 cells or hela cells expressing egfp-prrsv-n protein resulted in less loss of fluorescence when compared to egfp (figs. 7e and 8e) . the photo-bleaching analysis for egfp-prrsv-n protein also indicated that when the nucleus was photo-bleached loss of fluorescence in the cytoplasm was due to the trafficking of non photo-bleached protein into the nucleus rather than the rapid export of photo-bleached protein from the nucleus into the cytoplasm. thus the photo-bleaching analysis of egfp and egfp-prrsv-n protein in marc-145 and hela cells was similar to that found in 3d4/ 31 cells. thus we propose a model of n protein trafficking in which the nuclear import of n protein is faster than the nuclear export and n protein is apparently localised in greater amounts in the nucleolus (fig. 9) . the deficiency of the model is that the analysis was conducted in the absence of other viral proteins or rna (i.e. infected cells) which may associate with n protein and alter its relative trafficking rates. this may be certainly true for the cytoplasm, where n protein will complex with viral rna and possibly components of the replicase complex. although there is currently no published data that other prrsv macromolecules localise in the nucleus/nucleolus, the eav non-structural protein 1 (nsp1) has also been observed in the nucleus (tijms et al., 2002) , and therefore prrsv nsp1 may have similar properties. prrsv n protein does not accumulate or is sequestered in the nucleus/nucleolus per se and in common with other cellular nuclear/nucleolar proteins is continuously being exchanged between the nucleolus and the nucleoplasm and indeed is as mobile as the cellular nucleolar proteins examined. the implication of this study is that all de novo synthesized n protein (when over-expressed) traffics to the nucleolus. together with molecular genetic analysis showing that virus replication is reduced when n protein nucleolar localisation is abolished (lee et al., 2006; pei et al., 2008) , suggests that the nucleolar trafficking of n protein is playing a crucial role in virus biology. what this role is remains to be elucidated. cell culture 3d4/31 cells were grown at 37°c with 5% co 2 in dmem and rpmi 1640 (50:50). marc-145 and hela cells were maintained in dmem supplemented with 10% foetal bovine serum and grown at 37°c with 5% co 2 . 30mm glass bottom tissue culture dishes were seeded with 2 × 10 5 cells 24h prior to transfection. the plasmid pegfp-prrsv-n, which expressed prrsv n protein fused c-terminal to egfp, was constructed by amplifying the n gene using the amplicon fl12 (truong et al., 2004) (genbank accession number ay545985) as template. primers were designed to the 5′ and 3′ end of the n gene and contained appropriate restriction enzyme sites; 5′ hindiii and 3′ ecori respectively. the amplification product was ligated into the topo vector pcr2.1 and then cloned into hindiii and ecori restricted pegfp-c2 (clontech). the insert was sequenced in both directions to confirm authenticity and western blot analysis to confirm expression. plasmids which led to the expression of the nucleolar marker proteins egfp-nucleolin, egfp-b23.1, dsred-b23.1 and efp-fibrillarin have been described previously (reed et al., 2006; you et al., 2005) . transfected cells were imaged at 37°c in co 2 -independent media (gibco) supplemented with 5% fbs on an inverted lsm510 meta confocal microscope (carl zeiss) using a 63× objective and a 4 factor zoom. five images were captured before a short period of photobleaching using conditions that had negligible effects on total cell fluorescence. photo-bleaching was performed on an area of 12 by 12pixels (approx 20.16μm 2 ) with a 25mw argon laser at 100% power, bleaching took approximately 1.2s. fluorescence was analyzed with lsm510 software and raw data was imported into microsoft excel for analysis and generation of graphs. raw data were averaged before normalizing. normalization of the data was performed in such a way as to ensure that the recovery profiles were set to zero at the initial time point post bleaching, and set to 1 at the final time point; as previously described (marcelli et al., 2006; stenoien et al., 2002) . time to half life recovery (t 1/2 ) was performed on the mean raw data, determined by the equation (i e − i o ) / 2, where i e was the intensity at the final time point, and i o the intensity at the time point immediately following photo-bleaching. diffusion coefficient (d) values were calculated from the half life recovery (t 1/2 ) using the following diffusion equation d =(w 2 / 4 t 1/2 ) × 0.88 where w is the width of the bleach area, approx 1.68μm, and a constant factor of 0.88 was used for a gaussian beam profile (axelrod et al., 1976) . data from 5 experiments were used per construct and standard deviation was calculated in the usual manner using microsoft excel. transfected cells were imaged in glass base dishes as outlined above. imaging and photo-bleaching was performed with the same laser settings as detailed in the frap microscopy. in each flip experiment cells were imaged 5 times followed by a period of photobleaching for a total time of 3min. photo-bleaching was performed on a defined area (12 by 12pixels (approx 20.16μm 2 )) within the cell for 50 iterations (mean bleach time 2.1s). parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses nucleolar proteome dynamics directed proteomic analysis of the human nucleolus mobility measurement by analysis of fluorescence photobleaching recovery kinetics the multifunctional nucleolus cell cycle dependent localisation of the coronavirus nucleocapsid protein trafficking mechanism of west nile (sarafend) virus structural proteins visualization of intracellular transport of vesicular stomatitis virus nucleocapsids in living cells are nidoviruses hijacking the autophagy machinery? cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus 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infection and after expression as a recombinant protein in vero cells disruption of the nucleolus mediates stabilization of p53 in response to dna damage and other stresses intranuclear ataxin1 inclusions contain both fast-and slow-exchanging components potential subversion of autophagosomal pathway by picornaviruses crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus model of the trafficking of prrsv n protein within the cell where the width of arrows denotes the relative movement of n protein and shading is relative concentration of the protein. trafficking from the cytoplasm to the nucleus and localisation to the nucleolus is fast compared to export of n protein from the nucleus to the cytoplasm which is slower nuclear/nucleolar localization properties of c-terminal nucleocapsid protein of sars coronavirus a highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cdna clone retains the in vivo virulence and transmissibility properties of the parental virus nuclear localization of flavivirus rna synthesis in infected cells the in vitro rna synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor the interaction of cytoplasmic rna viruses with the nucleus continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility viral entry into the nucleus duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus localisation to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rnaassociated protein fibrillarin subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein trafficking motifs in the sars-coronavirus nucleocapsid protein this research was supported by a national pork board grant to jah. drs amanda stuart and t. david k. brown at the university of cambridge are thanked for providing the materials and reagents. key: cord-319517-denczc6t authors: salipalli, sandeep; singh, prafull kumar; borlak, jürgen title: recent advances in live cell imaging of hepatoma cells date: 2014-07-08 journal: bmc cell biol doi: 10.1186/1471-2121-15-26 sha: doc_id: 319517 cord_uid: denczc6t live cell imaging enables the study of dynamic processes of living cells in real time by use of suitable reporter proteins and the staining of specific cellular structures and/or organelles. with the availability of advanced optical devices and improved cell culture protocols it has become a rapidly growing research methodology. the success of this technique relies mainly on the selection of suitable reporter proteins, construction of recombinant plasmids possessing cell type specific promoters as well as reliable methods of gene transfer. this review aims to provide an overview of the recent developments in the field of marker proteins (bioluminescence and fluorescent) and methodologies (fluorescent resonance energy transfer, fluorescent recovery after photobleaching and proximity ligation assay) employed as to achieve an improved imaging of biological processes in hepatoma cells. moreover, different expression systems of marker proteins and the modes of gene transfer are discussed with emphasis on the study of lipid droplet formation in hepatocytes as an example. microscopy has contributed immensely to our understanding of cellular structure and morphology. however, traditional microscopic tools provide only limited information in terms of dynamical processes occurring in a living cell which are of great interest for biomedical researchers. advances in optical methods, in vitro culture systems and molecular biology led to the advent of live cell imaging techniques. this non-invasive technique provides better insight into the biological role of target molecules by allowing researchers to investigate the dynamic processes occurring in living cells in real time. the technique has many potential applications in various fields of biomedical science including developmental biology, cell biology and tumor biology and provides opportunity to study the dynamic behaviour of living cells in context to gene expression, protein-protein interaction, co-localization, cell division, chromosomal dynamics and intracellular transport of bio-molecules. the success of live cell imaging relies on various factors including the specific imaging system, climate controlling devices for cultured cells under investigation, construction of recombinant plasmid dna, transfer and expression of candidate genes and/or fluorescent proteins in mammalian cells. these factors greatly influence the fluorescent/bioluminescent signals obtained from the cultured cells. the gene transfer methods should not only be efficient in delivery and in ensuring stable expression but at the same time should exert minimum toxic effects to the cultured cells. furthermore, the chosen fluorescent or bioluminescent markers should be minimally phototoxic to the cells at their highest expression levels. amongst the bioluminescent markers, atp dependent and independent luciferases from various sources have been extensively used in imaging experiments [1, 2] . the use of bioluminescent markers is not only limited to in vitro assays or live cell imaging but is also applied to in vivo molecular imaging experiments. various lines of luciferase expressing transgenic mice and cells have so far been developed and are frequently employed in biomedical research, and a major breakthrough in the field of fluorescent protein imaging was the discovery of green fluorescent protein (gfp) by osamu shimomura who received the nobel prize in chemistry in 2008 together with martin chalfie und roger tsien [3, 4] . after the advent of gfp the technique of live cell imaging has taken a leap in understanding the detailed and complex cellular dynamics. apart from gfp and its variants, many other fluorescent proteins have been isolated from a variety of sources and are successfully used in imaging experiments of various cell types and their organelles. in this regard, live cell imaging has been employed to study functional genetics of liver specific diseases including steatosis, which results from accumulation of lipid droplets in hepatocytes [5] . efficient gene delivery in mammalian cells is another aspect of our review with appropriate choices of cell type specific promoters and their use for targeted gene delivery to hepatoma lines such as hepg2 and hep3b. nonetheless, the concept of gene transfer through plasmids started in bacteria via both, physical and chemical methods. similar approaches have been used in hepatoma cells and other higher eukaryotes and mammalian cells and include lipofection, deae-dextran, calcium-phosphate, viral vectors, peptides and electroporation [6] . lipofection has been used to achieve transient as well as steady transfection in hepatoma cells resulting in an improved and stable expression of transgenes even after several passages [7] . to develop protocols for cell type specific reporter activity, we discuss the use of alternate promoters and vectors for stable expression in actively dividing cells. bioluminescence is the phenomenon of the production of light by a chemical reaction within a living organism. it was first discovered in firefly (lampyridae species) and since then has been used for various screening and staining activities with an advantage of observing the cells under a compound microscope. firefly luciferase (fluc) emits luminescence (up to 560 nm) without the requirement of any external light excitation and uses atp for the conversion of its substrate luciferin to oxyluciferin in a luciferase enzyme catalyzed oxidation reaction. initially, fluc was used only in luminometery based reporter assays using cellular lysates. later luciferase expressing cells and mouse lines were developed for noninvasive imaging of rodents. injection of the luciferin substrate in mice produces luminescent signals that can be easily detected by in vivo imaging modalities. apart from beetle, luciferase has been isolated from members of the coelenterazine species, i.e. gaussia, renilla, pleuromamm and oplophorus. the luciferase from renilla sp. (rluc) uses a different substrate "coelenterazine" and produces a higher and stable luminescent signal as compared to the fluc [1] . rluc has an added natural advantage of being an atp independent enzyme, and thus requires less energy to produce luminescence. however, a major limitation of fluc and rluc is their short life span and therefore these luminescent proteins cannot be used for long duration imaging assays. this led to the development of a more photostable and robust luciferase in the form of an enhanced beetle luciferase (eluc) which again uses luciferin as the substrate and is an atp dependent enzyme [8] . a comparison between eluc and fluc showed phenomenal differences in the intensity and photostability of the two luciferases. however, the km values (michaelis constant) of the enzymes in regard to atp consumption were found to be almost equal, and it was concluded that the atp use is not the reason for a better performance of eluc. moreover, eluc was tested in cell lines such as nih/3t3 using various promoters, and it was shown that the luciferase primarily localizes in nucleus, cytosol and in peroxisomes. certain vectors and promoters also play an important role for their suitable expression namely pcmv vector used in mice cells along with the astrocyte specific promoter mper2 which outperformed fluc by a factor of 10-and 16-fold in cell extracts and live cells, respectively [8] . in contrast to bioluminescence where the enzyme catalyzed reaction initiates the excitation of luminescent molecule, fluorescence is triggered when photons from an external source excites the light absorbing pigments. green fluorescent protein (gfp) was the first fluorescent protein to be discovered in jelly fish (aequorea victoria). wild type gfp (wtgfp) is a 26.9 kda protein with a major and a minor excitation peak (395 and 475 nm, respectively) and a single emission peak at 509 nm. the chromophore of this fp is formed by three amino acid residues consisting of ser65-tyr66-gly67 and held by a single αhelix surrounded by 11 β-barrel sheets to prevent its quenching by water [8] . several modifications have been made to wtgfp to enhance its fluorescence intensity as well as stability by minimizing photobleaching. in the year 1995, heim et al. [9] introduced a single point mutation at the s65t residue that resulted in enhanced fluorescence at the same emission spectra but with a shift in excitation peak from 395 nm to 488 nm. the mutation also significantly reduced the time required for formation of the fluorophore from 2 hours to 0.45 hours. several additional mutations in the protein helped the molecule to fold optimally at 37°c while the protein expression was improved by codon optimization of the wtgfp according to the host organism [10] . additional mutants were generated replacing the serine65 with threonine, alanine, glycine, cysteine or leucine leading to a protein with a single absorbance peak at~489 nm [9] [10] [11] [12] . expression of gfp at high concentrations in the cells posed the problem of dimerization which was resolved by side directed mutations at a206k, l221k or f223r residues [13] . the enhanced gfp (egfp) was developed with the modification and mutations described above at various positions of wtgfp. proteins tagged with enhanced gfp can be visualized in cells with low light intensities causing less photobleaching to enable imaging and quantification of intracellular proteins and its pathways effectively [14] [15] [16] . to tackle the problem of mixed chromophores in live cell imaging experiments, several mutants of wtgfp including enhanced gfp (egfp) and emrald gfp were generated which retained its fluorescent properties but with non-overlapping spectral properties [17] . neutral chromophore gfp has also been generated with a shift in the excitation and emission wavelengths to uv and green light, respectively [12, 18] . the proteins were again mutated to create sapphire which displays increased emission at 37°c [10] . the new mutant of sapphire called turbo sapphire has better ph-stability with uv excitation and thus has a larger separation between the excitation and emission wavelengths. table 1 summarizes the development of different variants of fps and their physical properties. several fluorescent proteins from different species have been discovered with various excitations and emission spectra ranging from ultraviolet to far-red or infrared [10] . these have been used in several cells and organelles, and some of them are discussed in this review. development of different variants of gfp with different absorption and emission spectra provided the opportunity for simultaneous multi-color staining of the cells. early variants included the blue fluorescent protein (bfp), developed by introducing a point mutation (y66h) to shift the absorbance and emission spectra to 384 nm and 448 nm, respectively, and cyan fluorescent protein (cfp) which was discovered from mutants of a.victoria [17] . since the spectra of bfp and egfp are distinguishable, a combination of these two fps was first used for multicolor imaging in cells and fluorescent (förster) resonance energy transfer (fret) analysis [9, 31, 32] . however, a major drawback of bfp was its low intensity and faster photobleaching [33] . therefore, enhanced bfp (ebfp) was developed by using codon optimisation for human cells [34] . notably, azurite, an ebfp, was developed by the dauherty's group and was reported to be 40-fold more photostable and brighter than the conventional bfp. ebfp was generated by incorporation of two additional point mutations (f64l and s65t) in the bfp and was considered to be the brightest bfp for the time until further improvements were made [24] . subsequently, ebfp 1.2 was developed based on mutations at s30r, y39n, t65s, s72a, n105t, i171v, n198s and a206v in ebfp and was 4-fold brighter than ebfp [23] . further modifications of ebfp 1.2 led to the development of ebfp 1.5 which was more photostable than its predecessor ebfp 1.2 but with no increase in fluorescence. azurite, as previously mentioned, had increased photostability as compared to ebfp 1.2 and ebfp 1.5 due to the mutations at v150i and v224r, respectively. however, this caused a decrease in the fluorescence by 30% [24] . screening of a library of mutants generated by random mutagenesis identified ebfp2 with enhanced fluorescence and photostability when compared to ebfp and azurite. ebfp2 was reported to be 4fold brighter and 550-fold more photostable than ebfp and 1.4-fold brighter and 2.9-fold more photostable than azurite [23] . however, both ebfp2 and azurite tend to dimerize when expressed in cells at higher concentrations. this was overcome by replacing the alanine residue at position 206 by valine [35] . bfp was further modified to develop a stable red fluorescent protein (rfp). site directed and random mutagenesis of rfp generated mtagbfp with a shift in excitation spectrum and blue emission [36] . although mtagbfp is the brightest among all the bfps produced, it is 1.4-fold less photostable than ebfp2 [23] . therefore, mtagbfp was mutated at i174a to give rise to mtagbfp2, i.e. a 1.5 fold more photostable bfp with a similar spectral profile and maturation half time as that of the parent protein [37] . cfp and yellow fluorescent protein (yfp) were developed as a multi-color pair. cfp was also brighter and more photostable when compared to bfp [9] and was created by a point mutation (y66w) with a spectra intermediate between bfp and egfp. further improvements were also made to increase the stability and intensity of cfp [18, 32] . mutation at t203 position of wild type gfp with an aromatic amino acid resulted in shifting of excitation and emission over 20 nm into yellow wavelength. this led to the development of enhanced yellow fp (eyfp). although eyfp is a dimer and sensitive to chlorine, three variants of eyfp were developed namely citrine (monomer), venus -a fast maturing fp at 37°c and the yellow fluorescent protein for energy transfer (ypet)used in fret along with cyan fps [29, 30, 38] . red fluorescent protein (rfp) was first isolated from an anthozoan, diascoma, with excitation and emission of 558 and 583 nm, respectively [39] . the major advantage of rfps over gfps is their longer excitation wavelength causing less damage to the cells and much less auto fluorescence in cells at the red spectrum. also the maturation of fps obtained from anthozoan is more efficient as compared to that of jelly fish fps at 37°c. native rfp has the disadvantage of forming tetramers which at times tends to aggregate in the cells and also cause false oligomerization of target proteins to hinder their native function [40] . dsred, the common rfp available, has prolonged maturation time often taking days to turn red from a greenish complex; this feature has been used to observe aggregate formation in target cells [41] . monomeric rfps have also been developed from dsred through a series of mutations, and the resultant proteins exhibited different maturation rate as well as fluorescent properties, i.e. morange, mko, mstrawberry, mcherry, tag rfp, mplum, mkate and tdtomato [42] . due to its fast maturation rate, high photostability and wide ph tolerance mcherry is a widely used long wavelength fp. however, it tends to dimerize that limits its experimental use. later the protein was modified to prevent its dimerization in the cells. it is considered to be the brightest rfp with enhanced photostability, and it has only a 3 nm longer excitation and emission wavelengths compared to its predecessors [43] . similarly, tag rfp was developed from wtrfp isolated from sea anemone entacmaea quadricolo by introducing mutations at five different sites (r162e, q166d, s180n, f198v and f200y) [44] . as of today, live cell imaging has advanced mainly due to the advent of fps tagged biomolecules with specific functions in the cell. fps have successfully been cloned into bacteria to mammalian cell lines either alone as a marker or along with a gene of interest as a fusion protein [45, 46] . several organic dyes have been tagged to cell organelles to visualize the dynamics under a microscope, however, not all dyes are readily accepted by the cell organelles and in such conditions fp tagged proteins are used to target the organelle. multi-color live cell imaging is an effort to observe multiple organelles, their spatial organisation, interactions between organelles and their constituent components. therefore, the successful application of the technique demands fps and dyes with non-overlapping emission spectra [47] . in recent developments organelle staining dyes such as hoechst 33342, mitored, dioc 6 , syto 9 and rhodamine b were tested along with several other dyes for imaging of various organelles in live cells and for the study of host pathogen interactions [48] . in this regard, lipid droplet staining has been done since a long time but only with organic dyes such as oil red, and none of the fps mentioned earlier have been used for successful imaging of lipid droplets. the different fluorescent proteins widely used for staining of different organelles of a live cell are listed in table 2 . fluorogen activating proteins (fap) are proteins/peptides such as bovine serum albumin and igg that can bind and activate the fluorescence of target fluorogen molecules and may also alter their spectral properties. conjugation of 9-(2-carboxy-2-cyanoviny1)julolidine (ccvj) fluorogen to either bsa and/or igg not only improved its solubility but also the fluorescence intensity by 6.3-fold when compared to the unconjugated molecule [49] . the use of larger fps such as gfp in reporter assays hinders the function of the fusion proteins in the cells. therefore, small tag systems such as tetracysteine motif (ccxxcc) were genetically inserted into the target proteins that can bind with high specificity to biarsenical dyes, resorufin arsenical helix binder (reash) and fiash (fluorescein arsenical helix binder) in live cells and lead to an enhanced fluorescent signal [50] . of note, flash, a derivative of 4' , 5'-bis (acetoxymercuri) fluorescein, is a cell permeable non-toxic ligand that binds specifically to four cysteine residues (i, i + 1, i + 4 and i + 5) of the target proteins α helical structure to emit fluorescence [51] . out of the fourteen different flash ligands screened, flash-edt2 showed strong affinity for hexamer peptides and positive emission at 635 nm. fret studies in hela cells with fiash-edt2 genetically fused to the cooh-terminus of an enhanced cyan fluorescent protein (ecfp-fiash) evidenced a 3-fold increase in fluorescence at 635 nm when compared to the ecfp alone transfected cells [32, 51, 52] . however, emission of ecfp at 480 nm declined to about 30% in fiash-edt2 transfected cells due to fret interactions. reash-edt2, a resorufin based fluorescent complex with excitation and emission maxima of 593 nm and 608 nm, respectively, was successfully tested for fret studies with gfp and yfp probe pairs in hela cells [53] . similar protein-ligand complexes were synthesized and used namely crash-edt2, sflash-edt2, f2flash-edt2, choxash-edt2, splash-edt2-alexa594, cag flash-edt2 and ascy3-edt2. several modifications have been carried out to improve the quantum yield, tetracysteine binding affinity and to reduce the cytotoxicity of these complexes. specifically, malachite green (mg) and thiazole orange (to) derived intercalating faps were developed which bind to rna aptamer and dna, respectively, and lead to enhanced (2,360-and 550-fold, respectively) and bleaching stable fluorescent signal from the fluorogens [54] [55] [56] . several unique membrane permeant and impermeant faps were developed by screening a library of human single-chain antibodies (scfvs) using derivatives of thiazole orange and malachite green. the screened clones have comparatively smaller size (the smallest being 110 amino acids long that is almost half the size of gfp) and thousands-fold higher brightness as compared to the typical fps. moreover, different spectral variants of mg and to could be easily generated by different combination of the screened fluorogens and faps. the fluorescence and differential interference contrast imaging microscopy was performed in nih3t3 cells with the membrane impermeable (mg-11p) and membrane permeable (mg-ester) forms of mg fluorogens and fap hl4-mg scfv tagged to membrane protein plateletderived growth factor receptor (pdgfr). the mg-ester signal was detected from the plasma membrane while mg-11p emitting fluorescence inside the cells suggested a localization near endoplasmic reticulum [54] . figure 1 illustrates the uses of membrane permeable and impermeable mg fap fluorogens and their application with a fluorescent tagged protein to study trafficking of a transmembrane protein. additionally, these faps were also tagged along with gfp and other fluorescent proteins to study plasma membrane and transporter proteins such as the insulin regulated glucose transporter (glut4), β2 adrenergic receptor (β2ar) and cystic fibrosis transmembrane conductance regulator (cftr). these genes were designed such that the fap and gfp were present as intracellular and extracellular reporters and the fluorogens, being membrane permeable and impermeable, were used to observe the mobility and placement of the target proteins tested in nih3t3, c2c12 and hek293 cells [57] . faps have been tested and proved to be reliable fluorescent markers for detecting and imaging various activities in a cell like protein motility studies and even for transmembrane protein activity. faps provide wide scope in developing alternatives to traditional fluorescent proteins where size hinders the activity of the target proteins. bacterial transformations have led to the expression of various genes in the prokaryotic system with certain [45, 46] limitations as compared to the eukaryotic cells. however, the transfer of genes in the mammalian cells requires much more sensitive approaches, and several methods have been developed for effective transfer and stable expression of genes in these cells. with the advent of viral vectors much progress has been made in the field of gene transfer. the use of cell type specific promoters allows greater control over the expression of foreign genes. liver cell specific promoters such as glycerol-3-phosphate acyltransferase (gpat i and gpat ii) and phosphoglycerate kinase 1 (pgk-1), albumin, human alpha1-antitrypsin and hemopexin are shown to be active in hepatocytes of human and mice origin [7, 58, 59] . similarly, viral promoters and vectors have been tested in different cell lines for their specificity and stability of expression. pcns and pcns-d2 vectors were reported to express cloned genes in cell lines and can also act as a shuttle vector between bacteria and mammalian cell lines to facilitate an easy cloning process. this vector system has been tested for stable expression of luciferase in hepg2, hep3b, hela, snu638 and snu668 cell lines. the luciferase expression was higher in hepg2 and snu638 cells as compared to other cell lines. cytomegalovirus (cmv) and simian virus 40 are known to infect human and other primate cells and have been shown to induce a stable expression of transgene in various cell lines [60] . during the study of cmv promoter systems, a 5' untranslated region (5' utr) was identified which was shown to boost the expression of viral proteins facilitating the rapid propagation of viral particles in the host cell. incorporation of utr regions in the promoter of viral vectors boosted the expression of genes of interest in host cells such as cho or hepg2. notably, the use of two copies of the utr regions further improved the expression of target genes in actively dividing cells [61] . later, the utr regions were found to be the internal ribosome entry sequence (ires) being responsible for uninterrupted expression of genes. ires were first discovered in rna of poliovirus in 1988 by pelletier and sonenberg [61] and facilitate the viral replication machinery by providing means for viral rna to bind with the 40s subunit of the host ribosomal complex thus eliminating the need for eukaryotic translation factors [62, 63] . since then, several viral vectors have been developed including the recently explored corona virus of the sars virus family. this rna based vector provides a stable and prolonged expression for more than 30 passages in different cell types [62] . insertion of ires site in expression vectors also offers the possibility of separate expression of the cloned genes under a single promoter ( figure 2 ) [61] . table 3 summarizes the list of various promoters and plasmids that have been used extensively in mammalian cell lines. among all promoters used cmv and sv 40 are most efficient in a wide range of cell lines. apart from these cell specific promoters such as pgk1 and gpat enhance expression levels in hepatoma cell lines. a variety of methods have been developed for a robust and efficient gene transfer without causing toxicity to the host cell. earlier physical methods such as electroporation and microinjections [64, 65] were primarily used for gene transfer studies but have been sidelined gradually due to the obvious disadvantages of being time consuming and the associated technical difficulties. more advanced methods have come into place for gene transfer according to the need for expression as a plasmid entity or as an integrated system in genome which also can be used for gene knockouts. non-pathogenic viral vectors are commonly used for gene transfer studies. baculovirus of the autographa californica multicapsid nucleopolyhedrovirus (acmnpv) and bombyx mori nucleopolyhedrovirus (bmnpv) genus provide a safe and efficient means of gene delivery to target cells [66] . baculoviral vectors have been modified to infect mammalian cell lines either as the whole baculovirus or as a helper cell mediated gene delivery in cells. adenovirus terminal repeats were used for the replication of the target gene, and the whole plasmid was packed into the baculoviral vector bac-b4 for transfer in cell lines such as 293b5b1. the transfected cells showed not only stable expression but also a 100-fold increase in the vector titer during subsequent passages [67] . of note, dna fragments of approximately 38 kb were efficiently transferred into different cell lines using such a vector. direct incubation of oligodendrite and chicken muscle cells with baculoviral vector showed a transfection efficiency of 60% [68] . further increase in transfection and expression efficiency was achieved by additional modifications in baculoviral vectors such as an introduction of the metallothione promoter (bac-me). the bac-me vector showed a transfection efficiency of 90% with low leaky expression and reduced cytotoxicity in hela cells. the expression efficiency of these vectors was further enhanced by treating the transfected cells with znso 4 [69] . although the baculoviral vectors were successfully used for gene transfer experiments in several mammalian cell lines they were replaced by other viral vectors and methods due to the associated cytotoxicity specifically at higher cell to virus ratio. hence, non-viral vectors were developed for transfection of viral sensitive mammalian cell lines [70] . bacterial based transfection has also been employed by cloning sequences belonging to the pathogenic bacteria e.coli. the modified genes from pathogenic bacteria like listeria monocytogenes have also been used where the bacteria with its intracytoplasmic capabilities propagate into the host. likewise, e.coli was modified so that selflysis genes become activated as soon as the bacteria enter the host cell to release the vector into host cytoplasm [70] . other bacterial methods involve t4ss, a bartonella sp., for the introduction of single strand dna into the host cell. thus, after entry the bacteria releases ssdna into cytosol and along with the enzyme relaxase or integrase, which is bound to target dna, starts to replicate to finally produce plasmid dna as a whole by joining the loose ends present [70] . this mode of transfer is considered to be hazard free and is employed for a variety of cell lines. further methods of nonviral based gene transfer have been developed for higher efficiency and less toxicity to the cells. as mentioned earlier for efficient transfection the gene has to be integrated into the genome (constitutively) or is continuously expressed in the presence of a specific inducer reagent for the chosen promoter (inducible). owing to variable results the need remains to develop efficient methods as has been attempted in the form of a dual plasmid approach using the mice myogenic c2c12 cell line. according to this method t-rex plasmid system is first transfected into the cell line using a lipid based method with an efficiency of 70% and is activated by tetracycline. thereafter, a modified t-rex plasmid became available. after 18 days of initial transfection a second plasmid can be introduced with 40% efficiency that permits the study of complex biochemical reactions involving different factors [71] . in table 4 the various transfection efficiencies of different methods are summarized. bactofection seems to be an effective mode of transfection in cells without the risk of cytotoxicity as compared to some viral methods but much improvement has to be made for application of bactofection for a wide variety of cell lines. lipofection involves formation of a positively charged lipid-nucleic acid complex by suspending negatively charged dna/rna molecules with cationic lipids for a short duration of time. the positive charge on the complex facilitates attachment to the cell membrane and entry via endocytosis [70] . a comparative study concluded that lipofection is the safest method to transfer puast sv 40 expressed genes in yeast [60] genes into in mammalian cells amongst the various transfer methods [70] . different commercially available kits with a broad range of efficiency on different cell lines are available. the transfection efficiency of methods varies depending upon the cell type, cell number and ratio of dna and reagent used. using lipofection and as determined with a gfp marker an efficiency of about 60% was obtained in cho-k1 cell lines [72] . the major challenge faced by most of the non-viral delivery systems is the degradation of the transfected nucleic acid in the endosomes and/or lysosomes as they are mainly internalized by endocytosis. lysosomotropic agents like chloroquine and polyvinylpyrolidone (pvp) have been used effectively to increase the transfection efficiency of different delivery methods by reducing the lysosomic degradation of the transfected nucleic acid [73, [79] [80] [81] [82] [83] . however, the cytotoxicity of these agents raises concerns against their use. chloroquine is known to increase the intracellular ph leading to cytotoxicity and degradation of transfected dna at higher concentrations and at prolonged exposure times (>4 hours). amongst the two variants of pvp, pvp10 was reported to be cytotoxic at all concentrations while pvp40 showed negligible cell toxicity and even improved the transfection efficiency. pvp40 has been used for transfection studies in macrophages and hepatocytes [84] . sucrose is also known to cause intracellular swelling of vesicles in endosome and lysosomes due to osmotic pressure [85, 86] . sucrose in conjunction with lipid-dna complex or lipofection reagents was shown to increase their transfection efficiency in fibroblast cell. furthermore, no toxicity has been reported using sucrose as lysosomotropic agent and a concentration range of 5 to 500 mm was safe in various cell lines such as cho, cos7 and hek 293 t cells. beneficial effects of these lysosomotropic agents were compared against lipofectamine 2000 in cos7, cho and hek 293 cell lines. essentially, the transfection efficiency of lipofectamine was increased by 6-and 3-fold when used in conjunction with pv40 and sucrose, respectively. chloroquine, although being toxic showed an increase of 3to 6-fold when used at a concentration range of 0.01 to 0.1 mm as compared to lipofectamine alone [87] . poly(ethyleneimine) is another cationic polymer transfection reagent frequently used in a variety of cell lines. however high cytotoxicity is the major drawback associated with pei and therefore several changes in its molecular structure have been done to reduce its cytotoxicity and this includes pegylation and the introduction of carbohydrate, lipid and peptide moieties [75, [88] [89] [90] . a recent study showed the improved transfection efficiency and low cytotoxicity of an anionic glycopolymer derivative of pei in hepg2 and hek 293 t cells [91] . modified lipids were developed to enhance the transfection efficiency of vectors. specifically, cationic oligopeptide lipids are charged lipids with a linker molecule to provide cations and are said to be more efficient in the presence of the serum containing medium [74] . different ratios of nucleic acid and lipids were tested ranging from 1:1 to 9:1 and the ratio of 3:1 was found to be optimum in terms of cell viability and transfection efficiency [74] . other synthetic vectors have been tested for gene transfer in various cell lines. polyspermine imidazole-4, 5amide complex (psia) was employed with hepg2 as well as cos 7 cell lines for gene targeted therapy. psia transfection efficiency is of only 20-30% in hepg2. apart from the risk of psia degradation that hinders complex formation with plasmid dna the process of psia synthesis requires strict environmental (ph and temperature) conditions before dna can be introduced [92] . to improve transfection efficiency in various hepatoma cell lines like huh-7, hepg2 and hep3b different strategies were employed including lipofection, electroporation and microinjection. microinjection (100 μg/ml) and cationic lipids 47-60% 45-55% 65% 60% [73, 74] non-viral ---6 0 % [ 71, 75] e.coli (modified salmonells/listeris sp.) t4ss (bartonella sp.) 60-70% 50% >70% >60% 50-60% [76] [77] [78] lipofection (1 μg) methods were used for transfection of plasmid encoding fluorescent protein (pegfp and peyfp) tagged to human and mouse adrp genes in huh-7 cells as to study the pathways of lipid droplet metabolism. a transfection efficiency of 66% was achieved, and none of the methods affected the expression or the interaction of proteins in cells proving their reliability and safety. however, the transfection efficiency of these methods varied inversely with the size of the plasmid [93] . in this regard, adenoviral vectors have been employed in hepg2 cells to study the function of genes such as the cytochrome p450 monooxygenases in drug induced hepatotoxicity. notably, cyp1a2, cyp2d6, cyp2c9, cyp2c19 and cyp3a4 were transfected in hepg2 cells using recombinant adenoviral vector with a transfection efficiency of approximately 70% [94, 95] . in most cases transfection or transduction of primary cells proof to be difficult for several reasons like the stable state of non-dividing or quiescent cells. however, some viral vector-based methods have been used successfully such as the murine retroviral vector that can transfect primary hepatocytes stimulated by mitogens (epidermal or hepatocyte or keratinocyte growth factors). moreover, the associated high costs and varying efficiency of the growth factors make these transfection methods unfavorable [96] [97] [98] [99] . lentiviral vectors are capable of transfecting quiescent cells [100] [101] [102] and have been successfully used for gene transfer experiments in various hepatoma cell lines [76, 103, 104] . although the discussed methods and markers are compatible with various if not all cell lines, the cell lines chosen have to be noted to select the right kind of vector and promoter system. an overview of the various transfection methods in different cell lines is given in figure 2 . research in recent years significantly advanced an understanding of the molecular causes of fatty liver disease, and live cell imaging of hepatoma cells proved to be extremely valuable for studying lipid droplet formation. lipids are primarily stored in adipocytes as lipid droplets; however, under stressed conditions hepatocytes can also produce lipid droplets leading to pathological conditions like fatty liver disease [105, 106] . lipid droplet staining requires cell permeable dyes/fluorochromes which can bind specifically to the components of the lipid monolayer of droplets. the most commonly used dyes for lipid staining are oil red o, nile red and bod-ipy with an excitation-emission spectrum in the range of 400-500 nm and 500-600 nm, respectively. figure 3 depicts oil red o staining of lipid droplets in cultured human hepatocytes treated with palmitic acid (pa) and oleic acid (oa) and the cardiovascular drug amiodarone which is known to cause steatosis and steatohepatitis in patients. fps and dyes with non-overlapping fluorescence spectrum are required for differential staining of multiple biological molecules involved in lipid droplet metabolism. to address this issue, a long wavelength fp and a short range dye monodansyl pentane (mdh) was developed for lipid droplet staining with excitation and emission in the range of 405 and 480 nm [106] . such an approach allowed the study of perlipin 2 interactions on the lipid droplet as defined by the mdh stain. notably, in the early works of niemann et al. (2001) the use of mdh as a mean to study lipid droplets was reported [107] . mdh was also found to be photostable when compared to nile red and bodipy with a light emission of more than 15 minutes wherein the latter dyes were stable up to 10 minutes only [106] . mdh has a potential use for lipid droplet staining to facilitate an understanding of the formation of lipid droplets and their interaction with various proteins within hepatocytes. the lipid droplet monolayer has many associated proteins namely adipocyte differentiation-related proteins (adrp also known as perilipin 2/plin2) and perilipins (plins) which interact with other organelles in the cell. simultaneous detection of lipid droplets and adrp was achieved by transfecting huh-7 cells with adrp-gfp encoding plasmid and staining of lipid droplets with oil red o. although this method was useful in observing the cytoplasmic interaction of adrp/plin2 with lipid droplets the precise localization of gfp-adrp/plin2 at the surface of lipid droplet was distinctly observed only after incubating the cells with bodipy558/568 dodecanoic acid, a fatty acid sequestered in lipid droplets as evidenced by imaging by confocal microscopy in live and fixed cells conditions [93] . while this study was one of the first reports on lipid-protein interactions investigated by live cell imaging the use of bodipy hindered clear imaging due to background signals. to overcome these obstacles fret and frap methods were used as a means to study these complexes. moreover, mdh stain provides an efficient stain for lipid droplets which allows proteins to interact with the droplets to be tagged with longer wavelength fluorescent proteins and also mdh provides a more stable and robust stain for lipid droplets as compared to the traditionally used oil red o and nile red stains. fret is an approach to study protein-protein interaction and co-localization of organelles on the principle of energy transfer between two chromophores situated in close proximity (6-10 nm). the pair of fluorescent probes used has an overlapping spectral profile, and the emission wavelength of the donor probe (gfp) provides the required energy for the excitation of the acceptor probe (cfp). acceptor bleaching can be used to demonstrate whether the organelles are within the fret-distance (i.e. 6-10 nm), thus demonstrating true association on a molecular scale. fret was used to observe motility of certain proteins as well as organelles [108] . notably, laser scanning confocal microscopy based fret assays have been employed to study the co-localization of lipid droplets and mitochondria in porcine oocytes using the mitotracker green (mtg) and nile red (nr) fluorochromes. fret acceptor bleaching methods have also been used to examine mitochondria-lipid droplet co-localization [109] . to study protein-lipid interactions on the surface of the lipid droplets, perilipin 2 was tagged with cfp and followed by cyan fluorescence. since it forms a strong fret interaction with the lipid stain 22-(n-(7-nitrobenz-2-oxa-1, 3-diazo) aminostearic acid (nbd), the colocalized protein-lipid gave a signal of yellow-to-orange. the mouse perilipin 2 gene was cloned into the mammalian expression vector pecfpn1 and was stably expressed in mouse l fibroblast cells. subsequently, nbd was used to stain phosphatidyl choline to form nbd-pc complex, and a confocal microscopy based fret assay was employed to determine the interaction between lipids and proteins in the cell. similarly, co-localization of perilipin 2 and sphingomyelin (sm) near plasma membrane was demonstrated using nbd-sm and plin2-cfp vector constructs [110] . although cfp and yfp are most widely used probes yfp has the disadvantage of having low quantum yield and poor signal to noise ratio hampering the process of individual cfp intensity measurement. to counter this problem, two ecfp molecules were fused; however, the result was a further decrease in the intensity due to multiple excited states causing interconversion of excited states by homotransfer. notably, multiple excited states are due to two different crystal structure conformations of ecfp [111] . this problem was resolved by site directed mutagenesis of the his148 residue to aspartate, and the fluorescence intensity of the mutant was further increased by a factor of 2.5 as compared to ecfp. further mutations at s72a and y145a residues boosted the extinction coefficient of the monomer but caused a decrease in the quantum yield. the modified ecfp is known as cerulean and is proved to be a better fret donor than the conventional ecfp [111] . figure 4 depicts a basic understanding on how fret analysis can be done using ecfp and eyfp particularly for lipid droplet studies. fluorescent recovery after photobleaching (frap) is used to study single membrane bound structures, single cells and protein-lipid interactions. frap uses the concept of activating a fluorescent protein observing its intensity and subsequent bleaching by excess light to finally measure the bleached region for binding activity to the surface of the secondary structure or membrane. cfp and yfp have been commonly used to study the interactions of perilipin 1 (plin1) and perilipin 5 (plin5) with adipocyte triglyceride lipase (atgl) and its corresponding activator α-β-hydrolase domain-containing 5 (abhd5). on the basis of frap studies in cos 7 cells, it was concluded that plin5 interacts with atgl but not plin1 [27] . adrp/plin2 is shown to be located at the surface of cytoplasmic lipid droplets [112] , and localization studies for dna fragments encoding human and mouse adrp were ligated in-frame to the 3' end of egfp, thereby generating gfp-hadrp (expressed by plasmid pla4) and gfp-madrp fusion products. to examine whether the gfp-adrp products for both species retained the capacity to associate with these structures, constructs expressing the fusion proteins were transfected in huh-7 cells. after stimulation with fatty acid, the formation of lipid droplets in huh-7 cells can be identified by staining with lipophilic dyes and by antisera raised against adrp/ plin2. the signal from gfp-madrp was detected from the surface of spherical intracellular structures while egfp was seen throughout the huh-7 cells. the localization of endogenous hadrp and the gfp-madrp products coincided, indicating that gfp-madrp was located at the surface of lipid droplets therefore demonstrating that it is possible to detect tagged and untagged adrp on the same lipid droplet. to verify the localization of gfp-madrp, cells expressing the fusion protein were incubated with bodipy 558/568 dodecanoic acid, a fluorescent fatty acid analogue that is sequestered in lipid droplets. the dye was selected because of its spectral properties which allow differentiation from egfp and a better cell permeability as compared to oil red o. results in both live cells and paraformaldehyde fixed cells showed localization of gfp-madrp at the surface of lipid droplets stained with the bodipy dye. attempts to fuse egfp to the c terminus of adrp/plin2 yielded few cells that produced fluorescence of the chimeric protein but in this case the signal did not derive from the surface of lipid droplet. consequently, egfp fused to the n terminus of human and mouse forms of adrp/plin2 was used in further studies [93] . it is well recognized that caveolins are found on lipid droplets, but the functional significance of this association is poorly understood. adenovirus mediated transfer of cav1-gfp in nih3t3 cells demonstrated that caveolin-1-coated lipid droplets can grow larger than caveolin-1 devoid lipid bodies suggesting the importance of caveolins in determining the size of lipid droplets. this study provided the first detailed characterization of the impact of caveolins on molecular composition and the size of lipid droplets [113] . the most frequently employed tools (elisa, western blot, fret, frap co-immunoprecipitation) to study protein expression or protein-protein interactions rely on the use of antibodies tagged with either fluorophores or enzymes. the sensitivity of these techniques differs significantly, and the detection of proteins at low expression levels still remains challenging. the proximity ligation assay is a relatively inexpensive quantitative technique with high sensitivity. the technique is developed on the principles of antibody targeted assay, split reporter assay and polymerase based amplification of oligonucleotides. in this assay, the target proteins are recognized by specific antibodies (two or more) conjugated with short oligonucleotide strand. in case these probes are in near figure 4 fret studies to determine interaction between plin5 and the atgl protein on lipid droplet in adipocytes [39] . plin5 (1) and atgl (2) proteins were tagged with fret probes cfp (3) and yfp (4), respectively. the emission spectra of cfp act as excitation wavelength for the yfp. detection of yellow fluorescence after activation of cfp (436 nm) suggest possible interaction of the two tagged proteins. proximity, the oligonucleotides can be ligated by addition of complementary oligonucleotide and ligase enzyme and subsequently amplified using pcr or rolling circle amplification. this leads to the formation of more than 1000 copies of the complement of 100 bp in one hour using the phi29 polymerase [114] . the incorporation of multiple fluorescently labeled oligonucleotides in the polymerase catalyzed reaction causes amplification of the signal resulting in high sensitivity. apart from protein expression and proteinprotein interaction, this highly versatile technique can also be adapted to detect post-translational modifications, colocalization and interaction of proteins with other biomolecules ( figure 5 ). pla was recently used to demonstrate that the myc inhibitor 10058-f4 could prevent mycn/max interaction in situ and caused accumulation of lipid droplets in tumor cells [115] . the availability of a wide range of fluorescent proteins and organelle specific stains provides unprecedented opportunities for microscopic studies of diverse cellular processes including protein-protein interactions. however, for the visualization of certain cell components in live cells such as lipid droplets options are currently limited to organic dyes, i.e. bodipy, nile red and oil red o. the emission spectrum of these dyes overlaps with that of gfp and rfp; thus, it is not feasible to use them in combination for multi-color imaging. there is unmet need for the development of a new lipophilic dye which can be spectrally resolved from commonly used fluorophores. monodansylpentane, a cadaverin family dye with blue emission spectra, is the appropriate substitute for the red dyes for lipid staining. furthermore, this can be used along with far wavelength fluorescent proteins to obtain better insight into the lipid metabolism. discovery of novel proteins involved in the lipid droplet metabolism and their fusion with fluorescent proteins that can be spectrally resolved will help researchers to delineate the process of biogenesis of lipid droplets under live cell conditions. the advancements in culture systems to ensure long term metabolic activity of hepatocytes, the development of suitable transfection methods and reporter assays will be instrumental for the success of live cell imaging experiments. (2) formation of covalently joined circular oligonucleotide by ligase reaction: if the two probes are in close proximity, addition of two linear oligonucleotide leads to the formation of a covalently joined circular oligonucleotide molecule with the help of a ligase enzyme. (3) amplification via rolling circle mechanism: one of the probes linked to antibody act as a primer and addition of dna polymerase yields long single stranded concatemeric dna molecule composed of complements of the circular dna strands formed in the ligase reaction. (4) fluorophore labeling for detection: the amplified product is easily detected by addition of complementary oligonucleotides labeled with fluorophore. decision to publish, or preparation of the manuscript. we acknowledge support by deutsche forschungsgemeinschaft; the charge for this publication was covered by the dfg sponsorship "open access publication". optical imaging of renilla luciferase reporter gene expression in living mice enhanced beetle luciferase for high-resolution bioluminescence imaging the discovery of aequorin and green fluorescent protein discovery of green fluorescent protein association between nonalcoholic fatty liver disease and risk for hepatocellular cancer, based on systematic review construction of multi-purpose vectors, pcns and pcns-d2, are suitable for collection and functional study of large-scale cdnas identification and functional characterization of human glycerol-3-phosphate acyltransferase 1 gene promoters crystal structure of the aequorea victoria 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poly(l-lysine) and poly(ethylene glycol) for gene delivery cell line dependent uptake and transfection efficiencies of pei-anionic glycopolymer systems synthetic polyspermine imidazole-4, 5-amide as an efficient and cytotoxicity-free gene delivery system live cell analysis and targeting of the lipid dropletbinding adipocyte differentiation-related protein upgrading cytochrome p450 activity in hepg2 cells co-transfected with adenoviral vectors for drug hepatotoxicity assessment hepg2 cells simultaneously expressing five p450 enzymes for the screening of hepatotoxicity: identification of bioactivable drugs and the potential mechanism of toxicity involved retrovirus-mediated transduction of adult hepatocytes effects of keratinocyte and hepatocyte growth factor in vivo: implications for retrovirus-mediated gene transfer to liver hepatocyte growth factor induces hepatocyte proliferation in vivo and allows for efficient retroviral-mediated gene transfer in mice synergistic growth factors enhance rat liver proliferation and enable retroviral gene transfer via a peripheral vein development of lentiviral vectors for gene therapy for human diseases lentiviral vectors: turning a deadly foe into a therapeutic agent lentiviruses as gene transfer agents for delivery to non-dividing cells lentivirus-mediated overexpression of microrna-199a inhibits cell proliferation of human hepatocellular carcinoma cd81 is required for hepatitis c virus glycoprotein-mediated viral infection lipid droplets: a unified view of a dynamic organelle monodansylpentane as a bluefluorescent lipid-droplet marker for multi-color live-cell imaging fluorescence properties and staining behavior of monodansylpentane, a structural homologue of the lysosomotropic agent monodansylcadaverine jovin tm: fret imaging fluorescence resonance energy transfer analysis of mitochondrial:lipid association in the porcine oocyte direct interaction of plin2 with lipids on the surface of lipid droplets: a live cell fret analysis expansion of the genetic code enables design of a novel "gold" class of green fluorescent proteins adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases lipid droplet analysis in caveolin-deficient adipocytes: alterations in surface phospholipid composition and maturation defects proximity ligation assays: a recent addition to the proteomics toolbox myc inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells recent advances in live cell imaging of hepatoma cells we gratefully acknowledge support from the virtual liver network (grant 031 6154) of the german federal ministry of education and research (bmbf) to jb. the funders had no role in study design, data collection and analysis, the authors declare that they have no competing interests.authors' contributions jb initiated the review. all authors contributed to the writing and approved the final manuscript. key: cord-302514-rstvf3mc authors: abbas, wasim; kumar, amit; herbein, georges title: the eef1a proteins: at the crossroads of oncogenesis, apoptosis, and viral infections date: 2015-04-07 journal: front oncol doi: 10.3389/fonc.2015.00075 sha: doc_id: 302514 cord_uid: rstvf3mc eukaryotic translation elongation factors 1 alpha, eef1a1 and eef1a2, are not only translation factors but also pleiotropic proteins that are highly expressed in human tumors, including breast cancer, ovarian cancer, and lung cancer. eef1a1 modulates cytoskeleton, exhibits chaperone-like activity and also controls cell proliferation and cell death. in contrast, eef1a2 protein favors oncogenesis as shown by the fact that overexpression of eef1a2 leads to cellular transformation and gives rise to tumors in nude mice. the eef1a2 protein stimulates the phospholipid signaling and activates the akt-dependent cell migration and actin remodeling that ultimately favors tumorigenesis. in contrast, inactivation of eef1a proteins leads to immunodeficiency, neural and muscular defects, and favors apoptosis. finally, eef1a proteins interact with several viral proteins resulting in enhanced viral replication, decreased apoptosis, and increased cellular transformation. this review summarizes the recent findings on eef1a proteins indicating that eef1a proteins play a critical role in numerous human diseases through enhancement of oncogenesis, blockade of apoptosis, and increased viral pathogenesis. translation is the central event leading to protein synthesis and translation factors are key actors involved in the translation process (1) (2) (3) . recently, it has been suggested that translation factors could represent a new class of potential oncoproteins that could favor cellular transformation through protein translation infidelity, association with cytoskeleton alterations, and modulation of signaling pathways (3) (4) (5) . the eukaryotic translation elongation factors 1 alpha, eef1a1 and eef1a2, are the second most abundant protein (1-3% of total protein content) after actin and an important component of translation machinery. in its gtp bound form, eef1a1 and eef1a2 deliver the aminoacylated-trna to the a site of the ribosome for decoding of mrna by codon-anticodon interactions (6) . following the hydrolysis of gtp, eef1a (eef1a1 and eef1a2), gdp is released from the ribosome (6) . thus, eef1a1 and eef1a2 are gtp-binding proteins and consist of three domains namely domain i, domain ii, and domain iii. domain i spans over 1-240 residues, which made up of a rossmann-fold topology. domain ii (241-336 amino acids) and domain iii (residues 337-443) consist of beta strands and each domain contains two beta sheets that form the beta barrel (7-10) (figure 1) . there are two known isoforms of protein eef1a, i.e., eef1a1 and eef1a2. the cellular expression of eef1a is divided into three classes. first are the majority of cell types that express only eef1a1. second, neurons and muscle cells that express only eef1a2. third class belongs to certain tumor cell types and cell lines that express both eef1a isoforms (11) (12) (13) . eef1a1 protein has been mapped on chromosomes 6q14. eef1a1 protein shares homology with eef1a2 protein at the nucleotide level (75%) and amino acid level (96%). eef1a2 does not bind gdp and gtp with the same relative affinity as eef1a1. the gdp dissociation rate constant is seven times higher for eef1a1 than for eef1a2. in addition, the nucleotide preference ratio (gdp/gtp) for eef1a1 is 0.82 and for eef1a2 is 1.50 (14) (15) (16) (17) . furthermore, eef1a has been shown to be a novel component of the nuclear export machinery in mammalian cells and is involved in the nuclear export of specific proteins such as vhl van hippel-lindau (vhl) tumor suppressor and poly(a)-binding protein (pabp1) (18) . the eef1a2 gene is present in the common ancestor of eukaryotes (table 1; figure 2 ). human eef1a2 gene spans approximately 12 kb human genome sequence, which consists of eight exons and seven introns plus 2 kb upstream promoter region, and has been mapped on chromosome 20q13.3. analysis of the region −2064 to +220 reveals that the promoter region contains the binding sites for several important cis-regulatory elements (e-boxes, egr family proteins, gata motif, and mef binding site) with no tata elements (figure 3) . the core region of the promoter is mapped from position −16 to +92 (9, 19) . beyond its central role in translation machinery, eef1a2 plays an important role in cell cycle regulation, heat-shock response, aging, posttranslational modifications, and signal transduction (20) (21) (22) (23) . protein synthesis is one of the most sophisticated biochemical processes occurring in the cell, which requires hundred of proteins, including eef1a proteins, eef1a1 and eef1a2. besides the role of eef1a proteins in the translational process, an increasing series of data are presently emerging about the non-canonical roles of these proteins in oncogenesis, modulation of apoptosis and viral pathogenesis (1, 20) . breast cancer is the most common cancer in women both in the developing and developed world; there are an estimated 1 million new cases per year. the use of gene signature or the identification of changes in gene expression in breast tumors relative to normal surrounding tissue is of great importance in terms of prognostic indicators and therapeutic targets (24) . the eef1a2 is hardly detectable in normal human breast tissue but the expression of eef1a2 is strongly upregulated in most of breast tumors (25, 26) . high levels of eef1a2 proteins are detected in 60% of primary breast tumors and metastases, but not in normal epithelium ( table 2 ). the expression of eef1a2 is sufficient to stimulate the formation of filopodia in bt549 human breast cancer cells and non-transformed rat2 cells. moreover, its expression is sufficient to activate akt in a pi3k-dependent fashion, as down-regulation of eef1a2 by sirna reduces akt activity. in breast cancer cell line bt549, eef1a2 expression stimulates cell migration and invasion in a largely pi3k and akt-dependent manner, suggesting eef1a2 regulates oncogenesis through akt and pi3k-dependent cytoskeleton remodeling (27) (28) (29) . in fact, eef1a2 participates in the regulation of the phospholipids signaling pathway (figure 4) . phosphatidylinositols are negatively charged membrane bound phospholipids that participate in the pathways that regulate the cell proliferation, survival, cytoskeleton organization, vesicular trafficking, and oncogenesis (30, 31) . phosphoinositols are composed of an inositol ring in which one or more −oh groups at the 3-, 4-, and 5-position of inositol ring are esterified with a phosphate group in all possible combinations. specific kinase families (pi3k, pi4k, and pi5k) are responsible for phosphorylation at these sites (32, 33) . overexpression of eef1a2 protein up-regulates overall pi4k activity and cellular phosphatidylinositol 4-phosphate (pi4p) generation in human cells. furthermore, eef1a2 directly interacts with and activates phosphatidylinositol-4 kinase iii β (a subfamily of pi4k), an enzyme that converts phosphatidylinositol to pi4p. knockdown of eef1a2 using eef1a2 sirna results in downregulation of phosphatidylinositol-4-kinase activity indicating that eef1a2 is a physiological regulator of pi4kiiiβ signaling (34, 35) . in addition, eef1a2 expression up-regulates the generation of phosphatidylinositol-4,5-bisphosphate [pi(4,5)p2] in the cytoplasm and at the plasma membrane. the subsequent increase in pi(4,5)p2 at the plasma membrane stimulates the eef1a2-induced filopodia formation through binding and activation of pi4kiii β. therefore, eef1a2 is involved in phosphatidylinositol signaling and actin remodeling (34, 36) . moreover, the gene expression profiling of primary mouse b cell lineage showed the high expression of eef1a2 in plasmacytomas (pct), which results in progression frontiers in oncology | molecular and cellular oncology transcription start site +1 of plasma cell neoplasms in both mouse and human (29) . finally, the knockdown of eef1a2 expression delays or impairs the il-6induced activation of stat3 and akt signaling pathways suggesting that activation of stat3 and akt through eef1a2 involvement that favors cell proliferation, cell cycle progression, and inhibition of apoptosis (29, 37, 38) . altogether, eef1a2 protein activates the pik-akt-stat3 pathways that have been extensively shown to favor cellular transformation and oncogenesis (39) (40) (41) (42) (43) (44) (figure 4) . in breast cancer, the genes in 20q13 are frequently amplified and have been shown by using comparative genomic hybridization and fluorescence in situ hybridization (45) . the differential screening of cdna libraries from metastatic and non-metastatic cell lines derived from the same parental rat mammary adenocarcinoma showed a 1.5-fold overexpression of eef1a in the metastatic compared to the non-metastatic cells (46) . studying cancer cell lines derived from breast, lung, prostate, and skin, eef1a2 gene expression exhibited the highest alteration in the cancer cell lines compared to normal controls using a profiling array (47) (48) (49) (50) . ovarian cancer represents 4% of all female cancer. it has the highest fatality to case ratio of all gynecological cancers because the majority of cases are diagnosed in the late stage. despite of significant efforts to improve the early detection and advances www.frontiersin.org cell proliferation apoptosis inhibition cell survival jak/stat figure 4 | eef1a2 activates the phospholipid, jak/stat, and akt pathways. eef1a2 is a physiological regulator of phosphatidylinositol-4-kinase (pi4k). it directly interacts with phosphatidyloinositol-4 kinase iii β (pi4kiiiβ) and enhances its lipid kinase activity by converting the phosphatidylinositol (ptdins) into phosphatidylinositol-4-phosphate (ptdins4p). then pi4kiiiβ and ptdins4p increase the phosphatidylinositol-4,5 bisphosphate generation at the plasma membrane level, which results in the filopodia formation and actin remodeling. eef1a2 also directly or indirectly regulates the jak/stat and akt signaling pathways. in chemotherapy, metastasis remains a major challenge in clinical management of ovarian cancer (51) (52) (53) (54) . the eef1a2 gene is not expressed in normal ovary but highly expressed in ovarian cancer (55) . eef1a2 expression enhances the ovarian cell growth in vitro and favors the tumorsphere formation (56) , suggesting that eef1a2 could favor the development of ovarian primary tumor formation. high levels of eef1a2 expression was observed in 30% all the primary ovarian tumors, 50% of serous tumors, 30% of endometrioid tumors, 19% of mucinous tumors, and 8% of clear-cell tumors ( table 2 ) (56) . furthermore, the eef1a2 protein and rna expression levels are upregulated in clear-cell ovarian carcinoma by 75 and 33% respectively. the eef1a2 gene is unmethylated both in normal and tumor cells, suggesting that up-regulation of eef1a2 gene expression is not dependent on epigenetic modifications (at least for the methylation status), but instead that the inappropriate expression of trans-acting factor(s) could be involved (55) . the enhanced expression of eef1a2 protein in ovarian cancers correlates with poor prognosis (57) . the oncogenic properties of eef1a2 have also been studied in different ovarian tumors and established cell lines. in rodent fibroblasts, eef1a2 protein favors anchorage-independent growth and results in decreased doubling time during cellular proliferation. furthermore, the induced expression of eef1a2 in nih3t3 cells makes them tumorigenic and increases the growth rate of es-2 ovarian carcinoma cells xenografted in nude mice (40) . the transcription factor znf217 and eef1a2, both located on chromosome 20q13, are frequently amplified in ovarian epithelial carcinomas. the stable preneoplastic ovarian cell lines that over-express eef1a2 provides the evidence that up-regulation of eef1a2 expression contributes to the neoplastic properties of precursor cells of ovarian carcinomas mediated through znf217 (58) . resveratrol, a phytoalexin produced naturally by several fruit plants, has been extensively studied for its chemopreventive and chemotherapeutic effects in cancer and animal models. resveratrol blocks the angiogenesis, induces the autophagocytosis and apoptosis in proliferating cells, and is a well-known sirtuin 1 activator (59, 60) . the expression of eef1a2 is increased in the pa-1 ovarian cell line after serum or insulin stimulation. resveratrol up-regulates the caspase-3 level in pa-1 cells by down regulating the expression of eef1a2 via the blockade of upstream akt pathway. additionally, resveratrol suppresses growth of human ovarian cancer cells in culture and in a murine xenograft model with reduced expression of proliferating cell nuclear antigen and increased tunel staining (61) . all together resveratrol down-regulates eef1a2 in ovarian cancer cells and thereby favors apoptosis. lung cancer is the most common cause of cancer related death both in men and women. it accounts for 1.3 million deaths worldwide annually. in spite of continuous efforts and clinical frontiers in oncology | molecular and cellular oncology trials to develop new diagnostic and prognostic markers, a better understanding of the molecular pathways involved in lung cancer is essential for developing new therapeutic targets (62, 63) . four lung adenocarcinoma cell lines (hkulc 1-4) were established from patients with different clinical characteristics. comprehensive studies of these cell lines show that eef1a2 is a putative oncogene that is highly expressed in all the hkulc cell lines (64) . in addition, high-resolution analysis of genomic aberration by metaphase and comparative genomic hybridization array identify the involvement of the 20q region, suggesting the potential role of eef1a2 as a candidate tumor gene in lung cancer cell lines (41) . in another study, 183 genes with increment in both genomic copy number and transcript in six lung adenocarcinoma were analyzed. forty-two proteins were overexpressed in these cell lines as compared to the normal cells. comparing the 183 genes with the 42 proteins, the expression of four candidates, namely, prdx1, eef1a2, calr, and kcip-1 was correlated with increased dna copy number and transcript levels. furthermore, expression of sirna targeting eef1a2 and kcip-1 in these cell lines suppressed cellular proliferation and triggered apoptosis. therefore, the overexpression of eef1a2 and kcip-1 in lung tumor samples strongly suggests that both proteins could be involved in lung adenocarcinoma and could be potential therapeutic targets in lung cancer (42) . hepatocellular carcinoma (hcc) is one of the most common malignant tumors in humans (65, 66) . hcc cell lines, hepg2, huh7, and jhh6 show increased expression of eef1a2 as compare to normal liver tissue, but the mrna of eef1a2 is markedly increased only in jhh6 cells (43) . in addition, the mdm4 and eef1a2 proteins show increased expression in cryptogenic hcc (44) . eef1a2 gene silencing reduces cell viability, proliferation, and increases the apoptosis rates in hcc cell lines (44) . furthermore, eef1a2 is overexpressed in 43% of hcc and activates the akt pathway as observed in 40-60% of primary hccs ( table 2 ) (44, 67) . using array based comparative genomic hybridization, frequent amplification and gain of dna copy number at 1q, 6p, 8q, 17q, and 20q were observed in hccs, suggesting that these genetic aberrations might facilitate the malignant transformation (68) . pancreatic cancer is one of the unsolved health problems associated with aggressive malignancies and therefore there is need to develop improved early diagnosis tools (69, 70) . the expression of eef1a2 gene, located on chromosome 20q13 is significantly upregulated in pancreatic cancer. although little or no eef1a2 expression is present in normal pancreatic tissue, 83% of pancreatic cancers display increased expression of eef1a2, suggesting that eef1a2 plays an important role in pancreatic carcinogenesis (71) (72) (73) . cell proliferation and cell death are highly regulated and coordinated during the normal development, and both are crucial for the maintenance of tissue homeostasis. impaired apoptosis can lead to autoimmunity or malignancy (74, 75) . an increasing number of evidence suggests the involvement of eef1a2 at the onset of cell transformation. peroxiredoxin-1 (prx-1) is a protein that is ubiquitously expressed in all mammalian cells and protects the cells from oxidative stress by reducing the range of reactive oxygen species. eef1a2 interacts directly with prx-1 and protects the cells from stress-induced apoptosis by the down-regulation of caspase-3 and caspase-8 activation parallel to increased expression of the pro-survival factor akt (76, 77) . additionally, the eef1a expression varies in cells treated with various concentrations of hydrogen peroxide, a strong apoptotic inducer associated with decreased mitochondrial respiration and an inhibitor of prx-1 (78, 79) . finally, eef1a provides protection against all endoplasmic reticulum (er) stress-mediated cell death. fl5.12 cells are non-transformed murine b-cells that require il-3 for growth, survival, and proliferation. enforced expression of eef1a in these cells protects them from il-3 withdrawal without cellular transformation (80) . wasted mouse is a spontaneous autosomal recessive mutation associated with neurological defects, immune system abnormalities, and defective response to dna damage repair www.frontiersin.org in immune cells. thymus and spleen from wasted mice show extensive apoptosis. the loss of activity in wasted mice and immunological abnormalities are solely linked to the presence of a deletion in the eef1a2 gene (81) (82) (83) (84) . furthermore, eef1a2 is highly expressed in terminally differentiated cells such as neurons, cardiomyocytes, and myofibers. during skeletal muscle development, undifferentiated myoblasts are susceptible to serum deprived caspase 3-mediated apoptosis but become resistant to apoptosis after differentiating into myotubes. in muscle cells, the eef1a1 is expressed during embryonic development of myoblast but is replaced by eef1a2 after 2 weeks of birth indicating that the eef1a1 isoform plays a pro-apoptotic role while the eef1a2 isoform displays anti-apoptotic properties (85, 86) . the eef1a proteins play a critical role in several viral infections at distinct stages of the viral cycle [reviewed in ref. (87)] ( table 3) . among the most studied viruses, human immunodeficiency virus (hiv) interacts with numerous cellular proteins, including eef1a (88) . first, eef1a has been detected as part of the hiv virions as an actin-binding protein (89) . second, eef1a has been reported to plays a critical role at early stages of hiv replication (figure 5 ). eef1a and also ef1g are part of hiv reverse transcription complex (rtc) (90) . both eef1a and ef1g proteins coimmunoprecipitated with the p51 subunit of reverse transcriptase (rt) and integrase using an endogenous reverse transcription assay (90) (figure 5) . the depletion of eef1a and ef1g using sirnas decreased reverse transcription parallel to reduced levels of rtc in treated cells (90) . since integrase is also an rt binding protein, a tight interplay between rt, integrase, and eef1a could be involved in several stages of hiv replication. actually, integrase has been shown to interact with eef1a in vitro (91, 92) . eef1a also interacts with other viral proteins involved in early stages of hiv replication such as nef and tat. several reports indicate that hiv nef interacts with eef1a (77, 93) ( figure 5 ). abbas and colleagues have shown that the nef-eef1a interaction favors the nucleo-cytoplasmic shuttling of eef1a and ultimately inhibits stress-mediated apoptosis in monocyte-derived macrophages (77) . additionally, nef interaction with actin impairs human podocyte actin cytoskeletal integrity and eef1a could play a role in the development of podocyte phenotype in hiv-1 associated nephropathy (93) . the trans-activation response (tar) element is critical for the activation of hiv-1 transcription. eef1a stimulates the rna polymerase ii and trp-185 binding to tar rna, which in turn regulates the hiv-1 gene expression (94) . eef1a has been described to interact with the hiv gag protein (88) . finally, eef1a is responsible for the selection and binding of cognate aminoacyl-trnas to the a site of the ribosome. hiv nef interacts with two components of the 40s small ribosomal subunit, the rps10 protein, and the 18s rrna, and also to trnas, and has been reported to decrease translation using an in vitro translation assay (95) . we cannot exclude that eef1a that binds to both nef and trna participates in the control of translation in hiv-infected cells. interestingly, ef1-delta has been reported to interact with the second coding exon of hiv tat, and to result in reduced efficiency of the translation of cellular proteins, but not of viral mrnas (96) . since overexpression of eef1a has been reported in several cancers, we cannot exclude a link between oncoviruses and eef1a. among oncoviruses, hepatitis b virus expresses the hbx protein that has been previously involved in liver cancer, especially hcc (110) . hbx protein has been reported to interact with eef1a1 in huh-7 hepatoma cells infected with recombinant adenovirus expressing hbx protein (97) . interaction of hbx protein with eef1a1 blocks filamentous actin bundling (97) . interestingly, the hepatitis delta virus (hdv) that can propagate only in the presence of hbv has a rna genome that also interacts with eef1a1 (98) . a dozen of human papillomavirus (hpv) types including hpv16 and hpv18 are well-known oncoviruses that express two oncoproteins e6 and e7, and favor cellular transformation, oncogenesis, and the appearance of cervical cancers (111) . e7 protein of hpv38 has been shown to interact with both eef1a1 and eef1a2 proteins leading to cellular immortalization and transformation of primary keratinocytes probably through disruption of actin stress fiber formation, a critical event linked to tumor formation (99) . eef1a facilitates virus replication complex (rpc) assembly and favors replication of west nile virus and dengue virus (100) (101) (102) . similarly, eef1a interacts with the nucleocapsid protein of transmissible gastroenteritis coronavirus (tgev) and favors tgev replication (103) . since eef1a is found in highly purified virions of numerous rna and dna viruses including vesicular stomatitis virus (104), vaccinia virus (105, 106) , cytomegalovirus (107, 108) , severe acute respiratory syndrome coronavirus (sars-cov) (109) , and hiv-1 (88, 112) , its role in viral pathogenesis needs to be further investigated. recent findings in the field of cellular and molecular biology reveal that the eef1a proteins are not only involved in the translational process but display also non-canonical functions. the specific up-regulation of eef1a2 in numerous tumors and its transforming properties indicate that it could play a significant role in tumorigenesis. furthermore, eef1a2 activates the phospholipid and akt signaling pathways favoring cell survival. additionally, eef1a proteins block apoptosis and favors viral replication. all together, the non-canonical functions of eef1a proteins are involved at the crossroads of oncogenesis, blockade of apoptosis, and viral pathogenesis. therefore, the eef1a proteins might play an important role in the pathophysiology of tumors and apoptosis, especially in response to stress and viral infections. future studies need to be done to further highlight the role of eef1a proteins in human diseases. wa and ak were responsible for drafting and revising the manuscript. gh was involved in critical reading of the manuscript. not just for housekeeping: protein initiation and elongation factors in cell growth and tumorigenesis central dogma at the single-molecule level in living cells protein-protein interactions and cancer: targeting the central dogma modulation of molecular mechanisms involved in protein synthesis machinery as a new tool for the control of cell proliferation targets and mechanisms for the regulation of translation in malignant transformation regulation of peptide-chain elongation in mammalian cells structural models of human eef1a1 and eef1a2 reveal two distinct surface clusters of sequence variation and potential differences in 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cord-319754-5isw53wl authors: balgoma, david; gil-de-gómez, luis; montero, olimpio title: lipidomics issues on human positive ssrna virus infection: an update date: 2020-08-31 journal: metabolites doi: 10.3390/metabo10090356 sha: doc_id: 319754 cord_uid: 5isw53wl the pathogenic mechanisms underlying the biology and biochemistry of viral infections are known to depend on the lipid metabolism of infected cells. from a lipidomics viewpoint, there are a variety of mechanisms involving virus infection that encompass virus entry, the disturbance of host cell lipid metabolism, and the role played by diverse lipids in regard to the infection effectiveness. all these aspects have currently been tackled separately as independent issues and focused on the function of proteins. here, we review the role of cholesterol and other lipids in ssrna+ infection. the ongoing covid-19 pandemic is developing (july 2020) worldwide with devastating global consequences, both for social organization and healthcare systems. covid-19 illness is brought about by infection with the severe acute respiratory syndrome coronavirus sars-cov-2 [1, 2] , which is an enveloped positive single-stranded rna virus (ssrna+) [3] . the most abundant studies related to human diseases induced by ssrna-positive viruses refer to picornaviridae, coronaviridae, and flaviviridae [4] . this impact in a short time span has brought the biology and biochemistry of viral infection mechanisms to reach momentum. the infection mechanisms have been described for diverse unrelated viral families [5] , with the majority of them being dna viruses. within picornaviridae, coronaviridae, and flaviviridae, rhino and poliovirus (picornaviridae), sars-cov, middle east respiratory syndrome coronavirus (mers-cov), hepatitis c virus (hcv), west nile virus (wnv) and dengue virus (denv) fall within the viruses whose life cycle biology is better known. nonetheless, knowledge regarding virus entry mechanisms and other related features of the virus life cycle has been gained from the research on the influenza virus from the orthomysoviridae family and the human immunodeficiency virus from the retroviridae family. consequently, these and other unrelated viruses will be also considered in this review from the point of view of the different aspects that affect the lipidomics of the viral infection. all ssrna+ viruses initially infect mammal cells through the interaction of virus proteins with any given host cell protein. further fusion of the virus and host cell membranes is required for the viral genetic material to get into the cell. once inside the cell, the genomic and subgenomic viral rnas are translated into the virus proteins; these then lead the virus replication, which is a process that involves modulation of the host cell lipid metabolism [3, 5, 6] . consequently, along with other features, current lipid studies about the aforementioned virus infection focus their research on membrane fusion and modulation of the lipid metabolism of the host cell. these two processes are considered separated disciplines of the infection. the fight against the virus infection encompasses primarily the inhibition of the binding of the viral spike protein to the host cell's receptor protein. consequently, most of the current research focuses on the role played by viral proteins but the lipid environment, where the proteins carry out their function and regulation, is considered secondarily [7] . nevertheless, improving the knowledge on how the lipids are involved in the mechanisms of infection may provide clues to develop treatments and better counteract the virus-induced pathology [3] . to fill this gap, here, we review the main aspects regarding the lipidome regulation of the viral infection mechanism by ssrna+ viruses. the initial step in virus infection is the binding of any viral structural glycoprotein to a receptor of the host cell. the spike protein accounts for such function in coronaviruses (covs) and other enveloped viruses. after the virus is attached to the host cell protein, the process of membrane fusion starts to get the viral genome into the host cell. this process implies viral envelope and host cell membrane fusion, for which an energetically cost-effective barrier must be overcome. for example, in coronavirus, membrane fusion is driven by the fusion peptide (class i), which is localized within the spike protein (s protein) and becomes active after cleavage of the s protein at specific sites by host proteases or ph-dependent mechanisms [4, 6, 8] . a different mechanism of attachment and endocytosis drives the virus entry in the case of hcv. this mechanism is more complex than that of coronaviruses and involves interaction of the virus envelope e1 and e2 proteins (class ii fusion loop) with several host cell proteins [9] [10] [11] . however, a membrane fusion-driven pore is also required in hcv to deliver the viral genetic material into the host cell cytoplasm. two main mechanisms of membrane fusion have been described: viral endocytosis by host cell membrane (endocytic pathway), and both viral and host cell plasma membrane fusion (non-endocytic pathway). after docking of the virus to the attachment factor or the receptor on the host cell surface, the virus may internalize its genomic material or the entire particle [12] [13] [14] . the non-endocytic pathway encompasses the direct delivery of the genetic material through a pore formed in the cell membrane by the induction of viral proteins at neutral ph. this pathway is typical of non-enveloped viruses. the endocytic pathway is more complex and harnesses the host cell endocytosis machinery for the virus internalization. three main ways have been described in the endocytic pathway, namely: the clathrin-mediated endocytosis (cme), the caveolae-mediated endocytosis (cavme), and the macropycnocytosis. the best-known endocytic mechanism is the clathrin-mediated endocytosis. the cme is used by small to intermediate-sized viruses. this mechanism uses vesicles coated by the protein clathrin, which forms a polyhedral lattice that surrounds the cell membrane-derived vesicle where the virus is internalized into the cell cytoplasm through the early endosomes. clathrin coating is coordinated by the adaptor protein (ap-2) and other adaptors; it is less commonly ap-independent. the protein dynamin is involved in regulating the clathrin-coated vesicle (ccv) formation as well as its scission from the membrane. some viruses proceed to membrane fusion at this stage for releasing their genome into the cytoplasm. the early endosomes have a ph of about 6.0 to 6.5; therefore, it is considered that membrane fusion is not strictly ph-dependent. other viruses need a lower ph for the membrane fusion to be effective; thus, it is considered ph-dependent. a further step leading to endosome maturation to become late endosomes with a ph of about 5 has to proceed before the membrane fusion takes place and the genetic material is delivered to the cytoplasm. sequential acidification of the virus proteins from the early to late endosomes has also been suggested through the self-organized endosomal network. maturation of the early endosomes to late endosomes and trafficking between them is controled by the rab proteins, which are members of the ras superfamily of small g proteins. subsets of rab proteins differ between the early and late endosomes, and the rab subset change is accompanied for by formation of the phosphoinositide pi(3,5)p 2 from the precursor pi(3)p. regarding lipid composition, early endosome membrane lipids are primarily composed of unsaturated and short alkyl chains, whereas long and saturated alkyl chains, such as in gangliosides, are predominant in the membrane lipids of late endosmes. membrane fusion in some viruses requires a further step in which late endosomes are fused with lysosomes, this step giving rise to the late endosome/lysosome pathway. cholesterol depletion driven by its synthesis inhibition or extracting agents as methyl-β-cyclodextran (mβcd) is used to assess whether the virus entry takes place through the caveolae/raft endocytosis. this pathway in less known and encompasses the formation of initial endocytic vesicles enriched in cholesterol from lipid-rafts, with complex signaling routes that involve the activity of tyrosine kinases and phosphatases. thereafter, the cargo is transported to the endoplasmic reticulum (er) through early and late endosomes. most of the viruses using this endocytic pathway have different gangliosides as receptors, mainly gm1, which has a high concentration in caveolae. polyomavirus, which are non-enveloped dna viruses that replicate in the nucleus, use preferently this endocytic pathway, but picornaviruses and the coronavirus hcov-229e have also been reported to internalize through the caveolae-mediated endocytosis [15] . macropinocytosis is a phagocytic-like mechanism of virus entry that is currently utilized by the cell to internalized fluids; it is dependent on actin and implies the actin cytoskeleton rearrangement to enable internalization of the virus particle [14] . macropinocytic vacuoles (macropinosomes) are formed after membrane ruffles fold to reach at its end the membrane again, and the vacuole is closed through self-membrane fusion. these vacuoles containing the viral particle may traffick afterwards through the early and late endosome network. macropinocytosis is common to large-sized viruses. however, recent work [16] has shown that ebola virus (ebov) may use a macropinocytosis-like process to entry the host cell in a clathrin, caveolae, and dynamin-independent manner, but dependent of actin and a lipid raft. conversely, this virus may use as well an endocytic pathway that is dependent on clathrin, caveolae, and dynamin. which endocytic route is used by this virus depends on the host cell type. description of the current methodologies used to study the entry route by viruses can be found in reference [14] . some viruses may use different entry mechanisms, this feature being likely dependent upon the membrane lipid composition of the host cell they infect as well as the particular cell surface factor attachment used. cme is the entry route currently used by hcv, hiv-1, ebov, rotaviruses, and some coronaviruses, even though other routes can also be used as for ebov (see above). a reaction between clathrin and actin seems to be necessary for the effective entry of these viruses. regulation by microtubules of the cme has been reported for flaviviruses. denv, wnv, and semliki forest virus (sfv, alphavirus family, togaviridae) have been found to depend on early endosomes (rab5 protein marker) for entry but not late endosomes (rab7 protein marker), which means that they do not have strict low ph requirements or depend on different acidification mechanisms for membrane fusion. conversely, influenza avian virus (iav) needs both early and late endosomes to entry, thus reflecting low ph dependence for membrane fusion. marburg virus (marv) may use for internalization a cme through the endo/lysosomal pathway. coronaviruses differ in their internalization mechanism among strains. thus, while hcov-229e is known to use the cav-me route, sars-covs use an endocytic pathway that is clathrin-and caveolae-independent but receptor and ph-sensitive, with lipid rafts playing an essential role [17] . this endocytic mechanism implies internalization of the receptor protein angiotensin-convering enzyme 2 (ace2) along with the spike protein into the early endosomes, but the receptor is afterward recycled to the membrane via lysosomes. nonetheless, previous studies showed that sars-cov could enter through a ph-independent direct membrane fusion as it could infect cells that do not express ace2, such as enterocytes and hepatocytes [18] . recent research on the virus sars-cov-2 points to ph-independent direct cell and viral membrane fusion, which is a process that is driven by the subunit s2 of the spike protein after cleavage by the cellular serine protease tmprss2 [19] . on the contrary, the infectious bronchitis virus (ibv), a gamma-coronavirus, was reported to use the cme pathway to entry, with vesicle scission being mediated by gtpase dynamin 1, and a dependence on low ph and lipid raft localization of the receptor. tracking of the virus trip inside the cell was followed by using diverse inhibitors, cholesterol sequestering agents, and virus particles labeled with fluorescent markers. membrane fusion takes place at the late endosome/lysosome step of the endocytic pathway, with deep rearrangement of the host cell cytoskeleton being induced by the endosomal viral cargo [15] . accordingly, viruses may sequester on their own profit the diverse endocytic pathways that are currently used by the host cell, but variability of the proteins and even the general mechanisms may also exist as a consequence of virus specifity. membrane fusion has been described to proceed through the catalytic action of three different types of fusion peptides or fusion loops of class i, ii, or iii. these proteins afford the free energy necessary to overcome through conformational changes the kinetic barrier due to repulsive hydration strength. most of the knowledge on the viral and host membrane fusion has been gained from the influenza virus and its type i fusion peptide hemagglutinin. a detailed description of the three fusion peptide-guided mechanisms involved in membrane fusion has been previously reviewed in [20] [21] [22] . bringing the viral and the host membranes closer enough (c.a. 20 å) for inducing the membrane fusion is a process that entails membrane curvature and changes in the lipid bilayer phase. they are driven by the insertion of a hydrophobic region of the fusion peptide, which requires dehydration of the inter-membrane space. nonetheless, from experiments with no-protein fusogens, such as polyethilen glycol, it seems that membrane curvature stabilization is not a key player in membrane pore opening. the calculated displacement of lipids in the outer leaflet of the host membrane accounts for no more than 10% of the membrane area (about 3500 å 2 ), which does not represent a substantial energetic demand [21] . this energetic burden has been demonstrated to be afforded by the cooperation of three fusion peptides in influenza virus membrane fusion [23] , whereas two adjacent trimers of the fusion protein are required in west nile virus [24] . this result points to the fact that the viral membrane curvature may not actually impose a constraint for proceeding to the hemifusion step and the formation of a steep curvature stalk, where the outer leafleats are merged. by the mesurement of electron density profiles through x-ray reflectivity in stalks formed from bilayers in a lamellar state with different lipid compositions, aeffner et al. [25] determined that the inter-bilayer separation should attain 9.0 ± 0.5 å in order to facilitate dehydration and promote stalk formation. these authors also found that increasing the relative proportion of nonbilayer-forming, cone-shaped lipids, such as glycerophosphoethanolamine or cholesterol, favored the stalk formation by reducing the hydration energy barrier and, possibly, by contributing with their intrinsic negative curvature. as well, the energy required for dehydration was, in this study, found to decrease with the length of the acyl chains of the glycerophospholipids. however, the hemifusion stalk stage was not detected by gui et al. [26] using fluorescence and electron microscopy. the results of this study show that such a stage might be an unstable intermediate that is quickly resolved toward the postfusion stage. contrarily, localized point-like contacts were abundantly visualized in this study, where the dimples formed in the target membrane, about 5 nm wide, were drawn toward the virus surface. they were able to detect up to well-resolved four types of virus-target membrane contacts at ph 5.5 and 5.25 using liposomes of dioleylglycerophosphocholine, dopc, with 20% cholesterol. at the lowest ph, a tight contact of the two membranes through an extended length of about 100 nm (catalogued by the authors as type iii) was the predominant interaction, whose abundance was increased by about 3-fold in cholesterol-containing liposomes in comparison to only dopc liposomes. using synthetic peptides that resemble the fusion peptide hemagglutinin and electron spin resonance (esr), ge and freed [27] found that the most relevant effect of the synthetic fusion peptides was the induction of highly ordered membrane domains, which came motivated by virtue of electrostatic interactions between the peptide and negatively charged phospholipid headgroups. a similar effect was reported for two putative fusion peptides enclosed in the spike glycoprotein of sars-cov-1. it was found in this study that the inner water content in the lipid bilayer was dropped by the insertion of the fusion peptide as a consequence of increased lipid packing, but only in membranes containing negatively charged lipids, whereas the water content was only slightly altered in zwitterionic dipalmitoylglycerophosphocholine (dppc) liposomes [28] . additionally, the fusion peptides created opposing curvature stresses in the highly bended membranes containing nonbilayer-forming phospholipids. however, previous studies had pointed out that interaction with the lipid headgroups is not an essential factor in reaching the membrane hemifusion state [21, 29] . in sars-cov, the possibility of existing two fusion peptides that act in coordination has been suggested [7] ; one of the peptides would promote the dehydration process, while the other one would act in modifying/disturbing the lipid organization within the target membrane [26, 28, 30] . hence, the catalytic role of the fusion peptide(s) is likely to tackle three properties of the target membrane in the virus entry machinery: (i) dehydration of the intermembrane space for the fusing membranes coming into the required proximity, (ii) to promote negative curvature to form the hemifusion stalk, and (iii) to alter the lipid packing density, which will be generated in the highly curved local dimples of the stalk [22, 28] . the effectiveness of these three processes is likely to depend upon the membrane lipid composition. further research is devoted to this issue, and new clues are expected to come from electron and fluorescence microscopy [31] . since the dominant phospholipid in the outer leaflet of most membranes is the bilayer-forming, positive charged diacylglycerophosphosphocholine (pc), the idea was raised that the viral docking to the receptor on the target cell and, consequently, the membrane fusion were likely to take place at specific microdomains with particular lipid composition, the so-called lipid rafts [27, [32] [33] [34] [35] [36] . a special characteristic of the lipid rafts is the high content of cholesterol [37] [38] [39] . even though a high content of sphingolipids and gangliosides is also a defining characteristic of lipid rafts (figure 1 ), direct in vivo visualization still remains unresolved [39] . metabolites 2020, 10, x for peer review 5 of 22 altered in zwitterionic dipalmitoylglycerophosphocholine (dppc) liposomes [28] . additionally, the fusion peptides created opposing curvature stresses in the highly bended membranes containing nonbilayer-forming phospholipids. however, previous studies had pointed out that interaction with the lipid headgroups is not an essential factor in reaching the membrane hemifusion state [21, 29] . in sars-cov, the possibility of existing two fusion peptides that act in coordination has been suggested [7] ; one of the peptides would promote the dehydration process, while the other one would act in modifying/disturbing the lipid organization within the target membrane [26, 28, 30] . hence, the catalytic role of the fusion peptide(s) is likely to tackle three properties of the target membrane in the virus entry machinery: (i) dehydration of the intermembrane space for the fusing membranes coming into the required proximity, (ii) to promote negative curvature to form the hemifusion stalk, and (iii) to alter the lipid packing density, which will be generated in the highly curved local dimples of the stalk [22, 28] . the effectiveness of these three processes is likely to depend upon the membrane lipid composition. further research is devoted to this issue, and new clues are expected to come from electron and fluorescence microscopy [31] . since the dominant phospholipid in the outer leaflet of most membranes is the bilayer-forming, positive charged diacylglycerophosphosphocholine (pc), the idea was raised that the viral docking to the receptor on the target cell and, consequently, the membrane fusion were likely to take place at specific microdomains with particular lipid composition, the so-called lipid rafts [27, [32] [33] [34] [35] [36] . a special characteristic of the lipid rafts is the high content of cholesterol [37] [38] [39] . even though a high content of sphingolipids and gangliosides is also a defining characteristic of lipid rafts (figure 1 ), direct in vivo visualization still remains unresolved [39] . an unexplored possibility is that rafts do not have a permanent localized existence, but they arise under the induction of certain proteins such as the hydrophobic insert of the viral fusion peptide or the fusion loop. this fact might be also responsible for bringing negatively charged lipids from the inner leaflet of the bilayer to its outer leaflet by flip-flop mechanisms. this hypothesis would explain the promotion of virus entry by the interaction of the fusion peptide with the negatively charged phospholipid headgroups [25, 27] as well as the kinetics of the membrane fusion [25] . a number of studies have shown that the hemifusion step and pore widening are sped up after increasing the relative concentration of cholesterol in the bilayer composition, whereas either the depletion of cholesterol in the cell culture medium or the inhibition of cholesterol synthesis by an unexplored possibility is that rafts do not have a permanent localized existence, but they arise under the induction of certain proteins such as the hydrophobic insert of the viral fusion peptide or the fusion loop. this fact might be also responsible for bringing negatively charged lipids from the inner leaflet of the bilayer to its outer leaflet by flip-flop mechanisms. this hypothesis would explain the promotion of virus entry by the interaction of the fusion peptide with the negatively charged phospholipid headgroups [25, 27] as well as the kinetics of the membrane fusion [25] . a number of studies have shown that the hemifusion step and pore widening are sped up after increasing the relative concentration of cholesterol in the bilayer composition, whereas either the depletion of cholesterol in the cell culture medium or the inhibition of cholesterol synthesis by statins was able to halt the viral infection at the virus entry step [26] [27] [28] 40, 41] . the effect of cholesterol on promoting membrane merging has also been observed for bis-(monoacylglycero)-phosphate (bmp) [26] . this particular phospholipid was shown to be strictly necessary for dengue virus (denv) entry even at low endosomal ph [42] . as pointed out above, the exact role played by cholesterol is not known in detail, but its intrinsic negative curvature seems to be an essential characteristic in promoting the stalk formation during virus entry. however, a recent study shows that the cholesterol action is likely to involve a direct influence on the oligomeric state of the fusion peptide after insertion into the host cell membrane, as well as on the effects of the fusion peptide on the membrane reorganization and dynamics [43] . in another recent study, a new lipid-label-free methodology was used to measure the kinetics of influenza virus infection [44] . according to the results of this study, cholesterol is able to augment the efficiency of membrane fusion in a receptor binding-independent manner. nevertheless, the rate of membrane fusion was not altered. these results led the authors to conclude that the positive effect of cholesterol in membrane lipid mixing is related to its capability to induce negative curvature. since membrane mixing was achieved in this latter study without binding of the spike protein of the influenza virus to the host cell receptor, the catalytic effect of the fusion peptide might proceed in an independent way in this virus. cleavage of the spike protein in sars-cov-1 does not seem to be also necessary for the fusion peptide to become fusogenic, but rearrangement of disulfide bridges in the s1 peptide after receptor binding are likely involved in the conformational changes driving the fusion mechanism [43, 45] . contrary to these latter results, which point to the fact that membrane fusion is independent of viral protein attachment to its receptor, guo et al. reported lipid raft-dependent viral protein binding with the suppression of viral infection if the lipid rafts were disrupted with cholesterol drug-induced depletion; lipid rafts, as recognized by the caveolin-1 marker, were the membrane domain where structural proteins of the infectious bronchitis virus (ibv) co-localized but the nonstructural proteins did not [35] . the question regarding whether the lipid-raft domains may serve as platforms to concentrate the proteins required for viral entry and, even though some evidence exists, to activate signaling pathways inside the host cell still remains unsolved. sphingomyelins (sms) are also common lipids found in lipid rafts, which contribute to make these membrane microdomains detergent-resistant [34] . the structure of a representative of this lipid class is illustrated in figure 2 . the ganglioside gm1, a sphingolipid, is used as a marker of lipid rafts [34] . sphingolipids (sls) promote to an extent higher than chol the liquid-ordered phase in the outer leaflet of the membrane bilayer because of the long saturated acyl chains they currently contain (the r group in figure 2 may extend to a length of up to 26 c), in addition to their capability to form intermolecular hydrogen bonds [46] . a relevant function of the lipid rafts has been suggested to be the connection between the events outside the cell with the pathways inside the cell, thus acting as 'signaling platforms'. with the aim of this function to be properly accomplished, the lipid rafts would act as concentrators of specific transmembrane proteins, mainly receptors, whose compatibility with the membrane phase would determine their selectivity. thus, sls would account for a role in connecting the outer leaflet with the inner leaflet through their long saturated acyl chains. regarding virus entry, research has been primarily focused toward the role played by cholesterol, but a number of studies have also enlightened the sm influence on this early step of viral infection. the displacement of cholesterol by sms and the other way round has been demonstrated, with the bilayer liquid-ordered phase being preferentially determined by the interaction between sm and cholesterol. this interaction would be controlled to a certain extent by the intracellular actin meshwork, which would also be responsible for the compartmentalization of the membrane into lipid-specific domains [47] . furthermore, the actin role is possibly extended to the routing of the viral genomic material toward the replication place inside the host cell. the hydrolysis of sm by sphingomyelinases to render the corresponding ceramide in specific membrane domains is proposed to regulate the dynamics of cholesterol in the cell membrane, the effect of such regulation being the progressive disassembly of cholesterol from the liquid-ordered phase and its displacement. since the interaction of ceramides with cholesterol has been suggested to be an apoptotic regulator, it can be expected that viral proteins would act in recruiting cholesterol to displace the ceramide and to avoid the programmed cell death. this fact is added to the other characteristics conferred by cholesterol to the membrane mechanical properties discussed above. to study the influence of ceramide on membrane fusion during semliki forest virus (sfv, alphavirus family, togaviridae) infection, ceramide analogs have been used [48] . according to this experiment, in which cholesterol-containing pc plus pe liposomes were used, the roles played by the 3-hydroxyl group and the 4,5-trans carbon-carbon double bond of the sphingosine backbone ( figure 2 ) were found to be essential in the fusion process. in additon, ceramide was the simplest sl to accomplish this significant contribution in mediating the fusion, independently of the length of the acyl chain. more recently, a ca 2+ -dependent pathway of infection by the rubella virus (ruv, rubivirus family, togaviridae) was demonstrated to proceed through direct binding of the fusion loop in the viral e1 protein to sm/cholesterol-enriched membranes [49] . however, the treatment of host cells with sphingomyelinase proved that sm is exclusively required for viral entry but is not required for the further steps of viral replication. sm in the host cell membrane and acid sphingomyelinase (asmase) activity have also been shown to be required by the ebola virus (ebov), a negative single-stranded rna virus belonging to the filoviridae family, to get into the host cell. the asmase activity renders ceramide that provoques raft enlargement and membrane invagination [50] . this study also showed that the virus was able to recruit both sm and asmase to the raft where the viral attachment was happening. conversely, bovine herpesvirus 1 (bohv-1, herpesviridae family) seems to require sm in the virus envelope but does not in the host cell [51] . the role played by ceramides is contradictory as they may enhance or inhibit virus replication, but this sl action seems to be related to the viral replication phase rather than to the internalization phase [52] [53] [54] . in virus using the endocytic pathway, similar to the influenza virus or the ebola virus, it has been shown that activity of glucosylceramidase (gba) is required for viral entry and membrane fusion through the regulation of endocytosis, but in a virus-dependent manner. it was also shown that trafficking of the epidermal growth factor (egf) to late endosomes was impaired in gba-knockout cells, which negatively affects the virus entry through spoiling the endocytic pathway [55] . indeed, co-clustering of the ha attachment factor and egf in submicrometer domains that overlap partially has been reported recently [56] . accordingly, there is evidence that sls have a function in enveloped ssrna viruses at the early stage of infection that accounts for the viral entry modulation, but further research is still necessary to unveil the exact mechanisms of sl reactions. metabolites 2020, 10, x for peer review 7 of 22 displace the ceramide and to avoid the programmed cell death. this fact is added to the other characteristics conferred by cholesterol to the membrane mechanical properties discussed above. to study the influence of ceramide on membrane fusion during semliki forest virus (sfv, alphavirus family, togaviridae) infection, ceramide analogs have been used [48] . according to this experiment, in which cholesterol-containing pc plus pe liposomes were used, the roles played by the 3-hydroxyl group and the 4,5-trans carbon-carbon double bond of the sphingosine backbone ( figure 2 ) were found to be essential in the fusion process. in additon, ceramide was the simplest sl to accomplish this significant contribution in mediating the fusion, independently of the length of the acyl chain. more recently, a ca 2+ -dependent pathway of infection by the rubella virus (ruv, rubivirus family, togaviridae) was demonstrated to proceed through direct binding of the fusion loop in the viral e1 protein to sm/cholesterol-enriched membranes [49] . however, the treatment of host cells with sphingomyelinase proved that sm is exclusively required for viral entry but is not required for the further steps of viral replication. sm in the host cell membrane and acid sphingomyelinase (asmase) activity have also been shown to be required by the ebola virus (ebov), a negative single-stranded rna virus belonging to the filoviridae family, to get into the host cell. the asmase activity renders ceramide that provoques raft enlargement and membrane invagination [50] . this study also showed that the virus was able to recruit both sm and asmase to the raft where the viral attachment was happening. conversely, bovine herpesvirus 1 (bohv-1, herpesviridae family) seems to require sm in the virus envelope but does not in the host cell [51] . the role played by ceramides is contradictory as they may enhance or inhibit virus replication, but this sl action seems to be related to the viral replication phase rather than to the internalization phase [52] [53] [54] . in virus using the endocytic pathway, similar to the influenza virus or the ebola virus, it has been shown that activity of glucosylceramidase (gba) is required for viral entry and membrane fusion through the regulation of endocytosis, but in a virus-dependent manner. it was also shown that trafficking of the epidermal growth factor (egf) to late endosomes was impaired in gba-knockout cells, which negatively affects the virus entry through spoiling the endocytic pathway [55] . indeed, co-clustering of the ha attachment factor and egf in submicrometer domains that overlap partially has been reported recently [56] . accordingly, there is evidence that sls have a function in enveloped ssrna viruses at the early stage of infection that accounts for the viral entry modulation, but further research is still necessary to unveil the exact mechanisms of sl reactions. some covs (hcov-oc43 and hcovhku1), as well as influenza a virus (whose fusion loop is hemagglutinin, ha) and other non-related viruses (i.e., non-enveloped simian virus 40 sv-40, of polyomavirus family), use the sialoglycan moiety (9-o-acetyl-sialic acid) of gangliosides or glycoproteins located in membrane lipid rafts as receptors for the spike protein. the amino acid trp90 in the domain a of the hcov-oc43 s protein was shown to be essential for receptor binding. however, despite the fact that binding to 9-o-acetyl-sialic acid is required for membrane fusion, further interaction of the virus protein with other host membrane sialoglycans or proteins is also necessary to induce the conformational changes leading to membrane fusion [57, 58] . conversely, formation of the complex sv40 protein with the host cell ganglioside gm1 was found to be enough to induce the membrane curvature and invaginations required for membrane fusion [59] . as already discussed above, some studies have depicted the possibility that interaction of the fusion peptide or fusion loop with negatively charged phospholipids on the host membrane might be required for an efficient membrane fusion [25] . in this regard, phosphatidylserine (ps) contained in the virus envelope has been demonstrated to serve after externalization as a virus co-receptor through the t cell immunoglobulin mucin domain 1 (tim-1) receptor in ebov and other viruses, even in an indispensable fashion [60] [61] [62] [63] . in the study of nanbo et al. [63] , flipping of ps from the inner leaflet to the outer leaflet of the cell membrane for virion adquisition and incorporation to its envelope is proposed as a previous step to tim1 binding. in herpes simplex virus (hsv), phospholipid scramblase-1 (plscr1), after activation by hsv exposure, flips both ps and akt to the outside of the membrane in a ca 2+ -dependent mechanism. ps is restored to the inner leaflet 2 to 4 h after infection to avoid apoptotic triggering [62] , suggesting a different role for ps in relation to the tim-1 ps receptor. however, the function of tim-1 as an essential receptor for hav has been disputed [64] due to the finding that quasi-enveloped ha virions (ehav) were able to infect tim1-knockout vero cells to a similar extent to naked hav. hence, the authors proposed tim1 to be an accessory attachment factor by binding ps on the hav envelope rather than an essential virus protein receptor. in spite of these contradictory data, ps seems to act in any way in virus attachment and entry in certain virus families, at least contributing to the process efficiency, but the exact role may depend on every virus or it may be complementary to other factors. a phospholipid currently associated to the inner leaflet of the lipid rafts is phosphatidylinositol (pi), which is a negatively charged phospholipid with important and versatil signaling functions ( figure 2 ) [65, 66] . abundant data suggest that a derivative of pi, the phosphatidylinositol 4,5-biphosphate (pip2), accumulates preferently in liquid-disordered phases (l d ) [7] , where the cholesterol content is presumed to be low, and interplays with ps, which is rather localized in liquid-ordered phases (l o ). pis play an essential role also in endosome maturation, which is a requisite for efficient virus infection of those using the endosomal pathway [56, 66] . during hiv infection, pip2 has been proposed to coordinate the actin cytoskeleton changes required for efficient virus entry in cd4+ t cells [67] ; after virus attachment to the host cell receptor, pip2 is recruited to the binding membrane microdomain, and in this way, pip2 controls the protein reactions, leading to actin polymerization. as well in hiv-1, the requisite of pip2 accumulation for the virus gag protein to be properly anchored and stabilized in the inner leaflet of the cell plasma membrane has been pointed out [68, 69] . two isoforms, α and γ, of the phosphatidylinositol-4-phosphate 5-kinase family type 1 (pip5k1) have recently been shown to participate in gag stabilization by pip2 through targeting the gag precursor pr55 gag to the cell plasma membrane [70] . as commented above, interaction with the headgroup of negatively charged phospholipids such as ps or pi may also contribute to the dehydration process in the formation of the hemifusion stalk, with this contribution happening by promotion of the inverted hexagonal phase in the lipid bilayer and binding of ca 2+ [25] . in in vitro experiments with cos-7 cells and multilamellar vesicles (mlvs), unspecific binding of the marburg virus (marv) mvp40 protein to pip, pip2, and even pip3 species present in the mlvs, both in the presence or absence of ps, has been reported. in this study, it was also found that with increasing ps concentration, the association of mvp40 to mlvs rose up to a threshold. furthermore, the addition of sphingosine with the aim to reduce the negative charge load in the inner leaftet of the cos-7 cells led to a decrease in the binding level. these facts suggest that the electronic density, rather than the specific lipid species, is a determinant factor for binding [70] . activation of the pi3k pathway for signaling is one of the most relevant features taking place for both entry and budding during infection by a number of viruses [58, [71] [72] [73] . pi3k converts pip2 into phosphatidylinositol 3,4,5-triphosphate (pip3). in addition to stabilizing proteins or serving as a binding factor, pip2 has been shown to collaborate with akt through the signaling pathway pi3k/akt on avoiding apoptotic events, and in this way, keeping the host cell metabolically active for virus replication and budding [71] [72] [73] . all these results clearly bring evidence that the lipid environment surrounding proteins involved in virus infection has a relevant function in the virus entry mechanism. different lipids are essential for virus docking to the cell receptor either serving directly as (co)-receptors or providing the appropriate environment (lipid rafts) for the necessary reactions (e.g., membrane curvature). in addition, the virus, through specific protein conformational changes, takes advantage of several cell signaling pathways controlled by diverse membrane lipids. this process allows the virus to govern the cell metabolism following endocytosis of the viral genetic molecules. after the virus or its genome gets inside the infected cell, ssrna+ viruses and other enveloped ones that replicate in the cytoplasm manage the cell metabolism to develop the replication scaffold, this membrane structure bolstering the so-called 'virus factory' [5, 58, [74] [75] [76] [77] [78] [79] [80] . there is consensus on that the functions of these structures are (i) to compartmentalize the diverse processes involved in viral genome replication, its envelopment, and structural protein assembly; (ii) to increase virion concentration during budding before infecting naïve cells; and (iii) to create a protected environment to escape the innate immune recognition of the viral components. virus replication imposes an extra-energetic expenditure to the cell metabolism. hence, cell central metabolism is orchestated by viral proteins to redirect toward the generation of enough energy and metabolites that are required for virus replication. in particular, building the scaffold demands a high rate of new lipid synthesis. therefore, the lipid metabolism is hijacked by the virus proteins for the de novo synthesis of fatty acids in order to generate the scaffold membranes, the replication complexes (rcs), as well as for energy production in the β-oxidation pathway in the mitochondria. concurrently, the cell metabolism needs to be kept above a threshold level to avoid exhaustion of the host cell. full understanding of the mechanisms and related factors involved in virus-host interaction is a requisite for developing efficient antiviral infection therapies. the scaffold structure raised for building the viral factory varies between different virus in their morphology and possibly lipid composition. flaviviruses develop a so-called 'membranous network' (mn) in a spherule/invagination type, while coronavirus does through a quarter-like type delimited by 'double membrane vesicles' (dmvs). nonetheless, hcv (flaviviridae) uses dmvs instead [77] ; hence, this morphological separation may have exceptions or be somewhat diffuse. an extended review of the different virus family-related morphologies of the mns as well as diverse factors influencing their formation can be found in [22, 75, 76] . it should be remarked that the exact lipid composition of the rcs' membranes is not known in detail yet, although there is evidence that their lipid profile differs from that of the organelles from which they are generated. the enrichment of typical lipids such as cholesterol, sphingomyelins, and glycosphingolipids in the lipid rafts seems to be a common feature of these mns. the rcs' membranes may be originated from the endoplasmic reticulum (er) in the perinuclear area, as for example in sars-cov and faviviridae [75, 78, 80] , from the golgi, giving rise to cytopathic vesicles (cpvs) as in togaviridae and picornaviridae [75] , from mitochondria (nodaviridae) [79] , or from the cell plasma membrane (cpvs in alphaviruses) [75] . however, vesicle trafficking between the er and the golgi organelles may contribute to an undefinition in this regard. mns, and in particular dmvs, are connected to the cytosol through a pore, which is believed to serve as the gate to the replication scaffold for the requiered metabolites, in particular nucleotides. this pore-mediated gate has not been detected up to date in sars-cov's dmvs, which raises the concern of how the required metabolites get inside the rcs. there is evidence from a number of studies that dmvs are the site of replication, but it has also been shown that dmvs can be developed irrespective of whether rna replication takes place by the sole action of the viral proteins, at least for hcv [81, 82] . viral nonstructural proteins nsp3, nsp4, and nsp6 are involved in dmv development in sars-cov-1 in a time-dependent manner and correlating with rna replication. timecourse events have been shown to run with the initial formation of single membrane vesicles (smvs) during the first 2-4 h after cell infection. these futher evolve to dmvs 16 h after infection, and they ultimately turn into multimembraneous vesicles (mmvs) close to the cis-golgi at the budding stage 36-48 h after infection, this latter transformation being coincident with the formation of vesicle packets [75, 78, 79, 83] . in hcv, ns5a seems to be enough for dmv formation, but the collaboration of ns3-5b is required for completing efficient dmvs, whereas ns4b is likely responsible for inducing the formation of smvs [77, 80, 82] . even though particular hints can be likely associated to every particular virus, there are common features shared by all ssrna+ viruses regarding rcs' structure and buildup. enveloped viruses such as ssrna+ viruses have a membrane lipid whose profile is different to that of the original organelle membrane when the envelope is created. since the viral membrane is known to be enriched in cholesterol, sphingolipids, and phospholipids with saturated acyl chains, the dmv is believed to be also primarily composed of such classes of lipids. an unusual sphingolipid, dehydrosphingomyelin, along with ps and plasmalogens of pe were reported in the hiv envelope [84] . a role for sphingomyelin-to-ceramide conversion has been proposed in wnv budding, as its envelope was found to be highly enriched in sphingomyelin [85] . more recently, using multi-color super-resolution microscopy and mass spectrometry analysis, a substantial increase in pip2 (from 11% to 51%) and pip3 (from 0.01% to 0.13%) was reported in the hiv membrane as compared with the plasma membrane of the host cell [69] ; this fact is related to the recruitment of gag protein for efficient membrane fusion as aforementioned ( figure 1) . however, the most striking and known lipid-related factor associated to the mns' development is the pi4kiii signaling pathway. the pi4pα isoform, which is mainly expressed in the er, has been shown to be a key factor for hcv replication, whereas the pi4kiiiβ is found in the golgi and is required by picornaviruses and some hcv strains [75] . this enzyme interacts with the viral protein ns5a, and disrupting this interaction prevents virus replication. the product of the pi4k enzyme is pip4; enrichment in this pi has been shown to act in different processes regarding virus replication: membrane curvature, directly or indirectly through recluting cholesterol [86] , glycosphingolipid transport to the rcs by the action of the fapp2 protein [87] , and protein concentration. however, conversely to these studies, it has been shown that currently used inhibitors of pi4kiiiα, enviroxime and bf738735, actually exert their inhibition against pi3k [88] . thus, this result points out a genomic dependence on the pi kinases in hcv; otherwise, the action on pi3k is required only at the entry stage (see above). enviroxime-like inhibitors have been shown to halt enterovirus replication through the action against pi4kβ [89] . the de novo lipid synthesis has also been evidenced for wnv, from the flaviviridae family as hcv, to proceed in a pi4p-independent fashion and, concurrently, it is not related to pi4kiii signaling [90] . there is no clear evidence on the fact that the pi4k signaling pathway has a relevant function in mns' development. hence, while pi4kiiiβ was shown to be important for sars-cov's dmv formation [91] , another study did not find its metabolite, pi4p, within the host factors involved in sars-cov replication, and the authors attibute to pi4p a function rather in virus entry. however, the authors of this latter study acknowledge that sirna methodology may provide false negatives [92, 93] . since dmvs are not common in healthy cells but they can be observed during authophagy, it has been suggested that sars-cov and other coronaviruses use the autophagy pathway for development of the dmvs; indeed, it has been shown that nsp6 in mhv or the equivalent nsp5-7 in arteriviruses, which hits the er, can activate such a pathway [79, 94] . nonetheless, dmvs are smaller than autophagosomes, and hence, they might be rather endoplasmic reticulum derived vesicles (edesomes) enriched in pi3p and not follow exactly the same synthetic route [94] . further work on coronaviruses and autopahgy found that only the lc3-i protein, the microtubule-associated proteins 1a/1b light chain 3b, is localized on the replication membranes, but the active protein lipidated with phosphatidylethanolamine lc3-ii inserted into the autophagosome membrane is absent. accordingly, present knowledge on coronaviruses in regard to autophagy suggests that they take benefit of the autophagocytic components but do not develop autophagosomes per se [95] . the autophatocytic pathway has also been associated to the start of hcv infection, but it seems not to be necessary for the infection to go on [82] . later on, it was shown that autophagy was key in rna replication at the onset of hcv infection [96] , but the virus life cycle can go ahead afterward without the autophagy system intervention. further work has shown that hcv, and possibly denv, uses the autophagy system to evade the innate immune system [97] . using immortalized human hepatocytes defective of the autophagy-related proteins either beclin (bcn1) or atg7, it was shown in the latter study that disruption of the autophagy machinery elicites activation of the interferon signaling pathway and leads to apoptosis of the infected cells. triggering of the autophagy pathways takes place after binding of the virus to the cell surface via the downregulation of mtor and inactivation of akt signaling [95] . conflicting results have been reported for the induction of autophagy by hcv in regard to the unfolded protein response (upr) [95] . recent work [98] has bound the induction of autophagy by hcv to golgi membrane fragmentation to render vesicles that colocalize with the hcv replicons. the immunity-related gtpase m protein (irgm) mediates the phosphorylation of the early autophagy initiator ulk1 as well as the golgi membrane fragmentation in response to hcv infection. the protein lc3 has also been detected in the replication membranes of the hiv-1, and the association of lc3-ii with gag-derived proteins seems to be a requisite for the efficient maturation of the gag subunit p24 [14, 99] . members of the picornaviridae family, non-enveloped viruses, have been reported to subvert the autophagosome pathway as a means to exit the infected cell without membrane lysis; support for this spreading mechanism comes from the finding of numerous extracelular vesicles that are enriched in phosphatidylserine phospholipids [14] . the best studied virus regarding autophagy is the dengue virus (denv). even though it was initially suggested that the denv replication complexes are developed from autophagosomes, further work pointed out that the replication of denv took place on invaginations arising from the endoplasmic reticulum (er), while autophagy was rather used by denv to modify the lipid metabolism in a way that is known as lipophagy [100, 101] . lipophagy was first shown to be an active way to get energy under starvation [102] through the association of autophagic components with lipid droplets (lds). recently, lipophagy has been demonstrated to regulate the fatty acid availability for the β-oxidation through contact sites between the mitochondria and the er [103] . regarding virus-associated hijacking of the cell lipid metabolism, heaton and randall [100] early showed that increased β-oxidation and the depletion of triglycerides was concurrent with and necessary for denv replication. then, these features were linked to the action of autophagy through the association with lipid droplets. a recent study by zhang et al. [104] has found that aup1, a type iii protein with signals for lds and er, plays a relevant role in lipophagy induced by denv and other flaviviruses such as wnv. unmodified aup1 is required for lipophagy triggering. a 10-fold increase in the content of diacylglycerophosphocholines (pcs) was measured in this study in infected cells containing unmodified aup1, this increase being concomitant with a depletion of triacylglycerols and cholesterol esters, whereas the contents of free fatty acids and unesterified cholesterol rose. conversely, smaller lds, but not a reduction of their abundance, were observed in aup1-knocked-out cells. thus, these data point to an augmented consumption of lds in the infected cells. this study unveils the mechanism that leads to the commented results; after the denv protein ns4a associates with aup1, the complex is relocalized from lds to autophagosomes, where the acyltransferase domain of aup1 is activated for the generation of phospholipids. this process was found to be dependent on the aup1 ubiquitylation status, with ns4a inhibiting the ubiquitylation of aup1. similar to viral entry, cholesterol has been found to be also relevant in the rcs' membranes [79, 82] . up to a c.a. 9-fold enrichment of cholesterol was found in hcv-developed dmvs as compared to its content in the er membranes from which dmvs were originated [77] . a key protein in cholesterol metabolism associated to non-vesicular transport is the oxysterol-binding protein (osbp). this protein has been described to transport cholesterol to pi4p-enriched membranes, which would agree with its collaboration in delivering cholesterol to dmvs with an abundant content of this pi [77] . the ceramide transfer protein (cert) and the four-phosphate adaptor protein 2 (fapp2) are known to undergo a similar fate in hcv infection [82] . an important protein involved in cellular lipid homeostasis is the sterol regulatory element binding protein (srebp), a bhlh-zip transcription factor with three isoforms; srebp1c regulates the expression of fatty acid (fa) biosynthesis genes [105, 106] , whereas srebp2 transactivates genes implied in cholesterol biosynthesis, intracellular lipid transport, and lipoprotein import [107] . a recent study shows that the inhibition of srebp with the retinoid derivative and rar-α agonist am580 prevents mers-cov infection by avoiding the formation of functional dmvs [105] . in this study, the lipid metabolism was the most affected pathway, with sterol biosynthesis being strengthened at expense of the glycerophospholipid metabolic pathways. fast activation of the lipid biosynthesis enzymes acetyl-coa carboxylase (acc), fatty acid synthase (fas), and hmg-coa synthase (hmgcs) was observed in such study, whose activity was partially blocked by am580 inhibition of srebp enzymes. promotion of lipid biosyntheis after infection had already been pointed out for hcv in an elegant proteomics and lipidomics study [108] . hcv infection elicites changes in the proteome of host cells that resembled the warburg effect described in cancer cells toward lactate production and the support of continuous glycolysis; concurrently, the up-regulation of citrate synthase (cs) and other lipogenic enzymes 24 h after infection was interpreted by the authors of the latter study as indicative of re-routing of the tricarboxylic acid (tca) cycle for cytosolic accumulation of citrate, which would be used in fa synthesis. the up-regulation of peroxisomal and mitochondrial fa oxidation pathways is concurrent with the other metabolic changes. an increase in pro-apoptotic ceramides was observed in the latter study as well; two possible interpretations were attributed to this finding, either a cytopathic effect after cell cycle arrest over time enough to complete virus offspring or a defense response of the host cell to avoid infection spread. blocking cholesterol suitability for the membraneous network or endosomes used for the virus replication and internalization has been demonstrated to inhibit the virus life cycle in a number of unrelated viruses. disruption of the srebp pathway restrains the andes virus (andv), an ssrnavirus, internalization, although it does not bind to the cell surface receptor [109] . in addition to srebp2, other components of this pathway were found to be necessary. the dependence of viral entry on the sterol regulatory element binding protein cleavage activating protein (scap) and the site 1 protease (s1p) was evidenced in cells null for these proteins. thus, in the study of petersen et al. [109] , the virus was not internalized in cells lacking s1p, this result pointing out that a complete cholesterol biosynthesis pathway is required. infectivity was also reduced 10-fold when the cells were treated with methyl-β-cyclodextrin (mβcd), a cholesterol sequestering agent, and comparable results were obtained after cell treatment with mevastatin or the s1p inhibitor pf-429242. however, the s1p dependence of virus infectivity does not seem to affect other viruses, thus this route being likely selective for hantaviruses [110] . in this study, the genetic or pharmacological disruption of the srebp pathway at the site of the regulatory element membrane-bound transcription factor peptidase/site 1 protesase (mbtps1/s1p) dramatically reduced viral infection, which is a feature that confirms the essential dependence of hantavirus on the high membrane cholesterol content for membrane fusion and effective infection. the down-regulation of sterol synthesis at the gene level after infection was found to be controlled by an interferon regulatory loop, in which a type i interferon-dependent mechanism down-regulates the expression of srebp2 [111] , this result showing a link between the innate immune response and cholesterol biosynthesis after viral infection. this type i interferon response toward cholesterol synthesis down-regulation was dependent on the mevalonate-isoprenoid branch as a supply of mevalonate completely blocked the cholesterol synthesis, whereas a supply of cholesterol did not. additionally, in the presence of geranylgeraniol, the type i interferon inhibition of sterol biosynthesis was severely diminished. further research has shown that interferon may regulate the sterol synthesis pathway in multiple forms through micrornas [112] . in particular, mir-342-5p was found to hit multiple srebp-independent targets of the mevalonate-sterol synthesis pathway after viral infection. the type i interferon response was also observed in regard to the impairment of the formation of double membrane structures induced by arteriviruses as replication sites [113] . host cell fight against viral infection by a reduction of cholesterol availability has been also pointed out to come from the antiviral effector protein interferon-inducible transmembrane protein 3 (ifitm3). this protein interacts with vesicle-membrane-protein-associated protein a (vapa), impeding its association with the oxysterol binding protein (osbp), and consequently, altering the normal function of osbp. as a result of the ifitm3 action, virus release into the cytosol is blocked by the accumulation of cholesterol in multivesicular bodies and endosomes. this effect restrains the membrane fusion of the intraluminal vesicles and that of the multivesicular bodies, which is a requisite for virus budding and release to the cytosol [114] . the viral accesory protein of hiv nef competes with the cholesterol transporter abca1 to prime the transport of cholesterol to lipid rafts as a viral strategy to raise the replication membranes, thus overcoming the antiviral properties of abca1 [115] . the replication of rabies virus (rabv), an ssrna virus, is halted by the action of viperin (virus inhibitory protein, endoplasmic reticulum-associated, ifn-inducible) in raw264.7 cells. this protein is induced by the rabv, ifv, hiv, or hcv infection through promotion of the innate immune response bound to the tlr4 signaling pathway. the inhibitory activity of viperin on virus budding is related to its capability to substantially drop the contents of cholesterol and sphingomyelin in the replication membranes [116] , thus pointing out the relevance of the membrane lipid composition for efficient virus replication. the induction of viperin has also been proven for hcv and ifav [111] . however, viperin does not intervene in the inhibition of arterivirus-induced double membrane formation [113] . remodeling of the lipid metabolism by virus infection may leave signals at the organism level even some years after healing. the metabolome profile of patients undergoing sars-cov-1 infection during the outbreak of 2002-2003 was assessed 12 years after overcoming the pathology [117] . an outstanding result of this study regarding disturbed lipid metabolism was the elevation of phosphatidylinositol (pi) and lysophosphatidylinositol (lpi) species concentrations in serum, which in turn correlated positively with the levels of very low-density lipoproteins (vldl); higher concentrations of products of the phospholipase a 2 (pla 2 ) such as lysophospholipids (lppls) and free arachidonic acid (aa) were also found in patients as compared to healthy voluntiers, with a correlation between the level of aa and the ratio of lpi(18:0) to total 18:0-pis being observed as well. these results show a potential high sensitivity of sars-cov patients to pla 2 activity. in the general context, the metabolome of these patients pointed to hyperlipidemia, cardiovascular abnormalities, and glucose metabolism alteration as a delayed efffect of the viral infection. nonetheless, the authors acknowledge that some of the related metabolic disturbations are likely owed to the pharmacological treatment. high levels of pla 2 group iid (pla2g2d) in lungs of middle-aged mice as compared to young mice had previously been associated to a fatal or worse outcome [118] . the authors of this study conclude that the negative influence of this enzyme in sars-cov infection was to increase the concentration of anti-inflammatory lipid mediators, mainly protaglandin d 2 (pgd 2 ), which impaired the efficient function of the immune system [119] . in the recent sars-cov-2 outbreak (covid-19), mortality has mostly affected aged people above 60 years old, thus showing an age-related fatality as for sars-cov-1 and mers-cov [120] . using a lipidomics approach, the effect of hcov-229e and mers-cov infection on the host cell lipid profile was recently investigated in cell culture [121] . the main conclusions of this study agree with the raised content of aa and lppls through plase activity, which indicates that the possible virus-induced activation of cpla 2 favors virus replication as a factor required for dmvs' formation. in this study, linoleic acid (la) or aa supplementation to the culture cells suppressed replication, which is a result that may be interpreted as a demonstration of the perturbation of the la/aa axis of the lipid metabolism. in the covid-19 outbreak, it has been suggested that increasing the levels of vitamin d could help fighting against the sars-cov infection [122] . this suggestion is based on the fact that 25-hydroxyvitamin d3 was found to protect huh7 cells against mers-cov [105] . vitamin d is a lipid-related compound belonging to the group of fat-soluble secosteroids, with the most important form in humans being vitamin d3 (cholecalciferol) [123] . in a recent study, high doses of vitamin d have shown protective effects against denv infection through regulation of the toll-like receptor expression as well as the modulation of pro-inflammatory cytokines release, which suggests that its action is focused toward the immune system modulation rather than to lipid metabolism [124] . however, evidence on the beneficial effects of vitamin d uptake is still poor, and more studies are devoted to this issue. lipids, as components of membranes, are related to viroporins, which are specific viral proteins that are known to create ion channels for ion trafficking [125] [126] [127] . the effect on cell metabolism of diverse viroporins differs among them, but there is evidence that they are closely related to viral pathogenity [125] . viroporins may play a relevant role during virus infection, as they are involved in membrane permeability and calcium homeostasis. their participation in the development of vacuoles from the er during the dmvs' formation has been suggested, but data on this issue are still scarce. the regulation of ca 2+ flux by viroporins might favor the membrane fusion through the interaction of this cation with the phospholipid headgroups and concurrently facilitate the required dehydratation reaction. viroporins are not required for virus replication with the exception of rotaviruses and picornaviruses; thus, whether this function is exerted through the ion channels or another property of viroporins remains an issue still unknown [125] . the lipid composition of the membrane may influence the viroporin activity, leading to different versions of ion channels, which depends on the electric charge that the phospholipids confer to the membrane and curvature [127] . a viroporin from rotavirus, nsp4, was shown to co-localize with the autophagy marker protein lc3 in membranes accomodating virus replication; this viroporin is implicated in the sequestering of autophagy for the transport of proteins from the er to the replication sites [128] . further research is necessary to understand the role played by viroporins in virus infection in order to consider them as potential therapeutic targets. remodeling of the virus-induced host cell lipid metabolism is a remarkable feature of the viral infection that affects viral entry, replication of the genomic material, and the releasing of progeny. a comperhensive view of the process is illustrated in figure 3 . the main actors are well known to be cholesterol, sphingolipids, and pis, but other lipid species and their related pathways such as the la/aa axis are also relevant. how to target the lipid metabolism in a safe manner to avoid virus infection or reduce its pathogenity is a promising therapeutic tool, but it demands improving the knowledge on the actual pathways that are affected over the virus life cycle. the exact mechanism through which the enzyme inhibitors act on the key enzymes of the lipid metabolism is additionally required to develop more efficient and safe therapeutic drugs. since the lipid metabolism is essential for proper 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supplementation on dengue virus replication, toll-like receptor expression, and cytokine profiles on dendritic cells viroporins: structure and biological functions viral membrane channels: role and function in the virus life cycle autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-signaling is required for rotavirus replication this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank m. s. crespo for helpful reading of the manuscript. the authors declare no conflict of interest. the authors thank m. s. crespo for helpful reading of the manuscript. the authors declare no conflict of interest. key: cord-316745-n10ia3j3 authors: liu, hongde; wang, rui; lu, xiaoquan; chen, jing; liu, xiuhui; ding, lan title: a new approach to the prediction of transmembrane structures date: 2008-05-23 journal: chin sci bull doi: 10.1007/s11434-008-0055-5 sha: doc_id: 316745 cord_uid: n10ia3j3 about 20%–30% of genome products have been predicted as membrane proteins, which have significant biological functions. the prediction of the amount and position for the transmembrane protein helical segments (tmhs) is the hot spot in bioinformatics. in this paper, a new approach, maximum spectrum of continuous wavelet transform (mscwt), is proposed to predict tmhs. the predictions for eight sars-cov membrane proteins indicate that mscwt has the same capacity with software tmpred. moreover, the test on a dataset of 131 structure-known proteins with 548 tmhs shows that the prediction accuracy of mscwt for tmhs is 91.6% and that for membrane protein is 89.3%. as the important components of nerve signal molecule, hormone, acceptors and all kinds of ion channels, membrane proteins play an important role in the life activity of the cells [1] . they are also the binding sites of some drugs [2] . however, directly measuring 3-d structure of the membrane proteins with experimental way (x-ray crystal diffraction) is not easy because their stable natural conformations often need the assistant of the biology membrane. therefore, it is one of the important bioinformatics subjects to develop a highly effective and accurate approach to predicting the structure of the membrane protein. in literature [3] , protein sequence was converted into a digital signal by substituting amino acid residues with their hydrophobic free energy. then, a sliding window was used to scan the signal. the probable tmhs were predicted by a reasonable threshold. von heijine proposed the well-known 'positive-inside rule' [4] , which provided a further guide for prediction. multiple sequence alignment information of membrane proteins was also used in such prediction [5] . from view of method used, besides of neural networks and sliding window technique [4, 5] , markov model was also used [6] . in a markov model, seven states were designed to re-spond to seven regions of membrane protein, namely, mohelix core, cap cyt, cap non-cyt, loop cyt, short loop non-cyt, long loop non-cyt and globular [6] . in literature [7] , the inclinations that each amino acid occurred in seven regions were computed. then, the prediction was achieved by dynamic programming algorithm. similarly with literature [7] , literature [8, 9] also achieved the prediction based on amino acid occurrence frequency. the improvement was the prediction for transmembrane directions. wavelet transform is called "the mathematical microscope" [10, 11] , which has the good capacity in dealing with non-stationary signal and singular signals. its first application in bioinformatics was the prediction for the protein hydrophobic core [12] . it was also found that wavelet transform has the capacity of recognizing the different structures of proteins [13] . the detection of protein motifs can also be achieved by wavelet transform [14] . recently, a wavelet-based transmembrane prediction was reported, in which the prediction was based on the single-scale continuous wavelet transform (cwt) [15] . in this paper, mscwt was proposed to predict tmhs of membrane proteins. the method has been successfully applied in resolving overlapped signals including high performance liquid chromatography signals, differential pulse voltammetric signals, ultraviolet-visible spectrum signals and so on [16] . differing from single-scale maximum detection, mscwt is based on multi-scale cwt. it has the capacity of detecting the maximum on different scales simultaneously, which is very helpful in analyzing multi-frequency constituent signals. the test dataset is retrieved from the latest mptopo database (http://blanco.biomol.uci.edu/mptopo/). the protein structures have been resolved by crystallography. eight sars-cov membrane protein sequences are retrieved form the website http://athena.bioc.uvic.ca/sars/ map/diagram.html. they are orf3, orf4, orf7, orf8, orf9, orf14, m and s. given a signal f(t) of time domain, its cwt is expressed with eq. (1), where ψ is the wavelet, a and b is the scale and the translation, respectively. wf(a,b) is a function of a and b. mscwt is derived from cwt. it is designed to locate signals' maximum positions, especially for multifrequency constituent signals. mscwt of a signal could be obtained from the following steps: firstly, choose an appreciated mother wavelet and an appreciated scale range to perform cwt; then, detect and record the cwt maximum at every translation; finally, plot the recorded maximum value to its position (translation). the following is the mscwt pseudocode: ***************************** for b =1 to length of signal for a =a1 to a2 'a1 and a2 form the range of scale' find and record the maximum of wf(a, b) next a plot the maximum to b next b notes: a1 and a2 refer to the bottom and top limit of the scale range, respectively; wf(a,b) are cwt coefficients. ***************************** in this paper, morlet function is chosen as the analytical wavelet. to get mscwt with a higher resolution, scales are set from 1 to 64. the amounts and positions of tmhs are obtained as the following steps: step 1: convert protein sequence into a raw signal by substituting amino acid residue with its hydrophobic free energy [3] . step 2: perform cwt for the raw signal and obtain mscwt. step 3: set 1.5 as threshold and find regions more than the threshold in mscwt as the candidates of tmhs. if the length of the candidate is less than 20 amino acid residues, the candidate is not tmhs because it is too short. if two or more tmhs are very close (less than three amino acids), they are combined a whole tmhs. the prediction is evaluated at three aspects: amino acid, tmhs and protein sequence [17] : (1) the prediction accuracy of amino acid residues the prediction accuracy rate of amino acid residues is calculated by f aacor =(n aacor /n aaall )×100% where n aacor is the number of correctly predicted amino acid residues; n aaall is the total number of amino acid residues predicted. (2) the prediction accuracy of tmhs i. fp (false-positive): the number of wrongly predicted tmhs; ii. fn (false-negative): the number of not predicted tmhs; iii. prediction accuracy of tmhs: p q m c = × × 100%, where m=n cor /n obs (n cor is the number of correctly predicted tmhs, n obs is the number of observed tmhs), and m can be regard as a measure index of sensitivity; c = n cor /n prd (n prd is the total number of predicted tmhs), and c is regarded as a measure index of specificity. (3) the prediction accuracy of membrane protein sequences once all the transmembrane regions of a membrane protein sequence are predicted properly, the whole membrane protein sequence is considered to be predicted correctly. the prediction accuracy of membrane articles chemical biology protein sequences is: q t = (n tt /n tor ) ×100%, where n tt is the number of correctly predicted membrane protein sequences and n tor is the number of membrane protein sequences in the test sets. the predictions by mscwt are compared with those by other two software, tmpred (http://www.ch.embnet. org/software/tmpred_form.html) and das (http:// www.sbc.su.se/~miklos/das/). mscwt can be used to predict the existence of tmhs and provide the information about the beginning position and end position. moreover, it can be used to analyze the finer structures of proteins. figure 1 shows the tmhs predicted by mscwt and tmpred for sars-cov protein orf9 and orf14. for the predictions of tmpred (subplot a' and b'), solid line and dotted line represent two model signals. i→o and o→i indicate the direction from inner membrane to outer membrane and vice versa, respectively. the predictions for eight sars-cov proteins are listed in table 1 . from figure 1 and table 1 , it is concluded as follows: (1) mscwt amplifies the profile of hydrophobic and hydrophilic residues, which is helpful in observing the changes of protein hydrophobia. the great peaks in mscwt respond to the changes of hydrophobias, which is the characteristics of tmhs. in fact, the mscwt curve more than the threshold (1.5) denotes tmhs. figure 1 shows that the predictions by mscwt are in consistence with those by tmpred, indicating mscwt has a good precision. (2) in this paper, cwt is performed on a scale range, which makes that the large scale corresponds to lower frequencies of signals while small scale to high frequencies. thus, mscwt can be used to study the global properties of signals as well as local properties. in mscwt, large span peaks are not very smooth and contain some small peaks, indicating the hydrophobias vary with residue positions in tmhs, which suggests that the protein has fine folding structure in tmhs and such information can be observed by mscwt. this is why those small peaks are not filtered. our experifigure 1 comparison of mscwt and tmpred in predicting the sars-cov proteins orf9 and orf14. the longitudinal axis is defined by forecast signals, and the transverse axis is defined by points at transmembrane protein sequences. (a) mscwt for orf9, tmhs predicted is from 8 to 30; (b) mscwt for orf14, tmhs predicted is from 36 to 58; (c) tmpred for orf9, tmhs predicted is from 9 to 30; (d) tmpred for orf14, tmhs predicted is from 36 to 58. in tmpred, solid line and dotted line represent two signals suggested, i→o indicates the direction from inner membrane to outer membrane, and o→i indicates the reverse direction. for the accuracy rate of tmhs, tmpred is 93.1%; mscwt is 89.7%; and das is 89.0%. mscwt has the highest accuracy rate of membrane protein sequence (84.6%), while tmpred and das are 75.4% and 80.0%, respectively. all the above indicates that mscwt has a relatively high performance among the existing methods. in this paper, the proposed new method mscwt shows a good efficiency in predicting the positions and amounts of tmhs of membrane protein. the limit of mscwt is that it cannot provide information about the direction of tmhs. how protein chemists learned about the hydrophobic factor computational searching and mutagenesis suggest a structure for the pentameric transmembrane domain of phospholamban a simple method for displaying the hydropathic character of a protein hydrophobicity analysis and the positive inside rule transmembrane helices predicted at 95% accuracy a hidden markov model for predicting transmembrane helices in protein sequences a model recognition approach to the prediction of all-helical membrane protein structure and topology prediction of transmembrane segments in proteins utilizing multiple sequence alignments topology prediction of membrane proteins wavelet analysis as a new method in analytical chemometrics analysis techniques in analytical chemestry the hydrophobic cores of proteins predicted by wavelet analysis wavelet transformation of protein hydrophobicity sequences suggests their memberships in structural families wavelet transforms for the characterization and detection of repeating motifs prediction of transmembrane proteins based on the continuous wavelet transform maximum spectrum of continuous wavelet transform and its application in resolving an overlapped signal performance analysis of methods that predict transmembrane regions key: cord-333966-st6gyozv authors: taherkhani, reza; farshadpour, fatemeh; makvandi, manoochehr title: design and production of a multiepitope construct derived from hepatitis e virus capsid protein date: 2015-03-17 journal: j med virol doi: 10.1002/jmv.24171 sha: doc_id: 333966 cord_uid: st6gyozv the aim of this study was to design a high density multiepitope protein, which can be a promising multiepitope vaccine candidate against hepatitis e virus (hev). initially, conserved and antigenic helper t‐lymphocyte (htl) epitopes in the hev capsid protein were predicted by in silico analysis. subsequently, a multiepitope comprising four htl epitopes with high‐affinity binding to the hla molecules was designed, and repeated four times as high density multiepitope construct. this construct was synthesized and cloned into pet‐30a (+) vector. then, it was transformed and expressed in escherichia coli bl21 cells. the high density multiepitope protein was purified by ni‐nta agarose and concentrated using amicon filters. finally, the immunological properties of this high density multiepitope protein were evaluated in vitro. the results showed that the high density multiepitope construct was successfully expressed and purified. sds‐page and western blot analyses showed the presence of a high density multiepitope protein band of approximately 33 kda. approximately 1 mg of the purified protein was obtained from each liter of the culture media. moreover, the purified multiepitope protein was capable of induction of proliferation responses, ifn‐γ elispot responses and ifn‐γ and il‐12 cytokines production in a significant level in peripheral blood mononuclear cells (pbmcs) isolated from hev‐recovered individuals compared to the control group. in conclusion, the newly produced multiepitope protein can induce significant t helper type 1 responses in vitro, and can be considered as a novel strategy for the development of hev vaccines in the future. j. med. virol. 87:1225–1234, 2015. © 2015 wiley periodicals, inc. hepatitis e virus (hev) is a positive-stranded rna virus which has been classified as the member of the genus hepevirus in the family hepeviridae [yamashita et al., 2009 ]. at least four major hev genotypes have been identified, which have different geographical distributions and host ranges [xing et al., 2011] . despite classification into four geographically distinct genotypes, the hev strains antigenically belong to a single serotype and share cross-reacting epitopes due to the high degree of sequence conservation [wang and zhuang, 2004; xing et al., 2011] . genotype 1 includes most of human hev strains which is endemic in asia and north africa, genotype 2 is identified in human and includes the mexican strain and few strains of african countries. genotypes 3 and 4 have been identified in pigs and other animals; however, humans can be infected with these two genotypes too. genotype 3 predominates in industrialized countries, and genotype 4 is endemic in asia, particularly china, taiwan, and japan yamashita et al., 2009] . hev causes acute hepatitis in one-third of the world's population [zhu et al., 2010] , with mortality rate of 1-15% in the general population [xing et al., 2011] . however, sporadic and epidemic outbreaks of the acute hepatitis usually lead to a self-limited disease, but the infection is potentially fatal and causes fulminant hepatitis in susceptible individuals such as pregnant women and patients with underlying liver disease [zhu et al., 2010; xing et al., 2011] , and has high mortality rate of up to 30% among pregnant women [xing et al., 2011] . therefore hev is a significant public health problem worldwide especially in endemic areas and primary prevention of the hev infection seems essential. currently, no protective vaccine against hev infection has been licensed and primary prevention remains limited [wang and zhuang, 2004] . development of an effective vaccine, which can significantly reduce the prevalence of this disease, is a desirable goal. killed and live attenuated vaccines are not available due to the lack of a permissive cell culture system for hev replication . therefore the main focus for vaccine design is on molecular approaches, especially expression of viral proteins in different expression systems jimenez de oya et al., 2012] . the hev genome ($7.2 kb) contains three partially overlapping open reading frames (orfs), including orf1, orf2 and orf3 [zhou et al., 2004; jimenez de oya et al., 2012] . orf2, which encodes capsid protein, is highly conserved and immunogenic among hev species . due to hydrophobicity and insolubility of full-length capsid protein (72 kda) [tsarev et al., 1994; zhang et al., 2001] , new generation vaccines such as multiepitope vaccines are of particular interest. therefore, the present study was undertaken to design a high density multiepitope protein compromising four htl epitopes with high-affinity binding to the hla molecules using the in silico analysis, and to evaluate the immunological properties of this protein in vitro. overall, this study represents a novel strategy for the development of hev vaccines in the future. construct from hev orf2 protein the amino acid sequences of the orf2 from standard strains of hev genotype 1 including pakistan/ sar-55 strain (accession number p33426), china strain (accession number q81871), burma strain (accession number p29326), myanmar strain (accession number q04611) and india strain (accession number q68985) were obtained from uniprot knowledgebase (www. uniprot.org). the sequences were aligned by mega software version 4.0 (biodesign institute, tempe, az) [tamura et al., 2007] , and then the conserved sequences among these strandard strains were determined. the highly conserved hev-derived hla-dr-restricted htl epitopes of these strains were predicted by syfpeithi online software (http://www.syfpeithi.de/ home.htm). hla-dr binding affinity of the predicted epitopes was determined by iedb analysis resource online program (http://tools.immuneepitope.org/analyze/html/mhc_ii_binding.html). genetic diversity of mhc molecules is very high in human population; therefore htl epitopes with high population coverage are needed for this study. four epitopes with highaffinity to the most common hla-dr alleles in iran and world population were selected. therefore it is predicted that this construct could cover the most world population. the four htl epitopes (15 amino acids) derived from hev capsid protein including hla-drb1 ã 1501 (a150-164), hla-drb1 ã 0701 (b320-334), hla-drb1 ã 1101 (c370-384), hla-drb1 ã 0401 and hla-drb3 ã 0101 (dr52) (d491-505)restricted epitopes were binded to each other as a linear multiepitope (abcd). to avoid creation of junctional epitopes, the construct was designed with amino acid spacer sequences (gly-pro-gly-pro-gly) between sequential htl epitopes [livingston et al., 2002; depla et al., 2008] . the four times repeated multiepitope was constructed as high density multiepitope (fig. 1a) . based on e. coli codon usage, the nucleotide sequence of the high density multiepitope gene was optimized using genscript rare codon analysis tool (genscript, piscataway, nj) for efficient expression. in order to simplify the purification of the target protein, a sequence encoding 8-his tag was added at the c-terminal of the optimized gene followed by adding two stop codons (taa and tag) to terminate protein synthesis. two restriction-enzyme digestion sites, ndei and xhoi, were placed in the both ends of the codon-optimized gene to subclone it into the pet30a (þ) vector. ndei digestion site (catatg) with starting codon (atg) was inserted at the n-terminal of the construct. the xhoi digestion site (ctcgag) was inserted at the c-terminal of the construct after the second termination codon. in silico digestion was performed using clone manager basic software version 9 (sci-ed software, cary, nc), and was checked by vector nti software (invitrogen, carlsbad, ca). finally, the optimized high density multiepitope sequence was synthesized and cloned into the commercial cloning vector, pbluescript ii sk (þ) by biomatik company (biomatik corporation, cambridge, canada). both pbluescript ii sk (þ) vector carrying the high density multiepitope gene (pbluescript ii sk-high density multiepitope) and the pet-30a (þ) plasmid (novagen, madison, wi) were digested by ndei and xhoi restriction enzymes (new england biolab, ipswich, ma). after ligation by t4 dna ligase (new england biolab, ipswich, ma), the recombinant plasmid pet30a-high density multiepitope was generated and transformed into e. coli dh5a competent cells (novagen) by electroporation [chassy et al., 1988] . the subcloning was confirmed by colony pcr, restriction-enzyme digestion, and dna sequencing. the recombinant plasmid was then transformed into competent e. coli bl21 (de3) cells to enhance protein expression (novagen). e. coli bl21 (de3) cells containing the recombinant plasmid were cultured overnight, then inoculated in terrific broth (himedia, mumbai, india) supplemented with kanamycin (50 mg/l) in a 1:100 volumetric ratio, and grown with shaking at 250 rpm at 37˚c until the optical density at 600 nm (od 600) reached 0.5. the bacterial culture was induced by adding various concentrations of iptg (0.1-1 mm) (sigma-aldrich, st. louis, mo), and grown with shaking for different induction times (2, 4, 6, 8, and 16 hr) at different induction temperatures (37˚c, 30˚c, and 25˚c) to determine the optimal conditions of protein expression. after the large-scale protein expression, the target protein was purified by ni-nta (qiagen, hilden, germany) under denaturing conditions using 8 m urea . the purified protein was dialyzed in pbs containing 10% glycerol with a linear urea gradient of 6, 4, and 2 m at 4˚c for 8 hr in order to decrease the amount of urea, and concentrated using amicon ultra-4 centrifugal filter unit (emd millipore, billerica, ma). sds-page and western blot were performed to confirm the presence of the target protein [laemmli, 1970; hao et al., 2013; taherkhani et al., 2014] . concentration of the protein was measured by the bradford method [bradford, 1976] . to remove bacterial endotoxin contamination in the purified protein, toxin eraser endotoxin removal kit was used according to the manufacturer procedure (genscript). the truncated orf2 protein (aa 112-608) of hev strain sar-55 ( fig. 1b) was expressed in e. coli bl21 host cells using the recombinant plasmid pet-30a-orf2 (aa 112-608) and purified by ni-nta (qiagen) as described previously . this protocol was approved by the ethical committee of ahvaz jundishapur university of medical science. a statement on ethical committee approval and the informed consent was taken from all participants. ten recovered individuals from acute hepatitis e infection (6 males and 4 females; mean age ae sd, 40.30 ae 13.76 years) with positive anti-hev igg antibody were enrolled in this study. the control group consisted of 12 healthy individuals (7 males and 5 females; mean age ae sd, 38.08 ae 15.35 years) with no history of acute hepatitis, and with negative anti-hev igm/igg antibodies. the commercial elisa kits (hev igg and hev igm elisa kits, dia.pro, milan, italy) were used for all the participants. all the participants were negative for hepatitis b surface antigen, anti-hav igm antibody and anti-hcv antibodies (hbs ag elisa kit, hav igm elisa kit, hcv ab elisa kit, dia.pro) with normal alt level. peripheral blood mononuclear cells (pbmcs) of the each individual were isolated from 10 ml heparinized venous blood by density gradient centrifugation using ficoll-hypaque (lymphoflot, biotest, dreieich, germany). the isolated pbmcs were washed and resuspended in complete rpmi 1640 medium (invitrogen). the viability of the cells was assessed by trypan blue staining. the cell proliferation assay was performed using non-radioactive mtt (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) assay [majumdar et al., 2013] . in brief, approximately 1 â 10 5 cells/well of pbmcs of each sample in rpmi 1640 and 10% fcs were added to four wells of round-bottom 96-well plates in total volume of 180 ml/well, stimulated with 20 ml/well of truncated orf2 protein (10 mg/ml), high density multiepitope (10 mg/ml) and phytohemagglutinin (pha) (5 mg/ml) (sigma-aldrich) separately, and incubated at 37˚c for 4 days. the unstimulated well of each sample was used as negative control. rpmi 1640 (20 ml) was used as blank. after 4 days, 10 ml of mtt solution (5 mg/ml) was added to each well and incubated at 37˚c for 4 hr. then, 100 ml of isopropanol containing 0.04 n hcl was added to each well and mixed thoroughly. finally, the absorbance of the wells was read at 570 nm with a reference wavelength of 630 nm by an elisa reader (tecan sunrise elisa reader; tecan trading ag, mannedorf, switzerland). the results were showed as proliferation index (pi), calculated as od570 stimulated sample/od570 unstimulated sample. the elispot assay for hev-specific ifn-g-secreting cells was carried out by human ifn-g elispot ready-set-go kit (ebioscience, san diego, ca) according to manufacturer's protocol. briefly, a 96-well pvdf bottomed elispot plate (millipore, bedford, ma) was coated with 100 ml/well of capture antibody solution and incubated overnight at 4˚c. approximately 2 â 10 5 cells/well of pbmcs of each sample suspended in rpmi 1640 and 10% fcs along with 10 mg/ml high density multiepitope protein and 10 mg/ ml truncated orf-2 protein separately were added to 2 wells in total volume of 150 ml/well. for negative control unstimulated pbmcs was used. for positive control, 5 mg/ml of pha (sigma-aldrich) was used. after 24-hr incubation at 37˚c, cells and medium were decanted from the plate and the plate was washed three times with elispot wash buffer. 100 ml of biotinylated detection antibody was added to each well and incubated overnight at 4˚c. after incubation, the plate was emptied and washed as described above. 100 ml/well of avidin-horseradish peroxidase reagent was added, and the plates were incubated at room temperature for 45 min. after incubation, the plates were washed three times with elispot wash buffer and two times with 1x pbs; then, 100 ml/well of freshly prepared aec (3-amino-9ethyl carbazole) substrate solution (sigma-aldrich) was added to the wells and incubated at room temperature until dark purple spots appears. the reaction was stopped by washing wells three times with 200 ml/well distilled water. plate was air-dried and the number of ifn-g-producing cells in each well was counted by a dissection stereoscope (nikon, tokyo, japan) and expressed as spot-forming cells (sfcs) per 10 5 cells. at a concentration of 1 â 10 5 cells/well, pbmcs of each sample in rpmi 1640 and 10% fcs were added to the four wells of 96-well plates, and stimulated with truncated orf2 protein (10 mg/ml), high density multiepitope protein (10 mg/ml), and pha (5 mg/ml) separately at 37˚c. the unstimulated pbmcs of each sample were used to evaluate spontaneous production of cytokines. culture supernatants were collected after 48 hr (24 hr for il-12). levels of four cytokines (il-12 p70, ifn-g, il-4 and il-10) were measured in duplicate by commercial elisa kits (ebioscience) according to the manufacturer's instructions. the elisa results were expressed as picograms per milliliter (pg/ml). statistical analysis was performed using spss 17 software (spss, chicago, il) and p values of less than 0.05 were considered statistically significant. discrete variables were compared using the pearson's chi-square test or fisher's exact test. intergroup comparisons (recovereds vs. controls) were performed using independent t test or mann-whitney u test. intragroup comparisons (high density multiepitope versus orf2) were made by paired t test or wilcoxon test. all data were presented as mean ae se. the high expression of the protein was induced by iptg at a final concentration of 1 mm at 30˚c for 4 hr. sds-page and western blotting analysis of the resultant protein showed a protein band of approximately 33 kda corresponding to high density multiepitope (fig. 2) . approximately 1 mg of purified protein was obtained from each liter of culture media. the cell proliferative responses in hev-recovered group following stimulation with high density multiepitope protein and truncated orf2 protein were found higher than control group (p < 0.001); and these responses were observed higher with high density multiepitope protein than truncated orf2 protein in hev-recovered group (p ¼ 0.013) ( table i, fig. 3 ). ifn-g elispot responses to high density multiepitope protein and truncated orf2 protein were found significantly higher in hev-recovered individuals than control group (p < 0.001). these responses to high density multiepitope protein were observed slightly higher than truncated orf2 protein in hevrecovered group, with no significant observation (p ¼ 0.220). table i and figure 4 show the frequency of ifn-g-secreting cells following stimulation with high density multiepitope protein and truncated fig. 2 . a: sds-page analysis of the expressed and purified high density multiepitope protein in e. coli. the high density multiepitope protein band is visualized at approximately 33 kda. lane 1, uninduced control; lane 2, induction with 1 mm iptg for 4 hr; lane 3, induction with 1 mm iptg for 8 hr; lane 4,5, flow-through; lane 6-8, washing steps of the ni column; lane 9-11, the first, the second, and the third purified eluates of high density multiepitope protein from the ni column; lane 12, prestained protein ladder (cinagen, tehran, iran). b: western blot analysis of the expressed and purified high density multiepitope protein in e. coli. the high density multiepitope protein band is visualized at approximately 33 kda. lane 1, prestained protein ladder (cinagen); lane 2, flow-through; lane 3, wash; lane 4-7, the first, the second, the third, and the fourth eluates. orf2 protein in hev-recovered individuals and control group. the levels of ifn-g and il-12 p70 responses to truncated orf2 protein and high density multiepitope in the recovered group were found significantly higher compared to control group (p < 0.001). however, the levels of these stimulated cytokines with high density multiepitope protein were found higher than truncated orf2 protein with no significant observation. the levels of specific il-10 for high density multiepitope protein and truncared orf2 protein were found moderate among the hev-recovered group, while the levels of specific il-4 for the both proteins were low (table i, fig. 5 ). a better understanding of the hev-specific cellular immune responses may help us in designing and development of vaccine for the prevention and control of the hev infection. although some progress has been achieved in developing hev vaccine, but due to some limitations in current approach for vaccine preparation such as the duration of the response and cost effectiveness of vaccine, the attempt was made to develop an alternative hev vaccine [jimenez de oya et al., 2012] . the virus-specific t-helper cell immune responses have been shown to be essential for controlling and clearing viral infection by activating cytotoxic t cells and producing cytokines especially inf-g which can suppress viral replication. thus, viral proteins comprising helper t-lymphocyte (htl) epitopes may be useful as protective vaccines [aggarwal et al., 2007] . it has been shown that hev capsid protein has several immunogenic epitopes [gu et al., 2004] . shata et al. identified dominant and subdominant epitopes of hev capsid protein such as amino acids 271-286, 199-214, 463-478, 583-598, and 631-646 [shata et al., 2007] . aggarwal et al. mapped cd4 t-cell epitopes in hev orf2 protein, and found the immunodominant t-cell epitopes of capside protein are encompassed amino acids 73-156, 289-444, and 505-588 [aggarwal et al., 2007] . khudyakov et al. reported the antigenic regions in hev orf2 protein are encompassed amino acids 319-340, 631-648, and 641-660 [khudyakov et al., 1993] . selection of the epitopes is the most important part of the design of the epitope-based vaccines that can guarantee the effectiveness and extent of the desired immune responses. this study focused on the consereved epitopes with high-affinity binding to the different hla molecules to cover the majority of the human population, on this basis the multiepitope protein comprising these epitopes was constructed. this multiepitope design is a new strategy and may counter the limitations present in current vaccine against hev, but it requires further investigation. to date, no application of multiple hla-dr-restricted epitopes was reported to induse htl responses against hev infection. oany et al. designed a multiepitope vaccine candidate compromising b cell and t cell epitopes from spike protein of human coronavirus by in silico analysis. in their study, sequences of the spike proteins were collected from the uniprotkb database and the consereved epitopes with high-affinity binding to the different hla molecules were predicted using in silico tools to cover the majority of the world population. this computational design was a novel study to determine antigenic epitopes in spike protein and to design an epitope-based vaccine against human coronavirus [oany et al., 2014] . these type of vaccines were reported to have many advantages including the stability, lack of carcinogenic, large scale production with the least equipments, no need for cold chain storage, the ability to stimulate the immune system against a wide variety of strains and genotypes of a microorganism, and use in different races or high population coverage [elliott et al., 1999; livingston et al., 2002; sette and fikes, 2003; jackson et al., 2006] . meanwhile, some disadvantages like the creation of junctional epitopes in the sequential arrangement of the epitopes have limited using these constructs [livingston et al., 2002; depla et al., 2008] . junctional epitopes is created by the juxtaposition of two epitopes, and can suppress the immune responses to the main epitopes [livingston et al., 2002] . this problem was overcome through application of the spacer residues between the epitopes to improve appropriate epitope processing. furthermore, it has been reported that if the epitope is expressed with high density will enhance protective immunity [liu et al., 2004; lu et al., 2008] . lu et al. designed constructs consisting of various v3 epitope densities from hiv-1 gp120, and found that high epitope density in one single protein molecule could enhance protective immunity [lu et al., 2008] . liu et al. reported that high epitope density from m2 protein of influenza virus enhanced specific humoral immunity against flu virus [liu et al., 2004] . vaccine development is dependent not only on selecting antigens and epitopes but also on defining the immune responses against target epitopes. by evaluating the antigenicity and immunogenicity of the high density multiepitope protein, it was indicated that the high density multiepitope protein stimulates slightly stronger t helper cell responses compared to truncated orf2 protein. it has been shown that during acute infection, virus-specific t helper cell responses can clear viral infection but lack of its activity leads to chronic infection [macdonald et al., 2002] . in this study, the hla typing was not carried out and the frequencies of various hla alleles among the participants were not determined. so it is not clear which type of hla alleles among the hev-recovered individuals had a low or highaffinity hla binding epitopes against the multiepitope protein. li et al. designed a multiepitope construct comprising six low-affinity hiv gag and pol ctl epitopes restricted to hla-a ã 0201 and the hiv p24 antigen by bioinformatics analysis. they used cryptic epitope modification to enhance the immunogenicity of lowaffinity hla-a2.1-binding epitpes, and found the multiepitope can induce a strong cd8þ t cell immune responses in mice [li et al., 2013b] . jafarpour et al. designed and synthesized a multiepitope construct encoding conserved and immunogenic epitopes from hiv-1 tat, pol, gag, and env proteins by bioinformatics analyses as a new dna vaccine candidate. the designed multiepitope plasmid was able to induce proper immune responces in balb/c mice [jafarpour et al., 2014] . li et al. designed a multiepitope construct encoding t and b lymphocyte epitopes from latent membrane protein 2 (lmp2) of epstein-barr virus (ebv) based on mammalian codon usage. the recombinant plasmid pcdna3.1þ/ ebv-lmp2 multiepitope was constructed and transfected into cos-7 cells. this multiepitope plasmid dna was able to stimulate strong cellular and humoral immunity in mice [li et al., 2013a] . in the present study, high level of specific ifn-g and il-12 responses (t helper type 1 cytokines) and low level of il-10 and very low level of il-4 responses (t helper type 2 cytokines) against high density multiepitope protein and truncated orf2 protein were observed among the hev-recovered individuals. thus, both proteins can be considered as promising vaccine candidate against hev infection. hashish et al. designed a multiepitope fusion protein compromising two b cell and t cell epitopes from the e2 glycoprotein of bovine viral diarrhea virus (bvdv), and also k99 major subunit fanc and sta toxoid of enterotoxigenic escherichia coli (etec). in their study, fanc-sta-e2 gene construction was cloned into the expression vector pet28 and transformed into e. coli bl21 cells. fanc-sta-e2 fusion protein was well expressed by iptg-induction (1 mm) at 37˚c for 4 hr and purified by ni-nta agarose using his-tag. this multiepitope fusion protein could develop neutralizing antibodies against etec and bvdv in vitro [hashish et al., 2013] . beside vaccine development, multiepitope technology can be used for diagnosis. several studies have been performed on multiepitopes proteins for development of diagnostic kit. the results of these studies showed that the use of multiepitope proteins can increase the sensitivity and specificity of the tests, since this kind of proteins has great efficiency to expose conserved and immunodominant epitopes [dipti et al., 2006; de souza et al., 2013] . de souza et al. designed a high density multiepitope protein containing three copies of four conserved epitopes from hepatitis b core protein (hbcag), and developed an elisa using this recombinant protein. the developed elisa was useful for hepatitis b diagnosis [de souza et al., 2013] . dipti et al. designed a novel recombinant multiepitope protein comprising six conserved and immunodominant epitopes from hepatitis c virus, and developed an anti-hcv diagnostic kit using this protein. the multiepitope protein was able to detect anti-hcv antibodies in human plasma with high degree of sensitivity and specificity [dipti et al., 2006] . although several microorganisms and cell lines are currently used for expression of foreign genes, but still e. coli are routinely used for expression of target proteins [vincentelli and romier, 2013] . fast cultivation, well-known genetics and high-level expression capabilities with high purity are some other advantages of e. coli expression system [fakruddin et al., 2013] . despite all these advantages, expression of a foreign gene in e. coli may be hampered by the presence of rare codons and high gc contents within the gene-coding region which result in decrease of the expression level or production of insoluble and nonfunctional proteins [mondal et al., 2013; elena et al., 2014] . in the present study, e. coli bl21 cell was used for expression of the high density multiepitope protein. the target genes were cloned in to the pet30aþ plasmid and expressed by iptg-induction under control of strong bacteriophage t7 promoter and t7 rna polymerase of the e. coli bl21 cell. the iptg-induction is economically desirable method and increases protein production [mondal et al., 2013] . previous studies have reported high yield protein purification by ni2þ chelate affinity chromatography using his-tag [glynou et al., 2003; farshadpour et al., 2014] . the application of affinity tags is simple, rapid, cost effective and also efficient for purification of proteins, and also has no effect on the structure, folding and function of the protein [glynou et al., 2003; carson et al., 2007] . in the present study, histag was used for the purification. the results of this study show that e. coli is a good system for expression of recombinant proteins with high yield. in conclusion, a high density multiepitope protein capable of induction of t helper type 1 responses was successfully produced and evaluated in vitro. the results of this study provide much information for the design of hev multiepitope vaccine. however, further works will be needed to evaluate protective immune response of the high density multiepitope protein in vivo. t-cell epitope mapping of orf2 and orf3 proteins of human hepatitis e virus a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding his-tag impact on structure transformation of bacteria by electroporation a recombinant multiepitope protein for hepatitis b diagnosis rational design of a multiepitope vaccine encoding t-lymphocyte epitopes for treatment of chronic hepatitis b virus infections a novel recombinant multiepitope protein as a hepatitis c diagnostic intermediate of high sensitivity and specificity expression of codon optimized genes in microbial systems: current industrial applications and perspectives peptide based cytotoxic t-cell vaccines; delivery of multiple epitopes, help, memory and problems recombinant vaccines for hepatitis e critical factors affecting the success of cloning, expression, and mass production of enzymes by recombinant codon-optimized expression and purification of truncated orf2 protein of hepatitis e virus in escherichia coli one-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography selection of a peptide mimicking neutralization epitope of hepatitis e virus with phage peptide display technology cloning and prokaryotic expression of cdnas from hepatitis e virus structural gene of the sw189 strain a multiepitope fusion antigen elicits neutralizing antibodies against enterotoxigenic escherichia coli and homologous bovine viral diarrhea virus in vitro totally synthetic peptidebased vaccines that target dendritic cells and induce potent antibody or ctl responses clustered epitopes within a new poly-epitopic hiv-1 dna vaccine shows immunogenicity in balb/c mice characterization of hepatitis e virus recombinant orf2 proteins expressed by vaccinia viruses epitope mapping in proteins e virus cleavage of structural proteins during the assembly of the head of bacteriophage t4 immunogenicity of a multiepitope plasmid dna encoding t and b lymphocyte epitopes from latent membrane protein 2 (lmp2) of epstein-barr virus as a vaccine in mice immune responses induced in hhd mice by multiepitope hiv vaccine based on cryptic epitope modification high epitope density in a single recombinant protein molecule of the extracellular domain of influenza a virus m2 protein significantly enhances protective immunity a rational strategy to design multiepitope immunogens based on multiple th lymphocyte epitopes v3 ctl epitope density in a single recombinant molecule antigen differentially affects the number and activity of primary and memory cd8þ t cells cd4 t helper type 1 and regulatory t cells induced against the same epitopes on the core protein in hepatitis c virus-infected persons evaluation of antigenicity and cell mediated immunity of hepatitis e virus patients: using non radioactive mtt assay high yield expression of proteins in e. coli for nmr studies design of an epitope-based peptide vaccine against spike protein of human coronavirus: an in silico approach epitope-based vaccines: an update on epitope identification, vaccine design and delivery characterization of hepatitis e-specific cell-mediated immune response using ifn-gamma elispot assay development of enzyme-linked immunosorbent assays using 2 truncated orf2 proteins for detection of igg antibodies against hepatitis e virus mega4: molecular evolutionary genetics analysis (mega) software version 4.0 successful passive and active immunization of cynomolgus monkeys against hepatitis e expression in escherichia coli: becoming faster and more complex hepatitis e: an overview and recent advances in vaccine research spatial configuration of hepatitis e virus antigenic domain biological and immunological characteristics of hepatitis e virus-like particles based on the crystal structure immunogenicity and protective efficacy of a vaccine prepared from 53 kda truncated hepatitis e virus capsid protein expressed in insect cells an elisa for putative neutralizing antibodies to hepatitis e virus detects antibodies to genotypes 1, 2, 3, and 4 vaccine efficacy and safety of a recombinant hepatitis e vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled key: cord-321275-7haq0e38 authors: renzi, fabiana; panetta, gianna; vallone, beatrice; brunori, maurizio; arceci, massimo; bozzoni, irene; laneve, pietro; caffarelli, elisa title: large-scale purification and crystallization of the endoribonuclease xendou: troubleshooting with his-tagged proteins date: 2006-02-28 journal: acta crystallographica section f structural biology and crystallization communications doi: 10.1107/s1744309106006373 sha: doc_id: 321275 cord_uid: 7haq0e38 xendou is the first endoribonuclease described in higher eukaryotes as being involved in the endonucleolytic processing of intron-encoded small nucleolar rnas. it is conserved among eukaryotes and its viral homologue is essential in sars replication and transcription. the large-scale purification and crystallization of recombinant xendou are reported. the tendency of the recombinant enzyme to aggregate could be reversed upon the addition of chelating agents (edta, imidazole): aggregation is a potential drawback when purifying and crystallizing his-tagged proteins, which are widely used, especially in high-throughput structural studies. purified monodisperse xendou crystallized in two different space groups: trigonal p3(1)21, diffracting to low resolution, and monoclinic c2, diffracting to higher resolution. intron-encoded small nucleolar rnas (snornas) are non-coding rnas that play essential roles in eukaryotes and archaea. they are transcribed as longer precursors and are generally released through splicing (reviewed by filipowicz & pogacic, 2002) . nevertheless, some snornas can also be processed by endonucleolytic cleavage in the intron sequences flanking the snorna (fragapane et al., 1993) . xendou is the first endoribonuclease involved in the processing of snornas in higher eukaryotes to be described and is involved in the processing of at least two snornas in xenopus laevis oocytes (fragapane et al., 1993; caffarelli et al., 1994; renzi et al., 2002; laneve et al., 2003) . it is a u-specific enzyme that, uniquely among known endoribonucleases, releases 2 0 -3 0 cyclic phosphate termini using mn 2+ as an essential cofactor. xendou is conserved among higher eukaryotes and has no homology to any known cellular ribonuclease, although it shows significant homology to proteins tentatively annotated as serine proteases. a viral counterpart of xendou, called nendou, has been reported. it is a distinctive marker of the nidoviruses, which include the sars coronavirus (snijder et al., 2003) : the sars homologue shows biochemical characteristics that are similar to those of xendou, although it prefers doublestranded substrates, and is essential in virus replication and transcription (ivanov et al., 2004) . xendou was isolated from a cdna bank, cloned with an extra 12 n-terminal amino acids including a non-cleavable his 6 tag, expressed in bacteria, affinity purified and tested for activity in vitro (laneve et al., 2003) . mutational studies allowed the identification of the residues (glu161, glu167, his162, his178 and lys224) essential for rna cleavage, while enzyme-rna interaction assays led to the definition of the sequence required for mn 2+ -independent binding (gioia et al., 2005) . the xendou coding sequence was cloned downstream to the his 6coding region of the pqe30 vector (qiagen; laneve et al., 2003) . in order to obtain large amounts of protein for crystallization screening, xendou was overexpressed in bacteria [escherichia coli m15 (prep4) strain] in lb medium at 303 k to an optical density (od 600 nm ) of 0.55 and was induced with 0.1 mm isopropyl -dthiogalactoside (iptg) for 4 h with continuous agitation and good aeration. subsequent manipulations were carried out at 277 k. harvested cells were resuspended in lysis buffer (50 mm na/phosphate ph 7.8, 300 mm nacl), 10 mm imidazole and 1 mg ml à1 lysozyme while stirring for 2 h. after sonication, the cell lysate was centrifuged for 30 min at 18 000g and the clear supernatant was filtered (0.2 mm filter, sartorius) and applied onto a 5 ml ni-nta column (amersham) previously equilibrated with lysis buffer. the column was washed with 1 l washing buffer 1 (10 mm hepes ph 7.8, 300 mm nacl, 10 mm imidazole) and 30 ml buffer 2 (10 mm hepes ph 7.8, 50 mm nacl, 75 mm imidazole). the protein was eluted in 10 mm hepes ph 7.8, 50 mm nacl, 100 mm imidazole. repeated steps of ultrafiltration and dilution across 10 kda cutoff filters (vivascience) eliminated the imidazole. the his-tagged protein was purified by hplc gel filtration using a polymeric resin gel (pwxl-g3000 60 cm â 17 mm column; tosoh biosciences) and concentrated to 0.5 mm (17 mg ml à1 ) in 10 mm hepes ph 7.8, 50 mm nacl, 20 mm edta. sds-page analysis showed a protein purity of greater than 95%. xendou was also cloned into pgex (amersham biosciences) to create a gst-xendou fusion protein. the resulting expression plasmid was transformed into e. coli bl21 (de3). cells expressing the cleavable gst-tagged protein were grown and lysed following the same protocol used for the his-tagged xendou. the supernatant from the cell lysate was applied onto a glutathione-derivatized column (fft-gst; amersham biosciences) equilibrated with lysis buffer (10 mm tris-hcl ph 7.8, 50 mm nacl). the column was washed with 30 volumes of washing buffer 1 (10 mm tris-hcl ph 7.8, 300 mm nacl) and ten volumes of washing buffer 2 (10 mm tris-hcl ph 7.8, 50 mm nacl) and subsequently incubated with bovine thrombin (sigma; 0.5 u per milligram of protein) at 277 k overnight to allow tag proteolysis on the column. a fft-paba-derivatized resin column (amersham) that binds proteases using the protease inhibitor para-aminobenzoic acid (paba) was connected in line with the fft-gst column. a single elution step allowed the thrombin to be retained on the paba column and the untagged protein to be recovered in the flowthrough. the untagged xendou was hplc purified by gel filtration (pwxl-g3000 60 cm â 17 mm column; tosoh biosciences) and concentrated to 0.5 mm (17 mg ml à1 ) in 10 mm hepes ph 7.8, 50 mm nacl. sds-page analysis showed a protein purity of greater than 95%. the purity and monodispersity of the protein were analyzed by hplc gel filtration both on methacrylate resin gel, which is resistant over a wide range of ph, and silica resin gel, which is inert toward analytes (pwxl and swxl-g3000 columns, respectively, 30 cm â 0.7 mm; tosoh biosciences) (see fig. 1 ). the his-tagged xendou used in crystallization screening was solubilized in buffers containing 20 mm edta. the hanging-drop vapour-diffusion method and index screen (hampton research) were used for initial crystallization trials, with a drop volume of 1 + 1 ml and a reservoir volume of 0.5 ml. initial crystals grew in hanging drops with 30%(w/v) peg 4000, 0.2 m sodium citrate ph 5-6 at 293 k; these crystals were thin and fragile and did not diffract even at a synchrotron source. subsequent screening resulted in the growth of crystals (300 â 300 â 300 mm) in 40% (nh 4 ) 2 so 4 , 0.2 m sodium citrate ph 6 at 293 k which belonged to space group p3 1 21 but diffracted poorly; the resolution was improved by cocrystallization with 50 mm uridine 5 0 -monophosphate (5 0 -ump), which may bind to the active site, possibly inducing the stabilization of flexible regions (fig. 2a) . crystals diffracting to higher resolution in space group c2 were obtained by the substitution of sodium citrate by 0.2 m phosphate ph 5.5; these crystals were larger but were not single (fig. 2c, top) . single crystals were obtained by tuning the growth speed and nucleation by (i) depositing silicon oil above the precipitating solution to slow down phase equilibrium and (ii) using macroseeding and microseeding techniques. this led to the growth of large platelet-shaped single crystals (500 â 500 â 30 mm; fig. 2c , bottom) which diffracted to higher resolution (2.2 å ). crystals grown in 40% (nh 4 ) 2 so 4 , 0.2 m sodium citrate ph 6.0 were cryoprotected by soaking in mother liquor with the addition of 25% glycerol and tested for diffraction at 100 k. although they did not diffract when exposed on a cu k rotating-anode x-ray generator, they diffracted to 3.3 å resolution at a synchrotron source and proved to belong to the trigonal lattice, space group p3 1 21 (fig. 2b) . to overcome the spot overlap arising from the long c axis, effect of chelating agents in the solubilization of aggregated his-tagged protein. hplc gel-filtration analysis shows that 250 mm (red) and 100 mm (blue) imidazole yield monodisperse his-tagged protein. 20 mm (light green), 10 mm (dark green) and 5 mm (purple) edta also improve monodispersity in a concentrationdependent fashion. addition of 20 mm mn 2+ salt with and without edta (brown and pink) causes the protein to aggregate again. elution profiles were analyzed using the csw program. the radiation wavelength was increased to 1.2 å , the crystal-todetector distance was increased to 210.00 mm and diffraction images were collected with a thin oscillation range (á' = 0.4 ). crystals grown in 40% (nh 4 ) 2 so 4 , 0.2 m phosphate ph 5.5 diffracted to 2.2 å resolution, although not in all crystal orientations (fig. 2d) ; this is probably correlated to the thin third dimension and to imperfect or loose packing as suggested by the tendency of crystals to split into several thin platelets upon manipulation and to be heterogeneous in unit-cell parameters. monoclinic crystals in fact displayed at least two different unit cells (a and b forms) . different protocols of dehydration and iterated cryocooling techniques did not improve either the anisotropy or the resolution limit (samygina et al., 2000) . all diffraction data were indexed and scaled with the hkl package (otwinowski, 1993) , mosflm and scala (from the ccp4 package; collaborative computational project, number 4, 1994) . data-collection details, scaling statistics and main crystallographic parameters are summarized in table 1 . large-scale expression of his-tagged xendou resulted in soluble protein that was heterogeneously aggregated, a condition that affects crystallization (wilson, 2003) . to obtain monodisperse protein, several conditions were explored. only the addition of chelating agents (100 mm imidazole or 20 mm edta) yielded homogeneously disperse protein. we attributed the polydispersity to the formation of intermolecular bridges between his tags mediated by divalent metal ions present as contaminants. consistently, the addition of mn 2+ to a protein preparation which had been made monomeric using edta was sufficient to induce aggregation again (fig. 1) . to confirm the hypothesis of intermolecular bridges involving his tags via divalent metals and to exclude that possibility that these interactions could involve a site on the protein other than the his tag, we expressed a cleavable gst-fused xendou under the same conditions as the his-tagged enzyme. analytic hplc gel filtration both on methacrylate and on silica gel showed the protein to be completely monodisperse even after addition of mn 2+ , thus demonstrating that aggregation was dependent on the his tag and not on other metal-binding sites on xendou. screening for crystallization of non-tagged xendou after gst cleavage only yielded crystals using the same conditions as for the tagged proteins, but with poorer or unimproved diffraction. in summary, it was possible to reverse the aggregation mediated by the his tag through the addition of edta or imidazole, allowing the growth of crystals suitable for structural analysis. use of chelating agents would be a less expensive and time-consuming alternative to specific proteolytic cleavage of the his-tagged protein, which in the case of xendou was not effective for crystal improvement. this information may be valuable especially in high-throughput structural projects, where a large number of proteins are expressed, purified and screened for crystallization. data were collected at the elettra (trieste, italy) and esrf (grenoble, france) synchrotron facilities. this work was partially supported by miur of italy (firb/rbla03b3kc-004, firb/ rbne015mpb and prin/rbne01kxc9 and centro di eccellenza bemm). proc. natl acad. sci. usa proceedings of the ccp4 study weekend. data collection and processing key: cord-329149-1giy1fow authors: martinez-martin, nadia title: technologies for proteome-wide discovery of extracellular host-pathogen interactions date: 2017-02-22 journal: j immunol res doi: 10.1155/2017/2197615 sha: doc_id: 329149 cord_uid: 1giy1fow pathogens have evolved unique mechanisms to breach the cell surface barrier and manipulate the host immune response to establish a productive infection. proteins exposed to the extracellular environment, both cell surface-expressed receptors and secreted proteins, are essential targets for initial invasion and play key roles in pathogen recognition and subsequent immunoregulatory processes. the identification of the host and pathogen extracellular molecules and their interaction networks is fundamental to understanding tissue tropism and pathogenesis and to inform the development of therapeutic strategies. nevertheless, the characterization of the proteins that function in the host-pathogen interface has been challenging, largely due to the technical challenges associated with detection of extracellular protein interactions. this review discusses available technologies for the high throughput study of extracellular protein interactions between pathogens and their hosts, with a focus on mammalian viruses and bacteria. emerging work illustrates a rich landscape for extracellular host-pathogen interaction and points towards the evolution of multifunctional pathogen-encoded proteins. further development and application of technologies for genome-wide identification of extracellular protein interactions will be important in deciphering functional host-pathogen interaction networks, laying the foundation for development of novel therapeutics. the plasma membrane constitutes a critical biological interface between the cytosol and the extracellular environment of the cell, and consequently membrane-tethered proteins and secreted molecules (collectively termed extracellular proteins) are essential regulators of cellular communication. from high affinity cytokine-receptor interactions to low affinity cell-cell adhesion contacts, extracellular protein-protein interactions (eppis) are key for information processing and coordination of virtually all processes in a living organism. furthermore, given their fundamental functions and their accessibility to systemically delivered drugs, extracellular proteins are particularly suitable targets for therapeutic intervention. in fact, despite these proteins being encoded by approximately one-fourth of the human genes, at least two-thirds of the existing drugs target either secreted or membrane-bound proteins [1] . thus, the elucidation of the eppi networks on a global scale has become crucial for the biomedical research. however, in spite of their relevance and abundance, eppis are remarkably underrepresented in available large-scale datasets. this discrepancy is due to the low sensitivity and limited compatibility of current high throughput technologies to detect extracellular interactions because of the unusual biochemical nature of the membrane proteins and the intractability of their binding partners [2] [3] [4] . in particular, transmembrane domain-containing proteins are amphipathic, making it difficult to solubilize them in their native conformation, and often contain posttranslational modifications such as glycans and disulfide bonds, which are not properly added in common heterologous expression systems [5] . in addition, interactions between cell surface proteins are often characterized by fast dissociation rates and therefore weak binding affinities, and in consequence well-established ppi methods such as yeast-two-hybrid or affinity purification-mass spectrometry (ap/ms) largely fail to detect these interactions. over the last decade, several innovative technologies have been developed to overcome the aforementioned technical challenges and allow for sensitive 2 journal of immunology research detection of eppis [2, [6] [7] [8] [9] [10] . nevertheless, the mapping of eppis remains a major challenge in biology. infectious diseases result in millions of deaths each year and therefore identifying new candidate targets for improved therapeutic development remains a pressing health concern. pathogens have evolved a myriad of elegant and often complex strategies to invade the host and commandeer host immune responses to allow pathogen replication, spread, and persistence in the infected organism. many cell surface molecules serve as entry receptors for initial host cell invasion, and concerted responses to the pathogenic challenge critically rely on cell functions mediated by receptors and secreted proteins. to allow host colonization, pathogens encode highly optimized protein modulators, in the form of secreted molecules or receptors expressed on the plasma membrane of the infected cells or the surface of the pathogen [11, 12] . interactions between these proteins and extracellular host molecules form the foundation of communication between a host and a pathogen and play a vital role in the initiation and outcome of the infection [13, 14] . characterizing host-pathogen eppi networks is therefore of utmost importance to gain a better understanding of the infection process and to inform the development of novel or improved therapeutic strategies. excellent studies on mapping hostpathogen interactions, particularly ms-based analysis of viral infection, have provided a wealth of insight into infectious diseases [15] [16] [17] [18] [19] . nevertheless, similarly to host eppis, a significant hurdle to the elucidation of host-pathogen biology has been the shortage of datasets of extracellular interactions between host and pathogen proteins, partly due to the technical challenges that these proteins present. moreover, an additional consideration when studying pathogen-encoded molecules is that these proteins often lack any recognizable homology with any host molecules, thus precluding prediction of their functions [11, 20] . robust methodologies that permit unbiased characterization of eppi in the absence of preexisting hypotheses are thus needed to elucidate the binding partners and molecular functions of most pathogenencoded molecules. excellent reviews have recently revisited the currently available technologies for proteome-wide eppi discovery [4, 8, [53] [54] [55] . here we discuss the application of some of these technologies to the study of host-pathogen interaction and describe some of the major findings that have recently impacted research in the field of extracellular host-pathogen recognition. protein microarrays and functional genomics approaches are highlighted here as emerging techniques with unique potential for the elucidation of host-pathogen eppi networks at a genome-wide scale. classical biochemical and biophysical approaches are particularly suitable for detection of high affinity host-pathogen interactions, such as those mediated by a viral capsid protein and a host cell surface receptor, or a pathogen-encoded glycoprotein and a secreted host factor. typically, these approaches have relied on the utilization of recombinant pathogen proteins as baits to probe for host binding partners, followed by immunoprecipitation and ms, or biophysical techniques for analysis of ppi such as surface plasmon resonance (spr). spr requires prior knowledge of the possible interacting partners and is therefore unsuitable for unbiased ppi discovery, whereas immunoprecipitation and ms approaches usually fail to detect weak interactions, which often characterize eppis, particularly those that take place on the cell surface. notwithstanding, the identification of the receptors for some of the most prominent pathogens, such as the severe acute respiratory syndrome coronavirus (sars-cov) or the new world arenaviruses, was made utilizing standard immunoprecipitation techniques [56, 57] . despite their inherent limitations, biochemical approaches continue to provide relevant insights into host-pathogen interactions, such as the discovery of the dipeptidyl peptidase 4 (dpp4) as the receptor for the mers-cov just within a few months after the emergence of this virus [58] . in addition, more recently kabanova and colleagues identified the cell surface receptor for the trimeric entry complex ghglgo encoded by human cytomegalovirus (hcmv) [21] . several studies have shown that the ghglgo trimer is involved in the infection of fibroblasts, whereas the ghglul128l pentameric complex is required for entry into endothelial, epithelial, and myeloid cells [59] [60] [61] . in this work, both the trimeric and the pentameric cmv protein complexes were generated as recombinant products and used as baits to perform binding experiments on biotinylated cell surfaces, followed by immunoprecipitation and ms identification of bait-cell receptor complexes. using this approach, the authors identified the cell surface protein pdgfr as a high affinity receptor for the ghglgo trimer and demonstrated that this interaction was required for infection of fibroblasts. interestingly, in the case of the pentameric cmv complex, multiple bands were detected upon protein immunoprecipitation from epithelial cells, suggesting the existence of multiple receptors on these cells, which so far remain unknown [21] (table 1) . different biophysical techniques for detection of ppi, in particular spr, have also proven valuable in the field of host-pathogen interactions. spr offers the advantage of label-free, sensitive detection of interactions between a diversity of ligands in real time, thus allowing calculation of kinetic parameters. spr has been widely utilized to monitor antibody binding to a variety of pathogen antigens, information that has informed vaccine development [62] [63] [64] . the spr technology has also been exploited for discovery of eppis. for example, viejo-borbolla and coworkers utilized spr to screen several secreted and membrane-expressed glycoproteins encoded by herpes simplex viruses (hsvs) for binding to chemoattractant cytokines (the chemokine family) and were able to identify a subset of human chemokines that bound to hsv glycoprotein g with high affinity [22] . recently, day and colleagues utilized a combination of glycan arrays and spr and identified over 60 host-bacterial glycan pairs characterized by a wide range of binding affinities, some of which participated in bacterial adherence to host cells in vitro, leading to the hypothesis that bacteria-host surface glycan interactions may mediate initial attachment to the target cell during infection [65] . despite spr and related methods offering higher sensitivity for detection of transient biochemical and ms pdgfr identified as a high affinity cell surface receptor for the cmv ghglgo protein complex [21] herpes simplex viruses (hsvs) biophysical secreted and plasma membrane-expressed glycoprotein g targets a specific set of human chemokines with high affinity [22] human immunodeficiency virus type 1 (hiv) monoclonal antibodies cd4 identified as the receptor for hiv infection of t cells [23, 24] rhinovirus monoclonal antibodies icam-1 as the common entry receptor for most rhinovirus serotypes [25, 26] plasmodium falciparum avexis basigin identified as the cell-surface receptor that mediates erythrocyte infection [27] human adenoviruses extracellular human protein microarrays elucidation of the extracellular interactome of adenovirus-encoded immunomodulatory proteins [28] pseudomonas aeruginosa extracellular pathogen protein microarrays (nappa) screening of patient sera against all p. aeruginosa extracellular proteins. 12 proteins identified as potent antigens [29] varicella zoster virus (vzv) extracellular pathogen protein microarrays (nappa) identification of 18 extracellular viral proteins that promote humoral responses upon screening of the entire vzv proteome [30] streptococcus extracellular pathogen protein microarrays identification of new streptococcal proteins that interact with fibronectin, fibrinogen, and c4bp factors [31] hepatitis delta virus lhdag antigen plasma membrane microarrays (mpa) 150 candidate interactions identified between viral antigen and plasma membrane proteins [32] simian virus 40 (sv40) plasma membrane microarrays (mpa) 99 candidate interactions between whole particles and plasma membrane proteins identified [32] vaccinia virus (vacv) triceps (ms) 7 candidate cell surface binding partners identified for vacv [33] viral pathogens computational studies insights into global principles of virus-host ppi networks [15] [16] [17] [18] [34] [35] [36] hepatitis c virus (hcv) cdna libraries cd81, claudin-1, and occludin as cell surface receptors and some of the players involved in hsv internalization [37] [38] [39] adenovirus and coxsackievirus b monoclonal antibodies and cdna libraries car identified as a common entry receptor for adenovirus 2/5 and coxsackievirus b [40] sindbis virus sirna screens nramp as cell surface receptor for entry into drosophila cells [41] murine norovirus crispr/cas9 cd300lf identified as a cell surface receptor that determines virus tropism [42] bacterial distending toxins (e. coli) haploid cell screens sphingomyelin synthase 1 and the putative g protein-coupled receptor tmem181 identified as toxin receptors [43, 44] clostridium difficile and perfringens toxins haploid cell screens lipolysis-stimulated lipoprotein and the low-density lipoprotein receptor-related protein 1 identified as the receptors for the bacterial toxins, respectively [45, 46] ebola virus haploid cell screens hops proteins and the niemann-pick c1 (ncp1) transporter identified as endosomal receptors that mediate cytosol access [47] lassa virus haploid cell screens lamp1, lysosomal-associated membrane protein 1 identified as an essential host factor mediating virus release to cytosol [48] adeno-associated virus (aav) serotype 2 haploid cell screens 46 cell host factors identified, including heparin sulfate proteoglycan biosynthesis and intracellular transport genes. the immunoglobulin domain-containing transmembrane protein kiaa0319l identified as aav receptor [49] 4 journal of immunology research population genomics analysis p. falciparum ebl-1 protein binds to the erythrocyte receptor glycophorin b, a highly polymorphic gene in malaria-endemic regions [50] streptococcus phage display 19 bacterial proteins identified as potential fibronectin-binding proteins [51] fusobacterium nucleatum transposon-based mutant libraries the bacterial protein fap2 binds to the receptor tigit and downregulates nk-mediated killing of tumor cells [52] avexis, avidity-based extracellular interaction screen; nappa, nucleic-acid programmable protein array; mpa, microfluidic-based comprehensive protein array; crispr, clustered regularly interspaced short palindromic repeat; ms, mass spectrometry; ppi, protein-protein interaction. ppis than most biochemical approaches, these biophysical techniques have not been exploited for large-scale eppi discovery, possibly due to the low throughput of the available instrumentation and the overall difficulties for generation of the relevant protein libraries. these studies, among many others, have demonstrated the power of the classical biochemical and biophysical techniques for detection of host-pathogen interactions. nevertheless, these approaches require previous knowledge of the pathogen-encoded proteins responsible for binding and the ability to produce such proteins as recombinant reagents, which may be challenging, as exemplified by the production of hcmv entry complexes [21, 66] . alternative methods have been utilized in those cases where there is no previous knowledge of the pathogen proteins required for interaction with the host cells. in this regard, the screening of large collections of monoclonal antibodies raised against membrane proteins has proven particularly useful to identify receptors that mediate viral entry. back in the early 80s, the discovery of cd4 as an entry receptor for the human immunodeficiency virus type 1 (hiv) significantly impacted our understanding of viral pathogenesis and subsequent development of therapeutics [23, 24] . in this case, the well-defined tropism of the virus determined the choice of over 100 antibodies directed against human leukocyte differentiation antigens, of which only antibodies that recognized the surface receptor cd4 blocked viral infection [23] . it is worth noting that similar monoclonal antibody screens have also been utilized for unbiased characterization of viral blockers. for example, colonno and colleagues performed a screen of more than 2,000 hybridomas from mice immunized with preparations of plasma membranes from human cells and were able to find one antibody that blocked rhinovirus binding to its cell surface receptor [25] , identified as the intercellular cell adhesion molecule 1 (icam-1) in subsequent studies [26] . despite the undoubted importance of the biochemical and biophysical approaches to the study of host-pathogen interactions, the aforementioned limitations have motivated the development of alternative technologies for large-scale analysis of eppis. from the initial utilization of microarrays for detection of ppi over a decade ago, human proteome chips containing thousands of recombinant proteins have been generated, some of which are now commercially available. protein microarrays offer the unique advantage of requiring minimal consumption of protein reagents, fast readouts, and relatively more affordable instrumentation. typically, a fluorescently labeled or tagged protein of interest (the bait) is generated as a recombinant product and screened against all proteins in the array [10, 53] . despite the increased availability of highcoverage protein arrays, very few are focused on extracellular proteins and therefore are not suitable for study of hostpathogen eppis. most existing microarray-based methodologies rely on multimerization of the bait protein for increased avidity and detection of weak eppis, mimicking the way these interactions occur in vivo, where proteins are arrayed in the crowded molecular environment of apposing plasma membranes. different multimerization strategies and protein microarray libraries have been developed and utilized for host-pathogen interaction discovery, some of which are described in more detail below. the wright lab developed a novel method for detection of low affinity eppis, termed avidity-based extracellular interaction screen (avexis) [2] . in brief, avexis consists of the expression of the extracellular domain (ecd) of the bait of interest as a recombinant protein, which retains its binding properties while removing the insoluble transmembrane region of the protein. these ecds are tagged with a coiled-coil sequence from the rat cartilage oligomeric matrix protein to allow for pentamerization of the bait and therefore increased binding avidity, alongside a -lactamase tag for detection of baitprey interactions upon incubation with the colorimetric substrate nitrocefin. this multivalent strategy has been used for medium-scale screens, allowing detection of weak interactions between human receptors with low false-positive rates [2] . notably, crosnier and colleagues applied avexis to search for the plasma membrane receptor responsible for plasmodium falciparum infection of erythrocytes [27] . the authors compiled a library consisting of most secreted or cell surface-expressed proteins in erythrocytes and systematically assayed more than 40 red blood cell proteins for binding to p. falciparum protein pfrh5, a parasite protein essential for blood stage growth, expressed as an avexis pentameric bait. notably, the ok blood group antigen basigin was identified as a unique receptor for pfrh5, and inhibition of this interaction was shown to be sufficient to block parasite invasion of the erythrocyte, findings that may importantly inform antimalarial therapies [27] . in later studies, avexis was miniaturized making this approach compatible with the protein microarray format, thus permitting more comprehensive and lower resource-intensive screenings [67] . although this technique should allow for high throughout and sensitive determination of eppis, this approach has not yet been applied to elucidation of pathogen-host interactions. over a decade ago, fueled by the recent completion of the human genome, genentech pioneered a significant effort to identify novel secreted or transmembrane domain-containing proteins, upon careful bioinformatics assessment and high throughput protein purification [68] . these efforts resulted in the generation of a comprehensive human protein library, which was subsequently utilized to develop an extracellular protein microarray platform, consisting of over 1,500 secreted or single-transmembrane domain containing proteins [10] . for the generation of this human protein library, secreted proteins or the ecd of single-transmembrane receptors were fused to different affinity tags and subsequently purified from cell culture supernatants by size-exclusion chromatography. mammalian cells or baculovirus-insect cells were preferentially used as expression systems, to maximize the likelihood of proper folding and glycosylation of the extracellular protein collection [10, 69] . sds-page and multiangle laser light scattering were used to analyze noncovalent aggregation and ensure high-quality protein production. subsequently, the purified proteins were spotted on epoxysilane slides using a nanoprint arrayer, and protein immobilization on the microarray was determined by probing the slides with the relevant anti-tag antibodies [10] . to enhance detection of low affinity interactions, a rapid method to assemble bait proteins (whose ecd was expressed as a fc tag-fusion protein) into multivalent complexes using fluorescently labeled protein a microbeads was developed. proof-of-concept assays showed high sensitivity for detection of weak eppis characterized by micromolar , a minimal off-target binding, and more than 70% true-positive to false-positive detection ratio [10, 69] . over the years, this extracellular protein microarray has successfully identified counterreceptors for a number of human molecules, providing relevant insights into novel pathways that coordinate a multitude of cell functions [70] [71] [72] . receptor discovery. recently, we applied this eppi discovery platform to the study of extracellular viral proteins ( figure 1 ), with a focus on human adenovirus-(hadv-) encoded immunomodulatory proteins [28] . despite the increasing relevance of hadv as both pathogens and therapeutic vectors, information on the interaction of these viruses with the host immune system remains scarce [73, 74] . interestingly, the immunomodulatory proteins encoded by these viruses, termed e3 proteins, show substantial diversity in their ecds across and within viral species and constitute one of the most divergent regions of the hadv genome [75, 76] . given this striking variability, the e3 proteins have been suggested to play a role in viral tropism and pathogenesis, yet the functions of virtually all e3 proteins have remained unknown [73] . in our study, we took advantage of such unique variability to evaluate the effect of viral immunomodulatory protein diversity in extracellular host targeting. screening of a substantial number of e3 proteins encoded by different hadv species using the extracellular protein microarray platform allowed identification of over 50 novel virus-host interactions encompassing 5 viral species, which were fully validated by orthogonal methods [28] . these findings revealed significant diversity in extracellular host targeting and, moreover, allowed identification of semiconserved host targets, pointing towards specific human receptors that may represent previously unrecognized hubs for viral perturbation. furthermore, most of the e3 immunomodulators were identified as multifunctional proteins, suggesting that viruses have evolved proteins capable of interfering with several cellular functions, a strategy consistent with the optimization of limited genomic resources. such economic targeting has been often observed in intracellular targeting [15] [16] [17] , but so far few examples of widespread targeting in the extracellular environment have been reported [28, [77] [78] [79] [80] , let alone a global elucidation of eppi networks, in part due to the technical challenges associated with eppi detection. remarkably, many of the hadv e3 proteins preferentially interacted with host receptors that exert known or predicted inhibitory functions during the immune response (as defined by the presence of intracellular immunoreceptor tyrosinebased inhibitory motif, itim), including lilrb1 [81, 82] , lair1 [83] , and mpzl1 [84] , suggesting previously unrecognized strategies of immunosuppression that may be utilized by other human pathogens [28] . moreover, several of the receptors identified as targets for the viral proteins in this study (including the prominent cell surface molecule cd45) do not have known counterreceptors in the host, supporting the longstanding hypothesis that pathogen molecules drive the evolution of immune receptors and in many instances may represent the most relevant modulators of host receptor function [85] [86] [87] . in summary, such unbiased, microarraybased study of immunomodulatory proteins represented the first large-scale analysis of the ppi landscape of a collection of extracellular immunomodulators encoded by viruses. future investigation of other pathogen-encoded molecules using similar extracellular protein microarrays will likely shape our understanding of the pathogen imprint in our immune system. microarrays. one of the main limitations of any protein microarray platform is the lower protein coverage relative to other genome-wide methods for ppi identification, due to the costs and difficulties for generation of comprehensive protein libraries to be deposited onto the microarrays. in an attempt to address this caveat, ramachandran and colleagues developed a method called nucleic-acid programmable protein array (nappa), in which dnas are directly deposited onto the array followed by protein synthesis in situ using an in vitro transcription and translation (ivtt) system, thus avoiding the need for protein purification [88] . although this promising approach has proven superior in generating transmembrane-containing molecules as soluble proteins, it still remains to be systematically addressed if the extracellular human proteins produced in this manner present the folding and posttranslational modifications necessary for protein functionality. nevertheless, emerging data support the utility of nappa as a useful tool for the study of bacterial proteins. for example, montor and colleagues used a bioinformatics approach to predict the pseudomonas aeruginosa proteins that reside in the outer membrane of the bacteria or are secreted to the extracellular environment of the infected cell [29] . in this work, the authors utilized the nappa approach to screen all predicted extracellular gene products for interaction with sera from cystic fibrosis patients, where p. aeruginosa establish a life-threatening lung infection. from 266 bacterial proteins initially selected, 12 proteins were recognized by antibodies in the sera, indicating that these bacterial proteins represent major antigens that trigger adaptive immune responses in humans. interestingly, robust antibody responses against three previously uncharacterized proteins were detected, suggesting this approach could help identify new extracellular proteins that exert unknown functions during the infection [29] . these results confirmed the utility of the microarrays to detect immune responses against membrane proteins encoded by pathogens, and supported the use of this methodology for diagnosis applications. in this regard, several groups have developed microarrays composed of pathogen-encoded proteins [30, [89] [90] [91] [92] [93] . such pathogen protein arrays have so far being exploited mainly for diagnosis purposes, to allow screening of antibodies present in patient sera for binding to extracellular bacterial or viral antigens on the array. nevertheless, their inherent high throughput and compatibility with multivalent bait approaches makes them a powerful tool for eppi discovery. for example, margarit et al. developed a streptococcus microarray to find novel microbial proteins capable of binding to the human proteins fibronectin, fibrinogen, and c4bp and were able to identify a set of streptococcal proteins that interacted with these factors [31] . nevertheless, despite such pathogen protein-based arrays offering great promise, this methodology remains to be systematically analyzed for eppi discovery. more recently, yu and colleagues applied nappa technology in combination with the halotag-halo ligand detection system to elucidate the interaction network of two effector proteins (sidm and lida) encoded by legionella pneumophila, a highly pathogenic bacteria that is the causative agent of legionnaire's pneumonia [94] . similarly to many 8 journal of immunology research pathogen proteins, these virulence factors lack significant homology to host molecules therefore complicating the assessment of their host targets and biological functions. in this work, the bacterial proteins of interest were tagged with a halotag, a modified haloalkane dehalogenase that covalently binds to synthetic halo-ligands (haloalkanes) that can be fluorescently labeled, thus allowing more robust detection of bait protein binding to interactors present on the array. in this study, more than 10,000 human proteins were expressed on the nappa array using different ivtt techniques, leading to identification of 20 and 18 binding partners for the lida and sidm effectors, respectively, most of them experimentally verified by pull-down [94] . although this study focused on identification of intracellular ppi, the applicability of the nappa-halotag technology for eppi determination should be explored in the future. moreover, bait multimerization strategies should be implemented in order to make this approach more suitable for detection of transient ppis. related to the nappa technology, glick and colleagues recently built a miniaturized platform focused on human membrane proteins. by integrating the microfluidics technology, protein microarrays, and an ivtt system, this group built a new device named microfluidic-based comprehensive human membrane protein array (mpa) [32] . a notable improvement introduced by these investigators was the addition of microsomal membranes to the ivtt system to allow for improved folding and posttranslational modifications in plasma membrane proteins, both common limitations of ivtt systems. in this work, a library of 2,700 human genes encoding for membrane proteins was built and subsequently utilized to screen the large-form delta antigen (l-hdag) encoded by the hepatitis delta virus (hda) and whole viral particles of the simian virus 40 (sv40), a nonenveloped human pathogen. proof-of-concept assays showed encouraging results, with over 75% true-positive rate within a small set of proteins with known interactors and, more importantly, indicated the feasibility of this approach for expression of multitransmembrane-containing proteins, a protein type that has proven challenging given their high hydrophobicity. the mpa screens identified 99 and over 150 interactions for sv40 particles and l-hdag viral protein, respectively, and around 35 interactions were validated by coimmunoprecipitation or protein-fragment complementation assays [32] . to our knowledge, this is the first study to assess eppis using a comprehensive human protein library and whole viral particles (sv40) as baits, a valuable approach that may provide important insights into pathogen tropism, alongside a molecular explanation for the cell surface receptors engaged by the pathogen. further utilization of this platform followed by a more systematic analysis of the candidate hits, including nonspecific binder determination, will be needed to assess the overall performance of the mpa technology. regardless, this platform provides an extended version of the nappa approach that focuses on mammalian eppis and may therefore provide relevant insights into extracellular hostpathogen interactions. the protein microarrays have represented one of the most fruitful approaches for unbiased determination of eppis, including host-pathogen interactions. nevertheless, one of the main limitations of this technology is the need to generate comprehensive libraries, a process that is resource consuming and often not available to many researchers [53, 93] . consequently, although some of the available arrays were designed to cover a significant fraction of the human proteome, any discoveries made using these platforms are limited to the proteins present in each array. the current libraries are likely to continue expanding alongside innovative approaches to facilitate sensitive detection of eppi using protein microarray formats. over the last decade, ms-based technologies have emerged as a versatile, powerful approach to decipher many aspects of the human proteome, including the characterization of protein complexes. excellent reviews on current ms-based technologies, recent improvements, and future prospects for elucidation of ppi networks are available [95] [96] [97] [98] . in this review, we briefly discuss the applications of some of these techniques to the study of extracellular host-pathogen interactions. proteins and interacting partners. the proteins expressed on the surface of pathogens mediate functions necessary for survival, replication, immunoevasion, and transmission and therefore are logical candidates for therapeutic and vaccine design. however, the study of the surface proteome in pathogens, particularly in bacteria is constrained by the fact that commonly used prediction algorithms fail to correctly predict the location of several proteins [29, [99] [100] [101] . despite the characterization of the extracellular proteins and their interactions still representing the achilles heel of most proteomics methods, ms has emerged as an invaluable approach to characterize the protein composition of plasma membranes [5] . to date, several studies have exploited ms-based techniques to gain insights into the extracellular protein composition of bacterial pathogens [101] [102] [103] [104] . for example, palmer and colleagues studied the surface proteome of the tick-borne intracellular pathogen anaplasma marginale (rickettsiales: anaplasmataceae) using liquid chromatography and tandem ms [105] . interestingly, the authors found that the surface proteome of a. marginale isolated from tick cells, despite being less complex than that of bacteria isolated from human erythrocytes, contained a novel protein, which the authors hypothesized to play a function in human cell invasion in spite of its human counterreceptor remaining uncharacterized. this interesting observation suggests a remodeling of the bacteria surface proteome during the transition between mammalian and arthropod hosts, an aspect of the infection that could be targeted to block transmission. similarly, several studies have pursued the identification of the proteins present in viral particles utilizing ms. although these analyses suffer from several drawbacks associated with membrane protein characterization, particularly the poor solubility of these proteins and the low abundance of many plasma membrane proteins, these studies have revealed a complex composition for most of the viruses studied, alongside incorporation of many host proteins in the virions, in most cases with undetermined functions [106] [107] [108] . an interesting observation from some of the studies referred above is the fact that certain bacterial proteins, predicted cytoplasmic by consensus, can be found in the extracellular environment of the cell, where they may play alternative functions. in fact, the number of proteins that are secreted through noncanonical signal sequence pathways is increasingly appreciated [99, 100, 109, 110] . little is known about these bacterial proteins originally described as cytosolic proteins but capable of exerting functions on the cell surface, which some authors have named moonlight proteins, in reference to their potential to exert multiple functions [111] . there is emerging evidence that protein moonlighting contributes to virulence of important bacterial pathogens including staphylococcus aureus or mycobacterium tuberculosis, sometimes in fascinating ways. for example, m. tuberculosis is known to encode two molecular chaperones, cpn60.1 and cpn60.2, which function as modulators of myeloid cells among other regulatory functions [112] . despite these chaperones being by definition cytosolic, cnp60.2 has been detected in significant amounts on the bacterial surface, and either recombinant cnp60.2 or antibodies against this protein efficiently block binding of m. tuberculosis to macrophages [112] , through a potential interaction with the receptor cd43 [113] . in addition, the protein dnak, a hsp70related protein encoded by m. tuberculosis, can locate to the bacterial surface and functionally interact with cd40 [114] and with the hiv coreceptor ccr5 [115] . notably, dnak appears to block hiv binding to ccr5 in vitro, an interesting observation given the co-occurrence of m. tuberculosis and hiv infection [116] . although a more detailed revision is out of the scope of this review, bacterial protein moonlighting, excellently revisited by henderson and martin [111] , is a thought-provoking phenomenon that suggests a much more complex extracellular landscape than anticipated. moreover, such protein moonlighting is in line with the hypothesis that pathogens have evolved multifunctional proteins as a prominent strategy for efficient use of limited genomic resources [17, 28] . another ms-based approach that holds great promise for host-pathogen eppi detection is the recently developed triceps [33] . triceps is a chemoproteomic reagent that consists of three moieties, one that binds the ligand of interest through its amino groups, a second one that binds glycosylated receptors on the cell surface, and a biotin tag for purifying the receptor peptides for subsequent identification by ms. notably, in the initial description of the method, triceps was successfully applied to the identification of receptors for extracellular ligands of diverse nature, such as secreted glycoproteins, small peptide ligands for g proteincoupled receptors, and therapeutic antibodies. importantly, this approach has also been utilized to study cell surface molecules targeted by vaccinia virus (vacv). interestingly, the analysis of vacv binding to hela cells revealed seven candidate binding partners, including the previously identified receptors axl, chondroitin sulfate proteoglycan 4, and laminin binding protein dystroglycan 1. further, downregulation of five out of the seven candidates using short interfering rna reduced vacv infection by 40-60%, supporting the functionality of the interactions identified, at least in vitro [33] . although this technology is still developing and no studies on other pathogens have been published yet, future triceps-based studies promise relevant insights into pathogen interaction with distinct components of the cell surface. as an addendum to the vast amount of knowledge acquired using ms approaches and some of the additional methodologies discussed in this review, bioinformatics offers an in silico systems biology approach that reveals a global perspective on host-pathogen interactions. advances in computation have been fundamental to dissect the complex datasets generated in many genome-wide msbased studies and have enabled the reconstruction of largescale host-pathogens ppi networks, providing fundamental insights into viral disease and hence host biology [15-18, 34-36, 117] . although the computational tools available for analysis of large datasets, in some cases developed in association with some of the high throughput screens mentioned in this review, certainly deserve a focused chapter, a couple of observations are specially notable. commonly observed in these studies is that the intracellular viral effectors preferentially target host proteins that act as hubs (proteins with many interacting partners) or bottlenecks (proteins central to many pathways in the network) [15, 16, 36] . for example, dyer and colleagues built a network of host-pathogen ppi by integrating published information from 190 pathogens [36] . supporting previous findings, this analysis indicated that pathogen-encoded proteins preferentially interfere with host molecules that control critical cellular processes, such as cell death or nuclear transportation, possibly as a strategy to maximize control of the host machinery given limited genomic resources. interestingly, this study highlighted a small set of extracellular host proteins recurrently targeted by several of the viral and bacterial pathogens analyzed, including cell surface receptors such as vegfr2/kdr and collagen, possibly indicating previously unrecognized roles in the immune response against pathogens. although informative, the analysis performed by dyer and collaborators was skewed towards viruses, with a prominent enrichment in hiv strains [36] . more extensive analyses encompassing other human viruses and bacterial pathogens may reveal general strategies of immunomodulation and potential human targets suitable to therapeutic intervention. interestingly, increasing evidence suggests that virus-host interactions are governed by principles distinct to those that dictate within-host interactions [20, 28, 85, 87, 118] . notably, detailed analyses carried out by the xia group highlighted significant differences between virushost and within-host (also called endogenous) interactions, such as the tendency of viral proteins to compete with host proteins for binding to a given receptor in the absence of sequence similarity with the host counterpart or the observation that viral molecules have evolved multiple short linear motifs capable of mediating a number of diverse interactions [20, 118] , features that are consistent with the multifunctional capabilities of some pathogen-encoded proteins [20, 22, 28, [77] [78] [79] [80] 118] . altogether, bioinformatics analysis of virushost interactions suggest that virus-mediated targeting of host proteins is characterized by signatures of pleiotropy, economy, and convergent evolution, conclusions that are supported by emerging experimental data. followed by thorough biological experimentation such computationalbased systems biology approaches will provide a unique tool to help decipher basic global principles of pathogenhost interaction and may reveal novel eppis amenable to therapeutic intervention. alongside protein microarrays-based technologies, ms, and computational analysis, the explosion of the functional genomics field in the last years has revolved the avenues to study pathogen interactions with their hosts, often in high throughput. in brief, genetic screens comprise gain-of-function and loss-of-function strategies, represented by complementary dna (cdna) libraries and rna-interference-(rnai-) based approaches, respectively. these methods were developed more than two decades ago and have been widely utilized by the scientific community, providing fundamental insights into the infection process. in particular, the cdna libraries have proven extremely successful in identifying viral receptors through a gainof-function approach, upon transduction of the cdna library from a susceptible cell line into nonpermissive cell lines. the use of cdna libraries is not reviewed in detail here in the interest of a more comprehensive revision of relative newer genomics-based approaches, such as the clustered regularly interspaced short palindromic repeat/ crispr-associated protein 9 (crispr/cas9) or the haploid cell screens. nevertheless, these libraries have represented one of the most significant technologies to further our understanding of the pathogen-host interaction. for example, early studies made use of cdna libraries to shed light on the complex mechanism exploited by hepatitis c virus for initial invasion of the cell [37] [38] [39] , identified car as a common receptor for adenovirus 5 and coxsackievirus b [40] , and were instrumental to identify slam1 and pvr as a receptors for measles and poliovirus, respectively [119, 120] . in turn, the rnai technology has yielded significant insights into virus-host interactions, such as the identification of the ion transporter nramp as the receptor for the mosquito-borne sindbis virus colonization of drosophila cells [41] . the main power of the rnai technology is that it allows high throughput genome-wide screens and therefore potential identification of essential factors that play roles in different aspects of the pathogen life cycle, including initial interaction with the host cell. although rnai screens have provided tremendous insights into host-pathogen interactions and remain widely utilized [121] , inefficient gene depletion and off-target effects are important limitations of this methodology [122] . technology. the increasingly popular crispr/cas9 technology overcomes some of the caveats often associated with genetic manipulation and holds enormous promise for genome editing and downstream applications, including host-pathogen interaction discovery [123] . although still early days, high throughput crispr/cas9 screens for genome-wide studies have already displayed remarkable results, with high levels of genomic modification, hit confirmation, and strong phenotypic effects [124] . the development of the crispr/cas9 technology has undoubtedly transformed the functional genetic analysis in mammals. recent studies have applied the crispr/cas9 technology to ablate expression of previously identified receptors for viral entry, such as the hiv coreceptors cxcr4 and ccr5, leading to resistance to infection in primary cells [125, 126] . an interesting additional application of crispr/cas9 is the direct editing of viral genes important for viral fitness. this approach has recently been used to target hsv-1, cmv, and epstein-barr virus (ebv) essential genes, leading to a significant decrease of viral replication [127] . these studies suggest the potential use of crispr/cas9 as an innovative therapeutic strategy, as aspect that will surely be further explored in the near future. another prominent example published recently is the identification of host factors that confer susceptibility to the evolutionary related type iii secretion systems, t3ss1 and t3ss2, encoded by vibrio parahaemolyticus [128] . the t3sss are highly complex nanomachines utilized by gramnegative pathogens to inject a variable repertoire of virulence factors into the cytosol of the eukaryotic cells, enabling pathogen adhesion and internalization of modulation of host processes. interestingly, using genome-wide crispr/cas9 screens, sulfation and fucosylation of cell surface components were identified as host determinants of t3ss1-and t3ss2mediated cytotoxicity, respectively. the authors hypothesized that interactions between sulfated cell surface molecules such as host proteoglycans and bacterial adhesins act as facilitators of t3ss1 activity, whereas fucosylated glycans on the surface may serve as receptors for t3ss2 components necessary for insertion of the complex in the host membrane [128] . the crispr/cas9 approach has just started to reveal its power as a tool for unbiased identification of novel eppis, elegantly exemplified by the identification of cd300lf as the cell surface receptor for noroviruses, which, strikingly, was identified as the main determinant for the tropism of the murine norovirus [42] . further optimization of this technology will unequivocally signify a tremendous advance for the discovery of extracellular host-pathogen ppis, the processes underlying host-pathogen interactions and its possible therapeutic applications. haploid cells, in turn, allow the study of recessive phenotypes that can be masked in diploid cells, due to the difficulties of creating true genetic knockouts in mammalian cells. despite yeast being a useful tool due to the simplicity of obtaining relevant mutants at its haploid life stage, the majority of human pathogens do not replicate in yeast therefore limiting the applicability of this approach [129] . in recent years, human haploid cells have been increasingly utilized for genome-wide loss-of-function genetic screens using insertional mutagenesis [43, 44, 47] . in initial studies, carette and colleagues took advantage of the kbm7 cell line, a derivative of the chronic myeloid leukemia cell line (cml) with a haploid karyotype except for chromosome 8 [130] . using gene-trap retroviruses for efficient insertional mutagenesis, the authors generated a genomewide collection of null mutants for most nonessential genes [43] . this approach was successfully utilized to identify host factors essential for the functions of the distending toxins or cdts, potent virulence factors secreted by a number of pathogenic bacteria. in particular, mutagenized kbm7 cells were treated with escherichia coli-derived cdts and resistant clones were isolated, leading to identification of insertions in the sphingomyelin synthase 1 and the putative g protein-coupled receptor tmem181, suggesting that this molecule may serve as a surface receptor for the toxin [43, 44] . similar haploid screens have identified novel receptors for a number of bacterial toxins, including the lipolysis-stimulated lipoprotein receptor for the clostridium difficile transferase [45] , or the low-density lipoprotein receptor-related protein 1 as a host receptor of the clostridium perfringens tpel toxin [46] . in a later study, carette et al. generated a kbm7-derived cell line named hap1, haploid for all chromosomes [47] . similarly to previous studies, the authors used the retroviral gene-trap approach to mutagenize hap1 cells followed by deep sequencing to map more than 800.000 insertions. in this study, using a replication competent vesicular stomatitis virus (vsv) carrying the ebola virus glycoprotein, a previously unknown entry receptor for ebola virus was identified. notably, these haploid cell screens identified six members of the hops complex, proteins known to play functions in endosomal/lysosomal trafficking, as well as the niemann-pick c1 (npc1) transporter as the most prominent hit of the assay. it is worth noting that npc1 is not a surface molecule but rather an endosomal receptor. these findings led the authors to propose a novel mechanism of entry by which ebola virus is internalized into the endocytic pathway, followed by endosome maturation and cleavage of the surface glycoprotein of the virus. endosome fusion, mediated by the hops complex, would allow interaction with ncp1 containing endosomes, triggering fusion and release of the viral genome into the cytosol. multiple cell surface receptors can lead to internalization of the ebola virus into the endocytic pathway [131] ; such redundancy in receptor usage likely explains why these receptors were not identified in the haploid cell screen [47] . notably, independent studies have confirmed that ncp1 acts an intracellular receptor for ebola, including a chemical screen approach, a study showing ncp1 dependence for infection of otherwise nonsusceptible cells, and more recently the elucidation of the crystal structure of this receptor bound to the ebola virus glycoprotein [132] [133] [134] . interestingly, after the aforementioned haploid genetic screens identified ncp1 as a noncanonical entry receptor (given its intracellular localization), other filoviruses have been shown to take advantage of this receptor [135] . the relevance of this intriguing mechanism of viral entry is further reinforced by recent work on lassa virus, an old world arenavirus that, similarly to ebola virus, causes severe to fatal hemorrhagic disease in humans [48, 136] . a genome-wide haploid screen using vsv pseudotyped with lassa glycoprotein was performed in order to identify host factors essential for viral entry. although -dystroglycan (dag1) was long recognized as the cell surface receptor for lassa virus, additional factors were suspected, given the observation that certain dag1-expressing cells are resistant to infection. the authors elegantly demonstrated that at a neutral ph, the lassa virus glycoprotein was bound to dag1, whereas upon exposure to lower ph (resembling the lysosome environment), a receptor switch occurred leading to strong association with the lysosomal-associated membrane protein 1 (lamp1) [48] . thus, similarly to ebola virus, in the model suggested the virus would be incorporated into the endocytic pathway after interaction with its surface receptor dag1, followed by increasingly acidic conditions that would result in interaction with lamp1 in the lysosomal membrane, triggering membrane fusion and release of the virus in the cytosol [48] . more recently, pillay and colleagues applied the haploid cell screening approach to the identification of host factors essential for the adeno-associated virus (aav) serotype 2 infection, one of the leading vectors for virus-based genes therapies [49] . notably, the most significantly enriched gene in these screens was kiaa0319l, a poorly characterized type i immunoglobulin domain-containing transmembrane protein named hereafter as the aav receptor. among the 46 host factors identified as hits, many were implicated in heparin sulfate proteoglycan biosynthesis as well as a number of proteins that participate in intracellular transport processes. aav is known to attach to the cells using heparin sulfate proteoglycans and hijacks endosomal trafficking to travel to the nucleus upon invasion of the cell; thus the authors hypothesized that these additional factors may influence virus tropism [49] . altogether, these studies elegantly demonstrate the power of genome-wide screens in human haploid cells and the power of this approach to study virus-host interactions. future studies should further assess the applicability of this method for general detection of interactions that take place at the pathogen-plasma membrane interface. it will also be important to generate additional haploid cell lines, in order to broaden the range of pathogens and pathogen-derived molecules that can be studied using these genetic tools. in this regard, a number of haploid cell lines have been generated in mammals [137] , unique tools to elucidate the basic aspects of human genetics. discovery. pathogens are among the most intriguing and prominent drivers of human evolution. humans have adapted to the pressure imposed by microorganisms through genomic diversification, particularly through variation of genes involved in immune system function, constantly challenged by the rapidly coevolving pathogen genomes. the advent of new technologies such as next-generation sequencing and the computational tools associated have opened new avenues for the study of human genetics, making it possible to evaluate the contribution of genetic diversity to susceptibility to infection at the genomic level. the emergence of datasets of genomic variation in multiple human populations, as well as pathogen genomes, allows detection of signatures of selection, which can be exploited to identify genes with major roles in immunity (for an excellent review see [138] ). remarkably, the cell surface-expressed receptors are among the most polymorphic gene families in mammals, subjected to strong positive selection and rapid evolution, in many instances possibly driven by pathogen molecules that remain unknown [87, [139] [140] [141] . polymorphisms in receptors and immunomodulatory genes contribute to the natural susceptibility of different individuals to infection [142] [143] [144] , as illustrated by protection against hiv infection in individuals carrying homozygous polymorphisms in the viral coreceptor ccr5 [145] . the identification and further study of genes under positive selection may represent a mainstream approach to dissect novel genes involved in disease and hostpathogen interaction. a notable example is the identification of glycophorin b as the erythrocyte receptor for p. falciparum protein ebl-1 through examination of highly polymorphic genes in populations from malaria-endemic regions [50] . further population genetics studies promise key insights into novel immunological mechanisms and have the potential to provide molecular details that will ultimately help design effective therapies. in addition to these encouraging technologies, the generation of phagemic libraries has also represented an important tool for deciphering ppis, in this case between particular binding partners and the whole genome of specific pathogens [146] [147] [148] . typically, pathogen-encoded molecules are expressed as fusions with phage envelope proteins, a method known as phage display that has been widely exploited to identify peptides with specific binding properties. for example, beckmann and colleagues built a phage display library to identify novel group b streptococci proteins capable of mediating adherence to fibronectin, a major component of the extracellular matrix often exploited for colonization of the host [51] . from this analysis, the authors identified 19 genes with homology to known bacterial adhesin proteins, genes involved in virulence, transport, or metabolic processes, along with genes with uncharacterized functions. interestingly, one of these genes showed significant homology with the scpb protein, a peptidase found in other streptococci that inactivates the member of the human complement system c5a, suggesting that this bacterial molecule acts as a bifunctional protein, similarly to other examples of multifunctional proteins discussed above [51] . more recently, a transposon-based insertion-inactivation mutant library was elegantly utilized to identify a bacterial protein capable of targeting the surface receptor tigit, an inhibitory molecule present in natural killer (nk) cells and t cells [52] . fusobacterium nucleatum is a common oral bacterium that has been associated with colon adenocarcinoma and rheumatoid arthritis among other malignancies. in this study, gur and colleagues showed that different strains of f. nucleatum blocked nk-mediated killing of human tumors. using a library of f. nucleatum mutants, the authors identified fap2 as the bacterial protein that directly interacted with tigit, leading to inhibition of nk cytotoxicity and downregulation tumor-infiltrating t lymphocytes activation. immunoevasion is a hallmark of cancer; however whether members of the microbiome found within the tumor provide cancer cells with immunoregulatory properties has remained a major matter of debate [149] . these interesting findings suggest that f. nucleatum present in the tumor niche may enhance tumor escape by inactivating nk-mediated killing upon interaction of the fusobacterial fap2 with the inhibitory receptor tigit. of note, transposon-based mutant libraries are readily available for other pathogenic bacteria and have been successfully applied to identification of bacterial genes implicated in bacterial physiology [150] [151] [152] . it would be of interest to employ these libraries for unbiased identification of eppis. notably, as mentioned above, we found that hadv immunomodulators preferentially target other immunoreceptors that, similarly to tigit, also play inhibitory functions [28] , suggesting this might represent a common immunosuppressive tactic evolved by pathogens. in fact, there is emerging evidence suggesting that may be the case, as a number of extracellular proteins from unrelated human pathogens have already been shown to target diverse immune receptors with inhibitory functions [28, 52, 82, 87, 153, 154] . further exploration of inhibitory receptor targeting by other pathogens warrants exciting biological discoveries. deciphering the human genome made possible the categorization of genes that encode for the human secretome; now, the challenge of the postgenomic era is to annotate the functions of those genes and their expression patterns during health and disease. a lot has been learnt from painstaking, highly focused experiments using classical biochemistry. in recent years, the impressive technological advances in proteomics, functional genomics, and computation have revolved our understanding of cell communication and function and have collectively created a versatile platform to enable biological discoveries, from mechanistic explorations to big data and systems biology analysis. nevertheless our understanding of the molecules and mechanisms of extracellular immunomodulation and pathogen invasion remains remarkably limited. extracellular ppis between host-and pathogen-encoded molecules orchestrate an enormous diversity of cellular processes, from initial colonization of the target cell to subsequent immune responses. the elucidation of these extracellular interactomes is integral to understanding the molecular basis of infection and will guide the development of more efficient or innovative therapeutics. improvements in proteomics and genomics approaches have exponentially increased our understanding of how pathogens, particularly viruses, modulate the intracellular environment of the cell. concomitantly, we and several other groups have implemented technologies directed towards elucidation of extracellular interactomes [2, 9, 10, 28, 32, 155] , which have begun to reveal fundamental principles of extracellular journal of immunology research 13 host-pathogen interactions. notably, recent studies have revealed extensive eppi networks in model organisms such as drosophila or zebrafish [2, 9] . these undertakings predict that, similarly to intracellular ppis, extracellular networks will be highly connected, with secreted and plasma membrane-expressed proteins having multiple binding partners. however, as discussed in this review, the identification of the host factors and in many cases the pathogen molecules that mediate eppis have largely defied molecular identification, in part due to the technical difficulties inherent to the study of these extracellular proteins. the elucidation of the global principles dictating extracellular pathogen-host ppis will require a coordinated effort to bring together the areas of biology and technology. there are now considerable opportunities for integrating multiple disciplines for eppi discovery, particularly proteomics and crispr/cas9 genome-wide screens, which should be powered by commensurable advances in bioinformatics and computation for big data analysis. the integration of orthogonal datasets coming from multiple "omics" approaches will be advantageous for elucidating the intricacies of the host-pathogen extracellular interactomes and will further enhance the rational identification of novel therapeutic targets by uncovering fundamental principles of biology. the journey from classical biochemical studies towards a systems biology approach has just begun and promises major technological breakthroughs and surprising biological findings. the development of powerful technologies for eppi discovery has already illuminated sophisticated 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immunodeficiency virus hsv:herpes simplex virus itim:immunoreceptor tyrosine-based inhibitory motif ivtt:in vitro transcription/translation lair1:leukocyte-associated immunoreceptor like 1 lilrb1: leukocyte immunoglobulin-like receptor 1 mpa:microfluidic-based comprehensive human membrane protein array mpzl1:myelin protein zero-like protein 1 ms:mass spectrometry nappa:nucleic-acid programmable protein array ncp1:niemann-pick c1 pdgfra: platelet-derived growth factor receptor alpha ppi:protein-protein interaction pvr:poliovirus receptor sars-cov: severe acute respiratory syndrome coronavirus sv40:simian virus 40 slam:signaling-lymphocytic activation molecule spr:surface plasmon resonance t3ss: type iii secretion system vavc:vaccinia virus. the author declares that they have no competing interests. key: cord-314567-purplsjn authors: fernández-ponce, cecilia; durán-ruiz, maria c.; narbona-sánchez, isaac; muñoz-miranda, juan p.; arbulo-echevarria, mikel m.; serna-sanz, antonio; baumann, christian; litrán, rocío; aguado, enrique; bloch, wilhelm; garcía-cozar, francisco title: ultrastructural localization and molecular associations of hcv capsid protein in jurkat t cells date: 2018-01-04 journal: front microbiol doi: 10.3389/fmicb.2017.02595 sha: doc_id: 314567 cord_uid: purplsjn hepatitis c virus core protein is a highly basic viral protein that multimerizes with itself to form the viral capsid. when expressed in cd4(+) t lymphocytes, it can induce modifications in several essential cellular and biological networks. to shed light on the mechanisms underlying the alterations caused by the viral protein, we have analyzed hcv-core subcellular localization and its associations with host proteins in jurkat t cells. in order to investigate the intracellular localization of hepatitis c virus core protein, we have used a lentiviral system to transduce jurkat t cells and subsequently localize the protein using immunoelectron microscopy techniques. we found that in jurkat t cells, hepatitis c virus core protein mostly localizes in the nucleus and specifically in the nucleolus. in addition, we performed pull-down assays combined with mass spectrometry analysis, to identify proteins that associate with hepatitis c virus core in jurkat t cells. we found proteins such as nolc1, pp1γ, ilf3, and c1qbp implicated in localization and/or traffic to the nucleolus. hcv-core associated proteins are implicated in rna processing and rna virus infection as well as in functions previously shown to be altered in hepatitis c virus core expressing cd4(+) t cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. thus, in the current work, we show the ultrastructural localization of hepatitis c virus core and the first profile of hcv core associated proteins in t cells, and we discuss the functions and interconnections of these proteins in molecular networks where relevant biological modifications have been described upon the expression of hepatitis c virus core protein. thereby, the current work constitutes a necessary step toward understanding the mechanisms underlying hcv core mediated alterations that had been described in relevant biological processes in cd4(+) t cells. hepatitis c virus core protein is a highly basic viral protein that multimerizes with itself to form the viral capsid. when expressed in cd4 + t lymphocytes, it can induce modifications in several essential cellular and biological networks. to shed light on the mechanisms underlying the alterations caused by the viral protein, we have analyzed hcv-core subcellular localization and its associations with host proteins in jurkat t cells. in order to investigate the intracellular localization of hepatitis c virus core protein, we have used a lentiviral system to transduce jurkat t cells and subsequently localize the protein using immunoelectron microscopy techniques. we found that in jurkat t cells, hepatitis c virus core protein mostly localizes in the nucleus and specifically in the nucleolus. in addition, we performed pull-down assays combined with mass spectrometry analysis, to identify proteins that associate with hepatitis c virus core in jurkat t cells. we found proteins such as nolc1, pp1γ, ilf3, and c1qbp implicated in localization and/or traffic to the nucleolus. hcv-core associated proteins are implicated in rna processing and rna virus infection as well as in functions previously shown to be altered in hepatitis c virus core expressing cd4 + t cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. thus, in the current work, we show the ultrastructural localization of hepatitis c virus core and the first profile of hcv core associated proteins in t cells, and we discuss the functions and interconnections of these proteins in molecular networks where relevant biological modifications have been described upon the expression of hepatitis c virus core protein. thereby, the current work constitutes a necessary step toward understanding the mechanisms underlying hcv core mediated alterations that had been described in relevant biological processes in cd4 + t cells. keywords: hepatitis c virus, immune evasion, proteomics, interactome, ultrastructure, regulatory t cells, immune tolerance introduction hepatitis c virus (hcv) infection is an important cause for chronic viral liver disease and one of the main indications for liver transplantation (anzola, 2004; dustin and rice, 2007) . hcv affects 80 million people worldwide and in more than 80% of the patients leads to chronicity (gower et al., 2014) . the high level of chronicity and the absence of a protective vaccine, makes hcv infection a significant public health problem (anzola, 2004; dustin and rice, 2007; gower et al., 2014) . the molecular mechanisms harnessed by hcv to establish a chronic infection and their implications in the innate and adaptive immune systems have not been fully elucidated. in this regard, several hcv viral proteins have been described as modulators of immunological phenomena (yao et al., 2007; krishnadas et al., 2010; tu et al., 2012; chen et al., 2015) . among them, hcv core protein has been widely associated with pathogenicity, virulence, immune evasion and immune regulation (dominguez-villar et al., 2007 , 2012a waggoner et al., 2007; tu et al., 2012; doumba et al., 2013; fernandez-ponce et al., 2014 , 2017 . however, the underlying molecular processes, as well as the behavior of hcv core protein or its interactions with host cell components, remain unclear. hcv core is a highly basic protein, which binds and protects the viral rna, multimerizing with itself to form the viral capsid (santolini et al., 1994) . in mammalian infected cells, hcv core interacts with endoplasmic reticulum membranes, lipid droplets and other cellular and viral proteins to promote assembly of new virions. however, several inhibitory molecules and the lack of some host factors can hamper viral production by disrupting the core multimerization process and the coordinated interactions with other viral proteins (mousseau et al., 2011; gawlik and gallay, 2014) . in this way, non-enveloped hcv core proteins can be directed to alternative subcellular locations and also be released into the extracellular space (maillard et al., 2001; polyak et al., 2006; tan et al., 2006) . according to these findings, in hcv chronically infected patients, hcv core protein, has been detected as non-enveloped isolated nucleocapsids, not only in hepatocytes (falcon et al., 2003) , but also in serum (maillard et al., 2001) , peripheral blood cd4 + t cells (fernandez-ponce et al., 2014) and other nonparenchymal liver cells, such as, lymphocyte, pit, endothelial, stellate, kupffer and polymorphonuclear cells (falcon et al., 2005) . in t cell lines, non-enveloped isolated nucleocapsids binding and internalization has also been described in vitro (doumba et al., 2013) . these data further support the presence of hcv core protein inside immune cells, including lymphocytes, during hcv chronic infection. interestingly, hcv core protein intracellular expression in cd4 + t lymphocytes has been shown to induce modifications in cell proliferation, cell cycle progression, expression of anergy genes, transcription of genes involved in cytoskeleton reorganization, vesicle trafficking, endocytosis, transcription and translation, cytokine production, cell death and generation of a t cell regulatory phenotype with exhausted features (bergqvist and rice, 2001; bergqvist et al., 2003; dominguez-villar et al., 2007 , 2012a doumba et al., 2013; fernandez-ponce et al., 2014) , characterized by an increased expression of foxp3 (forkhead box p3) and ctla-4 (cytotoxic t-lymphocyte antigen-4) (dominguez-villar et al., 2012a) , high levels of il-10 secretion, and decreased il-2 and ifn-γ production (doumba et al., 2013; fernandez-ponce et al., 2014) . it has been described for several viruses that the specific subcellular localization of viral proteins and their interactions with host molecules can alter the spatial distribution and organization of cellular proteins, and in this way, induce diverse molecular and cellular effects (chen et al., 2002; yoo et al., 2003; ning and shih, 2004; bertrand and pearson, 2008; ponti et al., 2008; hiscox et al., 2010; zhu et al., 2013; raval et al., 2015) . as studies using the whole virus do not allow for the elucidation of the specific molecular mechanisms in which each protein is implicated, in this work, we focused on a single viral protein, showing that in cd4 + t cells, hcv core protein mostly localizes in the nucleus and specifically in the nucleolus where it is greatly enriched. in addition, we performed pull down assays, combined with mass spectrometry analysis, in order to identify host proteins associated with hcv core, which could be implicated in the functional effect previously observed to be induced by the presence of hcv-core in t cells. we found several proteins implicated in important functions that are associated with hcv core protein. thereby, our results shed light on the molecular mechanisms underlying the alterations in biological cell processes and the generation of adaptive regulatory-like cd4 + t cells in the periphery by the intracellular presence of a single hcv viral protein. human embryonic kidney (hek) lenti-x tm 293t cell line (clontech) and jurkat cell line (american type culture collection, manassas, va, usa) were maintained in dulbecco's modified eagle's medium (dmem tm ) supplemented with 10% (v/v) heat inactivated fetal bovine serum (fbs), 2 mm l-glutamine, 10 mm hepes, 1% (v/v) sodium pyruvate, 50 µm 2-mercaptoethanol, 100 u/ml penicillin and 100 µg/ml streptomycin at 37 • c, 10% co 2 . human peripheral blood samples were obtained from healthy donors upon signature of an informed consent and following approval by the ethic sub-commission of the puerta del mar university hospital (dependent from the central quality commission), in accordance to spanish and european union regulations. peripheral blood mononuclear cells (pbmcs) were isolated by density gradient centrifugation using lymphocyte separation medium (eurobiotm, montpellier, france). cells were washed three times with pbs, subsequently stimulated with 1 mg/ml phytohemagglutinin-p (pha) (sigmatm, saint louis, missouri, usa) and cultured in dmem supplemented with 1% (v/v) sodium pyruvate, non-essential aminoacids, vitamins, larginin, l-asparragin, folic acid, 10 mm hepes, 50 mm 2-mercaptoethanol, 100 mg/ml streptomycin, 100 u/ml penicillin (life technologies, carlsbad, ca, usa) and 10% heat-inactivated fbs (gibco) at 37 • c, in a 10% co2 atmosphere. 40 u/ml il-2 was added to the cultures every 48 h, for a total of 5 days to obtain blasts. hek lenti-x tm 293t cells (clontech) were used as packaging cell lines to produce lentiviral supernatant by co-transfecting plasmids pcmvdr8.91, coding for hiv-1 gag and pol proteins and pmd2.g for the vesicular stomatitis virus g protein (vsvg) together with the transfer vector phrsincppt-sewhcv-core-gfp (hcv-core) coding for the first 191 amino acids of the hcv polyprotein (serotype 1a) corresponding to hcv-core protein. transfer vector phrsincppt-sewgfp expressing only gfp (green fluorescent protein) was used as a control (dominguez-villar et al., 2007) . cells were transfected in optimem tm medium using 10 cms diameter cell-culture dishes coated with collagen (collagen i, rat tail gibco r invitrogen cell culture), and polyethylenimine (pei), branched (sigma-aldrich) as cationic polymer. transfection efficiency was evaluated by facs analysis. supernatants were collected at 48 and 72 h, centrifuged to remove cells and debris and concentrated using lenti-x tm concentrator (clontech r ) to obtain a high-titer virus-containing pellet. pellets were frozen at −80 • c and stored until use. viral titer in supernatants was determined evaluating their efficiency in infecting jurkat cells. jurkat cells were transduced using hcvcore-gfp and gfp (as control) lentiviral supernatants, previously prepared, at a multiplicity of infection (moi) of 10. aliquots from the cultures were collected after 48 h to determine the transduction efficiency. gfp expression was monitored by facs (cyanadp-mle tm ; dakocytomation tm ) presenting a transduction range >90% in all experiments. untransduced, hcvcore-gfp and gfp transduced jurkat cells were collected 48 h post-transduction, counted, washed with phosphate buffer saline (pbs) and fixed for 24 h using 4% paraformaldehyde (pfa) in pbs, ph 7.37. subsequently, cell pellets were pre-embedded in agarose 1x, taken out of the tube with a needle and sliced into approximately 1 mm 3 pieces. pre-embedding immunogold was performed. agarose pieces were washed in pbs, permeabilized using 0.05% triton x−100 in pbs and incubated in aurion blocking solution containing normal goat serum (aurion r 905.002), for 60 min at 4 • c. sections were stained immunochemically with anti-gfp antibody (abcam ab6556) diluted 1:1,000 in pbs containing 0.2% bovine serum albumin (bsa) at 4 • c, overnight, and subsequently washed with the same buffer. for gold particle staining, sections were incubated with 10 nm gold-labeled anti-rabbit igg (sigma-aldrich) diluted 1:50 in 0.2% bsa/pbs at 4 • c, overnight. agarose pieces were washed in 0.1 m caco-buffer ph 7.37 at room temperature. sections were post-fixed in 0.5% osmium tetroxide and 0.1 m caco-buffer at room temperature, for 3 h and washed with 0.1 m caco-buffer. fixed sections were dehydrated by sequential incubation in ethanol 50-100% and propylene oxide, embedded in epoxy resin and polymerized at 62 • c for 72 h. semi (500 nm) and ultra-thin (50-70 nm) sections, were obtained using a leica em uc7 ultramicrotome. uultra-thin sections were collected onto plastic-coated nickel grids and immunochemically contraststained with uranium salts (uranyl acetate) and lead citrate to reveal cell ultrastructure. finally, samples were analyzed using a transmission electron microscope em902a fa.zeiss with the item soft imaging system software (olympus). untransduced jurkat cells and pbmcs cultured as was previously described, were collected, counted, washed with pbs and 10 6 cells were incubated 30 min with 100 µl lysis buffer (50 mm tris-hcl ph 7.4, 150 mm nacl, 1% nonidetp-40, 1 mm edta, 1 mm phenylmethylsulfonyl fluoride, 1 mm na 3 vo 4 , 1 mm naf, on ice. cell debris was discarded by centrifugation at 12,000 g for 20 min, 4 • c, and soluble proteins were stored at −80 • c before further analysis. protein levels were measured using bradford protein assay kit (pierce), according to manufactures recommendations. two hundred microliter magnetic beads (dynabeads r myone tm streptavidin t1 invitrogen) were washed following manufacturer's recommendations and conjugated with hcv core genotype-1b biotin-prospec (hcv-242) by incubating 100 µl (10 mg/ml) dynabeads with 100 µl (800 pmoles) hcv core protein in pbs, for 2 h at 4 • c, with gentle rotation. hcv core protein-coated beads were washed three times by resuspension in pbs containing 0.02% twin-20 and decanting the supernatant while placed on a magnet. for mass spectrometry analysis, coated dynabeads (or uncoated as control for unspecific binding) were incubated overnight, at 4 • c, with jurkat cells protein lysates, with gentle rotation, in the following ratio: 100 µl of coated beads with 150 µg of total protein lysate. for western blot experiments, coated dynabeads were incubated overnight, at 4 • c, with protein lysates from pbmc blasts, with gentle rotation, in the following ratio: 50 µl coated beads with 3 mg protein sample. 50 µl uncoated beads were incubated with 3 mg protein lysate, in the same conditions, as a control of unspecific binding. samples were washed twice with pbs and once with ultrapure water and incubated with 150 µl of 200 mm dithiothreitol (dtt) in 50 mm ammonium bicarbonate for 5 min at 65 • c. eluated proteins were quantified and 100 µg were precipitated with acetone during 1 h at −20 • c and centrifuged 30 min at 12,000 g. subsequently, proteins were resuspended in urea 6 m, reduced with 2 mm dtt in 100 mm ammonium bicarbonate at room temperature for 45 min and alquilated with 4 mm iodacetamide 45 min, in the dark, subsequently trypsin digestion was performed with sequencing grade trypsin (promega, madison, wi, usa) at 37 • c overnight (enzyme/substrate ratio 1:50). finally, the digestion was quenched adding 5% formic acid. the resulting peptides were transferred to a clean tube, dried in speed-vac and stored at −20 • c prior to analysis by mass spectrometry (ms). peptides were resolved by reverse phase chromatography using an eksigent ultra pump fitted with a 75 µm id column 25 cm length (acclaim pepmap 100 c18 2 µm 100 a). samples were initially loaded for desalting into a 2 cm length 100 µm id precolumn packed with the same chemistry as the resolving column prior to analytical chromatography. mobile phases used were 100% water 0.1% formic acid (buffer a), 100% acetonitrile 0.1% formic acid (buffer b). gradient was developed during 95 min up to 35% b. column was equilibrated in 95% b for 10 min and 5% b for 15 min, using a constant flow of 300 nl/min. peptides eluted from the column were analyzed using a sciex 5600 tripletof tm system. data dependent acquisition was collected upon a survey scan performed in a mass range from 350 to 1,250 m/z in a scan time of 250 ms. the top 25 most intense precursors were selected for fragmentation with a transmission window width of 0.7 da. minimum accumulation time for ms/ms was set to 75 ms giving a total cycle time of 2,125 ms. fragment ions were collected in a mass range from 230 to 1,500 m/z in order to have a single q2 transmission window. precursors were excluded from further fragmentation during 15 s. raw data files were processed using proteinpilot tm 4.5 software from sciex and mgf files were loaded onto proteinscape tm 3.1 software (bruker) for data grouping and protein identification via mascot program version 2.5 (matrix science ltd, london, uk). search parameters were: homo sapiens taxonomy. trypsin specificity was used and only one missed cleavage allowed, cysteine carbamidomethylation as fixed and oxidized methionine was used as variable modification. mass tolerance of precursor ion and fragment ions were 10 ppm and 0.05 da, respectively. peptides charges of 1+, 2+, and 3+ were selected and minimum peptide length was set at five residues. peptides and proteins were considered positively identified if their mascot score was higher than 20 and 30, respectively. proteins were identified with the minimum of one peptide and a false discovery rate <1%. proteins identified with one single peptide were only included if peptide scores were significant, and peptide spectra contained most "y" and/or "b" representative ions for the corresponding peptide sequence. examples are included in supplementary table 2 . subsequently, we compared the identified proteins that bound to the dynabeads vs. the ones identified binding to hcv core protein using the "compare results" option from proteinscape software 3.1. thus, 222 proteins were found to specifically associate with hcv core protein. proteome profiling data has been deposited in the peptideatlas repository, identified as pass00982. samples obtained from the pull down assay were washed twice with pbs using the magnet, and incubated with 2x laemmli buffer at 99 • c for 5 min. subsequently, beads were removed from the proteins using a magnet and proteins resolved by sds-page and transferred to pvdf membranes (immobilon-fl). membranes were incubated with the corresponding primary antibody, followed by the appropriated secondary antibody, conjugated to hrp. primary antibodies used were pp1 gamma antibody (pa5-21671) from thermo fisher scientific r and mypt1 antibody (#2634) from cell signaling technology r . reactive proteins were visualized using an ecl system and images were acquired in a chemidoc touch imaging system (bio-rad r laboratories, hercules, ca, usa). localization and network analysis of identified candidate proteins associated with hcv core protein string database version 10 (search tool for the retrieval of interacting genes/proteins) (http://string-db.org) (szklarczyk et al., 2015) was applied to visualize detailed information about the subcellular localization and the main interactions of the identified proteins. ingenuity pathways analysis (ipa; ingenuity systems, redwood city, calif.) software was used for further protein characterization according to known and predicted associations into function canonical pathways and networks recorded in the ipa library. nuclear localization sequence (nolss) in hcv core protein prediction was obtained by entering a protein sequence in fasta format on the nod web server http://www.compbio.dundee.ac. uk/www-nod/ (scott et al., 2011) . nolss were predicted if the average score output by the artificial neural network ann of 8 consecutive windows was at least 0.8 (scott et al., 2010 (scott et al., , 2011 . to analyze the localization of hcv core protein in t cells and to circumvent the lack of optimal anti-hcv core antibodies, we took advantage of a gfp-hcv core fusion construct that has been extensively used in our laboratory (dominguez-villar et al., 2007 , 2012a fernandez-ponce et al., 2014) and an unfused gfp expressing construct as a control. jurkat cells were efficiently transduced with lentiviral vectors expressing hcvcore-gfp or gfp as a control. percentage of transduced cells analyzed by flow cytometry was >98% in all cases (data not shown). jurkat cells transduced with gfp or hcv core gfp-expressing lentiviral construct or left untransduced were subsequently immunostained with anti-gfp antibody and a 10 nm gold-labeled anti-rabbit igg and analyzed by transmission electron microscopy. as shown in figure 1 , hcv core was mostly localized in the nucleus (figures 1b,d,e) and specifically in the nucleolus where it was greatly enriched (figures 1b-f) , although some immunostaining was observed in cytoplasm ( figure 1a) . hcv-core-gfp was detected in the nucleus in 35% out of 40 hcv core gfp expressing jurkat cells analyzed, 22% showed the presence of gfp inside both nucleus and cytoplasm, 5% of the cells only in cytoplasm (figure 1a) , while 18% showed gfp immunolabelling in nucleus with enrichment in the nucleolus (figures 1b-f) . 20% of the cells were not stained. in hcv core expressing jurkat cells which showed core protein nucleolar localization, the total raw number of gold particles counted inside the nucleoli was 196, with an average of 28 gold particles per cell and a standard deviation of 13.3. regarding gfp transduced control cells, gfp was localized in the nucleus of 84% from 40 gfp-expressing jurkat cells analyzed and in both nucleus and cytoplasm in 16%. there was no recognizable co-localizing with any organelle. nucleolus localization was not visible in any cell (figures 1g,h) . no immunogold staining was observed in untransduced jurkat cells (data not shown). thus, the study revealed specific immunolabeling of hcv core protein in the nucleolus of jurkat cells. based on the findings obtained with the electron microscopy assay, we decided to identify hcv core associated proteins that could mechanistically be correlated with hcv core protein nucleolar localization. thus, we performed pull down experiments using biotinilated hcv core protein as bait. further analysis with the string platform allowed us to classify the identified proteins depending on their localization (figure 2) . thus, from the 222 associated proteins identified (supplementary table 1 ), 128 have been previously described to localize in the nucleus (figure 2a) and 43 in the nucleolus (figure 2b) , while most of the 150 proteins described in the cytoplasm (figure 2c ) have also been identified in the nucleus and/or nucleolus. string also allowed us to determine potential protein functionalities according to previously published reports. interestingly, most of the identified hcv core associated proteins were found to participate in binding processes, as indicated in figure 2 . regarding protein function, we mainly focused on the functional classification given by the ipa software, which, based on biomedical literature and integrated databases, allows to determine the most probable pathways and/or functions in which identified proteins are involved. thus, ipa core analysis of our dataset (figure 3 ) revealed that the identified hcv core associated proteins were mainly described to participate in splicing and processing of rna, cell cycle progression, cell proliferation, apoptosis and rna virus infection. our findings support the concept that the subcellular localization of hcv core protein might have an impact on cd4 + t cells functions. in order to confirm, by an alternative method, some of the associations identified by mass spectrometry analysis, and most importantly to evaluate whether such associations were also present in primary t cells (pbmc), thus enhancing the relevance of our findings. postnuclear lysates from human pbmcs blasts (see methods), were subjected to hcv core protein pull down, using a biotinilated hcv core protein (800pmoles) bound to magnetic beads as a bait. uncoated magnetic beads were used as a control. experiments were first carried out in jurkat cells and inputs from jurkat and pbmc were loaded in parallel (1.5% from the amount used per pull down), to evaluate the relative amount of each target protein (data not shown). serine/threonine-protein phosphatase was selected due to its known localization (it has a dynamic and predominantly nucleolar distribution) and to its function (it has been implicated in the regulation of several biological pathways previously described in t cells transduced with hcv core protein) (aggen et al., 2000; ceulemans and bollen, 2004; nie et al., 2013) . it was also selected based on the fact that both its catalytic subunit (pp1γ) and its regulatory subunit 12 a (mypt1), were identified by mass spectrometry, showing a despairing mascot score of 37.6 and 336.5 respectively. as shown in figure 4 , western blot analysis of (pp1γ) and (mypt1), confirmed the presence of both frontiers in microbiology | www.frontiersin.org figure 2 | "actions" view, according to the "go cellular components" distribution option, in order to identify their subcellular localization. string analysis reported that proteins were nuclear (a) (128 from 222), nucleolar (b) (43 from 222), and cytoplasmic (c) (150 from 222). colored lines between proteins indicate the type of evidence for each interaction, with a minimum required confidence score of 0.4. proteins in pbmcs lysates obtained from the pull down of hcv core protein coated magnetic beads (figure 4) . in order to identify potential nucleolar localization sequences in hcv core protein, we used the nod web server (http://www. compbio.dundee.ac.uk/www-nod/), a program that predicts the presence of nolss in eukaryotic and viral proteins, based on a database of 46 statistically analyzed human nucleolar localization sequences (scott et al., 2011) . two nolss were identified in hcv core protein ( several biological and immunological consequences of hcv core intracellular expression in cd4 + t cells have previously been described by us and others, (bergqvist and rice, 2001; bergqvist et al., 2003; dominguez-villar et al., 2007 , 2012a doumba et al., 2013; fernandez-ponce et al., 2014) . these findings suggest an important role for the intracellular presence of hcv core in hcv pathogenesis and chronification. in this study, we have analyzed the ultrastructural localization of hcv core protein in cd4 + t cells. according to studies using cell lines unrelated to the immune system, hcv core protein has been shown to localize in the endoplasmic reticulum, mitochondrial outer membrane and nucleus of human embryonic kidney 293t cells (suzuki et al., 2005) ; associated to lipid droplets in cho, hepg2 and huh7 cells lines (barba et al., 1997; boulant et al., 2006; qiang and jhaveri, 2012) , in the cytoplasm, endoplasmic reticulum, in the proximity of the nuclear membrane, in the nucleus and nucleoli of hepatocytes isolated from chronically hcv infected patients (falcon et al., 2003) ; and in the cytoplasm and nucleus of non-parenchymal liver cells such as, lymphocyte-like cells, kupffer-like cells, polymorphonuclear-like cells, pit, endothelial, stellate, and fibroblast-like cells isolated from livers of chronically hcv infected patients (falcon et al., 2005) . in the present work, we found that in cd4 + t lymphocytes, hcv core protein mostly localizes in the nucleus and specifically in the nucleolus where it is greatly enriched and mainly organized in clusters (figure 1) . regarding these findings, nuclear localization signals (nlss) described previously in hcv core protein, could be responsible for nuclear localization (chang et al., 1994; suzuki et al., 1995 suzuki et al., , 2005 , while hcv core protein traffic and residence in the nucleolus, could be explained by the presence of two nucleolar localization sequences (nolss), identified using the bioinformatic nucleolar localization sequence detector web server for eukaryotic and viral proteins (scott et al., 2011) . nolss are showed in figure 4 . the statistical variation in core protein nucleolar localization shown by hcv-core expressing cells, could be due to differences in cellular cycle stage, as it has been described for other nucleolar resident proteins (chen and huang, 2001; stoldt et al., 2007; pirlot et al., 2016) . presence of nolss in cellular or viral proteins is a key factor in their dynamic traffic and residence within the nucleolus. presumably, nolss interact with nucleolar proteins and/or rna to mediate nucleolar targeting and retention. thus, in chimeric viral proteins with mutated nolss, trafficking to the nucleolus is abrogated, and heterologous nolss insertion restores their trafficking pattern (boyne and whitehouse, 2006; emmott et al., 2008) . interestingly, the function of hcv core protein inside the virus, can partly explain its subcellular localization as hcv core protein mainly interacts with hcv genomic rna, multimerizing around it and forming the capsid shell. while multimerizing inside the virus, we have not seen any multimerization in our experiments, which is in agreement with several studies showing that mammalian cell lines fail to produce capsid assembly (bukh et al., 2002; pietschmann et al., 2002; polyak et al., 2006; rouille et al., 2006; hourioux et al., 2007) , due to the lack of host cells factors that are essential for hcv core multimerization and assembly, or to the presence of inhibitory factors that induce the majority of hcv-core to be targeted away from the er (hope and mclauchlan, 2000; mclauchlan et al., 2002; polyak et al., 2006) . thus, un-multimerized hcv core protein traffics to alternate subcellular compartments. hcv core has been shown to bind rnas in addition to hcv genomic rna (kunkel et al., 2001; cristofari et al., 2004) , including ribosomal rna (santolini et al., 1994) and trna (kunkel et al., 2001) . since the nucleolus contains several 100 copies of rrna genes and it could be a recruiting site for trnas (carmo-fonseca et al., 2000) it is likely that the nucleolar rna constitutes another important molecular target structure for hcv core protein. in agreement with our findings, several dna viruses, retroviruses and rna viruses, as well as viral proteins, have been described to traffic to the nucleolus and be associated with nucleolar proteins wurm et al., 2001; chen et al., 2002; dove et al., 2006; michienzi et al., 2006; cawood et al., 2007; hiscox, 2007; emmott et al., 2010; lam et al., 2010; jarboui et al., 2012) . the nucleolus is a dynamic nuclear organelle whose proteome is continuously changing. it seems to be a temporary storage or a sequestration site for a multiplicity of proteins (emmott and hiscox, 2009; hiscox et al., 2010) . functionally, there is extensive evidence that the nucleolus is implicated in ribosome biogenesis (stoykova et al., 1985; warner, 1990; scheer et al., 1993) , cell cycle regulation, cell growth, senescence, stress response signaling (andersen et al., 2005; emmott and hiscox, 2009; tsai and pederson, 2014; lam and trinkle-mulcahy, 2015) and the pathogenesis of several diseases such as cancer (james et al., 2014; orsolic et al., 2015; yang et al., 2015) , cardiovascular disease (hariharan and sussman, 2014) and neurodegenerative disorders (payao et al., 1994; lu et al., 1998; rieker et al., 2011; tsoi and chan, 2013; lee et al., 2014a; parlato and liss, 2014; hernandez-ortega et al., 2015) . viral protein trafficking and localization into the nucleolus has shown implications in both viral life cycle and in host cell physiology and it has been narrowly related with the loss of essential nucleolar functions (hiscox, 2002) . in addition, it has been shown that accumulation of viral proteins in the nucleolus can cause volume exclusion and crowding effects, disrupting the nucleolar architecture (hancock, 2004; hiscox, 2007) . virus infection and some viral proteins from poliovirus, avian infectious bronchitis virus (ibv), coronavirus and human immunodeficiency virus-1 (hiv-1) induce disruption of the nucleolar architecture and changes on the subcellular distribution of nucleolar proteins or proteins that traffic to the nucleolus, such as nucleolin, p53, b23.1 (waggoner and sarnow, 1998; hiscox, 2002; dove et al., 2006) . these findings are closely related to the presence of perturbations in cell cycle, cytokinesis and apoptosis in the host cells (miyazaki et al., 1996; chen et al., 2002; galati et al., 2003; hiscox, 2007) . semliki forest virus nucleocapsid migrates to the nucleolus (jakob, 1994) and porcine reproductive and respiratory syndrome virus nucleocapsid specifically interacts with the small nucleolar rna (snorna)-associated protein, fibrillarin in virus infected cells (rowland et al., 1999; yoo et al., 2003) , while hepatitis b virus (hbv) core protein usually co-localizes with the nucleolar proteins, nucleolin and b23 (ning and shih, 2004) . in addition, it has been shown that coronavirus nucleocapsid protein localization in the nucleolus of infected cells and its association to the nucleolar protein b23.1 is related to cell cycle stage and could be involved in cell cycle delay or arrest to promote virus replication wurm et al., 2001; cawood et al., 2007) . thus, trafficking to the nucleolus and nucleolar residency time of hcv core protein in cd4 + t cells could be narrowly related with previous findings from us and others showing that the intracellular presence of hcv core in cd4 + t cells induces decreasing cell proliferation, delay in cell cycle progression and a differential expression pattern of genes with relevant function, including anergy-associated genes, genes involved in cytoskeleton reorganization, vesicle trafficking, endocytosis, cytokines production, cell death, transcription, and translation (dominguez-villar et al., 2007) . in agreement with the described localization, we found hcv core protein association with several nuclear, nucleolar proteins or proteins described to traffic to the nucleolus, which showed mainly binding connections (figure 2) . the wide range of proteins associated with hcv core, correlate with the findings obtained by dolan et al. who identified two computationally predicted molecular recognition features within the n-terminal intrinsically disordered region (idr) in the hcv core sequence. the identified molecular recognition features, mediate hcv core protein binding to hcv rna and to multiple host proteins, suggesting that hcv core protein exhibits hub protein properties (dolan et al., 2015) . interestingly, pathway and network-based analysis of proteins identified to be associated with hcv core protein, indicate that a wide range of these proteins are involved in several biological signaling pathways, such as, rna processing and splicing (figure 3a) , cell cycle progression, cell proliferation, apoptosis ( figure 3b ) and infection and replication of rna viruses, including hcv ( figure 3c) . with the integrated analysis of the present data, we confer a better description of the hcv core -human t lymphocyte relationship. concerning ribosomal biogenesis, cellular proliferation, cell cycle and apoptosis; several large (rpl) and small (rps) ribosomal proteins, and proteins involved in rna processing and splicing as dead-box rna helicases, precipitated with figure 5 | predicted nucleolar localization sequences in hcv core protein. graph obtained from the nucleolar localization sequence detector web server (nod), displaying nols prediction score for each residue of hcv core protein. pink shaded regions represent the range of scores within which a 20-residues segment is predicted to be a nols. thus, pink shaded regions represent the nols candidate segment, which highlights scores above 8.0. hcv core (figures 3a,b) (rocak and linder, 2004; xu et al., 2016) . interactions between viral and ribosomal proteins, splicing factors and dead-box rna helicases including ddx5 and ddx17, have been previously described and suggested as a key mechanism for viral replication and production as well as for life cycle progression and survival (bortz et al., 2011; naji et al., 2012; yasuda-inoue et al., 2013; cervantes-salazar et al., 2015; klymenko et al., 2016; li et al., 2016) . implication of other identified proteins, such as protein red (ik) and filamin a (flna) (figure 3b ) in proliferation and cell cycle progression, have also been widely demonstrated (lee et al., 2014b; sun et al., 2014) . thus, associations of hcv core protein in t cells could alter the function of the associated proteins, explaining some of the effects shown for hcv-core expression, including cell cycle delay and inhibition of cell proliferation (dominguez-villar et al., 2007 , 2012a fernandez-ponce et al., 2014) . in addition, we found that many host proteins associated with hcv core, are involved in replication and infection of rna viruses including hcv ( figure 3c) . proteins as dexd/hbox helicases have been extensively studied in virus infection and have been described as proteins hijacked by viruses for their benefit. specifically, dhx9 is involved in virus replication, innate immunity response to viral dsrna and participation in the expression of ifn-stimulated genes (fullam and schroder, 2013) . rna binding proteins (rbps) such as the host poly(rc)-binding protein (pcbp) and different heterogeneous nuclear ribonucleoproteins (hnrps), are also interesting as they stabilize viral rnas, co-localize with the viral replicase complex and facilitate viral rna template selection (li and nagy, 2011) . in addition, interleukin enhancer-binding factor 3 (ilf3) has been shown to be recruited to hcv replication complexes (li et al., 2014) and in other infections by rna viruses, ilf3 interaction with viral proteins has been described to affect virus replication among other virus life cycle stages (patino et al., 2015) . furthermore, the subcellular localization of hcv core and its association with nuclear and nucleolar proteins found in the present study, can aid in explaining the cd4 + t cell regulatory/exhausted phenotype described by us and others, in cd4 + t cells expressing hcv core protein (dominguez-villar et al., 2012a; doumba et al., 2013; fernandez-ponce et al., 2014) . some such associations are with serine/threonine-protein phosphatase catalytic subunit (pp1γ), protein phosphatase 1 regulatory subunit 12a (mypt1) (nie et al., 2013) , interleukin enhancer-binding factor 3 (ilf3) (shi et al., 2007a,b) , complement component c1q binding protein (c1qbp) (kittlesen et al., 2000) and runt-related transcription factor 1 (runx1) (klunker et al., 2009) . in conclusion, analysis of the association of hcv core with host proteins in cd4 + t cells and the study of its ultrastructural localization, open an extensive field of study poised to understand the mechanisms underlying functional findings previously described in cd4 + t cells expressing hcv core protein, that have demonstrated to be relevant for hcv immune system evasion and thus hcv chronification. conception, design and or interpretation of the work: fg-c, ea, and rl. wb (for localization studies). cb and md-r (for association studies). performed association studies: cf-p, md-r, and as-s. performed localization studies: cf-p, jm-m, and ma-e. run statistical analyses: rl and cf-p. wrote the paper: fg-c, cf-p, ea, rl, and md-r. performed confirmation co-ip experimentos: in-s and cf-p. all authors participated in critical revision and subsequently approved the manuscript. all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. for ea. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. regulation of protein phosphatase-1 nucleolar proteome dynamics hepatocellular carcinoma: role of hepatitis b and hepatitis c viruses proteins in hepatocarcinogenesis hepatitis c virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets transcriptional activation of the interleukin-2 promoter by hepatitis c virus core protein the hepatitis c virus core protein modulates t cell responses by inducing spontaneous and altering t-cell receptor-triggered ca2 + oscillations the conserved n-terminal domain of herpes simplex virus 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may disrupt host cell division the role of ribosomal proteins in the regulation of cell proliferation, tumorigenesis, and genomic integrity nucleolar repression facilitates initiation and maintenance of senescence direct binding of hepatitis c virus core to gc1qr on cd4 + and cd8 + t cells leads to impaired activation of lck and akt distinct ddx deadbox rna helicases cooperate to modulate the hiv-1 rev function colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rna-associated protein fibrillarin interaction of avian influenza virus ns1 protein and nucleolar and coiledbody phosphoprotein 1 we would like to thank christian hoffmann, evelyn janssen, beatrix martiny, jessicahausmann, mojgan ghilav and consuelo rivera for their excellent technical support, the plataforma andaluza de bioinformática (centro de supercomputación y bioinformática, university of málaga) for the use of the ingenuity pathways analysis (ipa) software and the core biomedical research facility of the university of cadiz for the use of core infrastructure. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2017.02595/full#supplementary-material key: cord-329840-f3dsu36p authors: hati, sanchita; bhattacharyya, sudeep title: impact of thiol-disulfide balance on the binding of covid-19 spike protein with angiotensin converting enzyme 2 receptor date: 2020-05-11 journal: biorxiv doi: 10.1101/2020.05.07.083147 sha: doc_id: 329840 cord_uid: f3dsu36p the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has led to an ongoing pandemic of coronavirus disease (covid-19), which started in 2019. this is a member of coronaviridae family in the genus betacoronavirus, which also includes sars-cov and middle east respiratory syndrome coronavirus (mers-cov). the angiotensin-converting enzyme 2 (ace2) is the functional receptor for sars-cov and sars-cov-2 to enter the host cells. in particular, the interaction of viral spike proteins with ace2 is a critical step in the viral replication cycle. the receptor binding domain of the viral spike proteins and ace2 have several cysteine residues. in this study, the role of thiol-disulfide balance on the interactions between sars-cov/cov-2 spike proteins and ace2 was investigated using molecular dynamic simulations. the study revealed that the binding affinity was significantly impaired when all the disulfide bonds of both ace2 and sars-cov/cov-2 spike proteins were reduced to thiol groups. the impact on the binding affinity was less severe when the disulfide bridges of only one of the binding partners were reduced to thiols. this computational finding provides a molecular basis for the severity of covid-19 infection due to the oxidative stress. the novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) or simply covid-19 is the seventh member of the coronavirus family. 1 the other two viruses in this family that infect humans are severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). these are positive-sense, single-strand enveloped rna viruses. the coronavirus particles contain four main structural proteins: the spike, membrane, envelope, and nucleocapsid. [1] [2] the spike protein protrudes from the envelope of the virion and consists of two subunits; a receptor-binding domain (rbd) that interacts with the receptor proteins of host cells and a second subunit that facilitates fusion of the viral membrane into the host cell membrane. recent studies showed that rbd of spike proteins of sars-cov and sars-cov-2 interact with angiotensin-converting enzyme 2 (ace2). ace2 belongs to the membrane-bound carboxydipeptidase family. it is attached to the outer surfaces of cells and is widely distributed in the human body. in particular, higher expression of ace2 is observed in organs such as small intestine, colon, kidney, and heart, while ace2 expression is comparatively lower in liver and lungs. [3] [4] the role of oxidative stress on the binding of viral proteins on the host cell surface receptors is a relatively underexplored area of biomedical research. [5] [6] [7] [8] [9] [10] previous studies have indicated that the entry of viral glycoprotein is impacted by thiol-disulfide balance on the cell surface. 5, [7] [8] [9] [11] [12] any perturbations in the thiol-disulfide equilibrium has also been found to deter the entry of viruses into their target cells. 5 the first step of the viral entry involves binding of the viral envelop protein onto a cellular receptor. this is followed by endocytosis, after which conformational changes of the viral protein helps the induction of the viral protein into the endosomal membrane, finally releasing the viral content into the cell. these conformational changes are mediated by ph changes as well as the conversion of disulfide to thiol group of the viral spike protein. 7 several cell surface oxidoreductases 9 regulate the thiol-disulfide exchange, responsible for conformational changes of viral proteins needed for virus entry into host cells. in the backdrop of significant mortality rate for sars-cov-2 (hereinafter referred to as cov-2) infection, it is important to know if the thiol-disulfide balance plays any role on the binding of the spike glycoprotein on to the host cell receptor protein ace2. a recent study with the spike glycoprotein of sars-cov (hereinafter referred to as cov) has exhibited a complete redox insensitivity; 7 despite the reduction of all disulfide bridges of cov to thiols, its binding to ace2 remained unchanged. 7 however, this study did not probe the redox sensitivity of ace2 receptor. thus, in the present study, we computationally investigated the redox state of both partners (ace2 and cov/cov-2) on their binding affinities. the structure of cov 1 3 and cov-2 14-15 complexed with ace2 are known and the noncovalent interactions at the protein-protein interface 16 have been reported recently. using these reported structures, molecular dynamics simulations and electrostatic field calculations were performed to explore the impact of thioldisulfide balance on cov/cov-2 and ace2 binding affinities. the structural and dynamical changes due to the change in the redox states of cysteines in the interacting proteins were analyzed and their effects on binding free energies were studied. the molecular basis of the binding of spike proteins to ace2 is known from x-ray crystallographic (sars-cov) 13 and cryo-electron microscopic (sars-cov-2) 16 studies. the sequence alignment of cov and cov-2 spike proteins showed high sequence identity (>75 %) indicating that their binding to ace2 receptors will be similar ( fig. 1 ). in both bound structures, the rbd of cov and cov-2 is found to be complexed with ace2 (fig. 2) . both ace2 and cov-2 possess four disulfide bridges, whereas cov subunit has only two disulfide linkages (table 1 and fig. 2a and 2b) . two large helices of ace2 form a curved surface (fig. 2, illustrated by the dashed curved line) that interacts with the concave region of cov or cov-2 subunit. structural change along the trajectory. in all cases, the simulation started with an equilibrated structure, which was obtained after minimizing the neutralized solvated protein complex built from the experimentally determined structures. the evolution of the protein structure along the md trajectory was monitored by calculating the root-mean-square deviation (rmsd) of each structure from the starting structure as a frame of reference following standard procedure. 17 briefly, during the md simulation, the protein coordinates were recorded for every 10 ps interval and a root-mean-square-deviation (rmsd) of each frame was calculated from the average root-mean-square displacement of backbone c α atoms with respect to the initial structure. then, the rmsd values, averaged over conformations stored during 1 ns time, were plotted against the simulated time (fig. 3 ). compared to the starting structure, only a moderate backbone fluctuation was noted in all protein complexes during 20 ns simulations and the maximum of rmsd was in the range of 2.0-3.8 å ( table 2 ). the evolution was smooth and its stability was demonstrated by the standard deviation of the computed rmsds, which was less than 0.3 å. taken together, results of the simulations showed no unexpected structural deformation of the sar-cov/cov-2⋅⋅⋅ace2 complex in both, reduced (thiol groups) and oxidized (disulfide bonds) states (table 2) . table 3 ). however, when all disulfides in cov/cov-2 as well as ace2 were reduced to thiols, the binding became thermodynamically unfavorable. in both cases, the binding free the study found that the reduction of all disulfides into sulfydryl groups completely impairs the binding of sars-cov/cov-2 spike protein to ace2. this is evident from the positive gibbs energy of binding (∆ bind g o ) obtained for both cov ox ⋅⋅⋅ace2 ox and cov-2 ox ⋅⋅⋅ace2 ox complexes. when the disulfides of only ace2 were reduced to sulfydryl groups, the binding becomes weaker, as the the redox environment of cell surface receptors is regulated by the thiol-disulfide equilibrium in the extracellular region. 12, 18 this is maintained by glutathione transporters, 19 a number of oxidoreductases 12 including protein disulfide isomerase, 8 and several redox switches. 12 under oxidative stress, the extracellular environment becomes oxidation-prone resulting more disulfide formation on protein surfaces. 12 therefore, under severe oxidative stress, the cell surface receptor ace2 and rbd of the intruding viral spike protein are likely to be present in its oxidized form having predominantly disulfide linkages. this computational study shows that under oxidative stress, the lack of reducing environment would result in significantly favorable binding of the viral protein on the cell surface ace2. in terms of energetics, this computational study demonstrates that the oxidized form of proteins with disulfide bridges would cause a 50 kcal/mol of decrease in gibbs binding free energy. furthermore, ace2, which the viral spike proteins latch on to, is known to be a key player in the remedial of oxidative stress. 20 binding of the viral protein will prevent the key catalytic function of ace2 of converting angiotensin 2 (a strong activator of oxidative stress) to angiotensin 1-7 thereby creating a vicious circle of enhanced viral attack. in summary, the present study demonstrates 1 0 that the absence of or reduced oxidative stress would have a significant beneficial effect during early stage of viral infection by preventing viral protein binding on the host cells. computational setup. setting up of protein systems and all structural manipulations were carried out using visual molecular dynamics (vmd). 21 disulfide groups were modified to thiols during setting up of structures using standard vmd scripts. molecular optimization and dynamics (md) simulations were carried out using nanoscale molecular dynamics (namd) package using charmm36 force field. [22] [23] [24] [25] [26] during md simulations, electrostatic energy calculations were carried out using particle mesh ewald method. 27 backbone root-mean-squaredeviation (rmsd) calculations were performed using vmd. protein-protein interactions were studied using adaptive-basis poisson boltzmann solver (apbs). 28 electrostatic field calculations were performed using pdb2pqr program suit. 29 molecular dynamics simulations. all simulations were performed using the structure of ace2 bound sars-cov (pdb entry: 3d0g) 1 3 and scar-cov-2 (pdb entry: 6m0j) 15 . in all simulations, setup of protein complexes systems was carried out following protocols used previous studies from this lab. 17, 30 briefly, hydrogens were added using the hbuild module of charmm. ionic amino acid residues were maintained in a protonation state corresponding to ph 7. the protonation state of histidine residues was determined by computing the pka using binding free energy calculations. gibbs free energies of binding between the ace2 and sars cov or cov-2 proteins were calculated using apbs using a standardized method of a treecode-accelerated boundary integral poisson-boltzmann equation solver (tabi-pb). 33 in this method, the protein surface is triangulated, and electrostatic surface potentials are computed. the discretization of surface potentials is utilized to compute the net energy due to solvation as well as electrostatic interactions between the two protein subunits, as outlined in thermodynamic scheme, scheme 1. following scheme 1, the free energy of binding of the two protein fragments in water, can be expressed as a sum of two components (eq. 1) where the ∆ coul g represents the coulombic (electrostatic) interactions between the proteins occurring at the protein-protein interface (scheme 1) and ∆ ∆ solv g is the difference of the solvation energies between the complex and the corresponding free proteins: however, the solvation calculation used only part of the entire spike protein as well as the ace2, therefore ∆ bind g tabi-pb was calibrated by correcting ∆ ∆ solv g using experimentally known binding free energy of ace2···cov-2: where k d is the experimental dissociation constant, which is equal to 37 nm. 16 the corrected free energy of the solvation using the ∆ g corr and eq. 4, the corrected binding free energy, ∆ bind g o of all protein complexes is expressed by as shown in eq. 4, the combination of last two terms in eq. 5 is equal to ∆ ∆ solv g corr. therefore, eq. 5 can be simplified as: figure 1 . sequence alignment (generated by clustal omega 35 ) between the receptor-binding domain of sars-cov and sars-cov2 proteins. the "*" represents the identical residues, ":" represents similar residues, and gap represents dissimilar residues. the cysteine residues are highlighted in yellow. rvqptesivrfpnitnlcpfgevfnatrfasvyawnrkrisncvadysvlynsasfstfk 60 cov ------------------pfgevfnatkfpsvyawerkkisncvadysvlynstffstfk 42 *********:* *****:**:**************: ***** cygvsptklndlcftnvyadsfvirgdevrqiapgqtgkiadynyklpddftgcviawns 120 cov cygvsatklndlcfsnvyadsfvvkgddvrqiapgqtgviadynyklpddfmgcvlawnt 102 ***** ********:********::**:********** ************ ***:***: the medium-scale fluctuations are shown in green. coronavirus envelope protein: current knowledge coronaviruses: an overview of their replication and pathogenesis structure analysis of the receptor binding of 2019-ncov high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent hiv-1 entry significant redox insensitivity of the functions of the sars-cov spike glycoprotein: comparison with hiv envelope cell surface protein disulfide isomerase regulates natriuretic peptide generation of cyclic guanosine monophosphate the role of cellular oxidoreductases in viral entry and virus infectionassociated oxidative stress: potential therapeutic applications implications of oxidative stress on viral pathogenesis cell entry by enveloped viruses: redox considerations for hiv and sars-coronavirus thiol-disulfide exchange reactions in the mammalian extracellular environment structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections cryo-em structure of the 2019-ncov spike in the prefusion conformation structural and functional basis of sars-cov-2 entry by using human ace2 structural basis for the recognition of sars-cov-2 by full-length human ace2 crowder-induced conformational ensemble shift in escherichia coli prolyl-trna synthetase from structure to redox: the diverse functional roles of disulfides and implications in disease plasma membrane glutathione transporters and their roles in cell physiology and pathophysiology ace2-mediated reduction of oxidative stress in the central nervous system is associated with improvement of autonomic function vmd: visual molecular dynamics charmm additive all-atom force field for glycosidic linkages between hexopyranoses charmm additive all-atom force field for glycosidic linkages in carbohydrates involving furanoses charmm additive all-atom force field for carbohydrate derivatives and its utility in polysaccharide and carbohydrate-protein modeling optimization of the additive charmm all-atom protein force field targeting improved sampling of the backbone (2) dihedral angles charmm additive all-atom force field for phosphate and sulfate linked to carbohydrates a smooth particle mesh ewald method improvements to the apbs biomolecular solvation software suite pdb2pqr: expanding and upgrading automated preparation of biomolecular structures for molecular simulations multiple pathways promote dynamical coupling between catalytic domains in escherichia coli prolyl-trna synthetase computer ""experiments" on classic fluids. i. thermodynamical properties of lennard-jones molecules scalable molecular dynamics with namd a treecode-accelerated boundary integral poisson-boltzmann solver for electrostatics of solvated biomolecules structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor the embl-ebi search and sequence analysis tools apis in 2019 we acknowledge computational support from the blugold super computing cluster (bgsc) of university of wisconsin-eau claire. figure. 2. structures of protein complexes of a) sars-cov⋅⋅⋅ace2 and b) sars-cov-2⋅⋅⋅ace2. all the disulfide bridges between cysteine residues are shown in green vdw spheres and thiol groups in cyan licorice. key: cord-004948-ad3i9wgj authors: nan title: 7th international congress on amino acids and proteins : vienna, austria, august 6–10, 2001 date: 2001 journal: amino acids doi: 10.1007/s007260170030 sha: doc_id: 4948 cord_uid: ad3i9wgj nan the raised concentration of protein bound homocysteine in homocystinuric (hcu) patients displaces protein bound cysteine and increases the free/bound cysteine ratio in plasma. this ratio is independent of albumin concentration. results from 31 hcu patients were compared to 40 controls. free cystine concentrations in hcu were poorly discriminated from the control range but the total cysteine results were almost invariably lower than control data. this appears to result from an increased free/bound cysteine ratio in hcu [mean (range) for control 0.50 (0.22-0.71) and for hcu 0.76 (0.32-1.75); p ϭ 0.0005]. ex vivo protein binding experiments in albumin solution revealed the free/bound cysteine ratio to be linearly related to the amount of homocysteine bound (r ϭ 0.907, p ͻ 0.001). we conclude that measurement of total cysteine is essential for assessment of the true cysteine status in hcu. however, any cysteine deficit, or alteration to free/bound cysteine ratios, does not obviously effect glutathione synthesis as assessed by measurement of plasma total glutathione. (339 nmol/g; 21.6%); sprague-dawley rat (rattus norvegicus, rodentia) (n ϭ 6; 98-381 nmol/g; 9.8-13.7%); rabbit (152 nmol/ g; 11.3%); pig (sus scrofa f. domestica, artiodactyla) (221 nmol/ g; 13.4%); bovine (bos primigenius f. taurus, artiodactyla) (284 nmol/g; 23.6%); seal (phoca vitulina, carnivora) (136 nmol/g; 3.8%), and rob (halichoerus grypus, carnivora) (218 nmol/g; 5.4%). from the date it is concluded that d-aas are common in body fluids and certain tissues of vertebrates. in order to determine the quantity of cyst(e)ine and methionine, the oxidation of cyst(e)ine and methionine (ϫ) refers to not detected or not determinable; asx ϭ asp ϩ asn; glx ϭ glu ϩ gln; his, arg, trp, cys not determined; a) feed fortified with dl-met; b) nmol/g lyophilized serum. mentation and subsequent purification. the separation of the enantiomers of cysteic acid, methionine sulphone, aspartic acid and glutamic acid is displayed on the chromatogram. into cysteic acid and methionine sulphone with performic acid is often applied before hydrolysis of protein. the authors examined the applicability of this process in case of quantification of cyst(e)ine and methionine enantiomers. the rp-hplc analytical method was developed for the determination of the amount of cysteic acid and methionine sulphone enantiomers. the rate of conversion during oxidation from cyst(e)ine into cystic acid and from methionine into methionine sulphone was determined. the racemisation of l-cyst(e)ine and l-methionine was negligible during oxidation with performic acid, therefore this process can be applied before hydrolysis during quantification of cyst(e)ine and methionine enantiomers. after the performic acid oxidation and the 6 m hcl hydrolysis of the protein, opa/tatg (o-phthaldialdehyde/tetra-o-acetyl-1-thio--d-glucopiranoside) precolumn derivatisation method was used, and the enantiomers of sulphur containing amino acids were separated by rp-hplc (lichrosphere 100 rp-18e, 125 ϫ 4 mm, 5 µm column, merck-hitachi lachrom hplc). the resolution of the peak of cysteic acid and methionine sulphone enantiomers was better than 1,5. the method was used to determine the amount of l-and d-cyst(e)ine and land d-methionine containing preparations prepared by ferd/l rate of aspartic acid and the individual age of specimens. a method for age determination based on d-aspartic acid content and on the racemisation of l-aspartic acid of teeth was developed. d-glutamic acid, beside d-aspartic acid, was found to be eminently suitable for the estimation of individual age, as it showed a sufficiently high sensitivity. calibration curves based on these investigations were used for the age estimation of 65 adults (39 males and 26 females) of unknown individual age from the avar period series of kereki-homokbánya (hungary). the age distribution of the sample was the following: 39 individuals (60%) belonged to the adult age group, 22 persons (34%) to the mature and 4 (6%) to the senile one. the correlation between our results and those obtained using standard paleoanthropological methods was over 0.9. quantitative determination of free and bound 3-nitrotyrosine in rat plasma and tissues using isotope dilution liquid chromatography-electrospray tandem mass spectrometry t. delatour, p. a. guy, j. richoz, j. vuichoud, and nestlé research centre, nestec ltd, vers-chez-les-blanc, lausanne, switzerland since 3-nitrotyrosine was reported to be readily formed in proteins by reactions with nitrite or nitrogen dioxide, it has been postulated to be a possible marker for investigating peroxynitrite-mediated nitration of proteins. thus, several methods were developed to assess nitration of tyrosine in proteins and determine 3-nitrotyrosine in physiological fluids. methods based on hplc or gc/ms techniques were described to quantify 3-nitrotyrosine within tissues or biological fluids. unfortunately, it has been demonstrated that an artifactual nitration of tyrosine occurs with gc/ms assays leading to an overestimation of the response. in the present work, lc-esi-ms/ms methods for quantification of free 3-nitrotyrosine in rat plasma as well as bound 3-nitrotyrosine in tissue samples are reported. plasma samples were spiked with 2,5,6-d 3 -3-nitrotyrosine and the following steps were applied prior to injection into the lc-esi-ms/ms system used in selected reaction monitoring (srm) mode (m/z 283 ae 181 for the analyte and m/z 286 ae 184 for the internal standard): protein precipitation, solid phase extraction on aminopropyl cartridge and derivatization in nbutanol in hcl 3 n. 3-nitrotyrosine butyl ester has lead to a dramatic increase of the sensitivity (ca. 5-fold) by comparison with 3-nitrotyrosine. under such conditions, calibration curves exhibited excellent linearity (r 2 ͼ 0.99) within concentration range 0.3 to 28.5 nm (equivalent to 47.3-4,730 fmol on column) and recoveries above 85%. inter-and intra-assay precision was determined below 15% over the concentration range 1.4 to 28.5 nm. no artifactual nitration of tyrosine occurring during sample clean-up was observed. this was unambiguously established by plotting experimental ratio of analyte response/ internal standard response versus expected within the range 0.3-28.5 nm. this curve strongly correlated with a linear model (r 2 ͼ 0.99) and slope was 1.07 ϯ 0.06 (mean ϯ sd). basal level of 3-nitrotyrosine in rat plasma was measured to be within concentration range ͻ lod to 1.5 nm. 3-nitrotyrosine basal level in rat plasma, kidney and liver proteins was established by performing enzymatic hydrolysis in order to avoid artifactual nitration of tyrosine which may occur under strong acidic conditions (hcl 6 n at 120°c). resulting hydrolysates were analysed by lc-esi-ms/ms and 3nitrotyrosine was monitored in srm mode (m/z 227 ae 181 for the analyte and m/z 230 ae 184 for the internal standard). t. guszczynski 1 , r. b. kapust 2 , d. s. waugh 2 , and t. d. copeland 1 1 basic research laboratory, and 2 macromolecular crystallography laboratory, national cancer institute at frederick, maryland, u.s.a. the set-can fusion gene was first detected as associated with acute undifferentiated leukemia. set (also called phap ii) is a nuclear phosphoprotein with a long acidic tail. set has been shown to inhibit phosphatase pp2a and is a substrate of human granzyme a. in order to determine any zn(ii) binding properties of set, we utilized affinity capillary electrophoresis (ace) to detect shifts in mobility as zn(ii) ions bind to the protein. we have earlier employed ace to measure the binding constants of zn(ii) to the nucleocapsid protein of hiv-1. with a constant concentration of recombinant set as a receptor and varying concentrations of zn(ii) as ligand in the sample buffer, we observed changes in electrophoretic mobilities of set when complexes were formed with zn(ii). scatchard analysis of the mobility provided the stoichiometry and binding constant of zn(ii) to set. interdisciplinary research center, institute of nutritional science, department of food sciences, university of giessen, germany peptaibols are defined as fungal polypeptides containing a high proportion of aib (α-aminoisobutyric acid) and a cterminal bound amino acohol. the mold trichoderma aureoviride (strain imi 91968; commonwealth mycological institute, kew, uk) was cultured in complex medium consisting of casein peptone, 17 g; soy peptone, 3 g; yeast extract, 5 g; dglucose, 2.5 g; nacl, 5 g; dipotassium hydrogen phosphate, 2.5 g in 1 l demineralized water adjusted to ph 6.8. fermentation was conducted in nineteen 2-l shake flasks, each containing 400 ml medium, for 7 d at 27°c. mycelia were obtained by filtration and extracted with meoh and meoh/chloroform. extracts were evaporated to dryness and subjected to sephadex and silica gel chromatography (eluent chloroform/meoh/acoh/water 65 : 25 : 3 : 4) yielding 2.83 g and 0.9 g, respectively, crude peptaibol mixture named trichoaureocins. the peptide mixture was uniform on tlc but could be separated by analytical (fig. 1 ) and semipreparative hplc (nucleosil 100 c-18; 250 ϫ 8 mm id; 3 µm). six peptides could be isolated each of which was subjected to sequencing using on-line hplc (fluorocarbon stationary phase) esi-ms/ ms (lcq, thermoquest, finnigan mat) as described for peptaibols trichovirins and antiamoebins. sequences are presented in fig. 2 . the 20-residue peptaibols represent a natural peptide library and cause hemolysis of sheep erythrocytes and exert antibiotic activity against bacillus subtilis and staphylococcus aureus. national institute of chemistry, ljubljana, slovenia wine consists of several hundred components present at different concentrations. the dominant ones are water, ethanol, glycerol, sugars, organic acids, and various ions, while amino acids are present at much lower concentration. the composition of amino acids is of great importance in wine production. they act as a source of nitrogen for yeast during fermentation, they influence the aromatic composition of wine and their composition can be used to differentiate wines according to vine variety, geographical origin, and year of production. among already established analytical methods high-field nmr has been shown to be a promising method for the nondestructive analysis of low-molecular mass compounds in complex mixtures like wine due to its selectivity and capability of simultaneously detecting a great number of compounds. 1 h and 13 c one-dimensional nmr spectra of wine are very crowded and many signals are overlapped. due to a great difference in concentration levels the signal intensities of particular compounds may vary for the factor of 25. the tails of the dominant frequencies of water, ethanol and glycerol obscure weak signals of minor compounds like amino acids in the near surroundings. the use of 2d homo-and heteronuclear experiments and the suppression of strong signals are a prerequisite for a successful 1 h and 13 c signal assignment. a complete assignment of 1 h and 13 c nmr resonances of seventeen amino acids commonly present in wine and of γ-aminobutyric acid at ph 3 was accomplished using gradient-selected cosy, tocsy, gradientselected hsqc and hmqc experiments with incorporated wet pulse sequence for the supression of large signals. unambiguous assignment of 1 h and 13 c nmr resonances of amino acids is necessary for the selection of appropriate signals in fast and simple one-dimensional nmr that can serve as parameters in the chemometric classification of wines according to the provenance, vine variety, and year of production. institute of medical biochemistry, jagiellonian university collegium medicum, kraków, poland highly sensitive colorimetric method for determination of aldehydes in the reaction with n-methyl benzothiazolone hydrazone (mbth) turned out to be not very specific for such carbonyl compounds. namely, it has been found that tryptophan and to higher degree its n-derivatives (n-acetyl-trp, ala-trp, gly-trp) and also tripeptides (gly-trp-gly and leu-trp-leu) in the reaction with mbth and fe 3ϩ are converted to coloured products, with maximum wavelength at 595 nm. the properties of the products and the kinetics of the reaction under defined conditions are described in the spectrophotometric procedure. proteins containing tryptophan are also substrates in the reaction with mbth. comparison of molar extinction coefficients of mbth-fe 3ϩ -treated various proteins with those of simple n-derivatives of tryptophan shows, that not all molecules of tryptophan in proteins are accessible to the reagents, and in order to determine all tryptophan moieties partial unfolding of protein has to be performed. it should be emphasized that aldehydes cannot be detected and accurately determined in the presence of tryptophan derivatives and protein, and also aldehydes interfere with determination of tryptophan derivatives. natural product laboratory, department of chemistry, the university of burdwan, w. bengal, india detection of protein amino is of utmost importance for the evaluation of protein structure and also their presence in numerous natural products. several specific and non-specific reagents have been used for their detection using thin-layer chromatography, an important tool for such purpose. of the reagents in general use, ninhydrin is the most popular for its high sensibility, however, nihydrin produces same purple color with most of the amino acids (only proline and hydroxproline produce yellow color). an endeavour has been made to resolve this color problem with a reagent which is capable of developing various distinguishable colors with many of the protein amino acids and also shows its high sensitivity comparable to ninhydrin. a probable mechanism for such color formation has also been proposed. measuring enrichments below the sensitivity range of conventional gc-ms. the gc-c-irms technique combines the resolution capabilities of gc with the accuracy and precision of irms. at low abundance gc-c-irms analysis it is superior in terms of time, labor, and sample requirement as compared to the conventional off-line analysis. we discuss some latest advancements and applications of gc-c-irms amino acid analysis related to nutrition research. plasma amino acids in omnivorous human subjects show a characteristic 15 n-isotopic pattern with phenylalanine and threonine showing the lowest abundance, whereas e.g. alanine and leucine are higher by 25‰ δ 15 n. in rats fed diets containing intrinsically labeled 13 c casein or the corresponding amino acid mixture labeled with 13 c leucine and 15 n lysine whole-body protein homeostasis is better supported by casein-bound than free amino acids. there is no adaptation to a low lysine diet by an enhanced bioavailability of intestinal microbial lysine to extra-splanchnic tissues in minipigs. highly selective hplc determination of tyrosine, tryptophan and their related compounds based on precolumn derivatization followed by intramolecular fluorescence resonance energy transfer detection h. nohta 1 , m. yoshitake 1 , h. yoshida 1 , t. yoshitake 2 , and m. yamaguchi 1 1 faculty of pharmaceutical sciences, fukuoka university, nanakuma, johnan-ku, fukuoka, and 2 chemical evaluation and research institute, ishii machi, hita, oita, japan we have developed highly selective hplc method for the determination of tyrosine, tryptophan and their related compounds (l-dopa, catecholamines, 5-hydroxytryptamine, etc.). the compounds were precolumn-derivatized with a commercially available fluorogenic reagent for amines by usual manner. each derivative afforded intramolecular fluorescence resonance energy transfer (fret) from the tyrosyl or tryptophoryl moiety (donor) to the labeled fluorophore (acceptor); the acceptor fluorescence was observed with the excitation of the donor at 280 nm. the derivatives were separated on a reversedphase column and then effectively detected by monitoring their fret. through the screening study of 11 fluorogenic reagents, o-phthalaldehyde (with 2-mercaptoethanol) and dansyl chloride gave the best results for the purpose. the fret detection method was highly selective and sensitive by comparison with the previous methods detecting native fluorescence of the compounds or typical fluorescence of the acceptor. the presented study was devoted to determination of the energetic effect of interactions in aqueous solutions between urea and neutral amino acid derivatives. the principal reason for studying of interactions of peptides with urea is the hope that such investigations will give insight into the factors affecting protein denaturation in aqueous solutions. the enthalpies of solution of n-acetylglycinamide, n-acetyl-l-alaninamide and n-acetyl-l-leucinamide were measured in water and in aqueous solutions of urea of molality 0.25 to 3.0 mol·kg ϫ1 using the "isoperibol" type calorimeter at 298.15 k. from the obtained standard dissolution enthalpies ∆ sol h ϱ m the enthalpic pair interaction coefficients h xy for urea-nacetylamino acid amide pairs in water were calculated. these parameters derived from mcmillan-mayer theory are regarded as a measure of effect of interactions between solute molecules in solution. the h xy values for the systems investigated suggest that the interactions between urea and amide molecules dominate the effects of dehydration of nonelectrolyte and of peptides. the replacement of the hydrogen atom in the hydrocarbon chain with a methyl group causes a positive change in the value of the enthalpic pair interaction coefficient. the obtained results were compared with those of earlier studies of interactions between electrolytes, namely sodium chloride, potassium chloride and sodium iodide and the same n-acetylamino acid amides. the effect of the solute type on the magnitude of the interaction parameter was also analysed. the side chains of amino acids in solution react in various ways with the water molecules which surround them as well as with other components of solution depending on the fact whether they possess non-polar, polar or ionic groups. many research laboratories carry out studies intended to describe precisely the intermolecular interactions with the participation of amino acid side chains. such a description may allow one to describe better the spatial structures of protein and the mechanisms of folding its surface area. the present work reports the results of calorimetric measurements of the dilution enthalpies of l-α-amino acids in water. using modified mcmillan-mayer's theory, these results served to calculate the enthalpic homogeneous interaction coefficients which characterise interactions between the amino acid zwitterions with the competitive participation of water molecules. thus, these coefficients illustrate the differences in amino acid molecules interactions both with the homogeneous amino acid molecules and water molecules around them, and consequently they may play the part of a parameter which differentiates the hydrophobic/hydrophilic properties of amino acid side chains. the enthalpic interaction coefficients of the homogeneous pairs of l-α-amino acids were compared also with the hydrophobicity parameters obtained by fauchere et al., which describe the side substituents of natural amino acids as well as aminobutiric acid (aba). based on the above statement, one may conclude that the obtained enthalpic homogeneous pair interaction coefficients of l-α-amino acids in water make it possible to systematise amino acid side chains according to their affinity to water or their hydrophobic-hydrophilic properties. thus the enthalpic homogeneous pair interaction coefficients may play the role of parameter describing the lipophilicity (hydrophobicity) of amino acid side chains. compounds (iii) with amino acid ligands. in this work we present results of x-ray investigation of fourth amino acid complexes of rhenium (iii), which have different coordination of amino acids around binuclear complexforming center -re 6ϩ . substances (glyh) 4 . 2h 2 o -in inner, but gaba has cisposition according to re -re bond. influences of fatty radical length in the amino acid ligand on week interaction between binuclear anion [re 2 cl 8 ] 2ϫ and protonized amino acid are discussed. role of hydrogen bonds in formation of crystal unit cell of investigated substances is shown. these two factors are the reason of formation of staggered conformation of an anion [re 2 cl 8 ] 2ϫ in the substance (glyh) 4 [re 2 cl 8 ]cl 2 together with existence of quadruple re -re bond that is described first. in the substance [re 2 (gaba) 2 cl 5 (h 2 o)]cl . 2h 2 o axial position of re 2 6ϩ fragment are substituted by ligands of different kind: h 2 o and cl ϫ -that says about possibilities to coordinate a substrate of biological nature exactly to these position. a precise, sensitive and reliable rp-hplc/uv method was developed to enable determination of α, and k caseins in cow's milk. the optimised method using a chrompack p-300-rp column allowed separation of caseins in 30 min. this column differs from conventional alkyl-bonded silica rp matrices in that it is an underivatised polystyrene-divinylbenzene matrix, a material which proved excellent chemical and ph stability. gradient elution was carried out at a flow rate of 1 ml/min and a temperature of 46°c, using a mixture of two solvents. solvent a 0.1% trifluoroacetic acid in water and solvent b was 95% acetonitrile-5% water-0.1% trifluoroacetic acid. the effluent was monitored by a uv detector at 280 nm. the determinations were performed in the linear range of 0.038-0.38 mg/ml for k-casein, 0.19-1.9 mg/ml for α-casein and 0.15-1.5 mg/ml for -casein. the detection limits were 0.037, 0.03 and 0.0075 mg/ml, respectively. the validity of the method was verified. the recoveries ranged from 91 to 100% for cow's milk. the precision of the method was also evaluated, the % cv being less then 3.67%. the developed methodology was also applied with success to the separation of caseins in ewe and goat milks. different chromatographic profiles were obtained for the three kinds of milk. department of aquatic biosciences, the university of tokyo, bunkyo-ku, tokyo, japan several aquatic crustaceans and bivalve molluscs accumulate a large amount of free d-alanine (3-50 µmol/g wet wt.) in their muscle tissues. during seawater acclimation from freshwater to 75% seawater, red swamp crayfish procambarus clarkii largely accumulated d-and l-alanine by 6.8-and 4.5fold, respectively, together with l-glutamine, l-proline, and glycine. the percentage of d-alanine to total alanine increased from 38% in freshwater to 48% in 75% seawater. these data indicate that d-and l-alanine are the major compatible osmolytes responsible for the intracellular isosmotic regulation of this species as well as other crustaceans. under anoxia stress for 12 h in freshwater, 50 and 75% seawater, crayfish increased d-and l-alanine in muscle and hepatopancreas in addition to the increase of lactate. the increase was much higher in seawater than in freshwater. thus, d-and l-alanine may be anaerobic end products during prolonged anoxia of this species. alanine racemase [ec 5.1.1.1] has been proved to catalyze the interconversion of d-and l-alanine in crustaceans and bivalve molluscs. this enzyme was isolated to homogeneity from the muscle of black tiger prawn penaeus monodon. the purification was 127,600-fold with 16% yield. the molecular weight of the enzyme was estimated to be 45 kda on sds-page and 90 kda on gel filtration, suggesting the dimeric nature of this enzyme. the amino acid sequences of the peptide fragments obtained from the isolated enzyme showed low homology below 50% with those of microbial enzymes. syntheses and immunological effect of thymic humoral factor-γ2 analogues research laboratory, global shinwa pharmaceutical co. ltd., yoriki, matsuomura, iwate-gun, iwate-ken, japan nine analogues of thymic humoral factor (thf)-γ2, were prepared by the solid-phase method and their in vitro restoring effect on the impaired blastogenic response of phytohemagglutinin (pha)-stimulated t-lymphocytes of uremic patients with infectious diseases were examined. the results were as follows: [arg6]-thf-γ2 exhibited higher restoring activity than that of our synthetic thf-γ2. phylos has developed a powerful combinatorial biology platform for peptide and protein selections. phylos' proprietary profusion tm technology enables the selection of peptides and proteins with desired properties. the fundamental advance represented in this unique platform is the in vitro covalent linkage of a peptide or protein (phenotype) to the encoding messenger rna (genotype). this linkage permits the selection of a protein based on its characteristics and allows the recovery and amplification of that protein through pcr, an efficient means of bring the desired proteins to easily detectable levels. profusion tm technology has routinely selected peptide and protein binders with affinity constants in the nanomolar to picomolar range. the starting library size of randomized peptide or protein profusion tm constructs is typically 10 13 . linear and constrained loop peptide libraries, for ligand generation, enzyme: substrate interaction, peptidomimetic design, and epitope mapping have been successfully used. randomized constrained loops have also been incorporated in a betasandwich scaffold, resulting in the successful selection of binders against targets of therapeutic interest. antigenic properties of three biological active de novo proteins were investigated by peptide scanning approach, using noncleavable multipin technology. a de novo protein albebetin (pid caa47376) was engineered to attain a pre-designed 3d structure and later modified by grafting short peptide fragments from human α 2 interferon (aag59605), and insulin molecules (aag59606). such protein constructs carrying important biological activities may be used in future as potential protein pharmaceuticals. despite artificial proteins are investigated for more than 10 years, immunological properties of these substances are not known. in our experiments we applied an innovative approach of raising antibodies in yolks of egg-laying hens. three continuous antigenic determinants with different immunogenic potentials have been revealed in two proteins with partially overlapping sequences. it was shown that the octapeptide interferon fragment is the immunodominant site in albeferon and albeferon-insulin molecules. on the contrary, the hexapeptides, corresponding to the insulin fragment displayed low immunogenic activity. thus we recognise that the fragments attached to the de novo frame could essentially govern immunological properties of resulting construct. no preference of any type of secondary structure was observed in antigenic determinants. nevertheless, all of them are located at the boundaries of the secondary structure elements and on the predicted surface-located sites of albebetin molecule. peptide fragments from human α 2 -interferon and insulin corresponding to the functionally important sites of their molecules were grafted into de novo protein albebetin (pid caa47376) engineered to attain a pre-designed tertiary structure with a unique topology that has not been observed in natural proteins. by means of genetic engineering the dna fragments corresponding to these peptides were inserted into the albebetin gene to obtain two variants of albebetin with antiviral fragment of human α 2 -interferon and two variants of albebetin with insulin-like peptide. the chimerical genes were expressed in escherichia coli in a fusion expression system with thioredoxin based on the plasmid pet-32 (novagen). the fusion proteins were digested by highly specific protease factor xa and the target chimerical proteins were purified and tested for their structure and biological activity. according to the cd spectroscopy study the chimerical proteins maintained the pre-designed structural properties of albebetin. toxicological testing of the proteins in the mtt-test did not reveal their cytotoxicity. antiviral activity of de novo proteins with human α 2 -interferon fragments was studied in vitro using human fibroblasts cell line l-41 and simian cell line vero. treatment of these cell lines with the proteins revealed the dose-dependent stimulated antiviral activity on fibroblasts and direct dose-dependent antiviral activity on the vero cells. one of two de novo proteins including insulin-like fragment (pid aag59607) acquired ability to stimulate glucose uptake by l-929 cells although the efficacy of stimulation was lower than that for the synthetic peptide and insulin. these results demonstrated that albebetin can be used as a scaffold for constructing of the functionally active de novo proteins possessing the pre-designed tertiary fold of albebetin and various biological activities. the identification of genes encoding unique tumor associated antigens (taas) has facilitated the development of novel immunotherapeutic strategies in cancer patients. clinical investigations have focused on targeting these cancer antigens for the generation of anti-tumor t-cell responses. taa epitopes come from differentiation antigens, from embryonal reexpressed or overexpressed proteins, from mutated proteins and from viral proteins in viraly associated tumors. we have recently developed a novel screening system for identification of immunogenic and antigenic ctl peptide epitopes using d b-/-x 2m -/double knockout mice, transgenic for a single-chain hla-a2-2m molecule (hhd mice). specific ctl were derived by immunization of hhd mice with tumor peptide extracts loaded on antigen presenting cells and with hhd transfected human tumor cell lines ctl induced against peptides from various tumors recognized tumor peptides more effectively than peptides extracted from normal tissues and also reacted with a serie of peptides derived from overexpressed candidate proteins, identified by differential display methods (sage, microarrays) comparison of ctl derived from hhd mice to ctl induced from patient's pbmc showed overlapping recognition of many candidate peptides. using these hhd mouse derived ctl we identified novel peptide sequences from prostate, bladder, breast and colon carcinomas, antigens pap and steap, from breast carcinoma antigens muc1 and ba46-1. analysis of tumor differentially expressed genes by the sage method in colon, followed by screening for hla-a2 binding peptides resulted in 500 candidate peptides for immunogenicity screening. we have identified 22 antigenic peptides of which 7 peptides were found to be immunogenic in hhd mice. interestingly 3 of these peptides are derived from the same protein. differential expression studies, using "dna chips" were performed on prostate and bladder tumors versus normal tissues. ten new candidate genes from tcc were analysed for expression and potential immunogenic peptides. novel peptides from uroplakins and from mage-8 were identified. surface plasmon resonance biosensing in the study of viral antigenic sites mimicked by synthetic peptides p. gomes 1 , e. giralt 2 , and d. andreu 2 1 centro de investigação em química da universidade do porto, portugal 2 department de química orgànica, universitat de barcelona, spain antigen-antibody binding has been regarded as one of the most representative examples of specific molecular recognition in nature. the simplistic view of antigenic recognition in terms of a lock-and-key mechanism is superseded, since it is now evident that both antigens and antibodies are flexible and can undergo substantial mutual adaptation. this flexibility is the source of complexities such as degeneracy and non-additivity in antigenic recognition. we have used surface plasmon resonance to study the effects of combining multiple amino acid replacements within the sequence of the antigenic gh loop of foot and mouth disease virus. our aim was two-fold: to explore to what extent can antigenic degeneracy be extended in this particular case, and to search for potential non-additive effects in introducing multiple amino acid replacements. combined analysis of one such multiply substituted peptide by spr, solution nmr and x-ray diffraction shows that antigenic degeneracy can be expected as long as residues directly interacting with the paratope are conserved and the peptide bioactive folding is unaltered. structural properties of creatine kinase from amphioxus, branchiostoma belcheri gray f. inoue, s. obase, t. suzuki, and t. imai department of physiological chemistry, faculty of sciences, toho university, funabashi, japan to further our knowledge of creatine kinase (ck) in the fields of molecular evolution and comparative enzymology, we analyzed the ck gene of the protochordate amphioxus. amphioxus is thought to be the phylogenetic predecessor of vertebrates and thus possesses characteristics, such as enzymological properties, that are associated with ancestral vertebrates. the results clarified the sequence of 789 bases including the active site. the homology of the active site and the surrounding 48 bases for the amphioxus ck gene to that of the human and electric ray ck-m gene was 89.6% and 87.5%, respectively. the amino acid sequence of this region of amphioxus ck was also identical to that of human and electric ray ck-m. in addition, the estimated secondary structure of amphioxus ck was compared to that of human and electric ray. there were no marked differences in the relative ratio of the α helix, sheet and turn structures for the peptide structure of ck consisting of 263 amino acid residues. there was a high degree of homology in the sequence of 25 amino acid residues (met271ϳhis295) near the active site of ck between amphioxus and other organisms, suggesting that this region of ck is functionally essential for transphosphorylation. gelsolin is a ca 2ϩ -activated and phosphoinsitide-regulated cytoskeletal actin-binding-and-severing protein, its fragments 135-142: ksglkykk (g135-142) and 150-169 khvvpnev vvqrlfqvkgrr (g150-169), are responsible for the binding of this protein to actin and the cellular messenger phosphatidylinositol 4,5-bisphosphate (pip2). the binding of peptides g135-142 and g150-169 to a cluster of four pip2 molecules in a dimyristoyl-phosphatidylcholine lipid was in vestigated by means of molecular-dynamics (md) simulations of 1,600 ps. the binding of the pip2 molecules to the peptides g135-142, g150-169 showed both electrostatic and hydrophobic nature: lysine residues of the peptides formed salt bridges with the phosphate groups of the pip2 molecules, while hydrophobic interactions occurred between the nonpolar residues of the peptides and the fatty-acid tails of pip2. during the binding some of the pip2 molecules were dragged out of the lipid, thus disrupting the bilayer. after the binding dissociated a draggen-out pip2 molecule tend to incooporate back to the lipid. division of applied physiology, institute of veterinary physiology, university of zürich, switzerland chemical modification of the proteins: bovine serum albumin, α-lactalbumin, -lactoglobulin and chicken egg white lysozyme by 3-hydroxyphthalic anhydride (3hp) yielded compounds which exerted antiviral activity in vitro as compared with the native unmodified proteins. of the three enveloped viruses tested: human herpes simplex virus 1 (hsv-1), bovine parainfluenza virus 3 (pi-3) and porcine respiratory corona virus (prcv), the 3hp proteins were shown to be active against human herpes simplex virus 1 only indicating that a perturbation of the viral envelope is unlikely. pre-incubation of vero cells with 3hp-albumin, 3hp--lactoglobulin and 3hplysozyme resulted in protection against hsv-1 infection whereas pre-incubation with 3hp-α-lactalbumin had no antiviral effect. however, all 3hp modified proteins showed a more significant inhibition when present during or after the viral infection step. thus multiple mechanisms appear to be involved in the inhibition of hsv-1 infection. the blocking of cell receptors may contribute to the antiviral activity as shown by the preincubation data. however, a direct interaction between the modified proteins and the hsv-1 glycoproteins responsible for viral entry and spread, seems to play a more important role, as indicated by the smaller ec 50 values obtained during and after the infection. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin-1, interleukin-6 and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. our previous investigations showed some biological activity of newly synthesized cluster rhenium compoundtetrachlorodi-µ-(γ-aminobutirato)dirhenium(iii) chloride -i such as antitumour activity, cell-stabilizing activity against osmotic hemolysis, changing of morphology of cells, and other. there exists some information about stabilizing effects of some metal-organic substances with antitumour properties on the isolated ishaemic-reperfused rat heart (leperre a. 1995) throughout decrease of malonaldehyde (mda) production. some new investigations showed the influence of metal-organic substances on apoptotic processes (winter b. 1998 , syrkin a. 1998 , that are considered now as the main mechanism of such tissue damages as ishaemia, myocardial infarct, etc. thus we tried to analyze such activity of i. two models of hemolytic anemia was used: a -on rabbits by introducing of pbac 2 -solutions; this model permits to investigate dynamics of anemia in one experimental animal; bon rats by introducing of phenylhydrazine chloride. i was administrated as in solution as in lyposomic (lyp) forms. all measurements and models were accomplished according to described procedures. administration of i led to: increase of hemoglobin and resistance of erythrocytes and to prolonging of life for hemolytic animals; significant decrease in quantities of mda and increase in quantities of reduced glutathion (gsh), glutathionreductase (gsr) and glutathionperoxidase (gsp) in myocardium, blood, brain, liver, splenic and entherocites of anemic animals. the most effective was i in lyposomic form. mechanism of antioxidant action of rhenium cluster compound is speculated and experiments with some well-known antioxidants to compare with i are working out. at present problem of finding remedies against the mostly dangerous human disease -aids is one of higher interest. the aim of this work was the investigation of inhibiting effect of high-pure l-lysine-α-oxidase (lo) e.c.1.4.3.2, extracted from trichoderma sp., on hiv-virus reproduction, comparatively to azidotymidin (azt), being now in use for treatment of aids-patients. for studying of inhibiting effect of lo, the mt-4 cells, sensitive to citopathical action of virus, were used. the experimental studying has shown, that the enzyme at concentration 7-70 ng/ml suppresses hiv reproduction and synthesis of virus' proteins, not exerting toxical effect on mt-4 cells. toxical dose of lo has been determined preliminary. a comparison with standard preparation -azidotymidin, which causes suppression of virus reproduction at concentration 3 mkm (1,2 mg/ml) not exerting toxical effect on mt-4 cells. the same effect is attained having used lo in doses 7-70 ng/ml. using lower concentrations of enzyme leads to partial increasing of virus' titre comparatively to control cultures. obtained data allow to conclude that lo from trichoderma sp. is more high specific agent than azidotymidin, because it needs 1000 times lower concentration for the same action. comparison of azt and lo action on synthesis of virus' antigens presenting in cultural media of mt-4 cells infected with virus, leads to conclusion, that lo has inhibitory action both on virus' reproduction and virus' protein synthesis. department of microbiology, dokkyo university school of medicine, mibu, tochigi, japan our previous studies showed that the cellular amino acid composition obtained by amino acid analysis of whole cells, differs in various organisms. these results suggest that the difference in the cellular amino acid composition reflects biological evolution. however, the basic pattern of cellular amino acid composition is relatively constant in all organisms, and the cellular amino acid compositions of the archaeobacteria are quite similar to those determined from codon usage data, based on the complete genomes. in the present study, the free amino acid compositions in archaeobacteria, eubacteria, protozoa, blue-green alga, green alga, slime mold, plants and mammalian cells were analyzed, to investigate whether changes in their free amino acid compositions reflected biological evolution. cell homogenates were treated with 80-90% ethanol to separate cellular proteins and free amino acids contained in the cells. rat hepatoma cells (r-y121b) were cultured in eagle's minimum essential medium (mem) containing 5% serum or in a modified mem lacking arginine, tyrosine and glutamine. no significant difference in the free amino acid composition was observed between the two cell groups cultured under two different conditions. the patterns of the free amino acid compositions differed completely from those of the cellular amino acid compositions, and from each other in various organisms. characteristic differences were observed between plant and mammalian cells, and between archaeobacteria and eubacteria. the patterns of the free amino acid composition in blue-green alga, green alga, protozoa and slime mold differed from each other and from those of eubacteria and archaea cells. it has been suggested that the free amino acid composition reflects apparent biological changes as the result of evolution. 2) catalyzes the hydrolysis of gamma-glutamyl compounds such as glutathione, and the transfer of their gamma-glutamyl moieties to amino acids and peptides. we previously developed enzymatic methods for the synthesis of various gammaglutamylamino acids using the transfer reaction of ggt from e. coli k-12 as a catalyst. it has been reported that gamma-lglutamyltaurine has a potent and long-lasting antiepileptic action, and its chemical synthesis has also been reported, but it required protecting and deblocking of reactive groups. thus, the purpose of this study was to develop an enzymatic method for the synthesis of gamma-l-glutamyltaurine using ggt. the optimum reaction condition was 200 mm l-glutamine, 200 mm taurine and 0.2 unit/ml ggt, ph 10, and 1-hr incubation of 37°c. forty-three mm gamma-glutamyltaurine was obtained and the yield was 21.%. gamma-glutamyltaurine was purified by dowex 1 ϫ 8 column and c18 column, and then identified with gamma-l-glutamyltaurine by nmr and polarimeter. in this study the yield of gamma-l-glutamyltaurine was comparatively low because synthesized gamma-lglutamyltaurine was promptly converted into the by-product, gamma-l-glutamyl-gamma-l-glutamyltaurine. the production of antimicrobial peptides is an important aspect of host defense in animals ranging from insects to mammals. they do not target specific molecular receptors on the microbial surface, but rather assume amphipathic structures that allow them to interact directly with microbial membranes, which they can rapidly permeabilize. they are thus perceived to be one promising solution to the growing problem of microbial resistance to conventional antibiotics. insects express a battery of potent antimicrobial proteins in response to injury and infec-tion. until now, approximately 170 immune peptides have been characterized from insects and other invertebrates. an antimicrobial gene (md-cecropin) belonging to cecropin family was cloned from the bacteria-charged adult house fly, musca domestica. expressed in the vector pgex-4t1. mrna was isolated and degenerated primers were designed according to the conserved sequences of cecropins. the full-length cdna encoding md-cecropin was cloned by rt-pcr and 5ј, 3ј-race and sequenced. the deduced amino acid sequence indicated that a prepeptide with 63 amino acid residues is first translated and then processed to a mature peptide with 40 amino acids. the dna encoding the mature peptide was subcloned into expression vector pgex-4t1, and expressed efficiently in e. coli bl21 as a fusion protein. the fusion protein was purified and specifically digested and the md-cecropin was further purified to homogeneity and the activity spectrum was investigated. escherichia coli with metabolic engineering methods l. yun, x. zhang, s. wang, q. xu, and l. ma biotechnology laboratory, institute of beijing radiation medicine, beijing, p.r. china a bioengineering escherichia coli strain was obtained by metabolic engineering method. three genes related to the biosynthesis of phenylalanine, arog, phea, and tyrb encoded key enzymes: 3-deoxy-d-arabino-heptulonate-7-phosphate synthetase (ds), a bifunctional protein-chorismate mutase (cm)/prephenate dehydratase (pd) and aminotransferase (at), respectively. in this work, the feedback inhibition of ds and cm/pd were relieved by site-directed mutagenesis on bases of homology comparison of related sequences of the key enzyme. the feedback inhibition resistant genes encoding ratelimiting enzymes in the main and terminal pathways were amplified by co-expressed in order of arog-phea-tyrb on the plasmid by their own operator plpr, pl, and pr. in the recombinant strain showed great resistant to the l-phenylalanine analogues, the specific activities of ds, cm, pd and at were increased by 3.10, 3.29, 4.91 and 8.16 folds, respectively. as the result, the amount of phenyalalnine biosynthesis of the bioengineered strain was increased greatly compared with that of the host strain. an enzymatic approach for the mapping of phosphoproteins resolved on two-dimensional polyacrylamide gels hiroshima proteome laboratory, regional science promoter program, kagamiyama higashihiroshima, japan an enzymatic approach for high-throughput mapping of phosphorylated proteins resolved on two-dimensional (2-d) polyacrylamide gels is presented. proteins of cultured rat skin fibroblasts were divided into two aliquots, one of which was dephosphorylated using recombinant 1 protein phosphatase. the two aliquots were then subjected to 2-d electrophoresis. the phosphoproteins could be mapped on the 2-d gel by com-paring the gels of the phosphatase-and non-treated samples, because the dephosphorylated proteins shifted to more basic positions on the gel. this technique revealed that approximately 5% of the detectable proteins were phosphorylated. fifteen phosphoproteins were identified by mass spectrometry, including proteasome component c8 and small glutamine-rich tetratricopeptide repeat-containing protein. furthermore, the extent of phosphorylation of two actin-modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. the presented technique can be applied to all biological materials because it requires no protein-labeling step, and is therefore useful for high-throughput mapping of phosphoproteins in proteome research. with the completion of the human genome sequence maldi-tof-ms is increasingly becoming an established method for identification of proteins separated by 2d gel electrophoresis. mono-isotopic peptide mass fingerprinting (pmf) has been previously shown to be amenable to full automation encompassing the process of acquisition, data processing and databank searching under full software control. until now the throughput of maldi-tof-ms for proteomics has been limited to several hundred samples in a working day and this represents approximately 5-10% of the total proteins resolved by a large format 2d gel. to reduce the number of proteins to be identified the 2d gels are imaged and analysed to determine differences in expression levels within a set of gels. although much of the image processing is semiautomated the comparison is labour intensive as manual pattern matching has a role in the gel alignments (land marking). increased ms sample throughput allows the possibility of identifying every protein spot in a 2d gel within a day. this could eliminate the potentially erroneous step of human gel image alignment, whereby land marking could be achieved using the ms data. increased sample throughput requires greater capacity and robust unattended instrument operation. in this poster we describe an integrated robotic multiple plate loader that allows overnight unattended ms operation. other improvements include an increased laser repetition rate that allows the data capture rate to increase four fold. sample tracking, data archiving and data reporting are essential attributes of this new technology and these aspects are outlined in the presentation. the proteinchip tm biology system for ciphergen biosystems: a novel proteomics platform for rapid biomarker discovery, validation and identification ciphergen biosystems ltd., surrey technology centre, the surrey research park, guildford, surrey, u.k. the proteinchip system uses seldi (surface enhanced laser desorption/ionization) proteinchip technology to perform the separation, mass detection and analysis of proteins at the femtomole level directly from biological samples. surfaces are based on either chromatographic based chemistry (ion exchange, reverse phase, imac etc.) that bind large classes of proteins or biologically defined surfaces (antibodies, dna, receptors, etc.) that are used to investigate specific proteininteraction events. as with conventional elution chromatography each type of surface is designed to bind a different subset of proteins from a crude mixture. sample complexity is reduced on the surface by washing with standard biological buffers compatible with the chosen proteinchip array. unlike elution chromatography, proteins are detected directly from the stationary phase using laser based mass spectrometry greatly increasing throughput whilst reducing sample loss and improving reproducibility. multiple proteinchip surface and wash conditions are explored with a small sample set to resolve hundreds of proteins and establish assay conditions that reveal candidate biomarkers or diagnostic protein profiles. the resulting custom built assay is then used to monitor disease processes or drug toxicity profiles by screening large banks of samples such as tissue extracts or physiological fluids (serum, urine, csf, etc.). pharmaceutical research, genomics technologies, f. hoffmann-la roche ltd., basel, switzerland to the present, samples representing the total protein mixture have been usually analyzed by proteomics technologies mainly only the abundant, hydrophilic components have been visualized. these proteins could be solubilized with reagents compatible with isoelectric focusing, for example urea and chaps. such an analysis provides us with a limited image of the proteome, which is insufficient for the detection of the majority of the proteins. in a 2-d gel, where about 1 mg of protein amount has been resolved, 1,000-3,000 protein spots can be detected, using coomassie blue staining. the spots represent the products of only 200-300 different genes. other gene products, not visualized, are most likely expressed at too low levels for detection or they can not be identified because of limitations of the current technology, they are too small, too large, basic or hydrophobic. here we will discuss protein enrichment approaches prior to the analysis, which we have applied for the enrichment of bacterial and eukaryotic proteins. proteomic analysis of the rat liver mitochondrial proteins m. fountoulakis 1 , j.-f. juranville 1 , and l. suter 2 1 genomics technologies, and 2 drug safety, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland subcellular fractionation increases the probability of detection of low-abundance proteins. we prepared mitochondrial, microsomal and cytosolic protein fractions from total liver of male rats. the proteins of the three fractions were analyzed by two-dimensional electrophoresis using broad and narrow ph range immobilized ph gradient strips. the proteins were identified by martix-assisted laser desorption ionization mass spectrometry. in the mitochondrial fraction, 190 different gene products were detected. approximately 70% of the identified mitochondrial proteins are enzymes with a broad spectrum of catalytic activities. most of the identified proteins had been detected before in other samples as well, analyzed in our laboratory. eight gene products were detected for the first time. these were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. in average, approximately 15 spots corresponded to one gene product. centre for molecular medicine, university college london, u.k. three kinds of experiments have been carried out successfully in our labs. (1) identification of post-translational modifications of the endothelin a and b receptors (etar and etbr) including both phosphorylation and acylation. we have developed new, very efficient methods for single step isolation of highly pure etar and etbr from cells. this has allowed us to obtain evidence that the post-translational modifications are very complex and result in multiple phenotypes showing different forms of modification for receptor. as with other systems, e.g. insulin-like growth factors, it is probable that these multiple phenotypes of the et receptors correspond to different forms of signalling dependent on cellular state, e.g. the cell cycle. it is, for example, already clear from the phosphorylation of the receptor that a series of different kinases must be involved. (2) following stimulation of fibroblasts with endothelin, phosphorylation/dephosphorylation signalling cascades involving several hundred proteins have been observed by use of high resolution 2d electrophoresis and detection of phosphorylated proteins labeled with 32 p by autoradiography or immunological methods. the large number of proteins involved are being identified by mass spectrometric methods such as mass fingerprinting or sequencing by mass spectropmetry. (3) differential gene expression has been followed by using 35 s met pulse chase labelling concurrently with endothelin stimulation. at least 50 proteins showed significant changes in expression of 2d gels and these proteins are also being identified. these experiments demonstrate that it is now possible to use proteomics methods to investigate the integration of response to an extracellular signal at the levels of the receptor itself, the subsequent signalling cascades and the ensuing gene expression. the proteomics technology permits concurrent monitoring of large numbers of protein phenotypes (the forms and amounts of individual proteins and is therefore able to provide a global overview of signalling processes which greatly augments more traditional investigations of individual proteins or pathways. furthermore, these new methods will allow quantitative determination of the changes in protein phenotypes, which is very important in view of the highly non-linear amplification properties of such signalling processes. an integrated approach to automated high throughput protein identification by 2d gel electrophoresis and mass spectrometry d. gostick 1 , s. cohen 2 , p. young 1 , b. karol 2 , j. langridge 1 , j. randell 3 , t. slyker 3 , and a. jacobson 3 1 micromass, manchester, u.k. 2 waters corporation, milford, massachusetts, and 3 bio-rad laboratories, hercules, california, u.s.a. establishing the function of gene products is the major challenge of the post genomic era. the rate-limiting step in this endeavour is the speed with which proteins can be isolated and identified. separation of proteins from cell lysates or sub-cellular domains by 2d gel electrophoresis is an established method of visualising these complex systems. recently mass spectrometry has proved to be a powerful method of further characterising these proteins. from the mass spectrum of the enzyme digest of a 2d gel spot, the resulting digest map is compared with the theoretical maps from the databases and the protein identified when these correlate. maldi-tof is of great benefit in these studies since it requires a minimal amount of sample, is relatively tolerant to salts and other contaminants arising from the gel and may be configured for automated sample analysis. high sample throughput with automated analyses including data processing and client-server database searching are already available. our system automatically acquires the data and processes the maldi mass spectrum into a monoisotopic peak list. this peak list is then automatically sent to a networked database for protein identification. when proteins are not identified from the maldi analysis or an ambiguous result is obtained, then further analysis of the sample by electrospray caplc-ms-ms is required. the development of a hybrid quadrupole orthogonal acceleration timeof-flight mass spectrometer (micromass, q-tof) has facilitated the generation of unambiguous amino acid sequences from the ms-ms analyses of tryptic peptides. these ms-ms spectra can be automatically searched against protein, nucleotide or est databases. thus enabling protein identification from gel spots, despite non-specific enzymatic cleavage, protein co-migration and post transitional modifications. for organisms who's genome sequences are poorly represented in the data bases de novo amino acid sequencing may be required. inferring de novo peptide sequences from ms-ms data is complex and is often the rate-determining step in this method. however, it is now possible to interpret the ms-ms spectrum automatically. in our approach the raw ms-ms spectrum is reduced to the plausible single-charge, monoisotopic mass spectrum. sequence interpretation is achieved by generating "trial sequences" consistent with the experimentally determined molecular weight. a probabilistic fragmentation model is used to transform the trial sequences to predicted spectra for comparison to the single-charge, monoisotopic spectrum and to calculate the likelihood that the trial sequence would account for the observed data. the possible number of trial sequences for any peptide is large, for example there are 20 10 possible sequences for a peptide containing any of the 20 naturally occurring amino acids and having 10 residues. to reduce the scale of the problem a terminated markov chain monte carlo algorithm is used to produce sequences. this bayesian method simulates an exhaustive search of all sequences having the correct mass. the huge increase in genomic sequence information available, combined with the increased sensitivity and selectivity provided by mass spectrometry, has allowed large-scale protein identification. however the analysis of the post translational modifications present on the identified proteins is a more challenging problem. currently the approach that offers the most expedient and specific solution, to determine modified peptides, is precursor ion scanning. this approach has primarily been performed on a triple quadrupole mass spectrometer where the rear quadrupole, (ms2) is set to transmit only the fragment ion of interest. the ms1 quadrupole is then scanned across the appropriate mass to charge range. in this paper we describe a method that allows specific post translationally modified peptides to be identified and sequenced during the course of an hplc experiment on the q-tof mass spectrometer. during the hplc run the instrument is switched alternately at one-second intervals between low and high collision energy with argon in the collision cell. the quadrupole, ms1 is not mass selective, operating in the rf only mode. the first data set at low energy (4ev) shows only the normal pseudo molecular ions. the second at higher energy shows their fragments. wherever a product ion of interest occurs in the high-energy data all its possible precursors are revealed by the corresponding 4ev data. since the two data sets contain the entire set of precursor and product ions that can be formed it is clearly possible to generate the equivalent of a constant neutral loss scan. this is invaluable in the case of phosphorylated peptides where the neutral loss of 98da (h 3 po 4 ) occurs via -elimination from the phosphoserine and phosphothreonine residues. this allows the q-tof mass spectrometer to switch from the ms mode to the ms/ms mode of operation when a potential pseudo molecular ion exhibits a neutral loss of 98 da between the high energy and low energy data sets. the product ion ms/ ms spectrum can then be acquired on the phosphorylated precursor ion. in the case of phosphotyrosine, neutral loss of the h 3 po 4 moiety is not observed, however a low mass immonium ion at m/z 216 can be detected. this characteristic ion (from the high energy data) is used to direct the mass spectrometer to fragment potential phosphopeptide precursor ions, which are selected from the low energy data. in this case several precursor ions may require ms/ms interrogation at one decision making time-point. with the first draft of the human genome completed largescale protein identification by mass spectrometry, even for samples originating from higher organisms has become relatively straightforward. this requires a high throughput facility to identify proteins that have usually been separated by 2d page. the approach providing the highest level of automated sample throughput, in terms of samples per hour, is currently maldi-tof-ms. this technique provides a peptide mass fingerprint of the protein digests and allows the rapid and accurate identification of the parent protein by comparison to a databank. however, under some circumstances, for example if the number of peptides detected is small or if the sequence coverage is poor, it is advantageous to be able to include even a short piece of sequence information to provide added specificity. in a conventional maldi-tof-ms instrument post source decay (psd) can be used to try and generate sequence information, however this approach is notoriously unreliable in producing good quality ms/ms data. one reason for this is that the peptide ions do not undergo fragmentation in a controlled environment such as a gas cell with selected collision gas and collision energy. an alternative approach is to use the predictable fragmentation obtained from a hybrid quadrupole ortho maldi source has been fitted to a hybrid quadrupole orthogonal acceleration time-of-flight (q-tof) mass spectrometer. in contrast to a conventional maldi-tof-ms instrument the resolution and mass measurement accuracy of the data is comparable between the ms and ms/ms modes. this allows superior data acquisition in the ms-ms mode compared to conventional maldi-tof-ms. a number of modifications have been made to optimise the system for high throughput proteomics. the maldi source has been configured with a high-density target plate, compatible with a 96 well microtiter plate. the acquisition software has been modified for automated data acquisition in both the ms mode and the ms to ms/ms switching mode. dedicated processing software has been developed to fully automate the post acquisition and databank searching. this software has been optimised to consider the unique nature of the data acquired from this configuration of instrument. in this paper we demonstrate the how an maldi-q-tof instrument can be used for high throughput proteomics. we also compare and contrast is functionality in comparison with alternative strategies for high throughput proteomics, namely conventional maldi-tof-ms and electrospray lc-ms/ms. pseudomonas putida is an ubiquitous, metabolically and physiologically extremely variable soil bacterium. it is kown to be a good colonizer of plant roots and a plant growth promoter. now, after the sequencing of the total genomic dna has been finished we have focused on the functional analysis of this strain. plant growth promotion is achieved in different ways. one is the inhibition of fungal and bacterial phytopathogens, which is known to be a multifactorial mechanism. an important factor of this mechanism is the production of siderophores (iron-transport-agents), small linear or cyclic peptides, which are synthesized in a ribosomal-independent manner by special synthetases. the siderophore production is induced by iron limitation. the regulation of this process was investigated by pulselabelling with [ 33 p] inorganic phosphate. 2d-protein patterns generated from cells grown with and without fesupplementation were compared. proteins which were phosphorylated under iron limitating conditions were analysed by maldi-tof peptide mass fingerprint. for the identification of the proteins we used an in-house peptide mass database which has been built based on the genomic sequence data. bio-rad laboratories, inc., hercules, california, u.s.a. worksbase software for proteomics is a platform independent information management system encompassing laboratory experimental workflow and bioinformatics for protein and biochemical research. the worksbase system is designed to allow direct internal integration between laboratory experimental data and background biological knowledge found in reference and in-house data, such as gene, protein and functional annotation databases. worksbase provides a crossdisciplinary research infrastructure for drawing together multiple lines of evidence for characterization of proteins, and integration of this data with domains such as gene expression, pharmacological screening, structure and related areas. while the focus is on the biology underpinning the experimental work, the system is also designed with the capability of providing a sample and workflow tracking system for use in the wet lab, effectively a proteome lims (laboratory information management system). as experimentation proceeds in the laboratory, worksbase software can be used for development of hypotheses on protein, biochemical pathway, and post-translational processing involvement in biological systems and disease processes. as such, identifications that are derived from lab work and user observation can be used to augment the reference data repository. however, unlike databases ands systems where the methods and reasoning for assignment of annotations are obscure, by maintaining the link between the source data and the biological roles derived from them, the accuracy and integrity of any information stored in the worksbase system can be directly ascertained. changes in the brain protein levels following administration of kainic acid k. krapfenbauer 1,3 , m. berger 2 , g. lubec 3 , and m. fountoulakis 1 1 f. hoffmann-la roche ltd., pharmaceutical research, genomics technologies, basel, switzerland 2 institute of cancer research, and 3 department of pediatrics, university of vienna, austria kainic acid (ka), a potent neurotoxin and excitatory amino acid, leads to derangements and modulation of brain proteins. no global brain protein expression pattern induced by ka-treatment has been reported yet. we studied the effect of systemic ka administration on the levels of brain proteins. rats were injected placebo or ka intraperitoneally and brain was taken after one week. the mitochondrial and cytosolic fractions of the brain proteins were analyzed by proteomics technologies. heat shock protein hsp 27 was exclusively detected in brains of animals treated with ka. the levels of neurofilaments and alpha-internexin were significantly decreased and a fragment of tubulin alpha-1 chain was manifold increased in ka-brains. the mitochondrial enzymes dihydrolipoamide dehydrogenase, atp synthase beta chain and isocitrate dehydrogenase were reduced and pyruvate kinase m1 was increased following ka treatment. the results indicate altered regulation of heat shock proteins, neuronal death, cytoskeletal disruption and mitochondrial derangement by systemic ka administration. this report confirms and extends previous studies on the effect of ka on the expression of brain proteins and suggests that our analytical system can serve as a model for neurotoxicological, neurobiological and neuropathological proteome studies. the rat brain mitochondrial proteins genomics technologies, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland we constructed a two-dimensional database for rat brain mitochondrial proteins. rat is a useful model of human diseases of the central nervous system. in order to detect alterations in the levels of the low abundance brain proteins, the mitochondrial, microsomal and cytosolic fractions were prepared. the proteins of each fraction were analyzed by two-dimensional electrophoresis, followed by martix-assisted laser desorption ionization mass spectrometry. approximately 500 proteins were identified in the mitochondrial fraction, which were the products of 165 different genes. about 75% of the identified proteins were detected in the mitochondrial fraction only and the rest were detected in the cytosolic and about 2% were found in the microsomal fraction as well. 98 of the 165 proteins had not been detected before in our laboratory. the identified proteins were in the majority enzymes or enzyme subunits with a broad spectrum of catalytic activities and heat shock proteins. whilst lc-ms/ms has been utilised for the identification of proteins from complexes and cell lysates (qualitative proteomics), the quantitative study of gene expression using differential display has until recently been the preserve of a 2d gel based proteomic experiment. however, recently a great deal of interest has been generated on the use of isotope coded affinity tags (icat) for the quantitative study of gene expression at the proteome level. the technique is based upon chemically modifying the cysteine residues of proteins isolated from cells in two different states with light and heavy isotopically labeled reagents. the two cell states are then combined, digested with trypsin and the cysteine containing peptides preferentially selected by binding to an avidin column, prior to analysis by mass spectrometry. the eluent from this column is then analysed by capillary lc esi-ms/ms. interrogation of the eluting peptides by tandem mass spectrometry and databank searching results in the identification of the associated protein. we describe how icat data analysis has been automated within a software environment. the ms and msms data acquired using the qtof instrument are processed and analysed using a new algorithm which recognises related isotope clusters and quantifies their relative intensities. based on a user defined ratio threshold the software will automatically carry out an lc-ms/ms experiment and databank search in a client-server mode and provide a report of the identified proteins and their expression ratio in the two cell states. deterioration of the transcriptional, splicing and elongation machinery in brain of fetal down syndrome b. lubec 1 and m. fountoulakis 2 1 department of neonatology, university of vienna, austria 2 gene technologies, cns research, f. hoffmann la roche, basle, switzerland perturbation of brain development i.e. regulation of gene expression, differentiation, growth and migration in down syndrom (ds) has been reported to occur early in life pointing to impairment of the complex system of transcription and or translation and indeed, altered expression of transcription factors has been reported in adult ds brain. we therefore decided to compare the transcriptional and translational machinery in cortex of brains of controls and fetuses with down syndrome in the second trimenon of gestation. we determined a series of transcription/translation factors by 2 d-electrophoresis followed by maldi -identification and quantification with specific software. the protooncogene c-crk, crk-like protein, elongation factor 1-alpha 1, elongation factor 2, elongation factor tu and two out of four spots representing ptb-associated splicing factor psf were significantly downregulated in brain of fetal ds fetuses as compared to controls. the finding of reduced transcription and translation factors may indicate deranged protein synthesis. the underlying cause for individual reduced transcription, splicing and translation factors may be explained by chromosomal imbalance or by posttranslational modifications as e.g. phosphorylation, known to be aberrant in ds. reduced expression of transcription factors in fetal ds during early life may be responsible or reflecting impaired brain development and deficient wiring of the brain in ds. r. mazzoli 1 , m. g. giuffrida 2 , e. pessione 1 , g. dellavalle 2 , c. barello 2 , e. griva 1 , and c. giunta 1 1 dipartimento di biologia animale e dell'uomo, università di torino, and 2 csaapz-cnr. c/o bioindustry park canavese colleretto giacosa (to), italy a fast phenol degrading acinetobacter radioresistens strain was isolated in our laboratories and selected for bioremediation applications. this bacterium is also able to grow on benzoate and catechol as sole carbon-energy sources, metabolizing them via the ortho route. in previous researches we detected, by means of proteome analysis, some marker enzymes of the phenol and benzoate degradative pathways. in the present work we extend the identification of the proteins involved in the aromatic-ring opening (the different components of the phenol hydroxylase and benzoate dioxygenase, the catechol dioxygenase isozymes) together with other satellite proteins specifically induced by the aromatic growth substrate. of these last proteins some are probably related to the cellular uptake of benzoate and phenol while others are ascribed to the groel family of heat-shock chaperonines, involved in proteins processing and folding. aromatic substrates may thus act as stress-agents like heat or cold. proteomic studies on rat body fluids i. miller 1 , r. wait 2 , l. sironi 3 , i. eberini 3 , m. gemeiner 1 , e. tremoli 3 , and e. gianazza 3 1 veterinärmedizinische universität, wien, austria 2 imperial college school of medicine, hammersmith, london, u.k. 3 universita' degli studi, milano, italy previously, we have characterized rat serum proteins, both under "normal conditions" and during experimental inflammation, using two-dimensional electrophoretic separation, densitometric quantitation and identification by mass spectrometry and immunological procedures (http://linux.farma.unimi.it/ homeframed.html). we have now extended these studies to the protein composition of cerebrospinal fluid (csf) and urine, and have identified several proteins specific to these fluids, including major urinary protein, uromodulin, and prostaglandin d synthase. these baseline data provide a useful comparison to the biological fluids of stroke-prone spontaneously hypertensive rats, an inbred strain, which develops cerebrovascular abnormalities following high blood pressure. our studies have detected signs of an inflammatory condition several weeks prior to stroke. we have confirmed the sharp rise in proteinuria preceding stoke onset, and have identified the excreted proteins. following stroke we observe a massive increase in csf protein concentration as serum proteins, even those of large molecular size, cross an impaired blood-brain barrier. as a first step to discover useful disease markers from the urinary proteome, we have developed a unique and systematic approach for detection of low molecular weight urinary proteins by using high resolution two-dimensional (2d) electrophoresis and mass spectrometric methods. unlike previous studies on urinary proteins, and most importantly as observed in present study, our results show that a large number of low molecular weight protein spots can be visualized in the 2d electrophoresis pattern. it was observed that protein concentration and fractionation methods were critical for our ability to detect many proteins in the gel pattern. therefore, several approaches were carefully considered to concentrate and fractionate proteins in urine samples. initially, urine specimens from normal individuals were concentrated by using centrifugation and ultrafiltration methods. the concentrated samples of urine proteins were then fractionated by size exclusion and immunoaffinity chromatography. the size exclusion method was used to generate two fractions of proteins based on their native molecular weights. further, this method allowed us to enrich concentrations of less abundant proteins for each fraction. the immunoaffinity method was used to specifically remove well-known abundant urinary proteins (such as albumin) from the above mentioned two fractions. that the 2d pattern includes many native low molecular weight proteins was confirmed by analyzing both protein fractions from size exclusion chromatography. a detailed mass spectrometric analysis of the protein spots is carried out to identify the proteins observed in 2d pattern. since urine is an ultrafiltrate of plasma, many factors in urine are present in proportion to their rate of synthesis in the body. these factors include many low molecular weight proteins that remain undiscovered due to their low abundance. therefore, the present analysis of urinary proteins would serve as the most useful guide for the discovery of novel diagnostic markers in urinary proteins. i. pucci minafra 1,2 , s. fontana 2 , p. cancemi 1 , g. alaimo 1 , and s. minafra 1,3 1 centre of experimental oncobiology, 2 department of cell biology and development, and 3 institute of histology and embryology university of palermo, italy breast cancer is one of the leading causes of death for cancer among women. there are different types of breast cancers, grouped as invasive and non-invasive types. among the invasive types "infiltrating ductal carcinoma" (idc) accounts for about 80% of all breast cancers. in order to study some biological properties related to this type of cancer, we have developed and well characterized an "in vitro" system, consisting of an idc-derived cell line, 8701-bc (minafra et al., br. j. cancer, 60, 185-192, 1989 ) and some of its cloned cell lines, selected for their high and low invasive activity in matrigel. using this model we are producing proteomic maps to compare with that of non-tumoral breast epithelial cells and with breast tissue fragments, existing in our collection or available at the expasy proteomics server. protein identification is currently done by means of gel matching, edman-microsequencing and immuno-detection. to rationalize data we grouped proteins into functional categories: a) cytoskeletal proteins, b) metabolic enzymes, c) chaperonins and other functionally related proteins, d) peptides and enzymes with regulatory functions. a fifth group consists of peptides with unknown identity. among these sets of proteins we found that glycolitic enzymes and some chaperonins are overexpressed in cancer cells. in addition, new isoforms of potential interest as biomarkers for breast cancer, were identified by means of microsequencing. a. santucci 1 , l. trabalzini 1 , d. soldateschi 2 , e. ferro 1 , a. paffetti 1 , and p. martelli 1 1 dipartimento di biologia molecolare, sezione di chimica biologica, universita' degli studi di siena, and 2 diesse diagnostica senese srl, siena, italy human cytomegalovirus (hcmv) is an ubiquitous virus, belonging to the herpesviridae family, betaherpesvirinae subfamily, able to induce morbidity in immunocompromised patients and congenitally infected new-borns. hcmv has the largest genome among the herpes-viruses (240 kbp): ad169 strain genome was completely sequenced, containing about 200 open reading frames encoding polypeptides, most of which are not characterized. the viral genes are activated in a cascade fashion: 1) alpha, immediate-early genes, coding for regulatory proteins necessary for the activation of 2) beta, early genes, needed for dna replication, and, finally 3) gamma, late genes, coding for structural proteins of the mature virions. this latter category includes the virus surface antigenic proteins responsible for the main immune response during hcmv infections. although the sequencing of hcmv genome has been completed, very little is known about the actual nature of the viral proteins. the most appropriate approach to characterize hcmv phenotype is to study its protein expression as it is carried out within the host cell. for this purpose, we analyzed by two-dimensional electrophoresis (2d-page) the protein phenotipic repertoire of human fibroblasts and compared it with that of the same cell type following infection with hcmv strain ad169. the phenotypic 2d map of human fibroblasts dramatically changes following infection with hcmv. a relevant amount of newly appeared spots is attributable to hcmv proteins, mainly of the structural category, since we analyzed host cells at the 7-9th day of infection, when the late, gamma genes are supposed to be the only to be activated. on the other hand, a marked decrease of protein synthesis can be easily evidentiated in the infected fibroblasts respecting to uninfected cells. a temptative mapping of the main structural viral proteins (those against which patients sera are directed) was carried out by immunoblotting, microsequencing and mass spectrometry. comparative proteomics of cultured cells: identification of genetic defects and molecular mechanism of apoptosis regulation v. seyrantepe 1 , k. landry 1 , s. taurin 2 , s. n. orlov 2 , and a. v. pshezhetsky 1 1 sainte-justine hospital research centre, and 2 research centre, chum, university of montreal, montreal, pq, canada we employed a comparative proteomics of cultured cells to study mechanism of genetic disorders and for identification of key proteins involved in cell proliferation, differentiation, and death. in particular, this technology proved to be very useful to understand molecular basis of severe inherited diseases resulting from deficiency of lysosomal membrane transporters, and a role of programmed cell death (pcd) of vascular smooth muscle cells (vsmc) in cardiovascular disorders. to increase sensitivity of the identification of cellular proteins we have either have isolated cellular organelles such as lysosomal membranes or performed the differential extraction of soluble, membrane and cytoskeletal proteins. by comparison of pro-teomic cell maps from normal controls and individuals affected with lysosomal transport disorders we have selected and identified several candidate disease-causing proteins, which have to be further studied by mutation analysis and functional expression. for the second group of disorders we identified proteins, which de-novo synthesis could result in survival of vsmc including a two members of hsp70 family, a molecular chaperone grp78, and so-called mortalin (grp75) highly expressed in non proliferative tissues and associated with mortal cell phenotype. two-dimensional polyacrylamide gel electrophoresis (2d-page) is the established technology employed for the separation of proteins from a cell lysate, sub-cellular organelle or tissue sample prior to identification of the excised protein spots by mass spectrometry. in the order of several hundred to several thousand proteins, can be separated and visualised on a 2d gel by conventional staining or utilising fluorescent labelling techniques. the advantage of performing a two dimensional gel based separation is the ability to obtain quantitative information by comparing and contrasting two samples in a differential display experiment, for example, between a healthy and diseased state. the last stage however stipulates that the gels are reproducible which can be both difficult and time consuming to achieve. the relativity poor dynamic range that the gels exhibit also limits quantification. other restrictions include the under representation of certain classes of proteins, such as membrane proteins, large or small proteins and very acidic/basic proteins. for these reasons, amongst others, alternatives to 2d-page are being investigated. advances in both lc and mass spectrometry instrumentation have allowed the analysis of protein complexes, which have not been separated on a 2d gel. in this case protein identification is achieved via database searching of esi-ms/ms data. this provides qualitative information on the proteins that are present and has recently been coupled with isotope dilution experiments to provide relative quantiative information. these experiments normally involve separation of the complex digest mixture by microcapillary liquid chromatography connected to an instrument capable of data dependant switching between the ms and ms/ms modes. using this approach it has been demonstrated that hundreds of ms/ms spectra can be acquired in a fully automated fashion, resulting in the identification of significant numbers of proteins, including low copy number proteins, from a single lc-ms/ms experiment. if, however, a complex protein mixture is to be investigated then a fractionation step prior to separation of the peptides on the basis of their hydrophobicity would be advantageous. we have, therefore, adopted a 2d lc-ms/ms approach using a capillary lc system (caplc) operating at nanoliter per min flow rates coupled to a q-tof 2 mass spectrometer. by replacing the standard sample loop within this system with a strong cation exchange (scx) cartridge followed by a c18 trap cartridge it is possible to pre-fractionate the peptides before separation on a c18 column. after loading the sample, discreet fractions are sequentially eluted from the cation exchange cartridge using a salt step gradient; the eluted peptides are then retained on the trapping c18 cartridge whilst they are desalted. finally the peptides are eluted from the c18 pre-column, at 200 nl/min, onto a 75 um id ϫ 10 cm waters symmetry analytical column for separation and elution into the mass spectrometer. this analytical approach will be discussed with examples where this methodology has been used for the analysis of standard protein mixtures and also for the analysis of cell lysates and sub-cellular fractions. monoclonal igg are commonly observed in various b cell disorders, the most clinically relevant being multiple myeloma. in a series of 73 serum samples, immunofixation identified igg 1 , igg 2 , igg 3 , and igg 4 in 63, 4, 5, and 1 cases, respectively. their light-chains were k in 45 cases and λ in 28 cases. these monoclonal igg were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis (2-de) with various isoelectric focusing conditions as well as by 3-de (2-de of the proteins extracted from agarose after serum protein agarose electrophoresis). after 2-de or 3-de, the monoclonal γ-chains were not visualized in 29 out 73 cases, whatever the isoelectric focusing conditions that were tested. in 6 cases, γ-chains were only detected using alkaline ph 6-11 gradients. monoclonal γ-chains and light chains were highly heterogeneous in terms of pi and mr. however, a good correlation (p ͻ 0.05) was observed between the index of migration of the monoclonal igg in agarose gels and the pi of their γand of their light-chains (r ϭ 0.735, multiple linear regression). because of the extreme diversity of the different γ-chains as well as of the k-and γ-chains, it appears that a classification of monoclonal igg based only on their electrophoretic properties is not possible. alzheimer's disease (ad) is one of disorders caused by protein conformational changes and recent studies have shown that several chaperone proteins are involved in this process. as information of chaperone expression in ad brain is limited, we aimed to study the expressional pattern of chaperones in several brain regions as this may be essential to understand how folding defects can lead to disease. we studied the concomitant expressional patterns of molecular chaperones in seven brain regions of adults with ad using two-dimensional polyacrylamide gel electrophoresis (2-de) and matrixassociated laser desorption ionization mass spectroscopy (maldi-ms). we unambiguously identified and quantified nine different chaperone proteins. six chaperone proteins, heat shock protein 60 (hsp 60), hsp 70 ry, heat shock cognate (hsc) 71, alpha crystallin b chain, glucose regulated protein (grp) 75 and grp 94 showed aberrant expressional patterns depending on brain region. hsp 70.1, grp 78 and t-complex 1 (tcp-1) epsilon subunit did not show any significant expressional change. these findings are compatible with neuropathological and biochemical abnormalities in ad brain and this report presents the first approach to quantify nine different chaperones simultaneously at the protein level in individual ad brain regions providing evidence for the relevance of aberrant chaperone expression to ad neuropathology. the mainstream approach to protein separation, visualisation and identification has been to use two-dimensional gel electrophoresis coupled to mass spectrometry for the identification of the separated proteins. however this approach is limited with the level of protein that may be loaded onto the 2d gel and the nature of the proteins that may be incorporated onto the first dimension (ipg strip). an alternative approach for the qualitative analysis of complex protein mixtures is the use of tryptic digestion followed by electrospray lc-ms/ms. this approach is dependent on a high degree of chromatographic separation prior to the mass spectrometer, such that ideally individual peptides are eluted into the source. if this is the case then the dynamic range of protein identification can be increased and low copy number proteins can be identified. often, however there is a large degree of redundant sequence information acquired, as in theory one peptide ms/ms spectrum is sufficient to identify a protein from a sequence databank. if a protein identification is obtained from a databank search of an ms/ms spectrum, it is potentially valuable to exclude the rest of the theoretical tryptic peptides to "mine" deeper into the protein complex being studied. we have introduced a new protein databank search engine capable of matching a tryptic peptide from the swissprot/ trembl databank to an ms/ms spectrum in one second. using this search engine we are able to generate dynamic tryptic peptide exclude and include lists, based upon the theoretical tryptic peptides from the identified protein, which can be passed to the acquisition software of our q-tof mass spectrometer in real time. thus, we are able to automatically steer the q-tof, during acquisition, to select and switch to the ms/ms mode only on those peaks that meet the modified selection criteria. experiments can be designed in which peaks that belong to a protein already identified during acquisition can be avoided. this exclusion list is based upon m/z, charge state and a user definable mass tolerance. the mass measurement of the data from the q-tof mass spectrometer is typically better than 10 ppm and as a consequence of this a tight mass tolerance can be selected, thus making the exclude list extremely specific. alternatively, in the case of samples derived from 2d gel spots, the mass spectrometer may abandon the current sample, re-equilibrate the lc column and move on to the next sample. to illustrate this methodology we show examples, both on standard samples and complex protein mixtures where q-tof data acquisition has been directed based upon the results from a databank search. this data will be compared and contrasted to data acquired in the normal automated lc-ms/ms mode. the specific anti-cancer activity of green tea (؊)-epigallocatechin-3-gallate (egcg) department of molecular biology, hebrew university-hadassah medical school, jerusalem, israel the effect of the green tea polyphenol-(ϫ)epigallocatechin-3-gallate (egcg) was tested in cultures of normal and transformed nih-patmras fibroblasts. in this system transformation can be induced at will by the addition of dexamethasone, which induces the expression of h-ras by activating the mammary tumor virus long terminal repeat (mmtv-ltr) promoter. this facilitates a reliable comparison of the susceptibility of normal and transformed cells to egcg. it has been shown that egcg inhibited the growth of transformed but not of the normal fibroblasts. in an attempt to elucidate the mode of the preferential inhibitory activity of egcg, its effect on growth promoting factors has been examined. the level of ornithine decarboxylase (odc, ec 4.1.1.17), which is a signal for cellular proliferation, was reduced by egcg in the transformed but not in the normal cells. egcg also showed strong inhibition of tyrosine kinase and mitogen-activated protein kinase (mapk) activities, without affecting the kinases in the normal cells. similarly, egcg also preferentially decreased the levels of the oncogenes ras and jun in transformed cell. egcg preferentially induced apoptosis in the transformed fibroblasts. in vitro chemosensitivity tests demonstrated that egcg inhibited the proliferation of leukemic cells. these findings suggest that egcg has a therapeutic potential in the combat against cancer. objectives: to develop a safo, affordable immune supportive therapy for hivϩ patients. design: a randomised, double blind, placebo-controlled study, testing an internationally patented l-methionine combination (lmc), in approximately 400 hivϩ patients; not yet on anti-viral treatment (cd4 count 200 to 500). methods: parameters measured included: cd4 count, total lymphocyte court, viral load, several clinical, as well as mechanistic parameters. the difference in the change from the baseline (active -placebo) was determined for each parameter. the study is ongoing. results: within 3 months, significant trends are noted. the cd4 count of the patients on the active therapy, presented with a slower rate of decrease, compared to the placebo group, mean difference (md) in this change from baseline; 15.1/cmm and 95% confidence interval (c1), this was confirmed by the total lymphocyte court values. after 12 months the placebo group was placed on active, causing the difference to disappear. conclusions: although further trials are needed, these results already indicate t-methionine as an important role player in the immune system of patients with impaired immune function. c. chiarla 1 , i. giovannini 1 , j. h. siegel 2 , g. boldrini 1 , and m. castagneto 1 1 centro di studio per la fisiopatologia dello shock cnr, catholic university, rome, italy 2 department of surgery, umdnj, newark, new jersey, u.s.a. in critical illness and sepsis, changes in amino acid plasma levels (aapl) have been assessed extensively, while little is known about the relationship with changes in other plasma components, such as those involved in fluid-electrolyte and osmotic balance; their investigation is also limited, in large clinical samples, by inter-patient variability. we analyzed the relationships between plasma sodium (na ϩ pl, meq/l) and aapl (µm/ l) in eighty consecutive measurements performed in one single patient with post-traumatic sepsis and severe, prolonged illness. unique feature of plasma taurine (tau) was maintenance of a highly significant inverse correlation with na ϩ pl (r 2 ϭ 0.48, p ͻ 0.001). all other aapl were correlated directly, or unrelated, to na ϩ pl, the only exception being a weak inverse correlation between tryptophan and na ϩ pl. tau was correlated, strongly and directly, also to phosphoethanolamine (pea), glutamate (glu) and aspartate (asp): tau ϭ 707.1 ϩ 7.3(pea) ϫ 4.6(na ϩ pl) r 2 ϭ 0.86, p ͻ 0.001 tau ϭ 710.2 ϩ 11.4(asp) ϫ 4.7(na ϩ pl) r 2 ϭ 0.61, p ͻ 0.001 tau ϭ 677.4 ϩ 30.7(logglu) ϫ 4.7(na ϩ pl) r 2 ϭ 0.59, p ͻ 0.001 and unrelated, or weakly and inversely related, to other aapl (measurements of beta-alanine were not included). co-variation of na ϩ pl and these aapl (particularly tau and pea) was influenced by severity of illness, and more complex regressions were needed to quantify this effect. these results provide useful information on interdependency of tau, na ϩ pl and other aapl in critical illness. the central nervous system (cns) shows an exceptionally high degree of vulnerability to reactive oxygen species. considerable evidence suggests that free radical formation and oxidative stress might play an important role in the pathogenesis of parkinson's disease (pd). moreover, it has been reported that the levels of glutathione and vitamin e increase in the brain of patients with pd as a compensatory mechanism to deal with oxidative stress. since vitamin e is an effective free radical scavenger in the brain, its neuroprotective function is the issue of new therapeutic approaches in neurodegenerative diseases. to elucidate the possible role of vitamin e in the pathogenesis of pd, we assessed the plasma levels of vitamin e, measured by high-performance liquid chromatography, in 54 patients with pd. vitamin e concentrations were also assessed in 93 age and sex matched normal individuals. the mean plasma levels of vitamin e did not differ significantly between these two groups (22.5 ϯ 8.15 mmoli/l for pd patients and 21.0 ϯ 7.9 mmoli/l for controls). the results of our study suggest that plasma vitamin e concentrations do not play a major role in the pathogenesis of pd. vitamin e and cardiovascular disease: nutritional and intervention approaches f. galli 1,2 1 institute of biological chemistry, university of urbino, italy 2 department of cardiovascular research, st thomas' hospital, london, u.k. vitamin e is represented by a family of eight natural vitamers (4 tocopherols and 4 tocotrienols) of which αtocopherol (α-t) form has the highest biological activity. this vitamin accounts for most of the lipid-soluble, chain-breaking antioxidant activity in mammalian tissues and plasma. in addition, it shows nonantioxidant properties through which it modulates cell signaling and the expression of specific enzyme in cell models playing a role in atherogenesis (e.g. endothelial and inflammatory cells). the preventive effect of vitamin e on acdv is still a matter of debate. the largest epidemiological investigations and 4 out of 5 main intervention studies at yet available have suggested a correlation between levels of vitamin e and incidence of atherosclerotic cardiovascular disease (acvd) and related mortality. an overall conclusion rising from these studies is that the major effect (if any) of vitamin e is to be found with intakes higher than 100 iu (100 mg all-rac α-tocopheryl acetate) per day. however, other investigations have failed to demonstrate a beneficial effect of vitamin e against acvd, suggesting the need for more studies on its metabolism and function. recently a family of tocopherol binding and transport proteins has been identified. they play a key role in the selective uptake and delivery of tocopherols to lipoproteins and tissues. genetic abnormalities of these proteins have been demonstrated to be responsible for conditions of vitamin e deficiency in humans. their tissue distribution and regulation are now under investigation. the information available on vitamin e metabolism and its response to supplements or diet changes are at yet poorly characterized. the synthesis of stable isotopes and the characterization of major metabolites of main vitamers provide important advances in this research. in the last years, both plasma levels and urinary excretion of relevant metabolites of α-t have been characterized. little information is available on metabolites formed by other vitamers. the emerging role of γ-t and its main catabolite 2,7,8-trimethyl-2-(b-carboxyethyl)-6hydroxychroman (γ-cehc) in the defense against nitrogen oxide species formed during the activation of inflammatory cells is now well established and suggests the need for further studies on the bioavailability and transformation of this homologue of vitamin e in humans. at the same time, an oxidation byproduct of α-t found in human plasma, namely α-tocopherylquinone, has been proposed to be also de novo synthesized from phenylalanine with a role in the genesis of a defective polyunsaturated fatty acid metabolism observed in phenylketonuric patients. this suggests a possible, and at yet unexplored relationship between vitamin e and phenylalanine/fatty acid metabolism which might have also a role in atherosclerotic process. r. gaspari 1 , s. mensi 1 , g. mercurio 1 , c. callà 2 , l. colacicco 2 , e. sacco 2 , and s. lippa 2 1 department of anaesthesiology and intensive care medicine, and 2 department of biochemistry and clinical biochemistry, catholic university of rome, italy four patients (3 females, 1 male; aged from 21 to 45 years) affected by severe liver failure, were treated by a new blood purification method, namely molecular adsorbent recycling system (mars). mars removes albumin-bound toxins using a specific membrane with a dialysate solution containing albumin. in the patients the plasma levels of methionine (meth), branched chain and aromatic amino-acids and liposoluble antioxidants were measured. the fischer's index did not show any significant variation, whereas the plasma levels of meth were well correlated with the levels of liposoluble antioxidants (vitamin e and coq10). in fact, in the patients receiving just branched chain amino-acids, the plasma levels of both meth and antioxidants progressively decreased. on the contrary, if meth and branched chain amino-acids were administered, the plasma levels of coq10 and vitamin e showed a positive correlation with the plasma meth levels (p ͻ 0.02; r ϭ 0.63 and p ͻ 0.005; r ϭ 0.77, respectively). since vitamin e and coq10 are mutually dependent-molecules, the administration of meth, essential substance for coq10 synthesis, may be effective to maintain a good antioxidant status in patients with severe liver failure undergoing mars treatment. we obtained new synthetic peptide preparation epitalon to be widely applied as a pharmaceutical due to its properties important in medical care. epitalon was found to stimulate repair processes in retinal diseases via restoring the retinal functions, in particular its photoreceptors. this promising peptide drug is a linear tetrapeptide of formula h-ala-glu-asp-gly-oh (alanyl-glutamyl-aspartyl-glycine). the substance was obtained by classic peptide synthesis in a solution (scheme: (1 ϩ 2) ϩ 1) with n-oxysuccinimide activated esters. coohgroups of lateral radicals of glutamic and aspartic acids were defended as benzyl esters, benzyloxycarbonyl (ala) and tert.butyloxycarbonyl (glu) n-defending groups were employed, deblockade conducted by trifluoroacetic acid and catalytic hydrogenolysis. preparative hplc on a reverse phase was applied for purification. the product was fully characterised by the data of analytical hplc (substance content -99%), amino acid analysis, ir-and hmr-spectra. the ready drug form is ampoules containing 10 µg of the substance in 1 ml of isotonic solution. epitalon application in patients with pigmented retinal degeneration stopped the pathology development in 100% and increased visual functions in 80% of the cases. in 70% of the patients visual acuity raised by 0.1-0.3. electroretinography confirmed the retinal functional activity increase. an increasing number of proteins are implicated in apoptosis and several of them have been shown to be altered in alzheimer's disease (ad) brain. because of this apoptosis is thought to be the underlying mechanism of neuronal cell loss in ad. to further substantiate this hypothesis we investigated the expression of a recently identified apoptosis related proteins and other apoptosis regulators in frontal cortex and cerebellum of ad by western blot and elisa techniques. quantitative analysis revealed unaltered levels of bax and raidd (receptor interacting protein associated ich-1 (caspase-2)/ ced-3 (caenorhabditis elegans death protease-3)-homologous protein with death domain) in both regions. zip (zipper interacting protein) kinase, bim/bod (bcl-2 interacting mediator of cell death/bcl-2 related ovarian death gene) and p21 were significantly increased only in ad frontal cortex (p ͻ 0.05, in all cases). cerebellar bcl-2 levels were significantly increased in ad (p ͻ 0.01) while in ad frontal cortex, although the levels tended to increase did not reach significance level. the results indicate that apoptosis indeed account for the neuronal loss in ad. however, it does not seem to involve bax and raidd. a. magyar 1 , m. brózik 3 , r. tóbi 1 , t. szabó 1 , j. szakonyi 2 , b. rojkovich 3 , p. gergely 2 , and f. hudecz 1 1 research group of peptide chemistry hungarian academy of science, budapest, 2 central laboratory of immunology, semmelweis university, budapest, and 3 national institute of rheumatology, budapest, hungary rheumatoid arthritis (ra) is a systemic autoimmune disease of unknown etiology. it is the most common of the inflammatory joint diseases, affecting 1-2% of the world population. anti-filaggrin antibodies (afa) directed against the epidermal protein, filaggrin, belongs to the most specific markers of ra. epitopes, containing citrulline within the sequence of filaggrin, have been recently identified as major antigenic sites recognised by afa. the aim of our study was to identify these epitopes of filaggrin derived-peptides targeted by ra specific antibodies to provide further information about the nature of the initial autoantigenic substance. the most immunogenic six sequences of filaggrin and further, on the n-and c-terminal, shortened version of the original peptide ( 306 shqestrgrsrgrsgrsgs 324 ) were synthesized. we used conventional solid-phase peptide synthesis (fmoc strategy) carried out on "multipin ncp" noncleavable kit. in elisa experiments the presence of afa was deter-mined using serum samples of ra patients and healthy blood donors. in conclusion our results provide further evidence that not simply the presence of citrulline but also the nature of its surrounding amino acids have important role in the creation of autoantigenic epitope reactive with anti-filaggrin antibodies. the autoimmune nature of multiple sclerosis (ms) has introduced cytokine genes as logical candidates for the loci determining susceptibility to the disease and/or influencing disease progression. interleukin (il)-1alpha and 1beta are major proinflammatory cytokines that have been related with several chronic inflammatory diseases such as ms. the il 1-receptor antagonist (il-1ra) is a protein structurally related to il-1beta that effectively inhibits the proinflammatory effects of il-1. a polymorphism in the 5ј-flanking regulatory region at ϫ889 of the il-1alpha gene, which may cause an overexpression of il-1alpha and a variable number tandem repeats (vntr) polymorphism in the il-1ra gene have been also associated with several inflammatory diseases. two biallelic base change polymorphisms in the il-1beta gene have been reported to influence the protein production: one is located in the promoter region at position ϫ511 and the other is in exon 5 at position ϩ3953. to analyze the contribution of il-1alpha, il-1beta and il-1ra genes in the genetics predisposition to ms, we have examined four polymorphic genetic markers in 132 italian patients with clinically definite ms and 130 healthy controls. in summary, no significant differences in genotypes and allele frequencies were found between ms patients and healthy controls. fibronectin -the extracellular matrix protein is oxidatively modified with oxygen reactive species (ros) in inflammation site. activated neutrophiles release the hypochlorite acid (hocl) and chloramines as products of myeloperoxidase/ h 2 o 2 /cl ϫ system. these reactive chlorine species chlorinate in turn matrix proteins. the resulting changes of tertiary protein structure could be evaluated by monitoring the antigen/antibody complex formation. the formation of the complexes between native/chlorinated fibronectin and igg class antibodies were examined by means of elisa with luminol chemiluminescence detection. the degree of fibronectin modification was monitored with spectroscopic methods. since the oxidation leads to the fibronectin aggregation -the tryptophane contents in resulting aggregates were evaluated with stern-volmer approach (acrylamide quenching). moreover, the aldehydes influence on the ag/ab complex formation was examined -since aldehydes are known products of amino acids n-chloramines deamination. also the native and modified fibronectin adherence to the matrix proteins was monitored with use of hrp labeled antifibronectin antibodies. the preliminary results suggest that chlorination impairs the ab/ag complex recognition but also prove that igg bounded chlorinated fibronectin promotes igg clusters formation. it was found also that mm concentration of the serine derived glycoaldehyde decreases the fibronectin/igg recognition and the effect could be attributed to the igg aggregates formation. we demonstrate also that hrp-labeled iggs detect the collagen and fibrynogen adherent fibronectin in a dose dependent manner-details of the elisa method are discussed. in subjects with rheumatoid arthritis (ra) oxidized low density lipoproteins (ldl) are supposed to serve as mediators for joint damage, further exacerbating the inflammatory process. to better understand mechanisms of ldl oxidation in ra a specific marker of oxidative modification of apolipoprotein (apo) b-100 proline and arginine residues, 5hydroxy-2-aminovaleric acid (hava), had been measured in plasma and synovial fluid ldl subfractions (ldl 1 , svedberg units (s f ) 7-12 and ldl 2 , s f 0-7) by gc-ms. paired knee synovial fluid and plasma samples were collected from 10 subjects with ra. additionally, plasma samples were collected from 10 healthy controls. the ldl 1 hava content in plasma was not different between the groups (ra, 0.004 ϯ 0.001 vs controls, 0.004 ϯ 0.001 mol/mol apob-100, p ϭ 0.748). the ldl 2 hava content in plasma was significantly higher in ra (0.145 ϯ 0.051 vs 0.013 ϯ 0.002 mol/mol apob-100, p ϭ 0.000). furthermore, synovial fluid ldl 1 and ldl 2 in ra contained elevated hava levels when compared with plasma concentrations (ldl 1syn , 0.023 ϯ 0.012 mol/mol apob-100 (p ͻ 0.001) and ldl 2syn , 0.434 ϯ 0.129 mol/mol apob-100 (p ͻ 0.001)). results suggest that proline and arginine residues of apob-100 are highly reactive toward oxygen radicals in both plasma and synovial fluid in ra. furthermore, susceptibility of apob-100 to oxidative modification increases along the lipoprotein metabolic cascade. particularly small dense ldl 2 were prone to direct oxidation of apob-100. correlation between hava content in plasma and synovial fluid ldl 1 and ldl 2 in ra may allow the use of hava as a clinical marker of antioxidant barrier impairment in ra. vascular collagen accumulation contributes to development of hypoxic pulmonary hypertension (ph). we have shown that injections of a polymer of the proline analogue cis-4-hydroxy-l-proline (chyp) in liposomes attenuated acute ph in rats (ajrccm 1997; 155 : 1384) . we now treated rats with established ph with a new polymer containing an increased "payload" of chyp. chyp was conjugated to a low mw poly(ethylene glycol)-lysine carrier {poly (peg1000)-lys-chyp} to increase the % by wt of the analogue. rats were exposed to 10% o 2 for 7 da to induce ph. on da 0, 7 and 14 after 7 da of hypoxia, animals were injected iv with chyp polymer in liposomes (hc) or bioinactive trans-hyp polymer in liposomes (ht). air controls received thyp polymer in liposomes (at). at 0 and 21 da, we measured mean right ventricular pressure (rvp) and hydroxyproline (hyp) content in main pulmonary arteries. on da 0, rvp (mmhg) was 9 ϯ 1 and hyp (µg/vessel) was 88 ϯ 6 in at. rvp and hyp increased to 17 ϯ 1* and 139 ϯ 4*, respectively, in hypoxic animals (n ϭ 4; *p ͻ 0.05 vs. at). on da 21, rvps were at 10 ϯ 1, ht 24 ϯ 1*, hc 15 ϯ 1* †; hyps were at 91 ϯ 9, ht 176 ϯ 15*, hc 122 ϯ 1* † (n ϭ 4; *p ͻ 0.05 vs. at; †p ͻ 0.05 vs. ht). from da 0 to 21, rvp did not increase and hyp decreased in the hc group vs. ht. we conclude that weekly injections of polymeric chyp prevented progression of established hypoxic ph and reversed hyp accumulation. targeted delivery of antifibrotic polymers may prevent and reverse the progression of ph. (support: phs, barbara cornwall foundation). glucosinolates are amino acid-derived natural plant products found throughout the capparales order, which includes agriculturally important crops such as oilseed rape, brassica vegetables and the model plant arabidopsis. glucosinolates and their degradation products have a wide range of biological activities, e.g. in plant defense as deterrents against insect and fungi and as attractants to insects that are specialized feeders in brassicaceae. the conversion of amino acids to oximes is a key step in glucosinolate biosynthesis. we have recently shown that cytochromes p450 belonging to the cyp79 family catalyze the conversion of aliphatic, aromatic as well as indole amino acids to the corresponding oximes. cyp71e1 catalyzes the oxime-metabolizing step in the biosynthesis of the cyanogenic glucoside dhurrin. we have recently shown that the oxime-metabolizing enzyme in the glucosinolate biosynthetic pathway is a cytochrome p450 homologous to cyp71e1. the post-oxime enzymes in the glucosinolate pathway have high substrate-specificity for the functional groups, and low substrate-specificity for the side chain. therefore, we have been able to metabolically engineer new glucosinolate profiles into arabidopsis by altering the level of endogenous cyp79s and by introducing new cyp79s. the approach has great potential for design of "biotech crops" with improved pest resistance and increased nutritional value. hypercalcemia as a potential threat in the dietary treatment of maternal phenylketonuria f. eyskens 1 and s. beernaert 2 1 pediatrician, metabolic diseases and 2 dietitian, azm-koningin paola childrens hospital, metabolic lab pcma, antwerp, belgium over 90% of infants born to mothers with blood phenylalanine (phe) concentrations above 1200 µmol/l exhibit evi-dence of foetal dammage, low birth weight, microcephaly, dysmorphic facies, slow postnatal growth and development and long-term intellectual impairment. keeping maternal phe concentrations below 250 µmol/l before conception and throughout pregnancy reduces significantly the risk of abnormalities in the offspring of women with phenylketonuria (pku). we describe a woman, 31 years old, who showed phe blood levels of 150-200 µmol/l under a strict diet (total protein content of 1.13 g/kg body weight/day with 0.54 g/kg natural proteins and 0.59 g/kg proteins provided by the aminoacid mixture pku3 (milupa, germany); 1,655 cal/day) at the beginning of her first pregnancy. the first weeks she developed vomiting which gradually increased in severity. at 8 weeks of pregnancy, she had diarrhea, severe bouts of vomiting and manifested a deficient nutritional status with intake of 0.2 g/kg bw proteins and 1,178 cal/day. she was hospitalized to start refeeding using continue drip feeding administered by nasogastric tube. after 2 days on this regimen she developed vomiting, heart palpitations and mental confusion. her serum calcium level, that was normal at admission in the hospital, showed an elevation to 6.5-7 meq/l (ref. value 4.2-5.1 meq/l). the feeding was stopped immediately and under an intravenous infusion and gradually introducing a feeding composed of pku3, carbohydrates and mct fats the serum calcium and the blood phe levels dropped to normal values. and volatile components of caramel obtained by heating commercial maltose solution for different time intervals. one sample containing maltose only was used as control, the caramelization was conducted at 130c° for total time period 90 minutes and subjected to sensory analysis and isolation of volatile components. the odour and colour sensory tests were evaluated according to the international standard methods (iso). the results showed that addition of lysine as a catalyst gave rise to a significant (p ͻ 0.05) increase in intensity of the whole flavour in comparison with the control sample. the sweet and caramel notes, the most characteristic attributes of caramel, showed remarkable increase. on the other hand the increase in heating time in presence of lysine as a catalyst resulted in high significant increase in browning rate of caramel solution. the volatile components of each sample were isolated by using the new technique, solid phase microextraction (spme) and subjected to gc and gc-ms analysis. over 250 volatile components were separated, however only the most important component for caramel flavour were reported. maltol and 5hydroxymethyl-2-furfural (hmf) and 4. h-pyran-4-one,2,3dihydro-3,5-dihydroxy-6-methyl (dihydro dihydroxy maltol), the main characteristic caramelization products were present in high concentration in samples contaning lysine heated for 60 minutes. in addition one pyrazine was only identified in the samples contaning lysine. a comparative study between the present results and those of our previous study concerning addition of alanine as a catalyst was carried out. short-term exposure of human umbilical vein endothelial cells (huvecs) to hyperglycemia increases l-arginine transport (system y ϩ /cats) and nitric oxide (no) production (via enos). it has been reported that enos could also be activated by a ca 2ϩ -independent mechanism involving phosphorylation of ser 1177 by a phosphatidylinositol 3-kinase (pi3-kinase) dependent pathway. we investigated the involvement of pi3kinase on the stimulatory effect of acute hyperglycemia on enos and l-arginine transport in huvecs. l-arginine transport, no synthesis and phosphorylation of ser 1177 in enos were increased by d-glucose (25 mm, 2 min). similar results were obtained in huvecs exposed to insulin. incubation of cells with wortmannin (pi3-kinase inhibitor) prevented the effects of d-glucose and insulin. no changes in the intracellular ca 2ϩ and enos protein levels were detected. thus, acute hyperglycemia increases l-arginine transport and enos activity through a pi3-kinase dependent, ca 2ϩ independent mechanism in huvecs. [ the hypercalcemia in this patient was due to a very high content in calcium of the feeding administered (2-3 times the adh value) associated with a high vitamine d concentration (see table) and a clinical state of dehydratation. the further pregnancy was uncomplicated and a healthy girl was born who developed normal. • the aminoacid mixtures used in the treatment of pku contain a high level of calcium, phosphate, magnesium and iron. they also contain a high concentration of vitamine d. • nutritional monitoring of pregnant pku patients should include the calcium, phosphate, iron, zinc and vitamins status. • vitamins a and d suppletion is contraindicated in these patients based on the high concentrations of these vitamins in the aminoacid mixtures used in the dietary treatment. flavour and aroma chemistry department, national research centre, dokki, cairo, egypt caramelization of various carbohydrates leads to product with a high tinctorial strength provided by different additives catalyzing the process. the present study was conducted to evaluate the catalytic effect of lysine on the sensory attributes administered pku3 (80 g ϭ adh 1, 2 g/kg/bw) lipoic acid is a prosthetic group of h-protein of the glycine cleavage system and e2 components of the pyruvate, 2oxoglutarate and branched-chain 2-oxoacid dehydrogenase complexes. in mammals, attachment of lipoic acid to these proteins requires two enzymes. lipoate-activating enzyme (lae) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. then, lipoyltransferase transfers the lipoyl moiety to the specific lysine residue of the proteins. we purified lae from bovine liver mitochondria. lae activated lipoate with gtp at a 1000-fold higher rate than with atp. the reaction absolutely required lipoate and mggtp, and the reaction product was lipoyl-gmp. lae activated both r-and senantiomers of lipoate to the respective lipoyl-gmp although preference for r-lipoate was observed. lipoyltransferase equally transferred both r-and s-lipoyl moiety from respective activated lipoate to apoh-protein. however, only h-protein carrying r-lipoate was active in the glycine cleavage reaction. cdna clones encoding a precursor lae with a mitochondrial presequence were isolated. amino acid sequence of lae was identical with that of xenobiotic-metabolizing/medium-chain fatty acid : coa ligase-iii, but an amino acid substitution due to snp was found. these results indicate that the medium-chain acyl-coa synthetase in mitochondria plays a novel function with gtp, the activation of lipoate. instituto di chimica biologica "g. fornaini", università di urbino, italy nitric oxide (no) can modulate red blood cells (rbc) glycolysis by translocation of the enzyme glyceraldehyde-3phosphate dehydrogenase (gapd) [e.c. 1.2.1.12] from the cytosolic domain of the membrane protein band 3 (cdb3) in the cytosol. in this study we have investigated which no-reactive thiols might be involved influencing gapd translocation, and which is the role of glutathione (gsh) in this context. two highly reactive cys residues (k 2 ϭ 73.7 m ϫ1 s ϫ1 and 101.5 m ϫ1 s ϫ1 , respectively) were identified by transnitrosylation with nitrosoglutathione (gsno) of cdb3 and gapd. the cys 149 in the catalytic site of gapd is exclusively involved in this gsno-induced nitrosylation. reassociation experiments carried out at equilibrium with preparations of rbc membranes and gapd revealed that different no-donors may form ϫsno on, and decrease the affinity between, gapd and cdb3. in intact rbc, both the no-donors 3-morpholino-sydnonimine (sin-1) and peroxynitrite (onoo ϫ ) significantly increased gapd activity in the cytosol and glycolysis measured as lactate production and energy charge levels. however, we obtained data suggesting that onoo ϫ is the main no-derivative able to cross the rbc membrane leading to gapd translocation and ϫsno formation. both in cell-free experiments and intact rbc, diamide (a thiol oxidant able to inhibit gapd activity) was observed to reverse the effect of sin-1 on gapd translocation. the results demonstrate that cdb3 and gapd contain reactive thiols that can be transnitrosylation mainly by means of gsno, these can ultimately influence gapd translocation/ activity and the glycolytic flux. abteilung für allgemein-viszeral-und gefässchirurgie, kliniken dr. erler gmbh, nürnberg, germany new surgical procedures like minimal-invasive-surgery brought many advantages for the surgical patient: less pain and shorter hospitalization. regarding nutrition, patients gets normal food on the ward still on the operation-day and need only saline-infusions overnight for fluid and electrolyte substitution but no hypocaloric parenteral nutrition. hypocaloric parenteral nutrition had been developped as a peripheral intravenous nutritional concept for patients with a normal body mass index over a period not longer than 4-5 days. multiple clinical studies showed that bowel movements increase earlier after an early postoperative enteral feeding which allows an earlier discharge of the patient. the result is a remarkable decrease of costs and an increase in patient benefit. still some years before surgeons preferred in visceral surgery parenteral nutrition over a period of 4-5 days under the opinion not to stress an anastomosis. this opinion changed in the last years under the aspect that about 1,000-1,500 ml of bile fluid, 1,000-1,500 ml pancreatic juice and 1,000-1,500 ml gastric juice per day are passing a small intestine anastomosis without any complications. concerning colon-anastomoses, the colon is preoperatively washed out, so it lasts until 5 days until defecation. multiple studies also showed a benefit for the patient regarding immunostimulation by early postoperative enteral feeding. conclusion: in our hospital with 244 surgical patients we recommend postoperatively either early normal enteral feeding or a high caloric parenteral nutrition if parenteral nutrition is needed for longer than 5 days. if artificial nutrition is necessary for more than 14 days we recommend enteral nutrition given by a tube or peg (percutaneous endoscopic gastrotomy). department of food technology, national research centre, dokki, cairo, egypt in the near east, "frekeh" has been known for many centuries as a stable food made from wheat. it is generally claimed that "frekeh" is better than wheat regarding its storage stability. the protein quality of parched immature durum wheat (frekeh) produced from 2 variety was evaluated. frekeh from four maturing levels during the dough stage of the seed development, were analyzed for approximate analysis. results showed that "frekeh" produced at the beginning of the dough stage was of better nutritional value than that produced at the following maturity levels, since the former was higher in protein, fat, minerals and crude fiber as well as in reducing sugar content. in addition, it was shown that these results confirm well with the sensory quality evaluation of the cooked product. further more, it was found that the cooking time was suitable to produce a "freqkeh" meal with high levels of acceptability. the observed decrease in protein content with increasing maturity level raised the question of how the protein quality of "frekeh" versus that of nature wheat grains varied. in this investigation, the amino acid of "frekeh" was determined. dietary treatment and carnitine supplementation has greatly improved long-term outcome of patients with ppa and (vitamin b12 unresponsive) mma. however, metabolic decompensation may be frequent and final outcome in most patients show various handicaps. to investigate the usefulness of measuring free carnitine and acylcarnitines in dried blood by tandem mass spectrometry, we investigated 9 patients with ppa and 5 with mma in a period of 8 months by weekly capillary blood punctures performed by the parents. age of the patients were from 0.5 until 18 years. clinical status at the time of blood drawing was evaluated by regular phone calls. free carnitine in all patients substituted by oral carnitine treatment (50-100 mg/kg/day bw) was normal. the parameter best reflecting clinical status was the c 3 /c 16 -acylcarnitine quotient. mean value in mma and ppa patients showed a range of 11.5-29.4 (normal 1.5 ϩ/ϫ 0.36, n ϭ 18), there was no difference between ppa and mma patients. individual mean values of the patients significantly increased when the patient was ranked higher in the clinical score system or during decompensation. since measurement of acylcarnitines in dried blood by tandem mass spectrometry is easy to perform, this method may be used for home monitoring of patients with mma and ppa. influence of acute treatment with 1,2,3,4tetrahydroisoquinoline on the levels of glutathione and reactive oxygen species, and on the enzymatic activity of γ-glutamyl transpeptidase in dopaminergic structures of rat brain e. lorenc-koci 1 , m. sokoĺowska 2 , m. zapaĺa 2 , and l. wĺodek 2 1 institute of pharmacology, polish academy of sciences, kraków, and 2 institute of medical biochemistry, collegium medicum, jagiellonian university, kraków, poland 1,2,3,4-tetrahydroisoquinoline (tiq) and its derivatives generated considerable interest as molecular species that may be implicated in the pathogenesis of parkinson's disease (pd). in pd, apart from the lack of dopamine in the striatum, a decreased concentration of glutathione (gsh) is found in the substantia nigra (sn). it is also known that gsh depletion potentiates the toxicity of mptp and 6-hydroxydopamine. however, there are no data available on the tiq influence on gsh metabolism. the aim of the present study was to exemine the effect of acute tiq administration on the levels of gsh and reactive oxygen species (ros), and on the enzymatic activity of γ-glutamyl transpeptidase (γ-gt) in dopaminergic structures of rat brain. the investigation was carried out 4 h after a single dose of tiq (100 mg/kg i.p.). at that time, a marked increase in the tissue gsh level and simultaneous significant inhibition of γ-gt were found in the structures studied. in tiq-treated rats, the production of ros was reduced in the sn, but it was markedly enhanced in the striatum. our results suggest that the increase in gsh level in dopaminergic structures stems from inhibition of γ-gt and refers to the extracellular pool of this peptide. apparently, the tiq-mediated alterations in the levels of gsh and ros may have some implications for the etiology of pd. tetrahydrobiopterin-responsive phenylalanine-hydroxylase deficiency with mutations distant from the tetrahydrobiopterin binding site z. lukacs 1 , r. steinfeld 1 , a. kohlschütter 1 , j. zschocke 2 , and k. ullrich 1 1 department of pediatrics, university of hamburg, and 2 university-children's hospital, heidelberg, germany phenylalanine hydroxylase (e.c. 1.14.16.1) catalyzes the hydroxylation of phenylalanine to tyrosine in the presence of oxygen and the cofactor (6r)-l-erythro-5,6,7,8tetrahydrobiopterin (bh 4 ). mutations in the phenylalanine hydroxylase gene may cause phenylketonuria or hyperphenylalaninemia. alternatively, disorders in bh 4metabolism also result in an increase in phenylalanine concentrations but simultaneously affect other bh 4 -dependent enzymes, consequently, causing a severe neurological disorder. recently, several patients with a phenylalanine-hydroxylase deficiency but with normal bh 4 -metabolism were reported who showed a significant decrease in blood phenylalanine concentrations upon treatment with bh 4 . indeed, two such patients in our hospital were also sensitive to daily oral doses of 5-10 mg bh 4 /kg. the subsequent molecular genetic analysis revealed that patient 1 was homozygous for the widespread mutation y414c and patient 2 was compound heterozygous for the mutations a104d and k320n. it is striking, that all mutations are located distant from the known bh 4 -binding site and thus, should not be associated with bh 4 -sensitivity. additionally, further patients who share the same genotype are not sensitive to bh 4 . therefore, it must be concluded that factors independent of the phenylalanine hydroxylase gene, like e.g. individual chaperone proteins, influence the three-dimensional structure of the enzyme and thereby, enhance enzymatic activity in the presence of elevated concentrations of bh 4 . retrotransposons are structurally similar to retroviral gag and pol which are required for their replication via reverse transcription, and seem to be an ancestral form of specialized retroviruses. reverse transcription of retrotransposons was assumed to occur in virus-like particles as well as in retroviruses. rna-packaging in this particles suggests a possibility of infection. presumably, the formation of functional virus-like particles requires the interaction of gypsy rna with a protein encoded by gypsy first open reading frame (orf1) or a product of its processing. the objective of this work was to study whether the protein by this frame can bind with nucleic acids similarly to retroviral gag-protein and how phosphorilation of that protein may influence to this interaction. then gypsy orf1 was cloned and expressed in escherichia coli, and its protein product was purified by ion-exchange chromatography on deae-cellulose and affinity chromatography on heparin-sepharose and tested electrophoretically. it was shown that recombinant protein bound with its own mrna and with dna. the affinity for ssdna bing higher than for dsdna. the binding constant was estimated with rna. the method utilizes the ability of nitrocellulose to bind proteins but not nucleic acids. binding of 50% gypsy rna was achieved with about 100 ng of the protein in 50 ml of the reaction mixture. the binding constant was 5&times; 108 m-1, which is consistent. the structure of the putative nucleic acid-binding domain suggests that the protein is more similar to the core proteins of spumaviruses of the family retroviridae that to those of other retroviruses. phosphorilation of gag-like protein encoded by first open reading frame of retrotrasposon gypsy (mdg4) affects to interaction with nucleic acid. tryptophan (trp) in humans is catabolized by several pathways leading to various metabolites of kynurenine and indolic compounds formation. a number of diseases are connected with abnormalities in its excretion, but relationship of cause and effect is usually unclear. we introduced a two-step procedure for the detection of defects in metabolism of trp: 1) tlc is employed when starting the investigation, 2) two hplc methods were proposed and used at the next step, when pathological findings are to be proved and the individual metabolites quantified. the first hplc procedure enables the assessment of tryptophan, indolylacry-loylglycine (iag) and other five indolic compounds. the second method is intended to the monitoring of kynurenine and seven of its catabolites. the same sep-pack pre-treated sample of plasma and urine is used for all methods. the reference values and the excretion pattern in some groups of patients (350 in total) were assessed. hepathopathy, gastrointestinal defects, myopathy and seizures with other neurological symptoms were the conditions connected with changes in the excretion of some metabolites of trp. significant decrease of iag excretion was found in burn patients early after the injury. urine analyses were performed at patient with hartnup disease and benign xanthurenic aciduria, inherited metabolic defects of trp. in other experiments, trp effect on the decarboxylation of other aromatic amino acids in the liver was investigated; only weak inhibition under physiological conditions was recognised. ( two hypothetical proteins of escherichia coli, ybbq and yhae, show high sequence similarity to d-threonine dehydrogenase from pseudomonas cruciviae ifo 12047. we cloned each gene encoding ybbq and yhae into e. coli jm109. both ybbq and yhae showed no d-threonine dehydrogenase activity and showed significant activities for d-serine in the presence of nad. ybbq and yhae were purified to homogeneity from the e. coli clones. ybbq consisted of two identical subunits with a molecular mass of 31 kda, whereas yhae was a tetramer (native molecular mass, 124 kda). ybbq showed the maximum activity at ph 11.0 for the oxidation of d-serine. whereas optimum ph of yhae was ph 10.5. they catalyzed oxidation of glycerate and 3-hydroxyisobutyrate. d-glycerate was the best substrate for both enzymes. both enzymes also catalyzed reduction of tartronate semialdehyde in the presence of nadh. at physiological ph, the rate of tartronate semialdehyde reduction was much higher than that of d-glycerate oxidation. the ybbq gene is in the operon of glyoxylate utilization and the yhae gene is in the operon for d-glucarate/galactarate utilization. these results suggest that both ybbq and yhae are dglycerate 3-dehydrogenases and function physiologically in conversion of tartronate semialdehyde into d-glycerate. a serine protease inhibitor model: synthesis and biology z. mucsi 1 , á. bódi 2 , l. gráf 2 , a. perczel 1 , a. patthy 3 , and g. orosz 4 1 department of organic chemistry, 2 biochemistry, eötvös university, budapest, 3 agricultural biotechnology centre, gödölló´, and 4 research group of peptide chemistry, hungarian academy of sciences, budapest, hungary sgci is structurally related to the pmpd-2 family of canonical serine protease inhibitors. in these peptides, there is a p1-p1ј position which is responsible for reversible binding to chymotrypsin. their structure is characterized by structural compactness: the molecule contains three -sheets and three disulfide bonds. in the sgci molecule the p1-p1ј corresponds to lys-leu bond, which is cleaved by chymotrypsin extremely slowly. the question arises why an excellent substrate behaves at the same time as inhibitor. it was assumed that the threedimensional structure of the molecule is responsible for the inhibitory activity. a model was designed to include all the known features of the inhibitor: the structurally necessary -sheet structure and the fragment containing the p1-p1ј environment. three model peptide were synthesized. two model peptides had no inhibiting effect and were cleaved by chymotrypsin. one of the cleavage points is the expected p1-p1ј position, while the other positions found to be chymotrypsin preferred positions after the first cleavage. the three-dimensional structures of the model peptides were mapped by nmr. on the basis of nmr structures obtained it has been shown that the cyclopeptide part is more flexible in the models than in sgci. the initial process in the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurttransferase: inactivation study by using chloropyruvate n. nagahara 1 , t. nakagawa 2 , and m. minami 1 1 department of hygiene and public health, nippon medical school, sendagi bunkyo-ku, tokyo, japan 2 institute for organic chemistry, darmstadt university of technology, darmstadt, germany to investigate the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurtransferase (ec 2.8.1.2, mst), inactivation kinetics with 3-chloropyruvate (chloropyruvate) was studied; each inactivation reaction was completed in a preincubation procedure. chloropyruvate is an analog of 3-mercaptopyruvate (mercaptopyruvate) and irreversibly inhibits mst. the inactivation depended on incubation time and the concentration of chloropyruvate and showed saturation kinetics. the plot for the logarithm of % activity remaining versus preincubation time showed pseudo-first-order. the kinact is 8.0 ϫ 10 ϫ2 min ϫ1 and k1 is 3.1 mm. these suggest that chloropyruvate serves as a mechanism-based inactivator. mercaptoethanol , so that chloropyruvate can approach cys247 via the donor substrate route and acceptor substrate one, and a ternary complex may be formed prior to the inactivation. these findings suggest that a donor substrate enters the catalytic cavity prior to an acceptor one in the initial process of the mst reaction: mst follows an ordered sequential mechanism. polyketides are natural products of bacteria, fungi, marine organisms and higher plants, many of which have clinical usage. actinorhodin (1) is an antibiotic produced by streptomyces coelicolor via an iterative type ii polyketide synthase (pks) system. this consists of a multi-enzyme complex with a single catalytic function for each enzyme. departments of 1 chemistry and 2 pediatric surgery, school of medicine, fujita health university, toyoake, japan it has been reported that, in rats with a single intoxication of α-naphthylisothiocyanate (anit), acute liver injury develops with enhanced lipid peroxidation and neutrophil infiltration in the liver tissue. melatonin functions as an antioxidant. melatonin is known to inhibit neutrophil infiltration into damaged liver tissues. therefore, we examined whether melatonin exerts a protective or preventive effect on anit-induced acute liver injury. male wistar rats received a single i.p. injection of anit (75 mg/kg) and oral administraton of melatonin (100 mg/kg) at 12 or 24 h after anit injection. animals administered with melatonin at 12 and 24 h after anit injection were sacrificed 24 and 48 h, respectively, after the injection. liver injury appeared 24 h after anit injection and developed at 48 h. melatonin administered at 12 h after anit injection prevented liver injury formation with attenuation of increases in hepatic lipid peroxide level and myeloperoxidase activity, an index of neutrophil infiltration. melatonin administered at 24 h after anit injection prevented liver injury development with attenuation of further increase in hepatic lipid peroxide level. thus, melatonin protects against and prevents anit-induced acute liver injury in rats possibly through its antioxidant action and/or its inhibitory action against neutrophil infiltration in the liver tissue. k. okamura-ikeda, s. katayama, k. fujiwara, and y. motokawa t-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. mutation in human t-protein (ht) gene results in clinical nonketotic hyperglycinemia (nkh). eight point mutations have been identified so far in nkh patients with t-protein deficiency. to understand the structure and function of ht, the wild-type (wtht) and three mutant t-proteins (g47r, g269d and r320h) were expressed in escherichia coli with chaperons groel and groes which facilitated the recovery of the expressed proteins as a soluble form. levels of expression of these proteins were similar but the recovered soluble forms of mutants were about one-third of wtht. g47r showed comparable specific activity to wtht, whereas g269d and r320h mutants exhibited remarkable reduction in specific activity. since homoallelism for g269d mutation and heteroallelism for g47r and r320h mutation were identified in typical and atypical nkh, respectively, these results suggest that g269 and r320h mutations are highly deleterious in the aspects of not only protein folding and/or stability but also catalytsis. on the other hand, g47r mutation might affect mainly on the protein stability. detailed characterization of these mutants is now in progress. laboratory of animal nutrition and biochemistry, miyazaki university, miyazaki-shi, japan the minimal actinorhodin pks, shown in black below, consists of the ketosynthase (ks), chain length factor (clf) and acyl carrier protein (acp) and is the minimum set of enzymes required for polyketide production. we have investigated the stoichiometry of the ks-clf complex and the ks-clf:acp minimal system using three methods: 1. native gel electrophoresis. 2. cross-linking of proteins using dibromoacetone. 3. radical cross-linking of proteins. this new method has also been used with wild type s. coelicolor cell free extract with 10% ks-clf in order to elucidate which proteins are in close proximity to ks-clf during in vitro actinorhodin production. in ruminant animals, essential amino acids have never been completely established, because of the difficulty of its estimation due to the presence of microorganisms such as bacteria and protozoa in the first stomach called rumen. in our previous paper, histidine was shown to be the first limiting amino acid in the rumen contents when evaluated by chemical score. recently we have also reported that rumen microorganisms cannot synthesize histidine from histidinol. on the other hand, there have been some reports which showed that nitrogen balance of ruminants was not improved by supplementation of histidine to rumen microbial protein together with methionine, lysine and threonine which had been known to improve. based on these facts, we have a hypothesis that histidine may not be an essential amino acid for ruminants. in the present paper, we will report about the abilities of cattle liver and kidney to synthesize histidine from histidinol comparing with those of swine liver and kidney. the ability was demonstrated by examining the activities of histidinol dehydrogenase (crude enzyme) by means of direct measurement of an increase in histidine and decrease in histidinol. the amount of histidine produced from histidinol by the enzyme seemed sufficient for meeting the histidine requirement of cattle. the browning reaction is the sequence of events which begins with the reaction of amino group in amino acids, peptides or proteins with glycosidic hydroxyl group of sugars; the sequence terminates with the formation of brown nitrogenous compounds or melanoidines. this reaction gives rise to tremendous number of components such as volatile alcohols, ketones, aldehydes, esters, ethers and sulfur and nitrogen containing heterocycles in addition to nonvolatile amadori compounds and complex brown pigments of medium to high molecular weights. the present study was designed to choose a currently occurring system (aspartic acid -fructose) as a model system, since aspartic acid was found to be one of the most important amino acids in many kinds of food varieties. the reaction was done under controlled conditions of reactants ratios, temperature and time. the reaction mixtures were subjected to successive extractions with suitable solvents where the obtained corresponding flavour concentrates were thoroughly investigated. the results indicated different classes of compounds such as aldehydes, furans, alcohols and alkylated pyrazines varying in quantities depending on the reaction conditions. these products were also investigated concerning their toxicological effects. so, such products of nonenzymatic reactions showed different chemical and biological properties. purification and characterization p. piyarat 1 , s. nagata 2 , h. misono 2 , and k. packdibamrung 1 1 department of biochemistry, faculty of science, chulalongkorn university, bankok, thailand 2 department of bioresources science, faculty of agriculture, kochi university, nankoku, kochi, japan nad ϩ dependent alanine dehydrogenase was purified 100 fold to homogeneity from aeromonas hydrophila. molecular mass of 230,000 daltons was estimated for alanine dehydrogenase by sephadex g-200 chromatography. sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified en-zyme showed 1 polypeptide band with molecular mass of 40,000 daltons, indicating that the enzyme is hexamer. the enzyme is highly specific for alanine and nad ϩ . sulfhydryl group of the enzyme plays an important role in the catalysis. the enzyme retained its activity on heating at 55°c for 16 h. optimum ph for reductive amination and oxidative deamination were 8.0 and 10.5, respectively. the steady state kinetic studies including product inhibition on the enzyme reaction indicated that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism in which nad ϩ binds first to the enzyme followed by l-alanine and products are released in the order of pyruvate, ammonia and nadh, respectively. the k m values for nad ϩ , l-alanine, pyruvate, ammonia and nadh were 0.17, 20, 1.33, 77 and 0.25 mm, respectively. an elevation of apolipoprotein (apo) b-100 concentrations is a particular feature of several metabolic disorders, such as type 2 diabetes (t2d), impaired glucose tolerance (igt), and familial combined hyperlipidemia (fchl). to further understand the in vivo turnover of apolipoprotein b-100 of very low density lipoprotein subfractions (vldl 1 , svedberg units (s f ) 60-400 and vldl 2 , s f 20-60) kinetic studies were performed in subjects with t2d, igt, fchl, and healthy controls using a tracer of either l-[ring-13 c 6 ]-phenylalanine or l-[5,5,5-2 h 3 ]leucine. these studies showed direct hepatic vldl 1 apob-100 secretion to be increased in patients with t2d and igt when compared with controls. in contrast, patients with fchl showed a discrete increase in hepatic vldl 2 apob-100 secretion. in all patients vldl catabolism is not essentially impaired. vldl 1 apob-100 secretion is associated with plasma insulin and free fatty acid (ffa) concentrations, resp., whereas vldl 2 apob-100 secretion is correlated with plasma mevalonate and lathosterol levels. in conclusion, vldl overproduction is supposed to be completely responsible for higher triglyceride (tg) levels found in patients with t2d, igt, and fchl. vldl 1 overproduction seems to be regulated by tg and ffa substrate and appears to be an indicator of decreased insulin sensitivity. in contrast, vldl 2 overproduction is more likely to be regulated by the availability of cholesterol substrate. these data give further in vivo evidence that vldl 1 and vldl 2 secretion is regulated independently. arabidopsis resulted in enhanced production of cysteine and glutathione graduate school of pharmaceutical sciences, chiba university, chiba, japan serine acetyltransferase (satase) catalyzes the formation of o-acetyl-l-serine (oas) which is the key intermediate of cysteine biosynthesis. oas is not only a dominant limiting factor but recently suggested as a possible signal molecule for gene expression in cysteine biosynthesis. in has been shown that the activity of cytosolic satase from watermelon was feedback inhibited by l-cysteine. to enhance the ability of cysteine biosynthesis in plants and to reveal the role of oas in the regulation of sulfur assimilation, we made the point-mutated watermelon satase gene (satg277c) whose product was not inhibited by cysteine, and introduced satg227c into arabidopsis. the contents of oas, cysteine, and glutathione in transgenic arabidopsis were increased significantly as compared to the wild-type arabidopsis. we are currently dealing with the expression analysis of sulfur-related genes in transgenic arabidopsis accumulating oas due to the overexpression of satase. certain amino acids as source of specific branched chain fatty acids in fish sauce manufacture n. g. sanceda 1 , e. suzuki 2 , and t. kurata 1 1 institute of environmental science for human life, and 2 department of human biological studies, ochanomizu university, tokyo, japan the source of some branched volatile fatty acids (vfa) during the fermentation process in the manufacture of fish sauce was investigated. we previously reported that straight chain volatile acids seemed to have been derived from fish fats but unlikely for branched fatty acids which was believed to be derived from other sources. to clarify the source of branched volatile acids, specific amino acids, alanine, leucine, iso-leucine and valine were used in this study. these amino acids were first mixed with salt and added to fish. the fish mixtures were then aerobically and anaerobically incubated for one and a half months. results showed that addition of valine significantly increased the production of iso-butyric and iso-hexanoic acids and leucine increased that of iso-valeric in the aerobically fermented fish mixtures. a similar tendency was observed in the anaerobically fermented fish mixture except that an increase in the amount of iso-hexanoic acid was observed in the leucine added mixture, which was not observed in the aerobically fermented one. it seemed that specific branched volatile fatty acids were derived from certain amino acids. glutathione (gsh) is an important component of the cellular defense mechanisms that protect cells from oxidative injury. in the retina, the glial (müller) cells have been shown to synthesize and transport gsh, and thus are likely to be involved in regulating gsh levels. in the present study, we have characterized gsh transport system in a müller cell line using 35 s-gsh uptake. the results showed that gsh was taken up in a na ϩ -and concentration-dependent manner with a k m of 0.31 mm. moreover, cellular gsh had no effect on the rate of gsh uptake. in related studies, we found that oxidative stress induced the expression of γ-glutamylcysteine synthetase (gcs) subunits, and that gcs mrna levels were correlated with the degree of gsh depletion. because organic anion transporters (oatps) have been implicated in glutathione cotransport, we examined expression of oatp members using rt-pcr. we found that the müller cell line expressed transcripts for oatp1, oatp2 and oatp3. these studies indicate that the müller cell plays important role in gsh homeostasis in the retina. in the active site of human porphobilinogen synthase (ec 4.2.1.24, pbgs), two zinc ions are coordinated by cys 122 , cys 123 and cys 132 , and his 131 and cys 223 , respectively. the fomer zinc ion, closer to catalytic site lys 252 , plays an important role in catalysis. on the other hand, a role of the latter (distal) one has not been clarified. interestingly, in human hep3b cell, his 131 was replaced with arg (h131r). to elucidate the role of his 131 in catalysis, the kinetic properties of wild type and h131r mutant enzymes were studied. these cdnas were cloned by rt-pcr with total rna from human peripheral lymphocyte and hep3b cell, respectively. each cdna encoding pbgs with 3ј non-coding region was inserted into pet-22b(ϩ) vector and then the constract was transformed into e. coli strain bl21(de3). the cells were cultured in lb medium containing 50 mg/ml ampicillin and 10 µm zn ion for 3 h at 37°c. after addition of 1 mm isopropyl--d(ϫ)-thiogalactopyranoside, cells were further cultured for 24 h at 20°c. the highly purified pbgss were obtained by ultora centrifugion, fractionation with ammonium sulfate and column chromatographies with deaecellulose, hydroxylapatite and superdex 200, serially. we are now investigating molecular properties of these pbgss. agriculture and agri-food canada, lacombe research centre, lacombe, alberta, canada handling and management procedures such as capture and restraint can be significant stressors for recently domesticated animals such as elk (cervidae elaphus). the objective of the current study was to investigate the use of pre capture nutritional therapy in attenuating hpa response and improving animal welfare. fourty eight adult male elk stags ranging in age from 2-4 years and raised on pasture were used in the study with 23 as control and 25 as nutritionally treated. twenty four hours prior to capture the elk were offered either 1 kg of a cereal grain based dietary supplement or 1 kg of a cereal grain based nutritional therapy product containing specified amino acids (usa patent # 5505968). the amino acid content of the nutritional therapy product was minimally 0.5 g per 500 kg animal weight of ala, lys, phen, meth, thre, isoleu, val and tryp plus 15 g per 500 kg weight of leu and 40 g/500 kg weight of glut. the animals were subsequently captured and held in appropriate facilities designed to handle elk. saliva samples were collected on all animals immediately following capture and salivary cortisol was monitored by ria. animals offered the nutritional therapy product containing the amino acid mixture displayed lower cortisol levels (11.8 nmol/l) compared to the untreated controls (14.9 nmol/l; p ͻ 0.05). the data suggest that amino acid therapy can be used to attenuate hpa response to a stressor in captured elk. department of bioengeneering and technology, delhi, new delhi, india resistance to analogues of methionine by corynebacterium lilium results in the partial de-repression of methionine biosynthetic enzymes. the levels of enzymes involved in methionine biosynthesis also increased step-wise by successive endowing the resistant markers, resulting in the overproduction of methionine. moreover, the repressibilities of the enzymes were also reduced by the addition of methionine analogue resistance. analogue resistant mutants were developed by uv induced mutagenesis of corynebacterium lilium (wild type) strain. the single analogue (norleucine) resistant mutant c. lilium nl-87 produced 372 µg/ml methionine in shake flasks with methionine yield at 0.068 g methionine/g glucose and specific methionine production at 0.237 mg/g dcw, while double analogue (norleucine and triazole) resistant mutant c. lilium nt-33 produced 521 µg/ml methionine. a triple analogue (norleucine, triazole and ethionine) resistant mutant c. lilium nte-99 produced 1.848 g/l methionine. the methionine yield was 0.248 g methionine/g glucose and its specific productivity was 1.04 g methionine/g dcw. clinical biochemistry, laboratory 22, luxemburg, grand duchy of luxemburg blood plasma glucose level was compared on fast and 60 minutes after oral administration of 1200 mg of acetylcysteine. in the group of 49 healthy persons the plasma glucose level feel by 7.6% over the 60 minute period. in the 30 diabetics on the contrary, the plasma glucose level observed 60 minutes after administration of acetylcysteine was 11.8% higher than in blood plasma taken on fast. similar tests were carried out "in vitro" to interpret these different results. the control group consisted of 1 ml of distilled water ϩ0.2 ml 10% glucose ϩ0.2 ml god pad (boringer mannheim gmbh). in the acetylcysteine group the distilled water was replaced by 1 ml 0.01% solution of acetylcysteine. in the glucagon group the distilled water was replaced by 0.01% solution of glucagen hypokit novo nordisk. spectrometric determination was carried out after 60 minutes of incubation. a 27% diminution of glucose was observed in the acetylcysteine group in comparison with the control group. a 32.2% increase in glucose was observed in the glucagon group in relation to the controls. the results with healthy persons and the tests "in vitro" indicate that acetylcysteine lowers the level of glucose. but it elevates the level of glucose in the blood plasma of diabetics. it may be presumed that acetylcysteine modifies the insulinglucagon balance in favour of glucagon. the objective of this study was to fortify yogurt with three oilseed protein hydrolysates prepared from soybean (glycine max), sesame (sesanum indicum) and rice bran (oryza sativa) flours. hydrolysis was carried with two enzymes one of plant origin (papain) and the other of microbial origin (alcalase). a yogurt fortification experiment was then carried using the previous hydrolysates. the hydrolysates were added to yoghurt at 5, 10 and 15% levels of fortification and the fortified yoghurt was analyzed fresh, and after 7 and 15 days of consuming period. fortified yogurt was chemically examined for fermentation activity (ph values, acidity and proteolysis) as well as its organoleptic properties. results of this experiment indicate that the addition of soybean hydrolysates with papain (8.9 units/g) for 5 minutes (tb) and rice bran hydrolysates with alcalase (27.6 units/g) for 5 minutes (te) to yoghurt can ex-ceed 5-10%, while fortification with sesame hydrolysed with papain (8.9 units/g) for 30 minutes (td) and soybean hydrolysed with papain (8.9 units/g) for 30 minutes (tc) can not reach up to 15%. it is well known that dna is fragile to reactive oxygen intermediates (rois) damage. evidences that dna fragmentation and apoptosis occur in cardiomyopathies, in the failing heart and in cultured cells under hyperbaric oxygen (hbo) stress, demonstrated that oxygen free radicals also play a critical role in heart failure. as a consequence, myocardial cell survival depends on response to oxidative stress. experimental data obtained in vitro suggested that polyamines, by acting as rois scavengers, play a role in prevention of endonucleasemediated dna fragmentation and inhibition of alkylating agents-mediated damage, potentially exerting a protective role against rois damage. thus we studied polyamine metabolism and superoxide dismutase (sod) expression in an in vivo model of heart oxidative stress, such as rats subjected to hbo. four experimental groups were used: 1) controls; 2) rats subjected to hbo for 15 min once and immediately sacrificed; 3) rats treated as group 2 but for 3 consecutive days and immediately sacrificed; 4) rats treated as group 3 but sacrificed 24 h later (recovery). northern blot analyses showed that odc mrna accumulation increased immediately (paralleled by activity) in groups 2-4, while ssat mrna decreased remarkably, thus leading to higher polyamine concentration in rois-stressed hearts. contrariwise, sod mrna level decreased rapidly in groups 2-4. this suggests that hbo-induced compensatory mechanism in rat heart is based on specific and rapid boosting of polyamine concentration, caused by coordinate induction of biosynthesis and inhibition of catabolism, and not of enzymes known to metabolise rois such as sod. amino acids oxidation was greater in tumor-bearing rats muscle. leucine is an important ketogenic amino acid that proves energy to the skeletal muscle. leucine supplemented diet was used to analyze the effects produced by walker 256 growing in pregnant rats which were distributed into six groups. three groups received normal diet (18% protein): control (c), tumor-bearing (w), pair-fed rats (cp). three groups were fed with diet supplemented with 3% leucine (15% protein plus 3% leucine): pregnant fed with leucine (l), tumor-bearing with leucine (wl) and pair-fed with leucine (lp). after 21 days, the animals were submitted to intestinal perfusion to measure leucine, methionine and glucose absorption. leucine absorption increased in w and wl groups. glucose absorption reduced in tumor-bearing. in pregnancy with cancer, metabolic changes provided both reduced fetal and tumor development. tumor-bearing rats showed increase in methionine and leucine absorption, probably diverting this nutrients to tumor cells. glucose absorption reduced in w and wl. leucine supplemented diet group promoted high leucine absorption which could be used by neoplasic cells, and mainly by fetus and host. probably, the transamination of the branch long chain amino acid provided energy substract for the skeletal muscle, keeping the nitrogen offered to host carcass. ( undernutrition cause several changes as body weight loss, in biochemical parameters, even microscopic alteration in absorptive epithelium. this means the nutrients absorption process has been harmfully and consequently increase the damages caused by malnourished. knowing leucine is used as a ketonic and oxidative amino acid our main propose was to recovery the malnourished young rats with normal (rc) and leucine supplemented diet (rl, 3% of leucine) for 60 days. it was measured body, liver, and muscle weight, intestinal absorption of glucose, methionine and leucine, and body chemical composition. the body weight gain in rc and rl was higher than control group, suggesting that nutritional replacement for these groups could provided nutrients to support the body weight recovery, reaching as the same weight as the control. methionine and glucose absorption was reduced in malnourished group, but it was recovered (glucose, methionine and leucine) after nutritional replacement. leucine supplemented diet promoted a good recovery of carcass collagen nitrogen, keeping the carcass structural nitrogen. further studies are necessary to investigate this mechanism. [financial support: fapesp ( we diagnosed the very rare autosomal recessive disorder hyperprolinaemia type ii (deficiency of ∆ 1 -pyrroline-5carboxylate dehydrogenase, ec 1.5.1.12) in a girl aged 20 months presenting with seizures and encephalopathy. l-∆ 1pyrroline-5-carboxylic acid accumulates in this disorder and there is a 10-15-fold increase in plasma proline. surprisingly, she also had vitamin b 6 deficiency. this was an unrecognised association, which was not explained by her diet or medications. we hypothesised that pyridoxal phosphate (vitamin b 6 coenzyme) was de-activated by l-∆ 1 -pyrroline-5-carboxylic acid. with high resolution 1 h nuclear magnetic resonance spectroscopy and mass spectrometry, we have shown that these two compounds react at ph 7.4 and 37°c in vitro to form three novel adducts, which we characterised. they are products of a claisen condensation (or knoevenagel type of reaction) of the activated c4 carbon of the pyrroline ring with the aldehyde carbon of pyridoxal phosphate. if this previously unreported interaction occurs in vivo, pyrroline-5-carboxylic acid is a unique endogenous vitamin antagonist. preliminary observations show that pyrroline-5-carboxylic acid also condenses with other biologically important aldehydes and ketones. some of these reactions may contribute to the brain disturbances in hyperprolinaemia type ii. we have already identified adducts with acetoacetic acid in urine from our child, which is evidence that condensation can occur in vivo. the kidneys are characterized by a high activity of γglutamyl transpeptidase (γ-gt), as well as by a high cysteine level. the present paper was aimed to obtain information on how the activity of γ-gt and the levels of non-protein sulfhydryl compounds (npsh) changed with age in rat kidneys. simultaneously, protein-bound cysteine (pb-cys) and sulfane sulfur compounds were estimated. the kidneys were from following rats groups: young (3-month-old), middle-aged (19month-old) and old (31-month-old). the obtained results showed that the activity of γgt and npsh levels in the kidneys fell with age. at the same time, a significant increase in the level of protein-bound cysteine was observed. on the other hand, the content of sulfane sulfur compounds was elevated in the group of the oldest animals. these findings indicate that -due to disturbances in the γ-glutamyl cycle -the capacity for extracellular glutathione degradation and, in consequence, the availability of cysteine for intracellular gsh biosynthesis may be impaired. the increased pb-cys level indicates potentiation of the thiolation reaction, i.e. development of protein-mixed disulphides, cysteine, sulfane sulfur compounds, oxygen reactive species. national research centre, dokki, cairo, egypt in the past few years, many attempts have been made to prepare a synthetic insulin. the biological activity of insulin is known to be closely related to the c-terminal octapeptide fragment of its b-chain. this does not necessarily mean, however, that each of the amino acid residues of the octapeptide fragment is essential for its activity. it was found that b 23 gly and b 24 phe were present in all insulins so far obtained from various animal species indicating the significance of these two residues. it would therefore seem desirable to study the effect of each of these two amino acid residues or both on biological activity of the octapeptiede fragment of the b-chain. weitzel et al. found that the substitution of arginine b 22 with another amino acid resulted in a very large decrease in biological activity, which indicates that it participates in the action of insulin. also it was found that the aromatic amino acid residues (b 24 -b 25 ) participate in the action of insulin. a heptapeptide arg-phe-tyr-thr-pro-lys-ala-och 3 , corresponding to (b 22 -b 30 ) insulin des gly 23 -phe 24 , and an octapeptide arg-phe-phe-tyr-thr-pro-lys-ala-och 3 , des gly 23 were synthesized using the solid phase method. the c-termenal ends of both peptide were converted to methyl ester by transesterification cleavage from the resin. the side chain protecting groups were removed by hf. manual counter current distribution method was used for purification of the free peptides. the way to solve the evaluation of tyrosine containing peptide was studied. the free methyl ester peptides were administrated for insulin-like activity test by glucose metabolism in the rat fat cells technique in vitro. nitric oxide synthase inhibitors influence dynorphin immunoreactivity in the rat brain following hyperthermia p. alm 1 and h. s. sharma 2 1 department of pathology, university hospital, lund university, lund, and 2 laboratory of neuroanatomy, department of medical cell biology, biomedical centre, uppsala university, uppsala, sweden nitric oxide (no) is a free radical gas that influences neuronal communication in the central nervous system (cns). recent reports suggest that no can influence dynorphin neurotransmission in the normal brain as well as in several pathological states. previous reports from our laboratory show that the enzyme nitric oxide synthase (nos) responsible for no formation is upregulated in several brain regions following hyperthermia. the present investigation was carried out to find, whether hyperthermia can influence dynorphin immunoreactivity in the brain, and if so, whether inhibition of nitric oxide synthesis will alter its distribution in heat stressed rats. rats subjected to hyperthermia at 38°c for 4 h in a biological oxygen demand incubator (bod) resulted in marked redistribution of dynorphin immunoreactivity in several brain regions e.g., cerebral cortex, hippocampus, cerebellum and brain stem. pretreatment with two potent nos inhibitors, l-name (50 mg/kg, i.p.) and l-nmma (30 mg/kg, i.p.) 30 min before heat stress significantly altered the dynorphin immunoreactivity in the brain. these drugs alone however, did not influence the peptide expression in normal rats. the results suggest that (i) hyperthermia has the capacity to influence dynorphin immunoreactivity in the brain, and (ii) inhibition of nitric oxide synthase considerably influences the dynorphin immunoreaction in hyperthermia, not reported earlier. the functional changes induced by uncompetitive and competitive nmda antagonists, memantine, amantadine and mk-801, and cgp 40116, respectively, were studied in both saline-pretreated and mptp-pretreated c57 bl/6 mice. the nmda antagonists were administered acutely by themselves or in combinations of either: nmda antagonist plus subthreshold l-dopa dose or nmda antagonist plus suprathreshold l-dopa dose, to either the mptp-pretreated or the salinetreated mice. activity-enhancing or functional restorative effects of the nmda antagonists were variable with memantine and mk-801 distinguished from amantadine and cgp 40116. in the study of long-term effect of nmda antagonists mk-801 was administered postnatally and spontaneous motor behaviour and motor activity in response to several pharmacological interventions was assessed. marked alterations associated possible with apoptotic penchance are discussed. t. archer 1 and a. fredriksson 2 1 department of psychology, university of göteborg, and 2 department of psychiatry, university of uppsala, ulleråkers hospital, uppsala, sweden synergistic antiparkinsonian actions of different classes of putative therapeutic agents co-administered with a subthreshold dose of l-dopa (5 mg/kg) in drug-naive mptp-treated mice as well as the restorative actions of those compounds in suprathreshold l-dopa-tolerant mptp-treated mice subjected to "wearing-off" of l-dopa efficacy were assessed in a series of experiments. the classes of compounds studied included the noncompetitive nmda antagonists, memantine, amantadine and mk-801, the anticonvulsive and putative anticonvulsive agents, lamotrigine, fce 26743, phenytoin, the monoamine oxidase inhibitors, l-deprenyl, amiflamine, α-ethyltryptamine, clorgyline and phenelzine, and the α 2 -adrenoceptor agonists, clonidine and guanfacine. in this final case, the restorative effects of clonidine and guanfacine were antagonised by the α 2 -adrenoceptor antagonist, yohimbine, but not the α 1adrenoceptor antagonist, prazosin. within each class of potentially therapeutic agents a differential restorative efficacy was obtained, but the combination of different doses of apomorphine with clondine failed to restore motor activity. in vivo proton mr-spectroscopy of the human brain: assessment of n-acetylaspartate (naa) reduction as a marker for neurodegeneration w. block 1 , f. träber 1 , s. flacke 1 , f. jessen 2 , ch. pohl 3 , and h. h. schild 1 1 department of radiology, 2 department of psychiatry, and 3 department of neurology, university of bonn, germany proton magnetic resonance spectroscopy ( 1 h-mrs) is a well accepted non-invasive method to investigate changes in brain metabolite composition in different types of cerebral disease. we performed proton spectroscopy in patients with dementia of the alzheimer's type (ad) and in patients with motor neuron disease (mnd) with the aim to detect a specific metabolic pattern for each of these two neurodegenerative disorders. overall, more than 150 spectroscopic data sets of patients with mnd and more than 100 data sets of ad patients were acquired within the last 5 years. in the mnd group we found a significant reduction of naa/tcr metabolite ratios in the central region, which correlates with the disease severity and the clinical lateralisation of neurological symptoms and increases in the time course of the disease. in ad patients a similar reduction in relative naa contents was observed in the medial temporal lobe. the observed regional metabolic alterations correlate well with the characteristic neurological symptoms in ad (dementia) and mnd (muscular palsy) and seem to follow the disease process over time. since naa is exclusively expressed in neurons as shown by immunohistochemical studies, reduced naa levels suggest neuronal loss or dysfunction in the observed regions. center for molecular imaging research, massachusetts general hospital, boston, massachusetts, u.s.a. non-invasive measurement of hemodynamic parameters and imaging neovasculature architecture during angiogenesis is highly important in determining tumor prognosis and in assessing treatment efficacy. we suggested a technique to map the tumor vascular (vvf) and interstitial volume fraction (ivf) noninvasively in vivo. a poly-l-lysine based macromolecular probe (mpeg-pl-gddtpa) with extended circulation in the bloodstream designed to shield chelated paramagnetic lantanide with poly(ethylene glycol) chains. we hypothesized that a magnetic resonance signal after intravenous administration of a vascular paramagnetic probe can be maximized so the signal change after administration of a second comound (gddtpa) reflects the ivf but not the vvf. the method and its assumptions were verified in animal models of cancer. tumoral vvf and ivf values were consistent with histology data and literature values. imaging showed heterogeneity of both parameters at submillimeter pixel resolution. this technique was used for characterizing differential angiogenesis in human mammary adenocarcinoma lines as well as for imaging anti-angiogenic drug effects. anti-angiogenesis was induced using synthetic d-reverse peptides derived from thrombospondin-1. this study showed that peptide treatment results in slower brain tumor growth due to inhibition of de novo blood vessel formation and synergistic anti-proliferative effect on tumor cells. in conclusion, in vivo mr imaging can be used for non-invasive treatment assessment of novel antiangiogenic drugs. wallenberg neuroscience centre, lund university, lund, sweden we have recently found that 6-hydroxydopamine lesioned rats gradually develop dyskinetic-and dystonic-like movements upon repeated administration of a therapeutic dose of l-dopa. such movements simulate the time course of peak-dose dyskinesia in parkinson's disease. in this rat model, the severity of l-dopa-induced dyskinesia is strongly correlated with an upregulated expression of the prodynorphin gene in striatal neurons. using antisense technology and gel-shift assay analyses, we have addressed the role of transcription factors which may mediate this response. we have found that the camp response-element binding protein (creb) is essential in maintaining a basal expression of prodynorphin mrna in the intact striatum, but it is not required for l-dopa to induce the prodynorphin gene in dopamine-denervated striatal neurons. we have thus addressed the role of fos-and jun family tran-scription factors, and found very high levels of fosb-and jundlike proteins in the striata of dyskinetic animals. these proteins could bind to both ap1 and cre sites in the prodynorphin promoter. moreover, intrastriatal fosb knockdown could inhibit both the upregulation of prodynorphin gene expression and the development of dyskinesias under chronic l-dopa treatment. we propose that dimers of fosb-and jund-like proteins mediate abnormal changes in striatal gene expression which are linked to the development of l-dopa-induced dyskinesia. department of pharmacology, grünenthal gmbh r&d, aachen, germany glutamate plays important roles in both normal and pathophysiological nocieception. upon physiological conditions, glutamate release from primary afferents in the spinal cord activates largely ampa receptors. as those are ubiquitously involved in fast transmission in the cns, ampa antagonists have a broad side-effect profile. prolonged activation of nociceptors by tissue damage, inflammation or nerve injury evokes a long-lasting release of glutamate and neuropeptides, activating nmda receptors in the spinal cord. this mechanism appears to play a key role in pain chronification. the nmda receptor is, therefore, an important target for chronic pain treatment. both animal and human studies confirm the efficacy of nmda antagonists in chronic pain, however, clinically available compounds are weak or have unacceptable side-effects. glycine b antagonists and compounds selectively blocking nr2b-containing receptors appear to be safer, the reasons for this remain unclear. central side-effects could potentially be avoided by using nmda antagonists with restricted central access. peripheral nmda receptors (as well as some other subtypes of glurs) could be activated by glutamate released from the site of injury, thus contributing to peripheral hyperexcitability. some other subtypes of glurs can also contribute to peripheral sensitisation. of ionotropic glurs, kainate receptors appear important in inflammatory and neuropathic pain. they can be activated by high intensity stimulation of nociceptive afferents, and may act as autoreceptors controlling release of glutamate. group i metabotropic glurs are also present on primary afferents and on second order neurones in the spinal cord, and may play a similar role. antagonists of these subtypes of glurs are active in some models of chronic pain. specific upregulation of group ii metabotropic glurs in some pain-relevant structures could reflect a possible adaptive role of these inhibitory receptors under chronic pain conditions; their selective agonists also have a potential for the treatment of chronic pain. we have performed a series of studies of the distribution and function of mglur subtypes in the basal ganglia that suggest that members of this receptor family could serve as targets for novel therapeutic agents that would be effective in treatment of pd. for instance, two group ii mglurs (mglur4 and mglur7) are localized on presynaptic terminals of striatal neurons in the globus pallidus where they could reduce gaba release. furthermore, activation of group i mglurs results in a depolarization and increased cell firing of neurons in the subthalamic nucleus (stn) and projection neurons of the substantia nigra pars reticulata (snpr). interestingly, this effect is mediated by mglur1 in snpr projection neurons and mglur5 in stn neurons. finally, activation of group ii mglurs results in inhibition of glutamate release from stn terminals in the snpr. furthermore, selective agonists of group ii mglurs inhibit haloperidol-induced catalepsy in rats, suggesting an antiparkinsonian effect of these compounds. the rich distribution and diverse physiological roles of mglurs in basal ganglia raises the possibility that these receptors may provide targets for novel therapeutic agents that could be used for treatment of pd and related disorders. a. cupello 1 , m. parodi 2 , and m. balestrino 2 1 centro di neurofisiologia cerebrale, cnr, genova and 2 dipartimento di scienze neurologiche, university of genova, italy in vitro rat hippocampal slices are commonly used to study the effects of hypoxia in the central nervous system, because they allow to differentiate the effects of hypoxia in the brain from that of systemic (e.g., respiratory and cardiac) failure that may accompany hypoxia. we used electrophysiology to monitor and evaluate the damage caused by transient hypoxia to the nervous tissue. a few minutes after oxygen deprivation brain tissue suddenly depolarizes. this event, which is termed ìanoxic depolarizationî is accompanied by dramatic metabolic changes: transmembrane ionic gradients disappear (na ϩ enters, k ϩ exits the neurons), neurons swell, there is intra-and extra-cellular acidosis. this event is caused by functional inactivation of (na ϩ / k ϩ )atpase caused by decreased atp content, as it is suggested by the fact that it is mimicked by ouabain treatment. one of us has contributed to show that if this event is not promptly reversed by reoxygenation it causes irreversible damage, mainly by determining a massive entry of ca 2ϩ into neurons. pretreatment of tissue with creatine (1 mm or more) both increases neuronal energy store by increasing neuronal phosphocreatine and protects brain tissue from irreversible damage. in vivo increase in phosphocreatine has been shown using lower (0.5 mm) creatine concentration, injected directly into the lateral ventricle. a different type of hypoxia-induced damage is observed when hypoxia is of shorter duration. in this case upon reoxygenation one does not observe disappearance of evoked potentials but their increase. this phenomenon, originally described as ìpost-hypoxic hyperexcitabilityî has been later called ìanoxic long-term potentiation (ltp)î. we showed that this event can be prevented by inactivating the nuclear protein s-100. while this damage is milder than that induced by anoxic depolarization, it may explain stroke-induced epileptic fits. we are currently investigating what role, if any, pretreatment with creatine may have in preventing also this type of damage. cytoskeleton is subject to continuous modification to yield changes in cell shape and function of plasmamembrane proteins linked to the cytoskeleton. gelsolin (gsn) depolymerizes filamentous actin and thus causes dynamic uncoupling of membrane ion channels. we have studied alteration of neuronal ca 2ϩ influx by the absence of gsn and its pathophysiological consequences during cerebral ischemia. cytosolic ca 2ϩ concentrations were determined ratiometrically in synaptosomes preloaded with fura-2am. glutamate release from synaptosomes superfused with krebs' buffer was measured by hplc. transient focal cerebral ischemia was induced by 2 h occlusion of the middle cerebral artery (mca). in gsn deficient mouse brain cortical synaptosomes [ca 2ϩ ] i increase in response to k ϩ (30 mm) depolarization was 42% higher than in wild-type. ω-agatoxin iva 0.2 µm decreased ca 2ϩ -influx in neocortical wild-type synaptosomes by 53%, and abolished differences between gsnϩ/ϩ and ϫ/ϫ genotype. k ϩinduced release of glutamate in neocortical synaptosomes was 78% higher and lesion size after mca occlusion was 45% higher than in wild-type. it is concluded that presynaptic ca 2ϩ influx is increased in gsn deficient nerve terminals which, together with subsequently increased glutamate release, increases neuronal vulnerability. in vivo assessment of tissue alteration in cerebrovascular and neurodegenerative diseases s. flacke, w. block, f. träber, p. mürtz, h. urbach, and h. schild the combined used of perfusion imaging (pi) and molecular diffusion imaging (dwi) are opening a new window into the processes that occur during the first hours of ischemia. dwi detects early changes of proton diffusion associated with cytotoxic edema. pi has the potential to characterize the degree of regional hypoperfusion. mismatches between dwi and pi, i.e. hypoperfused areas with normal adc are considered potentially salvageable. we present results of 11 patients with an angiographically defined thrombembolus in the middle cerebral artery and a spontaneous stroke evolution. whereas the infarct core was clearly visible on both dwi and pi, tissue at risk of infarction could only be detected by an increased blood volume and transit time. however only in a subgroup of patients (n ϭ 3) these areas were incorporated into the final infarct. in these patients perfusion parameter of tissue at risk of infarction were more pronouncedly altered than in those where the tissue at risk was spared from infarction (ratios of tissue at risk vs normal (rcbv 2.17 ϯ 0.59, mtt 1.67 ϯ 0.22, ttp 1.32 ϯ 0.11, p ͻ .05). these human data show that a detailed analysis of diffusion/ perfusion mismatches allow the identification of tissue at risk of damage. glucose deprivation enhances the sensitivity of cerebellar granule cells to die by excitotoxicity. we have previously reported that neither 70 min of glucose deprivation, a treatment that depletes cell energy resources, nor exposure to 20 µm glutamate (glu) for 30 min, induce significant cell death in cerebellar granule cell cultures, 24 h after treatment. in contrast, the combined treatment with 20 µm glu and glucose deprivation induces both cell death and choline (cho) release. we investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (pc) synthesis. we found that exposure to 20 µm glu alone for 30 min, glucose deprivation for 70 min, and the combination of both treatments inhibited pc synthesis when measured at the end of treatment by 71%, 92% and 91%, respectively. furthermore, we found that exposure to either 20 µm glu, glucose deprivation or 20 µm glu ϩ glucose deprivation decreased incorporation of [ 3 h]cho into phosphocholine and increased the intracellular content of free [ 3 h]cho, indicating that all these treatments inhibit the synthesis of pc by inhibiting choline kinase activity. since only the combined treatment with 20 µm glu plus glucose deprivation evoked cho release and excitotoxic cell death, the present results indicate that other factors in addition to inhibition of pc synthesis are required to induce cho release and excitotoxic cell death in cerebellar granule cells. (supported by cicyt, saf98-0063.) the role of striatal metabotropic glutamate receptors in degeneration of dopamine neurons k. goĺembiowska, j. konieczny, k. ossowska, and institute of pharmacology, polish academy of sciences, kraków, poland the present study was undertaken to characterize the effect of blockade of the mglu 5 receptor subtype by 2-methyl-6-phenylethynylpyridine (mpep), as well as the effect of stimulation of the mglu 2/3 receptor by (ϫ)-2-oxa-4aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid (ly379268) on spontaneous and stimulated dopamine (da) release in rat striatum using an in vivo microdialysis. mpep (100-500 µm), perfused through a microdialysis probe affected neither the basal nor the veratridine (100 µm)-stimulated striatal da release. however, mpep given intraperitoneally (5 mg/kg) diminished either the basal or the veratridine-evoked da release. ly379268 (100-250 µm) administered locally also inhibited the veratridine-evoked da release in rat striatum. antagonists of mglur-i and agonists of mglur-ii have been shown to have neuroprotective properties in several models of neurotoxicity in animals. we have approached this issue using a selective mglu 5 antagonist in an animal model of neurotoxicity induced by methamphetamine. in our preliminary experiments, methamphetamine (5 ϫ 10 mg/kg sc every two hours) decreased the tissue content of striatal da and its metabolites dopac and hva.mpep (5 ϫ 5 mg/kg ip) given before every methamphetamine injection reversed its action. the effect exerted by the mglu 5 antagonist mpep seem to be mediated by sites located outside the striatum due to relieving da neurons of the facilitatory influence of glutamate. in turn, the attenuation of da release from nigrostriatal terminals by ly379269 may be a consequence of activation of striatal mglu 2/3 receptors. reversal of the methampetamine-induced da depletion suggests a potential for neuroprotective activity of mpep. o. golubnitschaja 1 , h. h. schild 1 , and j. flammer 2 1 department of radiology, university of bonn, germany 2 university eye clinic, basel, switzerland glaucoma remains a major eye illness with unknown etiology. although elevated intraocular pressure has been shown to be the major risk factor, there is a cohort of relatively young patients developing normal-tension glaucoma (ntg). assymptomatic ischemic events in brain have been shown to be often attributable to galucoma. perfusion of the retina and optic nerve head suffering from observed vasospastic dysfunction may be further reduced by changes in the intraocular pressure. ocular ischemia developed due to these blood flow deficits may play a major role in initiation of glaucoma. possibly secondary to ischemia the autoimmunogenic capacity is activated by ntg patients having an increased prevalence of systemic autoimmune diseases. therefore, the determination of potential molecular markers in blood lymphocytes could be useful for early diagnostics of ntg. our recent study using "gent hunting"-techniques showed indeed altered gene expression in lymphocytes of ntg patients. the demonstrated downregulation of xpgc gene expression which subsequently leads to the accumulation of damaged dna and an elevated p53 expression, together with the upregulation of a new abc-transporter seem to be specific for the pathogenesis of ntg. molecular imaging of ntg provides insights in mechanisms of disease initiation and allows the early diagnostics and preventive treatment. (supported by "bio-rad" and "amersham pharmacia aggregate cell cultures prepared from fetal rat telencephalon were used to study neuronal amino acid consumption during glucose restriction. to that end, both mixed (neuronglia) and neuron-enriched cultures were grown in chemically defined medium and tested at an advanced maturational stage. it was found that 6 h of exposure to reduced glucose (0.25 mm instead of 25 mm) caused significant increases in the consumption of several amino acids and the accumulation of ammonia. it also greatly changed the intracellular level of several amino acids in neurons, particularly of aspartate and glutamate. irreversible neuron-specific damage was observed one week after the insult. elevated glutamine media concentrations (4 mm instead of 0.25 mm) during glucose restriction further increased ammonia production and neuronal damage, although the overall rate of glutamine metabolism remained practically unchanged. taken together, our findings suggest that glucose deficiency caused (i) the dysfunction of crucial transamination pathways; (ii) a shift towards the oxidative deamination of glutamine and several other amino acids used by neurons as alternative energy substrates; and (iii) the accumulation of neurotoxic ammonia levels. institute for brain research, university of vienna, austria the racemic (d,l) mixture of the naturally occurring neutral aromatic amino acid 3,4-dihydroxy-l-phenylalanine (l-dopa) was first synthetized in 1911. in 1913, the natural levorotatory isomer was isolated from vicia faba beans and declared to be biologically inactive. however, in 1930 l-dopa was observed to lower the blood pressure in the rabbit, an effect opposite to the vasopressor effect of adrenaline. following the discovery, in 1938, of the enzyme l-dopa decarboxylase, ldopa's conversion in tissues (by decarboxylation) to dopamine (da), the first biologically active substance in the biosynthetic pathway of catecholamines, was demonstrated. subsequent pharmacological studies, done between 1942 and 1957, showed that the biological actions of l-dopa were, in principle, due to da formed from it in the body. in 1957, the central antireserpine effect of d,l-dopa was described in mice and confirmed in 1960 with l-dopa in humans. following the demonstration of da's occurrence in the brain in the years 1957/58, d,l-dopa was found (in rabbits) to restore brain da levels, reduced by reserpine. in 1960, the severe brain da deficit, confined to patients with parkinson's disease (pd) was reported and a year later l-dopa's superior anti-akinesia effect in patients with pd demonstrated. finally, in 1967 the high-dose oral l-dopa regimen was successfully introduced into clinical practise. in contrast to these supreme achievements, two related early studies remained, for different reasons, without consequence. despite some initial doubts about its mechanism of action, there is now convincing evidence for l-dopa therapy being a classic example of a central neurotransmitter replacement therapy, with the severe brain da deficit furnishing a rational basis for the amino acid's clinical use and high efficacy in patients suffering from pd. b. jakobsen 1 , a. tasker 2 , and j. zimmer 1 1 anatomy and neurobiology, sdu-odense university, odense, denmark 2 department of anatomy and physiology, university of prince edward island, canada the toxicity of domoic acid (dom) was studied in rat hippocampal slice cultures, prepared from 7-days old rats and grown on semipermeable membranes for 2-3 weeks before exposure. dom (0.1-100 µm) was added to the medium, alone or together with the glutamate receptor antagonists ns-102, nbqx or mk-801, for 48 hrs followed by 48 hrs in normal medium. neuronal degeneration was monitored and ec 50 values estimated by densitometric measurements of the cellular uptake of the propidium iodide (pi) at 24, 48, 72 and 96 hrs. the lowest ec 50 values, obtained at 72 hrs, were: ca1 (6 µm), dentate granule cells (dg) and ca3ab (10 µm),ca3c (12 µm). protective effects of 10 µm nbqx at 72 hrs were seen against 3 µm dom in dg, ca1 and ca3c and against 10 µm dom in ca1 and ca3c. 10 µm ns102 and mk801 only displayed protective effects together with nbqx. mk801 thus significantly increased the protective effect of 10 µm nbqx in ca1 against 10 µm dom in combination with 10 µm nbqx and 10 µm ns102. we can confirm that dom neurotoxicity primarily involves ampa/kainate receptors, but also nmda receptors at high concentrations (glutamate release). department of physiology/neuroscience, medical university of south carolina, charleston, south carolina, u.s.a. although dopamine has been most clearly tied to the development of addiction to drugs of abuse, recent studies indicate that once addiction has been established the expression of addictive behaviors, such as drug craving, is mediated more by long-term neuroadaptaions in glutamate transmission. data will be presented which supports and extends this hypothesis. repeated cocaine injections were given for one week and three weeks after the last drug injection a number of molecular, neurochemical and behavioral neuroadaptations were measured. it was found that in the nucleus accumbens there is a increase in the expression of genes encoding mglur2/3 and glur1, and a decrease in the expression of mglur5 and its accompanying scaffolding proteins homer1bc. this was accompanied by an increase in the capacity of mglur2/3 receptors to regulate presynaptic glutamate release and a blunting in the effects of stimulating mglur1/5 receptors. in addition, there is reduced activity in the cystine/glutamate exchanger 3 wks after repeated cocaine. as a result of these changes there is a decrease in the basal release of glutamate, and a relative increase in the releasibility of glutamate upon stimulation. by using the reinstatement model of drug seeking behavior, it was shown that glutamate transmission in the projection from the prelimbic cortex to the core of the nucleus accumbens was particularly affected by the cocaine-induced changes in gene expression. taken together, these findings support the use of glutamate autoreceptor agonists as possible therapeutic adjuvants in treating the cravings associated with addiction. dopamine (da), a catechol that autoxidizes to an oquinone, is implicated as an endogenous pro-toxin, however, the following studies suggest that da has dual neurodegenerative and neuroprotective roles. in rats treated as neonates with 6-hydroxydopamine (6-ohda; 134 :g icv), there was a 99% reduction in striatal tissue da content in adulthood, and a 3 to 5fold increase in spontaneous hydroxyl radical (ho*) formation (indirect salicylate trapping method: dihydroxybenzoic acid analysis). additionally, systemic l-dopa (60 mg/kg i.p.) suppressed ho* formation. however, when glutamate (50 mm) was added to an in vivo microdialysate, ho* formation was increased substantially more in the microdialysate from dainnervated striatum. these findings indicate that da innervation is inherently neuroprotective, but in the presence of a high level of an excitatory amino acid, da innervation predisposes to formation of reactive oxygen species. ongoing neuronal activity is likely to interact with and to determine the role of da as a neurotoxic or neuroprotective substance. (supported by ns 39272.) the glutamate hypothesis of schizophrenia along with the dopamine hypothesis was intensively discussed in the past. the last years however suggest more and more that neither a hypofunction of the glutamatergic system alone nor a hypofunction of the dopaminergic system alone is responsible for symptoms found in schizophrenia. the basal ganglia (bg) as the critical structures mediating symptoms of schizophrenia are innervated by dopaminergic fibers from the mesencephalon as well as by glutamatergic fibers from limbic structures; like prefrontal cortex, hippocampus, entorhinal cortex and amygdala. thus, limbic input is able to modulate information processing in each structure of the bg and by this way control dopaminergic functions through feedback mechanisms. dysfunction in limbic structrues may result in an imbalance of information processing via the bg and terminates in behavioral symptoms of schizophrenia. we showed in recent neurochemical studies in combination with behavioral analysis that a simple, generalized hypofunction of limbic glutamatergic input on bg nuclei is not the key mechanism inducing schizophrenic behavior. a dysfunction of a particular limbic structure or pathway seems to be responsible for an imbalanced information processing via the bg and imbalanced behavioral adaptation terminating in schizophrenic symptoms. [ there is a need to identify subtype-specific ligands for mglu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. to date, most mglur antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular n-terminal domain. we have investigated novel subtype-selective mglur5 antagonists which are structurally unrelated to competitive mglur ligands. using a series of chimeric receptors and point mutations we demonstrate that these antagonists interact with novel allosteric binding sites in the tm domain via a noncompetitive mechanism of action. recent studies in animal models implicate mglu 5 receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety. vascular endothelial growth factor (vegf) is a major mediator in angiogenesis and vascular permeability. in central nervous system (cns) vegf plays pivotal roles such e.g., inductor of endothelial cell proliferation, migration and inhibition of apoptosis, as well as mediator of blood brain barrier (bbb) breakdown and subsequently of brain edema formation. these ubiquitous epiphenomena are major complications in several cns pathologies, including head trauma and stroke. reduced tissue oxygen tension (hypoxia) and hypoglycaemia triggers vegf expression that occurs in ischemic regions around postraumatic or postinfarct necrosis. after brain injury, the expression of vegf is increased contributing to disruption of the bbb. vegf increases the permeability of bbb via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. the immunohistochemistry shows an increase of stained astrocytes around a cortical micronecrosis. vegf participates in the response of the cns to injury in a dose dependent way. immunostaining correlates with infarct volume and clinical disability. vegf-antagonists reduce ischemic brain edema and injury, involving vegf in pathogenesis and eventually in treatment of stroke and related disorders. this cytokine also exerts a neuroprotective effect mediated by its receptor flk-1. functions related to the inflammatory response, co-expression with proteins of the ecm and interaction with the two main receptors, flk-1 and flt-1, will be discussed. n-methyl-d-aspartate (nmda) receptors can mediate excitotoxic or neuroprotective responses. one of the molecular mechanisms responsible for nmda neuroprotection involves the release of brain-derived neurotrophic factor (bdnf) which in turn binds to and activates its cognate receptor trkb. bdnf levels in the neuronal culture medium increased 2-fold when cells were preincubated for three hours with nmda. at three hours, the increase in bdnf protein levels in the medium was accompanied by a concomitant increase in bdnf mrna. thus, nmda elicited two temporally distinct responses: an early release of bdnf protein followed by a later transcriptional activation of dbnf mrna and protein release. these results suggest that nmda activates the trkb receptor via a bdnf autocrine loop resulting in neuronal survival. in addition, extracellular regulated kinases (erk 1/2) were rapidly activated, which peaked within six hours of nmda treatment. erk 1/2 activation is completely blocked by mk-801 and partially blocked by k252a, suggesting the nmda and trkb receptors act in a coordinated fashion to activate erk 1/2. as an extension of this work, we discovered a single nucleotide polymorphism in the human nr1 gene that, when transfected into hek cells, alters the electrophysiological properties of the nmda receptor complex. possible consequences of this nmda receptor variant in signaling will be discussed. this overview summarizes our recent knowledge of the role that tyrosyl radical (tyro • ) can play in neurochemical systems of brain and thereby lead to neural disorders (pd, ad, als). these could involve the interactions of tyrosine and tyro • with reactive oxygen species (ros) and reactive nitrogen species (rns), via radical mechanisms and chain processes in the presence of o 2 and endogenous brain antioxidants. concentrations of tyro • , ros and rns can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive. this in turn can directly damage (by lipid peroxidation) or indirectly damage (by protein oxidation and/or nitration) cellular substructures which ultimately can lead to apoptotic neuronal cell death or autoschizis. enzymatically (classical peroxidase mechanisms) or non-enzymatically formed tyro • can react with no • and this reversible and intrinsic "combination" acts to "buffer' tyro • concentrations. the reaction of tyro • with superoxide (o 2 •ϫ ) is a scavenging reaction which proceeds rather by addition, not by electron transfer; and major resultant products are tyrosine hydroperoxides (tyrooh). however, the decay of tyro • can be also terminated by self-termination (dimerization) resulting in dityrosine (dt) formation. tyro • can catalyze ldl oxidation, although the precise mechanisms of this reaction in vivo remain unknown. nitration of tyrosine to 3-nitrotyrosine (3-nt) requires a one-electron oxidation as a primary step, with formation of tyro • , followed by addition of the nitrogen dioxide radical (no • 2 ). the promoting effect of carbon dioxide on peroxynitrite-mediated tyrosine nitration (via radical mechanisms) (tyro • /no • /o 2 •ϫ /no • 2 system) is due to the selective reactivity of the putative carbonate radical anion, as compared to that of the oxidizing hydroxyl radicals ( • oh). moreover, once formed, 3-nt may act to promote repetitive redox cycling; it may be reduced to the corresponding nitroanion radical, which is then oxidized by molecular o 2 to o 2 •ϫ and parent 3-nt. one-electron oxidation of 3-nt can result in catalytically active imminoxyl radical. dt formation can outcompete tyrosine nitration at lowsteady state concentrations of peroxynitrite. it is unquestionable that very high fluxes of no • and o 2 •ϫ are requisite intermediates of peroxynitrite, a tyrosine nitration agent formed via tyro • . evidence for the existence of generalized stress within neurons includes the presence of protein peroxides (tyrooh), dt, and 3-nt. the nitration/denitration processes can be pathologic, but these also may play: 1) a signal transduction role; 2) a role of "blocker" for radical-radical reactions (scavenging of no • , no • 2 and co 3 •ϫ by tyro • ); or 3) a role of delimiting factors for peroxynitrite formation. it is still unknown whether oxidation/nitration of tyrosine (as dopamine precursor or protein residue) via tyro • formation, is a footprint of generalized stress and neuronal disorders, an important part of o 2 •ϫ and no • metabolism, or just a part of integral processes for maintaining neuronal homeostasis. the complete answer of these questions should be the first priority task of our recent search, wherein the problem of increased free radical formation in the brain and/or the imbalance of ratios: ros/rns/tyro • may be all important in determining neural cell and tissue injuries under pathological conditions resulted from generalized stress. [acknowledgements. this work was supported in part by kbn ( more than 50% of patients with type 2 diabetes have coronary heart disease, related to silent ischemia, caused by an autonomic denervation of the heart in diabetic patients. oxidative damage to dna has been well documented in cardiac cells isolated from diabetic patients and rats with streptozotocin-induced diabetes mellitus (dm) . this dmmodel shows already seven days after onset of disease structural changes in vascular tissue typical for the development of atherosclerosis. this study evaluates possible molecular mechanisms for early events in the development of dm-induced cardiomyopathy. methods: using "expression array" we examined the activation of cardiac cell death in heart of dm-rats. ms-pcr was used to examine a differential dna methylation. results: an increased expression of genes encoding renin, angiotensinogen and p53 was detected in heart of dm-rats. substantial changes in the methylation status of the p53dependent p21 waf1/cip1 -gene and the cyclin d1-gene were detected in dm-rats. conclusions: the renin-angiotensin system is upregulated with diabetes, and this may contribute to the development of cardiomyopathy via oxidative damage and p53-dependent activation of cardiac cell death. this pathway includes de novo methylation of the p53-inducible p21 waf1/cip1 -gene encoding a protein which binds to and inhibits a broad range of cyclincyclin-dependent kinase complexes. (supported by "bio-rad" and "amersham pharmacia biotech") department of pharmaceutical biosciences, uppsala university, uppsala, sweden during the past decade studies have indicated that growth hormone (gh) may exert effects on the central nervous system (cns). for instance, gh replacement therapy was found to improve the psychological capabilities in adult gh deficient (ghd) patients. furthermore, beneficial effects of the hormone on certain functions, including memory, mental alertness, motivation and working capacity have been reported. likewise gh treatment of ghd children has been observed to produce significant improvement in many behavioural problems seen in these individuals. studies also indicated that gh therapy affects the cerebrospinal fluid (csf) levels of various hormones and neurotransmitters. further support that the cns is a target for gh emerges from observations indicating that the hormone may cross the blood-brain-barrier (bbb) and from studies confirming the presence of gh receptors in the brain. it was previously shown that specific binding sites for gh are present in discrete areas in the cns of both humans and rats. in peripheral tissues gh is shown to elicit its effects through a second mediator insulin-like growth factor 1 (igf-1). igf-1 is well recognized as a protective agent against neural injury in the cns. the neuroprotective effect of this peptide has a broad spectrum affecting many brain regions and acts through its antiapoptopic effect. the production of igf-1 is upregulated in areas of brain damage and the igf-1 system may be an important part of an endogenous neuroprotective system. in spinal cord injuries, however, the content of igf-1 is reduced. we recently observed a neuroprotective effect of topical application of igf-1 in animals subjected to spinal cord trauma. the observed effect may be mediated via a mechanism involving nitric oxide. in the same animal model we have very recently observed a neuroprotective effect of gh. recent reports suggest that the level of gh is drastically reduced in patients with spinal cord injury. in victims of spinal cord injury the secretion of gh and igf-1, as well, is known to be decreased. therefore, exogenous substitution of gh and igf-1 might be a promising approach in the future therapy of spinal cord injury victims. in fact, there is one report indicating that prolonged treatment with synthetic gh of spinal cord injured rats attenuates some of the neurological motor dysfunction seen in these animals 3 weeks following trauma. in our animal model we observed that topical application of rgh significantly reduced traumainduced disturbances in the fluid micro-environment. we also noted that gh was capable of attenuating the trauma-induced depression of spinal cord evoked potentials. the mechanism by which gh exerts it neuroprotective effects will be discussed. chronically administered levodopa in parkinson's disease (pd) treatment is ultimately associated with alterations in motor response. in 6-hydroxydopamine lesioned hemiparkinsonian rats, chronic twice-daily administration of levodopa progressively shortens duration of contralateral turning and augments the period of turning at or below 20% of peak turning rate. the pathogenesis of the response alterations involves in part sensitization of the corticostriatal glutamatergic synaptic activity. characteristic changes involving interactions between striatal kinase and phosphatase signaling now appear to contribute to sensitization of spiny-neuron glutamatergic receptors. glutamate-mediated striatal dysregulation, subsequently, modifies basal ganglia output system in ways that favor the appearance of parkinsonian motor response complications. at a molecular level, transcriptional activation of striatal creb contributes to the persistent expression of the levodopa-induced motor response alterations. conceivably, a safer and more effective therapy for all stages of pd can be provided by drugs that target intracellularly on striatal kinases or phosphotases, or by agents that interact extracellularly on non-dopaminergic striatal receptors such as ampa and nmda, adenosine a2, adrenergic a2, opiod, and serotonergic 2b. the primary cause of parkinson's disease is a loss of dopamine in the corpus striatum. it has been postulated that this effect leads to disinhibition of the striopallidal pathway and, secondarily, to a functional shift towards glutamatergic stimulation. the aim of the present study was to find out whether inhibition of glutamatergic transmission at a level of metabotropic glutamate receptors (mglurs) in the striatum may alleviate parkinsonian-like symptoms in rats. the non-competitive antagonist of receptor subtype 5 (mglur5), mpep (1.0-10 mg/kg ip), or the agonist of group ii mglurs, ly354740 (5-10 mg/kg ip), reduced the haloperidolinduced muscle rigidity and catalepsy. intrastriatal injections of the antagonist of mglur1, (rs) aida (7.5-15 µg/0.5 µl), but not of the agonist of group ii mglurs, 2r,4r-apdc (7.5-15 µg/0.5 µl), inhibited the muscle rigidity induced by haloperidol. in order to search for an influence of mglurs on the striopallidal pathway, the effect of mpep or of the agonist of group ii mglurs, dcg-iv, on the preproenkephalin mrna expression in the striatum was examined. the obtained results suggest that blockade of group i mglurs, or stimulation of group ii mglurs may be important to the amelioration of parkinsonian symptoms. striatal mglurs may contribute to at least some of these effects. several lines of evidence suggest an important role of glutamate in depression. the involvement of group i mglurs in depression has also been proposed. thus, we decided to evaluate whether group i mglurs antagonists have antidepressantlike effects. we also investigated if antidepressant treatment influences group i mglu receptors in the brain. the experiments were performed on male wistar rats (200-250 g) and male c57bl/6 mice (22-26 g). aida (group i mglurs antagonist) given i.v. in the dose of 50 µg, decreased the immobility time in the despair test in rats. mpep (noncompetitive, systemically active mglur5 antagonist) given i.p., was not effective in the despair test in rats. however, in doses of 1.0, 10 and 20 mg/kg, it significantly decreased the immobility time of mice in the tail suspension test. moreover, the deficit in passive-avoidance learning, which was observed in bulbectomized rats, was reversed by chronic, but not acute mpep (10 mg/kg) treatment. prolonged imipramine treatment resulted in significant increase of the level of expression of mglu5 receptors in the ca1 field of the hippocampus, while prolonged electroconvulsive shock treatment (ect) enhanced significantly the chemiluminescence of mglu5 receptors in the ca3 field. the results indicate that group i mglu receptors are modified by chronic antidepressant treatment and that group i metabotropic glutamate receptors antagonists may play a role in the therapy of depression. (this study was supported by kbn grant no. 4.po5a.091.17) institute of pharmacology, polish academy of sciences, krakow, poland chronic exposure to nicotine, alcohol, opioids, sedatives, and cannabis results in development of drug dependence that becomes evident upon a cessation of drug administration and expresses itself as a withdrawal syndrome (with its physiological and motivational manifestations). adaptations at the nmethyl-d-aspartate receptor (nmda-r) complex have been observed in different brain areas during chronic exposure to, and upon withdrawal from, opioids, ethanol, benzodiazepines and barbiturates. behavioral studies employ the assessment of the effects of nmda-r antagonists on: a) the development of dependence (nmda-r antagonists are co-administered with the drug), b) the maintenance of dependence (nmda-r antagonists are administered to animals with pre-established dependence, and -most relevant to the clinical situation -c) on the expression of drug dependence (assessment of the withdrawal severity in subjects with nmda-r antagonists administered just before the expected emergence of withdrawal). the development of dependence to opioids and benzodiazepines is significantly retarded by nmda-r antagonists. studies from this laboratory demonstrate similar inhibition by nmda-r antagonists of the maintenance of opioid dependence. both in rodents and humans, the expression of opioid antagonist-precipitated as well as spontaneous (natural) withdrawal is inhibited by nmda-r antagonists, and animal data demonstrate similar inhibition of the expression of dependence produced by ethanol, barbiturates and benzodiazepines. the involvement of the excitatory amino acid, glutamate and the inhibitiory amino acid, gamma-amino butyric acid (gaba) in the pathophysiology of spinal cord trauma is not known in details. this investigation is focused on the involve-ment of glutamate and gaba in a rat model of spinal cord injury using immunohistochemistry. spinal cord injury induced by an incision into the right dorsal horn of the t10-11 segments resulted in profound edema formation and cell damage in the adjacent t9 and t12 segments at 5 h. pretreatment with h-290/51 (50 mg/kg, p.o.), a potent antioxidant compound, effectively reduced the edema formation and cell injury following trauma. at this time period, untreated traumatised rats exhibited a marked increase in glutamate immunoreactivity and a distinct decrease in gaba immunostaining in the t9 and t12 segments compared to the control group. the changes in glutamate and gaba immunoreactivity in traumatised rats were considerably attenuated by pretreatment with h-290/51. these results suggest that (i) oxidative stress contributes to alterations in glutamate and gaba in spinal cord injury, (ii) glutamate and gaba are contributing to edema formation and cell damage and (iii) the antioxidant compound h-290/51 has a potential therapeutic value in the treatment of spinal cord injuries. dov pharmaceutical, inc., hackensack, new jersey, u.s.a. both preclinical (i.e., behavioral despair models) and clinical studies indicate that compounds reducing transmission at nmda receptors are antidepressant. conventional antidepressants may be viewed as "monoamine-based", increasing the synaptic availability of serotonin, norepinephrine, and/or dopamine. however, chronic administration of of conventional antidepressants alters both mrna levels encoding nmda receptor subunits and radioligand binding to this family of ligand-gated ion channels in circumscribed areas of the cns indicating that nmda receptors may be a downstream target of these monoamine-based agents. we have recently reported (li, et al., neuropharmacology, in press ) that a class of ampa receptor potentiators also exhibits antidepressant-like actions in preclinical models. in this presentation, i will describe how these two distinct, and (at a cellular level) seemingly diametric approaches employing glutamatergic mechanisms converge on intracellular targets that are also impacted by chronic treatment with biogenic amine-based agents. kainic acid is an essential pharmacological tool for many forms of neurobiological research. until several years ago, all commercially available kainic acid was derived from a single biological source (digenia simplex). commercial isolation of kainic acid in japan ceased in 1999, creating a void in the marketplace. recently several different companies have become providers of kainic acid, but each uses a different source of the compound (2 biological and 1 synthetic) and different isolation procedures. our objective was to use three common assay systems to evaluate the comparative pharmacological and neurotoxicological properties of these three sources of kainic acid. dose response curves, both alone and in the presence of receptor selective antagonists, were constructed for each kainate formulation using (a) cerebellar granule neurons in culture, (b) isolated hippocampal slice preparations, and (c) whole animal behavioural toxicity studies. preliminary results reveal many similarities, but also distinct differences between the three formulations, especially when challenged with antagonists for different eaa receptors. full results will be presented and discussed with respect to their implications for both extending the known kainite literature and for future studies employing kainic acid as a ligand in both mechanistic investigations and in animal models of neurodegenerative disease. our results from in vitro studies further elucidate the role of cell-cycle related proteins in neuronal apoptosis induced by excitotoxins. exposure of primary cerebellar neurons to toxic concentrations of glutamate was found to produce a significant, short lasting increase in the expression of p53 and cdc2. transcriptional activity of p53 was shown by increased p53 dna binding activity and by the concomitant induction of the cdk inhibitor p21, the cell cycle regulator gadd 45 and the apoptotic induced bax. cell-cycle proteins are also expressed concomitantly to dna damage in neurons undergoing excitotoxic degeneration. we found that excessive activation of glutamate receptor by nmda results in the formation of 8-oh-deoxyguanosine, which is a marker of oxidative dna damage. in addition, the expression of the dna repair factor msh2 increases in cultured cerebellar neurons or in ca3 pyramidal cells that have been challenged with excitotoxins. excitotoxicity may thus provide a further example of how re-expression of cell-cycle proteins might be tightly connected to dna damage and repair in neurons. rush-presbyterian-st. luke's medical center, chicago, u.s.a. patients with parkinson's disease by definition benefit from levodopa therapy. however, after 5 years of therapy 50% of patients experience motor response complications (mrc's): the benefit from each dose becomes shorter (wearing-off), more unpredictable (on-off) and associated with involuntary movements (dyskinesias). when dyskinesias first arise, they are associated with high levodopa levels and may be prevented or minimized by lowering levodopa intake. later on, the therapeutic window of levodopa narrows progressively and dyskinesias occur at doses equal to those needed to induce an antiparkinson effect. while the pathogenesis of motor complications remains incompletely understood, recent clinical studies implicate mechanisms downstream from the degenerating nigrostriatal dopamine system, possibly involving glutamatergic projections to the basal ganglia. in a rat model of pd, blockade of striatal glutamate receptors of the n-methyl-d-aspartate (nmda) subtype reverses levodopa-induced motor fluctuations. similarly, in 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (mptp) lesioned primates, several nmda-antagonists reduce levodopa-associated dyskinesias. in parkinsonian patients the nmda-antagonists dextromethorphan, dextrorphan and amantadine improve dyskinesias as well. these findings have lead to the suggestion that hyperfunction of nmda receptors on striatal efferent neurons, as a consequence of chronic non-physiologic dopaminergic stimulation, contributes to the pathogenesis of motor response complications. protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including alzheimer's disease (ad), parkinson's disease, polyglutamine diseases, tauopathies, and prion diseases. in many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide spectrum of diseases. however, testing compounds preclinically will require diseaserelevant animal models. numerous transgenic mouse models of ad-like pathology have now been produced. our studies have found that tg2576 mice overexpressing human -amyloid precursor protein (huapp695k670n/m671l) produce copious deposits of diffuse and compact -amyloid as they age, and that females are more susceptible than are males (callahan et al., am. j. pathol. 158, 1173 -1177 , 2001 . recently, we also found that the overexpression of p25 protein, an activator of the kinase cdk5, results in tau hyperphosphorylation, axonopathy and severe motor deficits in transgenic mice, in the absence of neurofibrillary tangles. none of the existing transgenic models of -amyloidosis or tauopathy fully recapitulates the pathology of ad. in an attempt to more authentically model the human disease, we infused dilute ad-brain extracts into tg2576 mice at 3-months of age (i.e. 5-6 months prior to the usual onset ofamyloid deposition). we found that infusion of ad brain extracts results in: 1) earlier and more abundant deposition of -amyloid in app-transgenic mice (kane et al., j. neurosci. 20, 3606-3611); 2) evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and 3) tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. the seeding ofamyloid by ad brain extracts suggests pathogenic similarities between -amyloidoses such as ad and other cerebral proteopathies, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases. supported by warner-lambert/pfizer. purpose: the effects of essential amino acid deficiencies on function of cornea and lens were investigated. methods: dietary deficiencies of tryptophane and methionine were studied in young rats over 3 months. transparency of cornea and lens were evaluated using slitlamp microscope and scheimpflug camera. after sacrifice, lens fresh weight and crystallin patterns were determined to evaluate effects on lens growth and protein synthesis. results: methionine deficiency had no effect on the parameters investigated. tryptophane deficiency caused severe loss of body weight in both rat strains (brown-norway, bn; sprague-dawley, sd), sd rats also lost their hair. they developed corneal neovascularisations and cortical cataracts. bn rats developed faint neovascularisations and a discontinuity zone in the lens. diet intermission arrested pathological processes restarting when feeding diet again. this observation is supported by lens fresh weight data. dna staining evidenced that tryptophane deficiency arrested lens fiber maturation. conclusion: a difference has been found for 2 essential amino acids in their effects on transparency of cornea and lens. tryptophane deficiency stimulated corneal neovasculariseration, but arrested lens fiber cell maturation. the difference in reaction of cornea and lens to tryptophane deficiency between bn and sd rat eyes remains to be elucidated. dynorphin is a neuropeptide that is present in the dorsal horn of the spinal cord. the peptide is actively involved in pain processing pathways. however, its involvement in spinal cord injury is not well known. alteration in dynorphin immunoreactivity occurs following a focal trauma to the rat spinal cord. infusion of dynorphin into the intrathecal space of the cord results in ischemia, cell damage and abnormal motor function. antibodies to dynorphin when injected into the intrathecal space of the spinal cord following trauma improves motor recovery and reduces edema and cell changes. however, influence of dynorphin on trauma induced alteration in spinal cord bioelectrical activity is still not known. spinal cord evoked potentials (scep) are good indicator of spinal cord conduction that are altered following trauma. therefore, in present investigation, influence of dynorphin antibodies on trauma induced changes in scep was examined in our rat model. in addition, spinal cord edema formation and microvascular permeability disturbances were also investigated. our results show that intrathecal administration of dynorphin antiserum prior to injury has a beneficial effect on trauma induced electrical activity, microvascular permeability disturbances, and edema formation. these observations indicate that dynorphin is somehow involved in the altered bioelectrical activity of the spinal cord and participates in the pathophysiological processes leading to cell injury. fatty-acid binding proteins (fabps) are involved in the intracellular binding, targeting and transport of long-chain fatty acids (fas) to modulate cell growth and/or differentiation. fabp form a family of proteins displaying tissue-specific expression. the expression of brain type fabp (b-fabp) is spatially and temporally correlated with neuronal differentiation during brain development. heart type fabp (h-fabp) is widely distributed and present in skeletal muscles, kidney, lung, brain and endothelial cells. it is neuron-specific in postnatal brain and participates in neurite formation and synapse maturation. epidermal type fabp (e-fabp) is expressed at high levels during neurogenesis, neuronal migration, and terminal differentiation. although all three fabps could be involved in normal brain function in prenatal and postnatal life, a neurobiological role of fabps in neurodegenerative diseases has not been reported yet. these made us evaluate the protein levels of fabps in brains from patients with down syndrome (ds) and alzheimer's disease (ad) and fetal cerebral cortex with ds using two-dimensional (2-d) gel electrophoresis with subsequent matrix-assisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. in fetal brain, b-fabp and e-fabp levels were comparable between control and ds. in adult brain, b-fabp was significantly increased in occipital cortex of ds, and h-fabp was significantly decreased in ds (frontal, occipital, parietal cortices) and ad (frontal, temporal, occipital and parietal cortices). we conclude that aberrant expression of fabps, especially h-fabp in neurodegenerative diseases could be involved in impaired neurite outgrowth and synapse maturation. in our previous paper, it was shown that gaba-a receptor antagonist picrotoxin suppressed etoh (ethanol) selfadministration. recently, several authors indicated that systemic injection of dopamine or serotonine agonists reduced ethanol drinking in rats. therefore, in the present study we investigated the effects of thip (4,5,6,7-tetrahydroizokasazolo, 5,4-c pyridin-3-ol) gaba-a receptor agonist in naive and long-term ethanol-experienced rats on etoh selfadministration and on cardiovascular system. adult 13-17week-old male, normotensive wistar-kyoto (wky) and spontaneously hypertensive rats (shr) were used. naive rats were examined according to smith method. long-term ethanolexperienced rats were studied according to boyle method. thip was injected in naive rats at a dose of 8 and 16 mg/kg i.p. metabotropic glutamate receptors are coupled to phospholipase c stimulation and adenylyl cyclase inhibition through g-proteins. c6 glioma cells, that endogenously express the phospholipase c coupled metabotropic glutamate receptor type, were treated with different specific agonists of these receptors and the effect of these treatments on different components of metabotropic glutamate receptor pathway was studied by radioligand binding, phospholipase c activity and rt-pcr assays. agonists treatment caused a decrease in l-[ 3 h]glutamate binding to intact cells and membranes in a time dependent manner being maximum at 3-6 hours and recovered at 24-36 hours. this decrease was associated with a significant increase in the mrna level coding mglurs. no changes on g q/11 mrna level were detected in any case. however, a significant decrease in l-glutamate stimulated phospholipase c activity was detected after agonist treatments in both membranes and intact cells. this decrease was not associated to significant variations in mrna level coding phospholipase c 1 isoform. all these results suggest that agonist exposure causes a desensitisation of glial metabotropic glutamate receptor decreasing not only receptors number but its functionality. in this study the interaction between these two nuclei were investigated by means of microinjection and microdialysis techniques in sprague-dawley rats. steroetaxic surgery was performed by placing intracerebral parenchymal microinjection cannula into the right dmh and microdialysis probe into the left pvn. iliac artery was also cannulated to monitor the pulsatile blood pressure and heart rate by means of pressure transducer connected to a polygraph microinjection of 50 pmol nmda into the dmh was performed and microdialysis perfusates were collected simultaneously from the pvn in conscious rat model. γ-aminobutyric acid (gaba) and l-glutamic acid levels were analyzed by an isocratic hplc (high pressure liquid chromatography) method with the aid of a fluorescent detector. microinjection of 50 pmol nmda into dmh produced significant increases in mean arterial pressure and heart rate. nmda microinjection into the dmh produced significant increase in l-glutamic acid release in the pvn, but no significant change in gaba release was observed. these results suggest that stimulation of dmh by nmda results in subsequent stimulation of the pvn. [this study was sponsored by marmara university research foundation (project no: 1998/sag/38).] and in long-term ethanol-experienced rats only at a dose 16 mg/ kg i.p. control group (cg) received saline 3 ml/kg i.p. as can be seen in fig. 1 and table 1 the lower consumption of ethanol in shr in comparison to wky rats was observed. systemic injection of thip decreased dosedependently etoh intake in naive rats of both strains. this effect was more pronounced in shr (fig. 1) . similar phenomenon was observed after thip injection in long-term ethanolexperienced rats. there were no effect on systolic blood pressure and heart rate after thip treatment. born-bunge foundation, university of antwerp, and university of ghent, belgium increased neuronal excitability may underlie some of the neurological complications in uremic patients. in an effort to identify candidate neuroexcitatory compounds, 17 different uremic retention solutes, including several amino acids and amino acid derivatives, were applied to mouse spinal cord neurons in primary dissociated cell cultures. using the tight-seal whole-cell technique, a few of the candidate toxins were shown to evoke whole-cell currents in cells clamped at ϫ60 mv. in a first survey, each of the solutes was briefly applied in a concentration of 5 mm. significant inward whole-cell currents were evoked by guanidinosuccinate, spermine, and 3-indoxyl sulfate, whereas phenol evoked an outward current. further experiments indicated that guanidinosuccinate-evoked whole-cell currents were due to activation of nmda-type glutamate receptors in concentrations similar to those found in uremic patients. high (mm) concentrations of spermine activated voltage-gated calcium channels, whereas low (µm) concentrations were found to potentiate guanidinosuccinate-evoked currents through its action on the nmda receptor-associated polyamine binding site. whole-cell currents evoked by 3indoxyl sulfate or phenol seemed to be due to complex interaction with several different ion channels. we conclude that guanidinosuccinate-evoked nmda receptor activation, possi-bly potentiated by the neuroexcitatory effects of polyamines and other putative uremic neurotoxins, could be an important mechanism underlying the increased neuroexcitability in uremic brain. glutamine (gln) is one of the key metabolites in the cns (energy metabolite, precursor of neurotransmitter amino acids, end product of ammonia detoxication, osmolyte), and as such is a routine supplement of cns cell culture media. c6 glioma cells relatively easily adapt to culturing in a gln-deprived medium. the present study investigated the effects of gln deprivation on the characteristics of the different systems that mediate gln cell membrane transport in the cells. in contrast to a variety of cns and non-cns cells, the absence of gln did not derepress the methyl-amino-isobutyric acid (meaib)-sensitive ("system adependent") uptake. system asc became relatively more-, and system n less active than in cells grown in the presence of gln, but the ion -and substrate specificity of the uptake remained unaltered. system asc in c6 cells grown in a glnsupplemented medium shows two features distinct from most other cell types: a) strong ph sensitivity and b) partial tolerance of lithium substitution, pointing to domination of system asct2 -an asc variant strongly expressed in cultured astrocytes. cells grown in gln-deprived medium lost lithium tolerance, but not ph-dependence of the uptake, their properties thus resembling system glnt (sat1), a neuron-specific variant of system a. by contrast, transport of threonine, a standard asc system substrate, was not affected by gln deprivation and showed neither ph dependence nor lithium tolerance, which is typical of an asc in all the non-cns tissues. (supported by scsr grant no. 4 p05a 060 18.) the classical the hypothalamic-neurohypophysial system (hns) is comprised of neurons originating within the supraoptic nucleus (son) which project to the neurohypophysis to release the nonapeptides oxytocin (oxt) and vasopressin into the blood after appropriate stimulation. previous experiments have shown that a single social defeat experience triggers the release of oxt from somata and dendrites into the extracellular fluid of the son, but not from axon terminals in the neurohypophysis. to further investigate the regulatory mechanisms underlying this dissociated release, we exposed male wistar rats to a 30-min social defeat experience and monitored the release of the inhibitory amino acids gamma amino butyric acid (gaba) and taurine into the son using microdialysis. social defeat caused a significant increase of the intra-son pre-stress basal release). to reveal the physiological significance of the intrahypothalamically released gababicuculline, a specific gaba a -receptor antagonist -was administered into the son by retrodialysis. this treatment increased significantly the release of oxt both within the son (200%; p ͻ 0.05 vs. pre-stress basal release) and -as measured via chronically implanted jugular venous catheters -into blood under basal and stress conditions (up to 200%; p ͻ 0.01 vs. prestress basal release). however, bicuculline did not affect plasma vasopressin. these data demonstrate that gaba is released within the son during social defeat to act as an inhibitor of both, central and peripheral oxt secretion during emotional stress. the mechanism described here contributes to the regulatory capacity of the hns to ensure the appropriate involvement of oxt in the stress response of the animal (supported by dfg, en 366-21). down syndrome (ds) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21 and characterized clinically by somatic anomalies, mental retardation and precocious dementia. the phenotype of ds is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. reports that challenge this notion, however, have been published. to add to this body of evidence, the expression ofamyloid precursor protein (app), ets-2 and collagen α1 (vi) chain precursor, encoded on chromosome 21, was investigated in fetal brain by western blot and two-dimensional electrophoresis (2-de). western blot detected app and ets-2 that migrated at ϳ75 and 50 kda, respectively. subsequent densitometric analysis of app and ets-2 immunoreactivity did not produce any significant change between controls and ds. since the metabolic fate of app determines the propensity of amyloid production, the expression of the secreted forms of app (sapp) had been examined. neither the expression of sappα nor sapp showed any detectable changes among the two groups. collagen α1 (vi) chain precursor, a protein resolved as a single spot on 2d gel was identified by matrix associated laser desorption ionization mass spectroscopy. quantitative analysis of this spot using the 2d image master software revealed a significant decrease in fetal ds (p ͻ 0.01) compared to controls. linear regression analysis did not show any correlation between protein levels and age. the current data suggest that overexpression per se can not fully explain the ds phenotype. apoptosis is the mechanism by which cells are programmed to die under a wide range of physiological and developmental stimuli. accumulating evidence indicates that enhanced apoptosis (programmed cell death) in down syndrome (ds) may play a role in mental retardation and precocious neurodegeneration of the alzheimer-type. in this regard, alteration of several apoptosis related proteins have been reported in adult ds brain. fetal ds neurons exhibited increased reactive oxygen species leading to early apoptosis, however, expression of apoptosis related proteins in fetal ds, has never been considered. to address this issue, we investigated the expression of proteins involved in apoptosis including fas (cd95, apo-1), caspase-3, bcl-2 and annexins in the cerebral cortex of control and ds fetal brain by western blot and two dimensional electrophoresis. here, we report that no detectable changes were obtained in fetal ds brain in the expression of fas, caspase-3, bcl-2 and annexins (i, ii, v, and vi) compared to controls. in parallel experiment, we also examined the expression of neuron specific enolase (nse), a neuronal marker found to be decreased in adult ds brain, to see if there is any neuronal loss and no difference was observed between the two groups. protein expression did not correlate with age. the unchanged levels of fas, bcl-2 and annexins together with unaltered caspase-3 expression, a predominant caspase that executes apoptosis in the developing nervous system, suggest that enhanced apoptosis may not be apparent in fetal ds brain as demonstrated for adult ds brain. introduction. among the various metabolites indicating neuronal damage, amino acids are regarded particularly important. detection of amino acids by microdialysis is currently introduced as a neuromonitoring tool in patient care. here, we present changes in the extracellular concentrations of various amino acids in stroke patients and in experimental stroke in cats. method. cat focal ischemia was produced by occlusion of the middle cerebral artery (mca) for 1 h followed by 2 h reperfusion. glutamate, aspartate, gaba, taurine, glycine, serine, glutamine, methionine, threonine, tyrosine, asparagine, valine, phenylanaine, isoleucine and leucine were sampled by microdialysis in the ischemic core and subsequently analyzed by hplc. human microdialysis was performed in patients with large mca infarction. the microdialysis probes were inserted into primarily non-infarcted tissue in the border zone of the ischemic territory. results. transmitter amino acids rose immediately after occlusion in the cat model. correspondingly, these substances increased sharply in the human brain, when the tissue around the probes became infarcted, as shown by positron emission tomography (pet) and ct scan. in contrast, structural amino acids did not show marked increases or even decreased during severe ischemia in both, experimental ischemia and stroke patients. these substances did increase, however, when the brain tissue was only slightly ischemic, i.e. after reperfusion of the cat brain, when brain swelling occurred, or in human brain, when tissue did not show any infarction in the ct scan but hypoperfusion in the pet image. conclusion. extracellular amino acids detected by microdialysis can serve as markers for secondary ischemia. severe ischemia is reflected by rapid increases of transmitter amino acids, due to various mechanisms including synaptic release and reversal of reuptake systems. oligemia seems to be reflected by slow increases of structural amino acids, possibly due to a reduction in cerebral protein synthesis. apoptosis has been implicated in the selective neuronal loss of down syndrome (ds). apoptosis activates a family of cysteine proteases with specificity for aspartic acid residues referred to as, caspases that play a key role in dismantling a cell committed to die. caspases activity is regulated by a variety of proteins that possess a domain resembling the prodomains of caspases. little is known, however, about the changes of caspases and their regulatory proteins in ds. here, we investigated levels of nine such different proteins by western blot technique in frontal cortex and cerebellum of control and ds subjects. the protein levels of dff45 (dna fragmentation factor 45), and flip (fadd like interleukin-1 -converting enzyme inhibitory proteins) were significantly decreased whereas that of rick (rip-like interacting clarp kinase) increased in both regions of ds. in contrast, cytochrome c, apaf-1 (apoptosis protease activating factor-1), procaspase-9 and arc (apoptosis repressor with caspase recruitment domain) were unchanged. procaspase-3 and -8 were significantly decreased in frontal cortex but no significant change was observed in cerebellum. regression analysis revealed no correlation between postmortem interval and levels of the investigated proteins. however, inconsistent correlation was found between age and levels of proteins as well as amongst the density of individual proteins. these findings demonstrate that dysregulation of apoptotic proteins does exist in ds brain and may underlie the neuropathology of ds. the study further suggests that apoptosis in ds may occur via the death receptor pathway independent of cytochrome c. hence, therapeutic strategies that target caspase activation may prove useful in combating neuronal loss in this disorder. in order to examine the differential roles of nitric oxide (no) induced by either endothelial no synthase (enos) or neuronal no synthase (nnos) after transient cerebral ischemia, we investigated the effects of the relatively selective cnos inhibitor, l-n 5 -(1-iminoethyl)ornithine (l-nio), the relatively selective nnos inhibitor, 7-nitroindazole (7-ni) and the no scavenger, 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazole-1-oxyl 3-oxide (ptio) on hippocampal dysfunction caused by cerebral ischemia. we measured no concentration, mean arterial blood pressure (mabp), hippocampal blood flow, direct current potential, ca1 population spike (ps) and release of amino acids from rat hippocampus after transient forebrain ischemia, which was induced by 4vessel occlusion for 10 min. l-nio (20 mg/kg), 7-ni (25 mg/kg) and ptio (1 mg/kg) were administered intraperitoncally 20 min before ischemia. ptio, 7-ni and l-nio reduced ischemiainduced no production in the hippocampus during the early period of reperfusion. the rank order of inhibitory potency was ptio ͼ 7-ni ͼ l-nio. l-nio, but not 7-ni, reduced hippocampal blood flow during ischemia and increased mabp before, during and after ischemia, compared with the vehicle group. ptio increased mabp during and after ischemia. ptio and 7-ni, but not l-nio, reduced amplitude of anoxic depolarization induced by ischemia. 7-ni recovered in part ps amplitude 60 min after ischemia. 7-ni, but not l-nio, reduced ischemiainduced release of aspartate and glutamate, but not taurine. the present study provides further evidence for the idea that in the early stages of transient forebrain ischemia, enos-derived no has a neuroprotective effect in the hippocampus, while nnos-derived no has a neurotoxic effect. the estrogen affects brain protein synthesis in ovariectomized female rats k. hayase 1 , m. tanaka 1 , k. tujioka 1 , e. hirano 1 , and h. yokogoshi 2 1 department of home economics, aichi university of education, kariya, aichi, and 2 laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, japan the purpose of this study was to determine whether 17-estradiol affected the rate of brain protein synthesis in ovariectomized female rats. experiments were conducted on three groups of 12 wk old female rats: group 1. ovariectomized to reduce the level of plasma estradiol; group 2. ovariectomized and treated with estradiol; and group 3. sham-operated control. the fractional rates of protein synthesis in brain of ovariectomized rats treated with estradiol were significantly greater than in ovariectomized rats without estradiol treatment. in brain, the rna activity [g protein synthesized/(g rnaᮀd)] significantly correlated with the fractional rate of protein synthesis. the rna concentration (mg rna/g protein) was not related to the fractional rate of protein synthesis in any organ. the results suggest that estrogen treatment of ovariectomized female rats is likely to increase the rate of protein synthesis in the brain, and that rna activity is at least partly related to the fractional rate of brain protein synthesis. we have synthesized a series of new peptides that have demonstrated potent antidepressant activity in animal models for depression and in phase iia and iib clinical trials. mif-1 (prolyl-leucyl-glycinamide) an endogenous brain peptide has been reported to have some clinical activity in patients with unipolar depression with few apparent side effects. we have undertaken a study to determine the effect of molecular structural changes on the antidepressant activity of this peptide. we evaluated our new derivatives in a stress-induced animal model for depression, i.e. porsolt test, we have found that 4-f-phe-4-oh-pro-arg-gly-trp-nh 2 (inn 00835) is superior in all the statistical parameters used. in comparative testing inn 00835 was more active than prozac (fluoxetine) and zoloft (sertraline) in our antidepressant model. a u.s. patent has been granted on these compounds. the clinical results of inn 00835 show that it is effective in over 80% of depressed subjects when blood levels exceeded therapeutic threshold with no significant side effects. inn 00835 has a rapid onset of action, 3-5 days (vs. 2-6 weeks for currently marketed products) with sustained effects for months following 5 to 10 doses over 1-2 weeks. h. iwama 1 , 2 , a. umino 2 , a. hashimoto 2 , k. takahashi 2 , n. yamamoto 2 , and t. nishikawa 1,2 1 section of psychiatry and behavioral science, tokyo medical and dental university graduate school, and 2 department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan using an in vivo dialysis technique, we have studied the extracellular contents of endogenous free d-serine in comparison with that of l-serine, glycine and l-glutamate in the brain of the freely moving rat. a high amount of d-serine was detected in the dialysate obtained from the medial prefrontal cortex and striatum, whereas the cerebellar dialysate contained only a trace concentration of the d-amino acid. intra-medial prefrontal cortex perfusion of a sodium channel activator, veratrine, augmented the extracellular release of glycine and l-glutamate but a slight decrease in that of d-serine in a tetrodotoxin-sensitive manner in the prefrontal area. moreover, selective destruction of neuronal cell bodies in the medial frontal region by means of local infusion of an excitotoxin quinolinate resulted in a marked reduction of extracellular and tissue levels of d-serine in the infused prefrontal region. these findings suggest that endogenous d-serine might be liberated into the extracellular space from non-neuronal cells or certain exceptional neuronal cells probably by a carrier-mediated process in the mammalian prefrontal cortex. also, the endogenous d-amino acid has been indicated to be accumulated or synthesized, at least in part, in the neuronal cells. nucleoside diphosphate kinase (ndpk) catalyzes a transfer of the terminal phosphate from nucleoside triphosphates ((d)ntps) to nucleoside diphosphates ((d)ndps) and has been suggested to be involved in the regulation of wide variety of cellular functions. in addition, ndpk isoforms (a and b) are encoded by nm23 genes (h1 and h2) , which are related with the metastatic potential of some tumors. although ndpk/ nm23 has been also implicated to modulate neuronal cell proliferation, differentiation and neurite outgrowth, a neurobiological role of ndpk/nm23 in neurodegenerative diseases has not been reported yet. here we evaluated the protein levels of ndpk-a/nm23-h1 in brains from patients with down syndrome (ds) and alzheimer's disease (ad) using twodimensional (2-d) gel electrophoresis with subsequent matrixassisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. ndpk-a/nm23-h1 was significantly decreased in brain regions (frontal, occipital, parietal cortices) of both ds and ad compared to controls. we conclude that the down-regulated ndpk-a/nm23-h1 upon neurodegeneration could play a pivotal role in a wide range of neurobiological functions such as neurite outgrowth and consequently these could result in functional disturbance of the nervous system in ds and ad. brain α-endosulfine is manifold decreased in brains from patients with alzheimer's disease: a tentative marker? and drug target? α-endosulfine has the sulfonylurea-like ability to block atp-sensitive potassium (k atp ) channels and is expressed in a wide range of tissues. although the blockade of k atp channels has been reported to be involved in the release of neurotransmitters, the neurobiological role of α-endosulfine has not been studied yet. we examined the levels of αendosulfine protein in frontal cortex and cerebellum from patients with alzheimer's disease (ad). α-endosulfine was extremely decreased in both regions of ad compared to controls. this could result in the continuous opening of k atp channels with subsequent decrease of neurotransmitters release and change of potassium fluxes. this study is of great significance for providing a neurobiological function of α-endosulfine in brain and furthermore, α-endosulfine could serve as a useful marker for the diagnosis of ad and a target for drug treatment. children's hospital heidelberg, and department of pharmacology and toxicology, university of marburg, germany d-2-hydroxyglutaric aciduria is an inherited neurometabolic disorder of unknown etiology characterized by progressive neurodegeneration of vulnerable brain regions during infancy and early childhood, resulting in psychomotor retardation, hypotonia, seizures, macrocephaly, enlarged lateral ventricles, delayed cerebral maturation as frequent neurological presentation in affected children. the disease is biochemically characterized by the accumulation of d-2hydroxyglutarate (d-2), which is structurally similar to lglutamate (ϭ 2-amino-glutarate). we therefore investigated in primary neuronal cultures from chick and rat, whether d-2 induces excitotoxic neuronal damage. here we report that d-2 decreased cell viability in a concentration-and time-dependent fashion by disturbance of intracellular calcium homeostasis as determined by fura-2 measurement. furthermore, fluorescence microscopy using dihydrorhodamine-123 revealed an increased generation of reactive oxygen species (ros) elicited by expo-sure to d-2. n-methyl-d-aspartate (nmda) receptor blockade specifically prevented excitotoxic neuronal damage as well as increased calcium influx and ros production, suggesting that d-2 is an agonist at nmda receptors. patch-clamp investigation confirmed that d-2 activated recombinant nmda receptors in hek293 cells. furthermore, activity measurement of single respiratory chain complexes revealed a specific inhibition of complex v activity by d-2. we conclude that excitoxic mechanisms contribute to the neuropathology of d-2hydroxyglutaric aciduria, highlighting new neuroprotective strategies for this neurometabolic diseases. these studies were designed to determine the effects of aging and an aging intervention on nmda subunit expression. in situ hybridization and receptor autoradiography were performed on naïve or behaviorally-tested c57bl/6 mice of different ages (3, 10-15, and 26-30 months old) and diet groups (ad lib-fed and diet restricted). there were age-related decreases in both e2 and z1 mrna density in naïve, ad lib-fed mice. correlations were found between changes in e2 subunit mrna and agonist binding and z1 mrna expression and antagonist binding. diet restriction significantly improved learning ability, slightly spared glutamate binding to nmda sites and improved z1 mrna expression in older mice. significant correlations were found between agonist binding and both learning ability and e2 and e1 mrna density. learning ability in the old mice also correlated with the ratios of mrna expression for e1 and e2 and/or z1 subunits. these results suggest that changes in nmda receptor binding and the relationship between subunit expression levels are important for maintaining memory functions in older animals. extracts of st john's wort (hypericum perforatum l.) are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids. coronal cortical slices (400 mm) were cut and perfused with gassed (95% o2, 5% co2) acsf at 37°c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering 2 pulses of kϩ (60 mm) or veratridine (20 mm) 30 minutes apart. in control experiments the second kϩ pulse elicited glutamate release which was 80% of the first pulse. hyperforin (5 mm) perfused for 30 minutes prior to, and during, the second kϩ pulse significantly increased glutamate release to 170% (p ͻ 0.001, n ϭ 6-8). release elicited by the second veratridine pulse was 70% of the first pulse for both glutamate and aspartate. hyperforin (5 mm) increased this release to the second pulse to 160% and 130% respectively (p ͻ 0.001, n ϭ 6-8). when perfused on its own for 30 minutes, hyperforin (5 mm) increased the basal release of glutamate (p ͻ 0.001, n ϭ 4-5). in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca2ϩ or naϩ channels. glaxosmithkline group, glaxo wellcome s.p.a., medicines research centre, verona, italy n-methyl-d-aspartate (nmda) receptors are ligand gated ion channels widely distributed in mammalian brain, which play a crucial role in important physiological mechanisms, such as excitatory transmission, neuronal migration and memory formation. a peculiar feature of nmda receptors is the absolute requirement of l-glutamic acid and glycine for the opening of the channel. noteworthy, these two aminoacids reciprocally modulate binding at their respective recognition sites. aim of this work was to study nmda receptor glycine/glutamate interactions in rat and human brain. binding of the nmda antagonist [ 3 h]cgp39653 to rat cerebral cortical membranes was inhibited by glycine. the overall effect of glycine consisted in a decrease of [ 3 h]cgp39653 affinity, with a parallel increase of the receptor affinity for glutamate. the glycine antagonist gv150526a competitively reversed glycine inhibition, proving that the modulation was via the glycine binding site. [ 3 h]cgp39653 binding to rat brain sections revealed the existence of regionally distinct nmda receptor subtypes with difference glycine/glutamate interactions. in most regions of the human brain nmda receptors presented the same allosteric modulation of [ 3 h]cgp39653 binding, as revealed by large section autoradiography technique. nevertheless, detection of any regional variation was not possible, probably due to the high intersubject variability. the effect of long-term high k ϩ -treatment on neuronal survival, cellular maturation, nmda receptor (nr) splice variant expression, and receptor function was investigated in primary cultures of rat cortical neurones. long-term incubation (up to 15 days) with 25 mm k ϩ significantly increased neuronal survival and induced multiple morphological changes associated with promoted cellular maturation. cultures grown in medium containing 25 mm k ϩ also exhibited multiple changes in nr1 splice variant expression according to rt-pcr studies performed with primer pairs flanking the alternatively spliced regions, in order to estimate the ratios of the corresponding 3ј and 5ј splice variants. nr1-1 and nr1-3 (each containing exon 21) were decreased, whereas nr1-2 and nr1-4 (each lacking exon 21) were increased, accordingly. the predominant expression of nr1-b was further increased. after administration of ttx, each of the k ϩ -induced changes on mrna expression was virtually abolished. in voltage-clamp recordings (holding potential: ϫ70 mv), nmda induced inward currents in a concentration-dependent manner with a maximum effect of ϫ745 pa under control conditions. neurones treated with 25 mm k ϩ showed a significantly diminished response to nmda (max. response: ϫ368 pa). in conclusion, the present data indicate that a sustained increase in neuronal activity induces adaptive changes in nr1 splice variant expression and a decrease in receptor function. thus, alternative splicing associated with a diminished receptorcytoskeletal linkage may be important compensatory mechanism in preventing cellular damage due to long-term activation of excitatory nr. it seems conceivable that this mechanism contributes to the promoting effects of 25 mm k ϩ on neuronal survival and maturation. (supported by bmbf grant 01gg9818/0) there is accumulating reports that kainate-induced seizures elicit expression of various heat-shock proteins (hsps) in the brain, such as hsp27, hsp32, and hsp70. however, no investigation has been carried out on changes in level of apg-2, a member of hsp 110 family, after excitatory amino acid-induced seizures. by means of an immunoblot assay, we determined the levels of hsp70 and apg-2 in discrete brain structures of mice after a single intraperitoneal injection of kainate or nmda. apg-2 level was significantly decreased in the frontal cortex, hippocampus, and striatum 3 days after kainate administration, while hsp70 level was increased in these regions following the administration. decreased level of apg-2 returned to the control levels in the three regions 10 days after kainate administration. no significant changes were observed in levels of both hsp70 and apg-2 in hypothalamus, midbrain, medulla-pons, and cerebellum of kainate-treated mice. by contrast, nmda administration did not significantly affect both levels in any of the regions examined. these results suggest that, unlike the case of hsp70, apg-2 expression could be temporarily down regulated by signals peculiar to kainate, but not by those peculiar to nmda, in murine telencephalon. high concentrations of glucagon-like peptide-1 (7-36) amide (glp-1) and its specific receptor (glp-1r) have been found in the rat hypothalamus. in this study the actions of glp-1 and its related peptides, exendin-4 (glp-1r agonist), exendin (9-39) (glp-1r antagonist) and glp-1(9-36)amide (major glp-1 metabolite) on levels of amino acids (glu, asp, gln, gly, tyr, trp, gaba) in the hypothalamus were investigated. 125 i-glp-1 binding in rat hypothalamic membranes was competed by the peptides in the following order of potency; glp-1 ͼ exending-4 ͼ exendin (9-39) ͼ glp-1(9-36)amide. intracerebroventricular (icv) glp-1 (4 nmoles) produced a statistically significant reduction in levels of all measured amino acids compared with saline injected controls, whereas 4 nmoles of exendin (9-39) was ineffective. exendin-4 produced a statistically significant reduction in the levels of trp, glu, and tyr. glp-1(9-36)amide showed a statistically significant increase in the level all the amino acids tested in this study. prior administration of exendin (9-39) or glp-1(9-36)amide blocked the effects of glp-1 on the levels of the amino acids. these data are consistent with exendin-4 being a glp-1r agonist and exendin (9-39) being a specific glp-1r antagonist. glp-1(9-36)amide, a primary metabolite of glp-1, appears to act as an endogenous antagonist at the glp-1r. department of biophysics, instituto de fisiología celular, universidad nacional autónoma de méxico, méxico city, méxico cholecystokinin (cck), a family of neuropeptides, seems to be involved in anxiety. evidence from several laboratories indicates that the ansiogenic effects of cck are mediated by cck b receptors. however it has been reported that cck a receptors have been found in brain and cck a receptor antagonists have ansiolytic properties. the aim of this work was to study whether or not cck a receptors are also involved in the modulation of anxiety. male rats were cannulated in the lateral ventricle and cck (9 fmol) and/or cck antagonists (900 fmol) were injected 7 days after surgery. anxiety was evaluated in the elevated plus-maze test and locomotion in an open-field test. ansiogenic effects were observed when cck b receptor agonists (cck8ns; cck4) or a mixed cck b and cck a receptor agonist (cck8s) were injected. in contrast, cck33, a cck a receptor agonist or cck 1-21 and cck 26-29 were uneffective. furthermore, the ansiogenic effects of cck8s were prevented by the previous (15 min) administration of l365,260 (cck b receptor antagonist) but not by devazepide (cck a receptor antagonist). no effects on locomotion were observed in any condition. these results indicate that cck a receptors are not involved in anxiety, as measured by the elevated plus-maze test. congenital conditions (i.e. neural tube defects: ntg) have a multifactorial aetiology. deficiencies in the folate and transsulfuration pathways have, in recent years, been positively linked to ntd and other dysmorhogenic syndromes. efficient one-carbon metabolism is crucial for the synthesis of dna precursors, the remethylation of homocysteine and biomethylation of dna. more than 80% of the one-carbon units that flow through the metabolic system in mammals and birds are derived from l-serine and glycine, the natural substrates for shmt. the mitochondrial glycine cleavage enzyme system (gces) can potentially compete with shmt for tetrahydrofolate (thf) in the generation of the methylenetetrahydrofolate pool. valproate (depakene, epilim), an anti-epileptic agent, appears to be strongly associated with hyperhomocysteinemia, several other induced metabolic conditions, the inhibition of the gces and an increased incidence of ntd in epileptic women of child-bearing age. the exact mechanisms of valproate-induced ntd are not yet clear. we investigated the association of the teratogenic properties of valproate with the inhibition of shmt and/or the gces in developing embryos. chicken embryos were treated with sodium valproate (vpa) and pregnant female mice (c57bl) received intraperitoneal injections of vpa, during the critical period of embryonic neural tube development. control embryos were treated with sterilised saline solution. harvested embryos were subsequently investigated for congenital abnormalities and hepatic shmt and gces activities quantified with radiometric assays. the effect of vpa on hepatic dna synthesis was monitored ( 3 h-thymidine incorporation into embryonic dna) and the dna-methylation status determined (dna n 5 -methylcytosine levels). dose-responsive incidences of ntd were observed in vpa treated embryos. very few defects occurred in control embryos. shmt and gces appeared to be inhibited in liver extracts of vpa-treated embryos. hepatic dna synthesis was significantly compromised and 5-mc levels were altered in vpa-treated embryos. the inhibition of either shmt and/or gces activities appeared to be associated with valproateinduced ntd in the chicken and mouse embryo models. the primary mechanism of this effect can probably be ascribed to a restriction in the flow of one-carbon units through the metabolic system, decreased synthesis of dna precursors and alterations in the methylation status of dna. department of neuroscience, university of cagliari, italy ethanol is long known to cause dose-related biphasic effects and we recently found that ethanol bidirectionally affects also working memory. the euphoriant and excitatory effects produced at low doses are associated with the rewarding action of ethanol and are thought to be mediated by the activation of the mesolimbic dopamine (da) system. however, ethanol monophasically stimulates mesolimbic da release in the nucleus accumbens, even at doses that cause hypnosis and coma. in contrast, ethanol biphasically modulates mesocortical da release in the prefrontal cortex (pfc). the changes in da release induced by ethanol are time locked with corresponding changes in extracellular glutamate levels. these biphasic effects of ethanol on pfc da and glutamate are matched by biphasic changes in the performance in a spatial delayed alternation task -a working memory test that is sensitive to proper function of the pfc -suggesting a link between da and glutamate transmission in the cognitive effects of ethanol. focal application in the pfc of the competitive ampa/kainate receptor antagonist cnqx suppresses both da release and the improvement of working memory induced by low doses of ethanol. these results suggest that ethanol may increase da transmission in the pfc and enhance working memory functions by increasing the release of glutamate, thereby stimulating non-nmda glutamate receptors. the enhancing effect on working memory by low, excitatory doses of ethanol may be perceived as rewarding and could constitute an important neurobiological mechanism for excessive ethanol drinking. physiology department, faculty of medicine, al-quds university, jerusalem, palestine glutamate and asparate are considered as the main excitatory neurotransmitters in brain and spinal cord, in addition to their role in energy metabolism, synthesis of proteins and detoxification of ammonia. glutamate and aspartate are centrally involved in basic mechanisms generating epileptic seizures and in epileptogenesis. stimulated release of glutamate and aspartate was detected in vivo and in vitro following neuronal depolarization. photic stimuli has increased glutamate release from visual cortex, and afferent brachial stimulation has increased the endogeneous release of glutamate from contra-lateral sensorimotor cortex compared to ipsi-lateral side. similar results were achieved after local application of tityustoxin or veratridine to the sensorimotor cortex. implantation of cobalt powder over the right sensorimotor cortex of rats produced an epileptogenic lesions characterized by contra-lateral fore and hind limb jerks and an increase in the frequency of eeg spikes. the jerks started after 6 days with maximum myoclonic activity (15 jerks/min). the concentration of glutamate in the epileptogenic focus was decreased significantly by 29% (p ͻ 0.01) compared to the non-epileptogenic area on the left sensorimotor cortex, which was dissected but not treated with cobalt. part of the decrease in glutamate could be related to the enhancement of in-vivo release from the epileptogenic lesion to the extra-cellular fluid. kindling is the best model for studying the development of the epileptic focus (epileptogenesis), it could be achieved by repeated intra-cerebral micro-injection of glutamate (1.5 µ mol), aspartate (0.75 µ mol) or nmda (2-4 n mol), or repeated electrical stimulations of specific brain regions. in addition, glutamate antagonists particularly those specifically acting on the nmda receptor type e.g. 2-amino-5-phosphonovaleric acid (ap5) and 2-amino-7-phosphonopheptanoic acid (ap7) have been found to inhibit seizures in epileptic animals and inhibit the development of electrically kindled epilepsy. pre-synaptic glutamate receptor agonists like (1s, 3s)-acpd the agonist of group ii, and l-ap 4 the agonist of group iii receptors has reduced ca ϩϩ uptake and glutamate release, thus it has inhibited epileptogenesis by preventing the increase in both seizure score and after-discharge duration. injection into fully kindled animals has produced an anti-epileptic effect by reducing the mean seizure score and by increasing the mean generalized seizure thresholds. this results suggest the mechanism by which pre-synaptically active glutamate receptor agonists block the development of the chronically epileptic state induced by electrical kinding, and indicate that their anticonvulsive activity is due to inhibition of pre-synaptic glutamate and/or asparate release following blockade of pre-synaptic ca ϩϩ entry. testing the changes in glutamate release from hyperactive brain tissues, and the effect of different glutamate agonists and antagonists, supports the role of glutamate in initiating the process of epileptogenesis, and contributes in developing new anti-epileptic agents. (this project was supported by a grant from alexo) the functional roles of cl(ϫ) and divalent cations in the na(ϩ)/cl(ϫ)/gaba cotransport were examined in xenopus oocytes expressing the human gat-1 (hgat-1) gaba transporter cdna. our results showed that cl(ϫ) was not absolutely required for na(ϩ)/gaba transport via the hgat-1 (loo et al., j biol. chem. 275:37414-37422, 2000) . the cl(ϫ) interacted with the transporter to modulate the binding of external na(ϩ). although hgat-1 transported cl(ϫ) across the membrane with a stoichiometry of 2na(ϩ) : 1cl(ϫ) : 1gaba, the transported cl(ϫ) did not contribute to the net charge translocated across the membrane, suggesting a cl(ϫ)/cl(ϫ) exchange mechanism during the gaba transport cycle. the gaba transport via the hgat-1 is also modulated by divalent cations. the uptake of [3h]-gaba was inhibited significantly when both ca(2ϩ) and mg(2ϩ) were removed from the uptake buffer. several divalent cations tested were individually able to sustain the gaba uptake. in contrast to uptake, the gaba efflux was enhanced significantly upon removal of both ca(2ϩ) and mg(2ϩ) from the efflux buffer. the gaba transporter inhibitor skf89976a blocked the enhanced efflux, suggesting that the hgat-1 operated faster in the reverse mode in the absence of external divalent cations. these results suggest a regulatory role for the divalent cations in gaba transport. merck sharp & dohme, neuroscience research centre, terlings park, harlow, essex, u.k. the role of alpha5 containing gaba a receptors in hippocampal synaptic function has been investigated using pharmacological and electrophysiological techniques, as well as following disruption of the alpha5 subunit gene in knockout mice (ko). in the ca1 region of the hippocampus the induction of long-term potentiation (ltp) is powerfully regulated by gaba mediated synaptic currents (ipscs). agents that inhibit gaba-mediated transmission potentiate ltp induction, whereas allosteric agonists such as benzodiazepine-site agonists which slow the decay kinetics of ipscs suppress ltp induction. in alpha5 ko mice paired pulse facilitation of the amplitude of excitatory synaptic potentials is selectively enhanced in the ca1 region but not dentate gyrus. likewise, the frequency and rise time of spontaneous ipscs were similar in wt and ko slices. however their amplitude was significantly smaller in ko mice. furthermore, a significantly greater proportion of ipscs were best fitted to a mono exponential function in ko mice compared to wt animals. thus alpha5 containing gaba a receptors contribute to functional postsynaptic receptors on ca1 pyramidal cells in the hippocampus and modulate a postsynaptic component of synaptic facilitation. pharmacological research institute, volgograd medical academy, volgograd, russia the purpose of the study is to investigate effect of phenil (karphedon, mephebut, gammoxin) and circle (pyracetam) gaba derivatives on reproductive function of stressed male rats. the adult male rats were stressed by immobilization exposure (2 hours) twice in week during 6 weeks. four from five groups of stressed males were given substances (daily) at doses: karphedon -50 mg/kg, mephebut -10 mg/kg, gammoxin -40 mg/kg, pyracetam -200 mg/kg. the treated males were mated with intact females during 12 days. after the mate the treated males and more in 10 days all the mated females were sacrificed and investigated. analysis of our data indicates that the time of spermatozoa motion and epididymal sperm counts were decreased 73.2% (p յ 0.05) and 49.1% (p յ 0.05) respectively when compared with their intact controls. gaba derivatives have a softening effect on functional parameters of spermatozoa stressed males. karphedon and pyracetam increased the time of motion spermatozoa 86.3% (p յ 0.05), karphedon and mephebut drew near sperm counts to intact control level. the result of mate show that pregnancy rate was increased (p յ 0.05) by stress exposure and pregnancy rate of females mated with gaba stressed males was some more (p ն 0.05) than that of intact controls. the general embryonic morality was increased twice by stress and so the number of embryos was reduced 48.8%. the gaba derivatives exposure to stressed male rats reduced the embryonic mortality of their posterity and increased the number of embryos to intact control level. our findings demonstrate that gaba derivatives administration has a protective effect on reproductive function of stressed male. transmission on the brain. the realization that glutamatergic pathways are involved in such diverse processes in epilepsy, ischemic brain damage and parkinsons' disease, is of a great practical interest. there are at least three functional classes of ionotropic glutamate receptors: n-methyl-d-aspartate (nmda), α-amino-3-hydroxy-5 methyl-4-isoxazolepropionic acid (ampa) and kainate (ka). other central neurotransmitter systems are under nmda influence. some data point on neuroprotective action of nmda antagonist on nigrostriatal pathway. in the present study female wistar rats were exposed during pregnancy with daily injected mk-801 (dizocilpine) 0.5 mg/kg sc. control rats received tap water only. behaviour of 3 month old male offsprings was investigated by several psychopharmacological methods. oral activity, yawning, locomotor activity, stereotypy and catalepsy were recorded following respective central dopamine receptors agonists and antagonists administration (skf 38393, quinpirole, apomorphine, haloperidol). our results indicate that mk-801 applied during pregnancy modulate reactivity of the central dopamine receptors in adult offspring rats. [ the development of mammalian ingestive behavior is characterized by a transition from suckling to chewing, two distinct motor behaviors. we hypothesize that this transition is accompanied by changes in brainstem circuitry underlying these movements. since glutamatergic neurotransmission is critical for the proper functioning of brainstem circuitry responsible for mastication, we investigated the development of glutamate receptors in trigeminal motoneurons (mo5) and mesencephalic trigeminal neurons (me5); neurons comprising the circuitry responsible for jaw movements. we conducted a series of receptor immunohistochemistry experiments that characterized the expression of iontotropic and metabotropic glutamate receptors (mglurs) during early postnatal development. the functional roles of nmda, ampa and mglurs in neonatal mo5 were investigated using in vitro electrophysiological experiments. results demonstrated that the spatial and temporal expression of ampa, nmda and group i and ii mglurs are developmentally regulated within and between mo5 and me5 during early development. electrophysiological data demonstrate that mglurs function pre-and postsynaptically to modulate synaptic transmission between trigeminal premotoneurons and mo5. furthermore, nmda induced bursting is developmentally regulated and coincident with the transition from suckling to chewing behaviors. our studies suggest that the transition from suckling to chewing is accompanied by changes in the composition and function of glutamate receptors. fetal life in down syndrome starts with normal neuronal density but impaired dendritic spines and synaptosomal structure r. weitzdoerfer 1 , m. dierssen 2 , m. fountoulakis 3 , and g. lubec 1 1 department of pediatrics, university of vienna, austria information on fetal brain in down syndrome (ds) is limited and there are only few histological, mainly anecdotal reports and no systematic study on the wiring of the brain in early prenatal life exist. histological methods are also hampered by inherent problems of morphometry of neuronal structures. it was therefore the aim of the study to evaluate neuronal loss, synaptic structures and dendritic spines in the fetus with down syndrome as compared to controls by biochemical measurements. 2 dimensional electrophoresis with subsequent mass spectroscopical identification of spots and their quantification with specific software was selected. this technique identifies proteins unambiguously and concomitantly on the same gel. fetal cortex samples were taken at autopsy with low postmortem time, homogenized and neuron specific enolase (nse) determined as a marker for neuronal density, the synaptosomal associated proteins alpha snap [soluble n-ethylmaleimidesensitive fusion (nsf) attachment protein], beta snap, snap 25 and the channel associated protein of synapse 110 (chapsyn 110) as markers for synaptosomal structures and drebrin (drb) as marker for dendritic spines. nse, chapsyn 110 and beta snap were comparable in the control fetus panel and in down syndrome fetuses. drebrin was significantly and remarkably reduced and not even detectable in several down syndrome brain samples. quantification of snap 25 revealed significantly reduced values in ds cortex and alpha snap was only present in half of the ds individuals. we conclude that at the time point of about 19 weeks of gestation (early second trimester) no neuronal loss can be detected but drebrin, a marker for dendritic spines and synaptosomal associated proteins alpha snap and snap 25 were significantly reduced indicating impaired synaptogenesis. early dendritic deterioration maybe leading to the degeneration of the dendritic tree and arborization, which is a hallmark of down syndrome from infancy. pathfinding of growing axons to reach their target during brain development is a subtle process needed to build up contacts between neurons. abnormalities in brain development in down syndrome (ds) are described in a couple of morphological reports but the molecular mechanisms underlying abnormal wiring in fetal ds brain are not yet elucidated. we therefore performed a study using the proteomic approach to show differences in protein levels involved in the guidance of axons between control and ds brain in early prenatal life. proteins obtained from autopsy of human fetal abortus were applied on 2-dimensional gel, identified and quantified. we quantified 5 members of the semaphorin/collapsin family, the dihydropyrimidinase related proteins 1-4 and the collapsin response mediator protein-5 (crmp-5) in 8 ds and 7 control cortex samples. drp-1 and crmp-5 levels were comparable in the control and ds samples. evaluation of drp-2, drp-3 and drp-4 revealed significantly decreased levels of 2 of the 15 spots assigned to drp-2 and increased levels of one spot assigned to drp-3 and increased drp-4 in ds brain. we conclude that as early as from the 19 th week of gestation pathfinding cues of the outgrowing axons are impaired in ds. these findings may help to elucidate mechanisms leading to abnormalities in neural migration of ds brain. inflammatory processes play an important role in the degeneration of basal forebrain cholinergic cells alzheimer's disease. the proinflammagen lipopolysaccharide (lps) was infused chronically into the basal forebrain of young rats. we then determined whether the administration of two novel nonsteroidal anti-inflammatory drugs or a pancaspase synthesis inhibitor, zvad, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. we also determined whether the administration of the non-competitive n-methyl-d-aspartate (nmda) receptor antagonist, memantine, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. chronic lps infusions decreased choline acetyltransferase activity and increased the number of activated microglia within the basal forebrain. caspases 3, 8 and 9 activity was increased in ventral caudate/putamen. non-steroidal antiinflammatory drug therapy attenuated the toxicity of the inflammation upon cholinergic cells and reduced caspases 3, 8, and 9 activity in the caudate/putamen. zvad significantly decreased the levels of caspases 3, 8 and 9 but did not provide neuroprotection for cholinergic neurons. memantine significantly attenuated the cytotoxic effects of chronic inflammation upon cholinergic cells. these results suggest that prostaglandins contribute to the degeneration of forebrain cholinergic neurons in alzheimer's disease and that the cytotoxic effects of prostaglandins occur upstream to nmda receptor activation. intracranial administration of n-methyl-d-aspartate (nmda) receptor antagonists block learning of classical and avoidance conditioning in goldfish. studies with goldfish have shown that nmda receptors are mostly dense in the telencephalon and telencephalon ablation impairs avoidance learning. the present study investigated amnestic effects of microinjection of nmda receptor antagonist ap5 to the goldfish telencephalon in avoidance conditioning. in experiment 1, fish received no injection or microinjections of saline or various doses of ap5 to their telencephalon 20 minutes before three semiweekly training sessions. fish were tested without injec-tions in session 4. a one-way anova with multiple comparisons on the test scores showed that ap5 produced anterograde amnesia in a dose-dependent manner. in experiment 2, fish received several training sessions and a microinjection of various doses of ap5 20 minutes before testing. the test scores showed that ap5 did not decrease avoidance responses, suggesting that microinjection of ap5 did not impair performance processes. in experiment 3, fish received microinjections of ap5 or saline to their telencephalon immediately following three semiweekly training sessions and were tested without injections in session 4. a one-way anova on the test scores showed that ap5 did not produce retrograde amnesia. (supported by gvsu grant-in-aid.) tryptophan modulates striatal serotonergic activity relative to fatigue t. yamamoto 1 and e. a. newsholme 2 1 health science laboratory, tezukayama university, nara, japan 2 department of biochemistry, university of oxford, u.k. we have been reported that mechanism of fatigue in the brain relates to enhanced extracellular tryptophan and serotonergic function. brain concentration of tryptophan is not only dependent on the change of tryptophan which originates from the centarl nervous system, but also enhance tryptophan entering the brain from the blood-brain barrier and peripheral circulating tryptophan which is a trigger. supplementation of ltryptophan (2 um) into the incubation medium with the synaptosomal striatum causes tryptophan to the extrasynaptosomal release by high kϩ stimulation. injecting l-tryptophan (1 mm/ 30 min) into the left striatum by microdialysis method can induce early fatigue for running time of rats. on the other hand, tryptophan deficiency rats (body weight average 200 g) were made by tryptophan free feeding for 2 weeks, and the rat's running time increased (ͼ100 min difference). these results suggests that tryptophan is a potent active substance for fatigue in the brain. the active zone may be presynaptic terminal and the tryptophan itself may be releasing neuromodulators. (we appreciate that tryptophan free diet was provided by ajinomoto co., inc., japan.) our recent studies on the distribution of free d-serine, together with the d-serine action on the glycine site of the nmda type glutamate receptor, suggest that the d-serine can be an endogenous modulator of the nmda receptor. to explore the possible removal systems for brain d-serine signaling, we have evaluated the uptake of [3h]d-serine into the synaptosomal p2 fraction from the rat cerebral cortex. the cortical p2 fraction was able to accumulate [3h]d-serine in a temperatureand ph-dependent and saturable manner. the kinetic analysis indicates that cortical d-serine transport occurs by an apparent single-component system with km value of 283 µm and a vmax value of 207 pmol/mg protein/min. depletion of na ϩ and cl ϫ ions remarkably decreased d-serine uptake into the cortical p2 fraction. the pharmacological profile of the inhibition of dserine uptake by various amino acids was different from those of glycine uptake system and other amino acid transporters reported. d-serine uptake activity was preferentially observed in the brain tissues such as cerebral cortex and cerebellum to the peripheral tissues. the present data support the view that the endogenous d-serine is taken up mainly through a carriermediated transport system to regulate the extracellular concentration in the mammalian brain. a. bocheva et al. the mammalian brain contains all the urea cycle intermediates, whereas enzymes participating in the conversion of lornithine (l-orn) into l-citrulline (l-cit) are absent, resulting in an incomplete urea cycle. the discovery of nitric oxide (no) synthase that catalyses the formation of no and l-citrulline as a co-product from l-arginine (l-arg) in the brain has indicated an additional pathway for l-arg metabolism. l-canavanine (l-cav), is a potent antimetabolite and structural analog of larginine, produced by legumes such as the jack bean, canavalia ensiformis. l-canaline (l-can) is a potent inhibitor of ornithine aminotransferase. our previous results indicated that l-cav, l-cit, l-arg, and l-orn exerted an antinociceptive effect, whereas l-canaline induced hyperalgesia in rat. l-canavanine exert stronger antinociceptive effect than l-arginine, l-ornithine and l-citrulline. the aim of the present study was to investigate are d-arg, l-cav and naloxone reversed the analdesic effects of l-ornithine, l-citrulline and l-arginine. the experiments were carried out on male wistar rats. the changes in the mechanical nociceptive threshold of the rats were measured by the radall-selitto paw pressure test using and analgesimeter (ugo basile). the amino acids were applied intracerebroventricularly (i.c.v.) at a dose 20 µg/rat. the present results shown that d-arg, l-cav and naloxone reversed antinociception. the regulation of lysine metabolism in cereal crops r. a. azevedo 1 , p. j. lea 2 , s. a. gaziola 1 , a. p. pellegrino 1 , and s. m. g. molina 1 1 departamento de genética, escola superior de agricultura luiz de queiroz, universidade de são paulo, brazil 2 department of biological sciences, university of lancaster, u.k. a major nutritional drawback of cereal seeds is a deficiency in some amino acids, in particular lysine. biochemical, molecular and genetic studies have considerably increased our knowledge concerning the regulation of the aspartate pathway, by which lysine is synthesized. among the enzymes involved in lysine metabolism, aspartate kinase (ak) and dihydrodipicolinate synthase (dhdps) control the regulation of lysine biosynthesis, whereas lysine: 2-oxoglutarate reductase (lor) and saccharopine dehydrogenase (sdh), have been shown to play a key role in the breakdown of lysine. in general, lysine overproduction can be obtained by altering the sensitivity of dhdps to lysine, but accumulation of this amino acid in cereal seeds requires further manipulation of lor and/or sdh. this suggestion is strongly supported by five main points: (1) cereal mutant or transgenic plants do not exhibit any significant accumulation of lysine in seeds, but only in other tissues. (2) the enzymes of lysine degradation, lor and sdh, are endosperm specific in cereals only. (3) the opaque-2 mutant, which exhibits higher concentration of soluble lysine and protein lysine in the seed, contains several-fold lower lor and 2-fold lower sdh activity when compared to the wild-type maize. this reduction in activity in the opaque-2 mutant is due to a reduced protein lor-sdh concentration by reduction of the zlkrsdh gene transcript. furthermore, the opaque-2 maize gene has been shown to regulate ak and lor activity. (4) intermediates of lysine catabolism accumulated in the seeds of soybean and canola lysine overproducing plants, suggesting the presence of reduced lor and/or sdh activities. (5) among cereals and although still below the recommend values by fao, rice exhibits the higher concentration of lysine, but lor and sdh are present in much lower activities. also, in phaseolus vulgaris, lor and sdh activities were shown to be around 10fold lower then in maize endosperm. the regulation of the lor activity is complex and involves a calcium dependent phosphorylation/dephosphorylation mechanism. it remains to be seen whether this latter mechanism can be controlled, so as to allow the production of more crop plants that contain elevated concentrations of lysine in the seed. the genetic progress for nue can be accelerated with the use of secondary traits that possess high inheritance and correlation with productivity. several traits have been studied such as chlorophyll concentration, plant height, leaf senescence, anthesis-silking interval, kernel number, activities of enzymes of n assimilation and loci of quantitative traits for assisted selection. (we are grateful for financial support from fapesp, brazil and the british council.) (termed hyperaccumulators) that grow on metalliferous soils, are able to translocate cd from the roots and accumulate it in high concentrations in the shoots. cd may be detoxified in plants by combination with a family of sulphur rich peptides termed phytochelatins. cd has the capacity to inhibit a range of enzyme activities in plants, in particular those of the calvin cycle and chlorophyll biosynthesis. evidence that cd causes the production of reactive oxygen species (ros) has also been obtained. we have investigated the antioxidant responses of radish, soybean and sugarcane to cd treatment. seedlings were grown in increasing concentrations of cdcl 2 , ranging from 0.01-1 mm, for up to 96 h in a hydroponic system. analysis of cd uptake indicated that most of the cd accumulated in the roots, but some was also translocated and accumulated in the leaves. roots and leaves were analysed for catalase (cat), glutathione reductase (gr) and superoxide dismutase (sod) activities. gr activity increased considerably in the roots of all plant species tested after exposure to the metal, indicating a direct correlation with cd accumulation. cat activity also increased in roots but to a much lesser extent when compared to gr and also varied depending upon the plant species. the analysis of native page enzyme activity staining, revealed several sod isoenzymes in leaves of all plant species, however, only in radish was a clear increase in activity observed. the results suggest that in these plants, the activity of antioxidant enzymes responds to cd treatment. the main response may be via the activation of the ascorbate-glutathione cycle for the removal of hydrogen peroxide, or to ensure the availability of glutathione for the synthesis of cd-binding proteins. (we are grateful for financial support from fapesp, brazil and the british council.) all plant cells, tissues and organs provide the biosynthetic machinery and capacity to synthesise aliphatic polyamines. however, in physiological conditions only some organs and tissues synthesise polyamines, such as apical buds and sprouts, root apex, lateral buds of branches and secondary roots, as well as superficial layers of young stems and leaves, like epidermis, subepidermis and parenchyma cells. apical roots can also synthesise polyamines, but these activities in physiological conditions are lower than that of the shoots. this patterns recalls the one of auxins. polyamines are accumulated in high concentrations in storage organs, such as seeds, but not in tubers like helianthus tuberosus, potato or tuberised roots such as the carrot. also some fruit, e.g. oranges, contain high level of free polyamines, putrescine in particular. all other organs obtain polyamines through translocation via phloem tubes and xylem vessels. in plants, in addition to free polyamines, many polyamines are conjugated to hydroxycinnamic acids, the hydroxycinnamic amines, that only rarely represented outside the plant kingdom. this compounds are paticularly abundant in solanaceae family, where they can represent as much as 90% of the total polyamine pool, but they can be detected in different concentrations in many other families. the role of free and conjugated polyamines and their importance in food is discussed. drought, salinity or other environmental stressors promote the accumulation of free amino acids, amines and other organic n-metabolites with low molecular weight. in this contribution the influence of drought on the accumulation of amino acids, polyamines and trigonelline in growing barley plants and barley grains was examined. in comparison to non-stressed plants we obtained in stressed plants, exposed to drought before flowering, a higher concentration of proline (increase: 4-fold), n-trimethylglycine (2-fold), histidine (3-fold), tryptophane (1,7-fold), putrescine (1,4-fold), spermine (1,7-fold) and trigonelline (2-fold) in the dry matter of barley sprouts. in addition to this, drought caused an increase of the n-content in the plant biomass (35%) as a result of growth inhibition (34%). six weeks later the content of soluble n-metabolites and protein was analyzed in non-stressed and pre-stressed barley plants again. during this reproductive period of plant development all the test groups were cultivated under the same moisture conditions. the analysis of n-metabolites in the ripening grains showed, surprisingly an after-effect of the drought stress. for example, in grains of pre-stressed barley the concentrations of free proline, histidine, tryptophane and asxϩglx were threefold to fivefold higher than in grains of non-stressed barley. depending on the resistance of barley cultivars to drought the biochemical response was different: in plants with low resistance the increase of amino acids and amines was higher than in resistant cultivars. however, resistant cultivars have already high genuine concentrations of n-metabolities in non-stressed plants. by treatments with choline or 2-aminoethanol the stresspromoted accumulation of amino acids and trigonelline was diminished. consequently, different biochemical responses of cereals to drought result in changes of product quality and nitrogen use. our goal is to increase the lysine content in corn. we have used genetic engineering to increase lysine synthesis and to prevent metabolic breakdown of lysine. to increase synthesis we circumvented the normal feedback control of a key enzyme in the lysine biosynthetic pathway, dihydrodipicolinic acid synthase (dhdps). lysine-feedback-insensitive dhdps, encoded by the corynebacterium dapa gene, was expressed from seed-specific promoters in transformed corn seeds. expression of dhdps in the corn embryo, but not in the corn endosperm, resulted in a 20 to 30-fold increase in the accumulation of free lysine in the seeds and the total seed lysine content nearly doubled. lysine breakdown products have been observed in transgenic seeds that accumulate high levels of free lysine. we isolated a corn gene for the bifunctional enzyme lysine ketoglutarate reductase (lkr)/saccharopine dehydrogenase (sdh), which catalyzes the first two steps in lysine breakdown. knockout of lkr/sdh in corn by either mutation or genetic engineering results in a 10-fold increase in seed free lysine. combination of feedback-insensitive dhdps with knockout of lkr/sdh results in 2 to 3-fold higher levels of free lysine than dhdps alone. no adverse effects on seed or plant agronomic performance are associated with the high lysine trait. biotechnology center for agricultural and the environment and the plant science department, rutgers university, new brunswick, new jersey, u.s.a. 5ј-adenylylsulfate (aps) reductase catalyzes a key reaction in the plant sulfate assimilation pathway leasing to the synthesis of cysteine and the antioxidant glutathione. in arabidopsis thaliana aps reductase is encoded by a family of 3 genes. in vitro studies revealed that the enzyme product derived from one of the aps reductase genes (apr1) is activated by oxidation, probably through the formation of a disulfide bond. redox titrations show that the regulation site has a midpoint potential of ϫ327 mv at ph 8.5 and involves a 2-electron redox reaction. exposure of a variety of plants to ozone induces a rapid increase in aps reductase activity that correlates with the oxidation of the glutathione pool and is followed by an increase in free cysteine and total glutathione. during the response to ozone the level of immuno-detectable aps reductase enzyme does not increase. treatment of a. thaliana seedlings with oxidized glutathione or paraquat induces aps reductase activity even when transcription or translation is blocked with inhibitors. the results suggest that a post-translational mechanism controls aps reductase. a model is proposed whereby redox regulation of aps reductase provides a rapidly responding, self-regulating mechanism to control the glutathione synthesis necessary to combat oxidative stress. in aspergillus nidulans the structural genes coding for nitrate reductase (niad) and nitrite reductase (niia), share a common promotor region of 1,200 bp. we have previously characterized in vitro and in vivo the physiologically relevant cisacting elements for the two synergistically acting transcriptional activators, nira and area. we have further shown that area is constitutively bound to a central cluster of four gata sites and is directly involved in opening the chromatin structure over the promoter region and thus making additional cis-acting binding sites accesible. here we show that the asymmetric mode of nira-dna interaction determined in vitro is also found in vivo. binding of the nira transactivator is not constitutive as in other binuclear c 6 -zn ϩϩ -cluster proteins but depends on nitrate induction and additionally, on the presence of a wild type area allele. dissecting the role of area further, we found that it is required for intracellular nitrate accumulation and therefore could indirectly excert its effect on nira via inducer exclusion. but in a strain accumulating nitrate independently of area nira binding and chromatin rearrangement is not triggered by nitrate in the absence of area. v. nikiforova 1 , m. zeh 1 , o. kreft 1 , s. maimann 1 , h. hesse 2 , and r. höfgen 1 1 max-planck-institut für molekulare pflanzenphysiologie, potsdam, and 2 institut für biologie, angewandte genetik, freie universität berlin, germany higher plants, being a source of reduced sulfur for animal nutrition, assimilate inorganic sulfate into cysteine which is subsequently converted to methionine, another sulfur-containing amino acid. in order to investigate the possible regulatory points of the cysteine and methionine biosynthesis pathway a series of transgenic potato plants was engineered using clones encoding enzymes of the branched pathway from serine to cysteine as a pathway intermediate and from aspartate further on to methionine. increased cysteine levels were obtained in the leaves of serine acetyltransferase (sat) sense and cystathionine -lyase (cbl) antisense transformants. furthermore, glutathione levels were elevated in sat plants while downregulation of cbl was desastrous for plant growth, eventually. increased methionine levels were successfully obtained in potato by antisense inhibition of threonine synthase (ts). accumulation of free methionine was not only observed in source leaf tissues but as well in tubers. this enzyme competes with cystathionine gamma-synthase for the common substrate o-phosphohomoserine at the branchpoint between threonine and methionine synthesis, respectively. important control points of the biosynthesis of cysteine and methionine in potato, thus, turned out to be sat and ts, while further studies on overexpression of cystathionine gamma-synthase, cbl and ms did not reveal any substantial effect on potato methionine biosynthesis. dniepropetrovsk national university, board of biophysics and biochemistry, ukraine amino acids in root exudates of plants may be chelate agents as an alpha-amino acid can act like a bidentate ligand, forming a five-membered heterocyclic ring with suitable metal cations thus increasing mobility of metals. recently we have showed that application of growth regulators led to sharp increase of root exudative activity of some cultural (zea maize l.) and wild cereals (festuca rubra l., lollium perenne l.) during first days of germination. in this work we present results obtained in experiments with lollium perenne l., grown on sterile sand and on soils contaminated with great quantities of zn. detailed analysis of amino acid content of root exudates of several types of maize (hybrid, several lines, an opaque-2 mutant line) showed that the specie had more certain amino acids (cysteine, aspartic and glutamic acids and their amides, serine) in root exudates than cultural ones. these amino acids has more possibility for chelation due to existance of one more polar or ionogenic functional groop. seeds of lollium perenne l. were treated with growth regulater and planted on soils contaminated with salts of zink. it was shown that during 15 days of germination quantity of zn in primary leaves increased from 121,46 to 243,75% and decreased in soil: in upper layer from 95,74 to 85,07, midde layer from 95,83 to 82,04, lower layer from 85,72 to 72,74 mkg/kg correspondingly. thus, it was shown that stimulation of root exudative activity by pretreatment with a growyh regulator may be succesful in cleaning of soils and basicly this is a good method for phytoremediation. erenol exerted the strongest effect. exercise completely abolished the levels of cysteine in the atrial heart muscle. propranolol, isoproterenol, caffeine and pentylenetetrazol increased the ratio of cysteine to the total free amino acids in the atrial muscle, while physical stress and all cardioactive drugs tested increased this ratio in the ventricle muscle. disappearance of cysteine from the heart's atrial muscle after intensive exercise may be attributed to its utilization for atrial natriuretic factor and/or for endothelin synthesis, during stress. on the other hand it seems that hypoxia and isoproterenol are strong stimulants of no production, and consequently decrease the tissue levels of l-arginine, which is the major endogenous donor of no acting as the endothelin antagonist. measurement of serum levels of vitamin b12 is a screening test for detection of deficiency of this vitamin but low levels do not always indicate a deficiency of the vitamin. measurements of serum homocysteine and methylmalonic acid (mma) are used to confirm this deficiency because two enzymes involved in their metabolism have been shown to require vitamin b12, but these results can also be inaccurate. vitamin b12 deficient white cells exhibit ultrascopic nuclear appenages which have been shown to contain dna; this finding could possibly be used as another confirmatory test of vitamin b12 deficiency. twenty-seven patients (mean age -76.6 years) with low serum b12 were studied by electron microscopic determination of the percent of neutrophils exhibiting these appendages and routine clinical parameters. only one patient did not have nuclear appendages; the others had a range of 0.8%-17.6% of neutrophils examined. there was a significant correlation of homocysteine (r ϭ .46, p ͻ .05) and mma (r ϭ .53, p ͻ .01) with serum b12 levels but no correlation of appendage number (r ϭ .18) with serum b12. there was no correlation of appendage number with homocysteine (r ϭ .25) or mma (r ϭ .04). these results suggest that b12-deficient white cell nuclear appendages do not measure the same metabolic pathways as homocysteine and methylmalonic acid and may be useful in confirmation of vitamin b12 deficiency. further extensive clinical evalution would be necessary to explore this possibility. the hypothesis: "l-theanine has relaxing effects of central nervous system of human beings", was verified by electroencephalographical methods. methods: 14 male, healthy sport-students, free of drugs or stimulants, participated weekly in a cross-over study. after exhaustive bicycle-ergometer test as an individual, reliable, stress model, the subjects recovered by lying in a segregated shaded room. three testdrinks with different l-theanine content (d1 ϭ placebo, d2 ϭ 50 mg, d3 ϭ 200 mg) were given in a randomised, double-blind order. all test-conditions were standardized strictly. eeg-recordings (closed eyes) were carried out (m1 ϭ 3 min. after stress/before testdrink, m2 ϭ 30 min.-, m3 ϭ 45 min.-, m4 ϭ 60 min.-, m5 ϭ 120 min. after testdrink) with the cateem ® system. absolute and relative eeg-spectralpower were examined. results: significant reductions in all frequencies (exception theta-power) were found in early recovery, being not significant influenced by testdrinks. qualitative different behavior trends were found in frontal-, central-, occipital-regions with increased alpha1, theta (frontal) and decreasing beta1 relative-power earlier in recovery with d3. these findings were related to relaxing effects. after ingestion of l-theanine alpha2-, beta1-power at occipital regions decreased faster (m2) to placebo recovery levels (m3/m4). thus it may be concluded that l-theanine has no pharmaceutical effect on the down regulation system but supports the physiological mechanisms during recovery after physical stress in human brain. arginine and cysteine in muscle cytosol of rats' heart after exercise, hypoxia or challenge with six selected cardioactive drugs r. brus 1 , j. gabryś 2 , j. konecki 2 , and j. shani 3 1 department of pharmacology and 2 department of histology and embriology, medical university of silesia, zabrze, poland 3 department of pharmacology, the hebrew university school of pharmacy, jerusalem, israel levels of the amino acid l-arginine (a major endogenous donor of nitric oxide-no), cysteine (sulfur-containing amino acid, important for atriopeptins and endothelins synthesis), and of total free amino acids, were assayed by gas-liquid chromatography in cytosols of rats' atrial and ventricular muscle cardiomyocytes. the tissues were assayed after the rats had been exposed to either exercise (swimming), hypoxia or one of six cardioactive drugs such as propranolol, digoxin, pentylenetetrazol, reserpine, isoproterenol and caffeine. physical stress and the examined drugs significantly reduced the total amount of cytosolic free amino acids in both cardiac muscles. in the cytosol of the heart atrial muscle, reserpine, propranolol and pentylenetetrazol increased the relative content of l-arginine, while hypoxia and digoxin decreased it. in the cytosol of the ventricular heart muscle, hypoxia and all six drugs used, decreased the relative levels of l-arginine. hypoxia and isoprot-addition of somatostatin-14 or of some of its analogs was found to cause a selective inhibition, up to 50%, of the uptake of large neutral amino acids by isolated brain microvessels. although the luminal and abluminal sides of brain endothelial cells are both capable of taking up large neutral amino acids, only the uptake from the abluminal side was apparently inhibited by somatostatin. the involvement of a type-2 somatostatin receptor was suggested by assays with a series of receptorspecific somatostatin agonists, and was confirmed by the inhibition release caused by a specific type-2 receptor antagonist. a type-2 specific mrna was indeed shown to be present both in bovine brain microvessels ex vivo and in primary cultures of endothelial cells from rat brain microvessels. hemorphins represent a bioactive peptide class which contents between 4 and 10 amino acids and generated from the proteolysis of an hemoglobin "strategic zone". many activities have been related to hemorphins such as in vitro anti tumour effect, analgesia effects in vivo, and a potential role in the renin angiotensin system (ras). as far as their activity towards the ras is concerned, it was demonstrated that they could inhibit angiotensin converting enzyme (ace) and aminopeptidase n activity. so they could reduce angiotensin ii formation and angiotensin iv degradation. moreover some hemorphins, lvv-hemorphin-7 and vvhemorphin-7, could behave like angiotensin iv receptor binding competitor. further it could be interesting to study the angiotensin iv potentiality to interact with ace. inhibition studies showed that it was possible that angiotensin iv could behave like a competitive inhibitor of ace. so some hemorphins could interact at different ras steps to inhibit ace. additionally to their inhibition of angiotensin i conversion, they could inhibit angiotensin iv degradation and consequently cause ace feedback inhibition. inhibition studies have been checked with ras natural substrate (angiotensin i) and confirmed that angiotensin iv, vv-hemorphin-7 and mainly lvv-hemorphin-7 could be natural ace inhibitors. so the hemorphins regulatory role in the ras appears to be more and more probable. the role of administration of each of methionine and finastride on the testicular function of both normal and prostate precancerous old male rats was investigated. for normal animals, neither methionine nor finastride has exerted any significant change in the hormonal profile after teatment for 20 days. however methionine alone could exert a significant change in both testosterone and prostatic specific antigen {psa} levels after treatment for 40 days. on the other hand, both methionine and finasteride significantly increased the levels of testosterone and androstenedione, whileas markedly reduced the levels of dihydrotestosterone and prostatic specific antigen {psa} after treatment of prostate precancerous old male rats for a period of 20 days. noteworthy, continuation of treatment for aperiod of 20 days realized marked improvement of hormonal profile of the prostate precancerous old male rats. several observations in our department point to some role of glycine in fatigue and exercise. 1) in the framework of a study on the involvement of one-carbon matabolism in patients suffering from a polymorphic episodic psychosis, amino acid loading tests with serine, glycine and alanine were performed. a few hours after the administration of glycine, approximately 40% of the patients reported overwhelming feelings of fatigue and/or showed vegetative symptoms. 2) in patients suffering from chronic fatigue syndrome, we found increased plasma levels of glycine in 20% of the female patients. moreover, 60-70% of these patients omplained about a distorted sensory perception of objects. 3) young soccer players were observed during a period of 10 months, while in the course of this period eight blood samples were taken for amino acid analysis. based on the number and severity of injuries this population was divided into injury-prone and not injury-prone soccer players. it was found that in injury-prone soccer players plasma glycine levels during the whole observation period were significantly lower than in subjects who were not injury-prone. the consequences of the above mentioned observations will be discussed. institute of sportsmedicine, university of paderborn, germany 50 percent of amino acids in green tea leaves are represented by l-theanine (5-n-ethylglutamine). previous rat experiments demonstrated effects of l-theanine to act on metabolism of neurotransmitters. it was therefore suggested that is causes the relaxing effects of green tea. to examine its influence as a component of a drink on the sympathetic nervous system after maximal physical exercise skin resistance measurements through electrosympathicography (esg) were used. after individual maximal exercise on a bicycle-ergometer testdrinks with different amounts of l-theanine (0, 50 and 200 mg) were administered to 14 healthy volunteers in a randomised cross-over double-blind distribution on a weekly base. esg was monitored before and immediately after exercise as well as 13, 30, 45, 60, 75 and 135 minutes after end of exercise. all testconditions were standardized strictly. a characteristic esgcourse with subsequent qualities could be shown: 1. decreasing skin resistances after exercise could be established in each volunteer. 2. esg-activation levels before exercise could not even be reached again after a period of regeneration of 2 1 / 4 hours. 3. maximal electrodermal activity did not appear immediately after exercise, but after 13 minutes. however, l-theanine could not significantly influence peripheral sympathetic electrodermal activity during the regeneration after maximal physical exercise. a. mero 1 and h. pitkänen 1,2 1 neuromuscular research center, department of biology of physical activity, university of jyväskylä, and 2 rehabilitation center of kankaanpää, finland essential amino acid leucine has many important roles in the body. therefore the purpose of the present study was to investigate if leucine supplementation has effects on serum amino acid profile and performance following training period or following single training sessions. all experiments were carried out in a randomized double blind cross-over procedure during a training season. thirty six adult male track and field power athletes served as subjects. in experiment 1 ten of them were given leucine (50 mg/kg body weight per day) as tablets. the concentration of leucine decreased significantly (20%) in the placebo group (p; n ϭ 10) during 5 weeks but not when leucine was taken. also total amino acids (taas) decreased strongly (19%) during 5 weeks when dally protein intake was 1.26 g/kg body weight. in experiment 2 the subjects (n ϭ 16) carried out a single strength training seasion (sts) and consumed a drink containing leucine 100 mg/kg body weight. following sts leucine in serum increased by 11% (ns) when leucine was taken but decreased strongly (30%) in p, in experiment 3 the subjects (n ϭ 12) underwent at 7 days interval two maximal anaerobic running exercise (mare) tests on treadmill (n ϫ 20s with a recovery of 100s between the runs) until exhaustion. the subjects consumed drinks containing leucine (200 mg/kg body weight) or placebo 50 min before the test runs. following mare the concentration of leucine strongly increased by 28% whereas isoleucine (25%) and valine (15%) strongly decreased with the supplementation but no changes occurred in p. there were no improvements in physical performance either in mare or in explosive strength (experiment 2) with leucine supplementation. the date suggest that leucine supplementation during a training period and before single training sessions prevents decreases in serum concentration of leucine and may have also effects on some other single amino acids. this may be beneficial during intensive training although improvements in performance were not observed in this study. since there are only limited data regarding effects of training period or training sessions on serum amino acid profile, the purpose of this study was to investigate serum amino acid changes following training period and following three different training sessions. the subjects consisted of 31 track and field adult male power athletes. in experiment 1 eleven of them performed a 5-week training period including a training sessions per week, which included sprint work, speed endurance work, endurance work, weight training, and jumps. significant decreases in the fasting concentrations of total amino acids (taas) (19%), branched chain amino acids (bcaas) (21%), essential amino acids (eaas) (19%) and leucine (20%) were observed following training with the daily protein intake of 1.26 g/kg body weight. in experiment 2 eleven subjects performed a short run session (srs) of 3 ϫ 4 ϫ 60 m with recoveries of 120 s and 360 s, and a long run session (lrs) of 20 s runs with recoveries of 100 s until exhaustion. there were no significant changes in taas following the sessions but bcaas decreased by 8% in srs and by 7% in lrs. leucine decreased by 10% following srs but only by 5% (ns) following lrs. the peak blood lactate concentrations after srs and lrs were 13.8 ϯ 1.9 mmol/l and 16.4 ϯ 1.3 mmol/l, respectively. in experiment 3 sixteen subjects carried out a strength training session (sts), which consisted of jumps and heavy resistance exercises (speed and maximum strength) during 90 minutes. the taas decreased significantly by 14%, bcaas by 24% and leucine by 30% following sts, while the peak blood lactate concentration was 2.2 ϯ 0.4 mmol/l. these data indicate that remarkable decreases occur in the concentration of amino acids during a training period with the daily protein intake of 1.26 g/kg body weight. the decreases in serum amino acids are more pronounced following a strength training session than following lactic anaerobic running sessions. glutamine acts as a multipurpose regulator of amino acid and peptide transport across the blood-brain barrier departments of cellular biotechnology and haematology and of biochemical sciences, university "la sapienza", rome, italy isolated brain microvessels, the in vitro equivalent of the blood-brain barrier, have distinct na ϩ -independent uptake systems for the uptake of large hydrophobic amino acids, of enkephalins and of deltorphins, as shown by the absence of reciprocal inhibition. both d-and l-glutamine were capable, if added to the extracellular buffer, of exerting a competitive inhibition on the uptake of all these substrates. a trans-stimulatory effect was instead induced, in all cases, by l-glutamine preloading of the microvessels -the d-stereoisomer being instead ineffective, probably because of only l-glutamine could be taken up, in a concentrative manner, by some na ϩ -dependent concentrative system(s). all the na ϩ -independent systems present in brain microvessels seem therefore to share some structural feature responsible for their common susceptibility to interference by l-glutamine. this amino acid, whose synthesis can take place in the astrocytes, in the pericytes and also in the endothelial cells of the microvessels, plays a critical role in regulating the movements of several different substrates across the blood-brain barrier. department of applied bioorganic chemistry, division of life science, graduate school of agricultural science, tohoku university, aoba-ku, sendai, japan isolation and structure analysis of two amino acids from bovine ligamentum nuchae elastin hydrolysates revealed the presence of pyridine cross-links in elastin. the structures of these amino acids were determined to have 3,4,5-and 2,3,5-trisubstituted pyridine skeletons both with three carboxylic acids and a mass of 396 (c 18 h 28 n 4 o 6 ), identified as 4-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)pyridine and 2-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3carboxypropyl)-pyridine. we have named these pyridine cross-links, desmopyridine (desp) and isodesmopyridine (idp), respectively. structure analysis of these pyridine crosslinks implied that the formation of these cross-links involved the condensation reaction between ammonia and allysine. the elastin incubated with ammonium chloride showed desp and idp levels increased as the allysine content decreased. desp and idp were measured by hplc with uv detection and were found in a variety of bovine tissues. the desp/desmosine and idp/isodesmosine ratios in aorta elastin were higher than in other tissues. desp and idp contents in human aorta elastin were found to be gradually increased with age. the concentration of idp was significantly elevated in aorta elastin of rat with chronic liver cirrhosis induced by carbon tetrachloride when compared with normal rats. the provision of glutamine to marathon runners has resulted in a decreased, self-reported incidence of illness. increasing evidence -in vitro; and in vivo suggests that neutrophils in humans may benefit from exogenous glutamine. the provision of glutamine in vivo should replete the marked decrease in the blood concentration observed after stress such as clinical trauma or prolonged, strenuous exercise. beneficial effects of glutamine supplementation include increased phagocytic activity and reactive oxygen intermediate production in vitro; decreased neutrophilia and il-8 production (a chemoattractant for neutrophils) in vivo and ex vivo. the aim of the present study was to establish whether glutamine supplementation in vitro and in vivo affects neutrophil function at rest and after exhaustive exercise. in addition, it was planned to establish the presence of glutaminase in human neutrophils, which has not yet been achieved, although glutaminase is present in rat neutrophils. methods: blood samples were taken from marathon runners receiving either glutamine or placebo, immediately after and one hour after a race. measurements included the plasma concentration of glutamine (enzymatic assay), il-8 production (elisa), and neutrophil activity. the latter was measured with two different techniques for measuring oxidative burst in whole blood, one of which was a novel chemiluminescence assay (knight scientific ltd, u.k.) with the fluorescent label, pholasin, and two different stimuli, f-met-leu-phe (fmlp) and phorbol-myristate-acetate (pma). in addition, isolated whole cells and subcellular neutrophil fractions were assayed for the presence of glutaminase. results: the plasma glutamine concentration was reduced overall by 26% 1 hr after the race (p ͻ 0.02). there was an apparent decrease (only close to significance, p ͻ 0.13) in il-8 production in the glutamine group compared with the placebo group. neutrophil function did not change between groups at any stage. the incidence of illness was 46% higher in the placebo group than the glutamine group in the week after the race. neutrophils from four out of six subjects gave an increased response (39.2%) to fmlp when incubated with glutamine compared with no glutamine, and four out of four gave an increased response to pma (31.1%). in the fmlp experiments there were two individuals who did not respond to the addition of glutamine. however, the response was not diminished whether or not glutamine was present. in separate studies, the effect of glutamine on lipopolysaccharide-induced il-8 production was also monitored. conclusions: the provision of glutamine after prolonged, exhaustive exercise appears to modify exercise-induced neutrophilia via a reduction in il-8 production and to reduce the incidence of illness in the following week. in vitro data suggest a role for glutamine in neutrophil metabolism. disappointingly, little or no evidence of the presence of glutaminase was found in human neutrophils. the three different methods used, freeze-thaw, homogenisation, nebulisation were apparently not sufficient to break open the granules. current studies are addressing this problem. r. j. ward 1 and l. m. castell 2 departments of biochemistry, 1 university catholique de louvain, belgium 2 oxford university, oxford, u.k. it is essential that the developing muscle has adequate amino acids for the synthesis of actin and myosin as well as those required for a multitude of enzymes involved in muscle metabolism. with carbohydrates and lipids, the body is able to store a reserve as glycogen and triglycerides respectively; however this is not the case with amino acids creatine supplementation in increasingly being used as a dietary supplement by athletes during high intensity, short term exercise to improve physical performance since it is converted in the muscle to phosphocreatine. transporters which permit creatine to cross the muscle membrane namely crt1 and crt2 (a na ϩ and clј dependent mechanism) have now been identified. creatine uptake is enhanced by the ingestion of carbohydrate at the same time as supplementary creatine. this may be due to increased circulating levels of insulin or insulin-like growth factor 1. more recently attention has been focussed upon the various transporters for amino acids across the muscle membrane. certain criteria are needed for the amino acids to enter the blood which include the presence of specific carriers for its transport across cells of the gastrointestinal tract, such as enterocytes, as well as minimal metabolism within these cells. a wide number of different transporters has been identified, which include neutral amino acids and cationic amino acids. despite the evidence which suggests that supplementation with some amino acids can influence metabolism, and therefore athletic performance, much more experimental work is still required in this area. m. weiss 1 , t. barthel 1 , r. schnittker 1 , k. e. geiss 3 , w. falke 3 , and l. r. juneja 2 1 university of paderborn, germany 2 taiyo kagaku co., yokkaichi, japan, 3 isme gmbh mörfelden, germany in animal studies l-theanine was shown to influence neurotransmitter systems. thus it may be helpful in managing stress regulation. so we observed the down regulation after physical stress in the brain (measured by eeg-mapping) and in peripheral hormonal systems (plasma levels of catecholamines, cortisole, prolactine, serotonine, measured by hplc). n ϭ 14 healthy students consumed drinks containing either 0, 50 or 200 mg l-theanine in randomized double-blind trials in the min 6-14 after a near maximal bicycle step test. measurements were done directly after exercise (m1) and 30 (m2), 45 (m3), 60 (m4), 120 (m5) min after the drink. l-theanine seemed to accelerate the normalization of eeg spectral power in high frequency waves (barthel in this congress). the physiological return of increased hormon levels to basal levels / the circadianic rhythm up to m2 (catecholamines) or m5 (cortisole, serotonine, prolactine) was not influenced by the drinks. but in the l-theanine trials correlations between eeg spectral power and some hormones were altered (slow wave power/some catecholamines except norepinephrine/delta disappeared and new correlations with prolactine appeared). thus we conclude that l-theanine acts at the switch from the brain to the peripheral stress regulation and thereby supports physiological relaxing after severe exercise. polyamines the development from callus to plantlets, both activities increased, reaching the maximum at this latter stage. also sadenosylmethionine decarboxylase activity displayed a similar trend. all the activities were present in supernatant and in particulate fraction. higher activity of enzymes assayed in the small embryos rather than in the embryo with higher shape, was consistent with following polyamine accumulation. department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy intracellular transglutaminase (tg, ec 2.3.2.13), which catalyzes the formation of ε-(γ-glutamyl)lysine isopeptide cross-links between polypeptides, has been related to a variety of important biological processes and in the development of senile cataract. the majority of the dry weight of the eye lens is composed of protein called crystallins. in the mammalian lens, these proteins are divided into three major classes: α-, -and γcrystallins. native -crystallins are oligomers, which elute in two or more size classes during gel filtration, ranging from 200-50 kda. they contain 7 different types of subunits, named b1, b2, b3, a1, a2, a3, ba4, ranging from 31-32 kda. in the rabbit eye lens two -crystallin subunits ( b2 and b3), among the water soluble proteins, have been shown to act selectively as acyl donors substrates for lens tg. calpains are cytoplamic ca ϩϩ -dependent cystine proteinases. the cleavage of αand -crystallins, the main substrates of lens calpain ii, has been associated to the increase of lens turbidity, due to insolubilization of peptides. we observed that tg-induced post-translational modification of b2-and b3-crystallins with polyamines, enhances their cleavage by calpain ii. this finding suggests that the enhancement of calpain ii activity, after conjugation of polyamines into -crystallins, could represent an important regulatory mechanism which may contribute to the opacification process of the eye lens, conducting to cataract formation. transglutaminases represent a family of enzymes, widely diffused in nature, from bacteria to plants and higher animals. the present discussion will focus on 4 isoenzymes in mammals, which have been well characterized from the structural and functional point of view. they act on tissular proteins catalyzing crosslinkage through isopeptide bonds at peptidyl glutamine and lysine residues or incorporation of small molecular weight primary amines, usually polyamines, in an irreversible, calcium dependent reaction. in several instances the expression of transglutaminases is regulated at the transcriptional level. these enzymes help in maintaining structural integrity of tissues intervening in wound repair and in cellular homeostasis at the levels of cell activation, receptor signaling, cell proliferation, differentiation and death. these general roles involve bis(guanylhydrazones) are a class of compounds known to interfere with the metabolism of polyamines by virtue of their ability to inhibit s-adenosylmethionine decarboxylase (samdc), a key enzyme of polyamine biosynthesis. this property has made them useful tools to study the biological functions of these compounds. a curious feature of bis(guanylhydrazones) is their structural relationship with two molecules involved in polyamine biosynthesis, namely spermidine and s-adenosylmethionine. the methylglyoxal derivative of bis(guanylhydrazones), mgbg, has been actively studied both in animal and plant systems. in the present work the male pollen from actinidia deliciosa has been utilized to investigate the role of polyamines on the pollen tube growth. the effect of several bis(guanylhydrazones) was tested on pollen germination, length of pollen tube, levels of free and conjugated polyamines and samdc activity. all bis(guanylhydrazones) tested (glyoxal-bis-guanylhydrazone, gbg, methylglyoxal-mgbg, methylpropylglyoxal-mpgbg, ethylmethylglyoxal-emgbg) inhibit pollen germination and their effect is dose-dependent. a clear reduction of spermidine, both in free and conjugated form, was observed, as well as a pronounced decrease in samdc activity. these results suggest that the mechanism by which bis(guanylhydrazones) reduce the germination of kiwi pollen is related to their effect on spermidine biosynthesis. molecules structurally related to polyamines (1,8diaminooctane, 1,9-diaminononane, 1,10-diaminodecane) and other inhibitors of their metabolism (cyclohexylamine, cha) are also tested on kiwi pollen germination. n. bagni 2 , d. bertoldi 1,2 , e. candioli 1 , l. martinelli 1 , and a. tassoni 2 1 istituto agrario, san michele a/adige, and 2 dipartimento di biologia, università di bologna, italy in the frame of the study aiming to enlighten developmental programs during regeneration in grapes, polyamine content (free and conjugated to hydroxycinnamic acids) and biosynthetic enzyme activities were assayed during somatic embryogenesis. aliphatic polyamines are growth regulators affecting plant growth and development both in vivo and during in vitro cultures, being involved in several morphogenic processes related ti their action in cell division. the study was conducted on samples of callus, embryogenic callus, embryo at different stages and plantlets of vitis vinifera brachetto and chardonnay cultivars induced from anthers and ovaries. polyamine content (putrescine, spermidine and spermine) free plus conjugated to percloric acid soluble fraction, referred to unit, was higher in the cv. brachetto than in the cv. chardonnay, and reached the higher levels in the fullydeveloped embryo stage. besides, ornithine decarboxylase activity resulted higher than arginine decarboxylase and during multiple catalytic processes: the receptor signaling activity (demonstrated only for isoenzyme 2) is related to an intrinsic gtp-ase activity of type 2 transglutaminase; the processes leading to control of cell proliferation, differentiation and death are mainly related to the protein crosslinking activity, while the cell activation is tentatively considered dependent on the polyamidation of endogenous proteins at glutamine residues. the knowledge on this last aspect lies far back in comparison to the other roles of transglutaminases and requires further accurate investigation, which must further extend to the role of the enzyme in human pathology. the examination of polyamine metabolism at the present time suggests that vitamin b12 is implicated in polyamines metabolism. literature data speak that spermine and spermidine stimulate activity of cobalamin-dependant methionine synthase, the enzyme that catalyses the recycling of homocysteine to methionine; polyamines inhibit methionine adenosyltransferase. beside the wellknow significance of vitamin b12, in transmethylation reaction, the significance of 5,-deoxyadenozyl cobalamin, except the conversion of methylmalonyl-coa to succinyl coa, is not well elucidates. methionine as s-adenosylmethionine (sam) is essential amino acid for polyamine biosynthesis. sam has frequently usage in treatment of liver diseases. according the mentioned facts the aim of our experiments is to exanimate the significance of application of vitamin b12 alone and altogether with methionine to rats without and with experimentally induced cholestasis. our preliminary results speak about the disturbance of polyamine metabolism in hepatic tissue of rats with cholestasis. application of methionine alone increases the amount of polyamine in rat liver tissue, in-group without cholestasis and with bile duct obstruction. the animal treatment with cobalamin has higher amount of polyamines and lower activity of polyamine oxidase in liver tissues in both groups. the effects of vitamin b12 may be in direct relation with the formation of 5,-methylthio deoxyadenosine (mta), the by-product of spermidine and spermine biosynthesis. the explanation the exact roles of vitamin b12 in polyamine metabolism of liver tissue need the futher investigations. department of molecular genetics, the weizmann institute of science, rehovot, israel exposure of mouse myeloma cells that massively overproduces ornithine decarboxylase (odc) but not of parental cells to ornithine results in a massive increase in the intracellular concentration of putrescine, followed by rapid cell death. the treated odc overproducing cells display fragmented nuclei, chromatin condensation and an oligonucleosome-size dna "ladder"; consequently, their death can be described as apoptosis. the apoptotic process induced by the accumulated putrescine involves the release of cytochrome c from the mito-chondria, activation of caspases cascades demonstrated by the cleavage of caspase-2 and parp, a substrate of caspase-3. the general inhibitor of caspases, bd-fmk, effectively inhibited parp cleavage but failed to inhibit cell death. the intracellular ca 2ϩ chelator bapta/am and the antioxidant bha inhibit parp cleavage. however, only bapta/am inhibit the induction of cell death. it seems that bha subverted the death into caspase independent pathway. treatment with bapta/am did not interfere with the accumulation of putresine following ornithine treatment, suggesting that the accumulated putrescine induces the elevation in the concentration of intracellular ca 2ϩ which then activates the apoptotic process. the dominant anti-apoptotic effect of bapta/am over egta suggests that internal stores are the main source of the elevated ca 2ϩ , but that putrescine is also capable of inducing influx of extracellular ca 2ϩ . extensive small intestine resection results in the loss of absorptive surfaces, acceleration of intestinal transit and, as a consequence, in malnutrition, weight loss, diarrhoea and other complications of short bowel syndrome. the availability of human recombinant growth hormone rgh and its stimulatory effects on gut growth suggested its use in the treatment of short bowel syndrome. the trophic response of gi tract epithelium to hormones such as growth hormone is mediated by polyamines, which are vital in cell proliferation. this study was undertaken in rats to: 1/ evaluate the effects of rgh by monitoring polyamine and amine metabolism parameters in the adapting short bowel and 2/ determine whether erythrocyte (rbc) polyamine concentrations reliably reflect the proliferative activity of the remaining bowel. seventy per cent resection of the small intestine of wistar rats was performed under ether anesthesia leaving equidistant lengths of bowel from pylorus and ileocecal valve. recombinant human gh (0.2 iu, s.c., saizen, serono, switzerland) was administered once daily for 5 or 10 days, to randomly selected rats on the second postoperative day. animals were sacrificed 8, 13 and 21 days after the operation. enzyme activities were measured with radioassays or fluorimetry. polymines were determined as dansyl derivatives by hplc/fluorimetry. gh treated animals had significantly higher intestinal odc and sat and lowel dao activities; higher (non-significant) mucosal growth index and polyamine concentrations than in untreated counterparts on 8 th postoperative day. thereafter the two groups did not differ in the investigated parameters. rbc polyamine concentrations were higher in operated verses control rats; rgh treatment had no significant effect. however, rgh treatment significantly reduced hepatic mao a and b activities. our results suggest gh accelerated the adaptive growth of the bowel remnant. they justify use of erythrocyte polyamine concentration measurement as the marker of small bowel proliferative activity. however, side-effects of this treatment must be considered. tissue transglutaminase (ttg) activity has been evaluated in different neural tissues, such as brain, spinal cord and peripheral ganglia, and appears to be expressed in cerebellar granule cells (cgn) as well as in astrocytes. the role of ttg in neuronal functioning is likely to be quite complex. other than the role during development, significant changes of enzyme activity have been evaluated in different neurodegenerative conditions. it is well known that nmda receptor activation may be able to trigger excitotoxicity. the nmda-induced injury is mainly associated to ion influx and subsequent calcium overload. the effects of nmda application to both, cerebellar granule cells and glial cell cultures, have been assessed. in cgn, ttg activity increased rapidly after a brief stimulation with 100 µm nmda, whereas in glial cell cultures, high levels of enzyme activity was obtained after incubation of 24 h in presence of the same concentration of nmda. such results rule out the possibility that excitotoxicity can modify numerous proteins making them better substrates of ttg, and this could contribute to enhanced ttg-modifications of proteins in response to excitotoxicity. the pote protein can catalyze both uptake and excretion of putrescine. the km values of putrescine for uptake and excretion (putrescine-ornithine antiport) are 1.8 µm and 73 µm, respectively. amino acid residues, cys62, trp201, glu207, trp292 and tyr425 are strongly involved in both activities, and that glu77, tyr92, cys210, cys285, cys286 and glu433 are moderately involved in the activities. mutations of tyr78, trp90 and trp422 mainly affected uptake activity, indicating that these amino acids are involved in the high affinity uptake of putrescine by pote. mutations of lys301 and tyr308 mainly affected excretion activity, indicating that these amino acids are involved in the recognition of ornithine. the putrescine and ornithine recognition site on pote was found to be located at the cytoplasmic surface and the vestibule of the pore consisting of twelve transmembrane segments. the cadb protein has 30% sequence homology with pote protein. cadb can catalyze both uptake and excretion of cadaverine. the km values of cadaverine for uptake and excretion (cadaverine-lysine antiport) are 20 µm and 300 µm, respectively. it was found that two glutamate residues (glu76 and glu204) and four tyrosine residues (tyr73, tyr89, tyr90 and tyr310) are involved in the both activities. the difference of the substrate recognition site on pote and cadb is discussed. a. lentini, b. provenzano, and s. beninati department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy tissue transglutaminase (ttg, e.c. 2.3.2.13) is a protein cross-linking enzyme which catalyzes an acyl transfer reaction where the carboxamide group of a peptide-bound glutamine is the acyl donor, and a lysine residue the acyl acceptor. polyamines may act as acyl acceptors, leading to the formation of mono-and bis-(γ-glutamyl)derivatives. we provided evidence that ttg activity is directly associated to differentiation markers, and inversely related to cell proliferation and invasion. we have shown the in vivo reduction of experimental melanoma metastasis by i.v. injection of a plasmid (psg5) carrying the ttg gene sequence to c57bl6/n mice. tumor cell metastatization requires specific interactions with subendothelial basement membrane (bm) and migration through the endothelial wall, allowing the colonization of the target tissue. therefore, the investigation on the possible mechanisms responsible for ttg effects is focused on the posttranslational modification of bm proteins. we detected that "matrigel", a tumor-derived complex of bm proteins, modified with polyamines after ttg catalysis, reduces both melanoma cell adhesion and invasion in an in vitro metastatic assay. similar results were obtained using polyamines conjugated to laminin, one of the major bm components, as unique substrate. our findings suggest that the increase of bm proteins conjugated to polyamines may be responsible for impairments of the invasive properties of melanoma cells. we demonstrated that interferon-α (ifnα) induces apoptosis in human epidermoid cancer cells. tissue transglutaminase (ttgase) is an enzyme involved in the regulation of apoptosis through the inactivation of some cell components. among these eukaryotic initiation factor-5a (eif5a) is peculiar because its activity is modulated by the formation of the amino acid hypusine. recently, we found that growth inhibition induced by ttgase is paralleled by reduced hypusine levels. here we report the effects of ifnα on the apoptosis, ttgase modulation and eif5a activity in human epidermoid lung h1355 cancer cells. we have found that 48 h exposure to 2,500 iu/ml ifnα induces 50% growth inhibition and 15% apoptosis in h1355 cells. moreover, ifnα induced a 4-fold increase of ttgase activity and expression that already occurred after 24 h of exposure to the cytokine. this effect was paralleled by a 1.6-fold enhance of ttgase mrnas. ifnα induced also a 30% increased eif5a expression while an about 50% decrease of hypusine levels was observed. increased ttgase activity was paralleled by a decrease of hypusine content and of eif5a activity. therefore, ifnαinduced apoptosis could occur through an increase of ttgase activity and the mechanism by which ttgase regulates biological functions can be the reduction of eif5a activity. adometdc deficient mice k. nishimura 1 , f. nakatsu 2,3 , k. kashiwagi 1 , h. ohno 3 , t. saito 2 , and k. igarashi 1 1 graduate school of pharmaceutical sciences, chiba university, chiba, 2 graduate school of medicine, chiba university, chiba, and 3 cancer research institute, kanazawa university, kanazawa, japan the amd1 gene encodes s-adenosylmethionine decarboxylase (adometdc) that is one of the key enzymes of polyamine biosynthesis. to examine the physiological role of polyamines, we performed the targeted disruption of the gene in mice to generate spermidine-and spermine-free mice. although the level of adometdc mrna decreased by 50% in amd1 ϩ/ϫ mice, adometdc activity reduced only by 30% and spermidine and spermine contents did not change significantly. they showed normal phenotype and life span. to obtain amd1 ϫ/ϫ mice, we intercrossed amd1 ϩ/ϫ mice and determined the genotype of the resulting offspring. however, we could not obtain any amd1 ϫ/ϫ mice from 20 heterozygous intercrosses over. amd1 ϫ/ϫ embryos died early in development, between e3.5 and e6.5 days post coitum. in culture of blastocysts at e3.5, the shapes of all cell lines were normal, but amd1 ϫ/ϫ cells appeared to arrest the cell proliferation at day 3 after the onset of cell culture. the arrest of amd1 ϫ/ϫ cell proliferation was rescued by addition of spermidine. these data indicated that the lethal phenotype of amd1 ϫ/ϫ mice was caused by growth retardation by polyamine depletion at early developmental stage. the formation of active species such as h 2 o 2 and aldehydes during the oxidative deamination of biogenic amines by amine oxidases (ao) suggests for these enzymes a key role in cellular processes. the ability of bovine serum amine oxidase (bsao) to oxidase free amino groups of lysozyme and ribonuclease a has been observed indicating a possible ao involvement in the post-translational protein modification. furthermore, bsao inhibition by h 2 o 2 formed during substrate oxidation under limited turnover conditions was demonstrated, which may be relevant to cellular physiopathology. we have also observed that some inhibitors of mitochondrial amine oxidases (mao) protected human melanoma cell line (m14) against apoptosis. the protection by catalase of mao-substrates induced membrane permeability transition was also obtained in isolated rat liver mitochondria, thus confirming a role of mao-derived h 2 o 2 in apoptosis. enrichment in ao activity by treatment with vegetal ao has been obtained in a erythroleukemia cell line (k562), substaining the possibility to modulate the intracellular ao activity. an antiarrhythmic and cardioprotective effect of bsao has been also observed on isolated rat heart in reperfusion; a protective effect during anaphylaptic crisis has been shown "in vivo", thus suggesting aos as a possible therapeutic agents. tetrakis(3-aminopropyl)ammonium, a unique polyamine produced by an extreme thermophile, stabilizes nucleic acids at high temperature t. oshima and y. terui department of molecular biology, tokyo university of pharmacy and life science, hachioji, tokyo, japan an extreme thermophile, thermus thermophilus, produces tetrakis(3-aminopropyl)ammonium; a novel polyamine containing a quaternary ammonium nitrogen. to clarify the roles of the unique polyamine in thermophily, the effects of tetrakis(3aminopropyl)ammonium on biochemical reactions related to nucleic acids have been investigated. the unique polyamine stabilized both double and single stranded dnas and rnas. tm of a double stranded dna was raised by 8°c by the addition of 0.25 mm of tetrakis(3-aminopropyl)ammonium. at around the boiling temperature of water, depurination of dna takes place. other long polyamines produced by the thermophile such as caldopentaamine also stabilized dnas and rnas. we found that tetrakis(3-aminopropyl)ammonium prevents depurination most effectively. tetrakis(3aminopropyl)ammonium activated the protein biosynthesis catalyzed by a cell-free extract of the thermophile at high temperature. the effects of this unique polyamine on dna and rna polymerases are also being investigated and the results will be presented. tissue transglutaminase (ttg) catalyses the cross-link formation between glutamine (q) residues and nh 2 -donor molecules present in the cells (polyamines, lysine-donor proteins). recently, it has been correlated to neurodegenerative disorders characterised by polyglutamine (q n ) expansion, like huntington's disease. studies carried out on cell extracts revealed that glyceraldehyde 3-phosphate dehydrogenase (gapdh) was found covalently linked to q n domains. however, to date no structural data are available to solve the issue of which residues of gapdh are substrates for ttg. by coupling classical protein chemistry procedures and mass spectrometric techniques we achieved this goal by using as ttg substrates the substance p, an 11-aa peptide bearing the simplest q n domain (q 2 ), and polyamines of different size and shape as q-and nh 2 -donor, respectively. in the present study we report that out of the 26 lysines present in gapdh only three are sites of ttgasedependent cross-link formation in vitro. moreover, to characterize the ttg catalysed cross-link between gapdh and polyq protein we used a synthetic q 17 -peptide as ttg substrate in the catalysed reaction with polyamines. we found that any q residue is a potential ttg substrate, no matter the specific position in the sequence or the steric hindrance of the specific amine under investigation. cjf inserm 95-09, institut contre les cancers de l'apareil digestif (ircad), strasbourg, france as soon as the key role of odc in polyamine metabolism was recognised, it became the major target for selective inhibi-tion. s. harik presented in 1973 the first potent odc inhibitor, α-hydrazino ornithine. although efforts continued until today, with the aim to improve odc inactivation, 2-(difluoromethyl)ornithine (dfmo) remained the most important compound among all polyamine-directed drugs. a known anti-leukaemic drug, methylglyoxal-bis(guanylhdrazone), was recognised early on by g. williams-ashman and his collaborators as an inhibitor of adometdc, the other highly regulated biosynthetic decarboxylase, and served as matrix for more recent developments. in the course of the years selective inhibitors for all enzymes involved in polyamine biosynthesis and degradation were synthesised. moreover, a series of polyamineuptake inhibitors were reported. however, only some of these numerous compounds reached a stage above evaluation as growth inhibitors of cancer cells. owing to the sophisticated homeostatic regulation of the polyamines in cells and organs by de novo synthesis, degradation, uptake and release, and due to the fact that exogenous polyamines (i.e. gut polyamines) can be utilised by the vertebrate organism, the efficacy of selective enzyme and uptake inhibitors remained modest in cancer therapy. the fact the dfmo became the most important drug for the therapy of west and central african sleeping sickness relies on differences of vertebrate and parasite biochemistry. a novel approach, initiated by carl porter, involved the design and synthesis of structural analogues of spermidine and spermine, which do not share the growth-promoting effects of the natural polyamines. a very large variety of homologues, mostly of spermine, with different alkyl-substituents on the primary amino groups, have been studied systematically with regard to their ability to alter enzyme and polyamine patterns, and to inhibit cell growth. in addition polymine-like chains with interposed heteroatoms (0, s, si etc.), and analogues with rigid aliphatic chains (due to inbuilt double and triple bonds, or of small rings) have been explored. the structural analogues either mimic regulatory functions of the natural polyamines, and thus lead to the depletion of endogenous pools of putrescine, spermidine and of spermine, or they prevent growth effects of the natural polyamines by displacing them from functionally important binding sites. the later type may be considered as polyamine antagonists. the actual drugs usually exhibit to some extent polyamine mimetic and antagonist properties. at present several polyamine analogues are in clinical trial. however, after more than 25 years of active research, a polyaminerelated anticancer drug is still not available. one may conclude from this fact that the polyamines are an inappropriate target for cancer treatment. however, it is more likely that polyamine metabolism is a difficult target, because the differences between normal and cancer cells are mainly of quantitative nature. moreover, numerous mechanisms have developed in the course of evolution, which enable the vertebrate organism to prevent lethal polyamine losses. nine novel chemically modified polyamine (pa) analogs were evaluated for their ability to inhibit the pa biosynthesis in rat hepatoma g-27 cell-free system as well as the growth of caov tumor cells. the final concentration of oxy-and aminoadenosine pa analogs or two uracils modified pa analogs were 0.1 mm in the reaction mixture. bis(uracilyl)-analogs and 8-(2-oxyethyl)ami-no-9--d-xylofuranosyladenine supressed pa and putrescine synthesis and in the same conditions were more effective than dl-α-difluoromethylornithine (dfmo) -strong specific inhibitor of ornithine decarboxylase (odc). the other adenosine modified compounds could act both as activators of odc and inhibitors both diamine and polyamine oxidase activities in regenerating liver test system. in contrast to those mentioned above two uracils modified agents as well as dfmo were able to inhibit odc and to increase the rate of oxidative deamination of pa in the same system. thus bis(uracilyl) pa analogs were the most active and may be useful for further investigation as substances having potential antitumor and antiproliferative properties. several studies concerning the periodontal status in adult and adolescent patients treated with fixed ni-ti archwires have been performed, but until now it is not yet available any information about the influence of patient age on gingival tissue responses to ni-ti alloy. recently, researches by us demonstrated that the prolonged use for over 12 months of ni-ti appliances may contribute to local pathological proliferative processes early detectable only through salivary polyamine concentration increase. although other data from our laboratory showed that salivary polyamine amounts are age and sex-independent, nothing is known about the influence of the age on salivary polyamine content m subjects wearing ni-ti appliances. eighty patients, under orthodontic treatment for 12 months, were divided into four groups: the pre-, the mid-, the late-and the post-pubertal. salivary polyamine concentrations were determined by hplc. only the late pubertal group revealed a significant increase in both the spermine and spermidine content, while the other groups showed no modification. the results suggest that gingival pathological responses to a long-term appliance's use may be related to the endocrine modifications that occur in the late-pubertal age. sexual hormones appear to be in synergy with ni-ti alloy in promoting proliferative activity of gingival cells. the effects of polyamines on the synthesis of various σ subunits of rna polymerase were studied to determine how polyamines influence the functional specificity of transcription using western blot analysis. synthesis of σ 28 was stimulated 4.0-fold and that of σ 38 was stimulated 2.3-fold by polyamines, whereas synthesis of other σ subunits was not influenced by polyamines. stimulation of σ 28 synthesis by polyamines occurred at the level of transcription. since our hypothesis is that polyamines regulate macromolecular synthesis mainly at the translational level, we searched for a target protein, related to the polyamine stimulation of σ 28 synthesis, whose translation is altered by polyamines. stimulation of σ 28 synthesis was due to an increase in the level of camp, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. polyamines were found to increase the translation of adenylate cyclase mrna by facilitating the uug codon-dependent initiation. analysis of rna secondary structure suggests that exposure of the shine-dalgarno (sd) sequence of mrna is a prerequisite for polyamine stimulation of the uug codon-dependent initiation. antitumor quinones are approved for clinical use and others antitumor quinones are in different stages of clinical and preclinical development. the efficiency of the quinonic compounds in inhibiting cancer cells growth is believed to stem from their participation in key cellular redox mechanisms with consequent generation of highly reactive oxygen species (ros). the ros is turn modify and degrade nucleic acids and proteins within the cells. recently, quinonic drugs were attached to the neurodecapeptide lh-rh and evaluated as potential drugs in the treatment of different tumours. we have synthesized several series of n-quinonyl amino acids in which five ω-amino acids are attached to p-quinones with different values of redox potentials. the attachment was made via michael-like reductive addition of the amino acids to the quinonic ring or via substitution of a chlorinated atom. the n-ω-quinonyl amino acids were characterized as to their ability to form semiquinone anion radicals by epr and cyclic voltammetry technique. the preparative methods, the redox potentials as well as the physical and spectral data ( 1 h-nmr, ir, uv-vis and hrms) of these n-ω-quinonyl amino acids will be presented. the de novo design of biologically active peptides and proteins, mostly has involved consideration and design of backbone conformations (secondary structures) such as α-helix, -sheets, -turns, etc. (η/ψ space). however, for many bioactive peptides and proteins, especially those critical for information transduction such as neurotransmitters, hormones, antigens, neurocrines, etc. molecular recognition via side chain moieties is of paramount importance. thus far, the specific three dimensional orientations of side chain groups ( angles; chi space) in terms of biological activity has received only modest attention. in part this may be due to the energetics of chi space compared to ramachandran space. in order to overcome the current limitations of evaluating the importance of chi space in critical biological functions related to disease and behavior, we have designed amino acids with novel structures and unique constraints in chi space ( 1 , 2 , etc.), with special attention to their ability to mimic the chi space of native proteins and peptides. we have developed novel and simple asymmetric synthetic methods for such amino acids, often with ees greater than 98%. incorporation of these novel amino acids into bioactive polypeptide neurotransmitters has provided ligands with unique biological activities that effect unique behaviors including feeding, sexual, pain, and addictive behaviors. (supported by grants from the usphs and nida.) protein technology, wallenberg laboratory ii, lund university, lund, sweden we describe a method for comparative quantitation and de novo peptide sequencing of proteins separated either by standard chromatographic methods or by one and two-dimensional polyacrylamide gel electrophoresis. the approach is based on the use of an isotopically labelled reagent to quantitate (by mass spectrometry) the ratio of peptides from digests of a protein being expressed under different conditions. the method allows quantitation of the changes occurring in spots or bands that contain more than one protein, and has a greater dynamic range than most staining methods. since the reagent carries a fixed positive charge under acidic conditions and labels only the n-terminal of peptides, the interpretation of tandem mass spectra to obtain sequence information is greatly simplified. the sequences can easily be extracted for homology searches instead of using indirect mass spectral based searches and are independent of post-translational modifications. dehydroamino acids and their derivatives play important roles as constituents of various natural products and as synthetic intermediates for the preparation of optically pure amino acids. a large number of amino acid derivatives containing a pyrazol-4-yl, isoxazol-4-yl and other heterocyclic moieties has been prepared as potential agonists or antagonists for central glutamate receptors in connection with (r,s)-2-amino-3-(3hydroxy-5-methylisoxazol-4-yl)propanoic acid (ampa), a bioisostere of (s)-glutamic acid. -hetaryl-α, -didehydroalanines might be considered as conformationally constrained ampa analogs and might be potential candidates for the synthesis of novel types of ampa analogs, for example, via their hydrogenation. compounds containing 2h-pyran-2-one ring are also very useful synthons in selective synthesis. recently we have shown their use for the preparation of (e)-α, -didehydroα-amino acid derivatives containing a pyrazolyl moiety (vraničar l, polanc s, kočevar m (1999) tetrahedron 55: 271). as a continuation of our investigation in this field we report here a detailed study of the transformation of 2h-pyran-2-one derivatives 1 with hydroxylamine (2, x ϭ o) and various hydrazines (2, x ϭ nr 2 ) towards novel types of (e)-and (z)α, -didehydroamino acid derivatives 3. in most cases, the reactions were performed under basic conditions in a mixture of ethanol and pyridine. depending on the substrate and the reagent used the reaction could be controlled to give either (e)-or (z)-isomers; in some cases decarboxylation to the corresponding enamines 4 also occured during the reaction course. some attempts to hydrogenate compounds 3 towards α-amino acid derivaties 5 by homogeneous or heterogeneous catalysis were also performed. analogs of endomorphin 1 and 2 were prepared to investigate the effect of the positional and c-terminal amide replacements and modifications on the biological activity. modifications in position 2 and 4 were studied. in position 2 several hydroxy-and serine related amino acids were incorporated, whereas in position 4 the amide bond was replaced by hydroxymethyl and allyl group. protected peptide derivatives were synthesized on 2chlorotrityl resin and further transformed to the corresponding derivatives in solution phase. among the analogs tested, in in vitro tests the most effective compound found was d-ser 2 -endomorphin ϫ2. quite surprisingly, the partial agonist/antagonist properties of the derivatives in receptor binding and g-protein stimulation tests have been shown behave differently. the differences in efficacy and receptor binding properties of the compounds may explain the discrepancies between the in vitro and receptor binding tests. we have been assessing the possible applications of substituted 2h-pyran-2-ones 1 containing α, -didehydroamino acid unit in their structure as dienes in [4ϩ2]-cycloaddition reactions. as dienophiles we have been using different acetylene derivatives as well as n-phenylmaleimide and maleic anhydride. as it is evident from the structure of 2h-pyran-2-ones upon the cycloaddition of acetylene derivatives the first intermediate formed (2) still contains the carbon dioxide bridge. in many cases 2 easily expels co 2 and substituted benzene derivative 3 is produced. when the alkenes are used, the first part of cycloaddition is the same as when acteylene derivatives are used, but after the extrusion of co 2 from the adduct 4 there are two possible paths: so formed cyclohexa-1,3-diene (5) is either aromatized into benzene derivative (3) or it acts as another diene with favourably positioned double bonds and unusual double cycloadducts (6) are formed. since co 2 -containing adducts are thermally unstable it is advantageous to use high pressure techniques. with the acetylene derivatives we have not been able to isolate co 2 -containing adducts (2), while with alkenes we have isolated, depending on the structure pattern of the compound 1, all three types of products: aromatized 3, co 2containing 4 and double adducts 6. especially the type 4 is suitable for further transformations into other heterocycles containing amino acid moiety. research group of peptide chemistry, hungarian academy of sciences, budapest, hungary among the opioid receptors family, the cloning of µ, k and δ receptors was followed by another member, named lc 132 or orl 1 . searching for an endogenous ligand for this receptor resulted in successful identification of a peptide (fggft garksarklanq) called noc or ofq. in vitro and in vivo studies have demonstrated that noc mediates a variety of biological actions. results from structure-activity experiments suggest that the whole sequence of noc is not required for binding to the lc 132 receptor and for full biological activities. noc(1-13)-oh seem to be the minimum and essential sequence for good interaction with the receptor. this neuropeptide, similarly other peptides, are unresisting for enzymatic degradation and the releasing metabolites are very weakly active or inactive. some previous experiments refer to that the c-terminal amidation may protect the peptide from degradation. we purposed to synthesize carbamoyl analogues of noc(1-13)-nh 2 , hoping that these derivatives retain the ability to bind lc 132 receptor and are resistant against biological degradation: phe-nh-co--ala-noc(4-13)-nh 2 phe--ala-nh-co-phe-noc(5-13)-nh 2 phe-gly-nh-co--hphe-noc(5-13)-nh 2 the first step in the synthesis of the carbamoyl analogues was the preparation of the building block [r-co-nh-co-nh-hc(rј)-cooh] by the classical method and then it was incorporated into the peptide by solid phase peptide synthesis. [ nonproteinogenic amino acids and their derivatives are valuable compounds from their pharmacological and biochemical effects. they can be used also in synthesis of peptides, as biomarkers, as the ligands in catalitically active transition metal complexes and so on. it is possible to prepare such amino acids by asymmetric hydrogenation of their prochiral precursors. however high enantioselektivities was reached only in the case of chiral phosphine-rhodium catalysts. recently we showed that high diastereoselectivity in the hydrogenation of linear dehydrodipeptides may be achieved over achiral catalyst in the catalytic system substrate -salts of ca ore mg -pd/c due to formation of dehydrodipeptides complexes with ions ñà 2ϩ or mg 2ϩ and hence increasing of the conformational rigidity of substrates. this phenomenon may as well happen in other dehydrodipeptides, containing nonproteinogenic amino acids. among unnatural amino acids those bearing heterocyclic rings have attracted considerable attention due to the possibility of the heteroatoms participation in coordination with ions of metals. we have received some n-acyldehydrodipeptides, containing in the prochiral unit of dipeptides nonproteinogenic dehydroamino acids. all this n-acyldehydrodipeptides form in alcohol solution complexes with cax 2 and mgx 2 where one metal ion binds together several (up to 5) substrate molecules. this kind of complexation leads to the increase of conformational rigidity and to the diastereoface shielding of cϭc bond. moreover the combination of cations (ca 2ϩ or mg 2ϩ ) and anions (x) and the sequence of their mixing with a substrate determine the assembly inside complex particles and hence the sign and degree of asymmetric induction. indeed hydrogenation of these complexes formed in situ over achiral heterogeneous catalyst (pd/c) gives two diastereomers of corresponding n-acyldipeptides with the substantial increase of the reaction diastereoselectivity (up to 80%). in living cells, glutamine represents one of the main storage forms of nitrogen and is a major physiological source of ammonia for the biosynthesis of aminoacids, aminosugars, purine and pyrimidine nucleotides and coenzymes. glutamine-dependent amidotransferases perform nitrogen transfer from the amide group of glutamine to various electrophiles. when the latter is fructose-6p, the product of the reaction catalysed by glucosamine-6p synthase is d-glucosamine 6-phosphate, a structural building block of peptidoglycane (bacteria) and of chitin and mannoproteins (fungi). fluorinated analogues of glutamine are expected to interfere with this biological process due to the strong electron withdrawing effect of fluorine atom (without significant steric consequence), inducing modulation of binding and/or electronic properties. these compounds might therefore behave as reversible or irreversible active site-directed enzyme inhibitors. synthesis of optically active 1 from d-serine will be described and first results in the biological evaluation on glucosamine 6-phosphate synthase will be included. o. melnyk 1 , d. bonnet 1 , e. loing 2 , l. bourel 1 , and h. gras-masse 1 1-umr 8525, 2-sedac-therapeutics, biological institute of lille, france lipopeptides, owing to their ability to cross passively the cell membrane or biological barriers, are unique tools for the intracellular delivery of bioactive peptides. the structure of the lipophilic moiety is known to have a profound effect upon the interaction with the membrane and its alteration. the stepwise solid phase synthesis of lipopeptides is limited by the necessity to perform a complex rp-hplc purification following the cleavage and deprotection step. in addition, the harsh conditions used during the final acidolysis procedure does not allow the introduction of unsaturated or sensitive fatty acids. to speed up the access to large lipopeptides modified by various fatty acid moieties or cholesterol derivatives, we have designed novel synthetic methods which involve the chemoselective reaction of fully deprotected and purified hydrazinopeptides with fatty acid succinimidyl esters or glyoxylyl derivatives. application of these methodologies to the c-terminal 95-135 portion of interferon (ifn)-γ allowed the selection of the optimal lipopeptide ifn-γ agonist, as determined by its ability to induce the expression of surface mhc-ii molecules through interaction with the intracellular components of ifn-γ receptor. graduate school of science, osaka city university, osaka, japan glutamate receptors in mammalian cns are implicated in the construction of memory and early learning as well as in the pathogenesis of neuron damage to cause various neuronal diseases. in recent years, we have studied the conformational role of l-glutamate when it binds to the receptors through the synthesis of l-2-(carboxycyclopropyl)glycines (ccgs) and their related analogs. the works have demonstrated that not only the receptors require a specific conformation of l-glutamate, but also these analogs can be used as important tools for the neuropharmacological research. among them, dcg-iv, a 3јsubstituted analog of ccg-i, is used as a potent and selective agonist of mglur2. as an extension of these works, next program was focused on the synthesis of α-substituted glutamate analogs which would enable to develop potent and subtype-selective ligands for mglurs and transporters. α-alkoxymethylglutamate and ly354740 and its c5 epimer were chosen for the synthetic targets, since the former slightly restricts the glutamate conformation to an extended form and the latter rigidly fix to an extended or a folded form on its bicyclo[3,1,0]hexane skeleton. the key to the synthesis was a stereoselective construction of the quarternary carbon center, which was efficiently performed based on an asymmetric version of the strecker synthesis. details of the synthesis and their neuropharmacological activities will be described. using a genetically modified organism a broad variety of linear unsaturated amino acids are now accessible in enantiomerically pure form via this methodology, which can be used as starting materials for the synthesis of highly functionalized pipecolic acid derivatives. these compounds can be used to restrict conformations in polypeptides or can serve as scaffolds in synthesizing libraries for drug discovery. the synthetic approach involved both a pd-catalyzed amidopalladation reaction of alkoxy-allenes, in which the nh is added across one allene double bond and a ruthenium catalyzed ring closing metathesis step, to form a benzyloxypipecolic acid. further reaction of this n-sulfonyl-iminium-ion precursor with a nucleophile results in the formation of cis-substituted pipecolic acids. due to the unique electronic properties of fluorine, incorporation of α-fluoroalkylated amino acids is a new approach to design biologically active peptides with increased metabolic stability and defined secondary structure and provides a powerful nmr label for spectroscopic investigations. the application of proteases especially for cn-ligations is an attractive alternative to chemical methods, because the enzymatic formation of peptide bonds is highly regio-and stereospecific and, therefore, does not require large efforts to protect side chains of trifunctional amino acids. recently, the enzyme-catalyzed incorporation of α-fluoromethyl amino acids into the p 2 , p 3 and p 2 ј-position (nomenclature according to schechter and berger) of peptide fragments has been successfully performed. carboxypeptidase y was now shown to be suitable to catalyze the incorporation of α-trifluoromethyl alanine into the p 1 position of peptides. furthermore, the general applicability of the substrate mimetic concept in enzymatic peptide synthesis was expanded to the transfer of c-terminal α-fluoroalkyl substituted amino acids. generally, each trifluoromethyl-and difluoromethyl amino acid 4guanidinophenyl esters can be applied as acyl donor in trypsin and chymotrypsin catalyzed peptide bond formation independently of the acyl moiety and the natural enzyme specificity, respectively. via these two approaches, incorporation of αfluoroalkylated amino acids into the p 1 position of peptides using enzymatic methods was successfully applied for the first time. this investigation was performed in search of new 2јdeoxynucleoside analogues modified at 3ј-and 5ј-positions with amino acids and possessing antiviral activity. substrate mimetics strategy: an efficient approach to protease-catalyzed peptide ligation n. wehofsky 1 and f. bordusa 1,2 1 max-planck-society, research unit "enzymology of protein folding", halle, and 2 institute of biochemistry, university of leipzig, germany two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: (1) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. the latter can be overcome by the use of substrate mimetics. contrary to common acyl donors, substrate mimetics bear a binding site specific ester leaving group instead of having a specific amino acid moiety at the c-terminus of the acyl residue. this replacement mediates the acylation of the protease by nonspecific acyl residues. deacylation of the artificial acyl enzyme intermediate by the amino component added results in peptide bond formation regardless of the primary specificity of proteases enabling nonspecific coded and noncoded amino acid derivatives and even non-amino acid-derived acyl moieties to be coupled. the successful application of these artificial substrates for model peptide ligations catalyzed by the argspecific trypsin, the glu-specific staphylococcus aureus strain v8 protease (v8 protease), and α-chymotrypsin, which is specific for aromatic amino acid moieties, will be demonstrated. new development in the tritium labelling of peptides and proteins using solid state catalytic isotopic exchange with spillover-tritium yu. a. zolotarev 1 , a. k. dadayan 1 , b. v. vaskovsky 2 , and n. f. myasoedov 1 1 institute of molecular genetics, russian academy of sciences, and 2 shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia the reaction of high temperature solid state catalytic isotope exchange (hscie) of hydrogen in peptides and proteins with spillover-tritium was studied. the reaction ability of amino fragments in hscie was shown to depend both of their structure and on the availability and the mobility of the polypeptide chain. [ 3 h] peptide analysis using 3 h nmr spectroscopy was carried out, and the modified fragment [ 3 h]actc 4-10 (met-glu-his-phe-gly-pro), with molar activity of 80 ci/mmol and [ 3 h] zervamicin iib (ac-trp-ile-gln-iva-ile-trh-aib-leu-aib-leu-hyp-gln-aib-hip-aib-pro-phl, where aib ϭ 2amino-isobutyric acid) with molar activity of 70 ci/mmol was produced. the obtained preparations completely retained their biological activity. with the -galactosidase protein from termoanaerobacter ethanolicus as example, the interrelation between the protein's tertiary structure and the isotopic label distribution incorporated due to the hscie reaction was used. the labeled protein with the molecular mass of 83 kda was brought to fragmentation by glu-proteinase. peptide fragments were separated by hplc and were identified by maldi mass spectrometry. a correlation between the position of the amino acid fragment in the protein tertiary structure and its reaction ability in the hscie reaction was obtained. data on the retention of thegalactosidase enzymatic activity in condition of tritium label introduction are supplied. taurine chloramine modulates cytokine production by peripheral blood mononuclear cells m. chorą z . y 1 , e. kontny 1 , j. marcinkiewicz 2 , and w. maśliń ski 1 1 institute of rheumatology, warsaw, and 2 jagiellonian university, cracow, poland objective. proinflammatory cytokines are produced in a cascade fashion, where monocyte-derived tnfα and il-1 trigger production of il-6 and il-8 also in the other cell types. we reported recently that taurine chloramine (tau-cl) inhibits production of the latter cytokines in fibroblast-like synoviocytes. in present study the effect of taurine (tau) and tau-cl on tnfα, il-1 and il-6 production was examined. methods. peripheral blood mononuclear cells from healthy volunteers were stimulated with lps (24 h) in the presence of tau or tau-cl (100-500 µm). cytokine production was measured in culture supernatants (secreted) and cell lysates (intracellular) using elisa. results. in lps-stimulated cells both secreted and intracellular il-1 and il-6 were inhibited by tau-cl with ic 50 ϸ 300 µm and 425 µm, respectively. however, tau-cl exerted dual effect on tnfα production, raising it slightly (1.5 times) at low (100-200 µm) while reducing it (ic 50 ϸ 450 µm) at higher concentration. tau did not significantly affect cytokine production. tau-cl modulates proinflammatory cytokine cascade and eventually might down-regulate it when present at high (ͼ300 µm) concentration. department of biology, division of general physiology, university of oslo, blindern, norway every living cell must deal with osmotic and hydrostatic pressure changes between its environment and its interior and counteract volume changes. swelling activated channels is one group of effectors in the cell membrane that is important in preventing excessive volume increases by releasing inorganic ions and organic solutes that include taurine. such channels are associated with several physiological processes, but little is known about their activation mechanisms. we have used a rat thyroid cell line (frtl-5) to investigate the activation of a swelling sensitive [3h]taurine efflux pathway. hypo-osmolality and thyrotropin (tsh, 500 µm) increased transiently the rate coefficient for [3h]taurine efflux with a similar pattern of activation. the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (100 µm) increased the swelling activated efflux rate coefficient 6.4 times above the control level and the camp analogue dibutyryl-camp (500 µm) activated the pathway. these results indicate that both swelling and tsh activation of the taurine efflux pathway are mediated by camp. other aspects of the signal transduction pathway will be discussed. based on the inclination of n-chloroamines to disproportionate, the endogenous bactericidal agent n-chlorotaurine (nct), mainly at ph ͻ 7, is accompanied by n, ndichlorotaurine (ndct). since pure ndct could be synthesized as crystalline sodium salt, a first evaluation of its chemical and bactericidal properties was possible. ndct-na (melting point: 162-4°c, decomp.) is very well soluble in water and poorly in ethanol where it can be recrystallized from. on storage the initial ph 5 of the aqueous solution decreases which correlates with a decrease of oxidation capacity of 1.8% per day, probably originated by the elimination reaction r-ch 2 -ncl 2 ae r-chϭncl ϩ h ϩ ϩ cl ϫ as a first step. contrary to nct-na an immediate decomposition occurs when ndct-na comes into contact with undiluted dmso. in aqueous solution, however, ndct does not react with dmso. the bactericidal activity of 55 mm ndct at ph 7 and 5 against the gram-positive bacteria s. epidermidiand two strains of s. aureus was the same as with equimolar nct though ndct bears twice the oxidation capacity. against the gramnegative bacteria e. coli, p. aeruginosa, and p. mirabilis, however, a significantly higher activity of ndct was observed at both ph. the mechanism of taurine chloramine inhibition of fibroblastlike synoviocytes growth e. kontny 1 , m. kurowska 1 , j. kowalczewski 1 , i. janicka 1 , j. marcinkiewicz 2 , and w. maśliń ski 1 1 institute of rheumatology, warsaw, and 2 jagiellonian university, cracow, poland objective. in rheumatoid arthritis (ra) enhanced proliferation of fibroblast-like synoviocytes (fls) leads to hyperplasia of synovial membrane (sm). therapeutic approaches to inhibit an excessive growth of these cells are not satisfactory. thus, we investigated the effect of taurine (tau) or taurine chloramine (tau-cl) on ra fls growth. methods. fls isolated from sm of ra patients were stimulated for 72 hours with rhpdgf or rhtnf-α. tau or tau-cl were added at 100-250 µm concentrations. cell proliferation was determined by incorporation of 3 h-thymidine into dna. expression of proteins regulating cell-cycle progression or apoptosis, was estimated by western blotting. results. at 250 µm concentration tau-cl inhibited (by ϸ 70%) both pdgf-and tnf-α-triggered cell proliferation and similarly reduced expression of pcna (a cofactor for dna polimerase δ). however, tau-cl affected neither the expression of cell-cycle inhibitors (p21, p27) nor anti-apoptotic bcl-2 protein. tau has no effect on tested responses. conclusion. we report that tau-cl inhibits proliferation of ra fls by affecting expression of pcna, that is critical for cell cycle progression. e. kontny 1 , k. szczepań ska 1 , j. kowalczewski 1 , m. kurowska 1 , i. janicka 1 , j. marcinkiewicz 2 , and w. maśliń ski 1 1 institute of rheumatology, warsaw, and 2 jagiellonian university, cracow, poland objective. proinflammatory cytokines play critical role in the pathogenesis of rheumatoid arthritis (ra). we reported recently that taurine chloramine (tau-cl), but not taurine (tau), inhibits production of il-6 and il-8 by fibroblast-like synoviocytes (fls). in present study the mechanism of tau-cl inhibitory action was investigated. methods. fls isolated from synovial membrane of ra patients were stimulated with rhil-1 . tau or tau-cl were added at 250-500 µm concentration. after 0.5-2 h or 4 h the dna binding activity of nfkb and ap-1 (emsa) and the expression of il-6 and il-8 mrnas (rt-pcr) was examined, respectively. results. il-1 raised nfkb and ap-1 activity, followed by the elevation of cytokine mrnas expression. tau-cl, but not tau, reduced both the expression of cytokine mrnas (il-6 ͼ il-8) and the activity of transcription factors (nfkb ͼ ap-1). conclusion. tau-cl inhibits transcription of il-6 and il-8 genes due to its ability to diminish the activity of key transcriptional factors, that regulate these proinflammatory cytokine expression. institut für organische chemie, universität bremen, germany the synthesis of taurine and hypotaurine from cysteine can be followed up in astroglia-rich primary cultures obtained from brain of neonatal wistar rats. using 1 h and 13 c nuclear magnetic resonance spectroscopy cell extracts of glia cells incubated with 13 c labelled cystein show the label subsequently in hypotaurine, taurine, lactate and gluthathione. within 72 h, 35% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from cysteine. both metabolites were also released to the medium. neurones are capable to take up both metabolites from glia media to recruit their organic osmolite. part of newly synthesized glutathione and lactate are also exported to the medium. by this means lactate may serve as an energy substrate for neurons. in-vivo mrs of lactate is obscured by line splitting and signal overlay. using various two dimensional pulse sequences as spin preparation sequences prior to localized single voxel, in-vivo mrs or spectroscopic imaging sequences will provide homonuclear non-coupled resonance signal of taurine. these singlet signals are detectable and quantified. diffusion weighted spectroscopy is used to characterize the mobility of taurine in living tissue. department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. taurine depletion, whether by removing taurine from the diet or by using a taurine transport inhibitor, has demonstrated various pathologies in various animal models including man. the first reported pathology associated with dietary taurine depletion was in the retina of the cat. in this animal model, taurine deficiency resulted in disorganization of the tapetum (the light reflecting membrane), disruption of the outer segments, photoreceptor dysfunction, and cell loss. when allowed to proceed for a number of months the result was blindness. subsequent studies demonstrated that taurine deficiency also had a profound effect on cardiac physiology. echocardiograms of the left ventricle of the cat heart depleted in taurine showed a dilated cardiomyopathy reflected in an extended end-diastolic diameter and an extended end-systolic diameter. dietary taurine supplementation resulted in the above parameters returning to normal. the cat is a difficult animal model to use for a variety of reasons and thus the rat was chosen to further probe the consequences of taurine depletion. unfortunately, the tissue taurine levels in the rat do not respond to dietary taurine depletion, and thus other experimental means had to be designed. guanidinoethanesulfonic acid (ges), an analogue of taurine and a taurine transport inhibitor, has been utilized for the last 20 years to deplete rat tissues of their taurine content (j. e. shaffer and j. j. kocsis, methods of reducing tissue taurine levels, and r. j. huxtable, h. e. laird, and s. lippincott, rapid depletion of tissue taurine content by guanidinoethyl sulfonate. in: the effects of taurine on excitable tissues, spectrum publications, new york, 1981) . ges, when administered to rats in their diet in the drinking water as a 1-1.5% solution, usually produces a significant decrease in the taurine content of all tissues within one week of treatment. within 3-5 weeks the levels of taurine reach their nadir (20-50% of control) and continued feeding of ges does not further reduce the levels of taurine. unfortunately, ges replaces taurine and thus one must always consider the effects of ges on physiological events that occur within the tissues in question. again, as in the cat, taurine depletion manifested itself in retinal pathology: disruption of the photoreceptor structure, dissociation of the disc membranes, and abnormal electroretinograms (erg). other animals models such as the monkey have also demonstrated structural disorganization of the photoreceptors and abnormal ergs. finally, the ultimate test is whether taurine deficiency has an effect in man. in 1985, koppel and associates (geggel et al., n. eng. j. med. 312: 142-146, 1985) demonstrated that children on long term parenteral nutrition devoid of taurine had abnormal erg. supplementation of the parenteral nutrition with taurine restored the ergs to normal in the majority of the children. because of these definitive studies, all infant formulae in the united states, europe and japan now contain taurine. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. it has been demonstrated previously in our laboratory that taurine inhibits the phosphorylation of an ϳ44 kdalton protein present in the mitochondrial fraction of the rat heart (j. mol. cell. cardiol. 26: 1675 -1689 , 1994 . upon administering 1.5% guanidinoethanesulfonic acid (ges) in the drinking water of rats for 6 weeks, the taurine levels in cardiac tissue decline by 75%. however, the phosphorylation of a ϳ44 kdalton protein in the mitochondrial fraction of the heart tissue increased by 85% (j. mol. cell. cardiol. 26: 1675-1689, 1994) . reversal of these effects could be accomplished by feeding the rat 1.5% taurine in the drinking water for 6 weeks. the ϳ44 kdalton protein was isolated by 1-dimensional polyacrylamide gel electrophoresis (page) using traditional glycine buffers followed by re-electrophoresing the cut out portion of the gel, which corresponds to the ϳ44 kdalton protein, on a tricine-buffered gel resulting in sufficient pure protein for digestion and sequence analysis. it was determined that the ϳ44 kdalton was pyruvate dehydrogenase (amino acids 12: 139-144, 1997) which indicates a significant regulatory role for taurine in energy metabolism in cardiac tissue. these data are of significant interest in that taurine may be an additional effector of this enzyme or of the enzyme complex. studies are in progress to determine if taurine has a direct effect on either the kinase (inhibition) or the phosphatase (stimulation) associated with the pyruvate dehydrogenase complex. it has also been demonstrated and now reported that taurine depletion utilizing ges in vivo in rats affects the phosphorylation of myelin basic protein (mbp). in these experiments the animals were given ges (1%) for 6 weeks in their water and then killed; the hearts were removed and homogenized. the homogenate was then incubated with buffer containing mbp (50 ì) and radioactive atp for 6 minutes. animals were also treated with taurine (1%) in their drinking water for 4-5 weeks or treated with taurine following the ges treatment. page of the incubation mixture, autoradiography on the dried gel, and densitometry of the mbp band gave the following results: relative % activity ϯ sem (normalized to mg protein) control 100 ϯ 20 (n ϭ 6) ges-treated 147 ϯ 28 (n ϭ 6) p ͻ 0.05 (paired 5-test) control 100 ϯ 16 (n ϭ 5) taurine-treated 95 ϯ 17 (n ϭ 5) p ͼ 0.05 control 100 ϯ 32 (n ϭ 4) ges followed by taurine 95 ϯ 37 (n ϭ 4) p ͼ 0.05 these data confirm previous reports that it is easier to deplete animals of their cardiac taurine content than it is to raise the levels of taurine. these data on the effects of taurine depletion (increase in mapkinase activity) and taurine supplementation (no change in mapkinase activity) on mapkinase activity reflect these past observations. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) act additively, or in the case of mpo negated each other's effects. regarding our results there is significance to pharmacological regimens which enhance the supply of propofol or taurine in whole blood. these regimens influence considerably pmn intracellular amino acid concentrations and it is this pmn "labile free amino acid pool" which may be one of the determinants in cell nutrition positively or adversely affecting pmn immune functions. taurine supplementation to pmn seems to interfere independently from the effects of propofol on pmn free amino acids and on immune functions tested. institut für hygiene, universität innsbruck, austria n-chlorotaurine (nct) is a long-lived oxidant produced by activated human leukocytes during the oxidative burst. it has activity against a broad spectrum of pathogens including bacteria, fungi, viruses and helminths. as a special feature, the killing of microbes by nct can be increased significantly in the presence of ammonium and also of some amino acids (alanine, glycine). this is explained by transfer of the active chlorine ("transhalogenation") from nct to ammonium and amino acids to form the corresponding, stronger microbicidal n-chloro derivatives monochloramine and n-chloro amino acids, respectively. especially addition of ammonium to nct provokes rapid inactivation of fungi and even mycobacteria. because of its good tolerability, nct solution can be applied to human tissue to treat infections. in ammoniumcontaining body fluids like nasal mucus and urine, fungi and bacteria are killed within minutes. therefore, amino compounds of human secretions can be transformed to the above quoted endogenous and highly microbicidal chloramines by nct via transhalogenation -a unique property of an antimicrobial agent. successful treatment in cases of urinary tract and otorhinolaryngological infections and conjunctivitis in phase iia clinical trials provides strong support for this concept. the endogenous sulfonated amino acid taurine has numerous functions in the central nervous system, including positive modulation of gaba a receptor function. recently we found that mice lacking protein kinase c -epsilon (pkcε) are behaviorally and biochemically supersensitive to ethanol and other positive allosteric modulators of the gaba a receptor. in addition, these mice consume 50-75% less ethanol and wildtype controls in two separate self-administration paradigms. microdialysis studies in pkcε-deficient mice revealed elevated extracellular levels of taurine, which may account for the supersensitivity of gaba a receptors in these mice and resulting decreases in ethanol intake. in light of the fact that the taurine derivative acamprosate (calcium acetylhornotaurinate) is moderately effective in reducing craving and relapse in detoxified alcoholics, we examined the effect of taurine-related compounds on acute ethanol consumption in a two-bottle choice paradigm in rats. taurine (10-200 mg/kg ip) was only slightly effective in reducing ethanol intake but not preference, while the highest dose of taurine (200 mg/kg) also suppressed water intake. the taurine precursor hypotaurine (10-100 mg/kg ip) was also weakly effective in reducing ethanol intake but not preference or water intake. the most effective compound tested was homotaurine (10-100 mg/kg ip), which suppressed ethanol intake and preference by approximately 50% without altering water intake. these data indicate that endogenous taurine may regulate sensitivity to ethanol and subsequent ethanol self-administration, and that taurine-related compounds may be effective in reducing alcohol intake in humans. we are currently exploring whether taurine and related compounds are able to suppress ethanol-stimulated mesolimbic dopamine release, a primary neural substrate of ethanol reinforcement. (this work was supported by funds provided by the state of california for medical research on alcohol and substance abuse through the university of california at san francisco.) organic osmolytes, such as taurine, regulate a cell's osmotic balance without directly altering either the cell's ionic composition or the membrane potential. this property of the organic osmolyte often renders the cell resistant to damage during a pathological insult. indeed, ischemia is associated with a massive efflux of taurine from the cell, an event that minimizes the severity of the osmotic imbalance that develops from the accumulation of lactate, inorganic phosphate and sodium. however, taurine depletion also activates specific signaling pathways that provide further protection to the cell. among the signaling pathways activated by taurine depletion is a pi 3-kinase (phosphatidylinositol 3-kinase) linked pathway that catalyzes the phosphorylation and inactivation of the pro-apoptotic factor, bad. taurine depletion also activates protein kinase c, which in turn elevates the intracellular content of the antiapoptotic factor, bcl-2. increases in the extracellular osmolality by either addition of 20 mm taurine or 25 mm mannitol to the incubation medium activates similar pathways. however, pi 3kinase assumes a more important role in the mannitol treated cell than the taurine depleted cell. moreover, p38 map kinase is activated by mannitol treatment but not by taurine depletion. despite these differences, both taurine depletion and mannitol treatment protect the cell against hypoxia-induced apoptosis. the data suggest that osmotic stress protects the cell against apoptosis by increasing cellular levels of bcl-2 and promoting the inactivation of bad. this work was supported by a grant from the american heart association. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin-1, interleukin-6 and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. department of applied biological chemistry, the university of tokyo, japan the intracellular level of taurine is maintained not only by the taurine transporter that transports extracellular taurine inside cells but also by endogenous synthesis from methionine and cysteine. we therefore investigated the regulation of both the taurine transporter and the cysteine dioxygenase, one of the main taurine biosynthetic enzymes, in hepg2 human liver cells. the intracellular taurine content of hepg2 cells was extremely increased by culturing in a hypertonic medium. the activity of taurine transport was increased by hypertonic conditions, which was due to the increased expression of the taurine transporter gene. the expression level of the cysteine dioxygenase gene was also increased, suggesting that the expression levels of both the taurine transporter gene and the cysteine dioxygenase gene were regulated in harmony by hypertonic conditions to accumulate taurine inside cells. on the other hand, the activity of taurine transport in hepg2 cells was down-regulated on culturing the cells in taurine-rich medium, the expression level of the taurine transporter gene being also markedly decreased. however, the expression level of the cysteine dioxygenase gene was not significantly altered under taurine-rich conditions, indicating that the gene expression of the taurine transporter and that of the cysteine dioxygenase was independently regulated by extracellular concentration of taurine. the amino acid, taurine, is found in very high concentration in the heart. although its most important putative function is osmoregulation, it also serves as a regulator of cell growth. isolated cardiomyocytes exposed to medium containing 1 nm angiotensin ii undergo hypertrophy, a process blocked by 20 mm taurine. the amino acid also inhibits angiotensin iiinduced activation of c-fos, upregulation of atrial natriuretic factor and induction of tgf-betal. central to virtually all of these actions of angiotensin ii is the translocation and activation of key protein kinase c (pkc) isoforms. therefore, we proposed that taurine inhibited the hypertrophic actions of angiotensin ii by interfering with the translocation of one or more of the pkc isoforms. indeed, taurine and angiotensin ii exhibited different effects on the translocation of several pkc isoforms. while taurine promoted the translocation of pkcalpha, pkcdelta and pkcepsilon from the particulate fraction to the cytosol, the levels of the three isoforms in the particulate fraction were elevated following treatment with angiotensin ii. by contrast, both taurine and angiotensin ii increased the pkczeta content of the particulate fraction and the pkcbeta2 content of the cytosol. when the isolated cardiomyocytes were incubated with medium containing both angiotensin ii and taurine, the effects on pkc distribution were largely additive. these data support the notion that taurine prevents the hypertrophic effects of angiotensin ii by interfering with the translocation of either pkcalpha, pkcdelta, pkcepsilon or a combination of more than one of the isoforms. (the study was supported by a grant from taisho pharmaceutical co.) main final metabolites of l-cysteine in mammals are sulfate and taurine, and they are excreted in the urine. our previous studies in rats have shown that the ratio of urinary sulfate and taurine in rats fed diet containing sufficient methionine and cysteine is 10: 2-3. in the present study, we determined urinary sulfate and taurine in urine samples of 58 healthy japanese women after 12h starvation following usual meal. free (inorganic) and total (free ϩ ester) sulfate were determined with ion chromatography, and taurine by reversed-phase hplc after dabsylation. average excretions (micromols per mg of creatinine) were: total sulfate, 12.53 ϯ 3.85; free sulfate, 11.57 ϯ 3.69; ester sulfate, 0.96 ϯ 0.94; taurine, 0.78 ϯ 0.53; urea, 187.71 ϯ 66.13. the ratio of total sulfate and taurine was 10 : 0.62. this suggests that sulfate formation in humans is more dominant than taurine formation as in rats and this tendency is more evident in humans than in rats, which is in accordance with low cysteinesulfinate decarboxylase activity in humans. sum of sulfate and taurine excretions was significantly correlated with that of urea: correlation coefficient, 0.675. this indicates that sulfur metabolism in humans is in the state of sulfur equilibrium similar to that of nitrogen and reflects protein metabolism. h. yokogoshi 1 and h. oda 2 1 laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, and 2 department of applied biological sciences, nagoya university, nagoya, japan the effect of taurine on hypercholesterolemia induced by feeding a high-cholesterol (hc) diet to rats was examined. when various amounts of taurine (0.25-50 g/kg) were supplemented to hc for 2 wk, serum total cholesterol gradually and significantly decreased in a dose-dependent manner, compared with the control (cholesterol free) diet group. by contrast, serum hdl-cholesterol was elevated by taurine supplementation. in the hypercholesterolemic rats fed the hc diet, the excretion of fecal bile acids and hepatic cholesterol 7α-hydroxylase (cyp7a1) activity and its mrna level increased significantly, and the supplementation of taurine further enhances these indexes, indicating an increase in cholesterol degradation. agarose gel electrophoresis revealed that, in hypercholesterolemic rats fed the hc diet, the serum level of the heavier vldl increased significantly, but taurine repressed this increase and normalized this pattern. significant correlations were observed between the time-and dose-dependent increases of cyp7a1 gene expression and the decrease of blood cholesterol concentration in rats fed the hc diet supplemented with taurine. these results suggest that the hypocholesterolemic effects of taurine observed in the hypercholesterolemic rats fed the hc diet were mainly due to the enhancement of cholesterol degradation and the excretion of bile acid. in vitro studies have shown that ammonia, which is responsible for neurological symptoms associated with hyperamonemia, causes a massive release of taurine from cultured cns cells and brain slices. in this study, taurine (tau) release was measured in vivo in rat striatum following direct application to the microdialysis tube of 60 mm ammonium chloride which renders the final ammonia concentration in the extracellular space of ϳ5 mm. various in vivo stimuli evoke taurine efflux either by opening osmosensitive anion channels and/or by a mechanism secondary to glu accumulation and its interaction with nmda or ampa receptors. the following compounds were coadministered with ammonia to distinguish between these mechanisms: anion/cation transport inhibitors -dids and furosemide, a glu transport inhibitor-pdc, and nmda and ampa/ka receptor antagonists dizocilpine and dnqx. ammonia stimulated tau accumulation in the microdialysates to ϳ250% of basal value. dids and furosemide moderately inhibited the effect of ammonia (furosemide by ϳ30%), albeit dids added alone induced massive accumulation of tau with a delayed onset as compared to ammonia. ammonia-dependent tau accumulation was increased by ϳ50% in the presence of pdc and reduced in an equal degree (ϳ35%) by dizocilpine and dnqx. none of the agents affected tau accumulation in the absence of ammonia. the results show that ammonia in vivo evokes tau accumulation both via anion channels, possibly secondary to cell volume changes, and in consequence of stimulation of both nmda and ampa/ka receptors. (supp. by a scsr grant no 4p05a05519 and cimo, the acad. of finland) biosciences department, university of hertfordshire, hatfield herts, u.k. the discovery in 1987 that endothelium-derived relaxing factor is nitric oxide (no) was followed a year later with reports that the cationic amino acid l-arginine is the physiological precursor for nitric oxide. it has since been established that the terminal guanidinium nitrogen of l-arginine is metabolised via a series of oxidation reactions resulting in no production, with citrulline being formed as a co-product. of interest was the parallel observation that uptake of l-arginine was enhanced in inos expressing cells and that this was due to de novo synthesis of carrier proteins. the precise signaling pathway that regulates the enhanced expression of these carriers has been the subject of intense studies in recent years. current literature suggests that activation of upstream signaling molecules such as protein kinase c may be critical. in addition, downstream kinases thought to be points of convergence for various signals originating from cell surface receptors have also been implicated. two of these downstream targets include the 42 and 44 kda forms of mitogen-activated protein kinase (mapk) and the stressactivated 38 kda mapk. it is worth noting however that the involvement of these different transduction pathways in the regulation of the induction of l-arginine transporters is not universal, and likely to be different from system to system. as a result there has been conflicting data on the relevance of these signaling proteins in inducing l-arginine transport in different cell. these issues will be discussed and the individual signaling pathways assessed on a cell type and species basis. moreover, the role of downstream signaling molecules will be examined in more detail, looking in particular at the critical dependency on the p38 mapk. this kinase currently exists in four different isoforms which are p38α, , γ and δ. the involvement of individual isoforms of p38 in enhancing the expression of carrier proteins for l-arginine will be discussed. gw274150 is an acetamidine derivative of heterosubstituted lysine which has been shown to have a marked selectivity for the human inducible nitric oxide synthase isoform (young et al. 2000. bioorg. med. chem. lett., 10: 6, 597-600) . the systems associated with transport of this compound have been investigated using the macrophage cell line j774. prior to each study, j774 cells were seeded in 96-well culture plates and allowed to adhere for 24 h in dulbecco's modified eagle's medium (dmem). transport studies were carried out using hepes buffered krebs solution (50 µl; 37°c) containing l-[ 14 c]gw274150 (1 µciml ϫ1 ) in the presence of either 0.1 mm or 0.025-1 mm unlabelled substrate. in parallel studies transport (1 µciml ϫ1 , 0.1 mm) was monitored in the presence of 1 mm excess of various other amino acids known to be substrates for distinct transport systems. time course experiments revealed that transport of 0.1 mm of l-[ 14 c]gw274150 occurred in a time-dependent manner and was linear for up to 5 min. in addition, uptake was only marginally dependent on extracellular na ϩ . kinetic studies revealed that transport was saturable, and michaelis-menten analysis revealed single affinity entry with an apparent k t of 0.31 mm and v max of 5.15 pmol·µg protein ϫ1 min ϫ1 . at 1 mm, 2-methylaminoisobutyric acid (meaib), lalanine, l-valine and -2-amino-bicyclo-(2,2,1)-heptane-2carboxylic acid (bch) caused little or no inhibition of l-[ 14 c]gw474150 (0.1 mm) uptake. in contrast, transport of l-[ 14 c]gw274150 was inhibited markedly by l-arginine, llysine, l-leucine, l-methionine, 6-diazo-5-oxo-l-norleucine (don) and l-glutamine. with the exception of l-arginine and l-lysine, the inhibition caused by the other substrates was critically dependent on extracellular na ϩ and was completely reversed when extracellular na ϩ was replaced with choline. in parallel kinetic inhibition experiments, transport of 0.1 mm l-[ 14 c]gw274150 was inhibited in a concentration dependent manner by l-arginine (ki ϭ 0.04 mm), l-leucine (ki ϭ 0.06), don (ki ϭ 0.18 mm) and l-glutamine (ki ϭ 0.13 mm). taken together, these data suggest that gw274150 may be transported, at least in part, by system y ϩ . however, the marked inhibition caused by l-leucine, l-glutamine and l-methionine, substrates for the relatively high affinity cationic amino acid transporter system y ϩ l, would suggest that this system may also contribute to the uptake of gw274150; if so, the monophasic substrate kinetics imply that the two systems handle gw274150 with similar affinity. other systems such as b 0,ϩ could be ruled out on the grounds that this transporter is critically na ϩ -dependent while uptake of gw274150 is largely (ϳ80%) na ϩ -independent. similarly, b 0,ϩ , another broadspectrum aminio acid transporter that may be capable of transporting gw274150 does not interact with l-glutamine and thus unlikely to be involved in transport of gw274150, at least in j774 cells. although a large number of different amino acid transporters have been identified on a molecular basis, some of themfunctionally described in mammalian cells -are still missing. in search of mammalian est sequences, which contain the signature of the aaap (amino acid/auxin permease) family, we identified a murine full length cdna, which encodes a membrane protein with 10-11 putative transmembrane domains. the transporter mrna is expressed in various murine tissues, including lung, heart and kidney. for functional characterization we used the xenopus laevis oocyte expression system and employed flux studies and electrophysiological analysis. oocytes injected with the crna showed an increased uptake of 3 h-l-alanine and 3 h-l-proline. detailed electrophysiological analysis revealed an electrogenic transport mode, independent of sodium and chloride ions. lowering the extracellular ph increased significantly substrate induced currents in crna injected oocytes. out of the 20 proteinogenic amino acids the transporter recognizes only small amino acids, such as gly, ala, pro and ser. distinct structural analogues of these amino acids also interact with the transporters substrate binding site. in conclusion, we describe the molecular and functional characteristics of the first electrogenic proton driven amino acid transporter of mammals. pharmacology department, dr. willmar schwabe gmbh, karlsruhe, germany it is now well established that transport of amino acid neurotransmitters (like glutamate, aspartate, gaba and glycine etc.) from and to the neurones is essential for their proper functioning. like in the case of other neurotransmitters, specific pre-and post-synaptic as well as vesicular transporters are involved in such processes. extensive efforts to clarify the mechanisms and processes involved in the control and/or proper functioning of the amino acid transporters are now, therefore, being made in numerous laboratories. such efforts have not only led to the identification of a few specific ligands and/or modulators of neuronal amino acid transporters, but also have started unravelling the complex and diverse processes regulating their functions. aim of this communication is to point out potential usefulness of some neuroactive constituents isolated from therapeutically used medicinal herbs for clarifying the mechanisms involved in neuronal amino acid transport. our interest in such studies was initially triggered by the observations made with hyperforin, i.e. quantitatively the major neuroactive component of hypericum perforatum extracts widely used for the treatment of mild to moderate depressive disorders. this acyl phloglucinol derivative not only modulate synaptic transports of biogenic amines but also of glutamate, aspartate and gaba. since it does not interact with any of the till now described transporters for these neurotransmitters, efforts were made to clarify the mechanisms involved in their observed effects (both in vitro and as well as in vivo). the results of the in vitro studies available to date strongly suggest that its effects on neuronal amino acid transport processes is mediated via some novel extracellular mechanism controlling the h ϩ (and/or other ionic) concentrations of neurones. these observations not only demonstrate that hyperforin represent a structurally and mechanistically novel class of therapeutically useful agent but also suggest that it could be useful tool for clarifying the complex mechanisms involved in the control of neuronal amino acid transport. these observations stimulated us to screen other putative psychoactive herbal extracts and their active constituents on neuronal amino acid transport and on the consequences of disturbances caused by malfunction of specific transporters. observations made with several such agents indicate that either modulation of mechanisms and/or processes involved in neuronal amino acid transport or reversal of pathologies caused by anomaly of transporter functions could be involved in their modes of actions. these observations reinforce our conviction that studies directed towards clarifying the effects of herbal constituents on neuronal amino acid transport might not only be a feasible way for identifying novel types of therapeutically interesting molecules but also could expedite our knowledge on these complex processes. glutamate-regulated sodium dynamics in cortical astrocytes: implications for cellular bioenergetics j.-y. chatton, p. marquet, and p. j. magistretti the mode of na ϩ entry and the dynamics of intracellular na ϩ concentration (na ϩ i ) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. an elevation of na ϩ i was evoked by glutamate, whose amplitude and initial rate were concentration-dependent. the glutamate-evoked na ϩ increase was primarily due to na ϩ -glutamate cotransport. the rate of na ϩ influx decreased during glutamate application, with kinetics that correlate well with the increase in na ϩ i and which depend on the extracellular concentration of glutamate. a tight coupling between na ϩ entry and na ϩ /k ϩ atpase activity was revealed by the massive na ϩ i increase evoked by glutamate when pump activity was inhibited by ouabain. during prolonged glutamate application, na ϩ i remains elevated at a new steady-state where na ϩ influx through the transporter matches na ϩ extrusion through the na ϩ /k ϩ atpase. a mathematical model of the dynamics of na ϩ i homeostasis will be presented which precisely defines the critical role of na ϩ influx kinetics on the establishment of the elevated steady-state and its consequences on the cellular bioenergetics. indeed, extracellular glutamate concentrations as low as 10 µm approximately doubled the energetic demands of the astrocytes. department of biochemistry and molecular biology, faculty of biology, university of barcelona, spain in the last 5 years a new family of amino acid transporters composed by two different subunits has been defined. two heavy subunits (rbat and 4f2hc) and seven light subunits are known. rbat and the light subunits b0,ϩ at and y ϩ lat1 are responsible for the inherited aminoacidurias type i cystinuria, non-type i cystinuria and lysinuric protein intolerance, respectively. the heavy subunits are highly glycosylated type ii proteins, while light subunits are very hydrophobic unglycosylated membrane proteins, displaying a polytopic (generally 12 transmembrane domains) predicted structure. the specificity of the amino acid transport activity depends on the light chain expressed. this, together with its topology, indicates that the transport function mainly relies on the light subunits. i will summarize some of our current studies directed to the understanding of structure-function relationships of these heteromeric carriers, specially concerning their oligomeric structure and initial attempts to reconstitute them. ongoing work on the isolation of new rbat-associated light subunits and new b0,ϩ at-associated heavy subunits, which could also play a role in cystinuria, will also be discussed. department of pharmacology, joh. gutenberg university, mainz, germany mammalian cationic amino acid transporters (cats) catalyze the transport of basic amino acids through the plasma membrane. the cat family comprises at least five related carrier proteins (cat-1, -2a, -2b, -3 and -4) with cat-2a and -2b being splice variants. in humans, only the "old" members of the family have been characterized (hcat-1, -2a and -2b). hcat-1 and -2b exhibit high affinity for cationic amino acids and are sensitive to trans-stimulation, consistent with the classical system y ϩ . in contrast, hcat-2a is a low affinity carrier relatively insensitive to trans-stimulation. interestingly, hcat-2a and hcat-2b differ only in a region of 42 amino acids. cat-3, so far only identified in rat and mouse, exhibits also system y ϩ activity. however, the substrate recognition and maximal transport activity seems to differ from other y ϩ transporters. cat-3 expression has been reported to be restricted to the brain in adult animals. a cdna encoding for human hcat-4 has recently been isolated, however, the transport activity of hcat-4 has not been characterized. when optimally aligned, the amino acid sequence of hcat-4 shows only about 40% identity with the other hcat isoforms. in contrast, the amino acid sequences of hcat-1, -2(a or b) and -3 are about 60% identical. to elucidate which amino acids are responsible for the difference in the transport properties of the hcat proteins, we constructed chimeric proteins between hcat-1 and hcat-2a and performed site directed mutagenesis. using this approach, we identified two amino acid residues that are responsible for the different transport properties of hcat-2a compared to the high affinity cat-isoforms. to characterize the human cat-3, we cloned a cdna encoding hcat-3. when expressed in xenopus laevis oocytes, hcat-3 had a similar transport activity and affinity for l-arginine as hcat-1 or -2b. hcat-3mediated l-arginine transport was trans-stimulated and independent of extracellular na ϩ ions. expression studies demonstrated that hcat-3 is not only expressed in different regions of the human brain, but also in peripheral tissues. to investigate if hcat-4 also functions as an amino acid transporter, we measured the transport of cationic, neutral and acidic amino acids in xenopus laevis oocytes expressing hcat-4, but could not detect an transport activity for any substrate tested. a bright fluorescence could be detected in the plasma membrane of oocytes expressing hcat-4 with the green fluorescent protein attached to the c-terminus. therefore, hcat-4 might either need a complementary protein to function as an amino acid transporter or serve as a transporter for a yet unidentified substrate. renal amino acid reabsorption in immature and adult rats as a sensitive marker of heavy metal-induced nephrotoxicity (pt, cr, tl) institut für pharmakologie und toxikologie, klinikum der friedrich-schiller-universität jena, germany the effects of cis-platinum (cp; 0.6 mg/100 g b. wt. i. p.), sodium dichromate (cr; 1 mg/100 g b. wt. s. c.) and tl 2 so 4 (tl, 2 mg/100 g b. wt. i. p.) on renal amino acid excretion and plasma amino acid composition were investigated in 10-(both sexes) and 55-day-old (female) anaesthetised wistar rats (han : wist). on the basis of diuresis experiments on conscious rats (determination of urinary volume and protein excretion) the mentioned doses and times (1 st day after cr in both age groups and in 10-day-old rats after cp and 3 rd day after cp in adult rats; 2 nd [55-day-old rats] and 5 th -6 th day [10-day-old rats] after tl) were found out to be optimal for the characterisation of amino acid transport after heavy metal poisoning. interestingly, in conscious 10-day-old rats cr nephrotoxicity is not detectable after 1 mg/100 g b. wt. whereas all of the other experimental groups showed nephrotoxic effects of cr, tl and cp in conscious rats. urine volumes were lower, but not significantly, in anaesthetised immature rats, independently of the administered nephrotoxin. glomerular filtration rate (gfr) is significantly lower in 10-day-old rats compared to adults. after cp, cr and tl gfr is significantly reduced only in adult rats and age differences disappeared nearly completely. in principle the renal fractional excretion (fe aa ) of amino acids was distinctly higher in immature rats as a sign of lower amino acid reabsorption capacity. nevertheless, the amino acid plasma concentrations were relatively high in immature control rats. however, both cr and cp did not distinctly influence molecular cloning and functional characterization of ata3, a novel subtype of the amino acid transport system a medical college of georgia, augusta, georgia, u.s.a. recent molecular cloning studies have revealed that the amino acid transport system a consists of more than one subtype. two different system a subtypes, called ata1 and ata2, have been cloned and functionally characterized. ata1 is expressed primarily in the brain and placenta whereas ata2 is expressed ubiquitously. heterologous expression studies have shown that these two subtypes cannot be distinguished functionally based on substrate affinity nor substrate specificity. we have now cloned a third subtype of system a, designated ata3. it is expressed primarily in the liver. apart from the liver, detectable level of expression is noted only in the skeletal muscle. interestingly, ata3 can be easily differentiated from the other two subtypes of system a based on functional characteristics. we first isolated rat ata3 cdna from a skeletal muscle cdna library using rat ata2 cdna as the probe. rat ata3 consists of 547 amino acids and exhibits a high degree of homology in amino acid sequence to rat ata1 (47% identity) and rat ata2 (57% identity). interestingly, this new transporter also has a comparable degree of homology to sn1 and sn2, the two known subtypes of the amino acid transport system n. however, when expressed heterologously in xenopus laevis oocytes, rat ata3 transports α-(methylamino)isobutyric acid (meaib), a specific model substrate for system a, confirming that this transporter is definitely a subtype of system a. system n does not transport this system a model substrate. with two-microelectrode voltage-clamp technique, we have shown that exposure of rat ata3-expressing oocytes to neutral, short-chain aliphatic amino acids induces inward currents. the amino acid-induced current is na ϩ -dependent and phdependent. analysis of the currents with alanine as the substrate has shown that k 0.5 for alanine (i.e., concentration of the amino acid yielding half-maximal current) is 4.2 ϯ 0.1 mm and that the na ϩ : alanine stoichiometry is 1 : 1. subsequently, we have cloned the human homolog of rat ata3 from a liver cell line (hepg2) cdna library. human ata3 also contains 547 amino acids and shows 88% identity in amino acid sequence with rat ata3. the sequence identity of human ata3 with human ata1 and human ata2 is 47% and 57%, respectively. the homology of human ata3 with human sn1 and sn2 is also similar (56% and 51% identity, respectively). the gene coding for human ata3 contains 16 exons and is located on chromosome 12p13. in the human, ata3 is expressed almost exclusively in the liver. when expressed in mammalian cells heterologously, human ata3 mediates the transport of neutral amino acids, including meaib, in a na ϩ -dependent manner. interestingly, while characterizing the function of this clone, we have uncovered a unique feature of this system a subtype. human ata3 is capable of mediating the transport of cationic amino acids. in fact, the affinity of human ata3 for cationic amino acids is higher than for neutral amino acids. however, the human ata3-mediated cationic amino acid transport is na ϩ -independent. in this respect, ata3 is similar to transport system y ϩ l that also transports neutral amino acids in a na ϩ -coupled manner and cationic amino acids in a na ϩindependent manner. in contrast, ata1 and ata2 have not been shown to interact with cationic amino acids. in addition to this difference in substrate specificity, ata3 also differs from ata1 and ata2 in substrate affinity. ata1 and ata2 interact with meaib with a k t of ϳ0.3 mm whereas the affinity of ata3 for this model substrate is comparatively at least 20-fold lower (k t , ϳ8 mm). but, ata3 interacts with arginine with a k t value of 0.3 mm. since liver does not express any of the previously known high affinity cationic amino acid transporters, amino acid plasma concentrations. but in both age groups the administration of cr and cp significantly decreased amino acid reabsorption capacity (increase in fe aa ) as a sign of nephrotoxicity, most pronounced in adult rats after cp. on the other hand, after tl, the fe of amino acids was distinctly higher only in adult rats as a sign of lower amino acid reabsorption capacity and, thus, as a sign of higher nephrotoxicity. in immature animals fe aa was increased only for few amino acids. however, in both age groups tl administration significantly decreased plasma amino acid concentrations, more pronounced in immature rats. the investigation of renal amino acid handling confirmed: (1) cr, cp and tl were more nephrotoxic in 55-day-old animals compared to immature rats as could be demonstrated previously using other parameters for nephrotoxicity testing. (2) the extent of toxic effects of heavy metals on the kidney is related to the maturity of renal functions involved in the enrichment of the respective metal in renal tissue and in its toxicity mechanism. (3) changes in the fractional excretion of amino acids (reduction in renal amino acid reabsorption capacity, e.g. increase in fe aa ) and in amino acid plasma concentrations (especially decreases as a consequence of enhanced renal loss of amino acids) are early indicators of nephrotoxicity. (4) therefore, the determination of renal amino acid handling is a highly sensitive marker for nephrotoxicity testing, both in immature and in adult rats. the mammalian h ϩ /peptide cotransporter pept2 was initially identified in the brush border membrane of renal proximal tubular cells as a high affinity type ptr2-family member. here we describe the synthesis and functional analysis of novel high affinity inhibitors for pept2 that will be useful in further studies on structure and functions. starting from lys[z(no 2 )]-pro a series of different lysine-containing dipeptide derivates were synthesized and studied for interaction with pept2 based on transport competition assays in pichia pastoris yeast cells and in epithelial skpt cells, both expressing pept2. the twoelectrode-voltage-clamp technique in x. iaevis oocytes expressing pept2 was used to determine whether the compounds are transported electrogenically or block the uptake of dipeptides. synthesis and functional analysis of lys-lys derivates containing z(no 2 ) side chain protections provided a set of inhibitors that reversibly inhibited the uptake of dipeptides by pept2 with k i values as low as 10 nm. this is the highest affinity of a ligand of pept2 ever reported. moreover, based on the structure-function relationship we can conclude that the spatial location of the ε-amino protecting group in a lys containing dipeptide and its intramolecular distance from the alpha catom are key factors for the transformation of a substrate into an inhibitor of pept2. ata3 is likely to provide the major route for the uptake of arginine in this tissue. institute of pharmacology and therapeutics, faculty of medicine, porto, portugal the present study examined the nature and regulation of the l-dopa transporter in two functionally different clonal subpopulations of opossum kidney (ok lc and ok hc ) cells. the inward transfer of l-dopa was largely promoted through an energy-dependent and sodium-insensitive transporter, though a minor component (ϳ15%) was found to require extraceilular sodium. l-dopa uptake was insensitive to meaib, but competitively inhibited by bhc (ok lc , ic 50 ϭ 336 µm; ok hc , ic 50 ϭ 439 µm). l-and d-neutral amino acids and basic amino acids markedly inhibited l-dopa accumulation. l-dopa, lleucine, l-arginine, bhc or l-arginine plus bhc stimulated [ 14 c]-l-dopa efflux. the accumulation of l-dopa was significantly higher at an acidic ph, and incubation of cells with l-dopa (100 µm) resulted in marked intracellular acidification. modulators of pka, pkg, pkc and ptk failed to affect the accumulation of l-dopa. only the ca 2ϩ / calmodulin inhibitors inhibited l-dopa uptake. it is likely that system b 0,ϩ might be responsible for the sodium-dependent uptake of l-dopa in ok cells, whereas sodium-independent uptake of l-dopa may include systems b 0,ϩ and lat2, the activation of which results in trans-stimulation of l-dopa outward transfer. the trans-stimulation of l-dopa inward transfer by an imposed h ϩ gradient suggest that ok cells are provided with an l-dopa-h ϩ cotransport system. amino acids are essential nutrients for cell growth and maintenance. the essential amino acids arginine and lysine, are mainly transported via the cationic amino acid transporter 1 protein (cat1). the regulation of translation of the cat1 mrna during amino acid starvation was studied. an adaptive response to amino acid starvation and stress is a global decrease of protein synthesis, by phosphorylation of the translation initiation factor eif2a. translation of the transporter mrna increases when eif2a is phosphorylated, allowing synthesis of the essential for survival arginine/lysine transporter protein. the mechanism of increased translation of this mrna involves the induction of activity of a uorf-containing internal ribosomal entry sequence (ires). translation of the uorf and phosphorylation of eif2a are required for increased activity. we propose that eif2a phosphorylation triggers translational attenuation within the uorf, converting a relatively inactive, to a high activity ires. this study demonstrates that like yeast, mammalian cells have developed a sophisticated response to stress conditions: when expression of most genes decreases, synthesis of stress response proteins increases to support cell survival. amino acid transport, cell volume and the regulation of cell death f. lang, s. fillon, i. setiawan, p. lang, v. tanneur, d. häussinger, and s. bröer department for physiology, university of tübingen, germany cell volume regulatory mechanisms participate in a wide variety of cellular functions including regulation of epithelial transport, excitability, hormone and transmitter release, metabolism, migration, cell proliferation and apoptotic cell death. besides ion transport, polyols, betaine and glycerophosphorylcholine, cells utilize amino acids including taurine to balance extracellular osmolarity and regulate their volume. cells counteract shrinkage by uptake and swelling by release of amino acids including taurine. moreover, cell swelling stimulates synthesis and cell shrinkage favours breakdown of proteins which are osmotically less active than the sum of the amino acids thus generated. conversely, amino acid transport does influence cell volume. concentrative uptake of amino acids leads to cell swelling, amino acid release to cell shrinkage. through alterations of cell volume the amino acids participate in the regulation of protein metabolism. thus, concentrative amino acid transport inhibits and release of amino acids favours proteolysis. these mechanisms participate in the regulation of cell death. cd95 induced apoptotic death of jurkat t lymphocytes is paralleled by the release of taurine. the taurine release occurs with a delay of some 60 min following cd95 receptor triggering but immediately preceedes apoptic cell shrinkage and dna fragmentation. the signaling leading to taurine release is in large part elusive but requires at some stage activation of caspases. moreover, taurine release and apoptotic dna fragmentation are strongly inhibited by lowering of temperature. preloading of the cells with taurine retards cd95 induced dna fragmentation pointing to an active role of taurine in the regulation of apoptosis. peptide transporters of the ptr-family are integral plasma membrane proteins, that mediate the electrogenic protoncoupled transport of di-and tripeptides and peptide-like drugs across cell membranes. the physiological role of pept1, one member of this family in mammals, is mainly the uptake of small peptides into intestinal and renal tubular epithelial cells. in caenorhabditis elegans a homologue to mammalian pept1 is encoded by the pep-2 gene, which is expressed in the intestinal cells and a subset of sensory neurons in the head of the animal. to study the physiological role of the pep-2 transporter in vivo, a c. elegans pep-2 mutant was constructed. the animals deficient in pep-2 show a remarkable phenotype with pronounced signs of malnutrition, characterised by a delayed development, less eggs in the uterus, a smaller brood size and a prolonged mean life-span compared to wild-type animals. we rescued the phenotype by the expression of the wt pep-2 gene in the mutant. the observed starved phenotype in pep-2 mutants might be best explained by the reduced intestinal absorption of peptide bound amino acids that are required for protein synthesis and energy metabolism and provides the first direct evidence for the predominant role of the intestinal peptide transporter in amino acid absorption. adenosine is a potent vasodilator in many vascular beds and modulated tone via elevation of intracellular camp and/or release of nitric oxide (no). we have previously reported that adenosine (ado) stimulates l-arginine transport and no production in human cultured umbilical vein endothelial cells (sobrevia et al., j. physiol. 499, 135-140, 1997) , and here further characterise the signalling cascades. rt-pcr demonstrated that fetal endothelial cell possess mrna levels for a 2a , a 2b and a 3 -adenosine receptor subtype, whereas negligible levels were detected for the a 1 -receptor. adenosine (10 µm, 2 min) induced increases in l-arginine transport and no production were ca 2ϩ and camp independent and stimulated transport was abolished in cells depolarised with 80 mm k ϩ . whole-cell patch clamp experiments revealed that adenosine activated inward k ϩ currents, resulting in a membrane hyperpolarization and enhanced influx of the cation substrate l-arginine. adenosine induced l-arginine transport and no production were also abolished by inhibitors of tyrosine kinases (genistein), mek1/2 (pd98059, u0126) but unaffected by inhibitors of pkc (calphosin c) and pi-3 kinase (ly29002). these data suggest that adenosine induces membrane hyperpolarization by activating inward k ϩ currents, increasing the driving force for cationic amino acid influx via system y ϩ . the discovery of nocardicine a by aoki et al. and aztreonam showed that monocyclic -lactams, collectively known as monobactams, can have antibiotic activity. this activity is poor but compensated by the unique effect they can induce on certain microbial cell membranes. our quest for new non-conventional surfactants for various biomedical applications led us to synthesize bioactive compounds with structural similarities to nocardicins. we present here the preparation and the study of original trimodular biosurfactants of type i: spermine and amine oxidase induce a cytotoxic effect on multidrug resistant chinese hamster ovary cells e. agostinelli 1 , s. lord-fontaine 2 , e. przybytkowski 2 , and d. a. averill-bates 2 1 department of biochemical sciences "a. rossi fanelli", university of rome "la sapienza" and cnr, centre of molecular biology, rome, italy 2 department de chimie/biochimie and toxen (centre de recherche en toxicologie de l'environnement), université du québec à montréal, canada the occurrence of resistance to cytotoxic agents in tumor cells is a major obstacle to successful anticancer chemotherapy. multidrug resistance (mdr) is associated with several phenotypic alterations. cells with the mdr phenotype display decreased drug accumulation due to overexpression of pglycoprotein (p-gp), encoded by the mdr-1 gene, which acts as an energy-dependent pump involved in extrusion of drugs. we studied a new strategy to eliminate mdr cells using an enzyme, bovine serum amine oxidase, capable of forming cytotoxic products, h 2 o 2 and aldehyde(s), from polyamines (spermine). the involvement of both toxic products, formed by the bsao/spermine enzymatic system, in causing cytotoxicity was investigated in multidrug resistant chinese hamster ovary cells, ch r c5, at 37 and 42°c. we observed that hyperthermia, depletion of intracellular glutathione (by l-buthionine sulfoximine) and inhibition of glutathione s-transferase (by ethacrynic acid), sensitized ch r c5 cells to the cytotoxic effect of spermine enzymatic oxidation products. mdr cells showed no resistance to h 2 o 2 and aldehyde(s) relative to their drug-sensitive counterparts, auxb1 cells, in experimental conditions of: higher temperature, higher spermine concentration and longer incubation time. the inhibition of cellular detoxification systems led to increased cytotoxic effects of spermine enzymatic oxidation products on both mdr and sensitive cell lines. these results might be of great interest and suggest that toxic oxidation products formed from spermine and amine oxidase could be used in anticancer therapy, mainly against multidrug resistant tumor cells. [acknowledgements: this work was supported by cnr "target project on biotechnology", ministero della sanità tar these compounds present a hydrophobic part introduced by an ester or amide linkage with an aminoacid, a junction modulus which corresponds to -lactam, and a hydrophilic part which contains a triazole, well-known in pharmaceutical industry for its inhibitor effect against -lactamase. the compounds are synthesised from 2-hydroxymethyl-2methyl propionic acid in five steps. selective activation of one of the primary hydroxyl groups was accomplished by the formation of alkoxy tris(dimethylamino)phosphonium (atdp) salts 3 from the corresponding diol. treatment of 3 with excess potassium carbonate in refluxing anhydrous acetone yields the monobactams 4. activation by atdp salts followed by treat-ment with sodium azide and reflux in toluene gives the azido compound. the reaction with acetylenic derivatives allows to obtain the surfactants. the compounds show classical surfactant behavior and the evaluation of their biological properties give evidence for their antibacterial and antiviral activity, which corresponds apparently to antiprotease activity. a prodrug approach to glutathione derivatives with in vitro antiparasitic activity department of chemistry and materials manchester, faculty of science and engineering, metropolitan university, manchester, u.k. the potential chemotherapeutic activity of peptides are lost in many cases in vitro, due to their inability to cross cell plasma membranes. the recent identification of a series of glutathione diesters with high antiparastic activities in vitro against t.b.brucei (african sleeping sickness) lead us to investigate the determinants associated with their activity. a qsar study on some twenty-five diester derivatives against t.b.brucei and t.b. rhodesiense lead us to conclude that the mechanism of action of these compounds is related to membrane penetration and hydrolysis, controlled by hydrophobicity and steric factors. a hplc and sensor study have confirmed the de-esterified diacid as the active agent of these prodrugs. dietary taurine prevents oxidative stress and morphological alterations in the retina of diabetic rat f. franconi 1 , m. a. s. di leo 2 , s. caputo 2 , n. gentiloni silveri 2 , and g. ghirlanda 2 1 department of pharmacology, university of sassari, and 2 department of internal and geriatric medicine, catholic university, rome, italy diabetes mellitus can cause various complications including retinopathy, which is the earliest and most common complications of diabetes mellitus, affecting 90% of diabetics and progressing to blindness in about 5%. considerable evidence implicates oxidative stress in the pathogenesis of diabetic retinopathy. in fact, hyperglycemia generates reactive oxygen species and free radical defense is reduced in diabetic patients. thus, the prevention of oxidative stress may have important implications for pharmacological attempts to prevent diabetic retinopathy. at this regard, it has been found that taurine, a semi essential amino acid with antioxidant activity, is decreased both in type 1 and type 2 diabetes mellitus. moreover, taurine seems to have a peculiar role of taurine in terms of cellular physiology and pathophysiology of the retina. among others, taurine is thought to produce important physiological effects through osmoregulation, calcium modulation and antioxidant effects. therefore, we examined the effect of dietary chronic (4 months) taurine (2% and 5%) supplementation in diabetic rats in comparison with vitamin e (200 and 500 ui). dietary taurine supplementation, for 4 months, does not influence conjugated dienes (cd), lipid peroxides (lp) and na/k atpase activity in the retina of non diabetic rats. using rats streptotozocin (stz) induced diabetes of 4-month duration, we found that cd, lp are significantly increased and they remained elevated for 4 months. while, the na/k atpase is significantly decreased during the whole experimental time (4 months). moreover, an inverse correlation has been found among the cd and lp and atpase activity. in the retina of stz rats, these biochemical alterations are accomplished with marked profound morphological changes. in stz rats, taurine enriched diets decrease the lipid peroxidation and preserve the atpase activity, being 5% taurine more effective than 2% diets. the morphological examination reveals that in rats feed with 5% taurine no proliferative changes are present. moreover, the beneficial effects of taurine are more marked than of those of vitamin e. these results and previous findings encourage new investigations to evaluate the efficacy of taurine as an adjunctive agent ch ch 3 (ch 2 ) n xco iran applicated be (500 mg/kg -10 days) and the third -control. enzyme activities were determined spectrophotometrically in brain homogenate. results: polyamine oxidase activity decreased significantly lower dose of be didn't induce any significant change in diamine oxidase activity gaba-transaminase activity increased significantly (p ͻ 0.005; p ͻ 0.001) and dose dependently upon be treatment we have been examined the effects of propofol, taurine and propofol combined with taurine on free intracellular amino acid (aa) profiles, superoxide anion formation (o 2 ϫ ), hydrogen peroxide production (h 2 o 2 ) and released myeloperoxidase activity (mpo) in polymorphonuclear leucocytes (pmn). propofol led to significant changes in pmn free taurine, glutamine, glutamate, aspartate, methionine, basic, neutral (naa) and branched chain amino acid concentrations. exogenous taurine reduced pmn naa while increasing intracellular taurine. taurine supplemented to propofol significantly reversed the changes in taurine, naa and alanine only. regarding pmn immune functions propofol significantly decreased o 2 ϫ , h 2 o 2 formation and mpo. taurine decreased o 2 ϫ and h 2 o 2 production, while increasing released mpo. when propofol and taurine were combined they appeared to by reacting tyrosine with 1-nitroso-2-naphthol in the presence of nitric acid 1-2-benzyo-8-(alanyl)-3-phenoxazone (blp) an analog of actinomycin d is produced. the structural similarity of blp to actinomycin d prompted the national cancer institute (nci) to investigate its antitumor activities. the nci investigations revealed that blp exhibits growth inhibitory effects on various cancer cells and as a result blp has received the u.s. patent from the u.s. patent office. the purposed of this investigation was to synthesize similar benzo phenoxazone derivative by reacting 1-nitroso-2-naphthol with 4-(α-hydroxy -methylaminopropyl)phenol in the presence of nitric acid. during the study, it was found out that 1,2-benzo-3phenoxazone derivative is not produced but a hydrogenated form of 1,2-benzo-3-phenoxazone which is probably 1,2-benzo-8-(α-hydroxy -methylaminopropyl)-3-hydroxyphenoxazine (bhmhp) which has been suhhested from mass spectra obtained by electron ionization, ei, chemical ionization, ci and electro-spray ionization, esi, methods. 1 bhmhp was screened against various cancer cell lines by nci and has shown promising effect against three (3) breast cancer cell lines: mda-mb-435, mda-n and hs-578 t. the 50% growth inhibitory (gi 50 ) concentrations for these three cell lines were 4.60 ϫ 10 ϫ6 , 4.62 ϫ 10 ϫ6 and 5.74 ϫ 10 ϫ6 molar respectively. a. bocheva 1 and t. pajpanova 2 1 institute of molecular biology, bulgarian academy of sciences, and 2 institute of physiology, bulgarian academy of sciences, sofia, bulgariathe histamine is an endogenous substance with neurotransmitter and neuromodulator functions in the organism. its antagonists are used in the therapy of allergic diseases and inflammatory reactions and as antiulcer drugs.the limited potentialities of the antihistamine therapy together with the increasing number of the people suffering from allergic diseases give rise to the design and synthesis of new histamine analogues as a perspective area in the chemistry of therapeutic drugs.additionally, compounds containing the guanidine, oxyamino and sufonamide moieties are known to elicit a variety of pharmacological responses and are present in several marketing drugs or drug candidates.on the other hand, similar compounds, being a part of bigger structures (for instance peptides), can imitate the molecules of already known at ii-receptor antagonists.having in mind these data we aimed to synthesize new analogs of histamine containing sulfo-and oxy-guanidino groups with common formula: a. bocheva 1 , s. pancheva 2 , and t. pajpanova 2 1 institute of physiology, bulgarian academy of sciences, and 2 institute of molecular biology, bulgarian academy of sciences, sofia, bulgariathe problem of the efficient therapy of pain is important not only from clinical but from social and economic point of view. the great achievements in medicine are connected with the research on the development of antinociceptive drugs.melanocyte-inhibiting factor (mif) is a tripeptide (pro-leu-gly-nh 2 ) that was discovered in hypotalamus.the mif-1 exerted a weak analgesic effect. the synthesis of non-protein amino acids and their incorporation into biologically active peptides might become a powerful method for the design and development of modified analogues of natural peptides. having in mind these data we synthezied a number of new mif-analogues, containing unnatural amino acids such as cav, slys, sleu, slle and snie and in vivo experiments were performed to study their action on the nociception. the changes in nociceptive effects were examined in male wistar rats by the tail-flick (tf) and hot-plate (hp), as well as, the randall-seitto paw-pressure tests. the peptides were applied intaperitoneal (i.p) injection at a does 1 mg/kg. the results show that the newly sinthesized analogues exert an antinociceptive effects in all tests used. naloxone at a dose 1 mg/kg (i.p) antagonized the antinociceptive effects of mif-analogues. the interaction between platelets and fibrinogen is known to be mediated by the intergrin gp iib/iiia. the arg-gly-asp (rgd) sequence located on fibrinogen and other proteins of blood and extracellular matrix is the minimum requirement for cell attachment and adhesion. it has been found that peptides containing the rgd sequence can effectively inhibit the binding of fibrinogen to gp iib/iiia. in addition aspirin has been shown to be beneficial in the treatment of stable and unstable angina, acute myocardial infraction. aspirin acetylates and inhibits the enzyme cyclooxygenase, the first enzyme involved in thromboxane a 2 (txa 2 ) synthesis, an activator of platelet aggregation and adhesion.we have already reported that the combination in the same molecule of dipeptide amides, containing amino acid(s) of rgd sequence, with salicylic-residue 2-ro-c 6 h 4 -coϳ, {where rϭh or ch 3 co} at their n-terminal amino group have shown inhibitory activity on human platelet aggregation. continuing this research project on salicyl-peptides we have synthesized a series of rgd analogs, incorporating salicylic acid derivatives, by conventional solution techniques and/or by solid phase. the synthesized rgd analogs were identified by ir, nmr and es-ms spectra and tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation reagents (collagen, adp, thrombin) to citrated platelet rich plasma (prp). platelets were obtained from venous blood of healthy donors and the prp was isolated by centrifugation at 200 g for 5 min at 37°c. the aggregation was determined using a dual channel electronic aggregometer. malonyl dialdehyde (mda) production was measured using thiobarbituric acid reagent. in order to confirm these results, flow cytometry with monoclonal antibodies against gpib, gpiib/iiia, gpiiia and gmp140 was used. the ic 50 values of the synthesized and tested compounds, as well as their mda production and flow cytometry results will be discussed. amino acids have a long tradition as building blocks, chiral auxiliaries and/or ligands in advanced organic synthesis and catalysis. at dsm an enzymatic kinetic resolution process has been developed, based on an aminopeptidase catalyzed stereoselective hydrolysis of racemic amino acid amides to form a mixture of l-amino acid and unchanged d-amino acid amide.several small peptides currently are under investigation as possible anti-tumor agents. neuropeptides such as substance p (sp) and neuropeptide y (npy), have been studied for their ability to prevent tumor growth or the proliferation of several cancer cell lines. these neuropeptides have been investigated for their effect to prostate cancer, small cell lung cancer (sclc) and breast cancer. the synthetic sp analog [d-arg 1 , d-phe 5 , d-trp 7,9 , leu 11 ]sp (antagonist d) and the c-terminal analog [arg 6 , d-trp 7,9 , mephe 8 ]sp 6-11 (antagonist g) inhibit sclc cell proliferation in vitro and in vivo, while the analogs [glp 6 , glu(bu t ) 11 ]sp 6-11 and [glp 5 , glu(bu t ) 11 ]sp 5-11 showed significant inhibition in the proliferation of the cancer cell lines hela and t47d.in the present study the c-terminal analogs of sp [glp 6 , d-trp 7 , glu(bu t ) 11 ]sp [6] [7] [8] [9] [10] [11] (1), [glp 6 , d-trp 7,9 , glu(bu t ) 11 ]sp [6] [7] [8] [9] [10] [11] (2), [glp 6 , d-trp 7,9 , mephe 8 , glu(bu t ) 11 ]sp 6-11 (3), [glp 6 , d-trp 7 , mephe 8 , glu(bu t ) 11 ]sp 6-11 (4), [glp 6 , trp 7 , mephe 8 , glu(bu t ) 11 ]sp 6-11 (5), [glp 6 , mephe 7 , d-trp 8 , glu(bu t ) 11 ]sp 6-11 (6), [glp 6 , d-trp 7 , mephe 8 , glu(bu t ) 11 -oh]sp 6-11 (7), [glp 6 , d-trp 7 , cys(acm) 11 -oh]sp 6-11 (8), [glp 6 , d-trp 7 , mephe 8 , cys(acm) 11 -oh]sp [6] [7] [8] [9] [10] [11] (9), [glp 6 , d-trp 7,9 , mephe 8 , cys(acm) 11 -oh]sp 6-11 (10) have been synthesized and tested for their antineoplastic properties in several cancer cell lines. they were also examined for their cytotoxicity to normal cells.the analogs 1-6 are peptide amides whereas the analogs 7-10 are peptide acids. they were performed using the stepwise synthesis either in solution, using the method of mixed anhydrides with carbonic acids or in spps using the fmoc/bu t methodology. the fragment condensation method in solution, using phosphonium reagents, such as pybop, was also applied. the analogs were purified (hplc) and identified (ft-ir, es-ms, 1 h-nmr).the antineoplastic properties of the analogs were studied using sister chromatide exchange (sce) and proliferation rate index (pri). as it is known the sce method is an indicator of dna damages or its repair mechanism, while the method of pri is a sensitive marker of cytotoxicity. the experiments were carried out using cultured human lymphocytes from healthy donors and these results will be discussed.semiempirical quantum chemical investigation of some thymidine derivatives modified with amino acids and peptides at 3ј, 5ј-positions j. velkov 1 , i. stankova 1 , a. ivanova 2 , and a. tadjer 2 1 department of chemistry, south-west university "neophit rilski", blagoevgrad, and 2 department of chemistry, sofia university "st. kl. ohridsky", sofia, bulgaria optimized geometry and electron charge distribution for some thymidine derivatives (3ј,5ј-bis-o-n-α-benzyloxycarbonyl-alanyl-, 3ј,5ј-bis-o-n-α-benzyloxycarbonyl-valyl ,3ј,5ј-bis-o-n-α-benzyloxy-carbonyl-glycyl-glycyl-glycyl,3ј,5јbis-o-n-α-benzyloxycarbonyl-phenylalanyl,3ј,5ј-bis-o-n-αbenzyloxycarbonyl-glycyl) were calculated at the semiempirical (am1) level. the choice of method is limited by the molecular size. in addition, the differences between the ground state energy of the compounds and that of the hydrolysis reaction intermediates were compared to the experimentally found stability towards hydrolysis.with a few notable exceptions, attempts to crystallise integral membrane proteins have failed due to the difficulties in finding appropriate conditions for proteins that have both hydrophobic and hydrophilic domains. thus structural information is largely limited to predictions of secondary structure from the amino acid sequence and computer modelling, neither of which can as yet give high resolution detail. thus alternative approaches are required, and one that we have employed is to look at the substrate binding/transport characteristics of compounds and predict what features the binding site might have. the membrane transport protein that we are interested in is the proton-coupled di/tri-peptide transporter, which has a wide range of natural substrates and is known to transport therapeutically important non-peptides such as ᮀ-lactam antibiotics and angiotensin converting enzyme inhibitors.the initial question that interested us was what makes a di/ tri-peptide a substrate, but not an amino acid? while the obvious answer is the peptide bond, studies with 'space mimic' compounds (which have the space filling properties of a dipeptide but no peptide bond) gave the surprising result that the peptide bond was not essential for binding and translocation. although these space mimics had n and c termini, studies from our laboratory and others have shown that the presence of free amino or carboxyl groups are not a prerequisite for binding or translocation either. this leaves the question of what does distinguish a pept1 substrate from a non-substrate?computer modelling of a large number of pept1 substrates has allowed the development of a substrate template, whereby potential substrates can be scored according to their predicted binding affinity. from this it is clear that it is a sum of energies derived from a number of substrate-transporter interactions that determine binding affinity, including the n-and c-termini, the peptide bond components and the substrate side-chain groups. further studies aim to refine this model through the complimentary approaches of novel substrate design and sitedirected mutagenesis of the transporter protein.why are we interested in this? a large number of promising therapeutic compounds are found to have little or no bioavailability. compared with most membrane transporters pept1 has a wide range of potential substrates, and amongst its non-peptide substrates are a range of peptidomimetic therapeutic compounds. the recent finding that a peptide bond is not a prerequisite for transport opens up the possibility of designing prodrugs to be substrates for pept1, and this has found to be an effective strategy for example with the antiviral drug valacyclovir.(we thank the wellcome trust for their generous support.) nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyaim: the high amino acid metabolism of tumor cells allows tumor imaging with radiolabeled amino acids as 11 c-methionine (met) by positron-emission-tomography (pet). however in recent experimental and clinical studies met uptake was also found in inflammatory tissue thus leading to false positive results. the aim of the study was to compare [ 18 f]fluoroethyltyrosine (fet), a new amino acid analogue, with met to assess their suitability for differentiating between tumor cells and inflammatory cells in vivo and in vitro.methods: popliteal lymph nodes of balb/c and dba/2 mice were stimulated either by streptocotocin (stz), causing chronic lymphadenitis, or by concanavalin a (con a), causing in acute lymphadenitis. tumor infiltrated lymph nodes were induced by inoculating cells from a lacz transfected t-cell mouse lymphoma line into the footpads of syngenic dba/2 mice. the uptake of met and fet was determined quantitatively in tumor infiltrated and inflammatory lymph nodes as well as in the lymph nodes of untreated mice. in vivo imaging of tracer uptake in mouse lymph nodes was performed using a high resolution (2.4 mm) small animal pet (madpet). in vitro the uptake of the amino acids met and fet was investigated in different cells, such as sw707 human colon carcinoma cells and c6 rat glioma cells, stimulated human lymphocytes and macrophages. about 5 ϫ 10 5 cells of each cell line were incubated in a buffered medium containing either different concentrations of unlabeled amino acids or con a (stimulation of lymphocytes) or the transport inhibitors 2amino-norbornane-carboxylic acid (bch, l-system), α-(methylamino)-isobutyric acid (meaib, a-system) or l-serin (asc-system). 0.37 mbq of each amino acid tracer were added and incubated. uptake was stopped by using ice-cold pbs, cells were washed three times and uptake was analyzed.results: in tumor infiltrated lymph nodes uptake of both tracers was higher than in control lymph nodes. met showed an increased uptake in both lymphadenitis models, whereas fet did not accumulate significantly. met and fet uptake in tumor infiltrated lymph nodes was also seen in madpet images, however inflammatory lymph nodes could only be detected in met images.the amount of tumor uptake was different in the various cell types investigated. c6 cells showed the highest uptake of all cells investigated and a slightly lower uptake was found in sw707 cells. in con a stimulated lymphocytes, the uptake of fet was negligible, while met uptake was significantly higher than in both tumor cell lines. since bch reduced the uptake of fet and met to approximately 10%, fet seems to be also predominantly transported into tumor cells by the l-system. the results indicate, that fet appears to differentiate between tumor and inflammatory tissue, as a result of the low uptake of fet in inflammatory cells. nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyover the past few years numerous studies have documented the high diagnostic accuracy of positron emission tomography (pet) using the glucose analogue f-18-fluordeoxyglucose (fdg) for detection and staging of malignant tumors. a significant limitation of fdg-pet, however, is that increased uptake is not only observed in malignant tumors but also in activated inflammatory cells. due to the high glucose utilization of the normal brain and the lower protein synthesis in the normal gray matter the radiolabelled amino acid c-11-methionine (met) gives higher contrast between brain tumors and normal tissue than fdg-pet. rapid uptake of met has been documented for several malignant tumors like gliomas, lung cancer, bladder cancer and malignant lymphomas since amino acid transport and protein synthesis are generally increased in malignancies. the application of met-pet however has been limited by the short half life of the radioactive label c-11 (20 min) in contrast to f-18 (110 min). amino acid analogous labeled with f-18 like f-18-fluoro-α-methyltyrosine (fmt), f-18-fluoro-ethyltyrosine (fet), f-18-fluoro-phenylanaline, f-18-fluore-proline will allow a more widespread application of amino acid pet in oncology. an other amino acid analogue i-123-iodo-α-methyltyrosine (imt) is of clinical interest because the radionuclid i-123 allows it applicability for single-photoemission-computer-tomography (spect). the uptake of the amino acid analogues can only be regarded as a measure for the increased amino acid transport in the tumor cells because they are not incorporated into proteins. clinical data show that radiolabelled amino acids that are only transported into the cells are not inferior to those that enter protein synthesis. this tracers may also help to differentiate tumor lesions from inflammatory lesions when the expression of the transport systems for amino acids in tumor cells and inflammatory cells is different.lysinuric protein intolerance: understanding the pathophysiology of a multi-system disorder of dibasic amino acid transport m. p. sperandeo 1,2 , v. fiorito 2 , a. pietrosanto 2 , a. pepe 2 , g. andria 2 , and g. sebastio 2 1 telethon foundation, rome, and 2 department of pediatrics, federico ii university, naples, italy lysinuric protein intolerance (lpi; mim 222700) is an autosomal recessive disease, mainly found in finland and italy. clinical findings of lpi include: vomiting, diarrhea, failure to thrive, hepatosplenomegaly, osteoporosis, episodes of coma, and mental retardation. a life-threatening lung involvement (alveolar proteinosis) and renal insufficiency were also reported. metabolic derangement of lpi includes: reduced intestinal absorption of cationic amino acids (lysine, ornithine, arginine, caa), increased renal excretion of caa and dysfunction of the urea cycle leading to hyperammonemia and orotic aciduria. most of the clinical findings cannot be explained by a selective deficiency of amino acid transport, as indeed observed for cystinuria (mim 220100), a cognate disease of lpi. the molecular basis of lpi resides in an abnormal caa carrier functioning at the level of basolateral membrane of epithelial cells in the intestine and the kidney. caa transport is mediated by y ϩ l system, that is exerted by heterodimers consisting of the 4f2 heavy chain (4f2hc) and a light chain represented by either the solute carrier family 7a, member 6 (slc7a6) or 7 (slc7a7). after excluding the 4f2hc as the causative gene of lpi, we identified slc7a7 as the lpi gene and characterized mutations in twenty-five patients from 21 families (16 italian, 2 japanese, 1 moroccan, 1 greek, and 1 pakistani; 34 independent alleles) affected by lpi. thirty-two of the 34 independent alleles (94.1%) were characterized and fourteen mutations were identified. only five mutations (namely 1625insatca, w242x, 1425delctct, ivs3 ϩ 1gaea, s386r) were identified in more than one independent family. most mutations are located in the slc7a7 coding region, except for two splicing mutations. the pathogenesis of some clinical findings of lpi, namely alveolar proteinosis and renal involvement, remains mostly unknown. we are currently investigating the role of slc7a6 gene in lpi, which, in addition to slc7a7, is responsible of the y ϩ l activity. in fact, the regulation of the y ϩ l system, exerted by either 4f2hc/ slc7a76 or 4f2hc/slc7a7, is still unknown. hypothetically, the activation of 4f2hc/slc7a76 in all tissues might be the "simple" way to a lpi gene-therapy.[acknowledgements: m. p. s. is supported by telethon-italy (grant n.29cp) and is an assistant telethon scientist.] pre-eclampsia (pe) is a potentially life threatening complication of pregnancy and is one of the leading causes of maternal and fetal morbidity and mortality. pe is associated with endothelial cell dysfunction and inadequate placental perfusion. fetal plasma l-arginine levels are decreased in pe and there is controversy as to whether nitric oxide (no) production is altered. we have investigated whether the kinetics of l-arginine transport via system y ϩ and no production are altered in fetal umbilical vein endothelial cells (huvec) from pe pregnancies. kinetics of l-arginine transport were similar in huvec isolated from normal, preterm and pe pregnancies, however nethylmaleimide inhibited transport in normal but not pe huvec. basal and histamine-stimulated no production was similar in normal and preterm huvec, whereas pe increased basal (25 ϯ 5 vs 5.3 ϫ 3 pmol/10 8 cell/5 min) and histaminestimulated (70 ϯ 12 vs 20 ϯ 5 pmol/10 8 /5 min) no production. whole-cell patch clamp measurements revealed similar inward rectifying k ϩ currents in normal and pe huvec, with resting membrane potentials of ϫ65 ϯ 4 and ϫ80 ϯ 18 mv in normal and pe huvec, respectively. increased enos activity in pe endothelial cells may serve as a compensatory mechanism to counteract the hypertension observed in pe, however, elevated no production is apparently not associated with enhanced larginine transport. department of pharmacology, university of cambridge, u.k.over the past years, concerns have heightened over the escalating numbers of pathogenic microorganisms that are resistant to multiple antibiotics. this phenomenon poses major problems in the treatment of patients with hospital or community-acquired infections caused by bacteria, yeast, fungi and parasitic organisms. particularly intriguing are the so-called multidrug transporters, which have specificity of compounds with very different chemical structures and cellular targets. this lecture will focus on the molecular properties of the atpbinding cassette multidrug transporter lmra in the lactic acid bacterium lactococcus lactis. lmra is a close homolog of the human multidrug resistance p-glycoprotein, overexpression of which is one of the major causes of resistance of human cancers to chemotherapy. surprisingly, lmra can even substitute for pglycoprotein in human lung fibroblast cells. recent biochemical and pharmacological studies on lmra suggest that the protein may operate by a two-cylinder engine mechanism to transport amphiphilic drugs from the inner leaflet of the plasma membrane. this mechanism will be discussed in more detail. bone and bone marrow are important sites of metastasis formation in breast cancer; so, we studied the level of bone sialoprotein (bsn) and fibronectin (fn), two key connective tissue antigens, in patients with metastatic breast carcinoma. our data reveled that bsn have a statistically significant association with bone metastases in that disease. fn level was also significantly changed in metastatic breast carcinoma when compared to the non metastatic cases. kharkov national university, radiophysical department, chair of molecular and applied biophysics, kharkov, ukraine * present address: institute of cell and molecular biology, university of edinburgh, edinburgh, scotland, u.k.in the work the temperature dependencies of dielectric parameters of human serum albumin (hsa) and fibrinogen solutions (0.15 m nacl, ph 7.2) were obtained in the temperature interval 5-50 c degrees. the measurements of the dielectric parameters were carried out at the frequency of 9.2 hhz, i.e. in the range of free water molecules dispersion. in contrast to dependencies for poor solvent, temperature dependencies of dielectric parameters for protein solutions are of nonmonotonous character; they have a number of peculiarities in the temperature ranges of 8-10, 22-24 and 34-36 c degrees. this fact means that at these temperatures redistribution of free and bound water in protein-water system occurs due to structural changes in protein molecules. the dependencies of hydration of hsa and fibrinogen on temperature were obtained as well.in the work the mechanism of temperature changes of spatial organisation of protein molecules was proposed. perhaps, this mechanism is responsible for maintenance of thermal stability of the functionally active conformation of native proteins. as peculiarities on temperature dependencies of dielectric parameters of solutions of globular (hsa) and fibrillar (fibrinogen) proteins were in the same temperature regions, one may to assume that the mechanism of proteins thermal stabilisation in physiological temperatures interval has a general character. laboratory of cell pharmacology, university of leuven, medical school, campus gasthuisberg (o&n), leuven, belgium n-pomc was purified from conditioned medium of att20 cells using a sequence of concentration, fractionation by ion exchange, rp-hplc and gel-filtration. twenty isoforms of n-pomc, for both 11 and 13 kda, were identified by means of mass spectrometry and n-terminal sequencing. these isoforms are assumed to be pomc1-74 or pomc1-95 with heterogeneous glycosylation.the n-pomc isoforms were tested on prolactin (prl) gene expression and lactotroph mitosis in pituitary cell aggregate cultures. prl mrna content was quantified by means of real time rt-pcr. three 11 kda n-pomc fractions enhanced prl mrna levels by 33-36%, while all other isoforms were inactive. this effect was abolished by immunoneutralization with n-pomc monoclonal antibody. only one fraction stimulated lactotroph proliferation (38.2 ϯ 7.5%) as assessed by brdu incorporation in prl-immunoreactive cells. several (but not all) 13 kda n-pomc fractions stimulated prl mrna level and lactotroph mitosis. on the other hand, all 11 and 13 kda isoforms activated the mc-3 and mc-5 receptor in cell lines in which these receptors were transfected. thus, att20 cells produce various n-pomc isoforms, only a part of which display an effect on prl mrna expression. even fewer isoforms affect lactotroph proliferation. since all isoforms activate the mc-3 and mc-5 receptor, it is suggested that the effect of the few isoforms on lactotrophs is mediated by (a) different receptor(s). are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids.coronal cortical slices (400 µm) were cut and perfused with gassed (95% o 2 , 5% co 2 ) acsf at 37°c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering 2 pulses of k ϩ (60 mm) or veratridine (20 µm) 30 minutes apart.in control experiments the second k ϩ pulse elicited glutamate release which was 80% of the first pulse. hyperforin (5 µm) perfused for 30 minutes prior to, and during, the second k ϩ pulse significantly increased glutamate release to 170% (p ͻ 0.001, n ϭ 6-8). release elicited by the second veratridine pulse was 70% of the first pulse for both glutamate and aspartate. hyperforin (5 µm) increased this release to the second pulse to 160% and 130% respectively (p ͻ 0.001, n ϭ 6-8). when perfused on its own for 30 minutes, hyperforin (5 µm) increased the basal release of glutamate (p ͻ 0.001, n ϭ 4-5).in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca 2ϩ or na ϩ channels.glucagon-like peptide-1 (7-36) amide (glp1) is the main product of the glucagons gene expression in intestinal l cells into the cirulation in response to the ingestion of food and is the most potent stimulator of glucose-induced insulin secretion. glp1 receptors have also been detected in discrete areas of rat brain and intracerebroventricular glp1 has been shown to inhibit feeding in fasted rats. in this study hplc techniques were employed to evaluate the effects of glp1 on serotonin (5-ht) and γ aminobutyric acid (gaba) metabolism in rat brain. glp1 (0.5 µm) produced a significant decrease in levels of 5-ht by 20% after 15 minutes of incubation with combined hypothalamus and brain sterr. synaptosomes. levels of 5hydroxyindolacetic acid (5-hiaa), the principal metabolite of 5-ht, and tryptophan the amino acid precursor of 5-ht, were also decreased significantly by 21% and 37% respectively. gaba and its amino acid precursor glutamic acid were both measured at the same conditions as above, but a precolumn derivatization hplc technique was used. the increase in levels of gaba (14%) and glu (6%) by glp1 was not significant.the results suggest that decreased synaptosomal levels of 5-ht and 5-hiaa caused by glp1 are due to diminished availability of typtophan by the peptide. in experimental model of iron overload we obtained the following results: the concentration of carbonyl groups tended to increase, while mda level significantly increased after feso 4 treatment (1.66 ϯ 0.25 vs control 1.51 ϯ 0.50 µmol/mg prot.) and (2.13 ϯ 0.5 vs control 1.3 ϯ 0.3 nmol/mg protein p ͻ 0.01) respectively. it was associated with significantly increased iron content (0.89 ϯ 0.23 µg/mg prot. vs control 0.49 ϯ 0.17 p ͻ 0.001). it is clear that oxidative stress occurs in experimental iron overload, if sufficiently high levels of iron within hepatocytes are achieved. in group treated with feso 4 and spermine, iron content was significantly decreased (0.36 ϯ 0.07 p ͻ 0.01 compared with fe treated only) and carbonyl group content tended to be lower in comparison to feso 4 treated only (1.58 ϯ 0.24), but mda level didn't change (2.31 ϯ 0.72). in addition, treatment with spermine alone resulted in increase of mda level (2.74 ϯ 0.7 vs control p ͻ 0.01), iron content didn't change (0.59 ϯ 0.29), but carbonyl groups were decreased (0.99 ϯ 0.28 vs control p ͻ 0.05). feso 4 treatment increased gsh level (126.38 ϯ 34.11 nmol/mg prot. vs control 88 ϯ 22.77; p ͻ 0.05) while in combination with spermine this increase was more profound (235.48 ϯ 42.7; p ͻ 0.001 vs control, p ͻ 0.001 vs feso 4 ). spermine alone produced similar increase of gsh level (127.4 ϯ 34.11, p ͻ 0.05 vs control; p ͼ 0.05 vs feso 4 ). the results emanating from the human genome programme have required a reappraisal of protein science and have led to the rapid upsurge in interest in the area of proteomics. this sudden re-emergence of protein science, in fact, was predictable and should not have been surprising.recent experience of protecting group design with respect to lysine and aspartic acid will be discussed together with aspects of chemical synthesis of small proteins of biological significance and in the context of chemical synthesis methodology making contributions to the general field of proteomics. using a cell line permanently expressing the mouse taurine transporter (mtaut) as a fusion protein, we investigated the underlying mechanism by which the immunosuppressive drug cyclosporin a (csa) inhibits taurine transport. csa inhibited the recombinantly expressed mtaut function both in dose and time dependent manner. the inhibitory effect of csa was reversible. thus, washing out the csa resulted in almost complete recovery of taurine uptake. to obtain further insight, we examined the surface abundance of the mtaut as a function of csa treatment using a surface-labeling assay. our results demonstrated that csa treatment altered the surface expression of the mtaut without significantly altering its total expression level, and the reduction in the cell surface expression paralleled the decrease in taurine uptake. upon removal of csa, the virtual recovery in taurine uptake was due to the concomitant increase in the number of taurine transporters on the cell surface. taken together, our results suggest that csa induced inhibition of taurine uptake was either due to the impaired targeting of the taurine transporters to the cell surface or due to the removal of the transporters from the cell surface. polyamines are neuromodulators in a number of physiological and pathological conditions in cns. since application of ethylene glycols causes hypoactivity and lethargy of experimental animals, depression of cns and various neurological symptoms, the aim of this study was to examine the effects of 2butoxyetanol on polyamine and gaba catabolism, taking in account an alternative pathway of gaba synthesis from putrescine. methods: male wister rats were allocated into three groups: first treated by be (100 mg/kg -10 days), second key: cord-325230-3kg4oe4g authors: agol, vadim i.; gmyl, anatoly p. title: viral security proteins: counteracting host defences date: 2010-11-09 journal: nat rev microbiol doi: 10.1038/nrmicro2452 sha: doc_id: 325230 cord_uid: 3kg4oe4g interactions with host defences are key aspects of viral infection. various viral proteins perform counter-defensive functions, but a distinct class, called security proteins, is dedicated specifically to counteracting host defences. here, the properties of the picornavirus security proteins l and 2a are discussed. these proteins have well-defined positions in the viral polyprotein, flanking the capsid precursor, but they are structurally and biochemically unrelated. here, we consider the impact of these two proteins, as well as that of a third security protein, l(*), on viral reproduction, pathogenicity and evolution. the concept of security proteins could serve as a paradigm for the dedicated counter-defensive proteins of other viruses. supplementary information: the online version of this article (doi:10.1038/nrmicro2452) contains supplementary material, which is available to authorized users. all bona fide rna viruses encode at least two proteins, a capsid protein and an rna-dependent rna polymerase (rdrp). in this article, we do not consider 'abnormal' rna viruses such as hepatitis delta virus, which does not possess its own replication machinery and borrows a capsid from another virus 1 , or the capsid-less narnaviruses, which encode only an rdrp 2 ; hepatitis delta virus and narnaviruses are closer to viroids and plasmids, respectively, than to fully fledged rna viruses. rna viruses encoding only a capsid and an rdrp are known to infect protists such as giardia spp. 3 . some rna phages and fungal rna viruses express only three proteins. however, most rna viruses do not rely on such a scant protein repertoire. the proteomes of the majority of rna viruses comprise up to 12 different proteins, with the genomes of some coronaviruses encoding nearly 30 proteins. to infect, viruses must reach an appropriate intracellular environment and, if necessary, adapt this environment to their own requirements. thus, viral infection requires not only replication but also interactions with host defences. to carry out these tasks, rna viruses have only a few proteins at their disposal, with the available protein arsenal being limited by the genome size. as the proteomes of these viruses are strikingly small and specific viral functions generally require more than one protein, most proteins encoded by rna viruses are multi functional. at the same time, however, there is a certain division of labour between viral proteins. we consider some aspects of this problem using, as an example, the picornaviruses, a large family of small animal viruses with a medium-sized, single-stranded, positive-sense rna genome (box 1) . although they share a common genome organization, these viruses exhibit sufficient genetic variation to be separated into at least 12 distinct genera (box 2) . the family includes important human and animal pathogens that cause a range of disorders, including poliomyelitis, foot-and-mouth disease, the common cold, gastroenteritis, hepatitis, meningitis, myocarditis and uveitis. in addition to acute diseases, these viruses can also cause chronic persistent disorders. of the 12 'mature' (fully processed) picornaviral proteins (box 1) , the most ancient group includes the capsid proteins and a set of conserved proteins considered to be picornavirus signature proteins 4 , comprising the rdrp (3d pol ), the primer for rna synthesis (vpg), a protease (3c pro ) and an atpase (2c atpase ). the capsid and signature proteins are indispensable for viral viability. two less conserved proteins, 2b and 3a, are also essential for viability but are less directly involved in viral reproduction. the main functions of these two proteins are to target replicative proteins to the correct destinations and aid in the creation of a suitable replicative niche. finally, two other non-structural proteins that flank the capsid precursor in the polyprotein molecule, the leader (l) and 2a proteins, constitute a distinct group, although they have no common structural or biochemical features. they are the most variable among the picornavirus proteins, and some viruses even lack l altogether (fig. 1; tables 1, 2) . this group also includes the so-called l* protein, which is encoded in a different reading frame of the rna of certain cardioviruses. functionally, these proteins (l, l* and 2a) have been characterized in some detail for only a few picornaviruses, and in these cases their major function has been shown to be counteracting host defences, thereby ensuring optimal conditions for viral reproduction. in this review we argue that picornaviral l and 2a proteins that have yet to be characterized are likely to fulfil the same biological role. we have proposed that this group of proteins should be called security proteins 5 . they are sometimes also referred to as virulence factors 6 ; such a designation, although it may be more familiar, is somewhat less preferable to us for the reasons explained below. viruses have no special 'desire' to be virulent, that is, to harm, even less to kill, their hosts. the pathogenic properties of a virus are not a prerequisite for viral fitness. in fact, the most severe harm in a viral infection comes not from viral reproduction but from the (sometimes miscalculated) host defence response. host damaging or even suicidal defensive reactions include rna degradation, inhibition of translation, and the induction of endoplasmic reticulum stress, apoptosis, autophagy and inflammation, and these reactions are aimed at limiting viral reproduction and spread. as a natural response, viruses have evolved tools directed not only at overcoming the specific innate and adaptive immune responses but also at inhibiting the general host metabolic functions on which these specific defences are based -that is, processes such as transcription and translation, cell signalling and intracellular trafficking. inhibiting these processes is the major function of the security proteins. accordingly, the capacity to withstand host defences, rather than virulence (the ability to harm the host) per se, is the property that is selected for during viral evolution. the long-term co-evolution of a virus and its host will probably result in their mutual adaptation accompanied by a decrease in viral virulence. thus, in our opinion, the term 'security protein' is a better reflection of the evolutionary origin of the relevant proteins than the term 'virulence factor' . moreover, the possession of efficient security proteins does not necessarily make a virus particularly virulent. the aim of this review is to consider the properties and biological significance of picornavirus security proteins and, on the basis of this knowledge, to put forward a general view of security proteins as dedicated counterdefensive proteins that evolved primarily to overcome various mechanisms of host resistance. both l and 2a are extremely heterogeneous with respect to size, sequence and biochemical properties (fig. 1; tables 1,2). the length of l varies from ~70 amino acid residues in cardioviruses to ~450 residues in some sapeloviruses, and the variation is striking even in a single genus; for example, in sapeloviruses, the length varies from ~80 residues (one l protein) to ~450 residues (two l proteins). although certain sapeloviruses seem to express two l proteins (supplementary information s1 (figure)), approximately half the picorna virus genera lack l altogether. the length of 2a varies ~30-fold (from 9 residues in senecaviruses to ~300 residues in avihepatoviruses and some sapeloviruses), and it can comprise one to three separate polypeptides. the l proteins of cardioviruses and klasseviruses are strongly acidic and markedly basic, respectively, whereas the 2a proteins of these viruses exhibit the reverse ratio of acidity. the overall charge of the proteins will influence the choice of their interaction partners. the primary structures of l and 2a are, in most cases, totally unrelated when compared across different genera, and even intra-genus conservation among seemingly homologous proteins can be low, as is the case, for example, with the l proteins of kobuviruses and 2a proteins of cardioviruses and sapeloviruses. with regard to their biochemical activities, only a few security proteins have been characterized in any detail. 2a of enteroviruses (2a pro ) is a protease 7 with a chymotrypsin-like fold in which the catalytic serine residue has been replaced by cysteine 8 . 2a pro performs an essential function in viral reproduction through its involvement in the so-called 'primary' co-translational cis-cleavage of the polyprotein between the amino-terminal amino acid of 2a pro and the carboxy-terminal residue of the capsid protein vp1. aphthovirus l is also a protease (l pro ) with a papain-like fold 9 . translation of aphthovirus rna can start at two in-frame aug codons, generating distinct l proteins, lab (~200 residues) and lb (~170 residues) 10 , with lb predominating in vivo. l pro cleaves the bond at the l pro -vp4 boundary 11, 12 . erbovirus l pro exhibits similar picornaviruses possess a single-stranded positive-sense rna genome (see the figure) comprising approximately 7,000-9,000 nucleotides. the rna is packaged into an icosahedral capsid usually composed of four distinct proteins (vp1-vp4), but in some viruses vp4 and vp2 are fused (forming vp0). after productive contacts with specific cellular receptors, which differ between viruses, the genome is uncoated and enters the cytoplasm, where the main steps of viral reproduction occur. the viral rna, which contains a single orf (with one exception described in the main text), is translated in a cap-independent manner into a 2,200-2,500 amino acid polypeptide, which is eventually processed by limited proteolysis into a dozen 'mature' proteins. these proteins include: capsid proteins; an rna-dependent rna polymerase (3d pol ); a protein (vpg, or 3b) that serves as a primer for the initiation of rna synthesis; an atpase with a conserved superfamily 3 helicase motif (2c atpase ) and an essential but poorly defined role in viral rna replication; a chymotrypsin-like protease (3c pro ), which, as the mature protein or as a precursor, is a major factor in polyprotein processing; two hydrophobic membrane-binding proteins (2b and 3a) that participate in the generation of a virus-friendly environment; and, flanking the precursor of the capsid proteins, one or two highly variable proteins (l and 2a), the structure and functions of which are the subject of this review. the entire coding region of the rna is surrounded with untranslated regions (utrs) harbouring regulatory elements that control viral translation (for example, the internal ribosome entry site (ires)) and replication (the origins oril and orir), and this entire rna sequence is flanked by the covalently linked viral protein vpg and a poly(a) tract at the 5′ and 3′ termini, respectively. there are four structurally and functionally distinct types of ires 131 and many types of oril and orir. the mode of translation for which initiation is dependent on the presence of the so-called cap structure (m 7 gpppn) at the 5′ end of an mrna. specific cap-binding proteins (translation initiation factors) recruit the ribosome to the 5′ end of the mrna, and the ribosome scans the template until it encounters the initiation codon. this translation mode is exploited by most mrnas in eukaryotic cells. a protein that binds to the 3′ poly(a) tail of eukaryotic mrnas on the one hand and to cap-dependent translation initiation factors on the other hand. this dual binding results in a non-covalent circularization of the mrna template, which is accompanied by a significant increase in translation efficiency. enzymatic activity 13 . owing to the presence of a characteristic motif, the 2a proteins of some sapeloviruses have also been proposed to be proteases 14 . 2a of aphthoviruses is a short peptide that promotes co-translational interruption of polyprotein synthesis just downstream of its own coding sequence (and before the 2b moiety); cleavage at the aphthovirus vp1-2a boundary is accomplished by 3c pro . interruption of polyprotein synthesis (also known as ribosome skipping) is proposed to be caused by an interaction between the 2a peptide and the exit tunnel of the ribosome, preventing the interaction of the ribosome with pro-trna (proline is the amino-terminal residue of aphthovirus 2b) 15, 16 . to achieve this, a short peptide harbouring an npg(p) motif (the parentheses mark the interruption site) is sufficient. short 2a peptides from erboviruses 17 , teschoviruses 18 , senecaviruses 19 and cosaviruses 20 also possess npg(p) motifs and seem to be involved in polyprotein processing at the 2a-2b border. separation of these peptides from the vp1 capsid protein has been assumed but not yet clearly demonstrated. as these peptides possess no other known activities, their assignment as security proteins is not warranted; we refer to them here as 2a sp (2a short peptide). the ribosome-skipping npg(p) motif is also present in some 'composite' 2a proteins, such as those of ljungan virus 21 (a parechovirus), avihepatoviruses 22 and seal picornavirus type 1 (ref. 23 ), in which they seem to ensure self-separation from the downstream 2a moieties. in cardiovirus 2a, the npg(p) motif is located at the c terminus and interrupts polyprotein synthesis at the 2a-2b boundary 24 , which corresponds to the polyprotein 'primary' cleavage site in this genus 25 . apart from the npg(p)-containing peptides, 2a proteins of cardioviruses, avihepatoviruses, ljungan virus and seal picornavirus type 1 contain distinct but poorly characterized sequences. the cardiovirus encephalomyocarditis virus (emcv) encodes a 2a protein with rnabinding affinity 26 . the bipartite 2a of ljungan virus, the tripartite 2a of avihepatoviruses and the npg(p)lacking 2a proteins of kobuviruses, tremoviruses and some parechoviruses all have a ~140-150-residue h-nc domain, which contains a histidine with a downstream asparagine-cysteine dipeptide and a putative transmembrane domain 21, 27 . the observation that a similar h-nc domain is present in certain cellular tumour suppressors 21 suggests that these viral proteins are also involved in the control of host activities. between the npg(p) and h-nc motifs, 2a of the avihepatovirus duck hepatitis a virus possesses an additional moiety that contains the so-called aig1 domain 22 , which is also found in representatives of the ras-like gtpase superfamily. cardiovirus l proteins 28 , which are devoid of any enzymatic activity and exhibit noticeable intra-genus variability (table 1) , contain a non-classical but functional zinc finger (cys-his-cys-cys) motif 29,30 and a downstream acidic motif 31 that, in some of these l proteins, contains potential phosphorylation sites 31, 32 . certain strains of theiler's murine encephalomyelitis virus (tmev) are unique among picornaviruses in expressing a functional protein, l*, that is encoded in an alternative translation frame 33,34 that starts within the l coding sequence, goes through the vp4 coding sequences and terminates in the vp2 coding sequence of the main reading frame. the 2a and l proteins of other picornaviruses neither contain easily identifiable amino acid motifs nor have known specific biochemical activities. effects on general host metabolism security proteins contribute substantially to the shut-off of host macromolecular synthesis that occurs in response to infection with many picornaviruses. one could perhaps argue that such effects of security proteins contradict the key proposal of this review that these proteins are dedicated to counter-defensive functions. however, as already mentioned, inflicting harm on their hosts does not bring viruses any benefits per se. the only reason (or at least, the main reason) why viruses evolved the ability to damage infected cells is their need to incapacitate the cellular defensive machinery. this machinery includes several specific mechanisms (such as innate and adaptive immunity), the implementation of which requires general cellular functions such as translation, transcription and controllable nucleocytoplasmic trafficking. therefore, virus-induced impairment of these all-purpose metabolic functions can be regarded as a component of the viral counter-defensive strategy. the effect of security proteins on cap-dependent translation of cellular mrna is particularly important. 2a pro from diverse enteroviruses [35] [36] [37] [38] and l pro from aphthoviruses 12,39 cleave eukaryotic translation initiation factor eif4g. interestingly, erbovirus l pro does not seem to cleave eif4g and does not trigger translational shutoff 13 . poly(a)-binding protein 1 (pabp1; also known as members of the picornaviridae family are small (~30 nm), non-enveloped animal viruses with similar genome organizations and mechanisms of reproduction (box 1) . this family contains important human and animal pathogens, studies of which have made crucial contributions to our understanding of the molecular biology of viruses and the mechanisms of virus-cell interactions. the first animal virus (foot-and-mouth disease virus; fmdv) and the first human virus (poliovirus) to be discovered belong to this family. the current official classification of picornaviruses (see the international committee on taxonomy of viruses) includes 12 genera: aphthovirus (for example, fmdv), avihepatovirus (for example, duck hepatitis a virus), cardiovirus (including important model viruses such as encephalomyocarditis virus, mengovirus and theiler's murine encephalomyelitis virus, as well as human and animal pathogenic viruses such as the newly recognized saffold virus), enterovirus (a large genus that includes poliovirus and many other human and animal pathogenic viruses, such as coxsackieviruses, echoviruses and other 'numbered' enteroviruses, as well as rhinoviruses, the causative agents of the common cold), erbovirus (equine rhinitis b virus), hepatovirus (hepatitis a virus), kobuvirus (for example, aichi virus, which induces gastroenteritis in humans, as well as some animal pathogens), parechovirus (the causative agents of various human diseases, particularly in children; also isolated from rodents), sapelovirus (including an avian, a porcine and a simian pathogen), senecavirus (seneca valley virus, a candidate oncolytic agent), teschovirus (a porcine pathogen) and tremovirus (avian encephalomyelitis virus). in addition, there are two candidate genera, not yet officially approved, that are associated with human diarrhoea: cosavirus and klassevirus. finally, seal picornavirus type 1 is a distinct virus not assigned to any of the above genera. several new picornavirus genera have been identified recently, and it seems plausible that this number will grow, particularly with the breakthroughs in sequencing techniques and the achievements of metagenomics. cytoplasmic pabp), a host protein that is also involved in translational control, is another target of enterovirus 2a pro (refs 40, 41) . security proteins can inhibit host translation by mechanisms other than proteolysis. mutations of cardiovirus 2a (which is not a protease) alleviate virus-induced translational shut-off 42, 43 . it is thought that association of cardiovirus 2a with ribosomes 44, 45 , which seems to take place in the nucleolus 46, 47 and possibly occurs through the rna-binding activity of 2a 26 , might contribute to preferential use of the internal ribosome entry site (ires)-dependent viral templates. cardiovirus l was also reported to mediate translational shut-off 48 . however, this effect could largely be due to l-mediated inhibition of nuclear export of mrna rather than to inhibition of translation per se 49, 50 . on the basis of ectopic expression experiments, hepatitis a virus 2a has also been implicated in inhibition of cap-dependent translation 51 , but the relevance of this observation is uncertain, as hepatitis a virus does not exert translational shut-off. the effects of security proteins on cellular transcription are less well studied, although synthesis of the mrna for cytokines and chemokines is inhibited in certain cases (see below). individually expressed poliovirus 2a pro was reported to cleave the general transcription factors tata-box-binding protein 52 and cyclic amp-responsive element-binding protein 1 (ref. 38 ), but the biological significance of these effects is debatable 52, 53 . poliovirus 2a pro cleaves gemin3 (also known as ddx20), a protein that is involved in the formation of spliceosomes 54 . cardiovirus 2a has also been implicated in virus-triggered inhibition of host transcription 47 , but this effect was not investigated further. foot-and-mouth disease virus (fmdv) l pro (refs 55, 56) and human parecho virus 2a 57 accumulate in the nucleus during the course of infection and as a result of ectopic expression, respectively, but their nuclear effects are unknown. enterovirus 2a pro cleaves some cytoskeletal proteins, such as cyto keratin 8 (ref. 58 ) and dystrophin, a protein that connects the cytoskeleton to the plasma membrane 59 . the security proteins of several picornaviruses profoundly affect nucleocytoplasmic transport in infected cells. the targets of enterovirus 2a pro include nucleoporins, which are nuclear pore components that control nucleocytoplasmic exchange 60, 61 . as a result, bi directional passive diffusion through the nuclear envelope is facilitated. another manifestation of the perturbation of intracellular trafficking is the suppression of nuclear export of mrnas, ribosomal rnas and u spliceosomal small nuclear rnas 62 . passive nucleocytoplasmic diffusion of proteins is also facilitated by the cardiovirus l protein 63, 64 , and this effect seems to be caused by l-triggered phosphorylation of nucleoporins 50, 65, 66 . effects on innate immunity and viral pathogenicity one major consequence of the effects of security proteins on host metabolism is the downregulation of innate immunity. fmdv l pro suppresses interferon production 56,67 and action 68, 69 , largely through the inhibition of nuclear factor-κb-dependent transcription that is caused by the degradation of the p65 subunit of this transcription factor 55, 56 . the transcription of genes encoding various cytokines and chemokines (including tumour necrosis factor (tnf; also known as tnfa), t cellspecific protein rantes (also known as ccl5), myxovirus resistance protein 1 and interferon regulatory factor 7) is also suppressed by fmdv l pro (ref. 56 ). poliovirus reproduction, which is largely resistant to the effects of interferon, becomes interferon sensitive in 2a pro mutants. introduction of the poliovirus 2a pro gene into interferon-sensitive emcv facilitated its replication in cells pretreated with interferon 70 . the ability of rhinovirus 2a pro to cleave mitochondrial antiviral-signalling protein (mavs; also known as visa, cardif and ips1), an intermediate in the interferon generation pathway, might contribute to the insensitivity of these viruses to interferons 71 . 2a pro also cleaves the catalytic subunit of dna-dependent protein kinase, which, among other activities, is involved in the induction of pro-inflammatory cytokines 72 . cardiovirus l also suppresses interferon production by affecting the activation of interferon regulatory factor 3, but the exact mechanism of this interference has not yet been identified 32, 50, 73, 74 . tmev l* was proposed to suppress the antiviral cytotoxic t cell response in tmev-infected mice 75, 76 . in line with these observations, the functions of security proteins are less crucial for viral 'well-being' in hosts with innate immunity defects. mutations in cardiovirus l 48,77,78 or 2a 79 ) ), rather than one l protein. for tmev, an alternative l protein, l*, is encoded in an alternative reading frame that starts within the l coding sequence. ‡ according to the data in genbank. § values were calculated using protparam 132 . || for the viral genera with more than one species, the level of interspecies amino acid identity (and similarity, in parenthesis) was calculated with the aid of clustal_x2 alignments, using utilities implemented in bioedit and the blosum62 similarity matrix 133 . to focus on the core protein sequences, the internal insertions of >15 amino acid residues, as well as terminal insertions, were not taken into account. ¶ the peptidase_c28 motif was revealed by blast searches in the ncbi conserved domain database 134 . # the conserved non-canonical zinc (zn) finger domain (with a chcc motif) was found to be present. reproductive potential, but such mutations are less detrimental for viral growth in bhk-21 cells, which are deficient in interferon production, than in immunecompetent cells. similarly, cardiovirus mutants lacking l grow better in mice that have a deficient interferon system than in wild-type mice 50, 74 . for the l*-expressing tmev strains, functional l* is important for the ability of the virus to infect macrophages or microglia 80, 81 . one of the components of the innate immune response is apoptosis, which can potentially limit viral reproduction and spread, although certain viruses can subvert the apoptotic machinery to their benefit. conflicting results have been reported on the relationship between the security proteins and the apoptotic machinery. ectopic expression of enterovirus 2a pro triggers an apoptotic response 38,82,83 and enhances the sensitivity of cells to the apoptogenic activity of tumour necrosis factor 84 . however, in the context of the whole genome, 2a pro possesses anti-apoptotic activity 85, 86 . similarly, expression of tmev l in macrophage-like cells induces apoptosis, as occurs during tmev infection of these cells 87 , whereas emcv and mengovirus l are anti-apoptotic in the context of the whole virus (at least in hela cells, in which the virus itself elicits necrotic death) 5 . whether this apparent discrepancy is a result of the use of different assays or hosts or of the intrinsic peculiarities of cardiovirus l proteins remains unknown. tmev l* has also been reported to exhibit anti-apoptotic activity 88 . several illuminating examples strongly suggest that security proteins can markedly modulate viral pathogenicity. fmdv lacking l is highly attenuated 89 , 132 . || for the viral genera with more than one species, the level of interspecies amino acid identity (and similarity, in parentheses) was calculated with the aid of clustal_x2 alignments, using utilities implemented in bioedit and the blosum62 similarity matrix 134 . to focus on the core protein sequences, the internal insertions of >15 amino acid residues, as well as terminal insertions, were not taken into account. ¶ these are manually recognizable motifs. # one of the strains is reported to harbour an npr(p) motif instead of npg(p). and both l 73,90 and l* (refs 33, 81, 88) notwithstanding the low level of conservation of l proteins between emcv and tmev (table 1) , these proteins can be functionally exchanged with respect to their ability to inhibit interferon formation and to 'open' nuclear pores 90 . the replacement of l in the full-length cardiovirus genome with fmdv l pro generates a virus that can overcome host defences more efficiently than its leaderless counterpart, although it has lower fitness 92, 93 . such interchangeability of structurally and biochemically distinct proteins attests to the similarity of their biological functions. it is hardly by chance that viruses that encode long or multiple ls tend to have a short 2a or lack 2a altogether (assuming that 2a sp is not a security protein), and vice versa (fig. 1) . for example, simian and porcine sapeloviruses harbour a long 2a and a short l, whereas avian sapelovirus encodes the longest l identified to date and the predicted 2a orf is very short, if it encodes a protein at all. this tendency is consistent with the notion that l and 2a have similar roles in virus-host interactions. as already mentioned, some picornaviruses, for example, cosaviruses, encode no security proteins. even viruses that do encode security proteins can, under certain circumstances, survive and replicate after these proteins have been inactivated or eliminated. notably, cardiovirus l 31, 48, 77, 78 and l* (refs 34, 81, 94) and fmdv l pro (ref. 95) are not essential for viral viability, and extended deletions in 2a of cardioviruses 42, 79 and hepatitis a virus 96, 97 do not kill these viruses. furthermore, although some data suggest that poliovirus 2a pro has an essential replicative function 98 , recent experiments have demonstrated its dispensability for viral viability 86 . although this review focuses on the counter-defensive functions of security proteins, it should be kept in mind that other picornavirus proteins are also often engaged in similar functions. the targets of 3c pro (or its proteolytically active precursors) might include proteins that are involved in innate immunity [99] [100] [101] [102] . 2b and 3a are also important players in the virus-host struggle, being involved in the rearrangement of cytoplasmic membranes and in suppression of trafficking, secretion and antigen presentation on cellular plasma membranes 84, [103] [104] [105] . 2c can also participate in some of these activities 103 . even specialized proteins such as vpg 106 and capsid proteins 107 can sometimes assist in overcoming host defences. in all these cases, however, the 'security' functions are neither the main nor the conserved roles of these proteins. conversely, as well as representing a dedicated counterdefensive system, security proteins can be directly involved in viral reproduction. the products of the cleavage of eif4g, and possibly of some other host proteins, by fmdv l pro and enterovirus 2a pro can stimulate iresdependent cell-free translation 108, 109 ; the effect of 2a pro is partly the result of stabilization of the viral rna 110 . the physiological relevance of these phenomena is unclear, however. the poliovirus ires dysfunction that is caused by some mutations can be compensated for by mutations in 2a in a host-dependent manner 111 , suggesting the participation of host proteins. cardiovirus l was also implicated in control of ires activity, because emcv rna with deletions in the l-coding sequence exhibited a decrease in cell-free translatability 31 . poliovirus 2a pro was reported to stimulate strand initiation of negative-strand rna, and this was seemingly independent of its effects on rna stability and translation 110 . deletion of a carboxy-terminal sequence from this protein does not substantially affect its protease activity but does inhibit rna replication 112 . deletion of the amino-terminal region notably, but incompletely, suppresses replication of the relevant replicon 113 . similarly, deletions in 2a of aichi virus (a kobuvirus) inhibit viral rna replication 114 . however, the mechanisms responsible for these effects have not been elucidated, casting doubts on whether these 2a proteins affect viral replication directly or by modulating host cell activities. a role for hepatitis a virus 2a in virion assembly and maturation has been established 97, 115 . the primary cotranslational cleavage of the viral polyprotein takes place between 2a and 2b and is accomplished by the viral proteinase 3c pro (or its precursor), leaving the 2a sequence fused to the carboxyl terminus of capsid protein vp1 (refs 116, 117) . in immature virions, vp1 retains this 2a extension, which is eventually cleaved off by an unidentified enzyme 118 . the involvement of 2a in the maturation of some other picornaviruses cannot be excluded therefore. notably, the primary co-translational scission of the cardiovirus polyprotein generates a fusion between 2a and the capsid protein precursor 25 . origins and evolution of security proteins obviously, it is not possible to construct a genealogical tree of either l or 2a, because they are unrelated. these proteins are not considered at all in the hypothesis on the origin of picorna-like viruses 4 , and only the short, translation-interrupting 2a peptides (that is, the 2a sp peptides) are briefly mentioned in the proposal on picornavirus classification 119 . there is no correlation between the nature (or even the presence) of security proteins and either the type of ires that lies upstream of the l protein or the rdrp lineage (which is generally accepted as the most reliable indicator of viral relatedness) (fig. 2) . the diversity of the security proteins and their absence from many picornaviruses suggest that they are independent and late evolutionary acquisitions 5, 120 . it can be speculated that the most ancient of the 2a molecules are the 2a sp peptides, as hinted by their presence in most picornavirus genera, including those belonging to different lineages (fig. 2) . interruption of polyprotein synthesis after translation of the capsid proteins might be advantageous for viral reproduction. it might, for example, facilitate proper protein folding, ensure optimal kinetics of protein synthesis or control the ratios of structural to non-structural proteins, which could theoretically be achieved by incomplete translational re-initiation at the second proline residue of the npg(p) motif. it is worth noting that there is a difference in the translation factor requirements for translation of the emcv polyprotein upstream and downstream of the 2a-2b boundary 121 . strikingly, 2a sp peptides -or more accurately, the dxexnpg(p) motifs (where x is any amino acid) that are characteristic of these peptides -are found in some other picorna-like and unrelated viruses, where they seem to serve the same function [122] [123] [124] . assuming that the acquisition of npg(p) was an early event, this motif might have been lost by some parechoviruses (and other picornaviruses) at a certain step of evolution 21 . in contrast to picornaviruses, the acquisition of 2a sp by members of some other families of rna viruses has been proposed to have occurred at a late stage 122 . the idea that these peptides might have originated in picornaviruses is an attractive hypothesis. the npg(p) motif can also be found in several cellular proteins, but the current data do not allow researchers to determine whether the recoding ability of this motif was a viral or cellular invention. the non-npg(p)-containing regions of the 2a proteins are unrelated acquisitions. cardiovirus 2a possesses these additional moieties in its amino-terminal region, whereas other viruses of this subset (parechoviruses, avihepatoviruses and seal picornavirus type 1) contain these moieties in their carboxy-terminal regions (fig. 1) . this may suggest that these moieties were acquired after the npg(p) motif. in avihepatoviruses and some parechoviruses, these newly acquired sequences harbour the h-nc motif, which is also present in the npg(p)-lacking 2a proteins of other parechoviruses, kobuviruses and tremoviruses (that is, viruses of different rdrp lineages) (fig. 2) . a plausible hypothesis is that h-nc-containing proteins from cellular organisms were hijacked by certain picornaviruses on several independent occasions, but the possibility that 2a sp peptides were formed by deletions from larger 2a proteins 120 cannot be ruled out. taking into account the chymotrypsin-like fold that is found in enterovirus 2a pro , it is reasonable to assume that this protein was derived from picornavirus 3c pro or a cellular protease 125 . a similar assumption could perhaps be made for the putative sapelovirus 2a protease. there are no obvious clues to the possible origins of the 2a proteins of hepatoviruses and klasseviruses or of the non-npg(p) moieties of the 2a proteins of cardioviruses and seal picornavirus type 1. with regard to the picornavirus l proteins, one hypothesis is that the papain-like l pro proteases of aphtho viruses and erboviruses originated from cellular enzymes. no obvious relatives of other l proteins can currently be identified among cellular or viral proteins. only the origin of cardiovirus l* seems certain: the first l* probably came into being accidentally, by translation of an alternative reading frame, and was then shaped by mutations preserving the functional integrity of l, vp4 and vp2. it is worth noting that recently identified human tmev-like cardioviruses lack this alternative reading frame 126 . theoretically, several mechanisms could underlie the acquisition of security proteins. for example, one possibility is that viral genes were duplicated and subsequently substantially modified (such a scenario can be imagined for the origin of enterovirus 2a pro (ref. 125) ). other scenarios are: recombination with viral or cellular rnas encoding related proteins such as proteases; conversion of non-coding rna sequences into coding sequences (which could occur through different mechanisms, such as interspecies recombination 127 (v.i.a., a.p.g., e. v. khitrina and w. j. melchers, unpublished observations), or introduction or activation of an upstream inframe aug codon); frame shifting 120 (or double frame shifting, in the case of 2a proteins); and using an alternative reading frame. unfortunately, we can only pinpoint a definite mechanism for the case of l*, for which the last mechanism has obviously been operative. the acquisition of security proteins, by whatever mechanism, required that the new 'additions' did not interfere with the function of the adjacent viral proteins -vp4 (or vp0) in the case of l, and vp1 and 2b in the case of 2a. the new proteins should either be separated from these neighbours (by self-proteolysis, the action of other viral proteases or translation interruption) or, if they remain fused, they should not impair the functions of these neighbours (as is the case with the hepatitis a virus vp1-2a fusion). a separate issue is the evolution of the security proteins themselves. it was proposed that the l proteins of tmev and emcv diverged during evolution to adapt to the different replication fitnesses of these viruses 90 . the data are too scarce, however, for any generalizations at this point. l and 2a constitute a distinct and remarkable set of picornavirus proteins. they exhibit striking structural and biochemical diversity but (with the exception of the 2a sp peptides) accomplish similar biological functions by counteracting host defensive reactions. however, their abilities to solve similar problems might involve fundamentally different molecular mechanisms. this is illustrated in fig. 3 , which summarizes the properties of the three best studied security proteins. the biological functions of enterovirus 2a pro and cardiovirus l are strikingly similar: inhibition of host macromolecular synthesis, permeabilization of the nuclear envelope and inhibition of active nucleocytoplasmic transport, suppression of specific innate immunity mechanisms and control of the apoptotic machinery of the host cells. no less striking is the difference in the mechanisms by which the two proteins achieve these goals. conversely, aphthovirus l pro can solve only a subset of these problems. an intriguing figure 3 | major biological functions of the best studied but unrelated security proteins. there is a striking similarity between the functional activities of the enterovirus 2a protease (2a pro ) and the cardiovirus leader protein (l), but the underlying mechanisms by which these functions are carried out are fundamentally different. by contrast, the known functional activities of aphthovirus l, which is a protease (l pro ), seem to be more limited. irf3, interferon regulatory factor 3; nf-κb, nuclear factor-κb. question is how, and whether, the diverse security functions exhibited by a given protein (fig. 3) are related to each other. the fact that mutations inactivating one function usually also impair other functions suggests the existence of a common upstream target. most of the picornavirus l and 2a proteins still await researchers' attention. nevertheless, the data discussed here allow us to provisionally assign security functions even to those l and 2a proteins that have not yet been characterized. indeed, l and 2a do not definitely belong to the set of essential reproductive proteins that ensure translation and replication of the viral genome (although 2a of hepatitis a virus assists virion maturation). we propose that the concept of security proteins is of general relevance and can be applied to viruses other than picornaviruses. the hallmarks of these proteins are as follows: structural and biochemical unrelatedness or even absence in related viruses; the dispensability of the entire protein or its functional domains for viral viability; and, for mutated versions of the proteins, fewer detrimental effects on viral reproduction in immune-compromised hosts than in immune-competent hosts. possessing one of these features would make a viral protein a good candidate security protein, whereas a combination of these features would probably confirm this designation. viruses with large dna genomes possess impressive arsenals of security proteins. the complement of security proteins is much more limited in rna viruses but is sufficient for their evolutionary success. there are also tentative examples of security proteins in non-picornavirus rna viruses. coronaviruses have several so-called accessory proteins, which are neither conserved nor essential and exhibit the capacity to suppress host innate immunity by a range of mechanisms 128 . some, but not all, flaviviruses use ribosome frame shifting to express a non-essential non-structural protein, ns1′, mutational inactivation of which results in viral attenuation 129 . the nss protein of the rift valley fever phlebovirus suppresses the interferon system, and its inactivation does not kill the virus but attenuates its pathogenicity 130 . this list can readily be extended. the spectrum of picornavirus-induced diseases is extraordinarily broad. the reasons for this variability are poorly understood, although receptor compatibility and effects on viral protein and rna synthesis that are caused by differences in the availability of host factors are surely important contributors. however, the interaction between host defences and viral counterdefence is certainly one of the key factors underlying the pathogenicity of picornaviruses and other viruses. this interaction cannot be fully understood without elucidation of the roles of the security proteins. treatment and prevention of viral diseases may also markedly benefit from such elucidation. the study of security proteins is therefore an underdeveloped but highly promising research area. rna replication without rna-dependent rna polymerase: surprises from hepatitis delta virus persistent yeast single-stranded rna viruses exist in vivo as genomic rna·rna polymerase complexes in 1:1 stoichiometry giardiavirus double-stranded rna genome encodes a capsid polypeptide and a gag-pol-like fusion protein by a translation frameshift the big bang of picorna-like virus evolution antedates the radiation of eukaryotic supergroups the proposal to consider l and 2a proteins of picornaviruses as a distinct class of counter-defensive 'security proteins evading the host immune response: how foot-and-mouth disease virus has become an effective pathogen a review considering, among other topics, counter-defensive functions of aphthovirus l protein a second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications putative papain-related thiol proteases of positive-strand rna viruses. identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha-and coronaviruses structure of the fmdv translation initiation site and of the structural proteins the identification of the l protein in fmdv a second protease of foot-and-mouth disease virus the two species of the foot-and-mouth disease virus leader protein, expressed individually, exhibit the same activities conservation of l and 3c proteinase activities across distantly related aphthoviruses genomic evidence that simian virus 2 and six other simian picornaviruses represent a new genus in picornaviridae cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence site-specific release of nascent chains from ribosomes at a sense codon equine rhinovirus serotypes 1 and 2: relationship to each other and to aphthoviruses and cardioviruses sequence analysis of a porcine enterovirus serotype 1 isolate: relationships with other picornaviruses complete genome sequence analysis of seneca valley virus-001, a novel oncolytic picornavirus a highly prevalent and genetically diversified picornaviridae genus in south asian children molecular analysis of three ljungan virus isolates reveals a new, close-to-root lineage of the picornaviridae with a cluster of two unrelated 2a proteins molecular analysis of duck hepatitis virus type 1 a highly divergent picornavirus in a marine mammal the cleavage activity of aphtho-and cardiovirus 2a proteins the position of polypeptide g on the encephalomyocarditis virus polyprotein cleavage map rna-binding properties of nonstructural polypeptide g of encephalomyocarditis virus the 2a proteins of three diverse picornaviruses are related to each other and to the h-rev107 family of proteins involved in the control of cell proliferation the discovery of cardiovirus l protein, the first leader protein to be found for picornaviruses the leader peptide of theiler's murine encephalomyelitis virus is a zinc-binding protein nmr structure of the mengovirus leader protein zinc-finger domain leader protein of encephalomyocarditis virus binds zinc, is phosphorylated during viral infection, and affects the efficiency of genome translation the mengovirus leader protein suppresses a/β interferon production by inhibition of the iron/ferritin-mediated activation of nf-κb alternative translation initiation site in the da strain of theiler's murine encephalomyelitis virus a picornaviral protein synthesized out of frame with the polyprotein plays a key role in a virusinduced immune-mediated demyelinating disease the demonstration that enterovirus 2a pro cleaves a translation initiation factor poliovirus proteinase 2a induces cleavage of eucaryotic initiation factor 4f polypeptide p220 purification of two picornaviral 2a proteinases: interaction with eif4γ and influence on translation coxsackievirus b3 proteases 2a and 3c induce apoptotic cell death through mitochondrial injury and cleavage of eif4gi but not dap5/p97/ nat1 leader protein of foot-and-mouth disease virus is required for cleavage of the p220 component of the cap-binding protein complex cleavage of poly(a)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro cleavage of poly(a)-binding protein by coxsackievirus 2a protease in vitro and in vivo: another mechanism for host protein synthesis shutoff? genetic analysis of mengovirus protein 2a: its function in polyprotein processing and virus reproduction rapamycin and wortmannin enhance replication of a defective encephalomyocarditis virus virus-specific proteins associated with ribosomes of krebs ii cells infected with encephalomyocarditis virus cardiovirus 2a protein associates with 40s but not 80s ribosome subunits during infection encephalomyocarditis viral protein 2a localizes to nucleoli and inhibits cap-dependent mrna translation encephalomyocarditis virus (emcv) proteins 2a and 3bcd localize to nuclei and inhibit cellular mrna transcription but not rrna transcription mengovirus leader is involved in the inhibition of host cell protein synthesis a picornavirus protein interacts with ran-gtpase and disrupts nucleocytoplasmic transport inhibition of mrna export and dimerization of interferon regulatory factor 3 by theiler's virus leader protein inhibition of cap-dependent gene expression induced by protein 2a of hepatitis a virus poliovirus-encoded protease 2a pro cleaves the tatabinding protein but does not inhibit host cell rna polymerase ii transcription in vitro the effect of poliovirus proteinase 2a pro expression on cellular metabolism. inhibition of dna replication, rna polymerase ii transcription, and translation inhibition of u snrnp assembly by a virus-encoded proteinase degradation of nuclear factor kappa b during foot-and-mouth disease virus infection a conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function intracellular localization and effects of individually expressed human parechovirus 1 nonstructural proteins 2a proteinase of human rhinovirus cleaves cytokeratin 8 in infected hela cells enteroviral protease 2a cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy bidirectional increase in permeability of nuclear envelope upon poliovirus infection and accompanying alterations of nuclear pores differential targeting of nuclear pore complex proteins in poliovirus-infected cells rna nuclear export is blocked by poliovirus 2a protease and is concomitant with nucleoporin cleavage the leader protein of theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins nucleo-cytoplasmic traffic disorder induced by cardioviruses mengovirus-induced rearrangement of the nuclear pore complex: hijacking cellular phosphorylation machinery leader-induced phosphorylation of nucleoporins correlates with nuclear trafficking inhibition by cardioviruses ability of foot-and-mouth disease virus to form plaques in cell culture is associated with suppression of alpha/beta interferon inhibition of l-deleted foot-and-mouth disease virus replication by a/β interferon involves double-stranded rna-dependent protein kinase the leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mrna and blocks the host innate immune response proteinase 2a pro is essential for enterovirus replication in type i interferon-treated cells cleavage of ips-1 in cells infected with human rhinovirus proteolytic cleavage of the catalytic subunit of dna-dependent protein kinase during poliovirus infection the leader protein of theiler's virus inhibits immediate-early alpha/beta interferon production the mengovirus leader protein blocks interferon-a/β gene transcription and inhibits activation of interferon regulatory factor 3 a theiler's virus alternatively initiated protein inhibits the generation of h-2k-restricted virus-specific cytotoxicity cd4 t cells are important for clearance of the da strain of tmev from the central nervous system of sjl/j mice involvement of cardiovirus leader in host cell-restricted virus expression this work demonstrates that cardiovirus l is dispensable the leader polypeptide of theiler's virus is essential for neurovirulence but not for virus growth in bhk cells protein 2a is not required for theiler's virus replication l* protein of the da strain of theiler's murine encephalomyelitis virus is important for virus growth in a murine macrophage-like cell line influence of the theiler's virus l* protein on macrophage infection, viral persistence, and neurovirulence poliovirus 2a protease induces apoptotic cell death infection with enterovirus 71 or expression of its 2a protease induces apoptotic cell death poliovirus protein 3a inhibits tumor necrosis factor (tnf)-induced apoptosis by eliminating the tnf receptor from the cell surface bypass suppression of smallplaque phenotypes by mutation in poliovirus 2a that enhances apoptosis 2a protease is not a prerequisite for poliovirus replication theiler's murine encephalomyelitis virus leader protein is the only nonstructural protein tested that induces apoptosis when transfected into mammalian cells a protein critical for a theiler's virus-induced immune system-mediated demyelinating disease has a cell type-specific antiapoptotic effect and a key role in virus persistence pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle cardiovirus leader proteins are functionally interchangeable and have evolved to adapt to virus replication fitness an attenuating mutation in the 2a protease of swine vesicular disease virus, a picornavirus, regulates capand internal ribosome entry site-dependent protein synthesis construction of a chimeric theiler's murine encephalomyelitis virus containing the leader gene of foot-and-mouth disease virus differential ifn-a/β production suppressing capacities of the leader proteins of mengovirus and foot-and-mouth disease virus theiler's murine encephalomyelitis virus (tmev) l* amino acid position 93 is important for virus persistence and virus-induced demyelination the foot-and-mouth disease virus leader proteinase gene is not required for viral replication an article showing that aphthovirus l is dispensable hepatitis a viruses with deletions in the 2a gene are infectious in cultured cells and marmosets analysis of deletion mutants indicates that the 2a polypeptide of hepatitis a virus participates in virion morphogenesis studies on dicistronic polioviruses implicate viral proteinase 2a pro in rna replication molecular biology of picornaviruses proteolytic cleavage of the p65-rela subunit of nf-κb during poliovirus infection disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor rig-i is cleaved during picornavirus infection molecular biology of picornaviruses inhibition of cellular protein secretion by poliovirus proteins 2b and 3a coxsackievirus b3 inhibits antigen presentation in vivo, exerting a profound and selective effect on the mhc class i pathway role of nonstructural proteins 3a and 3b in host range and pathogenicity of foot-and-mouth disease virus early phosphatidylinositol 3-kinase/ akt pathway activation limits poliovirus-induced jnk-mediated cell death foot-and-mouth disease virus lb proteinase can stimulate rhinovirus and enterovirus ires-driven translation and cleave several proteins of cellular and viral origin the c-terminal domain of eukaryotic protein synthesis initiation factor (eif) 4g is sufficient to support capindependent translation in the absence of eif4e 2a pro is a multifunctional protein that regulates the stability, translation and replication of poliovirus rna coding changes in the poliovirus protease 2a compensate for 5′ncr domain v disruptions in a cell-specific manner the c-terminal residues of poliovirus proteinase 2a pro are critical for viral rna replication but not for cis-or trans-proteolytic cleavage replication of poliovirus rna and subgenomic rna transcripts in transfected cells aichi virus 2a protein is involved in viral rna replication intrinsic signals for the assembly of hepatitis a virus particles morphogenesis of hepatitis a virus: isolation and characterization of subviral particles identification and site-directed mutagenesis of the primary (2a/2b) cleavage site of the hepatitis a virus polyprotein: functional impact on the infectivity of hav rna transcripts the unique role of domain 2a of the hepatitis a virus precursor polypeptide p1-2a in viral morphogenesis picornavirales, a proposed order of positive-sense single-stranded rna viruses with a pseudo-t = 3 virion architecture origin and evolution of viruses translational barrier in central region of encephalomyocarditis virus genome. modulation by elongation factor 2 (eef-2) occurrence, function and evolutionary origins of '2a-like' sequences in virus genomes an article discussing viral translation-interrupting 2a peptides a case for ''stopgo'': reprogramming translation to augment codon meaning of ggn by promoting unconventional termination (stop) after addition of glycine and then allowing continued translation (go) euprosterna elaeasa virus genome sequence and evolution of the tetraviridae family: emergence of bipartite genomes and conservation of the vpg signal with the dsrna birnaviridae family molecular basis of viral evolution genomic features and evolutionary constraints in saffold-like cardioviruses mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation. microbiol ns1′ of flaviviruses in the japanese encephalitis virus serogroup is a product of ribosomal frameshifting and plays a role in viral neuroinvasiveness nss protein of rift valley fever virus induces the specific degradation of the doublestranded rna-dependent protein kinase divergent picornavirus ires elements the proteomics protocols handbook clustal w and clustal x version 2.0 cdd: specific functional annotation with the conserved domain database we thank f. van kuppeveld for critical reading of this manuscript. recent research in the authors' laboratory was supported by grants from the russian foundation for basic research (rfbr), the scientific school support program and the netherlands organization for scientific research-rfbr (nwo-rfbr). the authors declare no competing financial interests. see online article: s1 (figure)all links are active in the online pdf key: cord-314039-qkrmxvaj authors: houdebine, louis-marie title: production of pharmaceutical proteins by transgenic animals date: 2008-02-19 journal: comp immunol microbiol infect dis doi: 10.1016/j.cimid.2007.11.005 sha: doc_id: 314039 cord_uid: qkrmxvaj proteins started being used as pharmaceuticals in the 1920s with insulin extracted from pig pancreas. in the early 1980s, human insulin was prepared in recombinant bacteria and it is now used by all patients suffering from diabetes. several other proteins and particularly human growth hormone are also prepared from bacteria. this success was limited by the fact that bacteria cannot synthesize complex proteins such as monoclonal antibodies or coagulation blood factors which must be matured by post-translational modifications to be active or stable in vivo. these modifications include mainly folding, cleavage, subunit association, γ-carboxylation and glycosylation. they can be fully achieved only in mammalian cells which can be cultured in fermentors at an industrial scale or used in living animals. several transgenic animal species can produce recombinant proteins but presently two systems started being implemented. the first is milk from farm transgenic mammals which has been studied for 20 years and which allowed a protein, human antithrombin iii, to receive the agreement from emea (european agency for the evaluation of medicinal products) to be put on the market in 2006. the second system is chicken egg white which recently became more attractive after essential improvement of the methods used to generate transgenic birds. two monoclonal antibodies and human interferon-β1a could be recovered from chicken egg white. a broad variety of recombinant proteins were produced experimentally by these systems and a few others. this includes monoclonal antibodies, vaccines, blood factors, hormones, growth factors, cytokines, enzymes, milk proteins, collagen, fibrinogen and others. although these tools have not yet been optimized and are still being improved, a new era in the production of recombinant pharmaceutical proteins was initiated in 1987 and became a reality in 2006. in the present review, the efficiency of the different animal systems to produce pharmaceutical proteins are described and compared to others including plants and micro-organisms. human communities have extensively used for centuries plant extracts as pharmaceuticals. these pharmaceuticals did not include proteins which were unknown, denatured during extraction and not active by the oral route. the first protein used as a therapeutical molecule was insulin extracted from pig pancreas. although these preparations did not raised major problems for decades, human insulin was obtained from recombinant bacteria in the early 1980s. this protein which proved of better quality than the conventional insulin, has been adopted by all patients suffering from diabetes. human growth hormone was prepared by the same way soon after. both insulin and human growth hormone obtained from tissue extracts were not limiting but their quality was greatly improved by using recombinant bacteria. the major improvement for human growth hormone was that it was no more contaminated by human prions. the case of bovine growth hormone is somewhat different. this protein extracted from recombinant bacteria could not be contaminated by prions but this system was the only capable of providing farmers with enough quantity of the hormone to enhance milk production in ruminant herds. bacteria soon appeared severely limited by their poor capacity to synthesize some proteins and particularly those which have a complex structure. some proteins are so abundantly produced in bacteria that they form aggregates from which it is difficult to isolate the proteins of interest without denaturating them. bacteria cannot always fold proteins in an appropriate manner and assemble subunits to form biologically active molecules. moreover, bacteria can eliminate signal peptides but cannot cleave preproteins like native coagulation growth factors. bacteria are also unable to perform the posttranslation modifications of proteins, namely of glycosylation, g-carboxylation, phosphorylation, sulphatation and others. other systems have thus been implemented and proved more or less capable of producing at a low cost large amount of active proteins having an appropriate biochemical structure. the different systems to produce recombinant proteins which are being studied or used are summarized in table 1 . the advantages and limits of bacteria have been discussed above. yeast was expected to have some advantages over bacteria. indeed, these cells are eukaryotes and they may potentially synthesize and secrete large amount of matured recombinant proteins. yeast proved unable to fold and secrete monoclonal antibodies in an appropriate manner. moreover, some yeast did not glycosylate proteins or they added sugars which are not found in human proteins. interestingly, genetically modified yeast expressing foreign genes coding for enzymes responsible for glycosylation proved able to secrete substantial amounts of recombinant proteins having carbohydrates almost similar to those found in human proteins [1] . this suggests that yeast could become an essential system to produce pharmaceutical proteins in the coming years. however, recombinant hepatitis b vaccine prepared from yeast does not contain the disulphide bridges which have to be created in vitro. this modification is likely not possible with all the proteins. the baculovirus-sf9 insect cells system is very popular and used extensively in laboratories to prepare limited amounts of proteins. this tool still suffers from several limitations. the cells which harbour the baculovirus do not survive to the infection which must be repeated with new cells and virus preparation if large amount of proteins are needed. on the other hand, insect cells do not add to proteins all the sugars which are present in human proteins. for these reasons, the baculovirus system offers little advantage if any over cho cells and it is only marginally used to prepare pharmaceutical proteins. animal or human cells and particularly cho cells (chinese hamster ovary) have been used for two decades successfully. the major advantage of these cells is that the proteins secreted in the culture medium are post-translationally modified essentially as their native counterparts. it remains that the sugars appear not completely added to the recombinant proteins. the major drawback of the mammalian cell culture systems is their relatively low production levels. consequently, the use of these systems is limited by their high cost and their incapacity to produce more than a few kilograms of recombinant proteins per year. in addition, although the conditions of animal cell culture were improved, the production of recombinant proteins and their glycosylation may be not stable and reliable. transgenic plants offer interesting possibility to produce recombinant proteins as transgenesis is relatively easy in plants. a number of enzymes used for research or for diagnosis are currently being produced by transgenic plants at an industrial scale. producing pharmaceuticals in plants is a more ambitious project. this system offers several advantages but also serious limits [2] . various transgenic plant species can be obtained. foreign proteins may be stored in leaves, in seeds or both, according to the promoter used. leaves are very abundant but it may be difficult to purify the protein of interest from them due to the presence of proteases or substances like polyphenols which are not welltolerated by patients. the amount of recombinant proteins which can be prepared in plants is virtually unlimited and the production cost is low. moreover, agriculture techniques offer a great flexibility for scaling up. leaves and mainly seeds containing the proteins of interest can be stored easily. it is also simple to rescue the plant lines and establish master banks allowing a reproducible production of proteins. plant cells are able to fold proteins and associate subunits as efficiently as animal cells. on the contrary, plants cells add carbohydrates to protein chains but not as animal cells do. proteins synthesized in plant cells have no terminal sialic acid and they contain xylose which may induce deleterious immune response in patients (fig. 1 ). experiments are in progress to modify protein glycosylation by transferring various genes responsible for the addition of sugars to proteins in a way similar to what happens in mammalian cells [3] . proteins prepared from plants have very little chance to contain pathogens for humans or animals. using transgenic plants to prepare recombinant proteins raise little ethical problems. one major concern is the uncontrolled dissemination of the proteins thus of the antigens when plants are cultured in open fields [4] . low amount of antigens might induce a tolerance in humans or a basal unknown vaccination. this problem cannot be solved easily. plants may be sterile to prevent any dissemination of the transgene. another proposition which has been retained by several companies restricts the production of recombinant proteins by plants not used for human feeding, such as tobacco or alfalfa [5] . one possibility to suppress the problem consists of keeping the plants in greenhouses. this is technically possible but would enhance markedly the production cost, reducing the attractiveness of transgenic plants for this purpose. a satisfactory approach could be to use plants which can be cultured easily, in large quantity and at a low cost in confined areas. encouraging experiments have shown that duckweed and microalgae could provide humans with large amount of proteins produced in perfectly well-controlled conditions [6] . another possibility would be to use cultured plant cells. recent studies suggest that this perspective offers attractive alternative in some cases [7] . recently, a system called magnifection was shown to allow the rapid production (within 2 weeks) of grams of functional antibodies in plants [8] . this system involves the transient high-level co-expression of the transgenes (for example immunoglobulin heavy-and light-chains) through the use of plant viral vectors delivered by agrobacterium to the plant body. although encouraging, these results cannot predict when or if recombinant proteins prepared from transgenic plants will be able to reach the market. transgenic animals offer particularly attractive possibilities to prepare recombinant pharmaceutical proteins. the advantages are the high and low cost production as well as the high quality of the proteins. a drawback may be the difficulty to separate the human proteins from their animal counterpart. special care must also be taken to check that animal pathogens active in humans are not present in the protein preparations. moreover, some of the recombinant proteins may be active and deleterious for transgenic animals. the comparison of the different possible animal systems is depicted in the following section. milk is presently the most mature system to produce recombinant proteins from transgenic organisms [9] [10] [11] . blood, milk [9] [10] [11] , egg white [12] [13] [14] , seminal plasma [15] , urine and silk gland [16] , insect larvae haemolymph [17] are other theoretically possible systems (table 2) . silk gland is a promising system in particular cases. preliminary results indicate that active human factor vii can be found in different tissues of a transgenic fish (tilapia). it is not known if this system may be improved and scaled up (mclean unpublished data). blood cannot most of the time store high levels of recombinant proteins which are naturally too unstable. moreover biologically active proteins in blood may alter the health of the animals. milk avoids essentially these problems. several mammalian species (rabbits, pigs, sheep, goats and cows) are currently being studied or used to produce recombinant proteins in their milk. rabbits offer a number of advantages: easy generation of transgenic founders and offspring, high fertility, relatively high milk production, insensitivity to prion diseases, no transmission of severe diseases to humans. pigs are more costly but produce higher amounts of milk than rabbits. ruminants are potentially the most appropriate species to produce large amount of proteins but they need cloning or lentiviral vectors to integrate foreign genes, their reproduction is relatively slow, they do not glycosylate proteins as well as rabbits and pigs and they are sensitive to prion diseases (tables 3 and 4) . until recently, egg white was considered as a promising system strongly limited by the great difficulty of generating transgenic birds. this difficulty appears now surmounted. lentiviral vectors proved efficient in chicken as in mammals ( [14, 18] and this issue). more impressively, pluripotent cell lines from primordial germ cells (pg cells) have been established in chicken. these cells harbouring foreign genes can be reintroduced in early embryos and participate to the development of chimeric transgenic chickens [13] . in a previous experiment, the same group showed that chimeric transgenic chicken generated by using non-pluripotent cells was able to secrete a monoclonal antibody in egg white [12] . these experiments validate egg white as a source of foreign proteins including recombinant vaccines. the generation of transgenic farm animals may be achieved according to species by dna microinjection into embryo pronuclei, by using lentiviral vectors or transposons, by incubating sperm with dna followed in vitro fertilization using icsi (intracytoplasmic sperm injection), by transferring the foreign gene into pluripotent cells (embryonic stem cells or primordial germ cells) followed by the generation of chimeric animals harbouring normal and transformed cells, by transferring the foreign gene into somatic cells used for the generation of cloned animals using nuclear transfer. these methods have been described in recent reviews [19] [20] [21] . they are summarized in fig. 2 . microinjection into pronuclei is very poorly efficient in ruminants and some other species. it is still being used successfully in mice, rats, rabbits, pigs and fish. in order to increase the integration frequency, foreign genes can be introduced in vectors containing natural elements favouring dna integration such as transposons and lentiviral vectors ( [14, 18] and this issue). the latter proved highly efficient in ruminants and pigs. this technique is being adopted by experimenters even if these vectors have limited capacity to harbour foreign dna and if the number of integration sites in the same animal is presently difficult to control. dna transfer via sperm has been developed mainly in pigs and mice. it may simplify transgenesis in some cases. an improve protocol consist of initially degrading sperm membrane, to incubate sperm in the presence of dna and to fertilize oocytes using icsi ( [22] and this issue). icsi gave a good yield of transgenic pigs [23] . the utilisation of cells as carrier for the foreign genes has been used in mice for almost 20 years. in this case, pluripotent cells capable of participating to the development of chimeric transgenic animals are being used. this method is laborious and used only for gene targeting and in practise essentially to inactivate genes (gene knock out). for unknown reasons, it has not been possible to obtain and use pluripotent cells from embryos (es cells: embryonic stem cells) in species other than mice. a recent study has shown that, in chicken, it was possible to establish pluripotent cell lines (eg cells) from the pluripotent cells which are present in foetal gonads (pgc: primordial germ cells). this made it possible the generation of transgenic birds which are candidates to produce recombinant proteins in egg white ( [13] and this issue). the cloning technique used to generate dolly the sheep is being used to generate transgenic ruminants and pigs. this technique allows gene addition but also gene targeting by homologous recombination. this makes it possible gene knockout. gene targeting is also a way to integrate foreign genes in genomic sites known to favour their expression. the generation of transgenic animals remains relatively laborious and costly but it is no more a hurdle to the production of recombinant proteins. to be expressed in a reliable manner, a transgene must ideally contain a promoter, enhancers, insulators, introns and a transcription terminator [20] . expression in milk is achieved successfully with promoters from milk protein genes. expression in egg white is possible using the potent promoter of ovalbumin gene. using long genomic dna fragments containing the promoter of interest generally enhances greatly the expression of foreign cdna. this proved to be the case for the promoter of one milk protein gene, wap gene (whey acidic protein) [24] . this suggests that elements from long dna fragments will be identified and used in future to construct compact vectors expressing transgenes in a reliable manner ( [25] and this issue). constructing an efficient expression vector to produce a therapeutic protein is not a standard operation. two examples may illustrate this point. recombinant vaccines against malaria are presently under study [26] . one of the proteins was initially obtained in mouse milk [27] it is now being produced in goat milk. unexpectedly, the antigen produced in mouse milk lost its vaccinating properties when glycosylated. the second example is the production of vp2 and vp6 proteins from rotavirus in transgenic rabbit [28] . rotavirus has a genome formed of several independent rna fragments. this virus is replicated in cytoplasm and its proteins are not individually secreted. the following modifications of the vp2 and vp6 nucleotide sequence were performed: elimination of the splicing sites and of several n-glycosylation sites, addition of a peptide signal and adaptation of codons to optimize the expression of the two cdnas in the mammary gland of the animals. the modified cdnas were introduced into a vector designed according to the criteria defined above [20] . these gene constructs made it possible the co-secretion in milk of the two viral proteins at concentration up to 500 mg/ml. these proteins were able to protect mice against the virus completely or partially according to the mode of administration [42] . table 5 report some of the major projects in development based on production of recombinant proteins in milk. the projects implying production in egg white and depicted above could be added on this list. despite important progress in the design of vectors for the expression of transgenes in milk or egg white must still be improved. indeed, the level of foreign proteins found in milk or egg white is sometimes low for unknown reasons and it is not always strictly mammary specific. the position site effect must have a strong impact on transgene expression. the use of long genomic dna fragments depicted above is one way to obtain a more reliable expression of the transgenes. the use of episomal vectors independent of integration sites and foreign gene targeting to known active genome sites are other possibilities under study [20] . a level of 1 mg per ml of milk or even lower appears acceptable economically. at higher concentration, the recombinant proteins may not be fully matured and particularly glycosylated. the mammary cellular machinery is likely saturated and cannot fully glycosylate the extra proteins. the recombinant protein atryn (human antithrombiniii) produced in goat milk contains less sialic acid than its native counterpart is a case in point [29] . similarly, the human inhibitor c1 produced in rabbit milk is not fully sialylated [30] . monoclonal antibodies secreted in chicken egg white do not contain sialic acid [12] . this diminishes markedly the half-life of the proteins in patients and may complicate their use by clinicians. the transfer of genes coding for glycosylation enzymes improved carbohydrate transfer to proteins synthesized in yeast [1] but also in cho cells [31] . the optimization of glycosylation in plants is in course using the transfer of genes coding for the glycosylation enzymes [3] . the same could be achieved in mammals and chicken to improve glycosylation in milk and egg white, respectively. a more subtle glycosylation effect on antibody action has been recently observed. it has been shown that the addition of n-acetylglucosamine through gene transfer in cho cells allowing the formation of a third carbohydrate antenna in antibodies, enhances their capacity to induce the adcc (antibody-dependent cellular cytotoxicity) immune cellular reaction [32] . in fact, the absence of fucose in antibodies has a stronger effect on their capacity to stimulate adcc reaction [33] . it was also demonstrated that sialylated antibodies have a lower affinity for fc-fcgr receptors and a higher capacity to induce antiinflammatory reaction. the presence of some pathogens in animals induces a desialylation of antibodies to enhance their cytotoxic action. hence, recombinant monoclonal antibodies may be engineered to induce an anti-inflammatory reaction by adding sialic acid with or without fucose. similarly, monoclonal antibobodies may contain no fucose and no sialic acid to favour their cytotoxic effect and reduce proinflammatory reactions [33] . the last form of antibodies appears the most appropriate to destroy tumour cells. interestingly, the recombinant monoclonal antibodies found in egg white are devoid of sialic acid and fucose suggesting that they are potent tools to destroy tumour cells [12] . other post-translational modifications of proteins are crucial. this is namely the case for some specific cleavage and for g-carboxylation [34] . the complete maturation of human protein c found in the milk of transgenic mice was achieved by crossing these animals with others expressing the furin transgene in their mammary gland [34, 35] . this pioneer work indicates that living fermentors such as mammary gland can be engineering to perform the post-translational modifications of recombinant proteins. the modification of milk composition may have applications other than preparing pharmaceutical proteins. some of the studies in course aim at increasing the overall nutritional values of pig milk to provide piglets with enough feed. other modifications of ruminant milk composition may improve quality of the curd. several experiments led to the secretion in ruminant milk of antibacterial proteins such as human lactoferrin and lysozyme or lysostaphin having antibacterial activities. these molecules protect to some [19] and this issue). this shows that the techniques originally designed to produce pharmaceutical proteins in milk can be extended to improve milk as a food or to use it as a vehicle to provide consumers with molecules beneficial in some way for their health. the two major animal systems to produce pharmaceutical proteins in milk and egg white have recently been technically improved and their use as an essential source of new medicaments has become very likely. the different mammals which participate to this industrial activity are rabbits, pigs, sheep, goats and cow (tables 3 and 4 ). each of these species offers advantages and drawbacks. rabbits are sufficient to produce several kilos of proteins per year. this species is particularly flexible allowing a rapid generation of founders and scaling up. for very high protein production, larger animals are needed. it is now proven that the amount of human antithrombin iii obtained per year from transgenic goats is equivalent to this resulting from 90,000 human blood samplings. a possible alternative could be to use recombinant adenoviral vectors capable of infecting ruminant mammary gland [36] . this approach is laborious but could be helpful when a protein has a deleterious effect on animals and can be only transiently produced. the proteins which have been prepared in milk are mainly naturally secreted. in the case of lysostaphin, rotavirus vaccine and a few others, the proteins became secreted efficiently after adding a signal peptide and they were found in the aqueous phase of the milk. in two cases, cftr [37] and alkaline phosphatase [38] , the proteins were hydrophobic and concentrated in the membrane of milk fat globules. this may facilitate or complicate the purification of the recombinant proteins. as a general rule, it must be considered that the purification protocol of recombinant proteins from milk is not standard and must be designed on a case by case basis. recombinant proteins are stable in milk but they may be more or less trapped by caseins which are extremely abundant. rabbits and pigs are not sensitive to prions and the prp gene required for the development of prion diseases has been knocked out in cows [39] . guidelines to breed animals producing pharmaceutical proteins and to control the absence of pathogens in the purified protein fractions have been defined. it may thus be considered that the preparation of pharmaceutical proteins from milk is a safe process. the same is potentially true for the proteins which are being prepared from egg white [40] . the study of the biochemical and biological properties of the recombinant proteins is of paramount importance before they can be put in the market. this problem is particularly complex as natural and recombinant proteins often exist under different forms. this is particularly the case as far as glycosylation is concerned. the case of atryn is a good example [29] . indeed, atryn contains two forms of sialic acid: n-acetylneuraminic acid (nana) and n-glycosylneuraminic acid (ngna). only the nana form is present in humans. the second form ngna may induce an immune response in patients with not predictable side-effects. interestingly, recombinant proteins prepared in rabbit and pig milk contain only the nana form in human proteins. despite these imperfections, atryn was accepted by emea which argued that the protein has little chance to induce deleterious effects and could be helpful for patients. this decision suggests that emea does not consider that a recombinant pharmaceutical protein must absolutely be structurally similar to its native counterpart but it must meet satisfactory clinical and biosafety criteria on a case by case basis. the fact that atryn (human antithrombin iii) was accepted by emea is an important message to the involved biotechnology companies. using transgenic animals as a source of pharmaceutical proteins raise minor general biosafety and ethical problems. indeed, the escape of the animals in environment is extremely unlikely and in the majority of the cases, animals do not suffer from expressing a foreign protein in their milk or eggs. several proteins raised specific problems. human erythropoietin produced in rabbit milk altered their health [41] . less intense side effects were observed when human growth hormone was produced in rabbit milk and the high producers of human ec superoxide dismutase failed to lactate in a normal way [unpublished data]. the number of companies involved in the production of recombinant pharmaceutical proteins is expected to increase. this will result from the improvement of the different systems and from the fact that the oldest patents are becoming obsolete in the coming years. competition may thus become very intense. some experts consider that the demand of pharmaceutical protein production might increase faster than the capacity of the companies to produce proteins [10] . the different systems would be then all helpful for one or two decades. production of complex human glycoproteins in yeast transgenic rice for allergy immunotherapy biopharmaceutical production in plants: problems, solutions and opportunities risk analysis for plant-made vaccines plants as bioreactors: a comparative study suggests that medicago truncatula is a promising production system transgenic microalgae as green cellfactories plant cell cultures for the production of recombinant proteins rapid high-yield expression of full-size igg antibodies in plants coinfected with non competing viral vectors transgenic animal bioreactors antibody manufacture in transgenic animals and comparisons with other systems the methods to generate transgenic animals and to control transgene expression production of human monoclonal antibody in eggs of chimeric chickens germline transmission of genetically modified primordial germ cells oviduct-specific expression of two therapeutic proteins in transgenic hens making recombinant proteins in animals-different systems, different applications biosynthesis and cocoonexport of a recombinant globular protein in transgenic silkworms stable expression of human growth hormone over 50 generations in transgenic insect larvae lentiviral transgenesis-a versatile tool for basic research and gene therapy use of transgenic animals to improve human health and animal production transgenic animal models and target validation preparation of recombinant proteins in milk to improve human and animal health simultaneous removal of sperm plasma membrane and acrosome before intracytoplasmic sperm injection improves oocyte activation/embryonic development production of a transgenic piglet by a sperm injection technique in which no chemical or physical treatments were used for oocytes or sperm position-independent and tissue-specific expression of porcine whey acidic protein gene from a bacterial artificial chromosome in transgenic mice the potential benefits of insulators heterologous contructs in transgenic animals malaria vaccine initiative a recombinant vaccine expressed in the milk of transgenic mice protects aotus monkeys from a lethal challenge with plasmodium falciparum production of two vaccinating recombinant rotavirus proteins in the milk of transgenic rabbits transgenically produced human antithrombin: structural and functional comparison to human plasma-derived antithrombin n-and o-glycans of recombinant human c1 inhibitor expressed in the milk of transgenic rabbits engineering chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins engineered glycoforms of an antineuroblastoma igg1 with optimized antibody-dependent cellular cytotoxic activity a sugar switch for anti-inflammatory antibodies transgenic animal bioreactors in biotechnology and production of blood proteins proteolytic maturation of protein c upon engineering the mouse mammary gland to express furin high expression level of recombinant human erythropoietin in the milk of non-transgenic goats production of cystic fibrosis transmembrane conductance regulator in the milk of transgenic mice high level expression of tissuenonspecific alkaline phosphatase in the milk of transgenic rabbits production of cattle lacking prion protein the incredible, edible, and therapeutic egg the deleterious effects of human erythropoietin gene driven by the rabbit whey acidic protein gene promoter in transgenic rabbits recombinant rotavirus inner core proteins produced in the milk of transgenic rabbits confer a high level of protection after intrarectal delivery key: cord-034406-i1hbx3pz authors: matthews, abigail a.; ee, pui lai rachel; ge, ruowen title: developing inhaled protein therapeutics for lung diseases date: 2020-10-30 journal: mol biomed doi: 10.1186/s43556-020-00014-z sha: doc_id: 34406 cord_uid: i1hbx3pz biologic therapeutics such as protein/polypeptide drugs are conventionally administered systemically via intravenous injection for the treatment of diseases including lung diseases, although this approach leads to low target site accumulation and the potential risk for systemic side effects. in comparison, topical delivery of protein drugs to the lung via inhalation is deemed to be a more effective approach for lung diseases, as proteins would directly reach the target in the lung while exhibiting poor diffusion into the systemic circulation, leading to higher lung drug retention and efficacy while minimising toxicity to other organs. this review examines the important considerations and challenges in designing an inhaled protein therapeutics for local lung delivery: the choice of inhalation device, structural changes affecting drug deposition in diseased lungs, clearance mechanisms affecting an inhaled protein drug’s lung accumulation, protein stability, and immunogenicity. possible approaches to overcoming these issues will also be discussed. biological drugs are revolutionising the treatment and management of many serious illnesses including cancer, autoimmune disorders, and rare genetic diseases, with about a third of all new drug approvals by the food and drug administration (fda) consisting of biological drugs [1] . however, over the past decades, the development of inhaled therapeutics for the treatment of respiratory diseases has largely been focused on small molecules (corticosteroids, β2 agonists, and muscarinic antagonists), with only one inhaled protein biologic drug pulmozyme® being approved by the fda to date [2] . in the treatment of lung diseases via inhalation therapy, biological drugs such as proteins/polypeptides offer many advantages over small molecule drugs. proteins delivered via the pulmonary route could accumulate in the lungs while having a poor ability to traverse the airblood barrier due to their large molecular weight. this would result in higher target site accumulation (airway epithelial cells, alveolar macrophages, neutrophils etc) and minimise systemic toxicity, as compared to small molecule drugs that would pass easily into the systemic circulation after reaching the lungs [2] [3] [4] . in addition, protein therapeutics display higher potencies (picomolar to femtomolar range) than small molecules (nanomolar range), as well as highly specific receptor binding to reduce off-target effects [5] . notably, although peptide drugs (< 5 kda or < 40-50 amino acids in length) share some of the characteristics with protein/polypeptide drugs such as both are composed of amino acids linked via peptide bond and both having high target specificity, most of peptide drugs are much smaller in sizes, conferring them with some distinct differences including less enzyme stability, higher tissue penetration ability etc. in addition, many peptide drugs harbour chemical modifications in the form of peptidomimetics and/or cyclization, and some have direct cell membrane penetrating ability. these peptide drugs are not covered in this review. protein therapeutics are conventionally administered via the systemic route, although this has proven to be an inefficient approach for drug delivery to the lung, not mentioning the additional danger of exposing the rest of the body vulnerable to toxicity [4, 6] . for instance, monoclonal antibodies (mabs) are found at higher levels (500-10,000 times more) in serum than in bronchoalveolar lavage (bal) fluid following intravenous administration, a trend that has been demonstrated in all species [4] . by the same token, protein therapeutics delivered via the airways also pass poorly from the lungs into the systemic circulation. for example, only low amounts of anti-vegf-a g6-31 mab (5.1%) and cetuximab (11%), an anti-egfr mab, were present in the serum after aerosol delivery in mouse models of lung cancer [7, 8] . this means that high concentrations of the protein drug can be attained in the lung via pulmonary delivery, suggesting that lower doses of inhaled protein can have an equivalent or even superior therapeutic effect for lung diseases when compared to the higher doses that would be needed from systemic administration [9] . indeed, it was reported that the nebulised effective dose of avidinox-anchored biotinylated cetuximab was 1/25, 000 of the intravenous effective dose in a mouse model of advanced metastatic lung cancer [10] . although higher pulmonary levels of protein therapeutics can be achieved through inhalation compared to systemic administration, this could be offset by the short residence time of proteins in the lung compared to plasma. proteins and antibodies are mostly cleared from the lungs within 24 h, while plasma half-lives of full-length antibodies following intravenous injection can reach 3 weeks and more [11] . nevertheless, there are strategies that can be employed to increase the local residence time of protein therapeutics in the lungs, and these will be discussed in detail later on in this review. besides improving pharmacokinetic and toxicity profiles of protein therapeutics, the inhalation route is non-invasive and allows for self-administration, which could improve patient compliance [4, 5, 12] . in 1993, pulmozyme® (dornase alfa/deoxyribonuclease i), was introduced for the treatment of cystic fibrosis [9] . it has been almost three decades since then, and no other inhaled protein therapeutics for topical treatment of a lung disease has reached the market, despite the aforementioned advantages that inhaled proteins possess. presently, and to the best of our knowledge, ten inhaled protein therapeutics are being assessed in clinical trials for the treatment of a range of lung diseases including asthma, cystic fibrosis, lung cancer, copd and covid-19 (table 1) [13] [14] [15] [16] [17] [18] . there are also protein therapeutics such as mabs that are in the preclinical stages [19, 20] . in order to drive more of these therapies into clinical development and eventually to the market, it is crucial to take into account the challenges unique to the development of these agents into potential treatments so that they may be utilised successfully for pulmonary delivery. in this review, we will discuss the challenges in developing an inhaled protein therapeutic for lung diseases, as well as approaches that could help to circumvent these issues. the focus of this review is on the pulmonary delivery of protein drugs for local action in the lung, however, examples of inhaled proteins for systemic action will be mentioned wherever relevant. challenges and considerations in the development of protein therapeutics for local lung delivery choice and limitations of drug delivery device there are mainly three classes of inhalation devices namely dry powder inhalers (dpis), nebulisers, and metered dose inhalers (mdis). dpis deliver drug as a solid aerosol, and powder formulations possess inherent stability and shelf life benefits [4, 21] . however, the temperature and shear stress during the manufacturing processes needed to produce powders (e.g. freeze drying, spray drying) could lead to protein degradation [21] . dpis have been used for the marketed inhalable insulin formulations exubera® (approved in 2006, but was discontinued after 1 year due to large device size, high pricing, and safety concerns) and afrezza® (still commercially available) [22] . moreover, dpis have shown promising results in studies assessing their use for inhaled protein formulations. for example, weers et al. (2019) showed that dry powder formulations of csj117 (antithymic stromal lymphopoietin (tslp) mab fragment) could achieve a total lung dose (tld) of about 95% of the delivered dose (dd) with the use of particle engineering techniques such as via the introduction of surface corrugation through the addition of trileucine [23] . nebulisers (jet, ultrasonic, and mesh) generate aerosol droplets from a liquid solution of the drug [4] . the first and only inhaled protein formulation approved for pulmonary delivery to date, pulmozyme®, is administered via jet nebuliser. nebulised formulations are less expensive to produce and test, because the manufacturing process for these formulations does not include extra drying steps. nevertheless, prolonged storage of proteins in liquid solutions can lead to protein instability through degradation pathways (i.e. deamidation and hydrolysis), temperature and ph changes, and aggregation (through agitation of the aqueous carrier) [21] . furthermore, across all nebuliser types, the process of nebulisation exposes the protein to physical stresses such as shearing forces and heat, as well as the large air-liquid interface (ali) that could alter protein conformation and/or structure through denaturation, chemical modifications (oxidation, deamidation), and aggregation [4, 15] . device-specific limitations such as the shear forces generated by jet nebulisers, and the temperature increases that occur in ultrasonic nebulisers, can also lead to protein degradation. jet and ultrasonic nebulisers actually recycle 99% of the primary aerosol, and a molecule would typically be subjected to 10-15 cycles of nebulisation before leaving the nebuliser as a secondary aerosol [24] . this subjects the molecules to high shear stress in these devices, resulting in the denaturation of proteins, with the extent of protein denaturation and degradation varies depending on the characteristics of the individual protein [25] . for example, for jet nebulizer delivery of ldh and urease, there is a log-linear degradation with a fraction of protein degraded with every recirculation [26, 27] . in contrast, igg and g-csf has a rapid initial decline in native proteins in the first 5-10 min [26, 28] . for ultrasonic nebulizers, heating resulting from ultrasonic radiation in addition to aerosol recirculation generated various protein denaturation and degradation in different proteins [25] . for example, the degradation of ldh with ultrasonic nebulizer presented a sigmoidal progression instead, indicating different denaturation process and factors involved from jet nebulizers [29] . by comparison, vibrating mesh nebulisers employ single-pass technology, ensuring that there is no recirculation of droplets into the reservoir; they do not alter solution temperature, and produce less shear forces inside the drug reservoir during nebulisation, making them more suitable for the delivery of protein therapeutics [24] [25] [26] [27] [28] [29] [30] [31] . in fact, several studies have reported that jet and ultrasonic nebulisers produce lower levels of activity, lower amounts of protein monomers (because of partial degradation), and more aggregates (with or without excipients). on the other hand, mesh nebulisers appear to maintain protein integrity to a greater extent than other nebulisers [32] . safe aerosolisation with mesh nebulisers has already been demonstrated in several studies of labile drugs including proteins and mabs [33] [34] [35] [36] [37] [38] . the detailed designs and comparisons of various types of nebulizers have been reviewed previously and we will not elaborate further here [25, [30] [31] [32] 39] . recently, nanoengineered particles using metal-phenolic networks (mpns) with highly defined physical properties have been used to encapsulate both small molecule and macromolecules including proteins for pulmonary delivery via nebulisation. intratracheal nebulization delivery of fitc-labelled bovine serum albumin (bsa, 65 kda) in mice demonstrated that these capsules are biocompatible and biodegradable, showing > 85% of the capsules in the lung after 20 h, while only < 4% remaining after 30 days without causing obvious lung inflammation or toxicity. although still in early stage of development, these mpn particles may revolutionize the nebulization delivery of protein drugs and provide a more protected environment for effective pulmonary delivery [40] . moreover, new generation nebulisers are being developed such as surface acoustic wave (saw) nebulisers and the more recent hydra (hybrid resonant acoustics) nebulisers that provide new platforms for inhaled drug delivery. saw use surface waves to generate aerosols which can preserve macromolecule integrity that has been shown to be efficient in aerosolising proteins [41] . hydra uses a hybrid combination of surface and bulk sound waves to generate the aerosol droplets, and can overcome the low nebulisation rate of saw nebulisers and conventional nebulisers, while also avoiding the potential damage to proteins due to high shear (jet nebulisation) or cavitation (ultrasonic nebulisation). the first human lung deposition study using a prototype hydra nebuliser has been reported recently, indicating successful lung deposition of a radiolabelled small molecule [42] . it is probable that hydra nebulisers may be developed for pulmonary protein drug delivery. mdis deliver drug through an aerosol burst, and allow for the controlled delivery of specific amounts of drug to the lungs [21, 22] . however, as with nebulisers, the use of aqueous solutions is not ideal for protein storage [22] . there are also concerns that the hydrofluoroalkane (hfa) propellants used in mdis could denature proteins [21, 43] . despite this concern, there are examples of studies showing that proteins can remain stable in hfa-containing mdi formulations. quinn et al. (1999) utilised raman spectroscopy to analyse the secondary conformations of lysozyme in the hfa propellants tetrafluoroethane (hfa 134a) and heptafluoropropane (hfa 227), demonstrating that structural integrity of lysozyme was preserved in both hfas, and that there is potential for proteins to be developed as mdi formulations without compromising their conformational stability [44] . moreover, liao et al. (2005) demonstrated that spray-dried lysozyme and catalase that were stabilised with excipients (sugars and/or 80% polyvinyl alcohol) and then stored in hfa 134a at room temperature for 6 months showed retention of biological activity [45] . when choosing an inhalation device, it is important to be cognisant of the fact that not all devices in the same category are equivalent. for instance, although it is generally accepted that there is minimal heating of the drug reservoir in vibrating mesh nebulisers, and that heating occurs to a lesser degree than in ultrasonic nebulisers, considerable temperature increases have been reported in some brands of vibrating mesh nebulisers (pari eflow®, akita 2 apixneb®, and aeroneb go), with temperatures of up to 40°c being reached towards the end of nebulisation [24] . therefore, it is essential to choose the inhalation device carefully, bearing in mind that the best device type is the one that confers the most stability to the protein drug formulation. soft mist inhaler (smi), which also generate aerosols from liquid, is the newest type of inhaler which does not use any propellent. as only one medicine respimat uses smi, its suitability for protein drug delivery is not clear. another factor to consider is the aerodynamic diameter of aerosol particles, which is critical to control where the particles will be deposited in the respiratory track after inhalation. to be therapeutically effective, the drug containing particles need to be deposited into the correct location within the respiratory track. for example, for therapeutics for copd, drugs need to be delivered to the deep lung (the alveolar space) for which it requires the aerodynamic diameter of the particles to be between 1 and 5 μm. larger size particles will generally be deposited in the oropharyngeal region and be ingested, while small particles < 1 μm may be exhaled during the next breathing cycle. thus, suitable aerosol particle sizes need to be selected for precise drug delivery into the lung to enhance drug efficacy while simultaneously reducing harmful side effects [5] . the respiratory tract comprises of a series of branching airways, which can be categorised into two parts: the conducting zone and the respiratory zone. the conducting zone consists of the trachea, bronchi, bronchioles, and terminal bronchioles. the airway wall in the conducting zone is too thick for diffusion and this region does not contain alveoli. as such, no gas exchange takes place here, and the purposes of the conducting zone include transmitting air to the respiratory zone, as well as to warm, moisture and cleaning the inspired air. the respiratory zone consists of respiratory bronchioles, alveolar ducts, and alveolar sacs, and facilitates gas exchange between the air and the bloodstream. alveoli can occasionally be found in the walls of the respiratory bronchioles, and are abundant in the alveolar ducts and alveolar sacs [45, 46] . given the branched structure of the lungs, it is not only important to achieve high deposition rates, but also to obtain an appropriate deposition pattern for the respiratory disease in question i.e. the protein therapeutic would not only need to reach the lung, but would also need to reach the correct target site within the lung. for instance, a therapeutic for asthma would have to reach the large airway, as asthma mainly affects the bronchi, while a drug for emphysema in copd would need to go deeper and reach the small airways of the lung because emphysema affects the alveolar region [47] . the amount and pattern of lung deposition is not only affected by the device and the characteristics of the inhaled drug (particle size and physicochemical properties of the formulation), but also by factors that are influenced by the specific disease state, including breathing patterns, lung geometry (i.e. airway diameter, number of alveoli) and structure, and nasal, oral, and pharyngeal anatomy [45] . these factors need to be considered, and if possible, alterations to the drug formulation can be made to address these issues. for example, in certain lung pathologies (for instance cystic fibrosis, copd, and chronic sinusitis), the airway mucus becomes thicker. it has been reported that the thickness of the mucus layer ranges from 2 to 30 μm in normal lungs to more than 260 μm in cystic fibrosis and other obstructive airway diseases [48] . this presents a physical barrier that the protein drug would need to penetrate to reach its target site in the lung and exert its effects. the addition of anti-adhesive molecules (e.g. polyethylene glycol, peg) in the formulation may help to promote the translocation of the protein drug through the thickened mucus, although it should be noted that the adhesive properties of peg depend on peg molecular weight (mw) [12] . while high mw pegs display mucoadhesive properties, low mw pegs are able to prevent mucoadhesion, with pegs of mw up to 40 kda able to provide effective mucus penetration [49] [50] [51] . notably, as macromolecules, proteins have a relatively poor ability to penetrate the epithelial layer to reach the deep parenchyma lung. however, depending on the molecular weight and aerosol characteristics, a portion of the proteins would be able to reach the abluminal side of the epithelium or the air-blood interface in the thin alveolar wall, triggering local or even systemic immune signalling that may provide beneficial therapeutic effect in some cases [15] . a good lung deposition pattern would be worthless if the protein therapeutic cannot withstand the lung's clearance mechanisms. inhaled proteins would be subjected to clearance by three mechanisms. the first clearance mechanism is mucociliary clearance (mcc), which is the coordinated beating of cilia lining the nasal cavity, trachea, and bronchi, in order to move the mucus towards the larynx/pharynx, thereby pushing dust, microorganisms, and insoluble particles that are trapped in the mucus out of the lungs and into the upper airways to eventually be swallowed [46] . the surface lining of the airways in normal lungs consists of an aqueous layer adjacent to the epithelium and a surfactant containing film layer at the air-liquid interface. the peri-ciliary aqueous layer has a relatively low viscosity, while the surfactant film layer is more viscous. the surfactant film plays an important role in the displacement of airway particles towards the epithelium where they will be immersed and retained. the extent of particle immersion depends on the surface tension of the film. the lower the surface tension, the greater the immersion of particles into the aqueous layer adjacent to the epithelium [52, 53] . it is possible that some protein monomers could quickly reach the stagnant aqueous layer and not be subjected to mcc. on the other hand, some protein monomers would become aggregated during the inhalation delivery process and the aggregates may stay with the surfactant film layer at the air-liquid interface for some time for mcc to take effect. anti-adhesive formulations (achieved by using lower mw pegs for example) could be used to circumvent mcc clearance of inhaled therapeutics, thus increasing their lung accumulation. the mucus-penetrating ability of such pegs has already been demonstrated in multiple studies [12, [54] [55] [56] [57] [58] [59] [60] [61] . the second clearance mechanism is macrophage uptake, which is the primary clearance mechanism in the alveoli. proteins are taken up by alveolar macrophages in the deep lung via pinocytosis, and the uptake of particles is size dependent [5, 12, 62] . large proteins (≥ 40 kda) would have more time to be engulfed by alveolar macrophages by virtue of their slower transport and absorption across the alveolo-capillary barrier, while small proteins and peptides (≤ 25 kda) are absorbed rapidly from the airspaces and thus, may not be impacted by alveolar macrophage uptake as much. in essence, pinocytosis by alveolar macrophages could become significant for macromolecules with mw > 40 kda [62] . the use of excipients can help to reduce clearance of large proteins by alveolar macrophages. for example, koussoroplis et al. (2014) showed that pegylation conferred increased residence time to antibody fragments anti-il-17a f (ab ′)2 and anti-il-13 fab′ (unconjugated f (ab′)2 was 98 kda and unconjugated fab′ was 47 kda), and that the effect was due to mucoadhesion as well as evasion of alveolar macrophage uptake [11] . protein pegylation may potentially also alter its deposition pattern in the respiratory track, due to changes in molecular weight, hydrophilicity etc. however, systemic analyses of the effect of pegylation on protein deposition pattern in the respiratory track are still needed in order to know if a consistent deposition pattern and behaviour can be reached based on how pegylation is achieved. the third clearance mechanism is absorption into the systemic circulation. after deposition in the alveolar region, aerosol drug particles may dissolve in pulmonary epithelial lining fluid if the drug is water-soluble, and become available for systemic absorption and clearance [46] . for the purpose of topical lung treatment, the goal would be to minimise systemic absorption, which is greatly influenced by protein mw. the bioavailability of a protein after absorption from the lung decreases as protein mw increases. small peptides are absorbed rapidly from the lungs with 20-50% of the bioavailability for subcutaneous injection [63] . proteins with mw of 6-50 kda exhibit moderate absorption, with bioavailability ranging from 10 to 40%, although it should be noted that pulmonary absorption studies in animals may lead to an overestimation of bioavailability. for example, systemic bioavailability after aerosol administration in animals for growth hormone (gh) and interferon α (ifnα) was 45% and 70% respectively, compared to only 3-10% in humans [63, 64] . large mw antibodies (~150 kda) are not significantly absorbed across the lung, and bioavailability is negligible (<< 10%) unless an active transport system is included [63] . apart from high protein mw, the presence of obstructive lung diseases (e.g. asthma, copd, cystic fibrosis) can also reduce systemic absorption and bioavailability of proteins and other drugs [46] . for example, henry et al. (2003) reported that healthy subjects had significantly higher area under the curve (auc) and mean maximum concentration (c max ) after insulin inhalation than asthma patients, indicating that less insulin was absorbed into the systemic circulation in asthma patients [65] . in addition, diderichsen et al. (2013) reported that the c max of an inhaled long acting β 2 agonist (pf-00610355) was found to be reduced by 31% and 52% for copd and asthma patients respectively, compared to healthy volunteers [66] . additional possibility for the lower systemic absorption of drugs in lung disease patients could be due to altered drug deposition pattern in the diseased lung, for which further studies are needed. regardless the underlying mechanisms, effective local lung retention of protein drugs may not be a major issue for the successful inhalation treatment of obstructive lung diseases, since the protein can be relatively well retained in the lungs. inhaled protein therapeutics may undergo various degradation mechanisms during production, processing and/or storage. these degradation pathways may be physical (denaturation and non-covalent aggregation) or chemical (mainly covalent aggregation, deamidation, oxidation and/or glycation). denaturation is the result of physical stresses including low/high temperatures, high salt concentrations, organic solvents, and air/water or ice/water interfaces. removal of the stressor may be spontaneously reversible (for some single domain proteins), but is usually irreversible for most of the larger multi-domain proteins [67] . surface-induced aggregation is one of the common mechanisms of non-covalent aggregation, and one example of when it occurs is during the process of nebulisation [15, 67] . as most proteins are amphiphilic and surface active, they have a tendency towards adsorption at the ali. upon adsorption, conformation changes may occur, exposing hydrophobic residues to the interface to avoid contact with water, thus leading to aggregation and unfolding, which are the main factors contributing to protein instability [67, 68] . chemical degradation of proteins (deamidation, oxidation, glycation) may also cause aggregation (either covalent or non-covalent) [67] . aggregation has been extensively studied but chemical modifications have not, despite having implications on biological activity and immunogenicity [4, 15] . aggregation can result from both physical and chemical pathways; therefore, it is useful to also evaluate chemical changes in inhaled proteins. most studies assessing stability of inhaled protein formulations focus on formation of aggregates, while studies that also examine chemical changes are few and far between. one study did, however, consider chemical changes when evaluating the technical feasibility of delivering dornase alfa using perforated vibrating membrane devices for nebulisation. in this study, besides detecting protein aggregates, stability was also evaluated by measuring the percent deamidation of dornase alfa at asn 74 (the main chemical change for the protein), which was shown to be inversely proportional to dornase alfa potency [35] . another study looked at methionine 59 oxidation [met(o)] of nebulised insulin-like growth factor-1 (igf-i), and how that correlated with aggregate formation and bioactivity. highly aggregated samples displayed a complete loss of bioactivity, while samples with complete oxidation but minimal aggregation showed partial retention of bioactivity. limited met(o) formation and no aggregation was observed following delivery with air-jet or vibrating mesh nebulisers [36] . bandi et al. (2019) conducted a study to compare the effects of deamidation and oxidation on interferon alpha-2a (ifna2a), as deamidation of asparagine and glutamine residues, and oxidation of methionine residues are two of the most common chemical alterations that occur in pharmaceutical proteins that could compromise their efficacy and safety [68] . these findings revealed that deamidation destabilised ifna2a and enhanced its tendency to aggregate under stressful conditions, and reduced its function to a greater extent than oxidation. this is the first study that quantitatively compared the effects between deamidation and oxidation of a therapeutic protein [68] . it would be a good strategy to conduct such studies early on in the development of therapeutic protein candidates in order to identify the chemical modifications that a particular protein would be susceptible to, and to test out various excipients that could resolve specific stability issues. the protein therapeutic also needs to remain stable after reaching the lung, which can be challenging due to the high numbers of serine proteases and aminopeptidases present in the lung mucosa [69] [70] [71] [72] . these proteases could degrade protein drugs even before they reach their target sites within the lung. the use of appropriate excipients in the formulation such as peg, could help to enhance protein resistance to proteolysis by these lung proteases. for example, zhang et al. (2014) evaluated the stability of fibronectin (fn) preferentially pegylated at lysine residues using different mw pegs [2 kda (peg2), 5 kda (peg5) or 10 kda (peg10)] against the protease α-chymotrypsin. they showed that pegylation protected fn from proteolysis and that peg mw positively correlated with proteolytic stability (i.e. after 30 min of proteolysis, 4%, 34%, 43% and 65% of the starting amounts of native fn, fn-peg2, fn-peg5 and fn-peg10 respectively were remaining) [73] . one must also be aware of the possibility of protein aggregates forming in the lungs. lasagna-reeves et al. [74] demonstrated that mice exposed to inhaled insulin (exubera®) in a chamber twice daily for 1 week developed amyloid aggregates of insulin in both the proximal and distal airways, as well as the lung parenchyma (epithelium and muscle layer of the bronchi, bronchioles, and in the alveolar lining cells). the formation of insulin aggregates coincided with a significant decrease in respiratory flow rates, and also with caspase-9 activation. previous studies investigating the link between changes in pulmonary function and inhaled insulin use focused on formation of anti-insulin antibodies, or pulmonary inflammation and subsequent airway remodelling, but none of the published works before this looked at insulin aggregation in the lungs as a contributor to pulmonary dysfunction after inhaled insulin use [74] . indeed, exu-bera® was reported to cause cough, dyspnea, increased sputum and epistaxis [75] . this example highlights the possibility of inhaled proteins forming aggregates in the lungs, and thus the need for toxicity testing and safety studies examining this possibility to be done early on in the development of an inhaled protein candidate, during preclinical studies. in addition, proteins and other macromolecules have the potential to induce immunogenicity, with the production of anti-drug antibodies (adas) as the main immune response [5] . the development of adas in patients can alter pharmacokinetics, drastically reduce efficacy, and can also lead to severe adverse events or even lethal consequences [76] . immunogenicity is also linked to protein stability, as the presence of aggregates can render the protein immunogenic. as aggregates are typically composed of denatured molecules, they would exhibit no or decreased activity, but at the same time, aggregates are usually immunogenic leading to adas with important clinical implications [4] . aggregates are believed to be recognised and processed via non-specific uptake by antigen presenting cells and specific uptake by b cells. they may unmask neo−/cryptic/repetitive epitopes, and these differences may influence the mechanism by which they activate the immune system [76] . currently, only a few excipients have been approved by the fda for inhalation due to a dearth of toxicological studies for inhaled excipients [15, 67] . there is also very limited number of excipients that are approved by fda for biologics, rendering formulators limited choices to improve protein formulations when excipients are searched on the fda's inactive ingredient database guidance. furthermore, very few novel excipients have been investigated for biologic products; most are cyclodextrin-based excipients [77] . as such, there is a need for more extensive toxicity testing to identify novel excipients for pulmonary delivery. for excipients already known to increase protein stability, a trial and error approach needs to be taken in determining their suitability for a particular protein formulation, as an excipient may work for one protein but not for another for various reasons including sequence differences [15] . excipients that are commonly used in liquid formulations (nebulisers and mdis) include buffering or ph adjusting agents, and surfactants, and those that are commonly used in dry powder formulations (dpis) include sugars, polyols, and amino acids [67, 78] . buffering or ph adjusting agents such as sodium chloride, sodium citrate, hydrochloric acid, sodium hydroxide, and citric acid, are added to maintain the ph of the formulation. it is important to choose the right buffering agent at an appropriate concentration, as most proteins in solution only remain stable within a narrow ph range. different buffer systems and concentrations can also affect the aggregation pattern of proteins [67] . kim et al. [79] analysed the stability of a fusion protein, etanercept (marketed enbrel®), with changing ph and buffer concentrations. increasing the ph of etanercept from ph 6.6 to 8.6 resulted in a decrease in protein size and increase in aggregation. under high buffer concentrations (30 mm tris buffer), changes in protein size was reduced and irreversible aggregation was not observed, while in lower buffer concentrations (10 mm tris buffer), larger aggregates (~1 μm) were observed across the ph range [79] . surfactants (polysorbates, sorbitan esters, oleic acid, and soy lecithin) are frequently used to prevent aggregate formation, and they work by displacing protein molecules from the ali [46, 62] . polysorbates are the most commonly used surfactants, and are already being used to preclude aggregation in formulations of intravenously administered antibodies [34] . polysorbate 80 has been reported to lead to stabilisation in various inhaled protein formulations including those for granulocyte-colony stimulating factor (g-csf), lactate dehydrogenase (ldh), tissue plasminogen activator (t-pa) and aviscumine (recombinant mistletoe lectin) [68] . the ability of polysorbates and other surfactants to stabilise a protein and hinder aggregate formation is contingent on the protein-to-surfactant ratio. respaud et al. [34] examined the effects of various antibody and surfactant (polysorbate 20) concentrations to optimise the protein-to-surfactant ratio for a nebulised antibody formulation. the authors determined that high concentrations of either surfactant or protein could minimise the formation of medium and large-sized aggregates, without significantly affecting the volume mean diameter (vmd) of the aerosol cloud, ensuring suitability for inhalation. therefore, including surfactants and raising protein concentration to enhance the stability of inhaled protein formulations is a viable strategy, although it should be noted that this approach needs to be evaluated and optimised for each drug and device pairing being developed into an inhaled protein formulation [34] . sugars (sucrose, trehalose, raffinose and lactose) and polyols (mannitol) stabilise proteins through the preferential hydration of proteins via steric repulsion of sugar/ polyol molecules from the native protein [4, 68] . lactose is often used as a drug carrier in dpis, however, it may not be suitable for proteins because it is a reducing sugar, and it could interact with amino groups in proteins (maillard reaction) [67] . on the other hand, non-reducing sugars such as sucrose, trehalose and raffinose would not undergo the maillard reaction with proteins, and thus could be used as alternatives to lactose [81] . sellers et al. [82] demonstrated that sucrose could help to improve the stability of a dry powder formulation of ldh. supercritical fluid (scf) drying of ldh without excipients lead to irreversible loss of activity (only 15% recovered after rehydration). inclusion of 10% (w/w) sucrose during dehydration lead to an increase in activity recovered (to~60%), and there was almost complete retention of activity when polysorbate 20 was added in addition to sucrose [82] . trehalose and raffinose are currently not approved for any administration routes, but have been evaluated in experimental studies with promising results. for instance, ógáin et al. [81] incorporated lysozyme into nanoporous microparticles of trehalose and raffinose. lysozyme showed good retention of specific activity after storage for 12 weeks at either 4°c (98.2 ± 7.1% for lysozyme:trehalose and 99.1 ± 7.1% for lyzosyme:raffinose) or 25°c (92.5 ± 7.1% for lysozyme:trehalose and 90.8 ± 7.1% for lyzosyme:raffinose) [81] . mannitol was used as an excipient in the formulation of exubera® (table 2 ) [80] . small amino acids (histidine, arginine, alanine, glycine, lysine, isoleucine) are also used as stabilisers, and they work by the "water substitution mechanism" in which the amino acids hydrogen bond with the protein during drying to preserve the native protein structure in the dried state [4, 83] . ajmera and scherlieβ [84] screened different amino acids and their combinations for their ability to stabilize catalase during spray drying. when various ratios of arginine, glycine and histidine were mixed with catalase, some formulations were able to maintain close to 100% catalase activity [84] . despite encouraging results in studies such as this one, there is a lack of data on the local toxicity of the various amino acids following inhalation, which could limit their use. however, as they are endogenous substances, they may not present major safety issues for local lung delivery [67, 85] . the polyol, peg, could be used for both liquid and powder formulations of inhaled proteins. small mw pegs (< 10 kda) are often used as excipients in oral, intravenous and nasal formulations. larger pegs (up to 40 kda) may be used in pegylated biopharmaceuticals, and safety testing for these formulations are done during development on a case-by-case basis [86] . pegylation is a commonly used method to enhance solubility and stability, as well as to decrease immunogenicity of bioactive drugs including but not limited to proteins, peptides, antibody fragments, and enzymes, and is achieved by the covalent or noncovalent conjugation of peg to the biomolecule [87, 88] . pegylation can also help to reduce clearance and increase lung accumulation and residence time of inhaled protein therapeutics. for instance, conjugation of a peg chain to two antibody fragments (anti-il-17a f (ab′)2 and anti-il-13 fab′) increased their levels in mouse lungs following intranasal administration. fortyeight hours post-administration, levels of unconjugated antibody fragments in the lungs had dropped to 10% and 14% of the original deposited dose of f (ab′)2 and fab′ respectively, while this value was 40% for both pegylated fragments [11] . furthermore, conjugation of a peg chain to an anti-il-17a fab' antibody fragment increased pulmonary retention in all three species tested (mice, rats, and rabbits) following intratracheal administration. unconjugated fragments were cleared from the lungs within 24 h while large amounts of pegylated fragments still remained for up to 48 h [89] . the two biggest challenges in developing particle systems for pulmonary drug delivery are to maintain colloidal stability during aerosolisation and to achieve high delivery efficacy. encapsulation of proteins in carriers could provide multiple benefits such as protection from enzymatic degradation and specific targeting to the site of action through targeting ligands [5] . furthermore, carriers may also be used to provide sustained drug release, accumulating in the lungs and releasing therapeutic levels of the protein drug over extended periods of time. this would enhance efficacy while averting peaks in local drug concentrations that could cause pulmonary toxicity [90] . proteins, including insulin, calcitonin, and igg, have already been loaded into various carriers such as microparticles, liposomes, and solid lipid nanoparticles [90, 91] . indeed, afrezza® uses techno-sphere® technology, in which fumaryl diketopiperazine (fdkp), an excipient added into the formulation, selfassembles into microspheres, entrapping the insulin. upon reaching the alveolar zone of the lung, the tech-nosphere® particles rapidly dissolve in the ph-neutral environment and release the insulin for systemic absorption [75] . although this approach has not been extensively explored for topical lung delivery of proteins, and more work needs to be done on the use of carriers for the purpose of systemic delivery of proteins through the lungs, some promising results have been reported that support further development of this approach. tawfeek et al. [92] encapsulated a model mucinolytic enzyme, αchymotrypsin (which is very sensitive to unfolding and formulation conditions), in a novel biodegradable pegco-polyester microparticle carrier. the encapsulated αchymotrypsin exhibited retention of enzymatic activity and the results indicated suitability of the carrier for potential use in the delivery of macromolecules as dpi formulations for the treatment of lung diseases [92] . in another study by osman et al. [93] , various surface modifications were made to dnase i loaded microparticles using different excipients in order to provide higher lung deposition, enzyme stability and biological activity. surface modifying the microparticles with polyglutamic acid (pga) or dextran was found to provide high inhalation indices (emitted fraction (ef), respirable particle fraction (rp), and effective inhalation index (ei)) and increased mucolytic activity in cystic fibrosis sputum. this could be explained by the resulting surfaces of the particles after modification with pga (rough dented surfaces) or dextran (dimpled surfaces). compared to spherical particles with similar physical properties, corrugated particles have surface asperities that could reduce the true contact area between particles, decreasing powder cohesiveness and enhancing aerosol performance [93] . advancements in drug-loaded capsules for pulmonary delivery have been made in both inhalable dry powder or liquid drug formulations [94] [95] [96] . for dry powder drug particles, precise control of the particle size has been reported using the particle replication in nonwetting templates (print) technology [97, 98] . for control of aerodynamic particle size in liquid aerosols such as in nebulized liquid formulation, the recently reported mpns have presented promising possibilities. mpnbased drug-loaded capsules with highly defined physical properties can be generated for both macromolecular protein drugs and small molecule chemical drugs [54] . these new developments may transform inhalation drug delivery in the near future. one drawback with the use of carriers is their rapid uptake by alveolar macrophages [99, 100] . phagocytosis of carriers by alveolar macrophages can result in fast clearance and reduced residence time, limiting the therapeutic efficacy of the carrier-associated drug. this would be an issue for the treatment of chronic lung diseases such as asthma and copd, where the goal of using a carrier system would be to achieve controlled and continuous drug release over an extended period of time. however, various formulation design strategies may be employed to reduce the uptake of particulate carriers by alveolar macrophages including modulation of particle size, shape, surface charge and surface coating [101] . studies on the use of various polymer coatings demonstrate reduced alveolar macrophage uptake of coated carriers. for example, jones et al. [102] showed that respirable microspheres coated with dipalmitoyl phosphatidylcholine (dppc; a major component of lung surfactant) were able to significantly reduce phagocytic uptake by nr8383 in cultured alveolar macrophages compared to uncoated microspheres. the uptake of dppc coated microspheres was found to be only 24.1 ± 7.86%, 31.9 ± 3.74% or 36.6 ± 3.66%, of the uptake of uncoated microspheres for ratios of 5, 10 or excess microspheres per nr8383 cell respectively [102] . furthermore, shen et al. [103] demonstrated that surface coating of hydrogel nano-and microparticles with peg showed significantly reduced uptake by alveolar macrophages both in vitro (in mh-s cells) and in vivo (in mice) compared to unpegylated particles of the respective size. at 24 h post-dose, the fold difference between pegylated and unpegylated 80 × 320 nm, 1.5 μm, and 6 μm particles in bronchioalveolar lavage fluid (balf), was 1.5, 3.4 and 3.7 respectively [103] . on the other hand, drug-loaded particles may be advantageous for anti-tuberculosis drugs as efficient uptake of drugs into alveolar macrophages could potentially enhance the drug's efficacy to kill the parasitic mycobacterium tuberculosis that hide inside the cells [104] . if the usage of a carrier is to be included in the protein formulation, it should be noted that the formulation (i.e. combination of protein and carrier) would need to be optimised together with the choice of device, as the chosen carrier may not work well with all inhalation device types. for instance, liposomes may be delivered to the lungs either by dry powder inhalation or nebulisation of a liposome suspension. however, nebulised solutions of liposomes may cause instability as nebulisation has been reported to disrupt liposomal structure, leading to the release of loaded drug. these issues can be avoided with the use of dry powders of liposomes instead [90] . hence, although this review has presented a general overview for the various aspects of protein formulation design (such as choice of device, excipients), it is important to test out the formulation and device together to determine which combination works best. this review analyses the various obstacles that an inhaled protein drug would need to overcome in order to reach the lungs and exert its therapeutic effects. these obstacles include the physical and chemical stresses experienced by the protein during production/storage/ aerosolisation, the need to overcome mucociliary clearance and physical barriers arising from disease conditions in order to reach target sites within the lung, and the need to remain stable in spite of the presence of abundant proteases, and to evade clearance by alveolar macrophages after reaching the lungs (fig. 1) . all of these threats to the integrity of the protein need to be carefully considered, so that pre-emptive measures can be taken while designing the protein formulation to ensure its therapeutic efficacy. nevertheless, although the information provided here may serve as general considerations in developing pulmonary protein therapeutics, empirical testing of the formulation together with the device should still be performed to determine the best combination for a particular protein. several key areas will require further investigation in order to support the development of more successful inhaled protein therapies, and maintaining the stability of the inhaled protein is of paramount importance. firstly, more studies could look at other instability issues beyond protein aggregation. in depth studies on the specific chemical modifications that a protein would be susceptible to, such as the one conducted by bandi et al. (2019) , could be carried out on therapeutic protein candidates so that they may be developed into stable and effective treatments [68] . moreover, the scarcity of fdaapproved excipients for inhaled therapeutics further limits drug developers, and expanding this list through increased toxicological testing of new excipients would provide more options for formulation design. finally, innovative approaches such as the use of novel carrier systems should be employed for the purpose of topical lung delivery, as carrier systems could greatly enhance the stability and pharmacokinetic profile of proteins. these approaches would greatly benefit the field of pulmonary drug delivery, and will ultimately allow more inhaled protein therapeutics to reach the clinic. funding support for this work is provided by the grant moe2017-t2-2-122 awarded to ruowen ge from singapore ministry of education, republic of singapore. capturing the benefits of competition for patients current approaches to the discovery of novel inhaled medicines inhaled protein/peptide-based therapies for respiratory disease nebulization as a delivery method for mabs in respiratory diseases carriers for the targeted delivery of aerosolized macromolecules for pulmonary pathologies inhalation of immuno-therapeutics/ −prophylactics to fight respiratory tract infections: an appropriate drug at the right place! front immunol vegf neutralizing aerosol therapy in primary pulmonary adenocarcinoma with kras activating-mutations the airways, a novel route for delivering monoclonal antibodies to treat lung tumors inhaled therapy in respiratory disease: the complex interplay of pulmonary kinetic processes efficacy of aerosol therapy of lung cancer correlates with egfr paralysis induced by avidinox-anchored biotinylated cetuximab pegylation of antibody fragments greatly increases their local residence time following delivery to the respiratory tract repurposing of gamma interferon via inhalation delivery 2 phase ii clinical trial results of alidornase alfa for the treatment of cystic fibrosis protalix biotherapeutics announces phase ii clinical trial results for alidornase alfa in cystic fibrosis presented at the 40th european cystic fibrosis society conference. protalix biotherapeutics designing inhaled protein therapeutics for topical lung delivery: what are the next steps? inhaled gm-csf in a pulmonary alveolar proteinosis patient refractory to plasmapheresis combined with multiple whole lung lavages synairgen doses first patient in covid-19 trial ansun biopharma enrolls first patient in proof of concept trial of das181 for the treatment of covid-19 direct administration in the respiratory tract improves efficacy of broadly neutralizing anti-influenza virus monoclonal antibodies in a murine model of acute lung infection, airway administration of a therapeutic antibody confers greater protection than parenteral administration challenges and future prospects for the delivery of biologics: oral mucosal, pulmonary, and transdermal routes non-invasive delivery strategies for biologics idealhalers versus realhalers: is it possible to bypass deposition in the upper respiratory tract? that's cool! -nebulization of thermolabile proteins with a cooled vibrating mesh nebulizer protein stability in pulmonary drug delivery via nebulization protein nebulization: i. stability of lactate dehydrogenase and recombinant granulocyte-colony stimulating factor to air-jet nebulization stability of urease during aerosolization protein nebulization some factors associated with the ultrasonic nebulization of proteins nebulizers for drug delivery to the lungs the function and performance of aqueous aerosol devices for inhalation therapy devices for improved delivery of nebulized pharmaceutical aerosols to the lungs vibrating mesh nebulisation of pro-antimicrobial peptides for use in cystic fibrosis effect of formulation on the stability and aerosol performance of a nebulized antibody a technical feasibility study of dornase alfa delivery with eflow® vibrating membrane nebulizers: aerosol characteristics and physicochemical stability insulin-like growth factor-i aerosol formulations for pulmonary delivery development of a drug delivery system for efficient alveolar delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin effective nebulization of interferon-γ using a novel vibrating mesh factors to consider when selecting a nebulizer for a new inhaled drug product development program engineering of nebulized metal-phenolic capsules for controlled pulmonary deposition pulmonary monoclonal antibody delivery via a portable microfluidic nebulization platform in vivo deposition study of a new generation nebuliser utilising hybrid resonant acoustic (hydra) technology prospects of formulating proteins/peptides as aerosols for pulmonary drug delivery protein conformational stability in the hydrofluoroalkane propellants tetrafluoroethane and heptafluoropropane analysed by fourier transform raman spectroscopy computational modeling of lung deposition of inhaled particles in chronic obstructive pulmonary disease (copd) patients: identification of gaps in knowledge and data the impact of pulmonary diseases on the fate of inhaled medicines-a review drug delivery for traditional and emerging airway models. organs-on-a-chip nanodelivery in airway diseases: challenges and therapeutic applications addressing the peg mucoadhesivity paradox to engineer nanoparticles that "slip" through the human mucus barrier biodegradable polymer nanoparticles that rapidly penetrate the human mucus barrier nanoparticles coated with high molecular weight peg penetrate mucus and provide uniform vaginal and colorectal distribution in vivo surfactant displaces particles toward the epithelium in airways and alveoli pulmonary surfactant: surface properties and function of alveolar and airway surfactant transport of nanoparticles in cystic fibrosis sputum and bacterial biofilms by single-particle tracking microscopy protein nanocages that penetrate airway mucus and tumor tissue use of single-site-functionalized peg dendrons to prepare gene vectors that penetrate human mucus barriers rapid transport of large polymeric nanoparticles in fresh undiluted human mucus highly compacted biodegradable dna nanoparticles capable of overcoming the mucus barrier for inhaled lung gene therapy pegylated enhanced cell penetrating peptide nanoparticles for lung gene therapy lung gene therapy with highly compacted dna nanoparticles that overcome the mucus barrier the penetration of fresh undiluted sputum expectorated by cystic fibrosis patients by non-adhesive polymer nanoparticles preclinical models for pulmonary drug delivery formulation technology to repurpose drugs for inhalation delivery will pulmonary drug delivery for systemic application ever fulfill its rich promise? inhaled insulin using the aerx insulin diabetes management system in healthy and asthmatic subjects characterizing systemic exposure of inhaled drugs: application to the long-acting β2-agonist pf-00610355 inhaled proteins: challenges and perspectives 2d nmr analysis of the effect of asparagine deamidation versus methionine oxidation on the structure, stability, aggregation, and function of a therapeutic protein respiratory protease/antiprotease balance determines susceptibility to viral infection and can be modified by nutritional antioxidants role of proteases in lung disease: a brief overview role of proteases in chronic obstructive pulmonary disease proteases and antiproteases in chronic neutrophilic lung disease -relevance to drug discovery pegylation of lysine residues improves the proteolytic stability of fibronectin while retaining biological activity inhaled insulin forms toxic pulmonary amyloid aggregates technosphere®: an inhalation system for pulmonary delivery of biopharmaceuticals immunogenicity of different stressed igg monoclonal antibody formulations in immune tolerant transgenic mice the effects of substituted cyclodextrins on the colloidal and conformational stability of selected proteins formulation strategy and use of excipients in pulmonary drug delivery effects of ph and buffer concentration on the thermal stability of etanercept using dsc and dls practical, regulatory and clinical considerations for development of inhalation drug products particle engineering of materials for oral inhalation by dry powder inhalers. i-particles of sugar excipients (trehalose and raffinose) for protein delivery dry powders of stable protein formulations from aqueous solutions prepared using supercritical co2-assisted aerosolization mechanisms of protein stabilization in the solid state stabilisation of proteins via mixtures of amino acids during spray drying amorphous powders for inhalation drug delivery pegylation, an approach for improving the pulmonary delivery of biopharmaceuticals what is the future of pegylated therapies? from synthesis to characterization of site-selective pegylated proteins pegylation prolongs the pulmonary retention of an anti-il-17a fab' antibody fragment after pulmonary delivery in three different species delivery strategies for sustained drug release in the lungs multifunctional nanocarriers for lung drug delivery dry powder inhalation of macromolecules using novel peg-copolyester microparticle carriers inhalable dnase i microparticles engineered with biologically active excipients inhaled nano-and microparticles for drug delivery nanoparticles for drug delivery to the lungs advanced therapeutic strategies for chronic lung disease using nanoparticle-based drug delivery formulation of high-performance dry powder aerosols for pulmonary protein delivery microfabricated engineered particle systems for respiratory drug delivery and other pharmaceutical applications polymeric nanoparticles in development for treatment of pulmonary infectious diseases update on macrophage clearance of inhaled micro-and nanoparticles particle engineering to enhance or lessen particle uptake by alveolar macrophages and to influence the therapeutic outcome the inhibition of phagocytosis of respirable microspheres by alveolar and peritoneal macrophages distribution and cellular uptake of pegylated polymeric particles in the lung towards cell-specific targeted delivery drug delivery for tuberculosis: is inhaled therapy the key to success? publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-341324-f9g9gitn authors: rojas, josé m.; alejo, alí; martín, verónica; sevilla, noemí title: viral pathogen-induced mechanisms to antagonize mammalian interferon (ifn) signaling pathway date: 2020-10-21 journal: cell mol life sci doi: 10.1007/s00018-020-03671-z sha: doc_id: 341324 cord_uid: f9g9gitn antiviral responses of interferons (ifns) are crucial in the host immune response, playing a relevant role in controlling viralw infections. three types of ifns, type i (ifn-α, ifn-β), ii (ifn-γ) and iii (ifn-λ), are classified according to their receptor usage, mode of induction, biological activity and amino acid sequence. here, we provide a comprehensive review of type i ifn responses and different mechanisms that viruses employ to circumvent this response. in the first part, we will give an overview of the different induction and signaling cascades induced in the cell by ifn-i after virus encounter. next, highlights of some of the mechanisms used by viruses to counteract the ifn induction will be described. and finally, we will address different mechanism used by viruses to interference with the ifn signaling cascade and the blockade of ifn induced antiviral activities. innate immune responses are the first host defense against viral infections. conserved pathogen structures are recognized by pattern recognition receptors (prrs) on the host cells [1] , that recruit a variety of adaptor proteins to signal downstream and activate the ifn response. the ifn system is present in all vertebrates and is central to antiviral responses [2] . ifns are classified into three different families according to their receptor usage, mode of induction, biological activity and amino acid sequence [3] : type i, type ii and type iii ifns. type i ifns, originally identified by their antiviral activity [4] , include multiple ifn-α subtypes (13 in humans and 14 in mice), and a single ifn-β, and ifn-ε, ifn-κ, ifn-ω (humans) and ifn-ξ (mice) subtypes [5] . in mammals, type i ifn (ifn-i) response is essential for innate antiviral responses. they all bind to the same ubiquitously expressed receptor, ifnar receptor, but they differ in their biological functions, due partially to the different binding affinities to the ifnar receptor [6] . this differences in affinity results in different downstream signaling cascades [7] . for ifn-α subtypes, the quantity of the receptor on the surface of a target cell correlates also with their biological activities suggesting that the amount of ifnar expression might compensate the weak affinity of some ifn-α subtypes [6, 8] . type ii ifns include only one member, ifn-γ, secreted by activated t cells, natural killer (nk), nkt cells and dendritic cells with pro-inflammatory and immunomodulatory functions [9] . in general, type ii ifn acts as a link between the innate immune response and the activation of the adaptive immune response [10] . type iii ifns include ifn-λ1, ifn-λ2 and ifn-λ3, and, although genetically different to type i ifns and signaling through different receptors, they are induced by pprs and activates antiviral pathways similar to type i ifns [11, 12] . viruses use multiple mechanisms to by-pass the host ifn responses so that they can replicate and continue their infectious cycle. the present review will focus on how viruses interfere with ifn-i responses. viruses can act at different levels of the signaling cascades involved in ifn-i responses. they can inhibit the induction of the ifn response, block the ifn signaling, and/or interfere with the antiviral activities induced by ifn. we will review some of the emerging themes and new insights from the past years of the ifn evasion mechanism employed by viruses in these contexts. the antiviral state of an infected cells is attained by the initial induction of type i ifn expression, followed by ifn signaling transduction, which finally leads to the expression of multiple genes (fig. 1) . ifns are the main group of cytokines secreted by host cells in response to the presence of "aberrant" nucleic acids such as double-stranded rna (dsrna) molecules generated as viral intermediates during viral transcription in infected cells, to cpg dna, or uncapped ssrna with 5′ triphosphate present in some viruses. these elements are known as pathogen-associated molecular patterns (pamp) [13, 14] that can be recognized by prrs. four main types of prrs have been described to detect virus-derived genetic materials: toll-like receptors (tlrs) 3/7/8/9 [15] ; retinoic acid-inducible gene-i (rig-i) like receptors (rlrs) which include the cytosolic sensor rig-i, the melanoma differentiation-associated factor 5 (mda-5) and laboratory of genetics and physiology (lgp2) [16] ; and nucleotide oligomerization domain-like receptors (nlrs) [17] and the cytosolic dna sensors [18] . for a host to establish an antiviral state it first requires the production of type i (α/β) ifns in direct response to virus infection and recognition of virus-derived genetic material. hence, both rig-i and mda-5 sense cytosolic dsrna. rig-i also specifically senses 5′ triphosphate rna generated during infection, while mda-5 detects longer dsrna sequence generated during virus replication. rig-i and mda5 contain two caspase activation and recruitment domains (cards) at the n-terminal. activation of rig-i and mda5 liberates these card domains and drives the interaction of these tandem cards with the card of the mitochondrial activation signaling (mavs) protein [21] . mavs aggregates in filaments that provide a platform for the recruitment of the elements involved in the subsequent signaling cascade such as the tumor necrosis factor receptor-associated factor (traf) 3 and traf6, the tank binding kinase 1 (tbk1) and ikkε ultimately drives the activation of transcription factors as irf3/7, nfκb and ap-1, leading the production of type i ifn and pro-inflammatory cytokines [22] . irf3 is constitutively expressed in many cell types, and after phosphorylation, irf-3 forms a homodimer that translocates into the nucleus, activating the transcription of early transcribed type i and iii ifn genes, ifn-β, ifn all type i ifns signal through the same heterodimeric transmembrane receptor termed the ifnα receptor (ifnar), containing the subunit 1 and 2 (ifnar1 and ifnar2). in a first step, ifn-i binds with high affinity to ifnar1, and then recruit ifnar2 [34] . ifnar engagement with ifn-i promotes the induction of an antiviral state in cells. this involves the upregulation of products from a large subset of genes named ifn-stimulated genes (isg) that protect the cell from viral replication. broadly speaking, isg products modulate and mediate ifn activity in the cells. this includes for instance cooperating in prr recognition of viral pamps, stabilizing signaling complexes to improve their resistance to degradation, stopping virus entry, blocking viral capsid formation, impairing trafficking and budding of virions from the infected cells, but also modulating the ifn response to avoid the toxicity of these potent immune mediators. an important feature of the ifn signaling is the rapid speed at which the response happens, which is possible because protein synthesis is not required in an initial stage. the interaction of type i ifns with their universally expressed receptor (ifnar) elicits an intracellular signaling cascade through the janus protein kinase (jak) family members, jak and tyk2, that successively phosphorylate signal transducer and transcription activator (stat) family proteins [35] . the phosphorylated stat1/stat2 heterodimer associates with interferon regulatory factor 9 (irf9) to form the transcriptional factor complex isgf3, which translocate to the nucleus and binds the ifn-response elements (isre) in isg promoters leading to the expression of isg products [36] (fig. 2 the oligoadenylate synthetase (oas)-latent rnase (rnase l) pathway is another ifn-inducible pathway that provides the cell with an effector mechanism upon recognition of viral dsrna (reviewed in [44] ). when the oas senses dsrna activates the production of 2′,5′-oligoadenylates that act as a second messenger on the inactive monomeric rnasel [45] . the 2′,5-oligoadenylates binding to rnase l produces a catalytically active dimer that cleaves ssrna [46] . this leads to the translational arrest and prevent viral replication and spreading [47] . rna-activated protein kinase is an isg product that detects cytosolic dsrna. pkr recognition of its dsrna substrate leads to dimerization and autophosphorylation which in turn leads to the phosphorylation of the viruses has developed strategies to counteract dif-ferent steps on this signaling cascade. it is marked in red blades the main signaling targets of viruses eukaryotic initiation factor 2α (eif2α) required for translation initiation [48] . eif2α phosphorylation results in the shutdown of all translation of 5′capped mrna, thus preventing the synthesis of viral proteins. this also usually results in the formation of stress granules (sg) that consist of the accumulation of rna and proteins from the stalled translation complexes. sg formation has been linked to antiviral responses, and their formation is often inhibited in viral infections. in general, the antiviral activity of pkr is related to apoptosis induction [49], regulation of ifn-β synthesis and nf-κβ pathway [50] [51] [52] , serine kinase activity for stat1 that also regulate the ifn-i signaling pathway [53] . a family of proteins with a wide range of anti-viral functions are the interferon-induced proteins with tetratricopeptide repeats (ifits) [54] . these genes are expressed at very low basal levels, but their transcription is rapidly increased after activation by ifn signaling. ifit detect the lack of 2′-o-methylation on rnas species, a methylation absent in some viral rna but present in eukaryotic mrna [55] . ifit1 has also been shown to bind to the 5′-trip end of some viral rna [56] . ifit1 can sequester viral rna or interact with the eukaryotic translation initiation factor 3 to inhibit the translation initiation of ifit1-bound rna species. in addition to these isgs, one highly upregulated gene in the initial stage of the antiviral immune response is isg15 (interferon-stimulated gene 15), which encodes an ubiquitinlike protein involved in a post-translational process termed isgylation [57] . this process allows isg15 to bind covalently to a range of target proteins, both viral and cellular [58] , by a process that is reversible due to the action of the ubiquitin-specific protease 18 (usp18), an event regulated by type i ifn [59] . isgylation appears to modulate the activity of multiple elements involved in the ifn response. for instance, isgylation has been shown to sustain stat1 or irf3 activity [60, 61] , downregulate the turnover of ubiquitinated proteins [62] , but also to negatively regulate rig-i signaling [63] . isg15 acts during viral replication by interfering with the endogenous proteins that the virus needs to replicate. thus, isg15 conjugates to the eukaryotic factor 4e (eif4e) homologous protein (4ehp) that binds to the capped mrna, inhibiting in this way the viral rna translation of those viruses that contains a capped positivesense rna such as flaviviruses [64] . isg15 also exists as an unconjugated protein that acts as a cytokine, regulating viral replication and host responses [65, 66] . the role of the isgs members mentioned above clearly illustrates the breadth and diversity in the function of this protein group. a database has been created, called interferome (https ://www.inter ferom e.org/inter ferom e/home. jspx), in which isgs are catalogued and incorporated into a database, based on the information obtained from all published reports where cells were treated with ifn, thus, this database will allow to identify isg signatures from highthroughput data, having implications for determining the role of isgs. viruses employ mechanisms that impair isg activity to enhance their evasion from the ifn system. the mechanisms will be discussed in another section of this review. as discussed previously, prr activation leads to the production of ifn-i, -iii, and pro-inflammatory cytokines such as il-1β. the present section will be centered on how viruses affect ifn-i induction. typically, virus genetic material triggers ifn-i induction when recognized by viral nucleic acid sensors that are membrane bound or present in the cytoplasm/nucleus. this recognition leads to activation of signaling cascades that converge towards the induction of ifn-i production in infected cells. viruses are known to interfere at every point of this process. viruses can interfere with the sensing of their genetic material, impair the signaling cascade that leads to ifn-i induction, and/or antagonize the activity of the transcription factors involved in ifn-i gene expression. the present section aims at providing a nonexhaustive overview of some of the most commonly used viral mechanisms to counter ifn-i induction in the host with a focus on recent findings in the field. viruses use different mechanisms to counteract the recognition of their genetic material by the host cell so that ifns are not induced (table 1) . viruses can sequester, modify or even degrade their nucleic acids to avoid detection by prrs. for instance, during replication most flaviviruses create vesicular structures in the er membrane which physically shield the viral genetic material from cytosolic rlrs [67] [68] [69] . influenza a virus (iav) uses the nucleus for replication, atypically for an rna virus, so that its genetic material remains hidden from cytosolic rlrs [70] . vaccinia virus (vv), a large double-stranded dna virus has the peculiarity of replicating in the cytoplasm, where dna sensors like cgas are present, which potentially renders the viral genetic material susceptible to recognition by prr. vv replication, however, occurs in organelles similar to micronuclei [71] that probably render viral dna inaccessible to recognition by cytoplasmic dna sensors. rotaviruses (rv) concentrate their replication in a cytoplasmic structure called viroplasms where dsrna genome is generated for packaging so that it is not exposed to the cytoplasmic prr [72] . many viruses also encode proteins that help conceal their genetic material from prr. some viruses possess dsrna binding proteins that could potentially sequester these pamps from ppr recognition, such as vp35 from ebola virus or σ3 from reovirus [73, 74] . the encapsidation of the viral rna can also impair rlr recognition. for instance, iav nucleoprotein and polymerase prevents rig-i binding to viral rna during transit through the cytoplasm [75] . iav ns1 protein also possesses a dsrna binding site that prevents recognition by rig-i [76] . calicivirus and picornavirus ssrna ( +) is covalently linked to a capping protein that could prevent recognition of the 5′ viral rna extremity by rig-i [77] . lassa virus (lasv) nucleoprotein (np) can act as a capping enzyme with exonuclease activity specific for dsrna, which has been shown to antagonize ifn induction [78, 79] . other viruses can modify their 5′tri-p motifs recognized by rig-i to evade this cytoplasmic rna sensor. hantaan virus, crimean-congo hemorrhagic fever virus and borna disease virus can process their 5′ genome extremity to form 5′mono-p forms, evading rig-i recognition [80] . poxvirus decapping enzymes d9 and d10 can prevent the accumulation of dsrna, an intermediate necessary in viral replication, and thereby evade rlr recognition [81] . measles virus (mev) encodes for the non-structural c protein that can impair ifn response by modulating viral rna replication [82] and improving the polymerase processivity [83] , thus probably limiting the amount of viral material recognizable by cytosolic prr. another mechanism employed by viruses to limit detection by prr is to interact with these sensors to impair their activation. the kaposi's sarcoma-associated virus (kshv) uses the tegument protein orf52 to bind to cgas and inhibit cgamp production, the second messenger used for sting (stimulator of interferon response cgamp interactor 1) activation [84] . homologues of orf52 in other gammaherpesviruses have also been described to act similarly, indicating that inhibition of this prr pathway is probably shared by gammaherpesvirus. the herpes simplex virus 1 (hsv-1) tegument protein vp22 has also been shown to inhibit cgas enzymatic activity, indicating that other herpesviridae can directly target cgas [85] . other viruses can sequester prr so that they are unable to relocate to their activity site. for instance, the protein z of new world arenaviruses binds to rig-i and prevents its association with the signaling platform mavs protein [86] . severe acute respiratory syndrome coronavirus (sars-cov) m protein has been shown to associate with rig-i and can potentially sequester this prr [87] . viruses can also promote prr degradation, thus reducing the number of cellular sensors capable of detecting viral infection (fig. 3 ). this can be done directly by proteases encoded by the viral genome. foot and mouth disease virus (fmdv) l pro and 3c pro protease can reduce rig-i intracellular protein levels [88] . the 3c pro protease of other picornaviruses has also been shown to degrade rig-i [89] , indicating that this is a shared mechanism of rlr evasion by this viral family. rig-i is not the sole prr that picornavirus proteases can target; the poliovirus and enterovirus 71 (ev71) 2a pro protease can also degrade mda5 [90] . viruses also encode for proteins that indirectly promote prr degradation. the nuclear sensor of dna ifi16 is degraded during hsv-1 infection through a mechanism dependent on the viral icp0 protein that is not fully understood but probably involves targeting the dna sensor for proteasomal degradation [91] . a e3 ligase activity on the nss protein of the phlebovirus toscana virus was recently identified that allowed the ubiquitination of rig-i card domains and the subsequent proteasomal degradation of the prr [92] . the ns2b protein of the flavivirus dengue virus (denv) can target the cytosolic dna sensor cgas for lysosomal degradation. although this [98] [99] [100] mechanism could at first glance seem counterproductive for an rna virus, it actually allows denv to evade the recognition of the mitochondrial dna that becomes exposed during infection [93] . other viruses alter the post-translational modifications essential to prr signaling so that the ppr remains inactive. some viral-encoded proteins achieve this directly, as in the case of the deubiquitinase activity of coronaviruses papainlike protease (plp) that removes the k63 ubiquitin tail from rig-i that is essential for rig-i translocation to the mavs protein platform [94] . this deubiquitination activity has been characterized in several coronaviruses thus suggesting that this mechanism is central to coronavirus evasion from the ifn system [95] [96] [97] . paramyxovirus v protein binds to mda5 and impairs its dephosphorylation by blocking the atp hydrolysis necessary for mda5 folding to its active state, thus impairing the adequate activation of this prr [98] . viruses can also impair prr activity by interfering with the functionality of accessory cellular components required for prr activation. mev has been shown to act on the phosphatase pp1 required for rlr activation using two distinct mechanisms. the mev v protein can bind pp1 which prevents mda5 dephosphorylation [99] . mev infection in dendritic cells also produces recognition of the viral particle through the c-lectin receptor dc-sign [100] . this triggers a signaling cascade that results in raf-1 kinase activation and the association of the pp1 inhibitor i-1 with pp1 that prevented rlr dephosphorylation thus impairing ifn induction. several viruses have also been shown to interact with the dsrna binding protein pact that potentiates rlr activation [101, 102] . for instance, ebola virus vp35 protein, middle east respiratory syndrome viral interference with accessory cellular components involved in prr activation. mev can interfere with rlr activation by targeting the phosphatase pp1 using 2 distinct mechanisms. mev v protein can interact with pp1 to prevent the dephosphorylation of mda-5 required for activation. mev can interact on the cell surface with the c-lectin receptor dc-sign which results in the association of pp1inhibitor 1 with pp1 thus preventing rlr dephosphorylation. ebola virus vp35 protein, mers-cov 4a protein and arenavirus np can interfere with pact binding to dsrna, a mechanism that potentiates rlr activation. rlr ubiquitination is also essential for adequate activation and transport to mavs for subsequent ifn signaling events to take place. riplet and trim25 are critical to rig-i ubiquitination. iav-ns1 and denv sfrna can interfere with trim25 activity, whereas hepatitis c ns3-4a protease can cleave riplet to impair rig-i ubiquitination. the mitochondrial-targeting chaperone 14-3-3ε is responsible for rig-i translocation to the mitochondrial membrane. denv ns3 protein targets 14-3-3ε using a phosphomimetic domain that displaces activated rig-i from this chaperone. wnv ns3 possesses a similar domain coronavirus (mers-cov) 4a protein and arenavirus nucleoproteins have been shown to interfere with pact binding to rlr [103] [104] [105] . as previously stated, rig-i activation is associated to its ubiquitination with k63 ub chains that liberates its autorepressed n-terminal card domains, a mechanism dependent on the activity of the cellular proteins trim25 and riplet [106, 107] . this mechanism can be targeted by viral products such as the iav ns1 protein that impairs trim25-mediated rig-i ubiquitination [108, 109] , or the hepatitis c virus ns3-4a protease that cleaves riplet [106] . intriguingly, not only protein products appear to interfere with the rig-i activation complex. the subgenomic flavivirus rna (sfrna) generated during denv infection can bind to trim25 and prevent the deubiquitination step required for trim25 activity to take place [110] . denv uses yet another mechanism to avoid prr detection. denv ns3 protein possesses a phosphomimetic domain that binds the mitochondrial-targeting chaperone protein 14-3-3ε [111] . 14-3-3ε is responsible for rig-i translocation to the mitochondrial membrane where the subsequent steps of the ifn induction signaling cascade take place. by binding 14-3-3ε, denv ns3 displaces the activated rig-i complex and prevents ifn induction. interestingly, a similar phosphomimetic domain is also present in west nile virus (wnv) ns3 protein [111] . viruses not only interfere with the prr capable of detecting their presence during infection, they also commonly affect the activity of the signaling complexes in charge of signal transduction. indeed, this is a strategy employed by most viruses to limit ifn responses. viruses can antagonize these signaling cascades at multiple levels and through varied mechanisms (table 2 ). this can be achieved by impairing the post-translational modifications required for signaling, i.e. by altering the phosphorylation or ubiquitinylation status of signaling intermediates. for instance, sars-cov and human coronavirus nl63 (hcov-nl63) plps have been shown to prevent sting dimerization and thus subsequent activation of the tbk1 pathway possibly through the plp deubiquitinase activity [112, 113] , as sting dimerization is dependent on the attachment of k63ub chains [114] . similarly, sars-cov plp can inhibit tlr7 signaling by removing k63-ub chains from traf3 and traf6 and thus blocking tbk1 activation [115] . another strategy to prevent post-translational modification of ifn-i pathway signaling components consists of sequestering these components so that they do not reach the adequate cellular location for activation. the ns3 protein from the economically important orbivirus bluetongue virus (btv) binds to the ubiquitinbinding protein optineurin in the golgi apparatus [116] . this prevents optineurin from recruiting ubiquitinated tbk1 at the golgi apparatus, a necessary step for subsequent tbk1 phosphorylation to occur [116] . recently, it has been described that cgamp, the second messenger generated by cgas and that activates sting, can be cleaved by poxvirusencoded nucleases (named poxins) [117] . this allows poxvirus blockade of the cgas-sting signaling axis. viral proteins can also prevent the adequate formation of signaling complexes by steric hindrance. for instance, human adenovirus type 5 e1a protein has been shown to bind to sting and thus antagonize ifn signaling [118] . mers-cov and sars-cov m proteins can interact with traf3 and thus disrupt the traf3-tbk1 association [87, 119, 120] . mers-cov orf4b protein can associate with the tbk1/ikkε complex to impair signaling [121] , indicating that coronaviruses encode for multiple proteins that affect ifn induction at different stages of the signaling cascade. kshv encodes for viral interferon regulatory factor 1 (virf1), a protein that binds to sting and block the association of tbk1 with sting, thus hindering the consequent irf3 phosphorylation [122] . hsv-1 encodes for the icp27 protein that can interact with the active sting-tbk1 complex and inhibit the subsequent tbk1-mediated phosphorylation of irf3 [123] . the nss protein from some phleboviruses has also been described to antagonize ifn induction by targeting tbk1 [124] . tbk1 activity is thus commonly targeted by both rna and dna viruses to antagonize ifn induction. viruses can also promote the degradation of signaling proteins involved in ifn induction. virally-encoded proteases can directly cleave some of the components of these pathways. sting can be directly cleaved by the ns2b3 protease from several flaviviruses such as denv, zika virus, wnv, or japanese encephalitis virus (jev), but not others like yellow fever virus [125] [126] [127] . indeed, this specific cleavage could partially explain some of the host range and pathogenicity of these flaviviruses in humans, as the identified sting cleavage site for the ns2b3 protease is only partially conserved among species [125] . similarly, the 3c pro from several picornoviridae has been described to cleave mavs proteins, thus inhibiting signal transduction [128, 129] . the 3c-like protease from the arterivirus porcine reproductive respiratory syndrome virus (prrsv) and the ns3/4a protease complex from the flavivirus hepatitis c virus (hcv) have also been described to cleave mavs protein [130, 131] . other viruses encode proteins that promote the degradation of signaling complexes involved in the ifn responses. rv nsp1 and vp3 proteins have been shown to target mavs protein for proteasome-dependent degradation and thus impair ifn induction [132, 133] . sars-cov orf9b protein localizes in the mitochondrial membrane where it interacts with mavs protein and promote its degradation [134] . this mechanism probably involves the recruitment of the mavs protein cellular regulator pcbp2 [135] to the mitochondrial membrane by sars-cov orf9b protein, which favors mavs protein ubiquitination through k48-ub chains and thus subsequent proteasomal degradation. since mavs protein represents an important signaling platform in the ifn induction cascade triggered by rlr activation, some viruses use strategies to alter mitochondria structure to impair mavs protein assembly. denv ns2b3 protease can cleave mitofusins, which alters mitochondria dynamics and impair their fusion [136] . in other cases, viruses can also promote mitophagy to promote their replication and potentially impair ifn responses. mitophagy has been described in mev, hepatitis b virus (hbv) or newcastle disease virus infections [137] [138] [139] . the protein m from the human parainfluenza virus 3 has also been shown to promote mitophagy and thus trigger mavs protein degradation to antagonize ifn induction [140] . recently, iav has also been shown to employ a similar mitophagic strategy to reduce mavs protein levels through the expression of its pb1-f2 protein [141] . viruses can also act on the transcription factors that bind to the ifn-i promoter and trigger the expression of isgs. viruses use multiple strategies to impair the binding of the transcription factors to the ifn-i promoters (table 3) . they can impair the phosphorylation by the tbk1/ikkε complex of irf3/7 that activates dimerization and transport to the nucleus. for instance, the paramyxovirus c protein inhibits irf7 phosphorylation and thus its activation [142] . the denv ns2b3 protease can impair irf3 phosphorylation by masking the ikkε kinase domain necessary for irf3 activation [143] . lymphocytic choriomeningitis virus (lcmv) nucleoprotein was also shown to bind to the ikkε kinase domain, thus preventing irf3 phosphorylation [144] indicating that arenavirus np-mediated inhibition of irf3 phosphorylation [145] uses a similar mechanism. viruses not only target irf3/7 activation to antagonize ifn induction, they can also interfere with the activation of nf-κb, as this transcription factor collaborates with irf3 in the early activation of the ifn-β promoter [146] . most arenavirus nucleoproteins not only block irf3 phosphorylation but they can also impair nf-κb activity [147] . viruses can also target adaptor molecules that regulate nf-κb activity. the nf-κb essential modulator (nemo) is part of the kinase complex that controls nf-κb release from its inhibitor iκb. the 3c pro from hepatitis a virus or fmdv and the 3c-like proteases from porcine epidemic diarrhea virus or prrsv cleave nemo thus preventing iκb release from nf-κb and consequently antagonizing ifn-β production [148] [149] [150] [151] . the degradation of the transcription factors involved in ifn induction is also the target of different viral proteins, as in the case of the nsp1 from rv that promote the degradation of several irfs including irf3 and 7 [152] . rv nsp1 appears to target irfs by interacting with their dimerization domain and thus preventing their association in active form [153] . nsp1 has also been described to impair nf-κb by promoting the degradation of β-transducing repeat-containing protein (β-trcp) [154] , the protein responsible for substrate recognition of the e3 ligase complex that targets for degradation iκb, the associated inhibitor of nf-κb. similarly, to other viral products, such as hiv-1 vpu, vv a49, or epstein-barr virus lmp-1, rv nsp1 associates with β-trcp through mimicry of the phosphorylated iκb that requires degradation [155] [156] [157] [158] . rv nsp1, however, presents the particularity of not only blocking the interaction of iκb with the e3 ligase complex but also of targeting β-trcp for degradation. viruses can also impair irf3/7 activity downstream of their activation by phosphorylation. the virf1 encoded by kshv can block irf3 activity downstream of irf3 activation by impairing the recruitment of the cbp-p300 coactivators to the irf3 complex [159] . this can also be achieved by blocking the transport of activated transcription factors to the nucleus. for instance, some paramyxoviruses use their v protein to impair irf3 translocation to the nucleus [160] . jev ns5 blocks irf3 and p65 subunit from nf-κb transport to the nucleus by interacting with nuclear transport proteins [161] . the nss from the emerging bunyavirus severe fever with thrombocytopenia syndrome virus have also been described to sequester the tbk1-ikkε-irf3 complex in viral inclusion bodies to prevent the trafficking of irf3 to the nucleus [162] . some viruses also code for proteins that interfere with ifn induction in the nucleus. in many instances, the exact mechanisms of ifn antagonism by viral products in the nucleus are not fully resolved. mers-cov orf4b protein impairs ifn induction in the nucleus by a mechanism yet to be elucidated [121] . btv encodes non-structural protein 4 (ns4) that localizes in cell nucleoli and possess ifn antagonist activity [163] . mev c protein can interfere with ifn-β promoter activation in the nucleus and this property has been linked to the virus pathogenicity [164] . the nss protein from the phlebovirus sandfly fever sicilian virus can prevent irf3 activity by interacting with the dna binding domain of irf3 [165] . the protein orf36 from the murine gammaherpes virus 68 was shown to bind to irf3 and to prevent its association with the co-transcriptional activator cbp, thus impairing irf3 binding to the ifn-β promoter [166] . viruses have developed strategies to antagonize ifn induction at multiple stages of the signaling cascade. this includes limiting the recognition of their genetic material, impairing the activation of prrs or their signaling partners, promoting the degradation of key components of the signaling cascade, sequestering signaling complexes away from their site of action, or impairing dna binding of the transcription factors involved in ifn induction. in viruses, multiple mechanisms have also often evolved to target several elements in these pathways, and hence augment their capacity to evade innate immunity. viruses can suppress the ifn signaling at different levels ( table 4 ). in this section, we will discuss some of the mechanisms that viruses use to counteract the action of this signaling cascade. one of the first steps in the signaling cascade is the phosphorylation of jak1 and tyk2, relevant for initiating the jak-stat signaling. by directly promoting the dephosphorylation of the jak/stat pathway, viruses counteract the ifn response. for instance, sendai virus (respirovirus) c protein inhibits the phosphorylation of receptor-associated kinases jak1 and tyk2 by binding to the ifn receptor subunit ifn-α/β [167] . the ns5 protein of the jev blocks the tyrosine phosphorylation of tyk2 and stat1 [168] . stat1 phosphorylation is targeted by several viruses, using different mechanisms. the paramyxovirus family, that includes mev, peste des petits ruminants virus (pprv) and mumps, express v and p proteins that interact directly with stat1, inhibiting phosphorylation [169, 170] . the immediate-early protein icp27 of hsv-1 downregulate stat1 phosphorylation and prevent the accumulation of stat1 in the nucleus [171] . the denv proteins ns2a, ns4a, ns4b block the phosphorylation of stat1 [172] . the ns5 protein of hcv interacts with stat1, interfering with its phosphorylation [173] . among the reoviridae family, the nsp1 protein encoded by rotavirus block the phosphorylation of stat1 [174] , and the ns3 protein encoded by btv blocks stat1 phosphorylation [175, 176] . many viruses target stat2 to antagonize ifn signaling. thus, mev v protein binds to stat2 [177, 178] . yellow fever virus ns5 protein also binds stat2 but this interaction requires stat2 activation by ifn [179] . other example is denv ns5 protein acts as a bridge between ubr4 and stat2, driving stat2 to degradation through the proteasome [180] . the nsp11 protein of pprs degrades stat2 via proteasome [181] . the proteasome is not the only catabolic cellular machinery that viruses can highjack to degrade signaling components of the ifn signaling pathway. btv was recently shown to use ubiquitination of its ns3 protein to drive stat2 degradation by an autophagy-dependent mechanism [175] . another strategy used by viruses includes the interference with ifn signal transduction by modification of the constitutive or basal levels of molecules involved in the jak/ stat pathway that viruses members of the rubulavirus genus like simian virus 5, mumps virus or human parainfluenza virus type 2, use [182, 183] . the human papillomavirus-16 (hpv-16) expresses the viral e7 protein that binds the p48 protein blocking its translocation to the nucleus, impeding the association of irf-9 with the stat-1/stat-2 heterodimer (isgf3), and thereby inhibiting the induction of ifn-i inducible genes [184] . viruses involved in persistent infections, such as cytomegaloviruses (cmv), polyomaviruses, hcv [185] , or hsv-1 [186] use similar strategies. cmv affects the expression levels of jak1 and irf-9 [187] , and the viral large t antigen of murine polyomavirus binds to jak1 inactivating the transduction signal through ifn receptors [188] . viruses employ mechanisms that impair isg activity to enhance their evasion from the ifn system. in this section, we will discuss some of these mechanisms. as previously mentioned, some isg products enhance the recognition of viral pamps and provide the cell with effector mechanisms that block viral replication. thus, fmdv l pro cleaves g3bp1, an rna-binding protein essential to stress granules (sg) assembly, to impair the formation of these structures [189] . mev c protein has been involved in sg inhibition through blockade of pkr-induced sg by the activity of the adenosine deaminase acting on rna 1 (adar1) [190] . denv sfrna can bind to various components involved in sg assembly. this property of denv sfrna also led to impaired isg mrna translation, thus dampening ifn responses [191] . to overcome recognition by isg products, some viral mechanisms are discussed. the reovirus σ3 protein was shown to inhibit pkr activity probably through its ability to bind dsrna [192, 193] . nss protein from bunyavirus can promote pkr degradation [194] . poxvirus d9 and d10 decapping enzyme promote dsrna degradation, thus preventing pkr recognition [81] . viruses can also highjack regulatory isg pathways to evade isg product action. for instance, adar1 is an isg involved in rlr regulation. adar1 has an important physiological function as it edits adenosines to inosines in rna, a feature that destabilize the structure of complementary dsrna strands, thus preventing rlr or pkr recognition. this is an important mechanism in the prevention of autoimmunity, as it limits the recognition of cellular dsrna. viruses such as mev, vsv or hiv-1 have been shown to use adar1 function to block pkr activation and thus evade translation shutdown [195] [196] [197] . viruses can also interfere with the oas-rnase l pathway. rotavirus vp3 protein blocks rnasel activation [198] by cleaving the 2′,5′-oligoadenylates produced by oas. the ns2 protein from coronavirus murine hepatitis virus has been shown to act similarly [199] . the ifit is another isg that contribute to viral rna recognition. some viruses have developed 2′-o methyltransferase activities on their gene products to prevent translation blockade of their rna by ifit. this has been described for wnv, coronaviruses, rv and poxvirus [200] [201] [202] [203] [204] . the ifn-induced transmembrane proteins (ifitm) expression is greatly enhanced upon ifn activation, but these proteins are also expressed ubiquitously in the absence of ifn. the family of ifitm proteins has been shown to block iav, wnv and denv cell entry, a mechanism that probably involves viral hemagglutinin recognition [205] . hcov-oc43 has been shown to highjack ifitm2 and ifitm3 for cell entry. this mechanism could be important for virus entry in lower respiratory tract under inflammatory conditions induced by ifn [206] . in hiv-1, vpu and env proteins can mutate to increase infectivity and overcome ifitm1-mediated restriction of replication [207] . mutations to overcome the activity of the isg product mxgtpase have also been described for iav, indicating that multiple isg products probably exert a selective pressure on viruses. for instance, pandemic avian iav strains appear to adapt to human through evasion of the np recognition by mxa gtpase [208] . viruses have evolved strategies to dampen isg15 effects on ifn signaling. for instance, the plp from hcov-nl63, sars-cov and mers-cov have been shown to not only act as a deubiquitinase but also as a deconjugating protease for isg15 chains [94, 96, 97] . fmdv lpro has also been associated with cleavage of isg15, but instead of targeting the isopeptide bond used in isg15 conjugation, it hydrolyzes the peptide bond preceding the isgylation motif [209] . human cytomegalovirus (hcmv) tegument pul26 protein prevents isgylation [210] . pul26 activity appears to be supported by two other tegument proteins pul25 and pp65 [211] . hcmv also uses another tegument protein pul50 to affect isgylation by targeting ube1l, an important ligase responsible for isg15 linkage, for proteasomal degradation [212] . since isgylation also regulates the activation of ifnrelated pathways, some viruses have harnessed isgylation to favor their replication. hcv has also been described to use isgylation to favor its replication and develop persistent infections [213, 214] . recently, isgylation was also associated with increased replication in hbv infections [215] . multifaceted strategies have evolved in viruses to circumvent isg product activity and thus enhance their replication and spreading. these range from directly impairing the activity of isg products involved in host cellular defense to highjack the ifn modulating activity of some isg products. understanding these mechanisms of evasion will undoubtedly shed light on some of the pathogenic processes induced by viral infections. a unique strategy to inhibit the activity of ifn was described in 1995 with the identification of a poxviral secreted ifn type i binding protein (ifn-i bp) encoded by the b18r gene of vaccinia virus (vv) [216] a well characterized member of the orthopoxvirus genus that contains the strains used for the efficacious worldwide vaccination campaigns against smallpox. the protein was found to be a secreted glycoprotein of about 60 kda expressed early during infection. while its sequence is unrelated to either of the two subunits of the cellular ifn-i receptor, ifnar1 or ifnar2, the ifn-i bp was found to bind with high affinity (k d = 175 pm for hifnα2) to several subtypes of human ifn-is, inhibiting their binding to the receptor and thus abrogating their biological activity [217] . in stark contrast to the high species specificity observed for the cellular receptor, the viral protein is able to bind and inhibit the activity of ifn from different species, including mouse, rat, bovine and rabbit ligands, suggesting different interaction modes. currently, all human ifn-i molecules tested including 8 (out of 13) ifnα, ifnβ, ifnϖ as well as the more divergent ifnκ and ifnε are known to be bound and inhibited by b18 [218] [219] [220] , although with varying affinities. interestingly, murine ifnα, but not murine ifnβ are inhibited by the poxviral ifn-i bp in spite of being bound with high affinity, as assessed by surface plasmon resonance [219, 221] . competition studies using anti ifn monoclonal antibodies (mabs) showed that the binding interface of ifns with b18 is larger than the one with their cellular receptor, which could probably account for its broad species specificity and inhibitory capacity over a wide range of affinities [222] . further examinations substantiated an additional property of the poxviral ifn-i bp which is its saturable binding to the cell surface after secretion, where the protein is active and can inhibit ifn as efficiently as the secreted one [221] . this suggested that the main site of action is the cell surface, providing local tissue protection by protecting neighboring, still uninfected cells from entering into an ifn mediated antiviral state. examination of a truncated version of b18 expressed by the attenuated wyeth vaccv strain lacking its third, c-terminal immunoglobulin (ig) domain showed that cell binding capacity is mediated by the n-terminal regions of the protein [221] . additional transfection analyses with different constructs suggested that cell binding activity is mediated by ig domain 1, while ifn blocking activity requires ig domains 2 and 3 [223] site directed mutagenesis assays identified stretches of basic residues at the n terminus of b18 to mediate high affinity binding to cell surface sulfated glycosaminoglycans, preferentially heparan sulfate [224] and showed that mutants lacking gag binding activity could still bind and inhibit ifn efficiently. the ifn-i bp protein has been found to be conserved in other orthopoxviruses including cowpox virus and ectromelia virus (ectv), a natural mouse pathogen, as well as the two viruses causing significant disease in humans, monkeypox virus and variola virus, the causative agent of smallpox [219] . interestingly, the human viruses show an enhanced affinity for the human ligands, possibly reflecting the host adaptation of the virus, as occurs with other secreted cytokine binding receptors in this family. while possible orthologues can be readily found in virus species from several other poxvirus genera, these are frequently more distantly related, and their properties have not been extensively studied. the single exception to this is protein y136 of the tanapoxvirus yaba-like disease virus (yldv), a primate virus causing infection restricted to the skin. this protein, which shares only 27% aminoacid identity to the vacv b18, can bind and inhibit both human (and monkey) ifn-i as well as the more recently described family of type iii ifns [218] . the latter are a specialized group of ifns mediating antiviral response specifically at mucosal sites without compromising barrier integrity of the epithelia and promoting long-lasting humoral and cellular responses which signal through a distinct, specific heterodimeric cellular receptor (reviewed by [225] ). the authors have proposed that inhibition of these ifns might be related to the specific tissue tropism of yldv, although information on its role in vivo has not yet been provided. insights into the biological role of poxviral ifn-i bps comes from murine infection models using vaccv and ectv, the latter naturally causing fatal mousepox in susceptible mouse strains. early reports showed that deletion of ifn-i bp gene from vacv attenuated the virus in vivo both in intranasal [216] as well as intracranial [217] infection models. in ectv, absence of ifn-i bp resulted in a completely attenuated phenotype upon footpad inoculation (ld 50 reduction at least 10 7 -fold) with severely impaired dissemination of virus to its secondary replication sites, liver and spleen, as well as enhanced nk cell recruitment and both cd4 + and cd8 + t cell activation [226] crucially, these effects were shown to be dependent on ifnar signaling by the use of knockout mice. the ifn-i bp was found to bind to uninfected cells around infection foci in the liver and spleen protecting these tissues locally from ifn induced antiviral activity [227] . the biological relevance of tissue retention of this inhibitory protein was shown using recombinant ectv that express a mutated ifn-i bp unable to bind to the cell surface but still able to inhibit ifn-i efficiently. infection with these recombinants resulted in non-lethal infection as in the case of the virus lacking ifn-i bp altogether [228] . interestingly, it was found that immunization of mice with recombinant ifn-i bp could prevent the development of mousepox upon challenge [226] , probably through the development of antibodies capable of impairing its interaction with its ligands [227] and also pointing to a novel therapeutic target for the treatment of poxviral infections in humans. the structure of the complex of ectv ifn-i bp with murine ifnα-5 has been solved to high resolution (pdb entry 3oq3, deposited by fremont and lee, results to be published). comparisons with the ternary complexes of different ifn-i with their cellular receptor [229, 230] will be crucial to disentangle the structure function relationships in the interaction and inhibition of the biological activities of ifn-i ligands by the poxviral ifn-i bps. the particular properties of the poxviral ifn-i bp, especially its broad species and type specificity as well its high affinity have been instrumental to its use as a biotechnological tool. thus, b18 has been used to determine the implication of ifn-i in diverse processes, such as the monocyte-derived macrophage-mediated inhibition of human cytomegalovirus (hcmv) spread [231] . in addition, recombinant oncolytic herpes simplex viruses expressing b18 have been developed to improve their infectivity in the face of antiviral responses [232] . finally, b18 has been used to block ifnα mediated hiv associated encephalitis in a murine model [233] or to inhibit the detrimental ifn mediated effects produced by mrna exposure in induced pluripotent stem (ips) cell reprogramming [234] . secreted ifn-i bps have been exclusively found in poxviruses to date. a recent report described the murine norovirus ns1 protein, which is secreted by an unconventional caspase-3 mediated pathway, to be essential for tuft cell infection in gastrointestinal tissue through blockade of ifn type iii signaling [235] . while a direct inhibition of ifn type iii could not be demonstrated in the reporter assay used, the molecular mechanism employed by this protein remains unsolved and raises the question as to whether additional and different soluble ifn-i or ifn-iii bps might be identified in other virus species. ifn responses are a complex and important component of the innate immune system. this is reflected in the vastness and complexity of isgs roles, not only involved in antiviral responses but also in several immunomodulatory functions. viruses can disrupt ifn responses leading to the antiviral state to promote their successful replication. indeed, viruses often interfere with multiple pathways involved in the ifn response to evade innate immunity. the importance of the ifn system in host antiviral responses is highlighted by the fact that viruses dedicate some of their genetic material to encode for ifn antagonists. the viral mechanisms of ifn evasion can be mediated directly by viral gene products. viruses also often usurp components of the cellular machinery to carry out their ifn antagonistic activity. there is no doubt that understanding how viruses evade the ifn system will shed some light on the pathogenicity and allow for a better design of therapeutic approaches. the interaction of these pathogens with the ifn system can also shed some light on some of the regulatory cellular mechanisms that control the ifn response. studying the interaction of viral components with the ifn system remains essential to understand the pathogenesis of emergent viruses that threatened global health. innate immune sensing and signaling of cytosolic nucleic acids identification of the zebrafish ifn receptor: implications for the origin of the vertebrate ifn system how cells respond to interferons virus interference. i. the interferon cut, copy, move, delete: the study of human interferon genes reveal multiple mechanisms underlying their evolution in amniotes the human interferon alpha species and receptors type i interferon differential therapy for erythroleukemia: specificity of stat activation receptor density is key to the alpha2/beta interferon differential activities the dual nature of type i and type ii interferons functions of natural killer cells ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex il-28, il-29 and their class ii cytokine receptor il-28r pathogen recognition and innate immunity tlr-mediated innate immune recognition toll like receptors and viruses differential roles of mda5 and rig-i helicases in the recognition of rna viruses inflammasome recognition of influenza virus is essential for adaptive immune responses irf-1 induced cell growth inhibition and interferon induction requires the activity of the protein kinase pkr deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the pkr gene: role of irf-1 and nf-kappab physical association between stat1 and the interferon-inducible protein kinase pkr and implications for interferon and double-stranded rna signaling pathways ifits: emerging roles as key anti-viral proteins sequestration by ifit1 impairs translation of 2‫׳‬o-unmethylated capped rna structural basis for viral 5'-ppp-rna recognition by human ifit proteins isg15, a small molecule with huge implications: regulation of mitochondrial homeostasis interferon-stimulated gene 15 and the protein isgylation system high-throughput immunoblotting ubiquitiinlike protein isg15 modifies key regulators of signal transduction positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification acetaldehyde disrupts interferon alpha signaling in hepatitis c virus-infected liver cells by up-regulating usp18 identification and characterization of a novel isg15-ubiquitin mixed chain and its role in regulating protein homeostasis negative feedback regulation of rig-i-mediated antiviral signaling by interferon-induced isg15 conjugation isg15 modification of the eif4e cognate 4ehp enhances cap structure-binding activity of 4ehp immunoregulatory properties of isg15, an interferon-induced cytokine isg15 in antiviral immunity and beyond the dengue virus conceals double-stranded rna in the intracellular membrane to escape from an interferon response ultrastructure of kunjin virus-infected cells: colocalization of ns1 and ns3 with double-stranded rna, and of ns2b with ns3, in virus-induced membrane structures tick-borne encephalitis virus delays interferon induction and hides its double-stranded rna in intracellular membrane vesicles influenza a virus cell entry, replication, virion assembly and movement vaccinia virus dna replication occurs in endoplasmic reticulumenclosed cytoplasmic mini-nuclei regulated, stable expression and nuclear presence of reovirus double-stranded rna-binding protein sigma3 in hela cells relevance of ebola virus vp35 homo-dimerization on the type i interferon cascade inhibition influenza virus adaptation pb2-627k modulates nucleocapsid inhibition by the pathogen sensor rig-i a recombinant influenza a virus expressing an rna-binding-defective ns1 protein induces high levels of beta interferon and is attenuated in mice the genome-linked protein vpg of vertebrate viruses-a multifaceted protein cap binding and immune evasion revealed by lassa nucleoprotein structure structure of the lassa virus nucleoprotein reveals a dsrna-specific3‫׳‬ to5‫׳‬ e‫ٴٴ‬xonuclease activity essential for immune suppression processing of genome ‫׳5‬ termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction poxvirus decapping enzymes enhance virulence by preventing the accumulation of dsrna and the induction of innate antiviral responses measles virus circumvents the host interferon response by different actions of the c and v proteins the c protein is recruited to measles virus ribonucleocapsids by the phosphoprotein inhibition of cgas dna sensing by a herpesvirus virion protein herpes simplex virus 1 tegument protein vp22 abrogates cgas/ sting-mediated antiviral innate immunity z proteins of new world arenaviruses bind rig-i and interfere with type i interferon induction severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex foot-andmouth disease virus viroporin 2b antagonizes rig-i-mediated antiviral effects by inhibition of its protein expression rig-i is cleaved during picornavirus infection enterovirus 2apro targets mda5 and mavs in infected cells nuclear ifi16 induction of irf-3 signaling during herpesviral infection and degradation of ifi16 by the viral icp0 protein toscana virus non-structural protein nss acts as e3 ubiquitin ligase promoting rig-i degradation dengue virus ns2b protein targets cgas for degradation and prevents mitochondrial dna sensing during infection deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases structure-guided mutagenesis alters deubiquitinating activity and attenuates pathogenesis of a murine coronavirus structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease mers-cov papain-like protease has deisgylating and deubiquitinating activities a shared interface mediates paramyxovirus interference with antiviral rna helicases mda5 and lgp2 antagonism of the phosphatase pp1 by the measles virus v protein is required for innate immune escape of mda5 measles virus suppresses rig-i-like receptor activation in dendritic cells via dc-sign-mediated inhibition of pp1 phosphatases the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response pact facilitates rna-induced activation of mda5 by promoting mda5 oligomerization middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response mutual antagonism between the ebola virus vp35 protein and the rig-i activator pact determines infection outcome arenaviral nucleoproteins suppress pact-induced augmentation of rig-i function to inhibit type i interferon production a distinct role of riplet-mediated k63-linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i molecular mechanism of influenza a ns1-mediated trim25 recognition and inhibition dengue subgenomic rna binds trim25 to inhibit interferon expression for epidemiological fitness a phosphomimetic-based mechanism of dengue virus to antagonize innate immunity sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling ubiquitination of sting at lysine 224 controls irf3 activation sars coronavirus papain-like protease inhibits the tlr7 signaling pathway through removing lys63-linked polyubiquitination of traf3 and traf6 the golgi apparatus acts as a platform for tbk1 activation after viral rna sensing viral and metazoan poxins are cgamp-specific nucleases that restrict cgas-sting signalling dna tumor virus oncogenes antagonize the cgas-sting dna-sensing pathway middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets modulation of the cgas-sting dna sensing pathway by gammaherpesviruses hsv-1 icp27 targets the tbk1-activated sting signalsome to inhibit virusinduced type i ifn expression heartland virus nss protein disrupts host defenses by blocking the tbk1 kinase-irf3 transcription factor interaction and signaling required for interferon induction species-specific disruption of sting-dependent antiviral cellular defenses by the zika virus ns2b3 protease denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus targets the adaptor protein mita to subvert host innate immunity seneca valley virus suppresses host type i interferon production by targeting adaptor proteins mavs, trif, and tank for cleavage the coxsackievirus b 3c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling porcine reproductive and respiratory syndrome virus 3c protease cleaves the mitochondrial antiviral signalling complex to antagonize ifn-beta expression hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity mavs protein is attenuated by rotavirus nonstructural protein 1 rotavirus vp3 targets mavs for degradation to inhibit type iii interferon expression in intestinal epithelial cells sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/traf3/traf6 signalosome pcbp2 mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip4 dengue virus impairs mitochondrial fusion by cleaving mitofusins mitophagy switches cell death from apoptosis to necrosis in nsclc cells treated with oncolytic measles virus mitophagy promotes replication of oncolytic newcastle disease virus by blocking intrinsic apoptosis in lung cancer cells hepatitis b virus disrupts mitochondrial dynamics: induces fissi on and mitophagy to attenuate apoptosis the matrix protein of human parainfluenza virus type 3 induces mitophagy that suppresses interferon responses influenza a virus protein pb1-f2 impairs innate immunity by inducing mitophagy an anti-interferon activity shared by paramyxovirus c proteins: inhibition of toll-like receptor 7/9-dependent alpha interferon induction dengue virus subverts the interferon induction pathway via ns2b/3 protease-ikappab kinase epsilon interaction arenavirus nucleoprotein targets interferon regulatory factor-activating kinase ikkepsilon inhibition of the type i interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus convergence of the nf-kappab and irf pathways in the regulation of the innate antiviral response arenavirus nucleoproteins prevent activation of nuclear factor kappa b hepatitis a virus 3c protease cleaves nemo to impair induction of beta interferon footand-mouth disease virus 3c protease cleaves nemo to impair innate immune signaling porcine epidemic diarrhea virus 3c-like protease regulates its interferon antagonism by cleaving nemo arterivirus nsp4 antagonizes interferon beta production by proteolytically cleaving nemo at multiple sites diversity of interferon antagonist activities mediated by nsp1 proteins of different rotavirus strains rotavirus nsp1 mediates degradation of interferon regulatory factors through targeting of the dimerization domain rotavirus nsp1 inhibits nfkappab activation by inducing proteasome-dependent degradation of beta-trcp: a novel mechanism of ifn antagonism putative e3 ubiquitin ligase of human rotavirus inhibits nf-kappab activation by using molecular mimicry to target beta-trcp the human immunodeficiency virus type 1 vpu protein inhibits nf-kappa b activation by interfering with beta trcp-mediated degradation of ikappa b interaction of epstein-barr virus latent membrane protein 1 with scfhos/beta-trcp e3 ubiquitin ligase regulates extent of nf-kappab activation poxvirus targeting of e3 ligase beta-trcp by molecular mimicry: a mechanism to inhibit nf-kappab activation and promote immune evasion and virulence hhv-8 encoded virf-1 represses the interferon antiviral response by blocking irf-3 recruitment of the cbp/p300 coactivators inhibition of interferon regulatory factor 3 activation by paramyxovirus v protein japanese encephalitis virus ns5 inhibits type i interferon (ifn) production by blocking the nuclear translocation of ifn regulatory factor 3 and nf-kappab evasion of antiviral immunity through sequestering of tbk1/ikkepsilon/irf3 into viral inclusion bodies bluetongue virus ns4 protein is an interferon antagonist and a determinant of virus virulence measles virus c protein interferes with beta interferon transcription in the nucleus nss protein of sandfly fever sicilian phlebovirus counteracts interferon (ifn) induction by masking the dnabinding domain of ifn regulatory factor 3 conserved herpesviral kinase promotes viral persistence by inhibiting the irf-3-mediated type i interferon response respirovirus c protein inhibits activation of type i interferon receptor-associated kinases to block jak-stat signaling blocking of interferon-induced jak-stat signaling by japanese encephalitis virus ns5 through a protein tyrosine phosphatasemediated mechanism paramyxovirus v proteins interact with the rig-i/ trim25 regulatory complex and inhibit rig-i signaling the nucleoprotein and phosphoprotein of peste des petits ruminants virus inhibit interferons signaling by blocking the jak-stat pathway role for herpes simplex virus 1 icp27 in the inhibition of type i interferon signaling inhibition of alpha/beta interferon signaling by the ns4b protein of flaviviruses hepatitis c virus ns5a disrupts stat1 phosphorylation and suppresses type i interferon signaling rotavirus nsp1 protein inhibits interferon-mediated stat1 activation virus-induced autophagic degradation of stat2 as a mechanism for interferon signaling blockade induction and control of the type i interferon pathway by bluetongue virus the measles virus v protein binding site to stat2 overlaps with that of irf9 stat2 is a primary target for measles virus v protein-mediated alpha/ beta interferon signaling inhibition the interferon signaling antagonist function of yellow fever virus ns5 protein is activated by type i interferon dengue virus co-opts ubr4 to degrade stat2 and antagonize type i interferon signaling nonstructural protein 11 of porcine reproductive and respiratory syndrome virus induces stat2 degradation to inhibit interferon signaling the v protein of simian virus 5 inhibits interferon signalling by targeting stat1 for proteasome-mediated degradation association of mumps virus v protein with rack1 results in dissociation of stat-1 from the alpha interferon receptor complex the human papillomavirus type 16 e7 protein binds human interferon regulatory factor-9 via a novel pest domain required for transformation expression of hepatitis c virus proteins interferes with the antiviral action of interferon independently of pkr-mediated control of protein synthesis herpes simplex virus 1 gene products occlude the interferon signaling pathway at multiple sites human cytomegalovirus inhibits ifn-alpha-stimulated antiviral and immunoregulatory responses by blocking multiple levels of ifn-alpha signal transduction the polyoma virus t antigen interferes with interferon-inducible gene expression foot-and-mouth disease virus leader protease cleaves g3bp1 and g3bp2 and inhibits stress granule formation stress granule formation induced by measles virus is protein kinase pkr dependent and impaired by rna adenosine deaminase adar1 g3bp1, g3bp2 and caprin1 are required for translation of interferon stimulated mrnas and are targeted by a dengue virus non-coding rna inhibitory activity for the interferoninduced protein kinase is associated with the reovirus serotype 1 sigma 3 protein site-directed mutagenic analysis of reovirus sigma 3 protein binding to dsrna rift valley fever virus nss protein functions and the similarity to other bunyavirus nss proteins rna-specific adenosine deaminase adar1 suppresses measles virus-induced apoptosis and activation of protein kinase pkr double-stranded rna deaminase adar1 increases host susceptibility to virus infection adar1 interacts with pkr during human immunodeficiency virus infection of lymphocytes and contributes to viral replication silencing the alarms: innate immune antagonism by rotavirus nsp1 and vp3 homologous 2',5'-phosphodiesterases from disparate rna viruses antagonize antiviral innate immunity rotavirus open cores catalyze 5′-capping and methylation of exogenous rna: evidence that vp3 is a methyltransferase 2′-o methylation of the viral mrna cap evades host restriction by ifit family members attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2'-o-methyltransferase activity biochemical and structural insights into the mechanisms of sars coronavirus rna ribose 2'-o-methylation by nsp16/nsp10 protein complex middle east respiratory syndrome coronavirus nonstructural protein 16 is necessary for interferon resistance and viral pathogenesis the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus interferon induction of ifitm proteins promotes infection by human coronavirus oc43 hiv-1 mutates to evade ifitm1 restriction in vivo evasion of mxa by avian influenza viruses requires human signature in the viral nucleoprotein irreversible inactivation of isg15 by a viral leader protease enables alternative infection detection strategies consecutive inhibition of isg15 expression and isgylation by cytomegalovirus regulators the abundant tegument protein pul25 of human cytomegalovirus prevents proteasomal degradation of pul26 and supports its suppression of isgylation transmembrane protein pul50 of human cytomegalovirus inhibits isgylation by downregulating ube1l isg15, a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment the interferon stimulated gene 15 functions as a proviral factor for the hepatitis c virus and as a regulator of the ifn response interferon-stimulated gene 15 conjugation stimulates hepatitis b virus production independent of type i interferon signaling pathway in vitro vaccinia virus encodes a soluble type i interferon receptor of novel structure and broad species specificity vaccinia virus b18r gene encodes a type i interferon-binding protein that blocks interferon alpha transmembrane signaling inhibition of type i and type iii interferons by a secreted glycoprotein from yaba-like disease virus the highly virulent variola and monkeypox viruses express secreted inhibitors of type i interferon human interferon-and interferon-kappa exhibit low potency and low affinity for cell-surface ifnar and the poxvirus antagonist b18r the vaccinia virus soluble alpha/beta interferon (ifn) receptor binds to the cell surface and protects cells from the antiviral effects of ifn analysis of an interaction between the soluble vaccinia virus-coded type i interferon (ifn)-receptor and human ifn-alpha1 and ifn-alpha2 evaluating the orthopoxvirus type i interferon-binding molecule as a vaccine target in the vaccinia virus intranasal murine challenge model glycosaminoglycans mediate retention of the poxvirus type i interferon binding protein at the cell surface to locally block interferon antiviral responses type iii interferons: balancing tissue tolerance and resistance to pathogen invasion the orthopoxvirus type i ifn binding protein is essential for virulence and an effective target for vaccination antibody inhibition of a viral type 1 interferon decoy receptor cures a viral disease by restoring interferon signaling in the liver a virus-encoded type i interferon decoy receptor enables evasion of host immunity through cell-surface binding structural linkage between ligand discrimination and receptor activation by type i interferons structural basis of a unique interferon-beta signaling axis mediated via the receptor ifnar1 human monocyte-derived macrophages inhibit hcmv spread independent of classical antiviral cytokines incorporation of the b18r gene of vaccinia virus into an oncolytic herpes simplex virus improves antitumor activity the recombinant vaccinia virus gene product, b18r, neutralizes interferon alpha and alleviates histopathological complications in an hiv encephalitis mouse model highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mrna a secreted viral nonstructural protein determines intestinal norovirus pathogenesis acknowledgements this work was supported by grant rti2018-094616-b-100 from the spanish ministerio de ciencia e innovación; grant s2018/baa-4370-platesa2 from the comunidad de madrid (fondo europeo de desarrollo regional, feder) and vetbionet infraia-731014 from the euopean union h2020. key: cord-310847-63gh2tg4 authors: uversky, vladimir n title: the alphabet of intrinsic disorder: ii. various roles of glutamic acid in ordered and intrinsically disordered proteins date: 2013-04-01 journal: intrinsically disord proteins doi: 10.4161/idp.24684 sha: doc_id: 310847 cord_uid: 63gh2tg4 the ability of a protein to fold into unique functional state or to stay intrinsically disordered is encoded in its amino acid sequence. both ordered and intrinsically disordered proteins (idps) are natural polypeptides that use the same arsenal of 20 proteinogenic amino acid residues as their major building blocks. the exceptional structural plasticity of idps, their capability to exist as heterogeneous structural ensembles and their wide array of important disorder-based biological functions that complements functional repertoire of ordered proteins are all rooted within the peculiar differential usage of these building blocks by ordered proteins and idps. in fact, some residues (so-called disorder-promoting residues) are noticeably more common in idps than in sequences of ordered proteins, which, in their turn, are enriched in several order-promoting residues. furthermore, residues can be arranged according to their “disorder promoting potencies,” which are evaluated based on the relative abundances of various amino acids in ordered and disordered proteins. this review continues a series of publications on the roles of different amino acids in defining the phenomenon of protein intrinsic disorder and concerns glutamic acid, which is the second most disorder-promoting residue. intrinsically disordered proteins (idps) and intrinsically disordered protein regions (idprs) are new exciting members of the protein kingdom. 1, 2 they are highly abundant in nature, [3] [4] [5] [6] [7] possess numerous intriguing properties, 8 are intimately involved in various cellular processes [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] and are commonly found to be related to the pathogenesis of various diseases. 13, [24] [25] [26] [27] [28] [29] the common theme of protein disorder-based functionality is recognition, and idps/idprs are frequently involved in complex protein-protein, protein-nucleic acid and protein-small molecule interactions. some of these interactions can induce a disorder-order transition in the entire idp or in its part. 5, [9] [10] [11] [12] 15, 23, [30] [31] [32] [33] [34] [35] [36] furthermore, intrinsic disorder opens a unique capability for one protein to be involved in interaction with several unrelated binding partners and to gain different bound structures. 22, 37 some idps can form highly stable complexes; others are involved in signaling interactions where they undergo constant "bound-unbound" transitions, thus acting as dynamic and sensitive "on-off" switches. these proteins typically return to their intrinsically disordered state after the completion of a particular function. many of the idps/idprs can gain different conformations depending on the environmental peculiarities. 30, 37 all this constitutes an important arsenal of the unique physiological properties of idps/idprs that determines their ability to exert different functions in different cellular contests according to a specific conformational state. 8 the folding-at-binding principle is believed to help idps or idprs to obtain maximal specificity in a protein-protein interaction without very high affinity. 20 this combination of high specificity with low affinity defines the broad utilization of intrinsic disorder in regulatory interactions where turning a signal off is as important as turning it on. 10 although some partial folding during the idp/ idpr-based interactions is a widespread phenomenon, with significant fraction (~1/3) of the interacting residues in idps/idprs adopting α-helix, β-strand and irregular structures, 31, 32 there are still many other idps/idprs that are involved in the formation of "fuzzy complexes," where an idp/idpr keeps a certain amount of disorder in its bound conformation. 35, [38] [39] [40] often, the interacting regions in idps are observed as loosely structured fragments in their unbound forms. these disorderbased binding sites are known as molecular recognition elements or features (mores or morfs), 30, 31 preformed structural elements 41 or pre-structured motifs (presmos). 42 although the existence of such loosely structured regions suggests that idps can adopt their bound structure(s) at a free-energy cost that is not too high, it is important to remember that increasing the stability of the bound conformation does not necessarily enhance the binding affinity. 23 another important feature of the disorder-based interactions is their increased speed due to the greater capture radius and the ability to spatially search through interaction space (the so-called "fly-casting" mechanism) 43 and to the fact that fewer encounter events are required for the binding because of lack of orientational restrains. 44 linking all these form at phs greater than its pk a 4.6 (and thus glu is negatively charged at the physiological ph ranging from 7. 35-7.45) . therefore, glutamic acid is one of two acidic amino acids found in proteins that play important roles as general acids in enzyme active centers, as well as in maintaining the solubility and ionic character of proteins. in fact, glutamic acid residue has a nonpolar surface of 69 å 2 , and the estimated hydrophobic effect associated with the burial of this residue is 1.74 kcal/mol. 52 in ordered proteins, glutamic acids are predominantly located on protein surface so that they have access to the solvent. in fact, 93% of glutamic acids in known structures of folded proteins are classified as exposed since they have solvent exposed areas of > 30 å 2 , and only 4% of glutamic acids in folded proteins possess solvent exposed areas of < 10 å 2 and therefore are buried. 53 the carboxylate anions and salts of glutamic acid are known as glutamates. glutamic acid is one of the most common natural amino acids and the most abundant amino acid in the diet. besides being an important component of proteins and polypeptides (see below), being a substrate for the production of the krebscycle-related α-ketoglutarate intermediate, glutamine and proline, and being the precursor for the synthesis of the inhibitory γ-aminobutyric acid (gaba) in gaba-ergic neurons, glutamate is the principal excitatory neurotransmitter within the vertebrate nervous system. 54 in fact, glutamate is known to act on several different types of receptors and has excitatory effects at ionotropic receptors [such as n-methyl-d-aspartate (nmda), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (ampa), and kainite, which all incorporate ion channels that are permeable to cations] and modulatory effects at metabotropic receptors [which are g protein-coupled glutamate receptors (mglur) that modify neuronal and glial excitability through g protein subunits acting on membrane ion channels and second messengers such as diacylglycerol and camp]. 54 at chemical synapses of the glutamatergic neurons, glutamate is stored in vesicles and is released from the pre-synaptic cell by nerve impulses. in the opposing post-synaptic cell, binding of glutamate lead to activation of specific glutamate receptors such as nmda or ampa. glutamate plays an important role in synaptic plasticity in the brain and is involved in various cognitive functions, such as learning and memory. 55 in fact, long-term potentiation (one of the plasticity forms) takes place at glutamatergic synapses in the neocortex, hippocampus and other parts of the brain. 55 another important role of glutamate is its ability to generate volume transmission, where extrasynaptic signaling is created via the summation of glutamate released from a neighboring synapse. 56 in addition to glutamate receptors, neuronal and glial membranes contain glutamate transporters that are responsible for rapid remove of glutamate from extracellular space. 57 under stress conditions (such as brain injury or disease), glutamate transporters work in reverse leading to the accumulation of considerations with the recent report showing that idp affinities are tuned mostly by association rates 45 suggests that the degree of pre-adoption of binding conformations in idps has to be limited, but not unfavorable. all the functional and structural peculiarities of idps/idprs are encoded in their amino acid sequences. it was recognized long ago that there are significant differences between ordered proteins/domains and idps/idprs at the level of their amino acid sequences. 5, 10, 46 in fact, in comparison with ordered proteins, idps/idprs are characterized by noticeable biases in their amino acid compositions, 5, 8, 10, [46] [47] [48] containing less of so-called "order-promoting" residues (cysteine, tryptophan, isoleucine, tyrosine, phenylalanine, leucine, histidine, valine, asparagines and methionine, which are mostly hydrophobic residues which are commonly found within the hydrophobic cores of foldable proteins) and more of "disorder-promoting" residues (lysine, glutamine, serine, glutamic acid and proline, which are mostly polar and charged residues, which are typically located at the surface of foldable proteins) (fig. 1a) . glutamic acid is second of the most common disorder-promoting residues. figure 1b and table 1 represent the result of a statistical analysis of the amino acid compositions of proteins in four standard data sets (disprot, 49 uniprot, 51 pdb select 25 50 and surface residues 48 ) and shows that the glutamic acid content in these data sets is 9.89 ± 0.61%, 6.67 ± 0.04%, 6.65 ± 0.07% and 8.70 ± 0.17%, respectively (cprofiler.org/help. html). 48 in other words, idps/idprs contain 1.48-and 1.49times more glutamic acid residues than the average natural proteins from uniprot or ordered proteins from pdb, respectively. furthermore, the glutamic acid content in idps/idprs is 1.14times higher than that on the surfaces of ordered proteins. this article continues a series of publications on the intrinsic disorder alphabet dedicated to the exploration of the amino acid determinants of protein intrinsic disorder. i overview below some functions of glutamic acid in idps/idprs (as well as in ordered proteins and domains) and show that there is a variety of glutamic acid-specific functions in disordered proteins and regions. chemical structure of glutamic acid. glutamic acid (glutamate, glu, e, see fig. 2a ) is one of the 20 proteinogenic amino acids encoded by the standard genetic code and its codons are gaa and gag. glutamic acid is a dibasic nonessential amino acid that has a molecular mass of 147.13 da (molecular mass of glu residue is 129.12 da), surface of 190 å 2 , volume of 138.4 å 3 , pk a of side chain of 4.6 and pi 3.08 at 25°c. intriguingly, free glutamic acid is not very soluble, possessing solubility of 0.864 g/100 g at 25°c, which is significantly lower than the solubility of free prolines (162.3 g/100 g at 25°c), and the solubility of the vast majority of free amino acids (www.fli-leibniz.de/ image_aa.html). the side chain of glutamic acid contains two methylene group and the carboxylic acid functional group (see fig. 2a ) that exists in a negatively charged deprotonated carboxylate with stroke, autism, amyotrophic lateral sclerosis, lathyrism, some forms of mental retardation and alzheimer's disease. 58 the decreased glutamate release is associated with phenylketonuria leading to the developmental disruption of glutamate receptor expression. 59, 60 the excess glutamate in the extracellular space and promoting entrance of calcium to the cell via the nmda receptor channels. this process is known as excitotoxicity, and it results in neuronal damage and eventual cell death. the excitotoxicity might occur as part of the ischemic cascade that is associated figure 1 . amino acid determinants defining structural and functional differences between the ordered and intrinsically disordered proteins. (a) fractional difference in the amino acid composition (compositional profile) between the typical idps from the disprot database 49 and a set of completely ordered proteins 50 calculated for each amino acid residue. the fractional difference was evaluated as (c disprot − c pdb )/c pdb , where c disprot is the content of a given amino acid in a disprot databse, and c pdb is the corresponding content in the data set of fully ordered proteins. positive bars correspond to residues found more abundantly in idps, whereas negative bars show residues, in which idps are depleted. amino acid types were ranked according to their decreasing disorder-promoting potential. 47 (b) amino acid compositions of several data sets discussed in the text (disprot, 49 uniprot, 51 pdb select 25 50 and surface residues 48 ). linkages, or ion pairs. an electrostatic interaction is a non-covalent bond that is based on the attraction of two oppositely charged groups. it can easily be broken and reformed and is characterized by the optimal distance of 2.8 å between the interacting groups. the strength of these interactions depends on the distance of the two charges and the properties of the medium between them. in proteins, electrostatic interactions typically occur between coo − in the side chain of glutamic and aspartic acids and nh 3 + in the side chains of lysines and arginines. hydrogen bond (h-bond) is another non-covalent bond. this interaction depends on the sharing of one hydrogen atom (h-atom) between two other atoms, where the h-atom has a covalent bond to one of them (which therefore serves as the h-bond donor), and where the other atom, to which the h-atom has a weaker bond, serves as the acceptor, a. hydrogen bond is weaker than a covalent bond but stronger than a van der waals bond. similar to electrostatic interactions, h-bonds can easily be broken and reformed. among established geometrical criteria for h-bond are a set of optimal distances between the non-h atom of donor and acceptor (dono-acceptor < 3.9 å) and between the h atom of donor and acceptor (h-acceptor < 2.5 å). 65 being negatively charged at physiological ph, glutamic acid can serve as a hydrogen bond acceptor, whereas at acidic ph, it also can be a hydrogen bond donor. glutamic acid in the ramachandran plot. the structure of a protein can be described using torsion angles-φ and ψ-of its backbone that provides a simple view of the conformation of a protein. in sequence order, φ is the n i-1 -c i -cα i -n i torsion angle, and ψ is the c i -cα i -n i -c i+1 torsion angle. since most combinations of φ and ψ are sterically forbidden, the 2d plot of the torsion angles of the protein backbone, known as the ramachandran plot, 61 provides a simple view of the conformation of a protein, since the φ-ψ angles cluster into distinct regions in the ramachandran plot, where each region corresponds to a particular secondary structure. in the generic ramachandran plot (see fig. 2b ) that refers to the 18 non-glycine and non-proline amino acids, there are four distinct regions of density (the α (right-handed α-helix region), α l (mirror image of α), β s (region largely involved in β-sheet formation) and β p (region associated with extended polyproline-like helices but also observed in β-sheet). the shape of the generic ramachandran plot is determined mainly by the presence of specific steric clashes 61 and backbone dipole-dipole interactions. [62] [63] [64] glutamic acid in electrostatic interactions and hydrogen bonds. glutamic acid participates in electrostatic interactions, which are also known as ionic bonds, or salt bridges, or salt gcn4 leucine zipper dimer revealed that the free energy of helix stabilization associated with the hydrogen-bonding and hydrophobic interactions in this capping structure is −1.2 kcal/ mol, illustrating that helix capping might play a significant role in protein folding. 72 based on the analysis of 431 α-helices the normalized frequencies for finding particular residues at the c cap position, the average fraction of buried surface area and the hydrogen bonding patterns of the c cap residue side-chain were calculated. 74 this analysis revealed that the residue found in the c cap position is on average 70% buried and that there is a noticeable correlation between the relative burial of this residue and its hydrophobicity. 74 furthermore, c cap residues with polar sidechains were shown to be involved in hydrogen bonding, where the longer side-chains of glutamic acid, glutamin, arginine, lysine and histidine form hydrogen bonds with residues located more than four residues apart, whereas the shorter side-chains glutamic acid and protein secondary structure. although protein secondary structure is determined by hydrogen bonds between donor and acceptor groups in the protein backbone, different amino acids are known to favor the formation of different secondary structure elements, such as α-helices, β-pleated sheets or loops. the α-helix-formers include alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine, whereas valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine favor β-structure formation, and serine, glycine, uncharged aspartic acid, asparagine and proline are found most often in β-turns. it was pointed out that there is no apparent relationship between the chemical nature of the amino acid side chain and its secondary structure preferences. for example, although glutamic and aspartic acids are closely related chemically, glutamic acid is more likely to be found in helices and aspartic acid is predominantly located in β-turns. in fact, the helical propensity of glutamic acid is 0.40, whereas aspartic acid has an helical propensity of 0.69, the third largest value after proline and glycine. 66 note that the helical propensity is defined as the difference in free energy δ(δg) estimated in kcal/mol per residue in an α-helical configuration relative to alanine, which has been set to zero because it is usually the amino acid with the most favorable helix propensity. 66 here, the higher helical propensity values correspond to more positive free energies and therefore are related to residues which are less favored in α-helix. glutamic acid in α-helix caps. since α-helices in peptides and proteins have an overall dipole moments caused by the cumulative effects of all the individual dipoles from the carbonyl groups of the peptide bond pointing along the helix axis, the overall helical structure is destabilized due to the noticeable entropic effects. the effect of this helical dipole moment can be approximated by placing 0.5-0.7 positive unit charge near the n-terminus and 0.5-0.7 negative unit charge near the c-terminus of the helix. 67, 68 one of the nature's strategies to neutralize this helix dipole is the specific capping of the n-terminal ends of α-helices by negatively charged residues, such as glutamic acids. 67, 68 furthermore, careful analysis of α-helices revealed that their first and last four residues differ from the remaining residues by being unable to make intrα-helical hydrogen bonds. instead, these first four (> n-h) groups and last four (> c = o) groups in an α-helix are often capped by alternative hydrogen bond partners. [69] [70] [71] physico-chemical and statistical analysis suggested that certain residues are more preferable at the c-and n-termini of an α-helix (the helical c-and n-caps). 70 for example, based on the analysis of series of mutations in the two n-caps of barnase, it was concluded that a single n-cap can stabilize the protein by up to ~2.5 kcal/mol. 70 importantly, the presence of a negative charge of the n-cap was shown to add ~1.6 kcal/mol of stabilization energy mostly due to the compensation effects for the macroscopic electrostatic dipole of the helix. 70 from a global survey among proteins of known structure, seven distinct capping motifs are identified-three at the helix n-terminus and four at the c-terminus. 71 one of these motifs is the helix-capping motif ser-x-x-glu, a sequence that occurs frequently at the n-termini of α-helices in proteins. [71] [72] [73] thermodynamic analysis of this ser-x-x-glu motif from the ramachandran plots for backbone conformations of the 18 non-glycine and non-proline amino acids. marked regions of density correspond to the right-handed α-helix region (α), mirror image of α (α l ), region largely involved in β-sheet formation (β s ), and region associated with extended polyproline-like helices, but also observed in β-sheet (β p ). by the four glutamic acid residues located at homologous positions within each of the four pore-forming segments and which form a single or multiple ca 2+ -binding site(s) that entrap calcium ions, thus giving them a possibility to be electrostatically repulsed through the intracellular opening of the pore. 87 in the bacterial kcsa and inwardly rectifying k + (kir) channels, glutamic acid is also involved in the action of the selectivity filter. 88 here, the network of residues stabilizing the pore of kcsa involves a glu71-asp80 carboxyl-carboxylate interaction behind the selectivity filter, whereas the structure of the pore in kir channels is stabilized by a glu-arg salt bridge. 88 therefore, although glu is quite conserved among both types of channels, the network of interactions is not translatable from one channel to the other. this clearly shows that different potassium channels are characterized by diverse gating patterns. 88 the presence of a highly conserved glutamic acid residue in the middle of a transmembrane domain is a characteristic feature of a family of transmembrane glycoproteins with two immunoglobulin-like domains, such as basigin (bsg, also known as cd147 or emmprin), embigin and neuroplastin. 89 finally, a critical glutamic acid residue was recently identified in clc proteins, which constitute a large structurally defined family of cl − ion channels and h + /cl − antiporters which are found in prokaryotes and eukaryotes, 90 and which perform their functions in the plasma membrane or in various intracellular organelles such as vesicles of the endosomal/lysosomal pathway or in synaptic vesicles. 91 mutations in human clc channels are known to cause a set of very diverse diseases such as myotonia (muscle stiffness), bartter syndrome (renal salt loss) with or without deafness, dent's disease (proteinuria and kidney stones), osteopetrosis and neurodegeneration, and possibly epilepsy. 91 the side chain of the aforementioned critical glutamic acid occupies a third cl − ion binding site in the closed state of the channel and moves away to allow cl − binding. 90 glutamic acid valve. glutamic acid is known to play a unique role in regulation of the cytochrome-c oxidase (cco) activity. cco is the last enzyme of the respiratory electron transport chain in mitochondria (or bacteria) located in the inner mitochondrial (or bacterial) membrane, and it is responsible for reducing ~90% of the oxygen taken up in aerobic life. this protein powers the production of atp by generating an electrochemical proton gradient across the membrane via the catalysis of the oxygen reduction to water that takes place in the binuclear center (bnc) of the enzyme. cco uses four electrons taken up from the cytochrome c located at the positively charged p-side (outside) of the membrane and four "chemical" protons taken from the negatively charged n-side (inside) to reduce the dioxygen to two water molecules. in addition to this oxygen reduction reaction, four "pump" protons are translocated from the n-side to the p-side across the membrane against the opposing membrane potential, doubling the total amount of charge separated by the enzyme. [92] [93] [94] [95] therefore, the main role of cco is to serve as a proton pump and a generator of the electrochemical proton gradient or charge separation across the membrane, which is achieved via two separate processes. first, the reduction of oxygen to water by electrons and protons taken up from opposite sides of the membrane leads of aspartic acid, asparagine, serine and threonine form hydrogen bonds with residues located close in sequence. 74 finally, based on the analysis of α-helical propensity of a series of dodecapeptides containing alanine, asparagine, aspartate, glutamine, glutamate and serine at the n-terminus and arginine, lysine and alanine at the c-terminus, it was concluded that the α-helix-stabilizing abilities of these residues can be ranged as follows: aspartate > asparagine > serine > glutamate > glutamine > alanine at the n-terminus and arginine > lysine > alanine at the c-terminus. 75 glutamic acid and protein solubility. based on the analysis of solubility-changing substitutions in proteins it has been pointed out that together with two other hydrophilic residues (aspartic acid and serine) glutamic acid contributes significantly more favorably to protein solubility than other hydrophilic residues (asparagine, glutamine, threonine, lysine and arginine). 76 based on this observation, an important strategy for solubility enhancement was proposed, were the hydrophilic residues that do not contribute favorably to protein solubility can be replaced with the hydrophilic residues that contribute more favorably. 76 glutamic acids inside the pores of ion channels. being negatively charged at physiological ph, glutamic acid is perfectly suited for binding metal ions. this property is used in specific regulation of a variety of ion channels. for example, in cyclic nucleotide-gated (cng) channels (which are found in vertebrate photoreceptors and olfactory epithelium, 77 elsewhere in the nervous system [78] [79] [80] and in a variety of other cell types including kidney, testis and heart, 81 and whose activation represents the final step in the transduction pathways in both vision and olfaction [82] [83] [84] ), a single glutamic acid strategically located in the pore represents the binding site for multiple monovalent cations, the blocking site for external divalent cations and the site for the effect of protons on permeation. 82 this is not too surprising since the pore region of the channel controls both the singlechannel conductance and the pore diameter of the channel. 85 importantly, cng channels are permeable to ca 2+ , which is an important element in the activation of intracellular targets, and which in addition to permeating cng channels can profoundly block the current flow carried by monovalent cations through the cng channels. 83 this capability of ca 2+ to block the monovalent cation flow is determined by the high-affinity binding of ca 2+ to a single acidic amino acid residue located in the pore of the channel, which is glu363 for the rod cng channel and glu333 for the catfish olfactory cng channel. 86 this same glutamic acid residue is also responsible for the external rapid proton block of cng channels, another characteristic that the cng channels share with ca 2+ channels. 86 glutamic acid also plays an important regulatory role in the voltage-dependent calcium channels that are located in the plasma membrane and form a highly selective conduit by which ca 2+ ions enter all excitable cells and some nonexcitable cells. 87 for these channels to operate, ca 2+ ions must enter selectively through the pore, bypassing competition with other extracellular ions. the high selectivity of a unique ca 2+ filter is determined pathway for protons utilized in the catalytic no reduction; the carboxylate group of glu215, which is located at the backside of glu211, contributes to the electro-negative environment of the binuclear center of cnor, and to the low redox potential of heme b 3 iron; finally glu135 and glu138 are positioned in the loop connecting the transmembrane helices iii and iv, with glu135 serving as one of the ca 2+ ligands (which is crucial for maintaining the configuration of heme b and b 3 ) and assisting in the water-mediated proton transfer through interactions with a number of water molecules, and with glu138 serving as a key residue for maintaining the unique conformation of the long loop through interactions with the residues in transmembrane helix ii, which would stabilize the coordination of glu135 to ca 2+ . 100 mono-adp-ribosyltransferase, which is responsible for the mono-adp-ribosylation of proteins, possesses a critical glutamic acid at the catalytic cleft which functions to position nad for nucleophilic attack at the n-glycosidic linkage for either adpribose transfer or nad hydrolysis. 101 the pronounced na + /k + selectivity of na,k-atpase relies on the strategic positioning of glutamic acid residues. 102 here, intramembrane glu327 in transmembrane segment m4, glu779 in m5, asp804 and asp808 in m6 are essential for tight binding of k + and na + , whereas asn324 and glu327 in m4, together with thr774, asn776 and glu779 in the 771-ytltsnipeitp motif of m5 contribute to the na + / k + selectivity. 102 in the family of thiamin diphosphate enzymes, a highly conserved glutamate is known to promote the c 2 -h ionization and the thiamin diphosphate activation. 103 the direct catalytic role of glutamic acid can be seen in matrix metalloproteinases, which are ubiquitous endopeptidases characterized by an active site where a zn 2+ atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid that acts as a general base. 104 for example, one of the wellknown zinc-binding metalloproteases that uses a glutamic acid residue as the fourth ligand to coordinate the zinc ion is thermolysin. in thermolysin, glutamic acid is 20 amino acids downstream from the second histidine in the first motif and present in a small conserved motif (nexxsd). 105 in the zincin and pdf groups of metalloproteases, the catalytic zinc-binding site contains the hexxhxxg motif. 105 also, a glutamic acid residue may be catalytically active in the substrate-binding cleft of plant lysozymes. 106 each enzyme in the α-amylase family of multidomain hydrolases and transferases has one glutamic acid and two aspartic acid residues necessary for activity. 107 the irreversible dealkylation reaction catalyzed by the o 6 -alkylguanine-dna alkyltransferase (agt) that directly repairs alkylation damage at the o 6 -position of guanine is accomplished by an active-site cysteine that participates in a hydrogen bond network with invariant histidine and glutamic acid residues, reminiscent of the serine protease catalytic triad. 108 the spore germination protease (gpr) that degrades small, acid soluble proteins (sasp) protecting spore's dna against damage, is a structurally and functionally unique protease that utilizes glutamic acid residue to catalyze sasp degradation. 109 in the hydrolytic aldehyde dehydrogenases (aldhs), catalytic but flexible glutamic acid residues located within the active site serve as the general base that activates the hydrolytic water molecule in the deacylation step. 110 to the net translocation of one electrical charge across the membrane per electron consumed. second, an additional proton is translocated vectorially across the membrane for each electron consumed, resulting in a net transport of two electrical charges per electron. 96 the protons for the chemical reaction are extracted from the n-side of the membrane via two proton pathways, the d-and k-channels. the d-channel starts at a highly conserved residue, asp 91 (bovine numbering; subunit i) near the n side, and continues to another highly conserved residue glu242 that donates protons to the bnc, whereas the key residue in the k-channel is a highly conserved lysine (k319). 95 the d-channel is responsible for the delivery of four "pump" protons that are first transferred from glu242 to a "loading" site above the bnc and then delivered to the p side via a proton-exit channel. the mystery of this mechanism is in the ability of glu242 located at the end of the d-channel to somehow sort "pump" protons from "chemical" protons. 95 to explain this behavior, the glutamate valve model has been proposed according to which the side chain of glu242 shuttles between a state protonically connected to the d channel, and a state connected to the bnc and the pump site. 97 in this proton valve model, the glu242 motion depends on its protonation state, where the unprotonated residue remains predominantly in a "down" conformation, pointing toward the n side, and therefore facilitating the uptake of a proton, whereas protonation shifts the glu242 to the "up" conformation, where the side chain of this important residue is swung toward the p side by ~4 å. 97 glutamic acid in the active sites of enzymes. in addition to serve multiple structural roles and being involved in regulation of various channels, glutamic acid residues, being positioned within or in the close proximity to the active sites, might have roles in the catalytic activities of various enzymes. one of the illustrative examples of the functional roles of glutamic acid can be found in bacterial nitric oxide reductase (nor), which is a membraneintegrated enzyme that catalyzes the reduction of nitric oxide no to nitrous oxide n 2 o using a type of anaerobic respiration where cytotoxic no is immediately decomposed after its production from nitrite no 2 − via the nitrite reductase-catalyzed reaction. [98] [99] [100] three different nor types are found in bacteria, with the cytochrome c dependent nor (cnor) that consists of two subunits, norb and norc, being the most extensively studied enzyme. precise description of the complex catalytic mechanism of this important enzyme is outside the scopes of this review, and therefore only a small piece of the entire picture, where the roles of glutamic acid are emphasized, is briefly described below. the characteristic feature of cnors is the presence of five conserved glutamic acid residues (glu135, glu138, glu211, glu215 and glu280 in p. aeruginosa cnor) within the norb subunit consisting of 12 trans-membrane helices and containing the heme b and the binuclear center (heme b 3 /fe b ) buried in the hydrophobic interior of its trans-membrane region. 100 here, glu211 is involved in the coordination of fe b and its carboxylate functions as the shuttle for catalytic protons from glu280 to the bound-no; glu280, which interacts with glu211 but is not involved in direct interaction with fe b , is an important player of the thr330-ser277-glu280-glu211 network that acts as a delivery matrix communication) and their ligands, it has been concluded that divalent cations are critical for integrin interactions with almost all ligands. importantly, although divalent cations are bound to integrins, their coordination sphere is not completed and the interactions between integrin and its ligands typically involve completing the metal ion coordination with an acidic ligand residue. 115 for example, complexes between the human intercellular adhesion molecule-1 (icam-1) and the i domain of its integrin receptor αlβ2 are stabilized by a critical glutamate residue that completes the magnesium coordination in integrin. 116 similarly, in the crystal structure of a complex between the i domain of a2b1 integrin and a triple-helical collagen peptide containing a critical gfoger motif, glutamate residue from the collagen peptide completes the coordination sphere of the i domain metal ion. 117 based on these observations it has been concluded that a metalglutamate handshake represents a basic mechanism of integrin i domain interaction with its binding partners. 115 furthermore, it is believed now that the general mechanism by which integrins, these αβ-heterodimeric cell-surface receptors that are vital to the survival and function of nucleated cells, recognize their structurally diverse ligands relies on specific glutamic-acid-or aspartic-acid-based sequence motifs that function in a divalent cation-dependent and conformationally sensitive manner. 118 the levels of intracellular zinc in living cells are crucial for managing various cellular processes, such as growth, development and differentiation. zinc is involved in protein, nucleic acid, carbohydrate and lipid metabolism and also plays a role in the control of gene transcription and the coordination of other biological processes controlled by proteins containing dna-binding zinc finger motifs, ring fingers and lim domains. 119 the physiologically relevant intracellular levels of zinc are controlled by specific zinc transporters which mostly transport zinc into cells from outside. 105 members of one of the subfamilies of these transporters, liv-1 subfamily of zip zinc transporters (lzt), being similar to other zip transporters in secondary structure and ability to transport metal ions across the plasma membrane or intracellular membranes, possess a unique hexphexgd motif containing conserved proline and glutamic acid residues, that fits the consensus sequence for the catalytic zinc-biding site of matrix metalloproteinases (hexxhxxgxxh), and which is unprecedented in other zinc transporters. 105 in addition to this set of specific examples, one should keep in mind that all structures of the ca 2+ -binding domains have in common a high negative surface potential usually associated with asp or glu residues. 120 therefore, important glutamic acid residues responsible for calcium coordination can be found in various members of the major ca 2+ -binding proteins, such as ef-hand domains, egf-like domains, γ-carboxyl glutamic acid (gla)-rich domains, cadherin domains, ca 2+ -dependent (c)-type lectin-like domains and ca 2+ -binding pockets of family c g-protein-coupled receptors. 120 a particularly intriguing role was described for the n-terminal glutamic acid residues in the canonical ca 2+ -protein, α-lactabumin, 121 which is frequently used as a model protein in folding studies and in studies on the effect of calcium binding on protein structure, stability and folding. for example, in nudix hydrolases (which is a family of mg 2+ -requiring enzymes that catalyze the hydrolysis of nucleoside diphosphates linked to other moieties) there is a specific motif, nudix box (gx 5 ex 7 reuxeexgu, where u is a bulky hydrophobic residue), that forms a loop-α helix-loop structural motif that functions as a common mg 2+ -binding and catalytic site. 111 it was emphasized that the overall catalytic powers of nudix hydrolases consists in accelerating the reaction rate by 10 9 to 10 12 times. the reactions are accelerated 10 3 -10 5 -times by general base catalysis by a glutamate residue within, or beyond the nudix box, or by a histidine beyond the nudix box. the additional 10 3 -10 5 -fold rate acceleration is due to the lewis acid catalysis provided by one, two, or three divalent cations. one divalent cation is coordinated by two or three conserved residues of the nudix box, the initial glycine and one or two glutamate residues, together with a remote glutamate or glutamine ligand located outside the nudix box. 111 glutamic acids at various binding sites. hemopexin is an important multifunctional plasma protein involved in the sequestering of heme released into the plasma from hemoglobin and myoglobin as the result of intravascular or extravascular hemolysis and due to skeletal muscle trauma or neuromuscular disease. it also possesses hyaluronidase activity, serine protease activity, pro-inflammatory and anti-inflammatory activity and is involved in the suppression of lymphocyte necrosis, inhibition of cellular adhesion, and binding of divalent metal ions. finally, hemopexin possesses two highly exposed arg-gly-glu sequences that may promote interaction with cell surfaces. 112 glutamic acid plays an important role in defining the retinal binding site geometry of rhodopsin, which is the photoreceptor in vertebrate rod cells responsible for vision at low light intensities. 11-cis-retinal is the photoreactive chromophore located in the interior of the protein where it is covalently attached to a lysine side chain through a protonated schiff base (psb) linkage. 113 based on the 13 c-nmr chemical shift data, it was concluded that glu113 of rhodopsin is involved in charge interactions with the retinal psb, which are crucial for maintaining rhodopsin in the inactive state in the dark and whose breaking leads to the protein activation. 113 a centrally located glutamic acid residue in position 6 of transmembrane segment vii of the main ligand-binding crevice of the chemokine 7tm receptors (gluvii:06) is crucial for recognition and binding of small molecule non-peptide ligands that contain one or two centrally located, positively charged nitrogen atoms and are characterized by relatively similar elongated overall structure with terminal aromatic moieties. 114 furthermore, since this gluvii:06 is crucial for the binding and hence the function of a number of non-peptide ligands in several chemokine receptors, such as the ccr1, ccr2 and ccr5 receptors, it serves as a selective anchor point for the centrally located, positively charged nitrogen of the small molecule ligands. 114 glutamic acid and metal binding. the role of glutamic acid residues in coordination of various metal ions was already emphasized in sections discussing ion channels. a few other illustrative examples are listed below. based on the analysis of the complexes formed between integrins (which are central molecules in the adhesion processes that mediate cell-cell and cell-extracellular group giving rise to the pyrrolidone carboxylic acid (pyro-glu). however, it was emphasized that pyro-glu is exclusively found at the n-terminal end of the thermal polymers when glutamic acid is a predominant amino acid in a mixture of amino acids subjected to thermal polymerization. 129 another important glutamic acid-based ptm is gammacarboxylation catalyzed by the vitamin k-dependent carboxylase that transforms specific glutamate residues in proteins to gammacarboxy glutamic acid (gla) in the presence of reduced vitamin k, molecular oxygen and carbon dioxide. 130 this modification is widely distributed in the animal kingdom and has a wide range of physiological implications, such as hemostasis, bone calcification and signal transduction. 130 in addition to be a target for various ptms, glutamic acid itself can be used as an important protein modifier, giving raise to polyglutamylation, which is a specific ptm where polyglutamate chains of variable lengths are added to the modified protein. 131 polyglutamylation is evolutionarily conserved and is commonly found in the microtubule (mt) building block, tubulin. this ptm, being primarily found within the tubulin c-terminal tail that participates in binding of many structural and motor mt-associated proteins, is believed to be crucial for the functional adaptation of mts. polyglutamylation is catalyzed by a family of specific enzymes and in addition to tubulin can be found in some other proteins. 131 high content of charged residues is one of the tricks used by nature to make stable proteins in thermophilic and hyperthermophilic organisms. 132 in fact, based on the correspondence analysis of the 56 completely sequenced genomes available from the three domains of life (seven eukaryotes, 14 archaeal and 35 bacterial species) it has been concluded and the amino acid composition permits discrimination between the three known lifestyles (mesophily, thermophily or hyperthermophily). 132 the most specific amino acid compositional biases that represent specific signatures of thermophilic and hyperthermophilic proteomes are a relative abundance in glutamic acid, concomitantly with a depletion in glutamine and a significant correlation between the relative abundance in glutamic acid (negative charge) and the increase in the lumped "pool" lysine + arginine (positive charges). being absent in mesophiles, these correlations could represent a physico-chemical basis of protein thermostability. curiously, the distribution of the remaining charged amino acid, i.e., aspartic acid, appears to be quite homogeneous throughout all the species suggesting that this residue does not participate significantly in the aforementioned compensatory negative/positive (charged) correlation in thermophiles and hyperthermophiles. 132 on average, thermophilic and hyperthermophilic proteomes were shown to contain 1.9%, 7.8%, 4.8% and 12.6% of glutamine, glutamic acid, aspartic acid and lysine + arginine residues, respectively. importantly, some of these numbers are rather different from those found in idps/idprs, as shown in table 1 . α-lactabumin was shown to possess significantly different thermal and structural stability in its calcium-bound and calciumfree apo-forms, 122 with the apo-protein possessing molten globule-like properties at slightly elevated temperatures. 123, 124 this strong dependence of the α-lactabumin structural properties on metal-binding is determined by the simple fact that in the apo-form, many acidic side chains have unfavorable chargecharge interactions, with 11 residues (glu1, glu7, glu11, asp63, asp64, asp78, asp82, asp83, asp84, asp87 and asp88) possessing significantly unfavorable charge-charge repultion. 125 although calcium binding has the most pronounced effect on residues directly involved in cation coordination (asp82, asp87 and asp88) and strongly affects the other two residues in the ca 2+ -binding loop, asp83 and asp84, ca 2+ binding has relatively minor effects on residues more distant from the ca 2+ -binding site (glu1, glu7, glu11, asp63 and asp64), which mostly preserve unfavorable electrostatic interactions seen in the apo-form. 125 it was also shown that the mutation-induced neutralization of unfavorable charge-charge interactions in the n-terminus (residues 1-11 of which are characterized by a high proportion of negatively charged residues that cluster on the surface of the native protein) results in stabilization of both the apo-and ca 2+bound protein. 125 unexpectedly, the δglu1 mutant, where the glu1 residue was removed, leaving an n-terminal methionine in its place, possessed almost one order of magnitude higher affinity for calcium and higher thermostability (both in the absence and presence of calcium) than the native protein isolated from milk. 121 this unique tuning of the α-lactabumin structure and calcium binding suggested that the n-terminal region of this protein might have a direct effect on the calcium-binding loop (and perhaps other regions of the structure). 121 the side chains of glutamic acid residues are subjected to several ptms. some cytoplasmic and nuclear proteins are known to be methylated, i.e., enzymatically modified by the addition of methyl groups from s-adenosylmethionine. methylation reactions typically occur on carboxyl groups (such as the side chain of glutamic acid) and modulate the activity of the target protein. glutamate methyl ester formation plays a major role in chemotactic signal transduction in prokaryotes. for example, methyl-accepting chemotaxis proteins are a family of chemotactic-signal transducers that respond to changes in the concentration of attractants and repellents in the environment, transduce a signal from the outside to the inside of the cell, and facilitate sensory adaptation through the variation of the level of methylation. 126, 127 in some proteins and peptides, glutamic acids can be amidated. also, some glutamine residues in proteins undergo spontaneous (nonenzymatic) deamidation to glutamate with rates that depend upon the sequence and higher-order structure of the protein. functional groups within the protein can catalyze this reaction, acting as general acids, bases, or stabilizers of the transition state. 128 in rare cases, glutamate residues can be modified by cyclization via condensation of the α-amino group with the side-chain carboxyl neutral ph was shown to be accompanied by the instantaneous formation of a gel-like precipitate with intermolecular antiparallel β-structure. 139 in bacteria, pga may be composed of only d-, only l-or both d-and l-glutamate enantiomers, and pga filaments may be poly-γ-l-glutamate filaments (plga), pdga filaments or poly-γ-l-d-glutamate (pldga) filaments. 135 the production and maintenance of sufficient d-glutamate pool levels required for the normal bacterial growth is controlled by the glutamate racemase, which is a member of the cofactor-independent, twothiol-based family of amino acid racemases. 140 this enzyme is conserved and essential for growth across the bacterial kingdom and has a conserved overall topology and active site architecture. therefore, it represents an attractive target for the development of specific inhibitors that could act as possible therapeutic agents. 140 in gram-negative bacteria, the complex responsible for the polyglutamate synthesis is encoded in specific loci. if the pga is associated with the bacterial surface and forms a capsule, then the corresponding genes are named cap (for "capsule"); however, if the pga is released, then the corresponding genes are named pgs (for polyglutamate synthase). 135 the minimal gene sets contain four genes termed cap or pgs b, c, a and e, with all cap genes and the four pgs genes (pgsb, pgsc, pgsaa, pgse) being organized into operons. 141 since pga is an idp, whose biochemical and biophysical properties are environment-dependent, and since pga can be found in an anchored to the bacterial surface form or in a released form, this biopolymer can play different roles in different organisms and in different environments. 135 for example, when anchored to the bacterial surface, pga forms a capsule and act as a virulence factor. 135, 142 in fact, the virulence of bacillus anthracis (a gram-positive sporulating bacterium, which is the causal agent of anthrax) was found to be determined by its capsule composed solely of pga. 143 similarly, the virulence of staphylococcus epidermidis (another gram-positive bacterium that causes severe infection after penetrating the protective epidermal barriers of the human body) is dependent on the pga-based capsule. 144 furthermore, pga in capsules of these bacteria consists of either a mixture of l-and d-enantiomers (s. epidermidis) 144 or solely d-enantiomer (b. anthracis), 145 which makes them particularly non-immunogenic. 135 the released form of pga is used by the producing organism for rather different purposes, starting from the sequestration of toxic metal ions that increases the resistance of some soil bacteria to harsh conditions, 146 to serving as a source of glutamate for bacteria in a starvation state during late stationary phase, 147 to playing a role in decrease of the high local salt concentrations that helps extremophilic bacteria and archaea to survive in a hostile environment, 148, 149 and in hydra, to control explosion of the special stringing cells, nematocysts, that are used to capture prey, for locomotion and for defense. 150 in addition to have multiple functional roles, bacterially produced pga has found its way to serve as an important biodegradable component 151 with multifarious potential applications in foods, pharmaceuticals, healthcare, water treatment and other fields. 152, 153 a large commercial advantage of pga is that this although some amount of glutamic acid residues is crucial for the structure and function of ordered proteins/domains, when a protein or a peptide contains a large number of glutamic acid residues and, as a consequence, possesses a small number of hydrophobic residues, it is likely to be disordered at physiological ph due to strong charge-charge repulsion and weak hydrophobic attraction. an illustrative example of such charge-infused proteins is glurich human prothymosin α, in which 64 out of 111 residues are charged (there are 19 asp, 35 glu, 2 arg and 8 lys residues), the overall content of hydrophobic residues (leu, ile and val) is very low, and aromatic residues (trp, tyr, phe and his) and cystein are absent. 133 based on this amino acid composition, it was not a big surprise to find that prothymosin α behaved as a highly disordered coil-like chain, since one cannot expect that a highly charged polypeptide (that contains 60% of glu+asp residues) will have a strong tendency to fold under physiological conditions. 133, 134 the lack of stable structure also explains the extreme thermal and acid stability of prothymosin α, since one cannot break what is non-existent. 133 the peculiar amino acid composition of prothymosin α, this biologically active random coil, was one of the defining factors behind the charge-hydropathy plot (ch-plot) development. 5 in fact, based on the analysis of prothymosin α and of 90 other non-globular proteins that lacked almost any ordered secondary structure under physiological conditions in vitro, it was concluded that a combination of high net charge and low hydropathy represents the necessary and sufficient factor for a polypeptide to behave as a natively unfolded protein. 5 strategically positioned glutamic acid residues can modulate conformational stability and function of ordered proteins too. in fact, the role of a glutamic/aspartic acid cluster located outside the ca 2+ -binding site, and of the n-terminal glu1 residue in destabilizing the structure and weakening the calcium-binding capabilities of α-lactabumin has been already discussed (see above). 121, 125 therefore based on these observations, protein regions and whole proteins enriched in glutamic acids are expected to be substantially disordered. poly-γ-glutamate (pga) is a natural homopolymer synthesized by several bacteria, one archaea (natrialba aegyptiaca) and one eukaryote (cnidaria). 135 one of the most known sources of pga is the japanese specialty natto, a fermentation product made by bacillus subtilis grown on soybean. 135 pga is a highly soluble polyanionic polymer that sequesters water molecules and can be found in surface-bound and released forms. in structural studies, polyglutamic acid is traditionally used as a biopolymer with a well-characterized secondary structure response to changes in the environmental ph, where pga is in a random coil-like conformation at neutral ph, but gains monomeric α-helical structure at acidic ph and is transformed into a β-sheet structure at alkaline ph. [136] [137] [138] curiously, the addition of polylysine to an aqueous solution of polyglutamic acid homopolypeptide at ebd is not a structurally stable entity in the conventional sense, since for this protein region there are no folded states that exist for any appreciable amount of time. instead, the ebd represents a time-average 3d region of a protein derived from the thermally driven motion of certain polypeptide chains, including those that are part of an otherwise stable folded protein. 163 therefore, the ebd which is defined by the time-averaged occupancy of space by a polypeptide chain, can exclude lager molecules while allowing small molecules and water to move freely through it. it was proposed that since functions of ebd depend on the intrinsically rapid thermal motion of the polypeptide, and the free energy changes that result when that motion is confined, this domain can be used to control binding events, confer mechanical properties, and sterically control molecular interactions. 163 obviously, to be able to serve as an ebd, a given fragment of a protein has to possess specific amino acid composition that would preclude it from folding. therefore, ebds are expected to possess low hydropathy and high net charge; i.e., in the ch-plot, they can be found well above the boundary separating compact and extended disordered proteins. one of the illustrative examples of biologically active ebds (which are not tightly folded, but expected to have a very extended conformation) is given by side-arms of neurofilament (nf) proteins. 164 the side-arms of the nf heavy polypeptide, nf-h (which are ~600 amino acids long), were shown by rotary shadow electron microscopy to be ~85 nm long. since there was not enough mass to form a stiff folded structure to occupy such a volume, it was proposed that the side-arms were not folded but were in constant thermal motion. 164 analysis of the amino acid sequence of the porcine nf medium polypeptide (nf-m, which has an apparent molecular mass of 160 kda and is one of the two high molecular mass components of mammalian neurofilaments) revealed that this protein has several peculiar features. 165 the n-terminal 436 residues contain a non-α-helical arginine-rich headpiece (residues 1-98) with multiple β-turns followed by a highly α-helical rod domain that forms double-stranded coiled-coils (residues 99-412), followed by a c-terminal tailpiece extension (approximately 500 residues) that represents an autonomous domain of unique amino acid composition, being characterized by a high content of lysines and particularly glutamic acids. 165 in human nf-m, there are 185 glutamic acids (20.2%), most of which are concentrated within the c-terminal tail, where glutamate accounts for 26.4% (133 out of 504 residues). similarly, human nf-h (a polypeptide comprising 1,026 residues) has 189 glutamic acids, 143 of which are found in the 613 residues-long c-terminal tail of this protein, whereas in the human nf-l (nf light polypeptide which has 543 residues), there are 99 glutamic acids, with almost half of which (46) being located within the acidic c-terminal subdomain (the last 100 residues of the protein). in addition to neurofilament polypeptides, ebds were found in microtubuleassociated protein 2 (map2) 166 and numa. 167 analysis of the amino acid compositions of these proteins revealed that they follow the trend established by nfs and contain significant amount of glutamic acid residues (220 out of 1,827 residues in human map2 are glutamates and there are 291 glutamic acids in the 2,115 residues-long human numa). natural biopolymer is nontoxic, biocompatible and nonimmunogenic. it can be produced by various bacterial strains in a controllable way. 152 as a result, pga is commonly used in cosmetics/ skin care, bone care, nanoparticle for drug delivery system, hydrogel, etc. 154 for example, the pga-based medusa system has been recently developed for slow release of therapeutic proteins and peptides. 155 here, a poly l-glutamate backbone is grafted with hydrophobic α-tocopherol molecules, creating a colloidal suspension of nanoparticles in water that contain hydrophobic nanodomains suitable for the reversible binding of various drug molecules. 155 the potential multifarious applications of pga in the areas of biomedical materials, drug delivery carriers, and biological adhesives have been studied extensively. 156 in general, γ-pga is recognized now as an important biomaterial in drug delivery applications, with γ-pga-based nanoparticles being considered as promising delivery carriers for anticancer therapeutics. 157 recently, a high molecular weight γ-pga was shown to be used as an immune-stimulating agent. 154 finally, conjugation of paclitaxel, a widely used chemotherapeutic agent whose therapeutic index is limited by low tumor exposure and high systemic exposure, with biodegradable poly-lglutamic acid generates paclitaxel poliglumex (ppx, ct-2103). 158 this macromolecular drug conjugate enhances tumor exposure to the drug, since the release of paclitaxel from the polymeric backbone was shown to be dependent on the ppx degradation by the lysosomal protease cathepsin b, which is upregulated in many tumor types. 158 glutamic acid as a part of the protein degradation targeting signals, pest motifs. pest sequences (i.e., sequences enriched in proline (p), glutamic acid (e), serine (s) and threonine (t)) are known to serve as specific degradation signals. [159] [160] [161] [162] these degradation signals define cellular instability of many proteins and direct them either to the ubiquitin-proteasome degradation or to the calpain cleavage. 161, 162 this controlled protein degradation is important for activation and deactivation of regulatory proteins involved in signaling pathways that control cell growth, differentiation, stress responses and physiological cell death. [159] [160] [161] [162] pest-containing sequences were shown to be solvent exposed and conformationally flexible, which preclude them from been resolved in x-ray structures. 159 based on the comprehensive bioinformatics analysis of experimentally characterized disordered and globular regions and of pdb chains containing pest regions, it has been concluded that the pest motif is most frequently located within idprs. 161 furthermore, analysis of the prolinerich motif pro-x-pro-x-pro in pest sequences revealed that these sequences contain glutamic acids much more often than aspartic acids. 161 in addition to this pro-x-pro-x-pro motif, many pest sequences are highly enriched in negatively charged residues and are characterized by a very specific distribution of negative charged patterns. 161 glutamic acids in entropic bristle domains. the entropic bristle domain (ebd) concept was proposed to describe a characteristic behavior of some highly mobile protein regions. the several metals of the transition and main groups (ib-va, z = 29−83) of the periodic table of elements. phytochelatins are synthesized by a constitutive enzyme, γ-glutamylcysteine dipeptidyl transpeptidase, that uses glutathione (gsh) as a substrate and catalyzes the following reaction: γ-glu-cys-gly + (γ-glu-cys) n − gly→(γ-glu-cys) n+1 − gly + gly. 183 fertilization promoting peptide. another important glutamaterich peptide is fertilization promoting peptide (fpp; pglu-glu-pronh2), which is produced by the prostate gland and secreted into seminal plasma. 184 fpp was shown to stimulate capacitation, which is the penultimate step in the maturation of mammalian spermatozoa required to render them competent to fertilize an oocyte. furthermore, although fpp inhibits spontaneous loss of acrosome (an organelle that develops over the anterior half of the head in the spermatozoa), cells retain high fertility in vitro. 184 gala peptide. recently, a synthetic 30 amino acid-long gala peptide with a glutamic acid-alanine-leucine-alanine (eala) repeat was designed to analyze how viral fusion protein sequences interact with membranes. 185 this gala peptide was long enough to span a bilayer when in the α-helical state, and the eala repeat was adjusted so that the peptide would have a hydrophobic face of sufficient hydrophobicity to interact with the bilayer when the peptide was in an α-helix. glu residues were used in gala as a ph-responsive elements. 185 when the ph is reduced from 7.0 to 5.0, gala converts from a water soluble random coil conformation to an amphipathic α-helix that binds to bilayer membranes. functional analysis revealed that gala promoted fusion between small unilamellar vesicles and was able to form a transmembrane pore comprised of ~10 gala α-helical monomers that were oriented perpendicularly to the plane of the membrane. 185 based on these observations, it has been proposed that ph-controlled membrane permealization induced by gala can serve as a model for the design of environmentally responsive peptidic vehicles for drugs and genes delivery. 185 other type of pests: ptp-pests. protein tyrosine phosphatases (ptp) with proline-, glutamate-, serine-and threoninerich sequence, ptps-pest, are a ubiquitously expressed critical regulators of cell adhesion and migration. 186, 187 this family of ptps includes three intracellular phosphatases known as prolineenriched phosphatase (pep) in mice or lymphoid tyrosine phosphatase (lyp) in humans (also known as ptpn22 and ptpn8), ptp-pest (also referred to as ptpn12) and ptp-hematopoietic stem cell fraction (ptp-hscf, which is also known by several other names, such as also termed brain-derived phosphatase 1 (bdp1), ptp20, ptp-k1, fetal liver phosphatase 1 (flp1) and ptpn18. 186 all these phosphatases possess a common structural organization that includes an n-terminally located phosphatase domain, followed by a highly divergent central region that contains various motifs for interactions with other proteins, and a conserved c-terminal domain known as carboxyl-terminal homology (cth) domain. 186 human ptp-lyp (ptpn22/ ptpn8) is a 807 residues-long protein that contains 59 and 40 glutamic and aspartic acids and 45, 83 and 32 prolines, serines and threonines, respectively. human ptp-pest (ptpn12) consists of 780 residues and has 67, 49, 66, 72 and 54 glutamates, aspartates, prolines, serines and threonines, respectively, most of recently, we proposed that ebds can be used as protein solubility enhancers. 168 in fact, we showed that highly charged protein sequences (both natural and artificial) can act as ebds, and that translational fusion of such sequences to target proteins can serve as an effective solubilizing means by creating both large favorable surface area for water interactions and large excluded volumes around the partner. 168 this suggests that intrinsically disordered ebds (which extend away from the partner and sweep out large molecules) can enable the target protein to fold free from interference. 168 all artificial fusions used in our study had low sequence complexity and high net charge, but were diversified using distinctive amino acid compositions and lengths. 168 among successful solubilizers were artificial ebds containing the most disorder-promoting residues (glu, pro, gln and ser) in the proportion glu:pro:gln:ser = 2:2:1:1; i.e., sequences containing > 33% glutamic acids. 168 therefore, it seems that glutamic acid is crucial for the successful function of ebd-containing proteins. glutamic acids in intrinsically disordered chaperones. the high content of glutamic acids in artificial ebds designed as solubilization means was chosen because of the earlier observation that proteins with high net charge densities can function as effective intra-and intermolecular chaperones. 169-172 for example, polyglutamate among other polyanions was shown to act as a chaperone and to accelerate the in vitro refolding of the arc repressor protein. 173 small heat shock proteins (hsps) have flexible c-terminal extensions that, although variable in length and sequence, are rich in acidic amino acids. 169 the shsp α-crystallin can act as a chaperone on the fibroblast growth factor 1 (fgf-1), and this chaperone action is mediated by electrostatic interactions between the basic regions of the growth factor and acidic regions of α-crystallin. 174 nucleolar chaperone b23 (294 residues, 31 of which are glutamic acids) has two acidic regions (residues 120-132 and 161-188) that contain 8 glutamic residues each and that are necessary for the b23 chaperone-like activity. 175 tubulin has chaperone-like activity being able to suppress the aggregation of soluble lens proteins, equine liver alcohol dehydrogenase, malic dehydrogenase and insulin, but only if its acidic c-terminus (that contains 39% and 33.3% of glutamic acid residuess in the porcine αand β-tubulins, respectively) was intact. [176] [177] [178] many polyanionic propeptides were shown to serve as intramolecular chaperones to aid folding of the respective proteins. [179] [180] [181] [182] for example, propeptides of human neutrophil defensins contain up to 15.8% glutamic acids. also, the c-terminal solubilizing domain of human α-synuclein (residues 100-140) contains 24.4% glutamates, whereas erd10 (260 residues) and erd14 dehydrins (185 residues) from arabidopsis thaliana contain 19.6% and 21.1% glutamic acids respectively. some functions of glutamate-rich peptides. this section presents several illustrative examples of important biological functions attributed to glutamate-rich peptides. phytochelatins. heavy metal detoxification in higher plants is dependent on a set of heavy-metal-complexing peptides, phytochelatins, with structure of (γ-glutamic acid-cysteine) n -glycine (n = 2-11) [(γ-glu-cys) n -gly]. 183 the longest of these peptides possesses a molecular mass of 2.6 kda, a pi 3.26 and a net charge of −11. these peptides are induced by the exposure of plants to arglu1. transcriptional activators and rna polymerase ii are bridged via the central transcriptional coactivator complex, the mediator complex. it has been recently shown that the arginine and glutamate rich 1 protein (arglu1) colocalizes with the mediator subunit 1 (med1) in the nucleus, being in contact with the far c-terminal region of med1. 190 this arglu1-med1 interaction is crucial for the estrogen-dependent gene transcription and breast cancer cell growth. 190 human arglu1 is a 270 residues-long protein that contains 53 arginines and 54 glutamates. there are two regions with significant composition biases in this protein, an arginine-rich region (residues 3-74) that contains 25 arginines and a glutamic acid-rich region (residues 27-251) containing 49 glutamic acids. pelp1. proline-, glutamic acid-and leucine-rich protein-1 (pelp1) plays an important role in mediation of genomic and nongenomic signaling of β-estradiol. 191 this potential protooncogene functions as a co-regulator of estrogen receptor, and expression of pelp1 is deregulated during breast cancer progression. 192 pelp1 contains ten nuclear receptor-interacting boxes (lxxll motifs), which allow it to interact with estrogen receptor and other nuclear hormone receptors, a zinc finger, a glutamic acid-rich domain and two proline-rich domains. 191 there are several consensus pxxp motifs within the proline-rich regions, via which pelp1 couples the estrogen receptor (er) with sh3 domain-containing kinase signaling proteins, such as src and pi3k p85 regulatory subunit. 191 there are 148 glutamic acids in pelp1 (which is 1,130 residues long), and the majority of them (99) are concentrated within the glutamic acid-rich domain (residues 888-1101). eif5. eukaryotic translation initiation factor 5 (eif5) is a monomeric protein of about 49 kda that functions as a gtpaseactivating protein (gap) in translation initiation. eif5 is involved in initiation of protein synthesis in eukaryotic cells, where, after binding to the 40s initiation complex (40s-eif3-mrna-met-trna f -eif2-gtp) at the aug codon of an mrna, it promotes gtp hydrolysis. this initiates a cascade of events that starts from the release of bound initiation factors from the 40s subunit and ends with the joining of the 60s ribosomal subunit to the 40s complex to form the functional 80s initiation complex (80s-mrna-met-trna f ). 193 although eif5 binds gtp and is able to promote gtp hydrolysis reaction, it does not hydrolyze gtp by itself acting as a typical gtpase-activating protein (gap). in fact, eif5 forms a complex with eif2 via its glutamic acidrich c-terminal region that binds to the lysine-rich n-terminal region of the β-subunit of eif2 thus activating the gtpase activity of eif2. 193 in human eif5, the 3d structure is known for the n-terminal nucleotide binding domain (residues 1-150, pdb id: 2e9h) and for the w2 domain (residues 232-431, pdb id: 2iu1). the linker region connecting these two domains is highly disordered and contains one of the functionally important glutamic acid-rich regions (residues [196] [197] [198] [199] [200] [201] [202] . overall, there are 11.4% glutamic acid residues in the 431 residues-long amino acid sequence of human eif5. histone-interacting proteins. since histones are polycations, they are known to be involved in interactions with several polyanionic proteins, particularly with proteins containing glutamic which are located outside the catalytic domain, with respectively 44, 32, 53, 59 and 39 glutamates, aspartates, prolines, serines and threonines being found in the non-catalytic region (residues 294-780). finally, among the 460 residues of the human ptp-hscf (bdp1/ptp20/ptp-k1/flp1/ptpn18), there are 27 glutamic acids, 21 aspartic acids, 32 prolines, 29 serines and 25 threonines. importantly, glutamate-rich, non-catalytic regions of all these ptps are known to be involved in interactions with multiple binding partners. for example, ptp-lyp is involved in interaction with grb2, c-cbl, and the c-terminal src kinase (csk), which is the inhibitory protein tyrosine kinase (ptk). the interaction between the ptp-lyp and csk is mediated by the proline-rich motif in pep and by the src homology 3 (sh3) domain of csk. 186 ptp-pest promiscuously associates with various proteins involved in the organization of the cytoskeleton, such as cas (and cas-related proteins sin and casl), paxillin (and paxillin-related polypeptides hic-5 and leupaxin) and the ptks fak and pyk2. this protein also associates with shc, grb2 and csk. 186 finally, ptp-hscf is involved in association with csk and tec. 186 multifarious functions of glutamic acid-rich proteins. delta factor. in addition to γ-pga, bacillus subtilis produces another important polyanion, delta factor, which is an important component of the bacterial rna polymerase. 188 this delta factor is a 20.4 kda highly acidic (pi = 3.6) protein that contains two distinct regions, a 13 kda n-terminal domain with uniform charge distribution and a glu-asp-rich c-terminal region. the overall contents of glutamic and aspartic acids in delta factor are 20.8% and 17.9% respectively, whereas these numbers increase to 34.3% and 37.3% in the glu-asp-rich c-terminal domain. the ordered n-terminal domain contains 32% α-helix and 16% β-sheet, whereas the c-terminal 8.5 kda domain is highly charged (net charge of −47) and therefore is largely unstructured. 188 importantly, the c-terminal intrinsically disordered domain has an important biological function, since the ability of delta factor to displace rna from rna polymerase requires the activities of both the n-terminal core-binding domain and the polyanionic c-terminal region. 188 marcks. myristoylated alanine-rich c kinase substrate (marcks) is an abundant 32 kda protein which is unusually rich in alanine and glutamic acid, with glutamic acid and alanine in this proteins accounting for 16.0% and 30.7% residues, respectively. marcks is a very prominent cellular substrate for protein kinase c (pkc), and its 22 serine residues and 2 threonines are phosphorylated. human marcks is an acidic protein with a pi of 4.46 which in addition to ala-glu enriched n-and c-terminal domains possesses a compact "effector domain" (ed), which is responsible for interaction with calmodulin, is located near the middle of the sequence and is enriched in lysines, serines and phenylalanines. 189 marcks is a typical idp with a labile conformation and little ordered structure. in addition to calmodulin this protein can interact with synapsin and actin, and can serve as filamentous actin (f-actin) cross-linking protein. furthermore, being myristoylated, marcks is able to interact with membrane and serves as a cytoskeleton-membrane linkage crucial for controlling cell shape changes. 189 since although the molecular mass of the phosphoprotein was shown to be about 44 kda by sedimentation equilibrium analysis, it runs on 5-15% sds-page (sds-page) as a protein with a molecular mass of 75 kda. 197 later studies revealed that bsp is capable of nucleating the bone mineral hydroxyapatite and that this nucleation involves one or both of the glutamic acid-rich sequences suggesting that polycarboxylate sequences might represent a specific site for growth-modulating interactions between proteins and biological hydroxyapatite crystals. 198 similarly, the ability of another acidic, non-collagenous protein of bone and dentin, osteonectin (also known as secreted protein, acidic, rich in cysteine), to bind to hydroxyapatite crystals is determined by its n-terminal region containing glutamic acid-rich sequences. 199 sparc is a highly conserved acidic calcium-binding extracellular-matrix protein. 200 this matricellular glycoprotein is composed of three functional domains that are evolutionarily conserved in organisms ranging from nematodes to mammals. 201 starting from the n-terminus, these functional domains are: a ca 2+ -binding glutamic acid-rich acidic domain (domain i), a follistatin-like module (domain ii), and an extracellular ca 2+ -binding (ec) module that contains two ef-hands and two collagen-binding epitopes (domain iii). since domain i was not found in sparc isolated from the starlet anemone nematostella vectensis, it has been proposed that sparc first evolved as a collagen-binding matricellular glycoprotein. 201 human sparc is a 303 residueslong protein that contains 34 glutamic acids, 15 of which are located within the n-terminal calcium binding region (residues 22-69). although xenopus laevis sparc has a molecular mass of 32.6 kda, based on sds-page analysis this protein has a molecular mass of 43 kda. 200 nbp-45. in nuclei of mice cells, there is a nuclear protein nbp-45 related to the nuclear proteins hmg-14/-17. nbp-45 can function as a transcriptional activator, binds specifically to nucleosome core particles, 202 preferentially binds to euchromatin and modulates cellular transcription by counteracting linker histone-mediated chromatin compaction. 203 nbp-45 is composed of 406 amino acids and has several functional regions and domains: the n-terminal region (residues 1-85) contains three segments that are highly homologous to functionally important domains in the hmg-14/-17 protein family, namely a nuclear localization signal, a nucleosome binding domain and a chromatin unfolding domain, whereas the c-terminal region (residues 86-406) has 43.7% of negatively charged residues. 202 in fact, of the 110 glutamic acids and 44 aspartic acids found in nbp-45, 100 glutamic and 40 aspartic acids are located in this highly acidic region. garps in rod photoreceptors. glutamic acid-rich proteins (garps) are common in different organisms and have numerous biological functions. for example, rod photoreceptors contain three different glutamic acid-rich proteins (garps), two soluble forms, garp1 and garp2, and the n-terminal cytoplasmic domain (garp part) of the b1 subunit of the cyclic gmp-gated channel (also known as cyclic nucleotide-gated cation channel β-1, cngb1), that are involved in the control of the ca 2+ propagation from the site of its entry at the cyclic nucleotidegated channel to the cytosol of the outer segment. 204 the cyclic acid-rich domains or regions. for example, the non-epithelial intermediate filament (if) subunit protein (e.g., human vimentin, which is attached to the nucleus, endoplasmic reticulum and mitochondria, either laterally or terminally and that contains 11.8% glutamic acids) can specifically bind core histones with a stoichiometry of 8 core histones per a nonneuronal if protein dimer. 194 glutamic acids clearly play a crucial role in this interaction since the 68 kd neurofilament protein, which was already discussed in the ebd section and contains a glutamic acid-rich c-terminal extension, can bind more core histones per dimer (24 molecules of core histones) than the dimer of the non-neuronal if proteins. 194 in the nuclei of physarum polycephalum, there is an alanine, lysine and glutamic acid-rich nuclear protein (p2) with a molecular mass of ~19.5 kda that can specifically interact with histones and therefore is co-extracted with histones. 195 based on amino acid sequence analysis, it has been concluded that p2 is a hmg-like protein, which, according to cd measurements, contains only 5% secondary structure and is, therefore, essentially unstructured under in vivo conditions. 195 titin. the gigantic protein titin (there are 34,350 residues in the human protein) is a key component in the assembly and functioning of vertebrate striated muscles. among numerous cellular functions of titin (also known as connectin) are contribution to the fine balance of forces between the two halves of the sarcomere which is crucial for the elasticity of muscle cells, as well as participation in chromosome condensation and chromosome segregation during mitosis of non-muscle cells. the ability of titin to reversibly extend relies on a set of pevk segments, rich in proline (p), glutamate (e), valine (v) and lysine (k) residues. the single molecule analysis of the recombinant titin fragment, containing approximately 28-residue pevk repeats and glutamic acid-rich motifs, revealed that the bending rigidity of the pevk fragments can be reduced due to calcium-induced conformational changes. 196 furthermore, the glutamic acid-rich motif was shown to be critical for this process. based on these observations, it has been concluded that the glutamic acid-rich motifs embedded into the pevk segments make titin a calcium-dependent molecular spring that can adapt to the physiological state of the cell. 196 curiously, titin has 3,193 glutamic acids, 449 of which are found in the glutamic acid-rich region (residues 9974-11917) that contains 31 pevk motifs. glutamates are not evenly distributed within the glutamic acid-rich region; e.g., 42 glutamic acids are concentrated within the first 116 residues of this region (residues 9974-10,089). in other words, although the glutamic acid-rich region comprises just 5.6% of the whole titin, it has 14.1% of all the titin's glutamates. bone phosphoproteins. bone sialoprotein ii (bsp ii) is an important component of the bone mineralized matrix. this bone-specific glycoprotein contains phosphoserine and sulphotyrosine residues and two regions of contiguous glutamic acid residues (residues 77-84 and 156-169) . in one of the first studies dedicated to the analysis of bone phosphoprotein it has been shown that this glycoprotein can be purified from the mixture of proteins extracted by demineralization of rat bone with 0.5 m edta in 4 m guanidinium chloride. 197 it was also emphasized that this protein possessed an abnormal electrophoretic mobility this protein was shown to be involved in sensing infertile nutrient conditions in infected cells to promote a transfer from saprophytic to dormant microsclerotia for long-term survival. 213 vdgarp1 is a short (91 residues) extremely acidic protein with a pi of 3.3 that contains 52.8% negatively charged residues (31 glutamic acids and 17 aspartic acids). there are also several other garps in various organisms, the functions of which are not known as of yet. small garp (a 112-amino acid protein, with a molecular mass of 13.1 kda and an isoelectric point of 3.94, 29 residues of which are glutamic acids) was found in euplotes octocarinatus. 214 plasmodium falciparum garp consists of 679 residues, 169 of which are glutamic acids. 215 rhox8/tox. reproductive homeobox 8 protein (rhox8 or tox) is a homeodomain protein which is distantly related to the members of the paired/pax family belonging to the pepp subfamily of paired-like homeobox proteins. 216 in mice, tox is predominately transcribed in the testis and ovary and potentially plays an important role during gametogenesis. 216 this 320 residues-long protein contains 113 glutamic acids organized in two poly-glutamic acid stretches (residues 111-139 and 177-201) and several glu-rich regions, which together with 11 aspartic acids makes rhox8 highly acidic (pi 3.95). kibra. kidney and brain protein (kibra) is a large (1,113 residues) protein that serves as a potential regulator of the hippo/ swh (sav/wts/hpo or salvador/warts/hippo) signaling pathway that restricts proliferation and promotes apoptosis therefore being crucial for tumor suppression. 217, 218 kibra has 111 glutamic acids and possesses two n-terminal ww domains, an internal c2-like domain and a c-terminal glu-rich stretch (residues 819-873). 219 cellular functions of kibra are modulated via phosphorylation by protein kinase c zeta (prkcz). 220 some cellular activities of kibra may be associated with memory performance. 220, 221 furthermore, in mammalian cells, this protein co-activates functions of the dynein light chain 1, 222 is involved in regulation of the collagen-stimulated activation of the erk/ mapk cascade 223 and modulates directional migration of podocytes. 224 kibra interacts with histone h3 via its glu-rich region, and this interaction might play an important role in conferring an optimal transactivation function to the estrogen receptor-α (er) and also may be involved in the proliferation of ligand-stimulated breast cancer cells. 222 sh3bgr. sh3 domain-binding glutamic acid-rich protein (sh3bgr) is a highly acidic (pi 4.09) 239 residues-long protein that possesses 44 glutamates and 15 aspartates and that is expressed in heart and skeletal muscles. 225 the majority of glutamates are located within the c-terminal glu-rich region (residues 170-239), ~43% of which are glutamic acid residues. in addition to sh3bgr, several other members of the sh3bgr family were found in humans. these are the so-called sh3bgr-like proteins, such as sh3bgrl (114 residues, 12 glutamic acids), sh3bgrl2 (107 residues, 10 glutamic acids) and sh3bgrl3 (93 residues, 7 glutamic acids) encoded by chromosomes xq13.3 6q13-15, and 1p34.3-35, respectively. 226 it was shown that the sh3 domainbinding glutamic acid-rich-like protein 3 is upregulated in glioblastoma. 227 also, this protein was noticeably downregulated in nucleotide-gated (cng) cation channel of rod photoreceptors is a heterotetramer consisting of homologous subunits, α and β (also known as cnga1 and cngb1a). cnga1 is known to be indispensable for channel activation, whereas cngb1a plays mostly regulatory structural roles. 205 in fact, the n-terminal glutamic acid-rich protein (garp) domain of cngb1a and the soluble garp2 were shown to decrease the opening probability of the cng channel and therefore these garps serve as important autoinhibitors or molecular gate keepers that control the activation of heteromeric rod cng channels. 205 furthermore, cngb1 and garp2, in concert with a retinal tetraspanin (peripherin-2 or peripherin rds ), were shown to contribute to the organization of the specific organelle, outer segment (os), which possesses a characteristic membranous "stacked pancake" architecture that has to be partially renewed daily to maintain cell function and viability. 206 in fact, a mouse knockout of cngb1 and garp2 attenuated rod function and caused structural alterations and slowly progressive retinal degeneration. 207 bovine garp (or cngb1) is a 1,394 residues-long transmembrane protein which plays important roles in both visual and olfactory signal transduction. cngb1 has 209 glutamic acids. garp1 is a 590-residues-long cngb1 splice variant that possesses 141 glutamic acids. garp2 is another cngb1 splice variant that has 299 residues 38 of which are glutamic acids. native garp1 and garp2 purified from bovine rod photoreceptors were shown to be typical idps. 208 mgarp. mitochondria-localized glutamic acid-rich protein [mgarp, which is also known as ovary-specific acidic protein (osap), corneal endothelium-specific protein 1 (cesp-1) and hypoxia upregulated mitochondrial movement regulator protein (hummr)] is one of the highly expressed proteins in retina. 209 mgarp is highly enriched in steroidogenic tissues and the visual system, and early in development, this protein is mainly detected in the retina and adrenal gland. 210 during the estrous cycle, mgarp levels correlate with estrogen levels in the ovaries. furthermore, the expression of mgarp is regulated by estrogen in a tissue-specific manner and through a feedback regulatory mechanism. 210 as it follows from a long list of names, this protein has numerous important functions. in fact, among functions listed for this protein in the uniprot are (1) plays a role in the trafficking of mitochondria along microtubules, (2) regulates the kinesin-mediated axonal transport of mitochondria to nerve terminals along microtubules during hypoxia, (3) participates in the translocation of trak2/grif1 from the cytoplasm to the mitochondrion and (4) plays a role in steroidogenesis through maintenance of mitochondrial abundance and morphology. 211, 212 there are 283 residues in mouse mgarp, 49 of which are glutamic acids exclusively located in the glu-rich region (residues 79-277). based on the spectroscopic analysis of this protein, it has been concluded that mouse garp is an idp. 209 some other garps. the life cycle of the phytopathogenic fungus verticillium dahliae kleb causing wilt disease in a wide range of crops, including cotton, includes three vegetative phases: parasitic, saprophytic and dormant. 213 one of the genes tagged in a pathogenicity encoded a glutamic acid-rich protein (vdgarp1), which shared no significant similarity to any known proteins. 213 linked to a glutamic acid and alanine repeat (eg-ea repeat). 235 there are 171, 83 and 43 glutamic acids, glycines and alanines in this latency associated nuclear antigen. although there is low sequence identity between lana1, ebna1 and orf73, all three proteins determine the poor recognition of viruses by cd8 + cytotoxic t lymphocytes (ctl). however, the mechanisms of their action are rather different. in the epstein-barr virus and kaposi's sarcoma-associated herpesvirus the repeat domains were shown to enhance the stability of ebna1 and lana1 and decrease their translation rates, whereas the eg-ea repeat has no effect on the stability of hvs orf73 or its rate of translation, but results in decreased steady-state levels of orf73 mrna. 235 intriguingly, the motif eeaeeaeee of hvs orf73 was sufficient to cause a reduction in recognition of orf73 by cd8 + ctl, suggesting that the eg-ea repeat of hvs orf73 is crucial for the immune evasion. 235 nsp3a. the n-terminal domain of the severe acute respiratory syndrome coronavirus (sars-cov) nonstructural protein 3 (nsp3a) is a typical idp of 183 residues characterized by the presence of an ubiquitin-like globular domain (residues 1-112) and a flexible, highly extended glu-rich domain (residues . 236 nsp3a is a highly acidic protein (ph 3.72) that contains 40 glutamic acids, 28 of which are located within the c-terminal glu-rich domain. ppe antigens. proline and glutamic acid rich proteins (or pperepeat containing proteins, or ppe proteins) are important t-cell antigens produced by mycobacterium avium subsp paratuberculosis (map). 237 one of the ppes is a 34.9 kda protein (359 residues, pi 4.31) which following recombinant expression in e. coli was shown to elicit significant delayed type hypersensitivity skin reaction in mice sensitized with map, suggesting that this recombinant ppe protein of map was definitely associated with cellular immune response. 237 curiously, this ppe contains 73 alanines, 44 glycines, 37 prolines, 20 aspartic acids but just 10 glutamic acids. pt2l4. cassava storage roots differentially produce an interesting pt2l4 protein 238 with low sequence complexity characterized by a reduced amino acid alphabet (just 13 amino acids). this 107 residue-long protein contains 56 glutamic acids, 30 alanines, 24 valines, 20 prolines, 18 serines and 15 lysines, but does not have any arginines, asparagines, cysteins, histidines, phenylalanines, tyrosines and tryptophanes. glutamic acid-rich protein from cassava roots. based on the analysis of changes in the cassava root proteome during physiological deterioration of cassava root after harvesting, it has been concluded that the glutamic acid-rich protein was one of the proteins that were upregulated after harvesting. 239 cp190. eukaryotic genomes contain a set of specific functional elements, chromatin insulators or boundary elements that regulate gene transcription by interfering with promoterenhancer communication. 240 in drosophila melanogaster, the centrosome-associated zinc finger protein cp190 protein (cp190) is a component of the gypsy chromatin insulator complex, which is composed of cp190, mod(mdg4) and su(hw) and is required for the function of the gypsy chromatin insulator and other endogenous chromatin insulators organized by su(hw), ctcf the hippocampus and cerebral cortex of app(e693δ)-transgenic mice that are used as a model to study the pathological effects of aβ oligomers in alzheimer's disease. 228 abra. the acidic-basic repeat antigen (abra) is a 743-residues-long protein found in the vacuolar space surrounding merozoites in plasmodium falciparum-infected erythrocytes, being localized in the parasitophorous vacuole and associated with the merozoite surface at the time of schizont rupture. 229 due to its surface location, abra is one of the potential vaccine candidates against erythrocytic stages of malaria. 230 this protein is one of the antigens enriched in the clusters of merozoites formed with growth inhibitory immune serum and possesses chymotrypsinlike activity, 231 which can be inhibited with serine protease inhibitors such as chymostatin and phenyl methyl sulfonyl fluoride (pmsf). 232 it was shown that the n-terminal half of the protein is responsible for the protease activity, whereas the highly charged c-terminal part of the protein was not required for this activity. 232 furthermore, the n-terminus contains an erythrocyte-binding domain located within the cysteine-rich n-proximal region of abra. 229 there are 111 glutamic acids and 108 lysines in abra, and in agreement with its name, the amino acid sequence of this protein is characterized by the presence of eight tandem repeats of [vt]-n-d-[ed]-[ed]-d (residues 226-273) and by a lysinerich c-terminal region (residues 672-721). kerp1. the parasite entamoeba histolytica that colonizes the large bowel and provokes an asymptomatic luminal gut infection contains a peculiar lysine and glutamic acid-rich protein 1 (kerp1), which is associated to parasite surface, involved in the parasite adherence to host cells and plays a role in the entamoeba histolytica liver abscess pathogenesis. 233 an interesting feature of kerp1 (184 residues) is a very high content of lysines (25%) and glutamic acids (19%). proteins with long simple repeat elements from herpesviruses. one of the mechanisms employed by herpesviruses to evade the immune response, allowing them to persist life-long in their hosts, relies on the use of specific proteins that function as cis-acting inhibitors of antigen presentation. 234 among these inhibitors are the nuclear antigen 1 (ebna1) and pgzr in the epstein-barr virus (ebv) and the latency-associated nuclear antigen 1 (lana1) of the kaposi sarcoma herpesvirus. 234 the common feature of all these proteins is the presence of long simple repeat elements in their amino acid sequences. for example, pgzr is a 230 amino-acids long glycine, glutamine, and glutamic acid-rich repeat ("gz" repeat) protein that which is encoded by a large nested open reading frame located in the ebna1 mrna and is highly similar (65% amino-acid identity) to the acidic repeat of lana1. 234 latent nuclear antigen of human herpesvirus 8 (hhv-8) (kaposi's sarcoma-associated herpesvirus) is a large (1,036 residues) highly acidic protein (pi 3.81) that contains 237 glutamic acids, 179 glutamines, 114 prolines and 90 aspartic acids. in herpesvirus saimiri (hvs) that infects squirrel monkeys, the functional homolog of epstein-barr virus ebna1 and kaposi's sarcoma-associated herpesvirus lana1 proteins is the 501 residues-long product of the open reading frame 73 known as orf73 or latency associated nuclear antigen. 235 orf73 contains a repeat domain composed of a glutamic acid and glycine repeat mutation affects the ability of hla-dpb1 to present beryllium to pathogenic cd4 + t cells. 242 sickle cell anemia and glu6val mutation in hemoglobin. sicklecell (sca) or drepanocytosis is an autosomal recessive genetic blood disease with over-dominance, characterized by red blood cells that assume an abnormal, rigid, sickle shape. the disease is caused by a single point mutation in the β-globin chain of hemoglobin where the hydrophilic and negatively charged amino acid glutamic acid is replaced by the hydrophobic amino acid valine at the sixth position. as a result of this substitution, sickle hemoglobin polymerizes inside the affected erythrocytes. it was pointed out that such sickle hemoglobin polymerization occurs by homogeneous and heterogeneous nucleation mechanisms, which are both highly sensitive to macromolecular crowding. 243 in fact, the rates of homogeneous nucleation were shown to be enhanced by 10 10 when the initial concentration was augmented by 50% nonpolymerizing hemoglobin. 243 retinitis pigmentosa and mutations in a glu-rich domain of rpgr. retinitis pigmentosa (rp) is an inherited, degenerative eye disease associated with the progressive loss of photoreceptor genes that causes severe vision impairment and often blindness. 244 among other factors, rp is caused by mutations in the retinitis pigmentosa gtpase regulator (rpgr) gene which accounts for 15-20% of rp cases in caucasians. 245 genetic analysis revealed that of 240 rpgr mutations 95% are associated with x-linked retinitis pigmentosa (xlrp), 3% are found in cone, cone-rod dystrophy or atrophic macular atrophy, and 2% are related to syndromal retinal dystrophies with ciliary dyskinesia and hearing loss. 245 importantly, all disease-causing mutations occur in one or more rpgr isoforms containing the c-terminal exon open reading frame 15 (orf15), and 55% occur in a glu-rich domain within exon orf15, which accounts for only 31% of the protein. 245 rpgr (1,020 residues) contains 123 glutamic acids, more than half of which (70) are located within the c-terminal glu-rich domain (residues 530-903). pyoderma gangrenosum and glu250gln mutation in pstpip1. pyoderma gangrenosum is a condition that causes tissue to become necrotic, causing deep ulcers that usually occur on legs. pyoderma gangrenosum is one of the most common extra-intestinal manifestations of chronic inflammatory bowel disease. 246 the disease is caused by the alterations in the pathway that links the members of the proline-rich, glutamic acid-rich, serine-rich and threonine-rich (pest) family of protein tyrosine phosphatases (which are critical regulators of adhesion and migration) to their substrates. a major player in this pathway is a cytoskeleton-associated adaptor protein, namely proline-serine-threonine phosphatase-interacting protein 1 (pstpip1, also known as cd2-binding protein 1, cd2bp1). 246 defects in pstpip1 are the cause of papa syndrome (papas), also known as pyogenic sterile arthritis, pyoderma gangrenosum and acne or familial recurrent arthritis (fra). 247 papas is characterized by an autosomal dominant inheritance of early onset, primarily affecting skin and joint tissues. missense mutations glu250-gln and ala230-thr in pstpip1/cd2bp1 were identified in two families. 247 these mutations were shown to affect the ability of pstpip1to interact with its natural partners. 247, 248 and beaf32. 240 although cp190 is a large protein (1,096 residues) that possesses a complex multidomain structure, only three domains were shown to be essential for the insulator function and for the viability of flies: the btb/poz domain, an aspartic acid-rich (d-rich) region and a c-terminal glutamic acid-rich (e-rich) region. 240 here, the n-terminal cp190 fragment containing the btb/poz domain and the d-rich region was shown to be involved in regulation of the cp190 interaction with insulator complexes, whereas the c-terminally located e-rich region was necessary for the cp190 dissociation from chromosomes during heat-shock. 240 importantly, the 131 glutamic acids are not equally distributed within the protein, with the n-terminal half containing just 26 glutamic acids and with the remaining 105 glutamates being concentrated within the c-terminal half of cp190. therefore, although the overall glutamic acid content of this protein is 12%, its c-terminal half is especially enriched in these residues (19.2%). also, this uneven distribution is seen not only for glu, but for all the charged residues. in fact, the n-terminal fragment (residues 1-548) has a net charge of +18 (asp + glu = 25 + 26 = 51; arg + lys = 31 + 38 = 69), whereas the c-terminal half of cp190 (residues 549-1096) has a net charge of −120 (asp + glu = 62 + 105 = 167; arg + lys = 8 + 39 = 47). pcp4l1. purkinje cell produces two closely related proteins containing iq motifs, purkinje cell protein 4-like 1 (pcp4l1) and pcp4/pep-19. although pcp4/pep-19 is able to interact with calmodulin and inhibit calmodulin-dependent enzymes, and although the synthetic peptide constituting only the iq motif of pcp4l1 binds calmodulin and inhibits calmodulin-dependent kinase ii, the full-length pcp4l1 does not interact with calmodulin. 241 the lack of ability of the full length pcp4l1 to interact with calmodulin was ascribed to its nine-residue glutamic acid-rich sequence that lies outside the iq motif in pcp4l1. mutational analysis showed that calmodulin binding can be restored not only by the deletion of this inhibitory motif, but also by exchanging it with the homologous region of pep-19 and by simple point mutation converting a single isoleucine (ile36) within this motif to phenylalanine or to other aromatic residues. 241 therefore, although pep-19 and pcp4l1 possess noticeable sequence similarities, their functional properties are very different due to the presence of the glu-rich element in pcp4l1 that can functionally suppress an iq motif. 241 glutamic acid mutations and human diseases. chronic beryllium disease and lys96glu mutation in hla-dpb1. chronic beryllium disease (cbd) is a hypersensitivity disorder that affects 2-16% of workers professionally exposed to berillium in the workplace. cbd is characterized by a granulomatous inflammation and accumulation of beryllium-specific cd4 + t cells in the lung. 242 the susceptibility to this disease depends on both genetic factors (genetic susceptibility) and the nature of the exposure. genetic analysis revealed that a single point mutation at the 69th position of the human leukocyte antigen (hla) class ii histocompatibility antigen dp β 1 chain (hla-dpb1), where lysine is substituted by a glutamic acid, makes the carriers more susceptible to cbd. it has been proposed that the k→e point of enzymes, or be related to metal binding. in idps/idprs, overabundance of glutamic acids defines the extended conformation of native coils and native pre-molten globules. glutamic acid is an important part of the pest motif related to protein degradation. it is crucial for function of entropic bristle domains and several chaperones. stretches of glutamic acid residues have a lot of specific functions that range from unique metal binding properties of phytochelatins and bone phosphoproteins, to regulation of cell adhesion and migration, to defining specific immunochemical reactivity of several antigens. no potential conflicts of interest were disclosed. this review illustrates that glutamic acid is differently used in ordered proteins/domains and in idps/idprs. in ordered proteins, glutamic acid residues are crucial for protein solubility and, being strategically placed within protein structure, play several structure-forming and structure-stabilizing roles. here, glutamic acid is involved in electrostatic interactions and hydrogen bond formation, serves as an important α-helix former, and participates in the α-helix cap formation. glutamic acid is an important functional residue of ordered proteins, where it can be involved in the formation of specific electrostatic valves inside the pores of ion channels, or can play unique catalytic roles in the active sites intrinsically disordered proteins from a to z unstructural biology coming of age thousands of proteins likely to have long disordered regions intrinsic protein disorder in complete genomes why are "natively unfolded" proteins unstructured under physiologic conditions? prediction and functional analysis of native disorder in proteins from the three kingdoms of life orderly order in protein intrinsic disorder distribution: disorder in 3500 proteomes from viruses and the three domains of life understanding protein nonfolding intrinsically unstructured proteins: re-assessing the protein structure-function paradigm 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class i antigen presentation by the glutamic acid-rich repeat of herpesvirus saimiri open reading frame 73 nuclear magnetic resonance structure of the n-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome coronavirus expression of a gene encoding 34.9?kda ppe antigen of mycobacterium avium subsp isolation and characterization of the promoter sequence of a cassava gene coding for pt2l4, a glutamic acid-rich protein differentially expressed in storage roots itraq-based analysis of changes in the cassava root proteome reveals pathways associated with post-harvest physiological deterioration the chromosomal association/dissociation of the chromatin insulator protein cp190 of drosophila melanogaster is mediated by the btb/poz domain and two acidic regions proteomic analysis of the brain tissues from a transgenic mouse model of amyloid β oligomers amino terminus of plasmodium falciparum acidic basic repeat antigen interacts with the erythrocyte membrane through band 3 protein immunogenicity of recombinant fragments of plasmodium falciparum acidic basic repeat antigen produced in escherichia coli plasmodium falciparum: chymotryptic-like proteolysis associated with a 101-kda acidic-basic repeat antigen expression and characterisation of plasmodium falciparum acidic basic repeat antigen expressed in escherichia coli the lysine-and glutamic acidrich protein kerp1 plays a role in entamoeba histolytica liver abscess pathogenesis the nested open reading frame in the epstein-barr virus nuclear antigen-1 mrna encodes a protein capable of inhibiting antigen presentation in cis key: cord-033333-880jx1bt authors: salman, saad; shah, fahad hassan; chaudhry, maham; tariq, muniba; akbar, muhammad yasir; adnan, muhammad title: in silico analysis of protein/peptide-based inhalers against sars-cov-2 date: 2020-10-08 journal: nan doi: 10.2217/fvl-2020-0119 sha: doc_id: 33333 cord_uid: 880jx1bt aim: peptide/protein-based inhalers are excessively used to treat respiratory disorders. the molecular docking was performed for these inhalers including human neutralizing s230 light chain-antibody (monoclonal antibodies [mabs]), alpha-1-antitrypsin (aat), short-palate-lung and nasal-epithelial clone-1-derived peptides (splunc1) and dornase-alfa (da) against spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) to assess their inhibitory activity. materials & methods: hawkdock was used to dock these biologics against sars-cov-2 spike-glycoprotein. results: results showed that da, aat and mab were quite active against spike glycoprotein with a binding free energy of -26.35 and -22.94 kcal/mol. conclusion: mab and aat combined with da can be used in the treatment of coronavirus disease of 2019 as a potential anti-sars-cov-2 agent. ultimately reaching the brain. sars cov-2 spike glycoprotein consists of two subunits that are the s1 and s2. the s1 subunit consists of receptor binding domain (rbd) that binds with the host cell receptor to ace-2. the second subunit is s2 which arbitrate fusion between viral and plasma membrane of host. it contains protrusions that allow the virus to bind a receptor on the host cell. sars-cov-2 is transmitted by droplets and contact between a healthy individual and an infected person [4] . the spike glycoprotein is a 142.3 kda protein present on the surface of the virion that facilitates interaction by employing their 319-591 number amino acid residues to interact with the ace-2 receptor. after attachment, the infection develops, and the human body's immediate response is to produce an immune response against the infection and also to modulate the mucous secretion in the surrounding epithelial cells to deter the attachment of other freshly produced sars-cov-2 progenies. both sars-cov-2 and mucus aggravate disease progression by contributing to the production of a cough. it is imperative to develop aerosols that could help in the prevention and control of the disease. aerosols composed of monoclonal antibodies (mabs) are commonly used for respiratory disorders, such as asthma [5] . mabs are composed of transgenic animals or phage display technology, which benefit in several ways as to curtail immunogenicity, increase effector function and delay their serum half-life [6, 7] . cystic fibrosis (cf) airways manifest increased neutrophil elastase activity, which can likely destroy the lung, and also cleave and activate enac intensifying mucus dehydration and expediating mucociliary clearance. alpha-1-antitrypsin (aat) is an endogenous neutrophil elastase (ne) inhibitor that, by blocking ne, can improve pulmonary function [8] . the drug dornase-alfa (da) for cf reduces dna length, hydrolyzes dna polymer and makes a good mucolytic agent. making da a drug of choice for cf that is well tolerated, helps in the amelioration of lung function and reduces sputum viscosity [7] . so, short-palate lung and nasal-epithelial clone-1-derived peptides (splunc1) could be utilized in cf for the inhibition of enac [8] and regulation of th2 inflammation [9] . spx-101 is novel proteaseresistant peptide that inhibits enac, regulates mucosal secretions, and ameliorates rehydration [10] . however, the use of protein/peptide-based inhalers does have toxicogenic and immunogenic responses in the body [11] . the administration of these agents could be debilitating, if the dose is not controlled or with their chronic use [12] . some of these protein/peptide-based inhalers can precipitate pulmonary toxicity, cytokine-release syndrome, serum sickness, tumor lysis syndrome, toxic pulmonary-amyloid aggregates, antibodies generation and acute anaphylaxis, anaphylactoid reactions and in rare cases progressive multifocal leukoencephalopathy [13] [14] [15] . although the safety and efficacy of da [16] , splunc1 [17] , aat [18] and mab [19] are well established, still the detailed studies regarding their toxic and antigenic profiles remain elusive. these four inhalers containing mab, da, aat and splunc1 were tested through molecular docking against sars-cov-2 spike glycoprotein, their toxicity, antigenicity, water solubility and resistance to gastric enzymatic activity were evaluated. the protein ligands alpha-1-antitrypsin (pdb id:1atu), da (human recombinant dnase-i (pdb id: 4awn), angiotensin-converting enzyme-2 (ace-2) (pdb id:1r4l), human palate, lung and nasal epithelium clone protein (4n4x) and human neutralizing s230 light chain antibody (6nb6) and covid spike protein (pdb id:6vsb) was obtained from protein data bank [20, 21] . these protein structures were further refined with modrefiner [22] and hawkdock [23] was then used for docking studies coupled with discovery studio 4.0 for the visualization of docked complexes. to assess the in silico toxicity analysis of the protein/peptide-based inhalers, the uniprot database was used for the protein/peptide sequences. the toxicity was analyzed by utilizing toxinpred [24] while the prediction of allergenicity was calculated by utilizing allertop [24] . also, these samples were subjected to in silico prediction of gastrointestinal digestion resistance by utilizing peptidecutter [25] . the water solubility of these inhalers was checked through peptide property calculator, in order to establish its water solubility. the proposed mechanism of da, splunc1 and mab in asthma and cf are given in figure 1 . splunc1 is a protein that consists of the enac inhibitory domain-s18 region. splunc1-derived peptide, which serves in cf airways as an enac inhibitor, help in the regulation of lung function. (b) cf has high levels of dna and actin in the lung lumen, which are discharged by necrotic neutrophils. dnase1 cleaves extracellular dna in the lung lumen reducing dna length and sputum viscosity is decreased. mabs get attached to a wide variety of proteins with high affinity and specificity. in asthma, they bind to ige and fecri resulting in reduced exacerbations. i-iii: these three agents fight against sars-cov-2 by either binding (i, ii) or blocking (iii) its passage to reach out to the ace-2 receptor. cf: cystic fibrosis; mab: monoclonal antibody. protein-protein interaction (ppi) of covid-19 spike glycoprotein with alpha-1-antitrypsin (1atu), dornase-alfa (4awn), angiotensin-converting enzyme-2 (ace-2) (pdb id:1r4l), human palate, lung and nasal epithelium clone protein (splunc1) (4n4x) and human neutralizing the s230 light chain antibody was evaluated through hawkdock. the algorithm of hawkdock is composed of attract and hawkrank, which allows flexible protein-protein docking combined with mm/gbsa free energy decomposition of key protein residues involved in the interaction [23] . prior to ppi, we selected chain-a of sars-cov-2 receptor binding domain as the rest of the chain had identical sequence as compared with the rest of the protein chains (b, c). our strategy was to dock these selected proteins in the 319th to 591st number of amino acids within the chain-a of sars-cov-2 spike glycoprotein. these amino acids interact with the human ace-2 receptor which facilitates their entry inside the brain, hence inflicting neuronal and neurovascular disruption in affected patients [26] . after successful docking process execution, the top 10 models were downloaded and analyzed for amino acid interaction between sars-cov-2 spike receptor and protein ligands and for the type of bond formed by each other. the hydrogen bonds in ppi holds the participating amino acid residues of one protein in bond formation with the other protein more vigorous than van der waals forces [27] . by analyzing individual docked models, it was observed that, human neutralizing s230 light chain amino acid residues; serine 24 and serine 30 established h-bonds with the arginine 365, phenylalanine 388, tyrosine 267 of sars-cov-2 spike protein whereas valine 1 of s230 formed a h-bond with the tyrosine 312 having -22.94 kcal/mol of binding free energy (supplementary figure 1) . on the other hand, ace-2 formed van der waal interaction with sars-cov-2 spike proteins with the binding free energy lower than mab s230 as summarized in table 1 figures 4 & 5) . the results are summarized in table 1 . none of proteins/peptides: 6nb6, 4awn, 1atu, 4n4x and 1r4l demonstrate any kind of toxicity or allergic response in silico. only splunc1 demonstrated a 33.3% allergenic response in the in silico model. apart from the specific s18 of splunc1 peptide chain, they were nontoxic and nonallergenic. (table 2 ) none of the proteins demonstrated resistance to the digestive enzymes. the purpose of conducting the resistance to digestive enzymes test was to check the fate of these inhalers as ultimately, they reach to the stomach and may cause adverse drug reactions. these inhalers were checked against the enzymes, such as trypsin, pepsin (ph > 2) and chymotrypsin. these inhalers are not able to protect themselves from these digestive enzymes, and are broken down in the stomach, and so cannot illicit unwanted physiological effects. the poor solubility of these inhalers may be due to the fact that all of them are protein/peptides and are hydrophobic in nature. the greater the aqueous solubility of these drugs, the greater will be the chance for these inhalers to illicit a response in the systemic circulation [28] . sars-cov-2 spike glycoprotein is of great concern within the scientist community. these proteins can only reproduce by entering the cells. blocking such entry might be of great interest in terms of treatment and that could be a way of preventing sars-cov-2 infection and flattening the disease curve [4] . we attempted to address this issue by analyzing a variety of protein/peptide-based inhalers/antimucolytic agents and previously utilized mab (used in asthma) to observe their possible interaction with the sars-cov-2 spike protein. both mab and da established h-bonds with spike protein employed by sars-cov-2 for attachment. however, in comparison with da, the mab and aat showed considerable h-interaction with the sars-cov-2 spike protein. cf and chronic obstructive pulmonary disease (copd) have high levels of dna and actin in the lung lumen, which are discharged by necrotic neutrophils. the dna population and actin alter mucus rheology as well as increase mucociliary clearance. another way to boost mucociliary clearance in cf is to decrease mucus viscosity by cleaving extracellular dna in infectious lungs. da is a recombinant version of human dnase-1-protein which is used as a therapeutic moiety for cf [7] . dnase1 cleaves extracellular dna in the lung lumen, which results in reduced dna length/concentration and decreased sputum viscosity [6] . this agent can also help in the treatment of covid-19. splunc1, on the other hand, does not demonstrate good interaction with the spike protein but does inhibit enac by inducing endocytosis incongruous to traditional ion channel antagonists which the block enac's pore [8] . regulation of enac fails because of splunc1 in the cf lung, so, splunc1-derived peptide acts in cf airways as an enac inhibitor. this regulates cf airway surface liquid hydration, boost mucociliary clearance and decrease infection, and inflammation [6] . these peptides are heat stable and with maximum binding efficiency to accomplish a greater contact area with their target proteins. s18-derived peptides do not readily cross the respiratory epithelium. they do not reach the kidney, where they might cause hyperkalemia because s18-derived peptides are proteaseresistant as seen with small-molecule enac antagonists like amiloride. chronic inhalation therapy results in local immunogenicity and irritation from the peptides involved, but it has been well established that splunc1-derived peptides did not precipitate immunogenicity [6, 8] . our study highlights the toxic and antigenic profiles of the corresponding inhalers. although the safety and efficacy of these inhalers, such as da, splunc1, aat and mabs were established already [16] [17] [18] [19] , we report the in silico antigenicity of splunc1 protein while bearing in mind the potential side effects of mabs [13] . therefore, they could be utilized in the covid-19 therapy and as an adjuvant. antibodies with therapeutic and diagnostic potential for sars and mers have already been identified [3, 26] . the preparation of particular variants of antibodies that attach to sars and mers viruses are patented in 61 out of 99 registered agents. in 42 patents the s-protein in the viral infection exhibits an immune response. the scrutiny of patents associated with the establishment of therapeutic antibodies for sars has been provided by liu et al. [29] . these antibodies are 90% in opposition to s proteins along its rbd. moreover, compared with chemical drugs, mabs bind to multiple places on viral surfaces. this gives them an edge over chemical drugs. mabs become attached to a wide variety of proteins with high affinity and specificity. after breakdown, the products are amino acids, so the resultant compounds are not toxic [6] . an exciting property of the mabs is that they cling to their physical and immunological properties after the aerosolization, which implies that it is only a matter of time before mab inhalation is exploited therapeutically. mab and da might synergistically clear the mucus, alleviate coughing and modulate the immune response toward the virion of the spike glycoprotein eventually disrupting the viral attachment. these inhalers could be used in in low-income countries where people cannot afford convalescent plasma therapy and where there is a shortage of inhalers/nebulizers. on 24 march 2020, different pharmacists across the usa reported a shortage of metered-dose inhalers [30] . unfortunately, despite the demand, the production of these inhalers has not kept pace. the problem was further aggravated by people stocking up on inhalers due to fear of the pandemic. however, despite their usefulness, these inhalers are not commonly available in pharmacies due to shortage of supplies. pharmaceutical companies and health regulatory authorities in all the countries should ensure a proper supply of these inhalers. because of the apparent devastating situation caused by covid-19, computational studies can help in a quick drug repurposing analysis. mab and da demonstrated efficient binding with the spike protein of sars-cov-2. the protein/peptide-based inhalers are valuable assets due to their prolonged action, higher potency, lower systematic availability and minimum toxicity. therapeutics for covid-19 are in the infancy stage and no us fda approved vaccine is available. during the pandemic, the use of protein/peptide-based inhalers along with other vaccines for sars and mers could mitigate the disease and serve as both therapeutic and prevention. the general instructions for the use of these protein/peptide-based inhalers are: • follow all the manual instructions that are given by the manufacturers regarding the use and cleaning of the inhalers, compressors and nebulizers. • these inhalers are usually present in solution form in plastic/glass ampoules. • air compressor is connected to a jet nebulizer or erapid™ nebulizer system to convert the drug (pulmozyme r ) into minutely small particles [31] . • the patients should breathe in the spray through a mouthpiece for about 10-15 min or until all the nebulizer cup is emptied. • pari baby™ nebulizer must be used if the patient is unable to inhale or exhale. • never utilize a discolored or cloudy inhalation solution. • use it every day till your condition is ameliorated upon the direction of the physician. • the frequency/dose of the drug should be calculated by the pharmacist on an individual basis according to the strength of the drug, severity of the symptoms, disease progression and body mass index. • precautions should be taken to avoid drug-drug and drug-food interactions. • if you forget to take the medicine, do so when you remember, unless you are near the time of your following dose. in this case, do not take the dose you have missed, but return to the original schedule. never double the doses. • administer one drug at a time and do not mix it with any other agents [31] . • the prescribers are advised not to switch between the inhaler types during the treatment unless otherwise required. during the pandemic, certain areas of the world are experiencing shortages of the inhalers but it is not because of production problems. the spike in demand is due to patient overflow and the use of inhalers for respiratory difficulties experienced by the suspected and confirmed cases of the covid-19. it is also due to the panic among the 10 .2217/fvl-2020-0119 future virol. (epub ahead of print) future science group people and pharmacists who are ordering inhalers which are ultimately not needed in such high quantities. other reasons according to the fda are regulatory and logistical challenges in different countries. the manufacturers are not given incentives to produce economical inhalers [32] . this report provided some solutions to this issue including: • contracts with private companies to ensure a reliable supply of the inhalers. • incentives should be given to the manufacturers who provide a sustainable supply of good quality inhalers. • public sessions and seminars should be held to promote awareness of the impact of drug shortage on patients. different hospitals are moving away from the nebulizer use in order to avoid the spread of sars-cov2. so, the doctors are suggesting using these inhalers at home [30] . virtual screening of the inhalers should be subjected to clinical trials before the issuance of the necessary medical recommendations. the in silico studies cannot completely replace the experimental studies, so we do not favor using these protein/peptide-based inhalers while first-line therapy options are available, nor do we advise readers to do so. but given the shortage of albuterol and other inhalers according to the fda, the use of these peptide-based inhalers could be appropriate in the future. • molecular docking analysis of protein/peptide-based inhalers revealed that the s230 light chain antibody and dornase-alfa demonstrated a strong affinity for sars-cov-2 spike protein. • the binding free energy of the s230 light chain antibody and dornase-alfa were -22.94 and -26.35 kcal/mol indicating highly stable bonding with the spike receptor. • toxicity assessment of these inhalers suggest that they can be used as anti-spike attachment agents, except for splunc1, which showed a 33.3% allergic reaction in silico. • s230 light chain antibody, dornase-alfa and short-palate lung and nasal-epithelial clone-1-derived peptides are capable of impeding virus interaction with the human host cell. to view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/doi/suppl/10.2217/fvl-2020-0119 all authors equally contributed for this study. covid-19 -navigating the uncharted virtual screening of immunomodulatory medicinal compounds as promising anti-sars-cov-2 inhibitors emerging coronaviruses: genome structure, replication, and pathogenesis clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study convalescent plasma to treat covid-19: possibilities and challenges inhaled protein/peptide-based therapies for respiratory disease dornase alfa for cystic fibrosis splunc1 degradation by the cystic fibrosis mucosal environment drives airway surface liquid dehydration lung epithelial derived splunc1 regulates th2 inflammation in allergic asthma spx-101 is stable in and retains function after exposure to cystic fibrosis sputum chapter 2 -pulmonary delivery of peptides and proteins pulmonary delivery of therapeutic peptides and proteins the safety and side effects of monoclonal antibodies inhaled insulin forms toxic pulmonary amyloid aggregates the lung as a route for systemic delivery of therapeutic proteins and peptides dornase alfa ototoxic effects in animals and efficacy in the treatment of clogged tympanostomy tubes in children: a preclinical study and a randomized clinical trial evaluation of a splunc1-derived peptide for the treatment of cystic fibrosis lung disease efficacy and safety of inhaled alpha-1-antitrypsin in patients with severe alpha-1-antitrypsin deficiency and frequent exacerbations of chronic obstructive pulmonary disease monoclonal antibodies in type 2 asthma: a systematic review and network meta-analysis pubchem substance and compound databases cryo-em structure of the 2019-ncov spike in the prefusion conformation improving the physical realism and structural accuracy of protein models by a two-step atomic-level energy minimization hawkdock: a web server to predict and analyze the protein-protein complex based on computational docking and mm/gbsa in silico approach for predicting toxicity of peptides and proteins protein identification and analysis tools on the expasy server bt -the proteomics protocols handbook evidence of the covid-19 virus targeting the cns: tissue distribution, host-virus interaction, and proposed neurotropic mechanisms regulation of protein-ligand binding affinity by hydrogen bond pairing drug solubility: importance and enhancement techniques. isrn pharm research and development on therapeutic agents and vaccines for covid-19 and related human coronavirus diseases covid-19 pandemic sparking inhaler shortages dornase alfa (pulmozyme) drug shortages: root causes and potential solutions the authors thanked caleac for proofreading and editing the research design of this article. the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.no writing assistance was utilized in the production of this manuscript. key: cord-316258-7hucqcaj authors: henriques, elsa s; brito, rui m m; soares, hugo; ventura, sónia; de oliveira, vivian l; parkhouse, r michael e title: modeling of the toll-like receptor 3 and a putative toll-like receptor 3 antagonist encoded by the african swine fever virus date: 2011-01-28 journal: protein science doi: 10.1002/pro.554 sha: doc_id: 316258 cord_uid: 7hucqcaj african swine fever virus (asfv) is a large double-stranded dna virus responsible for a lethal pig disease, to which no vaccine has ever been obtained. its genome encodes a number of proteins involved in virus survival and transmission in its hosts, in particular proteins that inhibit signaling pathways in infected macrophages and, thus, interfere with the host's innate immune response. a recently identified novel asfv viral protein (pi329l) was found to inhibit the toll-like receptor 3 (tlr3) signaling pathway, tlr3 being a crucial “danger detector.” pi329l has been predicted to be a transmembrane protein containing extracellular putative leucine-rich repeats similar to tlr3, suggesting that pi329l might act as a tlr3 decoy. to explore this idea, we used comparative modeling and other structure prediction protocols to propose (a) a model for the tlr3–toll-interleukin-1 receptor homodimer and (b) a structural fold for pi329l, detailed at atomistic level for its cytoplasmic domain. as this later domain shares only remote sequence relationships with the available tlr3 templates, a more complex modeling strategy was employed that combines the iterative implementation of (multi)threading/assembly/refinement (i-tasser) structural prediction with expertise-guided posterior refinement. the final pi329l model presents a plausible fold, good structural quality, is consistent with the available experimental data, and it corroborates our hypothesis of pi329l being a tlr3 antagonist. african swine fever virus (asfv) is the etiological agent of an acute hemorrhagic fever of the domestic pig, with a mortality rate approaching 100%. although not a human pathogen, this virus is a serious concern to swine farming and livestock economy, as it is endemic in sardinia and sub-saharan countries. the increasing infections in africa and the worldwide commercial trade provide serious risk factors to the global pig industry; plus, there is no vaccine and so control is still based on diagnosis and the subsequent adoption of strict sanitary measures. 1 asfv is a large, cytoplasmic, double-stranded dna (dsdna) virus and the single member of the asfarviridae family, encoding many novel genes not encoded by other virus families. as it primarily infects porcine macrophages-a key cellular component of the innate immune system-this virus may have evolved immune evasion genes to manipulate innate immunity. the prediction is that half to two-thirds of the approximately 150 genes encoded by asfv are not essential for replication in cells but have an important role for virus survival and transmission in its hosts. so far, the major strategy of the known asfv proteins with roles in evading host defenses seems to interfere with intracellular signaling pathways and to inhibit transcriptional activation of important immunomodulatory genes. 2 this could in part explain the absence of an adequate host response on infection by asfv. understanding the viral proteins involved in this strategy can point the way to key drug targets or even be of therapeutical use themselves (or derivatives of them) to curtail inflammation. there are still a number of asfv-encoded proteins of unknown function that could be worth exploring for that purpose. the innate immune system is mediated by germline encoded pattern recognition receptors, each receptor having a broad specificity for conserved components of microorganisms, such as nucleic acids, polysaccharides, and lipids. the molecular signature of most viruses is double-stranded rna (dsrna), produced either as an intermediate of the viral replication cycle (e.g., for dsdna viruses) or as part of the viral rna genome. viral dsrna is recognized by the toll-like receptor 3 (tlr3), a member of the well characterized tlr family that comprises 10-20 pattern recognition receptor paralogs, all being type i integral membrane proteins. on dimerization subsequent to recognition of dsrna, tlr3 recruits the adaptor protein toll-interleukin-1 receptor (tir)-domain-containing adapter-inducing interferon-b (trif) to its cytoplasmic domain, thereby initiating a signaling cascade that results in the secretion of type i interferons and other inflammatory cytokines. 3 trif is actually the sole tlr adaptor that is able to engage mammalian cell death signaling pathways, and tlr3 is the only receptor in the tlr family that interacts directly with it. interestingly, asfv not only infects pigs but also ticks, both of which share tlr-mediated host defense systems. this, and the fact that asfv specifically infects macrophages, makes it only conceivable that some of the unassigned asfv-encoded proteins might well interfere with tlr3 or other tlr signaling mechanisms. in search for possible topological similarities and sequence homologies with already existing proteins, a preliminary computational screening of the asfv open reading frames (orfs) predicted a protein of unknown function, named pi329l after orf i329l, to be a transmembrane protein containing extracellular putative leucine-rich repeats (lrrs). as tlrs are also transmembrane proteins featuring an extracellular domain with lrr motifs, 4 this apparent similarity prompted for experimental testing to check for interference with tlr-signaling. the results showed that pi329l is a highly glycosylated protein expressed in the endoplasmic reticulum, the golgi, and the cell surface of recombinant lenti-virus transduced cells, and that it inhibits tlr3-dependent activation of two transcription factors (nfjb and irf3) responsible for the expression of interferon and chemokines (de oliveira et al. 24 ). and as presented here, the experiments also indicate that trif is the putative target for this viral host modulation gene. in more detail, tlr3 features a large glycosylated ectodomain (ecd) with multiple lrrs responsible for ligand recognition, a transmembrane a-helix and a cytoplasmic tir domain responsible for initiation of intracellular signaling. the recently proposed structures of two tlr3-ecds bound to a 46-bp dsrna (protein data bank [pdb] entry 3ciy 5 ) and of the tlr4-tir homodimer 6 provided a credible structural picture of how viral dsrna is recognized and how the signaling adaptors might be recruited. each of the tlr3-ecds binds dsrna at two sites located at opposite ends of the tlr3 horseshoe (refer to ref. 5 and fig. 5) , and an intermolecular contact between the two tlr3-ecd c-terminal domains coordinates and stabilizes the dimer. this juxtaposition should then mediate downstream signaling by the dimerization of the cytoplasmic tir domains. 5 despite the much shorter sequence of the viral protein, all the above information put together gave rise to the hypothesis that pi329l might form a heterodimer with tlr3, thus acting like a decoy receptor. here, we report a structural assessment of this hypothesis. using homology modeling and other structure prediction simulation protocols, we propose (a) a model for the so far experimentally unsolved tlr3-tir structure, assembled within the context of the overall tlr3-dsrna recognition complex, and (b) a structural fold for the pi329l intracellular extension that reinforces the idea of the viral protein being a tlr3 antagonist. the computational models are discussed and validated, are consistent with the available experimental data, and preliminary conclusions concerning the role of the asfv viral protein pi329l are put forward. structural assessment of the pi329l ecd the tmhmm posterior probabilities of inside/outside/transmembrane regions for the pi329l sequence are depicted in figure 1 , together with the equivalent analysis on tlr3 for comparison. for pi329l, the expected number of amino acids in transmembrane helices is 22.90714 within region 238-260 of the sequence; the number being larger than 18 and not in the n-first 60 residues (ruling out the possibility of a signaling peptide), it is very likely that pi329l is a transmembrane protein. the phyre server ranked the nogo receptor (ngr) ecd (pdb entry 1ozn) as the most apposite homologous fold of the pi329l putative ecd. ngr is a 470-residue, glycosyl phosphatidyl inositol-anchored membrane protein with a majority of the globular structure comprised of a lrr domain capped by n-terminal and c-terminal cysteine-rich modules. 7 as can be seen in figure 2 , the ngr structure easily superposes the tlr3-ecd membrane-adjacent region, despite the relatively low sequence homology (20% identity and 40% similarity, for the span of the ngr-ecd sequence). combining this structural superposition with the phyre-proposed alignment of pi329l to ngr, we devised the alignment presented in figure 3 . calculated (clustalw) sequence identity and similarity of pi329l are 15 and 34% to ngr (ecd only) and 11 and 25% to tlr3 (ecd þ transmembrane region). noticeable from the alignment is the pi329l's lrr pattern (a series of short segments rich in hydrophobic residues such as leucine, isoleucine, and valine 8 ). also worth mentioning is that the region aligned with the c-terminal cysteine-rich capping motif of ngr and tlr3 (underlined in fig. 3 ), delineates at least one of the conserved structural disulfide bonds of this motif. in addition, phyre predicted a high a-helix content for pi329l in this region (data not shown) in close agreement with the one observed in ngr 7 (highlighted in fig. 2 ). these results are consistent with our hypothesis of pi329l being a decoy of tlr3 through the formation of a heterodimer. the fact that the putative ecd of pi329l is considerably shorter than the tlr3 counterpart (note that the alignment in fig. 3 presents only the last $340 out of the 680 residues of tlr3-ecd) is not an issue, for it is the two c-termini of tlr3-ecd (i.e., the region with the aforementioned disulfide bonds, aligned with pi329l) that are brought into contact on binding to dsrna then followed by the association of the transmembrane helices and the dimerization of the cytoplasmic tir domains. 5 bearing this in mind, the subsequent modeling efforts were directed to the pi329l-cytoplasmic domain to consider a possible downstream inhibition of trif-induced signaling. to understand the structural implications of a possible heterodimer between the pi329l and tlr3 cytoplasmic domains, a necessary first step is to define the tlr3-tir dimer itself. as not even the structure of the monomer has been experimentally solved to date, our next step was to build a reasonable model of the tlr3-tir structure. a number of experimental structures for other tlr-tir domains are already available, including those of tlr1 (pdb entry 1fyv 9 ), tlr2 (pdb entry 1o77 10 ), and tlr10 (pdb entry 2j67 11 ), the latter in the form of a putative signaling dimer. these three receptors were found to be suitable homologous tem-plates for tlr3-tir, with values of identity and similarity of 26 and 57% (tlr1), 22 and 56% (tlr2), and 27 and 54% (tlr10), respectively, following the alignment in figure 4 . compared with the sequence homology among the three templates themselves (identity varies from 46 to 69%), these values emphasize the atypical character of tlr3 within the tlr-family. unfortunately, none of the available crystallographic structures that could serve as a template has the cytoplasmic linker region-the one immediately following the transmembrane domain, resolved (for the chosen templates, the n-terminal region displayed with the unresolved residues in italic in the alignment of fig. 4 ). this region is consistently predicted (phyre-server) to have an a-helix fold and, at least in the case of tlr3, it bears a few key functional residues that are required for tlr3induced activation of two transcription factors that promote the antiviral response. 4 these are residues phe732, tyr733, leu742, and gly743 (residue numbering according to ref. 4) marked with an asterisk in the alignment (fig. 4) , which are conserved across human, pig, mouse, and other species, respectively. the initial model structures of the tlr3-tir domain were built with modeller using the alignment presented in figure 4 . the most promising models were subjected to further restrainedmodeling for the cytoplasmic linker, by invoking the special restraints routine to impose an a-helix structure to it. the best quality refined structure presents all bonding parameters within the allowed limits, no bad contacts, and the corresponding ramachandran plot analysis (procheck) shows 94.0% of the residues in the core regions and 6.0% in the additional allowed regions. the model is of equivalent quality to the best quality template structure, the tlr10-one with 93.1% of its residues in the most favored regions. finally, the model was relaxed with 100 ps of molecular dynamics (md) under physiological conditions to ensure that it sustains the folding characteristics. as suggested by liu et al., 5 the dimerization of the cytoplasmic tir domains results from the interactions of the lrr c-terminal ecds on rna-binding, and similarly to what has been depicted by those authors, a construct of the entire tlr3-dsrna recognition complex dimer was next assembled to further assess the validity of our tlr3-tir model. two tlr3-tir monomers were first placed together using the structure of the tlr10 dimer as scaffold, and then connected to the experimentally determined structure of the ectodomains 5 via two a-helices-the transmembrane domains-which were built and positioned using distance-restrained modeling within modeller. the resulting model is depicted in figure 5 . the entire structure was positioned in a membranemodel to further ensure that each domain would fall in the appropriate cellular compartments. for the tlr3-tir dimer, the c-alpha root mean square deviation to the template protein is 0.90 å . as in the case of tlr10, the so called bb-loop and central distorted a-helix c (nomenclature used in previous works on tir domains 4,5,11 ) constitute the major part of the dimer's interface. the bb-loop (signaled in box 2 in the alignment of fig. 4) shares a conserved proline in all tlrs except tlr3, where this residue is replaced with an alanine (ala795 marked with an asterisk in the alignment). it should be pointed out that this is the binding-adaptor loop, which on dimerization shapes a twofold symmetrical exposed patch, 11 where the appropriate tlr adaptor is suggested to dock. in fact, experimental evidence demonstrates the importance of that particular alanine in the binding of tlr3 directly to the adaptor trif, while the other tlrs require mediator adaptors. 4 as for a-helix c, it has an extra arginine residue in tlr3 when compared with all other tlrs (the insertion also signaled in the alignment), which no doubt adds to the uniqueness of tlr3 within its family. in fact, this is to say that there might be some complementarity nuances in the dimer's contact region that may not be revealed by homology modeling alone. for the purpose of this work, however, it suffices to map the critical regions that the virus protein under study might interfere with. not surprisingly, attempts to sequence-align the pi329l putative cytoplasmic domain with the tlr3-tir domain resulted in very different alignments depending on the chosen clustalw scoring matrix and gap penalty. homology values were always too low for straightforward homology modeling to be considered (8% identity and 24% similarity at the best). the phyre server predicts a protein-fold featuring an initial a-helix and three to four small b-strands, with no match to their proposed set of weakly/distant homologous templates. a more sophisticated modeling approach was thus required, one possibility being the iterative implementation of (multi)threading/assembly/ refinement approach (i-tasser), the ''zhang-server'' that ranked as the no. 1 server in recent casp7 and casp8 experiments (http://predictioncenter.org/casp8/ groups_analysis.cgi). the i-tasser predicted secondary structure (nearly equivalent to phyre's) is presented in figure 6 . the server also proposed five crude models for the pi329l domain in question, the higher ranking one displaying a confidence score of à3.81 (range is typically within à5 to 2, the higher the value the higher the confidence 12 ). interestingly, a pdb-search by secondary structure content revealed a comparable motif for the recently solved nmr-structure of domain-c of the nonstructural nsp3e protein from the severe acute respiratory syndrome (sars) coronavirus (cov) (pdb entry 2kaf 13 ). the resulting alignment of these two viral protein domains within the secondary structure prediction context is also presented in figure 6 . the i-tasser best model was then used for further refinement within modeller, using the sars-cov domain as an additional template. the available tlr structures were used only to guide the modeling of a few localized segments. two apposite refined models for the pi329l-cytoplasmic domain were obtained in this way, both presenting a plausible fold and good structural quality. the ramachandran plot analysis showed 87.8% of the residues in the core regions (it is 88.1% for the best representative conformer in the 2kaf ensemble) and the remaining in the additional allowed regions. the alignment of the viral proteins with tlr3-tir, also depicted in figure 6 , was attained by superposition of the structural features assigned to each sequence. based on proximity criteria, both models of pi329l allow for two disulfide bonds (signaled in fig. 6) , which would be expected if such a small domain ($80 residues) is to sustain an ordered compact fold. the two models are practically equivalent, differing only in the n-terminal a-helix, which is ''straight'' in one case and ''distorted'' in the other (refer to fig. 6 ): both motifs are eligible considering that (1) this is the frontier fragment between the transmembrane and cytoplasmatic domains and (2) one such kink would not be uncommon in the region of a potential disulfide bond. figure 6 . the view in figure 7 (b) highlights the tlr3-tir residues that must come into play on homodimer formation, and how pi329l could ''ill-replace'' one of the monomers at the interface region. whether the outcome is mimic-binding or a more simple steric hindrance, either would suffice to impede the correct formation of the twofold symmetric region where the adaptor trif is expected to bind, and disrupt its corresponding signaling. moreover, at least one of the phosphorylation sites required for downstream regulation 4 (the tyrosine-759 in box 1, refer to the alignments in figs. 4 and 6) would be ousted in this scheme. arguably, the actual tlr3-region covered by the pi329l cytoplasmic domain would also depend on the positioning of the a-helix cytoplasmic linker in tlr3 (the first 25 residues in the alignment of fig. 4 , for which there are no template counterpart). the way it has been modeled [figs. 5 and 7(b) ], this linker accommodates on top of the tir domain, but even if it was somewhat detached from it, that would alternatively cause pi329l to mainly superpose over the linker. since (as mentioned in the previous subsection) the linker bares several residues required for tlr3-induced activation of two key transcription factors (nfjb and irf3), the very same that are inhibited by i329l expression, our proposed model still holds. one such signal inhibition hypothesis is also corroborated by the experimental observation that overexpression of trif does indeed reverse the inhibition caused by pi329l, as inferred from the results of the luciferase assay presented in figure 8 . the luciferase gene is controlled by the interferon-b (ifnb) promoter and the graph shows that the inhibition of the luciferase reporter activation induced by i329l is reverted in a dose-dependent manner by cotransfection of trif. remark that the possible inhibitory role of pi329l at the intracellular level does not exclude the potential decoy purpose of its extracellular domain, which is implied in the structural assessment of the i329l ecd presented in the corresponding subsection above. following the preliminary screening of the asfv orfs (data not shown), the sequence of the pi329l precursor-as taken from the ncbi genbank database (www.ncbi.nlm.nih.gov; accession number aaa65369)was further analyzed using the tmhmm program for predicting transmembrane regions. 14 it gives the most probable location and orientation of transmembrane helices in the sequence as found by the n-best algorithm that sums over all paths through the model with the same location and direction of the helices. 14 other possible similarities/homologies were investigated using phyre, the protein fold recognizer server, 15 which predicts protein structures by detecting remote homology to known structures. multiple sequence alignments were performed with clustalw2 16 and edited for manual refinement within jalview. 17 tlr3 structures were homology modeled with modeller 9v4, 18 based on the alignment presented in figure 4 . for the putative cytoplasmic domain of pi329l, a preliminary set of crude models was built with the i-tasser server for protein prediction. in short, i-tasser generates full length models of proteins by excising continuous fragments from local meta-threading-server multiple-threading alignments and then reassembling them using replica-exchange monte carlo simulations (for more details see ref. 12 and references within). it followed a more accurate modeling with modeller using the i-tasser results as starting input and an additional template (vide supra the modeling of pi329l) that resulted from a targeted pdb-search. 19 the quality of the models was assessed using procheck v.3.4.3. 20 the best quality modeled structures were regularized and md-relaxed using the namd program 21 with the charmm22 force-field. 22 the possible impact of i329l on trif signaling was further investigated through a luciferase reporter gene assay. vero cells were cotransfected with 300 ng of either the empty plasmid vector (pcdna3-ha) or the i329l expression vector (pcdna3-i329l-ha), and with increasing amounts of the trif plasmid vector (25-100 ng) along with 100 ng of the ifnb reporter construct [pifd(à125/þ72)lucter]. the cells were finally stimulated with 25 lg/ml of poly i:c for 5 h. luciferase activity was normalized to the b-galactosidase activity given by the cotransfected b-galactosidase internal control plasmid (pcmvb). the proposed fold and structural model of the pi329l viral protein was devised using both automatic tools and human expertise in template-based modeling, along with experimental guidance. one such strategy has been proven adequate, when only remote relationships between the target and the structural templates exist. 23 special attention has been paid in choosing the appropriate alignment(s) and template(s). the working hypothesis being that the viral protein is an antagonist of the tlr3, for which no experimental structure of its tir-domain exist, a model of this domain in the context of the tlr3-dsrna recognition complex was first assembled. it provided a background framework for the subsequent comparative modeling of the pi329l cytoplasmic domain. the modeling results substantiate the idea that pi329l may function as a tlr3 decoy, showing that the viral protein could hinder tlr3 dimerization, and in doing so, inhibit the downstream signaling pathway. in conjunction with the experimental evidence, the present modeling exercise allowed us to gain further insight into the strategies used by the asf-virus to evade the host immune response and the role of the nonassigned i329l gene encoded viral protein in this process. modeled structures are available on request. systematic analysis of longitudinal serological responses of pigs infected experimentally with african swine fever virus african swine fever virus proteins involved in evading host defence systems ticam-1, an adaptor molecule that participates in toll-like receptor 3-mediated interferon-beta induction sensing of viral infection and activation of innate immunity by toll-like receptor 3 structural basis of toll-like receptor 3 signaling with double-stranded rna a dimer of the toll-like receptor 4 cytoplasmic domain provides a specific scaffold for the recruitment of signaling adaptor proteins structure of the nogo receptor ectodomain: a recognition module implicated in myelin inhibition the leucine-rich repeat structure structural basis for signal transduction by the toll/interleukin-1 receptor domains an extensively associated dimer in the structure of thec713s mutant of the tir domain of human tlr2 the crystal structure of the human toll-like receptor-10 cytoplasmic domain reveals a putative signaling dimer 2008) i-tasser server for protein 3d structure prediction nmr assignment of the nonstructural protein nsp3(10661181) from sars-cov predicting transmembrane protein topology with a hidden markov model: application to complete genomes protein structure prediction on the web: a case study using the phyre server clustalw and clustalx version 2.0 jalview version 2-a multiple sequence alignment editor and analysis workbench comparative protein structure modeling using model-ler the worldwide protein data bank (wwpdb): ensuring a single, uniform archive of pdb data procheck: a program to check the stereochemical quality of protein structures namd2: greater scalability for parallel molecular dynamics all-atom empirical potential for molecular modeling and dynamics studies of proteins the use of automatic tools and human expertise in template-based modeling of casp8 target proteins vember 2010) novel tlr3 inhibitor encoded by the african swine fever virus (asfv). arch virol key: cord-320713-b37c8aye authors: roberts, lisa o.; jopling, catherine l.; jackson, richard j.; willis, anne e. title: chapter 9 viral strategies to subvert the mammalian translation machinery date: 2009-10-27 journal: prog mol biol transl sci doi: 10.1016/s1877-1173(09)90009-6 sha: doc_id: 320713 cord_uid: b37c8aye viruses do not carry their own protein biosynthesis machinery and the translation of viral proteins therefore requires that the virus usurps the machinery of the host cell. to allow optimal translation of viral proteins at the expense of cellular proteins, virus families have evolved a variety of methods to repress the host translation machinery, while allowing effective viral protein synthesis. many viruses use noncanonical mechanisms that permit translation of their own rnas under these conditions. viruses have also developed mechanisms to evade host innate immune responses that would repress translation under conditions of viral infection, in particular pkr activation in response to double-stranded rna (dsrna). importantly, the study of viral translation mechanisms has enormously enhanced our understanding of many aspects of the cellular protein biosynthesis pathway and its components. a number of unusual mechanisms of translation initiation that were first discovered in viruses have since been observed in cellular mrnas, and it has become apparent that a diverse range of translation mechanisms operates in eukaryotes, allowing subtle regulation of this essential process. viruses do not carry their own protein biosynthesis machinery and the translation of viral proteins therefore requires that the virus usurps the machinery of the host cell. to allow optimal translation of viral proteins at the expense of cellular proteins, virus families have evolved a variety of methods to repress the host translation machinery, while allowing effective viral protein synthesis. many viruses use noncanonical mechanisms that permit translation of their own rnas under these conditions. viruses have also developed mechanisms to evade host innate immune responses that would repress translation under conditions of viral infection, in particular pkr activation in response to double-stranded rna (dsrna). importantly, the study of viral translation mechanisms has enormously enhanced our understanding of many aspects of the cellular protein biosynthesis pathway and its components. a number of unusual mechanisms of translation initiation that were first discovered in viruses have since been observed in cellular mrnas, and it has become apparent that a diverse range of translation mechanisms operates in eukaryotes, allowing subtle regulation of this essential process. viruses are obligate intracellular parasites and therefore depend on host cells for their replication. viruses have evolved a number of ways in which to modify the translation apparatus of the host cell to ensure preferential translation of virus mrnas, and use alternative and novel mechanisms to initiate translation of viral mrnas under such conditions. the study of viral translation mechanisms has been a rich source of information about cellular protein synthesis and its regulation. viruses are diverse in the ways they interact with the host and replicate. for example, their genomes can be made up of dna or rna, and within the rna viruses the genomes may be positive-sense rna, negative-sense rna, or dsrna. production of mrna transcripts may take place in the nucleus and therefore the virus can make use of host cell enzymes. alternatively, this process may occur in the cytoplasm, in which case the virus must encode, or bring with it, its own transcriptional system. viral mrnas also differ in their structure; some viral mrnas are capped and polyadenylated, and these viruses often adopt novel ways in which to ensure the viral mrnas are preferentially translated over cellular mrnas, either through modification of the host cell translational machinery and/or adoption of novel translational mechanisms. there are also a large number of viruses that do not produce capped mrnas and have evolved novel strategies to direct initiation of protein synthesis. no virus has been discovered that encodes its own translation system, and in fact this is one of the main distinguishing features between a virus and a living cell. however, the recent discovery of the ''giant'' virus, acanthamoeba polyphaga mimivirus 1,2 has challenged this long-held belief. trnas have been discovered in a number of such giant viruses and this new mimivirus has also been shown to encode a number of translation factors and four aminoacyl-trna synthetases. the virus encodes homologs of eukaryotic initiation factor (eif)4e, eif4a, and eif1 and also possesses a homolog of the release factor erf1. although the virus therefore encodes proteins involved in all stages of translation, it does not encode any ribosomal components. it has been speculated 2 that these viral components probably represent the remains of a more complex translational system that has been gradually lost over time, rather than an acquisition of cellular components. in summary, to enable efficient synthesis of viral proteins a virus needs to be able to do one or more of the following: (1) modify the host translation machinery to favor the translation of viral rather than host encoded mrnas. many viral rnas are uncapped and/or contain highly structured 5 0 -untranslated regions (utrs) that would inhibit the scanning ribosome, so the viral rna would compete poorly with host encoded mrnas for the translation machinery. (2) use novel mechanisms that allow the selective synthesis of viral proteins. (3) circumvent the host defense mechanisms that function to inhibit translation following viral infection. a detailed molecular understanding of these three processes has the potential to provide unique insights into viral replication strategies and therefore highlight potential new targets for antiviral therapies. translation in mammalian cells is a multistep, highly regulated process (see chapter by fraser, this volume). it can be considered as three phases; initiation, elongation, and termination. all three phases are regulated, although initiation is thought to be the rate-limiting step for the whole process. for initiation to occur the eif4f complex, comprised of the cap-binding protein eif4e, the helicase eif4a, and the bridging protein eif4g, binds to the mrna. the 40s ribosomal subunit is recruited via an eif4g-eif3-40s interaction together with the ternary complex, which contains eif2, gtp, and initiator met-trnai. the resulting complex is known as the 48s complex. the scanning model of translation initiation predicts that this complex then scans along the mrna until the start codon is reached. 3 the poly(a)-binding protein (pabp) interacts with both the polya tail and the n-terminal half of eif4g to circularize the mrna (refs. 4,5; fig. 1 ). an interaction between eif4b, which binds to eif4a, and pabp further stabilizes this circularization. 6 the rate of translation initiation in mammalian cells is also controlled by sequence elements within the 5 0 -and 3 0 -utrs of mrnas which regulate this process by providing sites for interaction of regulatory proteins and rnas. these include upstream open reading frames (uorfs), microrna (mirna) target sites, and polyadenylation elements. 7, 8 transcription of mrnas from mammalian dna virus genomes such as herpesviruses and adenoviruses occur in the nucleus and results in the production of capped viral mrnas. these viruses need to establish conditions to permit selective translation of viral mrnas over cellular transcripts. it is therefore desirable to stimulate cap-dependent translation pathways and at the same time inhibit or downregulate the translation of host mrnas, which otherwise would be similarly stimulated, to provide the virus with a selective advantage. a major mechanism of regulation of cap-dependent translation is mediated by the eif4e-binding proteins (4e-bps), which regulate the formation of the eif4f complex ( the scanning model of translation initiation predicts that the 48s preinitiation complex moves along the mrna in a 5 0 -3 0 direction until it encounters an aug codon that is in a good context. many viruses produce proteases that cleave protein components of this complex to inhibit cap-dependent scanning. thus the cleavage of eif4g dissociates the ribosome binding ability of the complex from cap recognition. the cleavage of pabp by viral proteases will prevent the interaction of the 5 0 and 3 0 ends of the mrna and also reduce the stability of the complex. three 4e-bps in mammals, all of which act by binding to eif4e and inhibiting its interaction with eif4g, leading to an inhibition of cap-dependent translation initiation. hyperphosphorylation of 4e-bp1 releases eif4e and allows it to interact with eif4g in the eif4f complex. this hyperphosphorylation is stimulated by growth factors via the mammalian target of rapamycin (mtor) signaling pathway, and is a target of regulation for a number of viruses. how such regulation contributes to viral replication is described below. the herpesviruses are a large family of dsdna viruses and are responsible for a number of different diseases of vertebrates, such as cold sores caused by herpes simplex virus-1 (hsv-1) and chicken pox caused by varicella zoster virus (vzv) in humans. viruses of the herpesvirus family have adapted to colonize a variety of terminally differentiated cells 9 in which translation rates are generally low. these viruses establish latent infections during which a restricted subset of viral mrnas is expressed at a low level, so that the virus evades the host immune system. the virus replication may undergo periodic reactivation to a productive lytic infectious fig. 2. regulation of eif4e by 4e-bps. the availability of eif4e (the cap-binding protein) for eif4f complex formation (which also contains eif4g (the bridging protein)) and eif4a (a deadbox helicase), is controlled by interaction with its binding partners the 4e-bps which bind to and sequester eif4e. the interaction of eif4e with 4e-bp is regulated by phosphorylation and viral infection controls, either positively or negatively, the phosphorylation status of this protein. when bound to eif4g, eif4e can be phosphorylated by mnk1 and it has been suggested that this may increase the affinity of eif4e for the cap. the 100 k protein from adenovirus displaces mnk1 from eif4g and so prevents the phosphorylation of eif4e. viral strategies to subvert the mammalian translation machinery cycle, accompanied by major changes in viral gene expression. this switch from latent to lytic infection requires the induction of protein synthesis, and 4e-bp modification appears to play an essential role in this process. 10 the switch from latent to lytic infection in hsv-1-infected sensory neurons is accompanied by induction of 4e-bp1 phosphorylation. inactivation of 4e-bp1 in hsv-1-infected cells is increased further by proteasome-dependent degradation, and a decrease in the level of this protein accompanies the increase in phosphorylation. 11 the viral icp0 protein, an important master regulator of lytic reactivation, is required for both these processes. 4e-bp1 phosphorylation during hsv-1 infection is sensitive to the effects of rapamycin, suggesting that virus-induced signaling through mtor (see chapter by blenis and mahoney, this volume) is required for inactivation of this protein. 11 direct regulation of the mtor pathway occurs in cells infected with epstein-barr virus (ebv). the protein product of the lmp-2a gene activates mtor via pi3 kinase/akt signaling 12 and it has been suggested that the resulting phosphorylation and inactivation of 4e-bp1 may be required to increase translation in the transformed cells. 10 for other viruses in this family, such as human cytomegalovirus (hcmv), it would appear that additional mechanisms are also required, as rapamycin is not sufficient to abolish the 4e-bp1 phosphorylation induced in the virus infection. 13, 14 infection of b cells with kaposi's sarcoma associated herpesvirus (kshv) also stimulates hyperphosphorylation of 4e-bp1, although complete release of eif4e from 4e-bp1 is not observed in this case. 15 the best known poxviruses are smallpox virus and the smallpox vaccine virus, vaccinia virus. recently, the effects of poxvirus infection on the eif4f complex have been studied for the first time. poxviruses replicate in the cytoplasm of infected cells and manufacture capped viral mrnas using a viral methyltransferase complex 16 and therefore must effectively compete with host mrnas for the eif4f complex. it has now been shown that in vaccinia virus-infected cells 4e-bp1 is inactivated through its hyperphosphorylation. 17 in addition, the overall amount of 4e-bp1 decreases following vaccinia virus infection, so the virus is able to inactivate this protein through two different mechanisms. adenoviruses (adv) are also dna viruses and are widespread in humans and birds. these viruses have been shown to increase the phosphorylation of 4e-bp1, allowing eif4e to bind to eif4g and stimulate formation of the eif4f complex. 18 this suggests that the regulation of 4e-bp1 by multiple mechanisms is a common mechanism of regulation of translation initiation used by most dna viruses. the picornaviruses are a large family of positive-sense rna viruses including important animal and human pathogens such as foot-and mouth-disease virus (fmdv) and poliovirus (pv). the picornaviruses have uncapped mrnas that are translated by the cap-independent mechanism of internal ribosome entry. encephalomyocarditis virus (emcv) and, to a lesser extent, pv, affect 4e-bp activity by increasing the ability of this protein to bind to and sequester eif4e in order to silence cap-dependent translation of host mrnas and permit selective translation of emcv and pv mrnas. 19 upon infection with emcv, 4e-bp1 becomes dephosphorylated, and this coincides with the shutoff of protein synthesis that occurs. dephosphorylation of 4e-bp1 in pvinfected cells lags behind the shutoff of cellular protein synthesis, and it appears that in this situation protein synthesis inhibition is initiated by the cleavage of eif4g. further evidence of a role for dephosphorylation of 4e-bp1 in inhibition of protein synthesis during emcv and pv infections was demonstrated by addition of rapamycin, an inhibitor of 4e-bp phosphorylation, to virus-infected cells. this results in enhanced synthesis of emcv and pv viral proteins. 20 rhabdoviruses are negative-stranded rna viruses. inactivation of 4e-bp1 by dephosphorylation and downregulation of host protein synthesis is also observed in cells infected with vesicular stomatitis virus (vsv), despite the fact that vsv mrnas are capped. 21 however, multiple other factors control the translation of viral mrna including a relocalization of certain hnrnps from the nucleus to the cytoplasm. 22 c. other regulation of eif4f assembly in addition to their regulation of 4e-bp1, some herpesviruses stimulate the assembly of eif4f complexes in cells directly using a range of mechanisms. 11, 13, 14 for example, it has been shown that the icp6 protein produced in hsv-1 lytic infection is required for increased eif4f complex formation and interacts directly with eif4g, suggesting that it has a chaperone function. 10 hcmv infection leads to an increase in the abundance of eif4e, eif4g, and pabp, and to enhanced eif4f assembly. 14 reactivation from latency in kshvinfected cells also leads to a stimulation of eif4f assembly. interestingly, no viral strategies to subvert the mammalian translation machinery corresponding increase in the level of pabp association with the eif4f complex was observed, and pabp was seen to redistribute from the cytoplasm to the nucleus. this is perhaps surprising given the role of pabp in stimulating translation. 5 it was suggested that alternative eif4f complexes lacking pabp could selectively promote the synthesis of viral, but not host, proteins, so that kshv-encoded mrnas would compete more effectively for host translation machinery in infected cells. 15 poxvirus infection results in the reorganization of discrete cytoplasmic regions into replication factories. the poxvirus vaccinia virus induces the redistribution of eif4e, eif4g, and pabp to these replication compartments. it is not fully understood how redistribution of initiation factors occurs although it has been proposed that this may be important in selectively promoting translation of viral mrnas. 17 eif4e is phosphorylated on residue serine 209 by the map-kinase signalintegrating kinases mnk1 and mnk2 (reviewed in ref. 23 and the chapter by blenis and mahoney, this volume). the mnks bind to eif4g, bringing the kinase into close proximity to eif4e. [24] [25] [26] the exact role of eif4e phosphorylation in translational regulation is still unresolved, and although mnk1 and mnk2 are essential for constitutive and inducible phosphorylation of eif4e they are not required for cell growth or development. 27 however, it is thought that phosphorylation of eif4e leads to stimulation of cap-dependent translation 28 and this is associated with tumorigenesis. 29 changes in the phosphorylation state of eif4e are often seen in virus-infected cells and these have been shown to affect virus replication. adenovirus infection results in changes in eif4e phosphorylation that are important for virus replication. adenovirus mrnas are capped, but they are selectively translated during late viral infection. during this stage, the first protein to be synthesized is the 100 k protein, encoded by the l4 transcription unit, and this is produced in very large amounts. 30 the 100 k protein then binds to the carboxyl-terminus of eif4g at, or near, the site that is normally occupied by the eif4e kinase mnk1. by competing with mnk1 for binding, the 100 k protein acts as a direct inhibitor of mnk1 and displaces this protein from eif4g. 31, 32 the removal of mnk1 from eif4g results in the dephosphorylation of eif4e, which is thought to be associated with the inhibition of cellular capdependent translation, although the precise mechanism by which this occurs is not understood. late adv mrnas are capped but are still translated when cellular protein synthesis is inhibited as they possess a common 5 0 -noncoding region (5 0 -ncr) known as the tripartite leader. the tripartite leader allows the late mrnas to be selectively translated by an alternate mechanism of initiation known as ribosome shunting. during late infection, the 100 k protein enhances the binding of eif4g and eif4a to the tripartite leader complex at the 5 0 end of the adenovirus mrna. 33 the activity of the 100 k protein is stimulated by tyrosine phosphorylation, 33 and this phosphorylation event is necessary to promote viral translation following shunting and does not affect the binding of this protein to eif4g. similar decreases in eif4e phosphorylation are observed in vsv and influenza virus infections. 21, 34 in contrast, hsv-1 induces the phosphorylation of eif4e, which promotes its association with eif4g and may enhance capdependent (and therefore viral) protein synthesis, although it is not fully understood how selective translation of viral protein synthesis is achieved (reviewed in ref. 21 ). eif4g is the central component of the eif4f cap-binding complex and is frequently targeted during virus infection. there are a number of functional homologs of eif4g including eif4gi and ii. many picornavirus infections induce a rapid inhibition of host cell translation. in the case of the entero and rhinoviruses and fmdv, this shutoff is associated with the cleavage of eif4g. the entero-and rhinovirus 2a proteases cleave eif4gi such that the protein is separated into an n-terminal one-third, containing the eif4e-binding site, and a c-terminal two-thirds, to which eif3 and eif4a bind. [35] [36] [37] the bridging function of eif4gi between the capbinding activity of eif4e and the helicase and 40s recruitment roles of eif4a and eif3 is therefore lost. the fmdv l-protease similarly cleaves eif4gi at a site close to, but distinct from, the 2a protease cleavage site. 38 a secondary cleavage event is mediated by a second fmdv protease, 3c, in a species-specific manner. 39 both cleavage events result in separation of the eif4e-binding domain from the c-terminal portion of the protein, similar to the effects of the entero rhinovirus 2a protease (fig. 1) . the effects of eif4gi cleavage on host translation are more complex than was originally thought. experiments conducted in the presence of inhibitors of viral rna synthesis indicated that, although eif4gi is still cleaved under these conditions, host translational shutoff is minimal. 40 it was subsequently shown viral strategies to subvert the mammalian translation machinery that 2a protease also cleaves eif4gii. cleavage of both proteins is required for the virus to inhibit host translation. 41 in the case of pv, eif4gii is cleaved with slower kinetics than eif4gi, and eif4gii cleavage is therefore the ratelimiting step for induction of host translational shutoff. 41 fmdv l-protease also cleaves both eif4gi and ii, but with similar kinetics for each protein. 38 the significance of eif4gii cleavage is not certain, as it is much less abundant than eif4gi in cells and is no more active in supporting translation initiation. moreover, the central domain of eif4g, which lacks the eif4ebinding domain, can support translation initiation on capped mrnas. this eif4g p100 domain is fourfold less effective than intact eif4f in mediating translation initiation on capped mrnas, but is more active than intact eif4f for initiation on pv rna. 42 it is likely that, when viral rna synthesis increases the pool of pv rna in the cell, the p100 fragment of eif4g is redirected to pv rna at the expense of host translation. other effects of picornavirus infection, such as pabp cleavage, may also be involved in mediating the inhibition of host translation that occurs during picornavirus infection. picornavirus translation is directed by internal ribosome entry sites (iress) within the 5 0 -utrs of the viral rnas. the central one-third of eif4g, containing the eif3 and one eif4a-binding domain, is sufficient to support translation initiation from these iress. 43 this allows picornavirus rnas to compete effectively for the host translation machinery following infection, although the situation appears to be more complicated than this (see section iii). an exception to this is hepatitis a virus (hav), which does require full-length eif4g and eif4e for translation initiation, and hence does not cleave eif4g or induce shutoff. 42 the caliciviruses are an important family of viruses, being the main cause of outbreaks of viral gastroenteritis in man (noroviruses) and the causative agents of a number of animal diseases. infection with feline calicivirus (fcv) induces cleavage of eif4gi and ii, somewhat closer to the n-terminus than the picornavirus 2a protease cleavage site. 44 this cleavage occurs late in infection and correlates with host translational shutdown. despite this induction of cleavage, fcv requires intact eif4g to mediate translation of viral mrnas. it may be that in this case, cleavage of eif4gi results in a cleavage product that retains the eif4e-binding site, but removes the pabp-binding site. this would make sense as fcv mrna translation requires eif4e, 45 although the pabp requirement is currently unknown. however, translation of mrna from the related calicivirus murine norovirus (mnv) is insensitive to fmdv l-protease treatment and it therefore seems that intact eif4g is not required for translation of mnv mrnas. 46 it appears that even within the same family of viruses, different requirements for specific initiation factors in viral translation exist. retroviruses have rna genomes that undergo reverse transcription in infected cells to give a full-length dsdna copy, which then integrates into the host genome. human immunodeficiency virus (hiv) is a lentivirus responsible for acquired immunodeficiency syndrome (aids). proteases encoded by hiv-1 and -2 cleave eif4gi, but not eif4gii, in infected cells and in vitro. 47, 48 unlike the picornavirus proteases, this cleavage occurs at multiple sites and results in inhibition of hiv ires-driven translation, in addition to host translation. 47 several other retrovirus proteases cleave eif4gi and eif4gii at sites in a similar location when the protease is expressed in cells or introduced into cell-free systems. 49 the significance of this result is not clear, as most retroviruses other than hiv do not inhibit host translation. it is now well accepted that pabp plays a central role in stimulation of translation. by binding to poly(a) tails on capped mrnas, pabp can mediate the circularization of mrnas by simultaneously binding to eif4g at the 5 0 end of the mrna, thus promoting the recycling of ribosomes; this has been termed the ''closed loop model.'' 4,5 a number of viruses have been shown to target pabp as a mechanism of inhibiting host cell translation. it has been shown that infection of cells with the picornaviruses pv and coxsackie virus b3 (cvb3) results in the cleavage of pabp. 50, 51 furthermore, it was demonstrated that the viral 2a proteases directly cleaved pabp between m487-g488. 50,51 pabp contains four rna recognition motifs (rrms) that participate in eif4g-and rna-binding and a conserved c-terminal domain that interacts with other factors such as eif4b. 52, 53 cleavage of pabp by the picornavirus 2a proteases separates the rrms from the c-terminus and results in inhibition of protein synthesis, although pabp cleavage does not fully correlate with shutoff. in pv-infected cells pabp is cleaved by the viral 3c protease at different sites to the 2a proteases. 54 recent work has provided new information on the role of pabp cleavage in picornavirus infections. it is known that pabp also stimulates picornavirus ires-directed translation through its interaction with poly(a) tails and eif4g. 55, 56 one question that has been the focus of interest for picornavirologists for many years is the mechanism of switching from translation to replication of the viral rnas. as these two processes are occurring in opposite directions on the same rna, it is believed that something must stall translation to allow replication to occur. it has also been shown recently that hav 3c protease cleaves pabp in vivo and in vitro. 57 the resulting n-terminal cleavage product binds viral strategies to subvert the mammalian translation machinery to the hav 5 0 -utr (to the py1 region upstream of the ires) and suppresses translation of the hav mrna. hav does not induce host cell shutoff and infection does not result in cleavage of eif4g. a model has been proposed in which pabp binds to the poly(a) tail on the hav rna early in infection and stimulates translation. once enough viral proteins have accumulated, the 3c protease cleaves pabp and the n-terminal cleavage product binds to the py1 region of the 5 0 -utr of hav rna, inhibiting translation. the rna is then cleared of ribosomes to allow replication to occur in the opposite direction. 57 this model is also likely to apply to other picornaviruses, as it has now been shown that pabp cleavage by pv 3c protease also inhibits translation directed by the pv ires, both on rnas with and without poly(a) tails. 58 it was also demonstrated that expression of a pabp that is resistant to cleavage by 3c protease within cells resulted in reduced production of viral rna and reduced virus production. this suggests that pabp cleavage may be important in promoting the switch from translation to replication in picornavirus infections. it is not only in picornavirus infections that pabp cleavage is seen. the caliciviruses norovirus (nv) and fcv also induce pabp cleavage 54 and this results in inhibition of translation of polyadenylated rnas in vitro. the 3c-like protease is responsible for this cleavage. pabp cleavage does not occur until relatively late in fcv infection. as calicivirus rnas are also polyadenylated it is possible that pabp also stimulates translation of viral rnas and that pabp cleavage would inhibit viral translation, but this has not yet been demonstrated. in line with the model described above, it is tempting to speculate that pabp cleavage in calicivirus infections may also modulate the switch from translation to replication of the viral rnas. a different mechanism of targeting pabp that does not involve its cleavage has recently been described in rubella virus infection. the rubella virus capsid protein binds to pabp and there is an increase in pabp levels during infection. 59 addition of the rubella virus capsid protein to in vitro translation reactions inhibited translation of viral rnas, but this inhibition could be rescued by the addition of pabp. an inhibition of host protein synthesis was also observed, although this was not complete. the authors suggested that the binding of the capsid protein to pabp may mediate the switch between translation and packaging of the new genomes, in a similar manner to the model described for picornaviruses and caliciviruses above. rotaviruses belong to the reoviridae family and have segmented dsrna genomes. rotaviruses cause gastroenteritis and infect many animal species; in humans, they are responsible for severe diarrheal disease in infants which is a cause of high mortality in the developing world. these viruses replicate in the cytoplasm of infected cells where capped, nonpolyadenylated viral mrnas are made. 60 the rotavirus nonstructural protein nsp3 binds to the n-terminal region of eif4gi, both in vitro and in infected cells and also binds to the 3 0terminal sequence common to all rotavirus mrnas, similar to pabp binding to cellular mrnas. 61 rotavirus-infected cells also undergo inhibition of cellular protein synthesis and this has been attributed to the novel action of the nsp3 protein. the nsp3 protein specifically evicts pabp from the eif4f complex by competing for binding to eif4gi. in spite of this similar function, there is no sequence homology between pabp and nsp3. it is therefore believed that nsp3 binding to a consensus sequence in the 3 0 end of rotavirus mrnas recruits the eif4f complex to the viral mrnas via nsp3 binding to eif4gi, effectively circularizing the viral mrnas. it has been proposed that nsp3 functions in a similar way to pabp binding to polyadenylated eukaryotic mrnas. however, more recent data have questioned this model as it has been demonstrated that nsp3 (and its interaction with eif4gi) is not required for rotavirus mrna translation. 62 in this study, rnai-induced silencing of nsp3 in infected cells had no effect on viral protein production (except nsp3) or virus replication, although it did result in a less severe shutoff of host cell protein synthesis. in fact, viral progeny production was enhanced in the nsp3-silenced cells. similarly, these authors suggested that eif4gi is also not required for viral protein synthesis, as silencing of this factor also had no effect on virus production. a new model on the role of nsp3 was put forward that proposes that binding of nsp3 to the viral mrnas either protects them from degradation or prevents binding of the virus polymerase, thereby ensuring they are utilized for translation. 62 the exact role remains to be determined. bunyaviruses possess tripartite, negative-sense rna genomes and are responsible for a febrile illness in humans that is mosquito-borne. bunyavirus mrnas, like rotavirus mrnas, are capped but not polyadenylated. infection of cells with these viruses induces shutoff of host cell protein synthesis, possibly through the inhibition of transcription. it has been shown recently that a translational enhancer element (tee) within the 3 0 -utr of bunyamwera virus s segment mrna is able to substitute for a poly(a) tail. 63 a similar element has been shown to exist in dengue virus mrna. 64 translation of the bunyavirus mrnas requires eif4gi but does not require pabp, although the exact role of eif4gi is currently unknown. furthermore, bunyavirus infection of cells in culture resulted in a redistribution of pabp localization from the cytoplasm to the nucleus, and it was suggested that this may contribute to the inhibition of translation of host (polyadenylated) mrnas. the viral n protein binds to pabp in the cytoplasm but it is not yet known if this protein is directly involved in the nuclear relocalization. an alternative mechanism of translation initiation that is used in mammalian cells is termed internal ribosome entry. in this case, a complex, highly structured rna element (an internal ribosome entry site or ires) is formed in the 5 0 -utr of the mrna and the ribosome is recruited via the ires to an aug start codon that may be a considerable distance from the 5 0 end of the mrna. 48 accessory proteins termed ires trans-acting factors (itafs) are usually required by cellular iress and the data suggest that these act as rna chaperones that permit the ires to attain the correct structure to recruit the 40s ribosomal subunit. 48 in general, ires-mediated translation is used under conditions of pathophysiological cell stress which include genotoxic shock, temperature shock, hypoxia, and viral infection. 48 members of several different families of rna viruses are able to bypass the canonical, cap-dependent, translation initiation process by employing this strategy of internal initiation of protein synthesis. the translation initiation factors required, and mechanisms used, by different viral iress vary considerably. the picornavirus rnas are not capped, but are covalently linked to a small peptide known as vpg at the 5 0 terminus; this peptide is rapidly lost once the virus enters the cell, leaving an uncapped rna. picornavirus 5 0 -utrs tend to be long and structured, with many upstream aug codons that are not used for translation initiation. this suggested that a cap-dependent scanning mechanism of translation initiation was unlikely to be utilized, and led to the discovery of the first iress in the 5 0 -utrs of emcv 65 and pv rna. 66 these were identified by construction of dicistronic reporter rnas, in which the viral 5 0 -utr was placed between two cistrons and was able to promote translation of the downstream cistron. iress were subsequently identified in many different picornavirus rnas and divided into two major categories, within which there are common secondary structural and mechanistic features. type i iress are found in enteroand rhinoviruses, such as pv, and recruit ribosomes to an aug codon at the 3 0 end of the ires. a ribosomal scanning process then transports the 40s subunit and associated factors to the next aug codon further downstream, where translation initiation occurs. type ii iress, located in cardio-and aphthovirus mrnas (e.g., emcv), are similar in length to type i iress, at about 450 nucleotides (nt). they also recruit the 40s ribosomal subunit directly to an aug codon at the 3 0 end of the ires, but in this case this aug is the initiation codon 67 (fig. 3) . the fmdv ires is structurally related to the type ii iress, but only a minority of translation initiation occurs at the site of ribosome recruitment. the remaining ribosomes initiate translation at the next aug downstream following a scanning process, so this ires uses a hybrid of type i and type ii mechanisms. both type i and type ii iress require the entire canonical translation initiation machinery, with the exception of the cap-binding protein eif4e, and the eif4e-binding domain of eif4g. in vitro reconstruction of initiation complexes using purified and recombinant initiation factors and 40s subunits indicated that eif4g binds directly to the j-k domain of type ii iress, that this binding is stimulated by eif4a, and that this induces a conformational change in the region surrounding the initiation codon that is likely to promote 43s complex recruitment. 68 recently, similar experiments on the type i pv ires have indicated that an analogous mechanism is used to recruit eif4g-4a to domain v of the ires and to induce structural changes at the 3 0 border of the ires. 69 other structural features of both classes of ires are also required for initiation, and therefore eif4g/4a recruitment alone is not sufficient for ires activity. in addition to their requirement for components of the canonical translation initiation machinery, many picornavirus iress need to recruit noncanonical ires itafs to achieve optimal activity. a number of itafs that interact with specific iress have been identified, although in some cases the physiological role of these factors is questionable. well-characterized examples include the polypyrimidine tract-binding protein (ptb), which was first shown to interact with the human rhinovirus (hrv), 70 and was subsequently found to be required for efficient pv and emcv translation in cells. 71 a role for the autoantigen la in stimulation of pv ires activity has also been demonstrated in vitro and in cell culture. 72 itafs are thought to act by modulating the secondary structure of the ires such that canonical initiation factors are more effectively recruited, and such a role was demonstrated for ptb and itaf45 binding to the fmdv ires 73 and more recently in cells. 74 picornavirus infection is frequently associated with rapid shutoff of host translation, providing a rationale for the use of iress to maintain viral translation under these conditions. the enterovirus 2a and fmdv l-proteases, for example, cleave eif4gi and ii such that the n-terminal eif4e-binding domain is separated from the remainder of the protein. although picornavirus iress show unaffected or even enhanced activity in the presence of 2a protease, some of this stimulation is thought to be independent of inhibition of host translation or expression of the c-terminal fragment of eif4g. 75 stimulation of ires activity can still occur when 2a protease with a mutant active site is expressed, or when eif4g is resistant to 2a cleavage. 76 the effects of this protease on ires activity are therefore more complex than a simple competition between full-length and truncated eif4g mediating host and viral translation, and it is probable that other factors are regulated by the protease and have an effect on picornavirus iress. two further categories of picornavirus ires have also been identified. the hav ires forms a minor class of its own, and requires the full canonical initiation machinery including eif4e and full-length eif4g, although the viral rna is not capped. 42 it has been suggested, although not yet experimentally proven, that the requirement for eif4e may be due to its conformational effects on eif4g. 42 a fourth, and very distinct, class of picornavirus ires elements was recently identified in porcine teschovirus-1 (ptv-1), 77 and subsequently in several other picornaviruses such as avian encephalomyelitis virus. 78 these iress are distinct from other picornavirus iress in their initiation factor requirements and mechanism of action, and instead are very similar to the hcv and pestivirus iress. the ptv-1 and aev ires elements show sequence and structural homology to the hepatitis c virus (hcv) ires and act similarly to directly recruit the 48s complex in the absence of the eif4 factors. the ptv-1 ires has also been shown to interact directly with the 40s ribosomal subunit and eif3. 79 this suggests that exchange of genetic information between picornaviruses and flaviviruses has occurred at some point. the mechanism of initiation used by the ptv-1 ires and its relatives was initially identified in hcv and pestivirus rnas. 80, 81 hcv belongs to a subgroup of the flaviviridae family and is a major cause of human disease, causing blood-borne hepatitis that can result in the development of hepatocellular carcinoma. the flavi-and pestiviruses are positive-sense rna viruses with uncapped structured 5 0 -utrs that direct synthesis of the viral polyprotein. the hcv and pestivirus 5 0 -utrs are somewhat shorter than those of the picornaviruses, and almost the entire utr, approximately 330 nt, is required for ires activity. hcv and related iress can bind directly to the 40s subunit (in the absence of any initiation factors) such that the start codon is positioned close to the ribosomal p site. the iress then bind directly to eif3 and require this factor and the ternary eif2/gtp/met-trnai complex to form correctly positioned 48s* complexes. the ires can then recruit the 60s subunit and assemble functional 80s ribosomes. 82 these iress do not require any components of the eif4f complex, and it was recently demonstrated that at high magnesium concentrations the hcv ires is able to initiate translation independently of the eif2/gtp/met-trnai ternary complex. 83 eif2-and eif5-independent hcv ires activity was mediated by eif5b in a manner analogous to prokaryotic translation initiation. 84 this ability to function in an eif2-independent manner displays an intriguing similarity to the dicistrovirus intergenic region (igr) iress and is likely to allow the hcv ires to function under conditions of cell stress that induce eif2 phosphorylation. the minimal factor requirements for hcv ires activity have allowed close study in vitro and it has been possible to use cryo-electron microscopy (cryo-em) to determine the tertiary structure of the ires in complex with the 40s ribosomal subunit. 85 the hcv ires was shown to induce conformational changes in the 40s subunit, some of which are very similar to those induced by eif1 and eif1a binding to the 40s subunit. this suggests that the hcv ires may function in a similar manner to these factors to promote initiation. 86 a pathway for recruitment of hcv ires rna to the 40s subunit has been determined using directed hydroxyl radical probing, providing a further indication of similar, but distinct, effects of the hcv ires and eif1/1a binding to the 40s subunit. 87 the secondary structure of the hcv and pestivirus iress has been clearly defined (fig. 3) . domain iii is necessary for 40s binding and for subsequent eif3 recruitment, but domain ii is not required to recruit these components. however, deletion of domain ii results in severely impaired ires activity, and this region of the ires was shown to be important for 80s formation. 88 domain ii folds independently of the remainder of the ires, and is responsible for the conformational changes induced in the 40s subunit by hcv ires binding. 85 an analysis of the role of this domain in subunit joining indicated that it promotes eif5-induced gtp hydrolysis and release of eif2/gdp. 89 despite its ability to initiate translation by binding directly to the ribosome, there is some evidence for a role for several different itafs in hcv ires activity. several proteins have been shown to bind to the hcv ires, but the most convincing evidence is for a role for the autoantigen la. this factor interacts directly with the hcv ires at a site close to the start codon, and stimulates 40s binding in vitro. depletion of la in cultured cells led to a loss of ires activity. 72 the most recent class of ires to be identified was found in the igr of the cricket paralysis virus (crpv) genome and other members of the dicistroviridae family 90,91 of insect-infecting viruses that belong to the picornavirus superfamily. these viruses have a naturally discistronic genome and iress have been discovered in both the 5 0 -utr and the igr. the crpv igr ires is approximately 200 nt long, and adopts a high degree of secondary structure, with three pseudoknots (pki-iii) that are involved in different aspects of the ires activity. 92 the crpv igr ires and its relatives recruit the 40s and 60s ribosomal subunits independently of any host initiation factors or met-trnai. the igr ires binds to the 40s subunit via the pkii-pkiii domains, and is positioned correctly for elongation by pki, which occupies the p site of the ribosome. a gcu codon is positioned in the a site and recruits its cognate trna such that the first amino acid is alanine 91 (fig. 3) . the mechanism of initiation by the crpv igr ires has been analyzed in detail by in vitro reconstitution experiments. the ires mediates peptide synthesis following incubation with the 40s and 60s ribosomal subunits, the elongation factors eef1a and eef2, and aminoacylated trnas. 93, 94 the first translocation step on the ires occurs without peptide bond formation. the ires therefore mimics both the initiator and elongator trnas by its interactions with the ribosomal p site. the structure of the igr ires in complex with 40s and 80s ribosomes has been revealed by cryo-em and crystallography. 95, 96 these structures demonstrate the ability of the ires to mimic a trna in the ribosomal p site, and to form contacts with the a, p, and e sites. this binding pattern is very different to that of the hcv ires, which interacts predominantly with the solvent side of the 40s subunit, although there is some overlap in the e site. 85 despite this difference in binding, the crpv and hcv iress induce similar conformational changes in the 40s subunit, suggesting that these changes may be intrinsic to translation initiation. the ability of the crpv igr ires to initiate translation in the absence of initiation factors allows it to function effectively under conditions in which host translation is inhibited. the ires is relatively inactive in wild-type yeast, but is activated by eif2 phosphorylation, or by depletion of various other canonical initiation factors. 97, 98 this implies that the ires competes with host mrnas for ribosomes, and is able to do so effectively under conditions of cell stress. the ires found in the 5 0 -utr of the dicistroviruses is very different in both structure and mode of action to that of the igr ires. it has been demonstrated that the 5 0 -utr of the dicistrovirus rhopalosiphum padi virus (rhpv) is mostly unstructured and requires only a minimal set of factors for function, eif2, eif3, and eif1, although the addition of eif4f did stimulate 48s complex formation on the ires. 99 it may be that this simplified mode of action is responsible for the ability of the ires to direct translation initiation in mammalian, insect and plant systems. 100 two iress have been identified in hiv-1 rna. the first to be identified is located in the coding region of the gag gene and directs synthesis of an nterminally truncated gag protein. 101 the second ires is located in the 5 0 -utr and directs protein synthesis during the g2/m phase of the cell cycle. 102 an unusual form of internal ribosome entry has been described in hiv-2. hiv-2 viral particles contain gag p57 (the translation of which initiates at the first codon, aug1), and two shorter isoforms of p50 (which initiates at aug2) and p44 (which initiates at aug3). an ires that directs translation of all three isoforms of the gag protein is located in a highly structured region which is downstream of the authentic aug1 start codon and spans to aug3. 103 this ires is therefore required to deliver the preinitiation complex upstream to produce gag p57 polyprotein from the first aug codon. 103 h. mirnas mirnas have recently emerged as major regulators of gene expression (discussed in detail in the chapter by sarnow, this volume). metazoan mirnas function predominantly by binding via imperfect complementarity to sites in the 3 0 -utr of mrnas and repressing gene expression, both at the level of translation and by mrna degradation. this important mode of gene regulation has been found to affect the life cycles of a number of viruses in a variety of different ways (fig. 4 ). the first observation that viruses utilize the mirna pathway came from the herpesviruses. these large dna viruses encode their own mirnas within their genomes and express these mirnas during both latent and lytic infection. cloning and sequencing of small rnas derived from b cells infected with the -herpesvirus ebv led to the identification of the first five viral mirnas. 104 mirnas expressed by a number of other , , and -herpesviruses that infect a range of species have subsequently been identified by cloning and computational methods, 105 implying that this is a general mechanism used by this viral family. conservation of sequence or genomic location between mirnas derived from different viruses was generally not observed. 105 mirnas are also expressed by the small dna virus simian virus 40 (sv40) and several other polyomaviruses. [106] [107] [108] two mirnas, derived from opposite strands of a single pre-mirna, are expressed by these viruses. a number of small rnas derived from adenovirus va rnai, and predominantly from va rnaii, were identified and shown to associate with the rnai-induced silencing complex (risc), 109 although a functional role has not yet been ascribed to these small rnas. it is likely that viral mirna expression is limited to dna viruses, as drosha and dicer-dependent processing of mirna precursors results in the destruction of the parent rna, and would therefore be undesirable for a virus with an rna genome. cloning of small rnas from cells infected with hcv, yellow fever virus (yfv), or hiv-1 did not yield any mirnas derived from these rnas and viruses. 105 mirnas derived from both strands of the hiv-1 tar rna have since been detected in infected cell lines and primary cells. 110 however, hiv-1-derived mirnas were undetectable in another recent study, so it is questionable whether any expression that does occur is at a sufficiently high level to have functional consequences. 111 viral mirnas are similar to those of the host, as they are derived from longer hairpin transcripts expressed by rna pol ii or iii and undergo processing to yield mature, cytoplasmic mirnas. identification of novel viral mirnas is therefore amenable to computational analysis. 105, 112 ebv is now known to encode at least 23 mirnas, 112 located in two genomic clusters, within introns of the bart and bhrf1 genes. mirnas derived from each cluster show differential expression patterns across different stages of viral latency. 113 the temporal regulation of herpesviral mirna expression in general is not yet well understood, as most viral mirnas have been cloned exclusively from cells at a particular stage of the viral life cycle. however, hsv-1 mirnas show distinct expression profiles, with four mirnas expressed in latency, one during productive replication, and one throughout infection. 114 sv40 mirnas are only expressed in late infection. 107 the first identified target for a viral mirna was the sv40 large t antigen (tag). the viral mir-s1 is expressed antisense to the tag mrna and acts in an sirna-like manner to cleave and degrade the tag transcript. 107 this mechanism appears to function to mediate downregulation of antigen levels late in the viral life cycle and thus allows the virus to evade a cytotoxic t cell response. 107 the location and function of this mirna appear to be conserved in other polyomaviruses. 106, 115 regulation of a viral transcript by an antisense viral mirna has also been observed or predicted for some herpesvirus mirnas. mir-h2-3p, which is expressed during latent hsv-1 infection, is antisense to the viral icp0 transcript. surprisingly, despite its exact complementarity to its target, mir-h2-3p does not significantly affect icp0 mrna levels, but reduces the expression of the encoded protein at the level of translation. 114 this regulation is likely to be important for establishment and maintenance of viral latency, as the icp0 protein is important for productive replication and is thought to be involved in reactivation from latency. 114 a search for imperfect targets for viral mirnas within viral 3 0 -utrs yielded a number of attractive candidates, suggesting that viruses also use this mechanism to regulate their own gene expression. this was confirmed experimentally for hcmv mir-ul112-1, which downregulates expression of the major immediate early gene ie1 by binding to its 3 0 -utr. 116 ie1 protein is important for the switch from latent to lytic infection, as are some of the other predicted viral targets of herpesvirus mirnas, so viral mirnas may play a general role in regulation of herpesvirus latency. an important function of herpesvirus mirnas appears to be in the regulation of host gene expression. various host mrna targets of herpesvirus mir-nas have been detected, and the viral mirnas appear to function in a similar manner to cellular mirnas, by binding to the 3 0 -utr with imperfect complementarity and negatively regulating translation and rna stability. ebv mir-bhrf1-3 downregulates the chemokine cxcl-11, a t cell attractant, and may thus allow infected cells to escape the t cell response, 117 whereas mir-bart-5 targets the proapoptotic factor puma and protects infected cells from apoptosis. 118 several kshv mirnas repress expression of bclaf1, a protein involved in apoptosis (248), and others downregulate thbs1, a multifunctional protein which has a role in recruitment of monocytes and t cells to sites of infection. 119 despite a lack of sequence homology, mirnas from several different herpesviruses bind to adjacent sites in the 3 0 -utr of micb mrna and inhibit expression of micb protein, which is involved in immune surveillance. 120 the herpesvirus mirna targets identified to date appear to allow these viruses to regulate the host immune response or apoptosis, and thus are likely to be important in allowing effective infection. a particularly intriguing viral mirna is kshv mir-k11, which has an identical seed sequence to cellular mir-155, and has been shown to regulate many of the same targets. 121 mir-155 levels are upregulated in cells infected with ebv, 122 and a similar viral orthologue is encoded by another oncolytic herpesvirus, marek's disease virus type 1 (mdv-1). 123 mir-155 overexpression is linked to various cancers, so these results suggest that viral mirna expression, or virus-induced modulation of host mirna expression, may contribute to oncogenesis. viral infection can affect both the cellular mirna machinery as a whole, and the expression of individual mirnas. in addition to its effects on mir-155 expression, discussed above, ebv modulates the levels of multiple host mir-nas, with different effects in latent and lytic infections. 124, 125 hcmv also regulates the expression of specific host mirnas, some of which affect viral replication. 126 hiv-1-dependent downregulation of the mir-17-92 cluster had a positive effect on viral replication, mediated by the tat cofactor pcaf. 127 inhibition of the mirna and rnai pathways by viruses is well established as a mechanism of evasion of the host immune response in lower eukaryotes, but few examples exist in mammalian systems. adenovirus infection results in a global inhibition of host mirna expression and activity. expression of va rnai led to inhibition of processing of ectopically expressed pre-mir30, and to inhibition of rnai induced by short hairpin rna (shrna) precursors that require dicer cleavage for activity. this activity was mediated by va rnai competition with pre-mirnas and shrnas for binding to both exportin-5, required for pre-mirna export from the nucleus, and to the cytoplasmic processing enzyme dicer. 128 va-derived small rnas also compete with endogenous mirnas for incorporation into the risc. 109 va rnai is expressed at very high levels in infected cells, so it is possible that its interference with the mirna and rnai pathways may be a consequence of this expression and have little functional relevance for the virus. although some studies have suggested repression of the mirna pathway by retroviral transcription factors, 129,130 a recent analysis did not identify any such activity. 111 a direct effect of a cellular mirna on virus replication has been observed in the case of the liver-specific mir-122. mir-122 binds to the 5 0 -utr of hcv rna and is required to maintain viral rna abundance. 131 the mechanism by which this occurs has not yet been resolved. no effects of mir-122 on hcv translation were initially observed, suggesting that the mirna acts at the level of viral rna replication, 131 whereas translational stimulation mediated by mir-122 binding to the hcv 5 0 -utr was observed in a subsequent study. 132 it is possible that multiple mechanisms may operate. it is not yet known whether this mechanism is unique to hcv, or whether other viruses may use similar strategies. various mirnas, including several induced by interferon stimulation, have been shown to negatively regulate hcv, 133 and mirna-dependent repression of other viruses, including primate foamy virus 1 (pfv-1) has also been observed. 130 however, the extent to which these mirnas regulate virus replication in naturally infected tissues remains unclear. the strong requirement for conservation of complementary sequences to an mirna seed for regulation to occur, coupled with rapid viral evolution, would suggest that any mirna that has a detrimental effect on virus replication would quickly be evaded. tissue specificity is an important feature of the expression of many cellular mirnas. it is possible that viruses may have evolved to selectively avoid targeting by host mirnas that are expressed in the tissue they infect, and therefore results obtained outside normal target cells should be interpreted with caution. attempts to target viruses by introduction of artificial-binding sites for tissue-specific host mirnas were effective in restricting viral tropism, but some viral escape mutants evolved. 134, 135 in conclusion, the interplay between viruses and the mirna pathway is complex and varied, and has many important consequences for viral infection. viruses have evolved a number of unconventional mechanisms that allow the translation of distal orfs, contributing to the complexity of gene expression from compact viral genomes. these include (i) leaky scanning, where the aug of the 5 0 -most orf is poorly recognized and the ribosomes scan and initiate at a downstream orf, (ii) reinitiation, where a posttermination complex remains associated with the rna and reinitiates at a downstream orf, and (iii) frameshifting. although few of these mechanisms of translation initiation have been described in host mrnas thus far, it is interesting to speculate that, as in the case of iress, viruses could use or adapt a preexisting system to initiate synthesis of viral proteins. a number of rna viruses have a protein known as vpg covalently linked to the 5 0 end of the viral rna. in picornaviruses, the vpg protein is small and is removed from the rna following viral entry, such that an uncapped viral rna serves as a substrate for translation, which occurs by internal initiation. many of these novel mechanisms have been attributed to the presence of rna structures within the virus genome that are involved in translation initiation, however, recent work has described the presence of a proteinaceous ''capsubstitute'' on calicivirus rnas that directs translation of the viral mrnas. the vpg on calicivirus rnas is much larger than its picornaviral counterpart and has a different function, as it is retained on the rna and serves as a cap substitute that directs translation of the viral mrnas. the calicivirus vpg protein binds to eif4e at a site distinct from both the cap and 4e-bp1-binding sites. in the case of fcv, the vpg:eif4e interaction is required for translation initiation (at least in vitro) on the viral mrna, but in the case of mnv this interaction is not required for efficient translation, although the presence of vpg on the mrna is necessary. it has also been reported that human norovirus vpg binds to eif3, suggesting that vpg has multiple interactions with key components of the translational apparatus. 136 the interaction of calicivirus vpg with eif4e is unique amongst mammalian rna viruses but a similar interaction occurs on plant potyvirus mrnas (reviewed in ref. 137) . in this case, plants that do not express eif(iso)4e are resistant to infection with turnip mosaic virus. potyvirus vpg is thought to bind eif4e in competition with the cap structure. 138 hence, even though the principles of vpg-directed translation are common, the specific mechanisms by which the vpg proteins interact with eif4e are distinct. influenza virus is a major human health problem with worldwide prevalence. influenza virus is well known for causing pandemic outbreaks and is a zoonotic disease, infecting pigs, avian species and horses, as well as humans. influenza a viruses belong to the orthomyxoviridae family and have a singlestranded, negative-sense rna genome which is made up of eight segments. in influenza virus-infected cells there is a dramatic inhibition of host cell translation while the viral mrnas are selectively translated. 139 it is well established that influenza virus mrnas acquire their 5 0 caps through a process of ''cap snatching'' or ''cap stealing'' during which the virus polymerase complex ''snatches'' the 5 0 10-12 nt from host nuclear mrnas which then prime the synthesis of viral mrnas. the viral mrnas therefore contain host cell sequences at their 5 0 ends, which are followed by conserved viral sequences that are known to bind the viral polymerase. it is believed that polymerase binding to these conserved sequences prevents the snatching of caps from the viral mrnas 140 and contributes to the selective translation of viral mrnas during infection; the cellular mrnas being degraded once decapped. the polymerase itself is a complex of three subunits, pa, pb1, and pb2. the pb1 subunit is involved in binding to the caps on cellular mrnas that are then viral strategies to subvert the mammalian translation machinery thought to be cleaved by the endonuclease activity of the polymerase. the crystal structure of the pa subunit has recently been solved and the endonuclease active site was shown to reside in the amino-terminal end of the protein. 141, 142 as described above, influenza virus infection results in changes to the eif4f complex as eif4e is dephophorylated and eif4g is hyperphosphoryated. 34 a study to understand the components of the eif4f complex that are required for translation of viral mrnas has recently demonstrated that influenza virus translation has no requirement for eif4e, as viral mrnas are translated in cells depleted of eif4e and in cells treated with rapamycin. 143 the authors suggested that the polymerase is able to substitute for eif4e by binding to the conserved sequences in the 5 0 -utr of the viral mrnas and recruiting initiation factors. this seems to be another example of how some capped viral mrnas display a reduced requirement for eif4e in a similar manner to the adenoviruses. whereas influenza virus substitutes the eif4e component of the eif4f cap-binding complex with a viral protein, a recent report has demonstrated the unique ability of hantaviruses to replace the entire eif4f complex with just one viral protein. 144 the hantaviruses are rodent-borne viruses of the bunyaviridae family and the genome is made up of three negative sense, single-stranded rna molecules. hantaviruses include sin nombre virus and hantaan virus, viruses associated with high-mortality rates and symptoms including hantavirus pulmonary syndrome and haemorrhagic fever, respectively. the nucleocapsid (n) protein of sin nombre virus has been shown to uniquely possess activities that mimic all three components of the eif4f complex, eif4e (as n binds to the 5 0 end of capped mrnas), eif4g (as n recruits the 43s complex to the 5 0 cap), and eif4a (as n replaces the rna helicase). it was shown that n enhances translation of capped mrnas as a whole, but preferentially stimulates translation of capped mrnas that contained 44 nt of 5 0 noncoding sequence from the virus. following inhibition of translation by the addition of a picornavirus 2a protease to rrl such that eif4g is cleaved, n rescues translation of capped mrnas. finally, it was shown that n increased the rate of recruitment of the 43s complex to mrnas. 145 in summary, n protein increases the translation of both viral and cellular mrnas, although the enhancement is greater on viral mrnas. hantaviruses do not induce shutoff of cellular protein synthesis but the binding of n to viral mrnas is likely to permit their preferential translation over host mrnas. there are many examples of important mammalian virus pathogens including retroviruses (e.g., hiv-1) and coronaviruses (sars) that employ frameshifting during translation. in most of the systems examined to date, frameshifting is required for the expression of viral replicases. in retroviruses, frameshifting is necessary for the synthesis of gag-pol and gag-pro-pol polyproteins and the production of reverse transcriptases, whereas for the majority of other viruses it is essential to permit the synthesis of rna-dependent rna polymerases. 146 ribosomal frameshifting is a process that alters the triplet decoding of the mrna by the elongating ribosome. a specific signal in the mrna causes the ribosome to change reading frame from the 0 to the ã� 1 frame and translation then continues in the new frame. 146 in eukaryotes frameshift signals require two elements, a heptanucleotide ''slippery sequence,'' where the ribosome changes reading frame, and a stimulatory element that is located a few nucleotides downstream in the form of an rna pseudoknot 147 or a stemloop. 148 a spacer region of 6-8 nt between the slippery sequence and the stimulatory rna element is also required, and frameshifting efficiency is dependent upon the length of this sequence (fig. 5; refs. 147,149) . several models for how frameshifting occurs have been proposed (reviewed in refs. 146, 150) and the model that is most consistent with experimental data suggests that ribosomal pausing at the stimulatory rna element increases the time that the ribosome is held over the slippery sequence and this permits the trna to realign in the ã� 1 frame. 151, 152 1. coronaviruses coronaviruses are a family of animal viruses with a large positive stranded rna genome. in this family of viruses the replicase gene is composed of two partially overlapping orfs, 1a and 1b, and the fused polyprotein 1a/1b (pp1ab) is synthesized by programmed ã� 1 ribosomal frameshifting. 147, 153 the first characterized frameshift signal described for pp1a/pp1b in coronaviruses was in avian infectious bronchitis virus (ibv; ref. 147); subsequently, very similar mechanisms were shown to achieve this mode of translation elongation in other coronaviruses. it was shown by mutational analysis that the slippery sequence in ibv is uuuaaac 154 and that the downstream frameshift stimulatory element 6-7 nt away from this sequence is a hairpin (h)-type mrna pseudoknot ( ref. 147; fig. 6 ). to carry out detailed structural analysis of ribosomes paused on frameshift sequences, a variant of the frameshift sequence of ibv was generated in which the slippery sequence was changed to cgaggca and ribosomes, stalled in the act of decoding the frameshift signal, were purified and viral strategies to subvert the mammalian translation machinery subjected to cryo-em. 152 this allowed images to be generated of 80s ribosomes stalled over the 1a/1b ibv pseudoknot frameshift signal and important mechanistic details of the frameshift process have been derived from these basic mechanism of action of frameshifting. two elements are required in the viral rna for frameshifting to take place; a heptanucleotide slippery sequence and a structured downstream stimulatory element which is present in the form of either a rna pseudoknot or a hairpin-stem. ribosome pausing, due to the presence of the rna structure, over the slippery sequence permits the trna to realign in the ã� 1 frame. fig. 6. structures of the stimulatory sequences that are found in ibv and hiv. the structure of the stimulatory sequence found in ibv is an rna pseudoknot whereas the structured element in hiv forms a complex stem-loop. reconstructions. 152 importantly, these data indicate that translocation is the point in the elongation cycle at which frameshifting occurs. the rna pseudoknot interacts with the ribosome in close association with the putative 80s helicase at the entrance to the mrna channel as well as with the 18s rrna helix 16, rps9, rps2, and the ubiquitous eukaryotic ribosomal regulatory protein rack1. eukaryotic elongation factor 2 (eef2) was shown to be trapped in the a site of the ribosome and this would prevent binding of trna to this site until the frameshift has been completed. in the presence of the pseudoknot the p site trna is distorted and bent toward the a site that contains eef2. taken together these data allowed the following three step model for frameshifting to be proposed. 152 (i) the helicase at the entrance of the mrna tunnel on the elongating ribosome is unable to unwind the pseudoknot structure in the mrna, pausing the ribosome. (ii) the blockage imposed by the pseudoknot partially inhibits the movement of the trna, bound to the mrna by the codon-anticodon pairing, during translocation. the trna is unable to return to the a site due to the presence of eef2, and the resulting strain on the trna causes it to bend in a (ã¾) sense direction such that it now moves to the roof of the p site. (iii) due to the strain of these opposing forces the codon-anticodon interactions breaks. the trna then relaxes and moves in the (ã�) sense 5 0 direction and repairs with the mrna in the ã� 1 position. 152 this elegant model agrees well with earlier data and supports a number of other models that have been proposed. 146, 150, 155 severe acute respiratory syndrome (sars) in humans is caused by a novel coronavirus 156 and the frameshift element in this virus is highly related to those described previously although with some interesting differences. in sars-cov the site of frameshifting signal that allows the production of pp1ab is also a u_uua_aac stretch and this is found 12 bases upstream of the 1a stop codon. the frameshift is very efficient; using reporter-based systems, frameshift frequencies of between 14% and 27% were measured in vitro and in vivo. 157, 158 as with other coronaviruses there is a downstream pseudoknot, and disruption of base pairing in this element substantially reduces the efficiency of frameshifting. 157, 158 however, there are notable differences in this pseudoknot when compared to other coronaviruses. the pseudoknot conforms to the h type structure found in ibv, but there is extensive base pairing in loop 3. 157 most of loop 3 can be deleted without great effect. however, there appears to be an essential conformation that needs to be maintained to achieve maximum frameshifting efficiency. 146 frameshifting is essential to hiv replication and related lentiviruses (e.g., siv, hiv-2) as it controls both the expression of gag-pol polyproteins and importantly the precise ratio of the gag:gag-pol polyproteins. 160 even small variations in the gag and gag:pol ratio can have adverse effects on the virus in terms of infectivity. 161 the site of frameshifting for hiv is a u_uuu-uua stretch located within the gag/pol overlap 200 nt upstream of the gag termination codon. 151, 162 the downstream stimulatory element found at the gag-pol junction is a simple but very stable rna stem-loop. 148, [163] [164] [165] [166] for hiv-1 the nmr data suggest that the stimulatory rna is comprised of a two stem structure (fig. 6 ). there is an 11 bp helical stem and a highly ordered hairpin loop, the top of which contains an acaa tetraloop (fig. 6) . a less stable stem is also present which is separated from the upper loop. 148, 166 it has been suggested that the lower stem acts as a ''positioning element'' that permits the stem-loop to induce the ribosomal pausing required to perturb ribosome translocation. 166 in the longer term a full understanding of frameshift regions is likely to be of considerable importance in the development of antiviral drugs. for example, hiv has an absolute requirement for a ã� 1 ribosome frameshift during translation and this would provide an attractive new target to interfere with the viral lifecycle. it may be feasible to disrupt this process using small molecules, peptides, or oligonucleotides. 167 eukaryotic translation initiation generally occurs close to the 5 0 end of the mrna. in some mrnas, the 5 0 -proximal aug is followed by a short uorf, and if this is fewer than 30 codons a significant percentage of the ribosomes that have completed uorf translation may resume scanning (as 40s subunits) and reinitiate translation at a downstream aug codon. 168 since not all ribosomes resume scanning after termination, the presence of the uorf results in a decrease in translation of the major orf. 168 the uorf must be translated rapidly for reinitiation to occur, [169] [170] [171] suggesting that rescanning occurs only if some of the initiation factors that promoted initiation at the uorf aug remain ribosome-associated during uorf translation. although the mechanism of reinitiation is not fully understood, it has been shown that in mammalian systems efficient reinitiation following uorf translation only occurs if the complete eif4f complex, or eif4a, 4b, and the central domain of eif4g participated in the original initiation event. 172 some viral mrnas are able to mediate translation reinitiation following translation of a long orf, and special mechanisms have been developed to promote this event. the best studied case of translation reinitiation occurring following translation of a long orf is found in caliciviruses. the subgenomic mrna encoding the structural proteins of fcv is bicistronic with two overlapping cistrons. the first orf of the rna encodes the viral major capsid protein (vp1) and the second cistron encodes the minor capsid protein vp2. the two uorfs overlap by 4 nt in fcv (auga), one in norovirus (uaaug) and eight in rabbit hemorrhagic disease virus (augucuga). 173, 174 the expression of the downstream orf requires a termination-reinitiation event which is different from those identified in mammalian systems studied to date since it is independent of eif4g or the eif4f complex. instead reinitiation requires an interaction of eif3 and the 40 s ribosomal subunit with a sequence element that is present in the 3 0 -terminal 70 nt of the upstream orf, denoted the termination upstream ribosomal-binding site (turbs). 175, 176 two short sequence motifs present in turbs are required for reinitiation, the first of which is a pentameric uggga sequence that is complementary to the apical loop of helix 26 in the mammalian 18s rrna. evidence for a direct interaction between fcv mrna and 18s rrna was obtained using a yeast model system where mutations were introduced into both rnas. 128 thus when the yeast 18s rrna was mutated such that it was adapted to the fcv sequence or vice versa there was a dramatic increase in the translation of the downstream frame. 128 this ugga motif is conserved in caliciviruses. 175 the second motif is not conserved among caliciviruses but is located at an equivalent position in the turbs of fcv and rhdv, 175 and it is the secondary structure of these sequences, and not the primary sequence, that is important for function. 176 it has been proposed that the binding of posttermination eif3/40s complexes to turbs retains them in a position suitable for reinitiation once they have acquired the eif2/gtp/met trnai ternary complex. 175, 176 in agreement with this hypothesis it has been shown that eif3 is involved in termination and recycling of ribosomal complexes, thus providing in part an explanation for the interaction of eif3 with the 40s ribosomal subunit (ref. 177 and discussed in the chapter by fraser, this volume). in influenza b virus the genes encoding the m1 (matrix protein 1) and the bm2 proteins, both of which are important for virus viability, 178 are located on segment 7 of the viral genome. 179 the termination codon of m1 overlaps the start codon of bm2 (uaaug) and the bm2 polypeptide is expressed by termination-dependent reinitiation. 179, 180 the mrna sequence requirements for reinitiation are similar to those identified for the caliciviruses and are dependent on a 45 nt stretch of rna that is immediately upstream of the uaaug pentanucleotide and includes the uggga motif. the process of reinitiation and termination is thought to involve the interaction of a stemloop structure in this region that has complementarity with the apical loop of helix 26 in 18s rrna. 181 there are two genera of pneumovirinae: the pneumoviruses, including human respiratory syncytial virus (rsv) and the metapneumoviruses including avian pneumovirus (apv) and human mpv (hmpv). all these viruses cause acute respiratory infections in their hosts. the pneumovirinae direct the synthesis of eight mrna transcripts, encoding nine primary translation products and the m2 transcripts all contain two uorfs, m2-1 and m2-2, which are overlapping. 182 expression of the rsv m2-2 orf occurs via an unusual coupled translation event in which termination of translation of the m2-1 orf is required before translation of m2-2 can be initiated. [183] [184] [185] a number of regions in the rsv m2-1 orf have been shown to play a role in coupled translation. the most important region is not between the overlapping cistrons, but is located 150 nts upstream in the m2-1 orf and contains stable structural elements. 184, 185 a similar mechanism is used to initiate the translation of m2-2 transcripts of apv and hmpv, although there are differences in the efficiencies of the process, which appear to be due to lack of stimulatory sequences in the m2-1 orf. 185 the reinitiation mechanism is likely to be quite different from that found in influenza bm2 and calicivirus subgenomic mrnas, but may well involve the same principle of capturing some of the posttermination 40s subunits and restraining them in a position suitable for reinitiation. although translation initiation generally occurs at the proximal 5 0 aug codon that is reached after scanning of the 5 0 -utr, alternative sites of translation initiation also exist which contribute to the complexity of viral genomes. it is known that the sequences flanking the aug codon contribute to the efficiency of translation initiation. a purine, particularly adenosine, at ã� 3, and guanosine at ã¾ 4, when the a of aug is designated ã¾ 1, gives the greatest enhancement to the initiation event. in many viral mrnas the first aug codon is in a suboptimal context, so the scanning ribosome may migrate past this first aug codon and initiate at an alternative downstream aug. this process is termed ''leaky scanning.'' the process of shunting is occasionally observed following leaky scanning and examples of such a mechanism are provided by studies of adenovirus, sendai virus, and avian reoviruses. adenoviruses contain a double-stranded linear dna genome and the timing of the expression of mrna from this genome is dependent upon the stage of infection. during early stages of infection there is a production of proteins for viral dna replication whereas in late infection this switches to proteins that are required for the assembly of viral capsids. 186 most late adenovirus transcription is initiated from the major late promoter (mlp). mrnas derived from this promoter all possess a common 5 0 -ncr of 212 nt in length called the tripartite leader which arises from splicing of three small exons. 187, 188 there is an inhibition of host protein synthesis following adenovirus infection. the continued selective translation of viral mrnas under these conditions is due to the presence of the tripartite leader which is able to direct ribosome shunting. 189, 190 shunting involves the loading of the 40s subunit at the 5 0 end of the capped tripartite leader-containing mrnas, followed by linear scanning over a short distance and then a direct translocation of the 40s subunits via ''shunting elements'' to the start codon. 190 within the tripartite leader, the elements that are required for translation initiation during late viral infection are an unstructured 5 0 end to the mrna 189 and hairpin structures that have complementarity to the 18s rrna at the 3 0 end. 190 the s1 mrnas transcribed by the fusogenic avian (arv) and nelson bat (nbv) reoviruses encode three unrelated proteins from sequential partially overlapping orfs. the 5 0 -orf encodes the p10 fusion-associated small transmembrane protein that is responsible for the syncytium-inducing phenotype of these reoviruses. 191 the second orf encodes a nonstructural nucleocytoplasmic protein (p17) that has no known function. 192 the terminal orf encodes the sigma c protein. this is similar to the mammalian reovirus sigma 1 protein that has a function in cell adhesion. 193 analysis of the start codons of these three orfs revealed that the first aug codon is in a suboptimal context, whereas the sequence surrounding the aug of the second uorf provides a good context for translation initiation with a highly conserved a at the ã� 3 position. the start codon of sigma c is in a strong context but it is a considerable distance from the 5 0 -terminal cap structure. 194 by altering the sequences surround the augs of the uorfs that encoded the p10 and p17 proteins to optimal contexts it was shown that the coordinated expression of these proteins occurred by leaky scanning. 194 however, the translation initiation of sigma c was shown to be independent of leaking scanning, reinitation, and internal ribosome entry and it was proposed that sigma c translation is mediated by an atypical shunting mechanism. 194 viral strategies to subvert the mammalian translation machinery sendai virus belongs to the parainfluenza virus family, is a pathogen of mice, and has a negative-sense rna genome. in sendai virus p/c mrna there are five start codons that are located 81-201 nt from the 5 0 end of the mrna. a sixth start codon is located more than 1500 nt from the 5 0 end, and together these generate eight protein products, as alternative c-termini are also used. 195 initiation from the first three start codons, which are acg, aug, and aug, can be explained by the leaky scanning model. the sequence that surrounds the first unusual acg codon is in an otherwise optimal context (gccacggat) with a purine at ã¾ 4 and ã� 3. 196 if the acg sequence is mutated to aug there is increased expression of the encoded c 0 protein, but the expression of the p and c proteins initiated from the second and third start codons is ablated, presumably because there is no leaky scanning. the expression of the proteins from the downstream start codons is not affected under these circumstances, suggesting that the initiation of these proteins is independent of scanning. 197, 198 it was shown that in vitro translation of these downstream y proteins was initiated by ribosomal shunting, 195 but in vivo this occurred by both shunting and proteolytic processing of the c 0 and/or c proteins. 199 all proteins encoded by picornaviruses are present in a long single orf which must be ''cleaved'' to give the functional polypeptides. the fmdv 2a peptide is encoded between sequences that specify capsid and replicative functions of the virus and this is the major site for the processing of this polyprotein. 200 it has been shown that the 2a region of fmdv (and other picornaviruses) mediates ''cleavage'' of its own c-terminus to release it from the 2b region. interestingly, the 2a peptide is active when placed between reporter proteins and the cleavage reaction is not therefore dependent on viral (protease) sequences outside of this region. 200 instead the data suggest that the 2a peptide is able to interact with the ribosome and direct translational recoding. 201 a detailed analysis of this recoding reaction shows that the ribosomes pause over the final amino acid of the 2a peptide. the recruitment of release factors to this peptide catalyzes termination and releases the peptide, the sequence of which includes the penultimate amino acid encoded by this region (glycine), while the codon of the terminal amino acid (proline) remains in the a site of the ribosome. 201 these 2a like peptides have been termed chysel (cis-acting hydrolase element) peptides and their ability to act independently of any proteolytic activity in the ''host'' cell has lead to a number of biotechnological uses. 201 a. inhibition of pkr in mammalian cells there are four proteins that control the formation of ternary complex that is required to bring the initiator trna imet to the ribosome by inducing phosphorylation of eif2 (fig. 7) . these are the gcn2, perk, hri, and pkr kinases. these proteins are activated by different external stimuli, generally under conditions of cell stress, and the particular kinase that is activated is dependent upon the stress induced. pkr activation represents one of the major host responses to viral infection. exposure of cells to interferon stimulates the production of pkr 202 which then is activated in response to the presence of dsrna in cells. this often occurs as a result of viral infection by rna viruses, either because the virus has dsrna elements within its genome, or because viral replication induces the temporary formation of dsrna intermediates which could originate from bidirectional transcription. the interaction of pkr with dsrna initiates dimerization, autophosphorylation, and the subsequent activation of this kinase (fig. 8) . active pkr dimers then phosphorylate the alpha subunit of eif2 at serine 51 and this has the net effect of preventing further ternary complex formation (fig. 3) such that the translation of host and viral rnas is inhibited. 203 viruses have developed a number of mechanisms to counteract the effects of pkr and there are viral proteins that inhibit pkr activation, sequester dsrna, inhibit pkr dimerisation, activate antagonist phosphatases, or degrade pkr (fig. 9 ). many viruses employ mechanisms to inhibit pkr dimerization and thereby prevent pkr activation in the presence of dsrna. for example, the kshv-8 (hhv-8) protein virf-2 binds directly to pkr and prevents activation by inhibiting the autophosphorylation of the protein. 206 hcv encodes two pkr inhibitors, the e2 envelope and the ns5a protein. the cytosolic form of the e2 protein is unglycosylated, and has been shown to directly bind to and inhibit pkr function. 207 similarly, ns5a interacts with pkr and inactivates its kinase function. 208, 209 this interaction occurs via the interferon sensitivity determining region (isdr) and there is a correlation between a negative response to interferon (ifn) of some hcv-chronically infected patients and mutations in a region of ns5a, although the relevance of the ns5a/pkr interaction in this regard is still unclear. [210] [211] [212] interestingly, it has also been shown that the ns5a/pkr interaction may be involved in the development of liver carcinoma. 213 9 . mechanisms of inactivation of pkr by viral proteins. viruses have developed a wide range of mechanisms to prevent activation of pkr. these include the production of viral proteins that are able to prevent pkr activation by inhibiting pkr dimerisation and dsrna-binding proteins that sequester dsrna. activity. 218, 219 in addition, tat directly contributes to the downregulation of pkr 219 as this protein is a substrate for pkr and acts as a competitor of eif2. 220, 221 the phosphorylation of tat by pkr leads to more robust viral transcription since this phosphorylation increases the interaction of tat with tar. 222 the vaccinia virus k3l gene encodes an 88 amino acid protein that is 30% identical to the n-terminus of eif2. 223 the k3l protein acts as a competitive inhibitor of pkr and a pentapeptide motif is important for this effect. k3l is produced early in vaccina virus replication, suggesting that this serves to ensure continued translation once dsrna is produced by the virus. 224 ebv produces two small noncoding rnas eber-1 and eber-2. the eber rnas play an important role in downregulating pkr activity and preventing virusinduced apoptosis. 12 eber-1 inhibits the protein kinase activity of pkr directly in vitro by competing with dsrna activators for binding to the enzyme. 225 moreover, expression of eber-1 in ebv negative cells protects against ifn-induced apoptosis. 226 similarly, the adenovirus va i rna which is synthesized in large quantities following viral infection binds to pkr. 227 this rna blocks the activation of pkr in the presence of dsrna and as a consequence both the autophosphorylation of pkr and the subsequent phosphorylation of eif2a are inhibited. 134 many viruses synthesize dsrna-binding proteins that prevent activation of pkr by interacting with and sequestering any free dsrna molecules. 228, 229 the first such dsrna-binding protein identified in this regard was reovirus sigma 3. 230 the reovirus sigma 3 protein does not have catalytic activity and it was shown that inhibition of pkr by this protein could be overcome by the addition of excess dsrna, 229 suggesting that the protein acts as a competitive inhibitor by directly binding to dsrna. influenza ns1 protein is a multifunctional protein involved in both protein-protein and protein-rna interactions. 231 this protein dimerizes upon binding to poly(a), and ns1-rna complexes block pkr activation. 231 similarly, the e3l protein from vaccinia virus contains a dsrna-binding domain which binds to and sequesters dsrnas produced during virus infection and so inhibits pkr activation. 232 both ebv and hsv-1 encode proteins, sm protein and us 11, respectively, that bind to dsrna and interact directly with pkr. 233, 234 both of these proteins contain an rxp domain comprised of multiple copies of the amino acid sequence rxp. this domain has been demonstrated to be a dsrna recognition motif 234 and is involved in the interaction of these proteins with pkr. [233] [234] [235] in pv-infected cells pkr is activated by the presence of dsrna, resulting in an increase in the phosphorylation of eif2 that leads to a decrease in protein synthesis rates. to circumvent this, pv infection also initiates a series of events that lead to the degradation of pkr. 236 the detailed mechanism of this degradation is not fully understood, although it has been suggested that cellular rather than viral proteases are required. 237 finally, it has been shown recently that the nss protein of rift valley fever virus (rvfv) induces specific degradation of pkr. 238 following infection with rvfv there is a dramatic decrease in pkr protein levels with little change in the levels of the mrna. in the presence of proteasome inhibitors there is a decrease in rvfv growth and an increase in pkr, strongly suggesting that the degradation is mediated via the proteasome pathway. since other related viruses do not have this activity it was suggested that the extraordinary pathogenicity of rvfv is due to the acquired pkr degradation function of nss. 238 5. additional mechanisms to overcome pkr activation although many viruses have devised novel ways in which to circumvent the effects of pkr, it is interesting to note that viruses belonging to the dicistroviridae family which infect insects use a unique mechanism to initiate their translation which is independent of ternary complex. [91] [92] [93] in this case viral protein synthesis is stimulated when eif2 is phosphorylated. 92 an alternative mechanism is adopted by the alphavirus sindbis virus (sv) where activation of pkr by viral infection causes almost complete phosphorylation of eif2. however, under these conditions there is still efficient translation of sv 26s mrna. downstream of the initiation codon on the subgenomic rna there is a stable stem-loop, that is able to stall ribosomes on the correct site to initiate translation of sv 26s mrna. 81 the data suggest that eif2a delivers the met-trnai to the stalled 40s ribosome, bypassing the requirement for a functional eif2. 239 perk is a type i transmembrane protein that attenuates protein synthesis during endoplasmic reticulum (er) stress (see chapter by kedersha and anderson, this volume), including viral infection. virus infection has a profound effect on the er and this can result in activation of the unfolded protein response (upr) leading to perk-mediated eif2 phosphorylation [240] [241] [242] [243] [244] and transient translational arrest (reviewed in ref. 245) . viruses have developed a unique set of mechanisms to overcome the effects of perk activation and alternatively use the upr to selectively enhance the synthesis of viral proteins. the herpesvirus human cytomegalovirus (hcmv) is a widespread opportunistic pathogen that can cause disease and death of newborn infants or individuals that are immunocompromised. infection of cells with hcmv results in the production of large amounts of viral glycoproteins and an increase of ca 2ã¾ release from the er to the cytosol. this induces cell stress, which benefits the virus, as chaperone induction by the upr extends the protein folding capacity of the er. 246, 247 however, virus infection also induces the phosphorylation of perk and the translational upregulation of the transcription factor atf4, yet the virus prevents the activation of the atf4-dependent pathway that would lead to cell apoptosis. 246, 247 this is mediated by viral protein pul38 which promotes perk activation and atf4 production but suppresses the persistent c-jun n-terminal kinase (jnk) phoshorylation and er-stress-induced cell death. 244 it has also been shown that the lmp1 protein from ebv works in a similar manner. 240 again this protein induces the phosphorylation of perk and this translationally upregulates the production of atf4. atf4 then trans-activates the promoter of lmp1 leading to increased transcription and expression of this protein and enhanced proliferation of the infected b cells. 240 in hsv-1 infected cells perk is inactivated and not affected by the acute er stress that occurs as a result of the viral infection. 241 this is mediated by the viral glycoprotein (b) (gb) of hsv-1 that specifically associates with the luminal domain of perk and suppresses its activation. 241 interestingly, gb appears also to directly regulate viral protein accumulation in a perk-dependent manner. 241 similarly the hcv envelope protein e2, an er-bound protein, has been shown to directly bind to perk and inhibit its activation. mammalian cells that stably express e2 are resistant to the effects of er-stress inducers, and e2 can relieve the translational repression induced by perk. 243 as viruses do not possess their own translational machinery they are completely dependent on the host cell for this critical step in their replication cycle. it is clear that viruses have adopted a number of elegant mechanisms to inhibit host cell translation, or at least modify translation factors, in order to effectively compete with cellular mrnas (fig. 10) . viruses, especially rna viruses with their high rna replication error rates, are the best example we have of natural selection (as opposed to directed selection in agricultural and laboratory practice) operating in real time before our very eyes. the most ''successful'' viral strains or species are, by definition, those which are most abundant in the environment, and by this criterion h1n1 swine flu is proving a more successful virus than sars was, notwithstanding the much higher mortality rate (almost 10%) in sars infection. of course, such success may be short lived. for example, we expect swine flu to peak in the next couple of years and then decline, while other virus strains or species may be considered more successful because they maintain a more enduring high abundance, even though they never reach such extreme peaks. the drivers behind virus evolution seem relatively straightforward: (i) efficient transmission between host organisms, (ii) efficient redirection of host cell mechanisms, especially translation, toward viral multiplication at the expense of host cell functions, and (iii) an ability to evade the host cell defense mechanisms, both extracellular (e.g., immune system) and intracellular. against these, there is evolutionary pressure on the host to elaborate antiviral defense mechanisms, which in turn puts pressure on the viruses to develop mechanisms of negating or bypassing these host cell defenses. although this picture is almost certainly oversimplified, there is little doubt that virus evolution is relatively simple as compared with higher eukaryote whole organism evolution. viruses have contributed enormously to our understanding of eukaryotic protein synthesis mechanisms, and mrna structure and function. this is in part due to the fact that, irrespective of whether the viral genome is dna or rna, positive or negative strand, double or single stranded, all viruses are dependent on the cellular protein synthesis machinery, whereas they vary in their dependence on other facets of host-cell gene expression. the contribution of virus research to our understanding of mrna structure and function, and the mechanism of protein biosynthesis is highlighted by the following list of discoveries (in approximate chronological order) all made through exploiting viruses: eubacterial translation initiation sites (rna bacteriophages), poly(a) tails (vaccinia), 5 0 caps (reovirus), pkr (pv), translation of only the first cistron of polycistronic mrnas (tobacco mosaic virus), leaky termination (plant rna viruses), the scanning ribosome mechanism (reovirus), eif4g (pv), iress (picornaviruses), programmed-1 frame-shifting (retroviruses and coronaviruses), ribosome shunting (adenovirus), and reinitiation after translation of a long orf (pneumoviruses, caliciviruses, influenza b viruses). moreover, this list has not closed because there are unusual viral rna translation mechanisms that remain to be fully elucidated, for example, the extraordinary translational stop-restart mechanism promoted by the fmdv 2a peptide. some of the earlier discoveries in the above list exploited the fact that in the era before recombinant dna techniques and transcription vectors had been developed, viruses were often the best source of a single mrna species (e.g., pv, tobacco mosaic virus) or a very limited number of mrnas (reovirus), with the added advantage of being able to synthesize defined radiolabeled mrnas in some cases (e.g., reovirus). viruses also provide excellent tools with which to study cellular protein synthesis or novel viral mechanisms of initiation. for example, the picornavirus proteases that cleave eif4g are widely used to study capindependent mechanisms of initiation, and a range of viral ires elements that require different factors for function are routinely used to dissect translational control processes, such as mirna-mediated regulation of gene expression. however, the more recent discoveries are due to the fact that viruses, particularly positive-strand rna viruses with small genomes, have evolved to exploit the extremes of behavior (or what might be thought the weaknesses) in the host cell translation machinery (e.g., programmed frameshifting, leaky termination, shunting, reinitiation after a long orf). some of these viral ''tricks'' (e.g., iress, and perhaps shunting) have evolved to enable efficient viral protein synthesis to proceed despite the virus-induced shutdown of host cell protein synthesis, and, certainly in the case of iress, the discovery has proved highly relevant to mrna translation in the uninfected host cell. many other viral ''tricks'' have evolved to allow more than one protein to be expressed from a single rna: frameshifting and leaky termination give two proteins with the same n-termini, while reinitiation after a long orf gives two unrelated proteins as an alternative to relying entirely on proteolytic processing of a polyprotein by virus-encoded proteases, or synthesis of monocistronic subgenomic mrna(s). it is not yet clear whether these events also occur in the translation of some cellular mrnas. if they do, they certainly concern only a very limited number of cellular mrnas, and without the clues provided by the viral examples as to the sequence and structure of the required rna motifs, the task of identifying such cellular mrnas would be like trying to find the proverbial needle in a field of haystacks. even if they do not occur in any cellular mrna, the viral mechanisms are always instructive, because they provide insights into issues such as why does reinitiation after a long orf not normally occur with the vast majority of cellular mrnas. moreover, if the viral mrna motifs directing leaky termination, frameshifting, or reinitiation after a long orf, are absent from all essential cellular mrnas, they provide potential targets for the development of novel antiviral agents targeted against the intracellular phase of the infectious process (although the high mutation rate of rna viruses may well pose problems with this approach). thus, while the ''tricks'' are essential for viral multiplication, they are also a potential achilles heel. viruses not only interfere with host translational processes to improve competition for factors, etc., but they must also respond to a number of host antiviral ''attacks''-this means that viruses have evolved ways in which to manipulate the host to ensure their replication is not inhibited. viral countermeasures against host attack, and the delicate balance between the host and virus, are only beginning to be understood and it is likely that we will learn much more about this in the future. there is no doubt that a greater understanding of how viruses regulate host translation, and identification of viral strategies, will aid in the development of novel antiviral therapies; the diversity and ingenuity of viruses is also likely to further surprise us. a giant virus in amoebae the 1.2-megabase genome sequence of mimivirus regulation of translation initiation in eukaryotes: mechanisms and biological targets translational control by viral proteinases dissecting eif4e action in tumorigenesis disruption of the interaction of mammalian protein synthesis eukaryotic initiation factor 4b with the poly(a)-binding protein by caspase-and viral protease-mediated cleavages molecular mechanisms of translational control how do micrornas regulate gene expression? oncogenic gamma-herpesviruses: comparison of viral proteins involved in tumorigenesis phosphorylation and dephosphorylation events that regulate viral mrna translation regulation of the translation initiation factor eif4f by multiple mechanisms in human cytomegalovirus-infected cells epstein-barr virus: inhibition of apoptosis as a mechanism of cell transformation human cytomegalovirus infection induces rapamycin-insensitive phosphorylation of downstream effectors of mtor kinase human cytomegalovirus infection alters the expression of cellular microrna species that affect its replication activation of host translational control pathways by a viral developmental switch modification of rna by messenger-rna guanylyltransferase and messenger-rna (guanine-7-)methyltransferase from vaccinia virions phosphorylation of eif4e by mnk-1 enhances hsv-1 translation and replication in quiescent cells adenovirus overrides cellular checkpoints for protein translation activation of the translational suppressor 4e-bp1 following infection with encephalomyocarditis virus and poliovirus rapamycin stimulates viral protein synthesis and augments the shutoff of host protein synthesis upon picornavirus infection vesicular stomatitis virus infection alters the eif4f translation initiation complex and causes dephosphorylation of the eif4e binding protein 4e-bp1 hnrnps relocalize to the cytoplasm following infection with vesicular stomatitis virus eif4 initiation factors: effectors of mrna recruitment to ribosomes and regulators of translation mnk1, a new map kinase-activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates mitogen-activated protein kinases activate the serine/threonine kinases mnk1 and mnk2 circularization of mrna by eukaryotic translation initiation factors micrornas expressed by herpes simplex virus 1 during latent infection regulate viral mrnas does phosphorylation of the cap-binding protein eif4e play a role in translation initiation? cleavage of eukaryotic initiation factor eif4g and inhibition of host-cell protein synthesis during feline calicivirus infection rna-binding properties of a translational activator, the adenovirus l4 100-kilodalton protein adenovirus-specific translation by displacement of kinase mnk1 from cap-initiation complex elf4f a functional microrna-155 ortholog encoded by the oncogenic marek's disease virus ebv micrornas in primary lymphomas and targeting of cxcl-11 by ebv-mir-bhrf1-3 modification of eukaryotic initiation-factor 4f during infection by influenza-virus inhibition of hela cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex mapping of functional domains in eukaryotic protein synthesis initiation factor 4g (eif4g) with picornaviral proteases. implications for cap-dependent and cap-independent translational initiation mapping the cleavage site in protein synthesis initiation factor eif-4 gamma of the 2a proteases from human coxsackievirus and rhinovirus cleavage of eukaryotic translation initiation factor 4gii within foot-and-mouth disease virus-infected cells: identification of the l-protease cleavage site in vitro sv40-encoded micrornas regulate viral gene expression and reduce susceptibility to cytotoxic t cells lack of direct correlation between p220 cleavage and the shut-off of host translation after poliovirus infection proteolysis of human eukaryotic translation initiation factor eif4gii, but not eif4gi, coincides with the shutoff of host protein synthesis after poliovirus infection activity of the hepatitis a virus ires requires association between the cap-binding translation initiation factor (eif4e) and eif4g functional dissection of eukaryotic initiation factor 4f: the 4a subunit and the central domain of the 4g subunit are sufficient to mediate internal entry of 43s preinitiation complexes initiation of protein synthesis from the a site of the ribosome calicivirus translation initiation requires an interaction between vpg and eif4e caliciviruses differ in their functional requirements for eif4f components in vitro cleavage of eif4gi but not eif4gii by hiv-1 protease and its effects on translation in the rabbit reticulocyte lysate system translational resistance of late alphavirus mrna to eif2 alpha phosphorylation: a strategy to overcome the antiviral effect of protein kinase pkr the eukaryotic translation initiation factor 4gi is cleaved by different retroviral proteases cleavage of poly(a)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro cleavage of poly(a)-binding protein by coxsackievirus 2a protease in vitro and in vivo: another mechanism for host protein synthesis shutoff ? poly(a)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression interaction of rat poly(a)-binding protein with poly(a)-and non-poly(a) sequences is preferentially mediated by rna recognition motifs 3 ã¾ 4 calicivirus 3c-like proteinase inhibits cellular translation by cleavage of poly(a)-binding protein picornavirus ireses and the poly(a) tail jointly promote cap-independent translation in a mammalian cell-free system eukaryotic initiation factor 4g-poly(a) binding protein interaction is required for poly(a) tail-mediated stimulation of picornavirus internal ribosome entry segment-driven translation but not for x-mediated stimulation of hepatitis c virus translation a late adenovirus factor induces eif-4e dephosphorylation and inhibition of cell protein-synthesis cleavage of poly(a)-binding protein by poliovirus 3c proteinase inhibits viral internal ribosome entry site-mediated translation rubella virus capsid protein interacts with poly(a)-binding protein and inhibits translation capped and conserved terminal structures in human rotavirus genome double-stranded-rna segments rotavirus rna-binding protein nsp3 interacts with eif4gi and evicts the poly(a) binding protein from eif4f rotavirus nonstructural protein nsp3 is not required for viral protein synthesis bunyamwera orthobunyavirus s-segment untranslated regions mediate poly(a) tail-independent translation control of translation by the 5 0 -and 3 0 -terminal regions of the dengue virus genome a segment of the 5 0 nontranslated region of encephalomyocarditis virus rna directs internal entry of ribosomes during in vitro translation internal initiation of translation of eukaryotic mrna directed by a sequence derived from poliovirus rna internal initiation of translation in eukaryotes: the picornavirus paradigm and beyond eukaryotic initiation factors 4g and 4a mediate conformational changes downstream of the initiation codon of the encephalomyocarditis virus internal ribosomal entry site direct functional interaction of initiation factor eif4g with type 1 internal ribosomal entry sites polypyrimidine-tract binding protein (ptb) is necessary, but not sufficient, for efficient internal initiation of translation of human rhinovirus-2 rna the polypyrimidine tract binding protein is required for efficient picornavirus gene expression and propagation la autoantigen is necessary for optimal function of the poliovirus and hepatitis c virus internal ribosome entry site in vivo and in vitro a cell cycle-dependent protein serves as a template-specific translation initiation factor structural insights into the transcriptional and translational roles of ebp1 recognition of picornavirus internal ribosome entry sites within cells; influence of cellular and viral proteins competitive translation efficiency at the picornavirus type 1 internal ribosome entry site facilitated by viral cis and trans factors unique characteristics of a picornavirus internal ribosome entry site from the porcine teschovirus-1 talfan the picornavirus avian encephalomyelitis virus possesses a hepatitis c virus-like internal ribosome entry site element functional and structural similarities between the internal ribosome entry sites of hepatitis c virus and porcine teschovirus, a picornavirus secondary structure of the 5 0 nontranslated regions of hepatitis c virus and pestivirus genomic rnas mnk2 and mnk1 are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor 4e but not for cell growth or development a prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis c and classical swine fever virus rnas initiation factor-independent translation mediated by the hepatitis c virus internal ribosome entry site internal initiation in saccharomyces cerevisiae mediated by an initiator trna/eif2-independent internal ribosome entry site element hepatitis c virus ires rna-induced changes in the conformation of the 40s ribosomal subunit the eukaryotic translation initiation factors eif1 and eif1a induce an open conformation of the 40s ribosome the pathway of hepatitis c virus mrna recruitment to the human ribosome the pathway of hcv ires-mediated translation initiation hcv and csfv ires domain ii mediate eif2 release during 80s ribosome assembly translation initiation at the cuu codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro the 5 0 untranslated region of rhopalosiphum padi virus contains an internal ribosome entry site which functions efficiently in mammalian, plant, and insect translation systems factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus divergent trna-like element supports initiation, elongation, and termination of protein biosynthesis translation elongation after assembly of ribosomes on the cricket paralysis virus internal ribosomal entry site without initiation factors or initiator trna trna-mrna mimicry drives translation initiation from a viral ires cryo-em visualization of a viral internal ribosome entry site bound to human ribosomes: the ires functions as an rnabased translation factor translation initiation factors are not required for dicistroviridae ires function in vivo suppression of microrna-silencing pathway by hiv-1 during virus replication eukaryotic translation initiation machinery can operate in a bacterial-like mode without eif2 tethering of eif4g to adenoviral mrnas by viral 100k protein drives ribosome shunting the human immunodeficiency virus type 1 gag gene encodes an internal ribosome entry site the leader of human immunodeficiency virus type 1 genomic rna harbors an internal ribosome entry segment that is active during the g2/m phase of the cell cycle hiv-2 genomic rna contains a novel type of ires located downstream of its initiation codon identification of virusencoded micrornas identification of micrornas of the herpesvirus family evolutionarily conserved function of a viral microrna murine polyomavirus encodes a microrna that cleaves early rna transcripts but is not essential for experimental infection mrna helicase activity of the ribosome human cytomegalovirus protein pul38 induces atf4 expression, inhibits persistent jnk phosphorylation, and suppresses endoplasmic reticulum stress-induced cell death identification of functional micrornas released through asymmetrical processing of hiv-1 tar element analysis of the interaction of primate retroviruses with the human rna interference machinery a combined computational and microarray-based approach identifies novel micrornas encoded by human gamma-herpesviruses epstein-barr virus micrornas are evolutionarily conserved and differentially expressed hiv-1 protease cleaves eukaryotic initiation factor 4g and inhibits cap-dependent translation merkel cell polyomavirus encodes a microrna with the ability to autoregulate viral gene expression suppression of immediate-early viral gene expression by herpesvirus-coded micrornas: implications for latency adenovirus virus-associated rnaii-derived small rnas are efficiently incorporated into the rna-induced silencing complex and associate with polyribosomes an epstein-barr virus-encoded microrna targets puma to promote host cell survival identification of cellular genes targeted by kshv-encoded micrornas diverse herpesvirus micrornas target the stress-induced immune ligand micb to escape recognition by natural killer cells a viral microrna functions as an orthologue of cellular mir-155 generation of a conditionally replicating adenovirus based on targeted destruction of e1a mrna by a cell type-specific microrna tandem array-based expression screens identify host mrna targets of virus-encoded micrornas epstein-barr virus growth/ latency iii program alters cellular microrna expression epstein-barr virus-mediated dysregulation of human microrna expression the phosphorylation of eukaryotic initiation factor eif4e in response to phorbol esters, cell stresses, and cytokines is mediated by distinct map kinase pathways internal ribosome entry site within hepatitis c virus rna adenovirus va1 noncoding rna can inhibit small interfering rna and microrna biogenesis evidence that hiv-1 encodes an sirna and a suppressor of rna silencing a cellular microrna mediates antiviral defense in human cells modulation of hepatitis c virus rna abundance by a liver-specific microrna microrna-122 stimulates translation of hepatitis c virus rna interferon modulation of cellular micrornas as an antiviral mechanism engineering microrna responsiveness to decrease virus pathogenicity crystal structure of an avian influenza polymerase pa(n) reveals an endonuclease active site the genome-linked protein vpg of the norwalk virus binds eif3, suggesting its role in translation initiation complex recruitment translational control in positive strand rna plant viruses complex formation between potyvirus vpg and translation eukaryotic initiation factor 4e correlates with virus infectivity how does influenza virus regulate gene expression at the level of mrna translation? let us count the ways surprising function of the three influenza viral polymerase proteins: selective protection of viral mrnas against the cap-snatching reaction catalyzed by the same polymerase proteins the cap-snatching endonuclease of influenza virus polymerase resides in the pa subunit double-stranded rna-dependent protein kinase (pkr) is regulated by reovirus structural proteins influenza virus mrna translation revisited: is the eif4e cap-binding factor required for viral mrna translation? a protein that replaces the entire cellular eif4f complex characterization of the rna chaperone activity of hantavirus nucleocapsid protein programmed ribosomal frameshifting in hiv-1 and the sars-cov characterization of an efficient coronavirus ribosomal frameshifting signal-requirement for an rna pseudoknot structure of the rna signal essential for translational frameshifting in hiv-1 the sequences of and distance between 2 cisacting signals determine the efficiency of ribosomal frameshifting in human-immunodeficiency-virus type-1 and human t-cell leukemia-virus type-ii in-vivo frameshifting rna pseudoknots: structure and mechanism signals for ribosomal frameshifting in the rous-sarcoma virus gag-pol region a mechanical explanation of rna pseudoknot function in programmed ribosomal frameshifting mutational analysis of the rna pseudoknot component of a coronavirus ribosomal frameshifting signal mutational analysis of the slippery-sequence component of a coronavirus ribosomal frameshifting signal a crosskingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry presented at the 6th asia pacific congress of medical virology programmed ribosomal frameshifting in decoding the sars-cov genome a three-stemmed mrna pseudoknot in the sars coronavirus frameshift signal rna pseudoknots and the regulation of protein synthesis incorporation of pol into human immunodeficiency virus type 1 gag virus-like particles occurs independently of the upstream gag domain in gag-pol maintenance of the gag/gag-pol ratio is important for human immunodeficiency virus type 1 rna dimerization and viral infectivity the human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy programmed ribosomal frameshifting is siv is induced by a highly structured rna stem-loop pseudoknots: rna structures with diverse functions solution structure and thermodynamic investigation of the hiv-1 frameshift inducing element the murine doublestranded rna-dependent protein kinase pkr is required for resistance to vesicular stomatitis virus ribosomal frameshifting: an emerging drug target for hiv effects of intercistronic length on the efficiency of reinitiation by eukaryotic ribosomes ribosomal pausing at a frameshifter rna pseudoknot is sensitive to reading phase but shows little correlation with frameshift efficiency constraints on reinitiation of translation in mammals ribosomal pausing during translation of an rna pseudoknot what determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame? characterization of the sequence element directing translation reinitiation in rna of the calicivirus, rabbit hemorrhagic disease virus translation of the minor capsid protein of a calicivirus is initiated by a novel termination-dependent reinitiation mechanism the importance of inter-and intramolecular base pairing for translation reinitiation on a eukaryotic bicistronic mrna the mechanism of an exceptional case of reinitiation after translation of a long orf reveals why such events do not generally occur in mammalian mrna translation recycling of eukaryotic posttermination ribosomal complexes reduced incorporation of the influenza b virus bm2 protein in virus particles decreases infectivity eukaryotic coupled translation of tandem cistronsidentification of the influenza-b virus bm2 polypeptide characterization of the termination-reinitiation strategy employed in the expression of influenza b virus bm2 protein translational termination-re-initiation in viral systems animal pneumoviruses: molecular genetics and pathogenesis expression of the orf-2 protein of the human respiratory syncytial virus m2 gene is initiated by a ribosomal termination-dependent reinitiation mechanism coupled translation of the respiratory syncytial virus m2 open reading frames requires upstream sequences coupled translation of the second open reading frame of m2 mrna is sequence dependent and differs significantly within the subfamily pneumovirinae adenoviruses: update on structure and function spliced segments at 5 0 terminus of adenovirus 2 late messenger-rna analysis of rna cleavage at the adenovirus-2 l3 polyadenylation site translation by ribosome shunting on adenovirus and hsp70 mrnas facilitated by complementarity to 18s rrna rna interaction and cleavage of poly(c)-binding protein 2 by hepatitis a virus protease unshackling the links between reovirus oncolysis, ras signaling, translational control and cancer the second open reading frame of the avian reovirus s1 gene encodes a transcription-dependent and crm1-independent nucleocytoplasmic shuttling protein oligomerization and cell-binding properties of the avian reovirus cell-attachment protein sigma c leaky scanning and scanningindependent ribosome migration on the tricistronic s1 mrna of avian reovirus* sendai virus y proteins are initiated by a ribosomal shunt replication of paramyxoviruses scanning independent ribosomal initiation of the sendai virus-y proteins in vitro and in vivo scanning independent ribosomal initiation of the sendai virus x-protein intracellular processing of the sendai virus c 0 protein leads to the generation of a y protein module: structure-functional implications dissection of a co-translational nascent chain separation event site-specific release of nascent chains from ribosomes at a sense codon molecular cloning and characterisation of the human double-stranded rna-dependent protein kinase induced by interferon essential role for the dsrna-dependent protein kinase pkr in innate immunity to viral infection autophosphorylation of the protein-kinase dependent on doublestranded-rna sequential modification of translation initiation factor elf4gi by two different foot-and-mouth disease virus proteases within infected baby hamster kidney cells: identification of the 3c(pro) cleavage site latently expressed human herpesvirus 8-encoded interferon regulatory factor 2 inhibits double-stranded rna-activated protein kinase detection of a novel unglycosylated form of hepatitis c virus e2 envelope protein that is located in the cytosol and interacts with pkr interaction of the interferon-induced pkr protein kinase with inhibitory proteins p58(ipk) and vaccinia virus k3l is mediated by unique domains: implications for kinase regulation evidence that hepatitis c virus resistance to interferon is mediated through repression of the pkr protein kinase by the nonstructural 5a protein comparison of full-length sequences of interferon-sensitive and resistant hepatitis-c virus 1b-sensitivity to interferon is conferred by amino-acid substitutions in the ns5a region mutations in the nonstructural protein 5a gene and response to interferon in patients with chronic hepatitis c virus 1b infection mutations within protein kinase r-binding domain of ns5a protein of hepatitis c virus (hcv) and specificity of hcv antibodies in pretreatment sera of hcv-chronically infected patients and their effect on the result of treatment evolution of hepatitis c virus non-structural 5a gene in the progression of liver disease to hepatocellular carcinoma prevalence of wildtype in ns5a-pkr protein kinase binding domain in hcv-related hepatocellular carcinoma the oncogenic potential of hepatitis c virus ns5a sequence variants is associated with pkr regulation regulation of messenger-rna accumulation by a human-immunodeficiency-virus transactivator protein the integrity of the stem structure of human-immunodeficiency-virus type-1 tat-responsive sequence rna is required for interaction with the interferon-induced 68,000-mr protein-kinase tat-responsive region rna of human immunodeficiency virus-1 can prevent activation of the double-stranded-rna-activated protein-kinase control of the interferon-induced 68-kilodalton protein-kinase by the hiv-1 tat gene-product the tat protein of human immunodeficiency virus type 1 is a substrate and inhibitor of the interferon-induced, virally activated protein kinase, pkr hiv-1 tat directly interacts with the interferon-induced, double-stranded rna-dependent kinase phosphorylation of hiv tat by pkr increases interaction with tar rna and enhances transcription recombinant vaccinia virus k3l gene-product prevents activation of double-stranded rna-dependent, initiation factor-2-alpha-specific protein-kinase distinct patterns of ifn sensitivity observed in cells infected with vaccinia k3l(-) and e3l(-) mutant viruses comparative-analysis of the regulation of the interferon-inducible protein-kinase pkr by epstein-barr-virus rnas eber-1 and eber-2 and adenovirus va(i) rna epstein-barr virus-encoded poly(a)(-) rna confers resistance to apoptosis mediated through fas by blocking the pkr pathway in human epithelial intestine 407 cells adenovirus vai rna complexes with the 68,000 mr protein kinase to regulate its autophosphorylation and activity the e3l and k3l vaccinia virus geneproducts stimulate translation through inhibition of the double-stranded rna-dependent protein-kinase by different mechanisms selective translation initiation by ribosome jumping in adenovirusinfected and heat-shocked cells inhibition of pkr by rna and dna viruses binding of the influenza-virus ns1 protein to double-stranded-rna inhibits the activation of the protein-kinase that phosphorylates the eif-2 translation initiation-factor complementation of vaccinia virus deleted of the e3l gene by mutants of e3l characterization of rna determinants recognized by the arginineand proline-rich region of us11, a herpes simplex virus type 1-encoded double-stranded rna binding protein that prevents pkr activation identification of a lytic-cycle epstein-barr virus gene product that can regulate pkr activation the herpes simplex virus type 1 u(s)11 protein interacts with protein kinase r in infected cells and requires a 30-amino-acid sequence adjacent to a kinase substrate domain the cellular 68,000-mr protein-kinase is highly autophosphorylated and activated yet significantly degraded during poliovirus infection-implications for translational regulation degradation of the interferon-induced 68,000-mr proteinkinase by poliovirus requires rna nss protein of rift valley fever virus induces the specific degradation of the double-stranded rna-dependent protein kinase eukaryotic translation initiation factor 4f architectural alterations accompany translation initiation factor redistribution in poxvirus-infected cells the lmp1 oncogene of ebv activates perk and the unfolded protein response to chive its own synthesis maintenance of endoplasmic reticulum (er) homeostasis in herpes simplex virus type 1-infected cells through the association of a viral glycoprotein with perk, a cellular er stress sensor resistance of mrna translation to acute endoplasmic reticulum stress-inducing agents in herpes simplex virus type 1-infected cells requires multiple virusencoded functions protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis c virus envelope protein e2 through the eukaryotic initiation factor 2 alpha kinase perk microrna-155 is an epstein-barr virus-induced gene that modulates epstein-barr virus-regulated gene expression pathways transcriptional and translational control in the mammalian unfolded protein response production of infectious human cytomegalovirus virions is inhibited by drugs that disrupt calcium homeostasis in the endoplasmic reticulum human cytomegalovirus infection activates and regulates the unfolded protein response key: cord-333757-h12aozg2 authors: modis, yorgo title: class ii fusion proteins date: 2013-07-10 journal: viral entry into host cells doi: 10.1007/978-1-4614-7651-1_8 sha: doc_id: 333757 cord_uid: h12aozg2 enveloped viruses rely on fusion proteins in their envelope to fuse the viral membrane to the host-cell membrane. this key step in viral entry delivers the viral genome into the cytoplasm for replication. although class ii fusion proteins are genetically and structurally unrelated to class i fusion proteins, they use the same physical principles and topology as other fusion proteins to drive membrane fusion. exposure of a fusion loop first allows it to insert into the host-cell membrane. conserved hydrophobic residues in the fusion loop act as an anchor, which penetrates only partway into the outer bilayer leaflet of the host-cell membrane. subsequent folding back of the fusion protein on itself directs the c-terminal viral transmembrane anchor towards the fusion loop. this fold-back forces the host-cell membrane (held by the fusion loop) and the viral membrane (held by the c-terminal transmembrane anchor) against each other, resulting in membrane fusion. in class ii fusion proteins, the fold-back is triggered by the reduced ph of an endosome, and is accompanied by the assembly of fusion protein monomers into trimers. the fold-back occurs by domain rearrangement rather than by an extensive refolding of secondary structure, but this domain rearrangement and the assembly of monomers into trimers together bury a large surface area. the energy that is thus released exerts a bending force on the apposed viral and cellular membranes, causing them to bend towards each other and, eventually, to fuse. enveloped viruses acquire a lipid bilayer membrane when they bud across the plasma membrane or the membrane of the endoplasmic reticulum (er) during assembly of the virion. 1, 2 during infection, the viral membrane must be fused to the host-cell membrane to deliver the viral genome into the cytoplasm for replication (fig. 1) . the fusion of the viral and host-cell membranes is therefore the central molecular event during the entry of viral entry into host cells, edited by stefan pöhlmann and graham simmons. ©2013 landes bioscience and springer science+business media. enveloped viruses into cells. adjacent membranes do not fuse spontaneously; membrane fusion requires considerable energy (on the order of 100 kj mol -1 or 40 kt). 3, 4 envelope proteins anchored in the viral membrane provide this energy in the form of a conformational rearrangement that bends the apposed membranes towards each other, inducing them to fuse. [5] [6] [7] most 'fusion proteins' (or their cleavage products) also effect cellular attachment of the virus prior to the membrane fusion event by binding to a receptor on the cell surface, except the paramyxo-and alphaviruses, in which a second envelope protein binds the receptor. fusion proteins of enveloped viruses fall into two structural classes. the influenza virus haemagglutinin (ha) is the prototype of class i fusion proteins, 8 which encompass those of other orthomyxo-and paramyxoviruses such as measles virus, retroviruses such as human immunodeficiency virus (hiv), filoviruses such as ebola virus, and coronaviruses such as sars (see . class ii fusion proteins are a structurally and evolutionarily distinct class of proteins found in flaviviridae, such as dengue, yellow fever, and west nile viruses, and on alphaviruses, such as semliki forest and sindbis viruses. hepatitis c has a similar genomic organization to the flaviviruses, and therefore most likely relies on a class ii fusion protein as well. crystal structures of several class i and class ii fusion proteins before [9] [10] [11] [12] [13] [14] [15] and after 5, [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] their fusogenic conformational rearrangements have provided us with a detailed molecular understanding of the fusion mechanism ( table 1 ). the structures show that, despite the absence of similarities in the protein folds of the two classes, fusion proteins from both classes use the same physical principles and general topology to drive membrane fusion. first the fusion protein inserts a hydrophobic fusion anchor partway into the outer bilayer leaflet of the host-cell membrane. the fusion anchor is either an n-terminal peptide, 30 as in influenza and hiv, 31 or an internal loop, as in sars coronavirus, 32 avian sarcoma leucosis virus 33 and all class ii enveloped viruses. 34 second, the fusion protein folds back on itself, directing the (c-terminal) viral transmembrane anchor towards the fusion anchor. this fold-back forces the host-cell membrane (held by the fusion anchor) and the viral membrane (held by the c-terminal transmembrane anchor) against each other, resulting in fusion of the two membranes. in this chapter, i describe our current picture of how class ii fusion proteins drive viral membrane fusion, based on the structural and biochemical data available to date. three-dimensional structures of eight class ii fusion proteins in their native, or prefusion states, 10, [12] [13] [14] 35, [93] [94] [95] 97 that is, the conformation that they adopt on the surface of a mature virus particle, have been determined at near atomic resolution. figure 2 shows the three-domain structures of e 13 and e1, 12 the fusion proteins of dengue virus (a representative flavivirus) and of semliki forest virus (an alphavirus), respectively. the two proteins share a common molecular architecture, despite a lack of significant sequence similarity. domain i, an eight-stranded -barrel, organizes the structure. two long insertions between pairs of consecutive -strands in domain i form the elongated domain ii, which bears the fusion anchor, a fusion loop in class ii proteins, at its tip (figs. 2, 4) . domain ii contains twelve -strands and two -helices. domain iii is an igc-like module, with ten -strands. domain iii contains most of the antigenic sites on e, as well as most of the structural determinants of virulence and tropism. 10 this observation, and the widespread occurrence of immunoglobulin modules in cell-adhesion proteins, suggest that domain iii participates in attachment to a cellular receptor. 10 indeed, positively charged patches on the surface of domain iii in dengue virus have been suggested to promote attachment by binding heparan sulfate on the cell surface. 36 both e1 and e have one or more glycosylation sites. these glycans can aid viral attachment to the cell surface, in as found in the mature virus particle. 48 the fusion loop in a-c is marked with an asterisk. a second subunit of e, forming the dimer found on the viral surface and in solution, is shown in light gray. d) view rotated 90° relative to c, with the second subunit omitted for clarity. e) crystal structure of sfv e1 in the prefusion conformation, 12 as found in the mature virus particle. 50 the fusion loop is marked with an asterisk. f) view rotated 90° relative to e. the case of dengue virus by binding to the lectin dc-sign. 37, 38 as expected from their differ only in the length and structure of surface-exposed loops, some of which have been implicated in receptor binding. 10, 39, 40 despite these hints on the basis of cellular attachment, however, a cellular receptor that specifically recognizes an envelope protein on a class ii enveloped virus has yet to be conclusively identified, although candidate receptors for dengue virus type 1 41 and west nile virus 42 were recently suggested. it is important to note that all the crystal structures of fusion proteins determined so far, from both classes and regardless of their conformational state, lack the c-terminal viral membrane anchor. this anchor consists of one or two transmembrane helices, and has been intentionally omitted in constructs targeted for crystallization to facilitate expression and handling, and to promote crystallization. the crystallized species are therefore referred to as soluble fragments of the ectodomains of the full-length fusion protein. furthermore, all available crystal structures of class ii fusion proteins lack the 'stem' region, 43 a 30-55 amino acid linker between domain iii and the c-terminal transmembrane anchor (figs. 2a-b, 3). as i will discuss below, the stem region plays a key role in the final stages of membrane fusion. its function is analogous to that of the 'outer helix' in class i fusion proteins. 8 both class i and class ii fusion proteins rely on a proteolytic cleavage event to become primed to respond to the environmental conditions appropriate for fusion. these conditions are usually the acidic ph of an endosome ( fig. 1 ), but for some class i enveloped viruses, such as hiv, coreceptor binding is required instead. in contrast to class i fusion proteins, however, class ii fusion proteins rely on a priming proteolytic cleavage that does not cleave the fusion protein itself. instead, class ii proteins associate with a second, 'protector' protein, called m (for membrane protein) in flaviviruses or e2 in alphaviruses. the protector protein is cleaved by furin when immature virus particles assembled in the er reach the trans-golgi network. 44 the cleavage produces mature virus particles, which are then released from the host cell by exocytosis. the cleavage of the protector protein releases a conformational constraint on the fusion protein, which allows it to adopt its mature conformation (described above) in a large rearrangement on the viral surface. in the mature conformation, the fusion protein is primed to respond to acidic ph and induce membrane fusion with a further conformational rearrangement (described below). structures from electron cryomicroscopy of both immature 45, 46 and mature 47-50 flavivirus and alphavirus particles, provide a detailed picture of the rearrangement that accompanies maturation in these viruses. alphaviruses retain the t = 4 icosahedral packing of their envelope proteins, but domains that form spikes on the immature virion swing in towards the threefold symmetry axis, during maturation. 46, 50 the rearrangement is more dramatic in flaviviruses, in which the fusion protein e breaks the t = 3 icosahedral symmetry of the immature virion 45 to adopt an unusual icosahedral herringbone pattern in the mature virion. 47, 51 in both alphaviruses and flaviviruses, the fusion proteins form dimers in the mature virion albeit in different configurations. 10, 12 the key feature of the maturation process in both genera, however, is that cleavage of the protector protein allows the fusion loop to reposition itself so that it is poised to insert into the host-cell membrane in response to acidification of the solute in the endosome. mature virus particles are sequence identities (≥ 37%), flaviviral e proteins have very similar overall structures, and therefore infectious, 44, 52 unlike immature virions, 53, 54 which are insensitive to ph. the fusion loop is shielded from the viral surface in mature virions by e-e dimer contacts in flaviviruses, or by protein e2 in alphaviruses (figs. 3a,b, 5a). the three-dimensional structures of four class ii fusion proteins in their postfusion states 29,55,56,94 reveal striking differences from the prefusion forms (fig. 3) , and suggest a 13 and b) two sfv e1 12 molecules in the prefusion conformation as found on the viral surface, viewed perpendicular to the viral membrane. the fusion loop is buried, either in the dimer interface (a), or under e2 (b). the outer (proximal) bilayer leaflets of the cellular and viral membranes are shown to scale as solid rectangles. the thin outer layer within each leaflet represents the polar headgroup layer, and the thicker inner layer represents the hydrocarbon layer. the stem-anchor segments are absent from the crystal structure, but are represented here schematically as rods in the viral membrane. c,d) upon acidification of the solute in the endosome, the domain ii rotates 15-30° about a hinge in the domain ii-domain i interface. this exposes the fusion loop, which then inserts into the host cell membrane. the postfusion, trimeric structures of den e 29 (e) and sfv e1 (f). 55 after insertion of their fusion loops into the target membrane, the fusion proteins form trimers and fold back on themselves, bringing the fusion loops close to the c-terminal transmembrane anchors. molecular mechanism for membrane fusion (see below and fig. 5 ). like class i fusion proteins, flaviviral e proteins and alphaviral e1 proteins are both homotrimers in their postfusion conformations. class ii proteins form trimers from monomers on the viral surface, while class i proteins are trimeric in their prefusion state. 8 however, a comparison of the pre-and postfusion states of influenza ha-the only example in its class where both structures are known for the same protein-shows that, as in class ii fusion proteins, nearly all of the trimer contacts in the postfusion state are formed during the fusogenic conformational rearrangement. unlike influenza ha, which undergoes extensive refolding during membrane fusion, the three domains of class ii fusion proteins retain most of their folded structures (fig. 3) . instead, the domains undergo major rearrangements in their relative orientations, through flexion of the interdomain linkers. domain iii undergoes the most significant displacement in the fusion transition. it rotates by about 70°, and its center of mass shifts by 30-40 å towards domain ii. this folding-over brings the c-terminus of domain iii about 40 å closer to the fusion loop, at tip of domain ii (fig. 3) . domain ii rotates 15-30° with respect to domain i about a hinge region 13 in which mutations affect the ph threshold of fusion in various flaviviruses. [57] [58] [59] [60] [61] [62] these conformational rearrangements position the end of domain iii-and the beginning of the stem region that links domain iii to the c-terminal viral transmembrane anchor-towards the fusion loop ( fig. 3e-f) . 29, 55 a deep channel extends from the c-terminus of the crystallized fragment along the intersubunit contact between domains ii to the fusion loops, in both the dengue and semliki forest virus postfusion trimer structures. in the full-length fusion proteins, it is thought that the stem binds in this channel in an extended, but mainly -helical conformation. 29, 48 this proposed stem conformation places the viral transmembrane anchor in the immediate vicinity of the fusion loop, just as in the postfusion conformation of class i viral fusion proteins. the fusion transition in class ii viral proteins is irreversible. the refoldings just described may impart irreversibility by contributing a high barrier to initiation of trimerization and an even higher barrier to dissociation of postfusion trimers once they have formed. moreover, many new polar and nonpolar contacts are formed during the fusion transition, in several different areas along the threefold axis of the trimer. the total surface buried is 13,000-15,000 å 2,29,55 nearly four times more than is buried in the prefusion dimer. the stem, which is missing from currently available crystal structures, most likely forms additional contacts with the core trimer structure. the stem does indeed promote trimer assembly even in the absence of liposomes. 43 the process of viral membrane fusion in both class i and class ii enveloped viruses begins with the exposure of a fusion anchor, and its subsequent insertion into the host-cell membrane. fusion anchors from both viral classes vary in length but are in general rich in glycines and hydrophobic residues, particularly aromatic residues such as trp or phe. sequence conservation is poor between fusion proteins of both classes. the fusion anchor in class i fusion proteins-the 'fusion peptide'-is a region of approximately 20 residues at or near the n-terminus of the envelope protein. the crystal structure of the parainfluenza virus 5 fusion (f) protein in its prefusion form reveals the fusion peptide wedged between two subunits of the protein, in a partly extended, partly -sheet and partly -helical conformation. 15 structural studies on influenza ha in its postfusion conformation using nmr and other spectroscopic techniques show that the fusion peptide is mostly -helical in character and that its structure changes only subtly as it inserts partway into the outer leaflet of the host-cell lipid bilayer. 63, 64 none of the currently available postfusion class i protein crystal structures contain information on the fusion peptide. the recently determined crystal structures of class ii fusion proteins in pre 10,12-14 and postfusion 29, 55, 56 conformations offer the first direct views of fusion anchors-in this case, the fusion loops-as they insert into a target membrane (fig. 4) . like the class i fusion peptide, the class ii fusion loop penetrates only partway into the hydrocarbon layer of the target membrane. exposed carbonyls and charged residues prevent the fusion loop from penetrating further than 6 å. 29, 56 in flaviviruses, the fusion loop adopts a tightly folded conformation, which is stabilized by a disulfide bond (fig. 4a) . the structure of the fusion loop is essentially identical in the pre-and postfusion conformations of the protein, suggesting that membrane insertion has no effect on the structure of the fusion loop. during the fusion transition, three hydrophobic residues in the fusion loop (trp, leu, and phe) become exposed on the molecular surface. three fusion loops end up in close proximity at the tip of the trimer in the postfusion conformation, where they form a crater-like surface with a hydrophobic rim (fig. 3e) . electron cryomicroscopy 29 and mutagenesis studies 34 confirm that these hydrophobic, mostly aromatic residues on the crater rim insert into the host-cell membrane, acting as an 'aromatic anchor' for the fusion protein. the concave shape of the crater is thought to be important in generating distortions or perturbations in the host-cell membrane, 29 which are required for fusion. 65 in alphaviruses, the fusion loop is also rich in aromatic and other hydrophobic residues. unlike flaviviral fusion loops, however, alphaviviral fusion loops do not form trimer contacts (fig. 3f) . indeed, in the postfusion structure of the semliki forest virus e1 trimer, the fusion loops have high temperature factors and exhibit a high degree of flexibility despite the presence of two disulfide bonds. thus, the structures of the fusion loops are poorly defined, but each fusion loop seems to adopt a very different conformation (fig. 4b,c) . 55 the fusion loops in the postfusion semliki forest virus e1 structure form quite polar surfaces, with many mainchain carbonyls and some polar or charged sidechains exposed on the surface. this suggests that, in contrast to flaviviral fusion loops, alphaviral fusion loops either change their conformation upon membrane insertion to shield polar groups from the membrane, or the fusion loops only interact with the polar headgroups of the lipids, and do not penetrate into the hydrocarbon layer. semliki forest virus e1 trimers form irregular clusters, or 'rosettes' of about 40-60 trimers through contacts between fusion loops in adjacent trimers. 55 this is reminiscent of influenza virus ha, which aggregates into rosettes through interactions between the fusion peptide, at low ph and after proteolytic activation. 66 this fusion loop/peptide clustering may provide a mechanism for the direct coupling of several e1/ha trimers to work in concert around a single fusion site (see below). combined with previous knowledge, the structures of the fusion proteins from class ii viruses in their postfusion states 29, 55, 56 have led to a much better understanding of how conformational changes in the proteins drive membrane fusion. the structures confirm two major principles of membrane fusion machineries: (1) the fusion protein must insert an anchor into each of the two membranes to be fused, and (2) the protein folds back on itself in a thermodynamically favorable conformational rearrangement that drives membrane fusion by forcing the two anchors into close proximity. in the current model, viral membrane fusion proceeds as follows (fig. 5) . first, receptor binding by an envelope protein, which in flaviviruses is also the fusion protein, leads to clathrin-mediated endocytosis of the virus (figs. 1, 5a ). when the virus reaches endosomal compartments the low ph of the lumen (ph 6) causes an initial conformational rearrangement that leads to the exposure of the previously buried fusion loop 48, 50 at the tip of domain ii. in flaviviruses, domains i and ii flex relative to each other by 30°. 29 this hinge motion causes domain ii, and therefore the fusion loop, to swing away from the viral surface and towards the host-cell membrane (fig. 5b) . indeed, mutations at the domain i-domain ii interface in various flaviviruses alter the ph threshold of fusion. 13, [57] [58] [59] [60] [61] [62] as domain ii swings away from the viral surface, constraints imposed by the tight packing of e on the viral surface are released, allowing e to diffuse freely in the plane of the viral membrane. the stem may also be able to extend away from the membrane at this stage. in alphaviruses, constraints are released in response to low ph by the dissociation of the protector (and receptor-binding) protein e2. this exposes the fusion loop and allows domain ii of e1 to swing towards the nearest threefold symmetry axis in the virus particle in a 15° hinge motion relative to domain i, leading to the formation of trimer contacts with adjacent e1 molecules. 46 the second key step in the fusion process is insertion of the exposed fusion loop into the host-cell membrane (fig. 5c) . alphaviral e1 has already formed some trimer contacts at this stage, but flaviviral e proteins probably insert their fusion loops as monomers. membrane insertion probably catalyzes trimerization of the fusion loops, 67 by lateral rearrangement of e monomers. this trimeric prefusion intermediate (fig. 5c ) bridges host-cell and viral membranes, anchored by its fusion loops in the former and by the figure 5 . proposed fusion mechanism for fusion mediated by class ii fusion proteins. a) the virus binds to a receptor on the cell surface. in flaviviruses, the fusion protein e binds the receptor, while in alphaviruses, the 'protector' protein e2 binds the receptor. following attachment, the virus is internalized to an endosome. b) acidic ph in the endosome causes domain ii to hinge outward from the virion surface, exposing the fusion loop, and allowing e monomers to rearrange laterally in the plane of the membrane. c) the fusion loop inserts into the hydrocarbon layer of the host-cell membrane, promoting trimer formation. d) formation of trimer contacts spreads from the fusion loop at the tip of the trimer, to the base of the trimer. the protein folds back on itself, directing the fusion loop towards the c-terminal transmembrane anchor. energy release by this refolding bends the apposed membranes. e) creation of additional trimer contacts between the stem-anchor and domain ii leads first to hemifusion and then (f) to formation of a lipidic fusion pore. viral transmembrane anchors in the latter. this proposed intermediate is analogous to the 'prehairpin' intermediate postulated for class i viral fusion mechanisms. 68 upon insertion of the fusion loops into the host-cell membrane, formation of trimer contacts spreads from the fusion loops at the trimer tip to domain i at the trimer base. domain ii shifts and rotates, folding the stem and c-terminal anchor back towards the fusion loop (fig. 5d) , and burying additional protein surfaces. free energy released by this refolding drives the two membranes to bend towards each other, 5-7 as the c-terminal anchor is forced closer to the fusion loop, forming apposing nipples in the membranes (fig. 5d) . 3 fusion-loop insertion may induce positive bilayer curvature, which would stabilize the lateral surfaces of the nipples. the concave shape of the crater-like surface formed by the fusion loops at the trimer tip may also have a destabilizing effect on the membrane, as has been postulated for fusion peptides in class i fusion proteins. 65 based on the energy required to deform lipid bilayers, it seems likely that a ring of trimers refolding in concert is needed to provide sufficient energy to form nipples in the membranes. 3, 4 it is unclear exactly how many trimers are needed to drive membrane fusion in class ii viruses, nor how their conformational changes are coupled. in the case of influenza, fusion requires the concerted action of at least three ha trimers, 69 and is more likely driven by rings of 6-8 trimers. 70 the clustering of fusion loops may provide a mechanism for the direct coupling of several e1 trimers to work in concert around a single fusion site in alphaviruses, but such clustering has not been observed in flaviviruses. it is possible that coupling occurs via the membrane: only when several trimers fold back in concert can they overcome the resistance of the membrane to deformation and reach their final, most stable postfusion conformation (figs. 5d-f). as the fusion transition proceeds, the stem zippers up onto the core of the trimer, along a channel that spans domain ii, at the intersubunit contact regions (figs. 3, 5d-f) . the zippering up of the stem onto the domain ii forces the fusion loop and the viral transmembrane anchor closer and closer, until the proximal leaflets of the two membranes fuse to form a 'hemifusion stalk' (fig. 5e ). hemifusion is thought to be an essential intermediate of membrane fusion. 3, 4, 71 (fig. 5e ) illustrates the need for shallow penetration of the viral fusion anchor into the host-cell membrane: assuming several trimers do in fact act in concert around a single fusion site, fusion anchors from different trimers would collide if they inserted beyond the outer (proximal) lipid bilayer leaflet. this constraint on the length of the fusion anchor holds true for both class i fusion peptides and class ii fusion loops. hemifusion stalks can 'flicker' open into narrow fusion pores. 71 in order to prevent the transient fusion pores from closing, the stem must complete its zippering up onto the core of the trimer, and the c-terminal transmembrane anchor must migrate into the pore (fig. 5f) . indeed, the transition from hemifusion stalk to full fusion pore appears to require that the viral transmembrane anchor span the membrane completely, in all biological membrane fusion systems. thus, the replacement of the c-terminal transmembrane anchor of influenza ha with a glycosylphophatidylinositol (gpi) lipid anchor, [72] [73] [74] or with a half-length protein -helical anchor, 75 stalls the fusion reaction at the stage of hemifusion. other viral fusion proteins and cellular snare fusion proteins also require at least one transmembrane anchor. [76] [77] [78] [79] [80] [81] [82] [83] upon completion of fusion, the trimer has reached the conformation seen in the postfusion crystal structures. 29, 55, 56 the stems (not present in the structures) are docked along the surface of domains ii, and the fusion loops and transmembrane anchors lie next to each other in the fused membrane (fig. 5f ). some class ii fusion proteins, including those of alphaviruses, can only fuse membranes containing cholesterol and sphingolipids. 84 the structural basis for this requirement is still not well understood. several mutations in different regions of the semliki forest virus fusion protein e1 lower its dependence on cholesterol and/or sphingolipids for membrane fusion. 85, 86 it is unclear, however, whether the lower dependence on cholesterol of these mutants is due to an apparent destabilization of the e1 homotrimer, 87 or to the different physical properties of membranes lacking cholesterol and sphingolipids. in flaviviruses, cholesterol facilitates fusion, but neither cholesterol nor sphingolipids are essential for fusion. 88 many class ii viruses, especially the flaviviruses, represent important human pathogens such as dengue, hepatitis c, yellow fever, west nile, japanese encephalitis and tick-borne encephalitis viruses. 89 for most of these viruses, there are no specific treatments for infection, their control by vaccination has proved elusive, 89 and the number of infections is on the rise. recently determined three-dimensional structures of class ii fusion proteins suggest new strategies for inhibiting viral entry by blocking membrane fusion. one such strategy stems for the discovery in dengue virus e of a long, tapering channel lined with hydrophobic side chains. 13 in the crystal structure, the channel is occupied by a molecule of the detergent n-octyl--d-glucoside. in the absence of detergent, a -hairpin covering the channel swings towards the protein, and closes up the channel. 13 the location of this 'ligand-binding pocket' at the domain i-domain ii interface coincides with that of mutations affecting the ph threshold of fusion in various flaviviruses. [57] [58] [59] [60] [61] [62] most of these mutations involve side chains lining the ligand-binding pocket. the postfusion structure of dengue virus e shows that this region acts as a hinge between domains i and ii during the fusogenic conformational rearrangement (see above). 29 the opening up of a ligand-binding pocket just at the locus of a hinge suggests that compounds tightly inserted at this position might hinder the conformational changes required for membrane fusion (fig. 6a) . the mechanism of action of such compounds might resemble that of some of the well-studied antipicornaviral compounds, which block a concerted structural transition in the icosahedral assembly. 90 alternatively, small molecules that pry open the -hairpin on binding in the pocket may inhibit infection by facilitating the low-ph conformational change, causing premature triggering. knowledge of the structure of the binding pocket with a bound ligand will guide efforts to design derivative ligands with higher affinities for use as inhibitors of flaviviral membrane fusion. the postfusion structures of dengue 29 and semliki forest 55 viruses suggest a second possible strategy for fusion inhibition, related to an approach successful in developing an hiv antiviral compound. 91 peptides corresponding to the stem region of the gp41 fusion protein inhibit hiv entry by binding to the trimeric, n-terminal 'inner core' of the protein and interfering with the folding back against it of the stem and c-terminal viral transmembrane anchor. the way in which the stem is likely to fold back in class ii viral fusion proteins (figs. 3, 5d-f) suggests that an analogous strategy may be successful with class ii viruses. peptides derived from stem sequences could block completion of the fusogenic conformational change, by competing with the stem for interaction with surfaces on domain ii, at the trimer interface (fig. 6b) . stem-like peptides or peptidomimetic compounds could thus inhibit viral membrane fusion in class ii enveloped viruses by preventing the final folding back of the fusion protein that is required to drive the viral and host-cell membranes together to fuse. all viral membrane fusion proteins use the same physical principles and topology to drive membrane fusion. class ii fusion proteins are structurally and evolutionarily distinct class of proteins found in flaviviridae, such as dengue, yellow fever, and west nile viruses, and on alphaviruses, such as semliki forest and sindbis viruses. unlike class i fusion proteins such as influenza ha, which undergoes extensive refolding during membrane fusion, the three domains of class ii fusion proteins retain most of their folded structures. instead, the domains undergo major rearrangements in their relative orientations, through flexion of the interdomain linkers. class ii fusion proteins rely on a hydrophobic fusion loop to anchor themselves in the target cellular membrane. like the class i fusion peptide, the class ii fusion loop penetrates only partway into the hydrocarbon layer of the target membrane. class ii fusion proteins drive membrane fusion in a foldback rearrangement of a trimeric protein assembly. crystal structures of class ii envelope proteins have suggested two specific strategies for fusion inhibition, with hydrophobic small molecules and "stem"-like peptides or peptidomimetics, respectively. a) the discovery of a ligand-binding pocket at the interface between domains i and ii in dengue virus e, 13 just at the locus of a hinge motion required for fusion, suggests that compounds inserted in the pocket might hinder the hinge motion and hence inhibit the fusion transition. this approach would block the first step in the fusion mechanism ( fig. 5a-b) . b) peptides corresponding to the stem region of the fusion protein may inhibit viral entry by binding to the trimeric core of the protein in its postfusion conformation, 29, 55 and interfering with the folding back against it of the fusion protein's own stem. an analogous strategy has been successful with hiv gp41. 91, 92 this approach would block the last step in the fusion mechanism ( fig. 5e-f) . flaviviridae: the viruses and their replication togaviridae: the viruses and their replication a quantitative model for membrane fusion based on low-energy intermediates a mechanism of protein-mediated fusion: coupling between refolding of the influenza hemagglutinin and lipid rearrangements structural basis for paramyxovirus-mediated membrane fusion evidence that the transition of hiv-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion membrane fusion machines of paramyxoviruses: capture of intermediates of fusion receptor binding and membrane fusion in virus entry: the influenza hemagglutinin structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 a resolution the envelope glycoprotein from tick-borne encephalitis virus at 2 a resolution structure of the haemagglutinin-esterase-fusion glycoprotein of influenza c virus the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph a ligand-binding pocket in the dengue virus envelope glycoprotein variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein structure of the parainfluenza virus 5 f protein in its metastable, prefusion conformation structure of influenza haemagglutinin at the ph of membrane fusion retrovirus envelope domain at 1.7 angstrom resolution core structure of gp41 from the hiv envelope glycoprotein atomic structure of a thermostable subdomain of hiv-1 gp41 atomic structure of the ectodomain from hiv-1 gp41 crystal structure of the simian immunodeficiency virus (siv) gp41 core: conserved helical interactions underlie the broad inhibitory activity of gp41 peptides three-dimensional solution structure of the 44 kda ectodomain of siv gp41 crystal structure of the ebola virus membrane fusion subunit, gp2, from the envelope glycoprotein ectodomain crystal structure of human t cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins n-and c-terminal residues combine in the fusion-ph influenza hemagglutinin ha(2) subunit to form an n cap that terminates the triple-stranded coiled coil structural characterization of the human respiratory syncytial virus fusion protein core crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core structural basis for coronavirus-mediated membrane fusion. crystal structure of mouse hepatitis virus spike protein fusion core structure of the dengue virus envelope protein after membrane fusion purification of the fusion protein of sendai virus: analysis of the nh2-terminal sequence generated during precursor activation detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus s2 fusion protein structure and membrane interaction of the internal fusion peptide of avian sarcoma leukosis virus mutational evidence for an internal fusion peptide in flavivirus envelope protein e dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate dendritic-cell-specific icam3-grabbing nonintegrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses dc-sign (cd209) mediates dengue virus infection of human dendritic cells monoclonal antibodies that bind to domain iii of dengue virus e glycoprotein are the most efficient blockers of virus adsorption to vero cells an external loop region of domain iii of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells serotype-specific entry of dengue virus into liver cells: identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor interaction of west nile virus with alpha v beta 3 integrin mediates virus entry into cells mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein e proteolytic activation of tick-borne encephalitis virus by furin structures of immature flavivirus particles the first step: activation of the semliki forest virus spike protein precursor causes a localized conformational change in the trimeric spike structure of dengue virus: implications for flavivirus organization, maturation, and fusion visualization of membrane protein domains by cryo-electron microscopy of dengue virus structure of west nile virus cryo-electron microscopy reveals the functional organization of an enveloped virus, semliki forest virus placement of the structural proteins in sindbis virus cleavage of protein prm is necessary for infection of bhk-21 cells by tick-borne encephalitis virus fusion activity of flaviviruses: comparison of mature and immature (prm-containing) tick-borne encephalitis virions the murray valley encephalitis virus prm protein confers acid resistance to virus particles and alters the expression of epitopes within the r2 domain of e glycoprotein conformational change and protein-protein interactions of the fusion protein of semliki forest virus structure of a flavivirus envelope glycoprotein in its low-ph-induced membrane fusion conformation nucleotide changes responsible for loss of neuroinvasiveness in japanese encephalitis virus neutralization-resistant mutants mutations in the envelope protein of japanese encephalitis virus affect entry into cultured cells and virulence in mice changes in the dengue virus major envelope protein on passaging and their localization on the three-dimensional structure of the protein epitopes on the dengue 1 virus envelope protein recognized by neutralizing igm monoclonal antibodies attenuation of murray valley encephalitis virus by site-directed mutagenesis of the hinge and putative receptor-binding regions of the envelope protein single mutation in the flavivirus envelope protein hinge region increases neurovirulence for mice and monkeys but decreases viscerotropism for monkeys: relevance to development and safety testing of live, attenuated vaccines structure of an analog of fusion peptide from hemagglutinin membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin structure and function of membrane fusion peptides studies on the structure of the influenza virus haemagglutinin at the ph of membrane fusion membrane interactions of the tick-borne encephalitis virus fusion protein e at low ph hiv entry and its inhibition membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events hemifusion between cells expressing hemagglutinin of influenza virus and planar membranes can precede the formation of fusion pores that subsequently fully enlarge lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion gpi-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes meta-stability of the hemifusion intermediate induced by glycosylphosphatidylinositol-anchored influenza hemagglutinin the transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition the caenorhabditis elegans unc-64 locus encodes a syntaxin that interacts genetically with synaptobrevin close is not enough: snare-dependent membrane fusion requires an active mechanism that transduces force to membrane anchors mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation, and infectivity deletion of the cytoplasmic tail of the fusion protein of the paramyxovirus simian virus 5 affects fusion pore enlargement role of the cytoplasmic tail of ecotropic moloney murine leukemia virus env protein in fusion pore formation the role of the cytoplasmic tail region of influenza virus hemagglutinin in formation and growth of fusion pores truncation of the cooh-terminal region of the paramyxovirus sv5 fusion protein leads to hemifusion but not complete fusion functional analysis of the cytoplasmic tail of moloney murine leukemia virus envelope protein membrane fusion of semliki forest virus requires sphingolipids in the target membrane a single point mutation controls the cholesterol dependence of semliki forest virus entry and exit a conserved histidine in the ij loop of the semliki forest virus e1 protein plays an important role in membrane fusion novel mutations that control the sphingolipid and cholesterol dependence of the semliki forest virus fusion protein involvement of lipids in different steps of the flavivirus fusion mechanism the site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating inhibiting hiv-1 entry with fusion inhibitors potent suppression of hiv-1 replication in humans by t-20, a peptide inhibitor of gp41-mediated virus entry crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes crystal structure of dengue virus type 1 envelope protein in the postfusion conformation and its implications for membrane fusion glycoprotein organization of chikungunya virus particles revealed by x-ray crystallography structural changes of envelope proteins during alphavirus fusion structural insights into the neutralization mechanism of a higher primate antibody against dengue virus crystal structure of the japanese encephalitis virus envelope protein this chapter was originally written in 2006 and was updated in 2012 to add recent relevant advances. key: cord-323331-80d01l6f authors: li, jie; ahat, erpan; wang, yanzhuang title: golgi structure and function in health, stress, and diseases date: 2019-01-01 journal: the golgi apparatus and centriole doi: 10.1007/978-3-030-23173-6_19 sha: doc_id: 323331 cord_uid: 80d01l6f the golgi apparatus is a central intracellular membrane-bound organelle with key functions in trafficking, processing, and sorting of newly synthesized membrane and secretory proteins and lipids. to best perform these functions, golgi membranes form a unique stacked structure. the golgi structure is dynamic but tightly regulated; it undergoes rapid disassembly and reassembly during the cell cycle of mammalian cells and is disrupted under certain stress and pathological conditions. in the past decade, significant amount of effort has been made to reveal the molecular mechanisms that regulate the golgi membrane architecture and function. here we review the major discoveries in the mechanisms of golgi structure formation, regulation, and alteration in relation to its functions in physiological and pathological conditions to further our understanding of golgi structure and function in health and diseases. receive proteins and lipids from the endoplasmic reticulum (er) by the cis cisternae and export them from the trans cisternae and the trans-golgi network (tgn) to other intracellular membranes such as the endosomes, lysosomes, plasma membrane, and outside of the cell (tang and wang 2013; wang and seemann 2011) . while traversing the golgi stack, cargo molecules are modified and processed. the sub-compartments of the golgi stacks house a set of glycosidases and glycosyltransferases responsible for the synthesis of glycoproteins and glycolipids. in the tgn, many secretory proteins are proteolytically cleaved by lumenal proteinases zhang and wang 2016; huttner et al. 1995) . many efforts have been made to understand the mechanism of golgi structure formation. the formation of the golgi ribbon depends on golgi matrix proteins and an intact microtubule organization. cytosolic dynein moves golgi membranes along centrosome-derived microtubules toward the (-) end of the microtubules (matteis et al. 2008; rabouille and kondylis 2007; wei and seemann 2010) . subsequently, golgi-oriented microtubules maintain golgi stacks in the proximity and facilitate tubular connections between them (zhu and kaverina 2013) . while dynein and microtubules are required for the concentration of golgi stacks in the pericentrosomal region and golgi ribbon formation, they are not essential for the generation and maintenance of the stacked structure, as depolymerization of microtubules disrupts the golgi ribbon but not the stacks (thyberg and moskalewski 1999) . from the 1960s, morphological studies have shown connections in the space between cisternae that might be involved in the adhesion of cisternae into stacks (franke et al. 1972; mollenhauer 1965; cluett and brown 1992) , which were later identified as golgi matrix proteins. these include golgi stacking proteins and membrane tethers, as discussed below. in 1994, the concept of "golgi matrix" proteins was first introduced (slusarewicz et al. 1994) . since then, several golgi matrix proteins have been identified to be responsible for maintaining the unique architecture and function of the golgi apparatus. major components of the golgi matrix are summarized in table 19 .1. key proteins involved in golgi structure formation are discussed below. among all golgi matrix proteins, the golgi reassembly stacking proteins grasp65 and grasp55 (grasps, also called gorasp1 and gorasp2, respectively) are best characterized for their roles in golgi structure formation, including stacking , bekier et al. 2017 , shin et al. 2018 , barr et al. 1997) , ribbon-linking (puthenveedu et al. 2006; tang et al. 2016; feinstein and linstedt 2008) , cargo transportation (kuo et al. 2000; table 19 . -kctd5 (dementieva et al. 2009 ) • golgi stacking xiang and wang 2010; bekier et al. 2017) • golgi ribbon formation (feinstein and linstedt 2008) • cell cycle control (duran et al. 2008) • transport of specific cargo (kuo et al. 2000; d'angelo et al. 2009) • p24 cargo receptor retention • autophagy (zhang et al. 2018; zhang and wang 2018a, b) • unconventional secretion (dupont et al. 2011; rabouille and linstedt 2016; vinke et al. 2011; gee et al. 2011; piao et al. 2017) • (barr et al. 1997; xiang and wang 2010; bekier et al. 2017; shin et al. 2018) • golgi ribbon formation (puthenveedu et al. 2006; tang et al. 2016) • cell cycle progression (preisinger et al. 2005; sutterlin et al. 2005; yoshimura et al. 2005; duran et al. 2008; tang et al. 2010b) • mitotic spindle formation (sutterlin et al. 2005) (continued) • unconventional secretion (gee et al. 2011) • apoptosis (lane et al. 2002) golgins cis p rab1 (weide et al. 2001) -p115 (nakamura et al. 1997) -grasp65 -syntaxin 5 -akap450 (rivero et al. 2009 ) -zfpl1 (chiu et al. 2008 ) -tuba (kodani et al. 2009 ) • golgi ribbon formation (puthenveedu et al. 2006) • copii vesicle tethering (moyer et al. 2001; alvarez et al. 2001) • non-centrosomal microtubule organization (rivero et al. 2009) • centrosome regulation (kodani and sutterlin 2008; kodani et al. 2009) • spindle formation (kodani and sutterlin 2008) • cell migration (preisinger et al. 2004) • apoptosis (walker et al. 2004) • purkinje neuron development (liu et al. 2017) p115 cis p rab1 (allan et al. 2000) -gm130 (nakamura et al. 1997) -giantin (sonnichsen et al. 1998) syntaxin 5, gos-28 • post-mitotic golgi reassembly brandon et al. 2003; dirac-svejstrup et al. 2000) • snare assembly • membrane tethering nakamura et al. 1997; alvarez et al. 2001; seemann et al. 2000; allan et al. 2000) • apoptosis (chiu et al. 2002) • nuclear import (mukherjee and shields 2009) giantin/ golgb1 cis/medial tmd rab1/6 (rosing et al. 2007) -p115 (sonnichsen et al. 1998) -gm130 (sonnichsen et al. 1998) -gcp60/acbd3 ) • golgi stack organization (rosing et al. 2007; koreishi et al. 2013) • ribbon organization (koreishi et al. 2013) • membrane tethering (sonnichsen et al. 1998; linstedt et al. 2000; lowe et al. 2004; misumi et al. 2001; alvarez et al. 2001; munro 2011) • er-to-golgi trafficking • apoptosis (lowe et al. 2004) • cilia formation (bergen et al. 2017) golgin-45/ blzf1 medial/ trans p rab2 (short et al. 2001) -grasp55 (short et al. 2001 , zhao et al. 2017 ) • golgi stacking (short et al. 2001 , zhao et al. 2017 • membrane tethering (short et al. 2001) golgin-67/ golga8b tmd a • uncharacterized golgin-84/ golga5 cis tmd rab1 (satoh et al. 2003) -casp (osterrieder et al. 2017) -cog complex (sohda et al. 2010) • golgi stacking and reassembly (satoh et al. 2003) • golgi ribbon formation (diao et al. 2003) • er-golgi tethering and protein transport (osterrieder et al. 2017) • membrane tethering (malsam et al. 2005) (continued) (shin et al. 2017 (shin et al. , 2018 • tgn-to-pm trafficking of e-cadherin (lock et al. 2005) • endosome-to-tgn trafficking (lu and hong 2003; lu et al. 2004) • retrograde transport from recycling endosomes to the tgn (jing et al. 2010) • poxvirus morphogenesis (alzhanova and hruby 2007) golgin-160/ golga3/ gcp170 cis p -gcp60/acbd3 (sbodio et al. 2006; sbodio and machamer 2007) -gcp16 (ohta et al. 2003 ) -β1ar (gilbert et al. 2014 ) -romk, pist (bundis et al. 2006 ) • post-golgi trafficking (bundis et al. 2006; gilbert et al. 2014) • apoptosis (mancini et al. 2000 , maag et al. 2005 , sbodio et al. 2006 golgin-245/ golga4/ p230 trans grip arl1/3 (wu et al. 2004; sinka et al. 2008) rab2/6/19/30 (sinka et al. 2008 ) -tbc1d23 (shin et al. 2017 (shin et al. , 2018 • tgn-to-pm traffic (lu and hong 2003) • phagophore formation (sohda et al. 2015) gcp16 acylation -golgin-160/golga1/ gcp170 (ohta et al. 2003) • golgi-to-pm trafficking (ohta et al. 2003) gcc88 trans grip arl1/3 (sinka et al. 2008 ), rab6/19/30 (sinka et al. 2008) • tgn organization and endosome-to-tgn trafficking (luke et al. 2003; lieu et al. 2007) • golgi ribbon formation (gosavi et al. 2018) • actin organization (makhoul et al. -clasp (efimov et al. 2007 ) • golgi ribbon formation • golgi integrity (lin et al. 2011) • membrane tethering reddy et al. 2006; ganley et al. 2008) • mpr recycling (reddy et al. 2006) • attachment of non-centrosomal microtubules (efimov et al. 2007) acbd3 -poliovirus 3a protein (greninger et al. 2013) -numb (zhou et al. 2007 ) • golgi integrity and er-to-golgi trafficking • nuclear translocation of caspasegenerated golgin-160 fragments (residues 140-311) machamer 2007, sbodio et al. 2006) • asymmetric cell division (zhou et al. 2007) • aichi virus and poliovirus replication (greninger et al. 2012) bicaudal-d/ bicd2 p -dynactin, p50-dynamitin (hoogenraad et al. 2001 ) • recruitment of the dynein-dynactin complex (hoogenraad et al. 2001) (continued) • copi-independent golgi-to-er transport (matanis et al. 2002) • endosome-to-golgi transport (wanschers et al. 2007) • autosomal-dominant spinal muscular atrophy (neveling et al. 2013, martinez-carrera and wirth 2015) casp/coy1 cis tmd -golgin-84 (osterrieder et al. 2017) -cog3, sed5, sly1, gos1, sft1/gs15 (anderson et al. • er-golgi tethering and protein transport (osterrieder et al. 2017) • retrograde-directed copi vesicles (anderson et al. 2017) cg-nap (takahashi et al. 1999) • centrosome amplification (nishimura et al. 2005) • microtubule nucleation coh1/ vps13b -thyroid receptor (chen et al. 1999 ) • golgi ribbon formation (rios et al. 2004) • membrane tethering (drin et al. 2008) • er-to-golgi trafficking (gillingham et al. 2004) • γ-tubulin recruitment (rios et al. 2004) • sorting to primary cilia (follit et al. 2008) • interacts with thyroid hormone receptor beta (chen et al. 1999 • cell cycle progression (cao et al. 2003; fu et al. 2003; parisi et al. 2018) • protein aggregation (ghosh et al. 2018; ristic et al. 2018; yi and yuan 2017; kobayashi et al. 2007; song et al. 2007; nishikori et al. 2008) • autophagy (papadopoulos et al. 2017) • dna repair (van den boom et al. 2016; fujita et al. 2013; davis et al. 2012; meerang et al. 2011; partridge et al. 2003) • er-stress-induced gene transcription (marza et al. 2015) • antiviral signaling (hauler et al. 2012 , hao et al. 2015 acbd barr et al. 2001) , unconventional secretion (dupont et al. 2011; rabouille and linstedt 2016; vinke et al. 2011; gee et al. 2011; piao et al. 2017) , cell cycle regulation (preisinger et al. 2005; sutterlin et al. 2005; yoshimura et al. 2005; duran et al. 2008; tang et al. 2010b) , apoptosis (lane et al. 2002) , and autophagy (zhang et al. 2018; zhang and wang 2018a, b) , although the mechanisms are less well understood. both grasps share a similar structure: a conserved n-terminal grasp domain consisting of two pdz domains (pdz1 and pdz2) and an intrinsically disordered c-terminal serine/proline-rich (spr) domain with multiple phosphorylation sites (zhang and wang 2015b) ( fig. 19 .1). both grasp65 and grasp55 are peripheral membrane proteins that are attached to the golgi membranes via an n-terminal myristic acid modification and the interaction with their membrane-bound partner proteins (gm130 and golgin-45, respectively) and therefore are concentrated at the interface between the cisternae where stacking occurs (short et al. 2001; barr et al. 1998) . grasp65 is concentrated on the cis-golgi cisternae, whereas grasp55 localizes to the medial/trans-golgi cisternae (barr et al. 1997; ). both play complementary roles in golgi stack formation . mechanistically, grasp proteins form homodimers via the n-terminal pdz domains, and dimers from adjacent golgi cisternae further oligomerize in trans and function as the "glue" that tethers the cisternae into a stack (wang et al. , 2005 ). an in vitro study using modified grasp domain peptides indicated that insertion of the myristic acid moiety is required for the oriented association to golgi membranes, which ensures the protein-protein interaction in trans. furthermore, the conformational change caused by myristoylation affects the tendency of grasp domain for self-interaction (heinrich et al. 2014 ). depletion of either grasp65 or grasp55 reduces the number of cisternae per golgi stack, whereas depletion of both grasps leads to disassembly of the entire golgi stack (sutterlin et al. 2005 , bekier et al. 2017 ). the grasps are tightly modulated by a phosphorylation and dephosphorylation cycle during cell division, resulting in mitotic disassembly and post-mitotic reassembly of the golgi (feinstein and linstedt 2008; cervigni et al. 2015; lin et al. 2000; wang et al. 2003; preisinger et al. 2005; tang et al. 2012; xiang and wang 2010; tang et al. 2008; truschel et al. 2012) . recent studies have identified novel grasp-binding proteins involved in golgi biogenesis and morphology modulation. recent research of the crystal structure of grasp55 bound to the golgin-45 c-terminal peptide revealed that golgin-45 promotes the oligomerization of grasp55 by forming a new interaction between two neighboring pdz2 molecules to play an important role in golgi stacking (zhao et al. 2017) . meanwhile, using an optimized in vitro system, mammalian enabled homologue (mena) and dnaj homolog subfamily a member 1 (dja1) were identified as grasp65 binding partners with potential functions on golgi structure maintenance li et al. 2019) . mena is an actin elongation factor recruited to the golgi membranes by grasp65 to facilitate actin polymerization and grasp65 oligomerization and thus functions with actin as bridging proteins of grasp65 in golgi ribbon linking. dja1 is a co-chaperone of heat shock cognate 71 kda protein (hsc70), but the activity of dja1 in golgi structure formation is independent of its co-chaperone activity or hsc70, rather, through dja1-grasp65 interaction to promote grasp65 oligomerization. thus, dja1 facilitates golgi structure formation through an unconventional hsc70-independent pathway. these studies further confirmed grasp65 as a multifaceted protein in golgi structure formation and indicated that an array of grasp binding proteins could play important roles in golgi morphology maintenance ( fig. 19.1 ). in addition, grasp55 was reported to be involved in glucose starvation-induced autophagy (zhang et al. 2018 fig. 19 .1 structure, modification, and binding sites on grasp65 (a) and grasp55 (b). rat grasp65 and grasp55 sequences are used for illustration. both grasps share a similar structure: a conserved n-terminal grasp domain consisting of two pdz domains (pdz1 and pdz2) and a c-terminal serine/proline-rich (spr) domain with multiple phosphorylation sites (indicated by asterisks) that are involved in grasp modulation during the cell cycle. both grasp65 and grasp55 are peripheral membrane proteins attached to the golgi membranes via n-terminal myristoylation and the interaction with their membrane-bound partner proteins (gm130 and golgin-45, respectively). grasp65-binding proteins mena and dja1 have been identified to enhance golgi ribbon linking and stacking, respectively. grasp55 is regulated by o-glcnacylation depending on the glucose level and interacts with lc3 and lamp2 to facilitate glucose starvation-induced autophagy interacts with lc3 on autophagosomes and lamp2 on lysosomes to facilitate autophagosome maturation, which will be discussed in later sections. golgins are a family of golgi-associated coiled-coil proteins that are necessary for vesicle tethering at the golgi and maintenance of golgi integrity (muschalik and munro 2018; witkos and lowe 2015; gillingham and munro 2016) . most golgins are peripheral membrane proteins anchored on golgi membranes via their c-terminus and are associated with small gtpases of rab, arf, and arl families (table 19 .1) (munro 2011; sinka et al. 2008 ). these interactions mediate both membrane attachment and selective localization of a specific golgin to a specific sub-compartment of the golgi (witkos and lowe 2015) . golgins lack significant sequence homology between the family members and localize to different regions of the golgi to play distinct roles in tethering events, membrane traffic, and golgi organization. the coiled-coil regions provide the golgins with an extended structure required for the tethering function, while the interactions with rab gtpases control these molecules in their open (extended) or closed (folded) confirmation (cheung et al. 2015) . in addition, golgins often contain specific sequence and structural features at the n-and c-terminal ends, which allow them to recognize vesicles and golgi cisternal membranes based on the curvature and lipid composition of the membranes (drin et al. 2008; drin et al. 2007; magdeleine et al. 2016) . detailed information about golgins and their functions are summarized in table 19 .1. gm130 was the first identified golgi matrix protein and is predominantly found in the central region of the cis-golgi (nakamura et al. 1995) , where it forms a stable complex with grasp65 ( barr et al. 1998 ). depletion of gm130 results in the disruption of the golgi ribbon and causes protein glycosylation defects (puthenveedu et al. 2006) . there are two possible ways gm130 contributes to golgi ribbon formation. first, gm130 targets grasp65 to the rim of the cisternae where grasp65 promotes lateral linking of cisternae via oligomerization (puthenveedu et al. 2006) . mena and actin cytoskeleton may facilitate grasp65 in this action . second, gm130 recruits a-kinase anchoring protein 450 (akap450) onto the cis-golgi and allows golgi-associated nucleation of microtubules, which arranges golgi stacks in close proximity to form a ribbon (rivero et al. 2009 ). similarly, other golgins may also work with the microtubule cytoskeleton in a similar way to facilitate golgi structure organization. for example, gmap-210, another cis-golgi-localized golgin, recruits the γ-tubulin-containing complexes to the golgi membranes and promotes the formation of tubulin oligomers on golgi membranes. depletion of gmap-210 results in extensive golgi fragmentation, suggesting a role in golgi ribbon formation (rios et al. 2004 ). in addition to grasp65, gm130 also interacts with p115 and giantin to form the gm130-p115-giantin tethering complex (sonnichsen et al. 1998 ). the rab1 effector p115, when recruited to coat protein complex (cop) ii vesicles during the budding from the er, interacts with the soluble atpases n-ethylmaleimide-sensitive factor (nsf) attachment protein receptors (snares), a specialized set of copii v-snares, to form a cis-snare complex that promotes vesicle targeting to the golgi apparatus (allan et al. 2000) . meanwhile, p115 also binds giantin on copi vesicles and works with gm130 on golgi membranes to provide a bridging role for vesicle tethering (sonnichsen et al. 1998 ). thus, gm130 and p115 are two major tethering factors in er-to-golgi trafficking (munro 2011) . other than the well-studied grasp65-gm130 and gm130-p115-giantin complexes, the grip domain containing golgins are another group of proteins associated with the golgi structure. most grip domain-containing golgins localize to the tgn via their grip domains and are involved in golgi organization (luke et al. 2005) . gcc185 is reported to localize independently of arl1 on tgn and plays an essential role in golgi structure formation. depletion of gcc185 results in fragmentation of both cis-and trans-golgi that are dispersed throughout the cytoplasm ). on the other hand, another tgn golgin gcc88 is reported to play a role in tgn organization and ribbon-linking. overexpression of gcc88 causes a loss of the compact golgi ribbon and dispersal of mini-stacks throughout the cytoplasm, while knockdown of gcc88 results in a longer ribbon structure (gosavi et al. 2018) . a recent report suggests that gcc88-induces golgi ribbon dispersal via actin and non-muscle myosin iia. in addition, a novel gcc88-binding partner, the long isoform of intersectin-1 (itsn-1), a guanine nucleotide exchange factor for cdc42, is identified to be involved in this process (makhoul et al. 2019 ). besides the golgi matrix proteins and their cofactors described above, other proteins including snares, kinases, methyltransferases, and gtpases have also been reported to be related to golgi structural organization and function. a few examples are discussed below. vesicle-associated membrane protein 4 (vamp4), a v-snare protein located on the tgn, was first shown to play a role in retrograde trafficking from early endosomes to the tgn (steegmaier et al. 1999) . it was later reported that depletion of vamp4 led to fragmentation of the golgi ribbon, although golgi membranes remained in the juxtanuclear area. em studies revealed shortened golgi stacks with a normal arrangement. depletion of the cognate snares of vamp4, syntaxin 6, syntaxin 16, and vti1a also disrupted the golgi ribbon. these findings suggest that the maintenance of the golgi ribbon structure requires normal retrograde trafficking, which is likely mediated by the formation of vamp4-containing snare complexes (shitara et al. 2013) . serine/threonine-protein kinase h1 (pskh1) was primarily characterized with multiple intracellular localizations, including brefeldin a-sensitive golgi compartment, centrosomes, nucleus, and cytoplasm (brede et al. 2000) . pskh1 targeting to golgi depends on dual n-terminal acylation, myristoylation on glycine 2, and palmitoylation on cysteine 3. expression of palmitoylation site mutant pskh1 results in the disassembly of the golgi apparatus to a diffused cytoplasmic pattern without interrupting the microtubule cytoskeleton (brede et al. 2003) . the substrates of this kinase on the golgi are so far unidentified. protein arginine methyltransferase 5 (prmt5) localizes to the golgi apparatus and forms complexes with several components, including gm130, which was later identified as a substrate of prmt5. n-terminal methylation of gm130 does not affect its golgi localization but is critical for golgi ribbon formation. depletion of prmt5 and expression of methylation-defective gm130 mutants result in fragmentation and dispersal of the golgi ribbon (zhou et al. 2010) . in addition to the proteins mentioned above, rab small gtpases are another group of key regulators of mammalian golgi organization. many rab proteins, including but not limited to rab1, rab2 and rab8 (aizawa and fukuda 2015) , rab18 and rab43 (dejgaard et al. 2008) , rab6/41 (goud et al. 1990; martinez et al. 1997; liu et al. 2013), and rab30 (kelly et al. 2012) , have been shown to play a role in golgi structure organization and reviewed previously in detail (goud et al. 2018) . considering that rab proteins switch between inactive gdp-bound and active gtp-bound forms, it has been proposed that golgi organization-related rab proteins are divided into two categories. with class 1 rabs, the golgi ribbon is disrupted by rab inactivation but appears normal with overexpression, whereas with class 2 rabs, rab inactivation has little effect on golgi ribbon organization, while overexpression leads to the redistribution of golgi enzymes to the er (liu and storrie 2015) . these results indicate that rabs control the golgi structure through modulating membrane tethering and trafficking. the golgi undergoes a series of sophisticated cell cycle-dependent disassembly and reassembly processes, including the deformation and reformation of golgi ribbon, stacks, and cisternae. at the onset of mitosis, the golgi ribbon unlinks into ministacks, which further undergo unstacking and vesiculation. these processes ensure the equal distribution of golgi compartments into the two daughter cells (wang 2008; wei and seemann 2010) . in telophase, the golgi vesicles fuse into cisternae and form stacks. the new stacks then accumulate in the perinuclear region and further link into a ribbon. the molecular factors that control these processes include golgi matrix proteins, kinases and phosphatases, ubiquitin ligases and deubiquitinating enzymes, vesicle budding and fusion factors, and actin and microtubule cytoskeleton. an in vitro system has been developed to replicate the golgi disassembly and reassembly process through sequential treatments of purified golgi membranes with mitotic (mc) and interphase (ic) cytosol, or with purified proteins (tang et al. , 2010a . this system provides a powerful tool for testing key proteins in golgi structure formation, which makes it possible to identify the minimal machinery and key components that control mitotic golgi disassembly and post-mitotic reassembly . the first step of golgi disassembly at the onset of mitosis is golgi ribbon unlinking. at this step, golgi stacks in the ribbon are disconnected and dispersed. this step involves disconnecting the tubules between the stacks by the membrane fission protein ctbp/bars, which is crucial for g2/m transition (hidalgo carcedo et al. 2004; colanzi et al. 2007 ). further, grasps undergo mitotic phosphorylation which are also required for ribbon-unlinking. the extracellular-signal-regulated kinase (erk) directly phosphorylates grasp55 and blocks its activity in both golgi ribbon formation and trans-oligomerization (feinstein and linstedt 2008) , while grasp65 is phosphorylated by c-jun n-terminal kinase (jnk) on serine 277 (s277), which causes the separation of the golgi stacks (cervigni et al. 2015) . sequential phosphorylation of grasp65 on multiple sites by cyclin-dependent kinase 1 (cdk1) and polo-like kinase 1 (plk1) results in its conformational changes and subsequent de-oligomerization (lin et al. 2000; preisinger et al. 2005; wang et al. 2003; tang et al. 2012; vielemeyer et al. 2009 ). on the other hand, grasp55 is phosphorylated by erk and partially by cdk1 . in addition to the unique phosphorylation sites in the spr domains of grasps, s189 within the grasp domain of grasp65 is modified by plk1, which causes conformational change and impaired self-association (sengupta and linstedt 2010 ). an in vivo membrane-tethering activity assay using a construct of full-length grasp55 fused to the c-terminal mitochondrial anchoring sequence shows that the tethering activity is diminished by introducing a phosphomimic s189d mutant. this result suggests that s189 might be a plk1 target site on both grasps, although direct evidence remains to be provided (truschel et al. 2012 ). grasp65 is dephosphorylated by pp2a in late mitosis and the trans-oligomer reformation is therefore rehabilitated to promote cisternae stacking . the unstacked cisternae further disassemble into vesicles, which depends on copi vesicle formation and blockage of vesicle docking and membrane fusion. as mentioned above, the gm130-p115-giantin complex promotes copii vesicle docking to the golgi. cdk1 phosphorylation of gm130 on s25 during mitosis inhibits p115-interaction and therefore blocks vesicle docking ). inhibition of cdk1 causes golgi vesiculation failure, suggesting an essential role of cdk1 activity in mitotic golgi vesiculation. however, expression of the gm130 s25a non-phosphorylatable mutant in gm130-depleted cells causes no apparent defects in golgi vesiculation and mitotic progression, indicating the existence of gm130 s25 phosphorylation-independent pathways that ensure golgi vesiculation and mitotic progression in mammalian cells (sundaramoorthy et al. 2010) . recently, the recruitment and activation of aurora kinase family member aurora a at the centrosomes in m phase was reported to depend on golgi ribbon unlinking in g2 phase (barretta et al. 2016) . aurora a functions in centrosome maturation, mitotic entry, and bipolar spindle formation during mitosis (nikonova et al. 2013; carmena et al. 2009; kimura et al. 2013) . this finding indicates a potential link between aurora a activity and cell cycle-associated golgi structure modulation. indeed, it was later confirmed that knockdown or inhibition of aurora a induces golgi dispersal without affecting the gm130 protein level in interphase. further investigation revealed that interference of aurora a causes golgi dispersal only after mitosis via the dissociation of the golgi and centrosome (kimura et al. 2018) . these studies revealed a novel relationship between g2 phase golgi unlinking, m phase aurora a activation, and interphase golgi structure formation. two aaa atpases, nsf and p97/vcp, are involved in membrane fusion during post-mitotic golgi reassembly, and their activities are regulated by phosphorylation during mitosis . for nsf-catalyzed fusion, the p115-gm130 tethering complex is disrupted by gm130 phosphorylation during mitosis , while post-mitotic phosphorylation of p115 by a casein kinase ii (ckii)like enzyme is required for cisterna reassembly (dirac-svejstrup et al. 2000) . contemporarily, homotypic fusion of golgi membranes mediated by p97 is also blocked upon phosphorylation of p47 and p37. p97 uses these two distinct cofactors for its membrane fusion function: p47 is essential for the regrowth of golgi cisternae from mitotic golgi fragments (kondo et al. 1997 ), while p37 is required for the maintenance of the golgi structure in interphase as well as for its reassembly in late mitosis (uchiyama et al. 2006 ). both pathways are regulated by cdk1-mediated phosphorylation. phosphorylation of p47 on s140 abolishes its binding to golgi membranes, resulting in mitotic inhibition of the p97/p47 pathway (uchiyama et al. 2003) . phosphorylation on s56 and threonine 59 (t59) disables p37 from binding to golgi membranes and consequently blocks p97/p37-mediated golgi membrane fusion at late mitosis (kaneko et al. 2010 ). in addition to phosphorylation, p97/p47-mediated golgi membrane fusion is also regulated by ubiquitination (tang and wang 2013) . tang et al. discovered that the homologous to the e6-ap carboxyl terminus (hect) domain containing ubiquitin ligase hace1 is targeted to the golgi membrane through the interaction with rab1 and participates in post-mitotic golgi biogenesis ). depletion of hace1 or expression of an inactive mutant impairs post-mitotic golgi membrane fusion. the identification of hace1 as a golgi-localized ubiquitin ligase provides evidence that ubiquitin has a critical role in golgi biogenesis during the cell cycle . later, the golgi t-snare syntaxin 5 was identified as a ubiquitination substrate ). syntaxin 5 is monoubiquitinated by hace1 in early mitosis and deubiquitinated by the de-ubiquitinase vcip135 in late mitosis (wang et al. 2004 ). the monoubiquitination of syntaxin 5 at lysine 270 (k270) in the snare domain impairs the interaction between syntaxin 5 and the cognate v-snare bet1 but increases its binding to the p97 adaptor p47 through the uba domain of p47, which is required for post-mitotic golgi membrane fusion (meyer et al. 2002) . expression of the syntaxin 5 k270r mutant in cells impairs post-mitotic golgi reassembly. therefore, monoubiquitinated syntaxin 5 recruits p97/p47 to the mitotic golgi fragments and promotes post-mitotic golgi reassembly upon ubiquitin removal by vcip135 . vcip135 was originally identified as a p97 interacting protein (kano et al. 2005) . it was later shown to be a de-ubiquitinating (dub) enzyme involved in p97/p47-mediated membrane fusion (wang et al. 2004) . vcip135 dub activity, as well as its interaction with p97 and association with golgi membranes, is regulated by phosphorylation (zhang and wang 2015a; zhang et al. 2014) . in mitosis, vcip135 is phosphorylated at s130 by cdk1 and thus is inactivated, allowing syntaxin 5 to be ubiquitinated by hace1; in telophase, vcip135 is dephosphorylated and reactivated, removing ubiquitin from syntaxin 5 to allow p97-mediated membrane fusion wang 2008) . these studies revealed a novel mechanism that monoubiquitination regulates golgi membrane dynamics during the mammalian cell cycle. as stated above, the golgi apparatus in mammalian cells forms a unique stacked structure under normal growth conditions, which undergoes a regulated disassembly and reassembly process during the cell cycle. however, the golgi structure and function could be impaired under stress conditions, such as dna damage, energy and nutrient deprivation, and pro-apoptotic conditions. this could be attributed to perturbation of microtubule organization or phosphorylation, degradation, or cleavage of golgi structural proteins. additionally, many signaling molecules have been identified to be associated with the golgi. thus, it has been proposed that the golgi could sense and transduce stress signals and therefore serves as a hub in the cellular signaling network (farhan and rabouille 2011; mayinger 2011; makhoul et al. 2019 ). apoptosis, also known as programmed cell death, is a cell suicide mechanism carried out by organelle-directed regulators such as the bcl-2 proteins and ultimately executed by the caspase family proteases (nicholson and thornberry 1997) . organellar response to apoptotic initiation includes death receptor endocytosis, mitochondrial and lysosomal permeabilization, er calcium release, and golgi fragmentation. the golgi is one of the first organelles to be affected during apoptosis (mukherjee et al. 2007; aslan and thomas 2009) . during apoptosis, several golgi matrix proteins related to golgi structure maintenance are cleaved by caspases, leading to golgi fragmentation (hicks and machamer 2005) . apoptotic golgi fragmentation is one of the most extensively studied golgi stress responses. reported caspases-cleaved golgi proteins include grasp65, golgin-160, gm130, p115, syntaxin 5, and giantin (lane et al. 2002; mancini et al. 2000; walker et al. 2004; chiu et al. 2002; lowe et al. 2004; machamer 2015) , as summarized in table 19 .2 and discussed below. golgin-160 is a golgin that plays a role in vesicle tethering and trafficking . it is cleaved by caspase-2, caspase-3, and caspase-7 during apoptosis. under pro-apoptotic conditions stimulated by staurosporine, the golgi senses and transduces apoptotic signals using a local caspase, caspase-2. caspase-2 is special in a way that it has both the property of initiator caspases and the substrate specificity of executioner caspases (mancini et al. 2000) . although it is unclear how caspase-2 is activated by pro-apoptotic signals, in vitro and in vivo caspase cleavage assays showed that caspase-2 cleavage of golgin-160 at aspartate 59 (d59) happens prior to golgin-160 cleavage by caspase-3 and 7 at d139 and d311 (mancini et al. 2000) . expression of the d59a cleavage-defective mutant of golgin-160 delays golgi disintegration under staurosporine treatment (machamer 2003; hicks and machamer 2005) . golgi fragmentation (mancini et al. 2000; mukherjee et al. 2007; nozawa et al. 2002; hicks and machamer 2002; maag et al. 2005 ) grasp65 anisomycin; sts caspase-3 d320, d375, d393 golgi fragmentation (lane et al. 2002; cheng et al. 2010 (lowe et al. 2004; nozawa et al. 2002) ca carminomycin i, ch11 an anti-fas monoclonal antibody, d aspartic acid, sts staurosporine subsequently, it was shown that an n-terminal 85 amino acid fragment of golgin-160 contains both a golgi localization signal and a nuclear localization signal (hicks and machamer 2002) . expression of a non-cleavable golgin-160 mutant inhibits er stress or ligation of death receptor-induced apoptosis (maag et al. 2005) . latterly, yeast two hybrid screening revealed that gcp60 preferentially binds to one of the caspase cleavage products of golgin-160, aa 140-311, to inhibit its nuclear localization (sbodio et al. 2006) . overexpression of gcp60 sensitizes cells to staurosporineinduced apoptosis, while nuclear localization of a golgin-160 apoptotic cleavage fragment (aa 140-311) protects cells from apoptosis. however, another report indicates that golgin-160 depletion does not affect the golgi morphology nor constitutive secretion (williams et al. 2006) . therefore, the mechanism of how golgin-160 transduces apoptotic signals and regulates the apoptotic response needs to be further studied. grasp65 is cleaved in apoptosis induced by oxygen-and glucose-deprivation (ogd) as in ischemia-induced cerebral vascular endothelial injury (yin et al. 2010 ) and in staurosporine-or fas ligand-induced apoptosis (lane et al. 2002 , cheng et al. 2010 . in apoptosis, grasp65 is cleaved by caspase-3 on d320, d375, and d393. expression of a cleavage-resistant form of grasp65 delays golgi fragmentation in apoptosis and protects cells from fas/cd95-mediated apoptosis, whereas expression of an n-terminal caspase-cleaved fragment dramatically sensitizes cells to fas/cd95-mediated apoptosis (lane et al. 2002 , cheng et al. 2010 . further results revealed that the c-terminal fragments of grasp65 produced by caspase cleavage promotes fas/cd95-mediated apoptosis via being targeted to mitochondria by binding to bcl-x l (cheng et al. 2010 ). however, the mechanism of how the c-terminal cleavage fragment of grasp65 regulates apoptosis at the mitochondria and the role of bcl-x l in this process are still unknown. the golgi fragmentation phenotype induced by apoptotic grasp65 cleavage is similar to that of grasp65 phosphorylation in mitosis (warren 1995) . there is evidence that several kinases involved in mitotic grasp65 phosphorylation such as cdk1 and erk are activated during apoptosis and regulate apoptosis by phosphorylating caspase and bcl-2 family proteins (terrano et al. 2010; yamaguchi et al. 2008; lu et al. 2011) . whether these kinases directly regulate apoptotic golgi fragmentation by phosphorylating grasp65 or other golgi proteins remains unclear (ji et al. 2013 ). the er-to-golgi membrane tether, p115, is cleaved by caspase-3 and caspase-8 during apoptosis. expression of a caspase-resistant form of p115 delays golgi fragmentation in apoptosis. exogenous expression of a p115 c-terminal apoptotic fragment leads to apoptosis and golgi fragmentation (chiu et al. 2002) . the extreme c-terminal fragment, generated by caspase cleavage during apoptosis, translocates into the nucleus and further activates the apoptosis machinery. interestingly, translocation of the p115 c-terminal fragment happens prior to major golgi structural changes, indicating it as an early event (mukherjee and shields 2009) . the p115 c-terminus is sumoylated, which regulates its nuclear translocation and amplification of apoptosis signals in a p53-dependent manner (how and shields 2011; mukherjee and shields 2009) . in a high-throughput screen, carminomycin i (ca) was discovered to inhibit cell proliferation of von hippel-lindau (vhl) defective clear cell renal cell carcinoma (vhlà/à ccrcc) (woldemichael et al. 2011) . ca activates caspase-2 and caspase-3 to cleave p115, which inhibits ccrcc proliferation (woldemichael et al. 2011 ). several other golgi structural proteins are also involved in apoptosis or cleaved by caspases. the level of gm130 is reduced during fas-mediated apoptosis but not in staurosporine-induced apoptosis (walker et al. 2004 ). however, it is not clear whether the reduction is due to gm130 cleavage or degradation. syntaxin 5 and giantin are also cleaved by caspase-3 during apoptosis, which inhibits er-to-golgi transport (lowe et al. 2004 ). golgin-95 and golgin-97 are cleaved during necrosis but not apoptosis (nozawa et al. 2002) . cleavage of golgin-95 and golgin-97 during necrosis is also caspase-dependent, since pretreatment of the cells with pan-caspase inhibitor, zvad-fmk, abolished the cleavage of these two proteins (nozawa et al. 2002) . some of the golgi proteins are also reported to regulate apoptosis. the golgi snare gs28 is involved in cisplatin-induced apoptosis in a p53-dependent manner (sun et al. 2012) . overexpression of gs28 sensitizes hek293 cells to the apoptosisinducer cisplatin via the accumulation of p53 and bax and the stimulation of p53 pro-apoptotic phosphorylation at s46. it was also shown that gs28 forms a complex with the p53 ubiquitin e3 ligase murine double minute 2 (mdm2) to inhibit its function and consequential p53 ubiquitination and degradation (sun et al. 2012) . therefore, gs28 promotes cisplatin-induced apoptosis by stabilizing and regulating pro-apoptotic phosphorylation of p53. a well-studied mediator of intracellular vesicle fusion, nsf attachment protein α (αsnap), has been reported to have pro-survival functions (naydenov et al. 2012 ). depletion of αsnap triggers apoptosis in epithelial cells by reducing the antiapoptotic protein bcl-2. depletion of αsnap in p53 null or bax null cells still results in apoptosis, indicating that the anti-apoptotic function is independent of p53 and bax. interestingly, αsnap depletion induces apoptosis independent of the cleavage of golgi proteins such as grasp65, golgin-160, and p115 but rather by dysregulation of er-golgi vesicle cycling and possibly through er stress (naydenov et al. 2012) . some other golgi proteins, including human golgi antiapoptotic protein (h-gaap) (gubser et al. 2007; saraiva et al. 2013) and golgi integral membrane protein 4 (golim4) (bai et al. 2018) , are also reported to have anti-apoptotic functions. golim4 is overexpressed in some head and neck cancers, and depletion of golim4 reduces cell proliferation and cell viability by inducing apoptosis (bai et al. 2018) . although microtubule and actin filaments play important roles in golgi orientation and structure, golgi fragmentation in apoptosis occurs prior to cytoskeleton disorganization (mukherjee et al. 2007; yadav and linstedt 2011) . furthermore, the level of actin and tubulin did not change during apoptosis, while golgi structural proteins are cleaved as discussed above. therefore, golgi fragmentation in early apoptosis is independent of microtubule and actin filament disorganization. the golgi phosphoprotein 3 (golph3) is a peripheral membrane protein that regulates vesicle budding and tgn-to-plasma membrane trafficking (dippold et al. 2009 ). golph3 is localized to the tgn by binding to phosphatidylinositol 4-phosphate (pi4p). depletion of pi4p leads to golph3 dissociation from the tgn. golph3 also binds to the actin-based motor protein myo18a to link golgi membranes with the actin cytoskeleton. this bridging effect creates a tension required for vesicle budding, trafficking, and maintenance of the golgi ribbon. depletion of golph3 or myo18a leads to the loss of the tensile force, resulting in the shrinkage of the golgi ribbon and a reduction of vesicles formed at the tgn (dippold et al. 2009; bishe et al. 2012; ng et al. 2013) . golph3 is an oncogene known to be overexpressed in some solid tumors, including lung cancer and breast cancer (scott et al. 2009; zeng et al. 2012) . it is also reported that golph3 increases cell proliferation and cell size by regulating cell proliferation through the interaction with the retromer complex and activation of the mammalian target of rapamycin mtor (scott et al. 2009 ). golph3 induces cell proliferation in breast cancer cells by inhibiting the tumor suppressor transcription factor foxo1 through activating akt (zeng et al. 2012 ). these findings demonstrate that a trans-golgi protein can serve as an oncogene (scott et al. 2009; buschman et al. 2015; kuna and field 2018) . interestingly, dna damage causes golgi dispersal in a golph3-dependent manner. dna damage activates the dna-pk kinase to phosphorylate golph3 on t143/t148, which aberrantly increases the tensile force for the golgi to fragment. golgi fragmentation in this scenario increases cell survival with an unknown mechanism (farber-katz et al. 2014 ). depletion of golph3 or myo18a increases cancer cells' sensitivity to dna damage inducing agents, suggesting that golph3 phosphorylation-induced golgi fragmentation may serve as a protective mechanism (farber-katz et al. 2014) . considering that golph3 is overexpressed in many solid tumors, it is reasonable to speculate that this may be a mechanism of how cancer cells escape dna damage-induced apoptosis (farber-katz et al. 2014; buschman et al. 2015; li et al. 2016b) . in addition to its high expression level in some cancer cells, golph3 overexpression is also reported in mouse n2a cells under oxygen-glucose deprivation and reoxygenation (ogd/r), a model mimicking severe oxidative injury (li et al. 2016a) . in this ogd/r model, golph3 is overexpressed and forms puncta in the cytosol, which induces the formation of reactive oxygen species (ros) and lipidation of lc3. opposed to its anti-apoptotic role in cancer cells, depletion of golph3 in ogd/r desensitizes the cells to apoptosis (li et al. 2016a ). most recently, the golgi stacking protein grasp55 was reported to regulate autophagy upon energy deprivation (zhang et al. 2018; zhang and wang 2018a, b) . under normal growth condition, grasp55 is o-glcnacylated and localizes in the medial-and trans-golgi for stacking. however, under glucose starvation, a pool of de-o-glcnacylated grasp55 translocates to the interface between autophagosomes and lysosomes to facilitate autophagosome-lysosome fusion. after a short-term energy deprivation, the golgi structure is only mildly affected, possibly due to sufficient grasp55 molecules remaining in the golgi to maintain its structure. among over a dozen golgi proteins tested, only grasp55, but not grasp65, gm130, or golgin-45, is o-glcnacylated under growth conditions and targets to autophagosomes upon energy deprivation, indicating that grasp55 serves as an energy sensor on the golgi to regulate both intracellular trafficking and autophagy. significantly, the same scenario may be seen in autophagy induced by amino acid starvation and inhibition of mtor (zhang et al. 2018) . in addition to golgi fragmentation, gcc88 overexpression also induces autophagy via reducing the activity of mtor. a considerable pool of mtor is localized and activated on the golgi, which is dependent on the ribbon structure for recruitment but independent of lysosomal mtor activation (gosavi et al. 2018) . these findings indicate the golgi ribbon as an important location for the functional regulation of mtor activity. additionally, autophagosomes may directly form on golgi membranes (guo et al. 2012) or obtain membranes from the golgi . the only recognized transmembrane atg protein, atg9, localizes at the trans-golgi network and late endosomes and is essential for autophagosome formation (yamamoto et al. 2012) , although the detailed mechanism awaits further investigation (orsi et al. 2012) . recently, the endoplasmic reticulum-golgi intermediate compartment (ergic) is proposed to serve as a key membrane source for autophagosome formation . under normal condition, copii vesicles are generated from the er-exit sites (eres) for er-golgi membrane trafficking, while upon starvation, the copii assembly activator prolactin regulatory element-binding protein (preb)/sec12 relocates to the ergic and triggers ergic-copii vesicle formation as membrane templates for lc3 lipidation. golgi structure defects and dysfunctions have been observed in many diseases, including pathogen infection, neurodegenerative diseases, and cancer (aridor and hannan 2000) . generally, the mechanisms of golgi fragmentation include imbalanced membrane flux, altered microtubule dynamics, and posttranslational modifications or proteolytic cleavage of golgi structural proteins (wei and seemann 2017) . in many cases, the correlation between golgi defect and disease progression is unclear. a few interesting cases reported recently are discussed below. ad is an age-related neurodegenerative disease of the central nerve system characterized by progressive loss of cognition and memory. golgi fragmentation occurs in neurons of patients with ad since the earliest stages of disease development . some cases of early-onset ad are related to mutations in the amyloid precursor protein (app) or presenilin 1 and 2 (psn1 and 2). the amyloid-beta (aβ) peptide is a proteolytic product of app, which is considered to be the major inducer of ad (selkoe and hardy 2016) . aβ accumulation is likely the direct cause of golgi fragmentation, as aβ-treatment causes reversible golgi fragmentation in cultured neurons (joshi et al. 2014) . present research supports that activation of cyclin-dependent kinase 5 (cdk5) by aβ accumulation via the [ca 2+ ]-calpain-p25 pathway may be the major trigger of golgi fragmentation in ad joshi et al. 2015; joshi and wang 2015; evin 2015; ayala and colanzi 2017) . cdk5 may function in two ways. first, cdk5 phosphorylates gm130 at s25 and inhibits its interaction with the golgi tethering protein p115 (sun et al. 2008) . second, cdk5 phosphorylates grasp65 at t220/t224, which inhibits grasp65 function in golgi stack formation and ribbon linking (joshi et al. 2014) . furthermore, inhibiting cdk5 or expressing non-phosphorylatable grasp65 mutants both rescued the golgi structure and reduced aβ secretion by elevating the α-cleavage of app (joshi and wang 2015; joshi et al. 2014) , indicating that grasp65 phosphorylation may be the main reason for ad-induced golgi fragmentation. these studies not only provide molecular mechanisms for golgi fragmentation but also suggest golgi as a potential drug target for ad treatment (joshi et al. 2015; joshi and wang 2015; ayala and colanzi 2017) . als is a fatal neurodegenerative disorder specifically targeted to motor neurons. fragmented golgi has been observed in numerous models of superoxide dismutase 1 (sod1), tdp-43, fus, and optineurin-associated als (fujita et al. 2008; soo et al. 2015; van dis et al. 2014; wallis et al. 2018) . sod1 inhibits er-to-golgi transport and causes golgi fragmentation (atkin et al. 2014 ) by reducing the β-cop protein level, accumulating the er-golgi v-snares gs15 and gs28, and destabilizing microtubules by the upregulation of stathmins 1 and 2 (bellouze et al. 2016; atkin et al. 2014) . fus and tdp-43 impair the incorporation of secretory cargo into copii vesicles (soo et al. 2015) . furthermore, expression of als-related optineurin mutants impairs myosin vi-mediated protein trafficking from golgi to plasma membrane, which also induces golgi fragmentation . conclusively, impairment of distinct protein trafficking pathways by different als-linked proteins are specific triggers for golgi fragmentation in als. pd is pathologically characterized by the loss of dopamine-containing neurons and by the formation of intracellular protein aggregates known as lewy bodies in which α-synuclein has been recognized as a major constituent (forno 1996; wakabayashi et al. 1998) . golgi fragmentation can be detected in early-stage pd brains (fujita et al. 2006 ) and is strongly correlated to the presence of prefibrillar α-synuclein (gosavi et al. 2002) . since then, emerging studies have provided important insights into the mechanisms of how α-synuclein causes pathological golgi fragmentation and neuronal degeneration. the primary effect of α-synuclein aggregation is the inhibition of er-to-golgi transport (lashuel and hirling 2006) , which can be rescued by the overexpression of rab1 and rab8 and depletion of rab2 and syntaxin 5 (rendon et al. 2013; coune et al. 2011) . in a most recent report, mutations in the leucine-rich repeat kinase 2 (lrrk2), a major genetic cause of autosomaldominantly inherited pd, markedly enhance rab7l1 phosphorylation on s72, resulting in tgn fragmentation (fujimoto et al. 2018 ). golgi disorganization may be related to cancer progression and metastasis in the following aspects: aberrant glycosylation, abnormal expression of ras gtpase, dysregulation of kinases, and hyperactivation of myosin motor proteins (petrosyan 2015) . perturbation of the golgi morphology in cancers results in an increase of sialylation which is associated with a metastatic cell phenotype (schultz et al. 2012 ). overexpression of sialylated antigens is significantly correlated with tumor progression and therapy resistance due to an anti-apoptotic effect (lee et al. 2008; park et al. 2012; petrosyan et al. 2014) . over-activation of rabs, which coordinate with golgins in protein transportation and golgi structure maintenance, has been reported in different types of cancers (goldenring 2013) . furthermore, golgi disorganizationrelated kinases, including src, erk9, and p21-activated protein kinase (pak1), are found elevated in tumor cells (chia et al. 2014; ching et al. 2007; weller et al. 2010 ). the ubiquitin ligase hace1, which regulates p97-mediated golgi membrane fusion as discussed above, is reported as a tumor suppressor downregulated in multiple tumors including wilms' tumor (anglesio et al. 2004) , resulting in golgi fragmentation cui and wang 2012) . additionally, hyperactivation of golgi-associated myosins, including myo18a that directly binds to golph3 to promote golgi dispersal (dippold et al. 2009; allan et al. 2002) , is detected in many aggressive cancers. golgi fragmentation is one of the essential and earliest events in apoptosis, where several golgins and grasp65 are cleaved by activated caspases, as described in previous sections. several membrane structures including the golgi are used by viruses as viral factories to replicate, concentrate, and assemble the viral genome and proteins into viral particles (miller and krijnse-locker 2008; netherton et al. 2007; salonen et al. 2005) . as a highly dynamic organelle, golgi serves as a membrane scaffold for multiple viruses, including infectious hepatitis c virus, enteroviruses, poliovirus, foot-and-mouth-disease virus, dengue virus, coronavirus, kunjin virus, tick-borne encephalitis virus, rubella virus, and bunyamwera virus (miller and krijnse-locker 2008; harak and lohmann 2015; risco et al. 2003; salanueva et al. 2003; delgui et al. 2013; westerbeck and machamer 2015) , and is frequently fragmented after infection (campadelli et al. 1993; salanueva et al. 2003; yadav et al. 2016; avitabile et al. 1995; lavi et al. 1996; hansen et al. 2017; rebmann et al. 2016) . viruses use golgi membranes directly and/or hijack master controllers of golgi biogenesis and trafficking to generate vesicles that are used as the site of viral rna replication (quiner and jackson 2010; hansen et al. 2017; short et al. 2013) , wrapping (sivan et al. 2016; alzhanova and hruby 2007; alzhanova and hruby 2006; nanbo et al. 2018; lundu et al. 2018; procter et al. 2018) , intracellular transduction (nonnenmacher et al. 2015) , and secretion ). viral infection triggers golgi fragmentation via diverse mechanisms, ranging from phosphorylating key golgi structural proteins such as grasp65 (rebmann et al. 2016) , activating the src kinase to phosphorylate the dynamin 2 gtpase (martin et al. 2017) , targeting the immunity-related gtpase m (irgm) to the golgi to induce gbf1 phosphorylation (hansen et al. 2017) , modulating vesicular trafficking (yadav et al. 2016; johns et al. 2014) , to impeding the major histocompatibility complex (mhc) class i trafficking, antigen presentation, and/or cytokine secretion rohde et al. 2012 ). the golgi is the central hub in the secretory pathway, where proteins and lipids are processed, sorted, and dispatched to distinct destinations. as a well-organized polarized membrane structure, golgi function is tightly related to its structural integrity. thus, the first key question in golgi biology concerns how the stacked golgi structure forms. during the past decades, proteins with a variety of functions have been identified in the maintenance of golgi structure and regulation of golgi function, including but not limited to grasps, golgins, kinases, phosphatases, ubiquitin e3 ligases, and deubiquitinases, as summarized above. more detailed investigations need to be done to investigate how golgi structural proteins and their interacting molecules cooperate together to form the stacked golgi structure. golgi structural and functional defects have been increasingly reported in stress and disease conditions. in addition to its central role in protein sorting and trafficking, the golgi has been more recently recognized as a hub of signaling pathways, which facilitates golgi reaction upon stresses and diseases. thus, the second key question in golgi biology concerns how the golgi structure becomes defective in stress and disease conditions. in this regard, much effort and some progress have been made, such as golgi fragmentation in ad by cdk5-mediated grasp65 phosphorylation as discussed above. however, many questions remain. for example, is there an unfolded protein response (upr)-like mechanism at the golgi to cope with different stresses? is there a common stress sensor on the golgi? how do the signaling pathways on the golgi sense and transduce stress signals? apparently, a more systematic analysis of golgi response to different stressors is necessary. gene expression profile and posttranslational modification analysis of golgi structural proteins, golgi enzymes, and signaling molecules will fast forward the field and shed light on new directions. considering lipid organization and modification are important for golgi function and certain lipids can work as signaling molecules, more attention may be put on lipids at the golgi, in addition to proteins. the third key question in golgi biology concerns how golgi structure alteration affects its function in trafficking, glycosylation, and sorting. the consequence of golgi fragmentation in different diseases is likely different, but it has been reported that golgi cisternal unstacking by depleting grasp proteins enhances protein trafficking. this, however, impairs accurate glycosylation and causes missorting of lysosomal enzymes to the extracellular space (xiang et al. 2013; zhang and wang 2016; bekier et al. 2017; wang et al. 2008) . consistently, golgi fragmentation in ad enhances app trafficking and aβ production, while rescue of the golgi causes app accumulation in the golgi and reduces aβ secretion (joshi et al. 2014) . most recently, we have obtained evidence that golgi structure disassembly by grasp depletion reduces cell attachment and migration while accelerating cell growth and cell cycle progression (ahat et al. 2019 ). it will be interesting to investigate how golgi fragmentation enhances cancer cell proliferation and metastasis in the future. future efforts may also aim at developing small chemicals or molecular tools to rescue the golgi structure in diseases, which may delay the disease development. grasp depletion-mediated golgi destruction decreases cell adhesion and migration via the reduction of α5β1 integrin small gtpase rab2b and its specific binding protein golgiassociated rab2b interactor-like 4 (gari-l4) regulate golgi morphology a novel missense mutation in scyl1bp1 produces geroderma osteodysplastica phenotype indistinguishable from that caused by nullimorphic mutations rab1 recruitment of p115 into a cis-snare complex: programming budding copii vesicles for fusion motoring around the golgi the p115-interactive proteins gm130 and giantin participate in endoplasmic reticulum-golgi traffic a trans-golgi network resident protein, golgin-97, accumulates in viral factories and incorporates into virions during poxvirus infection a host cell membrane protein, golgin-97, is essential for poxvirus morphogenesis the golgin protein coy1 functions in intra-golgi retrograde transport and interacts with the cog complex and golgi snares differential expression of a novel ankyrin containing e3 ubiquitin-protein ligase, hace1, in sporadic wilms' tumor versus normal kidney traffic jam: a compendium of human diseases that affect intracellular transport processes death by committee: organellar trafficking and communication in apoptosis mutant sod1 inhibits er-golgi transport in amyotrophic lateral sclerosis redistribution of microtubules and golgi apparatus in herpes simplex virus-infected cells and their role in viral exocytosis alterations of golgi organization in alzheimer's disease: a cause or a consequence? golgi integral membrane protein 4 manipulates cellular proliferation, apoptosis, and cell cycle in human head and neck cancer purification and functional interactions of grasp55 with rab2 grasp65, a protein involved in the stacking of golgi cisternae mapping the interaction between grasp65 and gm130, components of a protein complex involved in the stacking of golgi cisternae golgi matrix proteins interact with p24 cargo receptors and aid their efficient retention in the golgi apparatus aurora-a recruitment and centrosomal maturation are regulated by a golgi-activated pool of src during g2 knockout of the golgi stacking proteins grasp55 and grasp65 impairs golgi structure and function stathmin 1/2-triggered microtubule loss mediates golgi fragmentation in mutant sod1 motor neurons the golgi matrix protein giantin is required for normal cilia function in zebrafish role of phosphatidylinositol 4-phosphate (pi4p) and its binding protein golph3 in hepatitis c virus secretion selective export of human gpi-anchored proteins from the endoplasmic reticulum membrane targeting of p115 phosphorylation mutants and their effects on golgi integrity and secretory traffic characterization of pskh1, a novel human protein serine kinase with centrosomal, golgi, and nuclear localization mutants of the protein serine kinase pskh1 disassemble the golgi apparatus involvement of golgin-160 in cell surface transport of renal romk channel: co-expression of golgin-160 increases romk currents rab and arl gtpase family members cooperate in the localization of the golgin gcc185 the golph3 pathway regulates golgi shape and function and is activated by dna damage fragmentation and dispersal of golgi proteins and redistribution of glycoproteins and glycolipids processed through the golgi apparatus after infection with herpes simplex virus 1 the aaa-atpase cdc48/p97 regulates spindle disassembly at the end of mitosis making the auroras glow: regulation of aurora a and b kinase function by interacting proteins aurrand-lions m (2017) genetic, structural, and chemical insights into the dual function of grasp55 in germ cell golgi remodeling and jam-c polarized localization during spermatogenesis jnk2 controls fragmentation of the golgi complex and the g2/m transition through phosphorylation of grasp65 thyroid hormone, t3-dependent phosphorylation and translocation of trip230 from the golgi complex to the nucleus caspase cleavage of the golgi stacking factor grasp65 is required for fas/cd95-mediated apoptosis erk8 is a negative regulator of o-galnac glycosylation and cell migration p21-activated protein kinase is overexpressed in hepatocellular carcinoma and enhances cancer metastasis involving c-jun nh2-terminal kinase activation and paxillin phosphorylation a caspase cleavage fragment of p115 induces fragmentation of the golgi apparatus and apoptosis zfpl1, a novel ring finger protein required for cis-golgi integrity and efficient er-to-golgi transport adhesion of golgi cisternae by proteinaceous interactions: intercisternal bridges as putative adhesive structures the golgi mitotic checkpoint is controlled by bars-dependent fission of the golgi ribbon into separate stacks in g2 rab1a over-expression prevents golgi apparatus fragmentation and partially corrects motor deficits in an alpha-synuclein based rat model of parkinson's disease identification and characterization of two novel (neuro)endocrine long coiled-coil proteins hace1 (hect domain and ankyrin repeat containing e3 ubiquitin protein ligase 1) grasp65 and grasp55 sequentially promote the transport of c-terminal valine-bearing cargos to and through the golgi complex dvc1 (c1orf124) recruits the p97 protein segregase to sites of dna damage rab18 and rab43 have key roles in er-golgi trafficking the endosomal pathway and the golgi complex are involved in the infectious bursal disease virus life cycle pentameric assembly of potassium channel tetramerization domain-containing protein 5 new insights into membrane trafficking and protein sorting the trans-golgi network golgin, gcc185, is required for endosome-to-golgi transport and maintenance of golgi structure the coiled-coil membrane protein golgin-84 is a novel rab effector required for golgi ribbon formation coordination of golgin tethering and snare assembly: gm130 binds syntaxin 5 in a p115-regulated manner golph3 bridges phosphatidylinositol-4-phosphate and actomyosin to stretch and shape the golgi to promote budding phosphorylation of the vesicletethering protein p115 by a casein kinase ii-like enzyme is required for golgi reassembly from isolated mitotic fragments a general amphipathic alpha-helical motif for sensing membrane curvature asymmetric tethering of flat and curved lipid membranes by a golgin autophagy-based unconventional secretory pathway for extracellular delivery of il-1beta the role of grasp55 in golgi fragmentation and entry of cells into mitosis asymmetric clasp-dependent nucleation of noncentrosomal microtubules at the trans-golgi network how accelerated golgi trafficking may drive alzheimer's disease (comment on human autoantibodies to a novel golgi protein golgin-67: high similarity with golgin-95/gm 130 autoantigen dna damage triggers golgi dispersal via dna-pk and golph3 signalling to and from the secretory pathway grasp55 regulates golgi ribbon formation the golgin gmap210/trip11 anchors ift20 to the golgi complex neuropathology of parkinson's disease inter-and intracisternal elements of the golgi apparatus. a system of membrane-to-membrane cross-links tmf is a golgin that binds rab6 and influences golgi morphology cdc48p is required for the cell cycle commitment point at start via degradation of the g1-cdk inhibitor far1p parkinson's diseaseassociated mutant lrrk2 phosphorylates rab7l1 and modifies trans-golgi morphology fragmentation of golgi apparatus of nigral neurons with alpha-synuclein-positive inclusions in patients with parkinson's disease anterior horn cells with abnormal tdp-43 immunoreactivities show fragmentation of the golgi apparatus in als a functional deficiency of tera/vcp/p97 contributes to impaired dna repair in multiple polyglutamine diseases a syntaxin 10-snare complex distinguishes two distinct transport routes from endosomes to the trans-golgi in human cells the er-golgi intermediate compartment is a key membrane source for the lc3 lipidation step of autophagosome biogenesis rescue of deltaf508-cftr trafficking via a grasp-dependent unconventional secretion pathway the golgi as a potential membrane source for autophagy post-golgi sec proteins are required for autophagy in saccharomyces cerevisiae the atpase vcp/p97 functions as a disaggregase against toxic huntingtin-exon1 aggregates three basic residues of intracellular loop 3 of the beta-1 adrenergic receptor are required for golgin-160-dependent trafficking finding the golgi: golgin coiled-coil proteins show the way the gtpase arf1p and the er to golgi cargo receptor erv14p cooperate to recruit the golgin rud3p to the cis-golgi a central role for vesicle trafficking in epithelial neoplasia: intracellular highways to carcinogenesis golgi fragmentation occurs in the cells with prefibrillar alpha-synuclein aggregates and precedes the formation of fibrillar inclusion the golgi ribbon in mammalian cells negatively regulates autophagy by modulating mtor activity small gtp-binding protein associated with golgi cisternae rab proteins as major determinants of the golgi complex structure the 3a protein from multiple picornaviruses utilizes the golgi adaptor protein acbd3 to recruit pi4kiiibeta acbd3 interaction with tbc1 domain 22 protein is differentially affected by enteroviral and kobuviral 3a protein binding a new inhibitor of apoptosis from vaccinia virus and eukaryotes ap1 is essential for generation of autophagosomes from the trans-golgi network hepatitis c virus triggers golgi fragmentation and autophagy through the immunity-related gtpase m a non-canonical role of the p97 complex in rig-i antiviral signaling ultrastructure of the replication sites of positive-strand rna viruses aaa atpase p97/vcp is essential for trim21-mediated virus neutralization multiple rab gtpase binding sites in gcc185 suggest a model for vesicle tethering at the trans-golgi myristoylation restricts orientation of the grasp domain on membranes and promotes membrane tethering gerodermia osteodysplastica is caused by mutations in scyl1bp1, a rab-6 interacting golgin distinct aaa-atpase p97 complexes function in discrete steps of nuclear assembly the nh2-terminal domain of golgin-160 contains both golgi and nuclear targeting information golgi structure in stress sensing and apoptosis mitotic golgi partitioning is driven by the membrane-fissioning protein ctbp3/bars mammalian golgi-associated bicaudal-d2 functions in the dynein-dynactin pathway by interacting with these complexes tethering function of the caspase cleavage fragment of golgi protein p115 promotes apoptosis via a p53-dependent pathway isolation and characterization of ara160 as the first androgen receptor n-terminal-associated coactivator in human prostate cells scyl1 binding protein 1 promotes the ubiquitin-dependent degradation of pirh2 and has tumorsuppressive function in the development of hepatocellular carcinoma structural basis for the interaction between the golgi reassembly-stacking protein grasp65 and the golgi matrix protein gm130 golgi structure formation, function, and post-translational modifications in mammalian cells monoubiquitination of syntaxin 5 regulates golgi membrane dynamics during the cell cycle biogenesis of neurosecretory vesicles identification and characterization of a novel golgi protein, golgin-67 the role of grasps in morphological alterations of golgi apparatus: mechanisms and effects golgin-84-associated golgi fragmentation triggers tau hyperphosphorylation by activation of cyclin-dependent kinase-5 and extracellular signalregulated kinase fip1/rcp binding to golgin-97 regulates retrograde transport from recycling endosomes to the trans-golgi network rab6 dependent post-golgi trafficking of hsv1 envelope proteins to sites of virus envelopment golgi defects enhance app amyloidogenic processing in alzheimer's disease abeta-induced golgi fragmentation in alzheimer's disease enhances abeta production caspase-mediated cleavage of the stacking protein grasp65 is required for golgi fragmentation during apoptosis rescuing defective vesicular trafficking protects against alphasynuclein toxicity in cellular and animal models of parkinson's disease syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the golgi apparatus potential role for protein kinases in regulation of bidirectional endoplasmic reticulum-to-golgi transport revealed by protein kinase inhibitor h89 protein sialylation by sialyltransferase involves radiation resistance golph3 mediated golgi stress response in modulating n2a cell death upon oxygen-glucose deprivation and reoxygenation injury golgi phosphoprotein 3 inhibits the apoptosis of human glioma cells in part by downregulating n-myc downstream regulated gene 1 dja1 maintains golgi integrity via interaction with grasp65 the golgin gcc88 is required for efficient retrograde transport of cargo from the early endosomes to the trans-golgi network peripheral golgi protein grasp65 is a target of mitotic polo-like kinase (plk) and cdc2 arl4a acts with gcc185 to modulate golgi complex organization lrrk regulates the dynamic profile of dendritic golgi outposts through the golgin lava lamp binding relationships of membrane tethering components. the giantin n terminus and the gm130 n terminus compete for binding to the p115 c terminus how rab proteins determine golgi structure rab41 is a novel regulator of golgi apparatus organization that is needed for er-to-golgi trafficking and cell growth rab1a mediates proinsulin to insulin conversion in beta-cells by maintaining golgi stability through interactions with golgin-84 loss of the golgin gm130 causes golgi disruption, purkinje neuron loss, and ataxia in mice e-cadherin transport from the trans-golgi network in tubulovesicular carriers is selectively regulated by golgin-97 cdc2 kinase directly phosphorylates the cis-golgi matrix protein gm130 and is required for golgi fragmentation in mitosis caspase-mediated cleavage of syntaxin 5 and giantin accompanies inhibition of secretory traffic during apoptosis interaction of arl1-gtp with grip domains recruits autoantigens golgin-97 and golgin-245/p230 onto the golgi autoantigen golgin-97, an effector of arl1 gtpase, participates in traffic from the endosome to the trans-golgi network paclitaxel induces apoptosis through activation of nuclear protein kinase c-delta and subsequent activation of golgi associated cdk1 in human hormone refractory prostate cancer grip domain-mediated targeting of two new coiled-coil proteins, gcc88 and gcc185, to subcompartments of the trans-golgi network the trans-golgi network grip-domain proteins form alpha-helical homodimers targeting of severe fever with thrombocytopenia syndrome virus structural proteins to the ergic (endoplasmic reticulum golgi intermediate compartment) and golgi complex p24a, a type i transmembrane protein, controls arf1-dependent resensitization of protease-activated receptor-2 by influence on receptor trafficking caspase-resistant golgin-160 disrupts apoptosis induced by secretory pathway stress and ligation of death receptors golgi disassembly in apoptosis: cause or effect? the golgi complex in stress and death a filter at the entrance of the golgi that selects vesicles according to size and bulk lipid composition intersectin-1 interacts with the golgin, gcc88, to couple the actin network and golgi architecture golgin tethers define subpopulations of copi vesicles caspase-2 is localized at the golgi complex and cleaves golgin-160 during apoptosis herpes simplex virus type 1 neuronal infection perturbs golgi apparatus integrity through activation of src tyrosine kinase and dyn-2 gtpase gtp-bound forms of rab6 induce the redistribution of golgi proteins into the endoplasmic reticulum dominant spinal muscular atrophy is caused by mutations in bicd2, an important golgin protein genomewide screen identifies a novel p97/cdc-48-dependent pathway regulating er-stress-induced gene transcription bicaudal-d regulates copiindependent golgi-er transport by recruiting the dynein-dynactin motor complex the golgi ribbon and the function of the golgins signaling at the golgi the ubiquitin-selective segregase vcp/p97 orchestrates the response to dna double-strand breaks direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and ufd1-npl4 modification of intracellular membrane structures for virus replication molecular characterization of gcp170, a 170-kda protein associated with the cytoplasmic face of the golgi membrane an essential cytoplasmic domain for the golgi localization of coiled-coil proteins with a cooh-terminal membrane anchor inhibition of the secretory pathway by foot-and-mouth disease virus 2bc protein is reproduced by coexpression of 2b with 2c, and the site of inhibition is determined by the subcellular location of 2c an intercisternal structure in the golgi apparatus a putative nuclear receptor coactivator (tmf/ara160) associates with hbrm/hsnf2 alpha and brg-1/hsnf2 beta and localizes in the golgi apparatus rab1 interaction with a gm130 effector complex regulates copii vesicle cis-golgi tethering nuclear import is required for the pro-apoptotic function of the golgi protein p115 fragmentation of the golgi apparatus: an early apoptotic event independent of the cytoskeleton the golgin coiled-coil proteins of the golgi apparatus characterization of a cis-golgi matrix protein, gm130 the vesicle docking protein p115 binds gm130, a cis-golgi matrix protein, in a mitotically regulated manner epstein-barr virus acquires its final envelope on intracellular compartments with golgi markers loss of soluble n-ethylmaleimide-sensitive factor attachment protein alpha (alphasnap) induces epithelial cell apoptosis via down-regulation of bcl-2 expression and disruption of the golgi a guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication mutations in bicd2, which encodes a golgin and important motor adaptor, cause congenital autosomal-dominant spinal muscular atrophy golph3l antagonizes golph3 to determine golgi morphology caspases: killer proteases aurora a kinase (aurka) in normal and pathological cell division homologs from caenorhabditis elegans, cdc-48.1 and cdc-48.2, suppress the aggregate formation of huntingtin exon1 containing expanded polyq repeat centrosome-targeting region of cg-nap causes centrosome amplification by recruiting cyclin e-cdk2 complex syntaxin 5-dependent retrograde transport to the trans-golgi network is required for adeno-associated virus transduction fragmentation of golgi complex and golgi autoantigens during apoptosis and necrosis identification and characterization of gcp16, a novel acylated golgi protein that interacts with gcp170 dynamic and transient interactions of atg9 with autophagosomes, but not membrane integration, are required for autophagy stacks off tracks: a role for the golgin atcasp in plant endoplasmic reticulum-golgi apparatus tethering the arf-like gtpases arl1p and arl3p act in a pathway that interacts with vesicle-tethering factors at the golgi apparatus vcp/p97 cooperates with yod1, ubxd1 and plaa to drive clearance of ruptured lysosomes by autophagy the golgin lava lamp mediates dynein-based golgi movements during drosophila cellularization cdc48/p97 segregase is modulated by cyclindependent kinase to determine cyclin fate during g1 progression sialylation of epidermal growth factor receptor regulates receptor activity and chemosensitivity to gefitinib in colon cancer cells dna damage modulates nucleolar interaction of the werner protein with the aaa atpase p97/vcp common variants in or near znrf1, colec12, scyl1bp1 and api5 are associated with diabetic retinopathy in chinese patients with type 2 diabetes tmf/ara160 is a bc-box-containing protein that mediates the degradation of stat3 onco-golgi: is fragmentation a gate to cancer progression? restoration of compact golgi morphology in advanced prostate cancer enhances susceptibility to galectin-1-induced apoptosis by modifying mucin o-glycan synthesis sec16a is critical for both conventional and unconventional secretion of cftr ysk1 is activated by the golgi matrix protein gm130 and plays a role in cell migration through its substrate 14-3-3{zeta} plk1 docking to grasp65 phosphorylated by cdk1 suggests a mechanism for golgi checkpoint signalling the hcmv assembly compartment is a dynamic golgi-derived mtoc that controls nuclear rotation and virus spread gm130 and grasp65-dependent lateral cisternal fusion allows uniform golgi-enzyme distribution fragmentation of the golgi apparatus provides replication membranes for human rhinovirus 1a golgi ribbon unlinking: an organelle-based g2/m checkpoint grasp: a multitasking tether an nsf-like atpase, p97, and nsf mediate cisternal regrowth from mitotic golgi fragments cdc48/p97 promotes reformation of the nucleus by extracting the kinase aurora b from chromatin golgins and grasps: holding the golgi together a novel 165-kda golgin protein induced by brain ischemia and phosphorylated by akt protects against apoptosis env7p associates with the golgin protein imh1 at the trans-golgi network in candida albicans phosphorylation of golgi peripheral membrane protein grasp65 is an integral step in the formation of the human cytomegalovirus cytoplasmic assembly compartment a functional role for the gcc185 golgin in mannose 6-phosphate receptor recycling rab6 and rab11 regulate chlamydia trachomatis development and golgin-84-dependent golgi fragmentation golgi fragmentation is rab and snare dependent in cellular models of parkinson's disease gmap-210 recruits gammatubulin complexes to cis-golgi membranes and is required for golgi ribbon formation structural maturation of rubella virus in the golgi complex toxicity and aggregation of the polyglutamine disease protein, ataxin-3 is regulated by its binding to vcp/p97 in drosophila melanogaster microtubule nucleation at the cis-side of the golgi apparatus requires akap450 and gm130 orf virus interferes with mhc class i surface expression by targeting vesicular transport and golgi giantin interacts with both the small gtpase rab6 and rab1 polymorphism and structural maturation of bunyamwera virus in golgi and post-golgi compartments viral rna replication in association with cellular membranes human and viral golgi anti-apoptotic proteins (gaaps) oligomerize via different mechanisms and monomeric gaap inhibits apoptosis and modulates calcium acbd3-mediated recruitment of pi4kb to picornavirus rna replication sites golgin-84 is a rab1 binding partner involved in golgi structure identification of a redox-sensitive cysteine in gcp60 that regulates its interaction with golgin-160 gcp60 preferentially interacts with a caspase-generated golgin-160 fragment regulation of the metastatic cell phenotype by sialylated glycans golph3 modulates mtor signalling and rapamycin sensitivity in cancer the role of the tethering proteins p115 and gm130 in transport through the golgi apparatus in vivo cohen syndromeassociated protein, coh1, is a novel, giant golgi matrix protein required for golgi integrity the amyloid hypothesis of alzheimer's disease at 25 years mitotic inhibition of grasp65 organelle tethering involves pololike kinase 1 (plk1) phosphorylation proximate to an internal pdz ligand tbc1d23 is a bridging factor for endosomal vesicle capture by golgins at the trans-golgi author correction: tbc1d23 is a bridging factor for endosomal vesicle capture by golgins at the trans-golgi vamp4 is required to maintain the ribbon structure of the golgi apparatus a grasp55-rab2 effector complex linking golgi structure to membrane traffic providence virus (family: carmotetraviridae) replicates vrna in association with the golgi apparatus and secretory vesicles a role for the vesicle tethering protein, p115, in the post-mitotic stacking of reassembling golgi cisternae in a cell-free system golgi architecture and inheritance grasp55, a second mammalian grasp protein involved in the stacking of golgi cisternae in a cell-free system sequential tethering of golgins and catalysis of snarepin assembly by the vesicle-tethering protein p115 centrosomal anchoring of the protein kinase ck1delta mediated by attachment to the large, coiled-coil scaffolding protein cg-nap/akap450 golgi coiled-coil proteins contain multiple binding sites for rab family g proteins retrograde transport from early endosomes to the trans-golgi network enables membrane wrapping and egress of vaccinia virus virions isolation of a matrix that binds medial golgi enzymes identification and characterization of a novel golgi protein, gcp60, that interacts with the integral membrane protein giantin interaction of golgin-84 with the cog complex mediates the intra-golgi retrograde transport trans-golgi protein p230/golgin-245 is involved in phagophore formation characterization of the aggregation-prevention activity of p97/ valosin-containing protein a role for giantin in docking copi vesicles to golgi membranes rab1-dependent er-golgi transport dysfunction is a common pathogenic mechanism in sod1, tdp-43 and fus-associated als vesicle-associated membrane protein 4 is implicated in trans-golgi network vesicle trafficking novel genetic tools reveal cdk5's major role in golgi fragmentation in alzheimer's disease golgi-snare gs28 potentiates cisplatin-induced apoptosis by forming gs28-mdm2-p53 complexes and by preventing the ubiquitination and degradation of p53 mitotic golgi vesiculation involves mechanisms independent of ser25 phosphorylation of gm130 golgi fragmentation in amyotrophic lateral sclerosis, an overview of possible triggers and consequences the golgi-associated protein grasp65 regulates spindle dynamics and is essential for cell division characterization of a novel giant scaffolding protein, cg-nap, that anchors multiple signaling enzymes to centrosome and the golgi apparatus centrosomal proteins cg-nap and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex cell cycle regulation of golgi membrane dynamics molecular mechanism of mitotic golgi disassembly and reassembly revealed by a defined reconstitution assay reconstitution of the cell cycle-regulated golgi disassembly and reassembly in a cell-free system the role of grasp65 in golgi cisternal stacking and cell cycle progression the ubiquitin ligase hace1 regulates golgi membrane dynamics during the cell cycle sequential phosphorylation of grasp65 during mitotic golgi disassembly mena-grasp65 interaction couples actin polymerization to golgi ribbon linking cyclin-dependent kinase 1-mediated bcl-xl/bcl-2 phosphorylation acts as a functional link coupling mitotic arrest and apoptosis role of microtubules in the organization of the golgi complex identification and characterization of rat 364-kda golgi-associated protein recognized by autoantibodies from a patient with rheumatoid arthritis allosteric regulation of grasp protein-dependent golgi membrane tethering by mitotic phosphorylation structural and functional analysis of a novel coiled-coil protein involved in ypt6 gtpase-regulated protein transport in yeast the localization and phosphorylation of p47 are important for golgi disassembly-assembly during the cell cycle is a p97 adaptor required for golgi and er biogenesis in interphase and at the end of mitosis vcp/p97 extracts sterically trapped ku70/80 rings from dna in double-strand break repair golgi fragmentation precedes neuromuscular denervation and is associated with endosome abnormalities in sod1-als mouse motor neurons adp-ribosylation factors (arfs) and arf-like 1 (arl1) have both specific and shared effectors: characterizing arl1-binding proteins direct selection of monoclonal phosphospecific antibodies without prior phosphoamino acid mapping the multiple facets of the golgi reassembly stacking proteins accumulation of alpha-synuclein/nacp is a cytopathological feature common to lewy body disease and multiple system atrophy golgi fragmentation during fas-mediated apoptosis is associated with the rapid loss of gm130 sod1 mutations causing familial amyotrophic lateral sclerosis induce toxicity in astrocytes: evidence for bystander effects in a continuum of astrogliosis the golgi apparatus. state of the art 110 years after camillo golgi's discovery a direct role for grasp65 as a mitotically regulated golgi stacking factor vcip135 acts as a deubiquitinating enzyme during p97-p47-mediated reassembly of mitotic golgi fragments mapping the functional domains of the golgi stacking factor grasp65 golgi cisternal unstacking stimulates copi vesicle budding and protein transport interactions: a dynamic cycle of p115 binding monomeric snare motifs and releasing assembled bundles a role for the rab6b bicaudal-d1 interaction in retrograde transport in neuronal cells intracellular membrane morphology unraveling the golgi ribbon golgi ribbon disassembly during mitosis, differentiation and disease progression the golgi matrix protein gm130: a specific interacting partner of the small gtpase rab1b src kinase regulates the integrity and function of the golgi apparatus via activation of dynamin 2 a coronavirus e protein is present in two distinct pools with different effects on assembly and the secretory pathway golgin-160 is required for the golgi membrane sorting of the insulin-responsive glucose transporter glut4 in adipocytes the golgin family of coiled-coil tethering proteins carminomycin i is an apoptosis inducer that targets the golgi complex in clear cell renal carcinoma cells structural basis for recruitment of grip domain golgin-245 by small gtpase arl1 grasp55 and grasp65 play complementary and essential roles in golgi cisternal stacking new components of the golgi matrix regulation of protein glycosylation and sorting by the golgi matrix proteins grasp55/65 golgi positioning influenza infection modulates vesicular trafficking and induces golgi complex disruption endoplasmic reticulum (er) chaperone regulation and survival of cells compensating for deficiency in the er stress response kinase, perk atg9 vesicles are an important membrane source during early steps of autophagosome formation functional involvement of tmf/ara160 in rab6-dependent retrograde membrane traffic aggregation of a hepatitis c virus replicase module induced by ablation of p97/vcp peroxisome proliferator-activated receptor delta regulation of mir-15a in ischemia-induced cerebral vascular endothelial injury convergence of cell cycle regulation and growth factor signals on grasp65 acbd3 functions as a scaffold to organize the golgi stacking proteins and a rab33b-gap overexpression of golph3 promotes proliferation and tumorigenicity in breast cancer via suppression of the foxo1 transcription factor cell cycle regulation of vcip135 deubiquitinase activity and function in p97/p47-mediated golgi reassembly grasps in golgi structure and function glycosylation quality control by the golgi structure the golgi stacking protein gorasp2/grasp55 serves as an energy sensor to promote autophagosome maturation under glucose starvation grasp55 facilitates autophagosome maturation under glucose deprivation phosphorylation regulates vcip135 function in golgi membrane fusion during the cell cycle scy1-like 1-binding protein 1 (scyl1bp1) suppressed sciatic nerve regeneration by enhancing the rhoa pathway golgi protein 73 facilitates the interaction of hepatitis c virus ns5a with apolipoprotein e to promote viral particle secretion grasp55 senses glucose deprivation through o-glcnacylation to promote autophagosome-lysosome fusion structural basis for the interaction between golgi reassembly-stacking protein grasp55 and golgin45 the mammalian golgi regulates numb signaling in asymmetric cell division by releasing acbd3 during mitosis regulates golgi apparatus structure through methylation of the golgin gm130 golgi as an mtoc: making microtubules for its own good acknowledgment we thank members of the wang lab for stimulating discussions. this work was supported by the national institutes of health (grants gm112786 and gm105920), mcubed, and the fast forward protein folding disease initiative of the university of michigan to y. wang, and a university of michigan rackham predoctoral fellowship to e. ahat. key: cord-316273-vo6j8zb0 authors: cosset, françois-loic; lavillette, dimitri title: cell entry of enveloped viruses date: 2011-02-08 journal: adv genet doi: 10.1016/b978-0-12-380860-8.00004-5 sha: doc_id: 316273 cord_uid: vo6j8zb0 enveloped viruses penetrate their cell targets following the merging of their membrane with that of the cell. this fusion process is catalyzed by one or several viral glycoproteins incorporated on the membrane of the virus. these envelope glycoproteins (envgp) evolved in order to combine two features. first, they acquired a domain to bind to a specific cellular protein, named “receptor.” second, they developed, with the help of cellular proteins, a function of finely controlled fusion to optimize the replication and preserve the integrity of the cell, specific to the genus of the virus. following the activation of the envgp either by binding to their receptors and/or sometimes the acid ph of the endosomes, many changes of conformation permit ultimately the action of a specific hydrophobic domain, the fusion peptide, which destabilizes the cell membrane and leads to the opening of the lipidic membrane. the comprehension of these mechanisms is essential to develop medicines of the therapeutic class of entry inhibitor like enfuvirtide (fuzeon) against human immunodeficiency virus (hiv). in this chapter, we will summarize the different envelope glycoprotein structures that viruses develop to achieve membrane fusion and the entry of the virus. we will describe the different entry pathways and cellular proteins that viruses have subverted to allow infection of the cell and the receptors that are used. finally, we will illustrate more precisely the recent discoveries that have been made within the field of the entry process, with a focus on the use of pseudoparticles. these pseudoparticles are suitable for high-throughput screenings that help in the development of natural or artificial inhibitors as new therapeutics of the class of entry inhibitors. enveloped viruses have a core incorporating the genetic material of the virus surrounded by a lipidic membrane acquired from the cells they bud from. their genetic material enters target cells following the merging of their membrane with the membrane of the cells at either the plasma membrane or the membrane of another internal compartment. the process of membrane merging is called membrane fusion, which preserves the integrity of the cell membrane. the fusion of two separate lipid bilayers in a nonaqueous environment first requires that they come into close contact. this is followed by an intermediate stage characterized by the merger of only the closest contacting monolayer, a process called hemifusion. third, the fully completed fusion results in whole bilayer merging, followed by the opening of the pore. to achieve this process, the envelope glycoproteins (envgp) have evolved in order to combine two features. on the one hand, they acquired a domain to bind to a specific cellular protein, named "receptor." on the other hand, they developed in a different manner, according to the genus of the virus, a function of fusion that allows the destabilization of the membrane and the opening of a pore through which the genetic material will enter the cell. despite three different classes of fusion protein having been described so far, three common main steps are described for achieving the pre-to postconformational changes. the first one, after envgp activation upon receptor binding or acidification of the endosomal compartment, exposes the fusion peptide that is projected toward the top of the glycoprotein, allowing the initial interaction with the target membrane. the second one is the folding back of the c-terminal region onto a trimeric n-terminal region that leads to the formation of a postfusion protein structure. the final and third step also requires further refolding of the membrane proximal and transmembrane regions in order to obtain a full-length postfusion structure where both membrane anchors (fusion peptide and tm domains) are present in the same membrane. the viral glycoprotein-induced fusion must be controlled to allow the virus to leave the cell, to prevent the aggregation of the viruses, and to release genetic material next to a compartment that will permit the continuity of the infectious cycle. this regulation is executed principally by the inactivation or the masking of the fusion machinery that directly disorganizes the lipid bilayer. on one hand, most of these envgp are synthesized as a precursor that requires a cleavage by a cellular protease (like furin) and prepares the molecules for the subsequent necessary changes of conformation for the fusion process. on the other hand, the activation of the conformational changes is induced by interaction with the receptor and/or the action of the ph (that protonates some amino acids), modifying the interactions of the envgp and their structure. in addition, some proteases or other enzymes are necessary to achieve some complementary priming of the envgp to make it competent for fusion (such as cathepsin b and l). two types of fusion mechanisms can occur, namely, ph independent and ph dependent. in the first case, the recognition between virus and receptor directly triggers conformational changes in the envgp that leads to the direct fusion between the two membranes (viral and plasma) and to the liberation of the viral genetic material. this activation of envgp at neutral ph allows the fusion in vitro and in vivo of envgp-expressing cells with receptor-expressing cells. this fusion leads to the merging of cell cytoplasms and to the generation of multinucleated cells called syncytia. in the second case, for ph-dependent fusion, the interaction between the envgp and the receptor leads to the obligatory endocytosis of the virus-receptor complex, and the acidification of endosomes triggers conformational changes in the envgp. for the ph-dependent virus, such a fusion can be reproduced in cell culture in vitro or in a liposomevirus fusion assay in the tube after decreasing the ph, but cannot occur in vivo. research during the last few years has greatly advanced our understanding of the cell surface receptors for viruses and has provided many surprising insights. these advances were achieved largely by identification and molecular cloning of the cell surface or cytoplasmic proteins that have been subverted for use as viral receptors or cofactor, and by parallel advances in studies of the viral envgp that bind to the receptors. another key area of new insights concerns the physical-chemical process of viral adsorption and of pulling the virus closely onto the cellular membrane. indeed, adsorption is a severely limiting step in infections of cultured cells, and the initial attachment often does not involve the receptors that ultimately mediate infections (andreadis et al., 2000; guibinga et al., 2002; pizzato et al., 1999 pizzato et al., , 2001 ugolini et al., 1999) . thus, we need to distinguish cell surface molecules such as heparan sulfate proteoglycans, dc-sign, or integrins that can enhance infections by concentrating retroviruses onto cells (bounou et al., 2002; geijtenbeek et al., 2000; jinno-oue et al., 2001; mondor et al., 1998; pohlmann et al., 2001; saphire et al., 2001) from authentic receptors that induce conformational changes in envgp that are a prerequisite for fusion of the viral and cellular membranes. in contrast to other cell surface components such as lectins or proteoglycans that influence infections indirectly by enhancing virus adsorption onto specific cells, the true receptors induce conformational changes in the viral envgp that are essential for infection. however, it appears that more and more intracellular proteins have roles in controlling viral host ranges, and the proteins involved in traffic of intracellular vesicles like endosomes, play a critical role in entry. therefore, proteins from pathways specifically characterized might be considered as a cofactor as their role is not to mediate direct contact with envgp but is crucial for entry. in addition, some viruses are able to use more than one endocytic pathway and the cellular proteins involved thus direct the virus to the entry door beneficial for the virus under a particular condition. pseudoparticles are retroviridae cores which incorporate heterogeneous envelope protein from a different virus, possibly of a different family. their entry process mimics precisely that of the wild-type viruses from which the envelope glycoprotein was derived. they represent a useful tool to study molecular processes of envelope viruses as they are very flexible, allowing the analysis of numerous mutants. moreover, they allow a precise measurement of the infectivity that depends on the envelope glycoprotein for the entry step and they allow the establishment of virus-liposome fusion assays. we will first introduce envelope architecture and structure of the viral fusion machineries that have been developed to achieve the fusion and entry steps. despite many differences of structure, they share a common refolding process that activates different fusion domains found in most fusion proteins. we will then illustrate the different fusion regulation processes that have been developed by viruses to lead to functional virions, both in terms of cleavage activation of fusion proteins during exit and in terms of activation during binding and endocytosis. despite some differences between distinct players, some common principles can be proposed for all fusion processes. we will illustrate the molecular details characterizing the maturation of the different fusion proteins, defined by the following three characteristics: the cleavage of an envelope protein precursor, the presence and triggering of the exposition of a fusion peptide, and an association as a trimeric complex association in its active fusion conformation. the progression of these structural rearrangements slows down the kinetic barrier between hemifusion and fusion-pore formation. in a second part, we will present the different entry pathways and cell proteins that are used by viruses to infect cells. viruses have emerged as valuable tools for the study of endocytic mechanisms. these properties have been crucial for the development of pseudoparticles for their use in terms of vector for gene therapy and for their use in terms of tool for receptor cloning, transgenesis, or transduction. finally, we will give examples of strategies that have been developed in vivo and in vitro to inhibit the entry step of the enveloped viruses which have led, in the case of hiv, to the development of inhibitors that are used in the clinic. a. membrane fusion according to the stalk-pore model envgp are responsible for bringing the membranes closer, triggering the link and the destabilization of the outer leaflet, the merging of the whole membrane, and the opening of a pore through the cell and virus membrane to allow the core to enter the cytoplasm of the cell. the hypothesis of the pore model in viral membrane fusion mechanism is supported by experimental results. the first evidence for a hemifusion intermediate was achieved by studying influenza virus entry that occurs after the hemagglutinin glycoprotein binding to the host cell. the substitution of the hemagglutinin transmembrane domain by a glycosylphosphatidylinositol (gpi) revealed the importance of the transmembrane region for the fusion pore opening and expansion. hemifusion structures are connections between outer leaflets of apposed membranes, whereas the inner leaflets remain distinct. this is a transient structure that either dissociates or gives rise to the fusion pore (chernomordik and kozlov, 2008) . interestingly, the helix breaker residues within the tm domain are critical for the fusogenicity of different retroviral env, such as hiv (owens et al., 1994) and mo-mlv (taylor and sanders, 1999) , being important for both the hemifusion and pore opening step of the fusion process. in addition, hemifusion intermediate has been detected in the case of hiv env-mediated fusion (munoz-barroso et al., 1998) by using peptide inhibitors that target a prefusion or prehairpin structure such as hiv-1 gp41 t-20. once the pore is formed, it allows a connection between two compartments initially separated by the apposed membranes. the ability of the membrane to hemifuse and develop a fusion pore has been found to depend on the lipid microdomain composition, for example, cholesterol (chernomordik and kozlov, 2003) . indeed, a potential lipid dependence of virus entry processes was first deduced from experiments on influenza virus, implying a role for detergent-resistant lipidic microdomain (takeda et al., 2003) . for retroviruses, the tm palmitoylations which contribute to the env localization in detergent-resistant lipidic microdomain domains (li et al., 2002) indirectly influence the fusion process (ochsenbauer-jambor et al., 2001) . as an alternative to the lipidic pore hypothesis, a direct fusion mechanism has also been proposed. the fusion pore is a full proteic channel-like structure dependent only on the transmembrane domains of the glycoproteins. in this model, the pore is opened by the joining of two hemipores located on each membrane kozlov, 2005, 2008) . after fusion pore opening and enlargement (melikyan et al., 2005) , the genetic material enters the cytoplasm of the cell, instigating the virus cell cycle. the fusion of the viruses that enter directly at membrane plasmic level (as with the paramyxoviruses and most retroviruses) is triggered by the activation of the viral envelope protein at neutral ph. in this case, only the binding of the virus to its receptor activates the fusogenic potential of the complexes of envgp. this mechanism is ph-independent. in the case of retroviruses, the binding of the surface subunit (su) to its receptor induces conformational changes not only in itself but also in the tm with which it interacts, thus inducing fusion. this activation at neutral ph permits the envelope glycoprotein of ph-independent viruses, in certain experimental conditions or in vivo, to induce the fusion between the cells expressing the envelope glycoprotein and the cells expressing the receptor (harrison, 2008; kielian and rey, 2006; weissenhorn et al., 2007) . this intercellular fusion, by merging their plasma membranes, places the cell cytoplasms in continuity, and one or more cells become giant multinucleated cells named syncytia. in contrast, the fusion of fig. 4 .1 most other viruses depends strictly on their internalization into one of the numerous endocytic pathways such as the clathrin-dependent, clathrin-independent, and caveolae-independent, as well as the macropinocytosis (vaccinia virus) (mercer and helenius, 2008) or the phagocytose (as recently described for equine herpesvirus 1 virus (frampton et al., 2007) and the hiv virus (trujillo et al., 2007) , although in this case not resulting in a productive infection; marsh and helenius, 2006; sieczkarski and whittaker, 2002; figure 4 .1). as described so far, the enveloped viruses that use these itineraries have fusion reactions that require exposure to a moderately acid ph in the different endocytosis vesicles (ph-dependent viruses). classical examples of viruses that fuse at low ph with the membrane of endosomes or artificial membranes in the test tube include the influenza orthomyxovirus, the dengue or the tick-borne encephalitis (tbev) flaviviruses, and the semliki forest alphavirus (sfv) (harrison, 2008; kielian and rey, 2006; roche et al., 2008; weissenhorn et al., 2007) . interestingly, an intermediate mechanism has been described for two retroviruses, asls and jsrv. the proposed mechanism of aslv virion entry occurs in two steps involving a receptor-priming step that induces env conformational changes, thus allowing the env to become sensitive to the action of acid ph (mothes et al., 2000) . this hybrid mechanism does not lead to cell-cell fusion in vivo. jsrv also uses receptor priming for fusion activation of jsrv env at a low ph, but the mechanism differs slightly to aslv, requiring dynaminassociated endocytosis (bertrand et al., 2008) . so far, many different endocytosis pathways have been described (marsh and helenius, 2006; mercer and helenius, 2009; mercer et al., 2010b) as being used by both ph-dependent and ph-independent viruses. however, reinvestigations of these entry pathways are clearly needed for many ph-independent viruses that were originally thought not to rely on endocytosis. for example, nipah paramyxoviruses induce fusion between cells at neutral ph and were considered as a ph-independent virus not reliant on endocytosis for entry. however, recently, it was proposed that nipah viruses (nivs) use macropinocytosis for entry (pernet et al., 2009) . it should be noted that viruses that use a phindependent mechanism of activation of envgp may still enter the cell by endocytosis without any imperative requirement for acidification activation of envgp in the endosomes. clathrin-dependent endocytosis is the best-characterized pathway of entry and is the major itinerary of entry for ph-dependent viruses. this process is initiated by the formation of the characteristic invaginations of membrane, known as clathrin-coated pits (ccp) (fig. 4.1) . the ccp assembly takes place on the internal face of the plasma membrane following a signal induced by the activation of a receptor. this clathrin-dependent method of internalization is used by the semliki forest and sindbis alphaviruses, the rubella rubivirus, and the viruses exploit different endocytosis pathways to enter host cells. multiple mechanisms have been defined as pinocytic, that is, they are involved in the uptake of fluids, solutes, and small particles. these include clathrin-mediated, macropinocytosis, caveolar/raftmediated mechanisms, as well as several novel mechanisms. large particles are taken up by phagocytosis, a process restricted to a few cell types. though poorly documented, the entry of some viruses can be mediated by numerous cargoes which can be endocytosed by mechanisms that are independent of the clathrin coat protein and the fission gtpase, dynamin. these pathways include rhoa-(il-2 pathway), arf6-, and cdc42-regulated pathway (geec pathway). after binding of the particles to the cell surface entry receptor, genetic material is delivered into the cytoplasm of the cell via a specific endocytosis pathway. recently, macropinocytosis has emerged as an important entry pathway for viruses (1). the entry of most ph-dependent viruses is mediated by the use of clathrine-coated endocytic vesicles (2). other viruses penetrate host cells via the formation of vesicles covered by caveola molecule (3). using this pathway, the viral particles are targeted to neutral ph caveosomes or to early endosomes with moderately acid ph. alternately, the virus can use non-clathrin, non-caveolin-dependent pathways, both dynamin-dependent or independent (4, 5, 6). these pathways are differentiated by the implication of different gtpases (rhoa, cdc42, or arf6) and their different compositions (cholesterol, flotillin, tem, etc). hantaan hantavirus, for example (detulleo and kirchhausen, 1998; helenius et al., 1980; jin et al., 2002; kee et al., 2004) . macropinocytosis is another endocytosis pathway utilized by viruses belonging to vaccinia, adeno, picorna, and other virus families. macropinocytosis is an endocytic mechanism normally involved in fluid uptake induced by growth factors, phorbol ester. however, the binding of virus particles can also activate signaling pathways that trigger actinmediated membrane ruffling and blebbing. this is followed by the formation of large vacuoles (macropinosomes) at the plasma membrane, internalization of virus particles, and penetration by the viruses or their capsids into the cytosol through the limiting membrane of the macropinosomes (mayor and pagano, 2007; mercer and helenius, 2009) . a variety of evidences suggest that macropinocytosis can be a mechanism for hiv-1 and epstein-barr herpes virus entry in some primary cells or cell lines (marechal et al., 2001; miller and hutt-fletcher, 1992) . based on the more defined criteria of macropinocytosis (mercer and helenius, 2009) , these conclusions are probably premature and may depend on the experimental conditions that use very concentrated viruses which may influence the utilization of less specific pathways. it should be noted that different strains of the same virus can elicit dramatically different responses in host cells during entry, and different macropinocytic mechanisms are possible in the same cell line through subtle differences in the activating ligand (mercer et al., 2010a ). an additional endocytosis pathway, whilst badly defined, is known as a clathrin-and caveola-independent pathway. the dependence on different gtpase and cargo proteins (dynamin, rho, cdc42, arf6, etc.) defines this endocytosis pathway. several viruses are using this clathrin-and caveolaindependent pathway, including the influenza orthomyxovirus (as a second possible pathway), the sars-cov coronavirus, the lymphocytic choriomeningitis arenavirus (lcmv), and picornaviruses (madshus et al., 1987; matlin et al., 1981; wang et al., 2008) . finally, other vesicles (characterized by caveolin or caveosome, with a high concentration in cholesterol and sphingolipids) are clathrin-independent but dynamin-dependent and do not have acid compartments before their merging with early endosomes (mayor and pagano, 2007) . the nonenveloped sv40 virus and some human enteroviruses are the prototypes of the ph-independent viruses that use this endocytosis pathway. so far, only two enveloped viruses have been described to use this pathway, the newcastle disease paramyxovirus (ndv) (cantin et al., 2007) and ebola virus (empig and goldsmith, 2002; sanchez, 2007) . most viruses have been shown to use only one entry pathway; however, there are more and more recent examples that indicate that some viruses use multiple endocytosis pathways to enter their target cells (influenza, hiv). for example, ebola viruses are known to enter cells by clathrin-mediated endocytosis (sanchez, 2007) , but lipid raft-associated, caveolin-mediated endocytosis has also been proposed as a mechanism of ebola virus uptake (empig and goldsmith, 2002) . in terms of the activation process of the membrane fusion and the need for particular microdomains, it is not always clear why the viruses are using so many different pathways. it is possible that the viruses that evolved to fuse intracellularly have a selective advantage to release their genome to specific intracellular sites that will allow the rapid and efficient establishment of the infectious cycle. one selective advantage could also be the requirement for particular lipid microdomains, like those enriched in cholesterol. indeed, it was shown that contrary to the class ii flavivirus dengue virus and the yellow fever virus, the e1 fusion protein from semliki forest virus (sfv) binds cholesterol which explains its dependence for cholesterol and compartment containing cholesterol (umashankar et al., 2008) . for other viruses, the dependence on cholesterol is linked to bulk effects on membrane fluidity and the maintenance of particular microdomains where receptors are located and where they have some particular diffusion. in addition to these dichotomies of ph-independent or ph-dependent fusion process, of plasma or endosomes membrane fusion, the mechanisms of activation of the fusion proteins of the different enveloped viruses are very diverse. some reactions of fusion are triggered by the interaction of one envelope glycoprotein with a unique receptor. one example is the interaction of the envelope glycoprotein su-tm of the -retrovirus with the multitransmembrane receptor (table 4 .1). in another case, one unique envelope glycoprotein can interact with several receptors. for example, the fusion of hiv-1 is triggered by the sequential interaction of the su gp120 with the cd4 receptor, followed by interaction with a coreceptor such as ccr5 or cxcr4, chimiokine receptors of seven transmembrane domains (hunter, 1997) . previously, it was believed that binding to receptors directly triggered a series of conformational changes in the viral envgp culminating in fusion of the viral and cellular membranes. however, new evidence suggests that -retroviral association with receptors triggers an obligatory cooperative interaction or cross-talk between envgp on the viral surface for hiv and mlv (tailor et al., 2003) . if this intermediate step is prevented, infection fails. conversely, in several circumstances, this cross-talk can be induced in the absence of a cell surface receptor for the virus, in which case, infection can proceed efficiently. this new evidence strongly implies that the role of cell surface receptors in infections of -retroviruses (and perhaps of other enveloped animal viruses) is more complex and interesting than was previously imagined. in all these cases, the envgp have a double function attachment to the receptor and an exclusive role in fusion in the same protein. however, for other viruses, these two functions are filled by different proteins. the e2 protein of the glut-1 neuropilin 1 hs immune recognition (1 tmd) chimiokine receptor (7 tmd) na-pi cotransporter (10 tmd) na-pi cotransporter (10 tmd) na-pi cotransporter (10 tmd) g-protein coupled receptor (10 tmd) cotransport na-a.a. neutres (7 tmd sfv alphavirus binds the receptor that permits the endocytose of the virus in a compartment where the acid ph will activate the e1 protein that will induce the fusion (table 4 .1). for most paramyxoviruses (including newcastle parainfluenza virus, human parainfluenza virus type 3 (hpiv-3), mumps virus, bovine rsv (brsv), ovine rsv (orsv), and human rsv (hrsv)), the hypothesis is that the binding of hn or g/sh to their receptor induced not only conformational changes in the receptor but also in the f protein with which it interacts. these changes make f competent for the membrane fusion, with the exception of the f protein of the simian parainfluenza virus 5 (sv5), the brsv, orsv, and hrsv that do not strictly require hn or g/sh to induce the fusion (though the fusion is greatly increased by hn or g/sh, respectively). this complexity in the distribution of the functions is still more evident in, for example, the alphaherpesvirus (hsv-1, hsv-2) (rey, 2006) . in this case, the binding of the gd glycoprotein to one of its three receptors (hvem for herpesvirus entry molecule mediator, or nectin 1, or a specific heparan sulfate) activates a complex of gb trimers associated to gl and gh proteins, all the three being essential to the fusion (gianni et al., 2006; table 4 .1). in the same way as for the hepatitis c virus, the e1e2 heterodimer induces entry following the recognition of at least four receptors, cd81, srb1, claudin, and ocludine (zeisel et al., 2009) . another interesting variation of activation of fusion is the artificial reverse process. in some cases, it was described that some exosomes or pseudoparticle-incorporating receptors were able to enter cells expressing envelope. for example, the multitransmembrane receptor mcat-1 from ectropic murine leukemia virus (mlv) or the tva receptor for avian sarcoma-leukosis virus (aslv-a) can be used to infect cells that express their respective retroviral envgp (balliet and bates, 1998) . similarly, the incorporation of both cd4 and one other coreceptor of human immunodeficiency virus (hiv), ccr5 (endres et al., 1997) or cxcr4 (endres et al., 1997; mebatsion et al., 1997; schnell et al., 1997) , allows the production of viral particles infecting cells that express simian immunodeficiency virus (siv) or hiv envelope glycoprotein. interestingly, it was shown that nef, an accessory protein of human immunodeficiency virus type 1 (hiv-1) that enhances the infectivity of progeny virions when expressed in virus-producing cells, significantly enhanced the infectivity of cd4-chemokine receptor pseudotypes for cells expressing hiv-1 env. surprisingly, nef also increased the infectivity of hiv-1 particles pseudotyped with tva, even though nef had no effect if the ph-dependent env protein of aslv-a was used for pseudotyping (pizzato et al., 2008) . this process indicates that the difference of superficial tension between the cell (weak because the radius of the cell is big) and the virus (strong as the radius of the virus is small) is not crucial for the development of an oriented fusion process with envgp on the one side and receptor on the other. having said that, the cell is not an empty bubble, and this efficacy of the fusion process regardless of which membrane harbors the receptor or the envgp may reflect the adaptation of the virus to optimize the membrane merging independently of the superficial tension. cells are not completely round, and superficial tension depends on the localization where the virus binds. moreover, the plasma membrane has different lipid compositions and interacts with the cytoskeleton which will attenuate or increase the superficial tension at certain localizations. d. structural classification: class i, class ii, and class iii fusion proteins glycoproteins from enveloped viruses have evolved to combine two main features. first, they have the capacity to bind with a specific cellular receptor and second, they include a fusion domain (peptide fusion and transmembrane domain) that can be activated to mediate the merging (fusion) of viral and cellular membranes. three different classes of viral fusion proteins have been identified to date based on key structural features at pre-and postfusion stages. many studies have demonstrated that the structural transition from a pre-to a postfusion conformation leads to a stable hairpin conformation. this includes class i fusion proteins, characterized by trimers of hairpins containing a central alpha-helical coiled-coil structure, and class ii fusion proteins, characterized by trimers of hairpins composed of beta structures. a third class of fusion proteins has been described recently, that also forms trimers of hairpins by combining the two structural elements alpha-helix and beta-sheet structures (roche et al., 2006 (roche et al., , 2007 . the synthesis and the conformation of these classes i, ii, or iii fusion proteins are different. class i viral fusion proteins of diverse virus families, including retroviridae, filoviridae, orthomyxoviridae, paramyxoviridae, and coronaviridae, differ greatly in size and amino acid sequence, but their membrane-anchored domains share common structural features that are essential for membrane fusion, including two heptad repeats (called hr-1 and hr-2), preceded by a hydrophobic fusion peptide. class i membrane-fusion reaction is mediated by the refolding of the fusion protein to a highly stable rod-like structure with a central trimeric -helical coiled coil. such coiled-coil structures are emblematic of class i proteins, and physical demonstration or computer prediction of such a structure is frequently used to help define a fusion protein as belonging to class i. the envelope glycoprotein of a retrovirus is generated by the cleavage of its precursor in one su and one transmembrane subunit containing an anchoring sequence to the membrane (hunter, 1997) . this maturation, essential to the process of fusion, frees a hydrophobic sequence to the n terminal extremity of the tm, named fusion peptide, as it is supposed to insert into the target membrane and initiates the fusion. initially, the fusion peptide is masked inside the trimer of envgp, a form competent for the fusion (fig. 4.2a ). in general, the cleavage of the fusion proteins is mandatory for most class i proteins to render them competent for fusion (earp et al., 2005; harrison, 2005) . exceptions are the envelope glycoprotein from ebola or sars-cov viruses, classified as class i on the grounds of the three-dimensional structure of one of their fragments, which are not cleaved but yet are functional. though an nterminal fusion peptide is predicted in the potential transmembrane subunit (localized by analogy to homologous viruses), their functionality can be explained also by the presence of an internal fusion peptide (ebola). another explanation of their functionality is their requirement for the l or b cathepsins that cleave the envgp during endocytosis (see below). the number of complexes on the surface of viruses harboring class i fusion proteins is very variable. it seems that lentiviruses have only 10-20 su-tm trimers, whereas the coronaviruses have hundreds of trimers coating the surface, giving them their characteristic morphology of "crown" which is the origin of the name of this family (tables 4.1 and 4.2). the process of assemblage and biosynthesis of class virus ii is very different to the class i proteins (kielian and rey, 2006) (fig. 4.2b) . during biosynthesis, the alphavirus (e1) and flavivirus (e) fusion proteins fold cotranslationally with a companion or regulatory protein, termed p62 (or pe2) for alphaviruses and prm for flaviviruses . this heterodimeric interaction is important for the correct folding and transport of the fusion protein. both p62 and prm are cleaved by the cellular protease furin late in the secretory pathway, in a maturation reaction that is a crucial regulatory step for subsequent virus fusion (salminen et al., 1992; stadler et al., 1997; wengler, 1989; zhang et al., 2003a) . though this cleavage in the envelope glycoprotein complex is important, contrary to class i fusion protein, it is not crucial, since mutated alphavirus and flavivirus for which the regulating proteins are not cleaved have only a decreased infectivity (salminen et al., 1992; zhang et al., 2003b) . in the case of the sfv alphavirus, the fusion process induced by the uncleaved fusion proteins can be a trigger after treatment of the virus at ph5 or less, rather than the normal fusion threshold ph 6 of the wildtype virus (salminen et al., 1992; zhang et al., 2003a) . this variation in the threshold of ph of the fusion is necessary for the dissociation of the heterodimer. the class ii fusion proteins are either homodimers or heterodimers (e homodimer for the flaviviruses or e2-e1 heterodimer for alphaviruses) that form an envelope netting the viral membrane (lescar et al., 2001; rey et al., 1995) . the internal fusion peptide is masked at the interface of the dimers at the extremity of a long beta sheet. one important difference between these two groups of viruses is the budding site . in alphaviruses, the p62-e1 complex is transported to the plasma membrane, and the heterodimer interaction is maintained after p62 processing. new virions bud at the plasma membrane, in a process that is driven by lateral contacts between e2 and e1 heterodimers (e2 being the mature companion protein) to induce the required curvature of the lipid bilayer, in combination with interactions of the cytosolic tail of e2 with the nucleocapsid. budding results in the formation of icosahedral-enveloped particles of triangulation t ¼ 4, containing 80 trimeric e2/e1 spikes. by contrast, flavivirus particles bud into the endoplasmic reticulum as immature virions formed by 60 trimers of prm-e. the immature particles have an organization similar to that of mature alphaviruses, with each trimer forming a of the endosomal compartment, exposes the fusion peptide that is projected toward the top of the glycoprotein, allowing the initial interaction with the target membrane. the second step is the folding back of the c-terminal region onto a trimeric n-terminal region that leads to the formation of a postfusion protein structure. the third and final step also requires further refolding of the membrane proximal and transmembrane regions in order to obtain a full-length postfusion structure where both membrane anchors (fusion peptide and tm domains) are present in the same membrane. three different classes of fusion have been identified so far based on common structural motives. (a) the class i fusion proteins are characterized by trimers of hairpins containing a central alpha-helical coiled-coil structure. for retroviruses, receptor binding induces the movement of the su, allowing a loop-to-helix transition of a polypeptide segment of tm that was previously buried underneath the su heads, projecting the fusion peptide 100 å toward the target membrane, where it inserts irreversibly. this occurs by a "spring-loaded" mechanism. the hr2 c-terminal end (green) of the long tm -helix jackknifes back, reversing the direction of the viral-membrane-proximal segment of tm, which then interacts in an antiparallel fashion with the groove formed by the n-terminal hr1 (blue) trimeric coiled coil. the final postfusion conformation of tm is, therefore, a highly stable rod with the tm and fusion-peptide segments together at the same end of the molecule, a structure termed a "trimer of hairpins" or helix buddle (hb). (b) class ii fusion proteins are characterized by trimers of hairpins composed of beta structures. the red, yellow, and blue parts of each subunit correspond, respectively, to domains i, ii, and iii of the ectodomain. the fusion loop is at the tip of domain ii. monomeric transition between the prefusion dimer and the trimeric-extended intermediate is shown. after exposure to the low ph of the endosomes, domains i and ii swing outward, while domain iii and the stem remain oriented against the membrane roughly similar to the prefusion state. the fusion loop, at the top of the diagram, interacts with the target bilayer. domains i and ii associate into the trimeric core of the postfusion conformation, and domain iii must then zip back along the trimer core, thus reorientating the domain iii. (c) a third class of fusion proteins has been described recently, which also forms trimers of hairpins by combining the two structural elements alphahelix and beta-sheet structures. class iii fusion proteins are composed of five domains that give rise to a molecular architecture very distinct from any reported class i or class ii fusion proteins. interestingly, the ectodomain of g has been crystallized in its pre and postfusion (low-ph) state. during the conformational change that occurs upon low ph exposure, the domains of g radically change their position and orientation as a result of rearrangements that occur in the linker regions. domain i (yellow), carrying the fusion loops, and the transmembrane domain move 16 nm from one end of the molecule to the opposite ). only domain iii (blue) undergoes significant refolding with extension of the central helix f. to complete the process, the c-terminal helices of domain iv (red) insert into crevices formed by two other protomers in the postfusion form, reminiscent of the structural changes observed during refolding events of class i fusion proteins with hb formation. spike in which prm covers the fusion protein e. the newly formed virions are then transported to the external milieu through the exocytic pathway. processing of prm generates the mature m protein with a short ( 40 residues) ectodomain (kuhn et al., 2002; zhang et al., 2003b) . presumably because of the removal of a large portion of the prm ectodomain, the flavivirus surface dramatically reorganizes after processing to give 90 e-e homodimers arranged with icosahedral symmetry. the mature flavivirus particles display a smooth, spikeless surface, with e dimers ordered in a characteristic "herring bone" pattern. there is no correlation between these criteria for class i and class iii fusion proteins, but all the viruses harboring class ii fusion proteins are ph-dependent. finally, the four class iii fusion proteins resolved (vsv-g, gb of hsv1 and ebv and baculovirus gp64) in spite of their homology of trimeric structure have some major functional differences arising from their difference of biosynthesis. g protein is the only class iii fusion protein whose prefusion structure is known. the proteins of class fusion iii have a combination of the two structural elements, alpha helix like class i and beta sheet like class ii heldwein et al., 2006; kadlec et al., 2008; roche et al., 2006 roche et al., , 2007 (fig. 4.2c) . the trimers are maintained by interaction between central alpha helixes, but each domain of fusion exposes two buckles of internal fusion peptide placed at the extremity of a long beta sheet. the g protein is the only protein responsible for the binding and the entry of the vsv. the rhabdovirus vsv possesses 1200 molecules of the g protein on its surface and forms 400 trimers. in contrast, the hsv-1 virus incorporates 12 different envgp on its surface, of which 4 are essential for the entry step (gd, gb, and gh/gl) (turner et al., 1998) . during their biosynthesis, neither the g protein of vsv nor gb of hsv-1 are cleaved, and they present internal fusion peptides. however, whereas the g is functional by itself, the gb envelope glycoprotein alone is not sufficient to induce the entry of the virus or the membrane fusion. nevertheless, currently, no precise interaction between gd, gb, and gh/gl has been identified, even though the gd ectodomain has been shown to allow the entry of engineered hsv-1 virus particles that lack gd (that is gd-null mutants; cocchi et al., 2004) . gp64 is the major component of the viral envelope, and the sole fusogenic proteins that are triggered to induce the fusion in the low ph environment of endosomes for baculovirus. interestingly, the distinguishing feature between g and gp64 and any other fusion protein is that they can undergo a reversible conformational change, unlike class i and most class ii fusion proteins, for which the postfusion conformation is thermodynamically more stable at all ph values, and the conformational rearrangement is effectively irreversible. vsv or gp64 exposure to low ph inactivates the virus, but the fusion activity can be fully recovered when the ph is raised. it has been proposed that the reversibility of the conformational change allows g to avoid unspecific activation during transport through the acidic golgi vesicles. as seen in the previous sections, though there is much described variation in the manner of activation of the fusion proteins and additional mechanisms await discovery, only three classes of fusion proteins have been defined so far (weissenhorn et al., 2007) . as previously described, based on the main structural organization of their envgp, the viruses are now assigned to the class i, ii, or iii. in parallel, based on the activation mechanism of the fusion protein, viruses have been classified as ph-dependent or -independent. interestingly, the relationship between the mechanism of activation of the fusion and the class of protein of fusion is not correlated. the hiv or influenza envgp are both class i and yet they have a process of activation that is ph-independent and -dependent, respectively, for the entry of the virus. similarly, the class iii fusion proteins enclose the herpes virus simplex 1 (hsv-1) gb protein and the rhabdovirus vsv-g protein that are ph-independent and ph-dependent, respectively, for their activation. so far, all the class ii fusion proteins have been phdependent for their activation. although there are notable differences between the activation processes, the structural motives used, and the initial oligomeric states of the fusion proteins (the native trimeric conformation of class i and iii proteins in opposition to the homo-or heterodimers of the class ii fusion proteins), the common features of the final structures obtained after fusion seem to suggest some generic mechanism of conformational changes common to all envgp of enveloped viruses (see fig. 4 .2). first, the activation of the fusion protein following the interaction with the cellular receptor(s), coupled or not to the exposure to the acid environment of the endosomes, exposes the fusion peptide that is projected toward the top of the glycoprotein, allowing the initial interaction with the cellular target membrane. for class i fusion proteins, the proposed model indicates that the transition of conformation requires the transformation of a part of the molecule in alpha helix and the association of this in three helixes bundle (named the "coiled coil"; weissenhorn et al., 2007) . for retroviruses, this movement allows a loopto-helix transition of a polypeptide segment of tm that was previously buried underneath the su heads, projecting the fusion peptide 100 å toward the target membrane, where it inserts irreversibly. in the case of class i fusion proteins like retroviruses, this occurs by a "spring-loaded" mechanism. this initial change is proposed to result in a "prehairpin intermediate," an extended structure that is anchored both in the target membrane by the fusion peptide and in the virus membrane by the tm transmembrane segment. for the class ii fusion proteins, the projection of the fusion peptide requires the dissociation of the hetero-or homodimers and modifications in the "hinge region" unstructured before conformational changes (stiasny and heinz, 2006) . for the class iii (roche et al., 2008) , similarly to the fusion proteins of class i, the exhibition of the fusion peptide requires a rearrangement of the domains mediated by modification of the central helixes that do remain parallels. second, the folding back of the region including the fusion peptide onto a trimeric c-terminal region leads to the formation of a postfusion protein structure with the outer regions zipped up against an inner trimeric core. interestingly, it has been described that all the class i, ii, and iii peptides can inhibit this step by competing with the interaction of envgp-specific domain for the formation of this structure. third, the final steps require further refolding of the juxtamembrane and transmembrane regions to obtain a stable postfusion structure with the fusion peptides and the transmembrane domains at the same extremity of a stable stem of a protein complex anchored in the target membrane. this structure brings the two membranes proximal and provides free energy to overcome the barrier of membrane merging (melikyan, 2008) . membrane fusion occurs, which leads to pore formation and release of the viral genome into the cytoplasm. the refolding of class ii fusion proteins generates trimers from monomeric intermediate. the existence of monomeric intermediates for class i and iii is not well known. however, if the steps that led to exposition of the fusion peptide and its interaction with the membrane targets maintain a three-order symmetry, the refolding of the c-terminal region requires the destruction of the trimer at least to the juxtamembrane region. moreover, from the differences observed between the amino acids involved in the interface of the reconstituted resolved trimers in their pre-and postfusion conformations, it is probable that the conformational changes are going through a monomeric intermediate for the class iii g envelope glycoprotein of vsv and the class i f protein of paramyxovirus. on the contrary, the interface of the class i ha2 subunit of the ha envelope glycoprotein trimer is very similar between the pre-and postfusion conformation, shedding doubt on the existence of monomeric intermediates. in contrast, when fusion is initiated at low ph, the dimers of the class ii fusion proteins are broken, freeing monomers that reassociate in trimers. finally, precise structural information of the native metastable conformation (prefusion) and the final stable conformation (postfusion) is available only for a limited number of viruses (for envelope glycoprotein of influenza virus, sfv, tbev, vsv, and the parainfluenza 5 f protein). the structural conversion of the native metastable conformation in a final stable conformation is not precisely known and is highly speculative for most viruses, and the envelope glycoprotein domains implied in these molecular rearrangements are littlereferenced. clearly, more studies are necessary to identify the intermediaries of envelope glycoprotein conformations. these intermediates would identify the domains that interact during the conformational changes which will highlight ways to generate inhibitory peptides. f. domain organizations/fusion domains 1. acquisition of fusion competence: priming by cleavage during virus production, the host cell is basically preserved, since the expression of fusogenic competent glycoproteins is highly controlled for most viruses. however, for some viruses, the envgp induce a cytopathic effect that leads to the generation of multinucleated cells, called syncytia, which are induced by the fusion of cells that express the envelope glycoprotein and receptors. abundant glycoprotein at the surface of the cell could induce cellular death by syncytia formation, toxicity via receptor interaction, or immune recognition. for these reasons, the localization and the amount of the oligomerized envelope glycoprotein at the host cellular surface are highly modulated by fine trafficking and sequestration mechanisms. the receptor interference mechanism (either by saturation of binding site on receptor by envelope glycoprotein or by internalization of receptor by different viral proteins, as it is achieved by some retroviruses; hunter, 1997) can also limit the amount of receptors available for fusion between infected cells. the control of the cleavage of the envgp to free the fusion peptide is also a regulation process of the fusion protein fusogenicity (labonte and seidah, 2008) . finally, envgp fusion competency may be a late event that occurs during virus budding, as described for mulv retroviruses (rein et al., 1994) . proteolytic priming is a common method of controlling the activation of membrane fusion mediated by viral glycoproteins. the members of the proprotein convertase (pc) family play a central role in the processing and/or activation of various protein precursors involved in many physiological processes and various pathologies such as neurodegenerative pathology, cancer bacterial toxins activation, and viral infections. the proteolysis of these precursors that occurs at basic residues within the general motif (k/r)-(x)-(k/r) is mediated by the proprotein convertases pc1/3, pc2, furin, pace4, pc4, pc5 (also called pc6), and pc7 (also called pc8, lpc, or spc7), whereas the proteolysis of precursors within hydrophobic residues performed by the convertase s1p/ski-1 and the convertase narc-1/pcsk9 seems to prefer to cleave at a lvfaqsip motif (lahlil et al., 2009 ). the seven pcs have different, albeit partly, overlapping expression patterns and subcellular localization. they have conserved aminotermini with highest homology in the subtilisin-like catalytic domain. data on various infectious viruses revealed that the cleavage of their envelope glycoprotein precursors by one or more pcs is a required step for the acquisition of the infectious capacity of viral particles. indeed, various studies have demonstrated the capacity of the pcs to correctly cleave a variety of viral surface glycoproteins. these include the hiv-1 gp160 (decroly et al., 1996) and surface glycoproteins of hong kong, ebola virus, the severe acute respiratory syndrome coronavirus and chikungunya virus (basak et al., 2001; bergeron et al., 2005; ozden et al., 2008) . in parallel, other studies revealed that the inhibition of processing of these viral surface glycoproteins by the pc inhibitors such as dec-r-v-k-r-cmk completely abrogated the virus-induced cellular cytopathicity. the surface glycoproteins of other viruses, particularly the hemorrhagic fever viruses (arenaviridae family), such as lassa (basak et al., 2002; lenz et al., 2001) , crimean congo hemorrhagic fever (vincent et al., 2003) , and lymphocytic choriomeningitis (beyer et al., 2003) , were shown to be cleaved by the convertase ski-1 that cleaves at hydrophobic residues. similarly, blocking of ski-1 activity by a specific inhibitor has also shown to affect the processing and the stability of the glycoproteins of these viruses (pullikotil et al., 2004) . for all highly pathogenic avian influenza (hpai) viruses of subtypes h5 and h7 known to date, the cleavage of ha occurs at the c-terminal r residue in the consensus multibasic motifs, such as r-x-k/r-r with r at position p4 and k-k/r-k/t-r with k at p4, and leads to systemic infection. early studies demonstrated that the ubiquitously expressed furin and proprotein convertases (pcs 5 and 6) are activating proteases for hpai viruses (basak et al., 2001; stieneke-grober et al., 1992) . recently, ubiquitous type ii transmembrane serine proteases, mspl and its splice variant tmprss13, have been proposed as novel candidates for proteases processing ha proteins of hpai (okumura et al., 2010) . is it interesting to note that viral receptors can also be modified by proprotein convertase. indeed, pcsk9 impedes hepatitis c virus infection in vitro, modulates liver cd81 expression, and enhances the degradation of the low-density lipoprotein receptor (ldlr) (labonte et al., 2009) , bestowing on the proprotein convertase an additional role in controlling the fusogenicity of the envelope glycoprotein. the exhibition and insertion of a hydrophobic fragment of 10-30 residues in the membrane, named "fusion peptide" or "fusion loop" is a crucial step of the fusion process (epand, 2003) . the fusion peptides in an n-terminal position (such as for the retrovirus or the influenza virus) is liberated for most viruses after envelope glycoprotein cleavage, and it can insert into the external layer of the membrane in an oblique manner, whereas the fusion loop (for the class ii and iii viruses) remains probably more superficial (see table 4 .3). the fusion peptide of the class i and ii proteins is initially buried in the envelope glycoprotein trimer or dimer, respectively. for the class iii, the fusion loop is present outside of the structure, most likely because the fusion peptide of class iii fusion proteins is weakly hydrophobic and probably requires a cooperation between several loops to be functional and efficient. the simple picture of a viral fusion protein acting on cell and viral membranes by means of only two restricted segments, that is to say, the fusion peptide and the transmembrane domain, is too simplistic. instead, a more complex concerted action of different membranotropic segments of the fusion proteins is necessary. more conformational changes are required to achieve a complete fusion of the two lipid bilayers. as described previously, the class i-iii fusion proteins roughly share common refolding processes and formation of intermediates. several regions of the fusion protein complex indirectly aid the fusion process, as for example, the "stem" regions (see below). contrary to the relatively simple and canonical organization of the fusion peptides for the influenza virus or the flavivirus e protein, the recently resolved structures of the gb glycoprotein of the simplex herpes type 1 virus (heldwein et al., 2006) and g protein (roche et al., 2006 (roche et al., , 2007 of the vesicular stomatitis virus (vsv) indicated a bipartite fusion peptide composed of two hydrophobic loops, each loop being relatively nonpolar or very weakly hydrophobic (which rarely leads to the identification of a fusion peptide by fusion peptide prediction based on hydrophobic domain identification). these differences in the organization of the fusion peptides suggest differences of action of the fusion proteins, notably in the number required. according to experiments using neutralization assays, it seems that one hiv envelope glycoprotein is capable of inducing membrane fusion. however, it has been shown that the induced fusion by ha requires a collaboration of several complexes of envelope glycoprotein (8-9 trimers). in the same way, different networks of class ii fusion proteins have been proposed, such as the hexagonal organization observed on the surface of liposomes or an association of five trimers in a structure similar to a volcano based on structure predictions (stiasny and heinz, 2006) . concerning the fusion protein of class iii, a hexagonal structure has been proposed for rabies virus envelope glycoprotein and recently for vsv-g according to a modeling using its structure. the requirement for multiple fusion peptides may be compared to the number of receptors required. the assembly of a complex containing several receptors may be a prerequisite for the membrane fusion steps that require multiple envgp molecules to cooperatively participate in this process. for example, in the case of hiv-1, the presence of more than one cd4 in contact with the virus enhances the infectivity dramatically and reduces the concentration of coreceptors needed for infection (platt et al., 1998) . further investigation of this system has implied that a critical complex containing approximately four to six coreceptors is a requirement for infection, although it is not known whether this complex performs a transient role and then disperses or is maintained throughout the membrane fusion process (kuhmann et al., 2000) . despite some uncertainties, several lines of evidence have suggested that three to six hemagglutin trimers may cooperatively participate in the influenza a virusmediated membrane fusion reaction (blumenthal et al., 1996; boulay et al., 1988) and that multiple envelope glycoprotein trimers are required for rabies virus-mediated membrane fusion (roche and gaudin, 2002) . see table 4 .3. the cytoplasmic tails of envelope viruses harbor different motifs that are responsible for its trafficking and are variable in length. it is surprising to see that the cytoplasmic tails of fusion proteins are not exchangeable. for example, when the hcv e1 and e2 cytoplasmic tails or the f cytoplasmic tails of hrsv (human respiratory syncytial virus) are substituted for that of vsv-g envelope glycoprotein (or cd4), the fusogenicity of these envelopes in cell-cell fusion assays and virus-cell assay (infection) is destroyed (buonocore et al., 2002; oomens et al., 2006) . similarly, when the ha glycoprotein is anchored by a gpi, the entry process is stopped at the hemifusion step (kemble et al., 1994; markosyan et al., 2000) . the cellular localizations (e.g., cholesterol-rich microdomains) are sometimes modified, and biochemical modifications (glycosylation, oligomerization) can affect the properties of the vsv-g envgp (kemble et al., 1994) . nevertheless, in their initial context, these cytoplasmic tails allow fusion. for some viruses, regulation of fusion is mediated by the cleavage of the cytoplasmic tail. for -retrovirus, in addition to the su-tm cleavage, a significant fraction of virion-associated tm is further processed by the viral protease removing the c-terminal 16 amino acids of the cytoplasmic domain, the r peptide. -retrovirus virions assemble and bud from infected cells as immature particles that must undergo an additional proteolytic maturation to become infectious (green et al., 1981; lavillette et al., 1998 lavillette et al., , 2002 rein et al., 1994) . this maturation concerns the viral protease-dependent cleavage of the so-called peptide r at the c-terminus of the cytoplasmic tail. the r peptide inhibits fusion, and different hypothesis have been proposed. first, the r peptide contains an y-x-x-internalization motif, and the removal of this motif following the cleavage of the r peptide might result in a higher amount of envelope at the surface membrane and thus more fusion (song et al., 2003) . second, following the r peptide cleavage, the remaining cyt tail forms a membrane-embedded amphiphilic alpha-helix domain that destabilizes the membrane (rozenberg-adler et al., 2008; zhao et al., 1998) . third, it has been proposed that as the r peptide contains a palmitoylation, its removal induces the close trimerization of the cyt tail and drastic conformational changes in the ectodomain of env (aguilar et al., 2003) which might influence env fusogeneity by destabilizing su-tm complexes. these conformational changes are necessary for the isomerization of the su-tm disulfide mlv env (loving et al., 2008) . this r peptide cleavage is the last step leading to a fusion competent infectious mlv retrovirus, but this final modification does not exist in lentiviruses which harbor a long cytoplasmic tail. however, truncations of the long cytoplasmic domains of lentiviral env proteins occur under certain culture conditions (chakrabarti et al., 1989) and increase env fusogenicity in a similar way to mutated truncated versions of siv, hiv-1, and hiv-2 envs (mulligan et al., 1992; spies et al., 1994; wilk et al., 1992) . this regulation is a hallmark of adaptation of endogenous retroviruses, as this cleavage is fulfilled by a cellular protease that activates the endogenous envgp herv-w in relevant tissues involved in placenta development. it is interesting to note that for most viruses (and not only the -retrovirus), the truncation of the cytoplasmic tail increases the fusogenicity of the envgp, as is seen in paramyxoviruses (moll et al., 2002) . in many cases, this truncation seems to increase the cell surface expression (since cellular trafficking signals in the cytoplasmic tails are modified), which may explain the increase of fusogenicity. for certain other env, the truncation leads to conformational changes in the ectodomain which lowers the activation threshold for fusion, resulting in enhanced env fusion activity and kinetics (aguilar et al., 2003; cote et al., 2008; spies et al., 1994; zhao et al., 1998) . finally, the cleavage of the cytoplasmic tail sometimes allows, at least partially, a cell-cell fusion at neutral ph (ph-independent), although the entry of the virus remains ph-dependent. the notion of phdependence, seems in this case, to be due to a specific conformation or a particular density of envelope glycoprotein . numerous studies on several viruses have highlighted the critical role of the pretransmembrane sequence (ptm), also called the membrane proximale region or the juxtamembrane domain (jmd), which is rich in aromatic amino acids. the class i fusion glycoproteins of coronavirus, lentivirus (hiv, siv, fiv), ebola virus (munozbarroso et al., 1999; saez-cirion et al., 2003) , and many other viruses contain this short jmd region in the ectodomain between the end of hr-2 and the beginning of the transmembrane (tm) domain . the class ii (sfv, dengue, tebv) and class iii (vsv and the herpes virus) fusion proteins also possess these regions, although they are less rich in tryptophan than the class i jmd (jeetendra et al., 2002 (jeetendra et al., , 2003 roche et al., 2008) . although the jmds of fusion proteins of enveloped viruses are rich in aromatic amino acids, the number, spacing, and sequence of the aromatic amino acids are quite variable; however, the function remains the same. these jmds contribute to the conformational changes that occur during membrane fusion, interact with membranes, induce membrane destabilization, and/or facilitate membrane fusion (munoz-barroso et al., 1999) . therefore, the jmd of viral fusion proteins is a potential target for viral inhibitors. entry inhibitors that target the jmd of class i fusion proteins include monoclonal antibodies that bind the jmd of gp41 to the fiv and to the hiv (lorizate et al., 2006; purtscher et al., 1994) . some peptides that mimic the jmd have been designed for envgp of fiv (giannecchini et al., 2004) , hiv (moreno et al., 2006) , ebola virus (saez-cirion et al., 2003) , and the sars virus (howard et al., 2008) and inhibit viral entry. this strategy has been also broadly used against many other paramyxoviruses such as the sendai virus (joshi et al., 1998; rapaport et al., 1995) , the newcastle disease virus (young et al., 1999) , the human parainfluenza type 3 (hpiv-3) (yao and compans, 1996) , the respiratory syncytial virus (rsv), and the measles virus (mv) (lambert et al., 1996) . in the same way, peptides that mimic the jmd from class ii and iii fusion proteins have also been developed against infection by dengue virus (hrobowski et al., 2005) and cmv (english et al., 2006; lopper and compton, 2004) . in the case of hcv, some juxtamembrane domains have been proposed in e1 and e2 (drummer and poumbourios, 2004; drummer et al., 2007) . high-throughput screening (hts) of peptides derived from e1 and e2 sequences has identified inhibitory peptides close to the transmembrane domain of e2, though they are not among the most inhibitory (cheng et al., 2008) . however, these strategies suffer from certain limitations. the derived peptides of these jmd often have the capacity to oligomerize, which inactivates them, and some stratagems need to be implemented to make the peptides more bioreactive against viruses. moreover, these peptides that prevent the correct conformational change of the envelope glycoprotein, must act in a certain window of time and in a particular compartment compatible with their active structure. indeed, the acid ph of some compartment is not compatible with the bioactive structure of the peptide. for the sars-cov virus, the peptide is able to inhibit the entry of the virus in the presence of protease at the cellular surface, but the peptide has little effect on the entry of the virus by endocytosis or on the activation by cathepsin l in the acid conditions of the endosome (ujike et al., 2008) . in the case of influenza, it has not been possible to develop such inhibitory peptides targeting the juxtamembrane domain. reasons suggested for this include the entry through acid compartments which modify protein structures, the entry by different pathways of endocytose, and the rapidity of the conformation changes of ha. some modifications can be made to these peptides to increase their efficiency for viruses that fuse at the plasma membrane. for three paramyxoviruses, hpiv-3, a major cause of lower respiratory tract diseases in infants, and the emerging zoonotic viruses hendra virus (hev) and niv, which cause lethal central nervous system (cns) diseases, the addition of cholesterol to a paramyxovirus hrc-derived peptide (derived from the heptad repeat immediately preceding the transmembrane domain) increased antiviral potency by 2 log units (porotto et al., 2010) . this enhanced activity is the result of the targeting of the peptide to the plasma membrane. the cholesterol-tagged peptides on the cell surface create a protective antiviral shield, target the f protein directly at its site of action, and expand the potential utility of inhibitory peptides for paramyxoviruses. the definition of a receptor is very complicated and has limitations. it is sometimes difficult to distinguish between "simple" receptors that mediate adsorption or binding and that may not even initiate conformational changes, and "critical" receptors for fusion which, upon binding, will generate the conformational changes that will allow the exposure of the fusion peptide and lead to membrane fusion. some cellular molecules are also involved in the localization or trafficking of viral receptors and these are important cofactors of entry. another ambiguity is the role of enzymatic activities. some are necessary to process the fusion protein inside producer cells, or inside the endosomes of target cells, and they are necessary to activate the potential of the envgp for membrane fusion. while it is inaccurate to consider them as "receptors", they are certainly critical cofactors. all viruses likely bind at least weakly to multiple cell surface components such as heparan sulfate proteoglycans, dc-sign, integrins, or glycolipids (bounou et al., 2002; cantin et al., 1997; fortin et al., 1997; jinno-oue et al., 2001; mondor et al., 1998; saphire et al., 1999 saphire et al., , 2001 . although such binding substances probably do not induce conformational changes in envgp that are necessary for membrane fusion, they can enhance viral adsorption and substantially increase efficiencies of infections, thus contributing to pathogenesis (alvarez et al., 2002; bounou et al., 2002; geijtenbeek et al., 2000; jinno-oue et al., 2001; saphire et al., 2001) . because such binding proteins contribute to infections, it can be difficult to unambiguously distinguish them from receptors that directly mediate the membrane fusion process, especially for viruses that bind to their authentic receptors only weakly (e.g., for retroviruses, in the cases of felv-t or polytropic mulvs; anderson et al., 2000; marin et al., 1999; temin, 1988) . we emphasize this because pathogenic variants of different animal viruses have often been associated with abilities to bind to apparently novel cell surface components, and it has sometimes been inferred that the viruses have switched their receptor specificities. in these instances, it has generally not been established that the cell surface binding components are receptors that directly mediate infections. in the case of ebola, for example, the receptor(s) that mediates its entry has yet to be definitively identified. c-type lectins such as dc-sign and dc-signr are thought to serve as adherence factors for ebola or marburg virus (marzi et al., 2006; matsuno et al., 2010) . other plasma membrane-associated proteins have been implicated in ebov uptake, including folate receptor alpha and the tyrosine kinase receptor axl (chan et al., 2001; shimojima et al., 2006 shimojima et al., , 2007 sinn et al., 2003) , but the physical interaction of ebov gp and these proteins has not been demonstrated, and cells that do not express these proteins are permissive for ebov gp-mediated virion uptake. some previous studies have implicated the actin cytoskeleton in ebov entry, where agents such as cytochalasin d and swinholide a that impair microfilament function, inhibited gp-mediated entry (yonezawa et al., 2005) . similarly, vsv was shown to bind ubiquitously to cells via phosphatidylserine (ps) (schlegel et al., 1983) . however, a more recent study reports that ps is not a receptor for vsv, as no correlation was found between cell surface ps levels and vsv infection, and annexin v, which specifically binds ps, did not inhibit infection of vsv (coil and miller, 2004) . therefore, the cell surface receptors for vsv have not been identified, but it is generally thought that binding via the g-protein is rather unspecific and involves negative charges on the plasma membrane (carneiro et al., 2006; coil and miller, 2005) . adsorption of viruses onto cultured cells from the medium is usually a very slow and inefficient process, principally because of the slow rates of their diffusion into contact with the cell surfaces (allison and valentine, 1960; andreadis et al., 2000) . in general, the rate of contact cannot be significantly enhanced by mixing or stirring, because the boundary layer of the relatively stationary fluid that surrounds walls or other large objects (e.g., cells) in flowing liquids is substantial compared with the rate of virus diffusion, hence stirring does not increase the concentration of virus surrounding this boundary zone (allison and valentine, 1960) . in the case of retroviruses, it has become especially clear that adsorption is a severely limiting step in infection of cultured cells. in classic studies in which virus samples were incubated with cells for several hours before washing with fresh medium and subsequently detecting the foci of infection, it was estimated that only 1/1000 or fewer of the virions in the medium were infectious. in contrast to previous interpretations, studies suggest that this low infectivity-to-virion ratio is principally caused by the inefficiency of adsorption (andreadis et al., 2000) . accordingly, serial incubation of a virus-containing medium for 2-h periods with sequential cell cultures results in the same titers in each of the cultures after correction for spontaneous viral decay (kabat et al., 1994) . furthermore, centrifuging the virus down onto the cultured cells (i.e., spinoculation) often increases retroviral titers by 1-2 log orders of magnitude (bahnson et al., 1995) . studies aiming to count retrovirions adsorbed onto cell surfaces by confocal immunofluoresence microscopy or by quantitative pcr methods (marechal et al., 2001; pizzato et al., 1999 pizzato et al., , 2001 have demonstrated that receptors for viral entry are irrelevant for initial adsorption of retrovirions onto surfaces of most cells (pizzato et al., 1999 (pizzato et al., , 2001 . on the contrary, the initial steps of virus attachment seem to more critically depend on accessory cellular binding substances, such as heparin sulfates, integrins, or lectins, including dc-sign (bounou et al., 2002; guibinga et al., 2002; jinno-oue et al., 2001; mondor et al., 1998; saphire et al., 2001) . by forming multivalent weak reversible bonds with such abundant cell surface components, a virus would become efficiently bound in a manner that would allow it to "graze" until it makes appropriate contact with a true receptor (haywood, 1994; park et al., 2000) . it is well known that for several viruses (rubella togavirus, bvdv pestivirus, newcastle disease paramyxovirus, hiv lentivirus, mouse hepatitis coronavirus (mhv)), rearrangements of the thiol content and of the disulfide bridges, induced by thioredoxine or protein disulfide isomerase (pdi), are essential to induce some big conformational changes necessary for the membrane fusion (fenouillet et al., 2007) . interestingly, the mlv and htlv retroviruses developed an "internal" oxydoreduction activity by adapting a catalytic motif involved in disulfure bridge isomerization. the two su and tm subunits can be linked in either a covalent or noncovalent manner. for hiv-1, the existence of the soluble gp120 protein indicates a noncovalent link between su and tm (kowalski et al., 1987) . however, for most other retroviruses, a covalent link was described at one stage. in all cases, with the exception of mmtv and jsrv, a disulfide bond between the su and the tm is formed between the highly conserved cx6cc motif of the tm and the cxxc of the su (pinter et al., 1997; schulz et al., 1992; sitbon et al., 1991) . this cxxc motif is extremely rare in cellular proteins and is similar to a motif found in the catalytic site of enzymes involved in thiol isomerization, like pdi or thioredoxin (pinter et al., 1997; sanders, 2000) . this motif in the su has been shown to be part of an autocatalytic isomerization function of su to destroy the initial bond between su and tm generated during env synthesis and create an intra-su bond inside the cxxc motif wallin et al., 2004) . this disulfide bond isomerization is crucial for the fusogenicity of -retrovirus (mlv) (fenouillet et al., 2007) . some viruses use the protease activity of particular cellular enzymes localized in the endosomes or at the cellular surface for activating their envgp. the ebola filovirus (for which the gp1-gp2 envelope glycoprotein is cleaved by the furine in the producer cells) (chandran et al., 2005; schornberg et al., 2006) , the hev (pager and dutch, 2005) , niv (diederich et al., 2009) , or the sars-cov and mhv-2 coronaviruses (for which the s spikes is not cleaved in the producer cells) (huang et al., 2006; qiu et al., 2006; simmons et al., 2005) require the cysteins lysosomal proteases, the l or b cathepsine (catl or catb) for their entry process. after virus uptake following angiotensin-converting enzyme 2 receptor binding, cathepsin l-mediated proteolysis induces conformational changes in the sars-cov s glycoprotein to trigger the endosomal membrane fusion process (simmons et al., 2005) . likewise, cleavage of the ebola glycoprotein by catl cleavage removes a glycosylated glycan cap and mucin-like domain (muc domain) and exposes the conserved core residues implicated in receptor binding (chandran et al., 2005; hood et al., 2010; lee et al., 2008; schornberg et al., 2006) . entry of this virus is ph-dependent and associated with the cleavage of gp by proteases, including catl and/or catb, in the endosome or cell membrane, which is required for entry into the host cell. however, the precise role of the cleavage of ebola envelope glycoprotein, which is already cleaved intracellularly during its exit, is uncertain. the cleavage of the gp to a stable form of 18 kda of gp1 may increase binding, suggesting that the cleavage facilitates the interaction with a cellular receptor (dube et al., 2009; kaletsky et al., 2007) . another possibility is that the catb cleavage is required to facilitate the triggering of viral membrane fusion by destabilizing the prefusion conformation of ebola envelope glycoprotein (wong et al., 2010) . the cathepsins comprise a family of lysosomal protease enzymes whose primary function (i.e., protein degradation) plays a critical role in normal cellular homeostasis. cathepsin l is one of the 11 members of human lysosomal cysteine proteases (i.e., b, c, f, h, k, l, o, s, v, w, and x) that fall in the c1 family (papain family) of the ca clan (rossi et al., 2004) . these enzymes were traditionally linked to nonspecific proteolytic activity within lysosomes. more recently, cathepsin l has been implicated in regulatory events relating to cancer, diabetes, immunological responses, degradation of the articular cartilage matrix, and other pathological processes (vasiljeva et al., 2007) , including osteoporosis, rheumatoid arthritis, and tumor metastasis (palermo and joyce, 2008) . interestingly, several host cell proteases appear to be able to prime fusion activation in the case of sars-cov, including cathepsin l, trypsin, factor xa, thermolysin, plasmin, tmprss11a, and elastase. the proteolytic cleavage events in sars-cov s that lead to membrane fusion occur both at the s1/s2 boundary and adjacent to a fusion peptide in the s2 domain (belouzard et al., 2009) . elastasemediated activation of sars-cov was originally reported by taguchi and coworkers, and it has been proposed that elastase may have important implications for viral pathogenesis (matsuyama et al., 2005) . elastase is known to be secreted by neutrophils as part of an inflammatory response to a viral infection and is also produced by opportunistic bacteria (e.g., pseudomonas aeruginosa) that can colonize virally infected respiratory tissue. as such, it has been considered that elastasemediated activation of sars-cov might be an important factor in the severe pneumonia seen in sars-cov-infected patients (belouzard et al., 2010) . in conclusion, in order to better classify the receptors, the receptors must be differentiated according to their precise function: those that permit a nonspecific adsorption, such as the glycosaminoglycans (used by dengue, tbe, hsv-1), the type c lectin receptors, such as l-sign, dc-sign, the hmgl, the lsectin, and the asialoglycoprotein receptor (used by hcv, ebola, marburg); those that permit a specific adsorption receptor (binding receptor) allowing the sorting (toward particular endosomes and intracytoplasmic compartment) and the initial conformational changes (such as the cd4 to hiv, the integrins or the laminin receptor for the dengue, sindbis, or lassa viruses); and finally, those that permit the exposition of the domains implicated in the destabilization of the membrane (like the fusion peptide) and are the latest receptors to act (such as the hiv coreceptors). therefore, studying the kinetics of the conformation changes of the envgp and the kinetics of action and utilization of the receptors is essential to accurately categorize the receptors (nonspecific adsorption receptor, receptor of binding, receptor of fusion). all the cellular proteins that allow the exposition, localization, and the trafficking of these receptors and/or endosomes should be considered as cofactors. with all the sirna screens that are coming out, the list of such cofactors is dramatically increasing. the use of different receptors often correlates with the need of a virus to overcome barriers existing in the cell type or tissue that they infect. one wellstudied example is the binding of coxsackievirus b to decay-accelerating factor (daf) in the apical surface of epithelial cells, and subsequently to the coxsackievirus and adenovirus receptor (car), which is localized in the tight junction region. daf helps to bring the virus to the tight junctions, and car induces a conformational change and promotes endocytosis (coyne and bergelson, 2006) . almost all animal viruses use receptors exclusively containing a single tm sequence (see table 4 .1). in striking contrast, cell surface receptors for -retroviruses have multiple transmembrane (tm) sequences, compatible with their identification in known instances as transporters for important solutes. similarly, hepatitis c virus, in addition to ldl receptor, exclusively uses multitransmembrane receptors:the scavenger receptor class b type 1 (srb1 with two transmembrane domains), the tetraspanin receptors cd81 and claudins (4 tms), and another tight junction protein occludin-1 (4 transmembrane domains; . another surprise is that some viruses, including many -retroviruses, use not just one receptor but pairs of closely related receptors as alternatives (tailor et al., 2003) . this appears to have enhanced viral survival by severely limiting the likelihood of host escape mutations. all of the receptors used by -retroviruses contain hypervariable regions that are often heavily glycosylated and that control the viral host range properties, consistent with the idea that these sequences are battlegrounds of virus-host coevolution. however, in contrast to previous assumptions, it is probable that -retroviruses have adapted to recognize conserved sites that are important for the receptor's natural function and that the hypervariable sequences have been elaborated by the hosts as defense bulwarks surrounding the conserved viral attachment sites. the fact that all virus groups have been severely limited throughout evolution in the types of receptors they can employ, may initially appear inconsistent with evidence that some viruses can switch their receptor specificities with apparent ease. this has been most dramatically suggested by shifts of influenza a viruses between animal reservoirs, which involve single amino acid changes in the viral hemagglutinin, enabling recognition of different sialic acid structures (e.g., n-acetyl or n-glycolyl neuraminic acids in 2,6 or 2,3 linkages to galactose) that predominate in the different host species (baranowski et al., 2001; gambaryan et al., 2005; skehel and wiley, 2000) . similarly, slight changes in specificity for receptors accompanied the emergence, in 1978, of the canine parvovirus (parker et al., 2001) . however, these are small shifts in receptor specificities rather than global jumps to dissimilar receptors. similar slight shifts are involved in the change from the ccr5 to cxcr4 coreceptor usage during aids progression (scarlatti et al., 1997) . small shifts in usages of highly similar receptors have also been reported during cell culture selections of subgroup b, d, and e avian leukosis viruses that all use polymorphic variants of the same tvb receptor (taplitz and coffin, 1997) and during cell culture selections of hiv-1 variants (platt et al., 1998) . therefore, despite the rarity of receptor repertoire expansions throughout millions of years of retrovirus evolution, limited switches can occur within single infected animals. for example, there is an evolution of coreceptor usage in hiv-1/aids, some in vivo adaptations of ecotropic mulvs and formation of polytropic mulvs, and some evolution of altered receptor usages in domestic cats infected with felv-a (tailor et al., 2003) . several viruses have been reported to use multiple alternative receptors or even alternative pathways for infection of cells. for example, mv isolates appear to be capable of using cd46 or slam, which both contain single tm domains (baranowski et al., 2001; oldstone et al., 1999; tatsuo et al., 2000) . complex viruses such as herpesviruses that contain several distinct envgp are also typically able to bind to several cell surface components (baranowski et al., 2001; borza and hutt-fletcher, 2002) . the foot-and-mouth disease picornavirus (fmdv) may also use multiple receptors, including heparan sulfates and integrins, and may, in addition, be able to invade cells via immunoglobulin fc receptors when the virus is coated with antibodies (baranowski et al., 2001; mason et al., 1994) . this alternative entry route is also used by the dengue flavivirus, which may explain the extremely strong pathogenesis that occurs when it reinfects previously exposed individuals (baranowski et al., 2001) . in the case of fmdv, it has not been established whether heparin sulfate is a true receptor that directly mediates infection or merely a binding factor that influences infection indirectly by enhancing virus adsorption. hiv-1 infections are also strongly stimulated by accessory cell surface binding components including heparan sulfates, glycolipids, and dc-sign (bounou et al., 2002; geijtenbeek et al., 2000; pohlmann et al., 2001) . similarly, a paralysis-inducing neurotropic variant of friend mulv binds more strongly than the parental virus to heparan sulfate, and thereby becomes more infectious for brain capillary endothelial cells while still remaining dependent on the cat1 receptor (jinno-oue et al., 2001) . these examples illustrate how changes in affinities for accessory binding substances can dramatically alter cellular tropisms and pathogenesis of viruses, and why it has often been difficult to distinguish such accessory binding factors from true receptors or coreceptors that are essential for infections. on the basis of these considerations, we believe that the available evidence strongly supports our proposal that all virus groups have been severely constrained in the types of receptors they can employ for infection of cells. however, some viruses have evolved several pathways for infection, and viruses such as hiv-1 have evolved distinct sites in a single su glycoprotein for recognition of dissimilar receptors and coreceptors. the complexity of envgp is variable. an example of a very simple fusion protein is the fast proteins of orthoreovirus that do not belong, however, to the family of the enveloped virus (barry et al., 2010; shmulevitz and duncan, 2000) . the orthoreovirus genus includes some no-enveloped viruses that cause the cell-cell fusion of infected cells. this fusion activity is due to the small no-structural membrane viral proteins named fast proteins (fusion-associated small transmembrane protein), in size ranging between 10 and 15 kda. the fast protein ectodomains are very small, with extreme cases of only 20 residues, and contain hydrophobic short regions with/without acid properties, and a myristoylation site. though their small size challenges their ability to form a hairpin structure, these fast proteins expressed alone are sufficient to induce the membrane fusion (barry et al., 2010; shmulevitz and duncan, 2000) . surprisingly, they are able to traffic as far as the plasma membrane without inducing intracellular disorder. indeed, by opposition, the expression of retroviral tm or orthomyxovirus ha2 alone does not lead to cell surface membrane expression, and these subunits are blocked intracellularly. the fusion induced by the fast protein is very broad, which questions their use of a particular protein receptor. thus, some fusion proteins are simple and possess solely the fusion function, and others comprise several domains in addition to the domain involved in fusion. the function of some of these domains has remained more or less independent from the fusion domain, and sometimes, they can be naturally separated in different proteins (i.e., h and f from paramyxoviruses) or experimentally on different fragments, as explain below. such is the case of the -retroviruses for which the function of binding to the receptor can be separated from the function of fusion. this line of inquiry was initiated by the studies of bae et al. (1997) relating to a conserved phq motif that occurs near the amino-terminal ends of su glycoproteins in all -retroviruses. mutation of this phq motif blocked membrane fusion but had no effect on receptor attachment. subsequently, we discovered that noninfectious -retrovirions lacking this histidine could be transactivated by addition of a soluble su or an amino-terminal fragment of su, called the receptor-binding domain (rbd), to the cultured cells (lavillette et al., 2000) . interestingly, studies by overbaugh and coworkers have demonstrated that similar transactivation processes can occur in natural infections by -retroviruses. specifically, they found that infections by the immunosuppressive felv-t virus, which has a pro in place of his in its phq motif, require transactivation either by a soluble felv-b-related su glycoprotein termed felix that is endogenously expressed in cat t cells or by an felv-b su glycoprotein . this activating process parallels that of herpes simplex virus for the transmission of a fusogenic signal among the envgp of the herpes simplex virus on receptor binding by glycoprotein gd. the soluble gd ectodomain has been shown to allow entry of engineered hsv-1 virus particles that lack gd (i.e., gd-null mutants; cocchi et al., 2004) . the evidence reviewed provides very strong support for the hypothesis that attachment of viruses to their receptors initiates a pathway that obligatorily contains intermediate steps. these intermediate steps very likely include viral association with multiple receptors, cooperative conformational changes within env glycoprotein, and cross-talk between env on the viral surfaces. in the case of the retroviruses, this evidence suggests that virus binding to receptors does not directly induce irreversible structural changes in su-tm complexes as was previously believed. rather, it implies that the binding to receptors induces su-su interactions that are prerequisites for later steps in a highly coordinated membrane fusion pathway. we anticipate that similar intermediate steps are likely to be involved in infections by other groups of retroviruses and perhaps in infections by other membrane-enveloped viruses. other viruses can be activated by soluble receptors that, by interacting with the envelope glycoprotein, induce some of the conformational changes necessary to trigger fusion. this example indicates that binding and fusion steps can be separated and can take place at different locations. in these cases, mhv, avian retrovirus aslv, and the herpes simplex 1 (hsv-1) can infect some cells that do not harbor binding receptors provided that the soluble form is present in the infection media (the soluble ceacam1 receptor for mhvr, the soluble tva receptor for aslv, and the soluble nectin-1 for hsv-1; kwon et al., 2006; matsuyama and taguchi, 2002; mothes et al., 2000) . vectors derived from retroviruses such as lentiviruses and oncoretroviruses are probably among the most suitable tools to achieve long-term gene transfer, since they allow stable integration of a transgene and its propagation in daughter cells. lentiviral vectors should be the preferred gene-delivery vehicles over vectors derived from oncoretroviruses (mlv) since, in contrast to the latter, they can transduce nonproliferating target cells. moreover, lentiviral vectors that have the capacity to deliver transgenes into specific tissues are expected to be of great value for various gene transfer approaches in vivo (frecha et al., 2008b) . to achieve such in vivo gene transfer, innovative approaches have been developed to upgrade lentiviral vectors for tissue or cell targeting and which have potential for in vivo gene delivery. one strategy is to develop vectors that harbor envgp with selective tropisms. vectors derived from retroviruses offer particularly flexible properties in gene transfer applications, given the numerous possible associations of various viral surface glycoproteins (determining cell tropism) with viral cores. selective tropisms were achieved by taking advantage of the natural tropisms of envgp from other membrane-enveloped viruses. for instance, the use of surface glycoproteins derived from viruses that cause lung infection and infect via the airway epithelia, such as ebola virus or influenza virus, may prove useful for gene therapy of the human airway (kobinger et al., 2001) . exclusive transduction of retinal pigmented epithelium could be achieved following subretinal inoculations of some vector pseudotypes in rat eyes (duisit et al., 2002) . high transgene expression was detected in dermal fibroblasts transduced with vsv-g-, eboz-, or mulv-pseudotyped hiv vector and effectively targeted quiescent epidermal stem cells which underwent terminal differentiation resulting in transgene expression in their progenies (hachiya et al., 2007) . importantly, several viral envgp target lentiviral vector to the cns such as rabies (wong et al., 2004) , mokola (watson et al., 2002; wong et al., 2004) , lymphocytic choriomeningitis virus envelope (lcmv) (miletic et al., 2004; stein et al., 2005) , or ross river (kang et al., 2002) viral envgp that even permit transduction of specific cell types within the cns. likewise, screening of a large panel of pseudotyped vectors established the superiority of the gibbon ape leukemia virus (galv) stitz et al., 2000) and the cat endogenous retroviral-modified glycoproteins (rd114) for transduction of progenitor and differentiated hematopoietic cells. recently, a new lv carrying the mv envgp on its surface was able to overcome vector restrictions in both quiescent t and b cells (frecha et al., 2008a (frecha et al., , 2009 ). importantly, naive as well as memory t and b cells were efficiently transduced, while no apparent activation, cell-cycle entry, or phenotypic switching were detected, opening the door to a multitude of gene therapy and immunotherapy applications. vectors derived from hiv pseudotyped with sendai virus fusion protein f (kowolik and yee, 2002) or e1e2 from hepatitis c virus (bartosch et al., 2003) , and such vectors are able to transduce human hepatoma cells and primary human hepatocytes efficiently, although they are unable to enter nonliver cells. the gp64 glycoprotein from baculovirus autographa californica multinuclear polyhedrosis virus pseudotyped fiv efficiently and also showed excellent hepatocyte tropism (kang et al., 2005) . the screening of cdna libraries has emerged as a powerful tool to identify and clone viral entry receptors. it is an alternative to the use of human/chinese hamster radiation hybrid panels of cells. in order to clone an unknown receptor by complementation screens, cdna from a cell permissive to infection by a certain virus is introduced into a nonpermissive cell. as some recessive cell lines are poorly transfectable, the use of pseudoparticles provides a good tool to transduce the cdna library for this cloning strategy. a retroviral cdna library approach, involving transfer and expression of cdnas from highly infectable cells to nonpermissive cells, has been used to clone and identify the mulv polytropic x-receptor tailor et al., 1999a; yang et al., 1999) , the rd114 asct2 receptor tailor et al., 1999b) , felv-c flvcr1 receptor from human and domestic cat cdna libraries (quigley et al., 2000; tailor et al., 1999c) , and two closely related human proteins, phur-a1 and phur-a2, that function as receptors for perv-a (ericsson et al., 2003) . briefly, when a cell line that is not susceptible to a particular pseudotype retrovirus vector harboring an envelope for which the receptor is not known, a cdna library from a cell line highly susceptible to transduction, was constructed by cloning the cdna into a retroviral expression vector. afterward, the cdna retroviral expression library was transduced into nonsusceptible cells by infection at a relatively low multiplicity of infection so that the majority of infectants would contain single-copy provirus inserts. the library-containing cells were then screened for susceptibility to pseudotype vector transduction through selection of drug-resistant cells after exposure to the vector carrying a resistance gene. of drug-resistant clones obtained from the primary screen and using pcr primers specific for vector sequences, cdna products from clones with conferred susceptibility were identified after nested pcr was performed on dna extracted from reinfectable clones. however, for many viruses, initial attempts using a retroviral cdna library were unsuccessful due to an inherent background of nonspecific infection with pseudoparticles. in fact, no cell line was completely nonpermissive to even "no envelope" pseudoparticles bearing no viral envelope proteins, indicating the existence of nonspecific uptake mechanisms. in the screens, this resulted in a high background of drug-resistant colonies, independent of glycoproteinmediated cell entry. thus, unless the entry factor cdna was highly represented in the library, a single round of transduction/challenge would not suffice. to deal with the high background observed in screens, methods that would allow multiple rounds of selection and enrichment have been developed. recently, the use of a cyclic packaging rescue (cpr) system using non-self-inactivating vectors has been shown to increase the efficiency of receptor cloning with powerful iterative screening methods (evans et al., 2007; . most retroviral vectors commonly used for gene-delivery applications are self-inactivating vectors that contain deletions in the long terminal repeat (ltr) elements. no packaging competent retroviral rna transcripts are generated from such integrated proviruses; instead, transgene expression is driven by an internal nonretroviral promoter. in contrast, if the cdna library is constructed in a provirus that retains the complete ltr elements, the retroviral promoter is active in transduced cells and a full-length viral rna is expressed. expression of the packaging components, gag-pol and vesicular stomatitis virus vsv-g envelope (that will efficiently infect recessive cell lines), in these cells allows packaging of the rna into pseudoparticles capable of transducing naive cells. this approach, termed cpr (bhattacharya et al., 2002; koh et al., 2002) , allows retrieval of the library after selection has been performed, followed by transduction of a naive cell population, concluded by a new round of selection. this process can be repeated sequentially for an unlimited number of selection/ enrichment steps. for an additional level of selection, different challenge virus genomes can be used, each encoding a different drug-selectable marker (e.g., puromycin (puror) or zeocin (zeor) resistance), in self-inactivating retroviruses. thus, after challenge and selection of a library-transduced population with one pseudoparticle-packaged selection cassette (e.g., puror), the population can be pooled and rechallenged with the second-selectable pseudovirus (e.g., zeor). then, during cpr, only the full-length retroviral transcripts from the non-selfinactivating provirus that encodes the library but not the self-inactivating challenge virus genomes are repackaged and transferred to the naive cell population. this enables researchers to perform multiple rounds of selection, thereby overcoming the background of nonspecific pseudoparticle uptake. moreover, using this scheme, underrepresented cdna will be enriched. once the final round of selection of pseudotype-susceptible cells has been achieved, genomic dna can be prepared from selected clones and used as a template in a pcr across the provirus cdna-cloning site. the nonpermissive target cell line for the cdna screen adheres to several stringent criteria. as stated above, the primary requirement is that (1) to minimize nonspecific background, cell lines with minimal uptake of pseudoparticle of interest and "no envelope" pseudoparticles were preferred. (2) to ensure that nonpermissiveness was a phenotype due to the lack of a pseudoparticlespecific entry factor(s) rather than poor infection by pseudotypes in general, chosen cell lines should be highly permissive to an unrelated pseudoparticle (vsvgpp, rhabdovirus) infection. in addition, this also ensures that the target cell line would be easily transduced with the cdna library. (3) for selection, candidate cell lines also had to be susceptible to the desired drug selections. (4) to perform multiple rounds of screening involving cpr, the ideal cell line had to demonstrate this method well and be highly transfectable. (5) finally, to facilitate the screen, the chosen cell line needed to be relatively fast growing and clone efficiently. to gain insights into virus entry, it is necessary to examine several inhibitors of pathway-mediated endocytosis in terms of their role in blocking infection mediated by pseudotypes with different envgp. an advantage of pseudoparticles is that the entry process of pathogens of bsl4 can be studied in bsl2 conditions. for example, to gain insights into ebola virus entry, inhibitors against different endocytosis pathways have been examined for their ability to block infection mediated by hiv pseudotyped with the ebola envgp (bhattacharyya et al., 2010) . the use of control pseudoparticles (e.g., pseudotyped with vesicular stomatitis virus envgp (vsv g)) can be used as controls to assess cell viability and specificity of inhibition. inhibition of clathrin function traditionally relied on three principal approaches: drugs that inhibit acidification of endosomes (such as bafa1, a specific, nonreversible endosomal proton pump inhibitor), as well as commonly used lysosomotropic agents (such as ammonium chloride (nh 4 cl) and chloroquine); potassium depletion; and finally, treatment of cells with brefeldin a (bfa) or chlorpromazine (sieczkarski and whittaker, 2002) . as such, these drugs have multiple effects on cell function, and their use to inhibit virus infection should be treated with some caution. thanks to pseudotypes, some other studies have implicated the actin cytoskeleton in ebola virus entry, where agents such as cytochalasin d and swinholide a that impair microfilament function were shown to inhibit envgp-mediated entry (yonezawa et al., 2005) . ebola enters cells through a low-ph-dependent, endocytosis-mediated process. a large body of evidence indicates that ebola viruses enter cells by clathrin-mediated endocytosis (sanchez, 2007) , but lipid raftassociated, caveolin-mediated endocytosis has also been proposed as an alternative mechanism of ebola virus uptake (empig and goldsmith, 2002) . low-ph events lead to cathepsin-dependent cleavage of ebola virus envgp that is required for productive uptake of the virus (chandran et al., 2005; kaletsky et al., 2007; schornberg et al., 2006) . other low-ph-dependent events have been postulated to be required as well (schornberg et al., 2006) . furthermore, ebola virus likely uses a rho-mediated pathway, as is seen in vsv virus, suggesting that this may be a route of entry utilized by many different viruses (quinn et al., 2009 ). more recently, proteins interfering with endocytosis, such as the use of dominant-negative eps15, or rna interference (rnai) have been developed, and such approaches target the different pathways with higher specificity (mercer and helenius, 2009; mercer et al., 2010b) . the rnai approach allows researchers to perform screens to identify previously unrecognized host factors that are required for viral replication. rnai screens rely on either short interfering rnas (sirnas) or short hairpin rnas (shrnas) to knock down the function of a particular gene in a cell. researchers can then infect the cells with specific viruses and monitor levels of viral replication. if viral replication is reduced, then the knocked-down gene might be necessary for the virus to replicate itself or function within the host cell. some small-scale screens have been developed using wild-type viruses (kolokoltsov et al., 2007) or pseudoparticles (trotard et al., 2009) . pseudoparticles can be exploited to focus a sirna screen specifically on the entry step of a virus. indeed, only the entry steps are governed by the envgp, whereas all the uncoating, integration, and expression steps of the transgene depend on retroviral proteins. having said that, this suffers an inherent drawback in that some steps are dependent on retroviral proteins and therefore limits the scope of the screen. one limitation to the sirna screen is specificity and cell toxicity, which can be overcome by the use of pseudotypes. indeed, a sirna screen can be done with different pseudotypes in parallel. if, contrary to the pseudotypes of interest, some pseudotypes are not affected by sirna, this indicates that the sirna specifically inhibits the virus of interest and, moreover, that the sirna is not toxic. to better characterize the entry pathway of the hepatitis c virus, a small interfering rna library dedicated to membrane trafficking and remodeling was screened in the context of the huh-7.5.1 liver cell line cells infected by hcv pseudoparticles (hcvpp) (trotard et al., 2009) . results showed that the downregulation of different factors implicated in clathrin-mediated endocytosis inhibit hcvpp cell infection. in addition, knockdown of the phosphatidylinositol 4-kinase type iii-alpha (pi4kiiialpha) prevented infection by hcvpp, and the presence of pi4kiiibeta in the host cells influenced their susceptibility to hcvpp infection. this library screening using pseudoparticles identified two kinases, pi4kiiialpha and beta, as being involved in the hcv life cycle. these results have been confirmed in published works of the identification of cellular factors required for the hcv life cycle using sirna libraries screening either over 4500 drugable genes (ng et al., 2007; vaillancourt et al., 2009) , 140 cellular membrane-trafficking genes coller et al., 2009) , kinome (supekova et al., 2008) , or the entire genome (li et al., 2009; tai et al., 2009 ). these works are much more time-and cost-consuming, and even though pseudoparticles may appear to be an overly simple model to study and identify host factors necessary for viral infection, they are also powerful and flexible tools that have led to major discoveries, as these examples have shown. recently, however, in order to identify host factors necessary for viral infections, researchers are turning to genome-wide genetic screens. indeed, the sequencing of the human genome and the emergence of technologies that allow the silencing of individual genes in cells one by one using sirna, when combined with the use of genome-wide sirna libraries and automated hts methodology, allows molecular information to be gained about virtually every critical intracellular event occurring during virus infection. within the last few years, there have been several publications describing such genome-wide sirna screens that have been applied to virus infections in tissue-cultured cells. rnai screens have now been preformed for several viruses, including hiv konig et al., 2008; yeung et al., 2009; zhou et al., 2008) , west nile virus (krishnan et al., 2008) , influenza (karlas et al., 2010; konig et al., 2008) , and, in drosophila cells, dengue (sessions et al., 2009) and vaccinia virus (moser et al., 2010) . from the hundreds of cellular factors that have been implicated in the outcome of infection, some were already known from other studies, but the majority are entirely novel, and the validation and analysis of data is ongoing in many laboratories. however, a poor overlap between results from different screens published on the same viruses has been observed. the differences are probably linked not only to technical variations but also to the inherent risk of false positives through off-target effects and false negatives due to inefficient silencing. cell toxicity is a common complication, as well as cell-type specificity. following-up such screens with pseudoparticle studies is useful to validate the cell proteins that are involved in the entry step only and facilitate the analysis, compared to the use of a replication competent virus. resistance to individual antivirals is likely to develop for most specific therapeutics targeting particular viral proteins, thus making therapy consisting of a combination of drugs targeting different stages of the viral life cycle highly desirable. the entry process represents another series of potential targets for therapeutic intervention. it has not been extensively explored due to highthroughput experimental limitations for some viruses that require biosafety level 3 or 4 or for the viruses with no robust infection system. pseudoparticle infection systems, utilizing different reporter genes or proteins (i.e., firefly luciferase or gfp), can be developed for hts of small molecule libraries, peptide libraries, or neutralizing antibodies for their entry inhibitor properties. in order to facilitate the screen, assay performance can be improved by modifying the properties of both the parental host cell line and the pseudovirus. for example, cells with improved pseudoparticle entry and cell spreading can be selected. pseudoparticles can be improved by using envgp that increase infectivity either by selecting a specific variant or by expressing a human codon-optimized envelope glycoprotein sequence. finally, the pseudoviruses can be engineered to express a human codon-optimized reporter to improve the sensitivity of the assay. using these modifications, hts can be developed using 96-or 384-well plates. compounds found to inhibit pseudoparticle infection can then be counterscreened against pseudoparticles containing other variants to characterize the cross-reactivity of the molecules in a virus family. moreover, molecules can then be counterscreened against pseudoparticles harboring envgp from other virus families to evaluate the specificity of the inhibitor or its large spectrum. a wide variety of entry inhibitors, namely, peptides, chemical compounds, or antibodies, exist. they can interfere with the different steps of the entry process, such as the binding to receptors or the conformational changes of the fusion proteins, by acting on the envelope protein itself, on the acidification of the endosomes, if necessary, or on the endocytosis. for hiv, most marketed drugs for treating aids are inhibitors of hiv-1 reverse transcriptase or protease enzymes, but new targets include the integrase enzyme, cell-surface interactions, membrane fusion, and also virus particle maturation and assembly (kaushik-basu et al., 2008) . entry inhibition entails preventing hiv-1 breaching the cell, either as a strategy to prevent infection altogether or to curtail infection of new cells in an hiv-positive individual. several strategies have proved effective in hiv-1 entry inhibition either in vitro (using pseudoparticles or replication competent viruses) or in vivo: binding to viral surface proteins gp120 and gp41, binding to human cell surface receptor cd4, and binding to human cell surface coreceptors, ccr5 and cxcr4 (leonard and roy, 2006; liu et al., 2007) . in particular, the synthesized peptide t-20 is believed to act by binding to gp41 (wild et al., 1993 (wild et al., , 1994 and is currently in clinical use. yet, several more recent studies have revealed that t-20 does not block the six-helix bundle prehairpin formation (liu et al., 2005) . another peptide, c37, derived from the c-terminus of gp41 (and nearly identical to the widely reported c-peptide c34; liu et al., 2005; root et al., 2001) , is also described as having strong anti-hiv entry activity due to the tight binding of the gp41 n-terminal helices. maraviroc is a small antiretroviral compound known to be a ccr5 antagonist, which blocks r5-tropic hiv entry into cd4 cells (hunt and romanelli, 2009) . several studies have established that synergy can occasionally be observed when two fusion inhibitors are combined in a viral assay or in vitro fusion assay. for example, some synergy is observed in the combination of ccr5 antibodies with t-20 (ji et al., 2007; murga et al., 2006) , the combination of small molecular antagonists of coreceptors with t-20 (tremblay et al., 2000 , the combination of small molecular antagonists of ccr5 with ccr5 antibodies (ji et al., 2007; murga et al., 2006) , and the combination of small molecular antagonists of ccr5 with chemokines (murga et al., 2006; tremblay et al., 2002) . in the case of hcv, entry into hepatocytes is a multistep process, involving at least four cellular receptors, leading to virion endocytosis and fusion of the viral and cellular membranes. unlike the hcv replication process, these steps have not been thoroughly exploited as targets for antiviral intervention. recently, with the development of hcvpp and the jfh1 infectious molecular clone, it has become possible to test drugs against entry. in vitro, proof-of-concept studies for inhibiting the hcv entry process have been demonstrated using cyanovirin-n that targets the n-linked glycans of the viral envelope proteins and prevents e2-cd81 interaction (helle et al., 2006) , neutralizing antibodies directed against the hcv e1 and e2 proteins (broering et al., 2009; habersetzer et al., 1998; keck et al., 2008; law et al., 2008; owsianka et al., 2005; perotti et al., 2008; schofield et al., 2005; yu et al., 2004) , antibodies against cellular receptors cd81 ( bartosch et al., 2003; cormier et al., 2004; lavillette et al., 2005; molina et al., 2008) , sr-bi (catanese et al., 2007; zeisel et al., 2007) , claudin-1 (krieger et al., 2010) , and agents that block endosomal acidification (bartosch et al., 2003; hsu et al., 2003; koutsoudakis et al., 2006; lavillette et al., 2005) . in vivo studies using humanized trimera mice model or human liver-u-pa-scid mice have also demonstrated prophylactic efficacy of monoclonal anti-e2 (eren et al., 2006) and anti-cd81 antibodies (meuleman et al., 2008) , respectively. some broad-spectrum antiviral drugs have also been tested (pecheur et al., 2007) , but more recent studies have used the hcvpp system in order to undertake a screening campaign that led to the discovery of a class of small molecule hcvspecific inhibitors consisting of several structurally related compounds defined by a common triazine core (baldick et al., 2010) . inhibition of entry was confirmed by using time-of-addition experiments to demonstrate that inhibitor activity is confined to the first 3 h of infection, with inhibition occurring postattachment and closely linked to the inhibition kinetics of the endosomal acidification inhibitor bafilomycin. pseudoparticles can also be used to identify the mode of action of molecules identified using a replication competent virus in a cell-based screening system involving multiple rounds of infection in a 96-well format. after analysis of a publicly available library of 446 clinically approved drugs, the impact of 33 identified compounds on viral entry was tested using hcvpp infection to recapitulate hcv particle adsorption, internalization, and viral envelopemediated fusion (gastaminza et al., 2010) . many of the candidates were lysosomotropic compounds that inhibited hcv entry with differential efficacy. both pseudotype (larson et al., 2008) and infectious virus screening (bolken et al., 2006) identified broadly active arenavirus entry inhibitors. isolation and mapping of resistant viruses, as well as chimeras between sensitive and resistant strains, mapped the target of activity to the gp2 subunit of the g envelope protein complex, specifically to the interface between the c-terminal stem and tmd domains. mechanistic studies showed that these arenavirus inhibitors prevented low-ph-induced fusion by blocking reorganization between the gp2 stem with n-terminal domains of the g protein complex (york et al., 2008) . another alternative for developing entry inhibitor compounds is to target endosomal protease necessary for entry of certain viruses. inhibitors of cathepsin l block viral entry of severe acute respiratory syndrome coronavirus (sars-cov) and ebola virus and impair conversion of hev glycoprotein into the mature, active form (chandran et al., 2005; pager and dutch, 2005; simmons et al., 2005) . with respect to the development of antiviral agents, inhibitors of human cathepsin l are not subject to resistance arising from rapid mutations of the viral genome (shah et al., 2010) , making cathepsin l an attractive target for drug development. hts for cathepsin l inhibitors identified a novel thiocarbazate compound exhibiting potent inhibition against cathepsin l myers et al., 2008; shah et al., 2008) . this compound prevented 293t cell infection by the ebola and sars-cov pseudotypes, respectively. in addition, the thiocarbazate inhibited in vitro propagation of malaria parasite plasmodium falciparum and inhibited leishmania major . finally, another natural entry inhibitor is provided by monoclonal neutralizing antibodies. many monoclonal antibodies can be isolated from immortalized b-cells recovered from patients or from mice hybridomas following immunization. the antibodies can be selected by elisa assay using soluble envelope proteins or pseudoparticles. the neutralizing potential of these antibodies can be easily screened using pseudoparticles with high-throughput infection assays, and the inhibitory effect can be verified with replicating viruses. in the case of hcv, using an antibody antigen-binding fragment phagedisplay library generated from a donor chronically infected with hcv, of 115 clones showing specific binding to hcv e2 glycoprotein, 5 monoclonal antibodies presented neutralizing activities against cell-culture hcv (hcvcc), jfh-1 virus, and a panel of hcvpp displaying e1-e2 from diverse genotypes (law et al., 2008) . overall, neutralizing antibodies can inhibit the different steps of the entry process from binding to membrane fusion by targeting the domains involved in this step or by limiting the conformational changes of the envelope complex. interestingly, one study has recently highlighted the feasibility of targeting short-lived envelope glycoprotein intermediates for inhibition of membrane fusion using monoclonal antibodies (york et al., 2010) . this action is very similar to the peptides against hiv membrane fusion on the market. such strategies to effectively target fusion peptide function in the endosome may lead to the discovery of novel classes of antiviral agents, and screens using pseudoparticles will provide an easily wielded system to identify such infrequent antibodies. all viruses have developed varied mechanisms to reach the same goal. they vary greatly in structure, but all seem to have a common mechanism of action, in which a ligand-triggered large-scale conformational change in the fusion protein is coupled to apposition and merger of the two bilayers. in spite of the different mechanisms to activate the fusion peptide, fusion proteins are distributed into three classes based on their structural homologies. future experiments must aim to elucidate the molecular mechanisms and the dynamics of the conformational changes driving virus entry. this will require the development of new approaches to study the rapid conformational changes of a small number of membrane interacting protein molecules and domains. a more realistic goal is the determination of all the structures of proteins that mediate the entry of all human viruses, either at a prebinding or postfusion stage. another challenge will be the identification of the cognate cellular receptors. identification of all the cellular receptors for human viruses would be an important contribution to our understanding of virus tropism and pathogenesis. once known, the role of receptors in entry, as a specific or nonspecific binding factor, or as receptors needed for conformational changes, or for routing the virus to the right compartment, will have to be established. moreover, for most viruses, the envgp appear to function in an autonomous manner and can permit fusion without a requirement for receptors at acidic ph. thus, the role of varied receptors remains enigmatic for many viruses. it is difficult to predict the roles of a receptor in fusion, sorting and routing the virus toward a particular favorable compartment in the pursuit of the infectious cycle based upon its family and its structure. the activation process of envgp and the postbinding events are early steps that are crucial to understand, as they could provide targets for the development of therapeutics. the better understanding of the envelopereceptor interaction also raises hopes for the possibility of designing entry machines that can deliver genes and other molecules to any cell of choice. recently, the use of whole genome sirna screen has become more and more widely used for different viruses in order to identified factors important for entry. it should drastically increase our knowledge of the factors necessary for entry of viruses. similarly, many high-throughput interactome studies will identify cellular proteins interacting with the different viral components. altogether, the comparison of all these high-throughput screens should help us to identify cellular proteins and pathways common to different viruses which may help, with rational structure/mechanism-based design of entry inhibitors, to 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particles and vsv-g pseudotyped mlv-derived retrovirus vectors to target cells characterization of human monoclonal antibodies specific to the hepatitis c virus glycoprotein e2 with in vitro binding neutralization properties gene transfer in human skin with different pseudotyped hiv-based vectors mechanism of membrane fusion by viral envelope proteins viral membrane fusion virus receptors: binding, adhesion strengthening, and changes in viral structure crystal structure of glycoprotein b from herpes simplex virus 1 on the entry of semliki forest virus into bhk-21 cells cyanovirin-n inhibits hepatitis c virus entry by binding to envelope protein glycans biochemical and structural characterization of cathepsin l-processed ebola virus glycoprotein: implications for viral entry and immunogenicity aromatic amino acids in the juxtamembrane domain of severe acute respiratory syndrome coronavirus spike glycoprotein are important for receptor-dependent virus entry and cell-cell fusion peptide inhibitors of dengue virus and west nile virus infectivity hepatitis c virus glycoproteins mediate ph-dependent cell entry of pseudotyped retroviral particles sars coronavirus, but not human coronavirus nl63, utilizes cathepsin l to infect ace2-expressing cells maraviroc, a ccr5 coreceptor antagonist that blocks entry of human immunodeficiency virus type 1 viral entry and receptors. in "retroviruses the membrane-proximal domain of vesicular stomatitis virus g protein functions as a membrane fusion potentiator and can induce hemifusion the membraneproximal region of vesicular stomatitis virus glycoprotein g ectodomain is critical for fusion and virus infectivity ccr5 small-molecule antagonists and monoclonal antibodies exert potent synergistic antiviral effects by cobinding to the receptor hantaan virus enters cells by clathrin-dependent receptor-mediated endocytosis a unique heparin-binding domain in the envelope protein of the neuropathogenic pvc-211 murine leukemia virus may contribute to its brain capillary endothelial cell tropism a core trimer of the paramyxovirus fusion protein: parallels to influenza virus hemagglutinin and hiv-1 gp41 differences in cd4 dependence for infectivity of laboratory-adapted and primary patient isolates of human immunodeficiency virus type 1 the postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity in vivo gene transfer using a nonprimate lentiviral vector pseudotyped with ross river virus glycoproteins persistent expression of factor viii in vivo following nonprimate lentiviral gene transfer genome-wide rnai screen identifies human host factors crucial for influenza virus replication peptide inhibition of hiv-1: current status and future potential therapeutic control of hepatitis c virus: the role of neutralizing monoclonal antibodies effects of endocytosis inhibitory drugs on rubella virus entry into veroe6 cells lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion virus membrane-fusion proteins: more than one way to make a hairpin filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo novel retroviral vectors to facilitate expression screens in mammalian cells small interfering rna profiling reveals key role of clathrin-mediated endocytosis and early endosome formation for infection by respiratory syncytial virus global analysis of host-pathogen interactions that regulate early-stage hiv-1 replication characterization of the early steps of hepatitis c virus infection by using luciferase reporter viruses functional regions of the envelope glycoprotein of human immunodeficiency virus type 1 preferential transduction of human hepatocytes with lentiviral vectors pseudotyped by sendai virus f protein inhibition of hepatitis c virus infection by anti-claudin-1 antibodies is mediated by neutralization of e2-cd81-claudin-1 associations rna interference screen for human genes associated with west nile virus infection cooperation of multiple ccr5 coreceptors is required for infections by human immunodeficiency virus type 1 structure of dengue virus: implications for flavivirus organization, maturation, and fusion soluble v domain of nectin-1/hvec enables entry of herpes simplex virus type 1 (hsv-1) into hsv-resistant cells by binding to viral glycoprotein d pcsk9 impedes hepatitis c virus infection in vitro and modulates liver cd81 expression emerging viruses: risk of pandemic the potential anti-tumorigenic and anti-metastatic side of the proprotein convertases inhibitors peptides from conserved regions of paramyxovirus fusion (f) proteins are potent inhibitors of viral fusion identification of a broad-spectrum arenavirus entry inhibitor a prolinerich motif downstream of the receptor binding domain modulates conformation and fusogenicity of murine retroviral envelopes activation of a cell entry pathway common to type c mammalian retroviruses by soluble envelope fragments the envelope glycoprotein of human endogenous retrovirus type w uses a divergent family of amino acid transporters/cell surface receptors characterization of host-range and cell entry properties of the major genotypes and subtypes of hepatitis c virus broadly neutralizing antibodies protect against hepatitis c virus quasispecies challenge structure of the ebola virus glycoprotein bound to an antibody from a human survivor the lassa virus glycoprotein precursor gp-c is proteolytically processed by subtilase ski-1/s1p the hiv entry inhibitors revisited the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph palmitoylation of the murine leukemia virus envelope protein is critical for lipid raft association and surface expression intersubunit disulfide isomerization controls membrane fusion of human t-cell leukemia virus env a genomewide genetic screen for host factors required for hepatitis c virus propagation different from the hiv fusion inhibitor c34, the anti-hiv drug fuzeon (t-20) inhibits hiv-1 entry by targeting multiple sites in gp41 and gp120 hiv entry inhibitors targeting gp41: from polypeptides to smallmolecule compounds coiled-coil domains in glycoproteins b and h are involved in human cytomegalovirus membrane fusion recognition and blocking of hiv-1 gp41 pre-transmembrane sequence by monoclonal 4e10 antibody in a raft-like membrane environment r-peptide cleavage potentiates fusion-controlling isomerization of the intersubunit disulfide in moloney murine leukemia virus env effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of hep 2 cells by picornaviruses human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis polymorphisms of the cell surface receptor control mouse susceptibilities to xenotropic and polytropic leukemia viruses the lipid-anchored ectodomain of influenza virus hemagglutinin (gpi-ha) is capable of inducing nonenlarging fusion pores virus entry: open sesame the signal peptide of the ebolavirus glycoprotein influences interaction with the cellular lectins dc-sign and dc-signr rgd sequence of foot-and-mouth disease virus is essential for infecting cells via the natural receptor but can be bypassed by an antibody-dependent enhancement pathway infectious entry pathway of influenza virus in a canine kidney cell line different potential of c-type lectin-mediated entry between marburg virus strains receptor-induced conformational changes of murine coronavirus spike protein protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection pathways of clathrin-independent endocytosis a cxcr4/cd4 pseudotype rhabdovirus that selectively infects hiv-1 envelope protein-expressing cells common principles and intermediates of viral protein-mediated fusion: the hiv-1 paradigm imaging individual retroviral fusion events: from hemifusion to pore formation and growth vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells virus entry by macropinocytosis vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry virus entry by endocytosis anti-cd81 antibodies can prevent a hepatitis c virus infection in vivo selective transduction of malignant glioma by lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus glycoproteins epstein-barr virus enters b cells and epithelial cells by different routes serum-derived hepatitis c virus infection of primary human hepatocytes is tetraspanin cd81 dependent importance of the cytoplasmic tails of the measles virus glycoproteins for fusogenic activity and the generation of recombinant measles viruses human immunodeficiency virus type 1 attachment to hela cd4 cells is cd4 independent and gp120 dependent and requires cell surface heparans the membranotropic regions of the endo and ecto domains of hiv gp41 envelope glycoprotein a kinome rnai screen identified ampk as promoting poxvirus entry through the control of actin dynamics retroviral entry mediated by receptor priming and low ph triggering of an envelope glycoprotein cytoplasmic domain truncation enhances fusion activity by the exterior glycoprotein complex of human immunodeficiency virus type 2 in selected cell types dilation of the human immunodeficiency virus-1 envelope glycoprotein fusion pore revealed by the inhibitory action of a synthetic peptide from gp41 role of the membraneproximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion potent antiviral synergy between monoclonal antibody and small-molecule ccr5 inhibitors of human immunodeficiency virus type 1 design, synthesis, and evaluation of inhibitors of cathepsin l: exploiting a unique thiocarbazate chemotype identification of host genes involved in hepatitis c virus replication by small interfering rna technology palmitoylation of the rous sarcoma virus transmembrane glycoprotein is required for protein stability and virus infectivity novel type ii transmembrane serine proteases, mspl and tmprss13, proteolytically activate membrane fusion activity of the hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication measles virus infection in a transgenic model: virus-induced immunosuppression and central nervous system disease the cytoplasmic tail of the human respiratory syncytial virus f protein plays critical roles in cellular localization of the f protein and infectious progeny production mutations in the membrane-spanning domain of the human immunodeficiency virus envelope glycoprotein that affect fusion activity monoclonal antibody ap33 defines a broadly neutralizing epitope on the hepatitis c virus e2 envelope glycoprotein inhibition of chikungunya virus infection in cultured human muscle cells by furin inhibitors: impairment of the maturation of the e2 surface glycoprotein cathepsin l is involved in proteolytic processing of the hendra virus fusion protein cysteine cathepsin proteases as pharmacological targets in cancer cell surface heparan sulfate proteoglycans: selective regulators of ligand-receptor encounters canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells biochemical mechanism of hepatitis c virus inhibition by the broadspectrum antiviral arbidol nipah virus entry can occur by macropinocytosis identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis c virus e2 protein localization of the labile disulfide bond between su and tm of the murine leukemia virus envelope protein complex to a highly conserved cwlc motif in su that resembles the active-site sequence of thiol-disulfide exchange enzymes initial binding of murine leukemia virus particles to cells does not require specific env-receptor interaction evidence for nonspecific adsorption of targeted retrovirus vector particles to cells nef can enhance the infectivity of receptorpseudotyped human immunodeficiency virus type 1 particles effects of ccr5 and cd4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1 human occludin is a hepatitis c virus entry factor required for infection of mouse cells towards a small animal model for hepatitis c dc-signr, a dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans viral entry inhibitors targeted to the membrane site of action development of protein-based inhibitors of the proprotein of convertase ski-1/s1p: processing of srebp-2, atf6, and a viral glycoprotein a broadly neutralizing human monoclonal antibody against gp41 of human immunodeficiency virus type 1 endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry cloning of the cellular receptor for feline leukemia virus subgroup c (felv-c), a retrovirus that induces red cell aplasia rho gtpases modulate entry of ebola virus and vesicular stomatitis virus pseudotyped vectors a synthetic peptide corresponding to a conserved heptad repeat domain is a potent inhibitor of sendai virus-cell fusion: an emerging similarity with functional domains of other viruses the rd114/simian type d retrovirus receptor is a neutral amino acid transporter function of the cytoplasmic domain of a retroviral transmembrane protein: p15e-p2e cleavage activates the membrane fusion capability of the murine leukemia virus env protein molecular gymnastics at the herpesvirus surface the envelope glycoprotein from tick-borne encephalitis virus at 2 a resolution characterization of the equilibrium between the native and fusion-inactive conformation of rabies virus glycoprotein indicates that the fusion complex is made of several trimers crystal structure of the low-ph form of the vesicular stomatitis virus glycoprotein g structure of the prefusion form of the vesicular stomatitis virus glycoprotein g structures of vesicular stomatitis virus glycoprotein: membrane fusion revisited protein design of an hiv-1 entry inhibitor comprehensive search for cysteine cathepsins in the human genome membrane-proximal cytoplasmic domain of moloney murine leukemia virus envelope tail facilitates fusion pre-transmembrane sequence of ebola glycoprotein. interfacial hydrophobicity distribution and interaction with membranes membrane fusion process of semliki forest virus. ii: cleavage-dependent reorganization of the spike protein complex controls virus entry a conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for env-mediated fusion and virus infectivity analysis of filovirus entry into vero e6 cells, using inhibitors of endocytosis, endosomal acidification, structural integrity, and cathepsin (b and l) activity sulfhydryl involvement in fusion mechanisms lentiviral vectors pseudotyped with a modified rd114 envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes and cd34+ cells derived from human and nonhuman primates host cyclophilin a mediates hiv-1 attachment to target cells via heparans syndecans serve as attachment receptors for human immunodeficiency virus type 1 on macrophages in vivo evolution of hiv-1 co-receptor usage and sensitivity to chemokine-mediated suppression inhibition of vsv binding and infectivity by phosphatidylserine: is phosphatidylserine a vsv-binding site? construction of a novel virus that targets hiv-1-infected cells and controls hiv-1 infection human monoclonal antibodies that react with the e2 glycoprotein of hepatitis c virus and possess neutralizing activity role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein conserved structural features in the interaction between retroviral surface and transmembrane glycoproteins? discovery of insect and human dengue virus host factors kinetic characterization and molecular docking of a novel, potent, and selective slow-binding inhibitor of human cathepsin l a small-molecule oxocarbazate inhibitor of human cathepsin l blocks severe acute respiratory syndrome and ebola pseudotype virus infection into human embryonic kidney 293t cells tyro3 family-mediated cell entry of ebola and marburg viruses the mechanism of axl-mediated ebola virus infection a new class of fusion-associated small transmembrane (fast) proteins encoded by the non-enveloped fusogenic reoviruses dissecting virus entry via endocytosis inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha substitution of leucine for isoleucine in a sequence highly conserved among retroviral envelope surface glycoproteins attenuates the lytic effect of the friend murine leukemia virus receptor binding and membrane fusion in virus entry: the influenza hemagglutinin a tyrosine motif in the cytoplasmic domain of masonpfizer monkey virus is essential for the incorporation of glycoprotein into virions truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein alters the conformation of the external domain proteolytic activation of tick-borne encephalitis virus by furin the lymphocytic choriomeningitis virus envelope glycoprotein targets lentiviral gene transfer vector to neural progenitors in the murine brain flavivirus membrane fusion influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease lentiviral vectors pseudotyped with envelope glycoproteins derived from gibbon ape leukemia virus and murine leukemia virus 10a1 identification of human kinases involved in hepatitis c virus replication by small interference rna library screening a functional genomic screen identifies cellular cofactors of hepatitis c virus replication cloning and characterization of a cell surface receptor for xenotropic and polytropic murine leukemia viruses a sodium-dependent neutralamino-acid transporter mediates infections of feline and baboon endogenous retroviruses and simian type d retroviruses a putative cell surface receptor for anemiainducing feline leukemia virus subgroup c is a member of a transporter superfamily cell surface receptors for gammaretroviruses influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion selection of an avian retrovirus mutant with extended receptor usage virus entry is a major determinant of cell tropism of edmonston and wildtype strains of measles virus as revealed by vesicular stomatitis virus pseudotypes bearing their envelope proteins the role of the membrane-spanning domain sequence in glycoprotein-mediated membrane fusion mechanisms of cell killing/cytopathic effects by nonhuman retroviruses strong in vitro synergy between the fusion inhibitor t-20 and the cxcr4 blocker amd-3100 anti-human immunodeficiency virus interactions of sch-c (sch 351125), a ccr5 antagonist, with other antiretroviral agents in vitro kinases required in hepatitis c virus entry and replication highlighted by small interference rna screening noninfectious entry of hiv-1 into peripheral and brain macrophages mediated by the mannose receptor glycoproteins gb, gd, and ghgl of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a cos cell transfection system hiv-1 attachment: another look heptad repeat-derived peptides block protease-mediated direct entry from the cell surface of severe acute respiratory syndrome coronavirus but not entry via the endosomal pathway differential cholesterol binding by class ii fusion proteins determines membrane fusion properties identification of a lipid kinase as a host factor involved in hepatitis c virus rna replication emerging roles of cysteine cathepsins in disease and their potential as drug targets crimean-congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase ski-1 isomerization of the intersubunit disulphide-bond in env controls retrovirus fusion sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway targeted transduction patterns in the mouse brain by lentivirus vectors pseudotyped with vsv, ebola, mokola, lcmv, or mulv envelope proteins virus membrane fusion cell-associated west nile flavivirus is covered with e+pre-m protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release a synthetic peptide from hiv-1 gp41 is a potent inhibitor of virus-mediated cell-cell fusion peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection retained in vitro infectivity and cytopathogenicity of hiv-1 despite truncation of the c-terminal tail of the env gene product transduction patterns of pseudotyped lentiviral vectors in the nervous system a forward genetic strategy reveals destabilizing mutations in the ebolavirus glycoprotein that alter its protease dependence during cell entry receptors for polytropic and xenotropic mouse leukaemia viruses encoded by a single gene at rmc1 peptides corresponding to the heptad repeat sequence of human parainfluenza virus fusion protein are potent inhibitors of virus infection a genome-wide short hairpin rna screening of jurkat t-cells for human proteins contributing to productive hiv-1 replication studies of ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha ph-induced activation of arenavirus membrane fusion is antagonized by small-molecule inhibitors an antibody directed against the fusion peptide of junin virus envelope glycoprotein gpc inhibits ph-induced membrane fusion interaction of peptides with sequences from the newcastle disease virus fusion protein heptad repeat regions neutralizing antibodies to hepatitis c virus (hcv) in immune globulins derived from anti-hcv-positive plasma scavenger receptor class b type i is a key host factor for hepatitis c virus infection required for an entry step closely linked to cd81 hepatitis c virus entry: molecular mechanisms and targets for antiviral therapy furin processing and proteolytic activation of semliki forest virus structures of immature flavivirus particles functional domains in the retroviral transmembrane protein genome-scale rnai screen for host factors required for hiv replication our research is supported by inserm, ens lyon, cnrs, and ucb lyon-i and by grants from the european union (lshb-ct-2004-005246 "compuvac"), the european research council (erc advanced grant to flc no. 233130 "hepcent"), the agence nationale de recherches sur le sida et les hepatites virales (anrs), and the finovi foundation. we thank sarah kabani for helpful comments on this chapter. we apologize to the many authors of important original contributions that were not directly cited. lastly, we thank the past and present members of our laboratory for their many experimental and intellectual contributions. key: cord-329448-kxxy60x9 authors: kumari, sudha; mg, swetha; mayor, satyajit title: endocytosis unplugged: multiple ways to enter the cell date: 2010-02-02 journal: cell res doi: 10.1038/cr.2010.19 sha: doc_id: 329448 cord_uid: kxxy60x9 endocytosis occurs at the cell surface and involves internalization of the plasma membrane (pm) along with its constituent membrane proteins and lipids. endocytosis is involved in sampling of the extracellular milieu and also serves to regulate various processes initiated at the cell surface. these include nutrient uptake, signaling from cell-surface receptors, and many other processes essential for cell and tissue functioning in metazoans. it is also central to the maintenance of pm lipid and protein homeostasis. there are multiple means of internalization that operate concurrently, at the cell surface. with advancement in high-resolution visualization techniques, it is now possible to track multiple endocytic cargo at the same time, revealing a remarkable diversity of endocytic processes in a single cell. a combination of live cell imaging and efficient genetic manipulations has also aided in understanding the functional hierarchy of molecular players in these mechanisms of internalization. here we provide an account of various endocytic routes, their mechanisms of operation and occurrence across phyla. over the years, numerous modes of vesicular endocytic trafficking have been discovered that coexist in the same cell type and operate concurrently. the operation of an internalization route and its cargo specificity is determined by factors that vary in a context-dependent manner. these factors include one or more underlying principle in cargo enrichment, necessitating specific coat and coat-associated protein assembly, a scission mechanism, and a means to integrate these steps; several molecules and membrane parameters can influence and diversify an endocytic process. in this review, with the intention of summarizing present understanding of principles of endocytosis, we have surveyed different types of endocytic processes, focusing on their molecular attributes and mechanism of vesicle formation at the cell surface. the physical and chemical properties of cargo molecules usually dictate the nature of the primary vesicular structures that participate in their internalization; the physical process of membrane deformation at initial entry is likely to dictate the mechanism necessary for endocytosis. at first glance, the scale of this initial membrane invagination provides a natural means of classifying different pathways of endocytosis (figures 1 and 2) . ingestion of particles larger than 500 nm size typically occurs via triggered processes called 'phagocytosis or macropinocytosis' (figure 1 ) whereas cargo below this size limit is often internalized by any of the other diverse endocytic processes available at the cell surface ( figure 2 ). these include the well-characterized receptor-mediated clathrin-dependent as well as the less understood panoply of clathrin-independent 'pinocytic' pathways. there are endocytic processes that involve the internalization of large-sized particles or a large volume fraction of the extracellular bulk phase relative to the cell volume. these are termed phagocytosis and macropinocytosis, respectively ( figure 1 ). these processes are tightly regulated, distinct internalization events often resulting in phase-lucent structures inside cells when viewed in a light microscope [1] . they involve longrange remodeling of membrane and the cytoskeleton lying beneath it, and have been focus of some recent re-a cup-shaped membrane distortion around the particle, culminating in a phagosome. two well-described types of phagocytic entry are: (i) ig receptor, fcr-mediated engulfment of immunoglobulin g-opsonized particles and (ii) complement receptor cr3-mediated ingestion of c3bi-coated particles. both the means of phagocytic entry form morphologically distinct phagocytic cups and represent different types of phagocytic mechanisms ( figure 1a ). thus the morphology of forming invagination and resultant compartment is a crucial parameter for classifying these processes. in fcr-mediated phagocytosis (termed 'zipper like'), bound fcr receptors elicit local signaling responses mediated by cytosolic immunoreceptor tyrosine-based activation motifs. this leads to localized actin rearrangement, membrane extension around ligand, and thereby further engagement of receptors with ligand in a protruding cup with zipper-lock arrangement. in cr3-type phagocytosis (termed 'trigger-like'), on the other hand, the process is triggered by treatment with either phorbol esters or extracellular agonists [10, 11] and the particle is loosely encased in a large membrane vesicle. the site of phagocytosis has discrete patch-like distribution of f-actin, and occurs by a regulated and triggered depression in the cell surface. there are instances, for example, engulfment of necrotic cells, where the morphology is in between the two [1] . in either case formation of phagosomes is achieved by spatiotemporally controlled sequential action of various small actin-remodeling gt-pases [12] . the two modes also differ in their requirements for rho-gtpases, in that fcr-mediated phagocytosis relies on rac1 and cdc42 activities, whereas cr3mediated entry is specifically dependent on rhoa [12, 13] . the host phagocytic machinery can also be manipulated by bacterial pathogens to facilitate their own internalization. here too, two general models have been described that outline bacterial entry into cells: the 'trigger' or 'zipper' models. salmonella and shigella are examples of 'triggering' bacteria; salmonella injects its effector, sopb, into cells, which activates an exchange factor for rhog, leading to actin remodeling that causes internalization of the bacterium [14] . the 'zippering' model of entry of the bacteria is similar to fcr-mediated internalization, as it involves engagement of cell-surface receptors all around the particle being internalized. a good example of this 'zippering' kind of bacterium is listeria monocytogenes, where the bacterium interacts with host epithelial cells through its adhesins, inla and inlb, that bind host cell receptors e-cadherin and met, respectively [15] . live cell imaging using raw264.7 macrophages, a phagocyte model system, and forster's resonance en-ergy transfer sensor for the small, activated gtpases, adp ribosylation factors (arfs) showed that fcr-mediated phagocytosis accompanies dynamic and stepwise activation of arf6 and arf1 [16] . this correlated with activation of cdc42 and rac1 [17] . activity of the rho gtpases is presumably required for regulation of actin polymerization during phagocytic cup formation; simultaneous inhibition of both cdc42 and rac1 abolishes this. inhibition of these gtpases individually however only partially inhibits phagocytosis, suggesting they act in tandem during the process. these rho gtpases upon activation, recruit wasp and thereby the arp2/3 complex for actin nucleation and polymerization [18] . the action of gtpases is probably regulated by the composition of polyphosphoinositides (pips) of the phagosomal membrane. pips are also intimately linked to actin dynamics [19] . during early stages of cup formation, pi4,5p2 is enriched at the inner leaflet of the cup; during phagosome closure, rapid disappearance of f-actin at the base of the cup is correlated with the reduction in local concentration of pi4,5p2. consistent with this, overexpression of the pi-kinase (pipki), which converts pi4p into pi4,5p2 or inhibition of plc and phosphatidylinositol 3-kinase (pi3k, enzymes that produce ip3, dag, and pi3,4,5p3, respectively) abrogates phagosome closure [2, 20] . the shape of the phagocytic cup is usually dictated by the nature of ligand. macrophages, when subjected to an opsonized flat surface, exhibit pseudopod extension and abortive attempts to phagocytose the surface [21] . since the phagocytic cargo is large, sometimes as large as an apoptotic cell, internalization involves vesicular addition of membrane in the vicinity of cup. although the origin of this membrane remains controversial, the delivery of new membrane is dependent on the adaptor protein complex, ap1, and small gtpase, arf1 [22] , suggesting a secretory origin. the composition of phagosomal membranes has been extensively characterized. a proteomics approach helped identify more than 140 molecules present in or on latex bead-induced phagosomes in j774 cells [23] . these include, lysosomal proteins, lamp1, lamp2, lgp110, limpii; mitochondrial vdac1; several members of the ras superfamily of small gtpases, rabs (2, 3c, 5, 7, 11, and 14) ; as well as proteins found in late-endosomes, alix and flotillin-1. once formed, phagosomes are gradually acidified and the cargo is destined for degradation. this feature of phagosomes is sometimes manipulated by pathogens, prominently by bacteria [3] to evade degradation and gain intracellular access. further knowledge of the molecular machinery involved in phagosome formation will increase our understanding of the process as well as how www.cell-research.com | cell research sudha kumari et al. 259 npg it is manipulated by pathogens in a given context. macropinocytosis is another process whereby a relatively large amount of the fluid phase is engulfed with respect to the cell volume, in some instances along with particles such as bacteria. first described by lewis [6, 24] as large phase-bright organelles originating from plasma membrane (pm) ruffles, macropinosomes were initially thought to occur via non-selective membrane uptake. this view has changed with increased understanding of this process. owing to its specific characteristics such as its inhibition with na + /h + exchanger inhibitor, amiloride [25] , and dependence on growth factor (gf) receptor (gfr) signaling, macropinocytosis is now defined as a highly coordinated, triggered process with distinct molecular regulators. in a study performed using soil ameba, dictyostelium discoideum, an active phagocytic and macropinocytic organism, it was shown that during macropinosome formation, some membrane proteins were selectively depleted from the site at the pm [26] . it is now clear that there is sorting of membrane components during the formation of macropinosomes. macropinosomes are variable in size and can range from 0.2 to 10 µm in diameter [1] . the size and morphology of macropinosomes are typically independent of ligand, although their formation can be stimulated by agents such as phorbol esters, some pathogens, and gfs [25, [27] [28] [29] . in professional antigen-presenting cells, macropinocytosis operates constitutively in quiescent circulating cells such as immature dendritic cells and is downregulated after these cells begin to mature [6, 30] , suggesting a role in immune surveillance. as noted above, intracellular pathogens also exploit macropinocytosis as a means of entry into cells. pathogens such as shigella and salmonella inject virulence factors that modulate the cytoskeleton, induce ruffling and subsequently, macropinocytosis. infection rates of bacteria such as sphingomona [31] and viruses such as hiv type i [32] are reduced upon inhibiting macropinocytosis using amiloride. a common feature of macropinocytosis and phagocytosis is the dependence on actin-polymerization machinery. an archetypal example of gf-induced macropinocytosis is the upregulated fluid uptake in cells stimulated with epidermal growth factor (egf) [33] ( figure 1b) . activation of egfr signaling leads to an increase in general actin polymerization and cell ruffling. egf binding results in activation of rac1 gtpase as well as generation of the phosphoinositide, pi4,5p2, both of which together activate wasp/scar proteins. these in turn bind to the arp2/3 complex, hence modulating actin po-lymerization. most instances of macropinocytosis, with few exceptions (for example, bone marrow dendritic cells, where cdc42 is implicated [34] ), are dependent on rac1 activation). another molecular regulator of macropinosome formation is pak1 (p21-activated kinase); manipulation of pak1 activity correlates with macropinocytic extent in nih3t3 cells [35] . while pak1 is capable of phosphorylating and therefore activating cdc42/rac1, it also phosphorylates ctbp1/bars proteins. ctbp1/bars (c-terminal-binding protein-1/brefeldin a ribosylation substrate) proteins were originally demonstrated to regulate dynamin-independent fluid uptake in a variety of cell lines, and were later reported to localize to the site of and affect macropinosome membrane closure in a phosphorylation-dependent manner [36] . it is possible that bars proteins represent a link between dynamin-independent fluid endocytosis and macropinocytosis (see below). the nature of fission machinery involved in macropinosome scission from the pm is not yet clear. there is evidence that implicates isoforms of dynamin in specific contexts. although egf or serum-repletion-induced macropinocytosis in epithelial cell lines is insensitive to dynamin inhibition [37] , macropinocytosis is severely impaired in pdgf-stimulated dynamin-null embryonic stem cells [38] . in these embryonic stem cells, fluidphase tracer uptake, as an indicator of stimulated macropinocytosis, was rescued by reintroduction of dynamin-1 or 2 [38] , suggesting that dynamin is crucial for this form of macropinocytosis. however, at this stage it is not clear whether dynamin could serve as a scissoring protein for specific cases of macropinocytosis, or has other regulatory functions for endocytosis. dynamin is known to regulate cellular distribution of rac1 [39] , and therefore can indirectly influence macropinocytosis by regulating the availability of rac1, necessary for actin remodeling. even though phagocytosis and macropinocytosis differ in their nature of induction and detailed mechanisms, these processes share multiple operational similarities, pointing toward similar membrane remodeling pathways during internalization. both have slow kinetics, which incidentally enables the study of the formation of these vesicles in live cells. multiple studies have demonstrated localization of regulatory proteins to phagocytic cups and macropinosomes [16, 36] as well as the local and distinctive membrane composition during their formation [40] . both processes employ molecules such as the pi3k and other actin polymerization effectors, and are typically initiated by phosphoinositide-dependent processes. since dependence for endosome formation on the actin machinery is also exhibited by many other endocytic pathways, the distinctive feature of phagocytosis and endocytosis at a scale smaller than 200 nm poses specific demands on membrane machinery needed to bend membrane at this scale [41] . the eukaryotic cell appears to have resolved this in many ways. there are several modes of endocytic processes, distinguished by specific sets of molecular regulators and functional modules that are associated with their optimal operation. molecules involved in the generation of a vesicle can be part of a module assisting coat assembly, or involved in the pinching process, or may represent specific membrane lipids and lipid-modifying proteins. here we discuss these attributes and the endocytic processes that have come to be defined by the combinations of them. a convenient way of classifying some of the modes of microscale endocytosis is by the nature of the coat proteins that are associated with a specific process (table 1, figure 2 ). clathrin-mediated endocytosis initially identified in electron micrographs used to study yolk protein uptake in mosquito aedes aegypti, clathrin was one of the first endocytic coat proteins to be discovered as a major component of 'bristled vesicles' [42, 43] . studies on clathrinmediated endocytosis have dominated and defined the paradigm for endocytosis. morphological studies with clathrin indicated that it is capable of coating vesicles 100-200 nm in diameter. clathrin functions as a trimer of heterodimers, each unit consisting of one heavy and one light chain forming a triskeleton [44] . these triskelia can assemble into a lattice-like structure around the vesicles. adaptor proteins function to link up specific cargo with the clathrin coat [45] . simply put, clathrin-pit-mediated endocytosis involves cargo recognition and coat assembly, followed by membrane invagination, and finally pinching off of the dimpled deformation. although it was believed for a long time that clathrinmediated endocytosis is a cargo-induced process, it is now known that clathrin coats can spontaneously assemble at the pm and are stabilized by interactions with cargo [46] ; the presence of sorting motifs yxxf, dexxx-lli, fxnpxy, and polyubiquitination in transmembrane proteins that can bind to adaptor proteins often couple productive coat formation to capture of cargo [47] . clathrin-coated vesicle (ccv ) formation and its regulation have been reported to be associated with ~150 proteins, however, at this stage, with the exception of a few, their precise sequence of action during ccv formation remains unexplored. in a study carried out in live [48] uncovered the chronology of a few effector protein dynamics during the formation of a mature coat. more of such investigations will shed light on dynamics of critical molecular events that occur during ccv formation. imaging of clathrin-coat nucleation at pm suggests that it assembles randomly, mediated by cargo protein, ap, and accessory proteins [46] . in neurons, aps direct lattice formation to specific sites at pm via interaction with pi4,5p2 and synaptotagmin i and clathrin. this assembly can be further stabilized by epsin and ap180/ calm, which bind to ap2, pip2, and clathrin [49] . subsequent to this, stabilization of the clathrin-coated pit (ccp) requires cargo acquisition via subunits of ap2 and clathrin [46, 50] . this step proceeds via the recruitment of various ap2-binding partners that facilitate cargo binding, optimal clathrin polymerization, and membrane bending. the process of membrane bending is brought about in part by bar (bin amphiphysin rvs) domaincontaining proteins [9] , such as endophilin and amphiphysin, and is further facilitated by epsin and inherent curvature properties of assembled clathrin structures [51, 52] . mature clathrin-coated vesicle, while still attached to the pm, contains amphiphysin and recruits the pinching module that contains the protein dynamin. dynamin eventually promotes the scission of the vesicle (see later section for details). the prevalent view of clathrin assembly is of a homogenous, symmetric polygonal lattice [53] , the size of individual vesicle varying depending on the cell type of origin. ccps at the cell surface are of diverse composition with respect to the usage of adaptor and associated proteins [54, 55] , and offer distinct microenvironments for the regulated entry of specific combinations of cargoes [56] . interestingly, ligand concentration also affects the mode of this endocytic route: for example, egf, at low concentrations, is internalized by a clathrin-dependent endocytic route [57] , but at higher concentrations, seems to enter cells through clathrin-independent routes with consequences for signaling via the egfr [58] . how such fine-tuning and discrimination are regulated is a subject of current investigation [56] . several viral pathogens such as ebola virus, sars coronavirus, and some nonenveloped mammalian reoviruses enter cells by receptor-mediated endocytosis, targeting receptors internalized by the clathrin-dependent pathway [59] . for some viruses this route is obligatory, and for others the clathrin pathway is one of a few that support infection. studies on the entry of influenza virus in epithelial cells have indicated that a large fraction is endocytosed into ccps; viral particles are also endocytosed by non-clathrin pathway operating in parallel [60] . viruses that take a clathrin-dependent route more often induce formation of clathrin pit at the site of binding at the pm rather than entering a preassembled clathrin structure [59] . this suggests a flexibility in regulation of clathrin-coated vesicle formation. similarly, in case of bacterial uptake, the entry of the bacterium, l. monocytogenes, into hela cells was sensitive to depletion of clathrin heavy chain and other endocytic proteins such as dynamin, eps15, but not the adaptor ap2. this was unexpected because it suggests that cargo 20 times larger than a normal endocytic cargo could use the clathrinbased machinery to enter a non-phagocytic mammalian cell, reiterating the flexibility in clathrin-mediated endocytic processes, both in terms of size and associated machinery [61] . caveolin and the caveolar endocytic process another membrane coat at the cell surface is caveolin. caveolae were originally described as flask-shaped structures in ultrastructure of blood capillaries by palade in 1953 [62] , but it was not until much later that the coat component, caveolin, was identified [63] . caveolae are formed by assembly of caveolins, integral membrane proteins that bind directly to membrane cholesterol. there are three subtypes of caveolin proteins, caveolin-1, caveolin-2, and caveolin-3, the former two being responsible for caveolae formation in non-muscle cells and latter in muscle cells [64] . a recent study demonstrated that while morphological features of caveolae are widely conserved, the functions of individual caveolin proteins may be diverse [65] . many mammalian cell lines such as human colorectal adenocarcinoma cells, caco2 [66] , or tissues (e.g. many blood cell lineages) do not have detectable levels of caveolin, pointing toward the specialized tissuespecific function of caveolins. overexpression of caveolins in the cells lacking them induces de novo formation of caveolae [67, 68] . the precise function and regulation of caveolar assembly have been a matter of intense investigation. caveolins have a predicted hydrophobic stretch of potential hairpin structure composed of α-helices. insertion of transmembrane amphipathic helices in a wedge-like manner such that inner leaflet lipids are displaced more than outer leaflet lipids could induce membrane curvature [69] . moreover, caveolins are capable of oligomerization (caveolin-1 and caveolin-2) on membranes, which could further potentiate membrane curvature. consistent with this idea, the density and number of caveolae generally correlate with cellular caveolin expression [70] . caveolae formation is cholesterol dependent and loss in membrane cholesterol leads to disassembly of the caveolar structures. recent investigations have identified additional factors for the coat component of caveolae, polymerase i and transcript release factor (ptrf), and serum deprivation protein response (sdpr) [8, 65] . these molecules appear to be both part of the coat and necessary for the assembly of caveolae from distributed caveolins at the pm. loss of ptrf in mammalian cells as well as in zebrafish correlates with lack of caveolar structures [71] ; ptrf possibly acts by modulating relative amounts of caveolae-bound caveolin to free caveolin in the pm [71] , whereas sdpr is likely to be a protein that is required at the sites of caveolar assembly. the caveolar pathway is responsible for the endocytosis of ligands such as albumin [72] , autocrine motility factor [73] , tetanus toxin [74] , cholera toxin [75] , and www.cell-research.com | cell research sudha kumari et al. 263 npg viruses such as polyoma and simian 40 (sv40) [76, 77] ; to name a few. in many of these cases the receptors for specific cargoes have also been identified but the mechanism by which cargo is concentrated in caveolae is not understood. in addition, endocytosis via caveolae also remains poorly characterized. it is not clear if caveolar endocytosis is constitutive, albeit occurring at a low rate, and is massively up-regulated on specific induction. fluorescence recovery after photobleaching studies has indicated caveolae to be relatively stable structures on pm [78] , which can however be made to bud off by the interaction of pathogens such as sv40 virus [79] or can also be induced by incorporation of fluorescent analogues of sphingolipids, namely, lactosyl ceramide [80] [81] [82] . during integrin-dependent cell adhesion, deadherence induces removal of caveolin-coated membrane from the cell surface, and it has been shown that caveolin is required for this process [83] . this results in altered pm lipid composition, due to the loss of many lipids and proteins that partition into detergent-resistant membrane (drm) domains. this has significant functional consequences foremost of which is loss of rac1 activity at the cell surface, and consequently remodeling of the cortical cytoskeleton, and activation of anoikisis, a form of apoptosis induced by the loss of cell anchorage [83, 84] . caveolin knockout mice have proven to be useful genetic tools for studying the function of caveolin and ascertaining caveolar cargo specificity [85] . in mouse embryonic fibroblasts (mefs) obtained from caveolin-1 knockout mice, albumin uptake was found to be defective [86] , but the entry of sv40 [87] and cholera toxin b subunit (ctxb) [88] , two typical cargoes of caveolae, was not compromised. this could be explained by the possibility that caveolin, when present, induces formation of caveolae, but is dispensable for internalization of some caveolar cargoes. in addition, there is evidence that caveolins can negatively regulate entry of certain cellsurface proteins, including ctxb [89, 90] , where overexpression of caveolins prevents their internalization, suggesting that apart from their direct role in endocytosis, caveolins can indirectly influence other endocytic pathways. a novel role for caveolin-1 has been reported, in regulation of cdc42 activity during membrane trafficking events. caveolin-1 binds preferentially to cdc42 in its gdp-bound form and functions as a guanine nucleotide dissociation inhibitor (gdi) for cdc42. thus, by maintaining cdc42 in an inactive state, caveolin-1 regulates basal secretion in the absence of stimuli in pancreatic β-cells [91] . it is possible that caveolin-1 has similar indirect regulatory roles at pm via inhibitory interactions with mediators of other endocytic pathways. endocytosis of sv40 virus has been reported to increase in cells lacking caveolin-1 [87] , indicating an inhibitory role for caveolin-1 in this process. however in this study it was not clear whether this effect is due to squelching of effector molecules or due to sequestration of a specific kind of membrane components. studies exploring redundant endocytic roles of caveolin will be invaluable in understanding the regulatory cross-talk between caveolar and non-caveolar internalization pathways. once a coated patch of membrane has been generated, the formation of an endocytic vesicle requires fission of the budded membrane from the parent membrane. a large gtpase, dynamin, has been implicated in multiple fission processes in eukaryotic cells [92] [93] [94] . there are three dynamin genes identified in mammalian cells, each gene expressing at least four multiply spliced forms (isoforms). dyn1 is restricted to neuronal cells; dyn2 is ubiquitously expressed; and dyn3 may be limited to brain, lung, and testis tissues [95] . a wide variety of endocytic processes, including clathrin-dependent receptor-mediated endocytosis, require dynamin for the scission of endocytic intermediates to generate vesicles (figure 2a) . originally, dynamin was identified as a microtubule-binding protein and was found to be homologous to the drosophila shibire gene [96, 97] , mutations in which were shown to cause a temperature-sensitive paralysis. insights into dynamin function came primarily from morphological analysis of synapses in the shibire ts1 mutant. cycling of mutants through restrictive and permissive temperatures, koenig and ikeda observed a reversible loss of synaptic vesicles at neuromuscular synaptic junctions, implicating dynamin in synaptic vesicle endocytosis [16] . overexpression of dominant negative, gtp-binding mutants of dynamin also blocked receptor-mediated endocytosis in various cells, suggesting a role for the gtpase activity of dynamin in the clathrin-dependent endocytic process outside the nervous system. dynamin was found to localize to ccps at the pm and in isolated synaptosomal preparation [98, 99] , providing further evidence of a direct role for dynamin in endocytosis. dynamin is likely to be recruited during clathrin-pit assembly, a process where n-bar domain-containing proteins, such as amphiphysin, which can directly interact with dynamin [100] , are also recruited. at the molecular level, dynamins contain a pleckstrin homology (ph) domain, a gtpase effector domain (ged), and a cterminal proline/arginine-rich domain (prd). ged can interact with the gtpase domain of an adjacent dynamin molecule thus causing oligomerization of the protein [101] , and concomitant activation of the gtpase activity. the prd of dynamin can interact with a variety of proteins, including the ones that contain src homology sh3 domain, and ph domain binds to pi4,5p2 phosphoinositides. wide range of interaction partners for dynamin enables it to recruit and bind proteins during coated vesicle formation. at high local concentrations, dynamin is also capable of forming multimers and spirals on membranes. dynamin oligomerization and dynamics lead to constriction and eventually budding of the vesicle. the exact function of dynamin at the level of vesicle fission has been a subject of controversy. there are various hypotheses regarding the gtp-dependent mechanism of dynamin action for vesicle pinching. first is the conformational change in dynamin brought about by gtp loading (switch model), which can cause scission by recruiting other molecules that will fuse membranes and acts essentially as a timer for this reaction, whereas the other relies on the actual mechanical force generated at the vesicle neck (pinchase model). while there is evidence for both the gtp switch and mechanoenzyme functions of dynamin [102] , recent studies support a mechanoenzyme or motor-like function of dynamin. the in vitro demonstration of dynamin-coated lipid tubules twisting in response to supply of gtp was a direct evidence supporting a mechano-enzyme behavior where the authors argue that in addition to membrane tension, torsional strain generated at the neck of the vesicle can lead to pinching [103] . two other studies demonstrated that it is the kinetics of dynamin depolymerization and polymerization on 'tension-free' membranes that brings about changes in membrane curvature, necessary for fission [104, 105] . at this point both these possibilities have distinct implications on the dynamics of dynamin action during pinching as discussed [106] . apart from acting as a part of the scission machinery for the clathrin-dependent pathway, dynamin function is also implicated in vesicle formation in a set of clathrinindependent pathways, including caveolar endocytosis [107] and a class of macropinocytosis [38, 108] . other clathrin-independent pathways that utilize dynamin are the rhoa-dependent il-2 receptor endocytic route [109] and app endocytosis in primary neurons [110] . although ultrastructural studies indicate that il-2 receptors are not recruited to ccps, they were internalized by small uniform-sized invaginations. the uniform and small size (50-100 nm) of the forming endocytic pit at the surface that appears to concentrate the cargo suggests the presence of a protein coat that could aid in the recruitment of these proteins to the site of endocytosis. a lot more information is necessary to uncover the mechanism of operation of this type of endocytosis. similarly, there exists an unusual mode of endocytic clearance of egfr from dorsal surface of migratory epithelial cells. here, cells on stimulation with egf generate circular wave-like ruffles that on concentric closure internalize ~50% of cell-surface egfr. this process employs dynamin 2 but is independent of clathrin or caveolins [111] . overall, the function of dynamin seems neither to be strictly dependent on the presence of clathrin on buds, nor is limited by the size of invaginations. as indicated above, the mechanism by which dynamin is recruited to these endocytic sites at pm is unclear, and ought to be the subject of further investigations. however, a general theme that emerges is that most coat-dependent mechanisms of endocytosis use dynamin to assist in scission of the membrane deformation from the cell surface. on the other hand there are a number of coatindependent endocytic processes that appear to function in the absence of dynamin (table 1) . at this time there are a number of endocytic pathways where neither specific coat proteins nor a particular pinching system have been identified ( figure 2b ). in fact, the existence of a coat-independent route for endocytic uptake has taken a long time to be well accepted; careful experimentation coupled with genetic manipulations provided the necessary evidence. for example, the acceptance of clathrin-independent endocytosis was built on a number of studies: (i) injection of african green monkey kidney cells (cv1) with anti-clathrin antibodies abolished receptor-mediated uptake completely, but inhibited the uptake of a bulk phase marker (lucifer yellow) by only 40% [112] , and (ii) clathrin-dependent endocytosis could also be inhibited by hypotonic shock, cytosolic potassium depletion, or acidification of cytosol while under all these conditions, endocytosis of the plant toxin ricin continued to take place [113, 114] . together, these and other experiments paved the way to the idea that cells could exhibit one or more clathrin-independent endocytic routes. in some instances, ultrastructural visualization of endocytic intermediates did not reveal electron-dense coats [88] , suggesting that either these pathways do not have a proteinaceous coat or the coat assembly, if any, is too transient to be captured by current methodologies of cell fixation and processing. it is also possible, however, that lipid or protein accretions could initiate membrane deformation (vesicle formation) without defined coat proteins. while the pathways that require coats also have their specific lipid requirements [115, 116] , in some instances, lipid organization alone has proven to be sufficient for membrane invagination. shiga toxin entry is one such example where binding of toxin to the ganglioside, gb3, www.cell-research.com | cell research sudha kumari et al. 265 npg induces invaginations in cells as well as model membranes [117] . this was specific to toxin binding, since simply cross-linking gb3 with antibodies failed to result in invaginations. these studies provide evidence for the requirement of a specific lipid-binding architecture for this process. a lipid-compaction model was proposed for the segregation and membrane budding induced by the toxin. this process was inhibited by increasing membrane tension, indicating that membrane properties, tension, and bending rigidity might play a crucial role in the regulation of lipid-based invaginations. endocytosis of the lipid-anchored proteins such as glycosylphosphatidylinositol-anchored proteins (gpi-aps) also does not appear to require any of the well-characterized coat proteins [64, 88, 118, 119] . this pathway could be another example where the foundation for membrane deformation is laid by membrane lipids (see the clic/geec pathway below). an example where a protein platform suffices to support endocytosis is the process of human papillomavirus 16 (hpv16) virus infection, where tetraspanin-enriched domains are required for virus entry [120] . it was widely accepted that dynamin would be indispensable for all endocytic activities in the cell. in part this notion had been fuelled by the existence of a number of dynamin genes, and isoforms/splice variants [95, 121, 122] . however, this notion was difficult to reconcile with a few key observations: (i) overexpression of dominantnegative mutant form of dynamin-2 in hela cells [98] inhibited clathrin-dependent endocytosis but could not completely block fluid phase uptake, and (ii) hemocytes derived from the temperature-sensitive shibire mutants of drosophila exhibited a complete block in clathrindependent endocytosis of scavenger receptors, whereas fluid-phase and gpi-ap uptake remained unaffected [123] . the dynamin gene mutation in drosophila renders all dynamin splice forms mutant since the mutation lies in the common exon encoding the gtpase domain [124] . to assess a role for dynamin in a pathway, most studies have used dominant-negative inhibition of dynamin to test concomitant quantitative defects in endocytic cargo uptake. interpretation of such experiments is however, complex. it is difficult to ascertain whether lack of defect is due to lack of involvement of dynamin, or due to an alternative internalization pathway that is induced by inhibition of dynamin. alternatively, the ability of dynamin mutants to interfere with the activity of rac [39] suggests that inhibition of a particular pathway may not reflect a direct role for dynamin. from the existing examples under the canopy of dynamin-independent pathways, there are also specific requirements for small gtpases such as the arf family proteins, and the rho family proteins (table 1) . these gtpases could function as timers for regulation of the assembly and disassembly of actin-based endocytic mechanism or as scaffold to recruit other regulatory proteins. however, it is evident that while these gtpases can be utilized in combination, they have a diversity of functions not solely restricted to endocytosis [109, 125] . there is a growing understanding of the molecular mechanism involved in the operation of different endocytic pathways and the role for the actin cytoskeleton in its regulation [126] (see table 1 and the following sections). this necessitates a new understanding of the role that actin and its associated molecular network may play in different aspects of the endocytic process. actinmeshwork could function as a coat and/or help in the recruitment of adaptor proteins, thus allowing for cargo concentration and membrane deformation, leading to the formation of membrane buds. in addition, coupled with motor protein activity and dynamic polymerization, it could play a role in the generation of a scission force. in this context, extensive genetic, biochemical, and morphological studies in budding yeast have provided valuable insights. in a mutant screen for endocytic defects in yeast, a large number of identified genes were related to actin and its regulators [127] ; subsequently, the recruitment of actin to the sites of endocytosis was elegantly demonstrated in live cells [128] . one of the first identified genes involved in α-factor uptake was act1 (end7), the yeast actin gene. nearly a third of endocytic proteins identified in yeast bind actin or regulate its assembly. a specific kind of actin structure, called 'cortical actin patches' has been observed to be associated with endocytic sites. using real-time fluorescence microscopy and particle tracking, drubin and colleagues [129] demonstrated that there are different cortical actin patcheswith distinct protein composition and motility properties; and these were intermediates in the endocytic process. subsequently, using a live imaging screen of deletion mutants, where they dissected defects in coat dynamics and actin assembly, they provided evidence for the existence of distinct protein modules for coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin-or actin-mediated endocytosis [130] . in the same study, they also demonstrated that, in yeast, while clathrin facilitates endocytic site assembly, it is not required for membrane invagination or vesicle formation. based on these studies, a fission mechanism for such a pathway has been recently proposed [131] . this model suggests that endocytosis can be initiated at a site where the membrane invaginates, and then elongates into a tube through the encasement by active polymerization of actin, culminating in the scission of a membrane bud of different membrane and protein compositions via traction by myosin motors [132] . considering that clathrin and its effectors also associate with these vesicles, it is not yet established if actin alone acts as the coat [133] . in mammalian cells, specifically nih3t3 cells, actin and cortactin are asymmetrically associated with highly dynamic early endocytic structures and move correlatively with these vesicles, supporting the notion that actin could generate the force required for vesicle movement and thus fission [134] . disruption of the actin cytoskeleton also causes a reduction in endocytosis in a number of clathrin-independent pathways [64, 135] . large vesicles formed as a result of compensatory endocytosis in xenopus oocytes also have been shown to have actin coats around them, although the precise function of actin there has not been addressed [136] . it should be, however, noted that actin is a multifunctional structural protein and perturbation in actin and actin nanomachinery can also result in non-specific and indirect defects in endocytosis; nevertheless colocalization of f-actin with endosomes indicates a more direct role in endocytosis. in the following sections we discuss some of the pathways that utilize actin and its regulators, in further detail. the clic/geec pathway as discussed above, endocytosis of gpi-aps represents a clathrin-and dynamin-independent internalization route. gpi-aps are outer leaflet membrane proteins that lack cytosolic extensions and thus are unable to directly bind cytosolic adaptors. these proteins belong to a family of proteins with diverse functions and have been shown to be internalized by a dynamin-independent endocytic route, into a specialized early endosomal compartment, where a majority of the internalized fluid phase is also detected in many cell types [119] . endocytic structures containing nascent endocytosed gpi-aps are named gpi-ap enriched early endosomal compartments (geecs). geecs are proposed to result from fusion of uncoated tubulovesicular clics (clathrin-independent carriers) directly derived from the cell surface [88] . clics/geecs are selectively enriched for gpi-aps [119] although at this stage the sorting determinants at pm are not clear. it could potentially be lipid-based sorting, as alterations in cholesterol and sphingolipid levels as well as perturbation of the nanocluster state of the gpi-ap affect endocytosis through this pathway [64, 119, 137] . however, a recent study shows the size of extracellular moiety attached to the lipid tail as an additional determinant for inclusion into geecs [138] . it will be interesting to investigate the size range of proteins that allows them to be endocytosed via geecs and also, how the size-based regulation crosstalks to lipidmediated sorting into the geec pathway. another feature of endocytosis via clics/geecs is its dependence on the actin polymerization machinery [64] ; maintenance of dynamic cortical actin architecture appears to be required for geec endocytosis. this is brought about by the cycling of the rho family gtpase cdc42 and downstream activation of the wasp protein, since locking of cdc42 in gdp or the gtp state leads to inhibition of the geec pathway [135] . cdc42 dynamics on pm is regulated by arf1; activation of arf1 leads to recruitment of a rho-gap domain-containing protein, arhgap10, and inactivates cdc42, thus maintaining its cycling [135] . another recently reported effector of geec formation is a bar domain-containing protein, gtpase regulator associated with focal adhesion kinase-1 (graf1). endogenous graf1 localizes to clic/geec carriers in hela cells and silencing its expression results in reduced geec marker uptake [139] . graf1 colocalizes with active cdc42 and contains a rho-gap domain, so it may be involved in regulating cdc42 dynamics apart from its other functions. graf1 also contains a bar domain, which could aid in membrane curvature and an sh3 domain, capable of interacting with dynamin gtpase. it is interesting to note that while geec formation is dynamin-independent, dynamin appears to be recruited to geecs post internalization [88] ; this could potentially be mediated by geec-associated proteins such as graf1, which has been shown to interact with dynamin. although there is increasing information about the molecular regulation of geecs, the functional relevance of this pathway remains unexplored. since a variety of gpi-aps are internalized by the geec pathway, and in some cases endocytosis via geecs specifically aids in accomplishing their cellular function [140] , this pathway could be of vital importance for this whole family of proteins. considering its constitutive nature, fast kinetics and heterogeneous resultant vesicle size, the geec pathway could mediate the regulation of pm homeostasis. there are an increasing number of proteins internalized by pathways that bear a resemblance to the geecs. endocytosis of cholera toxin [88] , internalization of vaca toxin, and sorting nexin 9 (snx9)-regulated gpi-ap internalization routes are examples similar to geecs [141, 142] . another cargo for a geec-like pathway is dysferlin, a muscle repair protein, mutations in which are associated with several myopathies. in mefs devoid of caveolin-1, dysferlin is endocytosed into compartments containing gpi-aps and its cellular entry is independent of dynamin activity, indicating that internalization could potentially be carried out via the geec pathway [89] . arf6-dependent pathway cell-surface proteins such www.cell-research.com | cell research sudha kumari et al. 267 npg as mhc i and tac (the il-2 receptor α-subunit) are internalized in a dynamin (and clathrin)-independent manner into an arf6-positive tubular endosomal system. these are distinct from the clathrin-dependent cargo-containing endosomes [143] . overexpression of constitutively active form of arf6 was shown to increase endocytosis of tac; however, inhibition of arf6 via overexpression of a gtp-exchange defective form does not perturb endocytosis [143] . there have been several additions to the cargoes and effectors of this pathway, recently reviewed [144] . endosomes-containing cargo internalized via this pathway, remain associated with activated arf6 [145] ; hyperactivation of arf6 traps a pip2-enriched early endosomes of a vacuolated morphology, while its constitutive inactivation blocks the process of recycling back to pm from tubular compartments. recently, candidates for cargoes endocytosed via the arf6-pathway were identified using hyperactivated arf6-trapped endosomes [146] . while arf6 associates with endosomes containing a specific set of cargoes, it is not clear whether it is involved directly at the internalization level or the recycling step or both. the localization of arf6 on endosomes can result both from stable association during endocytosis and acquisition on endosomal membrane post-internalization. while arf6 regulates dynamindependent endocytosis of the herpes simplex protein vp22 [147] , it has also been demonstrated to control the recycling of membrane [148] . it will of interest to visualize the localization of arf6 in the context of cargo endocytosis at pma. although the arf6 pathway bears a resemblance to the geec pathway, it is likely that these two processes are distinct modes of endocytic trafficking. perturbation of graf1 in hela cells causes inhibition of fluid uptake (a geec marker which is internalized into tubular endosomes as well), but it neither affects mhc i internalization nor does it associate with mhc i-positive structures [139] . an important function of this pathway may be to regulate membrane availability for migratory cells, and recycle activated small gtpases to the pm [149] . it is also responsible for recycling of a prohormone sorting receptor, carboxypeptidase e, to the golgi [150] . role of flotillins flotillins appear to outline a dynaminand clathrin-independent endocytic process, since cargoes including fluid phase and a gpi-ap, cd59, in hela cells are endocytosed via this pathway [151] . in the cells depleted of flotillin-1, there is a defect in uptake of gpi anchor protein cd59, and ctxb internalization is shifted to a dynamin-dependent mode. however, the relationship of this pathway to the geec/clic pathway in other cell types is not yet established; a role for cholesterol and specific rhogtpases remains as yet unaddressed. there are two flotillin genes, flotillin-1 and flotillin-2 (identical to reggie-2 and 1, respectively) [152] . in live cells, flotillins are localized to small puncta. flotillin-1 and 2 are both required for induction of membrane invaginations in a dose-dependent manner [153] . similar to the caveolins, flotillins are associated with drms [23] . while flotillins were shown to form heteromeric complexes with caveolins, as demonstrated by co-immunoprecipitation experiments, it is yet unknown if this interaction is functional in the context of caveolae formation [154] . in mefs lacking caveolin-1, while majority of flask-shaped invaginations are absent, there are some structures that remain. this suggests a role for other molecules in the formation of caveolae (flask)-shaped invaginations [155] . by em and immunogold labeling, flotillin appears to decorate small membrane invaginations distinct from clathrin-or caveolin-1-labeled pits. however, in a recent study in fibroblasts lacking caveolin-1, overexpression of flotillin isoforms, flotillin-1 and 2 could not induce flask-shaped invaginations, suggesting that factors more than flotillins may be necessary for the generation of the flotillin-decorated invaginations [156] . a recent study has implicated flotillin-1 in cxcr4 recruitment to drms and signaling [157] . it is possible that flotillins generate specialized surface platforms that aid in the regulation of signaling of immunoreceptors and their endocytosis. the trigger (if any) for flotillin-mediated endocytosis has recently been shown to be initiated by fyn-mediated phosphorylation, suggesting that this endocytic process is regulated by non-receptor tyrosine kinase activation [158] . in summary, while flotillins may define a dynamin-independent endocytic process, the studies thus far raise interesting questions regarding the function of endogenous flotillins, their means of interacting with specific cargo and the mechanism of vesicle generation. role of tetraspanins another protein platform that supports endocytosis is the family of tetraspanins. dynamin-independent endocytosis of hpv16 is localized to tetraspanin domains (cd63, cd151) of the pm and cd151 knockdown in cells blocked hpv16 entry [120] . this mode of endocytosis is distinct from other dynaminindependent routes in that, it is not inhibited by perturbation of cholesterol levels in the cell [120] , nor does it rely on cellular levels of clathrin, caveolin, and flotillin. however, exact steps in this endocytic mechanism, as well as the associated molecular players, are still unclear. recently, it was demonstrated that endocytosis of a tetraspanin, cd82, is dynamin-independent and occurs via a cdc42-dependent geec-like pinocytic pathway [159] . it raises an interesting possibility that while tetraspanins regulate a distinct cholesterol-independent internalization route, they may utilize a different endocytic pathway for their own cellular entry [159] . another such example, where inhibition of dynamin and cholesterol depletion does not affect uptake is the antagonist-induced internalization of nicotinic acetylcholine receptor [160] . binding of the antagonist α bungarotoxin causes slow internalization of nachr, which proceeds via activation of rac1 gtpase; inhibition of rac1 activity or actin polymerization impairs the process [135] . these observations suggest that specialized protein-centric molecular platforms could assemble in the membrane to aid in this form of endocytosis. there are now numerous examples of dynamin-independent endocytic systems [9, 114, 161, 162] that offer ample variety in terms of molecules employed and cargo endocytosed. further study will enrich our understanding of principles behind mechanisms of endocytosis. undoubtedly, there are a variety of endocytic pathways available at the pm. based on the environmental adaptations and molecular divergence, different kingdoms seem to have evolved specializations in endocytosis. in prokaryotes, evidence for a well-defined endocytic system is missing. a recent report using cryoelectron tomography showed various stages of the potential formation of membrane buds, including magnetosomes with inner membrane in magnetospirillum magneticum [163] . these structures are reminiscent of endocytic membrane buds in higher organisms [163] . in unicellular eukaryotes, some subgroups have been shown to have spatially defined distribution of endocytic proteins, and specialized structures for endocytosis. for example, in paramecium, endocytosis is confined to the cytopharynx, in trypanosoma, assembly of endocytic machinery is restricted to flagellar pocket and in euglenoids, the ingestion tubule serves as the site of endocytosis [164] . in unicellular eukaryotes such as fungi, owing to the availability of genetic, pharmacological and biochemical tools, endocytosis (especially in the budding yeast, saccharomyces cerevisiae) has been dissected in great detail [128] , as discussed earlier. riezman and colleagues had identified several endocytic mutants (end mutants) that were defective in internalization of the α-factor [165] . emr and colleagues had isolated dim (defective in internalization) mutants, defective in internalizing fm464 dye [166] . a quantitative genome-wide screen performed in yeast recently, examined the internalization of the vamp homologue, snc1 [167] . the screen uncovered novel regulators of endocytosis. it also uncovered phenotypes for predicted endocytosis genes for which no defects had previously been reported. apart from these fairly extensive studies in s. cerevisiae, initial ultrastructural studies in neurospora and membrane dye uptake assays in pisolithus tinctorius yielded no recognizable endocytic structures [168] . however, different species of ustilago were found to contain homologs of endocytic proteins in their genome and could internalize lucifer yellow and the membrane marker fm4-64, suggesting the operation of endocytic pathways. these studies were carried out in cells after removal of the cell wall, and were therefore subject to validation in hyphae with an intact cell wall [169] . similarly in aspergillus nidulans, a cluster of vesicular structures at the tip of hyphae called spitzenkörper were identified [170] . there seem to be differences in endocytic machinery in filamentous fungi and budding yeast. for example, internalization relies on arf6 activity in a. nidulans, whereas in yeast, arf6 is involved in polarity and bud growth but not in endocytosis [170, 171] . in invertebrates, drosophila has been most extensively used to study endocytosis. a limited variety of endocytic routes are reported to operate in drosophila, typically demonstrated in cultured hemocytes, isolated wing disc, oocyte nurse cells, and cultured neurons and pericardial cells for selected cell-surface proteins, primarily signaling receptors and bulk volume components [123, 172, 173] . recently, increased usage of cultured cell lines derived from drosophila, and advancement in techniques of genetic manipulation using dsrna, have made it feasible to study endocytosis and other membrane-trafficking processes on a genome-wide scale [85, 174] . in nematodes, typified by c. elegans, a plethora of genes involved in yolk protein endocytosis and trafficking were uncovered in a genome-wide screen [175] . the function of these genes was further validated in mammalian cells, where mutation in some of these genes replicated transport defects as were observed in c. elegans. these studies also indicated that components of the molecular machinery underlying membrane trafficking may be conserved across systems. in a study that carried out extensive phylogenetic analysis of caveolin genes in metazoan (which provided evidence of extensive gene duplication), it was observed that vertebrate caveolin-1 and caveolin-3 isoforms and an invertebrate caveolin (apis mellifera) were able to form morphologically identical caveolae in caveolin-1 null mouse cells, while the c. elegans caveolin could not. this indicates diversity of function in the caveolin gene family [156] . primary and immortalized mammalian cell lines have proven to be an invaluable system to study endocytosis. while some internalization routes seem to be conserved in primary and immortalized cell lines, it is not certain whether their kinetics is conserved and if immortalization has consequences on overall endocytic activity of a cell type. genetic knockout of known endocytic proteins in mice allows for the derivation of primary fibroblasts that are now routinely used to dissect the process [86] . these fibroblasts serve as excellent tools to study the involvement of candidate proteins in endocytosis -this approach not only avoids complexities associated with overexpression of their dominant-negative isoforms and off-target effects, but also can reflect on the functional redundancy of a given protein in the system. in plants, taking into account the cell wall as a physical barrier to pm accessibility, and the high intracellular vacuolar pressure that builds up turgor pressure, the very existence of endocytosis was debated for a long time. in a study using electron microscopy, pm-associated invaginations containing electron-dense tracer and clathrin coats were visualized [176] . more recently, uptake of fm dyes was demonstrated in plant cells [177] , following which many studies reported endocytosis of membrane markers and specific proteins in plant root hair cells, pollen tube, and cultured tobacco cells [178] . now there is accumulating evidence about the diversity of endosomal pathways and endocytic compartments in plants. although most of the studies used fm dyes to monitor internalization in plants, advancements in probing techniques are revealing a diversity of endocytic routes in this kingdom. apart from clathrin-dependent receptor-mediated endocytosis, actin-sensitive fluidphase endocytosis in inner cortex cells of maize root [179] and pi3k-dependent bulk phase sucrose endocytosis in suspension culture cells of sycamore have been reported [180] . blockade of clathrin-mediated endocytosis using ikarugamycin (ika) did not inhibit uptake of positively charged nanogold particles, suggesting the operation of a clathrin-independent pathway [181] . however, in another study, dhonukshe et al., showed that total membrane uptake in by-2 tobacco cells could be inhibited by inhibiting clathrin using clathrin hub dominant negative form, suggesting that clathrin is absolutely required for any form of endocytosis [182] . it is possible that a diversity of endocytic portals are conserved in plant and animal systems, and several modes of endocytosis operate in plant cells in a cell type-and tissue-specific manner. given the diversity and complexity of endocytic routes available in different cellular context, we provide here only a simplified overview of these pathways. detailed understanding of the functioning of an individual pathway is complex in terms of its associated molecules and mechanism, and will require an individual focus. it is likely that there are many different biochemical and physical principles behind the formation of vesicles in cell membrane. for example, cell-surface proteins such as the gpi-aps, which lack cytosolic extensions, cannot directly associate with cytosolic coat and adaptor proteins. these proteins therefore need to utilize radically different principles for sorting, membrane deformation, and vesicle generation at pm. similarly, much less is known about endocytosis of membrane lipids. dissection of the endocytic process of lipids will give insights into a novel way of vesicle formation. also considering the multiplicity of functions of proteins, it will be interesting to investigate the overlapping functions of molecules known to be associated with specific trafficking pathways. the identification of an endocytic pathway as distinct has been primarily based on associated cargo proteins or lipids, and molecular regulators; the contribution of kinetics and detailed physical mechanism to such categorization is not generally available except in some wellcharacterized situations, namely clathrin-pit endocytosis or endocytosis by actin-dependent forces in yeast. considering the limited knowledge and vast diversity in molecular players that are employed for efficient functioning of endocytic trafficking pathways, it is difficult to clearly demarcate these mechanisms from one another. on a cautionary note, it should be recognized that numerous cell lines have been utilized to study different types of endocytosis. these studies have helped uncover complex endocytic networks in a given cell system. the extent to which observations in one cell system may be extrapolated to another, or whether basic components of specific endocytic pathway are conserved across cell lines, is not completely clear. the possibility that modes of immortalization of cell lines itself could have consequences on how the endocytic pathways are configured in these cells has not been addressed. extensive work has been carried out to understand and elucidate the mechanism of endocytosis in some eukaryotes, and a considerable amount of information is available in some free-living eukaryotes and pathogens [183] . the integration of known information from complex endocytic systems in metazoan and comparison with uptake mechanisms in other eukaryotes could provide a means of understanding the scope of evolution, conservation, and redundancy in the internalization process across phyla. furthermore, whether specific endocytic modes are conserved in their entirety across phyla is also a subject of lively debate [183] . what is clear is that cells have many ways to endocytose material from the extracellular milieu, and perhaps as many reasons to do it by a particular mode. until we understand the basic principles behind cargo concentration, membrane deformation, and scission in each of these modes it will be difficult to provide any rationale for the proliferation of the mechanisms of entry. regardless, the endocytic process, in addition to its role in uptake of nutrients and fluid, influences diverse key processes in metazoans, such as establishment of cellular asymmetry and modulation of signaling. in addition to its physiological roles, the endocytic process is also exploited by various pathogens. insights into the underlying mechanisms will reveal new targets for drug design, and selective employment of these entry routes for drug delivery will also allow for the manipulation of cellular endocytic pathways to alleviate effects of specific diseases. shaping cups into phagosomes and macropinosomes molecular mechanisms of phagocytic uptake in mammalian cells phagosome maturation: going through the acid test fusion, fission, and secretion during phagocytosis the phagosome: compartment with a license to kill defining macropinocytosis macropinocytosis: regulated coordination of endocytic and exocytic membrane traffic events sdpr induces membrane curvature and functions in the formation of caveolae mechanisms of endocytosis studies of the macrophage complement receptor. alteration of receptor function upon macrophage activation phorbol esters cause sequential activation and deactivation 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transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules localization of autocrine motility factor receptor to caveolae and clathrin-independent internalization of its ligand to smooth endoplasmic reticulum lipid rafts act as specialized domains for tetanus toxin binding and internalization into neurons cholera toxin is found in detergent-insoluble rafts/domains at the cell surface of hippocampal neurons but is internalized via a raft-independent mechanism bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae endocytosis via caveolae caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the er selective caveolin-1-dependent endocytosis of glycosphingolipids the glycosphingolipid, lactosylceramide, regulates 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synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval reversible blockage of membrane retrieval and endocytosis in the garland cell of the temperature-sensitive mutant of drosophila melanogaster, shibire ts1 mutations in human dynamin block an intermediate stage in coated vesicle formation differential distribution of dynamin isoforms in mammalian cells identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules multiple forms of dynamin are encoded by shibire, a drosophila gene involved in endocytosis induction of mutant dynamin specifically blocks endocytic coated vesicle formation tubular membrane invaginations coated by dynamin rings are induced by gtp-gamma s in nerve terminals tandem arrangement of the clathrin and ap-2 binding domains in amphiphysin 1 and disruption of clathrin coat function by amphiphysin fragments comprising these sites regulatory mechanisms of dynamin-dependent endocytosis dynamin: switch or pinchase? gtp-dependent twisting of dynamin implicates constriction and tension in membrane fission real-time visualization of dynamincatalyzed membrane fission and vesicle release gtpase cycle of dynamin is coupled to membrane squeeze and release, leading to spontaneous fission the long and short of membrane fission dynaminmediated internalization of caveolae negative guidance factorinduced macropinocytosis in the growth cone plays a critical role in repulsive axon turning interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway internalization of beta-amyloid peptide by primary neurons in the absence of apolipoprotein e a novel endocytic mechanism of epidermal growth factor receptor sequestration and internalization inhibition of endocytosis by anti-clathrin antibodies inhibition of coated pit formation in hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin regulated portals of entry into the cell distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate shiga toxin induces tubular membrane invaginations for its uptake into cells folate receptor endocytosis and trafficking gpi-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway clathrin-and caveolin-independent entry of human papillomavirus type 16 -involvement of tetraspanin-enriched microdomains (tems) analysis of dynamin isoforms in mammalian brain: dynamin-1 expression is spatially and temporally regulated during postnatal development three dynamin-encoding genes are differentially expressed in developing rat brain shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of drosophila hemocytes probable mechanisms underlying interallelic 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through clathrin-independent endocytosis cellular internalization of green fluorescent protein fused with herpes simplex virus protein vp22 via a lipid raft-mediated endocytic pathway independent of caveolae and rho family gtpases but dependent on dynamin and arf6 arf6 and microtubules in adhesion-dependent trafficking of lipid rafts activation of arf6 by arno stimulates epithelial cell migration through downstream activation of both rac1 and phospholipase d recycling of raft-associated prohormone sorting receptor carboxypeptidase e requires interaction with arf6 flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells dissecting the molecular function of reggie/flotillin proteins coassembly of flotillins induces formation of membrane microdomains, membrane curvature, and vesicle budding flotillins/cavatellins are differentially expressed in cells and tissues and form a hetero-oligomeric complex with caveolins in vivo. characterization and epitope-mapping of a novel flotillin-1 monoclonal antibody probe molecular mechanisms of clathrinindependent endocytosis evolutionary analysis and molecular dissection of caveola biogenesis cxcl12-induced partitioning of flotillin-1 with lipid rafts plays a role in cxcr4 function endocytosis of flotillin-1 and flotillin-2 is regulated by fyn kinase cd82 endocytosis and cholesterol-dependent reorganization of tetraspanin webs and lipid rafts cholesterol depletion activates rapid internalization of submicron-sized acetylcholine receptor domains at the cell membrane pathways of clathrin-independent endocytosis clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity magnetosomes are cell membrane invaginations organized by the actin-like protein mamk reconstructing the evolution of the endocytic system: insights from genomics and molecular cell biology protein and lipid requirements for endocytosis a novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15 regulators of yeast endocytosis identified by systematic quantitative analysis endocytosis in the plant-pathogenic fungus ustilago maydis kinesin from the plant pathogenic fungus ustilago maydis is involved in vacuole formation and cytoplasmic migration aspergillus nidulans arfb plays a role in endocytosis and polarized growth role for arf3p in development of polarity, but not endocytosis, in saccharomyces cerevisiae endocytosis, endosome trafficking, and the regulation of drosophila development macromolecular uptake in drosophila pericardial cells requires rudhira function phagocytosis of candida albicans by rnai-treated drosophila s2 cells genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic receptor-mediated endocytosis in plants is energetically possible uptake of a fluorescent marker in plant cells is sensitive to brefeldin a and wortmannin molecular dissection of endosomal compartments in plants actin-dependent fluid-phase endocytosis in inner cortex cells of maize root apices sucrose-inducible endocytosis as a mechanism for nutrient uptake in heterotrophic plant cells distinct endocytic pathways identified in tobacco pollen tubes using charged nanogold clathrin-mediated constitutive endocytosis of pin auxin efflux carriers in arabidopsis phylogeny of endocytic components yields insight into the process of nonendosymbiotic organelle evolution key: cord-310680-klywz85w authors: li, qihan; wang, lichun; dong, chenghong; che, yanchun; jiang, li; liu, longding; zhao, hongling; liao, yun; sheng, yi; dong, shaozhong; ma, shaohui title: the interaction of the sars coronavirus non-structural protein 10 with the cellular oxido-reductase system causes an extensive cytopathic effect date: 2005-04-06 journal: j clin virol doi: 10.1016/j.jcv.2004.12.019 sha: doc_id: 310680 cord_uid: klywz85w the pathological mechanism of sars-cov infection was investigated. the gene for the sars-cov non-structural protein 10, which is located in the open reading frame of pp1a/pp1ab gene, was synthesized and used to screen for the specific cellular gene coding for the protein interacting with this nsp10 protein in a human embryo lung cdna library using a yeast trap method. the results indicated that apart from the two subunits of cellular rna polymerase complex, btf3 and atf5, this nsp10 protein was also able to interact specifically with the nadh 4l subunit and cytochrome oxidase ii. further study revealed that the activity of the nadh-cytochrome was altered and the inner mitochondrial membrane was depolarized in the transfected human embryo lung fibroblast by the nsp10 protein gene. the cytopathic effect of the coronavirus 229e strain appeared more extensive in these cells than in the control cells. the newly identified severe acute respiratory syndrome coronavirus (sars-cov), caused an epidemic during the period february to june, 2003 , which spread to 32 countries infecting more than 8000 people of which 900 died (pearson, 2003) . this disease was typically characterized by fever, malaise, rigor, headache and dyspnea, followed by severe respiratory failure, which led to a 15% fatality rate among infected patients (booth et al., 2003) . the pathological analysis of the lung tissue from the deceased patients revealed severe abbreviations: sars-cov, severe acute respiratory syndrome coronavirus; btf3, basic transcription factor-3; atf5, activation transcription factor-5; nadh, nicotinamide adenine dinucleotide dehydrogenase; fbs, fetal bovine serum; dmem, double minimal essential media; qdo, quartdrop-out; nc, nitrocellulose; he, hematoxyline and eosin method; gfp, green fluorescence protein; gst, glutathione s-transferase * corresponding author. lesions of the pulmonary alveolar, flooding of the alveolar lumina with proteinaceous fluid and necrosis of the bronchiolar epithelium (peiris et al., 2003) . the extent of tissue lesions usually depends on cell damage caused mainly by the virus (lee et al., 2003) , or, by the immune cytotoxic effect against the viral antigens (tyler and fields, 1990) . to date, no data strongly supports the idea that the pathological effect of the sars coronavirus are due to autoimmune damage against the cells infected by the virus, though clinical treatment indicates that corticosteroids can inhibit the development of this disease. therefore, it could be hypothesized that the cytopathic effect produced by sars-cov in alveolar cells and bronchiolar epithelium is probably the main factor causing the severe pathology. however, there is insufficient data to interpret the replication process of sars-cov in cells, and the observation of the cellular morphology change by sars-cov in vitro only exhibits the cytopathic effect including rounded, swollen cellular organelle, like golgi sacs, and numerous smoothmembraned vacuoles in the cytoplasm (ksiazek et al., 2003) . these observations suggest that the cytopathic effect caused by the replication of sars-cov in cells could be involved. in this case, the sars-cov or the components of this virus are possibly interacting directly or indirectly on a specific cellular organelle during the virus replication in cells. to investigate the pathological mechanism of sars-cov during its replication in cell culture, we investigated several nonstructural proteins of sars-cov, which are produced by the 3cl pro cleaving pp1a/pp1ab in the sars-cov's infection (thiel et al., 2003) . the results, interestingly, suggested that the non-structural protein 10 (nsp10) could be involved in the pathological function of sars-cov in cells. using the yeast trap method (fields and sternglanz, 1994) , the synthesized gene of sars-cov nsp10 was used to screen the specific genes of proteins probably interacting with this protein in a human embryo lung cdna library. surprisingly, the results indicated that apart from the two subunits of cellular rna polymerase b complex, btf3 and atf5, this sars-cov non-structural protein 10 was able to interact specifically with the nadh 4l subunit and cytochrome oxidase ii. this result was also supported by the pull-down detection of nsp10-gst fusion protein and western blotting with antibody against cytochrome oxidase. to further investigate the contribution of this interaction to the cytopathic effect of sars-cov, a detection series of mitochondrial function and the activity of the oxido-reductase system in the human embryo lung fibroblast transfected with the nsp10 gene was performed. the results suggested that the interaction of this protein with nadh and cytochrome oxidase ii in cells infected by the coronavirus 229e interrupted the physiological function of mitochondria and caused severe damage to the cells. the results obtained in this work could provide some data to explain the possible mechanism of the sars-cov infection, which causes more severe damage in lung tissue than infection with other coronaviruses. human embryo fibroblast, kmb-17 strain, was originated from human fetal lung tissue and grown in dmem, 5% (v/v) fetal bovine serum (fbs) to form a monolayer in a culture plate (guo et al., 1974) . the cells used were in the 18-20th passage. coronavirus 229e strain was grown in these kmb-17 cells in serum free dmem and harvested from the supernatant of the culture (li et al., 2002) . the virus was titrated in kmb-17 cells. the sars-cov nsp10 gene with the length of 423bp encoding 141 amino acids (according to the genome of sars-cov, accession no. ay291315. the gene location is from 12955nt to 13371nt, and added atg and taa) was synthesized by eight oligonucleotides (tokara co.) in a pcr system (thiel et al., 2003) . the gene fragment cloned in puc-18 was identified using a restricted enzyme method and sequencing. plasmid pcdna-nsp10 was constructed by inserting sars-cov non-structural protein 10 gene into the ecori site of pcdna-3 and pgfp (takara co.) as the eukaryotic expression vectors. plasmid pgbk-nsp10 expressing gal4-nsp10 fusion protein was constructed by inserting an nsp10 gene into the ecori site of pgbk-t7 (invitrogen) to express a bait protein in a yeast two-hybrid system. all constructed plasmids were confirmed with restrictive endonuclease digestion and sequencing. the plasmid pgbk-sl was used to screen a cdna library of human embryo lung (clontech). the procedure was conducted according to the manufacturers' protocol. after screening twice, using the qdo plate and the ␤-galactosidase assay, the cdna gene fragments from the library encoded the proteins capable of interacting with the nsp10 protein, were isolated and identified by sequencing. gst, gst-nsp10 were expressed in e. coli bl21 and purified according to standard protocols. the gst and the gst-nsp10 fusion proteins were incubated overnight at 4 • c with a kmb-17 cell extract pre-labeled with [ 35 s] methionine (prepared in lysis buffer (50 mm tris-hcl, ph 8.0, 150 mm nacl, 1% nonidet p-40, 1 mm edta, 0.5% na deoxycholate, and 0.1% sds) containing phenylmethylsulfonyluoride (pmsf, sigma), and conjugated further with glutathione-sepharose 4b beads in a total volume of 500 l of buffer (20 mm tris, ph 7.5, 75 mm kcl, 50 mm nacl, 1 mm edta, 0.1% nonidet p-40, 10% glycerol, 1 mm dithiothreitol, and pmsf). after centrifugation, the beads were washed five times with incubation buffer and resuspended in pbs buffer, boiled for 5 min, and centrifuged. the supernatant was subjected to electrophoresis in a 12% sdspolyacrylamide gel. after drying, the gels were exposed to x-ray film. the monolayer kmb-17 cells grown in dmem was transfected with the plasmid of pgfp-nsp10 according to the protocol described below. in the 24-36 h after transfection, the cells were observed under a fluorescence microscope. the recombined plasmid pcdna-nsp10 containing sars-cov non-structural protein 10 gene was linearized by digestion with restriction enzyme hindiii. kmb-17 cells were transfected with linearized pcdna-nsp10 and lipotamine tm 2000 as described in the manufacturer's instructions. the control cells were transfected with linearized pcdna-vp3 (a structural protein gene of poliovirus) and lipotamine tm 2000 in the same condition. the transfected cells were maintained in dmem 5% fbs for 24-48 h. to confirm the expression of the nsp10 protein, a western blot was performed using a specific antibody against nsp10 protein produced in mice immunized by nsp10 protein expressed in escherichia coli. the transfected cells, where the expression of the nsp10 protein was detected, could be used for the next experiment. subconfluent, control cells transfected with pcdna-vp3 and the cells transfected with pcdna-nsp10, were collected, rinsed in pbs, resuspended in 5 m rhodamine-123 (eugene, co.), and incubated at room temperature for 30 min. the cells were then analyzed with a flow cytometer (becton dickinson) as described previously (li et al., 2002) . the data from the flow cytometer analysis were analyzed by cellquest software (becton dickinson). the cells transfected with pcdna-nsp10 or pcdna-vp3 were grown in dmem with 5% fbs and collected by scraping 24, 48 and 72 h after transfection. after rinsing twice with cold pbs, the cells were homogenated with a sonicator at 450 w, 10 s × 10 and centrifuged at 2500 rpm for 10 min to yield a pellet. fifty unit liters of the supernatant was used to detect its activity of nadh-cytochrome oxidase with cytochrome 1 ml (1 mg/ml), nadh 100 ul (6 mg/ml), pbs 500 ul and 0.9% nacl 500 ul at 25 • c for 20 min. the reactive solution was read in a spectrophotometer (bio-rad m-550) at od 550 . meanwhile, the protein in this supernatant was quantified by the standard lowry method (lowry et al., 1951) . the activity unit of the enzyme was determined according to od 550 value for each milligram of protein at 25 • c for 1 min. the transfected kmb-17 cells grown in dmem for longer than 24 h were scraped off and rinsed in pbs. the washed cells were lysed in rsb buffer (10 mm tris-hcl, ph 7.5; 1.5 mm, mgcl 2 ), np-40 1% at 4 • c for 1 h. then, the lysed cells were centrifuged at 12,000 rpm for 10 min. the components of the supernatant were separated by sds-page 12% (v/v) and transferred to a nc membrane. a western blot of the nsp10 antibody on this membrane was performed as described in the standard protocol (towbin et al., 1979) . meantime, the proteins collected in the pull-down test (see above) were separated in sds-page gel and transferred to a nc membrane for the western blot with the antibody against cytochrome oxidase. transfected kmb-17 cells, grown in dmem for 24 h, was infected by coronavirus 229e at moi 0.5. the control cells transfected by pcdna-vp3 were also infected with the virus using the same procedure. after 24, 48, 72, 96 and 108 h post-infection, the sample and control cells were harvested and treated according to described protocols (bonavia et al., 1997) . the harvested virus was titrated in kmb-17 cells according to the method described (racaniello and baltimore, 1981) . the transfected kmb-17 cells were grown on a glass plate and were infected by the virus as described above. twenty-four hour post-infection, the cells were observed under a light microscope (hehine, 1981) . for electron microscopy the same cells were grown on a 100-mm plate and treated as above. the cells were scraped off and further fixed in 75% ethanol for observation under an electron microscope according to standard protocol (slater, 1991) . using a gal-nsp10 fusion protein expressed in yeast as bait, a human embryo lung dna library was screened using the yeast two-hybrid method (more than 2 × 10 6 clones). after screening twice using sd/-leu/-trp/-his/+25 mm 3amino-1,2,4-triazole (3-at) plates, five clones were identified as corresponding to nsp10. among them, one gene of unknown function warranted further investigation. the other proteins encoded by the other four genes were nadh 4l subunit, cytochrome oxidase ii, rna polymerase b transcription factor 3 and activation transcription factor 5, through comparison with gene sequences in genbank. to define the binding specificity of the nsp10 protein to these five proteins, a yeast-mating assay was conducted, which indicated that the nsp10 protein had a higher affinity to the nadh 4l subunit and cytochrome oxidase ii (fig. 1) . as we know, the sars-cov non-structural protein 10, formerly known as a growthfactor-like protein, exhibits 55-58% homology with other coronaviruses. however, little is known about its function in the coronavirus replication process (marra et al., 2003) . some data suggest that it could be part of the viral replicase polyprotein (thiel et al., 2003; anand et al., 2003) . however, the possible interaction between this protein and the nadh subunit 4l and cytochrome oxidase ii did provide a clue for further understanding its contribution in the pathogenesis of sars-cov. a pull-down detection of nsp10-gst fusion protein in the extract of labeled kmb-17 cells indicates that the nsp10 protein is able to bind several proteins from kmb-17 cells (fig. 2a) , one of which is confirmed being the component of cytochrome oxidase complex in the western blot (fig. 2b) . the observation of nsp10-gfp fusion protein expressed in kmb-17 cells shows that nsp10 protein is accumulated as a distributed cluster in the cytoplasm (fig. 2c) . this illustrates that, to some extent, the nsp10 attaches to a specific cellular apparatus in the cytoplasm. these data suggest probably the interaction of nsp10 and the component of cellular mitochondria in vivo. fig. 1 . specific binding activities of the proteins identified using a yeast two-hybrid system with the sars-cov non-structural protein 10 in mating assays. the identified gene clone in the yeast trap was transfected into y187 strain together with pgbk-7-nsp10. this transfectant was grown in the selection media of sd/-leu/-trp/-his/+25 mm 3-at followed by growth in a 1.5 ml sd medium. the supernatants were collected after being centrifuged at 10,000 rpm in an eppendorf tube. the od 600 of the culture was recorded. one hundred microliters of the supernatant was reacted with o-nitropheng/␤-d-galacto-pyranoside (onpg) in z-buffer with ␤-mercaptoethanol. the final od 420 nm value was recorded after at least 30 min. the negative sample is supernatant of yeast y187 transfected with pgbkt7 and pgadt7. the positive control is from the yeast y187 transfected with pgbkt7-53 and pgadt7-t as provided by the manufacturer. the ␤-galactosidase units were calculated using the formula: units = 1000 × od 420 /(t × v × od 600 ); t, elapsed time of incubation; v, 0.1 ml × concentration factor. approximately 4 ug expressed and purified gst-nsp10 fusion protein and gst protein were respectively mixed with the 500 ul labeled extract of kmb-17 cells (5 × 10 6 cells), which were lysed in lysis buffer at 4 • c overnight. the gst and gst-nsp10 complex were conjugated with glutathione-sepharose 4b beads in a total volume of 500 l of buffer in 25 • c for 4 h. after centrifugation, the beads were washed three times with cold pbs and boiled at 100 • c with 20 ul sample buffer for 2 min. after centrifugation, the supernatants were loaded in a 12% sds-page gel for electrophoresis. lane a-1, gst protein interacting with the extract of kmb-17 cells; lane a-2, gst-nsp10 fusion protein interacting with the extract of kmb-17 cells. (b) western blot of antibody against cytochrome oxidase complex to detect the proteins collected from pull-down test. the protein samples collected from pull-down test were separated in sds-page gel and transferred to nc membrane. this membrane was interacted with the polyclonal antibody against cytochrome oxidase complex and visualized. lane b-1, proteins from the sample of gst-nesp10 fusion protein interacting with the extract of kmb-17 cells; lane b-2, proteins from the sample of gst protein interacting with the extract of kmb-17 cells. (c) the distribution of nsp10-gfp fusion protein in kmb-17 cells. the nsp10 gene was inserted into pgfp plasmid and expressed as a fusion protein of nsp10-gfp after the pgfp-nsp10 was transfected into kmb-17 cells. the distribution of nsp10-gfp fusion protein in cells was observed under a fluorescence microscope with the amplification of 400×. lane c-1, observation of nsp10-gfp fusion protein in kmb-17 cells; lane c-2, observation of mitochondria stained with rhodamine-123 fluorescence in kmb-17 cells. depending upon the result provided by the yeast two-hybrid test and other experiments, the nsp10 protein is hypothesized to be able to impact the oxido-reductase system in mitochondria as it was expressed in vivo. a detection series was performed to determine the activity of cytochrome oxidase and the physiological function of mitochondria with the kmb-17 cells transfected by the dna-nsp10 plasmid. the expression of the nsp10 protein was identified by western blot analysis using a specific antibody, in the kmb-17 cells transfected (data not shown). the activity of cytochrome oxidase was detected at different time points after transfection. the results indicated a decrease of this activity in the cells transfected by the pcdna-nsp10 plasmid compared with the control cells (fig. 3a) . meanwhile, a marked decrease in rh-123 fluorescence intensity in the rhodamine-123 fluorescence profiles of the cells at the 24th hour and the recovery at the 48th hour after transfection were noticed in the detection of the depolarization of the inner mitochondrial membrane in the kmb-17 cells transfected with the nsp10 gene (fig. 3b ). this suggests a loss in the cellular inner mitochondrial membrane potential. by combining these two results, we can probably conclude that the nsp10 protein impacts the oxido-reductase system of mitochondria via an unknown mechanism. to further investigate the role of the nsp10 protein in the process of viral replication, the kmb-17 cells transfected by pcdna-nsp10 were infected with the coronavirus 229e. the infected cells were observed under a light and electron microscope every 24 h from the 24th until the 72nd hour post-infection. extensive cytopathic effect was observed in the transfected cells infected by the virus at the 24th hour post-infection and remained continuous when compared to the control cells (fig. 4a) . the cytopathological analysis indicated an obvious cellular edema, severe vacuolar degeneration with the large nuclear, chromosome condensation, the necrosis of some cellular apparatus, and the damage of mitochondria or endoplasm membrane when viewed through the electron microscope (fig. 4b) . this was also supported by the observation of the stained cells by the he method (data not shown). compared with these pathological changes, the control cells transfected with the pcdna-vp3 plasmid in the same condition and infected by the same moi of virus showed only a slight change, such as a few cells rounded in their morphology during 24-48 h post-infection. in the latter time of the infection (72-96 h after post-infection of coronavirus 229e), the classic cytopathic effect appeared in the control cells. these results suggest that the nsp10 protein might have a role in viral replication, which amplifies the cytopathic effect of the virus. generally speaking, the cytopathic effect of the virus indicates that the viral replication in cells uses the cellular apparatus for virus synthesis and causes cellular structure damage. in this case, the cytopathic effect is an indicator of viral proliferation in cells. to analyze the severe cytopathic effect appearing in the cells transfected by the nsp10 gene during the infection of virus, a titration of the virus harvested from the transfected cells was performed at different time points. a lower yield of the virus was demonstrated in the cells transfected by the pcdna-nsp10 plasmid compared with control cells transfected by the pcdna-vp3 (fig. 5 ). this suggests that the nsp10 protein expressed in the cells inhibits replication of this virus in the unknown way. however, its mechanism remains unclear. as a new species of coronavirus, the sars-cov should have a similar method of viral replication as other coronaviruses. in fact, infection with sars-cov causes a severe pathological process with its own specific and unknown mechanism, which can bring about a severe respiratory syn-drome in patients (ng et al., 2004) . this clinical characterization suggests two possibilities in the pathogenesis of sars. one is immune pathogenesis, in which the damage of alveolar cells and bronchiolar epithelium could be caused by sars-cov specific cytotoxic t-cells. however, the data reported to date does not provide strong evidence to support this possibility. the results obtained from the sars animal model also indicate that no obvious lymphocytes accumulate in the lung tissue of those infected by sars-cov (fouchier et al., 2003) . the other possibility is that the extensive cytopathic effect of sars-cov leads to a wider necrosis in lung tissue and impairs the respiratory function. this study seems to support this hypothesis. however, some previous data indicated that the protein component of coronavirus targets the cellular apparatus, like the golgi complex for releasing virus particles (emily and machamer, 2002) . the sars-cov nonstructural protein 10, as it is produced from the viral polyprotein pp1a/pp1ab by 3cl pro cleavage during sars-cov infection in cells, should theoretically function in the viral replication while interacting with the specific cellular protein. the yeast two-hybrid system analysis of the human embryo lung tissue dna library identified potential targeting proteins of the nsp10, including the proteins involved in the cellular transcription process and the enzyme components of the oxidoreductase system in mitochondria. this result suggests that the nsp10 protein could affect the activities of nadh and cytochrome oxidase ii via a direct interaction while being involved in viral replication. in this investigation, the localization test of the nsp10 protein fusing with the gfp fluorescence protein also showed that the nsp10 protein was accumulated as a cluster in the cytoplasm. this illustrates, to some extent, that the nsp10 protein attaches to a specific cellular apparatus in the cytoplasm compared with the localization results of mitochondria with rhodamine-123 staining. the pull-down test and western blot with antibody against the cytochrome oxidase complex also suggest the interaction of nsp10 and oxidoreductase system in vivo. the most important evidence is the change of two physiological indicators of human embryo lung fibroblast transfected with the nsp10 gene, which include the decreased tendency of cytochrome oxidase activity of the transfected cells, and a loss in the cellular inner mitochondrial membrane potential exhibited by the decreased rhodamine-123 fluorescence intensity. therefore, we could infer that the nsp10 protein might contribute to the severe damage of the cells infected by the sars-cov with its effect on the enzyme, like nadh or cytochrome oxidase of the mitochondria. depending upon this hypothesis, we further investigated the possible biological properties of the nsp10 protein in the replication of coronavirus 229e. this virus is able to replicate and cause cytopathic effect in kmb-17 fibroblasts. interestingly, these results revealed that the expression of the nsp10 protein in the cells enhanced the cytopathic effect of coronavirus 229e. the morphological observations indicated that the cells transfected by the pcdna-nsp10, in which the expression of the nsp10 protein was confirmed by western blot analysis, appeared to show a severe cytopathic effect, while in contrast, the control cells transfected by the pcdna-vp3 did not show any obvious cytopathic effect within 24-72 h after infection with the virus, this suggested that the cytopathic effect was unrelated to the accumulation of the expressed exogenous protein in the cells. titration of the virus harvested from the cells at different time points demonstrated the obvious difference between the experimental sample and the control cells. there was little increase in titre of virus in cells transfected by the nsp10 gene, while the virus titre in the control cells transfected only with the pcdna-vp3 plasmid had increased by about 2-3 log. this result seems to contradict the obvious cytopathic effect of the cells transfected with the nsp10 gene. however, as the extensive cytopathic effect appeared rapidly, we could deduce that the blocking of virus replication in these cells is due to damage to the cellular apparatus probably caused by the nsp10 protein, and the possible interaction of nsp10 protein and btf3 or atf5 indicated in yeast trap test. these results suggest that the sars-cov non-structural protein nsp10 probably enhances the cytopathic effect of sars-cov in the cells by impairing the oxido-reductase system in the mitochondria, which could be one possible reason for the cytopathogenicity of the sars-cov in vivo. nevertheless, further experiments are required to explore the effect of the nsp10 protein in sars-cov replication before we can use these results to analyze the pathological changes seen in sars patients. coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs infection of primary cultures of human neural cells by coronavirus 229e and oc43 clinical features and short-term outcomes of 144 patients with sars in the greater toronto area the cytoplasmic tail of infectious bronchitis virus e protein directs gulgi targeting the two-hybrid system: an assay for protein-protein interactions koch's postulates fulfilled for sars virus the biological properties of the human embryo fibroblast (kmb-17 strain) block staining of mammalian tissue with hematoxyline and eosin sars working group. a novel coronavirus associated with severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong an sr-protein induced by hsv i binding to cells functioning as a splicing inhibitor of viral pre-mrna protein measurement with phenol reagent the genome sequence of the sars-associated coronavirus inflammatory cytokine profile in children with severe acute respiratory synchorme sars: what we learned was the fuss overblown hku/uch sars study group. clinical progression and viral load of sars coronavirus pneumonia in a community outbreak molecular cloning of poliovirus cdna and determination of the complete nucleotide sequence of the viral genome a method for the examination of the same cell using light, scanning and transmission electron-microscope mechanisms and enzymes involved in sars coronavirus genome expression electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some application pathogenesis of viral infections we would like to thank san francisco edit (www.sfedit.net) for its assistance in editing this manuscript. this work was supported by the natural science fund 30370065 in china and natural fund 2003c0002r in yunnan. key: cord-326015-ky4y2xjt authors: füllekrug, joachim; nilsson, tommy title: protein sorting in the golgi complex date: 1998-08-14 journal: biochim biophys acta mol cell res doi: 10.1016/s0167-4889(98)00048-2 sha: doc_id: 326015 cord_uid: ky4y2xjt even after one hundred years, the golgi apparatus remains a major challenge in the field of cell biology. this is particularly true in terms of transport and of protein sorting. for example, the question how cargo proteins are transported through this organelle is still a matter of debate. emphasis has been put on the role of anterograde and retrograde transport vesicles. these have been proposed to carry cargo from cisterna to cisterna and to recycle components needed for further rounds of transport. alternatively, anterograde movement of cargo takes place in cisternal membranes rather than transport vesicles. these membranes assemble and mature in a cis to trans direction. in this case, retrograde transport vesicles need to recycle all components of the golgi apparatus and this demands a highly dynamic and efficient sorting machinery. here we will discuss possible mechanisms for protein sorting in the context of cisternal maturation and propose that a common mechanism is sufficient to explain both transport of cargo and sorting of resident proteins. the main function of the exocytic pathway is to modify and deliver newly synthesised lipids and proteins to the cell surface. this forward movement of cargo takes place against gradients of resident proteins which occupy the pathway. these are enzymes which catalyse a variety of secondary modi¢cations such as processing of n-and o-linked oligosaccharides, addition of m-6-p residues to lysosomal proteins, sulfation, phosphorylation or acetylation. in addition, proteins that confer structure and promote motility, docking and fusion of transport vesicles or membranes all need to be targeted or recruited to their appropriate location. in this review, we will discuss sorting of resident proteins in the golgi complex mainly in the context of cisternal maturation [1, 2] . this model (see fig. 1 ), also mentioned elsewhere in this issue, provides an alternative to the current textbook model of vesicular transport between (sub-)compartments de¢ned by function and composition. several excellent reviews discussing vesicular transport and protein sorting have been published over the years (e.g. [3, 4] ) and though much attention has been given to this model, the cisternal maturation model has remained a good alternative reconciling many observations incompatible with the vesicular transport model. for example, macromolecules too large to enter transport vesicles still move through the secretory pathway and are readily found in cisternal membranes of golgi stacks. this is most obvious in some algae where a gradient of macromolecular (scale) assembly is revealed in a cis to trans direction strongly suggestive of a maturation process (reviewed by [5] ). morphologically, the golgi apparatus also exhibits the structural characteristics of an organelle in transit, that is, one which is assembled at the cis side and taken apart at the trans side (for review, see [6] ). one of the attractive features of the cisternal maturation model is that there are no pre-existing compartments apart from the er. this is in contrast to the vesicular transport model where the pathway is divided into several (sub-)compartments; the er, the er to golgi intermediate compartment (ergic), cis golgi network (cgn), cis, medial, trans cisternae and the trans golgi network (tgn). these are readfig. 1 . how cisternal maturation would work. importantly, this drawing depicts not spatial but time resolution. a given population of cargo molecules moves over time t0^t5 through the secretory pathway. t0: cargo is selected and concentrated at er exit sites with the help of cop ii components. t1: cop ii vesicles shed their coat and fuse with retrograde cop i vesicles carrying cis golgi proteins, forming the ¢rst cisterna of the golgi apparatus. t2: cargo is modi¢ed by cis golgi proteins, and these enzymes are recycling to fuse with cop ii vesicles; at the same time, retrograde vesicles containing medial golgi enzymes start to join the cargo containing cisterna. t3: cargo is modi¢ed by medial golgi proteins, and these enzymes are recycling to fuse with cis golgi cisternae; at the same time, retrograde vesicles containing trans golgi enzymes start to join the cargo containing cisterna. t4: cargo is modi¢ed by trans golgi enzymes, and these enzymes are recycling to fuse with medial cisternae; sorting and budding of vesicles result ultimately in the consumption of the trans cisterna. t5: vesicles move to lysosomes, secretory granules or the plasma membrane. the grey bars at the cytosolic face of the individual cisternae correspond to the intracisternal matrix. if cargo molecules have a certain a¤nity for retrograde vesicles, they would undergo more than one round of maturation thus explaining di¡erent kinetics for transport from er to plasma membrane. an essential feature of this maturation model is the prediction that golgi resident proteins are more concentrated in retrograde vesicles than they are in the cisternae of the golgi apparatus. furthermore, since new cisternae are only assembled at the er-golgi intermediate compartment, every cisterna has to move forward to provide space for newly forming cisternae. ily discernible morphologically and therefore serve as useful names. however, in the cisternal maturation model, they re£ect early to late maturation stages of the cisternal cargo carriers. these form near er exit sites and move towards the microtubule organising center (mtoc) where they are seen as stacks assembled laterally into a golgi ribbon. the number of cisternae in a given stack is usually 3^5 but higher numbers have been observed (for example, in nurse cells of the insect oniscus, the golgi stack comprises up to 36 cisternae [7] ). such variation is permitted in the cisternal maturation model as new cisternae are constantly being formed and old ones disassembled at the level of the tgn. thus the number of cisternae re£ects the number of cargo carriers put through the pathway at the same time. as cargo containing cisternae mature forward, resident proteins need to move in the opposite direction. this is mediated by retrograde transport carriers (rtcs) and requires a highly e¤cient sorting machinery. as there is no need to preserve and maintain compartmental boundaries (there are no pre-existing compartments) a simpler and more dynamic sorting process can be envisaged compared to sorting in the vesicular transport model. however, it is the process of protein sorting which drives cisternal maturation making this a fundamental one. as residents in the cisternal maturation model rely on constant recycling, they would be predicted to display gradient-like distributions when observed at steady state. historically, it was assumed that residents occupy particular (sub-)compartments of the pathway and do not exhibit such gradients (for review, see [8] ). this dates back almost 50 years when, in 1949, gersh [9] suggested that`it may be possible to conceive of the golgi apparatus as a framework whose structure is of such nature that it may accommodate certain enzymes or other activities in an orderly manner'. this was based on the fundamental observation that the golgi apparatus is directly involved in the processing of carbohydrates (con¢rmed shortly after by leblond, 1950 [10] ). this was done well before the use of electron microscopy revealing the cisternal-like structures of the golgi stack. nevertheless, it set the stage for a view which prevails even today that cisternae contain separate sets of modifying enzymes. as golgi cisternae di¡er slightly in density, from earlier ones being heavier to later ones being lighter, subcellular fractionation did indeed con¢rm this, demonstrating that enzymatic activities could be separated from each other (although only partially; [11, 12] ). this was also shown at the ultrastructural level revealing either the location of a particular enzyme or its product (using lectins) (for a comprehensive review, see [13] ). the ¢rst resident of the golgi apparatus to be mapped at this level was the glycosylation enzyme l1,4-galactosyltransferase (galt) which was shown to reside in the trans cisternae [14] and this was followed by the mapping of k2,6-sialyltransferase (sia-lylt) to the tgn [15] . as both galt and sialylt add terminal monosaccharides to glycoproteins with complex n-linked structures, there was clearly a correlation between function and localisation. two other enzymes, l1,2-n-acetylglucosaminyltransferase i (nagt i) and k1,3-1,6 mannosidase ii (mann ii), both acting earlier, were subsequently found in medial cisternae [16, 17] . as these enzymes were found in more than one cisterna, this was taken as evidence for cisternal duplication. the ¢nding that nagt i and galt were present together in one cisterna but separately in adjacent ones argued that rather than having unique sets of enzymes, cisternae contained unique mixtures [18] . this observation was later extended to include all four enzymes, nagt i, mann ii, galt and sialylt demonstrating overlapping distributions for all four enzymes [19] . formation of gradients was also suggested from low but signi¢cant amounts of enzymes in more distal cisternae. this was more obvious when examining the distribution of three enzymes involved in initiation of o-linked glycosylation. n-acetylgalactosaminyltransferase-t1, -t2 and -t3 were all found throughout the golgi stack as distinct gradients [20] . this shows that golgi resident enzymes do form gradients across the pathway and that these are unique to the particular protein. the constant recycling of residents via rtcs ensures that these are maintained in the pathway. however, it does not explain how residents exhibit di¡erential gradients in such a way that enzymes such as galt are found later than for example nagt i. to explain this, glick and co-workers [21] proposed an elegant yet simple hypothesis suggesting that residents have di¡erential abilities to enter rtcs. furthermore, they showed that if this was the case, competition between di¡erent residents is su¤cient to establish and maintain gradients in the context of cisternal maturation. residents with a high ability to enter rtcs would be found in early compartments whereas those with a lower ability would be found in later ones. we suggest that such di¡erential ability is achieved through a combination of two mechanisms: the ¢rst envisages a direct interaction between residents and coat components forming the rtc. this ensures and allows for their incorporation. the second envisages a mechanism which is milieu induced promoting incorporation of residents into rtcs. both mechanisms are related to current sorting models for er and golgi residents and we will discuss these below in the context of cisternal maturation. the surprising ¢nding that the membrane spanning domain (msd) of golgi residents su¤ced to localise reporter molecules to the appropriate part of the pathway suggested that this domain harboured important sorting information (for review, see [22] ). attention was focussed on this domain and di¡erent models were put forward explaining how an msd would mediate protein sorting (for review, see [23] ). in one model, bretscher and munro argue that membrane thickness determines how far resident proteins would travel into the pathway. they showed that, on average, resident proteins of the exocytic pathway have signi¢cantly shorter membrane spanning domains compared to those found on the plasma membrane [24] . furthermore, munro [25, 26] showed that the amino acid composition of the msd of golgi residents could be altered to poly-leucins without changing their intracellular distribution but that extending their lengths resulted in plasma membrane localisation. this suggested that length rather than amino acid composition is important. the notion that polar residues were more common in msds of golgi residents further argued that their hydrophobic stretches were shorter than those of plasma membrane proteins. a model was proposed based on a gradual increase of membrane thickness from the er towards the plasma membrane [24] . such a membrane thickness gradient would be due to a gradual increase in cholesterol and sphingomyelin concentration. indeed, early work suggested the presence of a cholesterol gradient across the exocytic pathway [27] . in a vesicular transport model, this gradient would be maintained by selective sampling of cholesterol into forward transport vesicles but not into rtcs. in the cisternal maturation model, the gradient would be maintained by exclusion of cholesterol and sphingomyelin from budding rtcs. however, in the context of the latter model, sorting through membrane thickness would not prevent residents to enter rtcs prematurely, and could therefore not explain observed gradients of residents in later parts of the pathway. so far, no correlation has been observed between the length of msds of golgi resident glycosylation enzymes and their corresponding intra golgi localisation (for discussion, see [28] ). furthermore, upon shortening the cytoplasmic domain of galt, this molecule is subsequently found on the plasma membrane showing that its msd is compatible with this membrane [29] . also, the high degree of conserved amino acids observed in msds of golgi residents would not be predicted if they merely serve as membrane anchors with a speci¢ed length. rather, as proposed earlier, msds aid in the formation of oligomeric protein complexes [29, 30] . this could be possible either through a direct interaction with spe-ci¢c lipids or lipid domains or through protein-protein interaction. arguments in favour of such a scenario come from the notion that golgi residents may be isolated as detergent insoluble complexes, in vitro (see below). however, such complexes do not £oat when subjected to gradient centrifugation. rather, they are readily sedimentable suggestive of complexes with a low ratio of lipid to protein held together by protein-protein interactions. that golgi residents are capable of mediating direct protein-protein interaction comes from several lines of evidence. first, an msd of the cis golgi resident m protein of avian coronavirus infectious bronchitis virus has been shown to directly promote oligomerisation as well as localisation [31, 32] . this domain su¤ces to localise the g protein of vesicular stomatitis virus to the cis golgi when expressed as a hybrid protein. when polar residues facing one side of the putative k-helix were mutated, it resulted not only in a loss of intracellular localisation but also in a marked decrease in oligomeric properties of the mutated hybrid protein. this showed, for the ¢rst time, a direct role of msds in oligomerisation of golgi residents and that loss of this property leads to mislocalisation. second, as mentioned above, golgi residents can be readily isolated as detergent insoluble complexes [33, 34] . these highly oligomeric structures can be disassembled/reassembled by the addition/removal of salt [34] . also, full-length galt can be crosslinked into high molecular complexes [35] . third, the medial enzymes, nagt i and mann ii form hetero oligomers (or kin oligomers) in vivo [36] . the domain su¤cient in mediating this hetero interaction was shown to reside in the stalk region of nagt i [37] . similarly, a family of small membrane proteins, the p24s, cycling between the cgn and the er, can be isolated as detergent insoluble complexes and as with nagt i and mann ii, they also oligomerise in vivo [34, 38] . in the context of cisternal maturation, we suggest that oligomerisation directly promotes incorporation of residents into rtcs and that this event is milieu induced. this would provide an explanation for individual gradients exhibited by di¡erent residents along the pathway. there is a ph as well as a lipid gradient (see above) in the golgi apparatus and these would over time trigger oligomerisation and subsequent incorporation of residents into rtcs. we further suggest that such oligomerisation results in an increased ability of the residents to bind coat components needed to form an rtc through cooperative binding. thus, residents would be distilled at various levels of the pathway depending on their particular ability to oligomerise and directly bind coat components. that such direct binding is possible has been shown for several residents although mainly in the early parts of the pathway (see below). following the`distillation hypothesis' which postulated selective retrieval of residents against a forward £ow of cargo [39] , er localisation signals were identi¢ed in both soluble [40] as well as membrane proteins [41, 42] . these were shown to confer er localisation when appended to reporter molecules acting as retrieval signals [43, 44] . for lumenal er residents, a carboxyterminal kdel motif was shown to bind a receptor [45] localised to the early part of the golgi apparatus [46, 47] . likewise, for type i membrane proteins, the retrieval motif k(x)kxx was thought to bind a receptor oriented towards the cytoplasm. then, a groundbreaking discovery by cosson and co-workers showed that the k(x)kxx motif interacts with coat components of the cop i coatomer directly [48] . moreover, genetic evidence cemented this ¢nding demonstrating that cop i coatomer functionally mediates retrieval [49] . as this form of coatomer is found on golgi associated transport vesicles [50] , cop i vesicles constitute rtcs. the presence of k(x)kxx or related signals on membrane proteins other than er residents allows for the possibility that this motif also acts later in the pathway. both ergic-53 [51] and some members of the p24 protein family display k(x)kxx retrieval motifs and reside in the interface between the er and the golgi apparatus [34, 52, 53] . surprisingly, both ergic-53 and the p24s not only use k(x)kxx related signals for their steady state localisation, but further dissection of their cytoplasmic domains revealed a signal which acts in the opposite direction. both ergic-53 and p24 family members interact directly with sec23, a component of the cop ii coat involved in budding from the er [34, 54] . this suggests that their cytoplasmic domains contain a positive export signal and mutation of this leads to accumulation in the er. thus, the combination of an export and a retrieval signal ensures that ergic-53 and the p24s localise to the interface between er and golgi apparatus through constant cycling. do other golgi resident proteins display similar signals? in terms of er export signals, it appears that golgi glycosylation enzymes do [34] . the cytoplasmic tails of these proteins also bound sec23, probably ensuring their rapid export from the er after synthesis. there is also evidence for retrieval [55^57] though future analysis will reveal whether this is mediated by cop i vesicles. 6. recycling through the er as golgi residents redistribute to the er upon brefeldin a treatment, it was proposed that this fungal metabolite highlights an already existing pathway of limited fusion between the golgi apparatus and the er [58] . this would allow golgi residents to enter directly into the er and subsequently appear at er exit sites. such a recycling model has also been proposed to explain how golgi redistributes upon depolymerisation of microtubules or mitosis (see above and storrie and yang, this issue). it is di¤cult to envisage how this could account for observed gradients of late glycosylation enzymes, particularly in the context of cisternal maturation. if the steady state distributions of nagt i, mann ii, galt and sialylt are the consequence of constant recycling through the er, their distributions would be expected to appear £atter than their observed sharp gradients. moreover, if glycosylation enzymes were to travel through the er, unless they were inactive, they would act on er resident glycoproteins. as this appears not to be the case, it is hard to reconcile this with a continuous £ow of late golgi residents through the er [59] . in summary, we favour the model that steady state distribution of resident proteins along the exocytic pathway relies on a combination of oligomerisation and coat binding. this allows for a machinery maintaining residents at various levels of the pathway whilst moving cargo forward in a vectorial manner. international cell biology protein sorting by transport vesicles coat proteins and vesicle budding scale formation in algae three-dimensional electron microscopy: structure of the golgi apparatus the cell (2nd edn progress in unraveling pathways of golgi tra¤c a protein component of the golgi apparatus distribution of periodic acid-reactive carbohydrates in the adult rat early and late functions associated with the golgi apparatus reside in distinct compartments compartmentation of asparagine-linked oligosaccharide processing in the golgi apparatus subcellular organization of glycosylation in mammalian cells immunocytochemical localization of galactosyltransferase in hela cells: codistribution with thiamine pyrophosphatase in trans-golgi cisternae paulson, demonstration of an extensive trans-tubular network continuous with the golgi apparatus stack that may function in glycosylation attachment of terminal n-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the golgi stack cell type-dependent variations in the subcellular distribution of alpha-mannosidase i and ii overlapping distribution of two glycosyltransferases in the golgi apparatus of hela cells mapping the distribution of golgi enzymes involved in the construction of complex oligosaccharides localization of three human polypeptide galnactransferases in hela cells suggests initiation of o-linked glycosylation throughout the golgi apparatus a cisternal maturation mechanism can explain the asymmetry of the golgi stack targeting and retention of golgi membrane proteins golgi localization of glycosyltransferases: more questions than answers cholesterol and the golgi apparatus sequences within and adjacent to the transmembrane segment of k-2,6-sialyltransferase specify golgi retention an investigation of the role of transmembrane domains in golgi protein retention heterogeneous distribution of ¢lipin^cholesterol complexes across the cisternae of the golgi apparatus membrane protein assembly the membrane spanning domain of l-1,4-galactosyltransferase speci-¢es trans golgi localization a golgi retention signal in a membrane-spanning domain of coronavirus e1 protein oligomerization of a membrane protein correlates with its retention in the golgi complex retention of a cis golgi protein requires polar residues on one face of a predicted k-helix in the transmembrane domain isolation of a matrix that binds medial golgi enzymes nilsson, gp25l/emp24/p24 protein family members of the cis golgi network bind both cop i and ii coatomer post-translational modi¢cations distinguish cell surface from golgi-retained l1,4 galactosyltransferase molecules. golgi localization involves active retention kinrecognition between medial golgi enzymes in hela cells the role of the membrane-spanning domain and stalk region of n acetylglucosaminyltransferase i in kin retention, kin recognition and structural maintainence of the golgi apparatus in hela cells erv25p, a component of cop iicoated vesicles, forms a complex with emp24p that is required for e¤cient endoplasmic reticulum to golgi transport the golgi apparatus: two organelles in tandem a c-terminal signal prevents secretion of luminal er proteins short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum identi¢cation of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum recycling of proteins from the golgi compartment to the er in yeast retrieval of transmembrane proteins to the endoplasmic reticulum erd1, a yeast gene required for the retention of luminal endoplasmic reticulum proteins, a¡ects glycoprotein processing in the golgi apparatus localization of sed5, a putative vesicle targeting molecule, to the cis-golgi network involves both its transmembrane and cytoplasmic domains localization of the lys, asp, glu, leu tetrapeptide receptor to the golgi complex and the intermediate compartment in mammalian cells coatomer interaction with di-lysine endoplasmic reticulum retention motifs coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum rothman, puri¢cation of a novel class of coated vesicles mediating biosynthetic protein transport through the golgi stack ergic-53, a membrane protein of the er-golgi intermediate compartment, carries an er retention motif a major transmembrane protein of golgi-derived copi-coated vesicles involved in coatomer binding involvement of the transmembrane protein p23 in biosynthetic protein transport the recycling of ergic-53 in the early secretory pathway. ergic-53 carries a cytosolic endoplasmic reticulum determinant interacting with cop ii a hypothesis on the tra¤c of mg160, a medial golgi sialoglycoprotein, from the trans-golgi network to the golgi cisternae warren, evidence for recycling of the resident medial/trans golgi enzyme, n-acetylglucosaminyltransferase i, in ldld cells localization of a yeast early golgi mannosyltransferase, och 1p, involves retrograde transport rapid redistribution of golgi proteins into the er in cells treated with brefeldin a: evidence for membrane cycling from golgi to er retention of membrane proteins by the endoplasmic reticulum key: cord-323768-r7jbm1et authors: lagarda-diaz, irlanda; guzman-partida, ana maria; vazquez-moreno, luz title: legume lectins: proteins with diverse applications date: 2017-06-12 journal: int j mol sci doi: 10.3390/ijms18061242 sha: doc_id: 323768 cord_uid: r7jbm1et lectins are a diverse class of proteins distributed extensively in nature. among these proteins; legume lectins display a variety of interesting features including antimicrobial; insecticidal and antitumor activities. because lectins recognize and bind to specific glycoconjugates present on the surface of cells and intracellular structures; they can serve as potential target molecules for developing practical applications in the fields of food; agriculture; health and pharmaceutical research. this review presents the current knowledge of the main structural characteristics of legume lectins and the relationship of structure to the exhibited specificities; provides an overview of their particular antimicrobial; insecticidal and antitumor biological activities and describes possible applications based on the pattern of recognized glyco-targets. currently, lectins are defined as carbohydrate binding proteins of non-immune origin that can recognize and bind simple or complex carbohydrates in a reversible and highly specific manner. for a long time, these proteins were named hemagglutinins, due to their ability to agglutinate red blood cells. at present, this concept is used when the specificity is unknown. it is also known that monomeric lectins do not exhibit this agglutinating activity [1] . lectins are ubiquitously present in fungi, animals, bacteria, viruses and plants. among plant lectins, those of legumes have been the most widely considered [2] . these lectins are abundant in the seeds and belong to a group of highly homologous proteins. however, their carbohydrate specificities and quaternary structures vary extensively [3] , which greatly influences the type of recognized targets. throughout the history of lectin research, legume seeds have been screened for lectin activity. the existence of binding sites for specific carbohydrates, the main characteristic of lectins, is undoubtedly an important factor for determining their activity and future applications based on their properties. this review centers on the lectins of legumes, highlighting their specificity, particularities, main reported activities and potential applications. legume lectins represent the largest family of carbohydrate binding proteins, and their physicochemical and biological properties have been broadly studied [4] [5] [6] [7] [8] . research of the plant lectin family using newer biochemical and biophysical strategies has significantly moved the field forward, and provided a model framework for studying protein-carbohydrate interactions. although plant lectins share high sequence homology and exhibit comparable physicochemical and structural properties, the diversity of their carbohydrate recognition specificity is remarkable. generally, the three-dimensional structure of plant lectins is characterized by β-sheets that are connected by α turns, β turns and bends. in addition to short loops, the quaternary interfaces are formed between β-sheets. the β-sheets are connected by loops forming antiparallel chains usually devoid of α helices ( figure 1 ) [4, 5, [9] [10] [11] [12] . analysis of legume lectin monomers reveals high similarity in sequence as well as structure, with only minor variations in the length of loops and strands. the monomer structures display a jellyroll motif best described as a sandwich of 25-30 kda containing a carbohydrate recognition domain (crd) and metal binding sites for divalent cations (ca 2+ and mn 2+ ). however, in spite of strong similarities in primary, secondary and tertiary structures, the quaternary structures show considerable variations that lead to changes in the monomer-monomer interactions and the presence/absence of protein modifications, such as glycosylation. the impact of these variations has functional implications as they dictate the specificity of multivalent glycan binding [1, 5, 11, [13] [14] [15] [16] [17] [18] . most legume lectins appear to assemble as homodimers or homo-tetramers (dimers of dimers), the stability of which is attributed to hydrophobic cooperation, hydrogen bonds and salt links [4, 9, 19] . legume lectins are subdivided into two categories, those with indistinguishable or almost indistinguishable subunits, and those with a variety of subunits [20] . phaseolus vulgaris lectins (pha) are well-studied lectins of the first group [21] [22] [23] [24] [25] [26] . there are more than forty structures of legume lectins reported in either the apo (ligand-free) or holo (sugar-bound) form in the protein data bank [15, [26] [27] [28] [29] [30] [31] [32] . however, data about lectins from wild legumes is rare. mexico has roughly 1800 species of wild legumes confined to various regions, and more than 33 percent are within the sonoran desert [5, [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] . the isolation and purification of lectins from seeds of native plants such as parkinsonia aculeata, olneya tesota, acacia constricta, prosopis juliflora, cercidium praecox, caesalpinia caladenia and phaseolus acutifolius has been described. lectins from these plants have monomers of either 30 or 66 kda that also can form tetramers or dimers. in addition, it has been shown that these lectins involve a group of different isolectins, compared to those found in lectins of the phaseolus genus [43] [44] [45] [46] [47] . on the other hand, the primary sequences of plant lectins have been used to infer evolutionary relationships, to reflect processing routes, to suggest folding patterns as well as to provide clues on how carbohydrate ligands fit into their binding pockets [20] . direct amino acid sequencing has revealed homology among the lectins from the wild desert legumes a. constricta [6] and o. tesota [5, 48] , and more domesticated species like p. vulgaris and p. coccineus [49, 50] . furthermore, antibodies raised against pha show reactivity with lectins from a. constricta, c. praecox and c. caladenia [6] . from these studies, it is clear that lectins have been conserved during the evolution of legumes and that the extensive homologies reflect taxonomical relationships among these plants [51] . legume lectins represent a crucial point in the study of the molecular basis of protein-carbohydrate interactions. each has a crd with a basic architecture accessible to both monosaccharides and/or oligosaccharides that confers remarkable divergence in their specificities [12, 52] . lectins can non-covalently interact with carbohydrates in a manner that is usually reversible and highly specific. recognition can occur in the terminal or intermediate position of the glycan structure [53] . in all legume lectins, four invariant amino acid residues participate in the binding of the carbohydrate, yet each lectin shows different specificity [18, 54, 55] . the crd pocket is formed by four loops adjacent to each other, but the loops are not close in sequence. these regions exhibit the highest residue variability and appear to be involved in specificity determination [56, 57] . the crd of legume lectins lies in close proximity to the metal binding sites, which may aid in binding activity [18] , but are not often directly involved in carbohydrate binding [18] . most lectins bind to mono-and oligosaccharides; however, more recently discovered lectins show specificity for complex sugars and glycoproteins. generally, lectins show specificity for di-, tri-and tetra-saccharides with association constants significantly higher than those for the corresponding monosaccharides [5, 18, 58] . some lectins have shown affinity towards carbohydrate structures not present in plants, such as the thomsen-nouveau antigen or complex n-glycan structures with terminal galactose and sialic acid residues [30] . the specificity of legume lectins for some typical animal glycans, has led to the suggestion that legume lectins play a role in plant defense against insects and/or predator animals [59, 60] . the legume lectins that have been purified and characterized as members of the complex type include, griffonia simplicifolia iv lectin (gs iv) [5, 61] , maackia amurensis lectin (mah and mal-1) [62] , cicer arietinum lectin [63] , saraca indica lectin [58, 64] and p. vulgaris e and l lectins (pha-e and pha-l) [5, 23, 26, [65] [66] [67] [68] [69] . wild legume lectins from the sonoran desert also have shown varying degrees of specificity for some complex carbohydrates of glycoproteins. the hemagglutinating activity of a. constricta isolectins (vls) and the p. juliflora lectin is strongly inhibited in the presence of asialofetuin and to a lesser extent with fetuin and thyroglobulin [6, 40] . c. praecox and c. caladenia lectins are inhibited by fetuin and mucine [37] . o. tesota pf1 and pf3 lectins are inhibited by free monosaccharides and/or by complex carbohydrates of fetuin, thyroglobulin, immunoglobulin, mucine and ovalbumin, whereas pf2 is only inhibited by the intact glycoprotein [5] . although glycan structures present in glycoproteins are known to have n and o linked oligosaccharides, desialylation of the glycan can result in an increase or decrease in lectin binding affinity [70] . the galactose residues found in glycosylated proteins are often capped with sialic acid, a characteristic that may alter the interaction with lectins, as is seen for amaranthus caudatus and abrus pulchellus [71, 72] . many studies on lectin-carbohydrate specificity have been published. unfortunately, often specificity analyses are used to "specifically" determine structural motifs in glycoconjugates without taking into account the rather complex data on this subject. furthermore, the choice of ligands used to assay lectin binding in some studies is limited either by natural or commercially available sources of glycans, or to simple mono-or disaccharides. therefore, a critical re-evaluation of lectin binding specificities is required. such studies using microarray techniques with immobilized glycans or immobilized lectins, dot blot assays to screen different lectins and antibodies with multiple oligosaccharides, combined with information gleaned using recombinant glycosyltransferases, are opening up new possibilities of generating a broader range of standard (neo)glycoconjugates with clearly defined structures [73] . antimicrobials have been used to treat diseases caused by bacteria and fungi and such treatments have significantly reduced the mortality rate from these infections in humans and animals. however, the extensive use of antimicrobial substances in medical, agricultural, and veterinary practices is a topic of great concern to clinical microbiologists all over the world due to the growing emergence of opportunistic microorganism strains resistant to drugs that cause serious infections [74] [75] [76] [77] . microbial resistance is a genetic phenomenon resulting in a reduction in effectiveness of drug therapy; it may be caused by mutations during the reproductive process of the microorganisms or by imported genes acquired through transduction, conjugation, and transformation mechanisms [78] . therefore, there is a growing interest in developing new strategies for inhibiting the growth or survival of microorganisms based on the screening of new sources of natural compounds as alternatives to commercially available drugs [74, 79] . a large number of proteins with antibacterial, antifungal and/or antiviral activity have been isolated from the seeds, tubers, and rhizomes of different plants, where they accumulate to high levels and may function as a reserve source of amino acids and metal ions [80] . these proteins may directly interfere with the growth, multiplication and spread of microbial agents by different mechanisms [81] , as in the case of lectins, by agglutination and/or microorganism immobilization. plant lectins often are present at potential sites of microbial invasion and their binding to fungal structures led to the inhibition of fungal growth and germination. studies carried out with soy bean and common lentil agglutinins provide evidence for these roles [51, [82] [83] [84] . a main characteristic of lectins is their ability to interact with glyco-components present on the cell membrane surface, in cytoplasmic and nuclear structures and in the extracellular matrix of cells and tissues from almost all kingdoms of life [85] . therefore, plant lectins are thought to play important roles in the plant immune defense and emerge as potential antimicrobial candidates for drug therapies. the antimicrobial effect of legume lectins is shown in table 1 . although many lectins show antimicrobial activity, clinical trials are needed to establish therapeutic efficacy, to optimize dosage, delivery and bioavailability, and to assess potential allergic reactions [86] . the antibacterial activity of lectins results from their interaction with a wide variety of complex carbohydrates of the bacterial cell wall, such as teichoic and teichuronic acids, peptidoglycans and lipopolysaccharides [2, 85] . lectins of triticum vulgare, dolichos lablab l., trigonella foenumgraecum, trifolium alexandrium l., bauhinia variegata l. and delonix regia have shown antimicrobial activity against mycobacterium rhodochrous, bacillus cereus, b. megaterium, b. sphaericus, escherichia coli, serratia marcescens, corynebacterium xerosis and staphylococcus aureus [86] lectins from the vicieae tribe strongly react with the bacterial cell wall components, muramic acid, n-acetylmuramic acid and muramyl dipeptides [87] . nevertheless, although legume seed lectins can recognize and bind to the constituents of the bacterial cell wall, such binding does not imply that these interactions occur in vivo, and certainly does not prove that these lectins are involved in the protection of seedlings against bacteria [51] . it is thought that plant lectins do not alter the membrane structure and/or permeability nor disturb the normal intracellular processes of invading microbes, since they exert an indirect effect that is based on interactions with cell wall carbohydrates or extracellular glycans [60] . however, electron microscopy studies revealed that treatment with araucaria angustifolia lectin promoted morphologic alterations, including the presence of pores in the membrane of gram-positive bacteria, and bubbling on the cell wall of gram-negative bacteria [88] . others reported that the antibacterial activity of lectins occurred through the interactions of the lectins with n-acetylglucosamine, n-acetylmuramic acid and tetrapeptides linked to n-acetylmuramic acid present in the cell wall of gram-positive bacteria, or to lipopolysaccharides present in the cell wall of gram-negative bacteria [87] . bind to the glycosylated envelope protein and block cellular entry (interfere with replication cycle) [98] [99] [100] [101] [102] [103] [104] [105] in addition to these findings, lectins from the legumes from canavalia ensiformis, t. foenumgraecum, arachis hypogaea, cajanus cajan, p. vulgaris and pisum sativum were shown to inhibit biofilm formation by streptococcus mutans, but the growth of planktonic cells was not affected [89] . since bacterial biofilms make disinfection procedures more difficult by increasing bacterial resistance to detergents and antibiotics, the inhibition of biofilm formation and development by legume lectins could be a useful characteristic of these proteins. furthermore, the rapid evolution of bacteria with antibiotic resistance (in particular, multidrug resistant bacteria or superbugs) has reduced the efficacy of conventional treatments against their biofilms. hence, development of non-antibiotic alternatives that could efficiently treat or reduce biofilms should become a priority. furthermore, research focused on finding lectins that reduce superbugs could represent an interesting alternative drug therapy that deserves further attention. regardless of the considerable number of lectins that have been characterized, only a small number have shown antifungal effects. because plant lectins do not penetrate the cell wall or the membrane to reach the cytoplasm of fungi, direct inhibition of fungal growth by lectins is unlikely. in spite of this, indirect responses produced by the attachment of lectins to chitin and other glycans on the fungal surface could affect fungal survival or other activities [105, 106] . lectin binding to hyphae could result in inhibition of fungi growth as a result of poor nutrient absorption as well as by interference with the spore germination process [107] . lectins can cause different morphological changes that render fungi more vulnerable to differing stress conditions [108] . for example, in one study, lectin interactions resulted in swollen hyphae, vacuolization of the cell content and improved lysis of hyphal cell wall, that, in turn, increased susceptibility of fungi to osmotic shock [107] . in another case, it was suggested that small antifungal lectins could penetrate the fungal cell wall and reach the cell membrane where blocking of active sites of enzymes could alter cell wall morphogenesis [108, 109] . some reports indicate that lectins from the legumes astragalus mongholicus, p. coccineus, archidendron jiringa nielsen, b. ungulata, glycine max, indigofera heterantha and a. hypogaea can exhibit antifungal activity against various species of phytopathogenic fungi, such as botrytis cinerea, fusarium oxysporum, f. moniliforme, f. solani, colletorichum sp., drechslera turcia, and exserohilum turcicum, as well as pathogenic fungi such as candida albicans, penicillium italicummm, and aspergillus sp. [82, [91] [92] [93] 110, 111] . additional studies are required to elucidate the molecular basis for the antifungal activity of individual lectins as the ability of lectins to inhibit growth differs among fungal species. plant lectins with antiviral activity are of substantial therapeutic interest. some of these carbohydrate binding proteins exhibit significant activity against human immunodeficiency virus (hiv) and other viruses [97] . retroviruses, such as hiv, have a surface covered by highly glycosylated virally-encoded glycoproteins, e.g., gp120 (that contains high mannose and/or hybrid glycans) and gp41. the carbohydrates on these proteins are particularly important because the glycans can be used as tools to render the virus easily recognized by the immune system, and thus susceptible to immunological neutralization [112] . the interactions between host and proteins from the viral envelop also can be altered by compounds that specifically recognize and bind glycans. furthermore, lectins can crosslink surface viral glycans and thereby prevent interactions with other co-receptors [83] . antiviral lectins used as therapeutic agents offer lower toxicity and can be included in topical applications. generally, lectins are odorless, and resistant to high temperatures and low ph, ideal properties for development of microbicide drugs [86] . most current antiviral therapeutics prevent the viral life cycle, or inhibit the entrance of the virus into host cells [113] . the antiviral action of plant lectins varies extensively depending upon their carbohydrate specificity. corona virus are highly susceptible to mannose specific lectins that interfere with the viral attachment in early phases of the replication cycle and suppress viral development by binding towards the end of the viral infection cycle [114] . other antiviral non-legume lectins with encouraging properties include griffithsin (grft), cyanovirin (cv-n) and banana lectin (banlec), and banlec has been recommended as an antiviral microbicide. most often antiviral lectins are recommended for incorporation into vaginal and rectal gels, creams or suppositories that act as a barrier to prevent hiv transmission. in such processes, lectins bind the virus preventing its entry and fusion to target cells, consequently averting contamination [115] . several legume lectins possess antiviral activity. lectins like c. ensiformis agglutinin (con a), lens culinaris agglutinin (lca), vicia faba agglutinin, p. sativum agglutinin (psa) and pha-e bind to the envelope glycoprotein gp120 and to inhibit fusion of hiv-infected cells with cd4 cells by a carbohydrate-specific interaction with the hiv-infected cells [103] . in addition, g. max agglutinins inhibit hiv-1 reverse transcriptase activity [116] . although glycan structures of viral proteins involve high mannose, further research in this area is needed to explore lectins that recognize structures with different sugars such as sialic acid, fucose and n-acetyl glucosamine with the aim of developing novel approaches and therapies in the field of virus biology. legume lectins are toxic to a broad spectrum of insects representing several orders including, coleoptera, diptera, lepidoptera, hymenoptera, isoptera, neuroptera and homoptera. in this regard, the family of leguminous lectins is the most studied due to its insecticidal potential. a great number of legume lectins have shown insecticidal activity including those from c. ensiformis, p. sativum, p. vulgaris, glechoma hederacea, g. simplicifolia, o. tesota (pf2) and b. monandra [8, 117, 118] . these lectins showed harmful effects in the different developmental stages of insects (larvae and adults). in this regard, they may increase mortality, delay development and/or adult emergence and reduce fecundity. usually, the effect of lectins on insects is evaluated by feeding larvae with artificial seeds containing the lectin or with transgenic plants that overexpress the lectin of interest. the expression of a given lectin in transgenic plants is only possible if the lectin molecular sequence is well characterized and also requires an available transformation protocol for the desired plant species [119] . in the last twenty years, research has focused on elucidating the mode of action of insecticidal lectins. in general, lectins can exert a toxic effect via binding to the peritrophic membrane (pm), peritrophic gel (pg) or the brush-border microvilli of epithelial cells (figure 2 ) [120] . the pm or pg is a film surrounding the food bolus in most insects and is composed of chitin and proteins. some non-legume lectins with insecticidal activity such as the rhizoctonia solani agglutinin and sambucus nigra agglutinin ii can pass through the pm of the red flour beetle, tribolium castaneum. this ability to reach the endoperitrophic space is governed by the dimensions of the molecule and the charge and size of the pm pores [121] . in the case of insects lacking a pm, the insecticidal effect of lectins may be exerted by their direct interaction with glycoconjugates present on the cell epithelium [122] . in fact, the insecticidal activity of lectins relies on their binding properties to sugars present on the surface of the insect gut epithelial cells. in insects, the surface of epithelial cells is rich in glycoproteins that, in addition to their determinant roles for the proper gut function, provide a number of available targets for lectin binding. the interaction of lectins with different glycoproteins or glycan structures in insects may interfere with important physiological processes ( table 2) . although the key receptors for lectins could be localized on the surface of gut epithelial cells, if lectins are internalized, they may interact with a new set of targets located in the intracellular space, allowing the lectin to interfere with particular metabolic pathways. such internalization has been more studied for non-legume lectins. for example, the garlic leaf lectin, which affects survival and development of the moth helicoverpa armigera, was found to be internalized by binding to a glycosylated alkaline phosphatase anchored to the insect midgut membrane. in addition, galantus nivalis lectin was reported to cross the midgut epithelium of nilaparvata lugens and co-localize in the fat body and hemolymph. such co-localization was proposed to occur via a carrier-receptor, ferritin. the internalization of the colocasia esculenta tuber agglutinin into insect hemolymph also was demonstrated, suggesting it could interact with various n-glycosylated intracellular targets, which may, in part, explain the lectin toxicity [122] . although the complete mechanism by which some lectins are able to cross midgut epithelial cells is not yet fully understood, available evidence has highlighted the implication of clathrin-mediated endocytosis as part of this process [123, 124] . regardless of the mechanisms involved in lectin interactions with insect cells, in order to be toxic, a lectin must avoid degradation by the digestive enzymes from the insect gut. the insecticidal effect of some lectins is attributed to the resistance of these proteins to proteolysis and to their properties of stability in a wide ph range. for example, pf2, an insecticidal lectin to zabrotes subfasciatus, is resistant to in vitro proteolysis by insect digestive enzymes. this is in contrast to pha-e lectin that is digested after 4 h of incubation with these enzymes, and therefore is not toxic for this insect [8] . other lectins with insecticidal activity that share the common feature of resistance to proteolysis by different digestive enzymes include, g. simplicifolia ii lectin (gs ii), ulex europeus agglutinin (uea) and b. monandra lectin [117, 118] . the degree of resistance to digestive enzymes depends on the capacity of the lectin to bind to glycoconjugates of the insect gut. gs ii mutant lectins lacking the ability to bind carbohydrates displayed sensitivity to proteolysis by digestive enzymes with a consequent loss of toxicity [125] . helicoverpa armigera [134] alanyl aminopeptidase n sucrase acyrthosiphon pisum [134] in summary, the insecticidal effect exhibited by several plant lectins is a complex phenomenon that relies on the capacity of the lectin to interfere with critical physiological processes during insect development. such interference often may be mediated by a lack of nutrient absorption that occurs when midgut epithelial cells are atrophied as a result of the interaction of lectins with glyco-targets located either on the surface or inside the cell. studies of the recognition of plant lectins by insect gut glyco-targets can allow one to develop hypotheses on which cellular pathways are affected by the insecticidal activity. these pathways vary depending on the type of insect and the lectin, and could be as diverse as the type of recognized glycan or receptors, and might include the effectors of energy metabolism, and redox and ionic homeostasis (summarized in table 2 ). finally, the study of potential non-yet-identified insecticidal plant lectins may contribute to the development of distinctive tools for sustainable pest control. however, in this regard, care must be taken considering the effect lectins might exert on beneficial insects as well. recently, an increased interest in the potential of plant lectins as cancer therapeutic agents has become evident. lectins can be used to study the metastatic distribution patterns, the expression profiles of cancer cells glycoconjugates and their biological effects in various tumors. it is well known that carbohydrates present on the cell membrane are important for cell recognition, communication and adhesion. in cancer, these interactions are essential for tumor progression and metastasis. in tumor cells, glycosylation is generally altered in comparison with normal cells and these alterations can be detected by lectins [135, 136] . litynska et al. compared the lectin-binding pattern in different human melanoma cell lines using the pha-l lectin and other non-legume lectins, including the galanthus nivalis lectin, s. nigra lectin, mal, datura stramonium agglutinin and wheat germ agglutinin (wga). studies demonstrated that acquisition of metastatic potential of tumor cells is correlated with the expression of branched and sialylated complex n-oligosaccharides [137] . interestingly, the binding of mal-i to gastric cancer cells was proportional to their metastatic capacity, and a high expression of α (2,3)-linked sialic acids was closely correlated with lymph node metastasis [137] . korourian et al. used g. simplicifolia lectin-i (gs i) and vicia vilosa agglutinin (vva) to establish an expression analysis of carbohydrate antigens in human breast ductal carcinoma in situ (dcis). both lectins showed a significant association with nuclear grade (cell size and uniformity) of dcis [138] . positive correlations also were found between the expression of vva-and gsi-reactive structures and tumor grade in dcis patients, suggesting that the glycoconjugates found in these antigens are associated with invasiveness in dcis [138] . pha can be used to discriminate between hepatocellular carcinoma and benign liver disease [139] . this suggests that lectins could potentially be used to develop prognostic tools for cancer diagnosis and the detection of metastasis, and to some extent to estimate the severity of the clinical case. in addition to these uses, plant lectins have shown in vivo and in vitro antitumor activity. lectins with the potential to induce inhibition of tumor cell growth, such as, con a, mistletoe type-i lectins and pha are currently in different phases of clinical trials [138, 140] . plant lectins can modify the expression of interleukins and some protein kinases and in this way modulate the immune system. furthermore, in cancers, lectins could alter the signaling pathways involved in the expression of members of the bcl-2, autophagy (atg) related, and caspase families, as well as p53, erk, ras-raf, and bnip3, and thereby induce both apoptosis and autophagy [141] . soybean lectin produces reactive oxygen species in a dose-dependent manner in hela cells inducing apoptosis, autophagy and dna damage in the cells [142] . the cell surface carbohydrates are key targets for lectins; in cancer, a general pathway involves recognition of carbohydrate receptors triggering the activation of enzymes such as mbl-associated serine proteases [143] . however, the binding of lectins to glycans on the cell membrane may not be sufficient to induce apoptosis. kim et al. showed that cell death requires lectin endocytosis that triggers signaling for apoptosis [143] . con a is cytotoxic to hepatoma cell lines. its toxicity is mediated primarily by its binding to the mannose moiety of cell membrane glycoproteins, with subsequent internalization and accumulation in the mitochondria. autophagy is then activated, leading to lysosomal degradation of the affected mitochondria and, ultimately, cell death [144, 145] . overall, these findings of cancer research have revealed the mechanisms responsible for the antitumor action of different plant lectins. these mechanisms are varied and depend upon different factors such as the tumor cell origin and type as well as lectin concentration. the screening and characterization of novel lectins from legume resources has allowed the discovery of proteins with a great capacity to distinguish specific glycans from a diversity of glyco-targets found in a variety of organisms. legume lectins could be potential candidates for developing tools based on protein-carbohydrate interactions with variable specificities. lectin-mediated drugs focused on targeting specific cells could lead to promising anticancer and antimicrobial treatments, which would directly impact areas of economic importance, such as the pharmaceutical and food industries and agriculture. additionally, the use of lectins against insect pests could provide a form of sustainable pest control. more research is needed to explore and support the therapeutic effect of lectins. studies of action mechanisms, the relationship between the role of the lectins and their molecular features, and finally, their effect in modulating the expression of proteins and genes are required to move forward the use of lectins for clinical applications. domesticated legumes provide an accessible and abundant source of lectins, unlike wild legumes, which, in some regions, may even be considered protected species. in some cases, successful recombinant production of lectins would be a key factor in whether a specific lectin could find use in a feasible industrial application. the overexpression of recombinant lectins remains a great challenge, even more so for those lectins where the lack of adequate post-translational modifications could compromise their activity. the authors thank joy winzerling for her critical reading and editing of the manuscript. author contributions: all authors compiled the information, wrote the review, read and approved the final manuscript. the authors declare no conflict of interest. history of lectins: from hemagglutinins to biological recognition molecules proteins with diverse applications legume lectins a large family of homologous proteins purification of complex carbohydrate specific lectins from olneya tesota seeds using tandem affinity chromatography purification and characterization of complex carbohydrate specific isolectins from wild legume seeds: acacia constricta is (vinorama) highly homologous to phaseolus vulgaris lectins proteomic approaches to study structure, functions and toxicity of legume seeds lectins. perspectives for the assessment of food quality and safety insecticidal action of pf2 lectin from olneya tesota (palo fierro) against zabrotes subfasciatus larvae and midgut glycoconjugate binding analyses of carbohydrate recognition by legume lectins: size of the combining site loops and their primary specificity carbohydrate binding, quaternary structure and a novel hydrophobic binding site in two legume lectin oligomers from dolichos biflorus how proteins bind carbohydrates: lessons from legume lectins structural features of the legume lectins novel structures of plant lectins and their complexes with carbohydrates weak protein-protein interactions in lectins: the crystal structure of a vegetative lectin from the legume dolichos biflorus determinants of quaternary association in legume lectins l-type lectins lectins: tools for the molecular understanding of the glycocode signature of quaternary structure in the sequences of legume lectins plant lectins: occurrence, biochemistry, functions and applications purification of the phytohemagglutinin family of proteins from red kidney beans (phaseolus vulgaris) by affinity chromatography comparison of phaseolus vulgaris cultivars on the basis of isolectin differences characterization of the structural determinants required for the high affinity interaction of asparagine-linked oligosaccharides with immobilized phaseolus vulgaris leukoagglutinating and erythroagglutinating lectins a one-step procedure for isolation and resolution of the phaseolus vulgaris isolectins by affinity-chromatography the crystallographic structure of phytohemagglutinin-l phytohemagglutinin from phaseolus vulgaris (pha-e) displays a novel glycan recognition mode using a common legume lectin fold a portal for structural glycosciences. glycoinformatics crystallographic structure of metal-free concanavalin a at 2.5 angstrom resolution the monosaccharide binding-site of lentil lectin: an x-ray and molecular modeling study the structure of the pea lectin-d-mannopyranose complex at a 2.1 angstrom resolution the crystal structure of a plant lectin in complex with the tn antigen molecular modeling of native and mutated lima bean lectin: dissection of lectin/blood group a trisaccharide interactions agroecosystem diversity: a model from the sonoran desert mexican leguminosae: phytogeography, endemism, and origins estudio de algunas semillas de leguminosas del desierto de sonora. factores antinutricionales y calidad de sus proteínas y aceites aislamiento y caracterización de las lectinas de leguminosas silvestres del desierto de sonora: cercidium praecox (palo de brea) y caesalpinia caladenia (palo dorado) caracterización de los oligosacáridos de las lectinas de olneya tesota pf2 y su isoforma más abundante (if2) y establecer la relación con la función de reconocimiento isoformas de la lectina pf2, características moleculares y reconocimiento de leucocitos de sangre periférica de adultos purificación y caracterización de la lectina de prosopis juliflora y evaluación del desarrollo larval de zabrotes subfasciatus en las semillas purification, biochemical characterization, and bioactive properties of a lectin purified from the seeds of white tepary bean (phaseolus acutifolius variety latifolius) identificación de la interacción de monocitos humanos con las lectinas de olneya tesota (if2) y phaseolus vulgaris (pha-l) por citometría de flujo phytohemagglutinin mitogenic proteins. structural evidence for a family of isomitogenic proteins biological and biochemical properties of phaseolus vulgaris isolectins subunit heterogeneity in the lima bean lectin lectin and lectin-related proteins in lima bean (phaseolus lunatus l.) seeds: biochemical and evolutionary studies lectin from phaseolus acutifolius var. escumite: chemical characterization, sugar specificity, and effect on human t-lymphocytes analysis of olneya tesota lectin peptides with liquid chromatography in two dimensions and tandem mass spectrometry (lc-ms/ms) characterization of 2 phaseolus-vugaris phytohemagglutinin genes closely linked on the chromosome lectin-related resistance factors against bruchids evolved through a number of duplication events antimicrobial activity of lectins from antimicrobial activity of lectins from plants pattern recognition in legume lectins to extrapolate amino acid variability to sugar specificity las lectinas vegetales como modelo de estudio de las interacciones proteína-carbohidrato lectin-carbohydrate complexes of plants and animals: an atomic view molecular modeling of the dolichos-biflorus seed lectin and its specific interactions with carbohydrates: α-dn-acetyl-galactosamine, forssman disaccharide and blood-group-a trisaccharide analysis of sequence variation among legume lectins: a ring of hypervariable residues forms the perimeter of the carbohydrate-binding site a lectin from platypodium elegans with unusual specificity and affinity for asymmetric complex n-glycans comparison of the nature of interactions of two sialic acid specific lectins saraca indica and sambucus nigra with n-acetylneuraminic acid by spectroscopic techniques lectins, lectin genes, and their role in plant defense lectins as plant defense proteins structures of the lectin-iv of griffonia simplicifolia and its complex with the lewis b human blood-group determinant at 2.0-angstrom resolution an unusual carbohydrate binding site revealed by the structures of two maackia amurensis lectins complexed with sialic acid-containing oligosaccharides properties of a lectin purified from the seeds of cicer-arietinum. hope-seyler's saracin a lectin from saraca-indica seed integument recognizes complex carbohydrates structure of a complex-type sugar chain of human glycophorin a mitogenic leukoagglutinin from phaseolus-vulgaris binds to a pentasaccharide unit in n-acetyllactosamine-type glycoprotein glycans structural determinants of phaseolus-vulgaris erythroagglutinating lectin for oligosaccharides purification and characterization of novel lectins from great northern bean, phaseolus vulgaris l. bba-gen the high specificities of phaseolus vulgaris erythro-and leukoagglutinating lectins for bisecting glcnac or beta 1-6-linked branch structures, respectively, are attributable to loop b presence of beta-linked galnac residues on n-glycans of human thyroglobulin isolation and characterization of amaranthin, a lectin present in the seeds of amaranthus-caudatus, that recognizes the t-antigen (or cryptic-t)-antigen characterization of the sugar-binding specificity of the toxic lectins isolated from abrus pulchellus seeds specificity analysis of lectins and antibodies using remodeled glycoproteins antimicrobial lectin from schinus terebinthifolius leaf fluconazole-resistant candida albicans vulvovaginitis screening of aqueous extracts of medicinal herbs for antimicrobial activity against oral bacteria antimicrobial property of extracts of indian lichen against human pathogenic bacteria antimicrobial activity of cladonia verticillaris lichen preparations on bacteria and fungi of medical importance antimicrobial peptides from plants antimicrobial peptides in mammalian and insect host defence the structural basis for carbohydrate recognition by lectins antifungal and antibacterial activities of lectin from the seeds of archidendron jiringa nielsen medicinal applications of plant lectins purification of a lectin from eugenia uniflora l. seeds and its potential antibacterial activity antimicrobial activity of legume seed proteins interactions of plant-lectins with the components of the bacterial-cell wall peptidoglycan effects of a lectin-like protein isolated from acacia farnesiana seeds on phytopathogenic bacterial strains and root-knot nematode lectins: to combat infections interaction of a legume lectin with two components of the bacterial cell wall. a crystallographic study evaluation of antimicrobial activity of a lectin isolated and purified from indigofera heterantha a novel homodimeric lectin from astragalus mongholicus with antifungal activity a novel sialic acid-specific lectin from phaseolus coccineus seeds with potent antineoplastic and antifungal activities bul: a novel lectin from bauhinia ungulata l. seeds with fungistatic and antiproliferative activities studies on growth inhibition by lectins of penicillia and aspergilli isolation of a homodimeric lectin with antifungal and antiviral activities from red kidney bean (phaseolus vulgaris) seeds carbohydrate profiling of fungal cell wall surface glycoconjugates of aspergillus species in brain and lung tissues using lectin histochemistry lectins with anti-hiv activity: a review plant as a plenteous reserve of lectin bacterial detection using carbohydrate-functionalised cds quantum dots: a model study exploiting e. coli recognition of mannosides isolation and characterization of myrianthus holstii lectin, a potent hiv-1 inhibitory protein from the plant myrianthus holstii effects of succinylated concanavalin a on infectivity and syncytial formation of human-immunodeficiency-virus correlation between carbohydrate structures on the envelope glycoprotein gp120 of hiv-1 and hiv-2 and syncytium inhibition with lectins development and applications of lectins as biological tools in biomedical research proteins with antifungal properties and other medicinal applications from plants and mushrooms fungal cell wall chitinases and glucanases. microbiology lectins in higher plants fusarium sp growth inhibition by wheat germ agglutinin hevein: an antifungal protein from rubber-tree (hevea brasiliensis) latex insecticidal and antifungal activity of a protein from pouteria torta seeds with lectin-like properties three dimensional structure of the soybean agglutinin-gal/galnac complexes by homology modeling virus glycosylation: role in virulence and immune interactions structural studies of algal lectins with anti-hiv activity plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle griffithsin: an antiviral lectin with outstanding therapeutic potential bun ng, t. biochemical and functional properties of a lectin purified from korean large black soybeans a cultivar of glycine max insecticidal action of bauhinia monandra leaf lectin (bmoll) against anagasta kuehniella (lepidoptera: pyralidae), zabrotes subfasciatus and callosobruchus maculatus (coleoptera: bruchidae) pea lectin expressed transgenically in oilseed rape reduces growth rate of pollen beetle larvae plant-insect interactions: what can we learn from plant lectins? arch biodegradable microparticulate system of captopril penetration through the peritrophic matrix is a key to lectin toxicity against tribolium castaneum binding of insecticidal lectin colocasia esculenta tuber agglutinin (cea) to midgut receptors of bemisia tabaci and lipaphis erysimi provides clues to its insecticidal potential fungal lectin, xcl, is internalized via clathrin-dependent endocytosis and facilitates uptake of other molecules mechanism of entomotoxicity of the plant lectin from hippeastrum hybrid (amaryllis) in spodoptera littoralis larvae functional mechanics of the plant defensive griffonia simplicifolia lectin ii: resistance to proteolysis is independent of glycoconjugate binding in the insect gut the insecticidal activity of recombinant garlic lectins towards aphids ferritin acts as the most abundant binding protein for snowdrop lectin in the midgut of rice brown planthoppers (nilaparvata lugens) ferritin acts as a target site for the snowdrop lectin (gna) in the midgut of the cotton leafworm spodoptera littoralis effect of myracrodruon urundeuva leaf lectin on survival and digestive enzymes of aedes aegypti larvae entomotoxic action of jackbean lectin (con a) in bird cherry-oat aphid through the effect on insect enzymes insight to the mode of action of allium sativum leaf agglutinin (asal) expressing in t3 rice lines on brown planthopper identification of membrane proteins of the midgut of zabrotes subfasciatus larvae associated with the insecticidal mechanism of pf2 lectin binding of pf2 lectin from olneya tesota to gut proteins of zabrotes subfasciatus larvae associated with the insecticidal mechanism receptors of garlic (allium sativum) lectins and their role in insecticidal action enhanced expression of α2, 3-linked sialic acids promotes gastric cancer cell metastasis and correlates with poor prognosis comparison of the lectin-binding pattern in different human melanoma cell lines expression analysis of carbohydrate antigens in ductal carcinoma in situ of the breast by lectin histochemistry alteration of protein glycosylation in liver diseases plant lectins: targeting programmed cell death pathways as antitumor agents mistletoe extracts standardised in terms of mistletoe lectins (ml i) in oncology: current state of clinical research plant lectins, from ancient sugar-binding proteins to emerging anti-cancer drugs in apoptosis and autophagy the biological functions of mbl-associated serine proteases (masps) antitumor effect of soybean lectin mediated through reactive oxygen species-dependent pathway lectin-induced apoptosis of tumour cells autophagy induction in t cell-independent acute hepatitis induced by concanavalin a in scid/nod mice this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-350309-j4oh1z8m authors: liu, d. x.; yuan, q.; liao, y. title: coronavirus envelope protein: a small membrane protein with multiple functions date: 2007-05-29 journal: cell mol life sci doi: 10.1007/s00018-007-7103-1 sha: doc_id: 350309 cord_uid: j4oh1z8m coronavirus envelope protein is a small membrane protein and minor component of the virus particles. it plays important roles in virion assembly and morphogenesis, alteration of the membrane permeability of host cells and virus-host cell interaction. here we review recent progress in characterization of the biochemical properties, membrane topology and functions of the protein. coronaviruses are enveloped viruses with a singlestrand, positive-sense rna genome of 27-32 kb in length. in coronavirus-infected cells, a 3-co-terminal nested set of six to nine mrna species, including the genome-length mrna (mrna1) and five to eight subgenomic mrna species (mrna2-9), is produced. the four major structural proteins, spike (s), envelope (e), membrane (m) and nucleocapsid (n), are encoded by different subgenomic mrnas. similar to most of other rna viruses, the genomic rna replication, mrna transcription and protein synthesis of coronavirus occur in the cytoplasm of the infected cells. the newly synthesized structural proteins and the rna genome are assembled into virions that bud at the er-golgi intermediate compartment [1, 2] . following translocation to the golgi apparatus for further modification and maturation, the mature particles are released from the infected cells through the secretary pathway. coronavirus e protein is a small envelope protein present in virions at low levels, and ranges in size from 76 to 109 amino acids [3] [4] [5] . the e proteins from infectious bronchitis virus (ibv) and mouse hepatitis virus (mhv) are translated from the third and second orfs of mrna 3 and 5 of the respective viruses by a cap-independent, internal ribosomal entry mechanism [6] [7] [8] [9] [10] [11] [12] . in some coronaviruses, e.g., in severe acute respiratory syndrome coronavirus (sars-cov), the e protein is derived from a monocistronic mrna [13] . there is a low degree of sequence identity among e proteins from three coronavirus groups, and in some cases, among members of the same group (fig. 1) . nevertheless a number of general features can be highlighted in all coronavirus e proteins. these include a short hydrophilic n-terminal region, followed by a long hydrophobic stretch of 21-29 amino acid residues, 2-4 cysteine residues immediately downstream of the hydrophobic stretch, and a relatively long hydrophilic c-terminal tail (fig. 1 ). in cells infected with some coronaviruses, such as ibv, mhv and bovine coronavirus (bcov), the e protein exhibits a granular or punctuate pattern on immunofluorescence staining [5, 14, 15] and a portion of the e protein was transported to the cell surface [3, 5, 15] . the ibv e protein was shown to be localized to the golgi complex when expressed on its own, coexpressed with m protein and in ibv-infected cells [16] . the c-terminal tail of this protein directs the golgi targeting [17] . the sars-cov and ibv e proteins may be also transiently localized to the er at early stages of the infection cycles [18] [19] [20] . the mhv e protein was found to accumulate in the er-golgi intermediate compartment as judged by their colabeling with antibodies against e protein and rab-1 [21] . biochemical characterization of coronavirus e protein showed that the protein may undergo posttranslational modifications. for example, the ibv and sars-cov e proteins are modified by palmitoylation on one or more cysteine residues [17, 18] . however, attempts to label the mhv and transmissible gastroenteritis virus (tgev) e proteins with 3 hlabeled palmitic acid were unsuccessful [3, 21] . it is still unknown whether this modification is a general characteristic of the coronavirus e protein. in addition to palmitoylation, a minor proportion of the sars-cov e protein was recently found to be modified by n-linked glycosylation on asn66 [22] . this modification is unique to sars-cov e protein, and it is still unknown whether the modification can also be detected in virus-infected cells and in virions. coronavirus e protein can form homo-oligomers [23] . molecular dynamic simulations predict that sars-cov e forms pentamers [23] , but the oligomeric status of other coronaviruses is still unknown. as mentioned earlier, different coronavirus e proteins share significant similarities in their biochemical properties as well as biological functions. however, they seem to adopt distinct membrane topology. early studies showed that cell surface staining could be detected in cells infected with tgev using c-terminal-specific antibodies against the tgev e protein, suggesting that the c-terminal region of the protein is exposed to the external surface of the cells [3] . immunofluorescence staining and biochemical analysis of cells expressing mhv e protein revealed that the c-terminal region is exposed to the cytoplasm and the n-terminal region is probably buried in the membrane near the cytoplasmic side (n endo c endo ) [21, 24] . this model is supported by the observation that treatment of mhv particles with proteinase k did not alter the molecular mass of the e protein [21] . consistent data on the topologies of mhv e protein in virions, in cells overexpressing the protein and in the in vitro translation systems in the presence of microsomal membranes have been reported [21, 24] . in the case of ibv e protein, immunofluorescence assay showed that c-terminal-specific antibodies could detect the protein in cells treated with either digitonin or triton x-100, but the n-terminal-specific antibodies can detect the protein only in cells treated with triton x-100, demonstrating that the ibv e protein adopts an n exo c endo topology [16] . this n exo c endo topology was confirmed by the proteinase k protection assay [22] . recently, the biophysical properties and membrane topology of the sars-cov e protein were studied by several groups [22, 23, [25] [26] [27] . based on in vitro biophysical studies, arbely and coworkers [25, 26] proposed an unusual short, palindromic transmembrane helical hairpin for the putative transmembrane domain of the protein. however, a regular a-helical structure with the n-terminal and c-terminal regions in opposite sides of the lipid bilayer has also been proposed based on similar in vitro biophysical studies and in silico data [23, 27] . the reason for this discrepancy is not clear. one possibility is that sars-cov e protein may adopt more than one topology. in fact, evidence for the presence of mixed membrane topologies for the sars-cov e protein has recently been reported [22] . immunofluorescence analysis and proteinase k protection assay demonstrated that the majority of the sars-cov e protein adopts an n endo c endo topology. intriguingly, an nlinked glycosylation on asn66 was found within the cterminal region of the protein, confirming that a minor proportion of the sars-cov e protein is exposed to the luminal side. however, the membrane topologies of sars-cov e protein in virions and in virusinfected cells are still unknown. considering the multiple roles of the coronavirus e protein in viral replication cycles, it would be of interest to relate different topology to each distinct function. pivotal roles in virion assembly and morphogenesis coronavirus e protein is crucial for virion assembly. co-expression of m and e proteins, but not expression of m protein alone, led to the formation of virus-like particles (vlp), which are morphologically indistinguishable from coronavirions [16, 19, [28] [29] [30] [31] . additionally, expression of e protein on its own results in the release of e-containing membrane vesicles [32] . it is generally believed that e protein is required for the formation of coronavirus vlp. however, sars-cov m and n proteins were reported to be necessary and sufficient for the formation of vlp [33] , although this is not consistent with a report showing that sars-cov m and e are sufficient for vlp formation [30] . it either indicates that sars-cov might adopt a unique mechanism for virion assembly, or simply suggests that formation of vlp may vary with the expression systems used. the precise function of e protein in virion assembly is not fully elucidated, but its low abundance in virions and in vlp implied that it might serve to induce membrane curvature or act to pinch off the neck of the viral particles at the final stage of the budding process. analysis of bhk cells expressing the mhv e protein by electron microscopy showed that the protein could induce the formation of tubular structures in the er-golgi intermediate compartment network, suggesting that this protein has a tendency to induce membrane curvature [21] . in vitro incorporation of the transmembrane domain of sars-cov e protein into lipid vesicles can also deform the vesicle and induce changes in the thickness of the lipid bilayer and acyl-chain ordering [25, 26] . more recently, the transmembrane domain of the ibv e protein was shown to be required for efficient release of virus particles [34] . coronavirus e protein is also involved in the morphogenesis of viral particles. mutations introduced into the c-terminal hydrophilic tail of mhv e protein by targeted rna recombination showed that one of the mutants was remarkably thermolabile and appeared to be aberrant and heterogeneous in virion morphology, exhibiting pinched and elongated shapes instead of the normal rounded shapes [35] . this phenotype suggests that e protein is essential for creating the membrane curvature needed to acquire the rounded and stable virions. however, a more authoritative and in-depth study of the virion structure and morphology is needed to exclude the possibility that the observed abnormal morphology of the mutant virus may be caused by artificial factors during sample preparation, as the mutant virus might be more fragile than wild-type virus. a mutant mhv with deletion of the e gene was recovered later, although the mutant virus produces tiny plaques and shows low growth rate and titer [36] . alanine scanning insertion mutagenesis of the hydrophobic domain of the mhv e protein suggested that positioning of polar hydrophilic residues within the predicted transmembrane domain is important for virus production [37] . substitution of the mhv e gene with heterologous e genes from viruses spanning all three groups of coronavirus family showed that the e proteins of group 2 and 3 coronaviruses could almost fully replace the mhv e protein [38] . however, e protein of the group 1 coronavirus, tgev, could functionally replace the mhv e protein only after acquisition of particular mutations [38] . as these e proteins share a low degree of sequence identity, it indicates that sequence-specific contacts with other viral components may not be essential. more recently, a recombinant sars-cov that lacks the e gene was rescued in vero e6 cells, and the recovered deletion mutant was attenuated in vitro and in the hamster model [39] . interestingly, visions & reflections (minireview) 2045 electron microscopy analysis showed that wild-type and the deletion mutant viruses are morphologically identical [39] . at variance for group 1 tgev, deletion of e protein is lethal, as shown in two independent reverse genetic experiments [40, 41] . in general, coronavirus e protein is not essential for viral replication, but seems to be a non-obligate budding enhancer. an intriguing question on coronavirus assembly is whether direct interaction between e and m proteins is required for virion assembly. physical interaction of the two proteins was demonstrated by co-immunoprecipitation in virus-infected or transfected cells [19, 32, 42] . however, studies of ibv e and m mutants showed that interaction between the two proteins is not sufficient for the vlp formation [42] . meanwhile, chimeric m protein was able to form vlp with tgev e, bcov e, and bcv-tgev chimeric e proteins [28, 42] . as mentioned above, the e proteins of group 2 and 3 coronaviruses were readily inter-changeable for that of mhv. these results suggest that sequencespecific contacts of e protein with m protein may not be essential for virion assembly. two coronavirus e proteins, mhvand sars-cov e proteins are apoptosis inducers [43, 44] . the apoptotic pathway induced by mhv e protein can be blocked by overexpression of bcl-2, suggesting that the initiation of the apoptotic pathway by this protein is upstream of bcl-2 [43] . sars-cov e protein was also able to induce apoptosis in the transfected jurkat t cells, which can be inhibited by overexpression of bcl-xl [44] . a bh3-like domain located in the cterminal region of the sars-cov e protein could mediate binding of the protein to bcl-xl [44] . however, induction of apoptosis does not appear to be a general feature of the coronavirus e protein and is cell-type specific. for example, mhv infection induced apoptosis in 17cl-1 cells but not in dbt cells [43] . constitutive expression of the tgev e protein in bhk cells using a sindbis replicon system did not induce apoptosis [41] . however, apoptosis induced by coronavirus e protein was observed in cells overexpressing the protein. this may not faithfully reflect the real situation in virus-infected cells. several small viral membrane proteins, such as the influenza virus m2 protein and the hiv vpu protein, could modify host cell membrane and form ion channels. recently, the sars-cov e protein was shown to form membrane channels with selectivity for monovalent cations [45] . moreover, a peptide corresponding to the n-terminal 40 amino acids of the sars-cov e protein had the same properties as the full-length e protein. similar to other viroporins [46] , expression of sars-cov and mhv e protein enhanced the membrane permeability of bacterial and mammalian cells [47, 48] . this channel-forming activity of coronavirus e protein was more recently extended to the e proteins of human coronavirus 229e (hcov-229e) , mhv, and ibv [49] , and may therefore be a general feature of the e protein from all three groups of coronavirus. interestingly, channels formed by e proteins of group 2 (mhv and sars-cov) and group 3 (ibv) coronaviruses show greater preference for sodium ions over potassium ions [45, 49] . by contrast, the ion channels formed by the e protein of group 1 coronavirus hcov-229e exhibit greater preference for potassium ions over sodium ions [49] . hexamethylene amiloride (hma), an amiloride analogue, blocks the ion channel activity of hiv vpu [50, 51] , hepatitis c virus (hcv) p7 [52] and dengue virus m protein [53] . this molecule could also inhibit the ion channel activity of the hcov-229e and mhv e proteins, but not the ibv e protein [49] , suggesting a more divergent structure of group 3 coronavirus e protein. furthermore, hma is able to inhibit the replication of hcov-229e and mhv, but not the replication of a recombinant mhv with deletion of the entire e gene [49] . these results indicate that the ion channel activity of coronavirus e protein is important for virus replication, especially in the case of some coronaviruses, such as mhv. however, the exact role of e protein in coronavirus life cycle is yet to be revealed. it is believed that small ion channels formed by virus-encoded proteins can help uncoating or release of mature virus particles. for example, the influenza virus m2 protein can form a proton channel to lower the interior ph of the virion. the lower internal virion ph is also thought to weaken protein-protein interactions between the viral matrix protein, the ribonucleoprotein core and the lipid bilayer, thereby freeing the viral genome from interactions with viral proteins and enabling the viral rna segments to migrate to the host cell nucleus. one possibility of how e protein could enhance the release of the mature viral particles is that dissipation of the ionic gradient in the er-golgi intermediate compartment as well as the golgi compartment may promote virion exit through the transport pathway. further exploration of the functional significance of e protein in coronavirus life cycles may reveal more detailed information of coronavirus replication mechanisms and pathogenesis. coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step tgev corona virus orf4 encodes a membrane protein that is incorporated into virions association of the infectious bronchitis virus 3c protein with the virion envelope mouse hepatitis virus gene 5b protein is a new virion envelope protein sequencing of coronavirus ibv genomic rna: three open reading frames in the 5 unique region of mrna d in vitro synthesis of two polypeptides from a nonstructural gene of coronavirus mouse hepatitis virus strain a59 detection of a murine coronavirus nonstructural protein encoded in a downstream open reading frame a polycistronic mrna specified by the coronavirus infectious bronchitis virus internal entry of ribosomes on a tricistronic mrna encoded by infectious bronchitis virus coronavirus mhv-jhm mrna 5 has a sequence arrangement which potentially allows translation of a second, downstream open reading frame internal ribosome entry in the coding region of murine hepatitis virus mrna 5 characterization of a novel coronavirus associated with severe acute respiratory syndrome 5 kda encoded between the spike and membrane protein genes of the bovine coronavirus identification of a new membrane-associated polypeptide specified by the coronavirus infectious bronchitis virus infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles the cytoplasmic tail of infectious bronchitis virus e protein directs golgi targeting biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein the missing link in coronavirus assembly. retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-golgi compartments and physical interaction between the envelope and membrane proteins differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins characterization of the coronavirus mouse hepatitis virus strain a59 small membrane protein e biochemical evidence for the presence of mixed membrane topologies of the severe acute respiratory syndrome coronavirus envelope protein expressed in mammalian cells the transmembrane oligomers of coronavirus protein e membrane topology of coronavirus e protein a highly unusual palindromic transmembrane helical hairpin formed by sars coronavirus e protein sars coronavirus e protein in phospholipid bilayers: an x-ray study model of a putative pore: the pentameric alpha-helical bundle of sars coronavirus e protein in lipid bilayers coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes the production of recombinant infectious di-particles of a murine coronavirus in the absence of helper virus assembly of human severe acute respiratory syndrome coronavirus-like particles nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes release of coronavirus e protein in membrane vesicles from virusinfected cells and e protein-expressing cells generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production the transmembrane domain of the infectious bronchitis virus e protein is required for efficient virus release analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly the small envelope protein e is not essential for murine coronavirus replication role of the coronavirus e viroporin protein transmembrane domain in virus assembly visions & reflections (minireview) exceptional flexibility in the sequence requirements for coronavirus small envelope protein function a severe acute respiratory syndrome coronavirus that lacks the e gene is attenuated in vitro and in vivo heterologous gene expression from transmissible gastroenteritis virus replicon particles generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome the cytoplasmic tails of infectious bronchitis virus e and m proteins mediate their interaction induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of e protein as an apoptosis inducer bcl-xl inhibits t-cell apoptosis induced by expression of sars coronavirus e protein in the absence of growth factors sars coronavirus e protein forms cation-selective ion channels expression of sars coronavirus envelope protein in escherichia coli cells alters membrane permeability viroporin activity of murine hepatitis virus e protein hexamethylene amiloride blocks e protein ion channels and inhibits coronavirus replication amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by hiv-1 protein vpu the vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels cation-selective ion channels formed by p7 of hepatitis c virus are blocked by hexamethylene amiloride dengue virus m protein c-terminal peptide (dvm-c) forms ion channels key: cord-346819-11fkgzaa authors: khan, mohd imran; khan, zainul a.; baig, mohammad hassan; ahmad, irfan; farouk, abd-elaziem; song, young goo; dong, jae-jun title: comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations and the effect of mutations on major target proteins: an in silico insight date: 2020-09-03 journal: plos one doi: 10.1371/journal.pone.0238344 sha: doc_id: 346819 cord_uid: 11fkgzaa a novel severe acute respiratory syndrome-related coronavirus-2 (sars-cov-2) causing covid-19 pandemic in humans, recently emerged and has exported in more than 200 countries as a result of rapid spread. in this study, we have made an attempt to investigate the sars-cov-2 genome reported from 13 different countries, identification of mutations in major coronavirus proteins of these different sars-cov-2 genomes and compared with sars-cov. these thirteen complete genome sequences of sars-cov-2 showed high identity (>99%) to each other, while they shared 82% identity with sars-cov. here, we performed a very systematic mutational analysis of sars-cov-2 genomes from different geographical locations, which enabled us to identify numerous unique features of this viral genome. this includes several important country-specific unique mutations in the major proteins of sars-cov-2 namely, replicase polyprotein, spike glycoprotein, envelope protein and nucleocapsid protein. indian strain showed mutation in spike glycoprotein at r408i and in replicase polyprotein at i671t, p2144s and a2798v,. while the spike protein of spain & south korea carried f797c and s221w mutation, respectively. likewise, several important country specific mutations were analyzed. the effect of mutations of these major proteins were also investigated using various in silico approaches. main protease (mpro), the therapeutic target protein of sars with maximum reported inhibitors, was thoroughly investigated and the effect of mutation on the binding affinity and structural dynamics of mpro was studied. it was found that the r60c mutation in mpro affects the protein dynamics, thereby, affecting the binding of inhibitor within its active site. the implications of mutation on structural characteristics were determined. the information provided in this manuscript holds great potential in further scientific research towards the design of potential vaccine candidates/small molecular inhibitor against covid19. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in the last two decades, three coronaviruses viz. severe acute respiratory syndrome coronavirus (sars-cov) [1] , middle-east respiratory syndrome coronavirus (mers-cov) [2] and sars-cov-2 have crossed the species barrier to cause deadly pneumonia in humans. in 2002, sars-cov emerged in the guangdong province of china and spread to five continents, infecting 8,098 people with 774 deaths. in 2012, mers-cov emerged in the arabian peninsula, transmitted to 27 countries, infecting a total of~2,494 individuals and claiming 858 lives. the current outbreak of coronavirus disease caused by sars-cov-2, was first reported in december 2019 in wuhan, hubei province of china [3, 4] and spread across 200 countries, infecting over 2.5 million people and killed more than 1.5 lakh as of april, 23, 2020. sars-cov-2 was declared a pandemic by the world health organization on march 12, 2020. sars-cov-2 belongs to the family coronaviridae of genus betacoronavirus, having positive sense strand rna genome of 26-32 kb size. sars-cov-2 genome has six major open reading frames (orfs) viz. replication enzyme coding region (orf 1a and 1b), e gene (envelope protein), m gene (membrane protein), s gene (spike protein), and n gene (nucleocapsid protein) that are common to coronaviruses and a number of other accessory genes (orf 3a, 6, 7a, 7b and 8) (fig 1) [3] . the structural proteins: envelope protein, nucleocapsid protein, spike protein and membrane protein are essential for producing the structurally complete viral particle [5] [6] [7] [8] . entry of coronavirus into host cells is guided by spike glycoprotein. orf 1a and 1b encode replication enzyme consisting 16 non-structural proteins (nsp1-16) that are highly conserved among the coronaviruses. main protease (mpro, also known as 3clpro) is one of the important nsp encoded by orf 1a and 1b, play an essential role in the processing of polyproteins and control the replication of coronavirus [9, 10] . rna-dependent rna polymerase (rdrp) also known as nsp12, another important replicase catalyze the replication of rna using viral genomic rna template [11] . reports showed that mers-cov originated from bats, but the reservoir host fueling spillover to humans is unequivocally dromedary camels [12, 13] . both sars-cov and sars-cov-2 are originated from bats, which serve as reservoir host for these two viruses [3, 14, 15] . raccoon dogs and palm civets have been identified as intermediate hosts for zoonotic transmission of sars-cov between bats and humans [16] [17] [18] , however, the intermediate host of sars-cov-2 remains unknown. mutation rate is very high in rna viruses, up to a million times higher than their host, which enhance their virulence and evolvability (formation of new species) [19] . coronavirus replication is error prone as compared to other rna viruses and the estimated mutation rate is 4x10 -4 nucleotide substitutions/site/year [20] . the rate of sars-cov-2 mediated disease spread and the mortality varies from country to country. of several reasons affecting the rate of disease spread and mortality, mutations within the sars-cov-2 strains is also considered one of the major factors. this study was conducted to gather additional information on the sars-cov-2 sequences from different geographical locations infected with covid-19. the genome analysis of the sars-cov-2 strains from 13 different countries showed a large number of mutations within the major structural proteins. this is the first time we have comprehensively investigated these mutations and also discussed their potential roles in the pathogenicity, replication and entry of virus particle. this study provides a deeper insight into the emergence of these mutations within the major structural as well as nsp encoded by the sars-cov-2 genome from different countries. here, molecular dynamics and other in silico studies were also performed to investigate the effect of mutations on the dynamics of mpro. the findings of this study provide a clue for the futuristic development of potential vaccine candidate or therapeutic design against covid19. the genome sequence of orf1ab for sars with reference sequence id: nc_004718.3 and protein sequence with genbank id: aap41036. and visualization of sars and sars-cov-2 sequences from 13 different countries was performed using molecular evolutionary genetics analysis (mega) version 10.1.8. to delineate and analyze the mutation across different countries, an in-house script written in perl and python was used. mupro server was used to determine the effect of mutation on various sars-cov-2 proteins [21] . the crystal structure of mpro protein from sars-cov-2 in complex with boceprevir (pdb id: 7brp) was taken as a wildtype (wt). the structure of r60c was generated by inserting the point mutation and modelled using modeler 9v13 [22] . boceprevir was retrieved and redocked within the structure of mpro using ccdc gold. the rmsd for the crystal and redocked conformation of boceprevir were compared. further, boceprevir was subjected to dock within the active site of r60c mutant. the poses were visualized on pmv viewer [23] . the structure of boceprevir in complex with mpro (wt) and r60c mutant was subjected to energy minimization using gromacs-5 with the charmm27 all atom force field [24] [25] [26] . the models were solvated with a spc/e water model in a cubic periodic box with 1 nm distance from the edge of the complex atoms. the solvated system was neutralized by seven chloride ions. the system was, thereafter, minimized using steepest descent algorithm with convergence criteria of tolerance value 1000 kj mol −1 nm −2 . the complete simulation of minimized solvated proteins was performed under periodic boundary condition with time step of 2 fs. particle mesh ewald was used for long range electrostatic interactions with an interpolation order of 4 and a fourier spacing of 0.16. the first phase simulation was conducted under an nvt ensemble for 500 ps by keeping all bonds constrained using the lincs algorithm for temperature equilibration. the system was heated to 300 k using leap-frog integrator while pressure coupling was set off. a v-rescale thermostat was used to maintain constant temperature for each system, followed by pressure equilibration at 300 k using parrinello-rahman pressure coupling algorithm under an isothermal-isobaric ensemble for another 500 ps at 1.0 bar. isothermal compressibility of the solvent was set to 4.5e -5 bar −1 . further, simulation of 50000 ps production run was carried out at 300 k and 1 atm pressure for trajectory analysis. the final models obtained at the end of md were validated and taken for structural analysis. the complete nucleotide sequences of 13 sars-cov-2 reported from 13 different countries showed~82% sequence identity with sars-cov. also, all 13 sequences shared more than 99% sequence identity to each other. replicase polyprotein (orf 1ab) of 13 isolates, which are most conserved in all coronaviruses shared maximum identity (87%) with sars-cov (nc_0047180), which is less than the threshold value (90%) for demarcation of betacoronavirus species [27, 28] . phylogenetic analysis revealed that all 13 sars-cov-2 identified from different geographical locations clustered together in a single clad as compared to sars-cov (fig 2a and 2b ). further, we checked the mutation in all major proteins of 13 sars-cov-2 sequences and compared with sars-cov. orf 1a and 1b showed 11 changes among all 13 sars-cov-2. indian sars-cov-2 sequence showed three changes at 671 (isoleucine to threonine), 2144 (proline to serine) and 2798 (alanine to valine) compared to all other 12 isolates. here, we also noted two amino acid mutations (in orf1ab) in each sars-cov-2 sequences isolated from china (2708: asparagine to serine; 2908: phenylalanine to isoleucine), south korea (902: methionine to isoleucine; 6891: threonine to methionine) and sweden (818: glycine to serine; 4321: phenylalanine to leucine). brazil and vietnam isolate showed only one change at 3606 (leucine to phenylalanine) and 3323 (arginine to cystine), respectively (table 1) . 996 changes have been reported when orf 1a and 1b amino acid sequences of all 13 sars-cov-2 was compared with sars-cov (s1 file). the viral mpro controls the replication of coronavirus and is a key protein responsible for its life cycle [29] [30] [31] . mpro is an attractive drug discovery target. the analysis of mpro reveals that there was only one point mutation (r60c) in the vietnam strain of sars-cov-2 ( fig 3a) . rdrp, which is another important target for antiviral drugs functions by catalyzing the viral rna synthesis [32] . only one mutation (a406v) was observed in the rdrp of indian sars-cov-2 isolate (fig 3b) . spike proteins are the key surface glycoproteins and are well reported for their prominent role in interaction with host cell receptors [33, 34] . here, we analyzed the mutations in the spike protein of sars-cov-2 from different countries. it was found that this glycoprotein carried five different amino acid mutations at various positions within the investigated sars-cov-2 isolates. for instance, india, finland, australia, south korea and sweden sars-cov-2 isolates showed one amino acid change at 408 (arginine to isoleucine), 49 (histidine to tyrosine), 247 (serine to arginine), 221 (serine to tryptophan) and 797 (phenylalanine to cysteine), respectively ( table 2 and fig 3c) . the value of δδg show that the mutant r408i (0.49732107 kcal/mol) mutation was having stabilization effect on spike protein. it was found that the mutation on the receptor binding domain (rbd) of spike protein increases the stability. when these 13 sars-cov-2 isolates were compared to sars-cov sequence, 1338 changes have been reported (s1 file). the analysis of orf3a showed 3 mutations within different sars-cov-2 strains: w128l (south korea), l140v (japan), g251v (australia, south korea, brazil, italy, sweden) ( table 3) . one amino acid change occurred in each envelope protein of south korea sars-cov-2 isolate at 37 (leucine to histidine) and nucleocapsid protein of japan sars-cov-2 isolate at 344 (proline to serine) when compared among 13 sars-cov-2 isolates (tables 4 and 5 i i i i i i i i i i i i 818 t t m t t t t t t t t t t https://doi.org/10.1371/journal.pone.0238344.t001 when compared to sars-cov. mem glycoprotein did not show any amino acid change among 13 sars-cov-2 isolates, while 24 changes occurred when compared to sars-cov (s1 file). all the other point mutations occurring within the structural proteins of sars-cov-2 isolates from different countries were found to decrease protein stability (table 6 ). in the present study, we performed the md simulations for the boceprevir bound complexes of sars-cov-2 mpro and its r60c mutant to study the effect of mutation on the protein dynamics. the root mean square deviations of the backbone were calculated to analyze the trajectories of mpro from sars-cov-2 and its r60c mutant. in the complex form, a slight fluctuation in the comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations backbone rmsd was also noticed (fig 4a) . it was found that the rmsd of mutant was comparatively more stable than its wt. this variation in the average rmsd values suggests that this mutation was affecting the dynamic behavior of mpro. rmsf values of native as well as r60c mutant mpro were calculated to determine the impact of mutation on dynamic behavior of protein at residue level. rmsf plot clearly indicates the fluctuation in residues and showed the existence of higher degree of flexibility in r60c mutant mpro. it was found that the maximum amino acid fluctuation was in the region 50-76 and 127-222 ( fig 4d) . the binding studies also confirm that the residues falling within this region were very much involved in accommodating the inhibitor within the active site of mpro [29, 31] . throughout the md trajectory, the interaction energy of ligand in complex with the surrounding protein residues of wt and mutant mpro were calculated. the lennard-jones shortrange (lj-sr) and coulombic short-range (coul-sr) potential energies were calculated throughout the course of 50 ns of md simulation (fig 4e) . the average interaction energy for 50 ns are shown in table 7 . it was found that the binding of inhibitor within the active site of mpro (wt) was stronger as compared to the r60c mutant. the analysis of hydrogen bond network revealed that the r60c mutation also cause disturbance in the interactions with inhibitor as well as other surrounding active site residues of mpro. a large fluctuation was noticed in the hydrogen bond network of mpro and its r60c mutant (fig 5a) . it was found that r60c mutation results in the changes in local environment that cascade further to the short helix and loop of the catalytic active site of mpro. till date (april 23, 2020), 2.6 million cases of covid-19 have been reported worldwide. in this study, we analyzed 13 complete sequences of sars-cov-2 reported from 13 different countries and compared with sars-cov. phylogenetic analysis showed that sars-cov-2 sequences clustered together in a single clad irrespective of their geographic origin, whether from the same continent or neighboring countries. replicase polyprotein, which are most conserved among coronaviruses, shared 87% amino acid sequence similarity to sars-cov, less than the threshold value (90%) for demarcation of betacoronavirus species [27] . they belong to new virus species severe acute respiratory syndrome-related coronavirus of genus betacoronavirus [28] . among all known rna viruses, coronaviruses consist of the largest genome (26.4 to 31.7 kb) [36, 37] . the large genome size provides more plasticity in accommodating and modifying genes [36] [37] [38] . mutation frequency is very high in rna viruses, which enhances virulence and responsible for the formation of new species [19] . the high frequency of mutation within the viral genome at different geographical locations may be one of the reasons that sars-cov-2 is responsible for change in mortality rate and symptom of the disease [39] . the comparison of amino acid sequences of replicase polyprotein of 13 sars-cov-2 showed mutations in india, china, south korea, sweden, vietnam and brazil strains at different amino acid locations. earlier report showed the similar result i.e. a single mutation in replicase polyprotein at 3606 (l to f) [40] . we could identify few more single amino acid mutations at different positions in above mentioned sars-cov-2 strains ( table 1 ). the replicase polyprotein codes for nsp2 and nsp3 and it has been suggested in previous research that the mutation in nsp2 and nsp3 play a key role in infectious capability and are responsible for the differentiation mechanism of sars-cov-2 [39] . the rbd of spike protein is the region which specifically interact with ace2 leading to viral entry into the host cell [41] [42] [43] . the indian isolate of sars-cov-2 showed mutation within this region where at 408 position, arginine is replaced by isoleucine. for several years, the prediction of protein stability via theoretical or experimental approaches has been a profound area of research [44] . earlier findings suggest that a single point mutation at rbd is responsible for disrupting the antigenic structure, thereby, affecting the binding of rbd to ace2 [45, 46] . the mutation within this region of spike protein may affect the binding of rbd to its receptor, thus, affecting the viral entry within the host cells. further, in silico studies revealed that this point mutation within the rbd of spike glycoprotein was having stabilization effect on spike protein and found to increase the protein stability (δδg 0.49732107 kcal/mol). single amino acid mutation was observed in both mpro (r60c) of sars-cov-2 vietnam isolate and rdrp (a408v) of sars-cov-2 india isolate. the in silico findings revealed that the mutations in both strains decrease the stability of protein. the md simulation studies on mpro further confirmed that the point mutation on mpro affects the stability of proteins as well as the binding of inhibitor. our in silico study found that the catalytic active site of mpro is surrounded by amino acid residues of a loop (142-145, 175-200), short helix (40) (41) (42) (43) (46) (47) (48) (49) (50) , and beta sheet regions (25) (26) (27) (164) (165) (166) (167) . the r60c mutant lies at helix adjacent to the short helix (h2) that forms the catalytic channel (fig 5) . substitution of an amino acid with charged side chain to uncharged cysteine residue leads to loss of conserved ionic bond interaction and the effect cascades to other conserved ionic interactions. loss of conserved ionic interaction was observed between amide nitrogen of arginine and carboxylic oxygen atom of aspartic acid at position 48 of the catalytic channel. it was found that the short helix h2, that form the catalytic channel, have attained a more flexible loop like conformation in the mutant protein. conserved hydrogen bonded interactions that stabilizes the catalytic channel l1 loop between tyr54 oh� � �asp187 oδ1, tyr54 oh� � �asp187 o and leu50 o� � �arg188 ne were lost in the mutant enzyme, thereby, increasing the flexibility of structure forming the binding pocket ( fig 5b) . therefore, the local change of an ordered secondary structure to a more disordered loop like structure have increased the overall flexibility of the secondary structure elements forming the catalytic pocket, thereby, effecting the binding of ligand to residue in the catalytic channel. this is quite evident from the reduced lj-sd and coulombic-sr interaction energies between the enzyme and ligand in wild and mutant complexes ( fig 4e and table 7 ). the role of important active site residues, discussed in this study, has been reported before [47, 48] . the rmsf plot also reveals the key role of these residues in accommodating the inhibitor within the active site of mpro. envelop protein plays an important role in the assembly of viral genome and the formation of ion channels (ic), responsible for virus-host interaction, which is mainly associated with pathogenesis [5, 49] . we detected one amino acid mutation l37h in transmembrane domain (tmd) of envelop protein of sars-cov-2 south korea isolate. the tmd is hydrophobic in nature consisting mainly hydrophobic amino acids, while the mutation in tmd at 37 position changes hydrophobic to hydrophilic amino acid which changes the integrity of tmd. earlier report showed that mutations within the tmd domain of envelop protein completely disrupted ic activity [50] . this might be one of the reasons for slow spreading/low pathogenicity of sars-cov-2 in south korea. nucleocapsid protein of coronavirus is necessary for rna replication, transcription and genome packaging [51, 52] . a mutation p344s in nucleocapsid protein has been detected in sars-cov-2 japan strain. the p344s mutation on nucleocapsid was found to decrease the protein stability (δδg -1.2252261). this mutation is located in carboxy-terminal rna-binding domain (ctd) of nucleocapsid protein. earlier studies showed that ctd is responsible for oligomerization [53] . it was also revealed that among all the genomes studied in this study, the indian sars-cov-2 isolates were carrying maximum mutation. the indian isolates were carrying the r408i on the spike protein while a406v on the rdrp and several mutations on the replicase polyprotein of sars-cov-2. it is expected that these large number of mutations among the sars-cov-2 may affect the vaccine/inhibitor development against these isolates. to conclude, sars-cov-2 complete sequences from 13 countries were analyzed and compared with sars-cov. we identified country specific mutations in major proteins (replicase polyprotein, spike protein, envelop protein and nucleocapsid protein). further, molecular dynamics and other in silico studies revealed that mutations decrease the stability of protein and also hinders the binding of inhibitor. mutation r408i in spike protein of indian strain has significant influence on rbd domain of spike protein and this point mutation has a stabilization effect on the spike protein. the findings of the present study could help for the design of potential vaccine candidates/small molecular inhibitor against covid19. supporting information s1 file. 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an attempt to do so for residue 133 of t4 lysozyme using a combination of free energy derivatives, profec, and free energy perturbation methods structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor unravelling lead antiviral phytochemicals for the inhibition of sars-cov-2 m(pro) enzyme through in silico approach in silico screening of natural compounds against covid-19 by targeting mpro and ace2 using molecular docking the coronavirus e protein: assembly and beyond severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis nucleocapsid protein-dependent assembly of the rna packaging signal of middle east respiratory syndrome coronavirus coronavirus genomic rna packaging oligomerization of the carboxyl terminal domain of the human coronavirus 229e nucleocapsid protein the authors are very grateful to prof. indranil dasgupta, university of delhi south campus, new delhi, india, for giving suggestions to improve the manuscript. key: cord-190540-zf5ksac2 authors: rakshit, kausik; chatterjee, sudip; bandyopadhyay, durjoy; sarkar, somsekhar title: an effective approach to reduce the penetration potential of sars-cov-2 and other viruses by spike protein: through surface particle electrostatic charge negotiation date: 2020-06-18 journal: nan doi: nan sha: doc_id: 190540 cord_uid: zf5ksac2 the objective of this paper is to provide a mathematical model to construct a barrier that may be useful to prevent the penetration of different viruses (eg. sars-cov-2) as well as charged aerosols through the concept of electrostatic charge negotiation. (fusion for the opposite types of charges and repulsion for the similar types of charges). reviewing the works of different authors, regarding charges, surface charge densities ({sigma}), charge mobility ({mu}) and electrostatic potentials of different aerosols under varied experimental conditions, a similar intensive study has also been carried out to investigate the electron donating and accepting (hole donating) properties of the spike proteins (s-proteins) of different rna and dna viruses, including sars-cov-2. based upon the above transport properties of electrons of different particles having different dimensions, a mathematical model has been established to find out the penetration potential of those particles under different electrostatic fields. an intensive study have been carried out to find out the generation of electrostatic charges due to the surface emission of electrons (see), when a conducting material like silk, nylon or wool makes a friction with the gr iv elements like germanium or silicon, it creates an opposite layer of charges in the outer conducting surface and the inner semiconducting surface separated by a dielectric materials. this opposite charge barriers may be considered as inversion layers (il). the electrostatic charges accumulated in the layers between the gr iv ge is sufficient enough to either fuse or repel the charges of the spike proteins of the rna, dna viruses including sars-cov-2 (rna virus) or the aerosols. ) the data from where e is the elementary charge of an electron and a is the median number of elementary charges of magnitude e present on a particle of diameter 1 μm., it is found that, the value of b is nearly equal to 2 and also at per as suggested by jonhston. (range between 1 and 2). this expected range of b is also applicable for calculating the charge of any small particles. the accumulation of charges on the surface of sars-cov-2 can be attributed by the following four factors: • (depending upon their shapes and sizes). the ultimate structural understanding of a protein comes from an atomic-level structure obtained by x-ray crystallography or nuclear magnetic resonance (nmr) spectroscopy. however, structural information at the nanometer level is frequently invaluable. hydrodynamics, in particular sedimentation and gel filtration, can provide this structural information, and it becomes even more powerful when combined with electron microscopy (em). a hypothetical visualization is that if we consider one charged site per 'n' amino acids at a particular ph, the number of free electrons present per nano meter length of spike proteins remain in the order of (1400/n) and the value of the charges per nano meter length of the spike may be of the order of 1.6 x 10 -19 x (1400/n) = (2240 x 10 -19 /n) c. (experimental determination of the value of n is beyond the scope of this paper and is strongly recommended by the authors). therefore, at a particular ph considering the above values, a mathematical model has been established in 1.3 to find out the charges of the spike proteins of different lengths ranging from 8.0 nm to 10.0 nm for sars-covtherefore, it can be predicted that, the spike proteins of sars-cov-2 shows measurable electron donating or accepting capabilities. if we look at the enveloping proteins of the sars-cov, refereeing to the following research by dewald schoeman et al 45 ., we have found that, the envelope proteins (e-proteins) have a capability to maintain a neutrality through the membrane potential of their own, till a considerable amount of charge difference is created when the structure of whole envelope collapses. the cov e protein is a short, integral membrane protein of 76-109 amino acids, ranging from 8.4 to 12.0 kda in size. the primary and secondary structure reveals that, e protein has a short, hydrophilic amino terminus consisting of 7-12 amino acids, followed by a large hydrophobic trans membrane domain (tmd) of 25 amino acids, and ends with a long, hydrophilic carboxyl terminus, which comprises the majority of the protein. the hydrophobic region of the tmd contains at least one predicted amphipathic α-helix that oligomerizes to form an ion-conductive pore in membranes. comparative and phylogenetic analysis of sars-cov e revealed that, a substantial portion of the tmd consists of the two nonpolar, neutral amino acids, valine and leucine, lending a strong hydrophobicity to the e protein. the peptide exhibits an overall net charge of zero, the middle region being uncharged and flanked on one side by the negatively charged amino (n)-terminus, and, on the other side, the carboxy (c)-terminus of variable charge. the c-terminus also exhibits some hydrophobicity but less than the tmd due to the presence of a cluster of basic, positively charged amino acids. an investigation regarding maintenance of this membrane potential had been done in case of here, it can be seen that, the charge of the s-protein is predicted to vary linearly with spike length at a particular ph. the effects due to various factors affecting the charge are listed below in the form of equations stated beforehand: at the electrostatic charges form due to the surface emission owing to the frictions between the layers of semiconducting materials like ge with the conducting materials like nylon, wool. when the conducting materials like wools make friction with a semiconducting gr iv materials, it shows a surface emission of electrons and the charges remain accumulated at the outer conducting layers since the middle layer of the mask is a semiconductor and is separated by an effective dielectric medium. the charge densities per square cm area(σ) follow empirically the growth of charges equation in a capacitor. where, ε is the permittivity of the medium, μ is the coefficient of friction between the two layers, nf is the normal force acting over the surface depending upon the rate of movement, 4πr 2 is the surface area and dx is the separation distance between the conducting and semi conducting surface separated by the dielectric materials. given below is a graphical representation of 3. from the above studies it is clear that, the accumulation of charges per square cm area of a conducting bi layer due to the surface emission of electrons in normal movement is at least 10 6 times more than the charges on surface of viruses including sars-cov-2 and also the charge density is much higher than the charges accumulated in the concentrated dust particles of normal ranges. therefore, it may be concluded that, due to the surface emission of electrons, the il of a mask can effectively trap, neutralize or repel the the viruses and/or the charged aerosols which might be carrying air borne viruses. the charge accumulation on the surface of a virus is established to be in accordance with the equation which has been simplified with some basic assumption to using a derivation from this mathematical model, the model of a protective barrier has been established as follows: a three layer mask can be produced to prevent the immediate infections of dna, rna viruses including sars-cov-2 and others because the electric charge accumulation of the rna viruses is much less than the electrostatic charges accumulated in the layers of the mask within few minutes. in the inner surface, a cotton type non conducting material can be used which will work basically as a nonconductor so that the electrostatic charges produced inside the two layers does not drain out through surface of the (human)body. additionally, the cotton layer may be effective to protect the skin from electrostatic thermal radiation. frictions between the middle and the outer layers, where the hydrophobic conducting and semiconducting materials are used, effectively lead to accumulation of the static charges. the rate of accumulation of charges increase with the increase of friction but come to a saturation level following the model of growth of charges in a capacitor per square cm of area. from the above studies, authors strongly recommend the following investigations: • experimental determination of the value of 'n' as specified in equation 1.2.5. • experimental determination of the value of 'k' as specified in equation 2.2.2. • experimental determination of parameters 'χ' and 'c' as specified in this article. • conducting of further experiments to find out the applications of the same for the preparation of ppe or gloves. measurement of the electrostatic charge in airborne particles: ii-particle charge distribution of different aerosols a study on electrical charge distribution of aerosol using gerdien ion counter particle charge measurement for commonly generated laboratory aerosols covid-2019: the role of the nsp2 and nsp3 in its pathogenesis adsorption of viruses to charge-modified silica departments of microbiology and immunology and nutrition and food science 065402; and department of veterinary diagnostic investigation effects of membrane potential and sphingolipid structures on fusion of semliki forest virus current protocols in protein science (2005) 2.10.1-2.10.8 copyright c _ 2005 by size and shape of protein molecules at the nanometer leveldetermined by sedimentation, gel filtration identification of potential binders of the main protease 3clpro of the covid-19 via structure-based ligand design and molecular modeling structure, function, and antigenicity of the sarscov-2 spike glycoprotein cryo-em structure of the 2019-ncov spike in theprefusion conformation electrostatic origin of the genome packing in viruses features, evaluation and treatment coronavirus (covid-19) an overview on coronaviruses family from past to covid-19: introduce some inhibitors as antiviruses from gillan's plants structure, function, and evolution of coronavirus spike proteins the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex electrostatics of capsid-induced viral rna organization structural variations inhumanace2mayinfluence its bindingwith sars-cov-2 spike protein importance of sars-cov spike protein trp-rich region in viral infectivity coronavirus genomics and bioinformatics analysis ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission negatively charged residues in the endodomain are critical for specific assembly of spike protein into murine coronavirus cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer pre-fusion structure of a human coronavirus spike protein discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin a new coronavirus associated with human respiratory disease in china structural basis of sars-cov-2 3clpro and anti-covid-19 drug discovery from medicinal plants a novel coronavirus from patients with pneumonia in china electrostatic charged nanofiber filter for filtering airborne novel coronavirus (covid-19) and nano-aerosols, separation and purification technology covid-19 infection: origin, transmission, and characteristics of human coronaviruses origin and evolution of pathogenic coronaviruses severe acute respiratorysyndrome coronavirus 2 (sars-cov-2) and corona virus disease-2019 (covid-19): the epidemic and the challenges a simple theoretical analysis of the effective electron mass in heavily doped iii-v semiconductor in the presence of band-tails on the magnetic susceptibilities in quantum wires of ii-vi materials a 0.5-v 1-msps track-and-hold circuit with 60-db sndr a 0.5-v 74-db sndr 25-khz continuous-time delta-sigma modulator with a return-to-open dac ultra-low voltage analog integrated circuits kinget, 0.5-v analog circuit techniques and their application in ota and filter design studies on novel dual filler based epoxidized natural rubber nanocomposite thermal and mechanical properties of polymer nanocomposites based on ethylene methyl acrylate and multiwalled carbon nanotube,polymar composites influence of acrylate content on the properties of ethylene methyl acrylate multiwalled carbon nano tube composites studies on the filler microstructures in thermal and dynamic mechanical property development in enr based ternary nanocomposites the study of einstein relation in quantum wires of biomaterials : simplified theory and suggestions for further experimental determination the study of einstein relation in quantumdots superlatices (qdsl) of nonparabolic semiconductors : simplified theory and suggestions for future experimental determination in biomaterials coronavirus envelope protein: current knowledge authors are highly indebted to prof. (dr.) samir kumar patra, department of life science, national key: cord-312996-qzu8pkyt authors: iles, r. k.; zmuidinaite, r.; iles, j. k.; carnell, g.; sampson, a.; heeney, j. l. title: a clinical maldi-tof mass spectrometry assay for sars-cov-2: rational design and multi-disciplinary team work. date: 2020-08-22 journal: nan doi: 10.1101/2020.08.22.20176669 sha: doc_id: 312996 cord_uid: qzu8pkyt the covid-19 pandemic caused by the sars-cov-2 coronavirus has stretched national testing capacities to breaking points in almost all countries of the world. the need to rapidly screen vast numbers of a countrys population in order to control the spread of the infection is paramount. however, the logistical requirement for reagent supply (and associated cost) of rt-pcr based testing (the current front-line test) have been hugely problematic. mass spectrometry-based methods using swab and gargle samples have been reported with promise, but have not approached the task from a systematic analysis of the entire diagnostic process. here, the pipeline from sample processing, the biological characteristics of the pathogen in human biofluid, the downstream bioand physical-chemistry and the all-important data processing with clinical interpretation and reporting, are carefully compiled into a single high throughput and reproducible rapid process. utilizing maldi-tof mass spectrometric detection to viral envelope glycoproteins in a systems biology multidisciplinary team approach, we have achieved a multifaceted clinical maldi tof ms screening test, primarily (but not limited to) sars-cov-2, with direct applicable to other future epidemics/pandemics that may arise. the clinical information generated not only includes sars-cov-2 coronavirus detection (spike protein fragments s1, s2b, s2a peaks), but other respiratory viral infections detected as well as an assessment of generalised oral upper respiratory immune response (elevated total ig light chain peak) and a measure of the viral immune response (elevated intensity of iga heavy chain peak). the advantages of the method include; 1) ease of sampling, 2) speed of analysis, and much reduced cost of testing. these features reveal the diagnostic utility of maldi-tof mass spectrometry as a powerful and economically attractive global solution. mass screening of populations for infection in epidemics, but particularly pandemics, requires clinically effective (high sensitivity and selectivity) testing methods, requiring minimally-invasive sampling techniques, with fast turnaround (low hours) and a low cost of analysis (< 1 usd) a key feature in the context of large-scale screening of populations and the need for differential diagnosis of other circulating common cold pathogens. indeed, when the need for large-scale screening is global, logistical challenges such as the technical manpower, expertise, equipment and reagent availability and cost of the frontline test are less surmountable for most economically-challenged countries facing the pandemic, rendering control of the epidemic in many regions beyond reach. currently, rt-pcr testing is at the frontline for the identification of sars-cov-2 rna. sars-cov-2 rna is generally detectable in upper respiratory swabs and bronchoalveolar lavage (bal) samples during the acute phase of infection. positive results are indicative of the presence of sars-cov-2 rna; while clinical plasma biomarkers such as d-dimer, crp, il-6 levels with patient history are necessary to determine the patient's clinical covid-19 status and level of therapeutic intervention. the 2020 global pandemic caused by novel coronavirus sars-cov-2 pandemic has created an enormous challenge for health systems, clinical laboratories, public health intervention strategies, and community control measures worldwide. testing limitations, including reagent shortages, remain a bottleneck in the battle to curtail covid-19 spread in even the wealthiest countries [1, 2] the development of new matrix assisted laser desorption time of flight mass spectrometry (maldi-tof ms) diagnostics for sars-cov-2 detection is driven by the need for greater diagnostic capacity and alternative applications to complement standard pcr and antibody based diagnostics. preparative design in the development of any new assay system can circumvent many of the inevitable hurdles and dead ends that are encountered in early studies that have high promise, but have not considered detailed real-world characteristics. these include sampling errors, pathogen biology, biochemistry and physical limitations of the technology; not to mention due regard for bioinformatic rigor needed for classification and identification of robust and relevant markers. this has often been observed in the field of mass spectrometry, following sweeping assumptions and unrepresentative standards. however, rational multi-discipline based approaches to address these challenges, with step by step robust tests can result in significant diagnostic and socio-health economic returns [3] . to develop maldi-tof ms based diagnostics to specifically identify sars-cov-2, while distinguish this pathogen from other respiratory viruses, we had to overcome hurdles in the initial stages, looking beyond the traditional approachs by using established protocols routinely employed for bacterial identification [4] . panel b). schematic of virion fusion mediated by spike protein attachment to the ace2 receptor and accessory cleavage of the spike protein quaternary complex, resulting in fusion peptide exposure and binding to target cell plasma membrane. focal, host cell and viral envelope lipid bilayer membrane fusion and viral rna ingress of the target host cell. shedding of nucleocapsid and early orf expression to take control of the cell and inactivate internal anti-viral proteins (e.g. ubiquitin and rnase). late orf expression: replication of viral rna genome and expression of, via posttranslational processing mechanisms of the endoplasmic reticulum and golgi apparatus (e.g. glycosylation), membrane embedded proteins that are responsible for infectivity including the spike protein complex. copies of sars-cov-2 vrna are packaged into lipid bilayer membrane envelopes containing multiple copies of s, e, m and n, glyco-and phosphoproteins. inclusion transport vesicle, packed with enveloped virions, fuses with host cell membrane releasing infective sars-cov-2 virion. panel c). electro micrographs of multiple virions attacking the cell membrane (i), viral genome expression subsuming all functions of the cell to form thousands of virion copies within inclusion vesicles (ii) and finally release of multiple virions by fusion of inclusion packaging vesicles with the cell surface plasma membrane of the infected host cell (iii). enveloped viruses have the unique biological feature of utilizing the host cell membrane as the outer coating covering the core containing the rna genome. the coronaviruses have 4 key structural proteins and a large number of important functional proteins coded by their genome (figure 1 ). key amongst these viral envelope proteins are the spike protein encompassing the receptor binding domain (rbd) and membrane fusion complex, domains that enable infection of target host cells. for the coronaviruses these have been termed spike or "s" proteins (see figure 1a ). large trimeric complexes, these contain functional domains that undergo, what has been described as, tectonic conformational changes upon receptor binding and triggered exposure of membrane fusion peptide sequences [5, 6] . these bring the viral envelope and target cell lipid bilayer membrane into close contact such that fusion can occur. only a minor pore is sufficient for the viral rna to enter and be expressed (figure 1b). early phase orf expression takes control of the cell machinery, circumventing cellular defences such as ribonucleases and ubiquitination of viral proteins. late phase orf expression makes more viral envelope proteins, nucleoprotein and spike complex transcribed and post-translationally modified by the host cells endoplasmic reticulum and golgi apparatus. together with the viral rna genome, these are packaged into membrane exosomes to form enveloped virions bristling with spike protein complexes (see figure 1b and 1c) . in maldi-tof mass spectrometry for bacterial identification, the measured signals represent intracellular proteins, mostly highly abundant ribosomal proteins. the mass range usually measured ranges from 2,000 to 20,000 th ; the proteins revealed are largely not glycosylated, although they may be post-translationally modified in other ways such as phosphorylation [7] . the most appropriate robust and widely used matrix for ionisation of such unique or characteristic microbial intracellular proteins is alpha-cyano-4-hydroxycinnamic acid (chca) [8] . in consideration of a maldi-tof ms test for virus detection, this mass range is unsuitably low; significant differences in virion particles include the absence of intracellular "housekeeping" proteins. the majority of the unique features of viral proteins observed in maldi are much larger (10,000 -300,000 th),. furthermore, many of the virus specific proteins are heavily glycosylated [9] and posttranslationally modified. this mass range is outside the effective limits for chca; other matrices have been shown to be more suited for the maldi detection of large and glycosylated proteins [10] . our method development investigated alternative matrices (data not shown);sinapinic acid (sa) (3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-enoic acid) proved to be the most consistent and versatile and was used in all subsequently developed protocols. currently, sampling for covid-19 screening in frontline testing is via nasopharyngeal and or oropharyngeal swabs, often an unpleasant experience for the individual being tested and a potential reason for poor compliance within regular testing regimes, particularly amongst children [11] . once taken the swab sample has to be made safe for handling by medical, transport and laboratory staff. current methods inactivate virus by destroying proteins and the viral envelope membrane, preserving nucleic acid. generally, nasal-pharyngeal swabs are either heat inactivated, disrupting the protein and viral envelope membrane, or stored in a transport media containing sds and or triton which destroys the viral envelope of the virus, but is detrimental to mass spectrometric detection methods [12] . for effective maldi-tof ms covid-19 testing, based on detection and quantification of viral proteins, clinical sample deactivation has to largely do the opposite, i.e. preserve viral membrane and proteins but destroy the functional nucleic acid. this is conveniently achieved by the uv-c irradiation of sample tubes, for as little as 15mins to ensure safe handling in the laboratory and by transport systems [13] . consequently studies where swab samples have been split for simultaneous analysis by rt pcr detection systems of sars-cov-2 rna and by maldi-tof mass spectrometry for viral proteins, are compromised [4] . as an alternative to swabs, saliva and/or gargle is an acceptable, and more easily obtained sample means of collection; 10mls of water was found to be the optimum collection volume media for gargle saliva testing (data not shown). whether viral culture media, saliva/gargle, serum or urine, an enrichment of the target (e.g. viral envelope proteins), by the removal of unwanted entities is often necessary to improve mass spectrometric detection. he presence of abundant host molecules (such as albumin) can not only mask key, less abundant target proteins, but also suppress the ionisation of other low abundance proteins, even if they are detected at a distant m/z. not only does enrichment, and clean-up improve the detection of lower abundance signals, but also improves shot-to-shot and sample-to-sample reproducibility [14, 15] . in this work, the sample clean-up exploited the biophysical size difference of several orders of magnitude between virion particle and most components of acellular fractions of human biological fluids, allowing the convenient separation of them by precipitation. separation by ultracentrifugation was ruled out as impractical for routine implementation, as was selective precipitation by salt addition or peg, due to their deleterious impact on ms analysis. acetone precipitation, using icecold addition, was found to be the most effective means of reproducible, facile selective precipitation for ms analysis. other assisted precipitation methods such salting out resulted in samples being contaminated with high concentration of ions that suppressed target protein matrix assisted ionisation; whilst polyethylene glycol (peg) assisted precipitation resulted in multiple interfering polymer peaks in the resultant ms spectra (data not shown). the optimised methodology for efficiency, speed a simplicity consisted of a 1 to 1 addition of ice cold acetone, spun in a refrigerated bench top centrifuge at 16,000g for 30mins. the supernatant (containing unwanted background proteins) is discarded and the pellet resuspended in a membrane dissolution and viral envelope protein solubilisation buffer (figure 2). much higher ratios of acetone . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. . and lower temperatures are commonly used but many more moderate to small proteins coprecipitate as a result and interfere with the final diagnostic mass spectra [16] . acetone is an ideal organic solvent for the purpose of virion enrichment for mass spectrometry as not only does its weaker hydrogen and dipole bonding destabilise the hydration cloud surrounding large molecules, leading to precipitation, but it conveniently evaporates at room temperature without leaving a residue. other organic solvents, including ethanol, similarly effect a precipitation but leave a residue [17] . this may account for acetone precipitation/evaporation having no detrimental effect on subsequent maldi-tof ms analysis. in addition, acetone denatures the viral envelope such that any virus is rendered biologically inert, but without completely destroying its structure. (figure 2a ) [18] . this is not the case for salting, peg and even methanol co-precipitation enrichment of virion particles, where they have detrimental effects on ionisation & spectra, and have been shown to leave the virus biologically active with increased pfu potencies within the precipitation [19, 20, 21] . the supernatant containing smaller non-precipitated solutes is discarded. the pellet is enriched with viral particles and kept for analysis. acetone treatment inactivates envelope virus by deformation and partial fragmentation of the viral envelope and embedded protein structures. panel b: the pellet is resuspended in 10 to 100ul of maldi -tof mass spectrometry compatible dissolution and solubilisation buffer. termed lbsd-x this buffer does not suppress ionisation and contains a detergent at a concentration optimised to release viral envelope embedded proteins together with non embedded viral proteins. it also contains dithiothreitol (dtt) in order to further reduce disulphide bonds so that quaternary and tertial structures are fully disrupted and monomers, polypeptide chains and glyco-polypeptides are liberated for detailed mass analysis. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. . comparable to the analytical challenge laemmli faced in his pioneering development of sds -page for viral protein analysis [22] ; here, large complexes have to be disrupted and the viral proteins solubilised as free proteins to be seen by maldi tof mass spectrometry (figure 2b). unfortunately, detergents like sds and triton and chaps, although widely used in molecular biology, largely suppress ionization [23] and rapidly build up as fouling components on both the primary lens and other internal components of the mass spectrometer (supplemental figure a) . a series of novel detergents which avoided ion suppression and reduced lens fouling properties, were developed for solubilisation of hydrophobic and viral membrane bound proteins. the best performing formulation (data not shown), termed "lbsd-x" was adopted for all subsequent experiments. in addition to solubilisation and ionization, the size of the target (glyco)proteins has to be considered with respect to the measurement range of the mass spectrometers. in our case best performance on the shimadzu maldi 8020 (shimadzu-kratos, manchester, uk) was a maximum mass limit of 250,000 m/z. in more parallel's to laemmli's work, reduction of disulphide bonds was necessary in order to completely disrupt the tertiary structure to component protein/peptide chains. the resultant protein-polypeptide chains and glyco-polypeptides chains, although large, being within the high resolution capacity of this bench top mass spectrometer. dithiothreitol (dtt) is well tolerated by maldi-tof ms, does not suppress ionization and thus was incorporated in the matrix dissolution buffer formulation. an important concern in test development for an infectious agent and particularly highly contagious, life threatening virus, is safety. the sars spike s protein, a unique target as a marker of the infection, could be used and titrated into a biological sample in order to develop an analytical test with no risk to the development team [24] . however, although several recombinant sars cov-2 viral proteins are available these are not optimal as the starting material in a clinical mass spectrometry viral detection system. they fail to represent the true nature of the molecule as it exists in a real clinical analysis: the spike protein exist as a trimer of s proteins partially embedded within the viral envelope. it is extensively glycosylated and is also proteolytically cleaved to prime and activate it for receptor binding and then exposure of fusion peptide domains to allow ingress of the viral genome [5, 25] (figure 1a and 1b). recombinant proteins do not reflect this context and structural complexity, both of which complicate sample processing to achieve marker release and correct detection and quantification. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. . pseudo-virus are constructs whereby virion-like particles, consisting of viral envelope exosomes, are made in vitro; lacking the viral genetic material as it is not replicated or incorporated (see figure 3 ) [26] . these are ideal non-infectious starting material upon which to develop a viral protein mass spectrometry tests. sars2 spike protein s genetic sequences were inserted into a target host human cell line, hek293; so that fully post-translationally modified, membrane embedded viral proteins are expressed and tertiary structures are functionally intact. it was found that co-transfection with tmp322 was necessary for pseudo-virus vectors constructed to express the spike proteins as a functional ingress effector, i.e. fusion with a target cell (see figure 3 ). through optimisation of the maldi-tof ms method we discovered that the s protein was cleaved into s1, s2b and s2a fragments although held together by disulphide bridges [27] . these fragments were then searched for in the cultures of live covid-19 virus grown on vero, african green monkey, cells and shed as functional virions into the culture media. using the optimised protocol developed on pseudo-virus cultures the same spike fragments could be seen, s1 (79,000m/z) being the most prominent (see figure 3) . virus grown in vitro and mass spectra of gargle/saliva spiked with culture media from cells infected with sars-cov-2: s proteolytic fragments s1 and s2 were seen in all preparations and s2b only in serum free samples. viral envelope proteins (veps) became more prevalent in live virus culture and ig light chains and iga heavy chain were additional peaks found in gargle/saliva samples. hek293t/17 cells were seeded into 6 well cell-culture plates and co-transfected after 24 hours with p8.91 (gag-pol expression plasmid), pcsflw (luciferase reporter plasmid), and a viral glycoprotein expression plasmid encoding the spike protein of either sars-cov-2. the transfection mixture was prepared in optimem using fugene-hd as a transfection reagent; cell culture media was replaced prior to transfection. the cell supernatant was collected via syringe 48 hours after transfection and filtered through a 0.45 âµm filter to harvest the lentiviral pseudo-type particles (26) . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. . https://doi.org/10.1101/2020.08.22.20176669 doi: medrxiv preprint including s protein fragments, extracted & solubilized using extraction formulation lbsd-x buffer and detected as mass peaks by maldi tof ms. along with s protein fragments other viral component protein peaks were identified as characteristic of virions and were prominent in the viral cultures. these other viral envelope proteins (veps) were characterised by their molecular mass, particularly a group of middle to high mass range broad peaks, indicative of variable glycosylation, which we have termed vep26-28k, vep34k, vep39-40k and vep45-47k. having confirmed the specific and secondary target ms signals, indicative of corona virus, as s protein proteolytic fragments (primarily s1 and s2a) and veps as mass spectra peaks; the effect of the gargle/saliva sample milieu was examined for 'matrix effect' potential detrimental effects on the spectra. one striking example of a negative effect of natural biological sample matrix was clearly demonstrated by serum albumin, found in fetal calf serum (fcs) and used in supplementation of pseudo-virus and live virus cultures media. the mass peak of albumin obscuring detection of the similarly-sized s2b proteolytic fragment (figures 3&4). saliva is a size selective transudate of numerous serum proteins and organic biomolecules, often revealing these molecules at 10 to 100 fold lower concentrations than found in serum [28] . albumin is fortunately not normally found at any significant levels in saliva and s2b should be evident in covid-19 positive gargle/saliva samples. however, other large molecules such as amylase and iga are actively secreted into saliva and iga which has a mass of approximately 360,000 th. the large proteins (such as iga) are indeed co-precipitated in the acetone precipitation process described in this method, while the naturally free small to medium seized transudate and secreted proteins at lower masses, are removed in the supernatant. . similarly, any other micro-organisms present in the sample along with any virus would also co -precipitated from the oral wash. to minimise potentially interfering effects, all gargle/saliva samples were sequentially prefiltered with 1.2 and 0.45âµm syringe filters before acetone precipitation, retaining most large micro-organisms on the filter but allowing through virions and immunoglobulins. as a result of disulphide bond reduction, large molecular weight immunoglobulins were visualised in the mass spectra of saliva samples, resolving as heavy and light chains (figures 3 &4 ) . their presence did not detract from spectral detection of the s1, s2b fragments or vep's. despite being significant components. however, by adding in live virus containing culture media to a control gargle saliva remained slightly compromised as a comparative testing model as bsa would always be present (figures 3&4) . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. . https://doi.org/10.1101/2020.08.22.20176669 doi: medrxiv preprint figure 4 -clinical diagnostic protocol of gargle saliva collection, laboratory processing and spectral data features: panel a illustrates patient (home) sampling gargling 10ml of water and spitting that into a sample pot. panel b illustrates rapid processing where received gargle sample is filtered through 1.2 and 0.45mm filters, 5ml is recovered and is acetone precipitated. the pellet is resuspended in viral envelope dissolution and protein solubilization buffer before being applied to maldi tof plates and analyzed (3mins per sample); output data being processed by appropriate software. panel c illustrates a reference spectra of sars-cov2 grown in vitro with spectral pattern of viral envelope proteins (veps) and fragments of corona virus spike protein labelled. panel d illustrates overall spectra of 5 unscreened volunteer samples overlaid. the spectra from subject id2 is indicated (blue line). regions corresponding to mass ranges for veps and large characteristic fragments of the spike protein are illustrated. these are illustrated in juxta position to gargle/salivary immunoglobulin light chain and ig a heavy chain peaks. also to note that s2a fragment is surrounded by a host of high intensity/abundance proteins, the origins of which may be a mix of other co-enriched (bacterial) micro-organism from the oral cavity. an additional high molecular mass glycoprotein type peak was also seen at with a maxima at 112,000m/z (marked **) was only seen in this sample. collection of volunteer gargle/saliva was by signed, informed consent and the study was approved by the irb of mapsciences and nisad to estimate sensitivity and specificity saliva/gargle (n=10) was spiked with spent vero cell culture media producing sars-cov-2 at a concentration of 10 4 to 10 5 pfu/ml. these were subject to maldi-tof mass spectrometry and the peak heights measured for the peaks of interest. two rt-pcr negative persons simultaneously provided gargle/saliva samples at the time of swab sampling. these confirmed pcr-negative gargle samples were analysed by maldi-tof mass spectrometry 40 times; the measured peak intensities of which acted as comparative controls to the viral spiked saliva/gargle. viral envelope protein peak intensities were clearly elevated in the spiked samples ( figure 5 ). gargle/saliva was collected from 35 volunteers who did not have simultaneous covid-19 pcr determinations, although some suspected they had suffered from the infection but were recovered or recovering. the intensity of s protein fragments s1 (79,000m/z), s2b (62,000m/z) (can is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. . https://doi.org/10.1101/2020.08.22.20176669 doi: medrxiv preprint be obscured by albumin if present) and s2a (13,500m/z) were measured along with peaks masses thought to represent other viral envelope proteins (veps). the maximal intensity was measured for these broad glycoproteins peaks at 26,000-28,000m/z (vep26-28k), 34,000m/z (vep34k), 39,000 -40,000m/z (vep39-40k) and 45,000-47,000m/z (vep45-47k). culture supernatant from vero cells infected with sars-cov-2 were spiked into ten gargle/saliva samples at 1-10%. the viral load in these culture media was determined as at 10 4 to 10 5 pfu/ml. gargle/saliva were collected from two individuals, with and without a cough, who were confirmed as rt-pcr covid-19 negative at the time of gargle/saliva collection. these were analysed 20 times each. a series of 30 gargle/saliva was collected from volunteers, but covid-19 pcr status at time of sampling was not known. these were analysed only once each. all samples were acetone precipitated, subjected to viral envelope protein solubilisation and analysed by maldi-tof mass spectrometry. target peak intensities were then measured. panel a) illustrates the levels of each analysed sample for spike protein fragment s1 peak at 79,000m/z; panel b) spike protein fragment s2b at 63,000m/z and panel c) spike protein fragment s2a at 13,500m/z. the maximal intensity was also measured for broad glycoproteins peaks, considered to originate from other viral envelope proteins (veps), at 26,000-28,000m/z (vep26-28k -panel d), 34,000m/z (vep34k panel e), 39,000 -40,000m/z (vep39-40k -panel f) and 45,000 -47,000m/z (vep45k -panel g) red dots represents levels of peak intensity for the spiked gargle/saliva samples, orange dots represents levels of peak intensity for covid-19 rt-pcr negative control 1 samples, grey dots represent levels of peak intensity for covid-19 pcr negative control 2 samples and blue dots represent levels of peak intensity for unscreened volunteer gargle/saliva samples. blue dots with red circle borders are the values recorded for volunteer subject id2 s1 peaks showed clear elevation in levels for the sars-cov-2 virus culture spiked samples, being on average 10 2 x higher than the covid 19 rt-pcr negative and volunteer gargle samples, where it was essentially not detected in 80% of samples. this marker alone could perform as a specific corona virus maldi tof ms screening test with near 100% detection and specificity at 10 3 -10 4 pfu sars-cov-2 virions in a 10ml gargle/saliva sample. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. . s2b peaks may also be another key indicator as, although undetectable in the culture media spiked samples due to interference from albumin present in the media from fcs. s2a peak intensity measures were more complicated. although highly elevated in comparison to the control and random sample gargle/saliva group measurement was compromised as a peak of mass similar to that of s2b at 13,500 could be detected in a significant number of individuals. many more peaks are seen in the lower mass range (below 20,000m/z) of saliva/gargle sample and probably arise from the abundant housekeeping proteins of residual bacterial flora. this "s2a like peak" of similar mass may characterize a specific bacterial species. vep protein peaks all showed elevated levels in the gargle/saliva spiked with sars-cov-2 culture media. however levels found in the control significantly overlapped, indicating these were not specific markers of covid 19 but more generalised markers of virion membrane embedded proteins reflecting their common importance to enveloped viruses. a notable sample from the volunteer group is that of sample id2 which showed clearly elevated s1 and s2b , vep26-28k, vep34k, vep39-40k and vep45-47k and moderate elevated measurement of s2a. the local mucosal tissue barrier response to microbiological infection, including virus such as covid-19, elicit secretion of iga into the mucous and/or covering secretion such as tears and saliva (29) . peak masses matching that of immunoglobulin light chains and the heavy chain of iga were seen in the vast majority of gargle/saliva samples examined. ig a and ig g light chain are identical and that from igm only slightly different on maldi-tof ms. however the heavy chain of igg , ig a and igm are distinctly resolved on the maldi-tof mass spectrometer because of their different chain lengths (data not shown). only total ig light chains and iga heavy chains were seen in the gargle/saliva samples. the intensity of these were measured in order to evaluate any correlation with other marker peaks (excluded in the analysis were the culture media virus containing "spiked" samples-as levels here were not physiological and reflected that of the base control gargle/saliva). the levels of total ig light chains were clearly differentiated between the two simultaneous covid-19 rt-pcr negative control subjects .to note the higher levels seen in figure 6a were also reflected in s2a like (13,500m/z) protein peak levels for the same person ( figure 5 ). within the subjects for whom covid-pcr status had not been determined, the levels varied enormously from undetectable to the highest determined (figure 6a ). subject id2, the person with highest s1, s2b levels, had the third highest total ig light chain levels. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. . iga heavy chain levels overlapped for samples from the two pcr-negative covid-19 subjects and similarly, within the subjects for whom covid-pcr status had not been determined the levels varied enormously from undetectable to the highest determined (figure 6a) . the highest level being seen in the sample from the person with the highest s1, s2b levels, subject id2. it was noted that iga heavy chains were only seen when elevated viral envelope proteins (veps) were detected in the mass spectra. to test a possible association we plotted total ig light chain and iga heavy chain levels against the levels measured for individual veps. total ig light chain levels did not correlate with the levels of veps detected; but iga heavy chain levels did for vep34k, vep39-40k and vep45-47k (figure 6b ). panel a are plots comparing the total ig light chain and total iga heavy chain peak intensity levels for two covid-19 pcr negatives control individuals (grey and orange dots respectively) repeated 20 times each (n=40), versus the same peaks intensity level of 30 volunteers (blue dots) who gave gargle/saliva samples but whose covid-19 pcr status was unknown at the time. blue dots bound by a red circle are the levels of total ig light chains and iga heavy chains found in volunteer id2. panel b are scatter plots of total ig light chains (upper plot) and iga heavy chain peak intensities (lower plot) versus veps peak intensities for the 30 volunteer gargle/saliva samples but whose covid-19 pcr status was unknown at the time. no vep level correlated with total ig light chain level. correlation were seen with ig a heavy chain levels for vep39-40k (grey dots r 2 =0.60), vep44-47k (gold dots r 2 =0.53) and vep34k (orange dots r 2 =0.53) but not for vep26-28k (blue dots r 2 =0.06) . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. . adopting a maldi-tof ms based technology has had dramatic effects on reducing the costs in microbiological identification of infections and estimates have been as high as an 80% reduction overall [30] . a full evaluation of cost to achieve screening of millions in a population using this technique have to take into account discount on bulk orders. however, given that this system is generally reliant on extremely low volumes of generic reagents, the major cost is the purchase of the mass spectrometer. we have worked a basic model that mass spectrometry reagent analysis costs per test are less than $1 per sample. all the sample collection tubes and syringe filters associated in the process are more expensive at approx. $5 per sample at retail prices. ignoring the latter peripheral costs of initial sample handling, we estimate that when including paying the cost of a mass spectrometer over 3 year in operation (at 100,000 samples a year per machine) the basic instrument-reagent analysis cost per sample is approximately $2. although simply applying the technique of maldi-tof mass spectrometry, as it has developed for identification of bacteria, for covid-19 detection has been reported as promising [4] , this approach is fraught with practical complications and analytical errors. not least that the biochemistry focus that for bacterial identification the maldi-tof ms technique is optimized recognition of microbial pathogen hydrophilic, low to medium molecular weight, high abundance housekeeping proteins. sars-cov-2, an envelope virus, has little if any equivalent proteins and its virion contains a high abundance of medium to large membrane associated/embedded (predominately hydrophobic) glycoproteins. to develop and maximise a clinical efficient and cost effective maldi-tof mass spectrometry screening solution for covid-19 and future viral pandemic screening, requires rational design based on an understanding of the chemistry, biology and physics of the pathogen-host interactions and also their unique marker biomolecules. this is further complicated by how such chemistry and proteins behave in maldi-tof mass spectrometry. by targeting maldi-tof mass spectral detection to viral envelope glycoproteins a multidisciplinary team approach has achieved a multifaceted clinically maldi tof ms screening test for covid-19 and future such pandemics that may arise. further validation in relations to pcr detection in patients is needed but the system described promises a multifaceted diagnostic tool as follows: 1. sensitive and specific identification of corona virus in gargle/saliva by measurement of s1 and probably s2b protein peaks. 2. together with s1 and s2b, very high level of a peak at 13,500m/z, presumed to be s2a, present in the in vivo sample is an additionally indicator of a corona virus infection (with high confidence); 3. more moderate elevation of s2a-like peak mass in the absence of elevated s1 and s2b peaks may be due to a similar protein/fragment arising from a microbiological infection of the oral respiratory tract, probably a bacterial housekeeping protein. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. . the final threshold for each component of the test are yet to be fully validated for computation software automation, but results are likely to be expressed as a heat map, the basic system being red, green and grey. the grey zone requiring retesting ( see table 1 ). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. . https://doi.org/10.1101/2020.08.22.20176669 doi: medrxiv preprint in the music entertainment industry, they reported having been bed bound with covid-19 symptoms, severe fatigue, loss of taste & smell and persistent cough in april (2020) 2 months prior to giving a gargle/saliva sample. at the time of sampling they reported to be 60-80% recovered from the symptom of fatigue which had persisted. in our follow-up they gave another sample 14days later and was also tested for covid-19 by the nhs pcr service the same day. the analysis of the repeat sample showed no detectable corona virus s proteolytic fragment protein peaks; total ig light chains and iga heavy chain levels had fallen dramatically but were still elevated (see end of table 1 id44*). the covid-pcr result was reported as negative. validation studies against saliva/gargle spiked with cultured virus are on-going and the current study indicate a close to 100% sensitivity if measuring s1 peaks alone as the indicator of corona virus infection. direct comparison of the maldi-tof ms testing with rt-pcr detection of covid-19 in clinical samples is needed. however, this maldi-tof ms technique cannot work on stored second sample nasal-pharyngeal swabs as they will have either been heat inactivated (cooking the protein and viral envelope membrane) or stored in a transport media which contains sds and or triton which supresses ionization and similarly destroys the viral envelope. in the absence of a freezer full of pre-collected 10ml gargle/saliva for which rt-pcr results are known, only a prospective study is possible. this is in process. we are working with several groups to collect gargle/saliva samples at presentation where a swab for pcr testing is being taken simultaneously. these groups will analyse the sample on their maldi-tof mass spectrometer instruments and with all the technical support and necessary key reagents provided. as detailed, other markers measured in this technique may give further valuable clinical information such as other viral infections and magnitude of the mucosal humeral immune response. conceptualization, rk iies. and jl heeney; methodology, rk iles and jl heeney, validation, jk iles is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august 22, 2020. . https://doi.org/10.1101/2020.08.22.20176669 doi: medrxiv preprint coronavirus and the race to distribute reliable diagnostics molecular biologists offer "wartime service" in the effort to test for covid-19 cost savings realized by implementation of routine microbiological identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry detection of sars-cov-2 in nasal swabs using maldi-ms distinct conformational states of sars-cov-2 spike protein what is new in clinical microbiology-microbial identification by maldi-tof mass spectrometry: a paper from the 2011 william beaumont hospital symposium on molecular pathology î±-cyano-4-hydroxycinnamic acid as a matrix for matrix-assisted laser desorption mass spectrometry global aspects of viral glycosylation the combination of simple maldi matrices for the improvement of intact glycoproteins and glycans analysis comparison of respiratory specimen collection methods for detection of influenza virus infection by reverse transcription-pcr: a literature review the impacts of viral inactivating methods on quantitative rt-pcr for covid-19 susceptibility of sars-cov-2 to uv irradiation ion suppression; a critical review on causes, evaluation, prevention and applications evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry modified tca/acetone precipitation of plant proteins for proteomic analysis effects of residual ethanol on the rate and degree of conversion of five experimental resins. dental materials : official publication of the academy of dental materials inactivation of sars coronavirus by means of povidoneiodine, physical conditions, and chemical reagents concentration of viral vectors by coprecipitation with calcium phosphate polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis a virus and human rotavirus, from oyster, water, and sediment samples laemmli uk. cleavage of structural proteins during the assembly of the head of bacteriophage t4 comparative analyses of different surfactants on matrix-assisted laser desorption/ionization mass spectrometry peptide analysis in silico approach to accelerate the development of mass spectrometry-based proteomics methods for detection of viral proteins: application to structure, function, and evolution of coronavirus spike proteins an optimized method for the production using pei, titration and neutralization of sars-cov spike luciferase pseudotypes maldi-tof mass spectrometry, detection of sars-1 and sars-2 (covid-19) fusion glyco-peptide ejected from spike proteins comparative human salivary and plasma proteomes oral microbial ecology and the role of performance and cost analysis the authors thank the willing volunteers who provided gargle saliva samples and in particular colleagues sharing pseudo virus and viral stains used in this study. key: cord-320790-ley91488 authors: fogg, m. j.; bahar, m.; alzari, p.; bertini, i.; betton, j.‐m.; burmeister, w. p.; cambillau, c.; coll, m.; canard, b.; carrondo, m.; daenke, s.; dym, o.; geerlof, a.; egloff, m.‐p.; haouz, a.; enguita, f. j.; jones, t. a.; ma, qingjun; manicka, s. n.; owens, r.j.; migliardi, m.; nordlund, p.; peleg, y.; schneider, g.; schnell, r.; stuart, d. i.; unge, t.; tarbouriech, n.; wilkinson, a. j.; wilmanns, m.; wilson, k. s.; zimhony, o.; grimes, j. m. title: application of the use of high‐throughput technologies to the determination of protein structures of bacterial and viral pathogens date: 2006-10-04 journal: acta crystallogr d biol crystallogr doi: 10.1107/s0907444906030915 sha: doc_id: 320790 cord_uid: ley91488 the structural proteomics in europe (spine) programme is aimed at the development and implementation of high‐throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. despite the challenging nature of some of these targets, 175 novel pathogen protein structures (∼220 including complexes) have been determined to date. here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens. # 2006 international union of crystallography printed in denmark -all rights reserved the structural proteomics in europe (spine) programme is aimed at the development and implementation of highthroughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. despite the challenging nature of some of these targets, 175 novel pathogen protein structures (220 including complexes) have been determined to date. here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens. the aim of the structural proteomics in europe (spine) programme was to develop and exploit technological advances in structural biology to tackle difficult problems related to human health and disease. to this end, spine workpackage 9 focused on proteins from human pathogens, especially bacterial and viral, whereas work on human targets was addressed by workpackages 10 and 11 (see banci et al., 2006) . this article reports on the progress made in the study of pathogen targets and provides highlights of a number of developments in high-throughput (htp) technologies which have proved crucial to successful structure determination, illustrated by reference to some of the structures solved. whilst relevance to human health and disease was the overarching criterion for target selection, a subset of targets was selected explicitly as so-called 'low-hanging fruit'. these, primarily exemplified by the bacillus anthracis proteins (au et al., 2006) , proved particularly valuable in validating htp pipelines and technologies. the methods used by the various spine partners for target selection and other informatics aspects are described in detail in albeck et al. (2006) . the bacterial organisms targeted for structural analysis represent a range of threats to human health, including food poisoning, respiratory diseases and potential bio-terrorism agents, of which all manifest antibiotic resistant strains. the two key organisms were mycobacterium tuberculosis (mtb) and b. anthracis, which together accounted for 81% of our bacterial targets and 47% of the bacterial protein threedimensional structures solved (table 1 ). the efforts on mtb involved six partners (paris, hamburg, uppsala, stockholm, york and the weizmann) and especially targeted proteins from strain h37rv. the b. anthracis project was a collaborative effort between the oxford and york partners and a fuller assessment of this is given in an accompanying paper (au et al., 2006) . in addition to these, smaller numbers of targets were selected from campylobacter jejuni (york, lisbon), two members of the neisseriae family (n. meningitidis and n. gonorrhoeae; oxford), escherichia coli (e.g. 0157:h7; stockholm, weizmann, florence), klebsiella pneumoniae (lisbon) and streptomycetes (stockholm) and shigella flexnin, salmonella entrica typhimurium and streptococcus pyogenes (weizmann). 1.1.1. m. tuberculosis. a number of slow-growing mycobacterial species have evolved into highly successful human pathogens and are responsible for diseases such as tuberculosis (tb; m. tuberculosis and m. africanum) and leprosy (m. leprae). the world health organization estimates that one-third of the human population is currently infected and that one person dies roughly every 18 s (http://www.who.int) from mycobacterial diseases. there are now forms of m. tuberculosis (mdr-tb; espinal, 2003) resistant to the two most powerful anti-tb drugs (isoniazid and rifampicin) and there is a pressing need for new drugs effective against persistent tb infection and mdr-tb. closely related forms of mycobacteria are pathogenic to other mammalian species, e.g. m. bovis. the mtb target-selection process in paris is illustrative of those used for this organism in spine and was based on comparative analyses of the complete genome sequences for two mtb strains with other bacteria and mycobacterial strains, such as m. bovis, m. bovis bcg and m. leprae, which provided detailed information about every gene in mtb. over 300 proteins were identified that were only found in mycobacteria or actinomycetes, but were conserved in the degraded genome of m. leprae and therefore presumed to be important for bacterial viability and hence potential drug targets (albeck et al., 2006) . in addition, the paris laboratory targeted proteins for which structures would provide insights into mtb biology. funding for the mtb project was far from exclusive to spine and came from several other sources, including other ec grants. 1.1.2. b. anthracis. york and oxford each selected initial sets of 48 targets from b. anthracis, with the dual aim of determining a reasonable number of structures and, in the process, helping to refine their protein-production pipelines . to be considered, the protein had, in general, to be reasonably small (<50 kda), lacking in regions predicted to be transmembrane moieties, signal peptides or disordered and usually amenable to structure solution by molecular replacement. york targets included proteins involved in nucleotide metabolism and sporulation, whilst oxford targets included members of protein families well conserved across a range of pathogenic bacteria ). an additional spine collaboration between york and utrecht resulted in two nmr structures of sporulation-associated aspartic acid phosphate phosphatases, neither of which could be crystallized (ab et al., 2006) , and extended the targets to the bofc (bypass of forespore c) protein from b. subtilis, an intercompartmental signalling factor expressed in the forespore (patterson et al., 2005) , also found in b. anthracis (ba4653). 1.1.3. neisseria. many species of this gram-negative -proteobacteria are found only in humans. two members of the neisseriae, n. meningitidis and n. gonorrhoeae, have evolved into highly successful pathogens responsible for bacterial meningitis and gonorrhoea, respectively. although the regulatory systems of all gram-negative bacteria share features in common with e. coli, neisseria species display several features which are unique. these include a restricted set of sigma factors, a relatively small number of transcriptional regulators and a large number of phase variable genes. oxford focused on the complete repertoire of transcription regulators in both n. meningitidis and n. gonorrhoeae, with a view to defining the structure-function relationships of these proteins and to provide key reagents for experimental studies of regulation. the four available complete genome sequences (parkhill et al., 2000; tettelin et al., 2000) of neisseria were assessed for homologues of dna-binding proteins with potential regulatory functions. representative orthologues of each regulator in each genome were identified, with the genes selected in order of preference from n. meningitidis strain mc58, n. gonorrhoeae strain fa1090, n. meningitidis strain z2491 and n. meningitidis strain fam18, to provide a cohort of 62 dna-binding and associated signal-transduction proteins. in the vast majority of cases, the entire protein was targeted for analysis. to address the role of copper in cellular processes, the florence laboratory targeted copper-binding proteins. bacterial and eukaryotic genomes were compared to identify prokaryotic orthologues of proteins in yeast and humans. the aim was to solve high-resolution nmr structures that could be used as models for human proteins and to identify differences exploitable for potential antibacterial drugs. additionally, target selection was limited to proteins of fewer than 200 residues, as they are more tractable for nmr (banci & rosato, 2003) . structures were solved from a range of bacterial species including b. subtilis, e. coli and pseudomonas aeruginosa (ab et al., 2006) . a number of different viruses were targeted, ranging from large double-stranded dna (dsdna) viruses such as epstein-barr virus (ebv; grenoble) and vaccinia virus (vacv; oxford), to single-stranded rna viruses such as the newly emergent human pathogen, the severe acute respiratory syndrome coronavirus (sars-cov; marseille and oxford). finally, some viruses of less direct relevance to human health were investigated as model systems, for instance bacteriophage p2 (marseille). 1.2.1. poxviruses. this group of very large viruses includes variola virus, the aetiological agent of smallpox, and vaccinia virus (vacv), used as the smallpox vaccine. their genomes each contain 200 open reading frames (orfs) and the sequences of more than 20 poxviruses reveal considerable sequence variability towards each end of the genome. these regions code for genes that affect virus virulence, host-cell susceptibility and the host response to infection (gubser et al., 2004) , enabling the virus to suppress the immune response (smith et al., 1997 . in vacv half of the genes are nonessential for replication and a significant number are immunomodulators; nine of which were targeted by oxford as being reasonably small but of significant potential biological interest. 1.2.2. herpesviruses. members of this family of dsdna viruses have large genomes (100 orfs) and are responsible for a diverse set of human diseases. examples include herpes simplex virus (hsv; causing genital herpes and herpes encephalitis), ebv (causing burkitts lymphoma and glandular fever), varicella zoster virus (vzv; causing chickenpox and shingles), human herpesvirus-8 (hhv-8; implicated in all forms of karposi sarcomas, commonly seen in aids patients) and human cytomegalovirus (cmv; causing aids-related retinitis and pneumonia). no effective treatments are generally available, with the exception of the acyclovir compounds active against hsv. thus, herpesviruses are of immediate interest to the biomedical community and in particular to the pharmaceutical industry. barcelona and oxford have interests in the molecular mechanisms of genome packaging and targeted a number of proteins that form the complex that packages the dsdna genome of herpesviruses. this work demonstrated the inadequacy of the current e. coli proteinproduction pipelines for such difficult proteins (five out of six of the components are greater than 70 kda in size and all are part of complex robust macromolecular assemblies), since all the expressed proteins were insoluble. soluble proteins have now been produced in insect cells for some of these targets, but no structures have been determined and this work will not be considered further here. in contrast, the grenoble group targeted ebv proteins with known enzymatic activity and ranked them based on a number of properties, prioritizing those that were small and predicted to be stable with a high secondary-structure content. surface glycoproteins and those proteins forming multi-component complexes were explicitly excluded. application of these selection criteria led to the structure determination of four proteins from ebv (tarbouriech et al., 2006) . 1.2.3. coronaviruses. sars emerged as a new human disease in southern china in late 2002. the first manifestation of infection is a febrile illness, with respiratory symptoms, headaches and myalgia, followed by progression to acute respiratory distress and progressive respiratory failure (peiris et al., 2003) . the disease is caused by a coronavirus (kuiken et al., 2003) , one of a group of enveloped positive-strand rna viruses commonly associated with enteric and respiratory disease (ziebuhr & siddell, 2002) . the unusual severity of sars-cov infection probably reflects the introduction of an animal coronavirus into a susceptible human population. in the first outbreak at least 8000 people were infected and there were more than 750 fatalities (donnelly et al., 2003) . and n), non-structural involved in viral rna synthesis (nsp or replicase) and proteins thought to be non-essential for replication in tissue culture but that clearly provide a selective advantage in vivo (the nspx or accessory proteins; marra et al., 2003; rota et al., 2003) . a number of proteins, primarily from the large replicase polyproteins orf1a and orf1ab of sars-cov, have been targeted by oxford and marseille to test the efficacy of a focused structural proteomics approach against a newly emerging human pathogen. proteins were excluded on the basis of certain criteria, such as size, glycosylation and the presence of transmembrane regions. as with all large-scale projects, the 'scoreboard' is only a snapshot of target progress through the various pipeline stages and is therefore not a definitive assessment. it does, however, allow an assessment of whether there are particular bottlenecks that need addressing. the numbers for this section are from a comprehensive snapshot of the project taken in september 2005 (overall summary presented in fig. 1 ), whereas the detailed numbers for individual projects are for early 2006 (table 1) . a comparison of the number of expression trials with the number of crystals or good hsqc data sets for the entire spine project shows that around 10% of constructs led to the production of a protein suitable for structural analysis. this attrition arises from approximately equal losses at the expression stage and crystallization or sample preparation stages (for nmr). most of the proteins were expressed in prokaryotic systems; however, there is evidence, at least for human and viral proteins, that significant gains are possible by the use of eukaryotic expression systems, thus a global analysis of results from spine partners shows that the success rate for mammalian cell expression is more than twice that for soluble expression in e. coli (75% versus 30%), with insect-cell expression giving an intermediate success rate (44%). further development and uptake of these methodologies (see aricescu, assenberg et al., 2006; aricescu, lu et al., 2006) is likely to have a major impact on the study of difficult viral targets, as indicated by preliminary results for ebv proteins, where insect-cell expression yielded soluble protein for 50% of the proteins tested (tarbouriech et al., 2006) the production of soluble protein for nmr and crystallization studies is an absolute requirement, yet it remained a significant bottleneck for the spine bacterial targets. bacterial expression statistics from all contributing spine partners are illustrated in fig. 1 . over 1000 proteins were targeted and on average two or three constructs were produced for each target and each construct was subjected to two expression trials, with 29% of constructs producing soluble protein. a more detailed analysis reveals significant variability, with 77% of the first cohort of b. anthracis constructs expressed in soluble form, but only 33% for mtb and 15% for other bacteria. as of september 2005, almost 35% of these soluble proteins had produced protein crystals or data suitable for nmr; these had resulted in structure solutions for slightly more than 10% of the original 1127 targets (fig. 1) . however, target groups which were carefully selected for amenability for expression and structure determination, such as the b. anthracis cohort, fared better, with success rates in crystallization approaching 60% . the spine viral scoreboard (fig. 1) suggests that although viral proteins have a lower success rate for producing soluble proteins than b. anthracis proteins, they are comparable to more difficult bacterial and human proteins (27% of constructs resulted in soluble expression at a level sufficient for production and purification). however, the attrition rate from protein purification to structure determination is higher, with only about 30 crystal structures having been determined to date. it appears that this is in part a consequence of the difficulty of growing high-quality crystals. thus, 34% of soluble proteins crystallized compared with an equivalent rate of 58% for the york/oxford b. anthracis targets; furthermore, many crystals were not of diffraction quality. however, as for the bacterial targets, patterns of soluble protein expression vary between viral systems. for some groups of viruses, such as the poxviruses, the success rate for production in e. coli was reasonable, as discussed below. the protein-production technologies developed and used by spine partners are covered in detail by alzari et al. (2006) and aricescu, assenberg et al. (2006) and aspects of work on specific pathogen proteins are presented by au et al. (2006) and tarbouriech et al. (2006) . target genes were often amplified, cloned and screened for expression in parallel (e.g. 48 or 96 at a time). ligation-independent cloning strategies were widely (but not exclusively) used and the use of n-and c-terminal his 6 tags was almost universal. the automation of many procedures, including small-scale expression screening, was achieved using liquid-handling robots, with multi-channel pipettes providing an inexpensive option for less automated pipelines (expression-screening strategies are compared by berrow et al., 2006) . biophysical characterization methodologies usually included dynamic light scattering (dls) and mass spectrometry (esi-ms) (see geerlof et al., 2006) . protein crystallization on the nanolitre scale was used by many of the partners (berry et al., 2006) . here we illustrate the impact of a number of particular spine-based technologies on the structure determination of pathogen targets, namely (i) multiple-construct design for a single target, (ii) optimization of protein solubility, (iii) eukaryotic expression systems, (iv) standardized refolding protocols, (v) surface engineering and (vi) other biophysical characterization. the use of multiple constructs to define the correct n-and c-termini of a target domain has had a crucial impact on the successful structural analysis of both pathogen and human targets (banci et al., 2006; siebold et al., 2005) . an example of this in the context of viral proteins is the n-terminal domain of a viral methyltransferase, ns5, of murray valley encephalitis virus (mve), which was studied in oxford. the domain boundary was already reasonably well defined from the crystal structure of the related methyl-transferase domain from dengue virus (egloff et al., 2002) and 18 constructs, comprising of nine pairs of n-and c-terminal his 6 -tagged sequences, were designed that varied the c-terminal end of the protein domain. on the basis of automated small-scale expression screening, six constructs were selected for production. dls indicated that all protein samples were monodisperse and esi-ms confirmed the molecular weights for the samples. the his tags were cleaved (using the rhinovirus 3c cleavage site for n-terminal his tags or by carboxypeptidase a treatment to cleave back to a lys for c-terminal his tags) and these samples entered nanolitre-scale crystallization trials using the standard oxford set of 672 screens . since none of these screens gave crystals, the proteins were subjected to lysine methylation (as described below), resulting in crystals and a structure for one construct. efforts to improve the solubility of mtb proteins illustrate how the spine partners have tackled the problem of insoluble expression. mtb proteins are notoriously insoluble or provide poor yields when expressed heterologously in e. coli systems (bellinzoni & riccardi, 2003; alzari et al., 2006) , a possible consequence of the high gc content of mtb genes (65-70%) and unique codon preferences (de miranda et al., 2000) . to address such problems, hamburg contributed to the development of an mtb expression system based on the faster growing close relative m. smegmatis (daugelat et al., 2003) . proteins were expressed under close to physiological conditions in order to ensure the correct post-translational modifications such as glycosylation and methylation. the published protocol, using an inducible acetamidase promoter, was modified to be compatible with the embl petm vector system (geerlof et al., unpublished work) . using this system, the mtb lipb enzyme was expressed with a covalently bound ligand, which resulted in an x-ray structure at 1.08 å resolution (ma et al., 2006) . when expressed in e. coli, a mixture of native and ligand-bound enzyme was produced which failed to crystallize. m. smegmatis expression was also used by the weizmann for the mtb multidomain eukaryotic like fattyacid synthase i (fasi; zimhony et al., 2004) after e. coli expression had yielded a soluble but non-functional fasi. a recombinant m. smegmatis strain was constructed by deleting the native fas1 gene and replacing it with the mtb (h37rv) fas1 gene using a site-specific integrating cosmid carrying the mtb fas1 gene and its 5 0 and 3 0 flanking regions. this produced a faster growing non-pathogenic m. smegmatis (mc2155) derivative expressing mtb fasi. in paris, two strategies were adopted to enhance protein solubility. firstly, parallel cloning of orthologous mycobacterial genes from different species (i.e. m. tuberculosis, m. leprae, m. smegmatis and m. bovis) was used to identify the most soluble candidate. secondly, cell-free protein expression (roche rts) allowed the rapid evaluation of single gene constructs. in the latter strategy, n-terminal codons were optimized to identify the best silent mutation(s) for maximum expression (betton et al., 2004) . such cell-free systems provide a fast standardized method for screening potentially toxic protein expression without the inherent risks of using live cells. finally, in stockholm, the mtb nira protein could only be obtained in a soluble form in e. coli following cloning and co-expression with the cysg gene from salmonella typhimurium, a gene that codes for an enzyme catalysing three steps in the biosynthesis of sirohaem, a cofactor of nira. baculovirus-driven expression in insect cells has been used for a number of viral proteins that were resistant to soluble expression in e. coli. although screening for expression in such systems is at an early stage of development, the technology offers a viable alternative to bacterial expression for 'high-value' targets, as mentioned above for the ebv work. an example from oxford is the work on the capping enzyme of bluetongue virus vp4, which, owing to problems of toxicity in bacteria, was expressed in insect cells. this protein was problematic during both purification and crystallization, requiring the presence of high salt concentrations throughout. in addition, production of semet-derivatized protein proved difficult, prompting development of improved protocols for labelling in insect cells (sutton et al., unpublished work; aricescu, assenberg et al., 2006) . refolding from e. coli inclusion bodies was applied in a number of viral projects, with varying degrees of success. the extracellular immunomodulators of vacv studied in oxford provide a good example. these targets may be grouped according to how they act: (i) secreted proteins that bind to host factors that regulate complement, interferon (ifn), chemokines, cytokines and inflammation (smith & alcami, 2000) , (ii) intracellular proteins that modulate signalling pathways, apoptosis or the antiviral action of ifn tortorella et al., 2000) and (iii) others that are present on the infected cell's surface and modulate the interaction of the infected cell with host factors and other cells. nine secreted immunomodulators of vacv were targeted for expression, of which four were successfully refolded from inclusion bodies (for the protocol, see alzari et al., 2006) , resulting in the structure determination of two targets, one of which, a41l, is highlighted below. as noted above, a major bottleneck, particularly for the viral targets, was the growth of crystals that were sufficiently well ordered for structure solution. the crystallization of proteins is dependent on their surface properties and it is well established that flexible side chains (such as lysine residues) on the surface of proteins reduce the likelihood of successful crystallization (see, for example, rayment, 1997) . a straightforward method for changing the surface properties ('surface engineering') of proteins is the reductive methylation of lysine residues and this has been shown in some cases to yield samples that are more amenable for growth of high-quality crystals (kobayashi et al., 1999; kurinov et al., 2000; schubot & waugh, 2004) . to test the effectiveness of this method, oxford developed a simple, cheap and robust protocol and carried out a systematic study of eukaryotic, bacterial and viral targets. ten proteins which had previously proved refractory to structural analysis were successfully methylated and entered into crystallization trials (walter et al., 2006, in the press) . to date, this has led to three novel structures contributed to workpackage 9, one being the mve methyltransferase domain discussed above. the greatly enhanced use of ms for routine protein characterization was a significant outcome of spine and proved of value in numerous ways; for example, esi-ms aided a revised functional assignment of a c. jejuni extracytoplasmic solute receptor (cj0982), putatively annotated as a glutaminebinding protein from its amino-acid sequence. the esi-ms spectrum of purified cj0982 identified a peak with a mass of 125 greater than the mass of the protein alone. following crystallization and x-ray structure solution, a bound l-cysteine ligand (mass 125) was identified (mü ller et al., 2005) . standard procedures have been developed in oxford to use esi-ms to determine levels of post-translational modification such as glycosylation . nmr target proteins were checked for correct folding using 1 h-15 n heteronuclear single-quantum correlation (hsqc) spectroscopy and metal-binding properties checked by atomic absorption, using extended x-ray absorption fine structure (exafs) spectroscopy, uv-vis spectroscopy and (where appropriate) electron paramagnetic resonance (epr) spectroscopy. in several laboratories (marseille, oxford, stockholm), thermofluor analysis is routinely used to monitor the temperature-dependence of protein unfolding to define compounds to guide the optimization of crystallization conditions by stabilizing the protein. here, we present examples chosen to reflect different aspects of the broad target areas: nira from mtb and the dutpase of ebv are potential drug targets, while work on neisseria and copper-binding proteins attempts to use structure to further illuminate function and the work on c. jejuni, sars-cov and vacv shows how structure can shed light on the possible roles for proteins of unknown function. finally, the work on bacteriophage p2 shows how a test case, used to evaluate htp procedures, can provide biologically important data. overall, 31 mtb structures have been solved to date within spine and here we provide a single example. one structures of representative bacterial targets described in x4. all cartoons are drawn blue to red from the n-to the c-terminus. (a) schematic view of the structure of sulfite reductase, nira, from m. tuberculosis. the [fe 4 -s 4 ] cluster and siroheme cofactor are shown in ball-and-stick representation (schnell et al., 2005) . (b) schematic view of the structure of neisseria iiantr (nmb 0736) with the phosphoryl acceptor histidine residue (his67) indicated (ren et al., 2005) . of the mtb genes upregulated in its persistent state is the essential gene nira (rv2391), suggesting that it is a potential target for the development of anti-tuberculosis agents. the product of this gene, nira protein, has been targeted by the stockholm group. the amino-acid sequence of nira is homologous to ferredoxin-dependent sulfite reductases and the purified recombinant enzyme demonstrates a characteristic [fe 4 -s 4 ] absorption spectrum. the structure of the enzyme was determined using x-ray crystallography ( fig. 2 ; schnell et al., 2005) . for this and other difficult projects (including many mtb proteins), it was necessary to employ a hand-crafted approach, exemplified by the requirement to use streak-seeding to improve crystals and by the requirement for co-expression noted above (x3.2). at the active site, the side chains of tyr69 and cys161, located in close proximity to the sirohaem cofactor, form an unusual covalent bond. mutagenesis of these residues decreased the catalytic activity of the enzyme, with y69a and c161s having the most deleterious effect, suggesting that the covalent bond is important but not essential for enzyme activity. these residues are part of a sequence fingerprint which distinguishes ferredoxin-dependent sulfite from nitrite reductases. this is the first three-dimensional structure obtained of a ferredoxin-dependent sulfite/nitrite reductase. genes for the cohort of 62 dnabinding and associated signal-transduction protein were amplified, cloned and screened for expression in parallel and the proteins were expressed, purified and crystallized in parallel using the standard oppf pipeline. so far, the structures of seven have been solved, including the first bacterial leucine response regulator (lrp) protein (nmb1650), a global regulator of amino-acid metabolism (calvo & matthews, 1994 ) and a marr family regulator (nmb1585). in e. coli, lrp is a global regulator of amino-acid metabolism (calvo & matthews, 1994) , whereas marr is a specific regulator of the multiple antibiotic resistance operon (alekshun & levy, 1997) . studies are in progress to relate the structures of the neisseria transcription factors to their function in this organism and thermofluor analysis has been used to identify the amino-acid cofactor of lrp (nichols et al., manuscript in preparation). amongst the signal transduction proteins, it has been shown that the structure of the n. meningitidis nitrogen regulatory protein iiantr (nmb0736; fig. 2 ) confirms its assignment as a functional homologue of the iiantr proteins found in a range of other gram-negative bacteria (ren et al., 2005) . the gram-negative pathogen c. jejuni is the leading cause of gastroenteritis in humans and is responsible for 30% of guillain-barré syndrome cases, a debilitating polio-like autoimmune disease. c. jejuni colonizes the intestinal tract of many animals (svedhem & kaijser, 1981) and is commensal in poultry, cattle and swine (harris et al., 1986; kazwala et al., 1990) . the most promising route to reducing infections is to reduce the incidence in poultry via vaccination. the c. jejuni genome (parkhill et al., 2000) harbours several genes encoding highly antigenic proteins, three of which are the periplasmic binding component of an abc amino-acid transport system structures of representative viral targets described in x4. all cartoons are drawn with each monomer coloured from blue to red from the n-to the c-terminus. (a) the trimeric receptorbinding protein from lactococcal bacteriophage p2. the threefold axis is vertical and the separate architectural units of the molecule are labelled according to spinelli et al. (2006) . (b) the dimeric molecule of sar-cov nsp9, viewed orthogonal to the molecular twofold axis. (c) ribbon diagram of a41l of vacv (m. bahar, unpublished work). (d) ribbon representation of ebv dutpase in complex with an atomic representation of dump (c, o, n and p atoms drawn in yellow, red, blue and orange, respectively). and include cj0982, a major surface antigen and vaccine candidate (wyszynska et al., 2004) . such amino-acid uptake systems are crucial to c. jejuni since it relies on amino acids as a carbon source (its incomplete glycolytic pathway renders it unable to use sugars; kelly, 2001) . york determined the crystal structure of cj0982, revealing that the ligand-binding pocket contained a cysteine (fig. 2) , which was confirmed by esi-ms and tyrosine-fluorescence spectroscopy, suggesting that the protein belongs to a cysteine-binding abc-transport system (mü ller et al., 2005) . this is the first structure of a cysteinetransport protein. crystallization experiments were initially carried out at the oxford protein production facility and were refined using an in-house mosquito nanolitre-dispensing robot. five c. jejuni structures have been solved as part of the spine project. the florence group have solved 20 novel structures of copper-binding proteins by nmr. the copper homeostasis systems they have characterized by nmr include the p-type atpase copa from b. subtilis (banci et al., 2002) , a nonpathogenic close relative of b. anthracis that also contains a close copa orthologue (ba3859). an unusual feature of these p-type atpases is that the cytoplasmic n-terminus has one or more domains, depending on the complexity of the organism, each containing a metal-binding motif. b. subtilis copa has two domains (copaa and copab) with similar amino-acid sequences, whereas the copper atpases of other bacteria have only one domain. nmr analysis revealed one b. subtilis domain (copaa) to be largely unstructured, suggesting that it may be unfolded in vivo and not functional (banci et al., 2002) . sequence comparison of the two domains and orthologous proteins suggested that ser46 of copaa may destabilize the hydrophobic core of the domain, as hydrophobic residues (val and ala) occur frequently at this position. indeed, the single mutation s46v of copaa produced a completely folded domain. the structure of the copa nterminal domain was solved by nmr (fig. 2 ) and the interaction with its copper chaperone partner copz (from the same operon) was also characterized (banci et al., 2003a,b) . it is hypothesized that in vivo the folded functional domain is favoured over an unfolded state, in a manner analogous to the way that the n-terminus of the wilson's disease protein interacts with its atp-binding domain (banci et al., 2003a) . marseille have investigated, as a test case, the mechanism of cell entry by bacteriophage p2. this tailed phage infects lactococcus lactis, a gram-positive bacterium used extensively for the manufacture of fermented milk products. infection of l. lactis by tailed phages leads to serious financial losses. bacteriophage p2 infects specific l. lactis strains by using a receptor-binding protein (rbp) located at the tip of its non-contractile tail. as with the nira protein, described above, crystals suitable for x-ray diffraction experiments could only be obtained by seeding. bacteriophage p2 rbp is a homotrimeric protein, each subunit of which comprises three domains (fig. 3) : the shoulders (a -sandwich attached to the phage), the neck (an interlaced -prism) and the receptorrecognition head (a seven-stranded -barrel; spinelli et al., 2006) . the complex of rbp with a neutralizing llama vhh5 domain allowed identification of the area on rbp that attaches to the bacterial receptor , where it is able to bind various saccharides (tremblay et al., 2006) . the structural similarity between the recognition-head domain of bacteriophage p2 and those of adenoviruses or reoviruses, which invade mammalian cells, suggests that these viruses, despite being evolutionarily distant and having different chemical genomic composition (dna versus rna), may have a common ancestral gene. the sars-cov replicase gene encodes multiple enzymatic functions (snijder et al., 2003) . these include an rnadependent rna polymerase activity (rdrp, nsp12), a 3c-like serine proteinase activity (3clpro, nsp5, also known as the main proteinase, mpro), a papain-like proteinase activity (pl2pro, nsp3) and a superfamily 1-like helicase activity (hel1, nsp13). these types of proteins are common to the replicative machinery of many positive-strand rna viruses. in addition, the replicase gene encodes proteins that have domains likely to possess enzymatic activities associated with rna modification. marseille and oxford have studied sars proteins and have to date solved four separate structures using the standard protein-production and crystallization pipelines described by alzari et al. (2006) and berry et al. (2006) . one of these, the replicase protein nsp9, was solved by both partners (egloff et al., 2004; sutton et al., 2004) and we discuss this structure below. nsp9, encoded by orf1a, has no designated function but is most likely involved in viral rna synthesis. the protein comprises a single -barrel with a fold previously unseen in single-domain proteins (fig. 3) . the topology superficially resembles an ob-fold with a c-terminal extension and is related to both of the two subdomains of the sars-cov 3clike protease (which belongs to the serine protease superfamily). nsp9 has presumably evolved from a protease. the crystal structure suggests that the protein is dimeric and this was confirmed by analytical ultracentrifugation and light scattering. nsp9 binds rna and appears to interact with nsp8. the spine structural and functional analyses indicate that nsp9 may play multiple roles in the replicative cycle of coronaviruses. its interaction with other proteins may be essential for the formation of the viral replication complex together with its ability to interact with rna (in the absence of other proteins). the loops presented by the -barrel may principally confer the rna-binding capacity via non-specific interactions, while the c-terminal -hairpin and helix, which display a greater conservation across coronaviruses, are likely to be involved in dimerization and interaction with other proteins. to date oxford have solved four vacv protein structures, one of which, a41l, shares sequence similarity to several chemokine-binding proteins and is thought to play a role in reducing inflammatory responses to viral infection. mutants of vacv deficient in a41l are readily cleared by the immune system and the a41l protein has also been shown to reduce the infiltration of inflammatory cells into infected areas of host immune systems in animal models. oxford produced and purified a41l using the standard pipeline for expression in e. coli, followed by a generic refolding protocol, as mentioned above. refolded protein was quality-assessed by ms. crystal screens and optimizations were performed using the htp protocols developed in oxford (walter et al., 2003; brown et al., 2003; walter et al., 2005) . the structure was solved by the mad technique at the uk beamline bm14 (grenoble) using semet-labelled a41l protein (m. bahar, unpublished work). a41l is a single-domain protein with a core fold that adopts a distinct -sandwich topology. the -sandwich is defined by two -sheets arranged parallel to each other (fig. 3) and connected by an array of long loops. vacv a41l is structurally similar to the 35k chemokine-binding protein of the related cowpox virus. work is under way to identify the chemokines to which the molecule binds, but it is clear from the structure that the interactions are somewhat unusual since the protein does not possess the overwhelmingly negatively charged surface usually associated with chemokine-binding proteins. the ebv project in grenoble attempted a structural genomics approach to enzymes of this virus; however, most aspects of the activity were not performed in htp mode (tarbouriech et al., 2006) . the exception to this was the use for crystal screening of a cartesian robot dispensing nanolitre drops, which had a significant impact on the project (berry et al., 2006) and led to the determination of the structures of four proteins. of these, ebv dutpase is of particular interest. dutpases are ubiquitous enzymes hydrolyzing dutp in order to maintain a low intracellular concentration of dutp and to minimize its incorporation in dna. monomeric dutpases only occur in herpesviruses, whereas other organisms encode related trimeric or unrelated dimeric forms of this enzyme. for trimeric dutpases, the three different subunits contribute five conserved motifs to each of the three active sites located at the subunit interfaces. the structure of ebv dutpase represents the first example of a monomeric dutpase (tarbouriech et al., 2005) . the structure was determined in complex with the reaction product dump and the substrate analogue ,-imino-dutp. ebv dutpase (256 residues) consists of two domains, each structurally similar to the subunit of the trimeric dutpases, which contribute motif iii, and motifs i, ii and iv, respectively, to the creation of a single active site (fig. 3) . the c-terminal motif v is largely disordered in the solved structures but is tethered near the active site by an unexpected disulfide bridge between cys4 and cys246. the enzyme is a rare example of an evolution from a multimeric to a monomeric protein. presumably, a single gene-duplication event led to a molecule which maintained an active site extremely similar in structure to those of the trimeric enzymes. it is to be hoped that such subtle differences between the active sites of the viral and the human enzymes can be exploited in the design of specific inhibitors. the spine project has contributed to a shift in the way structural biology is now being carried out in europe through the democratization of the use of new technologies and the development of novel strategies at various steps of the structure-determination pipeline. since spine is driven by the notion of selecting 'high-value human health targets', it was natural that a number of human pathogens were targeted for analysis, from bacteria and viruses that cause 'established' human diseases to newly emerging threats to human health (sars-cov). overall, spine has solved a substantial number of structures from these targets (over 220, including 65 complexes), some of which are being evaluated as possible new drug targets. however, we have found no generic solution to the traditional problem of protein solubility; rather, the problem has been significantly ameliorated by the sheer numbers of potential constructs that can be screened through the establishment and development of parallel cloning and expression technologies both in bacterial and eukaryotic expression systems. for the future it seems that more comprehensive library-based approaches (sometimes termed 'directed evolution') may have a substantial impact in providing a much more extensive screening for soluble constructs and one such approach, the esprit method (see alzari et al., 2006) , is being applied to several viral targets of high value, including a sars-cov protein, in part as a collaboration between oxford and grenoble (c. meier, unpublished results). there is considerable scope for further developments. the analysis of a set of relatively straightforward bacterial targets was key to the benchmarking of the techniques used and developed in spine, especially for protein overexpression and purification. the york/oxford collaboration on b. anthracis identified a number of problems in the first generation of protocols, such as the insolubility of certain gateway constructs. the failure to overexpress significant amounts of soluble protein could easily have been put down to the challenging nature of human and human viral targets if only these problems had been used to test pipelines. the work on bacterial targets pinpointed the problems and provided a convenient and reliable benchmark for their resolution. in addition, the b. anthracis project has led to the determination of 45 structures to date, which demonstrates the high success rate which can be achieved with the refined pipelines (up to 30%). in addition, the bacterial projects facilitated the ready sharing of knowledge, technologies and technique, which has contributed greatly to the research papers establishment of european htp activities, and the dissemination of technologies to the wider community. the production of soluble proteins for certain eukaryotic viral targets was poor in e. coli and in terms of amenability to overexpression in soluble form at suitable concentrations for nmr or protein crystallography these proteins behave more like human proteins. for these targets, eukaryotic expression systems such as baculovirus and mammalian cells provide powerful alternative vehicles for the production of soluble protein. it is interesting to note that the pattern of soluble protein expression across different viruses can vary markedly. whereas herpes viral structural proteins were in general intractable in e. coli, there was a higher success rate with proteins from viruses such as vacv (from a small target set of ten, three crystal structures have been solved). proc. natl acad. sci. usa proc. natl acad. sci. usa encyclopedia of life sciences key: cord-325282-20l9xcmg authors: helal, mohamed a.; shouman, shaimaa; abdelwaly, ahmad; elmehrath, ahmed o.; essawy, mohamed; sayed, shireen m.; saleh, amr h.; el-badri, nagwa title: molecular basis of the potential interaction of sars-cov-2 spike protein to cd147 in covid-19 associated-lymphopenia date: 2020-09-16 journal: journal of biomolecular structure & dynamics doi: 10.1080/07391102.2020.1822208 sha: doc_id: 325282 cord_uid: 20l9xcmg lymphopenia is considered one of the most characteristic clinical features of the coronavirus disease 2019 (covid-19). sars-cov-2 infects host cells via the interaction of its spike protein with the human angiotensin-converting enzyme 2 (hace2) receptor. since t lymphocytes display a very low expression level of hace2, a novel receptor might be involved in the entry of sars-cov-2 into t cells. the transmembrane glycoprotein cd147 is highly expressed by activated t lymphocytes, and was recently proposed as a probable route for sars-cov-2 invasion. to understand the molecular basis of the potential interaction of sars-cov-2 to cd147, we have investigated the binding of the viral spike protein to this receptor in-silico. the results showed that this binding is dominated by electrostatic interactions involving residues arg403, asn481, and the backbone of gly502. the overall binding arrangement shows the cd147 c-terminal domain interacting with the spike external subdomain in the grove between the short antiparallel β strands, β1’ and β2’, and the small helix α1’. this proposed interaction was further confirmed using md simulation and binding free energy calculation. these data contribute to a better understanding of the mechanism of infection of sars-cov-2 to t lymphocytes and could provide valuable insights for the rational design of adjuvant treatment for covid-19. communicated by ramaswamy h. sarma in december 2019, an outbreak of a pneumonia-causing virus was first reported in wuhan, china (lu, stratton, et al., 2020; zhou et al., 2020) , and has been spreading globally ever since (sohrabi et al., 2020) . the virus was found to phylogenetically belong to the severe acute respiratory syndrome coronavirus (sars-cov) (lu, zhao, et al., 2020) . the virus was named sars-cov-2 (c. s. g. of the international, 2020; gorbalenya, 2020) , and the disease named coronavirus disease-19 . the entry of the virus into host cells is facilitated by binding of its transmembrane spike (s) protein with angiotensin-converting enzyme 2 (ace-2) receptor (hoffmann et al., 2020) . the spike protein is a class i viral fusion transmembrane glycoprotein that plays a dual role in viral entry and fusion with host cell membrane (bosch et al., 2003; li, 2016) . cryo-electron microscopy (cryo-em) revealed the structure of s protein as trimeric spikes composed of three s1 and s2 heterodimer subunits, protruding out of the viral envelope (gui et al., 2017; wrapp et al., 2020) . the s1 subunit facilitates viral binding to cell receptor through the receptor-binding domain (rbd), whereas the s2 subunit encloses a hydrophobic fusion domain, allowing viral fusion (song et al., 2018) . cleaving the s1-s2 boundary is primed by host cell proteases such as transmembrane protease serine 2 (tmprss2), and is required for viral activation and entry (hoffmann et al., 2020) . tmprss2 and ace-2 are highly expressed in the epithelial tissue of multiple organs and thus significantly contribute to viral pathogenicity, clinical manifestations, and mortality (sungnak et al., 2020) . one of the clinical indicators of the severity of sars-cov-2 is low lymphocytic count (lymphopenia) , especially in the cd8 þ , cd4 þ , and cd3 þ t lymphocyte populations (zeng, 2020; zheng et al., 2020) . lymphopenia is a risk factor in viral infections, and was reported to predict the severity of the disease in covid-19 patients (gui et al., 2017) . although several studies examined the causes of lymphopenia during viral infections (lalueza et al., 2017 (lalueza et al., , 2019 , the mechanisms underlying this feature are not fully understood in covid-19 patients. proposed mechanisms for the observed lymphopenia include direct invasion of lymphocytes , hyperlactic acidemia causing suppression of lymphocyte proliferation (fischer et al., 2007) , as well as an inflammatory cytokine storm resulting in lymphocyte apoptosis (liao et al., 2002) , similar to that reported earlier with mers-cov infection (chu et al., 2016) . angiotensin-converting enzyme 2, or ace-2 receptor, has been shown to provide an entry point to sars-cov-2 in different types of human cells zhou et al., 2020) . however, t lymphocytes were shown to be consistently negative for ace-2 receptors (hamming et al., 2004) , suggesting an alternate route for viral entry. cd147 was recently proposed as a probable route for sars-cov-2 invasion into vero e6 host cells (wang, 2020) . cd147 (also known as basigin or emmprin) is a transmembrane glycoprotein that was originally identified in 1992 as a t lymphocyte activation-associated antigen (kasinrerk et al., 1992) . further research confirmed its expression on activated t lymphocytes and its relatively weaker expression in resting t lymphocytes, and that its expression increases rapidly upon t lymphocyte activation (koch et al., 1999) . cd147 induces matrix metalloproteinase (mmp) production during physiological and pathological events including inflammatory response, wound healing, and tissue homeostasis (cruzat et al., 2018; jin et al., 2019; kato et al., 2009; ungern-sternberg et al., 2018) . interestingly, cd147 receptor is expressed on red blood cells (rbcs), providing a route for malaria entry (zhang et al., 2018) . it is worth mentioning that pfrh5 complex on the cell membrane of plasmodium falciparum, the causative agent of malaria, interacts with cd147 on human rbcs, a binding which was necessary for invasion by all strains when tested in vitro (crosnier et al., 2011) . anti-cd147 antibodies were shown to prevent entry of p. falciparum and sars-cov-2 into host cells (bian, 2020; zhang et al., 2018) . to understand the mechanism of interaction of the sars-cov-2 spike protein with the cd147 receptor, we have performed a four-stage in-silico study. first, the binding site of both proteins had to be accurately predicted, taking into consideration the few number of their available crystal structures. second, molecular docking was performed using three different protein-protein interaction servers. then, the best docking pose was analyzed in md simulation to check its stability. finally, the binding free energy was estimated, using snapshots of the md simulation, to get in-depth knowledge of the binding determinants. the study starts with determination of the most probable binding sites for spike and cd147. this was specially challenging for the latter because only few crystal structures were reported for this membrane glycoprotein. later, the molecular docking was performed using three different web servers and the best pose was selected using the pdbepisa website. then, the best complex was subjected to a 100 ns md simulation in npt ensemble. finally, free energy calculation was performed to gain insights into the major contributing forces in binding and the critical residues involved. the sars-cov-2 receptor binding domain (rbd) was obtained from its crystal structure complexed with human angiotensin-converting enzyme 2 (hace2) (pdb id: 6lzg), while that of cd147 was isolated from its crystal structure complexed with malaria invasion protein, reticulocyte-binding protein homologue (rh5) attained from the parasite plasmodium falciparum (pdb id: 4u0q). the crystal structures of both targeted proteins, sars-cov-2 and cd147, were closely examined for integrity and the active residues taking part in the interaction were identified. the surface of cd147 was examined using the computed atlas of surface topography of proteins (castp) server. the default probe radius used by castp is 1.4 å. we opted to use a smaller probe of 0.8 å to get more exhaustive coverage of all the potential binding sites on the cd147 surface. prior to the docking simulation, both proteins, spike and cd147, were checked for errors and prepared in the protein preparation wizard, within moe software. the preparation steps include removal of any bound ligands, water of crystallization, and salts. also, all the hydrogens were added and the protein termini were capped. both proteins were minimized individually in moe to a gradient of 0.001 kcal/mol.å 2 . first, all heavy atoms were kept fixed, and the hydrogen atoms were allowed to optimize their positions. second, the entire backbone of the protein was kept fixed and the side chains were minimized using the same parameters. finally, the whole molecule including the backbone was minimized (helal et al., 2011; helal & avery, 2012) . three different webdocking servers, haddock v2.2, zdock v3.0.2, and hawkdock 2018, were utilized to computationally predict the protein-protein interactions of the two unbound protein structures. the docking trial on each server was repeated twice using the default settings; in the first run no restraints was given to the server so that it can freely determine the binding region (interface) for each protein. this step was performed to find out to which extent the software would bias toward a specific docking pose. in the second run on each server, specific active residues were defined for the software to be considered as restraints (see the discussion part). these residues were selected based on our understanding of the crystal structures of both sars-cov-2-rbd/hace2 and cd147 rh5 as well as the prediction by castp server. molecular dynamics (md) simulation was performed using gromacs 2019 software package and the all-atom opls force field. the protein complex was solvated in a cubic box of spc water model of 100 â 100 â 100 å size and periodic boundary conditions were implemented. ionizable residues were assigned standard ionization states at ph 7. the system was then neutralized by the addition of five sodium ions. the total number of atoms in the system, including the water and ions, was 117,013. short-range interactions were treated with a 10 å cutoff for both lennard-jones and coulomb potentials, while the particle mesh ewald (pme) algorithm was used for long-range electrostatics. the md simulation was performed in four steps. during the first three steps, a force constant of 1000 kj mol à1 nm à2 was used to restrain all the heavy atoms to preserve the original fold of the proteins. first, the solvated protein system was minimized using a 5000 steps of steepest descent algorithm to resolve any steric clashes or inappropriate geometry. then, to ensure a reasonable starting structure, the system was equilibrated under constant number of particles, volume, and temperature (nvt) ensemble for 100 ps using berendsen thermostat (golo & sha k %tan, 2002) . the second round of equilibration was performed under constant pressure (npt ensemble) using the parrinello-rahman barostat for an additional 100 ps (tuble et al., 2004) . finally, the position restraints were released and the system was simulated in the production run under an npt ensemble (nos e-hoover thermostat and parrinello-rahman barostat) for 100 ns using a time step of 2 fs. simulation results were analyzed using the visual molecular dynamics (vmd) software, ver. 1.9.3 (humphrey et al., 1996) . pymol graphical software ver. 2.3 was used for the generation of the figures of protein-protein interactions. binding free energy was calculated using the molecular mechanics poisson boltzmann surface area (mm/pbsa) method with the g_mmpbsa script. this method calculates the binding energy using molecular mechanics potential energy (electrostatic and van der waals interactions) and free energy of solvation (polar and nonpolar solvation energies). a total number of 100 snapshots, extracted at regular intervals from the whole md trajectory, were used for calculating each energy term. the average for each term was then calculated. in addition, a per-residue binding free energy decomposition at each snapshot was performed and the average binding energy for each residue was calculated and depicted. the solvent-accessible surface area (sasa) model was used to compute non-polar solvation energy with a surface tension of 0.02267 (kj/mol 2 ) and a probe radius of 1.4 å. the recently reported crystal structure of sars-cov-2 spike protein complex with ace2 (pdb id: 6lzg) reveals that the virus utilizes the external subdomain of the spike receptor binding domain (rbd) to recognize the human ace2 receptor . this external subdomain is composed mainly of a flexible loop, s438-y508, connecting two anti-parallel small b strands. also, a part of this loop, the segment f486-g502, represents the most solvent accessible part of the spike rbd and makes direct contact with ace2 in the crystal structure. in addition, in a recent in-silico study, it was proposed that a small circular region of this loop, c480-c488, could be responsible for the interaction with the human grp78 (ibrahim et al., 2020) . collectively, these findings have convinced us to use the solvent-accessible f486-g502 segment as the interaction site of the spike protein in our docking simulation. regarding the cd147 receptor, the only crystal structure available of this protein, bound to another biological molecule, is for its complex with the pfrh5 protein (pdb id: 4u0q) (wright et al., 2014) . in this structure, the pfrh5 binds to the cleft between the two domains of cd147 assisted by a network of h-bond and salt bridge interactions. notably, the glycoprotein cd147 is a relatively small protein and few pockets could be noticed on its surface. to get an accurate prediction of the potential binding site on cd147, we have performed a comprehensive search for its surface pockets using the computed atlas of surface topography of proteins (castp) server (tian et al., 2018) . this tool enables an exhaustive and quantitative characterization of the protein topographic features, providing a complete list of the available pockets with the amino acid residues lining these pockets. figure 1 (a) shows the pockets with significant volume (>15 å 3 ) on cd147 surface. among them, pockets 1, 3, 4, and 6 are the most solvent accessible and constitute a nearly continuous grove extending at the interface of the two domains of cd147. furthermore, according to the crystal structure 4u0q, the pfrh5 protein uses pockets 1 (124.67 å 3 ) and 6 (72.57 å 3 ) for interaction with cd147. in addition to the castp server, we have also used the cprot tool (bonvinlab) which is an algorithm for the prediction of protein-protein interface merging six calculation methods into a consensus result (de vries & bonvin, 2011) . interestingly, the cprot results were very similar to those of castp with most residues of pockets 1 and 6 identified (figure 1(b) ). therefore, we have selected the residues of these pockets, 1 and 6, as the active residues in our docking study, namely ser78, asp79, gln81, trp82, gly103, pro104, pro105, arg106, val 107, lys108, lys127, ser128, glu129, ser193, and asp194. (table s1 , supporting information). molecular docking was performed using three different protein-protein docking servers, namely haddock v2. interacting, residues as the segment f486-g502 of the spike rbd and the residues of pockets 1 and 6 of the cd147 as illustrated in the previous section. in order to evaluate the docking results of the three software servers, the top ranking pose from each was examined using the pdbepisa online tool, developed by the european bioinformatics institute, embl-ebi, for the exploration of macromolecular interfaces (krissinel & henrick, 2007) . moreover, a molecular mechanics energies combined with generalized born and surface area (mm/gbsa) calculation, as implemented within the hawkdock server, was used to estimate the binding affinities (hou et al., 2011; zhong et al., 2020) . the results of the examination of the top complex generated by each software are illustrated in table 1 . interestingly, all the three servers returned very similar arrangements of the interacting proteins with rmsd of 2.23 for zdock/haddock and 0.24 for hawkdock/haddock. moreover, for each server, performing the docking run without specifying any constraints returned quite a similar docking pose, to the one with constraints, within the top ten out of more than one hundred solutions (data not shown). based on the rmsd values and visual examination, it was noticed that the poses proposed by haddock and hawkdock are very close and almost converging. we decided to use the pose generated by the haddock server as it shows a higher interface surface area of 1,069 å 2 and the best score of à68.00 in the mm/gbsa calculations (table 1 ). in addition, thorough investigations were carried out using pdbepisa tool to determine the key interacting residues in the selected docked complex generated by the haddock server. only interfacing residues within 4 å van dar waals distance cutoff between the sars-cov-2 rbd and cd147 were examined by the pdbepisa algorithm (table s3 , supporting information). it can be easily noted that the docked sars-cov-2 rbd adopted a similar orientation to that observed in its cocrystal structure with hace2, which highlights the binding role of the region that extends between the residues c480 and q505 (figure 2 ). the spike external subdomain forms a surface to which parts of cd147 domains dock. this surface is dominated by the interaction of the c-terminal domain of cd147 which binds to the spike external subdomain in the grove between the short antiparallel b strands b1' and b2' and the small helix a1' (figure 2(a) ). this grove makes contacts with nearly 50% of the c and f strands of cd147. on the other domain of cd147, parts of the a and g strands forms a complementary surface to the cyclic highly solvent-accessible segment c480-c488 of the spike rbd. in the predicted complex, a row of hydrophilic residues along the interface formed strong and intricate polar contacts ( figure 3 ). the most important interacting residues from both sides, together with the distances between each pair, are listed in table 2 . strong polar contacts were found to involve the spike rbd residues gln493 and ser494 with the side chain of thr135 in cd147. also, the side chain nitrogen of the spike gln493 makes a 3.00 å h bond with the hydroxy group of ser190 in cd147. in addition, the backbone carbonyl of spike glu484 formed a strong h bond (2.5 å) with the backbone nh of gln100 in cd147. moreover, the side chain nh 2 of asn481 on the cyclic segment of the spike b1'/b2' loop formed a bidentate h-bonds with the backbone of phe27 and side chain oh of thr28 in cd147. another set of adjacent hydrophilic residues located on the spike rbd external domain (gln498, thr500, asn501, gly502 and tyr505) were found to contribute a network of h-bonds with cd147 amino acids trp137, thr140, ser163 and thr188, which further augments the two proteins interaction. furthermore, two strong salt bridges were observed to stabilize the binding interface at both ends of the spike f486interface area, calculated as difference in total accessible surface areas of isolated and interfacing structures divided by two. c d i g p-value indicates the p-value of the observed solvation free energy gain. the p-value measures the probability of getting a lower than observed d i g, when the interface atoms are picked randomly from the protein surface. p < 0.5 indicates interfaces with surprising (higher than would-be-average for given structures) hydrophobicity, implying that the interface surface can be interaction-specific. g502 segment. the first one was noticed between arg403 and asp136 of the cd147 and the other was a stronger interaction (2.7 å) emerging from spike glu484 towards cd147 lys191. the latter was acting as a centrally located anchor point that might have a key role in stabilizing the complex. it is worth noting that the same residue, lys191, is an important player in the interaction of cd147 with the p. falciparum protein (pfrh5) (wright et al., 2014) . notably, fewer hydrophobic interactions were noticed at the interface as shown in table 2 . the most important of these interactions was the one between the spike tyr489 and his102 on the linker between the two domains of cd147. in addition, to further validate the proposed binding mode, we have docked the spike glycoprotein of sars-cov, the causative agent of the 2003 epidemic (pdb id: 5xlr) to the target protein cd147. this protein is known to bind with the human cell cd147 to facilitate entry of the virus responsible for the 2003 outbreak. we used haddock server and the same parameters exploited for docking of the sars-cov-2 spike. to our delight, this protein adopted a very similar binding pose to that of the sars-cov-2 predicted above, with a slight shift towards the c-domain of the cd147 ( figure s2 , supp. info.). also, cd147 protein made polar contacts to the sars-cov protein using the same residues, namely, gly100, his102, and lys191. this gives added validity to our proposed binding pose of the sars-cov-2 spike and cd147. the complex was then subjected to a molecular dynamics (md) simulation study in order to check the stability of the proposed binding mode and to study the time-dependent behavior of this system. in absence of 3d structure information, md simulations is especially useful for studying proteinprotein complexes as it can help the molecules "explore" their conformation space more efficiently than using the static "image" provided by the molecular mechanics energy minimization (karplus & petsko, 1990; zhao et al., 2013) . the system was solvated in a cubic spc water box and simulated for 100 ns in gromacs software package under npt ensemble using nos e-hoover thermostat (van der spoel et al., 2005) . the final frame was extracted, minimized to a gradient of 0.001 kcal.å à2 .mol à1 in moe software, and was further analyzed for key structural changes (vilar et al., 2008) . to our delight, the overall fold of both interacting proteins was preserved as well as most of the binding interface with an rmsd of just 1.25 å. the spike rbd has moved slightly towards the cd147 n-terminal domain, while most of the polar interactions were retained or replaced by similar interactions with nearby residues (figure 4(a, b) ). the backbone rmsd fluctuation of the whole complex as well as the interface residues were monitored during the md simulation to check the stability of the system (figure 4(c) ). both proteins were stable with some rmsd fluctuations that stabilized after 40 ns. to our delight, the residues of cd147 at the interface attained a significant stability over time and reached 1 å after 60 ns. distances of the most important polar contacts at the interface were calculated throughout the simulation and represented as a heatmap in figure 4(d) . it was noticed that the salt bridge between arg403 of the spike and asp136 of cd147 was stable during the simulation with only minor fluctuation for a short time between 20 and 36 ns. this ionic bond could be critical for anchoring the b-strand b of the cd147 c-terminal domain to the binding grove created by the spike rbd. similarly, the h bond between gln498 and thr188 at the end of stand f of cd147, although weak, could contribute to some extent to the interaction of this strand to the surface of the spike. on the other side, at the beginning of the spike f486-g502 segment, the h bond of asn481 to thr28 was replaced by a stronger and more stable one with thr44. the most prominent difference was the loss of the characteristic salt bridge between glu484 and lys191. in the starting complex, this interaction was centrally located below the loop connecting the two domains of cd147 and we initially thought it would be essential for stabilizing the interaction. as a compensation, both residues glu484 and lys191, upon liberation from the ionic bond, were able to form h bonds with gln100 and tyr449, respectively. finally, we noticed that tyr140 adopted a swing movement to bind with the backbone nh of gly502 instead of thr500, thus, stabilizing the strand d of cd147 to the edge of the spike f486-g502 segment. the residues proposed to be critical for the spike-cd147 interaction were mutated to alanine to validate their important role in the interaction. these residues are: arg403 and asn481 in spike, and tyr140 in cd147. the mutated proteins were docked using haddock server with the same parameters used for docking of the wild-type proteins. as expected, the number of residues at the interface and the interface surface area were lower than those of the wild-type proteins (table s4 , supp. data). also, the mm/gbsa score, as predicted by the hawkdock server, was à60.64 which is lower than the scores of the wild-type protein complexes generated by any of the docking servers we used. overall, the mutant proteins bind in a similar fashion to the wild-type one with an rmsd of 1.332 å. however, the critical salt bridges and h bonds formed by the mutated residues were replaced by weaker contacts involving phe486, tyr489, gln493, and asn501 from spike with val160, asp136, ser109, and gln100 from cd147, respectively ( figure s3 , supp. data). in addition, the complex with the mutated residues was energy minimized and then subjected to md simulation for 30 ns following the same protocol used for the wild-type complex. the purpose of this simulation is to compare the time-dependent behavior of both proteins and validate our proposed binding mode. it was noticed that the h bonds tyr489-asp136 and gln493-ser109 were maintained throughout the simulation. however, the complex was significantly less stable compared to the wild-type complex. this was evident from the higher fluctuation of the backbone rmsd of the whole complex and, especially, the interface residues ( figure s4 , supp. data). these findings highlight the role of the mutated polar residues, arg403 and asn481 in spike, and tyr140 in cd147, for the interaction of these proteins and give more confidence to our proposed binding mode. to get more insight into the nature of interaction between the two proteins and detailed information about individual ligand contribution, we have performed binding free energy calculation (cavasotto, 2020) . we have used the md-based method molecular mechanics poisson boltzmann surface area (mm/pbsa) as implemented in the g_mmpbsa tool. this technique can achieve similar accuracy to the free energy perturbation (fep) methods but at a smaller computational cost (kumari et al., 2014) . the calculation was performed with the single trajectory approach using 100 frames saved at regular intervals of the complex md simulation. according to the mm/pbsa method, the binding free energy of the interacting proteins is calculated using the following equations: where g complex is the total free energy of the complex and g protein is the total free energy of the isolated proteins. he mm i is the vacuum molecular mechanics potential energy. ts refers to the entropic contribution to the free energy where t and s denote the temperature and entropy, respectively. the last term hg solvation i is the free energy of solvation and is composed of the polar part g polar and the nonpolar part g non-polar ; the former could be estimated by solving the poisson à boltzmann (pb) equation and the latter could be calculated from the solvent-accessible surface area (sasa). the term c is a coefficient related to surface tension of the solvent and b is fitting constant (kumari et al., 2014) . the results of the binding free energy calculation are shown in table 3 . in accordance with our initial prediction, the binding energy is dominated by the electrostatic term with a contribution of 66% compared to 30% of the van der waals interaction. therefore, the attractive electrostatic term greatly exceeded the repulsive term and was the main driving force for the binding. the mm/pbsa method also returned a perresidue binding-free energy decomposition ( figure s5 , supp. data). this helps in identifying the key residues involved in the interaction. in our case, a contribution of more than 5 kj/ mol to the binding-free energy of the complex was considered significant. as expected, most of the residues participated in the binding of the initial and md-refined complexes showed a significant contribution. particularly, the calculation highlighted the role of the spike arg403, tyr449, asn481, val483, and tyr505 in the attachment to the cd147 receptor. the overall proposed binding mode of the host cell cd147 and the sars-cov-2 spike is illustrated in figure 5 . lymphopenia was found to be associated with the severity of sars-cov-2 infection in many clinical reports (guan et occurrence of lymphopenia corresponds to a nearly 3-fold increased risk of severe disease , supporting its potential usage as a clinical sign for serious morbidity . although the cause of lymphopenia during viral infection still needs further investigations, several studies have postulated potential mechanisms for the evident lymphopenia. these include direct viral invasion into t lymphocytes resulting in cell death , indirect upregulation of p53 apoptotic pathways in t lymphocytes by excessive secretion of pro-inflammatory chemokines and cytokines (il10, il7, il6, il2, tnfa, cxcl10/ip-10, ccl3/mip-1a ccl2/mcp-1, and ccl4/mip1b) liao et al., 2002; xiong et al., 2020) , and hyperlactic-acidemia-induced apoptosis (d ıaz et al., 2018) . notably, t lymphocytes barely express ace-2 receptor (hamming et al., 2004 ) -the currently accepted point of entry of sars-cov-2 . cd147 may thus present an alternative point for viral spike protein entry. indeed, cd147 was reported to mediate the entry of human immunodeficiency virus (hiv) into t lymphocytes (pushkarsky et al., 2001) . it was also reported to mediate entry of cytomegalovirus (cmv) into endothelial and epithelial cells (vanarsdall et al., 2018) , and plasmodium falciparum into red blood cells (zhang et al., 2018) . cd147, also termed as emmprin or basigin, is a pleiotropic molecule with various effects (grass & toole, 2016; iacono et al., 2007) . given the emerging use of bioinformatics in biological research (karim et al., 2020; mehmood et al., 2014) , it is worth mentioning that, through pathway analyses, cd147 was found to be a significant cell-surface protein contributing to chemoresistance and cell survival in cancerstem-cell-like cells (kang et al., 2013) , and the invasion of various cancers (li et al., 2015; liang et al., 2016; zheng & gong, 2017) . additionally, it was found to be highly expressed in activated regulatory t lymphocytes, enabling it to be a potential marker for distinguishing cytokine-producing activated t lymphocytes from resting t lymphocytes (landskron & task en, 2013; solstad et al., 2011) . the structure of transmembrane cd147 receptor consists of three domains: extracellular, transmembrane, and a short cytoplasmic tail (muramatsu, 2016) . those domains facilitate its interaction with several proteins, such as monocarboxylate transporters (mcts) and cyclophilins (cyps), especially cyclophilin a, to modulate specific functions (kirk et al., 2000; yurchenko et al., 2010) . cypa is a ubiquitously expressed chaperon protein that has a high affinity to bind to cd147 receptor for enhancing viral invasion and inflammation (chen et al., 2005; yurchenko et al., 2010) ; a study in 2013 reported that blocking of cypa inhibits sars-cov infection and replication (tanaka et al., 2013) . cd147 acts as the major chaperone protein of mct 1, mct 3 and mct 4 proton transporters, that control the export of lactic acid molecules outside the cell (kirk et al., 2000; philp et al., 2003) . activated t lymphocytes have been reported to undergo increased glycolysis with increased lactate production (cham & gajewski, 2005) , as they need high energy supplies for proliferation and producing cytokines (frauwirth & thompson, 2004) . the expression of mcts for lactate export helps t lymphocytes to cope with the increased lactic acid levels (merezhinskaya et al., 2004) , since the accumulation of lactic acid induces t cells apoptosis (d ıaz et al., 2018) . these data provide an insight into the possible impact of sars-cov-2 infection on the balance of cd147-mct interaction, and direct a need for further research on this role. angiotensin-converting enzyme 2, ace-2 receptor, has been proved to be the entry point of sars-cov-2 into the lungs and other types of human cells. however, t lymphocytes are rarely expressing this receptor. several recent studies have suggested that cd147 is a probable route for sars-cov-2 invasion into certain host cells. these findings encouraged us to design the current study in which we employed docking and md simulation to investigate this possibility. the results were also compared with the molecular modeling data of the mutant proteins interaction as well as the interaction of cd147 with the spike of the sars-cov, the causative agent of the 2003 outbreak. our model shows interaction between cd147 c-terminal domain with sars-cov-2 spike external subdomain in the grove between the short antiparallel b strands, b1' and b2', and the small helix a1'. we confirmed this proposition using md simulation and binding free energy calculation. this model provides an appreciation for targeting cd147 for adjuvant drug therapy strategies, to improve the prognosis of covid-19 pateint. our data is supported by the results of using meplazumab, an anti-cd147 humanized antibody, for viral clearance and lymphocyte count normalization (bian, 2020) . in addition, of proposing azithromycin as a therapeutic strategy to treat p. falciparum via interfacing in pfrh5/ cd147 interaction (muralidharan & striepen, 2015; wilson et al., 2015) . the mechanisms proposed in our study encourage research on cd147 involvement in sars-cov-2 in direct invasion to t lymphocytes and associated lymphopenia. no potential conflict of interest was reported by the author(s). meplazumab treats covid-19 pneumonia: an openlabelled, concurrent controlled add-on clinical trial the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 binding free energy calculation using quantum mechanics aimed for drug lead optimization glucose availability regulates ifngamma production and p70s6 kinase activation in cd8þ effector t cells epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study function of hab18g/ cd147 in invasion of host cells by severe acute respiratory syndrome coronavirus middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways basigin is a receptor essential for erythrocyte invasion by plasmodium falciparum colocalization of galectin-3 with cd147 is associated with increased gelatinolytic activity in ulcerating human corneas cport: a consensus interface predictor and its performance in prediction-driven docking with haddock unravelling the interplay between extracellular acidosis and immune cells haddock: a proteinprotein docking approach based on biochemical or biophysical information inhibitory effect of tumor cell-derived lactic acid on human t cells regulation of t lymphocyte metabolism dynamic attractor for the berendsen thermostat an the slow dynamics of biomacromolecules severe acute respiratory syndrome-related coronavirus-the species and its viruses, a statement of the coronavirus study group how, with whom and when: an overview of cd147-mediated regulatory networks influencing matrix metalloproteinase activity clinical characteristics of coronavirus disease 2019 in china cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis combined receptor-based and ligandbased approach to delineate the mode of binding of guaianolideendoperoxides to pfatp6 new insights into the binding mode of melanin concentrating hormone receptor-1 antagonists: homology modeling and explicit membrane molecular dynamics simulation study sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor assessing the performance of the mm/pbsa and mm/gbsa methods. 1. the accuracy of binding free energy calculations based on molecular dynamics simulations vmd: visual molecular dynamics cd147 immunoglobulin superfamily receptor function and role in pathology covid-19 spike-host cell receptor grp78 binding site prediction cd147 as a key mediator of the spleen inflammatory response in mice after focal cerebral ischemia proteomic analysis reveals that cd147/ emmprin confers chemoresistance in cancer stem cell-like cells a multi-omics analysis of bone morphogenetic protein 5 (bmp5) mrna expression and clinical prognostic outcomes in different cancers using bioinformatics approaches molecular dynamics simulations in biology human leukocyte activation antigen m6, a member of the ig superfamily, is the species homologue of rat ox-47, mouse basigin, and chicken ht7 molecule the e-selectin ligand basigin/ cd147 is responsible for neutrophil recruitment in renal ischemia/ reperfusion cd147 is tightly associated with lactate transporters mct1 and mct4 and facilitates their cell surface expression t cell activation-associated epitopes of cd147 in regulation of the t cell response, and their definition by antibody affinity and antigen density inference of macromolecular assemblies from crystalline state g_mmpbsa-a gromacs tool for high-throughput mm-pbsa calculations severe lymphopenia in hospitalized patients with influenza virus infection as a marker of a poor outcome impact of severe hematological abnormalities in the outcome of hospitalized patients with influenza virus infection cd147 in regulatory t cells structure, function, and evolution of coronavirus spike proteins cd147 reprograms fatty acid metabolism in hepatocellular carcinoma cells through akt/mtor/srebp1c and p38/ppara pathways expression of the sars-cov-2 cell receptor gene ace2 in a wide variety of human tissues e2f1 promotes tumor cell invasion and migration through regulating cd147 in prostate cancer il-19 induces production of il-6 and tnf-alpha and results in cell apoptosis through tnf-alpha analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease clinical and biochemical indexes from 2019-ncov infected patients linked to viral loads and lung injury outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding use of bioinformatics tools in different spheres of life sciences presence and localization of three lactic acid transporters (mct1,à 2, andà 4) in separated human granulocytes, lymphocytes, and monocytes teaching old drugs new tricks to stop malaria invasion in its tracks basigin (cd147), a multifunctional transmembrane glycoprotein with various binding partners loss of mct1, mct3, and mct4 expression in the retinal pigment epithelium and neural retina of the 5a11/basigin-null mouse zdock server: interactive docking prediction of protein-protein complexes and symmetric multimers cd147 facilitates hiv-1 infection by interacting with virus-associated cyclophilin a world health organization declares global emergency: a review of the cd147 (basigin/emmprin) identifies foxp3 þ cd45ro þ ctla4þ-activated human regulatory t cells cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes lymphopenia predicts disease severity of covid-19: a descriptive and predictive study suppression of coronavirus replication by cyclophilin inhibitors castp 3.0: computed atlas of surface topography of proteins an approach to developing a force field for molecular simulation of martensitic phase transitions between phases with subtle differences in energy and structure extracellular matrix metalloproteinase inducer emmprin (cd147) in cardiovascular disease gromacs: fast, flexible, and free cd147 promotes entry of pentamer-expressing human cytomegalovirus into epithelial and endothelial cells medicinal chemistry and the molecular operating environment (moe): application of qsar and molecular docking to drug discovery sars-cov-2 invades host cells via a novel route: cd147-spike protein structural and functional basis of sars-cov-2 entry by using human ace2 clinical features of 69 cases with coronavirus disease 2019 in wuhan, china hawkdock: a web server to predict and analyze the protein-protein complex based on computational docking and mm/gbsa macrolides rapidly inhibit red blood cell invasion by the human malaria parasite, plasmodium falciparum cryo-em structure of the 2019-ncov spike in the prefusion conformation structure of malaria invasion protein rh5 with erythrocyte basigin and blocking antibodies transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa cyclophilin-cd147 interactions: a new target for anti-inflammatory therapeutics mortality of covid-19 is associated with cellular immune function compared to immune function in chinese han population clinical characteristics of 140 patients infected with sars-cov-2 in wuhan disrupting cd147-rap2 interaction abrogates erythrocyte invasion by plasmodium falciparum mature hiv-1 capsid structure by cryo-electron microscopy and all-atom molecular dynamics lymphopenia is associated with severe coronavirus disease 2019 (covid-19) infections: a systemic review and meta-analysis cd147 expression was positively linked to aggressiveness and worse prognosis of gastric cancer: a meta and bioinformatics analysis functional exhaustion of antiviral lymphocytes in covid-19 patients improving the performance of the mm/pbsa and mm/gbsa methods in recognizing the native structure of the bcl-2 family using the interaction entropy method a pneumonia outbreak associated with a new coronavirus of probable bat origin the authors would like to thank the bibliotheca alexandrina, alexandria, egypt for providing access to the high-performance computing cluster which was used for the molecular dynamics simulation part of the study, asrt grant #7301 for partial funding, and internal funding zc003-2019 from zewail city for science and technology. key: cord-321441-t1v0pu0w authors: yang, yiming; gaspard, gerard; mcmullen, nichole; duncan, roy title: polycistronic genome segment evolution and gain and loss of fast protein function during fusogenic orthoreovirus speciation date: 2020-06-29 journal: viruses doi: 10.3390/v12070702 sha: doc_id: 321441 cord_uid: t1v0pu0w the reoviridae family is the only non-enveloped virus family with members that use syncytium formation to promote cell–cell virus transmission. syncytiogenesis is mediated by a fusion-associated small transmembrane (fast) protein, a novel family of viral membrane fusion proteins. previous evidence suggested the fusogenic reoviruses arose from an ancestral non-fusogenic virus, with the preponderance of fusogenic species suggesting positive evolutionary pressure to acquire and maintain the fusion phenotype. new phylogenetic analyses that included the atypical waterfowl subgroup of avian reoviruses and recently identified new orthoreovirus species indicate a more complex relationship between reovirus speciation and fusogenic capacity, with numerous predicted internal indels and 5’-terminal extensions driving the evolution of the orthoreovirus’ polycistronic genome segments and their encoded fast and fiber proteins. these inferred recombination events generated biand tricistronic genome segments with diverse gene constellations, they occurred preand post-orthoreovirus speciation, and they directly contributed to the evolution of the four extant orthoreovirus fast proteins by driving both the gain and loss of fusion capability. we further show that two distinct post-speciation genetic events led to the loss of fusion in the waterfowl isolates of avian reovirus, a recombination event that replaced the p10 fast protein with a heterologous, non-fusogenic protein and point substitutions in a conserved motif that destroyed the p10 assembly into multimeric fusion platforms. the fusogenic reoviruses are rare examples of non-enveloped viruses that induce cell-cell fusion and syncytium formation [1] . this diverse group of non-enveloped viruses, with segmented, dsrna genomes, are members of two distinct but related genera in the reoviridae family: the orthoreovirus genus, whose members infect a wide range of vertebrate hosts, and the aquareovirus genus, whose hosts are restricted to freshwater and saltwater fish [2] . three of the five recognized and sequenced species in the aquareovirus genus are fusogenic. of the seven currently recognized species of orthoreoviruses, only mammalian orthoreovirus (mrv) and piscine orthoreovirus (prv) lack isolates that are fusogenic. isolates of reptilian orthoreovirus (rrv), mahlapitsi orthoreovirus, (marv), nelson bay orthoreovirus (nbv), baboon orthoreovirus (brv), and avian orthoreovirus (arv) all induce syncytium formation [3, 4] . furthermore, three additional orthoreovirus species have been proposed, all of which are fusogenic: broome orthoreovirus (brrv) from a bat, neoavian orthoreovirus (arvn) from wild birds, and testudine orthoreovirus (rrvt) from a tortoise ( figure 1a ). the preponderance of fusogenic over orthoreoviruses are members of the spinareovirinae subfamily, whose virions have large turrets at the icosahedral vertices. the double-layered capsid contains eight structural proteins, five of which comprise the transcriptionally active core particle containing the viral rna-dependent rna polymerase (rdrp) [5, 6] . two of the three remaining proteins, the outer clamp and outer shell proteins, assemble into heterohexameric complexes and form the outer capsid shell [7] . during reovirus endocytic cell entry, cathepsins proteolytically degrade the outer clamp protein to expose the underlying outer shell protein that mediates the exit of the core particle from the endocytic pathway into the cytoplasm [8] . the rdrp functions from within the core particle to transcribe mostly table s1 for a list of viruses, proteins, and accession numbers. lowercase labels denote isolates within a species: gal and ans are landfowl and waterfowl isolates of avian orthoreovirus (arv); bu and co are bulbul and corvid isolates of neoavian orthoreovirus (arvn). bootstrap values (500 replicates) are shown for branch points where the associated taxa clustered together in less than 100% of the replicate trees. the tree with the highest log likelihood is shown. branch lengths are to scale and measured by the number of substitutions per site. (b) diagrams of the polycistronic s1 and s4 genome segments from the indicated orthoreovirus species, drawn to approximate scale. open reading frames are depicted by rectangles, labeled to indicate the protein product and color-coded to depict functional or evolutionary protein relationships: yellow-fast protein; blue-fiber protein; green-potential cell cycle inhibitory protein; purple-homologous proteins with no defined function; pink-nucleocytoplasmic shuttling protein. numbers refer to mrna length. (c) diagrams of the orthoreovirus fast proteins, depicting their topology in the plasma membrane. structural motifs contained within their n-terminal ectodomains and c-terminal cytoplasmic endodomains are color-coded as described in the legend at the bottom. numbers indicate the number of residues in each protein. orthoreoviruses are members of the spinareovirinae subfamily, whose virions have large turrets at the icosahedral vertices. the double-layered capsid contains eight structural proteins, five of which comprise the transcriptionally active core particle containing the viral rna-dependent rna polymerase (rdrp) [5, 6] . two of the three remaining proteins, the outer clamp and outer shell proteins, assemble into heterohexameric complexes and form the outer capsid shell [7] . during reovirus endocytic cell entry, cathepsins proteolytically degrade the outer clamp protein to expose the underlying outer shell protein that mediates the exit of the core particle from the endocytic pathway into the cytoplasm [8] . the rdrp functions from within the core particle to transcribe mostly monocistronic mrnas from the 10 genome segments that are capped but not polyadenylated by the turret protein during the exit from the core particle [9, 10] . at some stage, interactions between the viral rdrp, viruses 2020, 12, 702 3 of 20 packaging signals in the viral mrna, and the core shell protein result in the coordinated replication of the mrna and the encapsidation of the 10 dsrna genome segments inside the progeny virus core particles [11, 12] . these transcriptionally active progeny core particles drive mrna synthesis and amplification of the virus replication cycle. the three outer capsid proteins eventually assemble on the core particles to generate infectious progeny virions. during the progeny core assembly stage of the replication cycle in cells which have been co-infected with two different reoviruses, heterologous viral mrnas can be encapsidated and replicated inside a single progeny core particle to generate reassortant viruses. the genomes of these reassortants are mosaics of the parental virus genome segments and, as with antigenic shift in influenza viruses [13] , genome segment reassortment is a major driver of reovirus evolution [14] . the final structural protein is the fiber protein, referred to as σ1 in mrv and σc in the fusogenic reoviruses, that forms trimeric spikes at the capsid vertices and serves as the cell adhesin [15] [16] [17] . the reovirus fiber protein comprises a long n-terminal filamentous tail and an~140-residue c-terminal globular head that folds into a compact, eight-stranded b-barrel structure with receptor binding activity [18, 19] . the filamentous tail contains three structurally distinct subregions: the n-terminal 20-30 residues interact with the turret protein to anchor the spike to the virion capsid; a long α-helical coiled coil domain formed by heptad repeats zips together the trimeric fiber; and a region of triple β-spiral repeats connects the tail to the head [20] [21] [22] . in those fusogenic reoviruses that encode a fiber ( figure 1b) , the σc fiber is shorter than the mrv σ1 fibers (~320 versus 455-470 residues, respectively), due primarily to a reduced number of β-spiral repeats [19] . in some reovirus species (marv, brv, brrv), the fiber protein orf has been completely replaced by a heterologous sequence encoding a non-structural p16 protein of no defined function ( figure 1b ). it is unclear how these fiberless virus particles attach to cell receptors. the genomes of all fusogenic aqua-and orthoreoviruses contain an additional open reading frame (orf) which is not found in their non-fusogenic counterparts. this orf is present at the 5'end of a bi-or tricistronic mrna and encodes the fusion-associated small transmembrane (fast) protein responsible for cell-cell fusion and syncytium formation ( figure 1b ) [1] . fast proteins are a unique family of viral membrane fusion proteins that are structurally and evolutionarily unrelated to the fusion proteins that are responsible for enveloped virus entry into cells. these "accessory" proteins are non-essential for productive virus infection and they are non-structural proteins, hence they are not involved in cell entry. fast proteins are small, integral membrane proteins expressed inside reovirus-infected cells where they localize at adherens junctions and mediate cell-cell, not virus-cell, membrane fusion [23] . in so doing, fast proteins promote the rapid cell-cell transmission of the infection, a possible explanation for the positive selection pressure on acquiring or maintaining a fast protein. natural fusogenic reovirus infections are frequently associated with overt disease. arv is responsible for outbreaks of enteritis, tenosynovitis, and myocarditis in commercial poultry flocks [24] , rrv is associated with pneumonia and neurological dysfunctions in snakes [25, 26] , brv causes meningoencephalomyelitis in nonhuman primates [27, 28] , and nbv has been associated with acute respiratory infections in humans. studies in different experimental systems link fast proteins to the pathogenic potential of these viruses [29] [30] [31] [32] . there are currently six members of the aqua-and orthoreovirus fast protein family that share limited sequence conservation but share several hallmark biochemical features [1, 33] . the remainder of this discussion will focus on the orthoreovirus fast proteins ( figure 1c ). all fast proteins are small (~100-200 residues), acylated (shown or predicted to be palmitoylated or myristoylated), single-pass membrane proteins that position a very small (<40 residues) n-terminal ectodomain external to the plasma membrane and an extended (~40-140 residues) c-terminal endodomain in the cytoplasm [34] [35] [36] . fast proteins appear to have evolved via the assembly of diverse membrane interaction motifs into three small domains (ecto-, endo-, and transmembrane) that function as fusion modules. the ectodomains are amphiphilic, structurally dynamic peptides, with remarkably diverse structures, that function as fusion peptides to destabilize lipid bilayers [3, 29, [37] [38] [39] . fast protein tm viruses 2020, 12, 702 4 of 20 domains contain different motifs, adjacent to the ectodomain-transmembrane domain boundary, that are required for cell-cell fusion (e.g., polyglycine in p10, polyserine and hydrophobic β-branched residues in p15) [36, 40, 41] . the fast protein endodomains all contain a juxtamembrane cluster of basic residues and an adjacent amphipathic helix that are essential for syncytium formation. the polybasic motif functions as a golgi export signal for the rrv p14 fast protein [42, 43] , while the amphipathic helices in the p14 and brv p15 fast proteins partition into highly curved membranes to promote pore formation [44] . the c-terminal half of the fast protein endodomains are predicted to be disordered [45] , a common feature of protein regions that interact with multiple partners; annexin a1 is one such p14 partner [46] . lastly, fast proteins are modular fusogens, meaning that the exchange of modules between different fast proteins can generate functional recombinants, although not all combinations are tolerated. the noteworthy mechanistic implication of the above discussion is that specific combinations of different structural motifs can be assembled in order to generate a functional fast protein [3, 40] . the modular nature of fast proteins and the diversity of the functional motifs that they employ to drive membrane fusion raises interesting questions regarding the evolution of these non-enveloped virus fusogens. previous phylogenetic studies that included the six available sequenced species of orthoreoviruses suggested two separate gain-of-fusion events caused by a common ancestral, non-fusogenic reovirus led to the extant fusogenic aqua-and orthoreoviruses [2] . the evolutionary relationship between the orthoreovirus fast proteins was unclear. these studies also overlooked the waterfowl subgroup of arv typified by the muscovy duck reoviruses (mdrv). the mdrv infection of young ducklings results in high morbidity and mortality (10%-50% mortality), with necrotic focus formation in the liver, spleen, and kidneys [47] . early isolates of mdrv were reported to be syncytiogenic like their landfowl counterparts [47] , but no sequence information is available for these isolates. all the more recent mdrv isolates lack syncytium-inducing capabilities [48, 49] . mdrvs are divided into two subgroups, the "classical" and "novel" mdrvs. classical muscovy duck reoviruses (mdrvc) possess a bicistronic s4 genome segment encoding a truncated fiber protein of 269 residues and a p11 protein that lacks sequence similarity to the p10 fast proteins of arv, arvn, and nbv ( figure 1b ) [50] . the more recently identified novel subgroup (mdrvn) has a tricistronic s1 genome segment similar to other arvs [48] , encoding a p10 fast protein homologue, a p18 protein of unknown function, and a 326-residue fiber protein ( figure 1b ). there has been little discussion in the literature regarding the loss of fusion activity in mdrvs or the genetic events underlying the evolution of the orthoreovirus polycistronic genome segments. as we now show, using phylogenetic analysis that includes the current seven orthoreovirus species, three new proposed species, and previously excluded non-fusogenic mdrvs, the evolution of the orthoreovirus polycistronic genome segment from an inferred ancestral, monocistronic fiber-encoding genome segment involved polymorphic, species-specific indels at a "genetic hotspot" in the fiber orf. these genome segments further evolved by the addition of 5'-terminal extensions that added additional orfs upstream of the fiber orf, one of which encoded a fast protein homologue. evidence suggests these 5'-terminal recombination events occurred multiple times, both pre-and post-speciation, to generate the constellation of extant orthoreovirus polycistronic genome segments and fast proteins. we further directly demonstrate that point substitutions in a nine-residue multimerization motif are solely responsible for the loss of fusion capability in mdrvn isolates. these results highlight the underappreciated role of recombination, not just genome segment reassortment, in reovirus evolution, and support the concept that fast proteins evolved from small ancestral membrane proteins by independently acquiring the correct assembly of diverse membrane interaction motifs needed to generate a fusogenic fast protein. qm5 cells were maintained in medium 199 with earle's salts containing 100 u/ml of penicillin and streptomycin and 10% heat-inactivated fetal bovine serum. monoclonal mouse anti-flag (sigma-aldrich) and rabbit anti-myc (sigma-aldrich) antibodies, horseradish peroxidase (hrp)-conjugated goat anti-rabbit (santa cruz) and goat-anti mouse (santa cruz) antibodies, alexa fluor 488-conjugated goat anti-mouse (invitrogen) and alexa fluor 555-conjugated goat anti-rabbit (invitrogen), sulfo-nhs-lc-biotin (thermo scientific), maliemide-peg2-biotin (thermo scientific), neutravidin agarose resin (thermo scientific), and polyethylimine (pei, polysciences inc.) were purchased from the indicated commercial sources. arv p10, mdrv p10, and md-cm (mdrv p10 containing the 9-residue conserved motif of arv p10 created using sequential pcr reactions with custom oligonucleotide primers) were subcloned into pcdna3 mammalian expression vectors between the hindiii and ecori sites. a triple flag or single myc tag was added to the n-terminus, or egfp or mcherry were added to the c-terminus of the indicated p10 constructs by pcr amplification and cloning. custom oligonucleotide primers were purchased from integrated dna technologies (idt), and all constructs were confirmed by sequencing. qm5 cell monolayers at 50% confluency in 12-well plates were transfected with 0.5 µg of plasmid dna using pei as per the manufacturer's instructions. at 4 h post-transfection, the transfection mix was replaced with medium 199 (gibco), supplemented with 10% fetal bovine serum (sigma-aldrich). at the indicated times post-transfection, cells were fixed with methanol and wright-giemsa stained (siemens healthcare diagnostics), and the stained monolayers were imaged using a nikon diaphot microscope at 100× magnification. the numbers of syncytial nuclei were counted in five random fields of triplicate wells (n = 3 independent experiments) and the syncytial index reported as the mean ± sem. sds-page and western blotting were carried out as previously described [17, 49] . a 1:1000 dilution of primary anti-flag or anti-myc antibodies, followed by a 1:10,000 dilution of hrp-conjugated secondary antibody was used to measure overall protein expression levels. western blots were developed using clarity western ecl substrate (bio-rad) and imaged on a chemidoc imaging system (bio-rad). qm5 cells grown to approximately 50% confluence on coverslips were transfected with flag-or myc-tagged versions of arv or mdrv p10 constructs, as described above. at 24 h post-transfection, cells were fixed with 3.7% paraformaldehyde for 10 min at room temperature, blocked with 5% bovine serum albumin (bsa) in phosphate buffered saline (pbs) for 1 h, and then incubated with 1:200 dilutions of mouse anti-flag and rabbit anti-myc monoclonal antibodies overnight at 4 • c. cells were thoroughly washed with pbs and then incubated with 1:1000 dilutions of alexa 488-conjugated goat anti-mouse antibody and alexa 555-conjugated goat anti-rabbit antibody for 1 h at room temperature. coverslips were then mounted using prolong gold anti-fade reagent (invitrogen) and imaged using a zeiss lsm510 axiovert 200m confocal microscope. qm5 cells grown in 10 cm dishes (corning) to 50% confluency were transfected with the indicated flag-tagged constructs. at 24 h post-transfection, cells were washed three times with ice-cold pbs (ph 8.0), then incubated with shaking in 1 mg/ml sulfo-nhs-lc-biotin (a membrane impermeable biotinylation reagent) for 2 h at 4 • c to biotinylate primary amino groups on the cell surface. cells were washed with pbs containing 100 mm glycine to quench and remove excess biotin reagent. cells were washed twice with pbs, then lysed in a ripa buffer (10 mm tris ph 8.0, 150 mm nacl, 1 mm edta, 1% np40, 0.5% na desoxycholate) with protease inhibitors (pierce). cell lysates were incubated overnight with neutravidin agarose resin to pull down the biotinylated proteins, and pellets were boiled in a protein sample buffer with 100 mm dithiothreitol (dtt) to release the biotinylated proteins. samples were then analyzed by sds-page and western blotting. to detect the presence of an intramolecular disulfide bond in the p10 ectodomain, qm5 cells transfected with myc-tagged p10 constructs were washed twice with hanks buffered saline solution (hbss) at 24 h post-transfection and then incubated in hbss with or without 0.1 mm dtt for five min. cells were washed three times with hbss, then incubated with shaking in 1 mg/ml maliemide-peg2-biotin (a membrane impermeable biotinylation reagent) for 25 min at 4 • c to biotinylate free thiol groups on the cell surface. cells were washed four times with hbss to remove excess biotin reagent and once with hbss containing 1% bsa to quench residual biotinylation reagent. cells were washed twice with pbs, resuspended with 50 mm edta in pbs, and lysed in the ripa buffer with protease inhibitors, and the lysate was incubated overnight with neutravidin agarose resin to pull down biotinylated proteins. pellets were boiled in a protein sample buffer with 100 mm dtt to release biotinylated proteins and analyzed by sds-page and western blotting. a zeiss lsm510 confocal microscope was used in wide field mode to detect sensitized emission fluorescence resonance energy transfer (fret) from qm5 cells transfected with egfp-and/or mcherry-tagged versions of the indicated p10 constructs, as previously described [26, 51] . briefly, donor and acceptor spectral bleed-through (sbt) values were minimized using cells expressing egfp or mcherry alone, and donor and acceptor sbt ratios were modeled using exponential relationships with fluorophore intensity, after the exclusion of aberrant background values at low intensities and the application of a gaussian blur. using this microscope setup and the sbt values, three images were acquired from each cell that was imaged: (1) a donor image (donor excitation, donor emission), (2) an acceptor image (acceptor excitation, acceptor emission), and (3) a sensitized emission fret image (donor excitation, acceptor emission). the background subtraction and gaussian blur of the donor, acceptor, and fret channels were performed on each image stack prior to analysis. ten images were acquired for each sample from each of the two independent experiments (total of twenty images per sample). the pixfret image j plugin [46, 52, 53] was used to normalize the fret intensity of each pixel to the donor and acceptor expression levels by dividing the fret-channel pixel intensity by the square-root of the product of the corresponding donor-and acceptor-channel pixels, using the following equation: the gaussian distribution with the highest calculated r 2 value for the pixel amplitude distributions generated by pixfret from the 8-bit normalized fret (nfret) images was used to calculate the mean nfret (mnfret) for each image. phylogenetic analysis used clustal muscle to generate multiple sequence alignments of homologous proteins from the different orthoreovirus species that were analyzed for evolutionary relationships, using mega 7.0 and the maximum likelihood method based on the jones-taylor-thornton (jtt) matrix-based model [54, 55] . these methods were applied to concatenated protein sequences for the seven homologous orthoreovirus structural proteins from either the 10 orthoreovirus species or the proposed species (excluding the highly variable fiber protein), to the sigma-class outer capsid clamp proteins of either the eight fusogenic orthoreovirus species or the proposed species, and to the fast proteins encoded by these eight virus species. accession numbers are provided in the figures or figure legends and in the supplementary tables. phylogenetic analysis revealed species-specific features of the different orthoreovirus fiber proteins and the polycistronic genome segments encoding these proteins. the orthoreovirus fiber proteins share the same overall trimeric structure but with considerable variation in their lengths, ranging from 269 to 470 residues ( figure 2a ). all orthoreovirus fiber-encoding genome segments are polycistronic except the prv s4 genome segment. the prv genome segment is~400 nucleotides shorter than the corresponding s1 genome segment of mrv due to an internal indel of~400-550 nucleotides within the region of the fiber orf where the coiled-coil tail joins the β-spiral repeats ( figure 2a) . consequently, prv contains 17-18 predicted heptad repeats in its coiled coil tail and only two β-spiral repeats while mrv fibers contain 20-21.5 heptads and seven β-spiral repeats [18, 19, 21, 56] . viral polymerase errors frequently generate substitutions, but indels also occur at about a four-fold lower frequency [57] . excluding mrv, all other orthoreovirus species, including those whose hosts are more ancient (i.e., fish, reptiles, and birds), contain homologous short versions of the trimeric fiber. the logical inference is the ancestral fiber genome segment resembled that of prv, encoding a short fiber on a monocistronic mrna. the fiber orf indels are polymorphic but species-specific in their length, implying indels altered the fiber protein several times during orthoreovirus speciation. the extant fiber proteins cluster into four groups based on their total lengths and the approximate number of heptad and β-spiral repeats: the prv/arv/mdrvn/arvn/nbv fiber group (17-18 predicted heptad and two predicted β-spiral repeats), the mdrvc fiber protein (10 predicted heptad and two predicted β-spiral repeats), the rrv/rrvt clade (19 predicted heptad and three potential β-spiral repeats), and the mrv fibers (20-21.5 heptads and seven β-spiral repeats) ( figure 2a ) [49, 50] . three error-prone polymerase events, giving rise to indels with variable lengths, primarily during the synthesis of the β-spiral repeat region, would explain the evolution of the prv/arv/mdrvn/arvn/nbv fiber protein into the extant mrv, rrv/rrvt, and mdrvc fiber proteins ( figure 2b ). it would appear that this structural motif is highly tolerant to changes in the number of repeats without losing protein function, namely the cell attachment capacity. as occurs with recombination events in many rna viruses [58] , undefined rna secondary structures in this region might also play a role in making this region a hotspot for such genetic events. sequence analysis suggests these fiber orf indels continue to occur, as evidenced by an additional 18-residue indel detected in the fiber orf of a recent mdrvn isolate that creates a 303-residue mdrvn fiber protein lacking an additional β-spiral repeat [59] . figure 2a) . consequently, prv contains 17-18 predicted heptad repeats in its coiled coil tail and only two β-spiral repeats while mrv fibers contain 20-21.5 heptads and seven β-spiral repeats [18, 19, 21, 56] . viral polymerase errors frequently generate substitutions, but indels also occur at about a four-fold lower frequency [57] . excluding mrv, all other orthoreovirus species, including those whose hosts are more ancient (i.e., fish, reptiles, and birds), contain homologous short versions of the trimeric fiber. the logical inference is the ancestral fiber genome segment resembled that of prv, encoding a short fiber on a monocistronic mrna. the fiber orf indels are one major contributor to the variable lengths of the different polycistronic genome segments. the second is what can only be inferred are 5'-terminal extensions, assuming that the last common ancestor of the extant fiber-encoding genome segments resembled that of prv. these~400-700 nucleotide extensions in the fusogenic orthoreoviruses added additional reading frames upstream of the fiber orf, one of which encoded a fast protein homologue (figure 2a ). template-switching as the rdrp replicates the plus-strand into a minus-strand would generate such genetic hybrids with 5'-extensions, as previously reported in plant viruses [60] . this copy choice mechanism of recombination is common in many plant viruses and in plus-strand rna animal viruses including retroviruses, enteroviruses, and coronaviruses, and may simply reflect the stochastic association and dissociation of the rdrp from the rna template [58, 61] . rna recombination usually viruses 2020, 12, 702 9 of 20 occurs between regions of high sequence similarity. such homologous recombination between cognate genome segments has been noted in arvs, where as many as twelve recombination events between multiple cognate genome segments of different circulating virus strains have been predicted [62] . however, non-homologous recombination can also occur, with genetic exchange taking place between different virus genomes [63] or, more rarely, between viral and cellular rna [64] [65] [66] . the high degree of sequence divergence in the polycistronic orthoreovirus mrnas and the likelihood that ancient, non-homologous recombination events generated these 5'-terminal extensions confounds similarity searches to define specific breakpoints or even the potential source of the alternate template rna. however, additional analyses discussed below support the concept that such genetic events occurred more than once and were major evolutionary drivers of the fusogenic orthoreovirus polycistronic genome segments and the fast proteins that they encode. the distinct features of the p10 fast proteins and their tricistronic genome segments compared to the other orthoreovirus fast proteins and their bicistronic genome segments imply that at least two distinct recombination events occurred during the fusogenic reovirus evolution. despite their distinct host ranges (i.e., birds and bats), arv, arvn, and nbv form a monophyletic clade ( figure 3a ) and they share a tricistronic gene arrangement comprising three sequential, partially overlapping orfs encoding a p10 fast protein, a non-structural p17 protein, and the σc fiber protein ( figure 3b ). this includes the mdrvn, but not the mdrvc, subgroup of waterfowl arv isolates; the latter have a bicistronic gene arrangement and are further discussed below. the arv, arvn, and nbv p10 fast proteins also form a monophyletic clade that is distinct from the p13, p14, and p15 fast proteins, with a 94% bootstrap value at that branchpoint ( figure 3c ). in multiple sequence alignments, the p10 fast proteins of the three species (arv, arvn, and nbv) are essentially colinear (two small, terminal indels) and share~38%-65% amino acid identity in pairwise comparisons ( figure 4a ). this is not the case for alignments of p10 with the other fast proteins, where~20 indels are needed to attain amino acid identities of only~13%-23% (data not shown). the p10 proteins also contain several conserved features which are not found in the other orthoreovirus fast proteins-an unusual ectodomain cystine noose fusion peptide, a 9-residue ectodomain conserved motif, a palmitoylated endodomain di-cysteine motif instead of n-terminal myristoylation, and a short cytoplasmic tail ( figure 1c ) [36, 51, 52, 67] . based on the above considerations, we infer that the predicted 5'-extension that led to the evolution of the p10 fast proteins occurred after the rrv/brrv/brv/marv and arv/nbv orthoreovirus clades diverged from each other ( figure 3a ). in addition to p10, the arv/arvn/nbv clade acquired a second orf upstream of the fiber orf encoding a p17/p18 protein. the arv p17 protein is a nucleocytoplasmic shuttling protein that suppresses the cell cycle ( [68, 69] , but similar attributes have not been ascribed to other reovirus p17 proteins or to the mdrvn p18 protein. alignments of the p17 proteins from the nbv, arvn, and landfowl isolates of arv are mostly colinear ( figure 4b ), and in pairwise comparisons of the more distantly related species these proteins still share~23%-33% amino acid identity which is suggestive of a homologous relationship. however, this degree of amino acid conservation is considerably lower than that between the nbv and arv/arvn p10 proteins (~38%-65% identity). the p18 protein of mdrvn shares even less similarity with p17; protein alignments require 7-8 internal indels, totaling >100 nucleotides, in order to generate only~12%-20% amino acid identity (data not shown). it is therefore unclear whether one recombination event simultaneously added precursors of the p10/p17/p18 orfs that then evolved at very different rates or independent recombination events added these orfs separately ( figure 3a ). p10 proteins also contain several conserved features which are not found in the other orthoreovirus fast proteins-an unusual ectodomain cystine noose fusion peptide, a 9-residue ectodomain conserved motif, a palmitoylated endodomain di-cysteine motif instead of n-terminal myristoylation, and a short cytoplasmic tail ( figure 1c) [36, 51, 52, 67] . based on the above considerations, we infer that the predicted 5'-extension that led to the evolution of the p10 fast proteins occurred after the rrv/brrv/brv/marv and arv/nbv orthoreovirus clades diverged from each other ( figure 3a) . the brrv, marv, rrv, rrvt, and brv virus species form a monophyletic clade, as do their encoded p13, p14, and p15 fast proteins ( figure 3a-c) . these three diverse fast proteins are encoded by the first orfs of bicistronic genome segments that have two distinct gene arrangements. all reptilian orthoreovirus isolates, including the tortoise isolate of the new proposed rrvt species, encode homologous p14 fast proteins using the first orf and retain a truncated σc fiber protein orf ( figure 3b) . brv, brrv, and marv encode p13, p14, or p15 fast proteins, but their fiber orfs have been replaced by an unrelated orf encoding a p16 protein of no defined function. aside from terminal indels to accommodate length differences, these p16 proteins are mostly colinear (four single residue indels) and share 28%-30% amino acid identity clustered in subregions (figure 4c ), indicating a shared common ancestor. brv, marv, and brrv form a monophyletic clade when comparing their sigma-class outer clamp capsid proteins ( figure 3a ) or lambda-class core shell proteins [70] , consistent with a single recombination event replacing the fiber orf with the p16 orf prior to speciation of these viruses. since the three viruses are paraphyletic when comparing other homologous proteins ( figure 1a) , lateral gene transfer via genome segment reassortment between these evolving virus species presumably occurred after the p16 recombination event. recombination events and genome segment reassortment are well-linked to intra-species arv evolution, with some suggestion that similar inter-species genetic events may have occurred between orthoreovirus species [62] , possibly early post-speciation. a single recombination event that extended the 5'-end of the fiber orf with an orf encoding a shared ancestral protein could explain the evolution of the brrv, marv, rrv, rrvt, and brv p13, p14, and p15 fast proteins ( figure 3a ). evidence does support the divergent evolution of the brrv p13 and reptilian reovirus p14 fast proteins from a shared, fusogenic ancestor. phylogenetic trees based on fast protein sequences have weak bootstrap values at branchpoints separating the single isolates of brv p15, marv p14, and brrv p13, making evolutionary relationships unclear ( figure 3c ). however, the p14 fast proteins of rrv, rrvt, and marv are clear homologues. these proteins share conserved sequence motifs, including a conserved n-terminal decapeptide that is sensitive to substitution and contains the n-terminal myristoylation consensus sequence ( figure 4d ) [35] , and they share~27%-36% amino acid identity. brrv p13 fast protein possesses the same motifs as p14, including the n-terminal decapeptide but excluding a cytoplasmic ppxy motif of no defined function ( figure 4d ), and in mostly colinear, pairwise sequence alignments it shares~22%-29% sequence identity with marv and rrv p14. the hypothesis that the p13 and p14 fast proteins are homologues is consistent with all phylograms that show a monophyletic relationship between brrv, marv, rrv, and rrvt and their encoded p13 and p14 fast proteins ( figures 1a and 3a) . as indicated by the fast protein and concatenated sequence phylograms, brv and the p15 fast protein present as outliers. pairwise alignments of p15 with p14 or p13 require 7-8 large indels, totaling 50-80 residues, to generate~20%-24% sequence identity. a similar level of sequence conservation (~23% amino acid identity in alignments containing four large indels) is observed between brv p15 and a recently identified p11 fast protein (referred to as nsp1-1, the first orf on a bicistronic mrna) of rotavirus species b (rvb), a virus from a distantly related genus and different subfamily of reoviridae [71] . the brv p15 fast protein also contains several features not associated with p13 or p14; p15 lacks the conserved n-terminal decapeptide sequence present in p13 and p14, but it does contain a functional gxxxs myristoylation site [34, 72] . the~20-residue ectodomain is markedly truncated and contains a unique polyproline motif that forms a left-handed polyproline type ii helix required for fusion activity [38] , and the p15 endodomain is two to three times longer than the c-terminal cytoplasmic tails of the other orthoreovirus fast proteins ( figure 4d ). the absence of significant sequence conservation and dramatically different structural features of p15 relative to p13/p14, coupled with the more distant evolutionary relationships between the parental viruses encoding these proteins, suggests p15 may have arisen by an independent gain-of-fusion event ( figure 3a ). additional sequenced isolates of these interrelated orthoreoviruses are needed in order to more robustly test this hypothesis. the concept of independent gain-of fusion regarding p13/p14/p15 is not intended to exclude the possibility of recombination leading to acquisition of a shared non-fusogenic ancestor followed by an independent, post-speciation gain-of-fusion capability. for example, we previously speculated that fast proteins may have evolved from viroporins or other small, non-structural, non-fusogenic viral proteins that contain diverse membrane remodeling motifs (e.g., transmembrane domains, polybasic motifs, amphipathic helices) [2, 73, 74] . in the above discussion, a single recombination event could have added an orf encoding a non-fusogenic, viroporin-like fast protein precursor to the ancestral fiber-encoding genome segment and this precursor independently acquired fusion capability and evolved to become the p13/p14 and p15 fast proteins. while there have been reports of fusogenic mdrv isolates [47] , there are no recent reports of sequenced isolates that induce syncytium formation. as noted previously [75, 76] , the mdrvc p11 protein is not a fast protein homologue, implying that the loss of mdrvc fusion capacity reflects a post-speciation recombination event that replaced the arv p10 fast protein orf with a heterologous orf ( figure 3a ). this is not the case for mdrvn, which clearly encodes a p10 fast protein homologue, sharing the primary sequence conservation and almost all the hallmark features of a p10 fast protein. the exception is a nine-residue ectodomain conserved motif (cm; aggdlqats) present in all other arv and nbv p10 sequences ( figure 5a ). the mdrvn isolates have two sequence variants of the cm with two to three of the first four residues matching the consensus sequence (ahgnidsyt and asgdidsyt: boldface indicates sequence conservation with consensus motif; lowercase indicates mdrv sequence variants). the residues flanking the cm are also conserved between arv and mdrv p10 proteins ( figure 5a ). it seems most likely that a point substitution occurred in the cm after the classical and novel mdrvs diverged, destroying p10 function and allowing the remaining sequence to rapidly devolve from the consensus. whatever selective pressure there might be to acquire and maintain a fast protein, it did not prevent two distinct genetic events that resulted in the loss of fusion capability in mdrvs. the fact that mdrvn has retained almost all the hallmark fast protein features but is non-functional for syncytium formation suggests the loss of fusion capacity may have been a recent genetic event, consistent with earlier reports of fusogenic mdrvs [47] . we further explored the hypothesis that the loss of the cm is solely responsible for the absence of mdrvn's syncytiogenesis capacity. to generate syncytia, p10 proteins need to traffic to the plasma membrane in the correct n out /c in membrane topology. transfected cells expressing n-terminal flag-tagged versions of arv and mdrvn p10 were treated with membrane impermeable sulfo-nhs-lc-biotin to label the externally exposed lysine sidechains of plasma membrane proteins, and the western blots of the cell lysates were probed with anti-flag or neutravidin. as shown ( figure 5c ), both proteins were expressed and detected on the cell surface at similar levels. functional p10 proteins also need to form an intramolecular disulfide bond between the two conserved ectodomain cysteine residues in order to generate an 11-residue cystine noose fusion peptide ( figure 5a) , and the formation of this structure is highly sensitive to substitutions within and adjacent to the noose [37, 51, 67] . a previously described assay applying a membrane-impermeable biotinylation reagent (maliemide-peg2-biotin) to biotinylate free thiol groups on the cell surface [51, 52] determined that mdrv p10 forms a cystine noose; plasma-membrane localized mdrv p10 was only labeled if cells were treated with dtt prior to the thiol-specific biotinylation reagent ( figure 5d ). these results confirm that the formation of the p10 intramolecular disulfide bond occurs independent of the cm [52] and that the null syncytiogenic phenotype of novel mdrvs is not due to aberrant protein expression, trafficking, or formation of the essential cystine noose fusion peptide structure. substitution occurred in the cm after the classical and novel mdrvs diverged, destroying p10 function and allowing the remaining sequence to rapidly devolve from the consensus. whatever selective pressure there might be to acquire and maintain a fast protein, it did not prevent two distinct genetic events that resulted in the loss of fusion capability in mdrvs. the fact that mdrvn has retained almost all the hallmark fast protein features but is non-functional for syncytium formation suggests the loss of fusion capacity may have been a recent genetic event, consistent with earlier reports of fusogenic mdrvs [47] . in arv and nbv p10 proteins, the cm governs the cholesterol-dependent, reversible assembly of p10 multimeric fusion platforms in the plasma membrane [52] . single, conservative point substitutions in this motif, which presumably provides a p10 multimerization binding interface, ablated multimer formation. to determine whether this motif serves the same function in mdrv p10, and whether the absence of this motif is solely responsible for the cell-cell fusion defect in novel mdrvs, we constructed a recombinant mdrv p10 (md-cm) containing the nine-residue conserved motif (i.e., ahgnidsyt was converted to aggdlqats) ( figure 5a) . a time-course analysis of the syncytium formation revealed that the replacement of the cm restored mdrv syncytiogenesis to robust levels with only slightly reduced kinetics relative to arv p10 ( figure 6a,b) . furthermore, cell-surface immunofluorescence microscopy, using cells co-transfected with n-terminally flag-tagged and n-terminally myc-tagged p10 constructs, indicated that mdrv p10 failed to form the dual-color cell surface puncta that are typical of arv p10 fusion platforms, but replacement of the cm in md-cm p10 resulted in the extensive punctate colocalization of recombinant p10 proteins ( figure 6c ). to confirm the punctate staining pattern of the md-cm p10 reflected assembly of multimeric fusion platforms, we employed in cellula fret, as previously described [52, 77] . briefly, arv, mdrv, and md-cm p10 proteins were c-terminally tagged with either egfp or mcherry. donor and acceptor spectral bleed-through (sbt) values and normalized fret (nfret) intensities were calculated using the pixfret image-j plug-in and pixel-by-pixel analysis of sensitized emission fret [53] , and mean nfret (mnfret) values were determined for 10 cells in each of the two separate experiments. as shown in the fret images ( figure 7a ) and by mnfret quantification (figure 7b ), arvp10 formed the expected homomultimers in cells but mdrvp10 failed to multimerize. replacement of the cm in mdrv p10 generated positive fret signals equivalent to those observed with arv p10, indicating that point substitutions leading to the loss of a multimerization motif required for the fusion platform assembly destroyed the ability of novel mdrvs to induce syncytium formation. to confirm the punctate staining pattern of the md-cm p10 reflected assembly of multimeric fusion platforms, we employed in cellula fret, as previously described [52, 77] . briefly, arv, mdrv, and md-cm p10 proteins were c-terminally tagged with either egfp or mcherry. donor and acceptor spectral bleed-through (sbt) values and normalized fret (nfret) intensities were calculated using the pixfret image-j plug-in and pixel-by-pixel analysis of sensitized emission fret [53] , and mean nfret (mnfret) values were determined for 10 cells in each of the two separate experiments. as shown in the fret images ( figure 7a ) and by mnfret quantification (figure 7b ), arvp10 formed the expected homomultimers in cells but mdrvp10 failed to multimerize. replacement of the cm in mdrv p10 generated positive fret signals equivalent to those observed with arv p10, indicating that point substitutions leading to the loss of a multimerization motif required for the fusion platform assembly destroyed the ability of novel mdrvs to induce syncytium formation. viruses 2020, 12, x for peer review 16 of 20 current analyses support the concept that multiple recombination events were primary evolutionary drivers of the constellation of polycistronic orthoreovirus genome segments and directly contributed to the diversity of their encoded fiber and fast proteins. the four "classes" of extant orthoreovirus fiber proteins most likely arose via three polymorphic internal recombination events in a genetically tractable region of the fiber orf that encodes a structurally malleable repeat motif ( figure 2b ). genome segment reassortment may have influenced early inter-species evolution and the precise order of events, but nonhomologous copy choice recombination events are the most plausible explanation for the addition of 5'-extensions that generated fast protein-encoding polycistronic genome segments. one such event led to the tricistronic, p10-encoding arv/arvn/nbv clade, with a subsequent post-speciation recombination event leading to the heterologous replacement of the p10 fast protein orf and the loss of fusion capacity by mdrvc ( figure 3a) . a second independent genetic exchange led to the p14-encoding rrv/rrvt/marv clade and likely the related p13-encoding brrv. there is some evidence to suggest that brv may have acquired the p15 precursor from a third independent recombination event. at some point, a 3'proximal genetic exchange generated the brrv/marv/brv bicistronic s4 genome segments that have the entire fiber orf replaced with a heterologous p16 orf of no known function. lastly, we show that the extant mdrvs lost fusion capacity via two independent post-speciation genetic events: the recombinant loss of the entire p10 fast protein orf by mdrvc and point substitutions in the mdrvn cm that ablated multimeric fusion platform assembly. together, these results highlight the genetic tractability of the orthoreovirus polycistronic genome segments and the underappreciated role of recombination in dsrna virus evolution and the evolution of the fast protein family. current analyses support the concept that multiple recombination events were primary evolutionary drivers of the constellation of polycistronic orthoreovirus genome segments and directly contributed to the diversity of their encoded fiber and fast proteins. the four "classes" of extant orthoreovirus fiber proteins most likely arose via three polymorphic internal recombination events in a genetically tractable region of the fiber orf that encodes a structurally malleable repeat motif ( figure 2b ). genome segment reassortment may have influenced early inter-species evolution and the precise order of events, but nonhomologous copy choice recombination events are the most plausible explanation for the addition of 5'-extensions that generated fast protein-encoding polycistronic genome segments. one such event led to the tricistronic, p10-encoding arv/arvn/nbv clade, with a subsequent post-speciation recombination event leading to the heterologous replacement of the p10 fast protein orf and the loss of fusion capacity by mdrvc ( figure 3a) . a second independent genetic exchange led to the p14-encoding rrv/rrvt/marv clade and likely the related p13-encoding brrv. there is some evidence to suggest that brv may have acquired the p15 precursor from a third independent recombination event. at some point, a 3'-proximal genetic exchange generated the brrv/marv/brv bicistronic s4 genome segments that have the entire fiber orf replaced with a heterologous p16 orf of no known function. lastly, we show that the extant mdrvs lost fusion capacity via two independent post-speciation genetic events: the recombinant loss of the entire p10 fast protein orf by mdrvc and point substitutions in the mdrvn cm that ablated multimeric fusion platform assembly. together, these results highlight the genetic tractability of the orthoreovirus polycistronic genome segments and the underappreciated role of recombination in dsrna virus evolution and the evolution of the fast protein family. reovirus fast proteins: virus-encoded cellular fusogens bioinformatics of recent aqua-and orthoreovirus isolates from fish: evolutionary gain or loss of fast and fiber proteins and taxonomic implications 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declare that they have no conflicts of interest with the contents of this article. key: cord-338468-c0jv3i1t authors: kanduc, darja title: from anti-sars-cov-2 immune responses to covid-19 via molecular mimicry date: 2020-07-16 journal: antibodies (basel) doi: 10.3390/antib9030033 sha: doc_id: 338468 cord_uid: c0jv3i1t aim: to define the autoimmune potential of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. methods: experimentally validated epitopes cataloged at the immune epitope database (iedb) and present in sars-cov-2 were analyzed for peptide sharing with the human proteome. results: immunoreactive epitopes present in sars-cov-2 were mostly composed of peptide sequences present in human proteins that—when altered, mutated, deficient or, however, improperly functioning—may associate with a wide range of disorders, from respiratory distress to multiple organ failure. conclusions: this study represents a starting point or hint for future scientific–clinical investigations and suggests a range of possible protein targets of autoimmunity in sars-cov-2 infection. from an experimental perspective, the results warrant the testing of patients’ sera for autoantibodies against these protein targets. clinically, the results warrant a stringent surveillance on the future pathologic sequelae of the current sars-cov-2 pandemic. it is well known that coronavirus (re)infections have a pathologic immune potential. indeed: • progressive immune-associated injury is a hallmark of sars infection [1] and, since 2003 [2] , the association with hyper-immune inflammation and systemic immunopathology in the sars-cov-infected host has been well illustrated [2] [3] [4] [5] ; • in 2008, it was reported [6] that prior immunization with sars-cov nucleocapsid protein n causes severe pneumonia in mice infected with sars-cov; • in 2012, immunization with sars coronavirus vaccines was found to lead to lung immunopathology on challenge with the sars virus [7] ; • in 2016, agrawal et al. [8] demonstrated that immunization with inactivated middle east respiratory syndrome (mers) coronavirus vaccine leads to lung immunopathology on challenge with live virus. moreover, the authors documented that immunopathology with sars-cov vaccines occurred for whole-virus vaccines, subunit vaccines, different inactivation methods, different preparation substrates, and with recombinant surface spike protein. finally, the authors also underscored that, even if studies with vector vaccines point to the nucleoprotein n as responsible for the immunopathological effects and indicate that the s protein might be free of risk, nonetheless, also recombinant spike protein induced the immunopathology [6] [7] [8] [9] . however, the molecular and cellular basis for how sars-covs might impact on the host immune system resulting in dysfunctional immune responses, severe morbidity, and mortality remain obscure [1, 10] . following the current sars-cov-2 pandemic, cross-reactivity was proposed as a mechanism that might explain the immunopathology associated with the coronavirus infection [11] . the rationale is that the sharing of peptide motifs between sars-cov-2 and human proteins might evoke immune responses capable of hitting not only the virus but also the human proteins with consequent autoimmune pathologic sequelae in the human host. as a matter of fact, the study [11] called attention to a vast and specific peptide sharing between sars-cov-2 spike glycoprotein and alveolar surfactant-related proteins, in this way addressing the issue of why sars-cov-2 so heavily attacks the respiratory system. in the wake of such results, in order to validate (or, as well, invalidate) the cross-reactivity hypothesis, investigation was expanded here by analyzing the peptide sharing between the human host and immunoreactive epitopes that are also present in sars-cov-2. actually, the mere sharing of peptide sequences between pathogens and human proteins might be of little significance whether it remained sterile of cross-reactive autoimmune reactions. on the contrary, a peptide sharing between immunoreactive pathogen-derived epitopes and human proteins would assume the value of a proof of concept of cross-reactivity as a mechanism contributing to the pathogen-induced diseases. in this scientific framework, using the hexapeptide as an antigenic and immunogenic unit [12] [13] [14] [15] , immunoreactive epitopes that are present in sars-cov-2 were analyzed for matches with the human proteome. the results confirm a vast peptide commonality that involves human proteins implied in pulmonary insufficiency, neurological disorders, cardiac and vascular alterations, pregnancy dysfunctions, multiple cancers and anosmia, among others. analyses were conducted on an immunome composed of 233 linear epitopes that were experimentally validated, present in the proteins of sars-cov, and cataloged in the iedb [16] . the experimentally validated 233 epitopes have been described in detail by ahmed et al. [17] , map identically in sars-cov-2 proteins, and are listed in supplementary materials table s1 . the hexapeptide was used as a measurement unit to define minimal epitopic sequences. indeed, 5-6 amino acids (aa) can form a sufficient minimal determinant for epitope-paratope interaction [12, 13] . likewise, t-cell epitopes contain a core of 5-6 aa that are involved in antigenicity and immunogenicity [13] [14] [15] and are flanked by nh 2 -and cooh-terminus aa that act as anchor motifs. the sars-cov-2 epitope sequences were dissected into hexapeptides which overlapped each other by five aa residues. the resulting 733 hexapeptides were analyzed for occurrence(s) within human proteins using the pir peptide match program [18] . human proteins involved in the hexapeptide sharing were analyzed for function/diseases using uniprotkb, pubmed, and omim resources. the expected value for hexapeptide sharing between two proteins can be calculated by considering the number of all possible hexapeptides, n. since in a hexapeptide each residue can be any of the 20 aa, the number of all possible hexapeptides n is given by n = 20 6 = 64 × 10 6 . then, the number of the expected occurrences is directly proportional to the number of hexapeptides in the two proteins and inversely proportional to n. assuming that the number of hexapeptides in the two proteins is n and neglecting the relative abundance of aa, we obtain a formula derived by approximation, where the expected number of hexapeptides is 1/n or 20 −6 . table 1 reports that 230 out of the 733 hexapeptides composing the analyzed 233 immuno-reactive epitopes occurred among 460 human proteins. many of these shared sequences recurred more times. for instance, the zinc finger protein znf265-a splice factor that is important in renin mrna processing and stability [19] -shares the hexapeptide srsssr with the viral epitope sqassrsssr (iedb id: 60380). the hexapeptide srsssr recurs three times in the zinc finger protein, exactly at aa positions: 211-216, 241-246, and 256-261. then, including multiple occurrences, on the whole the hexapeptides shared with the human proteins amount to 505. for reasons of synthesis, table 2 reports the distribution of the shared hexapeptides relatively to a sample (n = 58) of sars-cov-2 epitopes. in the viral epitope sequences, the hexapeptides shared with human proteins are marked in capital letter format. the distribution of the shared hexapeptides throughout the entire set of 233 sars-cov-2 epitopes is described in supplementary materials table s2 . table 2 documents that numerous immunoreactive sars-cov-2 epitopes are composed mostly or, in many instances, uniquely of peptide sequences shared with human proteins. as an example, among the many, the epitope iedb id 4936, which is present in the viral nucleocapsid protein and corresponds to the aa sequence ategalntpk, results from the consecutive succession/overlapping of 6 mers present in human proteins too. taken together, table 1 , table 2 , and table s2 highlight the unexpectedness of the peptide overlap between the human proteome and the immunoreactive viral epitopes derived from iedb, described by ahmed et al. [17] and analyzed here. from a mathematical point of view, if one considers that the probability of a hexapeptide to occur in 2 proteins is~20 −6 (or 1 out of 64,000,000 or 0.000000015625), then the number of hexapeptides (namely, 505, including multiple occurrences) shared between the sars-cov-2 immunome and human proteins is surprisingly high and can be explained only on the basis of evolutionary processes [20] . in the context of autoimmunity, table 1 , table 2 , and table s2 concretize the effective possibility of cross-reactions between sars-cov-2 and the human proteome, given the fact that the immunological information unit in terms of both immunogenicity and antigenicity is contained in a space formed by 5-6 aa residues [12] [13] [14] [15] . as quantified in table 1 , hexapeptides from immunoreactive viral epitopes occur across 460 human proteins. the 460 human proteins are listed and synthetically described in supplementary materials table s3 . the 460 human proteins are involved in metabolic, developmental, and regulatory cellular functions and-when mutated, modified, deficient or, however, improperly functioning-may lead to altered functions and more or less severe pathologies in the human organism. obvious reasons of space prevent a protein-by-protein analysis of all of them and, here, only a few proteins (given by the uniprot name in italic and shared peptides in parentheses) and the related pathologies that could arise in case of cross reactivity are dealt with as follows. molecular mimicry between sars-cov-2 spike glycoprotein and alveolar surfactants-related proteins have already been described [11] . additional proteins that-if hit-can alter lung functions are: mothers against decapentaplegic homolog 9 protein (qassrs), alterations of which may lead to pulmonary hypertension with proliferating endothelial cells in pulmonary arterioles, right ventricular failure, and death [21] ; • phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (ikdlpk) that is deficient in pulmonary vascular endothelial cells of patients with acute respiratory distress syndrome [22, 23] ; • adenylate cyclase type 9 (kqlssn) that is expressed in multiple cells of the lung with expression highest in airway smooth muscle [24] ; • acetylcholinesterase (avlqsg) where imbalances in the neurotransmitter acetylcholine relate to neurological conditions, such as alzheimer's disease, parkinson's disease, and myastenia gravis; irreversible inhibition of acetylcholinesterase may lead to muscular paralysis, convulsions, bronchial constriction, and death by asphyxiation [25] . • endoribonuclease dicer (assrss) which is linked to pleuropulmonary blastoma [26] ; • protein naked cuticle homolog 1 (llpsla), downregulation of which is linked to poor prognosis in non-small-cell lung cancer and breast invasive ductal carcinoma [27, 28] ; • downregulated in multiple cancers 1 (aaeira) is not expressed in multiple human cancers [29] ; • ubiquitin carboxyl-terminal hydrolase bap1 (llsvll) relates to tumor predisposition syndrome; linked to mesothelioma [30] [31] [32] ; • in addition, the tumor suppressor proteins pinin (ssrsss) [33] , tripartite motif-containing protein 35 (sfkeel) [34] , unconventional myosin-ixb (llpsla) [35] , and cyclin-d-binding myb-like transcription factor 1 (aalqip) [36] . • low-density lipoprotein receptor-related protein 8 (lallll), alterations of which can lead to myocardial infarction [37] ; • dol-p-glc:glc(2)man(9)glcnac(2)-pp-dol alpha-1,2-glucosyltransferase (tltlav) is implicated in susceptibility to the long qt syndrome [38] ; • presenilin-2 (tlacfv) relates to dilated cardiomyopathy and heart failure [39] ; • nesprin-1 (llsagi) is involved in dilated or hypertrophic cardiomyopathy [40, 41] : • nuclear receptor coactivator 6 (pslatv) can cause dilated cardiomyopathy [42] ; • latent-transforming growth factor beta-binding protein 3 (lallll) can associate with skin thickening, cardiac valvular thickening, tracheal stenosis, and respiratory insufficiency [43] . • ephrin-b3 (lallll) is involved in blood pressure control and vascular smooth muscle cell contractility [44] ; • endoglin (lvlsvn) is required for normal structure and integrity of adult vasculature [45] ; • elastin (fgagaa) is a major structural protein of tissues, such as aorta and nuchal ligament, and relates to cutis laxa disease, may also, although rarely, lead to pulmonary artery stenosis, aortic aneurysm, bronchiectasis, and emphysema [46, 47] ; • filamin-a (pyrvvv), alterations of filamin-a can cause disorders related to the vascular system and to a large phenotypic spectrum of disorders such as deafness, urogenital defects, malformations, intestinal obstruction, constipation, recurrent vomiting, and diarrhea [48] ; • glomulin (rimasl) is crucial in vascular morphogenesis-especially in cutaneous veins [49] . • tumor necrosis factor receptor superfamily member 1a (gttvll) may associate with fever, abdominal pain, localized tender skin lesions, myalgia, and reactive amyloidosis as the main complication [50, 51] . • thrombomodulin (agaalq), a well-known natural anti-coagulant that acts as a linker of coagulation and fibrinolysis, is involved in disseminated intravascular coagulation, is a predictor of risk of arterial thrombosis and is essential for the maintenance of pregnancy [52] [53] [54] . • in addition, cross-reactive peptide sharing involving additional proteins can further affect the level of thrombomodulin. indeed, cross-reactions with retinoic acid receptor rxr-alpha (lgfstg) and prostaglandin g/h synthase 2 (tvllke) might completely eliminate thrombomodulin from blood circulation because (1) retinoic acid receptor rxr-alpha promotes the thrombomodulin gene transcription [55] and (2) prostaglandin g/h synthase 2 stimulates the expression of functionally active thrombomodulin in human smooth muscle cells [56] . adding up to such pro-thrombotic scenario, it is also worth of mention the potential cross-reactivity with tyrosine-protein kinase jak2 (llddfv) that is involved in myelofibrosis, myeloid leukocytosis, and thrombocytosis with excessive platelet production resulting in increased numbers of circulating platelets, hemorrhages, and thrombotic episodes [57] [58] [59] . • neuronal pas domain-containing protein 2 (assrss), which is highly polymorphic in autism spectrum disorder patients [60, 61] ; • circadian locomotor output cycles protein kaput (assrss) relates to bipolar disorder [62] ; • adenosine receptor a1 (vlppll), where sleep is significantly attenuated by the loss of adenosine a1 receptor expression [63] ; • bdnf/nt-3 growth factors receptor (sanlaa), alterations may cause temporal lobe epilepsy, memory impairment, anorexia nervosa, bulimia, alzheimer's disease [64] [65] [66] ; • calcium/calmodulin-dependent protein kinase kinase 2 (pslatv) is linked to disturbances of higher cognitive functions, such as working memory and executive function. as well as schizophrenia [67] ; • endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha-mannosidase (lafllf) can cause mental retardation [68] ; • glutaminase kidney isoform, mitochondrial (lqelgk) can associate with epileptic encephalopathy, infantile cataract, skin abnormalities leukocytoclasia at the surface of the dermis, focal vacuolar alterations, hyperkeratosis, parakeratosis, glutamate excess, and impaired intellectual development, global developmental delay, progressive ataxia, and elevated glutamine [69] [70] [71] ; • mitochondrial glutamate carrier 1 (rlqslq) relates to neonatal myoclonic epilepsy [72] . moreover, and remarkably, the voltage-gated calcium channel gamma subunits 2, 3, 4, 6, and 8 contain epitopic hexapeptides (table 3) . voltage-gated calcium channels control cellular calcium entry in response to membrane potential changes [73, 74] , and gamma subunits 2, 3, 4, and 8 regulate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (or ampa receptor or ampar) and are collectively known as transmembrane ampar regulatory proteins (tarps) [75] . ampars are involved in the fast synaptic transmission in the central nervous system and are altered in many psychological and neurological disorders such as schizophrenia, depression and parkinson's disease [76] . table 3 . hexapeptide sharing between sars-cov-2 epitopes and tarps. lsagif, srsssr gamma-2 subunit lsagif gamma-3 subunit srsssr gamma-4 subunit lgagcf gamma-6 subunit srsssr gamma-8 subunit as a final note, this results section closes with one disorder that is one of the first symptoms to appear in covid-19, namely, anosmia. the role of anosmia as a first symptom is clearly justified by the fact that seven olfactory receptors (i.e., proteins related to the smell) [77] share hexapeptides with the viral epitopes (table 4 ). this study shows that hexapeptides from immunoreactive epitopes present in sars-cov-2 are widespread among a high number of human proteins. such a peptide sharing implies the possibility of cross-reactions and, consequently, as discussed in the results (section 3), of a vast phenotypic constellation of diseases, from pneumonia and neurological disorders to cardio-vascular alterations and coagulopathies. hence, this study appears to offer scientific hints to explain the clinical fact that sars-cov-2 infection is capable of triggering so many and so different pathologies in so many and so different organs of the human host [78] . on the whole, the data indicate that-besides possible virus-induced multi-organ direct cytopathic effects and other possible pathogenic mechanisms-self-reactive antibodies may be at the root of the pathologic scenario that accompanies sars-cov-2 infection. in fact, previous research focusing on sars-cov, which similarly to sars-cov-2 causes a respiratory failure syndrome, showed that anti-spike protein iggs can cause lung damage by directly affecting inflammatory mechanisms, promoting the release of pro-inflammatory cytokines, such as il-8, as well as macrophagic tissue infiltration [79] . a similar effect of sars-cov-2 on inflammatory response has already been proposed [80] . then it appears reasonable to hypothesize that autoantibody-mediated macrophagic and complement activation and autoantibody-dependent cell-mediated cytotoxicity mechanisms might be some of the mechanisms behind the well-documented multi-organ damage in covid-19, thus representing one of many possible links between adaptive and innate immunity in the pathogenesis of the disease. moreover, as reviewed by vabret et al. [81] , it has been shown that high antibody response can associate with more severe clinical cases. this had also been seen in the previous sars-cov-1 epidemic, where neutralizing antibody titers were found to be significantly higher in deceased patients compared to patients who had recovered [82] and might lead to suspect antibody-dependent enhancement phenomena. of note, the present data also indicate that most possibly the damage caused by sars-cov-2 might not end with the end of the pandemic. indeed, the peptide sharing between sars-cov-2 epitopes and specific tumor suppressor proteins, and, in general, many tumor-related proteins [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] , theoretically predicts, in the absence of current clinical data, that a morbidity/mortality increase in various cancers might follow the current sars-cov-2 pandemic. as a conclusive note, it is mandatory also to observe that this study largely undervalues the potential risk of cross-reactivity between sars-cov-2 immunome and human proteins. indeed, analyses were conducted on linear epitopes and used the hexapeptide as an immune unit probe. then, if one considered conformational epitopes and used the pentapeptide as a minimal immune determinant [13, 83] , the number of viral versus human commonalities would increase exponentially. given this premise, the present results call researchers and clinicians for a common research effort to study the autoimmune pathogenicity connected to the anti-sars-cov-2 immune response and suggests, once more, that using entire antigens in anti-sars-cov-2 vaccine formulations might lead to autoimmune manifestations and adverse events [84] . actually, the present data further support the fundamental concept that only "non-self" peptides can lead to safe and efficacious immunotherapies [85] [86] [87] . supplementary materials: the following are available online at http://www.mdpi.com/2073-4468/9/3/33/s1, table s1 : sars-cov-2 immunome consisting of 233 immunopositive linear epitopes, assembled from iedb [16] , described by ahmed et al. [17] , and listed according to iedb id number. table s2 : hexapeptide sharing between 233 epitopes present in sars-cov-2 and human proteins. table s3 : list and short description of 460 human proteins that share hexapeptides with the 233 sars-cov-2 epitopes. funding: this research received no funding. the author declares no conflict of interest. human immunopathogenesis of severe acute respiratory syndrome (sars) lung pathology of fatal severe acute respiratory syndrome an interferon-gamma-related cytokine storm in sars patients pathogenesis of severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome prior immunization with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) nucleocapsid protein causes severe pneumonia in mice infected with sars-cov immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus immunization with inactivated middle east 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peptides in the design of vaccines immunogenicity, immunopathogenicity, and immunotolerance in one graph from hbv to hpv: designing vaccines for extensive and intensive vaccination campaigns worldwide key: cord-342118-fsmuqktd authors: dyakov, ilya n.; mavletova, dilara a.; chernyshova, irina n.; snegireva, nadezda a.; gavrilova, marina v.; bushkova, kristina k.; dyachkova, marina s.; alekseeva, maria g.; danilenko, valery n. title: fn3 protein fragment containing two type iii fibronectin domains from b. longum gt15 binds to human tumor necrosis factor alpha in vitro date: 2020-08-06 journal: anaerobe doi: 10.1016/j.anaerobe.2020.102247 sha: doc_id: 342118 cord_uid: fsmuqktd most species of the genus bifidobacterium contain the gene cluster pfna, which is presumably involved in the species-specific communication between bacteria and their hosts. the gene cluster pfna consists of five genes including fn3, which codes for a protein containing two fibronectin type iii domains. each fibronectin domain contains sites similar to cytokine-binding sites of human receptors. based on this finding we assumed that this protein would bind specifically to human cytokines in vitro. we cloned a fragment of the fn3 gene (1503 bp; 501 aa) containing two fibronectin domains, from the strain b. longum subsp. longum gt15. after cloning the fragment into the expression vector pet16b and expressing it in e. coli, the protein product was purified to a homogenous state for further analysis. using the immunoferment method, we tested the purified fragment's ability to bind the following human cytokines: il-1β, il-6, il-10, tnfα. we developed a sandwich elisa system to detect any specific interactions between the purified protein and any of the studied cytokines. we found that the purified protein fragment only binds to tnfα. the biomass of bl21(de3) strain containing the pet16b:fn3 plasmid was 126 resuspended in lysis buffer (50 mm nah2po4, 5 mm tris-hcl, 300 mm nacl, 10 mm 127 imidazole, 1 mm pmsf, 5 mm dtt, ph 8.0) containing lysozyme (1 mg/ml), 0.5% 128 triton x-100 and 20 mm 2-mercaptoethanol and was sonicated. insoluble particles were 129 removed by centrifugation (7500 g, 30 min, 4°c). the resulting lysate was filtered using 130 a 0.22 µm pore size millex-gp. 131 protein isolation and purification were performed using a biologic lp 132 chromatography system equipped with a fraction collector (bio-rad, usa). the 133 clarified lysate was loaded onto a bio-scale tm mini profinity tm imac cartridge (5 ml, 134 bio-rad, usa), then equilibrated and washed with the lysis buffer, followed by another 135 washing with the buffer (50 mm nah2po4, 5 mm tris-hcl, 300 mm nacl , 50 mm 136 imidazole, 1 mm pmsf, 5 mm dtt, ph 8.0). the bound protein was eluted from the 137 columns with buffer containing 300 mm imidazole. for further purification of the 138 protein, dialysis was performed in pbs buffer containing 10% glycerol and 1 mm pmsf. 139 the concentration of the isolated protein was measured by qubit fluorimeter 140 (invitrogen, usa). the average yield of purified protein was approximately 20 mg per 141 liter of e. coli culture. the purified protein was stored at -80°c. 142 the fn3-specific polyclonal antibodies were raised by immunization of rabbits, 144 which was carried out via subcutaneous immunisation ( freund's adjuvant. two weeks after the last immunization, 30-50 ml of blood was 151 collected from each immunized rabbit. blood serum was collected and stored at 4°c 152 after the addition of 0.1% sodium azide (nan3). 153 the fn3-specific antibodies were isolated by affinity chromatography. the serum 154 from immunized rabbits was diluted 4-fold in pbs, centrifuged and passed through 4b 155 tmb reagent were performed as described above. 195 in the second stage, we used 96-well polystyrene flat bottom plates coated with 197 cytokine-specific antibodies of from the human cytokine determination kits (vector-198 best, russia). we added in each well one at a time, first 100 µl of the cytokines il6, 199 il10 or tnfα at a concentration of 250 pg/ml, then 100 µl of the fn3 protein at a 200 concentration of 2 µg/ml and lastly 100 µl of the fn3-specific polyclonal antibodies, 201 obtained as described above, at a concentration of 20 ng/ml. in each case, the plates 202 were incubated for 1 hour at 37°c, followed by washing (4 times) with pbst. finally, incubation in carbonate/bicarbonate buffer for 1 hour at 37°c followed by washing with 210 pbst four times. to avoid nonspecific adsorption, the wells were incubated in a 1% 211 casein solution for 1 hour at 37°c. after washing, 100 µl of the fn3 protein solution at a 212 concentration of 2 µg/ml was added into the wells, incubated for 1 hour at 37°c and 213 washed four times with pbst. at the next stage, solutions of the cytokines at a 214 concentration of 250 pg/ml were added, incubated for 1 hour at 37°c and washed as 215 to reduce nonspecific interaction with various substrates of the protein fn3, which is 223 classified as an adhesive protein, it was introduced into wells in pbst containing 1% 224 casein (invitrogen, usa). 225 to search for nucleotide and amino acid sequences we used the databases available 227 on ncbi (http://www.ncbi.nlm.nih.gov/) and uniprot (http://www.uniprot.org/). to 228 calculate the molecular weight and isoelectric point of proteins, we used the protparam 229 program (http://web.expasy.org/protparam/). previously, based on the analysis of the genomes of 34 species of bifidobacteria, we 233 identified and characterized a species-specific cluster of pfna genes, which consists of to develop a sandwich elisa assay that would allow us to check the ability of the 298 recombinant fn3 protein to bind human cytokines, we raised polyclonal antibodies 299 specific to the recombinant fn3 protein. we obtained the fn3-specific polyclonal 300 antibodies from the serum of immunized rabbits. the process of antibody production 301 and isolation is described in the materials and methods section. the resulting affinity 302 purified and concentrated preparation contained 1 mg of fn3-specific rabbit polyclonal 303 antibodies per 1 ml and was highly active -specific interaction with the fn3 protein 304 was detected even after 1:820000 dilution. we used the bacterial aminoglycoside 305 phosphotransferase aphviii, purified previously at our laboratory, as a negative 306 control. the produced rabbit polyclonal antibodies interacted with aphviii only in the 307 smallest dilutions (1: 400), which is usually the case for nonspecific binding. adding 308 rabbit nonspecific γ-globulins instead of the polyclonal antibodies did not cross-react 309 either with the fn3 protein adsorbed to the wells of a 96-well polystyrene plate (see. 310 supplementary appendix table s1-s3). 311 to conduct an initial evaluation of the cytokine-binding capacity of the fn3 protein, 313 we first proceeded with elisa scheme 1, described in the materials and methods. 314 when conducting this study, we had to carry out a number of preliminary experiments, 315 the results of which are not shown. the studied fragment of the fn3 protein reacted 316 with cytokines in the same way. assuming that the capacity of the constructed system 317 with different cytokines may vary, we had to evaluate the effect of dose dependence. it 318 turned out that scheme 1 was not suitable for our task since the fn3 protein generated a 319 strong positive reaction with all the cytokines, and the reaction intensity did not 320 correlate with the concentration of cytokines bound to the solid phase (fig. 1) . further 321 testing showed (scheme 2 elisa) that the fn3 protein binds nonspecifically to virtually 322 any substrate attached to a solid surface, including the antibody-cytokine complex, 323 antibodies to cytokines, and even to non-treated polystyrene (fig. 2) . all while 324 nonspecific interaction of other reaction components with each other and with the solid 325 surface was excluded experimentally (see materials and methods). 326 to reduce nonspecific binding to substrates, 1% casein was added to the solution of 327 the fn3 protein. the fn3 protein was not able to bind specifically to immobilized 328 cytokines (either to directly adsorbed cytokines or to cytokines attached to a trap of 329 antibodies). this could be explained by the fact that adsorption onto a solid surface can 330 lead to the blockage of the binding sites of fn3-molecule. moreover, a strong 331 nonspecific interaction between the fn3 protein and the solid surface (see fig. 2 ) could 332 hinder any specific interaction. 333 to exclude the possibility of blockage of the cytokine-binding motif of the fn3 335 protein and its nonspecific adsorption onto the solid surface, we employed elisa 336 scheme 3 described in the materials and methods section: the fn3-specific rabbit 337 polyclonal antibodies were first adsorbed onto the solid surface after which the fn3 338 protein was added as a "second layer". then, we added into the wells the cytokine 339 solutions and the peroxidase conjugates from the commercial kits vector-best for 340 detecting cytokines. this design revealed a specific interaction between the fn3 protein 341 and the cytokine tnfα, but not with il-6, il-1, or il-10 (fig. 3) . this observation 342 suggests the possibility that the fn3 protein of b. longum subsp. longum gt15 binds 343 human cytokines, namely, the pro-inflammatory cytokine tnfα. as a control for tnfα, 344 we used a cytokine that does not bind specifically the fn3 protein in our elisa 345 scheme, il-6. 346 bifidobacteria as strictly anaerobic bacteria are considered to be some of the oldest 348 representatives of actinobacteria [36]. as they coevolved alongside their hosts (insects, 349 birds, and mammals), they appear to have developed a species-specific gene cluster 350 (pfna operon), which allowed them to adapt better to their niches and interact with 351 the host's immune system [19, 20] . the pfna cluster is unique to the genus the genes of the pfna cluster exhibit unusually high level of interspecific divergence 354 [20], suggesting that the functions of this operon are species-specific. 355 today, the functional role of most of the genes that make up the pfna operon and 356 their products remains unknown. nevertheless, at least two genes of this operon (pkb2 357 and tgm) are potential components of a signal system that accounts for a bidirectional 358 communication between the host cells and different species of bifidobacteria [19] . the presence of motifs in fn3-containing proteins similar in structure to cytokine 392 binding sites of mammalian receptors [23, 35], encouraged us to opt for fn3-containing 393 proteins as suitable candidates for this role. as mentioned before, one of these proteins 394 is encoded by one of the genes of the cluster pfna of b. longum. proceeding from these 395 premises, we aimed at detecting specific interaction between the fn3 protein and 396 human cytokines. 397 we set out to assess the ability of a fragment of the fn3 protein containing two fn 398 type iii domains to bind in vitro to various human cytokines. however, the protein's 399 strong adhesive properties rendered the task of revealing its specific interaction with 400 cytokines immobilized on a solid surface, impossible. 401 the fact that it was impossible to detect any specific interaction between the fn3 402 protein and cytokines attached to a solid surface suggests one of two things: 403 1) the fn3 protein undergoes conformational changes when adsorbed onto plastic, 404 which blocks its active site responsible for adhesion and cytokine-binding ability; 405 2) the cytokine-binding motif of the protein is directed inward or is located very 406 close to the adhesion site, which leads to spatial blockage during the adhesion of the 407 fn3 protein to a solid surface. to find out which of these two hypotheses is true, it is 408 necessary to evaluate the crystal structure of the fn3 protein. we managed to overcome the non-specific interaction between the fn3 protein and 410 solid surfaces by constructing an elisa sandwich system, in which the fn3 protein, 411 attached to rabbit polyclonal igg, acted as a cytokine trap. this construction allowed us course of evolution, bacteria of the genus bifidobacterium used proteins containing fn3 418 domains as an instrument for the identification of cytokines of the host organism. 419 it is well known that the components of the immune system can affect the growth of 420 bacteria [10-13], i.e. microorganisms not only affect the host organism, but are also able, 421 in turn, to perceive signals from it and accordingly respond to them. until recently, 422 such data for bifidobacteria were absent from the literature. 423 it is self-evident that for bifidobacteria to be affected by the host organism, they 424 need to be able to receive a feedback signal from the human body. table s1 . optical density of serum samples of rabbits immunized with the fn3 464 protein fragment bound to a specific antigen. table s2 optical density of affinity-465 purified rabbit antibodies specific to the fn3 protein fragment bound to specific and 466 non-specific antigens. table s3 optical density of affinity-purified rabbit antibodies 467 specific to the fn3 protein fragment and total igg of non immunized rabbits against the 468 protein fn3 of b. longum gt15. bifidobacteria and their role as members of the bifidobacteria and the infant gut: an example of co 483 evolution and natural selection mechanisms of action of antioxidant 488 properties of probiotic bacteria tumor necrosis 519 factor alpha binding to bacteria: evidence for a high-affinity receptor and alteration 520 of bacterial virulence properties recognition of host immune activation by pseudomonas 524 aeruginosa specific high affinity binding of 527 human interleukin 1 beta by caf1a usher protein of yersinia pestis molecular and structural 531 characterization of a novel escherichia coli interleukin receptor mimic protein functional and structural characteristics of bacterial proteins 534 that bind host cytokines positive selection in 20 longum: genetic environment and 542 substrate specificity signature proteins that are distinctive 583 characteristics of actinobacteria and their subgroups functional analysis 586 of an s-layer-associated fibronectin-binding protein in lactobacillus acidophilus ihalin r. a novel intrinsically disordered outer 590 membrane lipoprotein of aggregatibacter actinomycetemcomitans binds various 591 cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8 influence of probiotics on cytokine production in the in vitro and in vivo systems structure, function, and antigenicity of the sars-cov-2 spike glycoprotein pathogenic human coronavirus infections: causes 607 and consequences of cytokine storm and immunopathology the use of anti-inflammatory drugs in the treatment 611 of people with severe coronavirus disease 2019 (covid-19): the perspectives of 612 clinical immunologists from china specific interaction of fn3 protein with cytokines. cc -«conjugate» 632 control (no cytokines; secondary andibodies added after the fn3 protein 633 fragment). the values yielded by secondary antibodies against the cytokines il-634 6, il-10, il-1 and tnfα were relatively similar and grouped together key: cord-325559-di8lljoi authors: cappello, francesco; marino gammazza, antonella; dieli, francesco; conway de macario, everly; macario, alberto jl title: does sars-cov-2 trigger stress-induced autoimmunity by molecular mimicry? a hypothesis date: 2020-06-29 journal: j clin med doi: 10.3390/jcm9072038 sha: doc_id: 325559 cord_uid: di8lljoi viruses can generate molecular mimicry phenomena within their hosts. why should severe acute respiratory syndrome coronavirus 2 (sars-cov-2) not be considered one of these? information in this short review suggests that it might be so and, thus, encourages research aiming at testing this possibility. we propose, as a working hypothesis, that the virus induces antibodies and that some of them crossreact with host’s antigens, thus eliciting autoimmune phenomena with devasting consequences in various tissues and organs. if confirmed, by in vitro and in vivo tests, this could drive researchers to find effective treatments against the virus. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) induced disease (covid-19) is a planetary emergency that is urging many research groups to redirect their efforts and to channel their experience towards understanding its pathogenesis. despite many clinical reports and papers on viral genetics, detailed information on pathogenic mechanisms pertaining to covid-19 is still lacking. this type of information will no doubt help physicians in patient management and in providing treatment. the paucity of data on pathogenesis is due to a considerable extent to the very low number of autopsies that have been performed on covid-19 victims [1] . while histopathological and other data from laboratory tests and autopsies will accumulate as the pandemic persists in the next few months or so, some progress can be achieved applying bioinformatics and scientific reasoning. in this brief hypothesis paper, we have organized pertinent information available not only from the growing scientific literature but also from the chats of doctors and researchers on the web that cannot be ignored at this time, although they are not official instruments for dissemination of scientific data. these are temporarily useful channels for disclosing information as it is being generated at the "war front" (i.e., the doctors' offices and clinical departments) that under normal circumstances would be available in the form of scientific publications only many months after the fact. among the numerous articles consulted, some have caught our attention [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . by reading these and other publications, we arrived at the initial conclusion that covid-19 develops in three steps (figures 1 and 2 ). in the following considerations, we will focus on the disease caused when the virus invades the body via the upper respiratory tract disregarding the other ways of viral entry, which are considerably less frequent as per current data-nevertheless, it is very likely that the conclusions would have also applied to the latter. (1) the virus enters the body through the upper respiratory tract and invades the respiratory mucosa covering the nasal cavities, the paranasal sinuses, and the nasopharynx. here it replicates and encounters immune cells. the immune system, via the waldeyer's ring, recognizes viral antigens activating innate immunity. (2) if the virus is not eradicated at this stage, it reaches the lower airways and enters the bloodstream through the respiratory barrier. the architecture of the primary pulmonary lobules is rapidly subverted by the violent inflammatory response, including both innate and adaptive immune-systems activation (lymphocytes, macrophages, plasma cells, etc.). (3) plasma cells produce antibodies that by the bloodstream (the lung is a highly vascularized organ) can travel throughout the body. (the image of the human body is a courtesy of visible body atlas.). sars-cov-2: severe acute respiratory syndrome coronavirus 2. (1) the virus enters the body through the upper respiratory tract and invades the respiratory mucosa covering the nasal cavities, the paranasal sinuses, and the nasopharynx. here it replicates and encounters immune cells. the immune system, via the waldeyer's ring, recognizes viral antigens activating innate immunity. (2) if the virus is not eradicated at this stage, it reaches the lower airways and enters the bloodstream through the respiratory barrier. the architecture of the primary pulmonary lobules is rapidly subverted by the violent inflammatory response, including both innate and adaptive immune-systems activation (lymphocytes, macrophages, plasma cells, etc.). (3) plasma cells produce antibodies that by the bloodstream (the lung is a highly vascularized organ) can travel throughout the body. (the image of the human body is a courtesy of visible body atlas.). sars-cov-2: severe acute respiratory syndrome coronavirus 2. the first step consists of upper airway infection: the virus colonizes and multiplies in the ciliated columnar epithelial cells of the respiratory mucosa. this phase can be asymptomatic, paucisymptomatic, or symptomatic; in any case, an innate immune response against the virus is triggered. the disease can be resolved at this level (fortunately in most cases) or it can progress to the second step. . at this stage, the disease can be asymptomatic, paucisymptomatic or produce symptoms such as fever, cough, anosmia, ageusia, and shortness of breath. many subjects heal spontaneously. however, in a limited number of subjects the virus moves down to the lower airways, causing severe pneumonia. it is not clear why some patients develop pneumonia and other do not. however, cold weather, high humidity, and severe pollution can be considered prodisease factors because they may favor virus vitality outside the body and inflammatory status inside the airways. most of the patients with pneumonia manage to heal (for example, by ex juvantibus therapies, such as tocilizumab or hydroxychloroquine), however, some of them develop severe complications, i.e., a generalized activation of the immune system manifested as vasculitis, disseminated intravascular coagulation (dic), and other signs and symptoms of autoimmunity. at this point, the risk of developing a multiorgan failure (mof) is high, and the patient may die. the first step consists of upper airway infection: the virus colonizes and multiplies in the ciliated columnar epithelial cells of the respiratory mucosa. this phase can be asymptomatic, paucisymptomatic, or symptomatic; in any case, an innate immune response against the virus is triggered. the disease can be resolved at this level (fortunately in most cases) or it can progress to the second step. the second step is characterized by lung infection (bilateral interstitial pneumonia), which can be of varying severities. in the more fortunate cases, clinicians manage to contain the infection with antiviral and/or anti-inflammatory therapies (or the infection is self-limited, a possibility that cannot be excluded at this time). in more severe cases, for unknown reasons but which are probably related to a "hyperreactivity" of both innate and acquired immunities, the disease progresses towards the third step. all the pieces of information available on the internet agree in indicating that in the third step the disease is systemic (representative examples of clinical and laboratory studies are presented in table 1 ). the emerging picture is that of widespread microvascular damage, diffuse thrombosis, disseminated intravascular coagulation (dic) and, lastly, a multiorgan failure (mof), often leading to death ( figure 3 ). table 1 . examples of reports of generalized immune system activation in covid-19. highlight the association between covid-19 pathogenesis and excessive cytokine release from lungs, such as ccl2/mcp-1, cxcl10/ip-10, ccl3/mip-1a, and ccl4/mip1b. [12] clinical and laboratory compared with nonintensive care unit (icu) patients, icu patients had higher plasma levels of interleukin (il)2, il7, il10, gscf, ip-10, mcp1, mip1a, and tnfα. [13] laboratory sars-cov-2 infection significantly upregulated il6, mcp-1, cxcl1, cxcl5, and cxlc10/ip10. [14] clinical and laboratory a retrospective multicenter study of 191 patients reported more elevated il6 levels in nonsurvivors than in survivors; univariate analysis of the data revealed significant associations of elevated il6 serum levels with mortality. [2] clinical and laboratory compared to moderate cases, severe cases more frequently had dyspnea, lymphopenia, and hypoalbuminemia, with higher levels of alanine aminotransferase, lactate dehydrogenase, c-reactive protein, ferritin and d-dimer, as well as markedly higher levels of il-2r, il-6, il-10, and tnf-α. [15] clinical and laboratory elderly patients and with comorbidities showed higher plasma levels of il6, il10, lactate dehydrogenase, and c reactive protein. [16] clinical and laboratory inflammatory cytokines were more elevated in severe cases than the nonsevere ones, including il-2r, il-6, il-8, il-10, and tnf-α. immunoglobulins (ig) a, igg, and igm and complement proteins (c3 and c4) in patients with covid-19 were within normal range. there were no significant differences in the levels of iga, igg, and complement proteins c3 or c4 between the mild and severe groups, while igm slightly decreased in severe ones. [17] clinical and laboratory concentrations of alanine aminotransferase, aspartate aminotransferase, creatinine, creatine kinase, lactate dehydrogenase, cardiac troponin i, n-terminal probrain natriuretic peptide, and d-dimer were markedly higher in deceased patients than in recovered patients. [18] notes. ccl2: chemokine (c-c motif) ligand 2; mcp-1: monocyte chemoattractant protein 1; cxcl10: c-x-c motif chemokine 10; ip-10: interferon gamma-induced protein 10; ccl3: chemokine (c-c motif) ligand 3; mip-1a: macrophage inflammatory protein 1-alpha; gscf: granulocyte colony-stimulating factor; tnfα: tumor necrosis factor alpha; cxcl1: c-x-c motif chemokine 1; cxcl5: c-x-c motif chemokine 5. it is noteworthy that these patients do not show the typical features of disseminated intravascular coagulopathy (dic). typically, patients with dic present with a considerably prolonged prothrombin time and a major reduction in platelet counts. by contrast, covid-19 patients have a moderately prolonged prothrombin time and platelet counts are often in the lower range of the normal. this strongly indicates that blood-clotting activation in covid-19 is different from the standard dic clotting activation. furthermore, the moderately reduced platelet count clearly resembles an immune complex-mediated prothrombotic disorder, e.g., heparin-induced thrombocytopenia [19] . it is noteworthy that these patients do not show the typical features of disseminated intravascular coagulopathy (dic). typically, patients with dic present with a considerably prolonged prothrombin time and a major reduction in platelet counts. by contrast, covid-19 patients have a moderately prolonged prothrombin time and platelet counts are often in the lower range of the normal. this strongly indicates that blood-clotting activation in covid-19 is different from the standard dic clotting activation. furthermore, the moderately reduced platelet count clearly resembles an immune complex-mediated prothrombotic disorder, e.g., heparin-induced thrombocytopenia [19] . some reports of damage of extrapulmonary organs are listed in table 2 . in addition, at this writing there is growing evidence of autoimmune dermatitis, guillain barre syndrome, and kawasaki disease in some covid-19 patients, particularly the younger ones [20] [21] [22] . table 2 . clinical and laboratory evidence of damage to extrapulmonary organs during sars-cov-2 infection. heart blood tests on admission showed most patients had higher levels of creatine kinase isoenzyme-myocardial band (ck-mb), myohemoglobin, cardiac troponin i, and n-terminal probrain natriuretic peptide. [23] liver gastrointestinal tract sars-cov-2 rna was first detected in stool of the first reported covid-19 case in the usa, who also presented with the digestive symptoms of nausea, vomiting, and diarrhea. [4] 1 abbreviations: alt: alanine aminotransferase; ast: aspartate aminotransferase. mortality rate is very high if the patient is male, elderly, with other concomitant pathologies, especially those related to hypertension and/or diabetes [3, 5, 9, 18] . despite all the information summarized above, it is still a mystery what triggers the hyperactivation of the immune system, which is virtually always present. we have elaborated a working hypothesis [27] that we now would like to propose to the scientific community and, thus, provide food for thought and a basis to plan clinical and laboratory research. for many years our group has been studying a class of proteins highly conserved during evolution and organogenesis, the heat shock proteins (hsp), many of which are molecular chaperones. these are typically antistress proteins (asp) that have helped cells, since their origins at the beginning of life on earth, to survive environmental stresses of chemical and physical nature and have, therefore, played an important role in evolution [28] . typically, asps are overexpressed in cells exposed to various kinds of stressors including bacterial and viral infections. hsp/chaperones are essential for survival and maintenance of protein homeostasis in all organisms but, if abnormal can cause disease, the chaperonopathies [28] . understanding the role of these proteins can provide novel elements for researchers and clinicians useful in diagnosis and treatment [29] . in the course of our studies, we came to the conclusion that hsp/chaperones can be involved in molecular mimicry phenomena, most likely because of their long evolutionary history and high degree of structural conservation, which has produced a widespread sharing of various antigen within and across species. hsp/chaperones are very similar in all organisms, from the most primitive unicellular to the most complex multicellular, typically sharing many highly immunogenic epitopes. this situation sets the stage for immunological crossreactivity between hsp/chaperones from all over the spectrum of living organisms. for instance, hsp/chaperones from any organism (bacterium, virus, or protozoan) in the human skin, or gastrointestinal, respiratory, and genitourinary tracts can invade the blood and thus come in contact with the host's immune system. antibodies are formed against the foreign hsp/chaperone that will most likely crossreact with the equivalent molecule of the human host, and this would be a typical example of molecular mimicry [30] . the same can happen with other microbial and human molecules because there are epitopes shared not only between hsp/chaperones but also between them and other molecules with different functions [31] [32] [33] [34] . asps, including hsp/chaperones, are typically intracellular molecules, but following stress events that augment their intracellular levels, they undergo post-translational modifications (ptm) and translocate to the plasma-cell membrane with their antigenic epitopes exteriorized on the cell's surface [35] [36] [37] [38] . these human epitopes, in turn, can be recognized by circulating antibodies made against crossreactive microbial antigens; these antibodies behave like autoantibodies, causing the destruction of the stressed cells, representing a typical example of pathology caused by molecular mimicry and manifested as autoimmunity [30] . any asp can be affected by ptm, which may change its properties and functions and make it pathogenic against its own host, generating a chaperonopathy by mistake [39, 40] . we speculated that the progression of covid-19 from step 2 to step 3 relies on molecular mimicry phenomena, as already shown for other viruses (table 3) . table 3 . examples of molecular mimicry involving viruses in disease. alphavirus sequence alignment of structural polyproteins belonging to arthritogenic alphaviruses revealed conserved regions which share homology with human proteins implicated in rheumatoid arthritis. [41] cytomegalovirus human antibodies against ul44 (an obligate nuclear-resident, nonstructural viral protein vital for human cytomegalovirus (hcmv) dna replication) immunoprecipitated nuclear-resident systemic lupus erythematosus autoantigens (namely, nucleolin, dsdna, and ku70). [42] coronaviruses several t-cell lines isolated from multiple sclerosis patients showed cross-reactivity between myelin and coronavirus antigens. [43] enterovirus immunogenic epitopes in enterovirus capsid protein vp1 and procapsid protein vp0 have sequence similarities with diabetes-associated epitopes in tyrosine phosphatase ia-2/iar and heat shock protein 60. [44] epstein-barr virus anti-c1q in systemic lupus erythematosus (sle) patients can be induced by an ebv-derived epitope through molecular mimicry. [45] papillomavirus a potential antigenic mimicry between viral and human proteins may be causative of myalgic encephalomyelitis and chronic fatigue syndrome. [46] in active celiac disease, a subset of antitransglutaminase iga antibodies recognize the viral protein vp-7, suggesting a possible involvement of molecular mimicry in the pathogenesis of the disease. [47] varicella-zoster virus autoantibodies to protein s can induce vasculitis and direct endothelial damage. [48] west nile virus an in-silico analysis unveiled certain sequence similarities between viral antigens and receptor sequence fragments suggesting a molecular mimicry autoimmunization process. [49] zika and dengue viruses anti-non-structural protein 1 antibodies can cross-react with host platelets and endothelial cells. [50] the severe bilateral pneumonia that develops in some individuals causes a drop in the partial pressure of oxygen in the blood. this undoubtedly represents a systemic stress. all cells suffer hypoxia, and this can lead to an overexpression of stress proteins and, in turn, to their change by ptm and translocation to the plasma cell-membrane. this would trigger molecular mimicry phenomena and a pathogenic cascade leading to mof (figure 3 ). it should be clear that this cascade can be triggered also by other crossreactive proteins distinct from asp. therefore, the search for the protein responsible for molecular mimicry cannot be limited to asps but must be extended to a wider range of cellular proteins. this search, now, is really a conundrum calling for concerted efforts of many research groups worldwide (figure 4 ). it is important to bear in mind that, in addition to autoantibodies and their complexes with soluble or cell-surface antigens, other effectors of autoimmunity such as immunocompetent cells should also be sought for and characterized to obtain a comprehensive picture of the pathogenic mechanism underpinning tissue damage. , which may be irreversible without proper medical intervention. even with prompt medical intervention, the disease may follow its course and cause death. at the moment, there is no specific therapy for covid-19, but clinicians use ex juvantibus therapy based on anti-inflammatory drugs such as tocilizumab (that inhibits il6) and hydroxychloroquine (inhibits il-1 and tnf-alfa); it is noteworthy that both drugs are used with success in autoimmune diseases. we hypothesize that, at the basis of the generalized activation of the immune system, there are molecular mimicry phenomena: the antibodies produced against the virus could turn into autoantibodies against crossreactive proteins expressed on human cells, causing autoimmunity with cell destruction. what proteins? which cells? what are the predisposing factors? furthermore, can there be protective factors? all of these are open questions now, although there are several clues that show directions for research in the immediate future. for example, one possible pathogenic mechanism of tissue damage is antibody dependent enhancement (ade) of sars-cov-2 due to cross-reactivity. ade has been recently claimed as a mechanism favoring middle east respiratory syndrome coronavirus (mers-cov) entry into host cells [51] . however, in a sars-cov macaque infection model, anti-spike igg antibodies bind to the fcγr on alveolar macrophages and promote their activation with release of massive amounts of profigure 4 . working hypotheses. the establishment of generalized signs and symptoms of immune system activation indicates a serious aggravation of the covid-19, which may be irreversible without proper medical intervention. even with prompt medical intervention, the disease may follow its course and cause death. at the moment, there is no specific therapy for covid-19, but clinicians use ex juvantibus therapy based on anti-inflammatory drugs such as tocilizumab (that inhibits il6) and hydroxychloroquine (inhibits il-1 and tnf-alfa); it is noteworthy that both drugs are used with success in autoimmune diseases. we hypothesize that, at the basis of the generalized activation of the immune system, there are molecular mimicry phenomena: the antibodies produced against the virus could turn into autoantibodies against crossreactive proteins expressed on human cells, causing autoimmunity with cell destruction. what proteins? which cells? what are the predisposing factors? furthermore, can there be protective factors? all of these are open questions now, although there are several clues that show directions for research in the immediate future. for example, one possible pathogenic mechanism of tissue damage is antibody dependent enhancement (ade) of sars-cov-2 due to cross-reactivity. ade has been recently claimed as a mechanism favoring middle east respiratory syndrome coronavirus (mers-cov) entry into host cells [51] . however, in a sars-cov macaque infection model, anti-spike igg antibodies bind to the fcγr on alveolar macrophages and promote their activation with release of massive amounts of pro-inflammatory cytokines [52] . by analogy, anti-sars-cov-2/anti-asp cross-reactive antibodies may similarly mediate ade and contribute to tissue damage. this and other similar hypotheses need to be clarified. to test the proposed working hypotheses, several steps are necessary, for example: (1) in silico comparison of epitopes of viral and human proteins, considering all these as putative autoantigens; (2) screening the results from the in silico studies, using the clues provided by epidemiological and clinical data being generated as the pandemic continues, to identify the protein(s) candidates; (3) immunohistochemical and other molecular analyses of tissues obtained from autopsies of covid-19 fatalities to determine if, and where, these crossreactive molecules are expressed and are indeed reactive with pertinent antibodies. what are the main clues that epidemiology and the clinics provide to date? they are many and disparate. we have listed some of them in figure 4 and these can be classified into negative and positive prognostic factors. in brief, from an epidemiological point of view [6] , the main negative prognostic factors are the subject's advanced age, the presence of comorbidities (hypertension and dysmetabolism), and the male sex. conversely, main positive prognostic factors are young age (very few children are affected by the severe form of the disease), being female, and living in certain geographical areas. the latter might depend not only on the degree of environmental pollution or type of climate but also on genetic-driven protection that individuals might have developed by being exposed to other disease-causing agents. alongside these epidemiological indications, there are others that come from the clinic [3] [4] [5] 8, 11] . the disease, in the third step, involves endothelial cells and/or platelets and/or erythrocytes, more than other cells in the human body: this is suggested by signs of dic and anemia often found by clinicians in sars-cov-2 infected patients. furthermore, at this writing, it cannot be excluded that the renal failure that develops in many patients is not due either to the deposition of preformed circulating immune complexes or to the formation of immune complexes made by circulating antibodies bound to kidney antigens in situ. last but not least, a critical examination of the molecular mechanisms underlying the efficacy of drugs that are currently being tested with some success as ex juvantibus therapy should not be overlooked, since it may offer further cues to unveil unknown pathogenic mechanisms. all these clues, and others that may be revealed in the coming weeks from clinical and histological investigations, should guide researchers towards confirmation or exclusion of molecular mimicry as a determinant pathogenic factor. the understanding of sars-cov-2 phenotype using modern bioinformatics is critical to identify target proteins and shared epitopes between human and viral proteins. here, we want to provide some preliminary insights. many research groups have described the virus by performing structural studies or extrapolating information available pertinent to other coronaviruses. by resorting to previously known information on genome sequences and protein structures and functions as well, bioinformaticians have been successfully assisting virologists by structurally characterizing proteins of the novel virus, determining the evolutionary trajectories, identifying interactions with host proteins, and providing other important biological insights. the whole genome of sars-cov-2 was sequenced, and the sequence is available in genbank (accession number mn908947.3). structural and nonstructural proteins were identified (12 reported in genbank) and they are available in protein data bank (pdb) database and the universal protein resource (uniprot). moreover, many bioinformatics tools are currently used in the scientific literature to understand sars-cov-2 properties and in many cases are easily available online like clustal omega (embl-ebi, cambridge, uk), blast, modeller, mega-x, swiss-model (expasy, sib bioinformatics resource portal, lausanne, switzerland), just to mention a few examples. sars-cov-2 is a spherical or pleomorphic enveloped particle containing single-stranded rna associated with a nucleoprotein within a capsid comprised of matrix protein. the envelope bears club-shaped glycoprotein projections [53] . sars-cov-2, as other coronaviruses, has four structural proteins, known as the s (spike), e (envelope), m (membrane), and n (nucleocapsid) proteins. each of these proteins have a function since the n protein holds the rna genome while the s, e, and m proteins together make the viral envelope [54] . the spike protein, which has been visualized at the atomic level using cryogenic electron microscopy [55, 56] , is the protein responsible for allowing the virus to attach to and fuse with the membrane of a host cell and for this reason it has captured major interest in the scientific community [54] . experiments on the spike protein demonstrated that it has enough affinity to angiotensin converting enzyme 2 (ace2) on human cells, supporting the idea that ace2 is the cell entry receptor [10, 55, 56] . the s protein is composed of two functional subunits (s1 and s2) responsible for receptor binding and membrane fusion, respectively. the surface of the virally encoded envelope spike presents an array of host-derived glycans with each trimer displaying 66 n-linked glycosylation sites. this extensive glycosylation has important implications for vaccine design [57] . recently, a comparative analysis of sars-cov-2 proteins with human proteins was performed in search of high local homologous matches [58] . only one immunogenic epitope in sars-cov-2 had no homology to human proteins and it was concluded that, if all the parts of the epitopes that are homologous to human proteins are excluded from consideration due to risk of pathogenic priming, the remaining immunogenic parts of the epitopes may be still immunogenic and remain as potentially viable candidates for vaccine development. these results likely support our hypothesis and should prompt more investigations on this issue. covid-19 represents a global challenge for the medical community, researchers, and practitioners alike. we were not prepared from the health and social perspectives to face a pandemic, and states around the world are trying to adapt and find the best countermeasures and researchers are doing the same. at this moment, it is important not only to share the results, which may be few, but also the ideas, as these can serve as a stimulus to find solutions to the problems. with this short article, we wanted to offer our contribution, however small it might be, to face the challenge of the covid-19 pandemic, stimulating the scientific community to investigate the involvement of molecular mimicry in the pathogenesis of covid-19. this could be useful not only to reveal the pathogenetic mechanisms underpinning morbidity and mortality but also to direct the development of novel therapeutic strategies and a vaccine. acknowledgments: francesco cappello, francesco dieli and antonella marino gammazza were partially supported by the university of palermo. francesco cappello was partially supported also by iemest. everly conway de macario and alberto jl macario were partially supported by the university of maryland at baltimore-imet. this work was written under the umbrella of the agreement between iemest and imet. this is imet contribution number imet-20-011. authors declare no conflict of interest. covid-19 deaths: are we sure it is pneumonia? please, autopsy, autopsy, autopsy! 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anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection genotype and phenotype of covid-19: their roles in pathogenesis analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods cryo-em structure of the 2019-ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein site-specific analysis of the sars-cov-2 glycan shield pathogenic priming likely contributes to serious and critical illness and mortality in covid-19 via this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-337032-s4g4g80w authors: gupta, manoj kumar; vemula, sarojamma; donde, ravindra; gouda, gayatri; behera, lambodar; vadde, ramakrishna title: in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel date: 2020-04-15 journal: j biomol struct dyn doi: 10.1080/07391102.2020.1751300 sha: doc_id: 337032 cord_uid: s4g4g80w recent outbreak of coronavirus disease (covid-19) pandemic around the world is associated with ‘severe acute respiratory syndrome’ (sars-cov2) in humans. sars-cov2 is an enveloped virus and e proteins present in them are reported to form ion channels, which is mainly associated with pathogenesis. thus, there is always a quest to inhibit these ion channels, which in turn may help in controlling diseases caused by sars-cov2 in humans. considering this, in the present study, authors employed computational approaches for studying the structure as well as function of the human ‘sars-cov2 e’ protein as well as its interaction with various phytochemicals. result obtained revealed that α-helix and loops present in this protein experience random movement under optimal condition, which in turn modulate ion channel activity; thereby aiding the pathogenesis caused via sars-cov2 in human and other vertebrates. however, after binding with belachinal, macaflavanone e, and vibsanol b, the random motion of the human ‘sars-cov2 e’ protein gets reduced, this, in turn, inhibits the function of the ‘sars-cov2 e’ protein. it is pertinent to note that two amino acids, namely val25 and phe26, play a key role while interacting with these three phytochemicals. as these three phytochemicals, namely, belachinal, macaflavanone e & vibsanol b, have passed the admet (absorption, distribution, metabolism, excretion and toxicity) property as well as ‘lipinski’s rule of 5s’, they may be utilized as drugs in controlling disease caused via sars-cov2, after further investigation. communicated by ramaswamy h. sarma coronaviruses (covs) are responsible for causing numerous diseases in broad ranges of vertebrates, including humans. though earlier, covs were only associated with a common cold, for the first time in 2002, new covs related to the 'severe acute respiratory syndrome' (sars-cov) discovered in the human population of china and caused the death of 10% of the total cases worldwide (perlman & netland, 2009; rota et al., 2003) . recently, in december 2019, numerous patients from wuhan, china, reported regarding symptoms like pneumonia. however, initially it was identified as novel coronavirus, namely, 2019-ncov. later, the world health organization (who) renamed that virus as 'severe acute respiratory syndrome coronavirus 2' (sars-cov2) and the diseased caused by them is known as 'coronavirus disease 2019' . on march 11, 2020, the who officially strand rna genome of size 29.7 kb and encodes a viral replicase that is associated with the novel genome synthesis and generation of a 'nested set of sub-genomic messenger rnas, encoding both structural proteins present in all covs: spike (s), envelope (e), membrane (m) and nucleoprotein (n), and a group of proteins specific for sars-cov: 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b'(nieto-torres et al., 2014) . while m and s protein constitutes the major portion of the viral envelope, e proteins are reported to oligomerize and form ion channels (venkatagopalan et al., 2015) . the e protein is located along with s-spike glycoprotein (li et al., 2020) , where it played a significant role in the assembly of the viral genome (westerbeck & machamer, 2019) . 'sars-cov e' protein' is a short, integral membrane protein comprised of 76-109 amino acids and its size range from 8.4 to 12 kda. it start a with short hydrophilic terminal followed by large hydrophobic transmembrane domain and terminates with long hydrophilic carboxyl end. hydrophobic region oligomerise and form an ion-conductive pore in membranes. this protein play key role in various phases of the virus' life cycle, such as envelope formation, pathogenesis, budding and assembly (ashour et al., 2020; schoeman & fielding, 2019) . few studies have also suggested that the 'sars-cov e' proteins' ion channel activity is modulated via pentameric ion channel (pervushin et al., 2009 ). the 'sars-cov e' protein's ion channel activity is detected in the transmembrane region of the protein (verdi a-b aguena et al., 2012; wilson et al., 2004) . selectivity, as well as ion conductance associated with the e protein ion channel, are mostly modulated via the lipid membranes charge within which the pores aggregates. this, in turn, supports that 'lipid headgroups' are the main component of the structure of the channel facing the pore's lumen (verdi a-b aguena et al., 2012 (verdi a-b aguena et al., , 2013 . though the involvement of ion channels in covs pathogenesis remains a topic of debate, recently several studies have suggested that the absence of 'sars-cov e' protein results in an 'attenuated virus', thereby supporting that 'sars-cov e' protein is mainly responsible for pathogenesis (pervushin et al., 2009; regla-nava et al., 2015) . the involvement of other proteins of sars-cov in pathogenesis remains unclear to date (venkatagopalan et al., 2015) . few studies have also reported that mutations within the extra-membrane domain of 'sars-cov e' protein disrupt the normal viral assembly as well as maturation (fischer et al., 1998; torres et al., 2007; verdi a-b aguena et al., 2012) . in transmissible gastroenteritis virus, the 'sars-cov e' protein deletion led to virus trafficking blockage within the secretory pathway as well as virus maturation (curtis et al., 2002) . thus, the 'sars-cov e' protein server as a key biomarker for preventing pathogenesis associated with the sars-cov (pervushin et al., 2009) . for gaining detail insight into the structure & function, to date numerous 'three-dimensional' structures of the 'sars-cov e' protein have been deposited in the 'protein data bank (pdb)'. furthermore, as identifying novel drugs through laboratory approaches demands huge capital as well as investments, there is a continuous demand for screening drug molecules via high screeening computational methods that saves both money as well as times gupta & vadde, 2020b; gupta & vadde, 2020a) . nevertheless, admet ('absorption, distribution, metabolism, excretion and toxicity') as well as 'lipinski's rule of 5s' property passed natural drugs may have either minimum or no adverse-effects gupta & vadde, 2020a,b) . recently, genome of the first sars-cov2 (wuhan-hu-1) has also been successfully sequenced and submitted in genbank (accession no. mn908947.3) (shang et al., 2020) . by considering the above information, in the present study, authors employed computational approach for identifying the best possible structure of the 'sars-cov2 e' protein present in the pdb database to understand its structure and function as well as its behaviour towards various phytochemicals. this, in turn, may help us in identifying few phytochemicals that may inhibit the function of the 'sars-cov2 e' protein; thereby preventing the pathogenesis associated with sars-cov2. in the near future, these phytochemicals may serve as a good contestants for treating diseases caused by sars-cov2 after further laboratory investigation. the uniprot was utilized for downloading the human 'sars-cov e' protein sequence (id: p59637). the ncbi protein database was utilized for downloading the human 'sars-cov2 e' protein sequence (id: yp_009724392.1). multalin, a webbased tool was implemented to detect difference between 'sars-cov e' and 'sars-cov2 e' protein. subseqeuntly, sequence of 'sars-cov2 e' protein was subjected to ncbi's utility 'blastp' (altschul et al., 1990) . based on maximum sequence identity (81%), structure with pdb (protein data bank) id 5 â 29 is detected as the best homologous structure of human 'sars-cov2 e' protein. as the 'sars-cov e' protein function as a homopentamer (pervushin et al., 2009) , the complete structure of 5 â 29, comprised of five chains, was employed for downstream analysis. to gain detail insight into the structural characteristic of the downloaded protein and removing any conflicts present between its atoms of main and side chain gouda et al., 2020; gupta & vadde, 2020b) , mds were employed using 'gromos96-43a1 force field' of 'gromacs 5.1' for 200 ns (abraham et al., 2015) . as 'sars-cov2 e' protein resides is a transmembrane region, at first, downloaded structure of 5 â 29 was embedded in the 'equilibrated bilayer of dppc (dipalmitoylphosphatidylcholine)' using 'g_membed' tool of 'gromacs 5.1' (wolf et al., 2010) using the aid of 'berger lipids' derived parameters from 'berger, edholm, and jahnig' (berger et al., 1997) . further solvation of the entire system till energy minimization followed by equilibrating the complete system under nvt ('constant number of particles, pressure and temperature') and npt ('constant number of particles, volume, and temperature') conditions were carried out as stated in detail at http://www.mdtutorials.com/gmx/ membrane_protein/index.html. final molecular dynamic (md) trajectories as well as the quality of simulations were analyzed via 'gromacs 5.1' (gupta & vadde, 2020b) . 'principal component analysis' (pca) was also carried out for capturing the most flexible area and the motion of the a-helix & the b-strands present within the protein during 200 ns. equilibrated conformers from the md were used to produce the mean structure of the 'sars-cov2 e' protein (gupta & vadde, 2020b) . backbone atoms' free energy for the 'sars-cov2 e' protein was calculated using the 'gromacs 5.1' from 20 ns to 200 ns with 20 ns interval (gupta & vadde, 2020b) . 'three-dimensional' structure of 4153 phytochemicals having 'drug-like' features from our earlier published literatures (gupta & vadde, 2019a , 2020a were employed in the present study. the drugmint server the drugmint server (dhanda et al., 2013) was employed for detecting admet or 'drug-like' properties of each phytochemicals. drugmint server predicts admet or druglikelihood of any drug/phytochemicals using various classification models. all models were trained, tested and evaluated on a dataset comprised of 3206 experimental drugs and 1347 approved drugs of drugbank 2.5. all qsar models were developed using open source software packages like padel, weka, svm_light (dhanda et al., 2013) 'three-dimensional' structures of each phytochemicals were obtained from tipdb database and have either anti-tuberculosis, anti-cancer, anti-platelet, or no therapeutic properties (lin et al., 2013) . the active site pocket present in the 'sars-cov2 e' protein was calculated using the castp server (binkowski et al., 2003) . active pocket with the highest volume as well as area was considered for molecular docking studies with phytochemicals having 250 conformations gupta & vadde, 2020b) via the autodock (morris et al., 2009) tool. three best phytochemical having the minimal binding energy was considered for further study (gupta & vadde, 2020b) . complex formation was done employing the 'discovery studio' software (biovia, 2017) . amongst 250 conformations, the docking result of the 'sars-cov2 e' protein with three different phytochemicals having the minimal binding energy was considered for the 200 ns mds, separately . the prodrg server (sch€ uttelkopf & van aalten, 2004) was employed for generating the ligand's topology parameters. subsequently, complete mds was performed as described above in the mds: phase i. using the gmxapbs utility of the 'gromac 5.1', 2000 snapshots were retrieved from the md trajectories of all the three complexes, individually, to calculate the binding free energy from 20 to 200 ns with 20 ns interval (gupta & vadde, 2020b ). inter-molecular interactions present within the three complexes after 200 ns mds were performed via discovery studio (gupta & vadde, 2020b) . comparative sequence analysis via multalin reveals that 'sars-cov e' and 'sars-cov2 e' protein sequence share 94.74% identity amongst themselves. the 'three-dimensional' structure of one unit of 'sars-cov2 e' comprised of only seven a-helices and eight loops (figure 1(a) ). as there are five homo-units, the complete structure of 'sars-cov2 e' consists of 35 a-helices and 40 loops (figure 1(b) ). as the 'sars-cov e' proteins' ion channel activity is modulated via pentameric ion channel (pervushin et al., 2009) , complete structure comprising of five subunits was employed for the downstream analysis. to understand the structural characteristics of the 'sarsgupta et al., 2018; mehrnejad & chaparzadeh, 2008; rani & lakshmi, 2019; tandon et al., 2015) were employed for estimating the protein stability during the md analysis. the average rmsd value of the protein backbone atoms is estimated to be 2.74 å (figure 2(a) ). rmsd is found to be stable subsequently 170 ns. the value of rmsf fluctuates within 4 å & 6.3 å with an average value of 5.96 å. amino acids that undergo maximum fluctuation during 200 ns mds were val17, ala22, leu19, leu27, phe23, phe26, leu27, val24, val25, val29, ile33, ala36 and tyr42 (i.e. rmsf > 6 nm). as these residues play an important role during protein-ligand interaction, they may serve as a biomarker during the drug discovery process (gouda et al., 2020; gupta & vadde, 2019a, 2020a, 2020b) (figure 2(b) ). the rg values of the protein (figure 2(c) ), fluctuates within 5.81 å & 5.83 å with an average value of 5.82 å; thereby supporting its condensed architecture as well as size gupta & vadde, 2019b) . result obtained from the pca analysis suggests the random movement of the 'sars-cov2 e' protein ( figure 2(d) ) throughout the 200 ns mds. mean number of 'intra-protein' hydrogen bond & 'inter-hydrogen' bond formed between 'sars-cov2 e' protein & water is 1.98 and 1.98, respectively. 'cross-correlation matrix' of the c-a displacement revealed that all residues present within the 'sars-cov2 e' protein experience both negative (depicted via blue shades) as well as positive correlated motions (depicted via red shades) (figure 3(a) ), which in turn support random movement of the 'sars-cov2 e' protein. this finding is also the random movement of the 'sars-cov2 e' protein indicates its involvement in the ion channels. this is in accordance with earlier studies where authors have reported that the ion channel activity of 'sars-cov2 e' proteins is modulated via pentameric ion channel (pervushin et al., 2009) . it is pertinent to note that the structure of 'sars-cov2 e' protein becomes stable after 170 ns. hence, the average structure of the 'sars-cov2 e' protein was obtained from the stable plateau of the rmsd after 170 ns for downstream analysis. further, the castp server was employed for detecting the active site present within the 'sars-cov2 e' protein. the obtained result revealed that the highest volume and area of the binding cavity within the 'sars-cov2 e' protein is 7625.969 å 3 & 7163.067 å 2 , respectively. forty-four amino acids that are involved in the formation of the active site are glu8, thr11, leu12, val14, asn15, val17, leu18, leu19, phe20, leu21, ala22, phe23, val24, val25, phe26, leu27, leu28, val29, thr30, leu31, ala32, ile33, leu34, thr35, ala36, leu37, arg38, leu39, ala40, tyr42, ala43, ala44, ile46, val47, val49, leu51, pro54, val56, tyr57, ser60 , arg61, lys63, asn64 and leu65. subsequently, molecular docking between the protein and ligand with 250 conformations was performed using the autodock tool. 126 â 126 x 126 was assigned as a grid box with a grid center of à0.496, 0.0 and 0.0. the grids were selected very carefully for allocating active sites along with the surrounding surface's major area gouda et al., 2020; . subsequently, molecular docking of the 'sars-cov2 e' protein with ligands having 250 conformations using the autodock tool revealed that the best ten phytochemicals with minimal binding energy are tip006452 (belachinal), tip005365 (macaflavanone e), tip003272 (vibsanol b), tip003258 (14 r ã ,15-epoxyvibsanin c), tip005363 (macaflavanone c), tip000749 (luzonoid d), tip008605 (grossamide k), tip009461 ((-)-blestriarene c), tip005366 (macaflavanone f) and tip005783 (dolichosterone). binding energy of 'sars-cov2 e' protein with tip006452, tip005365, tip003272, tip003258, tip005363, tip000749, tip008605, tip009461, tip005367, tip005366 and tip005783 is à11.46 kcal/mol, à11.07 kcal/mol, à11.07 kcal/mol, à10.56 kcal/mol, à10.49 kcal/mol, à10.47 kcal/mol, à10.50 kcal/ mol, à10.40 kcal/mol, à10.40 kcal/mol, à10.36 kcal/mol and à10.31 kcal/mol, respectively (supplementary file i and table 1 ). binding energy of all 4153 phytochemicals is depicted in the supplementary file i. earlier one study has reported that macaflavanone c may be utilized for treating alzheimer's disease (gupta & vadde, 2019a) . in 2016, teponno and the team reported about the anti-melanogenic property of the grossamide k (bertrand teponno et al., 2016) . earlier seo and his team have also reported that compounds present in the ethyl acetate fraction of sanguisorba officinalis, namely euphormisin m3, arjunglucoside ii, pomolic acid-3-b-o-a-l-arabionopyranoside, 3,3 0 -di-o-methylellagic acid, 3,8-dihydroxy-4,10-dimethoxy-7-oxo-[2] benzopyrono [4,3-b] [1]benzopyran-7-(5h)-one, chikusetsusaponin ii, deglucose chikusetsusaponin iva, belachinal, irilone and ellagic acid, may have the inhibitory effect on inflammasome pathways and protective role in endotoxininduced septic shock. however, to best of our knowledge, no medicinal properties have been assigned to rest seven phytochemicals, namely, macaflavanone e, vibsanol b, '14 r ã ,15-epoxyvibsanin c', luzonoid d, (-)-blestriarene c, macaflavanone f and dolichosterone. as three compounds, namely, belachinal, macaflavanone e and vibsanol b, have the minimal binding energy with the 'sars-cov2 e' protein (table 1) , three separate complexes between 'sars-cov2 e' protein and each ligand were done separately using discovery studio software. the complex of the 'sars-cov2 e' protein with belachinal, macaflavanone e and vibsanol b, separately, will be known as complex a, b and c, respectively, henceforth. (figure 2(a) ). rmsd in all the three complexes is found to be stable subsequently 180 ns. the rg values of all three complexes ( figure 2 (c)) support their condensed architecture as well as size figure 2(d) ). this might be due to decreased potential energy and increased pressure in all the three complexes, which in turn supports that the random movement of the 'sars-cov2 e' protein decrease after binding with these phytochemicals (figure 2(d) ); thereby supporting that these three phytochemicals may function as drugs for treating or controlling diseases caused via sars-cov2 in human. the obtained result revealed that the final binding energy of complex a (à250.979 ± 0.272 kj/mol) <complex b (à214.938 ± 0.280 kj/mol) <complex c (à197.457 ± 1.236 kj/mol). the overall distribution of electrostatic, sasa (solvent accessible surface areas), van der waal, polar solvation, and final, binding energy from 20 ns to 200 ns with 20 ns interval of complex a to c complexes are depicted in table 2 . intermolecular interaction study in all the three complexes, separately, via discovery studio, suggests that amino acid involved in the intermolecular interaction of complex a with hydrophobic bond length ranges from 3.02 å to 5.90 å ( figure 5(c) ). no hydrogen and electrostatic bonds is found in either of the three complexes. in all the three complexes, amino acids present in the chain c and d mostly bind with ligands. amino acids, namely val25 and phe26 helps in bond formation in all three complexes. it is pertinent to note that these amino acids also experience the maximum rmsf during 200 ns simulation in the 'sars-cov2 e' protein; thereby suggesting their importance during protein-ligand interaction. thus, in the near future, these amino acids present in the chain c and d of the 'sars-cov2 e' protein may serve as a biomarker for controlling pathogenesis caused via sars-cov2 in human. in conclusion, the publically available structure of the human 'sars-cov2 e' protein was downloaded and employed to study its structure and function as well as its interaction with various phytochemicals using computational approaches. results obtained revealed that the human 'sars-cov2 e' is a pentameric protein comprised of 35 a-helices and 40 loops. during 200 ns, a-helix and loops present in this protein experiences random movement, which in turn modulate normal ion channel activity; thereby aiding the pathogenesis caused via sars-cov2 in human and other vertebrates. however, after binding with belachinal, macaflavanone e, and vibsanol b, the random motion of the human 'sars-cov2 e' protein gets reduced; this, in turn, inhibits the function of the human 'sars-cov2 e' protein. it is pertinent to note that, this is for the first time, authors are reporting about the medicinal property of macaflavanone e, vibsanol b. as these three phytochemicals, namely, belachinal, macaflavanone e & vibsanol b, have passed the admet test and 'lipinski's rule of 5s', they may be utilized 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site of assembly analysis of sars-cov e protein ion channel activity by tuning the protein and lipid charge coronavirus e protein forms ion channels with functionally and structurally-involved membrane lipids the infectious bronchitis coronavirus envelope protein alters golgi ph to protect the spike protein and promote the release of infectious virus sars coronavirus e protein forms cation-selective ion channels g_membed: efficient insertion of a membrane protein into an equilibrated lipid bilayer with minimal perturbation authors would like to thank mr. jan benzenberg, incident manager, carrier radio access networks (germany), for providing the computational facility to carry out a molecular docking studies. the authors of this manuscript declare no conflict of interest. http://orcid.org/0000-0003-1558-9471 ramakrishna vadde http://orcid.org/0000-0002-5223-7208 key: cord-336119-8g37xsys authors: nimgampalle, mallikarjuna; devanathan, vasudharani; saxena, ambrish title: screening of chloroquine, hydroxychloroquine and its derivatives for their binding affinity to multiple sars-cov-2 protein drug targets date: 2020-06-24 journal: j biomol struct dyn doi: 10.1080/07391102.2020.1782265 sha: doc_id: 336119 cord_uid: 8g37xsys recently chloroquine and its derivative hydroxychloroquine have garnered enormous interest amongst the clinicians and health authorities’ world over as a potential treatment to contain covid-19 pandemic. the present research aims at investigating the therapeutic potential of chloroquine and its potent derivative hydroxychloroquine against sars-cov-2 viral proteins. at the same time screening was performed for some chemically synthesized derivatives of chloroquine and compared their binding efficacy with chemically synthesized chloroquine derivatives through in silico approaches. for the purpose of the study, some essential viral proteins and enzymes were selected that are implicated in sars-cov-2 replication and multiplication as putative drug targets. chloroquine, hydroxychloroquine, and some of their chemically synthesized derivatives, taken from earlier published studies were selected as drug molecules. we have conducted molecular docking and related studies between chloroquine and its derivatives and sars-cov-2 viral proteins, and the findings show that both chloroquine and hydroxychloroquine can bind to specific structural and non-structural proteins implicated in the pathogenesis of sars-cov-2 infection with different efficiencies. our current study also shows that some of the chemically synthesized chloroquine derivatives can also potentially inhibit various sars-cov-2 viral proteins by binding to them and concomitantly effectively disrupting the active site of these proteins. these findings bring into light another possible mechanism of action of chloroquine and hydroxychloroquine and also pave the way for further drug repurposing and remodeling. communicated by ramaswamy h. sarma coronavirus disease pandemic is caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) (gorbalenya et al., 2020) , is a rapidly spreading disease globally and has so far claimed thousands of lives around the world and caused enormous damage to society and economy (cebm, 2020) . world health organization (who) has declared that the covid-19 is a pandemic, and a public health emergency of international concern (wee et al., 2020) . the novel coronavirus or sars-cov-2 has four transmission stages in line with other infectious diseases and is generally categorized into asymptomatic, moderate, extreme, and critical. sars-cov-2 exhibits different symptoms depending on the severity of the disease including fever, dry cough, dyspnea, pneumonia, hypoxemia, encephalopathy, heart failure, and acute kidney injury. there is currently no defined antiviral drug or therapy available for covid-19 treatment, and mostly the disease is managed symptomatically (yuki et al., 2020) . several medications are being tested in clinical trials for covid-19, including antiviral, antiinflammatory, anti-malarial and other pharmacologically active drugs (rabby, 2020) . however, recently chloroquine and its derivative hydroxychloroquine are being positioned as a possible treatment for covid-19. presently, multi-centric global clinical trials are underway to evaluate the therapeutic potential of chloroquine and hydroxychloroquine as a treatment for novel coronavirus infection. the food and drug administration (fda), usa, has, however, approved both chloroquine and hydroxychloroquine for covid-19 control and treatment for emergency purposes (scholz & derwand, 2020) . in order to address the virus infection and replication it is critical to understand proteins involved in the process. functionally, sars-cov-2 consists of two different types of proteins, which include structural proteins and non-structural proteins (nsps). the structural proteins are involved in the formation of the spherical shape of the virus, which including spike protein (trimeric), membrane protein, envelope protein, and the nucleocapsid protein. while sixteen non-structural proteins (nsps) are formed from the proteolytic cleavage of two polyproteins (pp1a and pp1b). these nsps are essential for the metabolic and molecular events include transcription and translation (prajapat et al., 2020) . in this context, key regulatory proteins and enzymes associated with the pathogenesis of sars-cov-2 were selected as drug targets for chloroquine and its derivatives. chloroquine and hydroxychloroquine are anti-malarial drugs, which are also used for the treatment of rheumatoid arthritis and lupus erythematosus (touret & de lamballerie, 2020) . these drugs are believed to be relatively safe when administered within the clinically advised limits with mild side effects. furthermore, chloroquine derivatives have been tested on pneumocystis pneumonia (pcp) for their therapeutic activity to repurposing antimalarial drugs for pneumonia (gomes et al., 2018; yeo et al., 2020) . pneumonia is a lifethreatening symptom for advanced stage coronavirus infected patients and clinicians are using chloroquine and its derivative hydroxychloroquine to treat the disease. currently, these two drugs are being reused for the treatment of covid-19 since the infection involves pneumonia (devaux et al., 2020) . recently, treatment with the combination of both hydroxychloroquine and azithromycin has shown a significant improvement within the covid-19 patients (gautret et al., 2020) . similarly, patients treated with chloroquine and hydroxychloroquine have also shown significant recovery from covid-19 (singh et al., 2020) . chloroquine and hydroxychloroquine is a cost effective drug that has long been a therapy of choice for malaria prophylaxis due to excellent results and good safety and tolerability. recently, world over chloroquine and its derivative analog hydroxychloroquine has garnered enormous attention as a possible treatment for sars-cov-2 infection. although in this context, the exact mechanism of action of chloroquine and hydroxychloroquine is still not known. at the same time some reports have cautioned against the use of chloroquine due to the known dose-related toxicity of chloroquine and its derivative. several adverse events mainly involving retinal and psychiatric symptoms are observed with chloroquine. however, such symptoms are dose-dependent and are observed when dosage levels exceed prescribed pharmacological dosage limits. an understanding of sars-cov-2 disease biology indicates that it is important to target the viral replication in order to effectively control the infection. also, it is perceived that chloroquine and its derivative can prevent the disease onset in covid negative and healthy subjects and treat sars-cov-2 infection in healthy but asymptomatic carriers. this can be effectively accomplished by understanding the detailed mechanism of action of chloroquine and hydroxychloroquine in preventing coronavirus infection. the mechanism of action of these two drugs is not well known, but it has been demonstrated in vitro that these drugs inhibit sars-cov-2 by elevating the endosomal ph, and alter ace-2 terminal glycosylation there by leading to the interruption of virus receptor binding (vincent et al., 2005) . however, if chloroquine and its derivative hydroxychloroquine acts exclusively by elevating the endosomal ph, then chloroquine should act as a broad-spectrum anti-viral agent since modulation of endosomal ph is a common strategy utilized by viruses for internalization. this appears to be doubtful since chloroquine is not effective against most of the viral diseases like dengue (tricou et al., 2010) , chickenguniya (lamballerie et al., 2008) , and hiv (savarino & shytaj, 2015) . however, both chloroquine and its derivative hydroxychloroquine were shown to be of some use in countering the sars virus (vincent et al., 2005) . it is a well-established fact that sars virus (sars-cov) and sars-cov-2 share almost 80% sequence similarity. with this, it can be postulated that chloroquine might be actively binding to one or more sars-cov-2 proteins to inhibit viral replication. to study this, multiple chloroquine derivatives reported earlier, were screened for their binding potential to various sars-c0v-2 virus proteins important for its binding, internalization, replication and budding in the host cell using the molecular docking studies. in this work, we have demonstrated the capability of chloroquine and its derivatives, reported for their anti-pcp potential earlier (gomes et al., 2018) for selective binding to different viral proteins. this work aims at increasing the information for anti-viral mechanism of chloroquine and hydroxychloroquine against the sars-cov-2 virus. this research can support new anti-viral drug discovery against sars-cov-2 virus and at the same time can support drug repurposing efforts around chloroquine. also these results can be used as a basis for modifying clinical dosage of chloroquine and thereby rendering it more effective against coronavirus infection. post the approval of hydroxychloroquine by the food and drug administration (fda), usa, the same has been used as a potential drug for the treatment and management of emerging disease covid-19. by using in-silico molecular docking studies, the binding potential of chloroquine and its derivatives with different sars-cov-2 proteins involved in viral replication was evaluated. the 2d-structures for chloroquine and its derivatives viz. hydroxychloroquine, chloroquine sulfate, chloroquine mustard, chloroquine pyrolidinyl, were taken from pubchem (https://pubchem.ncbi.nlm.nih.gov/) database. while, the 2d-structures of chemically synthesized chloroquine derivatives from gomes et al., 2018 were drawn using chemsketch software and named as cqn2a, cqn2b, cqn2c, cqn2d, cqn2e, cqn2f, cqn2g, cqn2h, cqn2i, cqn2j, cqn21a, cqn21b, cqn1a, and cqn1b (table 1 ). all the structures of chloroquine derivatives were organized as a compound library following energy minimization using the open babel module in pyrx software. all the compounds in this study were also assessed for their drug likeliness based on the lipinski's 'rule of five' using swissadme server. the key regulatory proteins and enzymes associated with the pathogenesis of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) were selected as potential targets for chloroquine and its analogues . specifically, the proteins table 2) . three-dimensional structures of the above drug targets were retrieved from the rcsb protein data bank (www.rcsb.org). before docking, the pdb structures were observed for sequence break and their ligand association by using pymol molecular visualization system (delano, 2002) . proteins (6m71 and 6svb) with sequence break were subjected to homology modeling using the swissmodel server (daina et al., 2017) , subsequently used for further studies. liganded form of protein (6lu7) were identified, retrieved, and employed for validation of docking (nimgampalle et al., 2019) . while non-liganded forms of proteins (6m71, 6vsb, 6vw1, 6vxs, and 6w4b) were subjected to active site prediction using the castp server (tian et al., 2018) . in this study, molecular docking was performed at known active site of ligand form of proteins (6lu7), whereas, both blind docking and predicted active site docking (site-specific docking) was performed to the remaining non-liganded form of proteins (6m71, 6vsb, 6vw1.e, 6vxs, 6w02, and 6w4b) to examine the binding affinities of chloroquine derivatives against each drug target of sars-cov-2. docking was performed using pyrx 0.8 software consist of autodock 4.2 (morris et al., 2009) . the structures of chloroquine and its derivatives were organized as a compound library after subjecting them to energy minimization using the open babel module in the virtual screening software, pyrx. later, the protein structures were refined for heteroatoms and water molecules to demarcate active sites of proteins. further, the gasteiger charges and hydrogen atoms were added to each drug target to maintain coordination between various interactions by using ucsf chimera-1.13.1 software (huang et al., 2014) . then, each drug target was saved in pdb format, and the chloroquine derivative compounds under examination were also uploaded and saved in ligand.pdbqt format. the grid was set around the active site of the drug target for site-specific docking, whereas the grid was maximized to surround the entire protein surface for blind docking. to generate possible binding conformation of chloroquine derivatives, empirical free energy force fields were applied to the lamarckian genetic algorithm (lga) with default parameters (lga: 2500,000 -energy evaluations) (bommu et al., 2019) . the grid spacing was generated around the active site residues of each drug target, and the grid parameters for each drug target are given in table 2 .1 as supplementary file. after completion of docking, the dock results were saved for the observation of binding affinities and binding interactions between ligand-target were analyzed by ligplot software (wallace et al., 1995) . prior to molecular docking studies, all the synthetic derivatives of chloroquine were tested for their "drug-likeliness" properties based on lipinski's rule of five. as shown in table 3 , all the compounds used in the present study follow lipinski's rule of five (molecular weight <500, hydrogen acceptor <10, hydrogen donor <5, and logp < 5) without any exception. these results demonstrate that all the chloroquine derivatives studied in this study are capable of eliciting pharmacological response through their absorption, distribution, metabolism, and excretion. the details of druglikeness and relevant molecular properties of compounds are listed in table 3 . it is important for a chemical compound to demonstrate drug-likeness properties since this would render in order to completely understand the binding potential of chloroquine and its derivatives to different sars-cov-2 proteins, it is important to identify their natural active sites. active sites of proteins can be predicted by ascertaining the geometric and topological properties of protein structures. castp server was used to predicted active sites for the nonliganded form of proteins (6w02, 6m71, 6vsb, 6vw1, 6vxs, and 6w4b). the amino acids that form the active site of each viral protein and their exact position on the polypeptide chain are listed in table 4 . it was presumed that chloroquine and its derivatives can bind to the active site of these viral proteins thereby inhibiting their natural function. the active site of each protein identified by this procedure was used for further molecular docking studies. in this study, nineteen compounds including chloroquine derivatives (chloroquine, hydroxychloroquine, chloroquine sulfate, chloroquine mustard, chloroquine pyrolidinyl) and other chemically synthesized chloroquine derivatives (cqn2a, cqn2b, cqn2c, cqn2d, cqn2e, cqn2f, cqn2g, cqn2h, cqn2i, cqn2j, cqn21a, cqn21b, cqn1a, and cqn1b) were studied to evaluate their binding potential against the various viral proteins and enzymes (6w02, 6lu7, 6m71, 6vsb, 6vw1.e, 6vxs, and 6w4b) through molecular docking approaches. the results clearly demonstrate that the ability of hydroxychloroquine and chloroquine to bind with different viral proteins albeit with different efficiencies. on the contrary some of the chemically synthesized chloroquine derivatives exhibit the higher binding affinities with tested viral proteins than the hydroxychloroquine (table 5 ). our results point to the fact that potential inhibitors against viral target proteins can be derived by further modifying chloroquine structures. in the results, blind docked ligandprotein complexes are illustrated as figures and the amino acid residues involved in the site-specific docking are listed in table 4 as predicted active sites of the non-liganded form of viral proteins. our results demonstrate that hydroxychloroquine can bind to nsp3 with a binding affinity of à5.6 kcal/mol and à7.3 kcal/mol in both blind and site-specific docking, respectively (table 5 ). in parallel, even stronger binding to nsp3 when compared to hydroxychloroquine was demonstrated by chemically synthesized chloroquine derivative, cqn2h showing the maximum binding affinity of à8.4 kcal/ mol and à8.8 kcal/mol in both blind and site-specific docking (tables 5 and 5 .1 supplementary). hydroxychloroquine shows hydrogen bonding with the residues gly47 and leu126, whereas cqn2h forms hydrogen bonds with amino-acid residues asn40, gly46, and gly130 ( figure 1 ). overall, chloroquine and its derivatives demonstrated a higher binding affinity for nsp3 viral protein when compared to other proteins included in this study, thereby signifying their potential as inhibitors of nsp3 function. the main protease is moderately inhibited by hydroxychloroquine with the binding affinity (à4.8 kcal/mol) (tables 5 and 5.1 supplementary). amino acid residues of the main protease involved in the formation of five hydrogen bonds with hydroxychloroquine are leu4, thr24, thr25, thr26, and thr45 ( figure 2 ). however, compared to hydroxychloroquine, both cqn2h and cqn1b show considerable higher binding affinity (à6.0 kcal/mol) to the main protease (tables 5 and 5 .1 supplementary). the compound cqn2h shows hydrogen interactions with the residues of thr24, ser46 ( figure 2 ). our results indicate that some derivatives of chloroquine and its derivatives are capable of binding to the main viral protease. the results obtained from both blind docking and site-specific docking demonstrate that hydroxychloroquine can effectively bind to rna polymerase with binding affinities à5.9 and à5.6 kcal/mol, respectively (table 5 ). the amino acid residues (asn52 and asn209) of rna polymerase are involved in formation of hydrogen bond with hydroxychloroquine ( figure 3) . interestingly, the compound cqn2h, demonstrate strong binding with rna polymerase with the highest binding affinity in both blind docking (à8.4 kcal/mol.) and site-specific docking (à7.0 kcal/mol) (tables 5 and 5.1 supplementary). cqn2h shows the ability to form hydrogen interactions with the rna polymerase residues thr319 and thr394 (figure 3 ). the high binding affinity of cqn2h indicates towards its potential as an inhibitor of viral rna polymerase. hydroxychloroquine exhibits considerable binding efficiency against spike glycoprotein during both blind docking and site-specific docking with binding affinities of à6.0 and à5.4 kcal/mole, respectively (table 5) . additionally, the compounds cqn1b, cqn2h demonstrated even stronger binding to spike glycoprotein with the binding affinities of à7.8 kcal/ mol. and à7.3 kcal/mol in both blind docking and predicted active site binding, respectively (table 5) . interestingly, only single amino acid (ile472) of the viral spike protein is involved in the formation of hydrogen bond with hydroxychloroquine, whereas cqn1b shows no hydrogen bonds formation rather it forms van dar waal interactions with various amino acid residues (figure 4) . the obtained results demonstrate that the compounds cqn1b and cqn2h may act as potential inhibitor of viral spike glycoprotein function. our studies demonstrate that hydroxychloroquine can also bind to the receptor-binding domain with a moderate binding affinity (à5.4 and à5.3 kcal/mol) in both blind docking and site-specific docking ( table 5 ). the amino acid residues of the receptor-binding domain involved in the hydrogen bonding are asp110, met112, phe197, and glu198 ( figures 5 and 5.1 supplementary) . on the contrary other chloroquine derivatives like cqn1a and cqn1b show a strong binding affinity (à7.6 kcal/mol) to the receptor binding domain of the viral spike protein in both blind docking and site-specific docking studies (table 5 ). the high binding affinities exhibited by these compounds represent their potential as inhibitors of receptor-binding domain. cqn1a shows hydrogen interaction with the residues ile154 and asn163 ( figure 5 ). molecular docking studies with chloroquine and its derivatives with adp-ribose-1 monophosphatase demonstrated the ability of hydroxychloroquine to bind with the mentioned viral protein with binding affinity à6.2 kcal/mol in both blind docking and site-specific docking studies (table 5) . a detailed analysis of the dock showed that hydroxychloroquine was able to form hydrogen bonds with leu126, ala129, and ala154 residues ( figure 6 ). two compounds cqn2e and cqn2i, also show effective binding with adp-ribose-1 monophosphatase with the binding affinity of à7.4 kcal/mol in blind docking (table 5 ). the amino acid residues involved in the formation of hydrogen bonding with cqn2i are val149 and ala154 ( figure 6) . the compound cqn2c also demonstrated a strong binding to adp-ribose-1 monophosphatase with the binding affinity à7.6 kcal/mol in site-specific docking studies. hydroxychloroquine shows moderate to minimal binding with replicase protein with binding affinities of à5.4 and à4.3 kcal/mol in blind docking and site-specific docking, respectively ( table 5 ). the residues of replicase protein, val42, and pro58 are involved in the formation of hydrogen bonds with hydroxychloroquine ( figure 7) . however, another derivative of chloroquine, compound cqn2h potentially inhibits replicase protein in both blind docking and site-specific docking with the binding affinities are à7.1 and à5.5 kcal/mol, respectively (table 5 ) and forms hydrogen bonds with arg40, pro58, and thr68 residues of the replicase protein (figure 7 ). in the present work, with the use of molecular docking techniques the molecular interactions between chloroquine derivatives and selective sars-cov-2 viral proteins were studied. since most of these viral proteins can be potential drug targets, this study aims at understanding the potential role of chloroquine and its potent derivative hydroxychloroquine in inhibition of sars-cov-2 viral infection and replication by assessing the binding efficiency of chloroquine and its derivatives to various viral proteins. these results obtained in this study can also be extrapolated to evaluate the therapeutic efficiency of chloroquine and hydroxychloroquine in controlling sars-cov-2 infection. also in this study we have attempted to screen for derivatives of chloroquine that can bind to sars-cov-2 viral drug targets more efficiently than chloroquine with the hope that this can open a case for chloroquine remodeling for better inhibition of sars-cov-2 multiplication. based on the recent reports, some of the essential regulatory proteins and enzymes associated with the pathogenesis of sars-cov-2 were selected as drug targets such as the spike glycoprotein that enables virus internalization, rna dependent rna polymerase that supports replication of viral genetic material, chimeric rbd (receptor binding domain) that interacts with the ace 2, main protease responsible for cleaving the viral polypeptide, non-structural protein3, nonstructural protein 10, non-structural protein 9 (replicase table 3 . drug-likeness properties of chloroquine and its derivatives predicted in swissadme web tool. protein) and adp-ribose-1 monophosphatase. in order to have a detailed understanding of interaction of chloroquine and allied compounds with sars-cov-2 viral proteins the potential active site of these proteins and the amino acid residues involved in formation of these active sites were identified. with the molecular docking studies, the hydrogen bonds and van der waals interactions formed as a result of binding of the drugs to the sars-cov-2 viral proteins were identified (table 5 .1 supplementary). the normal function of sars-cov-2 proteins can be inhibited as a result of the binding of these drugs to the active site of these sars-cov-2 proteins. our results clearly demonstrate that chloroquine and its derivatives can bind to sars-cov-2 proteins and thereby can disrupt the normal functioning of these proteins. also it was found that some chemically synthesized derivatives of chloroquine are more efficient in binding to viral proteins when compared with chloroquine or hydroxychloroquine. however, some of these derivatives can elicit toxicity and therefore better modeling of chloroquine can lead to the identification of an effective inhibitor of sars-cov-2 virus (supplementary table 3 .1). as we write this report, extensive research is ongoing around the globe to discover specific and effective drug(s) or drug combination that can halt the spread of covid-19 pandemic. with the help of computational methods for drug discovery and repurposing, several natural compounds, anti-viral compounds, anti-malarial drugs, antibiotics, and pharmacologically active compounds have been screened and are being investigated for their ability to inhibit sars-cov-2 protein function (elfiky, 2020; fantini et al., 2020; wu et al., 2020) . however, to the best of our knowledge not much research has been performed to probe the effect of chloroquine, hydroxychloroquine and their derivatives on various drug targets of sars-cov-2 virus. post internalization of the virus in the host cell, both nonstructural protein 3 & 5 are involved in the formation of multidomain m protease, which plays a key role in the replication process (stobart et al., 2013) . in this context, the binding potential of chloroquine and its derivatives was assessed for binding to non-structural protein 3 as this interaction can disrupt sars-cov-2 multiplication within the host cell. our results indicate that chloroquine and its derivatives can effectively bind to the nsp 3. moreover, it was found that some of the chloroquine derivatives such as cqn1b (à8.1 kcal/mol), cqn1a (à8.5 kcal/ mol) and cqn2h (à8.8 kcal/mol) can bind even strongly to nsp 3 than the binding affinity of hydroxychloroquine (à7.3 kcal/ mol) to nsp3. the main protease is also a critical enzyme in the life cycle of sars-cov-2 virus involved in the cleavage of polyprotein (pp) at the c-terminal end and leads to the formation of non-structural proteins (lindner et al., 2005) . targeting the main protease is one of the ways to prevent the replication of sars-cov-2. the obtained results clearly demonstrate that the chloroquine and its derivatives cqn2h and cqn1b can bind to the main protease and thereby can potentially inhibit its activity. non-structural protein 12 acts as rna dependent rna polymerase (rdrp). it is a vital enzyme in the replication of rna viruses. therefore, it has been studied in various viruses table 4 . predicted active sites for the non-liganded form of proteins using the castp server. active site predicted from castp 6w02 ala21, asp22, ile23, ala38, ala39, asn40, lys44, his45, gly46, gly47, gly48, val49, ala50, ala52, gly97, ly97, pro125, leu126, leu127, ser128, ala129, gly130, ile131, phe132, ala154, val155, phe156, asp157, leu160 6m71 val166, glu167, his439, phe441, asp452, tyr455, tyr456, ile494, asn496, asn497, leu498, asp499, ser501, lys511, arg513, thr540, met542, asn543, leu544, lys545, tyr546, ala547, ile548, ser549, ala550, lys551, arg553, ala554, arg555, arg555, thr556, thr556, val557, ala558, gly559, his572, leu576, lys577, ala580, val588, ile589, gly590, thr591, thr591, ser592, lys593, phe594, tyr595, trp598, gly616, trp617, asp618, tyr619, pro620, lys621, cys622, asp623, arg624, glu665, val667, lys676, ser681, ser682, gly683, ala685, thr686, thr687, ala688, asn691, leu758, ser759, asp760, asp761, phe793, ser795, trp800, trp800, glu811, phe812, cys813, ser814, gln815, pro832, arg836, ile837, ala840, val844, asp845, ile847, val848, thr853, leu854, arg858, val860, leu862, ile864, tyr903, ser904, asn911, arg914, tyr915. 6vsb ala27, tyr28, thr29, asn30, phe32, thr33, tyr38, pro39, asp40, lys41, val42, phe43, arg44, ser45, ser46, val47, leu48, his49, ser50, thr51, gln52, asp53, leu54, leu56, pro57, phe58, phe59, ser60, asn61, val62, thr63, trp64, phe65, pro82, val83, leu84, pro85, phe86, asn87, asp88, gly89, lys195, asn196, ile197, asp198, gly199, tyr200, lys202, ile203, tyr204, leu212, gln218, gly219, phe220, pro225, leu226, val227, asp228, ile233, asn234, ile235, thr236, arg237, arg246, ser247, tyr248, leu249, thr250, pro251, asp253, val267, val267, gly268, tyr269, tyr269, leu270, gln271, pro272, arg273, thr274, asp287 including the hepatitis c virus and the zika virus (elfiky, 2016; ganesan & barakat, 2017) . recently, the fda approved anti-rdrp drugs (ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir) have shown potential inhibitory activity against the rdrp of sars-cov-2 (elfiky, 2020) . in our study, chloroquine derivative cqn2h demonstrated strong binding affinity to rdrp (à8.2 kcal/mol) which is even stronger that that of hydroxychloroquine with rdrp protein. these results can be valuable in repurposing and redesigning cqn2h as a potential inhibitor of rdrp function. spike glycoprotein plays an essential role in the attachment of coronavirus with the ace2 on the host cell surface. hence, this protein is considered as an important drug target for drug discovery (prajapat et al., 2020) . our results demonstrate that hydroxychloroquine can effectively bind to both spike glycoprotein and chimeric receptor binding domain along with compounds cqn1b, cqn2h that can effectively inhibit spike glycoprotein. besides, the compounds cqn1a, cqn1b can also potentially inhibit receptor binding domain. these results point to the fact that the chloroquine chloroquine sulfate_chebi_50178 à3.9 (à4.0) à4.5 à5.5 (à5.2) à5.9 (à5.1) à5.0 (à5.1) à6.0 (à5.9) à5.1 (à4.9) 4. chloroquine pyrolidylin_zinc1666887 à7.0 (à8.0) à5.0 à6 (à5.8) à6.6 (à5.8) à5.5 (6.0) à6.3 (à6.7) à5.7 (à3.7) 5. chloroquine mustard_zinc5751278 à5.4 (à6.9) à4.2 à5.8 (à5.2) à5.5 (à5.5) à5.1 (à5.5) à5.5 (à5.6) à5.1 (à3.9) chemically synthesized chloroquine derivatives 6. cqn2a ( derivatives can bind to spike glycoprotein, which can potentially lead to disruption of spike protein interaction with the ace-2 on host cell surface. however, in another recent study is was demonstrated that both chloroquine and hydroxychloroquine can prevent the interaction of spike protein with sialic acids and gangliosides present on the host cell surface (fantini et al., 2020 matrosovich et al., 2013 . non-structural protein 9 (nsp9) acts as a replicase protein (rna-binding protein) and its dimerization is essential for viral propagation (prajapat et al., 2020) . therefore, targeting the dimerization of nsp9 can be an effective strategy to control virus multiplication (egloff et al., 2004 , hu et al., 2017 . our results show that chloroquine derivative cqn2h can bind to the replicase protein with higher efficiency than the hydroxychloroquine. further, the derivatives of chloroquine such as cqn2e, cqn2i, and cqn2c can also effectively inhibit adp-ribose-1 monophosphatase than the hydroxychloroquine. from these results, it is apparent that these chloroquine derivatives possess therapeutic activity against sars-cov-2 virus. our studies clearly demonstrate binding of chloroquine, hydroxychloroquine and their derivatives to various sars-cov-2 proteins. however, these results need to be substantiated with binding experiments to ascertain other drug-protein interaction parameters. at the same time, the specificity of these studies can be ascertained from the fact that one of the derivatives of chloroquine, cqn2f did not show binding to any viral protein included in the study. although chloroquine and hydroxychloroquine are losing their popularity due to possible mutagenic activity our literature survey points to the fact that chloroquine and hydroxychloroquine have well established toxicokinetic properties and might only elicit dosage dependent toxicity. also chloroquine and hydroxychloroquine represent an economical option to prevent the spread of covid-19 pandemic. we believe that from our study informed approach can be taken to understand the drug-protein interaction and then further modify the drug structure which will have a minimal toxic burden and maximal inhibitory potential against sars-cov-2 viral proteins. our results also enhance the basic knowledge of the interaction of chloroquine derivatives with the various drug targets of sars-cov-2 and point to a possible novel mechanism of action of the century old drug. from our results, it can be infered that hydroxychloroquine considerably inhibits all the drug targets of sars-cov-2. further, it was found that hydroxychloroquine might not only enhance the endosomal ph, but it could also interact with various proteins of sars-cov-2. chemically synthesized chloroquine derivatives reveal more effective inhibitory activity against all drug targets of sars-cov-2 than hydroxychloroquine. among the various derivatives of chloroquine, cqn2h and cqn1b show potential inhibition against all the drug targets. our results could also be used for further constructive research on chemically synthesized chloroquine derivatives to identify successful drugs for the treatment of covid-19. there is no conflict of interest. the current project being an in-silico work did not require any additional financial support. this is purely a working arising from "work from situation". otherwise, the authors are thankful for the financial support from their respective institutes. http://orcid.org/0000-0002-2052-400x ambrish saxena http://orcid.org/0000-0001-8620-7683 structural probing, screening and structure-based drug repositioning insights into the identification of potential cox-2 inhibitors from selective coxibs global covid-19 case fatality rates swissadme: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules pymol: an open-source molecular graphics tool new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? the severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded rna-binding subunit unique in the rna virus world ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov-2 rna dependent rna polymerase (rdrp): a molecular docking study zika viral polymerase inhibition using anti-hcv drugs both in market and under clinical trials structural and molecular modeling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov-2 infection applications of computer-aided approaches in the development of hepatitis c antiviral agents structure of the rnadependent rna polymerase from covid-19 virus hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial chloroquine analogues as leads against pneumocystis lung pathogens the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 structural basis for dimerization and rna binding of avian infectious bronchitis virus nsp9 enhancing ucsf chimera through web services structure of mpro from covid-19 virus and discovery of its inhibitors crystal structure of adp ribose phosphatase of nsp3 from sars cov-2. biorxiv on chikungunya acute infection and chloroquine treatment. vector-borne and zoonotic diseases the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme crystal structure of the sars-cov-2 non-structural protein 9 sialic acid receptors of viruses crystal structure of adp ribose phosphatase of nsp3 from sars cov-2 in the complex with adp ribose. biorxiv autodock4 and autodocktools4: automated docking with selective receptor flexiblity novel inhibitors of rho-kinase mediated neuroinflammatory pathways and their potential application in recovery of injured spinal cord drug targets for corona virus: a systematic review current drugs with potential for treatment of covid-19: a literature review chloroquine and beyond: exploring anti-rheumatic drugs to reduce immune hyperactivation in hiv/aids does zinc supplementation enhance the clinical efficacy of chloroquine/hydroxychloroquine to win todays battle against covid-19? medical hypotheses structural basis of receptor recognition by sars-cov-2 chloroquine and hydroxychloroquine in the treatment of covid-19 with or without diabetes: a systematic search and a narrative review with a special reference to india and other developing countries chimeric exchange of coronavirus nsp5 proteases (3clpro) identifies common and divergent regulatory determinants of protease activity castp 3.0: computed atlas of surface topography of proteins of chloroquine and covid-19 a randomized controlled trial of chloroquine for the treatment of dengue in vietnamese adults chloroquine is a potent inhibitor of sars coronavirus infection and spread ligplot: a program to generate schematic diagrams of protein-ligand interactions who declares global emergency as wuhan coronavirus spreads cryo-em structure of the 2019-ncov spike in the prefusion conformation analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods hydroxychloroquine may reduce risk of pneumocystis pneumonia in lupus patients: a nationwide, population-based case-control study covid-19 pathophysiology: a review the authors would like to thank iiser tirupati and iit tirupati for their support. key: cord-336542-6asieplk authors: tanco, sebastián; arolas, joan l.; guevara, tibisay; lorenzo, julia; avilés, francesc x.; gomis-rüth, f. xavier title: structure–function analysis of the short splicing variant carboxypeptidase encoded by drosophila melanogaster silver date: 2010-08-20 journal: journal of molecular biology doi: 10.1016/j.jmb.2010.06.035 sha: doc_id: 336542 cord_uid: 6asieplk abstract drosophila melanogaster silver gene is the ortholog of the coding gene of mammalian carboxypeptidase d (cpd). the silver gene gives rise to eight different splicing variants of differing length that can contain up to three homologous repeats. among the protein variants encoded, the short form 1b alias dmcpd1bs (d . melanogaster cpd variant 1b short) is necessary and sufficient for viability of the fruit fly. it has one single repeat, it is active against standard peptide substrates, and it is localized to the secretory pathway. in this work, the enzyme was found as a monomer in solution and as a homodimer in the crystal structure, which features a protomer with an n-terminal 311-residue catalytic domain of α/β-hydrolase fold and a c-terminal 84-residue all-β transthyretin-like domain. overall, dmcpd1bs conforms to the structure of n/e-type funnelins/m14b metallopeptidases, but it has two unique structural elements potentially involved in regulation of its activity: (i) two contiguous surface cysteines that may become palmitoylated and target the enzyme to membranes, thus providing control through localization, and (ii) a surface hot spot targetable by peptidases that would provide a regulatory mechanism through proteolytic inactivation. given that the fruit fly possesses orthologs of only two out of the five proteolytically competent n/e-type funnelins found in higher vertebrates, dmcpd1bs may represent a functional analog of at least one of the missing mammalian cps. the silver (svr) gene was discovered in drosophila melanogaster by bridges in the early 1920s and it maps near the distal end of chromosome x. [1] [2] [3] [4] adult fruit flies with mutations in this gene display cuticles that are pale and silvery in color due to reduced melanization, a finding that gave rise to the name of the gene. silver includes nine exons (1a, 1b, and 2-8), which code for eight different mrna splicing variants. these result in two short protein variants of 433 (protein svr-pe alias "1a short") and 435 [svr-pf alias "1b short" and dmcpd1bs (d. melanogaster carboxypeptidase d variant 1b short)] residues and in six long forms spanning between 1259 and 1439 residues (svr-pb, svr-pc, svr-pd, svr-pg, svr-ph, and svr-pi; see uniprot sequence database entry p42787 and fig. 1a ). [3] [4] [5] [6] the long variants possess the overall modular structure and sequence features of carboxypeptidase d (cpd), a glycosylated 180-kda enzyme studied since the middle 1990s in fruit fly, mouse, rat, duck, bovine, chicken, and humans. [7] [8] [9] cpd was localized to the trans-golgi network, the secretory and reuptake pathways, and transiently on the cell surface. 10 it was hypothesized to remove basic c-terminal residues from proteins and peptides supporting the endoproteolytic action of furin-like serine proprotein convertases of the subtilisin/kexin type. thus, cpd would contribute to processing of hormones (e.g., adipokinetic hor-mone and bradykinin), growth factors, neurotransmitters, and other bioactive peptides (e.g., dynorphin b-14) that follow the secretory and endocytic pathways. [10] [11] [12] [13] moreover, it has been suggested that arginine released by cpd from (a) protein isoforms described for the silver gene as a result of differential splicing (uniprot database entry p42787). each exon product is shown as a distinct bar with the residues it spans. the n-terminal fragments of 150 (encoded by exon 1a) and 152 (encoded by exon 1b) residues show ∼ 39% sequence identity; the extent of the signal peptide (sp) of the former was estimated with program signalp at www.expasy.ch. the short fragments of 5 and 10 residues, as well as the c-terminal fragments of 22 and 55, are unrelated to any other sequence of the protein. the extent of the transmembrane region (tm) is according to the uniprot entry. isoform 7 alias svr-pf, 1b short, herein dmcpd1bs, spans 435 residues, of which 27-420 correspond to the first funnelin + tld repeat studied herein. the flanking peptides protrude from the surface and are unstructured. the last residue encoded by exon 1b is gln152. see also ref. 5. (b) full-length amino acid sequence of dmcpd1bs in single-letter code. the 25-residue signal peptide is underlined, the residues involved in zinc coordination are labeled with full circles, and the two glycosylation sites are indicated by arrows. the cysteine pairing observed in the structure is cys156-cys309, cys236-cys237, and cys268-cys308. the sequential numbering employed throughout the text and the regular secondarystructure elements are shown above each sequence line. the tld is hallmarked by a light gray background. the segment of the regulatory loop, which is disordered in the structure, is shown in boldface and the experimentally verified serine protease cleavage sites are framed. (c) coomassie-stained sds-page analysis of the mature protein (residues tyr26-phe435) reveals two main bands at ∼ 52-58 kda, which indicates heterogeneity in the glycosylation pattern. incubation with trypsin produced a single cleavage within the regulatory loop at arg190 and gave rise to two cleavage product pairs of ∼35 and ∼ 23 kda. the cleavage products are already present in the non-treated enzyme due to incomplete inhibition of the p. pastoris serine protease during protein expression and purification. peptide substrates may stimulate the production of nitric oxide, an important regulator of cellular processes such as neurotransmission, vasodilatation, and tumor progression and survival. 14, 15 cpd belongs to the funnelins, a tribe of zincdependent cps also referred to as m14 metallopeptidases in merops database. 16, 17 its hallmark is an ∼ 300-residue α/β-hydrolase domain first described for bovine cpa, an a/b-type funnelin. in contrast to the latter, n/e-type funnelins such as cpd possess an additional ∼ 80-residue c-terminal transthyretinlike domain (tld). 10, 12 in addition to cpd, seven further n/e-type funnelins are found in humans and mice, of which five show enzymatic activity (cpd, cpe, cpm, cpn, and cpz) and three do not (aebp1/aclp, cpx1, and cpx2). 9 in contrast, drosophila comprises only cpd and cpm orthologs. 5, 6 in vertebrates, cpm binds to cell membranes, but it also circulates in body fluids. it is assumed to participate in the extracellular processing of peptides and thus to have an activity comparable to cpd. [18] [19] [20] cpd is unique in having three funnelin + tld repeats followed by a transmembrane anchor and a small cytosolic domain, which may be required for intracellular routing and export to nascent secretory vesicles. 9, 10, 16, 21, 22 only the first two funnelin domains are catalytically competent in mammals and duck. this also happens in drosophila variants encoded by exon 1b (see refs. 5 and 6 and fig. 1a ). when isolated, the two funnelin repeats show different ph optima and specificity. the n-terminal repeat is more active at neutral ph and prefers arginine over lysine at the c-terminus of peptide substrates. in contrast, the second repeat prefers slightly acidic environments (ph 5-6) and excises lysines slightly more efficiently than arginines. 6 variants encompassing both repeats have broader ph optimum and substrate specificity than singlerepeat variants. this is consistent with the conditions found throughout the secretory pathway, in which ph ranges from 5 to 7. 5, 6 the third funnelin domain of cpd is catalytically incompetent as it lacks critical residues, but it may maintain the overall α/β-hydrolase scaffold. 10 in the duck, it was reported to be recognized by the pre-s region of the large envelope protein of hepatitis b virus during infection. 23, 24 this is reminiscent of ace2, a cp belonging to another structural group, the cowrins, 25 which is the receptor for human sars coronavirus. 26 in contrast to the long, three-repeat variants, the two drosophila short forms have only the first repeat, and they are secreted and soluble ( fig. 1a and b) . among them, only dmcpd1bs is catalytically active. 5, 6 although both short forms are present in the golgi and may participate in physiological processes, the lack of membrane anchors makes them transient. moreover, the endocytic vesicles contain only the long forms. 5 in addition, while the membrane anchors can regulate the long forms through localization/compartmentalization, the soluble dmcpd1bs may be regulated in a different way along the secretory pathway. drosophila silver alias cpd is essential for life as mutants that do not express functional cpd are not viable and die early in the larval stage. the enzyme plays a major role in melanization and sclerotization of the insect cuticle, wing morphogenesis, catecholamine metabolism, phagocytosis, engulfment, memory, and sensitivity to cold and ethanol. [1] [2] [3] [4] [5] [6] 13, 27 interestingly, mutants that contain a functional dmcpd1bs but no three-repeat variant are viable, but they have a silvery body and minor defects in the wings. therefore, this short form is necessary and sufficient for viability. in order to investigate the detailed molecular basis for its activity and regulation, we studied the structure and function of dmcpd1bs and deduced a plausible hypothesis for its regulation in vivo. production and proteolytic susceptibility of dmcpd1bs initial heterologous recombinant overexpression trials of dmcpd1bs in the methylotrophic yeast, pichia pastoris, rendered sufficient protein for structural studies. however, although we assayed several purification strategies, the presence of several bands in sds-page could not be avoided. n-terminal sequencing of these bands revealed that they were cleavage products of the target protein occurring after leu189 that co-purified with the intact enzyme. subsequent assays with different broad-spectrum inhibitors against the major classes of endopeptidases revealed that addition of phenylmethanesulfonyl fluoride (pmsf) during protein expression significantly limited degradation, giving rise to mainly two major bands (fig. 1c , left lane). in contrast, inhibitors of metallo-, cysteine, and aspartyl proteinases had no appreciable effect. as a control, intact protein obtained in the presence of pmsf and purified was assayed for susceptibility to proteolysis by commercial trypsin and subtilisin. these experiments revealed similar degradation patterns to those observed during production in the absence of pmsf. in the case of subtilisin, cleavage occurred after leu189, and for trypsin, cleavage occurred after arg190 (fig. 1c , right lane). according to the provisional genome sequence ‡ (see ref. 28) , p. pastoris apparently encodes four soluble serine endopeptidases: a putative kex2 subtilisin-like proprotein convertase, a sequence relative of the mammalian omi/htra2 family of trypsin-like serine proteases, and two putative orthologs of saccharomyces cerevisiae yscb alias cerevisin (uniprot p09232). the latter enzyme belongs to the subtilisin/kexin family of serine peptidases and preferentially cleaves after arginine, tyrosine, or leucine according to merops data-base. 29 therefore, either of the cerevisin orthologs in p. pastoris may be responsible for the cleavage after leu189 observed during recombinant overexpression of dmcpd1bs. the two variant bands obtained (fig. 1c , left lane) corresponded to species of between 52 and 58 kda and did thus not coincide with the theoretical molecular mass of the protein (47 kda). treatment with endoglycosidase f and n-terminal sequencing revealed that they were glycosylation variants of the intact target protein. there are two potential glycosylation sites in the enzyme at asn133 and asn269. we tried to reduce heterogeneity by constructing two mutants, n133q and n269q. however, the expression yield of both single mutants was dramatically lower than that of the wild type. in addition, both mutants still displayed heterogeneous glycosylation. the double mutant n133q/n269q could not be expressed. these results underpinned the relevance of sugars for this protein, in good agreement with the importance of protein glycosylation in the secretory pathway. 30 the catalytic efficiency of the purified intact protein against the standard chromogenic type b cp substrate, hippuryl-l-arg (table 1) , was comparable to that of human cpe, human cpm, and human and bovine cpu (also known as activated thrombinactivatable fibrinolysis inhibitor, tafia). this is consistent with previous reports on dmcpd1bs produced in baculovirus, which was also active. 5, 6 reported values for human cpn evince slightly lower activity for this substrate due to its preference for c-terminal lysine residues (table 1) . we further produced and purified human cpb to provide a standard for an a/b-type funnelin for comparison and it had about 10-fold higher efficiency. this is in accordance with previous studies, which showed that "digestive" funnelins are more efficient cps than "regulatory" forms against small substrates. 9,34 moreover, intact dmcpd1bs previously incubated with commercial subtilisin or trypsin (see above) had an ∼ 70% decrease of activity. we conclude that cleavage compromises the proteolytic activity of dmcpd1bs. dmcpd1bs is inhibited by the small-molecule inhibitor 2-guanidinoethyl-mercaptosuccinic acid (gemsa) with an apparent inhibition constant (k i ) value of 0.67 ± 0.08 μm. this fits in the range reported for other funnelins of b-type specificity, which show k i values in the order of millimolar to nanomolar for this inhibitor. 9 in contrast, dmcpd1bs is not inhibited by protein inhibitors from potato, leech, tick, or mammal, even at micromolar concentrations. these inhibitors display high affinity for a/b-type funnelins, with low nanomolar inhibition constants, but they are inert against n/e-type funnelins. 16, 35 structure of dmcpd1bs in complex with gemsa dmcpd1bs was crystallized in the presence of gemsa, which significantly improved the crystal diffraction possibly by providing rigidity, as found in the case of tafi. 36 overall, the structure subdivides into an n-terminal catalytic funnelin domain (ile28-his336) and a c-terminal tld (ile337-glu420). the catalytic domain (cd) has a compact globular shape, which resembles the volume obtained when a cone is extracted from a sphere ( figs. 2a and b ). there is a funnel-like opening to the left, within which the active-site cleft lies, at the base of the opening. the cleft is rather shallow, which is compatible with the capacity of funnelins to cleave a large variety of substrates, as only few contacts between the enzyme and the c-terminal stretch of a substrate are required to fix the latter to the active site. the dmcpd1bs cd evinces an α 8 + 2 /β 8 topology conforming to an α/β-hydrolase or plees fold 37,38 ( fig. 2a and b ). it contains a central doubly wound eight-stranded β-sheet (β1-β8), which is strongly twisted and spans the molecule vertically bottom to top. the sheet has a mixed parallel/antiparallel topology and a strand connectivity + 1,+ 2, − 1x, − 2x, − 2, + 1x, − 2, and its core consists of four parallel coplanar central strands (β3-β5 and β8). the catalytic site is located at the c-terminal end of these strands, as observed in other funnelins. 16 these four strands are flanked by two parallel strands at the top (β6-β7) and a β-ribbon (β1-β2) at the bottom ( fig. 2a and b) . the sheet curvature leads to a concave front side, which shelters helices α5, α6, α8, and α9, as well as the active-site cleft. at the back of the molecule, the convex side of the sheet accommodates the surface n-and c-termini of the molecule, as well as helices α1-α4, α7, α8, and the cterminal helix α10 of the cd, which runs left to right across the molecule along the convex face of the βsheet and is bent by ∼ 40°. the funnel-like access to the active site is shaped by irregular segments of disparate length: the loop connecting strand β3 with helix α2 (lβ3α2), lβ5β6, lβ6α7, lα7β7, and, in particular, the 55-residue segment connecting strand β4 with helix α6 (segment β4α6 hereafter). this element contributes to the lower front of the molecule, shapes the funnel rim, and includes two short helices, α4 and α5. between five and nine residues are missing in each of the four molecules present in the asymmetric unit within the surface-located lα5α6 (hereafter referred to as "regulatory loop") within 16 showing the cd (left) and the tld (right). the repetitive secondary-structure elements are shown as green arrows (strands β1-β8 in the cd and β9-β15 in the tld) and coral ribbons (helices α1-α10) and labeled. the catalytic zinc ion is shown as a magenta sphere. the position of the regulatory loop is pinpointed by arrows. (b) close-up view of (a) displaying the active-site cleft and the funnel-like border that gives access to it. the zinc-binding residues are presented as sticks and labeled, as is the bound gemsa molecule. (c) initial σ a -weighted (2mf obs − df calc )-type electron density map centered on the gemsa-binding site. this map was calculated without the inhibitor molecule (omit map) and is shown contoured at 0.75 σ. the final refined model is superimposed (gemsa, magenta sticks; dmcpd1bs, cyan sticks). (d) the quaternary structure of dmcpd1bs in the crystal is a homodimer. (e) the crystal is made up of such dimers, shown in red, orange, yellow, and cyan. two dimers are found in the crystallographic asymmetric unit. the unit-cell box is shown for reference. segment β4α6. two disulfide bonds also contribute to the funnel rim by cross-linking loop lα4α5 with lβ8α10 (cys156-cys309) and lα7β7 with lβ8α10 (cys268-cys308). in addition, a conspicuous disulfide bond between neighboring residues is found within lβ6α7 (cys236-cys237; see fig. 2a and b) . the catalytic zinc ion resides at the bottom of the active-site cleft and is coordinated by the n δ1 atoms of his101 and his217 and, bidentately, by glu104 atoms o ɛ2 and o ɛ1 (fig. 2b) . these residues are provided by lβ3α2 and by the end of strand β5. the protein was co-crystallized in the presence of the small-molecule inhibitor gemsa (fig. 2c) , which mimics a product complex after substrate cleavage in which the c-terminal residue is still trapped in the s 1′ pocket. the carboxymethylene group of the inhibitor binds the catalytic zinc ion asymmetrically through two carboxylate oxygens, thus giving rise to an overall 6-fold metal coordination. the other carboxylate group of gemsa is anchored to arg165 n η2 , asn174 n δ2 , and, bidentately, to arg175 n η1 and n η2 . the guanidinoethylmercapto group, which imitates an arginine side chain, occupies the s 1′ pocket and is surrounded by ser224, gly279, trp282, tyr283, leu285, gln290, and thr303. the characteristic specificity for basic residues of n/e-type funnelins is provided in dmcpd1bs by the side chain of asp228, which binds one of the terminal guanidine nitrogen atoms of gemsa. the role of the general acid/base, essential for catalysis in funnelins and other metaldependent peptidases, 16, 39 is fulfilled here by glu305. the residues that give rise to the characteristic funnelin motif, hxxe + r + nr + h + y + e, 16 the c-terminal tld spans 84 residues in dmcpd1bs and is rod-shaped (fig. 2a) . its nand c-termini are on opposite sides of the rod, which folds into an all-β seven-stranded β-barrel or β-sandwich, with two layers of three mixed (β1, β4, and β7) and four antiparallel strands (β2, β3, β5, and β6), respectively, which are glued by a hydrophobic core. these strands are arranged as two subsequent greek-key-like elements related by a 2-fold axis perpendicular to the sandwich surface. 21, 40, 41 this domain has topological similarity with transthyretin, a conserved plasma protein that tends to aggregate, thus giving rise to several forms of amyloidosis. 42 however, transthyretin contains an eighth c-terminal β-strand that is absent in funnelins. 21 the tld is laterally attached to the cd and interacts with lβ2β3, lα6β5, lα9β8, and lβ6α7 of the latter through hydrophobic residues. it also interacts through a (bidentate) salt bridge formed by an aspartate (asp244) at the beginning of helix α7 of the cd, which is conserved among n/e-type funnelins, and an arginine (arg376) within the tld. as in the case of related n/e-type funnelins, the function of tld is uncertain. it may assist in folding, regulation of enzyme activity, or protein-protein interactions. 16, 21, 22, 35 hypothetical quaternary arrangement dynamic light-scattering and cross-linking experiments with glutaraldehyde showed that protein concentrations below 1 mg/ml rendered a monomer, whereas concentrations above 8 mg/ml gave rise to a monomer-dimer equilibrium. size-exclusion chromatography indicated that the protein is monomeric throughout the concentration range assayed (0.5 to 18 mg/ml, as injected into the fplc system). in the crystals, which resulted from highly concentrated protein solution (18 mg/ml), two pairs of dimers were found in the asymmetric unit ( fig. 2d and e) . inspection of the dimeric interface in the structure reveals that it measures ∼ 950 å 2 , which is larger than the range described for experimentally validated "weak" transient homodimeric proteins (740 ± 140 å 2 ; ref. 43) . such proteins form both monomers and dimers at physiological concentration. in dmcpd1bs, the dimer is symmetric, so that the same structural segments of each protomer are involved in complex formation: helix α6, strands β1 and β2, and loops lα5α6 and lβ2β3 of each cd and loops lβ11β12 and lβ9β10 of each tld. two symmetric bidentate salt bridges (asp353-arg85) and a total of nine hydrogen bonds are observed at the interface. in addition, the two activesite clefts are located on opposite surfaces of the dimer; that is, there would be no steric hindrance for substrate binding (fig. 2d) . however, there is no concluding evidence that the enzyme is a functional dimer in vivo since the protein seems to be monomeric in solution and the dimer could be an artifact of the crystallization process. moreover, we do not know whether the formation of a dimer could have functional implications. to date, no higher oligomerization states than a monomer have been reported for funnelins. only cpn is believed to function in vivo as a tetramer comprising two n/etype funnelin subunits linked to two non-catalytic regulatory domains, 44 but it is not known how these subunits are arranged within the heterotetramer. implications for the other splicing variants of the silver gene the splicing variants of silver give rise to two types of n-terminal repeat (fig. 1a) . these differ in the first 152 (encoded by exon 1b in dmcpd1bs, svr-pb, and svr-pg) or 150 (encoded by exon 1a in svr-pd, svr-pe, and svr-ph) residues, which share ∼ 39% sequence identity. 6 these stretches give rise to the lower half of the cd until the middle of segment β4α6. the second half of the first repeat until the end at glu420 (numbering hereafter is according to dmcpd1bs if not otherwise stated) is identical in all proteins. svr-pc and svr-pi do not possess an ∼ 150-residue n-terminal segment but have a short five-residue tail. as they lack a signal peptide, it is questionable whether they are secreted. they may play an intracellular role instead. a model of variant svr-pe, which had been shown to be catalytically incompetent by fricker et al., 5, 6 based on the coordinates of dmcpd1bs and a structure-based sequence alignment, reveals that the differences in sequence are compatible with maintenance of the overall structure upon minor readjustment of the main chain. most changes are conservative or are compensated for by differences in size of the side chains in neighboring positions. however, there is a slight trend towards smaller side chains at positions contributing to the central hydrophobic core of the funnelin domain, which might lower thermal stability: leu78ala, tyr96leu, ile97val, ile80ala, met100ile, leu111val, leu119ala, leu128val, met143cys, and ser151ala. in addition, the presence of a histidine replacing an asparagine at position 121 would entail a minor rearrangement of the n-terminal stretch up to phe34. in turn, the conservation of the n-glycoslyation site motif around asn133 (asn-leu-thr in exon 1a instead of asn-ser-thr in exon 1b) suggests that the asparagine is also attached to a sugar moiety. however, the presence of a glutamine instead of a glycine at position 129 may entail a rotation of the sugar moiety around the linking bond, n δ2 -c1, which would provide space for the glutamine side chain. in addition, only a four-residue insertion is found within lβ2β3, which should not disturb the interaction of the cd with the tld (see above) due to the maintenance of leucine residues at positions 89 and 90. the main difference between the exon-1a-and the exon-1b-encoded fragments is the substitution of glutamine (gln99 in uniprot entry p42787 isoform 6) for the zinc-binding histidine at position 101 in dmcpd1bs (fig. 1b) . although glutamine is a rare zinc ligand in proteins, 45, 46 in the case of funnelins, it may lead to metal-containing but catalytically impaired or even inert zinc sites, as reported for zinc-ligand mutants of human glyoxalase i. 47 a glutamine at the position equivalent to residue 101 in dmcpd1bs is also found in a series of (putative) proteins from related drosophila species, namely, d. ananassae (uniprot b3n0d7), d. willistoni (b4npc2), d. mojavensis (b4l2t3), d. sechellia (b4iew7), d. grimshawi (b4jx73), d. erecta (b3nwb3), and d. virilis (b4m2c3). these entries span between 1302 and 1495 residues and could represent three-repeat orthologs of the silver gene product, also encoded by an exon that gives rise to a non-functional funnelin domain. in addition to fruit fly species, a glutamine was found in a potential three-repeat ortholog of the starlet sea anemone nematostella vectensis (a7s4k6). in all these cases, it remains to be ascertained whether the glutamine-containing funnelin-like domains are functional, in particular taking into account that a recent report has shown that the third cpd domain greatly contributes to survival of transgenic fruit flies. 13 the six long constructs all have identical sequences for the putative second and third repeats, the transmembrane helix, and the first 51 residues of the cytosolic domain (fig. 1a) . this means that they all have the second catalytically competent and the third silent repeats. they differ only in the very last segments of the cytosolic domains: svr-pb stops after the common stretch; svr-pc and svr-pd have additional 22 residues; and svr-pg, svr-ph, and svr-pi have a further 55 residues, which are unrelated to the others. these differences allow differential trafficking of the protein between the trans-golgi network and the cell surface for the six membraneanchored forms. 5, 6 comparison with duck cpd domain ii dmcpd1bs shows 40% sequence identity with the second repeat of the duck ortholog, which is the only other cpd structure reported. 6, 21, 48 this sequence similarity redounds to close structural similarity, as reflected by an rmsd of 0.97 å for the 368 c α atoms deviating less than 3 å (see fig. 3a ). significant deviations are found, however, within lβ2β3, lβ6α7, lα7β7, and the regulatory loop, lα5α6 (fig. 3b) . these loops contribute to shaping the funnel rim that allows access to the active site (see above). this rim is responsible for interaction with large protein substrates in funnelins. 16 in addition, significant differences are found in two loops of the tld: lβ9β10 and, in particular, lβ14β15, which is on the back surface of the molecule. this region might be important for function in n/e-type funnelin tlds (see above) and structural differences might lead to disparate binding partners in duck cpd domain ii and dmcpd1bs. overall, the most noteworthy difference is found in the regulatory loop, which is partially undefined in dmcpd1bs (see above). as the protein found in the crystals is intact, this flexibility does not arise from cleavage but from intrinsic disorder, which, in turn, might have functional implications (see below). funnelins of a/b type are secreted as zymogens with an n-terminal 90-to 95-residue pro-domain in charge of latency maintenance until the environmental and temporal conditions for activity are given. this ensures that no undesired proteolytic activity occurs in the transit between the locus of biosynthesis and the final destination. 9, 16, 49 in contrast, n/e-type funnelins are not secreted as latent zymogens, and the regulation of the catalytically active forms is believed to rely on their localization: most of these enzymes are bound to membrane or to the extracellular matrix (e.g., long cpd forms, cpm, and cpz). however, soluble variants such as dmcpd1bs may be regulated by other mechanisms. cpd is palmitoylated at cysteine residues of its transmembrane domain in the duck, with implications for its localization and biological function. 50 covalent attachment of fatty acids enhances the hydrophobicity of proteins and contributes to their membrane association, thus providing a means of regulation through localization. the structure of dmcpd1bs has several exposed cysteine residues at the protein surface shaping the funnel rim, which could be potentially palmitoylated. in particular, adjacent residues cys136 and cys137, which were disulfide-linked in the crystal structure (see above), could be present as free thiols in physiological conditions and become palmitoylated. this could be a way to anchor dmcpd1bs to membranes and thus regulate its intracellular trafficking. another, more likely mechanism of regulation could be limited proteolysis within the regulatory loop. intrinsically flexible regions on the molecular surface of a protein directly correlate with instability, conformational changes and lability, and proteolytic susceptibility, 51 in particular in a proteaserich medium such as that of the secretory pathway. this would explain why the regulatory loop is a hot spot for cleavage by host proteases during recombinant heterologous overexpression (see above). the equivalent regions of all other n/e-type metallocarboxypeptidases thus far structurally analyzed, that is, duck cpd domain ii, human cpn, and human cpm, are well ordered and rigid, and they significantly deviate from the conformation found in dmcpd1bs, thus pointing to potential differences in the function of this loop (fig. 3c) . single cleavage at leu189 or arg190 was found to inactivate the protein, possibly due to disruption of the regulatory loop itself and the preceding structural elements α5 and lα4α5. therefore, a similar cleavage in vivo by peptidases along the secretory pathway would provide a means of selective regulation of dmcpd1bs activity. in contrast to mammals, the fruit fly has only two regulatory funnelins/m14b metallopeptidases, one cpd and one cpm ortholog, which is consistent with the fact that drosophila contains fewer genes. hence, the presence of eight splicing variants of cpd, which give rise to both single-repeat soluble and two-and three-repeat membrane-anchored forms, may be conceived as a strategy to compensate for this shortage. 5 this is reminiscent of the finding that only two matrix metalloproteinase paralogs are found in the fruit fly, while 23 have been described in humans. 52 of the five active n/etype funnelins in mammals, cpn is secreted into plasma and forms a dimer of heterodimers. cpm is membrane-anchored and functions extracellularly. cpz is partially targeted to the extracellular matrix and has a frizzled-like cysteine-rich domain that may make it a wnt-binding protein. 22 dmcpd1bs could have a homologous function to cpe, which is likewise a single-repeat funnelin and is found in secretory vesicles. 22 the fruit fly enzyme is active and cleaves synthetic substrates with similar efficiency to human cpe, although the former has a different ph optimum. 6 dmcpd1bs could function as a cpe-like enzyme in mildly acidic regions of the secretory pathway such as the trans-golgi network. however, further studies are required to validate this hypothesis. finally, the structure of dmcpd1bs conforms to the overall fold of n/e-type funnelins, as may be the case for cpe, but evinces two unique features that may correlate with its regulation: (i) two contiguous surface-located cysteines that may become palmitoylated, thus targeting the enzyme to membranes, and (ii) a surface hot spot for proteolytic inactivation. in this way, dmcpd1bs could participate in drosophila development through proteolytic processing of substrates previously targeted by furin-like proprotein convertases and would itself be inactivated at later stages by limited proteolysis. a clone encoding protein svr-pb of drosophila was kindly provided by dr. lloyd d. fricker (albert einstein college of medicine, new york) and was used as template to generate a pcr-construct encoding dmcpd1bs without the signal peptide, that is, encompassing amino acids tyr26 to phe435 (uniprot p42787, isoform 7). this construct was cloned into the ppiczαa vector with a c-terminal his 6 -tag, and transformed into p. pastoris strain km71h following the manufacturer's instructions (easyselect pichia expression kit, invitrogen). selected transformant colonies were used to inoculate 1-l shake-flask cultures, which were grown at 28°c for 36 h in buffered glycerol-complex medium until od 600 (optical density at 600 nm) reached 20-30. cells were collected by centrifugation at 3000g, gently resuspended in 100 ml fresh bmmy medium (buffered glycerol-complex medium supplemented with 1% of methanol instead of 1% of glycerol), and cultured at 28°c for another 24 h for protein expression. pmsf (0.3 mm) was added to the culture every 3-4 h to prevent cleavage of the recombinant protein. for protein purification, the culture supernatant was equilibrated with 30% ammonium sulfate, bound to a hydrophobic chromatography column (butyl-toyopearl 650 m, tosoh bioscience) connected to an äkta purifier system (ge healthcare), and eluted with a decreasing gradient of ammonium sulfate. the eluted sample was subsequently dialyzed against 20 mm tris-hcl, ph 8.0, and purified by anionexchange chromatography (tsk-deae 5pw, tosoh bioscience) by using a linear gradient of 0.4 m ammonium acetate (0 to 30%). dmcpd1bs was then loaded onto a hiload superdex 75 26/60 column (ge healthcare) previously equilibrated with 50 mm tris-hcl and 250 mm nacl, ph 7.5. eluted fractions were analyzed by sds-page, and the purest samples containing the active enzyme were pooled. the protein was bufferexchanged to 10 mm tris-hcl and 50 mm nacl, ph 7.5, and concentrated by using an amicon centricon centrifugal device (10-kda cutoff, millipore). the typical final yield was ∼ 0.3 mg of protein per liter of growth medium. western blot using an anti-his monoclonal antibody (ge healthcare) showed that this protein lacked the c-terminal his 6 -tag, possibly due to autolysis during production/purification, as described for other funnelins. 53, 54 point mutants n133q, n269q, and n133q/n269q were obtained by site-directed mutagenesis by using the quikchange site-directed mutagenesis kit (stratagene) and produced and purified in the same way as the wild-type protein. human pancreatic procarboxypeptidase b was overexpressed and purified as described for dmcpd1bs but without addition of pmsf. the enzyme was activated by controlled trypsin digestion as previously described. 55 dmcpd1bs was denatured in a solution of 0.1% sds and 50 mm β-mercaptoethanol for 5 min at 100°c. the protein was then incubated with endoglycosidase f (sigma) for 3 h at 37°c in the presence of 0.75% triton x-100 in 50 mm phosphate buffer, ph 7.5, and further analyzed by coomassie-stained 10% sds-page. activity was assayed by monitoring the rate of hydrolysis of hippuryl-l-arg (sigma) at λ = 254 nm on a cary 400 uv-vis spectrophotometer (varian) with 50 mm tris-hcl and 0.15 m nacl, ph 7.5, as buffer at 37°c. initial turnover rates were determined from the first 5-10% of the time trace of each reaction for substrate concentrations close to the k m value whenever possible. the kinetic parameters, k cat and k m , were calculated from at least six experimental points by direct fit to a michaelis-menten curve applying a nonlinear least-squares regression analysis with program grafit. three independent experiments were performed. the apparent k i of gemsa (calbiochem) against dmcpd1bs was determined by measuring the inhibition of the hydrolysis of the chromogenic substrate n-(4-methoxyphenylazoformyl)-arg-oh (bachem) at λ = 350 nm by considering linear competitive kinetics as previously described. 56 assays were performed with 100 μm of substrate in 50 mm tris-hcl, ph 7.5. similar inhibitory experiments were carried out by using the protein cp inhibitors from potato (pci), leech (lci), tick (tci), and human (latexin). these protein inhibitors were produced by heterologous overexpression in escherichia coli and purified as published elsewhere. 57, 58 proteolytic susceptibility assays dmcpd1bs (1.3 μg) was incubated with either 30 ng of bovine trypsin (sigma) or 70 ng of subtilisin carlsberg (sigma) for 30 min at 37°c in 50 mm tris-hcl, ph 7.5. equivalent samples without serine protease were used as controls. cleavage products were analyzed by coomassie-stained 10% sds-page, transferred to polyvinylidene difluoride membranes (millipore), and subjected to automated edman degradation analysis in a procise 492 protein sequencer (applied biosystems) at the in-house proteomics service at the universitat autònoma de barcelona. digested samples and controls were tested for cp activity as described for the inhibitory assays. each experiment was performed six times. dynamic light-scattering, cross-linking, and size-exclusion chromatography experiments dynamic light-scattering measurements were performed at six dmcpd1bs concentrations (ranging from 0.5 to 18 mg/ml) in 10 mm tris-hcl and 50 mm nacl, ph 7.5, in a zetasizer nanoseries apparatus (malvern instruments). each sample was measured 30 times and the results were analyzed with the dispersion technology software v5.1. for the cross-linking experiments, dmcpd1bs was prepared at six protein concentrations as above and treated with 0.8 mm glutaraldehyde for 2 min at room temperature in a final volume of 20 μl. the reaction was thereafter quenched with 12 μl of 50 mm tris-hcl, ph 7.5. samples were analyzed by coomassie-stained 10% sds-page. size-exclusion chromatography was carried out for the aforementioned protein concentrations in 10 mm tris-hcl and 50 mm nacl, ph 7.5, using a superdex 200 5/150 gl column (ge healthcare). crystallization and x-ray diffraction data collection crystallization assays followed the sitting-drop vapor diffusion method. reservoir solutions were prepared by a tecan robot and 100-nl crystallization drops were dispensed on 96 × 2-well mrc plates (innovadyne) by a cartesian (genomic solutions) or a phoenix (art robbins/rigaku) nanodrop robot at the high-throughput crystallography platform (pac) at the barcelona science park. the best crystals appeared in a bruker steadytemperature crystal farm at 4°c with protein solution (18 mg/ml in 10 mm tris-hcl and 50 mm nacl, ph 7.5) and 8% polyethylene glycol 4000, 0.2 m kscn, and 0.1 m sodium cacodylate, ph 6.5, as reservoir solution. these conditions were efficiently scaled up to the microliter range with 24-well cryschem crystallization dishes (hampton research). complex crystals with gemsa were obtained by soaking (10 mm inhibitor in reservoir solution) for 5 days. crystals were cryo-protected with 16.5% polyethylene glycol 4000, 15% glycerol, 0.2 m kscn, and 0.1 m sodium cacodylate, ph 6.5. a complete diffraction data set at 2.7 å resolution was collected at 100 k (oxford cryosystems 700 series cryostream) from a single liquid-n 2 flash-cryo-cooled gemsa-complexed crystal on an adsc q315r ccd detector at beam line id23-1 of the european synchrotron radiation facility (esrf, grenoble, france) within the block allocation group "bag barcelona". the crystal was primitive orthorhombic, with four molecules per asymmetric unit. diffraction data were integrated, scaled, merged, and reduced with programs xds 59 and scala 60 within the ccp4 suite of programs (see table 2 ). the presence of pseudomerohedral twinning following twin law "− h,l,k" was identified by using xtriage within the phenix suite of programs. 63 the structure of dmcpd1bs was solved by pattersonsearch methods with program phaser 64 by using the coordinates of duck cpd domain ii [protein data bank (pdb) accession code 1h8l 21, 48 ] as searching model. subsequently, manual model building on a silicon graphics workstation with program turbo-frodo 65 alternated with crystallographic refinement (including tls refinement) with program refmac5 within the ccp4 suite at initial stages and with program suite phenix under consideration of twinning (refined to a fraction of α = 0.079) at the final stages until the model was completed (see table 2 ). this model contained protein residues lys29-val419 (numbering according fig. 1b) for chain a, ile28-glu420 for chain b, glu30-val419 for chain c, and thr27-glu420 for chain d plus a zinc ion, an n-acetylglucosamine sugar moiety attached to asn133, and a gemsa molecule for each protomer. although there was evidence that asn270 was also glycosylated, the electron density maps were too weak to enable reliable modeling. segments 183-190, 185-191, 184-188, and 182-190 were disordered in each of the four chains, respectively. sds-page analysis of washed crystals revealed only intact protein. figures were prepared with programs setor 66 and turbo-frodo. interface analysis was performed with the pisa server §. model validation was performed with molprobity 62 and the whatcheck routine of program what if. 67 the model of variant svr-pe was constructed with turbo-frodo based on the structure of dmcpd1bs and a sequence alignment calculated with clustal-w, 68 which was modified to include structural restraints. the dimer interface was calculated as half of the total surface area buried by the complex. the final coordinates of dmcpd1bs are available from the pdb∥ (accession code 3mn8). the genetics of drosophila the genome of drosophila melanogaster the silver gene of drosophila melanogaster encodes multiple carboxypeptidases similar to mammalian prohormone-processing enzymes flybase: enhancing drosophila gene ontology annotations characterization of the molecular basis of the drosophila mutations in carboxypeptidase d. effect on enzyme activity and expression characterization of drosophila carboxypeptidase d purification and characterization of carboxypeptidase d, a novel carboxypeptidase e-like enzyme, from bovine pituitary tissue distribution and characterization of soluble and membrane-bound forms of metallocarboxypeptidase d metallocarboxypeptidases: emerging drug targets in biomedicine the enzymes. co-and posttranslational proteolysis of proteins cellular carboxypeptidases metallocarboxypeptidase d individual carboxypeptidase d domains have both redundant and unique functions in drosophila development and behavior prolactin and estrogen up-regulate carboxypeptidase-d to promote nitric oxide production and survival of mcf-7 breast cancer cells carboxypeptidase d is up-regulated in raw 264.7 macrophages and stimulates nitric oxide synthesis by cells in argininefree medium structure and mechanism of metallocarboxypeptidases merops: the peptidase database basic carboxypeptidases: regulators of peptide hormone activity structure and function of mammalian zinc carboxypeptidases 252. carboxypeptidase m crystal structure of avian carboxypeptidase d domain ii: a prototype for the regulatory metallocarboxypeptidase subfamily carboxypeptidases from a to z: implications in embryonic development and wnt binding a cell surface protein that binds avian hepatitis b virus particles gp180, a protein that binds duck hepatitis b virus particles, has metal§ www.ebi.ac.uk ∥ www.pdb.org locarboxypeptidase d-like enzymatic activity catalytic domain architecture of metzincin metalloproteases structure of sars coronavirus spike receptor-binding domain complexed with receptor the genetics of biogenic amine metabolism, sclerotization, and melanization in drosophila melanogaster genome sequence of the recombinant protein production host pichia pastoris merops: the peptidase database golgi linked protein glycosylation and associated diseases biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (tafi) comparison of a spectrophotometric, a fluorometric, and a novel radiometric assay for carboxypeptidase e (ec 3.4.17.10) and other carboxypeptidase b-like enzymes the role of the s1 binding site of carboxypeptidase m in substrate specificity and turn-over comparative studies on human carboxypeptidases b and n a carboxypeptidase inhibitor from the tick rhipicephalus bursa: isolation, cdna cloning, recombinant expression, and characterization crystal structures of tafi elucidate the inactivation mechanism of activated tafi: a novel mechanism for enzyme autoregulation the α/β hydrolase fold the plees proteins-a family of structurally related enzymes widely distributed from bacteria to humans caught after the act: a human a-type metallocarboxypeptidase in a product complex with a cleaved hexapeptide crystal structure of human carboxypeptidase m, a membrane-bound enzyme that regulates peptide hormone activity crystal structure of the human carboxypeptidase n (kininase i) catalytic domain evolutionary changes to transthyretin: structure-function relationships structural characterisation and functional significance of transient protein-protein interactions structure and function of human plasma carboxypeptidase n, the anaphylatoxin inactivator function and mechanism of zinc metalloenzymes zinc coordination sphere in biochemical zinc sites involvement of an active-site zn 2+ ligand in the catalytic mechanism of human glyoxalase i the crystal structure of the inhibitor-complexed carboxypeptidase d domain ii and the modeling of regulatory carboxypeptidases advances in metallo-procarboxypeptidases. emerging details on the inhibition mechanism and on the activation process palmitoylation of carboxypeptidase d. implications for intracellular trafficking probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: the case of minibody structural and enzymatic characterization of drosophila dm2-mmp, a membrane-bound matrix metalloproteinase with tissue-specific expression characterization of carboxypeptidase a6, an extracellular matrix peptidase expression and functional analysis of euglena gracilis chloroplast initiation factor 3 human procarboxypeptidase b: three-dimensional structure and implications for thrombin-activatable fibrinolysis inhibitor (tafi) purification and characterization of enkephalin convertase, an enkephalin-synthesizing carboxypeptidase structure of human carboxypeptidase a4 with its endogenous protein inhibitor mammalian metallopeptidase inhibition at the defense barrier of ascaris parasite chapter 25.2.9: xds. in international tables for crystallography scaling and assessment of data quality a new autocatalytic activation mechanism for cysteine proteases revealed by prevotella intermedia interpain a molprobity: allatom contacts and structure validation for proteins and nucleic acids phenix: building new software for automated crystallographic structure determination phaser crystallographic software turbo-frodo setor: hardware lighted threedimensional solid model representations of macromolecules what if: a molecular modelling and drug design program clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice this study was supported by the following grants from european, spanish, and catalan public agencies: key: cord-342639-vf9n2vf9 authors: chang, chung-ke; chen, chia-min michael; chiang, ming-hui; hsu, yen-lan; huang, tai-huang title: transient oligomerization of the sars-cov n protein – implication for virus ribonucleoprotein packaging date: 2013-05-23 journal: plos one doi: 10.1371/journal.pone.0065045 sha: doc_id: 342639 cord_uid: vf9n2vf9 the nucleocapsid (n) phosphoprotein of the severe acute respiratory syndrome coronavirus (sars-cov) packages the viral genome into a helical ribonucleocapsid and plays a fundamental role during viral self-assembly. the n protein consists of two structural domains interspersed between intrinsically disordered regions and dimerizes through the c-terminal structural domain (ctd). a key activity of the protein is the ability to oligomerize during capsid formation by utilizing the dimer as a building block, but the structural and mechanistic bases of this activity are not well understood. by disulfide trapping technique we measured the amount of transient oligomers of n protein mutants with strategically located cysteine residues and showed that ctd acts as a primary transient oligomerization domain in solution. the data is consistent with the helical oligomer packing model of n protein observed in crystal. a systematic study of the oligomerization behavior revealed that altering the intermolecular electrostatic repulsion through changes in solution salt concentration or phosphorylation-mimicking mutations affects oligomerization propensity. we propose a biophysical mechanism where electrostatic repulsion acts as a switch to regulate n protein oligomerization. introduction a fundamental part of viral self-assembly is the packaging of the viral genome into ribonucleoprotein (rnp) complexes called capsids. capsid formation requires two key activities: the interaction between protein and nucleic acid and the ability of the complex to oligomerize [1] . the severe acute respiratory syndrome coronavirus (sars-cov) nucleocapsid (n) protein is a phosphoprotein that performs both functions [2] . while the interaction between sars-cov n protein and nucleic acids has been examined at the genetic, biochemical and biophysical levels, understanding of sars-cov n protein oligomerization is relatively limited. although it is generally accepted that the n protein dimer serves as the basic building block of the nucleocapsid in coronaviruses, the structural and mechanistic bases of how n protein dimers oligomerize to form larger entities remain a mystery [3, 4, 5, 6, 7, 8, 9] . sars-cov n protein consists of two structural domains interspersed between intrinsically disordered regions (idr), a unique arrangement shared among coronaviruses [10] (fig. 1a) . the n-terminal structural domain (ntd, res. 45-181) adopts an ob (oligonucleotide binding)-like fold capable of binding to nucleic acids, whereas the c-terminal structural domain (ctd, res. 248-365) is responsible for dimer formation through a domain-swapping mechanism [3, 8, 9, 11] . we have shown that the ctd dimer is also capable of binding directly to nucleic acids [9, 12] . intriguingly, idrs also seem to modulate nucleic acid binding of sars-cov n protein, with at least one idr, the flexible linker region (lkr, res. 182-247), capable of direct interaction with rna under in vitro conditions [13] . like many capsid proteins, sars-cov n protein has a high isoelectric point (pi), with a large excess of positively charged residues distributed throughout the protein (+6 charges between residues 176-204, the sr-rich region; +7 charges between residues 249-267 in ctd region; +8 charges between residues 370-389 in the c-terminal idr region) that are important for the nucleic acid binding activity [11, 12, 13] . in the absence of nucleic acids, full-length sars-cov n protein predominantly exists as a dimer in solution [14] . however, a truncated mutant that includes part of the c-terminal structural domain and the c-terminal idr (res. 283-422) was shown capable of forming high-order oligomers in a concentrationdependent manner [15] . x-ray crystallography results of the ntd and ctd imply that the two structural domains also have oligomerization potential, even though they showed no indication of forming oligomers in solution [16] . in particular, we have shown that the ctd packed in a helical conformation with dimension and charge distribution that conforms to that observed in em structures ( figure 1b , 1c) [9, 17] . these studies suggest that sars-cov n protein could potentially form high-order oligomers by itself if protein concentrations were high enough, and small populations of oligomers could exist even at low concentrations, but could not be detected through conventional methods. closer inspection revealed that the interface area between the dimers in the helical packed crystal structure of ctd is small, consistent with the inability to detect higher order ctd oligomer in solution. nonetheless, such an oligomerization behavior may be relevant to rnp packaging. sars-cov n protein is highly phosphorylated in cells, and tentative phosphorylation sites have been mapped to the ser/argrich portion of the lkr [18, 19, 20] . notably, it has been suggested that the lkr is directly involved in n protein oligomerization [21] . a conflicting report, however, indicates that the lkr interfered with n protein oligomerization when the c-terminal domain is present [22] . peng et al demonstrated that phosphorylation of the lkr by the sr protein kinase-1 (srpk1) partially impaired the self-association of the full-length protein [19] . although there is a connection between phosphorylation and n protein oligomerization, whether this is due to a simple charge effect, conformational changes or involves interactions with other components of the virus-host system remains unclear. in this study, we used in vitro disulfide trapping and nmr spectroscopy to detect oligomeric species of the sars-cov n protein ctd in solution. we found that interactions between ctd dimers conform to that predicted from the helical crystal packing. extending our findings to a di-domain construct (dd, res. 45-365) which includes the ntd, ctd and lkr, we conclude that the dd is also able to form oligomers through the ctd in solution. introduction of negative charges to the lkr through phosphorylation-mimicking mutations in the dd construct resulted in enhanced oligomerization. we discuss the implications of our findings in the context of viral capsid assembly and propose a model where coronavirus n protein oligomerization is regulated by the modulation of charges on the protein. various protein domains were cloned and expressed as described previously [8, 10] . for disulfide trapping experiments, we chose mutation sites that would form disulfide linkages based on the crystal packing structures of the sars-cov n protein ctd ( figure 1 ) [9] . sites for the phosphomimicking mutations were chosen based on recent literature [18, 19, 20] . standard pcr techniques were employed to obtain mutant clones, which were then confirmed through dna sequencing. mutant proteins were expressed in escherichia coli bl21(de3) cells, followed by affinity and size-exclusion chromatography purification as previously described [8, 10] . isotope-labeled proteins for nmr studies were prepared using established protocols [8, 10] . purification of u-15 n, 2 h-ctd q290c tetramer was performed by collecting the tetramer fraction from a superdex 75/60 size exclusion column after treating the protein with 10 mm b-mercaptoethanol at 4uc for one hour and extensive washing with fplc buffer (50 mm sodium phosphate, 150 mm nacl, 1 mm edta, ph 7.4) in an amicon centrifugal concentrator (millipore, ma). nmr spectroscopy. samples contained 0.1-0.4 mm protein in nmr buffer containing 10 mm sodium phosphate, ph 6.0, 50 mm nacl, 1 mm edta, 0.01% nan 3 , 10% d 2 o and complete protease inhibitor cocktail (roche, germany). samples of cys mutants also contained 10 mm dithiothreitol (dtt) to avoid formation of disulfide bonds. all experiments were performed on bruker 600-mhz spectrometers equipped with cryoprobes at 30uc unless stated otherwise. the acquired data were processed with the topspin suite (bruker biospin, germany) or inmr (nucleomatica, italy). fixed amounts of purified proteins containing cysteine mutations (3 mg/ml for the ctd and 1.5 mg/ml for the dd) under various buffer conditions were allowed to react with 10 mm of bmercaptoethanol for 1 hour at 4uc, followed by extensive washing on an amicon centrifugal concentrator with fplc buffer to remove the b-mercaptoethanol. molecules in close contact and correct orientation spontaneously form disulfide linkages, which can be assayed through size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). controls in the form of dd q290c were performed for each experiment to ensure b-mercaptoethanol efficacy. experiments were conducted on an akta fast-performance liquid chromatography (fplc) system (ge healthcare, ca) equipped with a superdex 75/60 column at an elution rate of 1.0 ml/min. apparent molecular weights of the proteins were estimated from the elution profile calibrated with the lmw gel filtration calibration kit (ge healthcare, ca). elution volume and molecular weight have the following relationship: where mw is the molecular weight in daltons and e.v. is the elution volume in ml. peak quantification was achieved through analysis with igor pro software (wavemetrics, or), where each peak in the chromatogram was fit with a gaussian curve and the underlying area integrated in the program. area integration errors were estimated from the residual between fitted and experimental curves. disulfide trapping samples were boiled for 2 min at 94uc in the presence and absence of b-mercaptoethanol. the boiling time was kept short (2 min) to avoid protein aggregation. the samples were then loaded on a nupage 4-12% bis-tris gel (invitrogen, ca) and run at room temperature according to manufacturer's instructions. the gel was digitized on a uvp biodoc-it equipped with a fluorescent plate (uvp, ca). quantification of the disulfide-linked and unlinked bands was achieved through the imagej software (national institutes of health, ma). the solution structure (pdb id: 2jw8) and crystal structure (pdb id: 2cjb) of the ctd spanning residues 248-365 were obtained from the protein data bank (www.rcsb.org). all structure images were prepared with pymol (www.pymol.org). within the crystal asymmetric unit, the sars-cov n protein ctd packs as an octamer which stacks to form a helical arrangement with a continuous positively charged surface that could potentially allow the rna to bind to it through electrostatic interactions ( fig. 1 ) [9] . such organization suggests a rnabinding facilitated mechanism for the formation of a helical rnp particle. it is unclear whether the oligomer structure is biologically relevant, since there have been no reports of oligomer species being detected in solution. to test the possibility that the oligomer structure reflects the existence of transient interactions that have been trapped during the crystallization process, we applied an in vitro disulfide trapping technique in an attempt to capture these transient interactions in solution. since wild-type sars-cov n protein contains no cysteine residues, we engineered single-site cysteine mutations at various locations (t264c, q290c, r294c, r320c, and s328c) on the ctd dimer. q290 and r294 are located at helix 2 and s328 is located at the loop region connecting the two b-strands. based on the crystal packing of the ctd structure as shown in figures 1b-1e , both q290 residues (colored magenta) in a ctd-dimer are located at the interface of the adjacent dimers in the oligomer. the distance between the bcarbons of the q290-q290 pair is within 8 å , a suitable distance for disulfide trapping reactions ( figure 1c ) [23] . similarly, the bcarbons of the r294-r294 pair (colored green) are also within a distance suitable for disulfide bond formation. on the other hand, s328 residues (colored cyan) of the ctd dimer are located in the central cavity of the ctd super-helix and are in a favorable position to form inter-dimer disulfide bonds with another s328 residue. the rest of the mutations are not located at the interface and were engineered as negative controls. the mutants did not perturb the structure of the ctd when monitored through 15 nedited heteronuclear single-quantum coherence (hsqc) nmr spectroscopy as judged by the absence of chemical shift perturbation greater than 0.03 ppm other than that of the mutated residues. we then tested for the ability of these mutants to spontaneously form disulfide linkages at 150 mm salt concentration through sizeexclusion chromatography. none of the mutants are located close enough to form intra-dimer disulfide linkages, thus any disulfide linkage must be due to inter-dimer protein disulfide bond formation. the elution profile from a superdex-75/60 sizeexclusion chromatography (sec) column (molecular weight cutoff 75 kda) of the ctd mutants, shown in figure 2 , fall in two groups: ctd q290c , ctd r294c and ctd s328c had high oligomerization potential; whereas ctd t264c and ctd r320c had little to no oligomerization potential. we observed three major peaks with molecular weight of 32 kda, 64 kda and higher molecular weights for peaks d, t and h, respectively, in the chromatograms of ctd q290c , ctd r294c and ctd s328c . the first peak eluted out from the column, peak h, has a molecular weight outside of the resolution limit of the resin. these molecular weights correspond to dimer and tetramer for the d and t peaks. the populations of the oligomers (t + h) are plotted on figure 2b . our results are in general agreement with the proposed crystal packing structure of the ctd with gln290, arg294 and ser328 having higher populations of oligomers. these results qualitatively suggest that the ctd of sars-cov n protein is capable of transient self-association through the oligomer interface identified in the crystal structure. we also tested the oligomerization potential of the ntd since its crystal packing hinted at the possibility of ntd oligomerization [16] . we introduced cys mutations at arg69, asn127, glu137 and thr167; sites that have the potential to form spontaneous disulfide linkages based on the trimeric crystal packing structure of the ntd [16] . according to the structure, arg69-glu137 and asn127-thr167 pairs are close in space and should have high probability of forming spontaneous disulfide linkages. however, we did not observe significant oligomer formation from our chromatograms ( figure 2c) , suggesting that the ntd either does not form oligomers or forms oligomers through an unidentified intermolecular interface. electrostatic screening drives self-association of sars-cov n protein ctd sars-cov n protein is highly electropositive due to an abundance of basic residues. these charges are considered important for rna binding, but they are also potentially deterring the self-association of the protein through electrostatic repulsion [11, 12] . the inter-protein charge-charge interaction can be affected by salt concentration, thus we first studied the effect of buffer salt concentration on trapped self-associated species of ctd q290c and the results are shown in figure 3 . the systematic shift of the chromatograms toward higher retention volume (lower molecular weight) with increasing salt concentration ( figure 3a ) are due to non-specific adsorption of the protein to the superdex matrix [24] . the relative amount of tetramer and larger oligomers in solution increases with increasing salt concentration ( figure 3b ), suggesting that reducing charge repulsion by increasing salt concentration enhances self-association of ctd. salt-induced conformational changes, electrostatic screening or a combination of both potentially could promote self-association of the protein [25] . however, one would expect the resonance positions of the 15 n-edited hsqc of the ctd q290c in low-salt (50 mm nacl) and high-salt (500 mm nacl) conditions to be different if there were changes in the conformation. since the two spectra are virtually identical ( figure 3c ), one can rule out the possibility of conformational change-induced oligomerization. we further tested the oligomerization behavior of a q290c mutant of the di-domain construct consisting of the ntd, the linker region and the ctd (dd q290c , a.a. 45-365). we utilized a non-reducing denaturing polyacrylamide gel electrophoresis (page) assay due to the large molecular weight of the dd dimer (, 70 kd), which is close to the void volume of the sec column used for the ctd studies. because the positions of the two gln290 in the dimer are far apart, formation of disulfide bonds in the q290c mutant would require at least two dimers to draw close together in space, such as formation of tetramers of higher order oligomers. denaturation of the dimer under non-reducing conditions would form monomers, whereas under the same conditions, high-order oligomers would form a mixture of dimers (those that formed a disulfide bond) and monomers. so presence of dimer bands on a non-reducing denaturing gel would reflect the presence of tetramers or high-order oligomers. consistent with our previous results, we also detected oligomers of the dd q290c as shown in figures 4a and 4b . interestingly, we saw little effect on the oligomerization of dd q290c when changing solution salt concentration. although the reason for the discrepancy between dd q290c and ctd q290c is unknown, one possible explanation is that the additional positive charges of dd q290c require much stronger electrostatic screening (e.g. even higher salt concentrations) to have an observable effect on the oligomerization of the protein, akin to the electrostatic screening threshold observed in crystallization studies [26] . we further examined whether changing the electrostatic properties of the protein itself would affect transient selfassociation. we chose the putative phosphorylation sites on the flexible linker as our prime target, and assayed the effect of negative charges on n protein self-association by changing these sites from ser to glu in the dd q290c mutant [18, 19, 20] . in figure 5 , we observed that gradual introduction of negative charges on the unstructured linker had a positive effect on the oligomerization of the dd when compared to the dd q290c control, with maximum effect achieved when 3 negative charges were introduced per each chain (see figure s1 ). further increases in negative charges (s185/189/195/202/203/207e) were less effective in enhancing dd q290c oligomerization. we checked the overall structure of mutants mimicking putative phosphorylation with known kinases [19, 20] , i.e. s189e, s207e, s202/203e and s189/202/203/207e with 15 n-hsqc and found that the extra negative charges did not affect the overall structure of the dd (data not shown), suggesting that electrostatic interactions rather than conformational changes are responsible for the increased tendency to self-associate. overall, our results suggest that hyperphosphorylation of the lkr, which reduces the total positive charge of the n protein, can enhance and regulate oligomerization of dd through electrostatic effects. understanding the self-association of capsid proteins is a fundamental step towards unraveling the mechanism of viral self-assembly. the study of self-association of coronavirus n proteins poses a particular challenge due to the intricate arrangement of structured and disordered domains that have been implicated in protein self-interaction. the literature on how sars-cov n protein oligomerize is rather confusing and the mechanism by which n protein associate with rna to form the helical rnp is also far from understood. it has been previously established that the sars-cov n proteins exist as dimers in solution with little indication of forming higher order oligomers in the absence of nucleic acids [8, 10, 14] . through a combination of disulfide trapping and structure-based mutagenesis, we successfully trapped transiently interacting oligomers of the ctd, suggesting that transient self-association between dimers do occur at specific site(s). the q290c mutation proved to be particularly useful in this study because it had little effect on the structure of the ctd and was electrostatically neutral, yet allowed for the selective detection of direct physical interaction between dimers. it enabled us to explore the effect of various variables (e.g. salt concentration and phosphorylation) on the oligomerization of the sars-cov n protein in a controlled environment. this study provides biochemical evidence that the ctd, but not ntd, is a transient intermolecular self-association site of sars-cov n protein. our results further suggest that transient self-association can be regulated by lowering intermolecular electrostatic repulsion, either through environmental charge screening or charge modifications on the protein itself. interestingly, our data is contrary to what was found in a number of previous studies. luo et al. reported that oligomerization of a sars-cov n protein construct spanning residues 283-422 was not affected by salt [15] . peng et al. found that sars-cov n protein phosphorylated by srpk1 induced less oligomerization than its unphosphorylated counterpart [19] . the disagreement most likely lies in the differences between the cross-linking techniques employed by the different groups. our experimental design requires that two dimers must physically interact with each other close to the mutation site before crosslinking can take place, whereas the use of a chemical cross-linker in the previous studies did not distinguish between dimers that arise from intra-dimer or inter-dimer associations. in luo et al. 's construct, the loss of residues 248-282 not only strips away most of the positive charges on the surface of the dimer, but also results in the loss of three n-terminal helices, which could cause adverse effects on the structural integrity of the protein [12] . over all, we believe that our method provides a more direct measure of the selfassociation of the basic building blocks involved in capsid assembly by taking into account the structural modularity of sars-cov n protein. it should also be noted that although the free-form nterminal domain did not oligomerize in our system, under in vivo conditions it still could form oligomers in the presence of rna as proposed by fan et al for the n protein of the infectious bronchitis virus [5] . other parts of the coronavirus n protein, e.g. so-called c-terminal tail, have also been implicated in protein oligomerization [15, 27] . in fact, the multitude of regions found to contribute towards oligomerization of coronavirus n proteins suggests that n protein self-association is an intricate affair involving intermolecular interactions among multiple domains of the protein. our results show that it is possible to change the oligomerization potential of sars-cov n protein by tinkering with the electrostatic characteristics of the system (figures 3 and 4) . normally, the positively charged n protein exerts a repulsive force that prevents self-association. in the case of the ctd, we enhance the charge screening effect and reduce the intermolecular repulsion when increasing the salt concentration, thus resulting in enhanced oligomerization. however, this salt dependency was lost when we changed the system to the dd construct ( figure 4 ). this could arise from the large number of charges in the dd (+36 charges/dimer) versus in the ctd (+14 charges/dimer), which would make it more difficult to screen the charges using the salt concentrations in this current study. interestingly, our observations could be related to those by verheije et al [28] . they observed in situ that self-interaction of ctd was independent of rna, whereas homotypic interaction of full-length n protein required the presence of rna. one could speculate that the cellular environment provided enough charge screening for ctd selfassociation, whereas rna was required to counterbalance the excessive charges of the full-length protein. n proteins of coronaviruses are highly phosphorylated in infected cells [29, 30] . sars-cov has also been shown to be phosphorylated in cells by several kinases, mainly at the serine residues in the sr region [18, 19, 20] . however, the in vivo phosphorylation sites of sars-cov n protein in actual infection have not been mapped. furthermore, peng et al. reported that phosphorylation of sars-cov n at the sr-rich motif could modulate its translation inhibitory activity and also its multimerization activity [19] , thus phosphorylation may play a role in regulating the viral life cycle. our findings that electrostatic repulsion acts as a means of modulating nucleocapsid assembly and that an optimal number of charges is favored for multimer formation suggest a possible mechanistic roles for phosphorylation in the regulation of sars-cov nucleocapsid assembly. in a fully formed capsid, each n protein would be stabilized by a network of weak protein-protein interactions with adjacent n proteins and protein-rna interactions with the viral genome. the protein-rna interactions neutralize the excessive charges on the n protein, thus negating the effect of electrostatic repulsion. under these conditions, extensive phosphorylation of sars-cov n protein is not necessary and the protein can maintain a hypophosphorylated state within the virion as proposed in a past study [20] . this hypophosphorylated state does not necessarily imply complete dephosphorylation. in fact, it would be advantageous to ''tune'' the n protein such that it retained a small number of phosphorylated sites even in the assembled form (e.g. the ''diphosphorylated'' mimics in fig. 5b ). one could then envision that further dephosphorylation events would enhance uncoating of the viral rna due to electrostatic repulsion between n protein dimers (e.g. ''mono-phosphorylated'' mimics in fig. 5b ). such a possibility has been proposed for jhmv, a neurotropic strain of mouse hepatitis coronavirus (mhv), where dephosphorylation of the n protein by an endosomal-associated cellular protein phosphatase was found to facilitate viral infections [31] . newly produced n protein, on the other hand, would be able to bind to rna with high affinity, but capsid formation could be impaired due to lack of protein-protein interactions caused by residual electrostatic repulsion. in this case, hyperphosphorylation of n protein could become a critical assisting factor, i.e. a ''last straw'' in neutralizing excessive electrostatic forces, in nucleocapsid assembly (e.g. ''triphosphorylated'' mimics in fig. 5b ). electrostatic repulsions have been shown to play similar roles in other viruses, where n proteins often have excessive positive charges. hepatitis b virus (hbv) n protein, for example, has an isoelectric point of 9.93, and forms icosahedral capsids rather than helical ones found in coronaviruses. an increase in the solution salt concentration has been shown to induce the formation of empty hbv capsids, and theoretical studies have attributed the phenomenon to charge screening effects [32, 33] . recently, a ''charge balance hypothesis'' was proposed to explain hepatitis b virus (hbv) capsid stability, assembly, rna encapsidation, and dna replication [34, 35, 36] . this hypothesis emphasized the importance of a balanced electrostatic interaction between the positive charge from the arginine-rich domain (ard) of the core protein (hbc) and the negative charge from the encapsidated nucleic acid. our results reinforce the common theme that electrostatic repulsion is a universal mechanism of capsid assembly modulation in viruses. the observation of trapped transient oligomer shown in the present study does not mean that ctd acts as the nucleation center for the capsid formation. in fact, we had previously shown that n protein interacts with nucleic acid at multiple sites and with high affinity, thus n-rna interaction presumed to be the driving force for the ribonucleoprotein (rnp) formation [13] . in this rna binding-coupled rnp packing scenario, the multitude of weak protein-protein interactions reinforces the protein-rna interaction and contributes towards the assembly of a stable rnp, which guarantees the formation of nucleocapsids containing genetic material. the modular structure and multiple sites of moderate rna binding affinity of the n protein not only allow the packaging of a stable rnp but also offer an energetically favorable condition for the expression of the viral genomic information. one can envision an unzipping mechanism for unwinding of the viral rna molecule and dissociation of the rna molecule from the n protein in a stepwise manner, one module at a time, without the need to overcome a high-energy barrier. this is consistent with the concept for virus assembly that capsid proteins associate through locally weak interactions to form globally stable structures [25] . weak interactions between capsid proteins minimize formation of kinetic traps, allow a greater degree of regulation of assembly, and may be essential for viruses where dissociation is part of the virus life cycle. on the other hand, newly synthesized n protein would also be involved in many rna-protein or protein-protein interactions, most likely modulated by n protein phosphorylation state, affecting viral and host processes other than nucleocapsid assembly [2] . more works need to be performed to understand the effect of n protein phosphorylation on viral and host processes other than nucleocapsid assembly. by disulfide trapping technique we measured the amount of transient oligomers of n protein mutants with strategically located cysteine residues and showed that sars-cov n protein is capable of transient oligomerization in solution through the ctd in the absence of nucleic acids. the data is compatible with the helical oligomer packing model of n protein observed in crystal. this transient oligomerization process is driven by the neutralization of excessive charges on the protein, which can be accomplished through changes in environmental salt concentration and protein phosphorylation. the data is consistent with a general mechanism whereby the electrostatic repulsion between n protein molecules acts as an oligomerization switch. it also reinforces the concept of rna-binding coupled rnp packaging in sars-cov. figure s1 representative strips from sds-page of selected dd mutants after disulfide trapping. the arrow denotes the trapped species originating from tetramers or higher order oligomers. (pdf) theoretical aspects of virus capsid assembly the sars-cov nucleocapsid protein: a protein with multifarious activities. infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases recombinant severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein forms a dimer through its c-terminal domain crystal structure of the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona-and arteriviridae the nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its n-terminal domain and multimerization properties x-ray structures of the n-and c-terminal domains of a coronavirus nucleocapsid protein: implications for nucleocapsid formation characterization of n protein selfassociation in coronavirus ribonucleoprotein complexes the dimer interface of the sars coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure structure of the sars coronavirus nucleocapsid protein rna-binding dimerization domain suggests a mechanism for helical packaging of viral rna modular organization of sars coronavirus nucleocapsid protein structure of the n-terminal rna-binding domain of the sars cov nucleocapsid protein solution structure of the c-terminal dimerization domain of sars coronavirus nucleocapsid protein solved by the sail-nmr method multiple nucleic acid binding sites and intrinsic disorder of severe acute respiratory syndrome coronavirus nucleocapsid protein: implications for ribonucleocapsid protein packaging in vitro biochemical and thermodynamic characterization of nucleocapsid protein of sars carboxyl terminus of severe acute respiratory syndrome coronavirus nucleocapsid protein: self-association analysis and nucleic acid binding characterization nuclear magnetic resonance structure of the n-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome coronavirus supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization glycogen synthase kinase-3 regulates the phosphorylation of sars-coronavirus nucleocapsid protein and viral replication analysis of multimerization of the sars coronavirus nucleocapsid protein sr-rich motif plays a pivotal role in recombinant sars coronavirus nucleocapsid protein multimerization use of site-directed cysteine and disulfide chemistry to probe protein structure and dynamics: applications to soluble and transmembrane receptors of bacterial chemotaxis the critical role of mobile phase composition in size exclusion chromatography of protein pharmaceuticals are weak protein-protein interactions the general rule in capsid assembly optimization of salt concentration in peg-based crystallization solutions oligomerization of the carboxyl terminal domain of the human coronavirus 229e nucleocapsid protein the coronavirus nucleocapsid protein is dynamically associated with the replication-transcription complexes phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells synthesis and subcellular localization of the murine coronavirus nucleocapsid protein endosomal association of a protein phosphatase with high dephosphorylating activity against a coronavirus nucleocapsid protein weak protein-protein interactions are sufficient to drive assembly of hepatitis b virus capsids role of electrostatic interactions in the assembly of empty spherical viral capsids. physical review e, statistical, nonlinear, and soft matter physics exposure of rna templates and encapsidation of spliced viral rna are influenced by the arginine-rich domain of human hepatitis b virus core antigen (hbcag 165-173) testing the balanced electrostatic interaction hypothesis of hepatitis b virus dna synthesis by using an in vivo charge rebalance approach testing an electrostatic interaction hypothesis of hepatitis b virus capsid stability by using an in vitro capsid disassembly/reassembly system the nmr experiments were carried out with nmr spectrometers of the high-field nuclear magnetic resonance center (hfnmrc) supported by the national research program for genomic medicine, the national science council of the republic of china. key: cord-341564-fvuwick5 authors: qi, zhao-hui; li, ke-cheng; ma, jin-long; yao, yu-hua; liu, ling-yun title: novel method of 3-dimensional graphical representation for proteins and its application date: 2018-06-12 journal: evol bioinform online doi: 10.1177/1176934318777755 sha: doc_id: 341564 cord_uid: fvuwick5 in this article, we propose a 3-dimensional graphical representation of protein sequences based on 10 physicochemical properties of 20 amino acids and the blosum62 matrix. it contains evolutionary information and provides intuitive visualization. to further analyze the similarity of proteins, we extract a specific vector from the graphical representation curve. the vector is used to calculate the similarity distance between 2 protein sequences. to prove the effectiveness of our approach, we apply it to 3 real data sets. the results are consistent with the known evolution fact and show that our method is effective in phylogenetic analysis. with the number of available biological sequences developing rapidly, how to mine essential information from a huge amount of biological sequences effectively and reliably has become a critical problem. as a result, many methods in information extraction are proposed by researchers. among them, the graphical representation of dna sequences is an effective method for the virtualization and similarity analysis. graphical representation is a kind of alignment-free method. it provides intuitive information of data by visualization of biological sequences. what is more, it is more generally applicable because its mathematical description of data facilitates numerical analysis without difficult calculations. therefore, numerous works based on graphical representation have been presented by researchers. [1] [2] [3] [4] [5] [6] [7] [8] for example, randić et al 1 proposed a graphical representation of rna secondary structure based on twelve symbols. bielińska-waż et al 5 proposed a 2d-dynamic representation of dna sequences in 2007. after that they proposed more dynamic representations of dna sequences for generalization. 6, 7 however, the graphical representation of protein sequences is much more difficult because there are 20 amino acids instead of 4 nucleotides. various approaches have been proposed by researchers only until recently. [9] [10] [11] [12] [13] [14] [15] [16] among them, many approaches are based primarily on the physicochemical properties of amino acids. randić 9 early proposed a 2-dimensional graphical representation of proteins based on a pair of physicochemical properties in 2007. after that, yu et al 11 proposed a protein mapping method of protein sequences based on 10 physicochemical properties. wang et al 10 presented a graphical representation of protein sequences based on 9 physicochemical properties. in the works by he et al 15 and hu, 16 the physicochemical properties are also indispensable in information extraction from proteins because they have effects on the rate and pattern of protein evolution. from these, we can see that physicochemical properties are widely applied with graphical representation of protein sequences by these researchers and their results seem well. in this article, we propose a 3-dimensional (3d) graphic representation of protein sequences based on 10 physicochemical properties [17] [18] [19] [20] [21] of amino acids and the blosum62 matrix. 22 the representation can provide good visualization without degeneracy or circuit. then, we extract a specific vector from the graphical curve of a protein sequence. in addition, we proposed 2 applications based on the vector to analyze the similarity and evolutionary relationship of 3 data sets, respectively. the results are consistent with the evolution fact and works by other researchers. this shows our approach can be applied to hundreds of sequences with different lengths and perform well. as we know, a protein sequence is usually composed of 20 kinds of amino acids. every amino acid has its own particular physicochemical properties. therefore, to mine essential information from a protein sequence, we propose an effective graphical method combining physicochemical properties of amino acids and the blosum62 matrix. evolutionary bioinformatics that one amino acid is replaced by other amino acids. in their scoring scheme, a positive score represents a higher similarity between 2 amino acids and a negative score represents a lower similarity. here, we consider 10 primary physicochemical properties of amino acids, such as the pk1 (-cooh), 17 the pk2 (-nh3), 21 the polar requirement, 21 the isoelectric point, 18 the hydrogenation, 20 the hydroxythiolation, 20 the molecular volume, 19 the aromaticity, 20 the aliphaticity, 20 and the polarity values. 19 the 10 physicochemical properties of 20 amino acids are shown in table 1 . for each physicochemical property, we will use k-means clustering method 23 to classify the 20 amino acids into several groups. k-means clustering is an efficient unsupervised clustering method which is widely used in a diverse range of fields such as data mining, bioinformatics, and natural language processing. 24 however, there are some weaknesses in k-means. k-means needs to be given the number of clusters beforehand. silhouette 25 is a cluster validity index that can be used to determine the number of clusters. it considers 2 factors: cohesion and separation. its value ranges from −1 to 1 and a higher value represents a better effect of clustering. according to this index, we can obtain a valid number of clusters of the given data set. in this way, we can obtain 10 kinds of clustering classification based on the 10 different properties, which are shown in table 2 . according to the property pk1 (-cooh), we can divide the 20 amino acids into 7 groups: g1 (a, g, i, l, m, w, v), g2 (h, f), g3 (q, e, k, s, y), g4 (t), g5 (n, p), g6 (c), and g7 (d). if 2 or more amino acids are divided into the same group, it denotes that they are similar to each other by the property pk1 (-cooh). taking all the properties into consideration, we can obtain the number of similar properties between each pair of amino acids. if x denotes an amino acid and y denotes another amino acid, then we define the similar degree of 2 amino acids s xy as follows: where n xy is the number of similar properties between amino acid x and y . b xy is the value of amino acid x and y in the blosum62 matrix. then, we calculate all the values of s xy and the result is shown in table 3 . from table 3 , we can find that the similarity degree s xy of different amino acids can be numerically different from each other. to describe the similarity degree graphically, we will use a unitary linear regression to extract characters from every amino acid. here, we take i = {1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19} as independent variables of linear regression. for amino acid x, we take the 19 values in its corresponding row in table 3 as dependent variables. using unitary linear regression, we can obtain the corresponding slope and intercept of amino acid x. the slope and intercept can describe amino acid x effectively. all the slopes and intercepts of 20 amino acids are given in table 4 . we assume that p p p p n = 1 2 , , ,  is an arbitrary protein sequence composed of n amino acids. if x i , y i , and z i represent the 3d coordinates of p i in the protein sequence, then the 3d representation of a protein sequence will be constructed as follows: where a i and b i represent the slope and intercept of p i . in addition, the initial condition is x y 0 0 0 = = . next, we can convert the n points into a graphical curve. to demonstrate the effectiveness of the 3d graphical method, we take 2 protein sequences as an example. both the sequences are taken from yeast saccharomyces cerevisiae. 26 the graphical representations of 2 protein sequences are shown in figure 1 . based on the constructed graphical curve, we can get a specific vector from a protein sequence. using this vector we can analyze the similarity between 2 protein sequences effectively. characteristic vector is a common method to calculate the pairwise distance between 2 protein sequences. a good characteristic vector should avoid the problem about different lengths of sequences and complicated calculation. here, we define a 2-tuple ( , ) a b x x of amino acid x for characterization. given a protein sequence composed of n amino acids p p p p n = 1 2 , , ,  , we can compute the 2-tuple as follows: where a x and b x are, respectively, slope and intercept of amino acid x in table 4 . n x is the number of amino acid x in the sequence. from equation (3), we can see that n n x / table 4 . taking a short segment of 10 amino acids, aarrarrnnn, as an example, the numbers of amino acid a, r, and n in the segment are 3, 4, and 3. therefore, we can obtain the 40-dimensional characterizing vector (0.015, −0.738, −0.02, −0.16, −0.042, −0.369, 0, 0, . . ., 0, 0) according to table 4 and equation (3). the similarity/dissimilarity between 2 protein sequences can be represented by similar distance. there are several calculating methods for measurement of similar distance such as euclidean distance, city block distance, and manhattan distance. here, we use euclidean distance as a measure to represent the similarity/dissimilarity between 2 sequences. we will compute the similarity distance using the 40-dimensional characteristic vector. if the two 40-dimensional characteristic vectors are denoted as table 4 . slope, intercept, and linear equation of each amino acid similarity degree sequence. evolutionary bioinformatics , , , , , , their euclidean distance is calculated as follows: the smaller the distance d is, the more similar 2 protein sequences are. to show the effectiveness of the proposed similarity analysis method, we apply it to 9 nd5 protein sequences (provided as supplementary file 1): human, common chimpanzee, pygmy chimpanzee, gorilla, fin whale, blue whale, rat, mouse, and opossum (their accession number in ncbi [national center for biotechnology information] are ap_000649, np_008196, np_008209, np_008222, np_006899, np_007066, ap_004902, np_904338, and np_007105, respectively). according to the method given in section "similarity analysis," we can obtain the similarity distance matrix of these protein sequences. the corresponding result is shown in table 5 . on the basis of table 5 , we can find that the distance between fin whale and blue whale is the smallest. this indicates that they have a high degree of similarity. the distance between human, common chimpanzee, pygmy chimpanzee, and gorilla is relatively small, which means that they are similar to each other. besides, opossum is quite dissimilar to other species because the similarity distances between opossum and other species are large. all these results are consistent with the evolution theory and the recent studies. [14] [15] [16] that is to say the proposed method can analyze the similarities of proteins effectively. to further demonstrate the effectiveness of our method, we apply it to another data set which is widely used in many works. 10, 27 this data set consists of 29 spike protein sequences of coronavirus (provided as supplementary file 2) . the basic information of the protein sequences is shown in table 6 . we construct the phylogenetic tree for the 29 spike protein sequences based on our method using upmga method in figure 2 . from figure 2 , we can see that all the sequences are mainly classified into 4 groups by our method. this is consistent with the works 10,27 and the known biology fact that coronavirus are always classified into 4 groups: the group i (contains pedv, tgev), the group ii (contains bcov, mhv, rtcov), the group iii (contains ibv, tcov), and the sars-covs (severe acute respiratory syndrome coronavirus). in this section, we give an application for the similarity analysis of ha gene sequences of influenza a (h1n1) from march 1, 2009 to april 30, 2009 (available online at https://www.ncbi. nlm.nih.gov). we obtain a data set that consists of 560 gene sequences with full length (provided as supplementary file 3) . to further demonstrate the validity of our method, we apply the method to this data set. according to our method, for each virus isolate, we can get a corresponding 20-dimensional vector. thus, we can obtain a vector set of 560 vectors. by computing the similarity distance between pairs of these vectors, we can obtain a similarity distance matrix. next, we construct the phylogenetic tree based on our method in figure 3 . to analyze the results better, we mark 2 typical strains: a/california/07/2009 (h1n1) and a/ indiana/08/2009 (h1n1). from figure 3 , it is easy to identify that all virus isolates are mainly classified into 2 groups. this illustrates that there are 2 different kinds of influenza a (h1n1) virus isolates from march 1, 2009 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characterization a protein mapping method based on physicochemical properties and dimension reduction the graphical representation of protein sequences based on the physicochemical properties and its applications f-curve, a graphical representation of protein sequences for similarity analysis based on physicochemical properties of amino acids analysis of similarity/dissimilarity of protein sequences the genetic code and error transmission amino acid difference formula to help explain protein evolution relations between chemical structure and biological activity on the fundamental nature and evolution of the genetic code amino acid substitution matrices from protein blocks automated programming: the next wave of developer power tools recovering the number of clusters in data sets with noise features using feature rescaling factors silhouettes: a graphical aid to the interpretation and validation of cluster analysis novel 2-d graphical representation of proteins structure, function, and evolution of coronavirus spike proteins evolution trends of the 2009 pandemic influenza a (h1n1) viruses in different continents from mega6: molecular evolutionary genetics analysis version 6.0 the phylogenetic tree of the 560 influenza a (h1n1) isolates from march to this paper has not been submitted elsewhere for consideration of publication. z-hq conceived and designed the work that led to the submission. k-cl contributed significantly to analysis and manuscript preparation. j-lm and y-hy helped to perform the analysis with constructive discussions. all the authors reviewed and approved the final manuscript. this result is consistent with the works by qi et al. 14, 28 furthermore, the result is also consistent with the biology fact that a new influenza virus, a/california/07/2009 (h1n1)-like virus, appeared and showed a strong ability to infect human beings in april 2009. 23 the branch length in figure 3 is the similarity distance between 2 virus isolates.clustalw is one of the most widely used multiple sequence alignment method for nucleic acid and protein sequence in molecular biology. we construct the phylogenetic tree of the 560 gene sequences using clustalw method 29 in this article, a new 3d graphical representation of protein sequences is introduced based on 10 physicochemical properties and blosum62 matrix. on the basis of the graphical representation curve, we extract a specific vector and use the vector to calculate the similarity distance between 2 protein sequences. to prove the effectiveness of our method, we apply our method to 3 real data sets. the results show the validity of our method in phylogenetic analysis compared with related works and evolution facts. key: cord-352371-t54zftal authors: kumar, ravindra; kumari, bandana; kumar, manish title: prediction of endoplasmic reticulum resident proteins using fragmented amino acid composition and support vector machine date: 2017-09-04 journal: peerj doi: 10.7717/peerj.3561 sha: doc_id: 352371 cord_uid: t54zftal background: the endoplasmic reticulum plays an important role in many cellular processes, which includes protein synthesis, folding and post-translational processing of newly synthesized proteins. it is also the site for quality control of misfolded proteins and entry point of extracellular proteins to the secretory pathway. hence at any given point of time, endoplasmic reticulum contains two different cohorts of proteins, (i) proteins involved in endoplasmic reticulum-specific function, which reside in the lumen of the endoplasmic reticulum, called as endoplasmic reticulum resident proteins and (ii) proteins which are in process of moving to the extracellular space. thus, endoplasmic reticulum resident proteins must somehow be distinguished from newly synthesized secretory proteins, which pass through the endoplasmic reticulum on their way out of the cell. approximately only 50% of the proteins used in this study as training data had endoplasmic reticulum retention signal, which shows that these signals are not essentially present in all endoplasmic reticulum resident proteins. this also strongly indicates the role of additional factors in retention of endoplasmic reticulum-specific proteins inside the endoplasmic reticulum. methods: this is a support vector machine based method, where we had used different forms of protein features as inputs for support vector machine to develop the prediction models. during training leave-one-out approach of cross-validation was used. maximum performance was obtained with a combination of amino acid compositions of different part of proteins. results: in this study, we have reported a novel support vector machine based method for predicting endoplasmic reticulum resident proteins, named as erpred. during training we achieved a maximum accuracy of 81.42% with leave-one-out approach of cross-validation. when evaluated on independent dataset, erpred did prediction with sensitivity of 72.31% and specificity of 83.69%. we have also annotated six different proteomes to predict the candidate endoplasmic reticulum resident proteins in them. a webserver, erpred, was developed to make the method available to the scientific community, which can be accessed at http://proteininformatics.org/mkumar/erpred/index.html. discussion: we found that out of 124 proteins of the training dataset, only 66 proteins had endoplasmic reticulum retention signals, which shows that these signals are not an absolute necessity for endoplasmic reticulum resident proteins to remain inside the endoplasmic reticulum. this observation also strongly indicates the role of additional factors in retention of proteins inside the endoplasmic reticulum. our proposed predictor, erpred, is a signal independent tool. it is tuned for the prediction of endoplasmic reticulum resident proteins, even if the query protein does not contain specific er-retention signal. factors in retention of proteins inside the endoplasmic reticulum. our proposed predictor, erpred, is a signal independent tool. it is tuned for the prediction of endoplasmic reticulum resident proteins, even if the query protein does not contain specific er-retention signal. the endoplasmic reticulum (er) is an important organelle of eukaryotic cells. it participates in several essential cellular activities, such as quality control of protein production, recognition of mis-folded proteins, lipid biosynthesis, detoxification, steroids and xenobiotic compound metabolism, protection against cellular stress, intracellular calcium homeostasis, and intracellular signalling (lavoie & paiement, 2008; verkhratsky, 2002) . er malfunction might lead to many diseases like cystic fibrosis, cancer and juvenile pulmonary emphysema (robinson-rechavi et al., 2001) and neurological disorders like ischemia and epileptic seizures (paschen & frandsen, 2001) . er is involved in several co-translational and post-translational modifications like signal-peptide cleavage, disulphide bond formation, n-linked glycosylation, and glycosylphosphatidylinositol (gpi)-anchor formation (barz & walter, 1999) and is also essential for post-translational processing of secretory proteins that makes it one of the important components of eukaryotic secretory protein system (barlowe & miller, 2013) . secretory proteins are synthesized on rough er and routed across the er membrane into lumen through a co-translational process. inside the er, they undergo glycosylation and attain their specific 3d conformation before being further transported downstream in the secretory system. the whole process is achieved with the help of highly coordinated action of several proteins which includes molecular chaperones and different enzymes (nakatsukasa & brodsky, 2008) . proteins, which help er in carrying out above-mentioned functions, do not get secreted, and they are called as er resident proteins (errps). in addition to errps, mis-folded or unfolded proteins are also retained back in er and forwarded for proteasome-mediated degradation if the error could not be rectified (sitia & braakman, 2003; van anken & braakman, 2005; wang & hebert, 2003) . therefore, in order to remain localized in er, the errp must be recognized precisely and discriminated from the misfolded proteins or proteins of secretory pathway. the function of er is intricately bound with the golgi apparatus. for example, similar to er, from golgi apparatus, also the proteins automatically proceed downstream to the plasma membrane or vacuoles in absence of an active retention process. also a number of errps are forced to localize in er by continuous retrieval from the golgi complex. luminal er proteins may be retrieved by using retrograde transport with help of c-terminal kdel sequence while type i transmembrane proteins-including er residents and itinerant proteins of the er/golgi system-contain a c-terminal dilysine motif that signals their retrieval from post-er membranes (gaynor et al., 1994; jackson, nilsson & peterson, 1993; townsley & pelham, 1994) . the dilysine motif known as kkxx and kxkxx motifs, consists of a pair of lysine residues at the c terminus of the cargo protein (jackson, nilsson & peterson, 1990; nilsson, jackson & peterson, 1989) . many variants of the dilysine motif have been reported such as kxhxx retrieval motif in the tail of the spike protein of group i coronaviruses (lontok, corse & machamer, 2004) , and the rkxx motif present in the golgi-localized scyl1 protein (burman et al., 2008) . in past, a number of studies have focused on identification of proteins located in different subcellular organelles like nucleus (brameier, krings & maccallum, 2007; huang et al., 2007; kumar & raghava, 2009 ), mitochondrion (guda, fahy & subramaniam, 2004 kumar, verma & raghava, 2006 ), chloroplast (emanuelsson, nielsen & von heijne, 1999 , golgi body (chou, yin & xu, 2010 ), peroxisome (emanuelsson et al., 2003 neuberger et al., 2003) but errps remain unexplored. despite the fact that the functionality of golgi and er are intricately intertwined with each other, several golgi prediction methods have been developed (chou, yin & xu, 2010; jiao & du, 2016) . no attempt has been made to develop species neutral errps predictor. though in few subcellular localization prediction methods, er has been included as a location, but at present, to the best of our knowledge, a predictor which can specifically predict errps does not exist. moreover, predictors like prolocgo (huang et al., 2008) , knowpred site (lin et al., 2009 ), slocx (ryngajllo et al., 2011 ), iloc-animal (lin et al., 2013 ), iloc-euk (chou, wu & xiao, 2011 ), cello v-2.5 (yu et al., 2006 , hybridgo-loc (wan, mak & kung, 2014) , mgoasvm (wan, mak & kung, 2012) , hum-ploc (chou & shen, 2006) , euk-mploc (chou & shen, 2007) , pslt (scott, thomas & hallett, 2004) , euk-mploc 2.0 (chou & shen, 2010) , the method developed by cherian & nair (2010), and guo et al. (2016) considered er as one among many subcellular locations. but these have some shortcomings like (i) among the above mentioned predictors, none were designed specifically to predict errps; (ii) datasets used for training for prediction model were very old; (iii) subcellular locations were determined for a particular organism or groups (plant/animal/viral); (iv) many of them do not provide webserver/standalone software for scientific purpose and if some of them does so, they are not in working condition. we believe that identification of all errps is necessary to explore more about their functional associations and for better understanding of er functional machinery. in the present study, we report a dedicated method, erpred, to predict errps with 81.42% accuracy. erpred is based on the support vector machine (svm) and the individual amino acid compositions of 25 n-terminal, 25 c-terminal and remaining amino acids as svm input. we have also developed a freely accessible webserver and software that predicts errps using their amino acid sequence. we hope that the present work will help in better understanding of cellular secretory pathways as well as other er-mediated functions. dataset compilation is one of the most important steps during development of a prediction method. to avoid any ambiguity, the dataset that has to be used for training the predictor should contain experimentally annotated proteins of high quality. we found that there exists a database of human er proteins (both known and potential) named as hera (scott et al., 2004) . since hera is not updated and contains only human proteins hence we selected swissprot, the manually curated section of uniprot protein database, to download the data. to retrieve the errps from swissprot, we used following criteria (i) proteins should be of eukaryotic origin and reviewed; (ii) should be present only in er; (iii) existence proven by 'evidence at protein level'; (iv) protein should be full length and not fragmented (v) should have more than 50 amino acids because smaller proteins are mostly fragments; and (vi) protein location should be experimentally verified. for negative dataset, we tried to collect all types of non-errps so that the predictor is trained on a comprehensive dataset and it can easily differentiate errps from the non-errps. we selected two different types of proteins as non-errps (1) proteins which are n-glycosylated, since n-glycosylation occurs in er (bieberich, 2014; roth et al., 2010) ; (2) non n-glycosylated proteins, since not all er proteins are n-glycosylated. both types of non-errps were downloaded using the criteria earlier used to download er proteins. proteins downloaded for making dataset may contain similar or homologous sequences hence if they are used without reducing the redundancy among proteins, the obtained performance might not be a genuine performance but an over-estimation. hence most prediction methods have adopted the strategy of homology-reduced dataset to assess the performance of a predictor. in this work the redundancy was reduced to 40% using cd-hit (li & godzik, 2006) and 124 errps were obtained which were used as positive dataset. 1,200 eukaryotic non-errps were used as the negative dataset (table 1; table s1 ). the 1,200 non-er proteins were 10 times the positive dataset, which in our view should be sufficient for the predictor to model non-errps (kumar et al., 2015) . to assess the unbiased performance of a newly developed method, the evaluation must be carried out on a dataset which was not used during the training. so we created an independent dataset for this purpose. this dataset also contains both errps and non-errps. the source of errps was also swissprot (release: 2015_06) and contained proteins, which had existence at 'evidence at transcript level', 'inferred from homology' and 'predicted'. following criteria were used to download the errps: (i) location should be endoplasmic reticulum; (ii) protein length should be greater than 50 amino acids; (iii) protein should be non-membranous; (iv) proteins should not be experimentally verified. after removing sequences, which had a redundancy of more than 40% among independent or with the training dataset, finally we got 65 proteins (table 1; table s2 ). the non-errps were also downloaded from swissprot using following filters: (i) protein length should be greater than 50 amino acid; (ii) protein should be of eukaryotic origin and non-membranous; (iii) protein's existence should be experimentally verified; (iv) removed the low quality annotation by excluding sequences annotated as 'by similarity', 'fragment', 'uncertain' sequences from the dataset. the dataset obtained from above described step was made non-redundant by using cd-hit program with 40% threshold after which we got 13,271 sequences. after removing the protein sequences, which were also present as non-errps in training dataset, we selected only those non-errps, which had er targeting consensus sequence [krhqsa]-[denq]-e-l (raykhel et al., 2007) (searched by using standalone version of scanprosite (gattiker, gasteiger & bairoch, 2002) ) and got 2,900 non-errp sequences (table 1 ; table s3 ). in order to show the real life usage and efficiency of our method, we annotated six proteomes namely homo sapiens, mus musculus, saccharomyces cervisiae, caenorhabditis elegans, drosophila melanogaster and arabidopsis thaliana. their complete proteome set was downloaded from uniprot which had 68554, 45185, 5450, 26109, 22024 and 31527 proteins respectively. svm is a machine-learning algorithm to carry out pattern recognition and regression analysis on a given dataset (vapnik, 1995) . during training, svm maps input data to the higher dimension and generates model, which can be used for prediction of an unknown example. in this work we used freely downloadable svm_light package available at http://svmlight.joachims.org/ to implement svm. during training, svm needs only fixed-length feature along with their class as input. in the present work, fixed-length input feature of variable length protein sequence was obtained by amino acid composition, pseudo amino acid composition, dipeptide composition and different combinations of fragmented amino acid compositions. cross-validation is a way to estimate the performance of a prediction model during training. during cross-validation, whole data is divided into two distinct sets; one set (called as training set) is used to train the model and second set (called as test set) is used for performance evaluation of model on a dataset that was not used during training. in prediction methods, three cross-validation approaches are most frequently used: independent dataset test, subsampling test (n-fold cross-validation) and jack-knife test or leave-one-out cross-validation (loocv). among the three cross-validation approaches, loocv gives unique result for a given benchmark dataset and hence used in a number of prediction methods (kumar et al., 2014b; kumar et al., 2015; kumari, kumar & kumar, 2014; lin et al., 2011; xiao et al., 2013; xu et al., 2013) . in the present study, we have used loocv approach for the evaluation purpose, in which all except one sequence of dataset is used as training set and remaining one sequence as test set. this process is repeated till each sequence has been used for testing. at a selected parameter, svm was trained using the training set and predictive performance of model was evaluated on corresponding test set. the prediction performance of trained model was calculated by averaging over all test set predictions. to evaluate the performance of each trained svm model, we used standard parameters regularly used in other prediction methods for prediction evaluation namely sensitivity, specificity, accuracy and matthews correlation coefficient (mcc) (kumar, gromiha & raghava, 2008; kumar et al., 2014a; kumar et al., 2014b; kumar et al., 2015; panwar, arora & raghava, 2014) as formulated below: where tp represents true positive (proteins, which are actually errps and were also predicted as errps), tn represents true negative (proteins, which are actually non-errps and also predicted as non-errps), fp represents false positive (the number of non-errp predicted as errps), fn represents false negative (number of proteins, which are actually errps but predicted as non-errps). as svm needs fix length input to operate, hence we converted variable length protein sequence into fixed length vector. amino acid, pseudo amino acid, dipeptide, and split amino acid compositions were used to encapsulate the protein sequence information into fixed length vector. the amino acid composition is the fraction of each amino acid within a protein sequence. it has been used extensively in past for prediction of different protein features (kumar, gromiha & raghava, 2007; kumar, gromiha & raghava, 2011; kumar et al., 2014a; kumar et al., 2015; wang, xiao & chou, 2011) . the fraction of each amino acid was calculated by using the following formula: acomp(i) = total number of an amino acid(i) total number of amino acid in protein p ã� 100 (5) where acomp(i) is the percentage of amino acid 'i' in a given protein and (i) can be any amino acid of a particular protein (p). one of the major drawbacks of the amino acid composition is the loss of information about amino acid sequence order from the input features. to deal with this problem chou proposed a new way of amino acid composition called as pseudo-amino acid composition (chou, 2001) . pseudo-amino acid composition of a protein is actually a set of discrete numbers, which is derived from its amino acid sequence. it allows representation of both compositional and positional amino acid pattern in a discrete mode (limongelli, marini & bellazzi, 2015) . it also reflects sequence order information and length of the proteins (chou, 2005) . many tools (du et al., 2012; han, yu & anh, 2014; kumar et al., 2015; liu et al., 2014; mondal & pai, 2014) are available to calculate the pseudo-amino acid composition of protein sequences. here we used pseaac-builder (du et al., 2012) at default parameters (weight factor = 0.05 and lambda parameter = 1). dipeptide composition is a modified form of amino acid composition, which has been used in a number of protein classification methods like nuclear receptor family classification (bhasin & raghava, 2004; kumar et al., 2014b) , membrane protein prediction, ion channels prediction (lin & ding, 2011) , protein fold prediction (shamim, anwaruddin & nagarajaram, 2007) , heat shock protein and its class prediction (kumar, gromiha & raghava, 2011; kumar, kumari & kumar, 2016; reczko & bohr, 1994) . dipeptide composition calculates the number of all possible types of amino acid pairs in a protein sequence in a sliding window mode with step size 1 and window size 2. hence, it incorporates amino acid composition along with the local order information also. dipeptide composition describes each protein sequence in form of 400-dimension feature vectors, which can be calculated as where dipep(n) is the percentage of dipeptide 'n' in a given protein and (n) can be any dipeptides out of the all possible 400 dipeptides of a particular protein (p). secretory proteins are known to contain signal to guide them outside of the cell. this feature also distinguishes them from the intracellular proteins (wrzeszczynski & rost, 2004) . hence sequence encapsulation, which can highlight this difference, ought to be more informative. in view of this, we divided each protein sequence into different non-overlapping fragments, viz. 2-parts and 3-parts, and calculated amino acid composition of each part separately. fragment that contains the signal sequence would have amino acid compositions different from the remaining fragments, thereby highlighting the presence of signal sequence(s). hence split amino acid composition might provide more realistic information than amino acid and dipeptide compositions of whole protein. this approach has been used earlier to encode input protein features (afridi, khan & lee, 2012; kumar & raghava, 2009; kumar, verma & raghava, 2006; verma, varshney & raghava, 2010; hayat, khan & yeasin, 2012) . in this work, we used 2-parts and 3-parts-split amino acid compositions as input features. in 2-parts-split amino acid (saac-2-parts), first we divided each sequence in two different ways. we named these two split sets as n-terminal saac (n-ter-saac) and c-terminal saac (c-ter-saac) respectively. n-ter-saac had protein sequences in two parts, (i) 25 amino acids of n-terminal and (ii) the remaining residues of the sequence. similarly, c-ter-saac included (i) 25 amino acids of c-terminal and (ii) the remaining sequence amino acids. for both n-ter-saac and c-ter-saac, we calculated the amino acid composition of each part separately. in 3-parts-split amino acid composition (saac-3-parts), protein sequence was divided into three parts: n-terminal, middle part and c-terminal and then amino acid composition of each part was calculated separately. figure 1 depicts a protein sequence k, which is divided into three parts; k n , k c and k r where k n is the 25 n-terminal residues, k c represents the 25 c-terminal residues and k r represents remaining sequence. calculation of amino acid composition of each part provided 20 vectors, so the length of final input vector was 60 for each protein. due to the fractional nature of amino acid composition, it is likely to highlight the enrichment and depletion of specific amino acids in each segment and reveal the biasness in distribution of amino acids in a protein. the amino acid composition of proteins of each subcellular location is specifically adopted to suit its surrounding environment (andrade, o'donoghue & rost, 1998) . in order to understand this pattern in case of errps, we analysed the enrichment and depletion pattern of amino acids using composition profiler (vacic et al., 2007) . composition profiler detects the enrichment and depletion patterns of individual amino acid as well as group of amino acids classified on the basis of different physico-chemical and structural properties by using two groups of protein sequences as query and background sample. the fractional difference (in terms of enrichment and depletion) between distribution of a particular amino acid in query (d1) and background dataset (d2) is calculated as follows: in this work, during enrichment and depletion analysis, all errps were merged into a single group as a query sample while all non-errps were used as background sample. as per composition profiler analysis, at p-value â�¤ 0.05, aromatic (phenylalanine, tyrosine, tryptophan), negatively charged (aspartic acid and glutamic acid), polar (tyrosine) and hydrophobic (valine, leucine, phenylalanine, tryptophan) residues were enriched while positively charged (arginine) residues were depleted in errps (fig. 2) . it has been observed that er proteins are rich in transmembrane domain (schuldiner & weissman, 2013; scott et al., 2004) . the enrichment of aromatic and hydrophobic residues in errps is in accordance with this observation. first, we used simple amino acid composition as svm input. using loocv approach of training, the maximum accuracy we obtained was 73.34% (mcc = 0.29). when information of amino acids arrangement order was provided in the form of pseudo amino acid composition based input, prediction accuracy increased to 74.85% (mcc = 0.30). when dipeptide composition was used as svm input, we found a lower performance and the maximum accuracy decreased to 72.28% with mcc value 0.26 (table 2 ). these results show that pseudo-amino acid composition based svm module is better than the simple amino acid and dipeptide composition based modules. in saac based svm modules, we used three input features to train svm. with n-ter-saac, we found maximum accuracy 79.53% with mcc value 0.37. with c-ter-saac the accuracy reduced to 71.07% and mcc value to 0.27. but with saac-3-parts, the accuracy increased to 81.42% with mcc value 0.42 (table 2) . the performance obtained from different svm models can be explained by the fact that retention of proteins inside er is mediated by some signal sequences or related sequences (gomord, wee & faye, 1999) . hence, an input vector which resolves the signal sequences efficiently is expected to perform better than others. we feel that since saac-3-parts divide a protein into three parts hence, it can resolve the signal motif better, therefore, saac-3-parts based svm model has the maximum accuracy. receiver operating characteristics (roc) curve is a graphical way to illustrate the performance of a classifier. it is plotted as 'sensitivity' vs. '1-specificity' and shows trade-off between true and false positives (fawcett, 2006; hajian-tilaki, 2013) . the area under the roc curve is called auc value (bradley, 1997) which can be used to quantify the prediction performance of a classifier. higher the auc value better is the prediction. in this study, we used rocr package (sing et al., 2005) for plotting the roc curve and finding auc value. as shown in fig. 3 and table 2 , the roc curves and the auc values also confirms that the classifier based on saac-3 parts is better than classifiers developed using other sequence encapsulations. errps follow two basic mechanisms to stay inside the er: (i) signal dependent and (ii) signal independent. signal dependent proteins have er-retention signals while signal independent proteins do not have these signals, e.g., some cereal prolamin storage proteins (sophie pagny, faye & gomord, 1999) . in order to find out the efficiency of signal based approach, we evaluated er retention signal based prediction of errps using prosite motif database (hulo et al., 2006) . using standalone version of scanprosite (gattiker, gasteiger & bairoch, 2002) , out of 124 proteins of training dataset, we were able to find er retention signal ([krhqsa]-[denq]-e-l) in only 66 proteins, which shows that signal sequence is not present in all errps. this shows that signal based approach may not be appropriate for complete errp repertoire prediction of any proteome. similarly there are many non-errps, which also have er-retention signal but they do not reside into the er. efficiency of signal based approach also suggests that presence of er-retention signal may not be the only factor responsible to hold a protein inside er. it also implies that a good er predictor should also be able to discriminate and recognize errps that does not have er-retention signals and the non-errps that have er-signals. so, in order to evaluate our method on the above-mentioned two parameters, we analysed 65 errps of independent dataset for presence of er-retention signal by scanprosite. we found that only 21 proteins had er-signal while remaining 44 proteins did not have any er-signal. further, when we predicted whether these 44 errps localizes to er or not using erpred, 27 proteins were predicted as errps and 17 as non-errps. we also benchmarked the erpred using 2,900 non-errps of independent dataset containing er-retention signal sequences. erpred correctly predicted the non-errps with 83.69% specificity (table 3 ). this shows that the mere presence or absence of er-retention signal does not necessarily implies errp or non-errp respectively. wrzeszczynski & rost (2004) also observed that most of the known er-retention signals are too specific and/or too inaccurate to be used in alone for automatic protein annotation. their observation was also based on experimentally characterized short sequence motifs and sequence similarity search to experimentally characterized proteins. they also showed that either >80% pairwise sequence identity or <10 â��100 e-value can be used for homology-transfer based annotation and at very low coverage which severely limit the number of true positives. to annotate novel proteins, 'homology based function transfer' is the most popular approach. this approach involves blasting novel gene/protein against a gene and/or protein database to find well-annotated homolog(s) and transfer the annotation of top hit(s) to the query proteins (brown, krishnamurthy & sjolander, 2007; radivojac et al., 2013; rost et al., 2003) . since similarity search considers whole protein sequence rather than a short stretch of er-retention signal, this approach should be more effective than the signal based approach. in order to assess the efficiency of blast to discover new errps, we evaluated the performance of blast search using the proteins of training dataset. in this process, each protein out of 124 was used as query and remaining 123 proteins as database. at e-value threshold â�¥1e â�� 3, only 56 proteins found errp as first blast hit. remaining 68 proteins did not get any hit. it shows that finding homologous sequence using blast is also not sufficient to search all errps. our inference regarding usage of blast in prediction of errps is also supported by the observation noted by wrzeszczynski & rost (2004) that even very high levels of sequence similarity might not be sufficient to infer er localization without error. overall, the results show that performance of erpred does not depend either on er retention signal or sequence homology. therefore, we feel our approach can be an appropriate alternative to the annotation transfer on the basis of homologous protein and/or signal sequence based approaches for high throughput proteome annotation. to derive unbiased results we evaluated proposed prediction schema using proteins of independent dataset. the outcome of prediction was used to assess the sensitivity and specificity of erpred. we found erpred showed sensitivity 72.31% and specificity 83.69% on independent dataset (table 3 ). this shows that our method not only detects errps with high sensitivity but also recognizes non-errps with high specificity. to the best of our knowledge, there is no existing method which can specifically predict errps. though a few subcellular localization prediction methods considered er as one of many locations in the cell these predict er proteins along with proteins of other subcellular locations like, prolocgo (huang et al., 2008) , knowpredsite (lin et al., 2009 ), slocx (ryngajllo et al., 2011 ), iloc-animal (lin et al., 2013 ), cello v.2.5 (yu et al., 2006 , euk-mploc (chou & shen, 2007) , euk-mploc 2.0 (chou & shen, 2010) , hybridgo-loc (wan, mak & kung, 2014 ), mgoasvm (wan, mak & kung, 2012 , hum-ploc (chou & shen, 2006) and iloc-euk (chou, wu & xiao, 2011) . unfortunately, prolocgo (huang et al., 2008) , knowpredsite (lin et al., 2009) were not found in a working state and hybridgo-loc and mgoasvm considered only viral and plant proteins. hum-ploc is meant only for human proteins, slocx (ryngajllo et al., 2011) predicts only er proteins of arabidopsis thaliana and iloc-animal (lin et al., 2013) predicts only proteins of animal system. since erpred prediction is not organism/taxa specific hence we compared our method with those methods which are not restricted to a particular organism or plant/animal like cello v.2.5 (yu et al., 2006 ), iloc-euk (chou, wu & xiao, 2011 ) and euk-mploc 2.0 (chou & shen, 2010 (an updated version of euk-mploc (chou & shen, 2007) ) using independent dataset. the sensitivity of erpred on independent dataset was 72.31% with 83.69% specificity while for cello v.2.5 the corresponding values were 16.92% and 99.86% respectively and for iloc-euk the sensitivity and specificity was 15.38% and 99.76% respectively (table 3) . hence euk-mploc showed better performance than iloc-euk and cello v 2.5 in terms of sensitivity, which was 66.15%. as shown in table 3 , among all the predictors, erpred performed better in prediction of errps but its performance in terms of specificity or non-errps was lesser than other predictors. it might be due to the fact that all predictors, which we have used here for evaluation on the basis of independent dataset, were intended to predict multiple locations. the result shows that in majority of cases they were predicting locations other than er, due to which their overall efficiency was higher. in other words, in most cases, they were predicting errps and non-errps as non-errps, which increased their performance. but, if we carefully analyse the results, it can be observed that their performance is not equally good for the prediction of errps. this also vindicates our attempt to develop a dedicated predictor for errps. we also evaluated the performance of blast based annotation using sequences of training and independent dataset as database and query respectively. we found that only 40 errp sequences of independent dataset were annotated as errp. remaining 25 sequences did not get any hit. we selected six different proteomes ranging from less complex yeast to the most complex human for the purpose of annotation. in h. sapiens, out of 68,554 proteins, erpred predicted 2,293 proteins as errp, which is 3.34% of the proteome. in case of m. musculus, erpred predicted 1,781 errps out of 45,185, which is 3.94% of the proteome. in d. melanogaster, erpred predicted 707 proteins as errps out of total of 22,024 proteins, which is 3.21% of the proteome. in case of c. elegans, erpred predicted 1,014 proteins as errps out of 26,109 proteins, which is 3.88% of the proteome and 148 proteins are errps out of 5,450 proteins, which is 2.72% of the s. cerevisiae proteome. in a. thaliana, erpred predicted 1,089 proteins as errps (3.45% of proteome) out of 31,527 proteins (table 4) . since there is no method which can predict errp at proteome level so we compared our prediction method with two regularly updated existing databases er-golgidb (wrzeszczynski & rost, 2004) , the database which contained all the proteome which we had annotated and locate (sprenger et al., 2008) , the database containing only h. sapiens and m. musculus proteome. er-golgidb contains information about er and golgi localization based on sequence homology to experimentally annotated proteins. locate contains data about subcellular localization of proteins from the riken fantom4 mouse and human protein sequence set. the information content of locate was determined by a high-throughput, immunofluorescence-based assay and from peer-reviewed publications. the erpred estimated the existence of 2,293 errps in human, representing about 3.34% of human proteome. the estimated number was close to the 2,543 and 1,762 proteins of er-golgidb and locate databases respectively. in mouse, erpred estimated 1,781 proteins (3.94% of the proteome), which was in line with the estimation of 1,588 by locate and slightly lower than er-golgidb databases respectively. nearly identical fraction of errps in mouse and human proteomes was expected due to their close evolutionary relationship. we feel the difference in numbers might be due to the proteome size. we expect the number will increase, as more and more proteins will be added to the proteome. in case of c. elegans proteomes, the fractions of errps was almost equal while in d. melanogaster and a. thaliana the fraction was slightly less. this might be due to the fact that both er-golgidb and locate were last updated in 2010 and 2008 respectively. based on the schema described above, we have established a webserver erpred, which is freely available at http://proteininformatics.org/mkumar/erpred/index.html. this webserver allows users to predict er resident proteins. user can submit protein sequences in fasta format for prediction. the maximum limit of prediction is 25 sequences at a time. if a user submits more than 25 sequences, only the first 25 sequences will be processed for prediction. for batch prediction, we have also made a standalone version of this software, which is available at download page of the webserver. erpred also provides option to select different threshold values, which can be used to select the level of false and true positive predictions. even though the importance of endoplasmic reticulum resident proteins has been established undoubtedly, only a few methods have considered er as a separate subcellular locality. in these methods, the accuracy of prediction of errps is very less in comparison of other locations. the purpose of the present study is therefore to describe a tool, erpred, for the prediction of errps. the tool uses only the protein sequence information for prediction. while developing the method, we tried different forms of sequence compositions; namely amino acid, dipeptide, and pseudo amino acid compositions. better performance of a pseudo-amino acid composition based svm module than the simple amino acid and dipeptide composition based modules reveals that pseudo amino acid composition encapsulates amino acid ordering information better than dipeptide composition. but the best performance was obtained when amino acid compositions of different part of proteins were used. the amino acid composition based svm module gave 73.34% accuracy. the performance was decreased to â�¼1% giving 72.28% accuracy when we used dipeptide composition as input. dipeptide composition was used to incorporate composition as well as local order information. further, when we used pseudo-amino acid composition, it showed a better accuracy of 74.85%. in order to include information of enrichment and depletion of amino acids in a specific region of errps, we also used saac in which a protein sequence was divided into three different ways (n-ter-saac, c-ter-saac, and saac-3 parts). n-ter-saac contained two sets of amino acid compositions, one from 25 n-terminal residues and other from rest of the residues while c-ter-saac also had two sets of amino acid compositions firstly from 25 c-terminal residues and remaining residues. saac-3 parts had three sets of vectors from 25 n-terminal residues, 25 c-terminal residues and the remaining sequence residues. svm modules based on n-ter-saac, c-ter-saac and saac-3 parts respectively achieved accuracy of 79.53%, 71.07% and 81.42%. this shows that the svm module developed from c-ter-saac was least efficient whereas the saac-3 parts based module was better for discriminating errps from non-errps. the performance obtained from different svm models can be explained by the fact that retention of proteins inside er is mediated by some signal sequences or related sequences (gomord, wee & faye, 1999) . hence an input vector, which resolves the signal sequences efficiently, is expected to perform better than others. with use of machine learning techniques, we explored which form of composition can best help in high-level successful prediction of errps. the results show that split composition-based features were most predictive. kdel and its variants is the most common signal present generally at the c-terminal of errps. the other well-characterized retention signal is the di-lysine and di-arginine motifs present at c-and n-terminals respectively. but these signals are neither present in all errps nor sufficient to retain all to er (gao et al., 2014; ma & goldberg, 2013) . this also suggests that other parts of proteins also play a very important role in retention of errps in er (scott et al., 2004) . the most surprising result we obtained was the performance of dipeptide composition based svm models, which has shown least performance among all. ideally it should have picked up the signals responsible for retention of errps in er and should have shown better performance than at least amino acid and pseudo amino acid compositions based models. the higher performance of saac-3 parts in comparison to the c-and n-ter saac can also be explained in light of this fact that in saac-3 parts the presence of signal as well as other unknown factors present at non-terminal regions can be highlighted better than the c-and n-ter saac. we also benchmarked the performance of our method vis-ã -vis other predictors using an independent dataset. the results showed that erpred is better than other tools. we also observed that the performance obtained was roughly equal to what we have obtained earlier during the cross-validation. er is the one of the most important cellular organelles of eukaryotic cells. knowledge of complete er resident protein repertoire will help in better understanding of biological pathways of er. in the present study, a sequence-based tool for prediction of errps has been reported for the first time. we have also developed an open access web-server and standalone software for batch mode prediction of errps and it can be accessed at the recognition and retrotranslocation of misfolded proteins from the endoplasmic reticulum prediction of peroxisomal targeting signal 1 containing proteins from amino acid sequence short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum prediction and classification of ncrnas using structural information endoplasmic reticulum dysfunction-a common denominator for cell injury in acute and degenerative diseases of the brain a molecular specificity code for the three mammalian kdel receptors the def data base of sequence based protein fold class predictions how many nuclear hormone receptors are there in the human genome? automatic prediction of protein function the authors declare there are no competing interests. â�¢ ravindra kumar conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.â�¢ bandana kumari analyzed the data, wrote the paper, reviewed drafts of the paper.â�¢ manish kumar conceived and designed the experiments, analyzed the data, wrote the paper, reviewed drafts of the paper. the following information was supplied regarding data availability:the raw data has been supplied as supplementary material. erpred can be accessed at http://proteininformatics.org/mkumar/erpred/download. html. supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.3561#supplemental-information. key: cord-327199-ggomuomb authors: moerdyk-schauwecker, megan; hwang, sun-il; grdzelishvili, valery z. title: cellular proteins associated with the interior and exterior of vesicular stomatitis virus virions date: 2014-08-08 journal: plos one doi: 10.1371/journal.pone.0104688 sha: doc_id: 327199 cord_uid: ggomuomb virus particles (virions) often contain not only virus-encoded but also host-encoded proteins. some of these host proteins are enclosed within the virion structure, while others, in the case of enveloped viruses, are embedded in the host-derived membrane. while many of these host protein incorporations are likely accidental, some may play a role in virus infectivity, replication and/or immunoreactivity in the next host. host protein incorporations may be especially important in therapeutic applications where large numbers of virus particles are administered. vesicular stomatitis virus (vsv) is the prototypic rhabdovirus and a candidate vaccine, gene therapy and oncolytic vector. using mass spectrometry, we previously examined cell type dependent host protein content of vsv virions using intact (“whole”) virions purified from three cell lines originating from different species. here we aimed to determine the localization of host proteins within the vsv virions by analyzing: i) whole vsv virions; and ii) whole vsv virions treated with proteinase k to remove all proteins outside the viral envelope. a total of 257 proteins were identified, with 181 identified in whole virions and 183 identified in proteinase k treated virions. most of these proteins have not been previously shown to be associated with vsv. functional enrichment analysis indicated the most overrepresented categories were proteins associated with vesicles, vesicle-mediated transport and protein localization. using western blotting, the presence of several host proteins, including some not previously shown in association with vsv (such as yes1, prl1 and ddx3y), was confirmed and their relative quantities in various virion fractions determined. our study provides a valuable inventory of virion-associated host proteins for further investigation of their roles in the replication cycle, pathogenesis and immunoreactivity of vsv. vesicular stomatitis virus (vsv, family rhabdoviridae) is a prototypic nonsegmented negative-strand rna virus (order mononegavirales) serving as a model for other human and animal pathogens including other rhabdoviruses such as rabies virus. vsv is also being widely investigated as a vector for vaccines (reviewed in [1] ), gene therapy (reviewed in [2] ) and oncolytic (anticancer) virotherapy (reviewed in [3, 4] ). as a result, currently two phase i human clinical trials evaluating the safety of the vsv-based hiv vaccines (clinicaltrials.gov, trials nct01438606 and nct01578889) are currently in progress. in addition, a phase i human clinical trial using replication-competent oncolytic vsv against hepatocellular carcinoma is in progress (trial nct01628640). vsv replicates in the cytoplasm and contains a nonsegmented, negative-strand rna genome of approximately 11 kb encoding five viral proteins, all of which are included in the mature virion. the large polymerase protein (l) and phosphoprotein (p) together form the viral rna dependent rna polymerase (rdrp). in mature virions, the rdrp is associated with the nucleocaspid (n) protein encapsidated viral genome, together forming the ribonucleoprotein (rnp) complex. the matrix (m) protein is responsible for nucleocapsid condensation and is the primary driving force behind viral budding through the host cell plasma membrane, whereby the virion obtains its lipid bilayer envelope. embedded in that envelope is the transmembrane glycoprotein (g), which is essential for receptor binding and cell entry (reviewed in [5] ). vsv virions, like those of many viruses, contain not only virus encoded-proteins, but also host (cellular) proteins [45] . some of these proteins are enclosed within the virion structure, while others, in the case of enveloped viruses like vsv, are embedded in the host-derived membrane [6] . while many of these incorporations could be ''accidental'', others may reflect the mechanism of virus assembly, be necessary for viral function, impact host range and infection efficiency, or influence the outcome of subsequent infections. for example, a recent study showed that depletion of a number of host proteins normally incorporated into herpes simplex virus type-1 virions not only reduced virion production in those cells, but that new infections with the resulting virions also yielded fewer progeny virions even in cells expressing normal levels of the host protein [7] . the host-derived proteins of a virus may also affect the host immune response. this is an especially important consideration for viruses, including vsv, used in therapeutic applications where large numbers of virus particles are administered, as it may influence efficacy as well as the potential for adverse side effects. incorporation of icam-i into the envelope of human immunodeficiency virus type-1 (hiv-1) not only increased infection efficiency [8, 9, 10, 11] but also interfered with virus neutralization by host antibodies [12, 13, 14, 15] . in another example, the presence of host complement control proteins such as cd46, cd55 and cd59 in the viral envelope has been shown to protect against antibody dependent complement mediated virus lysis in several viruses including human t cell leukemia/ lymphoma virus type i [16] , human cytomegalovirus [16] , hepatitis c virus [17] , hiv-1 [18, 19] , extracellular enveloped vaccinia virus [20] , simian virus 5 [21] and mumps virus [21] . to comprehensively look at host protein incorporation, a number of purified viruses have been analyzed by mass spectrometry including poxviruses [22, 23, 24] , herpesviruses [25, 26, 27, 28, 29, 30, 31, 32] , orthomyxoviruses [33] , coronaviruses [34, 35] , retroviruses [36, 37, 38, 39] , paramyxoviruses [40, 41] , baculoviruses [42] , hytroviruses [43] and arteriviruses [44] . our previous study examined vsv virions grown in three different cell lines (bhk-21, 4t-1 and a549) originating from different species, and found a number of similarities and differences in the host protein content [45] . here we conduct an analysis not only of intact (''whole'') virions, but also virions treated with proteinase k (prok) to remove surface proteins to look at the localization of host protein incorporation into the virion. syrian golden hamster kidney fibroblast cells (bhk-21; atcc# ccl-10) were grown in monolayer cultures maintained in minimum essential medium (eagle's mem, cellgro) supplemented with 9% fetal bovine serum (fbs, gibco), 0.3% glucose (w/v), 3.4 mm l-glutamine, 90 u/ml penicillin and 90 mg/ml streptomycin, and kept in a 5% co 2 atmosphere at 37uc. infectivity [plaque forming units (pfu) per ml] of virus stocks was determined by standard plaque assay on bhk-21 cells. recombinant wild-type (wt) vsv (indiana serotype) was generated previously [46] using pbs-l, pbs-p, pbs-n, and pvsvfl(+) plasmids, and was kindly provided by john k. rose (yale university) [47] . to grow and purify virus vsv, bhk-21 cells were infected with at a multiplicity of infection (moi) of 0.001 and incubated at 37uc in media containing 5% fbs. virus containing media was collected around 24 hours (h) post infection (p.i.) when most cells were infected but significant cell detachment had not yet occurred in order to maximize exclusion of cellular debris. the media was centrifuged at 3,0006 g for 10 minutes (min) to remove large cellular debris. the virus was then purified as described in [48] , with slight modifications. in brief, clarified supernatants were underlayed with 5 ml 20% (w/v) sucrose in hen buffer (10 mm hepes ph 7.4, 1 mm edta, 100 mm nacl) and centrifuged at 28,000 rpm and 4uc for 3.5 h in a beckman sw32 ti rotor. the resulting virus-containing pellet was resuspended overnight in hepes buffered saline, ph 7.5 [hbs; 21 mm hepes, 140 mm nacl, 45 mm kcl, 0.75 mm na 2 hpo 4 , 0.1% (w/v) dextrose] and then centrifuged in a 7.5-27.5% continuous gradient of optiprep (axis shield) in hbs at 26,500 rpm and 4uc for 30 min using a beckman sw40 ti rotor. the virus-containing band was removed from the gradient, diluted with et buffer (1 mm tris-hcl ph 7.5, 1 mm edta), pelleted by centrifugation at 27,000 rpm and 4uc for 1.5 h using a beckman sw40 ti rotor and resuspended in et buffer. for protease treatment, purified virions were treated with 0.08 mg prok per 1 mg total protein, re-purified by centrifugation through a sucrose cushion as previously described [45] and resuspended in et buffer. for isolation of viral rnp complexes, purified virions not treated with prok were disrupted as previously described [49] . in brief, virions were disrupted in a buffer containing a final concentration of 10 mm tris-hcl (ph 8.0), 5% glycerol (v/v), 0.4 m nacl, 1.85% triton x-100, and 0.6 mm dtt, and pelleted through a 30% glycerol cushion onto a 100% glycerol cushion by centrifugation at 38,000 rpm and 4uc for 2 h using a tla 100.2 rotor. under these conditions, proteins with moderate to high affinity for the viral nucleocapsid, including the viral l/p polymerase complex, remain associated with the nucleocapsid [49] . the recovered rnp complexes were diluted 1:1 (v/v) with a buffer containing 50 mm tris-hcl (ph 8.0), 10 mm nacl, 5 mm mgcl 2 and 2 mm dtt. virions were absorbed to carbon-formvar coated grids (electron microscopy sciences) by floating grids on 5 ml drops of sample for 30 seconds (s). grids were blotted dry, stained with 2% uranyl acetate in water for 30 s, blotted to remove excess stain and airdried. samples were visualized using a jeol jem 2100 lab6 transmission electron microscope. total protein equaling 50 mg for purified virion samples and 60 mg for prok treated virions was separated on a 10% tris-glycine sds-page gel under reducing conditions and stained with coomassie brilliant blue r250. these quantities were chosen so that the total amount of viral n plus p protein was approximately equivalent for both sample types. each gel lane was cut into 13 or 14 gel bands for analysis. gel pieces were subjected to in-gel trypsin digestion and the resulting peptides were extracted from the gel matrix, separated using waters acquity ultra performance liquid chromatography (uplc) with nano-split and analyzed using a ltq-xl tandem mass spectrometer (thermofisher scientific, waltham, ma) as described previously [50] . briefly, samples were separated by a 65 min linear gradient from 95% solvent i (0.1% formic acid in water)/solvent ii (0.1% formic acid in acetonitrile) to 50% solvent i/ii at a flow rate of 500 nl/min on reversed phase chromatography using a trap/elute method with a in-house c 18 sample trap in line with a c 18 analytical column. each full ms scan was followed by eight ms/ ms scans of the most intense ions with data-dependent mode using the dynamic exclusion option (top 8 method). the spectra were searched using the sequest algorithm of the bioworks software (thermofisher, san jose, ca; version 3.3.1 sp1) against the ipi.human.v.3.18 and ipi.mouse.v.3.18 databases concatenated with a vsv and sendai virus protein database. a parent ion mass tolerance of 2.0 da, fragment ion mass tolerance of 1.0 da, and a 16 da differential modification for methionine oxidation were used for search parameters. protein identifications were accepted when the peptide probability was greater than 95.0% [51] , the protein probability was greater than 99.0%, and contained at least two unique peptides. scaffold software was used for data compiling of each group and calculating spectral counts, unique peptides, percent coverage and empai [52, 53, 54] . while a single set of gel bands was isolated for each sample, three replicate uplc-ms/ms runs were conducted for the whole and prok virion samples, although one set of runs could not be used for the whole virions due to poor data quality. protein enrichment was analyzed using the database for annotation, visualization and integrated discovery (david) v.6.7 [55, 56] . using the mus musculus genome as the background data set, protein sets were analyzed for enrichment using the terms from the gene ontology (go) biology process, cellular component or molecular functions fat databases. the go fat databases contain the more specific go terms while excluding the more general terms. the enriched terms were then subjected to cluster analysis using the default settings, to identify groups of related enriched terms, with overall enrichment scores based on the ease scores of the member terms. the broadest term, representing most if not all the proteins in the cluster, is used here to describe the cluster. cellular lysates were prepared by mock infecting bhk-21 cells or by infecting them with vsv at a moi of 0.05. cells were harvested at 18 h p.i. and lysed in ripa buffer (25 mm tris-hcl ph 7.6, 150 mm nacl, 1% np-40, 1% sodium deoxycholate and 0.1% sds). protein concentrations of cellular lysates, purified virions, prok treated virions and rnp complexes were determined by bradford assay. 50 mg of purified virions, 60 mg prok treated virions, 15 mg of rnp complexes and 10 mg of cellular lysates were separated on tris-glycine 10% sds-page gels, transferred to pvdf membranes and rapidly stained with the reversible dye ponceau s prior to the use of antibodies to confirm viral protein loading and the quality of protein transfer from gel to membrane. membranes were blocked in tbs (0.5 m nacl, 20 mm tris ph 7.5) with 0.1% tween 20 and 5% non-fat milk powder and then probed with antibodies against cc2d1a (a300-285a; bethyl laboratories), yes1 (3201; cell signaling), itch (32/itch; bd transduction laboratories), hsc70 (k-19; santa cruz biotechnology), prl2 (05-1583; millipore), ck1 (2655; cell signaling), or ddx3y (pa5-22050; thermo scientific). detection was with species-specific horseradish peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence plus (ecl+) protein detection system (ge healthcare) and captured using a chemidoc-it imaging system (uvp imaging, upland ca). recombinant wt vsv was grown in bhk-21 cells and purified using gradient centrifugation. the purity of the resulting material was then examined by electron microscopy (em). as seen in figure 1 , nearly all of the material present was clearly identifiable as vsv virions, although many of them were bent, a previously observed form that is generally believed to be infectious [57] and may represent an em processing artifact [58, 59] . the titer of these purified virions on bhk-21 cells was 1.4610 11 pfu/ml, demonstrating this preparation was highly infectious. a portion of these whole virions was used directly for proteomic analysis and additional confirmation assays, while the other portion was treated with prok and re-purified (fig. 2) . prok treatment cleaves all proteins on the exterior of the virions as well as the extracellular domain of all transmembrane proteins. however, it cannot penetrate the viral envelope, leaving all vsv proteins except for g intact, as well as all host proteins contained within the virion and the transmembrane and cytoplasmic domains of the host membrane proteins located in the viral envelope. this treatment also tends to alter the density of any remaining cell derived vesicles relative to the treated virions, facilitating their removal during the subsequent repurification step [60] . however, proteins on the interior of any residual cellular vesicles would still be detectable. for mass spectrometry (ms) analysis, total protein from 50 mg purified virions or 60 mg prok treated virions was separated by 1d-sds-page. these quantities were chosen so that the amount of viral n and p protein was approximately equal in both sample types as determined by coomassie staining (fig. 3a) to facilitate comparisons between samples. importantly, while n, p and l bands were similar in both virion preparations, no full length g protein was visible for prok treated virions, indicating the prok treatment was highly successful. after separation by sds-page, the resolved proteins were cut out in a series of bands as indicated in figure 3a . these bands were then subjected to in-gel trypsin digest and the resulting peptides were extracted, separated by uplc and analyzed by tandem ms (ms/ms). in this study, virions were grown on bhk-21 cells as it is the standard cell line for growth of vsv and the high virion yields aid in purification. however, a complete database of syrian hamster proteins is not available. instead, peptides were identified by matching them to either a human or a mouse database. the results from both searches were highly similar; therefore only those from the search of the mouse database are presented here. in addition to all five viral proteins, 257 different host proteins were identified in total (table s1) , with 181 proteins identified in whole virions and 183 proteins identified in prok treated virions (fig. 3b ). this is a considerably larger number of host protein than identified in our previous study where 30 proteins were identified in a different preparation of bhk-21 derived virions [45] . this is likely primarily due to dividing the samples into a larger number of bands following 1-d sds-page. in addition, in the present study we used enhanced sample separation through use of uplc, which should also allow for more sensitive detection. of the 30 proteins detected in bhk-21 derived whole virions in our previous study, 27 were detected in at least one sample type here, showing consistency in the proteins detected in independent virion preparations purified using two different methodologies (continuous iodixanol gradient versus discontinuous sucrose gradient). differences in the purity of the virions prepared using these two methods could also contribute to differences in the number of host proteins identified. however, em images, staining of total protein on sds-pages gels and infectivity (4.2610 7 pfu/mg total protein for the present method versus 2.4610 7 pfu/mg total protein for our previously published method [45] ) all indicate virions prepared using the method presented here are of equal or greater purity to those used previously. surprisingly, more than 40% of proteins found in prok treated virions were not found in whole virions even though the prok treated virions were derived from the whole virion preparation (fig. 2) . one possibility is that some of these proteins may have been masked by more abundant host or viral proteins that were removed by treatment. for example, 25% (19/76) of the proteins detected in prok treated virions but not whole virions were from the position correlating to that of the viral g protein in the whole virion sample (table s1 and fig. 3a) . another possible explanation is that these proteins are in low abundance or possess other properties making them difficult to detect consistently by ms. of the proteins found only in prok treated virions, 50% of proteins were identified based on detection of only two unique spectra (the minimum number allowed under our criteria), as opposed to 15% for proteins also identified in whole virions (table s1 ). unsurprisingly, ease of detection also seemed to be a major determinate of consistency of detection between technical replicates. proteins found in all technical replicates were identified based on only two unique spectra in 19% and 17% of cases for whole virions and prok treated virions respectively, while proteins found in only one technical replicate were identified based on only two spectra in 71% and 77% of cases (fig. 3c-d and table s1 ). in general, the position of the identified proteins on the gel was consistent with the predicted molecular mass of the proteins (table s1 and fig. 3a) although some appeared at higher molecular masses, likely due to post-translational modifications affecting protein mobility. exceptions to this general pattern were keratins, common environmental contaminants, which were found across a wide range of molecular weight and therefore excluded from this analysis. following prok treatment, 74 of the 181 proteins identified in whole virions were no longer detectable, likely due to their removal by the treatment. however, other proteins were still detected in the prok treated sample but appeared at a lower molecular mass versus the predicted molecular mass and/or that observed for whole virions, likely due to the removal of the extracellular domain. for example, hepatocyte growth factor receptor was found in a slice spanning approximately 140-225 kda in the whole virion while it was found in slices spanning approximately 50-80 kda in prok treated virions. this demonstrates the value of information about the approximate size of the proteins in the sample (as determined by the position of the gel band) in interpreting data from proteinase treated samples. although the primary focus of this study was determining host protein incorporation and localization in vsv virions, our search database also included vsv protein sequences allowing for their detection. in untreated samples, a mean of 1231-182 spectra per technical replicate were detected for each of the five vsv encoded proteins (fig. 4a) . the number of spectra went up upon prok treatment for all vsv proteins except g where there was a sharp reduction. this is consistent with our other data (fig. 3a) demonstrating that prok treatment is effectively removing the extracellular domain of the vsv g protein. removal of the extracellular domain was confirmed by determining the distribution of the spectra and peptides derived from the g protein (fig. 4b) . the mean number of spectra associated with the transmembrane domain and cytoplasmic tail of the g protein were unchanged between whole and prok treated virions, while prok treatment decreased the number of spectra associated with the extracellular domain of g. in looking at the distribution of the detected peptides in the g protein, prok treatment completely eliminated peptides associated with the n-terminal portion of the extracellular domain while a few were still detected in the more c-terminal portion of the domain, suggesting this region has a slight degree of resistance to proteinase cleavage, although the decrease in spectral counts suggests the majority the g proteins were cleaved. while ms analysis of unlabeled proteins cannot be used to make quantitative comparisons of protein abundance, spectral counts or measures derived from spectral counts (including empai values shown in table s1 ), can be used to make semi-quantitative comparisons [61] . based on spectral counts, few if any of the host proteins come close to matching the viral proteins in abundance within the virion. only one host protein, low-density lipoprotein receptor-related protein 1 (lrp1), had a mean of more than 100 total spectra per technical replicate in untreated whole virions, while only 17 (9.4%) had a mean of 20 or more (table s1 ). no proteins found only in prok treated virions had a mean spectral count of more than 20, indicating that these proteins are present in the virions in low abundance. as discussed in the previous section, proteins not associated with the interior of the virion, including proteins embedded in the host derived viral envelope, can be identified by their absence in prok treated samples or by a size shift upon prok treatment. here we wanted to examine the localization within the vsv virion of host proteins that did not display any of these patterns (i.e. proteins that do not appear to be associated with the viral envelope). five of the chosen proteins were found in both whole and prok treated virion samples and had similar spectral counts in both sample types (table s1 ). we also chose two proteins, proto-oncogene tyrosineprotein kinase yes (yes1) and protein tyrosine phosphatase type iva 2 (ptp4a2; also called prl2), detected in prok treated virions but not whole virions, as proteins found in the treated samples would also be expected to be found in the whole virions from which they were derived (fig. 2) . to identify proteins associated with vsv rnp complexes, we also isolated and analyzed viral rnp complexes from a separate preparation of purified vsv virions by treating them with detergent and salt to release the nucleocapsid from the envelope and m protein while maintaining its association with the l/p polymerase complex. because protein quantities were chosen so that the amount of n and p protein was approximately equal for all virion samples (fig. 5) , the host protein associated with whole virions that was retained in the treated samples could be estimated. based on this, proteins were determined to be associated with the viral rnp complex, associated primarily with the interior of the virion but not the rnp or associated primarily with the exterior of the virion. most proteins were barely detectable in the rnp complexes, suggesting they do not associate with the viral rnp or that the association was disrupted under the conditions used for rnp isolation. only for e3 ubiquitin-protein ligase itchy (itch) were substantial amounts of protein detected in rnp. the m protein of vsv, as well as other rhabdoviruses, contains a late domain (ldomain) including a proline rich ppxy motif known to bind ww domains found in a number of cellular proteins [62] . vsv m protein has been shown to bind the ww domain of nedd4 (also detected in this analysis) [63] , and can presumably also bind other hect domain-containing e3 ubiquitin ligases, including itch, as has been reported for other viruses with l-domains including ppxy motifs [64] . however, given that itch is present in rnp complexes at levels that nearly equal the other two sample types, while m protein is sharply reduced, it seems likely that itch may also interact with one of the rnp proteins (n, p or l). three of the tested proteins, yes1, casein kinase i isoform alpha (csnk1a1) and heat shock cognate 71 kda protein (hspa8; also known as hsc70), while barely detectable in rnp, were found at similar levels in whole and prok treated virions, indicated they are primarily associated with the interior of the virions. in contrast, levels of ptp4a2 and putative atp-dependent rna helicase pl10 (d1pas1; also known as ddx3y), dropped sharply in the prok and rnp samples indicating they are not primarily located in the interior of the virion. one protein, coiled-coil and c2 domain-containing protein 1a (cc2d1a), identified by ms could not be detected by wb, indicating either protein levels below the threshold of detection, a misidentification of the protein based on homology, or a false-positive identification. the latter seems the least likely as the protein was identified in both sample types, with at least 6 consecutive amino acids matched in manual inspection. of the 6 proteins that could be detected by western blot, only 4 were present at similar levels in whole and prok treated virions, despite similar spectral counts in both sample types and the absence of a size shift. therefore, while these characteristics may suggest a protein associated with the interior of the virion, localization should be independently confirmed. to obtain an overview of the types of proteins most commonly associated with purified vsv virions, an enrichment analysis was conducted using gene ontology terms. this analysis was done for all three go databases (biological process, cellular component and molecular function), first using all the host proteins found in our analysis then looking specifically at proteins associated with prok treated virions (fig. 6) , as this is the highest purity sample and excludes proteins found exclusively on the exterior of the virion. related enriched terms were then clustered together to give an overall enrichment score. the most highly enriched clusters, when looking at all the proteins, were vesicles and vesicle mediated transport, protein localization and nucleotide binding (fig. 6 ). these were also the most highly enriched clusters when looking specifically at proteins identified in prok treated virions. the cluster cell adhesion was also relatively highly enriched when looking at all proteins, but less so in prok treated virions as would be expected. enrichment of proteins involved in vesicles and vesicle mediated transport, protein localization and cytoskeletal organization is consistent with known features of vsv assembly and budding. more than one third (88/257) of the proteins identified are represented in at least one of these 4 clusters, indicating many virion-incorporated host proteins are likely involved in these functions. transport of the vsv nucleocapsid to the site of budding has been shown to be dependent on microtubules [65] and is an important step in vsv assembly. furthermore, as seen for many enveloped viruses (reviewed in [66] ), vsv budding appears to use the host proteins involved in multivesicular body (mvb) formation which are relocated from endosomal membranes to the plasma membrane in an m protein dependent manner (reviewed in [67] ). however, the budding of vsv and related viruses may still have some unique features. while the specific members of the mvb pathway utilized by vsv for its budding are not clear, it is known that, unlike what has been observed for many other viruses, tsg101 is not essential for the budding of vsv and rabies virus [68] , while contradictory reports exist regarding the importance of vps4a [68, 69] . in contrast, the host ubiquitinproteasome system does appear to be essential [63, 69] . the lipid composition of the vsv envelope is consistent with that of its host cell, although levels of cholesterol and sphingomyelin are elevated [48] , indicating that unlike some other viruses such as influenza virus and hiv-1 [70, 71, 72] , vsv does not bud through host membrane regions enriched in lipid rafts. instead, vsv g and m proteins appear to initially localize to separate microdomains of the host plasma membrane but then either merge or cluster together with each other and host protein containing microdomains at the site of virus budding [73] . therefore, vsv readily incorporates the membrane proteins of its host. in pseudotyping experiments using both mixed virus infections and expression of viral or host proteins from vsv recombinants, non-vsv proteins can make up a significant portion of the protein in the vsv envelope, with incorporation levels up to 31% of that seen for vsv g protein reported [74, 75] . consistent with these observations, 46% (118/257) of proteins identified in our study are associated with the cellular component go term ''plasma membrane'', suggesting they were acquired along with the envelope. the tendency of vsv to indiscriminately acquire relatively large numbers of host membrane proteins has the potential to be exploited to help fine tune therapeutic vectors through the choice of cell line used to generate the viruses. for example, growing oncolytic vsvs in a cell line naturally expressing or engineered to express high levels of complement control protein may improve efficacy by slowing the rate of clearance by the host immune system following administration. while many of the proteins identified in vsv virions appear to be associated with viral assembly, budding or the host-derived viral envelope, they may also have additional functions that affect virus replication. furthermore, proteins were also identified that do not figure 5 . confirmation of host protein incorporation in virion preparations. protein lysates from uninfected (bhk) and vsv infected (bhk+ vsv) bhk-21 cells were separated by sds-page along with total protein from whole virions, proteinase k (prok) treated virions, and viral ribonucleoprotein complexes (rnp). loading of the virion samples was confirmed by ponceau s staining of the membrane. the position of the viral glycoprotein (g), nucleocapsid protein (n), phosphoprotein (p), and matrix protein (m) is indicated. western blots using primary antibodies against coiled-coil and c2 domain-containing protein 1a (cc2d1a), protein tyrosine phosphatase type iva 2 (ptp4a2), putative atp-dependent rna helicase pl10 (d1pas1), proto-oncogene tyrosine-protein kinase yes (yes1), casein kinase i isoform alpha (csnk1a1), heat shock cognate 71 kda protein (hspa8) and e3 ubiquitin-protein ligase itchy (itch), were conducted as indicated to determine the presence of the host proteins. approximate molecular mass of the proteins is given on the left. doi:10.1371/journal.pone.0104688.g005 have known associations with these functions. our study provides a valuable inventory of virion-associated host proteins for further investigation into their potential roles in vsv replication cycle, pathogenesis, and immunoreactivity. table s1 cellular proteins identified in whole and prok treated vsv virions following 1-d sds-page and uplc-ms/ms. (xlsx) figure 6 . functional enrichment analysis. enrichment analysis and clustering based on gene ontology terms was conducted for (a) all proteins identified and (b) proteins identified in the proteinase k (prok) treated virions, as described in the materials in methods. the five clusters in each database with the highest enrichment score are depicted here. numbers above the bars indicate the number of identified proteins associated with each cluster. doi:10.1371/journal.pone.0104688.g006 nonsegmented negative-strand viruses as vaccine vectors recombinant rhabdoviruses: vectors for vaccine development and gene therapy vesicular stomatitis virus as a flexible platform for oncolytic virotherapy against cancer vesicular stomatitis virus as an oncolytic vector rhabdoviridae. in: in: knipe dm and plunder and stowaways: incorporation of cellular proteins by enveloped viruses analysis of virion-incorporated host proteins required for herpes simplex virus type 1 infection through a rna 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raft-derived microdomains: a quantitative analysis of the surface distribution of ha, na and m2 proteins evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides plasma membrane microdomains containing vesicular stomatitis virus m protein are separate from microdomains containing g protein and nucleocapsids foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles role of heterologous and homologous glycoproteins in phenotypic mixing between sendai virus and vesicular stomatitis virus the authors thank dr. sue moyer (university of florida college of medicine) for providing vsv reagents for this project and eric hastie for critical comments on the manuscript. key: cord-304306-rxjahqwh authors: vlachakis, dimitrios; papakonstantinou, eleni; mitsis, thanasis; pierouli, katerina; diakou, io; chrousos, george; bacopoulou, flora title: molecular mechanisms of the novel coronavirus sars-cov-2 and potential anti-covid19 pharmacological targets since the outbreak of the pandemic date: 2020-10-08 journal: food chem toxicol doi: 10.1016/j.fct.2020.111805 sha: doc_id: 304306 cord_uid: rxjahqwh the novel coronavirus sars-cov-2 has emerged as a severe threat against public health and global economies. covid-19, the disease caused by this virus, is highly contagious and has led to an ongoing pandemic. sars-cov-2 affects, mainly, the respiratory system, with most severe cases primarily showcasing acute respiratory distress syndrome. currently, no targeted therapy exists, and since the number of infections and death toll keeps rising, it has become a necessity to study possible therapeutic targets. antiviral drugs can target various stages of the viral infection, and in the case of sars-cov-2, both structural and non-structural proteins have been proposed as potential drug targets. this review focuses on the most researched sars-cov-2 proteins, their structure, function, and possible therapeutic approaches. coronaviruses (covs) are enveloped, positive-sense, single-stranded ribonucleic acid (rna) viruses that belong to the family coronaviridae. these viruses harbor the largest genome among rna viruses, specifically 26 to 32 kilobases . based on phylogenetic analyses, covs can be classified into four genera, which include alpha coronaviruses, beta coronaviruses, gamma coronaviruses, and delta coronaviruses (jaiswal and saxena, 2020) . out of those genera, beta-covs contain the majority of viruses that infect humans, with the aforementioned genera being subdivided into four lineages (a, b, c, d) . generally, covs can infect the respiratory, hepatic, gastrointestinal, and central nervous system of mammals, birds, and fish . there are seven covs known to cause human disease. four of those covs (hcov229e, nl63, oc43, and hku1), known as non-severe acute respiratory syndrome (sars)-like covs, induce mild diseases and are globally endemic, while three highly pathogenic covs (sars-cov-1, mers, and sars-cov-2) can lead to lethal disease (raoult et al., 2020) . out of the latter three covs, sars-cov-2, responsible for the coronavirus disease 2019 , has proved a serious threat against public health and global economies in 2020 (zheng, 2020) . sars-cov-2 is a beta-coronavirus of the b lineage with a diameter of approximately 60 to 140 nm (cascella et al., 2020) . its genome appears to encode as many as 14 open reading frames (orfs), with the 5'orf1a/orf1ab encoding polyproteins, which are autoproteolytically processed into 16 non-structural proteins (nsps) (gordon et al., 2020) . those nsps (nsp1-16) form the replicase/transcriptase complex (rtc). this complex is composed of various enzymes, including a papain-like protease (nsp3), the main protease (nsp5), an nsp7-nsp8 primase complex, a primary rna-dependent rna polymerase (rdrp/nsp12), a helicase/triphosphatase (nsp13), a 3'-5' exoribonuclease (nsp14), a uridine-specific endonuclease (nsp15), and n7-and 2'o-methyltransferases (nsp10/nsp16). the remaining orfs encode four structural proteins and nine putative accessory factors (gordon et al., 2020) . these structural proteins include the spike (s) glycoprotein, the small envelope (e) glycoprotein, the membrane (m) glycoprotein, and a nucleocapsid (n) protein (astuti and ysrafil, 2020) . coronaviruses entry into host target cells is dependent on the binding of s glycoprotein to a specific cell receptor and the subsequent priming of the aforementioned protein by host cell proteases. sars-cov-2 uses the angiotensin-converting enzyme 2 (ace2) host receptor for internalization and, mainly, the type ii transmembrane serine protease (tmprss2) for spike glycoprotein priming (kumar et al., 2020) . specifically, the s glycoprotein binding domain, which is present at 331 to 524 residues, attaches strongly to the ace2 receptor (astuti and ysrafil, 2020) . after attachment, tmprss2 and a number of lysosomal proteases cleave and activate the s glycoprotein leading to the entrance of sars-cov-2 into the cell through endocytosis or direct fusion of the viral membrane with the host membrane (romano et al., 2020; shang et al., 2020) . once inside the host cytoplasm, the viral rna undergoes translation, where the orfs that encode nsps are firstly translated and produce the polyproteins pp1a and pp1ab that are further cleaved by virus-encoded proteases into nsps (kumar et al., 2020) . the aforementioned proteases are the papain-like proteases (plpro) and the chymotrypsin-like protease (3clpro) (astuti and ysrafil, 2020) . these nsps have a great role in viral rna replication. specifically, the rna replication machinery of sars-cov-2 involves the rdrp (nsp12), the zinc-binding helicase (nsp13), and enzymes, such as a bifunctional 3′→5′ mismatch exonuclease and a cap ribose 2′-o methyltransferase, which are related to viral rna modification, such as messenger rna (mrna) capping and rna proofreading (viswanathan et al., 2020) . the accessory and structural proteins are translated by a specific set of subgenomic rnas (romano et al., 2020) . some of those, including m, s, e, are insulated in the endoplasmatic reticulum and then transported to the endoplasmatic reticulum-golgi intermediate compartment (ergic) (astuti and ysrafil, 2020) . the replicated genome has the ability to join the n protein to the nucleocapsid form and move into the ergic (astuti and ysrafil, 2020) . the virions are later assembled at ergic and are subsequently released through exocytosis out of the cell (kumar et al., 2020) . patients infected with sars-cov-2 were first reported in december 2019, and, since then, cases have risen to the point that as of 30 august 2020, this virus has led to 24,854,140 cases and 838,924 deaths worldwide (who, 2020; zheng, 2020) . the dominant clinical features of covid-19 include cough, shortness of breath or difficulty breathing, fever or chills, muscle or body aches, vomiting or diarrhea, and new loss of taste or smell (cdc, 2020) . person to person spread of sars-cov-2 via respiratory droplets, when a patient coughs, sneezes, or talks is the main way of covid-19 transmission, while the mentioned disease can also occur through indirect means such as touching objects contaminated with sars-cov-2 (lotfi et al., 2020) . this virus primarily affects the respiratory system, with respiratory symptoms of covid-19 being quite heterogeneous, varying from minimal symptoms to significant hypoxia with acute respiratory distress syndrome (ards) (yuki et al., 2020) . moreover, severe covid-19 patients display an acute inflammatory response. specifically, transcriptomic rna-seq analysis of covid-19 patients showed that the viral infection induced several immune pathways and pro-inflammatory cytokines, implying sustained inflammation and cytokine storm (wu et al., 2020b) . covid19 could be fatal with risk factors, including the co-existence of chronic diseases, age, sex, obesity, and smoking (michelozzi et al., 2020; petrakis et al., 2020) ). obesity, particularly, is considered a chronic inflammatory state with obese individuals showcasing high concentration of pro-inflammatory cytokines like interleukin 6 (il-6). with regard to covid-19, il-6 has been shown as an independent predictor of mortality (yadav et al., 2020) . the currently available antiviral option for hospitalized patients is remdesivir, which may inhibit the replication process by targeting the rdrp. previously proposed treatments for hospitalized patients included hydroxychloroquine, which thought to disrupt virus endocytosis, and lopinavir/ritonavir, which thought to inhibit sars-cov-2 main protease (astuti and ysrafil, 2020; magro, 2020) . the aforementioned facts have made the development of therapeutic molecules a necessity in the scientific community. spike is a transmembrane glycoprotein in covs that forms homotrimers that protrude from the viral surface . spike is the structural protein that gives cov viral particles their characteristic crown-like shape, from which their original name was coined (coutard et al., 2020) . spike is essential in viral entry, a process which is achieved through binding with the ace2 host receptor . the above has led to considering s a promising drug target, therefore the study of this glycoprotein is important in the development of novel therapeutics against covid-19. structure-wise, each monomer of the s glycoprotein contains two subunits termed s1 and s2, which mediate attachment and membrane fusion, respectively (ou et al., 2020) . the s1 is divided into the n-terminal domain (ntd), followed by the receptor-binding domain (rbd) and two structurally conserved subdomains termed sd1 and sd2. the rbd constantly switches between a standing-up position, associated with receptor binding, and a lying down position associated with immune evasion (shang et al., 2020) . the sd1 and sd2 subdomains cap the s2 subunit and, thus, protect the conserved fusion machinery (henderson et al., 2020) . the s2 subunit contains the fusion peptide (fp), the heptad repeat 1 (hr1), the heptad repeat 2 (hr2), the transmembrane domain (tm), and the cytoplasmic domain (cp) (xia et al., 2020) . viral entry requires both binding of s to the cellular receptor and s glycoprotein priming by cellular proteases (hoffmann et al., 2020) . this priming includes a cleavage at the boundary between the s1 and s2, for the majority of coronaviruses, plus a cleavage at the s2' site located immediately upstream of the fp, a process present in all coronaviruses (hoffmann et al., 2020; mccallum et al., 2020; walls et al., 2020) . the latter cleavage has been implicated in activating the glycoprotein for membrane fusion through extensive irreversible conformation changes (mccallum et al., 2020) . specifically, after cleavage, the exposed fusion peptide inserts itself into the plasma membrane while the hr1 and hr2 can interact to form the six helical bundle (6hb) fusion core, which eventually leads to the fusion of the viral membrane with the host plasma membrane (joshi et al., 2020) . the above priming is accomplished through proteases such as tmprss2 and lysosomal cathepsins (jaimes et al., 2020) . sars-cov-2 s differentiates from covs of the same clade since it features a novel furin-like cleavage upstream of the cleavage site 1 which seems to be cleaved during biosynthesis. this characteristic potentially allows for more efficient spreading of sars-cov-2 in the human population compared to other lineage b beta coronaviruses coutard et al., 2020) . while using the spike glycoprotein as a potential drug target, a researcher can focus on viral binding to host receptor or membrane fusion. as prime example, monoclonal antibodies can be used to tightly bind the spike glycoprotein's rbd in order to neutralize sars-cov-2. such a case is the monoclonal antibody cr3022, which binds to s rbd while not competing with the binding of ace2, allowing the monoclonal antibody to interfere with ace2 binding (huo et al., 2020) . another approach, this time focusing on membrane fusion, is ek1c4. ek1c4 is a lipopeptide which disrupts membrane fusion by targeting hr1 (xia et al., 2020) . on the other hand, interrupting s function could also be achieved by targeting host cell proteins with the use of drugs such as ace2 and tmprss2 inhibitors (mckee et al., 2020) . unfortunately, ace inhibitors, which are used for the treatment of hypertension and chronic heart failure, do not inhibit ace2. moreover, scientists are concerned that such drugs could increase the likelihood of severe covid-19 illness, though current data tends to provide no link between the use of ace inhibitors and increased severity of covid-19 (hippisley-cox et al., 2020; who, 2020) . all the above can be considered the first steps towards covid-19 therapeutics and indicate that the spike glycoprotein may indeed be the most promising drug target against sars-cov-2 infection. coronaviruses' nucleocapsid protein, or n protein, is the most abundant viral protein that can be detected in the human host from the early stages of infection (che et al., 2004) . its main function is to bind to viral rna to form a ribonucleoprotein nucleus, which contributes to its entry into the host cell as well as to the interaction with cellular processes after fusion, such as cell cycle regulation, and antigen processing and presentation of the virus (huang et al., 2004; wang et al., 2010) . the sequence of n protein of sars-cov-2 is approximately 90% similar to the same protein of sars-cov (gralinski and menachery, 2020) . the n protein genome comprises a serine-rich binding region (sr) between an n terminal domain (ntd) and a c terminal domain (ctd). n structural protein forms the replication transcription complexes (rtc), which play an important role in the synthesis of the genome virus (xia et al., 2020) . in sars-cov, the ν protein contributes to the activation of cyclooxygenase-2 (cox-2), thus promoting inflammation in the lungs (yan et al., 2006) . in addition, it participates in processes that affect the cell cycle, such as inhibiting the phosphorylation of phosphoprotein b23, a protein that is essential for cell cycle evolution during centrosome replication (zeng et al., 2008) . moreover, studies have shown the inhibitory effect of n protein on the cyclin-cyclin-dependent kinase complex (c-cdk), resulting in a reduction of s-phase progression (surjit et al., 2006) . a different function of the n protein is its interaction with the protease subunit p42, which degrades viral proteins . specifically, this interaction may impair proteolysis of viral proteins and their presentation to cytotoxic t-lymphocytes, thus promoting viral evasion from immune effectors . concerning the immune system, n protein causes limitations in the immune responses generated by the body due to viral infections as it inhibits type i interferon (ifn) (lu et al., 2011) . according to another study, due to the aggregation of the human elongation factor 1 a (ef1a) induced by the n protein, the proliferation of the cells of the cell line under study, specifically 293 t cells, was reduced (zhou et al., 2008) . in contrast, n protein interacts with heterogeneous nuclear ribonucleoprotein (hnrnpa1), causing an increase in viral rna synthesis (luo et al., 2005) . the structural study of the ntd of sars-cov-2 n protein showed that it resembles a wrist consisting of acidic moieties with a palm of basic components, and the core of the beta-sheet extends like fingers (kang et al., 2020) . the ntd of the n protein binds to the rna genome in the n45-181 region that exists as a monomer (chang et al., 2006) , while the presence of the amino acids arginine at position 94 and tyrosine at position 122 appears to be necessary for viral rna binding (mcbride et al., 2014) . according to further studies, the enhanced binding capacity of viral rna requires the combination of the binding region and ntd and ctd of n protein (chang et al., 2009) . n protein function regulation is mediated through the central linker region, which includes serine and arginine residues (sr region), having essential phosphorylation sites. in this region, the major phosphorylation site is formed, which enhances protein interactions in proteins and the localization of binding proteins within the cell (chang et al., 2014) . the ctd is hydrophobic and rich in helical region in which dimerization occurs as it contains residues that self-bond to form homodimers (mcbride et al., 2014) . structurally, each asymmetric ctd group consists of four individual homodimers from the compound of which an octamer is formed. the arrangement of these sections is schematically similar to the letter "x" which forms symmetrical folding structures, perpendicular to the middle point of the structure. the n terminus appears to be basic due to the positive charges in this region, which could be implied to be a site for nucleic acid binding (chen et al., 2007) . n protein, together with s protein, is a very promising area in the development of effective therapeutics to prevent the proliferation of viral offspring as it participates in activities necessary for the function and proliferation of the virus and is, therefore, another key ingredient after spike proteins (satarker and nampoothiri, 2020) . currently, the only potential drug targeting the n protein is pj34 which inhibits the viral protein's function and has shown some promise both in vitro and in animal studies (saxena, 2020) . the coronaviruses' envelope protein, or e protein, is a vital protein, which is tiny and consists of the n-terminal domain, a hydrophobic domain, and the c terminal domain (ruch and machamer, 2011) . the n terminal extends to the first nine amino acids, the hydrophobic region extends between 10-37 amino acids and the c terminal between 38-76 amino acids, where the first 11 amino acids are located in virion while the hydrophobic tail is in the cytoplasm, which through its oligomerization creates an ionic pore across the membrane (shen et al., 2003; verdia-baguena et al., 2013) . through structural studies, sars-cov-2 form is presented, which includes 35 α-helical regions and 40 looped regions (gupta et al., 2020) . a unique characteristic that occurs in sars-cov-2 e protein and not in other homologous sars-cov e protein is the replacement of the 69 th amino acid of the protein sequence, from arginine to alanine, glutamine, and aspartate. furthermore, a deletion specific to sars-cov-2 envelope protein flanks this position. these changes may have an effect on viral conformation, protein-protein interaction and, possibly, affect the oligomerization process necessary to form a transmembrane ion channel. in addition, at positions 55 and 56 of the amino acid sequence, amino acid valine and threonine have been identified (bianchi et al., 2020) . in coronaviruses, the e protein forms viroporins which are small hydrophobic proteins that are necessary for viral assembly and release, while also participating in pathogenic processes that cause cytotoxicity (ye and hogue, 2007) . the co-expression of m and e proteins facilitates the production of spherical infectious particles, while the heterotypic j o u r n a l p r e -p r o o f interactions of nsp2 and nsp3 cause the desired curvature in the endoplasmic reticulum (er) membrane (schoeman and fielding, 2019) . the hydrophobic tail of the protein in the cytoplasm through the proline residues incorporated in it targets the region of the cis-golgi complex, as well as the n-terminal of protein e targets the golgi complex through additional elements (cohen et al., 2011) . the diffusion of the ionic gradient from the e protein into the ergic and golgi may lead to the exit of the virion (liu et al., 2007) . the last four amino acids of the sequence that comprise the c terminal domain include a pattern called postsynaptic density protein / disc large / zonula occludans-1 (pdz) binding pattern (pdm). therefore, e protein through binding of protein associated with caenorhabditis elegans lin-7 protein 1 (pals1) to the pdm, aids in the disruption of the lung epithelium, thus being a potential pharmacological target for improved treatment against sars-cov-2 (teoh et al., 2010; satarker and nampoothiri, 2020) . regarding therapeutics, in silico studies have proposed a number of drugs that target the e protein, which include belachinal, macaflavanone e, and vibsanol (gupta et al., 2020) . membrane protein, or m protein, is present in greater amounts than all other proteins in coronaviruses (ea and jones, 2019). in coronaviruses, this protein has a short length n terminal domain, as its total length is 220-260 amino acids, belongs to the n-linked glycosylated proteins with a conserved region of 12 amino acids and is attached to tripartite transmembrane regions which are further linked to the c terminal domain (arndt et al., 2010) . according to structural studies, it exists in two forms, one compact and one long. initially, these two forms are homodimers of the n-terminal ectodomain and the c-terminal endodomain, where the endodomain can be elongated or compressed, resulting in a long and compact form. tyrosine residues at position 211 are necessary for the stability of the long form of the m protein, as well as the spike protein is observed mainly in the long form of the m protein, suggesting that this form promotes the establishment of the spike proteins. the long form of the m protein bends the membrane, thus creating a spherical structure surrounding the ribonucleoprotein (neuman et al., 2011) . m protein is organized in a two-dimensional lattice and provides a scaffold in viral assembly, while its translation takes place in the polysomes connected to the membranes following its fusion to the endoplasmic reticulum and transport to the golgi complex, where it interacts with the e proteins to create virions (neuman et al., 2006; tseng et al., 2010) . m protein appears to affect the immune response to pathogens, as it inhibits nuclear factor kappa b (nfkb) through interactions with ikkb (i kappa b kinase) leading to a decrease in cox-2 levels and resulting in an increase in the proliferation of the pathogenic virus (fang et al., 2007) . in addition, the c terminus of the m protein inhibits the interaction of 3-phosphoinositide-dependent protein kinase 1 (pdk1) and protein kinase b (pkb), thus leading to the release of the caspases 8 and 9, which eventually cause cell death or apoptosis (tsoi et al., 2014) . based on the above information regarding the m protein of coronaviruses, sars-cov-2 protein can be a potential pharmacological target for limiting and inhibiting the formation of virions and preventing inflammatory reactions in host cells, as the presence of the m protein is crucial in the viral life cycle (satarker and nampoothiri, 2020) . currently, broad spectrum antiviral drugs that target the membrane protein of coronaviruses, such as jl103, have been proposed for possible use against sars-cov-2 (khan et al., 2020). viruses in the coronaviridae family code for two types of cysteine proteases, including the 3c-like protease (3clpro) and up to two papain-like proteases. with these enzymes' use, the polyprotein generated through the translation of the viral genome of the virus is cleaved into sixteen nonstructural proteins. this processing step is crucial for the generation of the replicase complex that is required for rna replication. sars-cov-2, in particular, encodes a single papain-like protease (henceforth referred to as plpro) (freitas et al., 2020) . the plpro domain is part of the multi-domain nsp3 protein. it processes the nsp1/2, nsp2/3, and nsp3/4 cleavage sites. the plpro domain, which is membrane-associated, recognizes a cleavage sequence in the threshold areas between nsp1/12, nsp2/3, and nsp3/4. after the cleavage, the nsp1 nsp2, nsp3 are separated from the viral polyprotein and the next steps of the formation of the replicase complex may be excecuted, allowing the production of new virions and the establishment of the viral infection. studies on the enzymatic function of plpro have shown its capacity for recognition and hydrolysis of the ubl protein isg15 (interferon-induced gene 15) and the protein ubiquitin (ub), both of which are cellular proteins. thus, plpro constitutes a deubiquitinating (dub) and deisgylating enzyme (báez-santos et al., 2015) . this type of activity has important ties to the course of the innate immune response in the setting of sars-cov infection. numerous instances of ubiquitination and isgylation events are observed in various stages of the innate immune response. the interference of plpro in different parts of these pathways can be achieved through the recognition and the interaction with deisgylating and/or deubiquitinating proteins that exist therein. specifically, plpro appears to act as an antagonist, blocking the generation of cytokines of importance in the host innate immune response. these include type i interferon β (ifnβ), as well as ccl5 and cxcl10 (freitas et al., 2020) . given its crucial role in the virus replication cycle and the suppression of the host immune defenses, plpro has had a significant existing history as a drug target against a number of coronaviruses, such as mers cov and sars cov. similarly, amid the covid-19 pandemic, plpro constitutes a promising pharmacological target. within the scientific community, there is a race for the development of inhibitors against this particular viral enzyme, as well as for the possible employment of existing protease inhibitors. numerous studies are dedicated to the exploration of pre-existing, fda-approved drugs with the goal of repurposing them for the inhibition of sars-cov-2. through a number of methods, among which is molecular dynamics and virtual screening, fda-approved drugs, which offer the advantage of already completed preclinical and clinical phases, are combed through in order to select the best candidates for the inhibition of the sars-cov-2 papain-like protease. a prime example of a drug candidate provided through such an approach is phenformin (kandeel et al., 2020) . as mentioned above, the main protease, also known as 3c-like protease (3clpro), is among popular targets against sars-cov-2. 3clpro executes proteolytic cleavage of the overlapping pp1a and pp1ab translated viral polyproteins, allowing the production of functional proteins during the coronavirus replication process (ullrich and nitsche, 2020) . more specifically, 3clpro (nonstructural protein 5), following its autocleavage, processes the aforementioned polyproteins at their respective cleavage sites (du et al., 2004) . the 3clpro monomer contains three domains, with a long loop connecting the second and third domains. the enzyme's active site is located between the first and the second domain, containing a catalytic pair of cysteine and histidine. the histidine's imidazole attracts the side-chain proton of the cysteine, creating a thiolate nucleophile, which then attacks the amide bond on the enzyme's substrate. through proton abstraction from histidine, the n-terminal peptide product is released, while the cterminal product is released through the hydrolysis of the thioester, allowing the subsequent restoration of the catalytic pair (estrada, 2020) . in order for the enzyme to perform its catalytic activity, dimerization is necessary. the two protomers bind to each other through an n-terminal finger, which aids in the formation of the substrate-binding site . the mediation of nonstructural viral proteins' maturation by 3clpro makes it a very attractive target for the development of anti-coronavirus drugs. it is important to note that 3clpro executes cleavage exclusively of the polypeptide sequences that follow a glutamine residue. this ability offers a significant advantage, given that there are no known host-cell protease enzymes that share this particular specificity of substrate. in essence, inhibitors of the main protease would be unlikely to have off-target effects (ullrich and nitsche, 2020) . sars-cov inhibitors have so far steered towards peptide inhibitors and small-molecule inhibitors (wu et al., 2020a) . studies conducted on the design and screening of 3clpro inhibitors usually focus on cleavage sites, substratebinding sites, the catalytic pair at the enzyme's active center, as well as various reported residues of critical importance for enzyme function. significant conservation of the catalytic site has been observed between the 3clpro structures of the sars-cov, mers-cov, and sars-cov-2 viruses. this discovery allows the examination of already established inhibitors of the other coronaviruses' 3clpro enzyme, under the possibility that they may prove effecting against the 3clpro of the novel sars-cov-2 . furthermore, research groups have been establishing screening processes for the possible repurposing of various drugs. among those, anti-bacterial drugs, antihypertensive drugs, as well as natural compounds with antiviral properties have shown a high binding affinity to 3clpro, making them attractive candidates for the inhibition of the viral 3clpro towards the treatment of covid-19 (wu et al., 2020a) . another key enzyme necessary for the viral replication cycle that has been characterized as potent therapeutic target is the viral helicase (vlachakis et al., 2013c) . sars-cov-2 nsp13 has been shown to possess ntpase and rna helicase activity; it catalyzes the unwinding of double stranded rna in an ntp-dependent manner (shu et al., 2020) . due to its sequence conservation across coronaviruses, targeted inhibition of its enzymatic activity is recognized as a promising strategy for antiviral therapy (habtemariam et al., 2020) . sars-cov-2 nsp13 has a high structural and functional similarity with mers and sars nsp13, belonging to the sf1 helicase family. it consists of 5 domains that form a triangular pyramid shape; the two 'reca-like' domains, domain 1a and 2a, along with domain 1b form the triangular base, while the zinc binding domain (zbd) and stalk domain, that connects 1b and zbd domains, are located at the apex of the pyramid (hao et al., 2017; jia et al., 2019) . domains 1a, 1b and 2a are involved in ntp and nucleic acid binding, while it has been shown that the nsp13 enzymatic activity is enhanced by direct interaction of nsp12 at the zbd-1a interaction site (jia et al., 2019; romano et al., 2020) . a number of compounds have been reported as competitive inhibitors against sars coronavirus (scv) nsp13 activity that could pave the ground for clinical research for sars-cov-2 inhibition. myricetin and scutellarein have been identified to strongly interrupt atpase but not helicase activity, possibly by direct binding at the atpase domain (yu et al., 2012) . aryl diketoacids (adk) and dihydroxychromone derivatives demonstrated selective inhibition against the scv nsp13 duplex dna-unwinding activity in multiple binding sites in the target enzyme, while 2-arylmethyloxy-6-(3chlorobenzyloxy)-5-hydroxychromones were shown to inhibit both ntpase and helicase activities (lee et al., 2009a; lee et al., 2009b; kim et al., 2011) . another class of antiviral compounds, bananins, have been shown to inhibit viral transcription and replication by significantly reducing the viral rna levels whereas they did not affect viral entry. the compounds were tested against scv ntpase/helicase activity and a number of bananin derivatives were found to be potent atpase and helicase inhibitors at concentrations significantly below toxicity levels (tanner et al., 2005) . 3-[(2-nitrophenyl)sulfanylmethyl]-4-prop-2-enyl-1h-1,2,4-triazole-5-thione, ssya10-001, was recognized by adedeji and coworkers as a novel inhibitor of scv that interferes with the dsrna/dsdna activity of nsp13 without competitive inhibition of ntp and nucleic acid binding, but possibly by inducing conformational changes of the enzyme that disrupt the unwinding and translocation mechanisms (adedeji et al., 2012) . bismuth salts have been also characterized as strong inhibitors of both atpase and duplex-unwinding activities through binding to the zinc binding domain (zbd) of scv nsp13 at micromolar concentrations (yang et al., 2007a; yang et al., 2007b) . similarly, a recent study on sars-cov-2 identified inhibitory effects of bismuth salts against nsp13, and specifically, bismuth potassium citrate (bpc) and ranitidine bismuth citrate (rbc) exhibited effective inhibition of ntpase and helicase activity with the same binding mode, interrupting the sars-cov-2 replication cycle (shu et al., 2020) . in silico methods have been proven to be a crucial step for the rapid, cost-efficient identification of potent compounds with inhibitory effects in antiviral research loukatou et al., 2015; papageorgiou et al., 2016) . as such, computational approaches employing molecular modelling and virtual screening against known antivirals, active compounds and/or fda approved drugs for drug repurposing, have been applied for the identification of candidate sars-cov-2 helicase inhibitors (loukatou et al., 2015) . iftikhar et al. identified two compounds, meclonazepam and oxiphenisatin, that bind at β19-β20 loop on the 1a domain of sars-cov-2 helicase, that is directly involved in unwinding double stranded nucleic acids (iftikhar et al., 2020) . vapreotide, an aids-related diarrhea drug, was found to exhibit the lowest binding free energy amongst 23 approved antivirals in a computer-aided drug design pipeline (borgio et al., 2020) . similar approaches have identified cmp1, cmp3a, cmp11 and cmp15 to competitively bind at the ntp binding site (mirza and froeyen, 2020) , and elbasvir, approved for hcv treatment, to inhibit not only helicase activity by blocking ssdna binding, but also rna binding to rdrp (balasubramaniam and reis, 2020). rna viruses encode rna-dependent rna polymerase (rdrp), an essential enzyme for viral replication since it catalyzes rna synthesis, governing the initiation and elongation phase (ng et al., 2008) . rdrps share a similar core structure resembling the shape of a right hand with three subdomains, namely the palm, finger and thumb subdomains (vlachakis et al., 2017; venkataraman et al., 2018) . the catalytic site of rdrps is formed by seven conserved motifs (motif a-g), five of which are located within the most conserved palm subdomain (a-e), while motifs f and g are found in the fingers (vlachakis et al., 2013a; vlachakis et al., 2013b; shu and gong, 2016) . sars-cov-2 polymerase, or nsp12, recently resolved structure consists of a right-hand rdrp domain and a nidovirus-specific n-terminal extension domain, the niran domain. rdrp and niran domains are connected by an interface domain and an additional β-hairpin domain located at the groove formed by niran domain and palm subdomain stabilizes the structure (gao et al., 2020) . due to their high significance in viral life cycle and their conserved mode of function across viral families, rdrps have long been the target of antiviral research with a number of approved therapies targeting viral polymerases (tsai et al., 2006; ng et al., 2008; reznik and ashby, 2017; yao et al., 2018; peersen, 2019) . consequently, during the current covid-19 outbreak, nsp12 is considered as a promising target for antiviral therapeutics. identified nucleoside analogues that were originally developed to target viral rdrps and novel compounds are evaluated as antiviral drug candidates against covid-19 (neogi et al., 2020) . remdesivir is the most promising drug candidate to date. it is a prodrug of an adenosine nucleotide analog that is converted to active nucleoside triphosphate upon cell entry and competes with atp in the rdrp binding site, leading to chain termination (warren et al., 2016; yin et al., 2020) . remdesivir was developed for the treatment of ebola virus disease (evd), but it has not been approved after showing low efficacy in a phase iii clinical trial for evd (warren et al., 2016) . however, in vitro and in vivo studies of remdesivir against mers-cov and sars-cov and recent studies against sars-cov-2 showed inhibitory effects and antiviral activity with low cytotoxicity levels (sheahan et al., 2017; wang et al., 2020) . additionally, remdesivir was tested in a compassionate clinical trial for severe covid-19 patients, and after 10-day treatment an 68% clinical improvement was observed (grein et al., 2020) . several clinical trials have been reported to evaluate remdesivir safety and efficacy, while a number of patents have already been granted (eastman et al., 2020) . favipiravir, a guanine analog developed for treatment of influenza viruses, currently approved in china and japan, is also an anti-covid19 drug candidate (furuta et al., 2017) . favipiravir inhibits polymerase activity, yet the exact mode of action has not been elucidated. two possible mechanisms for chain termination have been proposed, either by inhibition of the atp catalytic site, or by misincorporation in a nascent viral rna (furuta et al., 2005) . clinical trials for favipiravir have shown efficient treatment for moderate covid-19 patients and several clinical trials have been registered and are ongoing (cai et al., 2020; du and chen, 2020; li and de clercq, 2020) . besides remdesivir and favipiravir, several nucleotide analogs have been reported as potential antiviral agents. ribavirin, a ribonucleoside analog that has been tested against several rna viruses but exhibits higher levels of cytotocisity and side-effects, is also on ongoing clinical trial for covid-19 treatment combined with interferon-α2b (falzarano et al., 2013) . jockush et al. have shown in the inhibitory effects of sofosbuvir, alovudine, zidovudine, tenofovir alafenamide and emtricitabine and termination of polymerase rna synthesis through in vitro assays (jockusch et al., 2020) . silibilin is predicted to have a dual activity against sars-cov-2 infection; silibilin can potentially reduce viral replication activity by targeting nsp12 as a remdesivir-like inhibitor, and modulate inflammatory responses by direct inhibition of stat3 (boschbarrera et al., 2020) . stat3 showcases both pro-and anti-inflammatory abilities; it mediates interleukin 10 (il-10) activity, a known anti-inflammatory cytokine. on the other hand, in the case of cancer, persistent activation of stat3 mediates tumor-promoting inflammation (hillmer et al.; yu et al., 2009) . as an alternative strategy, an isonicotinic acid derivative, enisamium and its putative metabolite, vr17-04, have been identified by in vitro assays as drug candidates against sars-cov-2. enisamium was shown to inhibit the activity of the sars-cov-2 rna polymerase and its active metabolite exhibits similar efficacy with remdesivir triphosphate, it is approved in 11 countries and does not require intravenous administration, unlike remdesivir (walker et al., 2020) . in silico approaches have also been used to identify novel inhibitors, either by screening clinically used polymerase inhibitors, or fda approved drugs and/or natural compounds (kar et al., 2020; pokhrel et al., 2020; ruan et al., 2020; zhang et al., 2020b) . it is critical to find ways to block the spread of sars-cov-2 and stop covid-19. research on sars-cov-2 structure and mechanisms of action has provided a number of intriguing drug targets. those targets, which include both non-structural and structural proteins, have an essential role in various stages of viral infection, from viral entry to viral replication and formation of viral vesicles. disrupting their action may have a therapeutic effect against covid-19. although research on the aforementioned targets is intense, and some results may seem promising, the current situation requires being precautious before declaring the uncovering of a sars-cov-2 therapy. nevertheless, specific studies, especially on the spike glycoprotein and viral proteases, allow some restrained optimism regarding the future. as in any therapeutic approach concerning human disease, toxicity is a critical component in the development of safe and effective drugs against sars-cov-2. ace2, as reviewed earlier, is a promising target for the development of therapeutic strategies. a recombinant form of the human ace2 protein was synthesized as a therapeutic treatment for covid-19, functioning as a decoy for sars-cov-2 and essentially preventing the virus from binding to the cell surface ace2 (schuster et al., 2010) . the approach of recombinant proteins carries the risk of off-target binding, which is the binding of the recombinant protein to other cell-receptors, leading to unwanted signaling cascades. furthermore, use of recombinant proteins may lead to the activation of the immune system, which may in turn result in the production of anti-drug antibodies and their deposition in the form of immune complexes, potentially hindering the function of important organs (de groot and scott, 2007) . lastly, this sort of treatment may lead to serum sickness, in the case that the immune system may view the recombinant protein as an antigen (schellekens, 2005) . in the case of viral replication inhibitors, instances of toxicity reported for other nucleoside analogs can provide insight into the possible toxicity of analogs such favipiravir or remdesivir, which are being evaluated for use against sars-cov-2. while inhibiting the viral polymerase enzyme, nucleoside analogs additionally inhibit mitochondrial dna polymerase-gamma, resulting in reduction of mitochondrial protein synthesis (moyle, 2000) . aspects of toxicity include myopathy and pancreatitis (johnson et al., 2001) , severe metabolic acidosis paired with elevated lactate, with significant observed mortality (falcó et al,. 2002 ) and bone marrow suppression in patients treated with 3'-azido-3',3'-dideoxythymidine (azt) (moyle, 2000) . as far as protease inhibitors are concerned, drugs such as ritonavir, an inhibitor of cytochrome cyp3a4, can have an effect on the metabolism of drugs-substrates of cyp3a4, which in turn may result in unpredictable interactions between the drug (rock et al., 2014) . as summarized by chary and colleagues, the use of humanized antibodies as a therapeutic method harbors dangers of hypersensitivity reactions, immunostimulation, which results in chills, fever and other reactions, and finally, immunosuppression, which may often become a window for opportunistic infections (chary et al., 2020) . to date, efforts are being made by the scientific community to develop a vaccine against sars-cov-2. vaccines that have been developed and are in the stage of clinical trials are based on one hand on the virus genome and on the other hand, on the structure of the viral s-protein tu et al., 2020) . one of the vaccines that started developing in early 2020 is moderna's mrna-1273. this vaccine is a synthetic clone of mrna that encodes the viral s-protein. the goal of the vaccine is to provoke an immune response specific for s-protein of sars-cov-2. another characteristic of this vaccine is that the virus is not required for its development, as the synthetic mrna is encapsulated in lipid nanoparticles (tu et al., 2020) . the chadox1 ncov-19 vaccine was developed by the university of oxford and is currently in clinical phase iii. this vaccine consists of a non-replicable adenovirus vector and the s protein sequence of the sars-cov-2 protein. characteristic of this vaccine is that due to the adenovirus-based vector, it can cover both the respiratory and gastrointestinal epithelium, which are the two main sites expressing the sars-cov-2 ace-2 receptor (folegatti et al., 2020) . finally, the university of queensland is developing a vaccine that is equally based on the molecular structure of the virus sars-cov-2. as sars-cov-2 is an enveloped virus, the fusion of its viral membrane with the host cell membrane is required for infection. for this purpose, the structure of the glycoproteins of the viral envelope alter from the pre-fusion form, which is more unstable, to the post-fusion form. thus, this vaccine is a stabilized subunit vaccine based on molecular clamp technology, such as the vaccines against influenza virus and ebola virus, and allows the recombinant viral proteins to remain stable in the pre-fusion form (coleman et al., 2014; tu et al., 2020) . the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid 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the nucleocapsid protein of sars-associated coronavirus inhibits b23 phosphorylation crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors novel coronavirus polymerase and nucleotidyl-transferase structures: potential to target new outbreaks sars-cov-2: an emerging coronavirus that causes a global threat the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha the authors are responsible for the choice and presentation of views contained in this article and for opinions expressed therein, which are not necessarily those of unesco and do not commit the organization. key: cord-319722-udqu5jub authors: ng, eddy w. y.; wong, melody y. m.; poon, terence c. w. title: advances in maldi mass spectrometry in clinical diagnostic applications date: 2013-04-07 journal: chemical diagnostics doi: 10.1007/128_2012_413 sha: doc_id: 319722 cord_uid: udqu5jub the concept of matrix-assisted laser desorption/ionization mass spectrometry (maldi ms) was first reported in 1985. since then, maldi ms technologies have been evolving, and successfully used in genome, proteome, metabolome, and clinical diagnostic research. these technologies are high-throughput and sensitive. emerging evidence has shown that they are not only useful in qualitative and quantitative analyses of proteins, but also of other types of biomolecules, such as dna, glycans, and metabolites. recently, parallel fragmentation monitoring (pfm), which is a method comparable to selected reaction monitoring, has been reported. this highlights the potentials of maldi-tof/tof tandem ms in quantification of metabolites. here we critically review the applications of the major maldi ms technologies, including maldi-tof ms, maldi-tof/tof ms, saldi-tof ms, maldi-qqq ms, and seldi-tof ms, to the discovery and quantification of disease biomarkers in biological specimens, especially those in plasma/serum specimens. using seldi-tof ms as an example, the presence of systemic bias in biomarker discovery studies employing maldi-tof ms and its possible solutions are also discussed in this chapter. the concepts of maldi, saldi, seldi, and pfm are complementary to each other. theoretically, all these technologies can be combined, leading to the next generation of the maldi ms technologies. real applications of maldi ms technologies in clinical diagnostics should be forthcoming. the history of matrix-assisted laser desorption/ionization (maldi) mass spectrometry (ms) can be dated back to 1985 [1] . karas et al. first reported the use of an organic molecule as a matrix to assist desorption/ionization of other small molecules under uv laser irradiation [1] . in 1987, koichi tanaka and his colleagues showed that coupling maldi to a time-of-flight (tof) mass analyzer allowed the detection of macromolecules, especially proteins [2] . koichi tanaka's maldi-tof ms method for analyses of macromolecules was highly regarded. it created new opportunities for application of ms to biomedical research. in 2002, koichi tanaka together with two other chemists were awarded the nobel prize in chemistry 2002 for their developments of soft desorption ionization methods for mass spectrometric analysis of biological macromolecules. in the past 15 years, while applications of maldi ms technologies to qualitative and quantitative analyses of proteins and metabolites have been investigated, these technologies have been widely employed in proteomic/metabolomic research, especially in biomarker discovery. because this chapter is aimed at updating readers on the advances in maldi ms technologies in clinical diagnostic applications, it is not going to provide a comprehensive review on all maldi ms technologies. this chapter will only cover maldi-tof ms, maldi-tof/tof ms, saldi-tof ms, maldi-qqq ms, and seldi-tof ms, which have great potentials in influencing the clinical diagnostic practices. basic principles and brief overviews of these technologies will be provided to an extent allowing readers to understand the potentials and limitations of these technologies in diagnostic applications. then the applications of these technologies to biomarker discovery and their potential uses in biomarker quantification will be reviewed. practical concerns and their possible solutions on applying maldi-based ms technologies, especially seldi, to quantification and discovery of serum/plasma biomarkers will also be addressed. maldi is regarded as a soft desorption ionization method because it can result in the formation of ions without significantly breaking any chemical bonds by using optimal laser irradiance [1] . this is particularly important in obtaining the correct mass of a biomolecule, especially for proteins, during ms analysis. subsequently, structure or sequence information can be obtained by tandem ms analysis. as indicated from the name maldi, a matrix is needed to assist desorption and ionization of an organic molecule under uv irradiation [1, 3] . desorption/ionization efficiencies of different types of biomolecules depend on the chemical used as the matrix. for example, cyano-4-hydroxycinnamic acid (chca) is a very good matrix for peptides [4] . sinapinic acid is good for intact proteins [4] . super-dhb (i.e., a mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid) is good for glycan analysis [5] . 3-hydroxypicolinic acid is commonly used for maldi-tof ms analysis of dna molecules [6] . an analyte or a mixture of analytes needs to mix with a chemical matrix in solution phase, and is added on a conductive ms sample plate. after drying, analyte-matrix co-crystals are formed. this work-flow is illustrated in fig. 1 . they are then subjected to maldi ms analysis. under uv irradiation the analytes will be desorbed and ionized [3] . in the absence of alkali metal ion and/or halide ion in the co-crystals, singly charged protonated and deprotonated molecules are usually formed. the mass (or molecular weight) of a molecule will be approximately equal to "m/z value à1.0073" and "m/z value +1.0073", respectively. in the presence of alkali metal ion and/or halide ion, such as na + and cl à , metal ion adducts and/or halide ion adducts may be formed. either positively or negatively charged molecules are transferred to the mass analyzer for separation according to their mass-to-charge (m/z) ratios. there are various types of mass analyzers. a tof or tof/tof mass analyzer is the most commonly used coupled with a maldi source. when kinetic energy is given to a group of charged molecules in direct proportion to their charge states under vacuum, the charged molecules will travel in a flight tube at a velocity inversely proportional to the square root of their m/z values [7] . in other words, charged molecules with larger m/z values have longer tof, and they are efficiently separated for generating a mass spectrum within 1 s. the resolving power of a tof mass analyzer depends on the length of the flight path. in contrast, other commonly used mass analyzers, such as ion trap, orbitrap, and fourier transform ion cyclotron resonance (fticr) mass analyzers, have resolving powers directly proportional to the time of the charged molecules staying inside the mass analyzers. the highthroughput nature of a tof mass analyzer makes it perfectly match with a maldi source. a single tof mass analyzer does not allow efficient structure/sequence elucidation of a targeted analyte in a mixture of analytes. this can be overcome by linking two tof mass analyzers in series, i.e., tof/tof. the first tof mass analyzer is used to resolve and select the precursor ion of a targeted analyte for later fragmentation, whereas the second tof mass analyzer is used to separate the fragment ions for generating a tandem ms spectrum [8, 9] . discovery of disease-specific biomarkers for assisting diagnoses is still a difficult but important task all over the world. one commonly used approach for identification of disease-specific biomarkers is to compare the quantitative biomolecule profiles of plasma/serum specimens from the patients with the target disease and control subjects without the disease. because of high heterogeneity in the baseline concentrations of various circulating biomolecules among both the patients and control subjects, it is important to obtain and compare quantitative plasma/serum biomolecule profiles from patient and control groups with a reasonable sample size. in such a case, high-throughput technologies are required in order to complete the analyses of the specimens within an acceptable period of time. the major advantage of maldi-tof ms is that it is high-throughput in nature. after preprocessing, samples for ms analyses are applied on a maldi sample plate as individual spots. maldi-tof ms analysis of each sample spot takes less than 1 min. one hundred samples can be automatically analyzed within 1 h. in contrast to electrospray ionization (esi), which is another commonly used soft ionization technology, a single preprocessed sample is usually subjected to liquid chromatography (lc) before esi ms analysis [10] , resulting in a turn-around time of 20 min to 1 h. therefore, it takes 30-100 h for analyzing 100 samples by esi ms. for a shotgun proteomic profiling approach, it will take a day for obtaining a coarse proteomic profile of a single specimen, or at least a week for obtaining a comprehensive proteomic profile. it is well known that maldi-tof ms and maldi-tof/tof ms have been widely applied to protein identification in proteomics laboratories. when subjected to maldi-tof ms, peptides and proteins are predominantly detected as singly charged protonated molecules at high sensitivities. on one hand, the detection sensitivity of maldi-tof ms depends on the chemical composition of a molecule. on the other hand, in general the detection sensitivity is inversely proportional to the mass of a molecule. for large proteins, e.g., albumin, usually at least an amount of 100 fmol to 1 pmol is required for a reliable ms signal. for a clean preparation, a peptide of 0.25 fmol can be readily detected. maldi-tof ms can efficiently obtain the masses of majority of peptides in a protein tryptic digest in the ms range of m/z 1,000-2,500 with high accuracy (<40 ppm for external m/z calibration; <5 ppm for internal m/z calibration). the resulted list of tryptic peptides' peak intensities and masses can then be subjected to a database search to obtain the protein identity by using the tryptic peptide mass fingerprinting algorithms, e.g., mascot [11] . for individual tryptic peptides, it can be further subjected to tandem ms to obtain a series of a-, b-, y-ions if one has a maldi-tof/tof ms instrument. the resulting list of fragment ions' peak intensities and masses can be further subjected to a database search to obtain the protein identity by using the ms/ms ion search algorithms [12] . as described in the previous section, analytes are embedded in analyte-matrix co-crystals before ms analysis. the majority of the matrices are derivatives of benzoic acid, cinnamic acid, and carboxylic acids [3] . however, in maldi-tof ms, these small molecules themselves form protonated ions, fragment ions, and cluster ions, and cause intensive chemical noises in the mass range below m/z 800 [13, 14] . these noises cause significant interference during the analyses of small molecules. this explains why there have been only a few reports on using maldi-tof ms for small molecule analysis in the past 27 years [14] . although analyses of small molecules are technically difficult, all these reports have provided concrete evidence that maldi-tof ms is a feasible tool for analysis of small biomolecules, including amino acids [15] , lipids [16] , o-linked glycans [17] , steroid hormone [18] , etc. in addition to matrix chemical noises in the low mass region, the prerequisite formation of analyte-matrix co-crystals causes uneven distribution of analytes on a sample spot. ms signals of various analytes vary significantly among the cocrystals. in common practice a representative mass spectrum of a single sample spot is generated from the summation of mass spectra obtained at different positions within a sample spot. as a result, conventional maldi-tof ms methods are usually considered not quantitative, or at most semi-quantitative [4] . however, the reproducibility of the peak intensities of biomolecules in a maldi-tof ms spectrum can be improved by using an unbiased automatic mass spectrum acquisition protocol across a sample spot and by providing a fine network (e.g., nitrocellulose coating) for formation of a layer of homogeneous small analyte-matrix co-crystals [4] . with an unbiased ms acquisition protocol and the use of nitrocellulose film, intra-assay and inter-assay coefficients of variation (cvs) of the normalized peak intensities of peptide/protein standards were found to be <15% [4] , suggesting that maldi-tof ms is a feasible tool for profiling and quantifying peptides and proteins in biological samples. solved by using chromatographic techniques to enrich a subgroup of proteins with matched physicochemical properties. this concept was first introduced by bruker daltonics inc. (bremen, germany) as a commercially available system called clinprot for semi-quantitative profiling of proteins/peptides in serum/plasma. in this system, various types of functionalized magnetic beads with different chromatographic properties are available, including hydrophobic interaction (c3, c8, c18), weak cation exchange, weak anion exchange, metal ion affinity (cu 2+ , fe 3+ ), and lectin affinity (concanavalin a). the clinprot magnetic bead technology is only licensed to be performed with maldi-tof ms instruments from the same manufacturer. the clinprot system users are supplied with a kit of standard protocol, together with specific buffers. the compositions of the binding and washing reagents are not disclosed. because maldi-tof ms is a sensitive technology for detection of proteins, only 5 μl of plasma/serum is required for the clinprot system, according to the supplier's instructions. the eluted proteins/ peptides are added on a thin-layer of chca, and subjected to maldi-tof ms for obtaining a semi-quantitative mass spectrum. the clinprot technology was first reported to be highly quantitative. the cvs for the normalized protein/peptide peak intensities were 7% [19] . however, later studies showed that the cvs were between 20% and 30% for both manual and robotic assays [20, 21] . there are about 40 reports on using the clinprot system in discovery of potential biomarkers of human diseases, such as oral cancer [19] , head and neck cancer [20] , and nephrotic syndrome [22] . in 2007, jimenez et al. reported an automated method comparable to the clinprot system by using c18 hydrophobic magnetic beads for profiling of serum peptides with masses in the range of m/z 800-4,000 [23] . the intra-assay and interassay cvs were 2-38% and 10-53%, respectively. later our group developed a strategy for quantitative profiling of both serum peptides/proteins and micropreparative purification of the corresponding peptides/proteins in parallel using c18 hydrophobic, strong anion exchange, and weak cation exchange magnetic beads [24] . in our method, only 2 μl of serum is required, and sinapinic acid is used as the chemical matrix. by using an automatic platform for the binding, washing, and elution steps and using a maldi-tof ms instrument optimized for quantitative proteomic profiling, both intra-assay and inter-assay cvs were found to be 4-30%. because the peptides/proteins corresponding to the potential diagnostic peaks are purified in parallel with the profiling experiments, the subsequent work for deciphering protein identities of the potential biomarker peaks is greatly simplified. using this method, we have recently identified proapolipoprotein cii (pro-apoc2) and a des-arginine variant of serum amyloid a (saa) as host response biomarkers for diagnosis of late-onset septicemia and necrotizing enterocolitis in preterm infants [25] . the aposaa score computed from plasma apoc2 and saa concentrations was effective in identifying necrotizing enterocolitis/late-onset sepsis cases in both independent case-control and prospective cohort studies. on the basis of the aposaa score, infants suspected with the diseases could be stratified into different risk categories. this enabled neonatologists to withhold treatment in 45% and enact early stoppage of antibiotics in 16% of non-sepsis infants. the combined use of hydrophobic magnetic beads and maldi-tof/tof ms allows both quantitative profiling of plasma/serum peptides and direct identification of the amino acid sequences of the peptides without the need for subsequent purification work. when villanueva et al. attempted to identify the serum peptide pattern associated with metastatic thyroid cancer by undertaking this approach, they found that the majority of the disease-associated peptides were derived from fibrinopeptide a, complement c3f, and fibrinogen-α as a result of exopeptidase degradation [26] . it was speculated that proteases produced by the thyroid cancer cells led to the formation of these disease-associated peptides [27] . this led them to develop further the sequence-specific exopeptidase activity test (sseat) test [27] . instead of identification of the disease-associated peptides, the test monitors degradation of artificial substrates in the presence of individual patients' sera by maldi-tof ms. double labeled, non-degradable peptides are spiked into the samples as internal standards at the same time to adjust for the adsorptive and processing-related losses. the peak intensity ratios of degradation products to the corresponding non-degradable reference peptides are used as biomarkers. the cvs of these ratios were reported to be 6.3-14.3%. using the sseat test, the group could classify 48 metastatic thyroid cancer patients and 48 healthy controls at 94% sensitivity and 90% specificity [27] . the major advantage of the sseat test is that reproducibility problems related to sample collection, storage, and handling in serum peptide profiling analysis can be greatly reduced. furthermore, theoretically, by using specific peptide sequences as substrates for different diseases, the diagnostic sensitivity and specificity of a sseat test may be further improved. immunosorbent assay and immunoturbidity assay are the most commonly used technologies for quantification of specific protein biomarkers in routine clinical chemistry laboratories. both technologies require the use of specific antibodies. the use of antibodies allows sensitive and specific quantification of a target protein biomarker. however, the use of antibodies can cause uncertainty in measurement. affinity and specificity of the antibody preparations against a specific antigen varies significantly from source to source. it is not uncommon for immunoassay kits from different manufacturers to produce disconcordant readings. furthermore, a specific protein can appear as different forms in biological specimens, including glycosylation variants, free subunits, and metabolized forms. a typical example is circulating human chorionic gonadotropin (hcg), which is a useful biomarker for diagnosis of pregnancy, hydatidiform mole, and certain poorly differentiated cancers. hcg is present in a number of forms in blood, including intact hcg, nicked hcg, hyper-and hypoglycosylated hcg, hcg missing the c-terminal extension, free alphasubunit, large free alpha-subunit, free beta-subunit, nicked free beta-subunit, and beta-core fragment [28] . for blood samples collected in normal pregnancy, only minor variations in the assay performance appear among the commercial immunoassay kits. however, for irregular gestations, immunoassay results can be significantly different among the kits [28] . when different forms of a protein biomarker have different molecular weights, they can be readily differentiated by mass spectrometry, resulting in more reliable measurements [29] . maldi-tof ms can be used alone for quantification of a protein biomarker in uncomplex biological specimens, such as urine. for example, maldi-tof ms has been used to semi-quantify albumin in urine for the diagnosis of albuminuria [30, 31] . this approach does not require any pretreatment of a urine sample [30] , and the results are not affected by the presence of interfering substances, such as drugs, detergents, and blood, which often cause false-positive and false-negative results in conventional urinary dipstick tests [31] . glycated and glutathionylated hemoglobin can be measured by direct maldi-tof ms analysis of hemolysate with both intra-assay and inter-assay cvs <10% [32] . the maldi-tof ms results correlated well with results obtained by using a validated routine assay for hba1c (correlation coefficient ¼ 0.92) [32] . in complex biological specimens like serum, direct maldi-tof ms analysis of low abundant proteins is not possible. the high and medium abundant proteins in serum will mask the signals of the targeted protein. in such a case, maldi-tof ms can be combined with immunoprecipitation or immunocapture techniques to enrich and unmask the signal of a protein biomarker. because it is difficult to control the amount of the target proteins recovered from the antibody beads, stableisotope labeled internal standard protein that has the same amino acid sequence must be added to specimens for normalizing the variations. for example, after immunoprecipitation of amyloid-beta peptides from the cerebral spinal fluid, different amyloid-beta isoforms as well as their corresponding stable-isotope labeled internal standards appear as individual peaks of expected m/z values in a maldi-tof mass spectrum, and their quantities can be measured with high accuracy with intra-assay cvs <10% [33] . the results obtained by this method correlated well with the results obtained by elisa with correlation coefficients of 0.89-0.95. using specific antibody coated beads, mason et al. has recently developed a sensitive method for quantifying angiotensin i and angiotensin ii in human plasma [34] . this assay has a limit of detection of 13 and 11 pg/ml for angiotensin i and angiotensin ii, respectively. the intra-assay cvs are <10%. the limitations of maldi-tof ms-based quantitative analysis of large intact proteins are low specificity, low sensitivity, and low resolution. an amount of 100 fmol to 1 pmol is required for generating reliable ms signal from an intact protein. for example, a concentration of 100 fmol/μl (i.e.,~6.5 μg/ml) is required for reliable measurement of intact bsa. in addition, maldi-tof ms does not have good resolution to resolve large intact proteins. the accuracy of a measurement can easily be affected by the presence of protein contaminants with close molecular weights. furthermore, wide-type proteins and corresponding mutant proteins cannot be efficiently resolved. to overcome these limitations one could digest a protein mixture first, capture the specific peptides that are commonly obtained by protease digestion (i.e., proteotypic peptides) with specific anti-peptide antibody coated beads, and finally quantify the peptides to reflect the protein concentrations. this approach is called imaldi [35] or siscapa [36] . for example, epidermal growth factor receptor (egfr) has a molecular weight of 180 kda. the detection sensitivity of this approach for egfr was shown to be 5 fmol [35] . if one has a maldi-tof/tof ms instrument, the identity of a detected target peptide can be further confirmed by tandem ms. this could help to avoid false positive test results [35] . by using synthetic proteotypic peptides of six proteins and corresponding stable isotope peptides as internal standards for proof-of-concept, this approach has been shown to have average intra-assay cvs of 2.5% at a loading amount of 11 fmol on the sample spots [36] . although the sensitivities of these methods are still at a magnitude of nanograms per milliliter, it is expected to be improved with the advancement of maldi-tof ms in the near future. furthermore, this approach has a great potential in specific quantification of mutant proteins resulting from sense mutation of a gene sequence, e.g., egfr with t790m mutation, which is a therapy response predictor for non-small cell lung cancer patients treated with egfr tyrosine kinase inhibitors [37] . the major shortcoming of the siscapa or imaldi approach is that it cannot differentiate different forms of a target protein if the proteotypic peptide selected for quantification does not cover the differences. for example, a proteotypic peptide lying in the n-terminal region of a target protein cannot differentiate its intact form from the c-terminal truncated forms. more details about imaldi can be found in chap. 6 ("mass spectrometry in high-throughput clinical biomarker assays: multiple reaction monitoring" written by parker et al.). there has been a long history in applying glycoprotein biomarkers for disease diagnosis and prognosis. alternations in glycosylation changes have been observed in various diseases, such as congenital disorders of glycosylation syndrome (cdgs) [38] , liver diseases [39] , kidney diseases [40] , and cancers [41] . a typical example of glycoprotein biomarkers for monitoring disease-associated glycosylation is circulating transferrin, which is still used in most hospitals for liver damage caused by chronic alcohol abuse [39] and identification of various types of cdgs nowadays [42] . as early as 1978, abnormal microheterogeneity of serum transferrin was observed in male alcoholics after alcohol intoxication [43] . in 1993, serum transferrin was first used to examine abnormal glycosylation in cdg patients [38] . alternation in glycosylation of glycoproteins and glycolipids is a common feature in various cancers, and is involved in numerous ways in carcinogenesis, such as progression, cell-cell interaction, and metastasis. tumor cells have different glycosylation machineries. changes of glycosylation machinery in the cancer cells can be reflected in blood circulation by tracing the changes in the glycosylation of the proteins released by the tumor [44] . the poor specificity of a tumor biomarker is often due to the fact that it is also produced by normal cells under other pathological conditions. however, this problem can be reduced by measuring the circulating levels of its variants carrying cancer-associated glycosylations. for cancer diagnosis, a typical example is alpha-fetoprotein (afp). compared to the total serum afp level, both fucosylated afp and monosialylated afp are more specific in the diagnosis of hepatocellular carcinoma (hcc) [45, 46] . elevated mrna expression of alpha1-6 fucosyltransferase in human hcc tissues was associated with the production of tumor-specific fucosylated afp glycoform [47] . serum levels of monosialylated afp were negatively correlated with the tissue levels of betagalactoside alpha-2,6-sialyltransferase [41] . maldi-tof ms can be used to identify and quantify disease-associated glycosylations carried by either a single protein or a mixture of proteins. for both cases, n-linked glycans or o-linked glycans can be cleaved from a protein preparation, cleaned up to remove interfering substances, and subsequently subjected to maldi-tof ms to obtain a mass spectrum of glycans (fig. 2a) . after normalization, the peak intensities of individual glycans can be used to estimate their relative levels in the preparation [5] . the intra-and interassay cvs of normalized peak intensities of n-glycans were reported to be <10% and <17%, respectively [5, 48] . the first application of maldi-tof-ms to analysis of n-linked glycans on transferrin preparations (fig. 2b ) that were affinity isolated from serum samples for diagnosis of type-i cdgs was reported in 1994 [49] . besides analyzing glycans cleaved from glycoproteins, one could use maldi-tof ms to examine diseaseassociated glycopeptides which are obtained by proteolytic digestion of affinity isolated proteins. maldi-tof ms analysis of glycopeptides from serum transferrin has been applied in cdg screening system to the diagnosis of type-ii cdgs in japan [50] . because maldi-tof is a sensitive technique, only 20 μl of serum is required for screening of type-i and type-ii cdgs [50] . when analyzing glycans released from all proteins in a tissue instead of a single protein, the concept of glycome appears. maldi-fticr ms was first used to obtain a semi-quantitative profile of o-linked glycome in serum, and identified potential glycan biomarkers for ovarian cancer [51] . one year later the same approach was used to discover potential glycan biomarkers for breast cancer [52] . despite these encouraging results, a maldi-fticr ms instrument is far too expensive to be acquired by most of the clinical laboratories for providing routine services. almost at the same time, another team and our team reported the use of maldi-tof ms for obtaining semi-quantitative profiles of serum n-linked glycome (fig. 2c) , and showed the potential use of serum n-linked glycome fingerprints in the diagnoses of metastatic prostatic cancer and liver fibrosis [5, 53] . similar maldi-tof ms approaches have been used to identify serum n-glycan biomarkers for diagnoses of various cancers, including hcc [54] , breast cancer [55] , esophageal adenocarcinoma [56] , and ovarian cancer [57] . in the case of breast cancer, a serum n-glycan at m/z 2,534 was found to be a potential predictor of patients' response to trastuzumab [58] . 150 e.w.y. ng et al. in 1992, nordhoff et al. first demonstrated the use of maldi-tof ms to detect and measure the masses of nucleic acids [59] . three year later, the research team led by charles cantor showed that maldi-tof ms was a useful tool for dna a sequencing [60] . since then, various maldi-tof ms-based methods have been being developed for applications of molecular genetics in clinical diagnosis. the successful application of maldi-tof ms in genotyping has been widely applied in the past 10 years. the most commonly used maldi-tof ms method is detection and quantification of single base primer extension products for qualitative and quantitative analysis of dna copies containing single nucleotide polymorphism (snp) by maldi-tof ms [61] . when the primers are well designed to achieve a good separation of the primer and the extension products in a mass spectrum, the genotyping assays can be combined to perform up to 15-fold multiplex snp analysis [62, 63] . the single base primer extension assay can be applied to diagnosis and screening of hereditary diseases such as cystic fibrosis and betathalassemia [64, 65] . in fact, any diseases/pathological conditions that are associated with mutations in a specific gene or a specific set of genes can be easily identified and quantified by this method. this has recently been applied to the detection and quantification of the frequency of egfr activating mutations in nonsmall-cell lung cancer tissues for prediction of patient's response to egfr tyrosine kinase inhibitor [37] . this method was shown to have detection limits of 0.4-2.2% [37]. in addition, when a known amount of an oligonucleotide having a welldesigned sequence is spiked into a biological sample for competition in the primer extension reaction, the primer extension method can be used for measurement of the exact number of copies of dna containing a mutation of interest. a typical application example is detection of 60 hepatitis b virus variants in four multiplex reactions [63] . the limit of quantification was 1,000 hbv copies/ml. besides dna, the primer extension method can be applied to the qualitative and quantitative analysis of rna [66] . in 2007 it was first shown that quantification of plasma placental rna allelic ratio permitted noninvasive detection of prenatal chromosomal aneuploidy detection [67] . this work has opened a new avenue for prenatal diagnosis. the matrix chemical noises in the low mass region (<m/z 800) make maldi-tof ms inferior for small molecule analysis. despite that, attempts have been made to use maldi-tof ms for direct quantification of biomolecules, such as amino acids [15] and lipids [68] , without the need for chemical derivatization. maldi-tof ms peak intensities usually increase with the amount of biomolecules. by spiking an internal standard and using an external calibration curve, one can use maldi-tof ms to estimate the concentration of a metabolite in a biological specimen through calculating the peak intensity ratio of the target metabolite to the internal standard. in gogichaeva et al.'s study, methyltyrosine was used as a universal internal standard for quantification of various amino acids [15] . the calibration curves exhibited linearity in a range between 20 and 300 μm with correlation coefficients >0.983. the between-day cvs for the majority of amino acids were <10%, with proline and arginine being exceptions with cvs of about 12% [15] . by using 4-cholesten-3-one as a universal internal standard, it was practically feasible to use maldi-tof ms to identify and measure the lipid composition (m/z 369.6-833.0) of vldl, ldl and hdl [68] . it has recently been shown that by ms acquisition at the negative ion mode and using 9-aminoacridine instead of the typical chemical matrices, matrix chemical noise can be greatly reduced [69] . this method allowed the detection and quantification of metabolites having acid protons, such as amines, alcohols, carboxylic acids, phenols, and sulfonates, with high sensitivity [70] . high linearity of the ms peak intensities of the deprotonated metabolites was observed at low concentration [69, 71] . the detection limits were in the femtomole range [69, 71] . by using 9-aminoacridine as the matrix and n-1-napthylphthalamic acid as the universal internal standard, spe-enriched various bile acid species from plasma specimens could be directly measured with the limit of detection within the range 0.25-4.60 μg/ml [72] . another method for reducing matrix chemical noises is the replace of the chemical matrix by a solid matrix. this approach is called surface assisted laser desorption/ionization (saldi) (fig. 1) [73] . the concept of saldi was introduced by sunner et al. in 1995. by using graphite to replace the chemical matrix, it was shown that peptides and proteins could be detected at high sensitivities [74] . moreover, the background signal at the low mass region was low [73] . since then, many other solid materials, such as silicon [75] , carbon nanotube [76, 77] , graphene flake [78] , reduced graphene oxide [79] , polymer matrix [80] , and gold nanoparticles [81] , have been shown to be useful matrices for saldi-tof ms analysis of small biomolecules, including carbohydrates [76, 81] , amino acids [77] , and lipids [82] . on one hand, the use of a solid-phase matrix alleviates the matrix chemical noises and interference problem at the low mass range. on the other hand, it solves the problem of uneven distribution of the analytes on a sample spot. by using graphene-based materials, the maldi-tof mass spectra of small molecules were found to be highly reproducible [78] . the within-spot spectrum-to-spectrum cv of peak intensities for spermin was 14% for the graphene matrix, compared to 40% for the chca matrix [78] . recently, lu et al. examined the shot-to-shot and spot-to-spot reproducibility of saldi-tof mass spectra for polypropylene glycol polymers. the shot-to-shot and spot-to-spot cvs of the signal intensities were 1.9-7.1% and <10%, respectively [83] . saldi-tof ms has great potential in quantitative profiling of small biomolecules, especially metabolites. in the post genome era, besides proteomics, metabolomics has been a hot topic in the past 10 years. many research groups have been attempting to use ms technologies to obtain quantitative profiles of metabolites in patients' specimens, and to identify potential metabolite biomarkers by comparing the profiles between subjects with and without the diseases. lc-esi ms has been the most commonly used technology in this research area [84] . esi is a kind of atmospheric pressure ionization-based method, resulting in occurrence of ionization suppression [84] . another disadvantage is that the use of lc limits the throughput. it can be very time consuming when one wants to obtain comprehensive metabolite profiles from over 100 biological specimens in a biomarker discovery study. because of the highthroughput nature of maldi-tof ms, the use of maldi-tof ms in metabolite profiling is a very attractive alternative. it has been shown that 9-aminoacridine can be used to obtain quantitative cellular metabolite profiles by direct mixing of cells and the matrix without any preprocessing [69, 85] . for example, by a single direct on-spot analysis of 2,500 human acute lymphoblastic leukemia jurkat cells, this method detected up to 150 metabolite peaks in the range of m/z 250-850 within 90 s [69] . it is important to note that signal suppression of a metabolite was observed when another metabolite with a similar chemical structure was present [86] . hence, when using maldi-tof ms for quantitative analysis of metabolites, the data should be interpreted carefully. in the near future it will be interesting to see whether metabolites in plasma/serum can be directly profiled with the use of 9-aminoacridine as the matrix. nowadays selected reaction monitoring/multiple reaction monitoring (srm/mrm) is the most widely accepted ms method for reliable quantification of small molecules, and typically implemented in an esi triple quadrupole (esi-qqq) mass spectrometer (see chap. 6 for details of the basic principle and instrumentation). the qqq tandem mass analyzer is used dedicatedly in the srm/mrm method because a quadrupole mass analyzer can be used as a mass filter, which only allows charged molecules of a specific m/z value to pass through the mass analyzer for either subsequent fragmentation or detection. by undertaking the filtering approach, the background noise can be greatly reduced, leading to high detection sensitivity. srm cannot be implemented in maldi-tof/tof ms instruments. few reports on using maldi-tof/tof ms in tandem ms mode for quantification have been available. gogichaeva observed [87] . although correlation coefficients and coefficients of variation of their maldi-tof/tof ms method were not reported, the study highlighted the potentials of applying maldi-tof/tof ms to biomolecule quantification [87] . recently, using citrulline for proof-of-concept, our team has developed a novel mald-tof/tof ms-based quantification method called parallel fragmentation monitoring (pfm) [88] . this method is comparable to srm. as in the srm method, the pfm method also requires at least two pairs of precursor and selected fragment ions of specific m/z values, one pair for the target molecule and one pair for the internal standard. a stable isotope analog of the target molecule only 1 mass unit heavier is used as an internal standard, so that precursor ions of both the target molecule and internal standard can be specifically isolated with the first tof analyzer at the same time, and undergo fragmentation simultaneously to yield a full range composite ms/ms spectrum. in both the srm and pfm methods, the peak area ratio of the selected fragments of the target analyte to internal standard was used for quantification. the use of a stable isotope analog should also be able to minimize the error due to systematic bias of the instrumentation, and normalize the recovery yield after enriching the analytes from the biological samples for quantification. to reduce the matrix noises in the low mass range, a carbon-based nanomaterial was used as the matrix. the performance of the pfm method appears to be comparable to those of the srm/mrm methods. both pfm and srm/mrm methods generated linear calibration curves with correlation coefficients >0.99 (fig. 3 ) [88] [89] [90] . moreover, both types of assays gave the within-and betweenday cvs 10% [88] [89] [90] . our results also showed that the calibration curves were highly reproducible. daily calibration or use of a stored calibration generated highly similar measurement values [88] . this suggests that pfm can potentially be a cost and time effective and robust technology for quantification of biomolecules in routine clinical chemistry laboratories. the major advantage of using maldi-tof/tof ms instead of maldi-tof ms for quantification is that maldi-tof/tof ms has higher detection specificity and sensitivity for direct quantification of a target biomolecule in a complex biological specimen or in a partially enriched preparation. maldi-tof ms does not have enough resolution to resolve two ion species with highly close molecular weights, but they can be easily differentiated by looking at the fragmentation pattern. even a highly advanced maldi-tof ms with ultra-high resolution, such as maldi-fticr ms, is not able to differentiate the naturally occurring isomers, such as leucine and isoleucine, by only focusing on the intact ions because of the exactly similar molecular weights. isomers can only be differentiated on the basis of fragment ions. furthermore, the background noises and interference from the other biomolecules in the preparation, like signal suppression by biomolecules sharing similar chemical structures, can be minimized by measuring ratios of the indicator fragment ions, resulting in higher detection sensitivity and measurement accuracy. although a qqq tandem mass analyzer is usually coupled with an esi source, it can also be coupled with a maldi source [91] . in maldi-qqq ms the identity of a biomolecule can be defined by a mass transition ion pair as in the case of srm [91] . by operating the qqq analyzer as mass filters for the targeted precursor ions and fragment ions, the matrix chemical noises at the low mass region can be greatly reduced [91] . comparing the quantitative results obtained by maldi-qqq ms and esi-qqq ms for 53 small-molecule pharmaceutical compounds, gobey et al. demonstrated the potentials of maldi-qqq ms for high-throughput quantification of small biomolecules [91] . when operating maldi-qqq ms in srm/mrm mode, the cvs for quantifications of small biomolecule or drug are typically around 10% [91] [92] [93] [94] . it has recently been shown that maldi-qqq ms can also be used to measure protein biomarkers in plasma by quantifying their proteotypic peptides in the presence of corresponding isotopically labeled peptide standards, as in case of typical mrm methods [95] . the measurement results are highly comparable to those obtained by using esi-qqq ms. both technologies are accurate (within-day cvs <20%) and precise (relative errors <20%) for protein quantification [95] . because mald-qqq ms is a relatively new technology, the currently available data have been limited. however, all the recent reports have demonstrated that maldi-qqq ms in srm/mrm mode, which combines the merits of maldi ionization technology and those of the conventional srm/mrm approach, is a reliable high-throughput technology for biomolecule quantification. more successful applications of maldi-qqq ms to quantification of biomarkers in human specimens should be forthcoming. surface-enhanced laser desorption/ionization tof mass spectrometry (seldi-tof ms) is a variant of maldi-tof ms, and is mainly designed for quantitative analysis of proteins in biological samples. this concept was first introduced by hutchen and yip in 1993 [96] . instead of spotting of a mixture of proteins on a maldi sample plate, a mixture of proteins is subjected to proteinchip array-based retentate chromatography before maldi-tof analysis. proteinchip arrays coated with different types of chromatographic materials (hydrophilic, hydrophobic, cationic exchange, anionic exchange, immobilized metal affinity, antibody affinity, ligand affinity, etc.) can selectively bind and concentrate proteins with the matched physicochemical or biochemical properties (fig. 4a, b) . those nonspecifically bound proteins and impurities are then washed away with suitable washing buffers [97] . retained proteins are finally co-crystallized with a chemical matrix (fig. 4c) , and subjected to maldi-tof ms for unbiased detection of protonated proteins (fig. 4d, e) . in the case of seldi-tof ms, sinapinic acid is the most commonly used matrix to assist desorption/ionization of proteins. most of the proteins are detected as singly charged protonated molecules, and presented as individual peaks in a mass spectrum, resulting in a proteomic profile (fig. 4f) . the combinations of specific m/z values and the physicochemical properties that are reflected by the type of the proteinchip arrays used provide unique identities for individual proteins [97] . this is why seldi-tof ms is commonly regarded as a proteomic fingerprinting technology. a series of follow-up experimental work is needed to purify the corresponding proteins and decipher the true identities [98] [99] [100] . it is worth noting that the concepts of saldi and seldi can be combined. a solid-phase matrix can both capture biomolecules with a particular physicochemical property and assist desorption/ionization of the captured biomolecules in maldi-tof ms analysis. immobilization of chca onto the hydrophobic proteinchip arrays allowed direct quantitative profiling of urine proteins by seldi-tof ms without the need for adding any chemical matrix after sample binding and washing [101] . recently, a graphene-based seldi probe has been developed for capture and direct detection of dna oligomer without addition of any chemical matrix [102] . as in the case of maldi-tof ms, with appropriate ms analysis conditions, seldi-tof ms is quantitative. on the proteinchip arrays, chromatographic resins are coated on a film of hydrogel, which provides a network for the formation of fine analyte-matrix co-crystals. furthermore, in a typical seldi-tof ms experiment, an unbiased automated ms acquisition strategy is used. ms signals from 60 to 120 laser shots are obtained from a sample spot in a linear sweep or from various positions with a regular spacing on the entire sample spot, and are summed up to form a representative mass spectrum. after normalizing the ms signals by either total ion current and/or total peak intensity, the intensity values of the protein/ peptide peaks is highly reproducible. the intra-assay and inter-assay cvs for the normalized intensities of majority of the seldi peaks are between 5% and 25% [103, 104] . with the use of standardized experimental protocol and quality control strategy, the inter-laboratory cvs of the normalized peak intensities are between 15% and 36% [105] . by using a combination of proteinchip arrays with different chromatographic coatings, seldi-tof ms can be used to obtain comprehensive semi-quantitative profiles of proteins with molecular weights between 2 and 250 kda [106] . similar to other affinity technologies, seldi-tof ms can be applied to various types of research projects where appropriate. it all depends on what chromatographic functional groups, affinity materials, or proteins are being conjugated covalently on the proteinchip array surface. it can be used to capture and profile transcription factors by coating with dna materials of a specific sequence [107] . it can also be used to study the effect of dna methylation on binding the transcriptional factors to a dna sequence [108] . such an approach could help to characterize transcription factors and to screen for differences in cellular regulatory networks. when a specific protein is coated, it could be used to study protein-protein interaction [109, 110] . for example, it has been used to search for protein-protein interaction partners for s100a8 [109] and glialcam [110] . when the proteinchip arrays are coated with a specific antibody, specific protein or protein complex can be purified for subsequent analysis. after a specific protein has been captured, seldi-tof ms can identify and provide quantitative information of individual variants. they could be structural variants with different amino acid compositions, e.g., amyloid beta peptide variants [111] and saa variants [112] , as well as variants with different post-translational modifications, e.g., s-glutathionylated and s-cysteinylated variants of transthyretin [113] and glycosylated variants of eosinophil cationic protein [114] . above all, seldi-tof ms has been more commonly used for quantitative profiling of biological samples to search for protein biomarkers. theoretically, seldi-tof ms can also be applicable to high-throughput quantification of other types of biomolecules, like metabolites and glycans. in the following sections the applications of seldi-tof ms to protein biomarker discovery will be reviewed in more detail. in a typical seldi-tof ms experiment, biological specimens can be directly added on the proteinchip arrays without any preprocessing, or only after several advances in maldi mass spectrometry in clinical diagnostic applications 159 simple steps for denaturation and dilution. the proteinchip arrays can be assembled in a 96-well plate format, and the binding and washing procedures can be performed as if carrying out an enzyme-linked immunosorbent assay. the use of the proteinchip arrays has greatly simplified the protein profiling assay workflow. another advantage of seldi-tof ms is its capacity for high throughput during mass spectrum acquisition, as in the case of maldi-tof ms. in addition, the combinations of specific m/z values and the type of proteinchip arrays used provide unique identities for individual proteins. these are the major reasons why seldi-tof ms has been widely used for analysis of biological specimens, especially for biomarker discovery, since it first appeared as a commercially available platform in 1997. as of today, there are at least 850 publications on applications of seldi-tof ms to protein biomarker discovery. it has been used to analyze a wide range of biological samples, for example, serum [100, [103] [104] [105] [106] , plasma [110, 113, 115] , urine [101, 113, 116] , tears [117] [118] [119] , cerebrospinal fluid [120] [121] [122] , amniotic fluid [123] [124] [125] , tissue/cell lysate [107, [126] [127] [128] , etc. it has been applied to the discovery of potential biomarkers for various types of diseases, for example, cancers [103-106, 112, 129] , infectious diseases [99, 100, 115, 130] , autoimmune diseases [118, 131] , eye diseases [117, 119] , neurological diseases [120] [121] [122] , perinatal and neonatal diseases [123] [124] [125] [126] , etc. in a typical proteomic profiling experiment the individual biological samples were first denatured with urea and non-ionic solvent to denature or destroy the non-covalent protein-protein interaction, and then diluted in an appropriate binding buffer for subsequent seldi-tof ms analysis [99, 100, 103-106, 112, 115, 129] . in such an approach only a very small amount of biological sample is needed. for serum/plasma specimens, as little as 2 μl of serum samples would be enough [99, 129, 130] . one reason for the popularity of seldi-tof ms in biomarker discovery is that it is complementary to two-dimensional polyacrylamide gel electrophoresis (2d-page) for the quantitative analysis of intact proteins or their fragments formed in vivo. the most important point is that 2d-page is the best for resolving proteins with molecular weight in the range of 10-250 kda, while seldi-tof ms has the best resolving range of 1-20 kda. furthermore, highly hydrophobic proteins, such as membrane proteins, and proteins with isoelectric points (pi) less than 3 and higher than 11, are usually poorly resolved by 2d-page, but can be satisfactorily analyzed by seldi-tof ms. another reason for the popularity of seldi-tof ms in biomarker discovery is that seldi-tof ms itself has a great potential to be used as a clinical diagnostic tool. the simplicity in the assay procedure and its short turn-around time allow this to be implemented in routine clinical chemistry laboratories. we could easily translate the laboratory research findings into clinical use. diagnosis/prognosis could be based on the intensity of a seldi peak at a specific m/z value and the usage of a specific type of proteinchip arrays, or based on a combination of specific protein peaks which have been identified with the use of several specific types of proteinchip arrays. 160 e.w.y. ng et al. proteins corresponding to the diagnostic/prognostic peaks could be purified by micro-scale chromatography with the same binding condition, separated by gel electrophoresis, and finally identified by using typical approaches, e.g., peptide mass fingerprint, tandem ms, etc. with clear protein identities, specific immunoassays could be developed. now it is clear that the majority of diseaseassociated proteomic fingerprints are composed of intact forms, fragments, and/or post-translationally modified forms of host response proteins, such as apolipoprotein a1, apolipoprotein a2, apolipoprotein c1, apolipoprotein c2, apolipoprotein c3, alpha-1 antichymotrypsin, complement component 3a, complement component 3c, fibrinogen, immunoglobulin kappa light chain, inter-alpha trypsin inhibitor heavy chain 4, haptoglobin, beta-2 microglobulin, platelet factor 4, saa, transthyretin, beta-thromboglobulin, etc. [99, 100, [132] [133] [134] [135] [136] [137] [138] [139] . in fact, host response proteins were also identified as potential biomarkers when the serum/plasma proteomic profiles were compared by using other techniques, such as 2d-page [140, 141] , magnetic beads-based maldi-tof ms [24, 25] , and even shotgun proteomic profiling by lc-esi-ms [142] [143] [144] . the use of signatures of hostresponse proteins as disease biomarkers has both pros and cons. the major advantage is that the host response of a patient helps to amplify the signal for the presence of a particular disease, which may help to identify a disease at an early stage [132] . the major disadvantage is that the specificity of those host-response signatures should be carefully validated before they can be claimed as disease-specific biomarkers. similar symptoms, which generate specific host-response protein signatures, can easily be found in other diseases. although seldi-tof ms has been a popular technology for biomarker discovery, there have been doubts about the reliability of this technology. before discussing this issue, i would like to emphasize this should not be a problem that is only restricted to the seldi proteomic profiling studies. such a problem has been observed in many seldi-tof ms studies. it may be because seldi-tof ms was the first high-throughput technology that allowed quantitative profiling and comparison of the serum/plasma proteins in a large number of patient samples within a very short period of time. after much more serum proteomic/metabolomic profiling, data obtained by using other technologies are available, i believe that similar problems will be observed. in this section, selected biomarker discovery studies employing seldi-tof ms technology will be used as examples for reviewing this issue. the typical example is the application of seldi-tof ms to the diagnosis of ovarian cancer. in 2002, petricoin et al. identified a pattern of seldi peaks that could completely differentiate ovarian cancer cases from non-cancer cases in the training set. for the masked set, the diagnostic pattern achieved a sensitivity of 100% and specificity of 95% [145] . unfortunately, when the data set was reanalyzed by other teams, it was found that there was significant non-biological experimental bias between the cancer and control subjects [146] , and the features in the noise regions of the seldi mass spectra allowed discrimination of control subjects from cancer patients [147, 148] . these analysis results suggested that (1) the cancer and control samples had been analyzed separately and (2) there was a change in the experimental protocol in the middle of the study. another important example is the identification of seldi peaks for diagnosis of prostate cancer. in 2002, by using the copper(ii) ion loaded metal affinity (cu 2+ -imac) proteinchip array, a pilot single-center study showed that serum protein fingerprinting was useful for diagnosis of prostate cancer [149] . when classifying the blind test samples, the sensitivity and specificity were found to be 83% and 97%, respectively. subsequently, a series of follow-up studies were performed to validate the value of serum proteomic profiling with cu 2+ -imac proteinchip arrays in the diagnosis of prostate cancer. to allow validation carried out by six research centers, a standard protocol and quality control system was developed [105] . then serum samples (181 prostate cancer patients, 143 benign prostatic hyperplasia cases, and 220 normal controls, who were age and race-matched) from eastern virginia medical school (evms) were used to construct a decision algorithm for classifying 42 prostate cancer patients and 42 normal controls provided by four institutions [150] . all test samples were distributed to six laboratories for analysis. the final conclusion was that the decision algorithm was unsuccessful in separating cancer from controls. analysis of the experimental data for biomarker discovery indicated that the sample source is the major factor affecting the results. inappropriate selection of control subjects (i.e., selection bias) is one of the major causes of systemic bias. selection bias and information bias will appear when the diseased and control subjects were recruited from two different populations, such as two different clinics. for example, three laboratories had attempted to use seldi-tof ms to identify the biomarkers for detection of severe acute respiratory syndrome (sars) in adults [99, 151, 152] . in two of the three studies, controls cases were recruited from other clinics [151, 152] . patients with other types of respiratory infections had been included as the controls. saa concentration was found to be significantly higher in the sars patients than in the controls. one study included saa into the diagnostic model for detection of sars [152] . in the third study which was performed by our team, both sars patients and control subjects were recruited from the same clinics. the control subjects were suspected sars cases, but were later proven to be negative for sars [99] . in this study, both the seldi-tof ms assay and immunoassay showed that saa was elevated in the sars patients [153] . however, saa levels were found to be much higher in the control group, indicating that saa was not a useful biomarker for diagnosis of sars [153] . these three studies clearly illustrate the importance of recruiting the diseased and control cases from the same clinic. for case-control biomarker study, confounding bias should also be controlled. if it is not controlled, the biomarkers found could be related to the characteristics of the disease group, but not related to the disease itself. patients with gastroenterological cancer may lose appetite, leading to under nutrition [154] . malnutrition will become one of confounding factors, and some differential seldi peaks could be related to the nutritional status. for hepatitis virus-related liver cancer, it is well known that gender is one of the confounding factors [155] . smoking is a wellknown confounding factor for lung cancer [156] . levels of considerable amounts of blood proteins can be changed in response to smoking [157] . for biomarker discovery, even though the diseased and control cases are recruited from the same clinics, unknown confounding factors still exist. in our recent gastric cancer study we attempted to use post-operative serum samples to verify the validity of the potential proteomic markers found by comparing the diseased and control cases from the same clinics [129] . surprisingly, over 80% of the potential biomarkers could not show a reverse in the serum levels after the removal of the tumors from the patients. this strongly suggested that most of the differential seldi peaks between the gastric cancer and control groups were not specifically associated with gastric cancer, but only associated with certain characteristics of the gastric cancer patients. in our sars study we identified the clinical and biochemical variables which were significantly altered in the sars patients, and attempted to verify the potential diagnostic seldi peaks by only considering those that were significantly correlated with at least two disease-associated biochemical/clinical parameters as sars-specific (fig. 5a) [99] . similar to the gastric cancer study, about 80% of the differential seldi peaks were rejected in the sars study. both the gastric cancer study and the sars study have highlighted the high risk of false discovery when we simply consider the differential seldi peaks as potential biomarkers. the presence of about 80% of the differential seldi peaks, which are likely caused by confounding bias, are not restricted to the studies employing seldi-tof ms. our group recently attempted to identify circulating host response biomarkers for diagnosis of late-onset sepsis or necrotizing enterocolitis in preterm infants suspected for the diseases by using hydrophobic magnetic beads and maldi-tof ms [25] . by using the longitudinal samples to verify the clinical relevance of the differential proteomic features, again about 80% of them were rejected (fig. 5b) . encouragingly, the diagnostic values of the verified proteomic features were subsequently confirmed in the prospective study [25] . while one can reduce the systemic bias in a single center study by verification with longitudinal samples or by correlation with known disease-associated changes, one can also reduce the systemic bias by using samples from multiple centers [158] . 39 fig. 5 the study designs which were used to identify biomarkers for diagnosis of sars in adults (a) and diagnosis of necrotizing enterocolitis/late-onset sepsis in preterm infants (b) by undertaking ms-based proteomic profiling approaches. in the sars study, potential diagnostic seldi peaks were filtered by only considering those that were significantly correlated with at least two disease-associated biochemical/clinical parameters as sars-specific (a) [99] . in the preterm infant study, the longitudinal samples were used to verify the clinical relevance of the differential proteomic features [25] . only the differential ms peaks showing statistically significant reverse of peak intensities upon recovery were retained (b). in both studies, about 80% of differential seldi peaks, which were obtained by case-control comparison, were rejected multi-center design provides an unbiased clinical validation of the proteomic diagnostic models. zhang and chan have proposed a multicenter design that helps to eliminate the systemic biases in samples and site-associated confounding variables in biomarker discovery [159] . in the biomarker discovery phase, cases from independent sites are used separately and independently to identify the potential biomarkers. the potential biomarkers from the different sites are crosscompared to produce a common set. in the validation phase, the clinical value of the common set is further validated using independent samples from additional sites. by using this multicenter design, a panel of ovarian cancer-associated protein biomarkers that were identified in blood samples by seldi-tof ms finally became the first in vitro diagnostic multivariate index assay (ivdmia) of proteomic biomarkers, which was recently cleared by the us fda (food and drug administration) [158, 160, 161] . the global research 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spectrometry (imaldi) assay for egfr diagnosis precision of heavy-light peptide ratios measured by maldi-tof mass spectrometry pretreatment epidermal growth factor receptor (egfr) t790m mutation predicts shorter egfr tyrosine kinase inhibitor response duration in patients with non-smallcell lung cancer carbohydrate deficient serum transferrin in a new systemic hereditary syndrome carbohydrate-deficient transferrin in serum: a new marker of potentially harmful alcohol consumption reviewed the pathogenic role of iga1 o-linked glycosylation in the pathogenesis of iga nephropathy correlation and prognostic significance of beta-galactoside alpha-2,6-sialyltransferase and serum monosialylated alpha-fetoprotein in hepatocellular carcinoma congenital disorders of glycosylation: a review abnormal microheterogeneity of transferrin in serum and cerebrospinal fluid in alcoholism a glycomic approach to hepatic tumors in n-acetylglucosaminyltransferase iii (gnt-iii) transgenic mice induced by diethylnitrosamine (den): identification of haptoglobin as a target molecule of gnt-iii afp-l3: a new generation of tumor marker for hepatocellular carcinoma quantification and utility of monosialylated alpha-fetoprotein in the diagnosis of hepatocellular carcinoma with nondiagnostic serum total alpha-fetoprotein gene expression of alpha1-6 fucosyltransferase in human hepatoma tissues: a possible implication for increased fucosylation of alpha-fetoprotein comparison of the methods for profiling glycoprotein glycans-hupo human disease glycomics/proteome initiative multi-institutional study diagnosis of carbohydrate-deficient glycoprotein syndrome by matrix-assisted laser desorption time-of-flight mass spectrometry mass spectrometry for congenital disorders of glycosylation, cdg profiling of glycans in serum for the discovery of potential biomarkers for ovarian cancer a serum glycomics approach to breast cancer biomarkers alterations in the serum glycome due to metastatic prostate cancer detection of hepatocellular carcinoma using glycomic analysis breast cancer diagnosis and prognosis through quantitative measurements of serum glycan profiles quantitative serum glycomics of esophageal adenocarcinoma and other esophageal disease onsets n-linked glycan structures and their expressions change in the blood sera of ovarian cancer patients identification of predictive biomarkers for response to trastuzumab using plasma fuca activity and n-glycan identified by maldi-tof-ms matrix-assisted laser desorption/ionization mass spectrometry of nucleic acids with wavelengths in the ultraviolet and infrared efficient preparation of short dna sequence ladders potentially suitable for maldi-tof dna sequencing detecting cftr gene mutations by using primer oligo base extension and mass spectrometry high level multiplex genotyping by maldi-tof mass spectrometry multiplex detection of 60 hepatitis b virus variants by maldi-tof mass spectrometry parallel minisequencing followed by multiplex matrix-assisted laser desorption/ionization mass spectrometry assay for beta-thalassemia mutations the suitability of matrix assisted laser desorption/ionization time of flight mass spectrometry in a laboratory developed test using cystic fibrosis carrier screening as a model maldi-tof mass spectrometry for quantitative, specific, and sensitive analysis of dna and rna plasma placental rna allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection analysis of human serum lipoprotein lipid composition using maldi-tof mass spectrometry highly sensitive matrix-assisted laser desorption ionization-mass spectrometry for high-throughput metabolic profiling aminoacridine as a matrix for negative mode matrix-assisted laser desorption/ionization analysis of low molecular weight acids by negative mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry advances in maldi mass spectrometry in clinical diagnostic applications quantification of bile acids directly from plasma by maldi-tof-ms recent advances in saldi-ms techniques and their chemical and bioanalytical applications graphite surface-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides and proteins from liquid solutions desorption/ionization on silicon time-of-flight/time-of-flight mass spectrometry immobilized carbon nanotubes as matrix for maldi-tof-ms analysis: applications to neutral small carbohydrates amino acids analysis by maldi mass spectrometry using carbon nanotube as matrix graphene as a novel matrix for the analysis of small molecules by maldi-tof ms reduced graphene oxide films used as matrix of maldi-tof-ms for detection of octachlorodibenzo-p-dioxin small molecule matrix-assisted laser desorption/ionization timeof-flight mass spectrometry using a polymer matrix gold nanoparticles as assisted matrix for determining neutral small carbohydrates through laser desorption/ionization time-of-flight mass spectrometry desorption/ ionization on self-assembled monolayer surfaces (diams) laser desorption/ionization on the layer of graphene nanoparticles coupled with mass spectrometry for characterization of polymers the future of liquid chromatography-mass spectrometry (lc-ms) in metabolic profiling and metabolomic studies for biomarker discovery metabolomic analysis of eukaryotic tissue and prokaryotes using negative mode maldi time-of-flight mass spectrometry quantitative detection of metabolites using matrixassisted laser desorption/ionization mass spectrometry with 9-aminoacridine as the matrix amino acid analysis by means of maldi tof mass spectrometry or maldi tof/tof tandem mass spectrometry quantification of citrulline by parallel fragmentation monitoring -a novel method using graphitized carbon nanoparticles and maldi-tof/ tof mass spectrometry certification of nist standard reference material 2389a, amino acids in 0.1 mol/l hcl-quantification by id lc-ms/ms simultaneous bioanalysis of l-arginine, l-citrulline, and dimethylarginines by lc-ms/ms characterization and performance of maldi on a triple quadrupole mass spectrometer for analysis and quantification of small molecules comparison of maldi to esi on a triple quadrupole platform for pharmacokinetic analyses determination of the antiretroviral drug tenofovir in plasma from hiv-infected adults by ultrafast isotope dilution maldi-triple quadrupole tandem mass spectrometry ultra-fast analysis of plasma and intracellular levels of hiv protease inhibitors in children: a clinical application of maldi mass spectrometry peptides quantification by liquid chromatography with matrix-assisted laser desorption/ionization and selected reaction monitoring detection new desorption strategies for the mass spectrometric analysis of macromolecules opportunities and limitations of seldi-tof mass spectrometry in biomedical research -practical advices independent validation of candidate breast cancer serum biomarkers identified by mass spectrometry serum proteomic fingerprints of adult patients with severe acute respiratory syndrome proteomic analysis reveals platelet factor 4 and beta-thromboglobulin as prognostic markers in severe acute respiratory syndrome proteomics profiling of urine with surface enhanced laser desorption/ionization time of flight mass spectrometry graphene-based seldi probe with ultrahigh extraction and sensitivity for dna oligomer proteomics and bioinformatics approaches for identification of serum biomarkers to detect breast cancer identification of gastric cancer patients by serum protein profiling evaluation of serum protein profiling by surfaceenhanced laser desorption/ionization time-of-flight mass spectrometry for the detection of prostate cancer: i. assessment of platform reproducibility comprehensive proteomic profiling identifies serum proteomic signatures for detection of hepatocellular carcinoma and its subtypes a rapid method to capture and screen for transcription factors by seldi mass spectrometry dna affinity capture and protein profiling by seldi-tof mass spectrometry: effect of dna methylation detection and identification of protein interactions of s100 proteins by proteinchip technology detection and identification of plasma proteins that bind glialcam using proteinchip arrays profiling of amyloid beta peptide variants using seldi protein chip arrays serum protein profiling by seldi mass spectrometry: detection of multiple variants of serum amyloid alpha in renal cancer patients characterization of the microheterogeneity of transthyretin in plasma and urine using seldi-tof-ms immunoassay a seldi-tof ms study of the genetic and post-translational molecular heterogeneity of eosinophil cationic protein serial analysis of plasma proteomic signatures in pediatric patients with severe acute respiratory syndrome and correlation with viral load profiling of urine using proteinchip® technology seldi-tof-ms proteinchip array profiling of tears from patients with dry eye diagnostic potential of tear proteomic patterns in sjögren's syndrome elevated levels of human alpha-defensin in tears of patients with allergic conjunctival disease complicated by corneal lesions: detection by seldi proteinchip system and quantification proteomic profiling of cerebrospinal fluid identifies biomarkers for amyotrophic lateral sclerosis identification of a novel panel of cerebrospinal fluid biomarkers for alzheimer's disease evaluation of two different albumin depletion strategies for improved analysis of human csf by seldi-tof-ms proteomic profiling of the amniotic fluid to detect inflammation, infection, and neonatal sepsis identification of proteomic biomarkers of preeclampsia in amniotic fluid using seldi-tof mass spectrometry different protein profile in amniotic fluid with nervous system malformations by surface-enhanced laser desorption-ionization/time-of-flight mass spectrometry (seldi-tof-ms) technology proteomic analyses associate cystatin b with restricted hiv-1 replication in placental macrophages proteomic profiles differ between bone invasive and noninvasive benign meningiomas of fibrous and meningothelial subtype proteomic analysis of laser microdissected ovarian cancer tissue with seldi-tof ms diagnosis of gastric cancer by serum proteomic fingerprinting prediction of liver fibrosis and cirrhosis in chronic hepatitis b infection by serum proteomic fingerprinting: a pilot study using seldi-tof ms to identify serum biomarkers of rheumatoid arthritis classification of cancer types by measuring variants of host response proteins using seldi serum assays discovery and identification of potential biomarkers of pediatric acute lymphoblastic leukemia identification of a 17-protein signature in the serum of lung cancer patients detection of pancreatic adenocarcinoma using circulating fragments of fibrinogen apolipoprotein a1 and c-terminal fragment of α-1 antichymotrypsin are candidate plasma biomarkers associated with acute renal allograft rejection identification of β2-microglobulin as a urinary biomarker for chronic allograft nephropathy using proteomic methods identification of novel serum biomarkers in child nephroblastoma using proteomics technology longitudinal analysis of maternal plasma apolipoproteins in pregnancy: a targeted proteomics approach proteome analysis and its impact on the discovery of serological tumor markers study of serum haptoglobin and its glycoforms in the diagnosis of hepatocellular carcinoma: a glycoproteomic approach altered proteolytic events in experimental autoimmune encephalomyelitis discovered by itraq shotgun proteomics analysis of spinal cord advances in maldi mass spectrometry in clinical diagnostic applications deglycosylation and label-free quantitative lc-maldi ms applied to efficient serum biomarker discovery of lung cancer label-free quantitative proteomic analysis reveals dysfunction of complement pathway in peripheral blood of schizophrenia patients: evidence for the immune hypothesis of schizophrenia use of proteomic patterns in serum to identify ovarian cancer a data review and re-assessment of ovarian cancer serum proteomic profiling reproducibility of seldi-tof protein patterns in serum: comparing datasets from different experiments signal in noise: evaluating reported reproducibility of serum proteomic tests for ovarian cancer serum protein fingerprinting coupled with a pattern-matching algorithm distinguishes prostate cancer from benign prostate hyperplasia and healthy men analytical validation of serum proteomic profiling for diagnosis of prostate cancer: sources of sample bias protein chip array profiling analysis in patients with severe acute respiratory syndrome identified serum amyloid a protein as a biomarker potentially useful in monitoring the extent of pneumonia proteomic fingerprints for potential application to early diagnosis of severe acute respiratory syndrome serum amyloid a is not useful in the diagnosis of severe acute respiratory syndrome the relationship between quality of life (eortc qlq-c30) and survival in patients with gastro-oesophageal cancer hepatocellular carcinoma: epidemiology and molecular carcinogenesis cannabis and respiratory disease research group (2008) cannabis use and risk of lung cancer: a case-control study serum parathyroid hormone (pth) levels in smokers and non-smokers. the fifth tromsø study three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer cancer proteomics: in pursuit of "true" biomarker discovery proteomic approaches to tumor marker discovery the road from discovery to clinical diagnostics: lessons learned from the first fda-cleared in vitro diagnostic multivariate index assay of proteomic biomarkers advances in maldi mass spectrometry in clinical diagnostic applications 175 acknowledgements the authors are grateful for the continuous support from the li ka shing foundation on developments and applications of mass spectrometry technologies to biomedical research. key: cord-315984-5gbhobw8 authors: isaacson, marisa k.; ploegh, hidde l. title: ubiquitination, ubiquitin-like modifiers, and deubiquitination in viral infection date: 2009-06-18 journal: cell host microbe doi: 10.1016/j.chom.2009.05.012 sha: doc_id: 315984 cord_uid: 5gbhobw8 ubiquitin is important for nearly every aspect of cellular physiology. all viruses rely extensively on host machinery for replication; therefore, it is not surprising that viruses connect to the ubiquitin pathway at many levels. viral involvement with ubiquitin occurs either adventitiously because of the unavoidable usurpation of cellular processes, or for some specific purpose selected for by the virus to enhance viral replication. here, we review current knowledge of how the ubiquitin pathway alters viral replication and how viruses influence the ubiquitin pathway to enhance their own replication. ubiquitin is a small 76 amino acid protein widely expressed in eukaryotic cells. viruses are connected to ubiquitin and ubiquitin-like modifiers in a variety of ways ( figure 1 ). many viruses encode proteins that can modify the host's ubiquitin and ubiquitin-like machinery, often altering substrate specificity to favor replication. viral proteins themselves can be directly modified by ubiquitin or ubiquitin-like proteins, and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. host cells utilize the ubiquitin-proteasome system to counteract viral infections through the generation of target structures recognized by t cells, whereas viruses can alter proteasome degradation machinery to interfere with class i major histocompatibility complex (mhc)-restricted antigen presentation and thus escape from t cell recognition. viruses, in turn, depend on the ubiquitination and degradation of host surface receptors as countermeasures to escape from t cell recognition. the ubiquitination pathway comprises e1, e2, and e3 enzymes, ultimately responsible for the conjugation of ubiquitin to protein substrates (for reviews see pickart, 2001; kerscher et al., 2006) . the e3 ligase usually determines substrate specificity, although the e2-conjugating enzyme can also play a role in substrate selection. the best understood function of the ubiquitin-conjugating system is to tag proteins for degradation by the proteasome. a classic example of viral-induced degradation of host proteins is the ability of human papilloma virus (hpv) to degrade the transcription factor p53 (scheffner et al., 1990) . this occurs by means of the e6 viral protein that activates an e3 ligase, e6ap, which then specifically targets p53 for ubiquitination. it is now known that ubiquitin and ubiquitin-like modifiers have a far greater impact on viral lifestyles. ubiquitin and ubiquitin-like modifiers are involved in the innate and adaptive immune response (figure 2) , transcription, signal transduction, membrane trafficking, and more. given the fact that viruses co-opt the biosynthetic and degradative apparatus of their host, extensive involvement of the ubiquitin-proteasome pathway is to be expected in almost every aspect of their life cycle. it is therefore a challenge to distinguish between adventitious modification of viral proteins by host ubiquitination machinery without immediate consequences for virus lifestyle and those events that are required for virus replication and viral gene transcription. for the viruses that encode their own ubiquitin ligases or ubiquitin-specific proteases, it seems reasonable to infer functional relevance, but even in these cases, the identification of the exact role of such viral gene products remains a challenge. nonetheless, the study of viral interactions with the ubiquitin system highlights the selective pressure to which these pathogens are exposed and the countermeasures required for them to ensure replicative success. furthermore, these examples have helped clarify some of the inner workings of the ubiquitin-proteasome system itself and have brought to light other functions of ubiquitin, such as its role in the assembly of virus particles. in this review, we discuss several mechanisms viruses use to enhance their own replication, including modulation of the cell cycle by dna viruses, interference with the innate and adaptive immune responses, and the requirement for ubiquitin in virion egress for many rna viruses. we further discuss the many unknowns that remain, including the emerging and often undefined role of deubiquitinating enzymes, the uncertainties of ubiquitin-related modifiers isg15 and sumo, and the potential role of urm-1 in viral replication. some viruses modulate the cell cycle to enhance their own replication (table 1) . these viruses are generally dna tumor viruses such as hpv, adenovirus, and simian virus 40 (sv40) (reviewed in levine, 2009) . a common mechanism shared by these viruses entails targeting cell cycle regulator proteins for degradation, often resulting in transformation. the hpv-16 and -18 e6 proteins co-opt the cellular e3 ligase e6ap to mediate ubiquitin-dependent proteasomal degradation of p53 (reviewed in beaudenon and huibregtse, 2008; mammas et al., 2008) . because p53 is a tumor suppressor protein, degradation of p53 by these hpv strains contributes to their oncogenicity by allowing uncontrolled cellular proliferation without inducing apoptosis. other substrates besides p53 can be targeted for degradation via hpv e6, including mdm7 (murine double minute 7) protein, proteins containing a pdz domain (a common structural motif found in signaling proteins), bak (a protein involved in apoptosis), and e6tp1 (e6 targeted protein 1) (reviewed in beaudenon and huibregtse, 2008) . for several substrates including the pdz domain proteins and p53, hpv e6 can act as an e3 ligase in the absence of e6ap, but the mechanism is unknown. hpv infection also leads to degradation of the retinoblastoma protein prb through the ubiquitin-proteasome pathway (reviewed in mammas et al., 2008) , mediated via the hpv e7-protein-induced generation of an e3 ligase complex consisting of the cullin2 processed ubiquitin (ub) or ubiquitin-like modifier (ubl) is activated with atp by an e1 ubiquitin-activating enzyme (1) and then transferred to an e2 ubiquitin-conjugating enzyme (2). the ub/ubl is then ligated to a substrate via the action of an e3 ubiquitin ligase (3). the e3 determines substrate specificity and can be either cellular or viral in origin. the ubiquitinated substrate is then released (4) and can be polyubiquitinated, often leading to proteasome-mediated degradation (5), or the ub/ ubl can be removed via the action of a cellular or viral deubiquitinating enzyme (dub) (6). alternatively, in the course of viral infection, a viral protein can bind to a cellular ubiquitin ligase complex, altering substrate specificity (7). the substrate is released after ubiquitin conjugation (8). class i mhc molecules are dislocated from the er in response to hcmv viral proteins us2/ us11 or mhv-68 mk3 protein. kshv k3 and k5 proteins mediate ubiquitination and downregulation of class i mhc molecules at the cell surface. hiv vif induces the formation of an e3 ubiquitin ligase complex, which binds to and ubiquitinates apobec3g, leading to its degradation and preventing its incorporation into hiv virions. expression of antiviral ubiquitin-like modifier, isg15, is induced upon infection of certain viruses, including influenza b. the ns1b protein of influenza inhibits the ube1l ligase and prevents conjugation of isg15 to substrates. some viruses also encode deubiquitinating enzymes that mediate removal of isg15 from substrates. abbreviations: isre, interferon sensitive responsive element; ub, ubiquitin. targeting both p53 and the mrn complex can lead to transformation of the host cell. although degradation of cell cycle proteins by co-opting cellular e3 ubiquitin ligase complexes is the most common mechanism used by viruses to interfere with the cell cycle, some encode proteins that can inhibit cellular e3 ligases and prevent degradation of particular host proteins. hpv encodes the e5 protein, which inhibits the degradation of activated epidermal growth factor receptor (egfr) (reviewed in blanchette and branton, 2009 ). e5 binds egfr and interferes with receptor binding to c-cbl (zhang et al., 2005) , an e3 ubiquitin ligase that downregulates activated phosphorylated receptor tyrosine kinases by ubiquitin-mediated degradation. the hpv e2 and adenovirus e4orf4 proteins can inhibit the cell cycle by interfering with the anaphase-promoting complex (apc), an e3 ligase required for progression through m phase (reviewed in blanchette and branton, 2009) . both viral e2 and e4orf4 ultimately inhibit the degradation of cyclin b and probably other substrates, leading to g2/m arrest (bellanger et al., 2005) . viral e2 is itself controlled by an as yet unknown cellular ubiquitin ligase that ubiquitinates the viral protein (bellanger et al., 2001) . sv40 interferes with the cell cycle via its large t antigen, which binds to the scf fbw7 ubiquitin ligase complex and inhibits degradation of cell cycle proteins such as cyclin e, probably by acting as a competitor (welcker and clurman, 2005) . the large t antigen may also induce the ubiquitination of new substrates not normally targeted by the scf complex. in addition to altering the cell cycle, viruses utilize the ubiquitin pathway to interfere with the immune response of the host cells. as depicted in figure 2 , viruses use a variety of methods to hijack the host ubiquitin-proteasome pathway to enhance their own replication and prevent detection by the immune system. virus infection rapidly induces an antiviral state in the host cell, as exemplified by the induction of an interferon response. the nuclear factor-kb (nf-kb) pathway is pivotal in activating both the innate and adaptive immune response. furthermore, the nf-kb transcription factor is itself regulated by the ubiquitinproteasome pathway (reviewed in bhoj and chen, 2009) . a variety of stimulators, including virus infection, induces the activation of nf-kb through the degradation of the ikb proteins, which normally sequesters nf-kb in the cytoplasm. upon ubiquitin-dependent proteasomal degradation of ikb, nf-kb is released, enters the nucleus, and activates a variety of genes involved in setting up an antiviral state in the cell. activation of nf-kb can also be controlled by a20, a dual enzyme with both deubiquitinating and ubiquitin ligase activity, and will be discussed in more detail later. the role of ubiquitin in activation of the innate and adaptive immune response has been extensively reviewed (bhoj and chen, 2009 ). many different host factors that control the innate and adaptive immune response pathways are often targeted by viral gene products to counteract it ( figure 2 ). human immunodeficiency virus 1 (hiv-1) and most other lentiviruses encode several accessory proteins required for pathogenesis of the virus in vivo. one such accessory protein is viral infectivity factor (vif), which acts as a substrate-recruiting subunit of a cellular e3 ligase complex (reviewed in goila-gaur and strebel, 2008; huthoff and towers, 2008) . vif is required for viral replication in ''nonpermissive'' cells such as t cells and macrophages, but vif is not required for replication in permissive cells such as epithelial cells. this is due to the (2005) hcmv us2/us11 induces dislocation from er into cytosol for ubiquitination and degradation of mhc class i molecules wiertz et al. (1996a wiertz et al. ( , 1996b ebv lmp1/lmp2a increases cellular dub activity to stabilize b-catenin and associates with nedd4 e3 ligase to ubiquitinate and degrade lyn and syk tyrosine kinases action of a cellular cytidine deaminase, apobec3g, present in nonpermissive cells, which acts as an intrinsic immune response modulator. apobec3g is normally encapsidated into budding virions and induces cytidine to uracil mutations in the singlestranded dna of hiv-1 during reverse transcription. this hypermutation leads to a block in viral replication (lecossier et al., 2003; mangeat et al., 2003; mariani et al., 2003; zhang et al., 2003) . however, when vif is present, it prevents encapsidation of apobec3g into viral particles by inducing its ubiquitin-dependent degradation (reviewed in goila-gaur and strebel, 2008; huthoff and towers, 2008) . vif recruits elongins b and c, cullin5, and the rbx1 e3 ligase (yu et al., 2003) . the entire complex then polyubiquinates apobec3g, leading to proteasome-mediated degradation (reviewed in goila-gaur and strebel, 2008; huthoff and towers, 2008) and prevents its encapsidation into the retroviral virion ( figure 2 ). another accessory protein of hiv-1, vpu, induces the downregulation of cd4 (reviewed in nomaguchi et al., 2008) , an entry receptor for the virus. it is likely that cd4 downregulation in the endoplasmic reticulum (er) is required for proper trafficking and maturation of the viral glycoprotein, gp160, which would otherwise interact with cd4 and prevent its incorporation into the virion during egress. vpu acts as an adaptor, interacting directly with cd4 in the er and with the f box protein btrcp, causing the transfer of ubiquitin via the skp1-cullin1 e3 ligase complex to cd4, which is then dislocated from the er to the cytosol where it undergoes proteasomal-mediated degradation (reviewed in nomaguchi et al., 2008) . vpu itself is not degraded during this process, but under certain circumstances vpu can be ubiquitinated and degraded by an unknown e3 ligase complex (estrabaud et al., 2007) . the role of ubiquitin in controlling various aspects of receptor endocytosis has been reviewed extensively (see miranda and sorkin, 2007; komada, 2008) . similar to hiv-1 vif, the rubulavirus v protein acts as an adaptor protein to alter the substrate specificity of a cellular e3 ligase (ulane and horvath, 2002) . rubulaviruses presumably seek to destroy stat (signal transducer and activators of transcription) proteins by degradation as a mechanism to dampen the antiviral response. stat-1 and -2 interact with irf-9 (interferon regulatory factor-9), which is activated in response to interferon (ifn) binding to the ifn receptor. viruses often activate the antiviral interferon response during infection, and thus degradation of stat1/2 helps frustrate this response. structurally, the mechanism for virusinduced degradation of the stat proteins is very well understood. the rubulavirus v protein binds to the ultraviolet damage-specific dna-binding protein-1 (ddb1). structural analysis has determined that ddb1 contains a three-propeller cluster that allows the v protein to bind between two of these propellers while the third recruits the e3 ligase cullin4a (li et al., 2006) . the rbx1 ring protein is then recruited and binds to an as yet unknown e2 conjugating enzyme. rubulavirus v proteins can then either directly target stat2 for ubiquitination (human parainfluenza virus type 2) or stat2 can be used to recruit stat1 (simian virus 5 and mumps virus) (precious et al., 2005) . regardless, the result is polyubiquitination of stat1/2 and degradation by the proteasome. mumps v protein, along with rbx1, directly targets stat3 for ubiquitination and degradation (ulane et al., 2003) . degradation of target proteins is mediated not only by viral adaptor proteins that hijack cellular e3 ligases, but also by viral proteins that induce dislocation from the er. human cytomegalovirus (hcmv) downregulates mhc class i, presumably to decrease antigen presentation to cd8 + t cells, thus effectively avoiding immune recognition ( figure 2 ). hcmv us11 and us2 proteins induce dislocation of mhc class i from the er into the cytosol where the class i molecules become polyubiquitinated and degraded by the proteasome (wiertz et al., 1996a (wiertz et al., , 1996b . for us11, the hrd1-sel1l complex serves as the e3 ligase, a multisubunit complex that acquires its ubiquitin from a tailanchored er resident e2, ubc6e. class i mhc products are frequent targets of viral evasive maneuvers (see figure 2 and van der wal et al., 2002; loureiro and ploegh, 2006 , for review). because a fraction of all newly synthesized proteins, regardless of whether derived from host or virus, fails to fold correctly, their disposal is essential and requires involvement of the ubiquitinproteasome pathway. accordingly, a fraction of newly synthesized viral proteins will undergo this fate as well. however, some viral functions require ubiquitin modification for purposes other than protein turnover (table 2) , as exemplified by the role of ubiquitin in virus budding. some early observations include the detection of free ubiquitin in retroviral particles, such as hiv, and the inhibition by proteasome inhibitors of the budding of retroviruses and other rna viruses, presumably because the store of free ubiquitin to be used for conjugation is tied up (martin-serrano, 2007) . the retroviral gag protein contains multiple domains, including matrix, capsid, nucleocapsid, and p6, all of which play a role in the (2008) hpv e2 ubiquitinated by unknown cellular ligase for degradation bellanger et al. (2001) assembly and budding process. the p6 domain is also known as the late budding domain (l-domain) and is thought to serve as an adaptor to recruit cellular proteins to mediate membrane fission, allowing release of the virion. conserved motifs, such as ppxy or ptap, are found in p6 and are thought to mediate its ability to recruit these proteins (strack et al., 2000) . the process of membrane fission that occurs during retroviral egress is similar to that of the multivesicular body (mvb) pathway (see morita and sundquist, 2004; martin-serrano, 2007, for reviews (babst et al., 2000) . binding of tsg101 to the ptap motif in gag occurs through the uev domain of tsg101. tsg101 is required for hiv budding: its depletion with rna interference (rnai) or overexpression of its uev domain both inhibit virion release (garrus et al., 2001; demirov et al., 2002) . the nedd4 (neuronal precursor cell-expressed developmentally downregulated) protein is a ww domain-containing hect e3 ubiquitin ligase required for budding of many viruses including retroviruses, ebola, and rhabdoviruses (reviewed in ingham et al., 2004) . this e3 is recruited to ppxy motifs in late budding domains, including the ebola vp40 protein, which is itself ubiquitinated (yasuda et al., 2003) . ebola vp40 contains both a ppxy and ptap motif. like hiv-1 gag, it binds tsg101 through the ptap motif and this interaction is required for virion budding and release. nedd4 binds to the ppxy motifs in retroviral gag proteins, including rsv, m-pmv, and mmlv. the ppxy motif in gag of m-pmv, vp40 of ebola, and m protein of rhabdoviruses (vesicular stomatitis virus [vsv] and rabies) controls virion budding and release. nedd4 may also function in the maintenance of ebv (epstein-barr virus) latency in b cells (reviewed in ingham et al., 2004) . the viral protein latent membrane protein 2a (lmp2a) contains two pppy motifs that recruit nedd4 family members. signaling pathways initiated by the b cell receptor (bcr) activate the viral bzlf1 immediate-early gene, which then activates viral lytic genes. lmp2a binds, sequesters, and causes the degradation of the lyn and syk tyrosine kinases, preventing them from interacting with the bcr, and possibly preventing bcr signaling and reactivation from latency (winberg et al., 2000) . the nedd4 e3 ligase ubiquitinates lyn, syk, and lmp2a itself and targets them for degradation (ikeda et al., 2000) . however, lmp2a with a mutated pppy motif still blocks the induction of viral lytic genes, questioning the importance of the recruitment of nedd4 through that motif. although viruses take advantage of ubiquitination to enhance their own replication, viral proteins can also be ubiquitinated by cellular ligases, leading to their degradation. hpv e7, in addition to modifying the ubiquitination of host proteins such as prb as discussed above, is itself modified by both an e3 ubiquitin ligase and a deubiquitinating enzyme. e7 is a short-lived protein degraded via the ubiquitin proteasomal pathway. the suppressor of cytokine signaling (socs1) protein is a member of the stat signaling pathway and is induced upon exposure to ifn-g. socs1 causes ubiquitination and degradation of e7, thus increasing prb levels (kamio et al., 2004) . e7 interacts with the scf complex, which ubiquitinates e7 with the help of the e2 enzyme ubch7 (oh et al., 2004) . the two pathways of e7 degradation are complementary: one occurs in the cytoplasm (socs1) and the other in the nucleus (scf). the action of these ligases is opposed by the usp11 deubiquitinating enzyme: it interacts with e7, removes ubiquitin, and extends the half-life of e7 (lin et al., 2008) . the list of viruses that encode ubiquitin ligases continues to grow (table 3) . given the parsimonious use of genetic capacity available to viruses, it stands to reason that these activities were selected for as a means to increase viral fitness. herpes simplex virus 1 (hsv-1) infected cell protein 0 (icp0) is a e3 ubiquitin ligase that induces polyubiquitination and degradation of a variety of proteins, including the promyelocytic leukemia (pml) protein, sp100 (another component of pml nuclear bodies) (chelbi-alix and de boutell et al., 2002) , cyclin d3 (van sant et al., 2001; hagglund et al., 2002) , p53, and the cellular deubiquitinating enzyme usp7. icp0 has two e3 ligase sites: a ring domain and a herpesvirus ubiquitin ligase-1 (hul-1) domain. the ring domain recruits the cellular e2 enzyme ubch5a as well as usp7, which is then ubiquitinated and degraded. icp0 also undergoes self-ubiquitination, but its interaction with usp7 can lead to the stabilization of icp0 by reversal of autoubiquitination. in the course of infection, the end result of these two opposing forces is the stabilization of icp0 by usp7. icp0 also likely plays a role in preventing activation of the antiviral response by suppressing interferon-stimulated gene (isg) expression (eidson et al., 2002) . it was first discovered that hsv-1 induces an antiviral response in the absence of viral replication, suggesting that expression of a viral protein normally counteracts this response. cells infected with an icp0 null mutant virus replicate much less efficiently and express higher levels of isgs than do cells infected with wild-type virus (harle et al., 2002) . icp0 inhibits irf-3-and irf-7-mediated activation of the isgs (lin et al., 2004) . this inhibition requires the ring domain of icp0 and the pml protein, but the exact mechanism is unknown. icp0 also mediates the degradation of dna-pk, which stabilizes irf-3, further inhibiting the activation of isgs, required for the induction of the ifn response. however, the ability of an icp null virus to replicate is not enhanced by the inhibition of the stat-1 or irf-3 pathways, suggesting that some other mechanism, such as pml degradation, may be responsible for this phenotype (everett et al., 2008) . the kaposi's sarcoma-associated herpesvirus (kshv) proteins k3 and k5 and murine gammaherpesvirus 68 (mhv-68) k3 are e3 ubiquitin ligases that conjugate ubiquitin to mhc class i proteins, leading to their downregulation from the cell surface or the er (figure 2 ; coscoy and ganem, 2000; ishido et al., 2000; boname and stevenson, 2001) . k3 and k5 require cellular components such as tap and tapasin, proteins that form a complex with the class i mhc molecules (the peptide loading complex), for successful ubiquitination to occur. k5 also downregulates other molecules important for the stimulation of t cells, such as icam-1 and b7 (coscoy and ganem, 2001) . kshv encodes yet another e3 ligase, the rta (replication and transcription activator) protein (yu et al., 2005) , a transcription factor encoded by the orf50 gene. the rta protein is essential for activation of viral dna replication upon initial infection of the virus. this activation occurs by transactivation of downstream lytic genes as well as the orf50 gene as well. furthermore, the rta protein is also required for reactivation from latency. the rta protein contains a cysteine-rich region that likely contains e3 ligase activity, as indicated by the fact that mutation of this region abrogates conjugation activity. rta can not only polyubiquitinate itself but can also ubiquitinate several rta repressors, including k-rbp (kshv-rta binding protein) and hey1, a cellular transcriptional repressor, leading to their degradation by the proteasome (yang et al., 2008; gould et al., 2009) this ability of the rta protein to induce the degradation of these transcriptional repressors is an important viral means of control and essential for the reactivation of lytic dna replication. irf-7 is also ubiquitinated by the rta protein, which leads to its degradation and prevents the generation of ifn-a, compromising the innate immune response (yu et al., 2005) . several ubiquitin-like modifiers play a role in viral infection. these include sumo and isg15. furthermore, recently discovered ubiquitin-like modifiers, such as urm-1, may also prove to be involved in viral replication and will be also discussed. sumo because sumoylation is a predominantly nuclear event, it is the nuclear proteins encoded by dna viruses that tend to be directly sumoylated (rodriguez et al., 2001) . for herpes-, adeno-, and papillomaviruses, the viral proteins that are sumoylated generally have roles in viral transcription or replication; sumoylation often causes changes in their subcellular localization but it has remained a challenge to determine the exact role of sumoylation in viral infection. some viral proteins can interact with sumo or with ubc9, the sumo-conjugating enzyme, but are not themselves sumoylated, perhaps as a means of recruiting the sumoylation machinery to other viral or cellular proteins, in a manner analogous to recruitment of e6ap to p53 by hpv e6. the first viral proteins found to be directly sumoylated was the immediate-early (ie) ie1 and ie2 proteins of hcmv (reviewed in rosas-acosta and wilson, 2004). the ie proteins of ebv (bzlf1) and human herpes virus 6b (hhv-6b, ie-1) are also sumoylated. the ie proteins of herpesviruses are essential in initiating viral gene expression upon virus entry. they often act as transcriptional regulatory factors that help jump-start downstream early viral gene expression during viral infection (hcmv ie2), but ie proteins also regulate the cell cycle and apoptosis and can disrupt the pml nuclear bodies (hcmv ie1 and ebv bzlf1). there is currently no phenotype associated with a sumoylation-resistant hcmv ie1 protein (lee et al., 2004) . hcmv ie2 interacts with prb, p53, as well as several cellular transcriptional activators. ie2 interacts with sumo-1, sumo-3, and ubc9. although a sumoylation-resistant ie2 shows no differences in subcellular localization or stability, it does not transactivate early viral promoters as well as wild-type ie2 (hofmann et al., 2000) . sumo may thus alter the ability of ie2 to interact directly with those viral promoters or transcription factors required for efficient viral gene expression. the exact role of sumo modification of the ie proteins in hcmv infection remains to be clarified. sumoylation of the hhv6 ie1 protein may increase its stability, but whether its sumoylation affects transcriptional activity is not known. similar to the other herpesviral ie proteins, the ebv review bzlf1 (z) protein is a transcriptional activator and can disrupt the pml nuclear bodies. the role of sumoylation in the viral life cycle is unclear, because a sumoylation-deficient z protein likewise disrupts nuclear bodies; however, sumoylation of z does appear to decrease the ability of z to transactivate certain promoters (adamson, 2005) . these examples once again illustrate the challenges in distinguishing between adventitious and purposeful ubiquitin and ubiquitin-like modifications by viruses. the role of sumo in adenovirus and papillomavirus replication is better understood than for herpesviruses (reviewed in rosas-acosta and wilson, 2004) . adenovirus e1b55k, together with e4orf6 and cellular proteins, ubiquitinates and promotes proteasomal degradation of p53 as discussed earlier. e1b55k contains a consensus site for sumoylation responsible for its nuclear localization (endter et al., 2001) . indeed, a k104r sumoylationresistant mutant shows altered localization and a reduced ability to interact with p53 and to transform cells (endter et al., 2001) . both the hpv and bovine papilloma virus (bpv) e1 proteins interact with ubc9 (yasugi and howley, 1996) and bpv e1 can be sumoylated. an hpv e1 mutant unable to bind to ubc9 fails to support viral replication (yasugi and howley, 1996) and demonstrates the importance of sumoylation for small dna tumor viruses. the ability to bind sumo and/or ubc9 is a property shared by numerous other viral proteins. the bunyaviridae and retroviridae families contain viral nucleocapsid proteins that interact with sumo and/or ubc9, but these are not themselves sumoylated. the hantaan virus nucleocapsid (htnv-np) interacts with both ubc9 and sumo-1, possibly to relocate np to the perinuclear region where viral replication and virion assembly occur (kaukinen et al., 2003) . retroviral gag proteins, required for virion assembly and budding, interact with ubc9, as exemplified by mason-pfizer monkey virus (mpmv) (weldon et al., 2003) . overexpression of ubc9 causes both ubc9 and gag to localize to nuclear and perinuclear regions and therefore may help direct gag to the site of virion assembly. a further layer of compelexity is provided by the existence of multiple isoforms of sumo (sumo-1, -2, -3), which appear to have different targeting abilities and only some of which have been explored in any detail in the context of virus infections. the distinct functions of these sumo isoforms remain to be established, particularly for sumo-2 and sumo-3, for which even their cellular roles are not well defined. examples of viral proteins that interact with specific isoforms of sumo include vaccinia early protein (e3l) (which interacts with sumo-1) and ebv nuclear antigen 3c (ebna-3c) (which interacts with both sumo-1 and sumo-3, but not sumo-2) (lin et al., 2002) . this is particularly surprising for ebna-3c, because sumo-2 and sumo-3 are 97% identical but sumo-1 and sumo-3 are only 46% identical. sumo proteases (senps) remove the sumo molecule from substrates, thereby reversing the effects of sumoylation. the adenoviridae, asfarviridae, and poxviridae encode cysteine proteases related to the mammalian senps: they share conserved catalytic cysteine and histidine residues. these include the adenovirus protease (li and hochstrasser, 1999) , the ps273r proteinase of african swine fever virus (asfv) (andres et al., 2001) , and the i7 proteinase of poxviruses (li and hochstrasser, 1999) . thus far, desumoylating activity remains to be demonstrated for any of these proteases and their role, if any, in viral replication is not known. by analogy with ubiquitination, however, where the importance of ubqiquitin-specific proteases is now firmly established, it is a reasonable supposition that these activities would be important, all the more so when the host cell already comes equipped with its own complement of senps. isg15 isg15 contains two ubiquitin-like domains. upon viral infection, interferon is induced, leading to the induction of isgs including isg15, one of the most highly induced igss (reviewed in sadler and williams, 2008) . isg15 can be secreted from cells that produce it by an as yet unknown mechanism and therefore may act as a cytokine to modulate the immune response (d'cunha et al., 1996) . isg15 has specific e1 (ube1l), e2 (ubch6 and ubch8), and e3 (herc5 and trim25) ligases. isgylation is reversible via the action of ubiquitin-specific proteases (usps), including usp18 (ubp43), usp2, usp4, usp13, and usp14 (reviewed in sadler and williams, 2008) . however, unlike ubiquitination, isgylation is not known to promote degradation, but instead acts as an antiviral effector. a complicating factor is the possibility of alternative modification of a given substrate protein either with ubiquitin or with isg15, or modification with both ubiquitin and isg15. recombinant sindbis viruses that drive the expression of a variety of isgs was used to infect ifn-a/b receptor ã�/ã� (ifn-a/br ã�/ã� ) mice to identify attenuators of infection (lenschow et al., 2005) . expression of isg15 had an antiviral effect, protecting against sindbis virus-induced death, and decreased viral replication. however, the antiviral state in the course of vsv and lymphocytic choriomeningitits virus (lcmv) infection in isg15 ã�/ã� mice was similar to wild-type controls (osiak et al., 2005) . viruses can interfere with isgylation. influenza b virus strongly induces expression of isg15, but the ns1 protein binds isg15, inhibits activity of the ube1l ligase, and prevents isgylation (yuan and krug, 2001) . surprisingly, influenza a ns1 protein lacks this activity, but in this case, viral infection fails to induce expression of isg15 (yuan and krug, 2001) . ovarian tumor (otu)-domain proteases from nairoviruses of the bunyaviridae family (crimean-congo haemorrhagic fever virus [cchfv] , large [l] protein) and arteriviruses (equine arteritis virus [eav], nonstructural protein 2 [nsp2]) are deisgylases as well as deubiquitinases (frias-staheli et al., 2007) . these otu domain proteases are present in eukaryotes, bacteria, and viruses. the viral otu domain proteases show much broader target specificity, unlike their host counterparts. expression of these proteases reverses the antiviral effect of isg15, increasing the host's susceptibility to sindbis infection (frias-staheli et al., 2007) . the viral otu domain proteases inhibit nf-kb-dependent signaling, likely through their deubiquitinating activity. expression of the otu domains of cchfv-l and eav-nsp2 inhibits the translocation of the p65 subunit of nf-kb to the nucleus (frias-staheli et al., 2007) . severe acute respiratory syndromeassociated coronavirus (sars-cov) expresses a distinct protease, plpro, which mediates deisgylation. although isg15 is its preferred substrate, plpro is also capable of deubiquitination (lindner et al., 2007) . isg15 inhibits virus-mediated degradation of irf-3, increasing ifn-b expression to enhance the innate immune response (reviewed in sadler and williams, 2008) . the transcriptional activity of irf-3, essential for activation of type i ifn genes, is tightly regulated by protein degradation. newcastle disease virus (ndv) infection or ifn treatment increases the conjugation of isg15 to cellular proteins, including irf-3 . this conjugation stabilizes irf-3, prevents its ubiquitin-mediated degradation, increases activation of the ifn-b promoter, and facilitates irf-3 translocation to the nucleus. after ndv infection of isg15 ã�/ã� mefs (mouse embryonic fibroblasts), the levels of irf-3 decrease more drastically than in wt mefs . isg15 conjugation may also help direct modified proteins to the proteasome, because the proteasome-associated deubiquitinating enzyme (dub), usp14, can recognize isg15-modified substrates, or it may function to attract components of the ubiquitin conjugation machinery. isg15 plays a role in resistance to ebola virus (also vsv and rabies virus) by blocking the activity of nedd4 via isgylation (reviewed in sadler and williams, 2008) . isg15 blocks the interaction of nedd4 with its e2, preventing transfer of ubiquitin from the e2 to nedd4 (malakhova and zhang, 2008) . the ubiquitination of hiv-1 gag and tsg101 and nedd4 are necessary for virion budding and release from the cell surface as discussed earlier. type i ifn inhibits the assembly and release of hiv-1 virions. this occurs through the ifn-induced expression of isg15, which inhibits the ubiquitination of gag and tsg101, preventing the interaction of the gag l domain with tsg101 . however, isg15 is not itself conjugated to gag or tsg101. ubiquitination of gag by the nedd4 ubiquitin ligase is necessary for the release of virions. overexpression of isg15 effectively decreases the ability of nedd4 to ubiquitinate the viral matrix proteins (vp40), thereby decreasing release of ebola virus-like particles (vlps) from cells (okumura et al., 2008) . other ubiquitin-like modifiers: urm-1 the role of ubiquitin-like modifiers is no longer confined to the modification of proteins and lipids. a recent series of papers describes an unexpected role for ubiquitin-related molecule-1 (urm-1) in transfer rna (trna) modification (schlieker et al., 2008; leidel et al., 2009; noma et al., 2009) . though only distantly related to ubiquitin, urm-1 has the typical b-grasp fold that characterizes members of the ubiquitin family (singh et al., 2005) and also possesses the typical diglycine motif at the c terminus of the mature polypeptide, common to ubiquitin and ubiquitinlike modifiers. the occurrence of proteins covalently modified by urm-1 (''urmylation'') has been reported (furukawa et al., 2000) , but thus far appears to be limited to a single protein of known identity, alkyl hydroperoxide reductase (ahp1), in yeast (goehring et al., 2003) . whether urmylation of proteins is a widespread modification, and if so, under which conditions (stress, growth conditions, and virus infection) remain open questions. urm-1 serves as a sulfur carrier essential for thiolation of certain trnas [(uuu) lys; (uuc) glu; and (uug) gln], which carry a thiolated uridine residue in the wobble position of the anticodon (schlieker et al., 2008; leidel et al., 2009; noma et al., 2009) . urm-1 can be isolated from mammalian cells as a c-terminal thiocarboxylate that is the immediate precursor for a sulfur transfer reaction involving a persulfidic intermediate (schlieker et al., 2008) . in the absence of urm-1, yeast cells can no longer carry out the requisite thiolation reaction and become sensitive to a variety of stresses (pedrioli et al., 2008) . the mechanistic link between a failure to execute thiolation and the increased sensi-tivity to stress observed in urm-deficient cells is not yet understood. for example, it is not clear how trnas that fail to undergo thiolation affect translational efficiency, fidelity, or the half-lives of the trnas themselves. a number of plant viruses, including brome mosaic virus, possess trna-like structures at the 3 0 end of their genomes. these structures are suffciently similar to trnas to serve as substrates for aminoacyl trna synthetases and other enzymes that act on trnas (dreher, 2009) . the mouse gammaherpesvirus mhv-68 encodes at least eight trna-like sequences that can be processed into mature uncharged trnas, the function of which in the context of the virus life cycle is obscure. clearly, viruses have co-opted trnas and trna-like structures to initiate replication, as is well established for retroviruses (reviewed in abbink and berkhout, 2008) , presumably for reasons that increase fitness, but we still lack an adequate mechanistic explanation. given the relationship between urm-1 and specific trna modifications, it is perhaps reasonable to suggest that aspects of translational control and fidelity, as well as virus replication itself, may be modified in a manner that involves urm-1, although for the moment this remains pure speculation. there are currently seven known classes of dubs that act to reverse ubiquitin and ubiquitin-like modifications: (1) the ubiquitin-specific protease (usp), (2) autophagin (atg), (3) ubiquitin c-terminal hydrolase (uch), (4) ovarian tumor (otu) domain proteins, (5) josephin-domain (jd) or machado-joseph disease (mjd) proteins, (6) ubiquitin-like protein-specific protease (ulp), and (7) jamm (jab1/mpn domain-associated metalloisopeptidase) domain proteins (see nijman et al., 2005; sulea et al., 2006, for review) . several viral proteins are known to interact with cellular dubs. one of the most well studied is usp7 or herpesvirus-associated ubiquitin-specific protease (hausp). icp0 of hsv-1 and ebna1 of ebv interact with usp7 (everett et al., 1997; holowaty and frappier, 2004) , which removes ubiquitin from p53, ebna1, and icp0, thereby preventing their degradation (li et al., 2002; canning et al., 2004) . ebna1 of ebv is required for multiple aspects of viral replication: maintenance of the viral genome, transcription and translation of the viral dna, viral persistence, and transformation of cells. the exact role of hsv-1 icp0 interaction with usp7 is not known, but as noted earlier, icp0 is an e3 ligase that ubiquitinates usp7, causing both to be degraded. icp0 is also a substrate for usp7 and is stabilized by interaction with this dub. in the course of infection, the net result of these two opposing forces is the stabilization of icp0 by usp7. upon ebv infection, several cellular dubs are known to increase in activity including usp5, -7, -9, -13, -15, and -22 (ovaa et al., 2004) . furthermore, the dubs recruited by ebv may stabilize b-catenin, a component not only of cellular junctions, but also a key component of the wnt signaling pathway that regulates growth and differentiation of cells. furthermore, its disregulation has been implicated in cancer development (reviewed in reya and clevers, 2005) . this stabilization occurs in latently infected b cells and may involve viral proteins lmp1 and lmp2a, although a contradictory report suggests that lmp1 is not sufficient to induce activation of the wnt pathway (webb et al., 2008) . further investigation is needed to determine the exact role ebv plays in activation of the wnt signaling pathway, because ebv is associated with several cancers, including nasopharyngeal carcinoma and burkitt's and hodgkin's lymphomas. measles virus also induces the upregulation of a cellular deubiquitinating enzyme, a20 (yokota et al., 2008) . a20 is an otu domain dub that inhibits nf-kb upstream by removing ubiquitin from traf6 and rip1, two signaling molecules of the nf-kb pathway. furthermore, a20 also has ubiquitin ligase activity and can ubiquitinate rip1, leading to proteasomal-mediated degradation. in certain cell types, measles virus infection dramatically upregulated a20. the expression of measles virus phosphoprotein (p protein) is necessary and sufficient to induce upregulation of a20, leading to the suppression of toll-like receptor signaling (yokota et al., 2008) . several viral proteins possess deubiquitinating activity (table 3) , including the adenovirus protease adenain sars-cov papainlike protease (plpro) and herpesvirus large tegument protein. deubiquitinating activity in herpesviruses was first discovered with activity-based ha-ubiquitin probes (haubvme) in hsv-1infected cell lysates (kattenhorn et al., 2005) . the n-terminal fragment of ul36, the large tegument protein, is the source of this activity. ul36 homologs from a-, b-, and g-herpesviruses all contain a putative cysteine box with a histidine box further downstream. ul36 lacks obvious similarity to any known dub, and the crystal structure of murine cytomegalovirus (mcmv) m48 dub domain has determined that the herpesviral dubs represents a new family of deubiquitinating enzymes (schlieker et al., 2007) . dub activity has been confirmed for several other herpesviruses including ebv, mcmv, and hcmv (schlieker et al., 2005; wang et al., 2006) . mutation of the putative active site cysteine in ul48 of hcmv produced lower virus yields but the activity is not essential for replication of hcmv in tissue culture (wang et al., 2006) . further work with marek's disease virus (mdv), an a-herpesvirus, has demonstrated a potential role for the herpesvirus dub in pathogenesis in vivo. mutation of the mdv dub active site cysteine caused a stark reduction in the formation of t cell lymphomas in chickens (jarosinski et al., 2007) . similar mutants were made for pseudorabies virus (prv): mice infected with a dub active site mutant virus survived twice as long and demonstrated a decrease in neuroinvasion compared to those infected with wild-type virus (bottcher et al., 2008) . the prv dub active site mutant demonstrates a defect in virion assembly and egress in vitro (bottcher et al., 2008) . the large tegument protein remains tightly bound to the nucleocapsid during transit to the nucleus, and could thus be important for entry of the virus, as well as for viron assembly and egress. as a key component of the virion, the tegumentencoded dub might participate in either or both of these events. more recently, two other active dubs (bslf1 and bxlf1) were discovered to be encoded by ebv via computer modeling to search for the conserved c-and h-boxes of the known dub families (sompallae et al., 2008) . homologs of these proteins exist in some of the other herpesviruses; therefore, it is likely that more functional dubs will be discovered in these viruses. the crystal structure of the sars-cov plpro enzyme demonstrates that it belongs to the usp family of dubs; moreover, it has dub activity in vitro (lindner et al., 2005) . plpro is part of the repli-case polyprotein and is involved in cleaving the nonstructural proteins (nsps), particularly nsps1, -2, and -3, to form the viral rna replication complex. whether the sars-cov plpro functions as a dub in the course of infection is not known, though it is probable. it might serve to protect the viral replication complex from ubiquitination and degradation by the proteasome. the adenovirus protease adenain is found both in the nucleus and the cytosol. upon activation in the nucleus, the adenain protease cleaves six viral capsid precursor proteins inside the viral capsids (ruzindana-umunyana et al., 2002) . in the cytoplasm, adenain is thought to cleave cytoskeletal proteins, aiding in cell lysis and release of the virions. adenain also contains deubiquitinating activity (balakirev et al., 2002) . during adenoviral infection, the levels of adenain correlate with a corresponding decrease in ubiquitinated proteins, particularly in the nucleus. the identity of the affected substrates is not known, a challenge common to most enzymes in this class, and the role of the adenain-associated dub activity in the viral life cycle remains to be clarified. hijacking of the ubiquitin system by viruses continues to emerge as a central theme around virus replication. not only is modification of ubiquitinated substrates during the course of viral infection a mainstream event, but examples of viruses that encode their own ubiquitin ligases as well as deubiquitinating enzymes are increasing rapidly. a continuing challenge that remains is the discovery of the substrates for these newly described viral enzymes. enzyme-substrate interactions may demonstrate high affinity but may also be transient. the relatively recent advances in mutagenesis techniques that allow rapid production of virus mutants, also for the large dna viruses, coupled with the ability to manipulate host protein expression, will help determine the function and substrates of these emerging viral and cellular ubiquitin modifiers. because of space limitations, we have been unable to exhaustively cover every aspect of ubiquitination and viral infection and have cited predominantly more recent publications or pertinent reviews on the subject. we apologize to those who have contributed to the field and whose work we were unable to recognize. hiv-1 reverse transcription initiation: a potential target for novel antivirals? effects of sumo-1 upon epstein-barr virus bzlf1 function and bmrf1 expression african swine fever virus protease, a new viral member of the sumo-1-specific protease family mammalian tumor susceptibility gene 101 (tsg101) and the yeast homologue, vps23p, both function in late endosomal trafficking stability of the human papillomavirus type 18 e2 protein is regulated by a proteasome degradation pathway through its amino-terminal transactivation domain high-risk but not low-risk hpv e2 proteins bind to the apc activators cdh1 and cdc20 and cause genomic instability ubiquitylation in innate and adaptive immunity manipulation of the ubiquitin-proteasome pathway by small dna tumor viruses mhc class i ubiquitination by a viral phd/lap finger protein mutagenesis of the active-site cysteine in the ubiquitinspecific protease contained in large tegument protein pul36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo herpes simplex virus type 1 immediate-early protein icp0 and is isolated ring finger domain act as ubiquitin e3 ligases in vitro a ring finger ubiquitin ligase is protected from autocatalyzed ubiquitination and degradation by binding to ubiquitin-specific protease usp7 herpes virus induced proteasomedependent degradation of the nuclear bodies-associated pml and sp100 proteins kaposi's sarcoma-associated herpesvirus encodes two proteins that block cell surface display of mhc class i chains by enhancing their endocytosis a viral protein that selectively downregulates icam-1 and b7-2 and modulates t cell costimulation immunoregulatory properties of isg15, an interferon-induced cytokine overexpression of the n-terminal domain of tsg101 inhibits hiv-1 budding by blocking late domain function role of trna-like structures in controlling plant virus replication expression of herpes simplex virus icp0 inhibits the induction of interferon-stimulated genes by viral infection sumo-1 modification required for transformation by adenovirus type 5 early region 1b 55-kda oncoprotein regulated degradation of the hiv-1 vpu protein through a betatrcpindependent pathway limits the release of viral particles a novel ubiquitin-specific protease is dynamically associated with the pml nuclear domain and binds to a herpesvirus regulatory protein stat-1-and irf-3-dependent pathways are not essential for repression of icp0-null mutant herpes simplex virus type 1 in human fibroblasts ovarian tumor domain-containing viral proteases evade ubiquitinand isg15-dependent innate immune responses a protein conjugation system in yeast with homology to biosynthetic enzyme reaction of prokaryotes tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding attachment of the ubiquitin-related protein urm1p to the antioxidant protein ahp1p hiv-1 vif, apobec, and intrinsic immunity kshv rta promotes degradation of the hey1 repressor protein through the ubiquitin proteasome pathway herpes simplex virus 1-infected cell protein 0 contains two e3 ubiquitin ligase sites specific for different e2 ubiquitin-conjugating enzymes the immediateearly protein, icp0, is essential for the resistance of herpes simplex virus to interferon-alpha/beta covalent modification of the transactivator protein ie2-p86 of human cytomegalovirus by conjugation to the ubiquitin-homologous proteins sumo-1 and hsmt3b hausp/usp7 as an epstein-barr virus target human papillomavirus type 16 e7 oncoprotein associates with the cullin 2 ubiquitin ligase complex, which contributes to degradation of the retinoblastoma tumor suppressor restriction of retroviral replication by apobec3g/f and trim5alpha the epstein-barr virus latent membrane protein 2a py motif recruits ww domain-containing ubiquitin-protein ligases the nedd4 family of e3 ubiquitin ligases: functional diversity within a common modular architecture downregulation of major histocompatibility complex class i molecules by kaposi's sarcoma-associated herpesvirus k3 and k5 proteins a herpesvirus ubiquitin-specific protease is critical for efficient t cell lymphoma formation socs1 [corrected] inhibits hpv-e7-mediated transformation by inducing degradation of e7 protein controlling receptor downregulation by ubiquitination and deubiquitination hypermutation of hiv-1 dna in the absence of the vif protein ability of the human cytomegalovirus ie1 protein to modulate sumoylation of pml correlates with its functional activities in transcriptional regulation and infectivity in cultured fibroblast cells ubiquitin-related modifier urm1 acts as a sulphur carrier in thiolation of eukaryotic transfer rna identification of interferon-stimulated gene 15 as an antiviral molecule during sindbis virus infection in vivo the common mechanisms of transformation by the small dna tumor viruses: the inactivation of tumor suppressor gene products: p53 a new protease required for cell-cycle progression in yeast deubiquitination of p53 by hausp is an important pathway for p53 stabilization structure of ddb1 in complex with a paramyxovirus v protein: viral hijack of a propeller cluster in ubiquitin ligase epstein-barr virus nuclear antigen 3c putative repression domain mediates coactivation of the lmp1 promoter with ebna-2 the herpes simplex virus icp0 ring finger domain inhibits irf3-and irf7-mediated activation of interferon-stimulated genes usp11 stabilizes hpv-16e7 and further modulates the e7 biological activity the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme selectivity in isg15 and ubiquitin recognition by the sars coronavirus papain-like protease antigen presentation and the ubiquitinproteasome system in host-pathogen interactions isg15 enhances the innate antiviral response by inhibition of irf-3 degradation isg15 inhibits nedd4 ubiquitin e3 activity and enhances the innate antiviral response human papilloma virus (hpv) and host cellular interactions broad antiretroviral defence by human apobec3g through lethal editing of nascent reverse transcripts species-specific exclusion of apobec3g from hiv-1 virions by vif the role of ubiquitin in retroviral egress regulation of receptors and transporters by ubiquitination: new insights into surprisingly similar mechanisms retrovirus budding a genomic and functional inventory of deubiquitinating enzymes mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at trna wobble positions role of hiv-1 vpu protein for virus spread and pathogenesis the papillomavirus e7 oncoprotein is ubiquitinated by ubch7 and cullin 1-and skp2-containing e3 ligase innate antiviral response targets hiv-1 release by the induction of ubiquitin-like protein isg15 isg15 inhibits ebola vp40 vlp budding in an l-domain-dependent manner by blocking nedd4 ligase activity isg15, an interferon-stimulated ubiquitin-like protein, is not essential for stat1 signaling and responses against vesicular stomatitis and lymphocytic choriomeningitis virus activity-based ubiquitin-specific protease (usp) profiling of virus-infected and malignant human cells protein modifications: beyond the usual suspects mechanisms underlying ubiquitination simian virus 5 v protein acts as an adaptor, linking ddb1 to stat2, to facilitate the ubiquitination of stat1 degradation of p53 by adenovirus e4orf6 and e1b55k proteins occurs via a novel mechanism involving a cullin-containing complex wnt signalling in stem cells and cancer sumo-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting viruses and sumoylation substrate specificity of adenovirus protease interferon-inducible antiviral effectors the e6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53 a deubiquitinating activity is conserved in the large tegument protein of the herpesviridae structure of a herpesvirus-encoded cysteine protease reveals a unique class of deubiquitinating enzymes a functional proteomics approach links the ubiquitin-related modifier urm1 to a trna modification pathway three-dimensional structure of the aah26994.1 protein from mus musculus, a putative eukaryotic urm1 epstein-barr virus encodes three bona fide ubiquitin-specific proteases a role for ubiquitin ligase recruitment in retrovirus release adenovirus oncoproteins inactivate the mre11-rad50-nbs1 dna repair complex structural aspects of recently discovered viral deubiquitinating activities paramyxoviruses sv5 and hpiv2 assemble stat protein ubiquitin ligase complexes from cellular components stat3 ubiquitylation and degradation by mumps virus suppress cytokine and oncogene signaling the hcmv gene products us2 and us11 target mhc class i molecules for degradation in the cytosol the infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 e2 ubiquitin-conjugating enzyme, and possesses in vitro e3 ubiquitin ligase activity high-molecular-weight protein (pul48) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its activesite cysteine or histidine are viable epstein-barr virus associated modulation of wnt pathway is not dependent on latent membrane protein-1 the sv40 large t antigen contains a decoy phosphodegron that mediates its interactions with fbw7/hcdc4 mason-pfizer monkey virus gag proteins interact with the human sumo conjugating enzyme, hubc9 the ebv deubiquitinating enzyme, bplf1, reduces ebv ribonucleotide reductase activity the human cytomegalovirus us11 gene product dislocates mhc class i heavy chains from the endoplasmic reticulum to the cytosol sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction latent membrane protein 2a of epstein-barr virus binds ww domain e3 protein-ubiquitin ligases that ubiquitinate b-cell tyrosine kinases kaposi's sarcoma-associated herpesvirus transactivator rta promotes degradation of the repressors to regulate viral lytic replication nedd4 regulates egress of ebola virus-like particles from host cells identification of the structural and functional human homolog of the yeast ubiquitin conjugating enzyme ubc9 measles virus p protein suppresses toll-like receptor signal through up-regulation of ubiquitinmodifying enzyme a20 induction of apobec3g ubiquitination and degradation by an hiv-1 vif-cul5-scf complex the kshv immediate-early transcription factor rta encodes ubiquitin e3 ligase activity that targets irf7 for proteosome-mediated degradation influenza b virus ns1 protein inhibits conjugation of the interferon (ifn)-induced ubiquitin-like isg15 protein the cytidine deaminase cem15 induces hypermutation in newly synthesized hiv-1 dna hpv16 e5 protein disrupts the c-cbl-egfr interaction and egfr ubiquitination in human foreskin keratinocytes key: cord-319563-9lwr6k8p authors: aitekenov, sultan; gaipov, abduzhappar; bukasov, rostislav title: review: detection and quantification of proteins in human urine date: 2020-10-14 journal: talanta doi: 10.1016/j.talanta.2020.121718 sha: doc_id: 319563 cord_uid: 9lwr6k8p extensive medical research showed that patients, with high protein concentration in urine, have various kinds of kidney diseases, referred to as proteinuria. urinary protein biomarkers are useful for diagnosis of many health conditions – kidney and cardio vascular diseases, cancers, diabetes, infections. this review focuses on the instrumental quantification (electrophoresis, chromatography, immunoassays, mass spectrometry, fluorescence spectroscopy, the infrared spectroscopy, and raman spectroscopy) of proteins (the most of all albumin) in human urine matrix. different techniques provide unique information on what constituents of the urine are. due to complex nature of urine, a separation step by electrophoresis or chromatography are often used for proteomics study of urine. mass spectrometry is a powerful tool for the discovery and the analysis of biomarkers in urine, however, costs of the analysis are high, especially for quantitative analysis. immunoassays, which often come with fluorescence detection, are major qualitative and quantitative tools in clinical analysis. while infrared and raman spectroscopies do not give extensive information about urine, they could become important tools for the routine clinical diagnostics of kidney problems, due to rapidness and low-cost. thus, it is important to review all the applicable techniques and methods related to urine analysis. in this review, a brief overview of each technique’s principle is introduced. where applicable, research papers about protein determination in urine are summarized with the main figures of merits, such as the limit of detection, the detectable range, recovery and accuracy, when available. urine is a readily available liquid for the medical diagnosis of patients. urine tests are noninvasive procedures that involve no pain or discomfort for patients to determine problems with kidney function since extensive medical research showed that patients, with high protein concentration in urine, have various kinds of illnesses of the kidney, referred to as proteinuria [1] . thus the precise and simple determination of urinary concentrations of total protein is important for diagnostic purposes. since urine is a complex matrix, it poses challenges for the analytical determination of proteins and other constituents. among the reasons that make urine challenging for researchers to analyze are: urine matrix is complex, it consists of various inorganic and organic compounds, from low-molar mass molecules to polymers; urine could contain cells, such as blood cells, or bacteria, which changes the composition of urine in time rapidly; an analytical method for diagnosis of proteinuria should cover protein presence in urine in a wide range from 0.01 mg/ml to 10 mg/ml. also, it is important to note that the concentration of protein in urine taken from patients can vary widely depending on dieting, exercising, and time of the day a patient urinated. it is widely accepted that analysis of urea taken throughout a day is the best representation of any illnesses in the human body if any. the so-calledthe urine 24-hour volume test, that measures the amount of urine the human body produces in a day. another option is to determine creatinine to protein ratio in urea since creatinine to protein ratio is positively correlated with 24-hour volume test for quantifying proteinuria [2, 3] . the noninvasive collection of samples and wide range of diagnostic targets found in urine makes urinalysis well suited for point-of-care (poc) monitoring applications, in which testing is done at the time and place of patient care [4] . table 1 shows constituents in urine taken from the urine 24-hour volume test. table 1 . composition of urine (averages of selected components in 24-hour collection test) [5] . average weight (mg) table 1 . composition of urine (averages of selected components in 24-hour collection test) [5] . albumin 90.0 in healthy individuals, tamm -horsfall protein (also known as uromodulin) is the most abundant protein in urine (50%), followed by albumin (20%) and immunoglobulin (5%) [6] . other constituents in the urine of either sick or healthy patients include, but not limited to: glucose; low abundant proteins, such as bilirubin, and urobilinogen; cells, such as erythrocyte, leukocyte, and other cells; bacteria [7] . the urine of a healthy individual contains up to 150 mg of protein in total measured throughout a day, of which approximately 20 mg is albumin (human serum albumin) [1] . albumin excretion of 30 to 300 mg a day, which is called microalbuminuria, is an early and sensitive marker of diabetic nephropathy [8] , cardiovascular and renal disease [9] . the 15-30 mg/l albumin concentration is a critical value that could indicate kidney problems when it is repeatedly exceeded [10] . though one of the main proteins in urine is albumin, there are thousands of other types of proteins. furthermore, the removal of albumin from the urine helps to identify the low abundant proteins [11] . marimuthu et al. by using high-resolution fourier transform mass spectrometry were able to identify 1823 proteins in the urine of healthy subjects [12] . using mass spectroscopy and sub-fractionating normal urine by successive steps (vesicle separation, cpll, and solvent treatments) santucci et al. were able to identify 3429 individual proteins [13] . all those discovered proteins could be potential biomarkers for diseases. in a review paper by röthlisberger et al. it was stated that besides albumin, other proteins such as cd14, hh-fabp, bnp / nt-probnp, ngal, orm1 are potential biomarkers of cardiovascular disease [14] . another biomarker in human urine, bence jones protein (bjp), has an important diagnosis and prognosis value for multiple myeloma, cancer formed in a white blood cell called plasma cell [15] . other types of chemical substances in urine can be important biomarkers as well, for example, urine micrornas have the potential to be a valid marker for bladder cancer detection [16] . proteinuria is the main clinical presentation of glomerular diseases (the glomerulus are complex capillary set that are located in the nephronsrenal cells) [17] . the glomerular diseases can be primarily when the disease caused by kidney diseases (such as glomerulonephritis), or secondary, when kidney glomerulus becomes target organ affected by different diseases such as diabetes and cardiovascular diseases, autoimmune and inflammatory disorders, amyloidosis and neoplasms, cancer j o u r n a l p r e -p r o o f and many other including genetic disorders [17, 18] . the levels of proteinuria, which usually measures in clinical settings, may vary depending on severity of disease and glomerular injury, despite any cause of disease. any detectable proteinuria in the urine between 30 -300 mg/24h called albuminuria (or microalbuminuria), above the 300 mg/24h -proteinuria. depending the amount of proteinuria they considered to named nephritic range proteinuria below 3.5 g/24h and nephrotic range proteinuria when protein loss excess the 3.5 g/24h (heavy proteinuria). the most of the cases, proteinuria caused by cardiovascular diseases limited with nephritic range proteinuria, whereas in renal diseases and cancer the proteinuria may reach nephrotic range proteinuria [19, 20] . since urine is in direct contact with kidneys, analysis of urine is important for diagnosis of renal diseases, such as chronic kidney disease. several viral infections such as epstein-barr virus, hepatitis b and c viruses, herpes zoster, hantavirus, human immunodeficiency virus, dengue fever, covid-19 and many others also may lead to proteinuria [21, 22] . the potential mechanisms of proteinuria are related to primary affect to glomerulus and/or secondary to autoimmune response to infection [23, 24] . chronic kidney disease (ckd) is a major public health problem. albuminuria, urinary sediment abnormality and other markers of kidney damage are criteria of ckd, according to international guidelines [25] . fassett et al. summarized biomarkers for ckd in urinecystatin c, β-trace protein, ngal, kim-1, nag and many others [26] . a more recent article on this topic is discussed in [27] . in the united states, it was estimated in 2003 that 11% of the adult population (aged 20 or older) has chronic kidney disease [28] . in a review paper about the prevalence of chronic kidney disease (ckd) by zhang et al., the authors studied relevant data across america, europe, asia, australia [29] . they reported that the median prevalence of ckd was 7.2% in persons aged 30 years or older. in persons aged 64 years or older prevalence of ckd varied from 23.4% to 35.8%. the authors concluded that worldwide, ckd is becoming a common disease in the general population. in a more recent paper, wei [45] . they concluded that it is important to perform an investigation using non-invasive approaches, such as urine can provide, for the diagnostic of emerging viral diseases. urinalysis is an abbreviation for clinical urine tests for diagnostic purposes. a urinalysis (ua) is one of the most common methods of medical diagnosis. there are three basic components to urinalysis [46] : • gross/physical examination targets parameters that can be measured or quantified with the naked eye (or other senses), including volume, color, transparency, odor, and specific gravity. • microscopic examination. the numbers and types of cells and/or material such as urinary casts can yield a great detail of information and may suggest a specific diagnosis. • chemical examination of urine measures quantitatively and qualitatively for ph, blood, nitrite, protein, glucose, ketones, bilirubin, urobilinogen, ascorbic acid. conditions for storage of urine samples is an important consideration for chemical analysis, since undesired changes in unpreserved urine occur, such as a decrease of concentration of glucose due to consumption of it by cells or bacteria; a decrease of concentration of bilirubin due to photo-oxidation and etc [47] . the most common form of preservation of urine samples is refrigeration, also chemicals j o u r n a l p r e -p r o o f can be utilized too. remer [55] . to date, different diagnostics methods in clinical settings can be used to ascertain urinary protein and albumin. urine dipstick test, technique with acidic buffer precipitation and immunoelectrophoresis are commonly used diagnostic methods for urinary total protein quantification [56] . the protein-specific dipstick and immunochemical techniques are the fastest and cheapest way to determine proteins in the urine for diagnostic purposes. modern automated urine analyzers are based on similar principles and are widely used in clinical routine tests [57] . a urine analyzer is a device used in the clinical setting to perform automatic urine testing. the units can detect and quantify a number of analytes including bilirubin, protein, glucose, and red blood cells. many models contain urine strip readers, a type of reflectance photometer that can process several hundred strips per hour. some analyzers can perform up to 240 tests an hour. prices of urine analysis are relatively low, but they j o u r n a l p r e -p r o o f depend on a location/country and they are available from the most clinical laboratories the highperformance liquid chromatography mostly used for scientific purpose due to its relatively high cost , though relatively inexpensive versions of mass spectrometers are now available [58] . prices of urine analysis are relatively low, but they depend on a location/country and they are available from the most clinical laboratories the high-performance liquid chromatography mostly used for scientific purpose due to its relatively high cost , though relatively inexpensive versions of mass spectrometers are now available [58] . several review papers are focused on urine proteomics. a review by albalat et al. focuses on urinary proteins as potential biomarkers for mainly urine diseases, and capillary electrophoresis coupled mass spectroscopy as an instrumental method [33] . the review also gives information on biomarkers on non-renal diseases obtained from the urine. kalantari et al. state that mass spectrometry as a detection technique is the most common [59] . the paper discusses technical aspects of urinary proteomics, proteomic technologies, and their advantage and disadvantages. as of 2015, several recent experiments are presented there which applied urinary proteome for biomarker discovery in renal diseases including diabetic nephropathy, immunoglobulin a (iga) nephropathy, focal segmental glomerulosclerosis, lupus nephritis, membranous nephropathy, and acute kidney injury. decramer et al. in their review paper focus on mass spectrometry-based urinary protein and peptide profiling [60] . the advantages and disadvantages of different mass spectroscopy methods are compared. applications of urinary proteome analysis to urogenital and non-urogenital diseases are also discussed there. this review, besides covering mass spectrometry, holds chapters discussing fluorescence spectroscopy, immunoassay, infrared, and raman spectroscopy that was not given much attention compared from the reviews on urinalysis mentioned in the previous paragraph. this review also covers separation techniqueselectrophoresis and chromatography. for each technique, a brief overview of the technique's principle is introduced. where applicable, research papers about protein determination in urine are summarized with the main figures of merits, such as the limit of detection, the detectable range, recovery, and accuracy. particularly, electrophoresis, chromatography, immunoassay, and fluorescence spectroscopy chapters are followed by summary tables with analytical parameters taken from individual research papers. it should be noted that not all analytical parameters are given in every paper, for example, sometimes recovery is unknown, and thus is not included in the following tables. since the volumes used for all techniques are 100 to 1000 times smaller than a typical urine probe available from one patient for analysis, usually 50-100 ml, urine volume is not introduced in the tables. for instance for proteomics study, a typical sample size for high performance liquid chromatography (hplc) ranges from few microliters to 0.1 ml [61] . a commercial immunoassay test kits developed by valle et al. require 10 μl of urine [62] . this review ends with a paragraph about urinary protein biomarkers. most of those biomarkers were identified by mass spectrometry or immunoassay methods. a summary table of experimental papers on urinary protein biomarkers with related diseases or health conditions is given. electrophoresis is an electrokinetic process, with a long history of development, which separates charged particles (macro-molecules, such as proteins) in a fluid using a field of electrical charge [63] . particles could be separated based on their charge or their mass. the electrophoretic method is a widely used method in a modern research laboratory, but not so much in a clinical laboratory. electrophoresis is commonly used for proteins, peptides and nucleic acid analysis [64] . it could be applied as a qualitative or quantitative analytical technique on its own if a reference substance is given, or it could be used as a separation technique for a sample to be further examined by other techniques, such as mass spectroscopy, fluorescence spectroscopy and etc. aguzzie et al. successfully used the electrophoretic technique to detect bence-jones protein (bjp) in human urine with the limit of detection lod of 1 mg/l to bjp, and lod of 3 mg/l to other types of protein [65] . these results were obtained by immunofixation using dako antisera on cellulose nitrate followed by further staining with gold. the authors claim that their technique does not require particular skill and is cheap. such low levels of lod allowed them to use unconcentrated urine samples, thereby eliminating the problem of the loss of low molecular weight proteins. usually, though it is often necessary to concentrate the urine before electrophoresis because of the low urinary protein level [6] . this is a time-consuming process. machii et al. used cellulose acetate membrane electrophoresis followed by colloidal silver staining on the urine of healthy subjects with success [6] . however, when they used silver staining on an agarose gel, urinary protein in healthy subjects was not detected. they estimated that urine has to be concentrated 10 times, furthermore, the pattern of the urinary fraction will change after the concentration procedure is conducted. giovannoli et al. developed a noncompetitive capillary electrophoresis immunoassay with laser-induced fluorescence detection was used to determine human serum albumin (hsa) in buffer and urine [66] . the injection of an excess of labeled antibody off-line incubated with the analyte allows the surface capture of the free antibody and consequently the immunocomplex detection. in buffer, authors were able to achieve the limit of detection lod of 0.60 mg/l, with the quantitative range up to 6.6 mg/l. recovery in the urine matrix was dependent upon sample dilution and hsa added and was as low as 91±8%. one of the variations of the electrophoretic method is called gel electrophoresis, where a gel is used as an anticonvective medium. particularly polyacrylamide gel page is used for separating [69] . the use of these preconcentration techniques makes it possible to increase sensitivity in the determination of analytes by a factor of 100 or higher. in another work by bessonova et al., they found that the lowest lod was obtained by the lvss approach, and it was 15 mg/l with uv-detection [70] . [68] . a gel column is utilized to achieve electrophoretic separation of proteins, analogous to sds-page, which are then eluted into the liquid-phase for manual collection. the fractionation can then be visualized by running a portion of the fractions on a sds-page gel. reprinted from [95] . electrophoresis is often used to be followed by another method, such as mass spectroscopy. another widely used separation technique in chemistry is chromatography. in chromatography, a sample is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. the various constituents of the mixture travel at different speeds, causing them to separate due to differential affinities (strength of adhesion, as an example) of the various components of the analyte towards the stationary and mobile phase results. then resulting constituents can be further analyzed by various techniques, such as mass spectroscopy, uvspectroscopy, fluorescence detection, etc. depending on the mobile phase methods in chromatography could be classified as gas-chromatography (gc) [74] , and liquid-chromatography (lc) [75] . typically, proteomic sample size ranges from few microliters to 0. [76] . particularly, they used 8-anilino-1-naphthalenesulfonic acid as a fluorophore to bind hsa. the sample size was 1 ml for urine, and the retention time was around 20 minutes. a linear relationship was observed between fluorescence peak and the amounts of hsa in both plasma and urine, as for the latter matrix, a linear relationship was observed up to 400 mg/l, with the correlation coefficient being 0.998. the lod for hsa was 0.2 mg/l, while the recovery was 94.5±3.8%. the same authors also compared their method with the immunological nephelometric method and obtained the correlation coefficient of 0.990 [77] . often researchers compare their method with the immunoassay technique that is used in clinical practice. brinkman et al. in their experiments using the immunonephelometric approach and hplc determined that hplc reveals higher values of hsa, especially in the lower concentration range, resulting in a higher prevalence of microalbuminuria [78] . [85] . aqps regulate numerous downstream effector signaling molecules that promote cancer development and progression [86] . in numerous cancer types, aqp expression has shown a correlation with tumor stage and prognosis. linearity is observed within the concentration range 0.5·10 -3 -50·10 -3 mg/l, intra and inter-assay precision ranged from 9 to 35% at the lower limit of quantification (lloq), and accuracy from 94 to 114%. overall, due to complex composition of biofluids, including the urine, liquid chromatog-raphy in particular hplc, which is frequently coupled with ms for detection, remains a common method in bioanalytical laboratories to separate complex mixtures. a review by novakova et al. states that in order to decrease time for an analysis without compromising resolution and separation efficiency, three main approaches in hplc existthe use of monolith columns, lc at high pressures and temperatures, and hplc at ultra-high pressures [87] . by employing those approaches, time for analysis of biofluids could be done within few minutes up to half an hourhe table 2 below shows a summary table of quantitative methods for application of electrophoresis and chromatography for protein determination in the human urine matrix. table 2 . summary table of quantitative methods for application of electrophoresis and chromatography for protein determination in the human urine matrix (unless otherwise stated in the method section). cv (coefficient of variation) parameters are taken from maximum readings, for recovery -the range is given. j o u r n a l p r e -p r o o f mass spectrometry is a powerful analytical technique that measures the mass-to-charge ratio of ions. firstly, molecules in a sample must be ionized, which causes a large molecule to fragment into smaller ions; then those ions are separated by a magnetic field depending on their mass to charge ratio. the results are typically presented as a mass spectrum, a plot of intensity as a function of the mass-tocharge ratio. usually, mass spectrometry is utilized after pre-treatment of a sample by electrophoresis or chromatography. mass spectrometry is a widely used technique for proteomics studies. the proteome is the entire set of proteins that is produced or modified by an organism. research on urine proteomics is important for the identification purposes of reliable biomarkers that help the diagnosis of disease [90] . in a review by kalantari et al. on urine proteomics is stated that common techniques for urinary proteome analysis are two-dimensional gel electrophoresis followed by mass spectrometry (2de-ms), liquid chromatography coupled to mass spectrometry (lc-ms), surface-enhanced laser desorption/ionization coupled to mass spectrometry (seldi-tof), and capillary electrophoresis coupled to mass spectrometry (ce-ms) [59] . by using mass spectroscopy protein constituents of urine were identified. also reviews by albalat et al. and decramer et al. focus on mass spectrometry-based urinary protein and peptide profiling [33, 60] . the advantages and disadvantages of different mass spectrometry methods are compared. as was mentioned in the introductory part of this review, marimuthu et al. by using high-resolution fourier transform mass spectrometry were able to identify 1823 proteins in the urine of healthy subjects [12] . santuccie et al. by using mass spectrometry and sub-fractionating normal urine by successive steps (vesicle separation, cpll, and solvent treatments) were able to identify 3429 individual proteins [13] . two ionization techniques in mass spectrometry are commonly used for protein studies: electrospray ionization (esi) and matrix-assisted laser desorption/ionization (maldi) [91] . in the esi approach, ions are produced in an electrospray in which a high voltage is applied to a liquid to create an aerosol. esi is different from other ionization processes (e.g. maldi) since it may produce multiplecharged ions, effectively extending the mass range of the analyzer to accommodate the kda-mda orders of the magnitude observed in proteins and their associated polypeptide fragments [92] . in maldi, a sample is dispersed in a certain solid matrix (e.g. aromatic compounds), which then irradiated by the laser that causes electronic excitation of molecules in the sample matrix [93] . both j o u r n a l p r e -p r o o f methods esi and maldi produce relatively high molecular ions due to mild fragmentation of those approaches. it should be noted a specific method of ms applied to biomolecules, which is called tandem mass spectrometry, also known as ms/ms. in ms/ms two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyze samples. in general, protein identification in mass spectroscopy is performed using either bottom-up or top-down approaches. bottom-up protein identification methods rely on enzymatic digestion to break down proteins into smaller peptides that are easier to ionize and fragment, resulting in higher sequence coverage [94] . however, in top-down methods, intact proteins are injected into the mass spectrometer and subjected to fragmentation without pre-treatment [95] . aside from better tracking of protein modifications, an advantage of top-down protein sequencing is that it complements imaging experiments by enabling mass measurement of the intact protein that relates more directly to the maldi ims generated signals [96] . analysis of a mass-spectrum, a plot of intensity as a function of the mass-to-charge ratio, could be a challenge itself, especially for big molecules like proteins, since they can be divided in many fragments resulting in a complex spectrum. usually, the mass-spectrum is compared by software with the existing database. several computationally feasible approaches have emerged for performing protein inference from such data [97] . recovery was between 97% and 108% with an average of 103%. in conclusion, quantitative mass spectroscopy assays for peptides and proteins are difficult to implement, and the future will see standardization of ms validation and quality control requirements. overall, mass spectrometry coupled with liquid chromatography is a powerful tool for the discovery and the analysis of biomarkers of diseases from biofluid samples, which includes proteomics study of urine as well [104] . many of urinary proteome biomarkers were discovered by this technique. however, quantitative protein determination represents a challenge by following this approach [105] . thus, mass spectroscopy could be used for biomarker discovery but not for clinical applications [60] . another consideration for an analytical technique is the cost of analysis. practical usage of lc-ms is limited by factors such as instrument size, cost, ease of operation, though, low cost compact mass spectroscopy detectors coupled with cinematographic separation are being developed, their costs equal to around $50000 [58] . immunoassay is a widely used technique in clinical and research laboratories to determine qualitatively and quantitatively macromolecules (usually proteins) generated from organisms [106] . immunoassay is based on a reaction between an antigen and an antibody, which is found in the adaptive immune system of vertebrates, that includes humans [107] . the reaction is carried out in-vitro (not in a living organism), and the result of it is the formation of a complex antibody-antigen formed by spatial complementary, like lock and key, thus such interactions are highly selective. antibody-antigen complexes are bounded by weak forces such as electrostatic forces, hydrogen bonds, hydrophobic interactions, and van der waals forces; which means that such bindings are reversible. the specific piece of the antigen to which an antibody binds is called the epitope. usually, in analytical papers, the analyte is the antigen while the reagent is the antibody. immunoassay methods have been widely used in many areas of pharmaceutical analysis such as diagnosis of diseases, therapeutic drug monitoring, clinical pharmacokinetics, and bioequivalence studies in drug discovery and pharmaceutical industries j o u r n a l p r e -p r o o f [108] . conventional immuno-chemical based urinary albumin assays for diabetic patients as well as nondiabetic patients with renal disease, including those who may suffer hypertension and cardiovascular disease, now amount to 100 million assays per year worldwide as of 2004 [79] . the widespread immunoassay methods in the pharmaceutical analysis is attributed to their inherent specificity, high-throughput, and high sensitivity for the analysis of a wide range of analytes in biological samples as well the relatively low cost of the instruments, tools, or the reagents [109] . immunoassay experiments could be classified into types -competitive and non-competitive [106, 109] . [109] . based on signal detection immunoassays could be classified as follows: • radioimmunoassay ria. detection is based on radioactive labeling of antigen with unstable isotopes such as i-125, c-14, h-3. • enzyme immunoassay eia with sub-categories: elisa -enzyme-linked immunosorbent assay; emit -enzyme-multiplied immunoassay technique; meia -microparticle enzyme immunoassay. • fluoroimmunoassay fia • chemiluminescence immunoassay clia • immunonephelometry in. detection is based on an antigen-antibody complex scattering light. • immunoturbidimetry it. detection is based on an antigen-antibody complex blocking light thus increasing the turbidity of the sample. as it was mentioned in the introductory part, the concentration of albumin higher than 2 mg/l could be indicative of potential kidney diseases [10] . subsequently for many immunoassays methods for albumin determination in urine the limit of detection (lod) of magnitude around 1 mg/l would be enough for clinical purposes, thus priority should be given to the parameters such as specificity, accuracy, costs, etc. however for low abundant proteins achieving lower lod is important [110] . in table 3 . table 3 . summary table of quantitative methods for application of immunoassay for protein determination in the human urine matrix (unless otherwise stated in the method section). cv (coefficient of variation ) parameters are taken from maximum readings, for recovery -the range is given. j o u r n a l p r e -p r o o f fluorescence is a luminescence phenomenon in which a compound emits light after absorption of electromagnetic irradiation [133] . in most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. often in chemical analysis, a sample is irradiated in uv or visible range, and as a result, fluorescence occurs in the visible and near-infrared region respectively. fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after. fluorescence spectroscopy is one of the most common techniques with applications in geology, chemistry, medicine, and astronomy [133] . because of the sensitivity that the method affords, fluorescent molecule concentrations as low as 1 part per trillion can be measured [134] . fluorescence spectroscopy can be used on its own, and also it is often used in conjunction with high-performance liquid chromatography as a detector. often fluorescence detection is used in immunoassays to either label antigen or antibody. usually, analyte molecules do not emit strong luminescence for their analytical detection. hence fluorescence quenching aptasensor [132] . when albumin was added to the complex go with the fluorescence-labeled aptamer, the aptamer detached from the complex to bind albumin, which resulted by an increase in fluorescence intensity, as shown in figure 4 . the limit of detection was 0.05 mg/l and the detection range is 0.1-14.0 mg/l. other quantitative works using the fluorescence spectroscopy are summarized in table 4 . j o u r n a l p r e -p r o o f figure 4 : schematic of graphene oxide-mediated fluorescence quenching aptasensor for the detection of albuminuria in urine and hsa in human serum. when albumin was added to the complex go with the fluorescence-labeled aptamer, the aptamer detached from the complex to bind albumin, which resulted by an increase in fluorescence intensity. reprinted from [132] . table 4 . summary table of quantitative methods for application of fluorescence spectroscopy for protein determination in the human urine matrix (unless otherwise stated in the method section). cv(coefficient of variation) parameters are taken from maximum readings, for recovery -the range is given. j o u r n a l p r e -p r o o f the absorption of infrared radiation ir excites vibrational transitions of molecules [148] . the infrared spectral region covers wavelengths from 780 nm to 1000 μm which can be further subdivided into the near-infrared region from 780 nm to 2500 nm, the mid-infrared region from 2500 nm to 50 μm and the far-infrared region from 50 μm to 1000 μm. since vibrational frequency and probability of absorption depend on the strength and polarity of the vibrating bonds, they are influenced by intra-and intermolecular effects [149] . the approximate position of an infrared absorption band is determined by the vibrating masses and the type of bond (single, double, triple), the exact position by electronwithdrawing or donating effects of the intra-and intermolecular environment and by coupling with other vibrations. information that can be derived from the infrared spectrum: • chemical structure of the vibrating group • chemical properties of neighbouring groups in a molecule • redox state • bond parameters • hydrogen bonding • electric fields • conformational freedom basically, all polar bonds contribute to the infrared absorption so there is no need to specifically label biomolecules to detect them. however, the infrared spectrum of biomolecules is challenging to analyze since they are complex with many overlapping bands. certain mathematical and statistical procedures have to be utilized to analyze the resulting spectrum. the ir spectroscopy is often used to identify organic structures because functional groups give rise to characteristic bands both in terms of intensity and position (frequency). the ir spectra of organic molecules are often interpreted as having two regions: functional group region (>1500 cm -1 =6.7 μm), and fingerprint region (<1500 cm -1 ). in the fingerprint region, there are many troughs that form an intricate pattern that can be used as a fingerprint to determine the compound. modern infrared spectrometers are usually fourier transform infrared (ftir) spectrometers [149] . the ftir spectrometer works differently than the dispersive spectrometer where a monochromatic light beam is absorbed by a sample. this technique shines a beam containing many frequencies of light at once and measures how much of that beam is absorbed by the sample. next, the beam is modified to contain a different combination of frequencies, giving a second data point. this process is rapidly repeated many times over a short timespan. afterward, a computer takes another analytical technique that relies on molecular vibrations is raman spectroscopy [150] . raman spectroscopy is a powerful technique to determine vibrational modes of molecules to provide a structural fingerprint, like infrared spectroscopy, by which molecules can be identified. infrared raman spectra for biomolecules is hard to analyze due to overlapping bands, the same issue as in the ir spectroscopy. certain mathematical procedures must be employed on raman spectra to analyze a sample. despite difficulties in the analysis of large biomolecules such as proteins, the ir, and raman spectroscopy make their advances in this regard. for the protein analysis with ir, the mir region plays an important role since it includes bands that predominantly arise from three conformationally sensitive vibrations arising from the peptide backbone-namely, amide i, amide ii, and amide iii [151] . amide group vibrations of the backbone have attracted much attention in protein ir spectroscopy, as they are native to all proteins and inform on secondary conformation and solvation. such bands include the amide i region, between 1600 and 1700 cm-1, primarily resulting from c=o stretching vibration, the amide ii band, related to cn stretch and nh in-plane bending, the amide iii vibration, associated with cn stretching, nh bending, and co in-plane bending, and, finally, the amide a (nh stretch) band. absorption spectroscopy (seiras) [152] , and surface-enhanced raman spectroscopy sers [153] [154] [155] improve sensitivity to overcome this problem. both seiras and sers utilize the surface plasmon effect from the interaction of light with metallic nanoparticles. sers has an enhancement factor (of the order of 10 6 -10 12 ) compared to the traditional raman signal [156] . for seira, the surface-enhancement is comparatively modest ( 10 1 -10 3 ) [157] . although the enhancement factor of seiras is smaller than that of sers, the cross-section for ir absorption is several orders of magnitude higher than the corresponding raman cross-section. despite the modest enhancement factor, the seiras technique may be sufficient for many applications [152] . [160] . pezanniti et al. used a polynomial based spectral smoothing method to the urine matrix [161] . in general, all the mentioned works requires no specific reagents, they are fast and are able to quantify multiple analytes with one spectrum. premasiri et al. investigated urine by raman spectroscopy [5] . shown in figure 5 [164] . the authors used the set of mathematical procedures on spectra which included spectral processing (e.g., truncation, baselining, and vector normalization); principal component analysis (pca); statistical analyses (anova and pairwise comparisons); discriminant analysis of principal components (dapc); and testing dapc models using a leave-one-out build/test validation procedure. their approach was able to identify "unknown" urine specimens as from pd patients or healthy human volunteers with better than 96% accuracy (with better than 97% sensitivity and 94% specificity). 1. for albumin -peaks at 1208 cm −1 and 1370 cm −1 are attributed to so 2 symmetric stretch and ch bond deformation of albumin. 2. for creatinine -the raman signature peaks are observed at 888 cm −1 , 958 cm -1 , and 1444 cm-1 which could be due to c=o stretching, c-c stretching and ch3 anisometric deformation respectively. 3. for urea -a very strong raman peak at 1018 cm −1 which corresponds to c-n stretching mode. throughout is important consideration for laboratory tests. zhu et al. performed quantitative sers detection of creatinine in urine, and then compared with a clinically validated enzymatic "creatinine kit" [168] . they wrote that sers detection process could be completed within 2 min compared with 11 min for the creatinine kit, indicating the practicality of the quantitative sers technique as high-throughput platform for relevant clinical and forensic analysis. human urine contains thousands of individual proteins [12, 13] . excessive presence of some of them are associated with certain diseases related not only to renal diseases, but also to cancer, diabetes, and infections. those molecules that are associated with a certain biological condition are called biomarkers. most of those biomarkers in human urine were identified by comparing patients with healthy individuals by the means of chromatography or electrophoretic separation followed by mass spectroscopy, or by the immunoassay. literature review of urinary protein biomarkers with related diseases is summarized in table 5 , corresponding diagnostic sensitivity and diagnostic specificity are given [169] . some of the listed research papers used western blot as a technique [170] . due to the usage of antibodies, this technique was counted as the immunoassay in table 5 . most of the methods mentioned followed established procedures. to get a general idea of those experimental methods, refer to the previous paragraphs about separation and detection techniques, and tables 2-4 . below are some observations and conclusions regarding table 5 about the content of the listed research papers: • urinary protein analysis are useful for not only renal diseases. urine proteomics can be used for detection of at least 15 conditions, including 8 kinds of cancer; 5 kinds of renal complications; and at least 3 different infections. the list of health complications that can be diagnosed from j o u r n a l p r e -p r o o f urine analysis is bigger if non-protein substances are included, such as peptides, nucleic acids, low molar mass metabolites, and etc; that can be found in reviews [14, 26, 27, 36] . • new potential urinary protein biomarkers are found primarily by mass spectrometry nowadays. however, further extensive validation on clinically representative population is required. often it is achieved through the immunoassay, if the appropriate method is developed with a sufficient analytical sensitivity, since the immunoassay methods are expressive, low-cost and quantitative compared to the chromatography coupled with mass spectrometry. • in total 34 references are included in table 5 ; 12 of them utilized mass spectrometry , 24 utilized the immunoassay, and 1 paper -fluorescence spectroscopy. • authors of these research papers utilized the mass spectrometry methods with further validation by the immunoassay [171] [172] [173] [174] . • all of the mentioned work (except [129] ) in the immunoassay followed manufacturer's protocol, or if a custom immunoassay was used, other established procedure from their own or previous works. • elisa-based immunoassay was the most popular method accounting for 13 references. • diagnostic sensitivity and specificity increased if a panel of several biomarkers was considered simultaneously. though, the panel should be carefully chosen as not all of the components provide increase in diagnostic sensitivity and specificity. urine is a readily available liquid for the medical diagnosis of patients. urine tests are noninvasive procedures that involve no pain or discomfort for patients to determine problems with kidney function since extensive medical research showed that patients, with high protein concentration in urine, have various kinds of illnesses of the kidney, referred to as proteinuria [1] . thus the precise and simple determination of urinary concentrations of total protein is important for diagnostic purposes. in this review, we focused on instrumental determination of abundant proteins in urine by electrophoresis, chromatography, mass spectrometry, immunoassay, fluorescence, ir, and raman spectroscopy. the mentioned techniques are not mutually exclusive, for example, high performance liquid chromatography is usually a separation step followed by mass spectrometry, another example is labelling with fluorophores in immunoassay. each technique has its own strength to analyze urine for specific information, hence, it is hard to compare them directly, and to find a common denominator for comparison. overall, number of cited experimental papers that were covered by this review in tables 2-5 : electrophoresis and chromatography -22 in total and 8 related to hsa detection; mass spectrometry -17 in total and 5 related to hsa; immunoassay -47 in total and 17 related to hsa; fluorescence spectroscopy -22 in total and 19 related to hsa; ir and raman spectroscopy -10 in total; all topics -101 papers. thus, almost half of the cited literature by the means of immunoassays, which is in a good correlation that immunoassays are widespread in clinics and science. the limit of detection is important parameter for evaluating an analytical method. in order to develop a method, the limit of detection should take into account which amount of analyte has clinical significance. the 15-30 mg/l albumin concentration is a critical value that could indicate kidney problems when it is repeatedly exceeded [10] . for the corresponding articles discussed above, the limit of detection for electrophoresis, chromatography, mass spectroscopy, immunoassay, fluorescence exceeded 2 mg/l for albumin, which is enough for the clinical purpose. for some methods, explained in [117, 119, 122, 123, 127, 132, 143] , the limit of detection does not exceed 0.1 mg/l for albumin in the urine matrix. for low abundant proteins the limit of detection must be obviously much lower. for example, in the article for aquaporin-2 determination by jaffuel et al. the limit of detection was 5·10 -4 mg/l [85] . in another work by zhao et al., the lod for nuclear matrix protein 22 was 1.7·10 -6 mg/l [110] . for the ir and raman spectroscopy methods the lod might not be so relevant factor. the ir and raman methods is better characterized by how accurate a particular method can distinguish whether a spectra belongs to a healthy subject or not, as evidenced from works [158, 164] . j o u r n a l p r e -p r o o f electrophoresis and chromatography are powerful separation techniques which are accompanied by further detection, usually uv-vis spectrometry or fluorescence. since the human urine matrix is very complex consisting of inorganic and organic compounds, those techniques are useful for urine analysis [7] . though certain methods might require preconcentration techniques to enhance sensitivity [69] . high-performance liquid chromatography with mass spectroscopy detection is a particularly powerful tool for proteomics studies, as it was able to identify 1823 proteins in urine [12] . mass spectroscopy in itself does not allow for quantitative analysis due to the different physicochemical properties of different peptides and proteins [98] [99] [100] . however quantification in ms is possible if an internal standard is used, for such there are approaches based on labeling with stable isotopes (such as n-15, o-18), involving artificial labeling of peptides and proteins, as in work by singh et al., specifically in the urine matrix [81] . the immunoassay is widely used technique both in clinical practice and in research papers for urine analysis. though different immunoassay methods can overestimate or underestimate an analyte concentration [79] . they can hurt patients due to inaccurate results [112] . the ir and raman spectroscopy have advantage of easy preparation of samples to analyze. they require no specific reagents, or no reagents at all, no preconcentration or dilution, as evidenced from [158, 162, 165] . they are rapid. on the other hand, due to overlapping bands of large biomolecules, and the complexity of urine components -hard to analyze their spectra that require mathematical analysis. overall, there is no "silver bullet" modern quantification method for the analysis of proteins in urine that has been reported and validated so far. however, at least for the routine clinical diagnostics of kidney problems, applications of relatively direct methods (such as sers or ir) spectroscopy may expand as instrumentation, substrates (at least sers substrates), software and computing power for data analysis (peak deconvolution) become progressively less expensive. j o u r n a l p r e -p r o o f [68] . a gel column is utilized to achieve electrophoretic separation of proteins, analogous to sds-page, which are then eluted into the liquid-phase for manual collection. the fractionation can then be visualized by running a portion of the fractions on a sds-page gel. reprinted from [95] . primary care approach to proteinuria correlation of urine protein/creatinine ratios to 24-h urinary protein for quantitating proteinuria in children diagnostic utility of protein to creatinine ratio (p/c ratio) in spot urine sample within routine clinical practice review-point-of-care urinalysis with emerging sensing and imaging technologies urine analysis by laser raman spectroscopy urinary protein fraction in healthy subjects using cellulose acetate membrane electrophoresis followed by colloidal silver staining urinalysis: a comprehensive review microalbuminuria as a predictor of clinical nephropathy in insulin-dependent diabetes mellitus update on microalbuminuria as a biomarker in renal and cardiovascular disease an overview of urinary proteomics applications in human diseases a comprehensive map of the human urinary proteome from hundreds to thousands: widening the normal human urinome (1) urine protein biomarkers for detection of cardiovascular disease and their use for the clinic history of multiple myeloma urine mirna as a potential biomarker for bladder cancer detection -a meta-analysis proteinuria in adults: a diagnostic approach proteinuria: potential causes and approach to evaluation classification of renal proteinuria: a simple algorithm pathophysiology of proteinuria proteinuria in children infected with the human immunodeficiency virus proteinuria in children: evaluation and differential diagnosis understanding the mechanisms of proteinuria: therapeutic implications chronic kidney disease blood and urine biomarkers in chronic kidney disease: an update prevalence of chronic kidney disease and decreased kidney function in the adult us population: third national health and nutrition examination survey prevalence of chronic kidney disease in population-based studies: systematic review prevalence of kidney damage in chinese elderly: a large-scale population-based study chapter 3: morbidity and mortality in patients with ckd global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the global burden of disease study urine proteomics in clinical applications: technologies, principal considerations and clinical implementation discovery and validation of new protein biomarkers for urothelial cancer: a prospective analysis virus bioresistor (vbr) for detection of bladder cancer marker dj-1 in urine at 10 pm in one minute update on urine as a biomarker in cancer: a necessary review of an old story urinary proteomic biomarkers in coronary artery disease urinary proteomics in diabetes and ckd the potential of albuminuria as a biomarker of diabetic complications an immunoassay cassette with a handheld reader for hiv urine testing in point-of-care diagnostics analysis of viruses present in urine from patients with interstitial cystitis development of universal and lineage-specific primer sets for rapid detection of the zika virus (zikv) in blood and urine samples using one-step reverse transcription loop-mediated isothermal amplification (rt-lamp) quantitative detection of hepatitis c virus rna in urine of patients with chronic hepatitis c using a novel real-time pcr assay detection and phylogenetic analysis of human papilloma virus in urine from a sample of iraqi women with vaginal discharge find the right sample: a study on the versatility of saliva and urine samples for the diagnosis of emerging viruses urinalysis: a review of methods and procedures fundamentals of urine and body fluid analysis long-term urine biobanking: storage stability of clinical chemical parameters under moderate freezing conditions without use of preservatives free light chains nephelometric assay: human urine stability in different storage conditions effect of common storage temperatures and container types on urine protein : creatinine ratios in urine samples of proteinuric dogs comparison of various methods for the determination of total protein in urine protein measurement with the folin phenol reagent a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding measurement of protein using bicinchoninic acid urinary protein as measured with a pyrogallol red-molybdate complex, manually and in a hitachi 726 automated analyzer assessment of proteinuria semiquantitative, fully automated urine test strip analysis the emergence of low-cost compact mass spectrometry detectors for chromatographic analysis human urine proteomics: analytical techniques and clinical applications in renal diseases urine in clinical proteomics automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column an enzyme immunoassay for determining albumin in human urine samples using an ultra-microanalytical system electrophoresis: the march of pennies, the march of dimes recent developments in capillary and microchip electroseparations of peptides high-sensitivity electrophoretic method for the detection of bence jones protein and for the study of proteinuria in unconcentrated urines a novel approach for a non competitive capillary electrophoresis immunoassay with laser-induced fluorescence detection for the determination of human serum albumin urine protein quantification in stacking gel by sds-page gel-eluted liquid fraction entrapment electrophoresis: an electrophoretic method for broad molecular weight range proteome separation preconcentration techniques in capillary electrophoresis electrophoretic determination of albumin in urine using on-line concentration techniques determination of peptides and proteins in human urine with capillary electrophoresis-mass spectrometry, a suitable tool for the establishment of new diagnostic markers mapping and identification of the urine proteome of prostate cancer patients by 2d page/ms cellulose acetate membrane electrophoresis based urinary proteomics for the identification of characteristic proteins: came-based urinary proteomics fast gas chromatography-mass spectrometry: a review of the last decade high-performance liquid chromatography combined with electron ionization mass spectrometry: a review high-performance liquid chromatographic determination of human serum albumin in plasma and urine by post-column fluorescence enhancement detection using 8-anilino-1-naphthalenesulfonic acid manual immunonephelometric assay of proteins, with use of polymer enhancement which method for quantifying urinary albumin excretion gives what outcome? a comparison of immunonephelometry with hplc differences in urinary albumin detected by four immunoassays and high-performance liquid chromatography performance characteristics of an hplc assay for urinary albumin a liquid chromatography-mass spectrometry method for the quantification of urinary albumin using a novel 15n-isotopically labeled albumin internal standard principles of micellar electrokinetic capillary chromatography applied in pharmaceutical analysis optimization and validation of a capillary mekc method for determination of proteins in urine field-amplified sample injection for the determination of albumin and transferrin in human urines by mekc optimization of liquid chromatography-multiple reaction monitoring cubed mass spectrometry assay for protein quantification: application to aquaporin-2 water channel in human urine water transport proteins-aquaporins (aqps) in cancer biology a review of current trends and advances in modern bio-analytical methods: chromatography and sample preparation highly sensitive reversed-phase high-performance liquid chromatography assay for the detection of tamm-horsfall protein in human urine detection of urine protein by a paper-based analytical device enhanced with ion concentration polarization effect, microfluid nanofluid proteomics approach and techniques in identification of reliable biomarkers for diseases interpretation of mass spectra electrospray ionisation mass spectrometry: principles and clinical applications matrix-assisted laser desorption/ionization mass spectrometry of biopolymers top down proteomics: facts and perspectives protein identification strategies in maldi imaging mass spectrometry: a brief review a review of statistical methods for protein identification using tandem mass spectrometry quantitative mass spectrometry-based proteomics: an overview quantitative mass spectrometry: an overview clinical peptide and protein quantification by mass spectrometry (ms) multiplexed quantification of 63 proteins in human urine by multiple reaction monitoring-based mass spectrometry for discovery of potential bladder cancer biomarkers state of the art for measurement of urine albumin: comparison of routine measurement procedures to isotope dilution tandem mass spectrometry a reference system for urinary albumin: current status ultra high performance liquid chromatography as a tool for the discovery and the analysis of biomarkers of diseases: a review practical considerations and current limitations in quantitative mass spectrometry-based proteomics immunoassays: tools for sensitive, specific, and accurate test results vertebrate and invertebrate respiratory proteins, lipoproteins and other body fluid proteins validation of immunoassays for bioanalysis: a pharmaceutical industry perspective immunoassay methods and their applications in pharmaceutical analysis: basic methodology and recent advances a highly sensitive label-free electrochemical immunosensor based on aunps-ptnps-mofs for nuclear matrix protein 22 analysis in urine sample comparison between immunoturbidimetry, size-exclusion chromatography, and lc-ms to quantify urinary albumin the fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry a solid phase fluorescent immunoassay for the measurement of human urinary albumin urinary albumin measurement by immunoturbidimetry laser immunonephelometry for routine quantification of urinary albumin excretion immunoturbidimetry of urinary albumin: prevention of adsorption of albumin a rapid and sensitive method for estimating low concentrations of albumin in human urine time-resolved fluorescence resonance energy transfer assay for point-of-care testing of urinary albumin comparison and development of two different solid phase chemiluminescence elisa for the determination of albumin in urine a combination of magnetic permeability detection with nanometer-scaled superparamagnetic tracer and its application for one-step detection of human urinary albumin in undiluted urine electrochemical immuno-biosensor for the rapid determination of nuclear matrix protein 22 (nmp22) antigen in urine samples by co(iii) phthlocyanine/fe3o4/au collide coimmobilized electrode resonance scattering detection of trace microalbumin using immunonanogold probe as the catalyst of fehling reagent-glucose reaction a quantum dot-based optical immunosensor for human serum albumin detection a homogeneous fluorescent sensor for human serum albumin chemiluminescence lateral flow immunoassay cartridge with integrated amorphous silicon photosensors array for human serum albumin detection in urine samples competitive amperometric immunosensor for determination of p53 protein in urine with carbon nanotubes/gold nanoparticles screen-printed electrodes: a potential rapid and noninvasive screening tool for early diagnosis of urinary tract carcinoma promptmas, determination of albumin in urine by a quartz crystal microbalance label-free assay a dual-label time-resolved fluorescence immunoassay for the simultaneous determination of cystatin c and β2-microglobulin in urine development of a fully automated chemiluminescence immunoassay for urine monomeric laminin-γ2 as a promising diagnostic tool of non-muscle invasive bladder cancer optical microchips based on high-affinity recombinant protein binders-human serum albumin detection in urine electrochemical elisa-based platform for bladder cancer protein biomarker detection in urine sensitive detection of albuminuria by graphene oxide-mediated fluorescence quenching aptasensor experimental methods in chemical engineering: fluorescence emission spectroscopy fluorometric assay using dimeric dyes for double-and single-stranded dna and rna with picogram sensitivity quantum dots versus organic dyes as fluorescent labels quantum dots in bioanalysis: a review of applications across various platforms for fluorescence spectroscopy and imaging determination of human serum albumin using an intramolecular charge transfer fluorescence probe: 4′-dimethylamino-2,5-dihydroxychalcone synchronous fluorescence determination of human serum albumin with methyl blue as a fluorescence probe synchronous fluorescence determination and molecular modeling of 5-aminosalicylic acid (5-asa) interacted with human serum albumin determination of albumin using cds/sio 2 core/shell nanoparticles as fluorescence probes protein determination using methylene blue in a synchronous fluorescence technique spectrofluorimetric determination of human serum albumin using terbium-danofloxacin probe determination of human albumin in serum and urine samples by constant energy synchronous fluorescence method an nir fluorescent probe of uric hsa for renal diseases warning a novel detection method of human serum albumin based on the poly(thymine)-templated copper nanoparticles a novel detection method of human serum albumin based on cuinzns quantum dots-co2+ sensing system a red-nir emissive probe for the selective detection of albumin in urine samples and live cells introduction infrared spectroscopy of proteins applications of raman spectroscopy in agricultural products and food analysis: a review mid-infrared spectroscopy for protein analysis: potential and challenges recent applications of atr ftir spectroscopy and imaging to proteins sers nanoparticles in medicine: from label-free detection to spectroscopic tagging sers: materials, applications, and the future probing single molecules and single nanoparticles by surface-enhanced raman scattering biochemical applications of surface-enhanced infrared absorption spectroscopy simultaneous determination of glucose, triglycerides, urea, cholesterol, albumin and total protein in human plasma by fourier transform infrared spectroscopy: direct clinical biochemistry without reagents quantification of albumin in urine using preconcentration and near-infrared diffuse reflectance spectroscopy near-infrared spectroscopic determination of serum total proteins, albumin, globulins, and urea preliminary investigation of near-infrared spectroscopic measurements of urea, creatinine, glucose, protein, and ketone in urine correlating the amount of urea, creatinine, and glucose in urine from patients with diabetes mellitus and hypertension with the risk of developing renal lesions by means of raman spectroscopy and principal component analysis estimating the concentration of urea and creatinine in the human serum of normal and dialysis patients through raman spectroscopy spectral characteristics of urine from patients with endstage kidney disease analyzed using raman chemometric urinalysis (rametrix) blu-ray dvd as sers substrate for reliable detection of albumin, creatinine and urea in urine quantitative analysis of metabolites in urine using a highly precise, compact near-infrared raman spectrometer development and validation of hybrid brillouin-raman spectroscopy for non-contact assessment of mechano-chemical properties of urine proteins as biomarkers of kidney diseases rapid and low-cost quantitative detection of creatinine in human urine with a portable raman spectrometer sensitivity" and "specificity" reconsidered: the meaning of these terms in analytical and diagnostic settings western blot: technique, theory, and trouble shooting identification of a three-biomarker panel in urine for early detection of pancreatic adenocarcinoma proteomic analysis of urinary extracellular vesicles from high gleason score prostate cancer exosomal proteins as prostate cancer biomarkers in urine: from mass spectrometry discovery to immunoassay-based validation urine α-fetoprotein and orosomucoid 1 as biomarkers of hepatitis b virus-associated hepatocellular carcinoma application of seldi-tof in n-glycopeptides profiling of the urine from patients with endometrial, ovarian and cervical cancer onco-proteogenomics identifies urinary s100a9 and grn as potential combinatorial biomarkers for early diagnosis of hepatocellular carcinoma deciphering the peptidome of urine from ovarian cancer patients and healthy controls discovery and validation of novel protein biomarkers in ovarian cancer patient urine prediction of chronic kidney disease stage 3 by ckd273, a urinary proteomic biomarker a urinary fragment of mucin-1 subunit α is a novel biomarker associated with renal dysfunction in the general population identification of unique blood and urine biomarkers in influenza virus and staphylococcus aureus co-infection: a preliminary study bladder cancer biomarker array to detect aberrant levels of proteins in urine fgfr3 and cyclin d3 as urine biomarkers of bladder cancer recurrence urinary cysteine-rich protein 61 and trefoil factor 3 as diagnostic biomarkers for colorectal cancer clinical evaluation of urinary transforming growth factor-beta1 and serum alphafetoprotein as tumour markers of hepatocellular carcinoma urine aquaporin 1 and perilipin 2 differentiate renal carcinomas from other imaged renal masses and bladder and prostate cancer an improved prediction model for ovarian cancer using urinary biomarkers and a novel validation strategy identification of o-glycosylated proteins that are aberrantly excreted in the urine of patients with early stage ovarian cancer kidney damage biomarkers and incident chronic kidney disease during blood pressure reduction tissue transcriptomedriven identification of epidermal growth factor as a chronic kidney disease biomarker associations of urinary epidermal growth factor and monocyte chemotactic protein-1 with kidney involvement in patients with diabetic kidney disease markers of early progressive renal decline in type 2 diabetes suggest different implications for etiological studies and prognostic tests development urinary epidermal growth factor, monocyte chemoattractant protein-1 or their ratio as predictors for rapid loss of renal function in type 2 diabetic patients with diabetic kidney disease urine aqp5 is a potential novel biomarker of diabetic nephropathy nonalbumin proteinuria is a simple and practical predictor of the progression of early-stage type 2 diabetic nephropathy infusion of autologous bone marrow derived mononuclear stem cells potentially reduces urinary markers in diabetic nephropathy urinary biomarkers to identify autosomal dominant polycystic kidney disease patients with a high likelihood of disease progression urinary biomarkers are associated with incident cardiovascular disease, all-cause mortality and deterioration of kidney function in type 2 diabetic patients with microalbuminuria urine epidermal growth factor, monocyte chemoattractant protein-1 or their ratio as predictors of complete remission in primary glomerulonephritis urinary tubular injury biomarkers are associated with esrd and death in the regards study development of a multiplexed assay for detection of leishmania donovani and leishmania infantum protein biomarkers in urine samples of patients with visceral leishmaniasis highlights: • urinary protein biomarkers are useful for diagnosis of many health conditions -kidney and cardio vascular diseases, cancers, diabetes, infections. • liquid chromatography -mass spectroscopy is a powerful tool for urine proteomics, but used mostly in research labs. many new biomarkers were discovered by this technique. • immunoassays are widely used in both clinical and bio-analytical laboratories, • infrared and raman spectroscopies are promising tools for analysis of urine and di-agnostics due to relatively simple sample preparation, low-cost and short time of analysis. no conflict of inrterst of any kind for the submitted manuscript review key: cord-333089-ufyzqgqk authors: aguilar-pineda, jorge alberto; albaghdadi, mazen; jiang, wanlin; lopez, karin j. vera; del-carpio, gonzalo davila; valdez, badhin gómez; lindsay, mark e.; malhotra, rajeev; lino cardenas, christian l. title: structural and functional analysis of female sex hormones against sars-cov2 cell entry date: 2020-07-29 journal: biorxiv doi: 10.1101/2020.07.29.227249 sha: doc_id: 333089 cord_uid: ufyzqgqk emerging evidence suggests that males are more susceptible to severe infection by the sars-cov-2 virus than females. a variety of mechanisms may underlie the observed gender-related disparities including differences in sex hormones. however, the precise mechanisms by which female sex hormones may provide protection against sars-cov-2 infectivity remains unknown. here we report new insights into the molecular basis of the interactions between the sars-cov-2 spike (s) protein and the human ace2 receptor. we further observed that glycosylation of the ace2 receptor enhances sars-cov-2 infectivity. importantly estrogens can disrupt glycan-glycan interactions and glycan-protein interactions between the human ace2 and the sars-cov2 thereby blocking its entry into cells. in a mouse model, estrogens reduced ace2 glycosylation and thereby alveolar uptake of the sars-cov-2 spike protein. these results shed light on a putative mechanism whereby female sex hormones may provide protection from developing severe infection and could inform the development of future therapies against covid-19. the novel coronavirus disease 2019 (covid-19) global pandemic caused by infection with the severe acute respiratory syndrome coronavirus 2 (sars-cov2) virus has infected nearly 15 million people worldwide resulting in nearly 600,000 deaths as july 21, 2020 1 . emerging data suggests that males are more susceptible to covid-19 infection and are at higher risk of critical illness and death than females [2] [3] [4] . there has been consistent evidence of an increased case fatality rate (cfr) among males in nearly every country with available sex-disaggregated data including peru, france, greece, italy, mexico, pakistan, philippines and spain amounting to a 1.7 times higher cfr than females 5 . understanding the mechanisms underlying enhanced covid-19 susceptibility and disease severity in males is key to developing new therapies and guiding vaccine development. changes in sex hormone concentration over an individual's lifetime and associated risk of comorbid conditions, such as cardiovascular diseases, may also contribute to variability in disease susceptibility and severity 6 . it has been postulated that the male-biased sex divergence in covid-19 deaths could be, in part, explained by the strict relationship between sex hormones and the expression of the entry receptor for sars-cov2, the angiotensin converting enzyme 2 (ace2) receptor 3, 7 . molecular studies have demonstrated that the male hormone testosterone regulates the expression of ace2 and the transmembrane serine protease 2 (tmprss2) which is an androgen-responsive serine protease that cleaves the sars-cov-2 spike (s) protein and facilitates viral entry via ace2 binding [8] [9] [10] . androgen-driven upregulation of ace2 levels may therefore be associated with increased vulnerability to severe infections in male patients with covid-19. paradoxically, ace2 plays an important role in lung protection during injury which is attenuated by the binding of sars-cov-2 11 . the presence of a male-biased dependence in covid-19 susceptibility may imply the presence of a protective factor against sars-cov-2 infectivity in women. in addition to the ability of sex hormones to modulate expression of proteins related to entry into host cells, both estrogens and androgens are also able to directly modulate immune cell function via receptor-mediated effects 12, 13 . additionally, sex chromosomes may mediate more favorable outcomes among women compared to men affected with covid-19 14 . x-linked genes associated with immune function tend to be expressed more often in females who generally have two x chromosomes compared to males 15 . additional clues to the possible protective effects of estrogens have been suggested by differences in dietary patterns among countries with different cfrs 16 . interestingly, countries with the lowest cfrs including japan and korea are the largest consumers of isoflavones-based foods, also known as phytoestrogens, that may also mediate favorable effects on ace2 expression and therefore covid-19 risk [17] [18] [19] . the observation that females and those individuals consuming higher levels of isoflavones may be protected from covid-19 infection and adverse consequences indicates a potential protective role of estrogens against sars-cov-2. here, we examine the role of two estrogen molecules (17β-diol and s-equol) to modulate the ace2-dependent membrane fusion protein and reduce cell entry of the sars-cov2 spike protein into lung cells. to the best of our knowledge, we report new findings regarding the importance of molecular interactions between hace2 and the viral spike (s) protein. furthermore, we provide insights into the molecular basis for our observations that estrogens impair sars-cov2 entry and highlight the potential for estrogens as an agent in patients with covid-19. glycosylation site-mapping of human ace2 and sars-cov-2 spike interactions. recent studies 20, 21 have shown the ability of the sars-cov2 virus to utilize a highly glycosylated spike (s) protein to elude the host's immune system and bind to its target membrane receptor, ace2, thus enabling entry into human cells. based on the structural complementarity and steric impediments between the s protein and human ace2 (hace2) protein membranes, we mapped the glycosylation sites of both models [21] [22] [23] [24] and performed molecular dynamics simulations (mds) by 250 ns to stabilize the glycosylated sars-cov2 spike (s) and hace2 complex (suppl. table 1 ., suppl. figure 1 and figure 1a ). these analyses revealed that glycosylation of the ace2 protein increases the affinity of the virus s protein to interact with the receptor via glycan-glycan interactions, glycan-protein interactions, hydrogen, and hydrophobic bonds (suppl. table 2 . and figure 1b) . notably, glycan-glycan interactions occur between the ace2 glycan at n322 and n546 and glycans found on the spike's receptor binding domain (s-rbd) at n165 and n343 (figure 1c , left panel). despite the close interaction between ace2 and s-rbd glycans, their affinity to anchor with highly negatively charged molecules such as the ace2 protein remains unalterable (figure 1c right panel) suggesting that glycan and electrostatic-dependent surface tethering may represent a plausible mechanism for ace-s-rbd binding and cell infection. the glycan-protein interactions occur between the ace2 glycan at n53 and the residues of the s-rbd at n437, s438, n439, l441, v445, g446, v483, q498, t500 and q506 (figure 1d ). while ace2 residues at d38, y41, w48 and g326 form hydrogen bonds with residues of the s-rbd at n440, d442, s443, n450 and e484 (figure 1f ). multiple distinct clusters of hydrophobic residues at the ace2 surface were also found to interact with the s-rbd protein (suppl. figure 2 ). importantly one key hydrophobic region on the ace2 surface at t334 interacts with five residues of the s-rbd (p479, g485, f486, g488 and y489), (figure 1g ). given the insights afforded by our in silico mds experiments, we sought to explore the impact of ace2 glycosylation on s-rbd cell entry using cultured human umbilical vein endothelial cells (huvecs). a variety of saccharide substrates were utilized for their ability to modulate glycosylation profiles in cells. the glycosylation pattern of the endogenous ace2 was increased in nearly all treated cells ( figure 1h ). notably co-incubation of huvecs with 10ug of recombinant s-rbd (rs-rbd) protein revealed that glucose (25mm) pre-treatment was associated with the greatest degree of rs-rbd entry into the cells by ~8 fold compared with hypoglycemic media (hbss or optimen) cells ( figure 1g ). this model indicates that glycosylated residues surrounding the cavity at the top of the ace2 molecule could increase binding by the s-rbd. given the possibility that occupancy at glycosylated residues or s-rbd binding sites by estrogens could modify the affinity of the sars-cov2 virus and alter entry into the cell thereby reducing infectivity, we sought to further examine these interactions using a range of complementary experimental approaches (see table s1 ). estrogens bind to hace2 and stimulate its stabilization and internalization. in an effort to explore the potential protective effects of female sex hormones against sars-cov-2 infection, we examined the impact of estradiol (17β-diol) and a dietary-derived phytoestrogen (s-equol) on hace2 structure and protein expression by a combination of in silico modeling, in vitro, and in vivo analysis. specifically, in light of the importance of glycan-glycan interactions that mediate virus-ace2 interactions, we sought to analyze the effect of estrogens on key molecular viral and receptor binding sites. in agreement with yan et al. 22 , we identified three important regions on the ace2 surface that are utilized for sars-cov-2 binding. the environment of these regions is composed of a high density of glycans, including a helix α1 from residues i21 to t52, a helix α2 from residues v59 to m82, and one loop from residues k353 to g354 (suppl. figure 3 and figure 2a ). we then homogenously solvated the glycosylated hace2 structure with 26.6mm of 17β-diol or 26.5mm of s-equol followed by 100 ns of mds. remarkably we found that the 17βdiol molecules interact with residues at f28, y41, q76, t78, q81, m82 and the s-equol molecules interacts with residues at q24, k26, t27, f28, k31, e35, l39, n64, d67, k68, a71, f72, e75, q76 and l79 (figure 2b , supp. table # ). both estrogen molecules energetically stabilized the α1 and α2 helices by physical interactions and thereby minimized the fluctuation of the ace2 chains a and b (figure 2c , supp 3#). importantly , our calculation of free-energy landscape (fel), demonstrated that the surface of chain b of ace2 (s-rbd's preferred interaction region) loses its interaction energy with the s-rbd protein from 10.2 kj/mol to 8.58 kj/mol (16%) for the 17β-diol system and to 9.18kj/mol (10%) for the s-equol system ( figure 2d ). in addition, binding of either estrogen molecules to the surrounding hydrophobic pocket of ace2 at the residue t334 promotes a decrease in energy by ~12% which may have a negative impact on the attachment of the s-rbd protein to the receptor (suppl. figure 4) . we also observed estrogen-glycan interactions particularly at the glycan-protein interactions between the ace2 (n53) and the s-rbd (n432) ( figure. 3a) . indeed, glycans are highly polar structures due to their high content of hydroxyl groups which make them suitable for attachment to the ace2 protein (mostly negatively charged) or the sars-cov-2 s-protein (polarly charged). the density functional theory (dft) calculation shows an important decrease of the glycan's molecular electrostatic potential (mep) due to the interactions with either estrogen molecules. therefore, estrogen-glycan interactions could decrease the adhesive effect of glycans that enhance s-rbd and ace2 receptor interactions (suppl. figure 5 and figure. 3b). these structural analyses suggest that estrogens could act as putative ace2 ligands due to their ability to bind to highly energetic pockets at the top of the ace2 surface protein which may increase its conformational equilibria and potentially boost its internalization to the cytoplasm. to support our in-silico analyses, we treated huvecs with 17β-diol (3nm) or s-equol (10nm) overnight under normal physiologic conditions. immunofluorescent staining demonstrated that estrogen-treated cells have less ace2 membrane cellular localization ( figure 3c ). immunoblot analysis revealed that endogenous and dietary estrogens promote ace2 internalization and degradation through the endocytosis process as assessed by lc3b 25 and lamp1 26 protein activation in treated cells ( figure. 3d). to test the hypothesis that lower levels of estrogens are associated with increased levels of ace2 protein in the respiratory tract, we administrated intratracheally either 17β-diol (0.3μm) or s-equol (1μm) to male mice. histologic analysis of lung sections demonstrated that both forms of estrogens decrease ace2 membrane expression levels in lung alveoli and also reduced the glycosylation of the ace2 receptor ( figure 3e & 3f) estrogens interfere with sars-cov-2 receptor binding and block entry into the cell. to determine if the decline of conformational gibbs free energy and gain in stabilization of ace2 due to estrogen binding could affect the ability of the s protein to interact with the ace2 receptor and thereby its entry into cells, we performed a refinement step of ace2-free or ace2-estrogen models with 100 ns of mds followed by molecular docking with the sars-cov-2 s protein. from 241 structures obtained, 57 with top scores were chosen for further analysis (suppl. table 4 ). the ace2-17β-diol model promoted the shift of s-rbds from the binding surface toward the lateral side of the ace2 protein decreasing the number of contact residues. notably s-rbds completely lose contact with key ace2-glycosylated residues at n53, n103, n432 and n690. we also observed that the contact between the s-rbd and the helix α1 and α2 of ace2 moved toward the n-terminal of the helix and thus affected the ability to bind the receptor. in the same manner, the ace2-s-equol model demonstrated that s-equol blocks the contact between the s-rbds and the receptor's surface, notably promoting novel interactions at the c-terminal of the helix α2 causing nonspecific contacts with the receptor at residues q429-i436 and p590-n601. interestingly, we found that the 17β-diol interacts with 66 residues on the surface of the receptor and notably forms a cluster on glycans at n546 (chain a) and n322 (chain b). on the other hand, the s-equol molecules tend to interact more widely accounting for a total of 145 interactions, including on 63 residues on the chain a and 82 residues on the chain b. (for better visualization, only the 5 top scored s-rdbs structures are shown in figure 4a ). the nonspecific binding by the s-rbds could be explained by the susceptibility of ace2 to interact with polar molecules and especially to electrophilic attacks. the fact that the 17β-diol or s-equol contain few polar groups but are deficient in negative charge renders them more susceptible to attack the surface of hace2 thereby blocking s-rbd from binding correctly. in addition, we computed the binding score of these models using the atomic energy contact function and in agreement with our previous docking results observed that both estrogen molecules significantly reduced the atomic energy contact between virus and receptor. remarkably, the 17β-diol reduced the atomic contact by 80% and the s-equol by 65% indicating that the entry of the virus may be affected by the presence of either estrogen molecules (figure 4b ). to validate our in-silico prediction, we pre-treated huvecs with either estrogen molecules followed by incubation with 10μg of rs-rbd protein overnight. importantly either low or high concentration of 17β-diol (low=1nm) & high=5nm) or s-equol (low=5nm & high=20nm) blocked more than 90% of the rs-rbd protein entry into the cell as assessed by immunofluorescence and colocalization with lamp1, a lysosome marker ( figure 4c ). in addition, immunoblot demonstrated a decrease of rs-rbd levels in the cytoplasm of huvecs for both estrogen-based treatments (figure 4d ). together these results suggest a potential molecular mechanism by which estrogens may provide protection against severe infection in covid19 among women and individuals with phytoestrogen intake. next, we sought to test the ability of estrogens to block key interactions between ace2 and the sars-cov-2 s protein and thereby infection of the respiratory tract. male wild-type mice were treated with 17β-diol (0.3μm) or s-equol (1μm) via intratracheal instillation for 24hrs before tissue collection. elisa-based binding assay showed a significant decrease of ace2 affinity to sars-cov-2 s protein in lungs from mice treated with either estrogen molecules compared with the control group (figure 5a ). we then evaluated in vivo whether intratracheal estrogen treatment would reduce internalization of the s protein in male mice. we observed that intratracheal instillation of both estrogen molecules 24hrs before intratracheal instillation of rs-rbd (20μg, overnight treatment) increased signal for the rs-rbd on the surface of lung cells which likely results from reduced binding to ace2 in estrogen-treated mice compared to the untreated group. in contrast control (dmso-treated) lungs showed normal ace2 membrane localization and cytoplasmic r-s-rbd signal indicating the unperturbed intake of the rs-rbd protein. the observed increase in extracellular rs-rbd in alveolar cells from lungs of estrogen-treated mice suggests estrogen-mediated reduction in internalization of the s-protein. indeed, we observed that pretreatment with estrogen resulted in rs-rbd protein accumulation on the surface of the alveolar cells (figure 5b ) rather than being internalized into the cytoplasm which would thereby support viral replication and disease progression. our data show that estrogens may interfere with sars-cov-2 infection in the respiratory tract through direct interaction with the ace2 receptor in vivo. increased susceptibility and risk of adverse clinical outcomes among males affected by covid-19 has been reported in multiple epidemiological studies [5] [6] [7] [8] . androgens can effectively upregulate viral target proteins that may increase viral entry and pathogenicity in patients following exposure to the sars-cov2 virus and sex-related hormones can modulate immune respose. a detailed understanding of the molecular and cellular mechanisms modulated by estrogen that contribute to viral pathogenicity is therefore critical to the development of new therapies to combat the covid-19 pandemic. beside the epidemiologic evidence suggesting that females are protected from severe infection, a recent study has demonstrated that the female reproductive tract, expresses very low levels of the ace2 receptor and almost undetectable tmprss2, suggesting that the virus is unlikely to infect the female reproductive tract, where female sex hormones are produced 27, 28 . in the current study, we utilized in silico, in vitro, and in vivo studies to characterize important glycosylation-mediated interactions between the sars-cov2 virus spike (s) protein and the human ace2 receptor that can be modulated by endogenous or dietary estrogens in a manner that may be protective against the sars-cov2 entry into human cells. previous studies have highlighted the critical role of glycosylation in viral pathobiology, host immune system evasion, and infectivity in a range of human viral illnesses 29 . in many of these viruses, the viral envelope and secreted proteins are extensively glycosylated which is necessary for structural integrity and functionality of these proteins. viral proteins may be glycosylated by the host cell as viruses are able to hijack cellular glycosylation processes. however little data exists on the impact of glycosylation of host proteins necessary for viral entry, such as ace2, on viral infectivity. using a novel molecular simulation approach, we demonstrated that ace2 glycosylation augments binding of the viral s protein by supporting multiple types of interactions including glycan-glycan and glycan-protein interactions, thereby facilitating the stability and affinity of viral binding to the target host receptor. we extend these in silico observations by also demonstrating that entry of the s-rbd can be augmented in vitro by exposure of cultured huvecs to a hyperglycemic environment that increases ace2 glycosylation. these observations provide insights into the enhanced susceptibility of diabetic patients to severe infections and death in covid-19 [30] [31] [32] . based on these findings that ace2 glycosylation enhances interaction with the viral s protein in silico, we explored whether the predominant endogenous form of estrogen, 17β-diol, may provide a protective effect as assessed using in silico modeling of viral s protein-ace2 interaction and in vitro and in vivo models of viral infectivity. in addition, we used an identical approach to understand the potential protective mechanisms of dietary phytoestrogens on sars-cov2 infectivity observed in populations with low cfrs where consumption of these foods is high. we found that both endogenous and dietary estrogens compete with the s-rbd protein to bind specific sites on hace2 that are used by the virus to bind the receptor. indeed, estrogens were found to bind at almost all sites including ace2 glycans causing a reduction of energy on the surface of the receptor rendering the receptor less susceptible to interact with other molecules via reduced cell surface expression including the viral s protein interactions. our findings that estrogens interfere with s protein and ace2 interactions in silico that is associated with reduced s protein uptake in an in vitro model of sars-cov-2 infectivity in cultured human endothelial cells are consistent with prior studies demonstrating that estrogens have antiviral properties against hiv, ebola and hepatitis viruses 33 . additionally, recent evidence indicates that decreased levels of estrogens in post-menopausal women are an independent risk factor for disease severity in female covid-19 patients 34 . the findings of the current study thus represent novel findings in our understanding of the molecular mechanisms underlying reduced susceptibility to sars-cov-2 among females or individuals with depressed estrogen levels and in countries where dietary estrogens are high. we then examined the ability of estrogen molecules to interfere with s protein uptake into pulmonary epithelial cells using an in vivo model of sars-cov2 infectivity. in agreement with our cellular experiments, lungs from mice treated with dietary or endogenous estrogens demonstrated a dramatic reduction in the uptake of s-rbd. in addition, we observed a remarkable reduction of ace2 binding possibly due to the low protein levels of ace2 in those lungs possibly in response to estrogen-mediated degradation. in conclusion we provide a molecular basis that helps elucidate the potential protective effect of estrogens in women infected by the sars-cov-2 virus which could inform the development of future therapeutic measures to protect against sars-cov-2 infection including the design of suitable blocking antibodies, estrogen-related treatments, and vaccine development. for immunefluorescence, huvec cells were cultured into 8-well lab-tektm ii chamber slides (nunctm) and were then treated with either 17β-diol at 3nm or s-equol at 10nm. cells were rinsed twice with ice-cold pbs, fixed with 4% paraformaldehyde in pbs (pfa, boston bioproducts) for 10 min at rt, and were permeabilized with 0.1% triton-x (sigma-aldrich) for 3 min. the slides were blocked with 10% donkey-serum, and 0.3 m glycine in pbs-tween 20 (0.1%) for 1 h at rt. subsequently, the antibodies anti-ace2 (1:100), s-rbd-his-tag (1:50), anti-lamp1 (1:50) and anti-lc3b (1:50) were added and slides were incubated overnight at 4°c. the slides were then washed 3 times for 5 min each with pbs-t and were incubated with secondary antibodies at 1:400 dilution for 1hr at room temperature. following immunostaining, slides were mounted with diamond mounting medium containing dapi (thermo fisher). slides were then visualized with the leica tcs sp8 confocal microscopy station and the pictures were digitized with the leica application suite x software. huvec cells were rinsed twice with ice-cold pbs and proteins were extracted with m-per for whole cell lysis, respectively (thermo fisher). these lysis buffers contained halt protease, phosphatase inhibitors and edta (thermo fisher). the protein concentration was determined by the colorimetric bicinchoninic acid assay (bca assay, thermo fisher). equal amounts of total protein from cell lysates were separated by sds-page (25μg or 40μg for ace2, lamp1, lc3b and rs-rbd-his-tag, respectively). proteins from the gel were then electro-transferred onto 0.45 μm nitrocellulose and 0.2 μm pvdf membranes. the membranes were then blocked for 1 h at room temperature, with either 5% non-fat powdered milk dissolved in tbs-t or 5% bovine serum albumin in tbs-t, for the nitrocellulose and pvdf membranes, respectively. following blocking, membranes were incubated overnight at 4°c with the primary antibodies anti-ace2 (1:1000), anti-lamp1 (1:2000), anti-lc3b (1:1000) and anti-his-tag (1:1000). the odyssey infrared western system was used to detect target proteins. band intensity was quantified using imagej software. all experiments involving mice were approved by the partners subcommittee on research animal care. personnel from the laboratory carried out all experimental protocols under strict guidelines to insure careful and consistent handling of the mice. mouse model of sars-cov-2 s protein entry. 9 weeks old male c57bl/6 were purchased from the jackson laboratories, usa. to induce the recombinant s-rbd protein. briefly, mice were anesthetized with sevoflurane inhalation (abbott) and placed in dorsal recumbency. transtracheal insertion of a 24-g animal feeding needle was used to instillate estrogen molecules, rs-rbd or vehicle (dmso), in a volume of 80 µl. mice were sacrificed 24 hrs after instillation of rs-rbs and lungs were removed for further analysis. histology. lungs were then fixed in formalin (10%) for 24 hours before transfer to 70% ethanol for photography prior to paraffinization and sectioning (7 μm) paraffin embedding. slides were produced for tissue staining) for quantitative analysis. saccharides treatment: hypoglycemic media was composed by hbss buffer or optiment media. normal media contained complete endothelial cell growth media. for hyperglycemic media, optiment was supplemented with d-glucose at 25mm, d-galactose at 50mm, d-ribose at 250μm, d-mannose at 300μm, or d-fructose at 20μm. huvecs at 60%-70% confluence were supplemented with hypoglycemic, normal or hyperglycemic media 24 hours before incubation with 10μg of recombinant s-rbd-his-tag overnight. estrogen treatment: huvecs at 60%-70% confluence were supplemented with opti-mem 24 hours before treatment with complete growing media containing 17β-diol at a concentration of 3nm or s-equol at a concentration of 10nm for 24 hrs. fresh media containing rs-rbd (10μg) was supplied the next day. prior to cellular collection, cells were washed with sterile pbs, protein extraction were performed as described above. rs-rbd-ace2 binding assay 500μg of total protein extracts from mouse lungs were cleaned up with iga/igg agarose beads for 1hr at 4c on a rotator followed by resuspension in assay diluent at 1x. then 100μl of each lysate containing 0, 5, 10, 15, 20, 25 or 30μg of total protein were placed into corresponding well of a covid19 s-protein microplate (cat#: cov-sace2, ray biotech,inc.) for overnight incubation at 4c on a rotator. then supernatant was removed, and wells were washed x 5 followed by incubation with 1x hrp-conjugated secondary antibody solution for 1hr at room temperature. then 100μl of tmb one-step substrate reagent was added to each well for 30 min at room temperature. before read 50μl of stop solution was added and microplate was read at 450nm. results are given as mean ± sd student's t test (2-tailed) was applied to determine the statistical significance of difference between control and treated groups (*p < 0.05, **p < 0.01 and ***p < 0.001). for all experiments, at least 3 experimental replicates were performed. violin plot graphs show mean ± sd. data were analyzed, and graphs were prepared with prism 6.0 (graphpad software). p values of less than 0.05 were considered statistically significant. the crystalline structures used in this work were pdb id:6vxx 23 for spike protein (sp, trimeric form) of virus sars-cov-2 and pdbid:6m17 22 (of rbd/ace2-b0at complex) for ace2 protein (dimeric form), both obtained in the rcsb protein data bank. the missing residues for sp located on n and c terminal domains (m-18 to p26 and f1148 to h1262, respectively) were not considered in the molecular simulations. therefore, each one sp chain was made up of 1121 residues (a27 to s1147). in our sp model, another disulfide bond was recognized between missing residues c499 and c507 and was considered in md simulations. for ace2, 29 residues on n-terminal domain were excluded (m-8 to t20) and on c-terminal domain only extramembrane residues were considered (to i21 to g732). ace2 structure contain two zinc ions in peptidase domain which were considered in this work. the remaining missing residues of both proteins were added using swissmodel server 34 (s-t1). the ace2 and sp structures are considered glycoproteins and the glycan-linked residues have already been reported [21] [22] [23] [24] . the oplsaa based doglycans software was used for building all models 35 (s-t1). for sp, there are 22 n-glycosylation residues on its surface, but n17, n1158, n1173 and n1194 sites were excluded due to residues considered in our sp model. oglycosylation sites was not included too. in ace2, all n-glycosylation sites were considered. the glycosylation process was carried out using the glycan glcnac2man3 model, a glycoside sequence composed of 2 n-acetyl glucosamines and 3 mannoses (s-f1b). this glycan type is the most common core sugar sequence on the n-glycans 36, 37 estrogen solvated systems. the systems were constructed with the average structure of glycosylated ace2, obtained in last 50 ns of mds trajectories. 17β-diol and s-equol structures were quantum optimized and their force fields were constructed using ligpargen server [38] [39] [40] . the previous simulation box of ace2 was augmented 0.4 nm in all directions and with the protein centered in box, was solvated two times using gmx solvate module. the first solvation was made homogeneous way with 60 17βdiol and s-equol molecules (26.6 and 26.5 mm solutions, respectively). in second solvation, explicit water molecules were added to fill the simulation box. in the solvation process, we made sure that the estrogen molecules were not close to the protein at the start of the md simulations. all quantum simulations were performed using density functional theory (dft) 41 at b3lyp/ tzvp level 42, 43 . the self-consistent reaction field (scrf) theory was used for describing the solvent effects on the molecules in water solutions. the calculations were performed in the electronic structure program gaussian 16 44 and results were visualized in gaussview v.6 45 . the molecular structures of 17β-diol and s-equol were optimized and it was ensured that they were at a global minimum through frequency analysis. these optimized structures were used in the molecular dynamic simulations. in meps analysis, single point calculations were carried out and total electron densities was mapped on molecular electrostatic potential surface. to address the structural interactions, we performed molecular dynamics simulations using gromacs (v.2019.4) 46 with the opls/aa force field parameters 47. the protein complexes were solvated with tip4p explicit water model 48 . in addition to na + counterions used to neutralize the total charge in the simulation box, we used a 150 mm nacl concentration to mimic physiological conditions. all molecular systems were built in a triclinic simulation box considering periodic boundary conditions (pbc) in all directions (x, y and z). minimum distance of the surface atoms of proteins to the edge of periodic box was 1.5 nm for ace2 receptor and sars-cov-19 spike protein, and 2.3 nm for ace2-estrogen solvated systems. the equations of motions were integrated with the leap-frog integrator 49 using a time step of 1 fs. temperature in the simulations was maintained at 309.65 k using modified berendsen thermostat (v-rescale algorithm) 50 with = 0.1 ps coupling constant with protein and water-ions coupled separately. pressure was maintained at 1bar using the parrinello-rahman barostat 51 with a compressibility of 4.5x10 −5 bar -1 and a coupling constant of = 2.0 ps. all simulations were carried out with a short-range non-bonded cut-off of 1.1 nm and the particle mesh ewald (pme) method 52 was used for computing long-range electrostatic interactions with a tolerance of 1x10 5 for contribution in real space. the verlet neighbor searching cut-off scheme was applied with a neighbor-list update frequency of 10 steps (20 fs). bonds involving hydrogen atoms are constrained using the linear constraint solver (lincs) algorithm 53 . simulations were first energy minimized using the steepest descent algorithm for a maximum of 100,000 steps. the equilibration was conducted by two steps. the equilibration was conducted by two steps. the first step, a 1 ns of dynamics in the nvt (isothermal-isochoric) ensemble and second step, was continued for another 2 ns in the npt (isothermal-isobaric) ensemble. production runs were performed in the npt ensemble for 300 ns for ace2 and sars-cov-19 spike protein and 150 ns for ace2-estrogen solvated systems. structure and data analysis. the structural interactions were obtained carried out a rigid-rigid body docking analysis using patchdock 54 server in order to obtain the contact residues between s-rbd and ace2 systems. the patchdock algorithm discard all unacceptable complex and results are assorted by geometry shape complementarity score. in addition, patchdock do calculate the effective atomic contact energies according to zhang et al. 55 the molecular docking was done take to ace2 protein as receptor molecule and spike protein as ligand molecule. clustering rmsd value and complex type they were selected according to the recommended parameters for protein-protein interactions (4.0ǻ and default mode). for ace2-estrogen systems, the docking was performed in the presence of the estrogen molecules bound to the ace2 structure, 40 17β-diol molecules and 47 s-equol molecules, respectively. from total results obtained in molecular docking, those structures that had steric impediments (intermembranal clashes) were discarded. the steric impediments were calculated based in sars-cov-2 virus size 56, 57 , whose diameter varies about 60 to 140 nm and its spike protein is about 9 to 12 nm length (100 and 10 nm on average, respectively). molecular interactions were analyzed with ligplot software 58 and the pdb files required was constructed with fortran based own computer programs and the statistical data results. statistical results, rmsd, rmsf, rg, sasa, hydrogen bonds, free energies, matches, structures, movies, b-factor maps, were obtained using gromacs modules and their different tool options. the analysis of structure properties was performed using md trajectories on the last 50 ns of each simulations and visualization of the md simulations was created using visual molecular dynamics (vmd) software 59 and the graphs were plotted by the xmgrace software 60 . each molecular conformation during an md simulation has an associated energy and this can be observed using fel maps. these maps are usually represented by two variables related to atomic position and one energetic variable, typically the gibbs free energy. this free energy can be estimated from probability distributions of the system with respect to the chosen variables that are then converted to a free-energy value by bolzmann inverting multi-dimensional histograms. when represented in three dimensions, the fel maps show the energy range of all possible conformations were obtained during a simulation. in this work, we considered two substructures of ace2 protein for fel map analysis, the alpha1-2 region (i21 to y83) and loops regions l2-3 and l3-4 (d303 to r357). the fel maps were plotted using gmx sham module while the rmsd and radius of gyration were considered as atomic position variables respect to its average structure and figures were constructed using wolfram mathematica 12.1 61 . . estrogens bind to ace2 glycans to promote its internalization. (a) glycanestrogen interactions stabilize ace2 structure through high-energy contacts involving ace2 glycan-residues at e57, n53, k341 and v339 (red color). (b) mep maps show the electrostatic impact of estrogen molecules on the surface of ace2 glycans. energy scale ranging from -0.075 μa (red) to 0.075 μa (blue). (c) immunofluorescence staining of human ace2 (magenta) and the lysosome marker lamp1 (green), shows loss of ace2 membrane levels in huvecs treated with 17β-diol or s-equol compared with control cells (dmso). (d) immunoblot of lysates isolated from huvecs showing decreased levels of total ace2 protein with estrogen treatment. reduced ace2 protein levels were associated with increased endocytosis activity as evidenced by immunoblot for lc3b and lamp1. (e) histologic analysis of mouse lungs after 48hrs of intratracheal installation with 17β-diol or s-equol shows loss of ace2 expression (red) on the membrane of alveolar cells. estrogen-treated lungs showed greater ace2-lamp1 colocalization (white arrows) indicating internalization of the receptor. (f) immunoblot showing decreased levels of total and glycosylated ace2 proteins in estrogen-treated lungs from male mice compared to control lungd. quantification of protein levels of three replicate experiments is shown. student's t-test, 2 tails. bar graphs are presented as mean with error bars (±sd). surface interacting with 5 top scored s-rbds (top 1-blue, 2-red, 3-orange, 4-purple and top 5yellow). s-rbds were scored based on shape complementarity principles. (b) heatmap of atomic contact energy between ace2 and 57 s-rbds, shows spontaneous energy structures from most favorable (green) to less favorable s-rbd structures (red). energy scale ranging from 500 kcal/mol to -500 kcal/mol. (c) immunofluorescence analysis of s-rbd entry into huvecs pretreated with 17β-diol or s-equol followed by treatment with 10μg/ml of recombinant s-rbd (red) demonstrate that estrogen-treated cells had reduced entry of s-rbd into cells in conjunction with a reduction in ace2 internalization as showed by colocalization with lamp1 (green). (d) immunoblot of isolated proteins from cultured huvecs shows a 90% reduction of s-rbd entry into cells in estrogen-treated cells. quantification of protein levels of three replicate experiments is shown. student's t-test, 2 tails. bar graphs are presented as mean with error bars (±sd). (a) elisa-based binding assay using lung protein lysates shows reduced sars-cov-2 s protein affinity for the ace2 receptor after treatment with either 17β-diol or s-equol. (b) immunofluorescence analysis of wild-type mouse lung treated with 17β-diol (0.3μm) or s-equol (1μm) compared with control lung (dmso) demonstrates rs-rbd protein accumulation on the surface of the alveolar cells rather than being internalized intracellularly where viral replication may occur. treatment with either estrogen also reduced ace2 prtoein expression (quantification in lower right panel). lamp1 (orange), ace2 (green) and rs-rbd (red). quantification levels of three replicate experiments is shown. student's t-test, 2 tails. bar graphs are presented as mean with error bars (±sd). a pneumonia outbreak associated with a new coronavirus of probable bat origin tripartite combination of candidate pandemic mitigation agents: vitamin d, quercetin, and estradiol manifest properties of medicinal agents for targeted mitigation of the covid-19 pandemic defined by genomics-guided tracing of sars-cov-2 targets in human cells circulating plasma concentrations of angiotensin-converting enzyme 2 in men and women with heart failure and effects of renin-angiotensin-aldosterone inhibitors predictors of mortality in hospitalized covid-19 patients: a systematic review and meta-analysis impact of sex and gender on covid-19 outcomes in europe are sex discordant outcomes in covid-19 related to sex hormones? sars-cov-2 and male infertility: possible multifaceted pathology covid-19 and androgen-targeted therapy for prostate cancer patients sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structural and functional basis of sars-cov-2 entry by using human ace2 ace2, much more than just a receptor for sars-cov-2. front the x chromosome in immune functions: when a chromosome makes the difference the x-files in immunity: sex-based differences predispose immune responses considering how biological sex impacts immune responses and covid-19 outcomes correction: sex hormones promote opposite effects on ace and ace2 activity, 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probing the glycosidic linkage: secondary structures in the gas phase potential energy functions for atomic-level simulations of water and organic and biomolecular systems 1.14* cm1a-lbcc: localized bond-charge corrected cm1a charges for condensed-phase simulations ligpargen web server: an automatic opls-aa parameter generator for organic ligands density-functional thermochemistry. iv. a new dynamical correlation functional and implications for exact-exchange mixing fully optimized contracted gaussian basis sets for atoms li to kr gromacs: high performance molecular simulations through multilevel parallelism from laptops to supercomputers development and testing of the opls all-atom force field on conformational energetics and properties of organic liquids temperature and size dependence for monte carlo simulations of tip4p water quiet high-resolution computer models of a plasma molecular dynamics with coupling to an external bath canonical sampling through velocity rescaling die berechnung optischer und elektrostatischergitterpotentiale a parallel linear constraint solver for molecular simulation patchdock and symmdock: servers for rigid and symmetric docking determination of atomic desolvation energies from the structures of crystallized proteins a novel coronavirus from patients with pneumonia in china science forum: sars-cov-2 (covid-19) by the numbers ligplot: a program to generate schematic diagrams of protein-ligand interactions vmd: visual molecular dynamics version 5.1.19 mathematica, version 12 key: cord-339558-li65qvq9 authors: rana, rashmi; rathi, vaishnavi; ganguly, nirmal kumar title: a comprehensive overview of proteomics approach for covid 19: new perspectives in target therapy strategies date: 2020-11-02 journal: j proteins proteom doi: 10.1007/s42485-020-00052-9 sha: doc_id: 339558 cord_uid: li65qvq9 world health organisation declared covid-19 a pandemic on march 11, 2020. it was temporarily named as 2019-ncov then subsequently named as covid-19 virus. a coronavirus is a group of viruses, known to be zoonotic, causing illness ranging from acute to mild respiratory infections. these are spherical or pleomorphic enveloped particles containing positive sense rna. the virus enters host cells, its uncoated genetic material transcribes, and translates. since it has started spreading rapidly, protective measures have been taken all over the world. however, its transmission has been proved to be unstoppable and the absence of an effective drug makes the situation worse. the scientific community has gone all-out to discover and develop a possible vaccine or a competent antiviral drug. other domains of biological sciences that promise effective results and target somewhat stable entities that are proteins, could be very useful in this time of crisis. proteomics and metabolomics are the vast fields that are equipped with sufficient technologies to face this challenge. various protein separation and identification techniques are available which facilitates the analysis of various types of interactions among proteins and their evolutionary lineages. the presented review aims at confronting the question: ‘how proteomics can help in tackling sars-cov-2?’ it deals with the role of upcoming proteome technology in these pandemic situations and discusses the proteomics approach towards the covid-19 dilemma. revolving from simple pneumonia to a pandemic, covid-19 has come a long way in contributing towards the mortality rate globally. the disease is caused by a novel coronavirus sars-cov-2 (severe acute respiratory syndrome coronavirus-2). apart from this, the other two viruses of the coronavirus family i.e., mers-cov (middle east respiratory syndrome coronavirus) and sars-cov (severe acute respiratory syndrome coronavirus) have also been the cause of mass destruction since the beginning of the 21st century (walls et al. 2020) . coronaviruses (covs) are the largest group of viruses belonging to the order nidovirales, which includes coronaviridae, arteriviridae, and roniviridae families. coronaviridae or covs includes a clan of positive-sense, single-stranded, non-segmented, enveloped rna viruses of vertebrates. these viruses contain the largest genomes of 25-31 kb and are infectious when introduced into permissive cells. they can be further classified into four other genera, i.e., alpha-coronavirus, beta-coronavirus, gamma-coronavirus, and delta coronavirus (yang et al. 2015) . six human coronaviruses (hcovs) are known to date, i.e., hcovs-nl63 and hcovs-229e (alpha covs), hcovs-oc43, hcovs-hku1 (beta covs), sars-cov, and mers-cov. due to their vast distribution, the frequent recombining ability of their genome, large genetic diversity, and zoonotic behaviour, novel coronaviruses have been evident to appear periodically in humans (wu and mcgoogan 2020) . the origin of this highly contagious disease is reported to be epidemiologically linked with a seafood market in wuhan, hubei province, china (zhou and yang 2020) . the zoonotic transmission of this beta coronavirus from the seafood market of wuhan has stormed the world by spiking death rates. it was reported in 213 countries and territories affecting more than 36,738,690 people, accounting for 1,066,412 deaths. at the time of writing this manuscript, as of 00:00 gmt + 0, 09 october 2020, india has reported 6,903,812 cases so far, 106,521 deaths and 894,084 currently active cases (https ://www.world omete rs.info/coron aviru s). similar to a chain reaction, the rapid human-to-human transmissibility has turned a majority of the world's population into viral carriers and incubators (chan et al. 2020) . the numbers of cases are increasing with more than 316,256 new cases being reported daily (https ://www.world omete rs.info/ coron aviru s). the infected individual might remain asymptomatic for days and the initial flu-like symptoms culminate into an elevated inflammatory response ). the sudden outbreak and accelerated spreading of infection have caused substantial public concerns. within about two months, close to one million individuals worldwide had been infected, leading to about 1,78,371 deaths (heng et al. 2020) . most covid-19 [sars-cov-2] studies have focused on its epidemiological and clinical characteristics (ghinai et al. 2020; li et al. 2020) . about 80% of patients infected with sars-cov-2 displayed mild symptoms with good recovering capabilities. they usually recover with or even without conventional medical treatment and, therefore, are classified as mild or moderate covid-19 (baden et al. 2020) . however, about 20% of patients suffer from respiratory failure and need immediate oxygen therapy or other inpatient interventions, including mechanical ventilation (murty et al. 2020; . these patients are classified as clinically severe and are mainly diagnosed empirically based on a set of clinical characteristics, such as respiratory rate (≥ 30 times/min), mean oxygen saturation (≤ 93% in the resting state), or arterial blood oxygen partial pressure/oxygen concentration (≤ 300 mmhg) (baden et al. 2020 ). however, patients exhibiting these clinical manifestations have already progressed to a clinically severe phase and require immediate access to specialized intensive care; otherwise, they may die rapidly. therefore, it is critical to developing new approaches to predict the cases which might progress to clinically severe condition. besides, effective therapy for severe patients remains hypothetical, largely due to limited understanding of covid 19 sars-cov-2 pathogenesis. the current review aims at analyzing the current status of clinical proteomics and metabolomics with particular emphasis on sars-cov2 biology and approaches for its treatment. therapeutic strategies majorly aim at proteins and not nucleic acids as drug targets. though the technologies available to date, such as microarray, succeeds in identifying an enormous count of differentially expressed genes but fail to justify their multiple protein products and functionality. on the other hand, proteome analysis aims not only in identifying the differential expression of proteins but also to take account of protein-protein interactions, post-translational modifications, the temporal pattern of expression, and cellular and sub-cellular distribution. hence, differential and functional proteomics generate information that leads to improved interpretation of the cellular pathways and how they are linked in cells and living organisms. for many decades, proteomics has proved its versatility and efficacy for the development of the novel potential drug targets for constantly appearing diseases posing challenges to humankind. currently, the world of proteomics has stuck with the dilemma to overcome the proposed oppositions of the sars-cov2 virus for drug and vaccine development. currently, reverse transcription-polymerase chain reaction is the most reliable method to detect ssthe viral genes in the covid-19-positive patients. the method is widely available and well established, exhibits many problems in its performance. due to the potent mutability of the viral genes, the technique might not be sensitive enough to detect the virus if it is mutated. furthermore, rt-pcr tends to exhibit low throughput due to intermediate and long reaction times (tahamatan et al. 2020) . beyond these facts, serological tests involving the presence of specific antibodies are also important as they identify false-negative rt-pcr responses. they also track how effectively the patient's immune system is working against the infection by quantifying the metabolites and antibodies and are essentially helpful for plasma transfusion therapies (funari et al. 2020) . these tests are based on the detection of antigens from nasopharyngeal swabs using antibodies. a positive test confirms the presence of the virus, however, a negative test is inclusive as the sensitivity of the antigen test is between 34 and 80% (bruning 2018 ). structural proteome analysis of earlier sars epidemic in 2003 revealed a large array of proteins that could be targeted for this pandemic too. the sars-cov genome encodes 28 proteins of which the structure of 16 proteins or their functional domain has been determined until today. interestingly, out of these 16 proteins, eight of them have novel functional domains that indicate the distinctiveness of coronavirus proteins (bartlam et al. 2007 ). stukalov et al. identified the interactions of sars-cov and sars-cov-2 with cellular proteins. they used affinity purification followed by mass spectrometry analysis and statistical modeling of the ms1level quantitative data which allowed the identification of 1484 interactions between 1086 cellular proteins and 24 sars-cov bait proteins. particularly, they discovered that sars-cov-2 targets several cellular regulators involved in innate immunity (orf7bmavs,-unc93b1), stress response components (n-hspa1a), and dna-damage response mediators (orf7a-atm, -atr). in a nutshell, sars-cov-2 interacts with specific protein complexes contributing to a range of biological processes (stukalov et al. 2020) . another study elaborated on the use of seldi-tof (surface-enhanced laser desorption/ionization) in early detection of the sars virus. the analysis results in an array of proteins that are specific to the infectious agents (marzulli et al. 2005) . wide arrays of techniques are available at hand for the segregation and identification of proteins from complex mixtures which could render a helping hand in this global pandemic. the most common separating methods include: one-and two-dimensional gel electrophoresis and highperformance liquid chromatography (hplc) whereas mass spectrometry (ms) is the yardstick in the world of protein identification (verills 2006) . the overall workflow of proteomics approaches for covid-19 patient shown in fig. 1 . the conventional techniques for purification and segregation of proteins include affinity-based chromatography, ion-exchange chromatography and size-exclusion chromatography. enzyme-linked immunosorbent assay (elisa) and western blotting techniques can be used for analysis of selective proteins. though, still in use but these techniques restrict the analysis to smaller number of proteins and also incapable to define expression level of proteins. sodium dodecyl sulfate-polyacrylamide gel electrophoresis, twodimensional and two-dimensional differential gel electrophoresis are used for separation of complex protein samples (aslam et al. 2017) . a current intervention in the 2d-gel electrophoresis has allowed the researchers to analyze the protein complement of the genome targeted. the technique now provides highthroughput and high-resolution separation of proteins. an improvement, such as the introduction of ipg (immobilized ph gradients) strips and their employment with a single ph range, has not only increased the sensitivity but also specify the location of proteins in a cell or tissue extracts (bjellqvist et al. 1982; scherl et al. 2002; cordwell et al. 2000) . a step ahead of 2d-ge, the introduction of cyanine fluorescent dyes for the pre-labeling of proteins has made this approach a valuable tool in proteomics. due to the presence of cye dye fluorophores which enable the co-migration of multiple proteins and their simultaneous detection, 2d-dige allows faster and more reliable gel matching. labeling before the fractionation prevents the alteration due to gel to gel variation and provides a good dynamic range. hence, multiple proteins can be analyzed directly in one gel (tonge et al. 2001; pasquali et al. 2017; eggeling et al. 2001 ). the efficacy of 2d-dige has been validated for numerous applications for various cancers and other diseases (seike et al. 2004; isaaq et al. 2007 ). protein identification and profiling through mass spectrometry techniques have now overpowered the loopholes of various other proteome technologies (including 2d gels) which require a large amount of purified protein for the analyses. maldi-tof or matrix-assisted ionization-time of flight ms has accelerated the identification of proteins isolated by 2d-gel electrophoresis and other methods to the elevated levels because of its sensitivity and speed (aebersold et al. 2003) . combining the latter with identification of protein sequence, searching through available databases has facilitated the process of drug discovery and development in an enormous amount. although a very powerful technique, 2d-ge sometimes fails to separate proteins of very high and low molecular weight, thus methods, such as free-flow electrophoresis (ffe), have been developed which enables the resolution of a complex mixture of proteins by combining 2d-ge with the liquid-based ief method (hoffmann et al. 2001) . mass spectrometry can also be seen as the backbone of the various proteomic technologies. it uplifts the studies, enlightens the new findings, and facilitates in taking a step ahead in the analysis. ms-based viral peptide detection has earlier been used for the detection of viral proteins which affect respiratory pathways (foster et al. 2015; majchrzykiewicz-koehorst et al. 2015) . it had been a phenomenal technique in the characterization of sars viruses in the later epidemic. a study employing maldi-tof ms used convalescent sera from several sars patients to detect proteins in the culture supernatants from cells exposed to lavage of another sars patient. the technique identified a very prominent protein of molecular weight 46 kda approx. which was found to be a novel nucleocapsid protein, further established to be the major immunogen. another 139 kda spike glycoprotein was also examined by maldi-tof which was the possible target for the prophylactic intervention in encountering sars (krokhin et al. 2003) . sars-cov-2-specific peptides have also been reported and shortlisted for targeted mass spectrometric studies by gouveia et al. they used infected cell lysate to identify these peptides and validated these peptides in a nasopharyngeal swab. (gouveia et al. 2020a, b) . another study using a gargle solution of three patients reported a peptide from nucleoprotein using a 180 min gradient. the study involves lc-ms analysis on a nano-hplc system coupled to an orbitrap fusion tribid mass spectrometer with nano-esi source after acetone precipitation and tryptic digestion of the proteins contained within the gargle solution (ihling et al. 2020) . three peptides have also been reported using 10.5 min run time with 83% sensitivity and 96% specificity (cardozo et al. 2020) . a simple and rapid method that could be used to detect the presence of the virus even in the recovered patients has also been developed. the technique employed multiple reaction monitoring mass spectrometry (mrm-ms) and detected two peptides, qiapgqtgk and aivstiqrkyk, from structural spike glycoprotein and replicase polyprotein 1 ab, respectively, in 2.3 min gradient with a sensitivity of 90% and specificity 100%. they used this method for asymptomatic recovered patients which tested negative for rt-pcr analyses. hence, suggested the possible application of this detection mechanism in asymptomatic subjects also (singh et al. 2020 ). one such study developed an automated antibody capture-based workflow coupled with targeted high-field asymmetric ion mobility spectrometry (faims)-parallel reaction monitoring (prm) assays on an orbitrap exploris 480 mass spectrometer. the study is based on analysis of 363 nasopharyngeal residual swab samples from patients. it involves low-flow lc method followed by a prm ms that incorporates ion mobility for selected viral peptides. the study confirms the nucleocapsid protein to be the major target antigen, based on the experiments done on nasopharyngeal swabs from covid-19 patients, recombinant viral proteins and purified virus. they established a collective machine learning-based model for identifying covid-19-positive samples employing fragment ion intensity in the prm data. particularly, their study resulted in 97.8% sensitivity and 100% specificity comparable to rt-pcr-based molecular testing (renuse et al. 2020 ). swath-ms (sequential windowed acquisition of all theoretical fragment ion mass spectra) that allows complete recording of all the fragment ions of the detectable peptide precursor present is a biological sample. to maintain sensitivity, selectivity and accuracy, swath-ms incorporates dataindependent acquisition and targeted data analysis. it has been used for biomarker discovery in various diseases and, hence, can be used for covid-19 as well. rosenberger et al. 2017 ). coupling basic protein analytical techniques with mass spectrometry enhances the sensitivity (to the femtomole levels), resolution, and accuracy of the examination. protein complexes are cleaved into smaller fragments and detected by the mass spectrometer. peptides can be detected in various ways using different mass spectrometers. time-of-flight (tof) ms instruments involve the peptides to fly down a flight tube and the time taken to reach the detector indicates the peptide mass. quadrupoles, ion traps, and fticr (fourier-transform ion cyclotron resonance) are some other types of mass analyzers that make the mass spectrometers more profound and reliable for peptide analysis (lim et al. 2004 ). the mass spectrum obtained gives the list of peptide masses that are extensively searched in genome databases, translated and trypsin digested in silico. another progression in the field of mass spectrometry is fractionating protein complexes multiple times (usually twice) into individual amino acids, it is termed as tandem mass spectrometry or ms/ms. this allows the determination of de novo peptide sequences and, hence, its identification through databases against known proteins (seidler et al. 2010 ). tagging protein fragments with heavy or light isotopes before exposing them to mass spectrometers makes the qualitative and quantitative analyses further easy. labeling techniques, such as isotope-coded affinity tags (icattm) (smolka et al. 2001 ), o18-water labeling (ye et al. 2009 ), isotope tags for relative and absolute quantification (itraq) (wiese et al. 2007) , and stable isotope labeling with amino acids in cell culture (silac) (mann 2006) , allow the subsequent identification and quantification effortlessly. a recent study involving chemical labelling identifies the host cell pathways that are modulated by sars-cov2 and thus showed that inhibition of these pathways prevents the virus from replicating. a human cell culture system was established for infection and further isotope labelled for the lc-ms/ms proteome analysis. their analysis revealed that sars-cov2 reshapes the central cellular pathways, such as carbon metabolism, proteostasis, nucleic acid metabolism, translation, splicing and carbon metabolism. viral replication was inhibited by small molecules that target these pathways. this study can be a boon in these situations as it reveals the cellular infection profile of sars-cov2 for which drugs could be developed. their finding provides insights for the development of therapies for the treatment of coronavirus (bojkova et al. 2020) . protein chips or protein microarrays could be an astonishing approach to detect and characterize the peptides available in a protein complex. chip technology has been widely used in the field of nucleic acids but its applicability in proteomics is not very well versed. as proteins are extensively heterogeneous, a simple chip for all types of proteins has not been achieved yet. while dna is stable, proteins tend to lose their three-dimensional structure and, hence, their functionality and specificity as the conditions fall out of a narrow range (eickhoff et al. 2002) . though, a variety of proteins and peptide arrays have been developed for the analysis of specific or group of proteins, surface-enhanced laser desorption-ionization (seldi) protein chipr, developed by ciphergen biosystems, inc., involves affinity capture of specific subgroups of proteins based on their biochemical or physical properties which is further analyzed with automated ms (merchant et al. 2000) . a low abundant protein that fails to be detected by 2d-ge can be efficiently detected by this technique. forward-phase arrays and reverse-phase arrays are the two microarray platforms being developed in this series. these two arrays work just opposite to each other. in forwardphase array, an antibody is immobilized on the solid surface and the sample protein mixture is poured on the latter whereas, in reverse-phase array, the sample is immobilized on the surface and then reacted with a specific antibody. bound molecules are detected by secondary antibody or direct labeling (liotta et al. 2003; phizicky et al. 2003) . functional microarray chips can be used to study protein-protein interactions, dna-protein interactions, and drug-target identification (phizicky et al. 2003) . despite all the challenges, protein microchips have also been used in the detection and characterization of sars viruses. a study involving screening of specific igg antibodies of 13 recombinant proteins associated with four structural proteins (s, e, n, and m and five putative uncharacterized proteins of sars-cov (3a, 3b, 6, 7a and 9b). the results suggested that anti-s and anti-n antibodies are diagnostic markers and s3 is immunogenic thus a potential candidate for vaccine development (qiu et al. 2005 ). zhu et al. has constructed a protein microarray that includes proteins from sars coronavirus (sars-cov) and five other additional coronaviruses. they also developed a computer algorithm that uses multiple classifiers to predict samples from sars patients and used to predict 206 sera from chinese fever patients. the test also identified patients with sera reactive against other coronavirus proteins. they further correlated these results with an indirect immunofluorescence test and suggested that viral infection can be scrutinized for months after infection. thus, this study proved protein microarrays can serve as a sensitive, rapid, and simple tool for large scale identification of viral-specific antibodies in sera (zhu et al. 2006) . a significant number of studies have been published that proves protein microarrays to be a valuable technique for diagnosis and screening of viral peptides (lu et al. 2005; reusken et al. 2013 ) a recently published study involves the development of an opto-microfluidic sensing platform to rapidly detect antibodies against sars-cov2 spike protein in diluted human plasma with high sensitivity. the technique was developed based on localized surface plasmon resonance (lspr) involving gold nanospikes in a microfluidic device which is further coupled with an optical probe. the platform achieves the limit of detection of 0.5 pm and takes up to 30 min to analyze a sample. this could be a great improvisation in the diagnosis of the covid-19 (funari et al. 2020) . taking immunohistochemistry a level ahead, tissue chenmicroarrays involve the analysis of protein expression in a multitude of tissue samples and ihc to be used in a highthroughput setting (andersson et al. 2006) . tma plays an important role in target validation of results from cdna arrays and expression profiling of tissues which have the major significance in proteomic research (avninder et al. 2008) . this technology has been proven significant in various types of cancers including breast cancer, adenocarcinoma, lung cancer, etc. (jacquemier et al. 2005) . techniques, such as nuclear magnetic resonance spectroscopy and x-ray crystallography, have been in use to determine the structural parameters since a long time. nmr studies for sars have provided countless insights in its structural factors. innumerable articles have been published elaborating various structural aspects of the virus (mahajan et al. 2015; johnson et al. 2010) . structural elucidation of sars-cov 2 involving nmr has also provided us with helpful perceptions about this new virus. one such study analyses glycan structures of the receptor-binding domain of sars-cov2 spike glycoproteins that are expressed in human hek293f cells. the study provides strong evidence for the presence of glycan structure which were not found in earlier ms-based analysis. a number of different possible interacting isotopes have been analyzed. the study proposed 3d models of these interacting complexes (lenza et al. 2020 ). metabolomics deals with the identification, quantification, and characterization of endogenous and exogenous metabolites. it is based on the idea that endogenous metabolites can accurately indicate even a minute variation in the metabolism. thus, due to its high sensitivity and specificity, metabolomics can be considered as a valid tool for exploring the interrelationship between the virus and the human body. when a virus enters the body, it alters the cellular metabolism and triggers new pathways; hence, the analysis of the metabolites produced turns out to be crucial for elucidating new features related to the infection and development of better non-invasive, early methods of diagnosis. although very few numbers of studies have been published regarding the metabolomics approach in viral infections, covid-19 has already been analyzed by this approach (yu et al. 2011; mussap et al. 2013; noto et al. 2014; beale et al. 2019) . metabolomic and lipidomic analyses performed on the plasma of covid-19 patients revealed a correlation between metabolite and lipid alteration and course of disease in these patients which suggested that the development of covid-19 affected their whole-body metabolism. particularly, malic acid (tca cycle) and carbamoyl phosphate (urea cycle) exhibit alterations in energy metabolism and hepatic dysfunction, respectively. moreover, carbamoyl phosphate is shown to be down-regulated in patients with severe conditions compared with patients having mild symptoms. interestingly, guanosine monophosphate (gmp) is significantly altered between healthy and covid-19 patients, alterations in gmp levels also differ among patients with mild and fatal symptoms. the findings of this study align with the severity of the disease and its progression. their work provides valuable knowledge about plasma biomarkers associated with sars-cov-2 against which therapeutic markers can be developed (wu et al. 2007 ). proteomics has been widely used in the treatment and analysis of cancer tumors, such as brain cancer ovarian tumors, breast and lung cancer, and various types of adenocarcinomas (petricoin et al. 2002; gast et al. 2009; okano et al. 2006; khalil et al. 2007) . their potential drug targets and novel biomarkers have been discovered (srinivas et al. 2002) . it has been established that infectious diseases have always been a major cause of death worldwide. the pandemic in which we are living now has been aroused earlier also. not just once, but sars viruses have ascertained that the same clan of virus can be the reason for contagion for thrice. hence, sars viruses have always been the topic of great concern and current proteome research. sera from patients have been analysed and a potential biomarker, truncated α-1 trypsin, has been identified. this protein marker is observed to be elevated in sars patients as compared to healthy individuals (yi 2004) . drug resistance mechanism and identification of the biomarkers that are resistant to the specific strains are some of the milestones which are required to be overcome for the effective treatment of sars and other infectious diseases. another major setback of these infections is the similarity of symptoms among sars and non-sars patients. a study overpowered this obstacle and suggested that serum proteomic fingerprints could be used to identify sars cases at their onset; thus, correct treatment could be administered simply by differentiating between sars and non-sars patients. by the application of seldi (surface-enhanced laser ionization) protein chip technology, they compared 39 patients with early-stage sars infection and 39 non-sars patients. their proteomic pattern was analyzed by bioinformatics and biostatistical analyses. the study showed twenty features to be significantly different in the two groups. fifteen were increased in the sars group and 5 were decreased (pang et al. 2006 ). an attempt to map the humoral antibody response to sars-cov-2 proteins can be made which may aid in the development of antibody-based assays for diagnostic and therapeutic purposes (srivastava et al. 2020) . a recent study demonstrated that some serological antibodies can neutralize the viral entry into the host cells through the angiotensinconverting enzyme 2 (ace2) receptor. essentially, the number of commercially available antibodies for sars-cov can also target sars-cov2 proteins . another study reported a comprehensive sars-cov2 human protein-protein interaction map using affinity-purification mass spectrometry. in this study, 332 high-confidence, sars-cov2 human protein-protein interactions were defined, 66 human proteins of them could be targeted by several existing fda approved drugs or drugs under clinical trials (gordon et al. 2020 ). integrated multi-omics studies in combination with host-pathogen interactomics analyses can lead to the recognition of therapeutic targets for this novel infection which is required for drug remodeling and development of new drugs and vaccines. host proteome and metabolome profiles should also be considered using a nasopharyngeal swab, bronchoalveolar lavage fluid, and blood samples from patients from sars-cov2 for understanding its complex pathogenesis and host immune response against the virus (srivastava et al. 2020) . another research observed dysregulation of macrophage, platelet degranulation, and complement system pathways and massive metabolic suppression in patients with severe covid-19 when compared to non-severe patients . comparative studies among patients at different levels of sars-cov2 (i.e., asymptomatic stage, early febrile, defervescence, and convalescent stages) must be made which are highly informative to determine how nonsevere or asymptomatic patients progress towards severe or deadly symptoms (srivastava et al. 2020) . recently a study identified 27 potential biomarkers that are differentially expressed depending on the who severity grade of covid-19 patients. the markers also check the level of inflammatory factors, coagulation factors, and inflammation modulators and indicate the upstream and downstream levels of interleukin 6 (messner et al. 2020) . another recent study uses single molecule array assays (simoa) for the quantitative proteome studies of the sars-cov-2 spike, s1 subunit, and nucleocapsid antigens in the plasma of coronavirus patients. the results showed the changes in 31 sars-cov-2 biomarkers in 272 longitudinal plasma samples obtained for 39 coronavirus patients. s1 and n antigens were detectable in 41 out of 64 coronavirus-positive patients. it is the first study to detect the sars-cov-2 antigens in the blood plasma of covid-19-positive patients. the data revealed that the viral antigens in the blood are associated with disease progression. (ogata et al. 2020) . a study revealed four pathways that are evidently modulated during sars-cov2 infection in vitro. the study involves an integrative proteo-transcriptomic analysis of sars-cov2infected huh 7 cells. western blot validation of effector molecules of identified pathways revealed a dose-dependent activation of akt, mtor, s6k1 and 4e-bpi at 24 h post infection (hpi). virus production was significantly reduced by the inhibition of the mtor signaling pathway using akt inhibitor mk-2206. further, the authors suggested profound studies are required for the treatment of covid-19 patients (appelberg et al. 2020). sars-cov2 is highly contagious which posed considerable difficulty for systems-level molecular studies and limits its processing in high-end research facilities. however, an urgent effort from molecular biology and proteomics researchers can eliminate this impediment. this would provide us an overview of the novel biomarkers and define point-of-care clinical healthcare 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proteome microarray for mapping covid-19 antibody interactions at amino acid resolution protein labeling by itraq: a new tool for quantitative mass spectrometry in proteome research characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72,314 cases from the chinese center for disease control and prevention ) plasma metabolomic and lipidomic alterations associated with covid-19 the sars-cov-2 outbreak: what we know the structure and functions of coronavirus genomic 3′ and 5′ ends 18o stable isotope labeling in ms-based proteomics the use of proteomics in the discovery of serum biomarkers from patients with severe acute respiratory syndrome viruses and metabolism: alterations of glucose and glutamine metabolism mediated by human cytomegalovirus a highly conserved cryptic epitope in the receptor binding domains of sars-cov-2 and sars-cov a pneumonia outbreak associated with a new coronavirus of probable bat origin severe acute respiratory syndrome diagnostics using a coronavirus protein microarray a novel coronavirus from patients with pneumonia in china acknowledgements authors are gratefully acknowledged to sir ganga ram hospital, delhi, india for providing necessary support.funding none. ethical approval this review does not contain any studies with human participants or animals performed by the author. key: cord-327883-s9nbr5y8 authors: nan title: section virology date: 1990-03-31 journal: zentralblatt für bakteriologie doi: 10.1016/s0934-8840(11)80039-3 sha: doc_id: 327883 cord_uid: s9nbr5y8 nan s71 contains a human endogenous retroviral element related to simian sarcoma virus (ssv) and its associated virus ssa v. by sequence analysis and comparison with the corresponding ssvlssav sequences the genomic organization of s71 element was determined to be 5' gag-s-nrs-pol-ltr 3'. s-nrs represents a region of 1130 bp in s71 that consists of nonretrovira!'sequences. a probe containing the complete s71 element was used to screen two human cdna libraries under low-stingency conditions. 21 clones were isolated which had yielded strong hybridisation signals with the s71 probe. hybridisation of these clones with different fragments of the s71 genome revealed that 12 of these clones contain s-nrs related sequences. five of the isolated clones were sequenced and compared with s71. one of them contains a homologous to the u3-region of the s71 l tr. the outer four clones sequences with 65-82% identity to s71 s-nrs on nucleotide level. the deduced amino acid sequences of the four s-nrs clones are only 30-70% homologous to s71 s-nrs. none of the sequences represent a contiguous open reading frame. these results indicate that s-nrss probably do not have protein encoding function. hybridisation of human genomic dna shows that s-nrs belongs to a multicopy family of related sequences. human immunodeficiency viruses, human t lymphotropic viruses, and the human spumaretrovirus (hsrv) constitute the natural occurring human retroviruses. molecular cloning and sequencing of hsrv revealed four open reading frames (orfs) additional to gag, pol, and env. three additional orfs, bel 1-3, are located berween the env gene and the 3'ltr, one additional orf, sl, is loacted in the central region of the genome. functional analysis of the bel and s genes requires an infectious molecular clone of hsrv, which was not available so far. we have constructed such a clone (phsrv). the biological activity of the clone was confirmed by a variety of criteria: induction of characteristic cpe in susceptible cells infected with cell-free supernatant from cultures transfected with phsrv; indirect immunofluorescence; radioimmunoprecipitation of viral proteins and electron microscopy. furthermore, it is shown that phsrv is able to transactivate the hsrv-ltr. glycoprotein iv of bovine herpesvirus 1 (bhv-l) is one of the major immunogenic glycoproteins of the viral envelope and is involved in absorption and/or penetration of the virus. it is esential for the production of infectious virus and cannot be deleted from the viral genome. in contrast to the analogous proteins of herpes simplex virus (gd) and pseudorabies virus (gp50) the giv of bhv-1 shows a size heterogeneity among different strains. this indicates that in at least one part of the polypeptide backbone the aminoacid composition is variable. comparison of the nucleotide sequences of four bhv-1 strains revealed that the size heterogeneity in these strains is due to different repetitions of a 90 bp sequence, which is located upstream the sequence coding the membrane spanning domaine of giv. we are currently testing the possibility to introduce heterologous sequences into this part of giv to analyse whether the giv can contain antigenic epitopes of different viruses without affecting the functional activity of the giv. the molluscum contagiosum virus (mcv), a member of the family poxviridae, induces epidermal proliferation in man. the genome of mcv type 1 (mcv-l; 188 kbp) had been characterized by physical mapping using a defined gene library of the viral genome and by dna-dna hybridization. the physical maps of the viral genome were constructed for the restriction endonucleases bamhi, clai, ecori, and hindiii. detailed hybridization experiments revealed the presence of repetitive dna sequences located within the terminal regions of the viral genome, e. g. bamhi dna fragment b (18 kbp; 0 to 0.095 mu) and e (10.9 kbp; 0.944 to 1 mu). the fine mapping of these particular regions indicates that the repetitive dna sequences are located within the hindiii dna fragments jl (4.2 kbp; 0.962 to 0.985 mu), k (4.0 kbp; 0.014 to 0.036 mu), pi (2.7 kbp; 0 to 0.014 mu), and p2 (2.7 kbp; 0.985 to 1 mu). nucleotide sequence analysis was carried out. it was found that the hindiii fragments jl and k each contained an inverted repeat of 1682 bp and 1675 bp, respectively. the homology between the both repetitive dna elements was found to be 99%. the analysis of the coding capacity of the determined dna sequences revealed the presence of 9 and 5 open reading frames in the hindiii dna fragment k and the corresponding region in the hindiii dna fragment jl, respectively. positive in-patients (n = 307) and outpatients (n = 30) showed an increasing prevalence of antibodies to herpes viruses. the difference was particularly evident for cmv-igg (100% prevalence in hospitalized, 91.8% in out-patients and 63.5% in hiv negative patients). anti-hbc, with a low prevalence in controls (16.5%) was markedly associated with progression of hiv infection (69.5% prevalence in out-patients, 84.6% in in-patients). the antibody pattern to opportunistic viral infections may be regarded as a marker of pathogenicity in hiv infected patients. because of the strong crossreactivity of the two serotypes of herpes simplex virus (hsv) it is very difficult to distinguish between hsv-l and hsv-2-typespecific antibodies in patients' sera. although genital herpes can be caused by hsv-l in 14% of the cases, hsv-2 is the cause of most recurrent genital herpes (86%). the serological differentiation of past hsv-2 from past hsv-l infections is necessary for identifying pregnancies likely to be complicated by recurrent maternal hsv-2 infection. by improving the enzyme-linked immunosorbent assay (elisa) for hsv-2-antibodies and additional testing of sera by western blot, we were able to specifically identify hsv-l-and hsv-2-antibodies in serum samples. -for the elisa, hsv-2-antigen was immobilized on 96-well micro titer plates (nunc), and patients' sera were added. antibodies were detected by biotin/streptavidin peroxidase. -for the western blot the electrophoretically separated hsv-2-antigen was used. the antigen was blotted to a polyvinylidenedifluoride (pvdf) membrane (immobi-10n™, pharmacia) and incubated with the test sera. antibodies against typespecific glycoproteins of hsv type 2 (gg-92), forming a sharp band, were visualized by the application of biotinlstreptavidin peroxidase. our results showed that the elisa and the western blot correlated in 91.6%. serum samples with high antibody titers against hsv-l showed false positive reaction in the hsv-2-elisa in 8.4% of the cases. the optical density of these sera ranged ±0,2 around the cut-off value of the elisa. these samples could easily be identified by western blot. -serological studies showed, that 30% of the prostitutes (n = 20) have antibodies against hsv-l and 25% against hsv-2. in 40% both types of antibodies could be found. furthermore 70% of female patients in gynecological treatment (n = 20) had antibodies to hsv type 1 and in 10% of the cases against both serotypes. 20% showed no antibody reaction. roughly the same prevalence of hsv-l and hsv-2-typespecific antibodies was found in 20 patients from the dermatological department. a. m. eis-hubinger, k. kurowski, and k. e. schneweis inst. f. med. mikrobiologie u. immunologie, univ., d-5300 bonn 1 a nonneutralizing monoclonal antibody (mab), directed against glycoprotein gc of herpes simplex virus type 1 (hsv-l) and neutralizing mab to glycoprotein gb were evaluated for their ability to protect mice from genital hsv-infection. the nonneutralizing mab had only little effect on virus replication in the mucous membranes, but completely protected the mice from peripheral skin lesions, neurological illness and death. progression of the virus to the central nervous system was obviously inhibited by a modified course of the ganglionic infection: the number of ganglia presenting infectious virus and final latency were reduced, although early latency was not induced. in addition to the effects of the nonneutralizing mab, the neutralizing mab effectively shortened viral shedding from the vagina and converted proliferative ganglionic infection into latency. the different modes of action of the monoclonal antibodies are discussed in context to the possible defense mechanisms of the humoral immunity against virus proliferation in mucous membranes and in ganglia. the thymidine kinase (tk) gene of bovine herpesvirus 1 (bhv-l) can serve as an integration locus for foreign dna into the viral genome, because expression of a functional active tk is not essential for viral replication. the tk -gene of bhv -1 was located by conversion of tk--negative cells to the tk+ -phenotype and marker rescue experiments. it contains an open reading frame of 1068 bp. sequence comparison with other herpesviral tk-proteinsequences revealed, that only certain parts of the amino acid sequence as the nucleotide binding loop are highly conserved. hybridization of rna from infected cells with tk-dna showed, that tk-mrna has a size of 5.1 kb, whereas the size of tk-mrnas of other herpesviruses measures about 2 kb. 5'-and 3'-end of the tk transcription unit were characterized using nuclease 51-analysis. an open reading frame, when translated into protein, is homologous to the glycoprotein h (gh) of herpes simplex virus type 1 and lies downstream the open reading frame of the tk-gene. we conclude, that the expression of,bhv-l-gh correlates with the expression of thymidine kinase. preliminary results suggest, that transcription of both genes is initiated from one common promoter. in foot-and-mouth disease virus (fmdv) infected cells disappearance of the nuclear protein histone h3 and the simultaneous appearance of a new chromatin associated protein termed pi can be observed. we have sequenced the amino terminus of pi and clearly showed that protein pi derives from histone h3 by proteolytic cleavage. the 20 n-terminal amino acid residues are specifically cleaved off early during infection. in addition using an in vitro transcription/translation assay with different fmdv clones we showed that the histone h3 -pi transition is fmdv 3c protease dependent. this protease until now has only been found to be responsible for the processing of the viral polyprotein. the 3c protease (i) is the only fmdv protein required to induce this histone h3-pi transition, (ii) no other viral protein can perform this specific cleavage, and (iii) no viral precursor fusion protein is necessary for this specific cleavage, as it is reported for the processing of the poliovirus p1 precursorpolyprotein. the 3c mediated histone h3 cleavage is not restricted to chromatin derived from natural host cells. as the deleted part of the histone h3 corresponds to the domain presumed to be involved in the regulation of transcriptional active chromatin in eucaryotes, it is postulated that this specific cleavage of h3 is a mechanism which fmdv utilizes to switch off host cell rna synthesis, as is reported for picornaviruses. in combination with the reported mechanism of host cell translation shut off by cleavage of the cap-binding protein complex, this specific histone h3 cleavage could contribute to the almost complete breakdown of host cell functions during infection. the tk of hsv -1 contains three regions of homology to highly conserved sequences of other nucleotide binding enzymes. these are the residues 49 to 66 (nucleotide binding loop), residues 161 to 168 and residues 317 to 319. comparison of the viral and cellular tk protein sequences and 3-d structure analysis led to the hypothesis that the asp 161 might be involved in binding of acyclovir which is used as a therapeutic for hsv infection. site specific mutagenesis which replaced asp 161 by asn led to a polypeptide with no tk activity. the same was found when residues 184 to 306, which are not present in cellular tks, were deleted. both mutant genes were integrated into vaccinia virus to study the biochemical properties of the resulting polypeptides and to analyze the importance of the mutated sequences for the functional activity of the enzymes. . in contrast the human glioma cell line u138 and the adenovirus transformed kidney cell line 293 revealed replication of both organspecific variants as early as 48 hours p.t. nevertheless the amount of replicated dna of the kidney variant was clearly reduced in glioma cells and replicated dna was detected later than with the cns variant. from these data we conclude, thatjcv replication is regulated not only host type specific, but also in a cell type specific manner. young human volunteers were vaccinated with a killed hepatitis a vaccine produced in human embryo fibroblast cells. different amounts of vaccine were administered by the intramuscular route. 4 weeks after the first vaccination with a 0.3!-lg vaccine 36.6% of the volunteers seroconverted. 4 weeks after the second injection the seroconversion rate was 95.1 %. anti-hav was determined quantitatively and results showed anti-hav titers 20 to 100 times higher than those after gamma globulin administration. one year after the vaccination 19/19 volunteers still had anti-hav titers in their sera 20 to 50 times higher than those after gamma globulin administration. human papillomaviruses (hpv) associated with the epidermodysplasia verruciformis syndrome (ev) represent a group of closely related viruses, clearly distinct from other hpvs. the most interesting features of ev-viruses are: (i) high oncogenic potential of some specific virus types, (ii) extremely narrow host range. to get some insight into the molecular basis of processes controlling the viral expression we studied the sequence-specific dna-protein interactions within the genomic regulatory regions. using the nuclear extracts of hela cells and a combination of exonuclease iii-and dnase i-footprinting techniques the protein binding maps have been constructed for hpv8 and hpv19, the prototypes of viruses with high versus low oncogenic potential. the sequences in question showed a complex array of protein binding domains, covering almost the entire length of the regulatory regions. a cluster of prominent binding sites, which overlapped with the sequences of the motif 44 (m44), a highly conserved element in most of the ev-viruses, was investigated in more detail. in transient cat assays the m44 motif of hpv8 as dimer or trimer was found to act as a strong expression activator. the analysis of m44 sequences in band-shift tests revealed partially different sets of dna binding proteins, interacting with the elements from hpv 8 and 19. one of these proteins, at least in case of hpv8, was shown to be the regulatory factor api. a part of m44 sequences in the vicinity of the ap1 binding site display a homology to enhancer elements in other small dna viruses. a. gerritzen, j.-p. kleim, and a. friedrich inst. for med. microbiology, univ., d-5300 bonn semiquantitative detection of hepatitis b virus (hbv) dna in sera of infected individuals has become an important means of modern serological hepatitis diagnostics. it enables investigators to draw conclusions on infectivity, prognosis and therapeutical success. molecular hybridization using radioactively (i. e. 32phosphorus) labeled dna probes with subsequent autoradiography is characterized by both high sensitivity and specificity. alternatively digoxigenin-labeled probes which are detected by enzyme immunoassay have been employed. using this method 1-2 pg of hbv dna in aqueous solution have been detected with high specificity on membranes made of cellulose nitrate. testing sera of hepatitis patients 1 pg of hbv dna has been detected on nylon membranes of 1.2 11m pore size e 2 p: 0.1 pg). unfortunately specificity remained disappointing even after centrifugation, digestion by proteinase k and treatment of the sera with phenole/chloroform. objective. to assess the risk for laboratory personnel resulting from patients with unknown hiv infection, sera sent in to the institute for medical virology and immunology (imvi essen) for immunologic and/or virologic testing were anonymously screened for hiv antibodies. methods. between july and december, 1988, a total of 6252 sera from different departments of the university clinics of essen were selected for screening under code on the basis that no hiv antibody test was requested for diagnostic purposes at the same time, and sera from patients found to be anti-hiv positive during the previous 4 years were disregarded (n = 61). sera reactive in elisa (enzygnost-anti-hiv, behring) were further tested in a second (dupont) and third elisa (pasteur) and in a confirmatory test (western blot -wb-, dupont). results. in 11 of 6252 sera from patients not suspected for hiv infection, hiv antibodies were confirmed by wb (0.176%) (table) . an additional 4 sera showed less than 2 specific we bands and were considered indeterminate. sera from unknowingly infected patients were sent in most often from the internal (n = 5) and dermatology departments (n = 3), whereas sera with indeterminate hiv antibody results mostly originated from patients who received tumor treatment (n = 2). conclusions. the prevalence of 0.176% unknown hiv infections reflects a higher rate than generally assumed but possibly results from a selection in a university of patients with immunological disorders and underlying hiv infection. an overall prevalence of 1 % anti-hiv positive sera suggests the strict adherence to infection control measures in medical laboratories. bovine viral diarrhea virus (bvdv) is a member of the pestivirus group. in culture of bovine cells, a non cytopathic (ncp) and a cytopathic (cp) biotype can be distinguished. using radioimmunoprecipitation a monoclonal antibody (mab) detected a 125 kd nonstructural (ns) polypeptide in cells infected with ncpbvdv. in cells infected with cpbvdv an additional 80 kd protein was precipitated. a second set of mabs was directed against the 48 kd minor glycoprotein. the distribution of both proteins in cells infected with the two biotypes was analyzed by immunofluorescence analysis (ifa). none of the two proteins was detected on the surface of live, infected cells. the ns protein was homogenously distributed in the cytoplasm of cells infected with both biotypes. the second set of mabs displayed different staining patterns in cells infected with each of the biotypes. in cells infected with cpbvdv, a homogenous cytoplasmic fluorescence was visible. in cells infected with ncpbvdv, the stain was largely restricted to the perinuclear zone, giving a distinct staining with each of the mabs. the possible significance of these results for cytopathogenicity are discussed. in sera of patients with a variety of inflammatory rheumatic diseases autoantibodies to selfantigens (autoantigens) are found. one of the hypotheses on the molecular basis of autoimmune diseases which is discussed intensively since decades, assumes that structure-or sequence-related epitopes of virus and host proteins might be involved in initiation of autoimmune processes (molecular mimicry). -to search for crossreactive or sequenceidentical epitopes of cell and virus proteins, we started to map in detail antigenic regions and individual epitopes on autoantigens. a most detailed study has been performed for the ulsnrnp specific p68 protein which is the major target of autoantibodies in systemic rheumatic diseases such as mixed connective tissue disease and systemic lupus erythematosus. -by immunoblotting and elisa assays performed with recombinant p68 fusion proteins and peptides four antigenic regions and many patient specific epitopes could be mapped. in one of the antigenic regions an epitope 5 amino acids long could be identified which is also present on the matrix protein m1 of influenza b viruses. with affinity purified p68 specific autoantibodies the reaction with the m1 influenza b virus protein and vice versa could be experimentally verified. -these results demonstrate for the first time that autoantibodies from patients with rheumatic diseases recognize an epitope of identical sequence on a highly prevalent and pathogenic virus and a major auto antigenic target. whether the existence of this common epitope is by chance or causually related is currently investigated. detection of antibodies against cytomegalovirus (hcmv) induced "early" -antigens by immunoblotting contradictory informations about the diagnostic assessment of antibodies against the "early" -antigens of human cytomegalovirus (hcmv) have been numerously published. we investigated the correlation between the incidence of antibodies against hcmv "early"proteins and the state of infection. ninety-six hcmv-igg-positive sera (elisa) were tested for specific igg-antibodies against hcmv "early"-antigens by immunoblotting. three groups were examined: (a) 29 renal transplantation patients, (b) 33 aids-patients and (c) 34 randomly selected healthy individuals. each group yielded approximately the same percentage of positive immunoblots (59%,58%,59%). sera belonging to group (a) or (b) reacted stronger and recognized a greater number of polypeptides (6±4; 5±3) when compared to healthy persons (4±3). all immunoblot-positive sera reacted at least with the 66kd protein (major "immediate early"-protein). fourteen out of 19 hcmv-igm-positive sera (elisa) belonging to group (a) or (b) recognized "early"-antigens (74%). twenty-two out of 43 hcmv-igm-negative sera (51 %) reacted as well. we conclude that an acute hcmv infection does not cause the formation of antibodies against "early" -antigens in all individuals and furthermore these antibodies can persist during a subclinical/latent infection. berne virus (bev), isolated from a horse, is the prototype of the toroviridae, a proposed new family of positive-stranded rna viruses. bev virions consist of a peplomer-bearing membrane which envelops a tubular nucleocapsid of helical symmetry; this structure can be straight (resulting in a tubular particle) or bent into an open torus (conferring the shape of a kidney or biconcave disc to the virion). the nucleocapsid contains a single polyadenylated rna molecule of > 25 kb and the most abundant polypeptide, an 18.3kd basic phosphoprotein with nucleic acid binding properties. upon infection, a set of 5'-coterminal sub genomic mrnas is synthesized by leader-independent transcription; only the unique 3' sequences of each mrna are translated. -the combination of virion structure, nucleocapsid protein size and leader-independent transcription is unique in virology and justifies a family status for bev and related viruses. however, coronaviruses have a similar genome organization, a 5'-coterminal nested set of mrnas and sequence similarities in the second orf of the polymerase gene of bev. since the latter suggest common ancestry, toro-and corona viruses together may be considered as a third evolutionary cluster of positivestranded rna viruses, in addition to the alpha-and picornavirus superfamilies. w. ]ilg, h. mairhofer, c. markert, and h. wolf max v. pettenkofer-inst. for hygiene and med. microbiology, univ., d-8000 munich in order to characterize viral and nonviral structures responsible for the recognition of ebv-infected lymphocytes by the immune system, we studied the reactivity of ebv-specific cytotoxic t cells towards autologous ebv-positive lymphoblastoid cell lines (lcls). lcls were established from different ebv-positive donors by either spontaneous outgrowth of cells transformed by the donors own virus or by infection with a laboratory strain of ebv (b 95-8). these cell lines were used for the generation of ebv-specific cytotoxic t cells by weekly stimulation and addition of interleukin 2; cells were cloned by limited dilution. in chromium release assays these cd8 positive t cell lines and clones were able to discriminate between two autologous lymphoblastoid cell lines infected by either the "own" virus strain or the b 95-8 strain, respectively. further experiments using various concentrations of effector cells showed that structures recognized by different t cell clones were expressed only in a certain percentage of cells (20-40%) of each lcl, and that nonviral components differently expressed on different lcls also seem to playa role ctlllcl interactions. the large-scale production and purification of recombinant gp 160 leads to a native and biologically active protein, which bounds specifically to the cd 4 receptor. anti-hbs response of hbs vaccine recipients within an ongoing immunization course was studied in vitro. antigen specific antibody production and proliferative response as well as the expression of cd23 and cd25 and the secretion of scd23 were detected. it was the aim of the study to analyze the effect of various cell stimuli (mitogens, lymphokines and antibodies against cell surface antigens) on the antigen-specific immune response in vitro. pwm driven enhancement in hbs specific antibody production was shown to be time dependent. in contrast to iii and il6, incubation with il4 led to a significant increase in anti-hbs antibody synthesis. low doses of 114 led to a significant increase in hbs induced cd23 expression. moreover, 114 induced scd23 secretion is enhanced by hbs antigen. il2 receptor expression of hbs vaccine non-responder pbmc is reduced as compared to the responder group. cd25 receptor expression of responder pbmc is influenced by antigen as well as iii whereas no modulation can be seen with non-responder pbmc. the capacity of nonresponder cells to respond to hbs antigen is reduced whereas the capacity of these cells to respond to iii is markedly enhanced. in summary, our data show that the hbs specific immune response in vitro is influenced by lymphokines. 38.9% of sera from women (n = 560) and 32.2% of sera from men (n = 96) were found to contain antibodies directed against the l2 gene products of the hpv types mentioned above. 9.6% of female and 12.5% of male sera exhibited antibodies directed against the hpv-16 e4 and/or e7 gene products. on the other hand in an individual female serum antibodies directed against hpv-16 el and e2 could be found, and another one harboured antibodies directed against hpv-16 e6. all of the antibodies were of igg type. in order to specify whether antibodies against papillomaviruses are associated with sexual activity, we used sera from 100 female individuals of age 14 years old. we tested also the sera from the same individuals which were subsequently taken at time intervals of 3 and 10 years. it could be shown that l2 antibodies were present in the sera over the whole period of time. however, also an increase in the frequency of antibodies directed against hpv -16 ll with growing age of the women was observed. these data indicate that beside the distribution of human papillomaviruses by sexual intercourse other routes of papillomavirus infection may exist, for instance perinatal infection. further experiments revealed differences in salt stability and ph dependence between gd, gb, and gc with respect to their binding ability. thus, binding of gd could be disrupted at 500 mm nacl and had a ph optimum between ph6 and ph7, whereas binding of gc and gb showed a ph optimum near ph7 and was stable even at salt concentrations above 1000mm nacl. the results given above provide direct evidence for a functional role of gc, gb, and gd in binding of hsv-1 to the cell surface. to 4 h. as demonstrated by electron microscopy, the mechanism of this effect is due to irreversible binding of the metal ions to the viral membrane, presumably to mercaptan groups of the viral glycoproteins. adsorption of zinc inactivated virus is reduced to some extent only whereas a heavy impact has to be presumed on virus penetration. only in a small concentration range (100 to 200 i!m zn in culture medium) zns04 is inhibitory to the virus replication in infected cells without serious toxicity to the cells themselves. -therefore the mechanism of the in vivo efficacy of zinc preparations against hsv lesions of the mucosa is mainly based upon inactivation of free virus and only partly due to virustasis or inhibition of hsv adsorption. experiments done either with synthetic peptides or with cells transfected with the gene encoding the ebv latent membrane protein bnlfi suggested that this protein is a target for ebv specific cytotoxic t-cells (ctls). as such experiments do not closely resemble the natural situation we wanted to expand on this topic and established a system which should better reflect in vivo conditions. we constructed two recombinant vaccinia viruses, one carrying the complete bnlf l-ma reading frame, the other encodes a truncated form of bnlf i-ma containing the third exon only which was found on ebv infected burkitt lymphoma in addition to the downregulated full length protein. the continuous coding sequence of the complete gene which is necessary for expressing foreign proteins in recombinant vaccinia was cloned from genomic fragments and synthetic oligonucleotides. after infection of several cell types including freshly isolated pbls the expression of both forms of lmp was clearly demonstrated in immunoblots and by immunofluorescence. -ebv -specific cytotoxic t-iymphocytes (ctl) generated by repeated stimulation with autologous ebv infected lymphoblastoid cells in the presence of il-2 were reacted with autologous pha stimulated blasts infected with the two vaccinia viruses and the wild type virus as a control. ctls of two persons recognized the full length bnlfi protein but not the shortened protein whereas ctls of two other persons did not recognize any of the two bnlfi proteins. from these experiments we conclude that 1., the n-terminus of the bnlfi protein contains a determinant which is recognized by some ebv-specific ctls and 2., that there are additional proteins which are targets for other ebv-specific ctls. the expression of the epstein-barr virus regulation gene bmlfi is regulated by a promoter/enhancer complex located upstream from the short orf bslfi (transcribed as a spliced unit). the region 5'-proximal to the tata box contains consensus sequences for binding transcription factors (e.g. nf-l, ap-l). we analyzed the activity of this control region in different cell systems using appropriate reporter assays (chloramphenicol acetyltransferase and hepatitis b virus surface antigen as reporter genes). -a 127 bp aluilbsteii fragment including the tata box was shown to be sufficient to promote the transcription in raji cells induced to lytic ebv expression by different procedures. responsiveness to phorbolester was shown in ebv negative cells (3t3). the upstream region of the bmlfi promoter (1333 bp bsteiiissti fragment) was identified as regulatory segment in transfection assays showing both positive and negative effects depending on the presence and state of ebv. -in ebv negative cells (hela), the upstream element clearly responded to the ebv transactivator brlfl. this specific trans-activation of the distal control region by brlfi (expression vector pksvr kindly provided by a. sergeant, lyon) was down-regulated in latently infected cells (raji) while in ebv producer cells (p3hr-l) trans-activity by brlfi was detected. following insertion into a heterologous expression system the bmlf1 upstream control region enhanced transcription of the sv40 early promoter independent of its orientation. we have previously shown that mutants of prv that do not express the nonessential glycoprotein gill adsorb less readily to cells than does wildtype virus. we show here that the first interactions of prv with target cells occur via binding of the virus to a heparin-like component on the cell surface and that the viral protein mediating this binding is glycoprotein gill. this conclusion is based on the following findings: 1) heparin inhibits adsorption of wildtype prv effectively as measured by plaque formation as well as by attachment of radioactively labelled virus to cells. however, it affects adsorption of gill-mutants only slightly. 2) while wild-type prv binds well to matrix-bound heparin binding of a giiimutant is dramatically reduced. 3) pre-treatment of cells with heparinase reduces plaque formation and adsorption of wildtype pry by 90% but does not affect gill mutants. 4) of the pry membrane proteins glycoprotein glii binds most abundantly and specifically to heparin sepharose beads. our results indicate that binding of pry via glycoprotein glii to a heparin-like cellular component promotes efficient adsorption. in the absence of glii or of the heparin-like cellular receptor the virus adsorbs by an alternative less efficient mode. a. e. metzler and r. wyler inst. of virology, univ., ch-8057 ziirich, switzerland bovine herpesvirus 1 (bhv1) causes two well established entities, namely infectious bovine rhinotracheitis (ibr) and infectious pustular vulvovaginitis (ipv). encephalitis, a third entity, is less well understood. whereas ibr usually follows horizontal virus spread by aerosols, ipv is acquired by venereal contact. it remains controversial if the conditions were associated with distinct viruses. restriction endonuclease analysis of viral dna or evaluation of viral proteins following separation in sds-polyacrylamide gels may be used to distinguish between bhv1.1, bhv1.2 and bhv1.3. the proteins of european field isolates, recovered from distinct disease episodes within the time period 1960 to 1985, were compared with the proteins of established laboratory strains. of 74 field isolates 43 were classified as bhv1.1, 31 as bhv1.2 and none as bhv1.3. bhv1.2 was most regularly recovered from cattle with genital afflictions. however, some bhv1.2 strains originated from animals with ibr and others from aborted fetuses as well. the first recognition of bhv1.1 among the field isolates in 1972 coincided with sequential waves of severe ibr outbreaks throughout europe. results obtained with laboratory strains indicated that bhv1.1, obviously of limited pathogenic potential, must have existed at earlier times. together with published data the results suggest that recognition of a strain as bhv1.1 or bhv1.2 does not necessarily reflect a specific pathogenic potential nor a distinct organ tropism. nevertheless, the severe ibr incidences were clearly associated with a virulent bhv1. it has been demonstrated that the late gene products ll and l2 of certain papillomavirus types exhibit dna-binding activity. hepatitis b virus core proteins are also an example for the interaction of viral capsid proteins with nucleic acids. in this case dna binding is mediated by carboxy terminally located clusters of basic aa residues. similarly, the ll gene product of hpv-16 contains also groups of basic amino acids at its carboxy terminus. to examine whether this region is in fact the dna binding site of ll, we expressed different parts of this gene in e. coli as ~-gal fusion proteins. the vector system which we used allows the cleavage of the viral protein from ~-gal by collagenase digestion. out of the different expression products only the whole l1 protein and its carboxy terminal part bound dna. to specify the dna binding site, the coding sequence for the last 30 amino acids of l1 was fused to ~-gal. by this measure dna binding activity could be transferred to ~-gal, which did not formerly exhibit such property. collagenase digestion and use of l1 specific polyclonal antibodies ensured that dna binding was a genuine attribute hpv-16 l1 gene products. the latter enzyme differentiates between a 2,3-and a 2,6-linkages, cleaving preferentially sialic acid attached to galactose via an a 2,3-linkage. na-treated cells were resialylated using specific sialyl-transferases and cmp-sialic acid. only a transferase attaching sialic acid in an a 2,3-linkage to gal was able to restore receptors for bfdv. accordingly, cultured chicken embryo cells were resistant to bfdv infection following destruction of this receptor by na treatment. detection of cytomegalovirus (antigen) in body fluids has become increasingly important.-for that purpose, in our study blood and urine specimens from 60 immunosuppressed patients (52 renal transplant recipients, 2 heart and 2 liver transplant recipients, 4 patients with aids) were tested for hcmv. indicator cell cultures (foreskin fibroblasts) were inoculated with urine and leucocytes, respectively, and 2 resp. 7, 11, and 18 days after inoculation, subjected to an immunoperoxidase staining (ips) using a monoclonal antibody (dupont, f.r.g.) directed against hcmv early antigen. as controls, cell cultures inoculated with leucocytes were examined for hcmv-specific cytopathic effects (cpe) for 60 days. additionally, leucocytes from 30 patients were subjected to in-situ-hybridization with biotin-labeled hcmv ad 169-dna (eco ri j-fragment) probes. serum samples were tested for the presence of hcmv-specific antibodies of ig classes g, m., and a by elisa (behring, f.r.g.). -hcmv was detected in 6 blood and 8 urine samples from 11 patients. 2 blood samples were positive by both ips and cpe, 3 by ips only, and 1 by cpe only. hybridization assays were all negative. in virologically positive cases, titres of hcmvspecific antibodies amounted to (reciprocal titres): igg ;::: 640 (10 cases, with a significant rise of titre in 1 case), igm ;::: 40 (4 cases), and iga ;::: 320 (3 cases). -our results indicate that, for laboratory diagnosis of active hcmv infections, the detection of hcmv early antigen in urine is, with regard to practicability and rapidity, superior to the test for viremia and is to be considered a helpful extension of serological testing. moreover, with urine samples being easily obtainable, the detection of hcmv early antigen in urine is especially appropriate, too, for controlling the course of active hcmv infections. kai-olaf netzer, axel rethwilm, and volker ter meulen inst. f. virologie u. immunbiologie, univ., d-8700 wiirzburg the human spumaretrovirus (hsrv) is a distinct member of the foamy virus subfamily of retroviridae. its genome of about 11 kilo bases has been sequenced. it comprises the typical retroviral genes gag, pol, and env, and at least three more open reading frames possibly coding for regulatory proteins. thus far, not much is known about the gene products of hsrv. by means of radioimmunoprecipitation, we have identified and partly characterized the major immunogenic antigens of hsrv. radiolabeled viral proteins precipitated by hsrv-positive sera (but not by hsrv-negative control sera) were in the range of 32 to 170 kilodalton (kda) apparent molecular weight. labelling with 14c-glucosamine, or with 35s_ methionine in the presence of tunicamycin led to the identification of three viral glycoproteins of 170, 130 and 48 kda apparent molecular weight, respectively. these glycoproteins most likely represent env gene products. a phosphorylated protein of 60 kda may be related to the gag gene of hsrv. the results of this study show that radio-immunoprecipiation provides a powerful diagnostic and research tool to identify hsrv-positive sera. thomas nowak! and gerd wengler inst. f. virologie, univ., d-6300 gief~en, !present address: behringwerke ag, d-3550 marburg the primary structure of the nonstructural proteins ns1, ns2a, ns2b, ns3, ns4b and ns5 of the wnv has been determined. the nonstructural proteins were isolated from nuclear membrane fraction of wnv infected bhk cells. aminoterminal sequence data of these purified proteins were determined. together with the published amino acid sequence of the nonstructural coding genom region (castle et ai., 1986, virology 149, 10-26) we obtained the sequences of the nonstructural proteins ns1 (50 kd), ns2a (19 kd), ns2b (14 kd), ns3 (70 kd), ns4b (27 kd) and ns5 (97 kd). the gene order, the sizes of the virus coded proteins and the processing of the nonstructural proteins appears to be identical between the flaviviruses. it is well accepted that the functional impairment and killing of infected cells, hypersensitivity and autoimmunity contribute to the symptoms and pathology of viral diseases. we will discuss evidence for an additional mechanism can be defined as the uncontrolled activation of host effector functions not focused on viral antigens. "autotoxicity" is evident in the following situations: (i), certain paramyxoviruses and influenza viruses are capable of activating the generation of reactive oxygen species in phagocytes in the absence of antiviral antibody. this reaction is triggered by the binding of viral surface glycoproteins to the plasma membrane of the phagocytes. when injected into the blood stream, these viruses exert toxic effects independent of viral replication. -(ii), liver damage is observed in a model of murine influenza despite the apparent lack of viral replication in this organ. this effect may be mediated by cytokines. -(iii), in canine distemper, demyelination occurs despite the apparent lack of viral replication in oligodendrocytes, the cells which form myelin. degeneration of uninfected oligodendrocytes is also observed in dissociated brain cell cultures. as measured by luminol-dependent chemiluminescence, antiviral antibody stimulates the generation of reactive oxygen in microglial cells by linking fe receptors with viral antigen expressed on the surface of infected cells, e. g. astrocytes. in these cell cultures, oligodendrocytes can readily be destroyed by reactive oxygen species generated by xanthine/ xanthine oxidase. about 112,000 sera (january, 1984 to august, 1989) were analyzed retrospectively for hepatitis a or hepatitis b markers; 83,000 of these were evaluable. age distribution and seasonal distribution of acute hepatitis a virus infections in groups of patients with german or foreign names were determined. most cases of acute hepatitis a in the foreign population occurred between september and december, and most patients were below 15 years of age. in the german population no seasonal peaks could be found. however, there was one peak between 5 and 9, and another between 20 and 49 years of age. -we did not see seasonal peaks of acute infections with hepatitis b virus. no age was preferentially affected, but infections occurred earlier in foreign population than in german people. -in both groups cases of acute infections with both hepatitis a and hepatitis b viruses were only rarely found. more foreign people than german had markers for hepatitis a or hepatitis b (81 % vs.59%). the glycoprotein complex gil belongs to the essential membrane proteins of pseudorabies virus (prv). therefore mutational analyses of viral gil require growth of the respective mutant in a complementing, gil-expressing cell line. a cell line capable of supplying gil in trans was isolated after co-transfection of a genomic pry-dna fragment encompassing the complete viral gil-expression unit and the plasmid psv2neo conferring resistance against the antibiotic g418. the viral expression unit is usually silent but can be transactivated after superinfection by herpes simplex virus (hsv-l). transient expression of the pry "immediate early" protein is not sufficient to induce transactivation. by immunofluorescence and radioimmunoprecipitation using gil-specific monoclonal antibodies expression of authentic gil could be demonstrated. attempts to isolate a constitutively gil-expressing cell line failed, presumably due to the toxicity of the expressed glycoprotein. results regarding processing of gil without the context of a pry-infection will be presented. furthermore, availability of this cell line enables us to specifically mutate the gil-gene in the pry genome and characterize the resulting mutants biologically. cellular and humoral immune reactions are important for the pathogenesis of viral infections. in our animal model of mv encephalitis, lewis rats develop a subacute cns disease process (same) in the presence of only low levels of neutralizing antiviral antibodies. the contribution of the mv-specific cellular immunity to this disease is yet unknown. therefore, the in vitro reactivity of polyclonallymphocyte cultures to mv structural components was determined. additionally, the possibility to replace purified viral antigens by bacterially expressed fusion proteins was studied. t cells were primed by immunization with antigens in emulsified in freund's complete adjuvant. t cells prepared from regionallymphnodes were taken into culture 9-12 days later and lymphoproliferation was measured by the incorporation of tritiated thymidine in the presence of specific and irrelevant antigens with a panel of antigen-specific t cell lines. with all polyclonal t cell populations except those primed with recombinant haemagglutinin (pbd-h) a specific proliferation was obtained when either whole inactivated mv or the immunizing antigen was used for restimulation. while the nucleocapsid (n) or matrix (m)-specific cell lines recognize equally well pbd-n and virion purified n (nv), or pbd-m and mv respectively, such substitution for pbd-h and hv was not found. our observations indicate the induction of a differential t cell dependent immune response against the procaryotically expressed h compared to the h glycoprotein purified from virion. k. rittert, b. nellent, h. eiffertl, h. kratzin 2 , and r. thomssen 1 in the course of acute epstein-barr virus (ebv) infection igm antibodies always occur against two cellular antigens that were characterized as proteins with a molecular weight of 26 kd (p26) and 29 kd (p29), respectively. purified p29 was identified as a monomer of human triosephosphate isomerase (htpi). p26 is a so far unknown protein that possesses a high homology with triosephosphate isomerase of rabbits (rtpi). -the two autoantibodies are produced only as igm class antibodies, there is no switch to igg. presumably these antibodies are monoclonal in 40 percent of the patients. -in 25 percent of the cases with acute hepatitis a virus (hav) infection anti-htpiirtpi antibodies were found, too. acute ebv infection as well as acute ha v infection may be complicated by hemolysis of different extent. igm anti-htpiirtpi antibodies purified by affinity chromatography caused an increased 5tcr release from human erythrocytes (rh negatice, group 0). the contribution of these autoantibodies to hemolysis in acute viral infections is likely and will be further investigated. in first attempts we tried to express hbv pre-s(2) epitopes as aminoterminal fusions with hbc, all attempts resulted in no detectable expression due to instability or toxicity. we then inserted oligonucleotides coding for two overlapping pre-s(2) epitopes (dprvrglyfpa) o. immunol. 137: 2703 immunol. 137: , 1986 ) at the site where in duck hepatitis virus core antigens an insertion of 39 amino acid is found compared to the mammalian hepatitis viruses and predicted to be at the surface of particles. the insertion results in the expression of stable fusion proteins with pre-s2 antigenicity in western-blots. the chromatographic behaviour of hbdpre-s(2) chimaera on sepharose 4b is similar to that of authentic recombinant core particles, suggesting that they assemble to particles. a rabbit anti-pres(2) 120-145 antiserum (kindly provided by r. neurath) recognizes the hbdpre-s(2) particles in a non-denaturing elisa type assay, a monoclonal antibody to the second epitope does not, which shows that a part of the inserted sequence is accessible on the particles. since we can now express hbc particles carrying pre-s2 epitopes in e.eoli the immunologic properties (induction of t-cell and b-cell responses) of purified particles can be investigated. w. the characterization of an enzyme-linked immunosorbent assay, ®enzygnost-hsv (ag), for the identification and typing of hsv is discussed. -differentiation of hsv was evaluated against 26 laboratory hsv strains. it was shown that the typing indices were valid over a broad dilution range, which is essential for samples of both low and high antigen content. -in a preliminary clinical study (n = 93 specimens) direct testing revealed an identification sensitivity of 89.5%, a typing sensitivity of 90.9% and overall specificity of 93.9%. in a confirmatory test, a 100% agreement for both identification and typing was obtained. -on the basis of these results, it was concluded that ®enzygnost-hsv (ag) is a suitable alternative to the cell culture method, since it overcomes the failure of virus isolation due to possible inactivation resulting from improper transport and/or storage of the specimens. -in comparison with conventional hsv detection and typing test systems, ®enzygnost-hsv (ag) offers the following advantages: -ease of performance (within 4h), -less expensive, -subjective reading of test results using elisa photometers, -computer-controlled automation using microtitre plates is possible. sera of 48 persons in the age between 20-to 30-years were tested parallel with the biotin! avidin western blot (b/awb), with the biotin!avidin enzyme-linked immunosorbent assay (b/aelisa) and with the complement fixation technique (cft). antigens used in all tests of the study were preparations of the human adenovirus type 2. specific igg-antibodies directed against two immunoblotted group-specific major adenoviral polypeptides, the hexon epitope a-antigen and the penton base ~-antigen, were found in more cases (85%) than antibodies found with the b/aelisa (81 %) and with the cft (48%). the prevalences of igg-and iga-antibodies to human adenovirus were studied by b/aelisa in 579 serum samples obtained from 12 different age-groups of frankfurt/main, west germany. the lowest igg-antibody prevalence (52%) was measured in the six-to 12-month age-group, increasing to 95% in the six-to seven-years age-group. a lot of adenovirus positive sera with specific iga-antibodies were measured in the one-to two-(30% and two-to three-(16%) years age-groups. a second peak of iga-antibody prevalence (12%) appeared in the six-to seven-years and seven-to eight-years age-groups, where the highest prevalence of anti-adenovirus igg was seen. reverse transcriptase is necessary for viral replication, therefore it is an attractive candidate for antiviral therapy. detailed functional and structural analysis like e. g. cristallographic studies or neutron solution scattering are predisposition for the development of new inhibitors affecting reverse transcriptase activity. -for this purpose we have constructed a plasmid which allows high level expression of the active reverse transcriptase after introduction into e. coli. by partially adopting the e. coli codon usage and adding the original amino-and carboxy terminal sequence by synthetic oligonucleotides we were able to produce the authentic enzyme in e. coli in considerable amounts (up to 10% of the total e. coli protein) and in a very stable form, as shown by coomassie staining and by western blot analysis. mutants with additional amino acids fused at the aminoterminal sequence show a lower expression and an increased accessibility to the bacterial proteases. -the activity of the authentic and of the amino terminally altered enzyme was compared by standard methods and by an activity gel procedure. enzyme activity could be detected only at 66kd and 130 kd, wereas no activity could be detected at 51 kd. the recombinant produced enzyme can be used for purification and crystallographic studies. the genome of hog cholera virus (hcv) consists of an rna of 12 kb in length. the rna contains one large open reading frame(l) which is probably translated into a polyprotein and then processed proteolytically. -metabolic labeling and radioimmunoprecipitation with a polyspecific antiserum led to the identification of four different glycoproteins -gp55, gp48, gp44 and gp33 -in hcv infected cells (2) . inhibition of n-linked glycosylation through treatment of infected cells with tunicamycin resulted in distinct changes in migration behavior of these proteins in sds-page. -various cdna fragments located in the region coding for hcv encoded glycoproteins were expressed as fusion proteins in bacteria. the purified fusion proteins were used to prepare antibodies specific for the respective hcv glycoproteins. these serological reagents were used in radioimmunoprecipitation assays and western blots. the following conclusions can be drawn: 1.) gp48 and gp44 represent differently processed forms of a single protein. in patients with epidermodysplasia verruciformis (ev) hpv8 induces lesions with a higher risk of malignant conversion. there is evidence that the virus occurs also in the normal population. as hpv8 can not be propagated in tissue culture capsid antigens are not readily available for seroepidemiologic studies. we therefore expressed the major structural protein l1 of hpv8 and fragments thereof as ~-gal fusion proteins in bacteria. the expression vector pros encodes a fxa-cleavage site, which allows the separation of viral and bacterial moiety by proteolytic digest. purified viral antigens were used to test 360 sera by western blot analysis. 12% of the sera revealed antibodies against hpv8 l1 at a dilution of 1 : 20. in some cases the titers exceeded 1: 150. the antibody prevalence was similar in all age groups. western blots with l1 fragments showed that the humoral immunoresponse of different persons is not always directed against the same epitopes. the molecular basis for the lack of hbeag in viremic sera of some acute and chronic infected patients is not clear. in this study it was investigated whether this group of patients is infected with hbv variants which cannot synthesize hbeag. -viral dna isolated from sera of 5 hbeag negative chronic carriers was amplified (per) and sequenced directly or after cloning. in all 5 patient's sera hbv variants were found which had in common a stop codon in the precore region. -since the precore protein is the precursor for synthesis of the classical hbeag, one can conclude that the precore mutation of the hbv variants are responsible for the lack of hbeag in the serum of these patients. these results also implicate that the expression of hbeag is not essential for the viability of hbv. at least under immunosuppressive conditions the precore mutation seems not to drastically effect viral replication since a high hbv-dna titer was observed in the serum of one patient after liver transplantation. -whether the lack of hbeag expression is also responsible for the frequent severe and progredient course of infection and the lacking response in interferon therapy is currently investigated with a test specific for precore variant's infection and with animal models. inst. of virology, univ., d-6300 giessen a highly purified nucleoprotein (np) preparation from influenza virus infected cells yielded in addition to the commonly known 56 kd protein a 42 kd component which could not be detected in virus particles. among a series of np-specific monoclonal antibodies some reacted with both proteins and others were only bound by the 56 kd protein. among both types of np-specific monoclonal antibodies only a limited number were bound at the surface of murine cells infected with any type a virus. another category of antibodies bound to cells infected with a given subtype, but failed to react with the surface of cells infected with a different subtype. the results indicate that only restricted antigenic domains of the native np and perhaps np fragments are exposed at the surface of infected murine cells. additionally, the protective capacity of cell-associated np was determined by immunization of mice with the purified np preparation. in parallel, and in order to determine the immunogenic potency of newly synthesized np, mice were immunized with a vaccinia virus recombinant containing the gene for np prior to challenge with infectious virus. although immunized mice produced monospecific antibodies and a cytotoxic t cell response to the employed forms of np, they were not protected from influenza virus infection. the s segment rna of nephropathia epidemica virus (nev) strain hiillniis b1 was isolated by molecular cloning of the corresponding cdna. therna is 1785 nucleotides long with the 3' and 5' termini being complementary for 23 bases. the viral messengersense rna contains one major open reading frame (orf) with a coding capacity of 433 amino acids encoding a 49 kda polypeptide. compared to the hantaan s segment cdna sequencing there is a nucleotide homology of 60% and 61 % homology at the amino acid level. many of the amino acid differences are conservative exchanges. the c-termini of the nev and hantaan nucleocapsid proteins are nearly identical. the hydrophilicity profiles are very similar and most of the potential kinase dependent phosphorylation sites have been conserved. in contrast, the following differences are significant: the calculated isoelectric points of the nev and hantaan nucleocapsid proteins are 5.6 and 6.7, respectively. the most prominent antigenic determinants predicted by the hydrophilicity profiles are located close to the c-terminus of nev and close to the n-terminus of hantaan virus nucleocapsid polypeptides. -bmft project 0318973a. foot-and-mouth diesease virus-infected cells suffer from cytopathic effects. one causative agent is the virus protease 3c, as shown by transient expression of respective cdna in baby hamster kidney cells. in contrast to other edna-encoded virus proteins 3c is not detectable by indirect immunofluorescence. it is, however, detectable as de novo synthesized protein 16 hours after transfection by radioimmunoprecipitation. the enzyme is then indistinguishable in size from that found in virus-infected cells, indicating similar autocatalytical release from fused protein. transcription of protease 3c-encoding edna fragments is inhibited, as well as that of co-transfected fragments which do not encode protease 3c, as analysed by northern blot hybridizations. the shut off of transcription which is one of the cytopathic effects observed in infected cells correlates thus to the production of active protease 3c. the inhibitory molecular mechanisms may involve truncation of the nuclear protein histone h3 at its n-terminus as found by western blotting. this protein is found similarly truncated in virus-infected cells. virusprotein-specific antigenicity and immunogenicity of tissue culture rabies vaccines o. thraenhart, 1. marcus, and k. ramakrishnan inst. f. med. virologie u. immunologie, univ., d-4300 essen pre-and postexposure treatment with inactivated rabies vaccines prevent rabies virus infection without complications in contrast to vaccines of nervous tissue origin. a considerable diversity concerning virus strains (pm, flury lep, era), cell strains (wi-38, mrc-5, chick fibroblast, bhk) and concentration procedures (ultrafiltration, ultracentrifugation) exists between vaccines of different producers. the influence of the various criteria on the antigenicity and immunogenicity were tested in in vitro experiments using mono-and polyclonal antibodies against rv proteins with antigen-elisa, western blot and immuneelectronmicroscopy in unfractionated and fractionated vaccines after rate zonal ultracentrifugation. the immunogenicity was tested in vivo by protection induction (pi) in mice, the results were correlated with the virusspecific antigenicity (vps-ag) in tissue culture supernatants. vps-, immunglobulin-specific and functional antibody-(ab) and interferon-(ifn) induction was tested in human vaccinees. -rv glycoprotein (gp) and nucleocapsid (np) concentration in vaccines were correlated with pi in mice. the gp : np ratio was > 1 in tissue culture vaccines in contrast to brain vaccines. the harvest of gp and np in tc depends mainly on the cell strain. the virion-associated : soluble gp ratio is productionspecific. the concentration virion tc vaccines contain gp, np, m, l, which induce in vaccinees an early, high and long lasting vps-, igg specific ab-response; anti-gp and -np is induced as early as 3-7 days. ifn-induction is correlated with the applied dosis. no nonresponder was observed and all vaccinees had protecting levels of neutralizing ab from 7-14 days onwards and still after 1 year. the frequency and specificity of antibodies to p-gene encoded proteins of human hepatitis b virus was tested in sera of acute and chronically infected patients with and without hepatocellular carcinoma (hcc). for antibody detection an immunoprecipitation gel assay was performed with radioactively labeled polypeptides produced by in vitro translation of rna of different p-gene regions. thus, five antigenic regions were identified. all anti-p antibody positive sera reacted with carboxy terminal p-polypeptides, a subset with polypeptides of the aminoterminal and middle region, and none reacted with p-protein derived from the most sequence variable region. anti-p antibodies were detected at very high frequency in sera of acute (73 %) and chronically infected patients without hcc (87%), but less often in hcc patients (27%). anti-p antibodies appear early in infection and decline prior to hbsag/anti-hbs seroconversion. -these data indirectly demonstrate the expression of most hbv p-gene sequences and the high immunogenicity of p-proteins in vivo. moreover, they establish anti-p antibodies as a frequent serological marker of infection and identify the carboxy terminal region of the p-proteins (s) as immunodominant. the human polyoma virus jc produces brain tumors in hamsters. in these tumors jcv tantigen, the major product of the early genes is generally expressed. the protein plays a central role in the regulation of polyomavirus replication. since tissue culture as a major source for t-antigen is limited to human primary fetal glial cells we characterized jcv tantigen in the hamster cell line hjc derived from a jcv induced medulloblastoma. -southern blot analysis revealed the presence of jcv dna in hjc cells. the physical state was predominantly integrated. the transcription of early genes was demonstrated by presence of jcv specific mrna in northern blots. the nuclear localisation of t-antigen could be established by immunohistochemical staining and by immunoprecipitation with a crossreacting monoclonal sv40 antibody (pab 416) a molecular weight of 85 to 90 kd was determined. furthermore from western blot analysis it could be assumed that high molecular forms of jcv t-antigen are present in the hamster cell line. taken together these data demonstrate that the molecular weight and nuclear localization were as expected from sv40 transformed cell lines. therefore jcv t-antigen in hjc cells shall be used to characterize its dna binding capacity to the origin region of jc virus dna. current laboratory diagnosis of a coxsackie-b-virus (cbv) infection is mainly based on virus isolation, supported by the detection of rising or high virus-specific neutralizing antibody titers. since such high titres have also been found in apparently healthy peopleprobably originated from a subclinical infection and persisting for a year or more -cbv-igm detection may be a more reliable criteria for serological diagnosis. attempts to identify these antibodies in routine context using conventional techniques (nt, immunodiffusion) or solid phase immunoassays (eia, ria, reverse elisa, immunoblot) have failed, despite high sensitivity and specificity, due to high costs or technical disadvantages. in the present study we developed a modified western blot technique (modi-western blot), which enables a rapid and reliable identification of cbv-igm antibodies. in this assay the antigen was electrophoretically separated using an integrated electrophoresis system (phastsystemunit) and transferred to a solid matrix by diffusion blotting. subsequent immunodetection was performed using a biotin-avidin amplification and monoclonal antibodies. to appraise the efficiency of the method 51 human serum samples were analysed for virus-specific antibodies (igm, iga and igg subclasses) to coxsackie-b-viruses. -group specific igm responses could be found in 22 of 31 (70.9%) of igm seropositive cases. type-predominant igm antibodies (9/31) were detected in 3 cases against cbv1, in 5 cases against cbv2 and in 1 case against cbv4. moreover igg/lga assay in 8 patients seropositive for igg and igm revealed in all cases igg3 and iga antibodies, which supports the evidence of an acute cbvinfection. improvement of diagnosis of cytomegalovirus (hcmv) infections in immunosuppressed patients by detection of hcmv using a monoclonal antibody directed against hcmv-early d-6000 frankfurt primary or recurrent hcmv infections can cause severe clinical problems in immunocompromised patients. as far as active hcmv infections in these patients are concerned, the diagnostic significance of serological data can be restricted tiel: virology, in press 2 an unsatisfactory reproducibility and comparibility was observed when six available enzyme immune test kits for anti-hbc were evaluated by four german red cross bloodtransfusion centers. only 62 of 1838 blood donors were consistently positive, but 228 samples produced discrepant results, although five of the assays used the same inhibition procedure of labeled anti-hbc (caspari et ai., j. clin. microbiol., in press). we reexamined the clearly and discrepantly positive samples of this study (besides some negative controls) by an assay which measures the binding of igg to hbc antigen by peroxidase labeled anti-igg. practically all consistently positive samples were confirmed by the different test principle while practically all discrepant samples were negative. this shows that consistent results by different inhibition assays are indeed reliable while divergent results can be neglected. anti-hbc is used in some countries as surrogate test for hcv carriers. using an experimental test kit from ortho diagnostics, 2 of 64 confirmed anti-hbc positiive and 2 of 389 confirmed anti-hbc negative donors reacted repeatedly as anti-hcv positive. these data do not provide evidence that anti-hbc is useful for elimination of hcv carriers. in this study we compared directly the detection level, sensitivity, and specificity of the most sensitive radioactive and the most sensitive non-radioactive method for detecting hepatitis b virus (hbv) dna in patient serum by dot blot hybridization, based on our previous experience with 6 different assay systems. the former employed the 32p-labeled hbv rna probe included in the hepprobe kit (gibco-brl), detected autoradiographically. an advantage of this kit is the low level of radioactivity of the pre-labeled probe « 10 f,tc), thereby permitting its use even in those laboratories lacking a license for radioisotopes. the non-radioactive method involved the use of an hbv dna probe sulfonated with sodium bisulfite (chemiprobe kit, orgenics), followed by immundetection and an enzymatic color reaction. the detection level of the 32p probe was found to be 0.3 pg hbv dna, corresponding to 3 x 10 4 genomes in 50 f,tl serum, compared with only 2 pg with the sulfonated probe. subsequently, sera from 159 patients with various constellations of hbsag, and anti-hbe were tested with both methods. the concordance rate was 71 % (r = 0.42). compared with 32p results, sulfonation showed a sensitivity of 80% and a specificity of 67%. radiolabeling, therefore, still allows the most sensitive and reliable detection of hbv dna in serum. sulfonation could eventually provide a feasible alternative to radioactivity if the viral dna in serum could be sufficiently amplified in vitro, for example with the polymerase chain reaction. such studies are being planned in our institute. key: cord-322955-7dw32xby authors: kathwate, gunderao h title: in silico design and characterization of multi-epitopes vaccine for sars-cov2 from its spike proteins date: 2020-06-12 journal: biorxiv doi: 10.1101/2020.06.03.131755 sha: doc_id: 322955 cord_uid: 7dw32xby covid 19 is disease caused by novel corona virus, sars-cov2 originated in china most probably of bat origin. till date, no specific vaccine or drug has been discovered to tackle the infections caused by sars-cov2. in response to this pandemic, we utilized bioinformatics knowledge to develop efficient vaccine candidate against sars-cov2. designed vaccine was rich in effective bcr and tcr epitopes screened from the sequence of s-protein of sars-cov2. predicted bcr and tcr epitopes were antigenic in nature non-toxic and probably non-allergen. modelled and refined tertiary structure was predicted as valid for further use. protein-protein interaction prediction of tlr2/4 and designed vaccine indicates promising binding. designed multiepitope vaccine has induced cell mediated and humoral immunity along with increased interferon gamma response. macrophages and dendritic cells were also found increased over the vaccine exposure. in silico codon optimization and cloning in expression vector indicates that vaccine can be efficiently expressed in e. coli. in conclusion, predicted vaccine is a good antigen, probable no allergen and has potential to induce cellular and humoral immunity. in december 2019 group of patients from wuhan city of china was found to have pneumonia like symptoms and diagnosed with the infection of beta coronavirus (1) . later it spread across the china and now spread all over the globe. these infections were named as covid 19 disease and the virus as sars-cov2 by who (2) . on 30 january 2020 due to its spread more than 120 countries declared covid 19 pandemic as a public health emergency of international concern. novelty of sars-cov2 is its rapid spread may be due asymptomatic patients (3, 4) and highly sophisticated, time consuming diagnostic methods(5-7). as of 13 may 2020, 7 million confirmed cases with more than 400,000 deaths worldwide (8) . in india due to lockdown positive cases are in control and spread is also marginal, but still after approximately four months' case reports are increases. as of 13 may 2020, a total of 71865 confirmed cases with 2415 deaths are reported by ministry of health and family welfare, govt. of india. till date, no antiviral drugs available to combat the sars-cov2 infections. there are few drug candidates in clinical trials but the process is time consuming (9) . in south korea, lopinavir/ritonavir combination was found significantly effective in lowering of viral load to no detectible or little sar-cov2 titer(10). but in another group study same combination was totally ineffective beyond standard care (11) . another drug pair drug, hydroxychloroquine, an antimalarial drug and azithromycin reported to found effectively associated with reduction of viral load (12, 13) . but qt interval prolongation that may cause life threating arrhythmia (14) and in large scale study both drugs and drug alone compared with neither drugs was not associated with mortality (15, 16) . in a drug repurposing study, remdesivir, lopinavir, emetine, and homoharringtonine have significantly inhibited replication of sar-cov2 (17) . but this was in vitro study, clinical trial reports are underway. similar determined attempts are going through the globe. to break the chain of infection prevention is much better option than treatments. several evidences suggest and support the importance of vaccine to eliminate covid19 epidemic (18) (19) (20) . various epidemiological surveys provide the evidences that naturally acquired immunity can eliminate the sars-cov2 titer (21, 22) . more than 80% patients develop mild and 14% develop severe symptoms of covid19 with zero case fatality rate (23) . presently there is no vaccine available against covid19 disease. efforts are being taken for the development of effective vaccine all over the globe (24) (25) (26) (27) . all these vaccines are under development (28) and may require at least 1to 1.5 year to hit the market (29) . bioinformatics approach can accelerate the process by predicting potential candidate peptides. vaccines against mers, ebola, and human papilloma virus were designed and successfully developed by using bioinformatics approaches (30) . sars-cov2 belong to beta coronoviridae family which includes four endemic viz hcov-hku1, hcov-oc43, hcov-229e, hcov-nl63, two epidemic viruses like middle east respiratory syndrome virus (mers), and sars (31) . this is a non-segmented positive strand rna virus with envelop. envelop proteins are categorized into structural, non-structural and accessory proteins. structural proteins are involved in protection and bind to the host. several proteins of sar-cov2 are important for virulence and pathogenesis. for example nucleocapsid (n) protein n, is essential for rna binding and its replication and transcription (32) . envelope (e) and membrane (m) proteins and instrumental in virus assembly and virulence promotion (33, 34) . e and m proteins are effective in induction of immune response (24) . spike (s), a structural protein is responsible for binding to receptor on host cell, angiotensin converting enzyme 2 (35) . thereby proteolytic cleavage by tmprss2 allows subsequent entry into cell through endocytosis (36) . s protein also found to be involved in activation of t cell response (37, 38) . entire s protein expressed in chimpanzee adeno 38 (chad)-vector showed protection against sars-cov2 in mice and rhesus macaques (39) . single dose of chadox1 ncov-19 vaccine is sufficient to elicit humoral and cell mediated immune response in both animals. viral load was found reduced compared to control animals and symptoms of pneumonia were absent. here we designed a multi-epitopes vaccine from s protein. this vaccine has all the ideal properties like stability at room temperature, immunogenic, antigenic and non-allergen. all the epitopes are good in stimulation of humeral as well and cell mediated immunity. complete spike protein sequence (p0dtc2) of sars-cov2 was downloaded in fasta format from uniprot protein database. this sequence was used for further analysis and obtaining potential epitopes for b and t cell receptors. there are various tools used to predict the epitopes that could be presented to t cell receptors. potential epitopes of various bacteria and viruses are predicted by using such online epitopes predicting tools. following tools were used for prediction of tcr (t cell receptor) epitopes iedb mhc-i processing predictions, mhc-np, netctlpan1.1, rankpep, and netmhcpan4.0. all these tools were used to screen the potential peptides as epitopes for tcr. these tools have various methods to predict the epitopes. iedb (instructor/evaluator database) is a web-based epitope analysis resource includes tools for t cell epitope prediction, b cell epitope prediction and other analysis tools like epitope conservancy etc. this resource use methods based on artificial neural network (ann), stabilized matrix method (smm), and combinatorial peptide libraries(comblib), predicts the peptides way that processed naturally and presented by mhc i (40) . immunogenicity, toxicity, allergen, conservancy, and antigenicity were analyzed for predicted tcr epitopes. iedb mhc class i immunogenicity server and conservancy tool were used for determination of immunogenicity and conservancy. immunogenicity of a peptide mhc complex (http://tools.immuneepitope.org/immunogenicity/ ) were assessed keeping all the parameters at default. protein sequence variants(40) used setting sequence identity 100% and other parameter default. toxipred (http://www.imtech.res.in/raghava/toxinpred/index.html ) online tool predicts the toxicity considering physicochemical properties of selected peptides(41). online server allergenfp v.1.0 (http://ddgpharmfac.net/allergenfp/ ) was used to predict peptides as allergens (42) . antigenicity of epitopes was analyzed by online server vaxijen v2.0 (http://www.ddg-harmfac.net/vaxijen/vaxijen/vaxijen.html ). threshold value was set to 0.5 (43) . this is alignment independent predictor based on auto-cross covariance (acc) transformation epitopes sequences into uniform vectors of principle amino acid properties. accuracy of this server varies in between 70 to 89% depending on targeted organism. six different method were used for the prediction of b cell receptor (bcr) epitopes. all these methods generate fragments the protein. for the prediction of b cell epitopes, it is necessary to find linear sequence of b cell epitope in the protein sequence. bepipred linear epitope predication server (http://www.cbs.dtu.dk/services/bepipred/ ) uses a hidden markov model and propensity scale method. similarly, other properties are also being consider to predict good b cell epitopes (44) . those properties are calculated by different methods at iedb server (http://tools.iedb.org/bcell/ )like kolaskar-tongaonkar antigenicity scale provide physiology of the amino acid residues(45), emini surface accessible score for accessible surface of the epitope(46), secondary structure of epitopes also has role in antigenicity. karplus-schulz flexibility score (47) and chou-fasman β turns methods (48) were used for flexibility and β turns prediction respectively. parker hydrophilicity prediction method (49) was used for determination of in silico hydrophilicity of peptides. high scored and common peptides predicted by various tools were selected for deriving sequence of potential vaccine candidate. different epitopes for t cell and b cell receptors were linked together by gpgpg and aay. to enhance the immunogenicity, ompa (genbank: afs89615.1) protein was chosen as adjuvant and was linked through eaaak at n terminal site of the vaccine. antigenicity of chimeric vaccine was predicted by vaxijen v2.0 (http://www.ddgpharmfac.net/vaxijen/vaxijen/vaxijen.html) and antigenpro. vaxijen 2.0 is alignment free antigenicity prediction tool utilizes auto cross covariance transformation of protein sequence into uniform vector vectors of principal amino acid properties (43, 50) . antigenpro (http://scratch.proteomics.ics.uci.edu/) is also online tool utilizes protein microarray dataset for the prediction of antigenicity. based on cross validation experiment accuracy of this server is estimated to be around 76 % using combined dataset. allertop v2.0 and allergenfp were two online tools utilized for the prediction of allergenicity of chimeric protein. amino acid edescriptors, auto-and cross-covariance transformation, and the k nearest neighbours (knn) machine learning methods are basis of allertop v2.0 (http://www.ddg-pharmfac.net/allertop) for allergenicity prediction (51) . allergenfp, is descriptor-based fingerprint, alignment free tool for allergenicity prediction. the tool use four step algorithm. in first step, proteins are described based on their properties like hydrophobicity, size, secondary structures formation and relative abundance. in subsequent step, generated strings are converted into vectors of equal length by acc. then vectors are converted into binary fingerprints and compared in terms of the tanimoto coefficient. applying this approach to known allergen and non-allergens can identify the 88 % of allergen/non-allergen with mathew's correlation coefficient of 0.759 (42) . for solubility prediction of multi-epitopes chimeric vaccine, proso ii server (http://mbiljj45.bio.med.uni-muenchen.de:8888/prosoii/prosoii.seam) was utilized (52) . proso ii server works on an approach of classifier which utilizes difference between targetdb and pdb and insoluble proteins of targetdb. the accuracy of this server is 71% at default threshold 0.6. protoparam, online web server was exploited for evaluation of physiochemical properties. the properties like amino acid composition, theoretical pi, instability index, in vitro and in vivo half-life, aliphatic index, molecular weight, and grand average of hydropathicity (gravy) were evaluated. psipred and raptorx property online servers were used to determine secondary structure of predicted vaccine. psipred is publicly available webserver, includes two feed forward neural networks works on output obtained from psi-blast. psipred 3.2 attains average q3 score of 81.6% obtained using very stringent cross approval strategies to assess its performance. raptorx property is another web based server prediction secondary structure of protein without template (53) . this server utilizes deepcnf (deep convolutional neural fields), a new machine learning model that predicts secondary structure and solvent accessibility and disorder regions simultaneously (54) . it accomplishes q3 score of approximately 84 for 3 state secondary structure and approx. 66% for 3 state solvent accessibility. tertiary structure of multi-epitopes chimeric vaccine candidate was built using i tasser online server (https://zhanglab.ccmb.med.umich.edu/i-tasser/). the i-tasser (iterative threading assembly refinement) web server utilize sequence-to-structure-to-function paradigm to build protein structure (55) . it is top ranked 3d protein structure web server in community wide casp experiments (56) . two web bases servers were used to refine the 3d structure of multi-epitopes chimeric protein. initially modrefiner server (https://zhanglab.ccmb.med.umich.edu/modrefiner/) and finally galaxyrefine server (http://galaxy.seoklab.org/cgi-bin/submit.cgi?type=refine) was used. modrefiner server is an algorithm for atomic level structure refinement utilizes c alpha trace, main chain or atomic model. output structure is refined in term of accurate position of side chains hydrogen bon network and less atomic overlaps (57) . on other hand galaxy server rebuild the 3d structure, performs repacking, and uses molecular dynamic simulations to accomplish overall protein structure relaxation. structure refined by galaxy server of best quality in accordance with community-wide casp10 experiments (58) . prosa-web (https://prosa.services.came.sbg.ac.at/prosa.php), the errat server (http://services.mbi.ucla.edu/errat/) and rampage (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) web servers were utilized for 3d structure validation obtained after galaxy server refinement (59) . prosa web server calculate the quality score as z score that should fall in a characteristic range (60) . z score obtained for a specific input protein is in context with protein structure available in public domain. errata server analyses non bonded atom-atom interaction the refined 3d structure of protein compared to high resolved crystallographic protein structures (61) . ramachandran plot displays energetically allowed and disallowed dihedral psi and phi angles of amino acids. the plot is calculated based on van der waal radius of the protein side chain. rampage server determines ramachandran plot for a protein that include percent residues in allowed and disallowed regions (62) . more than 90% b cell epitopes are discontinuous that is they are present in small segments on linear protein and the brought to proximity while protein folding. discontinues b cell epitopes for designed vaccine were predicted by ellipro online tool (http://tools.immuneepitope.org/tools/ellipro) at iedb. in this tool three algorithms implemented to determine the discontinuous b cell epitopes. 3d structure of input protein is approximated as number of ellipsoid shapes, calculate protrusion index (pi) and clusters neighboring residues. ellipro defines pi score of each residue based on the center of mass of residue residing outside the largest possible ellipsoid. in consideration with other epitope predicting tools ellipro gave an auc value of 0.732, best of all (63, 64) . molecular docking server haddock (https://bianca.science.uu.nl/haddock2.4/) was used to see the interaction of designed vaccine and tlr4. haddock (high ambiguity driven proteinprotein docking) is information derived flexible docking server (65) . galaxyrefine 3d model of multi-epitopes chimeric proteins, adjuvant tlr2 (pdb id: 6nig) and tlr4 (pdb id: 4g8a) were uploaded for docking at hddock server keeping all the parameter default. finally, top five models were downloaded and by prodigy (protein binding energy prediction) webserver (https://nestor.science.uu.nl/prodigy/) was utilized for prediction of binding affinities (66) . in silico cloning of vaccine construct was predicted by c-immsim server (http://150.146.2.1/c-immsim/index.php). c-immsim server is freely available web based server utilizes position specific scoring matrix (pssm) for the prediction of immune epitopes and machine learning techniques for immune interaction (67) . jcat (java codon adaptation tool) server was utilized for reverse translation and codon optimization. codon optimization is carried out in order to express the construct in e. coli host. jcat (http://www.prodoric.de/jcat) output display sequence of nucleotide, other important properties of sequence that includes codon adaptation index (cai), percent gc content essential for to assess the protein expression in host (68) . finally, vaccine construct was cloned in pet30a (+) plasmid vector by adding xhoi and xbai restriction sites at c and n terminus, respectively. snapgene tool was used to clone the construct to ensure the cloning and expression (69) . iedb recommend, mhc-np, netctlpan1.1, rankpep and netmhcpan3.0 server predicted the potential candidate epitopes from the spike glycoprotein sequence of sars-cov2. epitopes commonly predicated by at least four prediction tools were selected further for analysis parameter. there were four such epitopes with high score and predicted by four different tools (table 1a) . all the four epitopes were immunogenic and antigenic in nature, conserved 100 %, and predicted to be non-allergen (table 1b) . immunogenicity scores for the predicted epitopes ranges from 0.3858 to 0.0961. all the epitopes were with positive score hence considered for further analysis. all the peptides predicted to be nontoxic by toxipred online toxicity prediction tool. after provision of spike protein sequence to the bepipred server showed average score of 0.407, with 0.695 maximum and 0.163 minimum scores (fig1). other properties like physiology of amino acid residues, accessible surface, hydrophilicity, fexibility and β turns were also determined ( table 2 parker hydrophilicity method (1256-1262). three peptides high scored, predicted for t cell receptors and two peptides for b cell receptors were ligated together by gpgpg or aay. additionally, ompa (genbank: afs89615.1) was linked at n terminal end of the designed polypeptides by eaaak linker. for purification purpose sis histidine residues were added. final amino acid residues of designed vaccine were 454 when all the five peptides, linkers and adjuvant were ligated (fig 2) . antigenicity of the designed multi-epitopes vaccine predicted by vaxijen v2.0 server was 0.7048 with probable antigen annotation for virus as target organism at 0.5 threshold. antigenicity predicted for same sequence of vaccine by antigenpro server was 0.765946 with 0.953508 predicted probable solubility upon overexpression. antigenicity of peptide without adjuvant sequence was 0.671028 with 0.861451 probable solubility of peptides upon overexpression. the results obtained indicates that with and without adjuvant predicted vaccine sequence is potentially antigenic in nature. allergenfp and allertop servers predicted that both vaccine with adjuvant and without adjuvant are probable non allergen with 0.84 and 0.77 tanimoto similarity indexes, respectively. the computed molecular weight of designed vaccine was 48.49368 kda and theoretical pi is 7.96. predicted pi indicates the slight alkalinity of the vaccine ( table 3 ). the estimated half-lives were 30 hrs in mammalian reticulocytes (in vitro), >20 hours in yeast (in vivo) and >10 hours in escherichia coli (in vivo). vaccine is highly stable with instability index 24.58 that classify the protein as stable. estimated aliphatic index 79.02 indicating thermo-stability of the protein (70) . the predicted gravy score is -0.175 (table 3) . negative gravy score indicates that protein is hydrophilic in nature and will interact with water molecules (71) . secondary structure prediction: secondary structure predicted by online tool raptorx property includes 16% alpha helix region, 42% beta sheet region and 40% coil region. furthermore, solvent accessibility of amino acid residues predicted to be 42% exposed, 27% medium exposed and 24% buried. total 56 residues (12%) are predicted as residues in disordered region. pictorial representation of secondary structure predicted of final protein by psipred is shown in figure 3 . i-tasser predicted the top five 3d structure model of designed vaccine utilizing 10 threading templates. z score for these 10 templates was ranging from 0.95 to 5.50 indicating good alignment with sequence submitted. c score is critical in the quality of built model which is quantitatively measured. c score ranges in between -5 to 2 signifies the best model quality. c score of top five model ranging from -4.14 to -3.41. model with high c score i.e. -3.41, estimated tm score 0.34±0.11 and estimated rmsd 15.6±3.3 was chosen for further analysis ( fig 4a) (72) . for refinement purpose tertiary structure predicted by i-tasser is initially submitted to modrefiner and finally to galaxyrefine. among top five refined models, model 5 found to be the best based on various parameters like gdt-ha 0.8398, rmsd-.696, molprobit-3.526 and rama favored 77.1 (fig 4b) . quality of refined model 5 was validated by ramachandran plot at rampage webserver. 79.5 % residues of modelled vaccine are in favored region, 13.1% in allowed region and 7.3% are in outlier region (fig 4d) . error in the model are analyzed by prosa web and errat web server. z score is -1.64 ( fig 4e) and errat score index is 74.387 (fig 4c) . ramachandran plot z score and errat score showed that refined model 5 is of good quality and can be used further. four discontinuous epitopes include of 265 residues were predicted. the score of the predicted epitopes ranges from 0.58 to 0.749. shortest and longest discontinuous b cell epitope is of 14 and 97 residues long respectively. haddock web bases server has been utilized to dock the designed vaccine to tlr2 and tlr 4. tlr2/4 eliciting immune response toward designed vaccine was analyzed by conformational change with reference to adjuvant tlr2/4 complexes. top models from each group with lowest haddock score were selected. haddock score for tlr2/adjuvant and tlr2/vaccine were, -89.3 and -95.9 kcal/mol, respectively. in addition, relative binding energies were -9. (fig 5a, b) . similarly, tlr4/adjuvant complex have charged-charged 3, charged polar 10, charged-apolar 21, polar-polar 3, polarapolar 15 and apolar-apolar20. while tlr4/vaccine complex has charged-charged 1, charged-polar 9, charged-apolar 10, polar-polar 9, polar-apolar 15 and apolar-apolar 8 interface contacts were observed ( figure 5c, d) . enhance expression of nucleotide construct in escherichia coli, is reverse translated in jcat online webtool. total number of nucleotides in the constructs were1350. after codon optimization cai was 1.0 and average gc content of optimized nucleotide construct was 50.7340 indicating good probable expression of vaccine in e. coli k12. to clone the gene xho and xbai restriction sites are added at n and c terminus of the construct and gene was cloned in pet30a+ plasmid, by using snapgene software (fig 6) . c-immsim immune simulator web server was used for determining ability of vaccine to induce immunity. results obtained are consistent with the experimental results published elsewhere (73) . within first week of vaccine administration primary response was observed by high level of igm. secondary and tertiary immune response clearly seen with increase in b cell number, level of igm, igg1+igg2, and igm+igg with decreasing concentration of antigen. different b cell isotypes population were also found increasing indicating isotype switching and memory formation (fig 6a) . b cell activities were also found high, especially b isotype igm and igg1, was observed with prominent memory cell formation (fig 6b) . similarly, cell population of th and tc cells are found high along with memory development (fig 6c, d) . in addition, macrophage active was found to be increased consistently after each antigen shots and declined upon antigen clearance (fig 6e) . another cell from cell mediated immunity, dendritic cells were also found increased (fig 6f) . ifnγ and il2 expression was found high with low simpson index indicating sufficient immunoglobulin production, suggesting good humoral immune response ( fig 6g) . simpson index (d) is a measure of diversity. increase in d over time indicates emergence of different epitope-specific dominant clones of t-cells. the smaller the d value, the lower the diversity. till date there is no cure available for covid19, suggesting urgent requirement of drug or vaccine to control the spread of sar-cov2 infections. advancement in bioinformatics tools, process of vaccine development can be facilitated by identifying potential epitopes for t and b cells that will lead to vaccine for prevention of covid19. several reports and animal trials suggest that s-protein can be potential target for vaccine development (22, (73) (74) (75) (76) . there are several reports suggesting the importance of spike protein in activation of cells of immunity and vaccine development (77) (78) (79) . hla-a2 restricted epitopes from s protein of sars-cov2, elicit t cell specific immune response in sars-cov2 recovered patients (27) . similarly serum samples of sars-cov2 recovered patients were sufficient to neutralize epitope rich region on the spike s2 protein from sars-cov2 (80) . programing of live attenuated form of pathogen is commonly used strategy for the vaccine development. despite efficacy of such vaccines, reversion of virulence remains a challenge and not utilized in weak immune patients (81, 82) . here in case sars-cov2 due to its high communal transmission vaccine research requires highly skilled workers, sophisticated instrumentation and biosafety level iii facility. this may likely the slow down the process of vaccine development, enhanced cost leading to restriction on availability of vaccine for mass population. advancement in the field of bioinformatics and molecular biology techniques have provided opportunity of development of vaccine with high efficacy, less time for development, low cost of production that may lead to low cost vaccine in time for large population. we designed a multi-epitopes vaccine construct from s-protein of sars-cov2. several online tools were used for the designing of epitopes and thereby a vaccine. from various epitopes predicted by the online server based on common sequence and high score three tcr and two bcr epitopes were selected as part of covid19 vaccine. all the five tcr and bcr epitopes were linked by gpgpg and aay linker peptides (fig 2) . to enhance the antigenicity of tcr/bcr linked epitopes peptide, adjuvant ompa were linked by eaaak linker sequence for a complete vaccine design which was found to restore antigenicity and nonallergen status. (table 2 ). for successful vaccine candidate, secondary and tertiary structures characters play an essential role. secondary structure of designed vaccine contains 16% alpha helixes, 42% beta sheets and 40% coils. upon refinement tertiary structure of designed vaccine showed improvement to desirable level as indicated by ramachandran plot. designed vaccine showed high antigenicity score, no probable allergen. tlr are essential receptor proteins in activation of innate immune response. that recognize and respond by induction of immune reactions to pamps (pathogen associated molecular patterns). various tlrs have shown to activate immune response to virus through their interaction with nucleic acids and envelopes proteins. tlr3 and tlr7/8 involved in nucleic acids detection, while tlr2 and tlr4 recognize envelope glycoproteins (83) . interaction of s protein with tlr2 increase the production of il8 in human monocyte macrophages (84) . tlr 2 also involved in eliciting innate immune response upon recognition of several other virus components (85, 86) . in a comparative binding efficacy of s-proteins to tlrs, tlr 4 showed strongest proteinprotein interaction with hydrogen and hydrophobic bonds on extracellular domain (87) . in molecular docking analysis, tlr2 and tlr4 showed stable protein-protein interaction with designed vaccine compared to adjuvant protein. tlr 2 showed more stable interaction than tlr4, which is consistent with previous report (87) . stable interaction of designed vaccine indicates efficiency of vaccine for activation of tlrs, involved in dendritic cell activation, thereby subsequent antigen processing, and presentation on surface to t cells (88) . immune simulation showed typical natural immune response pattern upon multiple exposure to same antigen. server predicted elevated level of b cell and t cell for longer time on repeated exposure of antigen. increased level of antiviral cytokine ifnγ and il2 indicate potential of subsequent activation of t-helper cell thereby high level of ig production, supporting humoral immune response (89) . to validate the designed vaccine in the screening of immune reactivity by serological test, it is necessary to express the construct in preferred host like e. coli k12 for recombinant proteins. codon optimization of was performed to achieve high expression designed vaccine in e coli 12. codon adaptability index (cai=1.0) and gc content (51.66%) of the vaccine construct was optimum for high expression of recombinant protein in host. immediate step, here after is to express the protein in preferred host and validate results obtained in this report by performing the several immunological assays. inadequate number of drugs and time-consuming process of drug development, multiplication chain of sars-cov2 infection can be control only by vaccine. we utilized tools and techniques of immune informatics to design a potential vaccine candidate. this vaccine codes epitopes form s protein of sars-cov2 virus for t and b cell receptors. this is one more time showed that bioinformatics approach can effectively use to develop vaccines in short time and with incurred less cost. although in silico results point out the effectiveness of the vaccine, efficacy need to be analyzed by preforming laboratory experiments and animal model studies. properties of that peptides. in case of immunogenic properties peptide number 3 has negative score for antigenicity hence neglected for further analysis. the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak-a n update on the status world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) asymptomatic transmission, the achilles' heel of current strategies to control covid-19 presumed asymptomatic carrier transmission of covid-19. jamanetwork improved molecular diagnosis of covid-19 by the novel, highly sensitive and specific covid-19-rdrp/hel real-time reverse 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mechanistic link between men, obese and elderly designing a multi-epitopic vaccine against the enterotoxigenic bacteroides fragilis based on immunoinformatics approach key: cord-325943-3hvy7b7c authors: bouwman, kim m.; delpont, mattias; broszeit, frederik; berger, renaud; weerts, erik a. w. s.; lucas, marie-noëlle; delverdier, maxence; belkasmi, sakhia; papanikolaou, andreas; boons, geert-jan; guérin, jean-luc; de vries, robert p.; ducatez, mariette f.; verheije, monique h. title: guinea fowl coronavirus diversity has phenotypic consequences for glycan and tissue binding date: 2019-05-01 journal: j virol doi: 10.1128/jvi.00067-19 sha: doc_id: 325943 cord_uid: 3hvy7b7c guinea fowl coronavirus (gfcov) causes fulminating enteritis that can result in a daily death rate of 20% in guinea fowl flocks. here, we studied gfcov diversity and evaluated its phenotypic consequences. over the period of 2014 to 2016, affected guinea fowl flocks were sampled in france, and avian coronavirus presence was confirmed by pcr on intestinal content and immunohistochemistry of intestinal tissue. sequencing revealed 89% amino acid identity between the viral attachment protein s1 of gfcov/2014 and that of the previously identified gfcov/2011. to study the receptor interactions as a determinant for tropism and pathogenicity, recombinant s1 proteins were produced and analyzed by glycan and tissue arrays. glycan array analysis revealed that, in addition to the previously elucidated biantennary di-n-acetyllactosamine (dilacnac) receptor, viral attachment s1 proteins from gfcov/2014 and gfcov/2011 can bind to glycans capped with alpha-2,6-linked sialic acids. interestingly, recombinant gfcov/2014 s1 has an increased affinity for these glycans compared to that of gfcov/2011 s1, which was in agreement with the increased avidity of gfcov/2014 s1 for gastrointestinal tract tissues. enzymatic removal of receptors from tissues before application of spike proteins confirmed the specificity of s1 tissue binding. overall, we demonstrate that diversity in gfcov s1 proteins results in differences in glycan and tissue binding properties. importance avian coronaviruses cause major global problems in the poultry industry. as causative agents of huge economic losses, the detection and understanding of the molecular determinants of viral tropism are of ultimate importance. here, we set out to study those parameters and obtained in-depth insight into the virus-host interactions of guinea fowl coronavirus (gfcov). our data indicate that diversity in gfcov viral attachment proteins results in differences in degrees of affinity for glycan receptors, as well as altered avidity for intestinal tract tissues, which might have consequences for gfcov tissue tropism and pathogenesis in guinea fowls. closely associated with turkey coronavirus (tcov) (7) , with both causing gastrointestinal tract infections in their respective hosts (6, 8) . clinical signs related to gfcov infection in guinea fowl include prostration, ruffled feathers, and decreased water and feed consumption, and gfcov infection has resulted in a daily death rate of up to 20% in several farms in france (6) . upon necropsy of affected animals, whitish and enlarged pancreases were consistently reported. histopathological analyses revealed pancreatic necrosis and lesions of various intensities in the intestinal epithelium, with the most severe lesions found in the duodenum of affected animals (6) . genetic classification of avcovs is based on phylogenetic analysis of the s1 domain of its viral attachment protein spike (2) . the spike protein is the main determinant for tropism (9) , and the n-terminal part of the s1 of ibv has been shown to contain the receptor-binding domain (rbd) (10) . studies using recombinant ibv s1 and/or rbd proteins have demonstrated that the viral tropism is reflected by tissue binding of such proteins (11) . mutations in the spike proteins of ibv might result in either decreased (10) or increased (12) avidity for its receptor present on epithelial cells of the chicken trachea. in contrast to ibv, gfcov and tcov target the epithelial cells of the gastrointestinal tract (4, 6) , and recombinant protein binding of their s1 proteins reflects this viral tropism, with predominant staining of epithelial cells of the small intestine (4) . glycan array analysis identified elongated biantennary n-acetyllactosamine (di-lacnac) molecules on n-glycans as the host receptor for enteric avcovs, which are abundantly expressed on intestinal tissues (4) . clinical symptoms in guinea fowl similar to those reported in 2011 are continuously reported by veterinarians in france (personal communication). however, studies on newly emerging gfcovs are particularly hampered by the lack of models to grow the virus. more specifically, susceptible cell lines have not yet been identified, inoculation of embryonated guinea fowl eggs did not result in gfcov production (data not shown), and specific-pathogen-free (spf) guinea fowls are not available for experimental infection. here, we set out to study the consequences of gfcov genetic diversity for glycan and tissue interactions. we revealed that the gfcov spike gene from the 2014 -2016 outbreak in guinea fowl flocks in france was 89% identical to that of gfcov/2011 (7) . glycan and tissue binding analyses of gfcov/2011 and gfcov/2014 recombinant spike s1 proteins revealed that while both proteins had the same specificities, gfcov/2014 s1 had a much higher affinity toward glycan receptors and tissues of the lower gastrointestinal tract, in agreement with the observed replication of the virus in these tissues from field cases. taking these observations together, we demonstrate that gfcov diversity results in phenotypically different receptor binding properties. lesions and coronaviral protein expression in the gastrointestinal tract of diseased guinea fowls between 2014 and 2016. fulminating disease (peracute enteritis) in guinea flocks continued to be reported after the initial outbreak of gfcov infection in 2011 (6) . between february 2014 and november 2016, duodena from 29 diseased guinea fowls were collected and analyzed for lesions and coronaviral protein expression. histological analysis of tissues by hematoxylin and eosin (h&e) staining revealed lesions in all duodena, with clear infiltration of inflammatory cells in remnants of the villi (fig. 1, black arrowheads) . for seven animals, the entire gastrointestinal tract was available for histological analysis, showing lesions across the entire length of the intestinal tract, including the colon (fig. 1, black arrowheads) . viral protein expression using antibodies against the m protein of avian coronaviruses was observed in all duodena and in four out of the seven lower intestinal tracts by immunohistochemistry (ihc) (fig. 1, white arrowheads) . in the colons devoid of expression of viral proteins, the infiltration of inflammatory cells was noted, suggestive of a previous exposure to a virologic agent. in contrast to what we observed, virus replication of gfcov/2011 appeared to be restricted to the duodenum (6). unfortunately, we were unable to confirm the lack of infection of lower gastrointestinal tract samples in the previous outbreak due to unavailability of samples. nevertheless, we here hypothesize that genetically divergent gfcovs might have caused phenotypic differences in guinea fowls over the years. circulation of genetically diverse gfcov. gastrointestinal content collected from 20 affected animals between february 2014 and november 2016 was analyzed for the presence of gammacoronavirus genetic material by one-step real-time reverse transcription-pcr (rt-pcr) using pan-gammacoronavirus primers (13) . for all samples, threshold cycle (c t ) values obtained were below 35 (data not shown), confirming the presence of coronaviral rna in all tested samples (table 1) . next, overlapping conventional pcrs were performed with primers based on the spike gene of the gfcov/2011 virus (sequences available upon request). partial s1 sequences could be obtained from 10 of 20 rt-pcr-positive samples (893 to 1,841 nucleotides [nt]; 3,624 nt in the complete s sequence) ( table 1 ); the quality and/or quantity of the remaining 10 samples was too low to generate pcr products. sanger sequencing of the obtained fragments confirmed the presence of gfcov in the intestinal content of all 10 birds, confirming continuous gfcov circulation in france. phylogenetic analysis was performed to investigate the genetic diversity of the obtained partial s1 sequences using maximum likelihood analyses (fig. 2) . the results showed that the 2014 -2016 sequences clearly clustered with the s1 reference gene from gfcov/2011 (genbank accession number hf544506) supported by a bootstrap value of 100, while they were genetically more distantly related to tcov. each of the gfcov 2014 -2016 partial s1 sequences shared 85% to 88% nucleotide identity with the gfcov/2011 s1 sequence, and between the 2014 and 2016 partial s1 sequences, the variation was 0.1 to 8.0%. only from one sample could a full spike sequence be obtained (␥cov/avcov/guinea fowl/france/14032/2014) while for the other samples the amount and/or quality of the viral rna samples was too low for further analyses. comparison of the s1 gene of gfcov/2014 with that of gfcov/2011 using the kimura two-parameter distance model indicated that the genes had an 85% nucleotide and an 89% amino acid sequence identity. alignment of the amino acid sequences did not indicate clear mutation hot spots (data not shown), and the huge sequence diversity with the s1 of ibv strain m41 (the only avian coronavirus for which a cryo-electron microscopy [cryo-em] structure has been elucidated [14] ) precluded further suggestions on the implications of each of the mutations. gfcov/2014 s1 recognizes the enteric coronavirus dilacnac glycan receptor with higher affinity than gfcov/2011 s1. using the glycan array of the consortium for functional glycomics, we previously determined that s1 from gfcov/2011 specifically binds to the dilacnac glycan receptors (gal␤1,4glcnac␤1,3gal␤1,4glcnac) (4). to study whether the observed changes in the spike of gfcov/2014 resulted in differences in recognition of this glycan receptor, we recombinantly produced gfcov/2014 s1 and gfcov/2011 s1 and applied both proteins to dilacnac-polyacrylic acid (paa) conjugates in an enzyme-linked immunosorbent assay (elisa) as previously described (4). at similar protein concentrations, gfcov/2014 s1 showed improved binding to this receptor ( fig. 3) , indicating that the mutations in s1 did not affect the specificity but resulted in significantly higher affinity for this particular receptor. the genetic differences between gfcov/2014 and gfcov/2011 did not alter glycan specificity. next, we investigated whether the mutations in s1 resulted in recognition of additional n-linked glycans. to this end, both s1 proteins were applied to a novel glycan array containing n-glycan structures with their linear counterparts, either with terminal galactose or two differently linked sialic acid moieties (f. broszeit and r. p. de vries, submitted for publication). schematic representations of each of the glycans are given in fig. 4a . the data revealed that both gfcov s1 proteins bind to longer biantennary lacnac structures (fig. 4b , structures 3 and 4), including the dilacnac structure used in the elisa (fig. 3) . furthermore, both gfcov s1 proteins bound to longer linear lacnac repeats (fig. 4b , structure 1), which were not included in the previous array (4). finally, both gfcov s1 proteins bound longer linear and biantennary lacnac repeats with terminal alpha-2,6 sialic acid (fig. 4b , structures 9 to 12) but not those capped with alpha-2,3-linked sialic acids (fig. 4b , structures 5 to 8). erythrina crista-galli lectin (eca), sambucus nigra lectin (sna), and maackia amurensis lectin i (mal1) were used as controls. we observed, as expected, specific binding to galactose and alpha-2,6-linked and alpha-2,3-linked sialic acid terminal glycans, respectively (fig. 4c ). in conclusion, both gfcov s1 proteins show specificity for the same glycans, ending with either galactose or alpha-2,6-linked sialic acids on the glycan array. however, the relative fluorescence observed for gfcov/2014 s1 was consistently higher than that for gfcov/2011 s1, which is suggestive of differences in their affinities for glycan receptors, as observed for dilacnacs in the data shown in fig. 3 . gfcov/2014 s1 has higher affinity for glycan receptors than gfcov/2011 s1. to allow comparison of the binding affinities of the two proteins for each glycan, we applied 5-fold serial s1 protein dilutions onto the glycan array and compared binding intensities at various scan powers. at each concentration, for all glycans shown in fig. 4a , binding signals of gfcov/2014 s1 (fig. 5a) were consistently higher than those of gfcov/2011 s1 (fig. 5b) . detection of linear glycan binding (glycans 1 and 9) required higher concentrations and scan powers than the detection of biantennary lacnac structures (glycans 3 and 4 and glycans 11 and 12) for both proteins. interestingly, binding of gfcov s1 to the enteric coronavirus glycan receptor dilacnac. concentrationdependent binding of gfcov s1 proteins to gal␤1,4glcnac␤1,3gal␤1,4glcnac in elisa. the n-terminal domain of the s1 protein of ibv strain m41 was used as a negative control (10). 1, significant difference between gfcov s1 and ibv-m41; 2, significant difference between gfcov/2014 s1 and gfcov/2011 s1 (p ͻ 0.001). binding intensity of gfcov/2011 s1 to glycans with terminal alpha-2,6 sialic acids was less than the binding intensity to glycans with terminal galactose (fig. 5b , compare structures 3 and 4 to structures 11 and 12 at a protein concentration of 100 g/ml to those at 20 g/ml). this difference in preference for galactose-terminal glycans was not observed for gfcov/2014 s1 since binding levels to glycan structures 3 and 4 and to structures 11 and 12 were similar in each dilution applied to the array (fig. 5a) . taken together, the data indicate that gfcov/2014 s1 has a higher affinity than gfcov/2011 s1 for all glycans bound on the array. gfcov/2014 s1 has broader gastrointestinal tract tropism. to reveal whether the observed differences in glycan binding properties of the s1 proteins have biological consequences for tissue tropism, we first determined whether the identified glycans are indeed present on gastrointestinal tract tissues of healthy, uninfected guinea fowl. both sna and eca lectins stained the epithelial lining of the duodenum, jejunum, and cecum intensely, while intermediate staining of the proventriculus and colon was observed. in the pancreas, only limited binding of sna was observed, with no staining by eca; in contrast, in the ileum eca strongly bound whereas sna bound only to a limited extent. in conclusion, all tissues of the gastrointestinal tract, except cloaca, express gfcov glycan receptors (table 2) (15). next, we investigated the binding patterns of gfcov s1 proteins to gastrointestinal tissues. both proteins stained the epithelial cells of almost the entire gastrointestinal tract (duodenum and colon in fig. 6 , first column; results are summarized in table 2 ), indicating that receptors present on the tissues allow binding of s1. interestingly, staining intensities of the lower intestinal tract (ileum, cecum, and colon) were much more apparent for gfcov/2014 s1 than for gfcov/2011 s1. this prompted us to analyze avidity and specificity of gfcov s1 proteins to glycan receptors in the guinea fowl gastrointestinal tissues. we therefore pretreated tissue slides with arthrobacter ureafaciens neuraminidase (auna) and/or galactosidase to cleave off terminal sialic acids and/or galactose residues from host glycans, respectively. treatment of the tissues with glycan binding specificity of guinea fowl s1 proteins. (a) schematic representation of selected glycan structures present on the glycan array; numbers correspond to those shown in the graphs. numbers 1 to 4 represent glycans ending with galactose, numbers 5 to 8 represent glycans capped with alpha-2,3 -linked sialic acids, and numbers 9 to 12 represent glycans capped with alpha-2,6-linked sialic acids. yellow circle, galactose; blue square, glcnac; green circle, mannose. (b and c) glycan receptor specificity of gfcov s1 proteins and lectins eca, mal1, and sna in glycan array assay (broszeit and de vries, submitted). rfu, relative fluorescent units. auna had only a minor effect on the binding of both gfcov s1 proteins, with a slight decrease in binding intensity to the duodenum for gfcov/2014 s1 (fig. 6a , second column; table 2 ). sna lectin binding was completely abolished after pretreatment with auna, confirming that the treatment did effectively cleave off all sialic acids from the host glycans (table 2) . when galactose residues were removed from the tissues by treatment with galactosidase prior to application of eca, binding was severely reduced or totally absent ( table 2 ). binding of gfcov/2011 s1 to the tissue was completely abolished (fig. 6 , third column; table 2 ), indicating that gfcov tissue engagement is almost exclusively dependent on the presence of galactose-terminating glycans. on the other hand, gfcov/2014 s1 still clearly bound to the epithelial cells of the intestinal tract, indicating a significant difference in receptor binding avidity (fig. 6 , third column; table 2 ). finally, tissues were simultaneously pretreated with auna and galactosidase to remove both galactose and sialic acids from the glycans of the host. indeed, binding levels of both eca and sna were strongly reduced ( table 2 ). tissue binding of gfcov/2011 s1 was completely prevented, while gfcov/2014 s1 still clearly bound to the epithelial cells of the gastrointestinal tract (except pancreas) (fig. 6 , fourth column; table 2 ). these results suggest that either a minor amount of receptor is still present or that an additional (glycan) receptor is involved in tissue binding of gfcov/2014 s1. in this study, we demonstrated ongoing gfcov circulation in guinea fowl flocks in france. the sequence diversity between the viral attachment proteins of gfcov circulating in 2011 and 2014 resulted in differences in receptor binding properties with profound phenotypic consequences. the relationship between these findings and in vivo pathogenesis can only be elucidated in detail, however, when new models to study this virus have been developed. an amino acid sequence identity of 89% between viruses circulating only several years apart might indicate either that a novel gfcov strain was introduced in france from a yet unidentified source or that there was high evolutionary pressure on the 2011 gfcov strain. high mutation rates for avian coronaviruses are not uncommon (based on full-genome sequences, around 1.2 ϫ 10 ϫ3 substitution/site/year [16, 17] ). when the s1 sequences of gfcov/2011 and gfcov/2014-s1 are compared, the calculated mutation fig 6 binding of gfcov s1 proteins to guinea fowl duodenum and colon without and with enzymatic pretreatment of the tissues. (a and b) spike histochemistry was performed on uninfected, healthy duodenum and colon tissues without and with pretreatment of enzymes (auna and/or galactosidase) before application of gfcov/2014 s1 and gfcov/2011 s1. binding of proteins was visualized by red staining. rate was 5 ϫ 10 ϫ2 substitutions/site/year with a ratio of nonsynonymous to synonymous substitutions (dn/ds) of 0.45. similar mutation rates of the spike have been reported for ibv (18) and are believed to be driven by selective pressure after vaccination (19, 20) . however, no vaccine is available against gfcov or against the closely related turkey coronavirus, tcov. another driver for genetic diversity is the population size (21) ; however, this is unlikely to explain the observed high mutation rate of gfcov since flocks are considerably smaller than chicken flocks. it might well be that circulating antibodies against field strains of gfcov are the main drivers of the observed sequence diversity. unfortunately, retrospective studies to further elucidate the contribution of virus evolution, the circulation of other virus populations in the last years, or the introduction of novel strains via, for example, trade of birds between farms are impossible due to the lack of archived material. here, we revealed a novel glycan receptor for gfcov, the first coronavirus that binds n-glycans capped with alpha-2,6-linked sialic acids. alpha-2,6 sialic acid presence has been reported previously in guinea fowl large intestine (15) , along with the previously elucidated poly-lacnac expressed in guinea fowl small intestine (4). together, their expression patterns can explain in large part the tropism of gfcov, but this observation, combined with the results presented in the manuscript, does not exclude the possibility that yet another host factor plays a role in gfcov/2014 infection. initial attempts to show whether protein receptors, required for infection of many other coronaviruses (22) (23) (24) , are required have yet to be successful (data not shown). while spike protein binding analyses suggest phenotypic differences between these viruses in vivo, the reported gross clinical signs in field cases between 2011 and 2014 were not markedly different. unfortunately, and in contrast to a previous study (6) , attempts to study the pathogenesis of gfcov/2014 by inoculating commercial guinea fowls with gfcov-containing fecal samples did not result in manifestations of clinical signs or convincing detection of viral rna by reverse transcription-quantitative pcr (rt-qpcr) (data not shown). whether this was due to previous exposure of commercial birds to gfcov and, hence, circulating antibodies preventing the infection remains to be investigated. here, we have demonstrated that gfcov/2014 s1 has higher affinity for glycan receptors and increased avidity for the lower gastrointestinal tract than gfcov/2011 s1. the viral genetic diversity between these spikes and the implications for receptor recognition further add to our understanding of this virus for which models are basically lacking. collection of field samples. samples were collected from guinea fowls showing enteritis and concomitant high mortality (ͼ10%) in flocks in five regions in france (bretagne, pays de loire, nouvelle-aquitaine, occitanie, and auvergne-rhône-alpes) from february 2014 through november 2016. gastrointestinal content was collected and stored at ϫ80°c for viral rna isolation. tissues (duodenum, pancreas, air sac, lung, small intestine, large intestine, kidney, cloaca, trachea, and bursa) were collected during necropsy of euthanized or deceased animals in routine diagnostics by local veterinarians, fixed for 24 h in 4% (mass/vol) buffered formaldehyde, and stored in 70% ethanol. immunohistochemistry. paraffin-embedded tissues were sliced at 4 m, deparaffinized in xylene, and rehydrated in an ethanol gradient from 100% to 70%. antigen retrieval was carried out in tris-edta, ph 9.0 (preheated), before application of 1% h 2 o 2 in methanol. after two washes in normal antibody diluent (immunologic), the monoclonal antibody (mab) mouse anti-ibv m protein 25.1 (prionics, lelystad, the netherlands), cross-reacting with tcov and gfcov (5), was applied for 1 h at room temperature (rt). slides were washed in phosphate-buffered saline (pbs)-0.1% tween, and an envision kit (catalog no. k4001; dako) was used for anti-mouse secondary antibody staining according to the manufacturer's protocol. slides were washed three times in pbs, and viral m protein presence was visualized with aminoethylcarbazole (aec). the tissues were counterstained with hematoxylin and mounted with aquamount (merck). molecular characterization of gfcov. the gastrointestinal content collected from affected guinea fowl was clarified by centrifugation (30 s at 11,000 ϫ g), and rna was extracted using a qiagen viral rna extraction kit according to the instructions of the manufacturer. a one-step real-time rt-pcr targeting the avian coronavirus n gene was carried out to confirm the presence of coronavirus rna as previously described (13) . subsequently, the isolated rna was reverse transcribed using a revertaid kit with random hexamers (thermo fisher, waltham, ma), and overlapping conventional pcrs were performed to amplify the guinea fowl s gene (primer sequences available upon request). sanger sequencing of the resulting fragments was performed using pcr primers. contigs were generated with bioedit (version 7.0.8.0) (25) and submitted to ncbi. muscle (26) was used for the alignment, and mega (version 6.06) was used with a bootstrap value of 1,000 for the phylogeny (27) . selective pressure was calculated as dn/ds, and the hypothesis dn ϭ ds was tested using the pamilo-bianchi-li method (28), with a p value of ͻ0.05 considered statistically significant. construction of the expression vector. the codon-optimized sequence for gfcov/2014 s1 (␥cov/ avcov/guinea fowl/france/14032/2014), containing upstream nhei and downstream paci restriction sites, was obtained from genscript and cloned into the pcd5 expression vector by restriction digestion, as previously described (29) . the s1 sequence is in frame with a c-terminal gcn4 trimerization motif and strep-tag. the expression vector encoding gfcov/2011 s1 was generated previously (29) . production of recombinant proteins. recombinant s1 proteins were expressed by transfection of human embryonic kidney (hek293t) cells with pcd5 expression vectors using polyethylenimine (pei) at a 1:12 (wt/wt) ratio. cell culture supernatants were harvested after 6 days. the recombinant proteins were purified using strep-tactin sepharose beads as previously described (29) . elisa. gal␤1,4glcnac␤1,3gal␤1,4glcnac (consortium for functional glycomics) was coated in a 96-well nunc maxisorp plate (sigma-aldrich) at 0.5 g/well overnight at 4°c, followed by blocking with 3% bovine serum albumin (bsa; sigma) in pbs-0.1% tween. s1 proteins were preincubated with strep-tactin-horseradish peroxidase (hrpo) (1:200) for 30 min on ice. for each protein, 2-fold dilutions were made in triplicate in pbs and applied onto the coated well, followed by incubation for 2 h at room temperature. tmb (3,3=,5,5=-tetramethylbenzidine; thermo scientific) substrate was used to visualize binding, after which the reaction was terminated using 1 m h 2 so 4 . the optical density at 450 nm (od 450 ) was measured in a fluostar omega instrument (bmg labtech), and mars data analysis software was used for data analysis. statistical analysis was performed using a two-way analysis of variance (anova). glycan array. glycan structures were printed in six replicates on glass slides (nexterion slide h; schott inc.). prelabeled s1 proteins with alexa fluor 647-linked anti-strep-tag mouse antibody and with alexa fluor 647-linked anti-mouse igg (4:2:1 molar ratio) were applied to the slides (concentrations are given in figure legends) and incubated for 90 min, after which the slides were washed with pbs and deionized water, dried, and imaged immediately. as controls different lectins were applied: erythrina crista-galli agglutinin (eca), which is specific for glycans with terminal galactose, n-acetylgalactosamine, or lactose, and sambuca nigra agglutinin (sna) and maackia amurensis lectin i (mal1), which are specific for alpha-2,6-linked and alpha-2,3-linked sialic acids attached to terminal galactose, respectively. of the six replicates, the highest and lowest values were removed, and of the remaining four replicates, the total signal values and standard deviations (sd) were calculated and plotted in bar graphs or heat maps. spike histochemistry. spike histochemistry was performed as previously described (29) . s1 proteins precomplexed with strep-tactin-hrpo were applied onto 4-m sections of formalin-fixed paraffinembedded healthy guinea fowl tissues, and binding was visualized using 3-amino-9-ethyl-carbazole (aec; sigma-aldrich). proteins were applied onto slides at 5 g/ml. where indicated (on fig. 6 , its legend, and the text), the tissues were treated per slide with 40 u ␤-galactosidase (␤-gal) (megazyme, usa) or 2 mu of neuraminidase (sialidase) from arthrobacter ureafaciens (auna) (sigma, germany) in 10 mm potassium acetate and 2.5 mg/ml triton x-100, ph 4.2, and incubated at 40°c overnight (o/n) before protein application. lectin histochemistry. lectin histochemistry was performed as previously described (4) . biotinylated erythrina crista-galli lectin or biotinylated sambucus nigra lectin (both from vector laboratories) were diluted in pbs to a final concentration of 2 g/ml (eca) or 6 g/ml (sna) and applied to healthy guinea fowl tissue sections for 30 min. after samples were washed with pbs, the signal was visualized by an avidin-biotin complex (abc kit; vector laboratories) and counterstained with hematoxylin. data availability. contigs are available in genbank under accession numbers mg765535 to mg765542 and accession numbers mk290733 and mk290734. phylogenetic and phylogeographic mapping of the avian coronavirus spike protein-encoding gene in wild and synanthropic birds s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification coronavirus associated with an enteric syndrome on a quail farm novel receptor specificity of avian gammacoronaviruses that cause enteritis transmission kinetics and histopathology induced by european turkey coronavirus during experimental infection of specific pathogen free turkeys novel avian coronavirus and fulminating disease in guinea fowl platelet activation and aggregation promote lung inflammation and influenza virus pathogenesis enteric viruses in turkey enteritis recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus contributions of the s2 spike ectodomain to attachment and host range of infectious bronchitis virus polymorphisms in the s1 spike glycoprotein of arkansas-type infectious bronchitis virus (ibv) show differential binding to host tissues and altered antigenicity first full-length sequences of the s gene of european isolates reveal further diversity among turkey coronaviruses cryo-em structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins characterization of influenza virus sialic acid receptors in minor poultry species a large variation in the rates of synonymous substitution for rna viruses and its relationship to a diversity of viral infection and transmission modes the evolution and emergence of rna viruses origin and evolution of georgia 98 (ga98), a new serotype of avian infectious bronchitis virus location of antigenic sites defined by neutralizing monoclonal antibodies on the s1 avian infectious bronchitis virus glycopolypeptide development and characterization of neutralizing monoclonal antibodies against the s1 subunit protein of qx-like avian infectious bronchitis virus strain sczy3 genetic diversity and selection regulates evolution of infectious bronchitis virus receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus bioedit: an important software for molecular biology muscle: multiple sequence alignment with high accuracy and high throughput mega6: molecular evolutionary genetics analysis version 6.0 evolution of the zfx and zfy genes: rates and interdependence between the genes binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity key: cord-332948-h297ukuu authors: olotu, fisayo a.; omolabi, kehinde f.; soliman, mahmoud e.s. title: leaving no stone unturned: allosteric targeting of sars-cov-2 spike protein at putative druggable sites disrupts human angiotensin-converting enzyme interactions at the receptor binding domain. date: 2020-10-16 journal: inform med unlocked doi: 10.1016/j.imu.2020.100451 sha: doc_id: 332948 cord_uid: h297ukuu the systematic entry of sars-cov-2 into host cells, as mediated by its spike (s) protein, is highly essential for pathogenicity in humans. hence, targeting the viral entry mechanisms remains a major strategy for covid-19 treatment. although recent efforts have focused on the direct inhibition of s-protein receptor-binding domain (rbd) interactions with human angiotensin-converting enzyme 2 (hace2), allosteric targeting remains an unexplored possibility. therefore, in this study, for the first time, we employed an integrative meta-analytical approach to investigate the allosteric inhibitory mechanisms of sars-cov-2 s-protein and its association with hace2. findings revealed two druggable sites (sites 1 and 2) located at the n-terminal domain (ntd) and s2 regions of the protein. two high-affinity binders; zinc3939013 (fosaprepitant – site 1) and zinc27990463 (lomitapide – site 2) were discovered via site-directed high-throughput screening against a library of ∼1500 fda approved drugs. interestingly, we observed that allosteric binding of both compounds perturbed the prefusion s-protein conformations, which in turn, resulted in unprecedented hace2 displacement from the rbd. estimated δg(binds) for both compounds were highly favorable due to high-affinity interactions at the target sites. in addition, site 1 residues; r190, h207, k206 and k187, i101, r102, i119, f192, l226, v126 and w104 were identified for their crucial involvement in the binding and stability of zinc3939013. likewise, energy contributions of q957, n953, q954, l303, y313, q314, l858, v952, n953, and a956 corroborated their importance to zinc27990463 binding at the predicted site 2. we believe these findings would pave way for the structure-based discovery of allosteric sars-cov-2 s-protein inhibitors for covid-19 treatment. the novel coronavirus disease also referred to as covid-19 is caused by the sars-cov-2 (severe acute respiratory syndrome coronavirus 2), with incidences first reported in wuhan china in december 2019. 1 this disease has, however, persisted till mid-2020, spreading across 212 countries with over 3,513,507 cases reported coupled with increasingly high casualties numbering over 245,544 globally. 2 sars-cov-2 belongs to a large group of coronaviruses which are known to cause respiratory infections and related complications. these rna viruses are spherical, pleomorphic, positive-sensed, single-stranded and polyadenylated. 3 of all known viruses, coronaviruses (covs) have the largest rna genome 4 , with diverse pathogenic effects in animals and humans. this virus class is divided into four genera namely: alpha-cov, beta-cov, gamma-cov and delta cov [5] [6] [7] , with the beta-cov class prominent for their disease-causing effects in humans (hcovs). seven hcovs have been characterized to date [6] [7] [8] ; among which four (hcov-hku1, hcov-oc43, hcov-nl63 and hcov-229e) cause very mild respiratory symptoms. 9, 10 on the other hand, mers-cov, sars-cov, and sars-cov-2 cause severe respiratory and gastrointestinal infections which, in most cases, can be fatal. 11 although sars-cov-related infections were zoonotically transmitted into human populations, 12, 13 human to human transmissions has further contributed towards viral super-spread via respiratory aerosols. 14 the entry of sars-cov-2 coupled with its replication process in target human cells is achieved by the functionalities of a cohort of components, majorly non-structural and structural proteins, that make up the virus. generally, about 16 non-structural proteins (nsps) mediate diverse pro-pathogenic functions such as replication, processing and proof-reading of genomic frames, host immune evasion among many others, as previously reported. [15] [16] [17] more so, covs comprises of four major structural proteins that are integral to their pathogenesis. [18] [19] [20] these are the nucleocapsid (n), envelope (e), membrane (m) and spike (s) proteins. the n protein makes up the nucleocapsid and other viral genome-related processes 21 while the m protein is the most abundant of the four, playing major roles in maintaining viral structural integrity as well as coordinating other structural proteins. 22 e protein, on the other hand, is crucial to the maturation of the virus [23] [24] [25] [26] [27] while the trimeric s protein mediates viral entry into the host cell via the endosomal or non-endosomal route. 28 two domains make up the s protein namely the n-terminal s1 domain and the c-terminal s2membrane-anchored domain. the s2 region is extensively conserved in covs while constituent s1 region residues are highly diverge across the cov strains. 29 these domains have been further characterized into subdomains due to specific functionalities with respect to host receptor recognition and binding (s1), coupled with membrane fusion and entry (s2) (figure 1 ). similar to sars-cov architecture, some recent reports have sub-categorized the sars-cov-2 s1 ectodomain into the n-terminal domain (ntd), a conserved receptor-binding domain (rbd) which recognizes the human angiotensin-converting enzyme 2 (hace2), 30 and subdomains 1 and 2 (sd1 and sd2). during infection, proteolytic cleavage or priming of the s protein is crucial for viral fusion and entry into host cells, a process mediated by host cell proteases such as the transmembrane serine protease 2 (tmprss2) and cathepsin l, [31] [32] [33] at the s1/s2 (boundary between s1 and s2 subunits) and s2' (immediately upstream s2 fusion peptide -fp) cleavage sites. [34] [35] [36] the s protein primarily exists in a metastable prefusion complex prior to cleavage, after which notable conformational arrangements occur in order to fuse the viral membrane into j o u r n a l p r e -p r o o f the host cell. [37] [38] [39] in addition, the rbd adopts disparate conformational motions to engage the host cell receptor. 40, 41 conformations. [42] [43] [44] the up conformation corresponds to the hace2 accessible state while the down state cannot engage the host cell receptor. 44 the s2 domain, on the other hand, consists of the functionally important fusion peptide (fp), which is critical for viral fusion and formation of the post-fusion complex; heptad repeats 1 and 2 (hr1 and hr2); transmembrane domain (tm) and cytoplasmic tail (ct). the hrs of the s-protein trimer interact to form a fusion core of sixhelical bundle which helps bring the membranes of the virus and host cell in close proximity for fusion and entry. 42 therefore, the roles of sars-cov-2 s-protein present it as an important therapeutic target, which would enable the prevention of viral entry and fusion in host cells. numerous studies have been reported over the past months with regards to the possibility of blocking direct interactions between sars-cov-2 s-protein and hace2. most of these studies were aimed at targeting the s protein rbd domain with antibodies, peptide-based or small molecule compounds that binds with a much higher affinity to block s-protein-hace2 interactions. [45] [46] [47] [48] [49] [50] also, targeting host proteases such as tmprss2 was explored in a recent study, with consequential impediments on sars-cov-2 entry. 30 identification of other functional (allosteric) sites on the prefusion s protein could present another dynamic and effective approach of preventing sars-cov-2 infectivity relative to its interaction with the host cell ace2 and proteases. this alternative target approach for sars-cov-2 s protein is important because its rbd (similar to other covs) has been associated with a high mutational propensity which may in turn alter the affinity of small molecule inhibitors or peptide designed to bind therein. 51 allosteric targeting was explored in a recent study wherein the cov-conserved s2 hr1 region was identified as an important target site for the development of broad-spectrum inhibitors of human covs. the resulting peptide inhibitor (ek1) was evaluated in vivo and exhibited desirable safety and efficacy 52 . more so, the protein contact network (pcn) paradigm was used to map functional allosteric loci on sars-cov s protein. 53 relatively, this study was implemented to (i) identify potential druggable sites across the s1 and s2 domains of the sars-cov-2 s protein other than the rbd-hace2 interface (ii) perform high-throughput (virtual) screening of ~1500 fda approved drugs against the most druggable site(s) (iii) investigate the binding dynamics and interaction mechanisms of the compounds and their consequential effects on the s-protein rbd-ace2 complex. we believe this systematic study will be able to provide structural and molecular insights into possible allosteric sites on sars-cov-2 s protein suitable for selective targeting and structure computational methodologies the three-dimensional structure of sars-cov-2 s-protein (prefusion) was retrieved from pdb with entry 6vsb. 44 this, as previously reported, represents the s-protein rbd conformation in its up (open) state, which is most suitable for hace2 binding. also, to model binding interactions between the prefusion sars-cov-2 s-protein (s1/s2) and the hace2, a crystalized structure with pdb entry 6m0j 54 was separately retrieved. this complex depicts binding between the rbd domain (truncated) of sars-cov-2 s-protein and the protease domain (pd) of hace2. co-crystallized molecules not relevant to this study were removed while missing residues (gaps) in the structures were filled using the modeller algorithm. 55 this preparation was performed on the ucsf chimera graphic user interface (gui). 56 subsequently, using the structural superposition method, we were able to model a complex between prefusion s-protein (s1/s2) monomer (rbd -up conformation) and the hace2 protein ( figure 2 ). j o u r n a l p r e -p r o o f possible druggable sites other than the sars-cov-2 rbd interface were predicted using approaches previously reported. [57] [58] [59] [60] herein, we employed multiple tools for site identification and validation, which include sitemap 61 , fpocket 62 , discovery studio 2016 client 63 and prankweb. 64 sitemap is an exhaustive tool which ranks protein pockets based on properties such as druggability, surface exposure, hydrophobicity and hydrophilicity among others [65] [66] [67] . these details were then used to characterize the predicted pockets after which other predictive algorithms were used complementarily for cross-validation. two highly ranked sites were then selected for further analyses. furthering on the rationale of the study, we mapped out the two most druggable sites on the target protein and virtually screened against them a large chemical library of fda approved drugs (~1500 compounds) derived from the zinc repository (http://zinc.docking.org/substances/subsets/fda/). this screening was performed using highperformance computing-integrated autodock vina 68 prior to which coordinates of the predicted sites were mapped using gridboxes. corresponding binding scores were retrieved from the resulting .pdbqt files and were used to filter down to the topmost 20 compounds for each predicted sites 1 and 2. subsequently, two compounds with the highest binding scores (most negative) were selected for the two predicted sites yielding complexes that were subjected to further simulation studies. as explained in 2.1, the prefusion s-proteins (ligand-bound and unbound) were superimposed with the rbd-hace2 complex (6m0j) after which the single j o u r n a l p r e -p r o o f truncated rbd was removed. by so doing, we obtained models of allosterically-bound and unbound pre-fusion s-protein-ace2 complex. this, as aimed in this study, would provide structural and dynamical insights into the mechanistic effects of allosteric targeting on sars-cov-2 host entry machinery. although computationally expensive (1673 residues), we proceeded with long-timescale md simulation runs for the systems on amber18 graphical processing unit (gpu) using its embedded modules. 69 protein parameters were defined using the ff14sb forcefield while ligand parameters were generated with the antechamber and parmchk modules. likewise, the leap program was used to define coordinate and topology files for the ligand-bound and unbound protein complexes. this program, also, was used to neutralize (addition of counter-ions; na + and cl -) and solvate the systems in a tip3p water box of size 10å. structural minimization was first carried out partially for 5000steps with a restraint potential of 500kcal mol -1 . å 2 followed by another 100000 steps of full minimization with no restraints. a canonical (nvt) ensemble with a 5kcal mol -1 å 2 harmonic restraints was used to heat the systems gradually from 0 -300k for 50ps, after which the systems were equilibrated for 10000ps at a constant 300k temperature without restraints in an npt ensemble. atmospheric pressure was maintained at 1bar with a berendsen barostat 70 while each protein system was subjected to a production run of 350ns. studied systems include zinc3939013-s-protein-hace2 (allosteric site 1), zinc27990463-sprotein-hace2 (allosteric site 2), and unbound s-protein-hace2. corresponding trajectories were saved at every 1ps time-frame until the end of the simulation followed by data plot analyses using microcal origin software. 71 snapshots were also taken and analyzed to monitor structural events and ligand interaction dynamics across the trajectories on the ucsf chimera user j o u r n a l p r e -p r o o f interface (gui) and discovery studio client. 63 the molecular mechanics/generalized born surface area (mm/gbsa) method was used to evaluate binding affinities of the predicted allosteric s-protein binders at their target sites. binding energy profiles for both compounds, inclusive of their energy components, were estimated using 1000 snapshots from the terminal 30ns of md trajectories where conformational stabilities were visible. this approach was important in order to minimize the effects of conformational disorder or entropy on ligand interactions. the equations below mathematically express binding energy calculations: as shown, internal (∆e int ), electrostatic (∆e ele ) and van der waals (∆e vdw ) energies sum up the gas-phase energy (∆g gas ) while the solvation free energy (∆g sol ) is defined by the polar solvation (∆g ele,sol ) and non-polar contribution to solvation (∆g np,sol ) terms. the mm/gbsa method was used to estimate the generalized born (gb) for ∆g ele,sol while the linear relationship between the surface tension proportionality constant (γ = 0.0072 mol -1 å -2 ), solvent accessible surface area (sasa, å 2 ), and β constant was used to solve ∆g np,sol . furthermore, estimated ∆g bind was decomposed into individual residue energies, most especially those that constitute the predicted allosteric pockets where the ligands were bound. this method was essential to identify specific residues that contribute crucially to the stability and inhibitory activities of potential allosteric inhibitors. j o u r n a l p r e -p r o o f based on the study rationale, we set out to identify possible sites for drugging the target protein table 1 ). the architectures of these pockets are shown in figure 3 . furthermore, defining the druggability of a site on target proteins depends on the size (volume) and hydrophobicity (with minimal hydrophilicity) while, on the other hand, high hydrophilicity, reduced hydrophobicity, small pocket size and shallowness characterize "difficult-to-drug" and undruggable pockets 61, [65] [66] [67] . while large hydrophilicity could have repulsive effects on ligand mobility at the binding site, a small or shallow cavity would impede ligand access, fitness, optimal binding and stability. j o u r n a l p r e -p r o o f from table 2 , sites 1 → 3 ranks above the 0.83 halgren dscore threshold making them suitable for therapeutic targeting. relatively, site 1 appears to be highly surface-exposed with a score of 0.933 while a large pocket size and volume for site 2 could favor the use of large-molecule compounds. taken together, high surface-exposure coupled with relatively large volumes, hydrophobicity and favorable donor/acceptor properties for sites 1 and 2 could account for their suitability as targetable allosteric regions on the s-protein other than the rbd (figure 3 ). these presumptions are also reflected by the estimated dscore and sitescore values. in addition, since these predicted sites are highly functional, particularly the overlapping fp, hr1 and cr, targeting them could high-throughput screening and identification of potential allosteric binders to the predicted sites 1 and 2 high-throughput screening using a library of ~1500 fda approved drug compounds (http://zinc.docking.org/substances/subsets/fda/) were performed against the two predicted allosteric sites. results for the top 20 compounds with the highest binding scores are presented in supplementary table s1 and supplementary table s2 for sites 1 and 2 respectively. from the screening results, overall highest scores were estimated for zinc3939013 (-10 j o u r n a l p r e -p r o o f kcal/mol) at site 1 and zinc27990463 (-9.3 kcal/mol) at site 2. as highlighted in our methods, md simulations were performed for the prefusion s-protein-hace2 complexes bound distinctly at two potential allosteric sites. this approach was essential to investigate the likely effects of allosteric targeting on the entry/fusion mechanisms of sars-cov-2 via host hace2. however, this conformation appeared distorted the allosterically-bound s-proteins and could account for displacement motions of the interacting hace2 from the rbd interface. therefore, the allosteric-mediated disruption of sars-cov-2 s-protein rbd and its interaction with hace2, as reported herein, is a major finding that could indicate the viability of allosteric targeting in sars-cov-2 therapy. furthermore, we measured structural stabilities across the ligand-protein complexes relative to the unbound system using the rmsd metrics. as shown in figure 6 , structural instability was highest in the unbound s-protein while its associated hace2 was relatively stable compared to this could indicate the structural effects of allosteric targeting on the s-protein and its interaction with hace2. estimated mean rmsds, as presented in table 2 , corroborates conformational variations among the unbound and bound protein complexes. to minimize the effects of structural disorderliness (entropy) in our calculations, we selected, from the md trajectories, terminal time-frames (270-300ns) from which the systems appeared to relatively stabilize. these were defined as the finally equilibrated (fe) time-frames and were used for subsequent structural analyses ( table 2 ). from the resulting fe-rmsd plots, unbound s-protein was highly unstable while its associated hace2 exhibited low structural motion in line with the rmsd calculations, which could also imply that the binding of s-protein stabilized hace2. in contrast, the allosterically-bound sproteins (sites 1 and 2) were notably stable while their corresponding hace2 showed high structural instability that could correlate with their systemic motions at the s-protein rbd as earlier mentioned. structural analyses of ligand orientations at the respective allosteric sites of sars-cov-2 sprotein were performed using averaged structures from the md trajectories ( figure 9 ). findings reveal that the allosteric binding of zinc3939013 (fosaprepitant) was stabilized at the ntd. fosaprepitant contains a terminal triphosphate group that orients towards residues such as n99, k187, n188, r190 and h207. likewise, its trifluoromethyl group oriented towards d102 while constituent -o and -nh groups mediate interactions with q173 and n121, among others. these altogether could facilitate high-affinity interactions accountable for its stability and allosteric inhibitory effects against the sars-cov-2 and associated hace2. j o u r n a l p r e -p r o o f binding affinities of the compounds were determined using the mm/pbsa technique, which also allowed us to measure the energy contributions of interactive residues at the predicted allosteric sites. energy calculations, as presented in table 4 were performed using relatively stable time-frames (270-300ns) to minimize entropical effects that may interfere with ligand binding activities. in addition, we observed that electrostatic effects contributed most notably to the allosteric binding of zinc3939013 at the ntd region while van der waals contributions had the highest effect on the binding of zinc27990463 at the predicted site 2 pocket. electrostatic contributions at site 1 could be due to the high number of electropositive residues that constitute the pocket, as shown in figure 10 , which may form high-affinity interactions with electronegative moieties of the compound. calculations further revealed that ∆e vdw and ∆e ele were more favorable in the gas phase for zinc3939013 while polar solvation energies were more favorable for zinc27990463 j o u r n a l p r e -p r o o f at the s2 region of the s-protein. this could imply that while the former was buried in the deep hydrophobic pocket of the ntd, the latter was surface exposed due to its trans-domain binding activity as earlier reported. to understand the mechanistic binding of the compounds at both predicted sites, we decomposed the binding free energies into individual contributions of the interacting residues. these were juxtaposed with structural analysis that showed the type and (π-alkyl) interactions. more so, π-π stacked interaction between y313 and a benzene ring (of the 4-tri-fluoromethyl-1,1'-biphenyl group) could be highly crucial for the stability of the compound. taken together, electrostatic energies favored the binding of zinc3939013 at site 1 while vdw energies favored zinc27990463 binding at site 2, which consequentially, were able to perturb the s-protein rbd and allosterically disrupt hace2 interactions. the systemic entry of sars-cov-2 into the human host cell is a crucial process that underlies its virulence and pathogenicity in humans and other animals it infects. this mechanism is mediated by its interaction with the host ace2 (hace2) via attachment and fusion. potential intervention approaches in sars-cov-2 treatment include therapeutic strategies that could prevent sars-cov-2 s-protein binding to hace2. in this study, we implemented an exhaustive approach to identify drug molecules that could potentially bind to sars-cov-2 s-protein at other sites other than the rbd. pertinent to the allosteric targeting approach implemented herein j o u r n a l p r e -p r o o f was the identification of highly druggable sites inherent in the s-protein (s1/s2), which was carried out using multiple pocket prediction algorithms for identification and validation of possible allosteric sites. predicted pockets were then characterized based on their attributes after which two highly probable pockets were selected. these were then screened distinctly against a library of ~1500 fda approved drugs retrieved from the zinc database. amongst all, thermophoresis (mst) can be employed for further validation. these implementations will provide additional insights into the targetability and suitability of these pockets for novel covid-19 therapeutics. findings from this study paves way for novelty in the structure-based design of high-affinity allosteric inhibitors or disruptors of sars-cov-2 association with host hace2 thereby preventing viral entry. authors thank the college of health sciences, university of kwazulu-natal, south africa for providing infrastructural support and we also acknowledge the center for high performance computing (chpc), capetown, south africa, for providing computational resources. authors declare no conflict of 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conformers autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading amber 18 molecular dynamics with coupling to an external bath originpro 9.1: scientific data analysis and graphing software-software review structural basis for the recognition of sars-cov-2 by full-length human ace2 mapping allosteric communications within individual proteins the following information is required for submission. please note that failure to respond to these questions/statements will mean your submission will be returned. if you have nothing to declare in any of these categories then this should be stated. all sources of funding should be declared as an acknowledgement at the end of the text. authors should declare the role of study sponsors, if any, in the collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication. if the study sponsors had no such involvement, the authors should so state. studies on patients or volunteers require ethics committee approval and fully informed written consent which should be documented in the paper.authors must obtain written and signed consent to publish the case report from the patient (or, where applicable, the patient's guardian or next of kin) prior to submission. we ask authors to confirm as part of the submission process that such consent has been obtained, and the manuscript must include a statement to this effect in a consent section at the end of the manuscript, as follows: "written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor-in-chief of this journal on request".patients have a right to privacy. patients' and volunteers' names, initials, or hospital numbers should not be used. images of patients or volunteers should not be used unless the information is essential for scientific purposes and explicit permission has been given as part of the consent. if such consent is made subject to any conditions, the editor in chief must be made aware of all such conditions. even where consent has been given, identifying details should be omitted if they are not essential. if identifying characteristics are altered to protect anonymity, such as in genetic pedigrees, authors should provide assurance that alterations do not distort scientific meaning and editors should so note. please specify the contribution of each author to the paper, e.g. study design, data collections, data analysis, writing, others, who have contributed in other ways should be listed as contributors.this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.not applicable to this study.fao conceptualized, implemented, analyzed, interpreted and wrote the manuscript, kfo performed molecular dynamics simulation, while mes revised and approved the manuscript for submission. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests:j o u r n a l p r e -p r o o f key: cord-325825-0lyt8gfq authors: griffiths, samantha j.; koegl, manfred; boutell, chris; zenner, helen l.; crump, colin m.; pica, francesca; gonzalez, orland; friedel, caroline c.; barry, gerald; martin, kim; craigon, marie h.; chen, rui; kaza, lakshmi n.; fossum, even; fazakerley, john k.; efstathiou, stacey; volpi, antonio; zimmer, ralf; ghazal, peter; haas, jürgen title: a systematic analysis of host factors reveals a med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication date: 2013-08-08 journal: plos pathog doi: 10.1371/journal.ppat.1003514 sha: doc_id: 325825 cord_uid: 0lyt8gfq herpes simplex virus type 1 (hsv-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. to comprehensively investigate the complex interaction between hsv-1 and its host we combined two genome-scale screens for host factors (hfs) involved in virus replication. a yeast two-hybrid screen for protein interactions and a rna interference (rnai) screen with a druggable genome small interfering rna (sirna) library confirmed existing and identified novel hfs which functionally influence hsv-1 infection. bioinformatic analyses found the 358 hfs were enriched for several pathways and multi-protein complexes. of particular interest was the identification of med23 as a strongly anti-viral component of the largely pro-viral mediator complex, which links specific transcription factors to rna polymerase ii. the anti-viral effect of med23 on hsv-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to hsv-1, as a range of other viruses including vaccinia virus and semliki forest virus were unaffected by med23 depletion. we found med23 significantly upregulated expression of the type iii interferon family (ifn-λ) at the mrna and protein level by directly interacting with the transcription factor irf7. the synergistic effect of med23 and irf7 on ifn-λ induction suggests this is the major transcription factor for ifn-λ expression. genotypic analysis of patients suffering recurrent orofacial hsv-1 outbreaks, previously shown to be deficient in ifn-λ secretion, found a significant correlation with a single nucleotide polymorphism in the ifn-λ3 (il28b) promoter strongly linked to hepatitis c disease and treatment outcome. this paper describes a link between med23 and ifn-λ, provides evidence for the crucial role of ifn-λ in hsv-1 immune control, and highlights the power of integrative genome-scale approaches to identify hfs critical for disease progression and outcome. up to 90% of the global population is infected with the aherpesvirus herpes simplex virus type i (hsv-1). whilst hsv-1 is largely responsible for outbreaks of vesicular oral skin lesions (fever blisters, or cold sores), it can also cause a variety of more severe diseases including encephalitis, meningitis and keratitis [1, 2] . furthermore, the frequency of association with genital lesions (previously associated mainly with hsv-2 infection) is increasing. as co-infection with hsv is a significant contributing factor to transmission of the human immunodeficiency virus (hiv), our understanding of hsv disease, and herpesviruses in general, has wide implications for global healthcare. like all herpesviruses, hsv-1 establishes lytic (epithelial cells) and asymptomatic latent infection (sensory neurons in trigeminal and sacral ganglia) which undergoes periodic reactivation [3] . the equilibrium between these two infection states requires a fine balance between innate and adaptive immune responses, and viral immune evasion mechanisms [4] . whilst aspects of the hsv-1 replication cycle have been intensively investigated, there remain gaps in our understanding of the complexity of virus:host interactions. for example, a proteomics study identified over 100 changes in the cellular proteome within the first 6h of infection with hsv-1 [5] , and a recent analysis of virionincorporated cellular proteins found that about 30% of these directly affected virus growth [6] . to systematically identify host factors (hfs) required for viral replication, rnai screens have been performed with a range of different rna and dna viruses including hiv-1 [7, 8, 9] , influenza a virus [10, 11, 12] , hepatitis c virus [13] , west nile virus [14] , dengue virus [15] , enterovirus [16] and vaccinia virus [17, 18] . the overlap between the results of these studies is generally very low [19] , reflecting either differences in biology, or different experimental set-ups, cutoff and selection criteria. in addition, microenvironmental effects might also play a role for the differences of the results [20] . whilst loss-of-function sirna screens provide functional information on specific genes, protein interaction studies can provide insight into the mechanism of action by identifying physical interaction partners between pathogen and host. genome-scale virus-host protein interaction screens using the yeast-two-hybrid system have been performed for hcv [21] , influenza a virus [22] , epstein barr virus (ebv) [23] , vaccinia virus [24, 25] , sars coronavirus [26] and several non-human viruses [27] . based on these genome-scale studies and individual interactions found by literature curation, several virus-host interaction databases have been created including the hiv-1, human protein interaction database at ncbi [28] , virhostnet [29] , virusmint [30] , pig [31] and hpidb [32] . although there is little overlap between individual cellular interactors of different viruses, targeting of a number of cellular processes such as cell cycle regulation, nuclear transport and immune response appears to be conserved [33] . understanding the complex interplay between viral and host components is critical to the definition of herpesvirus infection and pathogenesis. as herpesviruses encode a large number of proteins, in contrast to small rna viruses such as hiv and influenza, many cellular processes may be directly affected by viral proteins, and whilst there exists a wealth of information on individual viral proteins, there remain large gaps in our understanding of the hsv-1 life cycle and its interaction with its host. here, we present data from the first integrative and systematic screening approach to characterise the role of cellular proteins in the hsv-1 life cycle. a genome-scale rnai knockdown screen to identify hfs functionally influencing hsv-1 replication was performed in parallel with a yeast two-hybrid (y2h) protein interaction screen to simultaneously gain insight into potential mechanisms of action. combined analyses confirmed the importance of known cellular proteins involved in processes such as cell cycle, proteins transport and gene expression important for virus replication. furthermore, we identified a subunit of the mediator multi-protein complex, med23, as a key regulator of ifn-l induction, which appears to be of crucial significance for the control of hsv-1 both in vitro and in vivo. these data demonstrate the power of a combined screening strategy to investigate pathogen:host interactions and identify novel host factors and cellular pathway targets for the development of essential clinical interventions. host factors (hfs) which positively or negatively regulate hsv-1 replication were identified by screening a druggable genome sirna library (4 sirnas per gene) targeting 7,237 human genes against a hsv-1 reporter virus expressing the enhanced green fluorescent protein (egfp; hsv-1 strain c12) in the epithelial hela cell line, due to their ease of transfection and susceptibility to hsv-1 infection [34] . to generate a robust and reliable dataset the screen was carried out three times in triplicate, with one replicate used in a cell viability assay to determine any cytotoxic effects of gene depletion and duplicates infected for the virus infection assay. the sirna library was reversetransfected into hela cells before infecting with hsv-1 and monitoring virus growth kinetics as a measure of gfp-fluorescence (figure 1b). by following virus growth over multiple rounds of replication, host proteins involved in all stages of the virus life cycle can be identified. replication slopes during linear growth were normalized to controls (mock-transfected cells, and cells transfected with a sirna unable to be processed by the rna silencing complex, rscf) and the mean of six replicates was calculated. sirnas found to be cytotoxic (81 in total) were excluded from further analyses, and a hitlist of 358 containing the top 2.5% inhibitory and the top 2.5% enhancing hfs was generated ( table s1 in text s2). the identified hsv-1 hfs were compared to datasets from published sirna depletion screens aimed at identifying cellular factors affecting hiv-1 [7, 8, 9] , west nile virus (wnv) [14] , hepatitis c virus (hcv) [13] , dengue virus [15] and influenza a virus [10, 11, 12] . of our 358 hfs, 54 cellular proteins (15.1%) overlapped with these other virus screens (influenza a, 29; hiv-1, 24; hcv, 6; wnv, 2; dengue virus, 1) ( figure 1c ; table s2 in text s2). a genome-scale yeast two-hybrid protein interaction screen identifies novel viral protein interaction partners hsv-1 is currently known to encode at least 84 proteins, expressed sequentially under strict temporal regulation during herpes simplex virus type 1 (hsv-1) infects the vast majority of the global population. whilst most people experience the relatively mild symptoms of cold sores, some individuals suffer more serious diseases like viral meningitis and encephalitis. hsv-1 is also becoming more common as a cause of genital herpes, traditionally associated with hsv-2 infection. co-infection with hsv-2 is a major contributor to hiv transmission, so a better understanding of hsv-1/hsv-2 disease has wide implications for global healthcare. after initial infection, all herpesviruses have the ability to remain dormant, and can awaken to cause a symptomatic infection at any stage. whether the virus remains dormant or active is the result of a finely tuned balance between our immune system and evasion techniques developed by the virus. in this study we have found a new method by which the replication of the virus is counteracted. the cellular protein med23 was found to actively induce an innate anti-viral immune response in the form of the type iii interferons (ifnlambda), by binding irf7, a key regulator of interferons, and modulating its activity. interferon lambda is well known to be important in the control of hepatitis c infection, and a genetic mutation correlating to an increase in interferon lambda levels is strongly linked to clearance of infection. here we find the same association between this genetic mutation and the clinical severity of recurrent cases of hsv-1 infection (coldsores). these data identify a med23-interferon lambda regulatory axis of innate immunity, show that interferon lambda plays a significant role in hsv-1 infection, and contribute to the expanding evidence for interferon lambda in disease control. infection. to gain further mechanistic insight into host factors involved in hsv-1 infection, in parallel to the sirna depletion screen we carried out a yeast two-hybrid protein interaction screen to identify cellular interaction partners of viral proteins. we generated a collection of 107 partial and full-length hsv-1 cdna constructs and tested them for interactions with proteins encoded by a library of 12,381 human cdna clones [35] . 231 hsv-1human protein interactions were detected once (low confidence), and 63 more than once (high-confidence)( table s3 in text s2) . using these high-confidence interactions, the previously reported hsv-1 interactome [36] was connected into a human interactome (62,310 published protein interactions) to generate a combined pathogen-host interactome ( figure s1a ). both degree centrality (which indicates the number of interactions a protein has, where high values represent highly interactive 'hubs') and betweenness centrality (which indicates the number of shortest paths between (3 replicates) or the capacity to influence replication of the hsv-1 gfp reporter virus c12 (6 replicates) from 24 to 80 h post-infection. virus replication slopes during the linear phase were calculated and normalized to mock-transfected cells. replication slopes were then compared to replication upon knockdown of essential (icp4, vp16) or non-essential (vp11/12) viral genes, a cellular receptor for hsv-1 (hvem) or control risc-free sirna (rscf). (c) overlap between the hsv-1 hfs identified in this study with those published in hiv-1 [7, 8, 9] , hepatitis c virus (hcv) [13] and influenza a virus [10, 11, 12] . doi:10.1371/journal.ppat.1003514.g001 any pair of proteins passing through the protein considered) were significantly increased for hsv-1 interactors, particularly in the high-confidence network (figure s1b-e). these data suggest hsv-1 proteins preferentially target highly connected central human proteins in the cellular interaction network, similar to other viruses [23] . analysis of this interactome for hfs identified by rnai found they were enriched in the fraction of cellular proteins that directly interact with viral proteins or that interact via one intermediate, in comparison to proteins that only interact via 2 or more intermediates (p = 0.036, fisher's exact test)( figure s1f) . a direct comparison of hsv-1 protein interaction partners and the sirna screen hfs found 215 genes in common. of those, ten (4.6%) were identified as a hit in both screens ( table s4 in text s2), suggesting that these technologies identify complimentary yet not necessarily overlapping hfs. an extended literature and database search identified 599 cellular proteins that interact with or are involved in infection with human herpesviruses. the overlap between the high-confidence y2h cellular interactors (63) and hfs (358) with this set was statistically significant (p = 0.008) (figure 2a; figure s1h ; table s5 in text s2). from this combined analysis, a subset of hfs was chosen for further validation. protein interactions were tested in a mammalian cell system by lumier pull-down assay [37] . of the 45 interactions tested, 26 (57.8%) were confirmed, with 15 strongly positive (z-score .2) and 11 weakly positive (zscore 1-2) ( figure s1g ). sirna deconvolution (4 sirnas per gene tested individually) was used to further validate 72 hfs (figure 2b ; figure s2 ). the replication phenotype could be confirmed ($2 or more sirnas gave the same or better replication slope than observed in the primary screen) in a high proportion (83.3%) of candidates, highlighting the reliability of the primary screen dataset. quantitative rt-pcr analysis of mrna expression levels found a minimum depletion of 60% (mean 88%) in a subset of 52 genes (data not shown; table s6 in text s2) confirming the observed effects on hsv-1 replication are genuine and not due to 'off-target' effects or insufficient gene knockdown. to further investigate the virus-specificity of our identified hfs, we tested this subset for their effect on the replication of an additional a-herpesvirus (varicella-zoster virus, vzv), the bherpesvirus cytomegalovirus (cmv), and a completely unrelated rna virus, semliki forest virus (sfv). none of the three proteins which enhanced hsv-1 replication upon knockdown had an effect on either vzv or cmv, and one (nr3c2) was even inhibitory for sfv ( figure 2c ). of the 64 sirnas which inhibited hsv-1, 27 (42.2%) were also inhibitory for vzv, 60 for cmv (93.8%) and 23 (35.9%) for sfv replication ( table s7 in text s2) . some functional groups (transcriptional regulators) were required by most viruses, but there were notable differences between other proteins. for example ifitm-1, previously identified as an inhibitor of influenza a, dengue virus and wnv [10] , inhibited vzv yet had a positive effect on hsv-1 replication. these data suggest that whilst there are some hfs which are broad in their effects on virus replication, a large proportion are species-specific. table s8 in text s2). pathways included those involved in gene expression, transcription, splicing and translational regulation (rnai screen), and protein transport, cell cycle, and transcriptional repressor activity (y2h screen). a combined analysis of hfs from both screens found dominant functional categories centred on the regulation of transcription (rna polymerase ii-associated genes, splicing factors, transcription activation and the mediator complex) ( figure s3c, d) . the physiological relevance of some hfs and pathways was confirmed by further biological validation. protein transport pathways (in the form of dynein microtubule networks) are exploited by hsv-1 early after infection to shuttle viral capsids to the nucleus. these screens confirmed known interactions between dynein subunits and viral proteins, and identified additional previously unknown interactions (text s1 and figure s4a ). several dynein chain subunits were found to be essential for virus replication, whilst the moderate effect of depletion of other subunits demonstrated a level of functional redundancy in hsv-1 capsid transport ( figure s4b -e) [38, 39] . intrinsic anti-viral host defense mechanisms, in the context of cellular e2 ubiquitin ligases, were also investigated. the immediate-early viral protein icp0, an e3 ubiquitin ligase, is crucial for blocking anti-viral defense mechanisms by degrading promyeloctic leukemia (pml) nuclear bodies (nd10 domains) in the presence of cellular e2-ubiquitin-conjugating enzymes (e2s). our sirna screen found multiple e2s were required for this, and suggests that hsv-1 icp0 is promiscuous in its exploitation of e2s to mediate pml degradation and ensure successful infection (text s1 and figure s5 ). combined bioinformatic analyses of protein interaction and sirna depletion screens found a significant functional enrichment for proteins involved in transcription, and identified multi-protein complexes enriched for pro-viral hfs which strongly inhibited hsv-1 upon depletion, including the rna-polymerase ii, eif3 and mediator complexes ( figure 3a) . the mediator complex links the cellular transcription machinery (rna polymerase ii) to specific transcription factors, and the identification of many mediator subunits as hfs in other viral sirna depletion screens highlights its significant role in viral genome transcription [7, 9, 11, 40] (table s2 in text s2) . further, several mediator subunits (med25, 29, 17 and 8) are known to interact with the hsv-1 transactivator vp16 (ul48) and other herpesviral proteins [41] ( figure s6a) . consistently, the mediator complex was found to be strongly required for hsv-1 replication, with depletion of the majority of subunits (med 4, 6, 7, 8, 14, 16, 17, 21, 25, 26, 27 and 28) leading to a severe reduction in virus replication in the primary screen ( figure s6b ) or in confirmatory deconvolution assays ( figure 3b) . however, depletion of the med23 subunit was striking in that it led to a significant enhancement of virus growth ( figure 3b ). flow cytometry quantification found that removal of med23 not only increased the total number of infected cells (combination of gfp lo and gfp hi cells; 75.5% in comparison to 49.6% in mock-transfected cells) but also the copy number of virus genomes (gfp hi cells; 44.8% in comparison to 24.4% in mocktransfected cells) (figure 3c med23 could exert anti-viral effects either by having an inhibitory effect on viral transactivators or by interacting with and having a positive effect on an existing anti-viral factor. we first tested whether med23 directly affects viral gene expression using luciferase reporters with hsv-1 promoters, however observed no inhibitory effect (data not shown). since the mediator complex and med23 in particular is known to be involved in jak/statmediated interferon signaling [42] , we used the lung epithelial cell line a549 and its stat-1-deficient derivative a549-v [43] to determine if med23 influences hsv-1 replication by modulating innate immunity. in the parental a549 cells the phenotype of hsv-1 replication was the same as that observed in hela cells, where depletion of med23 enhanced replication and overexpression inhibited virus growth. however, in the stat1-deficient a549-v cells hsv-1 replication was unaffected by both depletion and over-expression of med23 (fig. 4a) , indicating that med23 requires an intact jak/stat signalling pathway to exert its anti-viral effects. to determine which interferon may be responsible for the antiviral effects of med23, a549 cells were depleted for med23 and infected with hsv-1 following pre-stimulation with type i (ifn-a or ifn-b), type ii (ifn-c) or type iii (the distinct ifn-l1 or the almost identical ifn-l2 and -l3, termed ifn-l2/3) interferons. whilst treatment with ifn-a, -b and -c significantly decreased hsv-1 replication levels, the observed ,2-fold enhancement of hsv-1 replication following med23 depletion was still seen. however, pre-treatment with the both ifn-l1 and ifn-l2/3 blocked the enhancing effect of med23 depletion (figure 4b) . investigation into the effect of med23 on interferon induction by qrt-pcr found that whilst med23 over-expression induced ifnb (,3-fold increase), induction of ifn-l1 and l2/3 was considerably and statistically significantly higher (,26-fold induction; p = 0.003 and 0.002, respectively) ( figure 4c ). this induction was specific, as levels of other cytokines and interferon-regulatory factors (irfs) were unaffected by med23 overexpression ( figure s7a ). secretion of ifn-l2/3 protein was also increased in all cell lines tested, but most significantly to ,11-fold in a549 cells (figure 4d) , which is consistent with a recent report showing that type iii interferons are the dominant type of ifns expressed by primary airway epithelial cells [44] . furthermore, qpcr analysis found depletion of med23 inhibited the induction of ifn-l expression following hsv-1 infection of a549 cells in comparison to cells transfected with the rscf sirna control ( figure 4e ). together, these data suggest that ifn-l is responsible for the observed inhibitory effect of med23 on hsv-1 replication. as ifn-l expression is induced following activation of pathogen recognition receptors (prrs) by virus infection [45, 46, 47, 48] , we tested whether med23 induced ifn-l by directly interacting with an interferon-responsive transcription factor (irf). y2h and confirmatory co-immunoprecipitation experiments in mammalian cells with a panel of irfs found that med23 interacted with irf4 and irf7 (figure 5a ; figure s7b ). we also observed a weak interaction with irf9, which may explain the previously observed effect of med23 on jak/stat signalling [42] . to determine if this interaction had a functional effect, we looked at whether med23 influenced irf-mediated induction of ifn-l. in a luciferase reporter assay, neither irf4 nor irf9 led to a significant induction of the ifn-l1 promoter, either alone or in conjunction with med23 (data not shown). irf7 induced expression from the ifn-b and ifn-l1 promoters to similar levels (,7-fold and 9-fold higher than background, respectively), whilst the isre, induced by irf7 and also present in the irf7 promoter, was induced ,15-fold ( figure 5b ). whilst co-expression of med23 with irf7 had no further effect on ifn-b expression, a synergistic induction of the ifn-l1 promoter and, to a lesser extent, the isre, was observed (ifn-l1 doubled to ,18-fold, p = 0.02) ( figure 5b ). interestingly, a med23 mutant unable to induce immediate early gene expression via jun/fos (r617q, or r611q in med23 transcript variant 1 used here) synergistically induced isre expression with irf7, yet was unable to further enhance irf7mediated induction of ifn-l1 (data not shown). a similar synergistic effect of med23 and irf7 was seen at the protein level, where co-expression increased supernatant levels of ifn-l3 more than 2-fold those seen with med23 or irf7 alone ( figure s7c , d). successful disease and treatment outcome in hepatitis c virus infection (demonstration of a sustained virologic response) is strongly associated with a single nucleotide polymorphism (snp) in the ifn-l3 promoter (rs12979860; cc genotype over ct or tt) and higher plasma levels of ifn-l3 [49] , [50] . furthermore, ifn-l expression is impaired in a cohort of ethnically italian individuals suffering recurrent hsv-1-related herpes labialis reactivation [51] . to determine if the clinical severity of hsv-1 disease is due to the observed deficiency in ifn-l expression, we screened a subset of the recurrent herpes labialis (hl) cohort and additional subjects for the ifn-l3 promoter polymorphism. genotypic analysis found the presence of a t (ct or tt genotype) had a dose-dependent association with clinical severity, with the homozygous tt genotype being more prevalent as disease severity increases ( figure 6 ). in spite of the relatively small sample numbers in some clinical categories (table 1) , the association of a ct or tt genotype with the most severe recurrence of herpes labialis (h+) was statistically significant (p = 0.014; fishers's exact t-test). as the cc genotype is directly associated with increased ifn-l3 levels [51] , these data highlight a previously unknown association between the frequency/severity of recurrence of herpes labialis, the ct/tt genotype and subsequent reduction in secretion of ifn-l3. it is of importance to investigate this genotype association with a larger cohort of hl patients, as well as those suffering with other hsv-1-related disease, in order to determine the role of ifn-l in the full spectrum of hsv-1 pathogenesis. was selected for validation with deconvoluted sirnas to confirm the phenotype observed in the primary screen. the effect of the four individual sirnas (1-4) and a reconstituted smartpool (sp) were tested by reverse-transfecting into hela cells before infecting after 48 h with hsv-1-egfp (c12) and monitoring replication. replication slopes were calculated and normalized as described, and compared to the primary screen slope (1u). a heat map of replication slopes was generated where red represents inhibition (replication slope ,0.5) and green represents enhancement (slope .1). the phenotype was considered validated if $2 sirnas produced the same or better phenotype as the primary screen. (c) virus specificity of hsv-1 hfs. the effect of hf sirna smartpools on the replication of vzv (a-herpesvirus), hcmv (b-herpesvirus) or semliki forest virus (sfv; rna virus) was determined and compared to hsv-1. normalized replication 626stdev of the controls was considered inhibiting/enhancing. doi:10.1371/journal.ppat.1003514.g002 taken together, these data identify med23 as a novel anti-viral factor which acts as a key regulator of ifn-l expression by interacting with and enhancing the activity of irf7, a major transcription factor involved in innate immunity. our observation of a link between the clinical severity of hsv-1 disease and, a ct/ tt genotype at a snp known to regulate ifn-l3 secretion demonstrates the significance of ifn-l in the control of hsv-1 replication in vivo. whilst this study provides no direct link between the ifn-l3 promoter polymorphism and med23, these associations of ifn-l with hsv-1 disease, combined with our observations that med23 is required for the induction of ifn-l following hsv-1 infection, identifies for the first time a link between med23 and ifn-l, provides a clinical context for med23 regulation of ifn-l expression and underscores the potential biological significance of these data. the use of hsv-1 in a combined genome-scale screening approach has led to the identification of a regulatory axis in antiviral innate immunity, and this important finding not only highlights the power of such combined genome-scale screening approaches to identify novel host candidates for anti-herpesvirus drug discovery, but provides an invaluable dataset to the herpesvirus and scientific community at large. by nature of their scale, high-throughput screening technologies have limitations. rnai technology is limited by technical issues such as off-target effects, where an alternative gene to the intended target is degraded, and insufficient gene knockdown. similarly, y2h protein interaction screens can generate both false-positive interactions, due to 'sticky' proteins and auto-activation of the reporter gene used, and false-negative interactions. whereas the number of false positives can be considerably reduced by stringent screening and selection criteria, the low sensitivity of the y2h assay, which detects 20-30% of known interactions, is inherent to the system and can only be marginally improved. this poor sensitivity is caused by factors such as structural restraints of the y2h bait and prey fusion proteins, a lack of or existence of distinct protein modification in yeast cells, and cellular localization signals in bait and prey proteins preventing nuclear import [52] . however, as all other high-throughput methods for measuring binary protein interactions possess a similarly low sensitivity, but are considerably more laborious and expensive, the y2h system is still the most commonly used technology [53] . in this study we have exploited a combined genome-wide screening approach to investigate hsv-1 replication and interaction with its host. this identified 358 functional hfs modulating hsv-1 replication, and 63 cellular interaction partners. in validation experiments, 57.8% of the interactions were confirmed by co-immunoprecipitation assays in mammalian cells, and of the 358 functional hfs identified in the sirna screen, the phenotype of 83.3% was confirmed in deconvoluted sirna experiments. this, combined with qpcr data demonstrating a minimum gene depletion of 69%, suggests that the functional phenotypes on virus replication are genuine, and not due to 'off-target' effects. the confirmation of such a high proportion of selected validation candidates, in spite of the potential technical drawbacks, highlights the reliability of our primary screen datasets, and thus provides an invaluable resource for the herpesvirology research community. one interesting outcome of this study was the surprisingly low overlap between hits identified using these different technologies. of the 215 genes in common between the sirna and cdna libraries, only 10 (4.7%) were classified as a hit by both methods. this, however, is not unexpected, as even the overlap between studies using the same technology has been reported to be low. for example, the overlap between the three previously published hiv screens was only 7% [19] . furthermore, the degree of functional redundancy within the sirna library, and cellular pathways in general, the potential situation-specificity of virus-host interactions, and the possibility of indirect interactions between viral and host proteins, suggest that these methodologies detecting functional outcomes or physical interactions are linked, but complementary rather than confirmatory. the identified hfs were enriched for a range of cellular processes, such as transcription, gene expression, protein transport and cell cycle ( figure s2 and s3) , and involved at different stages of viral infection. we investigated hfs involved in capsid transport and ubiquitination of antiviral intrinsic host defence factors in more detail. incoming hsv-1 capsids are transported to nuclear pores via the microtubule-organizing centre (mtoc), mediated by capsid proteins vp26 (ul35) and ul46 binding to the dynein light chains dynlt1 (tctex1) and dynlt3 (rp3) [54, 55] . our y2h screen confirmed the known interaction between the capsid protein vp26 and the dynein light chain dynlt3 (text s1 and figure s4 ). combined with the sirna screen data, which found depletion of multiple light chain subunits had moderate anti-viral effects on hsv-1, these data confirm propositions of redundancy in the capsid transport process which ensures successful infection in the event of viral mutations [38, 39] , and provide further evidence that hsv-1 has evolved to be highly promiscuous in its exploitation of cellular pathways to its advantage. to overcome the intrinsic host defence, hsv-1 induces a proteasome-dependent degradation of anti-viral promyelocytic leukemia (pml) nuclear bodies (nd10 domains) by the ringfinger ubiquitin ligase icp0 expressed during early infection [56] . in vitro, icp0 is a biochemically active e3 ubiquitin ligase in the presence of e2 ubiquitin conjugating enzymes (e2s) [ube2d1 (ubch5a) and ube2e1 (ubch6)] [57] , but which e2s are used during infection has remained unclear. we identified 20 cellular e2s that are able to influence hsv-1 replication (text s1 and figure s5 ). depletion of ube2d1-4, ube2e1-3 and ube2n significantly increased the number of pml-positive cells postinfection, in an icp0-dependent manner, indicating that icp0 can use multiple e2s to degrade pml [57, 58] . one of the multi-protein complexes affecting hsv-1 replication was the mediator complex, a large (.30 subunits) complex which links specific transcription factors to the rna polymerase ii transcription machinery [40] . as the requirement of mediator subunits in the replication of herpes and other viruses is already well-known [7, 9, 11, 41] , it was striking that depletion of the med23 subunit exerted the opposite phenotype and led to a strong increase in virus growth. the mediator is composed of four distinct modules termed the head, middle, tail and kinase domains, which provide the mediator with some degree of active control over transcription [59] . as individual subunits of this large complex interact with and exert functional effects via specific transcription factors, it is not unexpected that the observed antiviral effects were specific to med23 [60] . within the mediator, med23 forms a tight sub-complex with med24 and med16 [60, 61] . the increase in virus replication observed upon depletion of med24 may be caused by the destabilisation of the structure of this sub-complex ( figure s6b) . investigations into the mechanism of action revealed med23 inhibits hsv-1 replication by preferentially inducing a type iii interferon response (ifn-l) at the mrna and protein level. this induction was mediated via a direct interaction with the transcription factor irf7, which resulted in a synergistic increase in ifn-l expression. med23 was unable, however, to further enhance irf7-induced levels of ifn-b, suggesting an additional level of complexity to the regulation of interferon signalling. interestingly, the inhibitory effect of med23 was specific to hsv-1, with replication of a range of other viruses including vaccinia virus and semliki forest virus being unaffected by med23 depletion. as vaccinia virus is resistant to ifn-l anti-viral activity [62] , this observation further highlights the importance of ifn-l, as opposed to ifn-b, in the anti-viral effect of med23. the r617q mutation in med23 (r611q in med23 transcript variant 1, used here) was unable to enhance irf7-induced ifn-l expression. this mutation causes hereditary dementia [63] , and the failure to induce ifn-l and thereby control hsv-1 in the brain may be a potential cofactor for the development of dementia, similar to alzheimer's disease [64] . there is mounting evidence for a role of the ifn-l family in the regulation of virus pathogenesis [45] , particularly in the case of hepatitis c infection where a polymorphism in the promoter region of ifn-l3 (il-28b; polymorphism rs12979860), which correlates with plasma levels of ifn-l3 [50] , is associated with disease and treatment outcome [49] . individuals with recurrent hsv-1 reactivation have been shown to be deficient in ifn-l expression [51] , and here we found the similar association between the ifn-l3 promoter polymorphism and ethnically italian patients suffering recurrent and severe reactivations of hsv-1-related oral herpes outbreaks, albeit with a small sample group (n = 58). furthermore, sporadic mutations and genetic polymorphisms in innate immune receptor and signalling molecules that lead to the induction of type i and iii ifns have also been shown to be associated with herpes encephalitis [65] , as well as oral and genital herpes [66, 67] . hsv infection controlled by a complex, interconnected and highly regulated network of cytokines expressed by innate immune cells. type i ifns mainly produced by hsv-infected keratinocytes [68] and pdcs [69] inhibit the spread from neurons to epithelial cells and between epithelial cells [70] , similar to ifn-c. type iii ifns are also able to directly inhibit hsv-1 infection in primary neurons, astrocytes, macrophages and dendritic cells [71, 72] . ifn-c levels produced by peripheral blood cd4+ t-cells correlate with the frequency of hsv-1 reactivation [73] . ifn-l is able to induce expression of both itself and the type i ifns, and a similar effect has also been observed for type i ifns which induce both type i and iii ifns [71, 72] . type iii ifns are mainly expressed by myeloid dendritic cells (mdc) and monocyte-derived macrophages [74] , and signal through the heterodimeric il10rb/il28ra receptor complex whose expression is largely restricted to cells of epithelial origin and plasmacytoid dendritic cells (pdc), in contrast to the broadly expressed type i ifn receptor (ifn-ar1/2) [75, 76] . since primary hsv-1 infection and reactivation affects skin and mucosa in the majority of cases, ifn-l may play a much greater role in the control of hsv-1 pathogenesis, likely in a complex network of coregulated type i and ii ifns, than previously thought. we hypothesize that hsv-infected dcs at the site of the lesion (such as skin langerhans dcs whose role in ifn-l production is currently unknown, or intruding myeloid dcs) in individuals with the rs12979860 t/t or c/t haplotype express reduced levels of type iii ifns, and, in consequence, of type i ifns, which leads to a reduced inhibition of local hsv-1 replication and the occurrence of fresh skin lesions. however, the relative contribution of ifn-l1 and l2/3 to the interferonmediated control of hsv-1 replication in vivo, and indeed the role of med23 in this, remains to be seen. in summary, this study provides a comprehensive and robust analysis of hfs that influence hsv-1 replication in vitro, which will benefit many future studies on hsv-1. the identification of med23 as a crucial cellular component for ifn-l expression, and evidence for the significant role of type iii ifn in the innate immune control of hsv-1 in vitro and in vivo, demonstrates the power of combined, genome-scale studies to identify physiologically important hfs for virus pathogenesis. future studies will clarify the role of genetic variations in both med23 and ifn-l in hsv-1-related diseases, such as meningitis, keratitis and orolabial/genital reactivations. sirna smartpools (4 sirnas per gene) at 0.3 mm were dispensed in 10 ml volumes using a rapidplate384 liquid handler (qiagen) into triplicate black 384-well plates (corning), sealed with adhesive seals (thermofisher) and plastic lids. plates were stored at 280uc until needed (minimum 24 h, maximum 48 h). on the day of transfection, assay plates were thawed at room temperature and 10 ml transfection reagent (dharmafect 1, dharmacon), diluted in hank's buffered saline solution (hbss, thermofisher) to give a final concentration of 0.1%, was added using a multidrop 384 (thermofisher). plates were incubated for 20 min at room temperature to allow formation of transfection complexes. during complex formation, low-passage (p20-22) hela cells (ecacc) from ,50% confluent flasks were washed in pbs and trypsinised in trypsin-edta (lonza) before diluting in phenol red-free, antibiotic-free transfection medium (dmem/f-12 1:1/5% fcs with 15 mm hepes and l-glu; gibco). cells were counted and 3610 3 cells in 40 ml were added to each well using the multidrop 384. plates were incubated for 48 h at 37uc in a humidified incubator with 5% co 2 . to infect, media was removed from plates by inversion, and 10 ml media (as for transfection, but containing penicillin-streptomycin; lonza) or virus (hsv-1-egfp strain c12, diluted to moi 0.5 in infection media) [34] was added using the multidrop 384. plates were incubated at 37uc for 1 h before 50 ml infection media was added and plates returned to the incubator. replication was monitored as a function of egfp fluorescence from 24 h to 80 h post-infection using the polarstar optima plate reader (bmg labtech). virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. cells were transfected as described above, and the cytotoxicity of sirnas was determined using the celltiter blue (ctb, promega) reagent, which gives a fluorescent or absorbance signal relative to the number of live cells. briefly, 5 ml ctb was added per well using the multidrop 384. plates were incubated at 37uc in a humidified incubator with 5% co 2 for 2 h before measuring fluorescence (polarstar optima plate reader). readings were normalized to viability of mock-transfected cells, per plate, and mean cell viability over three replicates was calculated. distribution analysis of cell viability values identified median viability as 60%, and values ,60% were considered cytotoxic. the hsv-1 clone collection was cloned by recombinatorial (gateway tm , invitrogen) and conventional cloning into the bait vector pgbkt7, and screened against a library pooled from 12,381 mgc clones [35] in the pgadt7 prey vector using a semiautomated y2h assay [77] . interacting prey cdnas were identified by sequential blasting of refseq, ensembl and unigene databases. blast hits with identical parameters (score, expectation value, length of alignment) were considered indistinguishable and counted separately. a high-confidence dataset was generated from interaction pairs isolated at least twice, or where the bait interacted with two highly related, non-promiscuous preys. interactions between hsv-1 and human proteins were connected to a network of human protein-protein interactions (a total of 62,310) taken from the databases hprd [78] (release 9), biogrid [79] , dip [80] , mint [30] and intact (downloaded may 18 th 2010). a high-confidence interaction set (9,829 interactions) was compiled from interactions identified in at least two studies. betweenness centrality (g(v) of a protein v was calculated as g(v) = gs {v{t (s st (v)/s st ), where s st is the total number of shortest paths from protein s to protein t, and s st (v) is the number of those shortest paths that contain v. betweenness centrality was normalized by dividing by the total number of protein pairs in the network. enrichment for functional annotations from gene ontology (go) [81] , kegg [82, 83] , reac-tome [84, 85] , and biocarta was performed using david [86] . data on known human protein complexes was retrieved from the corum database, and complexes with subunits showing consistently stronger effects (inhibiting or enhancing) than expected by chance were detected using wilcoxon's rank-sum test. genes included in the rnai screen were ranked by their distance from the median knockdown, with the most inhibiting and enhancing genes being ranked highest. fdr was used for multiple testing correction. the hsv-1 replication phenotype observed in the primary screen was validated for a subset of candidates by deconvoluting the assay smartpools. the four individual sirnas targeting different regions of each gene, as well as a reconstituted smartpool, were diluted to 0.3 mm in 16 sirna buffer and dispensed to black 384well plates. transfection and infection was carried out as described above. replication slopes were calculated and normalized as described, and a phenotype was considered validated if two or more of the four sirnas resulted in the same, or better, phenotype. for inter-viral comparison, sirnas were considered inhibitory or enhancing if normalized replication was 626stdev of the controls a) hsv-1 replication assays. selected sirna smartpools were diluted to 0.3 mm in 16sirna buffer and dispensed in black 96-well plates (corning). to this 10 ml dharmafect 1, diluted in hbss to a final concentration of 0.15%, was added using the multidrop 384. following a 20 min incubation to enable complex formation, 1610 4 hela cells in 80 ml transfection media were seeded on to the complexes bringing the final volume of transfection to 100 ml in each well. plates were incubated for 48 h at 37uc in a humidified incubator with 5% co 2 before infection. to infect, media was removed from plates by inversion, and 25 ml media (as for transfection, but containing penicillinstreptomycin; lonza) or virus (strain c12, diluted to moi 0.5 in infection media) was added using the multidrop 384. plates were incubated at 37uc for 1 h before virus was removed by plate inversion and 100 ml infection media was added. plates were returned to the incubator before replication was monitored as a function of egfp fluorescence from 24 h to 80 h post-infection. virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. b) vzv replication assays. selected sirna smartpools were diluted to 0.3 mm in 16sirna buffer and dispensed in black 384-well plates (corning). to this, 10 ml dharmafect 1 diluted in hbss to a final concentration of 0.07% was added using the multidrop 384. following a 20 min incubation to enable complex formation, 2.5610 3 mewo cells (atcc, htb-65 tm ) in 40 ml media (emem/10% fcs/1% non-essential amino acids) were seeded on to the complexes bringing the final volume of transfection to 60 ml in each well. plates were incubated for 48 h at 37uc in a humidified incubator with 5% co 2 before infection. to infect, media was removed from plates by inversion and ,25 colony forming units of vzv-egfp-infected mewo cells (vaccine strain oka) [87] diluted in mewo growth media were seeded on to the complexes using the multidrop 384. virus growth was measured in 3 h intervals as a function of egfp fluorescence from 44 to 72 h post-infection. virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. c) hcmv replication assays. selected sirna smartpools were diluted to 0.5 mm in 16sirna buffer and dispensed in 10 ml volumes in black 384-well plates (corning). to this, 10 ml dharmafect, 1 diluted in hbss to a final concentration of 0.2%, was added using the multidrop 384. following a 20 min incubation to enable complex formation, 3610 3 mrc-5 (atcc, ccl-171 tm ) in 40 ml growth medium (phenol red-free dmem/ 10%fbs/l-glutamine/1% non-essential amino acids were seeded on to the complexes bringing the final volume of transfection to 60 ml in each well. plates were incubated for 48 h at 37uc in a humidified incubator with 5% co 2 before infection. to infect, media was removed from plates by inversion, and 10 ml media (as for transfection, but containing penicillin-streptomycin) or virus hcmv-gfp (strain ad169) [88] , diluted to moi 0.5 in infection media, was added using the multidrop 384. plates were incubated at 37uc for 1 h before 50 ml infection media was added and plates returned to the incubator prior to monitoring virus replication. replication was monitored as a function of egfp fluorescence from 24 h to 80 h post-infection using the polarstar optima plate reader (bmg labtech). virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. d) sfv replication assays. selected sirna smartpools were diluted to 0.5 mm in 16sirna buffer and dispensed in 10 ml volumes in black 96-well plates (corning). to this, 10 ml dharmafect 1 diluted in hbss to a final concentration of 0.15% was manually added. following a 20 min incubation to enable complex formation, 4610 4 hela cells in 80 ml transfection media (dmem/5% fcs/l-glu) were seeded on to the complexes bringing the final volume of transfection to 100 ml in each well. plates were incubated for 48 h at 37uc in a humidified incubator with 5% co 2 before infection. to infect, media was removed from plates by inversion, and 25 ml media (as for transfection, but containing penicillin-streptomycin; lonza) or virus (sfv4(3h)-rluc [89] diluted in phosphate buffered saline (pbs)/0.75% bovine serum albumin to an moi 0.01) was manually added. plates were incubated at 37uc for 1 h before media (as for transfection media) was added manually to increase the volume to 100 ml per well. plates were incubated at 37uc for 24 h before cells were lysed using a passive lysis buffer and renilla luciferase levels measured with a microplate reader (promega) using a dual luciferase reporter assay kit (promega). luciferase activity, which is representative of virus genome replication, was normalized to mock-transfected cells and mean luciferase activity from six replicates used for subsequent data analyses. e) vaccinia virus replication assays. hela cells were transfected as described in primary sirna screen. plates were incubated for 48 h at 37uc in a humidified incubator with 5% co 2 before infection. to infect, media was removed from plates by inversion, and 15 ml media (as for transfection, but containing penicillin-streptomycin) or 15 ml media containing vaccinia virus strain wr with egfp-tagged a5 protein [90] , diluted to moi 0.05, was added using the multidrop 384. plates were incubated at 37uc for 1 h before 50 ml of media was added to each well, the plates inverted to remove the media and virus, and a final volume of 50 ml of media added to the plates before they were returned to the incubator. replication was calculated as a function of egfp fluorescence at 48 h post-infection using the polarstar opti-ma plate reader (bmg labtech). virus replication was normalized to mock transfected wells on individual assay plates, and the mean replication from eight replicates used for subsequent data analyses. hela cells were transfected with selected smartpool sirnas in 96-well plates, in triplicate, as described. after 48 h transfection, medium was removed, cells rinsed in pbs and lysed in 100 ml trizol (invitrogen). triplicate wells were combined, and rna extracted by standard phenol:chloroform extraction methods. mrna levels were determined by taqman qpcr, using the one-step rt-qpcr kit (thermofisher), with gene-specific primers ( table s9 in text s2), and probes from the universal probe library (roche). expression levels normalized to the housekeeping cellular gene hypoxanthine phosphoribosyltransferase 1 (hprt) and calibrated to mock-transfected cells. qpcr was carried out in duplicate for each sample, and the mean of normalized expression levels calculated. proteins were transiently expressed in hek293 cells as hybrid proteins with the staphylococcus aureus protein a tag or renilla reniformis luciferase fused to their amino termini. 20 ng of each expression construct were transfected into 1610 4 hek293 cells using 0.05 ml of lipofectamine 2000 (invitrogen) in 96-well plates. after 40 h, medium was removed and cells were lysed on ice in 10 ml of ice-cold lysis buffer (20 mm tris ph 7.5, 250 mm nacl, 1% tritonx-100, 10 mm edta, 10 mm dtt, protease inhibitor cocktail (roche), phosphatase inhibitor cocktail (roche), benzonase (novagen) 25 units per ml final concentration) containing sheep-anti-rabbit iggcoated magnetic beads (invitrogen, dynabeads m280, 2 mg/ml final concentration). lysates were incubated on ice for 15 minutes. 100 ml of wash buffer (pbs, 1 mm dtt) were added per well, and 10% of the diluted lysate was removed to determine the luciferase activity present in each sample before washing. the remaining sample was washed 6 times in wash buffer in a tecan hydroflex plate washer. luciferase activity was measured in the lysate as well as in washed beads. negative controls were wells transfected with the plasmid expressing the luciferase fusion protein and a vector expressing two copies of protein a. for each sample, four values were measured: the luciferase present in 10% of the sample before washing (''input''), the luciferase activity present on the beads after washing (''bound''), and the same values for the negative controls (''input nc'', and ''bound nc''). normalized interaction signals were calculated as follows: log(bound)/log(input) -log(bound nc)/ log(input nc). normalized interaction signals were z-transformed by subtracting the mean and dividing by the standard deviation. the mean and standard deviation were calculated from large datasets of protein pairs which were not expected to interact, i.e. from negative reference sets. selected sirnas smartpools were diluted to 500 nm in hbss and 40 ml was incubated with 40 ml dharmafect 1 diluted in hbss to a final concentration of 0.15%. after 20 min incubation, 3610 4 hela cells in 320 ml transfection medium were added, mixed with the transfection complexes and transferred to 8-well glass bottomed chamber slides (becton dickinson). plates were incubated for 48 h at 37uc in a humidified incubator with 5% co 2 before infection by removing medium and adding 100 ml hsv-1-egfp at a moi of 1. after incubation for 1 h at 37uc, virus was removed and replaced with 500 ml growth medium. images were acquired 48 h post-infection. select sirnas smartpools were diluted to 500 nm in hbss and 100 ml was incubated with 100 ml dharmafect 1 diluted in hbss to a final concentration of 0.15% in individual wells of a 12well plate. after 20 min incubation, 2610 5 hela cells in 800 ml transfection medium were added. plates were incubated for 48 h at 37uc in a humidified incubator with 5% co 2 before infection by removing medium and adding 500 ml hsv-1-egfp at a moi of 1. after incubation for 1 h at 37uc, virus was removed and replaced with 2 ml growth medium. after 48 h, medium was removed, cells rinsed in pbs and dislodged by trypsinisation. cells were washed in pbs and pelleted by centrifugation for 10 min at 199 g. supernatant was removed and cells fixed in 4% paraformaldehyde before analysing for egfp expression by flow cytometry (facs diva, bd biosciences) using the cellquest software package. for transient over-expression, 1.5610 4 hek293 cells were seeded in black 96-well plates. the following day, cells were transfected with 100 ng pcr3-med23 using lipofectamine tm ltx (invitrogen) and incubated for 48 h before infection with the recombinant hsv-1 reporter viruses c12 and vp26-yfp at moi 0.5. replication growth curves were monitored, and endpoint replication (as determined by fluorescence) was normalized to untransfected cells. for stable expression, hela cells were transduced with plenti-med23, generated using the virapower tm lentiviral expression system (invitrogen), as per manufacturers' instructions. stable cells were infected, and replication monitored, as above. for confirmation of overexpression, rna was extracted (qiagen rna-easy kit) and expression quantified by one-step rt-qpcr (thermofisher), with gene-specific primers (table s9 in text s2), and probes from the universal probe library (roche). expression levels were normalized to the housekeeping cellular gene hypoxanthine phosphoribosyltransferase 1 (hprt) and calibrated to mock-transfected cells. qpcr was carried out in duplicate for each sample, and normalized expression levels averaged. the vp26-yfp reporter virus was generated from a kos bac kindly provided by david leib using the red-mediated recombination system, where the first 4 amino acids of vp26 were replaced with yfp [91, 92, 93] . the effect of med23 over-expression and depletion was investigated in interferon-deficient cells. the human alveolar epithelial cell line a549, and a549-v, a stat1-deficient derivative cell line stably expressing the v protein from simian virus 5 [43] , were seeded at 2610 4 cells per well in a 96-well plate, and transfected with med23 sirna or pcr3-med23 and infected as described for hela cells. qrt-pcr analysis of interferon and cytokine induction a549 cells were transfected with 100 ng pcr3 or med23 overexpression plasmids, or rscf or med23 smartpool sirna (50 nm) in duplicates, in 96-well plates as described. rna was harvested 20 h post-transfection, and mrna expression levels quantified by qrt-pcr, as described. induction of ifn-l by med23 was determined in a range of cell types by seeding cells in 96-well plates to be ,80% confluent the next day. cells were transfected in duplicates with 100 ng pcr3 or pcr3-med23 using lipofectamine ltx with plus reagent (invitrogen), in antibiotic-free medium. ifn-l levels quantified 96-120 h post-transfection. the synergistic effect of med23 and irfs on ifn-l induction was determined by co-transfection of pcr3 or pcr3-med23 (50 ng) with pcr3-irf7 (50 ng) in a549 cells, with lipofectamine ltx with plus reagent. the effect of med23 depletion on ifn-l induction was determined by transfection of a549 cells in 96-well plates with 50 nm rscf or med23 sirna. ifn-l was quantified 120 h post-transfection. ifn-l protein expression was quantified in supernatants using a commercial ifn-l duoset elisa kit (r and d systems). a point mutation (r611q) was introduced into med23 (transcript variant 1) by pcr with specific primers (see table s9 in text s2) and the clone verified by sequence analysis. a549 cells were co-transfected with 60 ng of pcr3, pcr3-med23 or pcr3-r611q, 60 ng of pcr3-irf7, 20 ng of ifn-b-, ifn-l1-or isre-responsive luciferase reporter constructs and 10 ng prl-tk, a renilla luciferase transfection control, in antibiotic-free low serum (1%) medium. after 33 h cells were lysed, and firefly (promoter reporter construct) and renilla (transfection control) luciferase activity was measured (dual-luciferase reporter kit, promega). relative luminescence activity was normalized to renilla (as a transfection control). a549 cells were transfected in triplicate in black 96-well plates, as described. after incubation at 37uc for 24 h, cells were serumstarved for 24 h by growing in low serum (1%) medium, before cells were left untreated or stimulated with 50 ng/ml ifn-a, ifnb or ifn-c, or 100 ng/ml ifn-l1 or ifn-l2/3 in serum-reduced medium. after 6 h cells were infected with hsv-1 c12 diluted to moi 0.5 in ifn-containing serum-reduced medium. after 1 h incubation, virus was removed and media replaced with 100 ml serum-reduced growth medium containing no interferon, ifn-a/b/-c at 50 ng/ml, or ifn-l1/-l2/3 at 100 ng/ml. replication was monitored as described and normalized to replication in mock-transfected, unstimulated cells. potential interactions between med23 and the irfs were determined by yeast two-hybrid analysis and confirmed by coimmunoprecipitation (co-ip) in mammalian cells. a) co-immunoprecipitation. co-immunoprecipitation was performed using the epitope-tagged plasmids pgbkt7 (myc epitope) and pgadt7 (ha epitope) containing the t7 promoter, and recombinant vaccinia virus vtf-7 expressing the t7 rna polymerase (nih aids repository). 5610 6 hek293 cells were seeded in 10 cm dishes and the following day infected with vtf-7 (moi 10) for 1 h before transfecting 10 mg each of empty bait (pgbkt7) or med23-bait, and irf-prey (pgadt7) vectors with lipofectamine (invitrogen). after 48 h cells were lysed on ice for 30 min in np40 buffer, containing protease and phosphatase inhibitors. debris was removed by centrifugation, and protein quantified with a bca protein assay kit (thermo scientific, uk) as per manufacturers' instructions. equal protein quantities (650 mg per sample) were pre-cleared by continuous mixing with 50 ml preequilibrated protein g sepharose beads for 1 h at 4uc. samples were centrifuged and supernatants halved before overnight mixing incubation with beads pre-coated with 1 mg a-ha (roche, uk) or a-c-myc (santa cruz, uk) at 4uc. beads with precipitated proteins were washed three times with ice-cold np40 buffer, resuspended in 26 sds protein sample buffer and boiled for 10 min before sds-page separation on two 8% polyacrylamide gels. proteins were transferred onto nitrocellulose membranes overnight at 4uc before blocking with 5% milk in tbs-tween then incubating with either a-ha or a-myc antibody (diluted 1:1000 in 5% milk/tbs-tween). membranes were washed in tbs-tween before incubation with hrp-conjugated secondary antibody (diluted 1:3000 in 5% milk/tbs-tween). after further washes, proteins were detected by incubation with ecl western blotting detection system, exposing to x-ray film and developing films in an optimax x-ray film processor. b) yeast two-hybrid assays. haploid yeast strains ah109 and y187 were transformed with 1 mg prey (pgadt7 or pgadt7-irf1, -irf2, -irf3, -irf4, -irf5, -irf7 or -irf9) or bait (pgbkt7 or pgbkt7-med23) plasmid dna, respectively, and grown overnight in synthetic defined (sd) medium lacking either leucine (-l; prey) or tryptophan (-w; bait) before prey-and bait-expressing haploid yeast cells were mated overnight in sd-lw/5% ypda medium. haploids were selected in sd-lw for 48 h before transferring to triple-knockout, histidine-deficient sd-lwh liquid medium containing 3-aminotriazol (3-at) and 4methylumbelliferyl-b-d-galactopyranoside (4-mux) for 5-7 days. interactions were tested in quadruplicates, detected by growth of colonies on sd-lwh agar with 3-at, and quantified by measurement of fluorescence released from a-galactosidase cleavage of 4-mux upon protein interaction. relative fluorescence (rfu) was normalized to negative control interaction (empty pgadt7 mated with empty pgbkt7). ethnically italian subjects with or without a history of recurrent herpes labialis (hl) gave written voluntary informed consent and were enrolled in this study at the university of rome tor vergata with the approval of the ethical committee at the university of rome tor vergata. all subjects were interviewed by medically trained investigators using an appropriate questionnaire and agreed to provide saliva and/or blood samples. data and blood sample collection was carried out as previously described [51] . a total of 58 healthy immunocompetent individuals (17 men and 41 women) age 22-61 years (overall median age 38.5 yr; male median age 40 yr, range 22-61; female median age 38, range 23-60) participated in the study. none of the patients presented with an active lesion at the time of or in the 3 weeks preceding saliva sample collection. for the purpose of this study, patients were characterized into no recurrence of hl (nr), low recurrence (l; 1-3 hl episodes/yr, with a maximum extension of 1 cm, mild symptoms and healing time ,7 days), high recurrence (h; 4 or more hl episodes/yr, extension of lesions more than 1 cm), very high recurrence (h+; more than 4 hl episodes/yr, extension of lesions .3 cm and/or involving nose or cheek beyond the lip, more severe and long lasting associated symptoms including itch, burning, paresthesias and/or neuralgia, with healing times .7 days and who required antiviral therapy (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) ]). saliva samples (,3 ml) were obtained from each subject after an overnight fast and after rinsing the mouth twice with water, split into two aliquots and frozen at 220uc. samples were anonymised and stored with dual code labels before shipping on dry ice to the university of edinburgh for dna extraction. saliva and pbmc samples were thawed and dna extracted using a qiaamp dna blood mini kit (qiagen) as per manufacturer's instructions, quantified using a nanodrop and il28b genotype determined by melt-curve analysis pcr on a lightcycler480 (roche) using the lightmixh kit il28b (tib molbiol) as per manufacturer's instructions. significance of genotype association was determined by fisher's exact test, comparing the frequency of the cc, ct or tt genotype in the nr group versus the l (pvalue = 1), h (p-value = 0.12) or h+ (p-value = 0.015) clinical groups. below lists the geneid numbers for genes and proteins mentioned within the text of this manuscript: ifitm1 (8519) text s1 text for supporting figures. descriptive text for figures s4 and s5 validation of hsv-1-host y2h interactors. a subset of protein interactions identified in the hsv-1-host y2h screen were validated using the lumier pull-down assay in a mammalian cell system. strength of interaction was determined by z-score, where a score 1 to 2 represents a weak interaction and score .2 represents a strong interaction. (h) distribution of direct hsv-1 targets in the rnai screen. the proteins directly targeted by hsv-1 were taken from the y2h data set and from the literature curation. an enrichment of literature-derived targets could be observed in the top 5% most inhibiting knockdowns (2.4-fold enrichment; p = 0.008), but the same could not be observed for the y2h-detected direct targets. (tif) figure s2 validation of hf identified by rnai. hfs identified in the hsv-1 perturbation screen were validated with deconvoluted sirnas and qpcr: (a) chromoboxes, (b) homeoboxes, (c) general transcription factors, (d) nuclear receptors, (e) proteasome family members, (f) topoisomerases, (g) mediator complex subunits, (h) proteins involved in vesicle transport, (i) integrins, (j) y2h interactors, (k) ubiquitin e2 ligases, (l) vesicle transport -further candidates, (m) interferon-stimulated membrane proteins and (n) others. hsv replication is presented as normalized replication slope, and is the mean of six individual assay points. error bars represent standard deviation of the six data points. deconvoluted sirnas which had a sequence different to that in the original screen are highlighted in red. genes for which the primary screen phenotype was not confirmed in the deconvolution assay are shown in bold text. (tif) figure s3 hsv-1 hfs are involved in diverse cellular pathways and at multiple stages of the hsv life cycle. enrichment of protein functions among the hsv-1 hfs. the enrichment for gene ontology (go) terms and kegg, bio-carta or reactome pathway annotations among the hsv-1 hfs identified by (a) rnai and (b) y2h assay was performed using david bioinformatics software. (c) direct interactions between human and hsv-1 proteins. the protein-protein interactions (ppis) depicted are from the high confidence y2h data set and from literature curation. circles and diamonds correspond to human and hsv-1 proteins, respectively. human proteins detected in the y2h screen are drawn with red borders. human genes that showed the strongest effects in the rnai screen are colored yellow (extreme 10%) and orange (extreme 5%). (d) highly interconnected regions in the human interaction network composed of hfs. highly interconnected regions in the human interaction network composed of hfs. we assembled a human interaction network using data from the major ppi databases. a subnetwork consisting of hfs was then defined by limiting the network to the hfs detected in the y2h screen, the rnai (extreme 5%) screen, and the literature curation. highly interconnected regions in the subnetwork were sought out using the mcode algorithm. the top six scoring regions are shown. proteins displayed in red correspond to hfs that are known only from the literature, and those in green are those that were detected in either of the screens performed in this study. the three boolean values beside each gene symbol represent whether the hf is present in the y2h screen, the rnai screen (extreme 5%), or the literature curated set, in that order. dominant functional categories could be observed in each of the regions, including transcription (e.g., mediator complex, rna polymerase ii and associated genes), translation initiation, splicing, and intracellular transport. (tif) figure s4 hfs involved in viral entry and capsid transport. (a) diagrammatic summary of the role of dynein chains in hsv-1 infection. (b) microtubule transport is required for hsv-1 infection. the role of dynein microtubule transport components in hsv-1 replication was analysed by comparing the replication slope of hsv-1-egfp (c12)-infected cells depleted for a range of dynein chains from the primary sirna perturbation screen. error bars represent the mean of three independent experiments done in duplicate. (c) depletion of dyneins inhibits virus particle release. the effect of dynein chain depletion on hsv-1 particle release was determined by quantifying virus titer in supernatants of hela cells depleted of dync1h1 (heavy chain), dync1i2 (intermediate chain) and dync2li1 (light intermediate chains) in a high multiplicity (moi 5; hsv-1 kos) growth assay. titers were compared to control transfected cells (nt, nontargeting sirna). (d) depletion of dynein chains prevents immediate-early gene expression. immediate-early (icp0) and late (vp16) viral protein expression in cells depleted of dync1h1, dync1i2 or dync2li1 was analysed and quantified by western blot. levels were compared to control transfected cells (nt, non-targeting sirna). (e) quantification of icp0 and vp16 protein expression. protein levels of icp0 and vp16 in cells depleted of dync1h1, dync1i2 or dync2li1 were quantified with an odyssey imager and normalized to protein levels in control transfected cells (non-targeting sirna). (tif) figure s5 e2 ubiquitin conjugating enzymes in hsv-1 immune evasion. (a) depletion of e2 ubiquitin ligases inhibits pml degradation following hsv-1 infection. hela cells mounted on coverslips were depleted for a range of e2 ubiquitin ligases for 24 h before infecting with hsv-1 17+. cells were fixed and stained for icp0 (green) and pml (red), analysed by confocal microscopy and pml-positive cells were counted (5 fields of view per coverslip) and expressed as a mean percentage of pmlpositive cells remaining (3 independent experiments). error bars represent the standard deviation over 3 independent experiments. (b) inhibition of pml degradation by e2s is icp0-dependent. hela cells were seeded on coverslips, transfected as above and infected with an icp0 ring-finger deletion mutant (fxe). remaining pml-positive cells were quantified as above. (c) immunofluorescence staining for pml bodies in cells transfected with control sirna not incorporated into the risc complex (rscf) or cells depleted of the e2 ubiquitin conjugating enzymes e2d1 or e2l3. the arrows highlight cells that have wt pml levels remaining in them whilst containing wt icp0. (tif) figure s6 med23 is an anti-viral component of the largely pro-viral multi-protein mediator complex. (a) diagrammatic summary of the role of mediator complex subunits in virus replication. subunits are coloured according to whether hsv-1 replication was unchanged (grey), inhibited (top 5%, red; top 10%, orange) or enhanced upon gene knockdown (top 5%, light green; top 10%, dark green). subunits not included are white; herpesvirus proteins reported to target mediator subunits are violet; mediator subunits detected in other viral rnai screens are highlighted by coloured diamonds. (b) mediator complex subunits influence hsv-1 replication. individual subunits of the mediator complex and associated proteins were depleted by sirna knockdown and infected with hsv-1 c12 (moi 0.5). replication was monitored over multiple rounds and the slope of replication over the linear phase was calculated and normalized to controls (mock-transfected cells). grey, no significant effect; red, strongly pro-viral; green, strongly anti-viral. error bars represent the mean of six replicates. herpes simplex virus type 1 infection: overview on relevant clinico-pathological features herpes simplex virus infects most cell types in vitro: clues to its success the family herpesviridae: a brief introduction herpesviruses and immunity: the art of evasion proteomic analysis of cells in the early stages of herpes simplex virus type-1 infection reveals widespread changes in the host cell proteome analysis of virion-incorporated host proteins required for herpes simplex virus type 1 infection 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mediator is critical for activation by vp16 in mammalian cells role of metazoan mediator proteins in interferon-responsive transcription ns1 proteins of avian influenza a viruses can act as antagonists of the human alpha/beta interferon response toll-like receptor expression and induction of type i and type iii interferons in primary airway epithelial cells lambda interferon (ifn-lambda), a type iii ifn, is induced by viruses and ifns and displays potent antiviral activity against select virus infections in vivo activation of toll-like receptor-3 induces interferon-lambda expression in human neuronal cells ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex il-28, il-29 and their class ii cytokine receptor il-28r genetic variation in il28b predicts hepatitis c treatment-induced viral clearance interferon-lambda serum levels in hepatitis c interferon-lambda in immunocompetent individuals with a history of recurrent herpes labialis connecting viral with cellular interactomes an experimentally derived confidence score for binary protein-protein interactions herpes simplex virus type 1 capsid protein vp26 interacts with dynein light chains rp3 and tctex1 and plays a role in retrograde cellular transport walking the walk: how kinesin and dynein coordinate their steps replication of icp0-null mutant herpes simplex virus type 1 is restricted by both pml and sp100 herpes simplex virus type 1 immediateearly protein icp0 and is isolated ring finger domain act as ubiquitin e3 ligases in vitro the degradation of promyelocytic leukemia and sp100 proteins by herpes simplex virus 1 is mediated by the ubiquitinconjugating enzyme ubch5a the metazoan mediator co-activator complex as an integrative hub for transcriptional regulation transcription control by e1a and map kinase pathway via sur2 mediator subunit the trap100 component of the trap/mediator complex is essential in broad transcriptional events and development inhibition of type 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and neurons induction of cytokine expression by herpes simplex virus in human monocyte-derived macrophages and dendritic cells is dependent on virus replication and is counteracted by icp27 targeting nf-kappab and irf-3 the cycle of human herpes simplex virus infection: virus transport and immune control expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand-and cell-dependent interferon-lambda: a new addition to an old family ifn-lambda (ifn-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo automated yeast two-hybrid screening for nuclear receptor-interacting proteins human protein reference database-2009 update the biogrid interaction database: 2008 update the database of interacting proteins: 2004 update gene ontology: tool for the unification of biology. the gene ontology consortium kegg: kyoto encyclopedia of genes and genomes the kegg resource for deciphering the genome reactome: a knowledgebase of biological pathways reactome: a knowledge base of biologic pathways and processes systematic and integrative analysis of large gene lists using david bioinformatics resources varicella-zoster virus infection of a human cd4-positive t-cell line regulation of the transcription and replication cycle of human cytomegalovirus is insensitive to genetic elimination of the cognate nf-kappab binding sites in the enhancer properties of non-structural protein 1 of semliki forest virus and its interference with virus replication vaccinia virus cores are transported on microtubules live visualization of herpes simplex virus type 1 compartment dynamics construction and characterization of bacterial artificial chromosomes containing hsv-1 strains 17 and kos en passant mutagenesis: a two step markerless red recombination system we are grateful to tomozumi imamichi (national cancer institute, bethesda), rick randall (university of st andrews) and geoffrey l. smith (university of cambridge) for providing reagents, as well as george sorensen, tobias bergmann, gabriella siszler, frank schwarz, melanie ott, verena raschbichler, hannah striebinger, philippa beard and susanne bailer for assistance. key: cord-345712-gmzue6lj authors: palazzo, luca; mikoč, andreja; ahel, ivan title: adp‐ribosylation: new facets of an ancient modification date: 2017-04-26 journal: febs j doi: 10.1111/febs.14078 sha: doc_id: 345712 cord_uid: gmzue6lj rapid response to environmental changes is achieved by uni‐ and multicellular organisms through a series of molecular events, often involving modification of macromolecules, including proteins, nucleic acids and lipids. amongst these, adp‐ribosylation is of emerging interest because of its ability to modify different macromolecules in the cells, and its association with many key biological processes, such as dna‐damage repair, dna replication, transcription, cell division, signal transduction, stress and infection responses, microbial pathogenicity and aging. in this review, we provide an update on novel pathways and mechanisms regulated by adp‐ribosylation in organisms coming from all kingdoms of life. evolution shows remarkable examples of how living species adapt and survive in response to natural and environmental changes [1] . all living organisms have evolved molecular mechanisms that enable them to quickly adapt to nutritional, chemical or physical alterations. these adaptations are induced by cascades of molecular events involving qualitative and quantitative changes in the basic, cellular macromolecules, such as proteins, nucleic acids and lipids. ultimately, these signalling events will trigger the appropriate response. one of the most common tools to induce a rapid change in the cellular environment is the post-translational modification (ptm) of proteins by addition of chemical moieties, such as phosphate, acyl (most commonly methyl and acetate), small proteins or sugars [2]. one highly conserved ptm system is the adp-ribosylation, the addition of adp-ribose (adpr) groups from nicotinamide adenine dinucleotide (nad + ) to proteins [3] (fig. 1) . interestingly, adp-ribosylation can happen not only on proteins but also on other macromolecules such as dna, or small chemical groups [4-7] (fig. 1 ). the first discovered adp-ribosyltransferase (art) enzymes were identified as bacterial toxins, such as the cholera and diphtheria toxins [7, 8] . these toxins are released from bacterial pathogens to irreversibly modify host proteins to gain an advantage over the infected host [7] [8] [9] . later on, homologous transferases and modification-reversing hydrolytic enzymes have been discovered in organisms from all kingdoms of life [3] . moreover, many recent observations show how viral genomes evolved the genetic tools that enable them to modulate adp-ribosylation signalling of infected cells [10] [11] [12] [13] . adp-ribosylation seems to be particularly prominent in the highest organisms and it is best studied for the poly(adp-ribose) polymerase (parp) superfamily of art enzymes [3,7, [14] [15] [16] . altogether, adpribosylation is a widespread modification that controls a vast number of cellular processes, including dna damage repair, transcription, cell-cycle progression, cell division, unfolded protein response, aging, nitrogen fixation, microbial pathogenicity, cell death and many others [7, 14, [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] . however, our understanding of adp-ribosylation is still in its infancy, as can be seen from the current rapid rate of discoveries of previously unknown pathways regulated by adp-ribosylation. all so far characterized arts use nad + cofactor and transfer a single or multiple adpr moieties onto an acceptor molecule (termed mono-and poly(adpribosyl)ation, also called marylation and parylation respectively) combined with the release of nicotinamide (na; fig. 1 ) [3, 7, 16] . the most widespread super families of arts contain transferase folds evolutionary related to bacterial toxins. these proteins can be grouped into: (a) parp-like proteins, alternatively called diphtheria toxin-like art (artd) superfamily; and (b) cholera toxin-like art (artcs) superfamily. most of the transferases from these groups are known to modify proteins, however, some of them modify dna or small chemical groups such as phosphate [3-9,16,17,31] (fig. 1) . eighteen mammalian genes with a sequence homology to parps/artds have been described [3, 7, 14, 16, 27, 28] . the first and best characterized of these proteins was originally noted for its ability to synthesize adpr polymers upon dna damage and called poly(adp-ribose) polymerase 1 (parp1) [15, 23, 26, [32] [33] [34] . human parp1, parp2 and tankyrases (parp5a and parp5b), and close homologues from lower organisms and bacteria are able to produce long repeating chains of adpr on target proteins [3, 7, 16, 23, [35] [36] [37] . parp1 and parp2 can also catalyse the formation of branched chains of poly (adp-ribose) (par) on proteins and possibly dna [6, 35] . most of the other characterized parps are mono(adp-ribosyl) transferases (monoarts) [16, 24, 35] . another, highly diverged art may also belong to parp-like proteins, but it transfers a phosphate group from rna onto adpr (fig. 1 ). this is kpta/tpt1 protein, rna phosphotransferase found in all three domains of life [7, 38] . in yeast, tpt1 catalyses a nad + -dependent dephosphorylation of trnasplicing intermediates, generating adpr-1-phopshate through a cyclic intermediate [38] (fig. 1) . among the artc subfamily of transferases, pierisins are the only transferases able to act on dna [4], all the other enzymes in this group characterised so far act as transferases for proteins [7, 17] . several bacterial toxins can be included in this family, such as the c3 ectotoxin from staphylococcus aureus, vip2 from bacillus cereus, and spvb from salmonella typhimurium [7,39-41]. mammalian artc family includes four human proteins (hartc1, 3, 4, 5) that are glycosylphosphatidylinositol (gpi)-anchored or secreted proteins [17, 42] . hartcs have been reported to modify soluble and plasma membrane-associated protein targets and thus they are proposed to be involved in intercellular signalling, immune responses and inflammation [17, 42, 43] . evolutionary unrelated art enzymes to the previous group are sirtuins. sirtuins are best known as nad + -dependent protein deacetylaeses and they are found in proteins of all kingdoms of life [44, 45] (fig. 1 ). there are seven sirtuin proteins operating in human cells [46] , their primary enzymatic activity is protein deacetylation producing o-acetyl-adp-ribose (oaadpr) metabolite as a by-product of its art reaction [47] . sirtuins can sometimes directly modify proteins [48, 49] (fig. 1) . it has been suggested that the nonenzymatic adpribosylation of proteins may reach significant levels in vivo. this is due to the chemical reactivity of the free adpr with side chains of variety of amino acids, most notably lysine and cysteine [50] . as with other ptms, adp-ribosylation tags are recognized by cellular proteins in a timely manner in order to activate downstream events in the relevant signalling pathways [16, 26, 30, 51] . therefore, many proteins involved in these pathways possess adpr-binding domains within their protein structure [16, 30, 51] . among the evolutionary widespread adpr binding domains, the macrodomain has been studied the most extensively. macrodomains are found in proteins from all kingdoms of life supporting different cellular processes [3, 16, [51] [52] [53] [54] . macrodomain-containing proteins can recognize variety of substrates, including marylated and parylated proteins, different adpr metabolites (such as oaadpr) [16, [53] [54] [55] and rna [54, 56, 57] (fig. 1) . some macrodomains have also evolved enzymatic activity and are capable of hydrolysing adp-ribosylation (see below) [36, 49, 54, 58, 59] . as a consequence, macrodomain-containing proteins are involved in a diverse set of cellular functions, such as chromatin remodelling and dna-damage repair, oxidative stress response, metabolic processes and pathogenic mechanisms [3,5,10-13,30,37, 53, 54, [59] [60] [61] . in addition to macrodomain, several other widely distributed domains have been described as readers for adp-ribosylation, such as the par-binding zinc finger (pbz) [62] , the wwe (named after three of its conserved residues) [63] , the oligonucleotide/oligosaccharide-binding (ob) domain [64] and the par-binding motifs (pbm) which is abundant in dna-damage repair proteins [65] . as mentioned before, the adp-ribosylation is a reversible modification [66] . two evolutionary unrelated protein domains are known to support this catalytic activity: already mentioned macrodomains and the drag-like fold containing proteins [3, 67, 68] . the catalytic macrodomain fold is found in a number of proteins coming from all the kingdoms of life [54] . in humans four catalytic macrodomains have been identified: poly(adp-ribose) glycohydrolase (parg), macrod1, macrod2 and terminal adp-ribosyl glycohydrolase 1 (targ1/c6orf130) [16, 36, 54, 59, 69, 70] . parg efficiently cleaves the par-specific o-glycosidic ribose-ribose bonds, however, it is unable to remove the terminal adpr unit directly linked to a protein ( fig. 1) [36, 58] . the existence of parg-splicing variants ensure the presence of the enzyme both in the nucleus and in the cytoplasm or in membranous organelles [71] [72] [73] and allows for rapid turnover of par, ensuring tight control of this modification [66] . the terminal adpr moiety is removed by marylation preferring hydrolases, such as macrod1, macrod2 or targ1 [58, 59, 69, 70] (fig. 1) . the latter enzymes can also hydrolyse some other adpr metabolites, such as oaadpr [54, 55, 74] (fig. 1 ). an additional macrodomain containing protein is poa1p (ybr022) from saccharomyces cerevisiae, functionally characterized as a specific phosphatase that removes the phosphate group of adpr-1-phosphate in the trna-splicing pathway in yeast [75] . another class of de-adp-ribosylation enzymes includes the dinitrogenase reductase-activating glycohydrolase (drag) and related proteins [67, 68, 73, [76] [77] [78] [79] . drag is known to regulate, in conjunction with art called drat, the central enzyme of nitrogen fixation in several bacterial species [68, 76] . mammals carry distant homologues of drag (called arh1-3 in humans), whose functions are so far not fully understood. the arh1 protein shows efficient hydrolytic activity against marylated proteins on arginine residues [77] . adp-ribosylated proteins on arginine are found on cellular plasma membrane, in the lumen of endoplasmic reticulum (er) [42, 43, [78] [79] [80] [81] and in cytoplasm [82, 83] . arh3 was shown to hydrolyse the o-glycosidic bond of par chains and oaadpr [79, [84] [85] [86] . arh2 is believed to be catalytically inactive [79, 84, 85] . the released adpr by all the active hydrolases can be further recycled and eventually converted to atp by enzymes such as members of the nucleoside diphosphate linked to x-moiety hydrolases (nudix) family [87] [88] [89] . noncanonical enzymes able to perform the hydrolysis of adpr linked to proteins have been recently identified. these include two unrelated protein families, the nudix [87, 88] and ectonucleotide pyrophosphatase/ phosphodiesterase (enpp) [90] , both of which hydrolyse the adpr phosphodiester bond in mono-adpribose and par linked to proteins, thus liberating adenosine monophosphate (amp) and phosphoribose-amp and leaving ribose-5 0 -phosphate (phosphoribosylation; pr) tags bound to the protein [91] [92] [93] [94] (fig. 1) . the nudix enzymes able to perform this reaction are human nudt16 and escherichia coli rpph [91, 92] . within the enpp family of enzymes, vertebrate enpp1 proteins and phoshodiesterase i found in the poison glands of rattlesnakes (snake venom phosphodiesterase) exhibit the same activity [93, 94] . the physiological relevance of the activities of enpp1 and nudt16 enzymes against the protein adp-ribosylation remains unclear. phosphoribosylation of proteins is also a consequence of the activity of sde, an enzyme that couples the art with a phosphodiesterase domain, associated with the control of the host ubiquitination signalling by human pathogen legionella pneumophila (see detailed description below) [95, 96] . adp-ribosylation in mammals is known to regulate a number of different processes [14, 17, 24, [26] [27] [28] 30 ]. best understood is regulation of dna-damage repair pathways by parp1-3 that are activated upon binding to dna breaks [14, [26] [27] [28] [97] [98] [99] [100] [101] [102] parp1 also plays roles in transcription and metabolism [29, 30, 103] . the functions of other parps are comparatively much less understood [27, 28] . parp4 (also called vparp) is a component of the cytosolic ribonucleoprotein vault complex, however, its biological functions are unknown [104] . parps 5a and 5b (tankyrases) are best understood for their roles in mitosis [19, 25] and wnt signalling [105] [106] [107] [108] , but they also have roles at telomere and dna-damage repair [108] [109] [110] . parp6 and parp8 are poorly understood, however, parp6 has been shown to be involved in hippocampus neuronal development [111] . several parps (parp7 parp10, parp12 and parp13) are involved in mechanisms of post-transcriptional regulation of mrna, mediated either by rna-binding domains [112] or by adp-ribosylation of rna-binding proteins [20] . in addition, parp10 has been implied in the regulation of nf-kb [113, 114] , gsk3b [113, 115] and transcription [103, 113, 116] . also, parp9 and parp14 are suggested to act on transcription, in particular of genes required for macrophage activation [117] . parp16 regulates the unfolded protein response [18] . concurrent with its nuclear pore localization, parp11 modifies targets involved in the coordination of the nuclear envelope and the organization of nuclear pores [118, 119] . future work is needed to properly understand the physiological functions of most of the parps and the new potential functions for parps and other arts are continuously arising [120] . compared to parps, much less research has been conducted on members of the artc family in mammals. this family includes four proteins in humans (hartc1, 3, 4, 5) and six in mice (martc1, 2.1, 2.2, 3, 4, 5) that are glycosylphosphatidylinositol (gpi)anchored (hartc1 and martc1) or ecto-proteins (hartc3, 4, 5 and marct2.1, 2.2, 3, 4, 5) [42]. martc1, 2, and 5 have been reported to modify soluble and plasma membrane-associated protein targets on arginine residues, including the p2x7 purinergic receptor, and thus they can affect cellular processes such as intercellular signalling, immune responses and inflammation [43] . hartc1 has been detected in the lumen of er and its function in stress response has been suggested [81] . although all the artc proteins in mammals characterized so far modify substrate proteins on arginine residues [42], the situation for parp family is more complex and there is still no strong consensus in the field on the preferred amino acid targets for many parps. in this respect, the progress has been additionally hampered in the case of poly(adp-ribosyl) ating parps, as the current methods to identify sites do not make the difference between mono and poly (adp-ribosyl)ation for specific amino acid position. overall, acidic residues might be the main targets for most of the parps [35, [121] [122] [123] [124] but cysteine [35, 120, 123] , arginine [121, 123, 124] , lysine [83, 121, 123, 124] and serine residues [125] have been suggested as well. adp-ribosylation of acidic residues and lysines has been shown to be induced by oxidative stress [83] . however, additional evidence has revealed that many of these lysine residues may have been misannotated as modification sites, and that actual modification sites are proximal serine residues, that usually follow immediately after these lysine residues [126] . indeed, the ks motif has been identified as a preferred target for serine adp-ribosylation by several studies [125] [126] [127] . notably, serine adp-ribosylation seems to be specific for regulation of dna damage response and other pathways important for genome stability such as regulation of chromatin structure, transcription and mitosis [127] . hpf1/c4orf27 is the first protein identified acting as a specificity factor for the serine adp-ribosylation [127, 128] . it acts in conjunction with parp1 and parp2 proteins and directs modification of histones, parp1 itself, high-mobility group proteins and likely many other proteins [127] . the first discovered arts were secreted toxins that are found sporadically in bacteria and that irreversibly modify crucial host cell proteins [129] . however, the genomic evidence suggests that intracellular, reversible adp-ribosylation is much more common amongst bacteria, yet, there is little evidence on its physiological relevance. a notable exception is the drat/drag system of nitrogen-fixating bacteria from the azospirillum and rhodospirillum genera. drat homologues are restricted to several nitrogen-fixing bacteria, while drag homologues are distributed across all three domains of life [67] . endogenous adp-ribosylation has also been reported for some other bacterial species where this process probably regulates important cellular functions such as sporulation in bacillus subtilis [130] , development and cell-cell interaction in myxococcus xanthus [131, 132] , as well as differentiation and secondary metabolism in streptomyces [133] [134] [135] [136] . streptomycesbacterial model organism for the study of adp-ribosylation so far, the most evidence for intracellular endogenous protein adp-ribosylation has been found in streptomyces species. streptomyces are soil-inhabiting grampositive bacteria best known for their complex life cycle that includes morphological differentiation and the production of various secondary metabolites including antibiotics, anti-cancer drugs and immunosuppressors. adp-ribosylation has been discovered in streptomyces over 20 years ago [137] . first reports demonstrated considerable adp-ribosylating activities in cell extracts and suggested a role for adp-ribosylation in growth and differentiation processes in streptomyces griseus [133, 134] . in both str. griseus and streptomyces coelicolor, adp-ribosylation patterns change with morphological differentiation [138, 139] and several identified adp-ribosylated proteins in str. coelicolor suggested a connection between protein adp-ribosylation and the regulation of metabolic requirements of the cells [135] . streptomyces coelicolor genomic data (table 1) suggest that adp-ribosylation should be prominent in streptomyces. two arts have been characterized in streptomyces, sco5461 from str. coelicolor [136, 140] and scabin from plant pathogen streptomyces scabies [141] . these proteins are homologues of pierisins [4] and possess guanine-specific dna art activity, but they are not conserved across the streptomyces species and cannot be found in str. griseus, suggesting that the major protein arts in streptomyces have yet to be discovered. disruption of sco5461 leads to conditional pleiotropic phenotype characterized by defects of morphological differentiation, antibiotic production and secretion [136] . the sco3953 protein is a homologue of yeast trna 2 0 -phosphotransferase tpt1, an essential enzyme in yeast that catalyses the final step in trna splicing. this reaction includes dephosphorylation of trna 2 0phosphate in two steps; transfer of adpr from nad + to trna 2 0 -phosphate that generates a 2 0 -phospho-adpr-rna intermediate and release of mature trna together with adpr 1″-2″-cyclic phosphate [142] (fig. 1) . tpt1 homologues are found distributed across all domains of life including bacterial species that have no known intron-containing trnas (str. coelicolor and e. coli whose orthologue is called kpta are among them). therefore, bacterial tpt1/kpta homologues should have some yet uncovered substrate(s) and function(s). the sco2860 is a homologue of mycobacterium smegmatis art arr that modifies antibiotic rifampicin (fig. 1) and causes antibiotic resistance. mycobacterium smegmatis arr gene has been acquired from horizontal gene transfer [143] . it is upregulated after exposure to different kinds of stress and its endogenous cellular function has been proposed in a general stress response [144] . there is evidence of a much larger number of potential adpr hydrolases in str. coelicolor. eight of them are uncharacterized drag homologues and three are macrodomain proteins representing three different classes within the macrodomain superfamily ( table 1) . the sco0909 is a bacterial-type parg that cleaves the parylation in the same manner as mammalian pargs [36] . nothing is known about the function of sco0909, but in the radiation-resistant bacterium deinococcus radiodurans the sco0909 gene is one of the most highly induced genes after dna damage caused by ionizing radiation [145] . the sco6450 is a macrod homologue and it is predicted to remove protein marylation. sco6450 orthologues are found in most of the bacteria [54] . escherichia coli homologue ymdb appears to be a multifunctional protein that regulates variety of cellular processes; deacetylates oaadpr, hydrolyses marylated protein substrates, regulates rnase iii activity and modulates bacterial biofilm formation [55, 69, 146, 147] . sco6735 is a macrodomain protein closest to human proteins alc1 and targ1 [54, 148] . in vitro sco06735 can remove marylation from glutamate residues, yet structural and biochemical characterization indicate a mechanism distinct from any other known macrodomain hydrolases [148] . although sco6735 physiological substrate is still unknown, its expression is under the control of a reca-independent dna damage inducible promoter [149, 150] and upregulated upon uv-induced dna damage [148] , thus indicating a role in dna damage response. moreover, sco6735 is possibly involved in the regulation of antibiotic production and disruption of the sco6735 gene was shown to increase actinorhodin production [148] . two sirtuins, cobb1 (sco0452) and cobb2 (sco6464), have been identified in str. coelicolor. cobb1 is a sirt4 homologue that exhibits deacetylase activity on acetyl-coa synthetase and consequently regulates its activity [151] . auto-adp-ribosylation was demonstrated for the sirt4 homologue of m. smegmatis [152] . cobb2 appears to be related to sirt5 and its overexpression suppresses production of two pigmented antibiotics, thus creating a loss-of-colouration phenotype [153] . altogether, streptomyces represent a good model for the study of adp-ribosylation in bacteria and future studies on this model should help deciphering players and mechanisms of reversible adp-ribosylation process. since adp-ribosylation is involved in the control of antibiotic production in streptomyces, a better understanding of this process will also enable better exploitation of streptomyces biotechnological potential. studies looking either at the genomic context of adpribosylating systems or their evolution in bacteria suggest that adp-ribosylation might be involved in the regulation of many crucial cellular processes including bacterial persistence, oxidative stress response and adaptation to the host environment in general [5, 9, 49, 154] . a novel dna-ribosylating toxin-antitoxin (ta) system has been identified in a variety of different bacterial species including the human pathogens mycobacterium tuberculosis and enterohemorrhagic e. coli [5] . the toxin component of the ta system is a dna art (dart), which catalyses the modification of the second thymidine base in the tntc motif of ssdna. this modification is reversed by the dna adpr glycohydrolase (darg) activity of the antitoxin. the substrate specificity of dart led to the discovery that the adp-ribosylation interferes with dna replication and induces dna damage signalling via the sos response [5] . darg belongs to the alc1-like class of macrodomains and is structurally most similar to human targ1. in addition to the reversal of the dna adp-ribosylation by darg macrodomain hydrolytic activity, protein-protein interaction between dart and darg (resembling a type ii ta system) revealed a second layer of dart regulation [5]. all available data including the fact that darg is essential in m. tuberculosis suggest that targeting this adpribosylating ta system may have a therapeutic potential [5, 155] . protein adp-ribosylation carried out by a distinct class of sirtuins (sirtm) was described in sta. aureus and streptococcus pyogenes and was suggested to regulate oxidative stress response in these pathogens. sirtm is encoded within an operon containing a modification carrier protein (gcvh-l). gcvh-l becomes doubly modified by two different ptms through the actions of sirtm (adp-ribosylation) and another component of the operon that acts as a lipoate ligase (synthesising the protein lipoylation) [2, 49, 156 ]. yet another protein product of the same operon is a macrodomain protein (belonging to the macrod-type class), which specifically reverses the adp-ribosylation of gcvh-l [49] . it was suggested that the lipoylation acts as a scavenger of reactive oxygen species (either host derived or environmentally induced), while the reversible adp-ribosylation may regulate interactions with other proteins involved in the oxidative stress response that are part of this protein complex [49] . sirtm homologues are found in a number of fungal pathogens [49] . bacterial-induced adp-ribosylation has been implicated in regulation of host ubiquitination signalling, a eukaryotic-specific ptm via attachment of a small protein ubiquitin and associated with modulation of the target protein function or degradation [2, 157] . the pathogenic bacterium l. pneumophila uses ubiquitin effector proteins of the sde family, a new class of ubiquitin-specific monoart, to modulate the host ubiquitin signalling and create a favourable growth environment within the host cell [158] . one of the sde proteins, sdea, contains a monoart domain as well as phosphodiesterase domain (pde) [95, 96] . the monoart catalyses the adp-ribosylation of ubiquitin on arg42, while the pde hydrolizes the adprphosphodiester bond, thus establishing a 5 0 -phosphoribosyl modification (fig. 1) . subsequently, the phosphoribosylated ubiquitin is linked to a serine residue within target proteins, thus completing an e2/3 ligaseindependent ubiquitination system. sde-mediated ubiquitination of several er-associated rab proteins and reticulon 4 impairs several cellular processes, such as mitophagy, tnf signalling, tubular endoplasmic reticulum functions and proteasomal degradation, allowing better bacterial growth [95, 96] . among archaea only the tpt1/kpta art type can be found widely spread. considering its wide distribution in all three domains of life and structural simplicity, this protein could represent the ancestral version of the entire art superfamily [9] . other representatives of the art superfamily can be found, but these are limited to only a few species per homologue [9]. in the two methane-producing archaea methanobrevibacter smithii and methanospirillum hungatei homologues of the exotoxin alt/vip2 were identified. a gene encoding a fusion-protein homologue of the dart-darg ta system was found in nitrosopumilus maritimus. protein adp-ribosylating sirtuins (sirtm) have been found in the genomes of sulfolobus solfataricus and methanobrevibacter species [54, 159] . although parps-encoding genes could not been found in archaeal genomes, a parp-like protein adp-ribosylation activity has been detected in su. solfataricus [160] . enzymes capable of removing adp-ribosylation are represented in archaea by two classes, macrod-type and targ1-like. the best-studied archaeal macrodomain protein is af1521 from thermophile archaea arhaeoglobus fulgidus. this protein is capable of binding both adpr and par, and possesses enzymatic activity capable of hydrolysing appr-1-p and marylated protein substrates [52, 69, 70] . af1521 is also used as a tool to enrich adp-ribosylated protein for massspectrometry analyses of modification sites [83, 161] . targ1-like enzymes, as well as drag homologues are sporadically found in some archaeal species such as methanococcus janaschii (pdb code 1t5j). viruses can manipulate host adp-ribosylation machinery and marylation has been recognized as an efficient weapon in the bacteriophage arsenal that is successfully used against bacterial antiphage defence [155, 162] . alt, moda and modb are t4 phage artclike monoarts that modify the e. coli host proteins shortly after infection to overtake the control of the host transcriptional and translational machinery. these enzymes together modify over 30 e. coli proteins, including rna polymerase, ribosomal protein s1, ef-tu and mazf [162, 163] . of these mazf belongs to one of the best-studied type ii ta systems (maze/ mazf), which is involved in bacteriophage defence. maze is a rapidly degraded antitoxin and mazf is a stable toxin with rna cleavage activity (specific to aca rna sequence) that blocks protein synthesis. using alt, the t4 phage defends itself against this system by adp-riboysilating mazf, impairing the rna cleavage activity, and thus enables phage growth [163] . another type of arts that can be identified in a limited number of dsdna viruses are parp-like proteins. these are most likely acquired by horizontal gene transfer and their physiological role remains yet to be studied [3] . proteins encoding macrodomains are more frequently distributed in viral genomes and several different types of macrodomains can be found in both dsdna and positive-strand ssrna [10, 54] . viral macrodomains are usually part of larger proteins that contain additional domains. biochemical, structural and phylogenetic evidences showed that viral and cellular macrodomains are closely related. viral macrodomains bind adpr and par and can perform activities characteristic for cellular macrodomains. most viral macrodomains belong to macrodtype class, but besides their basic de-marylation activity, they are also capable to remove the whole par chain from parylated substrates resembling targ1 activity [10]. in coronaviruses, the macrodtype macrodomain is a part of the multidomain nonstructural protein 3 (nsp3). it has been shown that this macrodomain promotes virulence and suppresses the innate immune response during severe acute respiratory syndrome (sars) coronavirus infection [11] . in addition to the macrod-type of macrodomains, a highly diverged macrodomain sud-m was found as a part of the nsp3 in sars coronaviruses. this unique macrodomain binds nucleic acids, preferentially rna, and is crucial for viral genome replication/transcription [164] . numerous other examples show that viral macrodomains affect virus replication and interferon-response in humans [12, 13] . viral macrodomains may act against mammalian parps that are known to possess antiviral activity [10] . parps involved in the antiviral defence are interferon-inducible, bear the signature of accelerated evolution and inhibit virus replication [165] . specifically, parp7, parp10 and parp12 have been experimentally shown to act as inhibitors of virus replication [166] . another rapidly evolving parp with broad antiviral activity is parp13 (zinc finger antiviral protein), which specifically binds to viral rna sequences targeting them for degradation [167] . evidences for positive selection have also been found in macro-parps (parp9, parp14 and parp15) and parp4 [168] . in some cases, cellular parp activity can be beneficial for viral infection rather than inhibitory [169, 170] . numerous studies investigating adp-ribosylation have been performed in the last several decades. yet, our understanding of the molecular mechanisms governing adp-ribosylation signalling and the physiological and pathophysiological importance of the pathways regulated by adp-ribosylation are still poorly understood. thus, there are many exciting findings waiting to be discovered in this field of research and the scientific community researching the adp-ribosylation has been steadily growing in recent years. many researchers are now also investing great efforts in developing new platforms, tools, methods and pipelines to study the adp-ribosylation [83, 91, 122, 123, 125, 161, [171] [172] [173] [174] [175] these should greatly facilitate further understanding of the complexity of molecular and cellular mechanisms controlled by adp-ribosylation. 28 adp-ribosylhydrolases. sci rep new insights into the molecular and cellular 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activation in response to dsbs parp6 is a regulator of hippocampal dendritic morphogenesis rna regulation by poly(adp-ribose) polymerases intracellular mono-adp-ribosylation in signaling and disease regulation of nf-jb signalling by the mono-adpribosyltransferase artd10 artd10 substrate identification on protein microarrays: regulation of gsk3b by mono-adpribosylation parp-10, a novel myc-interacting protein with poly(adp-ribose) polymerase activity, inhibits transformation and parp14 cross-regulate macrophage activation via stat1 adp-ribosylation spermatid head elongation with normal nuclear shaping requires adpribosyltransferase parp11 (artd11) in mice identifying familymember-specific targets of mono-artds by using a chemical genetics approach chemical proteomics reveals adp-ribosylation of small gtpases during oxidative stress mapping parp-1 auto-adp-ribosylation sites by liquid chromatography-tandem mass spectrometry sitespecific characterization of the asp-and glu-adpribosylated proteome the promise of proteomics for the study of adpribosylation phosphoproteomic approach to characterize protein mono-and poly(adp-ribosyl)ation sites from cells serine is a new target residue for endogenous adp-ribosylation on histones combining higher-energy collision dissociation and electron-transfer/higher-energy collision dissociation fragmentation in a product-dependent manner confidently assigns proteome wide adp-ribose acceptor sites ahel i & matic i (2017) serine adp-ribosylation depends on hpf1 hpf1/c4orf27 is a parp-1-interacting protein that regulates parp-1 adp-ribosylation activity a family of killer toxins -exploring the mechanism of adp-ribosylating toxins adp-ribosylation of proteins in bacillus subtilis and its possible importance in sporulation endogenous adpribosylation during development of the prokaryote myxococcus xanthus adp-ribosylation by the extracellular fibrils of myxococcus xanthus the possible role of adp-ribosylation in sporulation and streptomycin production by streptomyces griseus evidence of a role for nad(+)-glycohydrolase and adp-ribosyltransferase in growth and differentiation of streptomyces griseus nrrl b-2682: inhibition by m-aminophenylboronic acid analysis and identification of adp-ribosylated proteins of streptomyces coelicolor m145 disruption of sco5461 gene coding for a mono-adp-ribosyltransferase enzyme produces a conditional pleiotropic phenotype affecting morphological differentiation and antibiotic production in streptomyces coelicolor adp-ribosylation of membrane proteins of streptomyces griseus strain 52-1 endogenous adp-ribosylation of proteins during development of streptomyces griseus changes in patterns of adp-ribosylated proteins during differentiation of streptomyces coelicolor a3(2) and its developmental mutants adp-ribosylation of guanosine by sco5461 protein secreted from streptomyces coelicolor scabin, a novel dna-acting adp-ribosyltransferase from streptomyces scabies structurefunction analysis of the yeast nad+-dependent trna 2 0 -phosphotransferase tpt1 rifamycin antibiotic resistance by adp-ribosylation: structure and diversity of arr catalytic and non-catalytic roles for the mono-adpribosyltransferase arr in the mycobacterial dna damage response transcriptome dynamics of deinococcus radiodurans recovering from ionizing radiation ymdb: a stress-responsive ribonuclease-binding regulator of e. coli rnase iii activity escherichia coli ymdb regulates biofilm formation independently of its role as an rnase iii modulator disruption of macrodomain protein sco6735 increases antibiotic production in streptomyces coelicolor transcriptional analysis of the reca gene in streptomyces rimosus: identification of the new type of promoter identification of a promoter motif regulating the major dna damage response mechanism of mycobacterium tuberculosis cobb1 deacetylase activity in streptomyces coelicolor a sirt4-like auto adp-ribosyltransferase is essential for the environmental growth of mycobacterium smegmatis use and discovery of chemical elicitors that stimulate biosynthetic gene clusters in streptomyces bacteria mechanisms of bacterial persistence during stress and antibiotic exposure discovery of functional toxin/antitoxin systems in bacteria by shotgun cloning the amidase domain of lipoamidase specifically inactivates lipoylated proteins in vivo ubiquitin and ubiquitin-like proteins as multifunctional signals ubiquitination independent of e1 and e2 enzymes by bacterial effectors the interaction of alba, a conserved archaeal chromatin protein, with sir2 and its regulation by acetylation purification and biochemical characterization of a poly(adp-ribose) polymeraselike enzyme from the thermophilic archaeon sulfolobus solfataricus combining affinity purification by adp-ribose-binding macro domains with mass spectrometry to define the mammalian adp-ribosyl proteome post-transcriptional control by bacteriophage t4: mrna decay and inhibition of translation initiation an adp-ribosyltransferase alt of bacteriophage t4 negatively regulates the escherichia coli mazf toxin of a toxin-antitoxin module a g-quadruplex-binding macrodomain within the "sars-unique domain" is essential for the activity of the sars-coronavirus replicationtranscription complex virus-host interactions and the artd/parp family of enzymes interferonstimulated poly(adp-ribose) polymerases are potent inhibitors of cellular translation and virus replication the zinc-finger antiviral protein recruits the rna processing exosome to degrade the target mrna rapid evolution of parp genes suggests a broad role for adp-ribosylation in host-virus conflicts regulation of epstein-barr virus orip replication by poly(adp-ribose) polymerase herpes simplex virus 1 infection activates poly(adp-ribose) polymerase and triggers the degradation of poly(adp-ribose) glycohydrolase identifying direct protein targets of poly-adp-ribose polymerases (parps) using engineered parp variants-orthogonal nicotinamide adenine dinucleotide (nad+) analog pairs analysis of chromatin adpribosylation at the genome-wide level and at specific loci by adpr-chap synthesis of well-defined adenosine diphosphate ribose oligomers synthesis of dimeric adp-ribose and its structure with human poly (adp-ribose) glycohydrolase synthesis and macrodomain binding of mono-adpribosylated peptides the authors are grateful to johannes rack and kerryanne crawford (university of oxford) for their constructive comments on the manuscript. key: cord-337825-ujq9mxk7 authors: chen, bin; tian, er-kang; he, bin; tian, lejin; han, ruiying; wang, shuangwen; xiang, qianrong; zhang, shu; el arnaout, toufic; cheng, wei title: overview of lethal human coronaviruses date: 2020-06-10 journal: signal transduct target ther doi: 10.1038/s41392-020-0190-2 sha: doc_id: 337825 cord_uid: ujq9mxk7 coronavirus infections of multiple origins have spread to date worldwide, causing severe respiratory diseases. seven coronaviruses that infect humans have been identified: hcov-229e, hcov-oc43, hcov-nl63, hcov-hku1, sars-cov, mers-cov, and sars-cov-2. among them, sars-cov and mers-cov caused outbreaks in 2002 and 2012, respectively. sars-cov-2 (covid-19) is the most recently discovered. it has created a severe worldwide outbreak beginning in late 2019, leading to date to over 4 million cases globally. viruses are genetically simple, yet highly diverse. however, the recent outbreaks of sars-cov and mers-cov, and the ongoing outbreak of sars-cov-2, indicate that there remains a long way to go to identify and develop specific therapeutic treatments. only after gaining a better understanding of their pathogenic mechanisms can we minimize viral pandemics. this paper mainly focuses on sars-cov, mers-cov, and sars-cov-2. here, recent studies are summarized and reviewed, with a focus on virus–host interactions, vaccine-based and drug-targeted therapies, and the development of new approaches for clinical diagnosis and treatment. coronaviruses are crown-like particles with spikes protruding from their surface. they are enveloped viruses with single-stranded, positive-sense rna (+ssrna) genomes of approximately 26-32 kb, which is currently the largest known genome size for an rna virus. 1 rna viruses are divided into five branches. 2, 3 certain groups in a particular branch might be related to and/or share features with viruses classified in another branch. in branch 1 are the leviviruses and eukaryotic relatives (e.g., mitoviruses, narnaviruses, ourmiaviruses); branch 2 represents several +rna virus families (of eukaryotes) (e.g., orders picornavirales and nidovirales, and the families caliciviridae, potyviridae, astroviridae, and solemoviridae) and several dsrna viruses (e.g., partitiviruses and picobirnaviruses); in branch 3 are certain +rna viruses such as the supergroups of alphaviruses and offlaviviruses, the nodaviruses, tombusviruses, and many small and novel groups; in branch 4 are dsrna viruses such as the cystoviruses, reoviruses, and totiviruses; branch 5 consists of −rna viruses. 2 coronaviruses were classified as most likely related to branch 2, and belong to the order nidovirales, and the family coronaviridae. in 2018, the international committee on taxonomy of viruses divided the coronaviridae into the orthocoronavirinae and letovirinae subfamilies. according to their host targets, coronaviruses can also be divided into animal and human coronaviruses. many diseases in domestic animals are related to animal coronaviruses, such as canine respiratory coronavirus, which causes respiratory disease in dogs. 4 the highly pathogenic human coronaviruses belong to the subfamily coronavirinae from the family coronaviridae. the viruses in this subfamily group into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. the classic subgroup clusters are labeled 1a-1b for the alphacoronaviruses and 2a-2d for the betacoronaviruses. to date, including the recently discovered sars-cov-2, there are seven coronaviruses that infect humans. human coronavirus (hcov)-229e, hcov-nl63, hcov-oc43, or hcov-hku1 cause only the common cold, 5 whereas the severe acute respiratory syndrome coronavirus (sars-cov) or the middle east respiratory syndrome coronavirus (mers-cov) cause relatively high mortality and emerged in 2002 6 and 2012, 7 respectively. notably, sars-cov-2 is currently causing a worldwide epidemic. sars-cov and mers-cov belong to subgroups 2b and 2c of the betacoronaviruses, respectively, 1, 8 whereas sars-cov-2 is a new member of the betacoronaviruses distinct from sars-cov and mers-cov. 9 figure 1 shows the phylogenetic tree of rna viruses. the estimated mutation rates of coronaviruses (covs) are moderate to high compared to those of other ssrna viruses. 1 there are two gene loci that are sites of variation in sars-cov. one of these sites is located in the spike (s) protein gene. the second major site of variation is the accessory gene open reading frame orf8. in mers-cov, the major sites of variation are located in the s, orf4b, and orf3 genes. 2 the major differences in the sequence of the s gene of sars-cov-2 are three short insertions in the n-terminal domain and changes in four out of five of the key residues in the receptor-binding motif. 9 the organizations of the genome and gene expression are similar for all coronaviruses. orf1a/b, located at the 5′ end, encodes 16 nonstructural proteins (nsps) (named nsp1-nsp16); other orfs at the 3′ end encode structural proteins, including the s, envelope (e), membrane (m), and nucleocapsid (n) proteins. the mutations in sars-cov-2 have become a hot research topic. the surface proteins of sars-cov and batcov-ratg13, the nearest potential bat precursors of sars-cov-2, have 76 and 97% sequence identity, respectively, to that of sars-cov-2. 10 compared to that of sars-cov, the antigenic surface of sars-cov-2 is highly divergent compared to other covs. since its outbreak began at the end of 2019, sars-cov-2 has acquired mutations throughout its genome, and there are already hundreds of virus strains distributed worldwide. according to gisaid.org, as of may 8, there were 16,004 full genome sequences available, and the s clade of the full genome tree contains the largest group of viruses, indicating that mutations in orf8-l84s are most frequent. the temporal resolution assumes an average nucleotide substitution rate of 5 × 10 −4 substitutions per site per year. based on these mutations, researchers found that sars-cov-2 can be divided into two subtypes. they reported that the snps at locations 8782 and 28,144 show strong linkage; the "ct" haplotype was defined as the "l" type because t28,144 encodes leucine, while the "tc" haplotype was defined as the "s" type because c28,144 encodes serine, 11 which is in accordance with another study that divided sars-cov-2 into two types. 12 the "l" type seems to be more prevalent and aggressive, but the "s" type may be ancestral, as sites 8782 and 28,144 were identical to the orthologous sites in the most closely related viruses. 13 patients may be infected by either or both types. 11 from the gisaid update, compared with ratg13, there are differences in the receptor-binding interface, which may have favored host switching. data related to the sequence and mutations of sars-cov-2 are also available and continuously updated online through the china national center for bioinformation (cncb) (https://bigd.big.ac.cn/ncov). the history of sars-cov, mers-cov, and sars-cov-2 the first case of sars was discovered in foshan, china, in november 2002. 6 infections occurred through either direct or indirect contact with patients. by july 2003, sars-cov had spread to over 30 countries, 12 causing 8096 reported cases and 774 deaths. after that, no additional infections were detected, and the sars pandemic was declared over. sars-cov was first isolated from himalayan palm civets (hpcs) from a live-animal market in guangdong, china. 12, 14 other animals, such as raccoon dogs (nyctereutes procyonoides), along with human workers from the same market, also showed evidence of viral infection. 12 multiple studies have demonstrated that the reservoirs of several coronaviruses, including sars-cov-like and mers-cov-like viruses, were bats. 8, 15 although palm civets might have been intermediary hosts of sars-cov, 16 researchers often concluded there was no evidence that these animals were the ultimate sources of sars-cov, and the viruses cannot circulate directly in palm civets in the wild. 17 in 2003, guan and zheng's team investigated a live-animal retail market in guangdong, focusing on recently captured wild animals and human consumption. the animals sampled included seven wild and one domestic animal species. they collected nasal fig. 1 phylogenetic tree of coronaviruses based on full-length genome sequences. all complete genome sequences of coronavirus were downloaded from the ncbi reference sequence database, refseq. the tree was constructed using maximum likelihood estimation (mle) by mega x, with clustal omega as the multiple sequence alignment method, and 1000 bootstrap replicates. only bootstraps ≥50% values are shown. the seven known human-infecting coronaviruses are indicated with a red star and fecal samples with swabs and then used reverse transcriptionpolymerase chain reaction (rt-pcr) to test for viral nucleic acid from the n gene of the human sars-cov. the rt-pcr assay results showed that samples from four of six hpcs were positive; the other seven species (beaver, chinese ferret-badger, chinese hare, chinese muntjac, domestic cat, hog-badger, and raccoon dog) sampled were negative. 12 mers-cov was first found in 2012 in a lung sample from a 60year-old patient who died of respiratory failure in jeddah, saudi arabia. on 15 september 2012, a similar type of virus named human coronavirus was isolated from a patient with severe respiratory infection. cases have also been reported in other countries. 18 mers has a 35% mortality rate, and since it emerged in the human population in june 2012, it has caused substantial morbidity and mortality. 19, 20 from 2012 until january 15, 2020, the total number of laboratory-confirmed mers-cov infection cases reported globally to the world health organization (who) was 2506, with 862 associated deaths, covering 27 countries (www. who.int/csr/don/31-january-2020-mers-united-arab-emirates/en). cases of mers from other countries were linked to travel to the middle east. 20,21 mers-cov was identified from the saliva of a patient with acute pneumonia and renal failure in jeddah (ksa). mers-cov can be detected in respiratory tract secretions, as well as in feces, serum, and urine. [22] [23] [24] in addition, it has been isolated from environmental objects such as bedsheets, bedrails, intravenous fluid hangers, and x-ray devices. 25 in the case of known camel infection, mers-cov was transmitted from a camel to a human, which was confirmed by rna sequencing. 26 at the end of december 2019, the first clusters of patients with pneumonia caused by sars-cov-2 were reported in wuhan, china. 27 the basic reproduction number of sars-cov-2 is approximately 2.2, indicating that each patient would on average spread the infection to 2.2 people. 28 human-to-human transmission of sars-cov-2 occurred rapidly, and the atypical symptoms during the early stage may be a further disadvantage. 29 moreover, human infection might have begun months prior to the official announcement of the outbreak. 30 to prevent further spread, stricter screening and surveillance are needed at travel hubs. 31 early detection, diagnosis, and treatment are also effective measures to contain the epidemic. 32 nonetheless, the fatality rate of sars-cov-2 is lower than that of sars, 33 with an estimated mortality risk of 2%. 34 sars-cov-2 was first isolated from clinical specimens using human airway epithelial cells and the vero e6 and huh-7 cell lines. 27 approaches to assess virions include isolation from lower respiratory tract specimens 35 and artificially infected specific pathogen-free human airway epithelial cells. 36 fluorescence quantitative pcr with primers designed according to specific sequences and serological testing aimed at igg and igm can be used to detect the virus. 9 sars-cov, mers-cov, and sars-cov-2 pose major challenges to global health, causing infections in large parts of the world. sars-cov was found in 30 countries and mers-cov in 27 countries, while sars-cov-2 was found in 213 countries by 8th may, 2020. details on the pandemics caused by sars-cov-2 are shown in box 1, highlighting the status of the coronavirus-caused diseases worldwide. epidemiological analysis and symptoms of sars, mers, and coronavirus disease 2019 (covid19) the clinical manifestations of sars include hypoxia, cyanosis, high fever (above 38°c), accelerated breathing or respiratory distress syndrome, and shortness of breath. x-rays show changes to the lungs to varying degrees. the who case definition (2003) includes the following: (1) fever higher than 38°c or history of such in the past 2 days, (2) radiological evidence of new infiltrates consistent with pneumonia, (3) chills, cough, malaise, myalgia, or known history of exposure, and (4) positive test for sars-cov by one or more assays. 37 similar to sars-cov infection, mers-cov infection manifests as a severe lower respiratory tract infection with extrapulmonary involvement and high fatality rates. 19 symptomatic patients may present fever, chills, stiffness, myalgia, discomfort, cough, shortness of breath, and gastrointestinal symptoms of diarrhea, vomiting, and abdominal pain. pneumonia is common, and severe infection with acute respiratory failure, renal failure, and shock is particularly frequent among older patients. 20 previous studies have estimated that approximately 12.5-25% of mers-cov infections may be asymptomatic. 20, 38 it must be noted that immunocompromised people are at high risk of mers-cov infection. 21 for covid-19, fever, cough, myalgia, and fatigue are most commonly observed at illness onset; less commonly observed are sputum production, hemoptysis, and headache, among others. 29, 35 similar to sars and mers, some patients develop acute respiratory distress syndrome (ards). 39 around 81% of infected patients only develop mild symptoms, with mild pneumonia or no pneumonia. 14 and 5% are severe and critical status, respectively. 40 patients requiring icu care tend to be much older and are more likely to have underlying comorbidities, such as hypertension and diabetes. 29 additionally, symptoms such as pharyngeal pain and anorexia are reported at higher rates among those admitted to the icu than among non-icu patients. 29 histopathologically, symptoms such as diffuse alveolar damage (dad), loss of cilia, squamous metaplasia, denudation of bronchial epithelia, and giant-cell infiltrate often occur in the lungs of patients with sars. the number of macrophages increases prominently in the alveoli and the interstitium. 41 according to the morphological changes, observed, most authors have subclassified lung lesions in sars into two or three consecutive phases: an acute exudative inflammatory phase, a fibrous proliferative phase, and a final fibrotic stage, although there is considerable overlap in histological findings among these phases. 42 the main features of the first phase include extensive hyaline membrane formation, edema, alveolar hemorrhage, and fibrin exudation in alveolar spaces. the second phase includes widening of septae, pneumocyte hyperplasia, and organizing fibromyxoid and cellular exudates. 42, 43 the third phase includes septal and alveolar fibrosis. 42 several autopsies of sars patients have demonstrated secondary bronchopneumonia, and the pathogens identified mainly consist of staphylococcus aureus, mucor sp., aspergillus sp., pseudomonas aeruginosa, and cytomegalovirus. 43 sars-cov also greatly affects the immune system. for example, hemorrhagic necrosis is usually obvious in the lymph nodes and spleen. 44 as described above, sars-cov attacks immune cells, especially t cells and macrophages, with approximately 50% of lymphocytes and 30% of monocytes in the circulation becoming infected. 42, 45 under the influence of the virus, the patient's germinal centers disappear, and both t and b lymphocytes are depleted. in the spleen, the white pulp shrinks, hemorrhage and necrosis occur in the red pulp, and the periarterial lymphatic sheaths decrease. 42, 45, 46 with regard to the immune system, sars-cov can infect the lymphoid component of the intestine. for this reason, the patient's submucosal lymphoid tissue atrophies. 47 sars-cov can also infect epithelial cells in the mucosa of the small and large intestines, and the digestive tract may undergo mild diffuse inflammation and atrophy of the submucosal lymphoid tissues. 47 according to previous reports, symptoms in the liver include extensive hepatocyte mitosis, balloon degeneration of hepatocytes, mild to moderate lymphocytic infiltrates, fatty degeneration, and central lobular necrosis. 42 additionally, apoptosis of liver cells has been confirmed in some cases. 48 on autopsy, the kidneys of some sars patients showed focal bleeding and acute tubular necrosis of different degrees. one of the major complications of ards is acute kidney injury. 42, 49, 50 in many other cases, the features are nonspecific, including benign hypertensive nephrosclerosis and autolysis. 42 moreover, researchers have detected viral particles and genome sequences in the cytoplasm of neurons of the hypothalamus and cortex in the brain, 51 which suggests that the virus can cross the blood-brain barrier and cause degeneration and necrosis of neurons, edema, extensive glial cell hyperplasia, and cellular infiltration. 42 pneumocytes and epithelial syncytial cells are important targets of mers-cov. the lung tissue presents dad. severe peripheral vascular diseases, patchy cardiac fibrosis, and hepatic steatosis have also been found in other organs. 52 another symptom is bronchial submucosal gland necrosis. 52 renal biopsy may show acute tubulointerstitial nephritis and acute tubulosclerosis with protein casts. 22 in covid-19 patients, a bilateral (sometimes unilateral) distribution of patchy shadows and ground glass opacity in the lungs is typical, based on ct scans. 29 plasma concentrations of interleukins (ils) 2, 7, and 10, granulocyte-colony stimulating factor, interferon (ifn)-γ-inducible protein 10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, and tumor necrosis factor α are increased in most dying patients. 39, 53 in addition, neutrophilia related to cytokine storms induced by virus invasion, coagulation activation related to a sustained inflammatory response, and acute kidney injury related to the effects of the virus, hypoxia, and shock, may be involved in mortality. 29 those who survive intensive care may experience long-term lung damage and fibrosis due to aberrant and excessive immune responses. 39 moreover, researchers found that as sars-cov-2 spread and changed across generations, clinical symptoms started to differentiate those infected previously from those infected more recently. the initial symptoms are becoming more insidious, which indicates that the virus may gradually evolve into an influenza-like virus and lie dormant for longer in asymptomatic patients. 54 in conclusion, in terms of outbreak times, sars-cov was the earliest, followed by mers-cov, then sars-cov-2. to date, mers-cov has the longest duration of infection, and sars-cov-2 appears to be the most infectious. sars-cov, mers-cov, and sars-cov-2 infections have similar symptoms, including fever, cough, myalgia, and shortness of breath, among others. the common transmission methods and symptoms of the three coronavirus infection are shown in fig. 2 . structural studies of sars-cov, mers-cov, and sars-cov-2 sars-cov is a coronavirus with a diameter of approximately 100 nm. coronaviruses are the largest +ssrna viruses and contain at least 14 orfs, 16 protein combines with viral rna to form a nucleocapsid, which is involved in the replication of sars-cov and is the most abundant protein in virus-infected cells. 56, 57 the m protein is a membrane glycoprotein that is involved in budding and envelope formation. the s protein of sars-cov is a type i transmembrane (tm) glycoprotein that is responsible for viral binding, fusion and entry into cells, as well as antibody induction. 16, 58 the s protein contains a signal peptide (sp: amino acids 1-12) and three domains called the extracellular domain (amino acids 13-1193), the tm domain (amino acids 1194-1215) and the intracellular region (amino acids 1216-1255). 16 the extracellular domain consists of two subunits: s1 and s2. the fragment located in the middle region of the s1 subunit (amino acids 318-510) is the angiotensin-converting enzyme 2 (ace2) receptor-binding region. the s2 subunit comprises a fusion peptide (fp) and two x 7-valent element repeats (hr1 and hr2) that are responsible for fusion between the virus and the target cell membrane. 58 thus, the s protein may be key to develop a vaccine to generate antibodies and block virus binding and fusion in the coronaviruses (fig. 3) . the 3′ end of the sars virus genome encodes 12 structural proteins and helper proteins, of which orf3a, orf6, orf7a, and orf7b have been proven to be viral structural proteins involved in the formation of viral particles. the 5′ end of the genome encodes 16 nonstructural proteins (nsps), which are important for virus assembly, and may enable the design of small molecule drugs and/or vaccines. mers-cov belongs to lineage c of the genus betacoronavirus (βcov) in the family coronaviridae, order nidovirales, and it is the first lineage c cov and the sixth cov known to cause human infection. mers-cov is an enveloped +ssrna virus similar to other covs, but the amino acid sequence homology between mers-cov and other known covs is less than 90%. 19 orf1a and orf1b located in the first two-thirds of the mers-cov genome are involved in virulence and encode nsps (nsp1-16). the remaining third of the genome encodes four structural proteins, the s, e, m, and n proteins, as well as five accessory proteins (orf3, orf4a, orf4b, orf5, and orf8b). [59] [60] [61] the flanking regions of the genome contain the 5′ and 3′ untranslated regions (utrs). 62 most mers-cov proteins are found in the cytoplasm. 63 to date, the ns4b protein and the orf3a-encoded protein are the only known coronavirus proteins to be detected in the nucleus. 64 nsp1 suppresses host gene expression in infected cells, and its rna cleavage-inducing function promotes virus assembly/budding. 65 nsp16 is a viral 2′-o-methyltransferase (2′-o-mtase) that encodes critical functions in immune modulation and infection, suggesting that nsp16 is a conserved universal candidate for rapid design of a live attenuated vaccine. 66 orf4a's protein can mask viral dsrna to play a role in immune evasion, including type 1 ifn and protein kinase r-mediated antiviral stress responses. 67 mers-cov nsp13 is a full-length coronavirus helicase that contains multiple domains, including an n-terminal cys/his rich domain with three zinc atoms, a beta-barrel domain and a c-terminal superfamily (sf) 1 helicase (sf1) core with two reca-like subdomains. this protein might interfere with the nonsensemediated mrna decay pathway to avoid degradation of viral rna. 68 the mers-cov s protein can recognize dipeptidyl peptidase 4 (dpp4) on the cell surface, promoting the fusion of the viral envelope and the cell membrane. the mers-cov s protein, a 1353-amino acid type i tm glycoprotein located on the viral envelope surface, is composed of s1 and s2 subunits. 69 the structure of the s protein consists of four parts: an sp located at the n terminus (amino acids 1-12), an extracellular domain (amino acids 13-1195), a tm domain (amino acids 1196-1215), and an intracellular domain (amino acids 1216-1255). 16 the s1 subunit contains two independent domains, an n-terminal domain (ntd, amino acids 18-353) 70 and a c-terminal domain (amino acids 367-588), both of which may function as receptor-binding domains (rbds); 71 these domains serve as critical targets for the development of vaccines and therapeutics 72, 73 and facilitate the involvement of the s1 subunit in cell receptor (dpp4) binding. the core structure of the mers-cov s protein rbd is a five-stranded antiparallel sheet with several short helices, in which three disulfide bonds stabilize the core by connecting c383 to c407, c425 to c478, and c437 to c585. 16, 73 the accessory subdomain of the c-domain is a hypervariable region for receptor recognition fig. 3 clustering of the spike protein of each coronavirus. fifty-four spike protein sequences filtered from each coronavirus coding sequences were clustered using the clans (cluster analysis of sequences) program on the website of mpi bioinformatics toolkit. each colored dot represents the spike protein sequence of each coronavirus. dots in the same color mean they are of the same genus, and each line shows the similarity of two sequences, with darker lines indicating higher similarity (lower e values). the coronaviridae family includes the following genera: alphacoronavirus (colored in green); betacoronavirus (red); gammacoronavirus (orange), and deltacoronavirus (blue). the indicated sars-cov, mers-cov, and sars-cov-2 belong to the genus of betacoronavirus and is called the receptor-binding motif (rbm). the accessory subdomains in sars-cov and mers-cov differ, resulting from divergent evolution and leading to the use of different receptors. 71 dpp4 consists of an n-terminal hydrolase and a cterminal β-propeller domain composed of eight lancets 74 and the rbd. the s2 subunit of mers-cov contains an fp (residues 943-982), an hr1 domain (residues 984-1104), an hr2 domain (residues 1246-1295), a tm domain (residues 1296-1317), and an intracellular domain (residues 1318-1353). 69 the s2 subunit is responsible for fusion between the virus and the target cell membrane. after the s1 subunit binds to dpp4, the s2 subunit inserts its fp into the target cell membrane to change its conformation. its hr1 helix then forms a homotrimer with an exposed surface that has three highly conserved hydrophobic grooves, and binds with hr2 to form a six-helix bundle (6-hb) core structure, which facilitates fusion by bringing the viral and cell membranes into close proximity. 69 the genetic material of mers-cov then enters the host cell via the fusion pore. 69 sars-cov-2, which causes covid-19, is a spherical betacoronavirus with a diameter of 60-140 nm and unique spikes ranging from 9 to 12 nm. 27 its rna has the typical betacoronavirus organization, containing a 5′ utr, a gene encoding the replicase complex (orf1a/b), the s gene, e gene, m gene, and n gene, a 3′ utr, and several unidentified nonstructural orfs. 27, 75 the length of the sequences is 265 nt for the 5′ terminal region, 229 nt for the 3′ terminal region, 3822 nt for the s gene, 228 nt for the e gene, 669 nt for the m gene, and 1260 nt for the n gene. 75 the 21,291nt-long orf1a/b gene contains 16 predicted nsps. 75 similar to sars-cov, there is a predicted orf8 gene that is 366 nt in length and located between the m and n orf genes. 75 nucleotides 1, 1029, and 1652 may be the recombination breakpoints because based on the fragments from nt 1 to nt 1029 and nt 1652 to the end, sars-cov-2 seems to be related to bat-sl-covzc45 and bat-sl-covzxc21; however, when considering the region from nt 1030 to nt 1651, sars-cov-2 is mostly likely to be grouped with sars-cov and bat sars-like covs. more research is required to analyze the recombination of the genome as a whole. 75 with over 10% of its conserved replicase complex being different from those of other betacoronaviruses, sars-cov-2 solely occupies a newly created subclade within the sarbecovirus subgenus. 27, 75 bats may be its natural host, but owing to several arguments including the complicated environment in the wet markets in wuhan, this remains unclear, and further research is needed. 36, [75] [76] [77] the free energy of the spike protein of sars-cov-2 is much lower than that of sars-cov, indicating that sars-cov-2 is more stable than sars-cov. 78 the high similarity of the rbd sequences and of the spike protein structures between sars-cov-2 and sars-cov, 36, 75, 79 also the simulation of the spike protein of sars-cov-2 binding to ace2, 80 the receptor of sars-cov, and results shown that ace2 plays a vital role in sars-cov-2 entry into hela cells, 76 indicating that sars-cov-2 also uses ace2 for cell entry. structural modeling of the ace2-b0at1 complex (b0at1 is used to obtain stable ace2) suggests that the complex can bind two s proteins simultaneously, providing important clues to the molecular basis of coronavirus recognition and infection. 81 the rbd-ace2 binding free energy for sars-cov-2 is significantly lower than that for sars-cov, in accordance with the fact that sars-cov-2 is more infectious than sars-cov. 78 a recent study found that cd147, a kind of tm glycoprotein, facilitates cell entry of sars-cov-2 functionally, and its affinity constant with the s protein is 1.85 × 10 −7 m. 82 table 1 shows a comparison of the structures of sars-cov, mers-cov, and sars-cov-2. the specific function of the sars-cov-2 proteins, including s, e, m, and n proteins, need further study. in conclusion, sars-cov, mers-cov, and sars-cov-2 are similar in structure. their major proteins are structural proteins, including the s, e, m, and n proteins, accessory protein, and nsps (nsp1-16). all these viruses rely on the s protein to identify cell targets and fuse with membranes. their s proteins are composed of s1 and s2 subunits. the s1 subunit contains an ntd and a c-domain, in which there is an rbd. the accessory subdomain of the c-domain is a hypervariable region for receptor recognition and is called the rbm. the difference is that sars-cov recognizes ace2, sars-cov-2 recognizes ace2 and cd147, but mers recognizes dpp4, which results from the difference between the accessory subdomains of sars-cov, sars-cov-2, and mers-cov. the function of the accessory and nonstructural proteins of these three viruses is related to viral replication and assembly, hence linked to virulence. pathogenesis of sars-cov, mers-cov, and sars-cov-2 sars-cov and sars-cov-2 are nearly identical in terms of invasion and self-replication, whereas mers-cov has different targets. the cellular receptor of sars-cov and sars-cov-2 is ace2, plus cd147 for sars-cov-2, while that of mers-cov is dpp4. the human ace2 protein is a typical zinc metallopeptidase that comprises 805 amino acids and contains a single catalytic domain. it is a type i integral membrane glycoprotein that is oriented with an extracellular n terminus and a catalytic site that functions in metabolizing circulating peptides. 83 it is believed that there is a virus-binding hot spot on ace2 that is not pre-existent or preorganized but is induced to form upon virus binding. 84 the hot spot consists of a salt bridge surrounded by hydrophobic tunnel walls. the general structural features of the hot spot favor virus binding: it is located in a region on ace2 that is furthest from the membrane, relatively flat, and free of glycosylation and is thereby easily accessible to viruses. the hot spot is mainly an intrinsic property of ace2, but it is also a dynamic structure and receives structural contributions from both ace2 and the viruses, with the contributions of ace2 being more pronounced. dpp4 is a type ii transmembrane glycoprotein that consists of approximately 766 amino acids. dpp4 contains an n-terminal hydrolase and a cterminal β-propeller domain composed of eight lancets. 74 the s protein rbd binds to the side surface of the dpp4 propeller and the amino acid residues in lancets 4 and 5, including k267, q286, t288, r317, r336, q344, a291, l294, and i295. 74 the pathogenic mechanism of sars-cov is believed to include two parts: the virus injures target cells directly, and subsequent immune system dysfunction leads to indirect injury. 42 sars-cov spreads via droplet and contact transmission and via the fecal-oral route. through droplet inhalation, the virus enters the respiratory tract and invades epithelial cells. infection and replication of the virus and local inflammatory changes contribute to lung tissue damage. subsequently, sars-cov infects circulating and resident immune cells. the key cells in this step consist mainly of t cells and macrophages. the viruses are then carried to other organs, including secondary lymphoid organs, by these circulating immune cells. 42 sars-cov resembles hiv because both viruses attack immune cells and cause immunodeficiency. 42 sars-cov causes lung injury mainly by inhibiting the function of ace2. the virus binds to ace2 via the spike protein, downregulating its function and contributing to lung injury. ace2 is an important component of the renin-angiotensin system (ras). in this system, angiotensinogen is first converted to angiotensini (angi) by renin, and then angiis converted to ang ii under the influence of ace2. ace2 downregulates the levels of angi and ang ii, which can bind to the ang ii type i (at1) receptor and cause certain types of lung injury, mainly pulmonary hypertension, pulmonary fibrosis, and acute lung injury. 85 ang ii can directly induce pulmonary artery smooth muscle cells to grow or proliferate rapidly through at1, thus causing pulmonary hypertension. 86 ang ii contributes to pulmonary fibrosis by promoting the expression of the profibrotic cytokine transforming growth factor-b1, causing fibroblasts to convert to myofibroblasts and accumulate collagen. 70 several strategies can be used by the virus to escape the innate immune response. normally, viral pathogen-associated molecular patterns (pamps) are detected by host pattern recognition receptors (prrs). pamps include viral dsrna and mrna, and prrs include retinoic acid-inducible gene i protein (rig-i) and melanoma differentiation-associated protein 5 (mda5). production of type i ifn is induced via the nuclear factor-κb (nf-κb) pathway. when ifn binds to the ifnα/β receptor, signal transducer and activator of transcription (stat) proteins are activated, which increases the production of other antiviral proteins and thus blocks sars-cov replication for further infection. 11 additionally, sars-cov could encode several proteins that hinder the signaling cascades downstream of prrs. the cell receptor of mers-cov is dpp4, which is widely expressed on epithelial cells in the prostate, alveoli, kidneys, liver, and small intestine and on activated leukocytes; thus, the range of mers-cov tissue tropism is broader than that of any other similar coronavirus. mers-cov infection of dendritic cells and macrophages can lead to the continuous production of proinflammatory cytokines and chemokines (such as tnf-α, il-6, cxcl-10, ccl-2, ccl-3, ccl-5, and il-8). 87 compared to sars-cov infection, mers-cov infection can cause higher expression levels of il-12, ifn-γ, ip-10/cxcl-10, mcp-1/ccl-2, mip-1α/ccl-3, rantes/ccl-5, and il-8. 87 induction of immune cell-recruiting cytokines/chemokines might lead to the infiltration of a large number of immune cells into the lower respiratory tract, causing severe inflammation and tissue damage. 87 mers-cov can also infect t cells and lead to apoptosis, avoiding the host immune response and facilitating faster spread. 88 mers-cov has also evolved a mechanism to escape the host cell immune system. when host sensors initiate signaling pathways, transcription of type i ifn genes begins, and the type i ifn response, which is an essential component of antiviral innate immunity, is initiated. [89] [90] [91] ifn activates the transcription of numerous ifn-stimulated genes (isgs) and synthesizes 2′,5′oligoadenylate (2-5a), which causes rnase l activation. [91] [92] [93] activated rnase l can cleave both viral and host ssrna, leading to translation stagnation and apoptosis and limiting virus replication and transmission in vitro and in vivo. 92, 94 however, the mers-cov protein ns4b has phosphodiesterase activity and antagonizes rnase l via enzymatic degradation, leading to inference with ifn signaling. although ns4b is mainly expressed in the nucleus, the expression level of the ns4b protein in the cytoplasm is sufficient to prevent activation of rnase l. 88 in addition, the mers-cov m, orf4a, orf4b, and orf5 proteins are reported to be strong ifn antagonists, among which the orf4a, orf4b, and orf5 proteins inhibit both type i ifn induction 61, 95, 96 and nf-κb signaling pathways, which are similar to the type i ifn signaling pathway, 96 providing a possible mechanism for mers-cov evasion of innate immunity. 88, 96 for sars-cov-2, the first cluster of patients was believed to be associated with a seafood market. 27 however, due to the identification of patients without direct exposure to the market, transmission between humans was proven. 97 hospital-related transmission has also been detected via infected medical workers and hospitalized patients. main routes of transmission include respiratory droplets, close contact, and extended exposure to aerosol environments loaded with high concentrations of virions. in addition to transmission through the respiratory tract, exposure of unprotected eyes to sars-cov-2 might cause acute respiratory infection. 98 notably, the virus can be transmitted during the incubation period. 99 the tm protease serine type 2 (tmprss2) primes the s protein of sars-cov-2 for cell entry. 100 based on analysis of the ace2 rna expression profile in normal human lungs, sars-cov-2 mainly infects type ii alveolar (at2) cells (only 0.64% of human lung cells can express ace2, 83% of which are at2 cells), and they express many other genes that facilitate sars-cov-2 infection; 101 moreover, cd147 was also reported that it probably functionally facilitates cell entry of sars-cov-2. 82 high expression of ace2 has also been detected in cells of the digestive system, such as upper esophageal cells; accordingly, this system is a potential route of infection. 102 in addition to the lungs and intestines, other organs, such as the heart, esophagus, kidney, bladder, testis, ileum, and adipose tissue, also express ace2, and the expression level is higher than that in the lungs. 103, 104 certain tumor tissues have higher expression of ace2, making cancer patients more vulnerable than other people. 104 in conclusion, sars-cov and sars-cov-2 bind cells expressing ace2. they affect the ras system due to the affinity for ace2 (much stronger with sars-cov-2 than with sars-cov), leading to the onset of symptoms. mers-cov causes symptoms by producing inflammatory cytokines and invading t cells. the specific pathogenic mechanisms of these three viruses are illustrated in fig. 4 . diagnostic methods in addition to clinical manifestations, definitive diagnosis and confirmation of a coronavirus infection requires specific testing. the criteria for the diagnosis of sars from the who include (1) fever higher than 38°c or history of such in the past 2 days, (2) radiological evidence of new infiltrates consistent with pneumonia, (3) chills, cough, malaise, myalgia, or known history of exposure, and (4) positive tests for by at least one assay. as for mers, the criteria for the diagnosis according to the who include (1) fever, cough, and hospitalization with suspicion of lower respiratory tract lesion, (2) history of contact with probable or confirmed cases, (3) history of travel or residence within the arabian peninsula, and (4) positive tests by a pcr-based detection method using respiratory samples, such as nasopharyngeal swabs, nasopharyngeal or tracheal aspirates, bronchoalveolar lavage (bal) and induced sputum, or by serological tests such as the rapid immunochromatographic assay using highly selective monoclonal antibodies at room temperature. the initial criteria for the diagnosis of sars included (1) a history of travel or residence in wuhan within 2 weeks before the onset of illness, (2) exposure to patients from wuhan with fever and respiratory symptoms within 2 weeks before the onset of illness or the existence of clusters of diseases, (3) fever accompanied by radiographic features of pneumonia, a normal or decreased total number of white blood cells, or a decreased lymphocyte count, and (4) fluorescence pcr testing positive for nucleic acids of sars-cov-2. 37, 59, [105] [106] [107] early diagnosis of mers is difficult, but several highly specific and sensitive molecular and serologic assays exist for diagnosis in both animals and humans. 108 among them, a rapid immunochromatographic assay can detect mers-cov antigens based on the detection of the mers-cov nucleocapsid protein in a short time frame; the relative sensitivity and specificity of this test are 93.9 and 100%, respectively. 105 nucleic acid tests, such as real-time pcr tests, were mostly used to detect virus in the early stage of the outbreak of sars-cov-2, although this approach requires a relatively long time and is not conducive to accelerating diagnosis or large-scale application. therefore, alternative, rapid diagnostic kits for sars-cov-2 may be used. for example, the colloidal gold detection kit uses serum, plasma, or whole blood samples to detect igm/igg antibodies for diagnosis on the 7th day of infection or the 3rd day after onset and requires only 15 min. the nucleic acid detection kit for six respiratory viruses, including sars-cov-2 and influenza a virus, uses thermostatic amplifier chips for diagnosis. by detecting different infections, it may help distinguish people with influenza from those with other viral infections. a newly developed fig. 4 pathogenesis of sars-cov, mers-cov, and sars-cov-2. sars-cov and sars-cov-2 play a pathogenic role by inhibiting ace2. under the influence of renin and ace, angiotensinogen is converted into ang ii. through the at1 receptor, ang ii acts as a lung injury-promoting factor, and in some cases, may cause vascular constriction, an inflammatory response, cell proliferation, fibrosis, and apoptosis of alveolar epithelial cells, resulting in diseases such as pulmonary hypertension, pulmonary fibrosis, and acute lung injury. ace2 converts ang ii into ang (1-7), and through the mas receptor, ang (1-7) play roles in vasodilation, antiproliferative activity, and antioxidant activity. sars-cov downregulates the activity of ace2 and causes an increase in the amount of ang ii and lung injury. mers-cov infection of dendritic cells and macrophages can lead to the continuous production of pro-inflammatory cytokines and chemokines, leading to a large number of immune cells infiltrating a patient's lower respiratory tract, causing severe inflammation and tissue damage. mers-cov can infect t-cells from human lymphoid organs and causes the peripheral blood inducing apoptosis by intrinsic and extrinsic pathways, thus avoiding host immune response detection method, nanopore targeted sequencing, also has the potential for efficiently detecting viruses in a reasonable time. moreover, it could identify 40 other respiratory viruses and monitor changes in virulence and transmissibility caused by viral mutations. 109 therapeutic agents all patients should be treated in isolation. 53 based on previous studies, drugs may be developed to inhibit cell entry. several strategies have been investigated to identify specific antivirals targeting the s protein, including mers-rbd-targeted nabs that block viral attachment, peptide inhibitors targeting s2 to prevent the formation of the fusion core, and small molecules without defined mechanisms. 110 sdpp4 has been found to bind mers-cov rbd with high affinity, suggesting that sdpp4 could serve as a blocker for mers-cov attachment to and entry into cells. 111, 112 one study reported a monoclonal antibody, 7d10, that binds to the ntd to inhibit cellular entry of mers-cov. 113 experiments have shown that 7d10 not only inhibits the binding of mers-cov to cells but also has effects after viral-cell attachment. 7d10 inhibits virus invasion by blocking the binding of the s1 subunit to the receptor and interfering with the conformational transformation of the s2 subunit when the virus fuses with the cell membrane. 113 a peptide fragment spanning residues 736-761 of the s protein exerts neutralization activity by inhibiting membrane fusion and the entry of mers-cov, suggesting that it is possible to develop vaccines targeting this neutralizing epitope. 114 griffithsin, a lectin derived from red algae, binds to oligosaccharides on the surface of sars-cov s protein and may also prove effective against sars-cov-2. 34, 115 researchers found that antibodies raised against sars-cov, such as css-2, -3, -4, and -5, could cross-neutralize sars-cov-2 by reducing s-driven cell entry, although the efficiency was lower than that observed for sars-cov. 100 meplazumab, an anti-cd147 humanized antibody, binds with the cd147 in competition with the s protein, 116 thus significantly inhibiting virus from invading cells and reducing the time of negative conversion. 82, 106 chloroquine could effectively inhibit sars-cov-2 in vitro, and hydroxychloroquine was even more potent than chloroquine. 117, 118 hydroxychloroquine could significantly help reduce the virus load, and azithromycin could reinforce its effect. 119 arbidol could also contribute to condition improvement. 120 note that these studies were generally limited and further trials and development are necessary. protease inhibitors also have the potential for coronavirus therapeutics. protease inhibitors, including disulfiram, lopinavir, and ritonavir, have been reported to be effective against sars and mers, and disulfiram, a drug for alcohol dependence, was reported to inhibit the papain-like protease (plpro) of mers-cov and sars-cov in cell cultures. 34, 108 in vitro, sars-cov can be inhibited by both 6-mercaptopurine and 6-thioguanine targeting plpro. 121 when used together with ribavirin, the protease inhibitors lopinavir and ritonavir have improved the outcomes of patients with sars compared with those achieved with the use of ribavirin alone. 122 such inhibitors are also being tested in patients infected with sars-cov-2, but whether these inhibitors effectively suppress 3clpro and plpro of sars-cov-2 remains controversial. 34 drugs such as colistin, valrubicin, icatibant, bepotastine, epirubicin, epoprostenol, vapreotide, aprepitant, caspofungin, perphenazine, prulifloxacin, bictegravir, nelfinavir, and tegobuvir bind to the main protease of sars-cov-2, and thus, are potential candidates. 123 lianhua-qingwen formula, a kind of traditional chinese medicine, has been also recently described theoretically as a potential treatment candidate against the main virus protease. 124 the serine protease inhibitor camostat mesylate could partly block sars-cov-2 infection of lung cells by inhibiting tmprss2. 100 viral rna synthesis blockers also have potential. remdesivir has broad-spectrum activity against mers-cov and sars-cov in cell cultures and animal models and is currently in clinical development for the treatment of ebola virus disease. 34, 125 it was also reported positively based on a patient with sars-cov-2 infection in the us, 126 and another study showed that it was able to inhibit the virus. 117 ribavirin is often used to treat sars and mers, but it is uncertain whether it is potent enough against covid-19. 34, 108 regardless, when used alone, ribavirin has minimal activity against sars-cov and may cause strong side effects. it is often used together with corticosteroids. 14,127 a preclinical study of galidesivir (bcx4430), an adenosine analog, revealed antiviral activity against sars-cov and mers-covcov. 115 a study of favipiravir (t-705), a guanine analog, proved its activity against sars-cov-2, and randomized trials of favipiravir plus interferon-α (chictr2000029600), and favipiravir plus baloxavir marboxil (chictr2000029544) are in progress. 34 corticosteroids may be used to treat sars patients to suppress lung inflammation; although this therapy is not associated with mortality in patients with mers, it is associated with delayed clearance of mers-cov rna. 14,128 moreover, clinical evidence does not support the use of corticosteroids for covid-19 treatment. 129 regarding antibodies, plasma therapy using convalescent plasma from fully recovered patients is effective against these coronaviruses, including sars-cov-2. 108, 130 tocilizumab (antihuman il-6 receptor) may curb immunopathology and trials are ongoing now. 131 a study of novel monoclonal antibodies against the mers-cov spike protein suggests that mabs can be utilized for the identification of specific mutations of mers-cov. 132 wang et al. provided an all-hydrocarbon-stapled peptide that likely mimics the native conformation of the c-terminal short α-helical region of the mers-cov s protein, which can block the formation of the hexameric coiled-coil fusion complex to inhibit viral-cell membrane fusion. 133, 134 current treatments for sars mainly include ribavirin, ifn α, plasma therapy, host-directed therapies, and systemic corticosteroids. 14, 41 due to the lack of clinical trial data, adequate supportive care supplemented with different combinations of drugs remains the main treatment for patients. 14, 41 for mers, despite many studies in humans, there is no consensus on the best treatment; thus, randomized clinical trials are needed to assess potential treatment options. 134 several therapeutics are in development, including convalescent plasma, lopinavir/ritonavir, ribavirin, ifn and novel therapies, including polyclonal antibodies and broad-spectrum antivirals. 108 antimicrobial peptides (amps) may be used as alternative therapeutic agents, 135 and many have been effective even against bacterial proteases agents. 136 antiviral drugs with effective activity in vitro include neutralizing monoclonal antibodies, antiviral peptides, mycophenolic acid, lopinavir, ifn, ribavirin, nitazoxamide, mycophenolate mofetil (mmf), alisporivir, silvestro, and corticosteroids. 19, 21, 59 although there is no agreement on the most ideal treatment option to cure covid-19, much research activities and pursued routes are underway. potential host-targeted agents the host-directed strategy is another approach for limiting viral replication. host proteases such as cathepsin b and cathepsin l can cleave the spike protein of sars-cov. drugs such as camostat inhibit host serine proteases and then interfere with the entry of sars-cov, but they may cause many significant side effects. 14, 135 pegylated ifn α-2a and -2b may be used to stimulate innate antiviral responses in patients, and chloroquine, an approved immune modulator, was reported to have inhibitory effects against sars-cov-2 under certain conditions. 34 researchers have also proposed that some commercially available drugs with suitable safety profiles, such as metformin, glitazones, fibrates, sartans, and atorvastin, as well as nutrient supplements and biologics, might reduce immunopathology, boost immune responses, and prevent or curb ards. in addition, ongoing cellular therapies using mesenchymal stromal cells from allogeneic donors have been shown to reduce nonproductive inflammation and affect tissue regeneration and are being evaluated in phase 1/2 trials in patients with ards; these therapies may be assayed in sars-cov-2-infected patients. 40, 137, 138 expansion of antiviral t cells as cellular drugs could aid in preparing t cell products for adjunct treatment of patients with severe infection. 40 overall, there are similarities and differences among the treatments for these three coronaviruses. notably, we summarize the potential drugs and vaccines in table 2 . vaccination could be used to prevent infection or to reduce disease severity. different kinds of vaccines, such as dna vaccines, recombinant proteins, subunit vaccines, and inactivated viruses, were described. 14 however, the highly sophisticated immune evasion mechanisms of viral pathogens and their high mutation rates make human vaccine development a major challenge. 136 the four nsps (3clpro, plpro, helicase, and rdrp), which are key enzymes in the viral life cycle, and the s protein, which is responsible for receptor binding during cell entry, are attractive targets for developing vaccines against coronaviruses. 115 among them, the s protein is most commonly targeted. 61 based on previous studies, it is believed that the s protein receptor-binding domain (s-rbd) located in the s1 subunit is an important target for the development of a sars vaccine, especially the key neutralizing region, cnd. indeed, cnd can induce a strong neutralizing antibody response and crossprotection against sars-cov mutants. one study showed that a recombinant fusion protein (designated rbd-fc) containing the 193-amino acid rbd and a human igg1 fc fragment can induce highly potent antibody responses in immunized rabbits. the antibodies inhibited sars-cov infection (serum dilution of 1:10,240), and they are believed to be safer than other types of vaccines. 139 additionally, with chloroplast transgenic technology, it is possible to combine the fusion gene s-rbd and the carrier molecule cholera toxin b subunit into the tobacco chloroplast genome to obtain a chloroplast transgenic tobacco plant that stably expresses the oral sars-cov subunit vaccine. 140 for mers, vaccines based on the viral s protein include full-length s or strimer protein-based vaccines such as a full-length s-based simian adenovirus vector vaccine (chadox1) and dna vaccine (gls-5300), 61 s1-based vaccines such as an ad vector encoding mers-cov s1 extracellular domain (ad5.mers-s1) and an rabv vector encoding an s1-elicited antibody, 141, 142 rbd-based vaccines, and vaccines based on other regions. it has been confirmed that the s proteins of sars-cov and sars-cov-2 are quite similar, but researchers recently found that the three monoclonal antibodies developed to bind to the s protein of sars-cov, s230, m396, and 80r do not cross-react with the s protein rbd of sars-cov-2, simian adenovirus vector vaccine (chadox1);an ad vector encoding mers-cov s1 extracellular domain (ad5.mers-s1); an rabv vector encoding s1 elicit antibody; rbd-based vaccines 61, 138, 139, 140 overview of lethal human coronaviruses chen et al. suggesting that tailored vaccines and antibodies against sars-cov-2 must be designed. 143 dna vaccines are also quite promising. specific igg antibodies against sars-cov can be promoted by the s gene dna vaccine. as mentioned above, the s protein of sars-cov plays an important role during the pathogenic process, and synthetic peptides induced by dna vaccine in escherichia coli elicit specific antibodies against the sars-cov s protein which might provide another approach for further developing sars-cov vaccines. 42, 144 to evaluate the safety and immunogenicity of a plasmid dna vaccine (gls-5300) that expresses the s protein of mers-cov, a phase i clinical trial on healthy volunteers was conducted in 2016, but the results were not reported. another phase i trial utilizing the viral vector, chimpanzee adenovirus, oxford university #1 (chadox1), containing the mers-cov s protein expression gene was started by oxford university in january 2018. 145 in addition, camel vaccines against mers-cov are a consideration. at present, at least two promising candidate camel vaccines are undergoing development, and field trial evaluation is in progress. 108, 146 one study found that the rbd fragment covering spike residues 377-588 is a key neutralizing receptor-binding fragment and an ideal candidate for mers vaccines. 147 another potential neutralizing epitope is a peptide fragment covering 736-761 residues of the s protein which blocks the membrane fusion and cellular entry of mers-cov 114 (fig. 5) . other approaches recombinant viruses may be employed to generate an immune response against the viruses. there are two kinds of recombinant viruses which are promising for designing a protective vaccine. the first is a defective or nonpathogenic vector capable of expressing viral proteins, and the second is a vector that can be assembled in a test tube to stimulate the assembly of virus-like particles. adenosine deaminase (ada), a kind of human enzyme, could interact with dpp4, therefore, ada could compete with mers-cov as a natural antagonist when the virus attempts to bind to cells. 148 in addition, anti-dpp4 can play a similar role, 149 however, it is not practical to use this method in vivo because dpp4 has important functions in the regulation of several different signaling pathways. 150 questions concerning the coronavirus-host interactions this topic has intrigued the community. understanding certain mechanisms about virus interactions will support drug research activities. for instance, it was found that sars-cov-2 has a stronger, more rapid spreading ability than many known viruses. is it possible that it invades host cells via additional novel routes, not limited to ace2? for example, cd147 is probably involved in virus invasion. 82 is there an s-like protein involved in invasion? the affinity of the human ace2 protein for the rbd of the new coronavirus is 10-20 times higher than it is for that of the sars virus, which likely explains why sars-cov-2 spreads more swiftly. there is a distinct insert present in the peptide fragment spanning of s1/s2 loop of the sars-cov-2 s protein, which is not shared the similarity with sars-cov or any sars-related viruses. does this peptide loop impact on the entry pathway type of sars-cov-2, compared to other known betacoronavirus lineage b? furthermore, the s protein is likely cleaved during virus assembly and delivery to the cell surface by golgi-resident proprotein convertases such as furin, 151 which is different from the behavior of its close relatives. furin is found in a variety of human tissues, including the lungs, liver, and small intestine. therefore, which are exactly the cell types that sars-cov-2 may attack? yet, in relation to the last two questions, it should be noted that studies are also necessary to investigate any possibility of receptor-independent virus entry. the sars-cov-2 genome encodes nonstructural proteins, structural proteins, and accessory proteins. 3clpro, plpro, helicase, and rdrp, which belong to the first type, and the s protein, which belongs to the second, have been recognized as promising targets for developing antiviral agents against sars-cov and mers-cov, which therefore may be applied to sars-cov-2. nonetheless, the rapid identification of effective interventions against sars-cov-2 is a major challenge. however, the subject of using currently known antiviral drugs has been frequently brought up by the community as a potential short-term strategy to combat sars-cov-2. drugs affecting such targets and their status for sars-cov-2 are listed below. the main candidates as inhibitors of 3clpro include other agents for sars-cov-2 fig. 5 the targets of the different drug candidates against the three coronaviruses. common targets against the three coronaviruses are mainly the s protein and the s1/s2 subunits, pl protein, rdrp, 3cl protein, and helicase. the figure shows drug candidates (in black) and vaccines (in red). among them, remdesivir has been trending in the news recently. it inhibits the rdrp, is in phase iii for sars-cov-2, and may have an effect on the three viruses. ribavirin in combination with a pegylated interferon may also have an effect against the three viruses. ritonavir and lopinavir, which inhibit the 3clpro and are in phase iii for sars-cov-2, have an effect on both sars-cov-2 and mers-cov. dna vaccines and vaccines based on the s protein or subunits of the s protein are in development lopinavir, ritonavir, darunavir, cobicistat, and asc09f (phase iii, in combination with oseltamivir, for sars-cov-2). 152 candidates as inhibitors of plpro include thiopurine analogs, compound 6 (preclinical), and remdesivir (phase iii for mars-cov). 121, 153 favipiravir, ribavirin (randomized trial for sars-cov-2), and remdesivir are among the candidate inhibitors of rdrp. 118 previously, compounds that may interfere with atpase and helicase activities were reported (before covid-19), such as bananins, 5-hydroxychromone derivatives, and triazole derivatives (preclinical). 34, 78 candidates that may suppress viral entry by targeting the s protein include peptide p9 and α-helical lipopeptides (preclinical), 154 and those targeting the s2 subunit of the s protein mainly include hr1p, hr1m, hr1l, hr2l, hr2p, and hr2l (preclinical). 155 due to the strong affinity between ace2 receptor and the rbd of sars-cov-2, another opportunity might be through the development of antibodies to block ace2. the second method is to use a large amount of soluble ace2 to directly block the spike protein. the third method is to find a drug that directly inhibits the spike membrane fusion process, such as the aids drug enfuvirtide. the fourth approach is to identify an agent that inhibits the activity of disintegrin and metalloproteinase 17 (adam17), which may also prevent viral release and proliferation in cells and protect ace2 and the lungs. 87 mrna vaccine technology sars-cov-2 vaccines are already under development. the mrna and dna vaccines are promising approaches to prevent cellular invasion by similar coronaviruses in the future. in january 2020, the coalition for epidemic preparedness innovations (cepi) announced three partnerships to develop a new coronavirus vaccine. among them, moderna and inovio presented mrna and dna vaccine technologies, respectively. the goal of the three teams is to have at least one candidate vaccine capable of preventing coronavirus infection in 16 weeks, to be tested in a phase i clinical trial. moderna's vaccine model is designed to use mrnas to safely pre-expose the immune system to a small amount of encoded proteins usually generated by the pathogen, so that the immune system becomes prepared to fight future pathogens and prevent infection. the candidate genes developed by moderna contain mrnas, and combining multiple mrnas into a single vaccine might be useful to rapidly induce responses, which is an approach that may be applied for future, emerging pandemic threats too. at the same time, because the mrna in the cell will be degraded over time, the mrna vaccine will not continue to produce antigen components for a long time, which can avoid the risk of continued stimulation of the immune system. generally, mrna vaccines are also described as simple to prepare and to yield remarkable results. additional advantages, as well as disadvantages, of nucleic acid-based vaccines were described in detail. furthermore, the basis of traditional vaccines, proteins or polysaccharides, cannot be straight forward applied at present against many pathogens. therefore, mrna vaccines would be suitable for achieving rapid, mass production of emergency vaccines in the event of a major epidemic. however, because rna is easily degraded, inducing cells to absorb mrna quickly is a challenge. in recent years, the method for delivery of mrna into cells has been greatly improved, and stemirna and moderna have adopted nanolipid (lpp or lnp (lipid nanoparticle)) drug-loading technology. however, this is still to be developed for mrnas. furthermore, ensuring safety and effectiveness is another challenge for viral mrna vaccines, and clinical validation will have to be carried out carefully. moderna is a worldwide leader in mrna therapy, with currently nine mrna-type vaccine candidates (e.g., mrna-1273 against sars-cov-2). the national institutes of allergy and infectious diseases (niaid) of the national institutes of health (nih) and the mrna vaccine giant moderna have teamed up to bring the new coronavirus vaccine possibly to phase ii within a few months, and phase iii late this year. simultaneously, in january 2020, the translational medicine platform of dongfang hospital affiliated with tongji university cooperated with stemirna (shanghai) biotechnology co., ltd. to rapidly promote the development of a new coronavirus mrna vaccine. in february 2020, the chinese scientific research team announced that animal testing of the newly developed coronavirus vaccine has begun. if animal testing goes well, this new vaccine will enter human clinical trials within a few months. also in february 2020, zhuhai livanda biotechnology co., ltd. announced that the first batch of new coronavirus mrna vaccine standard samples completed during the spring festival had been delivered to relevant national authorities on for animal testing and efficacy verification. the researchers detected the production of target antibodies in mouse serum at day 12 following immunization. the company claimed that this was also the first time worldwide that new coronavirus vaccines developed based on mrna technology have resulted in antibodies in animals. livanda is currently pushing ahead with the project through extensive cooperation with the academy of military medical sciences, the guangdong provincial institute of supervision, and the macau university of science and technology. the antigen used in its development is the same as that used by moderna. dna vaccine technology dna vaccine technology may have many advantages (e.g., safe, fast, less technical barriers), along with potential limitations too. 156 a phase i human clinical trial of the mers-cov vaccine has proven its safety and effectiveness. 157 the dna vaccine based on the sars-cov s protein developed by the team of barney graham and gary nabel of the us-nih vaccine research center (vrc) has achieved positive results in animal experiments and a clinical phase i trial. 158, 159 inovio pharmaceuticals has accumulated extensive safety and immunogenicity data for mers-cov vaccine studies. because of the high degree of similarity between mers-cov and sars-cov-2, lessons from such a vaccine development process may be beneficial for the development of a dna vaccine against sars-cov-2. since january 2020, inovio has been collaborating with several experts and companies (some in china), and has currently already begun phase i clinical trials of the dna vaccine ino-4800. china's cansino biologics might be the leader as it was announced it has moved to phase ii testing of a vaccine called ad5-ncov. the latter is currently often being reported as a dna vaccine, but to be precise, it uses viral vectors (adenovirus) to deliver dna related to sars-cov-2. part of the project is carried out currently with the chinese military medical research institute. the vaccine candidate is constructed using genetic engineering methods and defective human type 5 adenovirus as a vector that can express the sars-cov-2 s antigen, to stimulate the body to produce strong humoral or cellular immunity. sars-cov and mers-cov belong to subgroups 2b and 2c of the betacoronaviruses, respectively, and sars-cov-2 is a new member of betacoronaviruses, distinct from sars-cov and mers-cov. sars-cov and sars-cov-2 are similar in terms of invasion and self-replication, whereas mers-cov has different targets. the cellular receptor of sars-cov and sars-cov-2 is ace2, plus cd147 for sars-cov-2, while that of mers-cov is dpp4 (cd26). all of them evolved a mechanism to escape the host cell immune system. sars-cov and sars-cov-2 affect the ras system by suppressing ace2, leading to the onset of symptoms. mers-cov causes symptoms by producing inflammatory cytokines and invading t cells. sars-cov, mers-cov, and sars-cov-2 infections have similar symptoms, including fever, cough, myalgia, and shortness of breath, among others. current treatments for sars mainly include ifn-α, antiviral treatments (e.g., ribavirin), plasma therapy, host-directed therapies, and systemic corticosteroids. for mers, several therapeutics are in development, including convalescent plasma, lopinavir/ritonavir, ribavirin, ifn, and novel therapies, including polyclonal antibodies, broad-spectrum antivirals, and amps. for covid-19, candidates mainly include drugs targeting the s protein, nonstructural proteins (3clpro, plpro, helicase, and rdrp), and viral rna synthesis blockers such as remdesivir and ribavirin. vaccines such as dna vaccine, mrna vaccine, and recombinant vaccines are being rapidly developed. so far this century, human beings have experienced several epidemic outbreaks, and each outbreak had a negative impact at different levels, including health, economy, and even psychology and human behavior. in the future, more precautious measures should be available to guide 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to s1 protein that blocks receptor association potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies exceptionally potent neutralizationof middle east respiratory syndrome coronavirus by human monoclonalantibodies interaction between heptad repeat 1 and 2 regions inspike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors fusion mechanism of 2019-ncov andfusion inhibitors targeting hr1 domain in spike protein the authors gratefully acknowledge the financial support from the national natural science foundation of china (grant no. 31870836), and the 1.3.5 project for disciplines excellence, west china hospital, sichuan university (zyyc20005 to w.c.). competing interests: the authors declare no competing interests.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-332784-xkc89uaz authors: mishra, shashank shekhar; ranjan, shashi; sharma, chandra shekhar; singh, hemendra pratap; kalra, sourav; kumar, neeraj title: computational investigation of potential inhibitors of novel coronavirus 2019 through structure-based virtual screening, molecular dynamics and density functional theory studies date: 2020-07-15 journal: journal of biomolecular structure & dynamics doi: 10.1080/07391102.2020.1791957 sha: doc_id: 332784 cord_uid: xkc89uaz despite the intensive research efforts towards antiviral drug against covid-19, no potential drug or vaccines has not yet discovered. initially, the binding site of covid-19 main protease was predicted which located between regions 2 and 3. structure-based virtual screening was performed through a hierarchal mode of elimination technique after generating a grid box. this led to the identification of five top hit molecules that were selected on the basis of docking score and visualization of non-bonding interactions. the docking results revealed that the hydrogen bonding and hydrophobic interactions are the major contributing factors in the stabilization of complexes. the docking scores were found between −7.524 and −6.711 kcal/mol indicating strong ligand-protein interactions. amino acid residues phe140, leu141, gly143, asn142, thr26, glu166 and thr190 (hydrogen bonding interactions) and phe140, cys145, cys44, met49, leu167, pro168, met165, val42, leu27 and ala191 (hydrophobic interactions) formed the binding pocket of covid-19 main protease. from identified hits, zinc13144609 and zinc01581128 were selected for atomistic md simulation and density functional theory calculations. md simulation results confirm that the protein interacting with both hit molecules is stabilized in the chosen popc lipid bilayer membrane. the presence of lowest unoccupied molecular orbital (lumo) and highest occupied molecular orbital (homo) in the hydrophobic region of the hit molecules leads to favorable ligand-protein contacts. the calculated pharmacokinetic descriptors were found to be in their acceptable range and therefore confirming their drug-like properties. hence, the present investigation can serve as the basis for designing and developing covid-19 inhibitors. communicated by ramaswamy h. sarma in the twenty-first century, the novel coronavirus 2019 is a major cause of disaster due to a lack of vaccine, drugs to treatment. the pandemic has been born from the family of coronavirus and can cause illness such as elevated body temperature, common cold, pneumonia, bronchiolitis, rhinitis, pharyngitis, sinusitis, middle east respiratory syndrome (mers) and the severe acute respiratory syndrome (sars) (amanat & krammer, 2020) . in 2003, sars had killed 774 people, whereas mers killed 858 people between 2012 and 2019 (boopathi et al., 2020) . in 2019, a new virus identified in china namely novel coronavirus disease 2019 (covid-19) (boopathi et al., 2020) . coronavirus has a single-stranded rna genome (yang et al., 2006) , pleomorphic or spherical shape, and bears clubshaped projections of glycoproteins on its surface (80-120 nm diameter) (guo et al., 2010; belouzard et al., 2012) . the rna genome is made up of $30,000 nucleotides and covered by enveloped structure. it encodes four structural proteins, nucleocapsid (n) protein, membrane (m) protein, spike (s) protein, and envelop (e) protein and several non-structural proteins (nsp) (boopathi et al., 2020) . covid-19 packages its genome in a nucleocapsid (n) protein and forms a ribonucleoprotein (rnp) complex. the rnp is essential for viral replication, transcription, and assembly. several studies suggested that the modulation of n protein oligomerization by small molecules is a possible antiviral drug development strategy (chang et al., 2016; zumla et al., 2016) . the membrane (m) protein found in the surface of the virus and supposed to be the central organizer for the covid-19 assembly (sarma et al., 2020) . the spike (s) protein integrated throughout the viral surface and involves the attachment with the host cell surface receptors and facilitates viral entry into the host cell by fusion between the viral and host cell membranes (kirchdoerfer et al., 2016) . the s protein is composed of three identical chains with 1273 amino acid residue each and characterized by two welldefined protein domain regions: s1 and s2 subunits. these s1 and s2 subunits are involved in cell recognition and the fusion of viral and cellular membranes respectively (walls et al., 2020) . the envelop (e) protein, a small component of the virus particle, is a small membrane protein made up of $76-109 amino acid residues and involves in host cell interaction and virus assembly (boopathi et al., 2020; gupta et al., 2020) . the structural spike (s) protein of coronavirus binds with the angiotensin-converting enzyme 2 (ace2) receptors that are present on the surface of many human cells, including lungs (hasan et al., 2020) . the s protein is associated with proteolytic cleavages by host proteases, in two domain regions located at the boundary between the s1 and s2 subunits. in a later stage happens the cleavage of the s2 subunit to release the fusion peptide (boopathi et al., 2020; lin et al., 2020) . this process will trigger the activation of the membrane fusion mechanism (tahir khan et al., 2020) . therefore, inhibition of the activation of membrane fusion could be a possible target for antiviral drug development against covid-19. coronavirus entry, replication, transcription, and formation of rnp complex are depicted in figure 1 . furthermore, endosome opens to release covid-19 to the cytoplasm and translation of viral polymerase protein takes place (li, 2016) . the positive rna genome is translated to generate replicase proteins that use the genome as a template to generated full-length negative-sense rnas, which subsequently serve as templates in generating addition fulllength genomes (masters, 2006; van hemert et al., 2008) . the nucleocapsids are formed from the encapsidation of replicated genomes. the structural proteins s, m, and e are synthesized in the cytoplasm and further translation results in the transfer to endoplasmic reticulum-golgi intermediate compartment (ergic) (masters, 2006) . this ergic membrane coalesces with the nucleocapsids to self-assembly into new virions. finally, new virions are exported from infected cells by exocytosis, so that can affect other cells. the ongoing covid-19 threat that emerged in china has rapidly spread to the whole world and has been declared as a global public health emergency by the world health organization (who) (muralidharan et al., 2020) . many efforts have been directed to develop potential drugs or vaccine but no drug or vaccine has not launched. therefore, all nations implementing appropriate preventive and control measures only. so, it is an urgent need to develop effective and potential antiviral-covid-19 candidates for reducing disease severity, and transmission to block the covid-19 outbreaks. to accomplish the objective of the study, we have carried out the structure modeling of the crystal structure of covid-19. the prepared protein structure was used to perform high-throughput virtual screening (htvs) of chemical libraries. the novel hit molecules identified from docking study were selected based on the docking score, binding energy calculations, and their other interactions with amino acid residues. also, we performed molecular dynamics simulations and molecular orbital analysis for the best two hit molecules. pharmacokinetic parameters and bioavailability score were also assessed. the entire computational investigation was performed on windows 7 (64-bit) operating systems with 4 gb ram and 2.66 ghz intelvr coretm 2 quad q8400 processor except for molecular dynamics simulations. molecular dynamics simulations were performed on ubuntu 14.04.5 version in the linux environment with 4 gb ram by desmond. the three-dimensional crystal structure of the covid-19 main protease (pdb-id: 6y84) was retrieved from the protein data bank (http://www.rscb.org/pdb). the target protein has a resolution of 1.39 å and this indicates its good quality as a resolution of 2.0 å is the recommended maximum for a good structural protein for docking (hajduk et al., 2005) . the retrieved protein was pre-processed using the protein preparation wizard by adding missing loops and atoms, adding missing hydrogens and assigning bond orders. further, we removed non-essential water molecules and hetero atoms, before restrained minimization using opls-2005. the objective is to bind the ligands from the molecular docking to the binding site of the covid-19 main protease protein. sitemap tool of maestro was employed to recognize potential binding pocket by joining together 'sitepoints' that most probably subsidize to tie protein-protein or protein-ligand binding (halgren, 2007 (halgren, , 2009 ). the overall site score describes the rank of computed binding pocket using a linear combination of various terms. the active site of covid-19 main protease protein was located in region 3 site and the receptor grid files were generated using grid generation panel of glide module. this establishes two cubical boxes having a common centroid to organize the calculations: a larger enclosing and a smaller binding box (athar et al., 2016) . structure-based virtual screening is of major importance for the computational drug discovery process to speed up drug development. this approach emerged as an important tool in identifying small drug-like molecules through various computational tools, by using knowledge about the target protein, its constitutional information, or by known active compounds for target protein (kar & roy, 2013) . in this process of virtual screening, the selected dataset consists of all compound libraries of the zinc database in sdf format (rollinger et al., 2008) . the complete virtual screening was performed against the above said databases by the glide module of maestro. these database molecules were docked into the binding site of the protein utilizing htvs protocol to calculate ligand-protein binding affinities. molecules filtered from htvs were subjected to sp docking which can dock few tens to millions of ligands with high accuracy. a further precise amount of molecule was then subjected to glide xp docking where further elimination of false positives is carried out by more extensive sampling and advanced scoring resulting in higher enrichment. based on their glide gscore, the top five hit molecules were selected for further investigation. molecular mechanics-generalized born surface area (mm-gbsa) method was used for the calculation of binding free energy for the docked complexes (su et al., 2015) . this method used the following equation: where solv and sa represent the salvation energy and surface area associated energy and e represents the energy minimized states of ligand-protein used in the calculations. for this calculation, the top-scored ligand-protein complexes were selected with default parameters such as opls-2005 force field and vsgb solvent model. to analyze the structural stability of the covid-19 main protease protein-ligand complexes, molecular dynamics simulations were carried out by using desmond in the presence of the popc bilayer membrane (shekhar et al., 2019) . the protein-ligand complex was pre-processed using the protein preparation wizard panel to add missing atoms and refinement of side chains. the solvated model of a complex was prepared by selecting popc (300 k) as a membrane model. the solvated system was in an orthorhombic box to provide spc solvent model and the system was neutralized using na þ and cl à ions. the salt concentration was set as 0.15 m to maintain physiological conditions. to minimize the short range bad contacts, energy minimization was carried out using the hybrid method steepest decent and the limited-memory broyden-fletcher-goldfarb-shanno (lbfgs) algorithms (guo et al., 2010) for 2000 iterations. finally, these systems were subjected to 50 ns md simulation production runs at 300 k temperature and 1 bar as pressure. the model was relaxed before the production system run because it makes a series of predefined minimizations and md executions. the md simulations were analyzed in terms of rmsd and rmsf to estimate the variations in the position of the systems in terms of simulation time. protein-ligand contacts and torsion profiles were also examined during the simulation. in silico bioavailability and the synthetic feasibility of the topscored molecules were evaluated through the swiss adme tool. consideration of synthetic practicability produces a number, between 1for simply synthesized compounds, and 10for compounds that are challenging to synthesize. the assessment of pharmacokinetic parameters of top-scored molecules obtained from virtual screening protocol is believed to be an essential step of the computational drug discovery process. it is known that the major concern for the failure of drug candidates in clinical trials is poor pharmacokinetics single point energy calculations using density functional theory were performed using jaguar (bochevarov et al., 2013) to explain ligand-bound protein in electronic level. the structures were optimized using hybrid functional b3lyp parameters with 6-31 g ãã basis set. various properties such as electrostatic potential, average local ionization energy, gas-phase energy, canonical orbital, etc. were calculated. the positions of lowest unoccupied molecular orbital (lumo)-highest occupied molecular orbital (homo) orbitals of selected hit molecules were analyzed to evaluate the binding pattern at the quantum level. present computational work aimed to search novel, potential inhibitors of covid-19, and characterize the binding pattern to identify the structural features through structure-based screening and molecular dynamics simulation studies. subsequent in silico bioavailability and synthetic feasibility was also evaluated to strengthen the scope of work. the localization and identification of the binding site are necessary as the function and structure of the protein are related to specific interaction with binding site functionalities before structure-based drug design. for predicting the binding site, a comprehensive search was done by sitemap. it indicates the locations of hydrogen-bonded, van der waals, and hydrophobic regions in the representative covid-19 proteins. the calculated structural features of binding sites have shown in table 1 . the druggability of binding sites was determined which is given in the terms of site score. in all possible binding sites, site_1 has 0.961 site score along with 274.743 as volume. same as the site score of other site_2, site_3, site_4, and site_5 were found to be 0.946, 0.837, 0.591, and 0.571 respectively. based on the results, site_1 has the potential to mechanistically justify the binding pattern and answer for the protein-ligand interactions. therefore, site_1 was considered as an appropriate binding site to perform virtual screening covid-19 main protease protein composed of three regions of 306 amino acid residues. the first (blue), second (green), and third (orange) regions contain 8-101, 102-184, and 201-305 residues, respectively. the potential binding site lies between region 2 and 3 that is connected through a long loop (figure 2) . à2 to þ2 à2 to 6.5 à6.5 to 0.5 à3.0 to 1.2 <25 p >500 g 1-8 à1.5 to 1.5 a range: for 95% oral drugs. small molecules of the zinc database have been used for structure-based screening of covid-19 main protease protein. the active site with the best site score (top-ranked potential receptor binding cavity) was taken as a prerequisite for receptor grid generation. the executed approach of virtual screening was the hierarchal mode of elimination technique i.e. htvs followed by sp and further the xp docking. glide xp combines accurately energy-based scoring terms and thorough sampling that resulted in compounds with docking scores between à7.524 and à6.711 kcal/mol indicating strong ligand-protein interactions. final shortlisting of hit molecules was based on visual observation of the major amino acid residues involved in the binding that included hydrogen bonding with glu166, phe140, gly143, leu141, asn142, thr26 and thr190. from these observations, we selected the top five compounds, the docking score, binding free energies of hit molecules are provided in table 2 . the top hit molecule zinc13144609 (1,4-bis(1h-benzo[d]imidazol-2-yl)butane-1,2,3,4-tetraol) showed hydrogen bonding with amino acid residue glu166, leu141 and asn142 with a docking score of à7.524 kcal/mol. the selected five potential hit molecules in the binding site of protease protein, interacting with amino acid residues phe140, gly143, thr26, thr190, glu166, pro168, met165 and leu141 with a docking score of à7.524 and à6.711 kcal/mol. the number of hydrogen bond along with residue and other interactions is tabulated in table 3 . all hit molecules showed hydrogen-bonding interactions with main amino acid residues phe140, leu141, gly143, asn142, thr26, glu166 and thr190 with a well-fitted pose within the binding site in the hydrophobic pocket formed by phe140, cys145, cys44, met49, leu167, pro168, met165, val42, leu27 and ala191. the binding pattern within the binding site pocket of all top-scored hit molecules was quite same and additional p-p interactions contributed for the binding affinity of the molecules. the docking pattern of the docked hit molecule zinc13144609 and zinc01581128 is represented in figures 3 and 4 . the docking orientation of the remaining hit molecules zinc06062920, zinc03022586 and zinc00134422 are depicted in figures s1-s3. a molecular dynamics simulation time step involves a computationally intensive force calculation for each atom of a chemical system, followed by a less expensive integration step that advances the positions of the atoms according to classical laws of motion. it enables the atomic-level characterization of numerous biomolecular processes such as investigation of the stability of protein-ligand interactions associated with activation and deactivation of various molecular pathways. the atomistic md simulation was performed to obtain insights into the stability behavior of top hit molecules zinc13144609 and zinc01581128 at the binding pocket of 6y84. the complex was embedded within the popc bilayer chosen with default parameters and simulated for 50 ns. each complex was soaked in an orthorhombic box with spc water molecules. to maintain a neutral system, na þ and cl à ions were added with 0.15 m salt concentration. the trajectories generated were analyzed by plotting the rmsd, rmsf of each frame as a function of simulation time. for hit molecule zinc13144609, the rmsd value of the protein ca was found to increase up to a value of 2.6 å with respect to its starting coordinate (t ¼ 0) for first 10 ns and stabilize around an average value of 2.347 å for rest of the md trajectories which indicates no change in the protein backbone. further, the rmsf of the backbone at the binding site of 6y84 is found to be in the range of 1.0 å to 2.573 å which suggests lower degree of flexibility in binding region. from the above observation, it is proved that the ligand movement was stable during the simulation. the ligand-protein contacts are illustrated in figure 5 . it is found that the hydrogen bonds with glu166 and hydrophobic interactions with pro168, leu167, met 49, his41are major contributing factor for stabilizing hit molecule zinc13144609 at the binding site which is in accordance with our docking result. his41 found to exhibit p-p stacking with the benzene ring for 61% of the trajectory. among the six-hydrogen bond predicted by docking, only four are found to be preserved during the simulation. for hit molecule zinc01581128, the rmsd and rnsf plots are given in figure 6 . the analysis of 6y84 proteinzinc01581128 complex showed low rmsd value, 2.586 å indicating the stability of the atomistic molecular system. the plot showed that the system attained stability after 20 ns of simulations. the rmsf value is found to be in the range of 1.2-3.0 å which indicates low fluctuations in the binding site. the hydrogen-bonding interactions with gly143, phe140 and glu166 were retained throughout the simulations. it suggests the importance of these interactions in stabilizing the simulated complex. from the above results, it is clear that the protein interacting with both hit molecules is stabilized in the chosen popc bilayer membrane. the top hit molecules are stabilized through, hydrogen bonding, hydrophobic and p-p interactions with various residues indicate its better stabilization in 6y84 protein. in the drug discovery process, the molecules under consideration need to possess good pharmacokinetic profiles as well as potency to confirm their bioavailability and effectiveness. the various physicochemical and therapeutic significant descriptors were calculated and documented in tables 4 and 5, respectively. all the calculated physicochemical and therapeutic significant properties were found to be in their permissible range and therefore confirming their drug-likeness. the bioavailability of top scored hit molecules was determined through swissadme tool. the bioavailability radar chart of zinc13144609 and zinc01581128 is illustrated in figure 7 . the pink area shown in figure 7 represents most favorable area for each property like insatu (unsaturation), insolu (insolubility), flex (rotatable bonds), lipo (lipophilicity), size (molecular weight) and polar (polar surface area). the predicted bioavailability score is tabulated in table 6 . the synthetic feasibility of top scored molecules is shown in table 6 . the synthetic feasibility score is in the range of 1 for simply synthesizable and 10 for difficult to synthesize. all top hit molecules showed synthetic feasibility score around 3.0 that indicates all are easily synthesizable. furthermore, we forward to plan future synthesis with the best plausible route. the hit molecule zinc13144609 and zinc01581128 were chosen for energy calculations. the homo-lumo energy gap represents the chemical reactivity of molecules. the electronic properties of screened hit molecules were shown in table 7 . the position of homo-lumo orbitals of zinc13144609 (figure 8) suggests that the hydrophobic region of molecule alters the topology of homo-lumo orbitals and also energies are localized. homo and lumo covers mainly benzimidazole ring of hit molecule. the zinc01581128 molecule found to have lumo orbital over hydrophobic region and homo orbital mainly cover polar region (figure 9 ). therefore, the presence of homo and lumo orbitals near the hydrophobic part of the hit molecules is important to from stable ligand-protein complex. in the present investigation, structure-based virtual screening, dft calculation, mm/gbsa, pharmacokinetic parameters calculation, and molecular dynamics simulation studies in search of the antiviral drug against covid-19 were performed. the covid-19 main protease protein has made up of three regions of 306 amino acid residues. the predicted binding site lies between regions 2 and 3. virtual screening was executed through a hierarchal mode of elimination technique in the prerequisite binding site. further, the top five hit molecules were shortlisted based on docking score and non-bonding interactions. the reliability of docking analysis was confirmed by the low rmsd value of docked ligand and protein. the highest scoring hit molecule zinc13144609 (à7.524 kcal/mol) exhibited six hydrogen bonds with amino acid residues glu166, leu141 and asn142. the second hit molecule zinc01581128 showed a docking score à7.429 kcal/mol with six hydrogen bonding (gly143, leu141, phe140 and glu166) and p-p stacking interactions. the docking study suggested that the polar part of the molecules forms hydrogen bonding network. it is evident from the md simulation study that the ligand-protein complexes have equilibrated and changes were perfectly acceptable for small, globular proteins. the lumo-homo orbital study described the interaction pattern of hits with the protein at the quantum level. the pharmacokinetic properties calculation of top scored hits obtained from present work ensured their safe administration in the human body. lastly, all top-scored hits can be easily synthesizable based on the least synthetic accessibility score. therefore, investigated hits could be potent and efficacious drugs targeting covid-19 main protease protein for the covid-19 treatment. experimentally, the need to validate the molecular modeling results reported here with in vitro and/or in vivo inhibition evaluation is acknowledged but due to lack of funding, work is limited. therefore, investigated inhibitors could be potent and efficacious drugs targeting covid-19 main protease protein for the covid-19 treatment. sars-cov-2 vaccines: status report pharmacophore model prediction, 3d-qsar and molecular docking studies on vinyl sulfones targeting nrf2-mediated gene 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can virtual screening take us in drug discovery? pre-fusion structure of a human coronavirus spike protein in silico binding mechanism prediction of benzimidazole based corticotropin releasing factor-1 receptor antagonists by quantitative structure activity relationship, molecular docking and pharmacokinetic parameters calculation structure, function, and evolution of coronavirus spike proteins structure-based stabilization of non-native protein-protein interactions of coronavirus nucleocapsid proteins in antiviral drug design the molecular biology of coronaviruses computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov-2 protease against covid-19 virtual screening for the discovery of bioactive natural products. natural compounds as drugs, i in-silico homology assisted identification of inhibitor of rna binding against 2019-ncov n-protein (n terminal domain) computational investigation of binding mechanism of substituted pyrazinones targeting corticotropin releasing factor-1 receptor deliberated for anti-depressant drug design comparison of radii sets, entropy, qm methods, and sampling on mm-pbsa, mm-gbsa, and qm/mm-gbsa ligand binding energies of f. tularensis enoyl-acp reductase (fabi)) marine natural compounds as potents inhibitors against the main protease of sars-cov-2. a molecular dynamic study sars-coronavirus replication/ transcription complexes are membrane-protected and need a host factor for activity in vitro effect of hydrophobic and hydrogen bonding interactions on the potency of ß-alanine analogs of g-protein coupled glucagon receptor inhibitors structure, function, and antigenicity of the sars-cov-2 spike glycoprotein drug design targeting the main protease, the achilles' heel of coronaviruses coronaviruses -drug discovery and therapeutic options the authors are thankful to dr. sourav kalra, centre for human genetics & molecular medicine, central university of punjab for providing computational facilities to accomplish this research work. no competing interests exist. key: cord-329493-ueqlhgn0 authors: stadler, konrad; masignani, vega; eickmann, markus; becker, stephan; abrignani, sergio; klenk, hans-dieter; rappuoli, rino title: sars — beginning to understand a new virus date: 2003 journal: nat rev microbiol doi: 10.1038/nrmicro775 sha: doc_id: 329493 cord_uid: ueqlhgn0 the 114-day epidemic of the severe acute respiratory syndrome (sars) swept 29 countries, affected a reported 8,098 people, left 774 patients dead and almost paralysed the asian economy. aggressive quarantine measures, possibly aided by rising summer temperatures, successfully terminated the first eruption of sars and provided at least a temporal break, which allows us to consolidate what we have learned so far and plan for the future. here, we review the genomics of the sars coronavirus (sars-cov), its phylogeny, antigenic structure, immune response and potential therapeutic interventions should the sars epidemic flare up again. a new infectious disease, known as severe acute respiratory syndrome (sars), appeared in the guangdong province of southern china in 2002. it is mainly characterized by flu-like symptoms, including high fevers exceeding 38°c or 100.4°f, myalgia, dry nonproductive dyspnea, lymphopaenia and infiltrate on chest radiography. in 38% of all cases, the resulting pneumonia led to acute breathing problems requiring artificial respirators 1 . the overall mortality rate was about 10%, but varied profoundly with age -although sars affected relatively few children and generally appeared to be milder in the paediatric age group, the mortality rate in the elderly was as high as 50% [2] [3] [4] . although accurate information about the precise origin of the disease is not available, the chinese ministry of health reported an outbreak of unexplained pneumonia to the world health organization (who) on 11 february 2003. during the period from 16 november 2002 to 9 february 2003, 305 cases and five deaths due to atypical pneumonia, which were originally thought to be caused by chlamydia pneumoniae, occurred in the southern chinese province of guangdong 5 . a chinese doctor who had been treating patients in guangdong spread the infection outside of mainland china when he travelled to hong kong on 21 february 2003. there, while resident on the ninth floor of the hotel metropole, the patient developed symptoms and was transferred to the hospital on 22 february where he died the following day. subsequently, ten secondaryinfected guests of the hotel (eight from the ninth floor and two from the eleventh and fourteenth floors) boarded planes and carried the infection to singapore, vietnam, canada and the united states, making sars the first epidemic to be transmitted by air travel. when the epidemic finally waned after more than 100 days, the who counted a cumulative number of 8,098 probable sars cases and 774 deaths worldwide, in a geographical area spanning 29 countries 6 . carlo urbani, a who infectious disease expert who had been called to hanoi, was the first to realize that the chinese doctor in hong kong had suffered from a previously unknown disease and alerted the authorities. following unprecedented collaboration between laboratories and scientists worldwide, a previously unidentified coronavirus was isolated from frhk-4 and vero e6 cells that were inoculated with clinical (nasopharyngeal, oropharyngeal and sputum) specimens from patients 1, 7, 8 . the association of the virus with the disease was confirmed when macaques that were inoculated with the virus developed symptoms similar to those observed in human cases of sars 9, 10 . it is likely that scientists from the chinese academy of military medical science had observed what seemed to be coronavirus particles in an electron micrograph by 26 february 2003. however, with r e v i e w s indicating that this outbreak is the first introduction of sars-cov into the human population. at present, accidental infections of researchers handling the pathogen seem to pose the greatest risk for a renewed spread of the virus as a recent case in singapore has shown 15 . for now, the transmission of this emerging infectious disease has been stopped, but there is no information on when, or if, sars-cov will re-emerge in the human population. coronaviruses were named after their corona solis-like appearance in the electron microscope, which is caused by the club-shaped peplomers that radiate outwards from the viral envelope (fig. 1a) . the spherical capsid contains a positive-strand rna genome of about 30 kb -the largest of its kind. coronaviruses have been subdivided into three groups on the basis of serological and genetic properties 16 . their broad host range extends from man to turkey; where they are typically associated with respiratory, enteric, hepatic and central nervous system diseases. in man, they cause mainly upper-respiratory-tract infections, and are responsible for a large proportion of all common colds. sequence analysis revealed the organization of the 29,740 base (fra isolate; genbank acc.no. ay310120; see sars coronavirus fra in the online links) genome of the sars-cov (fig. 2) to show the characteristic features of coronaviruses [17] [18] [19] [20] (fig. 3) . nucleotides 1-72 contain a predicted rna leader sequence preceding an untranslated region (utr) spanning 192 nucleotides. two overlapping open reading frames (orf1a and orf1b), which encompass approximately two-thirds of the genome (nucleotides 265-21485), are downstream of the utr. a translational read-through by a −1 ribosomal frameshift mechanism allows the translation of the overlapping reading frames into a single polyprotein 20 . virus-encoded proteinases, namely the only a few samples available for analysis, they did not feel confident enough to challenge the authorities and did not publish their findings. close contact with very sick patients facilitates personto-person transmission of the virus -apparently, sars-cov spreads in droplets but its efficiency of infection seems to be low, with an infectivity index of about 3 (refs 11, 12) . in some cases, however, a single person can infect a high number of people but, as yet, there is no satisfactory explanation for this so-called 'superspreader' phenomenon 2 . a virus with close homology to sars-cov was isolated from palm civets and racoon dogs, which are considered delicacies in southern china, indicating that the virus could have jumped recently from these mammals to man 13 . however, a second search by another group failed to reveal any trace of sars-cov in more than 60 animal species, including 76 palm civets 14 . although unlikely, the possibility that humans infected these sars-positive animals cannot be formally excluded. in the meantime, over 30 different sars-cov isolates have been sequenced, which will hopefully allow us to trace the chain of transmission back to its origin. the sars epidemic, which caused global panic and huge economical damage to the asian economy, was contained primarily by aggressive quarantine measures. additional beneficial factors could include the attenuation of the virus following prolonged passage through the human population, or the onset of summer, as higher temperatures generally decrease the incidence of at least some respiratory infections. a natural reservoir for the virus could serve as the launch pad for another sars outbreak during the next winter-spring season. if we are lucky, however, the ability of this virus to cross the species barrier could be the result of a rare series of events that is unlikely to be repeated. a retrospective serological study did not detect anti-sars-cov antibodies in a normal population, assumptions about the function of the remaining proteins can be made by analogy with other coronaviruses, although our knowledge about the biological functions of these proteins is also incomplete (see table 1 for information on the encoded proteins). together with host factors, some of these proteins might form the viral replication-transcription machinery that is associated with the membranous structures in the infected cells 23, 24 . (the peculiar features of the replication cycle of coronaviruses are described in box 1.) the remaining 3′ part of the genome encodes four structural proteins that are arranged in the same order in all papain-like cysteine protease (plpro) and the 3c-like cysteine protease (3clpro), cleave the polyprotein into individual polypeptides, which provide all the proteins that are required for replication and transcription 20, 21 . wheareas group 2 coronaviruses contain two paralogous plpros (pl1pro and pl2pro), only one is found in the orf1a of the sars-cov genome. orf1b encodes a helicase with atpase and dna (and possibly also rna) duplex-unwinding activities, at least when it is expressed in escherichia coli 20 . computational analysis predicted that the carboxy-terminus of orf1b encodes a mrna cap-1 methyltransferase 22 . some finally, at the 3′ end of the genome, a second 340nucleotide utr, which is followed by a poly(a) tract, was found. this 3′ utr contains a 32-nucleotide stem-loop ii-like motif (s2m) motif, which has also been reported in astroviruses, in one equine rhinovirus and avian infectious bronchitis virus (ibv) 28 . a typical feature of coronaviruses is the presence of a transcription-regulatory sequence (trs) that is important in rna transcription and regulation (figs 2 and 3). this short motif is usually found at the 3′ end of the leader rna and, with a few exceptions, precedes each translated orf 29 . when thiel and colleagues 20 isolated one genomic and eight subgenomic rnas from the fra strain and sequenced their 5′ ends, they identified a conserved sequence (5′acgaac3′) that was located in coronaviruses: s, spike protein; e, envelope protein; m, membrane glycoprotein; and n, nucleocapsid protein. furthermore, the structural protein region of the sars-cov genome contains several genes that encode additional non-structural proteins that are known as 'accessory genes' (fig. 3) . some of these molecules seem to be dispensable for virus viability both in vitro and in vivo; their deletion creates viruses that are attenuated [25] [26] [27] . these accessory genes differ significantly among the three coronavirus groups -they are also referred to as group-specific genes. the sars-cov has eight predicted orfs of unknown function in this region of the genome. it lacks the haemagglutinin esterase (he) gene, which is encoded by almost all of the group 2 viruses. the analyses are based on the sequence of the sars-cov fra isolate (genbank accession number ay310120). transmembrane domains (tmds) were predicted using the program psort (threshold is less than −2); the glycosylation sites were predicted using the netnglyc server (see netnglyc in the online links). information on the functional predictions has been taken from refs 20,33. nsp, non-structural protein. coronaviruses contain the largest rna genomes described so far -the positive-sense, single-stranded rna molecule has a 5′ cap and a 3′ poly(a) tail. the life cycle of a coronavirus starts when the spike (s) protein, which forms the distinctive, eponymous crown that is observed with coronaviruses, interacts with a receptor through its s1 domain. the entry, which is probably mediated by the s2 domain, occurs by membrane fusion. the rna genome is then released into the cytoplasm where replication takes place. the host translation machinery translates the overlapping open reading frames orf1a and orf1b by a ribosomal frame-shifting mechanism to produce a single polyprotein. cleavage by virally encoded proteinases yields the components that are necessary to assemble the viral replication complex, which synthesizes full-length negative-strand rna. in addition, a discontinuous transcription strategy during negative-strand synthesis produces a nested set of sub-genomic negative-sense rnas. in this process, the transcription regulatory sequence (trs), which is present at the 3′ end of the leader sequence and, with a few exceptions, upstream of each translated gene, is important. it is postulated to fuse the 3′ ends of the nascent subgenomic minus strands to the antisense leader sequence. these discontinuously synthesized minus strands then act as templates for the synthesis of positive-sense mrnas. an alternative hypothesis proposes that these mrna molecules are generated by discontinuous transcription during positive-strand synthesis (the figure shows the positive-sense mrna products). in almost all cases, only the most 5′ orf is translated. nucleocapsid (n) protein and genomic rna assemble in the cytoplasm to form the helical nucleocapsid. this core structure acquires its envelope by budding through intracellular membranes between the endoplasmic reticulum (er) and the golgi apparatus. the membrane (m), envelope (e) and s proteins, all of which will be accommodated by the lipid bilayer, are transported through the er to the budding compartment, where the nucleocapsid probably interacts with the m protein to trigger assembly. during the transport of the virus through the golgi apparatus, sugar moieties are modified and in some, but not all, coronaviruses the s protein is cleaved into s1 and s2 domains. any s protein that is not incorporated into the virions is transported to the cell surface. finally, the virus is released from the host cell by fusion of virion-containing vesicles with the plasma membrane. the common leader sequence on the 5′ end of each mrna is shown in red. 13 . this observation raises the intriguing hypothesis that the 29-nucleotide deletion that has been observed in most human isolates could have increased the fitness of the virus in human hosts and allowed the spread to the human population. lacking a proof-reading mechanism, rna viruses are generally characterized by a high mutability. the high mutation rate results in continuously evolving viral species, which allow the virus to escape host defences. additionally, coronaviruses have a high frequency of rna recombination that has the theoretical potential of accelerating the emergence of new viral species 29 . owing to the limited number of sequenced isolates, however, it is too early to draw any reliable conclusions concerning the mutation rate of sars-cov. the most interesting amino-acid changes that have been reported so far are two recurrent nonconservative amino-acid substitutions (gly to asp and ile to thr) in the antigenic domain s1 of the spike protein 30 . gly and ile can be found in the hong kong front of nine predicted orfs, and which fitted the description of a trs (figs 2 and 3). by contrast, marra et al. 17 and rota et al. 18 proposed different trss (5′cuaaac3′ and 5′aaacgaac3′, respectively), but these sequences do not precede all predicted genes and no experimental evidence for their function has been provided. although the overall organization of the sars-cov genome is similar to other coronaviruses (fig. 3) , the amino-acid conservation of the encoded proteins is usually low (fig. 2; table 2 ). helped by the groundwork laid by previous generations of coronavirus researchers, the sars epidemic has been the first infectious disease outbreak to fully benefit from the revolutionary technologies of the post-genomic era. less than 1 month after the initial identification of the virus as the infectious agent of sars, two independent genome sequences of the virus had been obtained 17, 18 . within 3 months, the genome sequences of 20 independent clinical isolates were made available in the genbank database (see box 2 for details). comparative analysis of these isolates revealed more than than 99% sequence conservation. the few differences, however, have allowed a straightforward organization of all viral isolates into two families: those originating from mainland china and hong kong and those originating from the index case in the hong kong hotel 30 (fig. 4) . perhaps the most interesting observation was made in the the most important question following identification of the sars-cov was whether it represents a completely new group, a variant of one of the three known groups or a combination of these groups. phylogenetic analysis based on the first available 300 and 405 nucleotides of the highly conserved polymerase gene 7, 31 indicated that sars-cov was distinct from the three known groups. marra and rota also reached the same conclusion 17, 18 , and proposed that sars-cov be placed in its own group (fig. 5a) (fig. 5b) , which indicated that it is more closely related to members of group 2 and might share a common ancestor with them. moreover, this conclusion is corroborated by the striking observation that 19 out of 20 cysteine residues in the s1 domain of the sars-cov spike protein are spatially conserved when compared with the group 2 consensus sequence, whereas only 5 cysteines are conserved when compared with group 1 or 3 consensus sequences (fig. 6) . this analysis supports the conclusion that the sars-cov virus split early from other group 2 viruses and has evolved independently for a long period of time. although very little is known about the sars-cov proteins themselves, homologies with known coronavirus proteins can be used to predict the features of several sars-cov proteins that could be interesting targets for antiviral drugs or vaccines. viral enzymes that are essential for virus replication are the most attractive candidates for the development of small, antiviral molecules, whereas some of the structural proteins represent obvious targets for vaccine development. on the basis of the x-ray crystal structures of 3clpro from transmissible gastroenteritis coronavirus (tgv) 33 and human coronavirus 229e, a three-dimensional model of the corresponding sars virus protein has been proposed 34 . the model can be used to reduce the number of compounds to be tested to find an effective protease inhibitor. indeed, an active form of this protease has already been expressed in e.coli, which can be immediately used for drug screening. the helicase, which is already available in recombinant form, represents another attractive target for highthroughput screens 20 . plpro is another potential candidate for antiviral drugs, although no homologous index case group, whereas asp and thr are found in the mainland isolates. another, non-conservative substitution (gly to arg) in the s1 domain is only found in strains gz01 and bj02. it is tempting to speculate that these mutations represent adaptations to the new host or its immune response. additional information on ongoing variability studies can be obtained directly from the sars coronavirus resource (see online links). genbank database and were available to the scientific community. the 31 sequenced isolates come from china (10 strains), singapore (5 strains), taiwan (12 strains), vietnam (1 strain), germany (1 strain), italy (1 strain) and canada (1 strain), and are derived from patients representing both primary and secondary contacts. place of isolation accession number cov spike protein 39 . all group 1 coronaviruses can use apn as a receptor 40 , whereas the group 2 mouse hepatitis virus (mhv) uses glycoproteins belonging to the carcinoembryonic antigen family as receptors on their target cells 41 . whether the hapn binding domain is the receptor in vivo remains to be shown. the protruding s1 domain is adjacent to the s2 domain, which consists of a stem, a transmembrane region and a short cytoplasmic tail. analogous to other coronavirus s proteins, the sars-cov spike protein contains two heptad repeats that are located in the s2 domain. it is proposed that these repeats might trigger the fusion of the viral envelope with the cell membrane, as has been shown recently for mhv 42 . in some group 2 and group 3 viruses, the spike is cleaved during maturation into two subunits, s1 and s2, which stay non-covalently attached. the exact role of this cleavage process is unclear as it does not seem to influence infectivity; however, it may enhance fusion activity [43] [44] [45] [46] [47] . the mature sars-cov seems to contain uncleaved spike protein, but cleavage after binding to the target cell cannot be excluded. to conclude, the spike protein is a target for the development of agents that block the virus from binding to its cellular receptor and is the docking site for peptides that might inhibit fusion. several additional targets for further study have been identified. first, the 76-amino-acid e protein -computer analysis has predicted a long transmembrane domain close to the n-terminus and two n-terminal glycosylation sites with a very low level of amino-acid similarity to other coronaviruses. second, the m glycoprotein, which is a 221-residue polypeptide that consists of a short n-terminal ectodomain with a n-glycosylation site, three transmembrane segments and a c-terminus located on the interior side of the viral envelope, and which closely resembles m glycoproteins from other group 2 viruses. third, the n protein, which is a 397-residuelong phosphoprotein that interacts with viral genomic rna to form the helical nucleocapsid, and which has a low level of conservation with other coronaviruses. with the notable exception of β-interferon, which has been reported to interfere with the replication of the sars virus in vitro 48 , no licensed drug or vaccine is available. large-scale screening of existing antivirals or big chemical libraries for potential replication inhibitors has not been very successful. at present, the only promising substance is glycyrrhizin, a component of liquorice roots, which also has activity against hiv. this compound was identified during a small-scale experiment involving only a small number of known antiviral compounds 49 . the development of assays that are based on viral enzyme activity and which are amenable to high-throughput screening of existing and new chemical libraries, will likely identify effective compounds in the near future. nevertheless, unless these putative compounds are already licensed or are existing products in the advanced stages of development, they will not be available to treat patients in the structures are available. given its fundamental role in virus biology, the rna-dependent rna polymerase (rdrp) is high on the list of promising targets. of the different possible vaccine targets, the s glycoprotein is the most attractive candidate for exploitation. this protein forms the large surface projections that are characteristic of coronaviruses (fig. 1) , and which are most likely to be composed of homotrimers. the heavily glycosylated 1,255 amino-acid protein 35 contains an amino-terminal, bulbous head (s1), which comprises the receptor-binding domain and is also believed to be responsible for the host and tissue tropism of the virus 36 non-replicating viruses, or viral vectors that are based on adenovirus, canarypox, modified vaccinia virus ankara (mva) or alphavirus. in particular, the devlopment of non-replicating coronavirus-like particles that mimic the structure of native virions could prove promising in the search for a successful vaccine as they display a large repertoire of antigenic sites and discontinuous epitopes. however, each of these approaches, including passive immunotherapy, need to be carefully evaluated as some vaccines that have been developed against feline coronavirus actually exacerbated the disease when vaccinated animals were challenged with the wild-type virus 50 . killed-virus vaccines that are ready to be tested in phase i clinical trials are likely to be available soon. under normal circumstances, a vaccine takes 6-8 years of clinical development after entering phase i clinical trials. these timelines could be shortened considerably should the disease burden and a state of medical emergency induce the regulatory agencies to 'fast track' the approval process. in conclusion, the development of therapeutic strategies against this new coronavirus seems to be possible with the available technologies and is not an unreachable goal to the same level as hiv or hepatitis c virus. given enough time and economic pressure, antiviral drugs, human monoclonal antibodies, vaccines and sirnas that are active against sars-cov are all likely to become available. the remaining question is whether we will have the time to develop effective therapies before another epidemic emerges in the human population. should sars return this winter, we will still need to rely primarily on quarantine measures to contain the disease. equally important is the development of technologies that allow the rapid and simple diagnosis of sars. just as worrying, however, is a scenario in which the virus does not re-emerge for a couple of years, causing the economic incentive for companies to invest in sars to disappear, so none of the above measures will have been developed and implemented. near future. cellular proteins that are essential for virus replication should also not be overlooked as possible targets -new technologies such as double-stranded small interfering rnas (sirnas) have considerable future potential, but as yet, are still riddled with practical difficulties. at present, researchers are working on the development of efficacious delivery systems for sirnas. and following the successful conclusion of this research, they will still need to prove their potential in animal models of infection. antibodies that are able to neutralize viral infection are also likely to be an effective way to prevent and cure this disease. passive immunotherapy using sera from convalescent patients was initially proposed as treatment for disease. given the excellent track record of human monoclonal antibodies in the treatment of cancer and infectious diseases, this should prove a fruitful area for therapeutic development. indeed, monoclonal antibodies obtained from immortalized b lymphocytes of sars convalescent patients have been shown to neutralize virus infection in vitro and to prevent virus replication in a mouse model of sars-cov infection (a. lanzavecchia and r.r., unpublished observations). of course, a safe and effective vaccine would be the ideal solution, because not only would it prevent the disease in vaccinated people but a vaccine would also curtail the spread of the virus. although only a short period of time passed since sars-cov was identified as the infectious agent that was responsible for the epidemic, candidate vaccines based on killed virus are already available. their efficacy still needs to be shown, but our laboratory (and possibly others) are in the process of testing vaccines on the basis of inactivated sars virus in pre-clinical models. in addition to the traditional approach, a number of newer technologies are being used. these include subunit vaccines containing recombinant spike protein expressed in mammalian cells or yeast, either alone or in combination with other sars-cov antigens. alternatively, these antigens could be delivered by dna immunization by figure 6 | the s1 domain of sars-cov spike is structurally related to group 2 coronaviruses. schematic representation of cysteine positions in the s1 domains of group 1, 2 and 3 coronaviruses, compared with the sars-cov spike protein. horizontal bars represent the s1 amino-acid sequences (in the case of sars-cov and ibv) or the consensus profiles (generated from group 1, (g1 cons) and from group 2 (g2 cons)). the bars are drawn to scale. relative cysteine positions are indicated by rectangular bars. only cysteines that are conserved within each consensus are reported. coloured lines connect cysteines that are conserved between the sars-cov s1 domain and the consensus sequence generated from the group 1 (green), group 2 (red) and ibv s1 sequences (blue). coronavirus as a possible cause of severe acute respiratory syndrome epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong clinical presentations and outcome of severe acute respiratory syndrome in children clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study acute respiratory syndrome in china summary of probable sars cases with onset of illness from 1 identification of a novel coronavirus in patients with severe acute respiratory syndrome a nnvel coronavirus associated with severe acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions transmission dynamics and control of severe acute respiratory syndrome isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars in china: tracking the roots of a killer severe acute respiratory syndrome (sars) in singapore virus taxonomy the genome sequence of the sarsassociated coronavirus references 17 and 18 are the first reports of the complete genome sequences of two sars-cov isolates (tor2 and urbani strains, respectively) the complete genome sequence of severe acute respiratory syndrome coronavirus strain hku-39849 (hk-39) mechanisms and enzymes involved in sars coronavirus genome expression the complete genome sequence of a sars-cov isolate (fra) and experimental data on its key rna elements and protein functions are described. the enzymatic activity of the sars-cov helicase and two proteinases is shown viral replicase gene products suffice for coronavirus discontinuous transcription mrna cap-1 methyltransferase in the sars genome the molecular biology of coronaviruses the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system a common rna motif in the 3′ end of the genomes of astroviruses, avian infectious bronchitis virus and an equine rhinovirus comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection a novel coronavirus associated with severe acute respiratory syndrome unique and conserved features of genome and proteome of sars-coronavirus, an early splitoff from the coronavirus group 2 lineage the authors describe the genome organization and expression strategy of the sars-cov. a phylogenetic analysis of the orf1b indicates that the sars-cov represents an early split-off from group 2 coronaviruses structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra α-helical domain coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs on the basis of these structures, the authors provide a three-dimensional model of the main proteinase of sars-cov, and using molecular modelling mass spectrometric characterization of proteins from the sars virus: a preliminary report the coronaviridae retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier switching species tropism: an effective way to manipulate the feline coronavirus genome putative hapn receptor binding sites in sars-cov spike protein feline aminopeptidase n is a receptor for all group i coronaviruses receptor specificity and receptor-induced conformational changes in mouse hepatitis virus spike glycoprotein the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex the function of the spike protein of mouse hepatitis virus strain a59 can be studied on virus-like particles: cleavage is not required for infectivity fusiondefective mutants of mouse hepatitis virus a59 contain a mutation in the spike protein cleavage signal fusion formation by the uncleaved spike protein of murine coronavirus jhmv variant cl-2 the virulence of mouse hepatitis virus strain a59 is not dependent on efficient spike protein cleavage and cell-to-cell fusion proteolytic cleavage of the murine coronavirus surface glycoprotein is not required for fusion activity treatment of sars with human interferons this paper reports the first results obtained for the search of possible sars inhibitors. the authors show that a review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination the neighbor-joining method: a new method for reconstructing phylogenetic trees the authors are grateful to the fonds der chemischen industrie and the chiron vaccines cellular microbiology and bioinformatics unit for their continuous support. the authors have declared competing financial interests; see web version for details. key: cord-343791-0vykwml5 authors: hainline, kelly m.; fries, chelsea n.; collier, joel h. title: progress towards the clinical translation of bio-inspired peptide and protein assemblies date: 2017-11-08 journal: advanced healthcare materials doi: 10.1002/adhm.201700930 sha: doc_id: 343791 cord_uid: 0vykwml5 supramolecular materials composed of proteins and peptides have been receiving considerable attention towards a range of diseases and conditions from vaccines to drug delivery. owing to the relative newness of this class of materials, the bulk of work to date has been preclinical. however, examples of approved treatments particularly in vaccines, dentistry, and hemostasis are demonstrating the translational potential of supramolecular polypeptides. here we describe critical milestones in the clinical development of this class of materials and describe currently approved supramolecular polypeptide therapies. additional examples of not-yet-approved materials that are steadily advancing towards clinical use are also featured. spherical assemblies such as virus-like particles (vlps), designed protein nanoparticles, and spherical peptide amphiphiles are highlighted, followed by fiber-forming systems such as fibrillizing peptides, fiber-forming peptide-amphiphiles, and filamentous bacteriophages. finding utilization in biomedical applications ranging between immunomodulation, drug delivery, tissue regeneration, defect repair, cell delivery, and combinations thereof. many self-assembling systems have been investigated in preclinical research, but among these, relatively few have been successfully translated into approved devices and therapeutics. those that are in current clinical use tend to possess the following features that have enabled their success: -chemical definition-highly specified control over material composition, assembly properties, and bioactivity, in contrast with biologically sourced materials -manufacturability-the extent to which the platform can be manufactured with relative ease at minimum cost and with maximum reproducibility -tunability-the ability to adjust the amount of multiple selected active components in a material with precision and reproducibility. synthetic supramolecular systems tend to feature this property well beyond naturally derived materials. selected platforms are discussed based on these features, because they have had a meaningful translation into clinical practice, or because they have had steady progress toward clinical translation. these include spherical assemblies such as virus-like particles, designed protein nanoparticles, and peptide amphiphiles (figure 1) ; and elongated structures such as β-sheet nanofibers, fiber-forming peptide amphiphiles, and filamentous phage (figure 2) . a timeline of their development is shown in figure 3 . some of the first engineered supramolecular structures to be translated effectively have been virus-like particles (vlps), multiprotein constructs that self-assemble to mimic the organization, structure, and immunogenicity of native viruses but that lack infectious genetic materials ( figure 1a ). [7, 8] their development has preceded other supramolecular materials discussed here (see timeline in figure 3 ). vlps are potent immunogens, able to stimulate b cell/antibody responses, cd4 + t-cell responses, and cytotoxic t-cell responses. [17, 18] owing to their lack of a viral genome, vlp-based vaccines circumvent some risks associated supramolecular materials composed of proteins and peptides have been receiving considerable attention toward a range of diseases and conditions from vaccines to drug delivery. owing to the relative newness of this class of materials, the bulk of work to date has been preclinical. however, examples of approved treatments particularly in vaccines, dentistry, and hemostasis demonstrate the translational potential of supramolecular polypeptides. critical milestones in the clinical development of this class of materials and currently approved supramolecular polypeptide therapies are described in this study. additional examples of not-yet-approved materials that are steadily advancing toward clinical use are also featured. spherical assemblies such as virus-like particles, designed protein nanoparticles, and spherical peptide amphiphiles are highlighted, followed by fiber-forming systems such as fibrillizing peptides, fiber-forming peptide-amphiphiles, and filamentous bacteriophages. over the past few decades, researchers have built an expansive toolbox of self-assembling peptides and proteins that offer unique advantages over traditional small molecules and polymers for a range of biomedical applications. the first examples of synthetic self-assemblies were bioinspired derivatives of native proteins, but an increasing familiarity with the design rules governing supramolecular assembly has more recently facilitated the de novo design of this class of materials. the ability to create predictable and engineerable supramolecular structures has led to the implementation of several of these materials within biomedical applications. in this review, we highlight self-assembling polypeptide materials that have been clinically translated. we will also discuss the advantageous properties and features that have enabled their clinical implementation. for recent reviews on preclinical work relating to this class of materials including self-assembling immunomodulating materials, [1, 2] self-assembled materials for cell delivery, [3, 4] and self-assembled biomaterials as a whole, [5, 6] the reader is referred to the review articles indicated. properly designed polypeptides can self-assemble into a range of predictable structures including nanofibers, micelles, nanoparticles, and extended networks. these architectures are attenuated or inactivated live viruses, highlighting an advantage of supramolecular systems: they are more compositionally defined than the analogous biological structures, viruses. they also have advantages over subunit vaccines based on viral proteins or peptides conjugated to carrier proteins, which commonly require higher and more frequent dosing and adjuvants to be as effective as inactivated or attenuated viruses. [19] as alternatives to these previous platforms, vlps were developed to display an array of epitopes that mimic the surface of native viruses more effectively than subunit or peptide vaccines, thus improving their immunogenic properties. [20] vlps, like viruses, come in a range structures including those with a single capsid protein, multiple capsid proteins, or those without lipid envelopes. [21] vlps consisting of multiple capsid proteins are expressed and assembled via subsequent processing or by co-expression of polycistronic genes within a cell. several additional aspects of vlps have contributed to their clinical translation. whereas the first translated vlp-based vaccines raised antibodies against the naturally occurring virus capsid proteins of the assembled structure, vlps can also be used as vehicles to display heterologous antigens associated with other infectious diseases. this modularity of the platform is described [7] copyright 2007, asbmb. (a, right) adapted with permission. [8] copyright 2017, elsevier. (b) adapted with permission. [9] copyright 2006, elsevier. (c) adapted with permission. [10] become increasingly complex, they become increasingly more challenging to manufacture at a large scale. multi-subunit, chimeric, and other types of vlps must overcome these challenges. fortunately, a large assortment of expression systems (bacterial, yeast, insect, plant, mammalian, and cell-free) have been developed for the production of new vlp platform candidates. the idea of using noninfectious virus particles to develop prophylactic human vaccines first became attractive when noninfectious vlps composed of the surface antigen from the hepatitis b virus (hbsag, also known as the australia antigen), were discovered in human sera in 1965. [20] the first vlpbased human vaccine consisted of hbsag vlps derived from human plasma and was licensed in the united states in 1981 ( figure 3 ). subsequently, after the advent of hiv/aids rendered plasma-derived products more challenging, the hbsag particles were produced recombinantly in yeast, and the first recombinant human vaccine, recombivax hb, was licensed by merck in 1986. [22] three years later in 1989, engerix-b, a similar hbv vaccine, was licensed by glaxosmithkline. it took another 17 years before the next recombinant vlp-based vaccine was licensed for human use. [13] gardasil, licensed by merck in 2006, is a vaccine against hpv that protects against hpv-related diseases such as cervical cancer. in 1991, several laboratories concurrently observed that recombinantly expressed hpv l1 and l2 virus capsid proteins self-assembled into vlps resembling the native virion structure. [23] in early 1992, based on this observation, merck research laboratories (mrl) initiated an hpv vaccine program. at the time, a growing number of hpv genotypes were identified and associated with benign genital warts and or precursor lesions to cervical cancer. mrl devised a strategy to produce a quadrivalent hpv l1 vlp that would protect against cervical cancers (caused by hpv16 and 18) as well as genital warts (caused by hpv 6 and 11). several concurrent studies in the 1990s were additionally critical to the success of hpv vaccines. one important advancement was the development of an in vitro method of producing hpv virions. the hpv lifecycle is linked to human epithelial tissue differentiation which is difficult to achieve in vitro. kreider et al. overcame this challenge was overcome by developing a complex xenograft model in which human foreskin epithelial tissue was infected with hpv 11 and grown under the renal capsule of athymic mice. this method allowed for the production of infectious hpv 11. [23] in 1995, animal challenge studies were published that continued to advance hpv vaccines. two studies demonstrated measurable serum antibody responses to vlp immunization and subsequent protection in later challenges. [23] the third demonstrated the importance of maintaining the native vlp structure for optimal vaccine efficacy. [24] another key contribution to the success of mrl's hpv vaccine came from work developing a manufacturing process for hbsag production in saccharomyces cerevisiae. it provided a foundation for the development of methods to design, express, and purify large amounts of hpv vlps. [23] in 1997, hpv 11 l1 vlps were evaluated for the first time in humans as a monovalent vaccine during a phase i clinical trial to demonstrate safety and immunogenicity. the monovalent vaccine was chosen owing to the availability of robust models for the evaluation of immunogenicity, and because models for the other three hpv types had not yet been developed. the phase 1 study outcomes were promising, with no reported significant adverse effects. a second study tested the ability of monovalent hpv 16 l1 vlp efficacy in preventing hpv infection, providing the first demonstration that vaccination with hpv vlps could prevent disease. these favorable results supported the development of the multivalent vaccine and spurred the advancement of the program. in 2006, after impressive efficacy data and an acceptable safety profile, the first hpv vlp vaccine, gardasil, was licensed (see timeline in figure 3 ). [23, 25] another hpv vlp human vaccine for the prevention of cervical cancer, cervarix, was developed by glaxosmithkline and licensed in the united states in 2009. this vaccine includes hpv types 16 and 18 and has been produced using insect cells infected with recombinant baculovirus. [26] cervarix demonstrated a 90.4% efficacy against cervical intraepithelial neoplasia lesions containing hpv 16 and 18 and indicated cross-protection with the hpv types 31 and 45, leading to the protection against 80% of cervical cancers. [27] hecolin (xiamen innovax biotech), a recombinant vlp-based vaccine for prophylactic [11] copyright 2001, aaas. (a, right) adapted with permission. [12] copyright 2002, national academy of science. (b, left) adapted with permission. [13] copyright 2013, american chemical society. (b, right) adapted with permission. [14] (c, left) adapted with permission. [15] copyright 2014, elsevier. (c, right) adapted with permission. [16] use against hepatitis e virus infection, was licensed in china in 2011. [28] in the past decade, vlp vaccines have played a critical role in the improvement of human health and continue to be applied to new diseases. vlp-based vaccines are currently in clinical trials for the treatment of many infectious diseases: hbv, hiv, hpv, human parvovirus, influenza virus a, norwalk virus, ebola virus, and severe acute respiratory syndromerelated coronavirus. the inclusion of preclinical testing to this list broadens it even further, to diseases including chikungunya virus, west nile virus, and mumps virus. [29] as mentioned in the introduction, one of the key strategic strengths of supramolecular systems is the modularity that they tend to possess. in vlps, this modularity is exemplified by chimeric vlps, in which epitopes of choice from various diseases are inserted into the particle-forming proteins. provided that the new epitope can be inserted in a way that does not disrupt self-assembly, and that presents the epitope on the surface of the particle so that it is both antigenic and immunogenic, the modularity of the system is maintained. [20] by being able to insert epitopes of choice, the range of therapeutic applications becomes quite broad. for example, several clinical studies are currently evaluating chimeric vlp vaccines for the treatment of noninfectious diseases such as cancer (melanoma), [30] neurodegenerative diseases (alzheimer's disease), [31] autoimmune diseases (allergic rhinoconjunctivitis and asthmas), [32] and other disorders. another approach for including chosen epitopes/ antigens is separately expressing the vlp and target protein and conjugating them together. this approach is often preferable and necessary due to differences in optimal expression conditions for each component. thus, postproduction methods have been developed to link the vlp and target antigen. this is achieved through genetic manipulation, coupling via supramolecular or covalent bonds, or by encapsulation of cargo by disassembling and reassembling purified vlps in the presence of the desired molecule. [33] coupling chemistries range from the use of bifunctional crosslinkers, click chemistry, sortase-mediated attachment, polyhistidine/nta-ni 2+ , or affinity-tag interactions to conjugate the vlp and target antigen [34] notably, these postproduction-modified vlps have led to several platforms currently being tested in clinical trials. [35] [36] [37] although a range of chemical cross-linking strategies have been developed for vlps, [38, 39] conjugation of target antigens with multiple reactive sites can lead to heterogeneous coupling or unfavorable epitope display. [38] the bachmann group addressed this challenge using the spytag-spycatcher system. [40] spytag is a peptide that forms a spontaneous and irreversible isopeptide bond with spycatcher, its protein partner. [40] the spycatcher-vlp platform is expressed in escherichia coli and mixed with spytag-antigen to form "plugand-display" decorated vlps, another highly modular, tunable approach that allows for incorporation of a wide variety of antigens to the vlp surface. the group reported that spycatcher-vlps decorated with the cidr or pfs25 antigens generated a robust antibody response are only a single immunization without requiring adjuvant. this platform shows promise in application such as drug delivery, enzyme scaffolds, biosensors, and cancer immunotherapy, [41] and it mitigates the need for complex chimeric protein expression or the incorporation of unnatural amino acids, as would be necessary in coppercatalyzed azide-alkyne cycloaddition (click chemistry) or other chemoselective bioconjugation reactions. vlps, owing to their structural definition and flexibility in formulation, also make useful experimental tools for studying basic aspects of immunity. they usually range in diameter from 20-200 nm, an optimal size for drainage to lymph nodes and subsequent interactions with b cells. whereas the differentiation of naïve b cells into memory b cells has been extensively studied at the cellular and molecular level, the fate of memory b cells upon antigen re-encounter has been comparatively less well-studied. the bachmann group used a qβ vlp as a model system and were able to track vlp-specific b cells via flow cytometry and histology to follow naïve and memory b cell responses. [42] they unexpectedly found that, during secondary b cell responses, secondary plasma cells are generated, whereas naïve b cells are recruited into a parallel primary b cell response. this phenomenon allows a plasticity of the memory b cell repertoire upon multiple antigenic exposures. shortcomings of vlps include their requirement for a continuous and well-regulated cold chain, which negatively impacts their distribution to the developing world. to address this challenge, the chackerian group has developed a vlp-based vaccine candidate that is compatible with spray drying, thus enhancing its stability over a broad range of temperatures. [43] this platform targets a highly conserved, broadly neutralizing epitope from the hpv minor capsid protein, l2. not only does this vaccine elicit high-titer and long-lasting antibody immune responses, [44] but the spray dried vlps were highly immunogenic in a mouse model after being stored for 14 months at either room temperature or 37 °c. other constraints that vlps are subject to include the relatively small size of epitopes that can be accommodated within the particles. for example, large antigens such as hiv envelope and influenza hemagglutinin proteins are too large to be packaged within native vlps. the practicality of vlp platforms is also limited by manufacturing considerations. [21] for example, vlps derived from e. coli, the most widely used and most efficient expression system in the biotechnology industry, have a high degree of heterogeneity in their physical properties, including the shape and diameter of the particles. such particles require post-purification disassembly and reassembly in optimized conditions. in addition, it may be possible to produce highly immunogenic vlp preparations, but the antigen might not be viable in the vlp context until stable formulations can be developed. formulations must be resistant to aggregation upon exposure to low salt and protein concentration, as well as protection against surface adsorption and aggregation as a result of heat stress and physical agitation in order to achieve the multiple-year stability required for a marketed vaccine. [20] newer expression systems in conjunction with innovative purification strategies could determine the pace for the next generation of vlp-based vaccine candidates. for example, yeast, insect, and mammalian expression systems have been used to circumvent the limitations of bacterial expression such as sub-optimal ph and lipid compositions within the bacteria. additionally, the advancement of high-throughput biophysical and structural analyses of recombinant vlps may play a key role in the assessment of vlp candidates. electron microscopy, electrospray differential mobility analysis, atomic force microscopy, x-ray crystallography, and dynamic light scattering provide quantitative structural data for each vaccine candidate and play a valuable role in ensuring product robustness from the early clinical development stage and beyond. [20] as an alternative to particles inspired by natural virus capsid proteins, fully designed protein nanoparticles have received considerable interest. although "bottom-up" approaches for designing nanomaterials were popularized over 30 years ago, [45] the development of supramolecular polypeptide materials into successful medical technologies was initially slow, hindered by the sheer diversity of possible self-assembled structures and the lack of design rules. in 2001, the yeates group developed an approach based on molecular symmetry to fabricate protein assemblies having a range of predictable architectures. [46] their strategy was a breakthrough in rational self-assembling protein design. in the past few years, the capacity to model and predict protein structures and energetics has increased along with computing power, leading to the computational design of de novo self-assembling protein nanoparticles. [47] the baker group used naturally occurring oligomeric proteins as building blocks to design cage-like assemblies with accuracy. recently, they generated a hyperstable 60-subunit protein icosahedron via symmetric modelling coupled with computational protein-protein interface design. [48] this structure is robust to genetic fusions, making it a notably modular platform that could be used in multivalent epitope display as well as drug delivery. taking inspiration from nature has also proven invaluable in the design of mechanically and chemically stable nanoparticles. in 2006, raman et al. designed a self-assembling nanoparticle based on virus capsids via superposition of different protein oligomerization domains onto the symmetry axes of an icosahedron shown in figure 1b . the monomer building block consisted of a protein chain made of two coiled coils connected by a short linker region. the association between the coiled coils caused the assembly of monomers into a roughly spherical nanoparticle. [9] the self-assembling protein nanoparticle's versatile and flexible design allow for optimization of biophysical and immunological properties making them a desirable vaccine platform. whereas synthetic peptides are usually not sufficiently immunogenic and require adjuvants, the burkhard group has developed a self-assembling protein nanoparticle platform that displays both b and t cell epitopes to produce a vaccine with self-adjuvanting qualities. [49] this platform is currently under development to improve a malaria vaccine, "rts's" that is based on the circumsporozoite protein of plasmodium falciparum. [50] the self-assembled protein nanoparticles are able to stimulate high titer, high avidity antibodies and present cd8+ t-cell eptiopes to stimulate il-2 and ifn-γ producing longterm memory t-cells in mouse models. the vaccine candidate fmp014, based on this platform, is currently undergoing phase 1 clinical trials. a principal advantage of designed protein nanoparticles is their chemical definition. the ability to use computational methods to design nanoparticles of different geometries and sizes opens the door to applications such as drug delivery, in which the shape and size of the delivery vehicle are crucial. however, the manufacturability of these platforms, similar to vlps, is a challenge due to their multi-subunit nature and the need to be recombinantly expressed. micellar nanocarriers composed of amphiphilic molecules have had particular success in the pharmaceutical realm as a tool to increase bioavailability, retention, and solubility of various drugs. [51] however, in this review, we shall be focusing on peptide amphiphiles (pas) as they relate to our theme of the clinical translation of peptide-based materials. such structures self-assemble from molecules composed of a hydrophobic domain, usually an alkyl chain, and a hydrophilic peptide domain. [4, 52] pa assembly is driven by hydrophobic-hydrophobic interactions in water, and bioactivity is programmed into the hydrophilic peptide head groups (see figure 1c ). for a comprehensive review on pas used in biomedical applications, the reader is directed to acar et al. [6] targeting, diagnostic, and theranostic platforms have been derived from peptide amphiphile micelles (pams). to increase the size of hydrophobic head groups and push systems toward a micellar packing morphology, the micelles consist of a biologically active peptide attached to a hydrophobic alkyl tail via a bulky polyethylene glycol (peg) spacer. the peg spacer allows for enhanced blood circulation times while retaining the packing parameters necessary for micelle formation. [6] these molecules are shown to circulate through the blood stream without causing blockage, and are cleared via the reticulo-endothelial system and renal system with 90% clearance and no toxicity after 7 d. [53] pams are currently in preclinical development for both cancer and atherosclerosis diagnostic applications. for cancer applications, fluorescently-labeled pas with the fibrin-binding peptide creka were used to target glioblastoma cells. upon intravenous administration to gl261 glioma-bearing mice, nontargeting micelles passively accumulated at the fibrin deposits characteristic of tumor vasculature. these micelles displayed enhanced tumor homing as early as 1 h after administration without inducing cytotoxicity or tissue damage. [54] toward a therapy for atherosclerosis, the creka targeting peptide, an antithrombin peptide called hirulog, and fluorescence molecules were assembled into theranostic micelles. [55] this fibrin-targeting micelle could also be functionalized by the addition of the gadolinium chelator diethylenetriaminepentaacetic acid, allowing for plaque localization and visualization using t1-weighted magnetic resonance imaging (mri) imaging. [56] similar pams have been designed for the targeting of monocytes as a strategy to diagnose atherosclerosis via the areas of heightened immunological activity characterized by the disease. [10] similarly to previously described vaccine nanoparticles, pa micelles are able to elicit either humoral or cell-mediated immunity without additional adjuvant. an antigen is simply conjugated to the tail domain of the molecule prior to selfassembly. black et al. was able to assemble cylindrical micelles from monomers consisting of a dialkyl tail conjugated to a peptide containing the known cytotoxic t-cell epitope from the model tumor antigen ovalbumin. [57] these dic 16 -ova micelles were able to stimulate ova-specific t-cells, offering in vivo protection from tumors without any additional adjuvant. this observation spurred additional immunological studies to expand upon the potential of peptide amphiphile micelle vaccine platforms. in 2016, barrett et al. used a group a streptococcus (gas) b-cell antigen coupled to a dic 16 tail which drove self-assembly of cylindrical micelles, to induce a micelle-mediated immune response (without adjuvant) that was stronger than seen with a conventional gold-standard vaccine formulation. [58] although spherically assembled peptide amphiphiles have not yet reached regulatory approval for clinical applications, their versatility, modularity, and demonstrated success in preclinical work is encouraging. clinically translatable supramolecular materials are not limited to spherical morphologies. extended structures with high aspect ratios have also seen considerable development toward a variety of medical technologies (figure 2a-c) . prominent among these have been fibrillar assemblies of peptides and their derivatives. these structures have been investigated over the past 20 years and have made progress toward therapies in hemostasis, dentistry, wound healing, and immunology. while studying structural biology in alexander rich's research group, s. zhang discovered a self-assembling β-sheet peptide based on the dna-binding protein, zuotin. [59] the peptide formed amphiphilic tapes with two distinct surfaces, one hydrophobic and the other hydrophilic, and it also contained complementary charged residues that additionally favored this β-sheet folding (see figure 2b ). following this discovery, a mimic of the native peptide was designed by mutating the charged and hydrophobic residues, but leaving the pattern intact. [60] this mimic demonstrated that self-assembly of peptides with these motifs was not a sequence-specific anomaly, but could be recreated in similar systems, forming the first steps toward design rules for fibrillizing peptides. the designer peptide was shown to also form macroscopic membranes and support the attachment of mammalian cells, demonstrating its utility as a biomaterial. [61] subsequently, it was found that the original synthetic sequence rada16-ii (raradada) 2 and its modified form rada16-i (rada) 4 spontaneously formed hydrogels of entangled nanofibers in salt-containing solutions and cell culture media. [62] mixtures of peptides bearing multiple bioactive groups could be incorporated into a single macroscopic gel by dosing in various amounts of monomeric peptides to the gel mixture. additionally, the incorporation of ligands or epitopes within the materials simply required extension of the peptide at either terminus with the desired sequence, thus lowering the barrier of synthetic difficulty for biological researchers. these gels have been subsequently developed toward neuronal regeneration, cytokine delivery, as biotinylated scaffolds for versatile protein delivery, and other applications. [62] [63] [64] the release of proteins and small molecules from rada hydrogels largely depends on the size of the protein cargo regardless of charge and hydrophilic character, allowing a wide variety of biomolecules to be delivered in these gels. [65] besides physical entrapment, a biotin-sandwich approach has been used to tether insulin-like growth factor 1 (igf-1) to gels for long term localization of bioactive igf-1 in the scaffold. [63] biotinylation provides the advantage of modularity within the hydrogel delivery system, as any biotinylated protein can be immobilized to biotinylated fibers via a streptavidin linkage. biotinylated fibers have been investigated for myocardial regeneration following infarction and showed success in rats when injected into infarcted hearts along with cardiac progenitor cells. [66] although rada nanofibers have been investigated preclinically for a wide range of medical applications, they have had the most clinical success as a hemostatic agent. marketed under the trade name purastat by 3-d matrix ltd., [67] the product is composed solely of peptide dissolved in sterile water, which forms fibers when in contact with biological fluids. this self-assembly provides a physical blockage in order to limit bleeding at the site of application. although this peptide's use as a hemostat is still being actively developed as a component of layer-by-layer wound dressings, [68] the original rada16-i peptide has been useful as a surgical hemostat for multiple surgeries because of the dense mesh it creates at neutral ph. the rada16-i peptide which became purastat was first tested as a hemostatic agent in 2006 and demonstrated hemostasis in rats by stopping bleeding in under 15 s when applied as a 3%-4% aqueous solution to wounds in skin, brain, spinal cord, femoral artery, and liver. [69] this rapid induction of hemostasis circumvented the need for components of the clotting cascade to activate the hemostat, it avoided the use of heat or pyrogenic substances, and it was effective in the presence of anti-coagulant therapies [70] without causing pronounced tissue responses. [71] although the peptide is slightly acidic, it did not cause significant inflammation in any animal or clinical studies, even when applied to the brain. [72] also named tdm-621, the rada16-i peptide was first tested as a hemostat in human surgeries in japan during cardiovascular procedures, [73] and later during endoscopic mucosal resection, [74] with no treatment-related adverse events in either trial. purastat is limited to relatively low pressure hemorrhages in comparison to major arterial injuries, [75] but has general utility as a versatile, biodegradable hemostat. clinical trials to monitor the post-market performance of purastat for vascular surgery (nct03103282), and to test the use of purastat during endoscopic submucosal dissection are scheduled to begin soon (nct02833558). purastat has already achieved ce marking in europe and has currently received medical device product registration approval in a number of countries including thailand, mexico, and indonesia, and australia. initial studies of the rada family of peptides inspired the development of general design principles for forming nanofibers, during which other peptides were shown to have similar self-assembling properties. notably, peptide p 11, developed by aggeli et al., would eventually lead to the development of the enamel regeneration product curodont. the first sequence designed by aggeli et al. in 1997 was a mimic of a β-sheet transmembrane protein. [76, 77] this peptide was designed de novo based on patterns found in the transmembrane protein and was shown to form high aspect-ratio fibrils that tangled to form hydrogels independently of ph in a manner similar to the protein-inspired peptide on which it was based. [77] currently, this peptide is on the market in the european union (eu) and switzerland in the form of a treatment for dental enamel caries, and it functions by creating a local environment that enhances enamel mineralization. [77] upon injection, monomeric peptides assemble into nanotapes to create a 3d matrix that in some respects resembles the matrix environment necessary for enamel deposition. [78] a major design improvement preceding the final curodont peptide sequence was the introduction of ph-sensitivity. in 2003, aggeli et al. rationally modified the original peptide sequence so that it was negatively charged at ph > 8, thus creating electrostatic repulsion and allowing the peptides to remain in a monomeric, unassembled state. [78] upon switching to more acidic ph, however, the acidic residues became protonated and monomers assembled in to β-sheet tapes. [79] this ph-sensitivity allows for in situ assembly of peptides into nanofibers, which is hypothesized to improve the efficacy of curodont as the material is able to completely fill demineralized defects of varying shapes and sizes. this ph-sensitive assembly was specifically explored in the context of enamel remineralization in 2007, where simulated intraoral conditions were employed to assess the performance of self-assembled scaffolds which were administered in a basic solution, allowed to fibriliize, and then incubated under cyclic ph. [78] these studies were completed on enamel lesions formed on extracted human teeth and indicated that the p 11 scaffold caused hydroxyapatite mineralization where the peptide solution was applied. [78] thus, positive and significant results were obtained without the need for specialized application methods or a poorly translatable animal model, a significant advantage for the development of polypeptide biomaterials toward dental applications. after the enamel restorative properties of p 11 were developed in preclinical models, credentis was founded in 2010, curodont was launched as the company's first product, and its safety and efficacy were verified in a clinical trial to treat dental caries. [78] in 2012 it received market approval (ce-label) for medical devices in the eu. in keeping with the original peptide design, the final formulation of curodont is composed of only a monomeric 11-amino acid peptide (p 11 ) dissolved in water, with no additional bioactive agents. [80] following a single application, the size and color of lesions are significantly improved, as observed one month after treatment. [80, 81] peptide nanofibers have also increasingly received attention in immunological contexts, an area in which our group is active. however, because the focus here is on clinical development and these materials have not yet reached clinical trials, these applications will be only briefly described, and the reader is referred to other recent reviews for more expansive descriptions of this burgeoning field. [1, 2, 82, 83] after the discovery that β-sheet fibrillized peptides can raise strong antibody responses without the requirement of supplemental adjuvant, [84] these materials have been investigated preclinically toward a range of diseases and conditions including malaria, [85] staphylococus aureus infections, [86] influenza, [87] west nile virus, cancer, [88] and cocaine abuse. [89] immunogenic peptide nanofibers are produced by co-assembling fibrillizing peptides extended with specific epitopes or haptens along with other fibrillizing peptides bearing t-cell epitopes. they stimulate antibody/b-cell responses, cd4 t-cell responses, and cd8 t-cell responses [90] which can be raised in tunable magnitudes without causing inflammation. [85, 86, 89] other recent extensions of fibrillizing peptide technologies have included assemblies containing both peptides and other therapeutically active compounds. in these drug-peptide formulations, hydrophobic, π-π, and electrostatic interactions induce the assembly of the molecules into fibrillar networks much in the same way as pure peptide systems. [91, 92] a recently described example is a strategy in which the us food and drug administration (fda)-approved anticancer drug pemetrexed was conjugated to a four-amino acid peptide and used both as an mri contrast agent and to form drug depots near tumor sites. [93] the dual function of the material (contrast and depot formation) was possible because the peptide-drug conjugate formed fibrillar hydrogels at high concentrations to form the drug depot, while lower concentrations in the circulation acted as the mri contrast agent. a range of other strategies have likewise employed fibrillizing peptides to alter the delivery or pharmacokinetics of various drugs. for example, hartgerink and co-workers showed that several different multivalent drug molecules such as clodronate, heparin, and suramin could be used to stabilize β-sheet fibrillar hydrogels by shielding the surface charges on the peptides that would otherwise inhibit gelation, thus forming drug depots. [94] naphthalene-modified peptides have been explored to carry hydrophobic drugs such as curcumin, which require carrier transport due to their low solubility. [95] these examples highlight considerable future potential for peptide nanofibers toward clinical applications. alongside the use of β-sheet peptides, peptide amphiphiles composed of a peptide head group and an alkyl tail are a related class of materials that have seen considerable interest for creating supramolecular nanofibers (see figure 2a ). although the self-assembly of peptide amphiphiles into spherical structures had been known for some time, [96] the landmark 2001 paper by hartgerink, stupp, and colleagues catalyzed much interest in the ability of this class of molecule to form nanofibrous materials for specific biomedical applications. [11] in this report, nanofibers spontaneously assembled from peptide amphiphiles into parallel bundles and promoted mineralization of hydroxyapatite. [11] since then, fibrillar peptide amphiphile materials have been explored in a broad range of medical applications ranging between wound healing, [97] bone healing, [98] the delivery of proteins, [99] nervous tissue repair, [100] and others. additional chemistries have been developed to stabilize the materials. for example, toward applications where mechanical integrity is necessary, adhesive groups have been incorporated into the hydrophilic heads to render the fibrous gels self-healing after they are strained mechanically. [101] a recent example of clinically directed peptide amphiphile nanofibers used them to deliver bioactive proteins such as bone morphogenic protein (bmp) to induce osteogenesis in an environment mimicking native bone growth. [98] other examples have included nerve repair, which benefitted from the bioactivity and parallel alignment of fibrous scaffolds, [102] and burn injuries, where heparin-mimetic gels induced the formation of vascularized, collagen-rich tissues. [97] peptide amphiphiles have also been used to deliver bioactive cargo outside of the regenerative context, including the electrostatic complexation of antisense oligonucleotides to a cationic peptide head to form a depot for sustained release of oligonucleotide. [103] because networks of peptide-based fibers are morphologically similar to the extracellular matrix, they have also been highly useful as in vitro culture materials [104] and can be utilized as cell delivery vehicles, as demonstrated in the transplantation of islet cells [105] and bone marrow-derived pro-angiogenic cells cultured prior to transplantation. [106] although work with fibrous peptide amphiphiles has remained largely preclinical to date, is anticipated that the coming years will see many of these applications brought forward into approved therapeutics. filamentous bacteriophages (see figure 2c ) represent an additional step in the progression of nanofiber-forming biomaterials. although their production is considerably different than the chemical synthesis of pas and short peptides, their elongated morphology and polyamino acid composition are similar in some respects, and can be used to endow phage-based materials with similar properties to the other fiber-forming platforms discussed above. filamentous phages have highly engineerable coat proteins which allow the high density surface display of selected proteins. m13 phage are naturally filamentous, and they resemble peptide and peptide-amphiphile nanofibers in morphology as they are less than 10 nm in diameter yet almost 1 µm in length. because m13 bacteriophages are unable to infect mammalian cells, there is negligible risk of virulent infection when using these viruses in medical applications. for these reasons, they have been historically used as antimicrobial agents and are currently approved for use in food products, but have had seen limited use in clinical trials as therapeutics. a few examples exist where the tissue-targeting and tumor-homing abilities of full phage libraries were tested in humans. [107, 108] bacteriophages were initially investigated as nanomaterials when they were observed to form liquid crystals, and they proved useful for the templating of inorganic structures by incorporating metal binding peptides on the viral coat proteins. [109, 110] following the discovery of this functionality, their liquid crystalline behavior was utilized to form aligned matrices for neural cell culture by displaying arg-gly-asp (rgd) and ile-lys-val-ala-val (ikvav) peptides on the phage surface. [111] this work included the use of standard phage display to select optimal 8-amino acid sequences for receptor binding, and the resultant filamentous phages were produced in e. coli, a relatively simple manufacturing process that is likely to be scalable. in another recent example of preclinical materials development using filamentous phages, their self-templating properties proved useful for controlling the direction of osteoblast growth using the orientation of the phage-based substrate. [112] the use of both self-templated and fabricated directionality in phage substrates has allowed for the directional growth of human fibroblasts, [113] proliferation and elongation of neural progenitor cells, [114] and stimulated the differentiation of mesenchymal stem cells into osteoblasts when the phages displayed an osteogenic peptide on their surface. [115] as in vivo injectable materials, phages have been used preclinically as carriers for magnetic nanoparticles for targeted imaging of cancerous tumors by displaying a high density of targeting ligands and metal binding peptides on the phage surface. [116] a related study used a modified approach by conjugating a streptavidin-linked fluorophore to phages displaying tumortargeting and streptavidin-binding peptides on their surface for targeted cancer imaging without the use of metal particles. [15] these studies demonstrated the importance of directional patterning in combination with the display of specific peptides on the phages. moreover, the incorporation of multiple bioactive phage populations into a single material only requires adjustment of the mixture of various deposited phages, so in this way these materials feature the modularity characteristic of supramolecular systems. similar to other nanofibrous materials, filamentous phage materials have received significant attention for inducing osteogenesis because they can form collagen-mimetic bundles for the mineralization of hydroxyapatite similarly to pa and β-sheet peptide nanofibers. display of anioinic peptides caused parallel assembly of individual phage in the presence of cations, followed by formation of oriented crystals when counterions were introduced owing to the local supersaturation of inorganic ions. [117] this approach closely resembled the demonstration of pa mineralization presented by stupp and coworkers and illustrates conservation of biological processes across materials platforms. [11] since the demonstration of phage assembly mineralization, osteogenesis studies have expanded to include presentation of a hydroxyapatite nucleating protein, dentin matrix protein-1, as an alternative moiety for inducing crystal formation. [118] tobacco mosiac virus (tmv), another rod-like virus was shown to cause differentiation of mesenchymal stem cells into bone cells as culture on the tmv coated substrate caused an upregulation of bmp-2, osteocalcin, and calcium sequestration, which are all markers of bone development. [119] recently, rgd-bearing phages were 3d-printed into a ceramic scaffold to induce osteogenesis and angiogenesis concurrently without the addition of exogenous vascular endothelial growth factor. [16] because of the scale at which phage can be produced, they may have manufacturing advantages over other designer materials. over the past 50 years, supramolecular assemblies of peptides and proteins have developed from single vaccines to a broad range of technologies that spans the breadth of biomaterials applications as drug delivery vehicles, scaffolds for tissue regeneration, and other therapeutics. [1, 2, 5, 6] currently, several have achieved regulatory approval for clinical use ( table 1) , primarily in the vaccine space. the advancement of these platforms can be attributed to many factors. peptides and proteins are more economical than ever to produce, and manufacturing efficiencies continue to be developed. the design rules for each subclass of materials has been significantly mapped in recent years. and strategies have been optimized for incorporating disease-specific ligands, epitopes, or other moieties within each platform. we expect the coming decades to witness the implementation of many new examples of supramolecular polypeptide therapies. the immunogenic features of peptide assemblies are advantageous for the development of synthetic vaccines and other engineered immunotherapies, a topic which has been recently reviewed by our group and others, [1, 2, 5, 82, 83] but it is also important to note for other assemblies containing high densities of protein or peptide ligands/epitopes that such multivalent displays may induce unwanted immune responses. it remains to be seen whether such responses can be tolerated in specific applications, or if they could even be turned in the favor of the material's clinical performance. interestingly, neither curodont [78] nor purastat [67] contain specific ligands/epitopes, possibly avoiding immunogenicity that may be observed in trials of other nanofibrous materials containing additional protein or peptide functional components. additional future work may focus on investigating the biodistribution and pharmacokinetics of preclinical self-assembling materials. due to the dynamic nature of these materials, it is important to understand how the in vivo environment affects their long-term structural organization, retention, and clearance. in order to be clinically translated, these platforms must also be capable of large-scale production and stable storage. unlike traditional small molecules, these materials must generally be kept in monomeric or otherwise stable states of assembly prior to administration. each of these issues represents important considerations that have not yet been fully worked out for supramolecular materials. with several self-assembling platforms being discovered and developed over the past 50 years, it is worth considering the cross-roads that lay ahead: should we focus on the development of the promising platforms discussed here, or should we continue searching for new, novel platforms? on the one hand, several of the aforementioned platforms have shown encouraging preclinical data and are being investigated in new disease models. on the other, the discovery of a new, more efficacious and versatile platforms could spur unforeseen therapies based on the self-assembling concept. either way, we predict that self-assembling peptide and protein technologies will continue making strides in the preclinical and clinical realms. proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci biomate rials 2013 k.m.h. and c.n.f. contributed equally to this work. research on supramolecular peptide biomaterials in our group has been supported by the national institutes of health under grant numbers 5r01eb009701; 5r01ai118182; 5r21ca196434; and 7r21ar066244). this progress report's contents are solely the responsibility of the authors and do not necessarily represent the official views of these agencies. key: cord-313932-f0a1qh7p authors: chen, peng; zeng, zehua; du, hongwu title: establishment and validation of a drug-target microarray for sars-cov-2 date: 2020-07-21 journal: biochem biophys res commun doi: 10.1016/j.bbrc.2020.05.217 sha: doc_id: 313932 cord_uid: f0a1qh7p covid-19 has become one of the worst epidemic in the world, currently already more than four million people have been infected, which probably co-exist with human beings, and has a significant impact on the global economy and political order. in the process of fighting against the epidemic in china, the clinical value of a variety of herbal medicines has been recognized and written into the clinical application guide. however, their effective molecular mechanism and potential targets are still not clear. pathology and pharmacology research will gradually attract attention in the post-epidemic outbreak term. here, we constructed a covid-19 protein microarray of potential therapy targets, which contains the main drug targets to the sars-cov-2 virus and the anti-virus, anti-inflammatory cellar targets of the host. series of quality controls test has been carried out, which showed that it could be applied for drug target screening of bio-active natural products. the establishment of this microarray will provide a useful tool for the study of the molecular pharmacology of natural products. data from johns hopkins university updated that coronavirus disease-2019 (covid19) has infected more than four million people, which has become a rare pandemic in human history(https://www.jhu.edu/). in the early stage of the outbreak, diagnostic methods and virus traceability are the research focus. with the development of the disease, covid-19 is likely to become a kind of infectious disease coexisting with human beings. vaccine development, drug screening and related pharmacology will gradually become the research hot areas. in china, through thousands of years clinical application, herbal medicine has accumulated valuable experiences in the use of fighting against plague [1] . this includes artemisinin extracted from artemisia for malaria treatment [2] . in the early outbreak stage, the modern drug research and development system has also shown its disadvantages: it is impossible to develop new drugs and complete registration of clinical trials in a short period for a novel virus [3] . herbal medicine (experienced clinical safety evaluation) has the characteristics of compound multi-targets, non-specific adjustment of the human immune system and antiviral effect, which makes it possible for its rapid application [4] . in the practice of anti-covid-19, three chinese patent drugs were proved effective and written into the guideline for the treatment of covid-19 in china. lianhuaqingwen capsule has been shown to significantly inhibit severe acute respiratory syndrome-coronavirus 2 (sars-cov-2) replication [5] [6] [7] [8] . the pharmacological studies of herbal drugs and active natural products often lack compare to modern drugs [9] . the target and mechanism of clinically effective natural products are still largely unknown [10] . for protein target research of traditional chinese medicine, it is usually carried out through network pharmacology and bioinformatics, for example, by obtaining the main active ingredients of lianhuaqingwen capsule, molecular docking method was used to predict the possible targets [11, 12] . however, the researches that were not based on the real world are still not enough to reveal their mechanism of action by molecular pharmacology. there are two sources of proteins used in the study. one source was purchased from the company, including human ace-2 (from hek293), human cathepsin l (ctsl) (from hek293), jinmotang biotechnology (qingdao, china). another protein source were over-expressed in e.coli from the laboratory, the human anneixn a2, hmgb1, hnrnp a2/b1, eif4e2, nfκb1-p50 genes were inserted into the pet-28a vector with his-tag. bacteria were lysed by ultrasound and proteins purified by nickel column. all proteins were under unified quality control, which was carried out by gel electrophoresis. the reasons of target proteins selection are as follows: ace2 and cd147 are the main receptors of the virus [14] ; host cell protease cathepsin l/b is the key element of the lysosomal pathway [15] , and almost all of them are in lysosomes. 3c-like proteinase is main proteases of novel coronavirus [16, 17] . plpro is necessary for the virus to process polyproteins as mature subunits. guanine-n7-methyltransferase, nsp1/2/7/8/10 were involved in rna transcription and replication [18] . nfκb1 and hmgb1 are important lung inflammatory regulators [19, 20] , anneixn a2, hnrnp a2/b1 were proved as transcription regulators associated with pneumonia [21] [22] [23] . the latest research showed eif4e2 as an important host interaction protein with virus, potential to be a drug target for covid-19 [24] . the protein was mixed with sample buffer (capital-bio, beijing, china), then put into 384 well plates (axygen, ca), and made with microarray printing device (capital-bio, beijing, china). polymer 3d substrate (capital-bio, beijing, china) was selected, for each protein by three times of repetition. the proteins in the microarray after printed were quality checked by antibodies. human anneixn a2, hsp60, nfkb1-p50 and his-tag antibodies, goat anti-mouse igg-cy3 the protein structure files were downloaded from the pdb database, and the corresponding protein backbone was extracted, corresponding to pdbid as follows: ace2(6acg), annexin a2(2hyw), cathepsin b (3ai8), cathepsin l (2xu1), cd147 (3qqn), etif4e2 (5bxy), hmgb1 (2rtu), nfkb1-p50 (1u3y), nsp3 (6w6y), nsp12 (6m71), spike protein (6w9c), tnf-a (3alq). three small molecule files were downloaded from the pubchem database, corresponding to the compound cid of polydatin (5281718), andrographolide (5318517) and chlorogenic acid (1794427). the files were downloaded in the format of sdf (3d), and then converted into pdb files by openbabel (a tools to convert sdf format to pdb format) for subsequent analysis first, protein structure files (pdb files) were preprocessed with autodocktools (http://autodock.scripps.edu/), and the steps of pretreatment were "delete water", "add hydrogen", "compute gasteiger charges", "assign ad4 type atoms", and then the files were saved in pdbqt format. then use the adt autodocktools ligand (ver 4.2) function to preprocess ligand, steps of pretreatment which were "detect roots" and "choose torsion", also saves the file as pdbqt format, which will handle the good file with the autodock vina in the same folder, and then write the configuration file (https://github.com/starlitnightly/covid-19_protein/), run autodock vina (http://vina.scripps.edu/) to predict docking results. besides, it has been reported that the docking prediction accuracy of autodock vina is as high as 78 % in the training set [25] . snr (signal-to-noise ratio) of three repeat proteins was calculated based on the ratio of foreground value to the background value of each protein. statistical significance was set at p < 0.05. the proteins of the microarray come from virus and human, including the main functional targets related to the virus life cycle and host functional proteins. the microarray matrix design is shown in figure 1a . the functions they are responsible for are shown in figure 1b . all proteins used in microarrays were processed by gel electrophoresis and quality control. all the proteins were isolated in the gel (figure 2a) . the results of microarray scanning showed that the proteins were well fixed, the location of positive points were consistent with the design, and the repeat points were consistent with each other ( figure 2b ). then specific antibodies were used to identify proteins on the microarray, as shown in figure 2c , the positions of annexin a2 and hsp60 are completely consistent with the matrix design. in addition, these were also confirmed by the his-tag antibodies. all these showed that the protein samples and spotter system of the microarray have high quality. as shown before, the microarray was used to screen protein targets of natural products with biological activity. nfkb1-p50 protein is an important target of andrographolide, a traditional chinese medicine product with immunoregulation function, and their binding site has been widely verified [26] (figure 3a ). this result was repeatedly verified by the biotin-labelled andrographolide for testing this microarray. the results were shown in figure 3b , compared with biotin control, andrographolide and p50 protein have significant positive signals, and the signal-to-noise ratio (snr) after quantification showed a significant statistical difference. chlorogenic acid is the main antiviral and anti-inflammatory active substance in honeysuckle, and it is the main herbal medicine in lianhua qingwen capsule, which showed the clinical therapeutic effect and the ability to inhibit virus replication in vitro [6] . polydatin is the main active component of chinese traditional medicine polygonum cuspidatum, which has broad-spectrum antioxidant and antiviral effects. here, we use the method of network pharmacology molecular docking to predict the binding ability of these two natural products to the protein targets. the results showed that they have potential efficacy in the process of infection and may be used as the target for our further research. many prediction targets of them included in our microarray with potential application value (figure 4 ). it should be noted that this molecular docking only a prediction for helping microarray experiment and does not represent the actual efficacy of these molecules. the basic principle of protein microarray is to fix proteins on solid-phase carriers such as silicon and glass substrates. these proteins will react with biomolecules that can specifically combine with them in the samples to be tested, by which we can choose those proteins critical for our research purpose through analyzing the distinct signal of proteins on the chip obtained by special equipment such as chip scanner [27] . it provides an advanced high-throughput method for modern life science, especially molecular biology research. compared with affinity purification and separation based on mass spectrometry, protein microarray has been widely used in the field of drug target screening. compared with the traditional design of protein chip such as human proteome chip or virus proteome chip, this chip has its characteristic. it is not limited to the virus proteins but also combines the host targets, which expands the scope of drug screening, and many anti-inflammatory and anti-transcriptional drugs can also be applied. this idea may be more suitable to explain the pharmacological action of natural products from herb. the novel coronavirus leads to public health emergencies; some chinese medicines and natural products show certain efficacy. at present, the target selection of traditional chinese medicine is mainly based on bioinformatics network pharmacology. the rapid screening of these drugs can provide scientific and convincing experimental pharmacological evidences for the application and promotion of these drugs. the design and manufacture of this microarray provide a technical tool for the realization of this purpose. because this microarray contains antigens from virus, such as s protein, also annexin a2, hnrnp a2/b1 are classical endothelial cell antigens of host [28, 29] . this chip also has further value in studying the immune response after virus infection. however, there are still some limitations on this microarray. for example, the number of host protein targets is still small, which makes some potential new drug targets maybe ignored. at present, the proteins are not all derived from eukaryotic expression, which may have the problem of protein misfolding. we will continue to improve this microarray and provide more valuable and higher quality targets for researchers. at the same time, we hope to cooperate with other laboratories with this microarray as a starting point to make contributions to the fight against the epidemic. traditional chinese medicine: an effective treatment for 2019 novel coronavirus pneumonia (ncp) identification of 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docking with a new scoring function, efficient optimization, and multithreading andrographolide attenuates inflammation by inhibition of nf-kb activation through covalent modification of reduced cysteine 62 of p50 protein chip technology heterogeneous nuclear ribonucleoprotein a2/b1 as a target antigen in han chinese for bd patients annexin a2, autoimmunity, anxiety, and depression this study first proposed the idea of sars-cov-2 specific protein target microarray 2. the microarray was established and validated by practical test 3. network pharmacology suggest this chip has a broad application in pharmacology this work was supported by hebei provincial department of science and technology (no.19942410g). this study was supported by grants from the fundamental research funds for the central public welfare research institutes (zz2018007). the authors declare that they have no competing interests ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐ the authors declare the following financial interests/personal relationships which may be considered as potential competing interests:please note that all biochemical and biophysical research communications authors are required to report the following potential conflicts of interest with each submission. if applicable to your manuscript, please provide the necessary declaration in the box above.(1) all third-party financial support for the work in the submitted manuscript.(2) all financial relationships with any entities that could be viewed as relevant to the general area of the submitted manuscript.(3) all sources of revenue with relevance to the submitted work who made payments to you, or to your institution on your behalf, in the 36 months prior to submission. (4) any other interactions with the sponsor of outside of the submitted work should also be reported. (5) any relevant patents or copyrights (planned, pending, or issued). (6) any other relationships or affiliations that may be perceived by readers to have influenced, or give the appearance of potentially influencing, what you wrote in the submitted work. as a general guideline, it is usually better to disclose a relationship than not. key: cord-330715-olypwdoq authors: sun, zeyu; ren, keyi; zhang, xing; chen, jinghua; jiang, zhengyi; jiang, jing; ji, feiyang; ouyang, xiaoxi; li, lanjuan title: mass spectrometry analysis of newly emerging coronavirus hcov-19 spike protein and human ace2 reveals camouflaging glycans and unique post-translational modifications date: 2020-08-30 journal: engineering (beijing) doi: 10.1016/j.eng.2020.07.014 sha: doc_id: 330715 cord_uid: olypwdoq the covid-19 pandemic has led to worldwide efforts to understand the biological traits of the newly identified hcov-19 virus. in this mass spectrometry (ms)-based study, we reveal that out of 21 possible glycosites in the hcov-19 s protein, 20 are completely occupied by n-glycans, predominantly of the oligomannose type. all seven glycosylation sites in human angiotensin i converting enzyme 2 (hace2) were found to be completely occupied, mainly by complex n-glycans. however, glycosylation did not directly contribute to the binding affinity between hcov-19 s and hace2. additional post-translational modification (ptm) was identified, including multiple methylated sites in both proteins and multiple sites with hydroxylproline in hace2. refined structural models of hcov-19 s and hace2 were built by adding n-glycan and ptms to recently published cryogenic electron microscopy (cryo-em) structures. the ptm and glycan maps of hcov-19 s and hace2 provide additional structural details for studying the mechanisms underlying host attachment and the immune response of hcov-19, as well as knowledge for developing desperately needed remedies and vaccines. the current coronavirus pandemic, which has been ongoing since december 2019 (covid19) , has become a global health emergency [1] [2] [3] . the disease is caused by a newly identified betacoronavirus, hcov-19 (also known as sars-cov-2), which is closely related to severe acute respiratory syndrome coronavirus (sars-cov). like sars, hcov-19 causes lower respiratory tract infection (lrti), which may eventually progress to atypical pneumonia, requiring intensive care and mechanical ventilation [4] [5] [6] . compared with sars-cov, hcov-19 is more contagious and has infected far more people worldwide, thus causing substantially more casualties [4, 5] . although possible viral reservoirs including bats and pangolins have been suggested as sources of hcov-19 [3, 7] , more investigation is needed to ascertain its source and routes of zoonotic transmission [8] [9] [10] . to date, no vaccine or remedy specific for coronaviruses infection-including that of hcov-19-is available. similar to sars-cov, the virion surface spike glycoprotein encoded by the hcov-19 s gene is essential for target cell attachment and fusion processes [11] [12] [13] . given the importance of the s protein in covid-19 pathogenesis and in its potential immunogenicity for vaccine development, global efforts have been made to elucidate the structure of the s protein, beginning shortly after the first hcov-19 sequence was published [1, 13, 14] . the hcov-19 s glycoprotein is a 1273-amino acid precursor polypeptide; it can be cleaved by host cell proteases (cathepsin l, tmprss2) into the s1 fragment, which contains the receptor binding domain (rbd) to attach host receptor human angiotensin i converting enzyme 2 (hace2), and the s2 fragment, which is responsible for the subsequent membrane fusion [1, 12, 13] . the s protein is predicted to have a cleavable n-terminal signal sequence (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) , which presumably directs the protein toward the endoplasmic reticulum (er) for extensive glycan decoration prior to virion packing. glycosylation is one of the most prominent post-translational modifications (ptms) in many viral spike or envelope proteins, and has been shown to mediate host attachment, immune response, and virion packaging and budding [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] . in particular, the binding of coronavirus s proteins to their respective receptors has been shown to be mediated by viral oligomannose n-glycan [19, 20, 23] . in addition, c-type lectin dc-sign and l-sign can enhance viral entry via their binding to the s protein glycans [25, 26] . therefore, numerous antiviral strategies have been designed to interfere with protein glycosylation or glycan-based interaction [21, 24, 27, 28] . glycosylation is also considered to be a key aspect in the development of an effective vaccine [29, 30] . like the sars-cov spike protein, which contains 23 n-linked glycosylation sequons (n-x-s/t, x≠p), the hcov-19 spike protein is predicted to host 22 sequons per protomer or 66 per trimer [13] . however, the potential glycosite pattern in the s1 of the hcov-19 s protein is different from that of sars-cov, while the glycosylation sites in the s2 region are significantly conserved between hcov-19 and sars-cov. such a difference in the s1 glycosylation pattern may be associated with the fact that the biological and clinical characteristics of hcov-19 are profoundly different from those of other coronaviruses. in this study, we report a comprehensive n-glycosylation profile-as well as other ptms-of the hcov-19 s protein and hace2, elucidated by high-resolution mass spectrometry (ms) analyses. based on these analyses, we reinterpret the current hcov-19 s protein structural model to highlight important glycan features related to covid-19 pathogenesis. nonetheless, we demonstrate that the binding of the hcov-19 s protein and hace2 is independent of their glycosylation status. hcov-19 s gene (virus isolate: wuhan hu-1; genbank number qhd43416.1) was synthesized (genscript) with its codons optimized for insect cell expression. its ectodomain (val16-pro1213) was cloned into a pfastbac vector (life technologies inc., usa) with an n-terminal honeybee melittin signal peptide and c-terminal his6 and flag tags. hcov-19 s protein was expressed in sf9 insect cells using the bac-to-bac system (life technologies inc.) and harvested from the cell culture medium; this was followed by a purification procedure using a ni-nta column and superdex 200 gel filtration column (ge healthcare, uk) in tandem. the extracellular domain of hace2 (gln18-ser740, np_068576.1) with a c-terminal fc tag was expressed in hek 293 cells and was purified by protein a sepharose beads (ge healthcare). to test the glycosylation status of the hcov-19 spike and the hace2 protein, both proteins were deglycosylated by pngase f (neb, 1:50) overnight at 37 °c in phosphate-buffered saline (pbs). both the glycosylated and deglycosylated forms of the hcov-19 spike and the hace2 protein were then analyzed by 15% polyacrylamide gel electrophoresis (sds-page), followed by coomassie blue staining. the proteins were first digested into tryptic peptides according to ref. [31] . in brief, hace2 and the s protein were first reduced by 10 mmol•l −1 dithiothreitol in 50 mmol•l −1 ammonium bicarbonate (abc) for 45 min at room temperature and then alkylated by iodoacetamide for 45 min at room temperature in the dark. the proteins were then cleaned using acetone precipitation and resuspended in 50 mmol•l −1 abc. the alkylated glycoproteins were then digested for 14 h at 37 °c using sequencing-grade trypsin, chymotrypsin, or endoproteinase lys-c (all purchased from promega, usa), with a protein:protease ratio of 1:50 in 50 mmol•l −1 abc. the peptides were desalted using hydrophilic-lipophilic balance (hlb) columns (waters, usa). the peptide samples were separated into duplicates. the first duplicate was deglycosylated by pngase f (neb, 1:100) overnight at 37 °c in 50 mmol•l −1 abc prepared in pure h 2 o 18 . the second duplicate was used to enrich intact n-glycopeptides by hydrophilic interaction liquid chromatography (hilic). in brief, glycoworks (waters) cartridges were preconditioned with loading buffer comprising 15 mmol•l −1 ammonium acetate (ama) and 0.1% trifluoroacetic acid (tfa) in 80% acetonitrile (acn). peptides in the loading buffer were applied to the cartridges and the unbound flow-through fraction was collected. the column was washed two times with loading buffer. glycopeptides were eluted in 0.1% tfa. all peptide fractions were desalted by house-made c18 stage tips before liquid chromatography-tandem mass spectrometry (lc-msms) analyses were performed. about 500 ng of peptide was analyzed using an ultimate 3000 nanoflow liquid chromatography system (thermo scientific, usa) connected to a hybrid q-exactive hfx mass spectrometer (thermo scientific, usa). the mass spectrometer was operated in data-dependent mode with fullscan ms spectrum, followed by ms2 scans recording the top 20 most intense precursors sequentially isolated for fragmentation using high-energy collision dissociation (hcd). the ms and tandem mass spectrometry (ms/ms) spectra were recorded using xcalibur software 2.3 (thermo scientific, usa). detailed parameters for liquid chromatography (lc) separation and ms acquisition can be found in tables s1 and s2 in appendix a, respectively. the acquired ms raw files from the deglycosylated peptides were searched by maxquant (version 1.6.10.43; max-planck-institute of biochemistry, germany) against the human ace2 sequence from uniprotkb and the hcov-19 s sequence (yp_009724390.1_3) from the national center for biotechnology information (ncbi). cysteine carbamidomethylation was set as a fixed modification, while methionine oxidation and o 18 deamidation on glutamic acid were set as variable modifications. trypsin was set as having up to two missed cleavages. a mass tolerance of 15 and 4.5 ppm was set for the first and main search, respectively. for comprehensive ptm analysis, phosphorylation (s/t/y), acetylation (k), methylation (k/r/e), succinylation (k), crotonylation (k), farnesylation (k/n term ), myristoylation (k/n term ), palmitoylation or prenylation, glycosylphosphatidylinositol addition, and oxidation on proline were individually investigated in parallel database searches for respective variable modification. a peptide-level false discovery rate (fdr) of 1% was set to filter the result. confident identification of ptm was based on a localization probability of 99%. site occupancy for each ptm was calculated by dividing the peak intensities of the modified peptides (mp) and corresponding non-modified peptides (np) using the following equation: mp/(mp+np) ×100. to investigate the n-glycosylation forms, the acquired ms raw files from the hilic experiments were searched by pglyco (version 2.2.2, chinese academy of sciences, china) [32] against the same hace2 and hcov-19 s sequences. cysteine carbamidomethylation was set as a fixed modification, while methionine oxidation was set as a variable modification. trypsin was set as having up to two missed cleavages. a mass tolerance of 5 ppm and 15 ppm was set for the precursor and fragment mass tolerance, respectively. potential glycan fragments within the ms2 spectra were annotated by the built-in pglyco.gdb glycan structure database [32] . the precursor intensity of each glycopeptide was extracted by the maxquant feature-detection algorithm. all lc-msms raw data were deposited in an iprox database with the following link: https://www.iprox.org/page/psv023.html;?url=1588554427117yjft; password: b73w. glycan structures in protein databank (pdb) format were downloaded or predicted by glycam-web (https://dev.glycam.org/gp). n-linked glycan models of hace2 and the trimeric spike protein were made by manually adding the ms-identified glycan at each site based on previous models (pdb codes 6m18 for hace2 and 6vxx for spike protein) within coot [33] . the modified residues within each model were generated with coot [33] . in addition, models of the hcov-19 s protein and hace2 complex (pdb 6m0j) were used to show the location of the ptms surrounding the binding area. all structural analyses were performed by pymol (schrödinger inc., usa). binding of the hcov-19 s protein and hace2 was measured by bio-layer interferometry (bli) on an octet re96e (fortebio, usa) interferometry system. in brief, the hcov-19 s protein was immobilized using aminopropylsilane biosensors fortebio) . to evaluate the influence of the glycosylation of hcov-19 s on the binding, the s protein was first incubated with either 1000 u•ml −1 pngase f (neb) or deactivated pngase f at 37 °c for 14 h. after washing by kinetic buffer (1× pbs, ph 7.4, 0.01% bovine serum albumin (bsa), and 0.002% tween 20), the association between hcov-19 s and hace2 was measured for 180 s at 30 °c by exposing sensors to hace2 in kinetic buffer at concentrations of 6.25, 12.5, 25, 50, 100, and 200 nmol•l −1 , respectively. after the binding phase, the sensors were exposed to 1× kinetic buffer for dissociation for 300 s at 30 °c. the signal baseline was subtracted before data fitting using the equimolar binding model. mean association rate constant (k on ) and dissociation rate constant (k off ) were determined with a global fit model using all data. a parallel experiment was also performed using hace2loaded sensors incubated with hcov-19 s protein solutions at different concentrations. analogously, immobilized hace2 was pretreated by either pngase f or deactivated pngase f to evaluate the influence of hace2 glycosylation on binding. in all experiments, pngase f was deactivated by heating at 75 °c for 10 min. as shown in fig. 1(a) , pngase f deglycosylation resulted in a decrease in the molecular weight of both hcov-19 s protein and hace2 on sds-page. digestion by pngase f releases n-linked glycans from asn within the n-x-s/t motif, which also results in asn deamidation by losing one hydrogen and one nitrogen while incorporating one oxygen derived from the solvent [34] . to perform a thorough survey of glycosylated sites, protease-digested peptides from both proteins were subjected to pngase f deglycosylation in h 2 o 18 ; this enables the incorporation of o 18 and leads to a +2.98 da mass increment, which was used to mark the glycosylated sites. (table 1) . two remaining n-x-s/t sites-the n17 and n149 sites-were not identified in any deglycosylated peptide. however, a peptide with glycosylated n149 was identified directly without pngase f treatment, as described later, resulting in a total of 21 glycosylation sites out of 22 potential sites with n-x-s/t sequons in the hcov-19 s protein ( fig. 1(b) ). in addition, quantitative analysis of site occupancy showed that 18 sites were completely glycosylated, while n603 and n657 reached 43% and 74% occupancy, respectively (table 1) . fig. 1 (c) provides an example of mass spectra showing evidence of n331 and n343 glycosylation. quantitative analysis of site occupancy showed that all seven possible glycosylation sites in hace2-namely, n53, n90, n103, n322, n432, n546, and n690-were completely glycosylated (table 1) . fig. 1(d) provides an example of the mass spectra showing n90 glycosylation. these data suggest that both hcov-19 s protein and hace2 are highly decorated by n-glycans. to unveil possible ptms other than glycosylation, lc-msms data of deglycosylated peptides from both hcov-19 s protein and hace2 were subjected to several rounds of spectra-database matching for common ptms. the major ptms found in both proteins were methylation on lysine, arginine, or glutamic acid, as summarized in table 1 . using an occupancy greater than 50% as a criterion to identify sites dominantly decorated with ptms, we found that 78r, 224e, 654e, and 661e in hcov-19 s protein and 57e, 68k, and 329e in hace2 were highly methylated. in addition, the proline at sites 253, 263, 321, and 346 in hace2 was found to be prominently oxidized and converted to hydroxyproline. however, common ptms such as phosphorylation, acetylation, or other forms of fatty acid acylations were not found in hcov-19 s protein and hace2. to resolve glycan camouflage on the surface of the hcov-19 s protein and hace2, intact glycopeptides derived from protease digestion and fractionated by hilic were directly subjected to lc-msms analysis specifically designed to detect peptides with extra molecular weight due to n-glycan attachment. due to the highly heterogeneous nature of the glycan chain, a unique glycopeptide was designated in our study as a combination of both a unique peptide sequence and a specific n-glycan composition. according to these criteria, 419 and 467 unique n-glycopeptides were identified from the hcov-19 s protein and hace2, respectively (tables 2 and 3) . except for n1134, 19 of the 20 hcov-19 s glycosylated sides identified in the pngase f experiment were confirmed by intact glycopeptide profiling. furthermore, n-glycans were also found to present at site n149. although all seven glycosylation sites in hace2 were identified previously in the deglycosylated peptides labeled with deamidation, an n-glycan profile was obtained at five sites: n90, n103, n432, n546, and n690. fig. s1 in the appendix a provides an example of nglycopeptides spectra from hcov-19 s and hace2. a total of 144 n-glycans were found in the hcov-19 s protein, with the majority containing the common n-acetylglucosamine core ( table 2 ). all n-glycosites in the s protein were attached to multiple types of n-glycans, with n343 being decorated by the most diverse n-glycans. the hcov-19 s n-glycan composition was preferentially comprised of pauci-or high-mannose oligosaccharides, except in four n-glycosites (n73, n343, n717, and n1173) that contained a high proportion of complex and hybrid n-glycans (figs. 2(a, b) and table 2 ). based on lc-ms intensity, pauci-mannose hex3hexnac2 was the most common n-glycan in the hcov-19 s protein. interestingly, there was no evidence of a sialic acid component in the n-glycans of the hcov-19 s protein. a total of 220 n-glycans were found in hace2; of these, 78.2% were complex ( fig. 3(a) ), while hybrid, high-mannose, and pauci-mannose glycan only constituted 10.9%, 5.0%, and 5.9%, respectively ( fig. 2 (b) and table 3 ). based on lc-ms intensity, the most dominant hace2 glycan was neugc1hex5hexnac4, which was predicted to be a bi-antennary complex containing a sialic acid in the distal end. the n-glycan profile in all hace2 sites was primarily complex. it is notable that n90, n103, and n690 all contained more than 100 types of n-glycans, with their most dominant forms all containing sialic acid. collectively, our lc-ms/ms data confirm that both the hcov-19 spike protein and its receptor hace2 are indeed heavily n-glycosylated at most of their predicted n-x-s/t sequons. fig. 2 (c) provides a summary of the most dominant n-glycan composition and predicted structure for each site of the hcov-19 spike protein and hace2. by adding the chemical structures of the most abundant n-glycans at each site, based on the lc-ms/ms results, to the most updated cyro-em model of the hcov-19 s protein and hace2, we generated atomic models that represent the most likely spatial distribution of the n-glycans on both proteins (figs. 3(a) and (b) ). although four sites (n74, n149, n1158, and n1194) of the spike protein are not shown in the model due to the missing residues in model 6vxx, our model suggests that the camouflaging n-glycans shield more than two-thirds of the hcov-19 s protein surface, which could potentially lead to host attachment and immune evasion. in addition, both the hcov-19 s protein and the hace2 model showed that the glycans at n331 and n343 of the hcov-19 s protein and n90 of hace2 were in proximity to-albeit not exactly inside of-the binding area of both proteins (figs. 3(c) and (d) ). the model of the s-hace2 complex also showed that three methylation sites in hace2 (57e, 68k, and 329e) form a trident structure enclosing the contact area formed between k353-r357 of hace2 and n501 of hcov-19 s (fig. s2 in appendix a). to understand the contribution of protein glycosylation to the interaction between hcov-19 s and hace2, we compared the binding kinetics and affinity of the purified hace2 ectodomain to glycosylated and deglycosylated hcov-19 s ecd immobilized at the surface of biosensors in a bli experiment. we found that hace2 was bound to hcov-19 s that had been pretreated with active or inactive pngase f with the comparable equilibrium dissociation constants k d of 1.7 nmol•l −1 (fig. 4(a) ) and 1.5 nmol•l −1 (fig. 4(b) ), respectively. the affinity of hace2 binding to hcov-19 s that was determined in this study aligns with the previous report [13] . comparable affinity was also observed when hcov-19 s ecd was bound to the immobilized hace2 (k d = 16.7 nmol•l −1 , fig 4(c) ) or deglycosylated hace2 (k d = 18.2 nmol•l −1 , fig. 4(d) ). therefore, our data suggest that the glycosylation status does not contribute directly to the binding between hcov-19 s and hace2. a detailed summary of the bli binding kinetics can be found in table s3 in appendix a. fig. 4 . impact of glycosylation on binding between the hcov-19 spike protein and hace2. binding of the hcov-19 s protein and hace2 was measured by bio-layer interferometry. biosensors with (a) immobilized intact hcov-19 s protein and (b) its deglycosylated form were exposed to hace2 at six concentration levels. swap experiments were conducted using biosensors with (c) immobilized intact hace2 and (d) its deglycosylated form exposed to hcov-19 s protein at six concentration levels. red lines correspond to a global fit of the data using an equimolar binding model. glycosylation is a ubiquitous and complex ptm that greatly extends the structural and functional diversity of many proteins. however, comprehensive characterization of protein n-glycosylation is technically challenging. in this study, deglycosylated peptides, which are easier to analyze by lc-ms than their glycosylated precursors, were profiled to confirm the n-glycosylated sites in hcov-19 s and hace2. the o 18 deamidation modification ensured confidence in the glycosite identifications, as the isotope was incorporated during pngase f-mediated hydrolysis in h 2 o 18 . intact n-glycopeptide analyses were then performed to further verify the glycosites and to provide detailed information about the compositional and structural heterogeneity of the n-glycans associated with each site. our investigation showed that all sites except n17 were highly glycosylated in the hcov-19 s protein. according to the n-linked glycan model of the spike protein, a large proportion of the protein surface is covered by glycans, which is consistent with previous reports [13, 14, 35, 36] . when the spike proteins from hcov-19 and sars-cov were compared, it was noticeable that the majority of differences in the glycosylation sites occurred in the distal s1 subunit, resulting in a significant difference in the glycan profile in the outermost canopy of the virus formed by spike trimer clusters. this alteration in glycosites might be the result of host or environmental selective pressure, and could imply dramatic differences in viral infectivity, pathogenesis, and host responses. during our research, we noticed that several other research groups also made attempts to decode the glycan profile of the hcov-19 spike protein. all these studies have confirmed that the hcov-19 spike protein is heavily glycosylated [35, 36] . the n-glycans in the s protein are markedly heterogeneous and may greatly extend the conformational flexibility and epitope diversity for the s protein. although a small proportion of complex and hybrid n-glycans were found in the s protein, most sites were occupied by oligomannose glycans, which is in line with previous reports on the glycosylation profile of sars-cov [37] [38] [39] . many viral proteins are hallmarked by a high level of oligomannose glycans, probably as a sign of incomplete glycan maturation due to a high glycosite density that results in steric hindrance [40] . interestingly, n343, the glycosite closet to the binding surface, is decorated by the most diverse n-glycans and primarily by hybrid and complex forms. moreover, as in the case of sars-cov and marburg viral proteins [41, 42] , we found that sialic acid incorporation in the sars-cov spike protein glycans was negligible. in the ongoing global efforts to generate a vaccine, which primarily targets the s protein as the candidate antigen, vaccine developers should consider the diverse glycan forms decorating a large swath of the s trimer surface, as glycans could drastically modulate the protein immunogenicity. it was interesting to learn that different expression systems could affect glycan forms substantially, as we compared our data with other glycan profiling studies [35, 36] . in particular, mammalian cells prefer to assemble complex glycans to the s protein, as compared with insect cells. therefore, we should also expect that vaccines that are produced based on different s proteoforms with different glycosylation statuses could show varying efficacy against future infection. the binding of coronavirus s proteins to their respective receptors has been shown to be mediated by viral oligomannose n-glycan [19, 20, 23] . the sars-cov spike protein contains three rbdassociated glycosites (n318, n330, and n357), while hcov-19 only has two (n331 and n343) surrounding the binding pocket. contrary to our postulation that the n-glycan at these two sites might contribute polar interaction to receptor binding, the bli binding assay demonstrated that deglycosylation did not change the affinity of the sars-cov spike protein to hace2. however, this negative bli binding result does not exclude the possibility that glycosylation could affect other viral entry steps, including protease cleavage and glycan-mediated interactions with dc/l-sign, which were reported to enhance viral entry in sars-cov [25, 26] . further investigations are required to ascertain the role of glycosylation in the infectivity of hcov-19. antigen glycosylation greatly determines host immune responses. one of the prominent consequences of glycosylation is immune evasion by shielding off immunogenic epitopes in viral envelope or surface proteins [17, 43, 44] . the complete occupancy of glycans in most of the glycosites of the hcov-19 s protein suggests that the virus is able to invade the host in a stealthy fashion. successful innate immune evasion by hcov-19 during the early stage of infection might explain the long asymptomatic incubation period during which transmissible virions are produced [4, 6] . cases of hcov-19 reinfection have also been reported, suggesting that the virus is capable of escaping from antibody-mediated neutralization [45] . on the other hand, protein glycosylation can also facilitate immune response by means of sensor molecules or antibodies that are specific for glycan recognition. n-glycans are known ligands for galectins, and can trigger inflammation events such as cytokine release and immune cell infiltration [46, 47] -which may contribute to the hcov-19 pathogenesis and correlate with the disease severity. moreover, epidemiological studies have shown that abo polymorphism is linked to different susceptibilities to both hcov-19 and sars-cov infections [48, 49] . since the abh carbohydrate epitopes and viral protein glycans are likely to be synthesized by the same er-resident glycosylation enzymes, anti-a or -b antibodies can partially block the viral-host interactions [50] ; thus, individuals with blood type o would be more resistant to virus infection. it remains to be explored whether the glycosylation status of the s protein has implications toward inter-individual variation in terms of viral response and clinical outcome within the infected population. the glycosylation status of hace2 was also profiled in this study. all seven glycosites in hace2 were completely occupied by glycan, including n90, which is in the vicinity of the binding surface. however, we found that glycans did not directly contribute to the binding of hace2 with the hcov-19 s protein. this finding is in agreement with a previous finding that disruption of ace2 glycosylation did not affect its binding with the sars-cov spike protein [51] . however, deglycosylated ace2 did compromise the cellular entry and the subsequent production of infectious sars-cov virion [51] . therefore, ace2 glycosylation is still considered to be an important target for intervention of coronavirus infection. in fact, the ability of chloroquine to counter hcov-19 infection is thought to be due to its inhibition of ace2 glycosylation, in addition to its ability to increase the endosomal ph level [28, 52] . in addition to glycosylation, additional ptm forms were investigated in this study. methylation was identified in several sites in both the s protein and hace2. in particular, we found that the 57e, 68k, and 329e sites in hace2, which surround its binding site with the rbd of the s protein, are completed methylated. methylation causes a loss of charge and increases the hydrophobicity of these sites. four hydroxyprolines (at 253, 263, 321, and 346) were identified in the protease domain of hace2. the extra hydroxyl group is likely to increase the hydrophilicity of proline in this extracellular region of hace2. further study is needed to investigate possible biological roles of these ptms. we did not find phosphorylation or acetylation in our dataset, and previous ptm investigation of sars-cov also failed to identify these ptms [38] . fatty acid acylations were also considered in our study, since they are commonly found in surface viral proteins to facilitate membrane fusion and virus entry. however, our study found no evidence of an acylation ptm presence in the hcov-19 s protein. given that our multi-protease proteomic experiment provides almost full coverage for both proteins, we believe that the ptmome of the hcov-19 s protein and hace2 mainly consists of glycosylation, methylation, and proline oxidation. a new coronavirus associated with human respiratory disease in china an interactive web-based 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pathobiology characterization of filoviruses based on differences in structure and antigenicity of the virion glycoprotein identification of n-linked carbohydrates from severe acute respiratory syndrome (sars) spike glycoprotein cryo-em analysis of a feline coronavirus spike protein reveals a unique structure and camouflaging glycans virus glycosylation: role in virulence and immune interactions emergence of novel coronavirus and covid-19: whether to stay or die out? the sweet-side of leukocytes: galectins as master regulators of neutrophil function the role of galectins in virus infection-a systemic literature review abo blood group and susceptibility to severe acute respiratory syndrome relationship between the abo blood group and the covid-19 inhibition of the interaction between the sars-cov spike protein and its cellular receptor by anti-histo-blood group antibodies inhibition of endoplasmic reticulum-resident hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro this work was supported by the national key research and development program (2017yfc1200204, 2017yfa0504803, and 2018yfa0507700), emergency project of zhejiang provincial department of science and technology (2020c03123-1), fundamental research funds for the central universities (2018xzzx001-13) and independent project fund of the state key laboratory for diagnosis and treatment of infectious disease. we thank the proteomics and metabolomics platform in the state key laboratory for diagnosis and treatment of infectious diseases at zhejiang university for glycoproteomic analysis. we thank shuangtian shengwu biotech. co. ltd. for assistance of bil analysis. we thank pglyco team at institute of computing technology (chinese academy of sciences, beijing, china) for technical support for pglyco analysis. we thank dr. ma ping for consultation on protein expression and purification. the authors disclose no competing interests. supplementary data to this article can be found online. key: cord-336364-2ust3qoq authors: artigas, laura; coma, mireia; matos-filipe, pedro; aguirre-plans, joaquim; farrés, judith; valls, raquel; fernandez-fuentes, narcis; de la haba-rodriguez, juan; olvera, alex; barbera, jose; morales, rafael; oliva, baldo; mas, jose manuel title: in-silico drug repurposing study predicts the combination of pirfenidone and melatonin as a promising candidate therapy to reduce sars-cov-2 infection progression and respiratory distress caused by cytokine storm date: 2020-10-02 journal: plos one doi: 10.1371/journal.pone.0240149 sha: doc_id: 336364 cord_uid: 2ust3qoq from january 2020, covid-19 is spreading around the world producing serious respiratory symptoms in infected patients that in some cases can be complicated by the severe acute respiratory syndrome, sepsis and septic shock, multiorgan failure, including acute kidney injury and cardiac injury. cost and time efficient approaches to reduce the burthen of the disease are needed. to find potential covid-19 treatments among the whole arsenal of existing drugs, we combined system biology and artificial intelligence-based approaches. the drug combination of pirfenidone and melatonin has been identified as a candidate treatment that may contribute to reduce the virus infection. starting from different drug targets the effect of the drugs converges on human proteins with a known role in sars-cov-2 infection cycle. simultaneously, guildify v2.0 web server has been used as an alternative method to corroborate the effect of pirfenidone and melatonin against the infection of sars-cov-2. we have also predicted a potential therapeutic effect of the drug combination over the respiratory associated pathology, thus tackling at the same time two important issues in covid-19. these evidences, together with the fact that from a medical point of view both drugs are considered safe and can be combined with the current standard of care treatments for covid-19 makes this combination very attractive for treating patients at stage ii, non-severe symptomatic patients with the presence of virus and those patients who are at risk of developing severe pulmonary complications. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the identification of host key points of intervention has been done through manual curation of scientific publications and gene expression analysis of data included in the gene expression omnibus (geo) database [20] . this has provided 3 sets of proteins related with the infection process: 1) coronavirus-host interaction set (including sars-cov-2 entry points), 2) lungcells infection set, and 3) acute respiratory distress (ard) set. the 'coronavirus-host interaction set' is composed of human proteins with a relevant role in sars-cov-2 infection and a set of human coronaviruses host interactome retrieved through manual curation of scientific publications (s1 table) . the 'lung-cells infection set' has been populated through differential expression of the gse147507 [21] gene expression dataset using edger [22] package (p-value <0,05 and log2fc>1 or <-1). the 'ard set' has been defined using microarray data sets included in geo that define the role of different important cell types in ard pathophysiology: gse89953 (alveolar macrophages and monocytes ard) [23], gse76293 (neutrophils in ard) [24] and gse18712 (lung cells at different ard stages) [25] . the first 2 sets have been analysed with the geo2r tool [20] , and the last one with a statistical analysis method based on significance analysis of microarrays (sam) [26, 27] to determine the differentially expressed proteins. additionally, ard cytokine storm has been characterized by manual curation of scientific literature (s1 table) . the tpms technology employed has been previously described [12] and applied in different clinical areas with different objectives [8, 9, [13] [14] [15] [16] [17] [18] . tpms uses a human biological network that incorporates the available relationships (edges or links) between proteins (nodes) from a regularly updated in-house database drawn from public sources: kegg [28], reactome [29] , intact [30] , biogrid [31] , hprd [32] , and trrust [33] . drug targets and indications were obtained from drugbank [34] . the molecular description of the indications was obtained from a hand-curated collection of associations between biological processes and molecular effectors (defined as bed, biological effectors database, from anaxomics biotech). the method uses an artificial neural network (ann) to measure the potential relationship between the nodes of a network (i.e. proteins), grouped according to their association with a phenotype. the ann algorithm provides a score (from 0 to 100%). each score is associated with a probability (p-value) that the protein or group of proteins being evaluated, drug targets with molecular description of pathological processes, are functionally associated. scores greater than 91% indicate a very strong relationship with a p-value below 0.01; scores between 76-91% have p-value between 0.01-0.05; scores between 40-76% have medium-strong relationship and p-value in the range 0.05-0.25; and a scores lower than 40% have p-values above 0.25. the mechanism of action (moa) of pirfenidone and melatonin has been simulated by using the tpms technology. on the basis of the human protein functional network described above, mathematical models have been built. the models simulate the activation/inhibition status in the human protein network. as input, the model takes the activation (+1) or inactivation (-1) of the drug target proteins (s2 table) , and as output the protein states of the pathology of interest (s1 table) . it then optimizes the paths between the two protein sets and computes the activation and inactivation values of the full human interactome. the resulting subnetwork of proteins with positive and negative values defines the moa of the drug. sampling methods are used to generate mathematical models with stratified ensembles that comply with a set of validated benchmarks [12] . guildify v2.0 web server to calculate the effect of the covid-19 sets of proteins and repurposed drugs to the network guildify v2.0 [19] web server is used to extend the information of sets of proteins through the human biological network. guildify scores proteins according to their proximity with the genes associated with a drug or phenotype (seeds). in this study, guildify has been employed to calculate the neighbourhoods of the human biological network that are associated with the host-key points (for sars-cov infection) and at the same time affected by specific drugs. in this way, the mechanism of action of the treatments proposed by tpms can be corroborated using a completely different setting. guildify v2.0 has been run for each drug, using their targets as seeds (by introducing the name of the drug in the search box). the same has been done for each set of proteins: the coronavirus-host interaction set, the entry points set (a subset of the coronavirus-host interaction set containing only 5 host proteins relevant in the infection of sars-cov-2), the lung cells infection set and the ard set (by uploading a file with the gene symbols of the proteins). we included the entry points subset in the analysis to have a more specific neighborhood of the sars-cov-2 infection mechanism. there are two additional parameters to be chosen by the user in guildify web server: the human biological network and the scoring function. the human biological network used has been the protein-protein interactions network of i2d [35], as it is the more complete network in the web server (with 13,568 proteins and 224,675 interactions). the scoring function selected (used to score the proteins of the network according to the proximity with the seeds) has been netcombo [36], an algorithm that combines the performance of three network-based prioritization algorithms: netshort (considers the number of edges connected to phenotype-associated nodes), netzcore (iteratively assesses the relevance of a node for a given phenotype by averaging the normalized scores of the neighbors) and net-score (considers the multiple shortest paths from the source of information to the target). the top 1% scored proteins of each test have been selected to proceed with the analysis of each subnetwork. the overlap between the selected subnetworks has been used to calculate the number of common proteins between the top-scoring proteins associated with the drugs and the topscoring proteins associated to the host-key points (results detailed in s3 table) . human key molecular enclaves in covid-19 that could be good target areas for treatment have been identified through manual curation of scientific publications and gene expression analysis, describing molecularly aspects of the virus infection process from the host perspective and aspects related with the respiratory problems associated to the infection. we have defined three different main protein sets: 1) coronavirus-host interaction set, 2) lung cells infection set and 3) ard set. 1) 'coronavirus-host interaction set' is composed of human proteins with a relevant role in sars-cov-2 infection process. this set of proteins has been obtained from a bibliographic revision and it is composed of proteins that potentially interact with sarscov . in addition, a list of proteins reported to have a direct interaction with coronaviruses, retrieved from zhou et al. [6] has been included. in this study, the authors define a human coronavirus (hcov)-host interactome network including 119 host proteins with the aim of identifying antiviral drugs through repurposing strategies. it integrates known human direct targets of hcov proteins or that are involved in crucial pathways of hcov infection using the available information from four known hcovs (sars-cov, mers-cov, hcov-229e, and hcov-nl63), one mouse cov (mhv), and one avian cov (ibv, n protein). the final set contains 122 human proteins, the full list is provided in s1 table. 2) 'lung cells infection set' describes the transcriptional response of human lung epithelial cells to sars-cov-2 infection. it has been defined through differential expression analysis of human lung epithelial cells to sars-cov-2 infection, geo series reference gse147507. this set includes 47 proteins, the full list is provided in s1 table. 3) 'ard set' is composed of 412 unique protein entries defined by 6 subsets. it includes gene differential expression data on alveolar macrophages, monocytes and neutrophils in ard (gse89953 and gse76293), gene differential expression data on lung changes induced in the intermediate and late stages of the pathophysiology (gse18712) and the key players in ard cytokine storm extracted from literature research. all ard set data can be retrieved from s1 table. we have centred the drug repositioning analysis in the coronavirus-host interaction set. but the potential effects of the drug combination selected have been evaluated for the 3 data sets defined. an overall depiction of the whole procedure is depicted in fig 1. tpms-ann approach has been used to identify drugs that can be repurposed for set 1. the potential moa for these repurposed drugs has been identified by guiildify and tpms approaches, as well as moas of this repurposed drug candidates for sets 2 and 3. a systems biology based strategy has been used to screen the predicted relationship between 6,605 different drugs, as defined in drugbank [34] , and the coronavirus-host interaction set previously defined. drug candidates for repurposing were sorted and selected by the probability of its relationship with the coronavirus-host interaction set, 12 approved drugs scored higher than 74% (p-value <0.05). all candidates were grouped in two drug categories: (1) anti-inflammatory and immunosuppressant agents (ibuprofen, phosphatidyl serine, prasterone, vitamin c, pirfenidone, tacrolimus and pimecrolimus); (2) cardiovascular agents (isosorbide, icatibant, moexipril, irbesartan, phenindione). among them, 4 out of 12, are currently being tested in covid-19 clinical trials, these are: vitamin c (nct04264533, nct04323514, nct04323514), pirfenidone (nct04282902), tacrolimus (nct04341038) and irbesartan (nct04330300). two others appear described as potential treatments by third authors, moexipril [41] and icatibant [42], or somehow related to the disease outcome, such as ibuprofen [43] . among these candidates, pirfenidone (with score 75%) was selected because of its reported association with furin [39] and a good safety profile. the s protein of sars-cov-2 has a furinlike domain for cleavage and some recent works have addressed the potential implication of this domain in the s protein maturation and, as a consequence, in the host cell entry mechanism of the virus. furin, a member of the subtilisin-like proprotein convertase family processes proteins of the secretory pathway, is a type 1 membrane-bound protease that is expressed in multiple organs, including the lungs [44]. sars-cov-2, differently from sars-cov, presents a furin cleavage site between the s1/s2 subunits of the s glycoprotein, which is cleaved during the biogenesis of the virus to create a mature s protein [45] . inhibiting the expression of furin could then be a possible approach to diminish sars-cov-2 infectivity by making difficult s protein maturation [46] . in recent years, furin has emerged as a promising target for therapeutic intervention in a variety of infectious diseases as influenza a virus [47] or newcastle disease virus [48] . given this evidence, pirfenidone stands out as one of the most interesting repurposing candidates to treat covid-19. taking into consideration the drug repurposing analysis and the supporting evidence we have selected pirfenidone for further studies. combining different drugs can be an advantageous option as we can affect a larger number of key points of intervention and/or increase the effects against disease pathophysiology. we have applied ann of tpms technology to screen drug combinations composed by pirfenidone and other approved drugs listed in drugbank database. among all, 214 non-synonymous repurposed drug candidates have shown a predicted relationship value � 90%. experimental drugs, withdrawn drugs and those with poor safety profile or with opposite mechanistic effect than the desired one were further filtered out. lastly, we selected the combination of pirfenidone (predicted value 92% p>0.05) with melatonin as it fulfilled all criteria. furthermore, there is also mounting evidence that melatonin could be a good candidate to treat covid-19, due to its anti-inflammatory and anti-oxidative properties protecting against ards caused by other pathogens including viruses [6, [49] [50] [51] . to further characterize at a molecular level the action of the selected drug combination on the coronavirus-host interaction set, we have used the modelling strategy based in sampling methods of tpms. the models predict the most probable molecular routes that transmit the effect from the drugs' molecular targets to proteins in coronavirus-host interaction set. the main protein interactors identified are depicted in fig 2. pirfenidone inhibits furin, a protein effector included in the virus-host network, with a potential implication for the entry of the virus in the host cell. the inhibition of furin also results in the inhibition of transforming growth factor-beta1 (tgf-β1) [52] a gatekeeper of the immune response and one of the key proteins in set 1, as it is upregulated by the papain-like protease (plpro) from severe acute respiratory syndrome (sars) coronavirus (sars-cov) [53] . the inhibition of furin (a tgf-β1 converting enzyme) by pirfenidone reduces its expression [52, 54] . on the other hand, melatonin, through the suppression of estrogen receptor alpha [55] regulates the expression of ubc9 [56] , another key protein of set 1 that interacts with sars-cov n protein [57] . it has also been described that melatonin can lead to the activation of stat3 in some cell types [58, 59] , and it could be useful to counteract the stat3 dephosphorylation induced by sars-cov [60] . in the previous section, the drug combination pirfenidone and melatonin was selected as a potential treatment against the infection of sars-cov-2. the selection is fundamentally based on the mechanism of action of the combination, targeting the entry mechanism of the virus and specially furin, a protein that may have a key role in the infection process [39] . to corroborate the effect of pirfenidone and melatonin to the proteins that are relevant for the infection, we have used an alternative approach also based on the use of networks. we used guildify v2.0 [19] , a web server that extends the information of sets of proteins through the human biological network. guildify scores proteins according to their proximity with the genes associated with a drug or phenotype (seeds). using this web server, we can identify a list of topscoring proteins that are critical on transmitting the perturbation of certain proteins through the network (also known as network propagation). thus, applied to this case, we: (i) identify the proteins of the human biological network that are key on transmitting the therapeutic effect of pirfenidone and melatonin, (ii) identify the proteins of the network perturbed by the infection of the sars-cov-2, and (iii) calculate the network overlaps between the treatment and the infection, reflecting the potential effect of the treatment over the infection. the network used by guildify, obtained from i2d [35], is completely independent from the network used in the tpms, becoming an ideal, independent context to test the results of tpms. we have observed a significant overlap of 32 proteins between the top-scoring proteins achieved with the targets of pirfenidone (pirfenidone-network) and the top-scoring proteins of the sars-cov-2 infection cycle set (a subset of the coronavirus-interaction set specific for sars-cov-2, conformed by tmprss2, ace2, grp78, furin and cd147). additionally, there are 10 common proteins between the top-scoring proteins achieved with the targets of melatonin (melatonin-network) and the top-scoring proteins of the 5 entry point proteins. there are also 7 common proteins between the top-scoring proteins of pirfenidone and the top-scoring proteins of ard set. however, the rest of the sets (coronavirus-host interaction set and lung infection set) do not have a significant overlap with any of the two drugs (results detailed in s3 table) . the highly significant overlap between pirfenidone and the top-scoring proteins of the 5 entry point proteins of sars-cov-2 is however biased, because furin is in this set and it's also a target of pirfenidone (fig 3) . the therapeutic effect of pirfenidone on furin has a clear source effect on the neighbourhood of proteins that surround the entry points of sars-cov-2. melatonin has also a significant overlap with the entry points of sars-cov-2. this effect is explained because it targets the proteins mtnr1a and mtnr1b (melatonin receptors). melatonin affects sars-cov-2 grp-78 entry point (also called hspa5) because mtnr1a and mtnr1b are direct interactors of grp-78 (fig 3) . melatonin also affects sars-cov-2 furin entry point because mtnr1b interacts with the membrane protein itm2c, which directly interacts with furin. by identifying the interacting partners of mtnr1a and mtnr1b, we can understand better the potential mechanism of action of melatonin towards the entry of the virus. apparently, both pirfenidone and melatonin target close subnetworks but in different areas. this could explain the interesting additive effect predicted by tpms. as mentioned above, the overlap between pirfenidone-network and the sars-cov-2 entry points is biased by the fact that both sets of proteins contain furin. therefore, we have repeated the same analysis but removing furin from the set of sars-cov-2 entry points. here, we have observed that the overlap with pirfenidone is not significant, with only 4 common proteins, which proves that pirfenidone targets the sars-cov-2 network specifically by targeting furin. in contrast, the melatonin-network has a significant overlap of 7 proteins, affecting mainly neighbours of grp-78. guildify also reflects the effect of pirfenidone over 7 proteins related with ard, leaded by other targets of the drug: tnf-alpha and mapk13. according to the findings by guildify, we confirm the effect of the combination of pirfenidone and melatonin in the entry points of the sars-cov-2 infection, specifically the neighbours of furin and grp-78, and some proteins associated with ard. to further evaluate the impact of pirfenidone and melatonin as a novel therapy for covid-19, the relationship of the drug combination against the associated respiratory problems has been evaluated using various systems biology approaches. in a first approach, the relationship between the protein drug targets and the 2 sets of proteins defining respiratory problems associated with covid-19 (sets 2 and 3) has been evaluated using the tpms ann modelling approach. this type of measurement provides a probability of a relationship between groups of proteins. a weak relationship has been obtained for the combination with lung cells infection set while we obtained a strong relationship between the drug combination and ard set. specifically, with the subsets related to alveolar macrophages, monocytes, late phase ard and ard cytokine storm. a second approach using the sampling methods-based models of tpms has been used to evaluate at the molecular level the moa of pirfenidone and melatonin individually and in combination for treating ard set, evaluating each subset individually. this modelling approach identifies a probable molecular pathway between the protein drug targets and the ard subsets. the ability of each treatment to reverse the protein alterations occurring in these pathological mechanisms has been assessed, i.e. the ability of each drug to activate proteins that are inhibited in ard subnetworks or vice versa (according to the molecular characterization). we have calculated the percentage of proteins theoretically affected respect to the total number defining the subset (fig 4) . table. the main effect, for both drugs, is over the cytokine storm subset. pirfenidone and melatonin are able to affect 86% of the proteins defining the set individually. the drug combination increases the % of proteins affected to a 91%, providing a potential synergy in this area. the protein-network overlap indicates that most of the proteins (82%) in ard cytokine storm subset are modulated by both drugs. while the proteins modulated by one drug alone are 5% in both cases. the second subset with largest % of affected proteins, 27% for the drug combination, is alveolar macrophages set. the main effect is registered for melatonin, pirfenidone contribution is negligible for this subset. we measure also a minor modulation for the rest of ard subsets, mostly contributed by melatonin with pirfenidone slightly potentiating the effect in some of them. the anti-inflammatory role of both melatonin and pirfenidone has been described in the literature. they are able to attenuate nlrp3 inflammasome and il-1β [61] [62] [63] , being a possible starting point for the cytokine storm. studies have shown the potential of melatonin in decreasing the levels of inflammatory cytokines by reducing the activation of nf-κb, thus becoming an interesting candidate to treat different types of viral infections [49, 64, 65] . other studies have also shown the anti-inflammatory role of pirfenidone, modulating inflammatory cytokines [66, 67] . the anti-inflammatory effect of both treatments could potentially be useful to reduce the effects caused by the cytokine storm. pirfenidone has also been described as antifibrotic, reducing the rate of lung damage by 50% in patients with idiopathic pulmonary fibrosis [68] . antifibrotic therapy has been highlighted as a potential strategy to treat covid-19 patients and prevent fibrosis after sars-cov-2 infection [69] . in addition, both pirfenidone [70, 71] and melatonin [72, 73] have been reported to provide antioxidant effect, which has an important role in ard pathophysiology. multiple studies have reported reduced levels of antioxidant agents in ard patients [74, 75] , and remark the positive effects of therapies that include antioxidants in the treatment of ard [76, 77] . therefore, the antioxidant effect of both drugs could be one of the factors that explains the positive effect in ard predicted by tpms. the proposed drug combination has also a relevant effect modulating the expression of proteins associated to the phenotype of alveolar macrophages in ard. it has been suggested that these cells are key players in the pathophysiology of ard. they seem to have a proinflammatory role in early stage and anti-inflammatory effect at the late stage [78] . the combined mechanism of action (moa) of pirfenidone and melatonin in ard have been identified at molecular level through the use of sampling methods-based models [12] . tpms sampling-based methods trace the most probable paths, both in biological and mathematical terms, which lead from a stimulus (e.g. drug) to a response (e.g. disease) through the biological human protein network. in other words, tpms identifies the set of possible moas that achieve a physiological response when the system is stimulated with a specific stimulus. there is a certain variability in these moas, generating several possible pathways that represent patient heterogeneity. however, when identifying a graphical representation of the moa, only the mean and most represented paths among the set of possible solutions is represented. the moa of the combination of pirfenidone and melatonin in ard has been studied. as shown in fig 5, and as previously predicted through other strategies, the combination of both drugs counteracts the cytokine storm, which is responsible of the disease severity. melatonin and pirfenidone may reduce the levels of proinflammatory chemokines and cytokines that have been detected at high levels in patients with covid-19 [79] and which are well known for their role in the cytokine storm and contribution to ard: il-1b, il-6, cxcl10, il-8, ccl5 and tnf-alpha [79] [80] [81] [82] . these molecules are involved in both, early and late phases of cytokine storm. these effects have already been previously described in the literature for both drugs individually [49, 66, [83] [84] [85] [86] reinforcing the potential use of the drug combination to improve patient outcomes. this drug combination could modulate tgf-β1 and vegfa involved in processes associated to lung fibrosis, a late complication of ard. tgf-β1 is a profibrotic mediator known to be associated with the fibroproliferative response responsible of this disease complication [87] . pirfenidone inhibits furin, one of the proprotein convertases that mediate the maturation/activation of tgf-β1 [88] . on the other hand, it has been described that melatonin supress tgfβ1 expression through the inhibition of erk phosphorylation [89, 90] supressing pulmonary fibrotic response. one key component of the infrastructure in the lung that is damaged in ard is the vasculature. vegfa is involved in the excessive angiogenic response that may contribute to the initial tissue injury and drive the fibroproliferative response [87] . according to our model, melatonin could be downregulating vegfa expression through the inhibition of oestrogen receptor alpha (er1). it has been described that melatonin inhibits er transactivation [55] and that vegf expression in some cell types is regulated by 17β-oestradiol in a er dependent process [91] . there are some experimental evidences suggesting the inhibitory role of melatonin on vegf activity reinforcing the hypothesis of our model, although the pathway involved on this effect has not been described [92] [93] [94] . in summary, we predict that this drug combination could modulate the vast majority of effectors involved in the cytokine storm. furthermore, as detailed in fig 4, the drugs also act on other pathophysiological subnetworks of ard. it has also been described in the literature the anti-oxidant effect of both, pirfenidone and melatonin [70, 72, 95] . as reactive oxygen species (ros) play a central role in inflammatory responses and viral replication [96] , such antioxidant effect could also be beneficial for improving the pathophysiology of ard patients. the current situation with covid-19 prompts for easy and fast ways to control and reduce the burthen of the disease. the systems biology-based drug repurposing screening method has proved to be a good option, identifying most of the treatments already in clinical evaluation and highlighting others that may have not got sufficient attention on their potential. our analysis has highlighted pirfenidone as one of the best candidates for treating the sars-cov-2 host cell entry and melatonin as combination theraphy to increase the measured effect. the identification of the potential moa for this drug combination against the coronavirushost interaction set indicates that the principal effect is registered by the inhibition of furin by pirfenidone and the suppression of estrogen receptor alpha by melatonin. the effect of the two drugs in the key proteins surrounding the sars-cov-2 entry mechanism, furin and grp-78, has been corroborated with the web server guildify v2.0. we have also measured a potential effect of the drug combination over ard set. screening in a deeper detail the potential moa we have measured the strongest modulation over cytokine storm subset of ard with pirfenidone and melatonin contributing in a similar manner. the other ard subsets are not so strongly modulated and most of the effect comes from melatonin. several authors have suggested both compounds individually to treat covid-19 or in combination with other compounds. pirfenidone is already in clinical trials (nct04282902) to treat covid-19 associated lung injury. the fact that our study has identified also an effect on sars-cov-2 infection cycle and thus possibly contributing to reduce the viral load makes this drug combination very attractive in the treatment of covid-19. from a medical point of view both drugs are considered safe and can be combined with the current standard of care treatments for covid-19. the pharmacokinetics of the combination therapy can be altered respect to the individual drugs due to their competition (substratesubstrate) over cyp1a2. this substrate competition can lead to the drugs remaining longer in the organism than in monotherapy, potentially increasing the known pharmacologic effects of the drugs that in turn could lead to dose-dependent adverse drug reactions. the design of any clinical trial to test this combination therapy has to take into consideration this possibility and compensate by reducing the dosage of the drugs compared to their regular use in monotherapy. this drug combination is foreseen to tackle two important issues in covid-19, the viral load and the pulmonary associated complications making it very appropriate for treating patients' at stage ii [97] , this are non-severe symptomatic patients with the presence of virus and at risk of evolving to severe pulmonary complications. a clinical study to evaluate this drug combination is foreseen. supporting information s1 table. characterization of the different sets of proteins associated to covid-19. 1) coronavirus-host interaction set (including sars-cov-2 entry points), 2) lung-cells infection set, and 3) acute respiratory distress (ard) set that is composed of 6 subsets (alveolar macrophages, monocytes, neutrophils, intermediate phase ard, late phase ard and ard cytokine storm). proteins are listed by their uniprotkb identifier and under effect the type of action of the protein is described: 1 (being the protein more active contributes to the pathological effect of the motive) 0 (being the protein less active contributes to the pathological effect of the motive). coronavirus incubation could be as long as 27 days, chinese provincial government says centers for disease control and prevention (cdc). symptoms of coronavirus disease 2019 centers for disease control and prevention (cdc) clinical trials on drug repositioning for covid-19 treatment hopes rise for coronavirus drug remdesivir a sars-cov-2 protein interaction map reveals targets for drug repurposing network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 the imex coronavirus interactome: an evolving map of coronaviridae-host molecular interactions novel neuroprotective multicomponent therapy for amyotrophic lateral sclerosis designed by networked systems neuroprotective drug for nerve trauma revealed using artificial intelligence repurposed analogue of glp-1 ameliorates hyperglycaemia in type 1 diabetic mice through pancreatic cell reprogramming proximal pathway enrichment analysis for targeting comorbid diseases via network endopharmacology in-silico simulated prototypepatients using tpms technology to study a potential adverse effect of sacubitril and valsartan unveiling the role of network and systems biology in drug discovery proteome-wide alterations on adipose tissue from obese patients as age-, diabetes-and gender-specific hallmarks sardó n t. systems biology applied to non-alcoholic fatty liver disease (nafld): treatment selection based on the mechanism of action of nutraceuticals mechanisms of action of sacubitril/valsartan on cardiac remodeling: a systems biology approach network medicine framework for identifying drug repurposing opportunities for covid-19 melatonin as a potential compound against sars-cov-2 evidence that furin is an authentic transforming growth factor-β1-converting enzyme sars coronavirus papain-like protease induces egr-1-dependent up-regulation of tgf-β1 via ros/p38 mapk/stat3 pathway processing of transforming growth factor beta 1 precursor by human furin convertase melatonin inhibits estrogen receptor transactivation and camp levels in breast cancer cells estrogen receptor alpha and nuclear factor y coordinately regulate the transcription of the sumo-conjugating ubc9 gene in mcf-7 breast cancer cells sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus by interaction with ubc9 interleukin-6 autocrine signaling mediates melatonin mt(1/2) receptor-induced stat3 tyr(705) phosphorylation melatonin protects against ischemic stroke by modulating microglia/macrophage polarization toward anti-inflammatory phenotype through stat3 pathway the complex role of stat3 in viral infections melatonin attenuates airway inflammation via sirt1 dependent inhibition of nlrp3 inflammasome and il-1β in rats with copd pirfenidone ameliorates lipopolysaccharide-induced pulmonary inflammation and fibrosis by blocking nlrp3 inflammasome activation an open-label study with pirfenidone on chronic hypersensitivity pneumonitis melatonin possesses an anti-influenza potential through its immune modulatory effect melatonin: its possible role in the management of viral infections-a brief review antifibrotic activities of pirfenidone in animal models anti-inflammatory effect of pirfenidone in the bleomycin-hamster model of lung inflammation a phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis pulmonary fibrosis and covid-19: the potential role for antifibrotic therapy antioxidant activity mediates pirfenidone antifibrotic effects in human pulmonary vascular smooth muscle cells exposed to sera of idiopathic pulmonary fibrosis patients potent antioxidant role of pirfenidone in experimental cirrhosis melatonin as an antioxidant: biochemical mechanisms and pathophysiological implications in humans melatonin as an antioxidant: under promises but over delivers antioxidant status in patients with acute respiratory distress syndrome alveolar antioxidant status in patients with acute respiratory distress syndrome effect of enteral feeding with eicosapentaenoic acid, γ-linolenic acid, and antioxidants in patients with acute respiratory distress syndrome enteral nutrition with eicosapentaenoic acid, γ-linolenic acid, and antioxidants reduces alveolar inflammatory mediators and protein influx in patients with acute respiratory distress syndrome the role of macrophages in the pathogenesis of ali/ards. mediators inflamm cytokine storm in covid-19 and treatment the immunobiology of sars into the eye of the cytokine storm pathogenesis of covid-19 from a cell biologic perspective. the european respiratory journal. nlm (medline) proprotein convertase furin is preferentially expressed in t helper 1 cells and regulates interferon gamma furin-mediated protein processing in infectious diseases and cancer furin inhibition reduces vascular remodeling and atherosclerotic lesion progression in mice effect of pirfenidone (pfd) on cytokine/chemokine release from alveolar macrophages (ams) in interstitial lung diseases (ild): preliminary results the fibroproliferative response in acute respiratory distress syndrome: mechanisms and clinical significance regulation of tgfβ and related signals by precursor processing. seminars in cell and developmental biology beneficial effects of melatonin on nicotine-induced vasculopathy melatonin suppresses fibrotic responses induced by cigarette smoke via downregulation of tgf-β1 regulation of vascular endothelial growth factor (vegf) gene transcription by estrogen receptors α and β melatonin restricts the viability and angiogenesis of vascular endothelial cells by suppressing hif-1α/ros/vegf regulation of vascular endothelial growth factor by melatonin in human breast cancer cells melatonin modulates the expression of vegf and hif-1α induced by cocl2 in cultured cancer cells melatonin as a master regulator of cell death and inflammation: molecular mechanisms and clinical implications for newborn care. cell death and disease the cytokine storm of severe influenza and development of immunomodulatory therapy. cellular and molecular immunology covid-19 infection: the perspectives on immune responses. cell death and differentiation. springer nature; 2020 the authors acknowledge dr. pablo coto from the hospital vital alvarez-buylla (asturias, spain) for his collaboration about details about the covid-19 infection and specially the sars molecular characterization, and dr. esther ramírez for her contribution in the clinical trial design. key: cord-355477-7xd93aqv authors: satija, namita; lal, sunil k. title: the molecular biology of sars coronavirus date: 2007-04-23 journal: ann n y acad sci doi: 10.1196/annals.1408.002 sha: doc_id: 355477 cord_uid: 7xd93aqv abstract: severe acute respiratory syndrome (sars) is the first emerging infectious disease of the 21st century that has been highly transmissible and fatal and was caused by a previously unknown coronavirus (sars‐cov). the sars epidemic in 2003 resulted in more than 8400 sars cases and approximately 800 deaths. existing in non‐identified animal reservoirs, sars‐cov continues to represent a threat to humans although more than four years have passed since a large outbreak of sars, and no new cases have been reported. however, we cannot exclude the possibility of reemergence of sars. it is hence necessary to understand the biology of the sars‐cov to deal adequately with the next outbreak, whenever it happens. the sars‐cov is a novel coronavirus with a large (∼30 thousand nucleotides) positive‐sense, single‐stranded rna containing 14 functional open reading frames (orfs) of which 2 large orfs constitute the replicase gene which encodes proteins required for viral rna syntheses. the remaining 12 orfs encode the 4 structural proteins: spike, membrane, nucleocapsid and envelope; and eight accessory proteins. the viral genome and its expression within the host cell undergoes extensive translational and enzymatic processing to form the 4 structural, 8 accessory and 16 nonstructural proteins. in an effort to understand the molecular mechanisms or capsid assembly and viral pathogenesis, laboratories around the world have adopted a variety of approaches to answering these trivial questions. it has been our effort to consolidate all information known to date about the molecular mechanisms of the sars‐cov into this chapter to update our readership on the current status of research. severe acute respiratory syndrome (sars) is a newly emerged infectious disease that appeared in guangdong province, mainland china, in november 2002. by march 2003, the disease had spread globally and by july there were 8,447 probable sars cases including 811 deaths reported from 32 countries or regions worldwide to the who. the etiological agent was identified as a coronavirus (cov) 1 and the complete genome sequence of sars-cov tor2 (toronto) isolate was published in april 2003, which established it as a novel member of the family. 2, 3 the genome of sars-cov is a positive sense single-stranded ribonucleic acid (rna) consisting of 29,751 bases. phylogenetic analysis revealed that this cov was only moderately related to other known covs, including two human covs, hcov-oc43 and hcov-229e, known to cause common colds and lower respiratory tract infections and diarrhea in humans. the clinical symptoms of the syndrome include fever, chills, rigors, cough, and headache and the pathological aspects include lymphopenia, thrombocytopenia, elevated lactate dehydrogenase, and creatine kinase levels. although currently the spread of the virus seems to be confined to rigorous and timely quarantine measures (http://www.who.int/csr/sars/country/ table2003 0923/en/), it may still be circulating in the animal reservoir and it is impossible to predict when it will return. because this scare of recurrence of sars looms over mankind, better monitoring of sars outbreaks through accurate diagnostic tests and the development of effective antiviral therapies are urgently required. these in turn depend directly on better molecular understanding of the sars-cov infection cycle. research toward the understanding of the sars virus at a molecular level is therefore of vital importance to combat sars-cov infection if another sars pandemic was to occur in the future. sars mainly shows pneumonia-like symptoms and the lung is pathologically the most affected organ. detailed investigations, however, suggest that sars is a systemic disease with widespread extrapulmonary dissemination, resulting in viral shedding in respiratory secretions, stools, urine, and possibly even in sweat. 4, 5 immunohistochemistry and in situ hybridization of organs from four sars patients who died revealed that the virus was found not only in the lung and intestine, but also in the liver, distal convoluted renal tubules, sweat glands, parathyroid, pituitary, pancreas, adrenal gland, and cerebrum. 4 sars-cov rna was detected in the lung, small and large bowels, lymph nodes, spleen, liver, heart, kidney, and skeletal muscle, in descending order of viral load per gram of tissue using reverse-transcriptase polymerase chain reaction. 5 the virus seems to gain entry into the target cells by direct membrane fusion at the target cell surface. apart from this it has been postulated that the sars-cov may gain cell entry via ph-dependent endocytosis. the spike protein of the virus is the key player in this regard. 6 a metallopeptidase, angiotensin-converting enzyme 2 (ace2), the c-type lectin cd209l (also known as l-sign), and dc-sign bind sars-cov, but ace2 appears to be the key functional receptor for the virus. the ace2 protein is reportedly present in type 1 and type 2 pneumocytes, enterocytes of all parts of the small intestine, the brush border of the proximal tubular cells of the kidney as well as the endothelial cells of small and large arteries and veins of all tissues studied, and arterial smooth muscle cells. 7 this localization of ace2 explains the tissue tropism of sars-cov for the lung, small intestine, and kidney; however, notable discrepancies include virus replication in colonic epithelium, which has no ace2, and no virus infection in endothelial cells, which have ace2. other receptors or co-receptors such as l-sign may explain such discrepancies. the sars-cov genome has been predicted to contain 14 functional open reading frames (orfs). the comparison of different sars-cov orfs with those of other covs revealed a similar pattern of structural gene arrangement with the replicase and protease genes (gene 1a-1b) and the spike (s), envelope (e), membrane (m), and nucleocapsid (n) genes in a 5 to 3 order of appearance. interspersed between these well-characterized genes is a series of orfs, many of whose functions are yet unknown. there are two orfs situated between the s and the e genes and three to five orfs between the m and n genes (fig.1) . such gene organization most closely resembles that of group iii covs. also, the sars-cov genomic sequence does not contain a gene for the hemagglutinin-esterase (he) protein, which is present in most group ii covs. two-thirds of the sars-cov rna is organized in the genes 1a-1b. the sequence of these genes is highly conserved among all covs with the exception that sars-cov lacks the sequence coding for papain-like protease (pl1pro), one of the two papain-like proteinases operating on cleavage sites at the n terminus of the polyproteins. orfs 1a and 1b encode two polyproteins, pp1a and pp1ab, the latter is predicted to translate through a -1 ribosomal frameshifting mechanism. these two polyproteins are processed by virus-encoded proteinases, to yield 16 individual nonstructural proteins (nsp). many of the postulated nsp proteins to date have undesignated functions but it is suggested that they participate primarily in viral rna replication. most potential gene 1a-1b products are fairly well conserved between sars-cov and other covs. pl2pro of the sars virus is responsible for the cleavage of all the n-terminal proteins of gene 1a. the main proteinase (mpro), also called the 3c-like protease (3clpro), is responsible for the cleavage of all the remaining proteins encoded by gene 1a-1b. the 5 utr (untranslated region) of the sars-cov genome was initially characterized by 5 rapid amplification of the cdna ends and northern blot assays. these procedures elucidated the leader sequence and the transcription regulatory sequence (trs). the leader sequences found in the viral sg-mrna (subgenomic-mrna) transcripts were at least 72 nucleotides long. after aligning the leader sequence at the 5 end of the eight sg-mrnas, a minimal consensus trs was found with the sequence 5 -acgaac-3 , 8 which participated in the discontinuous synthesis of sg-mrnas as a signaling sequence. trs showed no clear relationship with the abundance of the sg-mrnas. a highly conserved s2m motif with 32 nucleotides was also identified in the 3 region of the genome, which has also been discovered in the avian infectious bronchitis virus. the replicase gene of the sars-cov encodes for two proteins as a consequence of the proteolytic processing of the large polyproteins (pp1a and pp1ab). the translation of segment 1b of such a polyprotein is interrupted by the -1 ribosomal frameshifting by a putative "slippery" sequence and a corresponding putative pseudoknot structure, which is required for stalling the translating ribosome at the slippery site to undergo -1 translational frameshifting. two functional domains, the papain-like cysteine proteinase (pl2pro) and the 3c-like cysteine proteinase (3clpro), were identified experimentally and are considered responsible for the proteolytic processing of the polyprotein into 16 subunits. a 375 amino acid sars-cov unique domain was identified upstream of the pl2pro domain, not found in any other known covs. in addition, seven more putative regions encoding rna processing enzymes were identified, namely, rna-dependent rna polymerase (rdrp), rna helicase (hel), poly (u)-specific endoribonuclease (nendou), 3 -5 exonuclease (exon), s-adenosylmethionine-dependent ribose 2 -o-methyltransferase (2 -o-mt), adenosine diphosphate-ribose 1 -phosphatase (adrp), and a cyclic phosphodiesterase (cpd). the translation of two polyproteins from the orf 1a and 1b starts the genome expression (fig. 1) . the polyproteins are then proteolytically processed by pl2pro and 3clpro into 16 units. pl2pro is responsible for the n-proximal cleavage and 3clpro is responsible for the c-proximal cleavage. the helicase is then released. atpase activity and dna duplex-unwinding activity were demonstrated by purified helicase, indicating that the protein has rna polymerase activity. 9 together with the m protein, the spike (s) protein is believed to be incorporated into the viral envelope before the mature virion is released. initial analysis of the 1255a.a. peplomer protein of the virus reveals the possible existence of a signal peptide that would most likely be cleaved between residues 13 and 14. 11, 12 it forms typical petal-shaped spikes on the surface of the virion and consists of three domains, the external n-terminal domain with its conserved s1 and s2 subdomains, a transmembrane domain, and a short cytoplasmic domain at the c terminus. s1 is the receptor-binding unit in the n terminus. molecular modeling of the s1 and s2 subunits of the s glycoprotein suggest that the former unit consists mainly of antiparallel ␤-sheets with dispersed ␣ and ␤ regions, in addition to the three domains identified in the s2 unit. entry of covs into target cells is initiated by binding of the viral s protein to receptor molecules. the s1 domain of sars-cov is significantly different from that of other human covs, implying that these viruses are using different receptors for host cell entry. the cellular receptor of several group i covs are aminopeptidase n and zinc metalloprotease, whereas mouse hepatitis virus (mhv) (group ii) uses carcinoembryonic antigen-related cell adhesion molecules as a cellular receptor. in the case of sars-cov, ace2 was demonstrated to be a functional receptor for the sars-cov in-vitro. syncytia were observed in cell culture expressing ace2 and the sars-cov s1 domain, which could be inhibited using anti-ace2 antibody. fine mapping on the n-terminal unit of the s protein indicates that the minimal receptor-binding domain is located between the residues 318-510. 10 antibodies specific for this s1 subunit of the sars-cov s protein were shown to neutralize sars-cov infection. the c-type lectin cd209l (also called l-sign), a human cellular glycoprotein has been reported to serve as an alternative receptor for sars-cov. the s protein of sars-cov contains 23 putative n-glycosylation sites, among which 12 have been described to be effectively glycosylated. 11, 12 the s protein, when expressed alone, acquires endoh-resistant complex n-glycans in the golgi within 30 min following expression. both high-mannose and complex glycan n-glycoforms have been detected on s trimers within er and golgi, respectively, suggesting that trimers form in the er and pass the quality control to move toward the golgi to acquire complex n-glycans. it has also been shown that the s glycoprotein is present all along the secretory pathway from the er to the plasma membrane. 13 recently, structure of sars spike protein's receptor-binding domain in complex with its receptor, ace2 has been determined using x-ray crystallography. the structure though determined at 2.9a, shows potential for usage in designing vaccines against the virus. 14 nucleocapsid or the n protein of sars-cov is a 46-kda phosphoprotein, which shows homology with other members of cov family. similar to other covs, it gets abundantly expressed during infection and antibodies against n have been detected in sars patients. [15] [16] [17] [18] n protein of most covs contains multiple epitopic sites lying most commonly at their c terminus. the epitope site for tgev is located around codons 360-382, 19 for mhv it is at 381-405 20 and for infectious bronchitis virus (ibv) it is located at 360-409, 21 all at the c terminus. likewise sars-cov, n protein has been found to contain its most antigenic site at the c terminus from codons 371-407. 22 this makes n an excellent candidate for diagnostic purposes. being a multipurpose protein, it plays a vital role during virion assembly. the process of virion assembly requires efficient packaging of the viral rna into the virion. for this, n protein associates with the viral genomic rna, and together they form the ribonucleoprotein. in mhv it has been shown that n protein does possess rna-binding activity. 16 the structure of the n terminal (49-178 aa) region of the n protein has been solved by a group at the abbott laboratories, illinois, usa. the structure reveals an rna-binding domain similar to u1a rna-binding protein. 23 studies have also revealed that packaging of sars rna into virus-like particles (vlps) is dependent on n protein. packaging signal within a domain from nucleotide 19715-20294 of genomic rna and the n terminal structure of n protein are essential for packaging and rna-binding activity, respectively. 24 purified sars-cov has been shown to have rna chaperone activity. 51 self-association of n protein governs the formation of viral capsid to a large extent and has been observed in many viruses. for sars-cov, two groups have shown that n protein does dimerize. the interaction domain responsible for dimerization was first shown to be residing in the c terminal 209 amino acid residues 25 and was further narrowed down to 138 amino acid residues (between 285 and 422) at the c terminus of the protein. 26 n's ability to form oligomers (trimers, tetramers, and hexamers) has been demonstrated to be residing in residues 343-402. 49 n also interacts with the membrane protein. amino acids 211-254, especially tetrad glutamines ( 240 qqqq 243 ), are essential for this interaction. 50 apart from interactions among viral proteins, interactions with the proteins of the host play a major role in the establishment of viral infection. hence mapping these viral host protein interactions provides vital hints about how infection actually takes place and progresses. it has been shown that n binds tightly to human cyclophilin a (cypa). 27 cypa is a family of proteins that has been extensively studied but their function remains unknown. recently, cyclophilins have been shown to play an important role in hiv infection. cypa specifically has been shown to play a vital role in viral core disassembly. 28 hence cypa interaction with n protein may provide new hints about how sars cov infects host cells. n protein has also been reported to bind to human cellular heterogenous nuclear ribonucleoprotein a1 (hnrnp a1) with high affinity. 29 the regions responsible for this protein-protein interaction have been shown to be residing in the region from amino acid 161-210 of sars cov and c terminus amino acid 203-320 of human hnrnp a1. studies have indicated that hnrnp a1 acts as a global regulator of alternative pre-mrna splicing and its activities result in inactivation of alternative distal 5 splicing sites and skipping of optional exons. 30 also a recent publication has demonstrated that the nucleocapsid protein of mhv could interact with hnrnp a1. 31 hence it may be postulated that interaction of sars-cov, n protein with hnrnp a1 may play a role in transcription and replication of viral genome. another aspect important for establishing an infection is modulation of host pathways by viral proteins. a report by he et al. in 2003 has shown that n protein activates ap1 signal transduction pathway. however, the mechanism underlying the observation is not known yet. neither is it known whether n interacts with the components of ap1 directly or through an intermediate. ap1 or activator protein 1 pathway is a regulator of a wide variety of cellular processes. these include cell proliferation, differentiation, and apoptosis. this makes this pathway an important target for modification by the viral proteins to gain control over the host cell cycle. 32 moreover, binding of n protein to cyclin-dependent kinase (cdk) complex leads to downregulation of s phase gene products, which in turn leads to inhibition of s phase progression. 33 sars-cov n protein has also been shown to induce apoptosis in cos 1 cells. cells transfected with n protein show significant cell death under conditions of stress, induced by serum starvation. n protein has been found to downregulate extracellular signal-regulated kinase (erk), but upregulate c-jun-n-terminal kinase (jnk) and p38 mitogen-activated map kinase (mapk). further, p38 mapk activation was also found to induce actin reorganization. 34 nucleocapsid protein of several covs exhibits cytoplasmic as well as nucleolar localization at some point of time during the infection cycle. in the case of sars-cov, the n protein had been predicted to contain as many as eight nuclear localization signal (nls) motifs, both monopartite and bipartite. a report by zhu et al. 35 showed that the wild-type n was distributed mainly in the cytoplasm. the n-terminal of n, which contains the nls1 (aa38-44), was localized to the nucleus. the c terminus of n, which contains both nls2 (aa257-265) and nls3 (aa369-390), was localized to the cytoplasm and the nucleolus. also the region containing amino acids 226-289 was able to mediate nucleolar localization. furthermore, deletion of the leucine-rich region (220-lalllldrlnrl) resulted in the accumulation of n to the cytoplasm and nucleolus, and when fusing this peptide to enhanced green fluorescence protein (egfp) localization was cytoplasmic, suggesting that n may act as a shuttle protein. 35 a report by surjit et al. (2005) provides evidence that n is indeed a nucleocytoplasmic-shuttling protein. 36 n proteins of many covs have been shown to be phosphorylated. 37 how-ever, the mechanism by which this happens and the functional significance of this process remains largely unknown. n protein of sars-cov has been shown to be a phosphoprotein, which is stable and localizes in the cytoplasm. the phosphorylation takes place at the serine residues and that too by multiple kinases. it has been shown to be a substrate for cdk, glycogen synthase kinase, mapk, and casein kinase ii. a possible mechanism for phosphorylation-dependent nucleocytoplasmic shuttling of the n protein has also been elucidated. it has been shown that phosphorylated n is translocated to cytoplasm with the help of 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein), a protein family that functions as adapters for modulating interactions between components of various cellular signaling and cell-cycle regulatory pathways through phosphorylationdependent protein-protein interactions. 36 it can be postulated that n phosphorylation may be serving an array of purposes ranging from acting as a deciding factor for localization of protein to aiding in creating a more favorable environment for replication of the virus. 36 cov matrix (m) protein is the most abundant structural protein at the surface of virus particles and contains three putative transmembrane domains, a short n-terminal ectodomain, and a long c-terminal endodomain. interaction of the m protein with n has been described for many covs. for sars-cov, the m-n interaction was first reported by fang et al. (2005) using glutathione-s-transferase pulldowns. laboratory experiments have shown that the 12 amino acids domain (194) (195) (196) (197) (198) (199) (200) (201) (202) (203) (204) (205) in the m protein is responsible for binding to the n protein. 39 linear epitope mapping of the m protein using synthetic peptides revealed that amino acid residues 2,137,158 interacted with sars patient sera by enzyme-linked immunosorbent assay, implying the potential capability of the m protein to induce an immune response. the m protein resides on the viral envelope and in the internal core. in the cells, the m protein is anchored in the golgi complex, thus dictating the site of virus assembly to the er-golgi complex. it has been shown that sars-cov m protein is n-glycosylated at asn 4 residue in mammalian cells. 40 also c-terminally tagged m glycoprotein strongly co-localizes with golgi markers and partially with the ergic-53 protein, a lectin that cycles between er, ergic, and cis-golgi. 41 the envelope (e) protein of the sars-cov is, as the name suggests, the main component of the virus envelope. it is an integral membrane protein, 76 amino acids long, and is highly hydrophobic in nature. in mammalian cells it is found to be localized to endoplasmic reticulum (er), golgi apparatus, and lipid rafts of cell membrane. the transmembrane domain of the e protein has been found to alter the membrane permeability. 42 orf 3a is also called sars x1 and was initially named u274. the 3a locus encodes an orf of unknown function and shows no homology to any known protein. the 3a mrna and 3a protein have been detected in sars-covinfected monkey kidney vero e6 cells. in addition, the 3a protein has been detected in the lung tissue of a sars patient. a signal sequence and three transmembrane domains have been predicted when 3a sequence was searched against smart server. 43 it is a novel sars-cov protein that has been shown to be transported efficiently to the cell surface of infected and transfected cells. its cytoplasmic domain contains sorting signals important for this process. the 3a protein expressed on the cell surface can also undergo endocytosis. it has been shown to interact with m, e, and s proteins suggesting that it may play a role in viral assembly and/or release of virus from infected cells. 44 also it has been shown to form ion channels in xenopus oocytes. formation of a pore in the infected cells may aid in virus release. the modulation of virus release may also be one of the important functions of 3a as reduction in 3a protein expression in sars-infected frhk4 cells led to a decrease in virus release. 48 3a has also been shown to upregulate expression of fibrinogen in lung epithelial cells. mrna levels of all three subunits of fibrinogen a␣, b␤, and ␥ were found to be upregulated. as a result of this intracellular levels as well as secretion of fibrinogen were found to increase. 45 in addition, 3a protein has also been shown to be expressed in the lungs and intestinal tissues of sars patients and that the protein localized to the er in 3a-transfected monkey kidney vero e6 cells. in vitro experiments of chromatin condensation and dna fragmentation suggested that the 3a protein may trigger apoptosis through a caspase-8-dependent pathway in vero e6 cells. 46 the orf 7a or x4 like orf 3a, sequence homology search yielded no significant result for any existing proteins, but the existence of a cleavage site (between residues 15 and 16) and a transmembrane helix were predicted for the 7a protein. it has recently been shown that overexpression of 7a induces apoptosis via a caspasedependent pathway in different cell lines derived from the lung, kidney, and liver. 47 four years since the discovery of sars, a great deal of scientific coordination across the globe has led to unfolding of various facts about this virus. but much needs to be learned about pathogenesis of the disease and biology of the virus. these aspects are not fully 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respiratory syndrome is associated with multiorgan involvement by coronavirus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis mechanisms and enzymes involved in sars coronavirus genome expression molecular biology of severe acute respiratory syndrome coronavirus a 193 amino acid fragment of sars coronavirus s protein efficiently binds angiotensin converting enzyme 2 mass spectrometric characterization of proteins from the sars virus: a preliminary report proteomic analysis on structural proteins of severe acute respiratory syndrome coronavirus differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins s, m and e structure of sars coronavirus spike receptor-binding domain complexed with receptor profile of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (sars)-associated coronavirus in probable sars patients diagnosis of severe acute respiratory syndrome (sars) by detection of sars coronavirus nucleocapsid antibodies in an antigencapturing enzyme-linked immunosorbent assay assessment of immunoreactive synthetic peptides from the structural proteins of severe acute respiratory syndrome coronavirus mapping of antigenic sites on the nucleocapsid protein of the severe acute respiratory syndrome coronavirus antigenic structure of transmissible gastroenteritis virus nucleoprotein location of antibody epitopes within the mouse hepatitis virus nucleocapsid protein localization of linear b-cell epitopes on infectious bronchitis virus nucleocapsid protein the epitope study on the sars-cov nucleocapsid protein structure of the n-terminal rnabinding domain of the sars cov nucleocapsid protein assembly of severe acute respiratory syndrome coronavirus rna packaging signal into virus-like particles is nucleocapsid dependent the nucleocapsid protein of the sars coronavirus is capable of self-association through a c-terminal 209 amino acid interaction domain recombinant severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein forms a dimer through its c-terminal domain nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a crystal structure of human cyclophilin a bound to the amino-terminal domain of hiv-1 capsid the nucleocapsid protein of sars coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein a1 crystal structure of human up1, the domain of hnrnp a1that contains two rna-recognition motifs the nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein a1 in vitro and in vivo activation of ap-1 signal transduction pathway by sars coronavirus nucleocapsid protein the nucleocapsid protein of sars-coronavirus inhibits the activity of cyclin-cdk complex and blocks s phase progression in mammalian cells the sars coronavirus nucleocapsid protein induces actin reorganization and apoptosis in cos-1 cells in the absence of growth factors nuclear/nucleolar localization properties of c-terminal nucleocapsid protein of sars coronavirus the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation phosphorylation of the porcine reproductive and respiratory syndrome virus nucleocapsid protein intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells peptide domain involved in the interaction between membrane protein and nucleocapsid protein of sars-associated coronavirus characterization of severe acute respiratory syndrome coronavirus membrane protein differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins s, m and e biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein the 3a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in vero e6 cells the severe acute respiratory syndrome (sars) coronavirus 3a protein may function as a modulator of the trafficking properties of the spike protein the severe acute respiratory syndrome coronavirus 3a protein up-regulates expression of fibrinogen in lung epithelial cells the 3a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in vero e6 cells overexpression of 7a, a protein specifically encoded by severe acute respiratory syndrome coronavirus, induces apoptosis via a caspase-dependent pathway severe acute respiratory syndromeassociated coronavirus 3a protein forms an ion channel and modulates virus release carboxyl terminus of severe acute respiratory coronavirus nucleocapsid protein: self-association analysis and nucleic acid binding characteristics nucleocapsid amino acids 211 to 254, in particular, tetrad glutamines, are essential for the interaction between the nucleocapsid and membrane proteins of sars-associated coronavirus coronavirus nucleocapsid protein is an rna chaperone key: cord-006230-xta38e7j authors: nan title: deutsche gesellschaft für experimentelle und klinische pharmakologie und toxikologie e.v. date: 2012-02-22 journal: naunyn schmiedebergs arch pharmacol doi: 10.1007/s00210-012-0736-0 sha: doc_id: 6230 cord_uid: xta38e7j nan nucleoside diphosphate kinases (ndpks) are multifunctional enzymes involved in a variety of cellular processes including cancer metastasis and heart diseases. the plasma membrane content of the three major ndpk isoforms ndpk a, b and c is increased in human heart failure. we have previously shown that the ndpk b isoform regulates camp levels and cardiac contractility through a receptor-independent gprotein activation involving direct g protein β subunit phosphorylation. the precise role of ndpk c in the heart is unknown and was the object of this study. ndpk c function was assessed in neonatal (nrcm) and adult (arcm) rat cardiomyocytes with real-time pcr, immunoblotting, and quantification of camp content. heart failure was induced by chronic treatment with isoproterenol (iso, 2.4 mg/kg/d 4 days) via minipumps. chronic iso increased mrna levels of ndpk c by 9.4±1.9-fold and its protein levels by 2.1±0.11-fold. immunoprecipitation of the g protein β subunit resulted in coimmunoprecipitation of ndpk c and the stimulatory gαs subunit: iso enhanced this interaction. upon iso stimulation, ndpk c translocated from the cytosol to the plasma membrane within 3 hours in both nrcms and arcms. adenoviral overexpression of ndpk c in nrcms caused a 1.5-fold increase in basal and iso induced camp synthesis, whereas sirna mediated knockdown of endogenous ndpk c decreased camp levels by ~50%. our results establish ndpk c as a novel and critical regulator of camp synthesis and gs signaling in the heart. the up-regulation of ndpk c and the increased responsiveness to iso in failing hearts point to ndpk c as a potential counterregulatory factor in the onset of heart failure. uptake and metabolism of methylated myricetin derivatives: studies in cell culture and c. elegans ackermann d. 1, 2 , büchter c. secondary plant compounds like flavonoids that are ubiquitary present in fruits and vegetables are believed to exert health protective effects in terms of lowering the incidence of widespread diseases such as cardiovascular diseases and cancer. besides their antioxidative effects, flavonoids may also modulate cell signaling pathways and thereby performing their disease-protective actions. though this class of dietary polyphenols has become increasingly popular as dietary supplements, only little is known about their metabolic fate in vivo. therefore, we investigated the absorption and metabolism of several flavonoids such as myricetin and its methylated derivatives laricitrin, syringetin, and myricetin-3',4',5'-trimethylether in the human colon carcinoma cell line hct116 and the human hepatoma cell line hepg2 as well as the model organism caenorhabditis elegans. all flavonoids were rapidly taken up by both cell lines as shown by hplc analyses. the intracellular amount of myricetin and laricitrin did not increase with time and was only half of that of syringetin and myricetin-3',4',5'-trimethylether. interestingly, no metabolites of these flavonoids could be detected which might at least in part be due to their low intracellular amounts. absorption as well as intracellular distribution was also evidenced by using the fluorescent dye "naturstoff reagent a" nsra) . fluorescence microscopy indicated a predominant cytosolic distribution of the employed flavonoids. in the model organism caenorhabditis elegans, the flavonoids were exclusively distributed in the intestine as visualized by nsra. the antioxidative capacity of the four flavonoids (measured by using the cell free teac assay and the h 2dcf-da assay in hct116 cells) decreased with increasing methyl groups in the b-ring. myricetin-3',4',5'-trimethylether (three methyl groups) was the least effective radical scavenging flavonoid in both the cell free system and in hct116 cells compared to myricetin (no methyl groups). in conclusion, for exerting their biological effects, uptake and distribution as well as metabolism of flavonoids in certain organs such as liver and gut are important and more research in that field is warranted. munich heart alliance, münchen, germany signaling through g protein-coupled receptors is affected by receptor polymorphisms, yet the molecular basis for the functional differences of individual receptor variants is unclear. to investigate the impact of the frequent gly389arg variant of the β1-adrenergic receptor (β1ar) on receptor conformation we used β1ar-sensors capable of fluorescence resonance energy transfer (fret). these sensors retained the pharmacological and functional characteristics of the native receptors. upon stimulation of the sensors we determined the activation characteristics of the polymorphic receptors in real time and in living cells. we found the β1ar variants to behave similar upon a single stimulation with an agonist, but to differentially respond with a change of their activation kinetics during subsequent stimulations. while the arg389-β1ar did not show altered activation kinetics after prestimulation, the gly389-β1ar became slower compared to the initial stimulation suggesting that β1ars possess a memory of previous activation. we then permeabilized β1ar-sensor-expressing cells with saponin to remove soluble cytosolic factors. upon permeabilization the β1ar variants did not display receptor memory, suggesting that the β1ar memory depended on the interaction of the receptors with soluble cytosolic factors upon their initial activation including the phosphorylation of agonist-bound receptors by protein kinase a or g protein-coupled receptor kinases. our findings suggest an intrinsic, polymorphism-specific property of βars that alters activation kinetics upon continued stimulation and that might account for individual drug responses. micro-rna replacement therapy: nanoparticle-mediated in vivo delivery of mirna-145 or mirna-33a exerts antitumor effects in colon carcinoma xenograft mouse models weirauch u. 1 micro-rnas (mirnas) control the expression of various genes, and under pathological conditions several mirnas are up-or downregulated. previous in vitro studies have established a pro-apoptotic and anti-proliferative role of mir-145, which shows decreased levels in colon carcinoma. in contrast, while mir-33a is only weakly expressed in several tumors as well, its role in cancer has not been analysed so far. in this study, we demonstrate the tumor-relevance of mir-33a and identify the protooncogenic kinase pim-1 as a target of mir-33a. pim-1 harbours a highly conserved mir-33a binding site within its 3'-utr, and seed mutagenesis of this target sequence abolishes the mir-33a-mediated downregulation of pim-1. the knockdown of pim-1 by rnai or mirna transfection inhibits proliferation in leukemia and in colon carcinoma cells by decelerating cell cycle progression, thus establishing a tumor inhibitory function of mir-33a. we furthermore introduce polyethylenimines (peis) for the therapeutic application of mirnas in vivo, which is critically dependent on the development of appropriate delivery tools. peis are able to form non-covalent complexes with mirnas, leading to mirna protection after systemic application in combination with an attractive biodistribution profile and the efficient uptake in target organs/cells. therapeutic effects of pei-mediated mirna delivery were demonstrated in subcutaneous colon carcinoma xenograft mouse models. the in vivo application of mirna-145 through systemic or local injection of pei/mirna complexes resulted in efficient mirna delivery and in antitumor effects, based on the concomitant repression of erk5. likewise, tumor growth inhibition was observed upon treatment of tumor-bearing mice with pei-complexed mir-33a. this is due to the mir-33a-mediated downregulation of pim-1 expression and resembles the pim-1 knockdown through rnai / pim-1 sirnas. taken together, in tumor xenograft mouse models we establish mirna replacement therapy through the pei-complexation of mirnas as a novel therapeutic strategy and demonstrate that mir-145 and mir-33a may be promising mirnas in colon carcinoma therapy. md 288, a hybrid of chloroquine and primaquine, is a potential drug against infectious diseases such as malaria. since one moiety of the hybrid, the known antimalarial drug chloroquine, is a known intercalator, the potential of md 288 to intercalate into dna was determined. due to the ability of intercalators to cause frame shift mutations, the mutagenic potential of md 288 was also investigated. the potential of md 288 to intercalate into dna was investigated by means of fluorescence based micro plate assay using ethidium bromide (eb) and isolated calf thymus double stranded dna. as a positive control chloroquine was used. the potential of md 288 to cause gene mutations was determined using the hypoxanthine-guanine phosphoribosyltransferase (hprt) test in chinese hamster v79 lung fibroblasts (v79 cells). v79 cells were treated with 0.4 µm, 0.8 µm and 1.5 µm md 288 or the positive control, the direct mutagen 4-nitroquinoline-n-oxide (nqo, 1 µm) for 24 h. on day 6, mutants exhibiting loss of hprt function were selected with 6-thioguanine . whereas at 1 µm chloroquine, a 38% decrease in fluorescence intensity of eb (indicating dna intercalation) was observed, 10 µm md 288 were needed to observe a similar decrease (28%) in fluorescence intensity of eb. therefore, md 288 is 10fold less potent to intercalate into dna than its moiety chloroquine. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells was 9 ± 2. as expected, 1 µm nqo caused a significant increase in the mutant frequency (mf, 168 ± 9) . in contrast, mf was not significantly affected by treatment with md 288 at both noncytotoxic (0.4 µm: 5 ± 4) and cytotoxic (0.8 µm: 9 ± 7) concentrations. in conclusion, md 288 is a less potent intercalator than the known antimalarial drug chloroquine. furthermore, md 288 does not cause gene mutations in the hprt test. since current studies show that various metabolites of md 288 are formed in vitro, their mutagenic potential is currently under investigation as well. -conducting channels but depends critically on the membrane potential. trp channels form cation entry channels thereby either contributing to ca 2+ entry or depolarisation. recently, we showed that trpm4 acts as a ca 2+ -activated non-selective cation channel and critically determines the driving force for ca 2+ influx in mast cells following fcεri-stimulation (1) . in addition to trpm4 we also identified the expression of other trp transcripts in bone marrow derived mast cells (bmmc) including those encoding trpc2, trpc3, trpc5, trpc6 and trpm7. in peritoneal mast cells (pmc), rt-pcr indicated expression of trpc1, trpc4, trpc5, trpc6, trpm2, trpm4 and trpm5. to identify the functional role of those trp channel proteins for mast cell activation we analysed ca 2+ signaling using microfluorimetry in bmmcs and pmcs after stimulation with substances known to activate trpc6 channels in other cell systems such as the diacylglycerol analogue oag, the hyperforin analogue hyp-9 and flufenamic acid (ffa), but could not evoke a rise in the [ca 2+ ]i in both pmc and bmmc. sphingosine 1phosphate and lysophosphatidylcholine, which were reported to activate trpc5 channels, induced only minor rise in [ca 2+ ]i in bmmcs, respectively. here, we will present our analysis of ca 2+ signaling following stimulation of the fcεri receptor and application of secretagogues that are supposed to affect ca 2+ -dependent mast cell activation such as adenosine, endothelin-1, substance p and compound 48/80 in bmmcs and pmcs derived from mouse lines with inactivation of trpc1, trpc3, trpc4, trpc5 or trpc6 since specific antagonists are still lacking for these trp channels. the α2a-ar is the main ar in the central nervous system and it plays a crucial role in regulating norepinephrine (ne) release from nerve terminals via presynaptic feedback inhibition. it is also associated with a number of physiological effects, including hypotension, pain perception, sedation and modulation of mood. ne, once released in the synaptic space, binds to α2a-ars and induces a rearrangement of the receptors from the inactive state into an active conformation. this allows the binding and activation of the cognate gi-protein and, hence, the transduction of the transmembrane signal to the downstream effectors. generally, α2a-ar activation has been deduced from the stimulation of a receptor-mediated biological response that could be easily followed experimentally. however, most of these approaches do not employ living cells and are normally applied under equilibrium conditions that need prolonged incubation periods incompatible with the physiological temporal dynamics of ne. here, we monitored the ne-mediated α2a-ar and gi-protein activation by using a fluorescence resonance energy transfer (fret)-based approach in living cells. to examine the effects of increasing concentrations of ne on the speed and extent of α2a-ar activation with very high temporal resolution, we took advantage of the previously described α2a-ar flash/cfp sensor [1] . the results indicate that in our system the efficacy of ne in eliciting α2a-ar flash/cfp activation increases in a time-dependent way and reaches the maximum with a half-life of ~ 70 ms. the ec50 values decrease in an exponential manner and arrive at ~ 2 µm with a half-life of ~ 330 ms. next, we analyzed the ability of increasing concentrations of ne to trigger a downstream intracellular response after α2a-ar stimulation by monitoring the kinetics and amplitude of gi activation in living cells. we applied the previously well characterized gi cfp/yfp sensor [2] . the results show that both the efficacy and the potency of ne in inducing gi activation reach the steady state slower compared to receptor activation (half-life ~ 700 ms and ~ 3,000 ms respectively). in conclusion, we were able to monitor ne-mediated events occurring in the millisecond time scale and reaching the equilibrium in a time interval compatible with physiological conditions. sphingosine-1-phosphate (s1p) is an immune modulator produced by sphingosine kinase 1 (sphk1) and sphingosine kinase 2 (sphk2) and de-phosphorylated or degraded irreversibly by s1p phosphatases and a lyase, respectively. we recently showed that tlr4-induced il-12p70 is selectively counter regulated by sphk1, s1pr1 and its extracellular ligand s1p. on the other hand, spiegel et al. have demonstrated that specific, sphk1-dependent, binding of s1p to traf2 enhances the tnf-alpha signaling. therefore we were interested whether the tlr/tir and tnf-alpha-signaling pathways are interfered with each other and are modulated by s1p. in a first approach we focused our investigations on sphk1 effects on both traf2 and traf6 stimulatory signals and cytokines produced downstream. experimentally, with gm-csf expanded, bone marrow-derived dcs we first desensitized the lps-tlr4 or cpg-tlr9 signal by a defined time period of costimulation with tnf-alpha. the initial results showed a partial decrease of il-12p70 secretion in tnf-alpha-co-stimulated dcs in contrast to lps stimulation alone. this might indicate that traf2 activated via tnf-alpha interacted with the traf6 pathway to reduce il-12p70. further series with dcs derived from sphk1-deficient mice confirmed our former results that il-12p70 in contrast to other cytokines is specifically sensitive to sphk1-s1p feedback, but did not change the effects of tnf-alpha on wt dcs il-12p70 release. in comparison, cpg-tlr9-induced il-12p70 release reached only 20% of lps-induced il-12p70 levels and was less sensitive to tnf-alpha costimulation. however, sphk1-deficiency strongly augmented cpg-dependent il-12p70 production. in ongoing experiments we started to analyze the details of traf2/rip1 and traf6/tak1 activation by ubiquitination blots in wt, sphk1-and s1plyase-deficient dcs. in conclusion, we hope to unravel possible mechanisms of the observed differential effects of s1p and its enzymes on inflammation and cancer-relevant cytokines. identification of the kh type splicing regulatory protein (ksrp) as a new important mediator of the anti-inflammatory effects of resveratrol art j. 1 , besche v. 2 , bros m. 2 , li h. 1 , handler n. 3 , bauer f. 3 , erker t. 3 , behnke f. 4 , mönch b. 5 , förstermann u. 1 , dirsch v. m. 6 , werz o. 5 , kleinert h. 1 , pautz a. university of vienna department of pharmacognosy, althanstr. 14, 1090 wien, austria resveratrol, a polyphenol derived from different plants, possesses multiple pharmacological functions such as anti-oxidative, anti-diabetic, cardioprotective, anticancer, neuroprotective and anti-inflammatory properties. many of these effects have been attributed to its anti-oxidative activity but resveratrol also modulates signal transduction pathways like the p38 mapk pathway or the activity of different transcription factors like nf-κb redox-independently. moreover, the histone deacetylase sirtuin 1 (sirt1) is an important mediator of resveratrol effects. nevertheless the direct molecular target of resveratrol remains unclear. in target fishing experiments we identified the rna-binding protein ksrp as direct resveratrol binding partner. ksrp is an rna-binding protein that controls proinflammatory gene expression on the post-transcriptional level by modulation of mrna stability. moreover, it is involved in the biogenesis of mirnas. resveratrol treatment of human dld-1 cells resulted in a decreased mrna expression of a number of well known ksrp target mrnas and enhanced mirna-155 function. downregulation of ksrp expression by sirna prevented the mrna destabilizing effect of resveratrol. as the activity of ksrp is mainly regulated on the post-translational level by phosphorylation of different serine and threonine residues we analyzed whether resveratrol changes ksrp activity by altering the phosphorylation of the protein. indeed, our immunoprecipitation experiments demonstrated that resveratrol reduces the p38 mapk-mediated phosphorylation of threonine residues in the ksrp protein and thus leads to an increase of ksrp activity. interestingly, resveratrol does not block p38 mapk activation or activity. in addition we have evidence that sirt1 is not involved in the resveratrol mediated activation of ksrp. so we believe that activation of ksrp by resveratrol is the major mechanism mediating the anti-inflammatory effects of resveratrol. in vitro testing of oecd reference nanomaterials (nm-series) in rat precision cut lung slices aumann a. 1 the oecd has defined reference nanomaterials (nm) to be tested in different endpoints concerning human health and environmental safety (1) in order to evaluate if the toxicity of nanomaterials can be linked to their physico-chemical properties. for nanomaterials, inhalation presents the major exposure route of concern and can be assessed using acute inhalation toxicity studies in rodents. however, these in vivo studies are resource intensive and animal consuming. the oecd working party on nanomaterials has named several alternative methods as being of particular interest for testing of nanomaterials; among them is the precision-cut lung slices model (pcls) to estimate respiratory toxicity. we have tested all 16 nm in pcls measuring cytotoxicity, apoptosis, oxidative stress and inflammatory response of the tissues as well as observing them histological. for in vitro exposure of pcls the test material was dispersed in medium. since it is the nature of these materials to change their surface characteristics and agglomeration state in different environments, a standardized dispersion method (nanocare) using bovine serum albumin as a stabilizing agent, was used. particle size-distributions of the nanomaterial dispersions were characterized via analytical ultracentrifugation and found the nanomaterials well dispersed. silver and zinc oxide but none of the other nm showed cytotoxicity to the lung tissue in the tested concentrations. however, differences in cytokine profiles among the nm were observed and showed several correlations to the results obtained in in vivo inhalation or instillation studies. universität des saarlandes institut für molekulare zellbiologie, gebäude 61, 66421 homburg, germany tmem2 proteins show similarities in their primary sequence to motifs that are conserved amongst various members of the trp protein family. based on hydropathy analysis these proteins exhibit 6 to 10 membrane spanning domains. in contrast to trp channels there is no evidence that these proteins form ion channels in the plasma membrane following overexpression of their cdna in hek293 cells. tmem2 -/mice are viable and show no obvious signs of disease, but exhibit increased pancreatic amylase and lipase plasma levels. microfluorimetric measurements using fura-2 revealed that the elevation of the cytosolic ca 2+ concentration after stimulation with carbachol and the cholecystokinin analogue caerulein is unchanged in tmem2-deficient acinar cells. tmem2 is expressed in several cell types including pancreatic acinar cells, cardiac myocytes, cardiac fibroblasts, but their subcellular localization is still unkown. we generated several constructs encoding tmem2 fusion proteins with fluorescence protein tags by fusing eyfp, mcherry and tagrfp-t to the n-and c-terminus of the protein, respectively. based on western blot experiments and expression in hek293 cells the tmem2-eyfp construct was most suitable for further colocalisation analysis and generation of viral vectors including adenovirus and semliki forrest virus. in contrast to the prediction by the psort ii algorithm tmem2-eyfp could not yet be identified in the plasma membrane of fibroblasts, cardiac myocytes or acinar cells but showed a vesicular subcellular localization pattern. we localized tmem2-eyfp in acidic compartments and predominantly in lysosomes (pearson coefficient (pcc) 0,79 ± 0,03, n= 4 using lysotracker ® dye). analysis of subcellular localization with independent tmem2 fusion constructs and additional vesicular markers will be presented as a framework to get insights towards the cellular function of tmem2 and to reveal the mechanisms underlying increased amylase release from acinar cells of tmem2 -/mice. the small molecule bcl-2/mcl-1 inhibitor tw-37 shows single-agent cytotoxicity in neuroblastoma cell lines bachmann h. s. 1 , akdeli n. high-risk neuroblastoma (nb) remains a therapeutic challenge in paediatric oncology. pro-survival bcl-2 family proteins critically regulate apoptosis, and may represent important therapeutic targets in nb. primary nb tumours heterogeneously express mcl-1 or bcl-2, with high expression correlating to high risk phenotype. co-expression can be detected in approximately 10% and is correlated to reduced survival. recent studies with two inhibitors that predominantly target bcl-2 and other proteins, but not or to a lesser extend mcl-1, elucidated the importance of mcl-1 inhibition for cytotoxicity in nb. tw-37 is a small molecule inhibitor that showed almost equal affinity to bcl-2 and mcl-1. to explore the effect of combined bcl-2/mcl-1 inhibition on neuroblastoma cells, four cell lines (sk-n-as, imr-5, sy5y and kelly) were treated with tw-37 and changes in growth properties were determined. furthermore, nude mice with kelly (human neuroblastoma cell line) xenografts were treated with tw-37. using sirna, we investigated the functional relevance of mcl-1 and bcl-2 in kelly cells. for in vitro cell viability we observed ic50 values of 0.59 ± 0.39 µmol/l. on treatment with 1 µmol/l dose of tw-37, all neuroblastoma cell lines analyzed showed significantly reduced proliferation and increased apoptosis rates. bcl-2 as well as mcl-1 knockdown induced apoptosis in kelly cells. interestingly, tw-37 was able to reduce, but not to abrogate growth of kelly neuroblastoma xenografts in nude mice. in conclusion, combined inhibition of bcl-2 and mcl-1 using tw-37 exhibits strong single-agent antitumor activity on human neuroblastoma cells in vitro, but limited single-agent activity in vivo. therefore, inhibition of bcl-2/mcl-1 may represent an interesting therapeutic strategy, most likely in combination with conventional chemotherapy and other specific inhibitors. localization and functional characterization of membrane transporters for sulfated steroid hormones in the human testis bakhaus k. 1 , wapelhorst b. circulating sulfated steroid hormones like estrone sulfate (e1s) or dehydroepiandrosterone sulfate (dheas) are delivered to the testis via membrane uptake carriers such as the sodium-dependent organic anion transporter (soat). inside the cell these sulfated steroids can be metabolized to active steroid hormones by the catalytic activity of the steroid sulfatase (sts), which shows high enzymatic activity in the testis ("sulfatase pathway"). in addition to soat, other candidate carriers like the organic solute carrier protein 1 (oscp1) and the organic anion transporting polypeptides oatp6a1 and oatp1c1 are predominantly expressed in the human testis and demonstrate transport activity for sulfated steroids. we aimed to evaluate the cellular expression of soat and the other steroid sulfate carriers and their co-localization with the steroid sulfatase (sts) in human testis. furthermore we want to perform functional transport studies with the steroid sulfate carriers in stably transfected hek293 cells. we detected soat by rt-pcr and western blot analysis in the human testis. single cell analysis and in situ hybridization revealed pachytene primary spermatocytes to express the soat mrna. soat expression in specimens showing maturation arrest at the level of early round spermatids seems to be severely reduced or absent. sts mrna was detected by rt-pcr in testis homogenates. preliminary immunohistochemical data showed that sts may be expressed in germ cells and interstitial leydig cells. hek293 cells stably expressing the soat carrier protein showed significant transport activity for dheas. this was demonstrated by using a radiolabeled [ 3 the hepato-intestinal induction of the detoxifying enzymes cyp3a4 and cyp3a5 by the xenosensing pregnane x receptor (pxr) constitutes a key adaptive response to oral drugs and dietary xenobiotics. in contrast to cyp3a4, cyp3a5 is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven disturbances of the steroid homeostasis. using cell lines and mice transgenic for a cyp3a5 promoter we demonstrate that the cyp3a5 expression in these organs is noninducible and independent from pxr. instead, it is enabled by the loss of a suppressing yin yang 1 (yy1)-binding site from the cyp3a5 promoter which occurred in haplorrhine primates. this yy1 site is conserved in cyp3a4, but its inhibitory effect can be offset by pxr acting on response elements such as xrem. taken together, the loss of yy1 binding site from promoters of the cyp3a5 gene lineage during primate evolution may have enabled the utilization of cyp3a5 both in the adaptive hepato-intestinal response to xenobiotics and as a constitutively expressed gene in other organs. our results thus constitute a first description of uncoupling induction from constitutive expression for a major detoxifying enzyme. they also suggest an explanation for the considerable tissue expression differences between cyp3a5 and cyp3a4. serum albumin adducts as biomarkers for systemic bioavailability of active metabolites of various glucosinolates in animal models and humans barknowitz g. 1 , engst w. glucosinolates (gls) are natural pesticides of brassicales, which comprise many important food and feed plants. upon physical damage to the plant, the enzyme myrosinase can convert gls to reactive metabolites (e.g. isothiocyanates). the same reaction can also be catalyzed by enzymes of the intestinal microbiota. modification of sensor proteins (e.g. keap-1) by some gls metabolites leads to adaptive responses, resulting in enhanced detoxification of reactive metabolites and other protective reactions. at least in experimental models, this mechanism can be exploited for chemoprevention of carcinogenesis induced by various chemical carcinogens. however, own research revealed that certain reactive gls metabolites can covalently bind to dna in vitro and in vivo, involving possible genotoxic and carcinogenic risks. animal models are useful for studying beneficial and adverse effects of gls. however, it has to be taken into account that the toxicokinetics of gls may differ between rodent and humans and that the active metabolites are short lived and thus difficult to detect and quantify. likewise, exposure of humans to gls and their breakdown products may enormously vary depending on (i) food preferences, (ii) cultivars, growth conditions and preparation of plants consumed and (iii) variations in human xenobiotic metabolizing system and composition of intestinal microbiota. in order to estimate individual levels of systemic exposure to reactive gls metabolites, blood protein adducts may be useful. we have developed lc-ms/ms methods for quantifying serum albumin adducts formed by glucoraphanin, glucotropaeolin and neoglucobrassicin. the method involves digestion of the protein to amino acids and the usage of isotope-labelled amino acid adducts as internal standards. serum albumin adducts were detected in mouse models after feeding broccoli and pak choi respectively, as well as after administration of purified gls and breakdown products. likewise, gls adducts were detected in human blood plasma after consumption of broccoli or cress. this work was financially supported by the bundesministerium für bildung und forschung (grant 0315370d) . barlow s. harrington house, 8 harrington road, brighton, bn1 6re, great britain values for cancer endpoints for the application of the ttc approach have been derived by linear extrapolation of results from animal carcinogenicity studies to calculate "virtually safe doses" (vsds). a vsd is defined as an exposure that represents an estimated upper bound increase in risk of 1 in a million of developing cancer during a lifetime. in 1995, from consideration of a range of vsds for carcinogens, the us food and drug administration (fda) proposed and adopted a value of 0.5 micrograms/kg of diet (0.5 ppb) as a threshold of regulation to be used for substances present in food contact materials. the fda considered that if exposure to a substance in the diet was below this value, consumers would be protected "with reasonable certainty of no harm" and no toxicological data on the substance need be submitted. the value of 0.5 ppb is equivalent to 1.5 micrograms/person per day, assuming that 1500 g of food and 1500 g of fluids diet might be consumed daily. this value was subsequently incorporated into the ttc approach to be used for assessment of substances without a structural alert for genotoxicity. in 2004, kroes and colleagues further explored cancer as an endpoint and recommended a lower ttc value of 0.15 micrograms/person per day for substances with a structural alert for genotoxicity and exclusion from the ttc approach of certain groups of high potency carcinogens (with vsds below this value). in this presentation, the data underpinning these ttc values will be discussed from the perspective of their reliability for risk assessment of substances with low exposures, for which there are no toxicity data, but which may in fact be genotoxic or non-genotoxic carcinogens. stable conjugates which are recognized by the immune system. in the current investigations, the ability of chemical pre-treatment to interfere with antibody-protein binding has been investigated using ovalbumin (ova) as the model protein and naturally occurring anti-ova igg from healthy human donors. preparation of conjugates: ova (1 mg/ml) was dissolved in sodium borate buffer (0.1m, ph 9.4) . for chemical treatment various amounts (50-200 mg) of 1-fluoro-2,4dinitrobenzene (dnfb; sensitizer) or 2,4-dichloro-1-nitrobenzene(dcnb; non-sensitizer) was added and stirred for 2 hrs at room temperature. unbound compound was removed by consecutive dialysis against phosphate buffered saline (pbs) and distilled water. inhibition elisa: plates were coated with 100 µg/ml ova and blocked with 10% fcs in pbs. ova samples (native or conjugated; 0.6 -10 µg/ml) were pre-incubated with polyclonal antibodies (ab) from pooled normal human serum for 30min and then added to the plates. human anti-ova igg abs were detected by colorimetric analysis using ortho-phenylendiamine as a substrate. the concentration of soluble native ova or conjugate required to displace 50% of ab to plate-bound ova (ic50) was calculated (minimum of n=3 independent experiments). treatment of ova with the chemical sensitizer and protein reactive dnfb resulted in increased ic50 value, whereas mock treatment resulted in comparable ic50 values to native ova. treatment with dcnb showed that the presence of a chemical per se (and possible denaturation) was not sufficient to alter the ic50 value; conjugation of the compound to the protein was required. the analysis is not test chemical specific (specific ab against compound-protein conjugates are not required) and the colorimetric analysis is unaffected by absorbance of the compound itself. thus, this method may have utility for the identification of chemical sensitizers which are directly protein reactive. 2´-deoxy-camp in human cell lines: another second messenger? beckert c. 1 , hinz c. we have shown that 2´-deoxy-camp (dcamp) is synthesized by recombinant human soluble adenylyl cyclase (sac). here, we report that dcamp can be detected and quantified by liquid chromatography coupled to mass spectrometry (lc-ms) in various human cell lines. in most cells a ratio of camp : dcamp of ~10 was observed. as a remarkable exception, in hl-60 promyelocytic leukemia cells, the dcamp concentration exceeded the camp concentration more than 3-fold. a differential regulation of camp versus dcamp was determined upon replacement of the incubation medium (proliferating condition with serum / serum-free resting condition). for example, camp was dramatically reduced in hek293 cells after 24 hours under resting conditions whereas dcamp was significantly increased. in cellular subfractions of hek293 cells ac assays (mn 2+ /forskolin-or mn 2+ /bicarbonate-stimulated) with either atp or datp as substrate revealed that comparable amounts of camp and dcamp accumulated. in addition to sac, membranous acs such as ac v were capable of forming dcamp with vmax and km values for datp comparable to those for atp. we also analyzed the substrate-specificity for several human phosphodiesterases. pde3 and pde4 hydrolyzed dcamp more effectively than camp. taken together, these data point to a putative second messenger role of dcamp in human cells. we are currently investigating the regulatory role(s) of camp and dcamp in apoptosis of hek293 cells. as a novel tool for these studies, we will use the cellpermeant dcamp-acetoxymethylester which penetrates the plasma membrane and releases dcamp intracellularly. the biogenic amine histamine is recognized by target cells via four different histamine receptors subtypes (h1r -h4r), which all belong to the family of g-protein-coupled seven-transmembrane receptors. histamine plays a crucial role in allergic reactions such as rhinitis or conjunctivitis and also in allergic asthma. previously, we showed an interaction of the effects of antagonists at the h1r and h4r in a mouse model of allergic asthma. however, not much is known about the signaling pathway activated by murine h1r and h4r. in order to analyze these signaling pathways, we established a cellular model using transfected hek 293 cells which stably express recombinant mh1r or mh4r. proper expression of the receptors was verified by western blot analysis and flow cytometry. in functional assays we demonstrated that histamine stimulation results in the increase of intracellular ca 2+ concentration ([ca 2+ ]i) in cells expressing either of both, mh1r (pec50 = 8.2) or mh4r (pec50 = 6.9). as a second readout, we analyzed the modulation of forskolin-induced camp-accumulation. in mh1r-expressing cells the intracellular camp concentration was increased by stimulation with histamine, while in mh4r-expressing cells forskolin-induced camp accumulation was reduced. the histamine-induced effects in h1r-expressing cells were blocked by the h1r antagonist mepyramine ([ca 2+ ]i: pkb = 8.6) and those in the h4r-expressing cells by the h4r antagonist jnj7777120 ([ca 2+ ]i: pkb = 8.8) or by pertussis toxin, which selectively blocks receptor gi-protein coupling. jnj7777120, which behaves as a partial mh4r agonist in the steady-state gtpase assay using membranes of infected sf9 cells, was without effect on [ca 2+ ]i and forskolin-induced camp-accumulation in the mh4r-expressing cells. currently, we are investigating mitogen-activated protein (map)-kinase pathways activated by the h1r and the h4r. using a phospho-map-kinase array, histamine dependent phosphorylation of erk1/2, p38, jnk, creb, pkb (akt), and mkk 3/6 were detected in cells expressing either of both, mh1r and mh4r. in summary, the hek 293 cell lines stably expressing selective histamine receptors are very useful tools to investigate hxr signaling pathways in-vitro. enhanced fibroblast motility in the absence of the β3 regulatory subunit of voltage-activated calcium channels belkacemi a. 1 cavβ subunits of voltage-activated ca 2+ channels are required for trafficking the poreforming cavα1 subunit to the plasma membrane and modulate the kinetics of its current. mouse embryonic fibroblasts (mefs), acutely isolated cardiac fibroblasts (cfs) and nih 3t3 fibroblasts do express cavβ2 and cavβ3 subunits, but we could not detect any voltage-activated ca 2+ influx. whereas in mouse cardiomyocytes or hek 293 cells coexpressing cavβ3 and cavα1 subunits a dihydropyridin-sensitive voltage-activated ca 2+ influx was readily detectable. apparently, cavβ subunits serve functions in fibroblasts unrelated to voltage-activated ca 2+ influx. among the proteins potentially interacting with cavβ3 are the inositol 1,4,5-trisphosphate receptors (ip3rs) [1, 2] . we therefore coexpressed mouse cavβ3 and mouse ip3r type 1, 2 or 3 in cos-7 cells and found coimmunoprecipitation of ip3rs using an antibody for cavβ3 and vice versa. to study the release of ca 2+ from ip3-sensitive stores we performed fura-2 measurements on fibroblasts isolated from wild type and cavβ3-deficient mice either in the presence of thapsigargin or after stimulation of gq-coupled receptors by par-1, lpa or bradykinin. receptor-activated ca 2+ release was more pronounced in β3-deficient mefs and cfs, whereas thapsigargin-induced ca 2+ release was the same in cells from both genotypes. in addition, ip3 production measured by a radioreceptor assay was already increased in β3-deficient cells under basal conditions. fibroblasts are migrating cells and involved in various physiological and pathophysiological processes. we therefore started in vitro assays for proliferation, migration and angiogenesis as well as in vivo assays for skin wound healing. angiogenesis and proliferation were apparently not different in both genotypes but migration (measured as transwell migration and in scratch assays) and wound healing were affected in different ways. fluorescent staining of cytoskeleton and quantification of the f-actin/g-actin ratio show similar results in both genotypes, suggesting that the increased migration rates and wound repair in β3 knockout may result, in part, from the increased amount of ip3-releasable ca 2+ . [1] berggren, yang, murakami, et al., removal of ca channel β3 subunit enhances caoscillation frequency and insulin exocytosis. cell 119, (2004) 273-284. [2] müller, haupt, bildl, et al., quantitative proteomics of the cav2 channel nanoenvironments in the mammalian brain. pnas 107, (2010) 14950-14957. bender-sigel j., closs e. i. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany human cationic amino acid transporters (hcat) are a family of multimembrane spanning proteins that mediate the transport of cationic amino acids through the plasma membrane. our earlier results have demonstrated that activation of either protein kinase c (pkc) by pma or cdc42 by egf leads to an internalization of these transporters. in addition, in a recent collaboration with the group of alexander sorkin (university of colorado denver) we found that ubiquitination and clathrin-dependent endocytosis are necessary for the down regulation of hcat-1-mediated arginine transport by pma (vina-vilaseca et al, j biol chem 2011 286:8697) . this mechanism requires nedd4 e3 ligases, but hcats do not contain a ppxy motif to bind the ligases, suggesting that an adaptor protein takes part in this process. however, an involvement of the adaptor protein beta-arrestin in this mechanism could be excluded. using sirna against pkc alpha we now show that pkc alpha is the major isoform that induces the reduction of arginine transport in human u373 glioblastoma cells overexpressing hcat-2a-egfp. in addition, sirna-mediated knock down of cdc42 prevented the decrease of hcat-2amediated transport induced by pma. taken together pkc seems to negatively influence the constitutive cycling of cats by activation the ubiquitination machinery and clathrinmediated endocytosis. cdc42 is part of this pathway. converging of the classical mitochondria-related pathway in parkinson and nuclear dna-repair signaling? scherr a. -l. 1 parkinson disease is the second most neurological disorder worldwide. despite the fact that most cases are idiopathic and only few can be traced back to specific genes, general progression between both tracks of the disease is comparable. the variety of clinical symptoms in motor control like tremor, rigor and postural problems all originate from loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. several proteins mutated in pd are involved in surveillance pathways, monitoring functionality and integrity of proteins and organelles either by the proteasome degradation machinery or by clearance of mitochondria via autophagy (mitophagy). disturbed calcium-and redox-homeostasis seems to play a major role in susceptibility to cell death signals in dopaminergic neurons, but if this is a preceding or successive event in cell death related to pd progression is not known. on the other hand, experimentally elicited parkinsonism by oxidative stress inducers like paraquat or rotenone and mptp (inhibitors of mitochondrial complex i) lead to damage in nuclear dna and activation of the dna repair protein poly(adp-ribose) polymerase 1. by consuming substantial amounts of its substrate nad + , this enzyme can drastically decrease energy levels and disturb the redox balance within a cell, also sensitizing it to stress induced cell death. inhibition of poly(adp-ribose) polymerase 1 has been proven to be beneficial to some extent in cell culture models as well as in experimental pd in mice. our research focuses on a putative crosstalk mechanism we recently discovered between the two pathways of experimentally induced cell death in culture models, i.e. mitochondrial signaling and parp1-dependent poly(adp-ribosyl)ation. both converge on two mitochondrial chaperones, mortalin and trap1. whereas mutations in mortalin have been reported recently to be responsible for some parkinson disease cases in humans, trap1 is a specific target of the kinase pink1 (pten induced putative kinase), which is often mutated in autosomal-recessive forms of the disorder. pink1 is a central regulator of the mitophagy process, tagging mitochondria with dissipated membrane potential for destruction. we could show now that both chaperones bind to short-chain poly(adp-ribose) specifically synthesized by poly(adp-ribose) polymerase 1. we will present our most recent findings about regulation of these two chaperones after application of parkinson-inducing toxins. aldrich). the effect was reversible within ten minutes when cells were re-incubated in regular cell culture medium. stimulatory effects were not due to osmolarity or cell stress due to medium exchange. analysis of different components of both media (table 1) revealed that bicarbonate stimulates accumulation of ccmp and cump besides cgmp and camp in a time-and concentration-dependent manner. bicarbonate is known to activate soluble adenylyl cyclase (sac) and particulate guanylyl cyclase g (pgc-g), regulating sugar metabolism, sperm motility and olfaction by synthesis of camp and cgmp, respectively. in order to identify a responsible cyclase for ccmp and cump generation after bicarbonate treatment, we are currently analyzing transiently and stably transfected hek293 cells overexpressing various known adenylyl and guanylyl cyclases (sac, mac1, mac3, mac9, soluble guanylyl cyclase, pgc-g, pgc-a, and pgc-d) for their pyrimidinyl and purinyl cyclase activity in vivo and their regulation by bicarbonate. in addition, cell fractions will be analyzed for the detection of specific cyclase compartments. question: long term ventricular pacing, especially at the right ventricle (rv), results in left ventricular (lv) failure. there are several lines of evidence that disturbed ca 2+ homeostasis is involved in the pathophysiology of human heart failure. in this study we examined if ventricular pacing affects the na + -and ca 2+ -channels and the expression of ca 2+ -handling proteins and investigated if there is a differential effect between right ventricular free wall (rvfw) pacing and left ventricular apex (lva) pacing. methods: after av-node ablation 14 minipigs underwent ventricular pacing at 120 beats/min (ddd mode) for one year. 7 minipigs were paced from the rvfw and 7 minipigs from the lva, respectively. 7 minipigs with normal sinus-rhythm served as control group. patch-clamp-experiments were studied to measure na + -and ca 2+ currents. western-blots were carried out to investigate the expression of the ca 2+handling proteins l-type ca 2+ -channel, serca2 and phospholamban. results: both rvfw-and lva-pacing led to significant decreased ca 2+ -currentdensities in cardiomyocytes of the lv compared to the control group. the plateau phase of the action potential was significantly shortened after ventricular pacing in relation to control minipigs. furthermore cardiomyocytes of rvfw-and lva-paced minipigs had significant lower na + -current-densities than control minipigs. the action potential amplitude was significantly decreased after rvfw-and lva-pacing whereas the diastolic potential remained unchanged. the expression of the l-type ca 2+ -channel was significantly reduced after ventricular pacing, regardless of the pacing site. in contrast rvfw-and lva-paced minipigs showed significant increased serca2-expression. the expression of phospholamban remained unchanged after rvfw-and lva-pacing compared to control minipigs. conclusion: in a chronic animal model ventricular pacing leads to remodeling of ionchannels and ca 2+ -handling-protein-expression, regardless of the pacing site. investigation on metabolic competence of dermal systems: native human skin, in vitro skin models and keratinocytes blatz v. 1 the implementation of reconstructed human skin equivalents (rhes) as an alternative method for dermal toxicity testing became very prominent in the last decades. their advantages are e. g. the human cell origin and an organ-like 3d structure. already regulatory accepted methodologies are widely in use for testing the skin corrosion (oecd 431) and irritation (oecd 439) within rhes. but there are still some questions open, one of them the metabolic competence of such dermal systems. in this context, enzyme activities of oxidizing (cyp; fmo; adh; aldh) and conjugating enzymes (nat; ugt) were investigated in subcellular fractions of in vitro systems such as keratinocytes and rhes (epidermis model epiderm tm (mattek), full-thickness skin models epiderm tm ft (mattek) and phenion ® ft (henkel ag)) and compared to those of native human skin. activities of cyp 1a, 2b and 3a isoenzymes were measured fluorometrically by oxidative desalkylation of alkoxyresorufines. fmo 1/3 activities were evaluated by hplc/fld detection of n-oxygenated product of benzydamine [1] . adh and aldh activities were investigated by photometrical detection of nadh generation during ethanol (adh) [2] or propanal (aldh) oxidation [3] . nat1 activity was followed by hplc/uv detection of acetylated p-aminobenzoic acid. ugt1 activity was quantified fluorimetrically by glucuronidation of methylumbelliferone [1] . during the course of this study the following results were observed: (loq = limit of quantification) since the metabolic competence of rhes is confirmed, these in vitro systems are estimated as suitable for further toxicity tests (e. g. genotoxicity by comet assay), where metabolic activation of substances may play a crucial role. however, for the data assessment, the determined metabolic profiles should be taken into account. we acknowledge bmbf funding this project (0315226d). signalling pathway indicates that in b104 cells the adenine receptor couples to a gqprotein followed by activation of phospholipase c pathway. these findings represent a new signalling pathway of the adenine receptor and allow the assumption that different adenine receptor subtypes exist in the rat brain. in the scope of the project lexukon ("foodborne exposure to environmental contaminants -data analysis to support and standardise exposure assessments based on nvs ii") exposure to the heavy metals cadmium (cd), lead (pb) and mercury (hg) via food consumption has been assessed for the german adult population based on the national nutrition study ii ( the updated intake assessments show that especially foods regularly consumed such as vegetables and grain contribute mainly to exposure of cd that is about 1.5 µg/kg body weight (bw) per week for average consumers over all food groups. this corresponds to 58% of the tolerable weekly intake (twi) of 2.5 µg/kg bw for cd defined by the european food safety authority (efsa) in 2009. beverages and vegetables are the food groups most relevant for exposure to pb. about 3.7 µg pb/kg bw is taken up by average consumers that is below the benchmark dose for renal toxic effects (4.41 µg/kg bw) defined by efsa for the weekly pb intake. for hg the intake amounts for all population groups examined were significantly below the toxicological reference values. for average consumers the weekly intake of hg is 0.49 µg/kg bw that is primarily taken up by eating fish and fishery products. however, individual population groups and high consumers reach and/or exceed the toxicological reference values for the daily intake amounts for cd and pb. high consumers almost reach the twi for the cd with 94%. for pb a weekly intake of 5 µg/kg bw was estimated for high consumers that exceeds the benchmark dose for renal toxic effects for the weekly pb intake. the results show that data collection should also focus on highly consumed and not only on highly contaminated foods. further, uncertainties in concentration levels should be reduced e.g. by lowering and standardizing the analytical limits. it's recommended to consider further measures in view of the reduction of contents of environmental contaminants in foods. however, other sources can also contribute to the intake of the mentioned heavy metals (e.g. smoking). major cell biological processes are regulated by rho-gtpases, actin-mediated processes in particular. amongst others, rho-gtpases are stimulated by the receptormediated activation of gα12/13 and gαq via specific rhogefs. the p63rhogef is activated by gαq and plays a major role in the acute response of vascular smooth muscle cells to angiotensin ii treatment. the aim of the present study was to establish a fret-assay between gαq-cfp and venus-p63rhogef and characterize the dynamics of p63rhogef-gαq-interaction in single living cells. the fusion of p63rhogef with venus resulted in a functional gαq-regulated p63rhogef-protein as determined by means of rho-luziferase-assays. whereas no specific fret signal was observed between the two interaction partners in the absence of receptor stimulation, a robust and rapid fret signal developed in response to stimulation of histaminergic h1and cholinergic m3-recptors. the onset of this signal after rapid application of agonist paralleled gαq activation kinetics. similar to the kinetics of gαq-protein deactivation the dissociation of p63rhogef and gαq after withdrawal of agonist was slow (tens of seconds). the specificity of the fret signal between gαq-cfp and venus-p63rhogef was verified by introducing point mutations rendering p63rhogef unable to bind to active gαq. furtehrmore we observed a robust acceleration of the dissociation of p63rhogef and gαq upon cotransfection of rgs2, suggesting a very short lifetime of the p63rhogef-gαq-complex or the ability of rgs2 to bind to p63rhogef-associated gαq. taken together, fret-based imaging of the interactions between p63rhogef and gαq revealed fast interaction kinetics closely resembling g-protein activation kinetics, both of which can be regulated by rgs2. toxicity of silver nanoparticles in intestinal cells boehmert l. 1 the rapid development of nanotechnology has been accompanied by an increased concern for the safety of nanomaterials. especially silver nanoparticles are used in many manufacturer identified consumer products including silver coated food contact materials or hydrosol silver supplements. these products lead to an intentional or unintentional oral uptake of silver nanoparticles and hence to a contact with the intestinal barrier. the human cell line caco-2 is a well established model system in studying effects on human enterocytes. although these cells are colon carcinoma cells and exhibit typical features of cancer cells when they are kept sub confluent, these cells have the capability to differentiate into polarized cells with morphological and biochemical properties of small enterocyte cells. we investigated the effects of silver nanoparticles on these colon carcinoma (proliferating) and small intestinal epithelium like (differentiated) caco-2 cells. the silver nanoparticles agpure were commercially available from rent-a-scientist gmbh. the behaviour of these silver nanoparticles in cell culture medium were characterised using asymmetric flow-fild flow fractionation (a4f), small ankle x-ray scattering (saxs) and dynamic light scattering (dls). we investigated the particle toxicity on both cell states using cell titer blue assay, xcelligence impedance measurements, annexin-v and caspase measurements, diclorofluorescein assay and antioxidant pre incubated cells. the agpure stock solution is an aqueous suspension of silver particles with a metal core radius of 7.2 nm stabilised with tween-20 und polyoxyethylenglycerol trioleate. agpure silver nanoparticles were toxic for proliferating as well as differentiated caco-2 cells in a time and concentration depending manner. the presence of foetal calf serum in the incubation medium has a minor influence on the toxicity. prior to cell death, morphological abnormal adherence characteristics and morphological changes in the cells were observed using microscopy and quantified by xcelligence impedance measurement. it is concluded that cell death is caused by an oxidative stress related mechanism rather than apoptosis. the release of sphingosine-1-phosphate from human platelets during acute coronary syndrome is attenuated by aspirin böhm a. 1 , polzin a. in addition to atherosclerosis ttp knock out mice develop more cardiovascular dysfunctions. in tail vein bleeding assays we monitored a significant difference in the bleeding times of ttp deficient mice in comparison to wildtype mice, triggered by a stronger granulopoeisis. our results leed us to the assumption that the chonic inflammation seems to be more improtant for the development of cardiovascular diseases in ra patients than the traditional risk factors. differentially expressed cardiac genes in a mouse model with heart-specific overexpression of pp2a bollmann p., makarova e. a., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06112 halle (saale) , germany in transgenic (tg) mice with cardiac myocyte-specific overexpression of the catalytic subunit of protein phosphatase 2a (pp2a) reduced cardiac protein phosphorylation, cardiac hypertrophy and impaired cardiac contractility were noted compared to wild type (wt) littermates. the hearts of tg mice also suffered from ventricular dilatation and a diminished response to β-adrenergic stimulation. analyses of mrnas expressed in tg and wt hearts (n=3) using affimetrix mouse genome microarray chips resulted in several candidate genes possibly differentially regulated. in this study, we focussed on verifying the mrna data of selected genes important for stress response and signal transduction on protein level in cardiac homogenates by western blotting. hearts from wt littermates were used as control. compared to wt heat shock protein 25 (hsp25) and calcium calmodulin dependent protein kinase type ii (camkii) mrnas were upregulated in tg but only hsp25 protein was increased (p<0.05, n=7-8) but not camkii (p>0.05). protein phosphatase type 5 (pp5) and superoxide dismutase (sod) were downregulated on mrna level in tg but on protein level this could be found only for sod (p<0.05, . in contrast, pp5 protein was upregulated (p<0.05, in tg compared to wt. for comparison the regulatory a-subunit of pp2a and hsp90 were studied. both genes were unchanged on mrna level in tg: western blotting revealed the same results for the corresponding proteins. in summary, mrna expression data could only partially be confirmed on protein level elucidating the importance of western blotting studies. these data indicate that increased pp2a activity is associated with modified gene expression in tg hearts possibly affecting stress response and regulation of cell signalling. (supported by the deutsche forschungsgemeinschaft) center for regenerative therapies, technische universtität, dresden, germany cell therapy in the form of beta cell replacement to cure diabetes has been practiced for decades without become a routine clinical therapy. more widespread clinical application is hindered by the scarcity of suitable organ donors, a dramatic loss of transplanted cells within the first days post-transplant, the requirement of long term immunosuppression to maintain graft survival, and despite this, a loss of graft function from a recurrence of autoimmunity in some patients. research is currently dedicated to overcome each of these limitations. additional beta cell sources investigated include embryonic stem cell derived insulin producing cells, human insulin producing cells lines, and xenogeneic beta cells. parallel to these efforts are the development of encapsulation devices to protect these sources from immune and inflammation mediated destruction, and transplantation into new sites such as the muscle and bone marrow to infuse beta cells. additional therapy to reduce immune suppression includes the infusion of t regulatory cells to control autoimmune and alloimmune response, and cytokine and chemokine receptor directed compounds aimed at blocking early inflammation or autoimmunity. these efforts are likely to lead to an expansion of clinical activity to replace beta cells in diabetes, and to novel pharmaceutical therapies that may be more generally applicable in patients with diabetes. pdgf-bb induces the h2s producing enzyme cystathionine-γ-lyase via a rosdependent mechanism in rat renal mesangial cells boosen m. 1 , hassan m. there is increasing evidence that hydrogen sulfide (h2s) that is endogenously produced in several cell types serves as a potent gasotransmitter in a wide variety of physiological processes involving vascular homeostasis and inflammation. in the present study we investigate the expression and the regulation of the hydrogen sulfide synthesizing enzyme cystathionine γ-lyase (cse) in cultured rat renal mesangial cells. as demonstrated by qpcr and western blot experiments, mesangial cells showed a marked time-and dose-dependent upregulation of cse mrna and protein levels after treatment with platelet-derived growth factor (pdgf-bb). the cse upregulation by pdgf-bb is accompanied by a marked increase in reactive oxygen species (ros) formation. interestingly, co-administration of the ros scavenger n-acetylcysteine, the glutathione peroxidase mimetic ebselen and the nadph oxidase inhibitor diphenylen iodonium chloride (dpi) drastically reduced pdgf-induced cse expression, indicating a role for endogenously produced ros in mediating regulation of cse. as demonstrated by electrophoretic mobility shift (emsa) experiments pdgf-bb induces binding of the redox-sensitive transcription factor nf-e2-related factor 2 (nrf2) to a consensus antioxidant response element and this effect was also diminished by co-administration of antioxidants (dpi, nac, ebselen) . furthermore, lps/ifnγ-as well as pdgf-bb-induced cse upregulation was nearly completely abolished in nrf2 -/spleen macrophages and mesangial cells, respectively. as a consequence of the elevated cse levels we could demonstrate increased h2s levels and a higher cse enzyme activity in mesangial cells after stimulation with pdgf-bb by using the colorimetric methylenblue method and a cse activity assay. importantly, in a rat model of anti-thy-1-induced proliferative glomerulonephritis we observed a marked upregulation of cse protein during the course of the disease paralleled by a stabilization of nrf2 protein. from our data, we hypothesize that pdgf-bb-mediated regulation of cse via a redox-mediated activation of nrf2 may constitute a protective mechanism during glomerular inflammatory disease. rac1 knockout protects from acute hepatic damage following doxorubicin treatment bopp a., wartlick f., fritz g. heinrich-heine-universität institut für toxikologie, universitätsstr. 1, 40225 düsseldorf, germany rac1 belongs to the best characterized members of the ras-homologous (rho) family of small gtpases, which are key regulators of the actin cytoskeleton. furthermore, rac1 is part of the activation of the nadph oxidase, which produces reactive oxygen species and regulates the activity of stress kinases (e.g. sapk/jnk) and transcription factors such as nf-κb and ap1. anticancer drugs cause dna damage, which in turn stimulates the dna damage response (ddr) regulating dna repair, cell cycle progression and, in case of non-repairable dna damage, triggers apoptosis. so far, a role of rac1 in the ddr has not been reported. based on its exceptional function as a regulator of transcription and because of its recently found ability to translocate to the nucleus, we hypothesize that rac1 may be involved in the ddr. to study the in vivo function of rac1 we used an inducible cre-based knockout mouse model (rac1 flox/flox/mxcre ). mice were treated with different doses of doxorubicin for different periods of time. we monitored gh2ax foci formation as a marker of dna strand breaks, used the masson-goldner staining for the detection of collagen accumulation, analyzed phosphorylated histone 3 as a marker of mitotic events and performed a tunel assay to detect apoptotic cells. in the absence of rac1 the basal mrna expression of pro-fibrotic ctgf was decreased. collagen levels were increased and mmp1 mrna expression was reduced in the liver of rac -/animals as compared with rac1 proficient animals. in addition we found more apoptotic cells in rac1 -/mice. 96 hours after treatment with the anthracycline derivative doxorubicin the number of gh2ax foci in rac1 -/animals was reduced in comparison to rac1 +/+ animals. we also found lower level of ctgf mrna expression and reduced amount of collagen in rac1 -/mice. none of these protective effects resulting from rac1 deficiency could be detected after administration of three consecutive doxorubicin injections over a time period of 21 days. there were no significant differences in the number of gh2ax foci or collagen accumulation. the mrna expression of ctgf was even higher in rac1 -/animals. furthermore the number of mitotic events was almost two times higher in the rac1 -/mice compared to the rac1 +/+ mice. summarizing, our findings show that impaired hepatic expression of rac1 protein is hepatoprotective against acute damage following doxorubicin exposure, but does not protect against doxorubicin-induced subacute toxicity. in vitro cytotoxicity of tbhq (tert-butyl-hydroquinone) braeuning a., vetter s., schwarz m. institut für experimentelle und klinische pharmakologie und toxikologie toxikologie, wilhelmstrasse 56, 72074 tübingen, germany at high concentrations, tert.-butyl-hydroquinone (tbhq), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. here we describe that treatment of murine 3t3 cells with tbhq in 96-well culture plates induces the death of untreated cells in neighboring wells on the same plate. the mechanisms underlying that effect were investigated. death of the seemingly untreated neighboring cells was caused by a more toxic and volatile tbhq oxidation product which was formed in a non-enzymatic process involving metal ions and oxygen. the unexpected perturbation of cytotoxicity testing by the volatile tbhq metabolite shows that not only metabolic processes, but also non-enzymatic mechanisms have to be considered as important parameters for in vitro assays. furthermore, our data show that even cells several wells distant from the site of treatment do not necessarily constitute proper "untreated" controls when cells are treated with tbhq, e.g. in assays aimed to analyze the activity of the tbhq-inducible nrf2 pathway. s14 056 angiotensin ii causes oxidative stress and dna damage in mouse kidneys via the angiotensin ii type 1 receptor brand s. 1 , amann k. 2 , schupp n. 1 1 universität würzburg institut für pharmakologie und toxikologie, versbacherstr. 9, 97078 würzburg, germany 2 universität erlangen-nürnberg institut für pathologie, krankenhausstraße 8, 91054 erlangen, germany angiotensin ii (ang ii), the reactive peptide of the renin-angiotensin-system, causes vasoconstriction and, in higher levels hypertension, which is connected with an increased cancer risk in the kidney. treatment of male c57bl/6 mice with ang ii results in the formation of superoxide radicals and dna damage in the kidney as well as in the heart. to answer the question if the dna damage is caused by hypertension or by elevated ang ii concentrations, mice were treated with different compounds: the angiotensin-converting-enzyme blocker ramipril, the ang ii receptor blocker ramipril, the ang ii receptor candesartan, the antioxidant tempol and the vasodilator hydralazine. the effect on blood pressure and renal function of ang ii-treated c57bl/6 mice was examined. treatment with ang ii led to a significant increase in blood pressure. candesartan and hydralazine led to a decrease, whereas intervention with ramipril and tempol had no effect. equal conditions could be found by examining renal function regarding the excretion of urinary albumin, which was ameliorated by candesartan and hydralazine. in addition, histopathological changes were investigated. there was significant glomerular damage and tubulointerstitial damage in ang ii-treated animals compared to control animals, which was significantly improved by candesartan and tempol. hydralazine and ramipril mitigated the observed renal damage but were less effective than candesartan. furthermore, the ang ii-induced formation of superoxide radicals in the kidney and the heart was slightly affected by all interventions. genomic damage, in the form of double strand breaks was prevented by the ang ii receptor antagonist candesartan and the antioxidant tempol. to sum up, the results from this study show that ang ii induces the elevation of markers of kidney failure and dna damage, which is prevented by substances lowering blood pressure like candesartan, showing the receptor responsibility for the induction of dna damage. actually by substances not lowering blood pressure like tempol, the oxidative stress and dna damage was ameliorated, showing the involvement of reactive oxygen species. optimization of the balb/c-3t3 cell transformation assay by coupling a drug metabolizing system brauneis m. d., steinberg p. stiftung tierärztliche hochschule hannover institut für lebensmitteltoxikologie und chemische analytik, bischofsholer damm 15, 30173 hannover, germany the analysis of the carcinogenic potential of chemicals plays an important role in toxicology. up to now the acquisition of such data requires a large amount of animal experiments. the aim of this study is to reduce the number of experimental animals being used by further optimizing the balb/c-3t3 cell transformation assay, an already well-established in vitro method. this method, which is also well suited for high throughput screening applications, allows a quantitative analysis of the aforementioned carcinogenic potential. the incubation of balb/c-3t3 cells (murine embryonic fibroblasts) with mutagenic compounds leads to a loss of contact inhibition between these cells, which results in the development of so-called foci. these foci can be distinguished by characteristic changes in cell growth behaviour, a result of the treatment with carcinogenic compounds, and their number is therefore directly related to the genotoxic potential of the latter. a major disadvantage of the "classic" balb/c-3t3 cell transformation assay is that a number of compounds initially require a metabolic transformation to gain their full genotoxic potential. hence, without prior metabolic transformation many chemicals are not detected as carcinogenic in the abovementioned test system. to overcome this drawback the balb/c-3t3 cell transformation assay has been coupled to a drug metabolizing system, in this case the so-called liver s9. in a first step the well-known genotoxic agents benzo[a]pyrene, aflatoxin b 1 and nnitrosodimethylamine were tested in this assay. all three compounds led to a concentration-dependent increase in the number of foci formed, whereby this concentration-dependent increase was observed in a non-cytotoxic concentration range. in a next step the balb/c-3t3 cell transformation assay will be coupled to further drug metabolizing systems as well as to the soft agar assay. this study is being financially supported by the stiftung set and the doerenkamp-zbinden foundation. adenylyl cyclases (acs) synthesize the second messenger camp. the family of acs consists of nine membranous and one soluble isoforms with ac5 and ac6 being the predominantly expressed isoforms in the heart. in the heart, acs integrate β-adrenergic (β-ar) signaling as the main physiological mechanism to improve cardiac performance. although ac5 and ac6 share high sequence homology, opposing effects on cardioprotection have been reported, where disruption of ac5, as well as overexpression of ac6 both exerting beneficial effects in heart failure. prospective pharmacological treatment of heart failure on the level of ac is under investigation. our study explored the impact of ac5 ko on ac-activities in the heart at a functional level. complementary, mrna expression studies of the β-ar-g-protein-ac signaling cascade were performed to detect possible compensatory alterations. hearts from 16-20 week old homozygote ac5 knockout and wild-type male littermates were examined in this study. ac activities where measured in cardiac membrane preparations from left ventricles. ac activities were assessed under β-ar and g-protein (g s) stimulation by isoproterenol, guanosine 5'-triphosphate (gtp) and 5'-o-(3thiotriphosphate) (gtpγs) as well as for direct activation by forskolin. relative mrna expressions for ac1-9, gs-, gi-a and β1-, β2-ar where measured by quantitative realtime pcr. surprisingly, assessment of basal, β-ar and g-protein-mediated ac-stimulation as well as direct activation by forskolin revealed no changes in ac activities. besides from detection of the ac5 knockout, mrna expressions analysis of ac1-9, gs-, gi-a and β1-, β2-ar did not detect any compensatory alteration. these findings suggest that proximal adrenergic signaling in the heart does not necessarily require ac5. whether physiological integration of beta adrenergic signaling in the heart is mediated by both isoforms ac5 and ac6, or can be attributed to one main isoform remains to be elucidated. melanocortin-promoted pka activation decreases ampk activity via erk-1/2 and lkb-1 in hypothalamic gt1-7 cells breit a., ellen d., gudermann t. goethestrasse 33, 80336 münchen, germany α-melanocyte stimulating hormone (α-msh)-induced activation of the melanocortin-4 receptor (mc4r) in hypothalamic neurons increases energy expenditure and inhibits food intake. intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of amp-activated protein kinase (ampk) that has recently been reported to enhance food intake in rodents. until now, it is not clear if α-msh affects ampk via direct intracellular signaling cascades or if the release of paracrine factors is involved. herein, we used a murine, hypothalamic cell line (gt1-7 cells) and monitored ampk phosphorylation at thr 172 which has been suggested to increase ampk activity. we found that α-msh dephosphorylated ampk at thr 172 and consequently decreased phosphorylation of the established ampk substrate acetyl-coa-carboxylase at ser 79 . inhibitory effects of α-msh on ampk were blocked by specific inhibitors of protein kinase a (pka) or extracellular-regulated kinases-1/2 (erk-1/2), pointing to an important role of both kinases in this process. since α-msh-induced activation of erk-1/2 was blunted by pka inhibitors, we propose that erk-1/2 serves as a link between pka and ampk in gt1-7 cells. furthermore, down-regulation of liver kinase b-1 (lkb-1), but not inhibition of calcium-calmodulin-dependent kinase kinase-β or transforming growth-factor-beta-activated kinase-1 decreased basal phosphorylation of ampk and its dephosphorylation induced by α-msh. thus, we propose that α-msh inhibits ampk activity via a linear pathway including pka, erk-1/2 and lkb-1 in gt1-7 cells. given the importance of the melanocortin system in the formation of adipositas detailed knowledge about this pathway might help to develop drugs targeting obesity. autism spectrum disorder (asd) is a complex neurodevelopmental disorder with dysfunction of social interaction and communication. a hitherto unknown complexgenetic principle of origin probably underlies asd. so far, more than 100 candidate genes were identified in literature. the patients affected with the monogenic timothy syndrome show multiorgan dysfunction including lethal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia and autism. this single gene disorder serves as a model disease for asd, giving insights in a possible pathophysiology. here, a point mutation in a highly-conserved region of the pore-forming subunit of the voltage-dependent calcium channel (ca v) cav1.2 gene (cacna1c) results in incomplete inactivation of the l-type calcium currents (splawski et al., cell 2004; 119:19-31) . functionally similar biophysical effects can be induced by structural variation β1-and β2-subunits of the voltagedependent calcium channels (herzig et al., faseb j. 2007; 21:1527-38; jangsangthong et al., pflugers arch. 2010; 459:399-411) . supported by findings in a meta-analysis of linkage data of asd patients (trikalinos et al., mol psychiatry. 2006; 11:29-36) , we are investigating a function-based candidate gene hypothesis linking the β2 subunit gene (cacnb2) with asd. we performed a case control study sequencing all exons and flanking intronic regions of cacnb2 in 155 patients with asd. we found three rare missense mutations in asd patients, but not in 259 unaffected controls. all three mutations occur at highly conserved positions and might alter protein function; additionally results one amino acid substitution highly probable in a post-translational modification by phosphorylation. so far, we characterized two of these mutations and also a phosphorylation-mimicking mutant in electrophysiological studies. all variants show a decelerated and incomplete time-dependent inactivation of the co-transfected cav1.2 subunit. furthermore, two variants exhibit a significant increased slope factor of voltage-dependent steady-state inactivation. we here present mutations in the β2 subunit gene of asd patients that result in a retardation of inactivation behavior, thus phenocopying the monogenic timothy syndrome mutations of cav1.2. β2 subunit mutations may influence neuronal function or development in some asd patients. jangsangthong, w., kuzmenkina, e., khan, i.f., matthes, j., hullin, r., and herzig, s. (2010) . inactivation of l-type calcium channels is determined by the length of the n terminus of mutant beta (1) subunits. pflugers arch 459, 399-411. herzig, s., khan, i.f., grundemann, d., matthes, j., ludwig, a., michels, g., hoppe, u.c., chaudhuri, d., schwartz, a., yue, d.t., et al. (2007) . mechanism of ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits. faseb j 21, 1527-1538. splawski, i., timothy, k.w., sharpe, l.m., decher, n., kumar, p., bloise, r., napolitano, c., schwartz, p.j., joseph, r.m., condouris, k., et al. (2004) . ca(v)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. cell 119, 19-31. trikalinos, t.a., karvouni, a., zintzaras, e., ylisaukko-oja, t., peltonen, l., jarvela, i., and ioannidis, j.p. (2006) . a heterogeneity-based genome search meta-analysis for autism-spectrum disorders. mol psychiatry 11, [29] [30] [31] [32] [33] [34] [35] [36] background: brain serotonin (5-ht) has been implicated in the regulation of food-intake. the ingestive effects of 5-ht are mediated by a number of different receptor subtypes under which the 5-ht1a-receptor plays a central role. former in vivo studies have shown an increased intake of food, elicted by 5-ht-receptor agonists. the aim of this behavioural pharmacologic project was to determine if the hyperphagic effect is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei or by postsynaptic 5-ht1a heteroreceptors in serotonergic terminal structures. methods: the effect of the 5-ht1a receptor agonist 8-oh-dpat (0.1, 0.5 or 1.0mg/kg) was investigated on feeding behaviour in non-food-deprived young-adult and adult nmri and transgenic l35 mice. l35 mice are characterized by an overexpression of postsynaptic 5-ht1a receptors. results: the administration of the 5-ht1a receptor agonist induced hyperphagia in all groups of mice, except for the adult transgenic mice which showed no drug effect. conclusion: the results confirm a key role of the 5-ht1a receptor in food intake. further, we make the assumption that the hyperphagic effect of 8-oh-dpat is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei which decreases 5-ht function in the central nervous system. it can be speculated that the aberrant feeding behaviour of the adult transgenic mice refers to a possible opposite role of the postsynaptic 5-ht1a receptors. these receptors might affect the release of neuropeptides in the hypothalamus. the efflux transporter abcc2 (mrp2) expressed at different compartment barriers is important for the elimination of various endogenous and exogenous compounds. with some evidence inflammatory processes regulate abcc2 expression and cause changes of absorption, distribution and clearance of a number of xenobiotics. the investigation of the influence of interleukin (il) 1β on abcc2 mrna and protein expression in various cell lines representing specific tissues is the aim of our study. a further aim is to characterize the signaling pathways regulating abcc2 expression while inflammation. three different cell lines a) hepg2 cells (liver tissue) and b) caco2 (colon tissue) both without naïve il1β expression and c) skhep1 cells representing physiological liver tissue with naive il1β expression, were stimulated with different concentrations of il1β (range 10 pg/ml to 10 ng/ml). over a period of 48h samples were taken at defined time points. abcc2 mrna and protein expression were quantified by qrt-pcr and western blot analysis, respectively. by using small molecule kinase inhibitors for signal transduction proteins (p38 mapk, akt, erk1 and jnk) we analysed the signal transduction pathways associated with il1β-mediated transcriptional abcc2 regulation. on abcc2 mrna level an up-regulation in caco2 cells (1, 35 fold) and hepg2 cells (3, 6 fold) within the first hour after stimulation with 1 ng/ml il1β was shown. in contrast skhep1 cells demonstrated a decreased abcc2 mrna expression (0,59-0,62 fold) in comparison to unstimulated controls. the abcc2 protein expression exhibited a time and il1β dependent regulation as well. the analysis for the signal transduction showed for p38mapk a moderat time dependet down regulated phosphorylation (15%) in hepg2 cells whereas it showed no effect in caco2 cells. concluding, the expression of abcc2 is regulated moderately by il1b in a concentration and time-dependent manner. interestingly, the effects are strongly tissue-dependent concerning abcc2 expression and signal transduction pathways and show partly contradictory results. the regulation of the different signaling pathways is currently subject of ongoing investigations. introduction: despite the remarkable success of imatinib treatment of chronic myeloid leukemia (cml), therapy resistance emerged as a major clinical problem. the aim of this study was to identify micrornas, which may serve as biomarkers for therapy response or predict pathways involved in pharmacoresistance of imatinib treatment. methods: blood was collected from 21 cml-patients, ten of whom responded to imatinib therapy. after rna extraction from leukocytes, we performed a taqman low-density array screen to determine the expression of 667 micrornas. statistical analysis using the 2 -∆ct method was performed. micrornas showing a p-value<0.01 and a fold change>2 were considered to be significantly differently expressed. in addition, by using microrna target prediction databases (targetscan 1 , mirdb 2 , pictar 3 , microcosm 4 , diana microt 5 ), selected putative target genes were further functionally investigated by the david bioinformatics database 6 . results: comparing treatment-naïve responders and non-responders four micrornas were identified to be deregulated that were predicted to target 97 genes, especially transcription regulators (21%). pathway analysis showed that six of the predicted genes are relevant in cancer pathways, four of which play a role in cml (smad4, nras, rb1, raf1). when comparing patients' expression profiles before and under treatment, seven micrornas were identified to be deregulated in responders and five micrornas in nonresponders. ninety-nine targets of the latter include transcription regulators (19%), but also cellular transporters (18%, especially uptake transporters of the slc-family). most target genes are involved in mapk signalling or endocytosis pathways. conclusion: analysis of microrna expression profiles revealed four micrornas involved in imatinib-response and 12 micrornas deregulated during imatinib treatment. predicted target genes code mainly for transcription factors as well as oncogenes relevant for cml and are involved in transporter expression and endocytotic processes. dissociations in the effects of beta2-adrenergic receptor agonists on camp formation and superoxide production in human neutrophils brunskole i. 1 , buschauer a. activation of the β2-adrenergic receptor (β2ar), a classically gs-coupled receptor, in neutrophil granulocytes results in an inhibition of inflammatory responses [1] , which could be further therapeutically exploited. the aim of the present study was to evaluate the effects of various β2ar ligands on cyclic adenosine 3',5'-monophosphate accumulation (camp assay) and n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced superoxide anion production (o2 •assay) in isolated human neutrophils, which are a physiologically relevant native test system. camp concentration in neutrophils was determined by hplc/tandem mass spectrometry, and o2 •formation was assessed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. (-)-isoproterenol, (-)-adrenaline, salbutamol and dobutamine were more potent in inhibiting fmlp-induced o2 •production than in stimulating camp accumulation. (-)-ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the camp assay, but could partially inhibit fmlp-induced o2 •production at higher concentrations. moreover, (-)-adrenaline and dobutamine were equi-efficacious in both assays whereas the efficacy of salbutamol was more than two fold higher in the o2 •assay. this suggests that salbutamol is able to stabilize a different receptor conformation than the other two ligands. thus, ligand-directed signaling via β2ar can also occur in human neutrophils. in addition, differences between the data from neutrophils and recombinant test systems [2, 3] were noticed, pointing to the problem of insufficient comparability of effects in recombinant and native test systems. the investigation of β2ar antagonists on neutrophil granulocytes is subject of ongoing work, in order to find out whether pkb values of β2ar antagonists in the camp assay and the o2 •assay are different. such differences were previously reported for β2ar antagonists in other test systems [4] . moreover, studies with protein kinase a inhibitors should give deeper insight into the signaling events in neutrophils that result in inhibition of fmlp-stimulated o2 •production and clarify how camp increase interferes with this events. agonist-selective internalization of the human 5-ht2a receptor buchborn t., kahl e., höllt v., koch t. otto-von-guericke-universität magdeburg institut für pharmakologie und toxikologie, leipziger straße 44, 39120 magdeburg, germany the serotonin 2a (5-ht2a) receptor is a g protein coupled receptor and the molecular target of lsd-like hallucinogens. downregulation of 5-ht2a receptors is an adaptive process considered relevant for the therapeutic action of diverse serotonergic antidepressants, such as ssris. since the antidepressant targeting of 5-ht2a receptors, however, is largely restricted to indirect agonists and/or antagonists, little is known about the mechanisms and implications of their regulation by direct agonists. in the present study we, therefore, investigated the capacity of various agonists to regulate the human ha-tagged 5-ht2a receptor by internalization. using immunocytochemical techniques in stably transfected hek293 cells, we show that agonists differ in their capacity to internalize the receptor. serotonin, quipazine and doi are the agonists most efficaciously internalizing the receptor, dmt and methysergide, on the other hand, hardly internalize; other agonists like psilocin, ergotamine and lsd induce low to intermediate internalization. the specificity of the agonistic effect was demonstrated by the 5-ht2a selective antagonist ketanserin, which blocked the agonist-induced internalization. in additional experiments, we show that the internalized 5-ht2a receptors colocalize with a488-labelled transferrin receptors, and that the internalization can be blocked by high molar sucrose; these results are indicative of a clathrin associated sequestration of 5-ht2a receptors in recycling endosomes. also, we demonstrate that the proteinkinase c activator pma efficaciously induces 5-ht2a internalization in the absence of an agonist, and that the doi-induced internalization can be blocked by the proteinkinase inhibitor staurosporine. we, thus, confirm previous findings that the activation of proteinkinases seems to be necessitated for the 5-ht2a internalization to occur. overall, we conclude that the internalization of the human 5-ht2a receptor is agonist-selective, and employs a proteinkinase (possibly pkc) dependent, clathrincoated endosome associated pathway. as there is recent evidence that the regulation of 5-ht2(a) receptors by agonists might have antidepressant (-like) properties, knowledge about the agonist-selective processing of 5-ht2a receptors could help to identify agonists most promising for future (pre-)clinical research. non-clinical safety assessment of homeopathic medicinal products: criteria for establishing a first safe dilution buchholzer m. -l., werner c., knoess w. bfarm bundesinstitut für arzneimittel und medizinprodukte zulassung 4, kurt-georg-kiesinger-allee 3, 53175 bonn, germany like all human medicinal products the homeopathic medicinal products for human use must demonstrate adequate safety. in general, they are regulated according to the analogue non-clinical safety principles (points to consider on non-clinical safety of homeopathic medicinal products of botanical, mineral and chemical origin, adoption by hma 2007). one particular approach is the recently introduced concept of a first safe dilution (fsd; introduction to the list of first safe dilutions, adoption by hma 2010) . this contribution summarizes the first experiences in establishing fsds of a selection of given homeopathic preparations by bfarm. for a given preparation the major toxicological concern and available data set is identified. this determines the safety assessment route: food regulation, permitted daily exposure (pde), threshold of toxicological concern (ttc) or lowest human recommended dose (lhrd/100). finally the acceptable amount/tolerable daily intake is derived and the respective fsd is calculated. for example the draft evaluation for reserpinum (ph. eur. method 4.1.1) and for atropine (ph. eur. method 3.1.1 or 4.1.1) based on lhrd leads in each case to a suggested fsd of d7 related to 10 g of preparation. furthermore, the draft evaluation for potassium iodide (ph. eur. method 3.1.1 or 4.1.1) based on food legislation emerged a proposed fsd of d6 related to 10 g of preparation. the concept of fsd combines a scientific and at the same time pragmatic approach in differentiated risk assessment of homeopathic medicinal products. impact of myricetin and its methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether in c. elegans büchter c. 1 , ackermann d. polyphenolic compounds ubiquitously present in herbal food are discussed to contribute to the health beneficial effects of a diet rich in vegetables and fruits. additional to a strong antioxidative activity of various flavonoids, most of these substances display a variety of other pharmacological properties. we investigated the flavonoid myricetin found in several species of berries, as well as the methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether. in this study caenorhabditis elegans was used as a model to explore the impact of myricetin and its methylated derivatives in vivo and to investigate molecular modes of action. myricetin (100 µm) caused an increase in mean and median adult lifespan of c. elegans. this longevity effect was associated with a decrease of the aging marker lipofuscin as well as a decrease in ros induction, measured by using the h 2dcf-da assay. however, myrictin failed to improve heat stress resistance, an attribute often associated with longevity in c. elegans. the methylated myricetin derivatives (100 µm) showed a decrease in lipofuscin accumulation and ros induction and they further improved the heat stress resistance. in order to elucidate the basis of the life prolonging action of myricetin, we investigated its influence on factors known to have important functions in stress response and the regulation of aging, namely the foxo homologue daf-16, the nad + -dependent protein deacetylase sir-2.1 and the heat-shock transcription factor hsf-1, respectively. lifespan extension by myricetin disappeared in daf-16 and sir-2.1 loss of function mutant strains, showing the effect is at least partially dependent on these signaling molecules. by using a hsf-1 loss of function mutant strain of c. elegans, it was further shown that the life prolonging effect of myricetin is independent of hsf-1. in conclusion, our results indicate that the life prolonging effect of myricetin is at least in part dependent on daf-16 and sir-2.1, probably due to a modified expression of target genes. stimulatory and inhibitory control of phospholipase c-gamma 2 bühler a., walliser c., becker l., gierschik p. universität ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany activation of phospholipase c-γ2 (plcγ2) upon b cell antigen receptor (bcr) stimulation has been implicated to be a critical step in the bcr-mediated calcium signaling. therefore it is important to understand the mechanisms of how the activity of plcγ2 is stimulated and inhibited. the mammalian plcs are divided into six subfamilies, designated β, γ, δ, ε, ζ, and η. within the plcγ subfamily, the two plcγ isoforms share a number of features that are distinct from those of the other plc subfamilies. the most striking difference is the insertion of additional domains between the catalytic subdomains x and y. this specific array (sa) contains a second, split pleckstrin homology (spph) domain, consisting of two halves separated by two src homology 2 (sh2) domains and one src homology 3 (sh3) domain. there is abundant evidence in the literature that plcs are autoinhibited in their basal state by structural elements within their x/y linker, pointing to a conserved role of the x/y linker in autoinhibitory regulation of plc isozymes. data from our group show that plcγ 2 is also regulated by autoinhibitory elements within its specific array (walliser et al., 2008; everett et al., 2011) . our recent data demonstrated that plcγ2 is negatively regulated by its sa. specifically, within the sh domain tandem, the c-terminal sh2 (sh2c) and the sh3 domain in combination, but not either one alone, cause the strongest autoinhibitory control of plcγ2. plcγ2 has been shown to be phosphorylated at tyrosine residues 753 and 759 upon bcr stimulation (kim et al., 2004) . both tyrosines are located in the linker between the sh2c and the sh3 domain, which we have shown to be the major elements involved in autoinhibitory regulation of plcγ2. interestingly, a novel phosphorylation site in plcγ2 was found in non-small cell lung cancer (nsclc) tissue which is located at tyrosine residue 733 (rikova et al., 2007) . in this work, we demonstrate, for the first time, the activation of plcγ2 by phosphomimetic mutations in these three positions and the functional interplay of the three tyrosine phosphorylation targets. most interestingly, mimicking phosphorylation of tyr733 is critical to fully activate the enzyme. the results not only point to a crucial role of plcγ2 in pulmonary tumorigenesis, but also prompt and stimulate the search for the protein kinase involved in phosphorylating plcγ2 at tyr733. molecular characterization of hepatotoxic effects of perfluorooctanoic acid (pfoa) buhrke t., scharmach e., lampen a. bundesinstitut für risikobewertung (bfr) lebensmittelsicherheit, max-dohrn-str. 8-10, 10589 berlin, germany perfluorooctanoic acid (pfoa) is an industrial chemical that is used for the fabrication of numerous products with oil-, dirt-and water-repellent properties. pfoa is resistant to chemical, thermal and biological degradation and has become a global contaminant of soil, water, air and food in the meantime. the toxicological data of pfoa give cause for concern as the substance was shown to damage the liver of rodents and to impair embryo development. currently, the hazard potential of pfoa for humans is controversially discussed. in this study the human liver cell line hepg2 was employed to analyse the hepatotoxic effects of pfoa on the cellular and on the molecular level. pfoa was shown to stimulate cellular proliferation at concentrations in a range between 5 µm and 25 µm. at concentrations higher than 25 µm the substance was cytotoxic to the cells (ic 50 47µm). cytotoxicity was not due to apoptotic mechanisms as no increase of caspase activity was detected up to a level of 100 µm pfoa. on the molecular level pfoa is known to act as an agonist of the peroxisome proliferator-activated receptor alpha (pparα), and the observed hepatotoxic effects in rodents are associated with pfoa-mediated pparα activation. here we show that pfoa has the capacity also to activate the human isoform of pparα. additional human nuclear receptors were tested for activation by pfoa, and pparγ as well as the pregnane x receptor (pxr) were shown to be activated at high concentrations of pfoa whereas pparδ and the liver x receptor alpha (lxrα) were insensitive to activation by pfoa. notably, we observed a significant inhibition of the activity of the hepatocyte nuclear factor 4α (hnf4α) by incubating the cells already with moderate concentrations of pfoa at a level of about 1 µm. these findings indicate that additional, pparα-independent mechanisms may contribute to the observed hepatotoxicity of pfoa. the elucidation of novel modes of action of pfoa is relevant for the ongoing risk assessment of the substance. s17 070 human breast stem cells as a toxicological model for endocrine disruptors, such as soy isoflavones stempin s. 1 , bumke scheer m. (kao et al., 1995) . two daughter cell lines were developed from m13sv1 after x-ray irradiation (m13sv1 r2) and an additional transfection with a mutated erbb2 oncogene (m13sv1 r2-n1), resulting in high and low tumorigenicity respectively and showing a change in estrogen response after growth in minimal media (wang et al., 2010) . isolated isoflavones are currently widely used in the treatment of postmenopausal symptoms of women. according to the stem cell theory of carcinogenesis, breast stem cells are the ideal target for the proposed research. however, the epidemiological data to the effect of isoflavone intake on breast cancer is contradictory. therefore, we want to develop a toxicological model using these human breast stem cell lines to test the effect of endocrine disruptors, such as soy isoflavones in a human relevant model. in the present study we analyzed the effect of the phytoestrogen genistein, the most intensively studied soy protein, on the differentiation of the 3 hbec lines. the expression of different luminal epithelial cell markers, estrogen receptors and stem cell markers was measured on the mrna level by quantitative real time pcr. the analysis of several of these markers was also performed on the protein level using western blot. additionally, a broad number of genes related to breast cancer and estrogen receptordependent signal transduction were studied using a commercial pcr-array. in parallel we are also analyzing the changes on the protein level using 2d gel electrophoresis. we want to use this panel of different markers to establish a toxicological model that can be used in the future to analyze a wide range of different endocrine disruptors. kao cy, nomata k, oakley cs, welsch cw, chang cc. (1995) bronchial asthma is a common inflammatory disease of the airways whose occurrence has increased dramatically over the past decades. histamine plays an important role in mediating the inflammatory response leading to characteristic symptoms like wheezing, coughing, chest tightness, and shortness of breath. since antagonizing the histamine h1-receptor (h1r) shows no ameliorating effects on asthmatic symptoms, h4r antagonists may be new drugs for asthma therapy. in addition to the h3r-and h4rselective antagonist thioperamide, the selective h4r antagonist 1-[(5-chloro-1h-indol-2yl)carbonyl]-4-methylperazine (jnj7777120) is used in pharmacodynamic studies. a correct interpretation of the collected data requires the detailed knowledge of the pharmacokinetics of the applied substances. for this reason, we developed a fast and robust method based on high performance liquid chromatography coupled to tandem mass spectrometry (hplc-ms/ms) which allows the simultaneous quantitation of thioperamide and jnj7777120 as well as the selective h3r antagonist 1-({4[3-(piperidin-1-yl) propoxy]phenyl}methyl)piperidine (jnj5207852) in murine plasma and lung tissue. the treatment of plasma samples based on protein precipitation performed with a mixture of methanol and 0.2 m znso4 using 30 µl of plasma. analyte extraction from lung tissue was achieved by treating 150 -200 mg of tissue with a mixture of ethanol and water followed by rigorous mixing using a fastprep-system. ten µl of the extracted samples were transferred to a synergy polar-rp 80a mercury column (10 x 2mm; 4µm) connected to a polar-rp security guard. chromatographic separation was performed via a gradient using an acetate buffer (ph 5) and methanol at a flow rate of 0.4 ml/min. the analytical run-time was 5 minutes. for plasma samples the assay was linear over a concentration range of 0.078 -40 µg/ml for jnj7777120 and jnj5207852, and 0.313 -40 µg/ml for thioperamide, respectively. in tissues, thioperamide could be quantified in a concentration range of 0.008 -2 µg/sample, jnj7777120 and jnj5207852 in a range of 0.004 -2 µg/sample. our results show that the developed hplc-ms/ms method is suitable for the quantitation of all tested histamine-receptor ligands in murine plasma and lung tissue. the functionality of the heart greatly depends on strict homeostasis and interplay of a range of signalling cascades. deregulation of either one is always harmful and eventually detrimental for life. some of the most relevant signals in the adult heart are triggered by the stimulation of g protein-coupled receptors such as adrenergic or angiotensin receptors. those in turn are modulated by a small subset of kinases, the g protein-coupled receptor kinases (grks). interestingly, grks, which for the longest time were believed to regulate only g protein-coupled receptors were shown to modulate also non-receptor-mediated signalling pathways. by now it is well documented that grk5 plays important roles in both the physiological as well pathological setting of the adult heart. in spite of the important functions of grks in the adult heart, it must be assumed that grk5, one of the two main cardiac grks, may also be involved in signal modulation in the course of heart development. deregulation of grk5-dependent pathways may very well be causal for impaired cardiac development up to congenital heart disease. in fact, grk5 is already expressed during embryogenesis and we can detect it in the developing heart. however, grk5 has not been studied yet for a potential function during embryonic development in general or heart formation in particular. we have established zebrafish as a very time and cost efficient vertebrate model to investigate the role of grk5 on cardiac signalling and development. tools for grk5 specific loss-as well as gain-of-function analyses have been developed in our lab. we revealed an unexpected role of grk5 in the development of left-right asymmetry in zebrafish. clinically, this has been associated with disorders such as heterotaxy and other syndromes linked to ciliary dysfunction. many of those disorders are known to affect proper heart development resulting for example in septum defects or in detrimental translocation of the outflow tract. precisely, depletion of the close homolog of human grk5 in zebrafish mirrors the human syndrome called heterotaxy by displaying randomized placement of inner organs, aberrant heart looping and disrupted valve formation in the heart. in addition, loss of zebrafish grk5 results in a lower heart rate as well as dilatation of the embryonic heart at later stages of development. therefore, we believe that grk5 may potentially serve as a candidate gene for congenital heart disease. identification of a hcn3 interacting protein in mouse brain cao-ehlker x., hammelmann v., zong x., fenske s., biel m. department of pharmacy, center for drug research ludwig-maximilians-universität, munich center for integrated protein science cipsm, butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization activated cyclic nucleotide-gated cation channels (hcn) pass a depolarizing current (ih) that is involved in cardiac pacemaking and the control of numerous basic functions in neuronal circuits. the four hcn channel types (hcn1-4) display specific expression pattern in brain suggesting that each channel fulfills a distinct physiological function. while hcn1, hcn2 and hcn4 channels have been studied in quite some detail there is only little information on the particular role of the hcn3 channel. as an important step towards achieving a better understanding of hcn3 function we set out in this study to identify proteins that are assembled with hcn3 in brain tissue. to this end we performed a yeast two hybrid screen with a mouse cdna library using the hcn3 c-terminal domain as bait. several proteins were obtained and confirmed for interaction with hcn3 using heterologous coexpression in hek293 cells. here, we provide an in-depth analysis of the functional interaction between hcn3 and one of the identified interacting proteins (hip3.1). we show that hip3.1 physically binds to hcn3 channels in vitro and in vivo. still several open issues remain to be clarified i.e. the precise function of tpc1 and its tissue-specific and subcellular distribution. therefore we established a mouse model with a general deletion of tpcn1 and generated a series of tpc1 antibodies. using these tools we investigate the closer molecular and vesicular environment by different biochemical approaches i.e. affinity purification from native tissue derived from wild-type and as a control from knock-out mice, density gradient based vesicle separation, fluorescence activated organelle sorting (faos), total internal reflection fluorescence (tirf) and confocal microscopy. so far, we confirmed the tpc1 knock-out model by our self-generated tpc1 antibodies. tpc1 knockout mice are viable and do not show any obvious deficits. to isolate tpc1 containing vesicles or protein complexes, tissue or cell culture derived material was prepurified by sucrose density gradient centrifugation. for a subsequent mass spectrometric analysis this preparation was taken as a source material for coimmunoprecipitation or faos respectively. in another approach the migration pattern of tpc1 containing endosomes on linear density gradients was compared with a series of endolysosomal markers i.e. different rab-, er-, golgi-and lysosomal antibodies. potentially interesting markers were then in turn analyzed for their co-localization with tpc1 by confocal microscopy/tirf. by combining these results with that from mass spectrometric analysis of faos samples we collect data to get detailed information on the precise endolysosomal distribution pattern of tpc1. foxos are involved in a wide spectrum of cellular functions, including cell proliferation, apoptosis and regulation of oxidative stress. in order to identify novel target genes of foxo transcription factors and to achieve further insight into their role in cancer cells, dna microarray analysis was performed using wild type mcf-7 breast cancer cells and mcf-7 cells overexpressing foxo. we found that several genes involved in the tnf receptor/nf-κb pathway were differentially regulated. one of the genes that was identified to be up-regulated in foxo4 overexpressing cells was a20 a negative regulator of nf-κb signaling pathway. at both mrna and protein level foxo4-dependent up-regulation of this ubiquitin modifying enzyme was confirmed. to determine whether a20 is a direct target of foxo4, a luciferase reporter containing a 1.2 kb of the a20 promoter was co-transfected with different amounts of foxo4 wild-type expression construct. foxo4 induced a dosedependent increase in a20 promoter activity, supporting the assumption that a20 is a direct transcriptional target of foxo4. overexpression of foxo4 led to decreased activity of nf-kb signaling pathway as confirmed by reporter gene and nf-kb specific elisa assays. in addition, mcf-7 cells can be sentisized to tnf-α mediated cytotoxicity which is assocciated with a dimineshed activation of nf-κb. altogether, we identified a20, an ubiquitin modifying enzyme, as a novel foxo4 target gene. our data implicate that sustained foxo4 expression may be involved in regulation of tnf receptor/nf-κb pathway and leading to reduced cell survival. trpm7 is a bi-functional protein consisting of a transient receptor potential ion channel segment linked to an α-type protein kinase domain. trpm7 is essential for motility, proliferation and cell growth. up-regulation of trpm7 function is involved in anoxic neuronal death, cardiac fibrosis and tumor cell proliferation. recently, we have demonstrated that the recombinant trpm7 channel is inhibited by the known modulators of sk1-3 channels such as antimalarial plant alkaloid quinine, cyppa, dequalinium, ns8593, ska31, ucl 1684. the most potent of these compounds, ns8593 (ic50 1.6 µm), interferes with the regulation of trpm7 by cytosolic mg 2+ . here we show that ns8593 (10 µm) fully and reversibly inhibits native trpm7-like currents in hek 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. furthermore, we examined whether targeting of the native trpm7 currents by ns8593 would impact cellular processes known to be affected by a genetic inactivation of trpm7. we found that ns8593 (10-30 µm) suppressed motility of hek 293 cells without a detectable effect on cell viability. taken together, our findings indicate that ns8593 is a potent and reversible inhibitor of endogenous trpm7 currents and may be a good candidate drug for pharmacological targeting of trpm7. sulfur mustard (2,2'-dichlorodiethylsulfide; sm) is a highly toxic and mutagenic warfare agent classified as a weapon of mass destruction. as soon as sm was first used as a warfare agent, research aimed at the development of an effective antidote was launched. early studies with first-generation inhibitors of poly(adp-ribose) polymerases (parp) have revealed promising therapeutic potential in sm-induced skin injury, but the underlying mechanism remains elusive. the current renaissance of parp inhibitors in cancer chemotherapy has revived the discussion on their use for treatment of sm injury. thus we established a comprehensive study aiming the elucidation of the role of parp in sm pathology based on model substance 2-chloroethyl ethyl sulfide (cees), which is not classified as warfare agent. we have recently demonstrated that parp becomes rapidly activated in living human keratinocytes (hacat) after treatment with cees. the maximal parp activity was observed 10 minutes after treatment with 3 mm cees. the activation was transient and dose dependent. to our knowledge this is the first demonstration of parp activation after treatment with mustards in the context of live cells. an important question is how parp-1 becomes activated upon treatment with mustards. parp-1 is a first-line protein involved in the cellular response to dna strand breaks. however, mustards do not directly induce large numbers of such lesions. one possibility is that parp-1 is activated by dna breaks incorporated as base excision repair (ber) or nucleotide excision repair (ner) intermediates. thus, we performed knockdown experiments of ape1 and ercc1, i.e. endonucleases involved in ber and ner, respectively. the reduction of ape1 expression had no effect on parp activity. surprisingly, the knockdown of ercc1 almost completely abolished the cellular par production after cees treatment. the functional consequence of the errc1-parp cross-talk with regards to adduct removal is under investigation. however, our present data indicate that parp activity is not obligatory for the survival of cells upon cees treatment, as revealed by the lack of effect of the potent parp inhibitor abt-888. expression and activity of g protein coupled receptor kinase 2 (grk2) are elevated in several conditions of compromised heart function. although grk2 inhibition has been characterized as a promising therapeutic strategy in heart failure, a specific grk2inhibitor is not available. raf kinase inhibitor protein (rkip) inhibits raf1 but it also acts as a physiological inhibitor of grk2 upon phosphorylation by pkc at serine153. a detailed understanding of the rkip/grk2 interaction may help to identify inhibitory compounds for grk2. since phosphorylation often induces homo-oligomerization of proteins, we investigated whether this could be implicated in switching rkip from a raf1-into a grk2-inhibitor. co-immunoprecipitation assays showed that rkip self-association was substantially increased after pkc-mediated phosphorylation of rkip. rkip mutants either lacking or mimicking s153 phosphorylation confirmed that this phosphorylation is indeed a prerequisite for rkip/rkip association. cross-linking experiments with myc-tagged rkip in living cells or with purified rkip revealed that rkip phosphorylation by pkc promotes rkip dimers -not oligomers. to test whether dimerization is a critical step for the association of rkip with grk2, we generated a peptide to inhibit rkip dimerization. intriguingly, the peptide did not only prevent rkip dimerization but also attenuated rkip/grk2 association. this implicates, that dimerization of rkip is essential to bind grk2. to determine whether rkip dimers consequently inhibit grk2 activity, we established rkip mutants with high tendency to form dimers. subsequent functional analyses demonstrated that enhanced dimerization of rkip indeed translates into increased grk2 inhibition. we conclude that pkc-mediated phosphorylation of rkip is important for dimerization and that these dimers are essential for grk2 binding and inhibition. our results reveal new insights in the molecular mechanism of rkip/grk2 interaction and will help to develop specific grk2 inhibitors. expression and function of trpm3 ion channels in epithelial mdck2 cells dembla s., meiser j., philipp s. university of saarland institute for experimental and clinical pharmacology and toxicology, kirrberger str. 1, 66421 homburg, germany trpm3 proteins build ca 2+ permeable cation channels [1] activated by steroids [2] and sensitive to increased temperatures [3] . trpm3 channels are expressed in pancreatic ßcells as well as neurons of the dorsal root ganglion, where they act as mediators of insulin release [2] or as nociceptors of noxious heat, respectively [3] . however, northern blots and in situ hybridization experiments revealed that trpm3 is also expressed in epithelial cells of the choroid plexus and the ciliary body [1] as well as in the kidney. pcr analysis of different epithelial cell lines indicated that trpm3 is also expressed in madin-darby canine kidney2 (mdck) cells. quantitative analysis of trpm3 expression by qrt-pcr revealed a ~ 5 fold upregulation in mdck2 cells grown in confluency compared to well separated, proliferating cells. in contrast the level of expression of trpm7, a related ion channel described as regulator of proliferation in other cell types, remained constant. hek293 cells overexpressing trpm3 channels did not proliferate in the presence of the trpm3 agonist pregnenolone sulphate. however, as indicated by impedance analysis, the proliferation of mdck2 cells in the presence pregs was only slightly affected. when we analysed the transepithelial resistance (ter) of mdck2 epithelial cells in transwells as a measure for the formation of tight junctions, we found that the ter of cells grown in the presence of pregs was reduced. interestingly, ca 2+ imaging experiments using fura2 revealed that pregnenolone sulphate induces ca 2+ entry in well separated mdck2 cells but not in cells growing in confluency. we hypothesize that trpm3 might act as a regulator of cell proliferation and/or the formation of tight junctions in mdck2 cells. inhibition of grk2 by rkip improves cardiac contractility and structure in a transgenic mouse model of heart failure denzinger s., schmitt j. p., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany the raf kinase inhibitor protein (rkip) has been identified as a physiological inhibitor of g-protein coupled receptor kinase 2 (grk2). grk2 initiates g protein coupled receptor (gpcr) desensitization. since expression and activity of grk2 are upregulated in human heart failure, it has been proposed that grk2 inhibition may resensitize badrenergic receptor activity in heart failure patients. in this study, we evaluated chronic grk2 inhibition by rkip as a potential strategy to improve cardiac function in heart failure. to analyse the effect of rkip on heart failure, rkip transgenic mice were crossed with mice carrying a mutation in phospholamban (pln r9c ). pln r9c causes severe heart failure and premature death in humans and transgenic mice. cardiac function was significantly improved in the presence of rkip as shown by left ventricular catheterization and echocardiography. expression of heart failure marker genes anf and bnp was indistinguishable between wild-type mice and mice co-expressing rkip and pln r9c . in line with these findings, the life span of double transgenic mice was significantly prolonged compared to pln r9c transgenic mice. slow calcium transport into the sarcoplasmatic reticulum was characterised as cause for dilatated cardiomyopathy of pln r9c transgenic mice. since western blot analyses of rkip transgenic heart lysates showed increased phosphorylation of important regulators of cardiomyocyte relaxation, we analysed calcium transients and contractility of isolated cardiomyocytes as possible mechanism of the rkip mediated rescue. in the presence of rkip, calcium reuptake into the sarcoplasmatic reticulum was accelerated and cardiomyocyte relaxation improved. furthermore, coexpression of rkip significantly attenuated pathological cardiac remodelling. interstitual fibrosis and apoptotic cells were quantified in histological sections after sirius red-and tunel-staining. this study revealed a protective function of rkip in a genetic mouse model of human dilated cardiomyopathy by improving cardiac contractility and attenuating interstitial fibrosis and apoptosis. a detailed understanding of this rescue may help to find a new therapeutic strategy to improve cardiac contractility in heart failure. gαi-proteins comprise a group of three highly related members characterized by specific expression patterns. based on previous work of gi-mediated signaling pathways in cardiomyocytes and platelets, we checked gαi expression in mouse heart and platelets. the analysis revealed the presence of gαi2 and gαi3 with gαi2 as the predominant isoform. gene-targeted mice lacking either gαi2 or gαi3 were analyzed to unravel the physiological role of gαi-proteins in the cardiovascular system. extraordinarily prolonged bleeding times in gαi2-deficient animals were an obvious phenomenon. detailed analysis using isolated platelets gαi2-deficient mice exhibited reduced platelet activation and attenuated aggregation in response to stimulation by various agonists accompanied by reduced thrombus formation and diminished stability on a collagen-coated surface. employing in vivo injury/thrombosis models revealed abrogated thrombus formation selectively in gαi2-deficient mice. comparable results were obtained in experiments using mice with megakaryocyte/platelet-specific gαi2deficiency. to assess the pathophysiological consequences of platelet gαi2 function, we challenged these mice in experimental models of myocardial and cerebral ischemia. the results clearly show that platelet-gαi2-deficient mice were protected from both, myocardial and cerebral ischemia. in contrast, conventional gαi2-deficient mice subjected to the heart ischemia/reperfusion model exhibited a significantly increased susceptibility to ischemic injury as compared to wild type controls. in contrast, gαi3deficient mice were strongly protected from injury. thus we suggest that gαi2 and gαi3 play distinct roles in major cardiovascular disorders pointing to specific, non-redundant functions of these two highly related gαi isoforms. the cgmp signaling pathway is activated by nitric oxide (no), natriuretic peptides (anp, bnp & cnp), and cgmp-elevating drugs. it regulates several physiological functions such as smooth muscle relaxation, platelet inhibition, and cell growth and differentiation. recent studies indicate that cgmp signaling might also play a role in tumorigenesis, but the cellular and molecular mechanisms of cgmp's potential pro-and/or anti-tumor activities are not well understood. this study has examined the expression and function of components of the cgmp pathway in melanoma cells of murine and human origin. we have found that mouse b16 melanoma cells specifically express the alpha isoform of the cgmp-dependent protein kinase type i (cgkialpha) but not the beta isoform. treatment of intact cells with the membrane-permeable cgmp analog 8-br-cgmp induced the phosphorylation of cgki substrates, vasodilator stimulated phosphoprotein and phosphodiesterase 5. anp and cnp, ligands of the membrane-bound guanylyl cyclase gc-a and gc-b, respectively, did also activate the endogenous cgmp/cgki pathway. however, b16 melanoma cells did not respond to dea-no, which stimulates no-sensitive soluble guanylyl cyclases. interestingly, activation of cgmp/cgkialpha signal transduction was associated with an increase in erk1/2 and p38 phosphorylation, growth and migration of b16 melanoma cells. similar results were obtained with wm1205 human melanoma cells. in conclusion, we have identified a gc-a/gc-b/cgmp/cgkialpha pathway in melanoma cells, which stimulates tumor cell growth and migration in vitro. pharmacologic inhibition of cgmp signaling may offer a promising strategy for the treatment of melanoma. an increasing body of evidence supports important roles for voltage-gated calcium channels in idiopathic generalized epilepsies (iges), however which calcium channels participate in ige pathogenesis and how has yet to be fully understood. recently, it has been proposed that cav2.3 (r-type) and t-type calcium channels jointly contribute to oscillatory bursting in the reticular thalamus (rt) 1 , which is associated with absence epilepsy. cav3.2 is one of the two t-type calcium channels known to be expressed in the rt 2 . it has been demonstrated that ablation of either cav2.3 or cav3.2 reduces susceptibility to experimentally induced epilepsy 3;4 and in addition that both channels share several pharmacological properties [5] [6] [7] . to gain further insight into interacting mechanisms of these two channels in epilepsy, we tested cav2.3(-|-), cav3.2(-|-) and cav3.2(-|-)xcav2.3(+|-) mice side-by-side in the kainic acid model of epilepsy. we provide first in vivo data supporting a synergistic mode of action for cav2.3 and cav3.2 calcium channels in epileptogenesis. the deubiquitinase cyld regulates mechanisms of rip1/rip3-dependent necroptosis in neuronal cells diemert s. 1 , krieg s. vivo model of cerebral ischemia, we found, that cyld -/-mice exhibit significantly reduced infarction volume compared to control littermates. overall, these data reveal a role for cyld in rip1/3-dependent mechanisms of necroptosis in a model of glutamate toxicity in neuronal cells and further suggests cyld-mediated mechanisms of neuronal cell death as a potential therapeutic target after acute brain injury in vivo. cyanamide-mediated inhibition of n-acetyltransferase 1 dierolf d., bonifas j., blömeke b. university trier department of environmental toxicology, universitätsring 15, bldg. n, 54286 trier, germany cyanamide has been used for decades for medical purposes in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolised in vivo mainly via coenzyme a dependent n-acetylation by n-acetyltransferases. reported to be a substrate for n-acetyltransferases, cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for nacetyltransferases. therefore a more detailed investigation of its interrelations with nacetyltransferases was performed. we analysed nat1 enzyme activities after incubation of thp-1 cells with cyanamide for 24h, and found that the metabolic conversion of the classic substrate para-aminobenzoic acid was significantly reduced at physiologically relevant concentrations. in detail a significant dose-and time-dependent nat1 protein inhibition was observed for 100 and 1000 µm cyanamide using over-expressed human recombinant nat1 (insect cell cytosol containing recombinant human nat1*4). however, no inhibition was found in the presence of recombinant nat2*4. as we also provide evidence that cyanamide is not metabolised via coenzyme a dependent nacetylation in vitro by human nat1 or nat2 cytosol, by thp-1 cells or by human liver cytosol, we can conclude that this inhibition is not based on substrate-dependent downregulation of nat1. further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of nat1, leading to its rapid inhibition. since the presence of the reduction agent dithiothreitol did not reverse the results it could be that it is possibly not caused by oxidative processes. in sum these data indicate that cyanamide is able to interact with cysteine residues of human nat1, which causes its inhibition but not by a substrate-dependent mode of action. taken together our results show, that cyanamide is not n-acetylated by human nats, but might modulate nat1 dependent detoxification and activation of arylamines. dissecting the signal transduction pathway of acute hypoxic vasoconstriction (hpv) in precapillary pulmonary arterial smooth muscle cells ( low levels of oxygen in the pulmonary airways induce acute hypoxic pulmonary arterial vasoconstriction (acute hpv) redirecting blood flow to normoxic areas of the lung to assure optimal uptake of oxygen during ventilation. acute hpv lasting several minutes occurs predominantly in the precapillary region of the pulmonary vascular tree [1] . therefore, precapillary pulmonary arterial smooth muscle cells (pasmc) have been suggested as sensor as well as effector cells and trpc6 a member of the classical transient receptor potential (trpc) ion channel family was identified to be essential for the initiation of ca 2+ influx and the subsequent contraction of pasmc [2] . however, the underlying oxygen sensor and the exact signal transduction pathway(s) in pasmc have not been fully elucidated yet. by using gene-deficient mouse models as well as downregulation of potential candidate proteins by specific small interfering rnas (sirnas), we aim to dissect signaling cascades of trpc6 channel activation in acute hpv. for pasmc isolation and culture from mice we use a technique based on magnetic separation of intrapulmonary arteries originally developed in rats [3] . trpc-expression in freshly isolated and passaged pasmc cultured in low (5%) and high fetal bovine serum (25%) was analyzed. interestingly higher passage numbers resulted in a significant down-regulation of trpc1 and trpc6 the most predominantly expressed channel monomers in pasmc, while different serum concentrations resulted in no significant changes in their expression rates. sirnas were designed, transfected and successfully tested in hek293 cells and pasmc. initial results of the dissection of the signal transduction pathway activating trpc6 and inducing acute hpv in pasmc will be presented. references [1] staub, n. c. (1985) . site of hypoxic pulmonary vasoconstriction, chest 88, 240s-245s. [2] weissmann, n. et al. (2006) . classical transient receptor potential channel 6 (trpc6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange, proc. natl. acad. sci. u.s.a. 103, 19093-19098 trpv5 is a highly selective calcium channel expressed in various tissues amongst others in placenta. the channel may be involved in transcellular calcium transport in epithelial tissues thereby playing some role in calcium homeostasis of the body. in the placenta trpv5 is assumed to contribute to the maternal-fetal calcium transport. most probably trpv5 is part of a multiprotein channel complex but most of the components of this complex are unknown so far. our aim is to find interaction partners of the trpv5 protein in the placenta that might contribute to the regulation of the trpv5 protein function. therefore we expressed the intracellularly located n-and c-terminal parts of trpv5 (aa 1-330 and 571-723) as trpv5-gst (glutathione-s-transferase)-fusion proteins and used the purified recombinant proteins for pulling down proteins from human placenta cell extracts. the proteins pulled down by this approach were analysed by mass spectrometry. we identified several potential trpv5 interacting proteins which were not associated with the gst protein only. one of the proteins which was highly enriched with the n-terminal part of the trpv5 protein is calpain 6. in contrast to the classical calpains (calcium activated cystein proteases), calpain 6 is unique in that it lacks the cysteine residue in the active site. calpain 6 is mainly expressed during embryogenesis and is reported to be involved in cytoskeleton stabilisation but with unknown function in placenta. the interaction between trpv5 and calpain 6 was confirmed in reciprocal pulldown experiments and the trpv5 binding region for calpain 6 was narrowed down by using overlapping n-terminal trpv5-gst fusion proteins. after injection of trpv5 crna into xenopus laevis oocytes calcium uptake into the oocytes was measured; this uptake was largely reduced after co-injection of the calpain 6 crna. in further experiments we want to study potential regulatory effects of the trpv5 protein on the calpain 6 function in cell culture models. comparative studies on the effects of the human carcinogen inorganic arsenite and its recently identified thiolated metabolite thio-dma v on human urothelial cells ebert f., leffers l., unterberg m., schwerdtle t. universität münster institut für lebensmittelchemie, corrensstr. 45, 48149 münster, germany it has been demonstrated that chronic ingestion of 50-200 µg/day inorganic arsenic is associated with an increased risk for cancers of the skin, the lung and the bladder, but until now the underlying toxic modes of action are still under debate. in this context, in the last five years one main focus has been given to the role of human inorganic arsenic metabolism and nowadays it is generally accepted that human biomethylation contributes to inorganic arsenic induced carcinogenicity. due to further improvements in arsenic speciation techniques recently a new thiolated arsenite metabolite, the thio analogue of the well known metabolite dimethylarsinic acid (dma v ), the so called thiodimethylarsinic acid (thio-dma v ), has been discovered in human biological samples. after synthesizing and analytically characterizing this metabolite (bartel et al. 2011 , j toxicol. 2011 we investigated its toxic effects in direct comparison with ias iii in human urothelial cells. thereby cell cycle studies revealed a g2/m-and s-phase arrest as well as subg1 peak formation in case of thio-dma v . moreover, thio-dma v induced apoptosis (subg1, caspase 3 activity) at lower concentrations and earlier time points as compared to ias iii . most likely this is partly due to the higher cellular bioavailability of thio-dma v (aas/icp-ms). regarding genotoxicity, a generation of dna single strand breaks (alkaline unwinding technique) as well as an increased formation of reactive oxygen species (ros, dcfh-da-fluorescence) occurred only at high cytotoxic concentrations. however, thio-dma v strongly increased h2o2 induced ros formation at very low nanomolar concentrations, which might result in cogenotoxic effects. since our earlier studies have shown a strong inhibition of h2o2 induced poly(adp-ribosyl)ation especially by trivalent methylated arsenic metabolites, actual studies investigate the impact of thio-dma v on cellular poly(adp-ribosyl)ation, parp-1 gene expression, protein level and cellular cleavage, which might hopefully give further hints regarding the mode of action behind thio-dma v induced apoptosis. mitochondrial dysfunction in models of alzheimer´s disease eckert a. universitäre psychiatrische kliniken basel neurobiology laboratory, wilhelm klein strasse 27, 4012 basel, switzerland the histopathological characteristics of alzheimer's disease (ad) are amyloid-ß (aß) containing plaques and neurofibrillary tangles (nfts) as well as neuronal and synaptic loss. until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. there is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several app and tau transgenic mouse models. recently, we examined mitochondrial function in a novel triple transgenic mouse model (pr5/app/ps2) -triplead mice -that combines both pathologic features of the disease in brain. using comparative, quantitative proteomics (itraq) and mass spectroscopy we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes i and iv of the oxidative phosphorylation system (oxphos). remarkably, deregulation of complex i was related to tau, whereas deregulation of complex iv was aß dependent, both at the protein and activity levels. the triplead mice showed synergistic effects of aß and tau already at the age of 8 months, resulting in a depolarized mitochondrial membrane potential. at 12 months, the strongest defects on oxphos, synthesis of atp and reactive oxygen species were exhibited in the triplead mice, again emphasizing synergistic, ageassociated effects of aß and tau in impairing mitochondria. evidences from ad post-mortem brain as well as cellular and animal ad models indicate that aß and tau protein trigger mitochondrial dysfunction through a number of pathways, such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. moreover, recent reports indicate that aß may also interact directly with intracellular proteins such as the mitochondrial enzyme abad (aß binding alcohol dehydrogenase) in executing its toxic effects. mitochondrial dysfunction occurs early in ad, and aß's toxicity seems to be in part mediated by inhibition of abad. in total, a vicious cycle as well as several vicious circles within the cycle, each accelerating the other, can be drawn emphasizing the synergistic deterioration of mitochondria by tau and aß. olesoxime is a novel mitochondrial-targeted compound that is orally active and crosses the blood brain barrier. the cholesterol-oxime targets proteins of the outer mitochondrial membrane and represents a promising drug candidate for neurodegenerative diseases 1 . we evaluated olexoxime's neuroprotective effects against mitochondrial dysfunction in an animal model for alzheimer`s disease (ad). dissociated brain cells (dbc) and mitochondria were isolated from brains of c57/bj6-thy1-appsl (ad-mice) mice that were fed with 600 mg olesoxime/kg feed for 3 months. drug plasma levels reached approx. 600 ng/ml. respiration of isolated mitochondria were significantly diminished in ad-mice due to reduced complex i, i+ii and cox activities. consequently, mitochondrial membrane potential (mmp) was significantly reduced in dbc from ad-mice. olesoxime normalized respiration chain complex activities and the mpp. to further evaluate the beneficial effects of olesoxime on complex i activity, we challenged dbc with rotenone ex vivo and observed that olesoxime treatment was protective. to further clarify the mode of action, we analyzed the ability of olesoxime to prevent opening of the mitochondrial permeability transition pore (mptp) in vitro using energized brain mitochondria by measuring ca 2+ -and atractyloside (atr) induced swelling. the opening of mptp precedes apoptosis and can be induced by mitochondrial dysfunction due to calcium overload, oxidative stress, elevated phosphate concentrations or adenine nucleotide depletion. olesoxime prevented ca 2+ -as well as atr induced mitochondrial swelling. atr inhibits the adenine nucleotide translocase (ant) that requires appropriate membrane properties to mediate mitochondrial permeability transition (mpt). since cholesterol (cho) and its derivates represent potent modulators of membrane viscosity, we related the effects of cho and olesoxime on mptp opening to membrane properties. both, cho and olesoxime reduced the flexibility of membrane acyl-chains in energized mitochondria and prevented atr induced mptp opening. however, cho didn`t prevent ca 2+ -induced mptp opening, indicating a different mode of action for olesoxime. our data confirm olesoxime as drug candidate against mitochondrial dysfunction, which is considered to play a pivotal role in neurodegenerative diseases. the work was supported by the european union under the 7th framework program for rtd -project mitotarget -grant agreement health-f2-2008-223388. several inflammatory glomerular kidney diseases are accompanied with a massive production of reactive oxygen species (ros) that may attack the glomerular filtration barrier by affecting podocyte function and may contribute to apoptotic or necrotic cell death of mesangial cells. otherwise, ros also trigger fine-tuned signaling processes that may result in cell proliferation or cell migration. to define such redox-driven signaling devices, we performed a non hypothesis-driven proteomic approach, to identify homo-or heteromeric protein complexes induced by ros. to this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250 µm) for 10 min. thereafter, the cell lysates were subjected to diagonal 2d gel electrophoresis and putative redox-affected proteins were analyzed by ms/ms-analysis. by this approach, we could identify a series of proteins that form interprotein-disulfide bonds in a redoxdependent manner. one of those proteins could be characterized as the regulatory subunit of protein kinase a (r-subunit of pka), which belongs to the family of serine/threonine kinases. to evaluate whether ros is capable to activate pka also in a more physiological setting, we treated rat mesangial cells with pdgf-bb to induce ros formation and we could demonstrate that pdgf-bb induces dimerization of r-subunits in a redox-dependent manner. to demonstrate whether pdgf-bb induces pkadependent pathways, we analyzed the effects of pdgf-bb on phosphorylation of serine 157 of vasodilater stimulated protein (vasp) a classical target of pka. in fact, pdgf-bb induced vasp phosphorylation independently of intracellular camp levels. moreover, elevating camp levels via activation of adenylate cyclase with forskolin did not change the dimerization state of r-subunits. pdgf-bb-induced dimerization of the r-subunits and subsequent phosphorylation of vasp was blocked by diphenyljodonium (dpi), indicating activation of a nadph oxidase is essential for pka activity. taken together, we demonstrate a redox-dependent activation of pka by pdgf-bb and this may hint also for a probably protective role of ros in rat mesangial cells. testing the potential sensitizing capacity of chemicals is currently done by using the murine local lymph node assay (llna). animal welfare and eu cosmetics directive demands alternative methods to animal tests. the purpose of this study was to establish an in vitro assay for the prediction of skin sensitizers. based on the finding that the majority of skin sensitizers are electrophilic or have the potential to be metabolized to electrophilic substances, it is assumed that they can activate the nrf2-keap1-antioxidant response element (are) regulatory pathway. here, we report the results obtained from the lusens assay that detects electrophilic chemicals using the nrf2 pathway. the cell line lusens was derived from immortalized keratinocyte hacat cells and carries a luciferase reporter gene under the control of an are-element from the rat nadph quinone reductase nq1. the lusens assay was in house validated with a panel of 54 chemicals and cosmetic ingredients including the 22 performance standard substances of the local lymph node assay. the predictivity of this assay was compared to the predictivity of the murine llna and to human patch test data and can be considered as reliable screening approach (accuracy of 83% compared to human data). however, in order to cope with the complex multi-step mechanism of skin sensitization, an integrated approach of in vitro assays mimicking several steps was designed; thereof, the are-dependent gene activation represents one module. time-resolved fluorescence ligand-binding assays for parathyroid hormone receptors emami-nemini a. 1 ligand-binding studies represent essential tools for pharmacological research on g protein-coupled receptors. in recent years, time-resolved fluorescence gained significant relevance as readout for ligand binding studies. however, ligand-binding assays for parathyroid hormone receptors (pthrs) utilizing fluorescent parathyroid hormone (pth) were missing. therefore, we generated various fluorescent pth analogues which exhibit properties of native pth in terms of affinity, potency and internalization. for the purposes of academic and commercial research, we utilized labeled pth to set up three time-resolved fluorescence assay formats: (i) classical separation binding assay, based on time-resolved fluorescence and suitable for native receptors; (ii) homogeneous timeresolved fluorescence resonance energy transfer (htrf) based on tag-lite technology for high through-put screening; (iii) htrf based on antibodies, a synergistic approach using htrf with minimized receptor modification. this work will facilitate the development of new drugs directed to the pthr as well as fundamental research on the pthr. anandamide production in eosinophilic granulocytes is independent of il-5 and eotaxin stimulation engeli s., reinke j., zörner a., tsikas d., jordan j. medizinische hochschule hannover institut für klinische pharmakologie, carl-neuberg-straße 1, 30625 hannover, germany introduction: some animal and in vitro studies suggest that endocannabinoids exert anti-inflammatory effects. specifically, inhaled anandamide reduced the obstructive effect of leukotrien d4 in airways, and a specific cannabinoid receptor agonist significantly reduced pulmonary inflammation in guinea pigs. we have recently shown that segmental bronchial allergen challenge is associated with significant increases of anandamide concentrations in bronchoalveolar fluid of patients with asthma. the concomitant increase in eosinophilic counts, eotaxin and il-5 concentrations in bronchoalveolar fluid led us to hypothesize that anandamide is produced by eosinophilic granulocytes in response to chemotactic stimuli. peripheral eosinophilic granulocytes were isolated from whole blood by means of percoll gradient centrifugation and magnetic separation employing cd16antibodies conjugated to magnetic beads. isolated cells were counted and anandamide measurements were typically performed in whole cell lysates of 1.5x10 6 eosinophils. we stimulated eosinophils with varying concentrations of il-5, eotaxin-1 (ccl11), eotaxin-2 (ccl24), and eotaxin-3 (ccl26). to prevent anandamide degradation, a specific fatty acid amide hydrolase (faah) inhibitor (oloxa) was employed. anandamide concentrations and faah activity were determined by stable isotope dilution using lc-ms/ms protocols. results: first, we confirmed the ability of eosinophilc granulocytes to synthesize anandamide. however, cellular anandamide content could only be measured when faah was effectively blocked with oloxa, and strong faah activity was demonstrated in eosinophils. with oloxa, typical anandamide concentrations were in the range of 2-4 pm/1.5x10 6 eosinophils. neither il5 (50-500 pg/ml), nor any of the eotaxins (50 ng/ml either alone or in varying combinations) did stimulate anandamide production after 30min of incubation. our results suggest that chemotactic molecules like eotaxin and il-5 are not responsible for increased anandamide formation in eosinophils during allergen challenge. in a next step, the effects of anandamide on eosinophils and bronchial epithelial cells need to be determined. the suprisingly high faah activity in eosinophils may point to an alternative pathway facilitating prostaglandin and leukotriene synthesis by production of arachidonic acid. screening methodology for estimatation of dermal absorption in vitro fabian e., goth c., guth k., mehling a., van ravenzwaay b., landsiedel r. basf se experimentelle toxikologie, carl-bosch-strasse 38, 67056 ludwigshafen, germany dermal absorption is used in the evaluation of the effectiveness of pharmaceutical or cosmetic formulations, but often dermal absorption is a critical parameter in risk assessment of pesticides or chemicals. therefore, knowledge of dermal absorption is e.g. helpful in formulation development. skin absorption is routinely measured in vivo or in vitro following oecd tg 427 or 428. however, these tests are complex, time consuming and expensive. therefore, a method was developed to allow simple and rapid screening. the experiment uses dermatomized skin in modified franz type diffusion cells. 10 µl of test substance preparation are applied to the skin preparation. after 6h, the skin is washed and the amount of penetrated substance is quantified. the receptor fluid and the washing solutions are optimized for subsequent analyses by lc-ms. we performed dermal absorption screenings in parallel to our routine guideline studies and demonstrated a good correlation of the results of both study types: the total recovery found in the screening studies is somewhat lower than in the corresponding guideline studies but is always in an acceptable range above 80%. the efficacy of the skin washing procedure is lower than under routine conditions, most probably due to the change to an lc-ms-compatible washing solution. overall, the dermal absorption screening is an easy, fast and cost-effective screening method for the estimation of dermal absorption of a wide variety of test substances and formulations. the p53 tumor suppressor protein is frequently inactivated in human cancers by diverse mechanisms. owing to its fundamental role in the maintenance of genomic stability and cancer prevention, p53 is an attractive target in cancer therapy and several approaches were pursued to restore p53 function in tumor cells. polyamidoamine (pamam) dendrons are positively charged molecules with a systematically branched structure that interact with the negatively charged cell membrane, inducing cellular uptake via endocytosis. in the present study, biotin-labeled p53 protein was attached to a dendronized streptavidin nanocarrier to facilitate its internalization into different tumor cell lines. first, biotin-substituted pamam dendrons were conjugated with streptavidin to allow formation of the dendronized streptavidin nanocarrier. the nanocarrier displayed uptake into hela cervix carcinoma and a549 lung adenocarcinoma cells without detectable cytotoxicity. biotin-labeled p53 was then conjugated to the dendronized streptavidin, preserving its specific dna-binding in vitro. immunoblot analysis revealed efficient internalization of biotin-p53 into hela cells in the presence of dendronized streptavidin. in line with this finding, specific cellular uptake of biotin-p53 was observed by confocal microscopy, which showed a cytoplasmic and peri-nuclear localization in hela, a549 and saos osteosarcoma cells. the internalized biotin-p53 also partially co-localized with early endosomes. importantly, the delivery of biotin-p53 into p53-deficient saos cells resulted in impaired cell viability and upregulation of caspase 3/7 activity, demonstrating its biological functionality. this study intriguingly demonstrate the efficient delivery of functional biotin-p53 into different tumor cell lines using the novel streptavidin nanocarrier, which can be further modified to allow cell-type specific targeting and combined with cytotoxic drugs such as doxorubicin. identification of novel ahr target genes in rat liver oval cells faust d. 1 , vondracek j. the aryl hydrocarbon receptor (ahr) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to 2,3,7,8tetrachlorodibenzo-p-dioxin (tcdd), some polycyclic aromatic hydrocarbons (pahs) and dioxin-like polychlorinated biphenyls (pcbs), such as pcb126. activation of the ahr by these ligands leads to its dimerization with arnt and transcriptional activation of several phase i and ii metabolising enzymes. while it is generally accepted that many pahs are thereby transformed to genotoxic metabolites, this classical signalling pathway so far failed to explain the tumour promoting effects of the nongenotoxic compounds tcdd and pcb126. thus, there is an urgent need to define genetic programmes orchestrated by ahr to unravel its role in physiology and toxicology. we have recently shown that treatment of rat liver oval cells with tcdd or pcb126 leads to a release from contact-inhibition involving activation of the ahr, elevation of jund protein levels and transcriptional activation of cyclin a (1, 2) . loss of contact-inhibition is one hallmark of tumour promotion. to better understand ahr-driven pathways we identified the transcriptional programme using high density microarrays in response to pcb126. already 6 h after treatment, 69 genes were found to be upregulated and 76 genes downregulated indicating that these are direct ahr-dependent target genes. david analysis revealed that these genes are involved, for instance, in drug and lipid metabolism, cancer pathways, tgf-b signalling and cell-cell communication. ten of the 69 genes were selected for confirmation by semi-quantitative rt-pcr. using the ahr inhibitor ch-223191 and sirna directed against ahr and arnt, we further demonstrated that ahr-and arnt-function is required for transcriptional activation of the selected genes. finally, we identified the transcription factor foxq1as a novel ahr target protein in rat liver oval cells. although the function of foxq1 is poorly understood, it has been shown very recently that foxq1 is overexpressed in colorectal cancer and is involved in epithelial-mesenchymal transition in breast cancer cells. its function in ahrmediated tumour promotion, however, remains to be determined. the practical relevance of histamine h1 and h3 receptors in the brain can be easily deduced since h1 receptor antagonists of the first generation have a sedative effect and an inverse h3 agonist, pitolisant (close to its introduction to the market), is active against excessive daytime sleepiness associated with narcolepsy. in this context the question arises whether also h2 and h4 receptors possess a practical relevance in the brain. to this end, we examined whether the electrically evoked 3 h-noradrenaline release in superfused human cerebral cortex slices is affected by agonists at the above receptors. the h2 agonist impromidine 10 µm failed to affect noradrenaline release in human cortex slices although it facilitated noradrenaline release in guinea-pig cortex slices; the maximum extent of facilitation was 50 %, the pec50 was 6.9 and the pa2 of the h2 antagonist ranitidine against impromidine was 7.2. with respect to h4 receptors there is some controversy in the literature whether they occur in the brain at all. however, we were able to detect h4 receptor mrna in the human and mouse cortex by the reverse transcriptase polymerase chain reaction. in cortex slices of either species, noradrenaline release was not affected by the h4 agonist 4-methylhistamine 10 -30 µm but inhibited by histamine 10 µm via h3 receptors by 28 and 55 %, respectively. in mouse cortex membranes, 4-methylhistamine 30 µm also failed to affect 35 s-gtpγs binding although r-α-methylhistamine 3 µm, acting via h3 receptors, increased it by 20 %. in conclusion, h2 receptors facilitating noradrenaline release are detectable in the isolated guinea-pig but not human cortex. despite the presence of h4 mrna in the brain, functional readouts of this receptor, i.e. modulation of noradrenaline release (humans, mice) and modulation of 35 s-gtpγs binding (mice), could not be shown. murine cx40 promoter activity is dependent on the transcription factor creb fels b., nunes f., schmitz w., müller f. u. westfälische wilhelms-universität münster institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany connexin 40 (cx40) is a gap junction protein expressed in atrial myocytes and the ventricular conduction system, mediating the electrical intercellular communication in the myocardium. alterations in cx40 function were linked to the pathophysiology of atrial fibrillation and heart failure. in the heart, camp dependent gene transcription is regulated by members of the creb/crem/atf family which bind to camp responsive elements (cres). similar to the human cx40 gene promoter, the murine promoter contains one cre. cardiomyocyte-specific overexpression of a crem-isoform (crem-ib∆c-x) led to cx40 down-regulation, suggesting that creb related transcription factors are involved in cx40 gene regulation. in order to study the functional role of creb in the regulation of the cx40 promoter we have generated a luciferase based murine cx40 promoter reporter gene construct and monitored its activity in a permanent cell line upon overexpression of constitutive-active creb (cacreb) and a non-phosphorylatable dominant-negative creb (dncreb) isoform. surprisingly, overexpression of cacreb and dncreb both lead to a reduction of cx40 promoter activity (cacreb 46% ± 5% vs control 100% ± 3%; p<0.05 vs control , n=15), dncreb 45% ± 2% vs control 100% ± 3%; p<0.05 vs control, n=18). the activity of the murine connexin 40 promoter is modulated by creb. both cacreb and dncreb led to cx40 down-regulation, which could be explained by induction of inhibitory transcription factors creb/crem/atf1 transcription factor family, which in turn could suppress cx40 promoter activity. (supported by the dfg) results: fxa increased par-2 mrna, protein and cell-surface expression and augmented par-2-mediated mitogenesis. par-1 expression was not influenced. the regulatory action of fxa on par-2 was concentration-dependent and mimicked by a par-2 selective activating peptide. the thrombin inhibitor argatroban or par-1 gene silencing did not influence fxa-stimulated par-2 expression. fxa increased oxidative stress and expression of the nadph oxidase subunit nox-1 in smc. nox-1 gene silencing prevented fxa-stimulated par-2 regulation, as did ebselen and catalase. exogenous hydrogen peroxide increased par-2 expression and mitogenic activity. fxa induced nuclear translocation and par-2 dna binding of nuclear factor kb (nfkb). inhibition of nfkb prevented fxa-stimulated par-2 expression. in separate studies, fxa promoted par-2 mrna stabilisation through increased human antigen r (hur)/par-2 mrna binding and cytoplasmic shuttling. hur gene silencing abolished fxa-stimulated par-2 expression. conclusion: expression and mitogenic activity of vascular par-2, but not par-1, is upregulated by fxa. this action involves transcriptional and post-transcriptional mechanisms mediated through nox-1-containing nadph oxidase and its downstream effectors hydrogen peroxide, nfkb and the mrna stabilising protein hur. continued generation of fxa by the mural thrombus, and the autoregulatory feedback control of par-2 may maintain the inflammatory and proliferative state of the injured vessel, thereby promoting vascular remodeling. the mrna stabilising factor hur is a critical regulator of human proteaseactivated receptor 4 aim: we recently reported that functional expression of par-4 thrombin receptors is induced in human saphenous vein smc exposed to high glucose. this effect could be attributed in part to transcriptional mechanisms mediated through nfkb but the contribution of post-transcriptional effects such as mrna stabilisation is not known. this study explored the potential role of the mrna stabilising factor human antigen r (hur) in the regulation of par-4. methods: human saphenous vein smc were serum-deprived prior to study. gene expression was determined by realtime pcr, protein expression by western blotting. gene silencing utilized commercially available sirna. hur binding to par-4 mrna was determined by immunoprecipitation ("pull-down") pcr. results: high glucose (25 mm vs 5.5 mm) slowed par-4 mrna degradation in the presence of actinomycin d. par-1 mrna decay was not affected. hur binding to par-4 mrna and nucleo-cytosolic shuttling was enhanced by high glucose, total hur protein expression was not affected. hur sirna abolished the high glucose-stimulated induction of par-4 mrna. hydrogen peroxide (h 2o2) also induced cytosolic hur shuttling and increased par-4 mrna and total protein expression. the role of endogenously generated h2o2 in the regulatory effect of high glucose on par-4 expression was investigated with the nadph oxidase inhibitors apocynin/diphenyliodonium (to prevent h2o2 generation) and cell-permeant catalase (to degrade cellular h2o2). both approaches prevented the stimulatory effect of high glucose on par-4 expression. cyclic amp has been reported to suppress hur activation and in the present study, the cyclic amp stimuli forskolin and cicaprost (prostacyclin analog) suppressed basal hur shuttling and par-4 transcript stability. cicaprost also attenuated high glucose-induced hur binding to par-4 mrna and as a consequence normalised par-4 expression and inflammatory signalling in high glucosetreated cell. conclusion: the regulation of par-4 thrombin receptors in human vascular smc is critically dependent on the mrna stabilising actions of hur. through activation of hur, high glucose and other hur stimuli such as ang ii and exogenous h2o2, increase par-4 expression, while cyclic amp agonists such as prostacyclin oppose this effect. such interactions could potentially represent a fine-tuning mechanism to control par-4 expression and ultimately also the mitogenic and inflammatory actions of thrombin in the vessel wall. nucleoside diphosphate kinase b (ndpk b) is a member of a family of ubiquitously expressed enzymes required for nucleoside triphosphate synthesis. thus, they are involved in the regulation of a variety of cellular processes, e. g. g protein mediated signal transduction. however, whether ndpk b has a specific role in the regulation of angiogenic processes in endothelial cells is unknown. therefore, we studied the function of ndpk b in the vasculature in a developmental, an ischemia-induced and an in vitro model of angiogenesis. firstly, depletion of ndpk b expression was achieved by morpholino-mediated knockdown in zebrafish embryos in which the developing vasculature can be visualized by egfp expression in the endothelium. 72h post fertilization, ndpk b knockdown larvae showed a dramatic inhibition of intersegmental and dorsal longitudinal anastomatic vessel formation compared to control injected fish. this phenotype could be rescued by early re-expression of ndpk b. secondly, ischemia driven angiogenesis was studied in ndpk b-depleted mice and wildtype littermates after excision of the left femoral artery. hind limb blood flow was assessed by laser doppler perfusion imaging immediately before and after ligation (day 0) and on postoperative days 3, 7, 14, 21, 28, and 35 . a significant reduction of recovery was observed in the ndpk b depleted mice at days 3 and 7. thirdly, in vitro-sprouting angiogenesis was analyzed in human umbilical vein endothelial cell (huvec) spheroids with and without sirna-mediated ndpk b knockdown. vascular endothelial growth factor (vegf)induced sprouting was significantly attenuated by ndpk b knock down by more than 50% in comparison with control transfected huvec. we conclude from these results that ndpk b is an essential regulator of angiogenesis. the loss of ndpk b may specifically interfere with the vegf-induced migration and proliferation during endothelial sprouting. ethylene oxide in blood of ethylene-exposed volunteers ethylene (et) is a commercially important high volume industrial chemical. inhaled and endogenous et is metabolized in mammals to ethylene oxide (eo), which is carcinogenic in rats and mice. until now, no data on the oxidation of et in et-exposed humans has been published. in the present study, we investigated the formation of eo in four male adult volunteers exposed for 4 hours to constant atmospheric et concentrations of 5, 20, or 50 ppm by means of a breathing mask. during exposure, et concentrations were measured in inhaled and exhaled air by gc/fid and eo concentrations in venous blood by gc/ms. rates of et metabolism were obtained from the product of the differences in the et concentrations with the pulmonary ventilation. in each subject, linear correlations were found between the et exposure concentration and the rate of et metabolism or the eo concentration in blood. mean rate of et metabolism was 5.5 ± 1.4 nmol/h/ppm/kg body weight. steady-state concentrations of eo in blood differed by a factor of 2 between the volunteers. these inter-individual differences likely reflect the polymorphism of glutathione s-transferase theta, the main eo metabolizing enzyme in human liver. mean eo concentration in blood at steady state was 1.5 ± 0.08 nmol/l blood per ppm of et. the data will be used for validating a physiological toxicokinetic model which will describe the et related eo tissue burdens in rodents and humans. the model predictions will support risk evaluations of et. financially supported by the lower olefins sector group of cefic. in vitro effect of stw11 on human dendritic cells fink c. 1 , bonaterra g. a. extracts of echinacea (purple coneflower) are used in the prevention and therapy of infectious diseases. the medicinal product stw 11 contains the extract from purple coneflower, and in addition, extracts of monkshood, venom of honey bee and bushmaster snake in homeopathic dilutions. previous studies showed a stimulation of the cellular and humoral immune response. dendritic cells (dcs) are antigen presenting cells that act at the interface of the innate and adaptive branches of the immune system. during stages of dc differentiation, the ability to internalize antigens varies and decreases during maturation. in this study, we determined the influence of stw11 on the expression of maturation related genes (cd1a, cd83), cytokines (tnfα, il-4, il-12), chemokines (ccr7), major histocompatibility complex ii(mhc-ii) and toll-like receptors (tlr2, tlr4). in mature (mdc) and immature dc (idc) using real-time rt-pcr. peripheral blood mononuclear cells (pbmcs) were isolated from buffy coats of human volunteers by densitygradient (ficoll ® ) and seeded in 6 well plates. non-adherent cells were eliminated. to induce idc development, 50 ng/ml il-4 and 50 ng/ml granulocyte macrophagecolony stimulating factor (gm-csf) were added. at day 6, maturation was induced by addition of lipopolysaccharide (lps) at a final concentration of 1µg/ml and cultured for additional 3 days. after incubation with different concentrations (0. in idc, compared to control, we found a significantly increased expression of cd83 (2.1-2.6 fold) and tnfα (2.6-3.3-fold) genes after treatment with 0.1-5% stw11, respectively, but no effect was found on the expression of cd1a, il-4, il12, adam19, cd11c, cd40,tlr2, tlr4, mhc-ii and ccr7. in summary, these data demonstrate a stimulatory effect of stw 11 in idc and especially in mdc, concerning an increase of various genes related to maturation (cd83), immunomodulation (tnfα, cd40), adhesion (ccr7) and antigen presentation (mhc-ii) and are in accordance with the therapeutic use in infectious diseases. waterproofing sprays are widely used consumer products containing for example fluorinated polymers or silicon based compounds dissolved in alcohols or volatile petroleum distillates. there have been repeated reports on cases of severe acute respiratory disorders especially when using products that newly entered the market. it is hypothesized that impairment of the pulmonary surfactant by deposition of inhaled respirable particles of the active compound is one of the main causes of the acute lung injury. since the inhalation toxicity cannot be predicted a priori based on the physical and chemical properties of the formulation, proper test strategies are required to ensure consumer safety. we propose to combine screening tests addressing both, exposure and acute lung toxicity. the exposure potential of the spray product is characterized by determining the release fraction of the active compound in the respirable particle size range under conditions relevant for the product application. this is carried out by spraying defined quantities of the product into a control volume and measuring the concentration of health related size fractions. this procedure takes into account spray ageing, especially size reduction of the droplets due to solvent evaporation. the isolated perfused lung is used as a model for testing acute toxicity. ventilated rat lungs are exposed to aged aerosols with proper particle size of approximately 1 µm mmad generated from the liquid spray formulation. lung compliance and lung resistance are continuously monitored during exposure. dose dependent deviations from the normal values (without exposure) are used as read-out parameters. using the combined procedure, different sprays could be ranked according to their realistic exposure risk and, most importantly, sprays with known lung toxicity could be uniquely distinguished from those that have been shown to be safe. in its current stage of development the simple test method is recommended for screening of substances only. induction of oxidative damage in calf thymus dna by the fusarium mycotoxin zearalenone after metabolic activation with liver microsomes fleck s. c., pfeiffer e., metzler m. kit -institute of applied biosciences chair of food chemistry, adenauerring 20a, 76131 karlsruhe, germany zearalenone (zen) is an estrogenic mycotoxin produced by fusarium species. the adverse effects of zen and its reductive metabolite zearalenol (zel) are often compared to those of 17-beta-estradiol (e2) and estrone (e1). these endogenous estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (i) hormonal activity and (ii) genotoxic effects by p450-catalyzed metabolic activation to catechols (wang et al., chem res toxicol 23, 1365 . like e2 and e1, zen and zel exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites (pfeiffer et al., mol nutr food res 53, 1123 . the aim of the present study was to examine the formation of catechol metabolites of zen by liver microsomes of various species and their potential for redox cycling. catechol metabolites are frequently associated with the generation of reactive oxygen species and subsequent oxidative damage of dna, for which 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxo-dg) is a common biomarker. the propensity of the catechol metabolites of zen and zel to cause the formation of 8-oxo-dg in isolated calf thymus dna was determined using a lc-esi-ms/ms method. to this end, zen was incubated with microsomes from human, rat, mouse, bovine and porcine liver as well as with human cyp1a2, and the incubations were extracted with ethyl acetate. the extract was analyzed with lc-ms and then added to a solution of calf thymus dna in the presence of copper(ii) ions and nadph. the formation of 8-oxo-dg could be demonstrated with each extract. the levels of 8-oxo-dg correlated directly with the extent of catechol formation, which increased from steer to swine to human to mouse to rat microsomes. 15-hydroxylated zen/zel, which is the major catechol, was more reactive than 13-hydroxylated zen/zel to form 8-oxo-dg. in conclusion, our study has shown that the catechol metabolites of zen are highly reactive and give rise to oxidative dna damage in vitro. in addition, recent research from our laboratory revealed that the catechols of zen are less efficiently inactivated by catechol-o-methyl transferase than the catechols of e2 and e1. the genotoxic potential of zen may constitute another biological activity in addition to the well-known estrogenicity. supported by deutsche forschungsgemeinschaft (grant me 574/32-1) and "food and health" of kit. thrombin regulates expression of sphingosine kinase-1 (sphk-1) in human vascular smooth muscle cells -inhibition by dabigatran reduces vascular sphk-1 expression and atherosclerotic burden in vivo flößer a. 1 results: thrombin induced a time-and concentration-dependent (1-100 nmol/l) increase in sphk-1 mrna and protein expression in human saphenous vein smc, n=6-7. this was mimicked by a synthetic par-1 ligand. inhibition of sphk-1 attenuated thrombin-induced smc proliferation but not smc migration (n=5). the regulatory action of thrombin on sphk-1 expression was suppressed by sirna against the mrna stabilisiserhur. in thrombin-stimulated smc, hur binding to sphk-1 mrna and subsequent nucleo-cytosolic shuttling was enhanced. accordingly, thrombin induced sphk-1 mrna stabilisation in smc in the presence of actinomycin d. in apoe-deficient mice, long-term treatment with the direct thrombin inhibitor dabigatran significantly reduced aortic sphk-1 expression by 50% (n=5) and plaque size by 35% compared to control animals (n=10). conclusions: thrombin induces sphk-1 expression and s1p synthesis in vascular smc via the mrna stabilising protein hur. this leads to increased smc proliferation. inhibition of thrombin by dabigatran treatment in vivo attenuates progression of plaques possibly by reducing sphk-1 expression. mycotoxin contamination and cytotoxicity of grain mill products typical grain mill products from north-rhine westphalia, i.e. grains, flour, wholemeal flour and bran (from wheat, rye and spelt) as well as typical by-products (outsourced fractions) from the milling process were analysed for their mycotoxin content by lc-ms/ms. the cytotoxicity of sample extracts with known mycotoxin composition was then assessed in v79 cell cultures by means of the neutral red uptake assay, in parallel with pure reference mycotoxin mixtures. extracts from flour and wholemeal flour samples with low levels of deoxynivalenol and enniatin b (from not detectable to 0.4 µg/g don and 0.5 µg/g ennb) induced no measurable cytotoxicity. on the other hand, although mycotoxin contamination levels were also rather low in bran, these samples induced strong cytotoxicity: extracts of bran derived from rye and spelt were more cytotoxic than those of wheat bran. the cytotoxic effects of the bran samples cannot be related to their mycotoxin content as comparable concentrations of pure mycotoxins and mycotoxin combinations tested in parallel were not cytotoxic. by-products from certain stages of the milling process (sorting and waste fractions) were found to contain mycotoxins at rather high levels: enniatin b was detected in nearly all samples, and also t-2 toxin, ht-2 toxin, ergotamine, ergocornin and deoxynivalenol were present. waste sample extracts with notable mycotoxin levels (up to 6 µg/g don, 7 µg/g ennb, 16 µg/g ergotamine, 50 ng/g ht-2 toxin) exert pronounced cytotoxicity in v79 cells. the cytotoxicity of these samples was somewhat stronger than expected when compared with mixtures of reference mycotoxins tested in parallel. in summary, the tested flour and wholemeal flour extracts contained only low levels of mycotoxins and were not cytotoxic. in contrast, bran samples showed cytotoxicity which cannot be explained solely by their mycotoxin content. this unexpected observation in real samples and combination effects of mycotoxin mixtures require further studies. the ahr is a ligand-activated transcription factor that mediates the toxicity of dioxins and related compounds. upon ligand binding the ahr translocates into the nucleus and dimerizes with arnt to modulate gene expression, e.g. of cyp1a1. recently, we have shown that uvb irradiation of human keratinocytes results in activation of the ahr and associated egfr signaling leading to an enhanced expression of cyp1a1 and proinflammatory cox-2, respectively. the initial step is the uvb induced intracellular formation of the tryptophan photoproduct 6-formylindolo [3,2b] carbazole (ficz), a high affinity ahr ligand. thus, the ficz activated ahr is an important mediator of the dna damage independent part of the uvb response. our current study aims to identify further aspects of ahr mediated uvb responses. therefore, we analysed changes in protein expression, proliferation and apoptosis in ahr+/+ and ahr-/-keratinocytes (nctc 2544) by western blot, flow cytometry and brdu incorporation. uvb exposure of nctc cells led to a dose-dependent increase in apoptosis. compared to ahr+/+ cells, ahr-/-cultures exhibited an increased amount of apoptotic cells. this finding was confirmed in irradiated ahr+/+ cells, pretreated with the ahr antagonist 3'methoxy-4'-nitroflavone. moreover, the proliferation of sham as well as uvb irradiated ahr-/-cells was significantly decreased. in ahr-/-cells we found a reduced expression of checkpoint kinase 1 (chk1), an important cell cycle regulator that arrests the cell in g2/m upon dna damage. interestingly, uvb exposure led to a higher net phosphorylation of chk1 in ahr-/-cells, indicating that this pathway is responsible for the observed ahr-dependent differences in proliferation and apoptosis. further expression analyses of chk1 client proteins emphasize our hypothesis. in conclusion our study identifies the ahr as an anti-apoptotic player in uvb irradiated human nctc cells. therefore, we propose that the ahr is a suitable target to prevent uvb induced skin diseases. synthetic progestins exert divergent effects on thrombosis in a murine model of atherothrombosis background: medroxyprogesterone acetate (mpa), a synthetic progestin often used in postmenopausal hormone replacement therapy, has previously been described to be pro-thrombotic in a murine model of accelerated atherosclerosis. however, nothing is so far known about effects of progestins with receptor profiles different from mpa (i.e. agonism or antagonism of mineralocorticoid-or androgen-receptors), such as drospirenone, levonorgestrel and norethisterone acetate. methods: apo -/mice were bilaterally ovariectomized (ovx) and substituted subcutaneously with mpa, drospirenone, levonorgestrel and norethisterone acetate as well as the respective placebo pellets for 90 days on a western-type diet. subsequently, thrombosis was induced by photochemical injury to the right carotid artery using rose bengal and a green light laser. results: compared to placebo, animals substituted with mpa showed significantly shortened times to occlusion of the right carotid artery (placebo mpa: 50.0 ± 5.8 min. vs. mpa: 33.4 ± 4.2 min., n = 6 -9, p < 0.05). in contrast, drospirenone, levonorgestrel or norethisterone acetate did not alter thrombotic responses. however, at least drospirenone (placebo drospirenone: 53.7 ± 5.4 min. vs. drospirenone: 47.5 ± 4.8 min., n = 7 -8) and levonorgestrel (placebo levonorgestrel: 47.0 ± 3.2 min. vs. levonorgestrel: 41.8 ± 2.1 min., n = 6) showed a trend towards shorter times to stable occlusion. furthermore, analysis of aortic gene expression revealed that in aortas of mpa-treated mice expression of matrix-metalloproteinase 9 (mmp-9) was induced as compared to placebo-treated mice. conclusion: mpa, a progestin with glucocorticoid effects, exerts a pro-thrombotic effect that is either progesterone-or glucocorticoidreceptor-dependent while progestins with receptor profiles different from mpa do not show a significant pro-thrombotic effect. furthermore, the pro-thrombotic effect exerted by mpa may be associated with increased expression of mmp-9, a metalloproteinase being known to destabilize atherosclerotic plaques and make them more prone to rupture. rapid screening for mitochondrial toxicity in vitro using an oxygen-sensitive phosphorescent probe freyberger a. bayer healthcare bph gdd ged toxikologie -p & cp, aprather weg 18, 42096 wuppertal, germany impaired mitochondrial function has been implicated with disease, aging, and druginduced toxicities. analyzing mitochondrial respiration (mr) rates is one of the most informative ways to assess mitochondrial function as it provides information on the the bioenergetic capacity of a tissue, however, previously measurements using polarography (clark electrode) were cumbersome with only low throughput. in this work we explored luxcel's water-soluble phosphorescent oxygen-sensitive probe mitoxpress tm for the assessment of mitochondrial toxicity in freshly isolated male rat liver mitochondria (rlm) in a 96-well plate format using glutamate/malate (20 mm/0.5 mm) and succinate (25 mm) as respiratory substrates. inhibition of mitochondrial complexes i to iv, adenosine triphosphate synthetase and the adenosine diphosphate (adp) / adenosine triphosphate (atp) antiporter by rotenone, thenoyltrifluoracetone (ttfa), antimycin a, potassium cyanide, oligomycin, and atractyloside was readily detected in adp-stimulated rlm. use of the two different substrates in parallel allowed to discriminate complex i inhibition by rotenone from complex ii inhibition by ttfa, whereas downstream of these complexes inhibition by the other model inhibitors occurred independent of the substrate used. decoupling of mr from oxidative phosphorylation by carbonylcyanid-p-trifluormethoxyphenylhydrazone (fccp) was detected best in the absence of adp. compared to polarographic measurement, the use of an oxygen-sensitive probe is superior with regard to assay cycle time and sample throughput and offers new opportunities to characterize and screen for mitochondrial toxicity, but also to support studies on mitochondria-mediated modes of action of new chemical entities. the murine local lymph node assay (llna) and the guinea pig maximization test (gpmt) have been used to study the sensitization potential of a series of unsaturated compounds by kreiling et al. (2008) . we have examined the same substances in the loose-fit coculture-based sensitization assay (lcsa), developed by our working group (schreiner et al., 2007) . eight unsaturated compounds [oleic acid (oa), linoleic acid (la), linolenic acid (lna), undecylenic acid (ua), fumaric acid (fa), maleic acid (ma), squalene (sq), 1-octyn-3-ol (oc)] and succinic acid (sua) were investigated using a coculture of keratinocytes and dendritic cell-related cells (dcrc). sensitization potential was quantified by flow cytometry measuring the increase of cd86 expressed on dcrc (ec50 = half maximal effective concentration). a pronounced induction of cd86 at low concentrations was seen with la, lna and oa (ec50: 3, 5 and 5 µmol/l, respectively). ua exhibited an intermediate response (ec50: 307 µmol/l). with oc and ma, we observed effects at higher concentrations only (ec50: 1340 and 2293 µmol/l). no significant increase of cd86 was observed with fa, sua and sq. because of poor solubility, sq could not be studied adequately. induction of cd86 was generally observed at concentrations which did not cause a major impairment of cell viability. our results show a high degree of concordance with those obtained by the gpmt, except for oa. in comparison with the results of the llna, those compounds which showed a strong effect in the llna (oa, la, lna) also induced an increase of cd86 at low concentrations, whereas those with low stimulation indices in the llna induced no significant increase of cd86 (fa, sua) or only at higher concentrations (ua). we observed a discrepancy between the tests with ma and oc, causing a strong stimulation of the murine lymph nodes, while the expression of cd86 was increased at high concentrations only. we assume that ma and oc might be false-positives in the llna, because they were also negative in the gpmt. background: the first step in elimination of many cationic drugs is their uptake from the blood into hepatocytes and renal proximal tubular cells by the organic cation transporter 1 (oct1) and oct2, respectively. the pivotal role of octs in the excretion of cationic drugs raises the possibility of drug-drug interactions in which one drug reduces octmediated elimination of a second drug. although many psychoactive drugs are cationic at ph 7.4 and some of these have already been recognized as oct inhibitors, a systematic screen of this class of compounds is missing. methods: we screened a drug library of 50 most frequently prescribed psychoactive drugs (inpatient prescriptions in germany, at least 4 million ddd each) for their inhibitory interaction with oct1 and oct2. human embryonic kidney (hek) cells stably overexpressing oct1 or oct2 and the prototypical oct substrate 1-methyl-4phenylpyridinium (mpp+) were used as a test system. cells transfected with the empty vector were used as controls. results: at 20 µm, 59% and 51%, respectively, of the tested compounds significantly decreased oct1-and oct2-mediated uptake of mpp+. the most potent inhibitors (inhibition >75%) of oct1 were chlorprothixen and clomipramine, whereas olanzapine, clomipramine and doxepin were the most potent inhibitors of oct2. in contrast, neither at 20 µm nor at 200 µm carbamazepin, haloperidol, lithium, moclobemide and valproic acid did significantly inhibit mpp+ uptake into hek-oct1 or hek-oct2 cells. there was a significant correlation between the degree of oct1 and oct2 inhibition (p conclusions: our results demonstrate that inhibition of oct function by psychoactive drugs has to be considered as a potential mechanism underlying drug-drug interactions. considering estimated peak sinusoidal concentrations e.g., of chlorprothixen and clomipramine between 30 and 80 µm in humans, inhibitory interactions of these compounds with hepatic oct1 have to be taken in account. our data will help to create a chemoinformatic model to predict potential oct-dependent interactions of psychoactive drugs with the hepatic or renal elimination of coadministered drugs. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. cardiac gene expression is altered during the development of hypertrophy and heart failure compared to the healthy heart. the molecular mechanisms controlling gene expression in cardiac failure are only partially known. dna methylation is one epigenetic mechanism that regulates long-term changes in gene-expression. to elucidate whether dna methylation is altered during the development and progression of chronic heart failure, genome-wide dna methylation profiles were determined in myocardial biopsies from control patients and patients with cardiac hypertrophy or failure. cardiac biopsies were obtained from patients with aortic aneurysm who served as control and did not show clinical signs of chronic heart disease (ef: 58±3 %, n=3) and from patients with aortic stenosis. the latter group was subdivided according to the ejection fraction into hypertrophic (ef: 63±7 %, n=3) and failing patients (ef: 28±1 %, n=3). after bisulfite conversion of extracted dna, the methylation status of genomic dna was quantified using the infinium® humanmethylation450 beadchip (illumina). this microarray allows analysis of more than 485,000 methylation-sites throughout the whole genome at single-base-pair resolution. these experiments identified 1280 cpg sites in hypertrophic samples and 1365 cpg sites in failing samples which were differentially methylated compared to control specimens (delta >15%; p<0.05). 523 cpg sites were significantly altered in both aortic stenosis groups compared with control hearts. from these cpgs, 496 sites were altered concordantly in hypertrophic and failing samples. analysis of regions harbouring distinct cpg densities revealed that most changes occured in shelf regions of cpg islands whereas the methylation status in the cpg islands was more stable. further analysis showed that differences in methylation were most frequent in gene body, enhancer and 3`utr regions. specifically 9 probes spanning a cpg-island at the promotor region of the muscle-specific serine kinase 1 (srpk3) showed diminished cpg-methylation in hypertrophic (-11.5±0.02%) and failing (-11±0.02%) as compared to control biopsies. remarkably, no alterations of dna-methylation were observed in loci of classic marker genes of chronic heart failure like nppa, serca, ctgf, myh6 or myh7. these results indicate that dna methylation is specifically altered in chronic heart disease but does not affect classic marker genes of chronic heart failure. gliomas are the most abundant type of primary brain tumor in the central nervous system in adults. the current standard of glioblastoma multiforme (gbm) therapy is surgery followed by radiotherapy and chemotherapy. however the morbidity and mortality of gbm remain very high and the median survival period is only 15 months even with treatment. therefore it is important to identify novel drugs to reduce gbm cell proliferation. purine-analogues (pa) are well known for their anti-proliferative effects on eukaryotic cells. in this study novel pa were synthesized and the library of substance-derivatives was tested using different gbm cell lines namely ln18, u87-mg and gl261. the effect on proliferation and viability was assessed by using brdu and resazurin assays. using these in vitro methods we were able to identify several compounds with cytotoxic and anti-proliferative effects in vitro showing ic50 values in the deeper µm range. cytotoxicity of selected compounds was further analyzed by assessment of caspase 3 and propidium iodide based cell cycle facs analysis to discriminate between apoptosis and cell cycle arrest. based on these data purine-derivatives might inhibit proliferation and induce apoptosis in glioma cells. as a result we hypothesize that these compounds could be potentially interesting for the drug-development of gbm therapy and therefore a clue for chemical modifications. further studies are required to identify the exact underlying mechanism of action of the tested purine-analogues. the biological role of adenosine receptors in brown adipose tissue gnad t. 1 brown adipose tissue (bat) is responsible for basal and inducible energy expenditure in mammals. bat contains large amounts of mitochondria and is highly vascularized. bat lipolysis and thermogenesis are stimulated by sympathetic neurons. importantly, recent findings indicate that adult humans possess metabolically active bat 1 . here, we analyzed the expression and function of adenosine receptors in bat. adenosine receptors (ador) are members of the superfamily of g protein-coupled receptors. there are four subtypes of adors in humans referred to as adora1, a2a, a2b and a3. they are widely expressed in tissues and mediate a variety of cellular functions, mostly due to their regulation of camp levels within cells. interestingly, it has been shown that adenosine can either inhibit or stimulate lipolysis in white adipocytes through adora1 or a2a, respectively 2 . however, the role of adenosine in the differentiation of brown preadipocytes to adipocytes and in bat function is not clear. to analyze the role of adors in bat, we use preadipocytes isolated from bat of newborn mice and subjected them to a differentiation protocol. 3 abundance of adora1, a2a, a2b and a3 mrna was measured using qpcr. all four receptor subtypes are present in preadipocytes with adora2b being the most abundant. adora1, adora2a and adora3 are significantly transcriptionally upregulated -albeit at varying degree -during differentiation. adora1 is upregulated 4.4 fold (+/-0.3 fold) and 11 fold (+/-0.49 fold) at day 4 and at day 7, respectively, as compared to preadipocytes (n=5). adora2a is 23 fold (+/-1.96 fold) upregulated at day 4 and 57 fold upregulated (+/-2.48 fold) at day 7, respectively (n=4). adora3 was found upregulated 3.6 fold (+/-0.46 fold) at day 7 (n=4). in contrast to this, ador2b was downregulated to 0.87 fold (+/-0.09 fold) at day 4 and to 0.69 fold (+/-0.04) day 7 compared to preadipocytes (n=5). to investigate the functional role of ador in bati cells, we analyzed lipolysis in mature cells after acute treatment with specific agonists and antagonists. we observed that adora2a activation by cgs21680 significantly increased lipolysis by 88% (+/-0.33%) compared to untreated control. moreover, adora1 antagonist psb36 increased lipolysis by 35% (+/-0.05%) (n=4). in conclusion, ador are highly regulated during brown fat cell differentiation. lipolysis of mature brown fat cells is significantly increased by adora2a agonist or adora1 antagonist, respectively. munich heart alliance, münchen, germany activation of the sympathetic nervous system and the subsequent activation of βadrenergic receptors (βars) through catecholamines represents the strongest mechanism to increase cardiac function. however, long-term activation of cardiac βars is clearly detrimental and β-blockers have been introduced as an effective treatment modality in cardiac failure. despite their central role in cardiac physiology and disease, our knowledge about the intracellular mechanism of βar stimulation is confined to a few targets and is likely incomplete. here, we report a functional proteomics approach to directly assess the entire phosphoproteome of βar-stimulated mouse hearts in vivo. to identify proteins that are phosphorylated in response to β-adrenergic stimulation in vivo, we treated mice with isoproterenol or, as a control, with propranolol. after lysis of hearts and tryptic digest, phosphopeptides were enriched by tio 2 or immobilized metal ion affinity chromatography (imac). subsequent analysis of eluated peptides by tandem mass spectrometry (ms/ms) mapped several phosphopeptides to cardiac proteins, among which known mediators of βar signaling such as phospholamban, troponin i and myosin binding protein c. we then employed multiple reaction monitoring (mrm) as a quantitative approach to assess changes of phosphorylation after βar stimulation. using this combination of ms approaches, we identified 39 peptides with pka consensus phosphosites that were more abundantly detected under βar stimulation. among those, we found myozenin-2 (myoz2) and g protein signaling modulator 1 (gpsm1, also termed ags3) as proteins previously not related to βar signaling. we validated the βar-dependence of phosphorylation at these sites in isolated cardiomyocytes by in vivo labelling or phosphoepitope-specific antibodies. current efforts aim at the functional characterization of these novel candidate mediators of βar signaling in the heart. taken together, we report the β-adrenergic phosphoproteome of the mammalian heart in vivo. we have identified several new targets of βar signaling that may represent essential factors in cardiac physiology and disease. background: drug measurement in autopsy material is normally used to investigate the cause of death. in our study it was possible to measure concentrations of drugs that were part of a regular treatment without connection to the cause of death. metamizole is used as an analgetic and spasmolytic agent. the active metabolite maa (4-methyl-aminoantipyrin) is metabolized by the liver and eliminated by the kidney. hepatic and renal dysfunction can therefore influence maa clearance. methods: maa concentrations were measured in different samples of the autopsy material (heart blood, venous blood, urine, liver, kidney and brain) using an hplc-ms/ms method. information about the dosage and time of drug application as well as information about existing renal or hepatic disorders were taken from the corresponding patient records. because of the low number of cases an explorative single-case study was necessary. results: 10 cases with oral intake of metamizole in a customary continuous dosage could be indentified. the maa distribution into body liquids and organs depended on the time between last oral intake and death. in two cases without renal or hepatic diseases maa blood levels were below 10 µg/ml. five cases with combined renal and hepatic disorders showed either increased blood levels of 40-50 µg/ml or prolonged maa elimination half-life of up to 12 hours. in one case with manifest hepatic insufficiency an maa concentration of more than 200 µg/ml was measured in venous blood. two cases with renal insufficiency alone had maa venous blood levels of less than 10 µg/ml. (pet) . pet detects the positron emission of neutron-deficient radioactive nuclides and allows their external localization in vivo. fet, a modified amino acid, is not incorporated in proteins but accumulates in glioblastomas. one pathway responsible for its accumulation is the preferential transport into the tumor cells, probably via amino acid transporters. we investigated in more detail (a) which individual, cloned amino acid transporters accept fet as substrate and (b) which transporter is responsible for the major fet transport into glioblastoma cells. studies with xenopus laevis oocytes, expressing individual human amino acid transporters, revealed that system l, y + l and b 0+ amino acid transporters recognize fet as substrate (lat1 and 2, y + lat2, and b 0+ at, respectively). in contrast, y + lat1 and atb 0,+ did not transport fet. rna expression studies using qrt/pcr revealed that lat1 is the dominant amino acid transporter in all glioblastoma cells investigated (ln229/u373/u87mg/u251/a172/t98g). a strong lat1 expression was also shown on the protein level. to find out whether lat1 is the main transporter responsible for fet accumulation, we first studied transport of the parent amino acid l-tyrosine in ln229 glioblastoma cells. [ 3 h] tyr uptake was completely na + -independent and inhibited by leu, phe and trp, but not by arg, pro or ser. sirna-mediated down-regulation of lat1 in ln229 cells led to a concomitant decrease of lat1 mrna and tyr transport (down to 3% and 20%, respectively). these results indicate that tyr is exclusively transported by lat1 in ln229 cells. we are currently performing transport studies using [ 18 f]fet to investigate whether fet transport is also exclusively mediated by lat1 in glioblastoma cells. a further question is if lat1, a sodium-independent transporter, can be responsible for the accumulation of fet observed in glioblastoma cells. if true, other amino acid derivatives that are lat1 substrates might also proof useful in cancer diagnosis. telmisartan reduces adipose tissue inflammation and biglycan accumulation in diabetogenic ldl-receptor knockout mice grandoch m., nagy n., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany in addition to lowering blood pressure some of the angiotensin ii at1 receptor antagonists (arb) such as telmisartan have additional beneficial effects on the onset of type 2 diabetes mellitus and obesity. this was contributed mainly to peroxisome proliferator activated receptor (ppar)γ modulating activity. hyaluronan (ha), a high molecular weight polysaccharide and the small leucine rich proteoglycans, decorin and biglycan, are known to be involved in atheroprogression. mechanistically these matrix components contribute to inflammatory processes via toll-like receptor-signalling and are supposed to modulate lipid retention. the aim of this study was to elucidate the effects of telmisartan in comparison to valsartan, an arb without pparγ activity, on extracellular matrix remodelling and inflammation in atherosclerosis and the interrelationship with adipose tissue inflammation using the ldlr-/-model of accelerated atherosclerosis. male ldlr-/-mice were fed either a diabetogenic diet alone or in combination with telmisartan (10 mg/kg), valsartan (25 mg/kg) or valsartan (50 mg/kg) from 8 weeks of age for 17 weeks. all treatment groups except of the lower valsartan dose showed significant effects on reducing the aortic plaque score. the content of ha, collagen and decorin in the aortic root were not changed. however, telmisartan reduced the content of biglycan in the aortic root significantly in contrast to valsartan. in addition, a trend towards decreased mac2-positive macrophages in abdominal adipose tissue was detectable after telmisartan treatment as well as a strong reduction in the adipose tissue mrna expression of biglycan. finally, telmisartan reduced the expression of hyaluronan catabolizing enzymes potentially leading to an increase of high molecular weight ha in the adipose tissue, which is thought to be homeostatic and antiinflammatory. in summary, the results of this study underline the pronounced anti-inflammatory capacity of telmisartan on atherosclerosis and adipose tissue inflammation in comparison to valsartan and strongly suggest that biglycan might be an additional target of telmisartan not only concerning matrix composition of atherosclerotic lesions but also concerning the structure of adipose tissue and metabolic effects of the compound. human primary malignant cancer cells derived from peritoneal effusions of a patient with colorectal carcinoma, as assessed by comet assay. the primary cancer cells were more efficient in dsb repair than ht-29 cells, and their doxorubicin ic50 was four times higher. comparative protein expression levels showed that the primary cells had less rad 51 and 52 as well as less topoiiα, while ku70 and 80 levels were similar. another very interesting protein is the mrn (mre11-rad50-nbs1) complex that initializes the phosphorylation of atm and thereby starts the signalling cascade. the newly described mrn-atm pathway inhibitor mirin interrupts mrn activity by inhibiting the exonuclease activity of mre11. the toxicity of mirin in ht-29 cells was measured using a luminescence-based assay detecting the amount of atp, which is correlated with cellular viability. mirin did not show any toxic effects up to a concentration of 100 µm and incubation times of 24 hours, indicating that mirin can be used under these conditions without detrimental effects. we are currently investigating the effect of mirin on the toxicity of topoiiα inhibitors. the inhibition of dna repair may be a valuable strategy to enhance the effect of dnadamaging anticancer drugs. since tumours (even of the same entity) are not only heterogeneous, but also polyclonal, a broad selection of response modifiers of anticancer drugs would be helpful to individually enhance chemotherapeutic effectiveness. evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. epigenetics play an important role in the control of gene expression. epigenetic mechanisms comprise modulation in dna methylation, histone modification and non-coding rna. several polyphenols have been reported to possess histondeacetylase (hdac) inhibitory properties [1] . histone deacetylation is generally linked to transcription repression. furthermore, hdac belongs to the group of small ubiquitin-related modifier (sumo) substrate proteins. sumoylation of hdac is associated with a modulation of its biological activity [2] . little is known so far about the mechanism by which hdac sumoylation mediates inhibition of gene transcription. we addressed the question whether sumo e1 and hdac 1 expression and whether potential hdac-sumoylation will be affected by polyphenols such as chlorogenic acid, genistein and (-)epigallocatechin-3-gallate (egcg). chlorogenic acid, genistein and egcg decreased sumo e1 protein level in the human colon carcinoma cell line ht29 after 24h of incubation measured with western blot analysis. egcg exhibited the most pronounced effect at concentrations ≥ 50 µm. hdac 1 expression was also affected by these polyphenols. the direct impact of polyphenols on the hdac sumoylation is detected by co-immunoprecipitation experiments with the respective antibodies against hdac-1 and sumo e1. these experiments are still under investigation. in conclusion, chlorogenic acid, genistein and (-)-epigallocatechin-3-gallate influenced the sumo and hdac expression in vitro. in further studies the direct impact on subtract-sumoylation will be investigated. these studies contribute to a better understanding of potential chemopreventive effects of dietary polyphenols on specific epigenetic alterations may provide chemopreventive strategies for reducing cancer risk. the no/cgmp cascade is thought to be essential for penile erection. within the smooth muscle of corpus cavernosum, nitric oxide activates the no-sensitive guanylyl cyclase (no-gc) which raises the intracellular concentration of cgmp. this second messenger activates the cgmp-dependent protein kinase i (pkgi) and subsequent phosphorylation of target proteins leads to relaxation of cavernosal smooth muscle. knock out of key enzymes of the no/cgmp cascade has led to discrepant results: the deletion of pkgi in the mouse has been shown to lead to erectile dysfunction whereas mice lacking neuronal no synthase are fertile. to investigate the role of the no receptor in fertility we have generated mice lacking no-gc (gcko), a bottleneck enzyme of the no/cgmp cascade. we have shown that lack of no-gc resulted in arterial hypertension concomitant with a totally abolished no responsiveness of vascular and gastrointestinal smooth muscle. in addition, we generated a mouse line in which no-gc was specifically deleted in smooth muscle cells (sm-gcko). using these ko strains we here examined the role of no/cgmp signaling with regards to the smooth muscle relaxation of corpus cavernosum. no failed to affect corpus cavernosum from gcko in organ bath experiments: neither exogenously produced no by no donors nor endogenous no release from neurons induced by electrical field stimulation led to relaxation. similar results were observed in the corpus cavernosum of sm-gcko mice. to our surprise, the gcko animals were fertile and produced offspring albeit at a reduced rate compared to wt animals. our data show that interruption of no/cgmp signaling results in complete absence of no-induced relaxation of penile corpus cavernosum in mice and reduces the ability to produce offspring but does not abolish fertility. novel modes of invasive cell motility regulated by the formin class of actin nucleators khan j., grosse r. philipps-universität marburg, pharmakologisches institut, karl-von-frisch-str. 1, 35034 marburg, germany pathological invasive cell migration essentially reqires actin polymerization. formins are the largest group of rho-gtpase effectors involved in actin nucleation and assembly as well as microtubule dynamics. here we studied the role of formins in cytoskeletal regulation during homotypic cancer cell invasion. we identified the actin-dependent steps and structures involved for this process. using live cell analysis we characterize the distinct actin dynamics controlled by formin-like 2 and rho function. the specific involvement of this signaling module will be discussed. formin-driven nuclear actin assembly controls mal/srf activity baarlink c., wang h. polymerization of actin in the cytoplasm is tightly linked to transcriptional activation of the srf cofactor mal (also known as mrtf-a) through release of actin/mal interactions and subsequent nuclear accumulation of mal. formins directly promote assembly of actin filaments thereby efficiently regulating mal-dependent transcription for cell shape, adhesion and motility. here we show that formins assemble f-actin and promote mal activation inside the mammalian nucleus. the rho-gtpase effector mdia2 rapidly enters the nucleus in a signal-dependent fashion and an active mdia confined to the nucleus potently promotes release of g-actin from mal to specifically activate srf. live cell imaging reveals formin-mediated nuclear actin dynamics. moreover, using actin assembly assays we find that inhibition of endogenous mdia formins controls f-actin turnover in isolated nuclear extracts. thus, formin activity is dynamically compartmentalized to the mammalian nucleus to potently regulate actindependent mrtf function. in women the placenta becomes the main source of maternal estrogens during pregnancy. placental estrogen biosynthesis is located in the syncytiotrophoblast, a syncytium that builds the main part of the placental barrier and limits the transfer of substances between the fetal and maternal compartment. since the human placenta is unable to convert cholesterol into 17-oh-pregnenolone, the placenta tissue highly depends on the supply of c-19 steroids for their conversion into c-18 estrogens. in contrast to lipophilic unconjugated steroids that penetrate the cell membrane passively via diffusion, circulating sulfated steroid hormones are delivered to the placenta via carrier-mediated transport, followed by their reactivation via the catalytic activity of the steroid sulfatase (sts). dheas of maternal and fetal origin contributes about equally to the placental formation of estrone (e 1) and estradiol (e2), while 16αoh-dheas supplied by the fetus contributes to over 90% of placental estriol (e3) synthesis. soat, a member of the slc10 family with highest expression in hormone-responsive tissues such as testis, placenta, and mammary gland has been shown to transport the sulfoconjugated steroid hormones dehydroepiandrosterone sulfate (dheas), estrone sulfate (e1s), and pregnenolone sulfate (pregs) [1] . aim of this project is to investigate the role of soat for placental estrogen synthesis by means of the choriocarcinoma cell line jeg-3 as in vitro model for the human syncytiotrophoblast. therefore, we characterized a jeg-3 cell line that transformed dhea into e2 and 16αoh-dhea into e3. by qrt-pcr we found expression of sts and aromatase, both essential for estrogen synthesis in these cells. upon transient transfection of soat the carrier was located in the cell membrane of transfected jeg-3 cells. currently we investigate the transformation of dheas of these soat-jeg-3 cells by lc-ms-ms. we could demonstrate transport of 16αoh-dheas for stably transfected soat-hek293 cells. in situ hybridization and immunohistochemistry showed coloured syncytiotrophoblasts and vascular endothelial cells in late term placenta. in conclusion, soat-mediated transport of sulfated steroids could play a pivotal role for placental estrogen synthesis from sulfated steroid hormones. developing non-animal test systems for evaluation of toxicity was important in the past and will remain essential in the future. here we present a toxicity test using the chicken yolk sac area vasculosa (cav) of fertilized white leghorn chicken eggs [1, 2] and compare it to hen's egg test on chorioallantoic membrane (het-cam) [3] for polymer toxicity testing. fertilized chicken eggs were incubated and after 72 h explanted shell less into sterile petri dishes. test substances were applied on the cav and the appearance of different effects (vascular lysis, haemorrhage, aggregation of blood components, lethality) was determined by light microscopy after 1 -48 h (fig.1 ). these effects were combined to a cav test score based on the irritation score calculation used for het-cam evaluation. different polymers like poly(ethylene glycol) (peg; neutral), poly(ethylene imine) (pei; cationic) and dextran sulphate (ds; anionic), as well as guideline-conform (recommended het-cam protocol from the interagency coordination committee on the validation of alternative methods) negative (0.9 % nacl) and positive controls (1 % sodium dodecyl sulphate (sds) and 0.1 n naoh) were investigated. additionally ld 50 values for different cationic polymers have been determined. within the selected incubation times (1 -48 h) , effects such as vessel lysis and blood component aggregation could be detected. additionally to het-cam, lethality as well as recovery of the cav could be observed. differences between neutral, positively and negatively charged polymers were obtained. pei showed strong vessel lysis and aggregation of blood components whereas ds and peg showed none of these effects. lethality was found to increase from peg < ds < pei and is concentration and time dependent. the results demonstrate that differences, regarding the toxicity of the used polymers, can be shown with this test. these findings in the cav test can be well correlated with already existing data. in summary, the cav test provides same data (testing control substances) and more information (recovery and lethality) compared to het-cam and could be a suitable model for toxicity testing of polymers. risk characterisation of chemicals consists of three steps (1) hazard identification and characterisation, based on substance-specific toxicological hazard data, (2) estimates of the level of exposure toward the substance and (3) the comparison between the toxicologically safe level and the exposure level. in contrast to the classical risk assessment approach, the threshold of toxicological concern (ttc) approach is developed as a tool to assess the risk of substances without toxicity data. its application requires (1) information on human exposure, for which it is essential that exposure is fully captured and (2) knowledge of the chemical structure to assess whether the chemical is not excluded from the application of the ttc concept. instead of chemical specific no observed (adverse) effect levels (noels/noaels), the ttc approach utilises knowledge on the empirical distribution of several hundreds of noels/noaels, originally 613, based on toxicological testing in animals (munroe et al., 1996) . with the basis on noaels, the ttc concept builds on the fundamental principle of toxicology, that toxicity is a function of dose and that a dose exists, below which no adverse effects of the substance can be detected. it is assumed that exposures below this level will not result in health risks. three separate ttcs were derived (munroe et al., 1996) by classifying the chemicals into three toxicity classes using a decision tree based on a series of 33 questions related to chemical structure, and on natural occurrence in food and in the body (cramer et al., 1978) . the ttc values are derived from empirical distribution of the noels/noaels in the class taking the 95th percentiles and dividing them by the default uncertainty factor of 100. it is assumed that the probability is very low that the unknown noael of a not tested chemical will be lower than the value of the 95th percentile in the distribution of the known noels/noaels. hence, at exposures below the ttc values, the probability of adverse effects on human health is considered to be very low. introduction: kibra, mainly expressed in kidney and brain tissue, is involved in brain development and memory formation as a postsynaptic scaffold protein. in podocytes, kibra is proposed to regulate cell motility as a linker between components of the cytoskeleton and polarity protein complexes (duning et al, jasn 2008) . furthermore, kibra has been identified as key regulator of the hippo pathway, which is involved in organ size control and tumorigenesis. in the current study, we focused on the identification of kibra gene expression regulation and functional promoter characterization. methods: serial promoter deletion constructs were generated by cloning 3627 bp of the 5'flanking region of kibra into the pgl3-vector system. deletion constructs were transiently transfected into human neuroblastoma cells (sh-sy5y) and immortalized human kidney epithelial (ihke) cells. potential transcription factors (tfs) were investigated in cotransfection experiments. transcriptional start sites (tss) were determined by rapid amplification of 5'cdna ends (5'race) . tss utilization between cell lines was assessed by semiquantitative pcr. transcriptional activity (ta) of the kibra promoter p1 was separated by ~1060 bp into two distinct regions, promoter p1a and p1b. deletion constructs harbouring promoter p1b were transcriptionally active only in ihke cells. 5'race revealed two alternative tss in both cell lines upstream of the annotated tss (nm_015238). exclusively in ihke cells, two additional tss were detected in intron 1, resulting in two alternative exons. deletion constructs harbouring the putative regulatory regions (p2 and p3) of both exons were transcriptionally active only in ihke cells. overexpression of full length tcf7l2 (transcription factor 7-like 2 [t-cell specific, hmgbox]) resulted in a ~3-fold increase of promoter p1a and intron promoter p2 ta. kibra gene expression is driven by a complex alternative promoter system comprising the constitutional promoter p1 and three alternative promoters p1b, p2 and p3. the tss utilization is cell type-specific. subsequent usage of an alternative translation start site within exon 3 could result in truncated kibra protein isoforms. tcf7l2 is involved in the differential kibra gene expression regulation. resulting kibra protein isoform and their cellular function will be assessed in further studies. skin absorption in vitro based on the study of human/animal skin ex vivo or reconstructed human epidermis, respectively, is an alternative method which is accepted by the oecd. guideline tg 428 and a corresponding technical guidance document (gd 28) give technical guidance how to perform valid experiments 1, 2 . the requirements include integrity evaluation tests for the skin samples. different tests are proposed to ensure an exclusively use of undamaged skin. to decide which test suites best to our routine test strategy, we investigated the correlation between integrity test results and absorption profiles of various penetrants (logp range: -0.07 -6.2). finite dose experiments using rat and human skin were performed with 14 c-labeled testosterone, caffeine, mcpa (4-chloro-2-methylphenoxyacetic acid) and its 2-ethylhexyl-ester mcpa-2ehe. for each experiment at least three of the five following integrity tests were conducted: transepidermal electrical resistance (teer), transepidermal water loss (tewl), transepidermal tritiated water flux (³h2o), transepidermal absorption of methylene blue (blue) , transepidermal absorption and flux of a ³h-labeled internal standard (istd); ³h-testosterone or ³h-mannitol was used as istd. the applied radioactivity of the ³h-istd was selected to show no analytical interference with the 14 c-penetrants. teer, tewl and ³h2o represent pre-study, istd concurrent and blue post-study tests. calculated maximal permeability constants (kp) and absorbed doses (ad) of the penetrants were compared to the results of the integrity tests. individual linear regression analysis was used to evaluate the correlation the correlations varied over a wide range for all five methods and four penetrants. the best correlations in average were achieved with the istd. no inverse correlations were obtained for the istd, but partly for tewl, teer, ³h2o and blue. in conclusion, the istd represents best the achieved absorption profiles of the test compounds and is based on that the most suitable integrity test for our dermal absorption studies. we will further confirm its effectiveness and generate a sufficient historical database in order to include the istd in our routine test protocol. investigation of mirna expression and dna methylation in focal and non-focal brain tissue of therapy-resistant epilepsy patients haenisch s. 1 background: resistance to anticonvulsants affects one third of all epilepsy patients. limited bioavailability of the drug at the target site caused by increased expression of efflux transporters on the blood brain barrier or alterations of target genes are potential mechanisms for therapy resistance. however, these mechanisms alone cannot completely explain the observed resistance and it is likely that multifactorial alterations lead to pharmacoresistance. there is increasing evidence that expression of micrornas probably caused by dna modifications is deregulated in many neuronal diseases. we hypothesize that mirna regulation of target genes is involved in drug resistance in epilepsy. methods: hippocampal focal and cortical non-focal brain tissue samples from 13 patients diagnosed with mts (mesial temporal sclerosis) who underwent neurosurgery have been screened for mirna expression using taqman low density arrays. in silico approaches for both a hypothesis-based (efflux-transporter and target gene) as well as a hypothesis-free approach were used to identify potential phenotype-relevant target genes. using the program r (bioconductor) a mann-whitney-u test was performed to compare mirna expression between brain regions. pyrosequencing was performed to investigate methylation status 5'-upstream of dna regions encoding for selected candidate mirnas. results: out of 754 mirnas, 150 were detected in both tissue types. the expression of one mirna was 7.2 fold higher (q=0.01) and another was 3.8 fold lower (q=0.01) in the hippocampus relative to the cortex. evidence could be found that down-regulation of the latter is possibly caused by hypermethylation of 5'-flanking region of its encoding dna locus. bioinformatic analysis has identified eight genes important for neuronal regulation and signal transmission (e.g. sox11, mecp2, bsn), as well as one abc effluxtransporter, as potential targets for these differentially regulated mirnas. conclusion: differential regulation of two mirnas could contribute to an altered function of several genes resulting in an imbalance between neuronal excitation and inhibition that is independent from mechanisms presently targeted by anticonvulsants. this work was supported by a fellowship from dfg and nih grant gm61390. recently, it has been reported that human b cells express and secrete the cytotoxic protease granzyme b (grb) after the combined stimulation of the il-21-and the b cell receptors. grb produced by b cells is enzymatically active and b cells deliver grb to sensitive cancer cell lines, thereby inducing apoptosis. to date, there is little experimental evidence on the mechanisms involved in grb expression, or its function in b cell biology. as experimental transgenic murine systems should enable us insights into these issues, we assayed for grb in c57bl/6 b cells using an extensive array of physiologically relevant stimuli, but were unable to detect either grb expression or its proteolytic activity, even when antigen specific transgenic b cell receptors were cross-linked. similar results were also obtained with b cells from dba/2, cba or balb/c mice. in vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of grb in cytotoxic t lymphocytes, but not in b cell populations. we also investigated a possible role of grb on the humoral immune response to np-klh, but grb-deficient mice produced normal amounts of antibody with typical affinity maturation and heightened secondary response, demonstrating conclusively the redundancy of grb for antibody responses. our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans and demonstrate the need to develop novel in vivo systems to study human humoral immune responses. investigations of the cholinergic neurotransmitter system in dyt1 mice hamann m. 1 early-onset torsion dystonia is an autosomal dominant inherited movement disorder associated with the dyt1 gene defect with deletion of a glutamic acid residue in the protein torsina. despite the gene defect, the pathophysiology is poorly understood. animal models can help to understand the underlying mechanisms and thereby to develop new therapeutic strategies. sharma et al. (2005, j. neurosci. 25 [22] , 5351-5355) initially described a transgenic mouse model (dyt1 mice) with overexpression of mutant torsina. previous studies in these mice pointed to alterations in the cholinergic system. to investigate the functional relevance of these in-vitro findings, we carried out pharmacological in-vivo experiments and determined the density of striatal cholinergic interneurons as well as the expression of choline acetyltransferase in different brain regions. the acute intraperitoneal administration of the cholinomimetic drug pilocarpine (75, 100 and 125 mg/kg) as well as a long-term treatment over 21 days (100 mg/kg/d) did not induce pronounced effects in dyt1 mice compared to wildtype controls. the higher incidence of epileptic seizures in dyt1 mice compared to controls after repeated local striatal applications of pilocarpine (25 and 50 µg/0.5 µl/hemisphere) let presume an altered synaptic plasticity in dyt1 mice. the immunohistochemical investigations revealed a moderately reduced density of striatal cholinergic interneurons in the dorsomedial subregion of dyt1 mice compared to wildtype controls, while significant differences in other striatal subregions were not detected. western blot analysis did not show clear differences in the expression of choline acetyltransferase between dyt1 and wildtype control mice. these results indicate that the cholinergic system seems not to play a key role in this line of dyt1 mice. ongoing receptor autoradiographic analysis of binding to different muscarinic receptors subtypes have to further clarify the existence of possible alterations within the cholinergic system of these dyt1 mice. inhibitors direct against cell cycle-regulatory kinases are being tested in clinical trials as anti-proliferative agents. thus, the atp-competitive kinase inhibitor pd332991 which inhibits cdk4 and cdk6 is currently tested in patients with solid tumors such as glioma. we found that pd332991 suppressed il-1-induced expression of il-8 suggesting that cdk4 or cdk6 may have unknown anti-inflammatory properties. to study the effects of cdk6 on the il-1-signaling network, we established a bidirectional doxycyline-inducible system to express a constitutively active mutant of cdk6, cdk6 s178p, in asynchronized hela cells. cdk6-expressing cells were identified by gfp which was expressed from the same promoter, isolated by laser-microdissection and analysed for mrna expression using a down-scaled rt-qpcr assay. compared to the uninduced state, cdk6 s178p enhanced il-1-induced il-8 and il-6 mrna expression. moreover, shrna-mediated suppression of endogenous cdk6 confirmed a role of this kinase in regulation of maximal il-1-induced gene expression of il-8. these data also revealed that the contribution of cdk6 to inflammatory gene expression is highest in g1, when activity of endogenous cdk6 is activated by d-type cyclins. these findings were corroborated in hela cells expressing fluorescent ubiquitin-dependent cell cycle indicator (fucci) proteins. hela-fucci cells from g1, g1/s, g2 or mitotic states were isolated by laser-microdissection and analyzed by rt-qpcr for tnf-inducible gene expression. stable knockdown of cdk6 in hela fucci or inhibition by pd332991 suppressed inducible il-8 expression. microarray experiments identified many additional genes that required active cdk6 for maximal il-1-or tnf-inducible gene expression. we also found that cdk6 co-immunoprecipitated with p65 nf-κb, colocalized with p65 in the nucleus and was recruited together with the p65 subunit to the proximal il-8 promoter as assessed by chip and re-chip experiments. collectively, these results suggest an unexpected control of inflammatory gene expression through a classical cell cycle regulatory pathway. these results also imply that pharmacological targeting of cdks may have effects and side-effects on the immune system in addition to inhibition of cell cycle progression. trp channels form a heterogeneous family of calcium-permeable channels, which play major roles in physiological functions ranging from sensory reception to cellular signal transduction. members of the trpc subfamily (classic transient receptor potential channels) are downstream targets of hormone receptors. of particular interest is the biological role of trpc6 channels. they are directly activated by diacylglycerol due to phospholipase c-driven signalling pathways which are involved in smooth muscle contractility, neuronal plasticity, keratinocyte differentiation and renal function. their impact in renal function became evident from analyzing patients suffering from familial forms of focal segmental glomerolusclerosis (fsgs) which could be linked to trpc6 mutations. since the first descriptions at least 17 different pathogenic mutations have been identified in humans to cause fsgs. in order to study the underlying pathophysiological mechanisms of trpc6 mutations, we have analysed all mutated trpc6 channels known to date heterologously expressed cells. one set of mutations showed a gain-of-function phenotype which has been previously suggested to cause an increased intracellular calcium load and subsequent cell death. hence, gain of function mutations fit to the current paradigm of fsgs pathophysiology. however, another set of mutations found in the patients showed a loss-of-function phenotype. our results enable a change in the current paradigm for the role of trpc6 in renal pathophysiology and may provide a basis for our understanding of the pathophysiology of loss-of-function mutations in familial focal segmental glomerolusclerosis. karlsruher institut für technologie (kit) institut für angewandte biowissenschaften, abteilung lebensmittelchemie und toxikologie, adenauerring 20a, 76131 karlsruhe, germany risk assessment for genotoxic carcinogens is an important challenge in toxicology. even though manifold attempts have been made to substitute carcinogens and to reduce exposures, their complete elimination appears to be not possible. thus, low concentrations of known or suspected genotoxic carcinogens are present at workplaces, in the environment and in food. in order to deal with this situation and to set priorities for risk management, different concepts have been established such as the alara principle (as low as reasonably achievable) and the margin of exposure (moe), based on the ratio between concentrations being carcinogenic in experimental animals and the actual exposure of humans for example via foodstuff. while usually linear doseresponse-relationships have been used as default assumption, analytical methods are now available to assess the induction and repair of dna lesions on low exposure conditions, including environmental background exposure, and to relate the extent of exposure-induced dna lesions to endogenous dna damage. this may be an important prerequisite to establish health-based limit values for selected genotoxic carcinogens. within this workshop, different examples will be discussed and research need will be identified. dendritic cells from h4r-deficient mice lose their ability to properly stimulate t lymphocytes hartwig c., seifert r., neumann d. mhh pharmakologie, carl-neuberg str. 1, 30625 hannover, germany the incidence of allergic airway diseases is increasing throughout the world, especially in western countries. although histamine (ha) is found at high concentrations in asthmatic lungs, a role for ha in bronchial asthma is still a neglected topic in clinical research. in particular, the capacity of ha to modulate the underlying immune reaction is far from being understood. the histamine h4-receptor (h4r) is involved in acute inflammation and th2 cytokine production. consequently, we intended to analyze the role of h4r in a murine th2 lymphocyte transfer-based model of asthma. specifically the ability of h4r expressed on dendritic cells (dcs) to modulate t cell function was analyzed. ova-specific cd4 + t cells were polarized in vitro under th2-favoring conditions with ova peptide-pulsed dcs, obtained either from wild-type or h4r -/mice. analysis of the polarized t cells after in vitro restimulation revealed a marked decrease of il-4 production in t cells polarized in the presence of h4r -/-dcs compared to those polarized in the presence of wild-type dcs. thus, on dcs, the h4r is essential for proper stimulation of spleen t cells and for directing their polarization towards a th2 phenotype. the transfer of in vitro polarized t cells into recipient mice and subsequent provocation elicited an asthma-like disease. the h4r on dcs not only affects in vitro polarization of t cells, but also the in vivo function of the obtained polarized t cells. a parameter indicating allergic inflammation is the enhanced influx of inflammatory immune cells into the lung tissue, mainly driven by eosinophils, which are virtually absent in non-asthmatics. when analyzing the number of eosinophils, a dramatic difference due to the polarizing conditions of t cells occurs. in bal fluids of mice that received t cells polarized in the presence of wild-type dcs, about 40% eosinophils were detected. in contrast, the transfer of t cells polarized in the presence of h4r -/-dcs yielded only about 10-20% eosinophils in bal fluids. in summery, the h4r on dcs plays an important role for t cell polarization and consequently affects the allergic reaction during sensitization. since the lack of the h4r on dcs reduced their ability to stimulate proper th2 polarization of cd4 + t cells, we conclude that ha via the h4r significantly affects the manifestation of asthmatic inflammation. antioxidant polyphenols and their effects on nrf2 (skn-1) signalling in a cell culture system and the model organism c. elegans havermann s., wätjen w. heinrich-heine-universität düsseldorf institut für toxikologie, p.o. box 101007, 40225 düsseldorf, germany oxidative stress has been connected with a variety of diseases, (e.g. alzheimer`s and parkinson´s disease), cancer and ageing over the last years. certain polyphenols were shown to have an antioxidant capacity as well as being able to activate the protective nrf2 signalling pathway. compared to direct radical scavengers modulators have the advantage of building up a permanent defense against oxidative insults whereas scavengers do not protect any more after consumption or may even cause stress due to redox cycling. we have employed cell culture based assays (dcf, western blot, gfp reporters) to analyse the effects of polyphenols. further we tested the coumpounds in vivo in the nematode c. elegans where skn-1 is the nrf2 homologue. baicalein and caffeic acid phenethylester (cape) protected cells and the nematode from ros accumulation after application of stress (shown by dcf assay). activation of nrf2 signalling is correlated with translocation of the transcription factor into the nucleus. in both systems nrf2::gfp accumulation in the nuclei could be observed after incubation with baicalein (fluorescence microscopy). but while cape is a potent activator of nrf2 in cells, it has no effect on skn-1 localisation. further the effect on the nrf2 protein amount was investigated by western blot analysis. the expression of target genes can be investigated by differing means: while pcr methods and western blotting are standard for in vitro studies, the vast number of available gfp reporter strains offers opportunities for research using c. elegans. we have performed congruent assays in a cell culture system and the model organism c. elegans to compare antioxidative capacity and effects of polyphenols on nrf2 signalling. therefore, depending on the substance tested, c. elegans is a suitable model system to investigate effects of natural compounds in an organism. being associated with adverse health effects, the human exposure to dehp is subject to concern. quantifying the population's exposure and determining the contributions of different exposure routes is a key task of environmental health risk assessment. the study presented comprises a review of the available data on dehp levels in foods, consumer products, and house dust. extensive survey data, e.g. from the current national nutrition survey ii and the eu rapex system were processed for modeling the exposure by the oral, inhalative and dermal path of the population in germany. the study also included analytical analyses of dehp levels in selected foods and consumer goods (incl. migration rates for mouthing). probabilistic techniques allowed elucidating the exposure's variation and the relevance of different routes. mean exposure estimates for german children and adults to dehp are 36 and 26 µg/(kg d), resp. for children, food accounts for 36% of the total exposure, followed by mouthing (30%) and house dust (19%). the adult exposure is almost entirely (82%) due to food. as dietary exposure is a result from concentration and consumption, foods exhibiting high contamination e.g. butter (8%) and dressings (mayonnaise) (14 %) as well as highly consumed foods e.g. bread and bakery (16%) and vegetables (6,8 %) contributed significantly. the mean estimate of children's dehp exposure via mouthing revealed 0,9 µg/(kg d). high exposures were estimated (95th percentile) up to 10,8 µg/(kg d). on average people in germany are exposed to dehp below the current tdi of 50 µg/(kg d). however, individual exposures exceeding the tdi still cannot be excluded. current data on dehp and other plasticizers in foods are scarce, which warrants broader monitoring. our findings highly facilitate further exposure modeling focusing on dehp substitutes and risks of combined exposure. this study was funded by the federal ministry for the environment, nature conservation and nuclear safety in the frame of the environmental research plan (umweltforschungsplan, förderkennzeichen (ufoplan) 3707 61 201). on the basis of the available measurements of dehp, the exposure assessment has been focused on 37 food categories characterising a selection of the most important food groups covering all major food classes of the german population. based on an extensive literature survey, the analysis considered the available data of dehp measurements in food, as well as the official german food control data taken from the national food consumption survey. the high amount of considered data allowed the consideration of several exposure assessment tiers (deterministic and probabilistic by using monte carlo simulation). a quantitative evaluation of the uncertainties of the estimate of the 37 food categories groups has been performed by means of a sensitivity analysis by using the methodology proposed within the 2008 who ipcs guidance document of characterising and communication uncertainty in exposure analysis. qualitative uncertainty analysis (tier 1) was applied to determine the most important sources of uncertainty, i.e. concentration of dehp in all food categories. the probabilistic monte carlo simulation (tier 2) was then used to rank the cumulative probability distributions of the exposure assessments of 37 food categories on the basis of the food categories that appear to dominate. sensitivity analyses were applied to prove the impact correlation of food groups for uncertainties. by a scenario based concept, the aggregation of the 37 food groups to 10 groups has been evaluated, as well as the sensitivities by characterising particular scenarios. for this purpose, particular "meals" have been described as fixed combined scenarios and. the aggregation leads to a considerable higher exposure estimate which can be explained by the combination of high contaminated foods with others of high consumption. the evaluation confirms the considerable role of possibly high contaminated foods e.g. fats, or mayonnaise. the evaluation shows that quantitative probabilistic sensitivity analysis is a suitable and pragmatic tool for uncertainty analysis in exposure assessment. the transcription factor camp response element (cre)-binding protein (creb) plays a critical role in regulating gene expression in response to activation of the campdependent signaling pathway, which is implicated in the pathophysiology of heart failure. we observed creb knock-out cardiomyocytes to be larger than wildtype cardiomyocytes (cell area in µm 2 ; mean±sem; creb-ko 4871±258 vs. wt 3396±171; n=68/69 cells, n=4 mice, p<0.01 vs. ctr.). the nuclear factor of activated t-cells c3, nfatc3, is another transcription factor involved in the development of heart failure and also a known positive regulator of hypertrophy. hence, we investigated whether inhibition of the cre-dependent transcriptional activation has an impact on the nfatc3 signaling pathway. we first studied the effects of an overexpression of a dominant negative creb mutant (dncreb) or of nfatc3 on the activity of a nfat-dependent model promoter in a permanent cell line. overexpression of dncreb evoked an 8.0±1.5 fold increase of the nfat model promoter activity (n=36; n=6 transfections), but had no impact on kv4.2 promoter activity which is known to be regulated by nfatc3 (1.2±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). nfatc3 overexpression led to a 4.5±0.7 fold increase of the nfat-dependent model promoter activity (n=12; n=2) and to an inhibition of kv4.2 promoter activity (0.8±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). we conclude that creb is a negative regulator of nfat-mediated gene transcription and that activation of nfatc3 might contribute to the observed hypertrophy of creb-ko cardiomyocytes. neuroleptika der perazin-klasse sind potente modulatoren des p2x7-rezeptors -perazine-type neuroleptic drugs are potent modulators of p2x7 receptors hempel c., nörenberg w., urban n., sobottka h., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany p2x7 receptors belong to a family of atp-gated, non-selective cation channels, which play an important role in immune cell activation, inflammatory hyperalgesia and neuropathic pain. they differ from other p2x family members by the low atp affinity, and by the ability to form or recruit dilated pores in the sustained presence of atp. owing to its involvement in many diseased states, p2x7 is a promising target for pharmacological intervention. accordingly, p2x7 blockers are currently tested in phase ii clinical trials. in an attempt to identify p2x7-modulating properties of approved drugs or natural compounds, we performed a medium-throughput screen, using an appropriate compound library (spectrum collection) and a stably transfected hek293hp2x7 cell line. with ic50 values of 1-2 µm, the tricyclic antipsychotics prochlorperazine and trifluoperazine showed a high potency and efficacy to block the atp (1 mm)-triggered increases in the intracellular ca 2+ concentration ([ca 2+ ]i) that was mediated by human p2x7 (hp2x7). the closely related phenothiazine-class neuroleptic drugs, such as chlorpromazine or triflupromazine did not have an appreciable effect on hp2x7mediated ca 2+ influx. whole-cell inward currents, measured at -60 mv, were blocked by more than 60% by 3-10 µm prochlorperazine. the inhibitory effects of perazines developed within about 200 ms, hinting to a direct mode of action by binding to the p2x7 protein. prochlorperazine added intracellularly via the patch pipette did not substitute for the extracellularly applied drug, indicating that its binding site is accessible from the extracellular side. in addition, both compounds blocked yo-pro-1 uptake when preincubated before p2x7 stimulation with 1 mm atp or when applied subsequent to the agonist. interestingly, when added to a hek293 cell line expressing the rat p2x7, perazines potentiated the atp-induced increase in [ca 2+ ]i. measurements in human monocyte-derived macrophages confirmed the ability of prochlorperazine and trifluoperazine to inhibit atp-evoked increases in [ca 2+ ]i, changes in yo-pro-1 permeability and whole cell currents. taken together, we conclude that perazine-type neuroleptics impede on p2x7 activity in a species-specific manner, presumably by binding to an extracellularly accessible binding site of recombinant or natively expressed p2x7. similarly, pre-treatment with lov also lowered dox-induced stabilisation of p53 and phosphorylation of chek1 and sapk/jnk. while lov had no influence on ir-induced initial dna damage formation in huvec and rat cardiomyoblasts (h9c2), it decreased dox-and eto-induced phosphorylation of histone h2ax, which is a surrogate marker of dna-double strand breaks. this indicates that lov specifically protects against the genotoxicity of topoisomerase type ii poisons. in an acute and subacute balb/c mouse model lov protected from ir-induced toxicity. this effect rested on inhibition of pro-inflammatory and pro-fibrotic processes as measured via quantification of mrna levels of il6, ctgf and tnfα. the same was true for dox-induced toxicity, i.e. heart and liver damage. similar to the in vitro experiments, dox-induced hepatic dna-damage was attenuated by lov treatment. overall, liver and heart toxicity were reduced by lov as mirrored by the serum levels of gldh/gpt and ctn-i, respectively. both in liver and in heart we observed collagen rich perivascular areas following dox treatment. under situation of lov-co-treatment these areas occurred more rarely and were less pronounced, pointing to a lowered level of fibrosis. pcr-array-based mrna analyses showed inhibitory effects of lov on dox-triggered expression of genes involved in oxidative stress response, drug transport, dna repair, cell cycle progression and cell death. for instance, up-regulation of p21, wee1, cjun/fos and hmox-1 following dox administration was attenuated by lov. altogether, we suggest that including lov in current cancer therapeutic regimen might widen the therapeutic window of anticancer therapeutics by lowering normal tissue damage. the p values. in addition, array data underwent cluster analysis for identification of substantial differences of gene regulation among the three different types of biopsies. results: of particular interest in our study was the expression of genes coding for metabolism and transport proteins. therefore 42 genes from the 156 significant differentially regulated genes, were selected for the qrt-pcr analysis. genes coding for abcb1 and abcg8 transport proteins showed higher expression in the jejunal tissue one year after surgery compared to the duodenal tissue (fold change 1.80 and 1.73). moreover, cyp7a1 mrna involved in metabolic processes is higher expressed in postoperative jejunum than in the jejunum tissue taken during the surgery (fold change 1.9). in conclusion roux-en-y gastric bypass operation leeds a change of mucosal gene expression profile in the jejunum during one year. there was also a significant differential gene expression between the original duodenum and jejunum one year after surgery. these results give strong evidence that jejunum not exposed to pancreatic but only to gastric fluids may change its gene regulation. background. numerous genome-wide association studies (gwas) identified polymorphisms located in transporter genes such as slc2a9, abcg2, npt1, and urat1 as predicitive for the serum levels of urate 1 . these genes encode membrane proteins expressed in the apical membrane of human kidney proximal tubule cells and are assumed to facilitate tubular exchange of urate 2, 3, 4, 5 . importantly several single nucleotide polymorphisms (snp) located in vicinity of slc2a9 have been identified as highly associated with serum urate levels. little is known about the transcriptional regulation of slc2a9. therefore, the aim of our study was to investigate which sequences in the slc2a9 gene harbour ciselements and regulate its gene expression. we also asked whether intronic snps influence gene expression at the transcriptional level. methods and results. performing dual luciferase reporter gene assays we found gene regulating modules in the slc2a9 gene. dna from human kidney samples was then genotyped for rs6449237 being part of this region. next total slc2a9 mrna-expression levels of the samples were determined using real-time quantitative rt-pcr assay. male samples with two minor alleles of snp rs6449237 showed lower slc2a9 mrna levels than samples with the wild type alleles. the effect was not seen in females. reporter gene constructs with either minor or major allele of rs6449237 were then used in luciferase assays, however showing no significant difference in activity. furthermore mrna-expression levels of other urate transporter genes were determined in kidney samples. after linear regression a positive correlation of mrna-expression of slc2a9, urat1, npt1, and oat10, respectively was observed. conclusion. our data suggest that the slc2a9 snps rs6449237 and rs74794351 might influence slc2a9 mrna-level without controlling the transcriptional activity. it needs to be elucidated whether those snps alter mrna stability. however, the mrna coexpression of slc2a9 and other urate transporter might be attributed to a common gene regulating pathway of an "transportosome" controlling urate homeostasis. sulfotransferases mediate the bioactivation of methyleugenol to a reactive sulfate ester binding to dna in vitro and in vivo herrmann k. 1 methyleugenol (me) is a secondary metabolite occurring in many herbs and spices. although me is hepatocarcinogenic in rodents, standard genotoxicity tests were negative. this may be due to the lack of critical activating enzymes responsible for the terminal bioactivation of me to a genotoxicant. me is initially hydroxylated by cytochrome p450 enzymes yielding 1´-hydroxymethyleugenol (1´-ohme). this alcohol can be further activated by sulfotransferases (sults) to an electrophilic sulfate ester that can be easily attacked by dna. the dna adducts formed could lead to mutation and further carcinogenicity observed in animals. the aim of the present study was to clarify whether individual human (h) and murine sult forms are involved in the activation of me to a genotoxicant. in order to identify critical sults, mutagenicity tests including bacteria expressing different sult forms were conducted. (±)-1´-ohme (separated into its enantiomers) served as test compound. we could show that hsult1a1, standing out due to its high expression level in many tissues, can efficiently activate both enantiomers even at low concentrations. furthermore, dna adduct formation in hsult1a1-proficient and sult-deficient bacteria was examined after incubation with 7 µm of (+)-or (-)-1´-ohme. for selective detection and quantification of me-derived 2´-deoxyadenosine (da) and 2´-deoxyguanosine (dg) adducts we developed a sensitive tandem mass spectrometry method including stable isotope dilution analysis. adduct formation was only observed in bacteria expressing hsult1a1. the concentration dependence of adduct formation in hsult1a1-proficient bacteria was examined for (+)-1´-ohme. both adducts turned out to be concentrationdependent. to check the extent and organ specificity of adduct formation in vivo we administered 54 mg/kg bw (±)-1´-ohme (i.p.) to mice carrying the hsult1a1/1a2 gene cluster. mice getting only the vehicle served as controls. animals were sacrificed and dna from eight organs was extracted. by means of tandem mass spectrometry adducts were measured and quantified. da and dg adduct formation was observed in all tissues studied, but not in untreated animals. furthermore, adduct levels were higher than in experiments using wild-type mice. altogether, we herein could show that sulfo conjugation leads to bioactivation of me to a dna-binding intermediate in vitro and in vivo. this work was financially supported by bundesinstitut für risikobewertung. objective: the soluble adenylyl cyclase (sac) activates the na + /k + -atpase in renal epithelial collecting duct cells. nuclear sac constitutes a functional complex with camp response element binding protein (creb), suggesting a more general role of sac in overall gene regulation. we determined the chromatin binding capacities of sac at cre sequences and its influence on genes, which play a role in aldosterone signalling. furthermore, we functionally characterised expression relevant promoter portions of sac and the influence of aldosterone and camp mediated signalling pathways on sac gene regulation. design and methods: in vascular endothelial cells (ea.hy926) and in human kidney cell lines (hek293t; ihke), we performed chromation immunoprecipitation (chip) assay with antibodies against sac and creb. we conducted transfection with a cre luciferase reporter vector and sac promoter constructs, following treatment with sac inhibitors and aldosterone. total rna of ea.hy926 cells, which were treated with sac inhibitors and aldosterone, was isolated and subsequently analysed by real-time pcr for expression of genes involved in aldosterone signalling. in vivo binding of sac at cre motifs was shown using cre consensus sequences in chip experiments. specific pharmacological inhibition of sac led to a significant decrease of transcriptional activity of the cre control vector in endothelial and kidney cell lines. furthermore, we were able to show the different effects of sac on the expression of downstream targets of aldosterone signalling, e.g. mineralocorticoid receptor and na + /k + -atpase alpha1 and beta1 and sac itself. regulation of sac itself is mediated by two different promoter portions, which are influenced by aldosterone and inhibition of sac and differentially accessed in kidney and endothelial cells. sac has transcriptional trans-acting properties as it interacts with cre sites and potentially influences the expression of genes, which play a role in aldosterone signalling. transcription of sac is regulated via aldosterone and camp. the location of promoter ta is cell type-and stimulation-specific. the role of the sodium-calcium exchanger (ncx1) in cardiac pacemaking herrmann s., stieber j., ludwig a. friedrich-alexander-universität erlangen-nürnberg institut für experimentelle und klinische pharmakologie und toxikologie, fahrstrasse 17, 91054 erlangen, germany the mammalian heart is driven by the sinoatrial node, the primary cardiac pacemaker. the unique feature of sinoatrial node (sn) cells is the ability to generate a spontaneous diastolic depolarization that periodically initiates action potentials which set the heart rhythm. the molecular origin of this cardiac pacemaker activity is still a matter of debate. recent findings point to a coordinated interplay between intracellular ca 2+ -cycling processes and plasma membrane-localized ion channels which determines the origin, periodicity and rate modulation of pacemaker potentials. in this study, we investigated the contribution of the cardiac sodium-calcium exchanger (ncx1) to pacemaking. ncx1 is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. it was speculated that the membrane depolarizing current incx, whose activity is dependent on intracellular ca 2+ -fluctuations, represents a main determinant of the spontaneous diastolic depolarization. we used an inducible and sinoatrial node-specific cre-transgene to delete ncx1 in the murine pacemaker system. the successful creation of a cardiac pacemaking and conduction system specific ncx1 knockout (cpncx1ko) was demonstrated by transcript quantification as well as immunofluorescence experiments. telemetric ecg recordings of cpncx1ko displayed a distinct cardiac phenotype. mutant animals were deeply bradycardic and lost their capability of maintaining a stable heart beat as demonstrated by various ecg abnormalities like sn arrhythmia, sn pauses, av block and ventricular tachycardia. analysis of the spontaneous activity of isolated sn preparations showed a slower and arrhythmic contraction rate of the mutant tissues strips confirming that the bradycardia and arrhythmia induced by deletion of ncx1 results from a slower and arrhythmic intrinsic pacemaker activity. a battery of experiments using different heart rate lowering as well as increasing drugs revealed an altered heart rate modulation in cpncx1ko animals as compared to controls. in conclusion, these initial results establish ncx1 as a major contributor to cardiac pacemaking. a wide variety of contaminants are ingested through food, among them the procarcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (bp) which is resorbed and partially metabolized in the enterocytes of the small intestine. previous in vitro studies revealed that bp phenols are excreted as phase ii metabolites including bp glucuronides and bp sulfates. this export is mediated by the breast cancer resistance protein (bcrp/abcg2). the ultimate carcinogenic phase i bp metabolite anti-bp-7,8dihydrodiol-9,10-epoxide (bpde) can be detoxified by glutathione conjugate formation catalyzed by various glutathione s-transferases. in the present study, differentiated human intestinal caco-2 cells were used as a model for the human small intestine to investigate the detoxification of bpde and the subsequent transport of the stereoisomeric glutathione conjugates in the presence of an inhibitor (acivicin) of the glutathione-cleaving enzyme gamma-glutamyl transpeptidase (ggt) at the surface of the cells. the results indicate that the glutathione conjugates of bpde are formed and excreted mainly to the apical and to a minor extent to the basolateral side of the polarized caco-2 monolayer. to stimulate the transport rate several inducers known to enhance gene expression of xenobiotic-metabolizing enzymes as well as transport proteins were used (quercetin, oltipraz, butyrate). however, solely oltipraz substantially increased the efflux of bpde glutathione conjugates after inhibition of ggt. inhibition studies revealed that the multidrug resistance-associated proteins (mrps/abccs) are involved in the transport of the bpde glutathione conjugates. stable abcc1, abcc2 and abcc3 knockdown cell lines were generated allowing to demonstrate that abcc1 mediates the basolateral, abcc2 the apical excretion of the bpde glutathione conjugates. in conclusion, the ultimate carcinogen bpde is detoxified via glutathione conjugation and subsequently excreted by caco-2 cells in both apical and basolateral directions. .this finding is equivalent to a transport into the feces as well as blood system in the in vivo situation. signaling via irag regulates store operated calcium entry (soce) in aortic vsmc hieke b., hüttner j., schlossmann j. university of regensburg department of pharmacology and toxicology, universitätsstr. 31, 93053 regensburg, germany the mechanisms involved in the activation of store operated calcium entry (soce) through depletion of intracellular stores and their regulation are not yet fully understood. we examined the effect of inositoltriphosphate-receptor associated cgmp-kinase substrate (irag) on soce. aortic vascular smooth muscle cells (vsmc) from wild type (wt) and irag-knock-out (ko) mice were loaded with the calcium indicator fura 2-am and soce was measured as a change in the intracellular calcium concentration. in experiments with vsmc from wt mice soce was attenuated by the application of 8-br-cgmp. this effect was not observed in vsmc isolated from irag-ko mice. these differences in the strength of the soce-signal were abolished by the replacement of extracellular sodium with n-methyl-d-glucamine. the observed sodium dependence of the soce regulation via irag suggests, that an alternated sodium conductance might be responsible to some extent for the differences detected in wt and irag-ko vsmc. as a change in sodium conductance might result in a changed membrane potential we tried to track these changes with the flipr membrane potential assay kit while executing the soce protocol with and without 8-br-cgmp. no significant differences in membrane potential could be detected in the various stages of soce. in conclusion, our results indicate that irag exhibits a dual action on calcium regulation as it inhibits not only the intracellular calcium release but also the extracellular calcium influx through soce. induction of the icer promoter in vascular smooth muscle cells hildebrandt i. 1 tokyo metropolitan institute of gerontology, tokyo japan several transcription factor isoforms are encoded by the crem (camp response element modulator) gene. one prominent isoform is the inducible camp early repressor (icer), which acts as a transcriptional repressor on so called camp responsive elements (cres) in its target gene promoters. the icer mrna expression is regulated by an intronic promoter of the crem gene. in vascular smooth muscle cells (vsmcs) crem/icer is involved in the regulation of cell proliferation and apoptosis with physiological consequences in vivo. for instance crem-knockout mice, in which none of the known isoforms can be expressed, exhibit an increased neointima formation after carotid ligation as well as an increased atherosclerotic plaque formation after high fat diet on an apoe background. these observations were associated with an increased proliferation rate in isolated crem deficient vsmcs. on this background we wanted to clarify the specific role of icer isoforms in the vasculature. in first experiments we examined the inducibility of icer in primary vsmcs and smooth muscle cell lines. reporter luciferase assays showed that the activity of the icer promoter is induced at the maximum of fourteen fold after 4 hours of stimulation with forskolin (fsk) in immortalized rat vsmcs (13.7 ± 0.93; n=12 from 3 isolations). in a7r5 rat smooth muscle cells the icer promoter showed a maximum stimulation of 3.2 ± 0.16 fold after two hours of fsk stimulation (n=20 from 4 isolations). these pilot experiments showed that the icer promoter is inducible in vsmcs by camp dependent pathways. further experiments have to be carried out to elucidate the role of icer in the vascular system for example by stimulation of primary vsmcs and analysis of icer knockout mice. (supported by the dfg) overexpression of transmembrane channel-like proteins (tmcs) uncouples receptor-mediated calcium mobilisation hill k., urban n., straub i., schaefer m. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany the family of transmembrane channel-like proteins (tmcs) consist of 8 members (tmc1-tmc8) all tmc genes are predicted to encode transmembrane proteins with at least six membrane-spanning helices. mutations of tmc1 cause deafness in human and mice whereas tmc6 and tmc8 (also referred to as ever 1 and 2) are linked to epidermodysplasia verruciformis (ev), a skin disorder, which is characterised by an enhanced susceptibility towards cutanous infections by human papillomaviruses. the cell biological and physiological functions of tmc proteins still remain elusive. we have overexpressed tmc6 and tmc8 in hek293 cells to get insights into their physiological function. all tmcs were located within the endoplasmic reticulum (er) after overexpression of yfp or cfp-tagged constructs. ratiometric calcium imaging revealed that after overexpression of tmc6 or tmc8, stimulation of gq-coupled receptors with carbachol and atp resulted in a greatly reduced amount of calcium release from the er. moreover, challenging tmc6-or tmc8-expressing cells with the serca pump inhibitor thapsigargin was also not followed by a release of er-based calcium within the cell. to test whether the lack of calcium release was caused by a reduced calcium content within the er, we investigated calcium dynamics within the er using an er-targeted fret-based calcium indicator (d1er cameleon). the experiments revealed that the amount of calcium within the er was reduced upon overexpression of tmc6 or tmc8. recently, it has been reported that tmc6 and tmc8 might influence intracellular zinc distribution in human keratinocytes. we could confirm the presence of tmc6 and tmc8 mrna in a human keratinocytes cell line (hacat). upon overexpression of tmc8, hacat cells revealed the same phenotype as described above for the hek293 cells with an uncoupling of the receptor-mediated calcium mobilisation due to a depletion of the er calcium store. the mechanism by which overexpression of tmc proteins causes a reduced calcium concentration within the er remains unclear. considering that the presumed topology of the tmc proteins distantly resembles those of other ion channel superfamilies such as anoctamins, one might speculate that a conductance through the tmc protein itself leads to a calcium leak from the er. terahertz radiation is defined as radiation between 0.1 thz and 10 thz. a number of applications are currently being developed using radiation in this frequency range. these applications will lead to exposure of the general public, making it very important to study potential effects on biological systems. historically, only a few studies on effects caused by terahertz radiation have been conducted because of the lack of suitable generators and detectors. during the last decade, a number of studies on effects caused by radiation with frequencies around 100 ghz have been published. the present study investigated the genotoxic potential of terahertz radiation at three different frequencies, 0.106 thz, 0.380 thz and 2.520 thz. two skin cell types were used, primary human dermal fibroblasts (hdf) and a keratinocyte cell line (hacat). the cells were irradiated applying different exposure times and different power intensities. two genotoxicity tests were applied: the comet assay quantifies dna strand breaks as well as alkali-labile sites whereas the micronucleus test quantifies chromosomal damage. all experiments were performed and evaluated under blinded conditions as three independent replicate experiments. positive (mms-treated) and negative (untreated, sham-exposed) controls were included. in the comet assay no dna damage was observed as a consequence of the exposure under all experimental conditions. the same was true for the chromosomal damage investigated with the micronucleus test. the latter finding was particularly interesting for the experiments at 0.106 thz, because this type of radiation had been reported to cause mitotic disturbances. therefore these experiments were extended, applying higher power intensities and longer exposure periods. also with these modifications, no genomic damage was observed in the form of micronucleus formation. all in all, terahertz radiation did not induce genomic damage under the applied experimental conditions. this result is in line with published findings on genotoxicity of low-frequency terahertz radiation around 0.1 thz. the question, why the reported mitotic disturbances do not lead to manifest genomic damage remains open and requires further research. introduction: tea flavonoids derived from camomile and green tea such as apigenin and epigallocatechin gallate (egcg) can inhibit intestinal neoplasia. recurrences of adenomas and cancers were reduced in patients with resected colorectal cancer by treatment with tea bioflavonoids after tumor operation [1] . to clarify the biomolecular pathway for suppression of neoplasia we investigated the anti-inflammatory effect of a nutritional supplement flavo natin® (fn) which had been used in the clinical study on tertiary tumor prevention and of egcg in a colon tumor cell line. the aim of our study was to investigate if tea flavonoids are capable to suppress the inflammatory markers produced by tumor cells after cytokine stimulation. method: we studied the cytotoxicity of fn in the colon cancer cell line t-84 by resazurin fluorescence and compared it with the placebo supplement. additionally, the t-84 cells were incubated with fn, egcg or placebo and stimulated with tnf-alpha, if-gamma and il-1-beta. after the cytokine stimulation the mrna expression of ip-10, il-8 and tnf-alpha was measured by quantitative real-time pcr (qrt-pcr). results: stimulation of t-84 cells increased the expression of ip-10 (gamma-interferon inducible protein 10), tnf-alpha and il-8. by preincubation with fn at 10 µm the mrna expression of ip-10 was strongly reduced (log2-ratio -14). the tnf-alpha mrna was also but less decreased by fn. egcg displayed an inhibition pattern similar to fn. placebo did not influence the mrna expression of the chemokines and tnf-alpha. discussion & conclusion: clinically useful dietary tea bioflavonoids inhibit the expression of inflammatory genes in a colon cancer cell line. down-regulation of inflammatory gene products could be achieved in vivo by botanicals without clinically relevant side effects. [1] h. hoensch, b. groh, l. edler, w. kirch (2008) . prospective cohort comparison of flavonoid treatment in patients with resected colorectal cancer to prevent recurrence. world j gastroenterol, 14, 2187-2193. the cxcr7 c-terminal domain mediates efficient cxcl12 uptake and degradation hoffmann f., müller w., schütz d., schulz s., stumm r. universitätsklinikum jena institut für pharmakologie und toxikologie, drackendorfer str. 1, 07747 jena, germany cxcl12-signaling mediated by the g protein-coupled cxcr4 receptor plays a key role during embryonic development and disease states including cancer and inflammation. the second cxcl12-receptor cxcr7 modulates cxcl12/cxcr4-signaling by acting as a cxcl12-scavenger. given the distinct functions of cxcr4 and cxcr7, we hypothesized that trafficking and receptor stability are differently regulated by the distinct c-terminal domains. here, we examined epitope-tagged wild type and c-terminal mutant receptors expressed in human embryonic kidney cells (hek293) with respect to trafficking, stability, 125 i-cxcl12 radioligand degradation, and g protein-coupling. we found that the 24 c-terminal residues of cxcr7 were sufficient for cxcr7 to undergo rapid spontaneous internalization. replacement of the cxcr7 c-terminal domain with that of cxcr4 (cxcr7-4tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization in conjunction with c-terminal phosphorylation. conversely, replacement of the cxcr4 c-terminal domain by that of cxcr7 caused ligand-independent internalization of cxcr4. receptor-mediated 125 i-cxcl12-uptake, release of 125 i-cxcl12-degradation products, and degradation of the receptor protein itself were accelerated with receptors bearing the cxcr7 c-terminus. while the cxcr7 c-terminus was sufficient to abolish g protein coupling in the cxcr4-7tail mutant, replacement of the cxcr7 c-terminus, cxcr7 second intracellular loop or both domains with the corresponding cxcr4 domain did not generate a g protein-coupled cxcr7 chimera. taken together, we provide evidence that the cxcr7 c-terminal domain influences the ligand-uptake/degradation rate, g protein-coupling, and stability of the receptor. this suggests that heterologous regulatory pathways targeting the cxcr7-c-terminal domain may effectively control cxcr7 functions. soluble guanylyl cyclase is a key mediator of brown adipocyte differentiation hoffmann l. s. 1 brown adipose tissue (bat) uses energy to produce heat by inducible thermogenesis. recent studies show that active bat is present in adults and involved in human energy balance, suggesting that the energy consuming property of bat might be exploited to fight obesity and related diseases. the no/cgmp signaling pathway is a key player in diverse physiological processes. recently, we have shown in bat that cgmp signaling is connected with insulin signaling and abrogation of cgmp signaling leads to impaired bat differentiation and function (haas, b. et al., sci signal, 2009 ). here we investigated the role of the cgmp generating enzyme soluble guanylate cyclase (sgc) in bat differentiation in vitro. mesenchymal stem cells isolated from bat of newborn sgcβ 1 -/mice and wt littermates were differentiated in vitro into brown adipocytes in the presence or absence of cgmp. abundance of sgc isoforms was determined by qrt-pcr and western blotting. bat differentiation was assessed by redo staining of accumulated intracellular lipids, measurement of triglyceride (tg) content, determination of expression of bat marker proteins pparγ, c/ebpα, ap2 and bat marker genes ucp1, pgc1α, cidea. the α2 and β1 isoforms of sgc were highly expressed in bat whereas α1 sgc could not be detected. redo staining of wt brown adipocytes showed basal differentiation which was increased upon addition of 8-pcpt-cgmp. in contrast, staining was lower in sgcβ1 -/cells compared to wt under control conditions and increased in the presence of cgmp. tg measurement showed that sgcβ1 -/brown adipocytes contain approximately 50% less lipids than wt cells under basal conditions. addition of cgmp doubled tg content in both genotypes. similar results were observed for marker protein expression. deletion of sgc resulted in 56-40% decrease in c/ebpα, pparγ and ap2 expression compared to wt. again, cgmp roughly doubled protein expression in sgcβ1 -/and wt cells compared to control. under basal conditions, bat marker gene expression was decreased by approximately 80% in sgcβ1 -/cells compared to wt cells. this decrease was prevented by addition of cgmp. these results show that sgc deletion leads to dysfunctional bat differentiation and emphasize the central role of cgmp signaling in bat differentiation. further investigation of sgc/cgmp signaling in bat might reveal new drugable targets bringing bat-dependent pharmacological therapy to treat obesity and related disease into closer reach. comparative inhalation toxicity of carbon-nanomaterials (multi-wall carbon nanotubes, graphene and carbon black) hofmann t. 1 carbon black is a spherical carbon anomaterial whereas multi-wall carbon nanotubes (mwcnt) are cylindrical and graphene is a laminar allotrope of carbon. processing and handling as well as abrasion processes can set free inhalable cnt particles. results of rodent studies collectively show that regardless of the process by which cnts were synthesized and the types and amounts of metals they contained, cnts were capable of producing inflammation, epithelioid granulomas, fibrosis, biochemical and or toxicological changes in the lungs (lam et al. 2004 , muller et al. 2005 , ma-hock 2009 , pauluhn 2010 . graphene possess similar physical properties as cnt but may different toxicological property. we performed short-term inhalation studies in rats to compare the toxic potency of four different cnt, two graphenes and one carbon black. the materials are characterized thoroughly according to the oecd list. the four mwcnt caused morphological changes as descriped above. several biochemical and cytological parameters in the broncho-alveolar lavage fluid were strongly increased consistent with the histological findings. two mwcnt exhibited a higher toxic potency than two other mwcnts and findings caused by one graphene typ were even less severe. the graphene with lower surface area as well as low surface area carbon black did not cause any adverse effects up to 10 mg/m 3 . the short-term inhalation studies were able to descriminate different toxic potencies of carbon-based nanomaterials and is hence used for the selection of less toxic materials for further product development as well as to define and prioritize higher-tier toxicological testing of nanomaterials. 165 synthesis and analytical assessment of possible dna adducts formed after activation of the tobacco alkaloid myosmine högg c., zwickenpflug w., gudermann t. walther-straub-institut abt.: toxikologie, nußbaumstraße 26, 80336 münchen, germany myosmine represents one of the minor tobacco alkaloids and its effective uptake from smokeless tobacco or tobacco smoke, as well as by consumption of food is not understood in detail. myosmine can be activated by peroxidation and n-nitrosation yielding 4-hydroxy-1-(3-pyridyl)-1-butanone (hpb) which is well known as reactive intermediate during activation of a variety of tobacco specific n-nitrosamines (tsna). therefore, myosmine may be a potential candidate for possible mutagenic or carcinogenic risk to human health. furthermore, myosmine n-nitrosation yields the tobacco specific nitrosamine n-nitrosonornicotine (nnn), which is classified as carcinogenic to humans. considerable efforts have been undertaken, especially in organic synthesis, to verify and elucidate the significance of the hpb precursor the pyridyloxobutyl (pob) intermediate and its dna-adducts, which were analysed only in animal experiments till now. the formation of 3-pyridylmethanol was observed under myosmine peroxidation and identified as a metabolite in rat urine after application of myosmine to rats. the formation of the 3-pyridylmethanol intermediate, might provide for the reactive electrophilic picolinium ion which could interact with dna. these possible adducts might be of special interest to elucidate the role of myosmine concerning its uptake by smokers and passive-smokers in contrast to non smokers ingesting the substance by consumption of food. dna adducts may help to obtain more information about possible risk assessment of myosmine and to differentiate between the activation from the other nicotinoids and tsna. the specific dna adducts, 5-methyl-1,3-bispyridin-3-ylmethyl-1h-pyrimidin-2,4-dion and 3-(3''-picolyl)thymidine have been synthesised as reference substances. the former was prepared by addition of diisopropylazodicarboxylate (diad) to a mixture of 3-pyridylmethanol, thymine and triphenylphosphine. the reaction product was identified using nmr ( 1 h, 13 c, cosy, hmqc, hmbc). for synthesis of 3-(3''-picolyl)thymidine the hydroxyl groups of thymidine were initially acetylated using acetic acid anhydride and 4dimethylaminopyridine (dmap). the existence of this thymidine adduct was confirmed using nmr. this adduct was labelled with 5-([4,6,-dichlorotriazin-2-yl]amino)fluorescin (dtaf) to enhance the sensitivity using hplc-fluorescence chromatography and used as reference substances for analysis of dna samples from biological tissue. supported by dfg grant (ty 81/1-1). cancer and cardiovascular diseases such as atherosclerosis are the most important causes of death in western societies. common to both diseases is a deregulation of cell death, with significant contribution of inflammatory processes. enhanced oxidative stress plays a dominant role in such events as it forms a vicious cycle with inflammation and controls multiple forms of cell demise. therefore, anti-oxidative enzyme systems gained considerable interest since control of reactive oxygen species (ros) has the capacity to regulate cell death in either direction. the human enzyme family of paraoxonases consists of three members, known as pon1, pon2 and pon3. while pon1 is found predominantly in the circulation, pon2 and pon3 are intracellular enzymes with established anti-oxidative functions. it has been shown that both pon2 and pon3 are protective against atherosclerosis. underlying mechanisms of their protective and antioxidative functions however remained elusive. here we demonstrate that both enzymes locate to the endoplasmic reticulum (er) and mitochondria where they fulfill vital functions in the control of ros generation. in particular, pon2 and pon3 were shown to interact with coenzyme q10 which diminishes mitochondrial ros formation. as a consequence, these enzymes reduce execution of mitochondrial apoptosis, such as cardiolipin peroxidation, cytochrome c release and caspase activation. moreover, pon2 and pon3 reduced er stress-triggered cell death, i.e. by diminishing jnk signaling and chop expression. while these results elucidate their protective role in cardiovascular diseases, it also establishes a relevant function in survival of tumor cells. in accordance, we demonstrate that both enzymes are frequently found overexpressed in various tumors. in cancer cell culture studies, overexpression of both enzymes granted considerable resistance against chemotherapeutics. in turn, knock-down of pon2 caused spontaneous apoptosis of several cancer cell lines. finally, our analyses also revealed that pon2-knockout mice show severe alterations of the hematopoetic stem cell compartment, which implies a significant role in leukemias where these enzymes are frequently found overexpressed. together, our results propose pon2 and pon3 as new putative anti-tumor candidates and demonstrate the efficacy of interventions targeting cellular redox-balance. steigerwald arzneimittelwerk gmbh wissenschaftliche abteilung, havelstr. 5, 64295 darmstadt, germany stw 5 (iberogast ® ), a multi-component herbal drug, is successfully used in the therapy of functional dyspepsia and irritable bowel syndrome (ibs). previous studies revealed effects of stw 5 on disturbed motility and inflammatory processes. although the antiinflammatory properties of stw 5 are well examined, the contribution each of the individual herbal constituents to the anti-inflammatory effect remains unclear. therefore, we studied the effects of stw 5 and its components on inflammation-induced cell death and on the release of the pro-inflammatory cytokine tnf-α. the aim of these investigations was to analyse additive or synergistic effects of the components. the experiments were carried out on caco-2 cells after stimulation with lps (10 ng/ml) for 2 hours. cytotoxicity was evaluated using a commercially available ldh (lactate dehydrogenase)-assay. furthermore, the release of tnf-α after lps (100ng/ml) stimulation of differentiated thp-1 cells was measured using a commercially available elisa. stw 5 (62.6 -500.5 µg/ml) reduced lps (10 ng/ml)-induced cell death in a concentration-dependent manner with a maximum inhibition of 50.0 %. the herbal components in equivalent concentrations contributed to the inhibitory effect of stw 5 to different extents. the maximum inhibition differed in a wide range between the components. stw 5 (500.5 µg/ml) reduced significantly the release of tnf-α by 87 % in lps (100 ng/ml)-stimulated differentiated thp-1 cells while having no effect in untreated cells. in concentrations equivalent to stw 5 caraway, milk thistle, lemon balm and greater celandine had no effect on the lps-induced increase in tnf-α release. bitter candytuft, peppermint, chamomile, liquorice and angelica reduced the tnf-α release, though less pronounced as compared to stw 5, indicating a possible synergistic effect. our results indicate a multi-target effect of stw 5. the anti-inflammatory effect may be due to a reduction of the cytotoxic effect on intestinal mucosa cells and to an inhibition of the release of the pro-inflammatory cytokine tnf-α from immune cells. the individual herbal components seem to contribute by different mechanisms of action to the overall effect of stw 5. immune cell-induced local steroidogenesis in the lung: implications for asthmatic disease and therapeutic intervention hostettler n. 1 , brunner t. glucocorticoids are steroid hormones with potent anti-inflammatory properties. synthetic glucocorticoids are frequently used for the therapeutic treatment of inflammatory disorders, such as asthma. endogenous glucocorticoids are predominantly produced in the adrenal glands in response to emotional, physical and immunological stress. recent years, however, revealed several alternative sources of these immunoregulatory steroid hormones. thus, we have found that the intestinal epithelium is a rich source of glucocorticoids and intestinal glucocorticoids contribute to the maintenance of local immune homeostasis (1) . as the intestinal and the pulmonary epithelium have much in common, i.e. barrier functions and transport of nutrients, resp. gases, we wondered whether the lung mucosa is also capable of synthesizing immunoregulatory glucocorticoids in response to immune cell activation. the murine lung was found to expresses all enzymes required for the synthesis of corticosterone from cholesterol. while most enzymes where expressed in a constitutive manner, cyp11a1, encoding p450ssc, was strongly induced in response to immunological tress. treatment of mice with t cell-activating anti-cd3 antibody of macrophage-activating lipopolysaccharides induced strong local glucocorticoid synthesis, which was effectively blocked by the corticosterone synthesis inhibitor metyrapone, indicating that glucocorticoids measured were produced bona fide in the lung tissue. surprisingly, allergen-induced allergic airway inflammation failed to trigger local glucocorticoid synthesis despite the massive infiltration of neutrophils, eosinophils and t cells. in contrast to that in the intestinal epithelium local glucocorticoid synthesis in the lung was found to be dependent on the presence of adrenal glands as adrenalectomy abolished pulmonary steroidogenesis. in line with the notion that the lung metabolizes steroid precursors we found that ex vivo cultured lung tissue metabolized 3 hduring the last decade small rna molecules has been identified as important regulators for gene expression. these micrornas (mirna) are single-stranded transcripts, which are expressed in many cell types, where they modulate rna stability and translation and, therefore, controlling various cellular mechanism and tissue development. against this background, in the present study the mirna expression and potential target genes were studied in the human placenta as a tissue demonstrating various developmental changes in a limited period of time. taqman®array microrna cards for profiling of 365 mirnas in placentas of different gestation times revealed a significant expression of 277 mirnas by comparing placentas of early gestation (<10.week), preterm (<30.week) and term (>37.week). when comparing the analyzed groups, 106 of these mirnas expose a continuous up-(including mir-20a, mir-379) or downregulation (including mir-9, mir-200a). while comparing placentas of early gestation to term placentas, 30% (e.g. mir-9, mir-429) were up-and 70% (e.g. mir-126, mir-373) were downregulated. by focusing on the latter group with early preterm placentas the ratio is opposed. here, 60% of mirnas showed higher (e.g. mir-107) and 40% lower expression (e.g. mir-627, mir-191). with emphasize on these mirnas, a computational prediction algorithm using the mirò database predicted a potential interaction between corticotropin-releasing hormone (crh) and the mirnas mir-9 and mir-429. specific expression analyses validate an inverse expression between mirnas and target with a reduced expression of mir-9 (93%) and mir-429 (94%) in term placentas, while crh is upregulated 114fold. this interaction was verified on functional level by reporter gene assay. a significantly suppressed luciferase activity of the reporter plasmid containing the 3´-utr sequence complementary to crh was exhibit for the predicted mir-9 binding site. here, the mirna-mrna-interaction reduces the luciferase activity by ~40%, whereas the decreased luciferase activity for the predicted mir-429 binding site is not significant. the results demonstrate a gestation dependent expression of placental mirnas, which may help to explain gestational changes in gene expression and highlight the potential of mirnas as biomarker for pregnancy-related pathologies. since homo sapiens era wound treatment by early civilizations was based on the use of local flora. scilla indica knuth liliaceae (s.i) is a perennial herb with a pear shaped, tunicated bulb, bearing fibrous roots, white flowers and leaves on the stem resembling u. maritima. it is used as cardiac tonic, against inflammation, ulcers and sinus diseases. traditionally the powdered bulb is used topically for warts treatment, while roasted and crushed bulbs are applied to corns of the feet soles. the plant contains steroid glycosides (bufadienolides 1-3%), glucoscillarene a, proscillaridine a, scillarene a, scillcyanoside ,scilliglaucoside ,mucilage and alkyresorcinol derivatives exerting skin healing properties similar to wheat bran products. the study is focused on the investigation of the restoration quality of a skin dorsal incision wound in wistar rats in three groups, control (a1) and experimental (a2,a3 ) of rats weighing 350-450g local application of dichloromethane extract of s.i in vehicle olive oil preparations of 50 and 100mg were used. animals were anaesthetized ( ether pro narcosi ), trauma 2 cm long and 2mm deep was performed by lancet on depilated dorsal area skin until the muscular aponeurosis and were treated for 4 days with 60λ of each preparation. group a3 showed increased remodelling of trauma area (limited width). granulomatous tissue was more pronounced in length, width and surface in group a2. the macroscopic ulcus dimensions were better in group a3 while the microscopic view were similar in a2 and a3 and better in comparison to control a1. a tendency to trauma remodelling was observed, without statistical significance (t =0.3) in recent years, it has been suggested that nanoparticles generated from combustion processes (e.g. diesel engine exhaust particles), may contribute to the pathogenesis of neurodegenerative diseases such as alzheimer's disease (ad). the aim of our current study was to investigate the effects of subchronic exposure to diesel engine exhaust (dee) in the 5xfad mouse model, which is characterised by progressive behavioural deficits as well as amyloid plaque formation and neuron loss. ten weeks old female 5xfad mice and their nontransgenic littermates were exposed by whole body inhalation to diluted dee (~1 mg particles/m 3 ) or clean air (controls) for 3 or 13 weeks (5 days/week and 6 hour/day). subsequently, all animals were subjected to a series of behavioural tests. at ten days post-exposure, mice were sacrificed to investigate lungs and brain tissues for pathological and biochemical and molecular-biological changes. in line with the expectations, the 5xfad mice displayed typically age-dependent behavioural deficits and amyloid plaque formation in cortex and hippocampus. a significant dee exposure-related effect was observed for the string suspension test, representing a measure of motor coordination/grip strength. dee exposure was also associated with mrna expression changes of markers of inflammation and oxidative stress in specific brain regions, including the olfactory bulb. histopathology of plaqueload in cortex and hippocampus (from a limited number of animals investigated so far) did not reveal clear evidence for increased plaque formation due to the dee exposures. further research is needed to evaluate the effects of long term exposure to nanoparticles in the central nervous system. this work is supported by funds from the research committee of the medical faculty of the university of düsseldorf (9772-365), the dfg graduate school grk1033 and the rivm -centre for environmental health, bilthoven, netherlands. mono-glucosylation of (h/k/n)ras by clostridium sordellii lethal toxin (tcsl) blocks critical survival pathways, including the pi3k/akt, the ralgef/ral, or the raf/erk, and results in apoptosis. in this study, ras glucosylation is presented to result in expression of the cell death-regulating small gtpase rhob based on transcriptional activitation. rhob expression depends on k-ras inhibition, as sirna-mediated knock-down of specifically k-ras (neither h-ras nor n-ras) provokes rhob expression. rhob expression further depends on inhibition of pi3k/akt, as activation of pi3k/akt using a pi3k activator prevents rhob expression downstream of inactivated ras. newly synthesized rhob is gtp-loaded and rapidly degraded in a proteasome-and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a rho family protein. although often characterised as a pro-apoptotic protein, rhob suppresses caspase-3 activation in tcsl-treated fibroblasts. conclusions: 1. rapid and efficacious ras inactivation by tcsl turns out to be particularly useful in the characterisation of ras inactivation-induced rhob expression as an immediate-early gene response. 2. the finding on the cytoprotective activity of rhob in tcsl-treated cells re-enforces the concept that rhob exhibits cytoprotective rather than pro-apoptotic activity on cellular background of inactive ras. the activity of the rhob promoter is suppressed by rhoa (through a not yet identified pathway or ras (through the pi3k/akt pathway. ras glucosylation by tcsl results in de-suppression of the rhob promoter and rhob expression. the calcium-binding protein annexin a4 (anxa4) is involved in diverse cellular processes including e.g. vesicular transport, ion channel regulation and transcriptional regulation. upon ca2+ binding anxa4 undergoes conformational changes, which lead to oligomerization of anxa4 to homotrimers and cause an increased affinity for membrane phospholipids, which in turn provokes the translocation from the cytosol and the nucleoplasm to plasma and nuclear membranes. in order to examine the effect of anxa4 on cre-and creb-(camp response element-binding protein) dependent transcription, a series of transient transfections using a luciferase reporter gene driven by the creb target promoter of the inducible camp early repressor (icer) was carried out. when the luciferase reporter construct was co-transfected with anxa4, there was significant reduction of basal (dmso) luciferase activity ( the implantation of drug eluting stents (des) after coronary artery intervention was an important step treating coronary artery disease. indeed, cytotoxic compounds like sirolimus used on des are responsible for reduced in-stent restenosis in comparison with bare metal stents. pimecrolimus, a potent anti-inflammatory drug has also been investigated for its efficacy on des. preclinical studies in pigs revealed promising antiproliferative effects of pimecrolimus on neointima formation. however, in humans, pimecrolimus coated stents exerted adverse effects. we hypothesize that compared to the highly active sirolimus, pimecrolimus may influence additional cellular processes leading to the worse outcome. in order to identify those processes we conducted in vitro studies in human coronary artery endothelial cells (hcaec) and smooth muscle cells (hcasmc). brdu in vitro assays of hcaec treated with pimecrolimus examined an ic50 value of 6.787 µm [5.745 to 8 .017] which is much higher than ic50 values of sirolimus described in literature [0.1nm to 1nm]. genechip array analysis comparing gene expression in pimecrolimus and sirolimus treated hcaec and hcasmc showed significant induction of several genes involved in interferon signaling. in detail, the expression of ifnβ activated genes like irf9 and ifitm1 was up regulated in cells treated with pimecrolimus while no or oppositional effects were observed with sirolimus. gene regulatory effects were validated by real time pcr. incubation with ifnβ itself showed similar effects in up regulation of genes involved in interferon signaling. furthermore, we were able to demonstrate a significant increase of ifnβ secretion in hcasmc and hcaec treated with pimecrolimus. however, comparison of ifnβ and pimecrolimus on proliferation of hcaec and hcasmc revealed different cellular responses. while ifnβ significantly decreased hcasmc and increased hcaec proliferation, treatment with pimecrolimus lead to anti-proliferative effects on both cell types. in conclusion, pimecrolimus activates pathways involved in interferon signaling but exerts different pharmacological effects, compared to the endogenous compound suggesting that infβ secretion is not the major factor contributing to the difference in pimecrolimus function. identification of novel phospho acceptor sites of the mu opioid receptor regulating receptor internalization illing s., just s., doll c., schulz s. uniklinikum der fsu jena pharmakologie und toxikologie, drackendorfer straße 1, 07747 jena, germany the opioid alkaloid morphine is among the most potent clinically used analgesics. however, the clinical utility of morphine to treat chronic pain is limited by its rapid development of tolerance and dependence. the stimulation of the mu opioid receptor (mor) with damgo or morphine results in different patterns of receptor phosphorylation and trafficking. so far, the three major phosphorylation sites of the mu opioid receptor namely serine 363 (s363) threonine 370 (t370) and serine 375 (s375) have been identified using phosphosite-specific antibodies. however, mutations of these three residues to alanine (s363a/t370a/s375a) did not prevent agonist-dependent internalization. in the present study, we have constructed a series of phosphorylationdeficient mutants showing that mutation of at least six residues (s363a/t370a/s375a/t376a/t379a/t383a) was required to completely block agonistdriven endocytosis. consequently, we generated phosphosite-specific antibodies to t376 and t379 which enabled us to provide direct evidence for agonist-dependent phosphorylation of these sites. our analysis of time-and dose-dependent phosphorylation of mor revealed that s375 was the primary phosphorylation site, whereas t370, t376 and t379 were secondary sites. moreover, the partial agonist morphine induced only the phosphorylation of s375 but not of t370, t376 of t379. our results show, that mor phosphorylation occurs in a hierarchical and agonist-selective manner directly regulating receptor sequestration. assessment of mda effects from toxicity studies with regard to endocrine modulation jäger r. 1 methylene dianiline (mda; cas no. 101-77-9) is on the usepa list 2 for endocrine disruption screen testing. in a yeast androgen screening (yas) assay (in vitro assay on receptor binding or blocking) mda revealed a significant anti-androgenic activity at a concentration of 0.01 mm. to assess the biological relevance of this observation under in vivo conditions the existing data from animal toxicity studies with mda were reviewed for indications of possible endocrine modulating (em) effects (weight-of-evidence approach). in addition, literature was searched for relevant studies on mda and structural analogues using the american chemical society scifinder client-server software. structural analogues indicated several drivers for em activity, notably the diphenolic ring structure. however, in vitro receptor binding assays showed mda had no androgenic or anti-estrogenic activitiy. one report described a weak estrogenic binding at 0.2 mm mda, while another described no effect at similar concentrations and a rat estrogen receptor binding study found no effect at 0.2 mm. overall it is concluded that mda does not bind to the estrogen receptor. endocrine related effects of mda were investigated in several species and dose routes in unvalidated research-type protocols. guideline subchronic and chronic toxicity studies with rodents revealed neither adverse organ weight changes nor histopathological alterations of sex organs. the main systemic effects from the oral 13-week studies were body weight reduction and histopathological changes of the thyroid and bile duct (lowest loael: 7.5 mg/kg bw). mda was carcinogenic to rats and mice (thyroid and liver) after oral administration over 2 years. non-neoplastic effects in the thyroid (rats and mice) were observed (lowest loael for systemic toxicity: 9 mg/kg bw). mda inhibits iodide oxidation which with concomitant decreased thyroid hormone formation is known to induce thyroid tumors. in summary, in vitro screening tests revealed no consistent endocrine related effects of mda. in vivo, there was an effect on the thyroid gland, possibly by enzyme inhibition. there were no histopathological changes of gonads and accessory sex organs and no evidence of sex hormone related em. the evidence from the full dataset on mda does not indicate androgenic or estrogenic effects. overall, based on a weight of evidence assessment there are insufficient alerts for em activity to suggest further testing should be done. the gtpase arfrp1 controls the assembly of apoa-i to and the lipidation of chylomicrons in the golgi of intestinal epithelium jaschke a. 1 background: the uptake and processing of dietary lipid by the small intestine is a multi-step process that involves luminal digestion, cellular uptake of fatty acids by the mucosa, and subsequent synthesis and export of chylomicrons. the gtpase adpribosylation factor related protein 1 (arfrp1) is a member of the arf-family and controls the arf-like 1 (arl1)-mediated golgi recruitment of grip domain proteins which in turn bind several rab-gtpases. the aim of the study was to define the role of arfrp1 in intestinal nutrient absorption. methods: for the generation of intestine-specific null mutants arfrp1 flox/flox mice were crossed with mice expressing the cre recombinase under the control of the intestinespecific villin promoter (vil-cre) and arfrp1 expression was suppressed by sirna in caco2-cells. the phenotype in respect to lipid absorption and chylomicron production in the intestinal epithelium and in caco2-cells, respectively, was analyzed. results: arfrp vil-/mice were viable but showed an early postnatal growth retardation (mean body weight of arfrp1 vil-/was 43.3±5% lower than that of control mice at the age of 28 days) arfrp1 vil-/mice displayed reduced triglycerides, free fatty acids and glucose plasma levels indicating that the growth retardation is the result of a malabsorption. uptake of glucose and amino acids were unaffected by the deletion of arfrp1. in contrast, lipid uptake as elucidated by oral fat tolerance tests was impaired in arfrp1 vil-/mice. arfrp1 vil-/enterocytes as well as arfrp1 mrna depleted caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triglyceride content. in addition, while the release of apolipoprotein a-i (apoa-i) was dramatically decreased apoa-i accumulated in the arfrp1 vil-/epithelium and was predominantly colocalized with rab2. our results demonstrate that the growth retardation of arfrp vil-/mice is a consequence of impaired intestinal fatty acid absorption. we suggest that arfrp1 is required for the assembly of aopa-i to the chylomicrons and for the further lipidation of chylomicrons in the golgi of intestinal epithelial cells. this finally leads to an secretion of chylomicrons with a markedly reduced triglyceride content. rhoa influences adhesion and spreading of cardiac fibroblasts via complex regulation of cytoskeletal proteins jatho a. 1 the monomeric gtpase rhoa is thought to be involved in the pathology of heart diseases, however, its role in cardiac cells is not well defined. therefore we intended to analyze the effect of rhoa in cardiac fibroblasts by using a lentivirus-based knockdown approach. by doing so, we could show that the knockdown of this gtpase by about 50% resulted in a massive change in cell morphology, but displayed no effect on cell viability. the appearance of the cells in the 2d-culture changed from a fibroblast-typical stretched morphology with intracellular stress fibers to a more epithelial-like cell morphology. by morphometrical analyses we demonstrate that fibroblasts with reduced rhoa expression display an increase in cell surface by 2.2-fold and in perimeter by 1.5fold. moreover, the depletion of rhoa significantly influences the adhesion velocity, as within the first hour after cell detachment about 20% of the rhoa knockdown cells reattach, but only 5% of the respective control cells. based on these findings, we investigated the distribution and composition of different cytoskeletal proteins by immunofluorescence stainings and immunoblot analysis. we found, that the amount of b-actin is not reduced in rhoa knockdown cells, however, the distribution is markedly changed. in these cells internal star-shape bundles of actin could be found instead of the commonly appearing stress fibers in control cells. in contrast, the cortical actin fibers, mainly consisting of g-actin, were not affected. in addition, smooth muscle-actin, which is characteristic for myofibroblasts, was clearly reduced in rhoa knockdown cells compared to control cells by 33%. this reduction might be responsible for the more relaxed cell shape. in summary, the knockdown of rhoa influences cell adhesion and the morphological characteristics of cardiac fibroblasts, without obviously affecting cell proliferation and viability. using site-specific fluorescence labeling to study uptake of pasteurella multocida toxin into eucaryotic cells jehle d., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abteilung 1, albertstraße 25, 79104 freiburg, germany pasteurella multocida is an opportunistic pathogenic bacterium living in the nasal pharyngeal space of animals. p. multocida is of particular importance in the livestock management of pigs. under special conditions infection of pigs with p. multocida leads to an atrophic rhinitis, which is characterized by the atrophy of nasal turbinate bones accompanied by a shortening and twisting of the snout. the causative agent of the atrophic rhinitis was found to be the bacterial protein toxin pmt (pasteurella multocida toxin). after entering the cell the 146-kda toxin activates various signal transduction pathways by stimulating heterotrimeric g proteins of the gαq, gα13 and gαi family. the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. the uptake of pmt into cells is not comprehensively understood. therefore, we utilized a recently described technique, called "sortagging" (popp mw et al. nat chem biol. 2007; 3: 707) , to specifically couple fluorescence tags to the n-or c-terminus of pmt. the enzyme sortase a (srta) from staphylococcus aureus attaches proteins to the bacterial cell wall. the substrates are recognized by an lpxtg motif. srta cleaves the peptide bond after the threonine and adds a glycine-containing nucleophile. we introduced these motifs into pmt to express srta-recognized toxin and coupled the toxin with fluorescence tags, respectively. fluorescently labeled pmt was used to study the uptake of the toxin into eucaryotic cells by laser scanning microscopy. munich heart alliance, münchen, germany cells within the myocardium communicate by secreted factors and this has been suggested to contribute to cardiac remodeling. to identify novel factors secreted by the myocardium, we have previously reported a genetic yeast screen which led to the identification of protease inhibitor 16 (pi16). here we report the generation of a mouse line where pi16 can be deleted globally or conditionally using the cre/loxp system. after electroporation of the pi16 floxneo targeting vector in embryonic stem cells and injection into murine blastocysts we gained a mouse line that carried the targeted modification of the pi16 allele. global pi16 deficiency (pi16 lox/lox ) per se did not lead to a cardiac phenoytpe (hw/bw (mg/g): pi16 +/+ = 5.4 ± 0.2 (n = 6), pi16 lox/lox = 5.9 ± 0.7 (n = 4), p > 0.05; fibrosis (%): pi16 +/+ = 3.3 ± 0.2 (n = 7), pi16 lox/lox = 3.9 ± 0.8 (n = 5), p > 0.05; fractional shortening (%): pi16 +/+ = 29.8 ± 0.7 (n = 7), pi16 lox/lox = 26.4 ± 2.5 (n=5), p > 0.05). in addition we carried out an immunohistochemical analysis of pi16 expression using pi16-deficient mice as negative controls. pi16 localized to cardiac fibroblasts, to the epididymis and the trachea. in the failing heart we detected accumulation of pi16 preferentially in fibrotic areas. we are currently applying cardiac stress models to gain a broader understanding of the function of pi16 and its potential as a therapeutic target molecule. enantioselective determination of r-and s-hyoscyamine in mammalian plasma and urine samples john atropine (atr) is the racemic mixture of the tropane alkaloids s-and r-hyoscyamine (hyo). s-hyo acts as a competitive acetylcholine antagonist at the muscarinic receptors (eutomer) inducing mydriasis, excitations, hallucinations, coma and ultimately death, whereas r-hyo does not (distomer). atr is used for clinical intervention of poisoning with organophosphorus (op) pesticides or nerve agents. despite well known differences in pharmacological behavior, individual pharmacokinetics of both atr enantiomers have rarely been addressed in the literature [1, 2, 3] . therefore, we initially developed a nonchiral liquid-chromatography-electrospray tandem mass spectrometric method (lc-esi ms/ms) that allows quantification of atr and additional natural and synthetic tropane alkaloids from plasma after simple deproteinization [4] . to discriminate both atr enantiomers the sample preparation step was expanded by an enzymatic pretreatment. samples were incubated either with diluted human serum (not containing atropinesterase, atre, procedure a) or with diluted rabbit serum (procedure b). rabbit serum contains atre (ec 3.1.1.10) which is suitable for stereospecific hydrolysis of shyo into tropine and tropic acid while r-hyo remains unaffected. after sample precipitation, hyoscyamines were quantified by the lc-esi ms/ms method. following procedure a the concentration of total hyo and following procedure b remaining r-hyo were determined. s-hyo was calculated by the difference between these concentrations [3] . the impact of potential matrix ingredients that may appear in samples from oppoisoned patients under atr therapy were evaluated (oximes, op agents, carbamates) [3] . the assay was applied to diverse toxicological and pharmacological samples. i) measurement of natural s-hyo in an extract of atropa belladonna leaves as well as in plasma and urine of a female patient who was poisoned after ingestion of such leaves revealed that no biotransformation to r-hyo occurred. ii) analysis of plasma obtained from an op-poisoned female patient under atr therapy revealed faster elimination of shyo when compared to r-hyo [3] . iii) in contrast, no enantioselective differences were obvious in healthy anaesthetized swine after intravenous injection of atr [5] . data indicate that the enzymatic enantioselective procedure represents a useful tool to characterize in vivo behavior of r-and s-hyo allowing to reveal individual kinetics. failing hearts are unable to adequately supply the body with blood and oxygen. common therapeutic strategies interfere with cardiac remodelling, reduce cardiac preand afterload or aim at direct improvement of cardiac contractility. cardiac contractility is mainly controlled by β1-adrenergic receptors. resensitization of b-adrenergic receptors by inhibition of g-protein coupled receptor kinase 2 (grk2) is discussed as a potential strategy to treat heart failure. we characterized raf kinase inhibitor protein (rkip) as a physiological inhibitor of grk2 and found rkip to increase contractility of neonatal cardiomyocytes. the present study evaluated the role of rkip in heart failure. we assessed its effects on cardiac function in pressure overload induced heart failure and determined the expression patterns of rkip in failing hearts of humans and mice. transverse aortic contriction (tac) was performed on 7-week-old c57/bl6 mice to induce heart failure by pressure overload of left ventricles. after three weeks of tac, echocardiography showed distinct signs of decreased cardiac function in wild-type mice. fractional shortening was reduced and left ventricular diameters were increased. histological analyses revealed increased interstitial fibrosis, caspase-and tunelassays indicated myocyte apoptosis. western blot analysis showed significant upregulation of rkip expression in failing hearts compared to non-banded control hearts. interestingly, this upregulation of rkip expression was also detected in failing human hearts and in samples of patients with aortic valve stenosis but not in healthy control samples. to assess the effects of rkip overexpression on heart failure, we analysed heart function and structure of rkip transgenic mice and wild-type mice 3 weeks after tac. while left ventricular hypertrophy was increased to similar extents in wild-type and rkip trangenic mice, rkip mice did neither develop dilatation of the left ventricle nor a decrease in fractional shortening. in contrast to wild-type mice, the expression of the heart failure markers bnp and anp was not upregulated in banded rkip transgenic mice after 3 weeks of tac. taken together, cardiac overexpression of rkip prevented left ventricular dilatation and loss of contractile function in a mouse model of pressure overload induced heart failure. therefore, increased rkip expression may be an interesting target to prevent detrimental effects from increased left ventricular pressure. the main focus of this study was the chromatographic separation. the most frequently prescribed antibiotic drugs clarithromycin, erythromycin, clindamycin, cefuroxim, doxycyclin, amoxicillin, levofloxacin, and ciprofloxacin were selected for the screening method. method: a relatively short operation time and a sufficient separation were reached by column, eluent, and gradient optimization with poplc (phase optimized liquid chromatography). in the first step columns with the five stationary phases c18 eps 2, c18 sh 2, phenyl 2, cn 2, and c30 were used to determine the retention times of the drugs in an isocratic mode. the stationary phases, the column length and the retention times were fed in the poplc software and the optimal column was calculated. this column contained different stationary phases and was compared with customary columns. results: using the optimal column a sufficient chromatographic separation of the eight antibiotic drugs was reached. that was not possible with the customary columns. with the optimal column the time of measurement was too long. using a mobile phase gradient the measuring time could be reduced. discussion: with lc-ms/ms a complete chromatographic separation of all analytes is not necessary. but when measuring many transitions in a biological matrix two problems should be excluded: ion suppression and a too small number of measurement points per peak. especially when using positive and negative ionization in the ms a good separation is mostly necessary. to determine only the eight antibiotic drugs an optimized column is not necessary, but for a screening method with more than twenty drugs the poplc system is very helpful. we have investigated the uptake mechanism of cdt and in particular the intracellular membrane translocation of cdta. our data indicate that cdt requires acidification of the endosomal lumen for translocation of cdta across endosomal membranes into the cytosol. bafilomycin a1, an inhibitor of endosomal acidification protects vero (african green monkey kidney) cells from intoxication with cdt. consistently, translocation of cdta was observed when the acidic conditions of the endosomal lumen were mimicked at the cytoplasmic membrane of intact cells. next, we tested whether host cell factors are involved in membrane translocation of the toxin. radicicol, a specific pharmacological inhibitor of the chaperone heat shock protein hsp90 as well as cyclosporine a, an inhibitor of cyclophilins delayed the intoxication of cells with cdt but not with toxins a and b [1] . this result was confirmed by analyzing the adp-ribosylation status of actin from such cells in the presence or absence of the inhibitors. in addition, we excluded that the inhibitors of hsp90 and cyclophilins have any effect on receptor binding, endocytosis or enzyme activity of cdt. the data strongly suggest that the participation of hsp90 and cyclophilin is crucial for translocation of cdta into the cytosol. comparable results were obtained for the related binary iota toxin of c. perfringens. in vitro purified immobilized hsp90 and cyclophilin a specifically bound to the enzyme components of cdt and iota toxins. in conclusion, the results imply a common hsp90/cyclophilin a-dependent translocation mechanism for the family of binary actin-adp-ribosylating toxins. our current investigations focus on the participation of fk506-binding proteins (fkbps), another group of peptidyl-prolyl cis/trans isomerases in the membrane translocation step of these toxins. [1] kaiser, e., kroll, c., ernst, k., schwan, c., popoff, m. r., fischer, g., buchner, j., aktories, k. and barth, h. (2011) . complex multicellular in vitro coculture models represent a promising tool regarding e. g. cellular interactions with nanoparticles, since they more closely mimic the cellular composition of the body. therefore, we used our developed coculture model of the alveolar-capillary barrier composed of lung epithelial cells (nci h441) on top and microvascular endothelial cells (iso-has-1) on the bottom side of a filter-membrane to study nanoparticle cytotoxicity and cellular uptake. with a coculture period of about 10 days the cells achieve a more differentiated and polarized state and develop a tight barrier, which can be measured via ter (transepithelial electrical resistance). regarding cellular uptake of fluorescently labeled amorphous silica nanoparticles (asnps, 30 nm) the coculture took up much less asnps than conventional monocultures. besides, we could not verify a specific uptake mechanism (e. g. clathrin-, caveolae-mediated endocytosis) via immunofluorescence staining of the cells. however, we detected asnps incorporated in flotillin-1 and -2 labelled vesicles. former studies concerning cytotoxicity (lactate dehydrogenase assay) of amorphous silica nanoparticles (asnps, 30 nm) revealed that our coculture behaved much more robustly compared to conventional monocultures. however, regarding inflammatory responses (e. g. sicam, il-8 increase) the coculture responded more sensitively than conventional monocultures. in a further development we added a third cell type, the alveolar macrophage (am), to our coculture. since ams embody the front-line of alveolar defense against inhaled pathogens and particles, they play a central role in regulating lung immunity. as model we applied the human acute monocytic leukemia cell line, thp-1 (prestimulated with 8 nm pma for 4d) apically to the epithelial monolayer of the coculture. our preliminary studies concerning inflammatory responses of the tripleculture (h441/iso-has-1 with thp-1) revealed a higher sensitivity of the triple-culture compared to the double coculture. the triple-culture responded with an increased il-8 release upon lps or tnf-a stimulation. in conclusion, this triple-culture model offers a promising prospect to mimic more closely realistic cell interactions with nanoparticles in the distal lung. ethanol is a component of many herbal fluid preparations [1] , since it is an excellent extraction solvent for the phytochemical components of herbal drugs and contributes to the stability of these medicines. toxicological and pharmacokinetic evaluations [2] have shown that the small amounts of ethanol applied with therapeutic doses are safe even in children. despite that these medicines have been used safely since many decades, they have occasionally been subject of discussion in the public, triggered by the increasing problem of recreational abuse of alcoholic beverages by children and young persons [3, 4] . therefore, there is a growing need of a systematic evaluation of pharmacovigilance data on these medicines. for evaluating the experience gained from the therapeutic use of these medicines, 16 pro-and retrospective studies with 10 herbal medicinal products containing ethanol at doses of 40 to 240 mg per single dose, depending on the age group, have been analyzed, covering 49 816 patients of 0-12 years of age. in these studies, altogether 15 adverse drug effects have been described, none of which was attributable to the ethanol content of the medicines. in a survey of the worldwide use of these and other 6 herbal medicinal products it was shown that during the past few years, more than 764 mio daily doses have been sold, corresponding to more than 33 mio of patients (data obtained from manufacturers; figures available partly from 1993 onwards, partly from 2003/4 onwards). from the packages sold in germany in the years between 2005 and 2009, 48.1 mio were attributable to self-medication, 10.8 mio to prescriptions reimbursed by health insurance (ims, frankfurt). as non prescription medicines are reimbursed in germany only in children, at least the latter part of the prescriptions can be attributed mainly to children. all of these medicines are registered or licensed by regulatory authorities. adverse effects are covered by the pharmacovigilance system, and no adverse effects attributable to the ethanol content have been reported. this set of data supports the conclusion drawn from the experience of a safe use over decades, i.e. that the ethanol content of herbal medicinal products does not give any causes for concern regarding their safety even in children. dedication: this contribution is dedicated to prof. dr. hilke winterhoff, institute for pharmacology and toxicology, university of münster, who died on 9 may 2010. she has initiated this work. prucalopride was introduced as a new selective 5-ht4 receptor agonist that is approved for treating obstipation. whereas one could expect -due to the fact that 5-ht4 receptors are functionally expressed in the human heart -in clinical trials prucalopride did not show cardiac effects. this is quite surprising because other 5-ht4 receptor agonists have been withdrawn from the market just for that reason. in this study we used prucalopride for in vitro studies with atrial preparations from transgenic (tg) mice with cardiac myocyte-specific overexpression of the human 5-ht4a receptor. isolated electrically driven (1 hz) left and spontaneous beating right atria of tg mice were compared with those of wild type (wt) littermates. moreover, we used isolated electrically driven (1 hz) human right atrial preparations from patients undergoing cardiac surgery. finally gr113808, a 5-ht4 receptor antagonist, was added. prucalopride exhibited a dose dependent positive inotropic effect in left atria and a positive chronotropic effect in right atria of tg mice with a logec50 of -6.8 and -7.1, respectively (p<0.05 vs. wt, n=3). in human atrial tissue prucalopride also acts as an agonist, leading to an inotropic effect. all effects could be antagonized by 10 µm gr113808. we could demonstrate that prucalopride acts as an agonist at the 5-ht4 receptor in our transgenic mice model and also in human right atrial preparations. these findings suggest that tachycardia and arrhythmias are possible side effects, which should be carefully looked for. the involvement of psychological factors, especially stress, are known to play an important role in functional gastrointestinal diseases (1, 2) probably by affecting the brain-gut axis. based on the good correlation between stress & functional dyspepsia (fd), many animal models for fd have been developed where animals are subjected to various kinds of psychological stress either during the neonatal period or in adulthood. this stress was found to induce gastric motor dysfunction resembling symptoms of fd. two models for stressinduced fd were performed in order to choose the more adequate one for studying sensitivity changes of the fundus to various mediators as well as changes in some relevant hormone levels in the blood for subsequently testing the efficacy of treatment with stw 5 (a 9 component herbal preparation of proven clinical efficacy in fd and in irritable bowel syndrome (3, 4) ). in one model, maternal separation (5) was performed on weanling rats starting from postnatal day 2 for 3 h each day for 3 weeks. rats were then allowed to mature to an adult age. the other model was that of restraint stress (rs) (6, 7) . adult animals were restrained for 90 min/ day for 1 week. the animals of both models were eventually sacrificed, the stomach fundus was isolated and its sensitivity in vitro to carbachol, potassium chloride, serotonin and adrenaline was tested. blood samples were taken to assess levels of ghrelin, corticosterone releasing factor (crf) and corticosterone. the sensitivity of fundus strips from restrained rats towards the agents tested, partly representing autonomic responsiveness, was more depressed than those from maternally separated ones. levels of ghrelin, crf and corticosterone were also more elevated in the rs model. that model was therefore chosen to test the efficacy of stw 5 in restoring the deranged parameters. a group of animals received stw 5 orally once daily starting treatment 1 week before exposing them to rs and continuing treatment for a further week during subjection to rs. stw 5 was effective in normalizing the depressed stomach fundus responses exhibited by animals subjected to rs and to normalize to a large extent the deranged blood levels of ghrelin, crf and corticosterone. the findings lend further evidence to the role of the brain-gut axis in fd and gives supportive evidence for the first time for the clinical usefulness of stw 5 in this condition. there is good evidence that oxidative damage to dna leads to down-regulation of transcription of affected genes and epigenetic gene silencing in a mechanism dependent on the 8-oxoguanine dna glycosylase (ogg1), which generates harmful repair intermediates [1, 2] . we have recently shown that the magnitude of inhibition of transcription of an egfp reporter gene by single 8-oxo-7,8-dihydro-deoxyguanosine (8-oxodg) varies between the opposing dna strands of the gene [3] . we now have addressed the question, to which extent the transcription inhibitory potential of 8-oxodg depends on its position in the gene and on the dna microsequence surrounding the modified nucleobase. to investigate the effect of position, we produced plasmid vectors containing single 8-oxodg or dg (underlined) in the second position of an agc trinucleotide. measurements of egfp expression in transfected mammalian host cells showed that a single 8-oxodg caused a strong inhibition of gene transcription. the magnitude of this effect for 8-oxodg situated in the transcribed dna strand of the gene was the same as in the non-transcribed dna strand. similarly, there was no quantitative difference between the effects of 8-oxodg present in the 5′-versus 3′-utr of the gene. the results thus indicate that inhibition of transcription by this base modification does not depend on the position in the gene. further comparisons were done between the effects of 8-oxodg nucleobases localised in the same dna strand but within different sequence contexts. gene expression analyses in the repair proficient host cells showed that the degree of inhibition of transcription caused by single 8-oxodg was dependent on the neighbouring nucleotides. among three tested sequence motifs, the minimal effect on the gene transcription was found to correlate with a significantly less efficient base excision by the purified human ogg1 protein. the results thus support the initiatory role of ogg1 in the mechanism of transcriptional repression. in addition, the finding of the effect of dna sequence on the base excision activity of ogg1 suggests that repair rates of single base modifications in genome could also be heterogeneous. allgayer j., kitsera n., epe b., khobta a. johannes gutenberg university of mainz institute of pharmacy and biochemistry, staudingerweg 5, 55128 mainz, germany interference of dna base modifications with gene transcription is an important biological consequence of genotoxic damage [1] . an efficient method for incorporation of a single 8-oxo-7,8-dihydroguanine (8-oxog) at a defined position in the egfp gene in a plasmid vector was recently developed in our lab. the method relies on the availability of adjacent sites for a sequence-specific nicking endonuclease, which allow the insertion of a synthetic oligonucleotide containing 8-oxog in a chosen position. we further showed that this single lesion inhibits transcription after excision by ogg1 [2] . in order to determine to which extend the observed effect depends on the position of the modified base in the gene, we constructed several new plasmid vectors which allow incorporation of the same dna oligonucleotide containing 8-oxog or g in different positions in different dna-strands. dna sequence cassettes were designed to contain a 5′-cattgcttcgctagcacg nucleotide sequence in different orientations, which was flanked by two unidirectional bsrdi recognition sites (5′-gcaatgnn). adapters containing the restriction sites for directional cloning into the 5′-or the 3′-utr of the plasmid-borne egfp gene were added. the produced plasmid vectors thus allow the insertion of a single 8-oxog in the same sequence context into opposing dna strands in the 5'utr and in the 3'utr. keratinocytes are the major cell type of epidermis and are responsible for the formation of an outer barrier, the statum corneum, to protect an organism against harmful environmental influences. for generation of this barrier, keratinocytes pass through a complex differentiation program that is accompanied by synthesis of lipids, like cholesterol and ceramides. finally, the differentiation of keratinocytes leads to apoptosis. another function of keratinocytes is to sense environmental factors, some of which are decoded by members of the transient receptor potential (trp) ion channel family. trpv3, for example, is predominantly expressed in keratinocytes, and decodes different chemical and physical stimuli like the terpenoid-derived ligands camphor and thymol or temperatures above 33°c. less is known about the influence of cholesterol on trpv3 signalling. we modified the cholesterol content of hek293 cells stably transfected with trpv3 and performed flipr-based calcium measurements. these experiments revealed that cholesterol enrichment robustly potentiates trpv3 by sensitizing it to lower agonist concentrations. we verified these results with whole-cell patch-clamp measurements. in contrast, trpv2, another heat-sensing channel, was not affected by cholesterol modification. since former studies showed a defective formation of epidermal barrier in trpv3 -/mice, our results imply that a cholesterol-regulated trpv3 signalling may contribute to the progression of differentiation or initiation of apoptosis of keratinocytes. ischemia-reperfusion injury causes severe problems in the early period after lung transplantation. since transient receptor potential (trp) channels are important regulators of vascular permeability and tone, we investigated the influence of a trpc6 blocker on pulmonary function after simulated transplantation, using an ex vivo model of isolated perfused and ventilated rabbit lungs. to this end, heart-lung blocks were excised and mounted in an artificial thorax chamber. negative pleural pressure ventilation was initiated, and lungs were perfused with albumin-containing tyrode-solution (100 ml min -1 ). after equilibration in a stable perfusion and ventilation mode for 30 min, lungs were flush-perfused with perfadex ® solution, followed by an ischemic storage for 4 h on ice. subsequently, ventilation and perfusion were re-initiated to simulate a transplantation situation. in the oxygenated group, pulmonary artery po2 was adjusted to 120 mmhg, in the deoxygenated group, the perfusate inflow was gassed with nitrogen to achieve a pulmonary artery po2 of 50 mmhg. both transplantation conditions were conducted in the absence or in the presence of the trpc6 blocker larixol acetate (5 µm). hemodynamic and ventilatory parameters were continuously monitored. the weight of deoxygenated lungs steadily increased during 2 h of reperfusion from 22.1 ± 1.32 g to 35.4 ± 4.23 g. this weight gain was inhibited by supplementation of the trpc6 blocker (from 21.4 ± 0.91 g to 27.4 ± 2.42 g 2 h after reperfusion). in contrast, oxygenated lungs showed no marked weight gain after reestablishment of perfusion (from 17.5 ± 1.51 g to 21.5 ± 2.29 g 2 h after reperfusion), and the trpc6 blocker had no additional effect (initial 19.5 ± 1.28 g, 2 h reperfusion 21.3 ± 1.22 g). we conclude that a trpc6-blocking compound to the lung perfusate during simulated transplantation counteracts the endothelial permeability increase and the resulting post transplant weight gain. the results indicate a role for trpc6 in the regulation of pulmonary vessel permeability and support the concept that perfusion of donor lungs with trpc6 blockers may prevent edema formation caused by ischemiareperfusion injury shortly after lung transplantation. regulation of human inducible nitric oxide synthase (inos) expression by noncoding rnas (ncrnas) kleinert h., schmitz k., koch k., hahn s., pautz a. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher str. 67, 55101 mainz, germany the transcriptome analyses of human cells showed that additionally to the 30.000 protein coding sequences the human dna codes for much more (450.000 ?) non-coding rnas 1 . beside the ribosomal, and transfer rnas (rrna and trna) involved in the mechanism of translation there are also short (snrna, sorna, mirna) and long noncoding rnas (ncrnas) implicated in regulation of gene expression (splicing, translation, chromatin packaging etc.). matsui et al. described regulation of il-1β-induced inos expression by an antisense rna (as-3-utr-rna) in rat hepatocytes 2 . a promoter located on the antisense strand 3' to the last exon (exon 27) of the rat inos gene drives the expression of an as-rna complementary to the 3'-utr of the rat inos mrna. using different sense primers with homology to the 3'-utr sequences of the human inos gene for specific rt-pcr reactions (detecting only an as-rna) we were not able to detect such an as-3-utr-rna in human cells. in addition, transient transfection analyses using constructs containing a 2.7 kb fragment of the 3'-flanking genomic sequences (as used by matsui et al. in the rat system) of the human inos gene in front of a luciferase reportergene into dld-1 cells revealed no promoter activity of these sequences. korneev et al. described the expression of a 2 kb anti-inos-ncrna in different brain tumors and showed reciprocal expression to the inos mrna in human embryonic stem cells 3 . analyzing the expression of this anti-inos-ncrna in cytokine-treated dld-1 cells also showed a basal expression of this as-rna and an enhancement of the expression by cytokine-treatment. downregulation of the anti-inos-ncrna by sirnas reduced whereas overexpression enhanced cm-induced inos expression in human dld-1 cells. this indicates that this anti-inos-ncrna regulates cytokine-induced inos expression in a positive manner. rna 14, 2030 rna 14, -2037 rna 14, (2008 . using directed evolution to improve functional expression of class b g-protein coupled receptors klenk c., scott d. j., plückthun a. universität zürich institut für biochemie, winterthurerstrasse 190, 8057 zürich, switzerland the class b of g-protein coupled receptors (gpcrs) comprises 15 peptide hormonebinding receptors which regulate important endocrine and neuroendocrine functions of the human body. several of these receptors are implicated in the pathogenesis of severe diseases such as diabetes, osteoporosis, growth disorders and depression, which makes them attractive targets for drug therapy. to develop new compounds targeting these receptors a detailed understanding of the molecular structure is required which has not been succeeded to date. structural studies of proteins by x-ray crystallography or nmr spectroscopy generally require large and homogenous quantities of protein. for gpcrs this prerequisite is difficult to achieve as the vast majority of gpcrs exhibits low endogenous expression and is very unstable in solution. therefore, improved expression conditions are necessary for the efficient characterization of new gpcr structures. here we present a method to optimize class b gpcrs for improved heterologous expression and increased thermostability by means of directed evolution. libraries of class b gpcrs were obtained by random mutagenesis and were expressed in a heterologous expression system in which functional gpcr is targeted to the inner membrane of e. coli. mutants that display increased receptor expression levels and ligand binding were selected by flow cytometry using fluorescently labeled ligands. repetitive cycles of randomization and selection allow to gradually increasing the level of protein expression and stability. with this evolutionary approach key residues within the receptor sequence can be identified rapidly that are responsible for improved biophysical properties without affecting the pharmacological features of the receptor. such gpcr mutants will become a valuable tool on the way to express high quantities of stable receptor protein for subsequent structural studies. pasireotide (som230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, cushing's disease and carcinoid tumors. whereas octreotide acts primarily via the sst2 somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst2 activity combined with enhanced binding to other somatostatin receptor subtypes. somatostatin and octreotide stimulate the complete phosphorylation of at least six carboxyl-terminal serine and threonine residues namely s341, s343, t353, t354, t356 and t359, which in turn leads to a robust endocytosis of the sst2 receptor. surprisingly, pasireotide failed to phosphorylate the four threonine residues and induced only a detectable phosphorylation of s341 and s343. somatoprim and ke108 are recently developed somatostatin analogs capable of binding to four of five somatostatin receptor subtypes. here, we performed an in vitro study comparing the effects of pasireotide, somatoprim and ke108 on sst2 somatostatin receptor binding, phosphorylation, internalization and signaling. further somatostatin, octreotide, pasireotide, somatoprim and ke108 were tested for functional selectivity at sst5 receptor mutants, which possess a carboxyl-terminal sst2tail. this approach allows detection of receptor activation by phospho-specific sst2 antibodies. compared to octreotide, somatoprim activates sst2 but has a higher activity on sst5. ke108 and pasireotide are partial agonists at the sst2 receptor. pasireotide, ke108 and somatoprim show comparable effects on sst5 receptor. however, none of these new pan-somatostatin analogs behaves like natural somatostatin on the sst5 receptor. cadmium modulates ahr-associated gene expression in the rat intestine after oral exposure kluxen f. m. 1, 2 , höfer n. cadmium has been shown to mimic steroid estrogen effects in vivo and in vitro. we have recently identified cross-talk of estrogen receptor (er) and aryl hydrocarbon receptor (ahr) in the rat uterus where 17beta-estradiol (e2) and cdcl2 modulate ahrassociated genes via er after i.p. injection in a similar fashion (kluxen et al., arch toxicol doi 10 .1007/s00204-011-0787-x). however, the predominant route of exposure to cadmium in non-smokers is via diet. moreover, uterus expresses mainly the receptor subtype eralpha, whilst small intestine and colon express mainly erbeta. thus, we now investigated by real-time rt-pcr the effects of cadmium (2mg/kg b.wt cdcl2 (cd2)) or steroid estrogen (0.1 mg/kg b.wt. 17alpha-ethinylestradiol (ee2)) on ahrassociated gene expression (i.e., ahr, cyp1a1, gsta2, nqo1) in the small intestine of rats after oral exposure (3 days gavage). the animals were also co-treated with cd2 and pure anti-estrogen (2.5 mg/kg b.wt zk191703 (zk)) or ee2, to asseess whether ermediated processes are involved. we also measured cyp1a1 mrna expression in two estrogen receptor negative colon cancer cell lines (ht-29 and caco2) treated for 5 days with cdcl2 (1µm) and e2 (0.01µm). the dose-dependency of cadmium induced ahr target gene modulation was studied in a second animal experiment, with administration of cadmium in drinking water for 28 days (0.4-9 mg/kg b.wt cdcl2 equivalent to 5, 50, and 150 ppm) and ee2 (0.08 mg/kg b.wt) as steroid reference. in summary we present two major results: the metalloestrogen cadmium modulates dose-dependently the ahr-associated gene expression in the intestine after oral exposure. yet, since the cadmium induced modulation of ahr target genes was not antagonized by anti-estrogen in the small intestine in vivo and was also found to occur in er-negative colon cells in vitro, we propose that er-independent mechanisms might play a role in this effect. meg) is the most cytotoxic lesion. if not repaired by o 6 -methylguanine-dna methyltransferase (mgmt), o 6 meg/t mismatch is recognized by the mismatch repair system (mmr) that performs futile repair cycles. during this process secondary lesions (i.e. dna single-strand brakes) are formed, which block dna replication in the next replication cycle, leading to dna double-strand breaks (dsbs). these dsbs eventually signal to apoptosis and other genotoxic endpoints. here, we wished to address the question whether autophagy is part of the cellular response triggered by o 6 meg. we also assessed whether autophagy influences apoptosis induced by tmz in glioma cells. we show that tmz induces autophagy in u-87 mg and ln-229 glioma cell lines. the maximum amount of autophagy was observed several days (96 h) after tmz treatment and mgmt proficient cells did not display significant autophagy. thus, the data show that mgmt protects against tmz induced autophagy, pointing to o 6 meg as the critical lesion responsible for induction of autophagy. using colon cancer cell lines proficient and deficient in mmr, we show that mmr is required for tmz-induced autophagy. because dsbs, which emerge during the processing of o 6 meg, are repaired preferably by homologous recombination (hr) ln-229 cells stably down-regulated for hr were tested for autophagy induction. the data indicate that dsbs are involved in tmz-induced autophagy. because autophagy following tmz treatment occurs earlier than apoptosis we hypothesize that autophagy protects glioma cells against apoptosis. using an early stage autophagy inhibitor 3-methyladenin we have shown, that autophagy inhibition sensitized glioma cells to tmz-induced apoptosis. taken together our data point out that tmz induces autophagy in glioma cells and that autophagy protects glioma cells against tmz-induced apoptosis. o 6 meg is the lesion responsible for autophagy induction. furthermore, the data also shows that mmr and dsbs are involved in the induction of autophagy after tmz treatment. work was supported by bmbf (02nuk016). retinitis pigmentosa (rp) is a severe human retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. in most cases, rp finally leads to legal blindness. mutations in the regulatory subunit of the rod cyclic nucleotide-gated (cng) channel (cngb1a) have been found in patients suffering from rp. we used cngb1-deficient (cngb1 -/-) mice to establish a gene replacement therapy as a potential treatment for rp by means of recombinant adenoassociated viral (raav) vectors. the packaging limitations of raav vectors required a capacity-optimized vector of the large cngb1a cdna (approx. 4 kb). therefore, we replaced regulatory elements within the expression cassette and used a short mouse rhodopsin promoter element for rod-specific expression. after injection of therapeutic raavs (serotype 8) into the subretinal space of 2-week-old cngb1 -/mice, we assessed the restoration of vision by analyzing i) protein expression and localization, ii) retinal function and morphology and iii) vision guided behavior. we found that treated cngb1 -/mice expressed full-length cngb1a and cnga1, which were previously downregulated. both proteins co-localized in rod outer segments and formed regular cng channel complexes in the treated area of the cngb1 -/retina. using electroretinography (erg) we observed a distinct rescue of rod-driven responses. moreover, cngb1 replacement significantly preserved outer segment morphology and delayed retinal degeneration. finally, treated cngb1 -/mice performed significantly better than untreated mice in a modified water maze task designed to test for rod-mediated, vision-guided behavior. in summary, this work provides a proof-of-concept for the treatment of rod channelopathyassociated retinitis pigmentosa by raav-mediated gene replacement. most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones. here, we present an alternative testing paradigm for detection of endocrine modes of actions that replace and reduce animal testing through refinement. receptor mediated endocrine effects were assessed using the yeast based receptor mediated transcriptional activation yes/yas assays and effects on steroid hormone biosynthesis were assessed using the human cell line h295r in the steroidogenesis assay. in our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile. the combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. thus, this proposal constitutes a potentially attractive alternative to epa's endocrine disruptor screening program. data on 14 reference substances for which the in vitro yes/yas and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine mode of action is presented here. the bovine corneal opacity and permeability (bcop) test has been adopted by oecd for the identification of corrosive and severe ocular irritants (ghs category 1) for single component substances and multi-component formulations. eye irritation tests (eit) using human reconstructed tissue models (such as epiocular) have been described to predict ocular non-irritants (ghs no category). thus the ultimate repaltement of the draize rabbit eye irritation test (oecd tg 405) by a combined or tiered testing strategy could be possible. the purpose of this study was to evaluate whether the bcop with additional corneal histology together with the eit could be used to predict eye irritancy of agrochemical formulations according to different classification schemes including un ghs and epa systems. we have performed the bcop (plus histology) and the eit of 50 agrochemical formulations for which in vivo eye irritation data were already available (for registration purposes). using the oecd tg guideline evaluation scheme for opacity and permeability in the bcop was not predictive for the agrochemical formulations assessed here, while corneal histology grades and the epiocular tissue viabilities were useful predictors of eye irritancy potencies and could be applied for the different classification schemes. the nanomaterials offers extraordinary opportunities. the nano-structure can change the physical and chemical properties, and often also alters the biological effects. hence the toxicity of a nanomaterial can differ from its larger-scale material; but as of today, no new quality of a general nano-specific toxic effect has been observed. therefore the established testing methods are generally suitable. it is, however, difficult to apply the nanomaterials to in vitro test systems, since it is the nature of these materials to change their surface properties and agglomeration stateand the uptake and distribution in the body may differ from their larger-scale materials. while the methods for topical effects may be used for nanomaterials without further modification, the in vitro methods for genotoxicity testing require the dispersion in culture media. the use of reproducible and well-documented dispersion protocols andthe characterization of its particle size distribution is de rigueur . [1] . for many nanomaterials published genotoxicity studies did not give a consistent picture [2] and therefore there are rather effects of individual nanomaterials than nano-genotoxicity per se. modern toxicology is based on the insight into toxic pathways. for nanomaterials a testing strategy will include testing for their primary effects (which might be only a handful: particle effects, catalyzing the formation of reactive molecules and ion release) and their uptake, distribution and clearance. the use of dermal penetration studies in vitro for the risk assessment of sunscreen nanomaterials has been demonstrated [3] . in vitro methods for specific effects (such as inflammation, pulmonary toxicity, sensitization) are currently awaiting validation (for both chemicals/molecules as well as nanomaterials). in the meantime alternative short-term in vivo studies with optimized biological readouts can deliver information on the toxic pathways as well as the biokinetics and dose-response relation of nanomaterials in the body. a short-term inhalation test for nanomaterials has already been used successfully [4] . testing strategies based on those methods engage less animals and provides more significant data than classical testing. moreover data from these methods will serve as a benchmark and a validation for the in vitro models under evaluation. nanoparticles (np) are increasingly used in various field of industry which necessitates evaluation of their safety. also in the food industry, nps have gained strong interest, for example as food additives or to improve food packing. however, the potential risks of ingested np have been rarely investigated. inhalation studies have revealed that inflammation and oxidative stress may represent unifying mechanism for the induction of adverse health effects of toxic np. in the present study, a co-incubation model of human polymorphonuclear neutrohils (pmns) and caco-2 human intestinal epithelial cells was used as a model to address potential genotoxic effect of np during intestinal inflammation. oxidative dna damage induction (measured by the fpg-modified comet assay) was induced in the caco-2 cells by activated pmn and this effect increased with increasing pmn to caco-2 cell ratio. the crucial involvement of the phagocyte nadph oxidase complex could be demonstrated using treatment of caco-2 cells with bone marrow-derived pmn from nadph oxidase deficient mice. dna damage by pmn as well as h 2o2 was increased in buthionine sulphoximine (bso) pre-treated caco-2 cells, illustrating the importance of the cellular glutathione (gsh) status in these target cells. gsh depletion in caco-2 cells could also be shown upon treatment with various types of np. our data suggests that ingested np may increase the susceptibility of the colon mucosa to genetic damage during the occurrence of intestinal inflammation. the ingestion of seafood contaminated by acute toxic doses of the marine toxin okadaic acid (oa) is responsible for diarrhetic shellfish poisoning. it is recently known that both the rat and the human hepatic cytochrome p450 monooxygenases (cyp) are able to metabolize this toxin. currently, there is a lack of data about the toxicity and mode of action of oa after xenobiotic metabolism. the aim of our study was the measurement of the toxicity and oxidative stress status in hepg2 cells incubated with oa in the absence and presence of s9 mix. pure oa, as well as oa pre-activated with liver homogenisates (s9 mix) were used to treat human hepg2 cells that have nearly undetectable levels of functional cyp but express phase ii enzymes. the experiments were performed with both human and rat s9 fraction plus cofactors of phase i enzymes. the cell viability was measured after 4 h using mtt-test and xcelligence real time cell monitoring system. furthermore, levels of intracellular reactive oxygen species (ros) were detected by 2´,7´-dichlorofluorescein diacetate and additionally by measuring the intracellular glutathione content. in the presence of both human and rat s9 mix oa showed a higher toxicity than the parental substance. oa pre-incubated in rat s9 mix was toxic at 75 nm oa. strong effects could be observed when oa was pre-activated with human s9-mix at a concentration of 50 nm oa. pure oa was non-toxic in that concentration range. we could also detect an increase of oxidative stress in hepg2 cells treated with oa in the presence of all investigated s9-mix. these results suggest that oa is activated after oxidative xenobiotic metabolism into metabolites which possess a higher cytotoxic activity and increase the amount of intracellular ros in hepg2 cells. ballast water treatment -emerging health risks werschkun b., banerji s., krätke r. bundesinstitut für risikobewertung, max-dohrn-strasse 10, 10589 berlin, germany the introduction of invasive marine species into new environments by ships' ballast water, via ships' hulls and other vectors has severe impacts on the oceans. in 2004 the international maritime organisation (imo) launched the international convention for the control and management of ships ballast water and sediments which requires ballast water to be treated in order to eliminate alien aquatic species. ballast water treatment may include mechanical, physical or chemical measures. any ballast water management system using active substances needs imo approval. therefore identification of active substances, relevant chemicals and submission of specified datasets on their physical, chemical and toxicological properties is required in order to assess the safety for the aquatic environment and for human health. the bfr is the german federal agency responsible for health risk assessment and has been involved in more than twenty assessment and approval processes so far. the majority of imo approved systems are based on oxidative principles such as chlorination and ozonation. these methods can generate disinfection by-products (dbps), which are a mixed group mostly of halogenated organic substances like trihalomethanes, haloacetic acids and haloacetonitriles. the formation of dbps is well known from the disinfection of drinking water. some dbps are regulated under drinking water directives because of their long-term toxicity but many are unregulated and have unknown toxicological properties. the formation of dbps may vary significantly depending on the treatment system as well as on environmental parameters like temperature, ph and composition of the organic matter within the aquatic environment. in sea water, sources for dbp formation besides ballast water treatment are aquaculture and the cooling systems of coastal power plants. in order to address possible health and environmental risks from dbps formed during ballast water treatment a conference on emerging risks from ballast water treatment was held at bfr in october 2011. here we summarise the main conference findings and identify areas for future research. two presentations corroborated that significant amounts of dbps can be formed in sea water and a presentation on the toxicological properties of dbps pointed out that many have genotoxic properties. accordingly, the determination of dbp species and generated concentrations under different ballast water treatment conditions was seen as a mayor task. different approaches for health and environmental risk assessments were also discussed. appropriate human exposure scenarios and methods for exposure assessment, taking into account common approaches used in risk assessment were presented during the conference. a suitable approach based on derived pec-values for exposure quantification was proposed in order to improve the procedure available for risk assessment of chemical agents used for ballast water treatment. agonist-selective signaling of µ-opioid receptors in t lymphocytes kraus j., börner c., lanciotti s., koch t., höllt v. inst. für pharmakologie und toxikologie, leipzigerstr. 44, 39120 magdeburg, germany opioids are the most potent analgesics and irreplaceable for the treatment of severe pain. in addition to their central effects, opioids modulate a great variety of immune effector cell functions, which may result in unwanted side effects during opioid treatment. the effects of most of the commonly used opioids are mediated by µ-opioid receptors, which belong to the superfamily of g protein coupled receptors. recent data support the concept that g protein coupled receptors function as dynamic entities, which may occupy multiple conformations and activate multiple signaling pathways in a ligand-dependent manner. consequently, different ligands activating the same receptor may have different cellular effects, which has been termed "agonistselective signaling". little is known about agonist-selective signaling of µ-opioid receptors in immune effector cells. in a first attempt to understand if and why such different profiles among different opioids occur we investigated effects of different opioids in human jurkat t cells. we report that the µ-opioid receptor ligands fentanyl, methadone, loperamide and betaendorphin induce internalization of a µ-opioid receptor-green fluorescent reporter construct, whereas morphine and buprenorphine did not induce internalization. the internalization was dependent on p38 mapk and phospholipase d2. in line with this, we observed marked phosphorylation of p38 mapk and activation of phospholipase d2 induced by the internalizing opioids, but no or little such activity by morphine and buprenorphine. as a physiological result, fentanyl, methadone, loperamide and betaendorphin treatment of primary human t cells and jurkat t cells resulted in a strong, up to 100 fold induction of il-4, which was dependent on p38. in contrast, morphine and buprenorphine only showed a weak, approximately one order of magnitude lower induction of il-4. by inducing il-4 opioids significantly modulate the t helper cell balance into the type 2 direction, which influences various immune responses, e. g. the antiviral, t helper cell type 1-mediated response. considering the vital necessity of opioid use in humans, it is an intriguing goal to identify analgetically feasible opioids that have little or no immunosuppressive or -modulatory effects. modulation of cgmp signals by phosphodiesterases in smooth muscle cells krawutschke c., koesling d., russwurm m. ruhr-universität bochum pharmakologie und toxikologie, universitätsstrasse 150, 44780 bochum, germany within the cardiovascular system, cgmp mediates smooth muscle relaxation and inhibition of platelet aggregation. cgmp is formed by particulate guanylyl cyclases and nitric oxide-sensitive guanylyl cyclases, that are activated by natriuretic peptides or nitric oxide (no), respectively. besides the cgmp-forming enzymes, the cgmp-degrading phosphodiesterases strongly determine amplitude and shape of cgmp signals. in vascular smooth muscle cells, three phosphodiesterases are considered to be responsible for cgmp degradation: pde5, the cgmp-specific phosphodiesterase is activated directly by cgmp binding to its gaf domains; this activation if further stabilized by cgmp-dependent protein kinase-mediated phosphorylation. pde1, the ca2+/calmodulin-stimulated pde constitutes the majority of cgmp-hydrolyzing activity in smooth muscle cells at least in the presence of high intracellular ca2+ concentrations. and lastly pde3, the cgmp-inhibited pde displays some cgmp-degrading activity, although cgmp binding to its catalytic domain is primarily thought to inhibit the campdegrading activity of pde3. cgmp signals measurable in radioimmunoassays require stimulation with cgmpincreasing vasodilator concentrations that are orders of magnitudes higher than those required for relaxation. thus, we developed fluorescent sensors for real-time measurement of cgmp signals in single cells. by using these indicators, we analyzed the contribution of different cyclic nucleotide-degrading phosphodiesterases to the modulation of cgmp signals elicited by physiologically relevant vasodilator concentrations. hyaluronan (ha) is a major component of extracellular matrices and is thought to control cellular phenotypes such as proliferation and migration. therefore, ha synthesis may play an important role in the pathophysiology of atherosclerosis. there are three major ha-synthase isoenzymes (has1-3). the has3 gene is alternatively spliced. has3 transcript variant 2 (has3v2) encodes the smallest has isoenzyme which has a different c-terminus and contains only two transmembrane domains compared to has3 transcript variant 1. the aim of the present study was to investigate whether has3v2 is expressed by vascular cells, how it is regulated and where it is localized in cells and whether it indeed synthesizes ha. has3v2 mrna expression was monitored by quantitative real-time rt-pcr. protein expression was determined by western blotting using a polyclonal antibody that was raised in rabbit. an n-terminal eyfp-has3v2 fusion protein and a ddk-tagged has3v2 construct were expressed for subcellular localization studies and co-immunoprecipitation (co-ip). endogenous has3v2 mrna was expressed in both vascular smooth muscle cells (vsmc) and endothelial cells. furthermore, western blotting revealed has3v2 protein expression in vsmc and platelets. in vsmc has3v2 mrna expression was strongly up-regulated in response to interleukin-1β (il-1β, 10 ng/ml) whereas stimulation with interleukin-10 (10 ng/ml), platelet-derived growth factor-bb (10 ng/ml), transforming growth factor β (10 ng/ml), tumor necrosis factor α (10 ng/ml) and interferon-γ (10 ng/ml) had no effect. transfection of hek cells with eyfp-has3v2 fusion protein revealed localization to the endoplasmic reticulum but not to the plasma membrane. furthermore, co-ip experiments showed that tagged has3v2 proteins were precipitated together suggesting formation of multimeric has3v2 complexes. transfection of has3v2 did not cause increased secretion of ha into the cell culture supernatant in hek cells. in conclusion, has3v2 is present in vascular cells and responds to inflammatory cytokines such as il-1ß. because of the intracellular localization and the lack of ha secretion in has3v2 transfected cells, has3v2 may serve intracellular functions apart from ha synthesis. the tubulin antagonist pretubulysin shows strong vascular-disrupting properties in vitro several epidemiological studies indicate a correlation of human exposure to ultrafine particulate air pollution caused by incomplete combustion processes and an increase in the incidence of pulmonary immune diseases like asthma. as a possible mechanism behind this pathological phenomenon, the adjuvant effect of lung inflammation induced by poorly soluble environmental particles has been hypothesised. the aim of our study was to investigate the causal link between carbon nanoparticle-induced lung inflammation and modulations of immune cell populations during processes leading to sensitization, and allergic immune responses of the airways. therefore mice were treated with ovalbumin (ova) alone or in combination with carbon nanoparticles (cnp) by pharyngeal aspiration. the induction of inflammation and the immune adjuvant activity were studied in the lungs and lung draining peribronchial lymph nodes (pbln) at the level of sensitization, and at the level of the immune response. ova-specific ige antibodies were measured in blood serum, and the development of allergic airway inflammation was studied after ova challenge. results at the level of sensitization showed that cnp-induced immediate airway inflammation had immune adjuvant activity resulting in an increase of specific cell populations in pbln and in a stimulation of asthma-specific th2 cytokines. a specific reduction of the neutrophilic lung inflammation by application of the compatible solute ectoine significantly reduced the adjuvant effects of cnp. in ova-sensitized mice, application of cnp 12 hours prior to allergen challenge, led to a significant increase in inflammatory cell infiltrate and respective cytokines in broncho-alveolar lavages. coapplication of 1 mm ectoine together with cnp reduced the particle-induced effects. our data show a link between neutrophilic lung inflammation and adjuvant effects of cnp. a specific reduction of neutrophils by the application of ectoine attenuated this np induced adjuvant effect, indicating that particle-induced lung inflammation rather than the direct interaction of nanoparticles with immune cells is the critical step in environmentally modulated pulmonary immune diseases like asthma. introduction: drug-eluting stents (des) are commonly used in the treatment of acute artery occlusion. however, even if released cytotoxic drugs reduced neointimal proliferation significantly there is still the risk of in-stent thrombosis. it is presumed that this is due to reduced reendothelialization. it has been suggested that coating the stent with biomolecules may provide a new approach to circumvent the lack of healing of the endothelial layer. one approach would be the use of biomolecular signals, such as (poly)peptides and growth factors. rgd and redv are peptide motifs, known to enhance cell attachment and spreading. aim: the aim of our study was to evaluate the efficacy of proteins, derived from elastin-like proteins (elp) and artificial modified by incorporating with the amino acid motifs rgd,redv and p15, in terms of endothelial healing on stents and other cardiovascular devices. results: in this work, we generated vectors encoding for different biopolymers consisting of various bioactive signal molecule sequences. the peptides, e.g. based on the elastinlike matrix (vpgig)2-vpgkg-(vpgig)2, were synthesized using heterologous expression. after optimizing culture conditions and extraction procedures their biological activity was assessed using human umbilical vein endothelial cells (huvec). elastin like proteins with differently incorporated bioactive signals (redv, rgd and a small p15 peptide) were linked covalently via carbodiimide coupling to poly(l-lactide) (plla) films. huvec growth was determined on these modified surfaces using the brdu assay (cell proliferation) and resazurin assay (cell viability). the chemically modified plla surfaces conferred higher cell viability after 1 h adhesion (60%) and an enhanced proliferation (63%, 1 h adhesion, 24 h cultivation) in comparison to the unmodified plla. these results indicate that the synthesized elp incorporated with amino acid motifs promote an accelerated endothelialization of the biodegradable stent material plla. discussion: in summary, we were able to generate elastin-like proteins modified by bioactive sequences. those sequences enhanced endothelial cell proliferation and adhesion. further studies are warranted to determine the activity on smooth muscle cells of these peptides. (1) the failing heart is characterized by excessive extracellular matrix production by myofibroblasts (myocfs) causing fibrosis and myocardial stiffening. myocfs represent phenotypically transformed cardiac fibroblasts (cfs) and are characterized by the expression of contractile proteins like a-smooth muscle actin (α-sma) and enhanced secretion of growth factors (e. g. ctgf). identification of intracellular enzymes that modulate this transformation process is desired to therapeutically modulate pro-fibrotic progression in heart failure. we show that pde2a, a phosphodiesterase isoform, that is able to hydrolyse cgmp and camp, is markedly upregulated in failing hearts from patients with end-stage heart failure (2-3-fold, p<0.05, n=8). notably, pde2a protein abundance is 4-fold higher in myocfs compared to cardiomyocytes from neonatal rat hearts (p<0.05, n=7). to this end we tested whether pde2a modulates the transformation of cfs isolated from neonatal rat hearts to myocfs. indeed, as assessed by immunoblotting and fluorescent microscopy (α-sma, phalloidin, dapi), adenoviral pde2a overexpression induced α-sma expression (3.2-fold p<0.05, n≥8) and to a lower extent ctgf synthesis (1.5-fold, p<0.05, n≥5). mechanistically, pde2a showed preferential subsarcolemmal localisation with diminished total cgmp levels (-56%, p<0.05, n≥5). consistently, parallel stimulation with atrial natriuretic peptide (anp), a selective activator of membrane-bound guanylyl cyclase, normalized ctgf synthesis indicating that pde2a controls cgmp in a discrete subdomain near the plasma membrane. moreover pde2a overexpression diminished the protein levels of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component (-60%, p<0.05, n≥5). these data implicate pde2a-dependent subsarcolemmal cgmp regulation in myofibroblast formation and potentially cardiac fibrosis. therefore, targeting pde2a may lead to regression of the fibrotic remodeling associated with heart failure. several anorganic nanoparticles (np) causedhigher inhalation toxicity than the corresponding coarse particles (oberdoerster et al. 2005) . we examined an organic pigment and a polymer dispersion each as nanomaterial and as the chemical identical coarse material in short-term inhalation studies in malerats. the polymer was an anionic acrylic ester copolymer containing free carboxylic groups. three different particle sizes were synthesized by varying polymerization conditions: 12, 80 or 250 nm. although polymeric acrylic ester was reported to be irritating to the respiratory tract at 3 mg/m3, all three tested polymers -including the np (12 and 80 nm) -did not cause any changes in lavage fluid and in histopathology at 10 mg/m3. the organic pigment was a poorly soluble pyrrol with an intense orange color. the np (10 to 50 nm width and 30 to 400 nm length) and the coarse pigments (70 to 200 nm width and 0.3 to 3 µm length) are both needle-like. they were tested at 1, 3, 10 and 30 mg/m3 for the np and 3, 10 and 30 mg/m3 for the coarse materials. mild and partly reversible morphological changes were observed in lung and lymph nodes at the highest concentrations, but the more pronounced effect were found in rats exposed to the coarse material. likewise there was an increase of lavage parameters in rats exposed to thecoarse material but not to the np. these data demonstrate that inhalation of finer np is not necessarily associated with higher toxicity compared to the coarse material. the results were obtained with two organic particles of rather different size and composition but are in contrast to the more severe effects seen with several anorganic np when compared to the corresponding coarse particles. within the nanocare project a standard short-term inhalation test to examine the toxicity of inhaled aerosols from nanomaterials has been developed. the inhalation toxicity of nano-andpigmentary materials was studied: baso4, zno, ceo2, al-doped ceo2, zro2, amorphous silica, surface-coated amorphous silica, titania, carbon black and three multi-wall carbon nano tubes, all with complete phys-chem-characterization as planned for the oecd sponsorship program. quartz dust tio2 and zno were tested as pigmentary materials. rats were exposed nose-only to three concentrations of one of these materials, 6 h a day for five consecutive days. positive controls were exposed to quartz dust or pigmentary zno, negative controls to clean air. a wide range of endpoints for pulmonary toxicity were evaluated immediately after the last exposure and after 3 days and 3 weeks after the last exposure. among these parameters, polymorphnuclear granulocyte count in bronchoalveolar lavage fluid is the most sensitive early parameter indicating inflammation process in lung, while histological examination reveals the type and localization of inflammation. among these substances, we identified baso4 as having the lowest toxicity. all mwcnts were most potent in producing progressive inflammation in the lung; granulomas in lung and lung associated lymph nodes were observed without indication for fibrosis. the noaecs of the 16 substances ranged between < 0.1 and >50 mg/m 3 . generally the material was only found in the lung (surface and macrophages) and in the draining lymph nodes. surface modified amorphous silica was also found in the spleen. the data demonstrate that the method is able to differentiate the toxic potential of different nanomaterials and to indicate regression or progression of the effects effects. moreover the lung burden and potential translocation to other tissues was detecable. comparing the material properties and effects of the 16 materials, no general relationship between the toxicity and either particle size, specific surface area or aerosol particle number concentration was found. hence we must not expect to find a gerneral "nanotoxicology" or a unifying dosimetry for all nanomaterials. we must rather be prepared to test individual nanomaterials for their effects. and to develop grouping concepts not only based on material properties but also on biopersistence, biokinetics and biological effects. part of this studes has been sponsored by bmbf (nanocare). endpoint-centric search for toxicological information and data to support the information retrieval for regulatory programs landsiedel r. 1 , wächter t. the eu reach regulation no 1907/2006 requires industry to ensure the safety of chemical use and manufacturing. all substances manufactured or imported in quantities above one tonne per year must be registered. information requirements for the dossiers increase with increasing tonnage or once hazards are suspected. searching for substance specific literature and the compilation of hazard data for safety assessments are highly challenging procedures. the novel web-based search engine go3r, accessible free of charge at www.go3r.org, has been created to allow quickly finding relevant hazard information and data. go3r provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information. furthermore, go3r specifically highlights information on animal testing alternatives. search results are presented automatically linked to an "intelligent table of contents" which enables the user to sort the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. retrieved documents are automatically organized in categories relating to the iuclid 5 chapters. hereby, the user can browse directly through the entire 21 million documents without even having to start the search with an initial query. the semantically enriched platform supports the user during query formulation, allows for bibliographic analysis, and specifically highlights information related to the replacement, reduction, and refinement of animal experiments. search results in go3r are shown in an dynamic table of contents (left) making them browsable for the contained information on animal testing alternatives and toxicologogical endpoints. towards a differentiation therapy of acute myelogenous leukemia with histamine h2-receptor agonists laue s., burhenne h., seifert r. hannover medical school institute of pharmacology, carl-neuberg-str. 1, 30625 hannover, germany acute myelogenous leukemia (aml) is a devastating malignancy characterized by a differentiation block of myeloid progenitor cells. recently, histamine dihydrochloride in combination with interleukin 2 has been approved as orphan drug for the consolidation treatment of aml (1) . it is assumed that histamine exerts its effects by activating the histamine h2-receptor (h2r) in human neutrophils, resulting in improved anti-tumor function of t killer cells by inhibiting nadph oxidase-catalyzed superoxide formation. previous studies had shown that histamine also induces myeloid differentiation (2) . considering the fact that all-trans-retinoic acids constitutes a powerful differentiation therapy of acute promyelocytic leukaemia, a specific subtype of aml (3), we initiated a study to explore the possibility that h2r-mediated myeloid differentiation provides an alternative or complementary strategy to treat leukemias associated with differentiation blocks. as model system, we used hl-60 cells. in hl-60 cells, histamine and various h2-receptor agonists induced concentrationdependent increases in camp levels. interestingly, ligands differentially increased cytosolic calcium concentration and extracellular receptor kinase (erk) pathways, indicative for ligand-specific h2r conformations. h2r activation resulted in myeloid differentiation as assessed by enhanced formyl peptide receptor-mediated increases in cytosolic calcium concentration. h2r agonists showed no signs of cytotoxicity. intriguingly, following h2r activation, the majority of the formed camp was exported into the extracellular space via multi-drug resistance protein (mrp) 4, indicating that export is a more important pathway for signal termination than cleavage of camp by phosphodiesterases. despite effective camp export, even a short-term exposure (30 minutes) of cells was sufficient to induce expression of functionally active formyl peptide receptors. these data indicate that in contrast to previously held dogma, induction of myeloid differentiation does not require continuous presence of a camp signal. from a therapeutic point of view this is very important since "spike" therapy with campincreasing substances may be sufficient to induce a therapeutic effect in aml, thereby also reducing toxic side effects. currently, we are systematically exploring the effects of h2r agonists on signal transduction pathways and differentiation in various myeloid cell types to identify highly efficacious compounds. introduction. activation of gαq/11 protein-coupled receptors of postsynaptic neurons can elicit the production of endogenous cannabinoids (endocannabinoids), which in turn inhibit transmitter release from axon terminals by activating presynaptic cb1 receptors. the aim of the present experiments was to study the mechanism of the endocannabinoid production. specifically, we wanted to clarify the role of ca 2+ release from intracellular stores in triggering endocannabinoid production. methods. patch-clamp-and ca 2+ imaging experiments were performed on purkinje cells in mouse cerebellar brain slices. glutamatergic excitatory postsynaptic currents (eepscs) were elicited by electrical stimulation of parallel fibers. the gαq/11 proteincoupled metabotropic glutamate receptor 1 (mglur1) was activated by superfusion of (rs)-3,5-dihydroxyphenylglycine (dhpg) or -more physiologically -by burst stimulation of the parallel fibers. results. both dhpg superfusion and burst stimulation of parallel fibers elicited an increase in intracellular ca 2+ concentration in the postsynaptic purkinje cells. dhpg superfusion and burst stimulation suppressed eepscs, and this suppression was abolished in the presence of the mglur1 antagonist cpccoet. the suppression of the eepscs was also sensitive to the cb1 receptor antagonist rimonabant, pointing to involvement of endocannabinoids and cb1 receptors. the suppression of the eepscs was attenuated after depletion of the endoplasmic reticulum ca 2+ stores by thapsigargin, cyclopiazonic acid and ip3. the results indicate that after activation of the gαq/11 protein-coupled metabotropic glutamate receptor 1 (mglur1) of the postsynaptic neuron ca 2+ is released from the endoplasmic reticulum. this ca 2+ release significantly contributes to the production of endocannabinoids. the endocannabinoids diffuse in the synaptic cleft retrogradely to the terminals of afferent axons and inhibit transmitter release there through presynaptic cb1 receptors. the guanine nucleotide exchange factor dock9 controls reelin dependent cdc42effects on radial migration pichler m. 1 the regulation of blood glucose levels is under tight control of a complex system including hormone and neurotransmitter signalling. many of these cellular signalling pathways are initiated by binding of the ligand to a g-protein coupled receptor (gpcr), e.g. noradrenaline inhibits insulin secretion upon binding to a gi-coupled receptor. upon gpcr activation the heterotrimeric g-protein is activated and both the α-subunit and βγdimers are released and interact with their specific target proteins. by the usage of bordetella pertussis toxin (ptx) as a common gαi inhibitor gαi-dependent signalling pathways are interrupted which leads to increased insulin secretion, and significantly improves glucose tolerance. since the gαi-isoform specific roles in the regulation of glucose homeostasis are still debated we studied the glycemic control in gαi2-deficient mice. surprisingly and in contrast to the ptx data, glucose tolerance was unchanged in the gαi2-deficient mice compared to wild type controls. however, the plasma insulin levels were significantly reduced upon glucose challenge. these findings point to disturbed islets function and improved peripheral insulin sensitivity. analysing gαi2deficient islets we show that islet size and number of nuclei are reduced. nevertheless, in vitro insulin secretion is improved at low (3 mm) and high (16 mm) glucose concentrations and can be further stimulated upon ptx-treatment. these data indicate that gαi2 proteins influence islet development and inhibit insulin secretion. in addition, these findings support our hypothesis that gαi2-deletion influences peripheral insulin sensitivity. therefore, we investigated glucose homeostasis and pakt-levels after two hours feeding ad libitum in gαi2-deficient mice. under feeding conditions no differences in plasma insulin levels were visible although blood glucose levels were significantly reduced in gαi2-targeted mice. pakt-levels of liver and skeletal muscle were unaltered, whereas akt phosphorylation in white adipose tissue was significantly increased, indicating improved glucose uptake of adipocytes. in conclusion, gαi2 is a negative regulator of both insulin secretion and peripheral insulin sensitivity and important for the maintenance of glucose homeostasis. 11689, 1992) in a radioligand binding test and to determine their functional effects with a membrane potential test using the dye r7260 (molecular probes, 0.125 mg/ml, excitation 505 nm, emission 530 nm). the affinity of compounds in radioligand binding was slightly higher in sur2b than in sur2a-type channels, but the enantiomeric ratio in sur2a channels matched that one determined for the sur2b-type indicating some conformity of the binding pockets of sur2a and sur2b-proteins. surprisingly, however, the membrane potential tests revealed that the (3r,4s)-enantiomer acted as agonist (a) whereas the (3s,4r)-enantiomer acted as antagonist (b): (3r,4s)-bms-191095 induced membrane hyperpolarisation whereas (3s,4r)-bms-191095 repolarised cells prestimulated with submaximally effective concentrations of diazoxide. concluding, bms-191095 is not selective for sur2a as compared to sur2b-type k atp channels. its enantiomers activate and block sur2-type katp channels in a stereospecific manner. thieno-thiadiazine derivatives with full agonistic activity at sur2b-type katp channels act as partial agonists at cardiac sur2a-subtypes oldenhage c., grittner d., schmidt c., lemoine h. heinrich-heine universität, inst. für lasermedizin, mol. wirkstoff-forschung, universitätsstr. 1, 40225 düsseldorf, germany new potassium channel openers (kco) of the thieno-thiadiazine(ttd)-type initially developed as agonists for the sur1-type katp channels (nielsen et al., j med chem 45: 4171, 2002) were characterized in sur2b-type katp channels as agonists and antagonists, if r contains a quaternary (methyl-cycloalkyl) and a tertiary (r = cycloalkyl) carbon, respectively (lemoine et al., this journal 375, r45, 2007) . to investigate the selectivity of ttd-derivatives for myocardial katp channels the membrane potential actions of compounds were tested in hek 293(kir6.2/sur2a)-cells and compared to hek 293(kir6.1/sur2b)-cells as a model for smooth muscle-type katp channels. membrane potential was measured by fluorescence (excitation 505 nm, emission 530 nm) using 0.125 mg/ml of the dye r7260 (molecular probes). standard-kco induced hyperpolarisation with ~10-fold smaller potency (pec50) in sur2a. ttd-compounds with ch3-cycloalkyl residues not only lost potency but also intrinsic activity for channel activation (emax) in sur2a. possibly, this loss of emax would be much greater in native heart cells with a normal channel density. in contrast, ttd-compounds with cycloalkyl residues acted as antagonists of cells pre-hyperpolarized with diazoxide with similar affinity in sur2a and sur2b-type katp channels. concluding, selectivity of kco for katp channel-subtypes cannot only be achieved by a different affinity but also by a selective stimulation of the channel of interest. small-conductance calcium activated potassium (kcnn/sk/kca2) channels maintain neuronal calcium homeostasis, shape synaptic functions and prevent excitotoxic neuronal death. so far, little is known about the function of kca2 channels in nonneuronal cells. the aim of this study was to investigate the expression of kca2 channels in microglial cells and their potential function in microglial activation and maintenance. expression of kca2 channel subtypes in microglial cells was assessed by mrna analysis, western blots and immunocytochemistry. lipopolysaccharide (lps)-induced microglial proliferation was evaluated by the xcelligence impedance-based system and mtt assays, and immunogenic activation of microglia was determined by measuring cytokines and nitric oxide (no) release into the cell culture medium. the kca2.2 and kca2.3 channel activator cyppa (25 µm) and specific inhibitory peptides (50 µm) were applied to distinguish effects mediated by the kca2 channel subtypes. all kca2 channel subtypes were detected on mrna and protein levels in resting and in lps-activated microglial cells. xcelligence real-time measurements and mtt assays demonstrated that lps (200 ng/ml) induced microglial proliferation. the kca2.2/kca2.3 channel activator cyppa reduced lps-induced microglial proliferation in a concentration-dependent manner. specific peptide inhibitors of kca2.3 channels, but not of kca2.2 channels, reversed the cyppa-effects on lps-induced microglial proliferation. cyppa alone did not alter the production of tnf-alpha or il-6, but strongly reduced the lps-dependent cytokine production. interestingly, chelation of extracellular calcium by edta induced differential cytokine kinetics by decreasing lps-dependent il-6 production while tnf-a production was not affected. moreover, using inhibitory sk3/kca2.3 channel peptides, we demonstrated that sk3/kca2.3 channels modulate lps-induced cytokine il-6 production in a calcium-dependent manner, while the tnf-a release was independent of extracellular calcium. in summary, the present study revealed that kca2.3 channel stimulation reversed microglial activation. thus, kca2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in cns diseases. (3r,4s)-(3s,4r)intracellular amyloid beta (aß) oligomers and extracellular aß plaques are key players in the progression of sporadic alzheimer disease (ad). still, the molecular signals triggering aß production are largely unclear. we asked whether mitochondria-derived reactive oxygen species (ros) are sufficient to increase aß generation and thereby initiate a vicious cycle further impairing mitochondrial function. complex i and iii dysfunction were induced in a cell model using the respiratory inhibitors rotenone and antimycin resulting in mitochondrial dysfunction and enhanced ros levels. both treatments lead to elevated levels of aß. presence of an antioxidant rescued mitochondrial function and reduced formation of aß demonstrating that the observed effects depended on ros. conversely, cells overproducing aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. again, the capability of these cells to generate aß was partly reduced by an antioxidant indicating that aß formation was also ros-dependent. moreover, mice with a genetic defect in complex i, or ad mice treated with a complex i inhibitor, showed enhanced aß levels in vivo. several lines of evidence show that mitochondria-derived ros result in enhanced amyloidogenic amyloid precursor protein processing, and that aß itself leads to mitochondrial dysfunction and increased ros levels. we propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic ad. comparison of methods to derive health-based guidance or limit values for chemicals licht o., voss j. -u., mangelsdorf i. fraunhofer item chemikalienbewertung, nikolai-fuchs-str. 1, 30625 hannover, germany health-based guidance or limit values are derived for chemicals to compare measured or estimated exposure concentrations with these values. if the exposure is below the limit value, adverse effect for human health can be regarded as negligible, e.g. the exposure is expected to be tolerable. in germany such values have been derived since years for chemicals that can be found in soil, water and air as well as human blood and urine (biomonitoring). in a research project sponsored by the german umweltbundesamt several methods used by the agency are compared to the method laid in reach guidance document r.8 to derive a derived no effect level (dnel). the aim was to identify possibilities for standardization as well as to figure out specific elements in individual methods. in addition to extrapolation factors the public availability of guidance and specific derivations as well as procedures for consensus on the limit value were evaluated. the comparison of extrapolation factors revealed that, although they are named differently such as extrapolation, safety or assessment factors, they are used in a comparable manner. factors for interspecies and intraspecies extrapolation are presented in more detail. the standard factor for such extrapolation is 10 in most cases. in the reach guidance this factor consists of a part for allometric scaling and remaining differences. other factors are only defined in some methods, like a factor for extrapolation from loael to noael, data quality or data gaps. a factor for data quality is not laid down in the basic scheme for setting of indoor air guidance values, but is used in some of the recent derivations of limit values. also the who guidelines for drinkingwater quality use comparable factors to account for adequacy of studies or database and nature and severity of effect. a transparent and documented derivation is necessary for acceptance of the value. the derivation methods as well as the evaluation document on a specific substance are available through publications or the internet in nearly all cases. for the dnel only the numeric value is available at the echa website, but not any information on starting point and extrapolation factors. although all guide or limit values are derived in a comparable way, differences, however, exist in some details. in most cases detailed explanation is lacking when deviating from standard or default assumptions. often such deviation is based on expert judgement. hepatocellular carcinoma (hcc) is the fifth most common cancer in the world and has a poor prognosis with limited therapeutic options. up to now, no curative systemic therapy exists emphasizing the high clinical importance of new therapies for hcc. therefore, the identification and characterization of novel drugable targets is a relevant goal. cyclin-dependent kinase 5 (cdk5) is well characterized for its function in cns development and disease. recently, few reports indicate functions of cdk5 in cancer. cdk5 was shown to regulate tumor growth, and our group discovered that cdk5 regulates angiogenesis. since hcc is a highly vascularized tumor and anti-angiogenic treatment (sorafenib) has shown some therapeutic benefit, we hypothesize that cdk5 is an interesting target for hcc therapy. the aim of this study was to characterize the function of cdk5 in hcc. histology of tissue micro arrays indicates an increased expression of cdk5 in human hcc tissue in comparison to healthy liver tissue of the same patient. to investigate the function of cdk5 in hcc, we analyzed the impact of both pharmacological inhibition of cdk5 and specific downregulation of cdk5 with rna interference on hcc cells. pharmacological inhibition of cdk5 with the small molecule roscovitine (r-roscovitine, seliciclib) decreased proliferation and clonogenic survival, induced g2/m cell cycle arrest and cell death, and reduced motility of huh7 and hepg2 cells. transient downregulation (sirna) and stable knockdown (shrna) of cdk5 also reduced proliferation, clonogenic survival, migration and invasion of huh7 cells. in a subcutaneous hcc xenograft model, treatment with roscovitine reduced tumor growth and angiogenesis, indicated by decreased tumor weight and volume, and reduced vessel density. moreover, cotreatment of hcc cells with roscovitine and tumor necrosis factor related apoptosis inducing ligand (trail) resulted in an over-additive additive effect on the induction of apoptosis. this coincided with reduced phosphorylation and activity of the anti-apoptotic transcription factor stat3 at ser727 that is directly phosphorylated by cdk5, and tyr705. in line with this, the expression of the antiapoptotic protein mcl-1 is reduced by inhibition of cdk5. our results point to an important function of cdk5 in hcc and suggest cdk5 as an interesting pharmacologically druggable target for hcc therapy. delivery of mono-biotinylated rnasea into macrophages with streptavidinconjugated clostridium botulinum c3 toxin lillich m. 1 , chen x. clostridium botulinum produces the adp-ribosyltransferase c3, which modifies and thereby inactivates exclusively the small gtp binding proteins rho-a,-b and -c. recently, we discovered a specific endocytotic internalization of c3 toxin in macrophages and myeloid leukaemia cells, but not in epithelial cells [1] . thus, c3 toxin provides a tool to target cells of the monocyte/macrophage lineage, which are involved in various diseases and are of great clinical interest. we used a biochemical crosslinking approach to design a delivery system based on an enzymatic inactive c3bot mutant (c3mut) and streptavidin. the c3 portion mediates uptake of the transporter into monocytes/macrophages and streptavidin allows for binding of biotinylated cargo molecules to the transporter. in vitro, the generated c3mut-streptavidin bioconjugate showed specific and concentration dependent binding to biotinylated oligonucleotides as demonstrated by electrophoretic mobility shift assay. cell fractionation experiments indicated an uptake of the bioconjugate into the cytosol of j774a.1 macrophages. in the next step, mono-biotinylated bovine pancreatic ribonuclease a (rnasea) was used as a model cargo for the delivery of macromolecules by the bioconjugate. rnasea is a highly stable, well studied protein which catalyzes the degradation of rna. mono-biotinylated rnasea interacts in a specific and concentration dependent manner with the c3mut-streptavidin bioconjugate in vitro as analysed with dot blot technique. the c3mut-streptavidin bioconjugate efficiently mediates the internalization of biotinylated rnasea into j774a.1 macrophages as analyzed with laser scanning microscopy in fixed cells. this finding was also confirmed by live cell imaging. furthermore, cell fractionation showed a cytosolic delivery of biotinylated rnasea in the presence of c3mut-streptavidin. as expected we could not observe a cytotoxic effect of biotinylated wild-type rnasea on j774a.1 macrophages, which is attributable to the presence of ribonuclease inhibitor protein in mammalian cells. in summary, the c3mut-streptavidin bioconjugate mediates the efficient internalization of biotinylated (macro)molecules into macrophage like cells, and therefore represents a useful tool for the transduction of exogenous molecules into macrophages. in addition, cytotoxic rnasea mutants are available and will be used in further studies. organometal compounds such as cisplatin or the second generation complexes carboplatin and oxaliplatin have become more and more important as antitumor agents. nevertheless there is still an increasing demand for novel metal-based compounds. this is necessary due to severe side effects and the occurence of resistent tumour cells. in this context we investigated the cytotoxic effects of imidazole-based phosphane gold(i) complexes as potential agents for cancer treatment. initially we have used the mtt-assay to examine the toxic potential of the gold complexes in h4iie rat hepatoma cells. in this context cw60 (a diphosphane ligand with azoyl substituents r2p(ch2)2pr2, r= thiazol-2-yl) turned out to be the compound with the highest cytotoxic potential with an ic50 value of 6,5mm (24h incubation). further investigations revealed that cw60 induced an apoptotic cell death in h4iie demonstrated by the activation of caspase 3/7 (48h incubation with 10mm cw60). in addition the induction of apoptosis was confirmed by the dna ladder formation (24h incubation with 5mm cw60). in connection with the molecular mechanisms of apoptosis induction we used the comet assay to analyse the generation of dna strand breaks as well as the dcf-assay to detect the formation of reactive oxygen species. however neither dna strand breaks nor increased levels of reactive oxygen species were detected after 1h of incubation. furthermore we analysed if the compound influences intracellular signalling pathways such as the jnk pathway and the pi3k/akt but after 24h of incubation neither pakt nor jnk were influenced. the imidazole based phosphane gold (i) complex cw60 shows strong toxic effects in h4iie cells and turned out to be a promising compound as a potential agent for cancer treatment. the high and inappropriate intake of loop diuretics in hypertensive elderly reported in former studies has again been confirmed. remembering that inappropriate intake of loop diuretics can lead to exsiccosis and electrolyte loss especially in elderly, better medical education has to follow these alarming results to improve the pattern of diuretic prescription. furthermore, our results lead us to assume a high estimated number of unreported cases of torasemide use in uncomplicated arterial hypertension in elderly. this loop diuretic agent shows a longer duration of action compared with furosemide (elimination half-life: 3-4 hrs vs. 1 hr) and is effective in decreasing blood pressure in subdiuretic doses. it must be pointed out that loop diuretics are still frequently inadequately prescribed because current guidelines recommend loop diuretics only in complicated arterial hypertension. the role of cgmp/cgki signaling and trpc channels in regulation of vascular tone loga f., domes k., hofmann f., wegener j. pharmakologie und toxikologie for 923, biedersteiner str 27, 80802 münchen, germany signaling by intracellular cgmp and cgmp-dependent protein kinase i (cgki) is the major pathway in vascular smooth muscle, by which endothelial no regulates vascular tone. recent evidence suggests that trpc channels are targets of cgki in smooth muscle and mediate, at least partially, the relaxant effects of cgmp. we tested this concept by investigating the role of cgmp/cgki signaling on vascular tone and peripheral resistance using cgki-, trpc6-, and trpc3-, and trpc3/c6-double knock-out mice. we found larger contractile responses to α-adrenergic stimulation in intact aorta from cgki-, trpc6-, and trpc3/c6-double knock-out mice as compared to aorta from ctr and trpc3-knock-out mice indicating a functional link between cgki and trpc6 channels. no differences were found if the vasodilator tone, provided by the no generation in the vascular endothelium, was inhibited by l-name. likewise, no differences were observed in the increase in peripheral resistance by α-adrenergic stimulation using the hind limb perfusion system. activation of cgki by 8-br-cgmp diminished aortic tone and peripheral resistance to a similar extent in control, trpc6-, trpc3-, and trpc3/c6-double knock-out mice. no effect of 8-br-cgmp was observed in preparations from cgki -/mice. to test the co-localization of cgki and trpc channels, we performed immunocytochemistry on isolated smooth muscle and endothelial cells from aorta of ctr, trpc3-, and trpc6-knockout mice. trpc3 could be detected in both smooth muscle and endothelial cells whereas trpc6 was only detected in endothelial cells. the results suggest that absence of cgki or trpc6 impairs the vasodilator tone induced by endothelial no production but that cgki and trpc6 channels are not functionally coupled in vascular smooth muscle. we thank profs birnbaumer (nih) and freichel (homburg) for providing us with trpc6 -/-, and trpc3 -/mice and prof. flockerzi (homburg) for the antibodies against the trpc channels. whole genome microarray analysis of the effects of tcdd and pcb 153 in human hepatic cell models lohr c. 1 , neser s. after the treatment with tcdd, however, a total of 281 genes were more than 2-fold up regulated in hepg2 e.g. cytochrome p450 1a1 (cyp1a1) (32-fold) a sensitive marker for ahr activation. additional up regulated genes in hepg2 were; arylhydrocarbon receptor repressor (ahrr) 15-fold, aldehyde dehydrogenase 3 a1 (aldh3a1) 11-fold, and cytochrome p450 1b1 (cyp1b1) 10-fold. 44 genes were more than 2-fold down regulated in hepg2 cells e.g. -proprotein convertase subtilisin/kexin type 9. markedly different findings were obtained in hheps, i.e., 117 genes were up regulated, the highest up regulated gene was cyp1b1 with a 95-fold increase in gene expression, followed by cyp1a1 (41-fold) and aldh3a1 (21-fold). only a small group of genes were significantly down regulated (17 in total), e.g., solute carrier family 2 (facilitated glucose transporter). comparing both human cell types, there was an unexpected small overlap of genes being up or down regulated. interestingly, in both cell types, only 30 in common genes were up regulated, including cyp1a1, cyp1a2, cyp1b1 and aldh1a3. only platelet-derived growth factor receptor, beta polypeptide, was down-regulated in both hepg2 and hheps. in conclusion, our data indicate pronounced differences in the patterns of tcdd-regulated genes between hepg2 cells and hheps. detection of redox modified proteins in nociceptive processing lorenz j. e. 1 , kallenborn-gerhardt w. recent data indicate that redox modifications of proteins induced by reactive oxygen species (ros) contribute to sensitization of pain pathways during persistent pain. however, little is known about the targets of ros in pain processing, because the relatively unstable nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. here, we used the quantitative thiol trapping technique termed oxicat to identify proteins which are redox modified during nociceptive processing. we investigated spinal cords of untreated mice, after zymosan injection into a hindpaw (inflammatory pain model) and after spared nerve injury (neuropathic pain model). we identified several proteins with marked changes in their redox states after nociceptive stimulation. our results show that the oxicat method is an efficient method to detect redox modifications in proteins and that redox modifications seem to play a role in pain processing. supported by the deutsche forschungsgemeinschaft (sfb 815/a14). additive antinociceptive effects of a combination of vitamin c and vitamin e after peripheral nerve injury lu r., kallenborn-gerhardt w., geisslinger g., schmidtko a. pharmazentrum frankfurt/zafes institute of clinical pharmacology, goethe university, frankfurt am main, germany accumulating evidence indicates that increased generation of reactive oxygen species (ros) contributes to the development of exaggerated pain hypersensitivity during persistent pain. in the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin c and vitamin e in mouse models of inflammatory and neuropathic pain. we show that systemic administration of a combination of vitamins c and e inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by complete freund's adjuvant. in contrast, vitamin c or vitamin e given alone failed to affect the nociceptive behavior in all tested models. the attenuated neuropathic pain behavior induced by the vitamin c and e combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. moreover, the vitamin c and e combination ameliorated the allodynia induced by an intrathecally delivered ros donor. our results suggest that administration of vitamins c and e in combination may exert synergistic antinociceptive effects, and further indicate that ros essentially contribute to nociceptive processing in special pain states. -206, -213 and -214) replaced by leucine residues. both amino acids are comparable in terms of hydrophobicity, volume and the preference for forming α-helices, but only methionine is oxidizable to a sulfoxide, in contrast to leucine. in the present study we examined the protein-protein interaction (ppi) of recombinant ac 1, expressed in sf9 insect cell membranes, with cam, cam-206, -213, -214 and -215 by measuring the catalytic activity of ac 1. cam-mutants show a 3-4-fold lower potency than cam, but they are more efficacious than cam. most prominently, cam-215 was 133 % more efficacious than cam. such striking differences between cam and cam-mutants have not yet been observed for other mammalian effector proteins. as a result of the exchange of all methionine against leucine residues in cam-215, it is more hydrophobic than cam and this leads to a better ppi with ac 1. in future studies we will examine the effects of cam inhibitors, antidepressants and antipsychotics on cam/ac 1 interaction. furthermore we will analyze the effects of oxidized cam and cam-mutants on the catalytic activity of ac 1. because oxidative stress is of great importance in aging, it is important to know more about the abovenamed interaction in view to the demographic change. taken together, our data point to a unique cam/ac 1 interaction that may be selectively targeted by small molecules. in particular, enhancers of these interaction could be useful to improve memory and learning. gender differences in fat distribution and diabetes prevalence in nzo mouse lubura m., scherneck s., zucker a., schürmann a. deutsches institut für ernährungsforschung experimentelle diabetologie, arthur-scheunert-allee 114-116, 14558 potsdam, germany background: excessive fat accumulation in visceral but not subcutaneous fat depots as well as ectopic fat storage in liver, skeletal muscle and pancreas are associated with an increased risk for the development of type 2 diabetes in humans. in this study we aimed to examine the influence of early fat distribution on onset of type 2 diabetes in mice. methods: nzo mice are regarded as insulin resistant model in which only males become diabetic. we used male and female mice fed with high-fat and standard diet. we determined fat distribution by computed tomography for three times and conducted oral glucose tolerance tests on two different time points. besides we assessed body weight and blood glucose levels on weekly basis. results: contrary to previous findings, we observed that not only male nzo mice on high-fat diet develop diabetes. blood glucose levels at the 16 th week of age and total pancreatic insulin content indicated diabetes prevalence of 68% in males and 25% in females these results lead to the conclusion that high-fat diet counteracts protective action of estrogens against diabetes. inversely to the findings in humans, female mice tend to store more fat in abdominal region than males. there was no relationship between early accumulation of fat in abdominal region and onset of type 2 diabetes. however, visceral fat was associated with liver fat in males as well as in females. furthermore, at the age of ten weeks hepatic fat content correlated with blood glucose levels (r² = 0.69) indicating that the early hepatosteatosis is a predictor for hyperglycemia. however, there was no correlation between hepatic insulin sensitivity (indicated by quantitative insulin sensitivity index-quicki) and amounts of hepatic fat we conclude that early hepatosteatosis does not predict for glucose intolerance in nzo mice. in the nzo mouse, the amount of liver fat but not the early fat distribution predicts for the later onset of type 2 diabetes. further experiments are needed to examine the gender dependent differences in the diabetes prevalence of this mouse strain. with a prevalence of about 20-30% non-alcoholic fatty liver disease (nafld) represents the most common liver disorder in europe. nafld manifestation ranges from steatosis through steatohepatitis (nash) to fibrosis and cirrhosis, followed in some cases by liver failure and hepatocellular carcinoma. fatty degeneration of liver cells, increased oxidative stress with concomitant lipid peroxidation and an induction of pro-inflammatory cytokines are proposed as possible causes for developing inflammation and fibrosis, but the exact pathogenesis of the progression of nafld into nash is still unknown. thus, besides life style modifications and weight reduction interventions, no established pharmacological therapy exists so far. to gain further insights into the pathogenesis of nafld and nash and to develop new therapeutic strategies, appropriate animal models are essential. thus, in the present study three different dietary animal models for nafld were evaluated and compared to the biochemical and metabolic alterations seen with nafld and nash in man. male adult lewis rats were given standard food or one of three different diets: fatty liver diet [fld] , methionine/choline deficient diet [mcd] or methionine/choline deficient plus high fat diet [mcd+hf] . after 1, 2, 4, 6 or 12 weeks of treatment, animals were sacrificed and body and liver weights, laboratory parameters (asat, alat) as well as histopathological changes in the livers and different parameters indicating oxidative stress or representing the biotransformation capacity of the livers were analyzed. with fld and mcd+hf a normal body weight gain was observed, whereas with mcd body weight gain was strongly impaired. liver weights were mainly increased after mcd+hf. elevation of asat and alat values and hepatic steatosis were more pronounced after mcd and mcd+hf than after fld. all three diets caused an increase in the oxidative stress in liver tissue, but especially with mcd a tremendous elevation in the hepatic levels of lipid peroxidation products was seen. with regard to liver biotransformation capacity, with all three diets mainly an induction of the cytochrome p450 2e1 and 4a1 isoforms expression and activity was observed, which was most pronounced after mcd and mcd+hf. in summary, the changes induced by mcd or mcd+hf most closely resemble the alterations described in literature for nafld in man and thus should be preferred over fld in future investigations on nafld and nash. ep3 receptors for prostaglandin e2 convey stimulatory and inhibitory effects. e.g., their stimulatory effect leads to vasoconstriction in the human pulmonary artery and their inhibitory activity to reduction of neurotransmitter release from neuron endings. the aim of our study was (1) the pharmacological characterization of ep3 receptors in human pulmonary arteries and (2) the examination of the involvement of these receptors in the regulation of the neurogenic tachycardia in pithed rats. l-826266 served as the ep3 antagonist. experiments were performed in human pulmonary arterial rings isolated from patients undergoing lobectomy during resection of lung carcinoma and in pithed and vagotomised rats. the ep1/ep3 agonist sulprostone (1 nm -100 mm) concentrationdependently contracted human pulmonary artery rings (pec50 and emax; 6.89±0.12 and 106.5±5.2%, relative to the contraction induced by kcl 60 mm). the concentrationresponse curve of sulprostone was not affected by the ep1 antagonist sc 19920 (10 µm) but shifted to the right by l-826266 (10 µm) (apparent pa2 6.22). extending the exposure time to l-826266 from 0.5 to 3 h increased its antagonistic potency to 7.39 (schild plot-based pa2; concentrations 0.1, 1 and 10 µm). in pithed rats electrical stimulation (0.66 hz, 1 ms, 50 v for 5 s) of the preganglionic sympathetic nerve fibers or intravenous isoprenaline (0.15 nmol/kg) increased heart rate (hr) by 55 beats/min. sulprostone (10 -1000 nmol/kg) did not affect the isoprenaline-induced increase in hr but inhibited the neurogenic tachycardia dose-dependently, maximally by 80%. l-826266 (3 µmol/kg) diminished the inhibitory effect of sulprostone 1000 nmol/kg on the neurogenic tachycardia by 20%. in conclusion, ep3 receptors (1) located postsynaptically strongly contract human pulmonary arteries and (2) located presynaptically on sympathetic nerve fibres supplying the heart of rats strongly inhibit the neurogenic tachycardia. -5-bromo-n[3-(5voltage-gated ca 2+ channels of the central nervous system control a multitude of ca 2+ dependent processes such as neurotransmitter release, neuronal excitability, neurite outgrowth, synaptogenesis, plasticity and neuronal survival. the cav2.1 ca 2+ channelalso known as p/q-type channel -belongs to the subfamily of high voltage activated ca 2+ -channels. ca 2+ influx via cav2.1 ca 2+ channels located at presynaptic nerve terminals triggers vesicle fusion and transmitter release at brain synapses and at the neuromuscular junction. thus, cav2.1 ca 2+ channels play a crucial role in synaptic transmission. the global cav2.1 knock-out phenotype is characterized by severe ataxia, dystonia and lethality during the first postnatal weeks and is therefore an unsuitable model to analyze the importance of cav2.1 ca 2+ channels for learning and memory. therefore, we crossed a floxed cav2.1 mouse line with nex-cre transgenic mice to establish a viable, forebrain specific knock-out mouse line (fbko-mice). results from western blot analysis confirmed an efficient knock out of cav2.1 in hippocampal and cortical preparations, whereas the expression level in the cerebellum was not altered. to investigate the specific role of cav2.1 channels in hippocampus and neocortex dependent behavior, we performed tests for motor functions and sensory abilities and in particular learning and memory tasks. mice with a forebrain specific cav2.1 knock-out show significant deficits in spatial learning & reference memory and a significant reduced recognition memory as revealed by the morris water maze and an object recognition task. the fbko-mice exhibit no obvious locomotor deficits during behavioral tasks in the open field test and elevated plus maze. some fbko-mice demonstrate episodes of seizures in the morris water maze and during different rotarod tasks. to assess motor-function of fbko-mice in a stress reduced environment, we performed home cage based running-wheel motor-learning tasks. in summary, the diverse phenotypes of the forebrain specific knock-out mouse line emphasize the critical importance of cav2.1 for learning and memory. helicobacter hepaticus-infected rag2 -/mice emulate many aspects of human inflammatory bowel disease (ibd), including the development of colitis and colon cancer [erdman et al., 2009 , pnas 106: 1027 -1032 . toward the goal of elucidating mechanisms of inflammation-induced carcinogenesis and developing biomarkers of inflammation, we undertook a comprehensive analysis of macromolecular damage products during disease progression in h. hepaticus-infected rag2 -/mice. infected mice developed severe colitis and hepatitis, accompanied by infiltration of myeloperoxidase-positive neutrophils and f4/80-positive macrophages, by 10 wks postinfection (pi), progressing into colon carcinoma by 20 wks pi. qpcr array-based gene expression profiling revealed that pathophysiological changes were associated with characteristic alterations in the expression of genes related to inflammation, dna repair, and oxidative stress response. to study inflammation-related macromolecular damage, colon and liver tissues were analyzed by isotope-dilution chromatography-coupled mass spectrometry to quantify a battery of 16 different dna, rna and protein damage products thought to represent the full spectrum of inflammation-related chemistries. our data revealed a significant predominance of chlorinated dna-, rna-, and protein damage products by 20 weeks pi. in contrast, levels of damage products arising from oxidation, nitration and nitrosation changed only modestly or remained unchanged. our analyses also revealed higher levels of damage products in rna than in dna and demonstrated organ-specific differences of oxidative damage products, such as 8-oxo-dg and its oxidation products spiroiminodihydantoin and guanidinohydantoin. collectively, these results suggest that neutrophil and myeloperoxidase-induced chlorination chemistry may serve as a biomarker of ibd and may play important roles in the pathophysiology of ibd and colitis-associated cancer. characterization of a membrane protein expressed in mouse heart and brain mannebach s. 1 recently, a novel membrane protein in drosophila was shown to be localized in presynaptic vesicles. it appears to mediate a ca influx after vesicle fusion with the plasma membrane. disruption of the corresponding gene leads to endocytic defects in drosophila [1] . apparently, this protein plays a role in exo-and endocytosis and could serve as a ca channel supplying ca required for endocytosis. we have identified a protein in mouse, c90rf7, which shares 26,3% amino acid sequence identity with the drosophila protein. it covers 171 amino acid residues. using rt-pcr the full length transcripts could be identified in brain, kidney, pancreas, heart, spleen, thymus and mast cells. coexpression of c90rf7 and the ca v2.2 channel in xenopus oocytes reduced the amount of the α1b and cavβ3 subunits of the ca 2+ channel in the plasma membrane but did not affect the gating properties of the cav2.2 channel. expression of c90rf7 alone did not yield any channel activity. we therefore started to produce recombinant protein using the his-sumo-prokaryotic expression vector. the protein was efficiently expressed as his-sumo-c9orf7-fusion in e.coli (yield 2mg at 1mg/ml). we are currently preparing the c9orf7 part of the his-sumo-c9orf7fusion protein by ulp1-protease digestion followed by various chromatographic steps. the purified recombinant protein will be used to immunize rabbits to get antibodies. in parallel we generated antisera by immunizing rabbits with peptide fragments derived from the c9orf7 sequence. we could not identify any homologues of c9orf7 in the mouse genome and to analyze its function we are currently generating c9orf7 deficient mouse lines by gene targeting. we have chosen a strategy for conditionally inactivation of the gene with the option to study the cellular localization of c9orf7 by expression of the bgalactosidase gene under the control of the endogenous c9orf7 promoter. by southern blot analysis we´ve already identified 210 homologous recombinant embryonic stem cell clones out of 300 analyzed ones and we will proceed with blastocyst injection to get chimeric mice and finally mice carrying the introduced mutations in the c90rf gene. parps are involved in various biological processes such as regulation of dna repair, cell cycle progression, and cell death. consequently, several parp inhibitors are currently in clinical development as chemo-and radiosensitizers as well as monotherapeutic agents following the concept of synthetic lethality. pharmacological and toxicological studies call for an accurate analysis of parp activity in terms of a detailed knowledge of the structure of par and a reliable method for its quantification. we have developed a sensitive, precise, and accurate bioanalytical method based on liquid chromatography coupled to electrospray tandem mass spectrometry (lc/ms-ms) to characterize and quantify par with femtmol sensitivity: par is extracted from cells and hydrolysed to specific monomeric units, i.e., ribosyladenosine, which is characteristic for linear par, diribosyladenosine, which is characteristic for branching points, and adenosine, which represents the terminal part of the polymer. using this method, we are currently analyzing par levels in different cell lines and in primary human peripheral blood mononuclear cells (pbmcs) both under physiological conditions as well as upon genotoxic stress and in the presence of potent parp inhibitors. we expect that after completing method validation this assay will be useful for a wide range of applications in pharmacology and toxicology. gene mutagenic potential and metabolite profile of 17β-estradiol in cultured v79 cells expressing human cytochrome p450 1a1 martínez jaramillo d., lehmann l. university of wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany oxidative metabolism of the female sex hormone 17β-estradiol (e2) is considered to play a major role in the initiation of hormone-induced carcinogenesis. in extrahepatic tissues, e2 undergoes metabolic activation by cytochrome p450-dependent monooxygenase (cyp) isozyme 1a1 to 2-hydroxy-(2-ho) and to a lesser extent to 4-ho-e2. if not conjugated, these catecholestrogens (ce) can further oxidize to electrophilic quinones (q), which may react with dna and induce thereby mutations. conjugation of these ce in extrahepatic tissues is mainly catalyzed by catechol-omethyltransferase. in order to identify possible mutagenic metabolites (i) the induction of gene mutations by e2 was determined in male chinese hamster lung fibroblasts (v79 cells) expressing human (h) cyp1a1 and (ii) the metabolite profile of e2 in these cells was analyzed via gas chromatography/mass spectrometry after solid phase extraction of the cell suspension in the culture medium. (i) gene mutations were assessed using the hypoxanthine-guanine phosphoribosyltransferase assay. the promutagen benzo[a]pyrene (bap) served as positive control requiring metabolic activation by hcyp1a1 and dimethylsulfoxide as solvent control. v79 hcyp1a1 were treated with 100 nm e2 for 3 weeks and the resulting 6-thioguanine (6-tg) resistant mutants selected at weeks (w) 2 and 3. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells ranged from 5 ± 1 (w2) to 9 ± 4 (w3). as expected, 0.25 µm bap induced a significant increase in mutant frequency (mf, 266 ± 13, w2 and 132 ± 46, w3) . treatment with 100 nm e2 resulted in a 3-fold (17 ± 2, w2) and a 2-fold (23 ± 4, w3) increase in mf, suggesting slight mutagenic activity. in culture medium of v79 hcyp1a1 treated with 100 nm e2, 2-ho-e2, 2-methoxy-(meo)-e2, 3-o-methyl-2ho-e2 and 4-meo-e2 (suggesting intracellular formation of 4-ho-e2) were detected. while 4-meo-e2 concentration remained constant over the exposure period, the concentration of the other metabolites increased in a timedependent manner. the maximum concentration increase was reached at w3 for methylcatechols and at w2 for 2-ho-e2, correlating with the maximum increase in mf, observed after 2 weeks as well. in conclusion, e2 possessed a slight mutagenic potential after hcyp1a1-mediated activation to 2-, 4-ho-e2 and their corresponding methylcatechols. cumulative effects of three triazole fungicides in a broad dose range in vitro rieke s., kneuer c., bumke scheer m., lampen a., hirsch-ernst k., marx-stoelting p. bundesinstitut für risikobewertung chemikaliensicherheit, max-dohrn-str., 10589 berlin, germany consumers are exposed to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. the aim of this work was to investigate potential combination effects of the three triazole fungicides epoxiconazol, tebuconazol and flusilazol for selected parameters in a broad dose range in vitro. parameters investigated were cytotoxicity, hormone synthesis (17-β estradiol, progesterone and β-hcg), expression of a panel of androgen-or estrogen-responsive genes in the human placental choriocarcinoma cell-line jeg-3 and transactivation via estrogen receptors α and β in stably-transfected hek 293 cells. the ability to inhibit steroidogenesis was analysed by measuring the concentrations of 17β-estradiol and progesterone in cell culture supernatants of jeg-3 cells. additionally, the placental peptide hormone β-hcg was measured. while no change in β-hcg and 17β-estradiol concentrations were observed, all triazoles induced a dose-dependent decrease in progesterone concentration and a cumulative effect was observed implying dose additivity at individual doses of >1.7 µg triazole/ml. significant activation of erβ by the three triazoles, especially by flusilazol, was observed at 5 µg triazole/ml and combined exposure showed additive effects, while no significant activation of erα was observed. based on the data, our findings suggest dose-additivity of triazole pesticides with the same mode of action for selected parameters in vitro. no significant effects were observed at lower doses [1ng -1µg triazole/ml] neither for substances applied individually nor in combination. transient receptor potential channels as mediators of catecholamine release mathar i. 1 trp proteins form cation channels that are regulated through strikingly diverse mechanisms. recently, genetic association studies identified many trp genes including trpm4 as risk factors for disease states such as arrhythmias, hypertension and cardiomyopathy. the melastatin trp channels trpm4 and trpm5 have distinct properties within the trp channel family; they form non-selective cation channels activated by intracellular calcium ions and are expressed in heart, aortic endothelial cells, kidney and adrenal gland. disruption of the trpm4 gene in mice leads to increased basal blood pressure without evidence for impairment of endothelium-or smooth muscle-dependent regulation of contractility of peripheral resistance vessels, the renin angiotensin aldosterone system, basal cardiac output or body fluid homeostasis. instead, trpm4-deficient chromaffin cells exhibit increased acetylcholine-induced exocytosis of catecholamines which is associated with elevated level of epinephrine in the plasma and its metabolites in the urine. this indicates that trpm4 serves as an inhibitory regulator of exocytotic catecholamine release, at least in chromaffin cells. whether catecholamine release is also regulated by trpm4 in other cells of the sympathetic nervous system such as perivascular neurons still needs to be clarified as well as the molecular mechanism underlying how trpm4 regulates catecholamine release. besides trpm4 we recently identified transcripts encoding additional trp channels including trpc5 and trpc6 in chromaffin cells isolated by laser capture microdissection but their functional role in these cells is still unknown. measurements of the time course of the intracellular calcium concentration before and during acetylcholine stimulation (10µm) of catecholamine release as well as the analysis of the number of released vesicles in chromaffin cells relvealed no changes in trpc1/c5/c6 triple knock out mice compared to wildtype controls. although it seems that these trpc proteins are not directly involved in catecholamine release from chromaffin cells induced by acetylcholine application in our hitherto existing experiments, their contribution to the modulation of catecholamine release by agonists of gq-coupled receptors still needs to be analysed. aims: sulfonylureas (sus) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. in addition, sus have pleiotropic effects on other tissues. regarding the effects of sus on adipocytes conflicting findings were reported. we have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. methods: primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. lipid accumulation was assessed by oil red o staining and determination of triglyceride content; gene expression was measured by real-time pcr and western blotting. results: we initially characterized the genes regulated during human preadipocyte differentiation by a global microarray analysis. treatment with glimepiride and glibenclamide caused a strong accumulation of lipid droplets and an increase in triglyceride content. genes involved in lipid metabolism were induced, chemokine expression was decreased. interestingly, the effects of sus were over all qualitatively and quantitatively similar to pioglitazone. in direct comparison glibenclamide was more potent than glimepiride in respect to the induction of fabp4 (ec 50 0.32 vs. 2.8 µm), an important adipocyte marker gene. su-induced differentiation was virtually completely blocked by the pparγ-antagonist t0070907 but not affected by diazoxide, indicating pparγ activation by sus. repaglinide, causing insulin liberation like sus but being structurally different, had no effect on adipocytes. conclusions: in primary human pre-adipocytes, glibenclamide and glimepiride strongly induced differentiation, apparently by activating pparγ . thus, sus but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation. the role of at1a and at1b receptors as mechanosensors in myogenic vasoconstriction blodow s. 1 , schneider h. arterial myogenic tone denotes the intrinsic property of vascular smooth muscle cells to constrict in response to an elevated intraluminal blood pressure. this physiological reaction is more distinct in small resistance arteries than in large conduit arteries. understanding the underlying mechanisms should provide useful information for the treatment of diseases like anaphylactic shock and systemic hypertension in which this reaction is altered. whereas the underlying signaling cascade has been extensively studied, the molecular identity of the mechanosensory elements still remains elusive. recent studies at the cellular level suggest a sensory function for a subgroup of gprotein coupled receptors (gpcrs) coupling to gq/11-proteins. by determining mrnaexpression levels of selected gpcrs in consecutive pairs of resistance and conduit vessels, we could identify a subset of gq/11-coupled receptors such as angiotensin ii at1b, vasopressin v1a, endothelin eta and etb and α1a adrenoceptor significantly enriched in resistance vessels. by pharmacological blocking of those highly expressed gpcrs by different antagonists and inverse agonists, we evaluated their influence on the formation or the intensity of myogenic tone, as measured in isolated murine mesenteric arteries ex vivo. while blocking of v1a receptor and α2a and α2ab adrenoceptors showed no differences of myogenic tone, blocking of at1a and at1b receptors by losartan and candesartan, eta receptor by bq123 and α1a adrenoceptor by prazosin caused significant reductions of the vascular response. analyzing the myogenic response of at1a -/mice with and without additional blocking of at1b receptors by candesartan suggested that especially at1b receptors play a dominant role for mechanosensitivity in mice. this was further supported by investigating the myogenic response of at1b -/mice. these findings suggest that mechanosensitive gq/11-protein coupled receptors, especially at1b receptors, play a dominant role for the development of myogenic vasoconstriction. trpm3 ion channels are activated by steroidal compounds and noxious heat and are considered to be involved in insulin secretion and pain perception. the expression of the trpm3 gene generates a variety of different transcripts which arise by alternative splicing and the use of different promoters [1] . they encode a substantial variety of isoformes and so far we have identified more than 20 distinct trpm3 proteins in mouse and rat each varying in exons 1, 2, 8, 13, 15, 17 and 24 . these variants differ enormously in their biophysical properties. for example splicing within exon 24 affects the channel pore and causes significant changes of the ionic selectivity of trpm3 channels [2] , whereas splicing of 54 nucleotides encoded by exon 13 leads to dormant trpm3 proteins. however, the frequency of these different isoformes in trpm3 expressing tissues is completely unknown. to get insight into the significance of the different trpm3 isoformes we investigated the abundance of alternative trpm3 transcripts in different tissues and cell types by reverse transcription quantitative pcr (rt-qpcr). we found that the frequency of splicing within exon 13 ranges from 5 up to 20 % in different cell types and tissues. furthermore we analyzed the trpm3 transcriptome in the choroid plexus of the brain and the pituitary gland, tissues in which trpm3 transcripts are most abundant. for that purpose we sequenced more than 120 clones, each. corresponding to our rt-qpcr result, we found a significant number of transcripts lacking exon 13. in cells of the choroid plexus nearly all (124 /126 clones) carried the short ca 2+ permeable pore. furthermore, we identified seven variants spliced in exon 20 encoding truncated trpm3 proteins. however, the composition of the trpm3 transcriptome in the choroid plexus and pituitary gland differed enormously, indicating the importance of alternative splicing for trpm3 function in different tissues. the concept of "thresholds of toxicological concern" (ttc) defines tolerable dietary intakes for chemicals without toxicity data and is widely applied to chemicals present in food in low concentrations such as flavorings. based on a statistical evaluation of the results of many toxicity studies and considerations of chemical structures, the ttc concept derives a maximum daily oral intake without concern of 1800, 540 or 90 µg/person/day for non-genotoxic chemicals depending on the allocation to so-called cramer classes i, ii or iii. for substances with a structural alert for genotoxicity a ttc value of 0.15 µg/person/day might be used. recently, it has been investigated, whether the ttc values, which were derived based on mostly chronic oral dietary rodent studies would cover all relevant toxicities (neurotoxic, repeated dose, reproductive and developmental, immune effects and endocrine-related effects). several authors using different specific databases have confirmed that the ttc values derived using cramer classes are also covering immunotoxic, neurotoxic, reproductive and developmental effects. a respective decision tree is going to be presented, also considering substances or substance classes which shall be excluded from the ttc approach. there are several areas in which the ttc concept is already used, or a ttc approach is considered useful, to assess low-level human exposures, or help in prioritizing toxicological testing; as for example the assessment of plant metabolites and degradates of pesticide active substances, feed and food additives, chemicals with a low exposure profile under reach, residues, metabolites and impurities in plants, chemicals, plant protection products or pharmaceuticals. if no structural alert for genotoxicity is given or standard genotoxicity tests are negative the cramer class iii value of 90 µg/person/day, which corresponds to a dose of 1.5 µg/kg bw is considered to represent a chronic tolerable daily intake of the test substance. examples for current and future uses of the ttc concept in regulatory toxicology are presented. objective: hyaluronan (ha), synthesized by three ha-synthases (has1, -2, -3), is a prominent matrix component of atherosclerotic lesions. the aim of the present study was to identify the has isoenzyme that is associated with ha-matrix remodeling in inflammatory regions of atherosclerotic plaques. furthermore the underlying regulatory pathways were determined and functional aspects of this regulation in vascular smooth muscle cell (vsmc) were addressed. methods and results: during atherosclerosis in apoe deficient mice the peak of macrophage invasion at 14 weeks coincided with ha deposition and induction of has3 in aortic root plaques. in human symptomatic carotid artery plaques has3 was by far the most prominent has isoenzyme as determined by quantitative real time rtpcr. in vitro, in human vascular smooth muscle cell (vsmc) has3 was specifically induced via activation of nfkb by interleukin-1β (il-1β) and tumor necrosis factor alpha (tnfa) as shown by chip assay and utilization of nfkb inhibitor bay 11-7082. has3 was also upregulated in a co-culture system by activated macrophages via paracrine release of tnfa and il-1β as verified by neutralizing antibodies. in human atherosclerotic lesions nfkb positive vsmc were frequently detected in close proximity with ha and f4/80 positive macrophages as shown by immunohistochemistry. to study the effects of has3 mediated ha synthesis in human coronary vsmc, lentiviral overexpression and knockdown of human has3 were employed. overexpression of has3 resulted in increased migration and proliferation whereas knock down had the opposite effect. the effects of has3 were mediated by both pi3k signaling and mapk signaling via hyaluronan receptors cd44 and rhamm. conclusion: the present results suggest that has3-dependent ha synthesis is induced in human vsmc by inflammatory cytokines released from activated macrophages. moreover, has3-mediated ha production induced phenotypic activation of vsmc. pulmonary inflammation and airway remodeling are major features of chronic obstructive lung disease (copd). in addition, pulmonary hypertension is a common comorbidity, which is associated with a poor prognosis of the disease. recent studies in a guinea pig model of allergic asthma have shown that increased arginase activity, which converts larginine into l-ornithine and urea and competes with nitric oxide synthases for the common substrate, contributes to allergen-induced airway inflammation, hyperresponsiveness and remodeling. there is evidence that cigarette smoke and lipopolysaccharide (lps), both involved in the pathogenesis of copd, increase the expression of arginase, however, its role in the pathogenesis of copd is currently unknown. this study aimed to investigate the role of arginase in pulmonary inflammation and remodeling, using a guinea pig model of lps-induced copd. to this aim, guinea pigs were instilled intranasally with lps or saline twice weekly for 12 weeks and were pretreated by inhalation of the arginase inhibitor (2)s-amino-boronohexanoic acid (abh) or pbs. repeated lps exposure increased lung arginase activity, resulting in increased lornithine/l-arginine and l-ornithine/l-citrulline ratio's. both ratio's were reversed by abh treatment. repeated lps exposure also induced increased il-8 levels, neutrophils, goblet cells, hydroxyproline and airway collagen content in the lung, which were all abrogated by abh. moreover, repeated lps exposure increased right ventricular mass, indicative of pulmonary hypertension, which was similarly prevented by abh. in conclusion, increased arginase activity contributes to pulmonary inflammation, airway remodeling and right ventricular hypertrophy in a guinea pig model of copd, indicating that arginase inhibitors may have therapeutic potential in the treatment of this disease. (supported by msd). behavioral abnormalities in hcn3-deficient mice michalakis s., schöll-weidinger m., mader r., cao-ehlker x., fenske s., wahl-schott c., biel m. center for integrated protein science munich (cipsm) department of pharmacy -center for drug research, ludwig-maximilians-universität münchen, butenandtstr. 5-13, 81377 münchen, germany hcn3 encodes a hyperpolarization-activated and cyclic nucleotide-gated channel, which is expressed in various brain regions including thalamic, hypothalamic and habenular nuclei as well as brain stem and olfactory bulb. in this study we performed a comparative analysis of hcn3 -/and hcn3 +/+ mice using a battery of behavioral tests and telemetric biopotential measurements to evaluate a potential role of hcn3 in central nervous system function. in general, the knockout mice showed normal motor function as assessed by the rotarod and open field tests. telemetric home cage activity and core body temperature measurements confirmed a normal circadian behavior, but revealed a lower basal activity that concurred with decreased body temperature during the light phase and the light-dark transition phase. hippocampus-dependent spatial learning was normal. by contrast, hcn3 knockout mice showed more immobility than control mice on day two of the porsolt forced swimming test, which could reflect increased depressionlike behavior. however, center exploration in the open field test as well as performance in the light-dark transition and the elevated-plus maze tests was normal in hcn3 -/mice. this suggests that general anxiety was not changed in the knockout mice. in addition, hcn3 knockout mice were less active on the second day of the open field test, which supports a habituation phenotype. finally, hcn3 -/mice had higher burying scores in the marble-burying test, which is a test for certain aspects of obsessive compulsive disorder in rodents. taken together, genetic deletion of hcn3 in mice results in distinct behavioral abnormalities related to behavioral despair and expression of repetitive behaviors in response to mild stressors. mielke h. 1 , gundert-remy u. alcohol consumption when breast feeding is discussed controversially. some groups recommend breast pumping before alcohol consumption and feeding the stored milk instead of breast feeding after drinking alcohol. this study was performed to simulate the blood concentration in the breastfed baby and to assess the health impact. method: we established a physiologically based kinetic model. its parameters were calculated (partition coefficients tissue/blood ; schmitt, 2008) silva et al.,1993) . we simulated 1. the alcohol concentration in a breastfed neonate and a 3-month-old suckling infant after the nursing mother had consumed alcohol,2. the alcohol concentration in utero/fetal compartment during pregnancy assuming the identical alcohol consumption of the pregnant woman 3. the alcohol concentration during infant´s treatment of bloating by an approved herbal drug containing alcohol. results: peak maternal alcohol concentration was 0.59 ‰ after consuming 0.25 l of wine, peak concentration was 0.0033 ‰ in the newborn, 0.0038 ‰ in the 3-month-old infant and 0.38 ‰ in the utero/fetal compartment. the peak concentration after herbal drug treatment was 0.015‰ in the neonate and 0.015‰ in the 3-month-old infant, respectively. we discuss the results of the simulations and compare it with doses and published concentrations measured in experimental animals or in vitro studies. conclusions: we conclude that the recommendation "1 to 2 glasses of wine on occasion" (agence nationale d'accréditation et d'évaluation en santé, assante 2002) is in accordance with the simulation results presented here whereas stricter rules are not scientifically sound. (2002) http://www.has-sante.fr/portail/upload/docs/application/pdf/ breastfeeding_guidelines.pdf da silva et al. (1993) adenylyl cyclases (ac) mediate physiological responses in virtually all cells, where their regulation through receptors and g proteins results in the modulation of camp. in the present study we focused on the kinetics of interactions between the alpha-subunit from inhibitory g protein type 1 (gαi1) and adenylyl cyclase type v (ac5). these proteins were labeled with cfp and yfp, respectively. the dynamics of their interactions was monitored by means of high temporal resolution fret imaging in hek cells expressing unlabeled a2a-receptor and gβg subunits. to activate the signaling pathway, we applied agonist using a rapid superfusion device. application of norepinephrine resulted in the development of a fret signal, indicating interaction between gai1-cfp and yfp-ac5. after withdrawal of agonist the fret signal recovered with a remarkably slow time course compared to the deactivation kinetics of gi proteins reported previously (bünemann et al. 2003) . to further analyze the properties of the dissociation between gai1 and ac5 we measured in parallel the offset kinetics of the interaction between gai1-yfp and gβg-cfp (gi1-fret) after agonist withdrawal under comparable conditions. in addition we tested to what degree the coexpression of rgs4 accelerated the deactivation of gi proteins and the dissociation of gai1-cfp from yfp-ac5. these experiments revealed that in the absence of rgs4 the dissociation of gai1 from ac5 after agonist withdrawal takes about 3 times longer than the deactivation of gi proteins. in the presence of rgs4 this difference is even larger due to the pronounced acceleration of g protein deactivation. the dissociation of gai1 from ac5 was only marginally accelerated by rgs4. these observations lead us to hypothesize, that ac5 might trap activated g protein-subunits and thereby affect the g protein cycle by shifting the equilibrium towards activated g proteins. if this hypothesis is true, it should result in a left-shifted dose response curve compared to g protein activation dose response. in support of this hypothesis we found that the concentration response curve for gai1-ac5 interaction was several-fold leftward-shifted compared to the concentration-response curve of gi-protein activation under very similar conditions. influencing the dynamics of the g protein cycle by effectors may represent a novel and powerful mechanism for finetuning the sensitivity of receptor evoked responses in an effector-specific manner. obesity, the excessive accumulation of white adipose tissue (wat), has reached pandemic dimensions. the factors that determine fat mass are not fully understood, but adipocyte hypertrophy and adipokine secretion are thought to be important. in present study, we investigated the role of the cyclic gmp (cgmp) signaling pathway focusing on cgmp-dependent protein kinase i (pkgi) in white adipocytes. pkgi is expressed in wat, preadipocytes and differentiated adipocytes as demonstrated by real-time pcr, western blot and immunochemistry. differentiation of pkgifl/fl preadipocytes, using an optimized protocol, resulted in an enhanced lipid accumulation as evidenced by oil red o staining. deletion of pkgi in pkgifl/fl adipocytes infected with a cre lentivirus (lv-cre, pkgi0/0) exhibited reduced differentiation. analysis of the triglyceride (tg) content revealed a significant decrease of tg levels by 65% ± 1% in pkgi0/0 as compared to pkgifl/fl adipocytes. western blot analysis of white adipocytes showed a significant decrease of c/ebpalpha (19% ± 4.8%), ppargamma (66% ± 2.9%) and ap2 (37% ± 10.6%) expression in pkgi0/0 cells as compared to pkgifl/fl. treatment of 3t3-l1 cells with cgmp resulted in increased lipid accumulation and enhanced expression of fat marker genes. lentiviral overexpression of pkgi further increased differentiation. importantly, pkgi significantly induced mitochondrial biogenesis in 3t3-l1 cells. concomitant activation of pkgi in 3t3-l1 preadipocytes and treatment with the demethylating agent 5-aza-deoxycytidine significantly increased expression of uncoupling protein-1 (ucp-1) -a unique protein of brown fat cells. we found rhoa as major target of pkgi signaling with increased phosphorylation of rhoa at ser-188 in pkgi overexpressing cells. moreover, pkgi-dependent phosphorylation counteracts the effects of rhoa on insulin signaling as well as adipokine expression. taken together, pkgi is a key player in white adipocyte differentiation that regulates cell size and has an anti-inflammatory effect. pkgi decreases the secretion of proinflammatory adipokines via inhibition of rhoa signaling. in addition, activation of pkgi can establish a brown fat cell like phenotype during white adipocyte differentiation if the ucp-1 promoter is accessible. the rag gtpases, raga, ragb, ragc, and ragd form a subfamily gtpases of the ras-related superfamiliy. rag proteins are characterized by a modified ras-like gtpbinding domain and a unique c-terminal region lacking a lipid modification motif. interestingly, rag proteins have been proposed to function as heterodimeric complexes consisting of raga or ragb associated with ragc or ragd. rag gtpases have been implicated in the control of mammalian target of rapamycin (mtor) function, in particular in regulation of the nutrient-stimulated and/or hormone-regulated mtor activity. the protein kinase mtor is found as the catalytic subunit of two larger protein complexes referred to as mtor complex 1 and 2, mtorc1 and 2. under amino-acidrich conditions, activated mtorc1 promotes protein synthesis and inhibits autophagy, while under starvation autophagy inhibition is released. increasing evidence suggests that activation of rag gtpases contributes to mtorc1 function. thus, rag proteins were found to be associated with a protein complex termed ragulator, a major regulatory protein of mtorc1 function and guanine nucleotide exchange of rag gtpases within the rag-ragulator-complex were described to promote mtorc1 translocation to its functional lysosomal compartment. however, the guanine nucleotide exchange properties of rag proteins are poorly characterized, and it is currently unknown, how amino acids promote rag proteins to facilitate the formation of the active, raptor-binding state of the rag heterodimers. to characterize the guanine nucleotide exchange properties of the rag gtpases as momomers or heterodimers in more detail, recombinant rag proteins were expressed in bacteria and purified from this source to near homogeneity. first, the parameters of gdp/gtp exchange of each of these proteins were compared using the non-hydrolysable gtp analogon gtpgs. the results showed that the rag isoforms are distinct in their guanine nucleotide exchange activities. in particular, nucleotide exchange on raga and ragc, but not on ragb and ragd, was only observed at low concentrations of gdp and mgcl 2 in extraction and assay buffers, i.e. conditions favoring the gdp/gtp exchange. these findings may indicate that guanine nucleotide exchange on raga and ragc is controlled by guanine nucleotide exchange factors and suggest specific functions of the individual rag gtpases within individual rag heterodimers. in in-vitro studies on rat and canine mast cells and human mast cell leukemia cells hmc1.2 bz at micromolar concentrations inhibited mediator release which appeared to be related to an inhibition of the intracellular camp pathway. in order to identify potential targets on/in mast cells at which bz may cause an inhibitory effect on mast cell activation, the 1,4-bz flunitrazepam (flu), clonazepam (clo) and 4chlorodiazepam (4-cd) were selected because of their different affinity and selectivity to/for the gaba-a-receptor and the translocator protein (tspo): flu and clo bind with nanomolar affinity to gaba-a receptors, whereas 4-cd is a selective ligand at tspo with nanomolar affinity to tspo but only micromolar affinity to gaba-a receptors. flu also possesses nanomolar affinity to tspo, whereas clo has no or only micromolar affinity to tspo. after incubation of hmc1.2 cells with 4-cd, flu and clo for 1, 6 and 24 hours up to 712 genes were significantly differently expressed in a substance-specific and timedependent manner. comparison of the genes differently expressed at 6 hours revealed that the expression of 217 genes was regulated by both flu and clo but only 8 genes were regulated by both 4-cd and flu suggesting that flu and clo induce gene expression by acting at a target site different from that of 4-cd. the difference between the gene regulation by flu and clo on the one hand and that of 4-cd on the other hand is also reflected in pathway analysis. since it was conceivable that the beneficial effects of the 1,4-bz could be mediated by the recognition sites targeted by the 2,3-bz, i.e. the gria2-encoded ionotropic glutamate receptor ampa2, we investigated by quantitative pcr whether hmc1.2 cells express gria2, tspo, the genes encoding the subunits of the gaba-a receptor and the gaba-forming enzyme glutamic acid decarboxylase. tspo, gabra3, gabrb3, gabre and gabrd were moderately expressed. in addition, there was a week or very week expression of gabra2, gabra4, gabrb2, gabrg3 and gabrr2. expression of gria2 was not detectable. taken together, it cannot be decided yet from our data whether the inhibitory effect of benzodiazepines on mast cell activation is due to an action at tspo or at gaba-a receptors of a novel subunit composition. monien b. h., glatt h. german institute of human nutrition (dife) department of nutritional toxicology, arthur-scheunert-allee 114-116, 14558 nuthetal, germany 5-hydroxymethylfurfural (hmf) and furfuryl alcohol (ffa) are common constituents of foodstuffs in which they are formed by heat-and acid catalyzed reactions from carbohydrates. hmf and ffa have been reported to induce the formation of hepatocellular adenomas in female mice and renal tubule neoplasms in male mice, respectively. we studied whether the carcinogenic effect of these hydroxymethylsubstituted furans may originate from sulfotransferase (sult)-catalyzed formation of electrophilic esters. hmf was inactive in in vitro mutagenicity tests using standard activating systems. in contrast, it was mutagenic in v79 cells genetically engineered for expression of human sult1a1 suggesting that hmf is converted into the reactive 5sulfooxymethylfurfural (smf). following incubation of mutagenic smf with porcine liver dna in vitro, specific methylfurfural adducts were detected using liquid chromatography tandem mass spectrometry (lc-ms/ms), i.e., n 6 -((5-formylfuran-2-yl)methyl)-2'deoxyadenosine (n 6 -ffmda) and n 2 -((5-formylfuran-2-yl)methyl)-2'-deoxyguanosie (n 2 -ffmdg). these adducts were also detected in dna from v79-sult1a1 cells incubated with hmf. in order to determine sulfo conjugation of hmf in mice in vivo, we conducted pharmacokinetic measurements showing that about 500 ppm of the hmf dose was converted to smf and reached the circulation. like hmf, ffa was negative in the standard ames test and various other in vitro genotoxicity tests. we showed that ffa is mutagenic in salmonella typhimurium ta100 engineered for expression of human sult1a1. the putative mutagen 2-sulfooxymethylfuran was synthesized and incubated with porcine liver dna, in which various nucleoside adducts were found. the main adducts, -mfda were detected in dna of ffa-exposed salmonella strain ta100-sult1a1 and in dna of liver, lung and kidney of fvb/n mice that had received about 390 mg ffa/kg body weight per day via the drinking water for 28 days. in summary, both furan derivatives form mutagenic sulfate esters in vitro and in vivo. in the future, we will use genetically engineered mice to characterize the role of single murine and human sult forms in the bioactivation of the furan derivatives and the contribution to tumor induction. background: micrornas are small non-coding rnas that can negatively regulate gene expression on a post-transcriptional level and have been shown to interact with epigenetic mechanisms like dna methylation. mecp2 (methyl cpg binding protein 2) is a protein that binds methylated dna cpgs in the promoter region of genes and can thus regulate their expression. otherwise, mecp2 is known to be a target gene for several micrornas including the cluster mir-132/212 in the brain. recently, our group could show that mecp2 expression is downregulated in human heart failure suggesting that mecp2 might be involved in cardiac pathogenesis. the aim of this project is to study the upstream regulation of mecp2 by the cluster mir-132/212 in the heart during cardiac hypertrophy in-vitro and in-vivo. methods and results: to test whether hypertrophic stimuli can induce mir-132/212 expression, we treated cultured nrcms with 40 µm norepinephrine for 72 hours. this induced cardiomyocyte hypertrophy and expression of the hypertrophy marker nppa, but also of mir-132 (1.79 ± 0.14-fold of untreated cells, p<0.01) and of mir-212-3p (1.73 ± 0.26-fold of untreated cells, p<0.05) and downregulated mecp2 mrna and protein levels (0.80 ± 0.04-fold of untreated cells, p<0.05). to check whether mecp2 downregulation also occurs by direct mir-132/212 activation we increased levels of mir-132 and mir-212 in cardiac myocytes by transfecting precursor mir-132 and mir-212-3p molecules. again, we observed nrcm hypertrophy, nppa mrna upregulation and mecp2 mrna and protein downregulation (0.75 ± 0.05-fold of control, p<0.05) after mir-132 overexpression. similar results were obtained by overexpression of mir-212-3p. to test the effects of adrenoceptor activation on the mir-132/212-mecp2 axis in-vivo, wild-type mice received isoprenaline and phenylephrine via osmotic minipumps (30 mg/kg/day each). after 7 days, cardiac ventricles were analyzed. nppa gene expression (2.81 ± 0.32 -fold of control animals), mir-132 and mir-212-3p levels (3.67 ± 0.5 and 2.62 ± 0.4 -fold of control animals, p<0.01 and p<0.05, respectively) were increased while mecp2 protein levels decreased to 77% (p<0.05) conclusion: these results suggest that in-vitro and in-vivo adrenoceptor stimulation leads to the activation of mir-132/212 expression and to downregulation of mecp2 in cardiac myocytes in-vitro and in-vivo. leopold-franzens-universität, innsbruck, austria at-1 receptor antagonists block the angiotensin ii-enhancing effect on noradrenaline release from sympathetic neurons. in a cell-free assay the binding affinity of the at-1 receptor antagonists telmisartan and valsartan to the gamma peroxisome proliferatoractivated receptor (pparγ) is close to that of the pparγ selective agonists thiazolidinediones (tzds). we tested whether the tzds rosiglitazone and pioglitazone would also modify the prejunctional facilitatory effect of angiotensin ii. left ventricular slices of rats were incubated with tritiated noradrenaline, perifused and electrically stimulated. the negative logarithm of the drug concentration that caused a 30% increase of control (pec30%) was calculated. angiotensin ii caused a concentration-dependent increase of tritium overflow induced by electrical stimulation [pec30%=8.6±0.2 (mean±sem, n=18); maximum increase=110±8%]. neither rosiglitazone nor pioglitazone (0.3-3 µm) had a direct effect. the concentrationresponse to angiotensin ii in the presence of fixed concentrations of rosiglitazone was shifted to the left with increase of the maximum (pec30%=8.8±0. 2, 9. 2±0.2 and 9.3±0.3; maximum increase=118±14%, 146±13% and 148±16%, in the presence of 0.3, 1 and 3 µm of rosiglitazone, respectively, n=4-6, each). in contrast, pioglitazone in concentrations up to 3 µm had no effect on the release-enhancing effects of angiotensin ii. results show that rosiglitazone but not pioglitazone potentiates the noradrenalinerelease enhancing effect of angiotensin ii. this action might contribute to the risk for myocardial infarction from rosiglitazone use but not from pioglitazone use. deleted in liver cancer 1 (dlc1) is a tumor suppressor whose allele is lost in 50% of liver, breast, lung and 70% of colon cancers. despite its significance, the molecular mechanisms that drive cancerous transformation upon dlc1 loss remain unclear. we found that the transcriptional coactivators megakaryoblastic leukemia 1 and 2 (mkl1/2) are constitutively localized to the nucleus in hepatocellular and mammary carcinoma cells that lack dlc1. moreover, dlc1 loss and mkl1 nuclear localization correlated in primary human hepatocellular carcinoma. nuclear accumulation of mkl1 in dlc1-deficient cancer cells was accomplished by activation of the rhoa/actin signaling pathway and concomitant impairment of erk-mediated mkl1 phosphorylation. dlc1 loss led to constitutive activation of the mkl-dependent, tumor-relevant target genes ctgf, cyr61, myl9 and myh9. furthermore, we identified a novel target gene, integrin a5, with a key role in cell migration and metastasis, that exhibited a dlc1-and mkldependent regulation. depletion of mkl1/2 suppressed not only cell migration, but also cell proliferation and anchorage-independent cell growth induced by dlc1 loss. our data provide insight into the mechanism by which dlc1 loss initiates tumorigenesis. as mkl1 and 2 have a key role in this process, this pathway may provide promising pharmacological targets for cancer therapy. universität bonn, pharma-zentrum bonn pharmazeutisches institut, pharm. chemie i, an der immenburg, 53121 bonn, germany membrane receptors activated by purine and/or pyrimidine nucleotides ("p2 receptors") are widely distributed in the body and constitute novel (potential) drug targets. they are subdivided into g protein-coupled p2y receptors (p2y1, 2, 4, 6, 11, 12, 13, 14) , and homo-or heterotrimeric ligand-gated ion channel or p2x receptors (subunits: p2x1-7). we have been interested in the identification and development of potent and subtype-selective ligands -as tool compounds and potential drugs -for the various p2y and p2x receptor subtypes. our strategy involves (i) establishment of a proprietary compound library consisting of synthetic small molecules and natural products; (ii) development of screening assays suitable for medium throughput screening; (iii) careful analysis of structure-activity relationships at each target and systematic optimization of the lead structures; (iv) pharmacological evaluation of selected compounds. this approach has led to new biological tools for several targets, including p2y and p2x receptors [1] [2] [3] [4] [5] [6] . fine particles in particulate matter (pm) are effective vehicles to transport toxicants into the lung; depending on their size, smaller particles may reach the bronchiolar or alveolar space. in recent years the pm fraction pm2.5 has especially been correlated with both pulmonary and cardiovascular diseases. in order to better characterize pm emission and distribution of environmental tobacco smoke (ets) from cigarettes (reference cigarette (rc), brand cigarette (bc)) we have developed an ets emitter to simulate human smoking emission and measured pm2.5 concentration in a telephone booth (1,75 m3 volume) as an example for small indoor spaces like cars. fine particulate matter was measured using an aerosol spectrometer with 6 sec time resolution; laser scatter allowed a size resolution from 0,25 µm to 32 µm. for the pm2.5 concentration the following values were calculated: cumulative pm2.5 concentration as auc-pm2.5 (µg/m3/sec), peak pm2.5 concentration as cmax-pm2.5 (µg/m3) and average pm2.5 concentration cmean-pm (µg/m3). in closed door condition both cigarettes produced particulate auc-pm2.5 values of 59 000 ± 15 000 µg/m3/sec (rc) to 85 000 ± 31 000 µg/m3/sec (bc after myocardial infarction (mi) inflammatory cells and cardiac fibroblasts (cf) determine the remodeling response. interleukin-6 (il-6) is induced in the ischemic myocardium and is known to stimulate the differentiation of fibroblasts to myofibroblasts. hyaluronan (ha) is an extracellular matrix component synthesized by ha-synthase isoenzymes (has 1-3) and is also known to control fibroblast phenotypes. however, it is presently unknown whether il-6 participates in the remodeling of the ha-matrix or whether the ha-matrix modulates the responses to il-6. therefore, the aim of the present study was to elucidate whether il-6 regulates the expression and function of ha-matrix in cfs. cells were isolated from c57bl/6j mice and used during passage 2-3 for experiments. cfs were stimulated with il-6 or hyper-il-6 which is a fusion protein of il-6 and soluble il-6 receptor (sil-6r). after 10 and 30 min signal transducer and activator of transcription 3 (stat3) was phosphorylated in response to hyper-il-6 but not in response to il-6. rt-pcr revealed rapid upregulation of has 1 (5.94 ± 2.49 fold of unstimulated control, 1h) in response to hyper-il-6. has2 was induced to a lesser degree (2.04 ± 0.55 fold of unstimulated control, 1h) whereas has3 was not responsive (1.49 ± 0.42 fold of unstimulated control, 1h). in contrast, il-6 had no effect on transcript levels of has isoenzymes. in turn, expression of has1 and has2 in response to hyper-il-6 was inhibited by ag490, which indicates the involvement of stat signaling. interestingly, despite induction of has1 and has2 the amount of secreted ha as determined by an elisa-like assay was not affected by hyper-il-6. this may indicate that il-6 regulates the cell surface associated ha-matrix of cfs. in conclusion, the present data demonstrate that cardiac fibroblasts respond to il-6 trans-signaling (hyper-il-6) via the soluble il-6r and subsequent stat3 signaling with increased ha-synthesis. the fact that il-6 had no significant effect suggests that the expression of the non-signaling membrane-bound il-6 α-receptor (il-6r) in cultured murine cardiac fibroblasts is not sufficient to induce has 1 and -2 gene expression. therefore, il-6 trans-signaling mediated by il-6 and the circulating sil-6r might be necessary to mediate the il-6-induced has expression in vivo. mrgprd receptor endogenously expressed in dorsal root ganglia: evidence for an activation by 3-aminoisobutyric acid müller s., hoffmann k., von kügelgen i. universität bonn institut für pharmakologie und toxikologie, sigmund-freud-straße 25, 53127 bonn, germany the gpcr mrgprd (mrgd) is highly expressed in small diameter dorsal root ganglion (drg) neurons and has been implicated to play a role in nociception. the receptor was previously shown to respond to β-alanine. in the present study we searched for agonistic activity of structural analogues of β-alanine. for further characterization of the receptor we used fura-2 fluorimetry, a nfat luciferase reportergene assay and the determination of the inhibition of forskolin-induced camp production ([ 3 h]-camp affinity assay). first, we confirmed the activation of the receptor by β-alanine and gaba. in reportergene experiments we then identified 3-dlaminoisobutyric acid as an agonist, with similar potency but weaker affinity when compared to β-alanine (ec50 165 µm). fura-2 fluorimetry showed an increase in intracellular ca 2+ levels by 3-dl-aminoisobutyric acid (300 µm). moreover, 3-dlaminoisobutyric acid reduced the forskolin-induced camp production by up to 65 % (ec50 195 µm). in addition to 3-dl-aminoisobutyric acid, we identified 3-dl-aminobutyric acid as a weak agonist acting at the mrgprd. other closely related substances failed to show significant responses. next to the agonists we further characterized antagonists inhibiting the response to βalanine mediated by mrgprd. chlorpromazine shifted the concentration-response curve of β-alanine to the right with an apparent pkb of 4.9 (nfat assay), thioridazine with an apparent pkb of 5.4 (nfat assay) and 5.3 (camp assay) and rimcazole with an apparent pkb of 5.2 (nfat assay) and 5.2 (camp assay). in conclusion we show for the first time that 3-dl-aminoisobutyric acid is an agonist at the mrgprd and that the structure-activity relationship of agonists at mrgprd is very close. the sdf-1-chemokine receptor cxcr4 plays a key role during embryogenesis and regulates functions of immune and stem cells in adult life. furthermore, cxcr4 is involved in disease states including inflammation and cancer. it is well established that sdf-1-stimulated cxcr4 receptors activate gi protein-dependent signal transduction pathways and undergo c-terminal phosphorylation and internalization. because the cxcr4 c-terminal domain contains 15 serine and 3 threonine residues, it is incompletely understood which of the potential phosphorylation sites contribute to homologous and heterologous regulation of cxcr4. here, we analyzed the phosphorylation pattern of cxcr4 at 3 c-terminal sites after stimulation of the receptor with sdf-1 and after pma-induced activation of the pkc pathway as a model for heterologous receptor phosphorylation. using phospho-specific antibodies against s324/325, s338/339 and s346/347 in immunoblot analyses, we showed that the 3 sites were phosphorylated after stimulation with sdf-1 or pma. stimulation with egf or forskolin did not induce phosphorylation at these sites. sdf-1-induced phosphorylation at s324/325, s338/339 and s346/347 was reversible after wash out of the ligand. time course analyses revealed that phosphorylation occurred first at s346/347 and then at s324/325 and s338/339. taken together, these results indicate that the c-terminus of cxcr4 is phosphorylated at multiple sites by homologous and heterologous pathways and that phosphorylation at the different sites may be hierarchically organized. human milk represents the best form of nutrition for infants early in life. however, it can also contain toxic contaminants that may adversely affect infant's development. the nephrotoxin ochratoxin a (ota) is present in human milk (tab. 1 in [1] ), but information on transfer from maternal blood to milk is scarce: published data [2] indicate that levels of ota in milk are roughly one tenth (0.1) of those in blood. but, the efficiency of the ota-transfer at various stages of breastfeeding may vary since studies in animals revealed that transfer of ota is apparently time-and dose-dependent. thus, the aim of this study was to assess the ota transfer from blood to milk at different stages of breastfeeding in humans. in a small chilean cohort, 18 lactating women were asked to provide blood and milk on the same day. these samples were collected on four different occasions within the first months after delivery and analyzed using hplc with fluorescence and/or tandem mass spectrometric detection [1] . the transfer of ota from blood to milk was quantitatively assessed by measuring the milk to plasma ratio (m/p). the average ota level in blood plasma was 235 ± 129 ng/l, and no major variations were observed over time (p = 0.19). on the other hand, ota levels found in colostrum (85 ± 60 ng/l) were higher than in mature milk (p < 0.05). in line with these data, higher m/p ratios (table) were obtained with samples collected in the first six days after delivery. this study showed that the transfer of ota from blood to milk was more efficient with colostrum (m/p 0.43 ± 0.26) than with mature milk. thus, a higher exposure to ota can be expected for neonates than for infants at later stages of breastfeeding. moreover, the lactating women have lower average ota levels in plasma than non lactating women from chile [3] , indicative of milk as additional excretion route. acknowledgement: this work has been supported by a stipend from conicyt/daad to km. exposure of infants to ochratoxin a (ota) deserves particular attention since ota is nephrotoxic, and one of the most potent rodent renal carcinogen studied to date [1] . moreover, infants may be more vulnerable to the toxic effects of contaminants than adults. ota-levels in plasma of infants are indicative of an early exposure in life [2] . but blood sampling is an invasive method not readily applicable for breastfed infants. thus, the aim of this study was to implement a non invasive biomonitoring method to assess ota-exposure in this group. to assess the exposure to ota, breast milk and infants' urine specimens were collected, from two different cohorts: chile (n= 28) and turkey (n=10, only urine). analysis of the samples was performed using enzymatic hydrolysis prior to extraction and hplc-ms/ms [3] . the magnitude of infants' exposure was assessed by calculating the ota-daily intake with human milk and relating it also to urinary ota levels. calculations of the daily intake with human milk [4] showed that infants may be exposed to ota at high levels, exceeding the tolerable daily intake (tdi) of 5 ng/kg-bw/day set for adults [1] . in both cohorts, most of the urine samples tested positive for ota (chile 72%, turkey 80%). ota levels observed in urine samples from the turkish infants (range: 116 -1,361 ng/l) were 10 fold higher than levels found in chilean samples (range positive samples: 17 -320 ng/l). further analysis of phase ii metabolites in urine confirm the excretion of ota as conjugate (glucuronide) in highly exposed infants. in conclusion, ota exposure of infants early in life was documented. given that otaintake by several infants exceeded the tdi for adults, further biomonitoring in this vulnerable group is advised including also suitable biomarkers of effect. a mixture of (e)-and (z)-clomiphene citrate is the first line therapy of female infertility. however, up to 30% of patients do not respond. (e)-clomiphene is structurally closely related to another selective estrogen receptor modulator, tamoxifen which is frequently used for the treatment of hormone receptor-positive breast cancer. like tamoxifen, clomiphene is extensively metabolised by the cytochrome p450 system. using the estrogen receptor response assay (e)-4-hydroxyclomiphene and (e)-4-hydroxy-n-desethylclomiphene (ec50: 2.5 and 1.4 nm, respectively) turned out to be 100 times more active at the er compared to the parent drug isomers and de-ethylated metabolites. using recombinant expressed human cyp isoforms and inhibitory antibodies, cyp2d6 revealed to be the major isoenzyme involved in the formation of 4-hydroxlated metabolites. n-deethylation was catalysed by cyps 3a4/5, 2d6, 2c19 and 2c8. rates of 4-hydroxylation in microsomes from 30 human liver donors correlated with the number of functional cyp2d6 genes. these in vitro results were confirmed in a pharmacokinetic study with female healthy volunteers receiving a single dose of clomiphene. in carriers of two non-functional cyp2d6 genes (poor metabolizers) cmax of (e)-4-hydroxyclomiphene and 4-hydroxy-ndesethylclomiphene was 8 and 12 times lower, respectively, when compared with subjects with at least one fully functional cyp2d6 allele. in contrast, half-life of (e)-clomiphene and (e)-n-desethylclomiphene was 10 and 50-fold higher, respectively, in poor metabolizers. our data provide first evidence of a pharmacogenetic rational for the variability in the response to clomiphene treatment. among the tested compounds, compound 1 proved to be the most active derivative, showing a significant toxicity at a concentration of 2,5 µm. compounds 2 and 3 showed significant toxic effects at a concentration of 5 µm. the compound 4 showed no toxicity up to a concentration of 100 µm. all derivatives 1, 2 and 3 have a ec50 between 10 and 15 µm. we further proved the induction of apoptosis by apo-one assay (caspase 3/7 activity) and life/dead-assay (fluorescence microscopy). in conclusion, these gold complexes exhibit an example of interesting potential candidates for future anticancer pharmaceuticals due to relatively high cytotoxicity. gene regulating effects in mouse liver subsequent to treatment with selected dioxin-like compounds and pcb 153 using whole genome microarray analysis neser s. 1 , lohr c. 1 , van ede k. i. 2 , andresen k. 3 interaction with the aryl hydrocarbon receptor (ahr), with 2,3,7,8-tetrachlorodibenzo-pdioxin (tcdd) being the most potent congener amongst the ahr agonists. recent risk assessments have employed the toxic equivalency factor (tef) concept. the current eu-project systeq aims at developing, validating, and implementing human systemic tefs as indicators of toxicity for dl-compounds. at present, the best known parameter of ahr mediated effects is the induction of cytochrome p450 isoenzymes (cyps), i.e., cyp1a1, 1a2, and 1b1. one of the major objectives of the systeq project is the identification of novel quantifiable biomarkers. in a three day study, female c57bl/6 mice were treated with single doses of six dl-congeners (tcdd, pcb 118, pcb 126, and pcb 156) , and the 'non-dioxin-like' (ndl) pcb 153. quality tested (agilent ® bioanalyzer) mrna isolated from livers was analyzed using the agilent ® mouse whole genome array (4x44k) system. the quantity of genes affected (≥ 2fold) was highly heterogeneous amongst the dl-compounds. whereas tcdd-treatment upregulated 89 genes, and down-regulated 66, 4-pncdf-treatment had impact on 3208 (up), and 2396 (down). treatment with pcb 118 led to marginal numbers of 16 up and 6 down-regulated genes. with 64 (up), and 38 (down) genes shared, the most extensive overlap occurred between tcdd-and 1-pncdd-treatment. no overlap was found due to treatments with the ndl pcb 153 (21 up, 1 down) and tcdd. when comparing the effects of all dl-congeners, minor numbers of genes of 19 up, and 5 down-regulated remain, most of them being related to drug metabolism. while pcb 118 regulated only genes involved in drug metabolism, omission of pcb 118-regulated genes resulted in consistently 51 (up), and 24 (down) regulated among dl-compounds. in conclusion, our findings suggest that the pattern of gene regulation in mouse liver elicited by pcb 153 was strictly different from tcdd, while a very limited coincidence of genes was found even among dl-compounds. comparison of these 'core' genes with data from human models is required with respect to determination of novel biomarkers. introduction: proper use of antibiotics is essential with regard to effective treatment of bacterial infections. providing adequate information for patients can contribute to achieve this aim. materials and methods: data was collected from the relational database of the drug information service at dresden university of technology. the patients, who used the service, were interviewed concerning socio-demographic characteristics, reason for enquiry, number and kind of drugs taken, and diseases. possible contact paths were phone, e-mail or letter. in the present evaluation, all enquiries from the years 2009 and 2010 were analyzed descriptively focussing primarily on systemic antibiotics as reason for the enquiry. results: in the evaluated period, 4454 enquiries were registered in total. in 4.7% of those enquiries systemic antibiotics were named with a total number of 283 drugs. 50.9% of those antibiotics were found to be the direct reason for the enquiry. most common information requested by patients corresponded to adverse drug reactions (50.7%), diagnosis/treatment (35.4%), drug application (29.2%) and drug-druginteractions (19.4%). the majority of the requesting patients (61.5%) was born before 1970. a correlation between incidence of enquiries especially concerning antibiotics and quarterly statistics could not be detected. conclusion: mainly patients aged 40 years or more seem to need or search for further information about antibiotic medication. advice is required especially regarding adverse drug reactions and diagnosis or treatment. in order to this, the advisory service can help patients to lose their insecurity and to gain more confidence in handling antibiotic drugs. colon cancer is one of the most frequent cancers in the industrialized nations. epidemiological studies show a correlation between highly processed meat and the development of colorectal tumours. it is assumed that the risk of developing colorectal cancer, among various different factors, is related to the uptake of toxic substances contained in food such as heterocyclic aromatic amines that arise during the processing of fish and meat. phip is the most abundantly formed heterocyclic compound, and therefore has the biggest impact. in a previous study, we measured the absorption of phip in different intestinal segments of the rat. in the present study we focussed on the potential mechanisms by which phip is reabsorbed. the unidirectional phip transport from the mucosal to the serosal compartment (j ms) and in the opposite direction (jsm) was examined using the ussing chamber technique and 14 c-phip as a radiotracer. the proximal jejunum and distal colon of male fischer 344 rats in short-circuit current chambers was clamped, so that mucosal and serosal compartments were built. the phip flux rates were determined at defined intervals over 120 min. the experimental conditions were selected in such a way that negative net flux rates (jnet = jms-jsm) were indicative of an active secretion. both intestinal segments showed large differences. while in the jejunum jms and jsm of phip were not significantly different, there was an active secretion in the colon. in a next step the transport proteins involved in this process should be examined. introduction: human organic anion transporter 2, oat2 (slc22a7), is abundantly expressed in kidney and liver and mediates the sodium-independent uptake of clinically relevant drugs like 5-fluorouracil, paclitaxel, bumetanide, tetracycline, and zidovudine. while immunohistochemical studies have localized human oat2 to the basolateral membrane of kidney proximal tubules, its hepatic localization is currently unknown. we, therefore, firstly determined oat2 localization in human liver. because interindividual variability of oat2 expression may affect hepatobiliary drug uptake and elimination, we next systematically investigated the influence of genetic and non-genetic factors on hepatic oat2 expression. methods: an expression profile of oat2 for 20 human tissues was determined by realtime quantitative polymerase chain reaction (taqman). oat2 mrna expression was analyzed in well-characterized human liver samples from 150 caucasians that were accompanied by detailed demographic and clinical data. oat2 was localized in human liver cryosections using a commercial rabbit polyclonal antibody and hepatic oat2 protein levels were determined. resequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and genome-wide single nucleotide polymorphism microarray technology served to genotype variants in the slc22a7 gene region. results: oat2 mrna was expressed in several human tissues, including liver. moreover, a new alternatively spliced variant of oat2 was identified in human liver. hepatic expression of full-length oat2 mrna and oat2 protein varied 37-fold and 41fold, respectively. oat2 mrna and protein levels did not correlate with each other. oat2 was localized to the sinusoidal membrane of human hepatocytes. no novel variants in the 9 exons, the 5'-flanking region, or the 3'-untranslated region of the slc22a7 gene were identified. univariate analysis showed that oat2 mrna is reduced in patients diagnosed for cholestasis (p=0.0012) and is affected by genetic variants. whereas the influence of genetic variants on hepatic oat2 expression appears to be limited, cholestasis significantly contributes to the variable interindividual oat2 expression. this indicates consequences for hepatic drug elimination of and response to oat2 drug substrates such as paclitaxel or tetracycline. the life threatening toxicity of organophosphorus (op) nerve agents is caused by the inhibition of the acetylcholine esterase (ache). oximes were shown to be potent reactivators of inhibited ache, but in poisoning by some compounds, e.g. soman, they have only a small therapeutic effect. for such cases, an alternative new strategy may be the intervention at nicotinic acetylcholine receptors (nachr). previous studies with the bispyridinium non-oxime mb327 demonstrated therapeutic effects against soman in vitro and in vivo which was partly attributed to its direct interaction with nachrs [1] . we investigated the interaction of mb327 and several structure analogous at the orthosteric binding site of human α7 nachr (hα7 nachrs), a subtype which appears to be widespread in the human body, and compared the results with data obtained from torpedo-nachrs, which show a high degree of homology with human muscle-type nachrs. interaction of compounds with the orthosteric binding site of hα7 nachrs were investigated with radioligand binding experiments performed as high-throughput method [2] . membrane preparations of gh 4c1 cells stably expressed hα7 nachrs were incubated with the nachr agonist [³h] epibatidine and appropriate concentrations of the unlabelled competitors e.g. bispyridinium compounds. after incubation, bound and free [³h] epibatidine were separated by rapid vacuum filtration. ki values of the competing compounds were calculated with nonlinear regression. three bispyridinium compounds, mb442, mb456 and mb770 exhibited ki values at micromolar concentrations while three other compounds, mb327, mb583 and the pharmacological inactive mb424 (negative control) did not show any interaction with the orthosteric binding site of hα7 nachrs. with torpedo-nachrs, ki values were in similar orders of magnitude -except mb442 which indicated significant subtype selectivity. interestingly, the affinity of monomeric pyridinium derivates did not correlate with their bispyridinium structure analogues. obviously, no correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission exists, although species-related differences cannot be excluded. in this study, we analysed the cytotoxic and clastogenic effects of the anticancer drug nimustine (acnu) in cells deficient in repair proteins involved either in homologous recombination (hr) or non-homologous end-joining (nhej). we show that hr mutants are extremely sensitive to acnu as measured by the induction of apoptosis and colony formation as well as the induction of chromosomal aberrations. the nhej mutants were slightly sensitive to acnu and differed in their sensitivity, with the ku80 mutants being moderately sensitive and the dna-pkcs mutant resistant, comparable to the wild-type (wt). cell death was mostly executed via the caspase-dependent apoptotic pathway with involvement of caspase-3 and -7, and necrosis was also induced. further, we investigated the kinetics of dna double-strand break (dsb) formation that resulted from the repair of acnu-induced interstrand cross-links by means of γh2ax and 53bp1 foci analysis in wt and mutant cells. cells mutant in hr did not repair dsb and went into the apoptotic or necrotic pathway, whereas wt cells were able to repair most of the dsb. cells deficient in ku80 formed at early times after acnu treatment less γh2ax and 53bp1 foci compared to the corresponding wt, which might be due to a reduced capacity of recognising dsb. at later times after treatment, ku80 mutant cells show foci levels similar to the wt indicating restitution of h2ax phosphorylation. we also analysed whether dsb formation after acnu treatment was replication-dependent using synchronised cells. we determined the formation of γh2ax and other dsb marker in wt cells that passed through the first cell cycle after demecolcine synchronization. the level of γh2ax foci increased significantly in the s-phase and remained at a high level during g2 where a fraction of cells remained arrested. rad51, atm, mdc-1 and rpa-2 foci were also formed and shown to co-localize with γh2ax. these foci were ameliorated significantly in s-and g2-phase, which was similar to the time course of γh2ax foci formation. in western blots, we confirmed a higher phosphorylation level of atm and chk2 and less phosphorylation of chk1 in hr mutants. the data indicate that acnuinduced dna cross-links give rise to cyto-and genotoxicity via the formation of dsbs that activate the cellular dna damage response. the endocannabinoid system has been established as a mediator of numerous central and peripheral biological functions. cannabinoids have emerged as attractive alternatives or supplements to therapy with opioids for chronic pain states. however, in human the activation of cannabinoid receptors is associated with side effects. for clinical exploitation of the analgesics properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without attenuating their analgesics effect. in animal studies, the anti-nociceptive efficacy of cannabinoids has been unequivocally demonstrated in several models of inflammatory and neuropathic pain. however, there are marked inconsistencies between different reports with respect to the locus of these pain-protective effects. we are working towards establishing the contribution of cb1 receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia. using cb1 globally knock-out animal as background, we induce the expression of cb1 specifically in nociceptive neurons localized in the peripheral nervous system and test the analgesic effects of cannabinoid systemical delivery in these mice. our results support the development of peripherally acting cb1 analgesic agonist with reduced central side effects. furthermore, we are utilizing proteomics approach to identify protein complexes that interact with cb1 receptor which hold promise in understanding cannabinoid signaling in health and disease. most chemoattractants, including chemokines, complement c5a, fmlp, and leukotriene b4 are signaling through heterotrimeric g proteins of the pertussis toxin (ptx)-sensitive gi family. the functional inactivation of all gαi proteins with ptx leads to a fulminant decompensation of the immune system, whereas the constitutive inactivation of a single gαi coding gene results in mild phenotypes in mice. we are mostly interested in the nonneuronally found gαi2 and gαi3 isoforms and their redundant and specific roles in immune function and infection. for this purpose cellular in vivo and ex vivo models and in vivo infection model with listeria monocytogenes are being used. macrophages were isolated from the peritoneal cavity of wild type (wt) and gαi-deficient mice 4 days after i.p. injection of 4% thioglycolate that induces peritonitis in vivo. we confirmed previous observations that in gαi2-deficient mice the migration of macrophages into the peritoneal cavity was reduced after induction of peritonitis. regarding the expression levels of gαi and gβ isoforms in the lavage samples, the predominant gαi isoform gαi2 was upregulated in gαi3-deficient macrophages. vice versa gαi3 was upregulated in gαi2-deficient macrophages. concerning gβ isoforms, both gβ1 and gβ2 were strongly reduced in the gαi2-deficient macrophages which resulted in a reduced total amount of gβ. surprisingly, the gαi3-deficient macrophages showed reduced gβ1 protein levels only which caused a change in the gβ2/ gβ1 quotient in favour of gβ2. we are currently establishing an in vivo infection model with l. monocytogenes in gαi2and gαi3-deficient mice. our previous in vitro infection studies in mice embryonic fibroblasts provided us with information about possible distinct roles of these two isoforms as far as the uptake of l. monocytogenes in the cells is concerned. challenging the immune system of gαi-deficient mice with this pathogenic organism will give us new insights into the systemic immune response in these mice upon bacterial infection. our data indicate that we may surmount the redundancy between these two isoforms and focus on their distinct and specific roles in pathogen defense. fret-based β-arrestin2 biosensors reveal conformational changes upon binding to the β2-adrenergic receptor in real time and living cells nuber s., zabel u., ziegler n., hoffmann c., lohse m. j. institut für pharmakologie und toxikologie pharmakologie, versbacherstr.9, 97078 würzburg, germany β-arrestins are multifunctional adapter proteins that regulate seven transmembranespanning receptor (7tmr) signaling and initiate also alternative signaling pathways. studies have shown that β-arrestins undergo conformational changes upon receptor stimulation, which are thought to be necessary for its downstream actions. to investigate these conformational changes in living cells we constructed fret based biosensors of β-arrestin2, in which cfp was fused to the c-terminus and the flashbinding motif (ccpgcc) was inserted to different positions within the n-or c-domain of β-arrestin2. upon β2-adrenergic receptor (β2ar) stimulation we observed a decrease of the intramolecular fret signal between cfp and flash at the n-domain (β-arrestin2flash2), indicating a conformational change moving the c-terminus and the ndomain of β-arrestin2 relative to each other. kinetic analysis revealed that this conformational change immediately follows β-arrestin2/β2ar interaction on a timescale of seconds. a β2ar mutant that was previously shown not to interact with β-arrestin2 was utilized as control and did not induce a conformational change in the β-arrestin2 molecule. our data provide evidence that β-arrestin2 changes it`s conformation upon binding to the activated β2ar in living cells. the β-arrestin2flash2 sensor could serve as universal biosensor for gpcr activation. studies on the physiological role of annexin a4 in the heart nunes f. 1 the calcium binding protein annexin a4 has been examined in the context of heart failure in the past. annexin a4 expression level was found to be elevated in ventricles of human failing heart in comparison to expression levels in non-failing ventricles. furthermore the intracellular localization pattern in atrial cardiomyocytes was found to be altered in the failing human heart (moravec and matteo, cardiovasc res 2000). in order to gain insight into the possible physiological significance of these findings we utilized an annexin a4 gene trap model (gt) in which the annexin a4 protein content was not detectable in ventricles and atria. measurements of sarcomere shortening and calcium transient kinetics in isolated ventricular cardiomyocytes revealed a prolonged calcium transient decay at stimulation frequencies of 0.5 hz, 1hz and 2 hz as well as an increased sarcomere shortening at 1 hz and 2 hz in anxa4 gene trap animals in comparison to wild type (wt) ( the effects of the β-adrenoreceptor agonist isoprenaline (iso) on the shortening of ventricular cardiomyocytes was increased in gt as compared to wt (0,2±0.013 vs. 0.17±0.012, *=p<0.05 vs. wt; n=14-18/5). western blot analyses indicated that the expression of the sarcoplasmic reticulum (sr) ca 2+ -atpase (serca2a) and the phosphorylation status of its regulator protein phospholamban (plb) did not differ between groups (n=7). however, co-immunoprecipitation experiments suggest, that anxa4 is able to interact with hax1, which acts as a repressor of serca2a (n=4). we performed force measurements in isolated and electrically stimulated left atria in response to rising isoprenaline concentrations (10 -9 m-10 -5 m). the positiv inotropic effect of isoprenaline was significantly increased in gt atria (rel. force at 10 -4 m iso [%]: wt: 433±76; gt: 699±63 *= p<0.05 vs wt; n=8-10). in conclusion, annexin a4 contributes to the regulation of cardiomyocyte contractility. the anxa4 up-regulation might therefore contribute to diminished cardiac performance in heart failure. matteo rg, moravec cs. immunolocalization of annexins iv, v and vi in thefailing and non-failing human heart. cardiovasc res. 2000 mar;45 (4) background: pregnane x receptor (pxr) is considered the most important sensor of natural and anthropogenic xenobiotics in vertebrates. in contrast, the amphibian ortholog is involved in neural development and irresponsive to xenobiotics. instead, the xenopus laevis constitutive androstane receptor (car) was recently found to possess pxr-like properties, featuring low basal activity and a pronounced ligand spectrum. thus a structural and functional characterisation of x. laevis car may provide further insights into human car basal and ligand-induced activity. methods: the time-point of origin of car genes was determined by macrosynteny analyses of car, pxr, and vdr (vitamin d receptor) gene loci, which form the nr1i subfamily of nuclear receptors. based on a 3-dimensional protein model of xenopus laevis car, docking studies with structurally diverse agonists were conducted. proteinligand-interactions as well as sequence comparisons were performed in order to select amino acids to be mutated towards human car. the organ response to car activators was determined in xenopus laevis using rna microarrays. results: car emerged together with pxr and vdr from an ancestral nr1i gene in early vertebrates via two whole-genome duplications. this was followed by losses of car from the fish lineage and of pxr from sauropsida (reptiles and birds). amino acids important for ligand binding were identified. structural features responsible for the pronounced basal activity in human constitutive androstane receptor are not present in x. laevis car. in human pxr the inter-helical loop in front of helix 3 is part of the ligandbinding pocket and supposed to be responsible for the wide substrate spectrum. in amphibian car this inter-helical loop plays no role in ligand binding. car agonists resulted in a pronounced induction of antimicrobial peptides in the ovary. conclusions: car emerged already in early vertebrates and it is conserved in land vertebrates, whereas xenosensing pxr is found only in the fishes and mammals. we provide a comprehensive modeling and mutational analysis of this first reported amphibian xenosensor. the induction of antimicrobial peptides by car activators suggests a link between xenosensing and innate immunity. the latter one may play a previously unrecognized role in the amphibian reproduction. background: retigabine belongs to a novel class of potent anticonvulsant drugs and is currently being investigated in clinical routine. the therapeutic range of retigabine serum concentration is unknown. a therapeutic drug monitoring (tdm) is used for most other anticonvulsant drugs. the aim of this study was to develop a method for the determination of retigabine in serum of patients and to compare the effect and the side effects of retigabine with the blood levels of the drug. method: a hplc method with tandem mass spectrometric detection for the sensitive determination of retigabine was developed. solid-phase extraction (spe) of 250 µl serum with oasis hlb cartridges allowed a reliable quantification down to 5 ng/ml. in order to develop an assay with high sample throughput and to obtain maximum response for the analytes we required the shortest possible retention time. to implement the determination of retigabine in a second step in the routine tdm of anticonvulsant drugs the corresponding hplc method was selected: a purospher rp18 column (55 mm x 2 mm; 4 µm, merck) and a mobile phase with a steep acetonitrile gradient. results: the great advantage of having analytes with different molecular masses and similar retention times in combination with ms/ms detection enabled us to aim at a minimum separation that might remove some salts or matrix components that can suppress or interfere with the analyses from the target components, while maintaining good sample throughput. the method was validated. the assay is precise, accurate, fast, sensitive, and selective. discussion: the developed method is suitable for therapeutic drug monitoring of retigabine. the correlation of the serum concentration and the effect of the drug and thus the necessity of tdm have to be tested. targeting inflammatory t lymphocytes with conditional chemokine receptor antagonist expression for a tissue-specific therapy of chronic inflammatory disorders ogrissek n., giegold o., pfeilschifter j., radeke h. h. uniklinikum der goethe-universität pharmazentrum / zafes, theodor-stern-kai 7, 60590 frankfurt am main, germany chemokines and their receptors are known to be involved in the pathogenesis of chronic inflammation and autoimmune diseases. several approaches tried to use chemokine receptor antagonists as therapeutics to reduce exagerrated immune response, however, due to compensation and systemic side effects clinical trials often failed. in previous experiments our group identified three promising antagonists. cxcl11(4-79) has antagonistic function for cxcr3, cxcl12(p2g2) is able to inhibit cxcr4 and the herpesvirus 8 encoded protein vmip-ii interferes with ccr1, -2 and -5 as well as with cxcr3, -4 and cx3cr1. their expression and secretion was confirmed in pichia pastoris and antagonistic function has been proven by a reduction of t cell migration. the aim of this project is to develop a cell-based therapy for chronic inflammation with a treatment that is based on the collective effect of cxcl11(4-79), cxcl12(p2g2) and vmip-ii. with targeting of stable transduced memory t cells these antagonists should be conditional expressed and secreted directly in the centre of inflammation, resulting in inhibition of further inflammatory t cell accumulation. to realize this project we first cloned constitutive lentiviral constructs containing these antagonists and optimized transduction of t cells, such as the ova-specific memory th-1 cell clone if12 with the potential to initiate antigen specific nephritis in scid mice. next we investigated expression and secretion of cxcl11(4-79), cxcl12(p2g2) and vmip-ii with pcr, western blot and elisa. at the moment we want to measure the inhibition efficiency of t cell migration in vitro with chemotaxis and flow chamber assays. construction of an inducible lentiviral vector plasmid to ensure expression of the antagonists only upon t cell activation, is also part of our current work. finally we would like to test the chemokine receptor antagonists in vivo in two relevant mouse models of type-1-diabetes and contact dermatitis. small heterodimer partner 1 (shp-1) is a member of the superfamily of nuclear receptors (nrs). in contrast to other nrs this orphan receptor lacks the dna binding domain. however, shp-1 is known to inhibit activity of several nrs by direct proteinprotein interaction. importantly several of the interacting nrs have been shown to directly regulate shp-1 expression, suggesting that shp-1 is involved in negative feedback loops of various metabolic pathways, such as cholesterol-, bile acid-and drug metabolism and glucose homeostasis. recently binding sites for nrs were identified in the promoter region of shp-1, including hnf4α, lrh1, lxr, fxr, srebp1c and pparγ. the aim of our study was to identify single nucleotide polymorphisms (snps) in the promoter region of shp-1 and to determine their impact on the transactivation of shp-1. 120 dna samples from subjects of the population based cohort study of health in pomerania were analyzed by sanger sequencing, thereby we identified four snps namely -594t>c (rs71636795), -413g>c, -423 c>t (rs78182695) and del-195ctga (rs145613139). subsequently those polymorphisms were tested for their functional consequence performing cell based reporter gene assays testing all above mentioned modulators (lrh1, lxr, fxr, srebp1c and pparγ) of shp-1 expression. only the transactivation by hnf4α was decreased in the presence of the -423 c>t polymorphism to 69% and the -413g>c polymorphism to 75%. in conclusion we described snps with impact on transactivation. it will be aim of future studies to determine the potential impact on physiological processes or disease development. autosomal recessive polycystic kidney disease (arpkd) is a rare genetic disease, afflicting about 1 in 20.000 individuals. arpkd is characterized by cystic fusiform dilatations of the renal collecting ducts leading to massive enlargement of the kidneys and ultimately loss of renal function. in addition, the patients suffer from congenital hepatic fibrosis (chf), possibly leading to portal hypertension and liver enlargement. so far, there is no cure for arpkd. therapy is focussing on controlling the disease symptoms [1] . mutations in the pkhd1 gene cause arpkd. more than 300 different mutations in this gene have been reported, all leading to the same phenotype, though there are differences regarding the severity of the disease [2] . in animal models of autosomal dominant polycystic kidney disease (adpkd) as well as arpkd elevated levels of camp were shown [1] [2] [3] . in isolated kidney cells camp stimulates cl-secretion and activates the b-raf /mek/erk pathway. these both are important factors for cyst development and disease progression [2, 3] . intracellular camp regulation is based on conversion of atp to camp by adenylyl cyclases (acs) and degradation by phosphodiesterases . referring to this, we asked the question if there are differences in the activation and expression pattern of acs in pck rats, an animal model of arpkd [4] and in sprague dawley rats. therefore, we examined membranes in a radioactive ac activity assay using various stimulatory compounds, e.g. forskolin, a direct ac activator, or hormones like glucagon and vasopressin to characterize acs. furthermore, we examined ac isoform expression on the mrna level via rt-pcr. we observed that in pck rats ac activity was decreased in general in comparison to sprague dawley rats. in future experiments we are aiming to obtain further knowledge about the influence various hormones exhibit on pck rat acs and to biochemically characterize acs. the major pathogenicity factors tcda and tcdb from clostridium difficile monoglucosylate and thereby inactivate small gtp-binding proteins of the rho subfamily after entering host cells via receptor-mediated endocytosis. although the intracellular mode of action of the toxins is well understood, far less is known about binding structure and internalization pathway of tcda and tcdb. since antibodies directed against the c-terminal located clostridial repetitive oligopeptides (crops) are able to neutralize toxin cytotoxicity the crop domain is acknowledged to mediate receptor binding. however, we recently demonstrated that crop deletion mutants of tcda (tcda 1-1874 ) and tcdb (tcdb 1-1852 ) enter host cells and exhibit full cytopathic potency though lacking the proposed receptor binding domain. we therefore refute the accepted opinion of a solely crop-mediated toxin uptake and re-evaluate the role of the crops in toxin endocytosis. tcda 1-1874 and tcdb 1-1852 induced time and concentration dependent cell rounding and rac1-glucosylation. however, depending on the cell line, truncated toxins exhibit up to 10-fold reduced potency towards host cells compared to the respective full length toxin. the observed difference in toxin potency might reflect the recognition of different receptor structures or the use of various endocytotic routes. interestingly, pre-incubation of cells with the isolated crop domain enhances binding as well as cytotoxicity of subsequent applied truncated tcda indicating that the crops primarily determine toxin uptake. in fact, competition experiments revealed that tcda and tcda 1-1874 predominantly use different receptor structures corroborating the notion of alternative internalization processes utilized by tcda. different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of c. difficile. thus, characterization of alternative endocytotic pathways used by the c. difficile toxins might therefore be the basis to investigate the opportunity of toxin uptake inhibition as therapeutic option. in neurodegenerative diseases, such as alzheimer´s disease and parkinson´s disease, mitochondrial pathways of apoptosis are considered as major features of the underlying neuronal cell death. such mitochondrial mechanisms of apoptosis are mediated by the bh3-only protein bid, a member of the bcl-2 family that triggers mitochondrial permeabilization and the subsequent release of death-promoting proteins into the cytosol. the pivotal role of bid in apoptotic cascades of neuronal cells has been shown in our previous studies showing a neuroprotective effect of bid sirna and small molecule bid inhibitors such as bi6c9 in vitro. in vivo, however, the available bidinhibitors failed to protect brain tissue likely because the compounds were not bioavailable or did not cross the blood brain barrier. therefore, chemical modifications of bi-6c9 were generated resulting in new structures and molecules with different pharmacophors. the aim of the present study is to identify novel potent bid inhibitors available for applications in model systems of brain damage in vivo. for the first screening of 80 compounds we used a model of glutamate toxicity in immortalized mouse hippocampal neurons (ht-22 cells). in this model system, glutamate induces a decrease of intracellular glutathione levels resulting in lipoxygenase activity and enhanced formation of toxic reactive oxygen species (ros). to investigate the compounds' ability to prevent glutamate induced cell death, we first analyzed the cell viability by the mtt assay. in addition, we examined the cell survival by using real time monitoring of cell impedance (xcelligence system) to determine the neuroprotective potency of the new structures. using these assays, we identified 10 novel molecules that significantly prevented glutamate-induced toxicity in ht-22 cells. further we were able to express and to purify recombinant bid in a high amount. in the ongoing study the purified bid protein will be used for co-crystallization with the identified neuroprotective structures for further optimization of novel bid inhibitors for therapeutic applications in experimental models of neurodegenerative diseases in vivo. polymorphic enzymes, urinary bladder cancer risk and structural change in the local industry ovsiannikov d. 1 , selinski s. in the 1990s, an uncommonly high percentage of glutathione s-transferase m1 (gstm1) negative bladder cancer cases (70 %) was reported in the greater dortmund area (golka et al., 1997) . the question arose whether this uncommonly high percentage of gstm1 negative bladder cancer cases was due to environmental and occupational exposure decades ago. thus, 15 years later, another study on bladder cancer was performed in the same area after the coal, iron and steel industries had finally closed in the 1990s. in total 196 bladder cancer patients from the st.-josefs-hospital dortmund-hörde and 235 controls with benign urological diseases were investigated by a questionnaire and genotyped for gstm1, gstt1 and the n-acetyltransferase 2 (nat2) tag snp rs1495741. the frequency of the gstm1 negative genotype was 52 % in bladder cancer cases and thus much lower, compared to a previous study performed from 1992-95 in the same area (70%). nat2 genotypes were distributed equally among cases and controls (63% slow acetylators). less gstt1 negative genotypes were present in cases (17%; controls 20%). apoptosis inducing factor (aif) has been identified as a key factor in intrinsic pathways of caspase-independent neuronal death in model systems of acute brain injury and neurodegenerative diseases, such as alzheimer's disease and parkinson's disease. aif is a mitochondrial intermembrane flavoprotein with the capacity to translocate to the nucleus where it induces chromatin condensation and large-scale dna fragmentation. previous studies revealed that aif deficiency leads to protective effects in different models of neuronal death in vitro and in vivo. however, aif also plays an important physiological role for the integrity and function of the mitochondrial respiratory chain. thus, aif deficiency may significantly alter mitochondrial functions and metabolic homeostasis thereby preconditioning the cells to tolerate subsequent stress stimuli. the present study addresses this hypothesis and investigates whether neuroprotection by aif depletion was attributed to a preconditioning effect, i.e. protecting mitochondrial function and integrity. as model system we use glutamate induced oxytosis in immortalized mouse hippocampal ht-22 neurons. silencing of aif expression by sirna (20nm) protected mitochondrial morphology and integrity against glutamate induced damage. microscopy analysis of the mitochondrial morphology revealed that aif sirna prevented mitochondrial fission. furthermore, facs analysis confirmed that mitochondrial membrane potential was stable in cells with aif silencing. this protection of mitochondrial morphology and integrity by aif depletion was associated with preserved atp levels and inhibition of cell death as detected by an mtt assay. pronounced formation of lipidperoxides as another indicator of mitochondrial damage was also attenuated in cells preconditioned by aif sirna. these protective effects of aif sirna were highly similar to effects obtained with low doses of rotenone (20nm), which was applied as an inhibitor of complex i and mediated comparable preconditioning effects in the ht-22 cells. overall, these findings support the conclusion that aif depletion mediates a preconditioning effect protecting neuronal cells from a subsequent glutamate toxicity. in order to link these preconditioning effects to complex i functions, protein expression and functional analysis of complex i are being analysed to identify the molecular mechanisms of aif dependent control of neuronal life and death. -dependent inactivation and display very slow voltage-dependent inactivation. both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained ca 2+ influx through cav1.4 channels is required to couple slowly graded changes of the membrane potential with tonic glutamate release. mutations in the gene coding for cav1.4 cause severe impairment of retinal circuitry function and have been linked to congenital stationary night blindness type 2a (csnb2), aland island eye disease (aied) and cone-rod dystrophy type 3 (cordx3). the clinical phenotypes of these eye diseases vary substantially regarding the ratio of rod to cone functional impairment. the reasons for this variability are not known. to gain more insights into the pathophysiology caused by loss of cav1.4 function we analyzed the visual phenotype of cav1.4-deficient mice. to this end, we combined immunohistochemistry, electroretinography (erg) and vision-dependent behavioral testing. immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in cav1.4-deficient mice. heterozygous cav1.4 mice showed mosaic synaptic defects most probably caused by random x-chromosomal inactivation of the healthy allele. electroretinography revealed a loss of scotopic and photopic photoreceptor function. this loss of retinal network function resulted in impaired performance of cav1.4 knockout mice in a water maze-based behavioral test of rod and cone function. in conclusion, loss of cav1.4 channels strongly impairs rod and cone retinal function and vision in mice. lsr is the host cell receptor for clostridial iota-like toxins papatheodorou p., aktories k. albert-ludwigs-universität freiburg institut für experimentelle und klinische pharmakologie und toxikologie, albertstr. 25, 79104 freiburg, germany the human enteric pathogen clostridium difficile is the most serious cause of antibioticassociated diarrhea and pseudomembranous colitis. hypervirulent strains of the pathogen, associated with more severe disease and increased death rates, produce the binary actin-adp-ribosylating toxin cdt (c. difficile transferase) in addition to the rho glucosylating toxins a and b. cdt is member of the family of clostridial iota-like toxins, including c. spiroforme toxin (cst) and the eponym c. perfringens iota toxin. the toxins induce depolymerization of the actin cytoskeleton and the formation of microtubulebased cell protrusions that increase adherence and colonization of clostridia. using a haploid genetic screen, we identify the lipolysis-stimulated lipoprotein receptor (lsr) as the target molecule for entry of cdt into host cells. in addition, we present evidence that lsr is shared as a cell entry point by all members of the iota-like toxin family. identification of the toxin receptor provides a most valuable basis for antitoxin strategies. bisphenol a (bpa) is a chemical of high interest due to its endocrine activity. controversy exists concerning the blood concentration due to normal exposures. some authors claimed to have measured concentrations in the ng/ml range which is in contrast to kinetic properties of bpa. bpa is excreted in the urine as glucuronide and sulfate metabolites. recently, data on the in vitro metabolism of bpa by recombinant udpglucuronyltransferase 2b15 enzymes (ugt2b15) revealed that ugt2b15.2 and ugt2b15.5 had markedly lower intrinsic clearance as compared to ugt2b15.1 (hanioka et al., 2011) . using the in vitro metabolism data, we scaled the kmand vmaxvalues in an established human physiologically based toxicokinetic (pbtk) model (mielke and gundert-remy, 2009, mielke et al., 2011) to the values of the variants. for oral doses at relevant exposure levels, the maximum blood concentration (cmax) for the ugt2b15.2 variant (v2) was 5 fold and those of the ugt2b15.5 variant (v5) was 7 fold higher than that of the ugt15.1 variant. with dermal exposure at a relevant exposure level, the cmax values were 1.4 (v2) and 1.6 fold (v5) of ugt15.1 variant. a combined exposure of oral and dermal exposure, an exposure scenario, which occurs in daily life, resulted in 2.4 fold (v2) and 3.2 fold (v5) higher cmax values as compared to ugt15.1 variant. the values for the area under the blood concentration time curve (auc) were for a relevant oral dose 5.7 fold (v2) and 8.6 fold (v5), for relevant dermal exposure 1.4 fold (v2) and 1.6 fold (v5), and for combined exposure 1.9 fold (v2) and 2.5 fold (v5) of ugt15.1 variant. from the results we conclude: (1) polymorphism of udpglucuronyltransferase (2b15.2 and 2b15.5) has an impact on the blood concentrations which, however, is less than 10 fold for cmax and for auc. the effect is more pronounced for oral as compared to dermal or combined exposure. (2) polymorphism of metabolism does not explain the blood/plasma concentrations in the ng/ml range measured by some authors. hanioka n, oka h, nagaoka k, ikushiro s, narimatsu s. effect of udpglucuronosyltransferase 2b15 polymorphism on bisphenol a glucuronidation. arch toxicol. 2011, 85(11) :1373-81. mielke h, gundert-remy u. bisphenol a levels in blood depend on age and exposure. toxicol lett. 2009 8; 190(1) :32-40. mielke h, partosch f, gundertthe heart responds to maladaptive pro-hypertrophic stimuli by stimulating intrinsic signals that contrast and dampen the onset and development of hypertrophy. cyclic guanosine monophosphate (cgmp) and its downstream effector cgmp kinase i (cgki) have been suggested to be an important anti-hypertrophic signaling pathway (1) . intracellular levels of cgmp can be raised by the action of nitric oxide (no) and natriuretic peptides (anf, bnf), or by inhibiting cgmp-degrading phosphodiesterases (pde). a growing body of evidence suggests that the pde5 specific inhibitor sildenafil (sil) prevents and reverses hypertrophy and chamber remodelling in the heart of mice subjected to thoracic aorta constriction (tac) by elevating cgmp levels and cgki activation (1) . in contrast, using a mouse model that lacks cgki expression in every cell type except smooth muscle cells (βres mice; see ref. 2), we recently showed that the absence of this kinase does not alter the onset of hypertrophy induced by tac or isoproterenol infusion (2) . sil is believed to increase cardiac cgmp levels, although it is unclear, if its target (pde5) is expressed in cm (2). sil may act on other pdes, such as pde1c which is abundant in cms. it is also unclear if sil effects are mediated by other cardiac cell types, in particular by cardiofibroblast. to answer these questions, we are currently investigating whether sil is able to prevent hormone induced cardiac hypertrophy in the absence of cgki in cm. preliminary results on βres mice show that even in the case of chronic angii infusion, lack of cgki in cm does not alter the induction of hypertrophic response, at least in the initial phase (7days of angii infusion at 2mg/kg/day). interestingly, βres mice showed impaired cardiac function, as indicated by decreased fractional shortening. sil was able to partially block the onset of cardiac hypertrophy in wt animals, but not in βres mice, indicating a requirement of cgki in this process. in particular, sil was able to block the transcription of pro-fibrotic genes such as tgfβ, ctgf, collageni and fibronectin. the overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of the stable somatostatin analogs octreotide and pasireotide. whereas the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (t333) and 347 (t347), which enabled us to selectively detect either the t333-or the t347-phosphorylated form of sst5. we show that agonist-mediated phosphorylation occurs at t333, whereas t347 is constitutively phosphorylated in the absence of agonist. we further demonstrate that the pan-somatostatin analog pasireotide and the sst5-selective ligand l-817,818 but not octreotide or ke108 were able to promote a clearly detectable t333 phosphorylation. however, none of these compounds was able to stimulate t333 phosphorylation and sst5 internalization to the same extent as the natural somatostatin. agonist-induced t333 phosphorylation was dose-dependent and selectively mediated by g protein-coupled receptor kinase 2 (grk2). like that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. however, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were complete within minutes. we also identify protein phosphatase 1g (pp1g) as sst5 receptor phosphatase. together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal t333. in addition, we identify grk2-mediated phosphorylation and pp1g-mediated dephosphorylation of t333 as key regulators of rapid internalization and recycling of the human sst5 receptor. termination of signaling of activated g protein-coupled receptors (gpcrs) is essential for maintenance of cellular homeostasis. although the regulation of agonist-induced phosphorylation has been studied in detail for many gpcrs, the molecular mechanisms and functional consequences of receptor dephosphorylation are far from understood. recent studies have shown that phosphatase inhibitors, such as okadaic acid and calyculin a, can block the dephosphorylation of a number of gpcrs including the ß2 adrenergic receptor, d1 dopamine receptor, parathyroid hormone receptor 1, thromboxane a receptor and the vasopressin receptor 1. however, a specific phosphatase has not been identified so far. in present studies, we have examined the mechanism and function of receptor dephosphorylation using the sst2a somatostatin receptor and the µ-opioid receptor (mor) as models. within those analyses, we have identified protein phosphatase 1beta (pp1ß) as the phosphatase for the cluster of phosphorylated threonines (353ttetqrt359) within the sst2a somatostatin receptor carboxylterminus using sirna knock down screeening. those phosphorylation sites mediate ß-arrestin binding. we have also identified protein phosphatase 1gamma (pp1γ) as mor phosphatase that catalyzed t370 and s375 dephosphorylation at or near the plasma membrane within minutes after agonist removal. here, we show the different activated phosphatases with functional selective mutants. we examined tailswap mutants which specify the different phosphatase activities. therefore we produced a mor-rsst2a chimera with the rsst2a c-terminal tail and a rsst2a-mor chimera with a mor c-terminal tail. detoxification by conjugation of glutathione? formations of dna adducts of patulin activated by glutathione pfenning c., lehmann l. university wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany as a frequent contaminant in apple juice, the mycotoxin patulin (pat) has shown mutagenic potential in cultured mammalian cells at concentrations which are equivalent to those found in marketable foods. this fact is in contrast with the assumption that conjugation to the major intracellular nucleophile glutathione (gsh) leads to detoxification of the electrophile pat. although pat reacts readily with gsh, previous studies showed that co-incubation of pat with model thiols and amine compounds increased the reactivity of pat towards amines forming mixed-type adducts. thus, we hypothesise that the potential to react with dna bases after being activated by gsh might contribute to the mutagenicity of pat. adduct formation of dna bases (adenine, guanine or cytosine) with pat in the presence and absence of gsh was studied under neutral conditions. liquid chromatography coupled with electrospray ionization tandem mass spectrometry was applied for identification and structure elucidation of putative adducts. besides published as well as hitherto unknown pat-gsh adducts, several pat-dna base adducts were formed both in presence and absence of gsh. in addition, with each of the three dna bases one product exhibiting a mass to charge ratio and fragmentation pattern suggesting a mixed thiol-amine adduct was detected. based on the fragment ions of adducts formed with gsh and chemically modified derivatives, we postulate a cyclic structure of the pat-gsh-dna base adducts, resulting from the reaction of the α-amino group of the glutamic acid residue with the c7-carbonyl function of pat. the exocyclic amino group of the dna base is linked to c1 of the pat backbone by an amid bond. thus, the present study demonstrates the reactivity of pat towards dna bases and the participation of gsh in adduct formation. the postulated structures of dna adducts could be used as biomarkers for the determination of the internal exposure to pat in humans. prescribing information detailed in the summary of product characteristics (spc) forms the officially approved basis for safe prescribing of drugs. in a project funded by the german federal ministry of education and research (bmbf) we aimed to derive an internationally valid data set for safe prescribing of psychiatric drugs and therefore analyzed and compared the content of internationally available prescribing information. a team of pharmacists and clinical pharmacologists performed an in-depth comparison of the german, swiss, british and us-american spcs of 10 top prescribed psychiatric drugs. for 7 drugs (of identical pharmaceutical form) the spcs from the same manufacturer were available for all countries, whereas for three drugs spcs were only available from different companies. in these cases the most recent prescribing information from each country was included in the comparison. in 40 spcs 2220 individual data points (55.5±17.4 per individual spc) were compared. between countries the timeliness of prescribing information for an individual drug varied by a median of 18.5 (range: 6-134) months. the respective spcs covered on average 71.4±30.3% (range: 12.5-100%) of all mentioned indications and 70.1±24.4% (range 15.4-100%) of all mentioned contraindications. the warnings and precautions section of an individual spc covered on average 59.5±17.1% (range: 12.5-93.3%) of all mentioned warnings and precautions for that drug. the variation observed was only marginally improved when restricting the analysis to the 28 spcs of the 7 drugs available in all four countries from the same manufacturer. across countries, the summary of product characteristics of individual psychiatric drugs show substantial variation in crucial prescribing information. as different manufacturers are unlikely to explain much of the observed variation, these data argue for a better international cooperation and standardization of the content of summary of product characteristics. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. protein kinase ck2 (former name 'casein kinase 2') is a highly conserved serine/threonine kinase, which acts as a component of regulatory networks implicated in many cellular processes but is also linked to various types of human cancer [1, 2] . elevated ck2 activity has been associated with aggressive tumor behavior and results in growth advantage, enhanced survival and dynamic adaption to stress of cancer cells [3] . ck2 is a heterotetramer consisting of two catalytic subunits (ck2α) attached to a dimer of regulatory subunits (ck2β) [4] . ck2β stabilizes ck2α against denaturation and modulates the substrate specificity of the catalytic subunit [5] . due to the relatively small and hydrophobic ck2α-ck2β interface (832 å²) [4] , low molecular weight inhibitors are able to interfere with the ck2 subunit interaction and thus affect the kinase activity [6] . such inhibitors might exhibit an increased specificity in comparison to those compounds interacting with the conserved atp binding site [3] . we have developed an elisa-based ck2α-ck2β binding assay using recombinant human ck2 subunits. different blocking reagents were analyzed to minimize nonspecific binding. the optimized binding assay was then applied to screen for inhibitors of the ck2 subunit interaction. primary hits were further characterized by determination of the parameters ic50 and ki as well as by comparing the results from the binding assay with literature-known or recently obtained crystal structures. numerous epidemiological studies have shown associations between exposure to ultra fine particles and an increase in cardiovascular diseases such as atherosclerosis, coronary heart disease and myocardial infarction. ultra-fine particles have an aerodynamic diameter of < 0.1 µm and are highly diverse with impurities of transition metals and organic compounds (e.g. polycyclic aromatic hydrocarbons). posttranslational modification (ptm) of proteins, particularly phosphorylation, is a key element in the regulation of cell function and any disturbance can lead to multiple diseases. the present study focused on the proteomic-based identification of phosphorylated proteins to understand the mechanism behind ultrafine particle exposure and cardiovascular disease development. as one of the major sources for ufp emissions are diesel exhaust, therefore to mimic the diesel particles, carbon black (cb) and benzo[a]pyrene loaded carbon black (cb+) were used in the present study. cells of the endothelial cell line ea.hy926 were exposed for 14 days to 100 ng/ml cb and cb+. phosphoprotein extraction of whole cell lysates was carried out by the method developed in our lab 1 . the obtained proteins were then separated by two-dimensional gel-electrophoresis followed by maldi-tof-ms (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of differently expressed proteins. to further validate the results invasive potential of cells were monitored by plating exposed cells for 24 hrs on top of matrigel-coated inserts. differential expressions of 20 phosphoproteins were found in cb-treated cells while an altered expression of 33 phosphoproteins was observed in cb+-treated cells. the maldi-tof analysis revealed proteins involved in the regulation of the endothelial permeability and the cellular plasticity such as vimentin, actin and transitional endoplasmic reticulum atpase. further, the invasion assay supported these results as the cb-exposed cells showed a high invasive potential as compared to control. [1] pink m. et. al. , precipitation by lanthanum ions: a straightforward approach to isolate phosphoproteins, j.protomics, 75, 2011, [375] [376] [377] [378] [379] [380] [381] [382] [383] 302 application of the ttc approach in cosmetics platzek t. bundesinstitut für risikobewertung, max-dohrn-straße 8-10, 10589 berlin, germany regulatory toxicologists in europe have been discussing the ttc approach since more than a decade, e.g. the previous scientific committee on food in 1996. since then, the concept was further developed and is now applied in the eu for the assessment of flavouring substances by efsa. two committees are discussing possible applications: the efsa scientific committee prepared an opinion exploring options for the application in food and feed, e.g. for impurities of food additives, thermal reaction products, food contact materials, contaminants etc. in addition, an eu non-food expert committee consisting of members of sccs, scher and schenir discussed the ttc concept in general as well as additional possible fields of application with the focus on cosmetics and an opinion was already published for public consultation. the proposal to apply the ttc approach also for cosmetic ingredients was introduced by a paper published in 2007 by a colipa (the european cosmetics association) supported working group. major aspects to be considered are the following: 1. applicability domain. the chemical space of the ttc dataset (> 600 compounds) has to be compared with that of cosmetic ingredients (> 10 000 compounds). route to route extrapolation. since no adequate dermal toxicity database is available both data on oral intake used in the ttc approach and on dermal exposure to cosmetic ingredients have to be transfomed to internal exposure figures. gastrointestinal and dermal bioavailability as well as route specific differences in metabolism have to be integrated. exposure. the reliability of exposure estimation is the second pillar of the ttc approach. compared to food, data on exposure to substances in cosmetics and consumer products is scarce. a pragmatic step forward is the comparison of ttcs and noael-derived safe exposure levels for cosmetic ingredients. this work was already done with substances in food contact materials and chemicals from the elincs list. for cosmetic ingredients a similar european project is ongoing. further refinement of the ttc approach is needed taking into account the up-to-date toxicological knowledge. with cosmetics specific problems may arise in praxi: according to the new eu cosmetic legislation the safety of cosmetic products available on the market has to be assessed by the manufacturer or importer and also assessors with limited toxicological experience may apply the ttc approach, e.g. by running the toxtree software. plöttner s., marczynski b., käfferlein h. u., welge p., groth h., engelhardt b., schmitz k., erkes a., brüning t. institut für prävention und arbeitsmedizin der deutschen gesetzlichen unfallversicherung -institut der ruhr-universität bochum (ipa) toxikologie, bürkle-dela-camp-platz 1, 44789 bochum, germany polycyclic aromatic hydrocarbons (pahs) comprise several hundred compounds with different carcinogenic potentials, typically occurring in complex mixtures. due to a lack of data risk estimations for pah mixtures are usually based on those for benzo[a]pyrene (b[a]p). the aim of our present study was to explore the suitability of a permanent human lung cell line as tool for future studies on genotoxicity of pah mixtures. in this pilot study we investigated the time-and concentration-dependent generation of specific anti-benzo[a]pyrene -7,8-diol-9 ,10-epoxide (anti-bpde)-dna adducts as well as cytochrome p450 (cyp) 1a1/1b1 enzyme activities after b[a]p-incubation in vitro. we used metabolically competent a549 lung carcinoma cells which display several characteristics of alveolar epithelial type ii cells. after 24 h and 48 h incubations with different b[a]p-concentrations cytotoxic effects were assessed with the neutral red assay and cyp1a1/1b1 activities using luminescent tests. the formation of specific anti-bpde-dna adducts was determined by hplc with fluorescence detection. a time-and concentration-dependent formation of anti-bpde-dna adducts was observed with maximum rates of 340.5 ± 39.1 and 599.6 ± 132.4 anti-bpde/10 8 nucleotides after incubation with 1 µm (24 h) and 3 µm b[a]p (48 h), respectively. however, the mean adduct rates decreased at higher b[a]p-concentrations. the reduction was more pronounced after 24 h than after 48 h. increased cyp1a1/1b1 activities were observed at > 0.1 -1 µm (24 h) and > 0.1 -3 µm (48 h). a clear decrease of enzyme activities was observed at higher concentrations for both incubation times. in the neutral red assay no more than 10% cytotoxicity in relation to the negative control were found after 24 h incubation with ≥ 10 µm b[a]p and after 48 h with ≥ 1 µm b[a]p. overall, incubation of a549 cells with b[a]p resulted in a time-and concentration-dependent increase of cyp-activities and anti-bpde-dna adducts. this clearly shows that a549 cells are able to generate mutagenic dna-adducts. thus, the in vitro model used in the present work appears suitable for genotoxicity studies of individual pahs and pah mixtures, and therefore may be a useful tool for research on syncarcinogenesis. the β subunit of the cav1.2 channel complex has been shown to play a key role in cav1.2 channel trafficking and channel characteristics like opening probability. furthermore, the last exon of the cacnb2 gene coding for cavβ2, exon 14, contains several potential phosphorylation sites, e.g. for protein kinase a or ca 2+ /calmodulin dependent camkii. pka-dependent phosphorylation mediates β-adrenergic stimulation of cav1.2. potential phosphorylation sites are ser478 and ser479 (in the β2 subunit in rat, perez-reyes et al. 1992 a ) and ser1928 in the c-terminus of the poreforming α1c subunit (lemke et al. 2008 b ) . in cardiomyocytes camkii regulates ca 2+ release and reuptake from and into the sr and is involved in the facilitation of the calcium channel. potential interaction sites between the cav1.2 channel complex and camkii are thr498 in the β2 subunit (in rat, grueter et al. 2006 c ) and ser1512 and ser1570 in the cterminus of the α1c subunit (blaich et al. 2010 d ) . however, the exact pathways remained widely unclear up to now. to clarify these mechanisms and to identify the relevant phosphorylation sites for pka and camkii we established a mouse line carrying a stop codon in exon 14 after aa pro501. this mutation prevented translation of the cavβ2 c-terminus containing the corresponding potential phosphorylation sites mentioned above (cavβ2stop mouse). these mice were viable, showed unaltered expression of the truncated of cavβ protein and unchanged ecg and echocardiography. electophysiological analysis of isolated cardiomyocytes showed no differences in current density, the effect of isoproterenol, the time course of inactivation and the facilitation property when compared to cells isolated from littermate controls. for further investigations we bred the cavβ2stop mice with s1928a mice (lemke et al. 2008 b ) lacking the pka phosphorylation site s1928 in the α1c subunit (s1928aβ2stop mouse) or with sf mice (blaich et al. 2010 d ) lacking the camkii phosphorylation sites s1512 and s1570 in the α1c c-terminus (sfβ2stop). both mouse lines were viable and showed unchanged echocardiography recordings compared to their control littermates and unaltered ecg for s1928aβ2stop mice. electrophysiological investigations on cardiomyocytes of s1928aβ2stop mice showed unchanged β-adrenergic stimulation with isoproterenol compared to littermate controls. these results suggest that β adrenergic regulation of the cardiac cav1.2 channel is not mediated by these phosphorylation sites. introduction. chronic atrial fibrillation (caf) is marked by increased fibrosis which contributes to the perpetuation of the disease. in addition to the role of fibrosis in structural remodeling of cardiac tissue, fibroblasts can couple with cardiomyocytes via gap junction thereby altering the electrophysiological properties of the later and potentially participating in atrial electrical dysfunction. in order to understand the importance of fibroblasts in the pathophysiology of caf, we compared the electrical properties of atrial fibroblasts isolated from patients in sinus rhythm (sr) and caf. methods. fibroblasts were isolated by outgrowth culture from right atrial biopsies and cultivated in medium containing 10% fetal calf serum. we used whole-cell patch clamp techniques to investigate ion currents and membrane potential. results. sr and caf fibroblasts showed similar capacitance (sr: 43.6 ± 4.6 pf, n=33; caf: 54.7 ± 5.1 pf, n = 17) and membrane potential (sr: -21.0 ± 4.3 mv, n = 14; caf: -27. 4 ± 4.8 mv, n = 16) . in both groups, we observed fast activating outward currents with a mean threshold at -20 mv. interestingly, current amplitude was significantly larger in sr than caf cells (sr: 23.8 ± 4.2 pa/pf, n = 15; caf: 6.1 ± 1.0, n = 6; p < 0.05). when maintained in culture for 3-5 weeks, cells from both groups developed na + currents. surprisingly, the fraction of caf cells displaying such currents was larger than the sr counterpart (caf: 38%; sr: 15%). furthermore, na + current amplitude was significantly larger in caf fibroblasts (sr: 6.1 ± 2.0 pa/pf, n = 5; caf: 17.4 ± 4.4 pa/pf, n = 6; p < 0.05). na + currents were not altered by 100 nm tetrodotoxin (ttx), but 10 µm ttx reduced current amplitude to 42% of control, suggesting that the channel involved is the cardiac ttx-resistant isoform nav1.5. conclusion. in the context of caf, fibroblasts undergo electrophysiological changes which need to be thoroughly described. understanding whether those changes contribute to the af substrate might provide new therapeutic targets for the treatment of caf. the initial recruitment of gi to the alpha2a-adrenergic receptor is affected by gprotein-coupled receptor kinases and arrestins prokopets o. s., krasel c., 35043 marburg, germany most g-protein-coupled receptors undergo homologous desensitization after agonist stimulation. in this process, agonist-activated receptors are phosphorylated by gprotein-coupled receptor kinases (grks), followed by binding of arrestins to the still agonist-occupied, phosphorylated receptors. it is assumed that arrestin competes with heterotrimeric g-proteins for the receptor molecule and thereby causes desensitization of g-protein-mediated responses. we tested this idea by investigating the effect of grks and arrestins on the recruitment of g-proteins by the alpha2a-adrenergic receptor. hek293t cells were transfected with yfp-tagged alpha2a-adrenergic receptor and the three subunits of gi1. the g(beta) subunit was cfp-tagged. upon stimulation with noradrenaline, a very rapid, robust increase in fret between the receptor and the g(beta) subunit was observed, confirming previous observations that the alpha2aadrenergic receptor recruits gi with a half-life of around 150 milliseconds. when arrestin3 and grk2 were also co-transfected, the half-life of gi recruitment was substantially delayed, increasing to around 2 seconds. there seemed to be no effect of grk2+arrestin3 cotransfection on a second stimulation; neither the kinetics nor the extent were altered compared to the first stimulation. interestingly, grk2 alone seemed to cause a similar but slightly less pronounced delay of g(beta) recruitment to the alpha2a-adrenergic receptor. in corresponding experiments, we measured the recruitment of arrestin3-cfp to yfp-tagged alpha2a-adrenergic receptors in the presence of grk2. upon stimulation with noradrenaline, we observed a robust increase in fret between the receptor and arrestin3, confirming previous observations that the alpha2a-adrenergic receptor recruits arrestin3. co-transfection of the three subunits of gi1 had no effect on the kinetics of arrestin3 binding to the alpha2a-adrenergic receptors. based on previous results with other receptors, an attenuation of gi recruiting to the alpha2a-adrenergic receptor is quite unexpected. our data suggest that the relation between receptors, g-proteins, grks and arrestins may be more complex than previously postulated. introduction: human urinary bladder expresses mrna of the three known badrenoceptor (b-ar) subtypes (b1, b2, b3) in detrusor and urothelium. we have shown previously that only b3-ar is involved in human detrusor relaxation. to investigate the urothelium-induced modulation of b-ar-mediated relaxation, we have examined systematically whether other b-ar subtypes are involved. human detrusor tissue samples were obtained from patients undergoing radical cystectomy for the treatment of bladder cancer. detrusor strips were studied with and without an intact mucosa layer. muscle strips were precontracted with 1 µm carbachol and relaxation was studied in response to the b-ar agonist ne. a-ar mediated processes were blocked with the a-ar antagonists phentolamine and prazosin. selective b-ars antagonists were used to investigate b-ar mediated relaxation. at the end of each experiment 10 µm forskolin was used to determine maximum camp-mediated relaxation. the presence of intact urothelium reduces potency but not effectivity of ne indicating involvement of urothelium-mediated processes not only during detrusor contraction but also during relaxation. ne-mediated detrusor relaxation is mediated through b3-ar in the absence of urothelium. but in intact detrusor strips b2-ars seem to have an additional inhibitory effect. affinity of the selective b3-ar antagonist l748,337 is unchanged, therefore the intact urothelium does not interact with the function of b3-ars. aldosterone causes oxidative stress and dna damage independent of blood pressure in vivo queisser n., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany background: an inappropriate increase of the mineralocorticoid aldosterone (ald) can be induced by a stimulated renin-angiotensin-aldosterone system. epidemiological studies exploring the connection between hypertension and cancer found higher cancer mortality and an increased risk to develop kidney cancer in hypertensive individuals. we recently showed that ald produces oxidative stress, activates transcription factor nf-kb and is genotoxic in kidney tubule cells. objectives: this study investigated the capacity of ald to induce oxidative/nitrosative stress, dna damage, dna repair, apoptosis, cell proliferation and the activation of nf-κb in rat kidneys. methods: mineralocorticoid-dependent hypertension was induced by ald/salt in sprague dawley rats. dna damage and oxidative/nitrosative stress markers were detected immunohistochemically. results: ald/salt treatment caused increased blood pressure compared to untreated rats. tempol, an antioxidant, and hydralazine, a vasodilator acting independent of the renin-angiotensin-aldosterone system, could lower the blood pressure, while the mineralocorticoid receptor (mr) antagonist spironolactone was administered in a subtherapeutical dose not lowering the blood pressure. ald/salt treatment caused oxidative and nitrosative stress, structural dna damage, double strand breaks, dna repair and nf-κb activation. spironolactone decreased these markers significantly. tempol was also able to reduce these markers, while hydralazine had no effect. ald/salttreated kidneys showed a tendency to lower apoptosis and to increased cell proliferation compared to control rat kidneys. discussion: this study provides a first hint of blood pressure-independent effects of ald. the mr and the production of ros seem to be crucial for the damaging effects of ald. an aberrant or long-term activation of nf-κb by persistently high ald levels could support resistance to apoptosis and the survival of cells with damaged dna, and increase cell proliferation. these actions could contribute to the increased cancer incidence in hypertension by initiating carcinogenesis. grant support by the dfg is gratefully acknowledged. creb regulating transcriptional coactivator 1 (crtc1) is a transcriptional coactivator of the transcription factor creb. we have recently shown its expression in cardiomyocytes and its activation by beta-adrenergic signaling. beta-adrenergic signaling contributes to the pathogenesis of cardiac hypertrophy, leading to heart failure, as evidenced by the therapeutic success of the beta-adrenoceptor antagonists. in order to investigate if crtc1 is involved in this process, we investigated the expression of crtc1 in hypertrophied myocardium from mice and humans. methods: protein lysates from mouse and human samples were investigated for crtc1 protein expression. we distinguished between an acquired and an inherited form of cardiac hypertrophy. acquired cardiac hypertrophy is an adaptation of the heart to an increased cardiac workload and can be found in patients with an aortic valve stenosis. in mice this kind of hypertrophy can be evoked by transverse aortic constriction (tac). the inherited form of cardiac hypertrophy is caused by mutations in genes coding for proteins of the sarcomeric apparatus and is referred to hypertrophic cardiomyopathy (hcm). as a model for hcm, transgenic mice with a mutation in the mybpc3 gene, coding for the sarcomeric protein cardiac myosin-binding protein c (cmybp-c), were used. these transgenic mice were characterized by a hypertrophied heart. in case of human samples we distinguished between patients with an acquired form of hypertrophy due to an aortic valve stenosis, and patients with a hypertrophic obstructive cardiomyopathy (hocm), a special form of hcm. results: in the tac mice and in the myppc3 mutant mice the expression of crtc1 was significantly higher than in the controls (2.1-and 1.9-fold, respectively; n=3). in both forms of human hypertrophy, we found a significantly 5-fold upregulation of crtc1 protein level in comparison to human samples from non-failing myocardium or samples from patients with a dilatative or ischemic cardiomyopathy (n=7-10 for each group). conclusions: crtc1 is upregulated in cardiac hypertrophy with acquired or geneticbased origin. the evaluation of the role of crtc1 in the heart may help to elucidate the role of beta-adrenergic signaling in the development of cardiac hypertrophy. microarray gene expression profiling reveals up-regulation of the cardiac lipid metabolic process at the onset of heart failure fu x., abd alla j., quitterer u. eth zürich molekulare pharmakologie, winterthurerstrasse 190, 8057 zürich, switzerland atherosclerosis and chronic pressure overload are major cardiovascular risk factors for the development of heart failure in patients. to mimic those risk factors in experimental models we used atherosclerosis-prone apolipoprotein e (apoe)-deficient mice, and chronic pressure overload was imposed by abdominal aortic constriction (aac). cardiac function was monitored by echocardiography. severe atherosclerosis in aged apoedeficient mice or chronic pressure overload induced signs of heart failure as evidenced by a significantly reduced cardiac ejection fraction (<30%). to investigate pathomechanisms underlying the development of heart failure, microarray gene expression analysis and qt-rt-pcr were performed of failing heart tissue relative to age-matched controls. gene ontology analysis of the microarray data revealed that the onset of heart failure, in two different experimental models, was characterized by a strong up-regulation (≥2-fold) of the cardiac lipid metabolic process and lipid overload. lipid metabolism genes were involved in lipid synthesis, storage and oxidation. the major palmitate-synthesizing enzyme, fatty acid synthase, was causally related to the development of cardiac dysfunction by enhancing cardiomyocyte apoptosis. taken together the data support that the onset of experimental heart failure is characterized by a dysfunction of the cardiac lipid metabolism promoting cardiomyocyte death. at1-receptor blockers (arbs) are established for the treatment of high blood pressure and new onset of diabetes is reduced by arbs. in the past years evidence increased that body weight may also be lessened particularly in rats and mice. however, less data are available whether arbs still reduce weight, when treatment was initiated not until animals became obese by diet. prior to drug treatment, spontaneously hypertensive rats were fed for 6 months with chow but also with a cafeteria diet (cd) to develop obesity. controls received only chow (conchow). cd-fed shr were treated for 3 months with telmisartan (tel 8mg/kg/d) or amlodipine (aml, 12 mg/kg/d), whereas controls received vehicle (concd). systolic blood pressure (sbp), feeding behaviour, body weight, abdominal fat mass (by mrt) and energy expenditure (by indirect calorimetry) was monitored. leptin sensitivity was assessed by measuring energy intake and expenditure after repetitive injections (s.c.) of leptin. insulin sensitivity was functionally determined by glucose and insulin tolerance tests. due to cd feeding body weight was increased after 6 months by more than 60 g. tel normalized sbp whereas it remained >200 mmhg in concd and conchow. tel additionally reduced cd-induced increase of body weight and abdominal fat mass. food intake was diminished during the first 4 weeks, but raised beyond control levels during the last 4 weeks of treatment. the shift of the respiratory index to lower levels indicated improved energy expenditure. in response to exogenous leptin, the food intake of concd was higher compared to conchow, indicating a leptin resistance. this assumption is further supported by high triglyceride concentrations of concd. after tel, leptininduced food intake was reduced and energy expenditure was increased compared to concd, indicating that leptin sensitivity was at least partially restored. accordingly, triglycerides were reduced. compared to concd, the insulin sensitivity was improved by tel since maximal increases in plasma concentrations of glucose and insulin in response to glucose challenge were reduced, but glucose response to insulin challenge was diminished. even though reduction in blood pressure was almost similar between tel and aml, metabolic and antiobese efficacies of aml were markedly attenuated. we conclude that telmisartan reveals wide efficacies in improving all symptoms of the metabolic syndrome. the pleiotropic effects are not related to the hypotensive action of tel. a β2-adrenoceptor -camp mediated, immediate stimulation of β2-adrenoceptor gene expression in human lung fibroblasts is opposed by a delayed up-regulation of inhibitory factors racké k., lamyel f., kämpfer n., schütz i., warnken m. univ. bonn dept. pharmacol. &toxicol., 53105 bonn, germany based on their bronchodilatory effects, β2-adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and copd. however, treatment with long-acting β2-adrenoceptor agonists has been associated with possible worsening of airway hyper-reactivity, possibly because of loss of β-adrenoceptor function. therefore, the molecular regulation of β2-adrenoceptor expression was addressed here. mrc-5 human lung fibroblasts were cultured for up to 48 h in absence or presence of test substances, followed by β2-adrenoceptor mrna determination by qpcr. β2-adrenoceptor mrna decreased with a half-life of 25 min after inhibition of mrna synthesis with actinomycin d (30 µm), but increased by 333±85%, 502±52% and 640±165% (means±sem) within 1.5, 4 and 6.5 h, resp. after inhibition of protein synthesis by cycloheximide (30 µm). the β2-adrenoceptor agonists formoterol and olodaterol (1-100 nm) induced a rapid increase in β2-adrenoceptor mrna (maximally within 1 h by 100±19% and 110±19% at 10 nm, resp.). however, after 4 h exposure to 10 nm formoterol or olodaterol a reduction in β2-adrenoceptor mrna by 59±8% and 58±6%, resp., was observed. both, the stimulatory and inhibitory effects of β2adrenoceptor agonists were mimicked by forskolin (10 µm, increase by 88±14% and inhibition by 49±4%) and cholera toxin (5 ng/ml, increase by 76±12% and inhibition by 77±7%). the formoterol-induced up-regulation of β2-adrenoceptor mrna was blocked by actinomycin d, but not by cycloheximide. moreover, in presence of cycloheximide, β2adrenoceptor agonist induced inhibition was converted into a marked stimulation. in conclusion, expression of β2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. the observations with cycloheximide indicate that the β2-adrenoceptor gene is under strong inhibitory control of short-living, not yet identified suppressors. although both, the time-dependent up-and down-regulation of the β2adrenoceptor gene expression by β2-adrenoceptor activation appears to be mediated via adenylyl cyclase -camp signalling, only the stimulatory effect appears to be a direct action on the β2-adrenoceptor gene. et-1 appears to be involved in the pathogenesis not only of pulmonary hypertension, but also in fibrotic remodeling associated with chronic obstructive airway diseases. since human lung fibroblasts (hlf) are a source of et-1 and have been shown to be controlled by muscarinic receptors and β-adrenoceptors, a possible muscarinic and βadrenergic modulation of et-1 expression in hlf was explored. mrc-5 hlf were cultured for up to 24 h in absence or presence of test substances, followed by prepro-et-1 (ppet-1) mrna determination by qpcr. the muscarinic agonist oxotremorine (10 µm) induced an increase in ppet-1 mrna by 180%, an effect prevented by 10 nm tiotropium. the β2-adrenoceptor agonist olodaterol (up to 100 nm) caused a reduction of ppet-1 mrna expression by 45%. the effect of 10 nm olodaterol was prevented by ici 118,551 (1 µm), but not affect by cgp 20712 (3 µm) . the pka agonist 6-bnz-camp (500 µm) caused a reduction in ppet-1 mrna expression by 65%, whereas the epac agonist 8-cpt-2'-o-me-camp (100 µm) caused only a marginal inhibition by 22%. olodaterol (10 nm) strongly opposed the stimulatory effect of 10 µm oxotremorine. an increase in ppet-1 mrna expression by 185% caused by 0.3 ng/ml tgf-β was effectively opposed by 10 and 100 nm olodaterol, resulting in an inhibition comparable to that in absence of tgf-β. however, the increase in ppet-1 mrna caused by a maximally effective concentration of tgf-β (1 ng/ml, increase by 620%) was not significantly affected by 10 or 100 nm olodaterol. likewise, the pkaagonist 6-bnz-camp (500 µm) opposed the increase in ppet-1 mrna expression caused by 0.3 ng/ml tgf-β, but not that caused by 1 ng/ml tgf-β. tgf-β caused, with an ic 50 of 0.3 ng/ml, a marked down-regulation of β2-adrenoceptor mrna expression, maximally by 90% within 6 h. et-1 expression in hlf is stimulated by muscarinic receptors and inhibited by β2adrenoceptors. the effect of β2-adrenoceptors may be mediated via pka. et-1 expression in hlf is markedly up-regulated by tgf-β, but only effects of sub-maximally effective concentrations of tgf-β are opposed by the β2-adrenoceptor -pka pathway, in part because of tgf-β-induced down-regulation of β2-adrenoceptors. since et-1 can promote pro-fibrotic features in hlf, inhibition of et-1 expression could contribute to long-term beneficial effects of long-acting β2-adrenoceptor agonists such as olodaterol and long-acting muscarinic antagonists such as tiotropium. cardiac hypertrophy leads to up-regulation of dipeptidyl aminopeptidase-like proteins in human and rat radicke s., hutschenreuther a., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstr. cardiac hypertrophy is a major risk factor for heart failure and associated morbidity and mortality. functional down-regulation of k + currents is a prominent feature of cells isolated from failing ventricles. a marked decrease in the transient outward potassium current ito has been shown in various models. changes in the k + channel expression differ depending on the species, and the mechanism of induction of heart failure. to study the regulation of ito channel subunits we compared the hypertrophic responses in human ventricular tissues from failing hearts with doxorubicin-induced hypertrophy in rats and in h9c2 embryonic rat cardiac cells. specifically, we quantified mrna expression of the pore-forming subunits kv4.3 and kv4.2, the cytosolic β-subunit kchip2 and the transmembrane subunits dpp6 and dpp10 using rt-pcr. treatment with doxorubicin (2 µm) induced hypertrophy and increased the mrna expression of the hypertrophy marker genes anf, bnp and beta-mhc in h9c2 cardiac myoblasts. while kv4.3 was detected in h9c2 cells and hearts from human and rat, kv4.2 mrna was only expressed in adult rats. during hypertrophy kv4.3 was downregulated in human tissue as well as in doxorubicin-treated h9c2 cells compared to the controls. in rat hearts kv4.2 expression was increased after doxorubicin treatment. interestingly, kv4.2 was also found to be up-regulated in rat heart tissues and h9c2 cells after treatment with doxorubicin. as previously shown, kchip2 mrna expression was significantly reduced in tissues of failing hearts. in contrast, kchip2 mrna was upregulated in hypertrophic rat hearts and h9c2 cells. the expression of dpp6 and dpp10 was observed only in human hearts. but mrna levels of both were significantly increased in failing tissues. dpp6 and dpp10 were not expressed in the adult rat heart or h9c2 cells, whereas in rats with doxorubicin-induced cardiac hypertrophy and in doxorubicin-treated h9c2 cells, the mrna of dpp6 and dpp10 was up-regulated. h9c2 cells showed almost identical hypertrophic responses to those observed in human ventricles and rat hearts. this finding validates h9c2 cells as a model for in vitro studies of cardiac hypertrophy. in further studies we will investigate the consequences of a knock-down of dpp6 and dpp10 in doxorubicin-induced hypertrophic h9c2 cells. in preliminary experiments specific short-hairpin rna, targeting dpp6 and dpp10, has been designed and tested in heterologous expression systems. the nonsynonymous c.521t>c germline genetic variation in the liver-specific organic anion transporter slco1b1 is associated with methotrexate pharmacokinetics in pediatric acute lymphoblastic leukemia radtke s. 1 background: methotrexate (mtx) plasma concentration is related to its clinical effect. transport proteins, such as abcc2, slco19a1, and slco1b1, have been implicated in the disposition of mtx. here we investigated whether common reduced-function variants in abcc2, slco19a1, and slco1b1 contribute to the interindividual variability in methotrexate pharmacokinetics in children with acute lymphoblastic leukemia (all). we analyzed mtx pharmacokinetics (mtx plasma concentration at the end of infusion c24h, mtx auc24-48h, and mtx clearance cl) in an unselected population of 419 children with all from the all-bfm 2000 trial (clinicaltrials.gov: nct00430118) who received 1676 courses of mtx at 5 g/m 2 as 24 h infusions. the contribution of genes (genetic component, rgc) to the interindividual variability in mtx pharmacokinetics was estimated according to the method of kalow et al. (1998) . abcc2 c.-24c>t (rs717620), slco19a1 c.80g>a (p.his27arg, rs1051266), slco1b1 c.521t>c (p.val174ala, rs4149056) and slco1b1 388a>g (p.asn130asp, rs2306283) genotypes were analyzed by taqman polymerase chain reaction. there was substantial interpatient variability in average (± sd) mtx c24h (50.95 ± 24.15 µmol/l), auc24-48h (57.44 ± 37.52 h*mg/l), and cl (390.72 ± 223.95 ml/min/m²). the rgc values of c24h, auc24-48h, and cl ranged from 0.61-0.71 suggesting that variation in mtx pharmacokinetics has a substantial genetic component. after adjustment for age and sex by multiple regression, the slco1b1 c.521t>c snp was significantly associated with c24h (p<0.001), auc24-48h (p<0.001), and cl (p=0.011) of mtx. compared with the wildtype genotype, in patients with the tc genotype c24h and auc24-48h increased by 18% (p=0.009) and 28% (p=0.003), respectively, whereas cl significantly decreased by 15% (p=0.012). pharmacokinetic variables significantly changed with increasing number of variant c.521t>c alleles (p<0.02, jonckheere-terpstra), suggesting a per allele effect consistent with a co-dominant model of association. in contrast, the abcc2 c.-24c>t, slco19a1 c.80g>a, and slco1b1 388a>g polymorphisms did not show an association with mtx pharmacokinetics. conclusions: the nonsynonymous c.521t>c polymorphism in slco1b1 contributes to the variability of mtx pharmacokinetics in this study of high-dose mtx in pediatric all. this project is supported by the johannes und frieda marohn-stiftung. the antitumorigenic mechanism of the selective cyclooxygenase-2 (cox-2) inhibitor celecoxib is still a matter of debate. using human lung cancer cell lines (a549, h358, h460), the present study investigates the contribution of cox-2 and peroxisome proliferator activated receptor γ (pparγ) to apoptosis elicited by celecoxib. celecoxib was found to cause apoptotic cell death in a concentration-dependent manner (10 -50 µm), whereas structurally-related cox-2 inhibitors (etoricoxib, rofecoxib, valdecoxib) were inactive in this respect. apoptotic cell death by celecoxib was suppressed by preincubation of tumor cells with the selective cox-2 inhibitor ns-398, the pparγ antagonist gw-9662 and by sirna targeting cox-2 or pparγ. celecoxib-induced apoptosis was paralleled by a time-and concentration-dependent upregulation of cox-2 and pparγ at both mrna and protein level. using an established cox-2 activity assay monitoring immediate conversion of exogenously added arachidonic acid to the respective prostaglandins (pgs), ns-398 was shown to suppress celecoxib-induced cox-2 activity when added prior to arachidonic acid. among the cox-2-dependent pgs analyzed, pgd 2 and its dehydration product 15-deoxy-∆ 12,14 -pgj2 were found to induce cytosol-to-nucleus translocation of pparγ as well as pparγ-dependent apoptosis. celecoxib-elicited translocation of pparγ was inhibited by preincubation of cells with ns-398 which itself did not alter celecoxib-induced total pparγ protein expression. finally, a cox-2-and pparγ-dependent proapoptotic mechanism of celecoxib was confirmed in primary tumor cells obtained from brain metastases of two lung cancer patients. together, our data demonstrate a proapoptotic mechanism of celecoxib involving initial upregulation of cox-2 and pparγ and a subsequent nuclear translocation of pparγ by cox-2-dependent pgs. uncoagulated ppp contained 40-60 nm thrombin throughout. fxa was initially measured in clots at 10 nm. this level declined over time while clot-conditioned pbs accumulated fxa. exposure of human aortic smc to clots or native unclotted ppp for 24h only marginally influenced smc apoptosis but increased mitogenesis over 15-fold. this was reduced by all 4 inhibitors. clot-stimulated induction of tnfα and interleukin-6 mrna was also attenuated by the inhibitors. denatured ppp (no protease activity) increased smc mitogenesis to a level seen in smc exposed to clot and combined hirudin + dx9065a, reflecting the well-known mitogenic actions of serum alone. conclusion: coagulation of human plasma generates nanomolar amounts of thrombin and fxa, sufficient to stimulate the proliferative and inflammatory properties of adjacent smc. our observations validate the use of purified thrombin and fxa at nanomolar concentrations for in vitro studies, and support the individual and coagulationindependent roles of these proteases in cell proliferation and inflammation. antithrombotic therapy with argatroban or rivaroxaban may limit the cellular effects of clot-derived thrombin and fxa, while normal anti-platelet therapy would not. this aspect should be considered in the clinical use of these agents, specifically in healing processes after vessel injury. rhogef17 mediates cgmp/cgk induced adherence and relaxation of vascular smooth muscle cells rauch j. 1 , stephan-schnatz k. the guanine nucleotide exchange factor rhogef17 is the only gef known so far to be directly activated by cgmp-dependent kinase. it is expressed in various types of smooth muscle cells and has been shown to play a role in the regulation of cell integrity. in a previous investigation we showed that the knockdown of rhogef17 by a shrna approach caused a loss of actin stress fibers and a subsequent change of smooth muscle cell morphology that finally resulted in cell rounding. we now provide evidence that the expression level of rhogef17 influences the re-attachment of cultured rat aortic smooth muscle cells (rasmc) to a surface after detachment. although rhogef17 depleted rasmc were still able to adhere and spread, their cell surface area remained considerably smaller than that of control cells in the first 24 hours after seeding. cell counting revealed that 6 to 12 hours after seeding the percentage of adherent cells was significantly lower in the rhogef17 knockdown group compared to the control group. these data indicate a delay in attachment. interestingly, the knockdown of rhogef17 was paralleled by a loss in rhoa and cadherine expression. as rhogef17 mediated a cgmp-induced activation of the small gtpase rhoa in rasmc, we studied the effect of a stable cgmp analogon (8-pcpt-cgmp) on the adhesion process. in accordance with previously published data, cgmp treatment accelerated the attachment of rasmc to the surface within the first 12 hours. in contrast, the adhesion of rasmc after rhogef17 knockdown was no longer stimulated by cgmp. as these data indicate that rhogef17 mediates cgmp-dependent signalling in a physiological process we wondered whether this protein might also play a role in the regulation of cgmpdependent relaxation of vascular smooth muscle cells. thus, we performed a collagenbased contraction assay with rhogef17 depleted rasmc. while the contraction in response to serum was not affected by the depletion of rhogef17, we observed a slight decrease in basal contractility. interestingly, cgmp was not able to counteract the serum-driven contraction of rhogef17 knockdown cells. there was no cgmp-induced relaxation in these cells. we conclude that rhogef17 is involved in the cell adhesion of vascular smooth muscle cells and likely promotes the expression of specific proteins. its activation is required to mediate cgmp-induced signalling in terms of vascular smooth muscle cell adherence and relaxation. human eosinophil and neutrophil granulocytes are cells of the innate immune system. they both express formyl peptide receptors (fpr) und histamine h2 receptors (h2r). activation of fpr leads to a release of reactive oxygen species (ros). h2r activation results in an increase of intracellular 3'-5'-cyclic adenosine monophosphate (camp) concentration and inhibition of fpr-mediated ros release via adenylyl cyclase activation. in this study we compared the effects of various h2r ligands on camp accumulation and formyl peptide n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced ros release in isolated eosinophils and neutrophils. camp concentration was determined by hplc/tandem mass spectrometry, and ros release was assessed by monitoring superoxide dismutase-inhibitable reduction of ferricytochrome c. in eosinophils, histamine, amthamine and 5-methylhistamine exhibited similar potencies and efficacies with regard to camp accumulation and inhibtion of ros release. in marked contrast, in human neutrophils, we observed dissociations in potencies and efficacies of ligands at increasing camp accumulation and inhibition of ros production. our data suggest that in human eosinophils, but not neutrophils, camp mediates inhibtion of ros production. in a broader context our data provide a compelling example of the context-dependency of the pharmacological properties of g-proteincoupled-receptors. specifically, one has to be cautious when extrapolating experimental observations from one cell type to another, even when they are very closely related to each other. comonomers and monomers are used as dental restorative materials (e.g. in dental composites). unconverted compounds can be released from dental composites and can enter the body in humans. comonomers can induce various side effects in humans. this study was evaluated to qualify and to quantify eluted compounds from various dental composites. following composites were tested (producer in parentheses): els extra low shrinkage (saremco), synergy duo shade (coltène), grandio (voco), tetric evo ceram (vivadent), venus (kulzer), gradia (g.c.), and premise (kerr).polymerized composites (100 mg) were incubated in gc vials with 1 ml dest. water or 1 ml methanol, each at 37 °c for 72 hours. aliquots were taken, and eluted compounds were analyzed with the method of gas chromatography/mass spectrometry (gc-ms) and liquid chromatography/mass spectrometry (lc-ms).from all composites 18 different compounds were found. following comonomers were quantified (µg/ml; mean ± s.d.; n=4)( fig. 1 ).following range of the eluted and detected comonomers from dental composites was found (dest. water; decreasing elution): venus > gradia > synergy duo shade > tetric evo ceram premise > grandio > els extra low shrinkage. * n.d. = not detectable (below limit of detection). triphenylstibane was detected in tetric evo ceram (5 ± 2 µg/ml). reimann c., lupp a., schulz s. institut für pharmakologie und toxikologie universitätsklinikum jena, drackendorfer str. objective: cxcr4 is a plasma membrane chemokine receptor which is involved e.g. in organogenesis, hematopoesis and inflammation. in the adult organism cxcr4 is physiologically expressed on various cell types, in particular on lymphocytes. with respect to neoplastic tissues, in the current literature an over-expression of cxcr4 is described in different types of tumors, especially in breast and prostate cancer. additionally, an involvement of cxcr4 in tumor metastasis is discussed. subsequently, detection of cxcr4 expression in a given human tumor sample would provide a valuable predictive information on disease prognosis and possible therapeutic intervention. however, previous attempts to localize cxcr4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have yielded predominant nuclear and occasional cytoplasmic staining, but did not result in the identification of cell surface receptors. thus, the aim of the present study was to reassess the cxcr4 expression in a panel of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples by means of immunohistochemistry using the well characterized novel rabbit monoclonal anti-cxcr4 antibody umb-2. methods: in comparison to negative and positive control samples (cxcr4-knockout and wild-type mouse embryos) the extent of staining in the different normal and neoplastic tissue specimens was scored from zero (no expression) to three (high expression). results: cxcr4 was found to be expressed in all neoplastic tissue entities analyzed. in many cases, the receptor was predominantly localized at the plasma membrane of the tumor cells. however, in all cxcr4-expressing tumor entities a huge interindividual variability both in the percentage of positive cells and in the intensity of staining was noted which strongly differed also between the various types of cancer. the most intense (score three) staining was found in the samples of (small cell) lung cancer, ovarian cancer and of pheochromocytoma. additionally, lymphatic organs such as lymph nodes, spleens and tonsils were cxcr4 positive, with mainly b cells displaying a distinct staining of the plasma membrane. the rabbit monoclonal antibody umb-2 may prove to be of great value in the assessment of cxcr4 expression in different human tumor entities and of the mechanisms underlying the formation of metastases, thus helping to find new targets and strategies in cancer therapy. link between β2-adrenoceptor-mediated inhibition of formyl-peptide-induced o2 β2-adrenoceptor (adrb2)-agonists are in daily use for asthma therapy. although most cases of asthma are controlled by standard medication, a subpopulation of asthmatics remains difficult to treat. the adrb2 gene contains a total of 49 polymorphisms. this variability could cause part of the ~70 % genetically-determined differences in therapy response. genetic and corresponding functional data on adrb2 can help to understand the complex disease and, in cases of severe asthma, optimize therapy with adrb2agonists for each individual. (ortega et al. 2007; chung et al. 2011) our present study connects sequence data with pharmacological data of prototypical adrb2-ligands, namely, (r)-isoproterenol, (r,r)-and (s,s)-fenoterol, (r)-and (s)salbutamol and (r,r)-formoterol. as a pathophysiologically relevant cell type, we analysed human neutrophils from peripheral blood of healthy volunteers. formyl-peptide-induced o2 .production and its inhibition by the agonists are examined in a 96-well cytochrome-c assay. characteristic pharmacological values (pic50, emax) are obtained for each individual. the data-set for each individual is supplemented by a differential blood cell analysis and an asthma-related questionnaire. most importantly, each volunteer's adrb2-sequence variant is determined by sanger-sequencing. complete determination of a 1,490 bp sequence, including the entire adrb2 exon and part of the flanking 5'-and 3'-untranslated regions, allows mapping of the most common, but also of new or rare polymorphisms. first data demonstrate the inter-day and intra-individual robustness of the functional data. in the next step, we will link sequence variants and functional differences within a population of sixty volunteers with sufficient statistical power. collectively, this study represents a straightforward approach to link functional and genetic data of a clinically relevant receptor. cyclic nucleotide phosphodiesterases (pdes) are classified into eleven families and are essential for second messenger metabolism in human cells. 1 recently, we have shown that several human pdes possess a much broader substrate-specificity than previously assumed, being capable of hydrolyzing not only the purine nucleotides cyclic adenosine 3',5'-monophosphate (camp) and cyclic guanosine 3',5'-monophosphate (cgmp), but also pyrimidine nucleotides such as cyclic uridine 3',5'-monophosphate (cump). 2 these data were obtained using a highly sensitive hplc mass spectrometric assay which is quite expensive and whose technical requirements are available only in few laboratories. in our present study we developed a fluorimetric pde activity assay using 2'-o-(n'methylanthraniloyl) (mant)-substituted cyclic purine and pyrimidine nucleotides that can be used more broadly in the scientific community. human pde3a is important for the regulation of platelet aggregation, oozyte maturation, vascular smooth muscle relaxation and contractility of cardiac myocytes. 1 moreover, this pde shows a broad substrate-specificity, hydrolyzing camp, cgmp, cump and cyclic inosine 3',5'-monophosphate (cimp). 2 using this enzyme, here, we demonstrate that various mant-substituted cyclic nucleotides are substrates of pde3a and undergo a significant change in fluorescence whilst being hydrolyzed, thus allowing a quantitative analysis of catalysis via fluorescence detection. in fact, not only native cump but also mant-cump is a substrate of pde3a. this finding is consistent with data published by hardman and sutherland 3 who described a cump-degrading pde activity in homogenates from beef and dog heart, leading to the assumption that the pde activity described there could be attributed to pde3a. as cump has furthermore been proven to be present in mammalian cells 4 , to differentially activate camp-and cgmp-dependent protein kinases 5 and to be synthesized from utp by mammalian soluble guanylyl cyclase 6 , our present study supports the hypothesis that this cyclic nucleotide could play an important role in cell metabolism. the newly established fluorescence assay with mant-cump facilitates future studies on pde3a and the assumed second messenger function of cump. rhoa is reportedly involved in stat-dependent transcription. however, the pathway connecting the gtpase and stat signaling has not been characterized. we made use of bacterial toxins, which directly activate rho gtpases to analyse this pathway. cytotoxic necrotizing factors (cnfs) are produced by pathogenic escherichia coli strains and by yersinia pseudotuberculosis. they activate small gtpases of the rho family by deamidation of a glutamine, which is crucial for gtp hydrolysis. we show that rhoa activation leads to phosphorylation and activation of stat3 and identify signal proteins involved in this pathway. rhoa-dependent stat3 stimulation requires rock and junkinase activation as well as ap1-induced protein synthesis. the secretion of one or more factor/s activate/s the jak-stat pathway in an auto/paracrine manner. we identify ccl1/i-309 as an essential cytokine, which is produced and secreted upon rhoa activation and which is able to activate stat3-dependent signaling pathways. the knowledge about the connection between rhoa and stat signaling is crucial for understanding several deseases, especially cancer. acid sphingomyelinase-deficient mice are protected from the lethal cardiovascular effects in tnf-induced septic shock reiss l. k. 1 christian-albrechts universität institut für immunologie, michealisstrasse 5, 24105 kiel, germany introduction: the cytokine tumor necrosis factor (tnf) is a mediator of septic shock. sepsis is a major cause of acute respiratory distress syndrome (ards), a heterogeneous lung disease with a mortality of about 50%. the present study was designed to investigate the effects of high systemic tnf-levels on the lung and on the systemic circulation in wildtype and acid sphingomyelinase-deficient (asm -/-) mice. the enzyme acid sphingomyelinase generates the signaling molecule ceramide that plays a critical role in edema formation and vasodilatation. material and methods: asm -/and wildtype mice were ventilated mechanically at vt=8ml/kg and f=180min -1 with fio2=0.3 and peep=2cmh2o while lung mechanics were followed. half of the mice received 50µg of murine tnf intravenously. blood pressure was stabilized by intra-arterial fluid support and body temperature was kept at 37°c to prevent lethal shock and to allow investigation of blood gases, lung histopathology, pro-inflammatory mediators and microvascular permeability 6 hours after tnf application. results: tnf induced septic shock in wildtype mice, as indicated by metabolic acidosis, high serum levels of the sepsis marker procalcitonin, decreasing blood pressure and reflex tachycardia. interestingly, asm -/mice were protected from the tnf-induced cardiovascular effects and mortality. in the present study, circulating tnf failed to cause lung injury. lung mechanics stayed stable during ventilation in all groups and also pulmonary histopathology, cytokine levels and microvascular permeability were unaffected. conclusion: circulating tnf alone is not sufficient to cause acute lung injury. we conclude that the cardiovascular effects in tnf-induced septic shock are partly mediated by acid sphingomyelinase. cyclophilin a sirna provides mitoprotection and prevents aif-dependent neuronal cell death reuther c. 1 cyclophilin a (cypa) is a peptidyl-prolyl-cis-trans isomerase which is localized in the cytosol. recent data suggested that neuronal cell death involved cytosolic cypa translocation to the nucleus, where it formed a pro-apoptotic complex with apoptosis inducing factor (aif). this cypa-aif complex induced caspase-independent chromatin condensation, dna degradation and cell death in various paradigms of apoptosis. on the basis of these data, the selective inhibition of the aif-cypa complex was proposed as a potential strategy to prevent aif-dependent cell death in neurons. therefore, the aim of this study was to determine effects of cypa silencing in a model of glutamate toxicity in immortalized hippocampal ht22 neurons. first, we addressed the interaction of aif and cypa by immunoprecipitation and their translocation to the nucleus by immunohistochemistry and confocal fluorescence microscopy. after exposure of ht-22 cells to glutamate the translocation of aif and cyp a occurred prior to cell death. cypa sirna attenuated glutamate-induced cell death as detected by the mtt-assay, impedance measurements (xcelligence system), and by facs analysis after annexin v/ propidium iodide staining. most intriguingly, cypa sirna also preserved the mitochondrial membrane potential as shown by facs analysis after tmre staining. further, confocal microscopy showed that cypa silencing prevented mitomorphology alterations and blocked the release of mitochondrial aif to the nucleus. the inhibition of the aif translocation to the nucleus was also shown by western blot analysis. in summary this study demonstrates that silencing of cypa prevents mitochondrial disruption and attenuates glutamate toxicity in vitro. thus, cypa is a promising target for mitoprotection as a basis for novel strategies of neuroprotection. up to now the question is unresolved how the ingested dose influences the absorption and metabolism of chlorogenic acids (cga) from food. so far no studies have been performed on the impact of the dose on cga absorption, circulation and excretion. recently we performed a dose-response study in a randomized, double-blinded, crossover design with five ileostomy subjects. in three trials the volunteers consumed after a two day polyphenol free diet coffee with varying cga content (high 4525 µmol; medium 2219 µmol; low 1053 µmol). the cga concentrations in plasma, ileal effluent and urine were subsequently identified and quantified by hplc-esi-ms, hplc-esi-ms/ms and hplc-dad. the results showed that the consumption of higher cga concentrations lead to a faster ileal excretion measured in the ileal effluents. this corresponded to the renal excretion of 8.0 ± 4.9% (high), 12.1 ± 6.7% (medium) and 14.6 ± 6.8% (low) of total cga and metabolites. we found that cgas with a caffeic acid moiety are predominantly sulphated and those with a ferulic acid moiety are predominantly conjugated via glucuronidation prior renal excretion. furthermore, in the ileal effluents, sulphation of both structural units dominated. in plasma samples (after enzymatic deconjugation) the auc values were determined by the major cga classes in coffee, the caffeoylquinic acids: 551.5 ± 93.8 nm*h -1 (high); 299.3 ± 79.6 nm*h -1 (medium) and 222.8 ± 91.3 nm*h -1 (low). no major differences in the metabolic pattern were observed. additionally, we were able to identify new metabolites of cga in urine and ileal fluids. we conclude that the consumption of high concentrations of cga via coffee might influence the gastrointestinal transit time and consequently affect cga bioavailability. this study was supported by the nestlé research centre (lausanne, switzerland). interaction of antagonists with the atp binding pocket at the human p2x3 ion channel helms n., riedel t., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany the homomeric p2x3 receptor (p2x3r) is a rapidly activating and desensitizing cation channel, gated by extracellular atp. it consists of three homomeric subunits. this representative of the p2x receptor family is highly expressed on sensory afferent neurons and plays a significant role in chronic pain, bladder reflexes and taste sensation. therefore, the development of selective antagonists for p2x3 receptors and knowledge about the binding of these antagonists are of great significance for future pain therapy and therapy of urge incontinence. to simulate the shape of the rapidly desensitizing agonist-induced current responses via p2x3 receptors, we created a specific markov model to describe the binding of agonists and competitive antagonists. this model can be used to prove the competitive character of inhibition and to calculate the association and dissociation constants of the antagonists. furthermore we use this model to fit current responses at p2x3 wild type receptors and their mutants to α,βmethylene atp in the presence of different antagonists. whole-cell patch-clamp recordings were performed on hek 293 cells, heterologously expressing the human p2x3 receptor, to determine the concentration-response relationship of different antagonists. by applying increasing concentrations, differences of antagonist potency could be observed at the wild type receptor. afterwards, we chose amino acid residues for replacement by alanine, which seem to be important for agonist binding and should be so for competitive antagonist binding as well, based on our homology model, developed from the zebrafish p2x4r crystal structure and previous mutagenesis studies. we intend to identify those amino acids which are important for competitive antagonist binding by monitoring the altered antagonist potency on the mutated receptor when compared with the wild-type receptor. analysis of the p2x3 agonist binding site by double mutant cycles riedel t., wiese s., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany purinergic p2x receptors belong to the family of ligand-gated ion channels. they are non-selective cation channels, activated by extracellular atp. one of the seven members of the p2x receptor family, the p2x3 receptor, is localized at the plasma membrane of sensory neurons and is involved in pain perception. therefore, this receptor is a possible target for new drugs in pain treatment. the development of such drugs can be supported by an exact knowledge of the receptor structure and function. there are many hints to the atp binding site, but the interaction of the p2x receptor with its agonists and antagonists remains still unknown. in this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed atp binding site of the homomeric human p2x3 receptor on the effect of nucleotide analogues. the mutant receptors were expressed in hek293 cells and the nucleotide effects were measured by means of the whole-cell patch-clamp method. modifications in the receptor binding site changed the concentration-response dependency as well as the current kinetics during fast pulsed agonist applications. based on this fact, we were able to distinguish binding from gating, conductance, and desensitisation, using a markov model that describes the complete channel behaviour by a matrix of rate constants. the results were also checked for consistency with a structural hp2x3 model that we developed from the known zebra fish p2x4 crystal structure in the closed state. voltage-dependent modulation of alpha2a adrenergic receptor signaling rinne a., birk a., bünemann m. philipps-universität marburg institut für pharmakologie und klinische pharmazie, karlvon-frisch str. 1, 35034 marburg, germany g protein-coupled receptors (gpcrs) are proteins that regulate numerous signaling pathways by activation of intracellular g proteins. gpcrs are activated by extracellular stimuli, such as light, hormones and neurotransmitters. recent evidence suggests that some gpcrs exhibit voltage-sensitivity leading to a modulation of their activity by the membrane potential (vm). we used a fret-based biosensor of the α2a adrenergic receptor to analyze receptor activation at defined membrane potentials in hek 293 cells by means of voltage-clamp recording. the biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (ne) and receptor activation was measured as decrease in the ratio of acceptor-/donor-fluorescence. receptor stimulation by ne was inhibited at depolarizing membrane potentials but enhanced by hyperpolarization. inhibition of ne activated receptors was strong at low concentrations (500 nm: 60 % inhibition) but almost absent at saturating agonist concentrations (100 µm: 9 % inhibition). both agonist-induced and hyperpolarizationinduced receptor activation exhibited a similar monoexponential time course and speed of activation was primarily dependent on agonist concentration for both activation modes. the latter indicates that depolarization lowers the apparent affinity of the ne receptor interaction and thus causes receptor deactivation by means of ne release. application of clonidine (1 µm, vm=-90 mv) resulted in a fret response that was inhibited by 40 % at +60 mv. in contrast to ne, strong receptor inhibition at +60 mv was present even at super-saturating concentrations of clonidine (100 µm), suggesting that voltage alters the equilibrium between active and inactive conformations of the receptor. voltage-dependence of the a2a adrenergic receptor also modulated downstream receptor signaling: g protein activation or the recruitment of arrestins, which we determined in fret assays that directly detect gαi protein activation or receptor-arrestin interactions, were both substantially inhibited at vm = +60 mv. therefore we conclude that negative membrane potentials promote active conformations of the a2a adrenergic receptor, increase affinity of full agonists and enhance receptor signaling. in conclusion the present data show that scc cells extrude more ha, possibly related to increased levels of has3, in comparison to keratinocytes. increased amounts of ha appear to be essential for the uvb induced tumourgenesis of sccs in mice. this effect might be related to the pro-proliferative property of high molecular weight ha. furthermore biological active ha fragments derived from ha degradation by hyaluronidases (hyal1,2) are thought to be pro-angiogenetic, anti-apoptotic and proinflammatory, thus possibly also promoting tumour growth and malignancy. estradiol induced paracrine release of egf from keratinocytes protects the dermal hyaluronan/versican matrix during photoaging röck k. 1 , meusch m. hyaluronan (ha) and versican are key components of the dermis and are responsive to uvb induced remodeling. the aim of the present study was to investigate the molecular mechanisms of estrogen (e2) mediated effects on ha-rich ecm during actinic aging. 10 weeks of uvb irradiation (3 x 1 med (80 mj/cm 2 ), weekly) of hairless skh-1 mice caused a marked decline of dermal ha, which was aggravated by ovariectomy (ovx). subcutaneous substitution of estrogen (e2) by means of controlled release pellets abolished these effects confirming the stimulatory role of e2. the increase of dermal ha correlated with induction of ha synthase has3 by e2. in addition the ha-binding proteoglycan versican was induced by uvb and further increased by e2. however in cultured skin fibroblasts e2 reduced the expression of versican and had no effect on has3. therefore, direct upregulation of has3 and versican in fiborblasts by e2 was excluded. however, e2 increased the expression of egf in uvb irradiated skin in vivo and in keratinocytes in vitro. egf in turn upregulated the expression of has3 and versican in dermal fibroblasts. furthermore the supernatants of estradiol treated keratinocytes led to the same effects in dermal fibroblasts, which could be abolished by previous treatment of the supernatant with neutralizing egf antibody or treatment of the fibroblasts with egf receptor blocker erlotinib. functionally, dermal ha and versican induction by e2 correlated positively with proliferation and negatively with accumulation of inflammatory macrophages in the dermis. collectively these data suggest that e2 treatment increases the amount of dermal ha and versican via paracrine release of egf which may be implicated in the pro-proliferative and anti-inflammatory effects of e2 during photoaging. differential pro-and eukaryotic toxicity of silver released from nanocomposite surfaces increases the therapeutic window of silver in antibacterial treatments röhl c. 1 , hrkac t. silver has been used since ancient times as antimicrobial agent. recently, silver gained new attention due to its higher effectiveness in its nanoform. this led to new developments of silver nanomaterials, e.g., for medical devices and consumer products. though, it is generally assumed that silver is less toxic for eukaryotes than for prokaryotes, concern is raised, if nanosilver at the same time might also increase mammalian cytotoxicity. in our study we examined the toxicity of silver released from nanocomposite surfaces with that of silver from agno3 solutions for adherent bacteria and mammalian cells. therefore, we established an in vitro reference system which enabled us to compare the therapeutic window between prokaryotic toxicity and eukaryotic integrity in both exposure settings. we focussed especially on the comparability of the bacterial and mammalian cell systems and the development of characterized ag/tio2 nanocomposite coatings with well-defined silver filling factors and silver surface release, which could be varied over a wide concentration range. as reference cells the e. coli sar 18 strain and human dermal fibroblasts, which are of special relevance in the context of medical devices like implants or wound dressings, were chosen. bactericidal effects were determined by direct growth visualization of the gfp-producing e. coli strain by epifluorescence microscopy. mammalian cell growth and toxicity was determined by the mtt assay, protein measurements and phase contrast microscopy. the ag/tio2 samples were prepared by sputter co-deposition from two separate magnetron sources. the silver surface concentration release was determined by xps and the silver release by icp-ms. in solution a concentration-dependent constant silver concentration could be determined between 2 and at least 72 hours at the surface. while lowest bactericidal and cytotoxic concentrations of ag + from agno3 solutions with 0.64 and 0.95 mg/cm 2 , respectively, differed only slightly, the therapeutic window increased significantly if ag + was released from the nanocomposite surface. while the toxicity on the fibroblasts was unchanged the bactericidal potency increased at least one order of magnitude. taken together, it can be concluded that local exposure factors i) can be modulated by silver nanocomposites and ii) play an important role for the differential toxicity of surface silver on bacteria and mammalian cells. charite -universitätsmedizin institut für integrative neuroanatomie, funktionelle zellbiologie, philippstr. 2, 10115 berlin, germany c3 exoenzyme (c3bot) a clostridial adp-ribosyltransferase does not possess a cellbinding/-translocation domain. nevertheless, c3 is able to efficiently enter intact cells, including neuronal cells but the mechanism of uptake is not yet understood. in the present work, binding of c3bot to the hippocampus-derived ht22 cell line was characterized by means of binding and blot overlay assays as well as mass spectrometry analysis to identify binding partners of c3bot. the binding assays established that c3bot bound in a concentration-dependent manner to ht22 cells. in the overlay assay we detected one clear band of 55 kda. to elucidate whether glycosylation is important for the c3bot-protein interaction, ht22 cells were incubated with glycosidase f resulting in a decreased binding of c3 to the 55 kda band. to explore the involvement of phosphorylation in the binding of c3 to the putative binding protein, blot was pre-treated with cip (calf intestinal phosphatase) before overlay with c3bot. pretreatment greatly reduced the c3bot-protein interaction. moreover, inhibition of dephosphorylation by vanadate before in intact cells showed an increased level of c3botprotein interaction in the following overlay. thus, interactions between c3bot and ht22 cell proteins may require phosphorylation. to further characterize the 55 kda band as binding target of c3bot, the 55 kda band was digested with trypsin and then subjected to lc-orbitrap mass spectrometry analysis. from this 55 kda single gel band 141 proteins were identified. further analysis of the identified proteins will provide a possible interaction partner of c3bot. in sum, protein overlay assays revealed that phosphorylation and glycosylation are critical for efficient c3bot-protein interaction. s76 335 solvent effects on enzyme kinetics in vitro rokitta d., pfeiffer k., gerwin h., streich c., fuhr u. uniklinik köln institut für pharmakologie, gleueler str. 24, 50931 köln, germany kinetic parameters provide essential quantitative information for characterisation of drug metabolising enzymes. such enzymes are located in an a partially queous environment, but to solve potential lipophilic substrates for in vitro measurements organic solvents are regularly needed. to preserve the enzymes from denaturation and other solvent related effects, the concentration of these solvents must be kept low. data on nature and extent of such solvent effects is sparse. in this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethylsulfoxide (1% to 4%) on the assessment of k m, vmax and clint with regard to the 1-hydroxylation of midazolam via cyp3a4 and the cyp1a2 catalyzed metabolism of caffeine to paraxanthine in vitro. the presence of acetonitrile showed the highest vmax value for paraxanthine formation but the lowest values for 1-hydroxymidazolam formation. the km value for midazolam showed no systematic effects of organic solvents, while for caffeine km was up to eightfold lower for solvent free samples compared to solvent containing samples. the present example suggests that the presence of organic solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. these effects are differing between enzyme-substrate systems and solvents. it remains to be determined to which extent such effects compromise in vitro -in vivo extrapolations, and which solvents are most appropriate. atrial remodeling and arrhythmia induced by the transcription factor er81 rommel c. 1 , rösner s. introduction: the transcription factor er81 (ets related 81) belongs to the large family of ets-transcription factors that are involved in developmental processes and in the pathogenesis of cancer. er81 is activated by gq-and gs-coupled receptors leading to a phosphorylation of the transcription factor by map-kinases and protein kinase a, respectively. cardiac er81 mrna expression is increased in failing human hearts. however mechanical unloading by a left ventricular assist device leads to normalization of er81 expression. thus, the aim of the present study was to investigate the cardiac function of er81 in genetically modified mouse models. we previously generated transgenic mice overexpressing er81 under control of the cardiomyocyte-specific α-myosin heavy chain gene (αmhc) promoter by pronuclear injection and established independent transgenic lines. electrocardiography (ecg) was assessed in mice at day 5 after birth (p5) and in adult mice (3 months) during isoflurane anesthesia and by ecg telemetry in awake mice, respectively. ecg analysis revealed no differences between the genotypes at day 5 after birth. however, we found a decreased heart rate, a replacement of regular p-waves by an undulating baseline and frequent supraventricular extrasystoles in adult er81 αmhc transgenic mice. next, isometric contractile force measurements on isolated left atria were carried out in organ baths. while wt left atria responded to increasing concentrations of isoprenaline, nkh477 and calcium with an increase in contractility, the maximal positive inotropic responses to these substances were severely blunted in er81 αmhc atria. we performed western blots to identify potential aberrations of calcium handling and regulatory proteins. phosphorylation of serine 16 of phospholamban (pln) was reduced in er81 αmhc mice. in addition, protein phosphatase 1 (pp1) expression was significantly increased in er81 αmhc mice, which is consistent with the increased dephosphorylation of phospholamban. furthermore, we found a decreased expression of calsequestrin and serca2a protein in er81 αmhc atria. electron microscopy revealed the significant structural remodeling of er81 αmhc atria at 3 months of age. conclusion: increased cardiac expression of the ets-transcription factor er81 leads to structural and electrical remodeling of the atria. thus, er81 may play an important role in the pathogenesis of cardiac arrhythmias in chronic heart failure. signal transduction pathway of atp and utp in neonatal rat cardiac myocytes rothkirch d., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06097 halle (saale), germany extracellular atp and utp can be released from the heart during pathological conditions such as ischemia or hypoxia. in humans, atp and utp levels are increased during myocardial infarction. atp and utp can act via p2-purinoceptors which are further divided in p2x1-7 and p2y1-14-receptors. as previously shown atp and utp can induce inotropic effects in cardiac preparations of mice and man. for rat articular chondrocytes and human intestinal cells it has been demonstrated that the mapk cascade can be activated by atp and utp. therefore, the cardiac effects of atp and utp on force of contraction probably occur via the mapk pathway. to investigate the signal transduction pathway involved, we studied the effects of atp and utp on mapk phosphorylation in isolated neonatal rat cardiac myocytes using phosphorylation-specific antibodies. 100 µm atp as well as utp transiently increased phosphorylation of erk 1/2 and p38 mapk with a maximum effect at 5 to 10 minutes after application of atp and utp in neonatal cardiac myocytes (n=3 preparations each). the maximum phosphorylation of p38 increased with atp to 284% ± 68% (p<0.05) at 10 minutes and with utp up to 204% ± 21% (p<0.05) at 5 minutes. the phosphorylation with erk 1/2 mapk increased with atp to 234% ± 46.5% (p<0.05) at 5 minutes and to 337% ± 27% (p<0.05) with utp at 10 minutes of basal values, respectively. after 20 minutes, predrug values of mapk phosphorylation were reached again. in summary, we noted an atp-and utp-induced phosphorylation of erk 1/2 and p38 mapk in isolated neonatal rat cardiac myocytes. the involved receptor subtype(s) and the link between mapk phosphorylation and inotropic effect of atp and utp need to be elucidated. hameel: use of elearning in teaching pharmacology and toxicology -the halle experience rulf k. 1 , gergs u. introduction: during the past three years, our faculty has started to integrate items of elearning into the standard curriculum of a classical medical school: the "hallesches medizinisches elearning -hameel". our hypothesis was that these new elearning tools would improve the willingness of students to spend more time into learning and this would lead to an improved outcome (in multiple choice tests). methods: hence, we offered medical students (5 th or 10 th semester) additional learning environments. the courses for students (experimental pharmacology and toxicology or clinical pharmacology) were existed of a weekly lecture and in addition tutorials (problem-based-learning style, paper cases) or classical seminars. furthermore, we offered the possibility to use an online multiple choice quiz (involving 8 -12 previously used tests) and/or an online module on heart failure each week. we used the learning management system ilias software in combination with the content management system stud.ip. all students were subjected to an introductory test (to assess knowledge prior to our teaching section and allowing us to exclude a conceivable bias due to previous knowledge, involving basic items from prior teaching opportunities), a mid-term test and a final test to assess gain of knowledge. a maximum of 60 points could be obtained as a sum of both tests. results: in the means 40% of students used the new elearning tools (quizzes, heart failure module). however, there was no association between the use of self-assessment quizzes and examination results. the usage of the online quizzes increased in the periods before the exams. however, usage of the heart failure module was accompanied by significantly increased scores in exams. moreover, in a formalized evaluation system, students positively commented on our elearning efforts. conclusions: while usage of our quizzes did not improve test marks, another more sophisticated clinically oriented elearning module seemed to be improving test outcomes marginally. targeting of erk thr188 phosphorylation attenuates cardiac hypertrophy but preserves the anti-apoptotic effects of erk1/2 ruppert c., vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany background and aims: the extracellular regulated kinases 1 and 2 (erk1/2) play an important role in cardiac hypertrophy and cell survival. erk1/2 are phosphorylated at the so-called tey motif, which in turn activates erk1/2. hypertrophic stimuli lead to an additional autophosphorylation threonine188 (thr188). this autophosphorylation of erk1/2 stimulates activation of nuclear erk targets, which are known to induce hypertrophy. the aim of this study is to investigate whether specific targeting of erk thr188 phosphorylation affects both erk functions -erk mediated hypertrophy and cardioprotective cell survival. methods and results: for the analysis of cardiomyocyte hypertrophy in vitro, we stimulated cardiomyocytes with phenylephrine and measured the incorporation of tritiated isoleucine. cardiac hypertrophy was assessed by echocardiography before and after transverse aortic constriction (tac). for analysis of cell survival, caspase activity and dna fragmentation was determined upon hydrogen peroxide stimulation in vitro and in response to tac in vivo. to differentiate between inhibition of erk1/2 activity and prevention of erk thr188 phosphorylation, we either inhibited erk activity with pd98059 or overexpressed a mutant of erk2, which cannot be phosphorylated at thr188 phosphorylation. while inhibition of overall erk activity with pd98059 attenuated cell survival and hypertrophy in vitro, specific targeting of erk thr188 phosphorylation by overexpression of the phosphorylation deficient mutant (erk2 t188a ) attentuated phenylephrine induced hypertrophy, but preserved the anti-apoptotic effects of erk. cardiac overexpression of erk2 t188a significantly reduced tac-induced hypertrophy compared to wild-type erk2 overexpressing mice. in line with the in vitro experiments, erk thr188 inhibition only prevented hypertrophy in the tac model without promoting apoptosis. conclusions: these results show that blockade of erk thr188 phosphorylation attenuates cardiomyocyte hypertrophy but preserves anti-apoptotic effects of erk1/2. therefore, specific targeting of erk thr188 phosphorylation might be a promising strategy for the treatment of pathological hypertrophy. intracellular camp levels are determined by interplay of camp formation by adenylyl cyclases and camp degradation by phosphodiesterases (pde). eleven families of pdes are known. one of the most recently identified pdes is pde10, a pde in principle capable of hydrolysing camp as well as cgmp. pde10 contains a tandem of so called gaf domains in its n-terminal regulatory domain that mediate activation by camp. because current knowledge about the tissue distribution of pde10 was mostly based on the analysis of mrna distribution, we generated antisera against pde10 to analyze tissue distribution of the protein level. using these antibodies, we found a prominent occurrence of the enzyme in testis and in brain, where it was confined to the striatum. thus, pde10 displays a comparably restricted tissue distribution which is in contrast to that of many other pdes. low camp levels in so called medium spiny neurons of the striatum have been implicated in schizophrenia. furthermore, studies using the nonspecific pde10 inhibitor papaverine as well as specific pde10 inhibitors suggest pde10 as a target for the treatment of schizophrenia. here we set out to analyze the contribution of pde10 to camp degradation in striatum, to identify the physiological pathways pde10 is involved in and to clarify the functional impact of the proposed phosphorylation of the enzyme. identification of cgmp-dependent kinase i substrate complexes salb k., schlossmann j. universität regensburg lehrstuhl pharmakologie, universitätsstrasse 31, 93053 regensburg, germany the cgmp-dependent kinases (cgks) are components of the no/cgmp/cgk-signalling pathway and have a great physiological importance in a multitude of tissues and organs such as smooth muscles and platelets. two isoforms of the cgki and the cgkii are known. cgkiα and cgkiβ differ only in their first ~ 100 amino acids which constitute the leucine zipper and the autoinhibitory domains. the n-terminal leucine zipper domains mediate homodimerization of the kinase and the interaction with diverse substrate proteins. since cgkiα and cgkiβ express different n-termini they interact with different substrates. the cgkiβ isoform is assembled in a macrocomplex at the endoplasmic reticulum (er) with the intracellular calcium release channel inositoltrisphosphate receptor i (insp 3r-i) and the inositol-trisphosphate receptor associated cgmp kinase substrate (irag). we investigated, whether irag also interacts with the insp3r-ii and the insp3r-iii in murine platelets and tissues. additionally, we analyzed the interaction between the 52 amino acid peptide phospholamban (plb), which is also located at the er and regulates the er calcium reuptake by the sarco/endoplasmic reticulum ca 2+ -atpase (serca), and the two cgki isoforms. we performed cgmp-agarose experiments with murine wt and irag-ko platelets to examine the irag-insp3r interactions. the insp3r-ii isoform was neither bound to cgmp-agarose nor detected in the anti-irag immunoprecipitate. on the other hand, insp3r-iii from wt but not from irag-ko platelets was bound to cgmp-agarose. hence, insp3r-iii interacts directly with irag but not with cgkiβ in murine platelets. however, in colon smooth muscle lysate, insp3r-iii not only interacted with the irag protein but was also detected in the anti-cgkiα-immunoprecipitate. phospholamban from wt and irag-ko platelets was also bound to cgmp-agarose. subsequent immunoprecipitation experiments with the respective antibodies against the two cgki isoforms revealed that plb interacted both with cgkiβ and cgkiα. these results were supported by analysis of colon smooth muscle tissue from wt and irag-ko mice. in conclusion, irag interacts with insp3r-i and insp3r-iii but not with insp3r-ii in murine platelets and colon smooth muscle tissue. moreover, phospholamban is an interacting partner of both the cgkiα and the cgkiβ isoform. the human immunodeficiency virus type 1 enhancer binding protein 1 (hivep1) is regulated by proinflammatory stimuli and statins salomon a. 1, 2 , schmitz b. objective: hivep1 binds nf-ĸb and other proinflammatory consensus sequences, and is suggested to be involved in inflammatory processes. we recently identified two tagging snps, one positioned 90 kb upstream (rs169713) and another in exon 4 (rs2228220) of the hivep1 gene, to be replicatively associated with venous thrombosis in gwas and follow-up studies (ajhg, 2010; plos one, 2011) . methods: total rna isolation was performed after treatment of vascular endothelial cells (ea.hy926) with proinflammatory cytokines or statins (24h). serial hivep1 promoter deletion constructs were cloned into the pgl3-basic vector, a potential enhancer fragment, harbouring rs169713c/t, into the pgl3-promoter vector. in ea.hy926 cells and thp1 monocytes, reporter gene assays were performed by transient transfection and overexpression of transcription factors. chip and bandshift assays were performed to identify candidate transcription factors. results: in ea.hy926 cells, endogenous hivep1 expression was increased by proinflammatory cytokines tnfα and il-1β. simvastatin (1.2 and 2.4 µm) and atorvastatin (9 µm) -but not pravastatin or aspirin -both dose-dependently decreased basal and tnfα-stimulated hivep1 expression. the construct harbouring rs169713t exerted significantly higher transcriptional activity (ta) compared to rs169713c (p<0.001). for an intronic modulator, reporter gene assays demonstrated a regulatory effect on hivep1 expression in ea.hy926 and thp1 cells. cotransfection of sp1 and egr1 led to an increase in ta, while wt1 exclusively upregulated ta of constructs comprising the intronic modulator. chip and bandshift assays combined with specific antibody detection revealed binding of sp1 to the 5'-flanking region and the intronic modulator of hivep1. conclusion: increased hivep1 expression during inflammatory conditions can be repressed by simvastatin and atorvastatin, and not by pravastatin or aspirin. basal hivep1 expression is regulated by sp1 combined in a transcription factor module with egr1 and wt1 under basal and/or inflammatory conditions. the rs169713 site harbours potential activational capacity for hivep1 gene transcription and may communicate with the sp1/egr1/wt1 module. to date, the treatment of various movement disorders of the central nervous system is still insufficient. in most cases this is due to the sparse knowledge of the pathophysiology. l-dopa-induced dyskinesias (lid) represent a severe complication of long-time pharmacotherapy in parkinson's disease that deserves novel therapeutics. an increased activity of striatal projection neurons, which express kv7.2/3 channels, seems to be involved in the pathophysiology of these spontaneous involuntary dystonic and choreatic movements. previous studies demonstrated an antidyskinetic effect of the kv7.2-7.5 channel opener retigabine after acute and chronic treatment in a rat model of lid. in order to clarify if this effect was based on the modulation of kv7.2/3 channels, we examined the acute effects of the preferred kv7.2/3 channel opener ica 27243 on lid in this animal model. four weeks post 6-ohda lesioning of the left forebrain bundle, dyskinesia was induced by chronic treatment with 10 mg/kg l-dopa and 15 mg/kg benserazide for 20 days. three subtypes of dyskinesia (limb, axial and orolingual) were rated according to a score system from 0 to 4 over 180 min. for drug testing, ica 27243 (5, 10 and 15 mg/kg) was administered intraperitoneal additionally to l-dopa (or vehicle). effects of drug action in comparison to vehicle controls were detected by adding up the severity scores of each observation time. additionally, effects on parkinsonian symptoms were examined 20 min after drug administration using the block and the stepping test. ica 24273 reduced the severity of dyskinesia significantly at all doses while no negative impact on the antiparkinsonian effect of l-dopa was observed. whereas the antidyskinetic effect was restricted to the first 20 min after the application of 5 mg, it lasted up to 110 min in rats treated with 10 mg ica 27243. a higher dose of 15 mg did not further enhance the antidyskinetic effect. the results of our study suggest that the antidyskinetic effect of the k v7 channel opener retigabine was based on its action on striatal kv7.2/3 channels. in line with the results of previous studies with retigabine, this action does not seem to interfere with the antiparkinsonian effect of l-dopa. this study was supported by the micheal j. fox foundation. background: skeletal muscle toxicity is the major side effect of hmg-coa-reductase inhibitors (statins) and can be simulated in engineered skeletal muscle. statins are known to exert "pleiotropic" effects, e.g. reducing endothelial dysfunction by inducing no synthases and no production. the role of no synthases in skeletal muscle under statin treatment is largely unknown. interestingly, some skeletal muscle pathologies (e.g. duchenne muscular dystrophy) may be exacerbated by increased inos activity. here we tested whether or not statin-induced skeletal muscle toxicity would be associated with enhanced no synthesis. we generated engineered skeletal muscle (esm) from rat skeletal muscle cells, matrigel and collagen. esms displayed typical skeletal muscle properties (differentiated muscle fibres, tetanic contractions). under baseline conditions esm expressed enos most abundantly, followed by inos and nnos (n=4-5). myotoxic cerivastatin (0.01, 0.1, 1 µm for 5 days) caused a concentration-dependent decrease of contractile force (p<0,05, n=17-20) paralleled by an increase in inos transcript (mean±sem: 0.01 µm 4±0.9-fold, n=3 p<0.05; 0.1 µm 9.3±2.8-fold, n=3 p<0.05) and protein (0.01 µm 5.1±2-fold, n=4 p<0.05; 0.1 µm 7.8±0.8-fold, n=3 p<0.05). mevalonic acid fully prevented the inos increase suggesting that the induction is hmg-coa reductase-dependent. to test whether inos may contribute to the decrease in contractile force we co-treated esm with 1400w, a specific inos inhibitor. we applied 5 µm of 1400w, a concentration found to potently reduce lipopolysaccharide (lps)induced no-production in cultured myotubes. however, we did not observe a rescue effect (n=9-15). also, l-name (10 mm), an unspecific nos inhibitor, did not improve contractile function, instead we observed increased myotoxictiy (n=6-13, p<0.05). to further investigate the role of no for muscle function we treated the esms with increasing concentrations of the no-donor snp. only high concentrations of snp (10 µm) caused a reduction of contractile force. combined treatment with cerivastatin and 0.1 µm snp showed a tendency towards improved force development in esm. conclusions: statins increase inos activity in our skeletal muscle model (esm). however, this does not seem to functionally contribute to myopathy in esm. increased production of no may in fact be a protective measure. esm may help to dissect clinically relevant functional changes in statin myotoxicity. characterization of primary skin fibroblasts of patients with 3m syndrome and mutations in the cul7 gene meyer k., hieber m., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany 3m syndrome is an autosomal-recessive disorder characterized by pre-and postnatal growth retardation (< -4 sd), facial dysmorphism and skeletal anomalies. the majority of patients harbor missense mutations of the cul7 (76%) or obsl1 (16%) gene, respectively. cul7 constitutes an e3 ubiquitin ligase that is involved in the regulation of the insulin-like growth factor 1 (igf-1) signaling pathway via ubiquitin mediated degradation of insulin receptor substrate 1 (irs-1). to investigate the role of cul7 mediated irs-1 degradation in the pathogenesis of 3m syndrome. primary skin fibroblasts of seven 3m syndrome patients (six with cul7 mutations, one with a obsl1 mutation) and control fibroblasts were analyzed for proliferation rate (cell counter), cell cycle profile (facs), cell morphology and cellular senescence (histochemistry), irs-1 protein concentrations and activation of the igf-1 signaling pathway (western blot). the proliferation rate of 3m patient fibroblasts was significantly increased when compared to control cells. in contrast, irs-1 protein levels and activation of the pi3k/akt and erk mapk pathway were only increased in a subset of 3m cells that carried cul7 mutations, but not in cells from a patient with the obsl1 mutation. no significant differences in cell cycle profile, cell morphology or cellular senescence were observed in 3m patient fibroblasts when compared to control cells. to determine the pathogenetic contribution of increased irs-1 levels to the observed phenotype, human imr90 fibroblasts were stably transfected with retroviral vectors encoding irs-1. despite 20-fold overexpression of irs-1 compared to empty vector controls, no significant effect of igf-1 stimulation on proliferation rate or pi3k/akt and erk mapk signaling was observed. skin fibroblasts of 3m patients with cul7 mutations displayed an increased proliferation rate and enhanced activation of the igf-1 signaling pathways. despite accumulation of irs-1 in fibroblasts from a subset of 3m patients with cul7 mutations, no pathomechanistic role for irs-1 could be demonstrated. collectively, our data indicate that a dysregulated igf-1 signaling may contribute to the pathogenesis of 3m syndrome, yet in an irs-1 independent manner. pharmacases.de -a student-centered elearning project of clinical pharmacology zollner b., berg c., gros n., muß n., oestreicher d., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany pharmacases.de is a novel e-learning website of clinical pharmacology that presents clinically relevant aspects of pharmacology and toxicology in an interactive and multimedial manner. the aim of the project pharmacases.de was to develop an innovative concept for creating high quality elearning content that i) integrates and promotes the theoretical and cooperative skills of final year medical students and ii) is easily adoptable by cooperating institutes and hospitals. a peer-teaching concept was developed in which final year medical students with the elective pharmacology (pj wahlfach pharmakologie) independently researched and wrote elearning lessions ("pharmacases"). subject-specific expertise was acquired by consulting elective students of other disciplines. at present (11/2011) , this "peer network" consists of elective students of nine cooperating institutions (pathology, microbiology, radiology, cardiology, psychiatry, dermatology, neurology, ophthalmology, pediatrics) at the technische universität münchen. the average time for the generation of one elearning lession by the peer network was 10 days. to date, the website consists of 49 pharmacases that are available to all students online (http://www.pharmacases.de). the website also contains a discussion forum and evaluation form for direct feedback. on average, pharmacases.de has 1000 visitors per month with the following evaluation results: "excellent": 76%, "good": 15% and "satisfactory": 9% (n=33). the didactic concept of pharmacases.de enabled the efficient generation of high quality elearning content in a student-centered and interdisciplinary manner. the peer-teaching approach supports the collaborative skills of final year medical students and facilitates the transfer of theoretical pharmacological knowledge into clinical practice. improved glucose tolerance, less chronic adipose tissue inflammation and reduced adipose tissue mass in mice with adipocyte-specific loss of tak1 sassmann a., offermanns s., wettschureck n. max-planck-institut für herz-und lungenforschung pharmakologie, ludwigstr. 43, 61231 bad nauheim, germany tgf-β activated kinase 1 (tak1) is known to be involved in numerous inflammatory processes by linking receptors for inflammatory stimuli like lps, interleukin-1 or tnfa to ikk, p38 and jnk activation. chronic inflammation of white adipose tissue is one of the major causes for the development of insulin resistance and impaired glucose tolerance in states of obesity. to investigate the role of tak1 in white adipose tissue, we crossed the tamoxifen-inducible white adipocyte-specific cre mouse line adipoqcreer t2 with animals carrying floxed alleles of the tak1 gene. adipoqcreer ; tak1 fl/fl animals and cre negative control littermates are viable and fertile and do not show any developmental defects. after tamoxifen induction and high fat diet feeding adipocytespecific tak1 knockout mice show improved glucose tolerance and lower fasting insulin levels compared to control animals. in line with this, serum levels of the adipose tissuespecific hormone resistin are reduced in adipocyte-specific tak1 knockout mice. these findings are accompanied by a lower state of chronic inflammation of adipose tissue as indicated by a dramatic reduction of adipose tissue macrophage number and lower serum levels of tnfα and interleukin-6. stimuli like tnfα, interleukins and tgf-β released from macrophages and adipocytes are known to promote obesity-related adipose tissue inflammation. when stimulated with these substances tak1 deficient adipocytes show reduced activation of jnk and p38 which both play an important role in the development of insulin resistance. interestingly, we observe a lean phenotype in adipocyte-specific tak1 knockout mice when fed a high fat diet which reflects a reduction of white adipose tissue mass. currently we are investigating the molecular mechanisms underlying the reduced adiposity and lower state of chronic inflammation in adipose tissue. growth of small cell lung cancer (sclc) cells is regulated via the autocrine stimulation of g protein coupled receptors (gpcrs), i. e., neuropeptide and muscarinic acetyl choline (ach) receptors. the activation of gq/11 and calcium-dependent gpcr pathways results in the stimulation of erk signaling which is necessary for the mitogenic effects of neuropeptides or ach on sclc cells. in contrast, the role of calcium-independent gpcr signaling and its interplay with gq/11-regulated pathways in sclc cells are less well defined. the aim of our studies was to characterize the molecular make-up and the interaction of these pathways, and to delineate the phenotypic effects of calciumdependent and -independent signaling cascades in sclc cells. using a panel of sclc cell lines, we found that the stimulation of neuropeptide receptors led to an increase of calcium which was independent of extracellular calcium and could be prevented by depleting internal calcium stores. this calcium increase was sufficient to activate the tyrosine kinase pyk2 and subsequently the erk1/2 cascade. the role of pyk2 for the growth of sclc cells was further supported by the fact that inhibition of pyk2 using a sirna approach or a novel specific inhibitor, pf431396, exerted pronounced cytotoxic effects on sclc cells, whereas non-sclc cells were less sensitive. interestingly, the inhibition of g 12/13 signaling by sirna-mediated g(alpha)12 or g(alpha)13 knockdown also markedly reduced the growth of sclc in vitro or in subcutaneous tumor xenografts, and increased the sensitivity of sclc cells towards certain cytostatics. to further define the role of calcium-dependent signaling via pyk2 versus the role of calcium-independent signaling via g12/13, we tested the effect of pyk2 inhibition in cells with impaired g12/13 signaling. notably, pyk2 and g12/13 double inhibition led to an even increased proliferation. thus, we propose that dysbalanced g protein signaling favoring either pyk2 activation or g12/13-dependent cascades inhibits the growth of sclc cells, whereas the parallel inhibition of both pathways restores again the balance and the growth capacity in this tumor entity. dendritic cells (dcs) are essential for the initial immune response and for the defence against inhalated pulmonary toxins and carcinogens in lung. to differentiate dcs, the cell line thp-1 were used for 7 days and stimulated with various cytokines (il-4, gm-csf, tnf-a, ionomycin). the dcs were characterized by flow cytometry with different typical dendritic cell markers (for example cd11c, cd209, cd83) and by immunfluorescence compared to monocytes. the bronchial tract contains up to 800 dcs per mm² and therefore we established a triple culture model to mimic the situation in vivo. the triple culture consists out of primary human epithelial cells from small bronchi (hbec) and lung fibroblasts which are cultured under air-liquid conditions on filter membranes for 4 weeks and dcs which were added after the differentiation phase of the bronchial cells. during the cultivation time the hbec formed an epithelial layer expressing both tight and adherens junctions. they also produced mucus, formed functional cilia with a beat frequency of between 16 to 20 hz and the transepithelial resistance values were stable between 600 to 800 ω·cm². pathomechanisms of pulmonary toxicity in vivo are difficult to investigate, so the tripleculture model is the basis for investigations of the toxic effects at cellular level. lungtoxic substances such as organophosphates are usually absorbed through inhalation. organophosphates are dangerous nerve agents for the human organism. at high concentrations organophosphates damage in the coculture without dcs the cell-cell contacts of the epithelial layer. in the triple culture dcs firstly respond to inhaled organophosphates and seem to compensate effects on the other cells. in summary, it is very important to understand the pathogenic mechanisms of lung injury in relation to the role of dendritic cells in lung. they could play an essential role in therapy against damage of organophosphates in the lung. co-purification of arf gtpase-activating protein git1 and cavb3 schalkowsky p., wissenbach u., fecher-trost c., flockerzi v. universität des saarlandes institut für experimentelle und klinische pharmakologie und toxikologie, kirrbergerstraße, 66421 homburg, germany high-voltage activated ca channels are assembled from pore-forming α1 subunits and two distinct types of auxiliary subunits, cavβ1-β4 and, maybe, α2δ1-δ4. by a cavβ3-specific antibody based affinity chromatography the cavβ3 protein was highly enriched from rat brain microsomal membranes. proteins associated with cavβ3 were identified by mass spectrometry (lc-esi-ms/ms) and include α1-subunits, α2δ-subunits and β-subunits. in addition to these expected interacting proteins additional proteins were co-purified with the cavβ3 protein, including the g protein-coupled receptor kinase-interactor 1 (git1). the 770aa git1 is a ubiquitously expressed multidomain protein which may serve as a scaffold to bring together molecules to form signaling modules controlling, for example, vesicle trafficking, cytoskeletal organization and cell migration. in rat brain lysates the git1 and cavβ3 proteins were co-immunoprecipitated by the antibodies for cavβ3 and git, respectively. we cloned the git1 cdna from mouse brain and co-expressed it with the cavβ3 subunit in hek cells. like in brain lysates the git protein was retained by cavβ3 precipitated by the antibody for cavβ3 and cavβ3 was retained by the git1protein precipitated by the antibody for git1. both proteins, cavβ3 and git1 are endogenously co-expressed in mouse embryonic fibroblasts (mef). we could not observe potassium-induced voltage-activated ca influx in these acutely prepared cells. accordingly, mefs can be used as a model system to study the impact of cavβ3-git1 interaction in the absence of functional cav channels. in addition, using mefs from cavβ3-deficient mice enables us to control the impact of cavβ3 on git1 function. vice versa down-regulation of git1 by specific sirnas might allow to control the impact of git1 on cavβ3 function. as read-outs we use cell migration assays and monitor receptor-dependent and receptor-independent calcium signaling in these cells. effects of sphingosine-1-phosphate and fty720 on epidermal hyperproliferation and inflammation in an imiquimod induced mouse model of psoriasis the sphingolipid sphingosine 1-phosphate (s1p) is a mediator that modulates various physiological functions of skin cells. s1p has distinct direct effects on keratinocytes as it diminishes proliferation and induces differentiation which is a classical goal of psoriasis therapy. furthermore, s1p modulates the function of various immune cells, mainly to an anti-inflammatory direction. thus, the strategy of targeting immune cells with locally acting s1p was explored in an experimental animal model of psoriasis vulgaris, the recently established imiquimod induced psoriasis mouse model and in the mouse tail test. topical administration of imiquimod onto back and ear skin led to a distinct inflammatory response characterized by epidermal hyperproliferation, scaling and redness which was scored with a modified pasi (psoriasis area and severity index). the positive control diflorasone diacetate and s1p, but not fty720 reduced the epidermal hyperproliferation by topical administration onto ear skin, indicating a mode of action for s1p via the s1p2 receptor, which is not activated by fty720. there was also a moderate reduction of inflammatory cell influx and edema formation in ear skin by s1p treatment, which was even more pronounced by treatment with diflorasone diacetate. the pasi determined on back skin was, however, only significantly reduced by diflorasone diacetate. the discrepancy between outcome on ear and back skin remains elusive. in the mouse tail assay, the influence of s1p in stratum granulosum formation (orthokeratosis) was tested compared to the positive control calcipotriol. whereas topical administration of calcipotriol led to the expected significant increase of stratum granulosum in mouse tail epidermis, s1p lacked such an effect, indicating a different mode of action in epidermal differentiation. taken together, these results imply that topical administration of s1p might be a new option for the treatment of mild to moderate psoriasis lesions. inhalation of toxicants such as sulphur mustard (sm), an alkylating chemical warfare agent, cause pulmonary complications like respiratory failure, pulmonary edema and secondary pneumonia. in order to investigate pathomechanisms of pulmonary toxicity, an in vitro alveolar-capillary co-culture model has been established recently by our group. in this model the human lung adenocarcinoma epithelial cell line (h441) is mimicking the epithelial site of the alveoli while the human hemangiosarcoma cell line (iso-has) represents the endothelial site. acute respiratory injuries are accompanied by disruption of the alveolar-capillary barrier that can be detected by the use of biochemical markers (e.g. ldh) and electrochemical indicators (e.g. transepithelial resistance). sm-mediated pulmonary injury is characterized by the increased secretion of proinflammatory mediators (e.g. il-6). a shortcoming of this model is the missing inflammatory component in the lung. aim of the present project is the addition of macrophages to the established co-culture model to improve the model and to investigate the relevance of inflammatory processes in toxic lung injury. the effect of sm on this triple-culture model is characterized with special regard to the interaction of epithelial cells and macrophages. the human acute monocytic leukemia cell line (thp-1) was stimulated to allow differentiation into macrophages. validation of the cellular differentiation was checked by specific clusters of differentiation (e.g. cd206) using flow cytometric analysis. after successful differentiation into macrophages, these inflammatory cells were added to the co-culture model before and after exposure with sm, respectively. the cytotoxicity of sm on the triple-culture model was evaluated by xtt assays and ter measurements. furthermore, immunohistochemical staining of tight junction proteins (e.g. zo-1) and of adherens junction proteins (e.g. e-cadherin) was conducted to enhance the knowledge of the function of the intercellular junction in injured and rejuvenated regions as well as the interaction of epithelial cells and macrophages. for the contact allergen para-phenylenediamine (ppd) we showed that concentrations above 50 µm are accompanied with inhibition of nat1 activity in human keratinocytes [1] . in the following we investigated the impact of ppd on nat1 activity in antigenpresenting cells using dendritic cell-like cells, namely the monocytic thp-1 cells. measured nat1 activity of thp-1 was comparable to those found in primary keratinocytes. a 24h treatment of thp-1 cells with physiologically relevant concentrations of ppd (10-200 µm) led to a 47% reduction of nat1 activity. comparable results were found for mono-acetylated ppd (mappd) whereas di-acetylated ppd demonstrated no inhibition. time-dependent studies found a significant decrease in enzyme activity already 8h after application of ppd or mappd while nat1 mrna levels were not modified. these results are indicative for a substrate-dependent inhibition. further investigations concentrated on the restoration of nat1 activity after treatment with ppd or mappd. here we found that n-acetylation capacities were restored after 24h cultivation of the treated cells in fresh medium. independent of the enzymatic activity, certain compounds are known to oxidise the catalytic cysteine or form adducts with the nat1 protein. therefore we studied whether ppd and/or oxidised ppd including the trimer bandrowski´s base interact additionally with recombinant nat1 protein itself in the absence of acetyl-coenzyme a. we found that all compounds but mappd bind to nat1 protein after 2h. the greatest inhibition was found for oxidised ppd (up to 50%). due to the greater inhibition by oxidized ppd we propose that oxidation products interact with the protein whereas ppd itself modulates nat1 enzyme activity in a substrate-dependent mode of action. overall we demonstrated that ppd can inhibit nat1 in two different ways. the work was partially financed by federal office of public health (foph), switzerland and stiftung zur förderung begabter studierender und des wissenschaftlichen nachwuchses objective: fabry's disease is a rare progressive multisystem disorder resulting from deficiency of the lysosomal enzyme alpha-galactosidase a (gla, ec 3.2.1.22). we hypothesize that genetic gla variants, especially those in its promoter region are of pathophysiological relevance for the development and progression of fabry's disease phenotypes. this study focuses on the characterization of the gla promoter, identification of functional genetic variants and impact of transcription factor eb (tfeb), a regulator of lysosomal genes. we screened 4011 bp of the 5'-flanking region of gla in 60 patients with fabry's disease and 60 controls for genetic variants. serial promoter deletion constructs for reporter gene assays were designed and identified genetic variants were introduced by site-directed mutagenesis. constructs were transiently transfected into immortalized human kidney epithelial (ihke) cells and human vascular endothelial cells (ea.hy926) to determine transcriptional promoter activity (ta). sequencing of patients' dna revealed five genetic variants in the 5'flanking region of gla, significantly more frequent in fabry's patients compared to control group (rs2071225; rs3027580; rs3027579; rs59647857; rs3027575; all minor alleles p<0.0027). we identified two regions, a proximal one between -110 and -425 and a distal region between 1106 and -1421 with significant ta, in both cell lines. cotransfection with tfeb activated ta of both regions significantly up to 5.3-fold (p<0.001). in ihke cells, insertion of the minor t allele (rs2071225) significantly enhanced basal ta of the proximal promoter region (p=0.0006), while insertion decreased basal ta (p<0.0001) of the distal promoter portion. the combined insertion of the minor c alleles (rs3027580; rs3027579), which were in complete linkage disequilibrium, significantly increased basal ta of the distal promoter region (p=0.0037). our results indicate that three genetic variants, overrepresented in fabry's patients, are located within transcriptionally active regions, possibly altering tf binding sites and therefore, affecting gla expression. future analysis will assess the impact of gla promoter variants and gla regulation by tfeb with respect to fabry's phenotypes. multiple sclerosis (ms) and its animal counterpart experimental autoimmune encephalomyelitis (eae) have a major inflammatory component that drives and orchestrates both diseases. ceramides (cer) are known as mediators of inflammatory processes, but until now their role in ms was not elucidated. we measured the ceramide levels in the cerebrospinal fluid of ms patients and control patients using lc-ms/ms. interestingly, the c16:0-cer levels were 1.9 fold increased in ms patients. this translates into the finding that c16:0-cer levels were also significantly elevated in the lumbar spinal cord of eae mice. the raised c16:0-cer levels in the lumbar spinal cord were caused by a transiently increased expression of ceramide synthase (cers) 6 in macrophages. nitric oxide (no) and tumor necrosis factor alpha (tnf-α) secreted by interferon gamma (infγ ) induced macrophages play an essential role in the development of ms. astonishingly, rnai experiments reveal that cers6 and its product c16:0-cer are mediators of inf-γ induced no/tnf-α release in raw macrophages. moreover, treatment of eae mice with l-cycloserine prevented the increase of c16:0-cer and of inos/tnf-α expression and caused a remission of the disease. in summary, cers6 plays a critical role in the initial phase of ms, most likely by regulating the no and tnf-α synthesis. this let us speculate, that a substance designed to inhibit cers6 and therefore to limit the inflammatory effects of c16:0-cer may represent a new drug in ms therapy. role of cgmp-dependent protein kinase i for kidney fibrosis schinner e. 1 cgmp is synthesized via nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions also in the kidney. hence, the integration of cgmp signaling via cgmp-dependent protein kinases (cgk) might play a critical role for renal physiology. both isozymes were detected in arterioles, mesangium and within the cortical interstitium. in contrast to cgkiα, the β isoform was not detected in the juxtaglomerular apparatus and medullary fibroblasts. here, we focused on the function of cgki in the renal interstitium emphasizing a functional differentiation of both isoforms. the interstitium exists mainly of fibroblasts playing a prominent role in the interstitial fibrosis. accordingly, cgki could also be involved in this pathophysiological process. therefore, we studied whether cgki influences renal fibrosis which was induced by unilateral ureter obstruction (uuo). at first we analysed the role of the no/cgmp signaling by application of cgmp increasing yc1 or isdn. thereby we detected antifibrotic effects of these substances. subsequently we tested whether these effects are mediated by cgki by using mutant mice. on the one hand we examined αsm-rescue mice (expressing cgkiα only in smooth muscle under the control of the sm22 promotor with a cgki-ko background) and cgki-ko mice (expressing no cgki). on the other hand we used tgtg mice expressing more cgkiα in smooth muscle than wt mice (transgenic cgkiα under the control of the sm22 promotor). due to the steeply increased use of nanomaterials for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. moreover, the continuous development of novel materials requires the implementation of hazard-predicting models to prevent potential health effects resulting from human exposure. in the present study, we studied the toxic potential of a set of nanoparticles (np) with varying physicochemical properties in human a549 lung epithelial cells, hepg2 liver epithelial cells and hk-2 proximal tubule epithelial cells. the used nanomaterials incorporated five tio 2 samples, two zno samples (i.e. uncoated and coated), two multi-walled carbon nanotube (mw-cnt) samples and a nanoparticulate ag sample. cells were treated with np at doses ranging from 0.3 to 80 µg/cm 2 for cytotoxicity and from 06 to 40 µg/cm 2 for genotoxicity. dna damage was evaluated using the alkaline comet assay while concurrent cytotoxicity was determined by the wst-1 assay. marked contrasts in cytotoxic and dna damaging properties were observed among the different materials. the overall strongest responses were observed with the uncoated zno-np sample and with ag-np, although effects were found to depend on the cell type. notably, the dna damaging effect of ag-np could at least partly be attributed to its dispersant. present results form part of a growing data set which are generated in the framework of the eu fp7 project enpra (fp7-nmp) to establish dose-response relationships and a mathematical model to predict the hazard of nanoparticles. increased spontaneous hprt mutant frequency in v79 cells expressing human cytochrome p450 1b1 schlechtweg a., esch h. , martínez jaramillo d., lehmann l. university of wuerzburg/institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany the hypoxanthine-guanine phosphoribosyltransferase (hprt) assay in chinese hamster v79 lung fibroblasts (v79 cells) represents a widely-used mammalian test system to detect gene mutations. since v79 cells do not express any cytochrome-p450dependent monooxygenase (cyp) isozymes, usually an activating system has to be added. therefore, v79 cells expressing human (h) cyp isozymes have been commercialized. to test these v79 cells for their use in the hprt test, v79 h1a1 and h1b1 cells were characterized regarding (i) spontaneous frequency of 6-thioguanineresistant clones per 10 6 clonable cells (smf), (ii) the stability of which over 4 weeks (w), and (iii) the mutational spectrum (ms) of cdna from mutant clones. ms of cdna was determined by isolation of total rna, reverse transcription/amplification of the coding region by polymerase chain reaction and sanger sequencing of the amplification product. activity of cyp isozymes was verified by ethoxyresorufin-o-deethylase (erod) assay. (i)/(ii) whereas the smf of v79 cells (w2:13±4; w4:2±1) and v79 h1a1 (w2:17±4; w4:3±0) only varied within the range of historical controls, smf of v79 h1b1 increased continuously over time (w2: 36±6; w4: 68±13). (iii) although the smf of v79 and v79 h1a1 were similar, the mutational spectrum of v79 cells was characterized by as many transversions as transitions and deletions of exon 4 or exon 4+5, whereas the mutational spectrum of v79 h1a1 was characterized exclusively by transversions and deletion of exon 7+8. surprisingly, with 57 out of 59 cdnas derived from v79 h1b1 mutant clones, no amplification product was detected. first results indicate that there is at least one gene mutation in the untranslated region before and behind the coding region precluding amplification with the original primers. (ii)to reduce the smf of v79 h1b1, cells with wildtype hprt activity were cloned and one clone with an erod activity which did not differ significantly from the original cell population was further characterized. initially, smf of the clone varied between 0.7±0.6 and 1.2±0.0. yet its smf was unstable reaching up to 104±6. in conclusion, the mutational spectrum differed between the v79 cell lines. furthermore, h1b1 expression seemed to enhance smf in v79 cells. even though a temporary reduction of the smf by cloning was possible, smf of v79 h1b1 cells was unstable. we wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adp-induced aggregation of human platelets by wild garlic, allium ursinum l. method: bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. for the bioassay, blood was taken from healthy human volunteers and platelet rich plasma (prp) was prepared for turbidimetric platelet aggregation tests. prp, stimulated with 20µm adp, was treated with extracts of different polarities, fractions and isolated single compounds from allium ursinum. the extracts were investigated by thin layer chromatography, hplc, mass spectroscopy, esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. for references the adt-antagonist mes-amp was used. result: fresh allium ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited adp-induced aggregation of human platelets. this ethanolic extract was subjected to liquid-liquid partition. whilst the aqueous phase containing the moiety of cysteine sulphoxide and thiosulphinate derivatives showed only weak activity on platelet aggregation, the ethyl acetate and especially the chloroform partitions showed highest aggregation inhibiting potency. thus, in our bioassay effects of alliins/allicins could be neglected. the chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient elution open cc on silica gel. the most active fractions 11-17 were separated again yielding 10 subfractions. this afforded 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol and β-sitosterol-3-o-β-dglucopyranoside, the structures of which were determined by esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. furthermore, the diminutive amounts of volatile oil of a.ursinum leaves obtained by steam distillation according to ph.eur. could be evaluated as a third aggregation inhibiting principle. conclusions: at the first time two active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on adp-induced aggregation in human blood platelets. as a major constituent, the galactolipid 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol, not yet found in allium spec., appears as a new, highly useful marker substance for a.ursinum drugs, or their pharmaceutical preparations. in recent years, public attention focused more and more on risk factors which may impair sperm quality and thereby human reproduction. in this context, for example pesticides, alcohol, cigarettes, and even mobile phones are discussed. a variety of parameters exists including sperm counts as well as sperm motility, which are considered to be two of the most important parameters to evaluate sperm quality in animal models with the final aim to assess human risk. in recent years computer assisted sperm analysis (casa) devices mostly replaced the formerly used manual counting and manual motility assessment. however, although casa offers multiple opportunities and can allow for an objective and more detailed evaluation, several pitfalls exist which can alter the results profoundly and consequently compromise the quality of the data and ultimately the validity of a study. the aim of the present study was to establish and validate the casa device tox ivos sperm analyzer from hamilton thorne and thereby to gain detailed knowledge about the practical advantages but also intricacies which may alter the obtained results. in this regard healthy adult male rats (10-12 weeks old) were used. ultrasonic sound resistant sperm heads were isolated from the testis and in addition, sperms were isolated from the cauda epididymis. testicular sperm head counts and sperm motility were assessed using different isolation procedures and/or instrument settings. results different instrument settings modulate both -sperm motility and testicular sperm counts. in this regard, a wide range of results including slight changes as well as false positive/negative results were obtained. in addition, the modification of the isolation procedure can lead to variable results especially for sperm motility. conclusion isolation procedures as well as instrument settings can alter the results. consequently, in an experimental setting, potential adverse effects can be confounded with methodologically mediated apparent findings exerted via inappropriate use of the device -depending on the respective conditions in the test laboratory. this study demonstrates the relevance of standardization of testing conditions adopted for computer assisted sperm analysis and the need for a robust validation prior to use in experimental settings. orai and stim proteins have been identified as central components of the highly ca 2+ selective, store-operated current in immune cells (icrac). the molecular basis of selective orai-mediated activation of the calcineurin/nfat pathway and the crosstalk with other channel and scaffold molecules of the trpc family are still incompletely understood. using patch clamp recordings complemented by fluorescence and tirf microscopy we investigated interactions between orai1 and trpc3 in plasma membrane microdomains of rbl-2h3 mast cells. orai1-mediated crac currents, activated by passive store depletion, were found significantly reduced by over-expression of trpc3. this negative impact of trpc3 on icrac was independent of channel function as the trpc3 pore dead mutant (e630k) inhibited icrac to a similar extent as wild type trpc3. importantly, despite a reduction in icrac, nfat translocation in trpc3 overexpressing rbl cells remained unchanged, or was even slightly promoted. store depletion-induced nfat translocation in rbl cells was as well unaffected by trpc3e630k but substantially reduced by trpc3 mutants with either i) eliminated fkbp12/calcineurin binding (p704q) or ii) deficiency in pkc phosphorylation (s712a). moreover, inhibition of pkc phosphorylation by (gfx109203x; 3 µm) strongly suppressed nfat signaling. we suggest trpc3 as a scaffold that links orai-mediated ca 2+ -entry to nfat/calcineurin signaling within plasma membrane microdomains. neurally-induced bronchoconstriction in human and guinea pig precision-cut lung slices schlepütz m. 1 , rieg a. d. introduction: precision-cut lung slices (pcls) are well suited to study peripheral airway responses in different species. airway tone is under close control of the autonomic nervous system and dysregulation may contribute to airway hyperresponsiveness as observed in human lung diseases such as asthma. hence, the aim of the present study was to characterize neurally induced bronchoconstriction (bc) in guinea pigs (gp) and to compare the results with those in human pcls. methods: pcls were prepared from gp or human lung tissue. nerve endings in pcls were activated by electric field stimulation (efs) or capsaicin addition. cholinergic nerve responses were proven by atropine. capsaicin was used to show excitatory nonadrenergic non-cholinergic (enanc) responses. ruthenium red or skf96365 were used to confirm transient receptor potential (trp) channel contributions upon enanc activation. results: gp and human pcls were both sensitive to efs and airways contracted to 39±26% of the initial airway area (%-iaa) and 63±21%-iaa, respectively. in frequency response curves half maximal response was found at 7.0±1.2 hz for guinea pig pcls and 8.1±0.8 hz for human pcls. efs-induced bc was inhibited by atropine in both species. capsaicin contracted gp to 18±15%-iaa. 60% of human pcls were responsive to capsaicin and airways contracted to 72±10%-iaa, respectively. in gp ruthenium red and skf96365 blocked capsaicin-as well as efs-induced bc. conclusion: gp and human pcls contain atropine sensitive cholinergic and capsaicin sensitive enanc nerve endings. since gp pcls were sensitive to trp channel inhibitors, the involvement of those channels can be characterized with respect to lung diseases. in conclusion, gp pcls resemble the human distal lung innervation and represent a useful model to study neural airway pharmacology. the erk1/2-pathway is involved in pkc-induced nox4 up-regulation schlufter f., xia n., förstermann u., li h. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55131 mainz, germany nadph oxidases (nox) are major producers of reactive oxygen species in the vascular wall and nox4 is the most abundant nox isoform in human endothelial cells. we have previously shown that treatment of human ea.hy 926 endothelial cells with phorbol 12myristate 13-acetate (pma) for 48 h leads to an up-regulation of nox4 expression. this effect of pma is mediated by protein kinase cα, because it is preventable by the pkc inhibitor gö 6983 and by pkcα-sirna. the present study is aimed to investigate the signal transduction cascade downstream of pkcα. pma-induced nox4 up-regulation can be attenuated by pd 98,059 (an erk1/2 inhibitor), but not by sp 600125 (a jnk inhibitor), indicating in the involvement of erk1/2. consistently, pma treatment leads to a sustained activation of erk1/2, and sirnamediated knockdown of erk1/2 markedly reduces the pma-induced nox4 up-regulation. h89, an inhibitor of the mitogen-and stress-activated protein kinases (msks) has no effect on the pma-stimulated nox4 expression, indicating that msks are not the target molecules of erk1/2 in this scenario. on the contrary, knockdown of the transcription factor elk-1 by sirna significantly reduces the pma-induced nox4 up-regulation. in conclusion, erk1/2 and elk-1 are involved in the pkcα-induced nox4 up-regulation. determination of spontaneous mutation frequencies in normal human mammary gland tissue using the random mutation capture technique schmalbach k., lehmann l. university of wuerzburg section of food chemistry, am hubland, 97074 wuerzburg, germany annually, over 57,000 women develop breast cancer in germany. the accumulation of genetic mutations in mammary gland tissue during lifetime may be reasonable for developing breast cancer. in particular mutations in tumor suppressor genes, e.g. p53, seem to play an important role in developing cancer. up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (smf) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p53 gene in epidemiological studies. the only test with the potential to determine low smfs was the random mutation capture (rmc) assay, a genotype selective method which detects mutants that render the mutational sequence non-cleavable by the taqi restriction enzyme after accumulation of the target sequence. therefore, the suitability of the rmc assay to determine smf in p53 gene in normal human mammary gland tissue was evaluated. thus, the rmc assay was optimized concerning (i) dna isolation, (ii) pcr conditions, and (iii) amount of mammary gland tissue. (i) genomic dna from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery for cosmetic reasons, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) the target sequence in intron 6 of p53 gene was captured by hybridization with a complementary uracil-containing dna-probe synthesized via polymerase chain reaction (pcr), followed by magnetic separation from the remaining genomic dna. the copy number of the target sequence was quantified by competitive pcr. the number of mutants was detected after cleavage of the target dna with taqi by means of pcr with a primer set flanking the restriction site. (iii) with 2 g of normal mammary gland tissue a smf of 2.2±1.4x10 -7 per base pair was determined indicating the rmc assay suitable for smf determination. in conclusion, the smf in the p53 gene in normal human mammary gland tissue was determined for the first time, enabling the future investigation of factors influencing the smf during breast cancer development. cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (il-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (asm) cells, may contribute to the development of chronic obstructive pulmonary disease (copd). despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic amp (camp), little is known on its exact mechanism of action. we report here that next to the β2-agonist fenoterol, direct and specific activation of either exchange protein directly activated by camp (epac) or protein kinase a (pka) reduced cigarette smoke extract (cse)-induced il-8 mrna expression and protein release by human asm cells. cse-induced iκbαdegradation and p65 nuclear translocation, processes that were primarily reversed by epac activation. further, cse increased extracellular signal-regulated kinase (erk) phosphorylation, which was selectively reduced by pka activation. cse decreased epac1 expression, but did not affect epac2 and pka expression. importantly, epac1 expression was also reduced in lung tissue from copd patients. in conclusion, epac and pka decrease cse-induced il-8 release by human asm cells via inhibition of nf-κb and erk, respectively, pointing at these camp effectors as potential targets for antiinflammatory therapy in copd. however, cigarette smoke exposure may reduce antiinflammatory effects of camp elevating agents via down-regulation of epac1. polycyclic aromatic hydrocarbons (key marker substance benzo[a]pyrene (bap)) have been assumed to play a role in the development of bladder cancer. the objective of the present study was to unravel cellular and in particular cytoskeletal response to bap. to follow the sequential steps of chemical carcinogenesis the differential proteomic profile was analyzed at early and late time points. the study was carried out in a superficial human bladder cancer cell line (rt4) exposed to 0.5 µm bap, a subacute concentration based on results of proliferation (brdu) and dna damage (tunel) tests. cells of a human bladder cancer cell line (rt4) were exposed to 0.5 µm bap for 24 h (n=5), 4 wk (n=7) and 8 wk (n=3). proteins of whole cell lysate were separated by twodimensional electrophoresis (fig. 1) . differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight analysis. cortactin, actin and tubulin were immunohistochemical stained. changes in migration and colony forming ability were assessed by scratch wound-healing assay and soft-agar colony formation. results: by using several databases (uniprot, reactome, panther) the identified proteins were categorized into different functional classes such as mrna processing, translation, protein metabolic process, or several areas associated with the organization of the cytoskeleton. 45 % of the differentially expressed proteins after 24 h of treatment are involved in the processing of pre-mrna (20 %) and protein metabolism (25 %). this pattern changed after 8 wk of treatment. then, 48 % of the proteins affected the cytoskeleton whereas still 26 % were categorized to protein metabolism and only 7 % to pre-mrna processing. in the immunhistochemical staining, the treated cells appeared after 8 wk of exposure larger and rounder predominantly due to the modifications of the actin cytoskeleton. merged images of actin and cortactin revealed that both proteins colocalized in invadopodiae. after 8 wk, a higher number of treated cells moved toward the centre of the wound and they formed more soft-agar colonies compared to vehicle conditions, suggesting a transformation of the cells. in conclusion, bap exposure causes in an early phase an activation of the spliceosome which can led to an epithelial-tomesenchymal transition. two coordinators of a cell-type-specific splicing program, epithelial splicing regulatory proteins 1 and 2, are currently being validate by pcr. fused master gel : representative 2-de gel of rt4 cells exposed to 0.5 µm bap for 8 wk. protein spots which were differentially expressed compared to control and identified were marked. cannabinoids stimulate mesenchymal stem cell migration via a mitogen-activated protein kinase pathway schmuhl e. 1 , ramer r. mesenchymal stem cells (mscs) are known to be involved in various regenerative processes such as cardiac, ocular, skin and bone tissue healing. however, little is known about the pharmacotherapeutical options aiming at tissue healing steps such as the mobilization and homing of mscs. here, we show that cannabidiol (cbd), a nonpsychoactive cannabinoid, stimulates the migration of human adipose-derived mscs in both boyden chamber and in vitro scratch wound assays. in boyden chambers cbd (0.01 -3 µm) was shown to promote cell migration in a time-and concentration dependent manner. this promigratory action was inhibited by am-630 (cb2 receptor antagonist) and by o-1602 (g protein-coupled receptor [gpr] 55 agonist). moreover, cbd activated the mitogen-activated protein kinase (mapk) pathway as evidenced by increased phosphorylation of extracellular signal-regulated kinase (erk) 1/2. blockade of erk activation by pd98059 prevented cbd-stimulated msc migration, whereas inhibition of p38 mapk by sb203580 was inactive in this respect. furthermore, am-630 and o-1602 were found to attenuate cbd-induced erk activation. an erk-dependent promigratory action was likewise demonstrated for the phytocannabinoid ∆ 9 tetrahydrocannabinol and for the hydrolysis-stable anandamide analogue r(+)methanandamide. we conclude that cbd promotes msc migration via receptordependent erk activation, possibly contributing to tissue healing. the duffy antigen receptor for chemokines (darc) binds promiscuously many inflammatory chemokines without showing intracellular signal transduction. it is mainly expressed on endothelial cells of postcapillary venules and on red blood cells, where it acts as a transendothelial transporter of chemokines and as a chemokine sink, respectively. surprisingly, as shown for human and mouse brain, darc is also expressed at high density in the cerebellum. however, nothing is known about the function of darc in this location. we addressed this question by subjecting c57bl/6 wildtype and darc-deficient mice to a series of behavior experiments including morris water maze-, elevated plus maze-, rotarod-and actometer tests. while the results from the water maze experiments are ambiguous, elevated plus maze trials show a strong aversion of darc -/mice to walk to the end of the open arm, which is consistent with anxiety-like behavior. moreover, darc -/mice show greatly reduced locomotor activity, which is at least partly caused by episodes of reduced mobility occurring more frequently than in the corresponding wildtype controls (elevated plus maze, actometer). finally, darc -/mice spend a significantly reduced time on the rotating rod compared to c57bl/6 wildtype controls, which may indicate an impaired cerebellar function. we conclude that darc in fact modulates brain function. surprisingly, this appears to be happening under homeostatic conditions, although darc binds for the most part to inflammatory chemokines. it remains to be elucidated, how this effect can be caused by a non-signaling chemokine receptor. it may be an indirect consequence of altered brain chemokine concentrations or of as yet unknown signaling pathways activated by darc. transporter gene expression in human head-neck squamous cell carcinoma and epigenetic regulation mechanisms schnepf r. 1 hals-nasen-ohren-klinik, kopf-und halschirurgie, friedrich-alexander-universität erlangen-nürnberg, waldstraße 1, 91054 erlangen, germany background: membrane transporters may affect the disposition and thereby treatment efficiency of anticancer drugs in human head-neck squamous cell carcinoma (hnscc). the gene expression profile of transporters in hnscc, however, is unknown and was evaluated in this study. moreover, we evaluated mechanisms by which transporters are regulated in hnscc. we focused on the role of the nuclear pregnane x receptors (pxr, nr1i2) and epigenetic mechanisms. methods and results: real-time rt-pcr revealed a significantly increased mrna expression of slco1a2 and slco1b3 and a significantly decreased expression of transporters such as slco2b1, slco2a1 and abcc3 in human hnscc tissue samples compared to adjacent normal mucosa. moreover, an association between slco2b1 mrna levels in tumor tissues and five-year survival of hnscc patients was observed (χ 2 =6.59; p=0.010; n=34). bisulfite sequencing revealed that promoter cpg islands of abcc3 and slco2a1 were not methylated and thus these genes were not epigenetically silenced in hnscc tissues. in the hnscc-derived umscc-1 and scc-15 cell lines, transcript expression of transporters (e.g., abcc3, slco2a1; p<0.001) and pxr (nr1i2; p<0.001) was markedly induced by the dna methyltransferase inhibitor decitabine. cotreatment with the prototypical pxr activator rifampicin significantly reversed decitabine-induced abcc3 and slco2a1 expression. conclusions: transporter expression profiles significantly differed between hnscc and normal mucosa and expression levels of slco2b1 may serve as a marker for prognosis. modulation of the epigenome with the anticancer drug decitabine substantially affects transporter expression in umscc-1 and scc-15 cells, suggesting epigenetic regulation mechanisms. moreover, interactions between epigenetic and nuclear receptor-mediated mechanisms in transporter regulation occur. this work was in part supported by the johannes und frieda marohn foundation. the role of hcn2 in neuropathic and inflammatory pain schnorr s. 1 , eberhardt m. the pacemaker current ih is carried by hyperpolarization-activated cyclic nucleotidegated cation channels (hcn1-4) and contributes to cellular excitability in the heart and the nervous system. hcn1 and hcn2 are the two most abundant hcn subunits in peripheral sensory neurons with hcn2 being the prevalent isoform in nociceptive small sized c-fibre dorsal root ganglion (drg) neurons. we examined the role of hcn2 for peripheral sensitization and spontaneous neuronal activity in neuropathic and inflammatory pain. we generated a conditional deletion of hcn2 by using a nociceptor specific cre-transgene driven by the nav1.8 promoter. the nociceptor-specific knockout of hcn2 in drg neurons (nosphcn2ko) was confirmed by quantitative rt-pcr and western blot. immunohistochemical staining revealed that the deletion of hcn2 was mainly restricted to small sized drg neurons. the conditional loss of hcn2 resulted in a significant reduction of ih positive small diameter drg neurons pointing to a central role of this isoform to the hcn current in nociceptive neurons. behavioral studies showed that the lack of hcn2 did not influence basal pain responses but led to a significant reduction in mechanosensation in both neuropathic and inflammatory pain models. however, thermosensation of the mutants was only decreased in neuropathic pain conditions. in wild-type animals, intraperitoneal, intraplantar and even intrathecal injection of the hcn channel blocker zd7288 nearly eliminated tactile allodynia caused by inflammation in contrast to thermal hyperalgesia which remained unaffected. in contrast, pain thresholds in nosphcn2ko mice did not significantly increase after pharmacological block of ih. additionally, experiments revealed that the inflammatory condition induced an upregulation of hcn2 protein in the spinal dorsal horn compared to saline injected mice. our results suggest that hcn2 might be a new target in the treatment of neuropathic and inflammatory pain. the proper functioning of the central, as well as the peripheral nervous systems is of outstanding importance to the survival and well-being of humans. yet, the integrity of neuronal systems is constantly challenged by a plethora of environmental and occupational toxins. some of these toxins preferentially target neural cells. these neurotoxins can exert their devastating effects by very different modes of action. neurotoxins may induce apoptosis or necrosis of neurons, or interfere with axon growth and elongation. these processes can be identified by specialized in vitro tests. furthermore, neurotoxins have been described to alter glial function which may compromise the viability of surrounding neurons. as another important mode of action, several neurotoxins act on neurotransmitter receptors, thereby altering signal propagation within neuronal networks. countless natural and synthetic substances have been characterized for their effects on neurotransmitter receptors and today can be used for detailed studies of receptor function. however, environmental toxins of anthropogenic origin and occupational toxins that both represent constant sources for human exposure are still poorly studied with respect to their effects on neurotransmitter receptors. thus, the need for a better understanding of the susceptibility of neurotransmitter systems for toxic effects exerted by these substances is of outstanding importance for the protection of human health. here, we introduce an imaging-based approach for the screening of the effects of potential and known neurotoxins on neurotransmitter receptors of intact cells in vitro. different neuronal cells were tested for their sensitivities for classical neurotransmitters using life-cell imaging experiments. in more detail, we examined the proportion of responding cells and determined the ec50 values for the most prominent neurotransmitters in cell lines widely used for in vitro neurotoxicity studies on the one hand, namely sh-sy5y and lumes cells, and primary mouse neurons on the other hand. with these data at hand, we are now able to identify and characterize the effects of neurotoxins on receptor function in chronic, as well as acute exposition paradigms. the use of an in vitro imaging-based physiological test system is at the interface between non-functional in vitro approaches and in vivo toxicity tests, thus, giving mechanistic insight into neurotoxic processes without requiring animal experiments. apomorphine acts on trpa1 channels scholze a., schaefer m., hill k. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany apomorphine is a non-narcotic derivative of morphine which acts as a dopamine agonist and is clinically used to treat "off-states" in patients suffering from parkinson´s disease. adverse effects of apomorphine treatment include dopaminergic effects such as nausea, but also ulceration and pain at the injection site. we wanted to test whether an activation of trp (transient receptor potential) channels in sensory neurones contributes to the perception of pain after apomorphine injection. while the warm/heat receptors trpv1, trpv2, trpv3, and trpv4 and the cold receptor trpm8 were insensitive towards apomorphine treatment, trpa1 could concentration-dependently be modulated by apomorphine. low micromolar apomorphine concentrations potently activated heterologously expressed trpa1 channels in a stably transfected cell line (hek293-trpa1), as well as natively expressed trpa1 in cultured dorsal root ganglion neurones. on the other hand, when using higher concentrations of apomorphine, we observed inhibition of trpa1 activity. previous studies have shown that subcutaneously administered apomorphine produces a biphasic dose response relationship in rats, inducing hyperalgesia at low doses whereas high doses of the substance cause antinociception. from our studies we conclude that such in vivo effects of apomorphine are presumably mediated by activation/inhibition of trpa1 expressed in sensory neurones agonist binding to a g protein-coupled receptor (gpcr) induces a conformational change of the receptor protein, which results in the activation of receptor-associated heterotrimeric g proteins [1] . in radioligand binding studies, conducted to investigate ligand binding to specific gpcrs, receptors are usually probed with radioantagonists. as in other gpcrs [2] , agonists of the muscarinic m2 receptor exhibit biphasic kinetics and biphasic competition curves with radioantagonists, indicating a more complex situation probably caused by g protein interactions. here, we present a detailed study of the binding of agonists to muscarinic m2 receptors including the novel super-high affinity agonist iperoxo and a differential chemical knockout of g proteins. in addition to membrane homogenates living cells were employed. we demonstrate that the high affinity fraction in biphasic curves does not differ between selected full agonist and is sensitive to pertussis toxin, thus indicating that this receptor population is associated with gi proteins. however, despite promiscuous signalling properties of m2 receptors, the low affinity fraction is not associated with any other g protein, since low affinity binding is insensitive to high concentrations of guanylnucleotides and cholera toxin. moreover, high affinity agonist binding appears solely in membrane homogenates but not in experiments conducted with living cells, probably due to their high intracellular concentration of guanylnucleotides. taken together the chemical knock-out of g proteins revealed that the high affinity binding of agonists in membrane homogenates is associated with the interaction of the muscarinic m2 receptor with gi proteins. the low affinity binding cannot be related to another g protein, although the muscarinic m2 receptor exhibits promiscuous g protein signalling properties. interestingly data obtained with living cells do not reveal any high affinity binding of agonists. prolonged stress leads to a dysregulation of the hypothalamus-pituitary-adrenal (hpa)axis and may affect the sensitivity of pain perception. however, it is not yet known whether the alterations of hpa-axis and increased pain sensitivity are related. to create a long lasting stressful situation, male wistar rats were exposed to a restraint-stress for 1 h daily over a period of two weeks. the effect of stress on the hpa-axis was determined by adrenal morphology and stress hormone levels, the influence on mechanical pain sensitivity was evaluated by the randall-selitto paw pressure test. on day 15 the animals exhibited a significant mechanical hyperalgesia. they also showed increased acth and corticosterone plasma levels and an enlarged zona fasciculata of the adrenal gland, indicating a dysregulation of the hpa-axis. for testing the correlation of hpa-axis dysregulation and hyperalgesia a persistent increase in plasma corticosterone in wistar rats was generated by the administration of corticosterone via the drinking water for two weeks. these animals also showed an increased mechanical nociceptive sensitivity with an accompanied decrease of the adrenal glands and reduced acth levels. the results show that chronic stress leads to a dysfunction of the hpa-axis with an accompanied mechanical hyperalgesia which can be mimicked by oral administration of corticosterone. thus, this in-vivo test system may provide a new animal-friendly pharmacological model for stress-related pain disorders. the alternaria mycotoxins aoh and ame induce cyp1a1 and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor mycotoxins are secondary metabolites of fungi including the genus alternaria (black mold). alternaria fungi are known to infest different types of foodstuffs and produce diverse toxins amongst them the mycotoxins alternariol (aoh) and alternariol methyl ether (ame) which are potential carcinogens. as planar compounds, aoh and ame are preferentially metabolized by cytochrome p450 (cyp) 1a1 and 1a2. the most prominent regulator of cyp1a1 is the dimeric transcription factor complex ahr/arnt, which is activated by planar ligands. therefore we studied the activation of ahr/arnt by aoh and ame and monitored cyp1a1 induction in murine hepatoma cells (hepa-1c1c7). indeed, aoh and ame enhanced the levels of cyp1a1 in hepa-1c1c7 cells but not in cells with inactivated ahr (hepa-1c1c12) or arnt (hepa-1c1c4). furthermore, we studied the cytotoxicity of aoh and ame. by using a fluorescence-based microscopic readout we measured effects on cell counts, apoptosis, senescence and micronuclei formation. both aoh and ame reduce the cell number and the cell nuclei show drastic morphological changes e.g. enlargement after aoh treatment or micronuclei formation. the observed effects where, except for the induction of apoptosis, independent of ahr/arnt. in summary, aoh and ame activate the ahr/arnt pathway to induce cyp1a1 expression and apoptosis. however, the predominant cytotoxic effect of aoh and ame in hepatoma cells is a profound reduction in cell numbers, which is independent of the ahr/arnt pathway. special purpose databases are the first place for researchers in the life sciences to obtain expert curated data. naturally, such resources are limited in terms of timeliness and comprehensiveness. the literature database pubmed alone lists more than 20,000,000 scientific abstracts, and 700,000 are newly added every year. the protein sequence database uniprotkb stores over 10,500,000 sequences, a hundred times more than ten years ago. turning these data into meaningful information and making it accessible to both humans and computers have become an essential part of biological discovery and biomedical research. text mining techniques have proven useful to extract the missing links in areas such as drug-target and drug-disease prediction, the extraction of mutation-phenotype associations, or the prediction of protein-protein and protein-ligand interactions. by systematically extracting information from available literature, reports or patents, text mining techniques can help to refine existing categorical knowledge stored in databases, and hence will support drug repositioning, the discovery of novel cancer biomarkers, or help to understand causes of hereditary diseases. in the area of regulatory toxicology we developed go3r, the first knowledge-based search engine for alternative methods to animal experiments. the system not only helps retrieving information on the availability of alternative methods that allows for replacing, reducing or refining animal experiments, but also provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information and data. the up-to-date taxonomicstructured "table of contents" provided by go3r allows for search in the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. the semantically enriched platform supports the user during the query formulation, allows for bibliographic analysis, and reveals existing relations to replacement, reduction, and refinement of animal experiments. impaired cardiac excitation-contraction-coupling in mice with complete inactivation of the crem gene schulte j. s., tekook m., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany the structurally related transcription factors camp response element binding protein (creb) and camp response element modulator (crem) mediate a regulation of gene transcription in response to camp and are expressed in the heart. mice with complete inactivation of crem display impaired cardiac contraction and relaxation, decreased expression of serca and down-regulation of β1-adrenoceptors. to elucidate the underlying functional mechanisms on the cellular level we here investigated cellular electrophysiology and ca 2+ -cycling in ventricular cardiomyocytes from crem ko mice (ko). adult ventricular cardiomyocytes were isolated from ko and wildtype (wt) mice (age 16-20 weeks) and subsequently used for experiments within 6 hours after isolation. action potentials (aps) were recorded from ventricular cardiomyocytes (perforated patch, whole cell current clamp inactivation of crem seems to have no consequences for ap duration and possibly associated ion channels but leads to impairment of ca 2+ cycling and sarcomere shortening under basal conditions well explaining the previous findings in vivo. our results show that crem is essential for a regular excitation-contraction coupling in the mouse heart. (supported by the izkf münster) new mechanistic insights in no/cgmp actions in the vasculature schulte k., koesling d., universitätsstr. 150, 44781 bochum, germany hypertension, a major risk factor for cardiovascular diseases, is associated with vascular changes resulting in increased vascular contractility und vascular peripheral resistance. a prominent factor in the development and maintenance of hypertension is the reninangiotensin-aldostrerone system. angiotensin ii (ang ii) is the main mediator of this system and a powerful vasoconstrictor. ang ii increases the intracellular ca 2+ concentration thereby activating myosin light chain (mlc) kinase, which enhances mlc phosphorylation and subsequent vascular contraction. opposite to angii-induced vascular contraction, no/cgmp pathway promotes vascular relaxation by decreasing ca 2+ concentration and lowering mlc phosphorylation. responsible for mlc dephosphorylation is the mlc phosphatase (mlcp), whose activity is regulated by different phosphorylations. phosphorylation of mlcp via rhoa-activated rho-kinase enhances phosphatase activity while phosphorylation via the cgmp-dependent protein kinase has been proposed to decrease enzymatic activity. to investigate the interplay of angii with the no/cgmp pathway, we treated wild-type and ko mice lacking the cgmp forming no receptor, no-gc1, with angiotensinii (1.44 mg / kg bw / d) for two weeks. in addition to various cardiovascular parameters, physiological changes in vascular reactivity of aortic rings of angii-treated wt and no-gc1 ko mice were assessed in organ bath experiments and correlated with biochemical parameters as the phosphorylation state of mlc, mlcp and rho-kinase activities examined by immunoblot analysis. analysis of cgmp levels revealed that angii treatment decreased cgmp in wt mice to levels comparable to those of the ko mice which were unaltered by the treatment. our study will provide further mechanistic insights in the molecular interactions between constrictor and dilator stimuli in the vasculature. nanomaterials are already used today and offer even greater use and benefits in the future. the progress of nanotechnology must be accompanied by investigations of their potential harmful effects. for airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. while standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. we have investigated two genotoxic endpoints -alkaline comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow -in a combined study 72 hours after a single instillation of 18 µg gold nanoparticles (np) into the trachea of male adult wistar rats. the administration of three test materials differing only in their primary particle size (2-, 20-and 200-nm) did not lead to relevant dna damage in the mentioned tests. the measurement of clinical pathology parameters in bronchoalveolar lavage fluid (balf) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. minor histopathology findings occurred in the lung of the animals exposed to 20-nm and 200-nm sized nanomaterials. in conclusion, under the conditions of this study the different sized gold np tested were non-genotoxic and showed no systemic and local adverse effects at the given dose. platelet dense granule secretion mediates platelet-dependent enhancement of tumor cell transmigration and formation of metastases schumacher d., strilic b., wettschureck n., offermanns s. mpi für herz-und lungenforschung offermanns, ludwigstr. 43, 61231 bad nauheim, germany tumor cell metastasis to distant organs is the primary cause of mortality in cancer patients. tumor cells leave the primary tumor, intravasate, survive in the circulation and extravasate through the endothelial cell layer to grow in the target organ. it has long been known that blood platelets play an important role in tumor cell survival and dissemination, but the mechanism by which platelets promote metastasis remained unclear. given that platelets are found closely associated with tumor cells shortly after vascular arrest, we explored whether platelets can facilitate the transmigration of tumor cells through the endothelium and thereby promote extravasation of tumor cells into the organ parenchyma. the ability of various mouse and human tumor cells like lewis-lung carcinoma cells (llc1), b16f10 melanoma cells or human neuroblastoma cells (sh-sy5y) to transmigrate through an endothelial cell layer was strongly enhanced by seeding tumor cells together with mouse or human platelets onto the endothelial cell layer. this indicates that platelets facilitate tumor cell transmigration in vitro. we found that platelet granule secretion is involved in this process as supernatant from platelets incubated with tumor cells but not from resting platelets was sufficient to enhance tumor cell transmigration. additionally, no platelet-mediated increase of tumor cell transmigration was observed in dense granule secretion-defective platelets of munc13-4 deficient mice. thus, dense granule secretion is required for platelet-dependent tumor cell extravasation in vitro. while the growth and weight of primary tumors after subcutaneous injection of llc1 and b16 cells was indistinguishable between wild-type mice and animals lacking munc13-4, the number of metastases in the lung was strongly reduced in munc13-4-deficient animals. the strong decrease in formation of metastases in munc13-4 deficient mice was also observed after i.v. injection of llc1 and b16f10 cells. thus, platelet dense granule secretion plays a critical role in tumor cell metastasis by enhancing tumor cell transmigration through the endothelial cell layer. formation of dna adducts in mouse tissues by the brassica ingredient 1methoxy-3-indolylmethyl glucosinolate and its break-down product 1-methoxy-3indolylmethyl alcohol schumacher f. 1 , engst w. glucosinolates are secondary metabolites present at substantial levels in cruciferous vegetables, such as broccoli and cabbage. after injury of plant tissue they are activated by the enzyme myrosinase to form various electrophilic degradation products like isothiocyanates. we previously showed that 1-methoxy-3-indolylmethyl (1-mim) glucosinolate (or neoglucobrassicin) is a potent genotoxicant in bacterial and mammalian cells after activation by myrosinase. the induction of mutations could be correlated with the formation of dna adducts [1] . we have identified and synthesized the major dna adducts n 2 -(1-mim)-dg and n 6 -(1-mim)-da. moreover, we developed a highly sensitive uplc-esi-ms/ms method for selective mrm quantification of these adducts using stable-isotopic labeled adducts as internal standards. while the plant enzyme myrosinase is probably almost completely inactivated after cooking the vegetables, the glucosinolates reach the gut mostly intact due to their good heat and ph stability. enzymes of individual intestinal bacteria are able to cleave the glycosidic bond of the glucosinolates, which leads to the formation of reactive metabolites within the gut lumen. we were able to detect significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in a dose-dependent manner in the large intestine of mice treated orally with isolated 1-mim glucosinolate. the peak levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in the murine large intestine were reached 8 h after a single administration of 600 µmol 1-mim glucosinolate/ kg body weight. the oral application of the relatively stable metabolite 1-mim alcohol to mice led to the formation of identical dna adducts. this benzylic alcohol can be activated by sulfotransferases to an electrophilic sulfo conjugate. in contrast to the intact glucosinolate the orally administered 1-mim alcohol generated significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da not only in the large intestine but also in other tissues, such as the liver, of mice. [1] h. glatt, c. baasanjav-gerber, f. schumacher, b. h. monien, m. schreiner, h. frank, a. seidel, w. engst, chem.-biol. interact., 192 (2011) human pregnane x receptor genotype of the donor but not of the recipient is a risk factor for delayed graft function after renal transplantation schwab m. 1, 2 , schaeffeler e. delayed graft function (dgf) is an important immediate complication in renal transplantation significantly contributing to decreased long-term allograft survival. in addition to donor-and recipient-related risk factors altered renal excretion of xenobiotics by membrane transporters may influence dgf as well. using recipients' and donors' dna, we assessed the impact of genetic variants on dgf for the transporter proteins, pglycoprotein (abcb1) and multidrug resistance protein 2 (abcc2), and the nuclear pregnane x receptor (pxr/nr1i2), regulating the transcription of drug metabolizing enzymes and membrane transporters. in our local cohort of transplant patients (n=178) dgf occurred in 27.5 %. logistic regression analysis using four different genetic models (i.e. co-dominant, dominant, recessive and log additive) indicates that only the donor's pxr rs2276707 8055tt genotype was significantly associated with dgf (recessive model: or, 9.0; 95%ci, p=0.004 unadjusted) , even after correction for multiple testing (p=0.035 holm-adjusted). when we performed multivariate analysis including genetic and 16 clinical co-variates (i.e. age, gender, hla mismatches, panelreactive antibodies, immunosuppression using cni, t cell-depleting agents, anti-il-2 receptor antibody and steroids, cold or warm ischemia time, living vs deceased donors or graft loss) again dgf was significantly associated only with the pxr rs2276707 tt genotype of the donor (recessive model: or, 16.08; 95% ci, 2.0-129.46; p=0.0046 unadjusted) which held true after correction for multiple testing (p=0.04). for abcc2 variants only the donor rs17222723 3563t>a genotype correlated with dgf by univariate (or, 4.65; 95%ci, p=0.005 unadjusted) as well as by multivariate analysis (or, 5.5; 95%ci, p=0 .01; table 4 ) but not after correction for multiple testing (p=0.12). for variants in the abcb1 gene no significant associations with dgf were detected for both the donor's and the recipient's genotype. in summary, our findings suggest for the first time that pxr may be a risk gene for the development of dgf independently from previously identified risk factors. supported by the robert-bosch foundation, stuttgart, germany, the bmbf grant 03is2061c (berlin, germany) and by the ferdinand eisenberger grant of the german society of urology (id krs1/fe-10). formation, morphology and structural requirements for formation of microtubule protrusions by clostridium difficile toxin cdt schwan c., kruppke a. s., nölke t., aktories k. institut für experimentelle und klinische pharmakologie und toxikologie i, albertstr. 25, 79104 freiburg, germany clostridium difficile is an anaerobe, gram-positive pathogen. it causes antibioticassociated diarrhoea and pseudomembranous colitis by production of the rho gtpaseglucosylating toxins a and b. recently emerging hypervirulent clostridium difficile strains additionally produce the binary adp-ribosyltransferase toxin cdt (clostridium difficile transferase). cdt is taken up via receptor-mediated endocytosis after binding to the lipolysis stimulated lipoprotein receptor (papatheodorou et al., pnas 2011) . in the cytosol, cdt adp-ribosylates actin at arg 177, thereby actin polymerization is blocked, resulting in disruption of the f-actin network. cdt and other binary actin-adp-ribosylating toxins induce redistribution of microtubules in the cell interior and formation of long (>150 µm) microtubule-based protrusions at the surface of intestinal epithelial cells which increase bacterial adherence (schwan et al., plos pathog 2009 ). the clostridial actin-adp-ribosyltransferases influence the dynamicity of microtubules and their capture at the cell cortex by indirectly affecting different microtubule regulating proteins like clasp2 and acf7. besides the influence of cdt on microtubule regulatory proteins, the formation of protrusions depends on plasma membrane composition. depletion of cholesterol, the breakdown of sphingomyelin or inhibition of sphingolipid-synthesis reduce the formation of microtubule-based protrusions. surprisingly, most of the cdt-induced processes contain membrane-tubules derived from the endoplasmatic reticulum (er). the remodeling of the er is microtubule dependent and is mainly mediated by stim1 that usually functions as a calcium sensor in the er and activates the store operated orai1 calcium ion channels in the plasma membrane. the data suggest that toxin-induced changes of the microtubule system including alterations of the er, may affect trafficking and er-dependent signalling. bilobalide is a neuroprotective constituent of ginkgo biloba with an unknown mechanism of action. in the present study, we first used microdialysis in mice to evaluate changes in the extracellular fluid of the brain during and after stroke. microdialysis probes were implanted into the striatum of cd-1 mice, and dialysates were obtained while a monofilament was inserted for 60 min via the common carotid artery (cca) to block perfusion through the middle cerebral artery (mca). while glucose levels dropped immediately upon middle cerebral artery occlusion (mcao), glutamate concentrations in the microdialysates -as measured with a cma 600 analyzer -rose extensively during ischemia to more than 2000% of baseline level. both glucose and glutamate levels recovered rapidly when mcao was terminated after 60 min. when bilobalide (10 µm) was perfused into the striatum through the microdialysis probe during mcao, glucose levels dropped but the neurotoxic rise of glutamate was significantly attenuated and reached only 500% of baseline level (p<0.01). in the following experiments, we investigated the activity of mitochondria in ischemic brain. ischemia was induced by mcao, and ischemic as well as "healthy" tissue from the opposite hemisphere was obtained. mitochondria were isolated and mitochondrial respiration was monitored using the oroboros ® oxygraph. significant deficits of respiration were observed after ischemia. in the healthy hemisphere, the respiratory states (leak i+ii, complex i+ii+iv, oxidative phosphorylation (oxphos) and electron transport system (ets) capacity) showed a decrease of oxygen consumption to 60-70% of sham-operated mice. in the ischemic hemisphere, several values were lower at 40% of sham-operated mice (leak i+ii, complex ii+iv,oxphos and ets) whereas complex i showed a remarkably low respiratory capacity of 16% of baseline. direct addition of bilobalide (10 µm) to post-ischemic mitochondria caused a 2-fold increase of complex i activity in vitro. pretreatment of mice with bilobalide (10 mg/kg i.p.) one hour before mcao caused a significant, 3-fold improvement of complex i respiration when measured ex vivo. these data clearly indicate that bilobalide targets mitochondrial processes within the respiratory chain, preserving complex i function during ischemia. this action likely explains its neuroprotextive activity in vivo. unreacted resin monomers such as 2-hydroxyethyl methacrylate (hema) are environmental stressors released from dental composites after incomplete polymerization. the production of reactive oxygen species (ros) is a major response of cells to monomer exposure. moreover, adverse effects of monomers including delayed cell differentiation or mineralization processes, dna damage or apoptosis are associated with increased ros production. the intracellular redox homeostasis is controlled by the major non-enzymatic antioxidant glutathione (gsh), and antioxidant enzymes. here, we hypothesized that cells exposed to hema responded by a differential expression of antioxidant enzymes such as superoxide dismutase (sod-1), catalase (cat) or glutathione peroxidase (gpx1/2). raw246.7 mouse macrophages were exposed to hema (0-8mm) for 24h, and protein expression was analyzed by western blotting. to study the influence of intracellular gsh on enzyme expression, gsh synthesis was reduced by the inhibitor buthionine sulfoximine (50µm bso), or enhanced by 2-oxothiazolidine-4-carboxylate (5mm otc) and n-acetyl cysteine (10mm nac). expression of sod-1 found in untreated cultures was decreased in the presence of hema and even further reduced by bso. in contrast, nac counteracted hemainduced inhibition of sod-1 expression. cat expression was not detected in untreated cells, however, the enhanced expression of cat in cells exposed to hema indicated the decomposition of abundant levels of hydrogen peroxide. the minor influence of bso or otc showed that expression of cat was independent of gsh levels while a decrease of hema-induced cat expression in the presence of nac indicated reduced oxidative stress. gpx1/2 was expressed in untreated cultures, and its down-regulation by bso indicated that this enzyme was primarily responsible for h2o2 decomposition. the inhibitory effect of hema on gpx1/2 expression was enhanced by bso but counteracted by otc or nac. these findings indicate that h2o2 is the predominant reactive oxygen species generated in the presence of dental resin monomers like hema. abundant h2o2 production leads to the activation of cat expression and a feed-back inhibition of sod-1 expression. the hema-induced reduction of gpx1/2 expression is most likely a consequence of reduced gsh levels because of the formation of glutathione disulfide (gssg) or by gsh-hema adducts. the life-threatening effects of certain organophosphorus compounds such as soman or fenamiphos cannot be antagonized adequately by the treatment with atropine and oximes. alternative approaches are necessary. since the adequate restoration of disturbed muscle function is considered to be life-saving, a model is needed for screening of potentially therapeutic substances. an established model for the development of such new therapies is the diaphragm preparation. however, this model requires a large number of animals and experimental available time frame is limited to some hours. here, the organotypic nerve-muscle co-culture may be an appropriate alternative, because a large number of specimens with low numbers of animals and a long period of investigation over several days is possible. in the present study, the restoration of vx paralysed muscle function with obidoxime was investigated by using both models. slices of spinal cord and muscle tissue were dissected from mice embryos (e 14-15) , fixed to coverslips and incubated in roller tubes for about 2-4 weeks. spontaneous muscle activity was recorded by video microscopic techniques and was quantified offline. muscle force production in mice diaphragm preparations was elicited by indirect field stimulation technique in a 12 chamber organ bath and quantified as time-force diagrams (auc) that were expressed as relative changes of the muscle force compared to the control data. application of the nerve agent vx (0.75 µm) resulted in a strong reduction of muscle activity in the co-culture and of muscle force production in the diaphragm muscle. after obidoxime (10 µm) was added spontaneous muscle activity in the co culture recovered from 0.45 ± 0.24 hz to 1.67 ± 0.24 hz (control 1.79 + 0.22 hz) . muscle force remained stable over the next days. the vx-blocked muscle force of diaphragm was restored to 69.9 ± 21.3 % by obidoxime compared to control. muscle force production after indirect stimulation was stable for 6 hours only. our results suggest that the organotypic nerve-muscle co-cultures may be an appropriate tool for the screening of new therapeutic approaches in restoration of blocked neuromuscular transmission. moreover, the model offers an additional advantage as long-term experiments may be performed and pre-and postsynaptic effects may be assessed directly. additionally, the number of experimental animals could be reduced. the modulation of gene expression by the transcription factor crem (camp responsiveelement modulator) represents a fundamental mechanism of gene control in response to elevation of intracellular camp levels. in vascular smooth muscle cells (vsmcs) crem is involved in the regulation of cell proliferation and apoptosis supporting its relevance for vascular proliferative diseases. mice with a global inactivation of crem (cko) showed a significant increase in neointima formation after ligation of the carotid artery and an increase of atherosclerotic plaque formation after high fat diet on an apoe knockout background compared to wildtype controls (wt). on the cellular level a crem deficiency was associated with a 1.3 fold increased proliferation rate of primary vsmcs after stimulation with the platelet-derived growth factor (pdgf; n=10 from 6 isolations). microarray analysis and subsequent realtime-rt-pcr validation revealed that the alpha-type platelet-derived growth factor receptor (pdgfra) the regulator of g-protein signaling 5 (rgs5) and peptidylprolyl isomerase a (ppia) were 1.5-2.5 fold upregulated in pdgf treated cko vsmcs compared to wt vsmcs (n=6). transcripts of rgs5 (2.6 fold, ) and ppia (1.8 fold, were also upregulated in the carotid artery of cko mice in comparison to wt mice (n=10-12). we conclude that crem deficiency is associated with transcriptional changes of rgs5, pdgfra, ppia, which might explain the increased proliferation rate in cko vsmcs and the increased responsiveness to pathophysiological conditions. (supported by the izkf münster). the role of non-catalytic p101 and p87 subunits in regulating phosphoinositide 3kinase γ by gβγ and h-ras shymanets a. 1 phosphoinositide 3-kinase γ (pi3kγ) controls a plethora of cellular responses. pi3kγ, a heterodimer formed by non-catalytic p101 or p87 and catalytic p110γ subunits, is regulated by gβγ and ras. earlier we speculated that p101 binds to gβγ to translocate cytosolic pi3kγ to the plasma membrane, enabling direct activation of p110γ (brock et al., j. cell biol. 2003) . however, the p87 subunit does not function as gβγ adapter (kurig et al., pnas 2009) . since the impact of each non-catalytic subunit in regulating p110γ by gβγ and ras still remains elusive, we studied their role in detail. gβ1γ2 variants harbouring mutations in positions involved in interaction with gα subunit were purified from sf9 cells and tested for their ability to activate pi3kγ. we observed that p101, but not p87, was able to rescue the stimulatory activity of gβ1γ2 mutants incapable to activate p110γ (shymanets et al., biochem. j. doi:10 .1042/bj20111664). to further study the functional impact of the non-catalytic subunits on pi3kγ regulation, we have designed phospholipid vesicles containing similar amounts of recombinant pi3kγ variants. although p87/p110γ exhibited stronger sensitivity to gβ1γ2 than p110γ, the activity of vesicles-associated p101/p110γ was significantly higher as compared to vesicles-associated p87/p110γ or p110γ in the absence and in the presence of gβ1γ2. to study an effect of ras proteins on pi3kγ variants, recombinantly expressed h-ras was purified from sf9 cells. the posttranslational processing and lipidation of the protein was verified by mass spectrometry analysis. the impact of h-ras on regulation of p87/p110γ and p101/p110γ differed, which may explain integration of pi3kγ variants in different signalling pathways. taken together, p87 and p101 subunits implement discrete functions in respect to (i) membrane recruitment of pi3kγ and (ii) regulation of enzymatic activity by gβγ and h-ras. preparation of consolidated exposure scenarios for mixtures under reach sica m., dorn s., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany under reach, formulators of mixtures need to include substance-related information into extended safety data sheets (esds), if mixtures are classified as dangerous according to the dangerous preparation directive (directive 1999/45/ec). one way to add information on substances into esds of mixtures is to generate exposure scenarios (ess) for mixtures. in order to fulfil this task, two approaches have been developed for the identification of the risk-determining substances (lead substances) in the mixtures: the critical component approach (cca) relies on dnels and pnecs for all substances, their concentrations in the mixtures as well as substance and use-specific availability parameters (echa, 2008) . in contrast, the dpd+ method is based on the legislation for classification of preparations (directive 1999/45/ec). the dpd+ method defines a lead substance for each route of human exposure and for the aquatic environment (cefic, 2010) . however, each of these methods has certain limitations. the aim of the present work is to improve the preparation of consolidated ess for a number of mixtures and provide information about their safe use. to this end, we first adopted information on risk management measures (rmms) and operational conditions (ocs) of the lead substances using the dpd+ methodology. at the same time, we considered the specific conditions of use of the mixtures (e.g. spraying, brush painting). we then conducted risk assessments by deriving dnels for the mixtures and using exposure modelling tools recommended under reach (e.g., ecetoc tra, riskofderm, art). we compared the outcome of these assessments with results obtained from the application of the dpd+ methodology. the work presents the results of application of dpd+ approach and the cca and indicates possible improvements for the risk assessment of mixtures. to check for seasonal and weather dependent influences in the prescription rate of drugs used to treat cardiovascular and respiratory diseases (atc codes c and r) a survey covering all prescriptions of a specimen german general regional health insurance (aok plus, data for saxony, largest health insurance service, approx. 50 % of all saxonian citizens are inscribed to this service) for the years 2005 to 2007 was analysed on a monthly basis. the number of prescriptions for cardiovascular drugs changed approximately +/-20 % around the mean for the different month without a clear seasonal pattern. for respiratory drugs only the systemic anticholinergics and drugs used to treat obstructive lung disease displayed a distinct seasonal pattern with a 50 -70 % above average prescription figure during spring time (february to may) and a 25 to 40 % trough in late summer/autumn (july to october). the data have to be analysed for further cofounders (e.g. influenza prevalence, environmental conditions etc.) to fully understand the fluctuations observed. the prescription rate for cardiovascular drugs and respiratory drugs seems to be influenced by multiple factors aside seasonal influences. pasteurella multocida toxin prevents osteoblast differentiation by activation of heterotrimeric g proteins siegert p., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abt. i, albertstr. 25, 79104 freiburg i. br., germany pasteurella multocida toxin (pmt) is a major virulence factor of pasteurella multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. the toxin modulates various signaling pathways by acting on the heterotrimeric g proteins gαq, gα12/13 and gαi. pmt activates gq to increase inositol phosphate production via phospholipase cβ and alteration of gene expression via the jak/stat pathway. the toxin also activates rhoa via gαq and gα12/13 family proteins. we showed that the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. because pmt is the causative agent to induce progressive atrophic rhinitis in pigs, which is characterized by loss of nasal turbinate bones leading to a twisting and/or shortening of the snout, we studied the effect of pmt on bone cells. here we studied the effect of the toxin on osteoblast differentiation in st-2 cells and in primary osteoblasts from rat calvaria. st-2 cells are stromal derived cells, which can be differentiated into osteoblasts or adipocytes. the toxin inhibits the differentiation of st-2 cells into osteoblasts studied by determination of specific osteoblast markers. additionally, pmt represses the induction of transcription factors essential for osteoblast differentiation. moreover, the principal pathways activated by pmt to induce these effects were investigated. ventilator-induced lung injury (vili) is a serious problem in intensive care medicine. its mechanisms are only incompletely understood, although it is widely accepted that ventilation-induced inflammation (biotrauma) makes an important contribution. the isolated perfused mouse lung (ipl) is a valuable tool to investigate the mechanisms of vili. several studies have shown considerable differences between various mouse strains with respect to lung mechanics and inflammatory responses. therefore, we hypothesized that the pulmonary responses to mechanical ventilation differ between c57bl/6 and balb/c mice. in addition, this study introduces the novel half lung technique that allows to obtain lung tissue from the same mouse at two different time points. isolated perfused mouse lungs from c57bl/6 or balb/c were subjected to high (25cmh 2o) or low pressure (8cmh2o) ventilation for 240 minutes. after 180 minutes the left lung was removed and used for western blot analysis. the right lung was ventilated for another 60 minutes. by the end of experiment the right lung was removed and qrt-pcr performed. during the whole experiment perfusate sample were taken from the venous catheter and used for protein quantification by elisa. it was possible to remove half of the lung and to further ventilate the other half without acute changes in lung mechanics. in both strains high pressure ventilation elicited a significantly higher cytokine release than low pressure ventilation. c57bl/6 mice showed higher tnf, il-1β and amphiregulin levels after high pressure ventilation, whereas balb/c exhibited increased production of cxc chemokines (cxcl-1, cxcl-2) and il-6. kinase activities (jnk, akt, erk1/2, p38 map kinase) were increased in high pressure ventilated animals, but were strain independent. the novel half lung technique builds on the well established ipl method. it permits to separately analyze the left and the right lung at different time points during continual ventilation. this method reduces animal numbers by 50% and allows statistical within subject analysis. using this method, the present study showed that inflammatory response to mechanical ventilation differ between c57bl/6 and balb/c. these findings show that the biotrauma response in mice depends on the strain that is studied. macrophages play an important role as an integral part of the first line of immune defense. two different macrophage populations have been described. m1 macrophages produce proinflammatory cytokines and are involved in inflammatory processes. by contrast, m2 macrophages release anti-inflammatory cytokines and extracellular matrix components. they can enhance wound repair and angiogenesis, but they can also promote tumor progression. recently, industrial nanoparticles have raised concerns because of their putative toxic effects. on the other hand, specifically designed nanoparticles can be used as clinical diagnostics and as drug carriers for pharmacotherapy. thus, investigations on the interactions of engineered nanoparticles with living cells and organisms are of great importance. macrophages as phagocytosing cells scavenge nanoparticles circulating in the bloodstream. therefore, we analyzed how nanoparticles with different surface functionalization might affect functions of human macrophages. monocytes were isolated from buffy coats and differentiated to macrophages with macrophage colony-stimulating factor. carboxy-(ps-cooh) and amino-(ps-nh 2) functionalized polystyrene nanoparticles were produced by the miniemulsion polymerization process and the average particle size, the polydispersity index and their zeta potential were determined by dynamic light scattering. the macrophages were cultured in the presence or absence of different concentrations of ps-cooh and ps-nh2 nanoparticles for up to 6 days. analysis of cell viability revealed that ps-nh2 but not ps-cooh concentration-and time-dependently reduced the macrophage viability. by annexin v/propidium iodide double staining we could show that ps-nh2 trigger apoptosis in macrophages. we further polarized macrophages to either m1 or m2 using ifn-γ and lps or il-4, respectively. these macrophage populations were characterized by their expression of extracellular markers by flow cytometry and their production of cytokines by elisa. the effects of functionalized polysterene nanoparticles on the cytokine production and surface marker expression of m1 and m2 macrophages were analyzed. our data indicate that surface functionalization is a critical parameter in the nanoparticle-induced toxicity in human macrophages. this work was supported by the dfg spp1313. loos c., lunov o., syrovets t., simmet t. ulm university institute of pharmacology of natural products & clinical pharmacology, helmholtzstr. 20, 89081 ulm, germany nanoparticles are currently used for various medical applications including imaging, diagnosis and drug delivery. due to particle size and surface area, their fundamental properties differ significantly from those of corresponding bulk materials. nanoparticles circulating in the blood are mainly sequestrated by the reticuloendothelial system that consists predominantly of phagocytic macrophages. macrophages express a variety of cellular receptors for sensing and internalizing particular material like viruses, microorganisms, and foreign particulate matter including nanoparticles. therefore, a detailed understanding of the intracellular fate and processing of the nanoparticles by macrophages is indispensable for controlled biomedical applications of nanoparticles. introducing distinct surface modifications, one might control nanoparticle uptake by different cell types and thereby target specific tissues and cellular compartments. tumor cell lines are frequently used as models for primary cells to analyze the effect of nanoparticles on cells. here we show that carboxy-(ps-cooh) and aminofunctionalized (ps-nh 2) polystyrene nanoparticles of ~100 nm in diameter are internalized by human macrophages, thp-1 monocytic leukemia cells, and by pmadifferentiated thp-1 cells via different mechanisms. in buffer, macrophages and thp-1 rapidly internalize both types of nanoparticles, yet, the carboxy-functionalized particles were taken up to a higher extent. the uptake of both nanoparticles was drastically reduced in media containing serum. using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between cd64 receptors and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization by thp-1 cells occurred via dynamin iidependent endocytosis. by contrast, pma-differentiated thp-1 cells took up the particles via macropinocytosis. in line with the in vitro data, more intravenously applied ps-cooh particles accumulated in liver tissue, whereas ps-nh 2 were preferentially targeted to tumor tissue. these data show that the amount of particle internalization, the uptake mechanisms, and kinetics differ significantly among primary cells and model tumor cells, whether differentiated or not, and that they are further critically dependent on the particle opsonisation by serum proteins. this work was supported by the dfg spp1313. specifically designed and functionalized nanoparticles hold great promise for a variety of biomedical applications. to ensure their safe application, such particles require a rigorous analysis of their effects on cell functions. here we demonstrate that aminofunctionalized polystyrene nanoparticles (ps-nh2) of ~100 nm in diameter in contrast to carboxy-(ps-cooh) and nonfunctionalized (ps) particles induce an nlrp3 inflammasome activation and the subsequent release of il-1β in human macrophages. amino-functionalized ps nanoparticles induced time-dependent lysosomal destabilization followed by release of lysosomal enzymes. this resulted in mitochondrial damage and formation of reactive oxygen species. accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (txnip). liberated txnip, in turn, interacted with the nlrp3 protein resulting in a conformational change of the pyrin domain of the nlrp3 protein as predicted by molecular modeling. txnip interaction with nlrp3 led to assembly of the nlrp3 inflammasome complex, to caspase-1 activation, and release of il-1β. using an in vitro knockdown approach, we showed that ps-nh 2 induced activation exclusively of nlrp3, whereas other inflammasomes remained unaffected. treatment of macrophages with n-acetyl-l-cysteine, a scavenger of reactive oxygen species, abolished both, the caspase-1 activation and the subsequent release of il-1β caused by ps-nh2 nanoparticles. these data reveal a novel mechanism of the nlrp3 activation induced by amino-functionalized nanoparticles and provide a strategy as to how such an effect can be functionally antagonized by supplementation with a radical scavenger. this work was supported by the dfg spp1313. the semi-permeable barrier of the endothelial cell lining of the blood vessels has important synthetic and metabolic functions including transport of cells and biomolecules, regulation of vascular smooth muscle tone, and control of hemostasis. plasmin is a serine protease, which is generated from its zymogen plasminogen under physiological and pathological conditions. small amounts of plasmin are produced in the context of contact activation during inflammation. consistently, increased generation of plasmin has been reported during atherosclerosis. we have shown previously that plasmin, in addition to its role in fibrinolysis, could induce proinflammatory activation of various cells including monocytes, macrophages, and dendritic cells. therefore, we analyzed how plasmin might affect the functions of endothelial cells, which could be relevant during inflammation and atherosclerosis. using flow cytometry, western immunoblotting and fluorescent microscopy, we show that endothelial cells of different origin express the plasmin receptor complex composed of annexin a2 and s100a10. addition of plasmin to human umbilical vein endothelial cells (huvec) induced timeand concentration-dependent cytotoxic effects in the cells. in addition, within 30 min plasmin triggered a rapid and prolonged expression of free radical oxygen species (ros) in endothelial cells as analyzed by microscopy and fluorometry using the rossensitive dye carboxy-h 2dcfda. the ros production in endothelial cells was accompanied by cell detachment. fluorometric and western blot analysis of caspase 3 activation in the cells treated with plasmin showed that plasmin induced apoptotic cell death in endothelial cells, which was evident already several hours after exposure to plasmin. thus, plasmin might induce production of ros in endothelial cells, their detachment and apoptosis, events which might be relevant for the development of atherosclerosis. this work was supported by the dfg. sesquiterpene lactones (stl) comprise a large group of secondary plant metabolites that constitute the active principle of a number of traditional anti-inflammatory phytomedicines. specifically helenalin and parthenolide have recently gained considerable attention as lead compounds or putative therapeutics for the treatment of inflammation and possibly cancer. both compounds have been shown to interfere with the signal transduction through inhibition of the nuclear factor κb (nf-κb). whereas the inhibitory effects of the stl on nf-κb are undisputed, their molecular mechanism of action remains a matter of debate. surface plasmon resonance (spr) analysis allows label-free measurement of molecular interactions. yet, analysis of the interaction of immobilized recombinant proteins with small molecular ligands remains a technically challenging task. in the present study we used spr technology to investigate the molecular interaction of the stl helenalin with putative intracellular target proteins such as the nf-κb protein p65/rela, the catalytic subunits of the ikk complex, namely ikkα and ikkβ, and the intracellular antioxidant glutathione (gsh). at physiological ph 7.4, helenalin interacts with rela (k d = 4.8 µm), yet it failed to bind either ikkα or ikkβ. hence, when dna with nf-κb binding consensus sequence was immobilized on sensor chips, the binding of rela was inhibited by helenalin with an ic50 of 5.0 µm. moreover, we provided several lines of evidence that stl may modify rela on cysteine 38 by a michael-type addition. this interaction was confirmed by molecular docking that identified the best matching interaction between rela and helenalin with predicted hydrogen bonding interactions between helenalin and residues arg35, lys37, gly44 and ile118 of rela. consistent with our hypothesis that helenalin interacts with sulfhydryl groups at ph 8.0, helenalin was also able to interact with reduced, but not oxidized, glutathione with a kd of 24 µm, though no significant interaction was observed at ph 7.4. thus, we showed that the sesquiterpene lactone helenalin interacts with the nf-κb protein rela but not with ikkα or ikkβ. moreover, at physiological ph, helenalin does not interact with glutathione to any significant extent. direct interaction of helenalin with rela leading to inhibition of rela-dna binding and transactivation might present the molecular mechanisms underlying the anti-inflammatory effects of stls. although nanosized materials are quickly taken up by macrophages, our understanding of the involved processes is still rather limited. therefore, we analyzed the uptake of diagnostically used carboxydextran-coated iron oxide nanoparticles of two different sizes, superparamagnetic iron oxide nanoparticles of 60 nm (spio) and ultrasmall superparamagnetic iron oxide nanoparticles of 20 nm (uspio), by human macrophages. by pharmacological and in vitro knockdown approaches, the principal uptake mechanism of macrophages for both particles was identified as clathrin-mediated, scavenger receptor a-dependent endocytosis. further, we created a mathematical model of the nanoparticle uptake by macrophages that permitted determination of key parameters of endocytotic process, such as the uptake rate, the mean uptake time, the number of particles taken up by a cell, and the correlation between the number of internalized particles and their extracellular concentration. the model also provided information on the individual and collective wrapping time of the nanoparticles and described the relation between biophysical parameters such as cytoskeletal forces, membrane elasticity, and the uptake time. finally, we gained information on the minimal linear spacing between simultaneously acting neighboring endocytotic pits that contain single nanoparticles and govern the collective uptake process. the calculated parameters were further confirmed experimentally using spinning disc confocal microscopy. thus, the new model provides important insights into the biophysical processes involved in endocytosis of nanoparticles by human macrophages. this work was supported by the dfg spp1313. prostaglandins (pg) are hormones which are formed during inflammatory processes from arachidonic acid by cyclooxygenases and prostaglandin synthases [4] . in the subsequent metabolism, in which the five-membered ring is dehydrated, α,β-unsaturated carbonyl compounds are generated [2, 3] . these come along with mercapto groups of amino acids in a michael addition reaction associated with activation of cellular enzyme cascades [1] that potentially contribute to their possessed antiinflammatory, antineoplastic and antiviral effects [5] . however little is known so far about possible adverse health effects.we addressed the question whether selected cyclopentenone prostaglandins (cypg) exhibit potential mutagenic and genotoxic properties in the hamster lung fibroblast cell line v79. induction of dna damage was investigated by single cell gel electrophoresis assay (scge). the impact of cypg on cellular redox status was detected by total glutathione (tgsh) assay. the induction of micronuclei and apoptosis was determined by staining with 4',6-diamidino-2-phenylindole (dapi). furthermore the hypo-xanthine-guanine phosphoribosyltransferase (hprt) assay was used for mutagenicity testing. , followed by prostaglandin a2 (pga2), showed the most distinctive genotoxicity, i.e., induction of micronuclei, and apoptotic effects. furthermore, the 15dpgj2 and pga2 -induced significant decrease in the tgsh level in v79 may contribute to the observed increase in oxidative dna-damage. however, none of the tested cypg exhibited mutagenic properties in the hprt assay. in conclusion, a potential in vitro genotoxicity of cypg has been observed which may be involved in carcinogenesis associated with chronic inflammation. parabens and methylisothiazolinone are used as preservatives in personal care products. sensitization to parabens and methylisothiazolinone is relatively rare considering their wide use in cosmetics, but only few quantitative or clinical data exist. therefore, we have tested methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, phenyl-and benzylparaben , and methylisothiazolinone in the loose-fit coculture-based sensitization assay (lcsa) developed by our working group. the coculture of primary human keratinocytes and allogenic dendritic cell-related cells (dc-rc) in this assay emulates the in vivo situation of the human skin. sensitization potential of the test substances was determined by flow cytometric analysis of the dc-rc maturation marker cd86. determination of the concentration required to cause a half-maximal increase in cd86-expression (ec 50) allowed a quantitative evaluation. the irritative potential of the substances was assessed by 7-aad (7-amino-actinomycin d)-staining. the concentration required to devitalize 50 % of the examined cells compared to a zero control was termed ec50%. parabens exhibited weak (methyl-, ethyl-, propyl-and isopropylparaben) or strong (butyl-, isobutyl-, pentyl-and benzylparaben) sensitizing potential, phenylparaben was found to be a moderate sensitizer, with ec50-values ranging from 16.67 µmol/l (pentylparaben) to 325.36 µmol/l (methylparaben). due to a pronounced cytotoxicity (ec50% = 70.94 µmol/l), we could not estimate an ec50-value for methylisothiazolinone. sensitization potential of parabens correlated with side chain length. parabens showed no (methyl-and ethylparaben) or weak irritative potential (propyl-, isopropyl-, butyl-, isobutyl-, phenyl-and benzylparaben) , only pentylparaben was rated to be irritative. apart from phenyl-and benzylparaben, irritative potential also correlated with side chain length but did not correlate strictly with the sensitization potential. overall, we were able to demonstrate and compare the sensitizing potential of parabens in this in vitro test. it was weak for methyl-and propylparaben, the most commonly used parabens. furthermore, we showed an irritative potential for most of the perservatives. thus the lcsa is a useful in vitro test to compare the sensitizing potential of xenobiotics. phosphorylation of the neurodegeneration-related septin 4 by protein kinase dyrk1a at serine 107 affects protein stability soppa u. 1 septins are gtp-binding proteins forming heterooligomeric complexes and filaments by interactions of the 13 family members. these complexes have important functions by building scaffolds for proteins involved in cell cycle or cell polarity but their subcellular distribution as well as their regulation remain largely unclear. septin 4 (sept4) was found in neurodegeneration related protein aggregates and is associated with migration of cortical neurons. here we first describe a potential mechanism for regulation of sept4 by phosphorylation via dual specificity tyrosine-phosphorylation-regulated kinase 1a (dyrk1a). dyrk1a is overexpressed in down syndrome and supposed to be involved in neurodevelopment and neurodegeneration. by site directed mutagenesis of flag tagged mouse sept4 and overexpression in hela cells we identified serine 107 as the major phosphorylation site of dyrk1a and generated a phosphospecific antibody. transient coexpression of sept4 and dyrk1a in hela cells increased phosphorylation of serine 107 by 50% in relation to basal phosphorylation. in contrast, cotransfection of the kinase deficient dyrk1a mutants k188r and d287n did not increase serine 107 phosphorylation. moreover we could show, that inhibition of kinase activity by the dyrk1a inhibitor harmine reduced phosphorylation of exogenous sept4 at serine 107 about 25% in hela cells. furthermore, down regulation of dyrk1a by rna interference lead to decreased phosphorylated serine 107. these results indicate that endogenous dyrk1a contributes to sept4 phosphorylation in hela cells. finally we analyzed protein stability of wild type sept4 compared to the phosphorylation resistant s107a mutant in hela cells by inhibition of translation with cycloheximide. we found that in living cells the sept4 s107a mutant is more stable than wild type sept4. in summary, our results suggest phosphorylation at serine 107 by dyrk1a as a novel mechanism to regulate sept4 stability and indicate a possible link of these proteins in cellular processes. is formaldehyde a good example for a "genotoxic carcinogen" with a threshold mode of action? speit g. institut für humangenetik, universität ulm, 89069 ulm, germany formaldehyde (fa) induces toxic and genotoxic effects in directly exposed cells (site of contact). several studies in which fa was administered to rats by inhalation showed evidence of tumor induction in nasal epithelium. there is also some epidemiological evidence that fa causes nasopharyngeal cancer in humans. although fa is a known mutagen, it is still a matter of discussion whether carcinogenesis is primarily mediated via a mutagenic mode of action. there is evidence that cytotoxicity and induced proliferation are the main causes for tumor formation. however, a decisive role of mutagenesis cannot be excluded and a mutagenic mode of action has to be considered for risk estimation. the basic assumption is that mutagens have a non-threshold mode of action. a threshold mode of action for a chemical is likely when a substance with a known mutagenic potential does not induce mutations at low concentrations due to a specific type of reaction with the genetic material and / or physiological protective mechanisms. because fa is a directly acting dna-reactive substance, a threshold mode of action may only be considered because of physiological protective mechanisms. after inhalation, pre-lesion protection occurs by unspecific binding to mucus, cellular proteins and glutathione. furthermore, fa is efficiently inactivated by enzymatic pathways. if fa reaches and damages the nuclear dna, dna repair mechanisms act as efficient postlesion protection mechanisms. in vivo inhalation studies with rats indicated that fa induces primary dna damage in the nasal epithelium but increased mutation frequencies were not measured. data are now available to show the relative distribution of endogenous versus exogenous dna adducts in different locations of the nose and other organs. considering the fa concentrations present in every living cell and the background levels of endogenous dna adducts, appropriate risk assessment and the identification of practical thresholds for fa-induced genotoxicity become feasible. fraud and misconduct in clinical trials steffen c. formerly federal institute for drugs and medical devices clinical trials unit, kurt-georg-kiesinger-allee 3, 53175 bonn, germany plagiarism in scientific publications has been the subject of public ("guttenplag") and scientific debate. plagiarism violates, however, "only" the intellectual property of its authors. in clinical trials, misconduct may also endager the health of patients. the suppression or falsification of data in clinical trials may mislead patients and doctors to use worthless treatments. clinicians may be provoked to repeat these trials, thereby wasting time and money. in clinical trials, misconduct includes everything from suppression or their repeated publication, the "correction" of unwanted results to the complete invention of the data, their intentional or negligent misinterpretation leading to the publication of biased conclusions from otherwise correctly performed clinical studies. the peer review system cannot protect against fraud, as few reviewers will have the means and the time to reevaluate original data. they will have to rely on these data, the calculations and statistics that are presented to them. some kinds of fraudulent behavior, such as double publications, can be found in the review process, but this is also time-consuming and depends on a helpful librarian. other kind of fraud, as the suppression of patient data in clinical trials (the deletion of "non-responders", according to cinderella: the good ones go into the pot, the bad ones go into your crop) can only be detected by the national competent authorities performing an inspection according to good clinical practice. even if the results of such an inspection become public as in the case of ukrain (gansauge et al. 2002) , there is no institution that will further analyze and publish the misconduct or initiate the retraction of the incriminated paper. a german agency similar to the us office of scientific integrity could foster good clinical practice in germany. although it is sometimes very difficult to decide whether improper results arise from fraud or error, a bias for the source of funding is obvious. trials funded by for-profit organizations were significantly more likely to recommend the experimental drug as treatment of choice (als-nielsen et al. 2003) . medicinal products with disputed efficacy such as orally applied enzymes for systemic action, bacterial preparations for irritable bowel syndrome and food supplements are to be reviewed with special attention. further examples from the author's experience will be presented. ]i favors further kca opening, kca may establish a feed-forward regulation of ca 2+ influx. in the present study we analyzed whether kca channels of sk4 and bk type play a role in sustaining ca 2+ oscillations at g1-to s-phase transition of primary mouse tumor cells and in human breast cancer cells, aiming towards a better understanding how kca modulate tumor cell proliferation. methods: kca expression was quantified by qpcr in human breast cancer biopsies, transgenic mmtv/c-neu + mouse mammary tumors and primary tumor cells derived thereof. the identity of the tumor cells was verified by gliolan staining. proliferation of the primary mammary tumor cells in the presence or absence of bk and sk4 modulators was tested using a real time cell monitoring system. the cellular dna content as a measure for cell ploidity was determined by propidium iodide staining and flow cytometry. changes in [ca 2+ ]i oscillations and peak amplitude were determined using ca 2+ indicator fura-2am. results: sk4, but not bk, expression is detectable in human and mouse breast cancer biopsies and in primary tumor cells derived from the mmtv/c-neu + mouse model. sk4 inhibition by tram-34 dose-dependently (0,1 to 10 µm) inhibits the growth of primary mammary tumor cells probably by a g1 cell cycle arrest. ca 2+ oscillations in proliferating mmtv/c-neu + tumor cells were ablated upon pharmacologic inhibition of sk4 channels. -dependent cell cycle progression is dependent on sk4 activity. blocking sk4 disrupts a feed-forward loop that coordinates ca 2+ influx via trp or crac channels in tumor cells. the consequences of sk4 inhibition in mammary tumors in vivo will be discussed. synthesis of a triphenylphosphonium substituted derivative of 5-hydroxymethyl-5-methylpyrroline n-oxide stolze k. 1 esr combined with spin trapping is a well-known analytical approach to detect free radicals formed in various biological systems, e.g. superoxide, hydroxyl and a series of carbon-centered free radicals, which are involved in oxidative stress. our aim was to modify the established spin trap 5,5-dimethyl-pyrroline n-oxide (dmpo) with a functional side chain, which can be used further as anchor for moieties enabling the spin trap to penetrate mitochondrial membranes, such as the positively charged triphenylphosphonium substituent. several synthetic routes were tested to introduce a 4-carboxybutyltriphenylphosphonium-substituent to the spin trap 5-hydroxymethyl-5-methylpyrroline noxide (hmmpo). while the activation of the carboxy group via the corresponding chloride was not successful, the use of a mixed anhydride with acetic acid appeared to be a promising way, although the reaction is considerably slower. preliminary spin trapping experiments have been performed with model systems generating superoxide, hydroxyl-, and carbon-centered radicals. othman e. m., stopper h. universität würzburg toxikologie, versbacher str. 9, 97078 würzburg, germany type 2 diabetes mellitus (dm2) is a growing health problem affecting more than 150 million people worldwide. it is associated with severe acute and chronic complications that negatively influence both the quality of life and survival of affected individuals. epidemiological studies clearly indicate that the risk of several types of cancer (including pancreas, liver, breast, colorectal, urinary tract and female reproductive organs) is increased in diabetic patients. diabetic patients are exposed to oxidative stress which plays a pivotal role in the pathogenesis of both micro-and macro-vascular complications. this is due to a decreased antioxidant capacity and chronic exposure to increased levels of reactive oxygen species (ros). since the insulin resistance in dm2 leads to hyperinsulinemia we studied the cellular consequences of the elevated insulin level and showed that it generates superoxide anions (o 2-) and dna damage by a nadph oxidase dependent mechanism in cultured cells. in addition, we found elevated genomic damage in the lymphocytes of diabetic patients as well as oxidative stress and genomic damage in kidneys of diabetic rats. this effect of insulin may contribute to the pathogenesis and progression of dm2 complications including the elevated cancer risk. the classical transient receptor potential (trpc) channel subfamily is regarded as nonselective, calcium permeable cation channels involved in a wide range of physiological events that require calcium signaling. until now, the specific roles of trpc channels in neuronal function are still elusive. given that trpc1 is able to form receptor-operated heterotetrameric channel complexes with other trpc channel subunits, we investigated the role of trpc1 for receptor-operated calcium influx in the heterologous expression system as well as in neurons. for this electrophysiological whole-cell measurements, fluorimetric calcium measurements, mn 2+ quenching and qpcr analysis were applied. furthermore, the effect of trpc1 knock-down on neuronal migration was monitored performing scratch assays, videomicroscopy and g-actin/f-actin assays. employing these techniques, we found that recombinant trpc1 was not able to function as a homomeric channel. instead, trpc1 subunits formed functional receptor-operated heteromeric channel complexes with trpc3, 4, 5, 6, and 7. heteromers containing trpc1 subunits showed significantly decreased calcium permeation in heterologous cell systems. mutation of amino acids in the putative pore forming region of trpc1 further reduced calcium permeability. in gnrh neurons endogenously expressing trpc1, 2, 5, and 6, downregulation of trpc1 by shrna resulted in increased basal cytosolic calcium concentrations and elevated calcium permeability. trpc1 was not involved in store-operated cation influx in gnrh neurons. moreover, trpc1 suppressed the migration of gnrh neurons without affecting cell proliferation. these findings suggest a novel regulatory mechanism relying on the expression of trpc1 and the subsequent formation of heteromeric trpc channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration. the transient receptor potential melastatin-3 (trpm3) is a calcium permeable nonselective cation channel that can be activated by the neurosteroid pregnenolonesulfate (pregs) or heat. trpm3 is expressed in various tissues, including insulin-secreting βcells and a subset of sensory neurons from dorsal root (drg) and trigeminal ganglia. the ability of pregs to evoke trpm3-like currents in pancreatic β-cells and to induce insulin secretion indicated its involvement in blood glucose regulation. however, trpm3 -/mice show so far no metabolic deficits but further investigations are recommended to evaluate its function in insulin secretion. further studies showed that trpm3 is a nociceptor channel involved in sensing heat and inflammatory thermal hyperalgesia. we performed a calcium-based screening of a compound library (spectrum collection) that identified several natural compounds as trpm3 blockers. the most potent blockers were the citrus fruit flavonoids hesperetin and naringenin as well as ononetin, a chalcon from ononis spinosa. the ic50 values of the substances are in the low micromoles ranges. electrophysiological whole cell measurements as well as calcium measurements confirmed the potency of the trpm3 blockers. furthermore, we could show that these blockers are effective on endogenous trpm3 in drg neurons from mice and isolated β-cells. by drinking grapefruit juice naringenin could be consumed in concentrations that are sufficiently high enough to block trpm3 activity in vivo. in sensory neurons, trpm3 may exert similar functions as trpv1. thus, trpm3 blocker could bear a therapeutic potential for analgesic treatment. xtt-based cell viability assay was used to determine the half-maximum effect concentration (ec50) for the investigated composite components in hgf. following concentrations of substances were used to determine the induced double strand dna breaks (dsbs): 1 /25× ec50, 1 /10× ec50 , 1 /3× ec50, and 1× ec50. each experiment was performed at least four times. hgf were incubated with various concentrations of substances for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative γh2ax analysis foci in cell nucleus were counted by eye down using a fluorescence microscope. each experiment was performed at least four times. in the xtt test following ec50 values of substances were found (mmol/l;mean +/-sem): tmp(eo)9ta a, b 0.087 ± 0.011; 1,6-hddma b, c 4.500 ± 0.700; etma a, c ; 12.000 ± 1.100; a significantly (p < 0.05) differently to 1,6-hddma, b significantly (p < 0.05) differently to etma, c significantly (p < 0.05) differently to tmp(eo)9ta. after six hours of exposure with tmp(eo)9ta at 0.00348 mm there were induced 0.55 γ-h2ax foci-formations in hgfs, at 0.00870 mm 0.67 foci, at 0.02900 mm 0.86 foci and at 0.08700 mm 0.97 foci. after exposure with 1,6-hddma at 0.180 mm there were induced 0.45 γ-h2ax foci, at 0.450 mm 0.64 foci, at 1.500 mm 0.94 foci and at 4.500 mm 1.32 foci. after exposure with etma at 0.48 mm there were induced 0.43 γ-h2ax foci, at 1.2 mm 0.50 foci, at 4.0 mm 0.61 foci and at 12 mm 0.71 foci. the negative controls dmso and medium cultures displayed 0.31 -0.34 γ-h2ax foci/cell. it was found that the induction of foci/cell were concentration-dependet for all xenobiotics in the order of: 1,6-hddma > tmp(eo)9ta > etma. these results show that dental composite components can induce dsbs in primary oral cells and therefore these substances demonstrate a genotoxic potential. effects of antioxidants on the dna-toxicity of dental (co)monomers in human gingival fibroblasts styllou p. 1 , scherthan h. unreacted (co)monomers can be released from restorative dental materials and may show biologic activity after ingestion in the human organism. in previous studies the mutagenic/carcinogenic effect of dental monomers/co-monomers (e.g. methacrylates) on the human dna was demonstrated. in this study the effects of the antioxidants vitamin c and n-acetylcysteine on the dna toxicity of the (co)monomers triethylenglycol-dimethacrylate (tegdma) and 2-hydroxyethyl methacrylate (hema) was investigated. the induction of dna double-strand breaks with (co)monomers alone and in combination with antioxidants was investigated in human gingival fibroblasts (hgf). hgf were incubated with substances without or with antioxidants for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative analysis of the γ-h2ax test, foci were counted by the same investigator by eye down the fluorescence microscope. each experiment was performed at least four times. the halfmaximum effect concentration ec 50 (mmol/l) of triethylenglykol dimethacrylat (tegdma) and 2-hydroxyethyl methacrylat (hema) was taken from of a previous study after using xtt-based cell viability assay. tegdma induced significantly (p < 0.05) higher dsbs compared to hema (1.91 ± 0.04 vs 1.66 ± 0.02). the mean number of cells scored and the standard deviation (sd) were calculated. when cells were exposed to tegdma in combination with the antioxidant vitamin c an increase of dsbs was observed (2.02 ± 0.06), compared to tegdma alone. when cells were exposed to hema in combination with vitamin c an increase of dsbs was observed (1.89 ± 0.07), compared to hema alone. when cells were exposed to tegdma in combination with the antioxidant nacetylcysteine a decrease of dsbs was observed (1.64 ± 0.04), compared to tegdma alone. when cells were exposed to hema in combination with n-acetylcysteine a decrease of dsbs was observed (0.76 ± 0.02), compared to hema alone. these results show that dental (co)monomers can induce dsbs in primary oral cells. it also shows for the first time that the genotoxic potential may be reduced by the addition of the antioxidant n-acetylcysteine. purpose: we aimed to investigate the role of superoxide and peroxynitrite generated by genetically destabilized enos for the development of endothelial dysfunction and vascular remodelling. methods: a mutant of bovine enos in which cys 101 was replaced by ala (c101a) resulting in destabilization of enos has been generated (enos-c101a). transgenic mice carrying c101a were generated on a c57bl/6 background using the endotheliumspecific tie-2 promoter. by breeding these mice with enos knockouts (enos-ko), mice that express enos-c101a (enos-ko/enos-c101a-tg) exclusively in the endothelium were obtained. unilateral common carotid artery ligation experiments were performed in c57bl/6, enos-ko, and enos-ko/ enos-c101a-tg to study a role of destabilized enos for vascular lesion formation. results: western blot analysis confirmed the expression of enos in enos-ko/enos-c101a-tg in aorta (37.1±8.4%, n=9), skeletal muscle (45.4±5.3%, n=10) and myocardium (17.4±4.9%, n=7) and revealed an increased phosphorylation of enos on ser1176/79 (470±47%) as compared to c57bl/6 (p<0.05, n=8). endothelium-specific overexpression of destabilized enos induced a large increase in superoxide and peroxynitrite formation in the aorta and the heart of enos-ko/enos-c101a-tg (p<0.05, n=5-8), which was abolished by nos-inhibitor l-nitroarginine (l-na) suggesting enos-c101a as a source of elevated radical generation. endothelium-specific introduction of enos-c101a at ~ 35% of c57bl/6 level almost completely restored aortic endotheliumdependent relaxation. experiments with l-na, soluble guanylyl cyclase inhibitor odq, peg-catalase and no-scavenger fe(detc)2 indicated that endothelium-dependent relaxation in enos-ko/enos-c101a-tg is nos-and cgmp-dependent and nomediated. four weeks after the carotid artery ligation, neointima formation, media thickening and luminal narrowing were observed in the ligated arteries of all studied genotypes (p<0.05, n=4-7). consistent with vasoprotective roles of enos, neointima formation was accelerated in enos-ko (n=4-7, p<0.05). despite significantly higher vascular levels of nitrotyrosine and peroxynitrite, neointima formation in enos-ko/enos-c101a-tg was substantially lower then in enos-ko and tended to be similar to c57bl/6. conclusions: increased vascular superoxide and peroxynitrite formation caused by destabilization of enos does not induce endothelial dysfunction in healthy mice and has negligible effect on neointima formation. fenton reactivity as a determining parameter for the interaction of manganese oxide nanoparticles with lung epithelial cells sydlik u. 1 , bieschke c. nanoparticles consisting of manganese oxide have been suggested for several innovative technological approaches, including the use in nanomedicine and diagnostics. therefore, the interaction of such nanoparticles with human target cells is of particular interest for the success of nanomedical approaches but also with regard to unintended side effects. to address this problem, we tested different kinds of manganese nanoparticles (mnnp) in an in vitro system which we earlier evaluated for proliferative, apoptotic, and pro-inflammatory endpoints induced by carbon nanoparticles (cnp). mnnp were synthesized by hydrothermal treatment of manganese salt solutions. the particles were subsequently characterized by scanning electron microscopy and dynamic light scattering. biological and toxic effects of the generated particles were studied in comparison to carbon nanoparticles (cnp) in experiments with rat and human lung epithelial cells (rle-6tn and 16hbe14o-). cytotoxicity was determined as measures of membrane damage (lactate dehydrogenase release) and metabolic activity (water soluble tetrazolium conversion). the oxidative capacity of the particles as well as the generation of intracellular oxidative stress was monitored using dichlorofluorescein diacetate in cell free experiments and flow cytometry assays (facs), respectively. the particle-specific phosphorylation of src family kinases (sfk) and mitogen activated protein kinases erk1/2 were investigated using western blot techniques. after physico-chemical characterization, a set of three mnnp consisting of mn3o4 or mno2 with significant differences in size and shape were selected. according to the different oxidation stages of manganese, the particles showed significant differences in fenton reactivity in the cell free system. these data did not reflect the capacity of the particles to induce intracellular oxidative stress. the characteristic to trigger membranedependent signaling processes, however, was correlated to the intrinsic oxidative capacity of mnnp than to the ability to induce intracellular ros. furthermore, the metabolic activity (wst) was negatively correlated with intracellular ros, indicating a link between mitochondrial activity and ros generation. none of the particles had effects on the membrane integrity of the cells. the data demonstrate that mnnp, unlike other poorly soluble nanoparticles (e.g. cnp), mainly trigger adverse health effects through ros production via the fenton reaction. acute ozone induced airway inflammation does not effect resting human sympathetic nerve traffic tank j. 1 numerous mediators released in inflammatory and neuropathic pain states activate gprotein-coupled receptors (gpcrs) and modulate nociception via activation of gs, gi/o, g12/13, or gq/11 g proteins. each of the g protein-coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of the individual signaling pathways in vivo has not yet been worked out. the gq/11 signaling branch is of particular interest in pain research because it leads to the activation of phospholipase c, protein kinase c, and the release of calcium from intracellular stores. using a conditional gene-targeting approach we generated double-deficient mice lacking gaq and ga11 selectively in nociceptors to investigate the contribution of the entire gq/11signaling pathway in nociceptors towards the regulation of pain. we observed that mice lacking gq/11 in nociceptive neurons show normal development of the nociceptive circuitry. the nociceptor-specific loss of gq/11 results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in gq/11 deficient mice, suggesting a novel function for gq/11 in tonic modulation of acute nociception. patch-clamp recordings revealed changes in voltagedependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in nociceptors upon a loss of gq/11, whereas potassium currents remained unchanged. our results indicate that the functional role of the gq/11 branch of g-protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain. provocation of arrhythmic events in single primary isolated adult mouse ventricular cardiomyocytes tekook m., fehrmann e., schulte j. s., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany ap duration and ca 2+ cycling are altered in cardiomyocytes of different genetic mouse models. here, we systematically tested various protocols to study the inducibility of arrhythmic events in mouse cardiomyocytes. adult ventricular cardiomyocytes were isolated from wildtype (wt) mice by enzymatic digestion and subsequently tested to trigger arrhythmic events within 6 hours after isolation. in patch clamp experiments (perforated patch, whole cell current clamp) aps were stimulated for 1 second (5hz; 10hz; 5hz + s2-stimulus after 50-80ms) followed by a 4s rest period. the resting-membrane-potential (rmp) was observed over 30 cycles. we observed rmp-fluctuations of different length (amplitude <5 mv; % of 13 observed cardiomyocytes, mean events/cell; <1s: 100%, 17.1; <3s: 92%, 11.5; >3s: 69%, 5.8), spontaneous depolarizations (>5 mv; 92%, 5.1) and spontaneous aps (62%, 1.9). intracellular ca 2+ transients and sarcomere shortening were measured after loading cardiomyocytes with indo-1/am. after preconditioning (10 min/1 hz) cells were measured under basal and continuous isoprenaline (10 -6 m) stimulation (iso). a 4 min pacing period was followed by a 1 min interval of no pacing. pacing frequency was reduced after each cycle (1 hz, 0.5 hz, 0.25 arrhythmic events were provocable with stimulation/rest protocols both by field stimulation and direct stimulation via patch pipette. however, low stimulation frequencies seem to lead to distinct destabilization of cardiomyocytes probably due to ca 2+ overload. we conclude that the tested stimulation protocols are able to provoke arrhythmic events even in wt single adult mouse ventricular cardiomyocytes and may serve as a tool to test for the relevance of potential proarrhythmic substrates in mouse models. ruhr-universität bochum pharmakologie ma nord1, bochum, germany cgmp is a second messenger involved in many (patho-)physiological processes such as smooth muscle relaxation, platelet inhibition, and the development and plasticity of the nervous system. however, it is not fully understood how cgmp regulates these and other processes on a mechanistic level. in particular, the existence and functional relevance of global and local cgmp signaling domains is not clear. recently, highly specific genetically-encoded optical biosensors for cgmp have been developed. these cgmp indicators are either based on fluorescence resonance energy transfer (fret), with cgmp-binding domains sandwiched between fluorescent proteins with overlapping spectra, or they consist of a single fluorescent protein fused to cgmp-binding domains. with these cgmp indicators, the spatiotemporal dynamics of cgmp signals, which result from the interplay between cgmp-producing guanylyl cyclases, cgmp-binding effectors, and cgmp-degrading phosphodiesterases (pdes), can be monitored in living cells. here, we report the generation of transgenic mice expressing the fret-based cgmp indicators cgi500 and cgi6000 with apparent cgmp affinities of 500 nm and 6000 nm, respectively. one mouse line expresses cgi6000 driven by a cmv promoter in neural cells. fret experiments were performed with isolated cerebellar granule neurons, hippocampal neurons, and astrocytes. we observed nitric oxide (no)-induced cgmp transients and analyzed the capability of other agents (natriuretic peptides, glutamate) to induce cgmp responses. in another mouse line, the sm22alpha promoter directs cgi500 expression specifically to smooth muscle cells (smcs). fret experiments have been performed with smcs isolated from aorta, bladder and colon, as well as with intact vessels in the retina and cremaster muscle of transgenic animals. in primary smcs we studied responses to no, atrial and c-type natriuretic peptide (anp,cnp). in different smc types we observed differences in the overall ability to react to these stimuli and in the kinetics of the induced cgmp transients. we also studied the effects of pde inhibitors on the no-, anp-, and cnp-induced cgmp signals. importantly, we were able to detect cgmp transients upon no stimulation in intact vessels of the retina and cremaster. we conclude that the cgi transgenic mouse lines are valuable tools to visualize cgmp signals in living cells in vitro and, possibly, also in vivo in the intact animal under physiological and pathophysiological conditions. current research data dealing with pharmacotherapy of α-ama intoxication shows a particularly high variability regarding the protective effect of silibinin. the aim of this study was therefore to evaluate the influence of the frequently used clinical antidotes benzylpenicillin, silibinin and their combination in human hepatocyte culture intoxicated with α-ama. cytotoxicity and apoptosis testing were performed after two and five days of simultaneously exposure to α-ama and/ or tested antidotes. to quantify apoptosis, necrosis and cell viability, we used cell death detection elisa plus®, toxilight® bioluminescence assay and cell proliferation kit ii (xtt). furthermore, we analysed the ways of apoptosis by using immunohistochemistry (differential detection of caspase 3, 8 and 9, activated caspase 3, and aif). exposure of hepatocytes to α-ama at concentrations of 0,2 µm, 0,5 µm and 1 µm resulted in disorder of cell cultures, apoptosis and reduction in cell viability compared with unexposed hepatocytes. in hepatocyte cultures treated with benzylpenicillin at concentrations of 30 µm and 1mm, silibinin at 50 µm and 100 µm and a combination of both (30 µm benzylpenicillin and 50 µm silibinin, 1mm benzylpenicillin and 100 µm silibinin), toxilight® values in the supernatant and xtt values were not significantly different from untreated cultures. simultaneous exposure to α-ama (at all tested concentrations) and benzylpenicillin, silibinin or combination of both showed higher cell viability and lower values of necrosis compared to the cultures exposed to α-ama alone (exept 50 µm silibinin at 0,2 µm α-ama); however, in both groups dosed with benzylpenicillin the highest hepatocyte viabilitiy was observed. this protective effect was particularly revealed at high α-ama concentrations (0,5 µm and 1 µm). in conclusion, our data suggest that benzylpenicillin in monotherapy is more effective than in combination with silibinin or silibinin alone. glucocorticoids (gcs) are important hormones in the regulation of metabolic homeostasis. synthetic gcs, such as dexamethasone (dex), play a fundamental role in the treatment of inflammatory diseases. there are numerous side effects of a dex therapy, e.g. the development of hypertension. in the pathogenesis of hypertension oxidative stress is a crucial factor. glucocorticoid-induced hypertension has been shown to be associated with an imbalance between nitric oxide (no) and superoxide. however, the source of this elevated superoxide production is unknown. we hypothesize that an uncoupling of the no synthase (enos), a key mediator of vascular homeostasis, may contribute to dex-induced oxidative stress. incubation of human endothelial cells (ea.hy 926) with dexamethasone led to a decrease in enos expression at mrna and protein levels. this effect of dex was timeand concentration-dependent. since the major cause of enos uncoupling is a deficiency of its co-factor tetrahydrobiopterin (bh 4), we analyzed the amount of bh4 in ea.hy 926 by hplc. a concentration-dependent reduction of bh4 and also bh2 (dihydrobiopterin) could be demonstrated in response to treatment with dexamethasone. bh4 can be synthesized endogenously by two different pathways -the de novo pathway (from gtp with gtp cyclohydrolase i, gch1, acting as the rate-limiting enzyme) and the salvage pathway (conversion of sepiapterin to bh4 involving dihydrofolate reductase, dhfr). treatment of ea.hy 926 cells with dex decreased mrna and protein expression of both gch1 and dhfr. because bh4 is the major "coupling switch", an enos uncoupling is likely to occur in dex-treated cells. in summary, we showed that dex treatment led to a reduced availability of the important co-factor bh4 which could lead to enos uncoupling. the uncoupled enos may possibly contribute to glucocorticoid-induced vascular oxidative stress. the cellular oncoprotein c-fos is a major component of the heterodimeric transcription factor ap-1 and has been commonly found over-expressed in tumors and cancer cells of different origin. previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (uvc) light. here we demonstrate that in human telomerase-immortalized vh10tert foreskin fibroblasts (behaving like primary cells) and sv40-immortalized gm637 fibroblasts, uvc-triggered induction of c-fos protein is a delayed and long-lasting event. sustained up-regulation of c-fos went along with transcriptional stimulation of the nucleotide excision repair (ner) gene xpf, carrying an ap-1 binding site in the promoter. c-fos mrna was induced in a biphasic manner. an immediate c-fos mrna expression (30-90 min after exposure) was not translated into the protein, the second wave of transcription (4-24h after uvc exposure) resulted in c-fos protein expression, 18-48h post-uv. the stress-activated/mitogen-activated protein kinases (jnk, p38k and erks) were immediately induced upon uvc exposure and stayed active for at least 24h. inhibitor experiments revealed that c-fos was phosphorylated by erks and jnk. the activation of c-fos preceded re-synthesis and the induction of xpf mrna, which was observed 24-40h post-uvc, resulting in the increased expression of the xpf protein. cells over-expressing c-fos showed an accelerated induction of xpf mrna, and consequently a faster repair of cyclobutane pyrimidine dimers (cpds). sirna-mediated silencing of c-fos (transient c-fos knockdown) resulted in abrogated uvc-triggered induction of xpf, attenuated repair of cpds and increased apoptosis. finally, we observed that the removal of cpds but not of photoproducts was significantly faster when cells were pre-exposed to a low uvc dose, indicative of an adaptive response to dna damage. the work was financed by deutsche forschungsgemeinschaft (dfg ch 665/2-1). the addition of clopidogrel to aspirin reduces ischemic events in patients with acute coronary syndrome and in those undergoing percutaneous coronary intervention (pci). however, recurrent ischemic event occurrence during dual antiplatelet therapy remains a major concern. variability in the pharmacodynamic response to clopidogrel is well recognized, and patients with higher platelet reactivity while receiving clopidogrel are at increased risk of ischemic cardiovascular events. clopidogrel is an inactive prodrug requiring biotransformation to form the platelet inhibiting metabolite. interindividual differences in clopidogrel metabolism are the major source of variability in antiplatelet response. polymorphically expressed cytochrome p450 (cyp) enzymes play a critical role in the metabolism of clopidogrel. these findings gave rise to the concept of personalized antiplatelet therapy -i.e. individual platelet function testing and correction of insufficient platelet inhibition to reduce ischemic events in patients with high on-clopidogrel platelet reactivity (hcpr). gravitas was the first study to test this concept by comparing double-dose clopidogrel to standard-dose clopidogrel in patients with hcpr. gravitas failed to correct hcpr consistently in the study arm, which coupled with a low overall event rate precluded demonstrating a substantial benefit from improved platelet inhibition. the trigger-pci trial tested the effectiveness of the more potent thienopyridine prasugrel versus clopidogrel in patients with hcpr after elective pci with implantation of drug-eluting stents (des). switching from clopidogrel to prasugrel in patients with hcpr afforded effective platelet inhibition. however, given the low rate of adverse ischemic effects using contemporary des after pci in stable ischemic heart disease, the clinical utility of this strategy could not be demonstrated and the study was terminated prematurely for futility. multiple studies have shown that both heterozygotes and homozygotes for loss-offunction cyp2c19 alleles have higher rates of adverse cardiovascular events as compared with noncarriers on approved maintenance dosing of clopidogrel (75mg qd), albeit carriage of cyp2c19 loss-of-function alleles accounted for only a minor proportion of the variability in on-clopidogrel platelet reactivity. results of ongoing studies with antiplatelet treatment stratified by cyp2c19 genotyping are awaited to assess the clinical benefit of this approach. the organic cation transporter novel type 2 (octn2/slc22a5) represents a high affinity uptake system for carnitine. besides metabolic disease like severe system carnitine deficiency, genetic variants within the slc22a5 gene have been associated with inflammatory diseases like colitis ulcerosa. against this background, we characterized octn2 expression in peripheral blood cells thereby identifying its expression in all cell types. in the present work we studied octn2 expression in monocytes and thp-1 cells as an in vitro model for this cell type. in addition we examined transcriptional regulation of the carnitine transporter in lps activated thp-1 and investigated the effect of carnitine and its analog mildronate on the respective cytokine response. octn2 expression was characterized on monocytes and thp-1 cells on mrna and protein level. transporter mrna expression could be shown in both cell types by realtime pcr. however, the protein expression was analyzed by western blot, flow cytometry and immunofluorescence microscopy demonstrating octn2 specific signals as well as a localization in the plasma membrane. following thp-1 cells were activated using lps (10ng/ml) for up to 6h, thereby indicating the expected cytokine response as demonstrated by increased tnfα (24fold induction) and il-1β (37fold induction) mrna levels. in addition, octn2 expression was analyzed identifying an initial reduction of around 60% compared to untreated cells. in parallel activated thp-1 cells were coincubated with increasing concentrations of the octn2 substrate carnitine or its analog resulting in reduced cytokine release as shown by elisa for tnfα. here, the tnfα effect was diminished by 64% in the presence of 50mm carnitine. this effect does not rely on a direct neutralization of lps by carnitine since it was also present in cells only preincubated with carnitine. in the present work we could show that thp-1 cells represent a useful model to study octn2 expression and function. in addition, we demonstrate immunosuppressive effects of octn2 substrates like carnitine. further experiments will be necessary to identify the underlying mechanism of this observation. castor oil has been used for more than 3000 years for its laxative effects and also to induce labor in pregnant women. despite its wide-spread use, the mechanism of action remained unknown. the active metabolite of castor oil is ricinoleic acid which is released from castor oil by intestinal lipases. we have found that exposure of meg-01 cells to ricinoleic acid caused an increase in [ca 2+ ]i, an effect which was dose-dependent and abolished by pretreatment of cells with pertussis toxin, suggesting the involvement of a g-protein coupled receptor. to search for a putative receptor, we determined ricinoleic acid-induced [ca 2+ ]i increases in cells transfected with a sirna library directed against human gpcrs. in this way, we identified prostaglandin e2 receptors ep3 and ep4 as mediators of ricinoleic acid-induced effects. to test if ep3 and ep4 receptors mediate pharmacological effects of castor oil in vivo, we analyzed laxative effects induced by castor oil in wild-type (wt) mice, ep3-deficient (ep3 -/-) or ep4-deficient mice (ep4 -/-). while ep4 -/mice responded similarly to the wt mice, ep3 -/animals were totally insensitive to castor oil-induced laxation. moreover, mice lacking the ep3 receptor only in the smooth muscle cells did not respond to castor oil, in contrast to mice which lack ep3 receptor only in epithelial cells of the intestinal mucosa. similarly, ricinoleic acidinduced contractions of isolated ileal segments were absent in segments lacking ep3, consistent with a preferential expression of the ep3 receptor in the longitudinal muscle layer of the intestine. also, ricinoleic acid-induced contractions of isolated uteri were dependent on the expression of ep3 receptor in the myometrium. these findings identify the cellular and molecular mechanism underlying the effects of castor oil and indicate a role of the ep3 receptor as a pharmacological target to induce laxative effects. introduction: patients seek health information from various sources. they are facing the challenge to differentiate between reliable and untrustworthy sources and at the same time identify the best drug therapy for them. furthermore generalised health information confuses more than they benefit or rather unsettle. patients are not necessarily qualified to assess the evidence of statements properly. there is thus a need for providing competent drug information, which is offered by the independent drug information service at the institute of clinical pharmacology in dresden, germany. for the present descriptive evaluation we selected 5 drugs (arimidex ® , cipralex ® pentalong ® , onbrez ® and pradaxa ® ), that were affected by new referenceprice formation, generic registration, warnings or directions in 2011. in specified time frames we assessed the increase in and the cause of enquiries. deductively we draw conclusions for a perspicuous presentation of patient information. since generic registrations of the aromatase inhibitor arimidex ® enquiries on side effects of this drug were stable, but 17 additional consultations were held on generic changeovers. the antidepressant cipralex ® as well as the long-acting β-agonist onbrez ® were assigned to reference-price groups, which resulted in an 8-times (cipralex ® : 5 → 40) and 5-times (onbrez ® : 2 → 10), respectively, increase in enquiries. main aspect was to give background information on reference prices and point out therapeutic alternatives (cipralex ® : 34 of 40; onbrez ® : 10 of 10). an additional amount of 22 conversations were carried on the fictive registered drug pentalong ® after health insurance companies advised practitioners to avoid recourse by not prescribing this organic nitrate. notable insecurity was aroused by media reporting on lethal bleeding after taking pradaxa ® for anticoagulation. every tenth enquiry in the evaluation period was focussing on these instigative reports (21 of 227). patients are confronted by current changes, but often do not get enough background information from their health care providers to become acquainted with the tidings. health seekers may find eligible data from media coverage. however the individual assessment as well as the risk-benefit-relation may not be feasible for them. the drug information service for patients is a convenient helpline to reduce lack of knowledge and uncertainties and therefore support shared decision making. insulin effects on hyaluronan production -a possible link between diabetes and cancer? twarock s., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany background: epidemiological studies have shown an elevated incidence of certain tumor entities in diabetes type 1 and type 2 patients. to reveal the underlying mechanisms we focused on the effects of increased glucose uptake in cancer cells with respect to matrix production. abundant production of hyaluronic acid (ha) in the vicinity of gastrointestinal cancer cells is a hallmark in tumor development. esophageal cancer is a rare but severe kind of gastrointestinal cancer which is differentiated in adenocarcinoma and squamous cell carcinoma (scc) of the esophagus. we studied the effects of increased glucose levels on ha production in an scc cell line (osc1). in starving and full media, elevated glucose concentrations increased the production of ha secreted to the medium in 24h as measured by an ha-binding protein linked assay (starved, 0g/l glucose: 100±4.7%; 1g/l: 185.7±44.8%; 4g/l: 362.6±58.3%; full medium 100±15.2%; 155.3±32.3%; 422.7±32.0%). surprisingly, total ha concentrations were about 2.2-2.8 fold higher under starved conditions. to investigate whether this effect might be due to insulin actions, starved cells were treated with 10 mg/l insulin for 24h. we observed a dosedependent decrease in ha production following insulin treatment (control vs insulin, 1g/l glucose: 100±6.5% vs 67.83±4.4%; 4g/l: 100±2.2% vs 71.8±6.6%). this finding might suggest that insulin directs glucose usage to the glycolytic pathway thereby diminishing ha synthesis. a premise to this assumption is the ability of osc1 cells for insulin independent glucose uptake. to verify this thesis, mrna expression levels of insulinindependent and insulin-dependent glucose transporters (glut1, glut4) were analyzed by qrt-pcr. the relative abundance was 24.32±3.49 in favor of glut1 indicating the presence of insulin-independent glucose transport. conclusion: in osc1 the absence of insulin actions caused increased ha production which might be due to diminished insulin driven glycolysis, thus leading to the use of early glucose metabolites for ha production instead of energy gain. this finding could be important in the context of diabetes type 2, where insulin actions are also diminished because of insulin resistance. since increased ha production is of critical importance for cancer growth and spread, the cellular shift in glucose usage from glucose catabolism to ha anabolism could therefore indicate a possible link between diabetes type 2 and cancer progression. schwarz m., unterberger e. universtität tübingen, institut für klinische und experimentelle pharmakologie und toxikologie abteilung toxikologie, wilhelmstraße 56, 72074 tübingen, germany chemical hepatocarcinogenesis is a multi-stage process triggered by an intitiating mutation in a gene encoding an important cell-regulatory protein. tumour initiation may be caused by genotoxic substances which directly interact with the dna, causing mutations. cells carrying permanent mutations experience clonal expansion which may be accelerated by exposure of the experimental animals to tumour promoters during the following step of tumour promotion. it has been shown that substances which constantly activate certain nuclear receptors act as tumour promoters in rodent liver, such as the model tumour promoter phenobarbital which, amongst others, activates constitutive androstane receptor (car). since these tumour promoters do not seem to directly interact with dna causing mutations they can be regarded as non-genotoxic carcinogens. however, the molecular mechanisms of non-genotoxic carcinogenesis are still widely unknown which also poses a major problem in preclinical drug-development. the aim of the marcar (biomarkers and molecular tumour classification for nongenotoxic carcinogenesis) project is to establish early biomarkers for non-genotoxic carcinogenesis by creating a comprehensive molecular profile of tumours generated by a regimen including model tumour promoters such as phenobarbital. the ultimate aim is to differentiate spontaneous liver tumours from tumours generated by non-genotoxic carcinogens. this molecular profile includes mutational analyses, immunostaining for known tumour-specific markers, phospho-proteome analyses, genome wide and promoter-specific dna methylation analyses, as well as mirna analyses. mutation analyses were carried out with mouse and rat tissue from phenobarbital promoted liver tumours to identify mutations which phenobarbital provides a growth advantage for. furthermore, real time pcr measurements show that the expression of a particular non-coding rna and mirna precursor is up-regulated in tissue isolated from phenobarbital promoted mouse liver tumours. additional in-situ-hybridisation experiments demonstrated the localisation of this transcript in ctnnb1-mutated tumours. larch-derived diterpenes are potent and selective trpc6 blockers urban n. 1 , kübler w. the transient receptor potential channel trpc6 is a poorly ca 2+ -selective cation channel that is activated by the membrane-resident second messenger diacylglycerol (dag). consistent with the major sites of trpc6 expression, its activation has been implicated in pulmonary and renal diseases, such as pulmonary hypertension, lung edema, chronic obstructive lung disease, allergic airway disease, and focal segmental glomerulosclerosis. amongst various plant extracts, conifer oils and resins are traditionally used to treat pulmonary ailments. therefore, we reasoned that they may contain constituents with a biological activity to modulate trpc6 activity. the true turpentines, oils and resins of various coniferous genera were tested with respect to a possible inhibition of dag-or receptor-induced activation of trpc6 and trpc3. indeed, turpentines and resins, but not coniphere oils blocked trpc6 and trpc3 in a concentration-dependent manner. interestingly, the larch-derived turpentine exerted a trpc6-prevalent inhibition. we identified larixol and its mono-and diacetates as the specific compounds that are contained in larch resin and give rise to a trpc6-selective block. larixol acetates displayed an ic50 towards the dag-or receptor-stimulated trpc6 activity of about 0.3-1 µm, but did not strongly inhibit a number of other trp channels, including trpv1, trpm2, trpm3, trpm8, or trpa1. selectivity for trpc6 compared to its closest relative, trpc3, was about 30-fold. unlike conipherous oils, which contain toxic pinenes, the resin constituent larixol ant its acetates exerted no significant cellular toxicity at concentrations that are required to block trpc6. electrophysiological analysis confirmed the highly potent block, which was voltageindependent and reversible. in a murine hypoxia-induced pulmonary vasoconstriction (hpv) model, larixol acetate abrogated the euler-liljestrand mechanism and, thus, mimicked the phenotype of trpc6 -/mice. we conclude that trpc6 blockers and, more specifically, larixol-related derivatives may provide novel therapeutic strategies to treat or prevent pulmonary diseases. dioxin is an environmental contaminant, believed to affect basic biological equilibria such as calcium and iron homeostasis. however, the molecular mechanisms underlying these effects are still largely unknown. this strongly hampers the estimation of the hazard to humans associated with dioxin exposure and necessitates further studies aimed at the clarification of these mechanisms. it has been suggested that nearly all biological and biochemical processes are mediated by protein complexes. the most commonly used technology for monitoring changes in the expression of complex protein mixtures is still 2d gel electrophoresis, but this method suffers from poor expression of low or moderately abundant proteins. blue native page and subcellular fractionation form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. the aim of the study is to identify and characterize multiprotein complexes by blue native page to elucidate the network of protein-protein interactions that regulate protein function after dioxin exposure. sample preparation and subcellular fractionation rt4 cells were cultured in mccoy's 5a medium. cells at confluence were harvested and fractionated into cytosolic, membrane/organelle and nuclear fraction by using the proteoextract subcellular proteome extraction kit. first dimension (bn-page) 50 mg of protein sample was mixed with 5% of coomassie blue g-250 (cbb g-250) and loaded in each lane of 4-15% polyacrylamide native gradient gels. the lanes from the first dimension were cut into individual strips and were placed into a 12% sds gel. the gels were stained with coomassie and the spots were picked up for mass spectrometry. bn/sds-page combined with ms led to the identification of proteins involved in the regulation of both calcium and iron homeostasis in dioxin-exposed cells. these results demonstrate for the first time that dioxin exposure simultaneously affects calcium and iron metabolism. since important iron and calcium requirement changes occur during the regulation of cell growth, the protein expression changes observed in our study may be associated with dioxin-dependent cell-fate decisions. the murine protease inhibitor serpina3n inhibits mechanical allodynia in a model of neuropathic pain vicuna l. 1, 2 , simonetti m. several chronic diseases are accompanied by strong, long-lasting pain. a majority of chronic pain diseases are not well understood yet and cannot be controlled by conventional analgesics or non-pharmacological approaches. therefore, there is a major need to develop novel therapeutic principles. using a genetic screen, we identified serpina3n, a serine protease inhibitor, which is homologous to human a1-antichymotrypsin, to be a determinant of low neuropathic pain. we found that serpina3n is expressed in the dorsal root ganglia (drg) and spinal cord and that it is upregulated in these tissues in mice developing neuropathic pain. importantly, we observed that spinal delivery of recombinant serpina3n inhibits mechanical allodynia in a mouse model of neuropathic pain. we identified a novel serine protease substrate for serpina3n, which is upregulated in the spinal cord in mice undergoing neuropathic pain ('enzyme e'). recombinant enzyme e delivered intrathecally to the spinal cord of mice elicited rapid and long-lasting allodynia, which was fully blocked by concomitant administration of serpina3n. our results suggest that serine protease-serpin signaling modulates spinal neuronal and glial cell networks involved in processing pain and that activity-induced spinal release of serpina3n constitutes an endogenous defence mechanism against establishing chronic pain hypersensitivity. these data have important implications for the pathophysiology of pathological pain and potentially hold therapeutic relevance. gβγ subunits are involved in β-adrenergic receptor induced cardiac hypertrophy vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany introduction. activated β1-adrenergic receptors and their g protein gαs induce the development of cardiac hypertrophy. however, the hypertropic effects of direct activation of downstream effectors, such as adenylyl cyclase, camp or pka, are controversely discussed. recently, a hypertrophic pathway involving a g protein βγ subunit induced phosphorylation of the mitogenic kinases erk1/2 at threonine 188 (erk thr188phosphorylation) has been described to mediate erk-induced hypertrophy. this study aims to investigate whether erk thr188 -phosphorylation is involved in cardiac hypertrophy triggered by β-adrenergic receptors. -phosphorylation was detected in hek cells overexpressing β1-receptors, murine hearts and neonatal rat cardiomyocytes after isoprenaline treatment. we performed [ 3 h]-isoleucine incorporation assays to assess cardiomyocyte hypertrophy in vitro. neonatal rat cardiomyocytes (nrcms) overexpressing wild-type erk2 showed a significant increase in [ 3 h]-isoleucine incorporation after isoprenaline treatment. in contrast, nrcms transfected with erk thr188 -phosphorylation deficient mutants (erk2 t188a and erk2 t188s ) or pretreatment with the erk inhibitor, pd98059, significantly attenuated cardiomyocyte hypertrophy. for in vivo studies, isoprenaline was given subcutaneously for 14 days to wild-type mice and transgenic mice overexpressing either wild-type erk2 t188t or erk2 t188s . echocardiography and histological analyses revealed that erk t188s mice developed less left ventricular hypertrophy than control mice. hypertrophic target proteins of erk (e.g. elk1) are located in the nucleus. western blot and confocal microscopy analyses showed that overexpressed erk2 t188a or erk2 t188s are retained in the cytosol and prevented elk1-phosphorylation after isoprenaline stimulation. co-immunopreciptation assays in hek cells and nrcms underlined the direct involvement of g protein βγ/erk interaction upon isoprenaline stimulation. in line with this finding, direct activation of adenylyl cyclase by forskolin did not lead to gβγ induced erk thr188 -phosphorylation. conclusion. taken together, gβγ-subunits participate in β1-adrenergic receptor mediated hypertrophy by enhancing erk thr188 -phosphorylation. these findings add important insight to the molecular signaling of g proteins in cardiac hypertrophy. the protein tyrosine kinase src and its role upon alpha-toxin stimulation of human platelets vogel k. 1 , burke m. introduction: alpha-toxin, a 34 kda calcium pore forming exotoxin, is a major virulence factor in the pathogenesis of staphylococcus aureus infections. alpha-toxin affects human blood cells such as platelets and induces aggregation that is accompanied by multiple changes in platelet protein tyrosine phosphorylation and dephosphorylation (1). in the present paper, we focused our interest on the protein tyrosine kinase src, the most abundant member of the src-family kinases present in platelets (2) . by the use of various inhibitors, we studied src and its role in α-toxin-induced platelet aggregation. methods: isolated human platelets from healthy volunteers were stimulated with α-toxin in the presence or absence of the src-family member inhibitors pp1, pp2 or su6654 (referred as src inhibitors). src and autophosphorylation of src were analyzed by sds-page and western blotting using specific antibodies against src and tyr-416-phospho-src from calbiochem and cell signaling, respectively (3). furthermore, calpeptin, an inhibitor of the calcium-dependent protease calpain, was used. platelet aggregation was measured by the method of born. staphylococcal α-toxin induced platelet aggregation in a concentration-dependent manner (0.18 -3.0 µg/ml of toxin). pre-incubation with 3 src inhibitors (pp1, pp2 or su6654) reduced α-toxin-induced platelet aggregation by about 50%. similar inhibitory effects have been observed by the use of calpeptin that acts as an inhibitor of the src degrading protease calpain. with respect to src itself, a-toxin induced autophosphorylation at tyr-416 followed by a fast and complete dephosphorylation within 10 min. while calpeptin modified the time course of dephosphorylation, only little effect of the src inhibitors has been seen on tyr-416 phosphorylation/dephosphorylation. the typical calpain-dependent degradation of src can be blocked by calpeptin (1µm), but also by depletion of extracellular calcium indicating that it is a calcium-dependent process. conclusion: taken together, our data demonstrate that α-toxin of staphylococcus aureus induces platelet aggregation accompanied by src degradation and autophosphorylation at tyr-416 typically observed in activated platelets. inhibition of the cellular tyrosine kinase src as well as the protease calpeptin reduces aggregation indicating an important role of src and/or other src-family members in α-toxin-induced platelet stimulation. the five subtypes of muscarinic acetylcholine receptors belong to the superfamily of gprotein coupled receptors. the even-numbered subtypes m2 and m4 prefer coupling to gi proteins, whereas the odd-numbered receptors m1, m3 and m5 prefer coupling to gq proteins. with respect to ligand binding and m2 receptor activation, the conserved epitope trp 7.35 at the beginning of tm7 displays remarkable functional features. it is located at the junction between the orthosteric and the allosteric binding site of the m2 receptor [1] . in the inactive m2 receptor, it provides subtype-independent baseline affinity for allosteric antagonists [1] . in the active receptor, m2 trp 7.35 affords binding affinity for the full agonist acetylcholine and intrinsic efficacy for the partial agonist pilocarpine [2] . to study the role of trp 7.35 for m3 receptor activation, agonist-induced formation of dmyo-inositol-monophosphate was measured in cho-cells transfected with the respective human receptor-cdna. surface receptor expression measured by radioligand binding was similar in hm3 wild-type-cells and hm3 trp 7.35→ala-cells, amounting to 0.07 and 0.11x10 6 receptors per cell, respectively. the intrinsic efficacy of acetylcholine was not influenced at m3 trp 7.35→ala relative to m3 wild-type, whereas potency was reduced about tenfold. these findings resemble those made previously in m2 and the corresponding mutant. in the case of pilocarpine, replacement of trp 7.35 by alanine in m3 did not reduce intrinsic efficacy. this finding is in contrast to m2, where the corresponding mutation induced a loss of pilocarpine's intrinsic efficacy. the potency of pilocarpine was diminished about tenfold at the m3 trp 7.35→ala mutant relative to m3 wild-type. this finding is also in contrast to m2, at which pilocarpine's potency was not sensitive to the trp 7.35→ala mutation. taken together, the diverging sensitivity of pilocarpine to the trp 7.35→ala mutation between the m3 and the m2 receptor suggests that the role of this epitope for receptor function may differ between even-and odd-numbered muscarinic acetylcholine receptors. in vivo experiments for inhalation toxicity are time and animal consuming. thus several in vitro methods aim to replace or reduce and refine the in vivo experiments. human 3dtissue models are commercially available reconstructed from different donors (normal, smokers, chronic obstructive pulmonary diseases), which show a normal human bronchiole tissue that reveals a pseudostratified epithelial structure, numerous microvilli and cilia on the apical surface of the cultures. the presence of tight junctions and mucus secretion has also been confirmed comparable to the in vivo situation. these 3d-models are cultured on a porous membrane as air-liquid interface. test substances can be applied apically, either as solution or with an aerosol-inducer. in our in house validation to test the strengths, handling and reproducibility of such 3dmodel systems as well as determining the correlation between in vivo inhalation data, we have assessed the epiairway tm model from mattek, usa. a set of 20 substances were selected with known in vivo toxicity data and mode of action. the substances were tested in the epiairway model an in parallel, in 3t3 and a549 cell lines to assess putative unspecific cytotoxic effects of the test substances. a comparison of toxicity data from the 3d-model and the in vivo data revealed, that the model is only predictive of respiratory toxicity in vivo for a subset of substances with specific modes of action. the epiairway tm model has proven to be robust, showing high reproducibility between pre-and main-tests as well as in the concurrent controls but it will need a strict definition of its applicability domain or further development of the test protocol to achieve a wider applicability. remodeling of intracellular ca 2+ handling and cyclic amp-dependent signaling in atrial myocytes from patients with chronic atrial fibrillation. voigt n. 1 background: in atrial myocytes ca 2+ entry through l-type ca 2+ channels (ica,l) triggers a larger ca 2+ release (ca 2+ transient,cat) from the sarcoplasmic reticulum activating contractile myofilaments. reduced ica,l is a hallmark of atrial remodeling in chronic atrial fibrillation (caf) and is supposed to contribute to action potential shortening and contractile dysfunction. however, the coupling efficiency between ica,l and cat and its regulation by camp-dependent signaling in caf patients are unexplored. methods: ica,l (voltage-clamp) and cat (fluo-3) were measured simultaneously in rightatrial myocytes from sinus-rhythm (ctl) or caf patients. a saturating concentration of the non-selective β-adrenoceptor (ar) agonist isoprenaline (iso, 1µm) and the nonselective phosphodiasterase (pde) inhibitor 3-isobutyl-1-methylxanthine (ibmx, 10µm) were used to increase cellular camp content. camp content was assessed with immunoassay. results: in caf amplitudes of ica,l (3.3±0.3pa/pf, n=12/6 [myocytes/patients] vs 6.2±0.8 pa/pf, n=15/10, p<0.01) and cat (182.2±26.8nm vs 307.5±30.9nm, p<0.01) were lower than in ctl myocytes, whereas diastolic [ca 2+ ]i levels were unchanged (caf, 312.3±56.7nm; ctl, 305.3±43.6nm). the coupling efficiency between ica,l and cat was similar in ctl and caf. application of iso increased ica,l amplitude to 10.4±2.0pa/pf (n=7/4) in caf and to 14.3±1.7pa/pf (n=9/7) in ctl. the corresponding cats increased to 493.0±132.3nm in caf and to 545.0±79.0nm in ctl. although the amplitudes of ica,l and cat also increased after pde inhibition with ibmx, the magnitude of these increases was smaller than the iso-induced enhancements. both iso and ibmx had no effect on diastolic [ca 2+ ]i and coupling efficiency. however, the relative iso-induced increases in ica,l (caf, +202.3±47.3% vs ctl, +98.4±16.1%, p<0.05) and cat (caf, +215.4±57.8% vs ctl, +101.9±20.3%, p=0.06) were significantly higher in caf compared to ctl myocytes and a similar tendency was found for ibmx. basal camp levels were higher in caf compared to ctl (caf, 9.9±1.5pmol/mg, n=6 vs ctl, 5.0±0.6pmol/mg, n=7, p<0.05), pointing to an increased camp-dependent signaling in caf patients. conclusions: these data point to remodeling of camp-dependent signaling in caf patients which likely contributes to the stronger relative increases of ica,l and cat amplitudes after β-ar stimulation and pde inhibition. remodeling of camp-dependent signaling might be a novel contributor to af pathophysiology. direct visualisation of g-protein-coupled receptors and heterotrimeric g-proteins using single-molecule microscopy wagner j. 1 g-protein-coupled receptors (gpcrs) form the largest family of membrane-bound receptors and mediate the effects of several extracellular stimuli. although the basic mechanisms of gpcr signalling have been extensively studied, a full characterization of the involved protein-protein interaction is still missing, largely due to technical limitations. in this study, we developed new methods for labelling gpcrs and g-protein subunits based on snap-and clip-tags and visualise them with single-molecule sensitivity. the snap-tag is a mutant of the dna repair protein o 6 -alkylguanine-dna alkyltransferase that reacts with fluorescent benzylguanine derivatives, whereas the clip-tag is reacting specifically with o 2 -benzylcytosine derivatives. these tags allow labelling proteins directly in living cells with very high specificity and low background. snap/clip-tagged receptors and g-proteins were covalently labelled with small organic fluorophores and visualised by total internal reflection fluorescence microscopy, which allows to selectively illuminate only fluorescent molecules located on or immediately underneath the cell surface. particles were automatically analysed with previously published as well as newly developed algorithms. the results indicated that both receptors and gproteins, although diffusing with high speed on the cell surface (diffusion coefficients: receptors ~ 0.05 mm 2 /s, g-proteins ~ 0.1mm 2 /s), can be visualised and correctly tracked. a variable fraction of receptors and g-proteins are immobile or show hop movements, possibly suggesting their interaction with cytoskeletal or other membranebound proteins. our data also suggest the feasibility of performing two-colour analyses with snap-and clip-tagged proteins aimed at directly visualizing transient interactions between receptors and g-proteins or among g-protein subunits. in-vitro screening systems are particularly well suited to preclinical toxicology testing at an early stage of drug development as they have the advantage of being fast and requiring only a small amount of test substance. the demands for in-vitro screening assays for systemic toxicity are multiple and include the need of organ specific cell systems, the use of optimal cell numbers, cell passages and incubation times. even minimal changes in the conditions of the test system may lead to significant changes of the biological system. therefore a reliable normalization compensating biological variability is crucial prior to any interpretation of results generated from a biological system. basf has developed an in-vitro metabolite profiling assay and a subsequently tuned normalization strategy allowing the prediction of specific organ toxicity. the in-vitro assay consist of exposing cells lines to test substances and to determine the metabolite profile using chromatography coupled to mass spectrometry systems. herein, we compare five different normalization strategies referring to their suitability in the application to in-vitro metabolite profiling data. the strategies comprise statistical approaches, approaches referring to reference values from each individual sample or samples generated in dependent batches. best results were achieved by an individual strategy using a new reference value correlating well over a large range of cell counts previously used for generating corresponding cell extracts. statistical analysis revealed the normalization based on the new reference value greatly improved the quality of the results compared to non-normalized samples as well as to all remaining strategies. generation and application of this new reference value and the corresponding normalization strategy will be presented the first time. validation will be featured on the basis of extracts of the human hepatocellular carcinoma cell line hep g2. molecular mechanisms of the inhibitory function of rhoh in phospholipase cmediated signalling walliser c., löschmann y., ziegler v., kühne e., schilling p., rasonabe z., bühler a., vatter p., gierschik p. universitätsklinikum ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany rho gtpases are a subfamily of ras gtpases regulating diverse signalling pathways, for example those regulating the reorganisation of the actin cytoskeleton. among them, rac2 and rhoh show an expression restricted to the hematopoietic lineage. rhoh is constitutively active, because it carries mutations in two positions (s13 and n62) known to be important for gtp hydrolysis. hence, rhoh is controlled on the level of protein expression and, possibly, by tyrosine phosphorylation. rhoh has been implicated in human malignancies, since the gene is subject to somatic hypermutation in its noncoding regions and to translocation to the gene encoding laz3/bcl6 or to other genes in human b-cell lymphomas. furthermore, rhoh is overexpressed in primary human chronic lymphocytic leukemia (cll) cells. these findings suggested that rhoh is involved in the initiation and/or progression of cll. we previously showed that rhoh acts as a potent inhibitor of both rac2-mediated phospholipase c-β 2 (plcβ2) and plcγ2 activation in intact cells. the aim of this study was to elucidate the molecular mechanisms of the inhibitory effect of rhoh on plc activity. the results showed that rhoh directly inhibited the activity of constitutively active variants of plcγ2, plcβ2, and plcδ1, but that it had little or no effect on the activity of plcγ1 and plcε. the amino acid residues s13 and n62, likely to be the cause for the gtpase-deficiency of rhoh, are not required for the inhibitory function of rhoh. furthermore, the switch-i and switch-ii regions of rhoh are not necessary for the inhibitory effect of rhoh, since rhoh mutants carrying switch-i or switch-ii regions of rac2 caused inhibitory effects on rac2-mediated plcβ2 and plcγ2 stimulation indistinguishable from wild-type. interestingly, rhoh seems to interact with regions of plcγ2 distinct from those which are necessary for rac2 interaction, as the split pleckstrin homology domain of plcγ2, which is essential for its interaction with activated rac2, is dispensable for the inhibitory effect of rhoh. in summary, our results indicate, that rhoh acts as a plc-isozyme-specific negative regulator of the activity of plcβ2 and plcγ2, both of which are specifically expressed in hematopoietic cells. these findings suggest a novel mechanism of plc isozyme regulation by rhoh. the results also suggest that rhoh plays an important role in b cell maturation, function, and leukemogenesis by modulating b-cell-receptor-mediated plcγ2 activation. effect of rac1 inhibition on doxorubicin mediated cell response wartlick f., fritz g. heinrich-heine-universität düsseldorf institut für toxikologie, universitätsstrasse 1, 40225 düsseldorf, germany background: the small gtpase rac1 is a well characterized member of the rashomologous (rho) family. rac1 is not only a key regulator of the actin cytoskeleton but also regulates the activity of nadph oxidase, stress kinases and transcription factors (e.g. nf-κb, ap1). furthermore, rac1 can translocate into the nucleus and interacts with topoisomerase type ii (topo ii). yet the general nuclear function of rac1 is still unclear. here, we address the question how rac1 influences the genotoxicity of the topo ii poisons doxorubicin and etoposide. methods: to study the function of rac1, human hepatoma cells were pretreated with the rac1-inhibitor eht 1864 before they were exposed to doxorubicin, etoposide or, for control, ionizing radiation (ir). to check the influence of rac1 inhibition on the outcome of genotoxin treatments, cell viability and cellular stress response were analyzed by the wst-assay, western blot (wb), co-immunoprecipitation experiments, facs-analysis and the alkaline comet-assay. results: as compared to the control, cells that have been pretreated with the rac1 inhibitor showed a higher viability, less phosphorylation of h2ax (s139) and a reduced dna damage formation (measured by alkaline comet-assay) after treatment with doxorubicin and etoposide but not after treatment with ir. furthermore inhibition of rac1 resulted in a reduced phosphorylation of topo iiα (s1106) and an increased interaction of topo iiα with hsp90 in doxorubicin treated cells. the data indicate that inhibition of rac1 protects human hepatoma cells against topo ii poisons due to interference with topo iiα function. the presence of drugs or other potential toxic substances in milk has enormous toxicological and nutritional consequences for consumers of dairy products. the atpbinding cassette (abc) transport protein breast cancer resistance protein (bcrp; abcg2) is expressed in alveolar epithelial cells of the mammary gland in cows, sheeps and goats. bcrp is known to play a major role in the active secretion of a variety of xenobiotics into human milk. so far there is little information about the transport activity and substrate specificity of dairy bcrp. therefore we aimed to establish a mdck cell in vitro model expressing bcrp of dairy animals. bcrp mrna was isolated from bovine, caprine and ovine mammary gland. full-length clones were generated using race (rapid amplification of cdna ends) pcr. the final full-length bovine, ovine and caprine abcg2 cdna-clone sequences were submitted to the ncbi genebank (eu570105, gq141082 and gq241418). stable transfection of bcrp in mdck cells was performed and the subcellular localization of bcrp at the apical plasma membrane was identified by confocal laser scanning microscopy. bcrp-mediated transport of the substrate hoechst 33342 was measured and the selectivity was determined by the bcrp inhibitor ko143. inhibition studies using hoechst 33342 identified various drugs including the antibiotic enrofloxacin or anthelmintic agents like oxfendazole as substrates of bovine, caprine and ovine bcrp. to further characterize bcrp carrier activity, bidirectional transport studies were performed with transwell® filter inserts that allow studying drug transport between an apical and basolateral compartment. cell monolayer integrity was checked by measuring teer values as well as by measuring the paracellular flux marker atenolol by lc/ms. bidirectional transport studies with enrofloxacin were performed to characterize the bcrp transporter activity. our results may contribute to increase the understanding of carrier associated drug transport into the milk of dairy cattle and therefore enlarge consumer protection. acrolein and acrylamide: excretion of mercapturic acids after consumption of potato chips watzek n., scherbl d., berger f., feld j., eisenbrand g., richling e. technische universität kaiserslautern fachbereich chemie; fachrichtung lebensmittelchemie & toxikologie, erwin-schrödinger-str. 52, 67663 kaiserslautern, germany acrolein (ac) and acrylamide (aa) may be formed from food constituents during heating of food. ac is supposed to be generated via heat induced formation from glycerides/glycerol, aa is known to arise during the maillard reaction from asparagine and reducing carbohydrates. ac also has also been suggested to be formed by endogenous metabolism as a side product of carbohydrate and/or amino acid turnover or by oxidative desamination of polyamines [1] . as an α,b-unsaturated aldehyde, ac forms 1,4-michael-adducts with biomolecular nucleophiles, such as sulfhydryl and amino groups. in the organism, ac and aa are preferentially conjugated to glutathione and are excreted as mercapturic acids (ma), n-acetyl-s-(3-hydroxypropyl)-cysteine , n-acetyl-s-(carboxyethyl)-cysteine (cema), (n-acetyl-s-(2-carbamoylethyl)-cysteine (aama), and (n-acetyl-s-(2-hydroxy-2-carbamoylethyl)-cysteine (gama). data on human exposure to ac and its occurrence in the diet are scarce. in general, contents in heat treated foods are considered to be in the low ppb range (µg/kg) [2] . nevertheless, in a pilot study in humans urinary 3-hpma excretion of 14 non-smokers was reported to be about three fold higher, as compared to aama [3] . in the present human intervention study we monitored the excretion of mas in five healthy volunteers (male) after ingestion of commercially available potato chips (175 g), equivalent to an uptake of 44 µg aa (absolute amount), together with an as yet unknown amount of acrolein [4] . urinary ma contents were monitored by hplc-ms/ms following solid phase clean-up of urine for up to 24 h after test meal uptake. the results demonstrated kinetics of 3-hpma and cema excretion in human urine to be clearly related to ingestion of the potato chip meal. on the basis of auc values, total excretion of 3-hpma plus cema exceeded that of aama plus gama by a factor of about four. the results confirm earlier findings on urinary mas, suggesting markedly higher human exposure to dietary ac / potential ac precursors than to aa. it is an as yet unresolved question, whether and to what extent concomitant substantial ac exposure may influence toxicology of such dietary heat-induced toxicants. [1] stevens, j.f. and maier, c.s. (2008) molecular nutrition & food research 52; 7 [2] osorio, v. m. and de lourdes cardeal, z., (2011) journal of chromatography a 1218; 3332 [3] schettgen, t., musiol, a., and kraus, t., (2008) rapid communications in mass spectrometry 22; 2629 [4] ewert, a., granvogl, m., and schieberle, p., (2011) lebensmittelchemikertag 2011 450 protective effects of increased nad + levels in human peripheral blood mononuclear cells exposed to dna damaging agents weidele k., beneke s., bürkle a. university of konstanz molecular toxicology group, department of biology, jacob-burckhardt-str.31, 78457 konstanz, germany the dna damage-activated enzyme poly(adp-ribose) polymerase 1 (parp-1) acts as a nick sensor and modifies target proteins by covalent attachment of poly(adp-ribose) [par] using nad + as substrate. the intracellular levels of par and nad + are important parameters for biological responses to genotoxic stress and influence diverse cellular functions including dna repair or maintenance of genomic stability. notably, loss of genomic stability is a hallmark of both carcinogenesis and the ageing process. here we analysed the impact of elevated nad + levels in human blood peripheral mononuclear cells (pbmc) with regard to (i) poly(adp-ribose) formation, (ii) cell death, (iii) initial dna damage and subsequent repair, as well as the influence on (iv) genomic stability under genotoxic stress. after ex vivo supplementation of pbmc with low concentration of nad + precursor nicotinic acid (na) intracellular nad + level significantly increased up to 2 fold in unstimulated [1] and 1.5 fold in mitogen-stimulated cells. after dna damage infliction, parp activity was dramatically increased in supplemented cells, necrotic cell death was reduced and dna strand break repair was significantly affected. furthermore the frequency of micronuclei decreased significantly after irradiation damage, emphasizing the fundamental role of adequate nad + levels in maintaining genomic integrity. the cyclic purine nucleotides adenosine 3':5' monophosphate (camp) and guanosine 3':5' monophosphate (cgmp) are well-examined second messengers with many proven biological functions. in a recent study, using a highly sensitive and specific mass spectrometry method, we have shown that cyclic 3':5' cytidine monophosphate (ccmp), a pyrimidine nucleotide, is naturally occurring in several mammalian cells [1] . ccmp activates both camp-and cgmp-dependent protein kinases with low potency [2] but the physiological function of ccmp is still very poorly understood. in an effort to delineate the function of ccmp, we analyzed expression of the early response gene egr1. we chose this gene because it is regulated by numerous stimuli including camp [3] . in our first study, we showed that dibutyryl-ccmp and ccmp failed to increase egr1 gene expression levels after stimulation of kb cells under various experimental conditions using real-time pcr (taqman®). we have now changed the experimental set-up using hela cells and the new ccmp analogue, ccmp-acetoxymethyl ester (ccmp-am), still focusing on gene expression of egr1. esterification of the negatively charged cyclic phosphate of ccmp allows better transport of the nucleotide across the cell membrane, thus augmenting possible intracellular effects. hela cells were stimulated in cell culture medium with extracellularly applied ccmp-am (3, 10, 33, 100 µm) over 15 to 240 min 24 h after seeding. analysis of real time pcr (taqman®) experiments, using β-actin as a housekeeping gene, showed a significant increase of egr1 expression in a time and concentration dependent manner. these effects were specific for stimulation with ccmp-am but not the control phosphate trisacetoxymethyl ester. hela cells were also cultured in serum free resting medium (mcdb 153, sigma) that induces growth arrest, one to eight hours prior to stimulation. here, even higher egr1 expression levels through ccmp-am stimulation could be seen. these results suggest that ccmp could function as a second messenger just as camp and cgmp do. studies are in progress to further examine the mechanisms of the ccmp-am effects on egr1 expression in hela cells. methyl-cpg-binding protein 2 (mecp2) recognizes methylated dna, it is involved in chromatin remodeling and it acts as a transcriptional repressor or activator. we have previously shown that expression of mecp2 is diminished in murine and human heart failure. prevention of mecp2 downregulation in transgenic mouse models aggravated cardiac hallmarks of heart failure. in patients with rett syndrome, which is caused by mutations in the mecp2 gene, mitochondrial function was found to be altered in the central nervous system. as the impact of mecp2 on mitochondrial function in the heart is unknown, the aim of the present study was to characterize the significance of mecp2 of cardiac mitochondria in mouse models with cardiac myocyte-specific expression or ablation of mecp2. in order to investigate the cardiac function of mecp2, two genetically modified mouse models were previously generated, including mice with inducible transgenic expression of mecp2 in cardiac myocytes under control of the tetracycline-system (mecp2-tg) and mice with targeted ablation of mecp2 in myocytes (mecp2 mlccre ). these mice were analyzed under basal conditions and after chronic transverse aortic constriction (tac). at baseline, cardiac-specific overexpression of mecp2 did not cause any difference in cardiac function as compared to control mice using millar catheterization. isolated interfibrillar mitochondria showed a decrease in citrate synthase activity. after chronic pressure overload, the decrease in cardiac mecp2 expression could be completely prevented by the mecp2 transgene. cardiac contractility and relaxation were significantly decreased in mecp2-tg animals. upon electron microscopical investigation, transgenic mecp2 expression was associated with a significant reduction of interfibrillar mitochondria, clustering of mitochondria in the perinuclear region and smaller mitochondrial cross sections as compared with control specimens. in contrast, cardiac myocyte-specific ablation of mecp2 caused a rightward shift in the size distribution of mitochondria as compared with mecp2-tg hearts. epigenetic processes, including the recognition of dna methylation by mecp2, may play an important role in the control of mitochondrial gene expression, structure, subcellular localization and function in the heart. thus, precise control of mecp2 expression and function is essential to prevent deterioration of metabolic function during chronic heart failure. normalisation of blood pressure does not prevent angiotensin ii-induced dna damage in kidney and heart of ren2 rats weissenberger s., hey v., lau d., schupp n. universität würzburg institut für pharmakologie und toxikologie, versbacher strass 9, 97078 würzburg, germany increased activity of the renin angiotensin system (ras) with enhanced levels of angiotensin ii (angii) leads to oxidative stress with endothelial dysfunction, hypertension and atherosclerosis. epidemiological studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. we could show in vitro and in vivo that angii causes structural dna damage dose-dependently in kidney cells and in the kidney. elevated angii levels therefore might contribute to carcinogenesis of the kidney. in a model of high angii organ levels, the transgenic ren2 rat, carrying an additional renin gene, dna damage in the kidney was analysed in animals of 13 and 32 weeks. untreated ren2 rats exhibit increased blood pressure from the age of 8 weeks on. therefore, the line is kept on angiotensin i converting enzyme inhibitor therapy, which normalizes blood pressure and kidney function to values of control sprague dawley rats. despite this normalized blood pressure of the ren2 animals, a significant higher superoxide production could be observed in kidneys already in 13 week old animals. also a higher frequency of structural dna damage and double strand breaks could be detected in the comet assay and with an antibody against the double strand break marker γ-h2ax in kidneys. further, fittingly, an increased dna repair activity exists in kidneys of ren2 rats compared to control rats. as another organ affected by hypertension the heart of the ren2 animals was analysed for oxidative dna damage. although only a marginal increase of superoxide production could be found, also in the heart a significant higher frequency of dna double strand breaks and cells positive for dna repair activity could be observed. our data let us conclude that normalization of blood pressure in a state of activated ras is not sufficient to prevent angii-induced genotoxicity. this further implies that also patients with treated hypertension still might suffer from endorgan-damaging effects of elevated angii levels. the d541a pore mutation leads to complete inactivation of trpv6 channels in epididymis weißgerber p. 1 , kriebs u. replacement of aspartate residue 541 by alanine (d541a) in the pore of trpv6 channels in mice disrupts ca 2+ absorption by the epididymal epithelium resulting in abnormally high ca 2+ concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilisation capacity raising the possibility of residual activity of channels formed by trpv6 d541a proteins (sci signal 4, ra27, 2011) . it is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared to the deletion of the corresponding protein. to gain insights whether the trpv6 d541a pore mutant still contributes to residual channel activity and/or channelindependent functions in vivo, we compared important fertility-parameters between trpv6 -/and trpv6 d541a/d541a mice: the fertilization rate observed in permanent matings, the in vivo fertilization rate as judged by the rate of embryos isolated from plug positive females of matings with males homozygous for either trpv6 mutation as well as the motility, in vitro fertilization capacity and viability of sperm were reduced to the same extent in both genotypes. also, no differences were observed in copulatory behavior between trpv6 -/and trpv6 d541a/d541a males. the profound reduction in ca 2+ uptake by the epididymal epithelium was identical in trpv6 -/and trpv6 d541a/d541a males. this direct comparison of these parameters indicate that deleting trpv6 does not further aggravate the phenotype observed in trpv6 d541a/d541a mice, and -in our opinion -allows the conclusion that the d541a pore mutant of the trpv6 protein leads to complete inactivation of the trpv6 channel activity or channel-independent scaffolding functions in epididymal epithelium. characterization of a naturally occurring c-terminal mutation (n996i) on herg channel function in hek293 cells sellmaier v. 1 , moretti a. long-qt-syndromes (lqts) are acquired or inherited disorders which predispose patients to cardiac arrhythmias and sudden death. in affected individuals, the electrocardiogram shows a prolongation in the qt interval, due to an unstable repolarization of the action potential. acquired forms of lqts are often the result of treatment with medications that block cardiac potassium channels, such as class iii antiarrhythmic drugs or antihistamines. inherited lqts are caused by mutations in cardiac ion channels. congenital long qt syndrome 2 (lqt2) is caused by loss-offunction mutations in the human ether-á-go-go-related gene herg (also known as kcnh2 or kv11.1). herg encodes the pore-forming α-subunit of the rapid delayed rectifier potassium current i kr, whose physiological role is to repolarize the late phase of cardiac action potentials. herg channel α-subunits exist as 2 isoforms (1a and 1b) that are identical except for structurally divergent n termini. native cardiac ikr channels are tetraheteromers containing 2 of each α-subunit types. a loss-of-function can be due to either defects in a) channel opening (gating), b) ion permeation or c) protein maturation and trafficking. we have identified a so far uncharacterized dominant missense mutation in the herg1 gene (n996i) in a patient with lqt. both herg1a and herg1b subunits were cloned from a human heart cdna library and the specific n996i mutation introduced by site directed mutagenesis. hek 293 cells were transiently transfected with equal amounts of mutated herg1a and wild type (wt) herg1b cdnas and the resulting potassium current compared to herg1a/b wt. whole-cell patch-clamp analysis showed similar current densities for wt versus mutated channels. also the voltage-dependence of activation was unchanged with a halfmaximal activation at -27 mv for wt and -29 mv for the mutated channel assembly. differences were found for the deactivation and inactivation kinetics. the deactivation was faster in the mutated channels with t fast = 62 ms and tslow = 480 ms versus tfast = 90 ms and tfast = 620 ms in wt channels, determined from the tail current at -40 mv after a 5-second pulse to + 50 mv. the half-maximal steady-state inactivation of the tail current was shifted by 20 mv to the depolarizing direction in the mutated channel compared to the wt. a defect in channel gating appears to be the most likely explanation. background: abcc2 is adjacent to p-glycoprotein the most important efflux transporter for various endogenous and exogenous compounds and is expressed at several compartment barriers. by increasing evidence it is shown that the abcc2 polymorphisms are of clinical significance. the aim of our study is to analyze the epigenetic regulation of distinct abcc2 haplotypes by the influence of micrornas. methods: abcc2 cdna clones containing five distinct haplotypes were generated by site-directed mutagenesis, cloned into pires-zsgreen expression vectors and transfected into different cell lines for further functional analysis. one modified vector set contained a short 3'utr sequence of abcc2 whereas the other contained the full length (fl) sequence. mirnas potentially interacting with abcc2 were identified form an mirna array after rifampicin stimulation of hepg2 cells. results: we could demonstrate that there is no difference in the basal protein expression level comparing the two vector types (fl 3'utr vs. mod. 3'utr) concerning the -24c/1249g/3972c (cgc) abcc2 wt in hepg2 cells. using the fl vector construct, the expression level of cac haplotype protein was increased (136,15% ± 20%). transfection assays with the mir-397, which was identified as a candidate mirna targeting abcc2 mrna, and the two different vector constructs harboring the cac or the cgc (wt) haplotype, confirmed that mir-379 was able to down regulate the abcc2 protein expression. there was no significance in downregulation for one abcc2 haplotype, respectively (modified 3'utr: cgc 22-34%; cac 20-40%, fl 3'utr: cgc 20-25%; cac 17-44% down regulation compared to mir-negative control). discussion: differences of abcc2 protein expression level are due to the epigenetic regulation of abcc2 haplotypes. to further characterize the effect of mir-379 on tcg, tgt and cgt abcc2 haplotypes, transfection assays are currently performed using cell cultures as well human primary leukocyte cultures. sex differences affect the pathophysiology and pharmacology of leukotriene biosynthesis werz o. friedrich-schiller-university jena, institute of pharmacy, philosophenweg 14, 07743 jena, germany inflammatory diseases affect more females than males. thus, women suffer more often from asthma, rheumatoide arthritis, alzheimer´s disease, and many autoimmune diseases than men. of interest, sex differences also exist in drug responses, with respect to both pharmacodynamics and pharmacokinetics. we have recently discovered a sex bias in the biosynthesis of pro-inflammatory leukotrienes (lts) due to testosterone, which may represent a molecular basis for gender differences in inflammation and asthma. interestingly, testosterone downregulates lt biosynthesis and also causes a sex bias in the efficiency of lt synthesis inhibitors, which demands for the clinical evaluation of a gender-tailored therapy with anti-lts. we found that certain inhibitors of lt biosynthesis were more efficiently in females than in males, and that androgens are responsible for these gender differences. in fact, the flap inhibitor mk886 effectively reduced ltb 4 pleural levels in female but not in male rats treated with carrageenan, and mk886 increased survival only of female mice in the lt-related disease model of paf-induced lethal shock. administration of testosterone to female mice abolished the protective effects of mk886. in view of the current active development of lt synthesis inhibitors as therapeutics in respiratory and cardiovascular diseases, our data prompt for consideration of gender issues in the development and use of such drugs, in order to optimize medical therapy both for men and women. considering the complexity of deposition and kinetics of air-borne nanomaterials in the lung, potential pulmonary toxicity of biopersistent nanomaterials should be evaluated by inhalation studies. those studies demand special equipment and large quantities of test material. intratracheal instillation appears as a simple and low substance-consuming alternative, although bolus dosing and the more central distribution of the particles in the lung are a well known trade-off. we compared the inflammatory response of the lung to amorphous silica (as) after instillation and inhalation. for inhalation the established short-term protocol for nanomaterials was employed (ma-hock et al. inhalation toxicology, 21:102, 2009 ): male wistar rats were exposed to the test items for 6 h/day on five consecutive days. the lungs were evaluated by analysis of bronchoalveolar lavage fluid (balf) and by histopathology three days and three weeks after the end of the exposure. in a parallel study, rats were intratracheally instilled and equally evaluated three days after instillation. assuming a deposition rate of 10%, the instilled dose corresponded to the aerosol concentration of 10 mg/m 3 used for inhalation. results show that inhalation and instillation of nominally equal mounts of amourphous silica elicit different results in the lung with inhalative treatment being less harmfull. this difference may be due to the bolus effect inevitable linked to instillation. instillation stuldies with amorphous silica may, therefore, be of limited value with respect to doseresponse assessment. sunscreen products containing uv filters protect consumers from the harmful effects of uv exposure. pigmentary grades of metal oxides like zno result in an opaque whiteness as a result of scattering visible light, whereasnanoparticles result in transparent products for better consumer acceptance and thus improved protection of human skin against uv-induced damage. in addition scatter uv light is most efficiently reflected at a nanosize of 60-120 nm. in the last 2 years the toxicological properties of nanozno in comparison with pigmentary zno were examined, the results of these comprehensive studies are presented. all tests were performed according to oecd guidelines, which were modified, especially in regard of substance preparation where appropriate. nanosized zno showed no acute toxicity after dermal application, in the bcop assay as well as in the epiderm assay it showed no corrosion / irritation potential. nanosized zno does not penetrate the intact as well as the sunburned skin. a dermal absorption test in rats (oecd 427) with 65 zn-labelled test item as well as penetration tests in weanling pigs after uv radiation did not show a penetration of the zno nanoparticles through the skin. genotoxicity was tested in vitro in the ames test, in the chromosomal aberration test in v79 cells, both showing negative results whereas the mouse lymphoma mutation test / l5178y/tk+/-cells was positive. in vivo no mutagenic effect was detected in two mouse micronucleus tests, on with intraperitoneally application and another after repeated inhalation. nanosized zno was tested in 5-days, 14-days and 90-days inhalation studies, in all studies the predominant effects were reversible local inflammatory changes in the nasal cavity and lungs, with a noaec of 1.5 mg/m3 in the 90-day study. in a prenatal developmental toxicity study according to oecd tg 414, with repeated inhalation exposure to female wistar rats from gestation day 6 through 19, maternal toxicity was observed by increase lung weights and inflammations in the lungs. but no substance-related effects on reproductive parameters (conception rate, corpora lutea, implantation sites, preimplantation loss, postimplantation loss, resorptions, dead fetuses) and no increase in external and soft tissue malformations and variations could be detected. the overall result of all the toxicological studies with nanosized and pigmentary zno is that the toxicological profile of both is very similar. studies were sponsored partly by cefci lri and partly by basf se. use of reach registration data for improving thresholds of toxicological concern (ttc) wieneke n., dorn s., jakupoglu c., schäfer c., sica m., wiegand c., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany the threshold of toxicological concern (ttc) concept is utilised to identify human exposures that are so low that in-depth toxicological investigations are expendable. this is called "exposure-based waiving". exposure-based waiving serves to focus available resources on substances with relevant human exposure potential. important work into establishing ttc values has been published by munro et al. (1996) . the initial report used a database of 613 organic substances compiled from publicly available sources. in total, 2941 noels were collected in this fashion. the munro concept used the cramer classification to categorise substances according to their hazard potential. we broadened the ttc database by including noaels published on the echa website as per 3 november 2011, containing data for more than 3900 registrations. only nongaseous mono-constituent substances with oral noaels were included in the ttc database. organophosphates and genotoxic substances were excluded from the database as well as noaels obtained for surrogate substances. noaels for all systemic endpoints (general toxicity, developmental toxicity, fertility, neoplasia) were taken into account. where appropriate, default assessment factors of up to 6 were used to establish chronic noaels for each substance. for every eligible substance, we collected the published clp category for acute oral toxicity as a potential predictor of overall hazard potential. this gives rise to five categories of acute oral toxicity. a ttc is calculated from the 5 th percentile of noaels in each of these categories using the reach rules for establishing dnels for workers and the general population. this poster presents the preliminary results for more than 1000 substances. the results indicate that the ttc concept becomes more robust when using the very broad echa database. it also suggests that acute oral toxicity categories can be used as a predictor for the overall hazard potential of a substance. comparison of different in-vitro models for inhalation toxicology with respect to the effects of cigarette smoke total particulate matter wiese j. 1 , schumann b. b-and l-moc can be cultivated up to 70 days without loss of viability, as determined by resazurin-assay. viability of cell cultures was determined by mtt-assay after incubation with increasing doses of tpm. exposure of h322 to tpm (30 mg/l) reduced viability to 95% or 83% after 24 or 72h, respectively. in a549 viability was 67% after 72h with tpm (30 mg/l). the same dose of tpm lead to a decrease in viability to 82% (24h) or 25% (72h) in nhbec and to 74% (24h) or 28% (72h) in plc. as a marker of oxidative stress the level of intra-cellular glutathione (gsh) was determined by hplc. in both the tumor cell lines analysed gsh-level was increased by tpm (5 mg/l). in h322 the induction was 1.6 and 1.2 fold after 24 or 72h, respectively. while in a549 it was 1.3 (24h) and 1.4 fold (72h). in nhec and plc tpm (5 mg/l) did not have a significant effect on gsh-levels after 72h. in n-moc tpm (5 mg/l) also did not modulate gsh after 24h, but it diminished gsh-level by 0.5 fold upon prolonged contact (72h). in b-moc gsh concentrations also decreased to 0.6 or 0.8 fold the level of controls after 24 or 72h incubation. the results presented show that in-vitro models of varying complexity and origin within the respiratory tract clearly differ in their response to tpm, which was used a model inhalative toxicant. the tumor cell lines used seem to be better adapted to chemical stress, while the models closer to the in-vivo situation are more vulnerable. the nongenomic effects of the mineralocorticoid receptor in transgenic mouse heart winter s. 1 , schreier b. within the renin-angiotensin-aldosterone system (raas), the mineralocorticoid receptor (mr) is important for the regulation of fluid and electrolyte balance in the kidney, salivary glands, sweat glands and colon. however, survival of patients with severe heart failure is increased when mr blockage is combined with standard therapy suggesting aldosterone, the mr ligand, as a key factor in the development of cardiovascular diseases, but the mechanism is not yet fully understood. in recent years, evidence accumulated that besides its function as a hormone-activated transcription factor the mr also functions via nongenomic pathways. to investigate the function of the nongenomic effects of the mr in cardiovascular dysfunction, we generated a transgenic (tg) mouse model expressing a truncated human mr lacking the dna-binding site (hmr def ) under control of the cardiac specific α myosin heavy chain promoter (αmhc), a model for nongenomic effects of the mr in the heart. in this mouse model no enhanced mortality could be observed. body weight (bw), heart weight and relative heart weight were not different compared to wild type (wt) while left atrial weight/bw was increased by 20 % (wt 0,25 ± 0,01 mg/g vs. tg 0,30 ± 0,02 mg/g, p<0.05, n=12). compared to wt mice neither surface electrocardiographic experiments nor echocardiographic experiments revealed modified parameters for tg mice under basal (i.e. unstimulated) conditons as well as under β-adrenergic stimulation by isoproterenol (iso, 100 µl 1 mm iso intraperitoneally applied). to uncover the role of aldosterone in the development of cardiovascular diseases treatment with aldosterone and high-salt diet (1%) was performed. after 28 days cardiac function and heart dimensions were analyzed, surface electrocardiographie uncovered increased p duration (16 ± 0.5 ms vs. 14 ± 0.7 ms, p<0.05) and qtc interval (61 ± 2 ms vs. 50 ± 3 ms, p<0.05) in tg (n=7) compared to wt (n=5) animals. these findings probably indicate more sensitive conduction pathways to aldosterone in tg mice. oligomerization is important for regulation of phospholamban activity wittmann t., lohse m. j., schmitt j. p. institut für pharmakologie und toxikologie, versbacher straße 9, 97078 würzburg, germany phospholamban (pln) is a heart specific protein located in the membrane of the sarcoplasmic reticulum. it inhibits the ca 2+ -atpase serca2a, thereby decelerating cytosolic ca 2+ clearance during diastole of the cardiac cycle. upon phosphorylation the inhibitory activity of pln on myocyte ca 2+ transport is attenuated. further, it is believed that phosphorylation favors the formation of (rather inactive) pentamers and that pln pentamers itselves were an inferior substrate for phosphorylation compared to monomers. this would suggest an important role of pln oligomerization in the regulation of pln activity. to prove this hypothesis, we are investigating the patterns and kinetics of pln phosphorylation in the context of alterations in pln structure. the introduction of specific point mutations into the transmembrane region of pln yielded mutants that are purely monomeric (l37a, i40a and c41f) or favor pentamer formation (i45a, v49a). transfected hek293 cells expressing these mutants or wildtype pln were stimulated with forskolin to induce pln phosphorylation before lysis of cells and western blot analysis using antibodies directed against phosphorylated pln. surprisingly, phosphorylation was increased for both monomeric and pentameric pln after stimulation with 0.25µm forskolin for less than one minute. at increasing forskolin concentrations phosphorylation signals increased in parallel for monomers and pentamers. for measurement of phosphorylation kinetics stimulation of cells with 2.5µm forskolin was stopped at different time points. we found phosphorylation of both pln monomers and pentamers within seconds of stimulation. differences in phosphorylation patterns became more pronounced when assays were performed at low temperature (14°c). intriguingly, preliminary analyses suggest that pka dependent phosphorylation occurs first in pentamers and that phosphorylation of monomers may catch up only after pentamer phosphorylation is almost complete. our data suggest that both pln pentamers and monomers are suitable substrates for pka dependent pln phosphorylation. unlike the prevalent assumption, kinetics of pentamer phosphorylation seem to be at least as fast as that of pln monomers in transfected hek293 cells suggesting an important role of pln pentamers in the regulation of pln activity. regulation of cardiac contractility by nucleoside diphosphate kinases in zebrafish wolf n. m. 1, 2 , abu-taha i. in the heart, nucleoside diphosphate kinases (ndpks) can interact with heterotrimeric g proteins, thus regulating camp synthesis in a receptor independent manner and thereby influencing contractility in cardiomyocytes. we further investigated the interaction of ndpk isoforms with heterotrimeric g proteins in the heart in vivo and in vitro using zebrafish embryos and embryonic fibroblast from ndpk a/b double knockout mice (ndpk a/b ko mefs). in zebrafish the morpholino-mediated knockdown of ndpk a did not lead to an obvious phenotype, although the total ndpk activity was reduced. depletion of ndpk b caused a cardiac phenotype characterized by severely impaired atrial and ventricular contractility and insufficient blood flow. the depletion of ndpk b was associated with a significant decrease of protein levels of the heterotrimeric g protein subunits gβγ, gα s and gαi. the knockdown of ndpk c led to a more restricted cardiac phenotype with markedly reduced pumping function of the ventricle, while the atrium was unaffected. in accordance to the reduced cardiac pumping function, camp levels were significantly diminished in the ndpk b and ndpk c morphants. similar findings were obtained in ndpk a/b ko mefs. the absence of ndpk a and b resulted in a decrease of the plasma membrane content of gβ and gαs and a significant reduction in camp synthesis. the protein expression of the isoform ndpk c was also significantly reduced in the ndpk a/b ko mefs. the re-expression of ndpk b but not ndpk a rescued the basal camp production and the membrane content of g proteins. interestingly, the overexpression of ndpk c led to a 5-fold enhancement of the camp level and a significant increase of the membrane content of gβ and gα, and thus rescued the knockout phenotype. our data indicate, that the ndpk isoforms b and c are essential for cardiac contractility, most likely by forming a signaling complex at the plasma membrane including ndpk b, ndpk c and heterotrimeric g proteins. the isoform ndpk c, with its n-terminal hydrophobic region, might serve as a membrane anchor for the ndpk/g protein complex. induction of apoptosis via pka-dependent and pka-independent pathways by cyclic purine and pyrimidine nucleotides in mouse lymphoma cell lines wolter s. 1 camp is a second messenger that plays an important role in intracellular signal transduction of various hormones and neurotransmitters. a major function of camp in eukaryotes is the activation of camp-dependent protein kinase a (pka). pka is involved in the control of a variety of cellular processes. pka exists as an inactive tetramer of a dimeric regulatory (r2) and two catalytic (c) subunits that releases the active c-subunits upon binding of camp. stimulation of the mouse t-lymphoma cell line s49 wild-type (wt) with dibutyryl (db)-camp induces apoptosis by an intrinsic, mitochondria-dependent mechanism. apoptosis induced by db-camp occurs via a pka-dependent mechanism, since s49 kincells lacking the catalytic subunit of pka are resistant to db-camp-mediated cell death. db-camp is cleaved by esterases into the biologically active compound n 6 -mb-camp and into 2'-o-mb-camp. other cyclic nucleotides (cnmps) in addition to camp, like ccmp and cump can also function as second messengers and activate pka and cgmp-dependent protein kinase (pkg) 1 . therefore, we investigated the effects of a series of membrane-permeable analogues of camp, cgmp, ccmp and cump in s49 wt und s49 kincells on apoptosis. stimulation with db-ccmp or db-cgmp induced neither apoptosis in s49 wt nor in s49 kincells. interestingly, we observed apoptosis in s49 wt and s49 kin cells after incubation with membrane-permeable nucleotide acetoxymethyl ester (am)-analogues of cgmp, ccmp, cump and also camp. induction of apoptosis occurs via pkadependent and also pka-independent pathways. a potential role of pkg and of the exchange protein activated by camp (epac) in the induction of apoptosis is unsolved and will be explored by specific pkg-and epac-activators in this system. investigations are on the way to identify the targets, the involved signal transduction pathways and the mechanisms of pro-apoptotic actions mediated by cnmp-ams. (1) wolter s, golombek m, seifert r (2011) differential activation of camp-and cgmpdependent protein kinases by cyclic purine and pyrimidine nucleotides. biochem biophys res commun. in press apoptosis in s49 cells : induction of apoptosis in s49 wt and in s49 cells lacking the catalytic subunit of pka (s49 kin-) with a) db-cnmps and b) cnmp-ams for 72h. nebivolol reduces vascular inflammation in spontaneously hypertensive rats wu z., xia n., förstermann u., li h. institut für pharmakologie, obere zahlbacher str. 67, 55131 mainz, germany nebivolol is a third generation β1 receptor blocker with additional effects on endothelial nitric oxide production. the aim of the present study is to investigate the antiinflammatory effects of nebivolol in vivo. 48 spontaneously hypertensive rats (shr) were divided into two groups: control or nebivolol treatment group. nebivolol treatment (5 mg/kg/day for 10 days) significantly reduced the blood pressure and the heart rate in shr. the drug had no effect on coagulation. aorta from nebivolol-treated rats showed significantly improved endothelial function. nebivolol did not change the expression levels of aortic nf-kb, but significantly reduced its dna binding activity. furthermore, nebivolol decreased the expression of adhesion molecules (e.g. icam-1, vcam-1) and pro-inflammatory cytokines (e.g. il-6). in conclusion, nebivolol reduces vascular inflammation in experimental hypertension. it inhibits nf-kb activity, decreases the expression of adhesion molecules and pro-inflammatory cytokine, and improves endothelial function. characterization of the cellular activity of pde4 inhibitors using two novel pde4 reporter cell lines wunder f., quednau r., barg m., tersteegen a. bayer pharma ag lead discovery wuppertal, aprather weg 18a, 42096 wuppertal, germany cyclic nucleotide-specific phosphodiesterases (pdes) play an essential role in cellular signal transduction by regulating the intracellular levels of camp and cgmp and, therefore, are important pharmacological targets. we report here the generation and pharmacological characterization of two novel pde4 reporter cell lines. plasmid constructs encoding human pde4b1 or pde4d3 were stably co-transfected with the beta1-adrenoceptor in a parental camp reporter cell line expressing a cyclic nucleotide-gated (cng) cation channel, acting as the biosensor for intracellular camp. in this reporter cell line, camp levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the cng channel. by using different pde4 and non-pde4 inhibitors, we could show that our novel pde4b1 and pde4d3 reporter cell lines specifically monitor pde4 inhibition with high sensitivity. pde4-selective inhibitors alone did not increase basal luminescence levels in this experimental setting. however, these inhibitors induced concentration-dependent luminescence signals in combination with the adrenoceptor agonist isoproterenol. in contrast, in a stable beta1-adrenoceptor reporter cell line with no recombinant pde4 expression, pde4 inhibitors had no effect on isoproterenol-stimulated luminescence signals. we compared the cellular activity of different pde4 inhibitors with the in vitro inhibition of full-length and truncated (catalytic domain) pde4d3 from cell lysates. two different groups of pde4 inhibitors could be identified. the first group, including the allosteric inhibitors pmnpq and d159153, showed high cellular activity and much better inhibition of full-length versus truncated pde4d3. the second inhibitor group, including classical competitive inhibitors like roflumilast, cilomilast and piclamilast, showed comparably lower cellular activity and similar inhibitory activity on full-length and truncated pde4d3. the results imply that these novel pde4 reporter cell lines are well-suited for the characterization of the cellular activity of pde4 inhibitors and may also support a better understanding of the complex pde4 pharmacology. plexin-b2 is required for kidney regeneration after acute renal failure xia j. 1 , gröne h acute renal failure is a common clinical problem with unsatisfactory therapeutic options and high mortality in humans. therefore, unraveling the mechanisms that promote kidney regeneration and repair may provide new therapeutic strategies for acute renal injury. plexin-b2 belongs to a family of transmembrane receptors which mediate the cellular effects of semaphorins. while plexins have first been described in the context of axon guidance, several recent studies have established them as key regulators of organogenesis, the immune system and cancer. we have recently found that plexin-b2 is highly expressed in the adult kidney, particularly in tubular epithelial cells which are most sensitive to acute ischemic injury. to study the role of plexin-b2 during kidney regeneration we generated mice lacking plexin-b2 specifically in tubular epithelial cells. under physiological conditions, these mice displayed normal kidney morphology and function. in contrast, following ischemia/reperfusion injury, plexin-b2 conditional knockout mice exhibited severely impaired kidney regeneration. while the renal function of control mice fully recovered within 3 weeks after injury, plexin-b2 knockout mice had strongly elevated serum creatinine and urea levels associated with increased morbidity and mortality. this was accompanied by hyperproliferation of tubular epithelial cells and obstruction of tubular lumina. we conclude that plexin-b2 is required for regeneration after acute ischemic renal injury and that pharmacological interventions activating plexin-b2 might represent a new therapeutic strategy in acute renal failure. the nadph oxidase enzyme complex consists of two membrane-bound catalytic subunits (a nox protein and p22phox) and several cytosolic regulatory components including p47phox, p67phox, p40phox and the small gtpase rac1. we have previously shown that treatment of apolipoprotein e knockout mice with resveratrol led to a downregulation of nox2 and nox4 in the heart. our recent data demonstrated that resveratrol also reduced the enzymatic activity of cardiac nadph oxidase. because activation of nadph oxidase enzyme complex is induced by translocation of the regulatory subunits, we studied whether the reduced enzymatic activity is due to an inhibition of such a translocation. indeed, resveratrol treatment prevented rac1 membrane translocation from cytosol. resveratrol is known as an activator and expression enhancer of the longevity gene sirtuin 1 (sirt1). we then wanted to find out whether the effect of resveratrol on rac1 was mediated by sirt1. sirt1 is a histone/protein deacetylase. in vitro incubation of rac1 with sirt1 led to a reduction of lysine acetylation. deacetylation of rac1 on lysine 166 could be identified by mass spectrometry analyses. the lysine 166 lies within the p67phox-binding region of rac1. consistently, in vitro incubation of rac1 with sirt1 markedly reduced its binding activity to p67phox. in conclusion, we provide evidence that rac1 is a direct target molecule of sirt1. sirt1 deacetylates rac1 on lysine 166 and thereby inhibits its interaction with p67phox. this is a novel mechanism of nadph oxidase inhibition by sirt1/resveratrol. mutational analysis of the effects of the cardioprotective drug dexrazoxane on topoisomerase ii beta in vitro yan t., deng s., frensch i., gödtel-armbrust u., wojnowski l. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany dexrazoxane (drz, icrf-187) is the only approved drug shown to protect against anthracycline-induced heart failure. the protection is usually ascribed to the ironchelation by drz, which is thought to reduce anthracycline-induced oxidative stress. however, similarly to anthracyclines, drz is also an inhibitor of the anthracycline target topoisomerase ii (top2). we hypothesized that the cardioprotective effects of drz are mediated by the prevention of the anthracycline-induced dna damage mediated by top2b, the dominant cardiac top2 isoform. this was investigated using top2b mutants resistant to drz, which were expressed in cells depleted of wild-type top2 isoforms. top2b-mediated double-strand dna breaks were assessed as γ-h2ax. the levesl of dsb generated by the top2b mutants in response to the anthracycline doxorubicine (dox) was indistinguishable from that mediated by a wild-type top2b. preincubation with drz depleted wild-type top2b and this was accompanied by a decrease in the dna damage following a subsequent exposure to dox. in contrast, neither top2b depletion nor the reduction of dsb by drz was seen in drz-resistant top2b mutants. furthermore, the cardially ineffective drz analog icrf-161, capable of iron chelation but not of top2 binding, affected neither the stability of top2b, nor the dox-induced dna damage mediated by this enzyme. these results indicate that drz may exert its cardioprotective effects by reducing the dna damage mediated by doxpoisoned top2b rather than by iron chelation. they also suggest a cardioprotective function of top2b, which is currently under investigation using cardiomyocyte-specific top2b mouse knockouts. aminoglycosides are important antibiotics in the treatment of life-threatening infections, especially those caused by gram-negative bacteria. their nephrotoxic and ototoxic potential is well-known, but little is known about the effects of aminoglycosides on the male reproductive system. we studied the effects of four aminoglycosides on sertolicells in vitro. rat sertoli-cells from the cell line serw3 were cultivated for 3, 6, and 9 days in dmem supplemented with three different concentrations of amikacin, streptomycin (30 mg/l, 100 mg/l, 300 mg/l), gentamicin or tobramycin (10 mg/l, 30 mg/l, 100 mg/l). we determined the expression of two junctional proteins (connexin 43, ncadherin) and one protein of the cytoskeleton (vimentin) by western blot. cells were solubilized in lysis buffer. lysates were separated by sds-page and electroblotted on a pvdf-membrane. after incubation with primary antibodies overnight and horseradish peroxidase-conjugated secondary antibody the visualization was achieved by a chemiluminescence-detection system. in addition, proteins were detected by immunohistochemistry. after three days in culture amikacin caused the most pronounced effect. at the lowest concentration tested (30 mg/l) connexin 43 and n-cadherin were reduced to 55±19% and 92±10% of the controls (n=6). no change was recognized for vimentin (102±16%). effects obtained with streptomycin were less pronounced for these these proteins (68±16%, 96±7%, and 102±13%, respectively). similar, but less pronounced effects were observed with gentamicin and tobramycin at a concentration of 10 mg/l (connexin 43: 88±15% and 81±15%; n-cadherin: 95±18% and 103±11%; vimentin: 81±12% and 102±19%) and 30 mg/l (connexin-43: 84±19% and 78±18%; n-cadherin: 98±19% and 103±13%; vimentin: 102±8% and 115±15%). after incubation for 6 and 9 days the effects occurred in the same range. the substances showed no influence on the viability of serw3 sertoli-cells up to 300 mg/l in the mtt assay. by immunohistochemistry we showed that the localisation of the proteins -connexin 43 and n-cadherin at the cell membrane and vimentin in the cytoplasm -was not influenced by the aminoglycosides. large conductance calcium-and voltage-gated potassium (bk) channels play an important role in controlling membrane potential and calcium influx, and are strongly modulated by protein kinases at multiple sites. the stress-regulated exon (strex) adds to the bk channel c terminus a cysteine-rich insert of 59 amino acids that inverts the channel regulation by protein kinase a (pka) from excitatory to inhibitory. here we investigated the mechanisms by which the strex insert influences bk channel regulation by protein kinase c (pkc). activity of bk channels without strex insert (bk-zero), transiently expressed in hek cells, was inhibited by pkc in inside-out membrane patches (~ 50% inhibition). bk channels with strex insert (bk-strex), however, were insensitive to pkc. phosphomimetic mutation of a pkc phosphorylation site (s700e) in bk-strex, resulted in a ~50% reduction of basal channel activity, whereas the s700a mutant retained normal activity. to examine whether palmitoylation, and thus association of the strex domain with the plasma membrane, prevents pkc inhibition of bk channel gating, palmitoylation was abolished by either site-directed mutagenesis (c12:13a) or by pharmacological inhibition of palmitoyl transferases with 2-bromopalmitate (2-bp). both, mutation and pretreatment with 2-bp resulted in the expression of bk-strex channels which were sensitive to pkc (~ 50% inhibition of channel activity). no inhibitory pkc effect was observed in patches of the bk-strex s700a channel mutant pretreated with 2-bp. in a clonal rat somatomammotroph pituitary cell line (gh3b6), in which pcr products without (zero) and with the 174 bp strex exon could be identified, the pkc activator pma blocked channel activity by ~25 %. this inhibition was increased to over 50% when gh3b6 cells were pretreated with 2-bp, indicating that both channel isoforms were functionally active. in summary, the present study demonstrates that palmitoylation of strex prevents bk channel regulation by pkc, which is mediated by phosphorylation of ser700, probably by steric hindrance. our results provide further evidence for a cross-talk between palmitoylation and phosphorylation as a crucial mechanism underlying the dynamic regulation of ion channels. human pleural mesothelial met-5a cells are a limited in vitro model system in determining potential asbestos-like genotoxic effects of multiwall carbon nanotubes ziemann c. 1 , reamon-büttner s. multiwall carbon nanotubes (mwcnt) are nanomaterials with important technological impact. depending on their diameter, length, and biopersistence, however, some mwcnt seem to exhibit a fiber-like cytotoxic and genotoxic potential, similar to asbestos. thus, a project funded by the german federal ministry of education and research (bmbf contract no. 03x0109a) focuses on potential adverse biological effects of different types of mwcnt to enlarge the knowledge base about toxicity determining parameters. this project comprises both in vitro (rat) and in vivo endpoints with long amosite asbestos as a positive control. as mesothelial cells are target cells for adverse effects of asbestos, in particular mesothelioma development, the human sv40transformed, non-malignant pleural mesothelial cell line met-5a was chosen as the main in vitro model in this project. in the present study part, met-5a cells were characterized concerning their usefulness as an in vitro model to study potential asbestos-like cytotoxic and genotoxic effects of different mwcnt varieties. using an mwcnt-optimized lactate dehydrogenase liberation assay and proliferation parameters derived from cell counts, concentration-dependent cytotoxicity of long amosite asbestos (2, 10, and 20 µg/cm 2 ) was demonstrated in met-5a cells. cells also showed asbestosinduced increase in dna-strand breaks and oxidative dna-damage in the hogg1modified comet assay. thus, met-5a cells were responsive to asbestos treatment. owing to asbestos potential to induce aneugenic effects and spindle fiber damage, micronucleus induction, determination of numerical chromosome aberration, and altered meta-, ana-, and telophase morphology were planned as in vitro endpoints. met-5a cells were thus initially characterized in this regard and were found to exhibit highly variable chromosome numbers with lower than 10% cells exhibiting a normal diploid chromosome set, an up to twentyfold higher spontaneous micronucleus frequency, as compared to polychromatic bone marrow erythrocytes in rodents, and a profound number of aberrant meta-, ana-and telophases with bridges, lagging chromosomes and multipolar divisions. in conclusion, met-5a cells are of only limited value as an in vitro model system to study potential asbestos-like effects of mwcnt and also biopersistent fibers. the cells are indeed responsive to asbestos, but unfortunately demonstrate marked genomic instability and thus limited significance concerning genotoxic effects. waixenicin a inhibits cell proliferation through magnesium-dependent block of trpm7 channels zierler s. 1 transient receptor potential melastatin 7 (trpm7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. they are abundantly expressed in a variety of human carcinoma cells controlling survival, growth and migration. these characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. we screened a chemical library of marine organism-derived extracts and identified waixenicin a from the soft coral sarcothelia edmondsoni as a strong inhibitor of overexpressed and native trpm7. waixenicin a activity was cytosolic and potentiated by intracellular free magnesium (mg 2+ ) concentration. mutating a mg 2+ -sensitive site on the trpm7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of mg 2+ . waixenicin a failed to inhibit the closely homologous trpm6 channel and did not significantly affect trpm2, trpm4, and crac (calcium release activated calcium) channels. therefore, waixenicin a represents the first potent and relatively specific inhibitor of trpm7 ion channels. consistent with trpm7 inhibition, the compound blocked cell proliferation in human jurkat t-cells and rat basophilic leukemia cells. based on the compound's ability to inhibit cell proliferation through mg 2+ -dependent block of trpm7, waixenicin a or structural analogs may have cancer-specific therapeutic potential, particularly since certain cancers accumulate cytosolic mg 2+ . release of metals from different sections of domestic drinking water installations zietz b. p., richter k., laß j., suchenwirth r., huppmann r. governmental institute of public health of lower saxony division of environmental medicine and environmental epidemiology, roesebeckstraße 4-6, 30449 hanover, germany different metals were used as important piping materials in the drinking water supply for a long time. due to corrosion metals can leach into the tap water. of special importance is the toxic element lead. however other heavy metals in drinking water such as copper, nickel and cadmium can also give reason for health concerns. in this study it was investigated in which amount relevant metals were released from different parts of domestic installations into the cold water. for the spatial allocation of the emission sources a sequential water sampling protocol was used after three hours of stagnation time representing the first 5 litre of the water column. after stagnation ten sample volumes were collected in series. existing facilities of domestic installations constructed with different plumbing materials were examined predominantly from residential buildings. the elements al, as, cd, cr, cu, fe, mg, mn, ni, pb, sb, se, u and zn were detected by means of icp-ms. in total 16 water pipe strands of 11 domestic installation systems were examined. they comprised 379 single water samples and 5306 single parameters. depending upon the type of plumbing different courses and concentration ranges of the elements could be measured in the tap water samples. terminal taps or installation parts were frequently responsible for a release of nickel and in several cases of cadmium. the concentration courses of the element zinc proved as a good indicator for the allocation of the emission source to a brass containing section of the installation (zinc as an alloy component of brass). one can conclude that an investigation by means of a sequential water sampling protocol and multi-element detection can be a valuable non-destructive method for drinking water-hygienic investigations of domestic installations. novel interaction partners of the murine trpc4 protein zimmermann j., beck a., flockerzi v. universität des saarlandes experimentelle und klinische pharmakologie und toxikologie, kirrberger str. 1, 66421 homburg, germany in this work novel interaction partners of the murine protein transient receptor potential canonical 4 (mtrpc4) were identified. the trpc4 protein is the major subunit of a cation channel, residing in the plasma membrane. it comprises six trans-membrane domains and cytosolic amino and carboxyl termini. two major splice variants of the trpc4 gene exist, trpc4a (974 aa) and trpc4b (890 aa), trpc4b lacks aa 781 to 864 of the trpc4a variant. both trpc4 variants are co-expressed in endothelial cells, intestinal smooth muscle and brain. to identify trpc4-interacting proteins a yeast two-hybrid system, cytotrap®, which allows identification of protein-protein interactions within the cytosol was used. a premade mouse brain cdna library was screened by the cytosolic amino and carboxyl terminal parts of mouse trpc4a (aa 1 to 324; aa 622 to 974). for the carboxyl terminal part fourteen proteins were identified. to independently prove the interaction, the fulllength cdnas of all fourteen proteins were cloned, fused to a flag-tag and coexpressed with trpc4 in hek 293 cells. co-immunoprecipitations were performed for all candidates using both the anti-flag-antibody and the antibody for trpc4. in addition, changes of cytosolic calcium were monitored and trpc4 currents were recorded in hek 293 cells expressing the candidate cdnas and stably expressing the trpc4a or trpc4b and the muscarinic receptor type 2 cdnas. the tarbp2 protein, one of the candidates shown to interact with trpc4, changed calcium influx when coexpressed with trpc4. in order to identify the domains of trpc4 responsible for its interaction with the tarbp2 protein, six trpc4-gst-fusion proteins covering the carboxyl terminal 353 aa of trpc4a were expressed in e. coli and used for pull-down experiments. by this approach two domains of trpc4 could be identified to interact with tarbp2. one of these domains is well conserved within the trpc5 protein, corresponding to the result, that trpc5 and tarbp2 effectively co-immunoprecipitate, too. the tarbp2 protein has been shown to be a component of the risc loading complex, also known as the micro-rna loading complex which is composed of dicer1, eif2c2/ago2 and tarbp2 (chendrimada et al. 2005) . by its interaction it may link trpc4 to pre-microrna processing. increased levels of angiotensin ii provoke dna damage and have influence on dna repair in mouse kidneys zimnol a., brand s., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany the renin-angiotensin system (ras) plays a crucial role concerning the blood pressure, electrolyte balance and cardiovascular homeostasis. angiotensin ii (ang ii), the active hormone of the ras, in higher concentrations leads to vasoconstriction, oxidative stress and hypertension. hypertensive patients have an increased risk to develop cancer, especially kidney cancer. we have shown in vitro and in vivo, that ang ii is capable to cause an elevation of blood pressure as well as dna damage dose-dependently. to investigate whether the high blood pressure or the enhanced levels of ang ii are responsible for dna damage, male c57bl/6-mice were equipped with osmotic minipumps, delivering ang ii in a concentration of 600ng/kg · min during 28 days. additionally they were treated with ramipril, an angiotensin-converting-enzyme blocker, with the ang ii receptor antagonist candesartan, the vasodilator hydralazine, and the antioxidant tempol. dna damage was analysed with the comet assay. we measured the base excision repair (ber)-related dna repair in the kidney with a comet-based in vitro repair assay. furthermore, the distribution and expression of the ang ii-type 1 (at1) receptor in the kidney was analyzed by immunohistochemistry. treatment with ang ii led to a significant increase of blood pressure, whereas the medication with candesartan decreased the systolic blood pressure. the intervention with hydralazine lowered the blood pressure only for a short time. the other substances had no effect at all on the blood pressure. genomic damage, quantified with the comet assay, was augmented by ang ii and improved by all interventions, particularly by candesartan and tempol. beyond that, ang ii showed a tendency to reduce dna repair. treatment with candesartan, hydralazine and tempol increased the repair capacity. furthermore, ang ii tended to result in a downregulation of the at1 receptor in kidney tubule cells. candesartan and ramipril, especially were able to augment the expression of the at1 receptor, whereas hydralazine achieved the opposite. these results demonstrate that ang ii leads to dna damage in the kidney independent of blood pressure. apparently elevated levels of ang ii affect dna repair and expression of at1 receptor. to confirm these findings we are going to examine more precisely the manifestation of other enzymes, which are implicated in dna repair. regulation of hcn channel activity by cyclic cytidine 3´, 5´-monophosphate zong x., krause s., chen c. -c., gruner c., cao-ehlker x., fenske s., wahl-schott c., biel m. lmu münchen, department pharmazie, pharmakologie für naturwissenschaften center for integrated protein science munich (cipsm), butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization-activated cyclic nucleotide-gated (hcn) channels play a key role in controlling cardiac pacemaker activity and are essential for normal function of neuronal circuits. hcn channels are principally gated by voltage but are coactivated by the cyclic nucleotides camp and cgmp which directly bind to a c-terminal binding domain. recently, cyclic cmp (ccmp) was shown to be present in various cell lines and tissues at concentrations that are comparable to cellular cgmp levels. moreover, there is recent evidence that ccmp can activate camp-and cgmp-dependent protein kinases in vivo. here, we examined whether ccmp exerts effects on hcn channels. to this end, we recorded hcn channel-mediated currents (i h) in hek 293 cells that stably express hcn1, hcn2, hcn3 or hcn4, respectively. currents were measured either with a standard patch-clamp setup or by employing the planar patch-clamp technology. in hcn2 and hcn4 channels, ccmp shifted the membrane potential for half maximal activation (v0.5) to more positive values. in addition, ccmp accelerated activation while it slowed down deactivation kinetics. the ec50 for ccmp was 30 µm which is about 5 times higher than the ec50 of cgmp. cyclic cmp is a partial agonist of hcn channels since it activates only 65 % of the maximal current obtained with camp or cgmp. to identify in vivo effects of ccmp we recorded ih of murine sinoatrial node (san) cells in the presence and absence of 1 mm ccmp. like for heterologously expressed hcn channels, ccmp shifted v0.5 of ih by about +5 mv. importantly, the steepness of the diastolic depolarization of san pacemaker potentials which is mainly determined by the amplitude of ih was profoundly increased by ccmp, compared to control conditions. as a consequence of the upregulation of ih the frequency of san pacemaker potentials was increased by about 20 % in the presence of ccmp. our results suggest that ccmp is a physiological regulator of hcn channel activity. a 3d actinic keratosis like construct for assessment of innovative tumour therapeutics zoschke c. 1 the incidence of actinic keratosis (ak) has increased dramatically in the last decades and it is considered the most frequent carcinoma in situ today. especially immunosuppressed patients are at high risk to develop invasive squamous cell carcinoma (scc) [1] which asks for most efficient and well-tolerated ak therapy. yet, current measures do not fit with these demands. nucleotide analogues, recently identified by molecular modelling [2, 3] , outperformed the current standard for the therapy of actinic keratosis, 5-fluoruracil, when tested in the tumour cell line scc25, in normal human keratinocytes and fibroblasts [4] . as next step in pre-clinical drug assessment, we aimed to characterise the effect of the most selective nucleotide analogue oxbu in reconstructed human tumour skin. based on the 3d construct of scc developed by höller and co-workers [5] for start we introduced several adaptations with respect to keratinocyte, fibroblast and scc12 seeding to grow an aklike construct with scc12 cells forming nests in particular in the epidermis. in addition, first experiments with the oxbu on the ak like constructs showed promising results for an efficient treatment of actinic keratosis. efficacy was derived from immunohistology (marker for proliferation: ki-67, marker for scc: cytokeratin-10, axl, marker for invasion: mmp2, marker for apoptosis: caspase-7, nuclei were stained with dapi) as well as the effects on the secretion of cytokeratin-18 and its caspase-induced cleavage product into the culture medium following drug exposure for up to 7 days. efficacy of a 0.05% oxbu solution proved close to or even better when compared to both a 0.1% 5fluorouracil and 0.025% aphidicolin solution. the former being the gold standard of current ak therapy, the latter is a frequently used inhibitor of human polymerase alpha and delta, however, failed to be introduced into clinical use. -in fact, in monolayer cultures aphidicolin proved most toxic for normal human keratinocytes which was not true with oxbu [4] . -therefore, these 3d tumour constructs offer a new approach to pre-clinical drug assessment and may be added to other 3d models of skin diseases currently gaining increased interest as test platforms. ima910, a multi-peptide cancer vaccine for advanced colorectal cancer, induces multiple cd8+ and cd4+ t-cell responses associated with improved survival walter s. 1 , kuttruff s. ima910 is a novel peptide-based vaccine consisting of 10 hla-a*02 and 3 hla-dr binding synthetic peptides that were identified based on natural presentation on human colorectal cancer (crc) samples. ima910 was characterized in a phase i/ii trial in advanced/metastatic crc patients being at least clinically stable after 12 weeks of first-line oxaliplatin-based therapy. all patients received a single application of low-dose cyclophosphamide for immunomodulation. this was followed by repeated ima910 vaccinations in combination with low-dose gm-csf (cohort 1; n=66) or ima910 / gm-csf plus topically applied imiquimod (cohort 2; n=26). before and post vaccination, patients were analyzed by hla-multimer assay and intracellular cytokine (ics) assay for cd8 + t-cell responses and by ics assay for cd4 + tcell responses. as immune status biomarkers, 6 phenotypically defined myeloid derived suppressor cell populations (mdsc1-6) were analyzed prior to immunotherapy. tumor status of patients was monitored repeatedly by ct/mri according to recist and corresponding tumor scans were reviewed centrally. clinical assessment included disease control rate (dcr), time to progression (ttp), progression-free survival (pfs) and overall survival (os). ima910 overall was immunogenic in 73/81 (90%) evaluable patients, with 43% and 65% of patients mounting multiple cd8 + and cd4 + t-cell responses, respectively. patients that received the immunomodulator imiquimod presented with significantly more multiple cd8 + cell responses as detected by ics (p=0.016). multiple cd8 + and multiple cd4 + responses were individually associated with significantly better clinical outcome. the association was most pronounced for patients with both multiple cd8 + and multiple cd4 + responses. these patients had significantly higher dcr at 6 months (p=0.002), improved ttp (p=0.006) and improved pfs (p=0.009) than other patients. most importantly, a trend for prolonged os was also observed in these patients (p=0.088, hazard ratio 0.53). in the study population, levels of 5 different mdsc phenotypes were significantly increased as compared to age/gender matched controls. high mdsc levels were associated with fewer immune responses and for mdsc4 and mdsc5 high frequencies were associated with shorter os (p=0.007 and p=0.019, respectively). to summarize, both hla-a*02 and hla-dr restricted peptides in ima910 were immunogenic. a significantly better clinical outcome of multi-tumap responders in comparison to patients with one/no tumap response strongly indicates clinical activity of ima910. acrylamide (aa), a genotoxic carcinogen (iarc class 2a) is formed in food by thermal treatment from different precursors. after oral ingestion, aa is metabolically epoxidized in the liver by cyp450 2e1 into glycidamide (ga). ga binds to dna, forming covalent adducts, primarily at n7 of guanine (n7-ga-gua). both, aa and ga undergo conjugation to glutathione (gsh) to be excreted via urine as mercapturic acids (ma), namely nacetyl-s-(2-carbamoylethyl)-cysteine (aama), and n-acetyl-s-(2-hydroxy-2carbamoylethyl)-cysteine (gama). in a dose response study, encompassing the dosage range from human dietary exposure levels up to 10 mg/kg bw, female sprague dawley (sd) rats on a diet devoid of detectable aa content were gavaged with single doses of aa. formation of urinary mas and of n7-ga-gua dna adducts in liver, kidney and lung was measured 16 h after application, a time point where cmax of n7-ga-gua was reached. the untreated control group was found to excrete about 0.8 nmol (aama plus gama) in the urine (16 h), indicating a background of endogenous aa formation. compared to untreated control, the lowest dosage of 0.1 µg aa/kg bw neither resulted in significantly enhanced ma excretion, nor in a detectable n7-ga-gua adduct levels in any organ tested (limit of detection, lod, 0.2 adducts/10 8 nucleotides). at the tenfold higher dose (1 µg/kg bw), adducts were found in kidney (about 1 adduct/10 8 nucleotides) and lung (< 1 adduct/10 8 nucleotides), but not in liver. at 10 and 100 µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at 1 µg aa/kg bw (about 1-2 adducts/10 8 nucleotides). the results of this in vivo study and of further recent research on aa toxicology will be discussed with respect to risk assessment. exposure of rats to single doses of aa in the range of human dietary exposure (0.1-10 µg/kg bw ) leads to n7-ga-gua adduct levels in the tissues monitored obviously not exceeding the range of steady state background dna lesions associated with endogenous/exogenous exposure to various genotoxic electrophiles. thus, the question of significant impact on human background dna damage resulting from exposure to a given genotoxic carcinogen, and on potentially ensuing biological consequences may become a highly relevant issue in risk assessment. pharmaco-economic impact of price, volume and demographic development böcking w., kirch w. institut für klinische pharmakologie, medizinische fakultät der tu dresden, fiedlerstr. 27, 01307 dresden health insurance costs in germany have grown by 3% p.a. over the last ten years and amount to approx. 280 bn eur in 2009. while costs for stationary treatment as the largest cost category have been intensely analyzed over the past years, pharmaceutical expenses have been analyzed in less detail, mostly focusing on the statutory health insurance side, even though pharmaceutical expenses have grown almost twice as much as costs for ambulant treatments. therefore, the question was asked how pharmaceutical expenses in a large german private health insurance company are allocated with respect to age and indication groups, and how those have developed from 2007 to 2011. the data of a private health insurance company with more than 600.000 customers was split into price and volume effects per age group to understand if price or volume drives the cost development. additionally, the two largest indication groups are analyzed in detail. as a result, both price and volume effects drive an overall cost increase. these effects are even stronger in older age groups. this cost increase is not sustainable for the german health insurance system over a longer period of time and will even further increase due to the ageing of the german population. a novel animal replacement system for the detection of endocrine disruptive capabilities in sexual development scheider j. 1, 2 , winter p. alternatives to animal testing for prediction of local toxicity and genotoxicity have been recently established. however, currently these methods are not suitable for measuring endocrine effects in developing organs such as e.g. embryonic gonads. here we present a phenotypic anchoring of a comprehensive study on sex-specific gene expression analysis accompanied by histological analysis of endocrine disruption in chicken embryo gonads, having the potential for an animal replacing system for endocrine disruptive toxicologic and ecotoxicologic examinations of chemicals. chicken embryos were inoculated with different amounts of tributyltin (tbt) and bisphenol-a (bpa). embryos were incubated and their gonads analyzed histologically 2 d prior to hatching. from identically treated embryos right and left testes and ovaries were separated and genome-wide transcription profiles generated using supertag digital gene expression (st-dge, supersage) profiling. male and female gonadal tissues both revealed histological aberrations in response to tbt and bpa. female gonads became masculinized in response to tbt and, viceversa, bpa-treated male gonads underwent feminization whereas in female gonads clearly visible structural aberrations occurred. in both chemicals mortality increased especially in the most affected sex (tbt: females, bpa: males). the expression profiles of more than 60 million mrnas revealed massive effects of both chemicals, tbt and bpa, on important cellular signaling pathways. gene expression differences were most pronounced in the phenotypically most affected sex. our results demonstrate that endocrine disruptive chemicals exert their effects on several levels including but not restricted to known hormone-based pathways. together with an ongoing study of gene expression differences in very young life stages and different chemicals these data will form the base for a blow-by-blow analysis of sexspecific gene expression of embryonic development. the project builds on already existing and further to generate data with the aim of the development of an in vitro method for testing chemicals at chicken eggs for 1) replacement of tests on juveniles and (sub-) adult rodents, 2) stages with impossibility of sensation of pain in the individuals, 3) highly sensitive prospects of modes of action of chemicals, which 4) might show consequences in the next generations. 3 channels are critical for oscillatory burst discharges in the reticular thalamus and absence epilepsy differential distribution of three members of a gene family encoding low voltage-activated (t-type) calcium channels hippocampal seizure resistance and reduced neuronal excitotoxicity in mice lacking the cav 2.3 e/r-type voltage-gated calcium channel transcriptional upregulation of cav3.2 mediates epileptogenesis in the pilocarpine model of epilepsy structure and functional expression of a member of the low voltage-activated calcium channel family a molecular determinant of nickel inhibition in cav3.2 t-type calcium channels histidine residues in the is3-is4 loop are critical for nickel-sensitive inhibition of the cav2.3 calcium channel substrate recognition and translocation by polyspecific organic cation transporters proton pump inhibitors inhibit metformin uptake by organic cation transporters (octs) structural determinants of inhibitor interaction with the human organic cation transporter oct2 (slc22a2) functional characterization of the human organic cation transporter 2 variant p.270ala>ser extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean? local glucocorticoid production in the mouse lung is induced by immune cell stimulation biomimetic materials in tissue engineering biomaterials offer cancer research the third dimension synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells directed growth of fibroblasts into three dimensional micropatterned geometries via selfassembling scaffolds novel pcl-based honeycomb scaffolds as drug delivery systems for rhbmp-2 tissue engineering spatio-temporal vegf and pdgf delivery patterns blood vessel formation and maturation presentation of rgds epitopes on self-assembled nanofibers of branched peptide amphiphiles controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces langmuir avintegrins as receptors for tumor targeting by circulating ligands heparin binding nanostructures to promote growth of blood vessels tirrell endothelial cell adhesion to the fibronectin cs5 domain in artificial extracellular matrix proteins design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes stimuli-responsive thin coatings using elastin-like polymers for epac as a novel effector of airway smooth muscle relaxation ) camp inhibits airway smooth muscle phenotype modulation functional roles of epac and pka in human airway smooth muscle phenotype plasticity assessment of the sensitizing and irritative potential of preservatives by loose-fit coculture-based sensitization assay (lcsa) sonnenburg a nsc-631570 (ukrain) in the palliative treatment of pancreatic cancer. results of a phase ii trial association of funding and conclusions in randomized drug trials: a reflection of treatment effect or adverse events a general method for the covalent labeling of fusion proteins with small molecules in vivo robust single-particle tracking in live-cell time-lapse sequences correlation of structural class with no-observed-effect levels: a proposal for establishing a threshold of concern trbp recruits the dicer complex to ago2 for microrna processing and gene silencing index a 009, 019, 035 011, 014 003, 044 objective: hypertension and arterial stiffness is influenced by environmental and genetic factors. high plasma sodium concentration leads to mechanical stiffening of endothelial cells resulting in endothelial dysfunction and elevated blood pressure. here we investigated whether endothelial cell stiffness of ex vivo preparations of human arteries is linked to plasma sodium concentrations and functional genetic variants of the mineralocorticoid receptor (nr3c2), rs2070951 modulating blood pressure, renin, and aldosterone levels, and rs5534, which alters a mirna binding site. design and methods: twenty patients were enrolled after a vein stripping procedure and collateral arterial blood vessels were prepared for atomic force microscopy (afm). plasma sodium concentration was routinely determined and dna for genotyping was extracted from edta blood samples. sodium levels >140 mmol/l were defined as 'high'. after application of 5 µm amiloride, a specific blocker of the endothelial sodium channel (enac) changes in endothelial cell stiffness, were defined as 'weak' (≤10%), or 'strong' (>10%). statistical analyzes were performed by anova. results: in ex vivo artery preparations of patients with high sodium levels (n=12), mechanical stiffness of endothelial cells was tend to increase (∆ amiloride) (p=0.06). both nr3c2 variants were associated with a change >10% in endothelial stiffness after amiloride treatment. the rs2070951 c allele was significantly associated with a strong amiloride response (p=0.024), while the rs5534 a allele only showed a trend towards stronger amiloride effects (p=0.06). conclusion: our findings indicate that high plasma sodium concentration results in an increased endothelial amiloride response and thus influencing mechanical stiffness, modulated by functional nr3c2 variants. our novel approach linking patients' sodium levels and genetic status to endothelial stiffness by afm will be further evaluated in larger clinical settings. protein expression changes in bap-exposed human bladder cancer cells from spliceosome activation towards redistribution of the cytoskeleton after long-term exposure to subacute concentration schmitz-spanke s., pink m., jeske e., stempelmann k., rehn s., verma n., rettenmeier a. w. universitätsklinikum essen institut für hygiene und arbeitsmedizin, hufelandstr. 55, 45122 essen, germany deregulation of the β-catenin signaling pathway plays an important role in the development of hepatocellular tumors. activating mutations in ctnnb1 (encoding β-catenin) are frequently observed in murine and human liver tumors (e.g. human hepatoblastomas). activation of β-catenin signaling induces an overexpression of several cytochrome p450 (cyp) enzymes, including cyp2e1. cytotoxicity of acetaminophen (aap) is based on its cyp2e1-catalyzed metabolism to the electrophilic compound n-acetyl-p-benzo-quinone imine, which forms covalent adducts with cellular macromolecules if depletion of glutathione occurs. treatment with aap should therefore lead to a selective damage of cyp2e1-overexpressing ctnnb1mutated hepatoma cells. mice were injected with a single dose of the liver carcinogen n-nitrosodiethylamine (den) and subsequently treated with the tumor promoter phenobarbital to select for ctnnb1-mutated tumors. administration of a single dose of aap (300 mg/kg of body weight) followed the tumor promotion protocol. two days after treatment immunohistological analysis of the livers showed about 90% necrotic tissue in the larger tumors which were positive for glutamine synthetase (gs), a marker for ctnnb1-mutated tumor cells. by contrast, gs-negative tumors remained unaffected. at later time points we observed regeneration processes with infiltration of the necrotic tissue by inflammatory cells followed by fibrotic cells. proliferation of normal hepatocytes surrounding the damaged areas could also be observed. however, repopulation of parts of the former tumor areas by remaining gs-positive tumor cells was also detected. these results suggest that treatment with aap might serve as a future therapeutic possibility to selectively poison cyp2e1-overexpressing hepatoma. release of 5,6-epoxyeicosatrienoic acid (5,6-eet) upon neuronal activity induces trpa1-dependent mechanical pain hypersensitivity sisignano m. 1 , epoxyeicosatrienoic acids (eets) are cyp-epoxygenase (cyp450) derived metabolites of arachidonic acid (aa) which act as endogenous signaling molecules in multiple biological systems. we investigated the specific contribution of 5,6-eet to transient-receptor potential-(trp)-channel activation in nociceptor neurons, and its consequence for nociceptive processing. we found that during capsaicin-induced nociception 5,6-eet-levels increased in the drg and it is released from activated sensory neurons in vitro. 5,6-eet potently induced a calcium flux [10 nm] in cultured drg-neurons which was completely abolished when trpa1 was deleted or inhibited. in spinal cord slices 5,6-eet dose-dependently enhanced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sepsc) in lamina ii neurons that also respond to mustard oil (aitc), indicating a presynaptic mechanism. furthermore, 5,6-eet-induced enhancement of sepsc frequency was abolished in trpa1 null mice, suggesting that 5,6-eet pre-synaptically facilitates spinal cord synaptic transmission via trpa1. finally, intrathecal injection of 5,6-eet caused mechanical hyperagesia in wild type but not trpa1 null mice. we conclude that 5,6-eet is synthesized upon acute activation of nociceptors and leads to mechanical hypersensitivity via trpa1 at central afferent terminals in the spinal cord. sisnaiske j. 1 , hardelauf h. introduction: neurite outgrowth and plasticity of neuronal networks are essential processes e.g. during brain development and learning. thus, morphological readouts of neuronal connectivity are thought to be important endpoints to assess neurotoxic effects of environmental chemicals as well as when discovering new drugs. to analyze neurite outgrowth and connectivity level rapidly and easily in vitro we developed the network formation assay (nfa) (pct/ep2010/002811). this platform requires a spatially standardized hexagonal array for culturing neuronal networks with no need to fix or stain the cells to visualize neuritic processes. methods: to demonstrate the feasibility of the nfa we performed experiments in which we disrupted mature neurite networks or inhibited generating networks of human sh-sy5y cells with different concentrations of acrylamide (acr). we also observed the counteracting effects of brain-derived neurotrophic factor (bdnf) and calpeptin in these systems. to create the hexagonal array we used a poly(dimethylsulfoxide) bilayer stencil comprising through holes for adhesion spots and interconnecting tracks. plasma stencilling a peg-coated glass substrate produces adhesive nodes for the neurons and micron-scale-tracks for guiding neurite outgrowth and connectivity. results: in both systems, the developing and mature network, we found not only a concentration dependant effect of acr and bdnf but also a time dependant effect with a limited capability of the developing system to regenerate, even in the presence of acr. the co-treatment of the cells showed that inhibition of calpains by calpeptin might reduce the effect of intracellular elevated ca2+, a known neurotoxic mechanism of acr. moreover, the neurothrophin bdnf acts via trkb receptors on pathways stimulating neurite outgrowth and thereby counteracting the adverse effect of acr. conclusion: with the nfa we provide a rapid and simple way to analyze neurite outgrowth and connection formation in real time. by spatially standardizing the array we provide assay coordinates to streamline the analysis process and bring it towards high throughput testing. furthermore preliminary data showed that modification of the surface with biomolecules allows cell adhesion of other neuronal celltypes (e.g. primary mouse neurons) and extracellular matrix proteins (e.g. laminin) stimulate neurite outgrowth via integrins. transcriptional regulation of nox4 by histone deacetylases siuda d. 1, 2 , zechner u. 3 , prawitt d. 4 nox4 is a member of the nadph oxidase family, which represents a major source of reactive oxygen species (ros) in the vascular wall. nox4-mediated ros production mainly depends on the expression levels of the enzyme. the present study is aimed to investigate the regulation mechanisms of nox4 transcription by histone deacetylase (hdac). in cultured human ea.hy 926 endothelial cells, treatment with the pan-hdac inhibitors (scriptaid, trichostatin a, tsa, and suberoylanilide hydroxamic acid, saha) leads to a drastic decrease in nox4 mrna expression. a similar down-regulation of nox4 mrna expression can be achieved with sirna-mediated knockdown of hdac3. hdac inhibition in endothelial cells is associated with enhanced histone acetylation in the human nox4 promoter region, with no significant changes in dna methylation. consistently, scriptaid-treated cells show increased chromatin accessibility in nox4 promoter. in addition, we provide evidence that c-jun plays an important role in controlling nox4 transcription. knockdown of c-jun with sirna leads to a marked downregulation of nox4 mrna expression. in response to scriptaid treatment, the binding to c-jun to the nox4 promoter region is reduced despite the open chromatin structure. in parallel, the binding of polymerase iia to the nox4 promoter is significantly inhibited as well, which may explain the reduction in nox4 transcription. in conclusion, hdac inhibition decreases nox4 transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the nox4 promoter. this is very likely because of a hyperacetylation-mediated steric inhibition. cyclopentenone prostaglandins induce oxidative dna-damage in hamster lung fibroblast v79 cells solecki g. m. 1 key: cord-348972-r94fhpe0 authors: gussow, ayal b.; park, allyson e.; borges, adair l.; shmakov, sergey a.; makarova, kira s.; wolf, yuri i.; bondy-denomy, joseph; koonin, eugene v. title: machine-learning approach expands the repertoire of anti-crispr protein families date: 2020-07-29 journal: nat commun doi: 10.1038/s41467-020-17652-0 sha: doc_id: 348972 cord_uid: r94fhpe0 the crispr-cas are adaptive bacterial and archaeal immunity systems that have been harnessed for the development of powerful genome editing and engineering tools. in the incessant host-parasite arms race, viruses evolved multiple anti-defense mechanisms including diverse anti-crispr proteins (acrs) that specifically inhibit crispr-cas and therefore have enormous potential for application as modulators of genome editing tools. most acrs are small and highly variable proteins which makes their bioinformatic prediction a formidable task. we present a machine-learning approach for comprehensive acr prediction. the model shows high predictive power when tested against an unseen test set and was employed to predict 2,500 candidate acr families. experimental validation of top candidates revealed two unknown acrs (acric9, ic10) and three other top candidates were coincidentally identified and found to possess anti-crispr activity. these results substantially expand the repertoire of predicted acrs and provide a resource for experimental acr discovery. a ll life forms evolve under constant pressure from numerous viruses and other parasitic genetic elements, and thus have evolved multiple defense systems 1 . the crispr-cas are adaptive immunity systems that are present in nearly all archaea and~40% of bacteria, and have been harnessed for the development of powerful genome editing and engineering tools [2] [3] [4] . in the incessant host-parasite arms race, viruses evolved multiple anti-defense mechanisms including diverse anti-crispr proteins (acrs) that are currently known to comprise 46 distinct families 5, 6 . the acrs employ different mechanisms to abrogate the activity of crispr-cas systems [7] [8] [9] [10] . most of the acrs that have been studied to date bind to functionally important sites of crispr-cas effector proteins and display high specificity toward a particular crispr-cas variant from a narrow range of bacteria or archaea. some acrs, however, have broader specificity 11 , for example, acting as nucleic acid mimics 12 . furthermore, recently, enzymatically active acrs, such as acetyltransferases and nucleases, have been discovered [13] [14] [15] . clearly, acrs have enormous potential for application as modulators of genome editing tools 16, 17 . despite the major interest of acrs for understanding the biology of the host-parasite interactions in prokaryotes and their potential to transform the use of crispr in dna editing, the discovery of acrs remains a formidable task. the amino acid sequences of acrs are extremely variable, which conceivably reflects the high variability and diversity of the crispr-cas systems in bacteria and archaea 2 . the combination of the small size and the high evolutionary variability of the acrs hampers their detection with even the most powerful sequence analysis methods 10 . the currently known acr families were discovered using a variety of customized approaches, the two primary bioinformatic ones being guilt-by-association and selftargeting 5, 12, [18] [19] [20] . guilt-by-association involves searching for homologs of hthcontaining proteins that are typically encoded downstream of acrs 18 . such proteins are known as anti-crispr associated (aca) and are notably more conserved among viruses than acrs themselves, which greatly facilitates their detection. the genomic neighborhoods encoding aca homologs are then searched for potential acrs. prokaryotic genomes containing crispr-cas systems that encompass spacers targeting regions of the same genome are known as self-targeting 20 . in this case, crispr-cas system should, in theory, target and kill the host cell. therefore, organisms with self-targeting genomes can only survive when they also carry acrs to prevent crispr-cas from functioning (or, perhaps, by employing an alternative strategy for keeping the crispr-cas silent) and thus keep the cell viable. despite the notable success of these two approaches, buttressed by experimental validation of many predictions, neither provides a comprehensive methodology for detecting acrs. in addition to their extreme sequence variability, acrs share few distinguishing characteristics outside of their common role in thwarting crispr. here, we describe a systematic machine-learning approach we developed to predict acrs, based on the few known acr attributes and a secondary screen using heuristics of known acrs, to further enrich for acr candidates. we show that this method is significantly predictive of acrs, compile a collection of 2500 previously undetected predicted acrs families and examine the top candidates in detail, including experimental validation. characteristic features of the known acrs. the general concept behind our approach is to combine the few characteristics acrs tend to share into a detection model. our first step was therefore to assemble and quantify features that previously discovered acrs appear to have in common. to keep track of the known acrs, we relied on a combination of curated acr databases 21, 22 , and our own manual data curation (supplementary table 1 ). at the time of our data curation, 39 acr families were known (supplementary table 1 ). we used this original set to iteratively search for homologs in the nonredundant (nr) database at the ncbi using psi-blast and to construct a multiple protein sequence alignment for each acr family. we then used each of these alignments as the query for a psi-blast search against our local protein sequence dataset 23 that includes prokaryotic and prokaryotic virus proteins, and consists of a total of 182,561,570 proteins. all hits with an e-value below the threshold of 10e−4 were manually curated to eliminate obvious false positives, such as partial false-positive hits to very large proteins or hits to proteins with unambiguously assigned functions (unrelated to anti-crispr activity), in an effort to create a high-confidence acr set. the final positive set consisted of 3654 acrs, spanning 32 families (seven of the known acr families were not represented in our database; supplementary table 1 , supplementary data 1). the most striking and obvious common feature of the acrs is their small size (weighted mean acr length: 104 aa, table 1 ), and the tendency to form sets of small proteins that are encoded by co-directional and closely spaced genes in (pro)virus genomes (hereafter directons; fig. 1 , table 1 ). we hypothesize that these directons are largely made up of co-transcribed early anti-defense genes. beyond these distinctive features, we considered other protein characteristics that we suspected might be predictive, such as the spacing of protein-coding genes within a directon ("directon spacing", table 1 ) or protein hydrophobicity 24 ("mean hydrophobicity", table 1 ). we also considered whether proteins had significant hits when searched against conserved domains from either the ncbi conserved domain database (cdd) 25 or prokaryotic virus orthologous groups (pvog) 26 using psi-blast (e-value < 10e−4, "protein is annotated", table 1 ), with the expectation that proteins with conserved domains likely perform other functions and therefore are unlikely to be acrs. in total, we constructed a set of 12 features (table 1 , see "methods" section for details) that, together, provided a compendium of quantifiable features that were used to identify acr candidates. training and test sets. to build a predictive model, a training set comprised of two components was required: a positive set, consisting of previously discovered acrs, and a negative set, consisting of proteins confidently inferred not to be acrs (non-acrs). for the positive set, the acrs were weighted by their family and interfamily similarities (supplementary data 1, see "methods" section for details), to ensure that related and highly similar acrs were not overrepresented in the training dataset. because there is no well-defined, standard set of known non-acr proteins, we constructed the negative set by randomly selecting viral and prokaryotic proteins, under the assumption that the majority of proteins are non-acrs. the negative training dataset was constructed by randomly selecting proteins from a combination of 1000 randomly selected prokaryotic virus genomes and 4000 randomly selected crispr-cas-containing prokaryote genomes. similar to the positive set, we sought to avoid oversampling particular protein families. therefore, these proteins were clustered by sequence similarity, and for each cluster, a single representative was selected. we randomly selected 3500 proteins from this set to constitute the negative, non-acr set. during our work on the predictive model, an additional set of acrs was published 27, 28 . we incorporated these into our analysis as an unseen test set, i.e., a set of acrs unavailable during the training stage that we could use to test our model against. thus, our training set consisted of all known acrs published before september 2018 (supplementary data 1; positive set: n = 2775, 26 families; negative set: n = 2600), and the test set consisted of the acrs published after that date (supplementary data 1; positive set: n = 879 proteins, 6 families; negative set: n = 600 proteins). building and evaluating a predictive model. given our relatively small positive set, we sought to identify a model that would tend toward low variance. thus, we chose a random forest of extremely randomized trees 29 . as an ensemble method with a highly random component, it is less likely than other machine-learning approaches to overfit the training data, while allowing a nonlinear mapping of features to label data and complex feature interactions. the model consisted of a random forest with 1000 decision trees. when training the model, each decision tree is built based on a random sampling of the training data. each split in the decision tree is determined by randomly selecting multiple values across a random subset of the features, and then setting the values that minimize the likelihood of misclassification as the thresholds for the decision tree split. thus, the final forest consists of 1000 decision trees, where each decision tree's leaf nodes correspond to members of the training set. when using the model to assess a candidate protein, the candidate traverses each decision tree. within each tree, it ends up in a leaf node that contains some mixture of acrs and non-acrs from the training set. the tree assigns the candidate a score that is equal to the fraction of acrs in its leaf. the score assigned by the model is the mean of the scores across all 1000 trees. using the model and the training set we developed, we assessed the performance of the model by five iterations of threefold crossvalidation. in each iteration, the model was trained on two-thirds of the acr families, and capacity to predict the families that were left out was assessed. for each protein in the test set, we predicted the likelihood of a protein being an acr using our random forest phage protein phage protein acrx acay phage protein fig. 1 characteristics of known acrs. a a cartoon of a sample directon. acr proteins characteristically fall upstream of an hth-domain-containing gene, termed aca. acrs are usually found in suspected mobile genetic elements, such as phages. the acr directon is highlighted in the gold color, while the surrounding proteins are indicated in blue. characteristically, acrs fall in directons with small, unidentified proteins. b a density plot of acr lengths. the xaxis denotes the common logarithm of the protein length, in amino acids. the y-axis denotes the probability density function estimated from the data across the values of x. c a density plot of the mean lengths of proteins in acr directons. the x-axis denotes the common logarithm of the mean length of proteins in an acr directon, in amino acids. the y-axis denotes the probability density function estimated from the data across the values of x. nature communications | https://doi.org/10.1038/s41467-020-17652-0 article model. given the imbalance in the weights of samples in the positive and negative sets, we down weighted the negative set in training the model, so that its combined weight was equal to that of the positive set. this weighting was applied to both model training and assessment. we relied on receiver operating characteristic (roc) area under the curve (auc) to assess the model performance and used a genetic algorithm for feature selection. the roc is plotted based on the true-positive rates (the proportion of acrs that are correctly identified) and the false-positive rates (the proportion of non-acrs that are predicted as acrs). on average, across all 15 cross-validation iterations, we found that our method was significantly predictive of acrs, with an auc of 0.93 (permutation p-value: 0.001). we next used the model to predict acrs in the unseen test set. the model was found to significantly distinguish acrs from non-acrs, with an auc of 0.83 (permutation p-value: 0.001; fig. 2 ). this result indicates that our method is indeed predictive of acrs that are not present in the training set. we converted the scores output by the model into binary predictions by setting a threshold for classification that maximizes the cross-validation balanced accuracy in the training set ( supplementary fig. 1a ). the binary model achieves a precision value of 78% and a recall value of 57% on the test set (permutation p-value: 0.001, supplementary fig. 1b, c) . the members of three of the six acr families assessed in the test set were detected most of the time (acrif12-if14), whereas members of the remaining three families were detected less than half of the time, with the single member of acrie7 in the test set not detected by the model (supplementary fig. 1d, supplementary table 2 ). using the model to predict acrs. having formally demonstrated the predictive power of our model on the test set of recently discovered acrs, we sought to leverage the model to generate a dataset that would be enriched for true acrs. we combined the model predictions with other enrichment approaches based on known acrs, under the expectation that this combination would ultimately enrich for true acrs, with the caveat that explicitly applying additional enrichment approaches skews the prediction performance away from that reported by the model, and might bias the resulting set to overlook acr families that are distant from known acrs. first, we sought to define an appropriate search space of proteins likely enriched for acrs. the initial dataset consisted of 182,561,570 proteins of that most (182,332,040) came from prokaryotes, and the rest were encoded by viruses (229,530). acrs are typically encoded either within prokaryotic virus genomes, or within prokaryotic genomic regions that appear to be integrated viruses (proviruses) or other mobile genetic elements (mges) 10, 19 . we therefore identified a subset of the prokaryotic database that consisted of genomes containing complete crispr-cas systems 30 , under the premise that these genomes are more likely to encompass prophages with acrs targeting the respective crispr-cas variants 16, 20 . we further sought to limit the prokaryote protein set to proteins encoded by (putative) proviruses. although there are many methods for predicting complete proviruses and their boundaries, these fall short of comprehensive identification of provirus regions in prokaryotic genomes, primarily, because numerous proviruses are inactivated and partially deteriorated 31, 32 . indeed, many of the known acrs are encoded in the vicinity of virus proteins 12 , but not necessarily within clearly active proviruses encoding hallmark virus genes and bounded by well-defined provirus boundaries. therefore, instead of explicitly predicting proviruses, we enriched for virus-related sequence, by filtering the prokaryote protein set to the proteins encoded in the vicinity of known virus proteins (see "methods" section for details). the resulting combined dataset of prokaryotic viruses and suspected proviruses consisted of 10,938,430 proteins. as these proteins are largely virus related, we expected this set to be enriched for acrs. this set of proteins was assessed with our random forest model that resulted in an initial set of 1,546,505 candidate acrs. we further filtered these to retain only those that had no significant hits to cdd 25 or pvog 26 protein clusters (supplementary data 2). heuristic filters were applied to each of these clusters, based on known acr characteristics, to further enrich the candidate set for true acrs. the hallmark characteristics of acrs are that they (i) are encoded upstream of hth proteins, and (ii) are found in self-targeting genomes 16 . we therefore required each family to have at least one member that fulfills each of these criteria. after this filtering, 11,304 families remained. of these families, 20 included known acrs from the initial positive set (supplementary table 3 ). following this filtering using the hallmark acr characteristics, we developed and applied additional heuristic thresholds based on our initial observations. as genes encoding acrs tend to form small directons, we sought to estimate a heuristic maximum threshold for the mean directon size in a candidate family that would enrich our protein set for true acrs. we therefore searched for the threshold that, when applied, retained the largest fraction of the known acrs in our set of 11,304, while filtering out as many of the candidate families as possible. to quantify this feature, we used the balanced accuracy metric, which is equal to the average of the fraction of correct classifications between the two groups. we found that a maximum mean directon size of five genes gave the highest balanced accuracy (see "methods" section for details). consequently, we removed protein families with an average directon size of more than five genes. after this filtering, 5507 families remained. of the remaining families, 18 included known acrs from the initial positive set (supplementary table 3) . to eliminate additional false positives, we performed a psi-blast search of each protein family alignment against our sequence dataset and, under the premise that acrs are highly variable, fast evolving proteins that are not known to be encoded outside the virus or provirus contexts, removed families with numerous homologs in diverse prokaryotes. we found that the heuristic cutoff value for the number of prokaryote homologs that maximized the balanced accuracy was 374. we therefore limited our set to clusters with no >374 significant hits to the prokaryotic protein set. next, we enriched for virus proteins by limiting to families that either include at least one homolog encoded in a virus genome or have a small ratio of prokaryote homologs to provirus homologs. we found that the cutoff value for the prokaryote to provirus ratio that maximized balanced accuracy was 3. finally, we sought to exclude families that have numerous annotations when assessed with hhblits and thus include wellcharacterized non-acrs 33 . we found the cutoff value that maximized balanced accuracy for the number of hhblits hits was 52. after this filtering, 2526 families remained. of the remaining families, 17 included known acrs from the initial positive set (supplementary table 3) . although, by applying these heuristics, we likely filter out some true acrs predicted by the model and bias our predictions toward the characteristics of known acrs, we expect that, overall, this approach enriches the resulting protein set for true acrs. after applying the above filters, our enriched set consisted of 2526 protein families (fig. 3, supplementary data 2) . characteristics of predicted acrs. we performed a psi-blast search of all 2526 candidate protein family alignments against a dataset of known acrs and acr-related sequences. for 26 of these families, significant hits to the acr set were detected. of these protein families, 22 included known acrs. the remaining four families with significant similarity to known acr-related sequences are homologous to uncharacterized proteins that are encoded within previously described acr directons, namely, in the genomic neighborhoods of acriia1-4 in listeria monocytogenes, and all have been suspected of acr activity although did not show such activity when tested 20 . these proteins were previously designated as orfa, orfb, and orfe. heuristic filtering of the acr candidates. a flowchart illustrating the heuristic filtering steps. the initial set consisted of 232,616 clusters and was first filtered for clusters that included at least one member with an hth-domain-containing protein encoded downstream, and at least one member from a self-targeting genome, two hallmark acr characteristics 20 . four additional filters were applied, for mean directon size, number of hhblits hits, number of homologs, and enrichment for virus homologs. the thresholds were set based on the data presented here. b bar plot indicating the percentage of candidates and known acrs from the initial positive set at each filtering step. the red bars denote the percentage of acrs remaining at each step, and the blue bars denote the percentage of all candidates remaining at each step. the raw numbers of remaining known acrs from the initial positive set are displayed above each red bar. nature communications | https://doi.org/10.1038/s41467-020-17652-0 article nature communications | (2020) 11:3784 | https://doi.org/10.1038/s41467-020-17652-0 | www.nature.com/naturecommunications after removing these 26 families, we obtained 2500 candidate acr families, consisting of 16,919 putative acrs. the mean size of a family was seven, the largest family included 319 members, and nearly half of the families (49%) were singletons (fig. 4) . given the different cluster sizes, each predicted acr was assigned a weight inversely proportional to the size of the respective cluster, in order to ensure that related and highly similar predicted acrs were not overrepresented in summary statistics. specifically, each predicted acr was assigned a weight of 1/n, where n is the number of predicted acrs in its cluster. the predicted acrs have a weighted average size of 109 aa, with a standard deviation (sd) of 71.6 (fig. 5a) . as expected by design, the acr genes tend to form small directons (weighted mean: 3.4; weighted sd: 1.47) consisting of short genes (weighted mean of the protein sizes in the predicted acrs directons: 200 aa; weighted sd: 155; fig. 5b ). the weighted mean isoelectric point of the predicted acrs is 7.73 with a weighted sd of 2.6, and the weighted mean hydrophobicity is −0.31 with a weighted sd of 0.5. per tmhmm and signalp predictions 34, 35 , a weighted 15% of predicted acrs have at least one putative transmembrane helix or signal peptide that, as expected, is substantially less than the expectation based on the negative set (28%, table 1 ). using jpred 36 , we predicted the secondary structure of the consensus sequences in the predicted acr set. the mean percentage of amino acids contributing to alpha-helices was 39%, and the mean percentage of amino acids contributing to beta-sheets was 13%. in the negative set, 96% and 88% of the proteins were predicted to contain at least one alpha helix or beta sheet, respectively, and the mean percentage of amino acids contributing to alpha-helices and beta-sheets was 39% and 15%, respectively. although these values do not differ substantially, we tested whether the distributions of the two categories differed significantly. we found a significant difference between the distributions of amino acids contributing to beta-sheets among the candidates and in the negative set (mann-whitney u test pvalue: 7.45e−13, supplementary fig. 2a) , but no such difference for alpha-helices (mann-whitney u test p-value: 0.823, supplementary fig. 2b) . the candidates are distributed across a diverse set of species (n = 1,770). escherichia coli accounts for the largest share of candidate acrs at 2.37%. peptoclostridium difficile (1.46%) and encoding at least one acr. of the analyzed virus genomes, 197 (71%) encode a single predicted acr, 66 (24%) encode two, and the remaining ones (5%) encode three or more acrs. archaeal viruses are also represented in this set, with 33 of the predicted acrs found in 21 archaeal viruses. the maximum number of predicted acrs in a single virus strain was five, observed in ruegeria phage dss3-p1, four of which fell in the same hth-containing directon. the viruses that were found to most commonly encode more than one acr were mycobacterium phages, followed by bacillus and synechococcus phages. among the archaeal viruses, the viruses that were found to most commonly encode more than one acr were sulfolobales mexican rudivirus followed by sulfolobus islandicus viruses. we sought to examine the genomic context of the largest predicted acr clusters and gauge how often they tend to appear in similar genomic neighborhoods. we examined the ten largest acr clusters and generated a presence-absence matrix for the members of these clusters in different genomic neighborhoods (fig. 6) , with a genomic neighborhood defined as the ten genes upstream and downstream of each acr. each column is a genomic neighborhood (ordered by similarity) and each row represents an acr family. whereas the larger acr clusters in this subset tend to appear in similar genomic neighborhoods, within these neighborhoods, we also find scattered predicted acr singletons. this pattern is similar to what has been observed in known acrs, where the acrs present in a given directon vary across closely related strains, with some acrs appearing in nearly all instances of the directon and others appearing sporadically 20 . case by case analysis of top acr candidates. we next examined in greater detail the top candidates from our acr candidate set. we constructed a set for in-depth examination, by filtering for clusters with more than four members and selecting the 30 clusters with the highest mean model score. these top 30 families were explored using hhpred 37 , psi-blast against nr and examination of the genomic context for each candidate (supplementary data 3). additionally, an overlapping but distinct set of 31 top candidates from proteobacteria possessing associations with type i-c, i-e, or i-f were selected for experimental interrogation against these subtypes in pseudomonas aeruginosa (see "methods" section for details, supplementary table 5 ). it has been previously shown that acrs are typically encoded in short directons consisting of small genes, usually including one gene encoding an hth-domain-containing protein 18, 20 . this configuration has been observed for multiple acr families and numerous virus and provirus genomes. one well-characterized example of this configuration involves the acriia1-4 families 20 . members of one of our top five candidate acr clusters, candidate 4338 (hereafter c4338), were found in suspected prophages and phages of l. monocytogenes, adjacent to acriia1, with three quarters of the members of this family found in self-targeting genomes. at the time of our analysis, c4338 was not found to be homologous to any of the previously discovered acriia genes. however, shortly after the completion of the analysis and while this manuscript was in preparation, preliminary results on testing c4338 for an anti-crispr function have been reported independently 38 . c4338 has been identified as an anti-lmocas9 protein (acriia12), supporting the utility of our approach to discover acrs. members of the c20391 cluster were identified in one phage (listeria phage b054) and four suspected prophages (one in listeria innocua and three in l. monocytogenes). all the prophage-encoded homologs were found in self-targeting genomes that carry cas-ii-a. three of these genomes also carry cas-i-b. all the prophage-encoded members of this cluster were found in bacterial genomes that also encoded acriia1, and two of these also encoded acrs iia2 and iia3. given that all the genomes encoding proteins of this family encompass cas-ii-a, we predict that this is the target of its anti-crispr activity, although targeting of cas-i-b is difficult to rule out. as is characteristic of known acrs, c20391 homologs are typically encoded in short directons consisting of three genes. one of these genes contains an hth domain and is homologous to orfd of l. monocytogenes. orfd has been previously identified as a marker for acr directons and is a distant homolog of acriia1 although in itself, this protein has not been shown to possess acr activity 20 . all members of this cluster are encoded adjacent to members of another predicted acr family, c12805. c12805 includes three additional members that are not adjacent to c20391, but are all found in a directon with acriia4 and an additional candidate, c42626, in prophages of listeria strains solely containing cas-i-b. in the genomic neighborhoods of c42626, one instance includes an expanded version of acriia4 (encoded in l. monocytogenes l99) that contains an hth, whereas the remaining two instances of acriia4 lack an hth domain. however, an examination of the nucleotide sequences immediately upstream of the truncated acriia4 indicates that this truncation is likely to be an error in the sequence annotation, and that the n-terminal of these instances of acriia4 can be extended to match the acriia4 homolog in l. monocytogenes 99, including the hth domain. furthermore, the region of acriia4 that contains the hth domain is similar to the portion of orfd that contains an hth domain (38% identity), so that extended version of acriia4 appears to be a fusion of orfd and acriia4. thus, candidates c20391, c12805, and c42626 all contain the hallmark characteristics of known acrs, including their tendency to fall in known acr neighborhoods and next to known acr markers. this corroborating evidence greatly raises our confidence that these are true acrs and further validates the predictive power of the methodology. members of the c23907 cluster, experimentally validated as acric9 (see below), were identified in one phage (rhodobacter rcapnl) and in three rcapnl prophages integrated in selftargeting genomes of rhodobacter capsulatus. c23907 belongs to a small directon of three genes, with the second gene in the directon containing an hth domain. this hth-containing gene is a distant homolog of aca3, a previously discovered gene associated with acrs, further supporting the prediction of anti-crispr functionality of c23907. the third gene in the directon is uncharacterized. the three self-targeting prophages containing the acr occur in genomes with two crispr systems, type i-c and type vi-a, either of that are potential targets of c23907. members of the c27905 cluster were found in clostridium. half of the homologs were found in genomes that are selftargeting. as is characteristic of acrs, c27905 genes typically belong to a small directon of 2-4 genes, where the second protein encoded in this directon contains an hth domain. the other proteins in the directon are uncharacterized. all the genomes in this set contain crispr i-c, a potential target of c27905. members of the c11640 cluster, experimentally validated as acric10 (see below), were found in xanthomonas. eight of these genes were identified in xanthomonas translucens and one in xanthomonas sp. shu199. eight of the nine homologs were found in self-targeting genomes. c11640 tends to fall in a small directon of two genes, as is characteristic of known acrs, where the second gene in the directon contains an hth domain. all the genomes containing c11640 have type i-c crispr systems, a potential target of c11640. to test these predictions, acr validation was conducted in p. aeruginosa strains expressing type i-c, i-e, or i-f crispr-cas systems targeting phage (see "methods" section for details). type i-e and i-f systems were expressed at endogenous levels with native spacers targeting phages jbd8 and dms3m, respectively, whereas the type i-c system was expressed heterologously in strain pao1, with an engineered spacer. the candidates associated with one of these three subtypes and present in pseudomonas, combined with members of the top 30 acr set (supplementary data 3) from proteobacteria, were selected for experimental validation. two candidates identified in this work (c40699 and c25827, supplementary table 5 ) were found to be homologous to two type i-c crispr inhibitors that have been independently identified via aca association 39 . c25827 is homologous to acric3 (100% amino acid identity, 100% coverage), and c40699 is homologous to acric4 (89.4% amino acid identity, 98% coverage). the remaining 29 genes were synthesized and 23 were successfully cloned into expression vectors (supplementary table 5 ). in addition to acric3 and acric4, anti-crispr activity was demonstrated for two proteins (acric9 and acric10) that also targeted the type i-c system (fig. 7, supplementary table 5 ). acric9 is 79 amino acids in length and highly acidic (pi = 3.96), much like previously described dna mimic acr proteins 9, 40 . this protein was found to be highly active, fully inactivating type i-c crispr-cas. acric10 is 94 amino acids and is a comparatively neutral protein (pi = 6.57) that displayed an~100-fold weaker activity than acric9, under our experimental conditions. acric9 and ic10 were the two highest confidence candidates tested, and acric4 and ic3 were the fourth and sixth highest confidence candidates tested, respectively. of the remaining 21 candidate acr proteins, four were toxic in the type i-c strain and therefore were not tested against i-c, and only six were tested against type i-f before the laboratory shutdown due to covid-19 (supplementary table 5 ). together, these results confirm that top-ranking predictions by our method are highly likely to be active acrs. the acrs are of major interest to a wide range of researchers, due both to their role in the evolutionary arms race between viruses and their prokaryotic hosts, and to their potential use as crispr-cas inhibitors in genome engineering applications. here, we demonstrate substantial predictive and discriminative power of a machine-learning approach for the identification of candidate acrs. this result appears unexpected given the paucity of distinctive features of the acrs. nevertheless, these few, rather generic features including the small size of the acr genes, type i-c crispr-cas type i-f crispr-cas type i-e crispr-cas fig. 7 identification of anti-crispr proteins acric9 and acric10. phage dms3m or jbd8 spot titration from left to right (tenfold serial dilutions) on lawns of p. aeruginosa strains expressing the indicated crispr-cas system (x-axis, type i-c, i-f, or i-e), with a crrna targeting the indicated phage and the indicated acr or empty vector (y-axis). during screening of all candidate acr proteins against all three systems, each combination was screened once. upon detecting inhibition, positive results were visually confirmed in triplicate. their arrangement in short directons that contain, additionally, genes for hth proteins, poor evolutionary conservation, association with viruses and proviruses, and self-targeting seem to be sufficient for apparently robust acr prediction. the underlying reason seems to be that, in viruses of prokaryotes, a substantial fraction, often, the majority of the genes that are not directly implicated in virus replication and morphogenesis are involved in anti-defense functions. a notable example can be found among archaeal viruses in some of which up to 40% of the genes appear to encode acrs 41 . hence a possible caveat of our predictions: some of the genes that we predict as acrs might target other, non-crispr defense systems. conversely, the possibility exists that, using the approach described here, we only detect one, albeit major, class of acrs, whereas others might exhibit distinct properties. the above caveats notwithstanding, the combination of sensitive database searches, machine-learning and heuristic filters applied here yielded 2500 previously undetected families of strong acr candidates that comprise an extensive resource, which we make accessible online (http://acrcatalog.pythonanywhere.com/), for structural and functional studies on acr-crispr interactions, with likely subsequent applications. the experimental validation presented here and elsewhere confirmed many of the top predictions. genes that tested negative for crispr-cas inhibition against single representatives of type i-c, i-e, and i-f in p. aeruginosa could lack inhibitory activity in this assay for many reasons. they might be acrs specific for different variants of the tested subtypes or different subtypes altogether, or acrs that act at a different stage of immunity, such as spacer acquisition. the three model strains used to represent the type i-c, i-e, and i-f systems do not necessarily reflect the potential interactions between the candidate acrs and diverse variants of these systems, or different crispr-cas types and subtypes present in the genomes where the acr candidate was found. future work will be required to test these candidates against relevant systems in the species of interest. lastly, some of these candidate acr proteins might inhibit other, non-crispr-based bacterial immune systems, given that, as recently shown, anti-defense genes show a strong tendency to cluster in mges 42 . the signatures of acrs described in this work might apply broadly to inhibitors of other prokaryotic immune systems. the current database of prokaryotic virus genomes is limited in scope but grows rapidly, thanks, largely, to metagenomic discovery of numerous viruses 43 . furthermore, so far, no targeted search for acrs in mges other than viruses, such as plasmids or transposons, has been performed. characterization of the distribution of acrs throughout the prokaryotic mobilome is a key next step to understanding the arms race that can be expected to lead to the discovery of numerous acrs. thus, the clear extension of this work involves searching the expanding virus genome databases, metagenomes, and other mge. iterative application of this strategy should greatly expand the diversity of acrs and, possibly, inhibitors of other defense systems. iterative search for acr homologs. for each acr family, a single representative sequence was selected, and a psi-blast search was run against the ncbi nr sequence database. iterative psi-blast was run to convergence, the identified homologs were aligned using muscle 44 , and the resulting alignment was searched against our prokaryote dataset 23 and our prokaryotic virus dataset from the ncbi viral genomes resource 45 , using psi-blast. we used a cutoff of e-value ≤ 10e−4 for homolog detection and manually reviewed each resulting alignment. of the 39 assessed families, seven were not detected in our database (supplementary data 1). as the database used in this study was curated in 2016 (ref. 23 ) and does not include all known proteins, and because acrs tend to be highly variable, with few homologs, it was not unexpected that these families were missed. all seven families not in our database were originally detected in strains that were not available at the time the database was constructed. weighting the acrs. for the positive set, we sought to weight each acr by its sequence similarity to the other acrs, in order to avoid oversampling closely related data points. initially, each acr family is assigned a weight of one. then, within each acr family, its member proteins were clustered using mmseq2, with the parameters c = 0.5 and s = 0.4 (ref. 46 ). each cluster is defined as a subfamily, and the initial weight of one given to the family is divided evenly amongst the subfamilies. following this, each subfamily's weight is divided evenly among its members. thus, each acr's weight is proportional to its similarity to other acrs in the set. for the negative set, an analogous procedure was followed. after randomly selecting a set of proteins as the negative set pool, these proteins were clustered using the same mmseq2 parameters as used for the acr families, and from each cluster, a single representative was selected. each representative protein was given a weight of one. in training and in assessing the model, the negative set was reweighted so that each class (acr and non-acr) had the same total weight. protein annotations. proteins in our dataset were annotated by applying a psi-blast search against cdd 25 and pvog 26 , with an e-value cutoff of 10e−4. proteins with hits to pvogs were classified as viral. when enriching for true acrs using heuristics, proteins with hits to either cdd or pvog were eliminated. self-targeting assemblies. self-targeting assemblies were detected by blasting the spacers 30 from each assembly in our dataset against the corresponding genome and filtering for exact matches. wherever an exact match was found, the respective assembly was classified as self-targeting (supplementary data 4) . defining the features for the model. overall, 12 total features were defined. some features related to the protein itself, while others relate to the protein's directon. a directon was defined as consecutive proteins on the same strand with a maximum of 100 bp between adjacent proteins. the features were defined as follows: protein size: the length, in amino acids, of the candidate protein. directon size: the number of genes in the directon. mean directon protein size: the mean length, in amino acids, of all proteins in the directon. protein hydrophobicity: the protein's hydrophobicity 24 . protein annotation: a binary score of whether the protein is annotated or not. we consider a protein as annotated if it has at least one significant hit to any alignment, outside of alignments annotated as hypothetical protein, putative predicted product, or provisional. fraction of directon that is annotated: the fraction of proteins in the directon that are annotated as defined above. hth-downstream: whether there is an hth-domain-containing protein encoded downstream of and adjacent to (within three genes) the acr candidate within the same directon. this feature was analyzed by running a psi-blast search of proteins against the subset of alignments from the pvog and cdd datasets containing in their name, or description either the term hth or helixturn-helix, with an e-value cutoff of 5e−3. self-targeting: whether the protein is encoded in a self-targeting genome. predicted membrane association: whether the gene is predicted to be transmembrane or contain a signal peptide using tmhmm and signalp, respectively 34, 35 . fraction of membrane-associated proteins in directon: the fraction of the proteins encoded in the directon that are predicted to be transmembrane or contain a signal peptide as defined above. directon spacing: the mean spacing between genes in the directon. whether genome is viral: whether the protein is encoded in a viral genome or in a prokaryotic genome. a genetic algorithm that selects subsets of features and creates different feature combinations while optimizing for the best feature set 47 was applied to the 12 features for ten generations, yielding the following feature set: (1) containing genome is self-targeting (2) directon annotated protein fraction (3) directon protein lengths mean (4) directon size (5) protein is annotated (6) protein has hth-downstream (7) protein length (8) protein hydrophobicity building the model. the model was constructed using scikit-learn (https://scikitlearn.org), specifically, the extratreesclassifier with the the n_estimator parameter set to 1000, meaning that the random forest consisted of 1000 trees. the rest of the parameters were left at default. a random forest was chosen to model the data given that it is an ensemble classifier that is less likely to overfit than other methods, while allowing a nonlinear mapping of features to labels data and complex feature interactions 29 . the model was trained on the training dataset described above, while downweighting the negative set so that each class (acr and non-acr) has the same total weight. the thresholds for each split in the random forest trees were selected to minimize gini impurity 48 , which measures how often misclassification would occur when a randomly selected member of the node is randomly classified based on the distribution of labels in the node, calculated as follows: where i g (n) is the gini impurity for node n, and p i is the fraction of samples for class i (either acr or non-acr) in node n. thus, the gini impurity reaches 0 when all samples in the node fall into a single category. predictive scores were calculated by using the extratreesclassifier function predict_proba. when calculating binary predictions, the threshold was set to the best value for differentiation in the training set when maximizing accuracy, which was equal to 0.09. defining the acr search space. the alignments of the pvog proteins were compared to the dataset of genomes containing crispr-cas 23, 30 . each directon containing a protein with a viral hit with an e-value <10e−4 was considered a provirus-related sequence, along with the adjacent directons on either side. adjacent blocks of prophage-related directons (within 500 bp of each other) were considered as provirus candidates. if the provirus candidate contained at least two virus hits within 3 kb of each other, it was considered a predicted prophage. the set of virus proteins was assembled from the ncbi viral genomes resource 45 , and subset to prokaryotic viruses based on taxonomy data (https:// www.ncbi.nlm.nih.gov/genomes/genomesgroup.cgi?taxid=10239). this virus set totaled 229,530 proteins encoded in 2291 genomes. permutation p-value calculation. to calculate permutation p-values, the model's predictions for the test set were shuffled. we then tested how well the model performed on this shuffled dataset. this procedure was repeated 1000 times, creating a null distribution of aucs. with this null distribution, a permutation pvalue was calculated as follows. let n p be the number of aucs in the null distribution that are greater than or equal to the actual observed auc. the permutation p-value, then, is equal to 1þn p 1001 . thus, when the actual auc was greater than any auc in the entire permuted set, the p-value was~0.001. clustering and weighting candidate acrs. candidate acrs were clustered using mmseq2, with the parameters c = 0.5 and s = 0.4 (ref. 46 ). a weight of 1/n c was assigned to each cluster, where n c is the number of acr candidate clusters. the weight of each cluster was then divided evenly among all protein members of the cluster, so that the weight of each acr was inversely proportional to the size of the cluster it belonged to. these weights were used when calculating summary statistics for the acr candidate set, to avoid oversampling closely related data points. psi-blast search against known acr and acr-related sequences. we created a sequence database of known acrs and acr-related sequences (supplementary data 5). this database included all known acrs, acas, and proteins previously suspected of possessing acr activity, but not showing any when tested. we included the group of previously suspected acr proteins as these are proteins that bear acr characteristics, and therefore may be detected by our method, but have already been tested for acr activity. a psi-blast search of each candidate acr cluster alignment as the query was performed against this dataset of known sequences, the clusters that produced hits with an e-value of <10e−3 were discarded as belonging to known acr families or families that have already been already tested for the acr function. heuristic filtering. to choose the thresholds for all the heuristics except for selftargeting and hth-downstream, ten evenly spaced threshold values were tested, between the minimum acr value and the maximum acr value. each of these ten thresholds were applied as cutoffs to the acr families, and for each threshold the balanced accuracy was calculated. the balanced accuracy is equal to the mean of the percentage of known acrs that passed the threshold and the percentage of all proteins that were filtered by the threshold, so that a higher balanced accuracy corresponds to better discrimination between the known acrs and the rest of the candidates. the final threshold was selected so as to maximize the balanced accuracy. the selected threshold was then applied to the dataset. six heuristics were defined to further enrich the acr candidate set. number of members that have hth-downstream: we required that at least one member of the candidate family have an hth-containing protein encoded downstream within the same directon. number of members in self-targeting or virus genome: we required that at least one member of the candidate family was either encoded in a self-targeting genome or encoded in a virus genome. mean directon length: the mean number of genes in the directon for all members of the family. number of homologs in prokaryotic dataset: a psi-blast search of the multiple protein alignment of each family was performed against the prokaryotic sequence dataset 23 , and filtered for hits with a maximum e-value of 10e−6, 50% identity and 50% query coverage. ratio of prokaryotic homologs to predicted provirus homologs: a psi-blast search of the multiple protein alignment of each family was performed against the predicted provirus sequence dataset and the virus sequence dataset, and filtered for hits with a maximum e-value of 10e−6, 50% identity and 50% query coverage. if a family produced at least one hit to a virus sequence, it was included. if not, it was required that the ratio between the number of hits to the prokaryotic sequence dataset to the number of hits to the predicted provirus dataset was less than or equal to three. number of hhblits hits: the alignment of each family was compared to pfam 49 and pdb70 (ref. 50 ) using hhblits 33 . families with >52 hits were discarded. construction of acr presence-absence matrix. to generate the presence-absence table, for the ten largest acr clusters, ten genes upstream and downstream were extracted where available (a maximum of 20 genes total). if within this set, an additional predicted acr was represented, the set was further extended to include the ten genes upstream and downstream of that additional predicted acr. the resulting gene arrays were considered the acr genomic neighborhood. a binary matrix was constructed where each column is a genomic neighborhood, ordered by content similarity, and each row is a predicted acr family. in addition to the acrs from the top ten largest clusters, those encoded within ten genes upstream or downstream of acrs from the largest clusters were included. each cell represents the presence or absence of a member of the respective acr family in the neighborhood. manual assessment of candidates. the multiple alignment for each of the top 30 candidates in supplementary data 3 was compared against the pdb, pfam, and ncbi cd databases using hhpred 37 . for each candidate, we calculated a consensus sequence, where the consensus letter for an alignment position was defined as the amino acid that has the highest blosum62 score among the amino acids occupying the position. a psi-blast search of the consensus sequence of each candidate family was performed against nr, and the genomic contexts of homologs were visually assessed using geneious prime. plasmid preparation. all candidate proteins were reverse translated and codon optimized for p. aeruginosa pao1 using idt codon optimization tool. gene fragments (twist biosciences) were cloned into the saci/psti site in the pherd30t vector using gibson assembly. the resultant plasmids were selected with 30 µg/ml gentamicin and propagated in e. coli strain dh5ɑ. transformation. all plasmids were transformed via electroporation into p. aeruginosa strains ll77 (a pao1 derivative with the type i-c cas genes integrated in the chromosome), smc4386 (native type i-e), and pa14 (native type i-f) to test for inhibition of the type i-c, type i-e, and type i-f crispr-cas immune systems, respectively. transformation was performed using p. aeruginosa cultures grown overnight in lb medium at 37°c with shaking. to make cells electrocompetent, 1 ml of overnight culture was pelleted, resuspended in 1 ml of 10% glycerol, and then pelleted and resuspended twice more, with the final resuspension done with only 100 µl of glycerol solution. a total of 1 µl (~300 ng) of plasmid was added to the electrocompetent cells, and the mixtures were allowed to sit on ice for 30 min. the cells/dna mixture was transferred to cuvettes and electroporated using the biorad gene pulser xcell electroporation systems preset p. aeruginosa setting. immediately after electroporation, 1 ml of lb was added to each cuvette. the cells were transferred from the cuvettes to 1.5 ml eppendorf tubes and recovered for 1 h at 37°c with shaking. the recovered cells were pelleted, the top 700-800 µl of supernatant was removed, and then the cells were resuspended in the remaining supernatant. a total of 150 µl of cells were then spread with glass beads onto lb agar plates with 50 µg/ml gentamicin. the plated cells were allowed to grow overnight at 37°c. plaque assay for crispr-cas activity. single colonies of the three testing p. aeruginosa strains with the candidate plasmids were grown overnight in 3.5 ml of lb medium with 50 µg/ml gentamicin. a total of 150 µl of each overnight culture were then mixed with 3.5 ml of molten top agar (supplemented with 1 mm iptg for ll77) in small glass tubes. the resultant agar-bacteria mixture was then poured onto circular lb agar plates with 50 µg/ml gentamicin, 0.1% arabinose, and 10 mm mgso 4 . after being left to dry for 10 min, tenfold serial dilutions of crisprtargeted bacteriophage, ranging from 1 to 10 −6 were pipetted onto the plates, and the plates were then incubated overnight at 30°c. phages jbd30, jbd8, and dms3m were used to assay type i-c, type i-e, and type i-f crispr-cas activity, respectively. plaque assays were conducted on standard petri plates, 10 cm in diameter. reporting summary. further information on research design is available in the nature research reporting summary linked to this article. supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-17652-0. correspondence and requests for materials should be addressed to e.v.k. peer review information nature communications thanks alexander hynes, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. peer reviewer reports are available. reprints and permission information is available at http://www.nature.com/reprints publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. this is a u.s. government work and not under copyright protection in the u.s.; foreign copyright protection may apply 2020 a virocentric perspective on the evolution of life diversity, classification and evolution of crispr-cas systems biology and applications of crispr systems: harnessing nature's toolbox for genome engineering major bacterial lineages are essentially devoid of crispr-cas viral defence systems bacteriophage genes that inactivate the crispr/cas bacterial immune system the discovery, mechanisms, and evolutionary impact of anti-crisprs multiple mechanisms for crispr-cas inhibition by anti-crispr proteins cryo-em structures reveal mechanism and inhibition of dna targeting by a crispr-cas surveillance complex structure reveals a mechanism of crispr-rna-guided nuclease recruitment and anti-crispr viral mimicry keeping crispr in check: diverse mechanisms of phageencoded anti-crisprs a broad-spectrum inhibitor of crispr-cas9 inactivation of crispr-cas systems by anti-crispr proteins in diverse bacterial species an anti-crispr protein disables type v cas12a by acetylation broad-spectrum enzymatic inhibition of crispr-cas12a an anti-crispr viral ring nuclease subverts type iii crispr immunity anti-crispr: discovery, mechanism and function disabling a type i-e crispr-cas nuclease with a bacteriophageencoded anti-crispr protein naturally occurring off-switches for crispr-cas9 a new group of phage anti-crispr genes inhibits the type i-e crispr-cas system of pseudomonas aeruginosa inhibition of crispr-cas9 with bacteriophage proteins a unified resource for tracking anti-crispr names anti-crisprdb: a comprehensive online resource for anti-crispr proteins systematic prediction of genes functionally linked to crispr-cas systems by gene neighborhood analysis a simple method for displaying the hydropathic character of a protein cdd/sparcle: functional classification of proteins via subfamily domain architectures prokaryotic virus orthologous groups (pvogs): a resource for comparative genomics and protein family annotation discovery of widespread type i and type v crispr-cas inhibitors systematic discovery of natural crispr-cas12a inhibitors extremely randomized trees the crispr spacer space is dominated by sequences from species-specific mobilomes prophages and bacterial genomics: what have we learned so far? a completely reimplemented mpi bioinformatics toolkit with a new hhpred server at its core predicting transmembrane protein topology with a hidden markov model: application to complete genomes signalp 4.0: discriminating signal peptides from transmembrane regions jpred4: a protein secondary structure prediction server the hhpred interactive server for protein homology detection and structure prediction listeria phages induce cas9 degradation to protect lysogenic genomes mobile element warfare via crispr and anti-crispr in pseudomonas aeruginosa phage acriia2 dna mimicry: structural basis of the crispr and anti-crispr arms race anti-crispr proteins encoded by archaeal lytic viruses inhibit subtype i-d immunity discovery of multiple anti-crisprs uncovers antidefense gene clustering in mobile genetic elements consensus statement: virus taxonomy in the age of metagenomics multiple sequence alignment with high accuracy and high throughput ncbi viral genomes resource mmseqs2 enables sensitive protein sequence searching for the analysis of massive data sets adaptation in natural and artificial systems: an introductory analysis with applications to biology, control, and artificial intelligence the pfam protein families database in 2019 rcsb protein data bank: biological macromolecular structures enabling research and education in fundamental biology, biomedicine, biotechnology and energy the authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. the ncbi nr sequence database is available at https://ftp.ncbi.nlm.nih.gov/blast/db/ under the file names "nr.*. tar.gz". other data are available from the corresponding author upon reasonable requests. source data are provided with this paper. the model code and sample data are available on github (https://github.com/ gussow/acr).received: 31 january 2020; accepted: 8 july 2020; the authors thank koonin group members for helpful discussions. this research was supported by the intramural research program of the national library of medicine at the nih. j.b.-d. is a scientific advisory board member of snipr biome and excision biotherapeutics, and a scientific advisory board member and co-founder of acrigen biosciences. key: cord-356019-k7gs1ohp authors: makhzoum, abdullah; benyammi, roukia; moustafa, khaled; trémouillaux-guiller, jocelyne title: recent advances on host plants and expression cassettes' structure and function in plant molecular pharming date: 2013-08-20 journal: biodrugs doi: 10.1007/s40259-013-0062-1 sha: doc_id: 356019 cord_uid: k7gs1ohp plant molecular pharming is a promising system to produce important recombinant proteins such as therapeutic antibodies, pharmaceuticals, enzymes, growth factors, and vaccines. the system provides an interesting alternative method to the direct extraction of proteins from inappropriate source material while offering the possibility to overcome problems related to product safety and source availability. multiple factors including plant hosts, genes of interest, expression vector cassettes, and extraction and purification techniques play important roles in the plant molecular pharming. plant species, as a biosynthesis platform, are a crucial factor in achieving high yields of recombinant protein in plant. the choice of recombinant gene and its expression strategy is also of great importance in ensuring a high amount of the recombinant proteins. many studies have been conducted to improve expression, accumulation, and purification of the recombinant protein from molecular pharming systems. re-engineered vectors and expression cassettes are also pivotal tools in enhancing gene expression at the transcription and translation level, and increasing protein accumulation, stability, retention and targeting of specific organelles. in this review, we report recent advances and strategies of plant molecular pharming while focusing on the choice of plant hosts and the role of some molecular pharming elements and approaches: promoters, codon optimization, signal sequences, and peptides used for upstream design, purification and downstream processing. a plant molecular pharming system has been thoroughly explored over the last two decades. molecular pharming has had positive impacts on the production of pharmaceutically important proteins. it has proven that some pharmaceuticals displaying advantageous clinical properties over their native counterparts could be produced in efficient ways by molecular pharming technologies. as molecular pharming platforms, plants are excellent biofactories for the production of drugs, antibodies, and vaccines in various host systems such as whole transgenic plants, cell suspension culture, hairy roots, and hydroponic culture [1] [2] [3] . however, based on the biochemical pathway features, plant species are different in their ability to express and accumulate recombinant proteins. various plant species have already been investigated for their relevance to produce bioactive molecules in an artificial way in plants. table 1 summarizes some human antibodies and vaccines produced in different plant species and eukaryotic algae. thanks to important advantages over other prokaryotic or eukaryotic systems, plants have gained great importance in molecular pharming because they offer (1) reduced contamination risks, (2) reduced costs, (3) scaling-up possibilities [4] [5] [6] , and (4) synthesizing of large and complex protein compounds while retaining their activities (post-translational modifications) [7] . there is, however, a major drawback to a plant molecular system regarding low expression and accumulation levels of some foreign proteins. to overcome this shortcoming, several studies have been conducted to improve the different aspects that allow increasing the yield of the recombinant proteins in plants. some of these studies have focused on the choice of plant hosts, others on the bioengineering strategies of the target proteins and the expression cassettes with their components such as promoters, terminal sequences, epitope tags, and signal peptides. these aspects must be taken into account when we attempt to maximize the expression and yield of the targeted proteins to increase their production. intensified efforts have also been done to improve heterologous gene structures and codon optimization. here, we review these aspects and report recent advances in the improvements of plant molecular pharming to increase protein yield and accumulation based on upstream and downstream processing studies and empirical essays. the choice of suitable plants for molecular pharming technology is an essential factor for the success of the plant molecular pharming approach. the choice of host plant depends on a broad range of criteria including the nature of potato [121] hbsag-env, gag epitopes of hiv-1 vaccine tomato hepatitis [122] norwalk virus capsid protein (nvcp) vaccine potato gastroenteritis phase i [123] major capsid protein vp6 vaccine potato severe viral diarrhea preclinical [124] hbsag hepatitis b surface antigen, hsv herpes simplex virus, icam intercellular adhesion molecule, lt-b heat labile enterotoxin b subunit, mab monoclonal antibody, tgev transmissible gastroenteritis virus the protein, ability for transformation and regeneration, post-translational modifications, scale-up of production and maintenance costs, span of production cycles, and the downstream processing requirements. a wide range of plant crops have thus been tested for molecular pharming purposes, including leafy crops, cereal and legume seeds, oil crops, plant cell suspensions, hairy roots, and microalgae. leafy crops are helpful in terms of biomass yield and high soluble protein levels [8] . additionally, leaf harvesting does not need flowering and thus significantly reduces contamination through pollen or seed dispersal [5] . however, there is a major problem of instability of the expressed proteins in leaves due to proteolytic degradation with aging of the leaves. in fact, the instability of proteins present in leaf cells, and also in cells of the other plant tissues, may start during the translation of the foreign proteins, which hold a natural tendency towards a structural heterogeneity in a heterologous environment [9] . despite this, one of the major causes of protein instability inside the leaf cells is the presence of numerous proteolytic vacuoles in their cytoplasm. in fact, the mature leaves possess very large extra cytoplasmic vacuolar compartments containing numerous active proteolytic enzymes that are involved in the degradation of native and foreign proteins, notably after harvesting or during downstream processing (extraction/ purification from freshly collected leaves). one main lifesupporting function of the vacuolar compartment is protein breakdown. indeed, numerous vacuoles are major sites of cellular proteolysis and contribute largely to amino acid recycling in the cell, in vivo [10] . to avoid this hurdle, the leaves must be processed immediately at the farm or transported as dried or frozen material [11] . moreover, protein expression in plant aerial parts could affect the growth and development of the host plant. tobacco is the most suitable leafy crop for many reasons such as high biomass yield, well-established technology for gene transfer and expression, year-round growth and harvesting, and the existence of large-scale infrastructure for processing. furthermore, tobacco has little risk in contaminating either food or feed chains because it is a non-food or non-feed crop. although many tobacco cultivars contain high levels of toxic alkaloids and phenolic substances [12] , these compounds can be removed during the purification process. the use of transgenic tobacco chloroplasts as an alternative bioreactor presents important advantages including high transgene-copy number and high level of accumulated proteins with reduced toxicity for the host plant [13] . many recombinant proteins have been produced at high levels in tobacco chloroplasts. for example, oey et al. [14] obtained a proteinaceous antibiotic by transformation of the tobacco chloroplast by up to 70 % of the total soluble proteins (tsp), which is the highest recombinant protein accumulation accomplished so far in plants. more recently, a proinsulin has been expressed in tobacco and lettuce chloroplasts with reduced cost and facility for oral delivery [15] . alternative leafy crops such as alfalfa, lettuce, and soybean are also being investigated as suitable hosts for molecular pharming [16] . alfalfa and soybean produce lower amounts of leaf biomass than tobacco, but they have a major advantage of using atmospheric nitrogen through symbiotic relations, resulting in reduced fertilizer inputs [17] . alfalfa is particularly useful because it can be harvested up to nine times a year and has high leaf protein content (up to 30 mg total protein per gram in fresh weight). medicago inc. company (http://www.medicago. com) has invested in the development of transformation methods for the production of recombinant proteins in alfalfa leaves in order to improve the yield of antibodies with human-like n-glycans [18] . lettuce is also being investigated as a production host for edible recombinant vaccines and it has been used in a series of clinical trials for vaccine production against hepatitis b [19] , pig edema disease [20] , pneumonic plague [16] , cholera [21] , severe acute respiratory syndrome coronavirus [22] , and porcine epidemic diarrhea viruses [23] . one disadvantage, however, of the chloroplast transgenic system is that plant plastids do not ensure some of the important post-translational modifications, such as glycosylation. the latter is required for the function of many human proteins; the only organelle that can achieve this process is the golgi apparatus. however, there are differences between the models of human and plant glycosylation, and these differences can cause an immune response in humans [24] . therefore, plastid-based expression technology is limited to the production of non-glycosylated products [25] . seeds of cereals and legumes offer many advantages, in particular the long stability of their expressed proteins because seeds have an appropriate biochemical and physiologic environment such as dormancy and low water content that promote stable protein accumulation [26] . indeed, the lost water by desiccation changes the ph ambient, consequently decreasing the activity of proteases inside seed cells and protecting the recombinant protein contents. hence, the protein storage in seed endosperm allows long-term storage for at least 3 years at room temperature with no detectable loss of protein activity [27] . cereal seeds also lack the phenolic substances that are present in tobacco leaves, increasing the efficiency of downstream processing [7] . the high seed protein content, ranging from 7 to 10 %, is translated into high expression for several seed-targeted recombinant proteins [28] [29] [30] [31] . however, the overall yields of recombinant proteins in seed crops are much lower than in tobacco. additionally, the transgenic plants must pass through a flowering cycle to produce seeds; therefore, there is a possibility of contamination that may occur by pollen transfer [5, 32] . maize, rice, barley, and wheat are the most important platforms for seed recombinant protein production. generally, maize seeds are excellent bioreactors because their kernel sizes are large (with 82 % of endosperm), which is the principal site of protein accumulation [33, 34] . maize also has several advantages such as having the highest biomass yield among food crops, and ease of transformation and scaling-up [7] . the foremost disadvantage of using maize as a bioreactor is that it is a cross-pollinating plant, and thus there is substantial risk of contamination of maize crops by their transgenic counterparts [35] . similar to maize, rice is easy to transform and to scale-up with a selfpollinating trait, reducing the probability of horizontal gene flow [36] . recent studies report that rice has the potential to offer an oral delivery system for vaccine antigens and therapeutic proteins [31, 32] . like rice, barley is a selfpollinating crop and could be considered a better choice than maize [7] . however, barley is less widely grown than maize and harder to transform [37, 38] . on the other hand, wheat shows the lowest yields per unit biomass and it is still under development and improving transformation efficiency [39] . legume seeds are also an interesting production system because they have exceptionally high protein content (20-40 %) , which could produce high yields of recombinant proteins too. soybean seeds offer the advantage of a low risk of contamination by pollen because soybean is largely self-pollinating, with a low production cost. furthermore, soybean has been explored for its efficacy in expressing human growth hormone [40] and humanized antibody against herpes simplex virus [41] . similar to soybean, peas present comparable advantages. however, only a few studies have reported on the use of pea seeds as a platform for commercial production of recombinant proteins. transgenic pea seeds are used as bioreactors for the production of a single-chain fv fragment (scfv) antibody [42] . on the other hand, saalbach et al. [43] have reported a high level of expression of scfv antibody with 2 % of the total seed protein. because of their inherently low associated proteolytic activity and simplified protein isolation via oil body separations, oleosin-fusion is an emerging approach as a promising system for recombinant protein production. the target recombinant protein is produced as a fusion with oleosin, accumulates in seed oil bodies, and can be purified using simple extraction and centrifugation procedures followed by the release of the target protein by proteolytic cleavage [26, 44] . however, oleosin expression levels are still not high enough for economical production. safflower seeds are also considered advantageous for their high protein yield, low acreage, and self-pollination [32] . plant cell suspension culture has been recognized as a promising alternative biosynthetic platform for valuable proteins, and combines the qualities of whole-plant systems with those of microbial fermentation and mammalian cell culture [45, 46] . suspension cells in particular increase safety compared with mammalian cell systems which can harbor human pathogens. additionally, it is well-suited for product isolation when it is secreted into the culture medium. the first plant-produced commercial therapeutic protein for human infusion, glucocerebrosidase, was produced in carrot cells [47] , though other recombinant proteins have previously been produced in plants for therapeutic purposes [48, 49] . insertion into the plant nuclear genome of the t-dna segment, carried on a voluminous and extrachromosomal ri-plasmid (ri for root-inducing) present in the phytopathogen gram-negative soil bacterium, agrobacterium rhizogenes [50, 51] , leads to the emergence of neoplastic adventitious roots, referred to as ''hairy root syndrome'' [52, 53] . the tumorous roots, resulting from an a. rhizogenes-mediated transformation, can be grown indefinitely in vitro and have become a viable alternative production platform for heterologous proteins, as well as for other plant metabolites naturally biosynthesized in wild roots [2] . similar to suspension cells, hairy roots can be axenically cultured in controlled and contained environments for the production of high-value pharmaceutical proteins. in addition, the possible extracellular secretion or rhizosecretion of expressed proteins from hairy root cultures [54] offers a simplified method for the recovery of foreign proteins from a low-cost process. the advantages of rapidly growing hairy roots over suspension cells include long-term genotype and phenotype stability, efficient productivity, and good growth on hormone-free media [50] . the swiss biotechnology company rootec (http://www. rootec.com) is currently focusing on the commercialization of plant-derived products with hairy roots grown in mist bioreactors. hairy roots appear to be an attractive in vitro expression system with several advantages compared with field-grown plants and suspension cultured cells. other systems, such as eukaryotic algae and higher aquatic plants (e.g., duckweeds) have been envisaged for heterologous protein expression. the main advantages of these platform types are fast growth, easy harvest, and high protein contents (up to 45 % dry weight) [55] . moreover, these eukaryotic systems are able to produce glycosylated proteins contrary to the microbial cultures and their production costs are lower than that of the mammalian cells [56] . biolex, inc. (http://www.biolex.com) is working to develop a duckweed-based expression system to produce recombinant pharmaceuticals and proteins. the leading product of biolex, a hepatitis c treatment, is a controlledrelease interferon (ifn)-a2b that is currently on the market [57] . monoclonal antibodies (mabs), i.e., anti-cd20 mab and anti-cd30 mab, represent another range of products that are actually in preclinical development [58] . microalgae also offer potential production systems of recombinant proteins because of their ease of genetic transformation, rapid growth, short life cycles, and cost effectiveness [59] . currently, most of the work is achieved with one green unicellular alga, chlamydomonas reinhardtii [11] . c. reinhardtii taken as a recombinant protein expression system combines, at once, the traditional bacterial fermentation or mammalian cell-culture methods. these microalgae offer post-translational modification systems that are not present in bacteria and essential for the biological activity of many therapeutic proteins [60] . furthermore, c. reinhardtii has a rapid growth rate with a doubling time of 10 h under optimal growth conditions and supports easy nuclear and chloroplast genome transformation. therefore, only the chloroplast is considered a realistic production system for a high level of recombinant protein accumulation [61, 62] . however, as mentioned earlier, the principal limitation of chloroplast recombinant protein production is that the expressed proteins lack posttranslational modifications such as glycosylation [11] . despite this limitation, functional recombinant proteins, including antibodies and growth factors [63] , vaccines [64] , and autoantigens [13] have been expressed in the chloroplast of c. reinhardtii. plant molecular pharming technology has attracted more attention in the corporate world and more international companies are specializing in this field. table 2 provides the names and web addresses of biotech companies working on molecular pharming to develop relevant plant platforms. the high costs of the production of drugs in transgenic plants can be minimized by increasing the expression levels of the target molecules. to improve protein expression levels, it is necessary to understand the functional gene structure and all genetic and epigenetic parts in spatio-temporal expression context. the prerequisite for making the system effective in molecular pharming is to improve the expression level based on the expression cassette structures to enhance the transcription and translation of recombinant proteins. to optimize the transcription of recombinant proteins, some approaches have been envisaged with a focus on promoter elements, translation and stability of the transcripts, leader sequences and exogenous or endogenous signal peptides. tandem repeats of recombinant proteins or peptides and rna interference (rnai) technology to suppress the proteinase activity have also been attempted in order to increase the yield of proteins of interest. gene promoter is an essential element in genetic engineering for plant molecular pharming. promoter can have ubiquitous expression (35s promoter) or spatiotemporal expression features as deacetylvindoline 4-o-acetyl transferase gene promoter from the medicinal plant catharanthus roseus [65] . for its strength and efficiency to express recombinant genes in living cells, the 35s promoter is widely used for driving the expression of various recombinant genes in the molecular pharming industry [5, [66] [67] [68] . the human growth factor placental lactogen (hpl) is an example transcribed by 35s promoter in transgenic tobacco cells [69] . the 35s promoter is used as single or multiple copies to increase gene transcription efficiency. it is used, for example, as a part of the vector prjc-htf to express the human serum transferrin (htf) in transgenic tobacco [70] , and in arabidopsis protoplasts and maize callus to study the localization of the escherichia coli heatlabile enterotoxin b subunit (lt-b) [34] . dual 35s promoter is used as a part of the pzp200 vector to increase the expression levels of the surface antigen 1 (sag1) [from 78 to 322] open reading frame (orf) antigen of toxoplasma gondii in tobacco leaves [71] . although 35s promoter is a good choice for molecular pharming systems, it has moderate expression levels in some seed crops expressing recombinant proteins due to the lack of cis-acting regulatory elements specific for promoters of mature seeds' genes and the elements on which specific transcription factors bind to activate or repress the transcription. moreover, 35s promoter is not as efficient in monocots as in dicots. for this reason, monocot seed-adapted promoters have to be used instead of 35s promoter. as such, the fatty acid elongase (fad) was employed as a seed-specific promoter to express a chimeric protein in canola seeds [72] . the maize 27 kda c-zein promoter with c-zein signal peptide was also used as a part of recombinant gene cassettes to drive the gene expression of the cholera toxin b subunit (ct-b) in maize seeds [34, 73] . another example of seed-specific promoters is the barley horde in promoter (0.45 kb), which is employed to express and produce an active recombinant human hematopoietic growth factor flt in pb22gl-12 in barley plants [74] . on the other hand, it was demonstrated that extending the length of the globulin1 promoter from 1.4 to 3 kb or using three tandem repeats of the 1.4 kb globulin1 promoter was a successful strategy to increase the expression level of the hepatitis b surface antigen (hbsag) in maize seeds. the obtained yield of this antigen shifted from 0.12 to 0.31 % of the tsp [75] . on the other hand, inducible promoters are a powerful approach to restricting the gene expression at the spatiotemporal level and expressing the recombinant genes when and where they are needed. for example, the rice gene promoter a-amy8, which is strongly inducible by sugar depletion, was successfully employed to produce the mouse granulocyte-macrophage colony-stimulating factor (mgm-csf) in rice suspension culture with a final yield up to 8 % of total secreted proteins [76] the microalgae c. reinhardtii has also employed for production of recombinant proteins based either on a nuclear or a chloroplast gene expression systems. for nuclear one, a few promoters have been used such as the promoters of the photosystem i complex, ribulose bisphosphate carboxylase, heat shock protein 70a genes while for chloroplast gene expression systems, other genes promoter were exploited as the rbcl (ribulose bisphosphate), atpa (alpha subunit of adenosine triphos-phatase), psbd (photosystem ii d1) and psba (photosys-tem ii psba) promoters are considered a good choice. these promoters have been found to be more efficient when they are used with their 5 0 -and 3 0 -untranslated regions (5 0 utr and 3 0 utr) [77] . chloroplast-based systems have the advantage of avoiding the nuclear gene silencing and methylation with enhanced increasing of the level of transgene expression. tobacco chloroplast is envisioned as a relevant host to produce native and synthetic insulin-like growth factor (igf)-1 (igf-1s), while employing its specific regulatory elements including 5 0 utr and 3 0 utr regions and 16s rrna (ribosomal rna) promoter as part of the expression cassette. the chloroplast recombinant protein is fully functional based on its ability to stimulate the growth and proliferation of the human hu-3 cells [78] . another example of using chloroplast as a model to produce human recombinant protein is the production of the ifn-a5 gene in tobacco chloroplasts [79] . in this study, the chloroplast rbcl light-inducible promoter was able to produce a yield up to 4,063 pg/g fresh weight [79] . table 3 summarizes some promoters and other elements in the expression cassettes used in molecular pharming systems. in addition to the importance of promoter architecture for gene expression in molecular pharming, other strategies based on using specific peptides at n-and c-termini have been employed to enhance the transcript level of recombinant proteins. these peptides are recognizable by the host system and can increase the level of transcription and translation of recombinant proteins with enhanced stability and protection against protease degradation by targeting recombinant protein in the secretory pathway to specific organelles [34] . the cereal a-amylase signal peptide is largely used in seed-specific vectors, e.g., to produce the mouse mgm-csf in a gateway vector under the control of the a-amylase gene promoter [76] . replacing the native signal with a-amylase signal peptide did not show significant effect on the expression and accumulation of the protein in transgenic tobacco and potato [80] . the signal peptide from the 2s2 seed storage protein was successfully used to direct the human recombinant proteins into a seed secretory pathway in arabidopsis, petunia, and tobacco [26, 81] . another signal peptide, the ap24 osmotin, is used to target the surface-expressed antigen 1 (sag1) and granule dense 4 (gra4) to the apoplast of tobacco leaves [71] . the bacterial lt-b from e. coli can drive the localization of recombinant proteins to the secretory pathway in arabidopsis protoplasts and maize [34] . the sequence kdel (lys-asp-glu-leu), encoding for endoplasmic reticulum (er) retention signal, was used to express the mouse (mgm-csf) into tobacco transgenic plants [82] . kozak consensus peptide plays an important role in eukaryotic gene expression system [72] . kozak and kdel (vacuole retention peptides) are largely exploited to enhance the expression, stability, and retention of recombinant protein [26] . ma and co-workers [80] showed that pleiotropic cytokine interleukin (il)-4 expression level under the control of 35s promoter in transgenic tobacco and potato was increased when using constructs containing the er sekdel (ser-glu-lys-asp-glu-leu) in c-terminal. hdel (his-asp-glu-leu) retention peptide was also shown to be crucial for er targeting of the igg-hdel fusion into transgenic tobacco plants [83] . the histidine tag (his-tag) is widely used for targeting and facilitating the purification of recombinant proteins [80, 84] . the zz linker from staphylococcus aureus is also used as an epitope tag in chloroplast expression cassette to facilitate the purification process of synthetic and native igf-1 [78] . other specific signals and peptides are used for their suitability to host plants and their positive roles in transcription and/or translation of the recombinant proteins. table 4 summarizes some of these specific signals and their usage as parts of the expression cassettes. re-engineering recombinant genes could generate active proteins with increased expression levels. the possibility 5 0 utr 5 0 -untranslated region, camv cauliflower mosaic virus, fw fresh weight, gad glutamic acid decarboxylase, hdel his-asp-glu-leu, hgad65 human glutamic acid decarboxylase 65, hfix human coagulation factor ix, hgm-csf human granulocyte-macrophage colonystimulating factor, hpv-8 human papillomavirus-8, il-10 interleukin-10, kdel lys-asp-glu-leu, mgm-csf mouse granulocyte-macrophage colony-stimulating factor, nls nuclear localization signal, rnai rna interference, sp storage protein, tsp total soluble protein of making a fusion between a few genes using linkers to assemble amino acids or orfs in a chimeric structure is a successful strategy in molecular pharming. using the optimal codon to match the endogen proprieties of the host plant can significantly increase the level of transcription and translation of recombinant proteins. amani et al. [72] , for example, employed linkers to create a chimeric structure of three virulence factors: espa, intimin, and tir receptor from e. coli o157:h7. the designed vaccine was able to reduce bacterial infection and colonization in mice. another example was constructed from two heavy-and light-chain sequences of lo-bm2 antibody (a therapeutic igg) and expressed in tobacco bright yellow (by)-2 cells [85] . the two chains were fused in tandem or inverted in a convergent orientation to be expressed as part of an expression cassette under the control of pma4 promoter with two 35s enhancer sequences. in this work, the tandem construct had a much higher level of expression than the inverted one in tobacco suspension cells and transgenic tobacco plants [85] . a number of chimeric proteins have been constructed from multiple hiv proteins to be evaluated as hiv vaccines. targets for hiv vaccines included the different categories of hiv proteins: structural proteins (ex. gag, env, glycoprotein [gp] 120, gp41, gp160); enzymes (pol); and regulatory proteins (nef, tat, rev, vpr). more information about potential vaccines reported as candidate plant-based vaccines for human is reviewed by soria-guerra et al. [6] . protein accumulation could also be increased by duplicating a small peptide to form a large multimeric protein. for example, ten sequential tandem repeats of the small peptide glucagon-like peptide-1 (glp-1) were combined into one large synthetic gene that was introduced to tobacco [86] . western blot analysis revealed that the multimerized glp-1 protein accumulated up to 0.15 % of the tsp in transgenic tobacco leaves. additionally, cerovska et al. [87] constructed a chimeric version of an epitope driven from the human papillomavirus type 16 l2 (amino acids from 108 to 120), fused to the coat protein of the potato virus x (pvx cp). then, the chimeric construction tamavidin 2/fungal avidin-like protein that binds biotin with high affinity, a versatile affinity tag psp24 sba-tamavidin (sba-s-tm2) fusion tobacco by-2 cells 2 mg/l highly purified (90-95 % purity) on the microbeads [132] thrombin protease/cleavage site ptz57 human interferon-a5 tobacco chloroplasts/4,372 pg/g fw [79] basb barley amylase signal peptide, ct-b cholera toxin b subunit, er endoplasmic reticulum, fw fresh weight, gfp green fluorescence protein, hbsag hepatitis b surface antigen, hfbi hydrophobin, hgh human growth hormone, orf open reading frame, sba soybean agglutinin, tsp total soluble protein was transiently expressed in transgenic tobacco with a resulting yield of the fused protein of 170 mg/kg of the fresh leaf tissue. codon optimization of the surface antigen, sag178-322 from toxoplasma gondii (protozoan parasite), was investigated in transgenic tobacco leaves [71] . the synthetic orf of sag1 was designed by overlapping oligonucleotides strategy with codon optimized by er signal peptide. the synthetic sag1 was then introduced into the plant binary vector, pzp200, and expressed in tobacco leaves. it was found that the tobacco leaves transformed with unmodified sag1 gene accumulated protein five-to tenfold more than the leaves transformed with a codon-optimized sag1 gene [71] . this may be due to the er localization signal that allowed high accumulation levels of the native sag1. while this study shows a negative impact of the codon optimization on the expression of synthetic, optimized gene, daniell et al. [78] found that igf-1s was expressed, and fully functional, in tobacco chloroplasts at similar levels of the native igf protein. in fact, in a comparison study between the expression levels of the native igf-1 and an igf-1s, the authors found that, after optimization of the igf-1 gene, the yield of the optimized igf-1 was 11.3 % of tsp, while the native igf-1 was 9.5 % of tsp. moreover, the expression of the igf-1s was detected by western blots in e. coli, while no native igf-1 protein was observed, suggesting that the translation machinery in chloroplast is more flexible than in e. coli, with regard to codon optimization and usage [71] . in plant molecular pharming, linkers are important elements for producing multimeric proteins, and introducing multiple and more complex traits. linkers are short peptide sequences composed of flexible residues such as glycine and serine between adjacent domains to ensure that these domains do not sterically interfere with each other. linkers are important elements assembling few proteins, orfs, or peptides together without interfering with the structure of fused units. linkers have no negative effects on the activity and stability of the assembled parts in the new structure. variable short peptides have been exploited as linkers in genetic engineering. for example, the linker eaaak was used to design a new synthetic gene encoding the carboxy-terminal fragment of intimin, the middle region of tir receptor and the carboxy-terminal part of the espa factor in e. coli [72, 88] . the new synthetic gene was translated into transgenic canola and tobacco plants with a yield up to 0.2-0.33 % tsp in transgenic canola seeds and 0.1-0.3 % tsp in transgenic tobacco. another example is the ag simple alanine-glycine linker (ag linker), consisting of six amino acids with three ag repeats (agagag). this linker was used as a part of the expression cassette in plm03, plm08, and plm09 vectors and resulted in a positive shift of yield from undetectable to 0.059 % extracted from maize kernels [34] . as a part of a cleavable polyprotein precursor with native kex2p-like protease activity, the kex2p linker (igkrgk) 3ef (amino acids leu/glu and glu/phe) was also successfully used to fuse red fluorescent protein (dsred) or human cytokine (gmcsf) to the green fluorescent protein (gfp) into the viral vector ttosa1-103spekcd43 and the two fusions were expressed in tobacco cells (referred to as rkg and gkg cells). the expression levels of rkg and gkg were 0.5 and 0.2 % of tsp, respectively [89] . to test the effect of the length of linker on the accumulation of recombinant proteins, two different lengths were compared by plchova et al. [90] . the first link was four amino acids in length (gpgp) and the second 15 amino acids in length (sgggg) 9 3. both of them were employed to link different mutagenized versions of the human papillomavirus e7 oncoprotein with pvx cp in c-and n-terminal [90] . the resulting fusions were then expressed in e. coli and tobacco (nicotiana benthamiana), but no significant difference was observed between the two lengths of linker on the accumulating protein constructs. however, the n-terminus fusions resulted in twofold higher protein yield than the c-terminus fusion (approximately 2.80 mg/ml culture medium against 1.35 mg/ml, respectively). more information about linkers and the choice of linkers are provided by soria-guerra [6] and crasto and feng [91] . the gateway recombination system is a relatively recent powerful tool used in molecular cloning and gene expression investigations. based on specific recombination sequences, the gateway system allows cloning and transferring of dna fragments in a high-throughput manner between different expression vectors while maintaining the orientation of the cloned reading frame [92] . the system is based on the insertion of a dna fragment, or a complete orf, between two flanking recombination sequences in a specific plasmid vector to create an ''entry clone''. once an entry clone is created, the cloned dna could be sub-cloned to any gateway-designed expression vector, independent of the vector function or the host background [92] . due to its advantage as a high-throughput cloning tool, gateway technology is suitable for creating 'molecular archives' containing the whole predicted orfs, orfeome, of an organism [93] . different orfeome projects have thus been generated using gateway technology for protein expression and functional analysis, such as the yeast orfeome [94] and the human orfeomes [95] . the gateway orfeomes also allow straightforward expression of native or n-terminal and/or c-terminal fusion proteins [96] . the use of gateway technology in molecular pharming is emerging at great pace. a recent study support this trend and reports the suitability of gateway-based vectors to transform plant chloroplast [97] . the author of this study converted a standard transformation vector into a gateway destination vector in which they cloned the gfp associated with a ribosome binding site, t7g10. then, they transformed the tobacco chloroplast by the biolistic method, and found that gfp was successfully integrated into the plastid genome and resulted in an accumulation level of gfp protein up to 3 % of the tsp. pcr testing and confocal laser scanning microscopy confirmed the presence of gfp in the chloroplast, suggesting that gateway system is suitable for plant pharming in this organelle. in another recent study, buntru et al. [98] report that multigene constructs carrying seven transgenes, up to 26 kb size, were successfully transformed into tobacco cells using gateway technology and were stably inherited for at least two generations. one of the implications of the findings in this study is the availability of a powerful and efficient tool for multigenes transformation that may facilitate the genetic engineering for the production of pharmaceutical compounds at an industrial scale. kagale et al. [99] have also constructed a series of tobacco mosaic virus (tmv) compatible with gateway technology and intended for orfeome exploitation with the facility of n-or c-terminal fusions with a wide range of epitope tags. similarly, liu et al. [76] have recently constructed a gateway-compatible binary t-dna destination vector to produce the mgm-csf in rice suspension cell culture under the control of the rice a-amy8 promoter with its signal peptide. a binary gateway vector, pk7wg2, has been successfully used to produce the human glutamic acid decarboxylase (gad65), an autoantigen implicated in type 1 diabetes mellitus, in transgenic tobacco with protein accumulation up to 2.2 % of tsp, but retaining its autoantigenecity [100] . morandini et al. [101] also investigated the use of gateway vectors such as the pphasgw for seedspecific transgene expression system. the pphasgw vector was successfully employed to express a few human recombinant proteins such as a chimeric version of gad67/65, an isoform of the glutamic acid decarboxylase (65 kda), a murine anti-inflammatory cytokine il-10 and proinsulin. these proteins were produced in arabidopsis and tobacco under the control of the specific seeds promoter phaseolin and resulted in a high yield of the chimeric gad67/65 in arabidopsis seeds (7.7 % of tsp) while il-10 and proinsulin reached up to 0.70 and 0.007 % of tsp, respectively [101] . the pphasgw vector was also a good choice to express a few other constructs harboring recombinant single-chain variable fragments to the crystallizing fragment of the antibody (scfv-fc) of two antiviral mabs (2g12 and ha78) in arabidopsis transgenic seeds and leaves under the control of the seed-specific bphaseolin promoter from phaseolus vulgaris; the maximal expression levels ranged from 0.8 to 9.4 mg recombinant protein per gram dry seed weight [81] . more information about gateway-compatible plants can be found elsewhere [102, 103] . 3.6 rna interference, gene silencing and suppressing of the proteases activity in molecular pharming the limitations to producing a high yield of recombinant proteins are sometimes related to the plant host system and its metabolism. this obstacle can be overcome by inhibiting some of the major metabolic activities in the plant host. for example, the production and accumulation of the recombinant human granulocyte colony-stimulating factor was significantly increased in transgenic rice suspension culture by an rnai approach designed to suppress the cysteine proteinase gene expression or by the inhibition of proteinase [104, 105] . the inactivation of atp/adp transporters in potato tubers by rnai methodology increased the accumulation level of human recombinant mab, scfv l1g6, in the tubers up to twofold, with 30 % increase in tuber biomass and 50 % more soluble protein than wild-type tubers [106] . in rice, it was found that the co-transformation of cell suspension culture with recombinant human granulocyte-macrophage colony-stimulating factor (rhgm-csf) and a serine proteinase inhibitor ii gene resulted in a reduction of the protease activity and an increase of the yield of the recombinant rhgm-csf by twofold compared with cells expressing rhgm-csf only [105] . the strategy of a gene silencing suppressor was applied on a few systems to enhance the gene expression and recombinant protein accumulation. for example, adding a suppressor of gene silencing can have a positive role on gene expression such as the derivatives of peaq-ht vector, which was used to express transiently the human papillomavirus under the control of 35s promoter in agroinfiltrated n. benthamiana leaves [107] . the anthrax receptor fusion protein co-expressed with viral gene silencing suppressors in agro-infiltrated tobacco leaves showed a tenfold increase in the recombinant protein compared with the absence of suppression, particularly with p1 suppressor [108] . rice seeds are an efficient model to produce pharmaceuticals for human health, particularly when storage proteins (13 kda prolamin and glutelin) have been silenced by the rnai technology, so the yield of the human growth hormone polypeptides reached the level of 470 lg/g dry weight [109] . protease activities, in turn, can be exploited to increase the level of recombinant proteins. the protease activities were investigated to express multiple secretory proteins in fusions between red fluorescent protein (dsred), or human cytokine (gmcsf), and downstream gfp as a single transgene by using a cleavable polyprotein precursors containing kex2p linkers such as leagg(igkrgk)3ef [89] . using protease inhibitors in a specific manner demonstrated its potential to increase the recombinant protein yield. the transient expression of tomato cathepsin d, inhibitors of a1 and s1 proteinases, led to improvement of the murine diagnostic antibody c5-1 accumulation and protection. it altered the a1/s1 protease activities in the targeted specific cell compartment (leaf apoplast) in n. benthamiana and increased the protein content by 40 % [110] . pharmaceutical proteins glycosylation represents the most important post-translational modification that corresponds to covalent linkage of an oligosaccharide side chain to the natural or biopharmaceutical proteins [111] . shared by all eukaryotic organisms, this fundamental mechanism significantly modulates folding, activity, yield, and immunogenicity of therapeutic glycoproteins [112] . nevertheless, there are interkingdom differences in the glycosylation patterns, particularly in the n-glycoform design. n-glycosylation starts in the er and continues in the golgi apparatus with the addition of a(1,3)-fucose and b(1,2)-xylose residues to n-glycans in plants and with the addition of a(1,6)-fucose moieties, glucose, and acid sialic residues to n-glycans of glycoproteins in mammals [17] . plant-specific xyloglucan is likely associated with allergenicity disorders limiting the use of therapeutic glycosylated proteins by parenteral administration. to overcome such hurdles, humanization of plant-made glycoproteins must be achieved. in recent years, promising strategies of glycoengineering have made humanizing plant n-glycolysation possible [113] , either by inactivation of the host plant's endogenous glycosyltransferases (a1,3-fucosyltransferases and b1,2xylosyltransferase) or de novo expression of heterologous glycosyltransferases (b (1,4) galactosyltransferase and sialyltransferase), similar to those of mammals [114] . besides, proteins resident of the er lumen possess high-man (mannose)-type n-glycans with structures common to plants and mammals [114] . thereby, the addition of the h/kdel sequence at the c-terminal end of a secreted protein makes its retention in the er possible and its glycosylation is limited to only high mannose-type n-glycans [111] . another non-immunogenic glycosylation approach, based on rna-interference, modulates the levels of xylosyl and fucosyl glycans and prevents the addition of undesirable sugar residues to biotherapeutic proteins [17] . many genetic, upstream and downstream aspects of the molecular pharming technology must be taken into account when we attempt to maximize the expression and translation of the recombinant proteins of interest. various plants species, organs, tissues, cells, and subcellular organelles have been extensively studied to optimize the yield of pharmaceutical recombinant proteins with more or less successful results. genetic transformation and regeneration systems are still not feasible or are unavailable for other plant species. this fact narrows the spectra of plant hosts available to explore new approaches and tools to enhance the potential of drug design, discovery, and production. the design of expression cassette in molecular pharming has profound effects on gene expression and protein accumulation. it is thus of high interest to elucidate the mechanisms underlying the transcription and translation process of the recombinant proteins. in this review, we attempted to bring into focus the involvement of some transcription and translation constituents. although a limited number of studies report the applications of gateway vectors in molecular pharming studies, these vectors facilitate the cloning and the expression in a short time in comparison with classical cloning techniques. epigenetic rnai or gene silencing suppressors can also have significant impacts on the whole plant metabolism and the expression and accumulation of heterologous proteins. the obstacle of plant glycosylation features can be overcome by humanization of plant-made glycoproteins. the plant-made pharmaceuticals industry will flourish in the near future when it is able to provide a high quality and quantity of pharmaceutical products at low costs and with basic infrastructure. it is currently in dire need of a standard regulation system to avoid all environmental and healthcare system troubles (biosafety and regulatory issues). the recent advancements and progress in molecular biology and genomic studies and molecular cloning techniques will certainly give more effective expression and purification systems for the plant molecular pharming platform. molecular farming for new drugs and vaccines. current perspectives on the production of pharmaceuticals in transgenic plants hairy root research: recent scenario and exciting prospectscommentary development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco plants and human health in the twenty-first century molecular farming in plants: host systems and expression technology two decades of plant-based candidate vaccines: a review of the chimeric protein approaches 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transient co-expression for fast and high-yield production of antibodies with human-like n-glycans in plants transgenic lettuce seedlings carrying hepatitis b virus antigen hbsag production of double repeated b subunit of shiga toxin 2e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease expression of a cholera toxin b subunit in transgenic lettuce (lactuca sativa l.) using agrobacterium-mediated transformation system accumulation of recombinant sars-cov spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines expression of a cholera toxin b subunit-neutralizing epitope of the porcine epidemic diarrhea virus fusion gene in transgenic lettuce (lactuca sativa l.) biopharmaceutical production in plants: problems, solutions and opportunities chloroplast biotechnology, genomics and evolution: current status, challenges and future directions seed-based expression systems for plant molecular farming production and localization of recombinant pharmaceuticals in transgenic seeds commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification the production of recombinant proteins in transgenic barley grains maize (zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants biopharming to increase bioactive peptides in rice seed plant seeds as bioreactors for recombinant protein production maize plants: an ideal production platform for effective and safe molecular pharming a bacterial signal peptide is functional in plants and directs proteins to the secretory pathway molecular farming on the rise-gmo regulators still walking a tightrope endosperm tissue is good production platform for artificial recombinant proteins in transgenic rice recent advances in barley transformation barley genomics: an overview advances and remaining challenges in the transformation of barley and wheat host limits to accurate human growth hormone production in multiple plant systems second-generation hiv microbicides: continued development of griffithsin transgenic pea seeds as bioreactors for the production of a single-chain fv fragment (scfv) antibody used in cancer diagnosis and therapy high-level expression of a single-chain fv fragment (scfv) antibody in transgenic pea seeds oleosins and oil bodies in seeds and other organs bioreactor engineering for recombinant protein production in plant cell suspension cultures a novel plant cell bioproduction platform for high-yield secretion of recombinant proteins production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of gaucher's disease using a plant cell system transgenic plants for therapeutic proteins: linking upstream and downstream strategies production of human lysosomal proteins in plant-based expression systems transgenic hairy roots: recent trends and applications hairy root: plasmid encodes virulence traits in agrobacterium rhizogenes dna from agrobacterium rhizogenes in transferred to and expressed in axenic hairy root plant tissues agrobacterium rhizogenes inserts t-dna into the genomes of the host plant root cells rhizosecretion of recombinant proteins from plant hairy roots the duckweeds: a valuable plant for biomanufacturing spirodela (duckweed) as an alternative production system for pharmaceuticals: a case study, aprotinin novel controlled-release lemna-derived ifn-alpha2b (locteron): pharmacokinetics, pharmacodynamics, and tolerability in a phase i clinical trial glycan optimization of a human monoclonal antibody in the aquatic plant lemna minor the microalga chlamydomonas reinhardtii as a platform for the production of human protein therapeutics micro-algae come of age as platform for recombinant protein production recent developments in the production of human therapeutic proteins in eukaryotic algae green factory: plants as bioproduction platforms for recombinant proteins production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of chlamydomonas reinhardtii microalgal vaccines functional analysis of the dat gene promoter using transient catharanthus roseus and stable nicotiana tabacum transformation systems identification of dna sequences required for activity of the cauliflower mosaic virus 35s promoter expression of a soybean b-conclycinin gene under the control of the cauliflower mosaic virus 35s and 19s promoters in transformed petunia tissues molecular farming of pharmaceutical proteins tobacco as biofactory for biologically active hpl production: a human hormone with potential applications in type-1 diabetes plant-derived recombinant human serum transferrin demonstrates multiple functions effect of codon optimization and subcellular targeting on toxoplasma gondii antigen sag1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice immunogenicity of a plant-derived edible chimeric espa, intimin and tir of escherichia coli o157:h7 in mice expression of the cholera toxin b subunit (ct-b) in maize seeds and a combined mucosal treatment against cholera and traveler's diarrhea barley as a green factory for the production of functional flt3 ligand production of highly concentrated, heat-stable hepatitis b surface antigen in maize production of mouse granulocyte-macrophage colony-stimulating factor by gateway technology and transgenic rice cell culture chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: current status and perspectives optimization of codon composition and regulatory elements for expression of human insulin like growth factor-1 in transgenic chloroplasts and evaluation of structural identity and function synthesis and expression of recombinant interferon alpha-5 gene in tobacco chloroplasts, a non-edible plant production of biologically active human interleukin-4 in transgenic tobacco and potato expression of antibody fragments with a controlled n-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of arabidopsis recombinant mouse granulocyte-macrophage colony-stimulating factor is glycosylated in transgenic tobacco and maintains its biological activity considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants the development, characterization, and demonstration of a novel strategy for purification of recombinant proteins expressed in plants different subcellular localization and glycosylation for a functional antibody expressed in nicotiana tabacum plants and suspension cells a proficient approach to the production of therapeutic glucagon-like peptide-1 (glp-1) in transgenic plants transient expression of human papillomavirus type 16 l2 epitope fused to n-and c-terminus of coat protein of potato virus x in plants in silico analysis of chimeric espa, eae and tir fragments of escherichia coli o157:h7 for oral immunogenic applications coordinate expression of multiple proteins in plant cells by exploiting endogenous kex2p-like protease activity expression of human papillomavirus 16 e7ggg oncoprotein on n-and c-terminus of potato virus x coat protein in bacterial and plant cells linker: a program to generate linker sequences for fusion proteins dna cloning using in vitro site-specific recombination gateway recombinational cloning: application to the cloning of large numbers of open reading frames or orfeomes orfeome cloning and global analysis of protein localization in the fission yeast schizosaccharomyces pombe from orfeome to biology: a functional genomics pipeline constructing orfeome resources with removable termination codons a novel chloroplast transformation vector compatible with the gateway recombination cloning technology delivery of multiple transgenes to plant cells by an improved version of multiround gateway technology tmv-gate vectors: gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins recombinant human gad65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant non-food/feed seeds as biofactories for the high-yield production of recombinant pharmaceuticals gateway-compatible vectors for plant functional genomics and proteomics gateway technology: a universal technology to clone dna sequences for functional analysis and expression in multiple systems improvement of recombinant hgm-csf production by suppression of cysteine proteinase gene expression using rna interference in a transgenic rice culture coexpression of proteinase inhibitor enhances recombinant human granulocyte-macrophage colony stimulating factor production in transgenic rice cell suspension culture the development of a high-yield recombinant protein bioreactor through rnai induced knockdown of atp/adp transporter in solanum tuberosum comparative analysis of recombinant human papillomavirus 8 l1 production in plants by a variety of expression systems and purification methods transient co-expression of post-transcriptional gene silencing suppressors for increased in planta expression of a recombinant anthrax receptor fusion protein production of human growth hormone in transgenic rice seeds: co-introduction of rna interference cassette for suppressing the gene expression of endogenous storage proteins a protease activity-depleted environment for heterologous proteins migrating towards the leaf cell apoplast from planta to pharma with glycosylation in the toolbox pharming and transgenic plants down-regulated expression of plant-specific glycoepitopes in alfalfa plant-specific glycosylation patterns in the context of therapeutic protein production is the drought over for pharming? rice-based mucosal vaccine as a global strategy for coldchain-and needle-free vaccination analysis of a cholera toxin b subunit (ctb) and human mucin 1 (muc1) conjugate protein in a muc1-tolerant mouse model plant-derived vaccines against diarrheal diseases plant molecular farming: opportunities and challenges regulatory issues for plant-made pharmaceuticals and vaccines clinical trials fuel the promise of plant-derived vaccines immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis b and human immunodeficiency viruses human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes expression of rotavirus capsid protein vp6 in transgenic potato and its oral immunogenicity in mice accumulation of functional recombinant human coagulation factor ix in transgenic soybean seeds high-level expression of human interferon alpha-2b in transgenic carrot (daucus carota l.) plants expression of the major mugwort pollen allergen art v 1 in tobacco plants and cell cultures: problems and perspectives for allergen production in plants production of the 42-kda fragment of plasmodium falciparum merozoite surface protein 1, a leading malaria vaccine antigen, in arabidopsis thaliana seeds influence of elastin-like peptide fusions on the quantity and quality of a tobacco-derived human immunodeficiency virus-neutralizing antibody hydrophobin fusions for high-level transient protein expression and purification in nicotiana benthamiana bioseparation of recombinant proteins from plant extract with hydrophobin fusion technology tamavidin, a versatile affinity tag for protein purification and immobilization acknowledgments abdullah makhzoum thanks prof. mark bernards for his helpful to publish the manuscript. no sources of funding were used to assist in the preparation of this review. the authors have no conflicts of interest that are directly relevant to the content of this review. key: cord-350935-p6euuop3 authors: doğan, tunca; atas, heval; joshi, vishal; atakan, ahmet; rifaioglu, ahmet sureyya; nalbat, esra; nightingale, andrew; saidi, rabie; volynkin, vladimir; zellner, hermann; cetin-atalay, rengul; martin, maria; atalay, volkan title: crossbar: comprehensive resource of biomedical relations with deep learning applications and knowledge graph representations date: 2020-09-15 journal: biorxiv doi: 10.1101/2020.09.14.296889 sha: doc_id: 350935 cord_uid: p6euuop3 systemic analysis of available large-scale biological and biomedical data is critical for developing novel and effective treatment approaches against both complex and infectious diseases. owing to the fact that different sections of the biomedical data is produced by different organizations/institutions using various types of technologies, the data are scattered across individual computational resources, without any explicit relations/connections to each other, which greatly hinders the comprehensive multi-omics-based analysis of data. we aimed to address this issue by constructing a new biological and biomedical data resource, crossbar, a comprehensive system that integrates large-scale biomedical data from various resources and store them in a new nosql database, enrich these data with deep-learning-based prediction of relations between numerous biomedical entities, rigorously analyse the enriched data to obtain biologically meaningful modules and display them to users via easy-to-interpret, interactive and heterogenous knowledge graph (kg) representations within an open access, user-friendly and online web-service at https://crossbar.kansil.org. as a use-case study, we constructed crossbar covid-19 kgs (available at: https://crossbar.kansil.org/covid_main.php) that incorporate relevant virus and host genes/proteins, interactions, pathways, phenotypes and other diseases, as well as known and completely new predicted drugs/compounds. our covid-19 graphs can be utilized for a systems-level evaluation of relevant virus-host protein interactions, mechanisms, phenotypic implications and potential interventions. systemic analysis of available large-scale biological and biomedical data is critical for developing novel and effective treatment approaches. parts of this big-data are continuously being updated and maintained by different organizations and institutions, thus, data is scattered across individual computational resources. although these entities are biologically related and complementary to each other, the connections between datapoints at different resources are not well-established and explicit at all. in addition to the connectivity problem, another issue related to biomedical data is the incompleteness in knowledge space (e.g., possible ligands of a target biomolecule, or the disease related implications of a critical mutation). there is a clear requirement for innovative computational approaches to integrate available biomedical big-data and to complete missing information with in silico predictions, to serve the ultimate aim of proposing novel treatment options (especially) for complex diseases. there are numerous studies in the literature that aimed to integrate the available biomedical data [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] . these studies provided useful tools and methods to the life-sciences research community; however, many of them miss important functionalities that prevent them from becoming widely adopted tools/services (supplementary information section 1). in this project, we aimed to address the current shortcomings by developing a comprehensive open access biomedical system entitled crossbar via integrating various biological databases to each other, inferring the missing relations between existing data points, and constructing informative knowledge graphs based on specific biomedical components/terms such as a disease/phenotype, biological process, gene/protein and drug/compound, or specific combinations of them. to construct the crossbar system, we accomplished multiple sub-projects: (i) the construction of the crossbar database to house, and its api service to serve the integrated biomedical data, (ii) development of deeplearning based drug/compound-target protein interaction (dti) prediction models, crossbar database (crossbar-db) comprises carefully selected features from various data sources namely uniprot, intact, interpro, reactome, ensembl, drugbank, chembl, 3 pubchem, kegg, omim, orphanet, gene ontology, experimental factor ontology (efo) and human phenotype ontology (hpo). extract-transform-load (etl) pipelines were developed for heavy lifting of data from these resources by persisting specific data attributes with the implementation of logic rules. these pipelines fetch, cleanse, validate and consolidate the data, and thus, implement a multi-omics data integration approach to release a single resource based on mongodb collections. crossbar-db, which provides a broad spectrum of information such as biomolecular functions, domains, interactions, pathways, diseases, phenotypes, drugs, compounds, etc., is hosted and maintained by the embl-ebi. current data statistics of the crossbar-db and the database schema are shown in fig. 1b and in fig. s1 , respectively. crossbar-db is periodically updated on demand/request basis via an automated procedure, which makes the underlying data up to date most of the time. crossbar-db can be queried via a public restful api at: www.ebi.ac.uk/tools/crossbar/swagger-ui.html, which provides a multi-faceted view of the stored data through 12 endpoints (fig. s3) . as a part of the crossbar project, we developed two novel deep-learning-based predictive systems: deepscreen and mdeepred, with the aim of enriching bioactivity data by identifying unknown interactions between drugs/drug-candidate compounds and target proteins. deepscreen employs convolutional neural networks to process 2d structural images of drugs/compounds in 704 individually optimized high performance target-based prediction models, suited for well-studied targets 11 . mdeepred utilizes both compound and target protein features within a pairwise input hybrid deep neural network architecture to produce real valued bioactivity predictions, especially for targets with a few or no training instances 12 . we trained both systems using carefully filtered and integrated data in crossbar-db, and ran our trained-models on large compound and human protein spaces to obtain comprehensive bio-interaction predictions, which are included in our knowledge graphs. we also developed an accompanying computational tool, ibioprovis, which is an unsupervised-learning-based visualization system for exploring large drug/compound-target interaction datasets in reduced dimensions 13 . the term knowledge graph (kg) defines a specialized data representation structure, in which a collection of entities (nodes) are linked to each other (edges) in a semantic context 14 . in this study, we chose to represent heterogeneous biomedical data in kg structures. in crossbar knowledge graphs (crossbar-kg), biological components/terms (i.e., drugs/compounds, genes/proteins, bio-processes/pathways, phenotypes/diseases) are represented as nodes, and their known or predicted pairwise relationships are annotated as edges (a protein and its coding gene is treated as one merged term/entry/node). the logic behind the construction of a knowledge graph is centered around queried biological 4 components/terms, as shown in fig. 1c with a work-flow diagram and with an example disease term query. at each step of the process, an overrepresentation-based enrichment analysis has been performed to select the terms that are significantly associated with the growing graph, and to discard the rest. this analysis comprises a series of hypergeometric tests, based on the recorded relations in the crossbar database. here, we applied a layered construction approach, always taking the genes/proteins at the centre of the enrichment analysis. finally, additional relation types are incorporated to the graph as edges between the existing nodes (e.g., drug-disease, disease-pathway and disease-hpo), to further enrich the provided information. during the construction of graphs, terms (nodes) and their pairwise relations (edges) are directly obtained from the crossbar-db. crossbar-kgs clearly display the direct and indirect relations between all of the terms in the graph. these intensely-processed heterogeneous biological networks are expected to aid biomedical research, especially to infer mechanisms of diseases in relation to biomolecules, systems and candidate drugs. we developed the crossbar web-service (crossbar-ws) to make the crossbar-kgs available to the public in an easily interpretable and interactive way (https://crossbar.kansil.org). kgs are presented visually on web-browsers as flexible cytoscape 15 networks. users can create queries with biomedical terms, individually or in combination, to obtain the relevant graph. combinatory term query is especially critical as it provides the ability to investigate the indirect biological relationships between the terms from both the same and different biomedical components. since there are billions of different ways to query crossbar, it was not feasible to pre-calculate the resulting graphs; therefore, they are set to be constructed on-the-fly, in real-time. several options are provided to users to customize the procedure both before the search, such as the uniprot databases to be used (uniprotkb/swiss-prot or uniprotkb/swiss-prot+uniprotkb/trembl), taxons to be included, and the number of terms/nodes to include from each entity type (selected from enrichment score-based ranked lists). it is also possible to display the graph using a variety of layout options, including our in-house crossbar-layout (fig. 2a) . saving options let users to store the graph in different formats, including json, figure-ready snapshots and protein-centric delimited data-tables. the interactive visualization also lets users prepare a custom display by relocating the nodes/edges as desired. as a use-case, we present coronavirus disease 2019 (covid-19) crossbar-kgs (https://crossbar.kansil.org/covid_main.php). starting from the end of 2019, the new coronavirus (sars-cov-2) pandemic has ravaged the entire globe and caused immeasurable damage 16 . as of july 2020, the scientific endeavour to develop effective drugs and vaccines is at peak, and systemic evaluation of the current knowledge about sars-cov-2 infection is 5 expected aid researchers in this struggle . to demonstrate the capabilities of crossbar, we have constructed two different versions of the covid-19 knowledge graph, (i) a large-scale version including nearly the entirety of the related information on different crossbarintegrated data sources, which is ideal for further network and machine learning based analysis or a detailed inspection (fig. 2b) , and (ii) a simplified version distilled to include only the most relevant genes/proteins as provided in uniprot-covid-19 portal (https://covid-19.uniprot.org), which is ideal for fast interpretation (fig. 2c) . it is interesting to observe the indirect relations between the diseases/phenotypes in the kgs and covid-19 over the incorporated host proteins and enriched pathways, and between covid-19 and our in silico predicted drugs, as they may reveal further evidence to be utilized against covid-19 (fig. 2b ,c). for this, we conducted a short literature-based validation study and found that many of these drugs have already been experimented at both preclinical and clinical stages for new covid-19 treatments (supplementary information section 2). although covid-19 is a respiratory disease and lung lesions have been considered the major damage caused by sars-cov-2, liver injury has also been reported in about one-third of hospitalized patients infected with the virus and the majority of covid-19 patient deaths are associated with cytokine storm/release syndrome resulting in multi organ damage 17 . hence, with the aim of indicating the biological relevance of the information in crossbar-kgs, we conducted in vitro experimentation on drug treated liver cancer cell-lines and comparatively analysed the results on both covid-19 kgs. chloroquine (cq) phosphate was reported to be used in treatment of covid-19 with controversies 18 . cq is an antiinflammatory drug that has been used in autoimmune diseases and can significantly alter the production of pro-inflammatory and anti-inflammatory cytokines. we investigated the effect cq on normal hepatocytes like huh7 cells and poorly differentiated mahlavu cells. cells were treated with cq and the differentially expressed gene (deg) data were acquired from a large multiplex panel of genes using the nanostring platform (fig. 2d ). our experimental data indicated significant alterations in jak/stat, pi3k, ras, mapk pathways involving cytokine production in liver cells (full pathway list: table s .3). these pathways were also presented in both kgs along with additional cytokine related pathways, such as interleukin signaling, along with dense connections to other biological components in covid-19 crossbar-kgs, which is an expected output considering the mode of action of cq in covid-19 (fig. 2c) . additional use-cases are provided in the supplementary information section 3. we believe that the crossbar system can be utilized towards the systematic analysis of the pharmacological effects of drugs as it brings relevant pieces of biological data together, that is relevant to the user's query, which can be manually explored by the expert to build new hypotheses. 6 integrated data resources and the crossbar-db crossbar-db has developed its bespoke etl pipelines in java 8 using the spring batch framework to structure the jobs. the latter are executed on state-of-the-art embl-ebi lsf clusters powered by ibm in a parallel distributed fashion to reduce the processing time. the data are finally stored in mongodb in the form of independent data collections, thus, providing schemaless flexibility and faster development, while sustaining data relationships in the form of nested documents. the pipelines have been both unit and integration tested using spock framework in groovy language. the public databases integrated in the crossbar system can be listed along with the type of the biomedical data it contains as follows; the statistics regarding the number of terms and annotations incorporated to crossbar-db from each resource listed above is given in fig. 1b supplementary information fig. s3 , where the 12 crossbar-db collections are shown. here, the collections entitled "activities", "assays", "molecules" and "targets" correspond to bioactivity, bioassay, compound and target entries in the chembl database, respectively. "drugs" corresponds to drug entries in drugbank, "efo disease terms" corresponds to the disease entries in the experimental factor ontology, "hpo" corresponds to phenotype entries in the human phenotype ontology, "proteins" correspond to a subset of the protein entries in the uniprotkb. the remaining four collections belong to the pubchem data. there is no one-to-one correspondence between the incorporated data resources and the crossbar-db collections since some of the resources had to be split to multiple collections for easier query (e.g., chembl and pubchem). also, some of the sources are directly incorporated from the uniprot database, thus, reside in the proteins collection (e.g., both terms and annotations for interpro, reactome, and only annotations for intact, omim and orphanet). it is possible to obtain cross-collection relational data (i.e., integrated relational data from multiple collections) by writing programmatic queries and submitting them to the api, as it is applied in crossbar-ws to construct the knowledge graphs. however, it is not possible to obtain this complex relational data in a single query using the swagger graphical user interface. currently, crossbar knowledge graphs do not include pubchem data due to both elevated computational demand (the sizes of pubchem collections are large) and high redundancy (a large portion of bioactivity data points in pubchem and chembl databases are shared). however, it is possible to query the crossbar-db using the provided api 8 service, to obtain data entries from pubchem database collections. the database and api construction work has been handled by the protein function development (uniprot database) team at embl-ebi, utilizing their expertise in biological database development and maintenance together with the available strong computational infrastructure, the team managed to build a huge but stable resource. the professional service providing approach applied by the team allowed the proper and constant maintenance of both the database and the entire crossbar system. the the current knowledge corresponds to less than 0.001% of the whole compound-target space 19 . the high rate of missing dti data negatively impacts the integrated biomedical resources as well. in the crossbar project, we aimed to address this issue by producing machine learning based dti predictions and incorporating these predictions to the crossbar resource. the studies specifically about the development of these tools have already been published or under review 11, 12 , however, we used our tools to produce dti predictions to be incorporated in our knowledge graphs in the framework of this study. one critical topic in developing dti prediction models is the source dataset to be used in system training procedures. it is especially critical to construct large-scale dti datasets to train deep-learning models. to address this issue, we prepared a dti dataset from the chembl database that is suitable for training machine learning systems, with standardized filtering operations on targets, compounds and bioactivities. the dataset is periodically updated with each chembl database release. we employed this dataset for the training and validation of the deep-learning based dti prediction models we developed in the framework of the crossbar project, and also as the source dataset for drug/compound-target 9 interaction space visualization (the methods are described below). it can also be used for developing new dti prediction models. the current version of the bioactivity dataset (chembl v27) is available for public use in: https://github.com/cansyl/crossbar/blob/master/crossbar_db_api/chembl27_preproc essed_activities_sp_b_pchembl.zip. details regarding the dataset can be found in our recent article 11 . deep learning base predictor 1 -deepscreen deepscreen was the first dti prediction system that we developed in this endeavour. deepscreen is a high-performance drug-target interaction predictor that utilizes deep convolutional neural networks and 2-d structural compound representations (i.e., simple images) to predict their activity against intended target proteins. deepscreen system is composed of 704 target protein specific prediction models, each independently trained using experimental bioactivity measurements against many drug candidate small molecules, and optimized according to the binding properties of the target proteins. the main novelty of deepscreen is employing readily available 2-d structural representations of compounds at the input level instead of conventional drug/compounds descriptors (e.g., molecular fingerprints) that display limited performance. deepscreen produces binary predictions, meaning that a compound is either predicted as active or inactive against a target protein. during the development of this method, we also carried out cell-based in vitro wet-lab experiments on computationally generated dti predictions, with the purposes of both validating the accuracy of the prediction models, and for gaining biological insight in the framework of health and disease, especially to contribute to the understanding of processes active in different cancer subtypes. deepscreen can be used for the fast screening of the chemogenomic space, to provide completely new dtis that can later be investigated experimentally in the fields of drug discovery and repurposing 11 in crossbar, the data is stored in a non-relational database (mongodb), as separate collections for easy maintenance and fast querying. as a result, the database itself is not a knowledge graph. instead, biologically relevant small-scale knowledge graphs are constructed on-the-fly, triggered by users' queries with a single or multiple term(s) such as the names or ids of genes/proteins, diseases/phenotypes, compounds/drugs and/or pathways/biological processes of interest. in crossbar knowledge graphs, biological entities are represented as vertices/nodes. distinct types of nodes are defined for: (i) biomolecules (i.e., genes/proteins), (ii) biological mechanisms (i.e., processes/pathways), (iii) pathologies (i.e., diseases and phenotypes), and (iv) small molecule ligands used for treatment (i.e., drugs and drug candidate compounds). relations between different types of biological entities are expressed by the edges of the graph. edge types vary according to the defined relations. for a relation between; (i) two proteins, the edge is labelled as "interacts_with", (ii) for a gene/protein and a disease, the edge label is "related_to", (iii) for a drug/compound and a protein, the edge label is "targets", (iv) for a gene/protein and a pathway, the edge label is "involved_in", (v) for a gene/protein and a phenotype term, the edge label is "associated_with", (vi) for a drug and a disease, the edge label is "indicates", (vii) for a disease and a pathway, the edge label is "modulates", and (viii) for a disease and a phenotype term, the edge label is "associated_with". the incorporation of pathway-related information (both signalling and metabolic pathways) in crossbar is done based on a membership-based approach, where pathways are expressed on the graph as single nodes and the nodes of those member proteins are connected to them via edges. this approach leaves out the detailed reaction-based mechanistic information provided in pathway databases such as reactome and kegg pathways; however, the inclusion of this information via applying a pathway resource styled network approach would prevent the generation of large heterogeneous networks composed of tens of different pathways and other components. nevertheless, it is possible to explore these pathways in detail using the provided links, which takes the user to the corresponding page on that pathway database. both reactome and kegg pathways provide the same type of biological information at the level of large-scale biological processes; however, reactome also divides these processes into sub-pathways, whereas kegg only provides the pathway information at a generic level. in crossbar, due to the way the overrepresentation analysis is done, only specific sub-pathways are incorporated from the reactome database, in most cases. as a result, pathway information in the knowledge graphs is displayed at different levels of specificity, and thus, not redundant. a simplified form of the knowledge graph construction work-flow is displayed in fig. 1c . in would not be feasible. to address this problem, we applied a multi-staged overrepresentation-based enrichment analysis process during the construction of graphs. in this analysis, we calculate an independent enrichment score for each biological entity in the database (i.e., a disease, phenotype, drug, compound, gene/protein or pathway), to be considered as its relevance to the graph that is being constructed. the calculation of enrichment score and its statistical significance is done using a modified version of the hypergeometric test for overrepresentation 20 , which also corresponds to a one-tailed fisher's exact test, and it is based on the statistics of the relations/connections with gene/protein nodes. for example, the enrichment score (ed,w) and its significance (sd,w), in terms of p-value, for a disease term d, for graph w is calculated as follows: (1) (2) where ed,w is the enrichment score calculated for the disease term d for graph w; md 2 represent the square of the number of genes/proteins in graph w that are associated with disease d; nw represents the total number of gene/protein nodes in graph w; md is the total number of genes/proteins (not necessarily in graph w) that is associated with disease d; and n represents the total number of reviewed human gene/protein entries (i.e., uniprotkb/swiss-prot entries) in the crossbar database that is annotated with any disease entry. sd,w represents the significance (p-value) for the disease term d for graph w calculated in the hypergeometric test. while constructing the graph, an enrichment score is calculated for each disease entry in the crossbar database and these scores are used to rank disease entries according to their biological relevance to graph w. a cut-off value k is employed to include the top k relevant disease entries to graph w. the default value for k is 10, which means that only top-10 relevant diseases will be included in the graph. apart from diseases, the same methodology is used to filter out the nodes of neighbouring genes/proteins, phenotypes, drugs, compounds and pathways. significance values are not directly used in the filtering operation, since the main objective here is not only including significantly over-represented terms, but just reducing the number of nodes in the graph by filtering out the ones that are least relevant. in the traditional way of calculating an enrichment score, md is without square. the reason behind taking the square of md here is to break the tie between the scores of terms in favour of the one with a higher md value. formalizing the graph construction around gene/protein entries during the construction of a knowledge graph, first, the gene/protein entries that are directly connected to the query term (i.e., core proteins) are fetched, such as the member genes/proteins of the queried signalling pathway. after that, neighbouring/interacting genes/proteins are added to the graph by calculating enrichment scores for each interacting protein, using the equation above, and filtering out based on the selected cut-off value. this is followed by the enrichment-based filtering and addition of other entity types; however, this time, both core and neighbouring genes/proteins are taken into consideration together to calculate the enrichment scores. if the user starts a heterogenous search that contains multiple terms from different entity types, both core and neighbouring genes/proteins are independently collected for each non-protein query term, queried gene/protein entries are added to this list (if there is any), and the entity collection process is continued using the union of these genes/proteins (fig. 1c) . this approach enables the exploration of direct and indirect relations between the queried terms. bioactive compound and bioactivity selection procedure small molecule compounds are selected and incorporated to kgs based on their reported bioactivities against target proteins. in a kg, a compound is represented as a node and a bioactivity is represented as an edge between a compound node and a gene/protein node. we start the bioactive compound collection procedure with a set of target gene/protein entries at hand (gathered in a previous step of the kg construction process), and obtain the compounds that are reported to be bio-actively interacting with these proteins, as their target biomolecules. despite having a simple logic, this procedure is extremely complex due to practical reasons. since there are more than 15 million bioactivity measurements in the chembl database (v26), we rigorously filter these data points with the aim of providing only the most relevant bioactivity/compound information in crossbar-kgs. since crossbar is a gene/protein centric system, we first filter out the data points where the target is not a single protein. we also set an organism filter for the targets, where the default selection is human. additionally, we filter out bioactivities if their standard (activity) type is not one of these: ic50, ec50, ac50, xc50, ki, kd, potency; since these standard types provide roughly comparable measures of half-maximal response. furthermore, we eliminated data points 15 without a pchembl value, which standardizes the above-mentioned standard types under one value in the negative logarithmic scale. bioactivity data points with an assigned pchembl value have usually received additional curation, and thus, they are more reliable. finally, with the aim of only taking the data points at the active binding range (i.e., high affinities between the ligand and the target) we discard the data points with a pchembl value less than 5 (i.e., xc50 > 10 µm). despite these filtering operations, we still usually end up with tens of thousands of compounds before the compound enrichment analysis, which significantly increases the kg construction run time. exploiting the fact that it is a better choice to include a compound with higher binding affinity compared to a compound with a lower binding affinity for the same target protein, we set the pchembl value cut off value to 8, at the beginning of the compound collection procedure. then, we reduce the cut off value and re-run the query if the total number of gathered compound entries is less than 1000 in the first run. we iteratively do this procedure until we obtain at least 1000 compound entries. similarly, if the number of returned compounds is more than 2500 in the first run, we further increase the cut off iteratively until we obtain less than 2500 compound entries. this number (i.e., 1000 to 2500) is still much higher than the number of compounds we incorporate to a kg, which is between 0 and 50; however, we aim to enter the enrichment analysis with a high amount to be able to select the compounds that are interacting with multiple proteins in the network, not just one. another reason is to be able to select diverse compounds, in terms of their scaffolds/structures, which is explained below (under the compound clustering sub-section). there are more than one million compound entries in the chembl database, most of which have bioactivity data points against target biomolecules. since it is not feasible to include each and every bioactive compound node in a kg (otherwise the graph would be extremely crowded), only the most overrepresented compounds are tried to be incorporated. we observed that some of the compounds with the same (or a very similar) enrichment score are also structurally very similar to each other. these are mostly molecules with matching scaffolds, which are screened against the same target and produced similar results in the same bioassay. since their enrichment scores are similar as well, they are either selected or discarded together. to provide a better selection of compounds in the graph, we incorporated a structural property-based filtering in the enrichment analysis. the aim here is to select overrepresented compounds that are as diverse from each other as possible in terms of molecular structures, so that users will be provided with a variety of ligands for the target proteins in the graph. to achieve this, we calculated the pairwise molecular similarities between all compounds in crossbar-db using circular fingerprints (ecfp4) and the tanimoto coefficient. after that, we clustered the compounds based on a predefined similarity cut-off value of 0.5, meaning that each cluster is composed of compounds that are at least 50% similar to each other. the cluster information is pre-calculated and recorded on our server. each time a knowledge graph is being constructed, enrichment score ranked compounds are checked one by one in terms of their cluster membership and if there already is a compound from the same cluster in the graph, the compound in turn is discarded (i.e., not incorporated to the graph). the same clustering-based selection approach is applied to incorporate compounds that are computationally predicted to interact with the proteins in the graph. following the finalization of the compound nodes, we check whether some of these compounds correspond to drugs that are already incorporated to the kg (since chembl also contains bioactivity measurements belonging to approved or investigational drugs), using the identifier mapping between chembl and drugbank databases. when a positive case is detected, we merge these two nodes and set the node type as a drug, since drugs are as a result, predicted relations comprise the least reliable part of the dti information provided in crossbar-kgs. with the aim of transmitting this evidence-based relation confidence information to users, we used edge labels in the kgs. in terms of visualization, these labels are encoded on the graphs as colours, such that green colour corresponds to dtis obtained from approved or investigational drugs, blue colour corresponds to experimental bioactivity measures obtained from chembl, and the red colour corresponds to computationally predicted interactions. during the generation of kgs, if a specific relation is obtained from multiple sources (e.g., when the same relation is reported both in drugbank and in chembl) the edge label of the more reliable relation is incorporated. to accomplish this task, kg construction process comprises an edge label update procedure. another process we applied at this step is the edge addition. some drugs possess bioactivity data points in chembl in addition to their approved targets in drugbank. to detect this, we first do a mapping between chembl compound entries and drugbank drug entries, to find the equivalent chembl entry for each drug. after that, we identify the reported chembl bioactivities between that compound and all of the proteins presented in the kg. for those relations, which were not already incorporated into the kg via drugbank, we added blue coloured edges. the same procedure is applied for adding red coloured edges to the drugs and compounds that have extra computationally predicted target interactions. a user query initiates the entity (node) gathering procedure first from the related database collections, using the crossbar restful api. together with the entities that match the search term, the information regarding the related/connected entities are obtained from the corresponding collection. after that, the next database collection is queried with the terms gathered at the previous step. the order of the api queries follows the logic defined for the construction of the kgs, as given in fig. 1c (simplified version) , and in fig. s2 . (full version). following the initiation of a query, the growing knowledge graph is displayed on the web-browser in real time (using cytoscape web), starting from the collection and filtering of core and neighbouring genes/proteins. the process is continued with the collection, filtering and addition of phenotype/pathway/disease terms, drugs, bioactive compounds and predicted interacting compounds to the kg (as nodes), together with their relations with gene/protein nodes (as edges). the construction process is finished with the addition of respective edges between non-protein nodes. an important subject in graph/network visualization is the layout. in crossbar-ws, we incorporated the standard layouts of cytoscape web, such as circle, cose, grid and concentric. however, none of these layouts were sufficient for communicating highly heterogeneous graphs with 7 different types of nodes and 9 different types of edges. to address this problem, we developed the crossbar layout, in which biological terms (nodes) from a specific biomedical component (e.g., diseases, pathways, ...) are placed on circular points within a fixed radius. with the aim of preventing overlapping nodes, the radius of each circle is selected as a different value. curved edge style (i.e., unbundled-bezier) is applied to reduce the amount of edge crossing. more information regarding the usage of crossbar-ws and its user interface can be found at https://crossbar.kansil.org/tutorial.php. crossbar web-service queries run in linear time and the actual duration of the process is correlated with the total number of core genes/proteins obtained within the query, together with the annotation volume of these genes/proteins. highly studied genes/proteins usually have high number of associations, which in turn, extends the actual query runtime in practice. according to our tests, most of the queries with disease, gene/protein, drug and compound terms (in terms of both single and combinatory term searches) take between 1 and 3 minutes to complete (from job submission to the display of the whole graph). however, most pathway and some phenotypic term queries take longer, especially when the number of directly associated genes/proteins is over one hundred. with the aim of creating a better user experience, we applied a procedure in which the collected nodes and their edges are instantaneously and interactively displayed on the screen, before the end of the job. this way, users do not have to wait for the whole job to be finished before starting to explore the kgs. the entire crossbar web-service including the web-site and the underlying api queries can be found in the github repository of the project (https://github.com/cansyl/crossbar). we constructed two versions of covid-19 knowledge graph. first, the large-scale version that includes nearly the whole of the covid-19 related information recently accumulated in 19 the scientific literature, organized and presented in an interpretable way. second, the simplified version, which is suitable for quick exploration. the aim behind constructing the simplified version was that the large-scale kg is not easily explorable visually due to its huge size. since most of the covid-19 related data has still not been integrated into the regular releases of biological databases, the data could not be pulled to the crossbar database at the time of writing this manuscript. as a result, we had to make manual interventions to obtain the data from crossbar data resources. we applied the same knowledge graph construction methodology incorporated in crossbar; however, we also conducted manual curation to a certain extent, since, unlike the rest of the crossbar data, covid-19 related information has not been extensively curated yet. we saved the pre-constructed kgs, which are directly accessible and viewable through the links given on our web-service (https://crossbar.kansil.org/covid_main.php). it is also important to note that, due to the integrated data resources, crossbar heavily contains rare and complex disease data, and mostly leaves infectious diseases out. nevertheless, the constructed covid-19 graphs provided rich biomedical information. below, we describe the methodology followed for the generation of crossbar covid-19 kgs. construction of the large-scale covid-19 graph started with acquiring the related efo disease term named: "covid-19" (id: mondo:0100096). we also incorporated the disease term for "severe acute respiratory syndrome" (id: efo:0000694) (the original sars) into the graph since sars is better annotated compared to covid-19. the full-scale covid-19 kg construction is continued as follows: we obtained related genes/proteins and their interactions from the intact database's covid-19 dataset. unlike a genetic disease, human genes/proteins represent only a portion of an infectious disease. due to this, we aimed to incorporate sars-cov and sars-cov-2 genes/proteins, as well as, the host genes/proteins into the graph. without any filtering, the kg contained 1,172 gene/protein and metabolite nodes from various organisms and 2,214 edges. due to the high number of genes/proteins in the kg, there was a risk of incorporating non-specific/irrelevant terms from the other biological components at later steps. to address this risk, we applied several filtering operations on this data. first, we eliminated all non-gene/protein nodes and we discarded the genes/proteins if the corresponding organism is not human or sars-cov/sars-cov-2. second, we eliminated the protein entries that are not reviewed (i.e., not from uniprotkb/swiss-prot) except sars-cov-2 orf10 (accession: a0a663dja2), which currently is an unreviewed protein entry in uniprotkb/trembl. we also filtered out a portion of the host genes/proteins using 20 interaction-based information, according to their confidence scores reported in intact. we discarded the edges between host proteins and sars-cov and/or sars-cov-2 proteins if the confidence score was less than 0.35. we also discarded the edges between host proteins in the kg (i.e., neighbouring proteins) if their interaction confidence score is less than 0.6. we removed the disconnected components made up of host proteins, which were formed due to the edge filtering operation. orthology relations between sars-cov and sars-cov-2 genes/proteins were annotated with "is ortholog of" edge type. the subunits of large protein complexes such as the nsps of replicase polyprotein 1ab of sars-cov/sars-cov-2 were mapped to their corresponding protein complex nodes with "is subunit of" edge label. after these operations, the finalized number of genes/proteins/subunits is 539 ( pathways of covid-19 related host genes/proteins signaling and metabolic pathway information was taken from reactome (via crossbar database) and kegg pathways data sources. the most relevant pathways were determined by the overrepresentation analysis and mapped to the related genes/proteins in the kg. some of the incorporated pathways are directly related to sars-cov-2 infection such as "viral mrna translation" (r-hsa-192823) or "isg15 antiviral mechanism" (r-hsa-1169408) and innate pathways of the host such as "endocytosis" (hsa04144), "cell cycle" (hsa04110) or "nf-kappa b signaling pathway" (hsa04064). we also incorporated pathway-disease the resource for the phenotype terms is the human phenotype ontology (hpo) database. for each phenotype term that is associated with at least one gene in the kg according to hpo data, we calculated an enrichment score and p-value via overrepresentation analysis. from the score-ranked hpo term list we selected phenotype terms that are not in a close parent-child relationship with each other in the hpo direct acyclic graph. hpo also has a curated list of sars related phenotype terms. these terms were also added into the network and mapped to "covid-19" and "severe acute respiratory syndrome" disease the authors declare no competing interests. supplementary information is available for this paper. genes/proteins that are associated with the query disease (i.e., core genes/proteins), (3) collects additional genes/proteins (i.e., first-neighbours) using ppis of core genes/proteins, (4) identifies biological processes (pathways), of which these genes/proteins (core+neighbouring) are members, (5) gathers phenotypic terms (hpo) associated with the whole gene/protein set, (6) obtains known drugs and drug candidate compounds targeting these genes/proteins, together with our deeplearning based interaction predictions, and (7) revisits the disease collection to make another query with all collected genes/proteins, to obtain the disease entries that have similar implications as the query disease. bioportal: enhanced functionality via new web services from the national center for biomedical ontology to access and use ontologies in software applications the ontology lookup service: bigger and better biograph: unsupervised biomedical knowledge discovery via automated hypothesis generation systematic integration of biomedical knowledge prioritizes drugs for repurposing biograph: a web application and a graph database for querying and analyzing bioinformatics resources constructing biomedical knowledge graph based on semmeddb and linked open data expanding a database-derived biomedical knowledge graph via multi-relation extraction from biomedical abstracts knowlife: a versatile approach for constructing a large knowledge graph for biomedical sciences kabob: ontology-based semantic integration of biomedical databases science forum: wikidata as a knowledge graph for the life sciences high performance drug-target interaction prediction with convolutional neural networks using 2-d structural compound representations mdeepred: novel multi-channel protein featurization for deep learning based binding affinity prediction in drug discovery interactive visualization and analysis of compound bioactivity space knowledge graph embedding by translating on hyperplanes cytoscape: a software environment for integrated models of biomolecular interaction networks a new coronavirus associated with human respiratory disease in china liver diseases in covid-19: etiology, treatment and prognosis controversial treatments: an updated understanding of the coronavirus disease recent applications of deep learning and machine intelligence on in silico drug discovery: methods, tools and databases enrichment or depletion of a go category within a class of genes: which test? a sars-cov-2 protein interaction map reveals targets for drug repurposing crossbar web-service (including a tutorial on how to use the service) is available at dr aybar can acar (faculty member, metu, turkey) and dr tugba suzek (faculty member, mugla university, turkey) for helpful discussions, comments and support. we also thank dr ian dunham (director, open targets, uk) and dr andrew leach ): (b) the large-scale kg (987 nodes and 3639 edges) and (c) the simplified kg (178 nodes and 298 edges). both of these graphs reveal the most overrepresented biological processes during a sars-cov-2 infection (i.e., cell cycle, viral mrna translation, endocytosis, interleukin signaling, etc.), as well as, the potential treatment options with covid-19 related pre-clinical/clinical results (e.g., chloroquine, remdesivir, favipiravir, dexamethasone, etc.) and our novel in silico predictions we checked the interaction between the significant degs (table s.2) and genes in the large-scale covid-19 kg, and applied fisher's exact test to analyse the significance of the presence of degs on the kg (table s.4) as opposed to the non-degs in the multiplex panel of the gene expression analysis platform (nanostring) key: cord-338980-pygykil7 authors: rahaman, jordon; siltberg-liberles, jessica title: avoiding regions symptomatic of conformational and functional flexibility to identify antiviral targets in current and future coronaviruses date: 2016-11-09 journal: genome biol evol doi: 10.1093/gbe/evw246 sha: doc_id: 338980 cord_uid: pygykil7 within the last 15 years, two related coronaviruses (severe acute respiratory syndrome [sars]-cov and middle east respiratory syndrome [mers]-cov) expanded their host range to include humans, with increased virulence in their new host. coronaviruses were recently found to have little intrinsic disorder compared with many other virus families. because intrinsically disordered regions have been proposed to be important for rewiring interactions between virus and host, we investigated the conservation of intrinsic disorder and secondary structure in coronaviruses in an evolutionary context. we found that regions of intrinsic disorder are rarely conserved among different coronavirus protein families, with the primary exception of the nucleocapsid. also, secondary structure predictions are only conserved across 50–80% of sites for most protein families, with the implication that 20–50% of sites do not have conserved secondary structure prediction. furthermore, nonconserved structure sites are significantly less constrained in sequence divergence than either sites conserved in the secondary structure or sites conserved in loop. avoiding regions symptomatic of conformational flexibility such as disordered sites and sites with nonconserved secondary structure to identify potential broad-specificity antiviral targets, only one sequence motif (five residues or longer) remains from the >10,000 starting sites across all coronaviruses in this study. the identified sequence motif is found within the nonstructural protein (nsp) 12 and constitutes an antiviral target potentially effective against the present day and future coronaviruses. on shorter evolutionary timescales, the sars and mers clades have more sequence motifs fulfilling the criteria applied. interestingly, many motifs map to nsp12 making this a prime target for coronavirus antivirals. severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov are two closely related zoonotic coronaviruses. both have successfully crossed the species barrier to allow animal-to-human transmission, and further to allow human-to-human transmission (song et al. 2005; reusken et al. 2016) . the sars outbreak in 2003 had a mortality rate of 10% (anderson et al. 2010) , and sars-cov was considered the most aggressive coronavirus compared to other human coronaviruses that commonly cause mild to moderate infection in their hosts (van der hoek 2007) . mers-cov is the cause of an ongoing outbreak of the respiratory illness mers (de groot et al. 2013) . at the time of writing, 1791 mers cases have been confirmed with a mortality rate of approximately 35% (world health organization 2016). both mers and sars have higher mortality rates in elderly and immunosuppressed populations (gralinski and baric 2015) . the host changes by mers-cov and sars-cov suggest that other coronaviruses can potentially cross the species barrier, become zoonotic, and enable human-to-human transmission, ultimately causing high morbidity and mortality. sars-cov and mers-cov exploited mechanistically different approaches to overcome the human species barrier, but these two viruses have a lot in common (lu et al. 2015) . here, we aim to identify the vulnerable regions in the proteomes of coronaviruses that neither sars-cov nor mers-cov nor their contemporary and forthcoming relatives can proliferate without, and address how to mobilize a defense against the present and future coronaviruses by targeting these regions. sars-cov and mers-cov are positive (+)-strand rna viruses encoding approximately 25 protein products. the mers-cov proteome is primarily composed of two polyproteins, orf1a and orf1ab; the latter is generated by a -1 ribosomal slippage frameshift. these proteins are cleaved into 16 nonstructural proteins (nsps). nsps 1-10 are products of both polyproteins, whereas nsps 12-16 are only yielded by orf1ab. nsp11 is unique to orf1a ( van boheemen et al. 2012) . structural proteins envelope (e), spike (s), membrane (m), and nucleocapsid (n) are elements of the physical structure that encloses the viral genome and come from distinct reading frames, unlike orf1a and orf1ab, which come from overlapping reading frames. additionally, the structural proteins are the product of subgenomic mrnas that are joined during discontinuous negative rna strand synthesis (van boheemen et al. 2012) . finally, ns3 protein (ns3), ns4a protein (ns4a), ns4b protein (ns4b), ns5 protein (ns5), and orf8b protein encompass the remainder of the proteome and also arise from distinct reading frames (van boheemen et al. 2012) . our approach utilizes genomic sequence data, which is readily available for viruses known to cause disease. however, because most viruses pose no major threat to their host, they pass by unnoticed leaving the majority of virus genome space uncharted. with the availability of costefficient genome sequencing technology, and recent developments in the field of viral metagenomics, large-scale identification of viral genome space is on the rise (rosario and breitbart 2011; mokili et al. 2012) . by exploring viral diversity, critical components constituting a viral genus' fitness can be evaluated. examples such as the common influenza virus illustrate the rapidity of viral gene mutation and in order to maintain immune protection, an annual flu vaccination is recommended. underway efforts aim to generate broadly neutralizing vaccines whose design accounts for the genomic sequences of multiple types of influenza virus to eliminate frequent re-vaccination against the flu ross 2011, 2012) . development of broadly neutralizing vaccines often relies on the consensus or ancestral sequences of extant viral sequences in order to provide greater coverage for related viruses (kesturu et al. 2006) . unfortunately, consensus sequences can be misleading, and ancestral sequence reconstruction is error-prone for quickly diverging sequences (mccloskey et al. 2014 ). in addition, viruses with compact genomes often express proteins with structural disorder that may undergo structural transformations. although these transformer proteins, like vp40 in ebola, are masters at changing their structure, and thus expanding their functional repertoire as needed for the life cycle of the virus (bornholdt et al. 2013) , flexible regions are potentially important in rewiring protein-protein interactions between the virus and its host (le breton et al. 2011; ortiz et al. 2013; gitlin et al. 2014) . the flexibility trait of many viral proteins is a complicating factor in vaccine development. for instance, dengue virus exhibits serotype-specific antibody affinity that causes antibodydependent enhancement, an obstacle in the development of dengue vaccines that protects against all four serotypes (flipse and smit 2015) . to overcome the hurdle posed by structural flexibility, we propose an additional screening step in identifying potential vaccine or antiviral targets that considers the structural flexibility of the viral proteins. the structural genomics initiatives increased their success rate by excluding proteins predicted to be structurally disordered (slabinski et al. 2007) . a similar approach can perhaps benefit vaccine development. furthermore, to make this approach robust to potential mutations, minimizing loss in efficacy or resistance, the evolutionary context of sequence and structure must be considered. thus, we suggest expanding the concept of broadly neutralizing vaccines/antivirals by increasing the diversity of viruses considered if possible. sites conserved for sequence, structure, and with low disorder propensity among diverse virus protein homologs are very likely to be constrained from 1) changing sequence on evolutionary time scales and 2) undergoing real-time structural transitions. these sites have potential as targets for broad-specificity antivirals or vaccines because conservation makes them broad-specificity and low dynamics avoids targeting a conformational ensemble, which is not only difficult (yu et al. 2016) , but that may change as the sequence diverges (siltberg-liberles et al. 2011) . a recent large-scale study of structural disorder in >2,000 viral genomes in 41 viral families found the amount of disorder in different virus families varying from 2.9% to 23.1% (pushker et al. 2013) . it was reported that coronaviridae has very low disorder content (mean disorder 3.68%) (pushker et al. 2013) . coronaviridae contains two subfamilies: coronavirinae and torovirinae. sars-cov and mers-cov are part the coronavirinae subfamily, from here on referred to as coronavirus (cov). the lack of disorder is intriguing because it may be important for rewiring interactions between viral proteins and host proteins (ortiz et al. 2013 ) and providing opportunities to acquire novel functional sequence motifs (gitlin et al. 2014) . structural disorder has also been proposed to be important for viral viability, enabling multifunctionality and vigor in response to changes in the environment (xue et al. 2014) . given the low fraction of structural disorder reported across coronaviridae, we set out to investigate the conservation of structural disorder and secondary structure across cov. sites identified as conserved for structure and lacking disorder can be considered to be vulnerable and druggable in the proteomes of coronaviruses. the structural divergence capacity of these regions is limited, leaving a wider range of the present and emergent coronaviruses susceptible to the effects of potential broadly neutralizing anti-cov therapies targeting these sites. we will refer to these sites as target sites. protein sequences were identified by individual blast searches with mers-cov (taxonomy id: 1335626) proteins orf1ab (yp_009047202.1; polyprotein), s protein (yp_009047204.1), m protein (yp_009047210.1), e protein (yp_009047209.1), and n protein (yp_009047211.1) against coronaviruses. blast searches of the orf1ab protein were performed, using start and end positions as detailed in the orf1ab ncbi reference sequence file, against the refseq_protein database. the sequences retrieved from the blast output maintained the following cutoff: >30% sequence identity and >50% coverage relative to mers-cov sequence query. the 30% sequence identity and 50% query coverage cutoff strikes a balance between alignment quality and at least 10 sequences for most protein families. nsp1 (yp_009047202.1; 1-193), nsp2 (yp_009047202.1; 194-853), ns3 (yp_009047205.1), ns4a (yp_009047206.1), ns4b (yp_009047207.1), ns5 (yp_009047208.1), orf8b protein (yp_009047212.1), and nsp11 (yp_009047203.1; 4378-4391) are not included in this study due to <10 blast hits. multiple sequence alignments were constructed for the selected blast hits using mafft (katoh et al. 2002) . phylogenetic trees were constructed using mrbayes 3.2.2 with a four category gamma distribution and the mixed model for amino acid substitution (huelsenbeck and ronquist 2001; ronquist and huelsenbeck 2003) . each tree ran for five million generations, with a sample frequency of 100. the final tree was constructed from the last 75% of samples, discarding the first 25% of samples as the default burnin, and using the half-compatible parameter, to avoid weakly supported nodes (i.e., with a posterior probability <0.5). all trees were midpoint rooted. for every protein family, the amino acid substitution rate per site in its multiple sequence alignment was calculated using empirical bayesian estimation as implemented in rate4site (mayrose et al. 2004) . substitution rates were calculated using 16 gamma categories, the jtt substitution matrix (jones et al. 1992) , and the reconstructed phylogenies. the rates were normalized per protein family with an average across all sites equal to zero and sd equal to 1. this means that sites with a rate <0 are evolving slower than average, whereas sites with a rate >0 are evolving faster than average. intrinsic disorder propensity was inferred using two different predictors: iupred (default settings; "long" option) (dosztá nyi et al. 2005a (dosztá nyi et al. , 2005b and disopred2 (ward et al. 2004 ) for all proteins. for iupred, the site-specific continuous disorder propensities for each protein were mapped onto their corresponding position in the multiple sequence alignment as raw disorder propensities and as binary states, order or disorder, using two cutoffs of 0.4 and 0.5. disorder propensities below the cutoff were assigned order and disorder propensities at the cutoff or above were assigned disorder. for the disopred2 predictions that were inferred using the nr database, the continuous disorder propensities for every site in a protein were mapped onto their corresponding position in the multiple sequence alignment as raw disorder propensities and as binary states, order or disorder, using a cutoff of 5. consequently, for every protein family (a multiple sequence alignment and its corresponding phylogenetic tree), two continuous matrices and three binary matrices resulted: iupred 0.4, iupred 0.5, and disopred2. an additional matrix was generated to indicate sites where the binary order and disorder assignments differ between iupred 0.4 and disopred2. a similar methodology was employed to analyze secondary structure predicted by psipred (mcguffin et al. 2000) and jpred (drozdetskiy et al. 2015) . for both predictors, the uniref90 database was used and sites were classified as loops, alpha helices, or beta strands and mapped back onto their corresponding sites in the multiple sequence alignment. this resulted in two three-state matrices for each protein family alignment, one for each predictor, and two binary matrices displaying secondary structure elements (alpha helix and beta strand) or loops. an additional matrix was generated to indicate sites where the secondary structure assignments differ between psipred and jpred. for every protein family, the binary matrices resulting from the different disorder predictions and from the different secondary structure predictions were analyzed in the corresponding evolutionary context using gloome. gloome (gain-loss mapping engine) analyzes binary presence and absence patterns in a phylogenetic context . in this study, the rate4site option in gloome was used to analyze the binary matrices (iupred 0.4, iupred 0.5, disopred2, psipred, and jpred) with the corresponding phylogenetic trees to map change of state across sites in each individual protein phylogeny . gloome was run with 16 gamma categories and a substitution matrix set to equal rates within each state and transitions between states treated equally. from the binary disorder and order matrices, transition rates between disorder and order or vice versa (dot) were estimated. from the binary structure and loop matrices, transition rates between structure and loop or vice versa (slt) were estimated. similar to rate4site, the rates were normalized per protein family with an average across all sites equal to zero and sd equal to 1. this means that sites with a rate <0 are evolving slower than average, while sites with a rate >0 are evolving faster than average. protein families were visualized in an integrative manner with a phylogenetic tree, any matrix (multiple sequence alignment or predictor based) displayed as a heatmap, and site-specific sequence transition rates using python packages ete3 (huerta-cepas et al. 2016) and matplotlib (hunter 2007) . amino acid evolutionary rates (seq) for all sites across all alignments were aggregated and binned into four possible categories characterized by the distribution of psipred predicted secondary structure at each site. sites predicted to have a loop across all sequences are "conserved loops; c(l)" and sites predicted to have a helix across all sequences or a strand across all sequences are "conserved helix-strand; c(hs)" (table 3) . sites predicted to have all three states (helix, strand, and loop) or any combination of loop and one other state are "non-conserved helix, loop, strand; nc(hls)" and sites predicted to have a mixture of helix and strand are "nonconserved helix-strand; nc(hs)" (table 3) . in all cases, gaps were ignored when classifying combinations of secondary structure at a site or if secondary structure conservation exists at a particular site. phylogenies were built for all protein products encoded in the mers-cov single-stranded rna genome, except for nsp1, nsp2, ns3, ns4a, ns4b, orf8b protein, and nsp11, all of which had insufficient sequence data (<10 sequence hits with blast). nsp12 is often used as a measure for newly identified coronaviruses. according to the international committee of taxonomy of viruses, a major criterion in determining if a coronavirus is considered novel is pairwise sequence identity below 90% for nsp12 in all comparisons to previously known coronaviruses (bermingham et al. 2012) . four main clades, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus ( fig. 1) , are identified in agreement with the taxonomic classifications described by the ictv (international committee on taxonomy of viruses 2015). coronaviruses not listed by the ictv are assumed to be a part of the clade in which representatives with known classifications are situated in our nsp12 phylogeny. the mers clade and sars clade are sister clades in the nsp12 phylogeny. the hku1 clade and equ clade are also sister clades. together these four clades form the betacoronavirus clade, in accordance with the ictv classification (international committee on taxonomy of viruses 2015). betacoronavirus is represented in all phylogenies although the order of the individual subclades varies. alphacoronavirus is often found as the sister clade or outgroup to betacoronavirus. deltacoronavirus or gammacoronavirus are the most distantly related to the betacoronavirus. in the nucleocapsid phylogeny, gammacoronavirus is the first outgroup clade to betacoronavirus, and alphacoronavirus is the most distant outgroup. most nsp trees exhibit some unresolved nodes at junctures immediately preceding terminal nodes. as an effect of the 50% majority rule, most of the 546 resolved nodes are well supported with posterior probability >0.9 for 82% and >0.99 for 68% (supplementary fig. s1 , supplementary material online). most trees follow the nsp12 topology for the main clades, with minor clade rearrangements. it should be noted that for nsp5, the entire alphacoronavirus clade is placed within the betacoronavirus clade, as a sister clade to the mers clade (supplementary fig. s1 , supplementary material online). this may be due to increased sequence divergence rates or due to recombination. recombination events are rather frequent in coronaviruses (su et al. 2016) , and the mers clade potentially underwent multiple recombination events as part of the host change (zhang et al. 2016) . the phylogenies for membrane protein, spike protein, nsp5, and nsp8-nsp16 demonstrate (with the given blast cutoffs) recoverable protein homologs such that all coronaviruses are represented (i.e., all coronaviruses represented in the nsp12 phylogeny). nucleocapsid, nsp4, and nsp7 have recoverable homologs in all clades except deltacoronavirus. nsp3 and nsp6 homologs are too divergent in deltacoronavirus and/or gammacoronavirus relative to mers-cov. envelope appears specific to betacoronavirus ( fig. 1) , but it is a short protein that has been found to diverge rapidly and is likely present outside betacoronavirus (fehr and perlman 2015) . because different protein families yield slightly different phylogenies, for the remaining evolutionary analyses, every protein family was analyzed in the context of its own phylogeny. for all protein families, structural disorder propensities were predicted using iupred (dosztá nyi et al. 2005a (dosztá nyi et al. , 2005b and disopred2 (ward et al. 2004) . to verify the robustness of the binary iupred and disopred2 predictions, the binary assignments were compared on a site-by-site basis (table 1) . when converted to binary (i.e., two states per site disordered or ordered) iupred 0.4 and iupred 0.5 are in good agreement with the larger differences seen for nsp8, nsp9, and nucleocapsid (7.5%, 6.5%, and 19.0%, respectively) (table 1). comparing iupred 0.4 or iupred 0.5 to disopred2, large differences are in particular seen for nucleocapsid (38.7% and 29.7% respectively) and nsp8 (23.5% and 25.9%, respectively) (table 1) . for nucleocapsid, regions that are found to be disordered by iupred 0.4 are found to be ordered by iupred 0.5 and disopred2 ( fig. 2 and supplementary fig. s2 , supplementary material online). for nsp8, regions that are only slightly disordered in a few sequences according to iupred 0.4 and iupred 0.5, disopred2 predicts disorder to be conserved for all sequences (fig. 3) . to quantify the fraction of disordered sites per protein family, we report the iupred 0.4 results only for simplicity (table 1). in general, iupred 0.4 predicts more disorder than disopred2, but several protein families have almost no disordered sites. nsp3 and nsp8-10 have some variation in disorder content for different viruses. based on the fraction of disorder, nucleocapsid is the only highly disordered protein among the covs in this study, even if nsps 8-10 have outliers that are >20% disordered. to compare the disorder-to-order transition rates (dot) for all protein families where the binary matrices of disorder and order include both states, the quadrant count ratio (qcr) was estimated as a measure of association in assigning slower than average vs. faster than average transition rates. for iupred 0.4 vs. iupred 0.5, for iupred 0.5 vs disopred2, and for iupred 0.4 vs. disopred2, the qcrs (table 2) . for nucleocapsid and nsp8, the positive associations are weaker, suggesting that many sites have iupred disorder propensity in the 0.4 to 0.5 range and large differences between iupred and disopred2, in accordance with the large disagreement between the binary assignment of these predictors (tables 1 and 2). for all protein families, secondary structure elements were predicted using psipred (mcguffin et al. 2000) and jpred (drozdetskiy et al. 2015) . for most protein families, the disagreement between secondary structure predictors is greater than for the disorder predictors (table 1). in fact, 15 of the 17 protein families compared disagree at more than 10% of alignment sites, and two of these disagree at more than 20% of sites. to compare the binary structureto-loop transitions (slt), qcr was estimated as a measure of association for slt based on the different predictors. in general, there is a moderate positive association between slt for psipred vs. slt for jpred that is weaker than for the different dot comparisons (table 2) . it should be noted that slt does not differentiate between alpha helix and beta strand, but considers both as "structure." this is a correct assumption if protein structure is conserved and consistently predicted, but for some protein families that is not the case. four protein families (nsp3, nsp12, nsp13, and spike) have more than 40% of their sites found within the nc(hls) category with non-conserved helix, strand, and loop (two or three states present at the same site) (table 3). for nsp13, jpred predicts 72% of all sites to be a mixture of helix, strand, and loop, or any combination of loop and one other structural element ( fig. 4) . envelope and nsp6 have 13% and 12% of their respective sites in the nc(hs) category. considering only the psipred predictions, the nc(hs) category has 245 sites across all 17 protein families. that is one-tenth the size of the next smallest set which is c(hs) with 2275 sites. next, c(l) has 3344 sites, and the largest category is nc(hls) with 4257 sites. comparing the evolutionary sequence rates for the sites in the different categories, based on psipred predictions only, reveals that sites in the c(hs) category are evolving at a slower rate than all other categories. nc(hs) is only just significantly different (p = 4.62eà03) from c(hs), and is not significantly different from nc(hls) and c(l) (p = 1.85eà02 and p = 8.33eà01, respectively). however, nc(hls) and c(l) are significantly different from each other, and both are significantly different from c(hs) (p = 1.82eà46 and p = 2.33eà21, respectively) ( fig. 5 ). for regions with five or more consecutive sites that were 100% conserved in sequence across 1) all cov or 2) across the mers and sars clades, the information of structural disorder prediction from iupred and disopred2 was used to identify all ungapped sites that were consistently predicted to have 100% conserved order. next, the information of secondary structure prediction from psipred and jpred was used to narrow down this list further by only including sites that are not changing their predicted secondary structure state for both predictors. applying the aforementioned filters to the initial 10,000 sites resulted in one (1) region of five residues or more conserved across all cov within the n-terminal domain of nsp12: dnqdl (table 4) . interestingly, this region is in the vicinity of sites found important for nucleotidylating activity across the order nidovirales (lehmann et al. 2015) . considering only the sequences in the sars and mers clades, 21 sequence regions of five residues or more were found in seven protein families (table 4) . for nsp5, nsp7, and nsp14, experimentally determined structures show that most regions are surface accessible ( fig. 6) . some of the identified target sites are known for their functional importance. for instance, c145 in the middle of gscgs in nsp5 is part of the catalytic dyad in the nsp5 protease (yang et al. 2003) . for nsp12 and nsp13, which have the majority of all sites, no structures are available. the sites adjacent to dnqdl are also conserved in the sars and mers clades, and five additional target sites, conserved for the sars and mers clades, are found in the c-terminal direction relative to the dnqdl motif (table 4) . continuing into the rna-dependent rna polymerase domain (rdrp) in nsp12, four additional regions of target sites are found, and the last three regions are found in the c-terminal part. importantly, in rdrp and in the c-terminal part are sites that are also conserved across all covs in this study. nsp13 has four regions of target sites distributed across the protein. 3. -the evolutionary context of intrinsic disorder in nsp8. the phylogenetic tree was built using the multiple sequence alignments for nsp8. (a) the multiple sequence alignment is colored by amino acid according to scale, arranged based on top-idp disorder promoting propensity of the amino acids (campen et al. 2008) , and gray denotes gaps. (b) iupred disorder propensity per site in the multiple sequence alignment. blue-to-white-to-red shows disorder propensity according to the scale for iupred 0.4. (c) iupred disorder propensity per site in the multiple sequence alignment. blue-to-white-to-red shows disorder propensity according to the scale for iupred 0.5. (d) disopred2 disorder propensity per site in the multiple sequence alignment. blue-to-white-tored shows disorder propensity according to the scale. above the multiple sequence alignment, the normalized evolutionary rates per site for amino acid substitution (seq) and the dot for the binary transformations of b-d are shown. heat maps visualized with the python packages ete3 (huerta-cepas et al. 2016) and matplotlib (hunter 2007) . see supplementary figures s2 and s4, supplementary material online for additional graphics for every protein family. we have analyzed the protein evolution of the genetic components that make up the mers-cov proteome. as previously established, mers-cov has the same genomic makeup as hku4-cov and hku5-cov in the mers clade (woo et al. 2012) . some protein products are only found in the mers clade, and these were excluded from this study due to insufficient data. furthermore, for other protein products, some clades may not be represented in our protein families if their proteins were too divergent. this was an important factor in determining the applied blast hit cutoffs, as relaxing cutoffs produced alignments with more gaps and increasing stringency reduced the representative pool. because alignment quality is important due to the sensitivity of both rate4site and for phylogenetic reconstruction, the chosen cutoffs are suitable. we note some clade-specific differences in recoverable homologs between different cov, but many components are shared among them ( fig. 1 ). viral proteins often possess multifunctionality, mediated by a conformational change in response to environment-specific factors (xue et al. 2014) . although conformational flexibility is important for function, it also offers flexibility in what sequence motifs are on display. if these sequences are rapidly diverging, different sequence motifs will be displayed, reinforcing the notion that flexible regions are potentially important in rewiring protein-protein interactions between virus and host (gitlin et al. 2014) . although most cov proteins have almost no intrinsic disorder, several cov protein families have homologous sites that display loop in some sequences, helix in others and strands in some (table 3, supplementary fig. s3 , supplementary material online). these sites are not necessarily disordered but they may be conformationally flexible in realtime (with secondary structure transitions in the same sequence, making them difficult to predict) or on evolutionary time-scales (so that different secondary structure elements actually are present in different sequences). the c(hs) and c(l) sites make up approximately 50-80% of most multiple sequence alignments. with the common expectation that protein structure is more conserved than sequence these numbers are surprisingly low. neither psipred nor jpred consistently predicts the same state for 20-50% of all sites in these multiple sequence alignments. the accuracy of psipred and jpred's secondary structure predictions are about 80% (bryson et al. 2005; drozdetskiy et al. 2015) . psipred has been found to rarely predict an alpha helix instead of a beta strand and vice versa, and most of the psipred errors are due to secondary structure not being predicted (li et al. 2014) . when secondary structure is not conserved for the same site in a multiple sequence alignment, it suggests that the secondary structure prediction may be 1) inaccurate, 2) not predicted with high confidence, or 3) the regions are indeed metamorphic; they can transition from one element to another. although (1) is difficult to address without experimentally determined structures for all sequences, (2) and (3) are not necessarily incompatible interpretations because low confidence secondary structure prediction could indicate metamorphic secondary structure regions. metamorphic secondary structure regions have interesting consequences for conformational and functional flexibility. it should be noted that, despite the low amount of disordered sites in most cov proteins, several regions are not conserved in disorder propensity across all sequences, but sometimes the different predictors disagree as in the case of nsp8. clade-specific disordered regions resulting from indel events suggest that they are not essential to the critical functions of the protein, but could cause gain-and-loss of interactions with its hosts. however, when disorder propensity is only mildly fading for a region that is present across the protein family, it may be important for the fundamental function of the protein. the virus structural proteins that interact to form the virion commonly include an envelope protein, a membrane protein, and a capsid protein that together form the machinery that encases, transports, and releases the virus. the interactions between the structural proteins are often regulated by conformational changes like vp40 in ebola (bornholdt et al. 2013 ) and envelope protein from dengue virus (zheng et al. 2014 ). conformational changes to another. it should also be noted that two mers clade specific inserts around position 241 and toward the c-terminal are consistently predicted to be highly disordered. with inserts and changing structural dynamics between clades or viruses, the questions become 1) which sequence motif are displayed and 2) to what extent are these sequence motifs displayed? furthermore, based on the inconsistent prediction of secondary structure elements, the possibility that covs are more conformationally flexible than their intrinsic disorder content implies is noteworthy. altogether, this suggests that various mechanisms for rewiring conformational and functional space are operating in the coronaviruses studied here. if regions symptomatic of conformational and functional flexibility can be avoided in order to identify broad-specificity antiviral targets with potential to be effective against coronaviruses of today and in the future, coronaviruses as a group may become more attractive drug targets for the pharmaceutical industry in the event an additional coronavirus changes host to include humans or increase its virulence. supplementary tables s1 and s2 and figures s1-s5 are available at genome biology and evolution online (http://www. gbe. oxfordjournals.org/). update on sars research and other possibly zoonotic coronaviruses the protein data bank severe respiratory illness caused by a novel coronavirus structural rearrangement of ebola virus vp40 begets multiple functions in the virus life cycle flavivirus ns3 and ns5 proteins interaction network: a high-throughput yeast two-hybrid screen protein structure prediction servers at university college london top-idp-scale: a new amino acid scale measuring propensity for intrinsic disorder gloome: gain loss mapping engine inference and characterization of horizontally transferred gene families using stochastic mapping middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group iupred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content the pairwise energy content estimated from amino acid composition discriminates between folded and intrinsically unstructured proteins jpred4: a protein secondary structure prediction server coronaviruses: an overview of their replication and pathogenesis the complexity of a dengue vaccine: a review of the human antibody response target sites shown in 3d context. (a) nsp5 dimer, based on pdb id 1uk4 (yang et al. 2003). (b) nsp7, based on pdb id 5f22 (unpublished). 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wu, ling-yun; wang, yong; zhang, xiang-sun; chen, luonan title: bridging protein local structures and protein functions date: 2008-04-18 journal: amino acids doi: 10.1007/s00726-008-0088-8 sha: doc_id: 346965 cord_uid: 0oq2n0af one of the major goals of molecular and evolutionary biology is to understand the functions of proteins by extracting functional information from protein sequences, structures and interactions. in this review, we summarize the repertoire of methods currently being applied and report recent progress in the field of in silico annotation of protein function based on the accumulation of vast amounts of sequence and structure data. in particular, we emphasize the newly developed structure-based methods, which are able to identify locally structural motifs and reveal their relationship with protein functions. these methods include computational tools to identify the structural motifs and reveal the strong relationship between these pre-computed local structures and protein functions. we also discuss remaining problems and possible directions for this exciting and challenging area. dna sequences can be called 'the blueprint of life', while proteins represent the fulfillment of this blueprint in terms of structures and functions. a fundamental goal of functional genomics research is to understand how proteins carry out functions in a living cell (eisenberg et al. 2000; brenner 2001; goldsmith-fischman and honig 2003) . in addition to experimental methods, computational methods have been extensively applied with the aim of developing hypotheses in terms of assigning specific functions to specific proteins and providing valuable biological insights. the basic rationale behind such research is that the gene sequence determines the amino acid sequence, and the amino acid sequence determines the protein structure, which, in turn, determines the protein function (whisstock and lesk 2003) . many proteins, even among those in the protein data bank (pdb), have not yet been annotated, although we have succeeded in deriving their structures watson et al. 2005) . we review here the in silico annotation methods currently used to determine protein function from protein local structures. generally speaking, proteins are the main catalysts, structure components, signal transfers and molecular machines in a biological organism. as such, they are the basic elements of functions. however, the definition of function means different things to different people since it is an evolving concept associated to an abundance of interpretations. in general, these functions can be described at many levels, ranging from the biochemical functions at the molecular level (e.g. catalytic or binding activities) to biological processes at the level of biomolecular cooperation (e.g. signal transduction or cellular physiological process) to the cellular components at the cell level of an organ (e.g. nucleus or rough endoplasmic) (devos and valencia 2000; watson et al. 2005) . several schemes/tools/ databases have been developed in recent decades for measuring protein functions in a systematic model with the aim of annotating the functions of proteins ; these include ec (barrett 1997) , mips (ruepp et al. 2004) , go (the gene ontology consortium 2000; camon et al. 2004 ) and kegg (kanehisa and goto 2000) , as shown in table 1 . using the existing function annotations as 'gold standard' data, researchers have been able to develop many protein function annotation methods in recent years based on protein relationships. we summarize the existing function annotation methods in the framework of fig. 1 , which shows the basic tendency for the functional inference methodology-i.e. to explore sequence similarity, structure similarity, protein interaction and their integration. we briefly review these in the following list: • using sequence information. the methods in this category often utilize a blast, fasta or psi-blast score to detect the sequence similarity and annotate the functions to a target protein from its homologous protein (whisstock and lesk 2003; watson et al. 2005) . in the safe zone (rost 1999 ) of sequence similarity, the sequence-based methods can provide putative annotations with high confidence (wilson et al. 2000) . a number of papers have tested the global performance between the relationship of the sequence similarity and function similarity. shah and hunter (1997) tested the sequence similarity among enzymes in many ec classes at various thresholds and concluded that the functional similarity could not be detected perfectly when the sequences are not similar enough. wilson et al. (2000) and devos and valencia (2000) obtained similar results. joshi and xu (2007) presented a systematic analysis on the sequencefunction relationships in four model organisms. • using structure information. protein structures are more conserved than protein sequences (orengo et al. 1999; hou et al. 2005) . a number of methods have been developed with the aim of assessing protein structure similarity (kolodny et al. 2005) ; these can be grouped as coordinate-based [such as structal (gerstein and levitt 1998) , samo , tm-align (zhang and skolnick 2005) and prosup (lackner et al. 2000) ], distance-matrix-based [such as dali (holm and sander 1993) , ce (shindyalov and bourne 1998) , fatcat (ye and godzik 2004) , ssap (orengo and taylor 1996) ] and secondary-structure-based [such as vast (gibrat et al. 1996) , ssm (krissinel and henrick 2004) , lock (singh and brutlag 1997) and fast (zhu and weng 2005) ]. classifying the proteins into different classes or families based on global structure similarity will assist researchers in determining the relationships among different proteins and provide a foundation of functional organization (brenner 2001) . scop (murzin et al. 1995) , cath (orengo et al. 1997) and fssp (holm and sander 1996) comprehensively cluster all proteins with known structures. based on the safe zone means that pairwise sequence identity is higher than 40%, the twilight zone, about 20-30%, the midnight zone below 20% those clusters, the functional relationships among the proteins can be roughly detected. • using interactome information. proteins always interact with other molecules to carry out their functions (sharan et al. 2007 ). information on protein-protein interactions or other interaction maps among molecules, such as dna binding with protein, can be explored to annotate the protein functions from complexes and pathways of the biochemical processes. the network-based methods extend the functional inference from the single molecular level to a systematic level by considering interactions among genetic components and transferring functions among them (vazquez et al. 2003 ; barabasi and oltvai 2004; zhang et al. 2007 ). sharan et al. (2007) cataloged the methods to direct methods and module-assisted methods individually. • using integrated information. another sensible strategy is to use many different data sources to increase the chances of obtaining function annotations for any given protein. for example, in marcotte et al. (1999) , proteins are grouped by experimental data, such as metabolic function, phylogenetic profiles, rosetta stone results and correlated messenger rna expression patterns to determine the functional relationships among proteins of the yeast. in fact, many methods are in this framework (sanishvili et al. 2003; george et al. 2005; pal and eisenberg 2005; zhao et al. 2008a, b) , especially when data integration becomes the focus of the systems biology study. in this review we highlight the relationships between protein local structures and protein functions since it is commonly believed that local regions on the structures are responsible for the performance of the particular functional tasks (russell 1998; ferre et al. 2005) . well-known examples include the ser-his-asp triad in enzymes and other known special structural frameworks that carry out certain functions of catalysis (torrance et al. 2005) . it is now widely recognized that some fold similarities suggest an 'analogous' rather than a 'homologous' relationship (russell 1998) . proteins can adopt similar tertiary folds while performing different functions at different binding site locations. given the existing status that the midnight zone functional linkages escape from the sequence and global structure similarity, only the local structures can be used to analyze detailed relationships with functions by determining the protein-protein interaction, protein-dna interaction or other global performance from the physical perspective. also, the local structures of protein provide more detail information on protein function not only from the single targeted action of that protein, but also from the integrative process due to the detailed components and the three-dimensional architecture. the local structures are also important in the design of drugs and bioengineering. in an interesting paper, schnell and chou (2008) convincingly provided nuclear magnetic resonance (nmr) data showing that the m2 proton channel of influenza a virus is typically controlled by the local conformational change with a phgated mechanism. the discovery provides sound evidence that the local structures are crucial for determining protein function, and it is vitally important in the search for effective anti-influenza drugs (borman 2008) . bridging protein local structures and protein functions can timely provide useful information for structure-based drug design [e.g. see the methods in chou et al. (2003) and wang et al. (2007a) against severe acute respiratory syndrome (sars), and that in du et al. (2007) against chicken influenza a virus h5n1, as well as a review paper (chou 2004) ]. thus, it a key task of researchers in this field is to investigate the relationships between protein functions and protein local structures. this review is organized into four parts. first, we will describe the main molecular functions related to protein local structures. this is followed by a description of existing definitions and methods for detecting similarities in local structures. in the third part, the detailed methodologies to bridge local structures with functions are reviewed. some discussion and future directions are summarized in the last part. to bridge the relationship between local structures and functions, we first catalog the molecular functions of proteins strongly related to local structures. the local structures are often regarded as the protein-protein interfaces, catalytic sites, ligand-binding sites, metal-binding sites, post-translational modification sites or other miscellaneous active sites. table 2 lists some of the important functional categories (chakrabarti and lanczycki 2007) . a protein generally interacts with other proteins in performing and regulating many processes in a cell. the pace of discovery of protein-protein interactions has recently accelerated due to rapid advances in new technologies (salwinski and eisenberg 2003; chou and cai 2006) . the basis of protein-protein interactions often lie in local planar patches on the protein surface. the factors that influence the formation of protein-protein complexes can be cataloged into four different types-i.e. homodimeric protein, heterodimeric proteins, enzyme-inhibitor complexes and antibody-protein complexes (jones and thornton 1996) . from the structural perspective, structural characterization of macromolecular assemblies usually poses a more difficult challenge than structure determination of individual proteins (russell et al. 2004) . effective approaches for the prediction of protein-protein interactions at physical interaction levels are also strongly in demand (wodak and mendez 2004) . zhou and qin (2007) reviewed the methods currently being applied for interface prediction. the characteristics between interface and noninterface portions of a protein surface, such as sequence conservation, proportions of amino acids, secondary structure, solvent accessibility and side-chain conformational entropy, are often used to distinguish the specificity of local structures relating to protein binding function. in the transcription and translation process, proteins always bind to dna and rna to fulfill various functions. proteinnucleotide binding is a fundamental function of proteins. luscombe et al. (2000) classified the dna-binding proteins into eight different structural/functional groups. the helix-turn-helix (hth) motif is one of the most common structures used by proteins to bind dna, while protein-rna binding involves a number of different structure specificities. a comparison between protein-rna and protein-dna complexes revealed that while base and backbone contacts (both hydrogen bonding and van der waals) are observed with equal frequency in protein-rna complexes, backbone contacts are more dominant in protein-dna complexes (jones et al. 2001) . the positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all play key roles in the sites for the rna-binding function. ligand binding is a key aspect of protein functions. proteins recognize their natural ligands for transportation, signal transduction or catalysis (campbell et al. 2003) . the cleft volumes in proteins have strong relationships with their molecular interactions and functions. the ligands are always bound in the largest clefts (laskowski et al. 1996) . metal ions have a role in a variety of important functions, including protein folding, assembly, stability, conformational change and catalysis (barondeau and getzoff 2004) . in order to leverage the wealth of native metalloprotein structures into a deep understanding of metal ion site specificity and activity, high-resolution analyses of metal site structures and metalloprotein design are increasingly being performed. one of the most ubiquitous zinc-binding motifs is the c2h2 zinc finger motif, which was first identified in transcription factors (ebert and altman 2008) . another broad concept for protein local structures is the active site. active sites of a protein are comprehensively related to functionally important local regions of the protein. the special features of functional local structure are to provide deep insights into the relationship between structure and function. for example, the catalytic triads provide a target of structure for finding the catalytic function of the proteins. to date, many different types of local structures have been defined or identified based on the geometry of the local regions, protein surface patterns, chemical groups or the electronic features. local structure features are believed to be the factors related to concrete functions. at the sequence level, the local regions may be scattered on the primary sequence, forming special motifs. alternatively, at the folding level, they form locally spatial shapes. we can simply catalog the types of methods used to identify the local structures as follows: methods to detect profiles of sequences with special local shapes, and methods to detect the substructures with special features based on folding. the primary sequence of a protein consists of (combinations of) 20 different amino acids, which fold and pack together to constitute a special three-dimensional structure. sequence motifs are conserved segments in protein primary sequences. multiple sequence alignment is often used to identify the common patterns in several protein sequences, especially in the homology family. more advanced sequence comparison algorithms can detect the profiles of the functional residues in the primary sequence. of these algorithms, one of the most common methods is the hidden markov model (hmm). there are a number of important sequence pattern databases, which are publicly available from the internet (table 3) . local three-dimensional structural patterns, such as the surface cavities of protein (e.g. the clefts and pockets) also have conserved structural features. table 4 lists a number of methods currently used to identify local structure patterns. the procedure of recognition can be generally divided into two parts. the first is to construct the local structures. the geometric structure patterns and biochemical properties can be used to segment the protein architecture into small substructures. the second is to search the annotated sites from the literature and databases. the analysis of the protein surface is an active area of research in terms of the study of local structures. to date, two aspects of protein surface patches have attracted the most attention. the first is based on the defined features, such as surface curvature, surface cavities, electrostatic potential and hydrophobicity. castp (binkowski et al. 2003b ) uses the weighted delaunay triangulation and the alpha complex for shape measurements. the local regions are defined by computational geometry, which identifies and measures surface accessible pockets as well as interior inaccessible cavities for proteins and other molecules. computational geometry also measures analytically the area and volume of each pocket and cavity, both in solvent accessible surface (sa, richards' surface) and molecular surface (ms, connolly's surface). castp provides an online resource for locating, delineating and measuring concave surface regions on the three-dimensional structures of proteins. these include pockets located on protein surfaces and voids buried in the interior of proteins. pvsoar (binkowski et al. 2004 ) provides an online resource to identify similar protein surface regions. kinoshita and nakamura (2003) provided a molecular surface database of proteins' functional sites, named the ef-site. the method displays the electrostatic potentials and hydrophobic properties of proteins together on the connolly surfaces of the active sites for analysis of the molecular recognition mechanisms. the connolly surfaces are made by using the molecular surface package program, and the electrostatic potentials are calculated by solving poisson-boltzmann equations with the self-consistent boundary method. the second aspect of protein surface patches is based on a predefined segmentation size of the surface. the method uses a segmentation procedure to divide the surface into small segmentations that correspond to certain physical modules of the surface. surfnet (laskowski 1995) generates molecular surfaces and gaps between surfaces from three-dimensional coordinates supplied in a pdbformat file. the gap regions can correspond to the voids between two or more molecules or to the internal cavities and surface grooves within a single molecule. the program visualizes molecular surfaces, cavities and intermolecular interactions by segmenting the surfaces. based on the surfnet algorithm, surface (ferre et al. 2004) identifies clefts and explores the cleft boundaries called the surface patch. a non-redundant set of protein chains is then used to build a database of protein surface patches. lig-site (hendlich et al. 1997 ) is a program for the automatic and time-efficient detection of pockets on the surface of proteins that act as binding sites for small molecule ligands. pockets are identified with a series of simple operations on a cubic grid. the special features of catalytic sites or other types of functional sites are also detected as local structures. some functional annotations of residues can be found in databases and the literature, and the location of these residues can be represented as potential structural motifs. although it is difficult to define just precisely what is the active site in protein structures, there are a number of methods for identifying active sites or functionally important residues. wallace et al. (1997) described a geometric hashing algorithm, called tess, to derive three-dimensional coordinate templates for motifs. tess has been used to create a database of enzyme active site templates called procat (wallace et al. 1997) . procat provides facilities for interrogating a database of three-dimensional enzyme active site templates. it has been superseded by the catalytic site atlas (csa). the csa (porter et al. 2004; torrance et al. 2005 ) is a database documenting enzyme active sites and catalytic residues in enzymes with a threedimensional structure. it contains the original annotated entries derived from the primary literature by hand and the homologous entries found by the psi-blast alignment. a hetatm and all annotated sites in the pdb also provide patterns of protein local structures strongly related to protein functions. stark and russell (2003a) reported patterns in non-homologous tertiary structures (pints) that can be used to uncover the recurring three-dimensional side-chain patterns based on the algorithm in stark et al. (2003c) . sitebase (gold and jackson 2006a) is a database of known ligand-binding sites within the pdb. the search for an annotated position in the pdb constructs the location information of the ligand-binding sites. a collection of known sites from mining the annotations in the pdb has been designated as the pdbsite (ivanisenko et al. 2005) , which collects amino acid content structure features calculated by spatial protein structures, and physicochemical properties of sites and their spatial surroundings. the pdbsitescan (ivanisenko et al. 2004 ) provides an automatic search of three-dimensional protein fragments similar in structure to known functional sites. a comparison of local structures in the pdb also provides valuable information for constructing the structural motifs. kleywegt (1999) presented two programs, spatial arrangement of side-chains and main-chains (spasm) and rigor, for recognizing spatial motifs in protein structure. spasm can be used to find matches in the structural database for any user-defined motif. the program also has a unique capability to carry out ''fuzzy pattern matching'' with relax requirements on the types of some or all of the matching residues. rigor, on the other hand, can compare a database of pre-defined motifs against a perhaps newly determined structure. rigor scans a single protein structure for the occurrence of the pre-defined motifs from a database. zemla (2003) presented a method for finding three-dimensional similarities in protein structure. this algorithm is able to generate different local superpositions between pairs of structures and to detect similar fragments. it allows the clustering of similar fragments and the use of such clusters to identify sequence patterns that represent local structure motifs. sumo (jambon et al. 2003) can detect the common site, which corresponds to the catalytic triad. the general procedure of bridging the local structures with functions lies in constructing a candidate pool of local structures, identifying important features of function-related local structures and validating their functional importance. the existing methods can be grouped into two categories, i.e. unsupervised and supervised methods, as shown in fig. 2 . the unsupervised methods directly mine those local structures with special features and then detect their functional implications. the supervised methods use known function-related structures as the templates and match these similar patterns by comparison. there are strong relationships between the two kinds of methods. most of the proposed methods are based on physical and/or biochemical patterns of the protein, and some particular patterns of local structures are strongly related to functions. in the unsupervised methods, the patterns are derived directly from a group of local structures without known functions. their functional importance and characteristics are identified by analyzing the conserved factors in the common features of local structures. the identified function-related local structures can then be used to enlarge the pool of functional templates, which in turn can be used to measure the potential functional importance of the new substructures. figure 2a shows these relations. these functionally important local regions can be referred to as functional motifs. the functional motif is the particular local structure pattern with factors that are the determinations of performing particular functions. note that the functional motif is very important for studying the relationship between structure and function in theory, and it is of practical importance to the protein design of drug targets and other bioengineering fields. we can investigate the functional patterns of the local structures in multiple ways. more specifically, we group existing methods to bridge protein local structure and function into three categories based on the hierarchical perspective, as shown in fig. 2b . 1. element-based methods. these identify the local structures from sequence, structure and/or other important amino acid residues information. the methods detect the common or conservation patterns in these elements of proteins and bridge the gaps between the local structures and functions at the micro level. during the bridging process, if prior knowledge is used to identify the functional importance or guide the detection, the method belongs to the supervised category, otherwise it belongs to the unsupervised division. 2. feature-based methods. these investigate the putative features between the local structures and functions. this category can be further divided into two subcategories-i.e. scoring methods and learning methods. the identified functional features of local structures provide templates of functional motifs. in the scoring methods, the features of local structures are scored by a defined function, and then the scores are used to decide whether the targets are functionally important. thresholds are often then chosen to provide guidance for detecting the importance of target local structures. in the learning methods, some features are chosen and learned from the known function-related local structures. the learned features in the trained machines can be used as the classifier to decide whether the testing targets are strongly related to the function. these methods belong to the supervised division. 3. network-based methods. these are based on graph theory and network topology. the methods can be divided into two subcategories. the first is at the individual level and the second is at the mapping level. at the individual level, the protein can be represented as an interactive graph of the residues, with linkages representing the close distance among them. cliques of the graph, hub residues and residues with other special topology measures may correspond to functionally important regions and residues. at the mapping level, a network represents the similarity relations among the local structures. the functional motifs are mined from informative subgraphs. this approach lies in between the other two methods mentioned above and can be regarded as being semi-supervised because it uses some heuristic knowledge. element-based methods are based on a basic intuition that the conserved part of a sequence and structure is an important functional motif (aloy et al. 2001; jones and thornton 2004) . the first step is a discovery process, which mines similar local structures from the sequences or structures of the target proteins. when similar local patterns of structures in some proteins are identified, the identified structure features of local regions will be the determinants of similar functions among the proteins. the second step is to match the process by comparing the target to the known functional templates. based on the similarity between these, the function relationship is inferred. this method is also a basic tool for developing more advanced techniques to bridge the relationship between local structures and functions. the sequences, structures or other elements of the proteins are considered in the comparison. table 5 lists the main methods that are currently being used. depending on whether or not some prior knowledge is used in the assessment, the method is classified as being supervised or unsupervised. similar patterns of local structures can be identified in different proteins, even in proteins of the midnight zone with neither sequence homology nor structure homology. in this case, the alignment of the sequences and/or structure (2007) tertiary side-chain patterns subgraph-isomorphism matching assam artymiuk et al. (1994) segments can imply similar functions of the local structures. these similar local structures of the proteins are important prognostic factors of their similar functions. multiple sequence alignment ma et al. (2003) used ten protein interface families selected from two-chain interface entries in pdb, identified surface residues and filtered out contact residues. the alignment results of the residue properties revealed that polar residue hot spots occur frequently at the interfaces of macromolecular complexes, thereby distinguishing binding sites from the remainder of the surface. using multiple structure alignment, these authors also showed the correspondence between energy hot spots and structurally conserved residues. three residues (trp, phe and met) were observed to be significantly conservative in binding sites. these identified local structures are linked with binding functions. all residues in a protein are not equally important. some are essential for certain structures or functions, whereas others can be readily replaced. conservation analysis is one of the most widely used techniques for predicting these functionally important residues in protein sequences. capra and singh (2007) proposed a method focusing on the analysis of a multiple sequence alignment of the homologous sequences in order to find columns that are preferentially conserved. the results show that conservation is highly predictive in identifying catalytic sites and residues near bound ligands, while it is much less effective in identifying residues in protein-protein interfaces. structure alignment: geometric hashing rosen et al. (1998) proposed a surface comparison algorithm in search of active sites and functional similarity. these authors first represents the surface by a face-center critical point technique and then derive active sites using geometric hashing to match the two surfaces. finally, a clustering process is used to obtain the functional active sites. this method addresses the question of the usefulness of geometric comparisons and concludes that pure geometric surface matching is capable of obtaining biological meaningful solutions. based on the geometric hashing algorithm, leibowitz et al. (2001) presented a multiple structural alignment algorithm to detect a recurring substructural motif. given an ensemble of protein structures, the algorithm automatically finds the largest common substructure (core) of c a atoms that appears in all of the molecules in the ensemble. the detection of the core and the structural alignment are carried out simultaneously. fischer et al. (1994) also presented an approach using geometric hashing to compare spatial, sequence-order independent atoms. it automatically detects a recurring three-dimensional motif in protein molecules without any predefinition of the motif. pairwise alignment of constructed local structures there are several methods that detect the functional relationship between local structures by structure alignment in an allagainst-all manner. pazos and sternberg (2004) presented an automatic method to extract functional sites (residues associated to functions). the method relates proteins with the same go functions through structural alignment in an all-against-all manner and extracts three-dimensional profiles of conserved residues. based on the identified local structures derived from geometry or physicochemical features, the functional relationship of these local regions can be detected and the comparison result is stored in a database. when querying a local structure, similar hits imply functional relationships. binkowski et al. (2003a binkowski et al. ( , 2005 described such an approach for inferring functional relationships of proteins based on the pvsoar by detecting sequence and spatial patterns of the functional relationship of pockets on protein surfaces. the pvsoar database provides a pairwise comparison of the pockets in the pocket database castp. similar pockets in different match degrees are searched for in an advanced analysis of the function relationship among the local structural motifs. with respect to the pockets on the protein surface, schmitt et al. (2002) developed a similar method based on a clique detection algorithm by comparing the query against the whole database. kinoshita and nakamura (2003) also provided an analogous method for comparing molecular surface geometries and electrostatic potential on the surfaces based on ef-site. their method bridges the protein surface electronic features of the local region with the specific functions. jambon et al. (2003) designed a new but similar approach for finding similarities using pairwise matching to detect common three-dimensional sites in proteins. the basis for their method is a representation of the protein structure by a set of stereochemical groups. protein surface regions with similar physicochemical properties and shapes may perform similar functions and bind similar partners. shulman-peleg et al. (2005) constructed two web servers and software packages for use in recognizing the similarity of binding sites and interface-siteengine and interface-to-interface (i2i)-siteengine. the input into the two methods is two protein structures or two protein-protein complexes; the output is the surface of the proteins for a region similar to the binding sites or the interfaces. the methods are efficient for large-scale database searches of the entire pdb. obviously, the two locally identified structures are related to functions by searching similar local regions of their protein structures. pairwise alignment of annotated local structures information on functional sites obtained from databases or the literature can be used to construct the function-related local structure database, while the pairwise alignment method is used to detect the functional relationships. stark and russell (2003a) developed pints to uncover the recurring three-dimensional side-chain patterns based on the algorithm in stark et al. (2003c) . their method queries the structural motif database constructed from the annotation mining from pdb to find similar three-dimensional motifs by a recursive, depth-first search algorithm, i.e. to find all possible groups of identical amino acids common to two protein structures independent of sequence order (russell 1998 ). the search is conducted with distance constraints by ignoring those amino acids unlikely to be involved in the protein function. stark et al. (2003b) identified some functional sites and compared these with procat and rigor. moreover, pints provides a measure of statistical significance based on a rigorous model for the behavior of rmsd (stark et al. 2003c) . sitebase (gold and jackson 2006a ) is a database of known ligand-binding sites within the pdb. gold and jackson (2006a) provided a method that automatically identifies ligand-binding sites by searching for hetatm keywords in pdb files and constructing a database by excluding protein/peptide ligands and treating het-groups as individual ligand-binding sites. protein atoms within a 5-å radius of any ligand atom were defined as its binding site in this work, and the ligand-binding was identified by comparison in an all-against-all way with geometric hashing. similar functions of binding sites were detected regardless of the sequence and folding similarity (gold and jackson 2006b) . pdbsitescan (ivanisenko et al. 2004) provides an automatic search of three-dimensional protein fragments that are similar in structure to known functional sites. a collection of known sites has been designated as the pdbsite (ivanisenko et al. 2005) , which is a database of amino acid content, structure features calculated by spatial protein structures and the physicochemical properties of sites and their spatial surroundings. protein-protein interaction sites are also generated by an analysis of contact residues in heterocomplexes. the algorithm is developed based on an exhaustive examination of all possible combinations of protein positions. the bid (fischer et al. 2003 ) database searches the primary scientific literature directly for detailed data on protein interfaces by text mining and stores the characterization of protein-protein binding interfaces at the amino acid level. the bid also organizes protein interaction information into tables, graphical contact maps and descriptive functional profiles. evolutionary tracing protein functional sites have a number of similar and unique features. in order to explore the information fully, one can incorporate both sequence and structure data in a functional site prediction method. the evolutionary trace (et) method is one such method that relies on both sequence and structure information. the most basic form of the algorithm requires a multiple sequence alignment of a protein family and an evolutionary tree, based on sequence identity, which can approximate the functional classification of the protein sequences (lichtarge and sowa 2002) . yao et al. (2003) proposed an automatic et method that ranks the evolutionary importance of amino acids in protein sequences. this was the first method to quantify the significance of the overlap observed between the best-ranked residues and functional sites. the information inherent in a phylogenetic tree is added to the analysis of conserved sequences, often revealing the more subtle aspects of protein function. starting with a multiple sequence alignment, a representative structure and a phylogenetic tree, this method evaluates conservation at each position in the alignment for different sequence similarity cut-offs. in its original implementation, residues were classified as variable, conserved or a group-specific set that is specific to one branch of the phylogenetic tree. this analysis can be further expanded by the use of amino acid substitution matrices to evaluate conservation. in either case, a representative structure is used to visualize the distribution of scores at the end of the analysis. based on the et method, landgraf et al. (2001) presented a three-dimensional cluster analysis that offers a method for predicting functional residue clusters. this method requires a representative structure and a multiple sequence alignment as input data. individual residues are represented in terms of regional alignments that reflect both their structural environment and their evolutionary variation, as defined by the alignment of homologous sequences. the overall and regional alignments are calculated from the global and regional similarity matrices, which contain scores for all pairwise sequence comparisons in the respective alignments. three-dimensional clustering analysis is an easily applied method for the prediction of functionally relevant spatial clusters of residues in proteins. armon et al. (2001) proposed the consurf method, which takes into account the evolutionary relationships among the sequence homologues by closely approximating the evolutionary process and by considering the phylogenetic relationships among the sequences and the similarity between amino acids. consurf maps evolutionary conserved regions on the surface of proteins with a known structure; it also aligns sequence homologues of the protein and uses the alignment to construct phylogenetic trees. the trees are then used to infer the presumed amino acid exchanges that occur throughout the evolution. each exchange is then weighted by the physicochemical distance between the exchanged amino acid residues. the results show that the patches of conserved residues correlate well with the known functional regions of the domains and are more sensitive than the et method. to obtain an indication of the validity of functional inheritance, aloy et al. (2001) proposed a method to evaluate the reliability by exploiting the conservative functional sites predicted by the et method. their method first used a fully automatic procedure to carry out the et method, and then was benchmarked in terms of required sequence divergence and the resultant selectivity and specificity of the prediction. finally, the results that were obtained using the prediction of location of functional sites to assist in filtering putative complexes were evaluated. the functional importance of local structures can be detected by empirical methods or by computational methods. the identified functional motif can then be used as the structure template to detect the functional regions in other protein structures. the chosen method often consists of a comparison process, and the structure and physicochemical features can be considered in the comparison to the templates. in addition, a measurement of the similarity to the template is used to assess the functional importance of the testing of local structures. wallace et al. (1997) described a three-dimensional template matching method based on geometric hashing for automatically deriving three-dimensional templates from the protein structures deposited in pdb. in their paper, these researchers described a template derived for the ser-his-asp catalytic triad. their results showed that the resultant template provides a highly selective tool for automatically differentiating between catalytic and noncatalytic ser-his-asp associations. goyal and mande (2007) described the generation of three-dimensional structural motifs for metal-binding sites from known metalloproteins. using three-residue templates and four-residue templates, the method scans all available protein structures in the pdb database for putative metalbinding sites. the search of the whole pdb database predicted many novel metal-binding sites, which are the identified functional motifs. chakrabarti and lanczycki (2007) recently performed a detailed survey of compositional and evolutionary constraints at the molecular and biological functional levels for a large set of known functionally important sites extracted from a wide range of protein families. they compared the degree of conservation across different functionally important sites. the compositional and evolutionary information at functionally important sites was compiled into a library of functional templates. in their paper, these researchers developed a module that predicts functionally important columns of an alignment based on the detection of a significant 'template match score' to a library template. benchmark studies showed good sensitivity/ specificity for the prediction of functional sites and high accuracy in attributing correct molecular function type to the predicted sites. the comparison between potential sites and the templates is very important in these kinds of methods. artymiuk et al. (1994) developed a program called assam, which represents a motif-by-distance matrix between pseudo-atoms and uses the subgraph-isomorphism algorithms to find matches. this is an elegant method for the detection of common tertiary side-chain patterns based on the use of the ullman subgraph isomorphism algorithm. singh and saha (2003) formulated the problem of identifying a given structural motif (pattern) in a target protein and discussed the notion of complete and partial matches. they described the precise error criterion that has to minimized and also discussed different metrics for evaluating the quality of partial matches. they also presented a novel polynomial time algorithm for solving the problem of matching a given motif in a target protein. the functions of a protein are strongly related to the physicochemical features of that protein. the physical features (such as geometry, size, depth and shape) and the chemical features (such as energy, hydrophobicity, amino acid propensity and conservation) of the local structure are often measured by a score function or learned by a machine learning algorithm. the functional importance and specificity of a protein can be identified from the evaluation score or the trained standards of features. the main methods are listed in table 6 . the scoring method can often calculate an explicit value for the features, while the learning method can reveal the patterns inexplicitly. the properties of local structures are believed to be conserved in terms of determining their functions. the identified local regions of structure are analyzed based on the variations in their properties, which are investigated using the identified functionally important sets of local structures. the method to predict the functions of the local structures is often based on a scoring scheme that is used to analyze the properties of the targets. in particular, the scores of the features are used as the measurements to determine whether the local structure has functional importance, for example, for a particular function. scoring by physical features first, the physical features of the local structures, such as size, depth and shape, are (2003) considered for scoring the function-related features. the shape features alone may provide basic information for the analysis of the functional features related to the protein function. siggers et al. (2005) introduced a new method to structurally align interfaces observed in protein-dna complexes. their method is based on a procedure that describes the interfacial geometry in terms of the spatial relationships between individual amino acid-nucleotide pairs. they subsequently provided a yet newer method to study the determinants of binding specificity. kawabata and go (2007) proposed a new definition for pockets using two explicit adjustable parameters, the radii of small and large probe spheres, which correspond to the two physical properties, 'size' and 'depth'. a pocket region was defined as a space into which a small probe can enter, but a large probe cannot. based on the geometric standards of large probe spheres, this method identified the binding site positions. from the geometrical viewpoint, the methods described above need further improvement to describe or compare the global shape and the local structures. morris et al. (2005) presented a novel technique for capturing the global shape of a protein's binding pocket or ligand. this method uses the coefficients of a real spherical harmonics expansion to describe the shape of a protein's binding pocket. shape similarity is computed as the l2 distance in coefficient space. kahraman et al. (2007) used a recently developed shape matching method to compare the shapes of protein-binding pockets to the shapes of their ligands. their results indicate that pockets binding the same ligand show greater variation in their shapes than those which can be accounted for by the conformational variability of the ligand. this result suggests that geometrical complementarity in general is not sufficient to derive molecular recognition. scoring by chemical features chemical features of local structures are very important for determining their functional specificity. these feature scores of local structures can be used as standards to determine their functions. the structural locations of functional sites are conserved between homologous proteins because functionally important residues tend to cluster together in space, forming threedimensional residue clusters or surface patches. panchenko et al. (2004) presented a method to assign each residue a score that depends on its own conservation in homologs and the conservation of residues in its spatial neighborhood. the high-scoring sites are more likely to be involved in specific binding or catalysis. functionally important residues in a protein are known to be those computed to have energy among experimentally destabilized residues. elcock (2001) proposed a method to predict functionally important residues based solely on the computed energetics of a protein structure. the energetic properties of binding surfaces in proteinprotein interfaces and protein-ligand sites were shown to be different (burgoyne and jackson 2006) . the pockets from qsitefinder (laurie and jackson 2005) were ranked by the scores of these properties-i.e. hydrophobicity, desolvation, electrostatics and conservation-which are used to determine binding sites. jones et al. (2003) developed a method to detect dnabinding sites on a protein surface. the surface patches and the dna-binding sites were initially analyzed for accessibility, electrostatic potential, residue propensity, hydrophobicity and residue conservation. in general, dnabinding sites are among the top 10% of patches with the largest positive electrostatic scores. this knowledge was used to make predictions. jones et al. (2001) presented a similar computational analysis of protein-rna interactions. there are a number of differences between dnabinding sites and rna-binding sites. for the rna-binding sites, van der waals contacts play a more important role than hydrogen bond contacts. as to the protein-dna binding local structures, luscombe et al. (2001) investigated hydrogen bonds as well as van der waals contacts and water-mediated bonds to assess whether there are universal rules that govern amino acid-base recognition. in a subsequent study, luscombe and thornton (2002) also identified the amino acid conservation and the effects of mutations on binding specificity. in liang et al. (2006) , an empirical score function consisting of a linear combination of the energy score, interface propensity and residue conservation score is used to predict interface residues. the top-ranked patches are predicted to be the potential interface sites. the accuracy of prediction has been improved significantly, relative to any single or pairwise combination, by combining the three terms. cheng et al. (2005) presented a method to predict protein function site using sequence alignment information as well as rosetta protein design and rosetta free energy calculations. logistic regression with the generalized linear model has been used to the determine weights of the sequence conservation, natural/designed sequence profile difference and natural/optimal residue free energy gap, all of which optimize the separation between functional and non-functional residues. innis et al. (2004) presented conserved functional group (cfg) analysis to predict function sites in proteins. the method relies on a simplified representation of the chemical groups found in amino acid side-chains to identify functional sites from a single protein structure and a number of its sequence homologs. scoring by physicochemical features those features based only on physical geometry or chemical energy often can not represent functional features comprehensively. most of the methods are used to integrate several important features together and then score these features for bridging the gaps between local structures and functions. the ligsite algorithm is based only on the geometry. huang and schroeder (2006) presented an extension and implementation method, ligsite csc , which is based on the notion of surface-solvent-surface events and the degree of conservation of the involved surface residues. the use of the connolly surface has led to slight improvements, whereas the prediction re-ranking significantly improved the binding site predictions. glaser et al. (2006) improved previous approaches by combining two known measures of 'functionality' in proteins, i.e. cleft volume and residue conservation, to develop a method for identifying the location of ligand-binding pockets in proteins. neuvirth et al. (2004) proposed a structure-based algorithm to identify the location of protein-protein interaction sites. the sites are defined based on connolly's molecular dot surfaces. the method defines an interface score that combines the chemical and geometry features of the interaction sites. interfacial residues are considered to be those with the 10% highest scores. geometry and energy properties have also been used to analyze the pocket functions for docking (li et al. 2004 ). hoskins et al. (2006) considered the use of solvent accessibility, residue propensity and hydrophobicity in conjunction with secondary structure data as prediction parameters to predict proteinprotein interaction sites. the influence of residue type and secondary structure on solvent accessibility is analyzed, and a measure of relative exposedness is defined. the highscoring residues are clustered as a basis for predicting interaction sites. tsuchiya et al. (2004) provided a method for analyzing protein-dna complexes, focusing on the shape of the molecular surface of the protein and dna, along with the electrostatic potential on the surface, and calculated a new evaluation score. based on the score, the method was used to classify dna-binding from non-dna-binding proteins. taroni et al. (2000) provided an analysis of the characteristic properties of sugar-binding sites. for each site, six parameters were evaluated-i.e. solvation potential, residue propensity, hydrophobicity, planarity, protrusion and relative accessible surface area (asa). three of the parameters were found to distinguish the observed sugarbinding sites from the other surface patches. these parameters were then used to calculate the probability of a surface patch being a carbohydrate-binding site. the total score of the properties was used to determine whether the surface patch was a carbohydrate-binding site. the features of the local structures play crucial roles in predicting protein function. to identify the relationship between protein local structure and protein function, the structural and/or physicochemical features can be learned implicitly using machine learning methods, such as the support vector machine (svm) and neural network. the support vector machine uses a linear model to implement nonlinear class boundaries through the input of a number of nonlinear mapping vectors into a high-dimensional feature space. it is based on mathematics theory and has many successful applications in statistical learning fields (vapnik 1998 ). these methods have been confirmed to be able to learn the features of local structures with functional importance. the features can first be investigated in the learning process and used to detect whether these features relate some specific functions. koike and takagi (2004) proposed an svm method to identify protein-protein interaction sites. the profiles of sequentially/spatially neighboring residues, plus additional information, constitute a feature vector, and the interaction site ratios are calculated by svm regression. the predictive performance is evaluated and compared in different quantitative features. cai et al. (2004) proposed an svm algorithm to predict the catalytic triad of the serine hydrolase family. bordner and abagyan (2005) proposed a similar svm to predict protein-protein interfaces. the local surface properties with a combination of an evolutionary conservation signal were used to train the machine on a large nonredundant data set of protein-protein interfaces. an svm learning protocol was provided by bhardwaj et al. (2005) for the prediction of dna-binding proteins. the characteristics, including surface and overall composition, charge and positive potential patches on the protein surface, were derived, and the svm was trained as a classifier to detect the dna-binding proteins. the high accuracy value has been achieved in a large set of testing proteins regardless of their sequence or structure homology. chung et al. (2007) recently exploited the svm approach to detect whether identified potential proteinbinding sites interact with each other. the information related to sequence and structural complementary across protein interfaces were extracted from the pdb. this work also built a pipeline to predict the location of binding sites. neural network the neural network is a learning method which adapts the relationships of neurons; as such, it is a simplified model of the neural processing of the human brain (zhang 2000) . based on the analysis of the both structures and sequences, gutteridge et al. (2003) used a neural network to identify catalytic residues in enzymes. the locations of the active sites were predicted by the neural network output and spatial clustering of the highest scoring residues. in most testing cases, the likely functional residues were identified correctly, as were a number of potentially novel functional groups. ofran and rost (2003) described a neural network to identify protein-protein interfaces from sequences. since the compositions of contacting residues of the interaction sites were believed to be unique, the features of this known interaction sites were used to train the neural network. zhou and shan (2001) trained a neural network to predict protein-protein interactions. their method combines conservation and structural properties of individual residues. fariselli et al. (2002) reported a neural network-based system using information on evolutionary conservation and surface disposition. chen and zhou (2005) also provided a neural network method to predict interface residues in a protein-protein complex. there are also neural network methods for predicting nucleic acid-binding (na-binding) sites. stawiski et al. (2003) presented an automatic neural network approach to predict na-binding proteins, specifically dna-binding proteins. this method uses an ensemble of features extracted from characterization of the structural and sequence properties of large, positively charged electrostatic patches. structural and physical properties of dna provide important constraints on the binding sites formed on the surfaces of the dna-targeting proteins. the characteristics of dna-binding sites may form the basis for predicting dna-binding sites from the structures of proteins alone. tjong and zhou (2007) used a representative set of protein-dna complexes from the pdb to analyze characteristics and to train a neural network predictor of dna-binding sites. the input to the predictor consists of psi-blast sequence profiles and solvent accessibility of each surface residue and 14 of its closest neighboring residues. ferrer-costa et al. (2005) provided a web-based method to detect if a protein structure contains a dnabinding helix-turn-helix (dbhth) motif. the method uses a neural network with no hidden layers, i.e. a linear predictor, to classify whether a protein is dna-binding with the hth motif. the linear predictor was trained on a nonhomologous set of 79 structures of protein chains with a dbhth motif and 490 without the motifs. sodhi et al. (2004) used a neural network to predict metal-binding sites residues in low-resolution structural models. the method involves sequence profile information combined with approximate structural data. several neural networks were proposed to distinguish the metal sites from non-sites and then to detect these functionally important regions. in keil et al. (2004) , the patches of the molecular surface were segmented into overlapping patches. the properties of these patches were calculated based on the physical and chemical properties. a neural network strategy was then used to identify possible binding sites by classifying the surface patches as protein-protein, protein-dna, protein-ligand or nonbinding sites. kuznetsov et al. (2006) applied an svm method to predict dna-binding sites using the features including amino acid sequence, profile of evolutionary conservation of sequence positions, and low-resolution structural information. the results indicate that an svm predictor based on a properly scaled profile of evolutionary conservation in the form of a position specific scoring matrix (pssm) significantly outperforms a pssm-based neural network predictor. such results imply that the combination of the two methods may improve the accuracy. passerini et al. (2006) introduced a two-stage learning method for identifying histidines and cysteines that participate in binding of several transition metals and iron complexes. the first stage is an svm, which is trained to locally classify the binding state of single histidines and cysteines. the second stage is a neural network trained to refine local predictions. the methods use only sequence information by utilizing position-specific evolutionary profiles. statistical methods statistical learning also provides an effective way to link the features of local structures with their functional implication. liang et al. (2003a) provided a supervised learning algorithm, feature, for the automatic discovery of physical and chemical descriptions of protein microenvironments. the calculated feature vectors were used to predict functional motifs based on bayesian inference. the method has also been proposed as an interactive web tool, webfeature, for identifying and visualizing functional sites . bradford et al. (2006) developed a method to predict both protein-protein binding site location and interface type (obligate or non-obligate) using a bayesian network in combination with surface patch analysis. two bayesian network structures, naive and expert, were trained to distinguish interaction surface patches. wang et al. (2007b) proposed a computational method learned by the expectation maximization (em) algorithm, insite, to search for motifs whose presence in a pair of interacting proteins determined which motif pairs have high affinity that would lead to an interaction between proteins. yan et al. (2004) also provided a two-stage method consisting of an svm and a bayesian classifier for predicting the surface residues of proteins that participate in protein-protein interaction. the method exploits the fact that interface residues tend to form clusters in the primary amino acid sequence. in addition, chou and cai (2004) provided a covariant discriminant algorithm to predict active sites of enzyme molecules. the high accuracy of prediction shows the effectiveness of the method. protein-dna interactions are critical for deciphering the mechanisms of gene regulation. yan et al. (2006) presented a supervised machine learning approach for the identification of amino acid residues involved in protein-dna binding sites. a naive bayesian classifier was trained for predicting whether a given amino acid residue is a dna-binding residue based on its identity and the identities of its sequence neighbors. mclaughlin and berman (2003) developed statistical models for discerning protein structures containing the dbhth motifs. the method uses a decision tree model to identify the key structural features required for dna binding. these features include a high average solvent-accessibility of residues within the recognition helix and a conserved hydrophobic interaction between the recognition helix and the second alpha helix preceding it. the adaboost algorithm was used to search the pdb with the aim of identifying the structure containing the motifs with high probability. metal ions are crucial in facilitating the function of a protein. identifying the features of metal binding sites provides crucial knowledge of the function performance of the local structures. because the residues that coordinate a metal often undergo conformational changes upon binding, the detection of binding sites based on simple geometric criteria in proteins without bound metal is difficult. however, aspects of the physicochemical environment around a metal-binding site are often conserved, even when this structural rearrangement occurs. ebert and altman (2008) developed a bayesian classifier using known zinc-binding sites as positive training examples and nonmetal-binding regions as negative training examples. babor et al. (2008) reported an approach that identifies transition metal-binding sites in proteins by combining the decision tree and svm. in the first step, the geometric search of structural rearrangements following metal binding was taken into account by a decision tree classifier. a second classifier based on svms was then used to identify the metal-binding sites. nayal and honig (2006) proposed a comprehensive method to identify drug-binding sites in which 408 attributes were first computed for each cavity, and these were then used to distinguish drug-binding sites by the random forest classification scheme. the cavity properties cover eight broad categories, such as cavity size, cavity shape, hydrophobicity, electrostatics, hydrogen bonding, amino acid composition, secondary structure and rigidity. an interesting method to identify function motifs is based on the graph theory and the network concept. the main methods are listed in table 7 . one subcategory of the method represents the protein structure as a complex network. a node represents a c a of the backbone, and an edge linking two nodes represents the physical distance or the functional relationship between the nodes. greene and higman (2003) viewed protein structures as network systems. the systems are identified to exhibit small-world, single-scale and, to some degree, scale-free properties. using the network model, amitai et al. (2004) identified active site residues. the method transforms a protein structure into a residue interaction graph, where graph nodes represent amino acid residues, and links represent their interactions. the active site, ligand-binding and evolutionary conserved residues are identified typically with a high closeness value, from which the functional residues are filtered out. del sol et al. (2006) also represented a protein liu et al. (2007b) structure as a small-world network and searched the topological determinants related to functionally important residues. the method investigates the performance of residues in protein families. the results indicate that enzyme active sites are located in surface clefts, and hetero-atom binding residues have deep cavities, while protein-protein interactions involve a more planar configuration. wangikar et al. (2003) reported a method for detecting recurring side-chain patterns using an unbiased and automatic graph theoretic approach. the method first lists all structural patterns as subgraphs. the patterns are compared in a pairwise manner based on content and geometry criteria. the recurring pattern is then detected using an automatic search algorithm from the all-againstall pairwise comparison proteins. similarly, huan et al. (2006) defined a labeled graph representation of a protein structure in which edges connecting pairs of residues are labeled by the euclidian distance between the c a atoms of the two residues. based on this representation, a structural motif corresponding to a labeled clique occurs frequently among the graphical representation of the protein structures. the paper further presented an efficient mining algorithm aimed at discovering structure motifs in this setting. in studies on protein structure and function, identifying calcium-binding sites in proteins is one of the first steps towards predicting and understanding the role of calcium in biological systems. calcium-binding sites are often complex and irregular, and it is difficult to predict their location in protein structures. deng et al. (2006) reported a rapid and accurate method for detecting calcium-binding sites. this algorithm uses a graph theory algorithm to identify oxygen clusters of the protein and a geometric algorithm to identify the center of these clusters. a cluster of four or more oxygen atoms has a high potential for calcium binding. a potential calcium-binding position is a clique and can be detected by a clique-detecting algorithm. the high accuracy of prediction shows that the majority of calcium-binding sites in proteins are formed by four or more oxygen atoms in a sphere center with a calcium atom. the above network methods all focus on individual proteins and represent a protein structure a complex network. the specific topology features clearly imply a particular function module (zhang and grigorov 2006; zhang et al. 2007 ). recently, a novel category of networkbased analysis of the protein local structures at the macro level has been proposed (liu et al. 2008) . the similarity of the local structures, specifically the pockets on the protein surface, is mapped to constitute a similarity network. the nodes represent the pockets, and the edges represent the certain similarity relationships among the pockets. the properties of the pocket similarity network are like other complex networks (liu et al. 2008) . the similar pockets are identified by the clusters and community structures, and the special features of the network are helpful in clustering the pockets into similar groups (liu et al. 2007b) , which may imply clusters of structure motifs and correspond to special functional implications (liu et al. 2008) . with the network concept, the pockets can also be used to characterize and predict protein functions by annotating the topology neighbors. in this way, the accuracy of the prediction is better than that with the global structural similarity approach (liu et al. 2007a ). prediction of functions at the cellular level most of the methods used to annotate protein functions that are listed above are based on molecular function at the biological processing level. at the cellular component and location levels, the importance of protein local structure is also critical. in fact, information on the subcellular locations of proteins is important because it can provide useful insights into protein functions as well as how and in what kind of cellular environments they interact with each other and with other molecules. such information is also fundamental and indispensable to systems biology because a knowledge of the localization of proteins within cellular compartments can facilitate our understanding of the intricate pathways that regulate biological processes at the cellular level. from this perspective, the functions of proteins at different levels are strongly inter-related to each other. at the cellular component level, local structures are still crucial in determining the roles of proteins and specific functions. many methods for predicting the subcellular location of proteins have been proposed recently because the location of such proteins in the cell can provide useful insights or clues about their functions . one of the more powerful methods applied in location prediction is based on an important descriptor of the protein sample, i.e. the pseudo-amino acid (pseaa) composition (chou 2001) . this descriptor can be used to represent a protein sequence with a discrete model yet without completely losing the sequence-order information. since the concept of pseaa composition was introduced, various pseaa composition approaches have been developed, all with the aim of improving the prediction quality of protein attributes (gao et al. 2005; zhou et al. 2007a, b; diao et al. 2008; fang et al. 2008; li and li 2008) . the pseaa method has been widely used and extended. a very flexible pseaa composition generator (pseaac) was established which enables users to generate 63 different kinds of pseaa composition. a web server called cell-ploc has recently been developed that allows users to predict the subcellular locations of proteins in various different organisms. pseaa composition and pssm have also been combined in various algorithms to improve the prediction quality for membrane protein type (i.e. memtype-2l: chou and shen 2007a) , enzyme main-functional class and sub-functional class (i.e. ezypred: shen and chou 2007a) and protein subnuclear localization (i.e. nuc-ploc: shen and chou 2007b) . a comprehensive review published recently provides a summary of these topics. in addition to sequence information, local structural information is useful, interesting and important in protein localization function prediction. a quality assessment of the results is necessary at all three levels of function prediction. the predicted functions of proteins can be taken as indicators of the directions to be taken by researchers when carrying out experiments to validate the functions of proteins. many of the computational methods used to annotate protein functions as well as those used to predict functionally important local structures use cross-validation methods to assess the performance of a prediction; these include the independent dataset test, subsampling test and jackknife test (chou and zhang 1995) . however, as elucidated by chou and shen (2008) , of these cross-validation methods, the jackknife test is considered to be the most objective and has been increasingly used by investigators to examine the accuracy of various predictors (zhou 1998; zhou and assa-munt 2001; zhou and doctor 2003; xiao et al. 2005; zhou and cai 2006; chen et al. 2007; shi et al. 2008) . it is important to consider the relationship among the functional terms and the semantic similarity with the aim of avoiding biases in the assessment of functional similarity (liu et al. 2007a ). the global structure similarity-based methods provide a straightforward approach to annotate protein functions. however, since the relationships between structures and functions are so complex, local structure-based methods can be used to predict protein function directly by identifying the local structures carrying out particular functions. laskowski et al. (2005) proposed a novel method of predicting protein function using local three-dimensional templates. the authors build a template database and use four types of templates-enzyme active sites, ligandbinding residues, dna-binding residues and reverse templates-to construct the relationship between templates and functions. ferre et al. (2005) described a method for the functionrelated annotation of protein structures based on the detection of local structural similarity with a library of annotated functional sites. an automatic procedure was used to annotate the function of the local surface regions, and then a sequence-independent algorithm was developed to compare exhaustively these functional patches with a larger collection of protein surface cavities. after tuning and validating the algorithm on a dataset of well-annotated structures, the results are able to provide functional clues to proteins that do not show any significant sequence or global structural similarity with proteins in the current databases. binkowski et al. (2005) provided similar methods to annotate protein functions from the protein surface similarity. pockets are identified by castp from several proteins. these pockets are queried in the pvsoar to locate similar pockets corresponding to annotated proteins. the conservation among the pockets can be detected by the sequence identities and other similarity metrics. tseng and liang (2006) developed a bayesian markov chain monte carlo method for rate estimation of the special substitution rates of the short sequence of local structure. moreover, a method for protein function prediction is presented by surface matching using scoring matrices derived from estimated substitution rates for residues located on the binding surfaces. the method is effective in identifying functionally related proteins that have overall low sequence identity. the method provided by pazos and sternberg (2004) first identifies functional sites in proteins by bridging the local structures and functions, then the functions of a target proteins can be inferred from the similarity of the functional sites in the position-specific scoring matrices. information on the functional importance of local structure can facilitate the annotation of protein function more precisely. george et al. (2005) proposed an effective method to annotate protein function through the use of functional clues of conservation among the catalytic residues. this method improves the precision of annotation significantly. the advantages of predicting protein functions from local structures are based on the fact that such methods can be implemented without any prior homology hypothesis. the methods can be used in proteins in midnight zone without sequence similarity, and local structures often provide concrete and specific functional annotations. to compare the precision and coverage of the global structural similarity and that of local structures, liu et al. (2007a) proposed a novel method to predicted protein from the pockets on the protein's local surface region. the similarity of regional local surface pockets and the global similarity of proteins are all represented by networks. the prediction is based on the network topology. a comparison of the results show that the local-structure-based prediction is better than the global-structure-based prediction (liu et al. 2007a ). in this paper, we have reviewed protein function prediction methods at different levels, i.e. sequence, structure, interaction and integration. we have mainly focused on the importance of local structures and the method used to predict functionally important local structures. in summary, we discuss possible future directions. the interaction between proteins provides high-level information on protein function, especially in various biological processes. although there are thousands of known interactions, a tiny fraction of these are available in precise molecular details. if we are able to examine structural details, systematic representation of the interaction would accurately reflect biological reality. for example, we can predict which part of the structures is most likely to be involved in interaction with other macromolecules, proteins, dna or rna by analyzing the properties of different local patches on the protein surface. the patch analysis, which considers properties of the surface such as flatness, hydrophobicity, charge and, in particular, residue conservation, is effective in identifying protein-protein interaction surfaces and has also been shown to successfully identify dna-binding sites (aloy and russell 2006) . structural systems biology is a very effective approach that combines protein interactions and protein three-dimensional structures. the mechanisms of protein and protein interaction lie in the local structures between the two protein surfaces. from this perspective, structural systems biology provides us with a new direction in the fields of structural biology and systems biology. it combines the key features of the two directions to provide more insight into linking the single protein and systematic interaction between proteins. the relationships between local structures and functions are expected to play important roles in structural systems biology. the computational methods used to bridge the relationship between local structures and functions can be further improved. the community of computational biology has a strong need for comprehensive feature selection in concise and effective ways. in addition, there is still much room for improvement in terms of the accuracy of the methods used to align the features between two local structures. the validation of the functions of structural motifs should also be conducted more carefully and by more reliable biological experiments. recent advances in the field inspired by developments in sequences and structures demonstrate the great potential of such research in protein science in elucidating essential functional roles of the local structures. in our opinion, research aimed at bridging the gaps between local structures and function is still in its infant stage, and further advances in such areas will greatly enhance our ability to study the fundamental properties of proteins at a system-wide level. in other words, we expect to gain deep insight into essential mechanisms of biological systems from both structural and functional perspectives. different methods based on the local similarity, global similarity and interaction require and use different information, and they have different aspects, intentions and advantages. to our knowledge, the function annotation problem is still in its developing period and needs more comprehensive or hybrid approaches. none of the existing methods are likely to be successful in all cases to annotate a protein with its functions correctly and comprehensively. one reason for this is that protein functions not only rely on the sequence and/or folding characteristics, but also on the cell environment, the cycle of the biological processes and other chemical compounds. there are still many difficultto-decipher proteins that researchers have been unable to annotate correctly by any existing method. hence, a sensible strategy is to use different methods to incorporate data from multiple sources and to extensively utilize existing function annotations. future directions include using combinations of different methods at different levels so as to efficiently explore the overall sequences, global structures and local structures and to obtain more information on interactions between the target proteins and others in the cellular context. although computational methods generally cannot directly validate protein functions, the predefined tentative annotations provide valuable information as a basis for further efficient validation experiments. network analysis of protein structures 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b-cell epitopes using amino acid pair antigenicity scale revealing divergent evolution, identifying circular permutations and detecting active-sites by protein structure comparison improvement in protein functional site prediction by distinguishing structural and functional constraints on protein family evolution using computational design prediction of protein cellular attributes using pseudo amino acid composition (erratum: ibid structural bioinformatics and its impact to biomedical science a novel approach to predict active sites of enzyme molecules predicting protein-protein interactions from sequences in a hybridization space memtype-2l: a web server for predicting membrane proteins and their types by incorporating evolution information through pse-pssm recent progresses in protein subcellular location prediction cell-ploc: a package of web-servers for predicting subcellular localization of proteins in various organisms prediction of protein structural classes binding mechanism of 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for active sites and functional similarity twilight zone of protein sequence alignments the funcat, a functional annotation scheme for systematic classification of proteins from whole genomes detection of protein three-dimensional side-chain patterns: new examples of convergent evolution a structural perspective on protein-protein interactions computational methods of analysis of protein-protein interactions integrating structure, bioinformatics, and enzymology to discover function-bioh, a new carboxylesterase from escherichia coli a new method to detect related function among proteins independent of sequence and fold homology structure and mechanism of the m2 proton channel of influenza a virus predicting enzyme function from sequence: a systematic appraisal network-based prediction of protein function ezypred: a top-down approach for predicting enzyme functional classes and subclasses nuc-ploc: a new web-server for predicting protein subnuclear localization by fusing pseaa composition and psepssm pseaac: a flexible web-server for generating various kinds of protein pseudo amino acid composition using pseudo amino acid composition to predict protein subcellular location: approached with amino acid composition distribution protein structure alignment by incremental combinatorial extension (ce) of the optimal path siteengines: recognition and comparison of binding sites and protein-protein interfaces structural alignment of protein-dna interfaces: insights into the determinants of binding specificity hierarchical protein structure alignment using both secondary structure and atomic representations identifying structural motifs in proteins predicting metal-binding site residues in low-resolution structural models annotation in three dimensions. pints: patterns in non-homologous tertiary structures finding functional sites in structural genomics proteins a model for statistical significance of local similarities in structure annotating nucleic acid-binding function based on protein structure analysis and prediction of carbohydrate binding sites gene ontology: tool for the unification of biology displar: an accurate method for predicting dna-binding sites on protein surfaces using a library of structural templates to recognise catalytic sites and explore their evolution in homologous families estimation of amino acid residue substitution rates at local spatial regions and application in protein function inference: a bayesian monte carlo approach structure-based prediction of dna-binding sites on proteins using the empirical preference of electrostatic potential and the shape of molecular surfaces global protein function prediction from protein-protein interaction networks tess: a geometric hashing algorithm for deriving 3d coordinate templates for searching structural database. application to enzyme active sites virtual screening for finding natural inhibitor against cathepsin-l for sars therapy insite: a computational method for identifying protein-protein interaction binding sites on a proteome-wide scale functional sites in protein families uncovered via an objective and automatic graph theoretic approach predicting protein function from sequence and structural data prediction of protein function from protein sequence and structure assessing annotation transfer for genomics: quantifying the relations between protein sequence, structure and function throng traditional and probabilistic scores prediction of protein-protein interactions: the capri experiment, its evaluation and implications using complexity measure factor to predict protein subcellular location a two-stage classifier for identification of protein-protein interface residues predicting dna-binding sites of proteins from amino acid sequence an accurate, sensitive, and scalable method to identify functional sites in protein structures fatcat: a web server for flexible structure comparison and structure similarity searching lga-a method for finding 3d similarities in protein structures neural networks in optimization similarity networks of protein binding sites tm-align: a protein structure alignment algorithm based on tm-score discovering functions and revealing mechanisms at molecular level from biological networks prediction protein homo-oligomer types by pseudo amino acid composition: approached with an improved feature extraction and naive bayes feature fusion gene function prediction using labeled and unlabeled data protein domain annotation with integration of heterogeneous information sources an intriguing controversy over protein structural class prediction some insights into protein structural class prediction predicting protease types by hybridizing gene ontology and pseudo amino acid composition subcellular location prediction of apoptosis proteins interaction-site prediction for protein complexes: a critical assessment prediction of protein interaction sites from sequence profile and residue neighbor list improved prediction of subcellular location for apoptosis proteins by the dual-layer support vector machine using chou's amphiphilic pseudo-amino acid composition and support vector machine for prediction of enzyme subfamily classes fast: a novel protein structure alignment algorithm acknowledgments this work was supported by the national natural science foundation of china (nsfc) under grant no. 10631070 and no. 60503004. lyw and xsz are also supported by the grant no. 5039052006cb from the ministry of science and technology, china. the research was also supported by nsfc-jsps collaborative project no. 10711140116. the authors are grateful to the anonymous referees as well as editors for comments and for helping to improve the earlier version. we recognize that this review is far from comprehensive, and we apologize for any papers related to the subject that were not mentioned. key: cord-338485-4zqeq1se authors: aiking, harry; de boer, joop title: the next protein transition() date: 2018-07-27 journal: trends food sci technol doi: 10.1016/j.tifs.2018.07.008 sha: doc_id: 338485 cord_uid: 4zqeq1se background: meeting the un sustainable development goals requires a relatively rapid transition towards a circular economy. therefore, a multidisciplinary perspective is required to sketch why a transition from diets based primarily on animal proteins towards diets based primarily on plant proteins products is extremely urgent for both food security and sustainability. scope and approach: this review starts out by identifying ecological, economic and social aspects of sustainable food consumption. subsequently, it is argued how protein supply is underlying and linking the top-3 of anthropogenic impacts based on the planetary boundaries concept, i.e. 1) biodiversity loss, 2) nitrogen cycle acceleration, and 3) carbon cycle acceleration (resulting in climate change). these environmental impacts associated with current western food consumption need to be reduced urgently. in order to address the inefficiencies inherent to current dietary patterns, therefore, a ranked list of more sustainable options is proposed, based on their order of magnitude. addressing consumers, industry, and governmental stakeholders plus cultural aspects, challenges and options are sketched. key findings and conclusions: clearly, a dietary transition from primarily animal towards plant protein products is required. fortunately, new dietary guidelines are increasingly taking sustainability into account and the contours of a diet transition are slowly emerging. in september 2015, the united nations (un) general assembly adopted the 2030 agenda for sustainable development. as an integral part, 17 sustainable development goals (sdgs) present a novel approach and incentive to prioritize and integrate a number of pressing issues, including food security, food sustainability, climate change, and the broader aim of staying within planetary boundaries. it is evident that a transition towards a circular economy is urgently required to meet the sdgs. since many of the planetary boundaries are strongly interlinked with protein supply, the goal of the current review is to sketch novel trends, resuming where the preceding one (aiking, 2011) left off. after summarizing and evaluating the impacts of protein production by sustainability pillar, options to reduce these impacts will be identified and ranked with the help of a framework for priority setting. the role of stakeholders including governments, industry and consumers will be highlighted. the 17 sdgs form a perfect starting point for this review. among the 17 sdgs adopted (un, 2015) , food security (sdg 2) evidently ranks higher than climate action (sdg 13) . that is only natural, because in 2017 the global population reached 7.5 billion, but even in the medium growth scenario the un (2017) project a world population of 8.5 billion by 2030 and almost 10 billion by 2050. furthermore, increasing urbanization and incomes will result in diets containing more animal products (fao, 2017a) . the fao project a 60% food demand increase by 2050 (alexandratos & bruinsma, 2012) , while many others project a doubling (see : tomlinson, 2013) . during the previous millennium, crop yield increases more or less kept up with demand, but yield increases are slowing down (ray, mueller, west, & foley, 2013) . in contrast with the fao's optimistic view, therefore, several financial institutions foresee that food prices may rise rapidly during the next decades, which might fuel conflict and migration (natalini, jones, & bravo, 2015) . kpmg, a global auditor, in fact forecast a 70-90% food price increase by 2030 (de boer & van bergen, 2012 . analysis of the trends since the previous review will highlight changes at the production and consumption side. food production is accelerating, and so are its environmental impacts. still, smallholders produce half the world's food (johnson, dudley, & alexander, 2017) , but development financing remains a bottle-neck (fao, 2017a) . on the consumption side, there is a dual diet transition in progress. booming economies are increasing their consumption of meat (china) and dairy (india) like western europe did half a century ago (grigg, 1995) . in europe, a reverse transition away from animal products is about to break through (geijer, 2017) . with respect to sustainability, this is none too early, for there is a growing consensus that animal protein has disproportionate environmental impacts, particularly when produced in intensive production systems employing massive use of feed crops (aiking, 2014; mcmichael, powles, butler, & uauy, 2007; smil, 2001; steinfeld et al., 2006; westhoek et al., 2011) . in 2009, rockström et al. quantified a dozen planetary boundaries (rockström et al., 2009) , including biodiversity loss, nitrogen cycle disruption, climate change (i.e. carbon cycle disruption), phosphate cycle disruption, ocean acidification, land-use change, freshwater use and stratospheric ozone depletion. subsequently, they were ranked (aiking, 2011) and put in the above order based on their current transgression (aiking, 2014) . taken together, the rockström boundaries can be seen as the carrying capacity of planet earth, in terms of safe rates of natural resource use and emission of pollutants. human society may be the primary target of phosphate depletion, butwithout exception -earth's ecology is targeted by each of the eight anthropogenic impacts listed above, either by pollution (such as acidifying emissions), or by resource appropriation (such as taking freshwater and land away from natural habitats). in fact, eutrophication, climate change, ocean acidification, hunting, fishing, land conversion, and freshwater use all contribute to biodiversity loss in unison, explaining its high ranking. with respect to biodiversity loss, intensive livestock farming plays a major role both through the immense land area in use for feed crops (over 400 million hectares; one third of all arable land) (tilman, cassman, matson, naylor, & polasky, 2002; van zanten, mollenhorst, klootwijk, van middelaar, & de boer, 2016) and by ammonia emissions from livestock manure (erisman, sutton, galloway, klimont, & winiwarter, 2008; fao, 2017a; machovina, feeley, & ripple, 2015) . the magnitude of livestock's impact on biodiversity was illustrated by zalasiewicz (2016) : "we humans have obviously left our mark on the earth's biological landscape as well. in particular, our speciesa very minor player amid the planet's biota even a few thousand years agois now the dominant predator on land and sea. we appropriate roughly a quarter of the earth's total biological production for our needs. as a result, we make up about a third of the mass of all land vertebrates (based simply on body weight), and the handful of species we have engineered to become our food make up most of the other two thirds. wild animals, pushed to the margins, constitute 5 percent or less." in other words, the ratio wildlife: humans: farm animals = 5: 30: 65 (by weight). considering the projected doubling in meat demand from 229 million tons in 2000 to 465 million tons in 2050 (steinfeld et al., 2006) , a rough estimate shows that land vertebrates living in the wild may be reduced to 1-2 percent by 2050. not surprisingly, even the intermediate ssp2 scenario by unccd foresees severe deforestation and land conversion in south asia, middle and south america, and disastrous damage to tropical forests in sub-saharan africa (johnson et al., 2017, p. 112) . half the world's food is produced by smallholders, for example, the share of farms smaller than 2 ha is 97% in china, 85% in south asia, 68% in the near east and north africa (fao, 2017b, p. 54). nevertheless, the associated economic power does not rest with smallholders. lack of land ownership and availability of capital are important bottlenecks driving them out, and critical parts of the food system are becoming concentrated in fewer hands (fao, 2017a) . a few powerful trade corporations, supermarket chains and financial institutions play important parts. animal protein products such as meat and dairy are important to them from an economic perspective, but when employing intensive production systems these are wasteful of plant protein from an environmental point of view and inherently, therefore, wasteful of the resources required to grow feed crops, such as land, water, phosphate and fuel. more and more, food and feed crops are competing for these increasingly scarce resources. in fact, by 2030 "peak oil" has been foreseen. by the same year, "peak phosphate" has been forecast (cordell, drangert, & white, 2009 ). in addition, "peak water" has been defined (gleick & palaniappan, 2010) , and a globally operating bank projected a 40% freshwater deficit by 2030 (crowder, fumasi, soccio, twomey, & williamson, 2016) . finally, the pressures on land resources are increasing steeply, also leading to compaction by heavy equipment, erosion, and deteriorating soil quality (fao, 2017a; johnson et al., 2017; pimentel & burgess, 2013) , with negative feedback on further production increases. in light of the above it is no wonder that kpmg, a global auditor, forecast a 70-90% food price increase by 2030 (de boer & van bergen, 2012) , because every little thing seems to converge then. since it is their core business, it stands to reason that financial institutions, primarily, issue such a warning of impending danger. interestingly, however, the latter is confirmed by an unexpected ally. unprecedentedly, in their ar5 (5th assessment report), the ipcc incorporated a chapter on h. aiking, j. de boer trends in food science & technology xxx (xxxx) xxx-xxx food security (porter et al., 2014) , showing a diagram with a time series of food price, cereal price, and crude oil indexes (fig. 1 ). as shown, these indexes behaved in parallel, and initially they were rather constant, fluctuating only slightly. in 2006 an increase started, with spikes in 2007-2008 and 2011-2012. apart from increased volatility, extrapolation of the baseline shows roughly a doubling of the food price index from about 200 in 2012 to about 400 in 2030, in line with the kpmg forecast. the volume and type (animal vs. plant protein; intensive vs. extensive production systems) of protein production have evident impacts on society, primarily on food security. moreover, undernutrition is often caused by a lack of protein, rather than a lack of calories. in children under 5 years of age, protein undernourishment is a leading cause of death (mcleod, 2011, p. 6) . although hunger and famines are roaming in developing countries primarily, food insecurity in developed countries should not be underestimated. for example, browning et al. note that "food affordability is a critical issue that is likely to rise in prominence. many millions of people currently struggle to be able to afford to eat enough, let alone to eat well and healthily. there are over 4 million people in the uk currently living in food poverty." (browning, hirsch, & lang, 2013) . with respect to poverty alleviation and rural development, of course, sustenance farming by smallholders (johnson et al., 2017) and smallholder inclusion by multinational enterprises (sjauw-koen-fa, blok, & omta, 2016) constitute important aspects. in addition, land grabbing and increasing conflict take their toll (johnson et al., 2017) . the situation in africa is particularly alarming, because most of the world's population growth in the next few decades will take place there. such will no doubt lead to unprecedented migration and urbanization (johnson et al., 2017) . the latter generally leads to diet change entailing increased proportions of processed foods and animal products. these are associated with malnourishment, obesity and related diseases such as diabetes and certain types of cancer (mcmichael et al., 2007) . in addition to health impacts deriving from consumption, there are health impacts resulting from the ever-increasing production of animal protein. the latter include emerging zoonotic diseases, such as avian influenza, human cjd (creutzfeldt-jakob disease) related to bovine bse (bovine spongiform encephalopathy, or mad cow disease), q fever, ehec (enterohemorrhagic e. coli), sars (severe acute respiratory syndrome), mers (middle east respiratory syndrome), etc. (browning, pennington, & rooker, 2009; world bank, 2010) . primarily due to indiscriminate use of antibiotics in livestock farming, increasing microbial antibiotics resistance, such as by mrsa (methicillin-resistant staphylococcus aureus), is another effect of intensive animal protein production on human health requiring rapid improvement. of particular concern is the fact that by 2030, antimicrobial consumption has been projected to double in countries such as china and india (van boeckel et al., 2015) , and that globally two-thirds of the estimated future growth of usage of antimicrobials is expected to occur within the animal production sector, with use in pig and poultry production predicted to double (fao, 2016) . another societal issue may be animal welfare (fao, 2017c; godfray & garnett, 2014) , which is stronger in western than in many other countries. finally, politicians anywhere tend to spare farmers and voters. consequently, environmental decision making is slow. the pivotal role of protein is becoming clear in several ways. with respect to food security, the metric of choice is generally calories per capita, even though undernourishment with minerals and vitamins is taken into account (fao, 2017c). however, as argued above, malnutrition is often caused by a lack of protein, rather than a lack of calories, and a leading cause of death in children under 5 years of age (mcleod, 2011, p. 6) . with respect to food sustainability, climate change is often considered paramount. however, about a dozen of the most important environmental issues were listed by rockström et al. (2009) , who identified and quantified several boundary values that should not be transgressed. subsequently, these were ranked by aiking (2014) , showing that food production is an important driver of all of them, without exception. moreover, he argued that rockström et al.'s top three (1. biodiversity loss, 2. nitrogen cycle disruption, 3. carbon cycle disruptionleading to climate change; transgressed by factors of > 10, 3.45 and 1.1-1.5, respectively) are strongly interlinked by protein production, with nitrogen cycle acceleration in a pivotal role. by the increasing amounts of animal proteins produced in intensive systems, i.e. with high input of feed crops, the global nitrogen use efficiency keeps dropping, and merely 17% of the nitrogen from fertilizers ends up in human mouths (erisman et al., 2008) . the remainder is lost to the environment, with increasing impacts on terrestrial biodiversity (resulting from ammonia emissions due to degradation of livestock manure) (sutton et al., 2011) , aquatic biodiversity (via eutrophication caused by manure and fertilizer run-off) (carstensen, andersen, gustafsson, & conley, 2014) , climate (due to the tremendous amounts of energy required for the production of nitrogen fertilizers) (goucher, bruce, cameron, koh, & horton, 2017; smil, 2001 ) and health (mcmichael et al., 2007) . conversion losses from plant to animal protein by livestock metabolism are 85% on average, of both (reactive) nitrogen and (embedded) energy (smil, 2000) . furthermore, intensive animal protein production requires 10-1000 times more water than plant protein (aiking, 2011; smil, 2000) . in addition to pollution by reactive nitrogen (see above), impacts on biodiversity result from resource appropriation (land and freshwater) and land-use change (johnson et al., 2017) . considering the anticipated trends in animal protein demand in the next few decades, continued increase in all of these impacts will be inevitable. in the next three decades, more food should become available in absolute terms. the good news is that there is a lot of slack in the food system. in fact, even food price increases may have a positive side in that they will reduce food waste, as well as demand of animal protein. furthermore, just following dietary guidelines would reduce ghg emissions significantly, in addition to being much healthier than current dietary habits (macdiarmid et al., 2011) . even cutting the climate change impact in half is feasible by adopting a culturally acceptable and cheap diet (van dooren, tyszler, kramer, & aiking, 2015) . nevertheless, the un fail to provide direction and initiate a concerted action, so it is up to stakeholders at the national level, i.e. governments, industry and consumers. in order to make progress swiftly, some priority setting is required. for the food system to become more sustainable it should be made more efficient. from early on it was noted that losses and waste should be reduced all along the global food supply chain and in the household, in particular (eberle & fels, 2016; fao, 2017a; kummu et al., 2012; parfitt, barthel, & macnaughton, 2010; ventour, 2008) . the consumer phase is important primarily because roughly one third of purchases in western supermarkets is discarded before consumption (ventour, 2008) . as a first step towards developing a framework to improve western consumption, alexander et al. (2017) established that the magnitude of over-consumption of food, however, surpasses that of food wasted in the household. within western over-consumption as such, protein is evidently more important than fats and carbohydrates, both numerically and environmentally, because the average protein intake in many western countries is 150-200% of recommended values (aiking, 2014; de boer & aiking, 2018) . therefore, reducing consumption of protein and calories should be ranked as first and second options, respectively, for turning current western consumption in a more sustainable direction by reducing household food waste should be in third place (table 1) . finally, replacing animal protein products (meat and dairy) with plant protein products (analogues and/or whole foods such as beans and nuts) should be ranked fourth, because their inputs and impacts are somewhat reduced, but not averted. in summary, table 1 provides a ranked list of potential improvements of current western consumption patterns. it has to be kept in mind that there is a dual protein transition in the world. in booming economies animal products are increasingly consumed, and in western countries a slow decrease set in. therefore, the appearance of the above table strongly depends on the dietary patterns extant in the countries under consideration. ethical arguments such as equity, animal welfare, and application of fresh fish and meat resources as pet food (de silva & turchini, 2008; okin, 2017) have cultural dimensions, as well. at any rate, in addition to quantitative impacts, qualitative aspects relating to feasibility also play a part, and will be addressed in the consumer section below. next to consumption-related improvements, the resource use and pollution associated with production should also decrease along the whole chain. to meet that goal, reducing losses, waste and other emissions are in order. in the production chain, the importance of nitrogen fertilizer has become evident once more, because in a wheat-to-bread supply chain it was shown that the ammonium nitrate fertilizer used in wheat cultivation contributed no less than 43 per cent of greenhouse gas emissions in a full life cycle assessment of a loaf of bread (goucher et al., 2017) . besides, the importance of food-demand management for climate mitigation had been argued explicitly, confirming that healthy and sustainable food largely go hand-in-hand (bajzelj et al., 2014) . in that respect it is interesting to note that the dutch gpa (green protein alliance) formulated some smart goals concerning the protein transition. according to this alliance of industry, academia, ngos and government, the current dutch consumption ratio of animal protein: plant protein of 60: 40 should be changed, via 50: 50 by 2025, into 40: 60 by 2050 (green protein alliance, 2016). within their programme for a circular economy (ministry for the environment & ministry of economic affairs, 2016), the dutch government instated transition teams in cooperation with industry, academia and ngos. even more interesting, therefore, is the fact that the gpa goals were adopted in full in the transition agenda food & biomass, also aiming for 10-15% total protein intake reduction per capita by 2050 (transition team biomass & food, 2018). consumer responses to a potential protein transition have only been studied in a number of (developed) countries, including australia (hoek, pearson, james, lawrence, & friel, 2017) , the united states (de boer, de witt, & aiking, 2016), member states of the european union (de boer & aiking, 2018), and switzerland (siegrist, visschers, & hartmann, 2015) . the studies show that most consumers in these countries are not yet prepared to make the associated diet changes (for a detailed review see also : hartmann & siegrist, 2017) . moreover, policy-makers in government, industry, and ngos are often reluctant to engage with this topic (laestadius, neff, barry, & frattaroli, 2014; lang, 2012 ). an additional table 1 improving current western dietary patterns (in descending order of magnitude). reducing over-consumption of protein 2 reducing over-consumption of calories 3 reducing food waste in the household 4 replacing animal protein with plant protein (analogues and/or whole foods) fig. 2 . targets of change strategies at the levels of diets, dishes, ingredients and bites. h. aiking, j. de boer trends in food science & technology xxx (xxxx) xxx-xxx difficulty is that food choice is a seemingly simple, but in fact very complicated behaviour that is influenced by many interacting factors, which cannot be captured by monodisciplinary, single-level research (köster, 2009) . key to a transition is reducing meat-centred meals and/ or replacing them with meals that are based on alternative protein options (e.g. pulses, vegetables, nuts, mushrooms, algae, seaweed, animal by-products, insects). such a change needs to take place at different levels of detail and context. as described in the literature (meiselman, 2009 ) and depicted in fig. 2 , these levels range from diets (i.e. the broad set of food items that is accepted by a population over a period of time), dishes (i.e. food items accepted on a plate in combination with each other), to ingredients (i.e. food items accepted as separate entities) and bites (i.e. single food exposures). each of these levelsand their interactionsshould be considered in order to develop and evaluate realistic, nutritionally, environmentally and culturally acceptable changes in dietary patterns of populations and subpopulations (masset et al., 2014; van dooren, marinussen, blonk, aiking, & vellinga, 2014) . fig. 2 demonstrates that alternative protein options should become an important part of the diet and consumers' repertoire of dishes as well as their set of appealing protein ingredients. various recent initiatives are exploring this approach. one of the few examples of an ambitious diet change programme was initiated by gastronomic, nutritional, and environmental specialists in the nordic countries, who developed a new regional (nordic) diet in accordance with healthy dietary recommendations, such as more calories from plant foods and fewer from meat, and more locally grown food in season, including food from the sea and the wild countryside (mithril et al., 2012) . by presenting the new diet under the nordic label, which already had positive connotations in other domains, the initiators tried to connect to lifestyle changes that are collective and highly visible (byrkjeflot, pedersen, & svejenova, 2013) . from a sustainability perspective, it has been calculated that the new nordic diet has, in theory, substantial environmental and socioeconomic advantages (saxe, 2014 ). an early study among a sample of overweight consumers found that the diet had high eating acceptance (tasty) but low practical acceptance, which the researchers attributed to perceived high price, low product availability, and time-consuming cooking procedures (micheelsen, havn, poulsen, larsen, & holm, 2014) . the latter might be mitigated through the development of more convenient and cheaper versions of the diet as well as by providing guidance regarding meal ideas, meal planning, and cooking skills. hence, the nordic initiative demonstrates that to effectively stimulate diet changes, the practical aspects at the level of dishes and ingredients are of utmost importance. moreover, other research shows that consumers may have the impression that they are unable to fit healthy eating into what they see as their complex lives, with constraints of time, work and social pressures (macdiarmid, loe, kyle, & mcneill, 2013) . these barriers make it important to ensure that the recommended changes do not depend solely on individual decisions but become an integral part of regional social and cultural processes with which individuals can identify themselves. at the level of protein ingredients and their interactions with dishes and bites, there is an increasing interest in mixed food concepts in which part of the protein consumed is from plant-based origin and the rest from animal-based origin. these concepts include flexitarian lifestyles, mixed (instead of meat-centred) dishes, and hybrid protein products. the term "flexitarian", coined in 1992 (glowka, melancon, & wyckoff, 2004) , is a union of the words "flexible" and "vegetarian", which has been used by individuals who saw themselves as vegetarians occasionally eating meat. from an alternative perspective, however, the term has recently been reframed to highlight the lifestyles of moderate meat-eaters who do not eat meat every day (de bakker & dagevos, 2012) . on a meat-free day, their dishes may involve a replacement of meat with a vegetarian version of a meat product, such as a veggie burger, or a variety of vegetables and legumes (schösler, de boer, & boersema, 2012) . a distinct but overlapping category of moderate meat-eaters is focussing more on smaller portions of meat, which may be of a better quality, such as organic or free-range meat (heerwagen, andersen, christensen, & sandøe, 2014; de boer, schösler, & aiking, 2014) . moderate meat-eaters may also like mixed dishes, based on recipes that involve a partial replacement of meat. for regular meat-eaters, however, a partial replacement may be associated with a loss of sensory appeal, which might be compensated by changes at the level of dishes, for instance, by stimulating sensory properties using the flavour of spices (spencer & guinard, 2018) . this type of research also shows that the combination of the protein ingredient and the rest of the dish has a large impact on consumer responses (elzerman, hoek, van boekel, & luning, 2011) . changes at the level of protein ingredients, interacting with bites, may include the development of hybrid products, such as beef burger and sausage analogues (neville, tarrega, hewson, & forster, 2017) . although these products may not be appreciated by "purists" who prefer authentic sources of either meat or environmentally-friendly proteins, such as lentils and seaweed, a hybrid meat product can be acceptable to consumers who are lowly involved with food (de boer, schösler, & boersema, 2013) . however, some pitfalls should not be ignored. although their current market share is very small, meat and dairy analogues are strongly on the rise (geijer, 2017; schmidinger, 2012 ). yet, their environmental impact reduction is limited compared to consumption reduction, and further improvement of their environmental performance is clearly required (smetana, mathys, knoch, & heinz, 2015; van mierlo, rohmer, & gerdessen, 2017 ). another point is that food choices may be influenced by fads and fashions. for instance, launching new products that contain a high level of proteins may be useful in professional sports, but for the general public experts consider this as a typical food fad (seidell & halberstadt, 2018) . the results of the recent initiatives can be translated in some strategic recommendations: increase awareness of the impacts of animal-based protein on the environment, the urgency of this issue and the availability of solutions, but take into account that science-based health and sustainability arguments in favour of a diet change do not sufficiently reach consumers or are too difficult for them to comprehend (de boer & aiking, 2017) . seek ways in which culinary, health and environmental aspects can complement each other. a regional approach may be necessary to bring gastronomic, nutritional, and environmental specialists together and to involve a range of commercial stakeholders, such as farmers, food processors, retailers, restaurant owners and new kinds of food networks. pay attention to mixed strategies and flexible options at the levels of diets, dishes, ingredients and bites. focus on alternative protein options and take into account that these should become an important part of consumers' diet, their repertoire of dishes as well as their set of appealing protein ingredients. develop change options that build on the familiar culinary principles of variety, balance, and moderation, as well as a moderate amount of novelty, and create an affordable diet that is convincingly healthier and more sustainable but not much different from the current one, for instance, due to correspondences in meal ideas, ingredient sourcing, culinary skills and social expectations. support consumers who find it difficult to adopt the diet and who need help with 'protein' literacy. ensure that the recommended changes do not depend solely on individual decisions but become an integral part of regional social and cultural processes with which individuals can identify themselves. the sdgs hold great potential in prioritizing and integrating the different dimensions of food production and consumption, which is urgently required. in order to meet the rapidly increasing demand, food production grew and intensified in parallel (godfray & garnett, 2014; tilman, balzer, hill, & befort, 2011; tscharntke et al., 2012) . so, for a h. aiking, j. de boer trends in food science & technology xxx (xxxx) xxx-xxx number of reasons, the current food system is not sustainable. first, food production is appropriating increasingly unsustainable amounts of natural resources (e.g. land, water, energy and nutrients) with harmful impacts on ecology, economy and society (also known as the three pillars of sustainability). second, food production leads to pollution (e.g. emissions of reactive nitrogen compounds, greenhouse gases, pesticides, antibiotics and biological agents) with unsustainable impacts on biodiversity (ecology) as well as on human health (society). third, intensive production leads to erosion and decreasing soil quality, with negative feedback on further increases (economy). finally, access to food is inequitable, leading to hunger and malnutrition, on the one hand, and to excessive food wastage, over-consumption and obesity, on the other hand. thus, food consumption does not have impacts on human health, exclusively, but huge overall impacts on ecology, economy and society. following the planetary boundaries concept (rockström et al., 2009; steffen et al., 2015) , aiking (2014) argued that their top-3 (1. biodiversity loss, 2. nitrogen cycle acceleration, and 3. carbon cycle acceleration) are strongly interlinked by protein production, with nitrogen cycle acceleration in a pivotal role. european protein supply (= production + importexport) in is over 100 g/capita/day (de boer & aiking, 2018) , which is almost twice the dri (daily reference intake). actual consumption may be 25-30% lower than supply, so conservatively reducing the average eu protein consumption by one third was recommended for health and sustainability as long as one decade ago (aiking, de boer, & vereijken, 2006) . in order to make western food consumption patterns more sustainable, a number of options were identified and ranked in order of decreasing magnitude. first, over-consumption of protein should be reduced, because ecologically it is the most costly nutrient and the average consumption in many western countries is over 150% of recommended values (de boer & aiking, 2018) . second, over-consumption of calories should be reduced, because health-wise it is the most costly nutrient, and because over-eating was found to be at least as large a contributor to food system losses as consumer food waste . third, in the whole chain, food waste should be reduced, in particular, consumer food waste in western countries (on average, about one third of supermarket purchase value). fourth, animal (protein) products (such as meat and dairy) should be replaced with plant (protein) products, because of the low conversion efficiency (ca. 15%) from crop protein to animal protein in intensive production systems involving massive use of feed crops (aiking, 2011; smil, 2000) . moreover, smil summarized: "thinking about the road ahead we must recognize several fundamental realities. solutions will not come from voluntary meatless diets, mass production of mock meat (transformed plant proteins) or muscle tissues cultured in bioreactors. substituting meat intakes by consumption of other high-protein animal foodstuffs is of marginal help. at the same time, meat production based only on truly sustainable grazing, feeding of forages rotated with food crops, and maximum use of crop and processing residues is inherently limited and although, once it is reoriented toward producing less beef and more pork and chicken, it could supply a surprisingly large share of today's meat consumption (as i will show, close to 70% of 2010 supply) it will not be able to satisfy global demand anticipated for 2030 and even less so for 2050. innovations and productivity improvements alone cannot prevent further increases in already significant environmental burden of meat production and to reduce them we will also need to moderate our meat consumption." (smil, 2014, p. 68) . we couldn't agree more. the problem is that few of the stakeholders involvedgovernment, industry, consumersare inclined to consumption reduction, or even promoting it. in fact, they are looking to one another to take the initiative (roberts, crossley, & barling, 2013) . fortunately, some consumer groups seem to be sensitive to flexible diet concepts offering a moderate amount of novelty, as shown above in the section on consumers. as mentioned before, it is important to seek ways in which culinary, health and environmental aspects can complement each other (de boer & aiking, 2017) . in that respect, it is important to note that dietary guidelines are increasingly addressing sustainability in addition to healthy nutrition. in 2015-2016, these included sweden, uk, germany, and the netherlands, as was shown in two reviews (behrens et al., 2017; fischer & garnett, 2016) . in the usa, the 2015 dietary guidelines advisory committee's recommendations to the same effect (dgac, 2015) were not ratified by the us senate. therefore, important sustainability issues (sabaté, harwatt, & soret, 2016) were omitted from the latest us guidelines. that is unfortunate, because health may be key to entice consumers to drop their conservative attitudes and progress towards a diet transition, which would benefit both the environment, and their health via reduction of obesity and sequelae (hadjikakou, 2017; you & henneberg, 2016) . in fact, there is increasing evidence that nutritionally healthy dietsi.e. according to dietary guidelinesgenerally go hand in hand with sustainable diets (buchner et al., 2011; mason & lang, 2017) . in that respect, a diet containing less animal products would benefit both public health and environment, but completely doing away with animal protein products would not be optimal for either (van kernebeek, oosting, van ittersum, bikker, & de boer, 2016) . western diets do not have to go completely vegetarian, therefore, but reducing total 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environmental impact of our food consumption pattern while retaining its nutritional value global food supply: land use efficiency of livestock systems the food we waste the protein puzzle -the consumption and production of meat, dairy and fish in the european union towards a one health approach for controlling zoonotic diseases meat in modern diet, just as bad as sugar, correlates with worldwide obesity: an ecological analysis a history of layers: what mark will we leave on the planet? this work reported here is based in part on results from the protein foods, environment, technology and society (profetas) research program funded by the netherlands organisation for scientific research (nwo). declarations of interest: joop de boer: none; harry aiking is a member of the scientific advisory committee of the european natural soyfoods association (ensa). key: cord-330337-d41imvo7 authors: basu, souradip; mukhopadhyay, suparba; das, rajdeep; mukhopadhyay, sarmishta; singh, pankaj kumar; ganguli, sayak title: impact of clade specific mutations on structural fidelity of sars-cov-2 proteins date: 2020-10-20 journal: biorxiv doi: 10.1101/2020.10.20.347021 sha: doc_id: 330337 cord_uid: d41imvo7 the sars-cov-2 is a positive stranded rna virus with a genome size of ~29.9 kilobase pairs which spans 29 open reading frames. studies have revealed that the genome encodes about 16 non-structural proteins (nsp), four structural proteins, and six or seven accessory proteins. based on prevalent knowledge on sars-cov and other coronaviruses, functions have been assigned for majority of the proteins. while, researchers across the globe are engrossed in identifying a potential pharmacological intervention to control the viral outbreak, none of the work has come up with new antiviral drugs or vaccines yet. one possible approach that has shown some positive results is by treating infected patients with the plasma collected from convalescent covid-19 patients. several vaccines around the world have entered their final trial phase in humans and we expect that these will in time be available for application to worldwide population to combat the disease. in this work we analyse the effect of prevalent mutations in the major pathogenesis related proteins of sars-cov2 and attempt to pinpoint the effects of those mutations on the structural stability of the proteins. our observations and analysis direct us to identify that all the major mutations have a negative impact in context of stability of the viral proteins under study and the mutant proteins suffer both structural and functional alterations as a result of the mutations. our binary scoring scheme identifies l84s mutation in orf8 as the most disruptive of the mutations under study. we believe that, the virus is under the influence of an evolutionary phenomenon similar to muller’s ratchet where the continuous accumulation of these mutations is making the virus less virulent which may also explain the reduction in fatality rates worldwide. what started off as an unusual outbreak of pneumonia in wuhan china has reached the farthest corners of the world as a global pandemic. till date there are over 60 million people affected by this novel strain of severe acute respiratory syndrome (sars) virus and has been named as sars-cov-2 with the disease named as covid-19. genomic structures and phylogenomic studies reveal that the causal agent of covid-19 belongs to genera beta coronavirus. it is similar to the human beta coronaviruses (sars-cov-2, sars-cov and mers-cov) but there are important differences at the genotypic and phenotypic levels which ultimately influence their pathogenesis. the sars-cov-2 contains a single stranded positive sense rna encapsulated in a nucleoprotein matrix. the rna genome of sars-cov-2 consists of approximately 29,800 nucleotides, encoding for 29 known proteins, though we find the expression of 28 to be constant with one remaining unexpressed. each of the 28 proteins that are reported to be encoded perform different functions in the viral pathogenesis cascade though we are still not fully aware of the plethora of functions that they perform (zeng et al., 2004) . four proteins make up the viral structure of which the most studied and important protein is the s protein or spike protein since they are the regulators of viral entry into the host. this protein binds to the angiotensin converting enzyme 2 (ace2) receptor and gains entry into the host cell. a pair wise amino acid comparison using the amino acid sequences reveal that the spike proteins of sars-cov-2 share more than 80% sequence homology to sars-cov. conservation is found in residues which interact with the ace2 receptor; however, the other residues are a bit different with a few insertions as well. till date the reports indicate that the interaction of the spike protein of sars cov-2 is much stronger as compared to the previous coronaviruses . a nonpeer-reviewed preprint posted on medrxiv hypothesises that since ace2 is overexpressed in the lung tissue of people with chronic respiratory disorders, they are likely to be more affected by this prevalent strain of the virus (pinto et al., 2020) . however, the infectivity of the virus is not restricted to its affinity for ace2. if we dig deeper into the structure of the s protein we shall find that they are composed of two segments, s1 and s2 that require a double cleavage to expose the peptide which initiates the fusion with ace2. the first cleavage is mediated by a host cell encoded enzyme furin. this ability to get human enzymes to do its work increases its infective ability as well. few non-structural proteins of the virus are involved in the evasion of the host immune system and also function towards the assembly and replication of the virus. these proteins are expressed as part of two large polyproteins which are produced from a ribosomal frameshift event and are cleaved by the protease into 16 different proteins each with specific functions (seah et al., 2020) . the rest of the proteins are the accessory proteins which are technically non-essential as they are not required for viral replication in vitro and are the least well understood of the total proteome (kim et al., 2020) . there have been several reports regarding the variation in severity and lethality of coronaviruses. nl63, 229e, oc43, and hku1 cause mild respiratory problems in humans and three others (mers-cov, sars-cov including the newly emerged sars-cov-2) have been observed to inflict severe respiratory syndromes (veriti et al., 2020; who 2020; who 2020 [mers-cov] ). sars-cov-2 infection was first reported from wuhan, china, on 24th december 2019 (veriti et al., 2020) and in less than three months, on 11th march 2020, who declared covid-19 as a pandemic (li, 2016) . by may, 2020, 213 countries had been already affected with almost 2.5 lakhs fatal cases .thus it was clearly visible that the infectivity of sars-cov-2 was much higher than sars-cov or mers-cov (folis et al., 2006) , though the fatality rate (0.9-3.3%) is still substantially lower than that of sars-cov (11%) and mers-cov (34%) (tang et al., 2020) . since this is a new pathogen, researchers worldwide are looking towards both primary and secondary prevention strategies in the form of vaccination as well as an over the counter medication which can be afforded by citizens around the world. whatever strategy is being envisaged, the threat lies in the appearance of new strains with resistant mutations to vaccines and therapeutic agents. as a large proportion of humanity is already infected and we have no strategy in place currently to identify the asymptomatic carriers due to the lack of infrastructural and manpower resources around the world, there is high possibility of the successful escape mutations in the genome of the virus. very few studies till date have focused on mapping the mutations on the important protein components of sars-cov-2. during the preparation of this manuscript we came across two studies which attempt to map the effects of significant mutations on the protein structure and stability. benvenuto et al (2020) reports in more recent isolates of sars cov-2,the presence of two mutations affecting the non-structural protein 6 (nsp6) and the open reading frame10 (orf 10) in adjacent regions. amino acidic change stability analysis suggests both mutations could confer lower stability of the protein structures. namely, at amino acid position 3691 (corresponding to nsp6 position 37), most of the sars-cov-2 sequences have a leucine residue while some more recent sequences from asian, american, oceanian and european isolates show phenylalanine. at the amino acidic position 9659 (corresponding to orf 10 position 3 to 4), most of the sars-cov-2 sequences have an arginine residue while some sequences from australian and american isolates have a histidine residue. in the second study by bhattacharya et al (2020) the 614 amino acid position in the s protein was found to be conserved among species (bat, pangolin, civet and human sars-cov and human sars-cov-2), until the a2a subtype defining mutation occurred (d614g) (bhattacharya et al., 2020) . authors report that an additional serine protease (elastase) cleavage site around s1-s2 junction was introduced due to d614g mutation in sars-cov-2. the glycine at 614 (aa) is predicted to be the nearest substrate site for elastase to perform proteolytic cleavage in the adjacent residue. this study focuses on mapping the effects of the clade specific mutations identified in the orf8, nsp2, nsp4, nsp6, nucleocapsid protein (n) and spike protein (s) on the conformation and stability of the structures. it also predicts the possible drug binding sites, along with the druggability of these protein structures and the conformational epitopes of b cell, t cell, mhc -i and mhc -ii alleles. clade specific mutations were identified using literature mining from the various sars -cov2 dedicated resources at pubmed and pubmed central and we identified a list of prevalent mutations across the major pathogenesis related proteins of the virus (table 1) . though currently there are reports of other mutations that have been identified by large scale viral genomic sequencing studies undertaken globally, (mercatelli and giorgi 2020) , the following dataset can be considered to be comprised of the initial founder mutations which resulted in the variations in the strain of the virus before the international borders were closed down (callaway, 2020) following which the selection pressure on the virus became more country and population specific. following the identification of the mutations we utilised the ncbi genome browser to extract the protein sequences using the wuhan strain as the ancestral strain. using a generic sliding window approach we were able to pinpoint the sites of mutations and we created two data sets -wild type protein sequences which were directly the ones from the wuhan strain and the mutant data set -which consisted of the protein sequences carrying the mutations in them prediction of physicochemical properties of the protein: to understand the exact conformational behaviour of a protein, physical and chemical parameters of the wild type and mutant protein sequences were analysed using the protparam protein analysis tool of expasy web server (https://web.expasy.org/protparam/) and pepstat protein sequence statistical tool of embl-ebi web server (https://www.ebi.ac.uk/tools/seqstats/emboss_pepstats/),to determine molecular weight, theoretical pi, extinction coefficient (gill & von hippel, 1989) , estimated half-life (bachmair et al., 1986) , instability index (guruprasad et al., 1990) , aliphatic index (ikai, 1980) , grand average of hydropathy (kyte & doolittle, 1982) , and isoelectric point (harrison, 2000) . the secondary structure of the wild type and the mutant proteins along with their degree of disordered residues and accessible surface area was predicted using the primary sequence of the protein. these were submitted as input to the netsurfp -2.0 server (http://www.cbs.dtu.dk/services/net-surfp/) (klausen et al., 2019) , which utilizes a neural network based approach trained on established protein structures as test sets. the accuracy of prediction is estimated to be 85% when compared to estimated structures. since the degree of disordered residues in a protein has its impact on overall structural and functional stability of a protein, the data obtained from netsurfp was cross(mészáros et al., 2018) . the possible epitopes were predicted for the 7 wild type and their corresponding mutant proteins selected for the study. linear b cell epitopes were predicted by using abcpred (crdd.osdd.net/ raghava/abcpred/) and lbtope (webs.iiitd.edu.in/raghava/lbtope/protein.php) servers. abcpred requires an amino acid sequence as input and generates epitopes using an artificial neural network (ann) (janahi et al., 2017) . a window length of 16 amino acids and threshold of 0.51 were considered for generating the results (kumar et al., 2018) . "lbtope: linear b cell epitope prediction server" was also used to reveal b cell epitopes of viral origin with a threshold probability score of 60% and above. the t cell epitopes of viral proteins binding to class i and class ii major histocompatibility complex molecules were mined using netctl 1.2 server (http://www.cbs.dtu.dk/services/ netctl/) and propred (https://webs.iiitd.edu.in/raghava/propred/). viral peptides capable of binding to cytotoxic t lymphocytes and mhc i molecules were recognized using netctl 1.2 server which facilitates the prediction by combining the scores for proteasomal cleavage, tap transport efficiency and mhc i binding (larsen et al., 2007) . the single letter code of amino acid sequence was entered in the user interface, the c terminal cleavage, weight on tap transport efficiency and threshold for epitope identification was set at values of 0.1, 0.05 and 0.75 respectively. the viral proteins were screened against 12 most recognized supertypes of hla alleles. the results were generated with a specificity of 97% and sensitivity of 80%, by integrating the scores based on multiple parameters as weighted sum with a relative weight on mhc i binding. for the prediction of mhc class ii epitopes, propred uses quantitative matrices methods which scan the antigen sequence against a profile of hla-dr binding pockets, each having a unique quantitative representation of the amino acid interactions within the peptide binding cleft (sturniolo et al., 1999) . amino acid sequence was provided as input at threshold value of 3, which enabled us to view the top 3% of best scoring peptides with respect to their probability of binding to different human leukocyte antigen (hla) molecules. epitopes were predicted for all the 51 hla-dr alleles that account for 90% of the mhc molecules on antigen presenting cells (palanisamy & lennerstrand, 2017) . the impacts of the mutations on the stability of the proteins were estimated using a sequence based approach at the i-mutant 2.0 (capriotti et al., 2005) web server. this is a program which classifies the effects of a particular mutation using a support vector machine based approach. the training set of the tool was the protherm database (bava et al., 2004) . the temperature and ph threshold used for this analysis were 25 and 7 respectively. no complete crystal structures of these proteins are currently available in the protein data bank. as a result, ab initio modeling was performed using the i-tasser server (https://zhanglab.ccmb.med.umich.edu/i-tasser/) (roy et al., 2010) and the resultant structures were subjected to molecular simulation for 40ns using a gromos96 43a2 force field in gromacs (bekker et al., 1993; abraham et al., 2015) . following the simulation, the structures were analysed for torsional/ conformational stability from the ramachandran plots of the models using the molprobity server (williams et al., 2018) . the integrity and validity of the 3d structure of the generated homology models were verified by q mean analysis. the variants prioritised by the sequence based analysis were inserted within the wild type models by using the mutate tool of deepviewv4.1.0 (guex & peitsch, 1997) following which they were simulated and validated using the protocols followed for the wild type proteins. the degree of evolutionary conservation of an amino acid in a protein is a signature of the balance between its natural tendency to mutate and the overall need for it to be retained in its original form to maintain the structural integrity and function of the macromolecule. in our analysis we used the consurf web server (http://consurf.tau.ac.il) (ashkenazy et al., 2016) , for predicting the evolutionary pattern of the amino acids of the macromolecule to reveal regions that are important for structure and function. the structures were submitted as inputs from which the sequences were extracted by the server and top 50 homologues were identified using three psi -blast iterations against the uniprot database. redundant homologous sequences were removed using the cd-hit clustering method (li &godzik, 2006; fu et al., 2012) . the resulting sequences were then aligned using mafft (katoh & standley 2013) and the generated multiple sequence alignment (msa) was used to generate the phylogenetic tree. the tree and the msa were then considered together and the rate4site algorithm (pupko et al., 2002) was used to calculate the position-specific evolutionary rates using an empirical bayesian methodology (mayrose et al., 2004) . the raw data obtained was then normalized and grouped into nine conservation grades where 1 represented the rapidly evolving positions, 5 denoted positions with intermediate rates, and 9 was the threshold for the most evolutionarily conserved position. these were then mapped on the 3d structure on a position specific basis. we performed this analysis using both the wild type and mutant structures. prediction of binding pockets and druggability: the alterations in potential ligand-binding sites and druggable pockets, in the wild type and mutant proteins were analysed using dogsitescorer (https://proteins.plus/) web server. the following parameters were assessed: • drug score-the total drug score was obtained by summing up the drug scores for each of the predicted pockets in the wild type and mutant structures. increase or decrease in the total drug score was an indication towards the alteration of the pocket conformation and druggability. • the number of ligand-binding pockets -the number of ligand-binding pockets in the wild type structure was considered as the reference and gain or loss of pockets in the mutant structure was considered for comparison. post simulation, structures of wild type and mutant pairs of each protein were aligned using tmalign tool (zhang & skolnick, 2005) . tmalign performs structural alignment by superimposing structures, it performs a pair wise alignment for individual residues of protein. the output is provided in terms of root mean square deviation (rmsd), tmalign score and aligned residues among structures. here rmsd value proves to very critical when it comes to structural variations, higher the rmsd value higher are the differences among structures under consideration. in case of this study, higher the rmsd between structures, more were implications of mutations in terms of structural distortion. the molecular structures were visualized using chimera. each sequence based and structural parameter analysed as a part of this study was given equal weightage since, the dataset was time limiting. a simple binary coding scheme was devised which was as follows: alteration in value of parameter from wild type to mutant = -1, when the parametric value was unchanged for wild type and mutants then the value was "0" indicative of a neutral impact of the mutation. using this scheme, a matrix was prepared and each mutation was provided with a cumulative score. this cumulative score was then arranged in a descending order and the mutation with the lowest score (most negative score) was considered to be the most disruptive. determination of protein sequences from genome: using the methods outlined above we identified the wild type and mutant sequences which would be used subsequently (table 2 ). throughout the course of results and discussion we would be using the names of the proteins for easier understanding. epitopes harboured in the viral proteins, that elicit immune responses in the host, can serve as potent vaccine candidates in near future. linear b cell epitopes, were ascertained using servers, abcpred and lbtope. lbtope organizes the antigenic determinants in descending order of their probability scores. epitopes with greater than 60% prediction scores were additionally screened for epitopes encircling the alteration site (supplementary table 3 table 5 : variation in average score of epitopes for the set of seven proteins using lbtope. for abcpred, the antigenic determinants are ranked with respect to their binding probabilities, from highest to lowest, thus endorsing the best binder the first rank and so on. in this work, we have analysed the epitopes having any rank within 10 for each protein, enumerated their numbers and computed their average score for wild type and mutant forms (table 6) . we observed a change in the number of epitopes and their mean binding score for n protein and nsp4 proteins. while the number of epitopes remained unaltered, the mean score showed discrepancy for the orf8 protein. an epitope holding the alteration site in wild type orf8 protein had a binding score of 0.64, which was altered to 0.67 post mutation. again, in proteins like nsp4 and n protein, mutation resulted in recognition of new epitopes covering mutation region, which was not predicted previously in wild type proteins. no fluctuation in epitope count and score was noted in case of orf3a, s protein, nsp2 and nsp6 proteins (figure 4) table 4 ). finally we summed up all the combined scores of all the epitopes of the individual proteins lying above their respective average score. the data of table y was represented through a histogram ( figure 5 ). propred was used to predict mhc class ii binding t cell epitopes from the set of 14 proteins. the top 3% of the best scoring epitopes generated from each protein were further shortlisted on the basis of the average score of the epitopes encompassing the mutation region in the wild type protein. the epitopes with probability scores equal to or greater than the average score was screened from the wild type and mutant proteins. the filtered epitopes were then, quantitatively and qualitatively compared and scrutinized between normal and mutant forms (supplementary table 5 ). in case of orf3a protein, 6 less epitopes were found to qualify the average score ( potential changes to stability of the proteins brought about by the mutations were analysed using the i-mutant server. according to the algorithm of the server the range of ddg values for a neutral mutation classification is : -0.5=<ddg=<0.5, while changes < -0.5 are classified as large decrease and changes > 0.5 are classified as large increase. thus in our analysis we found that n protein, orf3a and s protein could be categorised as having large decrease with orf8a having the highest negative value of -2.87, while nsp 2 had large increase in the value. alterations of nsp4 and nsp6 were predicted to be in the neutral change segment (figure7). stable structures were generated using ab initio modeling which were subsequently simulated [ qmeans values were found to differ in case of the wild type and mutant structures with significant changes observed in case of s protein, orf8a and orf 3a; while in case of nsp2 opposite trend was observed, though the differences in qmeans score was not very significant for nsp2 ( figure 9 ). all the fluctuation plots obtained following simulation [ figure 10 to further confirm the variations induced as a result of the mutations we aligned the protein structurally and distinct differences were observed ( figure 11 ). mutations led to differences in the pro-tein structure of every mutant -wild type pair. highest rmsd value was observed for s protein (5.67), while maximum number of mismatches was observed in case of n protein (24 out of 419). n protein also had a high rmsd of 4.93. the results of structural alignments do suggest that mutations altered the protein structures substantially (figure 12 ). the analysis of potential drug binding pockets and their druggability was further evaluated and all the proteins had alterations not only in the total number of drug binding pockets but also the average drug scores when wild type and mutant structures were compared. it was interesting to note that apart from nsp4 and orf8a all the other proteins, suffered a decrease in average druggability as a result of the mutations whereas the former two proteins exhibited an increase in potential druggability (figure 13 a and 13 b) . no significant variations were observed in our evolutionary analysis using consurf server. a simple binary scoring scheme was devised, where a parameter value alteration was indicated as -1 (indicating a change from wild type value) and unaltered parameter (indicating of no change from wild type parameter) was used for the analysis. for parameter values which involved decimal numbers, change was considered only if the first place after decimal had been altered (supplementary table 6 ). once the scoring scheme was populated with the corresponding values a heat map was generated to diagrammatically exhibit the contributions of each parameter under study towards the overall cumulative score obtained (figure 14 a) . the ranking of the mutations were then performed using a histogram in which the cumulative scores were plotted (figure 14b) . from the histogram we were able to clearly predict the l84s mutation in orf8a as the most disruptive of the mutations followed closely by nsp4 mutation (orf1a 3220v) and the mutations in n protein (s202n); or f3a (g251v) and s protein (d614g). the lowest ranked mutations were those of nsp 6 (l3606f) and nsp2 (v378i). according to kyte et. al. (1982) increased value of hydropathicity indicates the presence of more hydrophobic residues in the protein sequence and lins et. al. (2003) , points out that decrease in accessibility is more pronounced for hydrophobic than hydrophilic residues (kyte &doolittle, 1982; lins et al., 2003) . in our analysis s protein and orf3a have increased hydropathicity and decreased accessible surface area which suggest that the mutation have resulted into increased hydrophobicity. salahuddin and khan (2009) have reported that increase in hydropathicity can be a direct determinant of increased pathogenicity for viral proteins. this observation is supported by the observations of (sur et al., 2009 ). both of these works involve analysis of mutations and genome variations with flu viral genomes. s protein acts as a cellular receptor to induce the fusion of viral and host membranes (tan et al., 2004; tan et al., 2005) . it is important for the viral entry into the host cell and for inducing host immune responses. sequence comparison of isolates from different infections show both viral s protein and orf3a gets positively selected during evolution (yin, 2020) implying that orf3a plays an important role in the virus life-cycle and disease development. also, according to linding et. al. (2003) protein disorder is important for understanding protein function and protein folding pathway (linding et al., 2003) . in our study, orf3a protein exhibits decreased disorderedness in its mutant form which indicates towards a change in the structural conformation and rigidity. in our study, maximum change in accessible surface area was observed in s protein whereas in case of orf3a the intrinsic disorder was observed to decrease significantly which may affect the role of s protein's functional interaction along with orf3a. ikai et. al. (1980) , defined aliphatic index as the relative volume of a protein occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine) of amino acids which show a correlation with increase in thermostability of proteins (ikai, 1980) . in our analysis nsp2, nsp4 and orf3a were observed to show an increase in aliphatic index value from wild type to mutant which suggests probable increase in the thermostability of these proteins due to mutation. interestingly orf3a protein has a significant role in the functioning of s protein in the host system to increase infection which may be affected by the increased thermostability of orf3a. momen-roknabadi et. al. (2008) , in their study predicted that accessible surface area can be used to improve the prediction of secondary structure of a protein (momen-roknabadi et al., 2008) . according to lins et al (2003) , different secondary structures correspond to different accessibility of residues. in their study, they found that random coils, beta sheets were more accessible folds with average 30% accessibility, while the alpha helices have 20% accessibility. in our analysis, we found a similar phenomenon where loss of beta sheet, conversion of random coils to alpha helices have led to decreased accessible surface area and loss of alpha helix have caused an increase in accessible surface area. induction of adaptive immunity is largely dependent on recognition of viral epitopes by host b and t cell receptors. site specific mutations in the mentioned set of seven proteins from sars-cov2 were extensively analyzed for their effects on epitope recognition and binding efficiencies. while gain of function mutations can improve the binding affinities of epitopes to host lymphocyte receptors, loss of function mutations arising through positive selection can be crucial for b and t cell immune escape (ramaiah et al., 2019) . each of the seven mutant proteins investigated, exhibited either change in free energies of binding or recognition by specific alleles, thus necessitating further exploration to evaluate their impact on immune escape and viral persistence. besides, changes in the number of linear b-cell epitopes were spotted in proteins like nsp4 and n protein. in our study, we observed an epitope covering mutation region, recognized by a3 supertype in wild type of n protein but no changes in corresponding epitope was spotted by the same supertype in the mutant n protein (loss of function mutation); in contrast we spotted a mutation spanning epitope in mutant nsp4 protein perceived by a3 supertype, which was not detected by the same supertype in the wild type nsp4 protein (gain of function mutation). another significant finding was the decline in the number of mhc ii binding t-cell epitopes in the orf8 and s protein, where the mutant protein was found to have 4 and 3 less immunogenic determinants respectively. each of the seven proteins were assigned a score of either '-1' or '0', for each of the four computational tools used for epitope prediction, where '-1' corresponds to any change in number or binding efficacy of antigenic determinants, that may have surfaced because of mutation and '0' corresponds to no changes between wild type and mutant forms. the rmsf value measures the deviation between the positions of particle i and some reference position. rmsd and rmsf values can be differentiated remarkably. rmsf is averaged over time, depicting a value for each particle i, while in case of rmsd, the average is taken over the particles, giving time specific values. zhao et al (2015) have probed into the effect of mutations in the zinc binding b box1 domain protein (zhao et al., 2015) . in their study they have looked into the changes in the dynamics of the backbone atoms and also calculated root mean square fluctuation (rmsf) values at each time point of the trajectories of the native and mutant structures. they found that mutations induced higher rmsf which led them to suggest that the much larger side-chain of valine extends its steric hindrance to other zinc-binding residues. supporting the increased flexibility, they also measured the rmsf values for second zinc-binding residues (c134, c137, h150, h159); which led to the final conclusion that: larger rmsf values indicate increased random motions of residues leading to disruption in the structural integrity of the proteins. in our analysis we have also observed a similar phenomenon with the increase in the rmsf values in mutant structures ultimately causing randomness in the 3d structures which is evidenced by their increased rmsd and more negative q mean scores. the significance of q mean scores have been extensively reported by de carvalho and de mesquita (2013) in their work on human superoxide disputes 2 where they com-pared wild type and mutant structures and reported the effect of mutations (de carvalho & de mesquita 2013). using the tm align and q mean methodology datta et al (2015) have explored the effect of 124 single nucleotide polymorphisms of the ribonuclease l gene (rnasel) in prostate cancer (datta et al., 2015) . all the snp's were mapped on the protein structure and tm align was used to classify the most disrupting mutants. in our analyses we have been able to clearly show that mutations caused significant changes in protein structures where highest rmsd value was observed for s protein (5.67), while maximum number of mismatches were observed in case of n protein (24 out of 419). n protein also had a high rmsd of 4.93. thus from the structural comparisons of wild type and mutant proteins we were able to understand that almost all the mutations destabilised their native conformation. mutations in all the proteins were first matched with the alteration data and it was found that direct effect of the mutation residue was associated with the changes in the epitope site predictions (table 6 ). they were also associated with alteration in the accessible surface area values as predicted thus implying that the mutations resulted in the changes in both structural and functional aspects of the protein structures. the exposure-predominant type (likely introduced early in the assay) (huskova et al., 2017) . a score of 1 was added if the mutation was in a known human hotspot, if it was truncating or affected a splice site and so on. in our scoring scheme we prioritised the ability of the mutation to cause deviation in the structure of the mutant from the wild type and assigned scores as described in the materials and method segment. our final scoring revealed that orf8 protein mutation l84s was the most disruptive of the mutations under study. incidentally l84s mutation was also the most prevalent of the mutations under study as it was present in four different clades b, b1, b2 and b4 clades. incidentally bhattacharya et al (2020) (42), canada (40), and usa (446); b2 has been identified from china (10) and japan (4); while b4 finds its prevalence in australia (5), china (5) and usa (27). these data suggest that this mutation was one of the earliest of the mutations that could have occurred naturally as it does not exhibit any population specificity and may be attributed to a random event. this data was however consistent with the predictions made in the i mutant server which had assigned the most negative value for l84s and classified it as the most disruptive. this data justifies our scoring scheme and the parameters considered towards the prediction of the most disruptive mutation and thus can be explored further for future studies. recent works suggest that the lowering of the stability of viral proteins as a result of accumulation of mutations (banerjee et al., 2020) could be the reason behind the low national fatality rate in india. it may also be a phenomenon similar to muller's ratchet where this accumulation of mutations in fresh strains of the virus and lack of stabilising recombination events may result in the reduction in fitness of the virus as is being indicated by the effect of the mutations on protein stability. gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers con-surf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules in vivo half-life of a protein is a function of its amino-terminal residue spike protein mutational landscape in india: could muller's ratchet be a future game-changer for covid-19 protherm, version 4.0: thermodynamic database for proteins and mutants gromacs -a parallel computer for molecular-dynamics simulations global spread of sars-cov-2 subtype with spike protein mutation d614g is shaped by human genomic variations that regulate expression of tmprss2 the coronavirus is mutating -does it matter? i-mutant2.0: predicting stability changes upon mutation from the protein sequence or structure functional and structural consequences of damaging single nucleotide polymorphisms in human prostate cancer structural modeling and in silico analysis of human superoxide dismutase 2 furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry cd-hit: accelerated for clustering the next-generation sequencing data the proteomics protocols handbook calculation of protein extinction coefficients from amino acid sequence data swiss-model and the swiss-pdbviewer: an environment for comparative protein modeling correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence expression of soluble heterologous proteins via fusion with nusa protein receptor-binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine in vitro using barrier bypass-clonal expansion assays and massively parallel sequencing thermostability and aliphatic index of globular proteins in silico cd4+, cd8+ t-cell and bcell immunity associated immunogenic epitope prediction and hla distribution analysis of zika virus mafft multiple sequence alignment software version 7: improvements in performance and usability the architecture of sars-cov-2 transcriptome netsurfp-2.0: improved prediction of protein structural features by integrated deep learning structural and epitope analysis (tand b-cell epitopes) of hepatitis c virus (hcv) glycoproteins: an in silico approach a simple method for displaying the hydropathic character of a protein large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction structure, function, and evolution of coronavirus spike proteins cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences protein disorder prediction: implications for structural proteomics analysis of accessible surface of residues in proteins interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors comparison of site-specific rate-inference methods for protein sequences: empirical bayesian methods are superior geographic and genomic distribution of sars-cov-2 mutations iupred2a: context-dependent prediction of protein disorder as a function of redox state and protein binding impact of residue accessible surface area on the prediction of protein secondary structures computational prediction of usutu virus e protein b cell and t cell epitopes for potential vaccine development ace2 expression is increased in the lungs of patients with comorbidities associated with severe covid-19. medrxiv : the preprint server for health sciences rate4site: an algorithmic tool for the identification of functional regions in proteins by surface mapping of evolutionary determinants within their homologues evidence for highly variable, region-specific patterns of t-cell epitope mutations accumulating in mycobacterium tuberculosis strains i-tasser: a unified platform for automated protein structure and function prediction revisiting the dangers of the coronavirus in the ophthalmology practice generation of tissue-specific and promiscuous hla ligand databases using dna microarrays and virtual hla class ii matrices in silico analysis of evolution in swine flu viral genomes through re-assortment by promulgation and mutation characterization of viral proteins encoded by the sarscoronavirus genome a novel severe acute respiratory syndrome coronavirus protein, u274, is transported to the cell surface and undergoes endocytosis on the origin and continuing evolution of sars-cov-2 estimates of the severity of coronavirus disease 2019: a model-based analysis. the lancet. infectious diseases molprobity: more and better reference data for improved all-atom structure validation middle east respiratory syndrome coronavirus (mers-cov) update 49 -sars case fatality ratio genotyping coronavirus sars-cov-2: methods and implications characterization of the 3a protein of sarsassociated coronavirus in infected vero e6 cells and sars patients tm-align: a protein structure alignment algorithm based on the tm-score molecular dynamics simulation reveals insights into the mechanism of unfolding by the a130t/v mutations within the mid1 zinc-binding bbo -x1 domain supplementary table 2: total residue specific values of asa and disorder supplementary table 6: scoring table for impact assessment supplementary figure 1: images of ramachandran analysis for wild type and mutant structures the authors acknowledge the contributions of dr. souvik mukherjee, national institute of biomedical genomics, kalyani, west bengal for his insightful discussions regarding the data acquisition techniques as well as overall comments during the preparation of the manuscript. key: cord-347714-vxxhglx7 authors: abitogun, folagbade; srivastava, r.; sharma, s.; komarysta, v.; akurut, e.; munir, n.; macalalad, l.; olawale, o.; owolabi, o.; abayomi, g.; debnath, s. title: covid19: exploring uncommon epitopes for a stable immune response through mhc1 binding date: 2020-10-14 journal: biorxiv doi: 10.1101/2020.10.14.339689 sha: doc_id: 347714 cord_uid: vxxhglx7 the covid19 pandemic has resulted in 1,092,342 deaths as of 14th october 2020, indicating the urgent need for a vaccine. this study highlights novel protein sequences generated by shot gun sequencing protocols that could serve as potential antigens in the development of novel subunit vaccines and through a stringent inclusion criterion, we characterized these protein sequences and predicted their 3d structures. we found distinctly antigenic sequences from the sars-cov-2 that have led to identification of 4 proteins that demonstrate an advantageous binding with human leukocyte antigen-1 molecules. results show how previously unexplored proteins may serve as better candidates for subunit vaccine development due to their high stability and immunogenicity, reinforce by their hla-1 binding propensities and low global binding energies. this study thus takes a unique approach towards furthering the development of vaccines by employing multiple consensus strategies involved in immuno-informatics technique. coronaviruses are a large group of viruses that are rather common throughout the community, causing illness ranging from the common cold to more severe diseases such as severe acute respiratory syndrome (sars-cov) and middle east respiratory syndrome (mers-cov) (1) . the previous outbreaks of these coronaviruses in 2003 and 2015 respectively show similarities to the novel coronavirus (2) , which was first reported in december 2019 in wuhan (3) . it was later declared as a pandemic on 11th march, 2020 by the world health organization (who) having caused a global emergency across many countries and territories around the world. the causative agent of the covid-19 disease is the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which was initially tagged as 2019 novel coronavirus (2019-ncov) by the world health organization (4) before genomic studies revealed a 79.5% and 96% nucleotide level similarity with sars-cov and bat coronavirus respectively (5, 6) , hence necessitating its new identity. further analysis on its similarity with sars-cov and mers-cov including phylogeny revealed that it is categorized under the family coronaviridae, order nidoviralae and genus betacoronavirus alongside the other two viruses which have also caused pandemic situations in the past, causing thousands of death globally. (7, 8) the structural architecture of sars-cov-2 is an elaborate one, containing a positive single stranded rna as its genetic element with genomic length of about 29-30 kilobases, (9) which encodes the major structural proteins being the spike (s) glycoprotein, membrane (m) protein, envelope (e) protein, and nucleocapsid (n) protein. (10, 11) the structure of the spike glycoprotein of the virus is also an extended similarity with sars-cov, (4) which together with covid19: exploring uncommon epitopes for a stable immune response through mhc1 binding other proteins of the virus are candidates for vaccine development and are being explored in different settings due to the active roles of the proteins in the infectivity of the virus. (12) (13) (14) (15) developing an effective treatment for sars-cov-2 is a research priority. currently, different therapeutic strategies are being utilized by researchers to combat this dangerous covid-19, with much of the focus being on developing novel drugs or vaccines. however, in recent years, the development of vaccine design has been revolutionized by the reverse vaccinology (rv), which aims to first identify promising vaccine candidate through bioinformatics analysis of the pathogen genome. one of the successes of this method is the bexsero vaccine, discovered for group b meningococcus. (16) this immuno-informatics method includes the identification of peptide epitopes in the virus genome which can then be prepared by chemical synthesis techniques. these epitopes are easier in quality control; however, there is the need for structural modifications as well as the inclusion of delivery systems, and adjuvants in their formulation due to the low immunogenicity of the epitopes which is a result of their structural complexity and low molecular weight. (17) recently, a set of b and t cell epitopes highly conserved in sars-cov-2 were identified from the s and n proteins of sars-cov. (18) however studies have shown that full length spike protein vaccines for sars-cov may lead to antibody mediated disease enhancement causing inflammatory and liver damage in animal models (19, 20) which is why in this manuscript, we applied immuno-informatics "in silico" approaches to identify potential cd8+ cytotoxic t cell epitopes from proteins of sars-cov-2, sars-cov and mers-cov. in achieving this, the ~29.8kb genome of the sars-cov-2 which encodes for 28 proteins comprising 5 structural proteins, 8 accessory proteins and 15 non-structural proteins as well as the ~30kb genome of sars-cov-1 comprising 14 open reading frames (orf) were explored for some uncommon proteins of the viruses with therapeutic potentials. identified proteins were thus considered for highly antigenic epitope prediction and evaluation. the antigenicity and immunogenicity of all identified epitopes was estimated and their interactions with the human leukocyte antigen (hla) class i system were evaluated, owing to the wide usage of the hla system as a strategy in the search for the etiology of infectious diseases and autoimmune disorders, (21) as was explored in the case of sars-cov-1 after the first epidemic that broke out in east asia in 2002−2003. this is linked to the fact that hla genes are known to display the highest level of diversity in the genome, with thousands of different alleles now reported. each of these alleles is also a combination of multiple single nucleotide polymorphisms (snps). this, we also investigated the hla-class i system for evidence of disease association, with the hope that the discovery of disease susceptibility will help in developing protection such as vaccines against the virus. the uniprot (22) database was searched for the protein sequences inferred from the genome sequence analysis of different isolates of highly pathogenic human coronaviruses. the sars cov, mers-cov, and sars-cov-2 proteins without experimentally defined 3d structure or functional annotation were chosen for further analysis and their sequence files in the fasta format were downloaded. the fasta sequences were used to infer the physicochemical properties of the chosen each model was refined using a molecular dynamics simulation approach implemented by galaxy refine (36) tool or rapid energy minimization using discrete molecular dynamics with an all-atom representation for each residue implemented by chiron server. antigenicity prediction of the top eight uncharacterized coronavirus proteins selected after 3d molecular models' validation was performed using vaxijen tool. (44) a threshold value of 0.4 was taken into account. non-antigenic peptides having vaxijen scores less than 0.4 were discarded, while antigenic epitopes with scores higher than 0.4 were further prioritized for their immunogenicity. the proteins predicted to be non-antigens were discarded from the further analysis. antiviral immunity relies on the ability of major histocompatibility complex (mhc) class i molecules to bind antigen molecules and display them to t cells. each candidate is subsequently refined by restricted interface side-chain rearrangement and by soft rigid-body optimization. the side-chain flexibility is modeled by rotamers and the obtained combinatorial optimization problem is solved by integer linear programming. following rearrangement of the side-chains, the relative position of the docking partners is refined by monte carlo minimization of the binding score function. the refined candidates are ranked by the binding score which is a combination of atomic contact energy, softened van der waals interactions, partial electrostatics and additional estimations of the binding free energy. in order to identify functionally important regions in the selected proteins and the selection of epitopes with the desired degree of conservation, consurf (54) a total of 8 non-structural sequences were identified and retrieved from uniprotkb database. the mass, isoelectric-ph, basic amino acid %, cysteine %, polarity and hydrophobicity of the protein sequences were computed and outlined in table 1. the lengths of these protein sequences varied from 39 to 275 amino acid residues. cysteine was found to have the highest presence in the selected proteins with an average of 5.56% and its highest presence was observed in proteinq7tfa0 with 15.40%. arginine, lysine and histidine have an average presence of 2.35%, 3.69% and 4.36% respectively. the polarity and non polarity of the final selected proteins ranged from 27.907%to 63.839 % and from 36.161%to 72.093% respectively with m4svf1 having the highest percentage of polar amino acids (63.839%) while p0dtd8 has the highest percentage of non-polar amino acids (72.093%). likewise, the highest hydrophobicity index was recorded for p0dtd8. after obtaining the 3d structures of the proteins from modeling servers, the models were refined using galaxyrefine. upon refinement, the models were subjected to stereochemical and thermodynamic stability analysis. particularly, the models with highest ramachandran favored residues and lowest clash scores were used for energy analysis using chiron, scoop and errat servers as highlighted in table 2. all the protein structures had a ramachandran score of over 89% and an errat value of over 60, with q7tfa0, p0dtd8 and p0dtd3 all having a perfect 100% ramachandran scores. the free energy of the structures ranged from -28.7kcal/mol (q7tfa0) to -2.2 kcal/mol (a0a6h2lbe3). force field values ranging from -5955.989 (a0a6h2lbe3) to -435.783 (p0dtd8) were observed using gromos96 implementation of the swiss-pdb viewer. the models with lowest overall energies were determined to be stable. vaxijen antigenicity prediction was used to predict the overall antigenicity of the 8 proteins as outlined in table 3. two proteins: m4svf1_mers and q7tfa0 were predicted to be non-antigens with their antigenicity scores of 0.3373 and 0.1251 respectively while p0dtd8 and p0dtc8 showed very high antigenicity owing to their high scores of 0.8462 and 0.6502 respectively. t-cell epitope prediction was carried out using iedb, epijen, pepvac, rankpep, and netmhc. initially, 15,181 hla class i binding epitopes were predicted from 6 out of 8 stable proteins which were predicted to be antigenic. scrutiny on the basis of percentile rank filtered 112 peptide epitopes. considerable binding affinity for 5 hla-1 subtypes with high frequencies in asia and africa (hla-a*02:01, hla-a*11:01, hla-b*08:01, hla-b*27:05 and hla b*58:01) was observed from netmhcpan server which predicted high affinity binders (<0.5 percentile rank) using el-ranks. among predicted epitopes of sars-cov-2 virus, ten (10) epitopes showed considerably high immunogenicity towards the mhc-class 1 molecules which were then selected for further analysis (table 4 ). all the predicted epitopes were further subjected to allergenic and toxicity analysis, with all 10 coming out as non-allergens and non toxins (table 4) . epitopes including hlvdfqvti, rksapliel, and ylcflafll showed remarkably high antigenic tendencies, scoring 1.4119, 1.1591 and 0.9143 respectively. all of these epitopes, along with their features are reported in table 4 (table 5) . further docking analysis resulted in a total of 10 global binding energies predictions which were refined with firedock ( table 6 ). the global binding energies range from -68.71 to -11.19 indicating that all the proposed epitope sites from the modelled proteins can serve as good antigens. the conservation status of each residue in the selected t-cell epitopes in four (4) sars cov-2 proteins (q7tlc7, p0dtc6, p0dtd8, p0dtc8) were examined using consurf. results revealed that the highly conserved and exposed residues are found in all 4 proteins, with p0tdc6 and p0dtc8 having the larger amount of these residues. particularly, the epitopes without allergenicity and toxicity containing at least one predicted functional residue (highly conserved and exposed) included epitopes 'rksapliel' and 'hlvdfqvti' while t-cell epitopes 'rksapliel', 'flafllflv', and 'hlvdfqvti' contained at least one predicted structural residue (highly conserved and buried). interestingly, none of the epitopes from p0dtd8 contained any functional residues (highly conserved and exposed) while the highest concentration of predicted structural and functional residues was found in two epitopes hlvdfqvti (1 functional residue and 3 structural residues) and rksapliel (3 functional residues and 1 structural residue)-from the protein p0dtc6 . despite the continuous unrest caused by the covid19 pandemic, considering the 1,886,643 new cases recorded as of 7th september, 2020 which has spiked up the total reported cases to over 26.7 million and a total of 876,616 deaths globally, there is currently no available fda-approved vaccine against covid-19 (57). if a vaccine is successfully developed against covid-19, this will improve global human health. advancement in technology has led to various bioinformatic approaches such as reverse vaccinonology (rv) and immune-informatics being applied to revolutionize vaccine production in terms of production cost and time which has been a major setback in vaccine development. antibody response as well as cell mediated immunity can be established by using proper protein antigen. reverse vaccinology (rv) has been useful in identification of potential vaccine candidates against pathogens and the world is in a race to get one against sars-cov2. this approach presents an advantage over other vaccine approaches because it can identify all potential antigens coded by a genome irrespective of their abundance, phase of expression and immunogenecity. while most of the previous protein vaccines against sars-cov have focused on the full length spike protein of the virus, the vaccine development race against the covid19 virus have followed the same path, with a larger percentage of the 69 of the 210 vaccines currently in development utilizing the spike protein of the sars-cov-2 (58) not minding the reported negative side effects, including liver damage and antibody mediated inflammatory diseases, of the full length spike protein vaccines and other whole organism vaccines in the host. additionally, while novel technologies and approaches such as next-generation vaccines enabled through advances in nanotechnology which relies on the principle that nanoparticles and viruses operate at the same length scale are poised to make a clinical impact for the first time, many of these platforms may be several years away from deployment and therefore may not have an impact on the current sars-cov-2 pandemic, further necessitating the priority given to the quicker and safer vaccine development platforms. the current study focused on the hla-1 binding propensities of some uncommon proteins from covid-19 virus. obtaining these antigenic epitopes using the immune-informatics approach will help inform which epitopes to use in the construct of protein vaccines against sars-cov-2 using proteins other than the spike protein. from the 8 selected proteins obtained all the models with good stability analysis including the ramachandran analysis, which is a two dimensional plot drawn in the space of angles ϕ and ψ that quantify the rotation of the protein backbone and thus play a crucial role in the secondary structure of proteins and later the tertiary structure, were further analyzed for antigenicity and only highly antigenic proteins were selected for further analysis since the highly antigenic proteins will induce better immune response in the host. five (5) of the 35 most common hla-1 subtypes in africa and asia that were identified in this study were included in the analyses and their potential interactions between identified epitopes from the proteins were predicted using the netmhcpan 4.1 server. the binding affinities between these hla subtypes and the epitopes were indicated by the server which allowed the selection of epitopes that will have strong interactions with the hla alleles. the analysis of this interaction is important because vaccination is a proven strategy for protection from disease and an ideal vaccine would include antigens that elicit a safe and effective protective immune response. hla-restricted epitope vaccines, including t-lymphocyte epitopes restricted by hla alleles, embodies a newly developing and favorable immunization approach. research in hla restricted epitope vaccines to be used for the treatment of tumors as well as for the prevention of viral, bacterial, and parasitic infections have, in recent years, achieved substantial progress and this further strengthens the resolve of the hla-binding propensities analysis done in this study. the predicted strongest binding epitopes were thus further subjected to immunogencity evaluation which checked the ability of the epitope to induce an immune response and also predicted how strongly they will induce an immune response; allergenicity which verified the potential of our epitopes to cause sensitization or allergic reactions related to ige antibody responses; and toxicity which is its ability to cause bodily harm. using these criteria, 10 epitopes were identified and these could be potential epitopes for vaccine constructs against sars-cov-2. this was also evinced by the lower global binding energies between our identified epitopes and the hla molecules, ranging from -68.71 to -11.19, indicating stable complexes formed between them. our extensive approach towards the prediction of stable protein structures from previously uncharacterized proteins and the subsequent application of immune-informatics for the identification of immunogens has resulted in the classification of 10 different epitope sites from 8 different proteins that have high antigenicity and low binding energies with hla-1 alleles that are most commonly present in the continent of asia and africa. these epitopes have high tendencies to provide effective and strong protective cytotoxic immunity against the sars cov-2 if they are adequately exploited for vaccine production. a multiple consensus approach ensures the reliability and reproducibility of these results. this study thus provides further knowledge on the association of these hla-1 alleles with covid19, suggesting that good peptide coverage can be achieved by many different combinations of hla-1 alleles. multi-epitope based peptide vaccine design using three structural proteins (s, e, and m) of sars-cov-2: an in silico approach knowledge, attitude, practice and perceived barriers among healthcare professionals regarding covid-19: a cross-sectional survey from pakistan naming the coronavirus disease (covid-19) and the virus that causes it a novel coronavirus from patients with pneumonia in china a pneumonia outbreak associated with a new coronavirus of probable bat origin prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen honglong wuet al. genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding characterization of the coronavirus m protein and nucleocapsid interaction in infected cells shuliang zhouet al. clinical course and outcome of 107 patients infected with the novel coronavirus, sars-cov-2, discharged from two hospitals in wuhan raúl gómez román, stig tollefsen, melaniesaville. the covid-19 vaccine development landscape progress and prospects on vaccinedevelopment against sars-cov-2.vaccines(basel) sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor molecular biology of flaviviruses use of serogroup bmeningococcal vaccines in persons aged ≥ 10 years at increased risk for serogroup b meningococcal disease: recommendations of the advisory committee on immunization practices recent progress in adjuvant discovery for peptide based subunit vaccines preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets evaluation of modified vaccinia virus ankara based recombinant sars vaccine in ferrets hla studies in the context of coronavirus outbreaks uniprot: a worldwide hub of protein knowledge the proteomics server for in-depth protein knowledge and analysis the embl-ebi search and sequence analysis tools apis in 2019 peptide hydrophobicity/hydrophilicity analysis tool critical assessment of methods of protein structure prediction (casp)-round xiii swiss-model: homology modelling of protein structures and complexes lomets2: improved meta-threading server for fold-recognition and structure-based function annotation for distant-homology proteins intfold: an integrated server for modelling protein structures and functions from amino acid sequences the i-tasser suite: protein structure and function prediction falcon: a high-throughput protein structure prediction server based on remote homologue recognition the galaxy platform for accessible, reproducible and collaborative biomedical analyses template-based protein structure modeling using the raptorx web server ab initio protein structure assembly using continuous structure fragments and optimized knowledge-based force field protein structure refinement driven by side-chain repacking automated minimization of steric clashes in protein structures procheck: a program to check the stereochemical quality of protein structures verify3d: assessment of protein models with three dimensional profiles verification of protein structures: patterns of nonbonded atomic interactions deviations from standard atomic volumes as a quality measure for protein crystal structures scoop: an accurate and fast predictor of protein stability curves as a function of temperature the gromos biomolecular simulation program package a server for prediction of protective antigens, tumour antigens and subunit vaccines gold-standard data classification, open access genotype data and new query tools netmhcpan-4.1 and netmhciipan-4.0: improved predictions of mhc antigen presentation by concurrent motif deconvolution and integration of ms mhc eluted ligand data the immune epitope database (iedb): 2018 update silico approach for predicting toxicity of peptides and proteins pep-fold3: faster de novo structure prediction for linear peptides in solution and in complex ucsf chimera -a visualization system for exploratory research and analysis patchdock and symmdock: servers for rigid and symmetric docking firedock: a web server for fast interaction refinement in molecular docking using evolutionary data to raise testable hypotheses about protein function consurf: identification of functional regions in proteins by surface-mapping of phylogenetic information development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines coronavirus disease (covid-19) situation reports milken institute covid-19vaccinetracker table 3. immunogenicity analysis of predicted mhc class 1 epitopes we acknowledge the support of dnacompass, cmesbahf nigeria, biotrust scientific, galaxy project and neliref for this project. we also acknowledge the provision of figure images generated by rajavee srivastava and folagbade abitogun from ucsf chimera, discovery studio, pymol, mhc cluster server v 2.0 and consurf. we acknowledge t. thao, h. aminu, and v. ekundayo for their contribution to this project. folagbade abitogun is a research associate at the university college hospital, ibadan, nigeria.he is currently actively involved in antimicrobial resistant research and he has deep interest in the interplay between the human immune system and the clearance of infectious diseases, using molecular and bioinformatics approaches. coloured 'e' represents exposed residues, the green colored 'b' represents buried residues, the red colored 'f' represents functional residues (highly conserved and exposed) and the dark blue colored 's' represents structural residues (highly conserved and buried). the conservation scale represents the status of conservation from variable, average to conserved. key: cord-351559-az4pgi9k authors: turjya, rafeed rahman; khan, md. abdullah-al-kamran; islam, abul bashar mir md. khademul title: perversely expressed long noncoding rnas can alter host response and viral proliferation in sars-cov-2 infection date: 2020-06-29 journal: biorxiv doi: 10.1101/2020.06.29.177204 sha: doc_id: 351559 cord_uid: az4pgi9k background since december 2019, the world is experiencing an unprecedented crisis due to a novel coronavirus, sars-cov-2. owing to poor understanding of pathogenicity, the virus is eluding treatment and complicating recovery. regulatory roles of long non-coding rnas (lncrnas) during viral infection and associated antagonism of host antiviral immune responses has become more evident in last decade. to elucidate possible functions of lncrnas in the covid-19 pathobiology, we have utilized rna-seq dataset of sars-cov-2 infected lung epithelial cells. results our analyses uncover 21 differentially expressed lncrnas whose functions are broadly involved in cell survival and regulation of gene expression. by network enrichment analysis we find that these lncrnas can directly interact with differentially expressed protein-coding genes adar, edn1, kynu, mall, tlr2 and ywhag; and also akap8l, exosc5, gdf15, hectd1, larp4b, larp7, mipol1, upf1, mov10 and prkar2a, host genes that interact with sars-cov-2 proteins. these genes are involved in cellular signaling, metabolism, immune response and rna homeostasis. since lncrnas have been known to sponge micrornas and protect expression of upregulated genes, we also identified 9 micrornas that are induced in viral infections; however, some lncrnas are able to block their usual suppressive effect on overexpressed genes and consequently contribute to host defense and cell survival. conclusions our investigation determines that deregulated lncrnas in sars-cov-2 infection are involved in viral proliferation, cellular survival, and immune response, ultimately determining disease outcome and this information could drive the search for novel rna therapeutics as a treatment option. by mid-may 2020, the confirmed cases of covid-19 have crossed 4 million (1), making it 58 one of the largest pandemics in modern times. since the first reported case at wuhan, china 59 in december 2019 (2), this viral disease has been responsible for more than 300,000 deaths 60 worldwide (1), and the number is steadily rising. the causative agent behind the disease is a 61 novel beta-coronavirus (3), which has been named sars coronavirus 2 i.e. sars-cov-2 62 due to its similarity to the earlier sars-cov first detected in 2002 (4). the rapid spread of 63 the virus, the ever-increasing death toll, and absence of a sufficient treatment strategy has 64 affected societies and economies all over the globe. 65 the molecular mechanism of sars-cov-2 is complex and interrelated with host 66 mechanisms, as is common with most pathogenic viruses (5). it is possible that infected lung performing rna-seq analysis to identify differentially expressed (de) genes. but no such 103 study yet concluded the possible outcomes of the deregulated lncrnas in sars-cov-2. in 104 our present study we have identified lncrnas that are differentially expressed in sars-105 cov-2 infected cell's transcriptome compared to uninfected cells, and then correlated the 106 putative effects of the deregulated lncrnas in the tug-of-war between sars-cov-2 and the 107 host. we have also investigated the possible aftermaths of lncrna deregulation in covid-108 19 disease pathobiology. 109 to investigate the probable deregulation of lncrnas in sars-cov-2 infection, firstly we 112 have analyzed the 24 hours post-infection transcriptome data of sars-cov-2 infected 113 nhbe cells. analyzing the rna-seq data led to identification of 687 de genes and 114 intriguingly, amongst those de genes, we discovered 21 lncrna genes, 9 of them 115 upregulated while 12 were downregulated (figure 1 ). we now sought to elucidate the putative effects of these deregulated lncrnas in sars-119 cov-2 infection. in order to achieve that, we built a network of the deregulated lncrnas 120 along with their interacting protein coding target genes. among the differentially expressed 121 protein-coding genes, 6 were found to interact with the de lncrnas which (table 1) . there 122 are direct rna-rna interactions for 4 genes, and rna-protein interactions for the rest 123 ( figure 2 ). these lncrna interacting protein coding genes have potential roles during the 124 viral infections (table 1) innate immune response against the infection is inevitable, but the complex interactions 306 underlying its activation and effect may have been the target of lncrnas.hectd1 is an e3 307 ubiquitin-protein ligase that interacts with nsp8. as it is involved in class i mhc mediated 308 antigen processing and presentation and innate immune system, the binding may lead to 309 modulation of that response. in case of hectd1 mrna, absence of rna-rna interaction 310 with downregulated kcnq1ot1 lncrna can facilitate the response. ptges2 gene was 311 upregulated and also found to interact with sars-cov-2 nsp7 protein. as it is involved in 312 innate immune system and signaling pathways, this binding may exert an indirect influence. were analyzed using networkanalyst 3.0 (88) tools for functional overrepresentation and 386 network enrichment. mirnas that mostly targeted upregulated genes were finally selected. 387 not applicable. 395 publicly available data were utilized. analyses generated data are deposited as supplementary 397 files. 398 the authors declare that they have no competing interests. 400 funding 401 this research did not receive any specific grant from funding agencies in the public, 402 commercial, or not-for-profit sectors. 403 exosc5 is a non-catalytic component of the rna exosome complex which has 3' to 5' exoribonuclease activity and participates in a multitude of cellular rna processing and degradation events (121). protein-rna gdf15is a member of the glial cell-derived neurotropic factor (gdnf) family which binds to gdnf family receptor α -like (gfral) protein, a transmembrane receptor exclusively expressed in the hind brain (122). the protein is expressed in a broad range of cell types, acts as a pleiotropic cytokine and is involved in the stress response program of cells after cellular injury (123). increased protein levels are associated with disease states such as tissue hypoxia, inflammation, acute injury and oxidative stress (124, 125). meg3 rna-rna hectd1 is an e3 ubiquitin-protein ligase which accepts ubiquitin from an e2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates and mediates 'lys-63'-linked polyubiquitination of hsp90aa1 which leads to its intracellular localization and reduced secretion (126). the protein is involved in class i mhc mediated antigen processing and presentation and innate immune system. kcnq1ot1 rna-rna larp4bencodes a la-module containing factor that can bind au-rich rna sequence directly and promotes mrna accumulation and translation (127). it was deemed a candidate tumor suppressor gene in glioma, as it was consistently decreased in human glioma stem cells and cell lines compared with normal neural stem cells. larp4b overexpression strongly inhibited cell proliferation by inducing mitotic arrest and apoptosis andcdkn1a and bax were upregulated (128). nsp12 protein-rna larp7 works as a negative transcriptional regulator of polymerase ii genes, acting by means of the 7sk rnp system (129). this snrnp complex inhibits a cyclin-dependent kinase, positive transcription elongation factor b, which is required for paused rna polymerase ii at a promoter to begin transcription elongation (130). protein-rna prkar2a is the camp-dependent protein kinase type ii-alpha regulatory subunit, which works as the regulatory subunit of the camp-dependent protein kinases involved in camp signaling in cells (131). outbreak of a novel coronavirus a new coronavirus 416 associated with human respiratory disease in china epidemiology and 418 cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of 419 china the epidemiology and pathogenesis of coronavirus 421 disease (covid-19) outbreak host immune response and 423 immunobiology of human sars-cov-2 infection low expression of long 574 noncoding rna gas6-as1 predicts a poor prognosis in patients with nsclc low expression of long noncoding rna gas6-577 as1 as a novel biomarker of poor prognosis for breast cancer rotavirus 580 nsp4 triggers secretion of proinflammatory cytokines from macrophages via toll-like 581 receptor 2 pattern 583 recognition receptors tlr4 and cd14 mediate response to respiratory syncytial virus reading the viral signature by toll-like receptors and 586 other pattern recognition receptors lncrna kcnq1ot1 592 attenuates sepsis-induced myocardial injury via regulating mir-192-5p/xiap axis acute respiratory distress syndrome. american journal of 596 respiratory and critical care medicine kcnq1ot1 promotes migration and inhibits apoptosis 598 by modulating mir-185-5p/rab14 axis in oral squamous cell carcinoma lncrna meg3 aggravates palmitate-induced 601 insulin resistance by regulating mir-185-5p/egr2 axis in hepatic cells. european review for 602 medical and pharmacological sciences the long non-coding rna meg3/mir-let-7c-5p axis regulates ethanol-induced hepatic steatosis and apoptosis by 605 targeting nlrc5 607 ncbi geo: archive for functional genomics data sets-update tophat: discovering splice junctions with rna-610 initial 612 sequencing and analysis of the human genome biases in illumina transcriptome sequencing 614 caused by random hexamer priming the subread aligner: fast, accurate and scalable read 618 mapping by seed-and-vote differential expression analysis for sequence count data string 622 v11: protein-protein association networks with increased coverage, supporting functional 623 discovery in genome-wide experimental datasets cytoscape: a 626 software environment for integrated models of biomolecular interaction networks npinter v4.0: an integrated 629 database of ncrna interactions lncbook: a curated knowledgebase of 631 human long non-coding rnas mirtarbase 633 2020: updates to the experimentally validated microrna-target interaction database. nucleic 634 acids research diana-lncbase v3: indexing experimentally supported mirna targets on 637 non-coding transcripts networkanalyst 3.0: a 639 visual analytics platform for comprehensive gene expression profiling and meta-analysis. 640 adenosine deaminase acting on rna (adar1), a suppressor of double-642 stranded rna-triggered innate immune responses 645 rna editing by adar1 prevents mda5 sensing of endogenous dsrna as nonself new antiviral pathway 648 that mediates hepatitis c virus replicon interferon sensitivity through adar1 rna adenosine deaminase adar1 deficiency leads to 651 increased activation of protein kinase pkr and reduced vesicular stomatitis virus growth 652 following interferon treatment adenosine deaminase acting on rna 1 (adar1) 657 suppresses the induction of interferon by measles virus the large 660 form of adar 1 is responsible for enhanced hepatitis delta virus rna editing in interferon-661 alpha-stimulated host cells editing of hiv-1 rna by the 663 double-stranded rna deaminase adar1 stimulates viral infection pyk2 mediates 669 endothelin-1 signaling via p130cas/bcar3 cascade and regulates human glomerular 670 mesangial cell adhesion and spreading the role of endothelin-1 in pulmonary arterial 672 hypertension purification and biochemical characterization of some of the 674 properties of recombinant human kynureninase cloning and 677 recombinant expression of rat and human kynureninase bene, a novel raft-associated protein of the mal proteolipid family, 680 interacts with caveolin-1 in human endothelial-like ecv304 cells. the journal of biological 681 chemistry a novel gene 683 that encodes a protein with a putative src homology 3 domain is a candidate gene for familial 684 juvenile nephronophthisis the role of tlr2 in infection and 686 immunity mycobacterium 688 tuberculosis lipoproteins directly regulate human memory cd4(+) t cell activation via toll-689 like receptors 1 and 2. infection and immunity 691 tlr2 and its co-receptors determine responses of macrophages and dendritic cells to 692 lipoproteins of mycobacterium tuberculosis host defense mechanisms triggered by microbial lipoproteins through toll-like receptors. 695 science cell 697 activation and apoptosis by bacterial lipoproteins through toll-like receptor-2 14-3-3 700 γ associates with muscle specific kinase and regulates synaptic gene transcription at 701 vertebrate neuromuscular synapse resistance 704 exercise and insulin regulate as160 and interaction with 14-3-3 in human skeletal muscle. 705 diabetes 14-3-3gamma interacts with and is phosphorylated by 707 multiple protein kinase c isoforms in pdgf-stimulated human vascular smooth muscle cells. 708 dna and cell biology jnk phosphorylation of 14-710 3-3 proteins regulates nuclear targeting of c-abl in the apoptotic response to dna damage. 711 functional conservation of 14-3-3 713 isoforms in inhibiting bad-induced apoptosis kank regulates rhoa-dependent 716 formation of actin stress fibers and cell migration via 14-3-3 in pi3k-akt signaling mir-181b-3p promotes 719 epithelial-mesenchymal transition in breast cancer cells through snail stabilization by directly 720 targeting ywhag microrna-182 promoted esophageal squamous cell 722 carcinoma cell growth and metastasis via targeting ywhag wong-staal f. a novel shuttle protein 725 binds to rna helicase a and activates the retroviral constitutive transport element. the 726 wong-staal f. mapping the functional domains of 728 hap95, a protein that binds rna helicase a and activates the constitutive transport element 729 of type d retroviruses protein kinase a associates 731 with ha95 and affects transcriptional coactivation by epstein-barr virus nuclear proteins. 732 molecular and cellular biology the role of a-kinase anchoring protein 95-like 734 protein in annealing of trnalys3 to hiv-1 rna the 736 mammalian exosome mediates the efficient degradation of mrnas that contain au-rich 737 elements growth differentiation factor 15 (gdf15): a survival protein with 739 therapeutic potential in metabolic diseases cardiovascular diseases: a translational prospective tgf-b 743 superfamily cytokine mic-1/gdf15 is a physiological appetite and body weight regulator 746 gfral is the receptor for gdf15 and the ligand promotes weight loss in mice and 747 nonhuman primates hectd1 regulates intracellular localization and secretion of 749 hsp90 to control cellular behavior of the cranial mesenchyme 752 larp4b is an au-rich sequence associated factor that promotes mrna accumulation and 753 translation identification of rna-755 binding protein larp4b as a tumor suppressor in glioma la-related protein larp7 is a component of the 7sk ribonucleoprotein and affects 759 transcription of cellular and viral polymerase ii genes functional characterization of a 764 candidate tumor suppressor gene, mirror image polydactyly 1, in nasopharyngeal carcinoma human upf proteins target an mrna for 767 nonsense-mediated decay when bound downstream of a termination codon molecular mechanisms for the rna-dependent atpase activity of upf1 and its regulation by 771 upf2 binding 773 of a novel smg-1-upf1-erf1-erf3 complex (surf) to the exon junction complex triggers 774 upf1 phosphorylation and nonsense-mediated mrna decay 777 mov10 is a 5' to 3' rna helicase contributing to upf1 mrna target degradation by 778 translocation along 3' utrs 780 identification of novel argonaute-associated proteins apobec3g inhibits 783 microrna-mediated repression of translation by interfering with the interaction between 784 microrna silencing through risc recruitment of eif6 virus escape and manipulation of cellular 788 nonsense-mediated mrna decay provides antiviral activity against rna viruses by enhancing rig-i-mavs-independent 791 ifn induction not applicable. 409 key: cord-341378-pw60qx7c authors: armstrong, john; niemann, heiner; smeekens, sjef; rottier, peter; warren, graham title: sequence and topology of a model intracellular membrane protein, e1 glycoprotein, from a coronavirus date: 1984 journal: nature doi: 10.1038/308751a0 sha: doc_id: 341378 cord_uid: pw60qx7c in the eukaryotic cell, both secreted and plasma membrane proteins are synthesized at the endoplasmic reticulum, then transported, via the golgi complex, to the cell surface(1–4). each of the compartments of this transport pathway carries out particular metabolic functions(5–8), and therefore presumably contains a distinct complement of membrane proteins. thus, mechanisms must exist for localizing such proteins to their respective destinations. however, a major obstacle to the study of such mechanisms is that the isolation and detailed analysis of such internal membrane proteins pose formidable technical problems. we have therefore used the e1 glycoprotein from coronavirus mhv-a59 as a viral model for this class of protein. here we present the primary structure of the protein, determined by analysis of cdna clones prepared from viral mrna. in combination with a previous study of its assembly into the endoplasmic reticulum membrane(9), the sequence reveals several unusual features of the protein which may be related to its intracellular localization. in the eukaryotic cell, both secreted and plasma membrane proteins are synthesized at the endoplasmic reticulum, then transported, via the golgi complex, to the ceu surface 1 -4. each of the compartments of this transport pathway carries out particular metabofic functions 5-8, and therefore presumably contains a distinct complement of membrane proteins. thus, mechanisms must exist for locafizing such proteins to their respective destinations. however, a major obstacle to the study of such mechanisms is that the isolation and detailed analysis of such internal membrane proteins pose formidable technical problems. we have therefore used the el glycoprotein from coronavirus mhv • a59 as a viral model for this class of protein. here we present the primary structure of the protein, determined by analysis of edna clones prepared from viral mrna. in combination with a previous stu'!} of its assembly into the endoplasmic reticulum membrane , the sequence reveals several unusual features of the protein which may be related to its intracellular localization. the coronaviruses are a diverse class of enveloped rna viruses of considerable medical and agricultural significance; they also provide a model for the study of persistent viral infections (see ref. 10 for review). in contrast to many enveloped viruses, the corona virus mouse hepatitis virus (mhv) a59 buds inside the cell, into the lumen of the endoplasmic reticulum 11 the assembled virion then appears to travel, via the golgi complex, to the cell surface. of the two viral membrane proteins, the smaller one, el, is necessary for formation of the envelope, and is restricted to internal cell membranes; apparent?; it only reaches the cell surface as part of the budded virion 12 • 3 . thus, the el glycoprotein is potentially a convenient model for studying those features of a membrane protein that determine its arrest at a particular destination on the membrane transport pathway. the mrnas of mhv-a59 form a 'nested set': the seven rnas share the 3' region of the positive-stranded genome, but extend to different lengths towards the 5' end 15 -18 • from each rna, only the 5' gene is translated 19 ' 20 . in addition, a noncoding 'leader' sequence of approximately 70 bases, from the 5' end of the genome, is common to the mrnas 18 • 21 • 22 . the e1 gene is second from the 3' end and is therefore translated from the second smallest mrna, rna 6 (refs 19, 20) . the sequence of the 3' -terminal gene, encoding the viral nucleocapsid protein, has been determined previously 23 • 24 • copy dna clones spanning the e1 gene were prepared by two methods 23 -25 and sequenced in the vectors m13mp8 (ref. two versions were found, in two different clones, for the sequence immediately upstream from the e1 initiator codon. the shorter one is shown in fig. 1 ; in the second clone, an additional copy of the pentanucleotide a tct a was found between nucleotides 65 and 66, making the sequence similar to that of the region adjacent to the nucleocapsid gene of another strain of mhv 29 . this difference could represent a mutation; alternatively, it may reflect heterogeneity in the normal mrna population. indirect support for the latter possibility comes from the observation that a rnase-t 1 oligonucleotide from this region of rna 6, corresponding to the shorter sequence, was recovered in markedly lower yield than those from the rest of the molecule 30 • this site represents the point of fusion between the 5' leader sequence and the coding portion of the rna. the fusion is thought to occur by 'jumping' of the viral rna polymerase to particular sites on its genome-length, negativestranded template; the resumption of transcription then produces each of the subgenomic mrnas 22 " 31 ' 32 • thus, it seems possible that the polymerase may jump to more than one point on the template for each mrna, generating variable numbers of the repeated pentanucleotide aucua in the resulting transcript. figure 1 shows the amino acid sequence encoded by the e1 gene. the predicted molecular weight of the protein is 26,000, slightly higher than that observed by gel electrophoresis 19 • 33 but consistent with the unusual electrophoretic behaviour of this 33 , and other, hydrophobic proteins. several features of the protein, when assembled into membranes in the virus 33 , or in vitro 9 , are reflected in the sequence. first, in contrast to the majority of membrane proteins, el is known to lack a cleaved 'signal peptide' 9 : the n-terminal region of the sequence contains no good candidate for a cleava3e site 34 • second, the n-terminal region bears 0-linked sugars 5 ' 36 , which, uniquely among viral proteins so far studied, are the only known post-translational modification to el. assuming that the terminal met is removed 37 , then-terminal sequence is ser-ser-thr-thr, which is identical to the 0-glycosylated amino terminus of m-type glycophorin a (ref. 38 ). the 0-linked sugars of el are themselves identical to those found in glycophorin 39 . third, most of the protein is resistant to proteolysis when assembled in the membrane. only 2.5 kilodaltons of polypeptide from the nterminus are cleavable on the luminal side of the membrane (or outside the virion) and 1.5 kilodaltons from the c-terminus from the cytoplasmic (or intra-virion) side 9 , suggesting that the protein is largely buried in the membrane. in the sequence, a run of 22 uncharged residues from positions 26 to 4 7 represents a potential membrane-spanning region; residues 1-25 correspond to the portion removable by protease. a further sequence of uncharged residues, positions 57-106, is sufficiently long to cross the membrane twice more. if this region is divided in two, and each half plotted as an a-helical 'wheel', all the polar side chains of both sections cluster within 140°. thus, a plausible conformation for this region is two hairpinned helices in the membrane, with adjacent polar faces (fig. 2a) . there are no other long hydrophobic sequences, implying that the region from residues 107 to -190 is either folded in the membrane to neutralize charges, or, more likely, is adjacent to the membrane but resistant to proteolysis. these features are summarized in fig. 2b . which, if any, of these various features might be responsible for the protein's intracellular localization? we do not know, for example, whether the protein has an active 'signal' causing its arrest on the transport pathway, or, alternatively, if it lacks a signal for onward transport; nor do we know whether a sorting process might operate on one or the other side of the membrane. the availability of a edna clone for the protein presents the opportunity to investigate these questions by allowing expression of the cloned dna and in vitro mutagenesis. this approach has already been applied to two other viral glycoproteins, to investigate the importance of their cytoplasmic domains for transport to the cell surface, yielding opposite conclusions 40 • 41 • an intrinsic problem with the method, however, is the difficulty of distinguishing specific effects due to alterations at the site of mutagenesis, from a general structural disruption of the molecule. in this respect the e1 protein may be advantageous in that it provides the possibility of creating a more 'active' phenotype in the mutated molecule: specifically, particular alterations to the protein may result in its transport to the cell surface. we thank willy spaan for communicating results before publication, g. heisterberg-moutsis (g. b. f. braunschweig) for help with oligonucleotide synthesis, ben van der zeijst for discussion and annie steiner for preparing the manuscript. j.a. was supported by fellowships from the royal society and the outside (lumenl fig. 2 a, distribution of polar side chains in the hydrophobic regions of the el sequence. residues (1), 57-81 (2) and 82-106 (3) are plotted as a-helices and viewed endon. polar side chains are boxed: proposed hydrophilic faces of helices 2 and 3 are indicated. b, possible topologies of the el protein across the membrane. arrows indicate sites accessible to protease; broken arrows represent inefficient pro-teolysis9. european molecular biology organization, h.n. by the deutsche forschungsgemeinschaft, sfb4 7 (virologie), teilprojekt b3, and p.r. by a short-term embo fellowship. some of these results have been presented in preliminary form elsewhere 24 • 25 • proc. natn. acad. sc< u.s.a molecular biology and pathogenesis of coronaviruses molecular biology and pathogenesis of coronaviruses key: cord-335915-2apj4qy9 authors: melillo, alessandro title: applications of serum protein electrophoresis in exotic pet medicine date: 2013-01-22 journal: vet clin north am exot anim pract doi: 10.1016/j.cvex.2012.11.002 sha: doc_id: 335915 cord_uid: 2apj4qy9 serum protein electrophoresis (spe) is a useful diagnostic and prognostic tool in human and companion animals medicine: several experiences show that it can be useful in exotic practice as well. the fundamentals of spe interpretation as well as some normal and pathological patterns for the species most commonly seen in practice are provided. allowed to clot in a test tube for 15 to 20 minutes, the time needed for fibrinogen to be used in the coagulation cascade. in several exotic species, especially of small size, the need to work with small volumes of blood requires the clinician to collect the blood with an anticoagulant (frequently natrium heparin or lithium heparin, sometimes heparinizing the syringe to avoid the coagulation of the sample during collection); therefore, centrifugation is achieved in plasma. the main difference between the 2 products is the absence in the serum of fribrinogen, the protein involved in the processes of coagulation; the concentration of total solids of the plasma is thus slightly higher than that of serum (about 5%) and the electrophoretic pattern from it will result in a higher incidence of b-globulin fraction where the fibrinogen normally migrates. in clinical practice, the normal plasma protein concentration is measured very simply by using a refractometer: a few drops of blood are collected in a heparinized capillary whose centrifugation gives the value of the microhematocrit (percentage of blood occupied by cells compared with plasma) and a small amount of plasma. refractometry of that plasma allows easy estimation of the concentration of total solids. this method is simple and intuitive; however, it is not reliable if the sample quality is poor. among the factors that interfere with the measurement of total solids by refractometer include hemolysis, lipemia, hyperbilirubinemia, azotemia, severe hyperglycemia, hypernatremia, hyperchloremia, and administration of colloids, including synthetic hemoglobin-based oxygen carriers. in the absence of these factors, the amount of nonprotein solids present in plasma is relatively constant, therefore by subtracting the nonprotein component (1.5 g/dl) the value of total plasma proteins is obtained. an increase in the concentration of total solids (hyperproteinemia) must always be evaluated in correlation with the microhematocrit value and reflects mainly dehydration or increased synthesis of globulins for various pathologic conditions. the decrease in total solids (hypoproteinemia), vice versa, may reflect overhydration, decreased synthesis of albumin or immunoglobulin, or protein loss associated with hemorrhage, vasculitis, nephropathy, or enteropathy. with the exception of bleeding, which causes a loss of balanced albumins and globulins, in all pathologies involving protein loss albumin concentration falls to a greater extent than that of the globulins because of lower dimensions of the molecule, which allow easier migration through vascular endothelia. the measurement of total protein is certainly very useful, but for the purposes of a diagnosis that is as accurate as possible, it becomes essential to know the alterations of each protein fraction. for this purpose is used the characteristic of plasma proteins to be separated based on their charge and the consequent mobility in an electric field specially created. this method defines electrophoresis of proteins. the distribution of protein migrants on strips of cellulose acetate or agarose gel is represented graphically by a curve in which the extension and the height of each peak is corresponding to the breadth and the density of each protein fraction on the substrate migration. normally, 5 protein fractions are identified in the mammalian plasma, ordered according to the electric charge: albumin (high negative charge and low molecular weight, migrates to the anode and conventionally to the left of the track), a1-globulin, a2-globulin, and b-globulins and g-globulins that present the greatest molecular weight and the lowest negative charge and then migrate to the right end of the curve. a different protein fraction is present in most birds and reptiles, with an even more negative charge than albumin that is positioned at the left in the same electrophoretic pattern and is therefore referred to as prealbumin. conventionally, you find a point halfway between the beginning and end of the curve as the limit between the a2-globulin and b-globulins, and a point halfway to the start of b-globulins and the end of the track as the start of g-globulin. with the exception of albumin, each peak represents a large number of different proteins that can be further separated by more complex examinations. prealbumin is the protein fraction that migrates more rapidly and is positioned on the track before albumin. in species that have it, this is calculated with albumin in the ratio a/g. it is not normally detectable in mammals, except occasionally in cats, and it is normally found in human patients where it has the function of direct transport of thyroid hormones and indirectly of vitamin a by acting as a carrier protein that binds retinol. in the various avian species, it is present in variable amounts. for example, in the budgerigar (melopsittacus undulatus) or in the cockatiel (nymphicus hollandicus) it can constitute 75% of the total prealbumin 1 albumin, whereas in african gray parrots (psittacus erythacus), it represents no more than 10% of total albumin and can also be completely absent. 1 in reptiles, the study of plasma proteins is still sporadic and fragmentary data exist, but migrating prealbumin has been reported in some species of chelonians: caretta caretta, 2 geochelone radiata, 3 chelonia mydas, 4 and sauria (gallotia spp), 5 as well as crocodiles (caiman spp), but only with subsequent migration on polyacrylamide gels 6 apparently have similar transport functions, as in other orders. albumin constitutes the main fraction of the plasma proteins (in general between 35% and 50% of total protein and up to 60% to 65% in primates, including humans) and represents the main and more homogeneous peak in the electropherograms of all species. the main functions of albumin are to carry many molecules and maintain the oncotic pressure of the blood. 7 the group of globulins include the acute phase proteins (apps; a-globulins and b-globulins) and immunoglobulins (g-globulins). apps are the body's response to trauma, inflammation, or infection. the local activation of granulocytes and macrophages causes the release of cytokines (mainly interleukin-6) that stimulate the liver to produce a series of glycoproteins. alpha-1antitrypsin, a-1-acid glycoprotein, a2-macroglobulin, c-reactive protein, haptoglobin, and fibrinogen are positive apps, because their concentration increases in response to inflammation; albumin is considered a negative app, as its synthesis decreases during inflammation, diverting liver protein synthesis to the proteins mentioned previously. the electrophoretic pattern of the apps migrates to the peak of the a (a-1-acid glycoprotein, a2-macroglobulin, haptoglobin, protein c) and b (c-reactive protein, fibrinogen, complement, ferritin)-globulins, but with huge specific differences. the group of g-globulins is mainly composed of immunoglobulins, although some apps, such as degradation products of the complement, can migrate into this area as well, especially in avian species. immunoglobulins are quite impressive in size, consisting of 4 amino acid chains, 2 so-called "heavy" and 2 "light." depending on the composition of the heavy chains, immunoglobulins are divided into classes named by letters of the greek alphabet: gamma (igg), mu (igm), alpha (iga), and epsilon (ige). the igg antibodies are produced in response to various bacterial, viral, or toxic stimuli; ige (not present in all species) is mainly involved in allergic reactions; iga is primarily responsible for the defense processes located at the mucosal level; and igm has the characteristic of being active in pentameric form (ie, joining in groups of 5), thereby forming a protein complex of imposing size. igm is secreted by b-lymphocytes and early plasma cells and its main function is to activate complement, whereas igg is produced later and then plays a predominant action of opsonization, binding to applications of serum protein electrophoresis the pathogen and allowing phagocytosis by macrophages. it is normally not possible to differentiate igm from igg through normal electrophoresis, but distinguishing the ones by the others is very important because it allows us to date the pathologic process in acute (igm only) or chronic (igg with or without igm) and to differentiate an active process (igm) by a seropositivity alone (igg). electrophoresis serum is used as a "screening" of interpretation of the humoral response and during not specific processes. recent studies in rabbits have tried to bring to the surface the true diagnostic value of this report, in particular we tried to find a link between hypergammaglobulinemia and one of the diseases most commonly encountered in clinical practice veterinary medicine in lagomorphs: encephalitozoon cuniculi. the blood for sampling is collected in plain tubes (the use of tubes with accelerant is not possible because of the reduced volume of collectable blood in this species) and left to rest for about 30 minutes. the serum obtained is isolated and frozen at a temperature of -18 c to be examined in the shortest possible time. laboratory data have shown that a single cycle of freezing does not alter the electrophoretic pattern, whereas prolonged freezing determines a variation in the albumin/globulin ratio in favor of the latter. the serum of the rabbit contains, as in other mammals, various protein fractions: albumin, a-globulins, b-globulins, and g-globulins. the a-globulins and b-globulins are generally divided into subunits of a1 and a2, and b1 and b2 (fig. 1) . albumin constitutes the main fraction responsible for the oncotic pressure of body fluids, whereas the a1-globulin, a2-globulin, b-globulins, and g-globulins are a smaller fraction responsible for the humoral response of the organism. unlike the guinea pig, however, the area occupied by b-globulins is wider than that of the a-globulins. in rabbits, the normal serum protein electrophoresis (spe) pattern lacks a clear distinction between b1-globulins and b2-globulins, as present in dogs and cats, but when gammopathies in the b region occur, usually an extension of the electrophoretic band is seen, with consequent demarcation of the 2 peaks. the a1-globulins, together with the a2-globulins and b-globulins, increase in case of acute inflammation, expressing a more rapid response compared with that cell mediated. the g fraction includes circulating immunoglobulins, complement, the degradation products of complement, and to a lesser extent some proteins that characterize the acute phase. 8 fig. 1 . normal pattern for a rabbit. c-reactive protein is one of the first proteins produced by the body following an insult to any tissue. despite being defined as an app, it is also found in the course of chronic processes. 9 placed inside a magnetic field (principle of electrophoresis of proteins), because of its shape, weight, and molecular electrostatic charge, such protein migrates to the right (toward the anode or negative electrode) in the area of b-globulins and g-globulins to influence the path. other apps, such as aptoglobulina, a1 acid glycoprotein, and ceruloplasmin are normally found in the a and b fractions. in course of encephalitozoonosis we have seen a decrease of albumin, primarily because of the increased synthesis by hepatocytes of numerous immunoproteins (especially ig) with consequent widening of the electrophoretic band in the region of b and g. the b-globulin and g-globulin can be found in course of chronic inflammatory processes (like bacterial, viral, or fungal infections), parasitism (e cuniculi), cancer, or immunemediated diseases. in the specific case of protozoal infection by e cuniculi, healthy animals and seropositive animal spe patterns only differ significantly in the g fraction. 10 results are very similar to those obtained by didier in 1995, in a study of dogs showing clinical signs of encephalitozoon infection. 11 the hypergammaglobulinemia during encephalitozoonosis is usually mild to moderate; this value may be altered when rabbits with impaired immune response come in contact with the parasite, which may react with a decrease of circulating igg and a possible increase in igm. 12 the titration of igm and igg allows us to clarify the response against the parasite carried by the body at the time of collection. igm increases usually in the early stages when the parasite exerts its action, decreasing up to be absent on the 38th day, whereas igg increases more slowly in the course of infection but remains detectable for years. 13 the changes of g-globulin have been shown to not be significantly different in animals with igm titratable compared with animals with igg increased. this shows that the hypergammaglobulinemia is an artifact resulting in a chronic inflammatory process. in support of this theory, it is believed that the organism remains latent in the body until it occurs in a suppression of the immune system, a phase in which the body eliminates spores in the urine. 14 contrary to what occurs in avian species, and in accordance with what occurs in dogs, hemolysis increases the b fraction, not the g fraction. in the otherwise rich literature on diseases of the ferret, electrophoresis is quite neglected, mainly because the main source of information on ferret diseases is us research, where electrophoresis is relatively unknown: the only application is described as an aid in diagnosis of aleutian disease. actually, it is a useful diagnostic tool in ferret species and its interpretation is not very different from that of the dog and cat. being that the ferret is an animal of sufficient size, allowing access to the veins of good gauge as the cranial cava, it is recommended to draw blood without anticoagulant (heparin needle) to obtain serum instead of plasma and thus avoid the deposition of fibrinogen, which will inevitably alter the peak of b-globulins. in a healthy ferret, as in dogs, the proteins migrate in the electric field in dividing fractions: albumin, a1-globulin, a2-globulin, b1-globulin, b2-globulin, and g-globulin. albumins represent approximately half of circulating proteins, for which the a/g ratio is about 1. iga migrates in the field of a2 and b1 globulins, igm in the field of b2 globulin or "fast" g globulin, whereas the igg in the field of slow g-globulin (the far end of the track) (fig. 2) . in ferrets, dehydration progresses very quickly in case of insufficient intake or increased losses, so hyperproteinemia is a frequently encountered clinical sign (for example in diarrhea, intestinal obstruction, acute gastritis, or renal failure) but in the case of noninfectious diseases, proteins raise in their entirety for hemoconcentration, so the pattern will result basically normal; in the case of inflammatory diseases, globulins increase, altering the electrophoretic curve. the most characteristic alteration is described in patients suffering from aleutian disease. this disease, fortunately is infrequent (but certainly underdiagnosed) in italy, is caused by a parvovirus (adv) that affects the mustelidae family, including mink and ferret. it is transmitted by feces and all body secretions and is particularly insidious because it evolves as a chronic disease, debilitating the ferret to the invariably fatal outcome. aleutian disease causes lymphoplasmacytic infiltration and precipitation of immune complexes in various tissues from which a variety of symptoms is shown by sick animals. blood tests are often vague, showing nonregenerative anemia, abnormal liver enzymes, and uremia; however, electrophoresis is revealing. patients with adv in fact have a marked hyperproteinemia with values up to 10 to 12 g/dl or more, but at the same time an important hypoalbuminemia: g-globulins rise instead to constitute 20% to 60% of total protein, drawing a characteristic "horned" path and seriously altering the a/g ratio. other diseases can cause hypergammaglobulinemia, although not as marked as adv: other viruses such as influenza or distemper, pyoderma, kidney diseases with no protein loss, certain tumors, and systemic mycoses (blastomycosis, coccidioides spp). another, more frequent, viral disease is catarrhal enteritis (ece) caused by a coronavirus: kits are often asymptomatic carriers for adults who become instead gravely ill. in case of ece, electrophoresis is characterized at the beginning by moderate hypoalbuminemia, which becomes more and more severe during the following weeks. following the evolution of the disease, an increase in a2 or b1 may be noticed, meaning the production of iga in the acute phase, then a peak of gammaglobulines even if significantly below that of adv. after the second or third week, there is often hypogammaglobulinemia, evidence of the failure of the immune response. it is also interesting to note the changes in the electrophoretic pattern in the case of liver disease. in the case of hepatic neoplasia, often the protein curve does not differ much from that of a healthy animal, except for a modest elevation of a2-globulins. more marked is the alteration in case of hepatic lipidosis and acute cholangitischolangiohepatitis, when the increase of a2-globulins is greater and reflects an intense inflammation. chronic hepatitis and cholestasis phenomena, intrahepatic or extrahepatic, are reflected instead in a b-globulin peak, where transferrin, c-reactive protein, and generally the igm migrate. the decrease in the percentage of globulin is uncommon and is usually associated with renal failure or lymphoma. the hypoproteinemia usually indicates a defect in protein synthesis and has a negative prognostic value. 15 the electrophoretic pattern of the guinea pig (cavia porcellus) has been thoroughly studied in the experimental for which we know either paths in different normal farming conditions and adjustment of the normal bacterial flora (specific pathogen free subjects and the like) and the reaction to various pathologies experimentally induced; however, very little is described in the field of clinical practice about the response of animals living in uncontrolled conditions that get sick spontaneously. clinically healthy guinea pig serum proteins are separated into 7 sections: albumin, a1-globulin, a2-globulin, b1-globulin, b2-globulin (often divided into 2 peaks), and g-globulins, and in some subjects prealbumins have been occasionally signaled (fig. 3) . the area occupied by b-globulins and g-globulins in the complex is restricted in comparison with the well-developed area of the a-globulins and a/g ratio is high. by immunoelectrophoresis, up to 30 different protein migrants in those areas can be individuated, although not all were necessarily present in the same sample. an interesting study describes the changes in the electrophoretic pattern during the evolution of experimentally induced tuberculosis infection in a group of guinea pigs. 16 at the end of the first week, the electrophoretic pattern was still virtually unchanged, whereas at the fulfillment of the second, a large majority of individuals showed a marked increase in a2-globulins and the appearance of a new peak in the same region, characteristic of sick individuals and never found in healthy ones, named a2-t. at the third week, the a1 started to decrease whereas a2 increased further with increasing importance of a2-t peak, which persisted until the death of the subjects. more detailed studies have identified this protein as a glycoprotein, not macroglobulin. it is interesting to note that the response to tubercular infection occurs in the region of a2 in several species, including humans. guinea pig lipoproteins tend instead to migrate in the b-globulin peak. the same study also analyzed the serum of guinea pigs inoculated with killed mycobacteria: the electrophoresis of these subjects showed no increase of a2-globulins nor the appearance of a2-t, which can then be considered the sign of an organic reaction to the pathogen, but all the samples at the third week after inoculation showed a marked hypergammaglobulinemia. in another study, reaction to infection by entamoeba histolytica was investigated, which consisted of hypoalbuminemia and increase of a-globulins and b-globulins. 17 the importance of electrophoresis in the diagnosis of spontaneous disease in the guinea pig and chinchilla is currently studied: patterns from clinically healthy individuals are shown as a starting point for further observations (fig. 4) . the first studies of serum proteins in birds were performed on domestic chickens, showing many similarities with the layout of mammals (eg, the production of apps 18, 19 ), but also several differences: the widespread presence of prealbumin, for example, the lowest concentration of g-globulin, and conversely the more marked response to inflammatory stimuli in the b-globulin field. unfortunately for the needs of clinical practice, however, not only may the electrophoretic patterns of parrots differ from those of chickens, 20 but significant differences are found also among the different species of psittaciformes (figs. 5-10) . this variability has led some investigators to doubt the possibility of obtaining reliable results by electrophoresis in avian species, especially with regard to the differentiation between the various globulin fractions. 21 the answer to this question is probably a better standardization of the techniques of electrophoresis and graphical representation, as well as a better understanding of the normal values of the different species. normally, electrophoresis of birds is run on plasma samples obtained from heparinized blood: the percentages are derived from densitometric analysis of each protein fraction deposited on the gel and absolute values (g/dl) calculated by multiplying the percentage of each fraction for the value of total protein obtained from the refractometer. the pattern of the normal chicken shows a marked peak of albumin, and globulins are divided between the b-globulin (igm, iga) and g-globulin (igg): transferrin, app, migrates mainly in the b-globulins. in birds, the albumin protein fraction is undoubtedly preeminent; an interspecific important difference is noted in the group of prealbumin. whereas in some species, such as the african gray, prealbumin is nearly or totally absent, similar to chickens, other species, such as the budgerigar or the monk parakeet (myopsitta monachus), may have even more prealbumin than albumin. conventionally, the a/g ratio is calculated by dividing the sum of prealbumin 1 albumin for the sum of the globulins. according to some investigators, however, it may be more correct to subtract the prealbumin, because it is not properly albumin and even a globulin. the opinion of the author is that this precaution not only is of little practical value but also potentially counterproductive, being that prealbumin, as albumin, has a negative app and therefore its quantification, when present, has validity in the diagnosis of acute inflammation. the b-globulins are the predominant group of globulins in all psittaciformes: some species, such as the african gray, appear regularly divided into 2 subfractions, but the clinical value of this feature is still unclear. 22 the b-globulins increase from 15% to more than 35% in the psittaciformes with chlamydiosis, aspergillosis, or sarcocystosis. 23, 24 an increase of b-globulins has also been described in chickens experimentally inoculated with turpentine and other irritants, 18, 19 demonstrating that most apps (transferrin, fibrinogen, b lipoprotein, complement) in avian species migrate in this sector. 25 the transferrin appears to be the major app in avian species (in contrast to what happens in mammals) and, although it is not specific to a particular disease, has an important prognostic value: its short half-life (24-48 hours) makes the monitoring of b-globulins an excellent indicator of the effectiveness of a therapy. the a-globulins instead represent the group less defined and the whose measurement determines the highest margin of error. 22 altogether they represent only 4% to 8% of total protein, and already this modest quantity (in comparison with that of mammals and also of other avian orders, such as falconiformes) makes it difficult to precisely separate it from the 2 contiguous predominant peaks, albumins and b-globulins. they are divided into 2 fractions: the area of a1-globulins hosts mainly the a1 antitrypsin and in normal paths is not detectable as a well-defined peak; even in sick parrots an increase of this fraction has almost never been described, unlike what was observed in mammals. in some cases has been noted a selective increase dell'alfa1 antitrypsin, with or without moderate hypoalbuminemia, in psittaciformes with heavy parasitism. 25 the main protein in the a2 area is instead a2 macroglobulin. fig. 10 . bubo bubo. the pattern from this specimen looks normal, but the bird is hypoproteinemic, possibly as a consequence of a too severe food restriction for training. the characteristic of this protein is its large size (molecular weight > 400,000) for which it is still retained by the filter even in the presence of serious renal damage. one of the few cases when significant elevations of a2 are described is precisely acute nephritis, in which most of the proteins are lost through the kidneys and the percentage of macroglobulin increases accordingly: in response to other phlogistic stimuli, instead, increments much more moderate and inconstant are noticed. modest alterations of a-globulins have also been described in relation to the activation of the ovary, similarly as described in the human species. the increase of transferrin that occurs during this stage, however, also induces a concomitant peak of b-globulins. the g-globulins are a minor fraction in the electrophoretic pattern of the psittaciformes, on average 10% to 15% of the total protein. in the area of g-globulin migrate mainly igg so we see increments in this area only at a later stage of the pathology. diseases in which we expect massive increases in g-globulin are chronic diseases such as chlamydiosis or mycobacteriosis. aspergillosis also belongs to the group of chronic diseases but, because of its immunosuppressing activity, often patients suffering from chronic fungal forms have apparently normal electrophoresis, as the humoral response is inhibited. approximately 30% of patients suffering from aspergillosis show cyclical peaks in the b-globulins area, and only a small percentage show hypergammaglobulinemia. the normal response to a chronic inflammatory process is the production of a wide variety of antibodies that are expressed in a series of g-globulin peaks or, more commonly, in a single peak with a large base: this phenomenon is named polyclonal gammopathy. in some cases, however, a single or very few immunoglobulins are massively produced, a phenomenon that is expressed in a peak-to-narrow base similar to that of albumin and is defined oligoclonal or monoclonal gammopathy. normally behind this phenomenon there is a neoplastic proliferation of one or a few lines of b lymphocytes, even if in some cases, monoclonal gammopathies may occur in response to non-neoplastic stimuli as well, but anyway very serious. it is worth considering that the low reliability reported by some investigators about the avian electrophoresis is to be traced back to improper collection, retention, and selection of the sample. cray and colleagues 22 showed that serum refrigeration causes a discrete change in the albumin/globulin ratio, causing a progressive difficulty in defining and quantifying the a-globulins. conversely, a freezing of the sample did not show adverse effects. hemolysis is a phenomenon unfortunately present when sampling blood from small patients and difficult to control. in the dog, the products of hemolysis tend to migrate in the b-globulin region; conversely, those of birds seem to migrate primarily in the region of g-globulin. this may be attributable to a structural difference of the protein or by a different technique analysis. 22 lipemia is another frequent alteration especially in the serum of some south american species (amazona spp, miopsitta, pionus spp): the electropherogram of a lipemic sample shows an artificial elevation in the region of b-globulins. in contrast, the b-globulins would be below the normal values related in the literature, in which serum would be used in place of plasma because the fibrinogen (absent in serum) migrates essentially in the b region. 26 this is, however, the only significant difference between the tracks from the 2 different spin cycles. electrophoresis of birds of prey basically follows the same general considerations of psittaciformes: common use of plasma instead of serum, need to standardize techniques, and to study the parameters of normality among the different species (figs. 11 and 12) . compared with psittaciformes, most falconiformes do not present prealbumin or present it in a minimal amount: vice versa, apparently healthy falconiformes have levels of a1-globulins and a2-globulins, markedly higher than the parrots. major differences regarding the b-globulins and g-globulins are not described. electrophoresis is an important tool for the diagnosis of chlamydiosis and aspergillosis in falconiformes. a text antibody positive for chlamydia spp acquires a very different meaning in the presence of electrophoretic curve indicative of acute or chronic inflammation than in absence of any alteration; a marked alteration of electropherogram with prevailing in the region of the b-globulins can support a presumptive diagnosis of aspergillosis also in case of a negative antibody test. 27 the electrophoresis has proved useful as a diagnostic and prognostic tool in falconiformes even in the case of diseases such as mycobacteriosis, abscesses, and osteomyelitis. the use of electrophoresis in reptiles is still sporadic and most of the available data are anecdotal; nevertheless, they are interesting as a first element for future study. serum proteins of reptiles migrate in a manner quite similar to those of birds and are divided on the track in the fractions of albumin, a1-globulin, a2-globulin, b-globulins, and g-globulins. prealbumin appears in some species (eg, geochelone radiata and some turtles), in which it presumably has the same function of transport as in other animals. albumins are also the preponderant portion of the curve in reptiles, whereas the globulins as a complex, like in birds, are in minimal concentration in healthy subjects. 28 in the fraction of a-globulins and b-globulins migrate mainly apps and complement, whereas the g-globulins are circulating antibodies; both b-globulin and g-globulin can configure a single peak or two. even in the absence of standardization, it can be seen how the electrophoretic pattern changes in subjects markedly ill compared with healthy subjects in reptiles as well. in an iguana (iguana iguana) with evidence of osteometabolic disease, for example, is frequently observed the decrease of albumins and the marked increase in b globulins (apps) that indicates suffering hepatic and renal function as a cause or consequence of the metabolic problem. 28 the alteration of the ratio a/g is one of the most interesting parameters to observe and evaluate, as it can reveal a dysproteinemia even in the presence of a normal value of total protein, thus inducing the clinician to further investigation. an important obstacle to the use of electrophoresis, as well as the biochemistry as an assessment of the health status of a reptile, is the marked variability linked not only to the species but also to temperature, ambient humidity, photoperiod, and in general to the season, that for heterothermic animals has much more influence on homeostasis than in homeothermic animals. only the collection of an adequate volume of data will clarify the actual diagnostic value of this test in reptiles. applications of protein electrophoresis in avian diagnostics plasma protein electrophoresis of the atlantic loggerhead sea turtle caretta caretta biochemical and hematologic values for 18 clinically healthy radiated tortoises (geochelone radiata) on st catherines island immune status of free-ranging green turtles with fibropapillomatosis from hawaii comparative haematology and blood chemistry of endangered lizards (gallotia species) in the canary islands electrophoretic proteinogram reference interval from argentina northeastern captive caimans (crocodylia: alligatoridae) clinical biochemistry of domestic animals acute phase proteins in dogs and cats: current knowledge and future perspectives serum concentration of acute phase proteins in dogs with leishmaniasis new testing options for the diagnosis of encephalitozoon cuniculi in rabbits identification and characterization of three encephalitozoon cuniculi strains altered immune responsiveness associated with encephalitozoon cuniculi infection in rabbits acute and long term humoral immunity following active immunization of rabbits with inactivated spores of various encephalitozoon species encephalitozoon cuniculi in pet rabbits l'electrophorese des proteins seriques en pathologie du furet (mustela putorius furo) electrophoretic and immunoelectrophoretic studies of sera from normal, tuberculous and non infected tuberculin sensitive guinea pigs electrophoretic studies of blood serum proteins in guinea pigs inoculated with entoameba histolytica identification of ovotransferrin as an acute phase protein in chickens polyacrylamide gel electrophoretic patterns of chicken serum in acute inflammation induced by intramuscular injection of turpentine differences in electrophoresis patterns between plasma albumins of the cockatiel (nymphicus hollandicus) and the chicken (gallus gallus domesticus) assessment of the reliability of plasma electrophoresis in birds protein electrophoresis of psittacine plasma serologic and plasma protein eletrophoretic findings in 7 psittacine birds with aspergillosis serologic diagnosis of sarcocystosis in psittacine birds: 16 cases plasma protein electrophoresis: an update total protein determination in pigeon plasma and serum: comparison of refractometric methods with the biuret method protein electrophoresis as a diagnostic and prognostic tool in raptor medicine protein electrophoresis: a tool for the reptilian and amphibian practitioner key: cord-350423-yaeduwvb authors: james, claire d.; roberts, sally title: viral interactions with pdz domain-containing proteins—an oncogenic trait? date: 2016-01-18 journal: pathogens doi: 10.3390/pathogens5010008 sha: doc_id: 350423 cord_uid: yaeduwvb many of the human viruses with oncogenic capabilities, either in their natural host or in experimental systems (hepatitis b and c, human t cell leukaemia virus type 1, kaposi sarcoma herpesvirus, human immunodeficiency virus, high-risk human papillomaviruses and adenovirus type 9), encode in their limited genome the ability to target cellular proteins containing psd95/ dlg/zo-1 (pdz) interaction modules. in many cases (but not always), the viruses have evolved to bind the pdz domains using the same short linear peptide motifs found in host protein-pdz interactions, and in some cases regulate the interactions in a similar fashion by phosphorylation. what is striking is that the diverse viruses target a common subset of pdz proteins that are intimately involved in controlling cell polarity and the structure and function of intercellular junctions, including tight junctions. cell polarity is fundamental to the control of cell proliferation and cell survival and disruption of polarity and the signal transduction pathways involved is a key event in tumourigenesis. this review focuses on the oncogenic viruses and the role of targeting pdz proteins in the virus life cycle and the contribution of virus-pdz protein interactions to virus-mediated oncogenesis. we highlight how many of the viral associations with pdz proteins lead to deregulation of pi3k/akt signalling, benefitting virus replication but as a consequence also contributing to oncogenesis. psd95/dlg/zo-1 (pdz) domains are one of the most widely distributed protein-protein interaction domains; the human genome encodes over 320 pdz domain-containing proteins [1] . the domain itself is an evolutionarily conserved domain of approximately 90 amino acids folded into six β-sheets (βa-βf) and two α-helices (αa-αb), named for its presence in psd95, dlg, and zo-1 proteins. the domains are often found in combination with a number of other protein-protein interaction modules, for example members of the membrane-associated guanylate kinase (maguk) family include an sh3 and inactive guanylate kinase domain, as well as multiple pdz modules. the presence of modular domains within a single protein allows pdz domain-containing proteins (herein referred to as pdz proteins) to form interactions with many partner proteins simultaneously and act as a scaffold; thus these proteins are involved in myriad of functions, including organizing multiprotein signalling complexes at the immunological and neurological synapses, forming cell-cell junctions, cell proliferation and survival, intracellular trafficking, apico-basal and planar polarity. perhaps not surprisingly considering the wide remit of pdz protein functions, deregulation of a number of pdz proteins has been linked to tumourigenesis [2] . however, it is often not clear whether to define some pdz proteins as oncogenes or tumour suppressors, as their roles in tumourigenesis is context-dependent [3, 4] . whilst abundance and/or cellular localization is a contributing factor in pdz protein function, tissue specificity is also of importance; this is the case for the epithelial polarity protein scrib, which has a tumour suppressor role in epithelia but a pro-oncogenic role in myc-driven lymphomas [5, 6] . binding to the pdz domain is mediated by short peptide sequences-pdz-binding motifs (pbm)-often but not always found at the carboxy terminus of proteins. pdz domains are grouped into specificity classes based on the consensus sequence of the pbm recognized. early studies defined three main classes of specificity; class i s/t-x-ϕ-cooh, class ii ϕ-x-ϕ-cooh and class iii d/e-x-ϕ-cooh, where ϕ is an hydrophobic residue, but more recent analyses using protein arrays and peptide libraries have defined at least 16 distinct specificity classes (the reader should refer to the references for details of the consensus sequences) [1, 7] . in binding, the pbm sits into the hydrophobic groove of the pdz domain formed between one of the β sheets (βb) and the α helix αb, with a highly conserved carboxylate binding loop at the base. amino acids outside the pbm contribute to pdz substrate specificity of binding, and phosphorylation of the pbm can act as a negative or positive regulator of pdz binding [1, 7] . likewise, phosphorylation within the pdz domain itself is also a regulatory mechanism of ligand binding [1] . a number of diverse but highly pathogenic viruses encode proteins that target pdz domain containing proteins, often but not always, using a pbm. pathogenicity of both the sars coronavirus (sars-cov), a positive strand rna virus that causes severe acute respiratory infections, and the neurotropic rabies virus, are linked to pdz-binding functions of their envelope proteins. recombinant sars-cov expressing the envelope e protein lacking the c-terminal pbm had no effect on viral growth in a murine infection model, but caused less lung damage and mortality than in mice infected with the wild type virus [8, 9] . the e protein pbm binds to the tight junction associated pdz protein pals1, leading to disturbed tight junction formation of polarized epithelia. it can also induce proinflammatory cytokine expression, possibly through pbm binding of the pdz protein syntenin-1. both of these pbm functions could be determinants of the lung pathobiology associated with sars-cov infection [8, 10] . survival of rabies infected neuronal cells is associated with the ability of the viral envelope g protein to interact with the pdz domain-containing serine threonine kinase mast2, leading to the disruption of the mast2-pten complex that is intimately involved in the inhibition of neuronal survival [11] . in an attenuated strain, a mutation within the g protein pbm confers an expansion of the pdz proteins bound, and leads to apoptosis of neuronal cells [12] . also, the canonical class i pbm at the c-terminus of the ns1 protein of over 99% type a influenza viruses is a good target for attenuation of these pathogenic viruses [13] [14] [15] [16] . removal of the pbm decreases efficacy of influenza h1n1 virus transmission, as well as virus replication, and increases interferon expression in infected cells [17, 18] . moreover, switching the h1n1 pbm for the sequence of an avian-specific strain increased virus virulence [19] . the ns1 pbm of the highly pathogenic strain h5n1 interacts with the pdz domains of several pdz proteins, including the epithelial polarity regulators dlg1 and scrib and the tight junction protein magi-1 [20] [21] [22] . using models of influenza a infection, this motif has been shown to contribute to the pathogenesis of these viruses by disruption of tight junction integrity of polarized epithelia, also disruption of cell junctions may benefit viral dissemination within the host, and/or spread to other hosts [21] . furthermore, relocalization of scrib to cytoplasmic puncta in infected cells is associated with the abrogation of the polarity protein's pro-apoptotic function and may therefore protect infected cells from viral induction of apoptotic pathways, thus supporting viral propagation [20, 23] . targeting pdz protein function is also a common function amongst viruses classified as human carcinogens by the world health organization [24] . as well as having roles in virus infectivity, the virus-pdz protein associations are important in virus entry, replication and for some of these viruses, cell transformation. these include the hepatitis viruses b and c, kaposi sarcoma herpesvirus, human t cell leukaemia virus type 1, high-risk human papillomaviruses, and human immunodeficiency virus type 1. in this review, we examine the interactions between these cancer-causing viruses and pdz proteins and discuss their contribution to pathogenesis of this important group of viruses. whilst hiv-1 is defined as a group i carcinogen, it contributes indirectly to carcinogenesis via immunosuppression and infected individuals have higher incidence rates of tumours driven by other cancer-causing viruses, and so it is also included in the review. interactions with pdz proteins are also pivotal in the formation of mammary tumours in mice infected by the human adenovirus type 9 and although this virus is not linked to human cancers, the study of ad9 interactions with pdz proteins has given valuable insight in viral carcinogenesis. the different virus-pdz associations are listed in table 1 and common pdz targets are shown in figure 1 . table 2 for full list) yes (class i, s/txv/l) degradation via the proteasome, transcriptional downregulation, altered subcellular localization, complex formation to alter function see table 2 2 high-risk alpha-hpv e6* dlg1, magi-1, patj, scrib no degradation via the proteasome [45, 46] alter function 2 high-risk alpha-hpv e6* dlg1, magi-1, patj, scrib no degradation via the proteasome [45, 46] 1 interactions between virus proteins and pdz domains/proteins have also been identified from peptide/protein screens but they are not included in this table [47] [48] [49] ; 2 hpv e6* protein is an e6 isoform that lacks the pdz-binding motifs (pbm). (?) function/effect not proven. a common risk factor for hepatocellular carcinoma is chronic infection with hcv. the virus primarily infects the highly polarized epithelium in the liver and depolarization of this tissue by the virus is critical for virus entry and cell-to-cell transmission of virus. entry of hcv occurs at tight junctions and most likely requires disruption of these structures, contributing to depolarization of the tissue [50] . the tight junction protein claudin 1 and the major receptor for high-density lipoproteins, sr-b1 (class b scavenger receptor) are important entry factors for hcv and both proteins encode pbm at the c-termini. the interaction between sr-b1 and a pdz domain-containing adaptor partner pdzk1 facilitates virus entry [51] . expression of the hcv core protein disrupts the apicobasal polarity of polarized epithelia and leads to deregulation of components of the scribble polarity complex dlg1 and scrib at cell-cell contacts. dlg1 protein expression is decreased and there is mislocalisation of scrib from cell-cell contacts in liver epithelial cells [25] . the core protein does not target the polarity pdz proteins directly, but subverts the phosphoinositide (pi) phosphastase ship2, leading to a decrease in production of the pi phosphatidylinositol 4,5 bisphosphate (pip2) which has a role in the targeting of dlg1 and scrib to the basolateral membrane ( figure 2 ). such deregulation a common risk factor for hepatocellular carcinoma is chronic infection with hcv. the virus primarily infects the highly polarized epithelium in the liver and depolarization of this tissue by the virus is critical for virus entry and cell-to-cell transmission of virus. entry of hcv occurs at tight junctions and most likely requires disruption of these structures, contributing to depolarization of the tissue [50] . the tight junction protein claudin 1 and the major receptor for high-density lipoproteins, sr-b1 (class b scavenger receptor) are important entry factors for hcv and both proteins encode pbm at the c-termini. the interaction between sr-b1 and a pdz domain-containing adaptor partner pdzk1 facilitates virus entry [51] . expression of the hcv core protein disrupts the apicobasal polarity of polarized epithelia and leads to deregulation of components of the scribble polarity complex dlg1 and scrib at cell-cell contacts. dlg1 protein expression is decreased and there is mislocalisation of scrib from cell-cell contacts in liver epithelial cells [25] . the core protein does not target the polarity pdz proteins directly, but subverts the phosphoinositide (pi) phosphastase ship2, leading to a decrease in production of the pi phosphatidylinositol 4,5 bisphosphate (pip2) which has a role in the targeting of dlg1 and scrib to the basolateral membrane ( figure 2 ). such deregulation of the polarity pathways regulated by the scribble components may be a contributing factor in hcv driven oncogenesis [25] . also, it has been reported that the carboxyl sequence of the poorly characterized non-structural protein ns4b encodes a class i pbm-like sequence (x-t-x-c); however, as yet there is no experimental evidence that supports interaction between ns4b and pdz proteins [52] . nevertheless, propagation of other rna viruses within flaviviridae (tick borne encephalitis virus, west-nile virus and dengue virus) is dependent upon pbms within the non-structural ns5 proteins, and have been shown to bind to various pdz proteins, including those involved in polarity control and tight junction assembly and maintenance; and mutations in these pbm negatively affect viral replication [53, 54] . as yet there is no experimental evidence that supports interaction between ns4b and pdz proteins [52] . nevertheless, propagation of other rna viruses within flaviviridae (tick borne encephalitis virus, west-nile virus and dengue virus) is dependent upon pbms within the non-structural ns5 proteins, and have been shown to bind to various pdz proteins, including those involved in polarity control and tight junction assembly and maintenance; and mutations in these pbm negatively affect viral replication [53, 54] . hbv is another virus whose infection of the liver causes acute and chronic hepatitis with an increased risk of the development of hepatocellular carcinoma. like the hcv core, the activities of the core protein of hbv (hbvc) are linked to viral pathogenicity. yeast two hybrid screening for hbv core partners identified the pdz protein gipc1 (gaip-interaction protein, c-terminus, family member 1, also known as tip-2) as a robust binding partner. the interaction is mediated by the core's pbm, which has sequence similarities with other ligands that bind gipc1 (x-x-c-cooh, a nontypical pbm [56] ), and deletion of the carboxyl cysteine from the hbvc was sufficient to block the interaction [26] . the consequences of this hbvc activity in hbv pathogenesis is unknown, but as gipc1 is a scaffold protein important in the endocytic trafficking of multiple signal transduction receptors, the association may have an important role in facilitating movement of the hbv capsids to the nucleus [57] . it is possible that the association between the core and gipc1 is linked to the virus's role in carcinogenesis via disruption of interactions of cellular substrates with the pdz domain of gipc1. pertinent to this is the interaction between lysophosphatidic acid (lpa) and the pdz domain of gipc1, which is necessary to attenuate lpa signalling ( figure 2 ). loss of this control leads to increased cell proliferation and survival through the resulting upregulation of the akt pathway [57] . this activity may function synergistically with the virus's hbx activation of akt signalling to block apoptosis of infected hepatocytes, thus enabling persistence of this viral carcinogen [58] . hbv is another virus whose infection of the liver causes acute and chronic hepatitis with an increased risk of the development of hepatocellular carcinoma. like the hcv core, the activities of the core protein of hbv (hbvc) are linked to viral pathogenicity. yeast two hybrid screening for hbv core partners identified the pdz protein gipc1 (gaip-interaction protein, c-terminus, family member 1, also known as tip-2) as a robust binding partner. the interaction is mediated by the core's pbm, which has sequence similarities with other ligands that bind gipc1 (x-x-c-cooh, a non-typical pbm [56] ), and deletion of the carboxyl cysteine from the hbvc was sufficient to block the interaction [26] . the consequences of this hbvc activity in hbv pathogenesis is unknown, but as gipc1 is a scaffold protein important in the endocytic trafficking of multiple signal transduction receptors, the association may have an important role in facilitating movement of the hbv capsids to the nucleus [57] . it is possible that the association between the core and gipc1 is linked to the virus's role in carcinogenesis via disruption of interactions of cellular substrates with the pdz domain of gipc1. pertinent to this is the interaction between lysophosphatidic acid (lpa) and the pdz domain of gipc1, which is necessary to attenuate lpa signalling ( figure 2 ). loss of this control leads to increased cell proliferation and survival through the resulting upregulation of the akt pathway [57] . this activity may function synergistically with the virus's hbx activation of akt signalling to block apoptosis of infected hepatocytes, thus enabling persistence of this viral carcinogen [58] . the hbvc can also bind via its pbm to the non-receptor protein tyrosine phosphatase ptpn3; however the precise role of this association in hbv pathogenesis is not known. ptpn3 has been shown to have a suppressive role on hbv gene expression, but this control is independent of the pbm activity of the hbvc [27] . kshv (also known as human herpesvirus 8) is the aetiological agent of kaposi sarcoma, primary effusion lymphoma and multicentric castleman disease and kshv induced cancers are particularly common among patients with acquired immunodeficiency syndrome (aids). the route to carcinogenesis is not fully understood, but as with all cancer-causing viruses it involves dysregulation of signalling pathways important for host proliferation and survival. notably, a critical mechanism is the constitutive activation of the nuclear factor κb (nf-κb) and signal transducer and activator of transcription 3 (stat3) signalling pathways. in normal signalling, the tumour suppressor pdz-lim domain-containing protein (pdlim2, the lim domain is a protein:protein interaction domain composed of two contiguous zinc fingers) regulates turnover of rela (p65), one of the components of the nf-κb, and stat3 by ubiquitination and degradation via the proteasome [59] . this normal mechanism of turnover of stat3 and nf-κb is deregulated in kshv pathogenesis by the virus repressing pdlim2 transcription by increased dna methylation of the promoter region [28] . hiv-1 is a lentivirus that infects several different types of immune cells, but primarily cd4 + positive t cells, and causes aids, a condition that is characterized by immune suppression, and aids patients are at high-risk of opportunistic microbial infections, including cancer-causing viruses. interactions between hiv-1 and host pdz proteins have been reported to take place at early and late stages of virus infection. the hiv-1 gag polyprotein interacts with pdz proteins, although the interactions are not mediated by a classical pbm. the cytoskeletal protein pdzd8 interacts with gag involving those regions of the polyprotein associated with early post-entry events; knockdown of this cellular cofactor inhibits hiv-1 infection and negatively affects viral capsid stability, whereas pdzd8 overexpression enhances infection by hiv-1 [29, 60] . pdzd8 is a poorly understood protein that has been shown to regulate microtubule stability through an interaction with moesin and microtubule stability at the viral synapse is likely to be important for hiv-1 infection [61, 62] . however, recent findings using cell lines in which pdzd8 expression has been eliminated by the crispr-cas system indicated that hiv-1 infection is not affected by loss of this host protein [63] . the other known pdz target of gag is dlg1 [30] . binding to dlg1 is mediated through the nucleocapsid domain of gag and the viral protein associates with an isoform of dlg1 that preferentially locates to the plasma membrane, where the two proteins colocalize [30] . the gag-dlg1 association regulates hiv-infectivity, such that depletion of dlg1 enhanced infectivity and dlg1 overexpression reduced particle infectivity [30] . loss of dlg1 from infected cells leads to the redistribution of env and gag from predominantly plasma membrane sites to intracellular vesicle-like structures that function as specialized platforms for the generation of infectious particles and suggests that dlg1 regulates plasma membrane dynamics in hiv-1 infected t cells [30] . dlg1 is not the only pdz protein to act antagonistically in hiv-1 infectivity; syntenin-1 is recruited to the plasma membrane during hiv-1 attachment by the viral envelope glycoprotein (env) and associates with the main hiv-1 receptor cd4 [31] . over expression of syntenin-1 inhibits hiv production and silencing increases viral entry, most likely by inducing actin polymerization and accumulation at the viral attachment site to inhibit virus entry [31] . hiv-1 transmission occurs at mucosal epithelial surfaces and one of the consequences of hiv-1 infection is increased permeability of the intestinal tract leading to an enhancement in microbial infections. in polarized epithelial cell cultures, hiv-1 infection causes a displacement of the pdz protein zo-1 from the tight junctions in the apical region of the membrane, followed by a marked reduction in the transcription of zo-1 and other tight junction proteins [32] . the disruption of tight junction integrity is mediated by the hiv-1 glycoprotein 120, which induces upregulation of the inflammatory cytokine tnf-α, a known disruptor of tight junctions [32, 64] . htlv-1 is a retrovirus that is the aetiological agent of the highly aggressive adult t cell leukaemia and primarily replicates in cd4 + t cells. transmission of the virus occurs predominantly by direct contact between infected and target cells; the site of contact forms a virological synapse where the gag glycoprotein and envelope (env) protein, together with the viral rna become concentrated. the env protein contains at its c-terminus a class i pbm and the activity of this motif contributes to the ability of env to trigger cell-to-cell fusion as judged by analysis of syncytium formation-a marker of cell-to-cell fusion [33] . there is also evidence that the pbm regulates env trafficking since disruption of the motif causes accelerated endocytosis and degradation of the env protein [65] . thus, binding to pdz proteins may be important for the proper localization of env-directing it to specific microdomains in the plasma membrane. to date, only one pdz protein, dlg1 has been identified as binding to env in a pbm dependent manner. env and dlg1 colocalize at sites characteristic of virological synapses on htlv-1-infected t cells [33] . moreover, depletion of dlg1 reduced syncytium formation, although env expression was not affected suggesting that additional pdz proteins might be involved in regulating env trafficking [65, 66] . since there is a partial colocalization between dlg1 and the major htlv-1 receptor glut-1 (glucose transporter type 1) at intercellular contacts formed between infected and target cells, a possible function for this interaction may be to aid efficient clustering of glut-1 at the polarised synapse [33] . the htlv-1 tax transcription factor also contains a canonical class i pbm at its c-terminus. tax is the major oncogenic determinant of htlv-1; when expressed alone it induces the transformation of rat fibroblasts in culture and is capable of promoting leukaemia in transgenic mice [67] [68] [69] . studies of the contribution of the htlv-1 motif to the transformation functions of tax have shown that deletion of the motif reduces the transformation potential of tax in rat fibroblasts [67] . the domain is also necessary for t cell overproliferation and reconstruction of a tax pbm deletion mutation in the context of whole htlv-1 virus showed that the motif was critical for induction of proliferation of human lymphocytes following viral infection [70, 71] . using this same model of htlv-1 pathogenesis, the mutant tax pbm deletion virus, whilst not necessary for htlv-1 driven immortalization, was shown to be a requirement for acquisition of host genomic instability as judged by micronuclei formation, and the mutant virus was severely compromised in its ability to establish persistent infection in rabbits [70] . taken together, the function of the tax pbm plays a significant role in those characteristics of the virus necessary to induce t cell transformation. in further support of the role of the pbm in htlv-1 driven transformation, a pbm is absent from the tax protein of htlv-2, a virus that is not leukemogenic. interestingly, as for cellular pbm-pdz interactions, the function of the tax pbm is susceptible to regulation by phosphorylation [1] . the protein kinase casein kinase 2 phosphorylates the threonine residue within the tax pbm and negatively regulates binding to pdz proteins, suggesting that timing of pdz targeting may be important in the htlv-1 replication cycle and host transformation [72] . but what are the physiological targets of this domain? to date, scribble polarity complex components dlg1 and scrib have been identified as robust binding partners of the tax pbm and both polarity proteins have been shown to have critical roles in t cell signalling, including the formation and organization of the immunological synapse [34, 35, 71, [73] [74] [75] [76] [77] [78] . the subcellular localization of both proteins is affected by tax expression; in htlv-1 infected t cells, and in cells overexpressing tax, dlg1 and scrib are relocated from cell membranes to cytoplasmic puncti or granules and this is concomitant with an increase in the insolubility of the polarity proteins, effects that are dependent on an intact tax pbm [67, 71, 75, 79] . the relocalization of dlg1 and scrib is a likely mechanism of inactivation of their normal function. indeed, the ability of dlg1 to block cell cycle progression of fibroblasts is inhibited by tax overexpression and is pbm dependent [35] . the inhibition of cell cycle progression is associated with an increase in phosphorylation of dlg1 and it is well known that cell cycle mediated phosphorylation of dlg1 regulates dlg1 localization and function [35, 80] . so, is dlg1 inactivation necessary for t cell transformation by tax? whilst silencing expression of dlg1 in tax expressing t cells augments both growth and transformation of a mouse t cell line, depletion of dlg1 does not complement transformation in the absence of the tax pbm, altogether suggesting that htlv-1 transformation does not depend solely on tax binding and inactivating dlg1 [81] . correct localization of dlg1 and scrib is important for the formation of signalling complexes at specific sites in the cell and tax pbm mediated effects on these proteins is likely to lead to a disruption of these signalling pathways. two critical signal transduction pathways for t cell signalling are the t-cell receptor (tcr)-induced activation via the transcription factor nuclear factor of activated t cells (nfat), and the akt pathway. in t cells, dlg1 is required for p38-mediated activation of nfat and stabilization of pten to inhibit akt-mediated signalling ( figure 2) [71, 82] . moreover, scrib may also organize receptor signalling through the nfat pathway since overexpression of scrib attenuates nfat activity and in tax expressing t-cells, this activity of scrib was inhibited, whereas scrib orchestrated inhibition of nfat still occurred when the tax pbm was mutated [75] . in addition, in immortalized t cells, tax stimulates akt activating phosphorylation in a pbm dependent manner by a mechanism that involves decreased membrane localization of the phosphatases pten and phlpp (ph domain and leucine rich repeat protein), a critical requisite for both phosphatases to negatively regulate akt activity. in both cases, overexpression of membrane forms of the phosphatases overcame the effects of tax on akt phosphorylation. tax-mediated mislocalization of these phosphatases from the plasma membrane may involve tax disruption of dlg1 and scrib scaffolding functions; scrib is necessary for membrane localization of phlpp [83] and pten is a binding partner of dlg1 [84] . how dlg regulates pten activity is not altogether clear, but dlg1 increases pten stability and augments pten's inhibition function of the akt pathway [85] . interestingly, tax attenuates binding between pten and dlg1 in t cells and this requires the activity of the pbm [78] . the htlv-1 tax protein has been shown to bind to many other pdz proteins besides dlg1 and scrib (table 1 ), but as yet the physiological relevance of these interactions to htlv-1 infection and transformation is unknown. singling out one of these, the t-cell specific interleukin-16 precursor (pro-il-16) binds to tax via the pbm in human t-cells, where both proteins colocalize in the nucleus [86] . pro-il-16 is highly expressed in all t cells and is the precursor to il-16. the presence of an intact pbm enables tax-expressing cells to overcome pro-il-16 driven cell cycle arrest, suggesting a role for the interaction in overcoming the growth inhibitory activity of pro-il-16 and promoting cell cycle progression [86] . such progression is favourable for viral propagation and is likely to contribute to the overproliferative phenotype of htlv-1 transformed cells. whilst not human carcinogens, ad are potent carcinogens in rodent cells and are provocative agents for the study of carcinogenesis. this review will focus on ad type 9 (ad9); a human virus commonly associated with benign eye infections, but causative of mammary tumours in rats in the presence of high levels of the hormone oestrogen [87] . unlike other human ad which drive carcinogenesis by the actions of the products of the e1a and e1b genes, ad9's oncogenicity is determined by the e4 region orf1 gene (e4orf1) function alone [88] . three regions of e4orf1 are important in cell transformation, including a c-terminal class i pbm, which is known to bind to a number of pdz domain-containing proteins, including dlg1 and tight junction proteins, magi-1, mupp1, patj and zo-2 [38] [39] [40] [41] (table 1 ). in fact, in polarized epithelial cells, the expression of e4orf1 blocks localization of the tight junction proteins to the intercellular junctions and many of the proteins become sequestered to e4orf1-containing structures in the cytoplasm. the disruption of tight junction function and the associated polarity defects are entirely dependent on an intact viral pbm [41] . in contrast to the effects upon the tight junction proteins, dlg1 is not sequestered into the cytoplasm but driven to accumulate at the plasma membrane. a second intriguing function of the e4orf1 pbm is the activation of pi3k pathway at the plasma membrane, a function that is dependent on the e4orf1 pbm localizing the viral protein to sites in the membrane and triggering activation of downstream targets, including akt/pkb [89] . importantly, overexpression of individual pdz targets of e4orf1, including dlg1, suppressed e4-orf1 mediated akt stimulation, strongly suggesting a link between this activity and pdz targeting. since this function of the e4orf1 pbm was not required for tight junction disruption, this represents a distinct activity mediated largely (but not entirely [90] ) by the pbm. all together, these studies suggest that the different e4orf1 functions-disruption of tight junctions and apicobasal polarity, and activation of the pi3k signalling pathway-cooperate to transform mammary cells. the next part in this story went on to understand the mechanism of pi3k activation, and this led to a series of very elegant studies from the javier laboratory. an important finding was that the increase in pi3k activity was not a consequence of inactivation of dlg1 function as first thought, but that e4orf1 exerts a gain-of-function upon dlg1, by binding dlg1 and promoting a conformational change allowing binding to the plasma membrane to direct pi3k activation [42] . this led to the idea that dlg1, whilst widely regarded as a tumour suppressor can acquire oncogenic characteristics in a different cellular context [3] . the summation of these studies has shown that e4orf1 forms complexes with pi3k and dlg1 in the cytoplasm; complex formation leads to increased stabilization of pi3k and increasing pi3k catalytic activity and activation of akt. the complex is then recruited to the plasma membrane through the induction of changes to dlg1 conformation [91] . deregulated activation of akt thereby promoting cellular survival and metabolism, which are important for virus replication, but also necessary for adenovirus-mediated transformation ( figure 2) . indeed, the formation of the e4orf1/pi3k/dlg1 ternary complex was necessary for anchorage independent cell growth-a robust indicator of cell transformation. in addition, the complex of e4orf1 and dlg1 also deregulates epidermal growth factor receptor signalling leading to activation of nuclear myc; an action thought to aid virion production in infected cells, although this action is not solely driven by the function of the e4orf1 pbm [92] . the ability of e4orf1 to activate pi3k in a dlg1 dependent fashion was found to be a conserved function between human ad subgroups a through to d, including those members used as e4orf1-encoding vectors in human gene therapy and vaccination, raising possible safety concerns of their use in humans [93] . whilst some 200 hpv types have been identified, only a small subset (25 types) of these are defined as carcinogens, including some as possible or probable carcinogens [24] . these viruses-referred to as high-risk types-infect the mucosal surfaces of the anogenital and oropharyngeal tracts and are associated with cancers at these sites (cervix, vulva, vagina, penile, anal, tonsil and base of tongue). hpv types 16 and 18 are the most common high-risk types, present in~70% of cervical cancers which has a nearly 100% association with high-risk hpv infection, and hpv16 is found in over 90% of hpv driven oropharyngeal carcinomas. the e6 and e7 early viral proteins are the main drivers of oncogenesis and all express an e6 protein that contains a class i pbm at the c-terminus of the protein. the conservation of this motif amongst the high-risk viruses is a strong indication that a pdz targeting function is important in their life cycle and as an oncogenic signature. intriguingly, a recent analysis of the ability of different hpv types to target pdz proteins has revealed that some of the non-cancer causing types can degrade pdz proteins (e.g., hpv40 can degrade magi-1), suggesting that the ability to degrade pdz proteins was acquired prior to the oncogeneic trait and that the ancestor of both high-risk and low-risk types acquired this new trait [94] . the authors of this study propose a model in which this new trait allowed these viruses to colonize new cellular niches (e.g., the cervical transformation zone), but for viral fitness and survival adapted to this site by acquiring additional functions such as htert activation and p53 degradation, functions that are necessary for cell transformation [94] . several systems have been used to demonstrate the importance of this e6 domain to hpv pathogenesis. life cycle studies based on human keratinocytes transfected with whole hpv genomes in which the e6 pbm has been deleted have shown that pbm function is critical for the vegetative phase of the life cycle, with the mutant genome unable to support viral genome amplification or expression of the viral late proteins [95] . a role for the e6 pbm in viral episome maintenance was also identified in the life cycle models [95] [96] [97] . the various life cycle defects correlated with a marked reduction in proliferation of the mutant genome-containing cells, indicating that the e6 pbm regulates proliferation of the viral genome-containing and that this is important for multiple phases of hpv dna replication [95, 96] . interestingly, although e6 also mediates the degradation of the tumour suppressor p53 in infected cells, further depletion of p53 protein by using p53-targetted sirnas, or by the introduction of a dominant negative 53, into cells harbouring e6-pbm mutant hpv16 genome-containing cells, rescues maintenance of the viral genomes, suggesting that there is mutual cooperation between these two e6 functions in viral genome maintenance during the virus life cycle [98] . in agreement with the life cycle studies, expression of e6 in mammalian keratinocytes showed that the e6 pbm promotes cell proliferation, but also the acquisition of phenotypes linked to invasive and metastatic growth of tumours (epithelial mesenchymal transition and anchorage independent cell growth) [99] [100] [101] [102] [103] . in addition, the motif is necessary for the morphological transformation and induction of tumourigenesis of rodent cell lines and contributes to tumour development in a transgenic mouse model of cervical carcinogenesis [104, 105] . however, the motif was not required for the immortalization of keratinocytes suggesting that the e6 pbm function is of more importance in the later stages of hpv driven carcinogenesis [106] . the link between this hpv function and cell transformation is further supported by the discovery that the e7 oncoprotein of the rhesus papillomavirus type 1 virus (rhpv1, also known as macaca mulatta pv1 [mmpv1]), which drives mucosal neoplasia, including cervical cancer in its host (rhesus macaques), encodes a functional c-terminal pbm whose integrity is required for transformation of rodent cell lines [44] . but what of the targets of this virally encoded pbm? seventeen pdz proteins that interact with the e6 proteins have been identified to date and in the majority of cases (but not all) binding leads to degradation of the pdz protein via the proteasome [107, 108] (table 2 ). the pdz substrates are largely involved in defining cell polarity, cell-cell attachment, and organizing cell signalling pathways associated with these functions (table 2) . notably, components of the three apico-basal polarity complexes, crumbs, par and scribble are e6 targets, suggesting that deregulation of pathways controlled by these complexes is an important function (figure 1 ). the scribble component dlg1 was the first pdz protein identified as an e6 binding partner and is targeted for degradation [104, 109] . during epithelial differentiation, the localization of dlg1 changes; in the basal proliferating compartment it is cytoplasmic and nuclear but in more differentiated cells present at the cell periphery, indicating location specific functions [3, 123] . it is the nuclear phospho-dlg1 forms that are preferentially targets for proteasomal degradation by hpv16 e6 [124, 125] , suggesting that nuclear dlg1 may encode tumour suppressor activity [3] . but proteasomal degradation of dlg1 is perhaps not the only outcome of e6 binding since several studies have identified functional e6/dlg1 complexes. in hpv16-positive cervical cancer cells, rhog activity is upregulated and e6 contributes to this upregulation by forming a complex with dlg1 and a rhog guanine exchange factor (sgef) [110] . moreover, the e6-dlg1-sgef complex contributes to invasive phenotype of the cells, indicating that dlg1 acquires oncogenic functions in the presence of the viral oncoprotein [110] , as has been noted with ad9 e4orf1 (see section on adenoviruses). in hpv positive cervical cancers, the intercellular communication channels known as gap junctions are lost and e6 has been found to be in complex with dlg1 and the gap junction component connexin protein cx43 in cervical cancer cells, and this complex may act to inhibit normal trafficking of connexin to the cell membrane [111, 126] . gap junctions and their components, particularly cx43 play an important role in cell invasion and the e6-dlg1-cx43 complex may contributes to metastatic properties of cervical cancer cells (figure 1 ). therefore, in hpv infections the tumour suppressor forms of dlg that are involved in the negative regulation of cell proliferation might be the initial target of the e6 pbm, but during disease progression dlg1, either through mislocalization and/or the stabilization of specific pools, acquires oncogenic functions mediated by interaction with e6 [3, 127] . the other scribble component targeted by e6 is scrib and this interaction disturbs scrib localization at the periphery of epithelial cells and induces loss of tight junction integrity [112] . e6 also targets several other pdz proteins associated with tight junctions, including magi-1, mupp1, par3 and patj (table 1; figure 1 ). recent findings have shown that introduction of a mutant form of magi-1 that cannot be targeted by e6 for degradation (as it contains a point mutation in the critical residue in pdz1 to which e6 binds) into hpv18 positive cervical cancer cells enhances the ability of these cells to form junctional complexes [128] . these studies also identified roles for magi-1 in the negative control of cell proliferation and as a regulator of apoptosis, indicating that e6 may target different functional pools of the pdz protein; selective targeting of these may be relevant at different stages of the virus life cycle and/or disease progression [128] . protection of hpv infected cells against apoptosis also involves e6 pdz dependent activation of the pi3k-akt and nf-κb signalling pathways [120, 129] ; e6 targets the na + /h + exchange regulatory factor (nherf1), a pdz protein known to attenuate pi3k signalling through the negative regulator pten (figure 2 ). hpv e6 is known to stimulate the key regulator of cellular metabolism mtorc1 leading to activation of cap-dependent translation, and the e6 pbm function has been shown to contribute to the enhancement of translation [130] (figure 2) . one of the most difficult questions to answer is which of e6 pdz substrate(s) in the growing list are physiologically relevant targets? in metastatic cervical cancers, the reduction in expression of the polarity proteins dlg1 and scrib could be e6 mediated via the proteasome [127, 131, 132] . indeed, levels of both proteins are rescued upon silencing e6 expression in hpv-positive cervical cancer cell lines, although the most marked change was noted for magi-1, with membrane bound and nuclear pools of the protein restored and re-localization of magi-1 to tight junctions [133] . pard3, an e6 substrate that is not degraded but relocalized from apical domain tight junctions to the nucleus, is restored to the junctions upon e6 silencing in the cancer cells [121] . somewhat surprisingly therefore, the level of expression and localization of endogenous dlg1 and scrib proteins remain unaltered in normal human keratinocytes expressing e6 (e6 alone, or e6 and e7 together, or viral oncoprotein expression driven from intact viral genomes) and there are no changes in the expression or localization of these proteins when the e6 pbm is deleted [96, 134, 135] . therefore, it is likely that there is differential regulation of pdz substrates by e6 at the different stages of hpv pathogenesis. since the activity of the e6 pbm is regulated by phosphorylation this may be one layer of control. the threonine or serine embedded in the e6 pbm is phosphorylated by protein kinases pka and akt and negatively regulates binding to pdz domains, including those of dlg1 and magi-1 [136] . conversely, e6 pbm phosphorylation confers the ability to interact with a number of 14-3-3 isoforms [137, 138] . 14-3-3 proteins have a wide range of cellular functions, regulating key proteins involved in signalling pathways associated with transcription, cell cycle control and apoptosis, although the function of 14-3-3 binding to e6 is unclear at this stage. however, expression of a mutant form of hpv18 e6 that cannot be phosphorylated within the pbm (and therefore cannot bind 14-3-3 proteins), but can bind pdz substrates enhanced the e6 mediated morphological transformation of human keratinocytes [102] . when this same mutation was embedded in intact hpv18 genomes and transfected into primary keratinocytes, there was a marked increase in hyperproliferation of the epidermal keratinocytes [95] . thus, modulation of the pka and/or akt signalling pathways in hpv infections is likely to impact on e6 function and may be a contributing factor to tumour promotion and progression [95, 102] . the mechanism of hpv targeting of pdz substrates is complicated further by finding that e6*, a spliced isoform of e6 which lacks a pbm, is able to target dlg1, patj, magi-1, and to a lesser extent scrib, for proteasomal degradation [45, 46] (figure 1) . moreover, hpv16 e6 and e7 cooperate to induce the degradation of nherf1. hpv16 e7 promotes the accumulation of phosphorylated forms of nherf through activation of cyclin-dependent kinases and the modified forms of the pdz protein are the preferential degradation targets of the e6 pbm [120] . thus, multiple cooperative mechanisms exist in hpv infections to control pdz protein expression and function. targeting the various pdz proteins is not equal amongst the different high-risk hpv types and substrate specificity involves the pbm sequence as well as sequences upstream of the pbm [20, 116, 120, 133, 139] . for example, both hpv16 and hpv18 e6 proteins are able to target dlg1 and scrib, but exhibit different preferences in binding the pdz proteins; hpv16 e6 preferentially binds scrib over dlg1 and vice versa for hpv18 [133, 139] . the pbm of these two oncoproteins only differs by a single amino acid (the last amino acid) and, notably, the specificity for dlg1 and scrib can be switched within the context of the whole e6 protein by switching the last residue [139] . whether this has relevance to the carcinogenic strength of the virus (hpv16 is the most prevalent high risk type in cancers) is not clear, but it might indicate that the ability to target at least one protein of the polarity scribble complex is a key function. differential targeting of pdz proteins may also have relevance to the pathogenesis of the virus at different anatomical sites. one such target is ptpn13, a non-receptor tyrosine phosphatase (hpv16 and hpv18 e6 pbm also target another family member ptpn3 [103, 117] ). hpv16 is associated with over 90% of the hpv positive oropharnygeal tumours (tonsil and base of tongue). in human tonsil keratinocytes, the hpv16 e6 pbm contributes to anchorage independent cell growth and synergizes with the small gtpase ras for invasion in vivo, and these malignant hallmarks were abrogated by reintroduction of ptpn13 [119] . interestingly, up to 20% of hpv-negative head and neck cancers contain disrupting mutation in the ptpn13 gene. disruption of ptpn13 regulation of the ras/raf/mek/erk signalling pathways linked through ephrin b1 receptor-mediated signalling is likely to be important in both hpv positive and negative head and neck tumours [117, 140] . the list of e6 pbm targets is still growing with the use of various types of peptide and proteomic-based screening methods; for example the ubiquitin e3 ligase pdzrn3 was recently identified as a potential novel target of hpv16 e6 pbm in two separate studies [48, 49] . the interaction has been confirmed as a degradation target of the hpv16 and hpv18 e6 in a pbm dependent mechanism [122] . the e6 mediated loss of pdzrn3 was linked to activation of stat5β and this pathway is known to be important in amplification of the viral genomes in the upper layers of the infected epithelia by activating the atm mediated dna damage response [141] . thus, the e6 pbm targeting of pdzrn3 may function synergistically with e7 to activate stat5 and support viral amplification. other potential novel targets of the e6 pbm include α and β syntrophins, snta1 and sntb1, whose functions include regulating the activity of the gtpase rac during tight junction assembly, and sorting nexin 27 (snx27), important in endosomal recycling pathways [48] , but whether these are genuine targets of e6, and the function of these interactions in the hpv life cycle and in hpv driven cancers requires further investigation. intriguingly, papillomaviruses may also target pdz proteins by blocking their transcription. the e6 protein of hpv8, a betapapillomavirus implicated in the development of squamous carcinomas of the skin repressed transcription of the pdz protein syntenin-2, and hpv16 e6 was also able to repress syntenin-2 transcription but to a much lesser extent than the hpv8 protein [43] . loss of syntenin-2 expression was shown to inhibit differentiation and promote proliferation of human keratinocytes; possibly by deregulation of pip2 mediated nuclear signalling, a signal transduction pathway linked to epithelial cell growth and differentiation [43, 142] . it will be important to establish if other pdz proteins are similarly deregulated at a transcriptional level in hpv infected and transformed cells. one of the striking features of this group of viruses is the commonality in pdz targets ( figure 1 ). many are components of the polarity complexes and the deregulation of the cellular pathways they control is involved in virus infection, transmission, replication, survival and persistence in the host, and formation of new progeny. for many of the viruses, these interactions also play a part in their oncogenic actions and indicates that the deregulation of the pdz protein controlled pathways is a finely balanced process in the virus life cycle and once this balance is perturbed this can lead, or contribute to oncogenesis. this perhaps is not so surprising when it is known that many of the components of the polarity complexes behave as tumour suppressors in different systems, although this behaviour can be context dependent. the viruses deregulate the pdz proteins in many different ways-inducing cellular mislocalization, proteasomal degradation, or by binding to the protein to compete with host cell partners, and intriguingly, some viral-pdz protein associations lead to the pdz protein acquiring oncogenic functions. interestingly, many of the virus-pdz associations discussed here affect the pi3k/akt signalling pathway that is fundamental to cell growth and survival ( figure 2 ) and is thus an obvious target for the viruses to exploit for replication. indeed, many other viruses that are not oncogenic use an array of strategies to interfere with this cell signalling pathway and perhaps some of these may be relevant to the viruses discussed in this review [143] . the use of high-throughput proteomics and construction of large libraries to screen protein-protein interactions is leading to the identification of further pdz targets for these viruses. thus, one area to tackle is to confirm those interactions which are physiologically relevant, a problem particularly significant to the high risk hpv e6 proteins which already has a list of seventeen targets and is growing with addition of new potential targets from the large screening arrays; the use of hpv life cycle models will be an important asset in these investigations. it is important that we understand the intricacies of these interaction; when and where they occur during the life cycle and what interactions are involved in oncogenesis, because these interactions are attractive therapeutic targets that can be targeted using disrupting molecules [144, 145] . pdz domains and their binding partners: structure, specificity, and modification pdz domains: the building blocks regulating tumorigenesis the pdz protein discs-large (dlg): the "jekyll and hyde" of the epithelial polarity proteins differential regulation of cell-cell contact, invasion and anoikis by hscrib and hdlg in keratinocytes scribble acts as an oncogene in emu-myc-driven lymphoma mislocalization of the cell polarity protein scribble promotes mammary tumorigenesis and is associated with basal breast cancer a specificity map for the pdz domain family the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis the sars coronavirus e protein interacts with pals1 and alters tight junction formation and epithelial morphogenesis interference with the pten-mast2 interaction by a viral protein leads to cellular relocalization of pten attenuation of rabies virulence: takeover by the cytoplasmic domain of its envelope protein large-scale sequence analysis of avian influenza isolates species-specific contribution of the four c-terminal amino acids of influenza a virus ns1 protein to virulence amelioration of influenza virus pathogenesis in chickens attributed to the enhanced interferon-inducing capacity of a virus with a truncated ns1 gene the ns1 gene contributes to the virulence of h5n1 avian influenza viruses the pdz-binding motif of the avian ns1 protein affects transmission of the 2009 influenza a(h1n1) virus truncation of the ns1 protein converts a low pathogenic avian influenza virus into a strong interferon inducer in duck cells a new influenza virus virulence determinant: the ns1 protein four c-terminal residues modulate pathogenicity analysis of the pdz binding specificities of influenza a virus ns1 proteins the avian influenza virus ns1 esev pdz binding motif associates with dlg1 and scribble to disrupt cellular tight junctions pdlim2 selectively interacts with the pdz binding motif of highly pathogenic avian h5n1 influenza a virus ns1 the esev pdz-binding motif of the avian influenza a virus ns1 protein protects infected cells from apoptosis by directly targeting scribble a review of human carcinogens-part b: biological agents gassama-diagne, a. ship2 regulates epithelial cell polarity through its lipid product, which binds to dlg1, a pathway subverted by hepatitis c virus core protein interaction of hepatitis b virus core protein with human gipc1 suppression of hepatitis b viral gene expression by protein-tyrosine phosphatase ptpn3 oncovirus kaposi sarcoma herpesvirus (kshv) represses tumor suppressor pdlim2 to persistently activate nuclear factor kappab (nf-kappab) and stat3 transcription factors for tumorigenesis and tumor maintenance pdzd8 is a novel gag-interacting factor that promotes retroviral infection human discs large is a new negative regulator of human immunodeficiency virus-1 infectivity the pdz-adaptor protein syntenin-1 regulates hiv-1 entry exposure to hiv-1 directly impairs mucosal epithelial barrier integrity allowing microbial translocation human dlg protein binds to the envelope glycoproteins of human t-cell leukemia virus type 1 and regulates envelope mediated cell-cell fusion in t lymphocytes the c-terminus of the htlv-1 tax oncoprotein mediates interaction with the pdz domain of cellular proteins tax oncoprotein of htlv-1 binds to the human homologue of drosophila discs large tumor suppressor protein, hdlg, and perturbs its function in cell growth control human t-cell leukemia virus type 1 tax protein interacts with and mislocalizes the pdz domain protein magi-1 human t-cell leukemia virus type 1 tax oncoprotein induces and interacts with a multi-pdz domain protein interactions of the pdz-protein magi-1 with adenovirus e4-orf1 and high-risk papillomavirus e6 oncoproteins link of the unique oncogenic properties of adenovirus type 9 e4-orf1 to a select interaction with the candidate tumor suppressor protein zo-2 multi-pdz domain protein mupp1 is a cellular target for both adenovirus e4-orf1 and high-risk papillomavirus type 18 e6 oncoproteins viral oncoprotein-induced mislocalization of select pdz proteins disrupts tight junctions and causes polarity defects in epithelial cells oncogenic function for the dlg1 mammalian homolog of the drosophila discs-large tumor suppressor human papillomavirus type 8 e6 oncoprotein inhibits transcription of the pdz protein syntenin-2 human and primate tumour viruses use pdz binding as an evolutionarily conserved mechanism of targeting cell polarity regulators the human papillomavirus (hpv) e6* proteins from high-risk, mucosal hpvs can direct degradation of cellular proteins in the absence of full-length e6 protein patj, a tight junction-associated pdz protein, is a novel degradation target of high-risk human papillomavirus e6 and the alternatively spliced isoform 18 e6 large-scale interaction profiling of pdz domains through proteomic peptide-phage display using human and viral phage peptidomes the human pdzome: a gateway to psd95-disc large-zonula occludens (pdz)-mediated functions interpreting cancer genomes using systematic host network perturbations by tumour virus proteins hepatitis c virus infection reduces hepatocellular polarity in a vascular endothelial growth factor-dependent manner the sr-bi partner pdzk1 facilitates hepatitis c virus entry interaction networks of hepatitis c virus ns4b: implications for antiviral therapy two pdz binding motifs within ns5 have roles in tick-borne encephalitis virus replication flavivirus ns5 associates with host-cell proteins zonula occludens-1 (zo-1) and regulating synaptic membrane exocytosis-2 (rims2) via an internal pdz binding mechanism emerging theme: cellular pdz proteins as common targets of pathogenic viruses gipc binds to the human lutropin receptor (hlhr) through an unusual pdz domain binding motif, and it regulates the sorting of the internalized human choriogonadotropin and the density of cell surface hlhr the pdz protein gipc regulates trafficking of the lpa1 receptor from appl signaling endosomes and attenuates the cell's response to lpa the hepatitis b virus (hbv) hbx protein activates akt to simultaneously regulate hbv replication and hepatocyte survival pdlim2-mediated termination of transcription factor nf-kappab activation by intranuclear sequestration and degradation of the p65 subunit contribution of pdxd8 to stabilization of the human immunodeficiency virus type 1 capsid histone deacetylase 6 regulates human immunodeficiency virus type 1 infection pdzd8 is a novel moesin-interacting cytoskeletal regulatory protein that suppresses infection by herpes simplex virus type 1 efficient human immunodeficiency virus (hiv-1) infection of cells lacking pdzd8 tnf-alpha modulation of intestinal epithelial tight junction barrier is regulated by erk1/2 activation of elk-1 opposing effects of a tyrosine-based sorting motif and a pdz-binding motif regulate human t-lymphotropic virus type 1 envelope trafficking knockdown of synapse-associated protein dlg1 reduces syncytium formation induced by human t-cell leukemia virus type 1 pdz domain-binding motif of human t-cell leukemia virus type 1 tax oncoprotein augments the transforming activity in a rat fibroblast cell line oncogenic transformation by the tax gene of humant-cell leukemia virus type 1 in vitro development of leukemia in mice transgenic for the tax gene of human t-cell leukemia virus type 1 pdz binding motif of htlv-1 tax promotes virus-mediated t-cell proliferation in vitro and persistence in vivo scaffold protein dlgh1 coordinates alternative p38 kinase activation, directing t cell receptor signals toward nfat but not nf-kappab transcription factors the pleiotropic protein kinase ck2 phosphorylates htlv-1 tax protein in vitro, targeting its pdz-binding motif (dlg1) complexes in lymphocyte activation a network of pdz-containing proteins regulates t cell polarity and morphology during migration and immunological synapse formation the pdz domain-binding motif of the human t cell leukemia virus type 1 tax protein induces mislocalization of the tumor suppressor hscrib in t cells binding of human virus oncoproteins to hdlg/sap97, a mammalian homolog of the drosophila discs large tumor suppressor protein dlgh1 is a negative regulator of t-lymphocyte proliferation akt pathway activation by human t-cell leukemia virus type 1 tax oncoprotein the pdz domain binding motif (pbm) of human t-cell leukemia virus type 1 tax can be substituted by heterologous pbms from viral oncoproteins during t-cell transformation cdk phosphorylation of the discs large tumour suppressor controls its localisation and stability inactivation of tumor suppressor dlg1 augments transformation of a t-cell line induced by humant-cell leukemia virus type 1 tax protein scaffold protein disc large homolog 1 is required for t-cell receptor-induced activation of regulatory t-cell function scribble-mediated membrane targeting of phlpp1 is required for its negative regulation of akt threonine phosphorylation of the mmac1/pten pdz binding domain both inhibits and stimulates pdz binding a functional network of the tumor suppressors apc, hdlg, and pten, that relies on recognition of specific pdz-domains binding of htlv-1 tax oncoprotein to the precursor of interleukin-16, a t cell pdz domain-containing protein human adenovirus type 9-induced rat mammary tumors requirement for the adenovirus type 9 e4 region in production of mammary tumors selective pdz protein-dependent stimulation of phosphatidylinositol 3-kinase by the adenovirus e4-orf1 oncoprotein a new crucial protein interaction element that targets the adenovirus e4-orf1 oncoprotein to membrane vesicles the human adenovirus e4-orf1 protein subverts discs large 1 to mediate membrane recruitment and dysregulation of phosphatidylinositol 3-kinase adenovirus e4-orf1 dysregulates epidermal growth factor and insulin/insulin-like growth factor receptors to mediate constitutive myc expression hijacking dlg1 for oncogenic phosphatidylinositol 3-kinase activation in human epithelial cells is a conserved mechanism of human adenovirus e4-orf1 proteins degradation of human pdz-proteins by human alphapapillomaviruses represents an evolutionary adaptation to a novel cellular niche the role of protein kinase a regulation of the e6 pdz-binding domain during the differentiation-dependent life cycle of human papillomavirus type 18 role of the pdz domain-binding motif of the oncoprotein e6 in the pathogenesis of human papillomavirus type 31 stabilization of hpv16 e6 protein by pdz proteins, and potential implications for genome maintenance papillomavirus e6 pdz interactions can be replaced by repression of p53 to promote episomal human papillomavirus genome maintenance requirement of pdz-containing proteins for cell cycle regulation and differentiation in the mouse lens epithelium the pdz ligand domain of the human papillomavirus type 16 e6 protein is required for e6's induction of epithelial hyperplasia in vivo deletion of the pdz motif of hpv16e preventing immortalization and anchorage-independent growth in human tonsil epithelial cells activity of the human papillomavirus e6 pdz-binding motif correlates with an enhanced morphological transformation of immortalized human keratinocytes degradation of tyrosine phosphatase ptpn3 (ptph1) by association with oncogenic human papillomavirus e6 proteins binding of high-risk human papillomavirus e6 oncoproteins to the human homologue of the drosophila discs large tumor suppressor protein the human papillomavirus e6 oncogene dysregulates the cell cycle and contributes to cervical carcinogenesis through two independent activities binding of pdz proteins to hpv e6 proteins does neither correlate with epidemiological risk classification nor with the immortalization of foreskin keratinocytes hpv e6 degradation of p53 and pdz containing substrates in an e6ap null background the role of the ubiquitin ligase e6-ap in human papillomavirus e6-mediated degradation of pdz domain-containing proteins oncogenic human papillomavirus e6 proteins target the discs large tumour suppressor for proteasome-mediated degradation the invasive capacity of hpv transformed cells requires the hdlg-dependent enhancement of sgef/rhog activity hpv16 e6 controls the gap junction protein cx43 in cervical tumour cells human scribble (vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus e6 proteins and the e6ap ubiquitin-protein ligase oncogenic human papillomavirus e6 proteins target the magi-2 and magi-3 proteins for degradation the pdz protein tip-1 is a gain of function target of the hpv16 e6 oncoprotein human papillomavirus type 18 e6 protein binds the cellular pdz protein tip-2/gipc, which is involved in transforming growth factor beta signaling and triggers its degradation by the proteasome e6ap-dependent degradation of dlg4/psd95 by high-risk human papillomavirus type 18 e6 protein protein tyrosine phosphatase h1 is a target of the e6 oncoprotein of high-risk genital human papillomaviruses human papillomavirus type 16 e6 protein interacts with cystic fibrosis transmembrane regulator-associated ligand and promotes e6-associated protein-mediated ubiquitination and proteasomal degradation the pdz binding motif of human papillomavirus type 16 e6 induces ptpn13 loss, which allows anchorage-independent growth and synergizes with ras for invasive growth e6 and e7 from human papillomavirus type 16 cooperate to target the pdz protein na/h exchange regulatory factor 1 human papillomavirus (hpv)-18 e6 oncoprotein interferes with the epithelial cell polarity par3 protein pdzrn3/lnx3 is a novel target of human papillomavirus type 16 (hpv-16) and hpv-18 e6 changes in localization of human discs large (hdlg) during keratinocyte differentiation are [corrected] associated with expression of alternatively spliced hdlg variants hpv e6 specifically targets different cellular pools of its pdz domain-containing tumour suppressor substrates for proteasome-mediated degradation the high-risk hpv e6 oncoprotein preferentially targets phosphorylated nuclear forms of hdlg a functional interaction between the maguk protein hdlg and the gap junction protein connexin 43 in cervical tumour cells changes in expression of the human homologue of the drosophila discs large tumour suppressor protein in high-grade premalignant cervical neoplasias restoration of magi-1 expression in human papillomavirus-positive tumor cells induces cell growth arrest and apoptosis human papillomavirus type 16 e6 activates nf-kappab, induces ciap-2 expression, and protects against apoptosis in a pdz binding motif-dependent manner activation of cap-dependent translation by mucosal human papillomavirus e6 proteins is dependent on the integrity of the lxxll binding motif differential expression of the human homologue of drosophila discs large oncosuppressor in histologic samples from human papillomavirus-associated lesions as a marker for progression to malignancy analysis of the expression and localisation of a lap protein, human scribble, in the normal and neoplastic epithelium of uterine cervix a systematic analysis of human papillomavirus (hpv) e6 pdz substrates identifies magi-1 as a major target of hpv type 16 (hpv-16) and hpv-18 whose loss accompanies disruption of tight junctions roles of the pdz domain-binding motif of the human papillomavirus type 16 e6 on the immortalization and differentiation of primary human foreskin keratinocytes two distinct activities contribute to human papillomavirus 16 e6's oncogenic potential differential regulation of human papillomavirus e6 by protein kinase a: conditional degradation of human discs large protein by oncogenic e6 cancer-causing human papillomavirus e6 proteins display major differences in the phospho-regulation of their pdz interactions high-risk human papillomavirus e6 oncoproteins interact with 14-3-3zeta in a pdz binding motif-dependent manner the hscrib/dlg apico-basal control complex is differentially targeted by hpv-16 and hpv-18 e6 proteins impaired ptpn13 phosphatase activity in spontaneous or hpv-induced squamous cell carcinomas potentiates oncogene signaling through the map kinase pathway the jak-stat transcriptional regulator, stat-5, activates the atm dna damage pathway to induce hpv 31 genome amplification upon epithelial differentiation pdz domain-phosphoinositide interactions in cell-signaling make yourself at home: viral hijacking of the pi3k/akt signaling pathway targeting the two oncogenic functional sites of the hpv e6 oncoprotein with a high-affinity bivalent ligand an rna aptamer targets the pdz-binding motif of the hpv16 e6 oncoprotein acknowledgments: claire d. james was supported by the medical research council's doctoral training partnership awarded to the college of medical and dentistry (university of birmingham). we are grateful to all members of the roberts laboratory, past and present, and the funding agencies (wellcome trust and cancer research uk). we apologize to those authors we were unable to cite due to space constraints.author contributions: claire d. james and sally roberts wrote the manuscript. the authors declare no conflicts of interest. key: cord-327934-hjimlb6i authors: acar, delphine d.; stroobants, veerle j. e.; favoreel, herman; saelens, xavier; nauwynck, hans j. title: identification of peptide domains involved in the subcellular localization of the feline coronavirus 3b protein date: 2019-10-01 journal: journal of general virology doi: 10.1099/jgv.0.001321 sha: doc_id: 327934 cord_uid: hjimlb6i feline coronavirus (fcov) has been identified as the aetiological agent of feline infectious peritonitis (fip), a highly fatal systemic disease in cats. fcov open reading frame 3 (orf3) encodes accessory proteins 3a, 3b and 3 c. the fcov 3b accessory protein consists of 72 amino acid residues and localizes to nucleoli and mitochondria. the present work focused on peptide domains within fcov 3b that drive its intracellular trafficking. transfection of different cell types with fcov 3b fused to enhanced green fluorescent protein (egfp) or 3×flag confirmed localization of fcov 3b in the mitochondria and nucleoli. using serial truncated mutants, we showed that nucleolar accumulation is controlled by a joint nucleolar and nuclear localization signal (nols/nls) in which the identified overlapping pat4 motifs (residues 53–57) play a critical role. mutational analysis also revealed that mitochondrial translocation is mediated by n-terminal residues 10–35, in which a tom20 recognition motif (residues 13–17) and two other overlapping hexamers (residues 24–30) associated with mitochondrial targeting were identified. in addition, a second tom20 recognition motif was identified further downstream (residues 61–65), although the mitochondrial translocation evoked by these residues seemed less efficient as a diffuse cytoplasmic distribution was also observed. assessing the spatiotemporal distribution of fcov 3b did not provide convincing evidence of dynamic shuttling behaviour between the nucleoli and the mitochondria. coronavirus infections have emerged in various species of mammals and birds and are generally associated with a wide spectrum of respiratory, intestinal and systemic infections. feline coronaviruses (fcov) can cause both enteric and systemic diseases in domestic and wild felidae and occur as two pathotypes for which the disease-causing potential is determined by their cell tropism. the feline enteric coronavirus (fecv) is an enzootic enteropathogenic virus that replicates in intestinal epithelial cells after oral uptake [1] [2] [3] [4] [5] . most infections with fecv are subclinical and rarely give rise to symptoms such as mild diarrhoea or reduced appetite [5] . the highly virulent feline infectious peritonitis virus (fipv) arises within an individual cat by mutations in the fecv genome, which enables efficient viral replication in monocytes and macrophages [6] [7] [8] [9] . fipv spreads systemically and causes the highly fatal disease known as feline infectious peritonitis virus (fip), which is characterized by a fibrinous and granulomatous serositis, fibrinous effusions in the affected body cavities and/or multifocal granulomatous vascular lesions in several organs [10] [11] [12] . besides these two pathotypes, two fcov serotypes can be distinguished. serotype i fcovs are most prevalent in the field [13] [14] [15] [16] [17] [18] , but difficult to grow in vitro. to date, the only cell line that allows sustainable growth of serotype i fcov strains is a feline intestinal epithelial cell (fiec) line established by desmarets and colleagues [19] . the less prevalent serotype ii fcov arose by double recombination between serotype i fcov and canine coronavirus (ccov), resulting in a fcov with spike and open reading frame 3 (orf3) sequences of ccov origin [20, 21] . the fcov genome consists of a single-stranded positivesense rna strand of about 29 kb that codes for 16 non-structural, 4 structural and 5 accessory proteins. orf3 encodes accessory proteins 3a, 3b and 3 c. the orf3-encoded proteins do not seem to be essential for sustainable growth in vitro in felis catus whole foetus cells and feline blood monocytes [22, 23] , although virus titres were markedly lower after the inoculation of feline blood monocytes with a fipv orf3 deletion mutant compared to the wild-type [22] . moreover, inoculation of cats with recombinant fipv lacking orf3 failed to induce typical fip clinical signs. this loss of virulence in vivo suggests a potential role of the orf3-encoded proteins in virus-host interactions [23] . the fcov 3b amino acid sequence comprises about 72 amino acid residues and is well conserved within pathotypes [24] . it was recently reported that 3b localizes to mitochondria and nucleoli, both in the presence and absence of other viral proteins [25] . however, the function(s) of this accessory protein remain unknown. interestingly, the accessory 3b protein of severe acute respiratory syndrome coronavirus (sars-cov), which comprises 154 amino acids, also translocates to mitochondria and nucleoli [26] [27] [28] , even though it does not share any amino acid sequence homology with fcov 3b. sars-cov 3b induces apoptosis [29, 30] , necrosis [29] and cell-cycle arrest [30] , and suppresses type i ifn expression induced by retinoic acid-induced gene 1 and the mitochondrial antiviral signalling protein (mavs) [26] . many viral proteins are known to target the nucleolus and may interfere with cellular stress responses, cell cycle regulation and apoptotic processes. in addition, these proteins can modify cellular and viral transcription or recruit specific nucleolar components, such as nucleolin, to facilitate viral replication (reviewed in [31] [32] [33] [34] ). upon synthesis in the cytoplasm, nucleolar proteins first migrate to the nucleus and then subsequently to the nucleolus. small soluble proteins can passively diffuse from the cytoplasm through the nuclear pore complex into the nucleoplasm (reviewed in [35] [36] [37] ). transport of larger proteins to the nucleus, however, is an atp-dependent active process driven by nuclear localization signals (nlss), which are typically characterized by clusters of basic amino acids (reviewed in [38] [39] [40] [41] [42] ). these nlss are recognized by importins that mediate transport into the nucleus. similarly, shuttling of proteins from the nucleus back to the cytoplasm is established by nuclear export signals (ness) that are recognized by exportins such as crm1. nucleolar import of proteins can be achieved through association with nucleolar components such as rrna or nucleolar proteins [32, 34, 43, 44] . for various proteins, a nucleolar localization signal (nols) was identified that targets these proteins to the nucleoli. however, nolss are not well characterized and no consensus motif is available. in general, they display a high content of basic residues and thus resemble or incorporate nlss (reviewed in [32-34, 44, 45] ). viral proteins that target mitochondria have been implicated in ca 2+ homeostasis, energy production and apoptosis. furthermore, such proteins can cause oxidative stress and modulate the mitochondrial membrane permeability and potential, and antiviral responses that emanate from a mitochondria-associated complex such as mavs. some of these viral proteins mimic or hijack mitochondrial proteins to their advantage, or alter the dynamics or intracellular localization of mitochondria (reviewed in [46] [47] [48] [49] [50] [51] ). most mitochondrial proteins are encoded by nuclear genes, synthesized in the cytosol and subsequently imported into mitochondria. this process is typically driven by an n-terminal cleavable presequence, the mitochondrial targeting peptide (mtp), which interacts with the translocase of the outer membrane (tom) complex, more specifically with the tom20 and tom22 proteins in the outer mitochondrial membrane (reviewed in [51, 52] ). even though a consensus sequence for these mtps has not been established, there are common features with respect to amino acid composition and physicochemical properties. for example, mitochondrial targeting peptides are often rich in positively charged residues, such as arginine, almost always lack negatively charged residues and generally have the ability to form amphipathic α-helices with hydrophobic residues on one side and positively charged residues on the other [53] [54] [55] [56] [57] [58] . the hydrophobic surface is recognized by tom20 and the positively charged surface interacts with tom22 [59] [60] [61] . upon import into the mitochondrial matrix, the n-terminal presequence is cleaved off by the mitochondrial processing peptidase (mpp) and the protein subsequently translocates to one of the submitochondrial compartments [51] . in this study, we investigated the subcellular localization of fcov 3b and characterized the molecular determinants that drive intracellular targeting of this accessory protein. plasmid construction: pegfp-3b, p3b-egfp, p3×flag-3b, p3b-3×flag the fcov genome was amplified from viral rna present in faeces from cats that had been experimentally infected with fecv ucd [62] using the primers listed in table 1 . the faeces were collected 9 days post-infection and the complete viral genome sequence was obtained (genbank accession number ku215423). viral rna was extracted with the qiaamp viral rna mini kit (qiagen) according to the manufacturer's instructions and cdna was produced with the superscript iii first-strand synthesis system (invitrogen). next, the 3b open reading frame was amplified by pcr with herculase ii fusion dna polymerase (agilent technologies). to create p3b-egfp, where 3b is fused at its c-terminus with enhanced green fluorescent protein (egfp), the forward primer contained an ecori site, a kozak sequence (gccgccacc) and the 5′ end of the 3b gene. the reverse primer contained a bamhi site and the 3′ end of the 3b gene, but no 3b stop codon to allow readthrough transcription ( table 1 ). the restriction enzymedigested pcr product was cloned into pcdna3.1(−)-egfp with a mutated egfp start codon (ttg instead of atg). to construct pegfp-3b, where 3b is fused at its n-terminus with egfp, the forward primer comprised an ecori site and the 5′ end of the 3b gene with a mutated 3b start codon (ttg instead of atg), and the reverse primer contained a bamhi site and the 3′ end of the 3b gene ( table 1 ). the pcr product was cloned into pcdna3.1(−)-egfp in which the stop codon of egfp was deleted. similarly, p3b-3×flag and p3×flag-3b constructs were created using pcdna3.1(−)-3×flag. the correctness of the cloned expression constructs was confirmed by sanger sequencing. crandell rees feline kidney (crfk) cells were grown on coverslips in 24-well cell culture plates in minimum essential medium (mem; gibco brl) supplemented with 5 % (v/v) foetal bovine serum (sigma-aldrich) and 2 % (w/v) lactalbumine (bd biosciences) at 37 °c and 5 % co 2 . similarly, fiecs were grown in dulbecco's modified eagle's medium (dmem; gibco brl)/ham's f12 nutrient mixture (gibco brl) (1 : 1, v/v) supplemented with 5 % (v/v) foetal bovine serum (sigma-aldrich) and 1 % (v/v) non-essential amino acids 100× (gibco brl). when reaching 60-80 % confluency, 24 h post-seeding, cells were transfected with 1 µg of plasmid dna using lipofectamine 2000 (invitrogen). six hours post-transfection, cells were washed and fresh medium with antibiotics (100 u ml −1 penicillin, 0.1 mg ml −1 streptomycin and 0.1 mg ml −1 gentamycin) was added. twenty-four hours post-transfection, expression of the 3b protein was assessed by immunofluorescence. for the egfp fusion constructs, cells were fixed with 4 % (w/v) paraformaldehyde (pf) in phosphate-buffered saline (pbs) for 10 min at room temperature (rt) and nuclei were stained with hoechst 33 342 (invitrogen) for 10 min at rt. the coverslips were washed twice with pbs, mounted in 0.9 : 0.1 (v/v) glycerin/pbs with 2.5 % (w/v) 1,4-diazabicyclo (2,2,2) octane and analysed by fluorescence microscopy (leica microsystems dmrbe). for the constructs containing the 3×flag-tag, cells were fixed with 4 % (w/v) pf in pbs for 10 min at rt, followed by permeabilization with 0.1 % (v/v) triton x-100 for 2 min at rt. next, coverslips were incubated with rabbit anti-flag antibody (sigma-aldrich) dilutions containing 10 % (v/v) negative goat serum for 1 h at 37 °c, followed by incubation with fitc-conjugated goat antirabbit antibodies (invitrogen) for 1 h at 37 °c and hoechst staining of the nuclei. to evaluate the dynamic localization of fcov 3b, crfk cells transfected with p3b-egfp were fixed with pf at different time points (1, 2, 3, 4, 5, 6, 9, 12 and 18 h post-transfection) and nuclei were counterstained with hoechst. to assess mitochondrial localization, live cells were labelled with the mitotracker red cmxros (100 nm, cell signaling technology), followed by fixation and hoechst staining of the nuclei. to determine colocalization with nucleoli, cells were fixed and permeabilized and subsequently incubated with rabbit anti-nucleolin antibody dilutions (abcam) containing 10 % (v/v) negative rabbit serum. after two washes with pbs, texas red-conjugated goat anti-rabbit igg antibody dilutions (invitrogen) were added for 1 h at 37 °c. nuclei were stained with hoechst and coverslips were washed twice with pbs, mounted and examined for fluorescence. twenty-four hours post-transfection, cells were lysed for immunoblotting in radioimmunoprecipitation assay (ripa) lysis buffer (abcam) supplemented with the halt protease inhibitor cocktail (thermo scientific) for 1 h at 4 °c. after centrifugation, the supernatant was fractionated on a 12 % polyacrylamide gel by sds-page and then transferred to a hybond-p pvdf membrane. after blotting, the membranes were blocked in 5 % (w/v) non-fat dry milk in pbs with 0.1 % (v/v) tween-20 for 1 h at rt. the blots were further incubated overnight with abfinity recombinant rabbit gfp monoclonal antibodies (invitrogen) or rabbit anti-flag polyclonal antibodies (sigma-aldrich) at 4 °c. after being washed three times with tris-buffered saline with 0.1 % (v/v) tween-20, the blots were incubated for 1 h at rt with hrp-conjugated goat anti-rabbit antibodies (dakocytomation). after three washing steps, proteins were visualized by enhanced chemiluminescence (ecl prime, ge healthcare) and analysed with the chemidoc mp imaging system (bio-rad). the fecv ucd 3b protein sequence was run through the psort ii server (https:// psort. hgc. jp) to predict the protein localization sites in cells. potential nuclear export signals were identified using locnes [67] , meszaros et al. [25] and terada et al. [68] . the genbank accession numbers can be found in table s1 (available in the online version of this article). mega 7 software was used to perform multiple sequence alignment. serial 3b truncated mutants were engineered by designing several forward and reverse primers that flank the region to be deleted ( table 1 ). the p3b-egfp plasmid was amplified with these primers using q5 dna polymerase (new england biolabs), followed by dpni (new england biolabs) and t4 polynucleotide kinase (new england biolabs) treatment and ligation with the t4 dna ligase (promega). all plasmid sequences were confirmed by sequencing analysis. transfection and immunofluorescent stainings were performed as described above. bioinformatics analysis using the psort ii server predicted the 3b protein to localize to the cytoplasm (47.8 %), mitochondria (26.1 %), nucleus (21.7 %) or secretory vesicles (4.3 %). to examine its subcellular localization, the fcov 3b coding information was cloned in pcdna3.1(−)-egfp, fusing it either at the c-terminus (p3b-egfp) or at the n-terminus (pegfp-3b) with egfp (fig. 1a) . next, crfk cells were transfected with these constructs and the subcellular distribution pattern was evaluated by confocal microscopy (fig. 1c) . wild-type egfp, which functions as the control vector, showed a diffuse distribution throughout the cytoplasm and the nucleus, but was excluded from the nucleoli, as described by other authors (i.e. in [69, 70] ). transfection of the p3b-egfp expression vector resulted in a dotted or tubular distribution in the cytoplasm. visualization of the mitochondria using mitotracker red cmxros clearly demonstrated that fcov 3b-egfp colocalizes with this organelle (fig. 2) . as mitochondria are dynamic structures characterized by fusion and fission, they can occur either as individual structures or form a large interconnected tubular network [49] , which could explain the fact that some cells show a more dotted cytoplasmic 3b expression pattern and others a more tubular one. although the majority of cells exclusively expressed the 3b-egfp fusion protein in mitochondria, accumulation in certain regions of the nucleus was apparent in some cells (fig. 1c) . these subnuclear compartments corresponded with nucleoli, as confirmed by immunofluorescent staining (fig. 2) . western blot analysis using anti-gfp antibodies showed the migration of chimeric proteins at the expected molecular mass of approximately 36 kda (fig. 1b) . these results are in line with the observations from meszaros and colleagues, who investigated the subcellular localization of all fcov orf3-encoded proteins [25] . interestingly, fusion of 3b at the c-terminus of egfp abolished mitochondrial translocation and 3b expression was restricted to the nucleoli (fig. 1c) , which may indicate the presence of an important n-terminal targeting signal to allow mitochondrial localization. due to the lack of specific anti-3b antibodies and to exclude a potential effect of egfp on subcellular localization, 3b was also fused with a 3×flag-tag either n-terminally or c-terminally (p3×flag-3b and p3b-3×flag, respectively) (fig. 3a) and expression of the tagged 3b proteins was confirmed by western blot analysis, revealing bands with the expected molecular weight of approximately 12 kda (fig. 3b) . similar results were obtained as for the egfp fusion proteins, i.e. mitochondrial and nucleolar expression for p3b-3×flag and only nucleolar expression for p3×flag-3b (fig. 3c) . the fluorescent signal was, however, weaker compared to egfp. in addition, these egfp and 3×flag constructs were also introduced in fiecs to exclude cell type-dependent localization of 3b. these fiecs are in fact the target cells for fecv and to date the only cell line in which serotype i strains can be propagated in vitro [19] . even though the transfection efficiency is quite low in this epithelial cell line, the 3b expression pattern was comparable to that observed in crfk cells (fig. 4) . from these results, we can conclude that the 3b localization is not influenced by the fusion protein or tag to which it was fused nor by the cell type in which the protein is expressed, and thus most likely represents the actual subcellular localization of fcov 3b. furthermore, it was shown by meszaros and colleagues that the 3b expression pattern did not change when transfection was followed by infection with fipv df-2, although 3b accumulation in the nucleolus was more frequently observed in infected cells compared to transfection only [25] . proteins that target the nucleolus first migrate to the nucleus and then subsequently to the nucleolus. the active transportation of proteins through the nuclear pore complex is usually driven by nlss. classical nlss include the monopartite pat4 and pat7 motifs and the bipartite motif (reviewed in [38, 40, 41, 71] ). the pat4 motif is a continuous stretch of four basic amino acid residues (arginine or lysine) [β 4 ] or three basic amino acid residues followed by a histidine or proline [β 3 (h/p)]. the pat7 motif starts with a proline and is followed, within three residues, by a basic four-residue cluster containing at least three arginines or lysines [pχχ(β 3 χ)]. bipartite nlss have 2 clusters of basic amino acids separated by a 10-12 amino acid-long spacer, where the first cluster consists of 2 basic residues and the second cluster is a basic 5-residue region consisting of at least 3 basic residues [ββχ (10) (11) (12) (β 3 χ 2 )] [72, 73] . nls diversity keeps expanding as non-classical nlss (reviewed in [38] ) and additional nls classes have been identified [74] . it should be noted that, in the absence of an nls, proteins can still localize to the nucleus by co-transportation with other proteins that contain an nls [75, 76] . in addition, the presence of an nls does not guarantee nuclear localization, as the nls may be masked from the protein surface (reviewed in [42] ). nolss that drive nucleolar targeting are poorly characterized and usually closely associated with nlss, thereby hampering the identification of a consensus motif. it is important, however, to differentiate between (1) nls-only signals, which will target proteins to the nucleus but not to nucleoli, (2) nols-only signals, which will translocate proteins to the nucleoli, but are not capable of transporting them through the nuclear pore complex, and (3) joint nols-nls signals, which will target the proteins to the nucleus and subsequently induce nucleolar accumulation [45] . a potential nls is present in the fcov 3b amino acid sequence, as the psort ii server identified two putative (overlapping) pat4 motifs (amino acids 53 to 56 and 54 to 57) (fig. 5a) . in addition, a hopp-woods hydrophilicity plot (fig. 5b) shows that these overlapping pat4 motifs are located in a hydrophilic region and therefore the surface should be easily accessible for interaction with importins. to investigate if these residues are conserved amongst different strains, 178 (putative) serotype i fcov 3b protein sequences were compared. all of them contained at least one pat4 motif in the same region. thirteen (7.30 %) had a single pat4 motif and 165 (92.7 %) had 2 overlapping pat4 motifs. there is a 90 % amino acid homology between the 3b sequence described by meszaros and colleagues (isolate hu2009fc, genbank accession number awu66525.1), which was also shown to localize to nucleoli, and the fecv ucd 3b strain used in this study. both isolates have two overlapping pat4 motifs, with a slightly different amino acid sequence (krrkr and krrrr, respectively). the nucleocapsid proteins of several members of the order nidovirales, such as porcine reproductive and respiratory syndrome virus (prrsv, family arteriviridae) [70, [77] [78] [79] [80] , transmissible gastroenteritis virus (tgev) [81] , mouse hepatitis virus (mhv) [81, 82] and infectious bronchitis virus (ibv) [82] [83] [84] [85] (family coronaviridae), also target the nucleolus and have been shown to incorporate one or more potential nlss. similarly, the sars-cov 3b protein was predicted to have two overlapping nlss, a pat4 and bipartite motif, that were shown to be associated with nuclear (nucleolar) localization [27] . to examine which peptide domains are responsible for the observed nucleolar expression pattern of fcov 3b, a set of consecutive truncations, starting from both the n-terminus and the c-terminus of the 3b protein, were constructed and expressed in crfk cells. as shown in fig. 6 , deletion of residues 2 to 50 (p3b-egfp d2-15, d2-25, d2-35, d2-45 and d2-50) still resulted in egfp translocation to the nucleoli. however, deletion of residues 2 to 55 (p3b-egfp d2-55), which results in a deletion of the first three residues of the pat4 motifs, and deletion of residues 2 to 65 (p3b-egfp d2-65), whereby the complete pat4 motifs are removed, was associated with a complete loss of nucleolar accumulation of the corresponding egfp fusion protein. similarly, deleting the 12 c-terminal residues (p3b-egfp d61-72) did not alter nucleolar localization, but deletion of the 17 c-terminal residues (p3b-egfp d56-72), which results in a deletion of the last 2 residues of the pat4 motifs, or deletion of the 22 c-terminal residues (p3b-egfp d56-72), whereby both pat4 motifs are completely removed, abrogated nucleolar expression. in addition, deletion of the overlapping pat4 motifs (p3b-egfp d53-57), also resulted in a lack of nucleolar localization. these results suggest that, even though the 3b protein is theoretically able to passively diffuse through the nuclear pore complex, translocation to twenty-four hours after transfection with p3b-3×flag and p3×flag-3b, cell lysates were prepared and fractioned by sds-page. after blotting, the pvdf membrane was probed with polyclonal anti-flag antibodies. the expected molecular mass of p3b-3×flag and p3×flag-3b is ~12 kda. (c) cellular localization of the 3×flag constructs. crfk cells were transfected with p3b-3×flag and p3×flag-3b. twenty-four hours post-transfection, cells were fixed, permeabilized and incubated with rabbit anti-flag antibodies, followed by incubation with fitc-conjugated goat anti-rabbit antibodies. the nuclei were stained with hoechst (blue) and localization of the fusion protein (green) was assessed by confocal microscopy. the nucleus is likely to be an active process driven by a joint nols-nls signal. mitochondrial localization is often driven by the presence of an mtp. as mtps have no consensus motif, prediction is frequently based on (1) the amino acid composition of the n-terminal part of the protein, (2) the propensity to form amphipathic α-helices and (3) the presence of putative recognition motifs or mpp cleavage sites. a conserved motif ϕχχϕϕ in the n-terminal mitochondrial presequence, where ϕ represents hydrophobic residues (l, f, i, v, w, y, m) and χ any residue, that is recognized by the tom20 protein has been identified [86, 87] , with a preference for basic residues on position 3 (ϕχβϕϕ, with β representing either r, k or h) [87] . however, import into mitochondria can still be observed after substituting such hydrophobic residues with alanine, despite reduced tom20 binding [88] . in addition, 14 hexamers, such as ϕϕσβϕϕ, ϕϕβσϕϕ and βϕϕσσσ (where σ represents polar residues s, t, n or q), have been proposed to be involved in mitochondrial targeting, but their actual function as mitochondrial targeting sequences remains to be elucidated [89] . in the absence of an n-terminal mtp, proteins can still target the mitochondria through internal targeting signals or hydrophobic domains (reviewed in [51] ). mitochondrial targeting of the sars-cov 3b protein was shown to be determined by a region located in the middle of the protein rather than by n-terminal residues [26, 28] . furthermore, the outer membrane of the mitochondria contains proteins, called porins, that will freely allow molecules smaller than 10 kda to cross the mitochondrial membrane [46] . in addition, viral proteins can also be transported from the er into the mitochondria via the mitochondria-associated membrane compartment (reviewed in [51, 90] ). the n-terminal part of the fcov 3b protein mainly consists of hydrophobic and positively charged residues, whereas negatively charged residues are nearly absent. mitofates allocated the maximum positively charged amphiphilicity score to residues 10 to 19. furthermore, mitofates identified two putative tom20 recognition motifs [amino acids 13 to 17 and 61 to 65, further annotated as tom20 (1) and tom20 (2), respectively] with a consensus ϕχβϕϕ pattern (fig. 5a) . multiple sequence alignment revealed that 173 (97.19 %) and 176 (98.88 %) of a total of 178 aligned sequences respectively have a tom20(1) and tom20(2) recognition motif. the amino acid composition varied, however, amongst different strains: i/l-i/l/v-r-l-f/y for tom20(1) and y-r/g-k/r-i/v-a for tom20 (2) . five isolates (2.81 %) seemed to lack a tom20(1) motif and two isolates (1.12 %) a tom20(2) motif ( table 2) . comparison of the 3b sequence of isolate hu2009fc, which is known to locate to mitochondria [25] , and fecv ucd showed that the tom20(1) and tom20(2) recognition motifs were identical. in addition to these tom20 recognition motifs, two overlapping hexamers (amino acids 24 to 29 and 25 to 30) with a ϕϕϕβσϕ and ϕϕβσϕϕ pattern, respectively, and potentially associated with mitochondrial targeting, were identified by mitofates. of the 178 aligned 3b sequences, 155 (87.08 %) had at least 1 hexamer in this region. there was, however, great variability between isolates in terms of amino acid composition. finally, an mpp cleavage site between residue 16 and 17 was proposed (fig. 5a) . mpp cleavage sites are often characterized by the presence of arginine at position -2, -3 or −10 relative to the cleavage site (reviewed in [91] ). as for nucleolar localization, mitochondrial targeting was assessed after the transfection of different truncated 3b mutants fused to egfp (fig. 6 ). the constructs with c-terminal deletions of residues 21 to 72 (p3b-egfp d21-72, d31-72, d41-72, d51-72, d56-72 and d61-72) all continued to target egfp to the mitochondria, but p3b-egfp d11-72 was distributed diffusely throughout the cytoplasm and a mitochondrial expression pattern was no longer observed. for the n-terminal truncated mutants, deletion of residues 2 to 25 (p3b-egfp d2-15 an d2-25) did not seem to influence mitochondrial colocalization, but mitochondrial expression was abrogated when amino acids 2 to 35 (p3b-egfp d2-35) were deleted. these results suggest the presence of an n-terminal mtp with a critical role for residues 10 to 35, which comprise both the predicted tom20(1) motif and the identified hexamers, in mitochondrial localization. however, it remains unclear what specific role is exerted by the tom20(1) motif and the identified hexamers, as deletion of one of them has no effect on mitochondrial expression, but deletion of both constrains localization to the mitochondria. interestingly, after transfection of p3b-egfp d2-55, the fluorescent signal was diffusely present in the cytoplasm as for p3b-egfp d2-35, d2-45 and d2-50, but additionally the typical mitochondrial expression pattern could also be observed (fig. 6) . this could be a consequence of the presence of the tom20(2) motif that is now in the n-terminus of this truncated protein. furthermore, deletion of residues 2 to 65 (p3b-egfp d2-65), which includes deletion of the tom20(2) motif, no longer induced mitochondrial colocalization. it should be noted that as a diffuse cytoplasmic pattern was observed after the transfection of p3b-egfp d2-55, in addition to mitochondrial expression, there may be an indication that mitochondrial localization of this truncated protein is reduced compared to the wild-type 3b. this could be explained by the presence of an alanine at position 5 of the tom20(2) motif yrkia, as mukhopadhyay and colleagues have related that alanine substitution of the hydrophobic residues is associated with reduced tom20 binding [88] . to assess the dynamics of 3b expression and the potential trafficking of 3b from the nucleus to the cytoplasm or vice versa, 3b translocation was assessed at different time points posttransfection. faint expression in the nucleoli was observed 3 h post-transfection (fig. 7) . at 4 h post-transfection, the fluorescent signal in the nucleoli was found to increase and a vague cytoplasmic signal was detected, but it was not until 5 h after transfection that a mitochondrial pattern was clearly visible. as of 6 h post-transfection, the fluorescent signal was brighter and an increasing number of cells began to show exclusive mitochondrial expression of the 3b protein, as exemplified in fig. 2 . these results may indicate nucleolar-mitochondrial shuttling, as observed for the prrsv nucleocapsid [78] . like nuclear import, shuttling proteins from the nucleus back to the cytoplasm is established by a nuclear export signal (nes) that is recognized by exportins such as crm1. a wellcharacterized nes is the export motifϕχ (2) (3) ϕχ (2) (3) ϕχϕ (where ϕ represents one of the hydrophobic residues l, v, i, f or m and χ is any residue) [92, 93] , but this consensus pattern has the psort ii server identified two overlapping pat4 motifs that may be involved in nuclear translocation. a potential nes was predicted by the locnes and wregex. mitofates located two putative tom20 recognition motifs, which may be involved in binding of 3b to tom20 in the outer membrane of the mitochondria. additionally, a potential mpp cleavage site and two overlapping significant hexamers were observed. (b) hopp−woods hydrophilicity plot of fcov 3b protein. been progressively expanded and computational methods have been addressed to further model the sequence diversity of ness [93] [94] [95] [96] [97] [98] [99] [100] . a potential nes was identified in the fcov 3b sequence -fswilksrlii (amino acids 4 to 14) -by both the locnes and wregex prediction tools (fig. 5a) . however, despite the presence of this potential nes, the expression of the corresponding truncated 3b variants showed no indication of its actual functionality. indeed, deletion of the first 25 amino acids (p3b-egfp d2-15 an d2-25), including this potential nes, did not abrogate mitochondrial expression (fig. 6) . these results suggest that 3b may not migrate first to the nucleolus and subsequently to the mitochondria, or that another unidentified nes is present in the 3b sequence. an alternative hypothesis to account for the observed spatiotemporal kinetics is that nucleolar accumulation is much more concentrated compared to mitochondrial expression, thus facilitating the observation of fluorescent signal in the nucleoli. furthermore, it was described that proteins that [32, 34, 39] and it was suggested by garcia-bustos and colleagues that the most n-terminal-located signal dominates [39] . as the putative mtp of the fcov 3b is located more n-terminally compared to the nols, this may explain why many cells exclusively show mitochondrial expression. in the present study, we present data demonstrating that the fcov 3b protein directs the localization of a fusion protein (egfp) or tag (3×flag tag) to mitochondria and nucleoli, independently of the type of fusion protein and transfected cell type. nucleolar localization requires a joint nols-nls signal that encompasses two overlapping pat4 motifs. moreover, the presence of a pat4 motif is highly conserved among serotype i fcov strains. mitochondrial expression is mainly triggered by n-terminal residues 10-35, which contain a putative tom20 recognition motif 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polypeptide domain that specifies migration of nucleoplasmin into the nucleus nuclear transport of adenovirus dna polymerase is facilitated by interaction with preterminal protein peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus nucleolar-cytoplasmic shuttling of prrsv nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rna-associated protein fibrillarin a model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (prrsv) nucleocapsid protein based on live cell imaging localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell cell cycle dependent nucleolar localization of the coronavirus nucleocapsid protein changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus nmr identification of the tom20 binding segment in mitochondrial presequences peptide library approach with a disulfide tether to refine the tom20 recognition motif in mitochondrial presequences mito-fates: improved prediction of mitochondrial targeting sequences and their cleavage sites access of viral proteins to mitochondria via mitochondria-associated membranes processing of mitochondrial presequences protein sequence requirements for function of the human t-cell leukemia virus type 1 rex nuclear export signal delineated by a novel in vivo randomization-selection assay analysis and prediction of leucine-rich nuclear export signals prediction of leucine-rich nuclear export signal containing proteins with nessential nuclear export signal consensus sequences defined using a localization-based yeast selection system nesmapper: accurate prediction of leucine-rich nuclear export signals using activity-based profiles prediction of nuclear export signals using weighted regular expressions (wregex) nologo: a new statistical model highlights the diversity and suggests new classes of crm1-dependent nuclear export signals sequence and structural analyses of nuclear export signals in the nesdb database locnes: a computational tool for locating classical ness in crm1 cargo proteins the authors gratefully acknowledge ytse noppe and marthe pauwels for their skillful technical assistance. the authors declare that there are no conflicts of interest. five reasons to publish your next article with a microbiology society journal 1 . the microbiology society is a not-for-profit organization. 2. we offer fast and rigorous peer review -average time to first decision is 4-6 weeks. 3. our journals have a global readership with subscriptions held in research institutions around the world. 4. 80% of our authors rate our submission process as 'excellent' or 'very good'. 5. your article will be published on an interactive journal platform with advanced metrics.find out more and submit your article at microbiologyresearch.org. key: cord-031957-df4luh5v authors: dos santos-silva, carlos andré; zupin, luisa; oliveira-lima, marx; vilela, lívia maria batista; bezerra-neto, joão pacifico; ferreira-neto, josé ribamar; ferreira, josé diogo cavalcanti; de oliveira-silva, roberta lane; pires, carolline de jesús; aburjaile, flavia figueira; de oliveira, marianne firmino; kido, ederson akio; crovella, sergio; benko-iseppon, ana maria title: plant antimicrobial peptides: state of the art, in silico prediction and perspectives in the omics era date: 2020-09-02 journal: bioinform biol insights doi: 10.1177/1177932220952739 sha: doc_id: 31957 cord_uid: df4luh5v even before the perception or interaction with pathogens, plants rely on constitutively guardian molecules, often specific to tissue or stage, with further expression after contact with the pathogen. these guardians include small molecules as antimicrobial peptides (amps), generally cysteine-rich, functioning to prevent pathogen establishment. some of these amps are shared among eukaryotes (eg, defensins and cyclotides), others are plant specific (eg, snakins), while some are specific to certain plant families (such as heveins). when compared with other organisms, plants tend to present a higher amount of amp isoforms due to gene duplications or polyploidy, an occurrence possibly also associated with the sessile habit of plants, which prevents them from evading biotic and environmental stresses. therefore, plants arise as a rich resource for new amps. as these molecules are difficult to retrieve from databases using simple sequence alignments, a description of their characteristics and in silico (bioinformatics) approaches used to retrieve them is provided, considering resources and databases available. the possibilities and applications based on tools versus database approaches are considerable and have been so far underestimated. proteins and peptides play different roles depending on their amino acids (aa) constitution, which may vary from tens to thousands residues. 1 peptides are conventionally understood as having less than 50 aa. 2 proteins, on the contrary, would be any molecule presenting higher amino acid content and bothproteins and peptides-present a plethora of variations in plants. despite that, plant proteomes have been much more studied than peptidomes. it is well-known that the biochemical machinery necessary for the synthesis and metabolism of peptides is present in every living organism. from the variations of this machinery, a wide structural and functional diversity of peptides was generated, justifying the growing interest in their study. in eukaryotes, peptides are prevalent in intercellular communication, performing as hormones, growth factors, and neuropeptides, but they are also present in the defense system. 3 besides plants and animals, several pathogenic microorganisms, peptides can serve as classical virulence factors, which disrupt the epithelial barrier, damage cells, and activate or modulate host immune responses. an example of this performance is represented by candidalysin, 4 a fungal cytolytic peptide toxin found in the pathogenic fungus candida albicans that damages epithelial membranes, triggers a response signaling pathway, and activates epithelial immunity. there are also reports of defense-related fungal peptides. for example, the copsin, a peptide-based fungal antibiotic recently identified in the fungus coprinopsis cinerea 5 kills bacteria by inhibiting their cell wall synthesis. regarding bacterial peptides, certain species from the gastrointestinal microbial community can release low-molecular-weight peptides, able to trigger immune responses. 6 there are additionally peptides that act like bacterial "hormones" that allow bacterial communities to organize multicellular behavior such as biofilm formation. 7 some peptides are known for their medical importance, as defensins that 2 bioinformatics and biology insights present antibacterial, antiviral, and antifungal activities. for example, human alpha-and beta-defensins present in the saliva may potentially impede virus replication, including sars-cov-2, 8 besides other roles as protection against intestinal inflammation (colitis). 9 considering the roles of plant peptides, they can also be multifunctional, and have been classified into 2 main categories 10 (supplementary figure s1) : (1) peptides with no bioactivity, primarily resulting from the degradation of proteins by proteolytic enzymes, aiming at their recycling, and (2) bioactive peptides, which are encrypted in the structure of the parent proteins and are released mainly by enzymatic processes. the first group is innocuous regarding signaling, regulatory functions, and bioactivity. so far, it has been reported that some of them may play a significant role in nitrogen mobilization across cellular membranes. 11 the second group of bioactive peptides has a substantial impact on the plant cell physiology. some peptides of this group can act in the plant growth regulation (through cell-to-cell signaling), endurance against pathogens and pests by acting as toxins or elicitors, or even detoxification of heavy metals by ion-sequestration. comprising bioactive peptides, additional subcategorization has been proposed regarding their function. tavormina et al 12 figure s1 ) based on the type of precursor: • • derived from functional precursors: originated from a functional precursor protein; • • derived from nonfunctional precursors: originated from a longer precursor that has no known biological function (as a preprotein, proprotein, or preproprotein); • • not derived from a precursor protein: some sorfs (small open read frames; usually <100 codons) are considered to represent a potential new source of functional peptides (known as "short peptides encoded by sorfs"). a more intuitive classification of bioactive peptides was further proposed by farrokhi et al 10 receptors in leaves. 13 another example is the pls (polaris) peptide that acts during early embryogenesis but later activates auxin synthesis, also affecting cytokine synthesis and ethylene response. 14 regarding the second group, it includes peptides with signaling roles in plant defense, comprising at least 4 subgroups, including syst (systemin) (supplementary figure s1 ). the syst peptides were identified in solanaceae members, like tomato and potato 15 (acting on the signaling response to herbivory). the syst leads to the production of a plant protease inhibitor that suppresses insect's proteases. 16 stratmann 17 suggested that in plants, systs act to stimulate the jasmonic acid signaling cascade within vascular tissues to induce a systemic wound response. • • defense peptides or antimicrobial peptides (amps): to be fitted into this class, a plant peptide must fulfill some specific biochemical and genetic prerequisites. regarding biochemical features, in vitro antimicrobial activity is required. concerning the genetic condition, the gene encoding the peptide should be inducted in the presence of infectious agents. 18 in practice, this last requirement is not ever fulfilled as some amps are tissue-specific and are considered as part of the plant innate immunity, while other isoforms of the same class appear induced after pathogen inoculation. 19 plant amps are the central focus of the present review, comprising information on their structural features (at genomic, gene, and protein levels), resources, and bioinformatic tools available, besides the proposition of an annotation routine. their biotechnological potential is also highlighted in the generation of both transgenic plants resistant to pathogens, and new drugs or bioactive compounds. antimicrobial peptides are ubiquitous host defense weapons against microbial pathogens. the overall plant amp characterization regards the following variables ( figure 1 ): electrical charge, hydrophilicity, secondary and 3-dimensional (3d) structures, and the abundance or spatial pattern of cysteine residues. 20 these features are primarily related to their defensive role(s) as membrane-active antifungal, antibacterial, or antiviral peptides. regarding the nucleotide sequence, plant amps are hypervariable and this genetic variability is considered crucial to provide diversity and the ability to recognize different targets. for their charges, amps can be classified as cationic or anionic ( figure 1 ). most plant amps have positive charges, which is a fundamental feature for the interaction with the membrane lipids of pathogens. 21 concerning hydrophilicity, amps are generally amphipathic, that is, they exhibit molecular conformation with both hydrophilic and hydrophobic domains. 22 silva et al 3 with respect to their 3d structure, amps can be either linear or cyclic ( figure 1 ). some linear amps adopt an amphipathic α-helical conformation, whereas non-α-helical linear peptides generally show 1 or 2 predominant amino acids. 23 in turn, cyclic amps, including cysteine-containing peptides, can be divided into 2 subgroups based on the presence of a single or multiple disulfide bonds. a peculiar feature of these peptides is a cationic and amphipathic character, what improves their functioning as membrane-permeabilizing agents. 23 considering the secondary structures, amps may exhibit α-helices, β-chains, β-pleated sheets, and loops ( figure 1 ). wang 24 classified plant amps into 4 families (α, β, αβ, and non-αβ), based on the protein classification of murzin et al, 25 with some modifications. antimicrobial peptides of the "α" family present α-helical structures, 1 whereas amps from the "β" family contains β-sheet structures usually stabilized by disulfide bonds. 26, 27 some plant amps show an α-hairpinin motif formed by antiparallel α-helices that are stabilized by 2 disulfide bridges. 28 such amps present a higher resistance to enzymatic, chemical, or thermal degradation. 29 antimicrobial peptides from the "αβ" family having both "α" and "β" structures are also stabilized by disulfide bridges. an example of amp presenting "αβ" structures are defensins, usually with a cysteine-stabilized αβ motif (csαβ), an α-helix, and a triplestranded antiparallel β sheet stabilized mostly by 4 disulfide bonds. 30 finally, amps that do not belong to the "αβ" group exhibit no clearly defined "α" or "β" structures. 26 plant amps are also classified into families considering protein sequence similarity, cysteine motifs, and distinctive patterns of disulfide bonds, which determine the folding of the tertiary structure. 31 therefore, plant amps are commonly grouped as thionins, defensins, heveins, knottins (linear and cyclic), lipid transfer proteins (ltp), snakins, and cyclotides. 27, 31 these amp categories will be detailed in the next sections, together with other groups here considered (impatienlike, macadamia [β-barrelins], puroindoline (pin), and thaumatin-like protein [tlp]) and the recently described αhairpinin amps. the description includes comments on their structure, pattern for regular expression (regex) analysis (when available), functions, tissue-specificity, and scientific data availability. thionins are composed by 45 to 48 amino acid residues with a molecular weight around 5 kda, considering the mature peptide. they are synthesized with a signal peptide together with the mature thionin and the so-called acidic domain. 32 to date, there is no experimental information available about possible functions of the acidic domain, even though it is clearly not dispensable as shown by the high conservation of the cysteine residues. 33 the thionin superfamily comprises 2 distinct groups of plant peptides α/β-thionins and γ-thionins with distinguished structural features. 34 the α/β thionins have homologous amino acid sequences and similar structures. 35 besides, they are rich in arginine, lysine, and cysteine. 36 in turn, γ-thionins have a greater similarity with defensins, and some authors classify them within this group. 37 however, compared with the defensins, they present a longer conserved amino acid sequence. 31 regarding the cysteine motif, it can be divided into 2 subgroups, one with 8 residues connected by 4 disulfide bonds called 8c and the other with 6 residues connected by 3 disulfide bonds called 6c. 38 the general designation of thionins has been proposed as a family of homologous peptides that includes purothionins. the first plant thionin was isolated in 1942 from wheat flour and labeled as purothionin. 39 since then, homologues from various taxa have been also identified, like bioinformatics and biology insights viscotoxins (viscum album) and crambins (crambe abyssinica). 40 they have also been isolated from different plant tissues like seeds, leaves, and roots. 41, 42 thionins have been tested for different elicitors: grampositive 43, 44 or gram-negative bacteria, 45, 46 yeast, 38, 43 insect larvae, 47 nematode, 33 and inhibitory proteinase. 48 thionins are hydrophobic in nature, interact with hydrophobic residues, and lyse bacteria cell membrane. their toxicity is due to an electrostatic interaction with the negatively charged membrane phospholipids, followed by either pore formation or a specific interaction with a membrane. 38 it has been reported that they are able to inhibit other enzymes possibly through covalent attachment mediated by the formation of disulfide bonds, as previously observed for other thionin/enzyme combinations. 48 thionin representatives with known 3d structures determined by x-ray crystallography are crambin (pdb id: 1crn), α1and β-purothionins (pdb id: 2phn and 1bhp), β-hordothionin (pdb id: 1wuw), and viscotoxin-a3 (pdb id: 1okh). the first to be determined was the mixed form of crambin. 35, 49 it showed a distinct capital γ shape with the n terminus forming the first strand in a βsheet. the architecture of this sheet is additionally strengthened by 2 disulfide bonds. 50 after a short stretch of extended conformation, there is a helix-turn-helix motif. in crambin, there is a single disulfide involved in stabilizing the helix-tohelix contacts. at the center of this motif, there is a crucial arg10 that forms 5 hydrogen bonds to tie together the first strand, the first helix, and the c terminus. 50 the first plant defensins were isolated from wheat 51 and barley grains, 52 initially called γ-hordothionins. due to some similarities in cysteine content and molecular weight, they were classified as γ-thionins. later, the term "γ-thionin" was replaced by "defensin" based on the higher number of primary and tertiary structures of these proteins and also on their antifungal activities more related to insect and mammalian defensins than to plant thionins. 53 plant defensins belong to a diverse protein superfamily called cis-defensin 54 and exhibit cationic charge, consisting of 45 to 54 aa with 2 to 4 disulfide bonds. 53, 55 plant defensins share similar tertiary structures and typically exhibit a triple-stranded antiparallel β sheet, enveloped by an α-helix and confined by intramolecular disulfide bonds 1 (figure 2a ). this motif is called cysteine-stabilized αβ (csαβ). 56 the csαβ defensins were classified into 3 groups based on their sequence, structure, and functional similarity. defensins are known for their antimicrobial activity at low micromolar concentrations against gram-positive and gramnegative bacteria, 57 fungi, 58 viruses, and protozoa. 59 in addition, they present protein inhibition, insecticidal, and antiproliferative activity, acting as an ion-channel blocker, being also associated with the inhibition of pathogen protein synthesis. 60 instead, plant defensins act in the regulation of signal transduction pathways and induce inflammatory processes, in addition to wound healing, proliferation control, and chemotaxis. 61 in general, plant defensins do not present high toxicity to human cells, having in vivo efficacy records, with relevant therapeutic potential, and can be applied in treatments associated with traditional medicine. 62 cools et al 63 reported that a peptide derived from a plant defensin (hsafp1) acted synergistically with caspofungin (an antimycotic) (in vivo and in vitro) against the formation of candida albicans biofilm on polystyrene and catheter substrate, indicating that the hsafp1 variant presented a strong antifungal potential in the proposed treatment. other biotechnological applications of defensins are described, as in the case of ecgdf1, which was isolated from a legume (erythrina crista-galli), heterologously expressed in escherichia coli and purified. ecgdf1 inhibited the growth of various plant and human pathogens (such as candida albicans and aspergillus niger and the plant pathogens clavibacter michiganensis ssp. michiganensis, penicillium expansum, botrytis cinerea and alternaria alternate). 64 due to these features, ecgdf1 is a candidate for the development of antimicrobial products for both agriculture and medicine. 64 non-specific lipid transfer proteins (ns-ltps) were first isolated from potato tubers 65 and are actually identified in diverse terrestrial plant species. they comprise a large gene family, are abundantly expressed in most tissues, but absent in most basal plant groups as chlorophyte and charophyte green algae. 66 they generally include an n-terminal signal peptide that directs the protein to the apoplastic space. 67 some ltps have a c-terminal sequence that allows their post-translational modification with a glycosylphosphatidylinositol molecule, facilitating the integration of ltp on the extracellular side of the plasma membrane. the ns-ltps are small proteins which were thus named because of their function of transferring lipids between the different membranes carrying lipids (non-specifically, the list includes phospholipids, fatty acids, their acylcoas, or sterols). they consist of approximately 100 aa and are relatively larger in size than other amps, such as defensins. depending on their sizes, ltps may be classified into 2 subfamilies: ltp1 and ltp2, with relative molecular weight of 9 and 7 kda, respectively. 68, 69 the limited sequence conservation turned this classification inadequate. thus, a modified and expanded classification system was proposed, presenting 5 main types (ltp1, ltp2, ltpc, ltpd, and ltpg) and 5 additional types with a smaller number of members (ltpe, ltpf, ltph, ltpj, and ltpk). 66 the new classification system is not based on molecular size but rather on (1) the position of a conserved intron, (2) the identity of the amino acid sequence, and figure s2) . although this latter classification system is the most recent, the conventional classification of ltp1 and ltp2 types has been maintained by most working groups. lipid transfer protein nomenclature has been confusing and without consistent guidelines or standards. there are several examples where specific ltps receive different names in different scientific articles. the lack of a robust terminology sometimes turns it quite difficult, extremely time-consuming, and frustrating to compare ltps with different roles/functions. 67 therefore, an additional nomenclature was also proposed by salminen et al, 67 naming ltps as follows: atltp1.3, osltp2.4, hvltpc6, ppltpd5, and taltpg7, with the first 2 letters indicating the plant species (eg, at = arabidopsis thaliana, pp = physcomitrella patens); ltp1, ltp2, and ltpc indicating the type; while the last digit (here 3-7) regards the specific number given to each gene or protein within a given ltp type. for the sake of clarity, the authors recommend the inclusion of a point between the type specification (ltp1 and ltp2) and the gene number. for ltpc, ltpd, ltpg, and other types of ltp defined with a letter, the punctuation mark was not recommended. this latter classification system is currently recommended as it comprises several features of ltps and is more robust than the previous classification systems. lipid transfer proteins are small cysteine-rich proteins, having 4 to 5 helices in their tertiary structure ( figure 2b ), which is stabilized by several hydrogen bonds. such a folding gives ltps a hydrophobic cavity to bind the lipids through hydrophobic interactions. this structure is stabilized by 4 disulfide bridges formed by 8 conserved cysteines, similar to defensins, although bound by cysteines in different positions. the disulfide bridges promote ltp folding into a very compact structure, which is extremely stable at different temperatures and denaturing agents. [70] [71] [72] these foldings provide a different specificity of lipid binding at the ltp binding site, where the ltp2 structure is relatively more flexible and present a lower lipid specificity when compared with ltp1. 34 the first 3d structure of an ltp was established for taltp1.1 based on 2d and 3d data of 1h-nmr, purified from wheat (triticum aestivum) seeds in aqueous solution. 73, 74 currently, several 3d structures of ltps have been determined, either by nuclear magnetic resonance (nmr) or x-ray crystallography; in their free, unbound form or in a complex with ligands. the heveins were first identified in 1960 in the rubber-tree (hevea brasiliensis), but its sequence was determined later, whereas a similarity was detected to the chitin-binding domain of an agglutinin isolated from urtica dioica (l.) 75 with 8 cysteine residues forming a typical cys motif. 76 the primary structure of the hevein consists of 29 to 45 aa, positively charged, with abundant glycine (6) and cysteine (8-10) residues, 76 and aromatic residues. 31, 77 the chitin-binding domain is a determinant component in the identification of hevein-like peptides whose binding site is represented by the amino acid sequence sxfgy/sxygy, where x regards any amino acid. 76, 78 most heveins have a coil-β1-β2-coil-β3 structure that occurs by variations with the secondary structural motif in the presence of turns in 2 long coils in the β3 chain. 31 antiparallel β chains form the central β sheet of the hevein motif with 2 long coils stabilized by disulfide bonds ( figure 2c ). although the presence of chitin has not been identified in plants, there are chitin-like structures present in proteins that exhibit a strong affinity to this polysaccharide isolated from different plant sources. 79 the presence of 3 aromatic amino acids in the chitinbinding domain favors chitin binding by providing stability to the hydrophobic group c-h and the π electron system through van der waals forces, as well as the hydrogen bonds between serine and n-acetylglucosamine (glcnac) present in the chitin structure. 76, 77 this domain is commonly found in chitinases of classes i to v, in addition to other plant antimicrobial proteins, such as lectins and pr-4 (pathogenesis-related protein 4) members. 80, 81 it may also occur in other proteins that bind to polysaccharide chitin, 80 such as the antimicrobial proteins ac-amp1 and ac-amp2 of amaranthus caudatus (amaranthaceae) seeds which are homologous to hevein but lack the c-terminal glycosylated region. 82 plant chitinases (class i) have the hevein-like domains, called hlds. due to the similar structural epitopes between chitinases and heveins, they are responsible for the cross reactive syndrome (latex-fruit syndrome). 83, 84 among the several classes of proteins mentioned, the proteins with a high degree of similarity to hevein are chitinases i and iv. 76 chitinases are known to play an essential role in plant defense against pathogens, 85 also inhibiting in vitro fungal growth, 86 especially when combined with β-1,3-glucanases. 87 it also interferes with the growth of hyphae, resulting in abnormal ramification, delay, and swelling in their stretching. 81 however, it has been shown that heveins have a higher inhibitory potential than chitinases and that their antifungal effect is not related only to the presence of chitinases 88 ; pn-amp1 and pn-amp2 amps with hevein domains have potent antifungal activities against a broad spectrum of fungi, including those without chitin in their cell walls. 88, 89 modes of action of chitinases usually include degradation and disruption of the fungal cell wall and plasma membrane due to its hydrolytic action, causing extravasation of plasma particles. 21, 89 therefore, heveins have good antifungal activity, and only a few are active against bacteria, most of them with low activity. another role of hevein chitinases regards the antagonistic effect in triggering the aggregation of rubber particles in the latex extraction process in rubber trees. unlike heveins, other chitinases inhibit rubber particle aggregation. however, its action in conjunction with other proteins (β-1,3-glucanase) increases the effect of β-1,3-glucanase on rubber particle aggregation. 90 a study by shi et al 91 found that the interaction of the protein network related to the antipathogenic activity released by lutoids (lysosomal microvacuole in latex) is essential in closing laticiferous cells (cells that produce and store latex), not only providing a physical barrier, but a biochemical barrier used by laticiferous cells affected by pathogen invasion. knottins are part of the cysteine-rich peptides (crps) superfamily, sharing the cysteine-knot motif and therefore resembling other families as defensins, heveins, and cyclotides. 92 their structure was initially identified by crystallography of carboxypeptides isolated from potato, showing the cysteine-knot motif with 39 aa and 6 cysteine residues. 93 they are also called "cysteine-knot peptides," "inhibitor cysteine-knot peptides," or even "cysteine-knot miniproteins" because their mature peptide presents less than 50 aa, forming 3 interconnected disulfide bonds in the cysteine-knot motif, characterizing a particular scaffold. 92 this conformation confers thermal stability at high temperatures. for example, the cysteine-stabilized β-sheet (csb) motif derived from knottins presents stability at approximately 100°c with only 2 disulfide bonds. 94 the knottins may have linear or cyclic conformation. however, both exhibit connectivity between the cysteines at positions 1-4c, 2-5c, and 3-6c, forming a ring at the last bridge 92 ( figure 2d ). knottins have different functions, such as signaling molecules, 95 response against biotic and abiotic stresses, 96 root growth, 97 symbiotic interactions as well as antimicrobial activity against bacteria, 98 fungi, 99 virus, 100 and insecticidal activity, 101 among others. knottins antimicrobial activity has been attributed to the action of functional components of the plasma membrane, leading to alterations of lipids, ion flux, and exposed charge. 99 the accumulation of peptides on the surface of the membrane results in the weakening of the pathogen membrane, 102 resulting in transient and toroidal perforations. 99 in the course of a large-scale survey to identify novel amps from australian plants, 103, 104 an amp with no sequence homology was purified. its complementary dna (cdna) was cloned from macadamia integrifolia (proteaceae) seeds, containing the complete peptide coding region. the peptide was named miamp1, being highly basic with an estimated isoelectric point (pi) of 10 and a mass of 8 kda. the miamp1 is 102 aa long, including a 26 aa signal peptide in the n-terminal region, bound to a 76 aa mature region with 6 cysteine residues. 105 its 3d structure was determined using nmr spectroscopy, 104 revealing a unique conformation among plant amps, with 8 beta-strands arranged in 2 greek key motifs, forming a greek key beta-barrel ( figure 2e ). due to its particularities, miamp1 was classified as a new structural family of plant amps, and the name β-barrelins was proposed for this class. 104 this structural fold resembles a superfamily of proteins called γ-crystallin-like characterized by the precursors βγ-crystallin. 106 this family includes amps from other organisms, for example, wmkt, a toxin produced by the wild yeast williopsis mraki. 107 the miamp1 exhibited in vitro antimicrobial activity against various phytopathogenic fungi, oomycetes, and grampositive bacteria 103 with a concentration range of 0.2 to 2 μm generally required for a 50% growth inhibition (ic50). in addition, the transient expression of miamp1 in canola (brassica napus) provided resistance against blackleg disease caused by the fungus leptosphaeria maculans, 108 turning miamp1 potentially useful for genetic engineering aiming at disease resistance in crop plants. there are few scientific publications with macadamia-like peptides, maybe because they prevail in primitive plant groups (eg, lycophytes, gymnosperms to early angiosperms as amborella and papaver), being apparently absent in derived angiosperms (eg, asteridae, including brassicaceae as arabidopsis thaliana). on the contrary, they have been identified in some monocots (as zantedeschia, zea, and sorghum). 109 in fact, peptides similar to miamp1 appear to play a role in the defense against pathogens in gymnosperms, including species of economic importance (as pinus and picea) thus deserving attention for their biotechnological potential. 109 four closely related amps (ib-amp1, ib-amp2, ib-amp3, and ib-amp4) were isolated from seeds of impatiens balsamina (balsaminaceae) with antimicrobial activity against a variety of fungi and bacteria, and low toxicity to human cells in culture. these amps are the smallest isolated from plants to date, consisting of only 20 aa in length. the ib-amps are highly basic and contain 4 cysteine residues that form 2 disulfide bonds. interestingly, they have no significant homology with other amps available in public databases. sequencing of cdnas isolated from i. balsamina revealed that all 4 peptides are encoded within a single transcript. concerning the predicted precursor of ib-amp protein, it consists of a pre-peptide followed by 6 mature peptide domains, each one of them flanked by propeptide domains ranging from 16 to 35 aa in length (supplementary figure s3) . this primary structure with repeated domains of alternating basic peptides and acid propeptide domains has, to date, not been reported in other plant species. 110 patel et al 111 conducted an experiment to purify ib-amp1 from seeds of impatiens balsamina. after purification, this peptide had its secondary structure tested by circular dichroism (cd). the results revealed a peptide that may include a β-turn but do not show evidences for either helical or β-sheet structure over a range of temperature and ph. structural information from 2d 1h-nmr was obtained in the form of proton-proton internuclear distances inferred from nuclear overhauser enhancements (noes) and dihedral angle restraints from spinspin coupling constants, which were used for distance geometry calculations. owing to the difficulty in obtaining the correct disulfide connectivity by chemical methods, the authors had built and performed 3 separate calculations: (1) a model with no disulfides; (2) another with predicted disulfide bonds; and (3) a model with alternative connectivity disulfide, as assigned from the nuclear overhauser effect spectroscopy (noesy) nmr spectra. as a result, 2 hydrophilic patches were observed at opposite ends and opposite sides of the models, whereas in between them a large hydrophobic patch was identified. however, the study did not conclude which of the 3 models would be the most likely representative of ib-amp1, reporting only that cysteines are necessary for maintaining the structure. based on the experiment performed by patel et al, 111 the present work built 3 different models: model 1: without disulfide bonds, and the other 2 models with different disulfide connections-model 2: nmr prediction by patel et al 111 6-cys;16-cys and 7-cys;20-cys, and model 3: disulfide bond partner prediction by dianna 7-cys;16-cys and 6-cys;20-cys. calculations have shown that although the peptide is small, the cysteines constrain part of it to adopt a well-defined main chain conformation. from residue 4 to 20 (except 11), the main chain is well-defined, whereas residues 1 to 3 in the n-terminal region present few restrictions and appear to be more flexible (supplementary figure s4) . analyzing the rmsd (root mean square deviation), we observed that all the models lost the initial conformation and, among them, model 3 was the most stable. models 1 and 2 showed a similar pattern (supplementary figure s5) , as in the models of patel et al, 111 although model 1 was the most flexible. little is known about impatiens-like amps mode of action. lee et al 112 investigated the antifungal mechanism of ib-amp1 noting that when oxidized (bound by disulfide bridges), there occurs a 4-fold increase in antifungal activity against aspergillus flavus and candida albicans, as compared with reduced ib-amp1 (without disulfide bridges). confocal microscopy analyses have shown that ib-amp1 can either bind to the cell 8 bioinformatics and biology insights surface or penetrate cell membranes, indicating an antifungal activity by inhibiting a distinct cellular process, rather than ion channel or membrane pore formation. fan et al 113 reported the ib-amp4 antimicrobial activity dependent of β-sheet configuration to enable insertion into the lipid membrane, thus killing the bacteria through a non-lytic mechanism. 114 current approaches aim to make changes in ib-amp to improve its antimicrobial activity. as an example, synthetic variants of ib-amp1 were fully active against yeasts and fungi, where the replacement of amino acid residues by arginine or tryptophan improved more than twice the antifungal activity. 115 another study involving amp modification generated a synthetic peptide without the disulfide bridges (ie, a linear analog of ib-amp1), which showed an antimicrobial specificity 3.7 to 4.8 times higher than the wild-type ib-amp1. 116 puroindoline puroindolines are small basic proteins that contain a single domain rich in tryptophan. these proteins were isolated from wheat endosperm, have a molecular mass around 13 kda, and a calculated isoelectric point higher than 10. at least 2 main isoforms (called pin-a and pin-b) are known, which are encoded by pina-d1 and pinb-d1 genes, respectively. these genes share 70.2% identical coding regions but exhibit only 53% identity in the 3′ untranslated region. 117 both pin-a and pin-b contain a structure with 10 conserved cysteine residues and a tertiary structure similar to ltps, consisting of 4 α-helices separated by loops of varying lengths, with the tertiary structure joined by 5 disulfide bonds, 4 of which identical to ns-ltps. 117 the conformation of the 2 pin isoforms was studied by infrared and raman spectroscopy. both pin-a and pin-b have similar secondary structures comprising approximately 30% helices, 30% β-sheets, and 40% non-ordered structures at ph 7. it has been proposed that the folding of both pins is highly dependent on the ph of the medium. the reduction of the disulfide bridges results in a decrease of pins solubility in water and to an increment of the β-sheet content by about 15% at the expense of the α-helix content. 118 no high-resolution structure for any of the pin isoforms is available, bringing challenges to understanding the function of their hydrophobic regions, with some evidence coming only from partially homolog peptides. 117 however, wilkinson et al 119 proposed a theoretical model for several sequences of this amp. puroindolines are proposed to be functional components of wheat grain hardness loci, control core texture, besides antifungal activity. [120] [121] [122] [123] although the biological function of pins is unknown, their involvement in lipid binding has been proposed. while ltps bind to hydrophobic molecules in a large cavity, pins interact only with lipid aggregates, that is, micelles or liposomes, through a single stretch of tryptophan residues. this stretch of tryptophan residues is especially significant in the main form, pin-a (wrwwkwwk), while it is truncated in the smaller form, pin-b (wptwwk). [124] [125] [126] puroindolines form protein aggregates in the presence of membrane lipids, and the organization of such aggregates is controlled by the lipid structure. in the absence of lipids, these proteins may aggregate, but there is no accurate information on the relationship between aggregation and interaction with lipids. the antimicrobial activity of pins is targeted to cell membranes. charnet et al 127 indicated that pin is capable of forming ion channels in artificial and biological membranes that exhibit some selectivity over monovalent cations. the stress and ca 2+ ions modulate the formation and/or opening of channels. puroindolines may also be membranotoxins, which may play a role in the plant defense mechanism against microbial pathogens. morris 128 reported that the pin-a and pin-b act through similar but somewhat different modes, which may involve "membrane binding, membrane disruption and ion channel formation" or "intracellular nucleic acid binding and metabolic disruption." natural and synthetic mutants have allowed the identification of pins as key elements for antimicrobial activity. snakins are crps first identified in potato (solanum tuberosum). 129, 130 due to their sequence similarity to gasa (gibberellic acid stimulated in arabidopsis) proteins, the snakins were classified as members of the snakin/gasa family. 131 the genes that encode these peptides have (1) a signal sequence of approximately 28 aa, (2) a variable region, and (3) a mature peptide of approximately 60 residues, with 12 highly conserved cysteine residues. these cysteine residues maintain the 3d structure of the peptide through disulfide bonds, besides providing stability to the molecule when the plant is under stress 129, 130, 132, 133 (figure 2f; supplementary figure s6 ). snakins may be expressed in different parts of the plant, like stem, leaves, flowers, seeds, and roots, 134-137 both constitutive or induced by biotic or abiotic stresses. in vitro activity was observed against a variety of fungi, bacteria, and nematodes, acting as a destabilizer of the plasma membrane. 129, 138, 139 moreover, they were reported as essential agents in biological processes such as cell division, elongation, cell growth, flowering, embryogenesis, and signaling pathways. [140] [141] [142] [143] alpha-hairpinins as reported by nolde et al, 144 alpha-hairpin emerged as a new amp with unusual motif configuration. these peptides prevail in plants and their structure was resolved based on nmr data obtained from the ecamp-1 peptide isolated from barnyard grass seeds (echinoa crus-galli). 144 some α-hairpinins comprise trypsin inhibitors with helical hairpin structure and this group silva et al 9 was recently proposed as a new plant amp family. 145 similar to other amps, the amino acid sequences of α-hairpinins are variable. they share the conserved cysteine motif (cx3cx1-15cx3c) that form a helix-loop-helix fold and may have 2 disulfide bridges c1-c4 and c2-c3. 146 its structural stability is maintained by forming hydrogen bonds, so that the side chains have a relatively stable spatial orientation. 147 as reviewed by slavokhotova et al, 148 members of alphahairpin family have been described in both mono and dicot groups, including species as echinochloa crus-galli and zea mays (both poaceae, monocot), fagopyrum esculentum (polygonaceae, eudicot), and stellaria media (caryophyllaceae, eudicot). several transcripts with α-hairpinin motif exhibit similarities to snakin/gasa genes and are sometimes positioned within this family. although the α-hairpinins structure has been published, its mechanism of action is still not resolved ( figure 2j , pdb id: 2l2r). however, studies indicate they present a potential dna binding capacity. 149 the term cyclotide was created at the end of the past century to designate a family of plant peptides with approximately 30 aa in size and a structural motif called cyclic cysteine knot (cck). 150 this motif is composed by a head-to-tail cyclization that is stabilized by a knotted arrangement of disulfide bridges, with 6 conserved cysteines, connected as follows: c1-2, c3-6, c4-5. 151 cyclotides are generally divided into 2 subfamilies, mӧbius and bracelets, based on structural aspects. in addition to ccks, 2 loops (between c1-2 and c4-5) have high similarity between both subfamilies, while the other 2 loops (between c2-3 and c3-4) exhibit some conservation within the subfamilies 152,153 (supplementary figure s7) . to date, several cyclotides were identified in eudicot families such as rubiaceae, 154 violaceae, 155 fabaceae, 156 and solanaceae, 157 in addition to some monocots of poaceae family. 158 in general, cyclotides may act in defense against a range of agents like insects, helminths, or mollusks. in addition, they can also act as ecbolic (inducer of uterus contractions), 154 antibacterial, 159 anti-hiv, 100 and anticancer factors. 160 all these characteristics added to the stability conferred by the cck motif turn these peptides into excellent candidates for drug development. 161, 162 thaumatin-like protein thaumatins or tlps belong to the pr-5 (pathogen-related protein) family and received this name due to its first isolation from the fruit of thaumatococcus daniellii (maranthaceae) from west africa. 163 thaumatin-like proteins are abundant in the plant kingdom, 164 being found in angiosperms, gymnosperms, and bryophytes, 163 being also identified in other organisms, including fungi, 165, 166 insects, 167 and nematodes. 168 thaumatin-like proteins are known for their antifungal activity, either by permeating fungal membranes 169 or by binding and hydrolyzing β-1,3-glucans. 170, 171 in addition, they may act by inhibiting fungal enzymes, such as xylanases, 172 α-amylases, or trypsin. 173 besides, the expression of tlps is regulated in response to some stress factors, such as drought, 174 injuries, 175 freezing, 176 and infection by fungi 177, 178 viruses, and bacteria. 179 as to the tlp structure, this protein presents characteristic thaumatin signature (ps00316): 180, 181 most of the tlps have molecular mass ranging from 21 to 26 kda, 163 possessing 16 conserved cysteine residues (supplementary figure s8) involved in the formation of 8 disulfide bonds, 182 which help in the stability of the molecule, allowing a correct folding even under extreme conditions of temperature and ph. 183 thaumatin-like proteins also contain a signal peptide at the n-terminal, which is responsible for targeting the mature protein to a particular secretory pathway. 163 the tertiary structure presents 3 distinct domains, which are conserved and form the central cleft, responsible for the enzymatic activity of the protein, being located between domains i and ii. 184 this central cleft may be of an acidic, neutral, or basic nature depending on the binding of the different linkers/receptors. all plant tlps with antifungal activity have an acidic cleft known as motif reddd due to 5 highly conserved amino acid residues (arginine, glutamic acid, and 3 aspartic acid; supplementary figure s8 ), being very relevant for specific receptor binding and antifungal activity. 169, 185, 186 crystallized structures were determined for some plant tlps, such as thaumatin 187 (figure 2g ), zeamatin 169 ( figure 2h ), tobacco pr-5d 185 and osmotin, 186 the cherry allergen pruav2, 188 and banana allergen ba-tlp, 184 among other tlps. some tlps are known as small tlps (stlps) due to the deletion of peptides in one of their domains, culminating in the absence of the typical central cleft. these stlps exhibit only 10-conserved cysteine residues, forming 5 disulfide bonds, resulting in a molecular weight of approximately 16 to 17 kda. they have been described in monocots, conifers, and fungi, so far. 163, 189, 190 other tlps exhibit an extracellular tlp domain and an intracellular kinase domain, being known as pr5k (pr5-like receptor kinases) 191 and are present in both monocots and dicots. for example, arabidopsis contains 3 pr5k genes, while rice has only 1. 163 with the rapid growth in the number of available sequences, it is unfeasible to handle such amount of data manually. thus, amp sequences (as well as their biological information) have been deposited in large general databases, such as uniprot and trembl, which contain sequences of multiple origins. 192, 193 in this sense, the construction of databases that deal specifically with amps was an important step to organize the data. during the past decade, several databases were built to support the deposition, consultation, and mining of amps. thus, these databases can be classified into 2 groups: general and specific. 194 the specific databases can be divided into 2 subgroups: those containing only 1 specific group (defensins or cyclotides) and those containing data from a supergroup of peptides (plant, animal, or cyclic peptides) (supplementary table 1 ). in general, both types of databases share some characteristics such as the way that the data are available or the tools to analyze amps. the collection of antimicrobial peptides (campr3) is a database that comprises experimentally validated peptides, sequences experimentally deduced and still those with patent data, besides putative data based on similarity. [195] [196] [197] the current version includes structures and signatures specific to families of prokaryotic and eukaryotic amps. 197 the platform also includes some tools for amp prediction. the antimicrobial peptide database (apd) 198 collects mature amps from natural sources, ranging from protozoa to bacteria, archaea, fungi, plants, and animals, including humans. amps encoded by genes that undergo post-translational modifications are also part of the scope, besides some peptides synthesized by multienzyme systems. the apd provides interactive interfaces for peptide research, prediction, and design, statistical data for a specific group, or for all peptides available in the database. the lamp (database linking antimicrobial peptides) comprises natural and synthetic amps, which can be separated into 3 groups: experimentally validated, predicted, and patented. their data were primarily collected from the scientific literature, including uniprot and other amp-related databases. 199 the database of antimicrobial activity and structure of peptides (dbaasp) 200 contains information about amps from different origins (synthetic or non-synthetic) and complexity levels (monomers and dimers) that were retrieved from pubmed using the following keywords: antimicrobial, antibacterial, antifungal, antiviral, antitumor, anticancer, and antiparasitic peptides. this database is manually curated and provides information about peptides that have specific targets validated experimentally. it also includes information on chemical structure, post-translational modifications, modifications in the n/c terminal amino acids, antimicrobial activities, cell target and experimental conditions in which a given activity was observed, besides information about the hemolytic and cytotoxic activities of the peptides. 200 due to the diversity of amps and the need to accommodate the most representative subclasses, several databases were established, focusing on specific types, sources, or features. there are several ways to classify amps, and they can range from biological sources such as bacterial amps (bacteriocins), plants, animals, and so on; biological activity: antibacterial, antiviral, antifungal, and insecticide; and based on molecular properties, pattern of covalent bonds, 3d structure and molecular targets. 201, 202 the "defensins knowledgebase" is a database with manual curation and focused exclusively on defensins. this database contains information about sequence, structure, and activity, with a web-based interface providing access to information and enabling text-based search. in addition, the site presents information on patents, grants, laboratories, researchers, clinical studies, and commercial entities. 203, 205 the cybase is a database dedicated to the study of sequences and 3d structures of cyclized proteins and their synthetic variants, including tools for the analysis of mass spectral fingerprints of cyclic peptides, also assisting in the discovery of new circular proteins. 205 the phytamp is a database designed to be solely dedicated to plant amps based on information collected from the uniprot database and from the scientific literature through pubmed. 206 plantpepdb is a database with manual curation of plantderived peptides, mostly experimentally validated at the protein level. it includes data on the physical-chemical properties and tertiary structure of amps, also useful to identify their therapeutic potential. different search options for simple and advanced compositing are provided for users to perform dynamic search and retrieve the desired data. overall, plantpepdb is the first database that comprises detailed analysis and comprehensive information on phyto-peptides from a wide functional range. 207 biological data banks (dbs) are organized collections of data of diverse nature that can be retrieved using different inputs. the management of this information is done through various software and hardware resources, whose retrieval and organization can be performed in a quick and efficient way. 208 considering biological data, information can be classified into (1) primary (sequences), (2) secondary (structure, expression, metabolic pathways, types of drugs, etc), and (3) specialized, for example, containing information on a species or on a class of protein. 209 within this third group, some references to amps can be mentioned, such as campr3 196 and apd 198 that compile sequence data and structure retrieved from diverse sources, and also the defensin knowledgebase 203 and the cybase 205 which are dedicated to specific classes of peptides (defensins and cyclotides, respectively), in addition to phytamp, 206 a specific database of plant amps (supplementary table 2 ). the first step to infer the function of a given sequence (annotation) is to retrieve it in databases. for this purpose, 3 approaches have been used mostly: (1) local alignments, especially by using basic local alignment search tool (blast) 210 and fasta 211 ; by searching for specific patterns using (2) regex or (3) hidden markov model (hmm). 194 the first approach has been widely used, since most of the information is available in databases as sequences, together with tools to align them, whereas the blast is the primary tool for doing so. 212 this tool splits the sequence into small pieces (words), comparing it with the database. however, this approach has a limitation. small motifs may not be significantly aligned as they comprise small portions of the sequences that can be smaller than 20% of the total size. 31, 194 due to the high variability of amps, only few highly conserved sequences can be identified using this type of inference. to reduce the effects of local alignment limitations, other strategies based on the search for specific patterns were introduced, such as regex 213 (supplementary table 1 ) and hmm. 214 the regex is a precise way of describing a pattern in a string where each regex position must be set, although ambiguous characters (or wildcards) can also be used. for example, if we want to find a match for both amino acid sequences caiessk and waiesk, we can use the following expression: [cw]aies{1,2}k, this expression would find a pattern starting with the letter "c" or "w," followed by an "a," an "i," and an "e," 1 or 2 "s," and ending with a "k." the hmms are well-known for their effectiveness in modeling the correlations between adjacent symbols, domains, or events, and they have been extensively used in various fields of biological analysis, including pairwise and multiple sequence alignment, base-calling, gene prediction, modeling dna sequencing errors, protein secondary structure prediction, noncoding rna (ncrna) identification, protein and rna structural alignments, acceleration of rna folding and alignment, fast noncoding rna annotation, and many others. using hmm, a statistic profile is included in the model, which is calculated from a sequence alignment, and a score is determined site-to-site, with conserved and variable positions defined a priori. 194, 215 predicting antimicrobial activity the design of new amps led to the development of methods for the discovery of new peptides, thus allowing new experiments to be done by researchers. in this sense, the new challenge lies in the construction of new prediction models capable of discovering peptides with desired activities. the apd db has established a prediction interface based on some parameters defined by the entire set of peptides available in this database. these values are calculated from natural amps to consider features like length, net charge, hydrophobicity, amino acid composition, and so on. if we take as an example the net load, the amps deposited in the apd range from -12 to +30. this is the first parameter incorporated into the prediction algorithm. however, most amps have a net load ranging from -5 to +10, which then becomes the alternative prediction condition. therefore, the same method is applied to the remaining parameters. the prediction in apd is performed in 3 main steps. first, the sequence parameters will be calculated and compared. if defined as an amp, the peptide can then be classified into 3 groups: (1) rich in given amino acids, (2) stabilized by disulfide, and (3) linear bridges. finally, sequence alignments will be conducted to find 5 peptides of higher similarity. 198, 216, 217 the advent of machine learning (ml) methods has promoted new possibilities for drug discovery. in ml inferences, both a positive and a negative dataset are usually required to train the predictive models. the positive data, in this case, regard preferably experimentally validated amps that can be collected in databases, whereas negative data are randomly selected protein sequences that do not have amp characteristics. 197, 218 machine learning methods based on support vector machine (svm), random forest (rf), and neural networks (nn) have been the most widely used. svm is a specific type of supervised method of ml, aiming to classify data points by maximizing the margin between classes in a high-dimensional space. 219, 220 random forest is a non-parametric tree-based approach that combines the ideas of adaptive neighbors with bagging for efficient adaptive data inference. neural networks is an information processing paradigm inspired by how a biological nerve system process information. it is composed of highly interconnected processing elements (neurons or nodes) working together to solve specific problems. [221] [222] [223] evaluating proteomic data regarding the use of amps in peptide therapeutics, as an alternative to antimicrobial treatment, new efficient and specific antimicrobials are demanded. as aforementioned, amps are naturally occurring across all classes of life, presenting high active potential as therapeutic agents against various kinds of bacteria. 224 the identification of novel amps in databases is primarily dependent on knowing about specific amps together with a sufficient sequence similarity. 225 however, orthologs may be divergent in sequence, mainly because they are under strong positive selection for variation in many taxa, 226 leading to remarkably lower similarity, even in closely related species. in this scenario, where alignment tools present limited use, 1 strategy to identify amps is related to proteomic approaches. proteins and peptides are biomolecules responsible for various biochemical events in living organisms, from formation and composition to regulation and functioning. thus, understanding of the expression, function, and regulation of the proteins encoded by an organism is fundamental, leading to the so-called "proteomic era." the term "proteome" was first used by marc wilkins in 1994 and it represents the set of proteins encoded by the genome of a biological system (cell, tissue, organ, biological fluid, or organism) at a specific time under certain conditions. 227 protein extraction, purification, and identification methods have significantly advanced our capacity to elucidate many biological questions using proteomic approaches. 228, 229 due to the wide diversity of proteomic analysis, methods makes the choice of the correct approach dependent on the type of material and compounds to be analyzed. 213, 230 two main tools are used to isolate proteins: (1) the 2-dimensional electrophoresis (2-de) associated with mass spectrometry (ms) and (2) liquid chromatography associated with ms, each one with its own limitations. [230] [231] [232] obtaining native proteins is a challenge in proteomics or peptidomics, due to high protein complexity in samples, as the occurrence of post-translational modifications. alternative strategies applied to extraction, purification, biochemical, and functional analyses of these molecules have been proposed, favoring access to structural and functional information of hard-to-reach proteins and peptides. 233 based on 2d gel, al akeel et al 234 evaluated 14 spots obtained from seeds of foeniculum vulgare (apiaceae) aiming at proteomic analyses and isolation of small peptides. extracted proteins were subjected to 3 kda dialysis, and separation was carried out by deae-ion exchange chromatography while further proteins were identified by 2d gel electrophoresis. one of its spots showed high antibacterial activity against pseudomonas aeruginosa, pointing to promising antibacterial effects, but requiring further research to authenticate the role of the anticipated proteins. for amps, 2de is challenging due to the low concentration of the peptide molecules captured by this approach, their small sizes, and their ionic features (strongly cationic). in addition, the limited number of available specific databases and high variability turn their identification through proteolysis techniques and mass spectrometry, matrix-assisted laser desorption/ionization (maldi-ms) difficult. in addition, the partial hydrophobicity characteristics and surface charges facilitate peptide molecular associations, making analysis difficult by any known proteomic approaches. 232 in addition, peptides are most often cleaved from larger precursors by various releasing or processing enzymes. 235 furthermore, profiles generated do not represent integral proteome, as 2de has limitations to detect proteins with low concentration, values of extreme molecular masses, pis, and hydrophobic proteins, including those of membranes. 236 due to these limitations, multidimensional liquid chromatography-high-performance liquid chromatography (mdlc-hplc) has been successfully employed as an alternative to 2d gels. techniques and equipments for the newly developed separation and detection of proteins and peptides, such as nano-hplc and multidimensional hplc, have improved proteomics evaluation. 237 molecular mass values obtained are used in computational searches in which they are compared with in silico digestion results of proteins in databases. in silico approaches, usually by the action of trypsin as a proteolytic agent, may generate a set of unique peptides whose masses are determined by ms. 238, 239 these methodologies are widely adopted for large-scale identification of peptide from ms/ms spectra. 240 theoretical spectra are generated using fragmentation patterns known for specific series of amino acids. the first 2 widely used search engines in database searching were sequest 241 and mascot (matrix science, boston, ma; www.matrixscience.com). 242 they rank peptide matches based on a cross-correlation to match the hypothetical spectra to the experimental one. mascot is widely used for peptidomics and proteomics analysis, including amp identification in many organisms, or to evaluate the antibacterial efficacy of new amps. evaluating new amp against multidrug-resistant (mdr) salmonella enterica, tsai et al 243 used 2d gel electrophoresis and liquid chromatography-electrospray ionization-quadrupole-time-offlight tandem ms to determine the protein profiles. the protein identification was performed using the mascot with trypsin as cutting enzyme, whereas ncbi nr protein was set as a reference database. the methodology used in this study indicated that the novel amp might serve as a potential candidate for drug development against mdr strains, confirming the usability of mascot. in a similar way, umadevi et al 244 described the amp profile of black pepper (piper nigrum l.) and their expression on phytophthora infection using label-free quantitative proteomics strategy. for protein/peptide identification, ms/ms data were searched against the apd database 245 using an in-house mascot server, established full tryptic peptides with a maximum of 3 missed cleavage sites and carbamidomethyl on cysteine, besides an oxidized methionine included as variable modifications. the apd database was used for amp signature identification, 245 together with phytamp 206 and campr3. 197 to enrich the characterization parameters, isoelectric point, aliphatic index, and grand average of hydropathy were also used 246 (gravy) (using protparam tool), besides the net charge from phytamp database. based on label-free proteomics strategy, they established for the first time the black pepper peptidomics associated with the innate immunity against phytophthora, evidencing the usability of proteomics/ peptidomics data for amp characterization in any taxa, including plant amps, aiming the exploitation of these peptides as next-generation molecules against pathogens. 244 other tools use database searching algorithms, such as x!tandem, 247 open mass spectrometry search algorithm (omssa), 248 probid, 249 radars, 250 and so on. these search engines are based on database search but use different scoring schemes to determine the top hit for a peptide match. general information on database search engines, their algorithms, and scoring schemes were reviewed by nesvizhskii et al. 251 despite its efficient ability to identify peptides, database searching presents several drawbacks, like false positive identifications due to overly noisy spectra and lower quality peptides score (related to the short size of peptides). so, the identification is strongly influenced by the amount of protein in the sample, the degree of post-translational modification, the quality of automatic searches, and the presence of the protein in the databases. 252, 253 in this scenario, the knowledge about the genome from a specific organism is important to allow the identification of the exact pattern of a given peptide. if an organism has no sequenced genome, it is not searchable using these methods. 235, 240 once the sequences are obtained, bioinformatic tools can be used to predict peptides structure and estimate bioactive peptides. 254 more recently, an interactive and free web software platform, mixprotool, was developed, aiming to process multigroup proteomics data sets. this tool is compiled in r (www.r-project. org), providing integrated data analysis workflow for quality control assessment, statistics, gene ontology enrichment, and other facilities. the mixprotool is compatible with identification and quantification outputs from other programs, such as maxquant and mascot, where results may be visualized as vector graphs and tables for further analysis, in contrast to existing softwares, such as giapronto. 255 according to the authors, the web tool can be conveniently operated, even by users without bioinformatics expertise, and it is beneficial for mining the most relevant features among different samples. 24 the central tenet of structural biology is that structure determines function. for proteins, it is often said the "function follows form" and "form defines function." therefore, to understand protein function in detail at the molecular level, it is mandatory to know its tertiary structure. 256 experimental techniques for determining structures, such as x-ray crystallography, nmr, electron paramagnetic resonance, and electron microscopy, require significant effort and investments. 257 all methods mentioned have their own limitations, and the gap between the number of known proteins and the number of known structures is still substantial. thus, there is a need for computational framework methods to predict protein structures based on the knowledge of the sequence. 256 in addition, in recent years, there has been impressive progress in the development of algorithms for protein folding that may aid in the prediction of protein structures from amino acid sequence information. 258 historically, the prediction of a protein structure has been classified into 3 categories: comparative modeling, threading, and ab initio. the first 2 approaches construct protein models by aligning the query sequences with already solved model structures. if the models are absent in the protein data bank (pdb), the models must be constructed from scratch, that is, by ab initio modeling, considered the most challenging way to predict protein structures. 256 in the case of comparative modeling methods, when inserting a target sequence, the programs identify evolutionarily related models of solved structures based on their sequence or profile comparison, thus constructing structure models supported by these previously resolved models. 259 this approach comprises 4 main steps: (1) fold assignment, which identifies similarity between the target and the structure of the solved model; (2) alignment of the target sequence to the model; (3) generation of a model based on alignment with the chosen template; and (4) analysis of errors considering the generated model. 260 there are several servers and computer models that automate the comparative modeling process, with swiss-model and modeler figuring as the most used. 261, 262 although automation makes comparative modeling accessible to experts and beginners, some adjustments are still needed in most cases to maximize model accuracy, especially in the case of more complex proteins. 262 therefore, some caution must be taken regarding the generated models, considering the resolution and quality of the model used, as well as homology between the model and the protein of interest. threading modeling methods are based on the observation that known protein structures appear to comprise a limited set of stable folds, and those similarity elements are often found in evolutionarily distant or unrelated proteins. the most used servers based on this approach are muster, 263 sparks-x, 264 raptorx, 259 prosa-web, 265 and most notably the i-tasser. 266 in some cases, the incorporation of structural information to combine the sequence used in the search with possible models allows the detection of similarity in the fold, even in the absence of an explicit evolutionary relation. the prediction of structures from known protein models is, at first sight, a more straightforward task than the prediction of protein structures from available sequences. therefore, when no solved model is available, another approach is recommended, namely, the ab initio modeling. this method is intended to predict the structure only from the sequence information, without any direct assistance from previously known structures. the ab initio modeling aims to predict the best model, based on the minimum energy for a potential energy function by sampling the potential energy surface using various searchable information. 267, 268 such approaches turn it challenging to produce high-resolution modeling, essential for determining the native protein folding and its biochemical interpretation. on the contrary, later resolved structures and comparisons with previously predicted proteins point to a higher successful modeling generated by ab initio methods than those generated by pure energy minimization methods, classical or even pure methods. 256 among the most used servers and programs for ab initio modeling, we highlight the rosetta, 257 quark, 269 and touchstone ii. 267 the accuracy of the models calculated by many of these methods is evaluated by cameo (continuous automated model evaluation) 270 and by casp (critical assessment of protein structure prediction). 258 probably the first reasonably accurate ab initio model was built in casp4. since then, sustained progress was achieved in ab initio prediction, but mainly for small proteins (120 residues or less). in casp11, for the first time, a novel 256-residue protein with a sequence identity with known structures lower than 5% was constructed with high precision for sequences of this size. 271 in casp12, a significant improvement was reported in 4 areas: contact prediction, free modeling, template-based modeling, and estimating the accuracy of models. the authors report that this improvement is due to the accuracy of modeling and alignment methods, as well as increased data availability for both sequence and structure. 258 due to the number of amps deposited in the pdb (to date approximately 1099 structures), comparative modeling is the most used. however, when it comes to de novo peptide design, the most recommended choice would be ab initio 272 or a hybrid approach that uses more than 1 modeling method. 273 after the generation of a model, the amp stability should be evaluated using molecular dynamics (md). molecular dynamics comprises the application of computational simulations that predict the changes in the positions and velocities of the constituent atoms of a system under given time and condition. this calculation is done through a classical approximation of empirical parameters, called "force field." 274 if, on one hand, this approximation makes the dynamics of a system containing thousands of atoms numerically accessible, it obviously limits the nature of the processes that can be observed during the simulations. no quantum effect is visualized in a md simulation; just as no chemical bond is broken, no interactions occur between orbitals, resonance, polarization, or charge transfer effects. 275 however, the molecules go beyond a static system. thus, md is a computational technique that can be used for predicting or refining structures, dynamics of molecular complexes, drug development, and action of molecular biological systems. 276 molecular dynamics simulation is widely used for protein research, aiming to extract information about the physical properties of individual proteins. the results of such simulations are then compared with experimental results. as these experiments are generally carried out in solvents, it is necessary to simulate molecular systems of protein in water. these simulations have a variety of applications, such as determining the folding of a structure to a native structure and analyzing the dynamic stability of this structure. 277 the use of md to simulate protein folding processes is one of the most challenging applications and should be relatively long (in the order of microseconds to milliseconds) to allow observing a single fold event. in addition, the force field used must correctly describe the relative energies of a wide variety of shapes, including unfolding and poorly folded shapes that may occur during the simulation. 275 the considerable application potential led to the implementation of md simulation in many software packages, including gromacs, 278-280 amber, 281 namd, 282 charmm, 283 lammps, 284 and desmond. 285 in addition to the above mentioned, there are other simulation types available, such as the monte carlo method, stochastic dynamics, and brownian dynamics. 280 in the last decades, md simulation has become a standard tool in theoretical studies of large biomolecular systems, including dna or proteins, in environments with near realistic solvents. indeed, simulations have proven valuable in deciphering functional mechanisms of proteins and other biomolecules, in uncovering the structural basis for disease, and in the design and optimization of small molecules, peptides, and proteins. 286 historically, the computational complexity of this type of computation has been extremely high, and much research has focused on algorithms to achieve unique simulations that are as long or as large as possible. 278 the interplay between a given pathogen (eg, virus, bacteria, fungus) must be studied through a holistic approach. hostpathogen relationships are very complex and occur at diverse conceivable levels, including the cellular/molecular level of both, pathogen and host, under given environmental conditions. a most approximate understanding of these interactions at every level is the ultimate goal of "systems biology" (sb). it comprises a holistic approach, integrating distinct disciplines, as biology, computer science, engineering, bioinformatics, physics, and others to predict how a given system behaves under given conditions and what is the role of its parts. systems biology stands out because it is capable of correlating omics data for the understanding of plant-pathogen interaction. the construction of a plant-pathogen interaction network includes the reconstruction of metabolic pathways of these organisms, identification of the degree of pathogenicity, besides the expression of genes and proteins from both plant and pathogen. the networks can be classified into 5 types: (1) regulatory; (2) metabolic; (3) protein-protein interaction; (4) signaling and regulatory; and (5) signaling, regulatory, and metabolic. 287 each of these networks can be plotted according to computational approaches. also, further studies are required to contemplate the construction of evolutionary in silico models and the characterization of these molecular targets in vitro. 288, 289 studies of protein-protein interactions to understand the regulatory process are essential 290 and new computational methods are necessary for this purpose with more optimized algorithms, also to remove potential false positives. thus, in-depth studies on the orientation of molecules and their linkages to the formation of a stable complex are of great importance for understanding plant-pathogen studies and also to develop new drugs. 291 the understanding of the regulatory principles by which protein receptors recognize, interact, and associate with molecular substrates or inhibitors is of paramount importance to generate new therapeutic strategies. 292 in modern drug discovery, docking plays an important role in predicting the orientation of the binder when it is attached to a protein receptor or enzyme, using forms and electrostatic interactions, van der walls, coulombic, and hydrogen bond as parameters to quantify or predict a given interaction. 293, 294 molecular docking aims at exploring the predominant mode(s) of binding of a molecule (protein or ligand) when it binds to a protein with a known 3d structure based on a scoring function that has 3 main functions: the first is to determine the binding mode and the binding site of a protein, the second is to predict the absolute binding affinity between protein and ligand (or other protein) in lead optimization, and the third is virtual screening, which can identify potential drug leads for a given protein target by searching a large ligand or protein in database. 295 protein-protein interactions are essential for cellular and immune function. in many cases, due to the absence of an experimentally determined structure of the complex, these interactions must be modeled to obtain an understanding about their structure and molecular basis. 296 few studies on plant-pathogen interactions include docking approaches and most studies focus on drug development for medical purposes. drug research based on structure is a powerful technique for the rapid identification of small molecules against the 3d structure of available macromolecular targets, usually by x-ray crystallography, nmr structures, or homology models. due to abundant information on protein sequences and structures, the structural information on specific proteins and their interactions have become crucial for current pharmacological research. 297 even in the absence of knowledge about the binding site and limited backbone movements, a variety of algorithms have been developed for docking over the past 2 decades. although the zdock, 296 the rdock, 298 and the hex 299 have provided results with high coupling precision, the complexes provided are not very useful for designing inhibitors for protein interfaces due to constraints on rigid body docking. 294 in this context, more flexible approaches have been developed which generally examine very limited conformations compared with rigid body methods. these docking methods predict that binding is more likely to occur in broad surface regions and then defines the sites in complex structures of high affinity. 300 the best example is the haddock software, 297 which has been successful in solving a large number of precise models for protein-protein complexes. a good example of its use is the study of the complex formed between plectasin, a member of the innate immune system, and a precursor lipid of bacterial cell wall ii. the study identified the residues involved in the binding site between the 2 proteins, providing valuable information for planning new antibiotics. 301 however, the absolute energies associated with intermolecular interaction are not estimated with satisfactory accuracy by the current algorithms. some significant issues as solvent effects, entropic effects, and receptor flexibility still need to be addressed. however, some methods, such as moe-dock, 302 gold, 303 glide, 304 flexx, 305 and surflex 306 which deal with lateral chain flexibility, have proven to be effective and adequate in most cases. realistic interactions between small molecules and receptors still depend on experimental wet-lab validation. 294, 307 despite the current difficulties, there is a growing interest in the mechanisms and prediction of small molecules such as peptides, as they bind to proteins in a highly selective and conserved manner, being promising as new medicinal and biological agents. 308 while both "small molecule docking methods" and "custom protocols" can be used, short peptides are challenging targets because of their high torsional flexibility. 307 proteinpeptide docking is generally more challenging than those related to other small molecules, and a variety of methods have been applied so far. however, few of these approaches have been published in a way that can be reproduced with ease. [309] [310] [311] although it is difficult to use peptide docking, a recent focus of basic and pharmacological research has used computational tools with modified peptides to predict the selective disruption of proteinprotein interactions. these studies are based on the involvement of some critical amino acid residues that contribute most to the binding affinity of a given interaction, also called hot-spots. 312, 313 despite the number of docking programs, existing algorithms still demand improvements. however, approaches are being developed to improve all issues related to punctuation, protein flexibility, interaction with plain water, among other issues. 314 in this context, the capri (critical assessment of predicted interactions) is a community that provides a quality assessment of different docking approaches. it started in 2001 and since then has aided the development and improvement of the methodologies applied for docking. 315 an evaluation was carried out for capri in 2016, resulting in an improvement in the integration of different modeling tools with docking procedures, as well as the use of more sophisticated evolutionary information to classify models. however, adequate modeling of conformational flexibility in interacting proteins remains an essential demand with a crucial need for improvement. 314 different docking programs are currently available, 294 and new alternatives continue to appear. some of these alternatives will disappear, just as others will become the top choices among field users. molecular docking technique is not often used for amps, due to its standard mechanism of action based on the classical association with the external membrane of the pathogen. despite that, some amps have the ability to bind other proteins and/or enzymes, a feature still scarcely studied. in such cases, molecular docking can be useful. an example of success is the study performed by melo et al, 47 where they showed the specific binding of a trypsin to a cowpea (vigna unguiculata) thionine, revealing that this interaction occurs in a canonical manner with lys11, located in an extended exposed loop. therefore, further application of docking may bring new evidences about the antimicrobial mechanisms revealing other molecular targets of interest. it is clear that the combination of data bank information with bioinformatic tools (especially those allowing the identification of patterns, rather than sequence order) is able to revolutionize the identification of amps and prediction of their activity. the data may come from genomic, transcriptomic, or proteomic databases, or a combination of different information sources (eg, genomic and transcriptomics, transcriptomics and proteomics). supplementary figure s9 brings a schematic flowchart describing the steps for mining, annotation, and structural/ functional analysis of amps, in addition to some wet-lab analyses that can be integrated to assess/confirm candidate amps. similar bioinformatic approaches have been actually used to identify potential peptide candidates with anti-sars-cov-2 activity, especially those potentially able to interact with the spike protein and proteases involved in viral penetration. 316, 317 as emphasized, plant amps show greater diversity and abundance, when compared with other kingdoms. it can be speculated that plants shelter many yet undescribed amp classes, given their vast abundance and isoform diversity. the genomic and peptidic structure of amps can be variable, with few key residues conserved, which turns their identification, classification, and comparison challenging even in the omics age. nevertheless, advances in the generation of new bioinformatics tools and specialized databases have led to new and more efficient approaches for both the identification of primary sequences and molecular modeling, besides the analysis of the stability of the generated models. despite the large availability of omics data and bioinformatics tools, most new plant peptides have been discovered by wet-lab approaches regarding single candidates. high throughput in silico methods have the potential to transform this scenario, revealing many new candidates, including some new or "non-canonical" peptides. it may be also speculated that a myriad of new peptides may exist considering even smaller peptides, still less considered and more difficult to identify. finally, in silico approaches shall in future studies be mandatory to define the design of wet-lab studies, turning the identification more efficient and requiring reasonably less time to track, identify, and confirm new candidate amps. considering the actual pandemic scenario of covid-19, plant amps may be regarded as an important source of antiviral drug candidates, especially considering that some amp categories present not only antiviral effects but also a wide spectrum antimicrobial activity, act as anti-inflammatory, and also induce the immune response. of higher education personnel, biocomputational program), cnpq (brazilian national council for scientific and technological development), and facepe (fundação de amparo à ciência e tecnologia de pernambuco) for fellowships. the project is supported by the interreg italia-slovenia, ise-emh 07/2019 and rc 03/20 from irccs burlo garofolo/ italian ministry of health cass performed the literature review and whore the manuscript. lz, mol, lmbv, jpbn, jrfn, jdcf, rlos, cjp, ffa, and mfo wrote specific chapters, eak and sc critically revised the text and included relevant suggestions. ambi conceived the review, wrote the introduction and concluding remarks, besides critically revising the manuscript. all authors have read the manuscript and agree to its content. supplemental material for this article is available online. prediction of protein function from protein sequence and structure plant peptides in defense and signaling plant bioactive peptides: an expanding class of signaling molecules candidalysin is a 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discovery: protein-peptide binding affinities recent work in the development and application of protein-peptide docking peptide docking and structurebased characterization of peptide binding: from knowledge to know-how protein-ligand docking in the new millennium-a retrospective of 10 years in the field unal eb, gursoy a, erman b. vital: viterbi algorithm for de novo peptide design modeling protein-protein and proteinpeptide complexes: capri 6th edition docking, scoring, and affinity prediction in capri potential chimeric peptides to block the sars-cov-2 spike receptor-binding domain peptide-like and small-molecule inhibitors against covid-19 the authors are very grateful to capes (coordination for the improvement key: cord-342634-4ouhdjsr authors: semrad, katharina title: proteins with rna chaperone activity: a world of diverse proteins with a common task—impediment of rna misfolding date: 2010-12-26 journal: biochem res int doi: 10.1155/2011/532908 sha: doc_id: 342634 cord_uid: 4ouhdjsr proteins with rna chaperone activity are ubiquitous proteins that play important roles in cellular mechanisms. they prevent rna from misfolding by loosening misfolded structures without atp consumption. rna chaperone activity is studied in vitro and in vivo using oligonucleotideor ribozyme-based assays. due to their functional as well as structural diversity, a common chaperoning mechanism or universal motif has not yet been identified. a growing database of proteins with rna chaperone activity has been established based on evaluation of chaperone activity via the described assays. although the exact mechanism is not yet understood, it is more and more believed that disordered regions within proteins play an important role. this possible mechanism and which proteins were found to possess rna chaperone activity are discussed here. among all biological macromolecules, rnas represent one of the most functionally versatile players in the cell. rna molecules fulfill many different tasks such as coding and transfer of genetic information; they play regulatory functions in various cellular processes and catalyze chemical reactions (like cleavage and ligations). in addition to its functional versatility, rnas are also able to fold into countless different structures, many of which have similar stabilities as the native structure and therefore compete with the native fold. furthermore, rna molecules often undergo transition states during their folding pathways before they reach the native and active structure. these transient structures can represent traps along the folding pathway from which the molecules might have a hard time to escape and which then end up being long-lived intermediates. the reason for this structural versatility is the fact that rna consists of only four different bases which are easily capable of forming stable helices, that are not necessarily the native structure. the threshold for rna molecules to be able to perform their functions is usually the accomplishment of reaching its native and active structure. in the cellular environment, rna molecules do not appear as "naked" nucleic acids but always are found in conjunction with proteins. in some cases, the rna molecule helps the protein partner to fold correctly; in others, the protein stabilizes the rna structure. and last but not least proteins with rna chaperone activity aid during the folding process of rnas. proteins with rna chaperone activity open up misfolded rna structures and do not require atp [1] . furthermore, after the rna has been folded into its native structure, the protein becomes dispensable. although the term "rna chaperone" has been used routinely to describe various proteins that are capable to assist rna folding in vitro, the term rna chaperone is reserved to describe proteins whose rna folding activity has been verified on its natural target rna in vivo. therefore, most of the proteins in this paper will be referred to as proteins with rna chaperone activity if their rna folding activity was only determined in vitro and/ or on nonnatural rna targets. this paper will focus on the diversity of proteins with rna chaperone activity and which experimental assays are in use to determine whether a protein has rna chaperone activity. i will present examples for proteins with rna 2 biochemistry research international chaperone activity and discuss possible mechanisms of rna chaperoning. chaperone activity and rna misfolding 2.1. what are rna chaperones and proteins with rna chaperone activity? the list of proteins with possible rna chaperone activity is growing constantly. proteins with different activities that support rna folding are classified in this group. the definition of a protein with rna chaperone activity is that the protein prevents rna from misfolding by opening up misfolded structures. proteins with rna chaperone activity do not require atp, which distinguishes them from rna helicases, another group of proteins that facilitate rna folding (e.g., cyt-19) [2] . proteins with rna chaperone activity interact only transiently with rna molecules and are supposed to be dispensable once the rna has been folded correctly. this was shown for e. coli proteins s12 and stpa [3, 4] . a transient interaction and weak binding to rna might be difficult to define because many of the identified rna chaperones interact strongly with their target rnas and are found in rnp complexes like ribosomal proteins, hnrnps, la protein, and others. however, it has been demonstrated that a mutant stpa that shows stronger binding towards rna shows decreased rna chaperone activity suggesting that strong binding could also be detrimental to rna folding [5] . in that way, proteins with rna chaperone activity are also distinguished from "stabilizers" that are proteins that bind and stabilize an rna structure and are required to stay bound in order to keep the rna's native structure. cyt-18, the trna synthetase from neurospora crassa, is a "stabilizer" for the mitochondrial self-splicing group i intron: its presence is required to keep the native structure of the intron which otherwise unfolds readily. in the growing database of "proteins with rna chaperone activity", there exists an increasing number of proteins that simply possess rna annealing activity. a prominent and intensively studied member of this group is the bacterial host factor hfq that showed annealing activity on random substrates. hfq in addition is an rna chaperone as it was further demonstrated that hfq does possess unwinding activity upon its native substrates [6, 7] . in brief, the group of proteins with rna chaperone activity includes proteins that, first, open up misfolded structures without requirement of atp and that, second, are dispensable once the rna has been folded. 2.2. rna misfolding. rna molecules are prone to misfold in vitro and are usually prevented from misfolding in vivo. rna basically encounters two folding problems: a kinetic folding problem, where the rna molecule has to surmount kinetic barriers during the search for its native structure. secondly, rna molecules meet a thermodynamic folding problem as the final native structure often has to compete with alternative folds that have similar energetic stabilities [1] . rna folding is a hierarchical process, and first secondary structure elements have to form. secondary structural elements form between regions within the rna molecule that are in close proximity. they are a-form helices consisting of watson-crick base-pairs. secondary structures are very stable. the stability of a base-pair depends on the stability of both of its neighbouring base-pairs. already any rna of a reasonable length is able to form alternative base-pairs leading to alternative helices that become folding traps. tertiary structures are higher order structures that are built by assembling the secondary structure elements into a more complex collapsed fold. they can also involve formation of helices. this is the case in pseudoknots where either a loop region interacts with a distant single stranded region or with another distant loop. pseudoknots possess similar stability as secondary structures. but tertiary structural elements involve also other non-watson-crick interactions where for example, not only the watson-crick site of the nucleotide interacts with another nucleotide but also the hoogsteen edge or the sugar edge of the nucleotide is involved in hydrogen bonding [8] . an often reoccurring tertiary structure motif is the a-minor interaction where an adenine interacts with the minor groove of the a-form helix [9] . tertiary structures are often less stable and depend on the formation of secondary structures. finally, monovalent or divalent metal ions play an important role in tertiary structure formation. the first studies on rna structure and folding were done in the 1960s with yeast trna molecules. already then it was demonstrated that trnas are able to adopt two distinct conformations of which only one is the native structure which can be aminoacylated [10, 11] . the rna folding problem becomes even more prominent in the case of large rnas such as group i introns or in the context of large protein-ribonucleic acid complexes such as rnase p and the ribosome. it was demonstrated that the self-splicing group i intron of the thymidylate synthase gene of phage t4 misfolds in the absence of translation: when the ribosome does not prevent base-pairing between exon and intron sequences, the intron is not able to fold correctly and cannot perform the splicing reaction [12] . a similar observation was made with the group i intron of tetrahymena thermophila ribosomal rna: a subset of molecules misfolds and accumulates into an inactive population [13] . misfolding depends on exon sequences that form stable hairpins and intervene with 5splice-site formation. in vivo, however, some group i introns require the assistance of proteins to splice efficiently and prevent misfolding. for an example, the cyt-18 protein in neurospora crassa mitochondira is a trna synthetase which stabilizes the p4-p6 domain of group i introns and recruits cyt-19, an rna helicase, which then unwinds folding traps and promotes splicing [2, 14] . for large rnp complexes such as the ribosome, a growing body of evidence suggests that several additional factors such as helicases exist that assist during the folding process in vivo. as proteins with rna chaperone activity are very heterogeneous concerning their structure and their way to resolve the folding of rna molecules, there are various rna chaperone assays available to measure different activities. in principle, the assays can be divided into in vitro and in vivo assays that use either simple oligonucleotide annealing or displacement reactions or that measure catalytic activities of correctly folded ribozymes. measuring an activity that might be targeted more specifically to a certain subset of substrates in the natural environment of the putative rna chaperone makes it difficult to evaluate rna chaperone activity using a single assay. the substrates in the in vitro assay (e.g., oligonucleotides) might differ in sequence requirements or structure requirements from possible native substrates and might lead to negative results. strand unwinding assays might give positive results in the case of single-strand binding proteins. furthermore, in vivo assays to measure rna chaperone activity can be negatively influenced by possible toxicity of the putative rna chaperone when overexpressed or can lead to secondary effects in the cell that give false positive results. therefore, it is recommended to measure the rna chaperone activity at least in more than one chaperone assay to be certain that no non-specific activity is measured. figure 1) 3.1.1. oligonucleotide annealing. in this assay, two complementary oligonucleotides (oligos) present in concentrations above their dissociation constant are incubated together in the absence and in the presence of the protein to be evaluated for rna chaperone activity. an increase in the rate of duplex formation is observed when the tested protein has rna annealing activity. in principle two complementary short rna oligos are used in this assay, although it has been a habit to choose dna oligos instead. in order to measure rna chaperone activity, however, i think that rna oligonucleotides should be the preferred choice. detection methods of duplex formation include native gel electrophoresis where the two complementary rna strands can either be visualized by radioactive or fluorophor labelling and has been used in (besides many many other publications) [15] [16] [17] . rna annealing can alternatively be measured by observing the fluorescence resonance energy transfer (fret) upon the closing up of the two fluorescently labelled oligonucleotides (e.g., see [18, 19] ). activities. the ability of a protein to open up and unwind an already formed rna duplex is measured. to measure rna chaperone activity in contrast to helicase activity, this assay is performed in the absence of atp or an alternative energy source. analogous to the annealing assays, melting activity can be detected by using native gel electrophoresis or by measuring loss of fluorescence energy resonance transfer (fret) that occurs upon dissociation of the complementary fluorophor labelled rnas. in addition to under single turnover conditions, substrate to ribozyme annealing is measured. under multiple turnover conditions substrate dissociation is measured. (d) in the trans-splicing assay, the group i intron is split in two halves h1 (upstream exon, 5 -part of the intron) and h2 (3 -part of the intron and exon2), and splicing at low temperatures in the presence of chaperones is measured. (e) shows the cis-splicing assay where an enhancement of splicing at 37 • c is measured in the presence of chaperones. the construct (shosho) contains short exon 1 (27 nucleotides) and short exon 2 (2 nucleotides) sequences. the above-described detection methods, new approaches to detect folding or unfolding of single molecules emerge and include time-resolved nmr, which becomes a powerful tool to study folding of small rnas. cleavage. the hammerhead ribozyme cleavage reaction and folding of the ribozymesubstrate 3-way helical junction have been studied in a great detail. therefore, this assay represents a suitable tool to study rna chaperone activity upon folding of the hammerhead ribozyme-substrate construct. using the hammerhead cleavage assay, both annealing and strand displacement can be studied independently of each other [3, 20] . the advantage on the well-studied assay is that depending whether singleturnover conditions or multiple turnover conditions are employed, it is possible to distinguish between annealing and strand dissociation activities. using single-turnover conditions, where an excess of ribozyme and low concentrations of substrate are applied, substrate annealing is determined as substrate annealing becomes the rate limiting step. on the other hand, using multiple turnover conditions with an excess of substrate over ribozyme, the whole cleavage reaction is monitored consisting of annealing and product release. since product release represents the rate limiting step, product dissociation is measured. self-splicing of the thymidylate synthase group i intron (td intron) of bacteriophage t4 has been characterized, and the td intron has been used lengthily to monitor rna chaperone activity of various proteins. splicing of different group i intron constructs that do not fold readily into the splicing competent structure in vitro is tested with and without chaperones. cis-splicing assay. in this td intron construct both, 5 and 3 exons are shortened for the upstream exon down to 27 nucleotides and the downstream exon shortened to only 2 nucleotides. this short exon construct (td shosho) splices at 37 • c but rna chaperones increase folding and as a consequence the splicing rate of the short-exon construct is increased as well [5] . trans-splicing assay. here, the td intron is split into two halves in the center of loop l6 in the p4-p6 domain, where in the wild type group i intron an open reading frame for an endonuclease is present. the upstream in vitro transcribed construct contains 549 nucleotides of exon1 and 131 nucleotides of the 5 -part of the intron. the downstream construct consists of the remaining 147 nucleotides of the intron and 23 nucleotides of exon2. correct and efficient folding of the trans-intron-constructs is significantly impaired at 37 • c but works fine at elevated temperatures (55 • c), which is monitored through splicing [21] . chaperones with strong annealing and unwinding activities such as ribosomal protein l1 or l19 from e. coli are capable to catalyze trans-splicing at 37 • c or even at lower temperatures, for example, hnrnpi increases splicing at 25 • c [22] . in vivo rna chaperone activity assays (see figure 2) 3.2.1. in vivo folding trap assay in e. coli. splicing of the group i intron within the thymidylate synthase gene of phage t4 occurs efficiently in vivo. though, when splicing and translation are uncoupled by introducing stop codons in the upstream exon, splicing is significantly reduced. this is due to alternative base-pairing of exonic and intronic sequences which prevent the formation of the intron's native fold [12] . the mutant td precursor construct tdsh1 consists of an exonic stop codon and has an additional intronic point mutation (c865u) which further destabilizes the native intron structure. the tdsh1 construct is significantly impaired in splicing in vivo. overexpression of rna chaperones in the presence of the tdsh1 mutant is used to evaluate if the rna chaperone is able to rescue the misfolded intron and restore splicing [23] . transcription read-through of the chloramphenicol acetyl transferase gene (cat) is inhibited due to the preceding transcription terminator stem. the stable hairpin secondary structure of the terminator inhibits the polymerase to transcribe the cat gene and as a consequence no chaloramphenicol resistance is achieved. the cells are chaloramphenicol (cm) sensitive. proteins with rna chaperone activity are able to melt the terminator stem and as a consequence read-through occurs and the cells become chloramphenicol resistant. the transcription antitermination assay was used for assaying cold shock proteins or if1 from e. coli [24] [25] [26] . proteins that possess rna chaperone activity are very divers and span from viral to bacterial and human proteins that are involved in many different cellular processes. the list of rna chaperones is permanently growing. recently, a database for proteins with rna chaperone activity was established where every lab is able to contribute their data for a newly identified chaperone http://www.projects .mfpl.ac.at/rnachaperones/index.html [27] . the following selection of proteins with rna chaperone activity is not complete but points out the most important groups or single proteins. the first viral rna chaperone activities were reported in the early 1990s and showed that nucleocapsid protein 7 (ncp7) of hiv increases hammerhead ribozyme cleavage significantly [20, 28] . only a few years later, another virus-encoded protein with rna chaperone activity has been identified, the hdv delta antigen, which was also monitored in the hammerhead ribozyme assay and furthermore shown to be dispensable after folding has occurred [29] . flaviviridae core proteins were also monitored and shown to possess rna chaperone activity in a hammerhead cleavage assay and/or rna strand annealing activities [30, 31] . interestingly, many of the viral proteins show exceptional high degree of disorder. in the case of the flaviviridae core proteins, it was also reported that heat denaturation still retained strand annealing activity suggesting that the disordered domains of the proteins are involved in chaperoning. nucleocapsid proteins from two members of the coronaviridae family have been investigated and hammerhead ribozyme cleavage was shown to be enhanced in their presence [32, 33] . and again both nucleocapsid proteins show a high degree of disorder in in silico predictions. a growing body of evidence suggests that there exist many more viral proteins that possess rna chaperone activity. the list here is not complete but proteins that were shown to have only dna annealing activities were left out in this list. the majority of these small viral proteins show strong propensity for disorder which suggests that disorder might be a mechanistic requirement for chaperoning. interestingly, studies on nc proteins demonstrated that these proteins not only possess rna chaperone activity in vitro but also are required for strand annealing and strand displacement activities on their target rnas in vivo [33] . recently, a specific template switching assay designed to study strand displacement in a retroviral-derived system demonstrated that nucleocapsid protein from coronavirus shows rna chaperone activity and most likely is an rna chaperone in vivo [34] . stpa. the e. coli transcriptional regulator stpa, a 15 kd basic protein, was isolated as a repressor of a splicingdeficient group i intron in thymidylate synthase of phage t4 [4] . stpa was furthermore shown to possess strong rna chaperone activity in vivo in the folding trap assay [35] . the protein was tested in vitro in a strand-annealing and strand-displacement assay and exhibited strong activities in both tests [36] . more detailed studies on stpa revealed that the protein binds transiently to rna with a preference for unstructured regions and that binding to rna is diminished in the presence of high ionic strength [5] . in an elaborate study applying in vivo dms modifications to the rna, in vivo folding of the group i intron was evaluated in the absence and presence of stpa [37] : schroeder and coworkers demonstrated that stpa opens up tertiary interactions of the td group i intron. while the loosening effect is advantageous in wild type or misfolded introns, overexpression of stpa in the presence of introns that were already destabilized in their 3d structure was detrimental. structure prediction of stpa suggested that this protein exhibits more than 70% disorder and it was suggested that this unfolded regions of stpa might play a role in chaperoning [38] . ribosomal proteins are required within every cellular organism to build up the bacterial 70s or the eukaryal 80s ribosome. many ribosomal proteins further regulate transcription or translation of their own operons. in addition, ribosomal proteins are also involved in various very different cellular processes and fulfill extraribosomal functions [39, 40] . ribosomal proteins are highly conserved among various species and many ribosomal proteins have unusual long unstructured extensions that wind their way through the ribosome [41] . the first observation that ribosomal proteins are capable of chaperoning rna folding came from the belfort lab: screening for cellular factors that increase trans-splicing of the thymidylate synthase group i intron revealed that many ribosomal proteins possess chaperoning activity, with ribosomal protein s12 from the small ribosomal subunit having the strongest activity [3] . furthermore, s12 significantly increased hammerhead ribozyme cleavage [3] . a systematic study on large ribosomal subunit proteins from e. coli showed that 1/3 of the tested proteins possesses strong rna chaperone activity in vitro in the trans-splicing assay [21] . in addition, ribosomal protein l1 orthologs from eukarya, bacteria, and mesophilic archaea also exhibited strong transsplicing and cis-splicing activities in vitro [42] . although it makes sense that the rna chaperone activity of ribosomal proteins could play a role during ribosome assembly, a definite proof for the requirement of this activity in vivo has not yet been provided. recently, it was demonstrated that e. coli ribosomal proteins l15, l16, l18, and l19, that showed rna chaperone activity in vitro, further possess protein chaperone activity comparable to other protein chaperones such as hsp90 [43] . it was suggested that intrinsically unstructured domains of ribosomal proteins could play a role in chaperoning. the exact mechanism, however, still remains elusive (see section 5). cold shock proteins (csps) are conserved throughout bacteria and plants. they are expressed during cold-shock, when misfolding of rnas becomes a major problem for the organism and function as transcriptional antiterminator at low temperature. many experiments that have been performed to describe chaperone activity of cold-shock proteins utilize dna helices (and only sometimes in addition rna duplexes) and refer to the activity as nucleic acid melting activity. however, it has to be mentioned that there are no elaborate studies on whether there is a difference between rna duplex and dna duplex melting and whether dna melting activity automatically is the same as rna melting. e. coli contains nine members of the csp family and cspa, the major cold-shock protein and cspe were identified to interact non-specifically with rna molecules and to possess nucleic acid melting activities [44] [45] [46] . cold-shock proteins in higher plants are highly conserved. glycine-rich and zn-finger containing proteins from arabidopsis thaliana have been monitored for their nucleic acid melting activity, and it was shown that grp7 (glycinerich protein) and csdp1 (cold shock domain protein) possess rna chaperone activity [47] . a recent study also demonstrated that out of six glycine-rich proteins in rice (oryza sativa), which are likely to be involved in adaptation to cold-shock, three of them exhibit rna-(and dna-) melting activities suggesting that grps in plants fulfill a chaperoning role during low temperatures [48] . in e. coli translation, initiation factor 1 (if1) is a small 71 amino-acid long peptide, which contains 5 rigid β-barrels and belongs to the ob (oligomer-binding)-fold proteins such as the cold shock proteins. n-and c-termini of if1 are highly flexible. it was demonstrated that e. coli if1 is capable of complementing for a cspb and cspc double knock-out in bacillus subtilis suggesting that if1 and csps have at least partially overlapping activities [49] . e. coli if1 exhibits rna chaperone activity in various assays including rna annealing of complementary oligonucleotides, transsplicing, in vivo folding trap assay, and transcription antitermination in vivo and in vitro [25, 50] . heteronuclear ribonucleoproteins encompass a group of about 20 polypeptides that are predominantly nuclear in localization and are involved in rna processing. the first observation that hnrnps possess rna chaperone activity came in the early 1990s when fractionated hela nuclear extract was tested for annealing activity of an mrna and its antisense partner. three proteins, hnrnp a1, c1 and u, were identified and hnrnp a1 was further shown to enhance hammerhead ribozyme cleavage in vitro [16, 20] . later, a detailed study on possible functions of ro rnps, which are ro ribonucleoprotein complexes, composed of a small noncoding cytoplasmic rna, termed y rna and its protein partners was conducted: besides the permanently associated proteins ro60 and la, subpopulations of ro-rnps also contain hnrnp i and hnrnp k, both of which exhibited strong rna chaperone activity in vitro in the transsplicing and the cis-splicing assay [22] . hnrnp i is identical to poly-pyrimidine binding protein (ptb) isoform 4 and was identified as a splicing suppressor in mammalian cells [51] . it regulates cap-independent translation, localization of cytoplasmic rnas, and poly-a-site cleavage [52] . ptb belongs to the ires transacting factors (itafs), which are host factors (like la, hnrnp k, nucleolin, unr and many others) that interact with viral rnas and induce conformational changes that then lead to translation initiation [53] . it was further reported that calcivirus replication requires ptb but only at lower or at higher temperatures than the permissive 37 • c, suggesting a chaperoning role of ptb [54] . members of the group of itafs have been implicated in rna chaperoning like unr, a cold-shock domain containing protein [55] , human la protein, and hnrnp k [22] . hnrnp k is also a multifunctional protein that is a transcriptional factor for c-myc and c-src [56] [57] [58] ; it enhances splicing [59] and is a translational regulator [60] . la proteins primarily bind rna polymerase iii transcripts and protect them from nuclease attack [61] . they also interact with pre-trnas at their uuu-3 oh ends and facilitate their maturation. la contributes to assembly of rnp complexes by retaining rnas in the nucleus. la is also involved in translation regulation. and human la was demonstrated to possess rna chaperone activity in vitro in the cis-splicing assay and in vivo in the folding trap assay [22] . hfq. the bacterial protein hfq was first discovered in the end 1960s as a host factor for bacteriophage qβ replication [62] . the bacterial protein is a pleiotropic regulator for gene expression in bacteria. it interacts with many small rnas and their mrna targets and regulates posttranscriptional regulation of small noncoding rnas such as dsra, sodb, oxys, rpra, and spot42 [63] [64] [65] [66] [67] . hfq preferentially binds to a/u rich, unstructured regions. hfq encompasses an sm-domain, which is highly conserved among various species and usually is found in eukaryotic spliceosomal rnps. crystallographic studies of escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus hfq proteins showed that hfq forms homohexameric ring structures with a central cationic pore that forms the rna binding site [68] [69] [70] . using strand annealing and strand melting assays to measure rna chaperone activity of hfq, only strand annealing activity was observed [27] . however, a detailed study using rnase footprinting on hfq's interaction with its target rnas sodb mrna and the small noncoding regulator ryhb rna demonstrated that hfq indeed did loosen secondary structures within sodb mrna that lead to binding of its regulatory rna rhyb [7] . a similar observation was made using fluorescence labelled rpos mrna and dsra small noncoding rna [6] . by means of fret, it was shown that hfq induces annealing of dsra to rpos mrna and prior to the annealing event hfq disrupts rpos secondary structure elements. consequently, hfq is entitled to be called rna chaperone. the prion protein is a misfolded isoform of the essential component of prion diseases such as creutzfeldt-jakob disease in humans-one of several neurodegenerative diseases. the function of the human prion protein is not clearly understood. it was demonstrated that the prion protein has rna (and dna) annealing activity [71] ; however, it was not yet shown if it possesses also rna unwinding activity and may therefore be classified as an "annealer". interestingly, the prion protein contains an intrinsically unstructured n-terminal domain [72] . the fragile x mental retardation protein (fmrp) is linked to the fragile x syndrome as the disease is due to transcriptional silencing of the gene. fmrp possesses rna binding activity and its interaction partners include a large number of mrnas, micrornas, sirnas, and small noncoding rnas as well as a multitude of different proteins [73] [74] [75] [76] . it was demonstrated using hammerhead ribozyme cleavage that fmrp possesses rna chaperone activity [77] . and finally in line with many other proteins with rna chaperone activity, it is interesting to mention that fmrp consists of a highly disordered c-terminus suggesting that the substrate versatility of fmrp might be accomplished through its structural disorder [78] . chaperones provide a critical cellular activity. proteins with rna chaperone activity are very divers in structure as well as in function: stpa, a transcriptional activator and repressor of a multitude of bacterial genes, is a small (15 kd) bacterial protein with intrinsically unstructured regions. stpa has strong rna chaperone activity. on the other hand the bacterial protein hfq is a large multidomain protein complex (60 kd) and folds into a compact ring-like structure. among ribosomal proteins, many were shown to possess rna chaperone activity (e.g., one third of large ribosomal subunit proteins from escherichia coli show rna chaperone activity in vitro). ribosomal proteins are usually small proteins many of which have long unstructured domains and are highly basic proteins. proteins with rna chaperone activity do not require an external energy source as rna helicases do. this raises the question of how rna chaperones accomplish the rna folding task and where the energy for this process comes from. proteins with rna chaperone activity in most cases encompass two major activities: the annealing activity and the unwinding activity (see also figure 3 ). many proteins with rna chaperone activity are highly basic proteins and therefore interact readily with negatively charged rna molecules. in that way, they might stabilize folded states by bringing together distant regions of the rna molecule and as a consequence increase rna double-strand formation. this mechanism could be comparable to the action of chemical chaperones such as osmolytes which are small organic compounds, that do not interfere with the cellular metabolism but speed up folding processes enabled through a crowding effect [79] . another indication that a crowding effect might play a role at least to some extent during rna annealing is the following: when rna chaperone activity is measured in vitro, there is always an excess of protein over rna present in the assay. for example, in the trans-splicing assay, 200 nmols of rnas (leading to a 20 nm end-concentration) are tested for folding in the presence of 1-2 μm protein. it was shown that e. coli ribosomal protein l1 displays maximal rna chaperone activity starting from 400 nm up to 2 μm protein concentration [42] . this means that at least a 20-fold excess of protein to rna has to be present to achieve maximal chaperoning activity of ribosomal protein l1 from e. coli. in this line, it also has to be mentioned that in the in vivo chaperone assay, which uses the folding trap of a misfolded group i intron in the thymidylate synthase gene of phage t4, it is always necessary that the measured protein is overexpressed and available in higher concentrations [23] . for example, the e. coli protein stpa, which is found constitutively expressed in the bacterial cell, only shows its rna chaperone activity in vivo when stpa is additionally over-expressed from an expression vector, thus showing that the cellular concentration of stpa is not sufficient to increase folding of the misfolded group i intron. certainly, this observation might be due to the engagement of stpa in other regulatory functions in the bacterial cell; however, it also points to the direction that more than one molecule 8 biochemistry research international of stpa is required to assist folding of the td group i intron. as a consequence the question rises if and how it is possible to distinguish between rna chaperone activity and a nonspecific single-strand rna binding activity of the protein that might both prevent misfolding. using the in vivo folding trap assay, however, not only proteins with possible rna chaperone activity like stpa had been tested but also a viral single strand binding protein from influenza virus (np) was tested and did not show any increase in splicing suggesting that single strand rna binding might not be sufficient for chaperoning. furthermore, a detailed study on stpa wild type and mutants demonstrated that only the fulllength stpa was able to show rna chaperone activity by simultaneously interacting with two rna molecules [5] . rna chaperone activity of stpa has been studied for more than a decade. it was shown that stpa has strong in vivo and in vitro rna chaperone activities. in a mechanistical in vivo study of stpa, schroeder and coworkers demonstrated that stpa loosens tertiary contacts within the thymidylate synthase group i intron [37] . in contrast, the neurospora crassa trna synthetase cyt-18 that also increases group i intron splicing of td stabilizes tertiary interactions. but how is the opening of tertiary structure elements accomplished without the hydrolysis of atp? this strand unwinding activity is more difficult to explain as the question remains of how a protein can actively open up hydrogen bonds when no apparent source of energy is required. in the protein world, it became more and more visible that the classical structure-function paradigm does not necessarily hold for many proteins and their activities. a growing body of evidence suggests that a multitude of proteins do not fold into compact domains but are fully or at least partially unstructured [80] . in eukaryotes, for example, conservative estimations point out that 5%-15% of all proteins are completely disordered and 50% of the cellular proteins have at least long unstructured domains. an interesting study by tompa and csermely demonstrated that among chaperones a significantly high percentage of proteins show long unstructured regions [38] . among rna chaperones, the percentage of at least partially disordered proteins is even higher (54%) than in the group of protein chaperones (36.7%). disordered proteins and protein segments allow a broad versatility for interaction partners and in this case for interaction with different rna molecules. but it can also explain the ability of proteins with rna chaperone activity to multitask as so far no rna chaperone has been identified whose only task is to aid in rna folding. interestingly, it was recently demonstrated that some ribosomal proteins that possess rna chaperone activity and contain disordered regions are also capable to chaperone protein folding suggesting once again that disordered regions provide high versatility for substrate interactions [43] . the idea of disordered rna chaperones is especially attractive because there are many advantages of proteins with disordered regions over compact proteins: (1) the main advantage of a disordered region is that it can easily interact with a range of many different partners and is not limited to a single binding pocket or recognition element on a partner molecule. (2) the bigger surface of the unstructured protein might provide a "loosening effect" for the incorrectly folded rna molecule. (3) the troublesome question of where the energy for the rna unwinding might come from could be explained by the gain of compactness upon interaction with the rna and a simultaneous loosening of the rna structure (see figure 3 (b)). as a consequence, the rna gains another chance to fold correctly. (4) the intrinsically unstructured protein might provide a folding platform for the rna as the chaperone holds the rna molecule in close proximity. in future the research focus on rna chaperones will lie on the understanding of the molecular mechanism and how intrinsically unstructured regions in proteins might play a role in function. interestingly, two very closely related ribosomal proteins l1 from archaea, that encompass 70% aminoacid identity, possess opposite activities: the mesophilic l1 protein displays strong rna chaperone activity whereas the thermophilic one inhibits ribozyme assays [42] . a detailed mutation study will likely shed light on the different activities and explain the rna chaperoning mechanism at least for a subgroup of rna chaperones. the big challenge, however, will be to identify in vivo targets of rna chaperones. rna chaperones are evaluated in assays for their broad specificity but in vivo they might be specialized to supervise folding of only a subset of rna molecules. the specificity might possibly be conferred by a different domain than the chaperoning activity. rna chaperones and the rna folding problem a dead-box protein functions as an atp-dependent rna chaperone in group i intron splicing escherichia coli proteins, including ribosomal protein s12, facilitate in vitro splicing of phage t4 introns by acting as rna chaperones escherichia coli protein stpa stimulates self-splicing by promoting rna assembly in vitro rna chaperone activity and rna-binding properties of the e. coli protein stpa spectroscopic observation of rna chaperone activities of hfq in post-transcriptional regulation by a small non-coding rna hfq, a new chaperoning role: binding to messenger rna determines access for small rna regulator geometric nomenclature and classification of rna base pairs rna tertiary interactions in the large ribosomal subunit: the a-minor motif two interconvertible forms of tryptophanyl srna in e. coli native and renatured transfer ribonucleic acid a ribosomal function is necessary for efficient splicing of the t4 phage thymidylate synthase intron in vivo alternative secondary structures in the 5 exon affect both forward and reverse self-splicing of the tetrahymena intervening sequence rna a tyrosyl-trna synthetase recognizes a conserved trna-like structural motif in the group i intron catalytic core domain structure and rna annealing activity of the escherichia coli regulatory protein stpa rna annealing activities in hela nuclei hiv-1 nucleocapsid protein as a nucleic acid chaperone: spectroscopic study of its helix-destabilizing properties, structural binding specificity, and annealing activity coupling rna annealing and strand displacement: a fret-based microplate reader assay for rna chaperone activity dissecting rna chaperone activity an rna chaperone activity of non-specific rna binding proteins in hammerhead ribozyme catalysis rna chaperone activity of large ribosomal subunit proteins from escherichia coli rna chaperone activity of protein components of human ro rnps assaying rna chaperone activity in vivo in bacteria using a ribozyme folding trap escherichia coli cspa-family rna chaperones are transcription antiterminators transcription antitermination by translation initiation factor if1 assay of transcription antitermination by proteins of the cspa family rna chaperones, rna annealers and rna helicases protein enhancement of hammerhead ribozyme catalysis identification and characterization of the rna chaperone activity of hepatitis delta antigen peptides the hepatitis c virus core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand rna in vitro rna chaperoning and intrinsic disorder in the core proteins of flaviviridae role of rna chaperones in virus replication coronavirus nucleocapsid protein is an rna chaperone coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription assaying rna chaperone activity in vivo using a novel rna folding trap assays for the rna chaperone activity of proteins rna chaperone stpa loosens interactions of the tertiary structure in the td group i intron in vivo the role of structural disorder in the function of rna and protein chaperones how common are extraribosomal functions of ribosomal proteins? extraribosomal functions of ribosomal proteins atomic structures at last: the ribosome in 2000 biochemistry research international ribosomal proteins: phylogenetic conservation and splicing inhibition janus chaperones: assistance of both rna-and protein-folding by ribosomal proteins cspa, the major cold-shock protein of escherichia coli, is an rna chaperone cold shock induces a major ribosomal-associated protein that unwinds double-stranded rna in escherichia coli nucleic acid melting by escherichia coli cspe cold shock domain proteins and glycine-rich rna-binding proteins from arabidopsis thaliana can promote the cold adaptation process in escherichia coli glycine-rich rna-binding proteins are functionally conserved in arabidopsis thaliana and oryza sativa during cold adaptation process complementation of cold shock proteins by translation initiation factor if1 in vivo rna chaperone activity of translation initiation factor if1 regulation of alternative 3 splice site selection by constitutive splicing factors minireview: polypyrimidine tract binding protein antagonizes exon definition cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression feline calicivirus replication: requirement for polypyrimidine tract-binding protien is temperaturedependent the apaf-1 internal ribosome entry segment attains the correct structural conformation for function via interactions with ptb and unr heterogeneous nuclear ribonucleoprotein k is a transcription factor identification of the src pyrimidine-binding protein (spy) as hnrnp k: implications in the regulation of src1a transcription specific binding of heterogeneous ribonucleoprotein particle protein k to the human c-myc promoter, in vitro heterogeneous nuclear ribonucleoprotein (hnrnp) k is a component of an intronic splicing enhancer complex that activates the splicing of the alternative exon 6a from chicken βtropomyosin pre-mrna mrna silencing in erythroid differentiation: hnrnp k and hnrnp e1 regulate 15-lipoxygenase translation from the 3 end the la protein factor fraction required for the synthesis of bacteriophage qβ-rna a small rna regulates the expression of genes involved in iron metabolism in escherichia coli regulatory roles for small rnas in bacteria hfq: a bacterial sm-like protein that mediates rna-rna interaction hfq is necessary for regulation by the untranslated rna dsra the sm-like hfq protein increases oxys rna interaction with target mrnas structure of pseudomonas aeruginosa hfq protein sm-like proteins in eubacteria: the crystal structure of the hfq protein from escherichia coli structures of the pleiotropic translational regulator hfq and an hfq-rna complex: a bacterial sm-like protein the prion protein has rna binding and chaperoning properties characteristic of nucleocapsid protein ncp7 of hiv-1 scavenger, transducer, rna chaperone? what ligands of the prion protein teach us about its function microarray identification of fmrp-associated brain mrnas and altered mrna translational profiles in fragile x syndrome a drosophila fragile x protein interacts with components of rnai and ribosomal proteins rna and micrornas in fragile x mental retardation rna cargoes associating with fmrp reveal deficits in cellular functioning in fmr1 null mice the fragile x mental retardation protein has nucleic acid chaperone properties disordered rna chaperone proteins: from functions to disease protons, osmolytes, and fitness of internal milieu for protein function intrinsically unstructured proteins: re-assessing the protein structure-function paradigm both referees of the paper are thanked for their valuable comments. doris chen is thanked for critically reading and chaperoning the paper. k. semrad is supported by a hertha firnberg fellowship (t261) from the austrian science foundation (fwf). key: cord-199630-2lmwnfda authors: ray, sumanta; lall, snehalika; mukhopadhyay, anirban; bandyopadhyay, sanghamitra; schonhuth, alexander title: predicting potential drug targets and repurposable drugs for covid-19 via a deep generative model for graphs date: 2020-07-05 journal: nan doi: nan sha: doc_id: 199630 cord_uid: 2lmwnfda coronavirus disease 2019 (covid-19) has been creating a worldwide pandemic situation. repurposing drugs, already shown to be free of harmful side effects, for the treatment of covid-19 patients is an important option in launching novel therapeutic strategies. therefore, reliable molecule interaction data are a crucial basis, where drug-/protein-protein interaction networks establish invaluable, year-long carefully curated data resources. however, these resources have not yet been systematically exploited using high-performance artificial intelligence approaches. here, we combine three networks, two of which are year-long curated, and one of which, on sars-cov-2-human host-virus protein interactions, was published only most recently (30th of april 2020), raising a novel network that puts drugs, human and virus proteins into mutual context. we apply variational graph autoencoders (vgaes), representing most advanced deep learning based methodology for the analysis of data that are subject to network constraints. reliable simulations confirm that we operate at utmost accuracy in terms of predicting missing links. we then predict hitherto unknown links between drugs and human proteins against which virus proteins preferably bind. the corresponding therapeutic agents present splendid starting points for exploring novel host-directed therapy (hdt) options. the pandemic of covid-19 (coronavirus disease-2019) has affected more than 6 million people. so far, it has caused about 0.4 million deaths in over 200 countries worldwide (https://coronavirus.jhu.edu/map.html), with numbers still increasing rapidly. covid-19 is an acute respiratory disease caused by a highly virulent and contagious novel coronavirus strain, sars-cov-2, which is an enveloped, single-stranded rna virus 1 . sensing the urgency, researchers have been relentlessly searching for possible therapeutic strategies in the last few weeks, so as to control the rapid spread. in their quest, drug repurposing establishes one of the most relevant options, where drugs that have been approved (at least preclinically) for fighting other diseases, are screened for their possible alternative use against the disease of interest, which is covid-19 here. because they were shown to lack severe side effects before, risks in the immediate application of repurposed drugs are limited. in comparison with de novo drug design, repurposing drugs offers various advantages. most importantly, the reduced time frame in development suits the urgency of the situation in general. furthermore, most recent, and most advanced artificial intelligence (ai) approaches have boosted drug repurposing in terms of throughput and accuracy enormously. finally, it is important to understand that the 3d structures of the majority of viral proteins have remained largely unknown, which raises the puts up the obstacles for direct approaches to work even higher. the foundation of ai based drug repurposing are molecule interaction data, optimally reflecting how drugs, viral and host proteins get into contact with each other. during the life cycle of a virus, the viral proteins interact with various human proteins in the infected cells. through these interactions, the virus hijacks the host cell machinery for replication, thereby affecting the normal function of the proteins it interacts with. to develop suitable therapeutic strategies and design antiviral drugs, a comprehensive understanding of the interactions between viral and human proteins is essential 2 . when watching out for drugs that can be repurposed to fight the virus, one has to realize that targeting single virus proteins easily leads to the viruses escaping the (rather simpleminded) attack by raising resistance-inducing mutations. therefore, host-(1) we link existing high-quality, long-term curated and refined, large scale drug/protein -protein interaction data with (2) molecular interaction data on sars-cov-2 itself, raised only a handful of weeks ago, (3) exploit the resulting overarching network using most advanced, ai boosted techniques (4) for repurposing drugs in the fight against sars-cov-2 (5) in the frame of hdt based strategies. as for (3)-(5), we will highlight interactions between sars-cov-2-host protein and human proteins important for the virus to persist using most advanced deep learning techniques that cater to exploiting network data. we are convinced that many of the fairly broad spectrum of drugs we raise will be amenable to developing successful hdt's against covid-19. in the following, we will first describe the workflow of our analysis pipeline and the basic ideas that support it. we proceed by carrying out a simulation study that proves that our pipeline accurately predicts missing links in the encompassing drug -human protein -sars-cov-2-protein network that we raise and analyze. namely we demonstrate that our (high-performance, ai supported) prediction pipeline accurately re-establishes links that had been explicitly removed before. this provides sound evidence that the interactions that we predict in the full network most likely reflect true interactions between molecular interfaces. subsequently, we continue with the core experiments. we predict links to be missing in the full (without artificially having removed links), encompassing drug -human protein -sars-cov-2-protein network, raised by combining links from year-long curated resources on the one hand and most recently published covid-19 resources on the other hand. as per our simulation study, a large fraction, if not the vast majority of the predictions establish true, hence actionable interactions between drugs on the one hand and sars-cov-2 associated human proteins (hence of use in hdt) on the other hand. a b c d figure 1 . overall workflow of the proposed method: the three networks sars-cov-2-host ppi, human ppi, and drug-target network (panel-a) are mapped by their common interactors to form an integrated representation (panel-b). the neighborhood sampling strategy node2vec converts the network into fixed-size low dimensional representations that perverse the properties of the nodes belonging to the three major components of the integrated network (panel-c). the resulting feature matrix (f) from the node embeddings and adjacency matrix (a) from the integrated network are used to train a vgae model, which is then used for prediction (panel-d). for the purposes of high-confidence validation, we carry out a literature study on the overall 92 drugs we put forward. for this, we inspect the postulated mechanism-of-action of the drugs in the frame of several diseases, including sars-cov and mers-cov driven diseases in particular. see figure 1 for the workflow of our analysis pipeline and the basic ideas that support it. we will describe all important steps in the paragraphs of this subsection. this reduces the training time compared to the general graph autoencoder model. we tested the model performance for a different number of sampled nodes, keeping track of the area under the roc curve (auc), average precision (ap) score, and model training time in the frame of a train-validation-test split at proportions 8:1:1. table 1 shows the performance of the model for sampled sugraph sizes n s = 7000, 5000, 3000, 2500 and 1000. for 5000 sampled nodes, the model's performance is sufficiently good enough concerning its training time and validation-auc and -ap score. the average test roc-auc and ap score of the model for n s =5000 are 88.53 ± 0.03 and 84.44 ± 0.04. to know the efficacy of the model in discovering the existing edges between only cov-host and drug nodes, we train the model (with n s =5000) on an incomplete version of the graph where the links between cov-host and drugs have been removed. we further compute the feature matrix f based on the incomplete graph, and use it. the test set consists of all the previously removed edges. the model performance is no doubt better for discovering those edges between cov-host and drug nodes (roc-auc: 93.56 ± 0.01 ap: 90.88 ± 0.02 for 100 runs). the fastgae model is learned with the feature matrix (f) and adjacency matrix (a). the node feature matrix (f) is obtained from a using the node2vec neighborhood sampling strategy. the model performance is evaluated with and without using f as feature matrix. figure 2 shows the average performance of the model on validation sets with and without f as input for the different number of sampling nodes. we calculate average auc, and ap scores for 50 complete runs of the model. from figure 2 , it is evident that including f as feature matrix enhances the model's performance markedly. we use the node2vec framework to learn low dimensional embeddings of each node in the compiled network. it uses the skipgram algorithm of the word2vec model to learn the embeddings, which eventually groups nodes with a similar 'role' or having a similar 'connection pattern' within the graph. similar 'role' ensures that nodes within the sets/groups are structurally similar/equivalent than the other nodes outside the groups. two nodes are said to be structurally equivalent if they have identical connection patterns to the rest of the network 20 . to explore this, we have analyzed the embedding results in two steps. first, we explore structurally equivalent nodes to identify 'roles' and similar connection patterns to the rest of the networks, and later use lovain clustering to examine the same within the groups/clusters. the most_similar function of the node2vec inspects the structurally equivalent nodes within the network. we find out all the cov-host nodes which are most similar to the drug nodes. while it is expected to observe nodes of the same types within the neighborhood of a particular node, in some cases, we found some drugs are neighbors of cov-host proteins with high probability (pobs > 0.65). sars-cov-2 3cl protease 21 . some other drugs such as 'clenbuterol' and 'fenbendazole', the probable neighbor of ppp1cb and eef1a respectively, are used as bronchodilators in asthma. to explore the closely connected groups, we have constructed a neighborhood graph using the k-th nearest neighbor algorithm from the node embeddings and apply louvain clustering ( figure 3 -panel-c). although there is a clear separation between host proteins (including cov-host) cluster and drug cluster, some of the louvain clusters contain both types of nodes. for example, louvain cluster-16 and -17 contain four and two drugs along with the other cov-host proteins, respectively. figure 3 panel-d represents a network consisting of these six drugs and their most similar cov-host nodes. for drug-cov-host interaction prediction, we exploit variational graph autoencoder (vgae), an unsupervised graph neural network model, first introduced in 18 to leverage the concept of variational autoencoder in graph-structured data. to make learning faster, we utilized the fastgae model to take advantage of the fast decoding phase. we have used two data matrices in the fastgae model for learning: one is the adjacency matrix, which represents the interaction information over all the nodes, and the other one is the feature matrix representing the low-dimensional embeddings of all the nodes in the network. we create a test set of 'non-edges' by removing all existing links between drugs and cov-host proteins from all possible combinations (332 cov-host × 1302 drugs) of edges. the model is trained on the whole network with the adjacency matrix a and feature matrix f. the trained model is then applied to the test 'non-edges' to know the most probable links. we identified a total of 692 most probable links with 92 drugs and 78 cov-host proteins with a probability threshold of 0.8. the predicted cov-host proteins are involved in different crucial pathways of viral infection (table 4). the p-values for pathway and go enrichment are calculated by using the hypergeometric test with 0.05 fdr corrections. figure 4 , panel-a shows the heatmap of probability scores between predicted drugs and cov-host proteins. to get more details of the predicted bipartite graph, we figure 4 . drug-cov-host predicted interaction: panel-a shows heatmap of probability scores between 92 drugs and 78 cov-host proteins. the four predicted bipartite modules are annotated as b1, b2, b3 and b4 within the heatmap. the drugs are colored based on their clinical phase (red-launched, preclinical-blue, phase2/phase3-green and phase-1/ phase-2-black ). panel-b, c, d and e represents networks corresponding to b1, b2, b3 and b4 modules.the drugs are annotated using the disease area found in cmap database 22 a b c d e figure 5 . predicted interactions for probability threshold: 0.9. panel-a shows the interaction graph between drugs and cov-host. drugs are annotated with their usage. panel-b, c, d and e represents quasi-bicliques for one, two, three and more than three drugs molecules respectively. use a weighted bipartite clustering algorithm proposed by j. beckett 23 . this results in 4 bipartite modules (panel-a figure 4): b1 (11 drugs, 28 cov-host), b2 (4 drugs, 41 cov-host), b3 (71 rugs and 4 cov-host), and b4 ( 6 drugs and 5 cov-host). the other panels of the figure show the network diagram of four bipartite modules. b1 contains 11 drugs, including some antibiotics (anisomycin, midecamycin), and anti-cancer drugs (doxorubicin, camptothecin). b3 also has some antibiotics such as puromycin, demeclocycline, dirithromycin, geldanamycin, and chlortetracycline, among them, the first three are widely used for bronchitis, pneumonia, and respiratory tract infections 24 . some other drugs such as lobeline and ambroxol included in the b3 module have a variety of therapeutic uses, including respiratory disorders and bronchitis. the high confidence predicted interactions (with threshold 0.9) is shown in figure 5 panel-a. to highlight some repurposable drug combination and their predicted cov-host target, we perform a weighted clustering (clusterone) 25 on this network and found some quasy-bicluques (shown in panel-b-e) we matched our predicted drugs with the drug list recently published by zhou et al. 13 and found six common drugs: mesalazine, vinblastine, menadione, medrysone, fulvestrant, and apigenin. among them, apigenin has a known effect in the antiviral activity together with quercetin, rutin, and other flavonoids 26 . mesalazine is also proven to be extremely effective in the treatment of other viral diseases like influenza a/h5n1 virus. 27 . baclofen, a benzodiazepine receptor (gabaa-receptor) agonist, has a potential role in antiviral associated treatment 28 . antiinflammatory antecedents fisetin is also tested for antiviral activity, such as for inhibition of dengue (denv) virus infection 29 . it down-regulates the production of proinflammatory cytokines induced by a denv infection. both of the drugs are listed in the high confidence interaction set with the three cov-hosts: tapt1 (interacted with sars-cov-2 protein: orf9c), slc30a6 (interacted with sars-cov-2 protein: orf9c), and trim59 (interacted with sars-cov-2 protein: orf3a) ( figure 5 -panel-c). topoisomerase inhibitors play an active role as antiviral agents by inhibiting the viral dna replication 30, 31 . some topoisomerase inhibitors such as camptothecin, daunorubicin, doxorubicin, irinotecan and mitoxantrone are predicted to interact with several cov-host proteins. it has been demonstrated that the anticancer drug camptothecin (cpt) and its derivative irinotecan have a potential role in antiviral activity 32, 33 . it inhibits host cell enzyme topoisomerase-i which is required for the initiation as well as completion of viral functions in host cell 34 . daunorubicin (dnr) has also been demonstrated as an inhibitor of hiv-1 virus replication in human host cells 35 . the conventional anticancer antibiotic doxorubicin was identified as a selective inhibitor of in vitro dengue and yellow fever virus replication 36 . it is also reported that doxorubicin coupling with monoclonal antibody can create an immunoconjugate that can eliminate hiv-1 infection in mice cell 37 . mitoxantrone shows antiviral activity against the human herpes simplex virus (hsv1) by reducing the transcription of viral genes in many human cells that are essential for dna synthesis 38 . histone deacetylases inhibitors (hdaci) are generally used as latency-reversing agents for purging hiv-1 from the latent reservoir like cd4 memory cell 39 . our predicted drug list (table 3 ) contains two hdaci: scriptaid and vorinostat. vorinostrate can be used to achieve latency reversal in the hiv-1 virus safely and repeatedly 40 . asymptomatic patients infected with sars-cov-2 are of significant concern as they are more vulnerable to infect large number of people than symptomatic patients. moreover, in most cases (99 percentile), patients develop symptoms after an average of 5-14 days, which is longer than the incubation period of sars, mers, or other viruses 41 . to this end, hdaci may serve as good candidates for recognizing and clearing the cells in which sars-cov-2 latency has been reversed. heat shock protein 90 (hsp) is described as a crucial host factor in the life cycle of several viruses that includes an entry in the cell, nuclear import, transcription, and replication 42, 43 . hsp90 is also shown to be an essential factor for sars-cov-2 envelop (e) protein 44 . in 45 , hsp90 is described as a promising target for antiviral drugs. the list of predicted drugs contains three hsp inhibitors: tanespimycin, geldanamycin, and its derivative alvespimycin. the first two have a substantial effect in inhibiting the replication of herpes simplex virus and human enterovirus 71 (ev71), respectively. recently in 46 , geldanamycin and its derivatives are proposed to be an effective drug in the treatment of covid-19. inhibiting dna synthesis during viral replication is one of the critical steps in disrupting the viral infection. the list of predicted drugs contains six such small molecules/drugs, viz., niclosamide, azacitidine, anisomycin, novobiocin, primaquine, menadione, and metronidazole. dna synthesis inhibitor niclosamide has a great potential to treat a variety of viral infections, including sars-cov, mers-cov, and hcv virus 47 and has recently been described as a potential candidate to fight the 9/19 sars-cov-2 virus 47 . novobiocin, an aminocoumarin antibiotic, is also used in the treatment of zika virus (zikv) infections due to its protease inhibitory activity. in 2005, chloroquine (cq) had been demonstrated as an effective drug against the spread of severe acute respiratory syndrome (sars) coronavirus (sars-cov). recently hydroxychloroquine (hcq) sulfate, a derivative of cq, has been evaluated to efficiently inhibit sars-cov-2 infection in vitro 48 . therefore, another anti-malarial aminoquinolin drug primaquine may also contribute to the attenuation of the inflammatory response of covid-19 patients. primaquine is also established to be effective in the treatment of pneumocystis pneumonia (pcp) 49 . cardiac glycosides have been shown to play a crucial role in antiviral drugs. these drugs target cell host proteins, which help reduce the resistance to antiviral treatments. the antiviral effects of cardiac glycosides have been described by inhibiting the pump function of na, k-atpase. this makes them essential drugs against human viral infections. the predicted list of drugs contains three cardiac glycosides atpase inhibitors: digoxin, digitoxigenin, and ouabain. these drugs have been reported to be effective against different viruses such as herpes simplex, influenza, chikungunya, coronavirus, and respiratory syncytial virus 50 . mg132, proteasomal inhibitor is established to be a strong inhibitor of sars-cov replication in early steps of the viral life cycle 51 . mg132 inhibits the cysteine protease m-calpain, which results in a pronounced inhibition of sars-cov-2 replication in the host cell. in 52 , resveratrol has been demonstrated to be a significant inhibitor mers-cov infection. resveratrol treatment decreases the expression of nucleocapsid (n) protein of mers-cov, which is essential for viral replication. as mg132 and resveratrol play a vital role in inhibiting the replication of other coronaviruses sars-cov and mers-cov, so they may be potential candidates for the prevention and treatment of sars-cov-2. another drug captopril is known as angiotensin ii receptor blockers (arb), which directly inhibits the production of angiotensin ii. in 53 , angiotensin-converting enzyme 2 (ace2) is demonstrated as the binding site for sars-cov-2. so angiotensin ii receptor blockers (arb) may be good candidates to use in the tentative treatment for sars-cov-2 infections 54 . in summary, our proposed method predicts several drug targets and multiple repurposable drugs that have prominent literature evidence of uses as antiviral drugs, especially for two other coronavirus species sars-cov and mers-cov. some drugs are also directly associated with the treatment of sars-cov-2 identified by recent literature. however, further clinical trials and several preclinical experiments are required to validate the clinical benefits of these potential drugs and drug targets. in this work, we have successfully generated a list of high-confidence candidate drugs that can be repurposed to counteract sars-cov-2 infections. the novelties have been to integrate most recently published sars-cov-2 protein interaction data on the one hand, and to use most recent, most advanced ai (deep learning) based high-performance prediction machinery on the other hand, as the two major points. in experiments, we have validated that our prediction pipeline operates at utmost accuracy, confirming the quality of the predictions we have raised. the recent publication (april 30, 2020) of two novel sars-cov-2-human protein interaction resources 15, 16 has unlocked enormous possibilities in studying virulence and pathogenicity of sars-cov-2, and the driving mechanisms behind it. only now, various experimental and computational approaches in the design of drugs against covid-19 have become conceivable, and only now such approaches can be exploited truly systematically, at both sufficiently high throughput and accuracy. here, to the best of our knowledge, we have done this for the first time. we have integrated the new sars-cov-2 protein interaction data with well established, long-term curated human protein and drug interaction data. these data capture hundreds of thousands approved interfaces between encompassing sets of molecules, either reflecting drugs or human proteins. as a result, we have obtained a comprehensive drug-human-virus interaction network that reflects the latest state of the art in terms of our knowledge about how sars-cov-2 and interacts with human proteins and repurposable drugs. for exploiting the new network-already establishing a new resource in its own right-we have opted for most recent and advanced deep learning based technology. a generic reason for this choice is the surge in advances and the resulting boost in operative prediction performance of related methods over the last 3-4 years. a particular reason is to make use of most advanced graph neural network based techniques, namely variational graph autoencoders as a deep generative model of utmost accuracy, the practical implementation of which 19 was presented only a few months ago (just like the relevant network data). note that only this recent implementation enables to process networks of sizes in the range of common molecular interaction data. in essence, graph neural networks "learn" the structure of links in networks, and infer rules that underlie the interplay of links. based on the knowledge gained, they enable to predict links and output the corresponding links together with probabilities for them to indeed be missing. simulation experiments, reflecting scenarios where links known to exist in our network were re-established by prediction upon their removal, pointed out that our pipeline does indeed predict missing links at utmost accuracy. encouraged by these simulations, we proceeded by performing the core experiments, and predicted links to be missing without prior removal of links in our encompassing network. these core experiments revealed 692 high confidence interactions relating to 92 drugs. in our experiments, we focused on predicting links between drugs and human proteins that in turn are known to interact with sars-cov-2 proteins (sars-cov-2 associated host proteins). we have decidedly put the focus not on drug -sars-cov-2-protein interactions, which would have reflected more direct therapy strategies against the virus. instead, we have focused on predicting drugs that serve the purposes of host-directed therapy (hdt) options, because hdt strategies have proven to be more sustainable with respect to mutations by which the virus escapes a response to the therapy applied. note that hdt strategies particularly cater to drug repurposing attempts, because repurposed drugs have already proven to lack severe side effects, because they are either already in use, or have successfully passed the preclinical trial stages. we further systematically categorized the 92 repurposable drugs into 70 categories based on their domains of application and molecular mechanism. according to this, we identified and highlighted several drugs that target host proteins that the virus needs to enter (and subsequently hijack) human cells. one such example is captopril, which directly inhibits the production of angiotensin-converting enzyme-2 (ace-2), in turn already known to be a crucial host factor for sars-cov-2. further, we identified primaquine, as an antimalaria drug used to prevent the malaria and also pneumocystis pneumonia (pcp) relapses, because it interacts with the tim complex timm29 and alg11. moreover, we have highlighted drugs that act as dna replication inhibitor (niclosamide, anisomycin), glucocorticoid receptor agonists (medrysone), atpase inhibitors (digitoxigenin, digoxin), topoisomerase inhibitors (camptothecin, irinotecan), and proteosomal inhibitors (mg-132). note that some drugs are known to have rather severe side effects from their original use (doxorubicin, vinblastine), but the disrupting effects of their short-term usage in severe covid-19 infections may mean sufficient compensation. in summary, we have compiled a list of drugs, which when repurposed are of great potential in the fight against the covid-19 pandemic, where therapy options are urgently needed. our list of predicted drugs suggests both options that had been identified and thoroughly discussed before and new opportunities that had not been pointed out earlier. the latter class of drugs may offer valuable chances for pursuing new therapy strategies against covid-19. we have utilized three categories of interaction datasets: human protein-protein interactome data, sars-cov-2-host protein interaction data, and drug-host interaction data. we have taken sars-cov-2-host interaction information from two recent studies by gordon et al and dick et al 15, 16 . in 15 , 332 high confidence interactions between sars-cov-2 and human proteins are predicted using using affinity-purification mass spectrometry (ap-ms). in 16 , 261 high confidence interactions are identified using sequence-based ppi predictors (pipe4 & sprint). the drug-target interaction information has been collected from five databases, viz., drugbank database (v4.3) 57 , chembl 58 database, therapeutic target database (ttd) 59 , pharmgkb database, and iuphar/bps guide to pharmacology 60 . total number of drugs and drug-host interactions used in this study are 1309 and 1788407, respectively. we have built a comprehensive list of human ppis from two datasets: (1) ccsb human interactome database consisting of 7,000 genes, and 13944 high-quality binary interactions 61-63 , (2) the human protein reference database 56 which consists of 8920 proteins and 53184 ppis. the summary of all the datasets is provided in table 2 . cmap database 22 is used to annotate the drugs with their usage different disease areas. we have utilized node2vec 17 , an algorithmic framework for learning continuous feature representations for nodes in networks. it maps the nodes to a low-dimensional feature space that maximizes the likelihood of preserving network neighborhoods. the principle of feature learning framework in a graph can be described as follows: let g = (v, e) be a given graph, where v represents a set of nodes, and e represents the set of edges. the feature representation of nodes (|v |) is given by a mapping function: f : v → r d , where d specify the feature dimension. the f may also be represented as a node feature matrix of dimension of |v | × d. for each node, v ∈ v , nn s (v) ⊂ v defines a network neighborhood of node v which is generated using a neighbourhood sampling strategy s. the sampling strategy can be described as an interpolation between breadth-first search and depth-first search technique 17 . the objective function can be described as: this maximizes the likelihood of observing a network neighborhood nn s (v) for a node v given on its feature representation f . now the probability of observing a neighborhood node n i ∈ nn s (v) given the feature representation of the source node v is given as : where, n i is the i th neighbor of node v in neighborhood set nn s (v). the conditional likelihood of each source (v) and neighborhood node (n i ∈ nn s (v )) pair is represented as softmax of dot product of their features f (v) and f (n i ) as follows: variational graph autoencoder (vgae) is a framework for unsupervised learning on graph-structured data 64 . this model uses latent variables and is effective in learning interpretable latent representations for undirected graphs. the graph autoencoder consists of two stacked models: 1) encoder and 2) decoder. first, an encoder based on graph convolution networks (gcn) 18 maps the nodes into a low-dimensional embedding space. subsequently, a decoder attempts to reconstruct the original graph structure from the encoder representations. both models are jointly trained to optimize the quality of the reconstruction from the embedding space, in an unsupervised way. the functions of these two model can be described as follows: encoder: it uses graph convolution network (gcn) on adjacency matrix a and the feature representation matrix f. encoder generates a d -dimensional latent variable z i for each node i ∈ v , with |v | = n, that corresponds to each embedding node, with d ≤ n. the inference model of the encoder is given below: where, r(z i |a, f) corresponds to normal distribution, n ( z i µ i , σ 2 i ), µ i and σ i are the gaussian mean and variance parameters. the actual embedding vectors z i are samples drawn from these distributions. decoder: it is a generative model that decodes the latent variables z i to reconstruct the matrix a using inner products with sigmoid activation from embedding vector, (z). where, a is the decoded adjacency matrix. the objective function of the variational graph autoencoder (vgae) can be written as: the objective function c v gae maximizes the likelihood of decoding the adjacency matrix w.r.t graph autoencoder weights using stochastic gradient decent. here, d kl (.||.) represents kullback-leibler divergence 65 and p(z) is the prior distribution of latent variable. drug-sars-cov-2 link prediction 1. adjacency matrix preparation in this work, we consider an undirected graph g = (v, e) with |v | = n nodes and |e| = m edges. we denote a as the binary adjacency matrix of g. here v consists of sars-cov-2 proteins, cov-host proteins, drug-target proteins and drugs. the matrix (a) contains a total of n = 16444 nodes given as: where, n nc is the number of sars-cov-2 proteins. n dt is the number of drug targets, whereas n nt and n d represent the number of cov-host and drugs nodes, respectively. total number of edges is given by: where, e 1 represents interactions between sars-cov-2 and human host proteins, e 2 is the number of interactions among human proteins, and e 3 represents the number of interactions between drugs and human host proteins. the neighborhood sampling strategy is used here to prepare a feature representation of all nodes. a flexible biased random walk procedure is employed to explore the neighborhood of each node. a random walk in a graph g can be described as the probability: where, π(v, x) is the transition probability between nodes v and x, where (v, x) ∈ e and a i is the i th node in the walk of length l. the transition probability is given by π(v, x) = c pq (t, x) * w vx , where t is the previous node of v n the walk, w vx is the static edge weights and p, q are the two parameters which guides the walk. the coefficient c pq (t, x) is given by where, distance(t, x) represents the shortest path distance between nodes t and node x. the process of feature matrix f n×d generation is governed by the node2vec algorithm. it starts from every nodes and simulates r random walks of fixed length l. in every step of walk transition probability π(v, x) govern the sampling. the generated walk of each iteration is included to a walk-list. finally, the stochastic gradient descent is applied to optimize the list of walks and result is returned. 3. link prediction: scalable and fast variational graph autoencoder (fastvgae) 19 is utilized in our proposed work to reduce the computational time of vgae in large network. the adjacency matrix a and the feature matrix f are given into the encoder of fastvgae. the encoder uses graph convolution neural network (gcn) on the entire graph to create the latent representation (z). the encoder works on full adjacency matrix a. after encoding, sampling is done and decoder works on the sampled sub graph. the mechanism of decoder of fastvgae is slightly different from traditional vgae. it regenerate the adjacency matrix a based on a subsample of graph nodes, v s . it uses a graph node sampling technique to randomly sample the reconstructed nodes at each iteration. each node is assigned with a probability p i and the selection of noes is based on the high score of p i . the probability p i is given by the following equation: where, f (i) is the degree of node i, and α is the sharpening parameter. we take α = 2 in our study. the node selection process is repeated until |v s | = n s , where n s is the number of sampling nodes. the decoder reconstructs the smaller matrix, a s of dimension n s × n s instead of decoding the main adjacency matrix a. the decoder function follows the following equation: a s (i, j) = sigmoid(z t i .z j ), ∀(i, j) ∈ v s ×v s . at each training iteration different subgraph (g s ) is drawn using the sampling method. after the model is trained the drug-cov-host links are predicted using the following equation: where a i j represents the possible links between all combination of sars-cov-2 nodes and drug nodes. for each combination of nodes the model gives probability based on the logistic sigmoid function. a new coronavirus associated with human respiratory disease in china host-pathogen systems biology host-directed therapies for bacterial and viral infections network-based 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treatment of pneumocystis carinii pneumonia the antiviral effects of na, k-atpase inhibition: a minireview severe acute respiratory syndrome coronavirus replication is severely impaired by mg132 due to proteasome-independent inhibition of m-calpain effective inhibition of mers-cov infection by resveratrol structural basis of receptor recognition by sars-cov-2 angiotensin receptor blockers as tentative sars-cov-2 therapeutics next-generation sequencing to generate interactome datasets development of human protein reference database as an initial platform for approaching systems biology in humans drugbank 4.0: shedding new light on drug metabolism chembl: a large-scale bioactivity database for drug discovery therapeutic target database update 2016: enriched resource for bench to clinical drug target and targeted pathway information the iuphar/bps guide to pharmacology: an expert-driven knowledgebase of drug targets and their ligands towards a proteome-scale map of the human protein-protein interaction network a proteome-scale map of the human interactome network a reference map of the human binary protein interactome stochastic backpropagation and approximate inference in deep generative models on information and sufficiency. the annals mathematical statistics key: cord-350286-n7ylgqfu authors: giri, rajanish; bhardwaj, taniya; shegane, meenakshi; gehi, bhuvaneshwari r.; kumar, prateek; gadhave, kundlik; oldfield, christopher j.; uversky, vladimir n. title: when darkness becomes a ray of light in the dark times: understanding the covid-19 via the comparative analysis of the dark proteomes of sars-cov-2, human sars and bat sars-like coronaviruses date: 2020-04-03 journal: biorxiv doi: 10.1101/2020.03.13.990598 sha: doc_id: 350286 cord_uid: n7ylgqfu recently emerged coronavirus designated as sars-cov-2 (also known as 2019 novel coronavirus (2019-ncov) or wuhan coronavirus) is a causative agent of coronavirus disease 2019 (covid-19), which is rapidly spreading throughout the world now. more than 9,00,000 cases of sars-cov-2 infection and more than 47,000 covid-19-associated mortalities have been reported worldwide till the writing of this article, and these numbers are increasing every passing hour. world health organization (who) has declared the sars-cov-2 spread as a global public health emergency and admitted that the covid-19 is a pandemic now. the multiple sequence alignment data correlated with the already published reports on the sars-cov-2 evolution and indicated that this virus is closely related to the bat severe acute respiratory syndrome-like coronavirus (bat sars-like cov) and the well-studied human sars coronavirus (sars cov). the disordered regions in viral proteins are associated with the viral infectivity and pathogenicity. therefore, in this study, we have exploited a set of complementary computational approaches to examine the dark proteomes of sars-cov-2, bat sars-like, and human sars covs by analysing the prevalence of intrinsic disorder in their proteins. according to our findings, sars-cov-2 proteome contains very significant levels of structural order. in fact, except for nucleocapsid, nsp8, and orf6, the vast majority of sars-cov-2 proteins are mostly ordered proteins containing less intrinsically disordered protein regions (idprs). however, idprs found in sars-cov-2 proteins are functionally important. for example, cleavage sites in its replicase 1ab polyprotein are found to be highly disordered, and almost all sars-cov-2 proteins were shown to contain molecular recognition features (morfs), which are intrinsic disorder-based protein-protein interaction sites that are commonly utilized by proteins for interaction with specific partners. the results of our extensive investigation of the dark side of the sars-cov-2 proteome will have important implications for the structural and non-structural biology of sars or sars-like coronaviruses. significance the infection caused by a novel coronavirus (sars-cov-2) that causes severe respiratory disease with pneumonia-like symptoms in humans is responsible for the current covid-19 pandemic. no in-depth information on structures and functions of sars-cov-2 proteins is currently available in the public domain, and no effective anti-viral drugs and/or vaccines are designed for the treatment of this infection. our study provides the first comparative analysis of the orderand disorder-based features of the sars-cov-2 proteome relative to human sars and bat cov that may be useful for structure-based drug discovery. intrinsically disordered proteins (idps) and intrinsically disordered protein regions (idprs)), in order to better understand an interplay between the ordered and disordered components of the proteome. in classical structure-function-paradigm, it is believed that a unique, stable, and well-defined 3-dimensional structure is a prerequisite for a protein to accomplish its unique biological function. although this notion dominated scientific minds for over the hundred years, eventually an idea of the presence of functional intrinsic disorder in proteins came to the attention of the structural biologists. according to this "heretic" viewpoint, a noticeable amount of biologically active proteins (of protein regions) fail to fold into the well-defined structures and instead remain disordered, existing as highly dynamic ensembles of rapidly interconverting conformations under the physiological conditions. these proteins and protein regions are known now as intrinsically disordered proteins (idps) and intrinsically disordered protein regions (idprs), respectively. the propensity of being functional intrinsically disordered proteins (similar to the propensity of forming unique biologically active structures of ordered proteins) is determined by the amino acid sequences [11] [12] [13] . idps exhibit their biological functions in numerous biological processes commonly associated with cellular signalling, gene regulation, and control by interacting with their physiological partners [14] [15] [16] [17] [18] . these functions of idps and idps are regulated by their protein-protein, protein-rna, protein-dna interactions [19, 20] . molecular recognition features (morfs) are the regions in idps implicated in regulation of idps function by protein-protein interactions and serve as the primary stage in molecular recognition. zhang and colleagues have reported the genomic sequence of sars-cov-2 with genbank accession number nc_045512 having 29,903 nucleotides. the virus was isolated from the bronchoalveolar lavage fluid of a patient, went through a circle or renaming, from novel wuhan seafood market pneumonia virus to deadly wuhan coronavirus, to the 2019 novel coronavirus (2019-ncov) or the wuhan-2019 novel coronavirus (wuhan-2019-ncov, and was eventually named sars-cov-2 by the who [21] . it is known that the idps/idprs are present in all three kingdoms of life, and viral proteins often contain unstructured regions that have been strongly correlated with their virulence [22] [23] [24] [25] . in this report, we investigated the disordered side of the sars-cov-2 proteome using a complementary set of computational approaches to check the prevalence of idprs in various sars-cov-2 proteins and to shed some light on their disorder-related functions. we also have comprehensively analyzed idprs among the closely related viruses, such as human sars cov and bat sars-like cov. furthermore, we have also identified protein functions related to protein-protein interactions, rna binding, and dna binding from all three viruses. since these three viruses are closely related, our study provides important means for a better understanding of the sequence and structural peculiarities of their evolution. we believe that this study will help the structural and non-structural biologists to design and perform experiments for a more in-depth understanding of this virus and its pathogenicity. this also will have long-term implications for developing new drugs or vaccines against this currently unpreventable infection. sequence retrieval and multiple sequence alignment. the protein sequences of bat cov (sars-like) and human sars cov were retrieved from uniprot (uniprot ids for individual proteins are listed in table 1 ). the translated sequences of sars-cov-2 proteins [genbank database [26] (accession id: nc_045512.2)] were obtained from genbank. we used these sequences for performing multiple sequence alignment (msa) and predicting the idprs. we have used clustal omega [27] for protein sequence alignment and esprit 3.0 [28] for constructing the aligned images. for the prediction of the intrinsic disorder predisposition of cov proteomes, we have used multiple predictors, such as members of the pondr ® (predictor of natural disordered regions) family including pondr ® vls2 [29] , pondr ® vl3 [30] , pondr ® fit [31] , and pondr ® vlxt [32] , as well as the iupred platform for predicting long (≥30 residues) and short idprs (<30 residues) [33] . these computational tools predict residues/regions, which do not have the tendency to form an ordered structure. residues with disorder scores exceeding the threshold value of 0.5 are considered as intrinsically disordered residues, whereas residues with the predicted disorder scores between 0.2 and 0.5 are considered flexible. complete predicted percent of intrinsic disorder (ppid) in a query protein was calculated for every protein of all the three viruses from outputs of six predictors. the detailed methodology has been given in our previous reports [34, 35] . and disopred3 [42] . the protein residues with anchor, morfpred, and disopred3 score above the threshold value of 0.5 and morfchibi_web score above the threshold value of 0.725 are considered morf regions. idprs facilitate interactions with rnas and dnas and regulates many cellular functions [43] . thus, for predicting the dna binding residues in cov proteins, we have used two online servers: drnapred [19] and disordpbind [43] . for rna binding residues, we used pprint (prediction of protein rna-interaction) [44] and disordpbind [43] . the mean values of the predicted percentage of intrinsic disorder scores (mean ppids), that were obtained by averaging the predicted disorder scores from six disorder predictors (supplementary table 1 -3) for each protein of sars-cov-2 as well as human sars, and bat cov are represented in table 1 . * these sequences are based on genome annotations conducted by wu et al. [45] . proteins and their ppids are coloured to reflect their disorder status (ordered -blue, moderately disorderedpink, highly disordered -red) figures 2a, 2b , and 2c are 2d-disorder plots generated for sars-cov-2, human sars and bat cov proteins, respectively, and represent the ppid pondr-fit vs. ppid mean plots. based on their predicted levels of intrinsic disorder, proteins can be classified as highly ordered (ppid < 10%), moderately disordered (10% ≤ ppid < 30%) and highly disordered (ppid ≥ 30%) [46] . from the data in table 1 , figures 2a, 2b , and 2c, as well as the ppid based classification, we conclude that the nucleocapsid protein from all three strains of coronavirus possesses the highest percentage of the disorder and is classified as highly disordered protein. orf3b protein in bat cov, orf6 protein in sars-cov-2, human sars, and bat cov, and orf9b protein in human sars and sars cov belong to the class of moderately disordered proteins. while the structured proteins, namely, spike glycoprotein (s), an envelope protein (e) and membrane protein (m) as well as accessory proteins orf3a, orf7a, orf8 (orf8a and orf8b in case of human sars) of all three strains of coronavirus are ordered proteins. orf14 and orf10 proteins also belong to the class of ordered proteins. in ch-cdf plot of the proteins of (d) sars-cov-2 (e) human sars and (f) bat cov, the y coordinate of each protein spot signifies distance of corresponding protein from the boundary in ch plot and the x coordinate value corresponds to the average distance of the cdf curve for the respective protein from the cdf boundary. in order to further investigate the nature of the disorder in proteins of sars-cov-2, human sars, and bat cov, we utilized the combined ch-cdf tool that uses the outputs of two binary classifiers of disorder, charge hydropathy (ch) plot and cumulative distribution function (cdf) plot. this helped in retrieving more detailed characterization of the global disorder predisposition of the query proteins and their classification according to the disorder "favours". the ch plot is a linear classifier that differentiates between proteins that are predisposed to possess extended disordered conformations that include random coils and premolten globules from proteins that have compact conformations (ordered proteins and molten globule-like proteins). the other binary predictor, cdf is a nonlinear classifier that uses the pondr ® vlxt scores to discriminate ordered globular proteins from all disordered conformations, which include native molten globules, pre-molten globules, and random coils. the ch-cdf plot can be divided into four quadrants: q1 (bottom right quadrant) is an area of ch-cdf phase space that is expected to include ordered proteins; q2 (bottom left quadrant) includes proteins predicted to be disordered by cdf and compact by ch (i.e., native molten globules and hybrid proteins containing high levels of both ordered and disordered regions); q3 (top left quadrant) contains proteins that are predicted to be disordered by both ch and cdf analysis (i.e., highly disordered proteins with the extended disorder); and q4 (top right quadrant) possesses proteins disordered according to ch but ordered according to cdf analysis [34] . figures 2d, 2e and 2f represent the ch-cdf analysis of proteins of sars-cov-2, human sars, and bat cov and show that all the proteins are located within the two quadrants q1 and q2. the ch-cdf analysis leads to the conclusion that all proteins of sars-cov-2, human sars, and bat cov are ordered except nucleocapsid protein, which is predicted to be disordered by cdf but ordered by ch and hence lies in q2. molecular recognition features (morfs) are short interaction-prone disordered regions found within idps/idprs that commence a disorder-to-order transition upon binding to their partners [47, 48] . these regions are important for protein-protein interactions and may initiate an early step in molecular recognition [48] . in this study, we have analyzed and compared morfs (protein-binding regions) in sars-cov-2 with human sars and bat cov. the results of this analysis are summarized in table 2 , which clearly shows that most of the sars-cov-2 proteins contain at least one morf, indicating that disorder does play an important role in the functionality of these viral proteins. in addition to protein-protein interactions/protein-binding functions, idps and idrs also mediates functions by facilitating their interactions with nucleotides such as dna and rna [20, 49] . therefore, we have used a combination of two different online servers for locating protein residues that are showing the propensity to bind with dna as well as rna. nucleotide-binding residues in proteins of three studied coronaviruses are listed in supplementary coronaviruses encode four structural proteins, namely, spike (s), envelope (e) glycoprotein, membrane (m), and nucleocapsid (n) proteins, which are translated from the last ~10kb nucleotides and form the outer cover of the covs, encapsulating their single-stranded genomic rna. s protein is a large multifunctional protein forming the exterior of the cov particles [50, 51] . it forms surface homotrimers and contains two distinct ectodomain regions known as s1 and s2. in some covs, the s protein is actually cleaved into these subunits, which are joined non-covalently, whereas an additional proteolytic cleavage within the n-terminal part of the s2 subunit that takes place upon virus endocytosis generates spike proteins s2'. subunit s1 initiates viral infection by binding to the host cell receptors, s2 acts as a class i viral fusion protein that mediates fusion of the virion and cellular membranes and thereby promotes the viral entry into the host cells, whereas s2' serves as a viral fusion peptide [52, 53] . spike binds to the virion m protein through its c-terminal transmembrane region [54] . belonging to a class i viral fusion protein, s protein binds to specific surface receptor angiotensin-converting enzyme 2 (ace2) on host cell plasma membrane through its n-terminal receptor-binding domain (rbd) and mediates viral entry into host cells [55] . the s protein consists of an n-terminal signal peptide, a long extracellular domain, a singlepass transmembrane domain, and a short intracellular domain [56] . a 3.60 å resolution structure (pdb id: 6acc) of s protein from human sars complexed with its host binding partner ace2 has been obtained by cryo-electron microscopy (cryo-em the biophysical analysis reported in previous study has also revealed that the s protein from sars-cov-2 has a higher binding affinity to ace2 than s protein from human sars [58] . which has been calculated by averaging the disorder scores from all six predictors is represented by a short-dot line (sky-blue line) in the graph. the light sky-blue shadow region signifies the mean error distribution. the residues missing in the pdb structure or the residues for which pdb structure is unavailable are represented by the grey-coloured area in the corresponding graphs. (e) aligned disorder profiles generated for spike glycoprotein from sars-cov-2 (black line), human sars (red line), and bat cov (green line) based on the outputs of the pondr ® vsl2. msa analysis among all three coronaviruses demonstrates that s protein of sars-cov-2 has a 77.71% sequence identity with bat cov and 77.14% identity with human sars (supplementary figure s1a) . all three s proteins are found to have a conserved c-terminal region. however, the n-terminal regions of s proteins display noticeable differences. given that there is significant sequence variation rbd located at the n-terminal region of s protein, this might be the reason behind variation in its virulence and its receptor-mediated binding and entry into the host cell. according to our intrinsic disorder propensity analysis, s protein from all three covs analysed in this study are highly structured, as their predicted disorder propensity lies below 10% ( table 1 ). in fact, the mean ppid scores of sars-cov-2, human sars cov, and bat cov are calculated to be 1.41%, 1.12%, and 1.85%, respectively. figures 3b, 3c , and 3d represent the intrinsic disorder profiles of s proteins from sars-cov-2, human sars and bat cov obtained from six disorder predictors. finally, figure 3e shows aligned disorder profiles of s proteins from these covs and illustrates remarkable similarity in their disorder propensity, especially in the c-terminal region. it is of interest to map known functional regions of s proteins to their corresponding disorder profiles. the maturation of s protein requires specific posttranslational modification (ptm), proteolytic cleavage that happens at two stages. first, host cell furin or another cellular protease nicks the s precursor to generate s1 and s2 proteins, whereas the second cleavage that takes place after the viral attachment to host cell receptors leads to the release of a fusion peptide generating the s2' subunit. in human sars cov, the first and second cleavage site is located at residues r 667 and r 797 , respectively, whereas in bat cov, the corresponding cleavage sites are residues r 654 and r 784 . as it follows from figure 3 , these cleavage sites are located within the idprs. in human sars cov s protein, fusion peptide (residues 770-788) is located within a flexible region, is characterized by the mean disorder score of 0.232±0.053. similarly, in bat cov s protein, fusion peptide (residues 757-775) has a mean disorder score of 0.320±0.046. s protein contains two heptad repeat regions that form coiledcoil structure during viral and target cell membrane fusion, assuming a trimer-of-hairpins structure needed for the functional positioning of the fusion peptide. in human sars cov s protein, heptad repeat regions are formed by residues 902-952 and 1145-1184, which have mean disorder scores of 0.458±0.067 and 0.353±0.062, respectively. the analogous situation is observed for the s protein from bat cov, where these heptad repeat regions are positioned at residues 889-939 (0.44±0.11) and 1132-1171 (0.353±0.062). another functional region found in s proteins is the receptor-binding domain (residues 306-527 and 310-514 in human sars cov and bat cov, respectively) containing a receptor-binding motif responsible for interaction with human ace2. in human s protein of human sars cov this motif (residues 424-494) is not only characterized by structural flexibility, possessing a mean disorder score of 0.30±0.16, but also contains a disordered region (residues 461-466). since s protein is known as spike glycoprotein, it contains numerous glycosylation sites. due to rather close similarity of disorder profiles of s proteins analysed here, we can assume that all the aforementioned indications of the functional importance of disorder and flexible regions in s proteins from sars cov and bat cov are also applicable to sars-cov-2 s protein. finally, table 2 shows that s protein from sars-cov-2 contain one morf region at its cterminal (residues 1265-1272) by morfchibi_web, two morf regions ((residues 2-6) & (residues 819-823)) by morfpred, and one morf region at n-terminal (residues 1-10) by disopred3. these results indicating that intrinsic disorder is important for its interaction with binding partners. interestingly, the n-terminal region of s protein (residues 1-10) from all three viruses are observed to be a disorder-based protein binding region by two predictors (morfpred and disopred3). n-terminal morf displays its role in viral interaction with host receptor and c-terminal morf displays its role in m protein interaction and viral assembly. moreover, morf region mainly lies in the n-and c-terminal regions suggesting a possible role during cleavage as well. in addition to protein-binding regions, s protein also shows many nucleotide-binding residues. tables 9, 10, and 11 shows that numerous rna binding residues predicted by pprint in all three viruses and a single rna binding residue were predicted by disordpbind in human sars. further, drnapred and disordpbind predicted the presence of many dna binding residues in s protein of all three viruses. these results signify the role of s protein functions related to molecular recognition (protein-protein interaction, rna binding, and dna binding) such as interaction with host cell membrane and further viral infection. therefore, identified idps/idprs and residues/regions from s protein crucial for molecular recognition can be targeted for disorder-based drug discovery. envelope (e) protein is a small, multifunctional inner membrane protein that plays an important role in the assembly and morphogenesis of virions in the cell [59] [60] [61] . e protein consists of two ectodomains associated with n-and c-terminal regions, and a transmembrane domain. it homo-oligomerize to form pentameric membrane destabilizing transmembrane (tm) hairpins to form a pore necessary for its ion channel activity [62] . figure 4a shows the nmr-structure (pdb id: 2mm4) of human sars envelope glycoprotein of 8-65 residues [63] . msa results illustrate ( figure 4b ) that this protein is highly conserved, with only three amino acid substitutions in e protein of sars-cov-2 conferring its 96% sequence similarity with human sars and bat cov. bat cov shares 100% sequence identity with human sars. mean ppid calculated for sars-cov-2, human sars, and bat cov e proteins are 5.33%, 6.58%, and 6.58% respectively ( table 1) . the e protein is found to have a reasonably well-predicted structure. our predictions suggest that the residues of n-and c-terminals are displaying a higher tendency for the disorder. the last 18 hydrophilic residues (residues 59-76) have been reported to adopt a random-coil conformation with and without the addition of lipid membranes [64] . literature suggests that the last four amino acids of the c-terminal region of e protein containing a pzd-binding motif are involved in protein-protein interactions with a tight junction protein pals1. our results support literature as we identified long n-terminal region of approximately 30 residues long as disorder-based protein binding region in all three viruses (see table 2, supplementary table 7 and 8). pals1 is involved in maintaining the polarity of epithelial cells in mammals [65] . respective graphs in figures 4c, 4d , and 4e show the predicted intrinsic disorder profiles for e proteins of sars-cov-2, human sars, and bat cov. we speculate that the disordered region content may be facilitating the interactions with other proteins as well. in agreement with this hypothesis, table 2 shows that in e protein from sars-cov-2, the c-terminal domain serves as protein-binding region. we found that the residues from 45-75 is a long morf in e proteins of all three viruses as predicted by morfchibi_web ( table 2, supplementary table 7 and 8). as aforementioned, these randomly-coiled binding-residues at c-terminus may gain structure while assisting the protein-protein interaction mediated by e protein. one more morf region (residues [26] [27] [28] [29] [30] in the transmembrane domain was observed by disopred3 in the e protein of all three viruses. since these residues are the part of ion channel, we speculate that these residues do specific interactions and may be guiding the specifi functions of ion channel activity. few rna binding residues by pprint and disordpbind and several dna binding residues by drnapred are predicted for e protein in all three viruses. assembly by interacting with the nucleocapsid (n) and e proteins [66] [67] [68] . protein m interacts specifically with a short viral packaging signal containing coronavirus rna in the absence of n protein, thereby highlighting an important nucleocapsid-independent viral rna packaging mechanism inside the host cells [69] . it gains high-mannose n-glycans in er, which are subsequently modified into complex n-glycans in the golgi complex. glycosylation of m protein is observed to be not essential for virion fusion in cell culture [70, 71] . cryo-em and tomography data indicate that m forms two distinct conformations, a compact m protein having high flexibility and low spike density, and an elongated m protein having a rigid structure and narrow range of membrane curvature [72] . some regions of m glycoproteins might serve as important dominant immunogens. although no structural information is available for the full-length m protein as of yet, a short peptide of the membrane glycoprotein (residues 88-96) from human sars cov was co-crystallized with a complex between a-2 alpha chain of the hla class i histocompatibility antigen and β2microglobulin (pdb id: 3i6g) [73] . figure 5a shows that within this complex, the cocrystallized m protein region exists in an extended conformation. m protein of sars-cov-2 has a sequence similarity of 90.1% with bat cov and 89.6% with human sars m proteins ( figure 5b ). our analysis revealed that the intrinsic disorder levels in m proteins of sars-cov-2, human sars cov, and bat cov are relatively low since these proteins show the ppid values of 2.70%, 1.36%, and 1.36% respectively. this is in line with the previous publication by goh et al. on human sars hku4 where they found the mean ppid of 4% using additional predictors such as topidp and foldindex along with the predictors used in our study [74] . figures 5c, 5d , and 5e represent per-residue disorder profiles generated for m proteins of sars-cov-2, human sars cov, and bat cov and show that with the exception to their n-and c-terminal regions, these proteins are mostly ordered. the last 20 residues of mers-cov m protein are important for intracellular trafficking and contains a determinant that localizes it into the golgi network [75] . our results in table 2 illustrates that the disordered c-tail of the m protein is predicted to have disorder based protein-binding region and therefore can serve as a binding site for its specific partner required for its localization inside the host cell. a long morf region (residues 186-220) at the c-terminal of m protein in all three viruses were observed by morfchibi_web. two morf regions (one at n-terminus (residues 1-16) and one at c-terminus (residues 205-221)) was observed by disopred3 in human sars and bat cov. however, single morf (residues 117-132) observed in sars-cov-2 by disopred3. morfpred also predicts a short morf at c-terminus of sars-cov-2 (residues 214-222), human sars (residues 214-221), and bat cov (residues 211-221) ( table 2, supplementary tables 7 and 8) . furthermore, the m protein from all three viruses displays strong tendency to bind with rna (as predicted by pprint and disordpbind) and dna (as predicted by drnapred and disordpbind) (see supplementary tables 9, 10 , and 11). our understanding on m protein of covs (idps and morf at c-terminus and molecular recognition) elucidates its crucial role in interaction with the n and e proteins for viral assembly. nucleocapsid (n) protein: nucleocapsid (n) protein is one of the major viral proteins playing several significant roles in transcription, and virion assembly of coronaviruses [76] . it binds to viral genomic rna forming a ribonucleoprotein core required for the rna encapsidation during viral particle assembly [77] . sars-cov virus-like particles (vlps) formation has been reported to depend upon either m and e proteins or m and n proteins. for the effective production and release of vlps, co-expression of e or n proteins with m protein is necessary [78] . n protein of human sars consists of two structural domains, the n-terminal rna-binding domain (ntd: 45-181 residues) and the c-terminal dimerization domain (ctd: 248-365 residues) with a disordered patch in between these domains. n protein has been demonstrated to bind viral rna using both ntd and ctd [79] . figure 6a1 displays the nmr solution structure of the ntd of human sars cov nucleocapsid protein (45-181 residues) (pdb id: 1ssk) [80] . figure 6a2 shows an x-ray crystal structure of the ctd of human sars cov nucleocapsid protein (270-366 residues) (pdb id: 2gib) [81] . a model of the domain organization of the n-protein from sars-cov-2 is shown in figure 6b . the 419 amino acid-long n protein of sars-cov-2 shows a percentage identity of 88.76% with n protein of bat cov n protein and 89.74% with human sars n protein (supplementary figure s1b) . our analysis revealed that the n proteins of coronaviruses contain the highest levels of intrinsic disorder (see figure 6 and table 1 ). in fact, n proteins from sars-cov-2 human sars cov, and bat cov are characterized by the mean ppid of 64.91%, 71.09%, and 65.80%, respectively. in accordance with the previously evaluated intrinsic disorder predisposition [74] , n protein is highly disordered in all three sars viruses analysed in this study ( table 1) . graphs in figures 6c, 6d , and 6e depict the disorder profiles of sars-cov-2, human sars cov, and bat cov nucleocapsid proteins and show that their n-and c-terminal regions are completely disordered, and all three proteins also contain the central unstructured segment. as expected, the intrinsic disorder predisposition of the n protein of sars-cov-2 is remarkably similar to that for the n protein of human sars cov as reported in a previous study [74] . this is further supported by figure 6f , where pondr ® vsl2-generated disorder profiles of these three proteins are overlapped to show almost complete coincidence of their major disorder-related features. it is clear that in n-proteins, the n-and c-termini and a log central segment are completely disordered. figure 6c shows that in the n protein from sars-cov-2, residues 1-57, 64-102, 145-162, 166-289, and 362-422 are found to be disordered. many of these residues are lying within the ntd and ctd regions, and which, due to their structural plasticity, were not crystallized in human sars cov n protein. sars-cov-2 has a disordered segment from 168-289 residues while human sars has predicted to have an unstructured segment from 145-289 residues. overall, all three n proteins are found to be highly disordered. the n protein from human sars cov has one phosphorylation site (residue s 177 ) and several regions with compositional biases, such as ser-rich (residues 181-213), poly-leu, poly-gln, and ploy-lys (residues 220-225, 240-245, and 370-376), all predicted to be disordered. similarly, in n protein from bat cov, s 176 is phosphorylated, and this protein has ser-rich, poly-leu, and ploy-lys regions (residues 176-206, 219-224, and 369-375, respectively), all of which are disordered. it has been reported to interact using the central disordered region with m protein, hnrnp a1, and self n-n interaction [82] [83] [84] . the middle flexible region is also responsible for its rna-binding activity [85] . deletion of 184-196 residues, 169-308 residues, 161-210 residues of n abolishes its multimerization, rnabinding capacity, and hnrnp a1 interactions respectively. supplementary table 7 and 8, and table 2 shows that n protein is heavily decorated with numerous morfs, suggesting that this protein is a promiscuous binder. long disorder-based protein bonding regions at nand c-terminus of n protein of all three viruses were observed by all four predictors (morfchibi_web, anchor, morfpred, and disopred3). indeed, this is the single protein where we found many morfs as compared with the other structural, non-structural and accessory proteins of covs. the morfs present in these regions may mediate the abovementioned interactions of n proteins. figure 6a2 represents another important disorderrelated functional feature of the n protein. in fact, the ctd homodimer shown there is characterized by highly intertwined morphology, which is typically a result of bindinginduced folding [86] [87] [88] , indicating that a very significant part of ctd gains structure during dimerization. we identified numerous rna binding residues in all three viruses using pprint server. this finding supports the function of n protein as it interacts with genomic rna for a ribonucleoprotein core formation which is crucial step for rna encapsidation during viral particle assembly. in addition, drnapred and disordpbind predicts multiple dna binding residues for n protein in sars-cov-2, human sars, and bat cov. the long flexible (idprs) regions at n and c-terminus of sars-cov-2 have long protein-binding as well as nucleotide-binding regions that may have important role in its interaction with viral rna. these flexible regions can be targeted to inhibit interaction of n protein with viral genomic rna. literature suggests that some viral proteins are translated from the genes interspersed in between the genes of structural proteins. these proteins are known as accessory proteins, and many of them are proposed to be involved in viral pathogenesis [89] . proteins orf3a and orf3b. orf3a is a multifunctional protein with the molecular weight of ~31 kda that has been found to localize in different organelles inside the host cells. also referred to as u274, x1, and orf3, the gene for this protein is present between the s and e genes of the sars-cov genome [90] [91] [92] . the homo-tetrameric complex of orf3a has been demonstrated to form a potassium-ion channel on the host cell plasma membrane [93] . it performs a major function during virion assembly by co-localizing with e, m, and s viral proteins [94, 95] . orf3b protein can be found in the cytoplasm, nucleolus, and outer membrane of mitochondria of the host cells [96, 97] . in huh 7 cells, its over-expression has been linked with the activation of ap-1 via erk and jnk pathways [98] . transfection of orf3b-egfp leads to cell growth arrest at the g0/g1 phase of vero, 293, and cos-7 cells [99] . orf3a induces apoptosis via caspase 8/9 directed mitochondrial-mediated pathways, while orf3b is reported to affect only the caspase 3-related pathways [100, 101] . on performing msa, results of which are shown in figure 7d , we found that orf3a protein from sars-cov-2 is slightly evolutionary closer to the orf3a of bat cov (73.36%) than to the orf3a of human sars cov (72.99%). graphs in figures 7a, 7b , and 7c depict the propensity for disorder in orf3a proteins of novel sars-cov-2, human sars cov, and bat cov (sars-like), respectively. mean ppids in these orf3a proteins are 9.1% (sars-cov-2), 8.8% (human sars), and 6.2% (bat cov (sars-like)). orf3a of sars cov-2 shows protein-binding regions at its n-terminus (by morfchibi_web (residues 1-6), morfpred (residues 7-12), and disopred3 (residues [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] ) and at c-terminus (by morfchibi_web (residues 261-268) and morfpred (residues 259-263)) ( table 2) . similarly, orf3a of human sars and bat cov also shows morfs at n-and c-terminus with the help of morfchibi_web and morfpred (supplementary tables 7 and 8 ). these protein-binding regions in orf3a may have role in its co-localization with e, m, and s viral proteins. apart from morfs, it also displays several nucleotide-binding residues in all three viruses (see supplementary tables 9, 10 , and 11). in fact, this represents maximum number of rna and dna binding residues as compared with all other accessory proteins. these results indicate that the idps/idprs of this protein could be utilized in molecular recognition (protein-protein, protein-rna, and protein-dna interaction). according to the intrinsic disorder predisposition analysis of orf3b proteins, their mean ppid values in sars-cov-2, human sars cov, and bat cov are 0%, 7.1%, and 23.1% respectively, as represented in figures 8a, 8b , and 8c. msa results ( figure 8d ) demonstrate that orf3b of sars-cov-2 is not closer to orf3b protein of human sars and orf3b protein of bat-cov, having a sequence similarity of only 54.6% and 59.1%, respectively. as we can see in table 2 , there is not a single morf found in orf3b of sars-cov-2. however, for human sars we identified three morfs (residues 32-37, 41-70, and 125-153) and for bat cov one morf at n-terminus (residues 1-38) by morfchibi_web server. protein orf6. orf6 is a short coronavirus protein with just 63 residues. also known as p6, this membrane-associated protein serves as an interferon (ifn) antagonist [102] . it downregulates the ifn pathway by blocking a nuclear import protein, karyopherin α2. using its c-terminal residues, orf6 disrupts karyopherin import complex in the cytosol and, therefore, hampers the movement of transcription factors like stat1 into the nucleus [102, 103] . it contains a ysel motif near its c-terminal region, which functions in protein internalization from the plasma membrane into the endosomal vesicles [104] . another study has also demonstrated the presence of orf6 in endosomal/lysosomal compartments [104, 105] . msa results demonstrate that (figure 9d) , sars-cov-2 orf6 is closer to orf6 protein of human sars cov, having a sequence similarity of 68.85% than to the orf6 of bat cov (sars-like) (67.21%). novel sars-cov-2 orf6 is predicted to be the second most disordered structural protein, with ppid of 22.95%, and with especially disordered cterminal region. our analysis of the intrinsic disorder predisposition using six predictors revealed the mean ppid in orf6 proteins of sars-cov-2, human sars, and bat cov to be 22.95%, 20.63%, and 20.63%, respectively ( table 1) . graphs in figures 9a, 9b and 9c illustrate that orf6 proteins from all three studied coronaviruses are expected to be moderately disordered proteins with the high disorder content in their c-terminal regions. these disordered regions are important for the biological activities of orf6. as aforementioned, this hydrophilic region contains lysosomal targeting motif (ysel) and diacidic motif (ddee) responsible for binding and recognition during translocation [104] . however, the n-terminal region does not contain a noticeable disorder. the 1-38 residues of the n-terminal region of human sars cov orf6 was shown to be α-helical and embedded in the membrane, although orf6 is not a transmembrane protein [106] . a long morf region ((residues 26-61 in sars-cov-2), (residues 31-63 in human sars), and (residues 30-60 in bat cov)) is also present at cterminus of orf6 proteins which are tabulated in table 2 , and supplementary tables 7 and 8. no predictor other than morfchibi_web has located morfs in this protein. supplementary table 9 , 10, and 11 shows nucleotide-binding residues in orf6 of all three viruses. it represents very few rna binding residues by pprint and few dna binding residues by drnapred. orf7a and orf7b proteins. alternatively called u122, orf7a is a type i transmembrane protein [107, 108] . it has been proven to localize in er, golgi, and peri-nuclear space. the presence of a krkte motif near the c-terminal region is needed for importing this protein from the er to the golgi apparatus [107, 108] . orf7a contributes to viral pathogenesis by activating the release of pro-inflammatory cytokines and chemokines, such as il-8 and rantes [109, 110] . in another study, overexpression of bcl-xl in 293t cells blocked the orf7a mediated apoptosis [111] . on the other hand, orf7b is an integral membrane protein that has been shown to localize in the golgi complex [112, 113] . the same reports also confirm the role of orf7b as an accessory as well as a structural protein in sars-cov virion [112, 113] . figure 10d represents the 1.8 å x-ray crystal structure of the 14-96 fragment of the orf7a from human sars cov (pdb id: 1xak) and demonstrates the compact seven-stranded topology of this protein, which is similar to that of the ig-superfamily members [114] . importantly, in this crystal structure, residues 82-96 constituted the region with missing electron density, indicating high structural flexibility of this segment. in line with this hypothesis, the nmr solution structure of the 16-99 fragment of the orf7a from human sars cov (pdb id: 1yo4) showed that residues 81-99 are highly disordered [115] . at the domain level, the structure of the orf7a protein includes a signal peptide, a luminal domain, a transmembrane domain, and a short cytoplasmic tail at the c-terminus [95, 114] . we found that 121-residue-long orf7a protein of sars-cov-2 shares 89.26% and 85.95% sequence identity with orf7a proteins of bat cov and human sars cov, respectively ( figure 10e) . on the other hand, the orf7b of sars-cov-2 is found to be closer to orf7b of human sars than to orf7b of bat cov, showing sequence identities of 81.40% and 79.07%, respectively (see figure 11d ). as can be observed from table 1 , our disorder predisposition analyses resulted in the overall ppid for orf7a proteins of 1.65% for sars-cov-2, 0.82% for bat cov and 0.82% for human sars cov. mean ppids estimated for orf7b proteins are 9.30% for sars-cov-2, 4.55% for bat cov and 4.55% human sars cov. figures 10a, 10b , and 10c represent the residues predisposed for disorder in orf7a proteins of sars-cov-2, human sars cov, and bat cov, respectively. table 2 shows that orf7a protein is expected to have several morfs indicating the potential involvement of this protein in disorder-dependent proteinprotein interactions. at the n-terminus, we observed one morf region (residues 1-10) with the help of disopred3 in all three viruses. in addition to protein binding regions, orf7a also contains several rna and dna binding residues. analysis also represents, orf7b proteins from all three viruses have low disorder content, likewise, they are not predicted to contain any morf by any of the predictors used in this study ( table 2, supplementary table 7 and 8). although the orf7b does not contain protein-binding regions, it was found to contain nucleotide (rns and dna) binding regions in the protein figures 11a, 11b , and 11c depict the residues predisposed for disorder in orf7b proteins of sars-cov-2, human sars cov, and bat cov, respectively. according to our analysis, both proteins in all three studied coronaviruses have a mostly ordered structure. proteins orf8a and orf8b. in animals and isolates from early human infections, the orf8 gene codes for a single orf8 protein. however, in late infections, more specifically, at middle and late stages, a 29 nucleotide deletion in the orf8 gene led to the formation of two distinct proteins, orf8a and orf8b containing 39 and 84 residues respectively [116, 117] . both proteins have conformations different from that of the longer orf8 protein. it has been reported that overexpression of orf8b resulted in the downregulation of e protein while the proteins orf8a and orf8/orf8ab have no effect on the expression of protein e. also, orf8/orf8ab was found to interact very strongly with proteins s, orf3a, and orf7a. orf8a interacts with s and e proteins, whereas orf8b protein interacts with e, m, orf3a and orf7a proteins [118] . the disorder-based protein binding regions of this protein identified in this study may have important role in interaction with other proteins. orf8 protein found in early sars-cov-2 isolates having 121 residues and according to our analysis, it shares a 90.05% sequence identity with orf8 protein of bat cov ( figure 12c) . furthermore, figures 12a and 12b show that there is no intrinsic disorder in both orf8 proteins from sars-cov-2 and bat cov. therefore, these two proteins predicted to be completely structured having a mean ppid of 0.00%. in orf8a and orf8b proteins of the human sars, the predicted disorder is estimated to be 2.56% and 2.38%, respectively (table 1) . graphs in figures 13a and 13b illustrate the presence of some disorder near the n-and c-terminals of orf8a and orf8b proteins. table 2 shows the identified morf regions in orf8 of sars-cov-2. it shows three morf regions (residues 1-5, 26-52, and 69-91) by morfchibi_web and one morf region (residues 1-10) by disopred3. in human sars, the n-terminus of both orf8a (residues 1-39) and orf8b (residues 1-83) was found to be morf by morfchibi_web server (supplementary table 7) . further, four proteinbinding regions (residues 26-53, 70-91, 98-104, and 113-130) were identified by morfchibi_web server in bat cov (supplementary table 8 ). apart from protein-binding, orf8 of sars-cov-2, orf8a and orf8b of human sars, and orf8 of bat cov also comprise several nucleotide-binding residues (see supplementary table 9 , 10, and 11). this protein is expressed from an alternative orf within the n gene through a leaky ribosome binding process [119] . inside the host cells, orf9b enters the nucleus, which is a cell cycle-independent process and represents a passive entry. this protein was shown to interact with a nuclear export protein receptor exportin 1 (crm1), using which it translocate out of the nucleus [120] . our morfs analysis shows the presence of disorderbased protein binding regions in orf9b protein which may have role in its interaction with crm1 and further translocation outside the nucleus. a 2.8 å resolution crystal structure of orf9b protein from human sars cov (pdb id: 2cme) shows the presence of a dimeric tent-like -structure along with the central hydrophobic amino acids ( figure 14d) . the published structure has the highly polarized distribution of charges, with positively charged residues on the one side of the tent and negatively charged on the other [121] . based on the sequence availability of accession id nc_045512.2, the translated protein sequence of orf9b is not reported for the sars-cov-2 as of yet. however, based on the report by wu and colleagues [45] , the sequences of the sars-cov-2 are already annotated. therefore, we took the corresponding amino acid sequences from that study and conducted the intrinsic disorder analysis. according to the msa, results shown in figure 14e , orf9b protein from sars-cov-2 shares 73.2% identity with human sars and 74.23% identity with bat cov. our idp analysis ( table 1) shows that orf9b from human sars is a moderately unstructured protein with a mean ppid estimated to 26.53%. as depicted in figure 14a , 14b, and 14c, disorder mainly lies near the n-terminal end 1-10 residues and 28-40 residues near the central region with a well-ordered inner core of human sars orf9b protein. the x-ray crystal structure of orf9b has a missing electron density of the first 8 residues and 26-37 residues near the central region. this indicates that the corresponding regions are disordered, which are difficult to crystallize due to their highly dynamic structural organization. sars-cov-2 orf9b protein with a mean ppid of 10.31% has an n-terminal (1-10 residues) predicted disordered segment. orf9b of bat cov is shown to have an intrinsic disorder content of 9.28%, comparatively lower than that of the human sars orf9b protein. morfs lies in the n-terminal region of orf9b proteins ( table 2, supplementary table 7 and 8). in the absence of other viral proteins, its first 41 residues have been demonstrated to induce membranous structures similar to dmvs [106] . the available crystal structure also has the missing electron density in the n-terminal region suggests that these flexible amino acids are likely to interact with host lipids. the first 3-29 residues of sars-cov2 are identified as disorder-based protein binding region that may have role in its interaction with host lipids and formation of dmvs. supplementary tables 9, 10, and 11 represents nucleotide-binding residues for orf9b of sars-cov-2, human sars, and bat cov. the newly emerged sars-cov-2 has an orf10 protein of 38 amino acids. orf10 of sars-cov-2 has a 100% sequence similarity with orf10 of bat cov strain bat-sl-covzc45 [21] . however, we did not conduct the disorder analysis for orf10 from the bat-sl-covzc45 strain, since all our studies reported here are related to a different strain of bat cov (reviewed strain hku3-1). therefore, we report here only the results of disorder analysis for the orf10 protein from sars-cov-2, according to which this protein has a mean ppid of 0.00% (see also figure 15 for disorder profile of orf10). this protein contains a morf from 3-7 residues at its n-terminus as predicted by morfchibi_web. further, we found its tendency to nucleotides and found the presence of few rna binding sites, however, it does not contain dna binding residues. protein orf14. this is a 70-amino-acid-long uncharacterized protein of unknown function, which is present in human sars and bat cov. in sars-cov-2, orf14 is a 73-amino-acidlong protein. according to the msa, orf14 of sars-cov-2 has 77.1% identity with human-sars and 72.9% identity with bat cov as represented in figure 16d . we have performed the intrinsic disorder analysis to see the peculiarities of the distribution of disorder predisposition in this protein. figures 16a, 16b, and 16c show the resulting disorder profiles of orf14 of sars-cov-2, human sars cov, and bat cov. although these proteins have calculated mean ppid values of 0.00%, 2.86%, and 0.00% respectively, figure 16 shows that they have flexible n-and c-terminal regions. this protein can use intrinsic disorder or structural flexibility for protein-protein interactions since it possesses morfs. it mainly contains morfs at n-and c-terminal regions as tabulated in (table 2, supplementary table 7 and 8). it was also found to contain several rna and dna binding residues (supplementary table 9, 10, and 11) . these results indicating its vital role in protein function related to molecular recognition such as protein-protein, protein-rna, and protein-dna interaction. in coronaviruses, due to ribosomal leakage during translation, two-third of the rna genome is processed into two polyproteins: (i) replicase polyprotein 1a and (ii) replicase polyprotein 1ab. both contain non-structural proteins (nsp1-10) in addition to different proteins required for viral replication and pathogenesis. replicase polyprotein 1a contains an additional nsp11 protein of 13 amino acids, the function of which is not investigated yet. the longer replicase polyprotein 1ab of 7073 amino acids accommodates five other non-structural proteins (nsp12-16) [122] . these proteins assist in er membrane-induced vesicle formation, which acts as sites for replication and transcription. in addition to this, non-structural proteins work as proteases, helicases, and mrna capping and methylation enzymes, crucial for virus survival and replication inside host cells [122, 123] . global analysis of intrinsic disorder in the replicase polyprotein 1ab table 3 represents the ppid mean scores of 15 non-structural proteins (nsps) derived from the replicase polyprotein 1ab in sars-cov-2, human sars cov, and bat cov. these values were obtained by combining the results from six disorder predictors (see supplementary table s4-s6) . figures 17a, 17b , and 17c represent the 2d-disorder plots of the nsps coded by orf1ab in sars-cov-2, human sars cov, and bat cov, respectively. based on the mean ppid scores in table 3 , figures 17a, 17b, 17c , and taking into ppid based classification [46] , we conclude that none of the nsps in sars-cov-2, human sars cov, and bat cov are highly disordered. the highest disorder was observed for nsp8 proteins in all three coronaviruses. both nsp1 and nsp8 are moderately disordered proteins (10% ≤ ppid ≤ 30%). we also observed that nsp2, nsp3, nsp5, nsp6, nsp7, nsp9, nsp10, nsp15, and nsp16 have less than 10% disordered residues and hence, belong to the category of mostly ordered proteins. other non-structural proteins, namely, nsp4, nsp12, nsp13, and nsp14 have negligible levels of disorder (ppid < 1%), which tells us that these are highly structured proteins. the ch-cdf analysis of the nsps from sars-cov-2, human sars and bat cov have been represented in figures 17d, 17e , and 17f respectively. it was observed that all the nsps of the three coronaviruses are located within the quadrant q1 of the ch-cdf phase space, indicating that all the nsps are predicted to be mostly ordered. replicase polyprotein 1ab. the longer replicase polyprotein 1ab is a 7,073 amino acid-long polypeptide, which contains 15 non-structural proteins listed in table 3 . nsp1, nsp2, and nsp3 are cleaved using a viral papain-like proteinase (nsp3/pl-pro), while the rest of nsps are cleaved by another viral 3c-like proteinase, nsp5/3cl-pro. we mapped the cleavage sites of the replicase 1ab polyprotein from human sars cov to the disorder profile of this polyprotein. figure 18 represents the results of this analysis by showing zoomed-in regions surrounding all the cleavage sites with few residues spanning at both terminals. interestingly, we observed that all the cleavage sites are largely disordered, suggesting that intrinsic disorder may have a crucial role in the maturation of individual non-structural proteins. as the nsps of human sars cov are evolutionary close to the nsps of sars-cov-2, we hypothesize that the cleavage sites in the sars-cov-2 replicase 1ab polyprotein are also intrinsically disordered or flexible. to shed more light on other implications of idprs, the structural and functional properties of nsps and their predicted idprs are thoroughly described below. this protein acts as a host translation inhibitor as it binds to the 40s subunit of the ribosome and blocks the translation of cap-dependent mrnas as well as mrnas that uses the internal ribosome entry site (ires) [124] . figure 19d shows the nmr solution structure (pdb id: 2gdt) of human sars nsp1 protein (13-128 residues), whereas residues 117-180 were not included in this structural analysis [125] . sars-cov-2 nsp1 shares 84.44% and 83.80% sequence identity with nsp1s of human sars cov and bat cov, respectively. its n-terminal region is found to be more conserved than the rest of the protein sequence ( figure 19e ). mean ppids of nsp1s from sars-cov-2, human sars cov, and bat cov are 12.78%, 14.44%, and 12.85%, respectively. figure 19a , 19b, and 19c represent the graphs of predicted per-residue intrinsic disorder propensity of these nsp1s. according to the analysis, the following regions are predicted to be disordered: sars-cov-2 (residues 1-7 and 165-180), human sars cov (residues 1-5 and 165-180), and bat cov (residues 1-5 and 165-179). nmr solution structure of nsp1 from human sars revealed the presence of two unstructured segments near the n-terminal (1-12 residues) and c-terminal (129-179 residues) regions [125] . the disordered region residues) at c-terminus is important for nsp1 expression [126] . based on sequence homology with human sars cov nsp1, the predicted disordered c-terminal region of sars-cov-2 nsp1 may play a critical role in its expression. alanine mutants at k164 and h165 in the cterminal region of nsp1 protein is reported to abolish its binding with the 40s subunit of the host ribosome [127] . in conjunction with this data, several morfs are present in the unstructured segments of nsp1 proteins. these regions are tabulated in table 2, and supplementary tables 7 and 8 . this protein functions by disrupting the host survival pathway via interaction with the host proteins prohibitin-1 and prohibitin-2 [128] . reverse genetic deletion in the coding sequence of nsp2 of the sars virus attenuated little viral growth and replication and allowed the recovery of mutant virulent viruses. this indicates the dispensable nature of the nsp2 protein for sars viruses [129] . the sequence identity of the nsp2 protein from sars-cov-2 with nsp2s of human sars cov and bat cov amounts to 68.34% and 68.97%, respectively (supplementary figure s2a) . we have estimated the mean ppids of nsp2s of sars-cov-2, human sars cov, and bat cov to be 5.17%, 2.04%, and 2.03% respectively (see table 3 ). the per-residues predisposition for the intrinsic disorder of nsp2s from sars-cov-2, human sars cov, and bat cov are depicted in figures 20a, 20b , and 20c. according to this analysis, the following regions in nsp2 proteins are predicted to be disordered, residues 570-595 (sars-cov-2), residues 110-115 (human sars), and residues 112-116 (bat cov). as listed in table 2 , and supplementary tables 7 and 8, human sars cov does not contain morf while sars-cov-2 and bat cov have an n-terminally located morf region predicted by morfchibi_web. nsp3 is an almost 2,000-residue-long viral papain-like protease (plp) that affects the phosphorylation and activation of irf3 and therefore antagonizes the ifn pathway [130] . it was also demonstrated that nsp3 works by stabilizing nf-inhibitor further blocking the nf-pathway [130] . figure 21d represents the 1.85 å resolution x-ray crystal structure of the catalytic core of nsp3 protein from human sars cov (pdb id: 2fe8), which was obtained by andrew and colleagues [131] . this structure consists of the residues 723-1036 of nsp3. the structure revealed folds similar to a deubiquitinating enzyme in-vitro deubiquitinating activity of which was found to be efficiently high [131] . nsp3 protein of sars-cov-2 contains several substituted residues throughout the protein. it is equally close with both nsp3 proteins of human sars and bat cov sharing respective 76.69% and 76.31% identity (supplementary figure s2b) . according to our results, the mean ppids of nsp3 proteins of sars-cov-2, human sars, and bat cov are 7.40%, 7.91%, and 7.78% respectively ( table 3) . graphs in figures 21a, 21b , and 21c portray the tendency of nsp3 proteins of sars-cov-2, human sars, and bat cov for the intrinsic disorder. nsp3 proteins of all three studied sars viruses were found to be highly structured and characterized by rather similar disorder profiles. this is further supported by figure 21e , where pondr ® vsl2-generated disorder profiles of these three proteins are overlapped to show almost complete coincidence of their major disorder-related features. according to the mean disorder analysis (see figures 21a, 21b, and 21c) , nsp3 proteins are predicted to have the following idprs, sars-cov-2 (1-5, 105-199, 1221-1238), human sars (102-189, 355-384, 1195-1223) and bat cov (107-182, 352-376, 1191-1217) . the first 112 residues in nsp3 represent a ubiquitin-like globular fold while 113-183 residues form the flexible acidic domain rich in glutamic acid. it is thought to bind and ubiquitinate viral e protein using the n-terminal acidic domain [132, 133] . this unstructured segment has many morfs predicted by anchor and morfpred servers which may facilitate the protein-protein interaction ( table 2) . interestingly, nsp3 of all three viruses was found with highest number of rnabinding residues (supplementary tables 9, 10, and 11) . nsp4 has been reported to induce the formation of the double-membrane vesicles (dmvs) with the co-expression of full-length nsp3 and nsp6 proteins for optimal replication inside host cells [134] [135] [136] . it localizes itself in ermembrane, when expressed alone but is demonstrated to be present in replication units in infected cells. it was observed that nsp4 protein contains a tetraspanning transmembrane region having its n-and c-terminals in the cytosol [137] . no crystal or nmr solution structure is reported for this protein as of yet. nsp4 protein of sars-cov-2 has multiple substitutions near the n-terminal region and has a quite conserved c-terminus (supplementary figure s2c) . it is found to be closer to nsp4 of bat cov (81.40% identity) than to human sars nsp4 (80%). mean ppids of nsp4s from sars-cov-2, human sars, and bat cov are estimated to be 0.80%, 0.60%, and 0.60% respectively. the low level of intrinsic disorder is further illustrated by figures 22a, 22b , and 22c. with ppids around zero, nsp4 were classified as highly structured proteins, which, however, contain some flexible regions. likewise, table 2 shows the presence of only nand c-terminal morfs which possibly assist in cleavage of nsp4 protein from long polyproteins 1a and 1ab. also referred to as 3cl-pro, nsp5 works as a protease that cleaves the replicase polyproteins (1a and 1ab) at 11 major sites [138, 139] . x-ray crystal structure with 1.5 å resolution (pdb id: 5c5o) obtained for human sars cov nsp5 is shown in figure 23d . here, 3cl-protease is bound to a phenyl-beta-alanyl (s, r)-n-declin type inhibitor. another crystal structure resolved to 1.96 å revealed a chymotrypsin-like fold and a conserved substrate-binding site connected to a novel α-helical fold [140] . recently, the x-ray crystal structure (resolution 2.16 å) was solved for the sars-cov-2 nsp5 in complex with an inhibitor n3 (pdb id: 6lu7) ( figure 23e ). nsp5 protein is found to be highly conserved in all three studied cov viruses. sars-cov-2 nsp5 shares a 96.08% sequence identity with human sars nsp5 and 95.42% with nsp5 of bat cov (supplementary figure s2d) . therefore, it not surprising that our analysis demonstrated the identical mean ppid values of 1.96% for nsp5s from sars-cov-2, human sars, and bat cov ( table 3) . the predicted per-residue intrinsic disorder propensity of sars-cov-2, human sars, and bat cov nsp5s are presented in figures 23a, 23b , and 23c, respectively. as the graphs depict, nsp5s have several flexible regions and n-terminally idpr of six residues. due to the low flexibility of this protein, a single morf predicted by morfchibi_web is present in the n-terminal region (residues 3-8) in nsp5s of all three viruses (table 2, supplementary tables 7 and 8) . further, the identified nucleotide-binding residues in nsp5 of all three viruses are tabulated in supplementary tables 9, 10, and 11 . non-structural protein 6 (nsp6). nsp6 protein is involved in blocking er-induced autophagosome/autolysosome vesicle formation that functions in restricting viral production inside host cells. it induces autophagy by activating the omegasome pathway, which is normally utilized by cells in response to starvation. sars nsp6 leads to the generation of small autophagosome vesicles thereby limiting their expansion [141] . nsp6 of sars-cov-2 is equally close to nsp6s from both human sars and bat cov, having a sequence identity of 87.24% ( figure 24d ). according to our analysis, mean ppids for nsp6s are calculated to be 1.03%, 1.03%, and 1.03% for sars-cov-2, human sars cov, and bat cov, respectively. figures 24a, 24b, and 24c show the corresponding graphs of intrinsic disorder tendency of nsp6s from sars-cov-2, human sars cov, and bat cov and demonstrate that these proteins are highly ordered and show low flexibility. as it is a membrane protein, nsp6 proteins are predicted to have only a single morf near the nterminal region (residues 1-19 in sars-cov-2, residues 1-22 in human sars, and residues 1-21 in bat cov) by the disopred3 server (table 2, supplementary tables 7 and 8) . the role of these protein-binding regions for the induction of autophagy is need to be elucidated. nsp7 and 8) . the ~10 kda nsp7 helps in primaseindependent de novo initiation of viral rna replication by forming a hexadecameric ring-like structure with nsp8 protein [142, 143] . both non-structural proteins 7 and 8 contribute 8 molecules to the ring-structured multimeric viral rna polymerase. site-directed mutagenesis in nsp8 revealed a d/exd/e motif essential for the in vitro catalysis [142] . figure 25d depicts the 3.1 å resolution electron microscopy-based structure (pdb id: 6nur) of the rdrp-nsp8-nsp7 complex bound to the nsp12. the structure identified conserved neutral nsp7 and nsp8 binding sites overlapping with finger and thumb domains on nsp12 of the virus [144] . we found that nsp7 of sars-cov-2 share 100% sequence identity with nsp7 of bat cov and 98.80% with nsp7 from human sars (figure 25e) , while sars-cov-2 nsp8 is closer to nsp8 of human sars (97.47%) than to nsp8 of bat cov (96.46%) ( figure 26d ). due to the high levels of sequence identity, mean ppids of all nsp7s were found to be identical and equal to 9.64%. both sars-cov-2 and human sars nsp8 proteins were calculated to have a mean ppid of 23.74% and, for nsp8 of bat cov mean disorder is predicted to be 22.22%. figures 25a, 25b , and 25c display the intrinsic disorder profiles for nsp7s, whereas figures 26a, 26b , and 26c represent the predicted intrinsic disorder propensity of nsp8s. as our analysis suggests, nsp7s might have a well-predicted structure, while nsp8s are moderately disordered. nsp8s are predicted to have a long idpr (residues 44-84) in both sars-cov-2 and human sars, and a bit shorter idpr in bat cov (residues . furthermore, sars-cov nsp7 using its n-terminus residues (v11, c13, v17, and v21) forms a hydrophobic core with nsp8 residues (m92, m95, l96, m99, and l103). additionally, h-bonding takes place between nsp7 q24 and nsp8 t89 residues [143] . these amino acids are the part of morfs predicted in nsp7 and nsp8 proteins. the results are tabulated in both supplementary tables 9, 10 , and 11). nsp9 protein is a single-stranded rna-binding protein [145] . it might protect rna from nucleases by binding and stabilizing viral nucleic acids during replication or transcription [145] . our results on nucleotide-binding tendency of nsp9 shows the presence of several rna binding and few dna binding residues in nsp9 of sars-cov-2, human sars, and bat cov (supplementary tables 9, 10, and 11) . presumed to evolve from a protease, nsp9 forms a dimer using its gxxxg motif [146, 147] . figure 27d shows a 2.7 å crystal structure of the homodimer of human sars nsp9 (pdb id: 1qz8) that identified a unique and previously unreported for other proteins, oligosaccharide/oligonucleotide fold-like fold [145] . here, each monomer contains a coneshaped β-barrel and a c-terminal α-helix arranged into a compact domain [145] . nsp9 of sars-cov-2 is equally similar to nsp9s from both human sars and bat cov, having a percentage identity of 97.35%. the difference in three amino acids at 34, 35 and 48 positions accounts for these similarity scores ( figure 27e ). as calculated, the mean ppids of nsp9s of sars-cov-2, human sars cov, and bat cov are 7.08%, 7.96%, and 7.08% respectively. figures 27a, 27b , and 27c depict the predicted intrinsic disorder propensity in the nsp9 protein from sars-cov-2, human sars, and bat cov. according to our analysis, all three nsp9s are rather structured but contain flexible regions. nsp9 contains conserved residues (r10, k52, y53, r55, r74, f75, k86, y87, f90, k92, r99, and r111) of positively charged side chains suitable for binding with the negatively charged phosphate backbone of rna and aromatic side-chain amino acids providing stacking interactions [145] . these residues are a part of multiple disorder-based binding sites predicted by morfchibi_web server ( table 2, supplementary table 7 and 8) . nsp10 performs several functions for sars-cov. it forms a complex with nsp14 for dsrna hydrolysis in 3′ to 5′ direction and activates its exonuclease activity [148] . it also stimulates the methyltransferase (mtase) activity of nsp14 required during rna-cap formation after replication [149] . figure 28d represents the x-ray crystal structure of the nsp10/nsp14 complex (pdb id: 5c8t) [150] . in agreement with the results of previous biochemical experimental studies, the structure identified important interactions with the exon (exonuclease domain) of nsp14 without affecting its n7-mtase activity [148, 149] . sars-cov-2 nsp10 protein is quite conserved having a 97.12% sequence identity with nsp10 of human sars and 97.84% with nsp10 of bat cov (figure 28e) . mean ppids of all three studied nsp10 proteins are found to be 5.04%. figures 28a, 28b , and 28c represent disorder profiles of nsp10s and signify the lack of long idprs but presence flexible regions in these proteins. furthermore, [11] [12] [13] [14] [15] [16] [17] [18] was predicted by morfpred server. interestingly, the sars-cov nsp10 residues f16, f19, and v21 form van der waals interactions with many of the nsp14 amino acids [150] and one residue (f16) is located in morf region which we have identified. furthermore, many nucleotide-binding residues which are found in all three viruses (supplementary table 9 , 10, and 11) and above-mentioned residues are not found to interact with dna/rna. in coronaviruses, nsp12 is an rna-dependent rna polymerase (rdrp). it carries out both primer-independent and primer-dependent synthesis of viral rna with mn 2+ as its metallic co-factor and viral nsp7 and 8 as protein co-factors [151] . as aforementioned, a 3.1å resolution structure of human sars nsp12 in association with nsp7 and nsp8 proteins (pdb id: 6nur) has been reported using electron microscopy ( figure 25d ). nsp12 has a polymerase domain similar to "right hand", finger domain (398-581, 628-687 residues), palm domain (582-627, 688-815 residues) and a thumb domain (816-919) [144] . sars-cov-2 nsp12 protein has a highly conserved c-terminal region (supplementary figure s2e ). it is found to share a 96.35% sequence identity with human sars nsp12 and 95.60% with bat cov nsp12. mean ppid values for all three nsp12s are estimated to be 0.43% (table 3) . figures 29a, 29b , and 29c show that although these proteins are mostly ordered, they have multiple flexible regions. as rdrp protein is observed to be mostly structured, significant morfs in disordered regions are not found ( table 2, supplementary table 7 and 8). nsp13 functions as a viral helicase and unwinds dsdna/dsrna in 5' to 3' direction [152] . recombinant viral helicase expressed in e.coli rosetta 2 strain was reported to unwind ~280 bp per second [152] . figure 30d represents a 2.8 å x-ray crystal structure of human sars nsp13 (pdb id: 6jyt) [153] . this helicase contains a 19-20 loop on 1a domain, which is primarily responsible for its unwinding activity. furthermore, the study revealed an important interaction of nsp12 with nsp13 that further enhances its helicase activity [153] . the 601-amino-acid-long nsp13 of sars-cov-2 is almost completely conserved, as it shares 99.83% with nsp13 of humans sars and 98.84% with nsp13 of bat cov (supplementary figure s2f) . in accordance with our results, the mean ppids of all three nsp13 proteins are estimated to be 0.67%. figures 30a, 30b , and 30c show that nsp13s contain multiple flexible regions but do not possess significant disorder. as expected, being a low disorder protein nsp13 does not contain any morf region and not a single bindingregion is located by any server used in all three viruses ( table 2, supplementary table 7 and 8). it has many nucleotide-binding residues (rna and dna) which are tabulated in supplementary tables 9, 10, and 11. nsp14 is a multifunctional viral protein that acts as an exoribonuclease (exon) and methyltransferase (n7-mtase) in sars coronaviruses. it's 3' to 5' exonuclease activity lies in the conserved dedd residues related to exonuclease superfamily [154] . its guanine-n7 methyltransferase activity depends upon the s-adenosyl-lmethionine (adomet) as a cofactor [149] . as aforementioned, nsp14 requires nsp10 for activating its exon and n7-mtase activity inside the host cells. figure 28d depicts the 3.2 å crystal structure of human sars nsp10/nsp14 complex (pdb id: 5c8t), where amino acids 1-287 form the exon domain and 288-527 residues form the n7-mtase domain of nsp14. a loop (residues 288-301) is essential for its n7-mtase activity [150] . figure s2g) . mean ppid values for nsp14s from sars-cov-2 and human sars is calculated to be 0.38%, while the nsp14 from bat cov has a mean ppid 0.57%. predicted per-residue intrinsic disorder propensity of nsp14s from sars-cov-2, human sars, and bat cov is represented in figures 31a, 31b , and 31c, respectively. as can be observed from these plots and corresponding ppid values, all nsp14s are found to be highly structured. likewise, table 2 shows nsp14 contains two protein binding regions (residues 8-13 and 441-445) predicted by the morfpred server in all three viruses. as shown in supplementary tables 9, 10, and 11, the orf14 represents multiple nucleotide-binding residues. nsp15 is a uridylate-specific rna endonuclease (nendou) that creates a 2′-3′ cyclic phosphates after cleavage. its endonuclease activity depends upon mn 2+ ions as co-factors. conserved in nidovirus, it acts as an important genetic marker due to its absence in other rna viruses [155] . figure 32d represents a 2.6 å crystal structure of uridylate-specific nsp15 (pdb id: 2h85) that was deduced by bruno and colleagues using x-ray diffraction [156] . the monomeric nsp15 has three domains: nterminal domain (1-62 residues) formed by a three anti-parallel -strands and two α-helices packed together; a middle domain residues) that contains an α-helix connected via a 39-amino-acid-long coil to an ordered region containing two α-helices and five -strands; and a c-terminal domain (192-345 residues) consisting of two anti-parallel three -strand sheets on each side of a central α-helical core [156] . the nsp15 is found to be quite conserved across human sars and bat covs. sars-cov-2 nsp15 shares an 88.73% sequence identity with nsp15 of human sars and 88.15% with nsp15 of bat cov (supplementary figure s2h) . calculated mean ppids of nsp15s from sars-cov-2, human sars, and bat cov are 1.73%, 2.60%, and 2.60%, respectively. similar to many other non-structural proteins of coronaviruses, nsp15s from sars-cov-2, human sars, and bat cov are predicted to possess multiple flexible regions but contain virtually no idprs (see figures 32a, 32b, and 32c) . similarly, no significant disorderbinding regions are predicted in nsp15 proteins ( table 2) . sars-cov-2 contain one morf (residues 9-13) predicted by morfpred server. human sars do not have a single morf while bat cov possesses two very short binding regions (supplementary table 7 and 8). supplementary table 9 , 10, and 11 depicts the presence of many rna binding residues and few dna binding residues in nsp15 of all three viruses. nsp16 protein is another mtase domain-containing protein. as methylation of coronavirus mrnas occurs in steps, three proteins nsp10, nsp14, and nsp16 acts one after another. the first event requires the initiation trigger from nsp10 protein, after which nsp14 methylates capped mrnas forming cap-0 (7me) gpppa-rnas. nsp16 protein, along with its co-activator protein nsp10, acts on cap-0 (7me) gpppa-rnas to give rise to final cap-1 (7me)gpppa(2'ome)-rnas [149, 157] . a 2 å x-ray crystal structure of the human sars nsp10-nsp16 complex is depicted in figure 33d (pdb id: 3r24) [158] . the structure consists of a characteristic fold present in class i mtase family comprising of α-helices and loops surrounding a seven-stranded β-sheet [158] . nsp16 protein of sars-cov-2 is found to be equally similar to nsp16s from human sars and bat cov (93.29%) (supplementary figure s2i) . mean ppids for nsp16s from sars-cov-2, human sars, and bat cov are 5.37%, 3.02%, and 3.02%, respectively. in line with these ppids values, figures 33a, 33b, and 33c show that nsp16s are mostly ordered proteins containing several flexible regions. correspondingly, no significant morfs are present in this protein ( table 2, supplementary table 7 and 8). a single morf (residues [151] [152] [153] [154] [155] [156] were found with the help of morfpred in all three viruses. further, several rnabinding and few dna-binding residues are also identified (supplementary table 9 , 10, and 11). replicase polyprotein 1a. since replicase polyprotein 1a contains non-structural proteins 1-10 identical to those found in replicase polyprotein 1ab, we did not perform their disorder analysis separately. however, replicase polyprotein 1a has one additional non-structural protein designated as nsp11. nsp11 is a small uncharacterized protein cleaved from the replicase polyprotein 1a. this small protein with unknown function requires experimental insights to further characterize this protein. the intrinsic disorder predicting software used in this study requires amino acid sequences, which are at least 30-residue long. therefore, because of their short sequences (just 13 residues) nsp11s from all three studied coronaviruses were not checked for the intrinsic disorder, disorder-based protein binding regions, and nucleotide-binding residues. based on the msa outputs, nsp11 from sars-cov-2 was found to have a sequence identity of 84.62% with nsp11s from human sars and bat cov (figure 34 ). the emergence of new viruses and associated deaths around the globe represent one of the major concerns of modern times. despite its pandemic nature, there is very little information available in the public domain regarding the structures and functions of sars-cov-2 proteins. based on its similarity with human sars cov and bat cov, the published reports have suggested the functions of sars-cov-2 proteins. in this study, we utilized information available on sars-cov-2 genome and translated proteome from genbank, and carried out a comprehensive computational analysis of the prevalence of the intrinsic disorder in sars-cov-2 proteins. additionally, a comparison was also made with proteins from close relatives of sars-cov-2 from the same group of beta coronaviruses, human sars cov and bat cov. our analysis revealed that in these three covs, the n proteins are highly disordered, possessing the ppid values of more than 60%. these viruses also have several moderately disordered proteins, such as nsp8, orf6, and orf9b. although other proteins have shown lower disorder content, almost all of them contain at least some idprs, and all cov proteins analysed in this study definitely have multiple flexible regions. importantly, our study provides novel information on presence of intrinsic disorder at the cleavage sites of the replicase 1ab polyprotein of covs. this observation confirms the crucial role of idprs in maturation of individual proteins. we also established that many of these proteins contain disorder-based binding motifs. since idps/idprs might undergo structural transition upon association with their physiological partners, our study generates important grounds for better understanding of the functionality of these proteins, their interactions with other viral proteins, as well as interaction with host proteins in different physiological conditions. this will also guide structural biologists to carry out a structure-based analysis of sars-cov-2 proteome to explore the path for the development of new drugs and vaccines. the periodical outbreaks of pathogens worldwide always remind the lack of suitable drugs or vaccines for proper cure or treatment. in 2003, nearly 750 deaths were reported due to the sars outbreak in more than 24 countries. but this time, the outbreak of wuhan's novel coronavirus (sars-cov-2) has quickly surpassed this number, indicating more causalities soon. the lack of accurate information and ignorance of primary symptoms are major reasons, which cause many infection cases. although efficient transmission from human to human is confirmed, the actual reasons for fast sars-cov-2 spread are still unknown, but some assumptions were made by researchers and chinese authorities. the fast spread of sars-cov-2, covid-19 pandemic, and associated introduction of quarantine also have made major impacts on economy and education worldwide due to several restrictions, such as limited transportation, restrained or frozen traveling, halted attendance of mass events, the introduction of distant teaching and learning, etc. due to advancements in sequencing techniques, the full genome sequence of sars-cov-2 was made available in a few days of the first infection report from wuhan, china. however, massive subsequent research needs to be done to identify the actual cause of sars-cov-2 infectivity and to design suitable treatment in the coming future. certain possibilities can be explored with the available information. the mutational pressure study on this virus will be very interesting to see if this virus transforms from bat sars to human sars to sars-cov-2. more in-depth experimental studies using molecular and cell biology techniques to establish structurefunction relationships are required for a better understanding of the functioning of sars-cov-2 proteins. additionally, based on the sequence homology and information on proteinprotein interactions, the associated viral and host proteins should be explored, for finding means suitable for limiting replication, maturation, and ultimately pathogenesis of this virus. although structural biology techniques (so-called rational drug design) can be used in drug development utilizing high throughput screening of compounds virtually or experimentally, the applicability of these techniques is limited by the presence of intrinsic disorder in target proteins. therefore, the thorough disorder analysis of three coronaviruses conducted in this study will help structural biologists to rationally design experiments keeping this information in mind. authors contribution: rg: conception and design, interpretation of data, writing, and review of the manuscript, and study supervision. vnu: conception and design, acquisition and interpretation of data, writing, and review of the manuscript. ms, tb, pk, brg, kg: acquisition and interpretation of data, writing of the manuscript. table 6 . evaluation of intrinsic disorder in non-structural proteins of bat cov. table 7 : predicted morf residues in human sars proteins. supplementary table 8 : predicted morf residues in bat cov proteins. supplementary table 9 : predicted nucleotide-binding residues in sars-cov-2 proteins. supplementary table 10 : predicted nucleotide-binding residues in human sars proteins. supplementary table 11 : predicted nucleotide-binding residues in bat cov proteins. supplementary figures s1. multiple sequence alignment of structural proteins of all three studied coronaviruses are generated using clustal omega. the aligned images are created using esprit 3.0. figure s1a . msa of sars-cov-2, human sars, and bat cov spike glycoproteins. figure s1b . msa of sars-cov-2, human sars, and bat cov nucleoproteins. supplementary figure s2 . multiple sequence alignment of non-structural proteins of all three studied coronaviruses are generated using clustal omega. the aligned images are created using esprit 3.0. figure s2a . msa of sars-cov-2, human sars, and bat cov nsp2 proteins. figure s2b . msa of sars-cov-2, human sars, and bat cov nsp3 proteins. figure s2c . msa of sars-cov-2, human sars, and bat cov nsp4 proteins. figure s2d . msa of sars-cov-2, human sars, and bat cov nsp5 proteins. figure s2e . msa of sars-cov-2, human sars, and bat cov nsp12 proteins. figure s2f . msa of sars-cov-2, human sars, and bat cov nsp13 proteins. figure s2g . msa of sars-cov-2, human sars, and bat cov nsp14 proteins. figure s2h . msa of sars-cov-2, human sars, and bat cov nsp15 proteins. figure s2i . msa of sars-cov-2, human sars, and bat 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spike protein the role of severe acute respiratory syndrome (sars)-coronavirus accessory proteins in virus pathogenesis mitochondrial location of severe acute respiratory syndrome coronavirus 3b protein nucleolar localization of nonstructural protein 3b, a protein specifically encoded by the severe acute respiratory syndrome coronavirus sars-cov accessory protein 3b induces ap-1 transcriptional activity through activation of jnk and erk pathways g0/g1 arrest and apoptosis induced by sars-cov 3b protein in transfected cells over-expression of severe acute respiratory syndrome coronavirus 3b protein induces both apoptosis and necrosis in vero e6 cells the 3a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in vero e6 cells severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane enhancement of murine coronavirus replication by severe acute respiratory syndrome coronavirus protein 6 requires the n-terminal hydrophobic region but not c-terminal sorting motifs a putative diacidic motif in the sars-cov orf6 protein influences its subcellular localization and suppression of expression of cotransfected expression constructs the n-terminal region of severe acute respiratory syndrome coronavirus protein 6 induces membrane rearrangement and enhances virus replication characterization of a unique group-specific protein (u122) of the severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus 7a accessory protein is a viral structural protein augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/x1 and 7a/x4 proteins through nf-kappab activation chemokine upregulation in sars-coronavirus-infected, monocyte-derived human dendritic cells induction of apoptosis by the severe acute respiratory syndrome coronavirus 7a protein is dependent on its interaction with the bcl-xl protein the orf7b protein of severe acute respiratory syndrome coronavirus (sars-cov) is expressed in virus-infected cells and incorporated into sars-cov particles 7a protein of severe acute respiratory syndrome coronavirus inhibits cellular protein synthesis and activates p38 mitogen-activated protein kinase structure and intracellular targeting of the sars-coronavirus orf7a accessory protein solution structure of the x4 protein coded by the sars related coronavirus reveals an immunoglobulin like fold and suggests a binding activity to integrin i domains the 29-nucleotide deletion present in human but not in animal severe acute respiratory syndrome coronaviruses disrupts the functional expression of open reading frame 8 molecular evolution of the sars coronavirus during the course of the sars epidemic in china the human severe acute 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coronavirus the envelope protein of severe acute respiratory syndrome coronavirus interacts with the non-structural protein 3 and is ubiquitinated severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles mobility and interactions of coronavirus nonstructural protein 4 two-amino acids change in the nsp4 of sars coronavirus abolishes viral replication localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3clpro): implications for nsp5 regulation and the development of antivirals a novel mutation in murine hepatitis virus nsp5, the viral 3c-like proteinase, causes temperature-sensitive defects in viral growth and protein processing structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain coronavirus nsp6 restricts autophagosome expansion the sars-coronavirus nsp7+nsp8 complex is a unique multimeric rna polymerase capable of both de novo initiation and primer extension insights into sars-cov transcription and replication from the structure of the nsp7-nsp8 hexadecamer structure of the sars-cov nsp12 polymerase bound to nsp7 and nsp8 co-factors the severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a singlestranded rna-binding subunit unique in the rna virus world variable oligomerization modes in coronavirus non-structural protein 9 severe acute respiratory syndrome coronavirus nsp9 dimerization is essential for efficient viral growth rna 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex in vitro reconstitution of sars-coronavirus mrna cap methylation structural basis and functional analysis of the sars coronavirus nsp14-nsp10 complex biochemical characterization of a recombinant sars coronavirus nsp12 rna-dependent rna polymerase capable of copying viral rna templates mechanism of nucleic acid unwinding by sars-cov helicase delicate structural coordination of the severe acute respiratory syndrome coronavirus nsp13 upon atp hydrolysis discovery of an rna virus 3'->5' exoribonuclease that is critically involved in coronavirus rna synthesis major genetic marker of nidoviruses encodes a replicative endoribonuclease crystal structure and mechanistic determinants of sars coronavirus nonstructural protein 15 define an endoribonuclease family coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'o)-methyltransferase activity biochemical and structural insights into the mechanisms of sars coronavirus rna ribose 2'-o-methylation by nsp16/nsp10 protein complex key: cord-328483-sj8i9ss2 authors: jaegle, mike; wong, ee lin; tauber, carolin; nawrotzky, eric; arkona, christoph; rademann, jörg title: protein‐templated fragment ligations—from molecular recognition to drug discovery date: 2017-05-31 journal: angew chem int ed engl doi: 10.1002/anie.201610372 sha: doc_id: 328483 cord_uid: sj8i9ss2 protein‐templated fragment ligation is a novel concept to support drug discovery and can help to improve the efficacy of protein ligands. protein‐templated fragment ligations are chemical reactions between small molecules (“fragments”) utilizing a protein's surface as a reaction vessel to catalyze the formation of a protein ligand with increased binding affinity. the approach exploits the molecular recognition of reactive small‐molecule fragments by proteins both for ligand assembly and for the identification of bioactive fragment combinations. in this way, chemical synthesis and bioassay are integrated in one single step. this review discusses the biophysical basis of reversible and irreversible fragment ligations and gives an overview of the available methods to detect protein‐templated ligation products. the chemical scope and recent applications as well as future potential of the concept in drug discovery are reviewed. protein-binding,b ioactive molecules,t he starting points to future drugs,are in most cases identified and optimized in two clearly separated steps:first small molecules are synthesized chemically or isolated from natural sources,t hen their biochemical properties are investigated in bioassays (figure 1a) . both steps can be repeated iteratively during the optimization process.a ccordingly,t he classical process for lead discovery is characterized by the strict separation of the generation of chemical libraries and their screening for biological activities in aprotein-based or cellular assay. over the last two decades,h owever,a na lternative concept of drug discovery has emerged that aims at the integration of chemical synthesis and bioassays. [1] [2] [3] this approach exploits the molecular recognition of reactive small-molecule fragments by proteins both for assembly of the chemical ligand and for the identification of bioactive fragment combinations and is,t herefore,d enominated as "protein-templated fragment ligation". protein-templated fragment ligation is an attractive alternative to classical drug discovery for several reasons:t he combination of chemical synthesis and bioassay in one step considerably confines the effort for chemical synthesis to the most active fragment combinations and, thus,i sh ighly efficient, saving time,money,energy,and chemical resources. only small libraries of af ew hundred to at housand reactive fragments are required to cover the relevant chemical space and to test huge numbers of potential fragment combinations or fragment ligation products.m oreover,f ragment ligations enable the detection of low-affinity fragments for as patially resolved protein binding site,since the binding of the reactive primary fragment or protein probe amplifies the binding of asecondary fragment at ap recisely defined location. in this review we will present the current state and the future potential of protein-templated fragment ligations in drug discovery.for this purpose we propose acomprehensive definition of protein-templated fragment ligations: chemical reactions between two or more small molecules ("fragments") that utilize the proteinssurface as acatalyst to accelerate the formation of protein ligands with increased binding affinity are defined as protein-templated fragment ligations. this definition encompasses both reversible and irreversible ligation reactions.w ec onsider the combined coverage of both reaction types af itting approach, as all templated ligations share the same biophysical principles,pose the same challenges for the detection of products,a nd contribute excellent examples to the drug discovery process.m oreover, the definition creates ac lear distinction between templated fragment ligations and other catalytic transformations exerted by proteins similar to the turn-over of enzyme substrates.r emarkably,p rotein-templated fragment ligation reactions following this definition are the established mode of action of several clinically admitted drugs,which suggests that the reactions can indeed proceed efficiently under physiological conditions.f or example,t he anti-parkinson drug carbidopa binds to the enzyme dopad ecarboxylase and reacts with the cofactor pyridoxal phosphate to form ahydrazone as the active inhibitor ( figure 2a ). [4] likewise,t he anticonvulsive drug vigabatrin reacts in ap rotein-templated mode with the cofactor of gabat ransaminase to form am ichael acceptor intermediate,w hich leads to the irreversible inhibition of the enzyme ( figure 2b ). [5] other examples protein-templated fragment ligation is anovel concept to support drug discovery and can help to improve the efficacy of protein ligands. protein-templated fragment ligations are chemical reactions between small molecules ("fragments") utilizing aproteinss urface as areaction vessel to catalyzethe formation of aprotein ligand with increased binding affinity.t he approache xploits the molecular recognition of reactive small-molecule fragments by proteins both for ligand assembly and for the identification of bioactive fragment combinations.inthis way, chemical synthesis and bioassayare integrated in one single step.t his review discusses the biophysical basis of reversible and irreversible fragment ligations and gives an overview of the available methods to detect protein-templated ligation products.t he chemical scope and recent applications as well as future potential of the concept in drug discovery are reviewed. of protein-templated fragment ligations in admitted drugs are found for selegiline [6] (reacts with cofactor fadi nthe depression target monoamine oxidase) and for isoniazid (reacts with nad + in mycobacterium tuberculosis catalase). as fragment ligations are driven by thermodynamic interactions between the fragments and the protein, section 2 considers the biophysical basis of protein-templated fragment ligations to highlight the conceptual potential of the method. special emphasis will then be given to the detection of products of fragment ligation reactions,which is amajor and general challenge in fragment ligation assays (section 3). in section 4wewill give an overview of the chemical reactions used so far in templated ligations and also discuss possible future extensions of this reaction set. reversible reactions, which have been studied in the context of dynamic covalent chemistry [7] [8] [9] [10] [11] [12] [13] [14] and irreversible reactions,also denominated as target-guided synthesis (tgs), [15] are treated together,a sb oth reaction categories deliver examples of templated fragment ligations and in many cases it is difficult to categorize one reaction unambiguously.r epresentative recent applications of templated ligations in fragment-based drug discovery are reported in section 5, thus demonstrating how far the method has developed to date.f inally,i n section 6w ew ill discuss the current state of protein-templated fragment ligations,considering the strengths of this method and the requirements for it to succeed. thec omplementarity of the method with classical ligand screening and fragment-based methods will be considered, thereby leading to an outlook on the relevance and further development of proteintemplated fragment ligations for future drug discovery. in protein-templated fragment ligations the protein serves as acatalyst for the assembly of protein ligands from proteinmike jaegle, ee lin wong, carolin tauber,and eric nawrotzkya re phd students in the group, coming from academic backgrounds in chemistry, pharmacy,a nd molecular biology from universities in freiburg (m.j.), johor bahru, taipei, and seoul (e.l.w.), berlin (c.t.), and leipzig (e.n.). they all work on projects involving protein-templated reactions on various protein targets including proteases, phosphatases, and protein-protein interactions. christoph arkona studied biochemistry at the leibniz university in hannover and joined the group as asenior scientist after finishing ap hd in plant biochemistry and many years of drug development in the biotech industry. the photo shows from left to right:mike jaegle, eric nawrotzky,jçrg rademann,c arolin tauber,c hristoph arkona, and ee lin wong. binding small-molecule fragments.t he molecule fragments are chemically reactive and are linked covalently to yield fragment combinations with improved binding affinities and biological activities.t he process requires ac hemically reactive,d ynamic system that is able to adapt on the molecular level by the formation and-in the case of reversible reactions-recleavage of covalent chemical bonds and by evolving into at hermodynamically more favored state,t hus furnishing optimized protein ligands.s uch adaptive systems can be considered as examples of molecular learning, where the term "learning" is applied here to describe an adaptive, chemically evolving and self-optimizing system. theadditivity of free binding energies is the driving force of protein-templated fragment ligations ( figure 3 ). [16, 17] tw o fragments bind to the protein independently and without any overlapping of their free energies of binding, dg 1 and dg 2 . [18] thel inking of two fragments by ar eversible or irreversible chemical reaction forms aligation product with afree binding energy dg lig = dg 1 + dg 2 + x,w here x represents the deviation from the addition of the binding energies.provided that the covalent linking of the two fragments is additive (x = 0), the obtained fragment combination product will display abinding affinity that is the product of the binding affinities of its fragments: k d = e àdg/rt = k d1 k d2 . [19, 20] fore xample,t wo fragments with k d values of 1mm will result in af ragment ligation product of 1 mm.infact, fragment combinations can also be strongly superadditive,asaresult of the additional binding energy of the linker or entropic gain, which results in an even stronger enhancement of binding affinities. [19] likewise,t he linking of two fragments can reduce the binding energy of the ligation product below additivity if the linker contributes unfavorably to the binding. thea nalytical detection of proteinbinding fragments constitutes am ajor challenge and has been as ignificant limitation to the development and exploitation of fragment-based methods in drug discovery.t he main reason for this detection problem is the low affinity of protein-binding fragments requiring high fragment concentrations to generate and possibly saturate the detection signal. in some assays,s uch as fluorescence anisotropy or saturation transfer difference (std) nmr, high protein concentrations can be used instead to saturate the binding of the small-molecule ligand. compared to other fragment-based methods without ligation, the detection problem, however,i ss ignificantly reduced in protein-templated fragment ligations due to the higher affinity of the fragment ligation product formed. as aresult, fragment ligation assays can, in principle,detect even low-affinity fragments that would not be identified in other fragment assays,for example,because of concentration limits. in general, fragments have to be present at concentrations that exceed their dissociation constants (k d values) by afactor of at least 10 for as trong, saturated detection signal to be measured. in contrast, in the case of afragment ligation assay,c oncentrations of the starting fragment below the k d value are typically used to effect only partial inhibition, which can be saturated by the formation of the stronger binding ligation product. although the detection of fragment ligation products is thereby strongly facilitated compared to single fragments,i t remains ac hallenge for several reasons.a st he ligation products possess ah igher binding affinity than the starting fragments to the target protein, their formation is autoinhibitory and the amount of ligation product is limited strictly by the concentration of the protein template.a s ar esult, fragment ligation products have to be detected against astrong background of excess nonreacted fragments. other challenges can arise from the reactivity of fragments in the fragment ligation assays.although some reactive fragments such as those used in dipolar cycloaddition reactions are truly bioorthogonal, several fragment ligations rely on electrophiles that might also react with protein nucleophiles as reaction partners.c ontrol experiments that distinguish the effect of one fragment from the effect of af ragment combination are routinely conducted to avoid interference of such reactions with the assay read-out. in addition, it is generally recommended in hit validation to use independent secondary assays to detect false-positive hit fragments. several classical analytical methods have been widely applied and adapted to the detection of templated fragment ligations ( figure 4 ). most studies in the field have used liquid chromatography,usually in combination with mass spectrometry (lc-ms). [13, [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] in some examples,s pecific methods of nmr spectroscopy were developed. [31] [32] [33] [34] [35] [36] in addition, fragment ligations have been studied by x-ray crystallography. [37] [38] [39] [40] [41] [42] finally,asapowerful complement, the detection of fragment ligation products by various bioassays has been developed over recent years. [1, 10, [43] [44] [45] [46] [47] [48] thed etection of fragment ligation products by lc-ms has become substantially easier over the last two decades because of the rapid development of this method in terms of chromatographic separation and the sensitivity of mass detectors. [13, [25] [26] [27] [28] not only detection but also quantification and structure elucidation of ligation products is facilitated by using extracted-ion or single-ion chromatography and ms-ms techniques.s ome limitations remain, however.a s chromatographic separation takes some time,l c-ms detection is best suited for irreversible or quasi-irreversible ligation reactions. [15, 49] in the case of reversibly formed ligation products,t heir chemical fixation by ac hemical reaction or ap hs hift can be an option. this strategy was followed, for example,i nt he seminal report by huc and lehn on "virtual chemical libraries", in which the unstable imine ligation products were converted into stable amines by chemical reduction during the ligation reaction. [21] asecond limitation of lc-ms detection can arise from the buffer systems used in fragment ligation reactions.many buffer ions used in ligation assays interfere with the detection of ligation products in the mass detector,w hereas buffer salts such as ammonium formiate or acetate that are highly compatible with lc-ms may interfere with the ligation reaction by adding reactive nucleophiles in strong excess.inrecent years,the detection of protein-binding fragments by native protein ms has developed into am ature technology and it is very likely that this method will also be useful for the detection of fragment ligation products. [50] [51] [52] [53] nmr spectroscopy has the big advantage over lc that it can monitor ligation reactions directly in the assay solution. as standard nmr spectroscopy requires high (mm)c oncentrations of fragment ligation products in volumes of around 0.5 ml for the chemical analysis,l arge amounts of proteins are required. [54] therefore,t he method is only applicable to proteins that are available in large quantities and only af ew experiments with low throughput can be conducted. the throughput can be enhanced if mixtures of reactive fragments are employed that undergo reversible,c ovalent ligation reactions. [33, [55] [56] [57] these mixtures are denominated as dynamic combinatorial libraries (dcl), or dynamic covalent libraries, and can in principle form all possible combinations of the available fragment building blocks,that is,inthe case of n and m complementary reactive building blocks,anumber of n m possible products are obtained. thep rotein template shifts the equilibrium in favor of the best binding and, thus,m ost stabilized ligation product, which is then detected by one of the classical analytical methods. several nmr methods have been adapted specifically for the detection of fragment ligation products.r amstrçm and co-workers have investigated 1 hstd nmr spectroscopy for the detection of hemithioacetals formed by ap rotein-templated reaction in aqueous medium ( figure 5 ). [58] 1 hstd nmr spectroscopy is am ethod that exploits the selective transfer of proton magnetization from the protein to reversibly bound ligands and was used here to identify binding fragments from ad cl. foraproof of concept study, bgalactosidase was selected as the target protein that catalyzes the hydrolysis of o-b-galactosides and contains no cysteine residues in the active site.amixture of five thiols and two aldehydes was employed, thereby resulting in the potential formation of ten hemithioacetals.itwas demonstrated that 1b-mercapto-d-galactose binds more strongly to the target enzyme than do the four other thiols and strongly enhanced the signals of both aldehydes in the std spectrum-presumably through the formation of hemothioacetals. surprisingly,t he binding of the two sugars could not be suppressed by the addition of substrate,t hus suggesting additional allosteric binding sites for these fragments.s ignificant inhibition of substrate hydrolysis could be confirmed for one thiol-aldehyde combination, which also suggests the formation of hemithioacetals as active inhibitors. another application of std nmr spectroscopy was published recently by the hirsch research group. [36] recently, 11 bnmr spectroscopy was applied by claridge and coworkers for the detection of fragment ligation products (see figure 10 in section 5). [59] in addition to ligand-based nmr spectroscopy,t here have been intensive applications of protein-based nmr methods for the detection of protein-binding fragments. [60, 61] protein nmr spectroscopy uses in many cases proteins that have been produced with 13 cand 15 nisotopes to enhance the nmr signals.f ragment binding can then be observed, for example,in2dhsqc experiments from perturbations of the chemical shifts.t he method was introduced as "structureactivity relationships (sar) by nmr spectroscopy" and has the big advantage of furnishing information on the binding site of fragments if the signals in the nmr spectra are assigned to the protein structure. [62] protein-based nmr spectroscopy should also be av aluable method for the investigation of fragment ligation reactions, although we could not find applications of it so far. x-ray crystallography has found broad application in fragment-based drug discovery,a nd its attractiveness comes from the detailed structural information it reveals of the fragment-protein complex. [63] this information can be extremely helpful in the design of fragment combination products.s everal studies have so far used protein crystallography for the detection of fragment ligation products. [37] [38] [39] [40] [41] [42] ther apid and parallel detection of potent fragment ligation products formed in proteintemplated reactions was finally realized by the introduction of bioactivity-based assays.the initial strategy was denominated as dynamic ligation screening (dls), as reversible ligation reactions were first investigated. dls increased the sensitivity of ligand detection considerably,a nd the site-directed discovery of low affinity fragments with millimolar k d values was realized ( figure 6 ). [10, 43] bioactivity-based detection methods require only minimal amounts of protein;u sually low nanomolar concentrations of the protein are sufficient. they can be conducted using standard assay equipment such as microtiter plates and automated pipetting devices used for the handling of fragment libraries.i ne nzyme assays,t he sensitivity is further enhanced by the catalytic activity of the figure 5 . application of 1 hstd nmr spectroscopy for detecting the proteintemplated formation of hemithioacetals from adynamic combinatoriall ibrary and enzymatic selection of the best inhibitor. [58] protein targets resulting in the rapid turnover of many of the fluorogenic or chromogenic substrate molecules.f luorescence resonance energy transfer (fret) has also been used for the detection of fragment combinations. [44] to reach the highest sensitivity,t he primary reactive fragment has to be added to the enzyme assay at ac oncentration that results in 10-20 %i nhibition, thereby leaving am easurement window of 80-90 %f or detection of the enhanced inhibition after addition of the secondary fragment. va rious bioassay formats have so far been adapted to dynamic ligation screening ( figure 7) . as already described, fluorogenic substrates can be used in competition assays, competing with the fragment ligation product for the enzymesb inding site.i nt his assay the best fragment combination results in the strongest inhibition of the enzymatic reaction ( figure 7a) . alternatively,the substrate itself can be constructed with ar eactive group that allows for fragment ligations in which the ligated fragment can increase the affinity of the substrate to the target protein. these substrate-enhancement assays ( figure 7b )e nable the site-directed detection of proteinbinding fragments in secondary binding sites that lead to accelerated turnover of the substrate. [45] competitive binders can also be detected in this assay format. in the special case that the reactive,fluorogenic molecule is completely inactive without ligation of af ragment, it has been denominated as ap resubstrate and is especially sensitive to the detection of chemically unstable,t ransient ligation products such as hemiacetals and hemithioacetals. [46] them ethod has been used successfully for the identification of secondary-site binding fragments of serine proteases [46] and of protein tyrosine phosphatases (ptps), [45] where it was capable of detecting specific secondary-site binders as astarting point for the construction of ptp-specific inhibitors. thep rinciple of bioactivity-based detection of fragment ligation products has also been extended from enzymatic assays to protein binding assays,thus enabling the identification of ligands of non-enzymatic protein-binding sites.p rotein-binding assays are principally conducted either in homogeneous solution or heterogeneously after attachment of one binding partner to as olid phase or surface.a homogeneous protein-binding assay for protein-templated ligation reactions has been demonstrated by using fluorescence polarization (fp), also known as fluorescence anisotropy,for detection ( figure 7c ,d). [47] forthis assay,areactive protein-binding peptide fragment was labeled with af luoroangewandte chemie reviews phore.t he fp ligand (10 nm)w as dissolved in ab uffer containing the target protein at ac oncentration leading to about 50 %l igand binding.t hen al ibrary of fragments was screened for those modulating the fp.t he assay enabled the identification both of competing ( figure 7c )a nd of enhancing fragments ( figure 7d )i nas ingle experiment, and the templating effect exerted by the protein was quantified for the best enhancing fragment combination. reductive amination of the labeled fragment with the best enhancing fragment resulted in ap icomolar inhibitor.s imilar to the enzyme inhibition assays,h omogeneous protein-binding assays such as fp assays can be conducted in high-throughput equipment, in small assay volumes,a nd at low (nm)p rotein concentrations,p rovided aligand with sufficient affinity is available as astarting point. label-free,h omogeneous protein-binding assays will be an attractive complement for the detection of bioactive fragment ligation products.f or example,thermal shift assays are used increasingly for the detection of protein-ligand complexes. [65, 66] them ethod monitors the defolding ("melting") of proteins at increasing temperatures by using fluorophores that show increased fluorescence when they come into contact with the hydrophobic core of unfolding proteins. fragment binding can be observed by as ignificant enhancement of the proteinsmelting temperature because of the free binding energy of the ligand. even more thermodynamic information about the binding of fragments is revealed by isothermal titration calorimetry and future studies will most likely use this technique-although it requires al arge quantity of protein and can be conducted only at low throughput. [67] [68] [69] [70] heterogeneous protein-binding assays have been applied to the investigation of protein-fragment binding. these methods are labelfree with respect to the fragments;h owever,c ovalent immobilization or labeling with affinity probes such as biotin are required. [71] [72] [73] [74] recently,t he bioactivity-based detection of fragment ligation products has been further extended from protein assays to even more complex cellular experiments. [75, 76] ohkanda and co-workers have reported the intracellular generation of the inhibitor 3 of the 14-3-3 protein by intracellular oxime ligation (figure 8 ). the1 4-3-3 protein is involved in protein-protein interactions (ppis), which are especially difficult to inhibit, as interactions of large and dynamic interaction surfaces have to be disrupted by molecules that are still able to penetrate cells through cellular uptake.t he authors realized that the reactive aldehyde derivative of fusicoccin a( fc, 1)b inds in ah ydrophobic cavity adjacent to the binding site of peptide 2,which contains ah ydroxylamine functionality.t hey were able to demonstrate the templated formation of ah eterobivalent ligation product, the potent oxime inhibitor 3,i nvitro in the presence of 14-3-3 protein. next, the authors investigated the intracellular formation of 3 in stably transformed hek293 (flag 14-3-3 z cell line). hplc analysis showed the formation of 3 in cells treated with 1 and 2,w hich led to the highest cytotoxic effect. in contrast, chemically synthesized 3 was inactive in the cells,p ossibly because of the large molecular size that impedes their cell-penetrating properties (figure 8 ). [77] thus,t his study demonstrates that even large molecules that potentially modulate ppis can be generated through intracellular protein-templated fragment ligation. in summary,the considerable progress made in detection methods over the last few years,e specially in bioactivitybased methods,b ut also in the classical analytical methods and biophysical detection strategies,h as facilitated the identification of bioactive fragment ligation products in protein-binding,enzymatic,and cellular assays.this development will enable the discovery of further examples for templated fragment ligation reactions in the future and thereby contribute to the successful application of fragment ligation assays. numerous reaction types have so far been employed for templated fragment ligations.here,wewill give an overview of these reaction types,focusing on the recent additions to this repertoire and giving an outlook on further extensions that can reasonably be expected. from ap ractical viewpoint, the degree of reversibility of the ligation reaction is an important criterium, as it affects the detection and isolation of the ligation products.t hus,r eaction types used for templated fragment ligations are categorized here according to their reversibility ( table 1) . additions of heteronucleophiles to aliphatic or aromatic aldehydes/ketones typically represent reversible ligation reactions that equilibrate rapidly in aqueous solutions as aresult of low activation barriers.there are,however, marked differences with respect to reversibility and kinetics depending on the reacting nucleophile. hemiacetals and hemithioacetals are rapidly formed in water from aldehydes,alcohols,and thiols through templated fragment ligations,although they cannot be isolated as stable ligation products. [46, 58, 78, 79] theformation of both hemiacetals and hemithioacetals as ligation products is impeded by competition of the heteronucleophiles with ah igh molar excess of water (55 m), which furnishes hydrates as alternative reaction products.nevertheless,itcould be demonstrated by using nmr spectroscopy [58] and bioactivity-based assays [46] that aprotein template is able to shift the ligation equilibrium and, thus,t oenable the identification of hemiacetals and hemithioacetals as ligation products. [46] in contrast to the hemiacetals," full" acetals or dithioacetals (obtained by the addition of two alcohol or thiol nucleophiles to one carbonyl group) have not been reported as templated ligation products and cannot be expected to be formed because of the high activation barrier to the carbocation intermediates in this reaction. [80] [81] [82] many templated ligation reactions involve nitrogen nucleophiles that react with carbonyl electrophiles.t he addition of primary amines to aldehydes furnishes hemiaminals as intermediates,w hich react further to form imines. [126, 127] imine formation is also ap rocess that equilibrates rapidly and requires nonprotonated amines for reactivity. [10, 43, 44, 46, 47, 83, 84] thus,t he process depends on the pk a value of the amine nucleophile and the ph value of the reaction buffer.i mines are considerably more stable than hemiacetals,e specially those formed from aromatic amines, and can even be isolated in some cases. one consequence of the increased stability of imines is that the equilibration of dynamic combinatorial libraries may take significantly longer than the equilibration of hemiacetals or hemithioacetals.t he stability of amine-aldehyde ligation products can be further enhanced if the aldehyde carries acidic a-hydrogen atoms,w hich allow the formation of enamines.a nother possibility is the chemical fixation of imines by an irreversible reaction, for example,r eductive amination to yield secondary amines. [86, 99] thestability of the ligation products of nitrogen nucleophiles with aldehydes is further elevated in the case of hydroxylamines,h ydrazines, and acyl hydrazides. [14, 77, [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] 100] theformed products,oximes and (acyl) hydrazones,a re stable at physiological ph values and can be isolated by standard procedures,s uch as column chromatography.a ccordingly,t he ligation reaction is equilibrated very slowly or aniline has to be added as acatalyst for the formation of the hydrazine or oxime. [86, 87, 89, 99] alternatively,the equilibration of acyl hydrazones can be accelerated by the addition of acid. [98] further ligations of heteronucleophiles with aldehydes are awaiting further investigation. forexample,thiols can be added to imines to furnish n,s-acetals in al igation equilibrium that can possibly be shifted by interaction with aprotein template. [43, 46, 112] bioactivity data obtained from the investigation of ap resubstrate have suggested that the trimeric complexes of aldehyde,a mine,a nd thiol have as tronger affinity to the protein target, as expressed by ar educed k m value and increased turnover of the enzyme substrates. [46] likewise,t he observed superadditive binding and inhibition of peptide aldehydes and amines quantified by the fp assay can be interpreted by the formation of such thioaminal products. [43, 47] mannich bases,aspecial form of stable aminals, can also be expected to be suitable for protein-templated reactions,atopic which is currently under investigation in our group. further templated fragment ligations involve the addition of heteronucleophiles to celectrophiles other than aldehydes.t hio-michael additions have been reported in several cases,a nd the products can usually be isolated, even though the reaction can be reversible if the product favors retroaddition through b-elimination. [46, 117] alkylations are usually truly irreversible ligations;i nm ost cases thiolates are employed as nucleophiles for highest reactivity,and aliphatic halogenides,e poxides,o rs ulfonates have been used as electrophiles. [118] [119] [120] these studies suggest that alternative celectrophiles should be useful in protein-templated reactions.for example,nucleophilic substitutions at electron-poor aryl moieties could be an interesting extension of the reaction repertoire. boric acid and boronic acids have been used as alternative electrophiles.t he use of diols as bisnucleophiles furnishes boronate esters as ligation products with limited stability. [59, 125] other popular examples of reversible reactions that furnish stable products that can be isolated are disulfide formation and disulfide exchange reactions through the use of thiolates as the reactive nucleophiles under slightly basic and nonreducing conditions. [101] [102] [103] [104] [105] [106] [107] asimilar exchange reaction of thiolates has been reported for the thioester exchange reaction. [33, 55, 82, [108] [109] [110] c à cb ond-forming reactions are of special interest in templated ligations,a st hey can extend the choice and diversity of the accessible ligations products considerably.f ew examples have been reported to date. ramstrçm and co-workers have described ar eversible templated henry-aldol addition of nitroalkanes to aldehydes,i n which the formed, best-bound secondary alcohol was preferably acylated by the lipase as the protein target in the assay. [56] although no further reports on templated aldol reactions have yet been published, this reaction type should be highly suitable for fragment ligation chemistry considering the detailed literature on aldol reactions in water and under mild reaction conditions. [56, 111, 116, 128] likewise,a dditions of cyanide anions and isocyanides have been reported in water, which suggests that passerini and ugi reactions might be highly suitable for protein-templated reactions.t he same applies to the càcb ond formations by alkene and alkyne metathesis reactions,which have so far been demonstrated as ligation reactions in water. [112] [113] [114] [115] classical examples of irreversible templated reactions have been reported for dipolar cycloaddition reactions, including azide-alkyne ligations that furnish 1,4-disubstituted a) hemiacetals, b) hemithioacetals, c) acetals. d) thioacetals, e) n,s-acetals, f) imines, g) hydrazones, g')acylhydrazones, h) oximes, i) boronates, j) disulfides, k) thioesters, l) alkene metathesis, m) alkyne metathesis, n) nitroaldols o) alkylation, p) amidation between sulfonylazides and thioacids, q) 1,3-dipolarcycloaddition,r)addition, s) ring opening, t) amidation between amines and active esters. ,2,3-triazoles,a nd sulfonylazide-thioacid ligations that provide sulfonylamides through ac yclic intermediate. [24, 25, 76, [118] [119] [120] [121] [122] [123] [124] it is clear that numerous other ligations based on cycloaddition reactions may also succeed. besides cycloaddition reactions,h owever, irreversible ligations have not yet been investigated much and future extensions are necessary to provide ligation products that cover alarger part of the biologically relevant chemical space.i ng eneral, chemoselective reactions in water are required for asuccessful ligation. ideally,the reaction should provide alinker between the reacting fragments that supports binding or at least does not interfere with it. fore xample,t he structural analysis of the world drug index (a collection of molecules with reported bioactivity) revealed that amide linkages are privileged linkers in bioactive compounds. [129] thus,aprotein-templated amidation reaction can be considered an especially useful extension of the fragment ligation repertoire.v ery recently the first background-free protein-templated amidation reactions were discovered. [48] other reaction types that could considerably extend the opportunities of fragment ligations are reactions that are able to connect fragments without al inker remaining in the product, such as cross-coupling reactions or reactions forming heterocycles as the connection between two fragments instead of ac lassical linker moiety. having defined fragment ligation reactions,e xplained their biophysical background, useful detection strategies,a nd the underlying chemical transformations,w ea re now going to focus on the application of the concept for the discovery and optimization of protein ligands.w ew ill start with reversible ligation reactions and then proceed to the irreversible ones. many of the first applications of reversible,t emplated fragment ligations were reported for dynamic combinatorial libraries (dcls). as ar ecent representative example of this line of research, we highlight the studies by hirsch and co-workers. [36, 98] theauthors employed endothiapepsin as atarget and model enzyme for other aspartic acid proteases.n ine hydrazides (4-12)and one bisaldehyde were used to form adynamic library derived from two fragment hits [36] that bind to adjacent binding pockets of endothiapepsin ( figure 9 ). [98] to ease analysis, two sublibraries (dcl-1 and dcl-2) were formed that consisted of four and five hydrazides.r eversed-phase hplc and lc-ms were employed to analyze the templated formation of the bisacylhydrazone ligation products in the presence of endothiapepsin. out of the potential 78 different bisacylhydrazones and 12 monoacylhydrazones,o nly 6p ossible fragment combinations showed significant amplification of the hplc signal in the presence of the endothiapepsin template.t wo of the found combinations, 13 and 16,w ere synthesized and tested in af luorescence-based biochemical assay.b oth were reported to be active,and the best inhibitor, 13,showed a240fold increase in potencyc ompared to the starting fragments, thus illustrating as uccessful example of at emplated threefragment ligation reaction. thea uthors demonstrated in this study that, unlike other fragment optimization methods such as fragment growing or merging, templated fragment ligation is especially sensitive for discovering combined fragments with superadditivity,w here their ligand efficiencies were not only maintained but improved. in their approach, the challenge lay in preserving the binding mode of the fragments and finding aw ell-fitting linker that provides additional interactions to the target. despite the positive results,t he study also demonstrates the limitations of dcl, namely the tedious analysis of complex combinatorial mixtures and the limitation to few dynamic reactions that deliver non-druglike products such as acylhydrazones. figure 9 . protein-templated fragmentligations using adynamic combinatoriall ibrary. [98] a) isophthalaldehyde 3 and nine hydrazides, 4-12 form up to 81 bisacylhydrazones. bi)the model protein endothiapepsin is equilibrated with the dcl for the formation of potential inhibitors. bii) hplc analysis shows that the formation of the bisacylhydrazone 13 was amplified in the presence of the enzyme. biii)binding modes of 19 (purple) and 13 (cyan) were determined by xray crystallography (pdb ids:4 kup and 5hct respectively). c) the best compound 13 exhibits a240-fold improvement in potency and higher ligand efficiencyc ompared to the starting fragment 19. thes ame limitations are displayed in the study by claridge and co-workers who applied 11 bnmr spectroscopy for the detection of serine protease inhibitors from adcl. [59] foraproof-of-concept study, a-chymotrypsin (act) was recruited as amodel enzyme,a nd boronic acids were ligated with added sugar molecules to form improved enzyme inhibitors.t he formation of ternary complexes of enzyme, boronic acid, and sugar diol was monitored by 11 bnmr and 1 h-waterlogsy (water-ligand observed by gradient spectroscopy). these findings encouraged the groups of schofield and claridge to apply native mass spectrometry to detect hits from ad cl. reversibly formed boronate esters were identified as inhibitors of prolyl hydroxylase domain isoform 2( phd2), an enzyme of the 2-oxoglutarate (2-og) oxygenase family (figure 10 ). [130] phd2 is an fe ii -containing 2-og oxygenase which helps to regulate human hypoxic response, thus making it an excellent drug target for the treatment of anemia and ischemia-related diseases. in the experiments,aheterocyclic iron-binding boronic acid fragment was incubated with several pools of diol ligands and subsequently analyzed by native ms.t he best-binding fragment combinations were identified by the shift in protein mass and converted into stable,boron-free inhibitors by using suzuki cross-coupling reactions,w hich resulted in inhibitors of the enzyme being active down to the nanomolar range.the results of the fragment ligation assays were further validated in another study by using a 1 hnmr based method. [131] thetethering approach is aclassical example of reversible templated fragment ligations using disulfide exchange reactions. [132] this method exploits the reversible disulfide exchange reaction between an atural or an engineered cysteine residue on the protein surface and ad isulfidecontaining small-molecule fragment in solution. further developments by the erlanson group using so-called "extenders" made this approach even more applicable for fragment combination reactions. [133] in 2008 they reported an inhibitor screening that targeted the aurora kinase ( figure 11 ). [134] the first step was to introduce ac ysteine residue near the atp binding site.n ext, an extender was synthesized containing two disulfide-containing residues and ad iaminopyridine group,w hich is known to bind to the purine binding site. one disulfide residue was able to react and exchange with the introduced cysteine residue,t he other one was able to bind as econdary disulfide fragment. if this secondary fragment was able to interact with the adaptive region of the protein adjacent to the extender,athermodynamically stabilized disulfide bond was formed. this stabilized complex was subsequently detected by ms measurements through modification of the proteinsm ass.i nt heir experiments,alibrary containing roughly 4500 disulfide-containing fragments was screened in pools of 10 compounds,w hich resulted in the identification of several fragment combinations.afew modifications of the corresponding fragments led to the formation of astable inhibitor with affinity in the micromolar range.asimilar approach has been successfully applied to another kinase,3 -phosphoinositide-dependent protein kinase-1 (pdk1), by attaching the extender through an irreversible alkylation reaction. [135] dynamic ligation screening was introduced in 2008 to overcome the detection limits of combinatorial methods such as dcl. thef irst bioactivity-based detection of dynamically formed fragment ligation products has been demonstrated for figure 10 . a) schematic representation of the generation of boronate acid/ester leads using protein-directed dynamic combinatorial libraries containing diols and boronic acids. [130] b) the identification of potent boronate ester conjugates by using ab oronic acid "scaffold ligand" leads to the discovery of first and second generation stable analoguest hat inhibit phd2 in the nanomolar range. the sars coronavirus main protease (sars-cov m pro , figure 12 ). [43] sars-cov m pro is av irus-encoded cysteine protease essential for the replication of the virus inside the infected host cells.i nhibitors targeting m pro are,t herefore, relevant as potential antivirals. [136, 137] since proteases possess defined binding pockets for the side chains of peptides,itwas possible to develop ap eptide aldehyde inhibitor that positions an electrophilic aldehyde precisely at the active site of the protease.dynamic ligation screening of afragment library of 234 diverse nucleophiles revealed several fragment hits that targeted the s1' pocket of the enzyme with k i values in the millimolar range.t he best fragment hit was converted into the corresponding aldehyde fragment and assayed again for enhanced inhibition against an amine library,t hereby providing asecondary hit fragment for the adjacent s1 pocket of the enzymesb inding site.o nly two iterations of dynamic ligation screening starting from apeptide inhibitor resulted in the selection of two millimolar-active small-molecule fragments,a nd the covalent combination of these fragments by reductive amination revealed an entirely nonpeptidic inhibitor of sars-cov m pro with a k i value of 2.9 mm. further examples of bioactivity-based dynamic ligation assays are summarized in table 2 . caspase 3isaprotein that plays an essential role in the execution phase of cell apoptosis and, therefore,ap otential drug target in traumatic brain injury and amyotrophic lateral sclerosis as well as alzheimers and parkinsonsdisease.t he protein was tested with ananomolar fluorophore-labeled peptidyl ketoaldehyde inhibitor and atotal of 7397 fragments,including 4019 nucleophilic and primary amines in 384-well microtiter plates,byapplying the fp binding assay described above.weobserved no change for most of the fragments tested. 78 fragments led to lower fp signals (negative cooperativity) and 176 fragments evoked asignificantly stronger fp signal (positive cooperativity) than the control. 21 fragments were confirmed as competitive inhibitors of caspase-3 with k i < 10 mm.t he best cooperatively binding fragment b was linked covalently to the starting ligand a by reductive amination, thereby resulting in apotent inhibitor of caspase-3 with a k i value of 80 pm. dynamic ligation assays were further implemented for secondary-site screening of four closely related protein tyrosine phosphatases (ptps): human ptp1b,p tpn7, ptpn12 (= shp2), and mycobacterial mptpa. [45] in general, the development of specific ptp inhibitors is considered to be amajor challenge that raises doubts on the druggability of this physiologically relevant class of enzymes.b yu sing 4-formylfigure 11 . atethering approach using ad ynamice xtender has been applied to screen for fragments binding to aurora kinase. [134] angewandte chemie reviews phenyl phosphate as an electrophilic ptp substrate,s pecific secondary-site binding fragments could be identified for each of the ptps from al ibrary of only 110 primary amines.t he specific secondary site binders were detected using a"dynamic substrate enhancement" assay (see figure 7b ): active fragments form al igation product with the ptp substrate which results in ahigher stability of the enzyme-substrate complex and alowered k m value.this leads to an amplified release of phosphate ions,w hich was determined in am alachite green assay.r eplacement of the phenyl phosphate substrate by anoncleavable phosphotyrosine mimetic and covalent linking to the selected mptpa-specific amine fragment furnished inhibitors of protein tyrosine phosphatase a( mptpa) from mycobacterium tuberculosis,w ith no activity for the other three ptps.t his result suggests that second-site targeting of ptps enables the development of selective ptp inhibitors. dynamic ligation screening has been further extended toward aspartic proteases,t his time by employing af luorescence resonance energy transfer (fret) assay. [44] ap eptide aldehyde was used as adirecting probe to identify the activesite binding fragments of the aspartic protease b-secretase (bace-1). this enzyme is known to be the main culprit in the aggregation of amyloid-b-peptides,w hich is am ajor pathological hallmark of alzheimersdisease (ad). thus,considerable efforts have been made to discover bace-1 inhibitors for potential therapeutic treatment of ad.i nstead of reversible hemithioacetal formation, the aldehyde hydrate was formed to bind the catalytic aspartic acid dyad through formation of ah ydrogen bond. dynamic ligation of the peptide aldehyde with an amine nucleophile reversibly yielded an imine product. thep eptide aldehyde,w hich serves as ac hemically reactive inhibitor (cri), revealed 3-(3-aminophenyl)-2h-chromen-2-one to be ac ompetitive bace1 inhibitor.t he identified 3-(aminophenyl)coumarin fragment was used as astarting point for hit optimization and al ow micromolar (k i = 3.7 mm)b ace1 inhibitor was developed. another application of bioactivity-based detection has been reported recently for the aspartic protease endothiapepsin. [64] in the most recent application, reversible protein-templated fragment ligation has been extended toward the discovery of irreversible inhibitors of enteroviral proteases ( figure 13 ). [85] epoxyaldehyde 23,amodified partial structure of the known inhibitor of cysteine proteases e-64 and aweak irreversible inhibitor of the 3c protease of coxsackie 3b virus,w as tested with al ibrary of 850 primary amine fragments.5 -aminopyrazolone 24 was discovered as af ragment hit that led to asuperadditive inhibition of the protease and covalent modification of the enzyme by addition of both fragments.i terative optimization of ligation products of 23 and 24 finally led to sub-micromolar,b road-spectrum inhibitors of enteroviral proteases,w ith 300-to 500-fold acceleration of the protein deactivation rate and no significant crossreactivity with nonviral proteases. early studies in the area of irreversible protein-templated fragment ligations were conducted by the sharpless group and denominated as kinetic target-guided synthesis.t hey used dipolar cycloadditions of azides and alkynes for in situ click reactions templated by the protein acetylcholinesterase (ache), which is at arget in the treatment of alzheimers disease. [30] in af ollow-up study from 2005, the method was again applied to the same target, with the goal of identifying and synthesizing af ragment combination that occupied both the active center and the peripheral binding site ( figure 14 ). [123] as astarting point, the established ache inhibitor tacrine was used, which binds to the active center of the enzyme. tacrine was modified by introducing an azide functionality.a compound collection of 23 terminal acetylenes was composed of peripheral site binding fragments and tested to detect the formation of the 1,2,3-triazole by dipolar cycloaddition. the first lc-ms results confirmed the proteinstemplating effect during the ligation reaction of the bound fragments.o nly in the presence of the enzyme were 1,5-disubstituted (syn) triazoles formed, with only little or no background reaction. having confirmed the reaction with single fragment pairs, the authors turned their attention to reactions containing mixtures of up to 10 acetylene compounds.indeed the protein template induced the formation of highly potent fragment combinations with dissociation constants in the low picomolar or even femtomolar range. azide-alkyne ligations have also been applied to other protein targets. [76, [121] [122] [123] [124] [125] 138] fore xample,t he formation of aknown nanomolar inhibitor of hiv protease containing an anti-substituted 1,2,3-triazole was accelerated in the presence figure 12 . developmento fafragment-based, nonpeptidic sars-cov main protease inhibitor starting from ap eptide aldehyde. [43] dynamic ligation screening of alibrary of nucleophiles yielded the amine 20, which binds to the s1' subpocket of the protease. amine 20 was converted into the electrophile 21,w hich furnished asecondary hit fragmenti nthe next iteration. reductivea mination of fragment 21 and the best amine hit from the secondary screening yielded inhibitor 22. of the enzyme. [138] in this case,t he reaction was considerably slower than that reported for the pico-/femtomolar ache inhibitors,a nd this time ac lear background reaction that furnished the syn-triazole was observed. protein-templated azide-alkyne "click" reactions have also been successfully applied to the abl tyrosine kinase. [121] thef ukase research group has demonstrated the templated synthesis of functional mimetics of the grb2-sh2 domain which inhibited the growth of cancer cells in vitro. [76] to obtain the best inhibitor through the templated reaction of azide and alkyne fragments it is crucial to exclude even tiny amounts of copper ions from the reaction. theg roups of miyata and finn reported the in situ synthesis of an inhibitor of the histone deacetylase 8(hdac-8) from an alkyne fragment bearing ah ydroxamate and an azide fragment. [122] surprisingly,t he formed ligation product was not the best inhibitor by far and contained a1 ,4disubstituted anti-triazole instead of the more active syntriazol. ad etailed scrutiny of these results revealed that the reaction contained traces of cu i ions,w hich were bound by the metal-binding site of hdac-8 and were sufficient to catalyze the reaction to the less-favored cycloaddition product. in addition to azide-alkyne ligations,other dipolar cycloadditions have also been investigated. reactions of thioacids with sulfonylazide fragments,which provide acylsulfonamides via ac yclic intermediate,c onstitute especially successful examples and have been denominated as sulfo-click reactions. in 2011, the manetsch group demonstrated protein-templated, irreversible ligation reactions with ap articularly challenging target:the ppi domain bcl-x l . [120] theinteraction of bcl-x l with the bh3 peptide is ac rucial step in the regulation of apoptosis (programmed cell death). inhibitors of this interaction might be potent anticancer agents.i n ascreening for new modulators,the bcl-x l protein was used as atemplate for the protein-templated sulfo-click reaction of as ulfonylazide and at hioacid fragment ( figure 15 ). it could be shown that formation of the micromolar bcl-x l inhibitor proceeded in aprotein-templated reaction;less inhibitor was reviews 7372 www.angewandte.org formed without the protein or on blocking the binding site by the bh3 peptide,while addition of an inactive mutated bh3 peptide had no effect. protein-templated, irreversible fragment ligations were recently established for the formation of amide bonds (amidation reactions), one of the most relevant fragment linkages in bioactive compounds and clinically admitted drugs ( figure 16 ). [47] ac ollection of active esters 28-40 covering ab road range of chemical reactivity was incubated with 4aminomethylbenzamidine (41)a nd protein factor xa, adrug target from the blood coagulation cascade,t od iscover conditions for protein-templated amidation reactions.t wo active ester fragments,phenyl ester 39 and trifluoroethyl ester 40, displayed aclear protein-templated amidation of 41 in the substrate competition assay and in lc-ms,a nd afforded the nanomolar inhibitor 42 from two weakly binding fragments with millimolar affinities.extracted-ion chromatography with aq tofd etector was used to quantify the progress of the protein-templated formation of inhibitor 42.interestingly,the reaction of the trifluoroethyl ester 40 proceeded without detectable background reaction and was autoinhibited at as aturation concentration of 10 nm of the free inhibitor 42. theinhibitor displayed aremarkable superadditive enhancement of its free binding energy (the k i value was 29 nm instead of 3 mm for the additive case), which was proven to result from the relatively decreased entropy of binding because of fragment linking.t he protein-inhibitor complex was crystallized and the obtained high-resolution structure allowed the authors to rationalize the templated amidation reaction in steric and mechanistic detail. figure 13 . discovery of irreversible inhibitors of coxsackie virus b3 3c protease by using reversible protein-templated fragment ligations. [85] nucleophilic amine fragment 24 binds to the s1 pocket of the enzyme (a) to reversibly form af ragment ligation product with the biselectrophilic warhead 23 (b). next the epoxide is opened by attack of the cysteine side chain in the active site, as detected by lc-ms (c). the ligation product displays asuperadditive inhibitory effect in the fret assay and is optimized to potent broadband inhibitors of enteroviral and rhinoviral 3c proteases. figure 14 . developmentofache inhibitors by protein-templated fragment ligation reactions. [123] the ache inhibitor tacrine was modified with an azide functionality and acts as an anchoring molecule in the active site of the protein. mixtures of various acetylenes were added and the templated reaction furnished two highly potent syn-1,2,3triazoles. protein-templated fragment ligations have been established as an alternative and complementary route to access bioactive protein ligands over recent years.s ignificant progress has been realized on several fronts.i mproved and specialized analytical and bioanalytical methods have contributed both to ab road implementation and ap rofound understanding of the method. thechemical scope of ligation figure 15 . templated formation of sulfonamide inhibitor sz7ta2 of the protein bcl-x l from fragments sulfonylazide sz7 and thioacid ta2. [119] a) reference compound obtained by chemical synthesis. b) templatedr eaction:fragments incubated in the presence of target protein;b cl-x l serves as atemplate for the formation of sz7ta2. c) backgroundr eaction:f ragments incubated without bcl-x l lead only to small amounts of the product. d) blocking of the bcl-x l binding site by the bim-bh3 peptide suppresses the product formation.e )mutated bim-bh3 has only alow affinity towards bcl-x l ,t hereby leading to no inhibition of the templated fragmentl igation reaction. figure 16 . protein-templated amidation of active esters 39/40 with 4-aminomethylbenzamidine (41)f urnished the nanomolar inhibitor of the protein factor xa. [47] the protein-templated, background-free reaction was demonstrated in ab ioactivity-based assay and the autoinhibitory kinetics of inhibitorf ormation was proven by qtof-ms. the crystal structure of the protein-inhibitor complex enabled the pathway of the templated reaction to be modeled. reviews 7374 www.angewandte.org reactions has been continually extended, and further extensions to reactions that deliver templated fragment ligation products can be expected. after reviewing the biophysical background, the underlying chemistry,and actual applications of this method, we will now consider its strengths and limitations and finally give an outlook on the future possibilities and developments of this technique as part of the drug discovery process. theb iggest advantage of protein-templated fragment ligations compared to other fragment-based methods is that it enables the site-directed, spatially resolved identification of fragments that bind to ap recisely defined protein pocket. other fragment-based methods,f or example x-ray crystallography and some of the nmr-based methods,a lso deliver structural information on ligand binding,h owever, they cannot be used to screen specifically for one binding site. site-directed fragment detection is realized by the structure and by the defined binding of the reactive starting fragment. thes econd major advantage of protein-templated fragment ligation assays is their sensitivity caused by the (super-) additive binding enhancement of ligated fragments.a s ar esult, fragment ligation assays can identify fragments that are not detectable with other fragment-based discovery methods or only at considerably higher fragment or protein concentrations.thus,the method may provide protein ligands with alternative structures and with improved ligand efficiencies.a sathird advantage,p rotein-templated fragment ligations have practical and economic benefits,mainly arising from the integration of the synthesis of fragment combinations and the detection of bioactivity in one step.asaresult, the effort and the resources required for the chemical synthesis of bioactive ligands are reduced considerably,a s only bioactive fragment combinations need to be resynthesized for further structural and functional validation. whereas classical high-throughput screening uses large libraries of drug-like molecules to cover the chemical space,f ragment ligation screening needs only small libraries of reactive fragments to sample the chemical space of all potential fragment combinations. one general requirement of fragment ligation assays is the need for areactive starting fragment. this could be problematic if no suitable ligand of atarget protein is known. in such cases,c omplementary methods such as classical screening or structure-based design are indispensable to provide starting points for fragment ligation. another major requirement of protein-templated ligation assays is the availability of ligation reactions that cover the relevant chemical space and are compatible with the conditions of the protein assay.although, as shown in sections 4 and 5, the number of chemical reactions that have been adapted to fragment ligation screening is constantly growing, continuous research efforts will be needed to make more of the privileged drug-like fragment linkages accessible in ligation assays.i np articular, c à cb ond-forming reactions, formations of heterocycles,a nd direct connections between cyclic fragments,w hich are often constructed through crosscoupling reactions,need further investigation. thec ritical evaluation shows that protein-templated fragment ligations can currently be considered as ab roadly applicable method that can, and should, contribute to the modern drug discovery process by complementing the other established and successful methods when its specific virtues are required:s patially resolved and site-directed screening and high sensitivity for the identification of novel fragments as well as the further chemical development of known protein ligands. future research will show if the specific advantages of the method will be able to provide clinical candidates with improved properties.t he ultimate significance of proteintemplated fragment ligations will depend on to what extent they will contribute to fulfill the promise of fragment-based drug discovery:f ind better drugs with high potency, specificity,a nd fewer off-target related side effects.m ost likely, protein-templated fragment ligations will contribute to this goal in close collaboration with other fragment-based and classical lead discovery approaches.b eyond the practical success of the method in the drug discovery process,proteintemplated ligation reactions are also potent and attractive research and training tools that teach us how partial structures of am olecule can interact additively to create high-affinity protein ligands.t hrough this,t he method helps us to understand how molecular recognition makes the molecules of life work and how molecular evolution can proceed. howt ocite: angew.c hem. int rademann in fragment-based drug discovery lessons and outlook proc.n atl. acad. sci proc. natl. acad. sci proc.n atl. acad. sci proc.n atl. acad. sci proc.n atl. acad. sci expert opin. drug discovery angew.c hem. int angew.c hem. int drug discovery today dynamic combinatorial chemistry drug discovery today ramstrçm in dynamic combinatorial chemistry in drug discovery,b ioorganic chemistry,a nd materials science proc.natl. acad. sci proc.n atl. acad. sci revised manuscript received version of record online theauthors declare no conflict of interest. key: cord-342189-ya05m58o authors: banerjee, abhik k.; blanco, mario r.; bruce, emily a.; honson, drew d.; chen, linlin m.; chow, amy; bhat, prashant; ollikainen, noah; quinodoz, sofia a.; loney, colin; thai, jasmine; miller, zachary d.; lin, aaron e.; schmidt, madaline m.; stewart, douglas g.; goldfarb, daniel; de lorenzo, giuditta; rihn, suzannah j.; voorhees, rebecca; botten, jason w.; majumdar, devdoot; guttman, mitchell title: sars-cov-2 disrupts splicing, translation, and protein trafficking to suppress host defenses date: 2020-10-08 journal: cell doi: 10.1016/j.cell.2020.10.004 sha: doc_id: 342189 cord_uid: ya05m58o sars-cov-2 is a recently identified coronavirus that causes the respiratory disease known as covid-19. despite the urgent need, we still do not fully understand the molecular basis of sars-cov-2 pathogenesis. here, we comprehensively define the interactions between sars-cov-2 proteins and human rnas. nsp16 binds to the mrna recognition domains of the u1 and u2 splicing rnas and acts to suppress global mrna splicing upon sars-cov-2 infection. nsp1 binds to 18s ribosomal rna in the mrna entry channel of the ribosome and leads to global inhibition of mrna translation upon infection. finally, nsp8 and nsp9 bind to the 7sl rna in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. our results uncover a multipronged strategy utilized by sars-cov-2 to antagonize essential cellular processes to suppress host defenses. coronaviruses are a family of viruses with notably large single-stranded rna genomes and broad species tropism among mammals (graham and baric, 2010) . recently, a coronavirus, sars-cov-2, was discovered to cause the severe respiratory disease known as covid-19. it is highly transmissible within human populations and its spread has resulted in a global pandemic with more than a million deaths to date (andersen et al., 2020; zou et al., 2020) . we do not fully understand the molecular basis of infection and pathogenesis of this virus in human cells. accordingly, there is an urgent need to understand these mechanisms to guide the development of therapeutics. sars-cov-2 encodes 27 proteins with diverse functional roles in viral replication and packaging( bar-on et al., 2020; wang et al., 2020) . these include 4 structural proteins: the nucleocapsid (n, which binds the viral rna), and the envelope (e), membrane (m), and spike (s) proteins, which are integral membrane proteins. in addition, there are 16 non-structural proteins (nsp1-16) which encode the rna-directed rna polymerase, helicase, and other components required for viral replication (da silva et al., 2020) . finally, there are 7 accessory proteins (orf3a-8) whose function in viral replication or packaging remain largely uncharacterized (chen and zhong, 2020; finkel et al., 2020) . as obligate intracellular parasites, viruses require host cell components to translate and transport their proteins and to assemble and secrete viral particles (maier et al., 2016) . upon viral infection, the mammalian innate immune system acts to rapidly detect and block viral infection at all stages of the viral life cycle (chow et al., 2018; jensen and thomsen, 2012; wilkins and gale, 2010) . the primary form of intracellular viral surveillance engages the interferon pathway, which amplifies signals resulting from detection of intracellular viral components to induce a systemic type i interferon response upon infection (stetson and medzhitov, 2006) . specifically, cells contain various rna sensors (such as rig-i and mda5) that detect the presence of viral rnas, promote nuclear translocation of the transcription factor irf3 leading to transcription, translation, and secretion of interferon (e.g. ifn-α and ifn-β) . binding of interferon to cognate cell-surface receptors leads to transcription and translation of hundreds of antiviral genes. j o u r n a l p r e -p r o o f 4 in order to successfully replicate, viruses employ a range of strategies to counter host antiviral responses (beachboard and horner, 2016) . in addition to their essential roles in the viral life cycle, many viral proteins also antagonize core cellular functions in human cells to evade host immune responses. for example, human cytomegalovirus (hcmv) encodes proteins that inhibit class 1 major histocompatibility (mhc) display on the cell surface by retaining mhc proteins in the endoplasmic reticulum (miller et al., 1998) , polioviruses encode proteins that degrade translation initiation factors (eif4g) to prevent translation of 5'-capped host mrnas (kempf and barton, 2008; lloyd, 2006) , and influenza a encodes a protein that modulates mrna splicing to degrade the mrna that encodes rig-i (kochs et al., 2007; zhang et al., 2018) . suppression of the interferon response has recently emerged as a major clinical determinant of covid-19 severity , with almost complete loss of secreted ifn characterizing the most severe cases (hadjadj et al., 2020) . the extent to which sars-cov-2 suppresses the interferon response is a key characteristic that distinguishes covid-19 from sars and mers (lokugamage et al., 2020) . several strategies have been proposed for how the related sars-and mers-causing viruses may hijack host cell machinery and evade immune detection, including repression of host mrna transcription in the nucleus (canton et al., 2018) , degradation of host mrna in the nucleus and cytoplasm (kamitani et al., 2009; , and inhibition of host translation (nakagawa et al., 2018) . nonetheless, the extent to which sars-cov-2 uses these or other strategies, and how they may be executed at a molecular level remains unclear. understanding the interactions between viral proteins and components of human cells is essential for elucidating their pathogenic mechanisms and for development of effective therapeutics. because sars-cov-2 is an rna virus and many of its encoded proteins are known to bind rna (sola et al., 2011) , we reasoned that these viral proteins may interact with specific human mrnas (critical intermediates in protein production) or non-coding rnas (critical structural components of diverse cellular machines) to promote viral propagation. here, we comprehensively define the interactions between each sars-cov-2 protein and human rnas. we show that 10 viral proteins form highly specific interactions with mrnas or ncrnas, including those involved in progressive steps of host cell protein production. we show j o u r n a l p r e -p r o o f 5 that nsp16 binds to the mrna recognition domains of the u1 and u2 rna components of the spliceosome and acts to suppress global mrna splicing in sars-cov-2-infected human cells. we find that nsp1 binds to a precise region on the 18s ribosomal rna that resides in the mrna entry channel of the initiating 40s ribosome. this interaction leads to global inhibition of mrna translation upon sars-cov-2 infection of human cells. finally, we find that nsp8 and nsp9 bind to discrete regions on the 7sl rna component of the signal recognition particle (srp) and interfere with protein trafficking to the cell membrane upon infection. we show that disruption of each of these essential cellular functions acts to suppress the type i interferon response to viral infection. together, our results uncover a multipronged strategy utilized by sars-cov-2 to antagonize essential cellular processes and robustly suppress host immune defenses. we cloned all 27 of the known sars-cov-2 viral proteins into mammalian expression vectors containing an n-terminal halotag (los et al., 2008) (figure s1a , methods), expressed each in hek293t cells, and exposed them to uv light to covalently crosslink proteins to their bound rnas. we then lysed the cells and purified each viral protein using stringent, denaturing conditions to disrupt any non-covalent associations and capture those with a uv-mediated interaction ( figure 1a , methods). as positive and negative controls, we purified a known human rna binding protein (ptbp1) and a metabolic protein (gapdh) (figure s1a-e). we successfully purified 26 of the 27 viral proteins (figure s1a ; full-length spike was not soluble when expressed). we found that 10 viral proteins (nsp1, nsp4, nsp8, nsp9, nsp12, nsp15, nsp16, orf3b, n, and e protein) bind to specific host rnas (p-value < 0.001, figure 1b , table s1), including 6 structural ncrnas and 142 mrnas (table s1). these include mrnas involved in protein translation (e.g. cops5, eif1, and rps12,), protein transport (atp6v1g1, slc25a6, and tomm20), protein folding (hspa5, hspa6, and hspa1b), transcriptional regulation (yy1, id4, and ier5), and immune response (jun, aen, and j o u r n a l p r e -p r o o f 6 rack1) (fdr < 0.05, figure 1b , s1f). importantly, the observed interactions are highly specific for each viral protein, and each protein binds to a precise region within each rna ( figures 1c, s1f ). using these data, we identified several viral proteins that interact with structural ncrna components of the spliceosome (u1 and u2 snrna), the ribosome (18s and 28s rrna), and the signal recognition particle (7sl) (figure 1b) . because these molecular machines are essential for three essential steps of protein production -mrna splicing, translation, and protein trafficking -we focused on their interactions with viral proteins to understand their functions and mechanisms in sars-cov-2 pathogenesis. after transcription in the nucleus, nascent pre-mrnas are spliced to generate mature mrnas which are translated into protein. splicing is mediated by a complex of ncrnas and proteins known as the spliceosome. specifically, the u1 small nuclear rna (snrna) hybridizes to the 5' splice site at the exon-intron junction and the u2 snrna hybridizes to the branchpoint site within the intron to initiate splicing of virtually all human mrnas (séraphin et al., 1988) . we identified a highly specific interaction between the nsp16 viral protein and the u1 and u2 snrnas ( figure 1b) . because u1 and u2 are small rnas (164 and 188 nucleotides, respectively), we noticed strong enrichment of nsp16-associated reads across the entire length of each. to more precisely define the binding sites, we exploited the well-described tendency of reverse transcriptase to preferentially terminate when it encounters a uv-crosslinked protein on rna (konig et al., 2010) (figures 1a, s1d) . we determined that nsp16 binds to the 5' splice site recognition sequence of u1 (figures 2a-b, s2a based on the locations of the nsp16 binding sites relative to the mrna recognition domains of the u1/u2 spliceosomal components, we hypothesized that nsp16 might disrupt splicing of newly transcribed genes ( figure 2f ). to test this, we co-expressed nsp16 in human cells along with a splicing reporter derived from irf7 (an exon-intron-exon minigene) fused to gfp (majumdar et al., 2018) . in this system, if the reporter is spliced, then gfp is made; if not, translation is terminated (via a stop codon present within the first intron) and gfp is not produced ( figure 3a) . we observed a >3-fold reduction in gfp levels in the presence of nsp16 compared to a control human protein (figures 3b, s3a ). to explore whether nsp16 has a global impact on splicing of endogenous mrnas, we measured the splicing ratio of each gene using nascent rna sequencing. specifically, we metabolically labeled nascent rna by feeding cells for 20 minutes with 5-ethynyl uridine (5eu), purified and sequenced 5eu-labeled rna, and quantified the proportion of unspliced fragments spanning the 3' splice site of each gene (figure 3c, s3b) . we observed a global increase in the fraction of unspliced genes in the presence of nsp16 compared to controls ( figure 3d, s3c,d) . given that nsp16 is sufficient to suppress global mrna splicing, we expect that its expression in sars-cov-2-infected cells would result in a global mrna splicing deficit. to test this, we infected human lung epithelial cells (calu3) with sars-cov-2 and measured splicing levels of newly transcribed mrnas compared to a mock infected control. as expected, we observed a global increase in the fraction of unspliced transcripts upon sars-cov-2 infection, with ~90% of measured genes showing increased intron retention (figure 3e, s3e ). together these results indicate that nsp16 binds to the splice site and branch point sites of u1/u2 to suppress global mrna splicing in sars-cov-2 infected cells ( figure 3f) . although nsp16 is known to act as an enzyme that deposits 2'-o-methyl modifications on viral rnas (decroly et al., 2011) , our results demonstrate that it also acts as a host virulence factor. global disruption of mrna splicing may act to decrease host protein and mrna levels by triggering nonsense-mediated decay of improperly spliced mrnas (kurosaki et al., 2019) . consistent with this, we observed a strong global decrease in steady-state mrna levels (relative to ncrna levels) upon sars-cov-2 infection ( figure s3f ). j o u r n a l p r e -p r o o f 8 inhibition of mrna splicing suppresses host interferon response to viral infection because many of the key genes stimulated by interferon (ifn) are spliced, we reasoned that mrna splicing would be critical for a robust ifn response. to test this, we utilized a reporter line engineered to express alkaline phosphatase upon ifn signaling (mimicking an antiviral response gene). this ifn stimulated gene (isg) reporter line can be stimulated using ifn-β and assayed for reporter induction. we observed strong repression of this ifn responsive gene upon expression of nsp16 ( figure 3g ) and upon addition of a small molecule that interferes with spliceosomal assembly (figure s3g ). these results demonstrate that one outcome of nsp16mediated inhibition of mrna splicing is to reduce the host cells' innate immune response to viral recognition. consistent with such a role, we observed an increase in intron retention within multiple ifn-responsive genes (such as isg15 and rig-i) upon sars-cov-2 infection ( figure 3h , s3h-i). once exported to the cytoplasm, spliced mrna is translated into protein on the ribosome. initiation of translation begins with recognition of the 5' cap by the small 40s subunit (which scans the mrna to find the first start codon). we observed that nsp1 binds exclusively to the 18s ribosomal rna (figure 1b and s4a ) -the structural rna component of the 40s ribosomal subunit. several roles for nsp1 have been reported in sars-cov and mers-cov including roles in viral replication, translational inhibition, transcriptional inhibition, mrna degradation, and cell cycle arrest (brockway and denison, 2005; kamitani et al., 2009; lokugamage et al., 2015; narayanan et al., 2015) . one of the reported roles for nsp1 in sars-cov is that it can associate with the 40s ribosome to inhibit host mrna translation (kamitani et al., 2009; tanaka et al., 2012 ), yet it remains unknown whether this association is due to interaction with the ribosomal rna, protein components of the ribosome, or other auxiliary ribosomal factors. accordingly, the mechanisms by which nsp1 acts to suppress protein production remain elusive. j o u r n a l p r e -p r o o f 9 we mapped the location of nsp1 binding to a 37 nucleotide region corresponding to helix 18 ( figure 4a) , adjacent to the mrna entry channel (simonetti et al., 2020) (figure 4b ). the interaction would position nsp1 to disrupt 40s mrna scanning and prevent translation initiation (figure 4b) , and disrupt trna recruitment to the 80s ribosome and block protein production ( figure s4b) . interestingly, the nsp1 binding site includes the highly conserved g626 nucleotide which monitors the minor groove of the codon-anticodon helix for trna binding fidelity (ogle et al., 2001) . we noticed that the c-terminal region of nsp1 has similar structural regions to serbp1 (brown et al., 2018) and stm1 (ben-shem et al., 2011a) , two known ribosome inhibitors that bind within the mrna entry channel to preclude mrna access ( figure s4c ). consistent with this, a recent cryo-em structure confirms that nsp1 binds to these same nucleotides of 18s within the mrna entry channel (thoms et al., 2020) . given the location of nsp1 binding on the 40s ribosome, we hypothesized that it could suppress global initiation of mrna translation. to test this, we performed in vitro translation assays of a gfp reporter in hela cell lysates and found that addition of nsp1 led to potent inhibition of translation ( figure s4d ). we observed a similar nsp1-mediated translational repression when we co-expressed nsp1 and a gfp reporter gene in hek293t cells (figure 4c-d) . in contrast, we did not observe this inhibition when we expressed other sars-cov-2 proteins (nsp8, nsp9, m) or human proteins (gapdh) ( figure 4d ). to determine if nsp1 leads to translational inhibition of endogenous proteins in human cells, we used a technique called surface sensing of translation (sunset) to measure global protein production levels (schmidt et al., 2009) . in this assay, translational activity is measured by the level of puromycin incorporation into elongating polypeptides ( figure s4e ). we observed a strong reduction in the level of global puromycin integration in cells expressing nsp1 compared to cells expressing gfp (figure s4f-g) . because nsp1 expression is sufficient to suppress global mrna translation in human cells, we hypothesized that sars-cov-2 infection would also suppress global translation. to test this, we infected a human lung epithelial (calu3) or monkey kidney (vero) cell line with sars-cov-2 and measured nascent protein synthesis levels using sunset. we observed a strong reduction of to explore whether nsp1 binding to 18s rrna is critical for translational repression, we generated a mutant nsp1 in which two positively charged amino acids (k164 and h165) in the c-terminal domain were replaced with alanine residues (figure s4c ) (narayanan et al., 2008) . we observed a complete loss of in vivo contacts with 18s ( figure 4g) ; because this mutant disrupts ribosome contact, we refer to it as nsp1∆rc. we co-expressed gfp and nsp1∆rc in hek293t cells and found that the mutant fails to inhibit translation ( figure 4h and s4j) . in contrast, mutations to the positively charged amino acids at positions 124/125 do not impact 18s binding ( figure 4g ) or the ability to inhibit translation ( figure 4h ). together, these results demonstrate that nsp1 binds within the mrna entry channel of the ribosome and that this interaction is required for translational inhibition of host mrnas upon sars-cov-2 infection. we explored whether nsp1 binding to 18s rrna suppresses the ability of cells to respond to ifn-β stimulation upon viral infection. we transfected isg reporter cells with nsp1, stimulated with ifn-β, and observed robust repression of the ifn responsive gene (>6-fold, figure 4i ). to confirm that this nsp1-mediated repression occurs in human cells upon activation of double stranded rna (dsrna)-sensing pathways typically triggered by viral infection, we treated a human lung epithelial cell line (a549) with poly(i:c), a molecule that is structurally similar to dsrna and known to induce an antiviral innate immune response (alexopoulou et al., 2001; kato et al., 2006) (figure s4k ). we observed a marked downregulation of ifn-β protein and endogenous ifn-β responsive mrnas in the presence of nsp1, but not in the presence of nsp1∆rc ( figure s4l, m) . these results demonstrate that nsp1, through its interaction with 18s rrna, suppresses the innate immune response to viral recognition ( figure 4j ). j o u r n a l p r e -p r o o f 11 because nsp1 blocking the mrna entry channel would impact both host and viral mrna translation, we explored how translation of viral mrnas is protected from nsp1-mediated translational inhibition. many viruses contain 5' untranslated regions that regulate viral gene expression and translation (gaglia et al., 2012) ; all sars-cov-2 encoded subgenomic rnas contain a common 5' leader sequence that is added during negative strand synthesis (kim et al., 2020b) . we explored whether the leader sequence protects viral mrnas from translational inhibition by fusing the viral leader sequence to the 5' end of gfp or mcherry reporter genes ( figure s5a ). we found that nsp1 fails to suppress translation of these leader-containing mrnas ( figure 5a -b, s5b). we dissected the leader sequence and found that the first stem loop (sl1) is sufficient to prevent translational suppression upon nsp1 expression ( figure 5c) or sars-cov-2 infection ( figure 5d ). we considered three models for how the leader could protect viral mrnas: (i) it could compete with the ribosome for nsp1 binding, (ii) it could directly recruit free ribosomes or (iii) nsp1 could bind to the leader independently of its ribosome interaction to allosterically modulate the nsp1-ribosome interaction. we reasoned that if the leader competes for nsp1 binding or directly recruits free ribosomes, then the presence of sl1 should be sufficient for protection, regardless of its precise position in the 5' utr. in contrast, if the leader allosterically modulates ribosome binding then the spacing between the 5' cap (which is bound to nsp1-40s) and sl1 would be critical for protection. to distinguish between these models, we swapped the location of sl1 and sl2 in the 5' leader or inserted 5 nucleotides between the 5' cap and sl1 ( figure s5c ) and found that both mutants ablate protection ( figure 5e, s5d) . these results indicate that an mrna requires the 5' leader to be precisely positioned relative to the nsp1-bound 40s ribosome to enable translational initiation ( figure 5f ). while many aspects of this allosteric model remain to be explored, it would explain how leader-mediated protection can occur on an mrna only when present in cis. moreover, this model suggests that nsp1 might also act to further increase viral mrna translation by actively recruiting the ribosome to its own mrnas. consistent with this, we observe a consistent ~20% increase in 12 translation of leader-containing reporter levels upon viral infection ( figure 5d ) or expression of nsp1 ( figure s5e ). upon engaging the start codon in an mrna, the 60s subunit of the ribosome is recruited to form the 80s ribosome which translates mrna. the signal recognition particle (srp) is a universally conserved complex that binds to the 80s ribosome and acts to co-translationally scan the nascent peptide to identify hydrophobic signal peptides present in integral membrane proteins and proteins secreted from the plasma membrane (akopian et al., 2013) . when these are identified, srp triggers ribosome translocation to the endoplasmic reticulum (er) to ensure proper folding and trafficking of these proteins to the cell membrane (akopian et al., 2013) . we identified two viral proteins -nsp8 and nsp9 -that bind at distinct and highly specific regions within the s-domain of the 7sl rna scaffold of srp ( figure 6a , s6a). nsp8 interacts with 7sl in the region bound by srp54 (the protein responsible for signal peptide recognition, srp-receptor binding, and ribosome translocation) (akopian et al., 2013; holtkamp et al., 2012) ( figure 6b ). nsp9 binds to 7sl in the region that is bound by the srp19 protein ( figure 6b ), which is required for proper folding and assembly of srp (including proper loading of srp54) (akopian et al., 2013) . because srp scans nascent peptides co-translationally, we were intrigued to find that nsp8 also forms a highly specific interaction with 28s rrna (the structural component of the 60s subunit) ( figure 6c, s6b) . the binding site on 28s rrna corresponds to the largest human-specific expansion segment within the ribosome, referred to as es27 (parker et al., 2018) . es27 is highly dynamic, and thus has not been resolved in most ribosome structures (zhang et al., 2014) . however, when engaged by specific factors, es27 can become ordered, and was recently shown to be capable of interacting with the ribosome exit tunnel, adjacent to the 60s binding site of srp ( figure 6d , s6c) (wild et al., 2020) . together, these observations suggest that nsp8 and nsp9 bind to the co-translational srp complex. consistent with this, we find that nsp8 and nsp9 localize broadly throughout the j o u r n a l p r e -p r o o f 13 cytoplasm when expressed in human cells ( figure s6d ) or upon sars-cov-2 infection ( figure s6e -f). because nsp8 and nsp9 binding on 7sl are positioned to disrupt srp function, we hypothesized that they may alter translocation of secreted and integral membrane proteins ( figure s7a ). to test this, we expressed an srp-dependent membrane protein (nerve growth factor receptor, ngfr (izon et al., 2001a) ) fused via an internal ribosome entry site (ires) to a non-membrane gfp ( figure s7f ). in this system, if a perturbation specifically affects membrane protein levels we expect to see a decrease in the ratio of membrane to non-membrane protein levels. to ensure that the ngfr reporter accurately reports on srp function, we treated hek293t cells with sirnas against srp54 or srp19 and found that both lead to a dramatic reduction of the ngfrmembrane protein relative to the non-membrane gfp protein ( figure s7b) . similarly, we found that expression of nsp8 and nsp9 (alone or together) lead to a striking reduction in expression of ngfr relative to gfp ( figure 7a ). expression of control proteins did not specifically impact ngfr levels ( figure 7a, s7b) . to determine if there is a global effect on membrane protein levels, we utilized the sunset method to measure puromycin levels in membrane proteins using flow cytometry (see methods). we confirmed that disruption of srp leads to a global reduction in puromycin levels in the cell membrane ( figure s7c ). we observed a comparable global reduction of puromycin-labeled membrane proteins upon expression of nsp8 or nsp9 individually or together, but not with control proteins (figure 7b, s7c) . because nsp8 and nsp9 are each sufficient to suppress protein integration into the cell membrane, we anticipate that sars-cov-2 infection would lead to similar suppression. j o u r n a l p r e -p r o o f 14 however, determining whether sars-cov-2 infection specifically impacts membrane protein expression is confounded by the fact that nsp1 inhibits translation of membrane and nonmembrane proteins upon infection. to address this, we co-expressed a membrane protein reporter (ngfr) containing the 5' viral leader along with a non-membrane gfp reporter containing the viral leader. upon viral infection, we observed a strong reduction of membrane protein levels ( figure 7c ), but no reduction in non-membrane gfp levels ( figure 5d ). to ensure that these effects are specific to sars-cov-2 infected cells, we separated individual cells within the infected population into those expressing the viral spike protein (s+) and those not expressing the protein (s-). we found that the shift in membrane protein levels only occurs in s+ cells ( figure 7d ), while the spopulation resembled the mock infected samples ( figure 7c ). we observed a strong relationship between the level of spike protein -likely reflecting the amount of viral replication within each cell -and the level of membrane protein suppression ( figure 7c ). we observed this membrane protein-specific decrease upon infection of human lung epithelial (calu3, figure s7d ) and monkey kidney (vero, figure 7c -d) cell lines. together, these results demonstrate that nsp8 and nsp9 bind to 7sl to disrupt srp function and suppress membrane protein trafficking in sars-cov-2 infected cells. although nsp8 and nsp9 are thought to be components of the viral replication machinery (sutton et al., 2004) , our results indicate that they play an additional role as host virulence factors. because viral membrane proteins also require trafficking to the er, viral disruption of srp might negatively impact viral propagation, unless viral proteins are trafficked in an srp-independent manner ( figure s7e ) or if nsp8/9 selectively impacts host (but not viral) proteins. next we explored how disruption of srp might be advantageous for viral propagation. because secretion of ifn and other cytokines is dependent on the srp complex for secretion ( figure s7f ), a central component of the ifn response is dependent on srp. accordingly, we hypothesized that nsp8/9-mediated viral suppression of srp would act to suppress the ifn j o u r n a l p r e -p r o o f 15 response upon infection. to test this, we co-expressed nsp8 and nsp9 and observed a significant reduction in the ifn response relative to a control protein ( figure s7g ). together, these results suggest that sars-cov-2 mediated suppression of srp-dependent protein secretion enables suppression of host immune defenses ( figure 7e) . interestingly, many proteins involved in anti-viral immunity -including most cytokines and class i major histocompatibility complex -are membrane-anchored or secreted, and are known to use the srp pathway for transport (vermeire et al., 2014) (figure s7f ), suggesting that there may be other effects of srp pathway inhibition on sars-cov-2 pathogenesis. we identified several pathogenic functions of sars-cov-2 in human cells -including global inhibition of host mrna splicing, protein translation, and membrane protein trafficking -and described the molecular mechanisms by which the virus acts to disrupt these essential cell processes. interestingly, all of the viral proteins involved (nsp1, nsp8, nsp9, and nsp16) are produced in the first stage of the viral life cycle, prior to generation of double stranded rna (dsrna) products during viral genome replication. because dsrna is detected by host immune sensors and triggers the type i interferon response, disruption of these cellular processes would allow the virus to replicate its genome while minimizing the host innate immune response. disruption of these three non-overlapping steps of protein production may represent a multipronged mechanism that synergistically acts to suppress the host antiviral response ( figure 7f) . specifically, the ifn response is usually boosted >1,000-fold upon viral detection (through amplification and feedback, figure s4k ), yet each individual mechanism impacts ifn levels on the order of ~5-10-fold. accordingly, if each independent mechanism impacts ifn levels moderately, the three together may be able to achieve dramatic suppression of ifn (10 3 =1,000fold). this multi-pronged mechanism may explain the molecular basis for the potent suppression of ifn observed in severe covid-19 patients. interferon is emerging not only as a determinant of disease severity, but also a potential treatment option (zhou et al., 2020) . as such, our work identifies several therapeutic opportunities for boosting ifn levels upon sars-cov-2 infection. for example, disrupting the interaction between nsp1 and 18s rrna could allow cells to detect and respond to viral infection. because many small-molecule drugs target ribosomal rnas (liaud et al., 2019) , it may be possible to develop drugs to block the nsp1-18s and other interactions. additionally, disrupting the 5' viral leader may be a potent antiviral strategy since it is critical for translation of all viral proteins. because sl1 is a structured rna, it may be possible to design small molecules that specifically bind this structure to suppress viral protein production (hermann, 2016) . viral suppression of these cellular functions is not exclusive to the ifn response and will also impact other spliced, translated, secreted, and membrane proteins. many proteins involved in anti-viral immunity are spliced and/or membrane-anchored or secreted. for example, class i major histocompatibility complex (mhc), which is critical for antigen presentation to cd8 t cells at the cell surface of infected cells (hansen and bouvier, 2009 ). by antagonizing membrane trafficking, sars-cov-2 may prevent viral antigens from being presented on mhc and allow infected cells to escape t-cell recognition and clearance. in this way, interference with these essential cellular processes might further aid sars-cov-2 in evading the host immune response. more generally, we expect that insights gained from the sars-cov-2 protein-rna binding maps will be critical for exploring additional viral mechanisms. specifically, we identified many other interactions, including highly specific interactions with mrnas. for example, nsp12 binds to the jun mrna ( figure s1e ) which encodes the critical immune transcription factor c-jun which is activated in response to multiple cytokines and immune signaling pathways (weston and davis, 2007) . we also identified an interaction between nsp9 and the start codon of the mrna that encodes cops5 (figure 1c) , the enzymatic subunit of the cop9 signalosome complex which regulates protein homeostasis (cope and deshaies, 2003) , suggesting that it might disrupt its translation. interestingly, cops5 (also known as jab1) is known to bind and stabilize c-jun protein levels (claret et al., 1996) and several viruses are known to disrupt this protein (lungu et al., 2008; oh et al., 2006; tanaka et al., 2006) . while it remains unknown what, if any, role these interactions play in virally infected cells, the specificity suggests that they may provide a selective advantage for viral propagation. together, our results demonstrate that global mapping of rna binding by viral proteins could enable rapid characterization of mechanisms for emerging pathogenic rna viruses. we note several limitations of our current study that will need to be explored in future work. (i) our mapping experiments were performed in uninfected human cells expressing tagged viral proteins. accordingly, it remains possible that our maps may not fully capture all of the interactions that occur when human cells are infected, such as interactions that occur with viralinduced rnas, in specific viral compartments, or that require multiple viral proteins. (ii) while we characterized the functional and mechanistic roles of several viral proteins and structural ncrnas, we did not explore what roles viral protein interactions with mrnas might play. (iii) how the virus disrupts fundamental cellular processes while still maintaining its own production is still largely undefined. while we showed that the 5' leader is sufficient to relieve translational inhibition by nsp1, we still do not fully understand how this protection occurs and specifically how nsp1 might interact with the viral leader or allosterically modulate ribosome binding. similarly, viral membrane proteins are dependent on trafficking to the er and how nsp8/9 might selectively impact er translocation of host -but not viral -proteins remains to be explored. (iv) while we showed that viral disruption of these essential cellular functions can suppress ifn, what other roles host cell shutdown might play in viral pathogenesis and in suppressing other aspects of anti-viral immunity, including possible roles in adaptive immune responses, have not been explored. the authors declare no competing interests. further information and requests for reagents and resources should be directed to and will be fulfilled by the lead contact, mitchell guttman (mguttman@caltech.edu). all constructs and plasmids generated in this study will be made available on request sent to the lead contact with a completed materials transfer agreement. all datasets generated during this study are available at ncbi short read archive: bioproject prjna665692 (viral protein purifications) and prjna665581 (nascent and total rna-seq) essential medium (atcc) containing 10% fbs and 1% penicillin-streptomycin purchased from thermo fisher scientific. all cell lines were maintained at 37°c under 5% co 2 . cells were grown in a humidified incubator at 37ºc with 5% co 2 . all experiments using infectious sars-cov-2 conducted at the uvm bsl-3 facility were cloning of expression constructs. sars-cov-2 protein constructs (with the exception of nsp11) were a gift from fritz roth (see table s3 for addgene information) (kim et al., 2020a) and were lr-cloned (invitrogen gateway cloning, thermo fisher scientific) into mammalian expression destination vector pcag-halo-tev-dest-v5-ires-puror. note that following lr cloning, proteins were not v5-tagged because all entry clones contained stop codons. for nsp11, an entry clone was generated by bp cloning (invitrogen gateway cloning, thermo manganese/calcium mix (0.5mm cacl 2 , 2.5 mm mncl 2 ). samples were incubated on ice for 10 minutes to allow lysis to proceed. the lysates were then incubated at 37°c for 10 minutes at 700 rpm shaking on a thermomixer (eppendorf). lysates were cleared by centrifugation at 15,000× g for 2 minutes. the supernatant was collected and kept on ice until bound to the halolink resin (promega). of the 1ml lysis volume, 50ul was set aside for input, 20ul used for protein expression confirmation, and the rest for capture on halolink resin as described below. for qpcr analysis, cdna was generated from purified rna using maxima h-reverse transcriptase (thermo fisher scientific) following manufacturer's recommendations. amplification reactions were assembled with primer sets indicated in table s2 and lightcycler® 480 sybr green i master (roche) following manufacturer's protocols and read out in a roche lightcycler 480. library construction. rna-seq libraries were constructed from purified rna as previously described (van nostrand et al., 2016) . briefly, after proteinase k elution, the rna was dephosphorylated (fast ap) and cyclic phosphates removed (t4 pnk) and then cleaned using silane beads as previously described (van nostrand et al., 2016 ). an rna adapter containing a rt primer binding site was ligated to the 3' end of the cleaned and end-repaired rna. the ligated rna was reverse transcribed (rt) into cdna, the rna was degraded using naoh, and a second adapter was ligated to the single stranded cdna. library preparation was the same for input samples except that an initial chemical fragmentation step (90°c for 2 min 30 s in 1x fastap buffer) was included prior to fastap treatment. this chemical fragmentation step was designed to be similar to the fragmentation conditions used for purified halo bound samples. the for staining of infected cells, cells were fixed and permeabilized in 8% formaldehyde 1% triton, and subsequently labelled with primary antibodies raised in sheep to sars-cov-2 at 1/500 dilution, followed by incubation with a rabbit anti-sheep alexa 555 secondary antibody (abcam, ab150182) at 1/1000 dilution and mounted with dapi in the medium (thermo fisher scientific, cat# p36395). cells were imaged with a zeiss lsm 880 confocal microscope, with 1 airy unit pinhole for all primary antibody channel acquisitions and pixel size 0.07 µm x 0.07 µm. the objective lens used was a zeiss plan-apochromatic 63x/1.4na m27. structure modeling nsp1 homology model. the predicted model of sars-cov-2 nsp1 was generated using the transform-restrained rosetta (trrosetta) algorithm, a deep learning-based modeling method based on the rosetta energy minimization pipeline with additional distance and interaction restraints generated from co-evolution (yang et al., 2020) . all figures were generated using pymol (www.pymol.org). nsp1-ribosome model. the model of nsp1 bound to the ribosome was generated using modeller version 9.24 (webb and sali, 2016) . the c-terminal sequence of nsp1 (khssgvtrelmrelngg) was modeled using the structure of serbp1 bound to the ribosome (pdb id: 6mte, chain w) as a template. the default modeller parameters were used to create an alignment of nsp1 and serbp1 and to generate the model, and all atoms within 6å of serbp1 were included in the model to define the neighboring environment. twenty models were generated and the model with the lowest dope score was selected to visualize with pymol (delano, 2002) . x-ray crystal structures and cryo-electron microscopy structures were obtained from the protein data bank (www.rcsb.org) (berman et al., 2000) and visualized with pymol (delano, 2002) . for u1 and u2 structural analysis, we used a cryo-em structure of the pre-catalytic human spliceosome (pdb id: 6qx9). for 7sl structural analysis, we used an x-ray crystal structure of the human signal recognition particle (pdb id: 1mfq). to examine human srp in the context of the ribosome, we used a cryo-em structure of the mammalian srp-ribosome complex (pdb id: 3jaj). to analyze the ribosomal es27 expansion segment, we superimposed a cryo-em structure of the expansion segment (pdb id: 6sxo) onto the complete ribosome structure (pdb id: 3jaj) using the pymol command "super." finally, for nsp1-18s rrna structural analysis, we used multiple structures of the ribosome, including structures of the pre-40s subunit (pdb id: 6g5h), 48s late-stage initiation complex (pdb id: 6yal), 80s in complex with serbp1 (pdb id: 6mte), and 80s in complex with stm1 (pdb id: 4v88). j o u r n a l p r e -p r o o f 37 nsp1 was cloned into a bacterial expression vector resulting in n-terminally tagged halo-6xhistagged nsp1. the nsp1 sequence was pcr amplified from addgene nsp1 entry vector to add a n-terminal 6x his tag and restriction enzyme sites for digestion and ligation into n-terminal halo bacterial expression vector. this construct was transformed into bl21 de3 e. coli (agilent), expanded to a 500ml liquid culture, and grown until od 600 reached 1.0. iptg was added to a final concentration of 1mm. after 3 hours of iptg induction, bacteria was centrifuged for 15 min at 5000× g. pellet was lysed with binding buffer (50mm hepes, ph 7.5, 20mm mgcl 2 , 600mm nacl, 2mm tcep, 10mm imidazole, 2mm atp, 1% triton x-100) supplemented with atp (2mm), protease inhibitor cocktail (promega), benzonase (sigma) and triton-x 100 (sigma) using 5ml of lysis mix per gram of wet cell paste. cell suspension was rocked for 20 min at room temperature and then centrifuged at 16,000× g for 20 min at 4°c. supernatant was incubated with washed imac resin (bio-rad) and rocked for 20 min at room temperature. we loaded the resin-lysate mixture into an appropriately-sized column and washed with 5 column volumes of binding buffer (50mm hepes, ph 7.5, 20mm mgcl 2 , 600mm nacl, 2mm tcep, 10mm imidazole, 2mm atp, 1% triton x-100) followed by 10 column volumes of wash buffer (50mm hepes, ph 7.5, 600mm nacl, 2mm tcep, 20mm imidazole, ph 8). recombinant nsp1 (rnsp1) was eluted with 5 column volumes of elution buffer by adding 1 column volume at a time with column flow stopped, collecting eluate after each addition, and waiting 15 min between each elution buffer addition. we dialyzed these eluates with a 10ml spectra-por® float-a-lyzer® g2 (spectrum laboratories) into storage buffer (50mm hepes, ph 7.5, 150mm nacl, 10% glycerol) at 4°c using 2 exchanges, one after 2 hours and then overnight. pierce 1-step human coupled ivt-dna (thermo fisher scientific) in vitro translation kit was used to measure rnsp1-dependent translation inhibition. bovine serum albumin (bsa), and buffer only controls were used to control for the addition of excess protein or changes in buffer composition. to measure translation inhibition, 5µl in vitro translation reactions were assembled, scaled according to manufacturer's recommendations. the included control plasmid pcfe-gfp was used to measure translational output of the reactions. gfp fluorescence was measured on a biotek cytation3 plate reader using emission filters for gfp fluorescence. 1.5µm j o u r n a l p r e -p r o o f 38 stock dilutions of rnsp1 and bsa were made in storage buffer (50mm hepes, ph 7.5.,150mm nacl,10% glycerol). subsequent 10 fold dilutions were made in storage buffer to span a concentration range of 1000 nm to 1 nm for each protein in the final reaction. 10 µl of the diluted protein solution was added to the 5µl translation reactions, and incubated for 5 minutes at room temperature prior to the addition of the gfp reporter plasmid. duplicate reactions were made to measure variability for each condition. in addition, a buffer only control was included to measure the effect of dilution of the translation reaction by the storage buffer. after the 5 minute incubation, 50 ng of gfp reporter plasmid was added to each reaction and incubated at 30°c for 4 hours prior to fluorescence detection. two microliters from each reaction was measured in duplicate on a biotek cytation3 microplate reader using excitation and emission filters for gfp. sample readings were blanked by subtracting values obtained from the buffer only control. promega's rabbit reticulocyte lysate system was also used to assay translation inhibition. to measure translation inhibition, 10µl in vitro translation reactions were assembled, scaled according to manufacturer's recommendations. for each translation reaction, either 10µl of recombinant protein storage buffer or rnsp1 was added, followed by 500ng of mrna. after 4 hours of incubation at 30°c, luciferase was read out using the bright-glo luciferase assay (promega) or gfp fluorescence was measured, both on a biotek cytation3 plate reader. we assayed translation in hek293t cells transfected with mammalian expression vectors, mrnas, or combinations of these. for mrna transfections of fluorescence protein translation reporters (including unmodified, +sars-cov2 leader sequence, +sl1, +sl2-sl1, and +5nts), dna templates for in vitro transcription were generated with sequences appended to the 5' end of gfp and mcherry (see tables s4 and s5 for primers and templates, respectively) and transcribed using hiscribe™ t7 arca mrna kit with tailing (new england biolabs). for nsp1 mrna transfection, indicated primers from table s4 were used to add restriction enzyme sites for cloning into pt7cfe1-chis backbone provided in the pierce human 1-step coupled acquistion files were analyzed with flowjo analysis software. to assay global protein translation, a sunset assay was performed as previously described (schmidt et al., 2009) we transfected these mammalian expression vectors for nsp1 and gfp into hek293t using biot transfection reagent. after 3 hours, doxycycline (sigma) was added to a final concentration of 2µg/ml. after 24 hours, cells were incubated with puromycin (10µg/ml) for 10 min, then washed with fresh media, and harvested with cold pbs. pelleted cells were lysed for 10 min on j o u r n a l p r e -p r o o f 40 ice (mixing after 5 min) with 100ul ripa buffer supplemented with protease inhibitor cocktail (promega). insoluble debris was pelleted by centrifuging at 12,500 × g for 2.5 minutes and supernatant was run on a bolt™ 4-12% bis-tris plus gel (thermo fisher scientific). proteins were then transferred to nitrocellulose using the iblot transfer system (thermo fisher scientific) and western blotting carried out using an anti-puro antibody (clone 12d10, emd millipore). sunset in sars-cov-2 infection was performed as above with the following modifications. cells were infected or not (mock) with sars-cov-2, and 48 hpi cells were incubated with puromycin (10µg/ml) for 20 min. media was aspirated and cells lysed directly in 2x laemmli's buffer (biorad), heated at 95ºc for ten minutes and run on a 4-12% nupage gel (thermo fisher scientific). proteins were transferred to nitrocellulose using the iblot transfer system and probed as above. to assay srp-dependent membrane protein transport to the cell surface, we monitored surface arrival of exogenously expressed neuronal growth factor receptor (ngfr) by flow cytometry in the presence of nsps. mammalian expression vectors were exchanged for versions that contained an ires-ngfr to co-express a membrane reporter and thus, for these experiments, lr reactions were carried out with destination vector pb-6xhis-gfp-dest-ires-ngfr. resulting expression vectors drive protein expression by a dox-inducible promoter, contain the rtta needed for dox induction, and produce an n-terminally-tagged his-gfp fusion protein and a co-expressed ngfr. the gfp here is an enhanced gfp containing an amino acid substitution (a205k) to generate a monomeric variant based on previous literature (alberti et al., 2018) . we transfected these mammalian expression vectors for nsp8, nsp9, nsp1∆rc mutant and to knockdown srp19 and srp54, sirnas targeting each (dharmacon cat# l-019729-01-0005 and l-005122-01-0005, respectively) were transfected into hek293t cells using lipofectamine rnaimax (invitrogen) according to manufacturer's protocols. to validate knockdown, transfected cells were assayed by qpcr using primer sets (table s2 ) to amplify each target as well as normalizer calm3. transfections were carried out 48 hours prior to assaying cells, either by qpcr, membrane reporter, or membrane sunset (see below) experiments. calu3 and vero cells were transfected with mrnas encoding leader-ngfr and leader-gfp using transit-mrna transfection kit (mirus) and subsequently infected with sars-cov-2 at an moi of 0.1. after 24 hours, cells were washed with pbs, trypsinized and fixed in 4% pfa for 20 minutes before staining with biotinylated anti-ngfr (biolegend) and anti-sars-cov-2 spike antibody (sino) and subsequently stained with pe-labeled anti-rabbit (thermo, p-2771mp) and pacblue-labeled streptavidin (thermo, s1222). facs was performed on a macsquant flow cytometer and analyzed using flojo analysis software; facs distributions were compared using a 2-tailed kolmogorov-smirnov test. for these experiments, rna was transcribed from a pcr template (see table s4 ) using the hiscribe t7 arca mrna kit (with tailing). to assay transport to the cell surface of all plasma membrane proteins, the sunset assay was adapted to puro-label surface proteins as previously described (schmidt et al., 2009) , and read out by flow cytometry. briefly, cells were incubated with puromycin as described above, followed by two quick washes and a chase with fresh complete media for 50 min. cells were lifted with 1mm edta as described above and stained with an anti-puro antibody (clone 12d10, emd millipore) conjugated to alexa-647. for these experiments, nsp was expressed from the same vector described above for membrane reporter assays. fluorescence intensity j o u r n a l p r e -p r o o f 42 measurements were taken for gfp and alexa-647 on a macsquant flow cytometer and analyzed using flojo analysis software; distributions were compared using a 2-tailed kolmogov-smirnov. to assess splicing efficiency, exons 5-6 of mouse irf7 (enmust00000026571.10) containing its endogenous intron were fused upstream of 2a self-cleaving peptide and egfp and cloned into an mscv vector (pig, addgene) (mayr and bartel, 2009 ). this construct was co-transfected into hek293ts with nsp16 or gfp and measured 24 hours after transfection by flow cytometry (macsquant) and analyzed using flojo analysis software. sars-cov2 or mock infected calu3 cells and nsp16-or gapdh-expressing hek293ts were labeled with 5-ethynyl-uridine (5eu; jena bioscience) by adding 5eu containing media to cells for 20 min at a final concentration of 1mm, as previously described (jao and salic, 2008) . after the pulse label, cells were washed with warm pbs and lysed in rlt buffer (qiagen). total rna was isolated from cells using manufacturer's protocols for qiashredder and rneasy rna isolation (both qiagen), followed by turbo dnase treatment (ambion, thermo scientific), and zymo rna clean and concentrate. for each sample, 2µg of rna was used for ligation of a unique barcoded rna adaptor, following the relevant steps in the protocol described above in library construction of rna-seq libraries. samples were then pooled before proceeding to biotinylation steps. to biotinylate 5eu-labeled rna, samples were first mixed, in order, with water, hepes (100 mm), biotin picolyl azide (1 mm; click chemistry tools) and ribolock rnase inhibitor, then added to premixed cuso 4 (2 mm) and thpta (10mm), and finally added to freshly prepared j o u r n a l p r e -p r o o f 43 sodium ascorbate (12mm), as previously described (hong et al., 2009) . the click reaction was incubated for 1 hour at 25ºc with 1000rpm shaking on an eppendorf thermomixer followed by rna purification using >17nt protocol for zymo clean and concentrate. we completed three rounds of sequential capture on streptavidin beads to isolate nascent transcripts (see figure s3b ). to capture biotinylated rna, myone streptavidin c1 dynabeads (thermofisher scientific) were first washed three times in urea buffer (10mm hepes, ph 7.5, 10mm edta, 0.5m licl, 0.5% triton x-100, 0.2% sds, 0.1% sodium deoxycholate, 2.5mm tcep, 4m urea) followed by three additional washes in m2 buffer (20mm tris, ph 7.5, 50mm nacl, 0.2% triton x-100, 0.2% sodium deoxycholate, 0.2% np-40). washed beads were mixed with 3 parts 4m urea buffer and 1 part biotinylated rna and incubated for 60 min with 900rpm thermomixer shaking at room temperature. after magnetic separation, beads were washed 3 times with m2 buffer followed by 3 washes with urea buffer at 37 ºc at 750rpm for 5 min. rna was eluted from beads in 2 sequential elutions by incubating with elution buffer (5.7m guanidine thiocyanate , 1% n-lauroylsarcosine; both sigma) at 65 ºc for 2 minutes, repeating with more elution buffer for a second elution. the elutions were pooled, diluted with urea buffer, incubated with pre-washed streptavidin beads, washed, and eluted for 2 additional rounds exactly as described above for a total of 3 sequential captures. final elutions were pooled, cleaned with zymo rna clean and concentrate following manufacturer's protocols, and carried through rna-seq library preparation as described above starting with the reverse transcription step. hek-blue™ isg cells were seeded in 96 well plates, transfected with nsp1 mammalian expression vectors using biot and stimulated with 50 ng/ml human ifn-b (r&d systems). supernatants were assayed for alkaline phosphatase as per manufacturer instructions using j o u r n a l p r e -p r o o f 44 quanti-blue reagent (invivogen). hek-293t cells were seeded in 6 well plates, transfected with either halo-tagged gapdh, nsp1, nsp8 and nsp9 in combination, or nsp16 mammalian expression vectors using biot. 24 hours later, the media was replaced with media containing 50 ng/ml human ifn-β (r&d systems). expression was assayed using live cell halo-imaging. halo-tmr ligand was diluted 1:200 in media and added to the culture for a 1:1000 final dilution. samples were incubated 30 minutes at 37°c, 5% co 2 and then the media was aspirated. wells were rinsed twice with pbs, then media was added back to the wells. samples were incubated 30 minutes at 37°c, 5% co 2 to allow uncoupled ligand to diffuse out of the cells. media was then aspirated and replaced, and cells were imaged by widefield fluorescence microscopy. cultures were ultimately harvested for rna 24 hours later, or 48 hours post transfection. a549s were seeded in 6 well plates, transfected with nsp1 mammalian expression vectors using lipofectamine 2000 and stimulated with 1 µg/ml hmw poly(i:c) (invivogen) 24h after transfection. supernatant was assayed for secreted ifn-β by elisa (human ifn beta elisa, high sensitivity, pbl) 24 hours after stimulation, and rna from cells was purified and assessed for isg gene expression as normalized to gapdh expression (sybr green master mix, bio-rad). primers used for qpcr are listed in table s2 . sars-cov-2 leader sequence was appended to the 5' end of gfp and mcherry reporter templates via pcr. pcr templates were then transcribed using hiscribe t7 arca mrna kit (with tailing). leader mutants, including sl1 only, sl1/sl2 swap, and +5nts mutants were likewise appended to the 5' end of fluorescent reporter templates via pcr and transcribed using hiscribe t7 arca kit. mrna reporters were transfected in hek-293t cells with lipofectamine messengermax. to measure fluorescence of mcherry and gfp reporters, 24 hours post transfection cells were either lifted with pbs and transferred into black 96 well plates for fluorescence readout on a biotek cytation 3 or trypsinized and processed for flow cytometry. sequence alignment and analysis. for halo purifications and rna binding mapping sequencing reads were aligned to a combined genome reference containing the sequences of structural rnas (ribosomal rnas, snrnas, snornas, 45s pre-rrna) and annotated mrnas (refseq hg38) using bowtie2. to distinguish between the nascent pre-ribosomal rna and mature 18s, 28s, and 5.8s rrna, we separated each of the components of the 45s into separate sequence units for alignment (e.g. its, ets). we excluded all low quality alignments (mapq < 2) from the analysis. for mrna analysis, we removed pcr duplicates using the picard markduplicates function (https://broadinstitute.github.io/picard/). for each rna, we enumerated 100 nucleotide windows across the entire rna. for each window, we calculated the enrichment by computing the number of reads overlapping the window in the protein elution sample divided by the total number of reads within the protein elution sample. we normalized this ratio by the number of reads in the input sample divided by the total number of reads in the input sample. because all windows overlapping a gene should have the same expression level in the input sample (which represents rna expression), we estimated the number of reads in the input as the maximum of either (i) the number of reads over the window or (ii) the median read count over all windows within the gene. this approach provides a conservative estimation of enrichment because it prevents windows from being scored as enriched if the input values over a given window are artificially low, while at the same time accounting for any non-random issues that lead to increases in read counts over a given window (e.g. fragmentation biases or alignment artifacts leading to non-random assignment or pileups). j o u r n a l p r e -p r o o f 46 we calculated a multiple testing corrected p-value using a scan statistic, as previously described (guttman et al., 2009 (guttman et al., , 2010 . briefly, n was defined as the number of reads in the protein elution plus the number of reads in the control sample. p was defined as the total number of reads in the protein elution sample divided by the sum of the protein elution sample total reads and total reads in the control sample. w was the size of the window used for the analysis (100 nucleotides). the scan statistic p-value was defined using the poisson estimations based on standard distributions previously described (naus, 1982) . because rna within input samples are fragmented differently than the protein elution samples, we noticed that the overall positional distribution of protein elution samples was distinct from input distributions. accordingly, we used the remaining protein elution samples (rather than input) as controls for each protein. specifically, this enabled us to test whether a given protein is enriched within a given window relative to all other viral and control proteins. enrichments were computed as described above. these values are plotted in figure 1 and table 1 . igv (robinson et al., 2011) and were generated by either: (i) computing the enrichment for each nucleotide as described above. in this case, the read count for each nucleotide was computed as the total number of reads that overlapped the nucleotide. (ii) counting the number of rt stop sites at a given nucleotide. in this case, we compute the alignment start position of the second in pair read and computed a count of each nucleotide. we normalized this count by the total number of reads in the sample to account for sequencing depth generated. we then normalized this ratio by the same ratio computed for the control sample (merge of all other protein samples) for each nucleotide. heatmaps were generated using morpheus (https://software.broadinstitute.org/morpheus/). all values were included if they contained a significant 100nt window with a p-value<0.001 (see above) and minimum enrichment of 3-fold above the control sample. gene ontology analysis. the 66 non-n enriched mrnas were analyzed against the gene ontology biological processes and reactome gene sets using the molecular signatures database (msigdb) (liberzon et al., 2015) . significantly enriched gene sets with an fdr<0.05 were used. j o u r n a l p r e -p r o o f 47 to ensure that significant gene sets were not being driven by the multiple ribosomal proteins or histone proteins, these analyses were also carried out excluding these proteins. sequenced reads were demultiplexed according to barcoded rna adaptor sequences ligated to each respective sample. trimmomatic (https://github.com/timflutre/trimmomatic ) was used to remove any contaminating illumina primer sequences in the reads and low quality reads. demultiplexed and trimmed files were then aligned to a hg19 reference genome using the spliceaware star aligner (https://github.com/alexdobin/star). alignments were then deduplicated for pcr duplicates using picard markduplicates (https://broadinstitute.github.io/picard/). aligned read-fragments were defined as read1 and read2 contained within a paired-end read fragment along with the insert between these two reads. we defined a set of high-quality represented isoforms per gene using the appris database (rodriguez et al., 2013) . all readfragments that spanned any 3' splice site within an isoform of one of these genes was retained. for each 3' splice site spanning fragment, we classified the read-fragment as a spliced fragment if it spanned an exon-exon junction (e.g. aligned entirely within 2 distinct exons) or an unspliced fragment if it spanned an intron-exon junction (e.g. one of the reads was contained -or partially contained -within the intron). for each isoform, we computed an unspliced ratio by counting the total number of reads that were classified as unspliced divided by the total number of readfragments spanning 3' splice sites within that gene. to ensure that the splicing ratio that we measured is a reliable metric and not inflated/deflated due to low read counts, we only included genes that contained at least 10 read-fragments in each sample and where the total number of reads in the control and sample conditions (when merged together) contained a significant number of reads to reliably measure a difference between the two groups as measured by a hypergeometric test (p<0.01). because different genes contain different baseline splicing ratios due to gene length and coverage, we computed a change in the splicing ratio for each gene independently. to do this, we subtracted the unspliced ratio for each sample from the average unspliced ratio for that gene j o u r n a l p r e -p r o o f 48 in all of the control samples. we plotted the overall distribution of these differences in splicing ratios as violin plots for each sample. if there is no change in splicing ratio, we would expect that some genes would have higher splicing ratios and others lower splicing ratios but that the overall distribution would be centered around 0. total rna-seq libraries were generated from the same mock infected and sars-cov-2 virally infected calu3 samples treated with 5eu. prior to 5eu purification, total rna was taken and an rna-seq library constructed as described above using barcoded rna adapters. cytoplasmic ribosomal rnas (18s and 28s) were depleted using nebnext ribosomal rna depletion kit (neb e6310l) per manufacturers recommendations. demultiplexed reads were aligned using bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) to custom genomes encoding classical noncoding rnas (ncrnas) or human messenger rnas (mrnas). expression levels were computed for each mrna by counting the total number of sequencing reads aligned to the mature mrna. to normalize across the different libraries, we computed the read counts for each sample that align to non-spliced structural non-coding rnas -excluding rrna but including snrnas, 7sl, 7sk, etc. we then divided each mrna count by the sum of all ncrna counts. this normalized value for each gene per sample was then converted into a fold-change by dividing this normalized value to the mean value for both mock infected samples. the fold change of each gene relative to mock was plotted across all mrnas as a violin plot. and enrichment >3-fold for any of the viral proteins is reported. • nsp16 binds mrna recognition domains of u1/u2 snrnas and disrupts mrna splicing • nsp1 binds in the mrna entry channel of the ribosome to disrupt protein translation • nsp8 and nsp9 bind the signal recognition particle and disrupt protein trafficking • these disruptions to protein production suppress the interferon response to infection in brief -sars-cov-2 proteins directly engage host rnas and dysregulate rna-based processes to suppress the interferon response j o u r n a l p r e -p r o o f ); office of the vice president for research at uvm m.g. is a nyscf-robertson investigator signal recognition particle: an essential protein-targeting machine a user's guide for phase separation assays with purified proteins recognition of doublestranded rna and activation of nf-κb by toll-like receptor 3 visualizing late states of human 40s ribosomal subunit maturation the proximal origin of sars-cov-2 sars-cov-2 (covid-19) by the numbers innate immune 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mammalian srp-ribosome complex the genetic sequence, origin, and diagnosis of sars-cov-2 comparative protein structure modeling using modeller the jnk signal transduction pathway metap-like ebp1 occupies the human ribosomal tunnel exit and recruits flexible rrna expansion segments recognition of viruses by cytoplasmic sensors improved protein structure prediction using predicted interresidue orientations influenza virus ns1 protein-rna interactome reveals intron targeting viral and host factors related to the clinical outcome of covid-19 structural basis for interaction of a cotranslational chaperone with the eukaryotic ribosome interferon-α2b treatment for covid-19 sars-cov-2 viral load in upper respiratory specimens of infected patients we thank fritz roth for clones; marko jovanovic, bil clemons, shu-ou shan, and jamie key: cord-341701-zropd3mo authors: adhikari, subash; nice, edouard c.; deutsch, eric w.; lane, lydie; omenn, gilbert s.; pennington, stephen r.; paik, young-ki; overall, christopher m.; corrales, fernando j.; cristea, ileana m.; van eyk, jennifer e.; uhlén, mathias; lindskog, cecilia; chan, daniel w.; bairoch, amos; waddington, james c.; justice, joshua l.; labaer, joshua; rodriguez, henry; he, fuchu; kostrzewa, markus; ping, peipei; gundry, rebekah l.; stewart, peter; srivastava, sanjeeva; srivastava, sudhir; nogueira, fabio c. s.; domont, gilberto b.; vandenbrouck, yves; lam, maggie p. y.; wennersten, sara; vizcaino, juan antonio; wilkins, marc; schwenk, jochen m.; lundberg, emma; bandeira, nuno; marko-varga, gyorgy; weintraub, susan t.; pineau, charles; kusebauch, ulrike; moritz, robert l.; ahn, seong beom; palmblad, magnus; snyder, michael p.; aebersold, ruedi; baker, mark s. title: a high-stringency blueprint of the human proteome date: 2020-10-16 journal: nat commun doi: 10.1038/s41467-020-19045-9 sha: doc_id: 341701 cord_uid: zropd3mo the human proteome organization (hupo) launched the human proteome project (hpp) in 2010, creating an international framework for global collaboration, data sharing, quality assurance and enhancing accurate annotation of the genome-encoded proteome. during the subsequent decade, the hpp established collaborations, developed guidelines and metrics, and undertook reanalysis of previously deposited community data, continuously increasing the coverage of the human proteome. on the occasion of the hpp’s tenth anniversary, we here report a 90.4% complete high-stringency human proteome blueprint. this knowledge is essential for discerning molecular processes in health and disease, as we demonstrate by highlighting potential roles the human proteome plays in our understanding, diagnosis and treatment of cancers, cardiovascular and infectious diseases. a. establishing agreed, stringent, reliable standards, b. identifying >1 protein product from each proteincoding gene and c. detecting expression of the remaining missing proteins (see below). 2. to make proteomics an integrated component of multiomics studies to advance life sciences, biomedical sciences and precision medicine. comparisons with the hgp are numerous. both global projects are ambitious cooperative community efforts seeking to identify how genes (hgp) or proteins (hpp) help define the molecular mechanisms underlying health and disease. both groups have implemented exhaustive data sharing and stringent quality control efforts. however, we now know that sequencing human genomes is necessary but not sufficient to understand the complexity of human biology or pathology. knowledge of expressed proteins (including concentration, spatio-temporal localization, activities, protease-processed forms, transport, interactions, splice isoforms, ptms and the many proteoforms derived from the proteome) cannot be predicted by genome sequencing alone. hpp structure and achievements hupo launched the hpp in 2010 at the 9th hupo annual world congress in sydney, with gil omenn as the inaugural chair. the hpp started without long-term funding. financial support over the decade came through individual principal investigator projects, institutional infrastructure/core facilities and philanthropy, without large-scale, long-term, integrated multi-government strategic funding. the hpp grew from several archetypal hupo projects (plasma, liver, brain, cardiovascular, kidney/urine proteomes). at present, the hpp comprises two strategic initiatives-chromosome-centric (c-hpp; 25 teams) and biology/disease-centric (b/ d-hpp; 19 teams)-organized in a strategic matrix underpinned by four resource pillars: antibodies (ab), mass spectrometry (ms), kb and pathology (fig. 1a) . before explaining these elements in more detail, we will describe hpp's criteria underlying the establishment of the high-stringency human proteome. defining the human proteome at high-stringency. the hpp relies upon a coordinated system developed by nextprot and uniprotkb, which attributes five levels of supporting evidence for protein existence (pe) 6 . evidence at pe1 indicates clear experimental evidence for the existence of at least one proteoform, based on credible identification by ms, edman sequencing, xray, nuclear magnetic resonance (nmr) structure of purified natural protein, reliable protein-protein interaction and/or antibody data. pe2 indicates evidence limited to the corresponding transcript (cdna, reverse-transcriptase pcr, northern blotting). pe3 indicates the existence of orthologs in closely related species. pe4 refers to entries based on gene models without evidence at protein, transcript or homology levels. pe5 classifications indicate that coding evidence is doubtful and/or probably corresponds to an incorrect in silico translation of a non-coding element. as pe5 entries are largely non-coding, the hpp preferentially tracks only pe1,2,3,4 protein-coding entries. proteins classified as pe2,3,4 are colloquially referred to as missing proteins 7, 8 . since 2013, nextprot and the hpp (fig. 1b) have issued annual tallies of pe1,2,3,4,5 status (www.nextprot.org/about/protein-existence), which have been reported in annual collaborative metrics manuscripts (ref. 9 and references therein). the latest nextprot hpp reference release (https://www. nextprot.org/about/statistics, data release: 17-01-2020) designates credible pe1 evidence for 90.4% of the human proteome (17, 874 pe1s from the 19,773 pe1,2,3,4 protein entries excluding the dubious 577 pe5 entries). this leaves 1899 (9.6%) pe2,3,4 missing human proteome entries to be identified at highstringency. here, the term high-stringency refers to rigorous hpp standards for post-acquisition processing and any protein inference made from raw ms peptide spectral data 10 . this term avoids confusion with the pre-existing term 'high-accuracy' box 1 | hpp decadal achievements • generated a framework, plan and governance structure for community-based mapping of the human proteome. • confirmed nextprot as the hpp reference knowledgebase and supported creation and use of proteomxchange (px) to register and make proteomics raw mass spectrometry (ms) and metadata available and reusable under fair (findable, accessible, interoperable and reusable) principles. • engaged nextprot, peptideatlas, pride and massive as partners in the generation of annual nextprot hpp release and a high-stringency hpp knowledgebase (kb). • encouraged community support of high-stringency protein inference and proteomic data analysis. • built ms data interpretation guidelines that promoted the application of standardized analysis of community human ms and proteomic data to progressively complete the human proteome parts list. • aligned with the human protein atlasʼ (hpa) cell and tissue spatiotemporal maps in health/disease and supported community efforts to raise awareness of antibody specificity and quality assurance issues. • partnered with srmatlas to develop quantitative targeted proteomics assays for the analysis of key proteins and hallmark pathways/networks. proposed and built global collaborative initiatives to investigate the biology of human health and disease at a proteomic-wide scale. • raised the profile and visibility of proteomics, as an essential component of life sciences and biomedical research by promoting the development of instrumentation and methods for proteoform analysis, as well as activity/function that cannot be addressed by genomics. • initiated a programme to determine the biological function for uncharacterized pe1 proteins (see below) that currently lack functional annotation. • established a hupo early career researcher network to engage, mentor and highlight research from young scientists/clinicians, while actively promoting gender and regional balance. • establish a community initiative to systematically map all human proteoforms. • establish optimized workflows for human proteome detection, quantification and functional characterization, including low abundance and/or temporo-spatially restricted proteins. • continue to support and promote the provision of technical standards, metrics and stringent guidelines for confident protein identification and quantification. • create a comprehensive, accurate, publicly-accessible, reference human proteome knowledgebase, reusable under fair principles. • maintain education and training programmes in all aspects of proteomics including proteomic data analysis for early career researchers and clinical scientists. • be a focal point for life sciences researchers, pathologists, clinicians and industry communities seeking to translate and leverage proteomic and proteogenomic data to improve human health through: (i) greater understanding of the molecular mechanisms of common and rare diseases, (ii) identification of pathophysiological changes to generate disease and wellness diagnostic biomarkers, and (iii) development of new effective and safe personalized therapeutics. frequently used in ms parlance, which relates to the generation of high (mass)-accuracy spectra from modern instruments. the use of high-stringency influences the quality of all protein inferences derived from any raw ms data. the hpp routinely applies high-stringency protein inference analytics 10 , generated through the trans-proteomic pipeline 11 . claims for detection of new pe1s and/or detection of coding elements not previously in nextprot, should meet minimum evidence thresholds. the current hpp guidelines 10 require at least two uniquely mapping peptides (using nextprot's peptide uniqueness checker tool 12 ), which are at least nine amino acid residues long. peptides must be non-nested (i.e., one not fully contained within another) but may overlap partially so coverage exceeds >18 residues. the hpp also requires full declaration of false discovery rate (fdr) calculation procedures at peptide and protein levels, with a maximum allowable protein-level fdr of 1%. within hpp missing protein publications, further validation is required through synthetic peptide spectra matching 13 . going forward, the hpp also requests association of universal spectrum identifiers with every ms spectrum 10 . we emphasize that ms-based data from high-accuracy 14,15 ms instruments combined with subsequent high-stringency protein inference analysis provide definitive best-practice confirmation of protein identification and abundance. to ensure high-quality analyses, increasingly stringent criteria have been applied 10 spectral data findable, accessible, interoperable and reusable (fair) 17 . many previous studies use high (mass)-accuracy instruments with subsequent protein inference identifications undertaken at lower default settings, such as accepting single peptides or those only seven amino acids in length and/or not conforming to more rigid nextprot proteotypic analysis 18, 19 . these analyses can result in spurious identification of many more false positives 20 , with lower-quality single non-proteotypic spectra data colloquially referred to as one-hit wonders, better explained by sequence variation in other highly observed proteins 21 . chromosome-centric (c)-hpp. the c-hpp (https://www.hupo. org/c-hpp) aims to annotate all genome-encoded proteins 7, 8 in an unbiased and high-stringency manner. it explores proteins that have not previously been confidently observed by ms or other analytical methods 7, 8, 22 . international c-hpp teams are organized according to chromosomes (chrs), namely chrs 1-22, chr x, chr y and mitochondrial (mt) genome teams. from 2017, the c-hpp expanded its mission to include functional characterization of the 1899 pe2,3,4 proteins that have not been confidently observed and 1254 pe1s that have no nextprot curated function (uncharacterized pe1s or upe1s), cumulatively referred to as the dark proteome 23, 24 . biology/disease-centric (b/d)-hpp. the b/d-hpp (https:// www.hupo.org/b/d-hpp) measures and interprets human proteome data under a range of physiological and pathological conditions. it focuses on the following: (i) elucidating the hallmark protein drivers of biology/disease and (ii) promoting development of new proteomics analytical tools such as ab-based approaches and targeted selected/multiple/parallel reaction monitoring (srm/mrm/prm) assays. as an example, the initial hupo liver proteome project grew into a b/d-hpp team focussing on liver expression profiles, ptms, tissue expression, subcellular localization, interactions, physiology and pathologies 25 . the chinese cn-hpp have characterized four liver cell types, emphasizing benefits of acquiring cell-type specific maps to understand underlying biology/pathology 26 . in addition, they mapped landscapes of early hepatocellular and lung carcinoma, generating cancer subtype alterations where proteomic signatures identified poor prognosis patients and/or those benefiting from targeted therapy 27, 28 . in other studies, they analysed microdissected cell types with gross anatomical resolution using ms 26, 29 , revealing circadian cycles and spatio-temporal proteome expression in the liver, brain, heart and stomach 30 , providing resources to better understand organ biochemistry, physiology and pathology. significant discoveries continue to be made from all b/d-hpp teams across personalized cancer immunotherapy and therapeutic modalities (e.g., lymphoma 31 , ovarian 32 , liver 27 and lung 28 cancers), with ptms orchestrating many outcomes including response to therapy [33] [34] [35] . b/d-hpp resources include the human srmatlas 36 , a unique compendium of high-resolution spectra and multiplexed srm/ mrm/prm assays developed from 166,174 synthetic proteotypic peptides. this assay library enables targeted identification and quantification of a theoretical maximum of 99.7% of the human proteome 36 , provided proteins are expressed spatiotemporally at concentrations amenable to ms detection. for example, srmatlas supported the c-hpp chr x team's confident identification of missing proteins 37 . hpp resource pillars. the b/d-hpp and c-hpp are supported by four hpp resource pillars that ensure effective data generation, integration and implementation, including the establishment of metrics and guidelines, enhancement of technology platforms, reagent development and optimal use of existing and emerging data streams (fig. 1a) . the hpp ms resource pillar informs the community about ms technology/workflow advances, appropriate high-stringency standards and liaises with industry regarding instrument development, all leading to improved depth and accuracy of proteome identification, quantification and modification. these include methods like matrix-assisted laser desorption ionization time of flight (maldi-tof)-ms, electrospray-ms, bottom-up (shotgun) ms, data-dependent acquisition ms, data-independent acquisition (dia) ms, targeted srm/mrm/prm, top-down ms, crosslinking ms, ptm analysis, n-and c-termini measurement, ms data computational analysis and interactomics. the ms resource pillar previously undertook a swath/dia-ms reproducibility study 38 and are currently coordinating a phosphopeptide challenge involving >20 participating labs with partners synpeptide shanghai and resyn biosciences south africa, who have provided a human phosphopeptide standard set with unphosphorylated counterparts, as peptide mixtures spiked into a yeast tryptic digest background. this will result in a better understanding of phosphopeptide enrichment, ms data analytics and informatics tools. the hpp ab resource pillar, ostensibly led by the human protein atlas (hpa; www.proteinatlas.org), was initiated in 2003 and uses ab-based strategies to analyse spatio-temporal aspects of the proteome 39 . linking the identification of proteins with 'realtime' localization at tissue, cell and subcellular levels supports a more comprehensive understanding of biology, health and disease. this requires information at resolution not currently available by ms (see single-cell section below). approaches for spatio-temporal proteomics include single-cell in situ ms, fractionated cell lysates, proximity labelling or imaging-based proteomics 40, 41 . imaging-based proteomics has a clear advantage, namely analysing proteins in their native location at single-cell resolution. to this end, the hpa has developed industrial scale epitope-directed abs for community use. hpa also integrates multi-omics data. it contains extensive transcriptome data and nextprot pe assignments, and contributes to the open-access catalogue antibodypedia, containing >4 m abs (www.antibodypedia.org) 42 against >19,000 targets that assist the community to select application-appropriate abs. at hpp launch, the hpa had detected >50% of the proteincoding genome 43 . currently,~87% of the proteome is targeted by >1 hpa ab, detected though an encyclopaedia of >10 m annotated high-resolution digital images, partitioned into a number of sub-atlases that are interconnected [44] [45] [46] [47] [48] . these currently comprise the; tissue atlas (protein distribution across all major tissues), cell atlas (subcellular localization and heterogeneity in single cells), pathology atlas (correlations between gene expression and patient survival in major human cancer types), blood atlas (protein profiles across major immune cells and blood levels), brain atlas (protein distribution in the brain), and the metabolic atlas (various tissue metabolic enzymes localizations). over the decade, hpa's open-access database [44] [45] [46] [47] [48] has become one of the world's most visited biological resources (>3.6 m visits in toto annually). the hpa also plays an emerging role in establishing guidelines around the appropriate use of abs and ensuring immunoassay validation [49] [50] [51] . it recently spent considerable effort validating the selectivity of their abs, including championing efforts of the international working group for ab validation proposing many new approaches implemented across >10,000 abs 42 . in addition, hpa's massive collection of images has supported a multitude of publications and become a citizen science resource for developing ai classification learning models 52, 53 . the hpa is sustained perspective nature communications | https://doi.org/10.1038/s41467-020-19045-9 through community contribution to elixir 54 , that allows scientists from academia and industry to explore spatio-temporal aspects of the human proteome [55] [56] [57] . since 2018, the hpp pathology resource pillar has coordinated identification of areas of unmet clinical need, develops fit-forpurpose clinical assay guidelines/standards, promotes best-practice awareness, coordinates access to quality clinical samples/metadata and liaises with pathology organizations, diagnostic companies and regulatory agencies to promote professional application of proteomics in pathology. the hpp kb resource pillar captures, collects, collates, analyses and re-distributes all human proteome data. as cohesive knowledge transfer plays such a crucial role in big data science, including the hpp, the kb pillar's activities are addressed in the expanded section that follows. the human proteome in the nextprot hpp reference kb assembly and curation of nextprot. prior to the hpp, hupo established a protein standards initiative (psi; www.hupo.org/ proteomics-standards-initiative), emphasizing from the outset their priority for defining high-quality community standards, minimal requirements for experimental information 58 and highstringency data metrics. psi continues to work cooperatively with the hpp kb to inform hpp initiatives, pillars and teams. in 2013, nextprot 59,60 was officially designated as the hpp reference kb 61 . annually, a nextprot release is designated as the 'hpp release' and this serves as the basis for subsequent hpp high-stringency analyses, planning and reporting progress 10 . it receives and curates data from uniprotkb/swissprot 62 , adding ms evidence from peptideatlas 63 and since 2019 from mas-sive 64 . nextprot also curates ab-based, genome, transcriptome and other biological data to create an assembled snapshot of the human proteome 6, 65 . peptideatlas (http://www.peptideatlas.org) uses sequence search engines comet 66 , x!tandem 67 and spectrast 68 to reprocess publicly uploaded ms/ms data deposited through proteomexchange (px). data are aggregated using rigorous criteria including peptide spectral matching with fdr~0.0009% in the latest peptideatlas build to achieve ≤1% fdr at the protein level. massive searches public datasets using the ms-gf+ search engine 69 , also with strict criteria 70 to enforce global <1% fdr at the protein level for single-peptide identifications and stricter <0.01% fdr for proteins identified by >2 peptides. peptideatlas and massive peptide lists are integrated into current nextprot builds. nextprot then cross-references all peptides to protein entries and validates pe levels, requiring at least two msidentified uniquely mapping 9-mer non-nested peptides coming from either peptideatlas or massive. nextprot builds on uniprotkb/swissprot pe1 entries that include ms, partial or complete edman sequencing, x-ray or nmr structure, reliable protein-protein interaction data or ab detection by considering additional peptideatlas/massive data that meets minimum peptide uniqueness, number, length, nestedness and other requirements to upgrade entries to pe1. noticeably, nextprot is becoming more reliant on ms than non-ms data (e.g., 1860 non-ms data pe1s in 2016 down to 950 in 2020). to ensure only high-quality ab data are used, in 2018 nextprot/hpp ab pillar revised criteria to upgrade entries to pe1, including specificity and other rigorous criteria suggested by the international working group for ab validation 50 . as an example, nextprot recently analysed 41 ab-based publications and upgraded three pe2,3,4 entries to pe1. discussions regarding data provenance delivered by non-ms data for pe1 assignment actively continues in the hpp 71 . expansion of the high-stringency proteome. the baseline number of protein-coding genes in the reference proteome given by nextprot is managed by uniprotkb/swissprot. every proteincoding gene is assigned a protein entry (inclusive of all proteoforms), with chromosome location and other data organized under these entries. the number of protein-coding genes originally estimated by the hgp dropped from >100,000 72 to a relatively stable~19,700 at hgp draft release, a number that is close to the current nextprot's 2020 release of 19,773 proteincoding genes ( supplementary fig. 1) 60 . confident detection of the human proteome has consistently risen from 69.8% nextprot pe1 entries in 2011 to 90.4% in 2020 (fig. 2a) . however, progress has recently slowed (fig. 2a) , suggesting that it may be difficult to confidently identify all 1899 missing proteins. parenthetically, the hgp celebrated a provisional 90% completion ten years after its launch 5 and annotation of the human genome still remains incomplete or uncertain, especially in regions of repeat sequences and z-dna inserts. in each of the seven annual hpp metrics papers published to date ( 9 and refs therein), pe1,2,3,4,5 entries have been added, deleted, renamed, merged and/or de-merged, indicating ongoing fine-tuning of the hpp reference proteome. the sankey flow diagram 73 (fig. 2b) illustrates that significant fluidity has occurred across all pe classifications since 2011. the largest shifts occur with increases in pe1s (13,603 up to 17,874), followed by decreases in pe2s (5,587 down to 1,596), despite increasingly stringent hpp ms data guidelines adopted in 2015 (v2.1) and 2019 (v3.0). linear regression of pe1 increases against pe2 decreases results in a strong (r 2 = 97%) inverse correlation, suggesting new pe1s come from pe2s where mrna expression has been previously observed. extrapolation of this pe1 discovery curve suggests that 95% pe1s may be reached sometime between 2024 and 2027. minor conversions occurred between other pe categories, with downgrading of pe1 or pe2s and upgrading of pe4s, and even a few pe5s, to pe1s. although a plethora of studies show low linear correlations (40-60%) between mrna level and protein abundance 74,75 , our binary data (fig. 2 ) supports the contention that once a nextprot entry has mrna expression verified, those pe2s are amenable to upgrade to pe1, whereas pe3s, pe4s and particularly pe5s are more resistant to pe1 upgrade. missing protein analyses. nextprot protein descriptors and associated data can be used to analyse protein groups/families according to their chr location or pe classification. missing protein (pe2,3,4) analysis indicates that some groups/families have been upgraded to pe1 more successfully than others (fig. 3a) . for example, between 2011 and 2020, 372 zinc (zn) finger proteins, 171 transmembrane proteins, 93 carbohydrate metabolism proteins, 90 testes-, sperm-, prostate-associated proteins, 78 coiled-coil domain-containing proteins and 58 homeobox proteins have been upgraded from pe2,3,4 to pe1. these represent the six most prominent protein groups upgraded to pe1 over the decade (fig. 3a, green) . in contrast, two g protein-coupled receptor (gpcr) chemosensory families prove particularly resistant to pe1 upgrade: many olfactory receptors (ors) are still missing in 2020 76 (417 of the 2011 pe2,3,4 entries, including putative and uncharacterized ones) and 17 of 20 taste receptors remain pe2,3,4 s (fig. 3a, magenta) . in addition, some groups with a large number of pe1-upgraded protein entries still contain many pe2,3,4 entries (e.g., 85 non-gpcr transmembrane, 69 zn finger and 33 keratin-associated proteins remain pe2,3,4 s (fig. 3a) . when chr distribution of the most resistant (ors) or most discovered (zn-finger proteins) groups were plotted (fig. 3b) , difficult-to-find ors mapped mostly to chr 11 (~55%) and zn-finger proteins mapped mostly to chr 19 (~55%). our data demonstrate that 46% of all chr 19 proteins elevated to pe1 were zn-finger family members, illustrating this protein family has been highly amenable to confident ms detection over the decade. therefore, it was not surprising that most zn-finger protein members were coded on chr 19 (i.e., 255/698 total zn-finger genes), which has the highest chr pe1 reclassification rate over the decade (fig. 3b) . these data suggest that gene family duplication on particular chrs explains why some families are resistant or sensitive to pe1 reclassification. in agreement, pe1 discovery has occurred productively, but not uniformly, across all chromosomes (fig. 4) . missing protein decadal upgrade statistics range from 16% for chr y up to 29% for chr 1, with raw data representing 425 protein entries for chr 1 down to only 5 for chr y. a higher percentage of chr 19 proteins (29%) ascended to pe1 compared to those on chrs 11, 14, 21 or y (<17%). the hpa chromosome viewer (www.proteinatlas.org/humanproteome/proteinevidence) illustrates many recently-evolved protein family members are present on chr y, many ors on chr 11 and many keratinassociated proteins on chr 21. proteins on these three chrs have proven relatively resistant to pe1 reassignment. notably, there was just a single mt missing protein in 2011 and over the decade all mt protein-coding genes have now been identified. pe1 upgrade success by the 25 c-hpp teams could be predetermined by the presence of resistant or more easily-identified missing protein families on particular chrs. however, the current hpp kb processing pipeline utilizing peptideatlas, massive and nextprot (fig. 1b) makes it impossible to isolate decadal pe1 contributions from any particular c-hpp team as opposed to the overall community. although the hpp explores capturing full data provenance (i.e., from quantification/identification back to original data source) for fair data practices 17 , we can only historically estimate pe1s emanating from community deposition. in silico analysis reveals only 22 human proteins cannot produce the characteristic >2 high-stringency proteotypic peptides of the required length after tryptic digestion 36 . however, many missing proteins may be present at levels below detection limits or in under-studied cell types/tissues, expressed under particular conditions (e.g., stress/infection) or only found in developmental stages (e.g., embryo/fetus) 77 . equally, difficult-tosolubilize, hydrophobic multi-transmembrane domain membrane proteins may only generate short tryptic peptides that do not meet high-stringency guidelines or are indistinguishable from other family member sequences 76 . furthermore, transmembrane protein regions cut at single sites are unlikely to release embedded hydrophobic membrane-anchored protein strands 76 . we anticipate that finding the 9.6% remaining pe2,3,4 missing proteins will require exceptional future effort, including careful sampling of rare cells/tissues 78 combined with better sample fractionation and improved detection limits. low abundance proteins might be enriched using abs prior to ms. to this end, the hpa has developed abs against proteotypic sequences in many missing proteins 79 . several other labs are working on improved protocols for insoluble keratin-associated cross-linked missing proteins, non-tryptic or chemical digestion strategies to increase proteotypic peptide productivity 78,80 , higher efficiency search engines 81 , and compendia of missing protein biological evidence (e.g., missingproteinpedia) 71 . future hpp projects anticipate a shift to detection of biologically functional proteoforms 82 , noting their numbers are far larger and more difficult to measure 83 because of heterogeneous nuclear rna splicing, many ptms and detection of peptides with single amino acid variants (saavs). considerable ptm and splicing isoform data are already available through nextprot, including 190,938 ptm sites and 9,193,365 saavs 84 . community impact on the human proteome one barometer of community engagement in the hpp is the magnitude of investigator-submitted raw ms data that have been re-analysed. many journals require raw data submission and hpp action was a significant factor in journals adopting requirements aligned to hpp data deposition guidelines. raw data deposition occurs through px, which registers and standardizes capture/dissemination of public ms data from partner repositories, including founders pride 62 and peptideatlas and recent members mas-sive 64 , jpost 85 , iprox 86 and panorama public 87 . as of 2020, a total of 4634 human ms datasets have been received. each px dataset is branded with a unique pxd identifier with depositors, publications and voluntary metadata noted 88, 89 . illustrating the magnitude of this community data,~470 tbs of data have come from 5658 human datasets (~47% of 1 petabyte pride volume), with only 358 (6.3%) of these specifically tagged by depositors as from the hpp. the hpp encourages raw human ms data/metadata submission (including association to hpp) through px, and that journals request that pxd identifiers be published in accordance with fair principles 17 as discussed above. to provide an additional measure of global scientific impact, hupo commissioned the website construction of the human proteome reference library (hprl; https://hupo.org/hpp-hprl/), where all hpp-associated pubmed searches are hyperlinked and can be accessed and re-run routinely by the community. these hyperlinked searches automatically produce the latest pubmed outputs in a manner where all pubmed filtering, ranking and timeline tools can be applied subsequently by the hprl can also be used to measure impact (fig. 5) , where pubmed identifiers (pmids) from 2010 to 2019 references can be captured using web apis or software, e.g., ncbi e-utilities like https://www.ncbi.nlm.nih.gov/books/nbk25501 or the easy-pubmed r interface (www.rdrr.io/cran/easypubmed) that enable extraction and aggregation of pubmed bibliometric records. paralleling the low percentage of px datasets tagged as emanating from the hpp, hprl data show only~1000 pubmed title and abstract searches that specifically and cumulatively mention either the terms hpp, c-hpp and/or b/d-hpp (or their unabbreviated counterparts) out of a total of more than 50,000 'human and proteomics' title/abstract search outcomes over the decade. in contrast to this and more reassuringly, hprl searches of the community's biology/ or disease studies covered by nine of the b/d-hpp teams generated >5000 publications each since 2010 (fig. 5b) , with 'cancer proteomics' topping rankings at around 20,000 publications. although this may seem surprising, a pubmed search of 'human genome project' produced a similarly modest number of~1900 hits during the hgp's first decade. to visualize outputs from various hpp teams and the proteomics community in general, we have employed vosviewer 91 to construct and visualize bibliometric networks from collective pubmed searches (example shown in fig. 5c ). this particular analysis revealed a remarkable number of highly-interconnected relationships that have evolved between hpp teams, pillars and initiatives-providing evidence for the impressive level of established international collaboration developed over the decade. a key aspect of biomedical research lies in translating discovery into clinical use. protein assays remain a cornerstone of diagnostics. although individual proteins can be measured diagnostically with high precision (i.e., sensitivity and specificity), some assays suffer low specificity due to cross-reactivity with interfering substances including autoantibodies (e.g., thyroglobulin immunoassays). modern srm/mrm/prm assays allow multiple proteins to be measured simultaneously, accurately, sensitively and with high specificity. in addition, the use of liquid chromatography ms with immunocapture assays has been reported to eliminate interferences 92 . moreover, as most diseases are heterogeneous and multigenic, it is likely that multiplexed proteomic or multi-omics panels will achieve higher accuracy (e.g., optimized biomarkers for ovarian malignancy with adnexal masses 93 ). the hpp assists in the development of proteomic educational programmes with pathology societies to train the pathology community on the potential impact of proteomic technologies. below, in recognition of the impact human proteomics can and is having in precision medicine, we highlight examples demonstrating the role of the hpp and proteomics in tackling contemporary medical grand challenges. cancer precision medicine. as mentioned above, pubmed extracts~20,000 published human cancer proteome studies since 2011 (fig. 5b) . although genomics can routinely determine highrisk, predisposition and aspects related to tumour burden and recurrence, effective targeted cancer treatment is still not available for all cancers. for example, systematic genome-wide studies like the pan-cancer analysis integrated analyses of >2600 whole genomes from 38 tumour types with matching normal tissues, uncovered many cancer-associated genes 94 , chromosome rearrangements, some unknown drivers but few new therapeutic targets. this is mainly because mutations do not automatically cause predicted changes in the proteome, making it difficult to establish which changes are crucial biochemical drivers from those that are not. integrating genomic and proteomic data (i.e., proteogenomics) has the potential to provide insights into causes and mechanisms underlying diseases, including the hallmarks of cancer biology 95 . this can facilitate the implementation of effective therapeutic intervention. the value of a proteogenomic analysis of functional consequences of cancer somatic mutations has assisted in narrowing down candidate driver genes within large deletions and amplified regions 96 . reviewing the underlying causes of breast cancer also demonstrates that coupling genomic/transcriptomic data with proteomic/phosphoproteomic analysis was more insightful than any individual approach. of note, melanoma tumour genomic braf driver mutations match corresponding protein sequences 97 , illustrating that proteomic landscapes add value to genomic data, when considered with patient tumour histopathology and clinical metadata 98 . proteomics substantially benefits a comprehensive understanding of precision medicine. to illustrate this, nci's clinical proteomic tumor analysis consortium (cptac; proteomics. cancer.gov) in collaboration with the b/d-hpp cancer team, established guidelines, data sharing, and standards in analytical and computational workflows to ensure rigour in designing and performing research [99] [100] [101] [102] [103] . cptac applied these standards and workflows to tumours previously genomically characterized by the cancer genome atlas. in doing so, cptac pioneered the integration of proteomics with genomics (i.e., proteogenomics) to produce a more unified and comprehensive understanding of cancer biology and implemented its transition into cancer clinical research studies 96, 104, 105 . largely due to these efforts, nci hosts open-access repositories of unified proteogenomics datasets, assays and reagents, including the proteomic data commons (pdc.cancer.gov), a fit-for-purpose targeted assay site (assays. cancer.gov) 102, 106 and an ab portal (antibodies.cancer.gov) 107 . these activities empowered the recent creation of the international proteogenome consortium (icpc; icpc.cancer.gov) 108 . collectively, cptac and icpc collaborators have comprehensively characterized 13 cancer types at the proteogenomics level, with all datasets publicly accessible [109] [110] [111] [112] [113] [114] [115] [116] [117] . cardiovascular diseases. cardiovascular disease (cvd) research is challenged by daunting structural heterogeneity and molecular complexity. for example, cardiac circuitry function/dysfunction cannot be reduced to differentially expressed single genes. on the other hand, proteogenomics allows assessment of interactions, pathways and networks and informs diagnosis and therapy of multifactorial cvds. over the decade, cvd proteomics has broadened from identifying single canonical proteins to mapping proteoforms derived from combinations of alternative splicing, cleavage, and ptms 33, 83 . now, identification of proteoforms (e.g., genetic variations, alternatively spliced products, phosphorylation 118 , glycosylation 119 , oxidative 120 and other ptms 121 ) allow cvd sub-classification. in parallel, the heart is uniquely sensitive to alternative splicing (frequently altered in congenital heart diseases), explaining the b/d-hpp's interest in developing assays for splice isoform-specific changes in cardiomyocyte development and maturation 122, 123 . many technological developments have been prominent, including phospho-ptm analysis to identify pde5a targets in heart failure therapy 118 and proximity labelling to assess protein-protein interactions involved in β-adrenergic signalling of contractility in cardiac fight-or-flight responses 124 and evaluation of regenerative stem cell therapy efficacy in postinfarct hearts 125 . proteomic studies have also addressed the kilometres of vascular beds and extracellular matrix responsible for transporting blood, giving valuable insights into the molecular anatomy of aneurysms and atherosclerosis 126, 127 . targeted ms methods have been developed, again especially where interferences obfuscate immunoassays 128 or where additional biological context is required 129 . likewise, volumetric absorptive microsampling vams blood collection devices (e.g., mitra devices or dried blood spots) allow patients to mail samples from home to analytical labs to undertake srm/mrm/prm assays quantifying cvd risk-associated apolipoproteins 130 or other markers 131 . in summary, consumer-based cvd proteomics-based precision medicine testing services are now coming of age. microorganism detection. proteomics and the hpp have made fundamental contributions to understanding pathogenic infection, providing diagnostics and developing therapies 132 . the b/d-hpp infectious diseases team promotes international proteomics collaborations investigating viral, bacterial, fungal and parasitic diseases. maldi-time-of-flight (tof)-ms, once considered revolutionary, is now established as a routine tool in clinical microbiology 133 . classical phenotypic tests identify unknown and potentially pathogenic microorganisms, but may require incubation for several days, with misidentification resulting in adverse treatment consequences. maldi-tof-ms provides significantly shortened analyses (now minutes) with improved accuracy on single colony or bacterial pellets for difficult-todetect microorganisms, using automated spectra acquisition and extensive reference spectra databases 134 . minor spectral differences enable typing below species levels 135 , allowing subspecies identification through epidemiological analyses. bacteria and yeasts (most clinical identifications), mycobacteria 136 and moulds 137 can now be identified accurately and rapidly. further ms clinical diagnostic applications are being investigated (e.g., antibiotic resistance (art) and susceptibility testing (ast) based on hydrolytic β-lactamase activity 138 ), with kits under development commercially through star-carba, star-cepha and bruker daltonics. sars-cov-2 virology. the recent severe acute respiratory syndrome coronavirus 2 (sars-cov-2) outbreak that causes covid-19 disease represents a major threat to human health and our economies [139] [140] [141] . the pandemic underscores our need to understand virus pathobiology, identify host-pathogen interactions that support replication, find biomarkers correlative with clinical outcome and expand surveillance. many omics studies followed the 2003 sars-cov-1 and related mers and ibv coronavirus outbreaks [142] [143] [144] [145] [146] . the cell surface receptor for the cov-1 and cov-2 surface spike protein has been identified by affinity-ms to be angiotensin-converting enzyme 2 (ace2) 147 , which in a recent large-scale study based on antibodybased proteomics was shown to be mainly localized to the digestive system, kidney, heart, testis, placenta, eye and upper respiratory epithelia 148 . virus binding leads to proteolysis by the transmembrane serine protease tmprss2 expressed in airway epithelia 149 , thus a clinically approved tmprss2 inhibitor (camostat mesylate) is being investigated to block infections 150, 151 . furthermore, proteomics has characterized the infectious cov-1 viral particle 143 , temporal changes in host cells during infection 142 and virusinduced endoplasmic reticulum membrane remodelling into double-membrane vesicles 152,153 that house viral replication compartments 146 . proximity labelling revealed >500 host and 14 viral protein associations with the viral replicase nps2 146 , highlighting vesicular trafficking, autophagy and splicing proteins in coronavirus replication, which if shown to also be the case for cov-2, indicate potential drug targets. building on this knowledge of coronavirus infection, recent proteomics studies have focused on sars-cov-2, uncovering additional potential therapeutic targets 154, 155 . ms and array-based proteomics serology has screened for potential biomarkers and abs against infection 156, 157 . clinical isolate infection models have been developed using caco-2 cells 154 with temporal proteome changes identified during infection using multiplexed ms by combining metabolic labelling with tandem mass tagging methods. consistently, host vesicular trafficking, translation, rna splicing, nucleotide synthesis and glycolysis pathway proteins were upregulated following infection 143, 144 and targeting these processes with inhibitors revealed potential therapeutic targets 158, 159 . additionally, affinity-ms interactome studies examined 26 of 29 total sars-cov-2 proteins expressed within hek293t human cells 155 , suggesting 69 existing drugs merit further investigation. moreover, a recent phosphoproteome analysis pointed to the regulation of viral proteins through ptms 160 . the sars-cov-2 pandemic highlights the need for applying proteomic approaches to the development of serologic testing and preclinical and computational model systems to evaluate patient responses to infection 156 . serological biomarkers of asymptomatic/ symptomatic infection, disease severity, risk of re-infection and/or vaccine efficacy are being characterized 157, 161 . in addition to this accumulating omics knowledge, many aspects of sars-cov-2 pathobiology await further exploration including development of additional methods for clinical virus detection, identification of infection stage, and an in-depth understanding of functional spatiotemporal virus-host protein interactions and organelle remodelling [162] [163] [164] . for example, recent studies that utilize targeted ms for sars-cov-2 protein detection and proteomic characterization of serological immune responses 161,165-167 from patient samples may bolster pcr screening for the assessment of disease severity 168 . other proteomics approaches can also be deployed to further expand the understanding of sars-cov-2 biology. among these is the application of tails n-terminomics that promises to identify many sars-cov-2 protease substrates and those cellular pathways inactivated by viral proteolysis, as reported for other viruses 169 . in summary, proteomics plays increasingly important roles in understanding viral outbreak biology, accurate diagnosis and effective treatment and is positioned to continue to co-ordinate and drive international collaborations towards these goals. western and eastern cultures urge us to know thyself and thy enemy. these axioms resonate with precision medicine where future benefits arise from a detailed omics understanding of the hallmarks of health and disease. here, we reviewed the construction of a community-endorsed, high-stringency blueprint of the human proteome. the decadal nextprot hpp pe metrics shown here demonstrate the community's progressive success in pe1 identification from 13,588 in 2011 to 17,874 pe1s in 2020, marking the completion of >90% of the human proteome parts list (see strategic objective 1 above). we also present specific examples demonstrating proteomics will be an integrated component (with genomics and other omics) in future biomedical science discovery and precision medicine. hupo recommits to its original hpp strategic aims as well as the fair data principles 17 , while anticipating the following future priorities: 1. unearth credible proteomics data for the majority of current pe2 proteins: since most pe1 identifications come from former pe2s, our future strategy is to find credible data for 95-99% of current pe2s, allowing reclassification of these to pe1. pe3 also remain promising, as homologous proteins are detected in related species. 2. unravel currently unknown proteome functionalities: fill functional annotation gaps for all protein-coding elements, with a priority on credibly identified proteins 170 and develop, expand and apply function prediction tools 24, 171, 172 . 3. expand the hpp kb: maintain a sustainable knowledgetransfer hpp kb infrastructure with funding that captures/ displays high-stringency partner omics data streams and publication data (hprl) to researchers and the public in an accessible and compelling manner. 4. develop community-approved multi-omics technology guidelines: explore dia-ms, ab and aptamer-based multiplexed assays, top-down ms and other not yet invented multi-omics technologies. 5. champion collaborative multi-omics health/disease approaches: in addition to extensions in hpp kb partnerships, the hpp will collaborate with international and regional initiatives (including human variome, human cell atlas, hpop/ipop, motrpac, hubmap, cancer moonshot, htan, edrn, cptac, icpc and upcoming international initiatives) around multi-omics approaches to human disease processes, biomarker discovery and therapeutic development. 6. apply and improve single-cell proteomic technology: develop technologies that allow detection and quantification of proteomes in single cells to further understand cellular/tissue heterogeneity, differentiation, diseases and the intrinsic biological noise in health and disease 173, 174 . many single-cell proteomics advances will be explored to analyse cellular heterogeneity [175] [176] [177] [178] . sensitivity increases (analogous to pcr) and trade-offs maximizing coverage per cell with throughput/accuracy will be studied 179 . 7. champion dual ab-capture with ms identification: enhance the accuracy of ab-based epitope/antigen detection by confirmation using high-accuracy, high-stringency ms identifications across real-life spatio-temporal biological settings. collectively, there is recognition within the hpp that future stringent co-registration of ms with ab data are required to achieve the ultimate spatio-temporal human proteome expression atlas 180 . 8. exploit massively parallel ms 174 to increase throughput: for rapid, sensitive and higher-throughput ms analysis. 9. capitalize on human disease biobanks: the hpp will work with biobanking consortia to improve access for proteomics researchers to highly curated and accurately annotated clinical samples collected in a standardized manner. 10. encourage higher levels of community engagement: the hpp will continue to reach out to the community, supporting findable, accessible, interoperable and reusable (fair) data principles 17 , while encouraging and appropriately recognizing all contributions to the re-analysis of proteomics data. the post sars-cov-2 pandemic world will be different. it is likely that new paradigms to accelerate precision medicine will emerge. these will undoubtedly involve global collaboration (even between competing entities) using multidisciplinary approaches that enable the fast-tracking of novel diagnostic tests and precision therapeutics. almost certainly these outcomes will require knowledge involving the human proteome-celebrated here in the inaugural hpp high-stringency blueprint. received: 19 december 2019; accepted: 25 september 2020; a human proteome project with a beginning and an end building the 'practical' human proteome project-the next big thing in basic and clinical proteomics is a gene-centric human proteome project the best way for proteomics to serve biology first hpp publication after launch, outlining systematic global effort to map the human proteome (abundance, distribution, temporal-spatial and subcellular localisation, interactions and function) and decribing reource pillars (ms, ab and kb), with a biologically-driven and a chromosome-based mapping initiatives to deliver a protein parts list, reagents and tools human genome. finally, the book of life and instructions for navigating it metrics for the human proteome project 2013-2014 and strategies for finding missing proteins standard guidelines for the chromosome-centric human proteome project a first step toward completion of a genome-wide characterization of the human proteome 2019 annual hpp metrics publication (nextprot 2019-01-11) reporting progress in credibly identifying and characterising the human proteome details current high-stringency ms guidelines criteria used by the hpp for pe1 status (evidence for protein existence), where only protein entries with two or more nextprot uniquely-mapping, non-nested peptides with length 9 amino acids or greater are deemed to have sufficient evidence trans-proteomic pipeline, a standardized data processing pipeline for large-scale reproducible proteomics informatics the nextprot peptide uniqueness checker: a tool for the proteomics community looking for missing proteins in the proteome of human spermatozoa: an update on the proper use of mass accuracy in proteomics precision proteomics: the case for high resolution and high mass accuracy human proteome project mass spectrometry data interpretation guidelines 2.1 the fair guiding principles for scientific data management and stewardship a draft map of the human proteome mass-spectrometry-based draft of the human proteome analyzing the first drafts of the human proteome flexible and fast mapping of peptides to a proteome with proteomapper the chromosome-centric human proteome project for cataloging proteins encoded in the genome advances in the chromosome-centric human proteome project: looking to the future launching the c-hpp next-cp50 pilot project for functional characterization of identified proteins with no known function human liver proteome project: plan, progress, and perspectives a cell-type-resolved liver proteome proteomics identifies new therapeutic targets of early-stage hepatocellular carcinoma integrated multiomics example (proteome, phosphoproteome, transcriptome, and whole-exome sequencing) analyses leading to clinical outcomes, performed on 103 lung adenocarcinomas, revealing many cancer-associated features (e.g., tumourassociated variants, patient clinical outcome correlation with egfr or tp53 mutational status, proteomic stratification of lung cancer subtypes and potential drug targets proteome-wide profiling of activated transcription factors with a concatenated tandem array of transcription factor response elements a region-resolved mucosa proteome of the human stomach antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens the immunopeptidomic landscape of ovarian carcinomas precision profiling of the cardiovascular post-translationally modified proteome: where there is a will, there is a way in vivo phosphoproteome analysis reveals kinome reprogramming in hepatocellular carcinoma protein s-nitrosylation controls glycogen synthase kinase 3β function independent of its phosphorylation state generation and verification of a compendium of highly specific srm assays that enable quantification of >95% of all annotated human proteins. provides definitive ms data on 166,174 proteotypic peptides that facilitate the design of multiple, independent assays to quantify most human proteins, numerous splice-variants, non-synonymous mutations identification and validation of human missing proteins and peptides in public proteome databases: data mining strategy multi-laboratory assessment of reproducibility, qualitative and quantitative performance of swath-mass spectrometry a human protein atlas for normal and cancer tissues based on antibody proteomics spatial proteomics: a powerful discovery tool for cell biology getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles antibodypedia, a portal for sharing antibody and antigen validation data towards a knowledge-based human protein atlas a genome-wide transcriptomic analysis of protein-coding genes in human blood cells an atlas of the protein-coding genes in the human, pig, and mouse brain a subcellular map of the human proteome a pathology atlas of the human cancer transcriptome an atlas of human metabolism first of many encyclopedic hpa atlases that map the spatio-temporal expression human proteome based on integrated multi-omics data involving quantitative transcriptomics and ab microarraybased tissue immunohistochemistry and delivering spatial localisation of human proteins down to the single-cell level a proposal for validation of antibodies enhanced validation of antibodies for research applications deep learning is combined with massive-scale citizen science to improve large-scale image classification analysis of the human protein atlas image classification competition the elixir core data resources: fundamental infrastructure for the life sciences integration of transcriptomics and antibody-based proteomics for exploration of proteins expressed in specialized tissues cell type-specific expression of testis elevated genes based on transcriptomics and antibody-based proteomics the human cell atlas the minimum information about a proteomics experiment (miape) nextprot: a knowledge platform for human proteins the nextprot knowledgebase in 2020: data, tools and usability improvements nextprot: organizing protein knowledge in the context of human proteome projects the pride database and related tools and resources in 2019: improving support for quantification data state of the human proteome in 2014/2015 as viewed through peptideatlas: enhancing accuracy and coverage through the atlasprophet proteinexplorer: a repository-scale resource for exploration of protein detection in public mass spectrometry data sets the nextprot knowledgebase on human proteins: 2017 update comet: an open-source ms/ms sequence database search tool x!!tandem, an improved method for running x!tandem in parallel on collections of commodity computers development and validation of a spectral library searching method for peptide identification from ms/ms ms-gf+ makes progress towards a universal database search tool for proteomics assembling the community-scale discoverable human proteome community encouragement to identify biological data that complement high-stringency ms strategies to accelerate discovery and understanding of human proteome pe2,3,4 missing proteins. database allows unpublished, preliminary or proprietary data (e.g., antibody, ms, cell biology and genetic studies chess: a new human gene catalog curated from thousands of large-scale rna sequencing experiments reveals extensive transcriptional noise dynamic visualization of multi-level molecular data: the director package in r can we predict protein from mrna levels? proteomic and interactomic insights into the molecular basis of cell functional diversity in silico peptide repertoire of human olfactory receptor proteomes on high-stringency mass spectrometry toward completion of the human proteome parts list: progress uncovering proteins that are missing or have unknown function and developing analytical methods proteomic and n-terminomic tails analyses of human alveolar bone proteins: improved protein extraction methodology and lysarginase digestion strategies increase proteome coverage and missing protein identification the human protein atlas: a spatial map of the human proteome multiproteases combined with high-ph reverse-phase separation strategy verified fourteen missing proteins in human testis tissue open-pfind enhances the identification of missing proteins from human testis tissue proteoform: a single term describing protein complexity how many human proteoforms are there? what will nextprot help us achieve in 2020 and beyond the jpost environment: an integrated proteomics data repository and database iprox: an integrated proteome resource panorama public: a public repository for quantitative data sets processed in skyline proteomexchange provides globally coordinated proteomics data submission and dissemination the proteomexchange consortium in 2020: enabling 'big data' approaches in proteomics structural genomics: keystone for a human proteome project software survey: vosviewer, a computer program for bibliometric mapping measurement of thyroglobulin by liquid chromatography-tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies the road from discovery to clinical diagnostics: lessons learned from the first fda-cleared in vitro diagnostic multivariate index assay of proteomic biomarkers pan-cancer analysis of whole genomes details the hallmarks of cancer acquired during multistep development of human tumors (i.e., sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion/ metastasis, genome instability, inflammation, reprogramming of energy metabolism and evading immune destruction). creates a roadmap for the understanding of multi-omics based cancer data proteogenomics connects somatic mutations to signalling in breast cancer the hidden story of heterogeneous b-raf v600e mutation quantitative protein expression in metastatic melanoma-association with clinical outcome and tumor phenotypes clinical protein science in translational medicine targeting malignant melanoma repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-forpurpose approach protein-based multiplex assays: mock presubmissions to the us food and drug administration proteogenomic characterization of human colon and rectal cancer integrated proteogenomic characterization of human highgrade serous ovarian cancer cptac assay portal: a repository of targeted proteomic assays antibody characterisation-an essential researchers' resource revolutionizing precision oncology through collaborative proteogenomics and data sharing integrated proteogenomic characterization of clear cell renal cell carcinoma proteogenomic characterization of endometrial carcinoma proteogenomic characterization reveals therapeutic vulnerabilities in lung adenocarcinoma example of a cptac/icpc proteogenomics study highlighting new therapeutic insights into colorectal cancer. comparative proteomic and phosphoproteomic analysis of a prospectively-collected paired colon cancer tumour and adjacent normal tissue catalogue of colon cancer-associated proteins and phosphorylation sites, including known and putative new biomarkers proteogenomic characterization of ovarian hgsc implicates mitotic kinases, replication stress in observed chromosomal instability apobec3a is an oral cancer prognostic biomarker in taiwanese carriers of an apobec deletion polymorphism proteogenomic characterization of human early-onset gastric cancer integrated proteogenomic characterization of hbv-related hepatocellular carcinoma proteogenomics of non-smoking lung cancer in east asia delineates molecular signatures of pathogenesis and progression demonstrates the potential of proteomics and ptm analyses to increase our understanding of hypertrophic heart disease. transcription factor activation and phosphoproteome analyses of myocytes revealed pde9a regulates cgmp signalling independent of the no-pathway in stress-induced heart disease reference glycan structure libraries of primary human cardiomyocytes and pluripotent stem cell-derived cardiomyocytes reveal cell-type and culture stage-specific glycan phenotypes integrated dissection of cysteine oxidative post-translational modification proteome during cardiac hypertrophy cardiac troponins may be irreversibly modified by glycation: novel potential mechanisms of cardiac performance modulation splice-junction-based mapping of alternative isoforms in the human proteome an unbiased proteomics method to assess the maturation of human pluripotent stem cell-derived cardiomyocytes mechanism of adrenergic cav1.2 stimulation revealed by proximity proteomics cardiopoietic stem cell therapy restores infarction-altered cardiac proteome glycoproteomic analysis of the aortic extracellular matrix in marfan patients extracellular matrix proteomics identifies molecular signature of symptomatic carotid plaques targeted aproach to distinguish and determine absolute levels of gdf8 and gdf11 in mouse serum protein markers and risk of type 2 diabetes and prediabetes: a targeted proteomics approach in the kora f4/ff4 study application of volumetric absorptive microsampling for robust, high-throughput mass spectrometric quantification of circulating protein biomarkers concentration determination of >200 proteins in dried blood spots for biomarker discovery and validation the impact of mass spectrometrybased proteomics on fundamental discoveries in virology ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry how maldi-tof mass spectrometry can aid the diagnosis of hard-to-identify pathogenic bacteria-the rare and the unknown can maldi-tof mass spectrometry reasonably type bacteria? how to identify non-tuberculous mycobacterium species using maldi-tof mass spectrometry identification of molds by matrix-assisted laser desorption ionization-time of flight mass spectrometry susceptibility testing of bacteria using maldi-tof mass spectrometry a new coronavirus associated with human respiratory disease in china an interactive web-based dashboard to track covid-19 in real time how will country-based mitigation measures influence the course of the covid-19 epidemic? quantitative proteomics analysis reveals bag3 as a potential target to suppress severe acute respiratory syndrome coronavirus replication proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3 the cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology mers-cov and h5n1 influenza virus antagonize antigen presentation by altering the epigenetic landscape determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus the protein expression profile of ace2 in human tissues efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade illustrates the rapidity at which modern proteomics can contribute to the understanding of a recent pandemic, revealing insights into viral transmission and therapeutic targets. indicates sars-cov-2 uses the receptor ace2 for human cell entry and serine protease tmprss2 for sars-cov-2 spike protein priming. a clinically approved tmprss2 inhibitor blocked virus entry and sera from convalescent sars-cov-2 patients crossneutralized viral entry making of viral replication organelles by remodeling interior membranes proteomic approaches to uncovering virus-host protein interactions during the progression of viral infection proteomics of sars-cov-2-infected host cells reveals therapy targets a sars-cov-2 protein interaction map reveals targets for drug repurposing sars-cov-2 proteome microarray for global profiling of covid-19 specific igg and igm responses proteomic and metabolomic characterization of covid-19 patient sera inhibitory effect of mizoribine and ribavirin on the replication of severe acute respiratory syndrome (sars)-associated coronavirus nucleoside analogues for the treatment of coronavirus infections characterisation of the transcriptome and proteome of sars-cov-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein ultra-high-throughput clinical proteomics reveals classifiers of covid-19 infection post-translational modification control of innate immunity orchestration of protein acetylation as a toggle for cellular defense and virus replication temporal dynamics of protein complex formation and dissociation during human cytomegalovirus infection mass spectrometric identification of sars-cov-2 proteins from gargle solution samples of covid-19 patients proteotyping sars-cov-2 virus from nasopharyngeal swabs: a proof-of-concept focused on a 3 min mass spectrometry window a portrait of the human organelle proteome in space and time during cytomegalovirus infection multiplex targeted mass spectrometry assay for one-shot flavivirus diagnosis n-terminomics tails identifies host cell substrates of poliovirus and coxsackievirus b3 3c proteinases that modulate virus infection fusionpro, a versatile proteogenomic tool for identification of novel fusion transcripts and their potential translation products in cancer cells structure and protein interaction-based gene ontology annotations reveal likely functions of uncharacterized proteins on human chromosome 17 blinded testing of function annotation for upe1 proteins by i-tasser/cofactor pipeline using the 2018-2019 additions to nextprot and the cafa3 challenge single-cell analysis tools for drug discovery and development national cancer institute think-tank meeting report on proteomic cartography and biomarkers at the single-cell level:interrogation of premalignant lesions single-cell omics single-cell mass-spectrometry quantifies the emergence of macrophage heterogeneity multiplexed single cell protein expression analysis in solid tumours using a miniaturised microfluidic assay single-cell proteomics reveal that quantitative changes in coexpressed lineage-specific transcription factors determine cell fate transformative opportunities for single-cell proteomics investigating the applicability of antibodies generated within the human protein atlas as capture agents in immunoenrichment coupled to mass spectrometry was corresponding author, conceptualized hpp decadal blueprint, hupo proteomics timeline and hprl, and he designed/integrated all aspects of the manuscript made academic co-contributions to manuscript section drafting, editing, review and/or proof-reading health and human sciences australia. 3 institute for systems biology seoul 120-749, south korea. 8 faculty of dentistry usa. 12 science for life laboratory, school of engineering sciences in chemistry, biotechnology and health, kth royal institute of technology, 17121 solna, sweden. 13 rudbeck laboratory, department of immunology 23 cancer biomarkers research branch, national cancer institute, national institutes of health, 9609 medical center drive, suite 5e136 wellcome trust genome campus, hinxton, cambridge cb10 1sd, uk. 30 school of biotechnology and biomolecular sciences hupo acknowledges collaborators, proteomic scientists, independent partners, industry vendors and members of the scientific community who have contributed to the hpp. a full alphabetical listing of the human proteome project members appears in the supplementary information. in recognition of the many accomplishments, hupo has produced a publicly available hpp timeline available through https://hupo.org/ proteomics-timeline to be released with this hpp blueprint. parts of this work were supported by grants to s.r.p. is founder and chief scientific officer of atturos, a clinical diagnostics company. m.k. is an employee of bruker daltonics, a manufacturer of ms systems. all other authors declare no competing interests. supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-19045-9.correspondence and requests for materials should be addressed to m.s.b. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-341129-eo0vjcmk authors: kielian, margaret; rey, félix a. title: virus membrane-fusion proteins: more than one way to make a hairpin date: 2006 journal: nat rev microbiol doi: 10.1038/nrmicro1326 sha: doc_id: 341129 cord_uid: eo0vjcmk structure–function studies have defined two classes of viral membrane-fusion proteins that have radically different architectures but adopt a similar overall 'hairpin' conformation to induce fusion of the viral and cellular membranes and therefore initiate infection. in both classes, the hairpin conformation is achieved after a conformational change is triggered by interaction with the target cell. this review will focus in particular on the properties of the more recently described class ii proteins. enveloped animal viruses are covered -or 'enveloped' -by a lipid bilayer that is derived from the host-cell membrane during virus budding. this membrane protects the genetic material of the virus until it is delivered into the cytoplasm of a new host cell to initiate infection. delivery takes place by fusion between the virus membrane and the membrane of the host cell. this membrane-fusion reaction is a crucial step in virus infection and is mediated by transmembrane (tm) proteins that are anchored on the virus surface. virus membranes are relatively simple compared to hostcell membranes, and the process of virus-host-cell fusion is devoid of the additional regulatory and recycling proteins that are present in many cellular fusion processes 1, 2 . therefore, understanding the molecular mechanism of membrane fusion catalysed by viral membrane-fusion proteins is important not only for understanding virus infection, but also as a paradigm for cellular membrane-fusion reactions. virus membrane fusion can take place either at the plasma membrane or at an intracellular location following virus uptake by endocytosis 3, 4 . the fusion reactions of viruses that fuse directly at the plasma membrane are triggered by virus-receptor interactions at neutral ph, as discussed below. by contrast, the fusion of many other viruses is dependent on their internalization by receptor-mediated endocytic pathways such as clathrin-dependent, caveolae-dependent uptake or nonclathrin-dependent, non-caveolae-dependent uptake 4, 5 . viruses that use such routes frequently have fusion reactions that require exposure to mildly acidic ph within organelles of the endocytic pathway 6 . although it is unclear why viruses use such a variety of entry pathways, it is possible that those viruses that have evolved clathrin a cellular protein that is composed of three heavy chains and three light chains. clathrin is the main component of the coat that is associated with clathrin-coated vesicles, which are involved in membrane transport in both the endocytic and biosynthetic pathways. specialized regions that contain the protein caveolin and form flask-shaped, cholesterol-rich invaginations of the plasma membrane. to fuse intracellularly gain a selective advantage from releasing their genomes at specific intracellular sites 7 . virus membrane-fusion proteins drive the fusion reaction by undergoing a major conformational change that is triggered by interactions with the target cell. the specific trigger depends on the virus. for example, influenza viruses, alphaviruses and flaviviruses are classic examples of viruses that fuse upon exposure to low ph in the endocytic pathway or in the test tube 3, 6, 8, 9 . by contrast, fusion of hiv-1 occurs at neutral ph and is triggered by the sequential interaction of the virus fusion protein with the receptor cd4 and a co-receptor such as ccr5 or cxcr4, members of the 7-tm-domain chemokine receptor family 10, 11 . other variations include viruses with fusion reactions which seem to be triggered by interaction with a single receptor 3 , by the binding of the receptor to a separate attachment protein 12 , by a combination of receptor binding plus low ph 13, 14 , or by endosomal proteolysis 15 . given the recently described variations on the pathways of fusion-protein activation, it is probable that there are additional interesting twists to be discovered. to date, two classes of virus membrane-fusion proteins (fig. 1) have been defined, based on key structural features, as detailed below. viruses that are currently assigned to class i or class ii are listed in table 1. it is clear that for many viruses, the identity and/or functional features of the fusion protein have not yet been determined, making it difficult to classify them as class i or class ii, or perhaps even as the inaugural members of a new fusion-protein class (box 1) . this review will focus primarily on recent developments in the structure and function of the class ii fusion proteins (fig. 2) , using the alphavirus e1 glycoprotein as a paradigm. class i proteins will be introduced first a second receptor required for virus infection. in the case of hiv-1, fusion is triggered by sequential interaction of viral gp120 with the cd4 receptor, followed by a co-receptor. cytokines involved in specific inflammatory responses. they are differentiated into cc or cxc chemokines on the basis of their primary sequence. a single-pass transmembrane protein that contains an n-terminal external domain and a c-terminal cytoplasmic domain. to establish our current understanding of these fusion proteins, as they provide important context for the studies of the class ii proteins. structure and function. the class i membrane-fusion reaction is mediated by the refolding of the fusion protein to a highly stable rod-like structure with a central trimeric α-helical coiled coil. such coiled-coil structures are emblematic of class i proteins, and physical demonstration or computer prediction of such a structure is frequently used to help define a fusion protein as belonging to class i 16 . the class i proteins generally share several other important features, illustrated here by the example of the influenza-virus haemagglutinin (ha) 17 and summarized in table 2 and in refs 3, 11, 18. ha is a trimeric type 1 tm protein that contains both the receptor-binding and fusion activities of influenza virus. it is synthesized as a fusion-inactive precursor, ha0, which is processed by host-cell proteases to produce two disulphide-bonded subunits -ha1 (328 amino acids), containing the receptor-binding site, and ha2 (221 amino acids), the tm subunit responsible for fusion (fig. 1) . ha2 is maintained in a 'metastable' state at the virus surface 19, 20 . the n terminus of ha2 contains a conserved hydro phobic region known as the fusion peptide which inserts into the target membrane during fusion. the structure of the ha0 ectodomain reveals a trimer with a large globular head region that is composed mainly of ha1, and a long α-helical coiled-coil stalk region 21 . after processing, the overall structure of ha is almost unchanged, but the n-terminal fusion peptide of ha2 becomes buried at the trimer interface within the α-helical stalk 22 (fig. 3) . virus fusion is triggered by low ph, which destabilizes the ha1 trimer contacts at the head, causing the globular head domains to dissociate. this movement allows a loop-to-helix transition of a polypeptide segment of ha2 that was previously buried underneath the ha1 heads, projecting the fusion peptide ~100 å towards the target membrane, where it inserts irreversibly 23 . this initial change is proposed to result in a 'pre-hairpin intermediate' , an extended structure that is anchored both in the target membrane by the fusion peptide and in the virus membrane by the tm segment 11 . the c-terminal end of the long ha2 α-helix jackknifes back, reversing the direction of the viral-membrane-proximal segment of ha2, which then interacts in an anti-parallel fashion with the groove formed by the n-terminal trimeric coiled coil 23 . the final post-fusion conformation of ha2 is therefore a highly stable rod with the tm and fusion-peptide segments together at the same end of the molecule (fig. 4) , a structure termed a 'trimer of hairpins' 11 . the stability of the post-fusion conformation of ha is considerably higher than that of the metastable prefusion form, and class i membrane fusion is believed to be driven by the energy that is released during the conformational change 19, 20, 24, 25 . hairpin formation by ha2 is postulated to force the target and virus membranes into close apposition to trigger fusion, which occurs by the initial mixing of the outer lipid leaflets (termed 'hemifusion'), followed by mixing of the inner leaflets, opening of a small fusion pore, and widening of the pore to give complete fusion. membrane insertion. viral fusion peptides generally tend to be apolar regions, conserved within a virus family, relatively rich in glycine and alanine residues, and containing several bulky hydrophobic residues 3 . for influenza ha and several other class i proteins, the fusion peptide is located at the n terminus of the tm subunit, but other class i fusion peptides are located internally in the amino-acid sequence, and presumably insert into the membrane as a loop. structural studies of the ha fusion peptide indicate that it adopts a boomerangshaped structure formed by a kinked amphipathic α-helix lying roughly in the plane of the outer leaflet of the membrane at the polar-aliphatic interface 26 . membrane insertion seems to be primarily regulated by the initial release of the fusion peptide from its buried position to permit its amphipathic interaction with lipid bilayers. cooperativity. fusion by ha is positively affected by protein density, suggesting cooperative ha interactions during fusion 27, 28 . intriguingly, studies of cell-cell fusion mediated by influenza ha suggest that 'bystander' ha molecules outside the zone of cell-cell contact have effects on expansion of the fusion pore 29 . the mechanism by which ha might interact during fusion and the role of bystander fusion proteins are not well understood. structural information on class i fusion proteins has suggested several approaches for inhibiting fusion-protein refolding and membrane fusion 11, 30 . for example, the hiv-1 inhibitor t20/enfuvirtide is a peptide that corresponds to part of the c-terminal helix of the envelope glyco protein gp41 (refs 31, 32) . t20 blocks the interaction of the c-terminal segment of the fusion protein with the groove of the central coiled coil (fig. 4) , and blocks virus fusion and infection. importantly, small molecules that target crucial sites of interaction in the trimer of hairpins have also been shown to act as potent class i fusion inhibitors 33 . as small molecules can have higher bioavailability than peptides, they provide an important approach to develop more widely useful fusion inhibitors. although, to date, the class i inhibitors have mainly targeted viruses such as hiv-1 that are triggered at the cell surface, small molecules can also target viruses that fuse within endocytic compartments 34, 35 . the alphaviruses and flaviviruses are members of the togaviridae and flaviviridae families, respectively 36, 37 . these small spherically shaped viruses are composed of a nucleocapsid core that contains the single-stranded positive-sense genomic rna, surrounded by a membrane that contains the viral tm glycoproteins, which form an external icosahedral scaffold (fig. 2) . the x-ray structures of the fusion proteins of the alphaviruses and flavi viruses, described below, revealed that the ectodomains of these proteins have remarkably similar secondary and tertiary structures. this result strongly suggests that, despite the lack of any detectable amino-acid-sequence similarity, the corresponding genes were derived from a common ancestor. the three-dimensional structure of these fusion proteins is radically different from that of the influenza virus ha, a finding that introduced the concept of a separate class of fusion proteins, class ii 38 (table 2) . class i and class ii fusion proteins share common features of target-membrane insertion of a hydrophobic fusion peptide and refolding/hairpin formation to drive the membrane-fusion reaction, whereas their structural characteristics are markedly different. what other classes of viral fusion proteins might there be? one possibility would be proteins that mediate fusion using the same general mechanism but with a structural basis that belongs to neither class i nor class ii. for example, rhabdoviruses such as vesicular stomatitis virus and rabies virus have a single homotrimeric membrane protein, g, which mediates fusion in a low-ph-dependent reaction that involves membrane insertion of a hydrophobic region of g 93 and biochemically detectable conformational changes (reviewed in ref. 94 ). however, unlike the class i and class ii proteins discussed in this review, g is not proteolytically processed or synthesized with a companion protein, the g-protein conformational changes induced by the fusion trigger (low ph) are reversible and, as yet, no clear structural similarities to class i or class ii proteins have been identified. it is also probable that different overall mechanisms of fusion remain to be defined. for example, herpesvirus fusion involves several virus membrane proteins and might represent a different model from the single fusion proteins of class i and class ii (reviewed in ref. 95 ). likewise, recent data indicate that, in the case of the poxviruses, four proteins interact to induce fusion at low ph (see ref. 96 and references therein). the many open questions on the fusion of complex viruses such as herpesviruses and poxviruses will continue to be important and interesting topics in the future. the non-enveloped viruses of the orthoreovirus genus include some viruses that cause cell-cell fusion of infected cells. this fusion activity is due to non-structural membrane proteins termed fast proteins (fusion-associated small transmembrane proteins), which range in size from ~10-15 kda 97 . the ectodomains of fast proteins are small, in some cases comprising only ~20 residues, and can contain short hydrophobic regions and/or n-myristic acid 97, 98 . although their small size argues against hairpin formation, fast proteins are sufficient to mediate membrane fusion 99 , and it will be important to determine how they fit into our growing knowledge of the mechanisms of protein-mediated membrane fusion. small opening that allows flux between two membranebound compartments. fusion pores form at an early stage of membrane fusion and widen when they lead to full fusion. biosynthesis and assembly. during biosynthesis, the alphavirus (e1) and flavivirus (e) fusion proteins fold co-translationally with a companion or regulatory protein, termed p62 (or pe2) for alphaviruses and prm for flaviviruses 36, 37 (fig. 1) . this heterodimeric interaction is important for the correct folding and transport of the fusion protein. both p62 and prm are cleaved by the cellular protease furin late in the secretory pathway, in a maturation reaction that is a crucial regulatory step for subsequent virus fusion [39] [40] [41] [42] . one important difference between these two groups of viruses is the budding site 43, 44 . in alphaviruses, the p62-e1 complex is transported to the plasma membrane, and the heterodimer interaction is maintained after p62 processing. new virions bud at the plasma membrane, in a process that is driven by lateral contacts between e2-e1 heterodimers (e2 being the mature companion protein) to induce the required curvature of the lipid bilayer, in combination with interactions of the cytosolic tail of e2 with the nucleocapsid. budding results in formation of icosahedral enveloped particles of triangulation t = 4, containing 80 trimeric e2/e1 spikes 45, 46 . by contrast, flavivirus particles bud into the endoplasmic reticulum as immature virions formed by 60 trimers of prm-e. the immature particles have an organization similar to mature alphaviruses, with each trimer forming a spike in which prm covers the fusion protein e 47 . the newly formed virions are then transported to the external milieu through the exocytic pathway. processing of prm generates the mature m protein with a short (~40 residues) ectodomain. presumably because of the removal of a large portion of the prm ectodomain, the flavivirus surface dramatically reorgan izes after processing to give 90 e-e homodimers arranged with icosahedral symmetry 44, [48] [49] [50] . the mature flavivirus particles display a smooth, spikeless surface, with e dimers ordered in a characteristic 'herring bone' pattern. structure. crystal structures have been determined for the neutral ph ectodomains of the fusion proteins from the alphavirus semliki forest virus (sfv) 38,51 and the flaviviruses tick-borne encephalitis virus (tbe), dengue 2 virus, and dengue 3 virus 52-55 . the polypeptide chain of the class ii proteins follows a complex path, resulting in three globular domains -essentially constituted by β-sheets -organized so that the c terminus and the fusion peptide are found at the two ends of a rod-like molecule of ~120 å in length (fig. 2) . domain i, which contains the n terminus and a conserved glycosylation site, is a β-barrel with an 'up-and-down' topology. two of the connections between adjacent strands in this barrel are long and elaborated, and comprise the 'finger-like' domain ii with the fusion loop at the tip of the molecule. domain iii, which lies at the opposite end of domain i, has an immunoglobulin-superfamily fold and is connected to the c terminus of domain i by an ~12 amino-acid polypeptide. the tm domain of sfv e1 and the two tm domains of tbe were proteolytically removed to generate the soluble ectodomains. the sfv e1 ecto domain, referred to here as e1*, retains part of the 'stem' region that connects domain iii to the tm domain, although this flexible region is not visualized in the neutral ph structure. the stem regions of the flavivirus fusion proteins have been completely removed in the crystallized forms. conformational changes during fusion. unlike the class i fusion proteins, which are trimeric in both their pre-fusion and post-fusion conformations, class ii fusion proteins undergo an oligomeric rearrangement during fusion [56] [57] [58] , converting from the metastable prefusion dimer to a considerably more stable homotrimer conformation 59,60 (table 2) . formation of the targetmembrane-inserted homotrimer is required for class ii virus fusion 61, 62 . binding of protons in the acidic endosomal environment triggers a complete rearrangement of the surface of the class ii virus particles. the alphavirus e2-e1 heterodimers or the flavivirus e-e homodimers dissociate 58, 63 , resulting in disassembly of the icosahedral scaffold followed by a quaternary reorganization into e1 or e homotrimers inserted in the membrane by the fusion loops. importantly, as discussed below, the homotrimers seem to display lateral contacts between each other, forming a different type of surface lattice. in vitro studies using the ectodomains of both the alphavirus and flavivirus proteins showed that trimerization requires insertion of the fusion peptide into target membranes 64, 65 . solubilization of the trimers from the liposomes using non-ionic detergent led to their crystallization 66, 67 and to determination of their threedimensional structure [68] [69] [70] . the crystal structures of the class ii homotrimers showed a fold-back arrangement strikingly reminiscent of that of class i fusion proteins, despite the different architecture of the proteins (fig. 4) . the trimer is formed by a central parallel interaction of the subunits through roughly the n-terminal two-fifths of the molecule (including domains i and ii, with the fusion peptide at one end). the remaining c-terminal portion of the protein folds back along the sides of the core trimer, with domain iii moving ~30-40 å towards the fusion loop. the portion of the stem region that is present in the sfv e1 protein interacts closely with the trimer core and extends towards the fusion loop. this hairpin organization is therefore the same as that observed for class i fusion proteins, resulting in a trimeric protein rod with the fusion peptide loops and the c-terminal membrane anchors together at the same end. (fig. 2) . monoclonal antibodies to this region have been used to show that the fusion loop inserts into membranes and that insertion is required for fusion 65, 71, 72 . the fusion loop is composed of relatively apolar and conserved residues, and fusion is blocked by substitution of negatively charged amino acids into this crucial region 61,62 . the receptor-binding subunit, e2, is displayed as a sphere-filling model coloured grey, based on subtracting the e1 density from the cryo-electron microscopy reconstruction of the virus. the orange e1 fusion peptide (arrows) is buried at the e1-e2 interface. c | shows the pre-fusion complex (e) 2 of the flavivirus tick-borne encephalitis virus. the fusion peptide is buried at the e homodimer interface (arrows). all three panels are drawn at the same scale. the viral membrane would be tangential to a horizontal plane below the drawings. figure prepared using the ribbons program 100 . ha2 e1 e interestingly, the process of insertion of the alphavirus fusion peptide into the target membrane seems to differ from that of the class i proteins. for example, although heterodimer dissociation exposes the sfv fusion peptide, this alone does not seem sufficient for membrane insertion. the fusion loop is solvent-accessible in the ph-7 form of the e1* ectodomain 38 . the crystal structure shows that in the monomeric form, the e1 fusion loop (which is longer than its flavivirus counterpart) folds back on itself, burying most of the bulky non-polar side chains. its insertion into the target membrane apparently requires specific triggering by low ph and target-membrane cholesterol 64, 71 . this agrees with the cholesterol dependence of alphavirus membrane fusion, as discussed below. once e1* has stably inserted into the target membrane, it then requires detergent for solubilization, similar to the membrane-inserted class i proteins. the structure of the sfv detergentsolubilized e1* homotrimer shows that the fusion peptide unwinds, and the bulky non polar side chains are exposed, presumably within a detergent micelle in the crystal. liposome experiments show that alphavirus fusion is promoted by the presence of cholesterol and sphingolipid in the target membrane [73] [74] [75] . flavivirus-liposome fusion is also enhanced by cholesterol, although the stringency of the requirement seems considerably less than that of the alphaviruses 76, 77 . for both viruses, the action of cholesterol is more specific than its bulk effects on membrane fluidity or 'raft' formation, with the sterol 3β-hydroxyl group being particularly important for promoting membrane insertion, trimerization and fusion. interestingly, following membrane insertion, the sfv e1* ectodomain is strongly associated with cholesterol-rich membrane microdomains 72 . this could reflect a physical interaction of e1 with cholesterol, thereby coupling membrane insertion to the e1 conformational changes during fusion. in vivo studies show that membrane fusion and infection of the alphaviruses sfv and sindbis virus are decreased by 3-5 logs in cholesterol-depleted insect cells 78, 79 . this system was used to select for sfv mutants that have increased cholesterol independence 80, 81 . these srf (sterol requirement in function) mutants have single amino-acid changes in e1, located in the ij loop at the tip (the srf-3 p226s mutation) or in the hinge region (the srf-4 l44f and srf-5 v178a mutations) of domain ii (fig. 2) . the molecular mechanisms by which the srf mutations alter alphavirus cholesterol dependence are not yet known. for srf-3, the location in the ij loop suggests that the mutation could directly modulate the cholesterol dependence of the adjacent fusion loop. for srf-4 and srf-5, the location in the hinge area suggests that these mutations might affect the inherent flexibility of this region, indirectly altering the angle of interaction of the fusion loop with the target membrane. to demonstrate the suggested membrane interactions of the proteins, the final fused membrane is diagrammed in cartoon form. the class i proteins are drawn such that the n-terminal half of the hairpin is blue and the c-terminal part is red, with the missing fusion peptides and transmembrane domains indicated for one of the three trimer subunits as blue and red stars, respectively. for ha2, the residues are coloured as in the neutral ph form displayed in fig. 3 , and a membrane-proximal extended c-terminal 'leash' interacts with the central coiled coil of ha. in hiv glycoprotein 41 (gp41), the post-fusion structure is a six-helix bundle. the inhibitor t20 interacts at the site where the red helix is, effectively inhibiting the fusogenic conformational change. for the class ii proteins, an elongated red star indicates the predicted location of the tm segment, with a red arrow pointing to the c terminus of one of the subunits in the trimer of the crystallized class ii ectodomains, roughly showing the path of the missing 'stem' regions to complete the protein hairpin. figure prepared using the ribbons program 100 . in addition to being the site of the srf-3 mutation, several features of the alphavirus ij loop indicated its possible importance in the membrane-fusion reaction. although the e1 homotrimer is highly resistant to proteolysis, under defined conditions protease cleavage sites were identified around the fusion loop 82 . such protease cleavage releases the e1* homotrimer from the target membrane. other than the cleavage in the fusion-loop region, the only other cleavage site was immediately n-terminal to histidine 230 (h230) in the ij loop. sequence comparisons showed that, although the overall sequence of the ij loop is not highly conserved, a histidine residue is found in all of the reported alpha virus and flavivirus sequences 83 . the role of h230 in sfv fusion and infection was addressed by constructing an h230a mutation in the virus infectious clone 83 . the resulting mutant virus is not infectious and is completely blocked in membrane fusion, including the initial steps of lipid mixing. however, h230a virus undergoes apparently normal conformational changes upon exposure to low ph, including heterodimer dissociation and fusion-loop exposure, cholesterol-dependent target-membrane insertion, and formation of the e1 homotrimer. therefore, the h230a mutation demonstrates a crucial role for this residue within the sfv ij loop, and identifies a novel late-stage inter mediate in the class ii fusion pathway. two important observations indicate that class ii trimers interact cooperatively during membrane insertion and fusion. first, electron-microscopy studies of the membrane-inserted ectodomains of sfv and tbe show that insertion is highly cooperative and produces characteristic arrays of trimers organized in rings of five or six to form a lattice covering the liposome surface 67, 84 (fig. 5) . liposomes fully decorated with inserted sfv e1* display a fairly uniform diameter of ~120 nm, considerably smaller than the mean diameter of control liposomes without the protein. the surface-protein lattice therefore seems to control the curvature of the coated liposomes. because hexagonal arrays of e1* trimers result in a flat lattice, as shown in fig. 5, it is probable that rings of five e1* trimers are required to introduce the necessary overall curvature to make a fullerene-like 85, 86 , roughly spherical closed lattice at the liposome surface. second, the packing of sfv e1* trimers in the threedimensional crystals largely occurs through two-fold interactions between the fusion loops of adjacent trimers 68 . the trimer axes and the two-fold axis relating them intersect in space, making an angle close to the angle between three-fold and two-fold axes in an icosahedron. as in an icosahedron, the combination of these two operations (three-fold and two-fold about the corresponding symmetry axes) generates a five-fold axis also intersecting the three-fold and two-fold axes at the same point in space (at the centre of the body). the trimers in the crystals therefore interact through the fusion peptide in such a way that the contacts can be propagated -by repeating the observed trimer-trimer contacts through the fusion loops -to generate a closed ring of five e1* trimers, all associated by their fusion loops, just like the rings observed by electron microscopy. the fusion peptides in the five-fold rings form a 'crater' ~100 å in diameter and 40 å deep, with a base composed of five fusion loops and a rim composed of the remaining ten fusion loops. as all three fusion loops in each trimer are believed to interact tightly with the lipid heads, the resulting arrangement of trimers is such that it creates a dome-like distortion in the target membrane, which is postulated to be important in fusion, as shown in the model in fig. 6. therefore, electron-microscopy data and the crystal contacts provide support for two types of cooperative homotrimer interactions, producing rings of five and six trimers. we hypothesize that during fusion, five-fold interactions will occur between target-membraneinserted trimers at the side of the particle closest to the cell membrane. such five-fold interactions would act at the fusion site to induce formation of a 'dome' in the target lipid bilayer, whereas the refolding of e1 to a hairpin would pull the tm regions toward the centre of the five-fold ring, forming an opposing dome in the viral membrane and inducing hemifusion (fig. 6) . this 'lipid stalk' model for fusion is comparable to that proposed for the class i proteins 87 , and in keeping with recent studies which show that alphavirus fusion progresses through a transient hemifusion intermediate 88 . the six-fold trimer interactions might occur between trimers that do not directly interact with the target membrane and insert instead into the virus membrane. the more planar interactions of such trimers could have a role in the enlargement of the initial fusion pore by providing energy to flatten the viral membrane 89 . although these models are currently speculative, they provide a starting point for thinking about the geometry of the fusion reaction. the left panel shows a negatively stained sample of e1 ectodomain homotrimers (e1*ht) reconstituted at a low lipid-to-protein ratio to produce a planar hexagonal lattice. on liposomes, the e1*ht is proposed to display a fullerene-like architecture, in which insertion of rings of five instead of six trimers allows the required curvature to form a closed sphere (see text). images of the planar lattice such as that shown on the left were used to generate a three-dimensional reconstruction into which the atomic model of the e1*ht was fitted (right panel). direct lateral interactions between the trimer heads are observed from the fitting, as shown in this top view. interactions between the fusion loops of adjacent trimers could also occur through adjustment of the hinge region (indicated in fig. 2a) . figure how general is cooperativity? as discussed above, there is considerable evidence for fusion-protein cooperativity for members of the class i fusion proteins, particularly for the low-ph-induced influenza ha. interestingly, however, recent data indicate that some class i virus fusion reactions might be mediated by as few as one or two trimers. antibody-neutralization studies of hiv indicate that the target of neutralization is one trimer 90 , and detailed studies using viruses containing mixtures of wild-type and dominant-negative envelope proteins showed entry of viruses containing a single active hiv env trimer 91 . other class i viruses where fusion is triggered by receptor binding (murine leukemia virus) or by receptor plus low ph (avian sarcoma/leukosis virus) also showed entry properties that are suggestive of fusion mediated by a single trimer. by contrast, parallel studies of influenza ha fusion supported a requirement for eight or nine trimers 91 . further investigations will be required to determine if viruses with fusion directly triggered by low ph generally require multiple fusion proteins, the importance of concerted and cooperative interactions of those proteins, and whether fusion proteins with triggers that involve receptor binding typically activate fusion autonomously. implications and future directions studies of the class i and class ii fusion proteins indicate that, although their structures are very different, both classes refold during fusion to give analogous hairpin conformations, with the fusion peptides or loops and the tm domains at the same end of a stable protein rod. these observations indicate that the overall mechanism of membrane fusion is probably the same for both class i and ii, indicating that there might be a universal membrane-fusion mechanism, applicable to proteins that are clearly not homologous. although the structural and functional studies summarized here suggest an overall picture for class ii fusion, many important questions remain and new questions can now be identified. for example, we do not understand the mechanism by which the class ii fusion loop inserts into the target membrane, or the role of cholesterol in insertion. the current data indicate that insertion and fusion can be modulated by the ij loop and hinge region, but their mechanistic effects are unclear. we have a picture of the fusionprotein ectodomain before and after fusion, but we do not know how the fusion loop and tm domains are disposed in the fused membrane or how they might interact with each other. a series of conformational changes in class ii fusion proteins can be proposed (fig. 6) , but their order, kinetics and role in each of the steps of membrane fusion remain to be elucidated. we also do not understand how the refolding of the fusion protein to the trimer conformation takes place in the context of the whole virus particle, the fate of the companion protein (e2 in the case of alphaviruses) during this particle reorganization, and the role(s) of the observed five-fold and six-fold trimer interactions in the overall membrane-fusion process. an important question is whether the structure of the class ii fusion proteins can allow the development of inhibitors of specific steps in fusion. recent work indicates that exogenous domain iii blocks class ii membrane fusion and infection by binding to the fusion protein during the low-ph-induced conformational change 92 . similar to the class i proteins, such inhibitors could prove important for dissecting the molecular mechanism of fusion and, ultimately, for developing novel antiviral strategies. clearly, there is much exciting work to be done, and we look forward to future developments and discussions on the fusion of the class ii viruses. membrane fusion intracellular and viral membrane fusion: a uniting mechanism the many mechanisms of viral membrane fusion proteins how viruses enter animal cells viral entry on the entry of semliki forest virus into bhk-21 cells sfv infection in cho cells: cell-type specific restrictions to productive virus entry at the cell surface changes in the conformation of influenza virus hemagglutinin at the ph optimum of virus-mediated membrane fusion structures and mechanisms in flavivirus fusion hiv-1 entry cofactor: functional cdna cloning of a seven-transmembrane, g protein-coupled receptor mechanisms of viral membrane fusion and its inhibition paramyxovirus fusion: a hypothesis for changes retroviral entry mediated by receptor priming and low ph triggering of an envelope glycoprotein sequential roles of receptor binding and low ph in forming prehairpin and hairpin conformations of a retroviral envelope glycoprotein endosomal proteolysis of the ebola virus glycoprotein is necessary for infection a spring-loaded mechanism for the conformational change of influenza hemagglutinin receptor binding and membrane fusion in virus entry: the influenza hemagglutinin mechanism of membrane fusion by viral envelope proteins conformational changes in the hemagglutinin of influenza virus which accompany heat-induced fusion of virus with liposomes influenza hemagglutinin is spring-loaded by a metastable native conformation structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 å resolution first structure of a viral membrane-fusion protein first structure of the 'post-fusion' conformation of a viral membrane-fusion protein evidence that the transition of hiv-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion first demonstration that class i fusion is driven by hairpin formation membrane fusion machines of paramyxoviruses: capture of intermediates of fusion membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers synchronized activation and refolding of influenza hemagglutinin in multimeric fusion machines influenza hemagglutinins outside of the contact zone are necessary for fusion pore expansion the entry of entry inhibitors: a fusion of science and medicine a synthetic peptide from hiv-1 gp41 is a potent inhibitor of virus-mediated cell-cell fusion potent suppression of hiv-1 replication in humans by t-20, a peptide inhibitor of gp41-mediated virus entry targeting a binding pocket within the trimer-of-hairpins: small-molecule inhibition of viral fusion molecular mechanism underlying the action of a novel fusion inhibitor of influenza a virus structurebased identification of an inducer of the low-ph conformational change in the influenza virus hemagglutinin: irreversible inhibition of infectivity the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph prefusion structure of alphavirus e1 protein, showing unexpected similarity to flavivirus e protein, leading to concept of 'class ii' membrane-fusion proteins membrane fusion process of semliki forest virus ii: cleavage-dependent reorganization of the spike protein complex controls virus entry furin processing and proteolytic activation of semliki forest virus proteolytic activation of tick-borne encephalitis virus by furin cell-associated west nile flavivirus is covered with e+pre-m protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release budding of alphaviruses a structural perspective of the flavivirus life cycle cryo-electron microscopy reveals the functional organization of an enveloped virus, semliki forest virus placement of the structural proteins in sindbis virus structures of immature flavivirus particles first description of mature flavivirus surface organization, showing the striking herringbone arrangement of fusion protein homodimers visualization of membrane protein domains by cryo-electron microscopy of dengue virus structure of west nile virus crystal structure of the semliki forest virus envelope protein e1 in its monomeric conformation: identification of determinants for icosahedral particle formation the envelope glycoprotein from tickborne encephalitis virus at 2 å resolution a ligand-binding pocket in the dengue virus envelope glycoprotein variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein conformational changes of the flavivirus e glycoprotein membrane fusion process of semliki forest virus i: low ph-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells membrane fusion of semliki forest virus involves homotrimers of the fusion protein references 56 and 57 comprise the first reports of homotrimer formation for a class ii fusion protein oligomeric rearrangement of tickborne encephatitis virus envelope proteins induced by an acidic ph formation and characterization of the trimeric form of the fusion protein of semliki forest virus role of metastability and acidic ph in membrane fusion by tick-borne encephalitis virus mechanisms of mutations inhibiting fusion and infection by semliki forest virus mutational evidence for an internal fusion peptide in flavivirus envelope protein e the heterodimeric association between the membrane proteins of semliki forest virus changes its sensitivity to low ph during virus maturation membrane and protein interactions of a soluble form of the semliki forest virus fusion protein first demonstration that class ii ectodomain can form homotrimer when treated at low ph in presence of target membrane membrane interactions of the tick-borne encephalitis virus fusion protein e at low ph purification and crystallization reveal two types of interactions of the fusion protein homotrimer of semliki forest virus characterization of a membraneassociated trimeric low-ph-induced form of the class ii viral fusion protein e from tick-borne encephalitis virus and its crystallization conformational change and protein-protein interactions of the fusion protein of semliki forest virus structure of the dengue virus envelope protein after membrane fusion references 68, 69 and 70 are the first reports of low-ph-induced homotrimer structure of class ii fusion proteins multistep regulation of membrane insertion of the fusion peptide of semliki forest virus the fusion peptide of semliki forest virus associates with sterolrich membrane domains ph-dependent fusion between the semliki forest virus membrane and liposomes the role of cholesterol in the fusion of semliki forest virus with membranes membrane fusion of semliki forest virus requires sphingolipids in the target membrane membrane fusion activity of tick-borne encephalitis virus and recombinant subviral particles in a liposomal model system involvement of lipids in different steps of the flavivirus fusion mechanism cholesterol is required for infection by semliki forest virus the cholesterol requirement for sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence a single point mutation controls the cholesterol dependence of semliki forest virus entry and exit novel mutations that control the sphingolipid and cholesterol dependence of the semliki forest virus fusion protein molecular dissection of the semliki forest virus homotrimer reveals two functionally distinct regions of the fusion protein a conserved histidine in the ij loop of the semliki forest virus e1 protein plays an important role in membrane fusion visualization of the targetmembrane-inserted fusion protein of semliki forest virus by combined electron microscopy and crystallography physical principles in the construction of regular viruses graphitic cones and the nucleation of curved carbon surfaces protein-lipid interplay in fusion and fission of biological membranes class ii fusion protein of alphaviruses drives membrane fusion through the same pathway as class i proteins the protein coat in membrane fusion: lessons from fission stoichiometry of antibody neutralization of human immunodeficiency virus type 1 stoichiometry of envelope glycoprotein trimers in the entry of human immunodeficiency virus type 1 domain iii from class ii fusion proteins functions as a dominant-negative inhibitor of virus-membrane fusion photolabeling identifies a putative fusion domain in the envelope glycoprotein of rabies and vesicular stomatitis viruses reversibility in fusion protein conformational changes. the intriguing case of rhabdovirus-induced membrane fusion herpesvirus entry: an update the product of the vaccinia virus l5r gene is a fourth membrane protein encoded by all poxviruses that is required for cell entry and cell-cell fusion a new class of fusionassociated small transmembrane (fast) proteins encoded by the non-enveloped fusogenic reoviruses reptilian reovirus utilizes a small type iii protein with an external myristylated amino terminus to mediate cell-cell fusion liposome reconstitution of a minimal protein-mediated membrane fusion machine we acknowledge the important work of the many researchers whose contributions were not fully covered owing to space constraints. we thank j. lepault for the electron microscopy the authors declare no competing financial interests. key: cord-352172-g0jiaenw authors: stoevesandt, oda; taussig, michael j; he, mingyue title: protein microarrays: high-throughput tools for proteomics date: 2014-01-09 journal: expert rev proteomics doi: 10.1586/epr.09.2 sha: doc_id: 352172 cord_uid: g0jiaenw protein microarrays are versatile tools for parallel, miniaturized screening of binding events involving large numbers of immobilized proteins in a timeand cost-effective manner. they are increasingly applied for high-throughput protein analyses in many research areas, such as protein interactions, expression profiling and target discovery. while conventionally made by the spotting of purified proteins, recent advances in technology have made it possible to produce protein microarrays through in situ cell-free synthesis directly from corresponding dna arrays. this article reviews recent developments in the generation of protein microarrays and their applications in proteomics and diagnostics. protein microarray technology has rapidly developed into one of the most active areas in biotechnology today, and is being increasingly applied for the high-throughput discovery of molecular interactions, profiling of protein expression and monitoring of protein modifications. while genome sequencing and annotations provide comprehensive information on protein-encoding genes, and dna arrays analyze gene expression at the mrna level, these technologies need to be complemented by functional proteomic studies in order to better understand protein activities, expression levels and interactions at the system level. owing to alternative mrna splicing and a multitude of post-translational modifications (ptms), the number of cellular protein species far exceeds that of the genes. in addition, expression levels of individual proteins can range over as much as six orders of magnitude when cells are exposed to different environments [1] . it is clear that, for large-scale analyses, proteomics studies require sensitive, high-throughput tools with a wide dynamic range of detection. one such tool is the protein microarray, consisting of several thousands of protein species immobilized on a solid surface in miniaturized features, enabling sensitive, high-throughput screening with economical use of samples and reagents. in contrast to screening systems such as yeast two-hybrid, the in vitro nature of protein arrays allows for the control of the interaction conditions, protein concentrations, ptms and specific cofactor requirements [2] . the generation of a large diversity of proteins, such as the array elements, presents a major challenge for protein microarray systems, despite the develop ment of high-throughput proteinproduction methods. for many applications, the proteins also need to remain stable and functional on the array surfaces during long-term storage, which can pose an additional difficulty. a recent advance to overcome these problems is to produce protein microarrays through in situ cell-free synthesis directly from arrayed dna templates [3] [4] [5] . this article reviews the conventional methods, as well as more recent developments, in protein microarray techno logies, together with novel applications of these technologies in medical and proteomics studies. review stoevesandt, taussig & he or antigens (that may be nonfolded) are arrayed for profiling the expression of proteins [9, 10] or for the quantification of antibodies [11] in complex samples such as serum. applications of antibody arrays include biomarker discovery and monitoring of protein quantities and activity states in signaling pathways. antigen arrays are applied for profiling antibody repertoires in auto immunity, cancer, infection or following vaccination. moreover, antigen arrays are tools for controlling the specificity of antibodies and related affinity reagents. reverse-phase arrays comprise cell lysates [12, 13] or serum samples [14] . analysis is multiplexed by probing replicates of the arrays with different antibodies. reverse-phase arrays are particularly useful for studying the changes in the expression of specific proteins and protein modifications during disease progression and, thus, are primarily applied for biomarker discovery. figure 1 shows the two general strategies for making protein micro arays. in conventional approaches, individual proteins are expressed by recombinant technologies and are then purified and spotted onto a solid support, often glass slides. by contrast, in the newer in situ arrays, proteins are directly produced onto the array surface through cell-free protein synthesis. the most commonly used strategies for the production of proteins for arrays are heterologous expression in escherichia coli [15] or by cell-free systems [16] . the choice of protein production method depends on the downstream applications, in which protein yields, folding and modifications must be taken into account. clearly, high-throughput formats are desirable for generating proteins. the advantages of using e. coli are rapidity, low cost and ease of adaptation to high-throughput production. however, some eukaryotic proteins are poorly expressed in e. coli or are produced, but at low solubility, and eukaryotic ptms are lacking in prokaryotic cells. screening assays have been developed for the rapid assessment of protein solubility, including using reporter proteins to directly monitor protein expression and correct folding [17] [18] [19] . cell-free protein synthesis provides an effective and rapid alternative by using pcr dna fragments as templates, circumventing the need for e. coli cloning [16, 20] . cell-free methods are capable of making proteins that are difficult to express in in vivo systems. cell-free lysates can be prepared from different species, allowing protein synthesis to be carried out in prokaryotic or eukaryotic systems and at defined temperatures. these open systems can also be adjusted through the addition of further components, so as to provide environments suitable for protein folding or modifications [20] . in conventional methods, proteins expressed by e. coli or cell-free systems are purified prior to spotting onto the array surface. purification is usually performed using affinity tags attached at either the n-or c-terminus, of which the most popular tags are hexahistidine (his 6 ) and glutathione-s-transferase (gst) [3, 21] . by contrast, by spotting unpurified tagged proteins onto capture surfaces, the immobilization and purification steps can be combined into a single step [22] . proteins are often arrayed onto functionalized glass slides, using four main physicochemical principles, which may be combined: adsorption, covalent binding, affinity interaction by specific tags and physical entrapment into gels [23] . surface adsorption is generally mediated by electrostatic charges (e.g., on polylysine-coated slides) or hydrophobic interactions. despite its simplicity, the main drawback of this method is the possibility of denaturating proteins on the surface. nevertheless, studies have suggested that surface-adsorbed proteins may still retain their functionality [24] . surface adsorption is convenient where native protein structure and function are not essential; for example, antigen arrays. covalent binding of proteins to derivatized surfaces is a more efficient and robust approach [25] . the surfaces usually carry reactive groups, such as epoxides, aldehydes, succinimidyl esters or isothiocyanates, which react with nucleophilic groups (e.g., amino, thiol or hydroxyl groups) of amino acid residues. covalent immobilization via random attachment also tends to denature arrayed proteins. affinity interaction by specific tags provides a means of immobilizing proteins in a defined orientation on a tag-capture surface, often retaining full protein activity. examples include the use of biotin, his 6 -and gst-tagged proteins binding to streptavidin, ni-nta and glutathione-coated slides, respectively [3, 26] . proteins fused with a carbohydrate-binding module have been successfully immobilized on a cellulose-coated surface [27] . affinity-capture surfaces are also capable of immobilizing tagged proteins directly from complex mixtures without prior purification [22] . recently, an enzymatic method has been described for covalent immobilization of tagged proteins directly from cell lysates [28] . the use of 3d matrices (hydrogels) to entrap proteins into a structured environment maintains the function of arrayed proteins. usually, a layer of a polymer, such as polyacrylamide, agarose or gelatine, covers the slide and provides a porous structure filled with an aqueous buffer. the matrix can be additionally modified with functional groups for covalent or affinity immobilization of proteins. gel matrices permit the arraying of enzymes, while conserving their activities [29] . a systematic study comparing eight different commercially available array surfaces, combined with detection by different fluorescent dyes, has revealed critical parameters affecting the reproducibility and reliability of protein microarrays [30] . robotic spotting of proteins is generally carried out by either contact printing with solid or split metal pins, or via non contact delivery in an ink-jet printer-like fashion. unlike arraying of dna samples, procedures for spotting functional proteins must ensure that they are hydrated, stable and folded during the process. however, the distributed volumes of solutions are typically in the subnanoliter range and the spots are therefore prone to drying rapidly by evaporation. buffers for protein arraying therefore need to combine physiological conditions and compatibility with both the surface-immobilzation mechanism protein microarrays: high-throughput tools for proteomics and the robotic-arraying process in terms of viscosity and surface tension. typical buffer additives for protein stabilization against drying are hydroxylated cryoprotectants (e.g., glycerol, polyethylene glycol, trehalose and sucrose), but their effectiveness must be evaluated with the surface of choice [31, 32] . several new nonnucleophilic substances have recently been identified as buffer additives that improve spot morphology without interfering with immobilization [33] . an innovative method for preventing droplet evaporation was demonstrated by spotting under a layer of water-immiscible liquid fluorocarbon, coupled with a liquid-bridge, noncontact spotting technology [34] . depending on the types of slides and the viscosity of the buffer used, printing density can generally be up to 20,000 individual features on a standard microscope slide, corresponding to a center-to-center spacing of spots in the range of 250 µm [35] . to avoid separate expression and purification of individual proteins by cell-based methods, cell-free expression has been exploited to produce protein microarrays by synthesising proteins directly onto the surface from arrayed dnas (figure 2) . a number of different methods have been developed [36] . in the protein in situ array (pisa) method, pcr dna constructs and the cell-free expression system are co distri buted onto protein-capture surfaces, so that newly synthesized proteins are captured in situ and unbound lysate material can be washed away (figure 2a ) [5] . pisa first demonstrated the advantages of 'on-chip' synthesis of proteins, notably the ability to convert dna directly into functional proteins in an array format, without separate e. coli cloning, expression and purification. pisa also avoids the problem of maintaining protein stability on the array surface in long-term storage, since the proteins are only expressed as and when the protein array is needed. the pisa concept was first exemplified using an engineered double-his 6 tag, which permits single-step immobilization of the tagged proteins onto ni-nta-coated surfaces from the cellfree lysate [5, 37] . a highly miniaturized version, based on a multiple spotting technique (mist), has been developed, allowing in situ synthesis of proteins on the surface in subnanoliter volumes (350 pl). this makes it possible to produce protein microarrays with a potential density of up to 13,000 spots per slide [38] . instead of codistributing soluble dna and cell-free lysate as in pisa, the nucleic acid programmable protein array (nappa) method creates a protein array from immobilized dna templates [4, 39] . plasmid dna is immobilized together with a proteincapturing antibody onto a glass slide, which is then covered with a rabbit reticulocyte lysate cell-free system to express the proteins. the newly synthesized proteins become trapped by the antibody colocalized at each spot ( figure 2b ). nappa has been applied to produce protein arrays (up to 2000 spots per slide) identifying immune-response signatures of breast cancer autoantibodies in patient sera [39, 40] and to isolate target proteins with agonist activities from a cdna library [41] . as the proteins immobilized by nappa remain on the same surface as the dna array, the technology does not generate a 'pure' protein array, but rather one in which individual proteins are colocalized with both their encoding dna and the general capture reagent. in situ synthesis of protein array (see figure recently, a novel system has been introduced that converts a reusable dna array into multiple protein array copies ( figure 2c ) [ protein microarrays: high-throughput tools for proteomics dna array (but with a mirror-image layout). fully consistent with the concept that diffusion is the mechanism of protein transport to the capture slide, the protein-density profiles of dapa protein spots are well-defined gaussian distributions. dapa is capable of producing at least 20 copies of the same protein array by the repeated use of a single dna array template [3] . this technique may find particular use for the repeated printing of protein arrays in laboratories that do not have access to microarray spotters. dapa has been validated by arraying a number of different proteins, including antibody fragments, green fluorescent protein and transcription factors [3] . in situ puromycin capture from mrna arrays a puromycin-capture strategy has been described to fabricate protein arrays by capturing nascent polypeptides in situ [42] . it requires extra steps to make mrna-ssdna hybrid molecules, and protein yields are limited by the amount of mrna spotted. readout technologies to detect interactions of probes on protein microarrays can be grouped into label-based and label-free methods. to date, approaches based on fluorescent labeling are the most widely used, with fluorophores either directly attached to the interactors or introduced on secondary reagents, such as in sandwich-assay formats. label-free approaches, by contrast, measure an inherent property of the interactors themselves, such as mass or dielectric properties. fluorescence-based methods use widely available reagents and instrumentation, but the covalent introduction of fluorophores may affect the protein function or epitope being monitored. label-free technologies circumvent this problem and may allow real-time monitoring, but they require more sophisticated equipment. to date, the sensitivity of the routinely used label-based methods is higher than that of label-free approaches [43] . current microarray scanners are typically equipped for fluorophore excitation at 532 and 633 nm, allowing dual color fluorescence detection in the cy3 and cy5 ranges. scanners with a wider range of excitation wavelengths, including 488 nm suitable for gfp-fusion proteins, are also becoming increasingly available. direct labeling of proteins for interaction screens is usually by covalent conjugation of n-hydroxysuccinimide ester-linked fluorescent dyes to primary amines of the protein n-terminus or ε-nh 2 of lysine residues. alternatively, thiol-reactive dyes are used to couple to cysteine residues. these methods can be applied to purified proteins as well as to complex mixtures (e.g., sera). for sample comparison, ratiometric two-color labeling strategies are used, analogous to those used for expression profiling on dna microarrays [9, 44] . random chemical conjugation of dyes may interfere with protein function. cell-free expression systems provide alternatives for site-directed introduction of fluorophores at the protein n-or c-terminus. using a modified initiator trna, the n-terminal methionine can be replaced by a fluorophore-amino acid conjugate, with labeling efficiencies of 67% in an e. coli cell-free system [45] . c-terminal labeling proceeds through the incorporation of fluorophorecoupled puromycin derivatives, with labeling efficiencies of up to 90% [46, 47] . besides direct coupling of fluorophores, primary and secondary immunofluorescent staining protocols are often applied [48, 49] . in antibody arrays, such sandwich-detection strategies provide a second level of target recognition, significantly increasing the specificity and sensitivity (down to picogram per milliliter) of target detection. enhanced sensitivity can also be achieved with a number of amplification systems. tyramide signal amplification uses peroxidase-coupled reagents to catalyze the coupling of reactive fluorophore derivatives to tyrosine residues in the vicinity [3, 50] and can detect femtogram amounts of protein. dna-based technologies, such as rolling circle amplification (rca) [10, [51] [52] [53] , as well as proximity ligation of oligonucleotides coupled to antibodies recognizing different epitopes [54, 55] , provide high levels of amplification, essentially converting from protein to dna detection. quantum dots are emerging as the new-generation fluorophores for ultrasensitive readout of binding events [13, 56] . recently, single-walled carbon nanotubes coupled to antibodies were used as multicolor raman labels for the highly sensitive detection of proteins arrayed on raman-scattering gold surfaces [57] . it is likely that novel combinations of existing tools will continue to enhance the detection sensitivity of protein microarrays. current label-free methods applicable to array formats include mass spectrometry (ms), optical biosensors on metal surfaces (e.g., surface plasmon resonance [spr] and nanohole arrays) and conductance biosensors on carbon nanotube and nanowire surfaces [43] . mass spectrometry offers intriguing possibilities through its ability to directly identify proteins, and is 'hypothesis-free' compared with identification via the use of specific antibodies. the direct interfacing of 3d polymer-based protein arrays to maldi-ms for the detection of antigen-antibody interactions and enzymatic activities has been demonstrated [58] . porous silicon surfaces structured with arrays of nanovials have been applied for rapid tryptic digestion of biomarker proteins, subsequently identified by maldi [59] . biosensor-based approaches detect the binding of a protein to a sensor surface by virtue of the change of the local refractive index or conductivity on the surface. they typically enable the detection of real-time interactions to determine association and dissociation kinetics [60] . based on the principle of spr, gratingcoupled spr [61] allows higher degrees of multiplexing, with up to 400 arrayed elements [62] . a recently developed interferometric biosensor, based on optical-phase differences due to molecular associations, allows for real-time kinetics with picogram per square millimeter sensitivity [63] . nanohole-array sensors utilize the optical properties of light transmission through 150-200-nm diameter holes in metal surfaces, which are susceptible to changes in the local refractive index brought about by molecular interactions [64] . biosensors of carbon nanowires and nanotubes, which review stoevesandt, taussig & he detect changes in conductivity on the surface upon binding of molecules, can also be used to detect molecular interactions with proteins, small molecules and nucleic acids [65, 66] . interactions of proteins, both with other proteins and with nonprotein partners (e.g., nucleic acids, small molecules and lipids), form the basis of many cellular pathways. global identification of these interactions greatly contributes to our understanding of signal transduction and its aberration in disease, which, in turn, facilitates target discovery and drug design. with regard to identifying protein interactions and networks, protein microarrays will not generally be the sole definitive technique, but are likely to be complementary and confirmatory to a range of other methods, from isolation and characterization of protein complexes by pulldown and ms, to yeast two-hybrid screening and similar in vivo systems. nevertheless, protein arrays provide the benefit of studying defined individual protein components under controllable conditions. functional proteome arrays provide a high-throughput system for exploring protein interaction networks, as first exemplified by the landmark application of a yeast proteome array [26] . one-onone screening of interactions by functional protein micro arrays often identifies novel interactions. for example, screening of 49 arrayed coiled-coil domains from human leucine zipper transcription factors against each of the domains in their soluble form led to the discovery of a number of previously unknown pairwise interactions. their relative binding strengths, as determined on the array surface, were also in good agreement with those from solution-based studies [67] . in another study based on in situ arraying by nappa, a protein functional array containing 29 proteins involved in dna replication initiation was probed with each of these 29 proteins in turn. a total of 110 pairwise interactions were identified, of which 63 were previously unknown [4] . novel interactions with calmodulin and calmodulin-related proteins in the presence of calcium have been identified through the use of high-density functional protein microarrays prepared from 1133 open reading frames of arabidopsis spp. [68] . the regulatory functions of the proteins for a wide range of targets and cellular activities were revealed. unknown interactions were also identified directly from cell lysates using arrays of functional proteininteraction domains, such as sh2, sh3, pdz, ww, ff, ph, fha and 14.3.3 [24] . in an approach based on parallel capture of native protein complexes from lysates of differentially stimulated t cells, peptide microarrays were used for the ana lysis of signaling-dependent protein-interaction networks [48] . functional arrays of nuclear hormone receptors and coactivators were used for the systematic detection of receptor dimerization, functional classification of recruited coactivators and pharmaco logical characterization of receptor ligands [69] . recently, an interesting example has demon strated the use of human protein microarrays for identifying polyanion-binding proteins [70] . arrayed proteins, such as transcription factors, can be probed with dna or rna, enabling simultaneous identification of both the nucleic acid-binding proteins and their target sequences [71] . the first proteomic array employed for the ana lysis of dna-binding events contained 5800 yeast proteins. after screening with fluorescently labeled genomic ssdna or dsdna, 273 proteins that bound at least one of the dna forms were identified [26] . in another assay, genomic dna from saccharomyces cerevisiae was used to probe proteome chips, identifying 200 dna-binding proteins, including known transcription factors. this report also identified a novel dna-binding protein with a metabolic enzyme activity directly regulating eukaryotic gene expression [72] . recently, commercial protein microarrays have been used for the large-scale search of protein-dna interactions [73] . a microarray composed of 282 known and predicted yeast transcription factors detected numerous pairings of transcription factors and their binding dna sequences. target genes for one previously uncharacterized dna-binding protein were identified and shown to be involved in the stress response and oxidative phosphorylation [74] . the sequence-specific binding to dna of two bacterial transcription factors (argr repressor protein and camp-binding protein) and the dna-replication protein (dnaa) have also been demonstrated on protein microarrays [75] . using 33 p-labeled dna as the probe, p53 dna-binding variants were identified on functional arrays, and their relative affinities were simultaneously measured [76] . a screen for protein-lipid interactions was performed on functional yeast proteome arrays [26] . probing with several phospho inositides pointed to six new proteins associating with signaling lipids. the f irst example of using a protein array to analyze protein-small molecule interactions was between fkbp12 and three fluorophore-coupled small molecules, showing differences in the affinity of their interactions [77] . binding of gtp to gtp-binding protein on functional arrays has also been demonstrated [78] . a significant finding was that three different g-protein-coupled receptors arrayed together with their associated membrane lipids retained high-affinity, selective interactions with small-molecule ligands, demonstrating the functionality of arrayed membrane proteins [79] . screening of yeast protein microarrays with small molecules led to the discovery of a target protein required for the suppression of rapamycin growth arrest [80] . it is anticipated that, in the future, functional protein arrays will be further explored for screening a variety of biological and synthetic small molecules for their potential as drug candidates [81] . antibody and antigen arrays are ideal 'capture' tools for profiling protein expression or immune responses, with applications in diagnostics and biomarker discovery. protein microarrays: high-throughput tools for proteomics arrays comprising large numbers of immobilized anti bodies of different specificities are a sensitive means for qualitative and quantitative determination of protein expression [9, [82] [83] [84] . antibody arrays can be used to capture specific proteins from a variety of biological samples (e.g., lysates of cultured cells, tissue lysates and body fluids), enabling the identification of possible diagnostic signatures of disease-associated proteins [10, 85, 86] , and have been applied with success to cancer studies. a recombinant antibody (scfv) microarray allowed profiling of proteins from the sera of pancreatic adenocarcinoma patients, producing a protein signature of 19 nonredundant proteins that could discriminate cancer patients from healthy individuals [87] . in a further example of antibody array application to biomarker discovery, 29 out of 378 proteins were identified as having significant changes in their expression profiles in biopsies of human squamous cell lung cancer carcinoma compared with normal tissues [88] . the recent development of antibody microarrays in suspension using color-coded beads offers a fast, flexible, sensitive and multiplexed means for screening large numbers (>200) of clinical serum samples [89] . the content of antibody arrays, in terms of their range and quality, is a critical issue with regard to their applications. antibody arrays used in protein profiling must recognize the native conformations. antibodies have different affinities for their specific targets, so the ana lysis of ligand binding in parallel and on the same surface may require the optimization of antibody concentrations prior to spotting. the concentration range of the target proteins and the conditions under which different ligands bind may also require optimization of buffers. in addition, cross-reactivity of antibodies can cause a variable background. protein mutations, modifications and epitope changes may result in the failure of antibody arrays to detect these proteins. to help reduce these problems, the 'antibodypedia' database has been established, providing details of individual antibody validation from publicly available resources [90] . antigen arrays are used to profile antibody responses in autoimmunity, cancer, infection or following vaccination. proteomewide antigen arrays generated through cell-free synthesis of 185 proteins from vaccinia virus [91, 92] or 1700 proteins from francisella tularensis have been used to study humoral immune responses to infection with these agents [93] . they produced specific antibody profiles in sera from vaccinated or infected humans and animals that, in turn, facilitated the rational design of vaccines and diagnostic antigens [94] . other examples are the identification of immunodominant antigens from yersinia pestis [95] and potential vaccine candidates from plasmodium falciparum (a prominent malaria pathogen) proteins [96] . profiling autoantibodies has an important potential for biomarker discovery, with further uses in diagnostics and therapy selection. for example, the patterns of antibody reactivity in multiple sclerosis sera identified by antigen microarrays, comprising heat-shock proteins, lipid autoantigens and cns proteins, distinguished as different forms of the disease [97] . a remarkable 'myelin proteome' antigen microarray has been used to identify 'epitope spreading' through profiling the evolution of autoantibody responses that, in turn, guided the design of tolerizing dna vaccines for encephalomyelitis [98] . to address autoimmune events associated with neurological disorders, cerebrospinal fluid, rather than serum, has also been studied on antigen microarrays [99] . as well as in the established autoimmune diseases themselves, profiling autoantibodies may be a future means of cancer diagnosis, and the antigens identified may also be potential immuno therapeutics [100] . a conformational protein array containing 329 folded and functional autoantigens was capable of detecting serum antibody profiles from non-small-cell lung cancer patients [101] . reverse-phase antigen arrays, prepared by directly spotting various dilutions of lysates from 60 human cancer cell lines, have led to the identification of two promising pathological markers distinguishing colon from ovarian adenocarcinomas [50] . reverse-phase arrays allowed profiling of protein levels from as few as three cell protein equivalents in samples of primary leukemia and hematopoetic stem cells [102] . antigen arrays are also an ideal method for profiling specificity and cross-reactivity of affinity reagents such as antibodies. a proteome microarray comprising 5000 different yeast proteins was used to screen 11 polyclonal and monoclonal antibodies for a comparative profiling of antibody crossreactivity [103] . a commercial human-protein array comprising approximately 10,000 recombinant proteins has been found to be particularly useful for the rapid characterization of antibody specificity [104] . the activity of signal transduction pathways can be measured using arrays by determining the amounts and activation state of the signaling proteins downstream of receptors. this was demonstrated for the erbb receptor tyrosine kinase pathway using lysates of human tumor cell lines and antibody microarrays. this approach also enabled the characterization of the inhibitory profile of a small-molecule inhibitor on the pathway level [105] . to facilitate our understanding of pathways and their changes in disease, the effects of drugs and growth factors on the ptms of specific proteins in normal and tumor cells have been studied by antibody micro arrays [106, 107] . a reverse-phase micro array approach has been successfully applied to discover distinct pathway-activation differences among breast cancer cell lines, demonstrating the use of array-based ana lysis for multiple pharmacodynamic biomarkers responding to targeted kinase inhibitors [108] . additional signaling pathways or defects can be ascertained by exposing tumor cells or other diseased tissues to cytokines, chemokines or other bioactive molecules, using antibody arrays [85] or reverse-phase arrays [102] . in the future, it should be possible to reconstitute the complex pathways connecting the genome and the proteome in normal and diseased states through combining protein microarrays with genomicprofiling technologies. however, undertaking multiplexed quantitative ana lysis of protein-ligand interactions on microarrays, even with purified individual domains of the same functional families, requires careful standardization to avoid pitfalls due to review stoevesandt, taussig & he dissimilar behavior of the proteins and variable densities of active proteins. these considerations and the danger of relying on single concentration determinations have been discussed with respect to binding of phosphopeptides by an arrayed set of sh2 domains [2] . protein microarrays provide a high-throughput method for ana lysis of ptms such as glycosylation and phosphorylation. protein microarrays allow large numbers of different glyco proteins to be studied in a single experiment [109] . currently, lectin microarrays are used for high-throughput identification of protein glyco sylation [110] , while glycoprotein microarrays can be probed with a variety of lectins in turn to reveal glycosylation patterns in sera from healthy controls and patients with chronic pancreatitis and pancreatic cancer [111] . while phosphorylation controls much of the protein activity in living cells, linking particular kinases with specific substrates has been problematic. with functional protein microarrays, novel kinase substrates have been identified. for example, an array of 1690 proteins from arabidopsis thaliana was screened with mpk3 and mpk6, respectively, followed by antiphosphotyrosine antibodies, leading to the identification of 48 and 39 candidate substrates for mpk3 and mpk6, respectively [112] . in another study illustrating the power of functional yeast proteome arrays, substrate screening identified candidate targets for each of the 87 yeast kinases [113] . such discovery approaches are complemented by reverse-phase and antibody microarrays for the parallel ana lysis of known protein phosphorylations. using reverse-phase microarrays, site-specific phosphorylation of 62 signaling proteins from il-2 -stimulated t cells was detected, revealing differential protein phosphorylation [114] . antibody microarrays have been employed for the time-resolved monitoring of cell-signaling pathways by quantifying both the amount and the phosphorylation status of signaling proteins [115] . activities of phosphatases, cysteine proteases and serine hydrolases have been studied on array surfaces [116] and by using suicide inhibitors, which attach covalently to the active site [117] . sulfation reactions can also be studied on a chip surface [118] . significantly, enzymes constituting an entire functional pathway can be identified by enzyme arrays. this has been achieved by immobilizing mixtures of enzymes in different ratios on an array surface. activities and efficiencies of different enzymes were analyzed by product detection. two-step sequential reactions, catalyzed by either luciferase or nucleoside diphosphate kinase, and a five-step pathway for trehalose synthesis, have been demonstrated [119] . this approach provides a means of optimizing enzyme proportions for maximal pathway-output efficiency. an interesting application using metabolizing enzyme arrays for toxicologic screening of drugs has been described with arrayed p450 isozymes [120] . after exposing the array to a candidate drug, a cell monolayer was grown on the p450 drug array and analyzed for the proportion of living and dead cells. capture microarrays provide suitable systems for mapping protein epitopes recognized by antibodies or other affinity reagents. by arraying all overlapping peptides of defined length taken from a protein sequence, linear epitopes with the minimal sequence requirement for molecular recognition can be identified. individual amino acids within an epitope can be assessed for their contribution through the arraying of protein mutants. a libraryscanning strategy was described in which each individual position in a 25-mer peptide was randomized by the other 19 amino acids to generate 25 peptide libraries [121] . all libraries were then spotted and probed by the antibody. the detected signals indicated the role of particular residues in epitope recognition. identification of protective epitopes is essential for the design of effective vaccines. as there is no in silico method that can accurately predict the target proteins or epitopes that stimulate protective antibody immunity, screening with protein microarrays is useful for identifying immunodominant antigens from complex pathogens. in one such example, the immunogenic epitopes of the sars coronavirus were identified after screening 52 humansera samples from infected individuals on capture arrays of viral proteins [122, 123] . although a variety of array formats are increasingly used in basic research, biotechnology and medical applications, protein microarrays have not yet reached their full potential, particularly in academic research, for a number of possible reasons. current commercial array production methods are generally based on separate expression and purification of proteins; thus, they are prohibitively expensive, with costs of approximately us$1000 per slide. typically, academic applications require customized arrays displaying proteins of interest rather than complete proteome arrays. moreover, protein arrays for species other than humans and the most widespread model organisms are not commercially available. another problem is their limited shelf life, especially for functional protein arrays. we expect that the in situ methods for protein-array generation will contribute to changing this situation, especially approaches such as pisa, nappa and dapa, which offer cheaper and more flexible methods for academic laboratories. while small numbers of customized array slides can be economically produced through in situ synthesis when required, conventional methods that rely on spotting purified proteins are only cost effective if large batches of arrays are produced. the dapa technique is particularly useful for laboratories without microarraying equipment as it can generate multiple copies of the same protein microarray using a single dna array template. in current clinical applications, antigen microarrays and reverse-phase arrays are widely applied for profiling antibody repertoires from individual patient samples. protein microarrays: high-throughput tools for proteomics protein microarray technology will continue to be optimized in the coming years. the growing collections of cloned genes or gene sequences will facilitate the generation of 'proteome arrays' for many species. ongoing improvements in high-throughput protein expression and purification methods will enhance protein quality and quantity, increasing the functionality of arrayed proteins, with cell-free systems becoming a widely used alternative. technologies for making protein arrays by in situ synthesis will improve and accelerate array generation. further optimization of surface chemistries and structures, immobilization methods and readout technologies will allow for the generation of arrays with densities in the submicrometer scale. in proteomic applications, important areas of use will include high-throughput functional in vitro screens of molecular interaction networks, while for medical applications, arrays will be developed for diagnostics and the identification of relevant biomarkers. the increasing availability of affinity reagents through international initiatives such as proteomebinders will provide exciting possibilities in profiling cancer proteomes, to monitor characteristic protein signatures in patient sera or tumor extracts, leading to earlier diagnosis and prognosis before and during therapy [201] . it is expected that the multidisciplinary collaboration of scientists from biotechnology, biochemistry, material sciences, physics and bioinformatics will ensure the continued development of robust and reliable protein microarrays, their widespread uptake and novel applications. the babraham institute is an institute of the biotechnology and biological sciences research council (bbsrc), uk. this review is an activity of the proteomebinders consortium, a research infrastructure coordination action supported by the eu 6th framework programme. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no writing assistance was utilized in the production of this manuscript. • high-throughput protein production is desirable for making protein arrays. • arraying proteins in a defined orientation is a preferred approach, as it retains the protein activity on the array surfaces. • while current array readouts are mostly based on fluorescence, label-free detection methods have particular areas of use. • an emerging alternative to conventional protein arraying is the in situ synthesis and arraying of proteins by cell-free systems, as in the protein in situ array, nucleic acid programmable protein array and dna array to protein array technologies. • functional protein microarrays are increasingly used for high-throughput screening of molecular interactions, monitoring post-translational modifications and analyzing protein functions. • antibody arrays are valuable in profiling protein expression, thereby enabling the identification of biomarkers. antigen arrays are efficient tools for mapping protein epitopes and assist in the identification of vaccine candidates. • reverse-phase protein arrays are particularly useful for the screening of changes in the expression level of biomarkers and pathway targets involved in disease. • enzyme arrays provide a means for the large-scale ana lysis of enzymatic activities. pathway output efficiency can be enhanced by controlling the enzyme proportions spotted onto the array surface. papers of special note have been highlighted as: •• reports the use of a cell-free system to express the human proteome. in total, 33,275 clones have been constructed, of which 13,364 were tested by a cell-free, wheatgerm expression system. gene expression response to misfolded protein as a screen for soluble recombinant protein protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein cell-free protein synthesis: applications in proteomics and biotechnology proteomescale purification of human proteins from bacteria improved affinity coupling for antibody microarrays: engineering of double-(his)-6-tagged single framework recombinant antibody fragments protein and peptide arrays: recent trends and new directions a protein-domain microarray identifies novel protein-protein interactions multifunctional epoxy supports: a new tool to improve the covalent immobilization of proteins global ana lysis of protein activities using proteome chips -breaking paper that reports the first use of a yeast proteomic array for large-scale screening and identification of calmodulin and phospholipidbinding proteins versatile protein microarray based on carbohydrate-binding modules direct site-selective covalent protein immobilization catalyzed by a phosphopantetheinyl transferase • recent report describing the covalent immobilization of tagged proteins on solid surfaces from cell lysates semi-wet peptide/ protein array using supramolecular hydrogel systematic comparison of surface coatings for protein microarrays comparison of hydroxylated print additives on antibody microarray performance antibody microarrays: an evaluation of production parameters investigation of non-nucleophilic additives for the reduction of morphological anomalies in protein arrays non-contact protein microarray fabrication using a procedure based on liquid bridge formation advances in functional protein microarray technology in situ synthesis of protein arrays review protein microarrays: high-throughput tools for proteomics emerging tools for real-time label-free detection of interactions on functional protein microarrays antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers cell-free n-terminal protein labeling using initiator suppressor trna fluorescence labeling of the c-terminus of proteins with a puromycin analogue in cell-free translation systems rapid functional ana lysis of proteinprotein interactions by fluorescent c-terminal labeling and single-molecule imaging a network ana lysis of changes in molecular interactions in cellular signaling a new two-color fab labeling method for autoantigen protein microarrays proteomic profiling of the nci-60 cancer cell lines using new high-density reversephase lysate microarrays signal amplification by rolling circle amplification on dna microarrays multiplexed protein profiling on microarrays by rolling-circle amplification allergen-specific ige detection on microarrays using rolling circle amplification: correlation with in vitro assays for serum ige ligation-based molecular tools for lab-on-a-chip devices proximity ligation assays for sensitive and specific protein analyses high throughput quantification of protein expression of cancer antigens in tissue microarray using quantum dot nanocrystals protein microarrays with carbon nanotubes as multicolor raman labels analysis of protein interaction and function with a 3-dimensional maldi-ms protein array high-speed biomarker identification utilizing porous silicon nanovial arrays and maldi-tof mass spectrometry label-free reading of microarray-based immunoassays with surface plasmon resonance imaging present and future of surface plasmon resonance biosensors grating-coupled surface plasmon resonance: a cell and protein microarray platform label-free and dynamic detection of biomolecular interactions for highthroughput microarray applications high-throughput nanohole array based system to monitor multiple binding events in real time nanowire nanosensors for highly sensitive and selective detection of biological and chemical species protein recognition via surface molecularly imprinted polymer nanowires comprehensive identification of human bzip interactions with coiled-coil arrays differential binding of calmodulin-related proteins to their targets revealed through high-density arabidopsis protein microarrays a proteomic microarray approach for exploring ligand-initiated nuclear hormone receptor pharmacology, receptor selectivity, and heterodimer functionality a network-based ana lysis of polyanionbinding proteins utilizing human protein arrays high-throughput and multiplexed protein array technology: protein-dna and protein-protein interactions regulation of gene expression by a metabolic enzyme identifying protein interactions with metal-modified dna using microarray technology linking dna-binding proteins to their recognition sequences by using protein microarrays dissecting dna-protein and proteinprotein interactions involved in bacterial transcriptional regulation by a sensitive protein array method combining a near-infrared fluorescence detection functional protein microarrays for parallel characterisation of p53 mutants printing proteins as microarrays for highthroughput function determination microarrays to characterize protein interactions on a whole-proteome scale membrane protein microarrays finding new components of the target of rapamycin (tor) signaling network through chemical genetics and proteome chips identification of small molecule targets on functional protein microarrays antibody microarrays as an experimental platform for the ana lysis of signal transduction networks advances in recombinant antibody microarrays antibody arrays for determination of relative protein abundances profiling of cancer cells using protein microarrays: discovery of novel radiationregulated proteins antibody protein array ana lysis of the tear film cytokines detection of pancreatic cancer using antibody microarray-based serum protein profiling comparative application of antibody and gene array for expression profiling in human squamous cell lung carcinoma antibody suspension bead arrays within serum proteomics antibodypedia, a portal for sharing antibody and antigen validation data profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery proteome-wide ana lysis of the serological response to vaccinia and smallpox immunodominant francisella tularensis antigens identified using proteome microarray vaccinia virus h3l envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice protein microarray for profiling antibody responses to yersinia pestis live vaccine profiling humoral immune responses to p. falciparum infection with protein microarrays antigen microarrays identify unique serum autoantibody signatures in clinical and pathologic subtypes of multiple sclerosis protein microarrays guide tolerizing dna vaccine treatment of autoimmune encephalomyelitis autoantibody profiling on high-density protein microarrays for biomarker discovery in the cerebrospinal fluid immunotheranostics: breaking tolerance in immunotherapy using tumor autoantigens identified on protein microarrays seromic ana lysis of antibody responses in non-small cell lung cancer patients and healthy donors using conformational protein arrays reverse phase protein array: validation of a novel proteomic technology and utility for ana lysis of primary leukemia specimens and hematopoietic stem cells analyzing antibody specificity with whole proteome microarrays • first report using a proteome array comprising 5000 different yeast proteins to screen the specificity of antibodies rapid characterization of binding specificity and cross-reactivity of antibodies using recombinant human protein arrays profiling receptor tyrosine kinase activation by using ab microarrays antibodies immobilized as arrays to profile protein post-translational modifications in mammalian cells panorama ab microarray cell signaling kit: a unique tool for protein expression ana lysis proteomic ana lysis of breast cancer molecular subtypes and biomarkers of response to targeted kinase inhibitors using reverse-phase protein microarrays proteome chips for whole-organism assays a ratiometric lectin microarray approach to ana lysis of the dynamic mammalian glycome glycoprotein microarrays with multi-lectin detection: unique lectin binding patterns as a tool for classifying normal, chronic pancreatitis and pancreatic cancer sera high throughput identification of potential arabidopsis mitogen-activated protein kinases substrates global ana lysis of protein phosphorylation in yeast review protein microarrays: high-throughput tools for proteomics •• reports a large-scale ana lysis of protein phosphorylation by the use of proteome chips. over 4000 phosphorylation events protein microarrays for multiplex ana lysis of signal transduction pathways reports the first description of the use of reverse-phase protein microarrays to monitor site-specific phosphorylation of many signaling proteins from stimulated living cells quantitative protein microarrays for time-resolved measurements of protein phosphorylation developing a strategy for activity-based detection of enzymes in a protein microarray enzyme familyspecific and activity-based screening of chemical libraries using enzyme microarrays • describes an interesting thematic enzymemicroarray format to characterize family-specific enzymes on array surfaces enzymatic activity on a chip: the critical role of protein orientation a functional protein chip for pathway optimization and in vitro metabolic engineering reports the use of protein arrays to reconstitute an enzyme pathway, demonstrating that entire pathways can be studied by protein microarrays metabolizing enzyme toxicology assay chip (metachip) for high-throughput microscale toxicity analyses protein microarrays for antibody profiling: specificity and affinity determination on a chip screening of specific antigens for sars clinical diagnosis using a protein microarray antigenicity ana lysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein • review article for in situ synthesis of protein microarrays. 37 key: cord-333309-21czobqy authors: byun, hyewon; gou, yongqiang; zook, adam; lozano, mary m.; dudley, jaquelin p. title: erad and how viruses exploit it date: 2014-07-03 journal: front microbiol doi: 10.3389/fmicb.2014.00330 sha: doc_id: 333309 cord_uid: 21czobqy endoplasmic reticulum (er)-associated degradation (erad) is a universally important process among eukaryotic cells. erad is necessary to preserve cell integrity since the accumulation of defective proteins results in diseases associated with neurological dysfunction, cancer, and infections. this process involves recognition of misfolded or misassembled proteins that have been translated in association with er membranes. recognition of erad substrates leads to their extraction through the er membrane (retrotranslocation or dislocation), ubiquitination, and destruction by cytosolic proteasomes. this review focuses on erad and its components as well as how viruses use this process to promote their replication and to avoid the immune response. although endoplasmic reticulum (er)-associated degradation (erad) has been most thoroughly defined in yeast, recent studies in higher organisms have revealed the conservation of this process and its components. multiple diseases, including parkinson's, alzheimer's, cancer, and infectious processes, result from failure of erad, confirming its significance for correct cell function. predictably, viruses have exploited various aspects of this key cellular machinery to further their propagation. nonetheless, the complexity of erad and the number of players involved necessitates a review of its features prior to a description of how viruses have manipulated erad to their advantage. in understanding how viruses exploit erad, we learn more about the cellular process, but also how we might alter the outcome of viral diseases. a majority of newly synthesized proteins in mammalian cells are either misfolded or misassembled (hoseki et al., 2010) . approximately 30% of new proteins are synthesized in association with the er (brodsky and wojcikiewicz, 2009) . the er quality control system both senses and disposes of terminally misfolded proteins by erad, a process that is conserved in eukaryotes (vembar and brodsky, 2008; merulla et al., 2013) . this process detects misfolded proteins in the er lumen, and then extracts them through membrane channels in an energy-dependent manner for delivery to cytosolic proteasomes (olzmann et al., 2013) . protein extraction through er membrane channels is known as dislocation or retrotranslocation (hampton and sommer, 2012) . because protein folding depends on multiple cellular components (merulla et al., 2013) , protein overexpression or the presence of mutant proteins may sequester limiting components, leading to accumulation of misfolded proteins in the er lumen. a more general failure of the erad process may occur if proteins are unable to fold within a reasonable time, resulting in inefficient retrotranslocation and proteasomal degradation. levels of erad-associated factors also may be affected by the intraluminal concentration of misfolded proteins. inability of the erad system to destroy misfolded proteins is associated with more than 60 diseases, including neurological illnesses (alzheimer's and parkinson's), cystic fibrosis, infectious diseases, diabetes, and cancer (guerriero and brodsky, 2012) . particularly relevant to the subject of this review, viruses can produce large quantities of glycoproteins in a short period of time, which may overwhelm erad, leading to the accumulation of misfolded proteins, cell death, and associated pathology (franz et al., 2014) . although erad is vital to the maintenance of healthy cells, many parts of this process are not well characterized. multiple aspects of erad have been described in yeast (thibault and ng, 2012) , including the nature of the er channel and the components needed to identify misfolded proteins during and after translation. protein translocation across the er membrane is the prerequisite for erad. translation of many transmembrane proteins involves recognition of a hydrophobic signal peptide (sp) emerging from the ribosome by signal recognition particle (srp), which is associated with the trimeric sec61 complex. many of the sps are cleaved by signal peptidase, which is associated with the luminal side of the translocon (auclair et al., 2012) . the sec61 complex provides the aqueous channel for co-translational transfer of proteins across the er membrane (loibl et al., 2014) . recent evidence indicates that translocation across the er membrane can occur through an srp-independent process (denic, 2012; johnson et al., 2013) . based on recent experiments in yeast, more than 40% of signal-containing proteins fail to use srp, including tail-anchored (ta) proteins and short secretory proteins (johnson et al., 2012; ast et al., 2013) . instead, these proteins are targeted by the get pathway to the sec61 translocon that is associated with the sec 62/63 complex rather than through docking to the srp receptor (rapoport, 2007; ast et al., 2013) . one large class of srp-independent proteins includes the glycosylphosphatidylinositol (gpi)-anchored proteins, which contain both an n-terminal signal sequence and a c-terminal gpi anchor (ast et al., 2013) . this n-terminal signal is less hydrophobic than typical srp targets. furthermore, the sec61 translocon has been implicated as the channel for retrotranslocation (kiser et al., 2001) , and it has been proposed that protein transfer can be either forward or reverse with respect to the er lumen (johnson and haigh, 2000) . therefore, sec61 appears to complex with a number of different proteins, leading to a highly flexible and dynamic structure, where association with different proteins/protein complexes leads to transit in or out of the er (figure 1 ). reports in yeast indicate that proteins can be o-mannosylated prior to n-glycosylation (ecker et al., 2003) , and both types of glycosylation are believed to occur co-translationally (loibl et al., 2014) . these glycosylases also have been shown to be associated with the translocon (chavan and lennarz, 2006) , and experiments indicate competition for different glycosylation sites (loibl et al., 2014) . the protein o-mannosyl transferases (pmts) and the oligosaccharyltransferases (osts) are transmembrane proteins, but the latter catalyzes addition of oligosaccharides to nascent polypeptides on asparagine residues (breitling and aebi, 2013) . the osts prefer nxt/s sequences in an unfolded or flexible protein domain, and the unfolded state may be facilitated by the ost complex associated with the translocon (breitling and aebi, 2013) . glycosylation near the c-terminal end of the protein is less efficient, perhaps due to competition between osts and protein folding (ben-dor et al., 2004; breitling and aebi, 2013) . pmts also are essential for erad in yeast. a pmt mutant showed increased degradation of a typical erad substrate (arroyo et al., 2011) . moreover, addition of oligosaccharides can be prevented by nearby cysteines and disulfide bond formation (allen et al., 1995) . thus, glycosylation is one determinant of the correct folding of a protein in the er lumen (breitling and aebi, 2013 ; figure 1a ). the oligosaccharides on er luminal proteins are critical for their correct folding or selection for erad. the nascent n-glycosylated protein has a three-branch structure with glucose 3mannose 9 -n-acetylglucosamine 2 -asparagine (aebi et al., 2010; merulla et al., 2013) . trimming of the first two glucose residues on one branch then allows interactions with two er-resident chaperone/lectin proteins, calnexin and calreticulin, which may lead to protein folding (brodsky, 2012) . removal of the third glucose causes release from these lectins and exit from the er (smith et al., 2011; olzmann et al., 2013) , but re-addition of this glucose by udp-glucose:glycoprotein glucosyltransferase allows reassociation (shenkman et al., 2013) . proteins retry folding until removal of three or four mannose residues triggers erad (lederkremer and glickman, 2005; shenkman et al., 2013) . correctly folded proteins leave the er after one or two mannose residues have been cleaved (shenkman et al., 2013) . mannose removal is achieved using er mannosidase i (ermani), the er degradation-enhancing α-mannosidase-like proteins (edems) and/or the golgi-resident protein man1c1 (gonzalez et al., 1999; hirao et al., 2006; olivari et al., 2006; hosokawa et al., 2007) . several lectins, os-9 and xtp3-b, then interact via their mrh domains with the mannose-trimmed proteins, allowing their association with the retrotranslocon (bernasconi et al., 2008; christianson et al., 2008; hosokawa et al., 2008) . os-9 and xtp3-b also associate with different proteases, lonp2 and carboxypeptidase vitellogenic-like protein (cpvl), respectively, suggesting that some substrates may be partially degraded prior to dislocation (christianson et al., 2012; olzmann et al., 2013) . nonetheless, multiple attempts are made to refold proteins before their triage through erad. the role of chaperones includes recognition of inappropriate glycosylation as well as refolding efforts, but proteins delivered to the retrotranslocon may require unfolding and partial proteolysis to allow their transit through the narrow membrane channel (gogala et al., 2014) . non-glycosylated proteins can be subjected to erad, but detection of misfolding of these proteins does not involve calnexin and calreticulin (brodsky, 2012) . notably, the non-lectin chaperone bip is involved in erad targeting of both types of proteins (ushioda et al., 2013) , yet also serves to prevent leakage of calcium out of the er lumen (schäuble et al., 2012) . in addition, targeting of unglycosylated proteins to the proteasomes involves edem1 (shenkman et al., 2013) , which, like bip, recognizes misfolded glycoproteins, as well as the transmembrane herp protein (usa1p in yeast; okuda-shimizu and hendershot, 2007) . both glycosylated and their non-glycosylated derivatives are recruited to the erderived quality control compartment (erqc) near the nucleus in the presence of a proteasomal inhibitor (shenkman et al., 2013) . thus, these studies suggest that targeting of misfolded proteins for erad is similar for glycoproteins and non-glycosylated proteins (shenkman et al., 2013) . interaction of lectin-type and other chaperones with erad substrates allows association with members of the protein disulfide isomerase (pdi) family, which generally are characterized by one or more thioredoxin-like motifs (cxxc; brodsky and skach, 2011) . interestingly, these proteins can form, break, or rearrange disulfide bonds as well as act as chaperones (benham, 2012) . the yeast pdi family is composed of five members (pdi1, mpd1, mpd2, eug1, and eps1), although only pdi1 is essential (farquhar et al., 1991) . in mammalian cells, pdi is one of the best characterized family members, but there are at least 21 such enzymes (benham, 2012; grubb et al., 2012) . pdi family proteins are generally confined by a kdel retention sequence (benham, 2012) to the er, which has an oxidizing environment (costantini et al., 2013) . the oxidoreductase erp57, which is localized near the er-golgi intermediate compartment (ergic), may provide some protection for proteins that might be routed for erad by calnexin (frenkel et al., 2004) . in addition, some pdi members can escape the secretory system and appear at the cell surface (benham, 2012) . for example, a disintegrin and metalloproteinase (adam17; also known as tumor necrosis factor alpha-converting enzyme or tace) has been shown to be regulated by an extracellular activity of pdi (bass and edwards, 2010; willems et al., 2010; düsterhöft et al., 2013) . pdi members also have a role in erad, with different requirements for different substrates (grubb et al., 2012) . in hepatic cells, pdi promotes the folding of apolipoprotein b (apob) figure 1 | the erad process. (a) substrate recognition. many nascent polypeptides (curved line) have one or more high-mannose carbohydrates (shown as a branched structure), which must be recognized and processed in a timely manner to allow exit from the er. binding of these er-luminal proteins to substrates is affected by folding to their native conformations. folding involves formation and breakage of disulfide bonds by members of the pdi family, such as erp57 and erp72, and is facilitated by chaperone proteins, such as bip. specific carbohydrates are bound by different chaperones/lectins in the er lumen. these proteins include ermani, edem, os-9, xtp3-b, calreticulin, and calnexin. recognition of erad substrates probably results in assembly of the retrotranslocon (shown here as herp and the translocon/bip complex). herp is thought to facilitate oligomerization of the hrd1 e3 ligase. bip binds to a number of glycosylated and non-glycosylated erad substrates and provides a barrier on the er luminal side of the translocon. (b) retrotranslocation. recognition of misfolded or misassembled proteins triggers the assembly of the retrotranslocon. current evidence indicates that multiple types of retrotranslocons are possible (see text). a typical retrotranslocon/dislocon is shown containing derlin, the e3 ligase hrd1 and its partner sel1l, which then recruits the cytosolic atpase p97. derlin has 6 transmembrane domains with both the n-terminus and c-terminus in the cytosol. presumably some or all of the recognition components, such as pdi and ermani, disengage as the substrate passes through the translocon. all retrotranslocation events appear to involve p97. the retrotranslocon is shown with bip opening the sec61 channel for substrate passage into the cytosol. (c) ubiquitination of erad substrates. retrotranslocation exposes erad substrates to cytosolic e1 (unknown), e2 (shown here as ube2g1), and e3 enzymes (e.g., hrd1). a polyubiquitin chain is produced as the substrate is engaged by the e2 and e3 proteins. multiple e3s may be responsible for the polyubiquitin chains that then bind to the p97 partner proteins, ufd1 and npl4. the substrate is shown moving through the translocon into the center of the p97 hexamer. (d) proteasomal degradation. once the substrate has been retrotranslocated, the bip protein seals the luminal side of the translocon. the retrotranslocon may then be disassembled prior to engagement of a new substrate. the retrotranslocated proteins must be modified by removal of carbohydrate and ubiquitin chains for insertion into the narrow channel of the proteasome. it is possible that p97 substitutes for the 19s lid, which provides access to the proteasome channel and the energy for unfolding of substrates. degraded polypeptides are shown emerging from the 19s lid. this model suggests that there are retrotranslocon-specific proteasomes. www.frontiersin.org through its chaperone activity, whereas erp57 or erp72 expression leads to erad (grubb et al., 2012) . further, various cell types express different pdi proteins, allowing differential regulation of substrates (benham, 2012; pescatore et al., 2012) and, presumably, their erad targeting. protein folding involves both formation of disulfide bonds and cis/trans isomerization of peptide bonds preceding proline residues (hebert and molinari, 2007) . certain erad substrates appear to be dependent on proline isomerization (bernasconi et al., 2010b) , and such refolding events may be necessary for transit through the retranslocon by elimination of turns in substrate secondary structure (määttänen et al., 2010) . erad requirements for peptidyl-prolyl cis/trans isomerases (ppis) depend on whether the substrate is strictly in the er lumen or is tethered to the er membrane (bernasconi et al., 2010b) . the ppi protein cyclophilin b was needed for erad of a luminal target, but not the same target with a transmembrane domain (bernasconi et al., 2010b) . requirement for ppis during erad may depend on proline residues in the cis configuration (bernasconi et al., 2010b) , potentially by conversion into trans peptidyl-prolyl bonds, thus eliminating secondary structures that hinder retrotranslocation (määttänen et al., 2010) . mammalian cells have erad factors that are not present in yeast. as observed for other pathways (tsai and weissman, 2012) , erad components identified in yeast have multiple family members in higher eukaryotes; e.g., instead of a single derlin in yeast (der1p), mammalian cells have three proteins (derlin-1, -2, and -3; oda et al., 2006) . derlins are multiple membranespanning domain proteins that have been proposed to be part of the retrotranslocon channel and/or regulatory factors for retrotranslocation (brodsky, 2012; figure 1b ). in addition, derlin-3 has a cell-type specific distribution (oda et al., 2006) , suggesting that recognition of certain substrates may be involved in its function. derlins are related to rhomboid proteases, such as rhbdl4, which is an er-resident transmembrane protein that cleaves unstable single-membrane-spanning or polytopic membrane proteins (fleig et al., 2012) . rhbdl4 also is upregulated by er stress and binds to the cytosolic aaa atpase p97 (see below; fleig et al., 2012) . in contrast to the rhomboid proteases, the derlins lack proteolytic activity, suggesting that these proteins bind to erad substrates and target them to e3 ligases for ubiquitination and to p97 for membrane extraction (brodsky, 2012) . cleavage of erad substrates by rhbdl4 (fleig et al., 2012) , sp peptidase (spp; loureiro et al., 2006) , or proteases associated with os-9 and xtp3-b (olzmann et al., 2013) may occur prior to retrotranslocation of some substrates (tsai and weissman, 2012) . on the other hand, it has been proposed that derlins form a six-transmembrane structure with a gate that allows association and unfolding of substrates or access to other retrotranslocon components, such as p97 (see below; olzmann et al., 2013) . the p97 atpase (cdc48 in yeast) is bound to derlin-1 and derlin-2 through their shp domains (greenblatt et al., 2011) . suppressor/enhancer of lin12-like (sel1l) appears to link luminal factors that recognize misfolding and inappropriate glycosylation, such as os-9, xtp3-b, edems, erdj5, and the pdi protein erp90, to components of the retrotranslocon (olzmann et al., 2013; williams et al., 2013) . the transmembrane sel1l protein (hrd3p in yeast) also participates in regulation of erad by sequestering edem1 and os-9 into er-derived vesicles known as edemosomes (bernasconi et al., 2012a) . inducible knockout of sel1l in mice leads to death of adult mice from acute pancreatic atrophy (sun et al., 2014) . sel1l expression is required for stability of the e3 ligase hydroxymethylglutaryl reductase degradation protein 1 (hrd1), and its loss leads to er stress and attenuates translation, leading to cell death. other proteins have been described, such as erlins 1 and 2 and tmub1, which may act as adapters between polytopic membrane substrates and e3 ligases (olzmann et al., 2013) . the ubiquitin ligases (e3s) have been proposed to be a structural part of the retrotranslocon channel (brodsky, 2012) , but their role is considerably more complex ( figure 1c ). several e3 ligases associated with erad are multiple membranespanning proteins with cytosolic ring domains (smith et al., 2011; ruggiano et al., 2014) . in yeast, where erad has been studied most extensively, a prototypical transmembrane e3, such as hrd1p (also called syvn1; nadav et al., 2003; kikkert et al., 2004) , can promote erad of a luminal substrate (erad-l). the erad process also involves hrd3p (sel1l in metazoans) as well as usa1p and der1p (carvalho et al., 2010) . herp may assist with hrd1 oligomerization (carvalho et al., 2010) , nevertheless, the other components appear to be dispensable if hrd1p is overexpressed, consistent with a role for hrd1p in erad substrate transfer across the membrane (carvalho et al., 2010) , although such overexpression may be toxic due to inappropriate protein degradation (denic et al., 2006) . thus, protein adapters appear to be necessary to achieve substrate specificity (smith et al., 2011) . hrd1p-mediated erad requires oligomerization and transmembrane domains as well as ubiquitin ligase activity (carvalho et al., 2010) . overexpression of a dominant-negative ring mutant of the hrd1 ligase prevented erad of a non-glycosylated substrate, but a dominant-negative fbs2 mutant (a component of scf e3 ligases) did not (shenkman et al., 2013) . dependence on hrd1 also is affected by tethering of the substrate to the er membrane. splice variants of the human beta-site amyloid precursor cleaving enzyme (bace) with the same deletion mutation in the ectodomain are degraded through hrd1 if they are luminal (erad-l s substrates), but disposal occurs in a hrd1independent manner if the variant has a transmembrane domain (erad-l m substrates; bernasconi et al., 2010a) . therefore, hrd1 recognizes substrates for ubiquitination and, perhaps, modifies the translocon in the er membrane. multiple e3 ligases participate in erad. these ligases include the transmembrane proteins gp78/amfr (fairbank et al., 2009 ), trc8 (stagg et al., 2009) , rma1/rnf5 (el khouri et al., 2013) , march6/teb4 (doa10 in yeast; kreft and hochstrasser, 2011; olzmann et al., 2013) , and chip (matsumura et al., 2013 ). an additional 40−50 membrane-spanning e3s may be involved in erad (stagg et al., 2009) . other e3 ligases associated with erad frontiers in microbiology | virology are localized to the cytosol, where they recognize misfolded glycoproteins that already have been retrotranslocated (yoshida et al., 2005; shenkman et al., 2013) . these ubiquitin ligases are members of the cytosolic scf (s-phase kinase-associated protein 1 (skp1)-cullin 1 (cul1)-f-box) family, where the f-box components of the scf complex recognize the n-glycans of the retrotranslocated substrate, e.g., fbs1 and fbs2 (yoshida, 2007) . furthermore, e3s may work together to direct substrates for degradation (olzmann et al., 2013) . the p97 protein (cdc48 in yeast) is a member of the aaa atpase family (erzberger and berger, 2006 ) that functions during erad in a complex with several cofactors that have a ubiquitin-x (ubx) or ubx-like domain (schuberth and buchberger, 2008 ; figure 1 ). these cofactors include the heterodimer nuclear protein localization homolog 4 (npl4)-ubiquitin fusion degradation 1 (ufd1; meyer et al., 2012; wolf and stolz, 2012) , p47, ubxd1, ubxd7, ufd3/plaa, vcip135, and ataxin-3 (meyer et al., 2012) . the ufd1l and npl4 proteins are believed to form a heterodimer, where npl4 is needed to stabilize ufd1l (nowis et al., 2006) . the heterodimer acts as a substrate adapter to the p97 atpase associated with the retrotranslocon (bays and hampton, 2002) . ufd1l and npl4 bind to k48-linked and k63-linked polyubiquitin chains, respectively, which have been added by e3 ligases associated with the retrotranslocon (ye et al., 2003; komander et al., 2009) . in yeast, the cdc48 atpase binds to the hrd1 e3 ligase in a ring-dependent manner (hampton and sommer, 2012) , and the transmembrane ubx2 (sel1) protein acts as an adapter using a uba domain (neuber et al., 2005; schuberth and buchberger, 2005) . several other ubiquitin ligases bind p97 directly or through cofactors (alexandru et al., 2008) . the p97 cofactors act as ubiquitin-binding proteins, although p97 also has ubiquitin-binding activity (ye et al., 2003; meyer et al., 2012) . the adapter-p97 complexes may recognize different substrates and perform independent functions, such as membrane protein segregation and trafficking, as well as directing substrates to the proteasome (ritz et al., 2011) . alternatively, other models suggest that derlins are involved in unfolding of substrates as well as providing contacts with p97 and its associated factors (greenblatt et al., 2011) . the p97 atpase binds ubiquitin chain editors that can extend shorter chains as well as deubiquitinating enzymes (dubs; jentsch and rumpf, 2007; sowa et al., 2009) . two atpase domains (d1 and d2; meyer et al., 2012) within p97 form two stacked hexameric rings that provide the energy for protein remodeling and substrate extraction from the membrane or through the retrotranslocon (hampton and sommer, 2012) . mutations in the d2 domain result in dominant-negative proteins that bind, but fail to release, substrates (pye et al., 2006) . mutant proteins have been widely used to study p97 function in erad and its myriad other activities (meyer et al., 2012) . cytosolic chaperones, such as hsp70, also may provide energy for extraction of membrane proteins with misfolded cytoplasmic domains (erad-c substrates; taxis et al., 2003; hrizo et al., 2007) . once extraction from the er membrane has occurred, p97 recruits peptide n-glycanase (pngase) to cleave n-linked glycans from glycosylated substrates (hirsch et al., 2003; li et al., 2006 ; figure 1d ). in addition, p97 binds to a deubiquitinating enzyme yod1, presumably so that polyubiquitin chains will not interfere with insertion into the proteasome (ernst et al., 2009) . the proteasome is a highly complex structure with a 19s lid that has an atpase activity very similar to that of p97 (lipson et al., 2008; matouschek and finley, 2012) . these enzymes may function synergistically to deliver substrates to the 20s core (hampton and sommer, 2012) . alternatively, p97 may deliver certain substrates directly to the proteasome core (matouschek and finley, 2012) . the proteasome core is composed of 28 subunits arranged into four rings, each composed of seven subunits (bhattacharyya et al., 2014) . proteolytic activity is sequestered in the center of a narrow chamber formed by the rings and, therefore, only unfolded proteins can enter the chamber (groll et al., 2000) . the 19s lid, p97, or other activators provide docking for substrates and substrate modifying proteins as well as regulated opening of the chamber to allow access of unfolded proteins for degradation in the 26s core (bhattacharyya et al., 2014) . many questions remain about erad components and how they identify and interact with different substrates. similar to our analysis of other cellular and molecular biological processes through virology, studies of viruses that use erad are likely to prove insightful. the ability of viruses to cause persistent infections is a consequence of downregulation or subversion of the immune response. the herpesviruses are known to cause persistent infections. one well-studied example of herpesvirus manipulation of the immune response is reduced cell expression of major histocompatibility complex class 1 (mhc-i) molecules by the viral proteins us2 and us11 (wiertz et al., 1996) . both proteins are transmembrane glycoproteins and bind to newly made mhc-i to initiate retrotranslocation. despite their similar function, us2 and us11 use different pathways for mhc-i degradation (figure 2) . us2mediated degradation of mhc-i is independent of derlin-1 and involves spp (loureiro et al., 2006) , which cleaves many sps following their removal from nascent er-bound pre-proteins (voss et al., 2013) . using an sirna screen, trc8 was identified as the e3 ligase involved in mhc-i degradation by us2, but knockdown of this transmembrane ring-type e3 had no effect on us11mediated destruction of mhc-i (stagg et al., 2009 ). the us2 cytosolic tail interacts with spp and the p97 atpase (chevalier and johnson, 2003; loureiro et al., 2006) , whereas trc8 and us2 bind through their transmembrane domains (stagg et al., 2009 ; figure 2a) . unlike the derlin-independent mechanism proposed for us2, studies of the us11 protein facilitated identification of derlin-1 and sel1l as erad components (figure 1 ; lilley and ploegh, 2004; ye et al., 2004; mueller et al., 2006) . us11 does not require spp for mhc-i degradation (loureiro et al., 2006 ), but appears to interact with the e3 ligase marchvii/axotrophin (flierman et al., 2006) . the cytosolic domain of mhc-i is required for us11mediated erad targeting (story et al., 1999; barel et al., 2003) , and deletion of the c-terminal valine of mhc-i reduced interaction with derlin-1 (cho et al., 2013a) . the er luminal domain www.frontiersin.org also affects degradation (barel et al., 2003) . in addition, mhc-i substituted with the transmembrane domain of us11 caused interaction with derlin-1 and proteasomal degradation (cho et al., 2013b) . the p97 atpase does not appear to interact directly with mhc-i, but requires the interaction of mhc-i cytosolic domain with the c-terminal domain of derlin-1 (cho et al., 2013a) . cho et al. speculated that us11 recognizes mhc-i through its cytosolic domain and transfers it to derlin-1, which then interacts with the p97 atpase for membrane dislocation (cho et al., 2013a ; figure 2b ). therefore, studies of the herpesvirus us2 and us11 proteins revealed that the same substrate does not always use the same erad pathway, and presumably these viral proteins act as adapters that recognize different parts of mhc-i for targeting to the dislocon. herpesviruses use another mechanism to decrease levels of mhc-i. the mouse gammaherpesvirus 68 (mhv68) encodes an e3 ligase (mk3) that ubiquitinates newly made mhc-i heavy chains for proteasomal degradation (boname and stevenson, 2001) . the mk3 ligase also is associated with the transporterassociated with antigen processing (tap) as well as p97 and derlin-1 (wang et al., 2006) . polyubiquitination of mhc-i did not require lysines , but could occur on serine and threonine residues in the heavy chain c-terminal tail via the recruitment of the ube2j2 e2 enzyme (see figure 1 ; wang et al., 2007 wang et al., , 2009 herr et al., 2009) . these data indicate that multiple erad mechanisms can be used by viruses to diminish the adaptive immune response. like the herpesviruses, retroviruses also manipulate the immune system through erad. early studies indicated that human immunodeficiency virus type 1 (hiv-1)-infected cells had decreased levels of both cd4 mrna and protein (hoxie et al., 1986) . cd4 acts as the receptor for binding the viral envelope (env) protein (mcclure et al., 1987) . furthermore, cd4 participates in t-cell activation by binding to both the t-cell receptor and mhc class ii molecules on antigen-presenting cells. cd4+ t cells secrete cytokines that control antibody production, phagocytic cell function, and cytotoxic t-cell responses, making them crucial for adaptive immune responses (tubo and jenkins, 2014) . hiv-1 encodes a number of accessory proteins, including vpu, which are not required for virus replication in tissue culture, but contribute to viral pathogenesis (strebel, 2013) . expression of vpu and cd4 by transient transfection showed dramatic decreases in cd4 levels, and cd4 depletion was dependent on serines 52 and 56 in vpu (magadán et al., 2010) . vpu-induced cd4 degradation has been shown to involve the erad system. knockdown of both β-trcp1 and β-trcp2 largely prevented vpu-mediated cd4 loss (magadán et al., 2010) . β-trcp1 and β-trcp2 (also known as fbw1a, fbxw1, fbxw1a, or fwd1) are f-box proteins containing wd40 domains, which are associated with the scf family of ubiquitin ligases (figure 3) . these protein complexes are linked to regulation of multiple pathways involving cell cycle checkpoints, nfκb, and wnt (skaar et al., 2013) . in addition, knockdown of p97, ufd1l (also called ufd1) or npl4 (see figure 1c ) blocked depletion of cd4 (magadán et al., 2010) . mutations that prevented atp binding or hydrolysis by p97 failed to affect cd4 levels (magadán et al., 2010) . these experiments indicated that vpu uses erad to degrade cd4, but also prevents cell surface expression by retaining cd4 in the er, probably through transmembrane domain interactions (magadán et al., 2010) . moreover, vpu used an atypical e3 ligase to induce erad (margottin et al., 1998) , and this process involved scf β−trcp ubiquitination of the cd4 cytosolic tail on lysine, serine, and threonine residues (magadán et al., 2010) . thus, vpu may act as an adapter between cd4, retrotranslocon components, and a cytosolic e3 ligase. cd4 degradation promotes hiv-1 infection by preventing re-infection, facilitating virus release by avoiding env-cd4 interactions during their trafficking to the cell surface, and minimizing adaptive immune responses (lanzavecchia et al., 1988; willey et al., 1992; argañaraz et al., 2003) . hiv-1 vpu also targets another cellular protein, tetherin/bst-2, for erad (neil et al., 2008; mangeat et al., 2009) . tetherin is an unusual type ii membrane protein with an n-terminal frontiers in microbiology | virology knockdown of both β-trcp1 and β-trcp1 (shown to be contacting vpu) can prevent cd4 degradation, suggesting that either f-box protein can provide a functional scf complex for ubiquitination (magadán et al., 2010) . another e3 ligase (e3?) also may be involved. the p97 atpase with the adapters ufd1 and npl4 are required for cd4 degradation, but the ufd1l protein recognizes polyubiquitinated cd4. lysine and serine/threonine residues in the cd4 cytosolic tail are needed for ubiquitination (magadán et al., 2010) . transmembrane segment and a c-terminal gpi anchor (kupzig et al., 2003; sauter, 2014) . moreover, two tetherin monomers are bound together by disulfide bonds (ishikawa et al., 1995; kupzig et al., 2003) . using a unique method that only allows biotinylation of retrotranslocated molecules by cytosolic bira protein, recent experiments indicate that both cd4 and tetherin remain glycosylated and retain disulfide bonds during retrotranslocation (petris et al., 2014) . these data suggest that the typical sec61 channel used for translocation is insufficiently wide to accommodate retrotranslocation substrates modified with these structures (petris et al., 2014) , but an alternative model involving lipid droplet formation has not been confirmed . given the large number of proteins that have been implicated, a single mechanism for retrotranslocation is unlikely. despite common delivery of substrates to the proteasome via the p97 atpase, each of the previous examples of viral erad targeting involves different e3 ligases. recent evidence suggests that erad can target the retrovirus hiv-1 env (zhou et al., 2014) , a glycosylated transmembrane protein. studies of a human cd4+ t-cell line cem.nkr indicated that hiv-1 replication is restricted in these cells, which also are resistant to natural killer cell-mediated lysis (howell et al., 1985) . surprisingly, these cells overexpressed a mitochondrial translocator protein, tspo (braestrup and squires, 1977; papadopoulos et al., 2006) , and knockdown or knockout of this protein rescued env and hiv-1 production (zhou et al., 2014) . further experiments indicated that drugs inducing erad led to recovery of env levels and viral titers. these results suggested that the er and mitochondria communicate through juxtaposition of their membranes, so that conditions in the mitochondria influence protein folding and erad. in support of this conclusion, gp78 is an erad-associated e3 ligase (fang et al., 2001) localized to mitochondria-er membrane contacts (fu et al., 2013) . thus, mitochondria proteins may influence erad and modulate hiv-1 env presentation to the immune system. triggering of an innate immune response to viruses is affected by the erad process. some anti-viral signaling is controlled through mitochondria, which also cooperates with the er for lipid synthesis and calcium-controlled processes at the mitochondrialassociated membrane (mam; jacobs et al., 2014) . mitochondrial antiviral signaling protein (mavs; also called ips-1, visa, or cardif) binds to different retinoic acid-inducible gene-i (rig-i)-like receptor (rlr) proteins, which sense cytosolic viral rnas (kawai et al., 2005; meylan et al., 2005; seth et al., 2005; xu et al., 2005) . the mavs protein is present in the mitochondrial and peroxisomal membranes, and viral rna triggers both interferondependent or independent responses, respectively, (jacobs and coyne, 2013; jacobs et al., 2014) . the levels of mavs are affected by gp78, an e3 ubiquitin ligase that is localized to the ermitochondrial interface (mam; jacobs et al., 2014) . the gp78 ligase was detected by a high throughput rnai screen to identify genes that restricted enterovirus replication (coyne et al., 2011) . downregulation of gp78 was shown to decrease yields of vesicular stomatitis virus (vsv) and to increase type i interferon responses. some viruses, such as those inducing hepatitis b (hbv) or c (hcv), use erad to reduce the amounts of glycoproteins and particles produced. interestingly, both viruses partially induce the unfolded protein response (upr; li et al., 2007 li et al., , 2011 saeed et al., 2011) , which then increases the levels of certain erad components. hbv, a member of the hepadnaviridae, triggers upregulation of the glycoside hydrolase 47 family enzymes, edem 1 and 2. increased edem levels appear to bypass normal er folding of hbv glycoproteins to result in erad (lazar et al., 2012) . hcv, a member of the flaviviridae, induces primarily edem1 through the upr and splicing of x-box binding protein 1. further experiments suggested that elevated levels of edem 1 and 3 increase binding to sel1l, an adapter to the retrotranslocon (figure 1) . inhibition of edem binding to sel1l interfered with ubiquitination of hcv env protein, e2 (saeed et al., 2011) . interestingly, infections by another member of the flaviviridae, japanese encephalitis virus, did not result in edem binding to the env proteins, indicating that not all viral family members control env proteins by this mechanism. overall, manipulation of edem levels appears to be a common mechanism to reduce viral glycoprotein levels. lowered amounts of env proteins and virus particles then contribute to avoidance of innate and adaptive immunity, leading to chronic infections (saeed et al., 2011; lazar et al., 2012) . a number of pathogens harness the erad process to facilitate various replication strategies. the best known examples are the bacterial ab toxins, particularly cholera toxin, which is thought to hijack the erad machinery for delivery to the cytosol (hazes and read, 1997) . cholera toxin has a catalytic a chain divided into two subunits (cta1 and cta2) inside a pore composed of five receptor-binding b subunits (spangler, 1992) . the holotoxin www.frontiersin.org binds to the ganglioside gm1 on the surface of gut epithelial cells, which then triggers toxin internalization and trafficking through the golgi to the er (fujinaga et al., 2003) . the a subunits are bound to the b subunits by disulfide bonds, and the toxin complex interacts with the er-resident enzyme pdi (figure 1) . pdi is a redox-dependent chaperone that unfolds the toxin, which is then released in the oxidized state (tsai et al., 2001) . this unfolding event appears to be required for the ability of cta1 to retrotranslocate to the cytosol, where it induces the adp-ribosylation of the gαs protein and, ultimately, opening of chloride channels leading to massive diarrhea (muanprasat and chatsudthipong, 2013) . as noted above, retrotranslocation of erad substrates is preceded by a recognition step. the chaperone bip, which is known to be involved in identification of non-glycosylated erad substrates, and an er-resident atpase (torsin a) promote cta1 retrotranslocation (tsai et al., 2001; winkeler et al., 2003; forster et al., 2006; moore et al., 2010) . sel1l and erdj5, a co-chaperone of bip, also facilitate cta1 retrotranslocation, where the j domain of erdj5 is required (williams et al., 2013) . erdj5 also binds to sel1l, likely providing interaction with the hrd1 e3 ligase (see figure 1) . torsin a may provide the link to the membrane-resident derlin-1 protein (nery et al., 2011) . cta1 retrotranslocation appears to involve derlin-1 (bernardi et al., 2008) and the transmembrane ubiquitin ligases, hrd1 and gp78 . thus, multiple low affinity interactions are likely involved in the identification of cta1 as a substrate and its delivery to the retrotranslocon. similar to other retrotranslocated substrates, the cytosolic p97 atpase participates in cta1 extraction from the er membrane (abujarour et al., 2005; kothe et al., 2005) . nevertheless, cta1 subverts the normal erad process by avoiding polyubiquitination (rodighiero et al., 2002) . the hypothesis that cta1 avoids ubiquitination through the absence of lysines targeted for polyubiquitination was not substantiated by mutational analysis (rodighiero et al., 2002) . these results indicate that cta1 employs many of the typical components used for erad targeting, including the e3 ligase, but it is unclear how polyubiquitination and degradation of the substrate are avoided. therefore, retrotranslocon targeting and substrate extraction from the er membrane is not necessarily coupled to ubiquitination, although ubiquitination may be required for proteasomal degradation. viral pathogens also use erad. mouse mammary tumor virus (mmtv) is a betaretrovirus that subverts the erad process to complete its viral replication cycle. all retroviruses synthesize an unspliced viral rna that requires export from the nucleus to the cytosol for translation or packaging into virus particles (cullen, 2003) . the unspliced rnas of simple retroviruses have a highly structured cis-acting sequence, such as the constitutive transport element (cte) of mason-pfizer monkey virus (mpmv; bray et al., 1994) . the cte facilitates rna export through the typical tap/nxf1-mediated pathway used by cellular mrnas (grüter et al., 1998) . in contrast, the complex retroviruses encode an adapter protein, such as the rev protein of hiv-1 (hanly et al., 1989) , which binds to a structured rna element near the 3 end of the genome (daly et al., 1989; zapp and green, 1989) . mmtv also produces a rev-like protein, rem, for export of unspliced rna (mertz et al., 2005) , but rem binding to viral rna has additional translation-associated functions (mertz et al., 2009b) . unlike other complex retroviruses, rem is made from an internally deleted form of the env protein, and the export function resides in a long sp of 98 amino acids (indik et al., 2005; mertz et al., 2005) . interestingly, rem is a precursor protein that is directed to the er membrane for translation, where it appears to be cleaved by signal peptidase into the rev-like rem-sp and a c-terminal glycosylated product (rem-ct) of unknown activity (byun et al., 2010) . recent evidence indicates that rem-sp uses retrotranslocation for extraction from the er membrane, but, like cholera toxin, avoids proteasomal degradation (byun et al., 2010 (byun et al., , 2012 . dultz et al. (2008) first reported that rem is directed to the er membrane for translation and cleavage by signal peptidase. they also suggested that the rem precursor (the uncleaved protein) could be detected in the nucleus by fluorescence microscopy (dultz et al., 2008) . byun et al. (2010) showed that mutation of the predicted signal peptidase cleavage site prevented the appearance of rem-sp as detected by both western blotting and a highly sensitive reporter assay for rev-like function (mertz et al., 2005; byun et al., 2010) . this assay requires binding to a specific rna element in viral rna (müllner et al., 2008; mertz et al., 2009a) . fluorescence experiments indicated that only the cleaved rem-sp enters the nucleus, whereas the uncleaved form was highly unstable and localized to the cytosol (byun et al., 2010) . furthermore, rem-sp activity was inhibited by expression of a dominant-negative form of the p97 atpase required for retrotranslocation (byun et al., 2010) . rem-sp function also was reduced by the expression of a dominant-negative derlin-1, but not derlin-2 protein (byun et al., in preparation) . these results strongly suggest that rem must be cleaved by signal peptidase prior to sp retrotranslocation to the cytosol and import into the nucleus for rna binding (figure 4) . experiments indicate that an altered conformation of either the n-terminal rem-sp in the cytosol or the er-luminal portion of rem affect folding and accessibility to signal peptidase, which is associated with translocons (falk and gilula, 1998) . first, rem tagging at the c-terminus with green fluorescent protein (rem-gfp) resulted in a stable protein that was inefficiently cleaved and had little fluorescence (mertz et al., 2005; byun et al., 2012) . rem-gfp also had very low functional activity in reporter assays (mertz et al., 2005) . in contrast, rem tagged at the n-terminus with gfp was cleaved normally, and gfp-rem-sp localized to the nucleoli, a result typical of other rev-like proteins (cullen, 2003; mertz et al., 2005) . second, deletion mutations of the rem c-terminus greatly affected stability of the protein (byun et al., 2012) . removal of the 50 c-terminal amino acids had little effect on the cleavage or stability of the protein, but deletion of 100 or 150 amino acids produced a highly unstable precursor that could be rescued by the proteasomal inhibitor mg-132 (byun et al., 2012) . reduced cleavage of the precursor also was observed. surprisingly, further deletion to give only the sp (rem-sp) again yielded a stable protein (byun et al., 2012) . third, substitution of the leucine at position 71 in the sp gave a stable precursor protein that was poorly cleaved by signal peptidase (mertz et al., 2009a; byun et al., 2010 ). an independent report indicated that residues frontiers in microbiology | virology rem is a precursor protein that has an n-terminal signal peptide (rem-sp) that directs translation to the er membrane. the rem-ct enters the er lumen, where it is modified by n-glycosylation on two different sites. rem recognition for retrotranslocation is not understood, but appears to involve derlin-1 and, potentially, an e3 ligase, although ubiquitinated rem has not been observed. full-length rem is cleaved by signal peptidase, and rem-ct is released into the er lumen. similar to other retrotranslocation substrates, rem-sp is extracted from the er membrane using the p97 atpase. despite its dislocation into the cytosol, rem-sp escapes the proteasome and translocates into the nucleus for binding of mmtv rna. this figure is adapted from byun et al. (2012) . 80 through 98 act as the hydrophobic membrane anchor sequence, suggesting that position 71 is localized in the cytosol (dultz et al., 2008) . recognition of rem c-terminal sequences in the er lumen, presumably by their interaction or lack of interaction with specific chaperone proteins, prevent degradation by erad. the er-luminal chaperone bip has repeatedly been detected after purification and proteomic analysis of rem-binding proteins (gou et al., manuscript in preparation) . our preliminary data indicate that rem-sp is not ubiquitinated, and it is possible that this feature protects rem-sp from proteasomal degradation. since the rem precursor and c-terminal deletion mutants are subject to erad, cleavage and association with specific cellular proteins appear to be critical for avoidance of the degradative process. the idea that viral proteins manipulate e3 enzymes to form alternative complexes (olzmann et al., 2013) would be consistent with rem-sp escape from erad. the polyomaviruses have a unique entry method that uses retrotranslocation, while avoiding erad. the bk polyomavirus (bkv) first binds to the ganglioside receptors gt1b and gd1b and enters through caveolae (neu et al., 2009) , which are composed of membrane microdomains/lipid rafts that are enriched for sphingolipids and signaling molecules (head et al., 2014; figure 5) . particle delivery to the cytosol occurs through a phdependent step involving endosomal trafficking via microtubules to the er (eash and atwood, 2005; moriyama and sorokin, 2008; jiang et al., 2009) . other members of the polyomaviridae use caveolae-independent entry for er delivery (neu et al., 2009) . er localization of these viruses is necessary to access specific retrotranslocation components. the vp1 capsid proteins of polyomaviruses form pentamers during assembly that are held together by disulfide bonding (li et al., 2003) . each pentamer is associated with one molecule of either the minor capsid protein vp2 or vp3 (barouch and harrison, 1994) , which become accessible to antibodies after exposure to the unique environment of the er (norkin et al., 2002) . particle delivery into the er allows reduction and isomerization of disulfide bonds using erp29 (mouse polyomavirus; magnuson et al., 2005) or erp57 and pdi (sv40; schelhaas et al., 2007) to allow partial uncoating (jiang et al., 2009; tsai and qian, 2010) . the partially uncoated virion then engages the retrotranslocation machinery to allow cytosolic entry similar to cholera toxin (neu et al., 2009) . interestingly, different polyomaviruses use distinct derlin family members for retrotranslocation. sv40 uses derlin-1 and sel1l (schelhaas et al., 2007) , whereas mouse polyoma virus uses derlin-2 ; figure 5) . additional experiments indicate that exposure of vp2 hydrophobic sequences tethers virus particles to the er membrane, and that both bip and bap31 are needed for dislocation of sv40 to the cytosol (geiger et al., 2011) . bap31 may serve as a shuttle to the erqc that has been associated with enriched erad components (kamhi-nesher et al., 2001; wakana et al., 2008) . furthermore, use of epoxomicin or eeyarestatin 1, inhibitors of the proteasome or p97 atpase, respectively, blocked early events of bkv infection (bennett et al., 2013) . epoxomicin treatment of cells allowed accumulation of bkv in the calnexin-rich, bip-deficient erqc (bennett et al., 2013) . these results are consistent with erad extraction of polyomaviruses from the er to the cytosol, although it is has been suggested that www.frontiersin.org figure 5 | use of erad for polyomavirus uncoating. many polyomaviruses enter through caveosomes that are enriched for viral entry receptors, triggering particle uptake through endosomes. using the microtubule network, vesicles traffic the virus to the er, where the unique environment allows structural changes to the icosahedral capsids. studies of jcv, bkv, and sv40 indicate that viral particles interact with pdi and erp57 in the er lumen to rearrange capsid proteins. in contrast, the related mouse polyomavirus (pyv) uses the pdi family member, erp29, presumably for a similar function. the altered particles then appear to engage different retrotranslocons (dependent on either derlin-1 or derlin-2) to induce retrotranslocation to the cytosol, where the reduced calcium environment produces further capsid rearrangements. these particles then bind to the nuclear pore where uncoating occurs to allow passage of viral dna into the nucleus. this figure is adapted from neu et al. (2009). there are cell-type and virus-specific differences and that direct er to nuclear transport may occur (bennett et al., 2013) . low levels of calcium in the cytosol lead to further capsid destabilization and exposure of the nuclear localization signals on capsid proteins. the partially uncoated capsid then transits through the nuclear pores for initiation of viral dna replication (neu et al., 2009) . the preceding experiments indicate that erad is used by viruses to allow trafficking events that promote replication. mmtv rem trafficking through the er allows access to signal peptidase and cleavage of rem precursor into functional n-and c-terminal proteins. in contrast, the polyomaviruses use erad to partially uncoat virions on their path to the nucleus. importantly, both types of viruses avoid proteasomal degradation during erad, although the mechanisms remain unclear. erad may be regulated or "tuned" through the rapid turnover of specific components through the proteasomes or autophagosomes/vesicular trafficking to lysosomes (merulla et al., 2013) . normal secretory vesicles released from the er are 60-70 nm in diameter and have coatamer proteins, such as copii, whereas the er-derived tuning vesicles (edemosomes) lack coatamers and are 200-800 nm in diameter (bernasconi et al., 2012b) . tuning vesicles contain sel1l, edem1, and os-9, which are transmembrane or luminal proteins involved in erad (figure 1 ; olzmann et al., 2013) . edemosomes are believed to reduce erad by disposal in acidic organelles (bernasconi et al., 2012b) , favoring the correct folding of polypeptides (calì et al., 2008) . the coronaviruses are known to take advantage of erad tuning (reggiori et al., 2010) . many plus-stranded rna-containing viruses manipulate cellular membranes to further rna replication (paul and bartenschlager, 2013) . these membrane structures have been divided into invaginated vesicle/spherule type and double-membrane vesicle (dmv) type (two lipid bilayers). such vesicles allow viruses to concentrate their replication components, to separate distinct viral processes (e.g., translation, transcription, and replication), and to avoid immune detection (paul and bartenschlager, 2013) . severe acute respiratory syndrome coronavirus (sars-cov) and mouse hepatitis virus (mhv) induce dmvs for targeting their replication and transcription (reggiori et al., 2010) . the dmvs originate from er membranes and contain the non-structural transmembrane proteins nsp3 and nsp4 and viral double-stranded rna (stertz et al., 2007; reggiori et al., 2010) . nevertheless, dmvs lack markers typical of the ergic or the golgi (oostra et al., 2007) . recent experiments indicate that dmvs are coated with microtubule-associated protein light chain 3 [lc3; atg8 in yeast (reggiori et al., 2010) ], which is a ubiquitin-like modifier (van der veen and ploegh, 2012). lc3 can exist in a lipidated form (covalent linkage to phosphatidylethanolamine; also known as lc3-ii) or a predominantly cytosolic non-lipidated form (lc3-i). lc3-ii is believed to be involved in fusion of autophagosomes to lysosomes (van der veen and ploegh, 2012), but coronavirus dmvs display the non-lipidated lc3-i (reggiori et al., 2010) . these ubiquitinlike modifiers recognize specific receptors that target associated vesicles to particular cellular locations (van der veen and ploegh, 2012). the coronaviruses appear to be redirecting vesicles destined for autophagosomes to sequestered locations in the cytosol where replication will occur. the autophagy machinery is not required for coronavirus replication, and no colocalization of viral non-structural proteins was observed with lc3-ii-coated autophagosomes (reggiori et al., 2010) . coronavirus-induced dmvs and edemosomes both are coated with the non-lipidated lc3-i protein (calì et al., 2008; reggiori et al., 2010) , which is not covalently attached to membranes like lc3-ii (kabeya et al., 2000) . induction of autophagy in coronavirus-infected cells with rapamycin decreased the levels of edem1 and coronavirus (reggiori et al., 2010) . the viruscontaining dmvs had both edem1 and os-9, but not other erad-associated chaperones, and virus infection interfered with erad tuning by hijacking the edemosomes. nevertheless, lc3-i, but not edem1 and os-9, is necessary for coronavirus infection, and the hijacked edemosome cargo is not degraded by proteases in the endosomes/lysosomes (reggiori et al., 2010) . further, the erad transmembrane adapter protein, sel1l, is needed for dmv formation, capturing the er-resident edem1 and os-9 proteins (and possibly xtp3-b and edem3), while using its proline-rich cytosolic domain to bind to lc3-i. as expected, sel1l knockdown impairs coronavirus replication (bernasconi et al., 2012a) . the organizationally similar arterioviruses (classified with coronaviruses, toroviruses, and roniviruses into the order nidovirales; gorbalenya et al., 2006) subvert edemosome trafficking for their replication, although the size of the vesicles is smaller (monastyrska et al., 2013) . the mechanism for altering edem1containing vesicular trafficking is unclear, but likely involves expression of viral non-structural proteins that span the erderived membranes (monastyrska et al., 2013) , perhaps through their interaction with sel1l. these experiments indicate that viruses hijack edemosomes to sequester their double-stranded rna from cytosolic sensors that will trigger interferon production and innate immunity (zinzula and tramontano, 2013) . other components of the erad system, particularly chaperone proteins, also participate in the replication and transmission of both plant and mammalian viruses (verchot, 2014) . the erad system is a complex and highly regulated process controlling the disposal of misfolded or misassembled proteins that are directed to the er for translation. deregulation of this process results in pathogenic conditions, including infectious diseases. viruses exploit erad to decrease overall viral levels and allow establishment of chronic infections by minimizing antigen presentation to the immune system. trafficking of specific viral proteins or entire virion particles may involve erad for refolding or processing in the unique er environment. alternatively, viruses can use erad-associated components to form isolated lipid vesicles for replication and shelter from immune detection. virus-mediated subversion of erad can lead to degradation of molecules that are involved in innate or adaptive immunity. continued studies of viruses are certain to provide additional insights into both the erad process and the components that regulate it. further experiments may identify targets for viral therapeutics. p97 is in a complex with cholera toxin and influences the transport of cholera toxin and related toxins to the cytoplasm n-glycan structures: recognition and processing in the er ubxd7 binds multiple ubiquitin ligases and implicates p97 in hif1alpha turnover intracellular folding of tissue-type plasminogen activator. effects of disulfide bond formation on n-linked glycosylation and secretion enhanced cd4 down-modulation by late stage hiv-1 nef alleles is associated with increased env incorporation and viral replication functional and genomic analyses of blocked protein o-mannosylation in baker's yeast a network of cytosolic factors targets srp-independent proteins to the endoplasmic reticulum signal peptidase i: cleaving the way to mature proteins amino acid composition of alpha1/alpha2 domains and cytoplasmic tail of mhc class i molecules determine their susceptibility to human cytomegalovirus us11-mediated down-regulation interactions among the major and minor coat proteins of polyomavirus adams and protein disulfide isomerase: the key to regulated cell-surface protein ectodomain shedding? biases and complex patterns in the residues flanking protein n-glycosylation sites the protein disulfide isomerase family: key players in health and disease role of cell-type-specific endoplasmic reticulum-associated degradation in polyomavirus trafficking derlin-1 facilitates the retro-translocation of cholera toxin the e3 ubiquitin ligases hrd1 and gp78 bind to and promote cholera toxin retro-translocation stringent requirement for hrd1, sel1l, and os-9/xtp3-b for disposal of erad-ls substrates cyclosporine a-sensitive, cyclophilin b-dependent endoplasmic reticulumassociated degradation role of the sel1l:lc3-i complex as an erad tuning receptor in the mammalian er unconventional roles of nonlipidated lc3 in erad tuning and coronavirus infection a dual task for the xbp1-responsive os-9 variants in the mammalian endoplasmic reticulum: inhibiting secretion of misfolded protein conformers and enhancing their disposal regulated protein turnover: snapshots of the proteasome in action mhc class i ubiquitination by a viral phd/lap finger protein specific benzodiazepine receptors in rat brain characterized by high-affinity (3h)diazepam binding a small element from the mason-pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication rev-independent n-linked protein glycosylation in the endoplasmic reticulum cleaning up: er-associated degradation to the rescue protein folding and quality control in the endoplasmic reticulum: recent lessons from yeast and mammalian cell systems substrate-specific mediators of er associated degradation (erad) requirements for mouse mammary tumor virus rem signal peptide processing and function retroviral rem protein requires processing by signal peptidase and retrotranslocation for nuclear function segregation and rapid turnover of edem1 by an autophagy-like mechanism modulates standard erad and folding activities retrotranslocation of a misfolded luminal er protein by the ubiquitin-ligase hrd1p the molecular basis of coupling of translocation and n-glycosylation human cytomegalovirus us3 chimeras containing us2 cytosolic residues acquire major histocompatibility class i and ii protein degradation properties the c-terminal amino acid of the mhc-i heavy chain is critical for binding to derlin-1 in human cytomegalovirus us11-induced mhc-i degradation forced interaction of cell surface proteins with derlin-1 in the endoplasmic reticulum is sufficient to induce their dislocation into the cytosol for degradation defining human erad networks through an integrative mapping strategy os-9 and grp94 deliver mutant alpha1-antitrypsin to the hrd1-sel1l ubiquitin ligase complex for erad cysteineless non-glycosylated monomeric blue fluorescent protein, secbfp2, for studies in the eukaryotic secretory pathway comparative rnai screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers nuclear mrna export: insights from virology specific binding of hiv-1 recombinant rev protein to the rev-responsive element in vitro a portrait of the get pathway as a surprisingly complicated young man a luminal surveillance complex that selects misfolded glycoproteins for er-associated degradation the signal peptide of the mouse mammary tumor virus rem protein is released from the endoplasmic reticulum membrane and accumulates in nucleoli membrane-proximal domain of a disintegrin and metalloprotease-17 represents the putative molecular switch of its shedding activity operated by protein-disulfide isomerase involvement of cytoskeletal components in bk virus infectious entry o-mannosylation precedes and potentially controls the n-glycosylation of a yeast cell wall glycoprotein rnf185 is a novel e3 ligase of endoplasmic reticulum-associated degradation (erad) that targets cystic fibrosis transmembrane conductance regulator (cftr) the otubain yod1 is a deubiquitinating enzyme that associates with p97 to facilitate protein dislocation from the er evolutionary relationships and structural mechanisms of aaa+ proteins the complex biology of autocrine motility factor/phosphoglucose isomerase (amf/pgi) and its receptor, the gp78/amfr e3 ubiquitin ligase connexin membrane protein biosynthesis is influenced by polypeptide positioning within the translocon and signal peptidase access the tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum protein disulfide isomerase is essential for viability in saccharomyces cerevisiae ubiquitin-dependent intramembrane rhomboid protease promotes erad of membrane proteins e2-25k mediates us11-triggered retro-translocation of mhc class i heavy chains in a permeabilized cell system protein disulfide isomerase-like proteins play opposing roles during retrotranslocation create and preserve: proteostasis in development and aging is governed by cdc48/p97/vcp separate roles and different routing of calnexin and erp57 in endoplasmic reticulum quality control revealed by interactions with asialoglycoprotein receptor chains regulation of mitophagy by the gp78 e3 ubiquitin ligase gangliosides that associate with lipid rafts mediate transport of cholera and related toxins from the plasma membrane to endoplasmic reticulm bap31 and bip are essential for dislocation of sv40 from the endoplasmic reticulum to the cytosol structures of the sec61 complex engaged in nascent peptide translocation or membrane insertion identification, expression, and characterization of a cdna encoding human endoplasmic reticulum mannosidase i, the enzyme that catalyzes the first mannose trimming step in mammalian asn-linked oligosaccharide biosynthesis nidovirales: evolving the largest rna virus genome derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant α-1 antitrypsin from the endoplasmic reticulum a gated channel into the proteasome core particle protein disulfide isomerases contribute differentially to the endoplasmic reticulum-associated degradation of apolipoprotein b and other substrates tap, the human homolog of mex67p, mediates cte-dependent rna export from the nucleus the delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology finding the will and the way of erad substrate retrotranslocation comparative analysis of the htlv-i rex and hiv-1 rev trans-regulatory proteins and their rna response elements accumulating evidence suggests that several ab-toxins subvert the endoplasmic reticulum-associated protein degradation pathway to enter target cells interaction of membrane/lipid rafts with the cytoskeleton: impact on signaling and function: membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling in and out of the er: protein folding, quality control, degradation, and related human diseases role of the ring-ch domain of viral ligase mk3 in ubiquitination of non-lysine and lysine mhc i residues edem3, a soluble edem homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming a role for n-glycanase in the cytosolic turnover of glycoproteins mechanism and components of endoplasmic reticulum-associated degradation human xtp3-b forms an endoplasmic reticulum quality control scaffold with the hrd1-sel1l ubiquitin ligase complex and bip stimulation of erad of misfolded null hong kong alpha1-antitrypsin by golgi alpha1,2-mannosidases natural killing target antigens as inducers of interferon: studies with an immunoselected, natural killing-resistant human t lymphoblastoid cell line alterations in t4 (cd4) protein and mrna synthesis in cells infected with hiv the hsp110 molecular chaperone stabilizes apolipoprotein b from endoplasmic reticulum-associated degradation (erad) a novel, mouse mammary tumor virus encoded protein with rev-like properties molecular cloning and chromosomal mapping of a bone marrow stromal cell surface gene, bst2, that may be involved in pre-b-cell growth mechanisms of mavs regulation at the mitochondrial membrane regulation of mitochondrial antiviral signaling (mavs) expression and signaling by the mitochondria-associated endoplasmic reticulum membrane (mam) protein gp78 cdc48 (p97): a "molecular gearbox" in the ubiquitin pathway? early events during bk virus entry and disassembly the er translocon and retrotranslocation: is the shift into reverse manual or automatic? post-translational translocation into the endoplasmic reticulum trc40 can deliver short secretory proteins to the sec61 translocon lc3, a mammalian homologue of yeast apg8p, is localized in autophagosome membranes after processing a novel quality control compartment derived from the endoplasmic reticulum ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction human hrd1 is an e3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum expression and degradation of the cystic fibrosis transmembrane conductance regulator in saccharomyces cerevisiae molecular discrimination of structurally equivalent lys 63-linked and linear polyubiquitin chains role of p97 aaa-atpase in the retrotranslocation of the cholera toxin a1 chain, a non-ubiquitinated substrate an unusual transmembrane helix in the endoplasmic reticulum ubiquitin ligase doa10 modulates degradation of its cognate e2 enzyme bst-2/hm1.24 is a raft-associated apical membrane protein with an unusual topology t cells can present antigens such as hiv gp120 targeted to their own surface molecules activation of erad pathway by human hbv modulates viral and subviral particle production a window of opportunity: timing protein degradation by trimming of sugars and ubiquitins hepatitis b virus x protein (hbx) activates atf6 and ire1-xbp1 pathways of unfolded protein response the aaa atpase p97 links peptide n-glycanase to the endoplasmic reticulum-associated e3 ligase autocrine motility factor receptor subversion of cellular autophagy machinery by hepatitis b virus for viral envelopment characterization of self-assembled virus-like particles of human polyomavirus bk generated by recombinant baculoviruses murine polyomavirus requires the endoplasmic reticulum protein derlin-2 to initiate infection a membrane protein required for dislocation of misfolded proteins from the er a proteasomal atpase contributes to dislocation of endoplasmic reticulum-associated degradation (erad) substrates protein o-mannosyltransferases associate with the translocon to modify translocating polypeptide chains signal peptide peptidase is required for dislocation from the endoplasmic reticulum protein quality control in the er: the recognition of misfolded proteins multilayered mechanism of cd4 downregulation by hiv-1 vpu involving distinct er retention and erad targeting steps erp29 triggers a conformational change in polyomavirus to stimulate membrane binding hiv-1 vpu neutralizes the antiviral factor tetherin/bst-2 by binding it and directing its beta-trcp2-dependent degradation a novel human wd protein, h-beta trcp, that interacts with hiv-1 vpu connects cd4 to the er degradation pathway through an f-box motif cell biology. an ancient portal to proteolysis endoplasmic reticulum protein quality control is determined by cooperative interactions between hsp/c70 protein and the chip e3 ligase hiv infection of primate lymphocytes and conservation of the cd4 receptor mapping of the functional boundaries and secondary structure of the mouse mammary tumor virus rem-responsive element rev and rex proteins of human complex retroviruses function with the mmtv rem-responsive element mouse mammary tumor virus encodes a self-regulatory rna export protein and is a complex retrovirus specificity and regulation of the endoplasmic reticulum-associated degradation machinery emerging functions of the vcp/p97 aaa-atpase in the ubiquitin system cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus an autophagy-independent role for lc3 in equine arteritis virus replication the ero1alpha-pdi redox cycle regulates retro-translocation of cholera toxin intracellular trafficking pathway of bk virus in human renal proximal tubular epithelial cells cholera: pathophysiology and emerging therapeutic targets sel1l, the homologue of yeast hrd3p, is involved in protein dislocation from the mammalian er identification of the rem-responsive element of mouse mammary tumor virus a novel mammalian endoplasmic reticulum ubiquitin ligase homologous to the yeast hrd1 tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu torsina participates in endoplasmic reticulum-associated degradation the polyomaviridae: contributions of virus structure to our understanding of virus receptors and infectious entry ubx2 links the cdc48 complex to er-associated protein degradation caveolar endocytosis of simian virus 40 is followed by brefeldin a-sensitive transport to the endoplasmic reticulum, where the virus disassembles destabilization of the vcp-ufd1-npl4 complex is associated with decreased levels of erad substrates derlin-2 and derlin-3 are regulated by the mammalian unfolded protein response and are required for er-associated degradation characterization of an erad pathway for nonglycosylated bip substrates, which require herp edem1 regulates er-associated degradation by accelerating de-mannosylation of folding-defective polypeptides and by inhibiting their covalent aggregation lipid droplet formation is dispensable for endoplasmic reticulum-associated degradation the mammalian endoplasmic reticulum-associated degradation system localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication translocator protein (18kda): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function architecture and biogenesis of plus-strand rna virus replication factories protein disulfide isomerase is required for platelet-derived growth factor-induced vascular smooth muscle cell migration, nox1 nadph oxidase expression, and rhogtpase activation cd4 and bst-2/tetherin proteins retro-translocate from endoplasmic reticulum to cytosol as partially folded and multimeric molecules going through the motions: the atpase cycle of p97 protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication endolysosomal sorting of ubiquitylated caveolin-1 is regulated by vcp and ubxd1 and impaired by vcp disease mutations role of ubiquitination in retro-translocation of cholera toxin and escape of cytosolic degradation quality control: er-associated degradation: protein quality control and beyond role of the endoplasmic reticulum-associated degradation (erad) pathway in degradation of hepatitis c virus envelope proteins and production of virus particles counteraction of the multifunctional restriction factor tetherin bipmediated closing of the sec61 channel limits ca 2+ leakage from the er simian virus 40 depends on er protein folding and quality control factors for entry into host cells membrane-bound ubx2 recruits cdc48 to ubiquitin ligases and their substrates to ensure efficient er-associated protein degradation ubx domain proteins: major regulators of the aaa atpase cdc48/p97 identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 a shared endoplasmic reticulum-associated degradation pathway involving the edem1 protein for glycosylated and nonglycosylated proteins mechanisms and function of substrate recruitment by f-box proteins road to ruin: targeting proteins for degradation in the endoplasmic reticulum defining the human deubiquitinating enzyme interaction landscape structure and function of cholera toxin and the related escherichia coli heat-labile enterotoxin the trc8 e3 ligase ubiquitinates mhc class i molecules before dislocation from the er the intracellular sites of early replication and budding of sarscoronavirus the cytosolic tail of class i mhc heavy chain is required for its dislocation by the human cytomegalovirus us2 and us11 gene products hiv accessory proteins versus host restriction factors sel1l is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of erad the endoplasmic reticulum-associated degradation pathways of budding yeast cellular entry of polyomaviruses protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin a ubiquitin-binding rhomboid protease aimed at eradication cd4+ t cells: guardians of the phagosome glycosylation-independent erad pathway serves as a backup system under er stress ubiquitin-like proteins one step at a time: endoplasmic reticulumassociated degradation the er quality control and er associated degradation machineries are vital for viral pathogenesis mechanism, specificity, and physiology of signal peptide peptidase (spp) and spp-like proteases bap31 is an itinerant protein that moves between the peripheral endoplasmic reticulum (er) and a juxtanuclear compartment related to erassociated degradation requirements for the selective degradation of endoplasmic reticulum-resident major histocompatibility complex class i proteins by the viral immune evasion molecule mk3 ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce erad of mhc-i by viral e3 ligase mk3 ube2j2 ubiquitinates hydroxylated amino acids on erassociated degradation substrates the viral e3 ubiquitin ligase mk3 uses the derlin/p97 endoplasmic reticulum-associated degradation pathway to mediate down-regulation of major histocompatibility complex class i proteins sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction thiol isomerases negatively regulate the cellular shedding activity of adam17 human immunodeficiency virus type 1 vpu protein regulates the formation of intracellular gp160-cd4 complexes the erdj5-sel1l complex facilitates cholera toxin retrotranslocation bip-dependent export of cholera toxin from endoplasmic reticulum-derived microsomes the cdc48 machine in endoplasmic reticulum associated protein degradation visa is an adapter protein required for virus-triggered ifn-beta signaling function of the p97-ufd1-npl4 complex in retrotranslocation from the er to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains recruitment of the p97 atpase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane a membrane protein complex mediates retro-translocation from the er lumen into the cytosol f-box proteins that contain sugar-binding domains glycoproteinspecific ubiquitin ligases recognize n-glycans in unfolded substrates sequence-specific rna binding by the hiv-1 rev protein the mitochondrial translocator protein, tspo, inhibits hiv-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit we thank dr. jon huibregtse for helpful comments and suggestions on the manuscript and marianna grenadier for the figures. this work was supported by nih grants r01ca167053 and r21ai105710. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-349774-898tmq14 authors: zhang, haiyang; tu, jialu; cao, chulei; yang, ting; gao, liangcai title: proteasome activator pa28γ-dependent degradation of coronavirus disease (covid-19) nucleocapsid protein date: 2020-06-16 journal: biochem biophys res commun doi: 10.1016/j.bbrc.2020.06.058 sha: doc_id: 349774 cord_uid: 898tmq14 the nucleocapsid protein is significant in the formation of viral rna of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), accounting for the largest proportion of viral structural proteins. here, we report for the first time that the 11s proteasomal activator pa28γ regulates the intracellular abundance of the sars-cov-2 n protein (ncov n). furthermore, we have identified proteasome activator pa28γ as a ncov n binding protein by co-immunoprecipitation assay. as a result of their interaction, ncov n could be degraded by pa28γ-20s in vitro degradation assay. this was also demonstrated by blocking de novo protein synthesis with cycloheximide. the stability of ncov n in pa28γ-knockout cells was greater than in pa28γ-wildtype cells. notably, immunofluorescence staining revealed that knockout of the pa28γ gene in cells led to the transport of ncov n from the nucleus to the cytoplasm. overexpression of pa28γ enhanced proteolysis of ncov n compared to that in pa28γ-n151y cells containing a dominant-negative pa28γ mutation, which reduced this process. these results suggest that pa28γ binding is important in regulating 20s proteasome activity, which in turn regulates levels of the critical ncov n nucleocapsid protein of sars-cov-2, furthering our understanding of the pathogenesis of covid-19. as of may 31, 2020, nearly 6 million cases of coronavirus disease 2019 (covid19) and over 350,000 deaths from the disease, have been reported worldwide [1] . a novel coronavirus is the cause of covid-19. taxonomically, sars-cov-2 forms a clade within the subgenus sarbecovirus, orthocoronavirinae subfamily [2] . sars-cov-2 has a positive single-stranded rna genome, approximately 29.8 kb, including a various number (from 6 to 11) of open reading frames (orfs) [3] . the first orf, representing over 60% of the entire genome, encodes 16 non-structural proteins, while the remaining orfs encode auxiliary proteins and four structural proteins [4] . the four structural proteins are the small envelope protein (e), matrix protein (m), spike surface glycoprotein (s), and nucleocapsid protein (n) [5] . the sars-cov-2 nucleocapsid protein (hereafter, referred to as ncov n) accounts for the largest proportion of viral structure proteins and is the most abundant protein in virus-infected cells. its primary function is to package the viral rna genome into a ribonucleoprotein complex, the capsid [6] . the nucleocapsid protein encoded by sars-cov-2 can act as a viral inhibitory factor of rna interference in cells [7] . furthermore, it has been shown that the n protein of sars-cov can modulate the host cellular machinery and it may serve in a regulatory role during the viral life cycle [8] . therefore, the nucleocapsid protein is a crucial multifunctional protein, involved in the process of virus infection, replication, and packaging [9] . in general, viral nuclear proteins can enter the host nucleus and interact with a variety of host proteins to interfere with the life cycle of the host cell. it has been shown that the coronavirus n protein is not only localized in the cytosol but also, to a certain extent, translocated into the nucleus where it may interact with various cellular proteins that modulate cellular functions [10] . this process may depend on interaction of the n protein with the proteasome activator pa28γ, which is localized in the nucleus. pa28γ could be critical for degrading the sars-cov-19 ncov n protein in the nucleus as part of the 20s proteasome, which acts to degrade proteins in a ubiquitin-independent manner, such as seen in the hepatitis c virus (hcv) core protein [11] . the proteasome has an important role in the degradation of unneeded or damaged proteins by proteolysis. two distinct proteasomes differentially target proteins for degradation. the 26s proteasome, formed by association of the 20s catalytic core (composed of α and β subunits) with the 19s regulator, is responsible for degradation of the majority of proteins through a ubiquitin (ub)-and atp-dependent pathway [12] . additionally, the 20s proteasome, which is required for the ub-and atp-independent degradation of specific target substrates, is generated by a combination of one 20s catalytic core and one proteasomal activator 28 (pa28) member [13] . of the three pa28 family members, pa28γ (also called regγ, 11sγ, psme3, or ki antigen) is implicated in tumorigenesis because it regulates cell proliferation and apoptosis, and it predominantly exerts its function through nuclear proteolysis [14] . pa28γ has been known to target numerous intact proteins directly through proteasomal degradation. this establishes the function of pa28γ in a variety of biological processes with physiological and pathological relevance. in addition, pa28γ can also regulate some viruses such as the hcv core protein, hepatitis b virus x protein, and human immunodeficiency virus type 1 tat [15] . the nuclear retention and stability of pa28γ are regulated via a pa28γ-dependent pathway through which hcv pathogenesis may be exerted [16] . moreover, the hcv core protein can decrease 20s proteasome activity in the presence of pa28γ [17] . it has been proposed that the ubiquitin-proteasome system plays a critical role during various stages of the coronavirus infection cycle [18] . in turn, the proteasomal inhibitor mg132 strongly inhibits sars-cov replication by interfering with early steps of the viral life cycle [19] . however, the potential role of the ub-and atp-independent degradation pathway in the field of coronavirus research is unknown. a previous study found that the sars-cov n protein can interact with the host cell proteasome subunit p42, a subunit of the 26s proteasome [20] . the two activators, pa28 and 26s, can bind to the ub-and atp-independent 20s proteasome simultaneously [21] . thus, there may be a connection between the sars-cov n protein and pa28. owing to over 90% amino acid sequence similarity between sars-cov n protein and the sars-cov-2 n protein [22] , the latter ncov n protein may be presumed to play the same role in this process. the precise role of pa28γ in the degradation of coronavirus proteins is still unclear. in the present study, we found that the n protein of sars-cov-2 could be degraded by pa28γ in vitro. this may indicate that pa28γ is a regulator for sars-cov-2 n protein degradation. we also investigated the interaction between sars-cov-2 n protein and the pa28γ-20s proteasome system through a co-immunoprecipitation assay. this new finding provides a clue for understanding the previously unresolved physiological roles of the proteasome-dependent degradation of the sars-cov-2 n protein during pathogenesis of covid-19. plasmid flag-pa28γ, frt-pa28 γ , and frt-pa28 γ n151y were previously generated [23] . ha-2019-ncov-n was generated by polymerase chain reaction the 293t cells were purchased from atcc; the 293t (pa28γ-knockout) cell was constructed by talent. cells were cultured in dulbecco's modified eagle's medium. fetal bovine serum 10% and antibiotics were added to the media. the 293t cells (pa28γ wt and pa28γ knockout) were transiently transfected with the plasmid ha-psg5-ncov-n, and lipofectamine 2000 transfection reagent (invitrogen) was used depending on the manufacturer's instructions. the pa28γ and 20s proteasome proteins were purified well [24] . the sars-cov-2 n protein was created by in vitro translation using the tnt kit (promega). the day before the experiment, the cells were inserted into 60-mm dishes covered with sterile slides and maintained at 37 °c in 5% co2 until they were attach to the plate about 24 h later. the cells were then fixed using 4% paraformaldehyde for 10 min at room temperature. after washing with pbs. again, cells were blocked using 4% serum for 1 h. next, anti-ha was added and the cells were incubated at 4 °c overnight. subsequently, cells were incubated with fluorescent secondary antibody for 1 h at room temperature. cell images were visualized using a fluorescence microscope. 8 cells were collected and lysed on ice using the following lysis buffer. the 10% cell lysate was collected to be the input of co-ip assay. the previous studies have demonstrated that pa28γ can bind to the core protein of hcv and promote its degradation [11] . thus, to investigate whether exogenous pa28γ can interact with ncov n, we carried out a co-ip assay. the data support an interaction between pa28γ and ncov n. both plasmids flag-pa28γ and ha-ncov-n were transfected into 293t cells. pa28γ was immunoprecipitated with an anti-flag antibody (fig.1a) . ncov n could easily be observed in the immunoprecipitate by western blot analysis. similarly, pa28γ was readily detected using an anti-ha antibody (fig.1b) . our results demonstrate that exogenous pa28γ can physically interact with ncov n. next we determined whether endogenous pa28γ plays a role in the degradation of ncov n. the 293t cells (pa28γ-wt and pa28γ-ko) were transfected with the plasmid ha-psg5-ncov n and then treated with cycloheximide (chx) for the indicated times. we found that after 12 h, the expression of ncov n was higher in pa28γ-ko cells than in wt cells (fig.1c) , indicating that the protein was more stable in the absence of pa28γ in the cells. moreover, the stability of ncov n protein in pa28γ-wt was lower than that in the pa28γ-ko cell line over time (fig.1d) . taken together, the data show that endogenous pa28γ may have an effect on the degradation of ncov n protein through interaction. ev-psg5+pa28γ, n151y, and ha-ncov+ev-frt were transfected into cells as negative controls for comparison. as expected, the level of ncov n was significantly reduced when exogenous pa28γ was normally expressed in the cell. in addition, when the inactive mutant n151y pa28γ was transfected into the cells with ha-ncov, we found that the level of ncov n remained relatively equal to that of the control (fig.2b, c and d) . this confirmed that the degradation of ncov n could be mediated by pa28γ. we further investigated the effectiveness of the expression of pa28γ on the degradation of ncov n by adding an equal amount of egfp expression plasmid to detect the expression efficiency. we found that the increased expression level of pa28γ resulted in a marked reduction in ncov n protein in a dose dependent manner ( fig.2a) , confirming that it was the pa28γ that promotes the degradation of ncov n and its effectiveness is related to dosage. to more intuitively reflect the promoting effect of pa28γ on ncov n degradation, immunofluorescence staining assay was used. the same amount of ha-ncov-n was transfected into 293t cells lines (pa28γ-wt and pa28γ-ko). the expression level of ncov n protein (red) in the pa28γ-ko cell lines was observed to be prominently higher than that in the pa28γ-wt cell line ( fig.3c and d) , confirming that pa28γ deficiency promotes ncov n in the cell line. we then investigated the cellular localization and expression of ncov n in 293t cells under the regulation of pa28γ by immunofluorescence staining. the same amount of ncov n was transfected into 293t cell lines (pa28γ-wt and pa28γ-ko). in the pa28γ-wt cell lines, the ncov n protein (red) was prominently expressed in the cytoplasm, while in the pa28γ-ko cell lines it existed both in the nucleus and the cytoplasm (fig.3a ). in addition, the total expression of ncov n was quantified and, as expected, pa28γ-wt cell lines exhibited a decrease in ncov n (fig.3b) . pa28γ has been known to express in the nucleus [11] . therefore, our results indicate that the ncov n in the nucleus was degraded under the regulation of pa28γ, while that in the cytoplasm was not. 3.6. cartoon depicting the ncov n protein degradation process through the pa28γ-20s system a schematic diagram of the degradation process shows that pa28γ first binds with ncov n and transports it to the 20s proteasome subunit where internal degradation occurs (fig.4) . thus, the time of existence of ncov n in the nucleus is much shorter compared to that in the cytoplasm. an increasing number of studies have shown that the proteasome is associated with viral infection, and there is evidence that hcv core proteins can be degraded through the pa28γ-20s system. that is, the nuclear retention and stability of hcv core proteins are regulated by pa28γ-dependent pathways such that the pathogenicity of hcv may be achieved through this pathway [16] . in addition, the human immunodeficiency virus-1 (hiv-1) tat protein can inhibit the peptidase activity of the 20s proteasome by competing with the 11s/pa28 regulator (reg) for binding at the reg/tat-proteasome-binding (rtp) site and interfering with antigen processing [25] . it has been shown that the hepatitis b virus x protein-derived polypeptide, harboring the α4 proteasome subunit binding motif, impairs the activation of 20s proteasomes by pa28 [26] . the coxsackievirus infection can be enhanced by proteasome activator pa28γ promoting p53 degradation [27] , similar to the mechanism of degradation observed in the hbx virus [28] . furthermore, protein p30 interactions with pa28γ may also affect atm functions and increase cell survival [29] . on the other hand, pa28γ acts as a co-repressor of htlv-1 p30 to suppress virus replication and is required for the maintenance of control viral latency [30] . therefore, previous studies have shown that pa28γ is closely related to the htlv-1 virus and plays an indelible role in the formation and spread of the virus. additionally, the ability of pa28γ to promote viral protein degradation suggests its involvement in viral pathogenesis [31] . studies also have demonstrated that the novel coronavirus nucleocapsid protein (n) shares nearly 90% amino acid sequence homology with sars coronavirus. sars coronavirus n protein antibodies can cross-react with novel coronavirus, but cannot provide cross-immunity. similar to sars-cov, ncov n proteins can inhibit rna interference (rnai) to overcome the host defense [32] . early research revealed the involvement of the ubiquitin-proteasome system (ups) in multiple steps of the coronavirus infection cycle and identified ups as a potential drug target to modulate the significance of coronavirus infection [18] . in the present study, we found that a novel ubiquitination-independent pathway could regulate the protein levels of ncov n, and pa28γ could control the abundance of ncov n by regulating its stability. in addition, we showed that pa28γ interacts with ncov n and promotes its intracellular degradation. sars coronavirus nucleocapsid protein is a very important viral structural protein and an important indicator in early diagnosis due to its abundance and high conservation in cells. studying nucleocapsid protein structural function, how it participates in transcription and translation, the molecular mechanism in virus-infected cells, and the regulation of gene expression will help to thoroughly understand sars coronaviruses and find effective methods for prevention and treatment of related diseases [6] . when sars-cov-2 invades the human body, it makes contact with human immune cells, thus causing the immune cells to produce massive ifn-γ, and ifn-γ can induce pa28γ [32] . this in turn stimulates proteasome activity resulting in degradation of the coronavirus n protein. as a result, virus production is blocked and proliferation and diffusion are greatly amelioraten. in conclusion, our study substantiates that pa28γ could mediate the degradation of there is no conflict of interest. this work was supported by shanghai committee of science and technology. (no. 16dz2348900). (a) cells were transiently transfected with ha-ncov-n, fixed, and immunostained with anti-ha (red color). cell nuclei were stained with dapi (blue color). scale bar: 50 μm. (b) quantification analysis of ncov n expression as a percentage per cell of three independent experiments (n = 100; values represent the mean ± sem; *p < 0.05; **p < 0.01 versus control). (c) cells were transiently transfected with the same amount of ha-ncov-n plasmid, fixed, immune-stained with anti-ha (red color), and stained with dapi (blue color). scale bar: 100 μm. (d) quantification analysis of ncov n expression as a percentage in the same cell area. each data set represents three independent experiments (n = 300;. values represent the mean ± sem; *p < 0.05; **p < 0.01 versus control). who. coronavirus disease 2019 (covid-19): situation report-132 a novel coronavirus from patients with pneumonia in china from sars to mers, thrusting coronaviruses into the spotlight origin and evolution of pathogenic coronaviruses genome composition and divergence of the novel coronavirus (2019-ncov) originating in china the sars coronavirus nucleocapsid protein -forms and functions sars-cov-2-encoded nucleocapsid protein acts as a viral suppressor of rna interference in cells. sci china life sci the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks s phase progression in mammalian cells identification of a novel linear b-cell epitope on the nucleocapsid protein of porcine deltacoronavirus nuclear/nucleolar localization properties of c-terminal nucleocapsid protein of sars coronavirus proteasomal turnover of hepatitis c virus core protein is regulated by two distinct mechanisms ubiquitin-independent but pa28 gamma-dependent mechanism structure and functions of the 20s and 26s proteasomes identification, purification, and characterization of a protein activator (pa28) of the 20 s proteasome (macropain) arginine methylation increases the stability of human immunodeficiency virus type 1 tat proteasome activator pa28gamma-dependent nuclear retention and degradation of hepatitis c virus core protein n-terminal hcv core protein fragment decreases 20s proteasome activity in the presence of pa28gamma the ubiquitin-proteasome system plays an important role during various stages of the coronavirus infection cycle severe acute respiratory syndrome coronavirus replication is severely impaired by mg132 due to proteasome-independent inhibition of m-calpain interactions of sars coronavirus nucleocapsid protein with the host cell proteasome subunit p42 simultaneous binding of pa28 and pa700 activators to 20 s proteasomes covid-19 (novel coronavirus 2019) -recent trends reggamma contributes to regulation of hemoglobin and hemoglobin delta subunit purification procedures determine the proteasome activation properties of reg gamma (pa28 gamma) the rtp site shared by the hiv-1 tat protein and the 11 s regulator subunit alpha is crucial for their effects on proteasome function including antigen processing hepatitis b virus hbx peptide 116-138 and proteasome activator pa28 compete for binding to the proteasome alpha4/mc6 subunit proteasome activator reggamma enhances coxsackieviral infection by facilitating p53 degradation hepatitis b virus x protein activates proteasomal activator 28 gamma expression via upregulation of p53 levels to stimulate virus replication human t-lymphotropic virus type 1 p30 interacts with reggamma and modulates atm (ataxia telangiectasia mutated) to promote cell survival pa28gamma is a novel corepressor of htlv-1 replication and controls viral latency overexpression of the proteasome subunits lmp2, lmp7, and mecl-1, but not pa28 alpha/beta, enhances the presentation of an immunodominant lymphocytic choriomeningitis virus t cell epitope pa28γ can regulate sars-cov-2 nucleocapsid protein. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord-327744-5k8np850 authors: munteanu, cristian robert; magalhães, alexandre l.; uriarte, eugenio; gonzález-díaz, humberto title: multi-target qpdr classification model for human breast and colon cancer-related proteins using star graph topological indices date: 2009-03-21 journal: journal of theoretical biology doi: 10.1016/j.jtbi.2008.11.017 sha: doc_id: 327744 cord_uid: 5k8np850 abstract the cancer diagnostic is a complex process and, sometimes, the specific markers can interfere or produce negative results. thus, new simple and fast theoretical models are required. one option is the complex network graphs theory that permits us to describe any real system, from the small molecules to the complex genetic, neural or social networks by transforming real properties in topological indices. this work converts the protein primary structure data in specific randic's star networks topological indices using the new sequence to star networks (s2snet) application. a set of 1054 proteins were selected from previous works and contains proteins related or not with two types of cancer, human breast cancer (hbc) and human colon cancer (hcc). the general discriminant analysis method generates an input-coded multi-target classification model with the training/predicting set accuracies of 90.0% for the forward stepwise model type. in addition, a protein subset was modified by single amino acid mutations with higher log-odds pam250 values and tested with the new classification if can be related with hbc or hcc. in conclusion, we shown that, using simple input data such is the primary protein sequence and the simples linear analysis, it is possible to obtain accurate classification models that can predict if a new protein related with two types of cancer. these results promote the use of the s2snet in clinical proteomics. cancer is a leading cause of death worldwide, accounted for around 13% of all deaths in 2007 (who, 2008) . two of the leading types of cancer are the human breast cancer (hbc) and the human colon cancer (hcc). the estimated new cancer cases and deaths in us for 2008 shows that hbc will affect 26% of the women (15% will die) and hcc will involve 10% of the men/women (8% men and 9% women will die) (jemal et al., 2008) . therefore, simple and fast theoretical method can be very useful in the detection of cancer diseases. the actual work will use the protein quantitative proteomedisease relationship (qpdr) (ferino et al., 2008) , similar to quantitative structure-activity relationship (qsar) (devillers and balaban, 1999) . qpdr is one of the widely used analyse for predicting the protein properties and, in the present study, is using the macromolecular descriptors, named topological indices (tis), obtained with the graph theory. the branch of mathematical chemistry dedicated to encode the dna/protein information in graph representations by the use of the tis has become an intense research area (agü ero-chapin et al., 2006; bielinska-waz et al., 2007; liao and wang, 2004; liao and ding, 2005; randic, 2000; randic and basak, 2001; randic and balaban, 2003; randic et al., 2000) . the graphic approaches of the biological systems study can provide useful insights in qsar studies (gonzá lez-díaz et al., 2006 , 2007c prado-prado et al., 2008) , protein folding kinetics (chou, 1990) , enzyme-catalyzed reactions (chou, 1989; chou and forsen, 1980; chou and liu, 1981; kuzmic et al., 1992) , inhibition kinetics of processive nucleic acid polymerases and nucleases (althaus et al., 1993a, b; althaus et al., 1994 althaus et al., , 1996 chou et al., 1994) , dna sequence analysis (qi et al., 2007) , anti-sense strands base frequencies , analysis of codon usage (chou and zhang, 1992; zhang and chou, 1994) and in complicated network systems investigations (diao et al., 2007; gonzalez-diaz et al., 2007a , 2008 . recently, the ''cellular automaton image'' (wolfram, 1984 (wolfram, , 2002 has also been applied to study hepatitis b viral infections (xiao et al., 2006a) , hbv virus gene missense mutation (xiao et al., 2005b) , and visual analysis of sars-cov (gao et al., 2006; wang et al., 2005) , as well as representing complicated biological sequences (xiao et al., 2005a) and helping to identify protein attributes (xiao and chou, 2007; xiao et al., 2006b) . we have chosen the tis for these qpdr models based on the previous work results with similar qsar/qpdr models. even if the tis cannot be always interpreted, they demonstrate to encode the information that permits to create accurate qsar/qpdr models. other interesting fields to apply the graph theory are the oncology and clinical proteomics. a classification model for discriminating prostate cancer patients from control group with connectivity indices where constructed by gonzá lez-díaz et al. (2007b) . vilar's group designed a qsar model for alignment-free prediction of hbc biomarkers based on electrostatic potentials of protein pseudofolding hp-lattice networks (vilar et al., 2008) . the actual work is proposing a new cancer/non-cancer classification model based on protein embedded/non-embedded star graph tis such are the trace of connectivity matrices, harary number, wiener index, gutman index, schultz index, moreau-broto indices, balaban distance connectivity index, kier-hall connectivity indices and randic connectivity index. this classification can predict two types of cancer: hbc and hcc. the primary protein sequence is transformed in connectivity star graph's tis that are used by a statistical linear method in order to construct an input-coded multi-target classification model. two sets of protein primary sequences are used: a set of 189 hbc/hcc cancer proteins (sjoblom et al., 2006) and 865 noncancer proteins (dobson and doig, 2005; dobson et al., 2004) . the list of cancer-related proteins in our work is the same with the list obtained by the sjoblom group after the experimental analysis of 13,023 genes in 11 breast and 11 colorectal cancers. each protein sequence was transformed in a star graph, where the amino acids are the vertices (nodes), connected in a specific sequence by the peptide bonds. the star graph is a special case of trees with n vertices where one has got nà1 degrees of freedom and the remaining nà1 vertices have got one single degree of freedom (harary, 1969) . each of the 20 possible branches (''rays'') of the star contains the same amino acid type and the star centre is a non-amino acid vertex. a protein can be represented by diverse forms of graphs, which can be associated with distinct distance matrices. the best method to construct a standard star graph is the following: each amino acid/vertex holds the position in the original sequence and the branches are labelled by alphabetical order of the 3-letter amino acid code (randic et al., 2007) . the graph is embedded if the initial sequence connectivity in the protein chain is included. figs. 1a and b present the non-embedded/embedded star graphs of prps1 using the alphabetical order of one-letter amino acid code. thus, the primary structure of protein chains are transformed in the correspondent star graphs invariant tis. the resulted graphs are not depending on the three-dimensional structure or the shape of the protein. the comparison of the graphs is made by using the corresponding connectivity matrix, distance matrix and degree matrix. the matrices of the connectivity in the sequence and in the star graph are combined in the case of the embedded graph. these matrices and the normalized ones are the base of the tis calculation. the conversion of the amino acid sequences in star graph tis was made by using sequence to star networks (s2snet) application, developed by our group (munteanu and gonzá les-diá z, 2008) . s2snet is based on wxpython (rappin and dunn, 2006) for the gui application and has graphviz (koutsofios and north, 1993 ) as a graphics back-end. the present calculations are characterized by embedded and non-embedded tis, no weights, markov normalization and power of matrices/indices (n) up to 5. the results file contains the following tis (todeschini and consonni, 2002) : trace of the n connectivity matrices (tr n ) or the spectral moments: (1) where n ¼ 0-power limit, randic connectivity index ( 1 x): these tis and other derivate ones will be used in the next step to construct a cancer/non-cancer classification model by linear statistical methods. an input-coded multi-target classification model was created with general discriminant analysis (gda) method (kowalski and wold, 1982 ; van waterbeemd, 1995), statistica 6.0 package (statsoft.inc., 2002). this model can predict if a protein is hbc or hcc-related using a single equation. for this reason, in addition to the 30 star graph embedded and non-embedded tis are introduced other two types of continuous predictors (attributes) encoded specific information about each cancer types as following: 30 products of the hbc/hcc cancer probability with the embedded/non-embedded tis (pti ¼ prob hbc/hcc *ti) and 30 differences between the same tis and the average of the tis for each type of cancer [dti ¼ ti-average(ti) hbc/hcc ]. the cancer probabilities represent the fractions of proteins hbc/hcc-related from the entire sjö blom's proteins (cancer proteins) and have values of 0.639 (hbc) and 0.361 (hcc). for each protein there are two cases corresponding to both types of cancer. the dependent variable (cancerornot) takes 1 for cancer and 0 for non-cancer and the cross-validation (cv) variable has two values (train and val). the best cv methods to examine a predictor are the following: independent dataset test, subsampling test, and jackknife test (chou and zhang, 1995) . shen (2007, 2008) have shown that only the jackknife test has the least arbitrariness . thus, the jackknife test has been increasingly used by investigators to examine the accuracy of various predictors (chen and li, 2007a, b; diao et al., 2007; ding et al., 2007; jiang et al., 2008; li and li, 2008; lin, 2008; niu et al., 2006; xiao and chou, 2007; zhang et al., 2008; zhou et al., 2007) . in the actual work, the independent data test is used by splitting the data at random in a training series (train, 75%) used for model construction and a prediction one (val, 25%) for model validation (the cv column is filled by repeating 6 train and 2 val). all independent variables are standardized prior to model construction. the general qpdr formula contains embedded and nonembedded tis, ptis and dtis: where c/ncàscore is the continue score value for the cancer/noncancer classification (hbc or hcc), c 1 -c n are the tis coefficients (n ¼ number of tis), c n -c m ptis coefficients (nom; m-n ¼ number of ptis), c m -c 0 dtis coefficients (mo0; 0-m ¼ number of dtis) and c 0 is the independent term. we inspected the percentage of good classification and the number of variables to be explored in order to avoid over-fitting or chance correlation. the forward model type was tested for the embedded, non-embedded and both data, including tis, ptis, dtis and all indices. in addition, the dobson's set is use to select a subset of 61 noncancer proteins with cancer probability between 0.3 and 0.5 in order to proceed 17 single amino acid mutations with log-odds pam250 (dayhoff, 1978) greater or equal with 2 (see table 1 ). the best classification model predicted the probability of presence in hbc/hcc cancer for any of these mutated proteins and the results were analysed with two-way joining clustering analysis method (tw-jca) from statistica (statsoft.inc., 2002). fifteen classification models were tested with the aim of finding the best gda equation which is able to discriminate between proteins related with hbc and hcc. the attributes include 30 embedded/non-embedded star graph tis obtained with s2snet application and other 60 composed predictors, ptis and dtis. the values obtained for the training/predicting accuracies with the forward stepwise method are presented in table 2 . the forward stepwise selection variable method conjugated with the embedded tis and dtis provides the best results for our data set with values of correctly classified compounds of 89.9%, 90.3% and 90.0% for the training, cv and full sets, respectively, and using only six/five parameters/variables (eq. (14)). the embedded tis have the name of the non-embedded ones plus ''e'' as suffix. the simple linear mathematical form of the model has been chosen in the absence of prior information. c=nc2score ¼ à 4:4 þ 1:7 ã tr 3e þ 124:8 ã se à 126:5 ã dje þ 48:6 ã dx2e à 45:9 ã x5e (14) where n is the number of cases (c and nc), r c is the canonical regression coefficient, u is the wilk's statistics, f is the fisher's statistics and p is the p-level (probability of error). the above results are typically considered as excellent in the literature for lda-qpdr/qsar models (castillo-garit et al., 2008; estrada and molina, 2001; marrero-ponce et al., 2004; morales et al., 2006; vilar et al., 2008) . in order to check the variation of this model with the training/cv sets, we carried on a cv study by using ten totally random sets, including the initial one from the actual model (with the same 75% training and 25% cv). the classification values are presented in table s1 from the supple-mentary material and show an average of 90.2% for training and 89.2% for cv. these values demonstrate the stability of the model with the selection of the classification sets. in order to illustrate the performance of the approach when applied to a single set of cancer related proteins (e.g. either breast or colon), we obtained two equations, one for hbc and other for hcc. therefore, we have to consider that the eq. (14) represents an input-coded multi-target classification model that can evaluate if a protein is hbc or hcc-related by using the hbc or hcc average je and x2e values (contained in the dje and dx2e differences). eq. (14) can be reduced to two different equations, one for each type of cancer (hbc and hcc): hcc=nhcc2score ¼ à 20:8 þ 1:7 ã tr 3e þ 124:8 ã se2je þ 0:2 ã x2e à 45:9 ã x5e. the detailed classification results for each type of cancer obtained with eqs. (14a), (14b) are presented in table 3 . a similar input-coded multi-target classification model was obtained by using the forward stepwise method and the embedded ptis and provides values of correctly classified compounds of 90.3, 91.0 and 90.5 for the training, cv and full sets, respectively (using seven/six parameters/variables) (eq. (15)). c=nc2score ¼ à 4:1 à 118:6 ã p tr 0e þ 80:7 ã p tr 2e þ 1:4 ã p tr 3e þ 100:3 ã pse à 101:4 ã pje þ 39:7 ã px2e, in order to evaluate if a protein is hbc or hcc-related, it is necessary to use the hbc or hcc probability inside the ptis products. the classification values obtained for the individual equations are presented in table 3 . the equations obtained are the following: hbc=nhbc2score ¼ à 5:6 à 0:3 ã tr 0e þ 0:8 ã tr 2e þ 0:6 ã tr 3e þ 0:2 ã x2e (15a) table 1 single amino acid mutations and the corresponding log-odd pam250 value.. original aa mutated aa log-odd pam250 notation table 2 training/predicting accuracies of cancer (c)/non-cancer (nc) models using embedded (e) and non-embedded (ne) star graph tis, ptis and dtis.. cross-validation total eq. vars. hcc=nhcc2score ¼ à 5:6 à 0:2 ã tr 0e þ 0:5 ã tr 2e eqs. (14), (15) show similar results when the input data is containing probability of cancer (products with tis) or the tis averages for each type of cancer (differences with tis). in general, in the case of embedded, non-embedded and both indices, we obtained better results with dtis compared with the ptis (not mixed with the original tis). this difference can be explained by a superior recover of the cancer-related protein sequence information in the case of the differences between the original tis and the average of them for each type of cancer (dtis) compared with the products of the original tis and the cancer type probability (ptis). thus, we can conclude that the average of star graph structure for each type of cancer (dtis) is described better the actual qpdr model compared with the composition of the data sets for each type of cancer that generates the cancer probabilities. in addition, table 2 shows that better results are obtained using the original tis and the derived ones (ptis and dtis) compared with the isolated tis/ptis/dtis. this difference can be explained be the fact that each set of indices can contains different parts of the protein information that is cancer-related. therefore, the use of all these indices will sum all this information in a better qpdr model. another interesting aspect is the type of the indices (original or derived from the original) that are more frequent in all models presented in table s2 from supplementary material. thus, we can observe the importance of the wiener index (w) and kier-hall connectivity index x5 for the models based on the non-embedded tis. the embedded tis models contain more frequent the trace of the graph/sequence connectivity matrixes tr 3 and the non-trivial part of the schultz ti s (w is based on the distance matrix, x5 and s on the degree matrix, and tr 3 on the connectivity matrix). the most important type of index that is present in both embedded and non-embedded ti equations is j, the balaban distance connectivity index based on the node distance degree information. in order to compare two equations with the same number of table 3 accuracy of input-coded multi-target and individual hbc and hcc classification models based on the embedded tis (tie+dtie and ptie).. cancer tis, we have chosen the embedded models with ptie and {tie, ptie} that contain six variables and reduced the common terms (based on tr 3, s and j). thus, we can observe that the addition of the tie to the ptie will shift the preference from the low order traces (p tr 0e, p tr 2e) and kier-hall index (px2e) to high order trace (tr 5e), harary number (he) and gutman ti (s6e). the first embedded tis & dtis model was chosen to estimate the cancer probability for proteins mutants of non-cancer-related proteins. these values were analysed with tw-jca using 61 mutated proteins and 17 types of single amino acid mutations. in the case of hbc, we obtained 215 data groups, called input blocks. to detect the larger variability regions (mutants) we computed a tw-jca partition of input blocks (rearrange of blocks) setting the threshold value of variability at stdv/2 (see fig. 2 ). the value obtained was 0.059. the 215 input blocks are regrouped, for similarity, into 11 output blocks (see tables s3 and s4 in the supplementary material). we can observe that the proteins with number 24 to number 48 are very susceptible to become hbcrelated proteins for all studied mutations. the plot corresponding to the reduced values of the reordered data matrix (table s4) presented in fig. 3 . on the other hand, we carried out the same study for the hcc mutated proteins and found different susceptible proteins, with visible lower probability to be hccrelated (fig. 4) . the 184 input blocks were regrouped, for similarity, into 11 output blocks (stdv/2 ¼ 0.050) (see tables s5 and s6 in the supplementary material). the reduced data from table s6 are presented as a plot in fig. 5 . the tw-jca partition obtained in this way is statistically significant as reported by other authors that used this method to reach similar goals (ferino et al., 2008) . one interesting non-cancer chain protein is 1qrk b, the human coagulation factor xiii with strontium bound in the ion site (fox et al., 1999) , with eight single amino acid mutations that present hbc probability up to 71% as following: 70.8% for v-l, 68.8% for v-i, 62.0% for l-i, 59.3% for d-n, 58.3% for e-q, 55.9% for f-l, 54.8% for e-d and 51.0% for v-m. the most persistent mutation (log-odd pam250 ¼ 4), valine (v) to isoleucine (m), can be considered as the most dangerous one. the main calcium/ strontium binding site within each monomer involves the main chain oxygen atom of ala-457, and also the side chains from article in press fig. 6 . graphical representation of two-way joining cluster analysis of the probability of the mutated hbc-related proteins to turn into non-cancer proteins. fig. 7 . graphical representation of two-way joining cluster analysis of the probability of the mutated hcc-related proteins to turn into non-cancer proteins. residues . the mutations of glu (e) in q and d can affect the capacity of binding metals and the normal biological activity. this coagulation factor xiii is a transglutaminase which stabilizes blood clots by covalently crosslinking fibrin, being essential for normal haemostasis. fxiii deficiency due to the genetic mutations results in a life-long bleeding disorder with added complications in wound healing and tissue repair (anwar et al., 1998) . in addition, the abundant fibrinogen present in the tumor connective tissue might contribute to the structural integrity of breast or colon tumor tissues (costantini et al., 1991; takahashi et al., 2000; yee et al., 1994) . we can observe that, in general, the natural mutations with higher pam250 values are less frequent even for 1qrk b (y-f with pam250 of 7 is absent) because we cannot create a direct relation between the pam250 natural amino acid mutation frequency and the influence of the mutations in these types of cancer. the probability for a cancer-related protein to turn into a noncancer one was studied too. for each type of cancer, ten hbc/hccrelated proteins where mutated using the same pam250 values. the tw-jca plots are presented in fig. 6 (for hbc) and fig. 7 (for hcc), and correspond to data in tables s7 and s8 from the supplementary material. the results did not show important probability to obtain a hbc/hcc-related protein by using single pam250 natural mutations. activin beta e (inhbe, c_5) has the highest probability (around 50%) to turn into a hbc-related protein after almost all the mutations ( fig. 6 and table s7 ). this study is proposing two cancer/non-cancer input-coded multi-target classification models for hbc and hcc using the star network tis of the protein amino acid sequences. the results prove the excellent predictive ability (90.0%) of the simple and fast star network tis and gda statistics linear models in the case of the actual protein model. in addition, the prediction of cancer probability for mutated proteins was calculated. the human coagulation factor xiii (1qrk b), that normally do not generate hbc, if suffer several mutations, can become a hbc-related protein. this work can help in oncology proteomics or serve as a model for other studies. in addition, s2snet application is demonstrating his capacity to transform simple protein sequences in tis and to be the base of numerous protein studies. novel 2d maps and coupling numbers for protein sequences. the first qsar study of polygalacturonases; isolation and prediction of a novel sequence from psidium guajava l steady-state kinetic studies with the non-nucleoside hiv-1 reverse transcriptase inhibitor u-87201e kinetic studies with the nonnucleoside hiv-1 reverse transcriptase inhibitor u-88204e steady-state kinetic studies with the polysulfonate u-9843, an hiv reverse transcriptase inhibitor the benzylthio-pyrimidine u-31, 355, a 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sequences an application of gene comparative image for predicting the effect on replication ratio by hbv virus gene missense mutation a probability cellular automaton model for hepatitis b viral infections using cellular automata images and pseudo amino acid composition to predict protein subcellular location three-dimensional structure of a transglutaminase: human blood coagulation factor xiii analysis of codon usage in 1562 e. coli protein coding sequences prediction protein structural classes with pseudo-amino acid composition: approximate entropy and hydrophobicity pattern using chou's amphiphilic pseudoamino acid composition and support vector machine for prediction of enzyme subfamily classes supplementary data associated with this article can be found in the online version at doi:10.1016/j.jtbi.2008.11.017. key: cord-352481-iq3wor3w authors: postic, guillaume; janel, nathalie; tufféry, pierre; moroy, gautier title: an information gain-based approach for evaluating protein structure models date: 2020-08-18 journal: comput struct biotechnol j doi: 10.1016/j.csbj.2020.08.013 sha: doc_id: 352481 cord_uid: iq3wor3w for three decades now, knowledge-based scoring functions that operate through the “potential of mean force” (pmf) approach have continuously proven useful for studying protein structures. although these statistical potentials are not to be confused with their physics-based counterparts of the same name—i.e. pmfs obtained by molecular dynamics simulations—their particular success in assessing the native-like character of protein structure predictions has lead authors to consider the computed scores as approximations of the free energy. however, this physical justification is a matter of controversy since the beginning. alternative interpretations based on bayes’ theorem have been proposed, but the misleading formalism that invokes the inverse boltzmann law remains recurrent in the literature. in this article, we present a conceptually new method for ranking protein structure models by quality, which is (i) independent of any physics-based explanation and (ii) relevant to statistics and to a general definition of information gain. the theoretical development described in this study provides new insights into how statistical pmfs work, in comparison with our approach. to prove the concept, we have built interatomic distance-dependent scoring functions, based on the former and new equations, and compared their performance on an independent benchmark of 60,000 protein structures. the results demonstrate that our new formalism outperforms statistical pmfs in evaluating the quality of protein structural decoys. therefore, this original type of score offers a possibility to improve the success of statistical pmfs in the various fields of structural biology where they are applied. the open-source code is available for download at https://gitlab.rpbs.univ-paris-diderot.fr/src/ig-score. predicting the three-dimensional structure of a protein is only useful if the model produced is close enough to the native conformation of the macromolecule. according to anfinsen's hypothesis, the latter is assumed to be the one with the lowest free energy, in the native conditions [1] . therefore, being able to discriminate the best model among a set of predicted protein structures requires a scoring function that would behave like free energy, i.e. a scoring function whose global minimum would correspond to the native conformation. free energy estimation may be achieved by generating ensembles of protein conformations, from which the lowest free energy structure can be calculated by using physics-inspired molecular force fields. however, such conformational sampling is computationally costly, which makes these physics-based methods only applicable to a few proteins at a time. three decades ago, a faster approach has been proposed by m. j. sippl [2] , which consists in constructing scoring functions from interatomic distance (r) distributions observed in a dataset of experimentally determined protein structures, as: where ūi,j(r) is the estimated free energy of interaction between atoms i and j, fi,j obs (r) is the observed probability (i.e. frequency) of the atoms i and j being separated by a distance r (discretized into bins), fi,j ref (r) is a reference frequency aimed at eliminating the sampling bias, k the boltzmann constant, and t the temperature. the pseudo-energy of the whole protein is thus computed by summing the ūi,j(r) of every pairwise distance observed in the structure. in this article, for the 30 th anniversary of this knowledge-based approach, we present a new information-theoretic view of its functioning, and propose an improvement in both theory and practice. since 1990 [2] , these distance-dependent statistical potentials-also called "potentials of mean force" (pmf) by analogy with the potentials used in the physics of liquids [3, 4] -have been continuously applied to model quality assessment, as well as to various problems in structural biology, mainly ab initio protein folding [5] [6] [7] [8] [9] [10] [11] , molecular docking [12, 13] , and fold recognition [14] [15] [16] . in addition to interatomic distances, other structural features of proteins have been used, such as dihedral angle values, or solvent accessibility. then, with the emergence of machine learning approaches, such scores resulting from the statistics of various structural descriptors have been combined into composite scoring functions [17] [18] [19] [20] [21] . most recently, statistical potentials have drawn attention by their use in a deep learning-based approach for predicting protein structures [22] . the relatively good correlation of these scores with the free energy variation of protein folding, as well as the presence of a logarithm in the formula, have lead authors to describe this approach as resulting from the inverse boltzmann law. however, this physical explanation has been criticized and demonstrated as being invalid on several points [23, 24] , notably the fact that the atomic system of a polypeptide chain is not fairly comparable to that of a liquid. moreover, computing both fi,j obs (r) and fi,j ref (r) on native conformations does not allow interpreting the score as a free energy variation between the unfolded and the folded states. another point concerns the kt factor, which is (most often) not taken into account and replaced by an arbitrary value, thus further invalidating the physical interpretation. in 1997, baker and co-workers qualitatively showed that statistical potentials should actually be seen as an application of bayes' theorem to the conditional probabilities of pairwise distances [7] . in this view, the fi,j obs (r)/fi,j ref (r) factor in eq. 1 is equivalent to the ratio of the posterior to prior probabilities p(r|i,j)/p(r)-where p(r|i,j) and p(r) are the probabilities of observing two atoms at a distance r, with and without the knowledge of the atom types (i and j), respectively-thus quantifying the bayesian updating. hamelryck and co-workers later proposed a quantitative explanation [25] [26] [27] , according to which statistical pmfs approximate jeffrey's conditioning (or probability kinematics) [28, 29] , an alternative updating rule. a consequence of this is the non-necessity for data in the training set to follow a boltzmann distribution. despite the pertinence of this probabilistic framework, the misleading justification based on physics is still recurrent in the literature (e.g. [30] [31] [32] [33] [34] ), presumably because it does not interfere with the practical success of statistical potentials. here, we propose a new formalism that is conceptually advantageous over the popular 30-year-old statistical potentials, as it is disconnected from any physical interpretation, while being more relevant to probabilistic reasoning. as a proof of concept, we have built two scoring functions, respectively based on the new and the pmf equations, and compared their performance at ranking predicted structures of proteins by their quality. using the reference dataset 3drobot (n=60,200 structures) [35] , we show that the scoring function built with our new formalism is more accurate than statistical pmfs, based on three types of performance evaluation. finally, in our theoretical development, we also propose an explanation of what this new score measures regarding information-defined here as the quantitative property that is incorporated into the statistical model to update the prior probability. there are two critical elements in eq. 1, the first being how the reference state fi,j ref (r) is defined. the most straightforward way to do so is to calculate fi,j ref (r) as the weighted arithmetic mean of all fi,j obs (r) [36] . it is actually the same calculation as for fi,j obs (r), except that the atom types are indistinct. formally, the reference state could thus be written as fx,x obs (r), where x is an atom of any type. throughout the years, various improvements have been brought to this approach (see [37] for a review and comparative test), for example by taking into account the radius of gyration of each native structures, as the size of the proteins included in the training dataset is an obvious bias for the resulting interatomic distance distributions. the other critical part in eq. 1 is the logarithm, as it is a source of confusion between statistical potentials and boltzmannian statistical mechanics. boltzmann's entropy formula can be derived from classical mechanics. the logarithm thus appears when applying the second law of thermodynamics to the hamiltonian of a model system made of a single particle moving in a u-shaped potential [38] . in statistical potentials, the logarithm was presumably introduced for computational convenience, as it maps multiplication into addition, and for facilitating interpretation of the results: e.g. log(fi,j obs (r)/fi,j ref (r)) takes the values +1 and −1, for frequency ratios 10/1 and 1/10, respectively. however, to the best of our knowledge, it has never been raised-in the context of structural biology-that eq. 1 is also equivalent to a calculation of a relative difference between fi,j obs (r) and fi,j ref (r). indeed, the statistical pmf formalism in eq. 1 can alternatively be written (with kt=1) as: where . the latter would then properly play its part as a reference. therefore, we propose here to change the pmf formalism, and compute the score of a whole protein structure as: in addition of being more statistically sound, this new formalism avoids any confusion with the boltzmann distribution law, as it does no longer contain any logarithm. another non-negligible advantage is that it does not require any "pseudo-count" calculation procedure. the latter is otherwise necessary, as the logarithm function is undefined for zero. beyond its theoretical advantages, the practical validity of this new formalism is demonstrated in the present article through an extensive benchmarking of model quality assessment. since the new formalism is disconnected from physics, the score produced can no longer be viewed as an approximation of the free energy, and one may wonder what property is measured here. in what follows, we propose an explanation of how our scoring function works. in a distance distribution obtained from native conformations, in which all atom types are indistinct, observing two atoms (belonging to residues separated by at least three positions) at a distance of 2 å can be thought surprising. however, this observation becomes less surprising, when considering only the subdistribution of the cysteine atoms, as these residues can form disulfide bonds. this decrease in the surprise is measured in both eq. 2 and eq. 3 by the frequency difference δf(r) = fi,j obs (r)−fi,j ref (r) = fi,j obs (r)−fx,x obs (r). this change in the observed probabilities actually quantifies the information gain provided by the knowledge of the residue type. the more the surprise decreases, the more negative δf(r) is, and the more native-like is the observed interaction. conversely, an increase in how surprising the observation is (δf(r) > 0) after knowing the residue type indicates a non-native interaction. to evaluate an entire protein model, all the δf(r) for all atom pairs in the structure have to be added. however, summing all the δf(r) requires distinguishing, for example, a 0.2−0.4 difference from a 0.7−0.9 one. this is achieved through a relative difference calculation, i.e. through dividing fi,j obs (r)−fi,j ref (r) by a reference. in the pmf formalism as written in eq. 2, this reference is the unnecesary logarithmic mean between fi,j obs (r) and fi,j ref (r). in eq. 3, we have simply replaced this logarithmic mean and used, instead, fi,j ref (r) as a reference. we named the calculated score "total information gain" (tig), which is expressed as a dimensionless quantity. here, we call attention to the fact that, independently of the probabilistic framework, the property quantified by sippl's pmfs should also be interpreted as an information gain (rather than a pseudoenergy), but only when restricting the definition of information to the shannon "surprisal". indeed, eq. 1 can be alternatively written (with kt=1) as: where ii,j obs (r) and ii,j ref (r) are the surprisals of observing two atoms i and j at a distance r, for the observed and reference distributions, respectively, and δii,j(r) is the corresponding information gain. thus, a total information gain is calculated by summing every δii,j(r) for every combination of atoms i and j found in the evaluated structural model. also of note is the fact that the variation of shannon entropy (i.e. the average amount of information) is simply obtained by dividing this total information gain by the total number of interatomic distances in the evaluated protein structure. however, this aspect will not be developed further in the present article. finally, to further demonstrate the irrelevance of the logarithmic mean in eq. 2, we have built two "mock" scoring functions, in which this mean is replaced by either the arithmetic mean of fi,j obs (r) and fi,j ref (r), or only the highest of the two frequencies. we refer to these scores as "mck1" and "mck2", respectively. formally, they are expressed as: and their accuracies have been measured in the benchmarking procedure described below. residues; (iii) to ensure independence from the benchmark dataset, we have eliminated the 56 protein chains that share more than 20% sequence identity with any of the 200 proteins from the 3drobot dataset [35] . this last step has been carried out using the standalone version of pisces [40] . the resulting list of 1,917 protein chains is available in the supplementary materials. to compare the formalisms of eq. 1 and eq. 3, we have trained two scoring functions, to which we will refer as "pmf" and "tig", respectively. for both, the reference state fi,j ref (r) has been calculated as the weighted arithmetic mean of all fi,j obs (r) [36] , using all-atom representation of the native structures. the interatomic distance distributions have been computed for distance bins of 0.5 å, and a distance cutoff of 15.0 å. the distances between atoms belonging to residues i and i+1, i+2, or i+3 have not been taken into account [41] . the frequencies have been obtained by using kernel density estimations, as implemented in the r standard library (version 3.2.3). the bandwidths of the gaussian kernels have been selected with the scott's rule-of-thumb [42] . the same procedure has been followed for the mck1 and mck2 scores. each scoring function has been assessed based on its ability to rank structural models by quality as measured by their tm-score [43] to the native structure, which takes values between 0 and 1 (the higher the tm-score, the higher the model quality). as a benchmark, we used the 60,200 structures from the 3drobot dataset [35] , which represents 200 non-homologous proteins (48 α-, 40 β-, and 112 α/β-single-domain structures), each having 300 decoys and 1 native conformation. additionally, we used predicted protein structures from the casp13 experiment (2018). we selected models corresponding to targets in both the template-based modeling and free modeling categories, taking every model produced by every group. this represents a total of 52,296 models from 133 targets. each scoring function was evaluated through a pairwise ranking of the decoys for each of the 200 proteins from 3drobot, or each of the 133 casp13 targets. this allowed to calculate the accuracy of each method as the proportion (in %) of correct pairwise rankings. as the difficulty of ranking models may vary depending on their qualities, four subsets of the 3drobot and casp13 datasets have been defined, based on the tm-score to the native: "near-native", "good", "medium", and "poor" quality models are defined by three thresholds at 0.8, 0.6, 0.4, respectively. since comparing two very similar models is pointless, another threshold for the minimal tm-score difference between the compared models has been defined at 0.1. the other performance criterion used in this study is the average ranking, as predicted by the scoring function, for the aforementioned "nearnative" and "good" categories of models (the higher the rank, the better), as well as for the "poor" ones (the lower the rank, the better). the statistical significance of the observed differences between accuracies was determined by comparing the distributions of correct and wrong rankings, using the wilcoxon signed-rank test, with an α error of 0.05. the exact same procedure has been carried out using the global distance test total score (gdt_ts) [44] instead of the tm-score. since these two measures are calculated on the cα of the protein structures, the scoring with the statistical potentials was restricted to this atom type. to compare both the pmf and tig scores with an external reference from the literature, we have included the dope [45] and goap scores [46] into the benchmark. the former is the most cited of all model quality assessment programs, while the latter is a more recent and high-performing statistical potential. similarly to the scoring functions that we have built here, the only structural features that are quantified by dope are the interatomic distances, using the same distance bins and cutoff (0.5 å and 15.0 å, respectively; see above). goap is both distance-and angle-dependent: for each heavy atom in interacting pairs, it uses the relative orientation of the corresponding planes. for the computing fi,j ref (r), dope and goap take into account either the radius of gyration or the molecular volume of each protein structure from the training set, which eliminates the bias on the interatomic distances-the distance distributions may vary a lot, depending on the sizes of the proteins included in the dataset. thus, the difference between dope/goap and pmf/tig lies in (i) the training datasets, (ii) the calculation of the reference state and (iii), only in the case of goap, the dependence on orientation. the tig scoring function is supposed to be more accurate than sippl's statistical potentials (eq. 1), as it is built on the new approach (eq. 3). to demonstrate its practical superiority, we have compared its performance with those of our pmf score, through two different tests. the first one evaluates the ability of the method to rank pairs of models taken from the 3drobot dataset. the results of this benchmarking procedure are presented in table 1 . they include the accuracies of the dope, mck1, and mck2 scores (for goap, see the next paragraph). overall, it appears that tig is the best of these five methods, whereas pmf is the worst. to our surprise, the two mock scores systematically outperform pmf, although they were designed only to prove the irrelevance of combining fi,j obs (r) and fi,j ref (r) as a statistical reference. on the whole, the results obtained with the mock scores are also significantly better than those produced by dope. this indicates that the logarithmic mean could be advantageously replaced by other types of means, such as the arithmetic mean used in mck1. correspond to a random ranking. the "near-native", "good", "medium", and "poor" model qualities this article describes how sippl's formalism can be comprehended and improved, in light of probability and information theories. the simple tig and mock scores have been designed for that purpose. however, to give the reader an idea of the performance that a more complex scoring function can achieve, our benchmark includes a sixth method, goap, which ranks among the best statistical potentials [46] . the results obtained with goap are dramatically better than those produced with any of the other methods. in particular, goap outperforms tig by ~20% on the near-native decoys. on the "good" category, this difference is still >10%, when taking either the tm-score or the gdt_ts as a reference. on the two other categories of model quality, goap is also the most accurate method. this significant superiority is consistent with what has been previously observed on other datasets of decoys [46] , where goap outperformed the opus-psp potential [47] by ~15%. the latter was itself reported as more accurate than statistical potentials of lower complexity (i.e. which use less information), such as dfire [48] , rwplus [49] , and ddfire [50, 51] . thus, the results obtained with goap were expected and can be explained by the greater amount of stereochemical information it uses: the orientation and, in a lesser extent, the volume of the protein molecules. here, it should be highlighted that the only unbiased comparison-to provide insights into the improvement brought about by the new formalism-is the one between pmf and tig, as these scoring functions are equally complex. it is also the case for the two mock scores, which have been specially designed for the sake of fair comparison. the ranking of the methods is similar to the casp13 benchmark, although every accuracy is higher (table s1) . on this dataset, pmf is systematically outperformed by the two mock scores, which are themselves outperformed by tig. the latter is not significantly better than dope on this dataset, but goap is still far more accurate than the other five methods. compared to 3drobot, the differences between the methods are aggravated for the poor quality models, whereas they are nonsignificant for the near-native ones. moreover, the quality of the models appears to have a different influence over the accuracy: (i) the most difficult models to rank are those of near-native and poor quality in 3drobot versus those of medium quality in casp13; (ii) the easiest to rank are those of good and medium quality in 3drobot versus those of near-native quality in casp13. only the performances of goap are consistent between the two datasets: the higher the model quality, the higher the accuracy. all these discrepancies presumably arise from the different origins of the two datasets. the 3drobot set has been specifically designed for benchmarking purposes: for each protein, the native structure has been uniformly altered to generate exactly 300 decoys. models to confirm the above benchmarking results, a second test has been performed. it consists in observing how the models are ranked by a scoring function, depending on their actual quality as measured by either the tm-score or the gdt_ts. the results of this second benchmarking procedure are presented in table 2 . for the "near-native" and "good" models, the lower the value presented in table 2 (i.e. the higher the rank), the better; conversely, for models of "poor" quality, the lower the rank, the better. taken as a whole, these results further validate the new formalism, as tig significantly outperforms the other methods except goap. indeed, tig is better because it (i) ranks higher the near-native models, as well as the good models (although only when defined with the gdt_ts) and (ii) ranks lower the models of poor quality. again, the mock scores systematically outperform pmf, and dope is the only score that rivals or bests tig (at ranking good models). in agreement with the accuracies reported in table 1 , the performance of goap is far superior to those of the other methods, on the near-native and poor models. this is, however, not the case for the good models, which shows that the quality category influences the average predicted rank differently than the accuracy. in general, this second test is less discriminating than the pairwise ranking, since the observed differences are less significant. as for the first test, the results contain some discrepancies between the tm-score-and gtd_tsbased categories. this shows the difficulty of defining thresholds to categorize model quality. as both the tm-score and gdt_ts take values between 0 and 1, we used the same thresholds for these two scores. the >0.8 limit for the near-native models was defined based on a major and recent study, which defined conformations with a tm-score >0.7 as "high-accuracy" predicted structures [22] . we defined the <0.4 limit for the models of poor quality, based on the previously studied significance of a tm-score of 0.5 [52] . however, our categorization of the model quality seems appropriate, given that the tested methods have more difficulty ranking the near-native and poor models (for different reasons), than the ones of good or medium quality. table 2 . ranks predicted by the tig, dope, mock, and pmf scores, averaged for three categories of models. for the "near-native" and "good" models, the lower the value (the higher the rank), the better the performance. conversely, for models of "poor" quality, the lower the rank, the better. to further validate the information gain-based approach, the correlations between the scores produced by pmf or tig and the corresponding tm-scores have been investigated, for the decoys of 3drobot. averaged on the 200 proteins (×300 decoys), the pearson correlation coefficients are −0.719 and −0.782, for pmf and tig, respectively. this makes tig equal to ddfire, by referring to performances previously reported in the literature [53] . like goap, the ddfire statistical potential is both orientation-and distance-dependent, and uses protein molecular volume in the calculation of the reference state. however, unlike goap, it is based on a coarse grained representation of the protein structures. it should be noted here that ddfire is the lowestperforming program among those tested in [53] , where svmqa is the best, followed by opus-psp, goap, and rwplus. nevertheless, the outcome of this comparison is that tig can match a more complex method, such as ddfire. these results also confirm the improvement brought about the tig formalism over sippl's pmfs. finally, as the 200 proteins from 3drobot have been selected for their diversity, the difficulty to assess the decoys may vary from one protein to another. this is illustrated by figure 2 , in which values of the tm-score are plotted against those of the tig score, for proteins that show various levels of correlation. in these examples, the dispersion of the predicted quality goes higher, as the true quality goes lower. this is consistent with the intuition that the more altered is a decoy structure, the more uncertain is the prediction of its quality. as the new formalism has been designed to be independent of any physics-based interpretation, it is interesting to analyze the score profiles of our tig function, given that we will not attempt to draw any analogy with physical interatomic potentials (e.g. the lennard-jones or morse potentials). compared with the pmf profiles, a first observation is that most profiles are actually very similar. therefore, figure 3 presents the tig and pmf profiles for the four pairs of residues that differ the most, namely the cys-cys, asp-glu, val-val, and lys-arg residue pairs. strikingly, one can observe that the attractive part of the profile (i.e. the negative score well) is always stronger for tig than for pmf. this is simply due to the fact that, for x and y ∈ ]0, 1], the −ln(x/y) function from eq. 1 takes values that are always greater than those of −(x−y)/y from eq. 3. therefore, the scores computed are systematically lower with tig than with pmf. it is important to note that this difference is not related to the better performance of the new formalism, as our benchmark was only aimed at testing the ability of the scoring functions to rank models, rather than to assess their absolute quality. in other words, it is not valid to compare the two scores computed by tig and pmf for each structure, and conclude that tig always evaluates the structure as more native-like. setting a default value of the score (here equal to +10). however, due to the logarithm in the formula, only pmf is undefined for fi,j obs (r) = 0, which also requires a default value (again set to +10). for fi,j obs (r) = 0, the tig scoring function is defined and takes the value +1, which makes its repulsive part composed of two plateaus, at +1 and +10. the +1 plateau corresponds to an interatomic distance that has never been observed for the particular atom pair, but otherwise exists in the training set of native conformations. the +10 plateau, however, corresponds to an interatomic distance that has never been observed within the experimental structures, whatever the type of atoms. although it seems useful to distinguish these two cases, with a higher penalty for the second one, this repulsive part with either one or two plateaus presumably does not affect the results of our benchmarking procedure. indeed, only protein models of very poor quality would contain such abnormal interatomic distances. nevertheless, the importance of this parameter-which can be applied to both tig and pmf formalisms-remains to be investigated. interestingly, the right sides of the plots indicate that the information gain is limited for two residues spaced by more than 10 å, as the score fluctuates around zero. this suggests that similar performances could be achieved at a lower computational cost, by restricting calculations to shorter interatomic distances (see section below). finally, from a qualitative point of view, these profiles produced by tig do not seem to show any unexpected features. for the asp-glu and lys-arg residue pairs ( figure 1b and 1d, respectively), the positive peak at ~7 å is consistent with their presumably repulsive interaction. similarly, in the val-val ( figure 1c ) profile, the negative well at ~6 å can be attributed to the attractive interaction between two hydrophobic residues, and the very negative profile of the cys-cys ( figure 1a ) pair reflects the possibility of forming disulfide bonds. the new formalism presented here was developed to be more statistically relevant than sippl's pmfs. thus, the better performances observed on the benchmark were actually expected. through the inclusion of the two mock scoring functions, this study was also aimed at shedding light on how the statistical pmfs actually work by summing relative frequency differences, which correspond to information gains. it should be noted that we used here a general definition of information that quantifies the bayesian updating and is, therefore, different than the particular shannon surprisal (also called "self-information"). importantly, the conceptual improvement brought here is only valid however, the advantage of dividing by a logarithmic mean in eq. 2, rather than by a generalized mean (the special cases of which being the arithmetic, geometric, and harmonic means) still has to be demonstrated. in their original form, as devised by sippl 30 years ago, the statistical potentials used only the distance between each pair of atoms to represent protein structures. through the lens of probability theory, bakers's and hamelryck's research groups later showed how any other descriptors can be successfully used: typically, solvent accessibility or torsion angles. more exotic structural properties have also been exploited, e.g. lipid bilayer depth to build a potential aimed at evaluating structural models of transmembrane proteins [54] . nevertheless, in the particular case of scoring functions that are only based on interatomic distances (like tig), the performances might find their roots not only in bayes' theorem, but also in the representation of the problem. reducing a protein 3d structure to a set of pairwise distances allows the use of graph theory. protein conformations can thus be modeled as amino acid (weighted or unweighted) graphs and are referred to as "protein contact networks" (pcns; see [55] for a review). similarly to the tig formalism, methods based on pcns are not related to physics and, yet, are able to rank decoys [56] . authors have later combined such graph-theoretic approach with support vector machine in order to accurately assess the quality of structural models [57] . as a consequence of these results, generalizing the tig concept and confirming its relevance regarding statistics would require to rule out the pcn representation as a source of performance. this would mean evaluating the accuracy of tig scoring functions that would be built on other structural features than distances. the resulting scoring functions could be used as knowledge-based terms, combined with physics-based terms, into a composite energy function, such as that developed for the rosetta modeling software [58] . the weight of all terms would be optimized to fit experimental structural and thermodynamic data. alternatively, the elementary scores could be included in non-linear statistical models, thanks to machine learning and deep learning techniques, as it can yield highly accurate quality assessment programs [53, 59, 60] . the development of a distance-dependent scoring function relies on several parameters, such as the distance bin width, the minimum and maximum distance thresholds, and the minimum number of sequence positions separating the residues of the two interacting atoms. behind the setting of these values lies the question of how to treat long-range interactions and local contacts. here, we considered distances ranging from 0 to 15 å in order to be comparable with dope, but authors use a 4-8 å range, following the aforementioned pcns approach [55] . attempts to determine optimal values for these parameters have been made [61] . however, the training dataset and, more importantly, the benchmark then used were too small to draw any permanent conclusion. such a study could be redone with the computational tool used for developing the tig score, as it allows to create a custom scoring function, while setting the different thresholds with user-selected values. interestingly, the results obtained with goap show that a method based only on interatomic distances, orientation, and molecular volume can achieve high accuracy-especially for near-native decoys. this would indicate that a limited number of parameters are sufficient to model the process of protein folding and stability. such knowledge-based scoring functions, as estimators of protein free energy, could thus be considered as "sloppy models", i.e. models whose behavior depends on a relatively small number of combinations of parameters. although this theoretical framework has gained popularity in recent years for explaining phenomena in physics and biology (see [62] ), it remains unused as a means to study protein structures. we proved our concept for the ranking of predicted structures according to their quality. however, there are several other applications that could be explored-like protein-protein docking, for example. an interesting use of statistical potentials consists in training them on a particular type of native protein conformations, in order to gain insight into the rules that govern the relative positioning of residues within these protein structures. for example, this has recently been done for transmembrane protein structures [63] . as we provide here an open-source standalone version of our program, we hope that it will find usefulness in studying pairwise interactions within userselected protein structures. finally, this study focused on statistical pairwise potentials. similarly to their physics-based counterparts, these two-body potentials are inherently limited in their representation of the intra-protein interactions. further investigations should therefore be carried on many-body potentials. principles that govern the folding of protein chains calculation of conformational ensembles from potentials of mena force: an approach to the knowledge-based prediction of local structures in globular proteins knowledge-based potentials--back to the roots helmholtz free energies of atom 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the reference ratio method proteins, physics and probability kinematics: a bayesian formulation of the protein folding problem the logic of decision updating subjective probability korp: knowledge-based 6d potential for fast protein and loop modeling dispot: a simple knowledge-based protein domain interaction statistical potential protein thermal stability engineering using hotmusic shedding light on the dark matter of the biomolecular structural universe: progress in rna 3d structure prediction andis: an atomic angle-and distance-dependent statistical potential for protein structure quality assessment 3drobot: automated generation of diverse and well-packed protein structure decoys an all-atom distance-dependent conditional probability discriminatory function for protein structure prediction11edited by f. cohen what is the best reference state for designing statistical atomic potentials in protein structure prediction? derivation of the boltzmann principle mypmfs: a simple tool for creating statistical potentials to assess protein structural models pisces: a protein sequence culling server elucidating protein secondary structures using alpha-carbon recurrence quantifications multivariate density estimation: theory, practice, and visualization scoring function for automated assessment of protein structure template quality lga: a method for finding 3d similarities in protein structures statistical potential for assessment and prediction of protein structures a generalized orientation-dependent, all-atom statistical potential for protein structure prediction opus-psp: an orientation-dependent statistical all-atom potential derived from side-chain packing distance-scaled, finite ideal-gas reference state improves structure-derived potentials of mean force for structure selection and stability prediction a novel side-chain orientation dependent potential derived from random-walk reference state for protein fold selection and structure prediction specific interactions for ab initio folding of protein terminal regions with secondary structures ab initio folding of terminal segments with secondary structures reveals the fine difference between two closely related all-atom statistical energy functions how significant is a protein structure similarity with tm-score = 0.5? svmqa: support-vector-machine-based protein single-model quality assessment membrane protein orientation and refinement using a knowledge-based statistical potential protein contact networks: an emerging paradigm in chemistry graph theoretic properties of networks formed by the delaunay tessellation of protein structures svr_caf: an integrated score function for detecting native protein structures among decoys the rosetta all-atom energy function for macromolecular modeling and design deepqa: improving the estimation of single protein model quality with deep belief networks proq3d: improved model quality assessments using deep learning statistical potentials for fold assessment perspective: sloppiness and emergent theories in physics, biology, and beyond a comprehensive computational study of amino acid interactions in membrane proteins we present a new formalism that is conceptually independent of statistical mechanics • its practical validity is shown by the quality assessment of protein structures the authors gratefully acknowledge the financial support of the université de paris, the cnrs institute and the inserm institute. key: cord-340746-icuzy3vp authors: liang, yunfei; wan, ying; qiu, li-wen; zhou, jingran; ni, bing; guo, bo; zou, qiang; zou, liyun; zhou, wei; jia, zhengcai; che, xiao-yan; wu, yuzhang title: comprehensive antibody epitope mapping of the nucleocapsid protein of severe acute respiratory syndrome (sars) coronavirus: insight into the humoral immunity of sars date: 2005-08-01 journal: clin chem doi: 10.1373/clinchem.2005.051045 sha: doc_id: 340746 cord_uid: icuzy3vp background: the epidemic outbreak of severe acute respiratory syndrome (sars) posed a worldwide threat to public health and economic stability. although the pandemic has been contained, concerns over its recurrence remain. it is essential to identify specific diagnostic agents and antiviral vaccine candidates to fight this highly contagious disease. methods: we generated 14 monoclonal antibodies (mabs) specific to the sars coronavirus (sars-cov) nucleocapsid (n) protein and used these to thoroughly map the n protein antigenic determinants. we identified the immunodominant antigenic sites responsible for the antibodies in sera from sars patients and antisera from small animals and differentiated the linear from the conformational antibody-combining sites comprising the natural epitopes by use of yeast surface display. results: we identified 5 conformational and 3 linear epitopes within the entire n protein; 3 conformational and 3 linear epitopes were immunodominant. the antibody responses to the n protein fragments in mammalian sera revealed that 3 regions of the n protein are strong antigenic domains. we expanded the specificity of the n protein epitope and identified 4 novel conformational epitopes (amino acids 1–69, 68–213, 212–341, and 337–422). conclusion: the antigenic structures identified for the sars-cov n protein, the epitope-specific mabs, and the serum antibody profile in sars patients have potential use in the clinical diagnosis and understanding of the protective immunity to sars-cov. atypical pneumonia caused by the severe acute respiratory syndrome coronavirus (sars-cov) 3 (1-4 ) has spread through 30 countries on 6 continents since late 2002. although many of the clinical and epidemiologic features of sars remain to be elucidated, hematologic (5, 6 ) and serologic (7) (8) (9) data suggest that igg seroconversion plays a key role in virus growth inhibition and disease prognosis. serodiagnosis and serosurveillance of sars (10, 11 ) have revealed that nucleocapsid (n) protein-specific antibodies in the serum of sars patients have higher sensitivity (12) (13) (14) and longer persistence (9 ) than those of other structural proteins of sars-cov. this finding has led to the current focus on potential targets for antiviral therapy. the n proteins of other coronaviruses shed more antigen than other structural proteins in infected cells (15 ) and are among the most immunoreactive viral proteins. n-protein-specific monoclonal antibodies (mabs) have a protective effect in mice after lethal virus challenge (16, 17 ) , and immunization with the n protein of the avian infectious bronchitis coronavirus induces an immune response that protects chickens from infection by this virus (18, 19 ) . these findings suggest that the n protein of the sars-cov, or its fragments, is an efficacious immunoprotective antigen (20 ) . determination of the antigenic structure of the viral protein may lead to identification of the epitope responsible for the harmful vs beneficial effects on humoral immunity and provide valuable information for sars vaccine development. to characterize the immunogenicity and immunoreactivity of the sars-cov n protein and potential antigenic sites, several groups have used synthetic peptides (21) (22) (23) (24) and protein microarrays (25 ) to analyze sera from sars patients. the antigenic sites on the n protein of sars-cov identified in these studies were limited to strong immunodominant antigenic sites because few antibodies in the sera from sars patients recognized the less effective immunogenic determinants, which are usually obscured by the more numerous antibodies to stronger immunodominant determinants (26 ) . the identified antigenic sites were mainly linear epitopes (27 ) . the antigenic structure and the precise locations of epitopes on the sars-cov n protein are still unknown because of the absence of proteins expressed by the eukaryotic system and of n-protein-specific mabs that would enable characterization of the antigenic structure in the context of antigen-antibody interaction. to precisely map the epitopes of the sars-cov n protein, it is necessary to optimize the production of the n protein and its fragments as they occur in nature, along with n-protein-specific mabs. we generated 14 sars-cov n-protein-specific mabs and used these for epitope mapping, thus identifying a range of epitopes and the immunodominant antigenic structures of the sars-cov n protein. the sars-cov strain gd01 (genbank accession no. ay278489), isolated from patients with atypical pneumonia in guangdong province, was the viral source and was cultured in vero cell lines (ccl-81; atcc). the human coronavirus strains 229e (vr740; atcc) and oc43 (vr759; atcc) were propagated in mrc-5 cells (ccl-171; atcc) and bs-c-1 cells (ccl-26; atcc), respectively. all cell lines were cultured in dmem growth medium supplemented with 100 ml/l fetal bovine serum (gibco invitrogen) at 37°c in 5% co 2 . all experiments with sars-cov were performed in a biosafety level 3 laboratory. forty-seven serum specimens were collected from convalescent sars patients in 4 different hospitals in beijing and guangzhou province, china. all patients were classified according to the sars clinical diagnostic criteria released by the who. of the 47 serum samples, those from 20 adult males and 20 adult females were serologically confirmed by an immunofluorescence assay (ifa) and elisa, as described previously (28 ) . serum samples collected in 2003 from 40 healthy blood donors and 38 patients with upper respiratory symptoms but not infected with the sars virus were used as controls. generation and screening of sars-cov n-protein-specific hybridoma cell lines construction of recombinant sars-cov n protein and immunization procedures were performed as described previously (28, 29 ) . in brief, the cdna gene encoding the sars-cov n protein was extracted from the filtered supernatants of sars-cov-infected vero cells by use of the qiaamp viral rna mini kit (qiagen). the sequence coding for the sars-cov n protein was then cloned into the pgex-5x-3 vector (pharmacia biotech) in frame and downstream of the glutathione s-transferase (gst)-encoding sequence. the gst-n fusion protein was expressed and purified with a gst gene fusion system (pharmacia biotech). hplc analysis showed that the purity of this purified n fusion protein was ͼ95%. the recombinant n fusion protein was detected by western blot analysis with sera of convalescent-phase sars patients as the primary antibody. a sars-cov-infected cell culture filtrate was used as a positive control. horseradish peroxidase-labeled goat anti-human igg was used as the secondary antibody. aminoethyl carbazole single solution chromogen (zymed laboratories) was used for signal detection. the concentration of purified recombinant n fusion protein was determined by the coomassie plus protein assay (pierce biotechnology). six-week-old female balb/c mice were immunized with the purified recombinant n protein mixed with an equal volume of monophosphoryl lipid a and trehalose dicorynomycolate (mplϩtdm) adjuvant (sigma). splenocytes from the immunized mice were fused with ns-1 myeloma cells. the fused hybridoma cells were propagated in rpmi 1640 supplemented with 100 ml/l fetal bovine serum, hypoxanthine, aminopterin, and thymidine. the culture supernatants of the hybridoma cells were screened for antibody production by indirect elisa. the coating antigens used were recombinant gst (pharmacia biotech), recombinant gst-n protein, and viruscell lysates of the sars-cov. hybridoma clones that reacted with the gst-n protein and virus-cell lysates but not with gst were selected and subcloned twice by limiting dilution. fusion of splenocytes from mice immunized with ns-1 myeloma cells produced more than 100 positive hybridoma lines, of which 14 cell lines were selected on the basis of their strong reactivity with the n protein as shown by elisa. the cell lines were then frozen and stored in liquid nitrogen. the classes and subclasses of the 14 mabs were determined by use of the mouse monoclonal antibody isotyping kit (roche applied science) according to the manufacturer's instructions. clinical chemistry 51, no. 8, 2005 production and purification of mabs to produce mab-containing ascites fluid, fourteen 6-week-old female, specific pathogen-free balb/c mice were given 1-ml intraperitoneal injections of pristane 1 week before inoculation with 2.5 ϫ 10 6 mab-producing hybridoma cells. two weeks later, harvested ascites samples were examined for their anti-sars-cov n protein specificity by an elisa as described above. mabs were purified by protein g column chromatography (pharmacia biotech) according to the manufacturer's instructions. the purified sars-cov n proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. the membranes were then blocked in tris-buffered saline (150 mmol/l nacl, 50 mmol/l bis-tris, ph 7.5) containing 0.5 ml/l triton x-100 and 50 g/l nonfat milk for 2 h at room temperature and then incubated with purified mabs for 2 h. after a wash with tris-buffered saline-triton, the membranes were incubated with the goat anti-mouse igg-peroxidase conjugate. aminoethyl carbazole chromogen was used for signal detection. ifa vero cells infected with sars-cov, mrc-5 cells infected with human cov 229e, and bs-c-1 cells infected with human cov oc43 were first harvested and then washed twice in ice-cold phosphate-buffered saline (pbs). the cells were deposited on 8-to 10-well chamber slides, air-dried, and fixed in acetone. after a wash with pbs, the slides were incubated with purified mabs at 37°c for 1 h and incubated with fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse igg at 37°c for 30 min. the slides were stained with 0.25 g/l evans blue in pbs and analyzed with a leica eclipse epifluorescence microscope. preparation of n-protein-specific antisera for vaccination, four 6-week-old female balb/c mice were immunized intradermally with 100 g of purified recombinant n protein in complete freund's adjuvant emulsion (sigma) for the first injection on day 0. equalquality immunogen mixed with incomplete freund's adjuvant was used for the booster injections on days 7, 14, 21, and 28. antisera were collected 4 days after the final inoculation. the anti-n-protein antibody responses of the antisera were tested by elisa using the purified n protein as the coating antigen. the amino acid sequence encoding for the complete n protein was derived from swissprot/trembl (accession no. p59595). to analyze the molecular characteristics and structural information of the sars-cov n protein, we used the bioinformatics servers on the internet, including the protscale server (swiss institute of bioinformatics), jpred2 server (university of dundee school of life sciences), and the predictprotein server (columbia university bioinformatics center), and dnastar software (dnastar, inc.). the predicted results are shown in fig. 1a . on the basis of the predicted antigenic locations, putative epitopes within the n protein were referred for further plasmid construction. to construct the yeast expression vectors, a panel of series-truncated and nested-deletion mutations was designed according to the results of sequence analysis and epitope predictions (fig. 1b) . reverse transcription-pcr was performed with the forward primer 5ј-cggaattcat-gtctgataatggaccccaa-3ј, the reverse primer 5ј-cgtctagattatgcctgagttgaatcagca-3ј, and the sars-cov rna template described above. the restriction enzymes were purchased from new england biolabs, and the pyd1 yeast display vector kit was from invitrogen. the sequence coding the complete n protein was digested with ecori and xbai, ligated into the pcdna3.1 vector (invitrogen), which digested with the same restriction enzymes produced the pcdna3.1-n plasmid. to obtain the pyd1-n yeast expression plasmid, the full length of the n gene from the pcdna3.1-n plasmid was excised with ecori and apai and then ligated into pyd1. to produce series-truncated and nested-deletion mutations, 2 linkers (linker 1, ecori, xhoi, hindiii, nhei, eagi, hindiii; linker 2, ecori, nhei, bstxi, xbai, eagi, hindiii) were designed using vector nti 7.0.0 software (informax, inc.), synthesized (sangon co. ltd.), and then cloned into the pmd18-t vector (takara co., ltd.). a panel of n gene segments was subcloned into the pmd18-t linker 1 and pmd18-t linker 2 plasmids. the n gene segments were digested with ecori and eagi enzymes and then subcloned into the pyd1 expression vector. all constructed plasmids were identified by enzyme digestion and confirmed by sequencing (takara co.). after transformation into the escherichia coli strain xl1-blue and plasmid extraction, the pyd1 plasmids containing the full-length n gene and the various n gene fragments (fig. 1b) were prepared for yeast transformation and induction of protein fusion. the reagents used in this experiment were purchased from sigma-aldrich. the lithium acetate method was used for yeast transformation. briefly, the glycerol stock of eby100 was streaked out on a minimal dextrose plate containing leucine and tryptophan and then incubated at 30°c until colonies appeared 2 days later. the eby100competent cells were prepared with lithium acetate (1.0 mol/l), salmon sperm dna (50 g), and polyethylene glycol (500 g/l) admixtures, and transformed with pyd1, pyd1-n, and pyd1 containing various gene fragments of the n protein. the transformation reactions (100 l/ sample) were spread on leucine-containing minimal dextrose plates, which were then incubated at 30°c for 2-4 days until single colonies appeared. (a), hi1 to -6, hb1 to -6, and ac1 to -5 indicate the most hydrophilic regions, hydrophobic regions, and accessible residues of the n protein, respectively. (b), the black bar represents the full-length n protein, and the gray bars represent the n protein fragments that were cloned into the yeast expression vector pyd1 plasmid. the clinical chemistry 51, no. 8, 2005 induction of yeast surface-displayed fusion protein to express fusion protein on the eby100 yeast cell surface, transformants were grown in glucose-containing medium overnight, then switched to a medium containing galactose for induction of expression. the experimental procedures described below were applied according to the manufacturer's instructions and a previously published protocol (30 ) . briefly, a single yeast colony (i.e., untransformed eby100, eby100/pyd1, eby100/pyd1-n, and eby100/pyd1 containing various n protein fragments) was inoculated into 8 ml of yeast nitrogen-based casamino acid medium containing 20 g/l glucose and grown overnight at 30°c with shaking. when the a 600 readings reached 5, the cell cultures were centrifuged at 3000g to 5000g for 5 min at room temperature. the cell pellets were resuspended in yeast nitrogen-based casamino acids medium containing 20 g/l galactose to an a 600 of 0.5-1, ensuring that the cells continued to grow in a log-phase pattern. yeast cells were incubated at 20°c with shaking. the cell cultures were assayed over a 48-h period (at 0, 12, 24, 36, and 48 h) to determine the optimum induction time for maximum display. the maximum display of fusion proteins on the eby100 cell surface was observed 24 -36 h after induction. the optimum displayed yeast cultures were stored at 4°c for analysis. anti-xpress-fitc antibody was purchased from invitrogen and anti-6 ϫ his antibody from roche applied science. we obtained 14 mabs against the sars-cov n protein. fitc-conjugated goat anti-mouse igg and fitcconjugated goat anti-human igg were purchased from beijing zhongshan golden bridge biotechnology co., ltd. for the fluorescent labeling of yeast surface-displayed proteins, 1 ϫ 10 6 yeast cells were microcentrifuged. the cell pellets were resuspended in ice-cold pbs (0.01 mol/l, ph 7.2-7.4) containing 1 g/l bovine serum albumin and washed twice. the cell pellets were resuspended in 100 l of pbs containing either the primary antibody against the epitope tags (i.e., x-press or 6 ϫ his tag) or the sars-cov n-protein-specific mabs and incubated at 4°c for 30 min. the cells were then added to 100 l of pbs containing the fluorescently labeled secondary antibody (fitc-conjugated goat anti-mouse igg or fitcconjugated goat anti-human igg) at titers of 1:50, incubated at 4°c for 1 h in the dark, and then washed twice with 0.8 ml of ice-cold pbs. finally, the yeast cells were resuspended in 300 l of pbs and kept at 4°c until flow cytometry analysis. to distinguish the linear from the conformational epitopes of the sars-cov n protein, yeast cells were heated to 80°c for 30 min in a thermostatically controlled water bath and then chilled on ice 20 min before labeling with antibodies (30 ) . the specific antibody-binding assay was performed with a facscali-bur flow cytometer (becton dickinson inc), and the results were analyzed by cellquest macintosh software (becton dickinson). to identify the presence of epitope-specific antibodies against sars-cov n protein in sera, induced eby100/ pyd1 yeast cells (i.e., yeast containing the empty pyd1 vector) were pelleted and washed twice with ice-cold pbs. serum samples were diluted 1:50 in pbs containing 0.5 g/l sodium azide and mixed with 2 ϫ 10 6 yeast cells per 50 l of attenuated serum. this preadsorption mixture was agitated moderately at 4°c overnight to eliminate nonspecific binding to yeast surface molecules. after pelleting, the supernatant was recovered and diluted with pbs to a final serum dilution of 1:100. normal control serum was processed by the same procedure. the isotypes of the 14 mabs are shown in table 1 . the specificities and immunoreactivities of the mabs to the sars-cov n protein were determined by western blotting, elisa, and ifa. three mabs (a50, b30, and c31) against the sars-cov spike protein s1 domain at nterminal residues 249 -667 (31 ) were used as negative controls (table 1) . among yeast cell surface display of full-length sars-cov n protein and its overlapping fragments on the basis of the epitope prediction of the sars-cov n protein (fig. 1a) , we designed 7 series-truncated and 4 nested-deletion fragments (fig. 1b) . the yeast expression plasmid pyd1 containing the full-length n protein and a panel of fragments encoding various n genes were transformed into the yeast saccharomyces cerevisiae eby100 strain and then induced for protein expression. the his-tograms of detected sars-cov n protein and its fragments displayed on the eby100 yeast cell surface are presented in fig. 2 . in this yeast display system, a percentage of cells did not express protein on their surface, and as a result 2 histogram peaks occurred. the flow cytometry histograms of the positive yeast display showed a peak with background fluorescence equivalent in intensity to that obtained with negative controls, which also provided a good internal negative control of the labeling (30 ) . the full-length sars-cov n protein (amino acids 1-422) was expressed on the yeast cell surface, as indicated by reactivity of the xpress epitope tag with the anti-xpress antibody (fig. 2) . the n protein fragments nt1 (amino acids 1-69), nt2 (amino acids 68 -120), nt3 (amino acids 119 -213), nt4 (amino acids 212-341), nt5 (amino acids 337-422), nndc1 (amino acids 68 -213), nndc2 (amino acids 214 -422), nnd13 (amino acids 1-120), and nnd14 (amino acids 1-213) were also well expressed as detected by the xpress or 6 ϫ his epitope tags (fig. 2) . the fragment nnd15 (amino acids 1-341) was not expressed on the yeast cell surface; its absence suggests that this fragment corresponds to unstable protein-domain breakpoints that could not be properly folded or trafficked through the yeast secretory pathway (32, 33 ) , and it was removed from further functional studies. the well-expressed yeast-displayed fusion proteins and the well-characterized n-protein-specific mabs were used for antibody epitope mapping. antibody epitope mapping of the sars-cov n protein the 14 mabs were used in the same concentration (10 mg/l per sample) and diluted into pbs in a final volume of 100 l for the binding assay. all assays were performed in triplicate or more, and the individual fluorescence intensities were obtained from the mean value of the specific yeast-displayed protein. the ratio of the recombinant antigen expression on the yeast surface (rays) was calculated by dividing the fluorescence intensity obtained for the individual rays by the fluorescence intensity obtained from yeast expressing pyd1 (i.e., eby100/pyd1). a ratio ն2 was considered positive (34 ) . eby100 yeast cells displaying irrelevant antigens, decayaccelerating factor, and isotype-matched control antibodies were used as negative controls for the 14 mabs and 10 yeast-displayed fusion proteins. the negative controls did not show any specific binding (data not shown). the profile of the antibody epitope mapping of the sars-cov n protein is summarized in table 2 . the antigenic determinant sites located on the n protein were determined according to the specific binding of a mab to the particular yeast-displayed protein. flow cytometric analysis showed prominent immunoreactivity to the full n protein (amino acids 1-422) for 7 of the 14 mabs (m1, m3, m4, m5, m6, m7, and m9), demonstrating that yeast surface-display technology is a useful method for biochemical characterization of ligand-receptor interactions and antibody-binding epitopes. the antigen-antibody response of nnd14 (amino acids 1-213) was as strong as, or in some cases stronger than, that of the full-length n protein (amino acids 1-422). in addition to the full n protein (amino acids 1-422) and nnd14 (amino acids 1-213), nndc2 (amino acids 214 -422) showed specific binding to mabs m2, m8, m10, m11, m12, m13, and m14. on the basis of these experimental data, the 14 mabs were definitively divided into 2 different groups: mabs m1, m3, m4, m5, m6, m7, and m9, which recognized the full n protein (amino acids 1-422) as well as the n-terminal fragment of nnd14 (amino acids 1-213); and mabs m2, m8, m10, m11, m12, m13, and m14, which reacted only with the c-terminal fragment of nndc2 (amino acids 214 -422). mabs m3 and m9 displayed strong immunoreactivity with the full-length n protein (amino acids 1-422), igg2a a fourteen n-protein-specific mabs were generated and designated m1 to m14; 3 mabs (a50, b30, and c31) against the sars-cov spike protein s1 domain at n-terminal residues 249 -667 were used as negative controls in these assays. b the purified recombinant n protein (5 mg/l, 100 l/well) was used as the coating antigen and was reacted with purified anti-n-protein mab. the absorbance was measured at 450 nm: ϫ, negative result; ϩ, a 450 ϭ 0.2-1; ϩ ϩ, a 450 ϭ 1-2; ϩ ϩ ϩ, a 450 ͼ2. fragment nt2 (amino acids 68 -120) did not bind to any of the 14 mabs, indicating that the antibody-binding epitope may flank the breakpoint of the particular fusion protein tested or that we did not produce a mab specific to the antigenic site within nt2 (amino acids 68 -120). representative flow cytometry histograms depict the mean fluorescence signals for antibody labeling of the n-terminal xpress and c-terminal 6 ϫ his epitope tags of yeast-displayed fusion proteins. anti-xpress labeling of the n-terminal xpress and anti-6 ϫ his antibody labeling of the c-terminal 6 ϫ his tags expressed on eby100/pyd1 were used as positive controls (a1 and d1). eby100/pyd1 labeled with only the fitc-conjugated second antibody was used as a negative control (d4). the double peaks show the reactivity of the anti-xpress antibody with the n-terminal xpress epitope tag (a2-a4, b1-b4, and c1-c3) and the anti-6 ϫ his antibody with the c-terminal 6 ϫ his tag (d2 and d3) of eby100/pyd1 containing the full-length n protein and its fragments. eby100/pyd1 containing the fragment nnd15 showed no reactivity with the anti-xpress antibody labeling (c4), indicating that this fragment was not expressed by yeast cells. we used heat denaturation of the fusion proteins tethered to the eby100 yeast cell surface to categorize the specific linear and conformational sars-cov n protein mab epitopes (35, 36 ) . anti-xpress and anti-6 ϫ his label mabs were set as positive controls to identify the xpress and 6 ϫ his epitope tags flanking the fragment inserts before and after the yeast-displayed fusion protein denaturation and to verify that the protein and yeast cells were not compromised during the heat treatment. to further confirm the denaturation of yeast-displayed proteins on heat treatment and as a positive control, we simultaneously assayed the yeast cells binding to the specific mabs obtained. we calculated the rays ratio by dividing the fluorescence intensity obtained for the individual rays by the fluorescence intensity obtained for yeast expressing pyd1 before and after heat denaturation. a ratio ն2 was considered positive. the flow cytometry results are expressed as rays ratios to assess the antigenantibody binding and the rays ratios representing specific binding of yeast-displayed proteins to mabs before and after heat denaturation. the rays ratios before and after denaturation and the overall distinction between linear and conformational antigenic determinants of the sars-cov n protein are shown in fig. 3 . the binding of nt1 to mab m3 was abolished by heat treatment, indicating that mab m3 reacted with continuous or discontinuous conformational determinants. this explanation is supported by the abolition of the binding of the full-length n protein and fragments nnd14 and nnd13 to mab m3 after heat denaturation (fig. 3, a and b) . the mab m3-specific conformational epitope was identified and localized in the region of amino acids 1-69 on the n protein. the reactivities of mab m13 to nt4 and mab m10 to nt5 disappeared after denaturation, suggesting that m13-and m10-specific antibody epitopes located on the n protein fragments nt4 and nt5, respectively, are conformational determinants (fig. 3c) . except for the conformational epitopes identified above, mabs m1, m4, m5, m6, and m7 reacted with conformational epitopes located between amino acids 68 and 213 (fig. 3d ). heat denaturation abolished the specific binding ( fig. 3c ) of mabs m2, m8, m12, and m14, which had been raised against fragment nndc2 before heat treatment, showing that these reacted with discontinuous epitopes. mab m9 retained its immunoreactivity with the n protein and with nt1, nnd13, and nnd14 after heat treatment and showed binding to linear or sequential epitopes other than those bound by mab m3 (fig. 3, a and b) . similarly, the binding of mab m11 to nndc2 and nt5 was maintained after heat treatment, indicating that the mab m11 antibody-binding epitope is linear and different from that of mab m10 (fig. 3c ). the 2 identified linear epitopes localized in the regions of amino acids 1-69 and 337-422 on the n protein are consistent with previously published data from studies using the synthesis of overlapping or mutated peptides (21, 22, 24 ) and prokaryotic expression of sars-cov n protein segments (25 ) to probe sera from convalescent sars patients. although these studies found that heterogeneous specific synthetic peptides reacted with the sera from sars patients, the most immunoreactive oligopeptides were located mainly within the 2 separate regions (amino acids 1-69 and 337-422) of the n protein, as described above. immunodominant epitopes located on the n protein screened by antisera from immunized mice to distinguish the immunodominant linear from the conformational antigenic structures of the sars-cov n protein, 4 mice were immunized with the n protein and the antisera were collected. strong anti-n-protein antibody responses were detected by elisa in sera from the 4 immunized mice but not in sera from 2 nonimmunized control mice (data not shown). this result demonstrates that n protein is one of the immunoreactive coronavirus structural proteins that elicit the specific humoral immune responses in animals. eby100-pyd1 yeast cells were used to preadsorb mouse antisera to eliminate the nonspecific binding to yeast surface molecules in the reactions, as described above. the reactivity of the full-length n protein and its overlapping fragments before and after denaturation with the mouse antisera was detected by flow cytometry. no positive signals were detected in reactions containing control sera from 2 nonimmunized balb/c mice and the fusion proteins (data not shown). as shown in fig. 4 , yeast-displayed sars-cov n protein reacted with the antisera from the 4 mice. the immunoreactivity of the protein fragments with polyclonal antisera from immunized mice confirmed the identification of the antigenic sites on the n protein by mabs, as described above (fig. 3) . fragments nt1, nt3, nt5, nnd13, nnd14, nndc1, and nndc2 reacted with antisera both before and after denaturation (fig. 4) , indicating that both immunodominant linear and conformational epitopes are located in the regions containing amino acids 1-69, 70 -213, and 337-422. after denaturation, fragment nt4 gave positive signals in the antisera from 2 mice (fig. 4, antisera from mice 1 and 4) , and fragment nt2 was positive in the antisera from 3 mice (fig. 4, antisera from mice 1, 2, and 3) , suggesting that these regions also bear the linear epitopes. the reactivity of the n protein fragments with mouse antisera indicated that the regions containing amino acids 1-69, 121-213, and 337-422 are the dominant antigenic regions within the full-length n protein. the antigenic domains on the n protein (fig. 1d ) have potential as diagnostic markers and fig. 3 . linear vs conformational sars-cov n-protein-specific mab epitopes classified by mab labeling of the yeast-displayed fusion protein before (f) and after (ⅺ) heat denaturation. the fluorescence intensity was calculated as a rays ratio. a rays ratio of 2 was set as the baseline, and a rays ratio ͼ2 was considered positive. the xpress and 6 ϫ his epitope tags flanking the gene inserts on the yeast-displayed fusion proteins were determined to validate that the protein and yeast cell were not damaged during the heat treatment. mabs with known binding to specific yeast-displayed proteins before denaturation were labeled simultaneously as a parallel control. shown are representative interactions between yeast-displayed fusion proteins and the specific mabs tested. may represent potential antiviral targets for vaccine development. verification of identified immunodominant epitopes on the n protein with sera from sars patients we next validated the antigenicity of the identified immunodominant epitopes to gain insight into the humoral immune response to the sars-cov n protein in humans. forty serum samples from patients with atypical pneumonia identified by our assay were numbered from 1 to 40, and the sera of cases 21-40 were pooled for flow cytometric analysis. sera collected from 40 healthy blood donors and sera from 38 patients with upper respiratory symptoms who were not infected with the sars virus during the atypical pneumonia pandemic in 2003 were simultaneously assayed as individual or pooled samples to provide negative controls. all assays were performed in triplicate or more. no positive signals were detected in the yeast-displayed fusion proteins that reacted with the control sera in both individual and pooled samples (data not shown). the data for the 10 yeast-displayed fusion proteins before and after denaturation in cases 1-20 and in the pooled sera from sars patients are shown in table 3 and fig. 5 . both before and after denaturation, the eby100-dis-played n protein reacted with the antibodies raised in the sera from sars patients, as indicated by rays ratios ͼ2. twenty-one serum preparations (sera of cases 1-20 and the serum pooled from cases 21-40) were immunopositive, demonstrating that the n protein is an immunodominant antigen in sars-cov-infected patients with atypical pneumonia. in addition to the full-length n protein, both before and after heat treatment, fragments nt1, nt5, nnd13, nnd14, and nndc1 reacted with the pooled sera. nt3 also reacted with the serum pool from sars patients after denaturation (table 3 and fig. 5f ). fragment nnd14 reacted with sera from 19 of 20 cases before denaturation and all 20 cases after denaturation, a reactivity similar to that obtained by probing with the fulllength n protein. the reactivity of yeast-displayed proteins with various n protein fragments probed with the sera from sars patients suggests that 6 or more immunodominant epitopes are located on the n protein (fig. 5) . fragment nt1 reacted with the sera from 17 of 20 cases before denaturation and 16 of 20 cases after denaturation. fragment nnd13 reacted with the sera from 10 of 20 cases before denaturation and 8 of 20 after denaturation. fragment nt2 did not react with any of the sera tested. these data indicate that nt1 contains both the immunodominant linear and conformational epitopes. these data are consistent with the antisera analyses and with the mab epitope-mapping analysis showing that mab m3and m9-specific epitopes are located between amino acids 1 and 69 of the n protein (fig. 4) . fragment nndc1 reacted with pooled sera and with sera from 19 of 20 cases before denaturation and with 15 of 20 after denaturation. nt3 reacted with the sera of 3 of 20 cases before denaturation and 13 of 20 after denaturation. fragment nt2 did not produce a positive signal with any of the sera from sars patients. after comparing the analytical results of epitope mapping in the antisera and the current data, we deduced that the conformational epitopes are located between amino acids 68 and 213 on the n protein and that the epitope responsible for antibodies in the sera of sars patients lies in the region of amino acids 121-213 of denatured nndc1. because none of the 14 mabs reacted with the denatured fragments nndc1 or nt3, we did not obtain hybridoma antibodies raised against the linear epitope located in the region containing amino acids 121-213. fragment nt5 reacted with mab m10 before denaturation, with mab m11 before and after denaturation, and with antisera from the 4 immunized mice. this fragment was immunopositive with the pooled sera and the sera from 17 of 20 cases before denaturation and with the sera from 18 of 20 of cases after denaturation. in contrast, nndc2 was immunopositive with sera from only 2 of 20 cases before denaturation and 3 of 20 cases after denaturation and was not positive with the pooled sera. these data suggest that nt5 contains both linear and conformational immunodominant epitopes. fragment nt4 reacted with the sera from only 3 of 20 cases before denaturation, indicating that the mab m13specific conformational epitope located within nt4 (amino acids 212-341) is the minor immunodominant epitope. we detected 3 conformational immunodominant epitopes located in the regions containing amino acids 1-69, 68 -213, and 337-422 on the n protein and 3 linear immunodominant epitopes in the regions containing amino acids 1-69, 121-213, and 337-422 in the sera from sars patients (fig. 1c) . the immunodominant antigenic structures identified by the antibodies raised in the sera from sars patients are consistent with those obtained from the mabs and mouse antisera. these data demonstrate that the sars-cov n protein is one of the immunodominant viral proteins and that 3 domains (amino table 3 . serologic analysis of the anti-n-protein immune response obtained by reaction of the yeast-displayed fusion proteins with sera from convalescent sars patients. the sera from individual convalescent sars patients were numbered case 1 to 20. b rays ratios before/after denaturation of the particular displayed fusion protein: ϩ, rays ratio ն2 (positive); ϫ, rays ratio ͻ2 (negative). the rays ratio was calculated as described in the text. c ten proteins, including the full-length n protein and its fragments displayed on the yeast eby100 cell surface before and after denaturation, were used in this assay. the numbers in parentheses represent the amino acid numbers of the start and stop positions on the n protein. d pooled sera was obtained by mixing the sera from sars cases 21-40. acids 1-69, 121-213, and 337-422) are responsible for the immunoreactivity (fig. 1d ). we identified 5 conformational (amino acids 1-69, 68 -213, 212-341, 337-422, and 214 -422) and 3 linear (amino acids 1-69, 121-213, and 337-422) epitopes on the fulllength n protein (fig. 1c) . our subsequent determination of the antigenic structures of the n protein responsible for antibodies in polyclonal antisera from immunized mice and sera from convalescent sars patients demonstrated the immunogenic specificity of 3 conformational (amino acids 1-69, 68 -213, and 337-422) and 3 linear (amino acids 1-69, 121-213, and 337-422) epitopes (fig. 1c) . we also found 4 novel conformational antigenic sites (amino acids 1-69, 68 -213, 212-341, and 337-422; fig. 1c ). the antibody responses of the n protein fragments with mammalian sera revealed that 3 regions of the n protein are strong antigenic domains (fig. 1d) . diverse antigenic sites on the sars-cov n protein have been identified in the sera of sars patients and in mouse antiserum (21) (22) (23) (24) (25) . although the amino acid sequence of the immunodominant epitope may be involved in these peptides and in protein segments expressed by prokaryotes, it is unlikely that polyclonal serum is sufficient to distinguish the linear from the conformational antigenic structures of individual antigenic domains and the minor or nonimmunodominant epitopes of the n protein (26 ) . the linear epitopes of the n protein identified by probing the sera from sars patients with synthetic peptides or protein segments expressed by pro-karyotes (21) (22) (23) 25 ) were limited to the strongly immunodominant antigenic sites. for example, the linear epitope located in the region of amino acids 1-69 was not obtained by pepscan or microarray analysis of sera from sars patients (21) (22) (23) . mabs provide a more powerful tool than polyclonal serum or animal antiserum for defining the antigenic structures of a given protein in the context of the antigen-antibody interactions and play a vital role in studies of viral antigenicity and immunology (27 ) . using a combined approach involving the n-protein-specific mabs, mouse antisera, and sera from sars patients, we demonstrated that the mab m9-and m11specific linear epitopes were located in the regions containing amino acids 1-69 and 337-422 of the n protein, respectively. using polyclonal sera, we also observed that the third immunodominant linear epitope was located in the region containing amino acids 121-213 on the n protein. the linear epitopes on the n protein that we identified are consistent with those reported previously, although we were able to obtain a better specificity. we believe that it was necessary to use a eukaryotic system, such as the yeast surface display, to characterize the conformational epitopes of the n protein and specifically to differentiate antibodies raised to the linear from those raised to the conformational immunodominant epitopes in mammalian sera. the yeast surface display is a useful platform in directed evolution of protein expression (30 ) for the biochemical characterization of antibodybinding sites (35, 36 ) and for serologic analysis of antigens by recombinant expression cloning (serex) (34, 37 ) . the yeast-displayed fusion protein stimulates expression of the flow cytometry histograms represent the mean fluorescence intensities of the specific yeast-displayed fusion proteins before (dotted line) and after (solid line) denaturation. the yeast pyd1 cells that reacted with the pooled normal sera from healthy volunteers (a) and pooled sera from patients with upper respiratory symptoms but not infected with sars (b) were included as negative controls. the full-length protein n (c) and fragments nt1 (d), nt3 (f; after denaturation), nt5 (h), nnd13 (i), nnd14 (j), and nndc1 (k) were immunopositive with the pooled sera of the sars patients. the antibodies in the pooled sera from sars patients were not detected by probing with nt2 (e), nt3 (f; before denaturation), nt4 (g), or nndc2 (l). because the reactivities of the displayed n protein and its fragments with pooled control sera were negative, the flow cytometry histograms are not included. the native structure of the protein of interest. linear epitopes can be distinguished from conformational epitopes on a given protein by analysis of the antigenantibody interaction before and after heat denaturation of a particular yeast-displayed protein. these features make the yeast display a suitable tool to accurately map the antibody epitope in mabs, patient serum, and polyclonal antiserum from immunized small animals. for example, using protein microarrays to probe sera from sars patients, chen et al. (25 ) identified conformational epitopes in the regions covering amino acids 51-208 and 249 -422 of the n protein. we applied the yeast surface display (34 -36 ) in mabs and mammalian sera to further define the precise locations of the conformational epitopes within the entire n protein. we first identified mab m3-, m13-, and m10-specific conformational epitopes located in the regions covering amino acids 1-69, 212-341, and 337-422, respectively (fig. 1c) , and at least 2 other epitopes in the regions covering amino acids 68 -213 and 214 -422 (fig. 1c) . three of 5 (amino acids 1-69, 68 -213, and 337-422) conformational epitopes were immunodominant, as shown in the sera from sars patients and the mouse antisera (fig. 1c) . we have expanded the sars-cov n protein epitope profile with better specificity than that of previously identified epitopes and have also identified 4 novel conformational epitopes located in the regions covering amino acids 1-69, 68 -213, 212-341, and 337-422. numerous epidemiologic and serologic studies of sars-cov (9 -11, 13 ) have suggested that the sars-cov n protein could serve as a diagnostic marker because of its early appearance (11 ) , the long persistence of serumspecific igg (14 ) , and concordance in sensitive and specific immunoassays (9 -11, 14 ) during sars infection. the false-positive (38 -40 ) and false-negative (14 ) results reported by investigators using the recombinant sars-cov n-protein-based elisa and western blot analysis have raised concerns about using the recombinant full-length n protein, whole-virus antigen extracts, or virus-infected cells as reagents to diagnose sars-cov infections in humans and other animal species. thus, the definite antigenic sites located on the n protein and epitopespecific mabs identified in our study should enhance accurate serodiagnosis, serosurveillance, and retrospective studies of sars. recent reports of sporadic cases of sars and the uncertainty of sars recurrence have prompted the current interest in the pursuit of a sars-cov vaccine (41) (42) (43) (44) (45) . a clear-cut analysis of the humoral immunity induced by active (43) (44) (45) and passive (41, 42 ) immunization with the n protein in the formulation would be a prerequisite for the future use of these vaccines in immunoprophylaxis. preservation of the conformational antibody-directed epitopes by maintenance of conformational integrity is important in designing recombinant protein vaccines and may require expression in eukaryotic cells (46 ) , which is often neglected in the development of subunit vaccines. our data on the n protein epitopes should help uncover the gene products and mechanisms of protective immunity relevant to sars-cov. the experimental strategies we describe may facilitate the discovery of epitopes of other structural proteins of sars-cov, as well as other viral proteins in humans and other animal species, which may be useful in developing subunit vaccine candidates for antiviral therapy. the functions of the n protein, such as possible interaction with viral rna and the composition of the viral core and nucleocapsid (47) (48) (49) , are well documented in other coronaviruses, and several domains of the n protein play functional roles in viral biology. primary genomic structural analysis has revealed that the sars-cov n protein contains a highly conserved motif located in the region of amino acids 111-118 (fyylgtgp) (50 ) and a unique lysine-rich region around amino acids 362-381 (ktfpptepkkdkkkktdeaq) (51 ) , findings suggesting that the protein acts as a nuclear localization signal (fig. 1d ). the mab-specific epitopes located in the regions covering amino acids 68 -213 and 337-422 on the n protein suggest that mabs m1, m4, m5, m6, m7, m10, and m11 could serve as biochemical markers to analyze the functional roles of these regions in sars-cov pathogenesis in other experimental systems. although the nterminal (amino acids 45-181) structure of the sars-cov n protein has been determined by nuclear magnetic resonance imaging (52 ) and this region has been promoted as the putative rna-binding domain (53 ) , the structure of the full-length n protein has not yet been determined. we found that 7 of 14 mabs (m1, m3, m4, m5, m6, m7, and m9) reacted only with the yeastdisplayed full-length n protein (amino acids 1-422) and the n-terminal fragment (amino acids 1-213), but not with the c-terminal fragment (amino acids 214 -422), which specifically reacted with the other mabs (m2, m8, m10, m11, m12, m13, and m14). this unexpected finding led us to speculate that the c-terminal fragment (amino acids 214 -422) is buried within the entire n protein and provides a clue to further explore the native structure of the n protein. the identification of specific antibody-binding sites in our study may assist in determination of the 3-dimensional structure of the full-length n protein and interpretation of the putative role of the n protein in the life cycle of the sars virus. focusing on the epitope information, using n proteinspecific mabs we thoroughly mapped the antigenic determinants distributed on the sars-cov n protein. we identified the immunodominant antigenic sites responsible for antibodies in sera from sars patients and antisera from small animals and used yeast surface display to identify the antigenic structures involved in these natural epitopes. the precise positions of these antigenic structures in the sars-cov n protein should be studied further to identify the exact amino acids involved. these antigenic sites of the n protein responsible for humoral immunity and histopathology will undoubtedly provide significant insight into the pathogenesis of the sars-cov and will help in the design of potential molecular targets for new antiviral agents. coronavirus as a possible cause of severe acute respiratory syndrome the severe acute respiratory syndrome isolation and characterization of viruses related to the sars coronavirus from 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acute respiratory syndrome coronavirus severe acute respiratory syndrome (sars): a year in review sars virus: the beginning of the unraveling of a new coronavirus the transmissible gastroenteritis coronavirus contains a spherical core shell consisting of m and n proteins icosahedral rna virus structure characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus structure of the n-terminal rna-binding domain of the sars cov nucleocapsid protein high affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus rna key: cord-330852-n7j0c4ne authors: fischer, wolfgang b.; wang, yi-ting; schindler, christina; chen, chin-pei title: mechanism of function of viral channel proteins and implications for drug development date: 2012-02-23 journal: int rev cell mol biol doi: 10.1016/b978-0-12-394305-7.00006-9 sha: doc_id: 330852 cord_uid: n7j0c4ne viral channel-forming proteins comprise a class of viral proteins which, similar to their host companions, are made to alter electrochemical or substrate gradients across lipid membranes. these proteins are active during all stages of the cellular life cycle of viruses. an increasing number of proteins are identified as channel proteins, but the precise role in the viral life cycle is yet unknown for the majority of them. this review presents an overview about these proteins with an emphasis on those with available structural information. a concept is introduced which aligns the transmembrane domains of viral channel proteins with those of host channels and toxins to give insights into the mechanism of function of the viral proteins from potential sequence identities. a summary of to date investigations on drugs targeting these proteins is given and discussed in respect of their mode of action in vivo. all rights reserved. the cellular life cycle of viruses is tightly connected with lipid membranes. during viral entry, membranes have to be crossed, and within the cell, the viral life depends on subcellular membranes, since viruses build their proteins along lipid membranes. they also use the principle of compartmentalizing for optimized replication and therefore deform subcellular membranes. another strategy is to capitalize on electrochemical and substrate gradients formed across membranes. for the use of these gradients in the most sensible way, channel-forming proteins are encoded in the genome of the virus. channel-forming proteins are active at almost every stage of the viral replication. they are involved during the entry phase of the virus into the host and also within the infected cell. while the roles of some channels are well identified, for others this role still has to be elucidated. the assumption that similar tasks on a molecular level can only be accomplished by the same type of molecule in almost the same conformational and spatial arrangements inspires this review on the mechanism of function of viral channels in comparison with host channels. what makes knowledge of the mechanism of function so important? there is the obvious reason that we are simply curious about it. one simply cannot stop wondering how mechanics can be achieved in an environment in which we have to leave the concepts of movements in viscous media and can only rely on electrostatic and quantum mechanical principles to get a glimpse of what is happening. learning more about the time domain of the mechanics is highly desirable as well. maybe we can understand the biological "nanomachines" and design new ones, which are "longer lasting," capable of handling bigger loads, and show other improved characteristics. since the viral channel-forming proteins are smaller than their host companions, their architecture depicts miniature design in an already miniaturized world, or going down-scale in an already down-scaled world. on the other hand, there is crucial wish to secure and cure our health as well as to ease our life. how does knowledge about the mechanism of function fit into this wish? if we know the function of an enzyme, which cleaves a specific substrate, we can design a substrate which cannot be cleaved but rather jammed into the active site of the enzyme. if we know the conformation transition the molecule undergoes, we are able to generate more potent drugs. dealing with other proteins than enzymes, such as the channel proteins mentioned here, the site of drug action is not so easy to elucidate. in principle, we "see" the protein but do not know where to go with our drug molecule. consequently, an active site may be one of the many conformations the protein adopts during its "working cycle." from this particular perspective, one has to explore the entire conformational space of the protein as a prerequisite for improved drug development. crystal structures serve as an important starting point for computational drug development representing "frozen" conformations. computational modeling is able to screen and visualize more conformational space and in this respect possibly "link" individual crystal structures. the idea is to analyze the viral channels in respect to larger host channels. therefore, the architecture of those channels, which are anticipated to have a relation to the viral channels, is introduced. the relation is driven by sequence alignment of viral channel forming proteins (vcps) with those of host proteins . identifying related host proteins, specifically gating mechanics are elucidated and cross-related to the viral channels. in general, there are several mechanical movements especially within the lipid membrane known for ion channels, such as moderate conformational changes (cymes and grosman, 2008; miyazawa et al., 2003 ; and references therein) and sliding of helices (chang et al., 1998; kuo et al., 2003) . openings of pores designed by toxins seem to be made irreversibly by assembly (mueller et al., 2009) . finally, the idea of "drug hunting" is outlined in respect to small molecule drugs and peptide drugs. the proteins are introduced with respect to the mechanism of function. all citations are driven by the idea to deliver structural information. for extended references about molecular biological information, it is referred to respective reviews. the work on viral channel-forming proteins has been reviewed in great detail in the literature gonzales and carrasco, 2003 ; and referenced literature below). generally, the name "viroporin" or "vcp" is established in the literature for this class of proteins. so far, some of the channels are very selective for protons, others are permeable for small molecules, while the majority though are weakly selective for ions. as to date, the biophysical role of the vcps can be summarized to alter electrostatic repulsion of proteins so that lipid membranes can approach each other during fusion and budding, as well as alteration of cell homeostasis and membrane depolarization induced cell apoptosis (franco and bortner, 2006) . vcps comprise a series of proteins with different transmembrane (tm) topology. with the discovery of m2 from influenza a and vpu from human immunodeficiency virus type 1 (hiv-1), proteins with a single tm topology have been found. later, proteins such as 2b from polio virus and p7 from hepatitis c virus (hcv) have been proposed to harbor two transmembrane domains (tmds). the recently suggested viral ion channel from severe acute respiratory syndrome coronavirus (sars-cov), 3a, is now the longest channel with three tmds. other proteins found to form channels are belonging to either one of these classes of channels. how can a protein be defined as a channel? channels are made of subunits of proteins forming a circular tertiary structure around a symmetry axis. the channels per se could be either homo-or hetero-oligomeric. they are membrane embedded and conduct more or less selectively either one of the physiological relevant ions. the degree of selectivity comes with the diameter and side chain composition of the pore formed by the assembled proteins, the lumen of the pore. the wider the pore is, the less selective it should be. less selective channel proteins are likely to be called pores (gonzales and carrasco, 2003) , such as, 2b from polio virus, which is able to conduct besides ions also small molecules. in the following, the focus is on those vcps for which detailed structural information is available either from experiments or from computational studies. 2.1. m2, vpu, and p7 2.1.1. m2 from influenza a the genome of influence is spread over eight single-stranded rna segments. on the seventh segment, which encodes the matrix protein m1, a second overlapping reading frame has been identified coding a protein of about 11 kda called m2 (allen et al., 1980; lamb and choppin, 1981; lamb and lai, 1981; winter and fields, 1980) (fig. 6.1a ). its topology is found to be monotopic comprising an n terminal side on the extracellular side marking it a type iii integral membrane protein present at the plasmamembrane of infected cells (lamb et al., 1985) . the amino acid distribution indicates about 18-23 amino acids on the extramembrane side, 19 amino acids membrane spanning, and 54 amino acids toward the c terminus (zebedee and lamb, figure 6 .1 available structures of m2 and bm2 from experimental sources. the structures are shown in "gaussian contact mode" (moe software) indicating hydrophilic residues in blue and hydrophobic residues in green. in addition, helices are shown as yellow bands and specific amino acids are represented in a stick modus (histidines in red and tryptophans in violet). (a) m2 channel: 2l0j from solid state nmr (sharma et al., 2010) ; 2rlf from solution nmr (schnell and chou, 2008) , the drug rimantadine is shown in stick modus (blue); 3bkd from x-ray and expressed protein (stouffer et al., 2008) ; 3c9j from the same source as 3bkd with the drug amantadine shown in stick modus (green); 3lbw from spps (acharya et al., 2010, from spps) ; (b) tmd (2kix) and cytoplasmic domain (2kj1) of bm2 (wang et al., 2009b) . (c) structure of a synthetic peptide corresponding to pb1-f2 solved by nmr spectroscopy (bruns et al., 2007). 1988). it is also present in the virion with about 14-68 molecules (zebedee and lamb, 1988) . m2 assembles into cystein-linked homodimers which noncovalently associate into homotetramers (holsinger and lamb, 1991; sugrue and hay, 1991) . some of the m2 proteins of various subtypes have been identified to be palmitylated at cys-50 (sugrue et al., 1990) . amantadine resistance mutant studies of influenza viruses correlated resistance to mutations in the seventh segment, especially to the tmd of m2 (hay et al., 1985) . mapping the mutations on a helical wheel reveals that the mutations are along one side of the helical structure. the mapping supports the idea that the protein assembles around a kind of central axis which is relevant for its functioning as a proton channel (sugrue and hay, 1991) . experiments with a synthetic peptide construct representing the tmd of m2 reconstituted into artificial bilayers have verified its proton activity (duff and ashley, 1992) . also, whole-cell recordings of m2 and mutant m2 expressed in xenopus laevis show that m2 activity is enabled by low ph and that amantadine is interacting with the channel (wang et al., 1993) . channel activity has also been demonstrated in mammalian cells (wang et al., 1994) and vesicle-based fluorescence essays (schroeder et al., 1994) , with the latter technique confirming proton conductance (lin and schroeder, 2001) . reconstitution of the protein into artificial bilayers also reveals channel activity of m2 (tosteson et al., 1994) . electrophysiological experiments with m2 in mouse erythroleukemia cells have been conducted on ion selectivity confirming that m2 is 10 7 times more selective for protons than for monovalent cations (chizhmakov et al., 1996) . whole-cell recordings with m2 expressed in xenopus oocytes reveal a very low conductance of about 1-10 fa, which allows about 104 protons per second to pass (mould et al., 2000) . these measurements are flanked by results from studies with x. laevis (shimbo et al., 1996) . using the whole-cell patch clamp technique on x. leavis, first evidence is given that the protonation state of the one histidine within the tmd is important for conductance (wang et al., 1995) . proton conductance of full-length m2 expressed in bl21 cells and reconstituted into liposomes has also been shown to be independent on the presence of electrolytes (vijayvergiya et al., 2004) . using a fluorescence essay based study, it is reported that proton conductance is independent of the content of cholesterol in the liposomes (lin and schroeder, 2001) . with this finding, it is proposed that m2 is active in lipid raft free environment and would just be associated with a lipid raft due to its cytoplasmic part. the authors suggest that raft association may control the number of m2 proteins to be incorporated into the virion. experimental evidence that the tmd of m2 is helical has been achieved with m2 peptide reconstituted into dopc liposomes using cd spectroscopy . solid nmr spectroscopic measurements with m2-tmd peptides confirm the helical motif, deliver tilt angles of the peptide within dmpc bicelles of about 33 , and suggest a left-handedness of the putative bundle (kovacs and cross, 1997) . by adding data from functional studies like cys scanning and electrophysiological measurements as mentioned as well as computational modeling data (sansom and kerr, 1993; sansom et al., 1997; zhong et al., 1998) , an approximate structural model of the tetrameric assembly of the tmds of m2 with the histidines and tryptophans as important pore lining residues has been generated. in a combined study using ftir spectroscopy with sitespecific labeled amino acids and a dynamic molecular global search protocol, further strong support for the tetrameric model has been achieved . extensive solid state nmr spectroscopic investigations on chemically synthesized tmd peptides of m2 delivered "high-resolution" distance and orientation data sets of the residues at ph 7.0 (nishimura et al., 2002) . a precise proposal has been given for the orientations of the tryptophans and histidines in a tetrameric assemble. in addition, helix stability has been confirmed. according to models of assembled helices, a water filled cavity toward the n terminal side has been suggested. solid state nmr measurements of full-length m2 expressed in escherichia coli and reconstituted into dmpc bicelles confirm the findings on the studies with peptides (tian et al., 2002 (tian et al., , 2003 . these studies indicate a mode of assembly of the tmds which is independent of the rest of the protein . studies based on tryptophan fluorescence on fully expressed m2 and mutants reveal that trp-41 and his-37 work in concert upon low ph activation (czabotar et al., 2004) . together with other experimental wang et al., 1995) and computational studies schweighofer and pohorille, 2000) , it is proposed that several mechanisms of function shuttle the proton across a selective and narrow part around the histidines of the channel. in one mechanism, called "shuttle" mechanism, the incoming proton is taken up by his-37 on its d-nitrogen which induces a release of the hydrogen on the e-nitrogen. via a tautomerization, the sidechain of his-37 is reloaded. in another mechanism, the "water-wire" mechanism, the protonated histidines will repel each other allowing for an opening and transport via a hydrogen wire. computational modeling combining classical molecular dynamics simulations with multistate empirical valence bond (ms-evb) approach (schmitt and voth, 1998) has shown that a hydronium ion will not move through the histidine ring. it is rather anticipated that the proton could shuttle even if the histidines are not fully removed to generate a kind of "open" channel (smondyrev and voth, 2002) . the consequence of this study is that not all of the histidines need to be protonated and consequently charged to generate an "open" channel. solid state nmr spectroscopic investigations have shown that at high ph, the channel can exist in a closed state when a neighboring pair of the histidines share a proton, and consequently, a third one releases this pairing and opens for a putative water-wire to conduct the proton (hu et al., 2006) . with the detection of conformational distinct tryptophans upon low and high ph conditions by 19f nmr spectroscopy, a contribution of trp-41 during channel gating has been verified (winter et al., 2008) . finally, to date a cooperative gating model is proposed in which upon low ph on the n terminal side one histidine gets trapped via a cation-p interaction with the tryptophan while the other histidine is able to offer one of its protons to the water on the c terminal side (sharma et al., 2010) . structural models at atomic resolution are available based on nmr spectroscopy (pielak and chou, 2010; schnell and chou, 2008 ) and x-ray crystallography (acharya et al., 2010; stouffer et al., 2008; wang et al., 2011) (fig. 6.1a) . all structures reveal a left-handed bundle. for the nmr investigations, a tmd m2 construct with an amphiphatic helix at the c terminal side has been used to stabilize the helix bundle which otherwise could not have been investigated (schnell and chou, 2008) . the expressed m2 construct is reconstituted into dhpc detergent micelles and due to solution nmr spectroscopic investigations at high ph (ph 7.5). the construct is recorded in the presence of the antiviral drug rimantadine which interacts with the helix bundle at the lipid protein interface. the bundle forms a narrow pore which has a wider pocket of about 0.6 nm around gly-34 and is highly restricted around his-37 and trp-41. the structure is referred to as a potential closed form of the channel (pielak and chou, 2010) . solely, the tmd of m2 has been crystallized and its structure resolved to 0.2 nm in octyl-b-d-glucopyranoside (og) detergents at high ph (ph 7.3) (stouffer et al., 2008) . the pore is opened into a tepee-like shape with its restriction on the n terminal side and the trp-41 apart from each other. a mutant m2 (gly-34 is replaced by alanine) cocrystallized with amantadine at lower ph (ph 5.3) identifies a structure which is similar in shape as the one crystallized at ph 7.3. the binding site of amantadine is marked to be within the lumen of the pore. the shape of the bundle is considered to represent an open form of the channel. solution nmr studies reveal that at low ph the tmds are dynamic (hu et al., 2011; li et al., 2007; pielak et al., 2009) . it is a common theme of all models that, besides the his-x-x-x-trp motif, asp-44 and arg-45 play an additional role in the gating mechanism. similar to influenza a, segment 7 of the influenza b genome also harbors two proteins m1 and m2 with the latter to be a proton channel called bm2 (briedis et al., 1982) of about 15 kda (109 amino acids) (horvath et al., 1990) . the sequence of bm2 is highly conserved which indicates its importance for the virus (hiebert et al., 1986) . the protein is phosphorylated and localized in the cytoplasm but also transported to the plasma membrane where it is incorporated into virions (odagiri et al., 1999) . like m2, bm2 is a type iii integral membrane protein with an h-x-x-x-w motif but with no further sequence homology with m2 including cysteins in the ectodomain . its role is suggested to equilibrate the ph gradient between the golgi and the cytoplasm by forming proton channels (mould et al., 2003) . proton conductance of bm2 is found to be higher than for m2. mutation studies identify polar serine residues (serines 9, 12, and 16) facing the lumen of the pore which is discussed as an explanation for the resistance of bm2 against amantadine (ma et al., 2008; paterson et al., 2003) . the oligomerization state is experimentally confirmed to be a tetramer together with functional studies using the two electrode voltage clamp method (balannik et al., 2008) . despite functional similarity with m2 protein, bm2 cannot form oligomers with m2. not forming a tetramer via the "dimer of a dimer" concept, the route of assembly seems to follow the same as for other vcps such as vpu, p7, or 2b. solution nmr spectroscopy identifies a large left-handed coiled-coil tetramer consisting of two segments, separated by a 10-amino-acid region (residues 34-43) (wang et al., 2009b) (fig. 6 .1b). the "full-length" structural model is derived from experimental results of two overlapping constructs: a tmd containing construct bm2 1-33 (2kix) and a cytoplasmic domain containing construct bm2 26-109 (2kj1). with its large cytoplasmic domain which generates a large dipole moment, it is suggested that bm2 is also involved in the recruitment of matrix proteins at the cell surface and in combination with that involved in the viral assembly and budding process. the channel domain consists of two heptad repeats leu-8 to ile-14 and leu-15 to ile-21, and the data confirm serines 9, 12, and 16 as pore lining. ser-12 completely faces the pore while the other two are also involved in interhelix packing. the pore is occluded by a ring of four phe-5 and trp-23. interesting to note is that mutations of ser-12 and ser-16 into alanines affect proton conductance much more than mutations of his-19 and his-27 into alanine (wang et al., 2009b) . computational modes of the tmds of bm2 have been modeled using coarse-grained simulation techniques in combination with united atom simulations (rouse et al., 2009) . the suggestion in this study is that, also in this channel, a minimum of three histidines need to be protonated to induce conformational changes which lead to an open conformation. antiviral amantadine does not inhibit the activity of bm2 . the reason is found to be due to the polar serines facing the lumen of the pore. more polar drugs seem to be necessary to block the channel (ma et al., 2008) . recently, a monoclonal antibody has been identified to target the ectodomain of bm2 comprising antiviral activity successfully (wang et al., 2010b) . with pb1-f2 encoded by influenza a, a second channel protein has recently been identified (chanturiya et al., 2004; henkel et al., 2010) of which a structural model for its tmd is reported (bruns et al., 2007) ( fig. 6.1c ). this protein has been found with a strong tendency to oligomerize. this would be the second virus, next to sars-cov which encodes more than one channel protein. the studies have been done with synthetic peptides reconstituted into planar lipid bilayers and microsomes. the structural integrity has been demonstrated with a united atom md simulation in a fully hydrated lipid bilayer. to date, investigations on m2 are very advanced in respect to structural and functional studies. albeit, structural information about the cytoplasmic domain is still lacking, the tmd is very well characterized by nmr and x-ray data. the protein has been for a long time a potent drug target (davies et al., 1964) . in the late 1980, a novel open reading frame hosting a 16-kda protein has been detected independently by two groups (cohen et al., 1988; strebel et al., 1988) (fig. 6.2 figure 6 .2 available structures of vpu from experimental sources. the structures are shown in "gaussian contact mode" (moe software) indicating hydrophilic residues in blue and hydrophobic residues in green. in addition, helices are shown as yellow bands and specific amino acids are represented in a stick modus (histidines in red, tryptophans in violet, and serines in orange). 1pi7 tmd from solid state nmr (park et al., 2003) , 1vpu (willbold et al., 1997) and 2k7y (wittlich et al., 2009) of a frame shift mutation into the vpu gene, an up to 10-fold reduction of viral load has been observed (strebel et al., 1988) . the conclusion drawn from the experiments has been that vpu is involved in virus assembly and particle release. the new protein has not been detected in hiv-2 and siv (cohen et al., 1988) . vpu's involvement in inducing particle release (strebel et al., 1988 (strebel et al., , 1989 terwilliger et al., 1989) has been attributed to a downregulation of the receptor protein cd4 via the proteasome pathway (ruiz et al., 2010a; willey et al., 1992a,b) and the formation of ion channels (ewart et al., 1996; schubert et al., 1996b) . the two routes of action are attributed to two distinct domains of the protein (schubert et al., 1996a) : the cytoplasmic domain for the former (bour et al., 1995) and the tmd for the latter (schubert et al., 1996b) . the mechanism of how the protein enhances virus particle release is anticipated by the formation of a homooligomer at the site of the plasma membrane which renders the membrane permeable for ions (schubert et al., 1996b) . cd4 degradation is found to be due to the interaction of a short sequence, krllsekkt, in the cytoplasmic tail of cd4 with a helical domain of the cytoplasmic part of vpu (tiganos et al., 1997) . also, residues at the linker region between the tmd and cytoplasmic domain of vpu contribute to cd4 degradation . for cd4 downregulation, the two phosphorylation sites of vpu, ser-52 and ser-56, are identified to be essential (paul and jabbar, 1997; schubert et al., 1994) . downregulation via interaction with vpu has also been reported for btrcp (margottin et al., 1996) to hand over cd4 to the proteosomal degradation pathway. also, other host proteins such as vpu binding protein (ubp) (callahan et al., 1998) , cd74 (hussain et al., 2008) , and cd317 (bolduan et al., 2011; neil et al., 2008; van damme et al., 2008) are "marked" by vpu for downregulation. interaction of vpu with host factors at the site of the plasmamembrane are reported for task channels (hsu et al., 2004) and bst-2/tetherin (also called cd317) (neil et al., 2008; van damme et al., 2008) . in both host proteins, the tmd of vpu is responsible for the interaction (hsu et al., 2004; skasko et al., 2011) . for bst-2, it has been shown that vpu slows down bst-2 transport to the plasma membrane (dubã© et al., 2010) and that the vpu-bst-2 complex is retained in the er (skasko et al., 2011) . the interaction is reported to be due to the tmds of both proteins. bst-2 itself is known to form a dimer and crystallographic data identify for the extramembrane part an elongated helical motif which forms a coiled-coil toward the c terminal side and a flexible region able to assemble into a tetramer on the n terminal side (hinz et al., 2010; schubert et al., 2010) . the loop sequence, eyrkll, connecting the tmd of vpu with its cytoplasmic domain has been shown to be responsible of transporting vpu to the plasma membrane (ruiz et al., 2008) . at the site of the plasma membrane, it is anticipated that vpu forms channels via oligomerization. expression of vpu in amphibian oocytes induces conductance of cations measured under voltage clamp conditions (coady et al., 1998; schubert et al., 1996b) which shows marginal effect on divalent ions such as ba 2ã¾ and ca 2ã¾ . together with peptides representing the tmd and a scrambled sequence, it has been shown that solely the tmd exhibits cation specific channel activity. a series of experiments has been performed which show that expressed vpu in e. coli purified and reconstituted into artificial bilayers exhibit channel activity (ewart et al., 1996; mehnert et al., 2007) . recordings solely with the tmd of vpu reveal similar conductivity compared to full-length protein with minimal changes in the kinetics (ma et al., 2002; mehnert et al., 2007) . replacing trp-23 by leu in a synthetic peptide construct, vpu 132 , reconstituted into artificial lipid bilayers, alters the open time duration and shut kinetics of the peptide, while a change of ser-24 to leu abolishes channel activity completely (mehnert et al., 2008) . no affect has been reported when arg-31 is exchanged into val. channel recordings of the tmd of vpu in solutions of different ions reveal that the vpu channels which are only slightly selective indicate almost pore-like characteristics. it has been suggested that vpu shows a channel-pore dualism (mehnert et al., 2008) which means that vpu can either act as a more or less selective channel or nonselective pore depending on specific in vivo conditions. these conditions could possibly be due to changes in lipid environment. in a recent electrophysiological study, using whole-cell clamp conditions with 293t cells expressing full-length vpu mutant s24a does also not exhibit channel activity (bolduan et al., 2011) . in another study, in which vpu has been expressed in e. coli, the protein renders the membrane of the cells susceptible to a series of molecules as well (gonzales and carrasco, 1998) . 2-nitrophenyl-d-galactopyranoside, uridine, translation inhibitor hygromycin b, and lysozyme are reported to pass the membrane as well as hygromycin b and neurobiotin when expressed in eukaryotic cos cells. channel activity allows the change of electrochemical gradients, depolarization, across the lipid membrane. the effect of depolarization of the membrane is reported to affect the fission of the budding hiv-1 virion from the infected hela cells (hsu et al., 2010 ) (e.g., lowering electrostatic repulsion to enable fission): blocking two-pore k ã¾ (k 2p ) channel task virion release with vpu inactive hiv leads to an enhancement of virion release. since task channel activity in hela cells measured under wholecell voltage clamp conditions is not affected in the presence of vpu, it is concluded that vpu reduces the number of task channels at the plasmamembrane by interacting with the k ã¾ channel making it susceptible for degradation. sequence similarity of the tmd with the first tmd of task implies physical interaction of the two proteins (hsu et al., 2004) . it can be concluded at this state that vpu interacts with host proteins, which leads to downregulation via the proteasomal pathway or redirecting them. consequently, channel activity of vpu per se does not seem to be necessary to fulfill its auxiliary role in enhancing viral release. yet, vpu especially when inserted into lipid membranes shows channel activity which can be modulated by mutations. escape mutant studies as done for m2 from influenza a (hay et al., 1985) are lacking and a selective "channel blocker" is not yet identified (lamb and pinto, 1997) . the recent anti-channel drug bit225, n-[5-(1-methyl-1h-pyrazol-4-yl)naphtalene-2-carbonyl]-guanidine, seems to fulfill the role as a vpu channel blocker when administered in a concentration of 40 mm to vpu reconstituted into artificial lipid membranes of a channel recording device (khoury et al., 2010) . structural information of vpu has emerged from solution and solid state nmr spectroscopy ( fig. 6 .2). in the late 1990s, two structural models of the cytoplasmic domain of vpu have been published using solution nmr spectroscopy ( fig. 6 .2). the structural features discovered have been a helix-loop-helix motif followed either by another short helix (willbold et al., 1997) or a reverse turn (federau et al., 1996; wray et al., 1995) . while the former data derived from recordings of vpu expressed in e. coli purified and dissolved at high salt solution, the latter spectra have been recorded from a synthetic peptide dissolved in aqueous tfe solution. it is debated that the high salt solution induces the tertiary fold and thus the formation of the third helix. tfe is known to support helix formation but weakens the formation of a fold. more recent data in low salt solution confirm two helical motifs in the cytoplasmic domain (wittlich et al., 2009 ). measurements in the presence of micelles formed by dodecylphosphatidycholine (dpc) induce secondary elements (two helices) and a tertiary fold. secondary structures are supported by cd spectroscopy (wittlich et al., 2009; wray et al., 1995) . all structural investigations have in common that the two series 52 and 56 have not been phosphorylated. investigations on the structural implications of phosphorylation of the two serines to the structure have been done either on a synthetic peptide in aqueous tfe solution (coadou et al., 2001 (coadou et al., , 2002 or on a peptide expressed in e. coli, purified and measured in the presence of dpc micelles (wittlich et al., 2008) . without dpc upon posphorylation, parts of the cytoplasmic helices unwind into a b-strand (coadou et al., 2002) , while in the presence of dpc micelles, structural changes are limited to some loss of helicity of helix1 toward the c terminus and extension of helicity toward the n terminal side of helix2 (wittlich et al., 2008) . the helix motif for the tmd of vpu has first been suggested by solid state nmr spectroscopy (marassi et al., 1999; wray et al., 1999) . applying the same technique to extended constructs of the tmd of vpu with residues of the cytoplasmic domain indicates that the second helix is aligned parallel to the membrane surface (marassi et al., 1999; park et al., 2003) . x-ray reflectivity measurements done with full-length vpu on a lipid monolayer reveal that the second helix in the cytoplasmic domain is bound loosely (zheng et al., 2001) . the helical motif of the tmd of vpu has been supported by site-specific ftir spectroscopy . ssnmr data show that the tmd vpu construct shows rotational dynamics within the lipid membrane (park et al., 2006) . the extent of the tmd helix is still controversial, when comparing all the ssnmr data. in a recent study, the length of the helical motif toward the c terminal side of the tmd is reported to extend beyond the hydrophobic slab of the bilayer (sharpe et al., 2006) . the tmd of vpu is found to exhibit a weak kink around ile-17, a result obtained using a vpu 2-30ã¾ construct measured by solid state nmr spectroscopy (park et al., 2003) . md simulations using a vpu 1-52 construct support the findings reporting kink angles to vary between 7 and 15 around the same amino acids found experimentally (sramala et al., 2003) . computer simulations in various lipids identify a kink around ser-24 due to the compensation of hydrogen bonding of the side chain with adjacent backbone residues toward the n terminal side (krã¼ger and fischer, 2008) . further bending of the helix is found from ser-24 to ile-20. kinking is seen as a mechanism to compensate for varying lipid thicknesses. to summarize the findings, there could be hinge regions around ile-17 and ser-24 proposing modular segments of the protein within the lipid bilayer. according to the hypotheses of vpu being released after manufacturing into the er membrane, the protein could oligomerize prior to any interaction with other host proteins. oligomerization is also seen as a prerequisite of channel formation. earliest studies point toward an oligomeric assembly using sds-page (maldarelli et al., 1993) . on the bases of synthetic peptides linked together as either tetramers or pentamers and their channel activity, the latter state is reported to be the favored one (becker et al., 2004) . full-length vpu and its tmd alone, both expressed in vitro and analyzed using gel permeation chromatography, are found to be in a pentameric state (hussain et al., 2007) . computational modeling of the tmd based on ssnmr data on expressed tmd of vpu supports the pentameric state (park et al., 2003) but a tetrameric state is also proposed. a combined experimental study using bilayer recordings on synthetic peptide based on the tmd and computational modeling using md simulations is in favor of the pentameric assembly (cordes et al., 2002) . pentameric assemblies have been used for the computational modeling of the vpu ion channel embedded in hydrated lipid bilayers (cordes et al., 2001; grice et al., 1997; moore et al., 1998) . simulations of hexameric assemblies in a hydrated slab of octan, mimicking a lipid bilayer, have been reported to collapse and are disregarded as a potential assembly (lopez et al., 2002) . in another simulation study in a hydrated popc bilayer system, the tetrameric and hexameric assemblies have been ruled out based on the derived pore radii and the estimated conductance these pores would show (cordes et al., 2002) . simulations on extended models including the first cytoplasmic helix (sramala et al., 2003) and the entire cytoplasmic part have been reported with the proteins embedded either in a lipid monolayer at the air water interface (sun, 2003) or in hydrated popc sramala et al., 2003) . in one simulation of full-length vpu, the cytoplasmic domain has been generated artificially based on the available nmr data (sun, 2003) while for the other simulations experimentally derived cytoplasmic domain data have been artificially merged with simulated extended vpu ; tmd and first cytoplasmic helix taken from sramala et al., 2003) . vpu is predominantly acting via interactions with other host proteins. structural information of the tmd is in lack of x-ray crystallographic data, but in contrast to m2, the structural details of the cytoplasmic domain are well resolved. ion channel functionality is still being debated and it seems that vpu may emerge as a potential drug target. the genome of hcv is expressed as a large polyprotein and cleaved by cellular and viral proteases into 10 cleavage products among which is p7, a 63 residue 6-7 kda bitopic membrane protein (elbers et al., 1996; lin et al., 1994) (fig. 6 .3). the protein is preceded by the structural protein e2 from which it is incompletely cleaved and succeeded by the nonstructural protein ns2. it is not clear at this moment to which side structural or nonstructural p7 belongs to. abolishing the cleavage between e2 and p7 results in noninfections virions (harada et al., 2000) . expressing e2 and p7 independently recovers the production of infectious virions making p7 an essential part of the life cycle of hcv. also, functional relevant interaction of p7 with ns2 has been reported (tedbury et al., 2011) . ns2 is located to sites where viral replication and particle assembly take place. in the absence of p7, the location of ns2 to these sites is lost. this function of p7 is reported to be independent of its function as a channel protein. it has been suggested that p7 plays an accessory role in altering the topology of ns2 which consequently affects ns2 function (ma et al., 2011) . as a channel-forming protein, p7 is proposed to have the same role in the life cycle of the virus as m2 supporting cell entry (griffin, 2009) . it has been discovered that p7 is necessary during the early stage of particle assembly ( jones et al., 2007; steinmann et al., 2007a) . using a so-called j6/jfh chimeric genome which can be used to study the hcv life cycle in human hepatoma cells (huh-7.5), it has been shown that p7, but not its precursor forms e2-p7 and p7-ns2, is required for infectious virus production ( jones et al., 2007) . mutations in the basic loop between the two tmds (kr33/35qq or kr33/35aa) lead to a decrease of the amount of released infectious virions (steinmann et al., 2007a) . it is proposed that these mutations affect channel activity as they are at the mouth of the putative pore. other mutations within the tmds of p7 (tmd1 and tmd2), such as the highly conserved trp-30 and tyr-42 each of them being separately mutated into phenylalanine, impair the infectivity of the virions. mutations of his-31 also do not have an effect on rna replication (steinmann et al., 2007a) , similar to the wild-type p7 (lohmann et al., 1999) . concluding from these data, p7 is not essential for viral entry, supporting the essence of p7 within the later stage of infectivity which is linked to virus assembly. nevertheless, p7 is essential for the virus but the mechanism of ion channel activity is not yet linked to either of these events. the topology of p7 has been identified to contain two tmds connected via a short hydrophilic segment. both ends of the tmds are found to point into the er lumen (carrã¨re-kremer et al., 2002) . the protein is detected within the plasma membrane with both termini pointing toward the extracellular environment. experiments with p7 expressed in e. coli (clarke et al., 2006; griffin et al., 2003) , as well as p7 protein derived from solid phase peptide synthesis (spps) (pavlovic et al., 2003; premkumar et al., 2004) , both individually reconstituted into artificial lipid bilayers disturb the 2k8j tmd2 tmd1 his-17 tmd1 tmd2 tmd2 figure 6 .3 available structure of p7 from experimental and computational sources. the structures are shown in "gaussian contact mode" (moe software) indicating hydrophilic residues in blue and hydrophobic residues in green. in addition helices are shown as yellow bands with histidine residue represented in a stick modus (red). 2k8j: tmd2 from solution nmr (montserret et al., 2010) , boxed; top row to the right is a computationally assembled monomer, lower row a potential hexameric assembly of the monomers forming a channel. bilayer, making it susceptible to ion conductance. most recent data reveal that channel activity is dependent on lipid composition and they are discussed together with data from cd in terms of a change in topology of the protein (whitfield et al., 2011) . it is proposed that a lipid dependent equilibrium between an antiparallel alignment and a l-shape alignment (one helix parallel the other perpendicular to the membrane normal) of the two tmds exists. in another study of p7 purified from the same expression system p7, it has been reported using transmission electron microscopy (tem) that the protein assembles into a hexamer (griffin et al., 2003) . with a flag-p7 construct at the n terminal side also expressed in e. coli, purified and measured with tem, a heptameric assembly is proposed (clarke et al., 2006) . the heptameric assembly has been supported by sds-page and mass spectrometry. most recently, electron micrographs of p7 synthesized by spps are in support of a hexameric assembly of the protein (luik et al., 2009) . the first computational model of p7 has been generated using a coarse grained conformational search protocol (patargias et al., 2006 ) (see also fig. 6 .3). the two tmds and the short link between them have been identified as a consensus from the results of multiple secondary structure prediction programs (cuthbertson et al., 2005) . the antiparallel aligned p7 protein is hither forth called the monomer. the alignment as a monomer positions the tmds so that a bundle could be formed in which the histidines of tmd1 of all the monomers point into the putative lumen. consequently, a pivotal role for this particular histidine in gating has been suggested (patargias et al., 2006) . other groups have also challenged computational modeling to fill the data from electron microscopy (em) with structural information (griffin et al., 2003) . most recent md simulation studies with an extended p7 protein randomly positioned in a simulation box together with lipid molecules using coarse grained techniques support the antiparallel alignment of the tmds in the p7 monomer (luik et al., 2009) . the role of histidine in the conductance of ions across the protein bundle has been supported by mutant studies of chemical synthesized p7 reconstituted into lipid bilayers . studies with cu 2ã¾ containing solutions in combination with wild-type and h17a mutants have allowed the conclusion that histidine is pore lining, as predicted earlier (patargias et al., 2006) . solution nmr spectroscopic experiments with p7 expressed in e. coli and reconstituted into dhpc micelles deliver data which support its bitopic topology opella, 2010, 2011) . both of the two tmds are split into two segments with specific dynamics comprising four helical segments. the two unstructured n and c termini as well as the short and mobile loop segment between the tmds comprising residues 28-36 are outside of the membrane. residues 5-15 constitute a helical segment embedded into the micelles which is similar to the third segment in its dynamics. while the first helix has no membrane anchoring amino acids, the third helical segments including residues 41-48 seem to be affected by the dynamics of the loop segment. the second helix (residues 17-27) is found to be less mobile and it is suggested that this is due to the membrane anchoring aromatic residues trp-30 and tyr-31. similarly, low mobility is also found for the fourth helix which is flanked by two prolines, pro-49 and pro-58, containing a stretch of five leucines (leu-50-leu-54). in another solution nmr spectroscopic study, data of full-length p7 derived from peptide synthesis and recorded in a mixture of trifluoroethanol and water also mainly identify helical motifs (montserret et al., 2010) ( fig. 6.3, boxed) . flexible regions around residues gly-15 and gly-18 of tmd1 and at the c terminal end of tmd2 are observed. cd measurements in various membrane mimetic environments support the overall helicity of the protein in its monomeric state. distance restraints from the nmr measurements have been used as input for computational modeling using a docking approach in combination with extended molecular dynamics simulations to model the assembly of the two tmds. the model reveals that the aromatic residues phe-25, trp-30, tyr-42, tyr-45, and trp-48 form a staged p-system. channel characteristics of p7 reconstituted into asolecitin bilayers identify a voltage-dependent current behavior which is rectifying and showing an 11 times higher selectivity for cations (montserret et al., 2010) . main conductance levels are reported to be 22 and 41 ps at ã¾60 and ã¾140 mv, respectively. patch recordings with liposome identify slightly higher conductance levels at around 35, 57 120, and 184 ps when a pipette holding potential of ã¾140 mv is applied. channel recordings are reported to be blocked by hexamethylene amiloride (hma) but not by amantadine. the pore lining tmds is identified to be tmd1, since a major conductance level of about 60 ps has been found at a holding potential of ã¾100 mv when reconstituted into lipid bilayers. in contrast, there could be no channel recordings observed for tmd2. a synthetic full-length p7 construct derived from spps shows a reversal potential of 44.3 mv in a 10:1 (cis: trans) gradient buffer, suggesting a moderate cation selectivity (premkumar et al., 2004) . the experiments indicate that the protein bundle shows lower permeability for calcium ions and considerable chloride conductance. the oligomerization state of p7 is dependent on the protein:detergent ratio (c 12 e 8 ) when using sedimentation experiments (montserret et al., 2010) . at lower ratio, a hexameric assembly is proposed which can shift to slightly higher numbers at higher ratios. so far, crystallization trials failed to deliver any conclusive structural information ( jones et al., 2007) . his-17 has been identified to point into the lumen of the pore (patargias et al., 2006) . this residue has also been found in m2, which is identified as proton sensor involved in gating of the channel. conductance studies of p7 when reconstituted into artificial lipid bilayers reveal that the presence of cu 2ã¾ in the bath solution blocks the activity of p7 ). since copper ions can be complexed by histidines, this study confirms the orientation of these residues toward the pore. with histidines in the pore, it has also been suggested that this allows ph sensitivity in any kind of form (patargias et al., 2006) : is the p7 bundle just sensitive to protons but does not conduct them? or is p7 also a proton conductor similar to m2? it has been now suggested that protonation of the histidines will lead to an opening of the pore (foster et al., 2011) . with further analogy to m2, the question arises, can the protein also be built by less than the six units as it is proposed to date? of course, experimental evidence is given for larger units, such as hexamers and possibly heptamers. other channel-forming proteins 2.2.1. 6k from alphavirus a series of proteins in other viruses have also been reported as vcps (carrasco, 1995; fischer, 2005; fischer and sansom, 2002; gonzales and carrasco, 2003) . one of these proteins is 6k, which is found in several viruses of alphavirus genus belonging to the family togaviridae (carrasco, 1995; garoff et al., 1982; madan et al., 2005; schlesinger and schlesinger, 1986; strauss and strauss, 1994; wang et al., 2010a) . alphaviruses express their structural proteins in a polyprotein consisting of e3-e2-6k-e1-c which is proteolytically processed into its constituents (liljestrom and garoff, 1991) . the 6k protein is a 6 kda hydrophobic protein of 60 amino acids which is palmitylated (gaedigk-nitschko et al., 1990; lusa et al., 1991) . it is found to be transported to the site of the plasma membrane via a p62/e1 complex and only to a lower extend incorporated into the budding virion (lusa et al., 1991) . albeit sitespecific mutation in the 6k region identifies viruses with reduced budding capabilities, a full deletion of the protein does not result in blocking viral replication. the lack of 6k leads to an altered spike structure of the virion and it is concluded that the protein is important for the correct assembly of the virion (mcinerney et al., 2004) . these findings make 6k another accessory protein which amplifies the release of virions. two hydrophobic stretches in the sequence of 6k suggest two tmds one of which would be long enough to span the bilayer (liljestrom and garoff, 1991) . a bioinformatics approach using a hidden markov model reduces the number to only one tmd (sonnhammer et al., 1998) . a single tmd is also the conclusion drawn from mutation studies of semliki forest virus 6k protein in its interfacial region (sanz et al., 2003) . ross river and barmah forest virus 6k proteins expressed in e. coli and reconstituted into artificial lipid bilayers form channels with conductance ranging from 40 to 800 ps (melton et al., 2002) . the large conductance is attributed to the formation of larger oligomeric units of the protein. no other roles than permeabilizing the lipid membrane and being involved in the budding process are known to date. it has been discussed that topology and mechanism of function of 6k is dependent on lipid thickness and constitution which the protein experiences during trafficking (sanz et al., 2003) , a mode of action which has also been suggested for vpu from hiv-1 (mehnert et al., 2008) . 2.2.2. e, 3a, and 8a from severe acute respiratory syndrome-corona virus coronaviruses belong to the family of coronaviridae and replicate in various animal species (marra et al., 2003; stadler et al., 2003) . human coronaviruses have been known since the 1960s (myint, 1995) . in 2002, a member of the virus has been found to cross the species barrier and infected humans with a mortality rate of about 5%, and even higher for people above their sixties (zhong et al., 2003) . the symptoms are described by a sars which are almost flu like and come with severe fever (stadler et al., 2003) . the name of the family derives from a corona like shape of the virion when observed in electron microscopy (beniac et al., 2006) . coronaviruses belong to the enveloped viruses and harbor the largest positive-sense single-stranded rna genome of 30-32 kb amongst the rna viruses (narayanan et al., 2008) . the genome is transcribed into a large polyprotein which is cleaved by virus-encoded proteases. the structural proteins are spike (s), matrix (m), and nucleocapside proteins (n) as well as the envelope protein (e). a large series of accessory proteins is distributed toward the 3 0 end of the genome (narayanan et al., 2008) among several proteins exhibiting channel activity, as listed below. protein e has a length of 76 amino acids with a short n terminus of 7-9 amino acids, a long tmd of 21-29 amino acids and a c terminus (shen et al., 2003) . when e protein is synthesized and reconstituted into artificial lipid bilayers, channel activity with conductance in the range of 50 ps is observed . sds-page experiments identify e protein to form pentamers (parthasarathy et al., 2008) . electrophysiological measurements using whole-cell clamp conditions of e protein gene infected hek-293 cells reveal a current which is sensitive to the administration of hma (pervushin et al., 2009) . ftir spectroscopic investigations suggest a helical hairpin-like structure for the long tmd of e protein (arbely et al., 2004) . it is argued that, the peptide is highly helical with hardly any tilt and has a phenylalanine (phe-23) located toward the lipid head group region. in addition to that, an asparagine (asn-15) would support the n and c terminal ends to assemble. nmr spectroscopic investigations support a helical tmd without any turns (pervushin et al., 2009 ). merely, a leucine residue (leu-18) does not fit into the residual dipole coupling calculations proposing a non-helical section or a conformational flexible region. computational modeling studies of the e protein in an implicit bilayer model using monte carlo simulations support the experimental results of the secondary structure of the membrane traversing helical domain to be helical (chen et al., 2010) . the calculation starts from a random conformation of the tmd fully embedded in the hydrophobic slab of the bilayer. the presence of hma differences in the 1hn chemical shifts of two residues on either side of the tmd is found when compared to those without any drug (pervushin et al., 2009) . this is in contrast to measurements in the presence of amiloride. consequently, hma is suggested to be located within the pore most likely at the c terminal mouth. orf 3a of sars-cov is another protein identified to exhibit channel activity when expressed in xenopus oocytes (lu et al., 2006) . it is located between s and e proteins and built by 274 amino acids (zeng et al., 2004) . the protein is found at the site of the plasma membrane and in the cell (tan et al., 2004; yuan et al., 2005) as well as in intracellular virus particles (ito et al., 2005; yu et al., 2004; yuan et al., 2005) . the protein is also released in membranous structures from infected and 3a expressing cells (huang et al., 2006) . similar to other channel-forming proteins of other viruses, 3a also interacts with a series of other host and viral proteins (narayanan et al., 2008) . three tmds are proposed at the n terminal side preceding a longer c terminal cytoplasmic domain of about 148 amino acids. residues 127-133 harbor a cystein rich region which is involved in connecting to monomeric 3a proteins into a covalently linked dimer (lu et al., 2006) . two dimers assemble into another dimer via noncovalent bonds forming a tetrameric unit. experimental data of the structure of 3a are not yet available. up to now, only computational models are generated krã¼ger and fischer, 2009) . a variety of assembly protocols have been followed which can be categorized into protocols which screen the conformational space of the monomeric unit by a concerted movement of all tmds and in those which build up the monomeric unit in a sequential manner. the latter means, two tmds are assembled first following by the third tmd. finally, in both protocols, the tetrameric assembly is done in a concerted screening protocol. while a concerted protocol proposes the third tmd to be pore lining ), one of the sequential protocols (assembling tmd1 and tmd2 first followed by adding tmd3) proposes the second tmd to be pore lining in the tetramer . the rational for using the sequential assembly for the monomers is that it most likely resembles a biological relevant translocon based pathway of assembly. along these investigations, the finding of unusual residues lining the lumen of the pore such as tyrosines and histidines is a common feature. in addition for tmd2 lining the pore, a ring of histidines is formed in the tetramer . with histidines pore lining, the channel is getting in line with m2 of influenza a and computational models of p7 from hcv (patargias et al., 2006) . it needs to be evaluated whether 3a is sensitive to protons. the role of 3a in the life cycle of sars-cov is not elucidated yet. more recently, another open reading frame of sars-cov has been identified to exhibit channel activity. orf 8 is separated into two orfs 8a and 8b in human isolates with a 29-nt deletion (guan et al., 2003) . protein 8a has been identified to amplify viral release and to induce the hyperpolarization of the mitochondrial membrane (chen et al., 2007a) . consequently, it is concluded that 8a is involved in cellular apoptosis. patients with antibodies against 8a recovered from sars-cov infection. 8a is identified as a 39 amino acid transmembrane protein (guan et al., 2003) . secondary structure prediction programs suggest the n terminus to be transmembrane followed by an extramembrane domain containing approximately 15-20 . a peptide corresponding to full-length 8a derived from spps and reconstituted into artificial lipid membranes exhibits weak cation specific channel activity with conductance of around 9 ps at elevated temperatures (38.5 c). since the sequence of 8a is rich of cysteins, experiments have also been done under reducing conditions. computational modeling of the tmd reveals a sequence of cysteins on one side parallel to the helix axis and a hydrophobic stretch of serines and threonines on the opposing side. assembling the single tmd around a central axis into tetra-, penta-, and hexamers delivers potential structures for the putative pore. consequent molecular dynamics simulations show a pentameric model maintaining a continuous water column. this model shows ser-11 and thr-8 as well as cys-15 to be pore lining. in addition, the c terminal mouth is surrounded by summarizing the findings about sars-cov, with its large genome it seems that the virus hosts three potential channel-forming proteins, e, 3a, and 8a. all three of them are identified as auxiliary proteins, also interacting with a series of other viral and host proteins (narayanan et al., 2008) . all three proteins have in common, that their roles within the viral life cycle have yet to be identified. channel activity is found either with expression of the protein in other cells or by reconstitution of the proteins into artificial lipid membranes. the question emerges why the virus needs three channels while all other channel expressing viruses known to date proteins only need one type of channel protein. entero viruses among which poliovirus, coxsackie virus, and echo virus are listed encode a nonstructural 97-99 amino acid protein called 2b (aldabe et al., 1996; doedens and kirkegaard, 1995; van kuppeveld et al., 1995) . it has been identified to render the membrane of the endoplasmic reticulum and the plasma membrane permeable for small molecules and especially calcium ions (aldabe et al., 1996; doedens and kirkegaard, 1995; van kuppeveld et al., 1997a) . it has also been reported that 2b is retained in the golgi apparatus where it increases the release of ca ions, a role which is also found for the endoplasmic reticulum in the presence of 2b (de jong et al., 2006) . more recently, it has been reported that 2b is also able to conduct monovalent ions and its conductance can be blocked by a known chloride channel blockers such as 4,4 0 -diisothiocyanatostilben-2,2 0 -disulfonic acid (xie et al., 2011) . the measurements have been done using two electrode voltage clamp conditions and with 2b expressed in xenopus oocytes. 2b is composed of two hydrophobic domains (van kuppeveld et al., 1995 (van kuppeveld et al., , 1997b and is found to homooligomerize into tetramers (cuconati et al., 1998; de jong et al., 2002; van kuppeveld et al., 2002) . in a computational approach, a dimeric and tetrameric structure of 2b from polio virus have been proposed (patargias et al., 2009) . the individual tmds have been modeled into an ideal helix and inserted into a fully hydrated lipid bilayer to relax the structure during a short molecular dynamics simulation. the individual tmds have then been taken to form the monomer. after another md simulation, the tetramers have been produced and also relaxed for many nanoseconds of md simulations. from those models, it is suggested that both tmds contribute to form the lumen of the pore. it is further suggested that lysines contribute to the inner face of the pore. a short peptide based on 20 amino acids of the second tmd has been able to permeabilize the plasmamembrane of cells (madan et al., 2007) . the experiments show that tmd2 alone could form a pore and possibly is at least part of the pore lining motif of the bundle made of the full-length protein. for extended reviews, please consult fischer and krã¼ger (2009) and gonzales and carrasco (2003) . plant viruses such as green algae infecting p. bursaria chlorella virus (pbcv-1; yamada et al., 2006 ) express a 94 amino acid potassium selective channel, called kcv (gazzarrini et al., 2003; plugge et al., 2000) . sequence homology with the potassium kcsa (doyle et al., 1998) has been proposed and consequently a structural model is suggested to be similar to kcsa. in xenopus oocytes, the protein induces channel activity and it is suggested that the channel is important for viral entry (mehmel et al., 2003; neupã¤rtl et al., 2008) . it can be blocked by amantadine and barium ions (plugge et al., 2000) . a series of mutants have been analyzed in respect to the consequences on the physiology of the channel and its mechanism of function (gazzarrini et al., 2004) . extended physiological measurements reveal two types of kinetics of the channel (abenavoli et al., 2009 ). this identifies that despite of the small size and without any extramembrane domains, the protein undergoes diverse gating behavior similar to other k ã¾ channels. computational models of kcv built on homology modeling and implementation of nmr spectroscopic data have been generated (tayefeh et al., 2007 (tayefeh et al., , 2009 ). the simulations show functional relevant amino acids and facilitated ion movement through the selectivity filter. another k ã¾ channel has also been found in the genome of brown algae infecting ectocarpus siliculosus and called kesv . with its 124 amino acids, it is slightly longer than kcv. three potential hydrophobic regions have been identified by sequence analysis. the channel is blocked by barium ions and amantadine. structural studies are most advanced for m2 and vpu. also topological information is available for p7. these studies on the structure of the channel deliver a snapshot of the overall dynamics the protein exhibits. usually, special biochemical conditions are applied to grab the protein for structural studies or to trap the protein into a specific conformational state. the combination of the pictures allows assessment to the mechanics of the protein. inevitable to these investigations is the use of computational tools to catch a glimpse about the dynamics on an atomistic scale. simulations on gating are widely applied to m2 to identify the roles of the histidines chen et al., 2007b; khurana et al., 2009) . in case of other vcps, the studies are focusing on the generation of structural models . there are many questions linked with investigations on vcps. some of the vcps form covalently linked dimmers which assemble into proton conducting tetramers for which gating is achieved by a ring of histidines. other vcps assemble into oligomers and it is anticipated that any hydrophilic residue within the tmd will point toward a putative pore (vpu, e, 8a). these channels neither are covalently linked nor conduct protons. the question about what triggers a defined opening is unknown. to summarize the mechanics of the proteins are fully in the dark. for p7, it can be speculated that, with the suggestion of histidines facing a pore or being aligned at a helix-helix interface within the membrane, the protein is sensitive to the surrounding ph as well. in addition to that, histidines can interact with metal to stabilize the assembly. with the tritopic 3a forming covalently linked dimers which assemble into tetramers, speculations about more sophisticated gating mechanisms are allowed. reviewing the mechanism of function at this stage of research on viral channel-forming proteins is still very speculative due to a lack of structural details for most of the proteins. the accumulation of structural information for proton channel m2 and with it clues about its mechanics is definitively sparked by the fact that m2 has been very quickly identified as a target for antiviral therapy due to its importance in the viral life cycle (hay et al., 1985) . it is also this circumstance which has ignited the hunt for channel proteins in other viruses and their potency to serve as antiviral target. evaluating potential mechanics of these proteins (fig. 6 .5), comparative investigations are the route to go at this stage. in doing so, the sequence of the tmds of individual viral channels are compared with those of the host channels and toxins for which structural and more functional information are available schindler and fischer, 2011) . based on sequence alignment of the tmds of these proteins, speculation and links of how the viral proteins may work are given. in the following, host channel proteins and toxins are chosen to be reviewed which have been identified to show sequence identity with the tmds of selected viral channels such as vpu, p7, 2b, and 3a schindler and fischer, 2011) . the introduction of the host channels and toxins follows a review of ideas about referring to the mechanics of the respective viral channels. pore forming toxins (pfts) are a class of proteins which are expressed by bacteria as well as higher organisms (iacovache et al., 2008) and even human cells (agerberth et al., 1995) to protect them against or support an attack. their role is to punch holes into the membrane and enable the draining of the cytoplasmic interior and thereby leading to cell death. pores formed by this class of proteins tend to be highly unselective. they are considered in this review since in a recent study about sequence alignment of vpu from hiv-1 with clya identified an alignment of the tmd of vpu with the membrane spanning domain of clya, aa . there are two classes of pfts depending on the membrane spanning motif they use: those which adopt a helical motif, a-pfts, such as clya (mueller et al., 2009) , and those which adopt a b-barrel motif, b-pfts, such as a-hemolysin of staphylococcus aureus (song et al., 1996) . (for an overview, refer to http:// blanco.biomol.uci.edu/membrane_proteins_xtal.html.) the mode of action of pfts goes through various steps. the proteins are released, they will "see" an aqueous environment, have to attach to the membrane, and assemble to finally convert into a membrane protein which intrudes into the membrane. many of these modes are not comparable to the mode of action of vpu except when the toxins are embedded in the membrane to form unselective pores. clya belongs to the class of a-pfts. in its functional form, it forms a 400 kda pore of 12 monomers with a height of 13 nm and an inner pore diameter of 7 nm (mueller et al., 2009 ). the single protein consists of four helices, aa, ab, ac, and af which are oriented parallel to the membrane surface. upon membrane attachment of the monomer, its b-tongue, bt, named like that because of the short b-fold found in this region, initiates membrane anchoring and a rise of the monomer aligning the helices perpendicular to the membrane surface. at this stage, assembly is also initiated and the monomers align head-to-head into a dodecamer. assembly facilitates the interlocking of the 12 aas into an iris-like arrangement which intrudes into the membrane stabilizing the pore. due to the enormous size, this pore can then be considered to be "open" leading to the vanishing of the hydrophobic permeation barrier of the lipid membrane and with it of any electrochemical or substrate gradients across the plasma membrane. smaller pfts such as alamethicin, magainin, or melittin follow the same strategy with the only difference that they form either a pore fully mantled by protein (barrel-stave model) or a pore with the phospholipid head groups part of the mantling wall (toroidal model) (huang, 2000; shai and oren, 2001) . at this stage, there is no available information on a gating mechanism of the smaller pfts unless due to thermal fluctuations within the membrane, consequent membrane curvature, and stress alterations the pore may collapse. this may not be the case for the larger pfts such as clya or ahemolysin. due to the role of these proteins, any "sophistication" of the mechanics of gating would not be necessary anyway. due to the similarity of vpu with aa of clya, the following speculations are allowed: vpu assembles into homooligomers. numbers range from 4 to 5 as minimal assembly units. with the tetramer, most likely no channel activity should be possible adopting a tight helix packing motif. with a pentameric assembly, ions would be able to pass through and with hexameric or larger bundles ion selectivity should be lost and substrates may also be able to pass through. with 5 and more monomers, the pore could be mantled by the tmds. tilts of the tmd of vpu have been measured and range from about 6 (in dmpc; to 13 (park et al., 2003) , 18 (park and opella, 2005) , and lower than 20 (sharpe et al., 2006) with the latter three values obtained with peptide reconstituted into dopc. upon thinning of the membrane, the tilt of vpu will increase because of compensating for the mismatch with the lipid bilayer (park and opella, 2005) . the angles obtained for vpu are lower than the observed tilt in the crystal structure of clya 2wcd (mueller et al., 2009) which adopts values around 45 in detergents. it is possible that in a lipid bilayer this value may be lower as well. it is therefore speculated for vpu that possibly lipid composition and lipid dynamics may act as trigger to gate the protein bundle (mehnert et al., 2007 (mehnert et al., , 2008 (fig. 6.4) . conductance values for vpu are within a range of 30-60 ps (schubert et al., 1996b) and exhibit gating into subconductance states (ma et al., 2002; mehnert et al., 2008) . nevertheless, these values rather indicate ion channel activity than "brute" conductance through a hole in the bilayer. concluding from the above, gating may be driven by stochastic events triggered by the thermodynamics of the environment (fig. 6.5a and b) . assuming a "sliding mechanism" to rise the tilted tmds , possibly in combination with minimal rotational movements, since the peptide is dynamic in the lipid bilayer (park et al., 2006) , it can easily be triggered by membrane thickening or thinning. a concerted rotation around each of the helix axis (bour and strebel, 2003; montal, 2003) needs to cross large energy barriers but would on the other hand be mostly independent of lipid thickness. the overall number of monomers forming the pore is still a matter of debate since conflicting pore geometries and oligomeric states are proposed. the zipper motif (kim et al., 2005) composed by a line of alanines, formed when adopting a helical conformation (fischer, 2003) , is an important tool for membrane protein packing. nmr spectroscopic data reveal a splitting of nmr signals especially for ala-18 which is interpreted as that the 2wcd figure 6 .4 crystal structure of toxin clya (2wcd) (mueller et al., 2009) . the structure is shown in "gaussian contact mode" (moe software) indicating hydrophilic residues in blue and hydrophobic residues in green. the membrane inserted segment aa, which shows sequence alignment with the tmd of vpu, is either highlighted in orange or in red . left: side view and below view from the extracellular side; middle: view inside the pore, omitting several units and below view from the cytoplasmic side; right: backbone representation of the protein in side view and cytoplasmic view below. splitting is caused by interhelix packing (sharpe et al., 2006) . this information has to be taken into account when assembling vpu. with the emergence of the crystal structure (0.32 nm) of the bacterial (streptomyces lividans) potassium channel kcsa, a keystone for ion channel structure has been delivered (doyle et al., 1998; mackinnon, 2003) . its topology has been identified to be bitopic with an inner and outer helix. the two helices are linked by the "pore helix" and the selectivity filter. four subunits form the channel by lining a pathway for ions to cross the bilayer. the passage of an ion can be described as passing a gate on the cytoplasmic side and moving into a vestibule which stabilizes the ion via the dipole moment of a so-called inner helix. the ions need to pass the selectivity filer in a concerted motion with already potassium ions located within the filter already. with its high selectivity, kcsa shows similarity with voltage-gated k channels (kv) in terms of ion permeation and similarity in topology with inward rectifying k channels (kir). important to note is that kcsa channels are triggered by ph changes. deciphering the cause of the enormous selectivity of k channels for potassium over sodium is a challenge up to date (noskov and roux, 2006) . it is anticipated that the structure of kcsa represents a closed stage. with the structure (0.33 nm) of a prokaryotic (methanobacterium thermoautotrophicum) calcium-gated k channel, a model in the open stage is described ( jiang et al., 2002a,b) . with this discovery and in comparison with the ksca structure, a gating mechanism can be proposed . during activation, the inner helices push radial outward and open the constriction at the cytoplasmic side. this opening is accompanied by a hinge around a glycine residue at position 99 (g99). this is the most pronounced conformational change so far described for ion channels. the selectivity filter remains unchanged in its structural position and the outer helix only undergoes marginal conformational changes ( jiang et al., 2002b) . also, hydrogen bonding seems to be essential for the gating mechanism (rapedius et al., 2007) . as a conclusion for the context of this review and in respect to the findings for vpu in relation to kcsa , the four outer helices seem to fulfill the role as a sheltering corona toward the lipid environment. generation of computational models of a conducting pore is biased toward the idea, and findings in other channels, that hydrophilic residues have to face the lumen of a pore. sequence identity of the tmd of vpu is found with the outer helix of the k channel task (hsu et al., 2004) . it has been speculated that vpu results from molecular piracy. due to similarity with the outer helix of task, it could rather be interpreted that vpu also mainly acts on the lipid membrane. mechanosensitive channels (msc) belong to a class of ion channels which respond on the mechanical stress within a lipid bilayer (anishkin and sukharev, 2009; kloda et al., 2008; perozo and rees, 2003) . they are designed by nature to sense alterations in osmotic and mechanic pressures imposed onto the lipid membrane. by sensing the change, they enable the physiological relevant ions to diffuse passively through their interior to alter electrochemical and substrate gradients of relevant cells (zhou et al., 1991) . consequently, these membrane proteins convert the mechanical stress into readable signals for our nervous system. two types of msc can be distinguished according to the levels of conductance they generate: mechanosensitive channels of small (mscs) and large conductance (mscl). for both types of channels, crystallographic data from bacterial channels are available (bass et al., 2002; chang et al., 1998; liu et al., 2009; steinbacher et al., 2007; wang et al., 2008) . focusing on mscl and the crystal structure of a homologous protein from mycobacterium tuberculosis (chang et al., 1998) , the protein has been identified to consist of a pentameric homoassembly with two tmds. its water filled pore is aligned with hydrophilic residues and has a diameter of about 1.8 nm. it is assumed that this structure catches the closed state of the channel. other structures are reported to be due to intermediate states of the opening (liu et al., 2009 ). this structure (liu et al., 2009 ) includes a truncation in the cytoplasmic domain which allows the tmds to adopt an increased tilt compared to the "closed" and stable state of the protein. this protein has shown increased current and therefore supports the idea of now having a structure in a more open state, albeit the still very narrow pore diameter. however, the protein is reported to be a tetramer in contrast to the earlier studies of the mscl in the closed state (chang et al., 1998) . nevertheless, the larger tilt of the tmds in a partially open state compared to the tilt in the closed structure indicates a sliding mechanism for opening ( fig. 6.5b ). interesting to note is that similar to p7, over time, and with different experiments, several oligomeric states have been proposed. biochemical data (sukharev et al., 1999) and two-dimensional crystallography (saint et al., 1998) support the formation of hexameric assemblies of the protein. the crystallographic data identify pentameric (chang et al., 1998) and tetrameric structures (liu et al., 2009) . it is up to further investigations to decide about the implications of the findings on the mechanism of function of this channel protein. proteins p7 and 2b align particularly good with the tmds of the mechanosensitive channel mscl (schindler and fischer, 2011) . this ignites a discussion of whether the two proteins would also respond to mechanical stress profiles of the lipid bilayer and gate in a similar way (fig. 6.5b ). the nachr is part of the cystein-loop receptor super family of pentameric neurotransmitter-gated ion channels (le novã¨re and changeux, 1995) triggered by acetylcholine and susceptible to nicotine. the channel protein assembles symmetrically around a fivefold central axis. within the family, the channel can exist in homo-and heteromeric assemblies of about 290 kda (unwin, 2005) . upon acetylcholine binding, the channel converts into an open state enabling a flux of mostly monovalent cations across the lipid membrane. with its location at nerve-muscle synapses, its action induces fast chemical transmission of nerve signals. information about the morphology of the protein has mostly been derived from electron microscopy (unwin, 2003 (unwin, , 2005 unwin et al., 1988) . the channel can be divided into three parts, a large extracellular domain hosting the n termini of the subunits, a membrane spanning part which contains the gate, and a smaller cytoplasmic domain. the overall length of the protein is 16 nm, with a pore of about 2 nm in diameter. the two binding sites of the neurotransmitter are about 4 nm above the membrane surface and opposing each other. cryo-electron microscopy has been used to derive structural features of the receptor when briefly exposed to acetylcholine and immediately frozen (unwin, 1995 (unwin, , 1998 . the data yield a resolution of 0.9 nm. in comparison with earlier structures, which are related to the closed state, the major changes in the open state are due to an alteration of the ligand binding domain which initiates a small axial rotation in each of the two tmds of the respective subunits almost 5 nm away. imposing structural data into the models suggest that the closed state is due to a constriction of the pore in the middle of the membrane caused by leu-251 from each subunit, so to speak a ring of leucines. the residues are part of the pore lining m2 helix of each subunit. the constriction does not result from an almost overlap of the five residues; it rather follows from an approach of these residues. the consequence of this approach is a hydrophobic barrier which interrupts the water column which would otherwise fit through the pore. upon activation, the ring widens through a rotational motion, twist-to-open (miyazawa et al., 2003; unwin, 2003) , giving enough space so water can pass this barrier, which represents the gate of the channel. passing of water in the open stage has been shown using molecular dynamics simulations on the tmds of the receptor embedded in a hydrated lipid bilayer (beckstein and sansom, 2006) . it has been suggested that this mechanism is a blue print for other members of the ligand-gated ion channels (unwin, 2003) . the bacterium erwinia chrysanthemi also expresses a ligand-gated ion channel which belongs to the family of pentameric ligand-gated ion channels (elic). crystal structures of this channel in its potential closed state have been obtained (hilf and dutzler, 2008) . the elic channels show 16% sequence homology with nachra and comprise the closest to a structure representing this class of ligand-gated ion channels. in terms of the extramembrane and transmembrane part, this channel approximately adopts the same dimensions (9.5ã�11 nm) as the nachr. in contrast to the nachr, the channel has no cytoplasmic domain. the 10 b-strands of each of the subuntis mantle a cylindrical vestibule of approximately 1.6 nm diameter. within the membrane, the pore narrows down to a diameter of 0.7 nm lined by one of four helical tmds per subunit. toward the extramembranes segment, the transmembrane passage is confined by a ring of phenylalanines (f246) and leucines (l239). it is anticipated that these two constriction sites form a gate similar to the ring of leucines in the nachr. similar to the nachr, the respective tmds a1 and a3 are involved in inter-subunit contact, whereas a2 lines the pore. the tmd a4 is positioned at the periphery of the transmembrane subunit. since the ligand triggering of the channel is not known, a study about the potential open state cannot be conducted. cyanobacterium gloebacter violaceus encodes a pentameric ion channel (glic) (hilf and dutzler, 2009) homologous to elic which is activated by low ph and does not desensitize after activation. crystal structure of the channel at ph 4 with a resolution of 0.31 nm is therefore considered to show the open state of this channel. the structure of glic is "very similar" to the structure of elic and therefore the data are taken to identify a novel gating mechanism for the pentameric ligand-gated ion channels. the lumen of the pore in the transmembrane region confines gradually toward the cytoplasmic side. this is due to a2 and a3 helices which are rotated as a rigid unit around val-267 by about 9 . the extramembrane part of the pore is flanked by hydrophobic residues while the cytoplasmic part is flanked by polar side chains. both ends of the channel are guarded by rings of acidic residues. the narrow part at the cytoplasmic end is due to a ring of glutamate residues (e221). crystallization of a mutant channel in which the glutamate residues are replaced by alanine under the same experimental conditions (low ph) reveals a similar overall conformation of the protein backbone but with the restriction derived from the glutamic acids being removed. this indicates that the local change of the amino acid residues does not alter their conformation and that only minor changes of the side chains allow an increased pore radius. the conclusion is that the glutamates interact directly with the permeant cations. the studies on both elic and glic are considered as studies on a closed (elic) and open (glic) ligand-gated ion channel. the opening is shown to result from a change in the tilt of two of the tmds, a2 and a3, of each of the subunits around a pivot point two-third across the transmembrane pore. this is proposed for nachr in contrast to the rotational motion of m2, which is similar to a2. protein 3a identifies identity with ligand-gated channels such as the nachr and pglic/elic (schindler and fischer, 2011) . this finding proposes that this large viral channel could be triggered by a ligand and also allows more complex gating behavior (fig. 6 .5a and c) for viral monotopic proteins such as vpu, e protein, or 8a. there is no identity with the pore lining part of the two types of toxins, such as the helical toxin clya and the b sheet structured ahemolysin. with m2 and bm2 proteins of influenza a and b, respectively, known to conduct protons, it is tempting to compare the mechanism of function of these proteins with other known proton conducting or transporting membrane proteins. one of the proteins, which allow protons to move across the membrane, is the light driven proton pump bacteriorhodopsin (br) from halobacterium halobium (oesterhelt and stoeckenius, 1971) . bacteriohodopsin is part of a family of light activated membrane proteins such as halorhodopsin and rhodospin. albeit the fact that this protein needs light to activate a vectorial proton transport to build up a proton motif force, the movement of the protons along certain amino acids within the protein can give an idea of what is necessary for proton translocation. bacteriorhodopsin has been analyzed in great details using techniques like cryo-electron microscopy (henderson et al., 1990) , ftir spectroscopy (rã¶dig et al., 1999; rothschild, 1992) , nmr spectroscopy (herzfeld and lansing, 2002) , x-ray crystallography (edman et al., 1999) , kinetic analysis (kã¶tting and gerwert, 2005) , and other techniques. describing its mechanism in short, the proton translocation mechanism is triggered by light leading to an isomerization of a retinal linked via a schiff base to lys-216 from its all trans into 13-cis conformation. this very fast event is followed by structural rearrangements of the protein which are characterized by specific spectral changes known as the photocycle of br (neutze et al., 2002) . structural rearrangements include kinks in two of the helices (c and f). further, the action of localized water molecules (fischer et al., 1994) in combination with a series of well-aligned titratable amino acids are necessary to achieve vectorial grotthuss-type proton transport. another highly selective human voltage-gated proton channel is hv1 (decoursey, 2008). hv1 is polytopic membrane protein with four tmds (ramsey et al., 2010) . pore lining residues are also identified to be titratable ones. however, if these titratable residues are mutated, proton conductivity is not fully abrogated. computational modeling studies using homologous models of voltage dependent potassium channels reveal that the structure allows water molecules within the pore of the channel. combining the experimental and computational data, it is suggested that a water-wire exists to transfer the proton as selectively as this channel does, but that proton translocation does not necessarily require titratable residues. what is the mechanism of function of m2, a proton channel of minimal design (fig. 6.5d) ? in comparison to other proton translocating and channeling proteins, it is missing a sequence of titratable residues within the pore. localized and structurally defined water molecules are not yet reported. this may be due to the fact that a structure of full-length m2 protein is not yet at hand. the gating mechanism may find some relation to br in as much the histidines of m2 rotate or "flip," similar to cis/trans isomerization in br, and "shuffle" the proton to the cytoplasmic side to be picked up by trp-41 and asp-44. structural changes for m2 seem to involve a change of the overall tilt of the protein assembly. similarities are thus far speculative and it remains the question in how far the design of m2 resembles a novel structural concept of proton translocation. most of the vcps are smaller in size than their host channels. with about 100 amino acids in length, they harbor a maximum of two tmds. only 3a from sars-cov harbors three domains but is with 274 amino acids almost three times as long. with one or two tmds per protein which itself needs to self-assemble to form a channel, it is anticipated that the channel should be sensitive to the lipid environment, which is mentioned for vpu (mehnert et al., 2008) . additional tmds per protein, as found in many other host channels, could act as a "mechanical buffer" toward the lipid membrane. functional information is mainly done either with the proteins reconstituted into artificial lipid compositions or with the protein expressed in xenopus oocytes (table 6 .1). structural data from nmr or x-ray sources are also recorded in artificial environments. there is emerging evidence that lipid rafts are playing a highly important role in the cellular life cycle of enveloped viruses (brã¼gger et al., 2006; campbell et al., 2001) . lipid rafts are cholesterol and glycosphingolipid rich detergent-resistant membrane patches (simons and ikonen, 1997; stier and sackmann, 1973) . purification of m2 from infected cells or eukaryotic expression systems has identified the presence of cholesterol (schroeder, 2010; . therefore, it has been suggested that m2 from influenza a is attached to lipid rafts in vivo (lin and schroeder, 2001; . since all of the m2 channel recording data identify the function of m2, even when taking just its tmd and not even covalently linked together, it seems that rafts just only serve as a scaffold for in vivo function. to combine these findings, it is suggested that in the case of a raft attachment of a monomeric protein, or dimeric protein in case of m2, several of the raft patches can form a trapped cholesterol free "patch" in which the protein can function as a proton channel (lin and schroeder, 2001) . raft association has also been reported for vpu (ruiz et al., 2010b) . expression of vpu in human 293 cell lines reveals that it partitioned into detergent-resistant membrane microdomains. its partitioning could be abolished when residues in the tmd are substituted by alanines. mutations of trp22 and a sequence on the middle of the domain, ivv19, are key residues responsible for microdomain integration. taken together with computational modeling data in which it is suggested that vpu is flexible and adopts to its environment via residues in the region from ile-17 to ser-24 (krã¼ger and fischer, 2008; park et al., 2003) , replacement of these the code for structural information refers to the four letter code used by the rcsb protein data bank (www.pdb.org). c, cytoplasmic domain. for the x-ray structures the resolution is given in ã� . residues hampers essential flexibility necessary for domain integration. important to note is that raft association is not required for cd4 downregulation and that the above mutations can be correlated with a reduced particle release (ruiz et al., 2010b) . the exact placement within the microdomain is unclear and it could still be suggested, similar to m2, that channel activity of vpu, which is independent of the cholesterol or sphingolipid content, could be achieved with the protein "released" from the raft into the confined space of an entrapped cholesterol free patch surrounded by rafts. 3a has been detected in membranous structures from expressing and infected cells (huang et al., 2006) . membrane flotation essay identified 3a at relevant gradients indicating that the protein is associated with lipid membranes. when the membranous structures are treated with "raft" detergents, membrane floatation essay unravels that still some fraction of 3a is bound to membranes. it is therefore concluded that some of the 3a proteins are located within or at raft-like domains. additionally, e protein from sars-co has been identified to be located in lipid rafts expressed in hela cells as well (liao et al., 2006) . the role of lipid composition on the mechanism of function of these channels is still little explored. with the channel proteins being raft associated, novel routes for antiviral therapy can be envisioned as discussed below (ruiz et al., 2010b) . when blocking enzymes, the site of interference is well defined. the prime target site is the active region of the enzyme and eventually the outer side of the protein for, noncompetitive modulators. membrane proteins contribute in drug therapy as drug targets to a large extent. for these proteins, the sites of interference are similar to enzymes. they are, for example, the docking sites of the biological ligand relevant for the receptor or ion channel. in cases of the latter, physical blocking of the channel, like a plough, is another option. for the extramembrane parts of the membrane proteins, other allosteric sites are equally important to be targeted by modulators. anticipating vcps as targets, one can imagine that with their small size the lipid membrane plays an important role in the mechanism of function of these channels. the interaction of proteins with each other is also highly dependent on the dynamics of the lipid membrane (langosch and arkin, 2009 ). in other words, a potential drug does not necessarily need to interact with the protein but can also alter lipid dynamics which in turn modulates protein function. consequently, targeting, for example, lipid rafts could also have potential antiviral effects. for some of the channel proteins, it emerges that they are interacting with a series of host factors via protein-protein interactions. the interaction is not confined to the extramembrane parts of the viral protein but also includes the tmds. this is especially the case for vpu and its interaction with the host factor bst-2. hampering with protein-protein interaction is consequently a promising path for drug development. small molecule design is the way how man is trying to steer proteins for the cure of diseases. viruses do not use small molecules, they rather create their own proteins to interfere with the mechanism of function of the host proteins. this concept inspires the development of drugs based on specific peptide-peptide interaction, such as peptide drugs. the idea of a peptide drugs is to mimic the fold of the viral "protein ligand" which targets the host protein. the consequent blocking of the "protein ligand" interrupts the pathway of the viral protein within the cellular life cycle. the "coating" of the protein with peptides could either involve coating of the host or the viral protein. potential peptide candidates as "coats" can be designed specifically by analyzing protein mechanics of both host and viral protein. another option would be to search related proteins from data banks of phylomers , proteins/peptides known to interrupt protein-protein interactions and used for protein silencing to validate targets (watt et al., 2006 ). an example of such an approach which has been done for hiv-protease (park, 2000) and hiv-integrase (maroun et al., 2001; zhao et al., 2003) indicates that this approach is a valid one for globular proteins. up to now, peptide drugs are used in a limited number of cases (rishabh et al., 2009) . however, their potential use is hypothesized to increase in the near future. most striking is their highly specific mode of action which is expected to require a low dose to be administered. in addition, peptide drugs seem to be toxicologically safe and are found to exhibit lower side effects than their small molecule counterparts (agyei and danquah, 2011) . peptide drugs, despite the benefit, harbor some draw backs. even though they are now more comfortably manufactured, administering of the drugs is still a major hurdle for their use on a broader scale (shaji and patole, 2008) . injection still is the major route for entering the drug into the body and up to now mostly used for treating diabetes. among the routes of administering, oral or nasal routes are still the most favorable ones. once administered, they also face difficulties such as fast enzymatic degradation, membrane impermeability due to their size, and metabolic stability. thus, chemical modifications are also necessary not only to generate a deliverable form of the drug but also to increase the retention time of the drugs in the body to improve their therapeutic value. one of the routes to improve the therapeutic value is to use peptides and develop peptidomimetics. the latter step is used to develop antimicrobial peptides (godballe et al., 2011; scott et al., 2008) . one of the first peptide like drugs proposed for antiviral therapy against membrane proteins, called 5-helix, has targeted gp41 from hiv-1, (root et al., 2001) . the idea of this peptide is to mimic the trimer-of-hairpins state of gp41 which consists of six helical elements of gp41 tightly packed. with only five helices, it is anticipated that the steric vacancy for the 6th helix will be taken up by the c-peptide region of the viral gp41 protein under native conditions. this interaction hampers the fold of gp41 into the trimer-ofhairpin state. shorter peptides have been suggested to target the c terminal region further (eckert and kim, 2001) . also, conserved hydrophobic pockets in the n terminal region of gp41 are targeted by peptides including small molecules (debnath, 2006) . currently, computational methods such as md simulations have been applied to assess the binding and search for novel peptides (strockbine and rizzo, 2007) . these developments have led to the first antifusion drug enfuvirtide (poveda et al., 2005; steffen and pã¶hlmann, 2010) . d-peptides, which show better affinity and bioavailability, are also reported to interfere with gp41 (welch et al., 2007) . most recently, a peptide based on a 1 -antitrypsin has shown promising results in patients (forssmann et al., 2010) . this 20-amino-acid peptide targets the fusion peptide of gp41. another strategy is to use peptides as a "molecular shield" of the host protein to protect it against interaction with viral proteins (strangler et al., 2007) . this concept is introduced with studies on preventing membrane associated nef protein from hiv-1 to interact with the cellular transduction protein hemato-poietic-cell kinase (hck). an artificial 12 amino acid proline-rich peptide abolishes the interaction between these two proteins. amantadine (1-aminoadamantan) (davies et al., 1964) and its derivatives have been used as one of the first antiviral drugs targeting m2, from influenza (hay et al., 1985) (fig. 6.6 ). activity occurs in two stages. in a nonspecific way at higher concentrations (>0.1 mm), it affects the fusion event hampering the conformation of hemagglutinin to the fusion relevant conformational state. at a later stage of the viral life cycle and at much lower concentrations (0.1-5 mm), the drug acts strain specific against virus assembly. at this stage, it prevents the transformation of the fusion protein to forming a so-called high ph conformational stage. the nonspecific action can also be achieved by a series of amines (hay et al., 1985) . mutant escape studies have shown that a series of hydrophobic residues at positions 27, 30, 31, and 34 of m2 (hay et al., 1985) as well as 26 (wang et al., 1993) are mutated in respective strains. mapping the residues onto a helical wheel indicates that they are clustered on one side and assumed to face the inside of the bundle rather than the lipid environment (sugrue and hay, 1991) . thus, amantadine is assumed to intrude into the pore and block the protein by occlusion of the pore. computational modeling of the drug in the center of an in silico model of the pore have been undertaken along this line (sansom and kerr, 1993 ). the m2 model has been built by copy-rotation of the monomer so that the hydrophilic residues face the lumen of the pore resulting in a slightly wider pore at the n terminal side than on the c terminal side. by positioning amantadine along the center of the pore and calculating for each position the interaction energy, a profile has been generated, which indicates a favorable binding site at the level of ser-31 with the amantadine cube facing the n terminal mouth and the amino group the c terminal side. the same experiments with a cyclic-pentylamine have not revealed a favorable binding site at the same position. neutron diffraction experiments confirmed a position in the area between val-27 and ser-31 (duff et al., 1994) . in a similar approach, using molecular dynamics simulations, amantadine has been positioned in the same way as reported (sansom and kerr, 1993) within the pore of m2 (yi et al., 2008) . the computational model of m2 has been leaned on an assembly based on nmr spectroscopic investigations (hu et al., 2007) . during the molecular dynamics simulation of 15 ns, the drug remains at its position around ser-31 supporting the site of interaction of amantadine with m2. in another proposal, amantadine is modeled to be within the central cavity with the amino group closer to the ring of histidines (gandhi et al., 1999) . the proposal that the amino group is pointing toward the histidines from various sites within the n terminal side of the pore is verified by crystallographic studies (stouffer et al., 2008) . in this crystallographic study with a peptide corresponding to the tmd of m2 with only one mutation at position 34, where glycine is replaced by alanine, the location of amantadine is in the lumen of the pore on the n terminal side (3c9j; stouffer et al. 2008) . the crystal structure is obtained at low ph (ph 5.3) representing an open-like or conducting channel. residues such as val-27, ala-30, ser-31, and gly-34 "trap" the drug so that its amino group points toward the c terminal side. it has been shown that mutation of these residues makes the channel insensitive to amantadine (deyde et al., 2007) . since amantadine shows no cooperativity (wang et al., 1993) , this binding site is seen as the single site and the mode of action as an "occluding the pore." based on solid state nmr spectroscopic studies of a peptide corresponding to the tmd of m2 derived from spps and reconstituted into dlpc (1,2-dilauroyl-snglycero-3-phosphatidylcholine), a binding site near ser-31 has been suggested (cady et al., 2009) , which has been confirmed in a later experiment (cady et al., 2010) . also, at higher amatadine concentrations, solid state nmr experiments suggest a second site at the c terminal side which should be considered as a weak binding site with low affinity (cady et al., 2010) . molecular dynamics simulations over 15 ns with the amantadine bound m2 model from crystallography show that the drug remains at the position disrupting the continuous water column similar to the ring of valines (at position 27). therefore, it is concluded that the mode of amantadine blocking is by interfering with the water molecules at the mouth of the pore. in a solution, nmr spectroscopic investigation of a m2 peptide fusion construct corresponding to the tmd of the protein, which is reconstituted into dhpc (1,2-diheptanoyl-sn-glycero-3-phosphocholine), the amantadine derivative rimantadine has been detected to bind at the outside of the bundle at the c terminal side (schnell and chou, 2008) . the experiments have been performed at ph of 7.5 which implies that the structural model represents a "closed" or non-active state of the protein. rimantadine has a larger portioning coefficient between octanol and water than amantadine (belshe et al., 1989) and possibly diffuses from the site of the membrane toward the protein. however, in this experiment four rimantadines per bundle are reported. visualization of the binding site reveals that rimantadine sits within a pocket of hydrophobic residues ile-42, leu-40, and leu-43 at the helix-helix interface. a blocking mechanism is suggested in which the drug is hampering the transition of the bundle into the open or h ã¾ gating state. based on these studies and earlier suggestions , it is reasonable to conclude that there are allosteric effects (schnell and chou, 2008) involved in the binding of amantadine derivatives to m2. the mode of action for all drugs possibly involves an alteration of protein dynamics and based on the type of drug allosteric interactions rather than single-site blocking. amantadine is not reported to be active against bm2 . amantadine has been reported to block p7 when the protein is expressed in e. coli, purified and reconstituted into lipid bilayers (griffin et al., 2003) . the protein, a his-p7 construct, shows burst activities at a holding potential of ã�120 mv. adding amantadine to a final concentration of 1 mm into both chambers of the setup leads to a complete abrogation of the bursts after about 10 s. application of amantadine to hcv replication cell cultures (lohmann et al., 1999) revealed no effect of the drug on rna replication, virus release, and infectivity of the virions (steinmann et al., 2007b) . using a peptide derived from spps, corresponding to specific strain of hcv (gt 1a, isolate h77) which also is reconstituted into artificial lipid bilayer, addition of more than 10.3 mg/ml of amantadine has been necessary to affect channel activity of the p7 peptide. a full blocking, showing a zero channel activity, could not be achieved. a docking approach using auto-doc 3.0 has been used to dock amantadine onto a computationally derived hexameric model of p7 (patargias et al., 2006) . the study proposes a binding site of the drug within the lumen of the pore between residues h17 and ser21, with the amino group facing ser21. the binding constant has been estimated to be around k i â¼ 68 mm. since amantadine is located toward two monomers, it allows plenty of space for ions to pass the pore. so far, amantadine has shown little effect in clinical trials (deltenre et al., 2004; maynard et al., 2006) . currently, a series of derivatives of amantadine have been tested (foster et al., 2011) . channel activity of a peptide corresponding the n terminus of vpu including the tmd, vpu 1-32 , derived from spps, reconstituted into artificial lipid bilayers is not affected by amantadine . amantadine has also been reported to block kcv (plugge et al., 2000) . due to the emerging resistance of influenza strains against amantadines and derivatives and also due to the side effects caused in the central nervous system of the latter compounds, a novel class of drugs, spiropiperidines, is currently investigated (wang et al., 2009a ) (bl-1743, fig. 6 .6). solid state nmr studies reveal that the derivative 3-azaspiro[5.5]undecane hydrochloride alters the dynamics of the protein and affects a larger series of amino residues within the pore. using a docking approach (autodock) with the drug bound crystal structure of m2 (3c9j (stouffer et al., 2008) , a binding site similar to amantadine is proposed. iminosugars are more effective than amantadine against p7, as indicated by channel recordings measurements which is also confirmed by cell based essays (steinmann et al., 2007b) . especially, long alkyl chain iminosugars block p7 much stronger than iminosugars with short alkyl chains. it has been reported that vpu is sensitive to derivatives of amiloride . experiments have been done with vpu expressed in hela cells together with the gag protein of hiv-1. budding virus like particles have been observed by electron microscopy. experiments in the presence of hma in the culture medium after transfection of the cells with the expression plasmid lead to an almost complete inhibition of the budding process. channel recording of a recombinant vpu protein, expressed in e. coli, could be blocked with the addition of hma in a range of 25-125 mm. an allosteric blocking could be anticipated since it is reported that at lower hma concentrations blockage is not complete. blocking has also been dependent of the applied potential. therefore, it seems that blocking also depends on the side of which the drug approaches the channel. in the same type of experiments, a derivative dimethyl amiloride (dma) also affects channel recordings in a similar way. since blocking by dma has been less complete than by hma at higher concentrations, a lower potency for dma has been concluded. amiloride itself has shown no effect in this study. the data have been confirmed in a study using a vpu peptide (vpu 1-32 ) reconstituted into artificial "micro" bilayers which are spanning a porous silicon device (rã¶mer et al., 2004) . adding 100 mm of both hma and dma to a measurement setup results in a full blocking of channel activity, while amiloride does not show any effect on the activity. in a computational study with a helical model of the monomeric tmd of vpu and in an assembly of five and six tmds forming bundles, putative binding sites of hma and amiloride have been evaluated (kim et al., 2006) . most striking are calculations of an estimated free energy using the docking program autodock 3.0. the calculations reveal lowest binding constant for hma interacting with the pentameric bundle. binding sites of hma within the bundle identify an interaction of the guanidinium group of hma at the site of ser-24. interaction of hma with a monomeric tmd identifies trp-23 as a potential site for interactions, suggesting p-p interactions. the computational data support the currently emerging idea of multiple binding sites of antiviral channel drugs and with it the suggestion of allosteric binding modes (pielak et al., 2009; schnell and chou, 2008) . from investigations on vpu interacting with hma-two different inhibition levels at various drug concentrations in bilayer recording studies and two putative binding sites identified in a docking approach-it seems very likely that vpu also follows this path. hma is reported to block channel activity of full-length p7 when derived from spps, purified and reconstituted into artificial lipid bilayers (premkumar et al., 2004) . in repeatedly, recorded experiments hma has been added to the cis side of a bilayer recording setup which contained 500 mm kcl while the trans side contains 10 times less kcl. to date, n-[5-(1-methyl-1h-pyrazol-4-yl)-naphtalene-2-carbonyl]-guanidine (bit225, fig. 6 .6) is the second most advanced class of antiviral drugs targeting a vcp. synthetic p7 derived from spps and reconstituted into lipid bilayers can be blocked by bit225 at a concentration of 100 mm bit225 on both sides of the measurement chamber and both filled with 50 mm kcl ). an ic 50 in the submicromolar range has been reported with a cell-based essay system using the model virus bvdv (bovine viral diarrhea virus). in combination with interferon alpha-2b, (rifna-2b) even a synergistic affect has been observed. bit225 has now successfully completed phase ib/iia trials. the drug is also evaluated for its affection of vpu (khoury et al., 2010) . a synthetic peptide construct representing the first 32 amino acids of vpu including the tmd (vpu 1-32 ), reconstituted into lipid bilayers shows channel activity which is knocked out by a 40 mm solution of bit225. in monocyte-derived macrophages chronically infected with hiv-2, which does not encode vpu, no affect has been observed, which supports that vpu is the target. a wide range of hiv-1 isolates are susceptible to bit225 as well. based on a computational evaluation of several guanidines on a pentameric vpu 1-32 , bit225 has come out as a highly potential candidate (patargias et al., 2010) . the study uses models of vpu tmds in which the serines (ser-24) are facing the potential lumen of the pore. for this investigation, the docking software flexx (biosolvit) has been used. the small molecule 4,4 0 -diisothiocyanatostilben-2,2 0 -disulfonic acid has been reported to block 2b (xie et al., 2011) . amphotericin b methyl ester (ame) is a water soluble derivative of amphotericin b (waheed et al., 2008) (fig. 6.6 ). it is water soluble, shows low toxicity, and is able to bind to cholesterol within the membrane. viral membranes, especially those in hiv-1 virions, are found to exhibit some similarity in the composition to lipid rafts (aloia et al., 1993; brã¼gger et al., 2006) . with the rafts containing cholesterol, ame alters the properties of the viral membrane. when jurkat cells are infected with a plasmid containing an infectious full-length hiv-1 and continuously exposed to 10 mm ame escape mutants indicate mutations in an endocytoses motif in the cytoplasmic part of gp41, the viral fusion protein (waheed et al., 2006) . in another study, it has been shown that ame lowers virus production (waheed et al., 2008) . ame seems to be noneffective against the gagmembrane protein assembly and gag-association with detergent-resistant membranes. it rather lowers the amount of virion release by approximately fivefold. vpu-deficient hiv mutants are insensitive to ame treatment also in the presence of an overexpression of cd317/bst-2/tetherin. reinjection of the cells with vpu plasmid makes the cell sensitive to ame again. this indicates that ame most likely disrupts the vpu-cd317/dst-2/ tetherin interaction. it is speculated that ame blocks ion channel activity of vpu. further, it may be speculated that vpu may need raft association to be fully functional. this study (waheed et al., 2006 (waheed et al., , 2008 shows that there could also be a conceptual different pathway to block a membrane protein. it could be anticipated that ame locates itself at those sites of lipid rafts which "show" the cholesterol, and with this, the interaction of vpu or other viral proteins with these sites is not possible anymore. if ame does not interact with vpu, it cannot act as a "replacement" of cholesterol and vpu function is abrogated. lipid membrane composition is an integral part for the mechanism of function of some of the vcps such as m2 and vpu. especially, the latter is found in lipid rafts. since rafts are lipid patches with a high content of cholesterol, cholesterol depleting drugs are suggested to combat the virus (ruiz et al., 2010b) . experiments have shown that cells which express vpu but are treated with a combination of lovastatin and m-b-cd show reduced levels of vpu in detergent-resistant membrane microdomains. it has been shown that lipid composition and with it the existence of rafts is essential for viral entry and budding (brã¼gger et al., 2006) . anti-raft drugs have been proposed to target rafts and with it especially to combat the replication of hiv-1 (verma, 2009 ). plant-derived drugs have been found exhibiting potential preventive effects of hiv-1 progression. compounds such as o-3-fatty acids and plant-derived triterpenes are investigated. since involvement of rafts in the mechanism of function of the viral channels is gradually emerging, these drug candidates could potentially be affecting the vcps. other plant-derived drugs such as polyphenols are proposed to target extramembrane parts of channel proteins while lipophilic terpenoids act within the membrane either directly or indirectly (wink, 2008) . saponins target cholesterol and could also act in a dual mode as mentioned: directly by protein-drug interaction or by an indirect mode of action, such as distorting raft composition. it is up to further studies to modify these candidates to address them more specifically to viral channels. a promising plant-derived drug candidate is emodin (6-methyl-1,3,8trihydroxyanthraquinone, fig. 6 .6), targeting 3a not only of sars-cov but also hcov-oc43 (schwarz et al., 2011) . the binding constant has been calculated to be 20 mm using voltage clamp conditions on xenopus oocytes expressing 3a. to date, m2 is the only target channel protein used in antiviral therapy. protein p7 and eventually vpu are in closer reach. for other proteins, investigations are still very much on a laboratory level reporting interactions of known drugs with the channel proteins. the current findings on the viral channel-forming proteins are reviewed in respect of the mechanism of function and their role of affecting electrochemical or substrate gradients. while for m2, a detailed picture of the mechanics of proton translocation is emerging due to structural work, the reason for weak cation selectivity of most of the channels is still in the dark. based on sequence alignment studies, a relation to host channels and toxins is drawn for some of the channel proteins. with vpu relating to toxin clya, and ditopic channels like p7 and 2b relating to mcsl and tritopic 3a likely to ligand-gated channels, the picture emerges that this type of protein covers the range from pores to channels. m2 as a proton channel may "borrow" mechanisms from other proton conducting and translocating membrane proteins. the evidence is growing that some of these proteins are raft associated. also, the evidence emerges that channel functionality is not the only role these proteins inherit. many of them interact with host factors to steer the cell toward the benefit of the virus (stage of steering). questions arise whether this "steering of the host" is done as monomeric or oligomeric units. in addition to it, there is some kind of equilibrium between the "stage of steering" and the proper channel state as an oligomeric unit. or is the interaction with the host to be seen as a kind of "host based ligand" closing the channel while without the host protein the channel is open; or do other ligands trigger activity? more surprising, with influenza a and sars-cov two viruses emerge which harbor more than one channel protein. on the other end of this scale, for dengue virus there is no report of any channel protein up to now despite its membership to the same family, flaviviridae, as hcv. drug development is yet still to be fully explored. with m2 as the only target so far, two more channel proteins are on the horizon to be potential candidates. since structural information is sparse for most of the channels, development of pore occluding drugs is difficult. drug-protein interaction seems to obey, in the same sophisticated way as for any other host or protein of pathogens, allosteric binding modes. on the other hand, drug development is challenged, due to the fact that protein-protein interactions between viral and host proteins are also happening within the lipid membrane. thus, novel concepts for finding drugs are essential. this may put peptide drugs, peptidomimetics, and plant-derived drugs in an elevated position for lead discovery. fast and slow gating are inherent properties of the pore module of the kã¾ channel kcv structure and mechanism of proton transport through the 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cord-348360-20eq5meh authors: esposito, dominic; mehalko, jennifer; drew, matthew; snead, kelly; wall, vanessa; taylor, troy; frank, peter; denson, john-paul; hong, min; gulten, gulcin; sadtler, kaitlyn; messing, simon; gillette, william title: optimizing high-yield production of sars-cov-2 soluble spike trimers for serology assays date: 2020-06-04 journal: protein expr purif doi: 10.1016/j.pep.2020.105686 sha: doc_id: 348360 cord_uid: 20eq5meh the sars-cov-2 spike trimer is the primary antigen for several serology assays critical to determining the extent of sars-cov-2 exposure in the population. until stable cell lines are developed to increase the titer of this secreted protein in mammalian cell culture, the low yield of spike protein produced from transient transfection of hek293 cells will be a limiting factor for these assays. to improve the yield of spike protein and support the high demand for antigens in serology assays, we investigated several recombinant protein expression variables by altering the incubation temperature, harvest time, chromatography strategy, and final protein manipulation. through this investigation, we developed a simplified and robust purification strategy that consistently yields 5 mg of protein per liter of expression culture for two commonly used forms of the sars-cov-2 spike protein. we show that these proteins form well-behaved stable trimers and are consistently functional in serology assays across multiple protein production lots. the need for high quality protein reagents is an important aspect of the response to the covid-19 pandemic [1] . screening for the presence and extent of an immune response will be a critical tool in controlling the spread of the infection until the development of an effective vaccine. additionally, such monitoring will provide essential data to policy makers as they develop guidelines to limit virus spread in the population. with the ubiquitous presence of related coronaviruses in the population (e.g. sars-cov, mers-cov, common cold coronaviruses oc43 and hku1), the tests must be highly specific for sars cov-2. several serology assays, both published and in progress, are employing the s protein (hereafter referred to as spike) in enzyme-linked immunosorbent assays (elisa). the specificity inherent in the spike protein [1, 2] makes it an obvious target for therapeutic interventions and for use in serology studies to assess the prevalence of immune responses to a specific coronavirus. two spike antigens are currently widely used: the receptor binding domain (rbd), which interacts with the extracellular ace2 receptor, and a much larger soluble spike ectodomain modified for stability to mimic the prefusion native spike trimer conformation [2] . while robust production of spike rbd domain was recently reported [3] , high quality soluble spike trimers remain a difficult antigen to both express and purify. publication of one form of this protein cites a yield of 0.5 mg/l [2] , with another publication suggesting yields as high as 5 mg/l [3] . however, many unpublished reports indicate consistent yields only in the 1-2 mg/l range, providing a significant challenge for large-scale serology assay development. in addition, some of these publications provide limited information on purification details or protein quality assessment, which make interpretation of the yields challenging. to support an nih-led serosurvey on the extent of the coronavirus immune response in ~ 10,000 human samples, our lab was tasked to produce rbd and spike proteins for elisa assay optimization and deployment [4] . we found our rbd production yield to be similar to published reports, however, spike production was problematic with our initial attempts producing inconsistent results and lower yields than those reported in the literature. without a robust method in place in our lab for determining spike titer in culture supernatants, we were limited to the crude method of sds-page gel analysis for estimating expression levels. due to the size of spike (138 kda), heavy level of glycosylation, and low expression levels, sds-page gel analysis was inconclusive when comparing multiple lots of expression supernatants. thus, the work presented here is intended to provide a robust method for those wishing to reliably produce sars cov-2 spike protein in quantities sufficient for serology assays, structural biology, or simply to better understand some of the production variables affecting the yield. it is expected that this protocol could be further improved and perhaps eventually replaced by a stable cell line production platform. nevertheless, the approaches outlined here allowed us to improve the production yield of spike protein significantly by modifying cell culture temperature and harvest time, as well as improving the purification process. the final proteins produced were highly pure, formed appropriate trimeric structures, and were functional as antigens in elisa assays. taken together, these improvements allowed the production of sufficient spike antigen for more than 500 elisa plates per liter of culture and generated enough protein from a single 4-liter expression to support a robust serosurvey being conducted by the nih [4] . harvested culture supernatants were clarified by centrifugation (4000 x g, 20 min, 4°c) followed by filtration (catalog# 12993, pall corporation, port washington, ny). when used in column chromatography, clarified supernatants were concentrated and buffer exchanged by tff. specifically, a masterflex peristaltic pump (vernon hills, il) fed the clarified supernatant to a 30 kda molecular weight cutoff cassette (catalog# cduf002lt, milliporesigma, burlington, ma). the clarified supernatant was concentrated to 10% of the initial volume and then buffer exchanged with 5 volumes of 1x pbs, ph 7.4 (buffer a, diluted from 10x pbs, catalog #70011069 thermo fisher scientific, waltham, ma). following buffer exchange, the tff 6 cassette was rinsed with 200-250 ml buffer a, to collect any protein remaining in the cassette. clarified supernatants for use in batch purification were not buffer exchanged. chromatography was conducted at room temperature (~22°c) using ngc mediumthe sample was desalted into buffer a using a hiprep 53-ml 26/10 desalting column (ge healthcare #17-5087-01, chicago, il) with 14-ml injections at 9 ml/min for all steps. the final protein sample was created by combining the bulk elutions from multiple runs of the desalting column. the protein concentration was determined by measuring the a 280 using a nanodrop one spectrophotometer (thermo fisher scientific, ma, usa). final protein was dispensed as 0.5-ml or 0.05-ml aliquots, snap frozen in liquid nitrogen, and stored at -80°c. to assess the oligomeric state of the protein, a single 0.5 ml aliquot of the final protein was thawed and analyzed by analytical size-exclusion chromatography using a 10/300 superdex200 analytical column (ge healthcare, chicago, il), with a flow rate of 0.5 ml/min. for the batch purification from filtered culture supernatants, 20 ml of ni-charged magbeads (cat #l00295, genescript, piscataway, nj), previously equilibrated in 1x pbs adjusted to 300 mm final nacl concentration (buffer b), were placed on the bottom of 2 x 5-liter thompson flasks (10 ml per flask) and 2 liters of filtered culture medium was added to each flask. flasks were shaken at 105 rpm at 27°c for 3 hr. after incubation, the supernatant was decanted into a 4-liter glass beaker, a rare-earth magnet was used to capture the beads to the bottom of the beaker, and the medium was removed and saved as "flow through". buffer b (150 ml) was added to the beaker to suspend the beads, which were then transferred to 500-ml corning centrifuge bottles and shaken at ~22°c (room temperature for all subsequent steps) for 5 min. beads were collected, the wash removed, and a second 150 ml wash step was performed. the beads were washed an additional two times with 30 ml of buffer b in a 50-ml corning tube while shaking for 10 min for each wash (total of 4 wash steps). proteins were eluted from the washed beads by addition of buffer b with increasing imidazole concentrations of 25, 150, 300, and 500 mm. 20 ml of appropriate buffer was used for each elution and the beads were shaken in 50-ml corning tubes for 10 min before collecting. samples of each elution fraction were analyzed by sds-page and coomassie-staining and appropriate fractions were pooled. the pool was treated as above for buffer exchange, final gel analysis, and storage. transmission electron microscopy of the purified vrc and mt. sinai spike proteins was carried out by dilution of thawed final samples to 0.02 mg/ml in 20 mm tris-hcl, ph 8.0, 200 mm nacl followed by loading onto glow-discharged carbon support film grids (cf200-cu, electron microscopy sciences). grids were washed twice in buffer (20 mm tris-hcl, ph 8.0, 200 mm nacl) and stained with 0.75% w/v uranyl formate (ph 4.5) three times using filter paper to blot away the stain prior to immediate application of additional stain. a final staining step was carried out for 30 sec with the stain then removed by wicking with filter paper, and the grids were dried under an incandescent lamp. stained grids were imaged on a hitachi 7650 electron microscope at 40,000x magnification. in order to assess batch-to-batch reproducibility of spike antigens, purified vrc spike proteins from five different purifications were used as antigens in an elisa with positive control cov-2 serum at multiple dilutions. the elisa protocol was carried out as reported [4] using 8 100 ng per well of the various spike proteins and dilutions of 1:500, 1:10,000, and 1:100,000. absorbance measurements were made at 450 nm (signal) and 650 nm (background control) using a bmg labtech pherastar plate reader which is capable of linear absorbance readings up to an od of ~ 4. to produce sars-cov-2 antigens for the development of serology assays, we initially followed standard procedures for secreted protein production: transfection using the manufacturer's protocols, expression at 37°c, harvest at three days post-transfection, tangential flow filtration of the culture supernatant, immobilized metal ion chromatography with linear gradient elution, and size exclusion chromatography. however, this process resulted in very low yields (0.9 and 0.3 mg/l, respectively, for mt. sinai and vrc spike as seen in table 1 ). these yield levels were not high enough to support large serology studies without significant scaleup and associated high costs. the urgent need and the narrow time frame to provide protein support for nih serology studies limited our ability to perform extensive troubleshooting and optimization. rather, we assessed several parameters with internally controlled experiments (e.g. splitting an imac pool in two and passing the two aliquots over different size exclusion resins). this was essential as safety protocols for laboratory personnel during the pandemic limited the number of staff in the lab. using this approach, we were able to obtain information on the effect of multiple parameters including the temperature of cell culture, harvest time, and mode of target capture. post-harvest production process followed the procedure of tff > imac > desalting column. numbers represent yields from independent experiments. nd -not determined. inspection of the sds-page/coomassie staining analysis of the initial imac (not shown), suggested that we could improve the separation of spike from contaminant by adjusting the elution parameters. the relatively high affinity of the his-tagged spike proteins for the column enabled the use of a 6 cv linear elution gradient from 25 mm to 175 mm imidazole which eluted the majority of contaminants before spike was eluted in a step elution of 325 mm imidazole ( fig. 2a) . analysis of intermediate purification steps by the low-resolution method of sds-page/coomassie staining suggested significant protein loss was occurring during size exclusion chromatography (sec). this correlated with published methods of spike purification [3] and suggested that high concentrations of spike might lead to protein loss by precipitation. we found it difficult to quantitate the amount of target loss during these steps, likely due to heavy glycosylation, which is reported to interfere with bradford analysis of protein concentration [5, 6] . by using a desalting column for buffer exchange, rather than sec or diafiltration, our modified protocol is designed to minimize protein manipulations that might lead to protein loss especially during post-imac steps. thus, we compared purification yields from parallel experiments with either sec or a desalting column as the final purification step ( table 1) . the desalting column protocol led to a consistently higher yield of ~2 mg/l, indicating that this method improved protein recovery. in addition, very little difference in protein quality was observed in these samples, particularly in the case of the vrc spike protein, again arguing against the need for the sec step. as can be seen in fig. 2b , the vrc spike protein was consistently of higher purity than the mt. sinai spike protein. however, elisa data comparing these two proteins suggests that these minor impurities have no significant effect on the antigenicity in the assay [4] . nevertheless, for downstream processes which require higher levels of purity, mt. sinai spike may require additional purification steps. reducing expression temperature during recombinant protein expression, in both e. coli and the baculovirus/insect expression system, can significantly increase yields of soluble protein [7] . in transient cho systems, temperature reductions are commonly used to enhance protein production [8] . however, similar modifications in hek293-based systems are less frequently reported. previous work in our lab and others suggests one limitation to protein secretion in transiently transfected mammalian cell culture might be the secretion process itself. while multiple factors appear to be involved, a recent paper suggests that limiting components in the secretory pathway appear to be the major bottleneck [9, 10] . we did observe a significant amount of spike protein in the expi293 cell pellets (data not shown), suggesting that some protein was being held up in the endoplasmic reticulum, or was otherwise failing to completely mature through the secretory pathway. low cell viabilities (<70%) at harvest were another sign of potential toxicity caused by secretory failures. thus, we compared the purification yield of vrc and mt. sinai spike from transiently transfected cells incubated at either 32°c or 37°c after the addition of enhancers 18-hours post-transfection. as seen in table 1 and fig. 2b , the lower temperature expression led to a dramatic increase in yield to ~5 mg/l for both cov-2 spike proteins we tested. this yield is similar to that cited in a recent report of the mt. sinai spike protein produced at 37°c and using a diafiltration approach to buffer exchange [3] . however, unpublished results from other colleagues in the field using this construct suggest 1-2 mg/l is a more consistently observed result, suggesting that these improvements will make a marked enhancement to yield. published vrc spike protein yields are 0.5 mg/l [2] , suggesting that our modified procedures improved production of this protein by nearly 10-fold. we also investigated the time of harvest as a variable, as this is a common approach to improve yield from secreted protein platforms [11] . in initial experiments incubated at 37°c, increasing the time of harvest from 72 hours post-transfection to 96 hours resulted in some improvement in yield ( table 1) . we did not test production at the lower temperature of 32°c at 72 hours, as cell growth is considerably slower at this temperature and we anticipated this would not be enough time for high levels of expression. therefore, 96 hours was used as the standard for 32°c expression as noted above. however, we did explore longer growth times at the lower temperature and saw no further yield increase for either vrc or mt. sinai spike by harvesting 120 hr post-transfection (table 1, fig. 2b ). for these reasons, we chose 32°c and 96 hours as our optimal conditions for future expression. of note, we have also expressed and purified the analogous recombinant spike proteins from 4 other beta-coronaviruses using the original protocols described here as controls for the serology assay [4] . interestingly, our yields of the spike proteins from sars-cov, mers-cov, oc43, and hku1 (4.6, 10.6, 8.4, and 5.7 mg/l, respectively) in a single experiment from cells incubated at 37°c were considerably higher than that of the cov-2 spike under similar conditions. this argues that sars cov-2 spike is unique in some aspect of protein stability and/or recombinant protein expression, as these other coronavirus spike proteins were cloned in the same vector and using the same strategy as the vrc cov-2 spike. we anticipate that the new protocols outlined here might further improve the yield of those proteins as well. our observations of protein loss during steps in which spike is either concentrated or exposed to large surface areas, suggested that we might further improve purification by eliminating the tff, which is necessary to achieve maximum protein capture from the culture supernatant during subsequent column imac. thus, we used magnetized imac beads to capture vrc spike in batch mode from filtered lysates. the final protein purified with this approach was similar in terms of quantity and final purity ( table 1 and fig. 2c ) to proteins purified by the tff/imac/desalting protocol. this batch process has several distinct advantages over the more complex protocol. first, it eliminates the need for the labor-intensive tff process, which for large-scale transient cultures can take many hours, and the requirement for specialized equipment and consumables. second, when combined with a gravity flow approach to buffer exchange, this process completely obviates the need for a protein workstation, making the process accessible to many laboratories without fplc technology, and making it highly scalable. we evaluated the functionality of spike proteins by structural and conformation-based methods. unlike natural sars-cov-2 spike proteins, these engineered recombinant forms of the protein utilize an exogenous trimerization domain from phage t4 to form appropriate trimeric structures in the absence of cell membranes. to assess the oligomeric state, our soluble spike proteins were analyzed by electron microscopy and analytical size exclusion chromatography (ansec). negative-stain tem images of spike particles (fig. 3a) clearly show the expected trimeric structure and closely resemble previous published images of engineered soluble trimer spike proteins [2] . in addition, as seen in fig. 3b , recombinant spike proteins eluted during ansec at a volume expected for a protein of ~520 kda rather than that expected for monomeric spike (~180 kda). this result was consistent over all preparations of both vrc and mt. sinai spike proteins, and in no cases did we observe any detectable monomeric spike protein by ansec. these macromolecular based approaches support the conclusion that our purified spike proteins adopt tertiary and quaternary structures like those of previously published engineered spike trimers. an initial lot of vrc spike protein was previously used to develop and optimize a serology assay using an elisa format [4] . to assess whether spike proteins produced by our modified protocols had equivalent elisa sensitivity, multiple production lots of vrc spike were compared in the serology assay. fig. 4 demonstrates that independent batches of vrc spike proteins performed nearly identically in the assay at multiple concentrations of positive control sera. assay sensitivity was consistent across multiple lots of protein produced with the same expression conditions (duplicate bars of identical color) and also across protein produced at different expression times and temperatures. consistent lot-to-lot performance of spike protein is an essential part of a high-quality, sensitive serology assay. in summary, we presented multiple improvements to the production of sars cov-2 spike protein that will allow labs with modest protein expression and purification experience to consistently produce high-yield and high-quality protein for use in a variety of applications. while we optimized the process to generate proteins for serology assays, the high quality of these proteins makes them amenable for biochemical, biophysical, and structural studies, or as substrates for drug screening. we hope that the parameters we highlighted in this report will help others to overcome bottlenecks in spike production and guide future optimization work. serology assays to manage covid-19 cryo-em structure of the 2019-ncov spike in the prefusion conformation sars-cov-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup standardization of enzymelinked immunosorbent assays for serosurveys of the sars-cov-2 pandemic using clinical and at-home blood sampling comparison of the coomassie brilliant blue, bicinchoninic acid and lowry quantitation assays, using non-glycosylated and glycosylated proteins staining of glycoproteins/proteoglycans on sds-gels purify first: rapid expression and purification of proteins from xmrv mild hypothermia upregulates myc and xbp1s expression and improves anti-tnfα production in cho cells a consensus genome-scale reconstruction of chinese hamster ovary cell metabolism cho cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion a high density cho-s transient transfection system: comparison of expicho and expi293 key: cord-339333-7tpnbr8q authors: chen, yuxian; zeng, chun; zeng, hua; zhang, rongkai; ye, zhiqiang; xing, bangrong; hu, kunhua; li, mingtao; cai, dao zhang title: comparative serum proteome expression of the steroid-induced femoral head osteonecrosis in adults date: 2014-11-12 journal: exp ther med doi: 10.3892/etm.2014.2069 sha: doc_id: 339333 cord_uid: 7tpnbr8q steroid-induced osteonecrosis of the femoral head (sonfh) is a disabling, aseptic and ischemic disease that develops following steroid therapy. the pathogenesis of sonfh is unclear, so the early diagnosis and treatment for this disease is yet to be established. the purpose of the present study was to identify potential biomarkers for sonfh. the differential expression of serum proteins from patients with sonfh and healthy volunteers was analyzed by the proteomics method. the protein samples were labeled and subjected to isoelectric focusing and two-dimensional gel electrophoresis. the resultant protein spots were matched and quantified by an imaging analysis system. the differentially-expressed protein spots were subjected to in-gel trypsin digestion followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. significantly lower levels of complement component 3 (c3), c4, inter-α-trypsin inhibitor heavy chain h4 and α-2-macroglobulin were found in the serum of patients with sonfh. these proteins are reported to be actively involved in intravascular coagulation, apoptosis and reactive oxygen species imbalance, indicating that multiple pathological reactions occur in sonfh and these proteins may serve as potential biomarkers for the diagnosis of sonfh. the steroid-induced osteonecrosis of the femoral head (sonfh) is a devastating, irreversible and disabling disease developing following steroid therapy (1) . the functions of the hip joint are markedly impaired when the femoral head collapses. glucocorticoid (gc) therapy was the most common cause of onfh (2) . the onset of sonfh is within several months following the administration of steroids. regardless of continuous steroid administration, no expansion of the necrotic area was found, and recurrence was not identified. due to ischemia, patients have no symptoms when sonfh occurs. no pain is noted until the femoral head collapses. approximately 5-25% of patients using gc develop sonfh in the legs. notably, in china 53.5% of patients with severe acute respiratory syndrome prescribed gc developed femoral head osteonecrosis, and the sonfh comprises approximately half of non-traumatic femoral head necrosis. in total, >50% of patients with femoral head osteonecrosis admitted to the third affiliated hospital (sun yat-sen university, guangzhou, guangdong, china) between january 2000 and january 2010 had a history of using gc, the majority of whom were young males. at present, patients with sonfh already exceed 10 million in china. however, the pathogenesis of this disease remains largely unknown, so the prevention and treatment have yet to be established. with the progress of systemic biology, proteomic approaches provides a valuable tool to study the whole protein content of a biological sample in one set of experiments. one of the reliable technologies is two-dimensional (2-d) electrophoresis or 2-d difference gel electrophoresis (dige) analysis coupled with mass spectrometry identification (3, 4) . this technology has recently been employed to screen potential bioactive molecules underlying the pathogenesis of arthritis and osteopenia, and it is reported that the protein levels in serum, joint fluid and bone tissue correlate to the severity of rheumatoid arthritis (5), the autoimmune response in ankylosing spondylitis in animal models (6) , the loss of cartilage integrity in osteoarthritis (7) and estrogen loss-induced osteoporosis (8) . however, the changes detected in the serum protein profile of patients with sonfh have not yet been reported. the purpose of the present study is to find potential biomarkers of the sonfh by using proteomic technology to analyze serum protein profiles in patients with sonfh and the healthy control group. patients. the study was approved by the institutional review board of the third affiliated hospital, and informed consent was obtained from each patient. a total of 12 patients, including five females and seven males, fulfilled the inclusion and exclusion criteria. inclusion criteria included receiving a single short-course of corticosteroid medication within the three years prior to presentation, onfh identified by typical magnetic resonance imaging (mri) findings, stage 1 or 2 by the ficat classification system and no previous treatment of the femoral head (9) . the exclusion criteria for sampling were the history or evidence of metabolic bone diseases, including hyper-or hypoparathyroidism, paget's disease, renal osteodystrophy and the presence of cancers with bone metastasis. in total, 12 healthy volunteers, including five females and seven males, were enrolled in the control group. peripheral venous blood (5 ml) was drawn from each patient in the outpatient department or in the procedure room prior to general anesthesia for total hip replacement. the blood samples were processed to collect serum and stored at -80˚c until analysis. serum sample preparation. blood samples (3 ml) from each subject were collected in a drying tube early in the morning and allowed to clot for 1 h at room temperature. the samples were centrifuged at 2,500 x g for 15 min at 4˚c. the supernatant was dispensed into 0.5 ml aliquots and stored at -80˚c until use. all the serum samples were processed according to a standard protocol. all the samples were thawed at room temperature. high abundance proteins, such as albumin and immunoglobulin g (igg), were depleted with the proteoprep ® blue albumin & igg depletion kit (sigma-aldrich, inc., st. louis, mo, usa) as per the manufacturer's instructions. subsequently, samples were further cleaned with the clean-up kit (ge healthcare, piscataway, nj, usa) according to the manufacturer's instructions. proteins were quantified using 2-d quant kit (ge healthcare). the protein concentrations of samples were adjusted to 8 mg/ml and verified by sds-page and coomassie blue staining. fluorescent labeling. to limit experimental variation and ensure accurate in-gel matching, all the samples were labeled with fluorescence. the cydye dige fluor (minimal dye) labeling kit (ge healthcare) was used for tagging, following the manufacturer's instructions. cyanine 2 (cy2) was used for the internal standard sample that was generated by mixing together an aliquot of samples from the patient and control groups. cy3 and cy5 were used to label samples from the control and patient groups, respectively. the labeling reaction was carried out in the dark on ice. 2d-dige and in-gel trypsin digestion. all the labeled samples were subjected to 2d-dige with 24 cm immobilized ph gradient (ipg) strips (ph 4-7) (ge healthcare) for the first dimensional isoelectric focusing and subsequently the second dimensional sds-page with 12% polyacrylamide gels. the 2d-dige was run in triplicate for each sample to reduce the gel-to-gel variation. the gels were scanned using typhoon 9410 variable mode imager (ge healthcare) at the excitation/emission wavelengths specific for each cydyes immediately. subsequent to scanning, all the gels were stained with deep purple and stored for subsequent mass spectrometric identification. images were then processed with decyder differential in gel analysis v6.0 software (ge healthcare) to identify changes in spot fluorescence intensities. proteins were considered differentially expressed if the abundance showed >1.5-fold change between the patient and the control groups with p<0.05 using one-way analysis of variance. disparate points were excised using the ettan spot handing workstation (ge healthcare), then destained and subjected to in-gel trypsin digestion. peptides were extracted for subsequent mass spectrometry. matrix-assisted laser desorption ionization time-of-flight mass spectrometry (maldi-tof-ms/ms) and protein identification. following trypsin digestion, protein identification was carried out on the ettan maldi-tof mass spectrometer (ge healthcare). the trypsin auto-digestion peaks (m/z 842.509 and 2211.104 da) were used for internal calibration. each spectrum corresponded to the sum of 200 acquisitions for each of eight laser pulses, in which the threshold signal/noise exceeded a set value. the resulting data were then analyzed with mascot search engine (matrix science, london, uk) for protein identification and compared to the national center for biotechnology information (10) and swiss-prot (11) protein databases. the following keywords were used in the search: trypsin digestion, homo sapiens, 1-100 kda protein mass, 100 ppm peptide tolerance. western blot analysis. serum protein samples prepared as described above were diluted 1:25 in laemmli buffer and resolved by 10% sds-page (invitrogen life technologies, carlsbad, ca, usa). the separated proteins were transferred to polyvinylidene fluoride. the membranes were blocked with 5% skimmed dry milk in tris-buffered saline containing 0.05% tween 20 (tbst) for 1 h at room temperature and incubated with antibodies overnight at 4˚c. subsequent to washing two times in tbst, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. protein bands were detected using ecl plus (forevergen bioscience co., ltd., guangzhou, china) and the images were acquired by the imaging system (gel doc xr system, bio-rad, hercules, ca, usa). elisa. elisa assays using a microtiter plate assay were performed individually on the samples in each group chosen randomly and matched across the groups. primary and secondary antibodies were obtained from santa cruz biotechnology, inc. (santa cruz, ca, usa). a standard curve was generated by four-parameter curve-fitting using softmaxpro v 1.11 software, (molecular devices corp., sunnyvale, ca, usa). statistical analysis. the data analyses were performed by spss 15.0 (spss, inc., chicago, il, usa). statistical significance of the differences was determined using the student's t-test, and the tukey method was performed to correct multiple comparisons. all the values were reported as the mean ± standard deviation, and p<0.05 was considered to indicate a statistically significant difference. patients. in total, 12 patients with sonfh (age, 32.3±2.3 years; range, 25-40 years) and 12 healthy volunteers (age, 33±2.3 years; range, 28-41 years) were enrolled. there was no significant difference in the age (p= 0.827) between the sonfh and healthy volunteer groups. all the patients were diagnosed with onfh by mri. the patients classified as stage 1 or 2 by the ficat classification system were enrolled in the study. the medical records show that all of them had received glucocorticoid therapy due to arthralgia. the mean steroid dose in equivalent milligrams of prednisone was 850 mg (range, 290-3300 mg). the mean time from administration of steroids to the development of hip symptoms was 16.6 months (range, 6-33 months), but none of them fulfilled the diagnosis criteria of rheumatoid arthritis. 2-d dige analysis of differential protein expression. 2-d dige was performed to analyze differential protein expressions as previously described (12) . approximately 1,600 protein spots were detected across all four gels by the decyder image analysis software (ge healthcare). fig. 1a shows the superimposed images in pseudocolor from cy3-and cy5-labelled sera samples and the positions of the spots corresponding to the proteins revealed by 2-d dige analysis. fig. 1b presents the differences in protein expression by the decyder 3-d spot simulations. four protein spots with >1.4-fold decrease in abundance between the patient and control groups were identified by computer-assisted comparative analysis (p<0.05, student's t-test; table i ). to further confirm the change of these four protein spots, two preparative gels loaded with 500 µg protein from each extract were run in parallel and followed by deep purple staining. the decrease of the protein abundance was consistent with the result of cydye labeled images as analyzed by decyder software. four proteins were revealed to be downregulated in the sera of patients with sonfh. protein identification. the above four differential protein spots were excised and digested with trypsin in gel for maldi-tof peptide mass fingerprinting (pmf) analysis. the product spectra generated by maldi-tof-ms/ms were searched against the swiss-prot database for exact matches using the mascot by pmf (fig. 2) . the four proteins were respectively inter-α-trypsin inhibitor heavy chain h4 (itih4), complement component 4 (c4), a2mg and c3 (table i) . western blot analysis and elisa to confirm the differential expression. to validate the differential expressions of these four proteins, the eight serum samples used for the dige experiment were assessed by western blot analysis with the specific antibodies. the levels of c4, itih4, a2mg and c3 are significantly lower in the patient group than in the control group (fig. 3) , which is consistent with the results from the proteomic study. subsequently, it was determined whether proteins express differentially during femoral head tissue necrosis. to test if it is the case for a2mg, the proteins prepared from the necrotic femoral head tissue were analyzed by elisa. the expression of a2mg was significantly lower in patient group than in the control group (fig. 4) , which is consistent with the result of the serum samples. no change was observed in the levels of c3, c4 and itih4 in necrotic bone tissues (data not shown). the incidence of sonfh is increasing year by year, while the diagnosis of this disorder still relies on image examination, which often fails to detect the lesion at the early stage. therefore, a number of patients miss the opportunities for early treatment. it is important to find the diagnostic biomarkers for sonfh. since blood tests are commonly used in clinical practice, the protein profile in the serum from the patients with sonfh and healthy volunteers were analyzed using cutting-edge proteomic technology. the aim was to identify proteins whose level in the blood was significantly altered in patients with sonfh. to ensure the reproducibility, accuracy and objectivity of the experiments, the following steps were differentially-expressed protein spots were identified from the two-dimensional difference gel electrophoresis profiling of human plasma, with a lower abundance in the steroid-induced femoral head osteonecrosis group compared to the control group. mw, molecular weight. implemented to minimize the experimental errors between samples. first, the sample selection criteria were strictly followed. secondly, high abundance proteins were removed prior to the electrophoresis, which otherwise would mask the low abundance proteins. further, fluorescence labeled 2-d dige was employed. an internal standard was used for each protein spot in 2-d dige, and the software designed for 2-d dige automatically corrected the protein amount according to the internal standard. thus, these approaches significantly improved the reproducibility and the sensitivity. in the present study, four proteins (c3, c4, itih4 and a2mg) showed lower expression in the serum of patients with sonfh than that of the normal subjects. the changes were confirmed by western blotting. the expressions of these proteins were also examined in necrotic bone tissues. unlike serum, necrotic bone tissue did not have detectable amounts of c4 and itih4. in addition, c3 showed no difference in abundance between the two groups. only a2mg was downregulated in the protein levels in necrotic bone tissue, consistent with the result of the serum. the pathogenesis of sonfh remains unclear. several mechanisms have been proposed, including lipid metabolism dysfunction, intravascular coagulation, apoptosis and reactive oxygen imbalances. the present study shows that the expression of c3, c4, itih4 and a2mg were significantly altered in patients with sonfh. all four proteins are closely associated with apoptosis, and therefore the present study supports that apoptosis plays a major role in sonfh. c3, c4 and their degradation products are cytokines and acute phase reactive proteins that are produced by macrophages and hepatocytes. they are key factors in the activation of the complement system. the activation of the complement cascade can cause a variety of biological effects, including immune response, generation of sensitized lymphocytes and altered metabolism of blood sugar and lipid (13) . however, limited studies have systematically elucidated the mechanism by which the immune system affects sonfh. wu et al (14) showed that the complement factor c3 precursor is elevated in the serum of patients with onfh. this previous study showed that complement factor c3 precursor plays an important role in the homeostasis of inflammation, necrosis or apoptosis in onfh. the present results show that complement activation is reduced in patients with sonfh. this may be attributed to the immunosuppressive effect of steroids. excess steroids can suppress complement activation and immune complex formation (15) . familian et al (16) found that plasma levels of c3 and c4 increased in the majority of patients with rheumatoid arthritis prior to therapy, but significantly decreased following the start of infliximab (an immunosuppressive agent) treatment. the mechanism of complement inhibition involved in sonfh requires further study. itih4, when translated, is secreted into the blood, where it is cleaved by plasma kallikrein into two smaller forms. itih4 mrna is specifically expressed in the liver. the gene is part of a cluster of similar genes on chromosome 3. two transcription variants encoding different isoforms have been found. itih4 is also an acute phase reactive protein, but its biological function remains unknown. it was detected in swine, bovine and rat models with experimentally-induced acute inflammation (17) (18) (19) . pineiro et al (20) showed that in humans, itih4 mrna and the secreted protein are highly upregulated by il-6 in hepg2 hepatoma cells. bost et al (21) assumed itih4 may interact with components of the extracellular matrix and modulate cell migration and proliferation during the development of the acute-phase response. it is clear that onfh is accompanied by inflammation. aseptic inflammation presents in patients with onfh and it is conceivable that persistent consumptive inflammation and the effects of steroids lead to the decrease of serum itih4. further study is necessary to address the role of itih4 in the disease. a2mg is an inhibitor of matrix metalloproteases (mmp) (22) , which is mainly synthesized by hepatocytes in the liver. small amounts of a2mg are also produced by a number of other cells, including lung fibroblasts, macrophages, astrocytes and tumor cells (23, 24) . a2mg functions as a broad irreversible proteinase inhibitor and is involved in various physiological processes (25, 26) . a2mg regulates several key factors of sonfh. the conformational change can activate a2mg, resulting in exposure of binding sites for its cell surface receptor, including the low-density lipoprotein receptor-related protein. upon binding a2mg-proteinase complexes from the extracellular matrix are rapidly removed, which blocks lipid catabolism (27) . a2mg modulates blood coagulation. as reported by simpson et al (28) , a2mg significantly enhanced plasmin generation. however, a2mg binds vascular endothelial growth factor and the resultant a2mg-complex inhibits heparin activity, leading to elevated coagulation. human a2mg has been verified to effectively decrease the release of superoxide radicals by polynuclear leukocytes following radiation. the activity of superoxide dismutase in red cells can also be increased. the free radicals and mmp imbalance exist in the pathological process of sonfh. kerachian et al (29) demonstrated that the a2mg gene is significantly upregulated in avascular necrosis of the rat femoral head induced with steroids. along with those findings, the present study showed that a2mg was significantly lower in the bone tissue of patients with sonfh. lower a2mg may affect the process of sonfh through figure 4 . estimation of a2mg in necrotic bones. a2mg expression was lower in the necrotic bones of patients with sonfh (onfh) than that in the bones of subjects without sonfh (control). * p<0.05. sonfh, steroid-induced osteonecrosis of the femoral head; a2mg, α-2-macroglobulin. these aspects. consistent with the bone tissue, the serum a2mg level was also decreased. in conclusion, a2mg is involved in multiple mechanisms underlying sonfh, including blood coagulation, hyperlipidemia, free radicals and mmp degradation. this underscores the critical role of a2gm in the development of sonfh. therefore, a2gm may become a novel potential biomarker and a novel therapeutic target for sonfh. clinical condition of steroid-induced osteonecrosis of the femoral head glucocorticoids in osteonecrosis of the femoral head: a new understanding of the mechanisms of action proteome analysis of apoptotic cells calpain-truncated crmp-3 and -4 contribute to potassium deprivation-induced apoptosis of cerebellar granule neurons use of mass spectrometry to identify protein biomarkers of disease severity in the synovial fluid and serum of patients with rheumatoid arthritis identification of disease-associated proteins by proteomic approach in ankylosing spondylitis proteomic analysis of articular cartilage shows increased type ii collagen synthesis in osteoarthritis and expression of inhibin betaa (activin a), a regulatory molecule for chondrocytes proteome analysis for the identification of in vivo estrogen-regulated proteins in bone in brief: ficat classification: avascular necrosis of the femoral head characterization of proteins in human pancreatic cancer serum using differential gel electrophoresis and tandem mass spectrometry the adipsin-acylation stimulating protein system and regulation of intracellular triglyceride synthesis comparative serum proteome expression of osteonecrosis of the femoral head in adults predictive value of synovial fluid analysis in estimating the efficacy of intra-articular corticosteroid injections in patients with rheumatoid arthritis infliximab treatment reduces complement activation in patients with rheumatoid arthritis characterization of the acute phase serum protein response in pigs itih4 (inter-alpha-trypsin inhibitor heavy chain 4) is a new acute-phase protein isolated from cattle during experimental infection hepatic and extra-hepatic transcription of inter-alpha-inhibitor family genes under normal or acute inflammatory conditions in rat itih4 serum concentration increases during acute-phase processes in human patients and is up-regulated by interleukin-6 in hepatocarcinoma hepg2 cells inter-alpha-trypsin inhibitor proteoglycan family-a group of proteins binding and stabilizing the extracellular matrix progress in the development of matrix metalloproteinase inhibitors alpha 2-macroglobulin, a multifunctional binding protein with targeting characteristics metalloproteinase inhibitors: biological actions and therapeutic opportunities role of alpha-2 macroglobulin in oncologic diseases zinc-binding proteins (metallothionein and alpha-2 macroglobulin) and immunosenescence inhibition of bone morphogenetic protein 1 by native and altered forms of alpha2-macroglobulin simultaneous thrombin and plasmin generation capacities in normal and abnormal states of coagulation and fibrinolysis in children and adults new insights into the pathogenesis of glucocorticoid-induced avascular necrosis: microarray analysis of gene expression in a rat model the present study was supported by grants from the 863 scientific research of the national natural science key: cord-345088-krb1eidw authors: shen, s; law, y.c; liu, d.x title: a single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature date: 2004-09-01 journal: virology doi: 10.1016/j.virol.2004.06.016 sha: doc_id: 345088 cord_uid: krb1eidw the spike (s) glycoprotein of coronavirus is responsible for receptor binding and membrane fusion. a number of variants with deletions and mutations in the s protein have been isolated from naturally and persistently infected animals and tissue cultures. here, we report the emergence and isolation of two temperature sensitive (ts) mutants and a revertant in the process of cold-adaptation of coronavirus infectious bronchitis virus (ibv) to a monkey kidney cell line. the complete sequences of wild type (wt) virus, two ts mutants, and the revertant were compared and variations linked to phenotypes were mapped. a single amino acid reversion (l(294)-to-q) in the s protein is sufficient to abrogate the ts phenotype. interestingly, unlike wt virus, the revertant grows well at and below 32 °c, the permissive temperature, as it carries other mutations in multiple genes that might be associated with the cold-adaptation phenotype. if the two ts mutants were allowed to enter cells at 32 °c, the s protein was synthesized, core-glycosylated and at least partially modified at 40 °c. however, compared with wt virus and the revertant, no infectious particles of these ts mutants were assembled and released from the ts mutant-infected cells at 40 °c. evidence presented demonstrated that the q(294)-to-l(294) mutation, located at a highly conserved domain of the s1 subunit, might hamper processing of the s protein to a matured 180-kda, endo-glycosidase h-resistant glycoprotein and the translocation of the protein to the cell surface. consequently, some essential functions of the s protein, including mediation of cell-to-cell fusion and its incorporation into virions, were completely abolished. the coronaviridae family contains causative agents of a wide spectrum of diseases affecting humans, mammals, and birds. it is the etiologic agent of severe acute respiratory syndrome (drosten et al., 2003; fouchier et al., 2003) . the molecular basis of host specificity and tissue or cell tropism of coronavirus partially resides in the specific interaction between the surface spike protein and cellular receptor(s). this interaction would determine the outcome of infection: whether it is acute or chronic and persistent (rowe et al., 1997a (rowe et al., , 1997b , which organs or tissues are targeted, what symptoms are shown (ballesteros et al., 1997; leparc-goffart et al., 1997; navas et al., 2001) , and even whether host specificity is changed (kuo et al., 2000) . coronaviruses are a group of enveloped rna viruses with positive-sense, single-stranded genomes of 27 to 32 kb that are packaged into helical structures by the nucleocapsid protein (n). the viruses acquire their envelopes by budding of the structural components into the intermediate compartment (ic) between the endoplasmic reticulum (er) and the golgi apparatus (krijnse-locker et al., 1994; lai and cavanagh, 1998; tooze et al., 1984) . the membrane glycoprotein (m) and the envelope protein (e) mediate the formation of the envelope, as they are involved in the induction of virion assembly and incorporation of other structural components into virus particles (vennema et al., 1996) . the spike (s) glycoproteins incorporate into the envelope (de haan et al., 1999) in the form of homooligomeric, 20 nm projections, giving the distinctive morphology of the coronavirus virion. some coronaviruses also contain another envelope protein, the hemagglutinin esterase (he). the largest structural s protein is a type i integral membrane glycoprotein and spans the viral envelope once. in some cases, the s protein is proteolytically cleaved into the n-terminal s1 and the c-terminal s2 subunits of equal size by a host proteinase . the s1 and s2 subunits are noncovalently associated to each other. a hydrophobic transmembrane domain (tm) of s2 anchors on the envelope with a short cytoplasmic-tail inside. one leucine-zipper (heptad repeats) domain overlaps with the tm domain and another extends outside the envelope, folding into a-helices and coiled-coil structures involved in oligomerization (de groot et al., 1987) . it was found that the tm domain and endodomain mediated the incorporation of s into virions but the details of the s/m interaction remain to be elucidated. although s protein is dispensable for the formation of virus-like-particles (vlps), its incorporation into virion is critical to the assembly of infectious virus particles. the s protein is cotranslationally n-glycosylated in the er and also forms stable complex with m in the pre-golgi membrane (holmes et al., 1981; opstelten et al., 1995; sturman et al., 1985) . it was then transported to the golgi apparatus where its high mannose side chains are subsequently trimmed and further modified. the matured s protein is an endo-glycosidase-h-resistant (endo-h), 180-kda form (luytjes et al., 1997) . at the trans-golgi, the matured virions are encapsulated into vesicles of the constitutive secretory pathway and released from infected cells. a portion of s protein might be transported to the plasma membrane and responsible for the cell-to-cell membrane fusion, resulting in the formation of typical syncycia and spread of virus infection to neighboring cells. the tm, cysrich and leucine-zipper domains of s2 and the disparate region of s1 are associated with the membrane fusion activity of the protein (chang et al., 2000; gallagher et al., 1991; krueger et al., 2001; luo et al., 1999; tsai et al., 1999) . cleavage of the s protein enhances the membrane fusion, though not necessarily required. the s protein contains receptor-binding domains in the s1 subunit (kubo et al., 1994; godet et al., 1994; saeki et al., 1997; suzuki and taguchi, 1996) . upon binding of the s protein to the receptor(s), conformation changes induce the virus -cell membrane fusion and subsequently unloading of the ribonucleocapsid inside the cell. it has been demonstrated that the cell-to-cell spread of mhv infection does not need the presence of the receptor, but the s protein is required to mediate the cell-to-cell fusion (gallagher et al., 1992; nash and buchmeier, 1996) . for coronaviruses, however, the detail of molecular mechanisms of membrane fusion, especially those linked to the s1 region, is yet to be fully elucidated. in this study, ts mutants were generated by growing the beaudette strain of ibv at progressively lower temperatures from 35 to 28 jc in vero cells. two ts mutants were isolated from passages grown at 29 jc (ts291602) and 28 jc (ts282902). a revertant was also obtained by growing the ts mutant at the nonpermissive temperature (40 jc). sequence comparison revealed that mutations in the s1 subunit were responsible for the ts phenotype. comparative studies of viral protein synthesis and growth properties of the wt, ts mutant, and revertant viruses demonstrated that the ts mutants were extremely unstable at the nonpermissive temperature. if they were allowed to enter cells by absorption for 1 h at the permissive temperature (32 jc), the s protein would be core-glycosylated and partially modified at the nonpermissive temperature, but was unable to assemble into virions. the membrane fusion activities of the ts mutants were totally abolished in virus-infected cells and in cells overexpressing the mutant s protein at the nonpermissive temperature. the beaudette strain of ibv, grown in vero cells at 37 jc, was plaque-purified and was initially adapted to grow at 35 jc. after 4 passages, the virus was subsequently adapted to grow at progressively lower temperatures by culturing at 34 jc for 5 passages, 33 jc for 7 passages, 32 jc for 44 passages, 30 jc for 12 passages, 29 jc for 21 passages, and 28 jc for 29 passages. when the virus was initially adapted to a lower temperature, no cpe was observed within 72 -96 h postinfection. apparent cpe appeared usually after 2 to 5 passages. the virus was continuously passaged at the same temperature until cpe became visible within 24 -48 h postinfection, and then shifted to a lower temperature. plaque assays of different passages were performed at both 32 and 40 jc. it was found that viruses from early passages grown at 32 jc could form plaques at both temperatures at similar levels. but passages grown at 29 jc produced much fewer plaques at 40 jc than at 32 jc, suggesting the emergence of ts mutants during cold-adaptation. these ts mutants dominated quickly at 28 jc. two ts mutants, designated ts291602 and ts282902, were plaque-purified from passage 16 grown at 29 jc and passage 29 grown at 28 jc, respectively. plaque assays showed that both ts291602 and ts282902 formed plaques only at the permissive temperature, but not at all at the nonpermissive temperature. by growing the ts mutant at 37 jc overnight and then at 40 jc for 2 days, a revertant, rev-1, was obtained which produced plaques at both 32 and 40 jc at a similar level ( table 1 ). the titers of rev-1 were much higher than that of the wild-type virus wt6501 (isolated from passage 65 at 37 jc) at 32 jc, as it was derived from the cold-adapted virus (table 1) . to map the defects responsible for the ts phenotype described above, the complete nucleotide sequences of wt6501, ts291602, ts282902, and rev-1 were determined using rt-pcr products and cdna clones from each virus. comparison of the sequence of wt6501 with ts291602 revealed 22 point mutations, a 5-base-deletion, a 1-base-, 3-base-and a 9-base-insertion (table 2 ). these alterations caused 15 amino acid substitutions in eight mature viral proteins, a single amino acid insertion in the 3a protein, a truncated 3b (compared with early passages of wt virus) caused by a single base insertion in the 3b of ts291602, and a 3-amino acid insertion in the n protein. in ts282902, similar point mutations, deletion and insertion were found ( table 2 ). the most different feature of the two ts mutant is the insertion in the 3b gene. in ts291602, a single a insertion was found at a poly-a stretch at positions 24075 to 24080, causing frameshift of the 3b gene and resulting in a c-terminally truncated 3b protein (shen and liu, 2003) . however, a three-a-insertion at the same poly-a stretch was found in ts282902, which does not affect the open reading frame of the 3b gene. comparison of the sequence of rev-1 with the two ts mutants showed only one amino acid change (q 294 -to-l 294 ) in the s protein and one amino acid deletion in the nonstructural protein 3a (i 28 ), which exists in both ts291602 and ts 282902 but not in rev-1 and wt virus. the genotype of the rev-1 at these two positions (q 294 and i 28 ) are therefore the same as that of wt6501, but different from the two ts mutants. as both wt virus and the revertant could form plaques at 32 and 40 jc, these two mutations might be responsible for the ts phenotype. a blast search of genbank clearly showed that the amino acid at position 294 of the s protein was a conserved glutamine. the leu 294 residue in the two ts mutants is located immediately downstream of a hypervariable region and the upstream of a conserved region of the s1 subunit. one additional mutation, d 709 -n 709 , was found in ts291602 only, and an i 769 -m 769 mutation in the two ts mutants and rev-1. after mapping the potential determinants responsible for the ts phenotype, we would like to explore the mechanisms by which these mutations affect the propagation of the ts mutants at the nonpermissive temperature. synthesis of the s protein in cells infected with wt virus, ts mutants, and revertant was analyzed. two sets of experiments were carried out. first, vero cells were infected with wt6501, ts291602, ts282902, and rev-1 at 32 and 40 jc, respectively, and lysates were immunoprecipitated with anti-ibv antibodies. it was obvious that synthesis of the ts mutant s intergenic region between m and 5a 25,346 table 1 titers of wild type, mutant and revertant viruses at 32 jc and 40 jc (pfu/ml) titer 32 jc 4 0 jc wt6501 8.5 â 10 3 5.5 â 10 6 ts291602 2.0 â 10 6 0 ts282902 3.2 â 10 6 0 rev-1 7.0 â 10 6 5.5 â 10 6 protein was detectable at 32 jc but not at 40 jc (fig. 1a , lanes 3, 4, 5, and 6) under these conditions. in contrast, expression of the s protein was observed in cells infected with wt6501 and rev-1 at both temperatures ( fig. 1a , lanes 1, 2, 7, and 8), though the level of the proteins detected in cells infected with wt6501 was much lower at 32 jc. this was consistent with the lower titers of wt virus at 32 jc. in the second set of experiments shown in fig. 1b , cells were absorbed with each virus at 32 jc for 1 h and one of the duplicates was shifted to 40 jc. under these conditions, the two ts mutant s proteins were synthesized at both 32 and 40 jc (fig. 1b, lanes 3 , 4, 5, and 6), like those of the wt virus and revertant (fig. 1b , lanes 1, 2, 7 and 8). these results indicated that the viral structural protein could be synthesized at nonpermissive temperature if the mutants were allowed to enter cells. the viral subgenomic rna and one of the processed proteins, the 3c-like proteinase, were also detected in the same ways (data not shown), and the results were consistent with those described above. comparison of the nucleotide sequences and the s protein synthesis of wt, ts mutants and revertant indicate that the single amino acid mutation (q 294 -l 294 ) in the s protein may be responsible for the temperature sensitivity of the mutant virus. this possibility was studied by biochemical and functional characterization of the s protein from the revertant and a ts mutant. the s gene of rev-1 and ts291602 was cloned under the control of a t7 promoter to investigate whether membranefusion activity was affected by the mutations in the s protein. cells were infected with recombinant vaccinia/t7 virus and were transfected with plasmids containing the s gene from either ts291602 or rev-1. the expression of the s proteins was analyzed by western blotting using anti-ibv antibodies. as shown in fig. 2a , the s protein of both revertant and ts mutant was expressed at 32 and 40 jc (lanes 3, 4, 5, and 6), though the s protein of ts291602 was expressed at a relatively lower level than the rev-1 at 40 jc (compare lanes 4 and 6). the fusion activity of the s protein derived from the revertant and ts291602, respectively, was then examined. as shown in fig. 2b , membrane fusion was observed in cells expressing the s gene of ts291602 at 32 jc (panel c) but not at 40 jc (panel f), at 48 h posttransfection. in contrast, membrane fusion was observed in cells expressing the s gene of revertant at both 32 and 40 jc (panels b and e). interestingly, membrane fusion appeared at 72 h posttransfection, after the cells expressing the s gene of ts291602 were shifted from 40 to 32 jc at 48 h posttransfection (panel i). the recombinant s genes of the ts mutant and revertant were transfected into cells in 35 mm dishes at 32 jc and one of the duplicates was shifted to 40 jc at 4 h posttransfection. radiolabeled proteins were immunoprecipitated and treated with endo-h at 37 jc. as shown in fig. 3a , the ts mutant s protein, synthesized at 40 jc, was sensitive to endo-h digestion, as no 180-kda form was observed (lane 12). in contrast, a portion of the ts mutant s protein, synthesized at 32 jc (lanes 10), was resistant to endo-h digestion. the revertant s protein, synthesized at both 32 and 40 jc, was also resistant to endo-h digestion (lanes 6 and 8). these a b fig. 1 . analysis of the expression of the s protein from wt (wt6501), ts mutants (ts291602 and 282902) and revertant (rev-1) viruses. (a) cells were infected with each virus at 32 jc and 40 jc as indicated on the top for 1 h and were maintained at the same temperatures. (b) two dishes of cells were infected with each virus at 32 jc for 1 h. one of the duplicates was maintained at 32 jc (lanes 1, 3, 5, and 7) and the other one was shifted to 40 jc (lanes 2, 4, 6, and 8). radiolabeled cell lysates were immunoprecipitated with anti-ibv antibodies. the proteins were separated on 12.5% polyacrylamide gels and detected by autoradiography. numbers on the left indicate molecular mass in kilodalton and the position of the s protein is indicated on the right. results indicate that the ts mutant s protein synthesized at 40 jc was not a mature form of the glycoprotein. in addition, a novel glycosylated form of the s protein, migrating between the endo-h treated 130-kda and the matured 180-kda forms, was observed. it might represent trimming of an initial, core-glycosylated form of the s protein in the er and the cis-golgi. interestingly, this band was also observed in ts291602-infected cells (fig. 3b, lane 2) , when the incubation time at 32 jc was extended before the culture was shifted to 40 jc. this band is much stronger than the 180-kda form (lane 2), giving direct evidence that the mutations in the s protein of ts291602 dramatically hampered the maturation process of the s glycoprotein. compared with ts282902, the s protein of ts291602 contains another amino acid substitution, d 709 -n 709 (table 1) , which is not present in the s protein of rev-1 and wt6501. as this band was not observed in ts281602-infected cells (data not shown), it is not clear at this point if this additional mutation may also play a role in the maturation of the s protein. it was also noted that, at 40 jc for all viruses, the s1 and s2 subunits were hardly detected in cell lysates but abundant in the supernatants (fig. 3b) . at the nonpermissive temperature, the s protein was not cleaved at all (lanes 2 and 6). these results may suggest that only the mature form of the s glycoprotein could be cleaved into s1 and s2 and fig. 2 . membrane fusion activity of the s protein expressed from wt and ts291602. (a) vero cells were transfected with plasmids without insert (lanes 1 and 2) or with the s gene from the wt (lanes 3 and 4) and ts291602 (lanes 5 and 6), and were cultured at 32 jc (lanes 1, 3, and 5) or 40 jc (lanes 2, 4, and 6). the expression of the s protein was examined in western blot using anti-ibv antibodies, and h-tubulin was immunostained as loading controls. (b) the membranefusion activity of the s protein from the wt (panels b, e, and h) and ts mutant (panels c, f, and i) at 32 jc (panels b and c) and 40 jc (panels c and f) 2 days post-transfection was compared. panels h and i show induction of membrane fusion on cells transfected with the wt and ts291602 by shifting cells shown in panels e and f to 32 jc for 1 day. panels a, d, and g show cells transfected with empty plasmids. reinforce the conclusion that the majority of the s protein of the ts mutants synthesized at the nonpermissive temperature may represent a pre-mature form of the s glycoprotein. to detect whether the ts mutant s protein would be transported to the plasma membrane at the nonpermissive temperature, the s protein was transiently expressed in vero cells using the vaccinia virus-t7 expression system. the transfected cells were treated with cycloheximide at 3.5 h posttransfection for 30 min to inhibit further protein synthesis. the cells were then incubated at 32 and 40 jc, respectively. the subcellular localization of the s protein was analyzed by indirect immunofluorescent staining at 4, 8, 16, and 24 h postinfection, respectively. the confocal microscopy images of transfected cells are shown in fig. 4 . plasma membrane staining of the ts mutant s protein was not observed at any time points posttransfection at 40 jc, but was clearly seen at 32 jc at 16 and 24 h postinfection (fig. 4) . the revertant s protein was expressed on the cell surface at both temperatures at 16 and 24 h posttransfection. these results may explain the lack of membrane fusion activity of the ts mutant s protein in both infected and transfected cells. endo-h treatment of the s protein from rev-1 and ts291602. the s protein derived from rev-1 (lanes 5, 6, 7, and 8) and ts291602 (lanes 9, 10, 11, and 12) were expressed in vero cells at 32 jc (lanes 5, 6, 9, and 10) and 40 jc (lanes 7, 8, 11, and 12), using a t7-vaccinia expression system. the radiolabeled proteins were immunoprecipitated with anti-ibv antibodies, the eluted proteins were endo-h-(lanes 6, 8, 10, and 12) or mock-treated (lanes 5, 7, 9, and 11) and analyzed by sds-page. (b) detection of the less matured s protein from ts291602 at 40 jc and the defect in cleavage of the mutant s protein. cells were infected with ts291602 and rev-1 for 5 h at 32 jc, one of the duplicates was maintained at 32 jc (lanes 1, 3, 5, and 7), and the other one was shifted to 40 jc (lanes 2, 4, 6, and 8). cells were radiolabeled and viral proteins were immunoprecipitated with anti-ibv antibodies and analyzed by sds-page. lanes 1, 2, 3, and 4 refer to viral products detected from cell lysates and lanes 5, 6, 7, and 8 refer to viral proteins detected from virus particles released to the cultured media. quantitative analysis of surface expression of the wild type and ts mutant s protein was carried out by immunofluorescent staining with anti-s protein antiserum and the positive staining cells were sorted by flow cytometry. as shown in fig. 5 , 5.5% of nonpermeabilizing (panel a) and 0.7% of permeabilizing (panel f) cells expressing the empty plasmid showed background staining. when the cells were incubated at the permissive temperature, 3.8% (9.2 -5.5) of hela cells expressing the wt s protein (panel b) and 4.1% (9.6 -5.5) of cells expressing the ts mutant s protein (panel c) displayed surface staining. after permeabilizing with 0.1% saponin, 11.8% (12.5 -0.7) of cells expressing the wt s protein (panel g) and 10.9% (11.6-0.7) of cells expressing the ts mutant s protein (panel h) showed positive staining. when the cells were incubated at the nonpermissive temperature, 10.4% (15.9 -5.5) of cells expressing the wt s protein (panel d) and 2.3% (7.8 -5.5) of cells expressing the ts mutant s protein (panel e) exhibited surface staining. after permeabilizing with 0.1% saponin, 9.3% (10 -0.7) of cells expressing the wt s protein (panel i) and 9.7% (10.4 -0.7) of cells expressing the ts mutant s protein (panel j) showed positive staining. these results confirm that the ts mutant s protein could not be efficiently translocated to the cell surface at the nonpermissive temperature. to investigate whether the s protein of ts mutant was assembled into virion or not, ts291602 and rev-1-infected cells were radiolabeled and the virus particles were purified through sucrose gradients twice. immunoprecipitation of the purified virions using anti-ibv antibodies showed that the s protein of ts291602 was not detected at 40 jc (fig. 6 , lane 2), but was observed at 32 jc (fig. 6, lane 1) . the s protein was detected at both temperatures for rev-1 (fig. 6, lanes 3 and 4) . these results render support that the spike protein of the ts mutant may not be assembled into virus particles at 40 jc. coronavirus s protein is responsible for receptor binding and membrane fusion. it also induces neutralizing antibodies and bears determinants for virulence. a considerable diversity in s protein among coronaviruses exists, which contributes to host specificity, cell and organ tropisms, and pathogenesis. characterization of mutants with point or deletion mutations in the s protein has helped to establish links between the variations and the functions or altered antigenicity and virulence of viruses. through sequence analysis of wt virus, two ts mutants, a revertant and different passages of a cold-adapted ibv, data presented in this study not only mapped the defects of the ts mutants to the s gene, but also revealed the molecular events occurred during evolution of the ibv s gene. compared with the parental wt virus, mutations that are identical in the two ts mutants and the revertant might be associated with the cold-adaptation phenotype, while those identical only in the two mutants but different from the revertant and wt virus were considered to be linked to the ts phenotype. according to this criterion, the q 294 -to-l 294 mutation in the s protein that exists in the two ts mutants, and interestingly, occurred at a highly conserved domain among ibv viruses immediately downstream a variable domain in the s1 subunit may be responsible for the ts phenotype. the mutants with this mutation accumulated and became dominant under selective pressures, in this case, changes in the hosts and temperatures. the i 769 -m 769 mutation emerged earlier than the q 294 -l 294 mutation, which among variations in other gene products may be associated with cold-adaptation, suggesting potential segregation of mutations responsible for either cold-adaptation or ts phenotypes. further confirmation of the possibility that the q 294 -to-l 294 mutation may cause the ts phenotypic changes is currently being carried out by introducing the mutation to an infectious ibv clones and isolation of a recombinant virus containing this mutation only. the s protein is co-translationally n-glycosylated in the er, oligomerized (luytjes et al., 1997) if folded properly, and associated with the m protein in pre-golgi membrane (opstelten et al., 1995) . one of the essential steps in nlinked glycosylation is transfer of a preformed, 14-coreunit-oligosaccharide to a specific asn residue in the sequence asn-x-ser/thr (where x is any residue except pro, asp, or glu). the oligosaccharide chain is donated by dolichol-pyrophosphate-oligosaccharide to the protein chain in the er. it is trimmed down in the er and cis-golgi, and different external sugars are then added to the trimmed chain in the medial-and trans-golgi. glycoproteins with high mannose oligosaccharides in the er and cis-golgi remain sensitive to endo-h. they become endo-h resistant after being processed by the medial-and trans-golgi resident enzymes to glycoproteins with complex oligosaccharides. the acquisition of endo-h resistance is therefore an indication that viral n-linked glycoproteins are properly processed and transported to the golgi apparatus (luo and weiss, 1998; luo et al., 1999; luytjes et al., 1997) . examination of the amino acid sequence of ibv s protein showed that there are 13 and 9 potential glycosylation sites in the s1 and s2 subunits, respectively. the calculated molecular weight of the s protein is 128 kda. if all 21 sites are used, the initially transferred oligosaccharides account for 50 kda. in cells overexpressing s protein, core-glycosylation of the s protein in the er resulted in a 180-kda product as expected. trimming of the s glycoprotein in the er and cis-golgi led to a partially processed 155-kda form, larger than the endo-h-treated 130-kda form which only has one sugar residue left at each site. both the trimmed 155-and core-glycosylated 180-kda forms were sensitive to endo-h digestion. at the permissive temperature, further modification of the mutant s protein in the medial-and trans-golgi by addition of sugars to the trimmed chains resulted in the maturation of s, which co-migrates with the core-glycosylated 180-kda form but is resistant to endo-h digestion. at the nonpermissive temperature, however, no mature form of the fig. 6 . analysis of the structural proteins on purified ts291602 and rev-1 virions. immunoprecipitations were performed using virions, purified from ts291602-(lanes 1 and 2) and rev-1-infected (lanes 3 and 4) vero cells at 32 jc (lanes 1 and 3) and 40 jc (lanes 2 and 4) . the viral proteins were separated on 12.5% polyacrylamide gels and detected by autoradiography. numbers on the left indicate molecular masses in kilodalton and the positions of the three structural proteins are indicated on the right. mutant s protein was produced. in virus-infected cells, the endo-h sensitive, 155-kda form of ts291602 was abundant at the nonpermissive temperature, clearly indicating that the mutant s protein was trimmed in the er and cis-golgi but was not transported to the trans-golgi. furthermore, the mutant s protein was not cleaved into s1 and s2 at the nonpermissive temperature, which usually occurs in the trans-golgi shortly before the release of virions from cells (frana et al., 1985) . as only spikeless virions were produced, these results demonstrated that the core-glycosylated or the trimmed forms of s were unable to incorporate into virions. in addition, the mutant s protein fails to induce cell -cell fusion at the nonpermissive temperature. as the q 294 -to-l 294 mutation is not in the fusogenic domain of s, it renders support to the conclusion that the mutation causes the defect in its modification and intracellular transport, instead of having direct impact on the fusion process. taken together, these studies clearly indicate that the mutant s protein could not reach the site where the s protein is incorporated into virions. the reasons for the retention of the mutant s protein in the er and cis-golgi are yet to be explored. previous studies show that the retention of monomeric s protein in the er is due to misfolding induced by disruption of disulfide bonds (opstelten et al., 1993) , and also caused by lack of oligomerization of endo-h-sensitive s protein of ts mutants though the locations were not determined (luytjes et al., 1997) . it is not clear whether mutations in ibv s protein have an effect on its folding or oligomerization. nevertheless, in both circumstances, the intracellular transport, modification of the s protein and its incorporation into virion would have been affected. studies with some of the other enveloped viruses demonstrated that molecular chaperones (like calnexin and calreticulin) in the er transiently and specifically interact with partially trimmed, monoglycosylated form of viral n-linked glycoproteins (tatu and helenius, 1997) . for instance, calnexin interacts with the influenza hemagglutinin and vesicular stomatitis virus g protein (hebert et al., 1996; hammond and helenius, 1994) . these and other proteins could facilitate proper folding and ensure quality control for viral glycoproteins. proteins that fail to fold and oligomerize are prevented from transport and ultimately degraded. with the ts mutants, it is possible to explore the interaction of the s protein with molecular chaperones and folding enzymes. this approach would provide new insights into the maturation and assembly of the coronavirus s protein. the ts mutant quickly lost its infectivity at the nonpermissive temperature. this low thermostability indicated that even if the mutant s protein was synthesized, fully modified and assembled into virions at the permissive temperature, the resulting particles were relatively unstable and lost infectivity at the nonpermissive temperature. it suggests that the q 294 residue in the conserved s1 domain of ibv may also play an essential role in maintaining the thermal stability of the spike in the virion, resembling the s 287 residue in the s1 region of an mhv ts mutant (ricard et al., 1995) . in addition, if cells were incubated with the ts mutants at the nonpermissive temperature, viral proteins were hardly detected, indicating that at the nonpermissive temperature, the ts mutant s protein may be unable to either bind efficiently to the receptor or induce virus -cell membrane fusion in contrast to the s protein of the revertant and wt virus. this observation suggests that the mutation in the s protein might induce conformation changes of the spikes on virions at the nonpermissive temperature and hamper either process. vero cells were maintained in complete dmem medium (gibco brl), supplemented with newborn calf serum (10%), streptomycin (1000 ag) and penicillin (1000 units/ml). the beaudette stain of ibv was purchased from atcc and propagated in chicken embryonated eggs for three passages. the virus was then adapted to grow and passage on vero cells for 65 times at 37 jc. in this study, the viruses were further passaged on vero cells at progressively lower temperatures for 121 passages (at 35, 34, 33, 32, 30, 29, and 28 jc for 4, 5, 7, 44, 12, 21, and 29 passages) . virus stocks were used for plaque assays, plaque-to-plaque purification and viral rna extraction as described previously (shen et al., 1994) . the wild-type virus, wt6501, was plaque-purified from passage 65 grown at 37 jc, while ts mutants, ts291602 and ts282902, were purified from passage 16 grown at 29 jc and passage 29 grown at 28 jc, respectively. the revertant, rev-1, was plaque-purified from cells infected with ts 291602 for 12 h at 37 jc and then shifted to 40 jc for 2 days. recombinant vaccinia/t7 (v/t3) virus was propagated and was tittered on vero cells. virus stocks were kept at à80 jc until use. confluent monolayers of vero cells were infected with viruses at a multiplicity of infection (moi) of 0.1. after 2h absorption, the viruses were radiolabeled by replacing medium with methionine-free dmem supplemented with 30 aci/ml [ 35 s]-methionine. after 16-h incubation at temperatures appropriate for each virus, cells were harvested and virus stock was prepared by freezing and thawing three times. cell debris was removed by centrifugation at 5000 rpm for 15 min (beckmen, 25.50). the supernatant was centrifuged through a 20% sucrose cushion and the resulting pellet was resuspended in tne buffer (50 mm tris -hcl, ph 7.4, 100 mm nacl, 1 mm edta). the viruses were further purified by centrifugation through a 20-55% su-crose gradient in tne buffer at 45,000 rpm (beckmen, sw50) twice. fractions containing the virus were pooled together and used for protein analysis. viral structural proteins were labeled with [ 35 s]-methionine, immunoprecipitated and separated on 12.5% polyacrylamide gels as described previously (liu et al., 1998) . the anti-ibv serum was raised in rabbit against purified ibv virions and was used in immunoprecipitation and western blot as previously described (liu and inglis, 1991) . a portion of cell lysates was immunoprecipitated with anti-ibv serum. the pellets were washed three times with standard ripa buffer, dissolved in 20 al of digestion buffer (50 mm tris, ph 6.8, 0.25% sds) and incubated at 95 jc for 5 min. ten microliters of the supernatants were mixed with 10 al of digestion buffer with or without endo-h (0.2 mu, boehringer mannheim) and incubated for 4 h at 37 jc. confluent monolayers of vero cells were infected with recombinant vaccinia/t7 viruses at a moi of 0.1. after 1h absorption, cells (2 â 10 7 ) were trypsinized, centrifuged at 250 g and resuspended in 2 ml of pbs. the cells (0.3 ml) were mixed with 5 ag plasmid and electroporated (easyject, equibio) at 600 v in a 0.4-cm cuvette (biorad). the cells were then plated in a 35-mm dish containing 3 ml of dmem with 2% of newborn calf serum and incubated at temperatures indicated. viral rna was extracted from the purified viruses using the rneasy mini kit (qiagen) according to the manufacturer's instructions. reverse transcription and polymerase chain reaction (rt-pcr) were performed using the expand reverse transcription and high fidelity pcr kits (boehringer mannheim). annealing and extension times of pcr were optimized for amplification of pcr products with different sizes using different primers. more than 100 specific primers were used for amplification, sequencing and cloning. automated sequencing was carried out using pcr products or cdna clones and specific primers as previously described (shen et al., 1999) . sequence analysis was carried out using the gcg and blast suite of programs as described previously (shen and liu, 2000) . hela cells were infected with vaccinia/t7 virus, transfected with constructs encoding wt and ts mutant s protein using qiagen effectene transfection reagent, and incubated at the permissive and nonpermissive temperatures, respectively, for 18 h. cells were harvested, washed once with pbs, resuspended in blocking buffer (20% fbs and 1% bsa in pbs), incubated on ice for 30 min. a half of the cells were permeabilized with 0.1% saponin in facs washing buffer (2.5% fbs and 0.05% sodium azide in pbs) incubated for 10 min at room temperature, and stained with 1:100 diluted primary antiserum, the rabbit anti-ibv s protein. the other half was stained directly with the same primary antibody. cells were then washed two times with the facs washing buffer, stained with 1:20 diluted fitc conjugated swine anti-rabbit antibody (dako). after washing two times with the facs washing buffer, cells were then fixed with 1% ice cold paraformaldehyde and analyzed by flow cytometry. two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism coronavirus-induced membrane fusion requires the cysteine-rich domain in the spike protein evidence for a coiled-coil structure in the spike of coronaviruses mapping of the coronavirus membrane protein domains involved in interaction with the spike protein identification of a novel coronavirus in patients with severe acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus proteolytic cleavage of the e2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion alteration of the ph dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein cell receptor-independent infection by a neurotropic murine coronavirus major receptorbinding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein folding of vsv g protein: sequential interaction with bip and calnexin calnexin and calreticulin promote folding, delay oligomerization and suppress degradation of influenza hemagglutinin in microsomes tunicamycin resistant glycosylation of coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step variations in disparate regions of the murine coronavirus spike protein impact the initiation of membrane fusion localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier the molecular biology of coronaviruses altered pathogenesis of a mutant of the murine coronavirus mhv-a59 is associated with a q159l amino acid substitution in the spike protein association of the infectious bronchitis virus 3c protein with the virion envelope proteolytic mapping of the coronavirus infectious virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3c-like proteinase and identification of two novel cleavage products roles in cell-to-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein amino acid substitutions within the leucine zipper domain of the murine coronavirus spike protein cause defects in oligomerization and the ability to induce cell-tocell fusion characterization of two temperature-sensitive mutants of coronavirus mouse hepatitis virus strain a59 with maturation defects in the spike protein spike glycoprotein-mediated fusion in biliary glycoprotein-independent cell-associated spread of mouse hepatitis virus infection murine coronavirus spike protein determines the ability of the virus to replicate in the liver and cause hepatitis disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins envelope glycoprotein interactions in coronavirus assembly a conditional-lethal murine coronavirus mutant that fails to incorporate the spike glycoprotein into assembled virions evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted rna secondary structure identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the nsp2 gene with an unique insertion emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication rearrangement of the vp6 gene of a group a rotavirus in combination with a point mutation affecting trimer stability sequence analysis and in vitro expression of genes 6 and 11 of an ovine group b rotavirus isolate, kb63: evidence for a non-defective, c-terminally truncated nsp1 and a phosphorylated nsp5 proteolytic cleavage of the e2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90k cleavage fragments analysis of the receptor-binding site of murine coronavirus spike protein interactions between newly synthesized glycoproteins, calnexin and a network of resident chaperones in the endoplasmic reticulum replication of coronavirus mhv-a59 in sac-cells: determination of the first site of budding of progeny virions a 12-amino acid stretch in the hypervariable region of the spike protein s1 subunit is critical for cell fusion activity of mouse hepatitis virus nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes this work was supported by the biomedical research council, agency for science technology and research, singapore. key: cord-355377-b0rcg3rt authors: van der vlag, r.; hirsch, a.k.h. title: analytical methods in protein-templated dynamic combinatorial chemistry date: 2017-06-29 journal: comprehensive supramolecular chemistry ii doi: 10.1016/b978-0-12-409547-2.12559-4 sha: doc_id: 355377 cord_uid: b0rcg3rt protein-templated dynamic combinatorial chemistry (dcc) has emerged as a powerful method to identify new inhibitors given that it enables the protein to select its own best binder(s) from a library of interconverting compounds. since the first report of dcc applied to the discovery of binders for a protein, this elegant tool has been employed on a range of protein targets in early stage of medicinal-chemistry projects. a toolbox with various reversible and biocompatible reactions has become available, and the portfolio of analytical techniques is growing. despite progress, in most cases, the libraries employed remain of moderate size due to difficulties associated with their analysis. in this article, analytical methods used in protein-templated dcc are described using examples of recent developments and reports. in dynamic combinatorial chemistry (dcc), molecular building blocks are reversibly formed via covalent or noncovalent bonds. with only a few building blocks, a relatively large number of reversible products can be generated, forming a dynamic combinatorial library (dcl). given that the reaction between the building blocks is reversible, the members of the dcl continuously exchange to eventually reach a thermodynamic equilibrium. when an external stimulus, such as a protein target, is introduced, the dynamic system re-equilibrates upon selection and non-covalent binding of the library members with the strongest affinity for the target. the specific affinity of each library member is determined by the number, type, and strength of interactions (favorable and nonfavorable) it establishes with the protein target. examples of these intermolecular interactions are van der waals forces, p-pstacking, cation-p, dipole-dipole, hydrogen-bonding, and ion-dipole interactions, as well as ion pairing. ultimately, the best binders of the library are amplified at the expense of library members displaying a lower affinity for the target (fig. 1) . identification of the best binders circumvents the need for synthesis, purification, and characterization of every individual library member. however, up to date, it remains very difficult to analyze dcls of large size, preventing dcc from becoming a more widely used tool in drug development. 1, 2 in this article, analytical methods used in protein-templated dcc will be described. apart from the general theory, examples of recent developments and reports will be briefly discussed. 2 figure 1 schematic representation of protein-templated dynamic combinatorial chemistry. analytical techniques for the analysis of dcls can be divided into chromatographic and spectroscopic techniques. often, techniques are combined to obtain the benefits from both, for example, separating a complex mixture with lc and characterizing each member of the library using mass spectrometry (ms). prior to analysis, the sample has to be prepared. after "freezing" the equilibrium, the enzyme can be removed from the sample. denaturation of the protein is achieved by heating or adding an organic solvent such as acetonitrile or methanol. the denatured protein is subsequently removed by filtration or centrifugation. in the case of ms, it is not always necessary to remove the protein, and a direct analysis of protein-ligand complexes is possible. an overview of the steps in protein-templated dcc is given in fig. 2 . nowadays, access to chromatographic techniques such as high-pressure lc (hplc) is standard in the modern chemistry laboratory. owing to the large variety of columns available and a built-in diode array detector, mixtures of compounds can be separated and characterized. subsequently, the retention time and uv profile of the hplc chromatogram are analyzed and compared with experiments performed under different conditions. in the case of a dcl, comparison of the library in presence and absence of the target protein can lead to the identification of amplified and/or depleted species. in 1997, dcc was first applied to a protein target by the group of lehn using imine formation/exchange. 5 a dcl of three aromatic aldehydes 1a-c and four different amines 2a-d in ten-fold excess was synthesized with and without carbonic anhydrase (ca) ii as a model enzyme (0.4 mm) (scheme 2). in order to reduce the imines 1-2 to amines 3, nabh 3 cn (1.2 mm) was added to the reaction mixture, and the mixture was incubated at 25 c for 14 days. the reaction without enzyme was completed within 24 h. before analysis of the dcl, thermal denaturation of the enzyme (2 min. at 80 c) ensured release from the active site of possibly tightly bound ligands. using a cellulose membrane, the reaction products were separated from ca by microcentrifuge filtration. analysis of this first example of protein-templated dcc was performed by comparison of hplc traces of the dcl in the presence and absence of ca. it was shown that the presence of the enzyme favored the formation of those condensation products that were expected to display the strongest affinity for the enzyme's active site. the strongest binders were identified by comparison with retention times of separately synthesized reference compounds. scheme 2 formation of dynamic combinatorial library of aldehydes 1a-c and amines 2a-d in the presence of carbonic anhydrase (ca) results in imines 1-2, which are subsequently irreversibly reduced to amines 3. 5 a more recent example of analysis by lc was reported by greaney and coworkers, 6 who had demonstrated in 2010 the use of aniline as a nucleophilic catalyst for the reversible formation of acylhydrazones. 3 prior to this report, acylhydrazone chemistry required acidic ph, which is not compatible with most protein targets. besides a pre-equilibrated system, in which the dcl is formed first, followed by a "freezing" step and addition of the protein, acylhydrazone chemistry was considered to be of limited use for protein-templated dcc. with the introduction of aniline as a nucleophilic catalyst, acylhydrazones can be formed reversibly at ph 6.2 and reach a steady-state composition in 6 h instead of 7 days without aniline. by controlling the ph, the equilibrium of the dcls can be easily switched on and off. in the proof-of-principle study, two isozymes of glutathione s-transferase (gst) were used, which play an important role in cell detoxification 7 and are emerging targets for the treatment of drug resistance in chemotherapy (human (h) gst p1-1) 8, 9 and tropical diseases (schistosoma japonicum (sj) gst). 10 dcls were generated with aldehyde 4, related to the known gst substrate chloro-2,4-dinitrobenzene (cdnb), and a 2.5-fold excess of each of the ten hydrazides 5a-j in the presence of 10 mm aniline at ph 6.2 (scheme 3). the hydrazides were added in large excess with respect to the aldehyde to ensure pseudofirst-order behavior and to achieve faster equilibration rates. the dcls containing 4, 5a-j, and one of the isozymes were allowed to stand at room temperature, with occasional shaking, for 12 h. then, the ph was adjusted to 8.0 to "freeze" the dcl, and the protein was removed by ultrafiltration using a molecular weight cutoff filter of 10,000 da. subsequently, the library was analyzed by hplc and compared with the dcl in the absence of protein. both dcls showed a very clear amplification of acylhydrazones: t-butylphenyl and thiophenyl acylhydrazones 4-5c and 4-5g were amplified in presence of hgst p1-1 and sjgst, scheme 3 generation of acylhydrazone-based dynamic combinatorial libraries of (a) aldehydes 4 or 6 and hydrazides 5a-j. (b) structure of glutathione (gsh) and gsh-conjugated aldehyde 6. (c) acylhydrazones that were amplified in the presence of gst isozymes schistosoma japonicum (sj) gst or human (h) gst p1-1. 3 respectively. accurate ic 50 values of the resynthesized acylhydrazones could not be determined due to poor solubility. to solve this problem and at the same time increase the potency of the acylhydrazones, glutathione (gsh)-conjugated aldehyde 6 was synthesized and employed in a dcl with hydrazides 5a-j (scheme 3). the highly soluble gsh tripeptide motif was expected to function as an "anchor" at the gsh-binding site, enabling exploration of the adjacent hydrophobic site with a range of hydrazide fragments. again, the same hydrazide fragment was selected by the enzymes as the best binder: t-butylphenyl and thiophenyl acylhydrazones 6-5c and 6-5g for hgst p1-1 and sjgst, respectively. acylhydrazone 6-5j was also slightly amplified in the dcl with hgst p1-1. the most significant reductions in equilibrium concentration occurred for 6-5b, f, and i (sjgst) and 6-5f, g, and i (hgst p1-1). to confirm that the observed amplification was not caused by target-accelerated synthesis, sjgst was added to a preformed dcl. the same equilibrium distribution was obtained, indicating that the amplification is a result of genuine thermodynamic selection. to fully explore the isozyme-specific amplification of the two dcls, the ic 50 values of the separately synthesized acylhydrazone conjugates 6-5a-j were determined. the most amplified acylhydrazones were found to be the most potent in the biochemical assay. thiophenyl acylhydrazone 6-5g displayed the lowest ic 50 value against sjgst (22 mm), and t-butylphenyl acylhydrazone 6-5c had the lowest ic50 value among the four conjugates (6-5c, g, h, and i) against hgst p1-1 (57 mm). based on this initial study, greaney and coworkers developed stronger, bivalent acylhydrazone inhibitors of the gst isozymes. 6 this bivalent approach represents a novel concept in protein-templated dcc and provides access to much more (diverse) compounds than in a one-dimensional dcl using the same number of building blocks. the majority of gsts exist as homodimers, containing a conserved gsh-binding site and a hydrophobic substrate-binding site at the dimer interface. inhibitors featuring a long "arm" at either end of the central scaffold could be able to span the dimer interface and address both gsh-binding sites. an acylhydrazone-based dcl was generated comprising three nitro-substituted benzaldehydes 4, 6, and 7, which are derivatives of known binder cdnb, and four bivalent hydrazide linkers 8a-d in the presence of aniline as a nucleophilic catalyst (scheme 4a), resulting in 24 homo-and hetero-bis-acylhydrazones (plus additional mono-acylhydrazones), excluding e/z isomers. in order to have the opportunity to discriminate between the h-sites of different gst homodimers, the linkers were designed to have differing lengths and lipophilicity. four gst isozymes (murine (m) gstm1-1, hgstp 1-1, sjgst, and mgsta4-4) were used as template for the reversible formation of acylhydrazone binders. after incubation at room temperature for 48 h, hplc analysis of the adaptive dcls using two hplc columns in series was performed and compared with dcls generated in the absence of protein. each peak was identified by deconvolution in combination with lc-ms analysis. the highest amplifications were obtained for the m and sj isozymes, with an impressive amplification of over 600% by mgstm1-1 for hydrazone 6-8c-6 (scheme 4b). scheme 4 (a) aniline-catalyzed generation of acylhydrazone-based dynamic combinatorial libraries of aldehydes 4, 6, 7 and bis-hydrazides 8a-d. (b) amplified gsh-conjugated homo-bis-acylhydrazones in the presence of mgstm1-1. 6 the dcl containing mgstm1-1 showed strong amplification of acylhydrazones formed from gsh-conjugated aldehyde 6. the presence of mgsta4-4 did not lead to significant changes in the hplc traces and was later used as negative control to study specific binding-site interactions within the mgstm1-1 isoform. in contrast, hgstp1-1 appeared to interfere with the dcl equilibrium for certain compounds. for example, bis-acylhydrazones containing two units of 6 were degraded to mono-acylhydrazones by the enzyme. therefore, hgstp1-1 was not used further in the templating studies. a range of symmetrical bis-acylhydrazones, all containing gsh-conjugated 6, was selected and separately synthesized in order to assess the corresponding biochemical activities (scheme 4b and table 1 ). for mgstm1-1, the ic 50 data correlate well with the amplifications observed in the dcl: inhibitor 6-8c-6 (ic 50 ¼ 50 nm), which showed the greatest amplification, is nearly ten-fold more potent than the other bis-acylhydrazone products. the inhibition data for sjgst correlate less well with the amplification observed, presumably due to competition among the amplified library members for shared building block 8b, which reduces the extent of amplification. due to a lack of clear equilibration, the amplification factors observed could not be correlated with the ic 50 values for hgstp1-1. to elucidate the role of linker 8c, an overlay model was constructed based on the crystal structure of hgst m1a-1a, containing ligand s-2,4-dinitrobenzene. the model shows that the linker 8c can span the dimer interface of hgst m1a-1a, without introducing any unfavorable interactions or distorting the dimer structure, demonstrating that dcc with bivalent linkers is particularly suited for protein targets that are challenging to address by structure-based design (sbd). at the moment, lc techniques such as hplc are the most widely used techniques. lc is often combined with ms, in which case peaks can immediately be assigned to the corresponding library member. otherwise, first, the retention times and uv profiles have to be confirmed using separate building blocks and (side) products. new potential inhibitors that do not contain a strong absorbing chromophore can be overlooked when only uv detection is used. evaporative light scattering detectors (elsd) might solve this problem. however, the use of elsd requires different equipment and is therefore less commonly used. one of the major bottlenecks associated with hplc and lc-ms is the limited library size. although the analytical method possesses adequate sensitivity to detect subtle changes in concentration of compounds of interest, a mixture of many compounds can often not be fully resolved. this limits the size of the dcl to tens of compounds rather than thousands of possible ligands. in the future, it is expected that methods using ultra performance lc (uplc) will increase the potential library sizes, but even then, there will be a size limit. an additional downside of hplc analysis is the requirement for relatively large amounts of protein. in 2013, the group of guo reported a novel protocol based on size-exclusion chromatography (sec) and ms for direct isolation and identification of ligand-target adducts from dcls. 11 for the proof-of-concept study, hen egg white lysozyme (hewl) was chosen as a protein target, which is an important contributor to immune defense by attacking and degrading bacterial cell walls through hydrolysis. imine formation was selected as the reversible reaction, and a dcl of four amines 9a-d and ten aldehydes 10a-j was designed (scheme 5). the adaptive dcl was reduced to the corresponding amines using nacnbh 3 . after incubation with hewl, the library mixture was passed through an sec column to retain all the nonbinders in the library. the potential binders were released by denaturation of the eluted protein-ligand complexes using acetonitrile, and the eluent was analyzed by electrospray ionization (esi)-ms. three members of the dcl were found to bind hewl. in control reactions in the absence of protein no compounds were detected by ms, indicating that the sec column could retain all the unbound library members and that amines 9a-10b, 9a-10d, and 9a-10f are indeed binders of hewl. in case the library was not reduced to the corresponding amines, no imines were detected after sec, suggesting that amines rather than imine intermediates are the ligands of hewl. the amines were synthesized, and their mode of inhibition and relative potencies were determined by evaluating their effect on the lysis rate of micrococcus lysodeikticus. 12 the amines were found to adopt a competitive mode of inhibition, and the binding affinities follow the order 9a-10f (k m ¼ 0.203 ae 0.028 mg ml à 1 ) > 9a-10b (k m ¼ 0.163 ae 0.022 mg ml à 1 ) z 9a-10d (k m ¼ 0.162 ae 0.063 mg ml à 1 ). in 2015, building on their first report, guo and coworkers developed a sec-ms protocol for the identification of competitive inhibitors for bovine serum albumin (bsa). 4 serum albumins play an important role in the transport and delivery of fatty acids, metals, drugs, and other bioactive small molecules and are the most abundant soluble protein constituents of the circulatory system. two adaptive dcls (dcl1 and dcl2, scheme 6) were prepared using different catalysts consisting of 20 carboxylic acids (408 mm each) and 12 alcohols (500 mm) and 28 carboxylic acids (480 mm each) and 22 alcohols (550 mm). sulfuric acid and an immobilized lipase candida sp 99-125 were added as a catalyst to dcl1 and dcl2, respectively. after equilibration for 3 days, aliquots of the solutions were applied to a suitable sec column. after denaturing the protein by the addition of meoh and centrifugation, the supernatant was analyzed by esi-hrms. interestingly, the library was not "frozen" before denaturation. from both dcls, ethyl palmitate was identified as a new binder of bsa. the binding and affinity of the protein for ethyl palmitate were further examined via fluorescence spectroscopy. the thermodynamic parameters and association constants k a were determined using the van't hoff and stern-volmer equations, respectively. based on the thermodynamic results, the binding process is spontaneous (dg < 0), and ethyl palmitate is considered to be bound to bsa mainly through hydrophobic interactions. sec-ms protocols circumvent the need for resolution of all library members by lc, which is one of the major bottlenecks, preventing the use of significantly larger library sizes. a drawback of sec, however, is the fact that the concentration of the building blocks for dcl generation is limited to the micromolar or even nanomolar range. this can result in long equilibration times, which can cause problems with protein stability. another disadvantage are the large amounts of protein used (e.g., 30 mg hewl 11 or 6-10 mg bsa 4 per dcl) to ensure a sufficiently high concentration of binding inhibitors for proper detection by ms. with enzymes that are difficult to express or unstable over time, the sec-ms protocol is therefore not suitable yet. this problem could possibly be addressed with selected-ion monitoring ms, in which case selected m/z values are detected, but this will limit the library size or increase the analytical time significantly. nuclear magnetic resonance (nmr) spectroscopy is a commonly used tool in organic chemistry laboratories. since it is nondestructive, it can be performed directly in the reaction mixture and is extremely sensitive, making it an invaluable tool in chemistry. several nmr-based methods have been developed and used in protein-templated dcc, including saturation-transfer difference (std)-nmr, 11 b-nmr, water-ligand observed via gradient spectroscopy (waterlogsy), and competition-based 1 h-nmr spectroscopy. in std-nmr spectroscopy, the difference between a "normal" 1 h-nmr spectrum and an 1 h-nmr spectrum, in which the protein is selectively saturated, is studied (fig. 3) . 14 first, a reference spectrum is recorded with the irradiation frequency set at a value far away from any ligand or protein signal. this spectrum is also known as the off-resonance spectrum. in a second experiment, a spectrum is recorded while selectively irradiating the protein, without influencing the ligand(s). usually, this saturation step is performed between scheme 6 dynamic combinatorial libraries 1 (blue box) and 2 (red box) and the sec-ms protocol for analysis (green box). 4 scheme 5 generation of a dynamic combinatorial library of amines 9a-d and aldehydes 10a-j in the presence of hen egg white lysozyme (hewl), followed by imine reduction with nacnbh 3 and separation of protein-binder complexes by size-exclusion chromatography (sec). 11 0.0 and à 1.0 ppm, a region characteristic of aliphatic residues of the protein. the selective saturation is now transferred from the aliphatic side chains to the whole protein via spin diffusion through cross relaxation (intramolecular nuclear overhauser effect). fast exchange takes place between the free and bound ligands, which allows further transfer of magnetization from the protein receptor to the ligand protons, which are involved in binding to the protein's surface. the saturation that is transferred to the ligands is detected as a change in ligand signals in the saturated std-nmr spectrum. upon subtraction of the on-resonance spectrum from the off-resonance spectrum, only signals are observed from ligands that are in close contact with the protein. in 2014, we used de novo sbd in combination with acylhydrazone-based dcc and 1 h-std-nmr spectroscopy to identify new inhibitors of the aspartic protease endothiapepsin. 15 the family of aspartic proteases is involved in a wide range of diseases such as malaria (plasmepsins), alzheimer's disease (b-secretase), and hypertension (renin). 16 based on the interactions observed in two crystal structures of endothiapepsin (with and without a water molecule in the active site), a series of acylhydrazones were designed. retrosynthetic analysis of the acylhydrazones resulted in five hydrazides 11a-e and five aldehydes 12a-e (scheme 7). to facilitate analysis by 1 h-std-nmr spectroscopy, the full library was divided into five sublibraries, each consisting of one aldehyde and five hydrazides. after the addition of endothiapepsin to the preformed sublibraries, the acylhydrazones were identified by analyzing the imine-type (12a, 12b, 12d, and 12e) or a-carbon (12c) proton signals in the 1 h-std-nmr spectra. from the five sublibraries, featuring a total of 25 potential acylhydrazones 13 (50 isomers including e/z isomers), eight binders were identified (scheme 7, 13a-h). in order to confirm the binding mode, the competitive, potent inhibitor saquinavir (inhibition constant (k i ) ¼ 48 nm) was added to the 11a-e þ 12d sublibrary. the appearance of saquinavir signals and decrease of acylhydrazone signals in the 1 h-std-nmr spectrum confirmed specific binding of the acylhydrazones 13e and 13g. subsequently, the biochemical activity of the eight binders was determined using a fluorescence-based assay. seven of the eight acylhydrazones inhibit endothiapepsin with ic 50 values between 13 and 365 mm (scheme 7), confirming the outcome of the 1 h-std-nmr experiments (one acylhydrazone was insoluble under the biochemical assay conditions). the most potent inhibitors 13g and 13f display ic 50 values of 12.8 and 14.5 mm, respectively. in order to determine the predicted binding mode, endothiapepsin crystals were soaked with inhibitors 13g and 13f. cocrystal structure determination confirmed the in silico prediction that either direct or water-mediated interactions with the catalytic dyad can be achieved. 1 h-std-nmr spectroscopy has the benefits that it is nondestructive and can be directly performed using the sample. furthermore, since the std method indicates which hydrogen atoms of the small-molecule inhibitors are in close proximity of the protein, it provides some information on the binding mode. in addition, competition experiments with known binders can elucidate if the ligands bind in the same pocket as the known ligand (assuming that the binding mode of the latter is known). std-nmr spectroscopy only works if the ligand is used in excess, therefore requiring significantly less protein than lc-ms. it should, however, be noted that this technique does not discriminate between specific and nonspecific binders. 17 calculation of dissociation constants (k d ) is therefore highly overestimated, and additional experiments such as ic 50 determination are required. high-affinity binders that undergo slow chemical exchange on the nmr timescale are not observed. 14 1 h-stdnmr has only been applied twice in the analysis of a dcl, first in 2010 by the group of ramström in the analysis of b-galactosidase inhibitors based on hemithioacetals 18 and in 2014 by our group, 15 as discussed earlier. besides 1 h-std-nmr spectroscopy, more nmr methods have been developed for the analysis of protein-templated dcls. claridge and schofield reported the use of two nmr methods to analyze dcls with proteins as template for the first time, namely, 11 b-nmr spectroscopy and 1 h-waterlogsy. 19 using these nmr techniques, ternary complexes of enzyme, boronic acids, and sugars in the dcls were monitored to identify binders of a-chymotrypsin. the 11 b nucleus is quadrupolar (i ¼ 3/2), which can be exploited to determine the hybridization state of the boron atom: an 11 b nucleus in an sp 2 -hybridized trigonal planar environment exhibits relatively broad peaks compared with an 11 b nucleus in a highly symmetrical sp 3 -hybridized tetrahedral environment, which gives much sharper peaks. 20 although 11 b-nmr spectroscopy enables the direct observation of the boronate-enzyme complexes and is free from protein background signals, it is far from optimal for the analysis of dcls. the 11 b-nmr signals are broad, causing boronic acids with similar structures to overlap. additionally, 11 b-nmr spectroscopy suffers from low intrinsic sensitivity, which necessitates the use of high concentrations of boronic acids and protein to compensate for the loss in signal. 1 h-waterlogsy 21 features better sensitivity than 11 b-nmr spectroscopy. first, magnetization of the bulk h 2 o in the sample takes place, and after a variety of different transfer mechanisms, small molecules that interact with the target protein are detected. the protein concentration can be very low, but 1 h-waterlogsy 21 can be limited by the exchange kinetics of the reversibly formed protein-ligand complexes and might still suffer from overlapping signals, especially for large dcls. in 2013, the same group introduced a competition-based 1 h-nmr method to screen binders for human 2-oxoglutarate (2og)dependent oxygenases. 22 hypoxia inducible factor (hif) hydroxylases, specifically prolyl hydroxylase domain containing enzyme isoform 2 (phd2) and factor-inhibiting hif (fih), were used as model systems for this study. the natural cosubstrate 2og was used as a reporter ligand, and in order to block enzyme-catalyzed 2og turnover and to avoid oxidation of diamagnetic fe(ii), the native fe(ii) was substituted by paramagnetic and catalytically inactive zn(ii). when a competitive binder displaces bound 2og from the binding site into the bulk solution, the unbound 2og is observed via 1 h-nmr spectroscopy. by monitoring the intensity of the reporter signal as a function of inhibitor concentration, it is also possible to obtain dissociation constants (k d ). as a proof-of-principle study, the boronic acid scaffold and some diol hits from a previous study 23 using dynamic combinatorial ms (dcms; see section "dynamic combinatorial ms") were subjected to the competitive nmr analysis. the results obtained are in agreement with the nondenaturing dcms results. the competition-based 1 h-nmr technique only uses substoichiometric amounts of unlabeled protein and has additional advantages over other ligand-based nmr techniques: site-specific binding information and k d values for ligands with both high and low affinity can be obtained. as already briefly introduced in section "liquid chromatography," ms is a very useful tool for dcc, since compounds can immediately be assigned by their mass. prior to analysis, not only complex mixtures can be separated using lc but also direct analysis of dcls has been applied. the two main ms techniques, which are applied to protein-templated dcc, are discussed in this section "dynamic combinatorial ms" and "competitive ms". 24 in the dcl, one of the library members is known to bind strongly to the protein and serves in this way as an "anchor." next, the reversible reaction takes place with this "support ligand" to obtain more favorable interactions with the binding site of the enzyme. subsequently, nondenaturing esi-ms and ms-ms are used for the direct analysis of protein-ligand complexes. a recent example of dcms was reported in 2012 by the group of schofield enabling the identification of new inhibitors based on acids/boronate esters for phd2 (see section "other nmr-based methods"). 23 phd2 is an fe(ii) and 2og oxygenase that regulates human hypoxic response. involvement in treatment of anemia and ischemia-related diseases makes this human hydroxylase an important drug target. 25 first, based on modeling studies, "support ligands" 14 and 15 were designed to participate in both fe(ii) chelation in the active site and boronate ester exchange (scheme 8a). it was predicted that "support ligand" 14 fits well in a side pocket of the active site and 15 would function as a control, since it should not bind at the active site due to steric clash. four sublibraries of ten diols each were prepared based on molecular weight, and phd2-fe(ii) and "support ligands" 14 or 15 were added to each dcl (ph 7.5). subsequently, the eight dcls (four containing 14 and four containing 15) were analyzed with esi-ms in order to determine which boronate esters bind preferentially to phd2-fe(ii). of the 40 possible boronates with "support ligand" 14, seven boronate esters derived from seven diols 16a-g were found to bind (scheme 8b). esi-ms analysis of mixtures of 14 with the individual diols validated the dcms results. in the absence of 14, some of the individual diols (e.g., 16e and 16g) were also observed to bind to the enzyme, presumably through chelation with fe(ii). when 15 was employed as a control, no binding of boronate esters was found, and again, binding of some diols to the enzyme was observed. these results indicate that the mass shifts observed with 14 represent the boronate ester-phd2 complexes, instead of simultaneous binding of 14 and the diols. in order to validate the dcms results and to ensure that the results obtained were not biased by the ms technique, nmr-based water relaxation experiments (see section "other nmr-based methods") were performed, confirming that the dcms method (gas phase) is in agreement with the nmr studies (solution). verification of the potential of the boronic acid-/boronate ester-based dcms method was performed by determining the binding constants of stable analogs 17a-i of "support ligand" 14 and diols 16a-g (scheme 8 and table 2 ). all analogs bind significantly stronger to phd2 than boronic acid 14, but generally weaker than boronate esters 14-16a-g identified by dcms. in order to investigate the binding mode of the boronate esters, stable analog 17h was crystallized in complex with phd2, containing mn(ii) as a substitute for fe(ii). the protein cocrystal structure revealed that 17h binds to the metal in a bidentate fashion. to study its effect in cells, activity studies were performed with methyl ester derivative of 17g (18, scheme 8). compound 18 was found to upregulate hif1a by selectively inhibiting phds, but not fih. overall, the authors demonstrated that inhibitors based on boronate esters could be discovered using dcms in combination with nmr spectroscopy. the dcms method prevents the need for denaturing the protein, chromatography to separate the reaction mixture, and "freezing" of the dcl. the technique is very fast and enables direct analysis of the protein-ligand complexes. synthesis of all individual library members to validate spectral assignments is not required, and individual building blocks could be directly used in determining binding constants. despite the high sensitivity of ms, however, the library of 40 compounds had to be divided into sublibraries of ten possible compounds. schofield and coworkers also described some fragmentation of boronic acids, which is a limitation of the dcms approach. also not all proteins can be used for nondenaturing ms analysis, and during analysis, care should be taken, since not all of the noncovalent interactions might be translated during the transition from solution to gas phase in the same manner. 26 lastly, due to the soft conditions that have to be used, dcms methods are not that straightforward, and a lot of time and effort can be required to establish a method for analyzing a dcl with a novel protein target. a slightly different approach is the screening of dcls by competitive ms-based binding assays. in 2012, the group of wanner demonstrated an efficient way to screen hydrazone inhibitors using pseudostatic libraries and determined the binding affinity by competitive ms binding assays. 27 g-aminobutyric acid (gaba) transporter 1 (gat1) was chosen as a target, which is the most important subtype of the gaba transporters. decreased gabaergic neurotransmission is assumed to be a major cause of neurological disorders like epilepsy, parkinson's disease, and sleeping disorders. 28, 29 inspired by the work of andersen et al., 30 hydrazone derivatives 19 were designed that resemble mgat1 inhibitors 20a-c (scheme 9). nipecotic acid derivative 21 was used as the hydrazine building block, and 36 aldehydes 22 were divided over nine equal libraries (four possible hydrazones per dcl). in order to get a rough estimate of the equilibration time, five different aromatic aldehydes (40 mm to resemble four aldehydes at 10 mm) were reacted separately with hydrazine 21 (100 mm) in the absence of target, and the reaction was monitored by uv. in each case, almost complete equilibrium was reached within 4 h, and therefore, a 4 h incubation period was chosen for the dcc experiments in the presence of the target (adaptive dcc). to ensure nearly full conversion of the aldehydes toward the products, hydrazine 21 (100 mm) was used in a 2.5-fold excess with respect to the total aldehyde concentration (four aldehydes, 10 mm each). in case equal amounts of the structurally diverse aldehydes are added to the hydrazine, a library should be obtained with all constituents being table 2 binding constants (k d ) and ic 50 values for stable analogs 17a-i of boronic acid inhibitors 14-16a-g. 23 compound r x k d (mm) ic 50 after deconvolution experiments in which the aldehydes (10 mm) of the strongest binding dcls were separately tested in the presence of hydrazine 21 (100 mm), the most potent inhibitors were identified to be hydrazones 24a and 24b, which decrease marker binding to less than 10% ( table 3) . hydrazones 24c-e decrease marker binding between 20% and 40% and are therefore considered as binders of medium potency. interestingly, the five most potent hydrazones are all derived from an ortho-substituted aldehyde. the five best binders were synthesized, and the corresponding binding affinities (pk i ) and ic 50 values were determined using the ms binding assay for mgat1 31 and [ 3 h]-gaba uptake assay, respectively. 32 the order of inhibitory potencies is in good agreement with the results of the deconvolution experiments ( table 3) . one year later, in 2013, wanner and coworkers reported a follow-up study starting from one of the most potent inhibitors 24a. 29 competitive ms-based binding assays were employed in the search for optimized high-affinity gat1 binders. dcls were formed from hydrazine 21 and a total of 36 different aldehydes 25, in which different substituents were placed on the biaryl moiety (scheme 10a). the hydrazone-containing libraries were analyzed using the competitive ms method discussed earlier. all libraries led to reduced marker binding below 6%, and after deconvolution, 21 out of the 36 hydrazones 26 showed a higher affinity than the most potent mgat1 inhibitor of the previous study (24a). after synthesis of the hydrazones, the higher affinity compared with lead compound 24a (pk i ¼ 6.186 ae 0.028) was confirmed in the binding assay. the most potent hydrazone, 26a, displays a pk i value of 8.094 ae 0.098, which is 2 log units higher than the original lead compound 24a (scheme 10b). in order to develop the hydrazone hits into lead compounds, the carba analogs of five structurally diverse hydrazone derivatives were synthesized, and their inhibitory efficiency was determined. the pk i values of the carba analogs are roughly 1 log unit lower than those of the corresponding hydrazones, but the rank order of potency remained mostly unchanged. carba analog 27a of most potent hydrazone 26a was found to be also the most potent inhibitor of the carba analogs (pk i ¼ 6.930 ae 0.021). both studies show that the competitive ms-based binding assay of pseudostatic hydrazone libraries is an efficient hit-identification strategy and can ultimately be used as a starting point for the development of stable lead compounds with comparable binding affinities to the initial hydrazone hits. use of a native marker also prevents all drawbacks associated with radioisotopes. provided that the affinity of the marker is high enough, very low protein concentrations can be used, making this method suitable for proteins that are usually obtained in extremely low concentration, such as membrane-bound proteins like g protein-coupled receptors. table 3 marker-binding data (pk i ) and mgat1 activity (pic 50 one of the general problems associated with the analysis of protein-templated dcls is the need for large amounts of protein due to a lack of sensitivity. most analytical methods also suffer from difficult, time-consuming, and expensive detection of active compounds. in 2008, the group of rademann reported dynamic ligation screening (dls), in which reversibly formed ligation products from the dcl compete in a dynamic equilibrium with a fluorogenic reporter substrate for a target enzyme. 33 by using an enzymatic reaction to detect the fragments, only small amounts of protein are required, enabling high-throughput screening (hts) in microtiter plates. to demonstrate the dls method, severe acute respiratory syndrome (sars) coronavirus sars-cov m pro was used as a protein target, which is a cysteine protease that is essential for replication of the virus inside the host cell and responsible for sars. first, a fluorescence-based assay for sars-cov m pro was developed using substrate ac-tsasvlq-amca 28 ( table 4 ). enzymatic cleavage of 28 releases fluorophore 2-(7-amino-4-methyl-3-coumarinyl)acetamide (amca). next, peptide aldehyde inhibitor 29, which mimics the native protease substrate and acts as a directing probe, was synthesized on an oxazolidine resin. given that aliphatic aldehydes are less reactive toward imine formation in aqueous media than aromatic aldehydes, it was hypothesized that the imine products of the reaction between a nucleophile and aliphatic peptide aldehyde 29 could only be formed when stabilized by the protein surface defining the active site, enabling substrate competition. in each well on a 384-well microtiter plate, aldehyde 29 was mixed with the enzyme and an eightfold excess of one of 234 chosen nucleophiles 39 (a selection containing amines, thiols, and hydrazines). substrate 28 was added, leading to the release of fluorophore amca as a measure of enzymatic turnover which can be monitored by fluorescence spectroscopy. none of the individual nucleophiles showed inhibition of sars-cov m pro . for seven nucleophiles from the library, a stronger inhibition than with aldehyde 29 alone was observed ( table 5) . to confirm that binding took place in the active site of the protease and not, for example, at an allosteric site, derivatives of the most active amine 30 were studied. first, the reduced amination product (31, table 4 ) was tested in a hplc binding assay and showed a k i value of 50.3 mm. comparing the inhibitory activity of 31 with truncated amide 32 and those of the inactive peptides ac-dsfdq-oh, dsfdq-oh, and ac-dsfdq-nh 2 supported the directing effect of aldehyde 29 and the binding of fragment 30 to the s1 0 pocket. further evidence was provided by modeling studies and the inhibition results of aldehydes and 2-ketoaldehydes 33-36. these aldehyde derivatives of 30 were designed to interact with the active site of the protease using their electrophilic ends. dls showed that aldehydes 33-36 are active inhibitors of the protease and thus support the hypothesis that the fragments bind specifically to the s1 0 pocket of sars-cov m pro . in order to obtain a nonpeptidic inhibitor of the target protein occupying both the s1 0 and s1 pockets, dls was applied again, but now in a "reverted" mode. instead of using aldehyde 29, which binds to the s side, the s 0 side favoring 36 was employed. in this table 5 initial velocities v 0 of substrate conversion in the presence of sars-cov m pro (protease), substrate, peptide aldehyde 29 or 36 and nucleophiles 30, 37 and 39a-h 33 nucleophile n 0 (mm min -1 ) second screening, a library of 110 amines was used. in each well, electrophile 36 was incubated with one of the amines, the protease, and the fluorogenic substrate. three active fragments were identified in the presence of 36 ( table 5 ). the most active fragment 37 was then used for the reductive amination with 2-ketoaldehyde 36, affording amine 38, which displays a k i value of 2.9 mm. this proof-of-concept study shows that the dls method is an efficient way of identifying novel inhibitors with a k i value in the low micromolar range. the method uses a tiny reaction volume of only 20 ml per well, requires very small amounts of protein (1 mm sars-cov m pro per well), and works efficiently in a high-throughput setup. schmuck and coworkers showed in 2015 the use of a pre-equilibrated acylhydrazone-based dcl for the identification of b-tryptase inhibitors with low nanomolar affinities. 34 a pre-equilibrated approach was chosen, given that acylhydrazone formation requires acidic conditions that are not tolerated by the protein target. b-tryptase is the predominant serine protease in human mast cells and responsible for several allergic and inflammatory disorders. [35] [36] [37] the protease consists of four identical monomers and is stabilized by heparin. it features four active sites with a central access pore. it was proposed that when this pore is blocked, the substrate cannot access the active site. therefore, dcls were designed based on reversible acylhydrazone formation between tetrapeptide hydrazides and di-/trialdehydes. in this case, the two-and three-armed peptide acylhydrazones are large enough to (partially) block access to the four active sites, and the amino acids in the arms of the acylhydrazones can have multiple interactions with the protein surface. first of all, a dcl was generated with five di-and trialdehydes (40a-e) and five peptide-derived hydrazides (41a-e) (scheme 11). hydrazides 41a and 41b were based on earlier work, 38, 39 41c and 41d were derived from 41b, and negatively charged hydrazide 41e was chosen as a negative control, since the negative charge would be unfavorable for the inhibition of b-tryptase. the full dcl was generated by mixing the hydrazides with the aldehydes in acetate buffer (ph 4.0) and analyzed by a deconvolution strategy, which was first established by lehn and coworkers. [40] [41] [42] for the analysis, ten sublibraries, in which one building block is missing, were generated in the same way. the composition of the sublibraries can be compared with the full library. in case a building block is missing, which is normally part of a very active inhibitor, the corresponding sublibrary will be less active than the full library. equimolar concentrations of both building blocks were used, and the libraries were equilibrated at room temperature for three days. formation of the corresponding acylhydrazones was confirmed by analytical hplc and ms. when it is assumed that all of the aldehyde groups in the dcl have fully reacted to form an acylhydrazone, the dcl from five hydrazides, four di-aldehydes, and one tri-aldehyde can contain a total of 225 possible constituents. when the identical acylhydrazones are removed (all of the di-and trialdehydes are symmetrical), the library size is reduced to 95 different members. as for the sublibraries, the library size changes depending on the number of functional groups featured by the building block, which is removed. it could not be confirmed if all of the compounds were present in the libraries and in which relative amounts. therefore, it is possible that the inhibitors identified are not the best possible inhibitors. this limitation always applies to the use of pre-equilibrated libraries. scheme 11 (a) generation of acylhydrazone-based dynamic combinatorial libraries to discover new potent inhibitors for b-tryptase. (b) di-and trialdehyde building blocks 40a-e. (c) tetra-peptide hydrazides 41a-e. gcp, guanidiniocarbonyl pyrrole. 34 after the formation of the pre-equilibrated libraries, the effect on b-tryptase activity was determined. first, the dcls were "frozen" by changing the ph to 7.4, which stops the reversible acylhydrazone formation. then, analysis was carried out with a high-throughput enzyme-activity assay using the substrate toc-gly-pro-arg-amc, which generates fluorophore 7-amino-4methylcoumarin (amc) upon cleavage by the enzyme and enables monitoring of the rate of enzymatic hydrolysis. the hydrazides alone showed activity in the low micromolar range (scheme 11). hydrazide 41e (negative control) and the aldehydes were found to be weak inhibitors of b-tryptase (ic 50 > 1000 and > 10,000 mm, respectively). using the same assay, the activity of the enzyme in the pre-equilibrated sublibraries was determined and compared with the full library (building block concentration of 1.25 mm each) (fig. 4a) . the inhibition assay showed that hydrazides 41a-d are required for efficient enzyme inhibition. hydrazide 41c was shown to be the most active building block. when it was removed, 47% of the enzyme activity was restored compared with the full library. hydrazides 41a and 41b are of similar activity but less active than 41c. hydrazide 41d is the least active with approximately 13% of recovery. hydrazide 41e (negative control) shows a negative normalized activity, showing that inhibitory activity is increased when 41e is not included in the dcl. this was expected, since the relative concentration of active inhibitors increased. regarding the aldehyde building blocks, trialdehyde 40e emerged as the most active one. dialdehydes 40a-d show much smaller inhibitory effects (0-10% recovery when removed from the dcl). to further study the role of the aldehydes, a second library containing all five aldehydes and the most active hydrazide 41c and the corresponding sublibraries without one of the aldehydes were generated (fig. 4b) . the results confirmed that aldehydes 40c and 40e are the most active building blocks for hydrazide 41c. removal of hydrazide 41c completely restores the activity of the enzyme, confirming that the aldehydes alone do not inhibit b-tryptase. the representative acylhydrazones were synthesized in order to determine their k i values ( table 6 ). all individually synthesized acylhydrazones are inhibitors of b-tryptase with k i values in the low nanomolar range, which is three orders of magnitude more potent than the individual hydrazides. the two most active inhibitors 40e-41a and 40e-41c display k i values of 12 and 22 nm, respectively. using a dixon plot, it was verified that the compounds act as noncompetitive inhibitors, in which case k i is the same as the ic 50 value. negative control 40e-41e showed no inhibition of the protein at all. interestingly, individual hydrazides 41c and 41d, with a slightly different peptide structure, showed similar activity ( table 6) , while acylhydrazones 40e-41c and 40e-41d showed different k i values and different inhibition profiles (single mode for 40e-41c vs biphasic behavior for 40e-41d). these differences are likely caused by differences in binding mode, caused by the change in position of the guanidiniocarbonyl pyrrole group. molecular-mechanics calculations indicated that the inhibitors bind to two different types of binding sites on the protein surface. inhibitor 40e-41c blocks the pore completely, while 40e-41d does so only partially, resulting in weaker inhibition of the enzyme. additionally, to the pore-blocking ability, 40e-41d is $ 36 kj mol à 1 lower in energy in its unbound state than constitutional isomer 40e-41c, presumably due to more favorable stabilizing intramolecular interactions. the complex of b-tryptase table 6 inhibitory constants (k i ) of the individually synthesized acylhydrazones 40-41 (see also scheme 11 and 40e-41c is predicted to be 33 kj mol à 1 more stable than the complex with 40e-41d. this might suggest that the improved inhibitory activity of 40e-41c originates from a combination of stronger binding to the protein in a slightly different binding mode and the less stable ground-state conformation of the inhibitor itself. in 2015, our group reported the combination of fragment-based drug design, and in particular fragment growing, and dcc as a powerful and efficient strategy to convert a fragment into a hit. 43 dcls containing acylhydrazones and a fluorescence-based enzymatic assay were used to grow a fragment into a more potent, noncovalent inhibitor of the aspartic protease endothiapepsin. as a starting point, fragment 42 was chosen based on its favorable h-bond interactions between its amidine group and the catalytic dyad, promising physicochemical properties and the fact that it only contains 13 heavy atoms. 44 fragment 42 occupies the s2 and part of the s1 and s1 0 pockets of endothiapepsin and makes interactions with the catalytic dyad through charged h-bonding interactions. as shown in one of our previous studies, an acylhydrazone moiety is a suitable scaffold to address the catalytic dyad of endothiapepsin. 15 therefore, in order to grow into the s1 and s3 pockets, an acylhydrazone linker was proposed. based on molecular-modeling and docking studies, a series of nine potential acylhydrazone-based inhibitors were selected, corresponding to hydrazide 43 and aldehydes 44a-i (scheme 12a). nine dcls, each containing hydrazide 43 and one of the nine aldehydes 44a-i, were generated to form the corresponding acylhydrazones 45a-i. a fluorescence-based enzymatic assay was used with a fluorogenic substrate (2-aminobenzoyl-thr-ile-nle-phe(p-no 2 )-gln-arg-nh 2 , 46). hydrolysis by endothiapepsin of the fluorogenic peptide, affording fragments 47 and 48, leads to an increase in fluorescence (scheme 12b), allowing monitoring of enzyme inhibition by fluorescence spectroscopy (fig. 5) . analysis revealed two dcls displaying considerably higher inhibition of endothiapepsin than 42 (45% inhibition at 1 mm), namely, 43 þ 44a (75% inhibition and ic 50 ¼ 407 mm) and 43 þ 44g (79% inhibition and ic 50 ¼ 252 mm). since full conversion to the corresponding acylhydrazone is not ensured and hydrazide 43 is used in excess, the ic 50 values are expected to be lower for individually synthesized, isolated, and purified compounds. therefore, both identified acylhydrazone hits were synthesized and purified. after analysis by the same fluorescence assay, ic 50 values of 210 ae 32 mm and 85ae 8 mm were obtained for 45a and 45g, respectively. overall, it was shown that the combination of fragment growing and dcc is a powerful technique to generate novel hits for the aspartic protease endothiapepsin. the fluorescence-based assay enables direct screening of the dcls for active inhibitors. as shown by the three examples discussed earlier, fluorescence spectroscopy is a powerful method for protein-templated dcl analysis. by using an enzymatic reaction to detect inhibition, only small amounts of protein are required. given that fluorescence-based assays are fast, the enzyme needs to be in the assay mixture for a short time, making this type of analytical method ideal for precious and unstable proteins. the possibility for hts means that large numbers of compounds can be screened, decreasing the time required for analysis. in 2003, congreve et al. of astex technology reported the first and thus far only example of the direct detection of ligands from a dcl using x-ray crystallography. 45 up to date, the rcsb protein data bank reports over 100,000 protein crystal structures, which were elucidated by x-ray crystallography. 46 for the proof-of-concept study, congreve et al. used cyclin-dependent kinase 2 (cdk2) as a target due to its involvement in a number of human cancers. 47, 48 six hydrazines 49a-f and five isatins 50a-e were selected to form the corresponding hydrazones 49-50 (table 7) , which would present a variety of functional groups to occupy the lipophilic pockets in the atp-binding site. after a control experiment with lc-ms, which confirmed that all 30 possible products were formed in the dcl, individual crystals of cdk2 were soaked in reaction solutions containing monomer 49e and one of the isatin monomers 50a-e. in all cases, except for one (49e þ 50d), the resulting electron density in the atp pocket indicated that the corresponding ligand had bound. after synthesis and purification of each ligand, a cdk2 activity assay showed that each of the ligands binding to cdk2 was a potent enzyme inhibitor (ic 50 ¼ 30 nm for each binding ligand). then, in order to explore the potential of the x-ray crystallography method, soaking experiments of two dcls containing 50b þ 49a-d, 50b þ 49a-f, and cdk2 crystals led to the observation of electron density only in the latter case corresponding to hydrazone 49e-50b. subsequently, the complete dcl consisting of hydrazines 49a-f and isatins 50a-e was analyzed in the presence of cdk2. electron density in the atp-binding pocket confirmed 49e-50b as a potent inhibitor, clearly demonstrating that x-ray crystallography is an efficient and powerful method for the direct identification of binders from dcls while also providing details on the binding mode. for enzymes with a well-established crystallization or soaking protocol, the destructive x-ray crystallography method has several advantages over the previously discussed analytical methods: it is less time-consuming; it does not require large amounts of protein and provides the binding modes of the ligands identified. despite these benefits, this method has thus far only been used once for the analysis of dcls. this is probably due to the fact that it requires specific expertise and infrastructure, which is not readily available in a synthetic organic chemistry laboratory. table 7 generation of hydrazone-based dynamic combinatorial libraries of hydrazines 49a-f and isatins 50a-e to discover new inhibitors of cyclindependent kinase 2 facilitated by x-ray crystallography. 45 a modification of dcc is tethering, a strategy that can be exploited to identify weakly binding ligands, new allosteric regulatory sites, fragments that disrupt protein-protein interactions, and small-molecule stabilizers of conformationally malleable proteins to enable their structural characterization. [49] [50] [51] [52] [53] ligands that bind weakly to a specifically targeted site on a protein or macromolecule can be stabilized by the formation of a disulfide bond between the ligand and a cysteine residue in the protein target (fig. 6 ). this cysteine can be a naturally occurring one or it can be selectively installed using site-directed mutagenesis. if a compound has (weak) affinity for the protein, the disulfide bond will be entropically stabilized, and the equilibrium will shift toward the modified protein. a dcl can be formed from disulfide ligands and the protein target in the presence of a reducing agent, such as b-mercaptoethanol. the reducing conditions in the dcl promote rapid thiol exchange. after equilibration, the dcl can be analyzed by ms, as long as the potential binders have unique molecular weights. more recently, fluorescence-polarization assays were used to detect displacement of a peptide ligand from the protein target as indirect readout of disulfide formation. 52 tethering was first reported in 2000 by wells and coworkers. 49 as model system, the anticancer and anti-infective drug target thymidylate synthase (ts) was chosen. libraries containing 8-15 disulfide-containing compounds were added to protein-containing buffer in the presence of b-mercaptoethanol. after equilibration, analysis was performed by lc-ms, and the multiply charged ions arising from the protein were deconvoluted to afford the mass of the modified protein. after screening a total of 1200 compounds, some highly significant structure-activity relationships were observed. disulfide-linked n-tosyl-d-proline (51, scheme 13) was found to bind strongly to ts, and the tosyl group was found to bind approximately in the same position and orientation as methylenetetrahydrofolate, the natural cofactor for ts. therefore, the glutamate residue from the cofactor was grafted onto the methyl group of n-tosyl-d-proline, affording 52. the modified n-tosyl-d-proline (k i ¼ 24 ae 7 mm) showed an almost 50-fold higher activity than n-tosyl-d-proline alone (k i ¼ 1.1 ae 0.25 mm). the introduction of an ethylene linker (53) increased the activity even more, obtaining a k i value of 330 ae 40 nm. tethering can also be used to substantially increase the stability of conformationally dynamic proteins, which cannot be characterized by x-ray crystallography. wang et al. succeeded in obtaining the x-ray crystal structure of the co-activator gackix domain by taking advantage of this strategy. 53 the gackix domain of the co-activator cbp/p300 consists of 90 amino acids and is known to interact with > 10 distinct amphipathic sequences at two distinct binding sites. when activated, it stimulates transcription of hundreds of genes, including those regulating hematopoiesis, memory formation, and the inflammatory response. the flexibility of this class of proteins makes characterization by crystallography impossible, either in complex with a binding partner or alone. mapp and coworkers applied the tethering strategy to screen for small ligands that interact with the gackix domain. a leucine residue at the rim of the binding surface was mutated to a cysteine, affording gackix l664c. a fragment library scheme 13 optimization of ligands for thymidylate synthase starting from compounds selected by covalent tethering using a disulfide library. 49 figure 6 schematic illustration of screening using the tethering approach. a cysteine residue at the surface of the protein forms a disulfide bond with different small-molecule fragments. comprising 480 disulfides was screened in a high-throughput format using a ms assay. fragments 54 and 55 emerged from the screen with high tethering efficiency to gackix l664c (scheme 14). after characterizing the binding properties of the ligands, a selenomethionine-incorporated gackix l664c tethered to 54 was crystallized, and the structure of this conformationally dynamic protein was solved by x-ray crystallography. table 8 overview of the analytical methods used in protein-templated dcc discussed in this article. over the past 15 years, protein-templated dcc has emerged as a powerful tool that enables the identification of novel ligands for biological targets. it allows the in situ synthesis and amplification of new binders by the protein target. combined with efficient screening methods, protein-templated dcc holds the potential to accelerate the drug-discovery process significantly. nowadays, a whole range of analytical methods has been developed, such as hplc, lc-ms, and sec to separate the different members of a dcl and x-ray spectroscopy, nmr, and ms to characterize binders of the protein target ( table 8) . a major bottleneck of dcc, however, is the analysis of the dcls, and so far, most reported examples use libraries of only small to moderate size. these limitations have to be addressed in order to allow the use of larger and more complex libraries, which will transform dcc into an even more efficient and widely used tool in medicinal chemistry. furthermore, expanding the list of biocompatible, reversible reactions will give access to new potential binders and more structural diversity. it will also facilitate the development of doubly dynamic systems, in which fragments for two different pockets of the active site of an enzyme are screened simultaneously. 54 for these types of systems, comparable reactivity at a certain ph is required, which should also be in line with the stability of the protein target. chemical shifts and coupling constants for boron-11 and phosphorus-31 funding was granted by the netherlands organization for scientific research (nwo-cw and vidi grant to akhh) and by the dutch ministry of education, culture and science (gravitation programme 024.001.035). key: cord-340387-ohkjheat authors: wynne, james w.; di rubbo, antonio; shiell, brian j.; beddome, gary; cowled, christopher; peck, grantley r.; huang, jing; grimley, samantha l.; baker, michelle l.; michalski, wojtek p. title: purification and characterisation of immunoglobulins from the australian black flying fox (pteropus alecto) using anti-fab affinity chromatography reveals the low abundance of iga date: 2013-01-07 journal: plos one doi: 10.1371/journal.pone.0052930 sha: doc_id: 340387 cord_uid: ohkjheat there is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. immunoglobulins are major components of the adaptive immune system. early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. to further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, pteropus alecto. we employed a novel strategy, where igg was initially purified and used to generate anti-fab specific antibodies. immobilised anti-fab specific antibodies were then used to capture other immunoglobulins from igg depleted serum. while high quantities of igm were successfully isolated from serum, iga was not. only trace quantities of iga were detected in the serum by mass spectrometry. immobilised ligands specific to iga (jacalin, peptide m and staphylococcal superantigen-like protein) also failed to capture p. alecto iga from serum. igm was the second most abundant serum antibody after igg. a survey of mucosal secretions found igg was the dominant antibody class rather than iga. our study demonstrates healthy p. alecto bats have markedly less serum iga than expected. higher quantities of igg in mucosal secretions may be compensation for this low abundance or lack of iga. knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host. bats represent approximately one fifth of the world's mammalian species and are among the most diverse and geographically dispersed mammals. frugivorous and nectivorous pteropid bats (family pteropodidae, suborder megachiroptera) constitute approximately 170 species and are known colloquially as fruit bats or flying foxes. their capacity for long-distance dispersal through flight has undoubtedly contributed to their widespread distribution throughout tropical and sub-tropical asia and australia, and on islands of the indian and western pacific oceans. the black flying fox, pteropus alecto, is distributed mainly across the northern and eastern coasts of australia [1] . since 1994, this species has gained particular attention after it was identified as the natural host of hendra virus (hev) [2, 3] . a body of evidence exists implicating bats as a major source of zoonotic viruses [4] [5] [6] . bats have been shown to harbour and disseminate highly pathogenic viruses including ebola, sars-like coronavirus and the henipaviruses (hev and nipah) [4] . spillover events from bats to other species -including humans -have increased. in 1998, a spillover event of nipah virus from pteropid bats to farmed pigs caused a major disease outbreak in malaysia. more than 1 million pigs were culled, and of the 265 reported human cases, 105 were fatal [7, 8] . while the incidence of hev in australia has been more sporadic, an increase in spillover events from pteropid bats to horses has occurred. between 1994 and 2010, 14 hev outbreaks occurred in australia involving 48 confirmed equine cases (75% fatality rate) and seven human cases (60% fatality rate). in contrast, between june 2011 and january 2012, 19 hev outbreaks occurred [9] . the increasing incidence of hev, combined with its high fatality rate, means hev has the potential to cause further significant disease outbreaks in australia and abroad. recently, a novel paramyxovirus that is closely related to the henipaviruses, named cedar virus, was isolated from fruit bats in australia [10] . however unlike hev, cedar virus caused no clinical disease in experimentally infected ferrets and guinea pigs [10] . evidence of henipavirus infection in multiple bat species has also been identified in continental africa [11] . despite the risk of zoonotic diseases originating in bats, little is known regarding the bat immune system and very few diagnostic reagents are commercially available. despite their ability to carry highly pathogenic viruses such as hendra, nipah and ebola, bats appear asymptomatic and rarely show signs of disease following both experimental and natural infection with these agents [12] [13] [14] [15] [16] . the notable exceptions are rabies-like viruses, which are capable of causing clinical disease in bats and humans [17, 18] . anecdotal observations raise the possibility that the bat immune system may have atypical and possibly unique characteristics that permit asymptomatic virus persistence and spillover events. immunoglobulins, namely igg, iga and igm, are important and diverse secretory components of the mammalian immune system. antibodies of the igg class are the most abundant immunoglobulins in mammalian serum and consequently provide the majority of immunity against blood-borne infectious agents [19] . in contrast, iga is most abundant in mucosal secretions including milk, tears, and secretions of the upper respiratory tract and digestive tract. the abundance of iga in normal human serum is approximately one fifth of the igg level and accounts for about 15-20% of total immunoglobulins in serum [20] . in humans, serum iga is predominantly monomeric whereas iga within mucosal secretions is found as a dimer complex. polymerisation of the iga (and igm) is facilitated by the joining (j) chain which is expressed by immunocytes of various secretory tissues [21] . polymeric iga is secreted into the mucosa through its interaction with the polymeric ig receptor (pigr) which is expressed at the basolateral surface of secretory epithelial cells. the iga/pigr complex is transported via transcytosis to the cell surface where it is ultimately secreted in to the lumen [22] . mammalian iga plays a crucial role in mucosal immunity while in serum it interacts with the iga fc receptor (fcar) to control the inflammatory response [20] . indeed, in the absence of antigen, iga down-regulates igg mediated phagocytosis, chemotaxis, cytokine release and oxidative burst activity [23] [24] [25] [26] . igm is the first antibody produced following microbial invasion and exists predominantly as either a monomeric antigen receptor on b cells or as a soluble pentamer. igm can also exist as tetramer and dimers, however these forms are significantly less abundant. in healthy humans serum igm is slightly less abundant compared to serum iga. variation in the nature of n-linked glycosylation provides further diversity to immunoglobulins. these glycans play a vital role in maintaining structural integrity and in some cases act as ligands for immunologically important serum lectins such as mannan-binding lectin [27] . few studies have examined the immunoglobulin system of bats. recently baker et al. [28] sequenced mrna encoding the heavy chains of igg, igm and iga of p. alecto, revealing a diverse repertoire of variable heavy chain transcripts. transcripts resembling ige have also been indentified in different bat species, while igd appears specific to only insectivorous bats [29] . wild-caught bats are certainly capable of generating neutralising antibodies to viruses such as hendra, ebola and sars-like coronavirus [2, 30, 31] , but early studies have demonstrated pronounced differences in both the magnitude and duration of antibody responses in bats compared to other mammals [32] [33] [34] . the large frugivorous bat, p. giganteus, demonstrated a delayed primary antibody response following immunization with sheep red blood cells [32] . similarly, the magnitude and duration of the neutralising antibody response of the big brown bat (eptesicus fuscus) to the model antigen phix174 bacteriophage was lower than that of rabbits and guinea pigs [33] . the likelihood that bats have an atypical immunoglobulin response compared to other animals requires further attention. specifically, the abundance, tissue distribution and post-translational modification of the different immunoglobulin classes must be examined. given the importance of bats as viral reservoirs, understanding the bat immune system may facilitate development of methods to mitigate spillover events in the future. as a first step, this study aimed to purify and characterise the major immunoglobulin classes from healthy p. alecto biological specimens. considering that in other mammalian species, immunoglobulins igg, igm and iga are present in relatively high abundance in serum and tissues, we anticipated that bats would possess a similar immunoglobulin profile. however, while igg and igm appeared abundant in p. alecto serum, iga was not. iga was detected in the mucosal secretions of the small and large intestine lavages, milk and tears. diverse isoforms of igg and igm, suggestive of multiple subclasses, were identified. reagents developed within this study will aid future studies of this unique immunoglobulin repertoire, particularly in response to viral infection. all animal experimentation and sample collection was conducted following guidelines approved by the aahl animal ethics committee (permit no. 1302). p. alecto bats were captured in southern queensland, australia as described previously [35] and transported live by air to the csiro australian animal health laboratory (aahl). the animals were bled for serum and plasma and then euthanized for dissection of tissues. tissues were stored at 280uc in rnalater (ambion) for rna analysis or snap frozen in liquid nitrogen for downstream mass spectrometry (ms) analysis. lungs, small and large intestines were washed with 15-20 ml of cold phosphate buffered saline (pbs). washes (lavages) and tissues were stored at 280uc. faeces samples were collected from bat cages within 1-2 hours of being excreted and immediately resuspended in pbs containing protease inhibitors as previously described [36] . where indicated, serum was extracted from plasma according to the protocol described by salvador-morales et al. [37] . all chemical reagents were of the highest analytical grade available from merck, unless otherwise specified. the purification strategy employed in this study exploited the molecular characteristics of isotypes of mammalian immunoglobulins with the assumption that bat immunoglobulins would resemble those of other mammals. serum and plasma samples from p. alecto bats were used as a source of immunoglobulins and human serum was used as a control. immobilised proteins a, g and l were used in this study to capture igg from p. alecto serum. fab fragments, derived by papain digestion of purified p. alecto igg, were used to generate fab-specific antibodies in rabbits. in turn, immobilised anti-fab-specific antibodies were employed to capture remaining p. alecto immunoglobulins, from igg-depleted bat serum (fig. 1) . the final separation of igm from iga was attempted by size exclusion chromatography (sec) exploiting significant molecular mass difference between the two molecules or by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) separation of respective heavy chains. the identity of separated proteins was determined by comparing tandem mass spectrometric data with available protein sequence databases. isolation of igg from human, rabbit and p. alecto sera affinity chromatography purification of igg from mammalian sera using immobilised protein a, g or l was performed according to a method modified from that described by bjorck and kronvall [38] . protein a and protein g affinity columns (ge healthcare) or protein l affinity columns (thermo) were connected to a 280 nm uv detector and a chart recorder. serum samples were diluted 5-fold in binding buffer (20 mm phosphate buffer, ph 7.4) and loaded on to columns equilibrated with the binding buffer at a flow rate of 0.5 ml/min. columns were washed with 10 volumes of binding buffer and igg was eluted in elution buffer (100 mm glycine hcl, ph 2.7). collected fractions were immediately ph neutralised with 1 m tris-hcl, ph 9.0. protein concentration was determined using the micro bca protein assay (thermo) and protein samples were analysed by reducing sds-page. digestion of purified p. alecto igg with papain purified p. alecto and rabbit igg samples (1 mg) were diluted with pbs and concentrated 5-fold to 200 ml using 4 ml ultra-4 centrifugal filter devices with a 5 kda molecular weight cut-off (mwco) membrane (amicon). the concentrate was diluted with 100 mm sodium acetate buffer, ph 5.8, containing 50 mm cysteine and 0.5 mm ethylenediaminetetraacetic acid (edta). fifty microlitre aliquots were digested with increasing concentrations of papain (ranging from 0.01 to 0.05 mg/ml or 1:100 to 1:20 enzyme to substrate ratios, respectively) over different incubation periods (6 to 22 h) at 37uc. the reactions were stopped by addition of 30 mm iodoacetamide (sigma) and incubated at room temperature for 30 min. the extent of the digestion was assessed by reducing sds-page. the optimal digestion conditions obtained for bat igg (1:20 enzyme to substrate ratio, 18 h) were used for digestion of 20 mg of igg in scaled-up experiments. following large scale digestion, the papain digest was concentrated 5-fold to 200 ml using ultra-4 centrifugal filter devices with a 5 kda mwco membrane and buffer exchanged with protein g affinity chromatography binding buffer. this fraction contained igg fragments (fab and fc) and was diluted 25-fold with the binding buffer prior to fractionation on an immobilised protein a affinity column. p. alecto igg fc fragments bound to the column whilst fab fragments were not retained by the column. the fab and fc fraction was tested for purity by reducing sds-page and used to immunise rabbits to generate fab-specific antiserum. the igg fraction of this rabbit antiserum was obtained by purification on an immobilised protein a affinity column as described above. the purified p. alecto fab fragments (5 mg) obtained by digestion of p. alecto igg with papain were concentrated and buffer exchanged with 0.5 m nacl in 0.2 m sodium bicarbonate buffer, ph 8.3, using amicon ultra-4 centrifugal filter devices. coupling to a 1 ml hitrap nhs-activated hp column (ge healthcare) was accomplished according to the manufacturer's instructions. rabbit anti-fab antibodies were purified by affinity chromatography by applying the igg fraction to the immobilised-fab column using the procedure described for the purification of igg. the purified antibodies, designated anti-fab-specific antibodies (4 mg), were coupled to a hitrap nhs activated hp column as described above. igg-depleted p. alecto serum and plasma samples were obtained by repeated (2-3 times) affinity chromatography on immobilised protein g affinity columns. the flow through was assessed for the absence of igg by reducing sds-page and western blot analysis with protein g or whole p. alecto anti-igg rabbit serum as detecting agents. igg-depleted samples were fractionated by affinity chromatography on immobilised anti-fab-specific antibodies adopting the same procedure as that described for immobilised protein a and g except that the binding and washing buffer consisted of 0.3 m nacl in 50 mm phosphate buffer, ph 7.4. all fractions were assayed for protein concentration and analysed by reducing sds-page. affinity chromatography media reported to specifically interact with mammalian iga, including jacalin [39] , peptide m [40] and staphylococcal superantigen-like protein 7 (ssl7) [41] , were also used in attempts to purify p. alecto iga from serum, plasma and tissue lavages. the affinity procedures were first tested on human serum and plasma samples and proved suitable for isolation of human iga. the efficiency of purification steps was assessed by reducing sds-page and mass spectrometry. affinity-purified igg, fab and fc fragments from papain digests, and sec-purified igm fractions (250 mg) were separated by preparative reducing sds-page using large well 4-12% gradient bis-tris gels (invitrogen, carlsbad, ca, usa) under reducing conditions. the protein bands corresponding to heavy chain subunits of immunoglobulins were electroeluted from the gel using a bio-rad mini whole gel eluter according to the manufacturer's instructions with 0.01% sds, 60 mm tris, 40 mm n-cyclohexyl-3-aminopropanesulfonic acid (caps), ph 9.5, as eluting buffer or by passive elution overnight in pbs buffer containing 0.1% sds [42] . the eluted fractions were analysed by reducing sds-page, pooled and used for production of specific antiserum in rabbits. purified antigens (50 mg igg; fc fragment; fab fragment; igm h and igg h ) were emulsified with csiro triple adjuvant (montanide/quil a/deae-dextran; [43] ) to a final volume of 500 ml and injected intramuscularly at two sites at approximately 3 weekly intervals for 10 weeks. protein fractions were analysed using reducing sds-page under reducing conditions in precast gels (4-12% bis-tris gels in a mops/sds buffer or in 12% gels in tris-glycine/sds, invitrogen). protein bands were visualized after staining with either coomassie brilliant blue (cbb) or silver nitrate. serum samples (15 mg) were analysed by 2-de as described previously [44] . proteins were either visualized by silver nitrate or western transferred onto polyvinylidene fluoride (pvdf) membranes in 10 mm caps buffer, ph 11, containing 10% methanol for immunoblot analysis. membranes were blocked with 5% skim milk overnight at 4uc, then probed with specific rabbit antiserum diluted 1:10,000 or 1:20,000 followed by goat anti-rabbit igg horseradish peroxidase (hrp) conjugates diluted 1:20,000 or 1:40,000, respectively. visualization of reactive protein bands was achieved by enhanced chemiluminescence (ecl, ge healthcare) and exposure to x-ray film (fuji). samples of purified igg and igm from p. alecto and human sera were denatured with 0.25% triton x-100 (icn biomedicals) and treated with n-glycosidase f (pngasef, roche) or neuraminidase (from arthobacter ureafaciens, roche; relative rate of cleavage is a2r6.a2r3.a2r8) at 37uc overnight. samples were separated by reducing sds-page and western transferred onto low fluorescence pvdf transfer membranes (fluorotrans, pall) as described above and then blocked overnight in 50 mm tris-hcl, 150 mm nacl and 0.25% tween 20, ph 7.4 (tbs-t). after washing 3 times with tbs-t, membranes were incubated for 1 h with biotinylated lectins (vector labs burlingame, ca, usa): datura stramonium agglutinin (dsa; 1:3,000 dilution), galanthus nivalis agglutinin (gna; 1:3,000), maackia amurensis agglutinin ii (maa ii; 1:1,000), sambucus nigra agglutinin (sna; 1:30,000), peanut agglutinin (pna; 1:1,000) and ulex europaeus agglutinin i (uea i; 1:1,000). after lectin incubation, the blots were washed three times in tbs-t for 10 min and then incubated for 1 h with streptavidin-hrp conjugate (twice the dilution of lectin; dako, nsw, australia) in tbs-t followed by three 10 min washes, two in tbs-t then one in tbs. visualization of reactive protein bands was achieved by 5 min incubation with enhanced chemiluminescence substrate (ecl plus tm ) and scanning on a typhoon fla 9000 gel imaging scanner (ge healthcare). preparative sec was performed on a hrlc system (bio-rad) with uv monitoring of the eluate at 280 nm. the column used for separation was a bio-gel 40xl column (bio-rad) and the mobile phase consisted of 50 mm na 2 po 4 , 300 mm nacl, ph 7.4. eluted fractions were manually collected and subjected to analysis by reducing sds-page and mass spectrometry. purified proteins were analysed by mass spectrometry of bands excised from cbb-stained bands from reducing sds-page gels and subjected to in-gel digestion with trypsin [45] . resultant peptides were analysed by liquid chromatography tandem mass spectrometry (lc-ms/ms) using a surveyor high-performance liquid chromatography (hplc) system connected directly to the nano-spray ion source of an lcq classic quadrupole ion-trap mass spectrometer (thermo) as described previously [46] . identification of proteins was obtained using the turbosequest program [45] (v3.2) (thermo). ms/ms spectra of hplcseparated peptides from the in-gel tryptic digests was matched with theoretical mass spectra produced by in silico tryptic digestion of protein sequences from the ensembl pteropus vampyrus protein database (http://asia.ensembl.org/info/data/ftp/index.html) and published sequences [15] . search results were filtered by a single threshold where matches to peptides were reported only if the sequest cross correlation factor (xcorr) was .1.5 for a singly charged peptide ion, .2.0 for a doubly charged peptide ion or .2.5 for a triply charged peptide ion. furthermore, a protein was reported as identified only if 2 or more different peptide matches were found for that protein. total rna was extracted from lymph node, spleen, liver, lung, heart, kidney, small intestine, brain and salivary gland as previously described [47] . for each sample, 500 ng total rna was used as template in a 20 ml cdna synthesis reaction. quantitative pcr primers were designed using primer express 3.0 (applied biosystems) with default parameter settings. . qpcr reactions were preformed in triplicate as described previously [47] . copy numbers of target sequences were calculated using standard curves and normalised relative to 18 s ribosomal rna. purification of igg and production of antiserum. commercially available immobilised proteins g and a successfully captured whole igg from p. alecto serum and plasma. it appeared that p. alecto igg had greater binding affinity to protein g than to protein a ( fig. 2a and b) . washing removed significantly more igg from the immobilised protein a column compared to protein g. igg binding to protein l was negligible (data not shown). igg h was approximately 50-52 kda. a light chain immunoglobulin of approximately 25 kda was also visible in protein g purified fractions ( fig. 2a) . papain fragmentation of protein g purified igg revealed that the p. alecto immunoglobulin was significantly more resistant to protease cleavage compared to rabbit igg. the rabbit igg was completely digested after 12 h incubation (data not shown) while almost complete fragmentation of p. alecto igg could only be achieved after 18 h incubation with 5-fold higher enzyme concentration (fig. 2c) . following digestion, the fab and fc fragments were separated by protein a affinity chromatography. the fc (fcc) fragment was bound to the column and later eluted, whilst the fab fragment did not bind and was collected in the flow through fraction. this method isolated approximately 5.4 mg fab from 1 ml of serum. purified whole igg, igg h , fab fragments and fcc fragments were used as antigens for production of specific polyclonal antibodies in rabbits. igm enrichment by anti-fab affinity chromatography. immobilised anti-fab-specific antibody was used to capture non-igg immunoglobulins (igm and iga) from igg-depleted p. alecto serum and plasma samples. two major bands were detected by reducing sds-page in the eluate from both serum and plasma samples; a 66-70 kda band representative of igm h , and a 25 kda band representative of immunoglobulin light chain ( fig. 3a and 3b ). no band representative of iga h (predicted 52-55 kda from mrna sequence [28] ) was observed. to confirm the identities of these bands and others, the putative igm h band, its associated immunoglobulin light chain, and other minor bands (highlighted by arrows, fig. 3b lanes 1 and 3) were excised and subjected to lc-ms/ms (table 1) . bands 1 to 6 in serum and plasma contained protein corresponding only to cm, immunoglobulin light chain and/or j chain. however, band 4 in serum samples also contained sequence corresponding to ca. a similar result was obtained when the igm/iga enriched fractions from serum and plasma were pooled and subjected to sec ( fig. s1a and s1b). four fractions from each serum and plasma were analysed by reducing sds-page and lc-ms/ms. all fractions from plasma were found to correspond to cm, immunoglobulin light chain and/or j chain. one fraction from serum was also shown to correspond to ca (table s1 ). gel eluted igm h was used as antigen for production of specific polyclonal antibodies in rabbits. distribution and expression. the distribution of igg, igm and iga in healthy p. alecto was examined using lc-ms/ms analysis of gel-separated proteins from various tissue extracts and secretions (fig. s2) . no proteins of high molecular weight were detected in faeces (data not shown) and these samples were excluded from further analysis. igg h was detected in all samples with the exception of tears. in contrast, igm h was only detected in lung lavage while iga h was identified in the small and large intestine lavages, milk and tears ( table 2 ). due to the apparent low abundance of iga in tissues and secretions where it may be expected to be found, we examined the ighg, igha, ighm, igj and pigr mrna expression levels in ten tissues collected from three individual wild-caught p. alecto fruit bats by qpcr (fig. 4) . igha mrna was highly transcribed in the small intestine, lung and salivary gland. ighm was highly expressed in lymph node and spleen, and moderately transcribed in peripheral blood mononuclear cells (pbmc), small intestine and lung. igj and pigr were strongly expressed in small intestine, lung and salivary gland. brain, heart, kidney and liver expressed very low levels of all three mrnas. ighg was most abundant in the lymph node, spleen and pbmc. glycosylation. the glycosylation status of whole p. alecto igg and igm was assessed by treatment with glycosidases and lectin binding. human immunoglobulins were used as controls. electrophoretic analysis of immunoglobulin subunits following deglycosylation revealed greater glycan content associated with igm molecules than igg which was observed as a greater mass differential following deglycosylation ( fig. 5a and 5b ). this observation was confirmed by staining of igg and igm with a glycan specific stain (data not shown). following deglycosylation with pngasef, two bands of igm h were observed in p. alecto. it can be estimated from the mass differences that bat igg h and igm h have approximately 1-2 and 3-4 occupied glycosylation sites, respectively. glycan composition was analysed through lectin binding and revealed that the overall structure of igg and igm glycans was most likely n-linked di-antenary. reactivity with dsa and gna indicated the presence of 1,4-glcnac and 1,3-mannose component residues (fig. s3 ) and the reactivity with maa ii and sna indicated the presence of sialic acid residue(s), most likely in an a2r3 configuration (fig. s3c and s3d) . treatment with pna and uea i indicated the presence of galactose and fucose residues; however, these tests were less conclusive, possibly due to incomplete deglycosylation or nonspecific binding of the lectins. diversity of igg h and igm h isoforms. antiserum generated against igg h reacted predominantly with the two bands representative of igg h in p. alecto serum (fig. 6a ). the antiserum serum bands 1-8 from figure 3b are denoted ms1 to ms8, plasma bands 1-8 from figure 3b are denoted mp1 to mp8. doi:10.1371/journal.pone.0052930.t001 generated against igm h reacted predominantly with a single band representative of igm h in p. alecto serum (fig. 6a) . the anti-fab antiserum reacted only with immunoglobulin light chains (fig. 6a) . immunoblotting of 2-de separated p. alecto serum revealed the presence of a diverse range of igg h and igm h isoforms (fig. 6b, 6c and 6d) . igg h antiserum reacted with a variety of igg h isoforms ranging in pi from 5-9 (fig. 6d) . in contrast, the igm h antiserum reacted with a less complex igm h repertoire (fig. 6c) . reactivity of igg h and igm h antiserum. serum proteins for a number of species were separated by reducing sds-page (fig. 7a) . antiserum generated against p. alecto igg h showed reactivity to igg h of the fruit bats p. conspicillatus and, to a lesser extent, rosettus megaphylus (fig. 7b, lanes 2 and 3 respectively) . reactivity was also detected with horse igg h (lane 6). antiserum generated against p. alecto igm h reacted with p. conspicillatus and very weakly with ferret igm h (fig. 7c ). consistent with previous reports [39] [40] [41] , human iga was purified from igg-depleted human serum and plasma using immobilised ligands (jacalin, peptide m and ssl7) (data not shown). immobilised-peptide m and ssl7 bound no protein from p. alecto plasma and serum, and immobilised jacalin bound approximately 19 bands with varying molecular masses (fig. s4a, s4b, s4c) . protein fractions eluted from the jacalin column were analysed by reducing sds-page and 19 distinct protein bands were tested for the presence of iga by lc-ms/ms (fig. s4d) . none of these proteins contained peptides representative of iga (table s2) iga is the second most abundant immunoglobulin class in normal human serum, accounting for approximately 15-20% of the total immunoglobulin [20] . the immobilised anti-fab-specific antibody used within this study clearly enriched igm from igg depleted p. alecto serum. however, no visible enrichment of iga was observed. trace quantities of serum iga were detected only by lc-ms/ms. parallel experiments with anti-human fab antibody successfully enriched iga from human serum,. furthermore, affinity chromatography with ligands known to be specific to iga [39] [40] [41] failed to enrich this immunoglobulin from p. alecto serum. it must be kept in mind the approaches utilised in this study to isolate iga are based on the assumption that p. alecto iga would have similar characteristics to mammalian iga and the variable heavy chain regions would be shared between the major immunoglobulin classes. previous mrna sequencing studies of the p. alecto immunoglobulin repertoire would suggest this assumption is sound [28] . however, if p. alecto contains additional atypical iga-like molecules, our approaches may have failed to detect such variants. various iga-like equivalents have been identified across a range of species including teleost fish, amphibians and reptile [48] [49] [50] , highlighting the divergent evolutionary pathways that contribute to mucosal immunity. previous studies on a range of bat species found that the bat iga gene sequence more closely resembles human iga2. this similarity is due, in part, to the absence of a cysteine in the constant heavy 1 domain that is part of the covalent disulfide bond to the light chain [29] . the iga h sequence previously obtained for p. alecto is also missing this cysteine [28] . in humans, iga2 is the dominant iga subclass in mucosal secretions, whereas iga1 is more common in serum. if bats contain only a single iga2-like class, we may expect to find a higher concentration of iga in mucosal secretions compared to that found in serum. the apparent scarcity of serum iga led us to further investigate the abundance of iga in various mucosal secretions and tissues. lc-ms/ms analysis detected iga in the small and large intestine lavages, milk and tears. the absence of iga in the lung lavage and salivary gland may be the result of differential regulation of either iga itself or the j-chain or pigr. real-time pcr revealed both the igj and pigr transcripts co-localised with igha across most tissues. pigr and igj were most abundantly expressed in the small intestine where both iga transcript and protein was detected. in contrast the expression of pigr was markedly lower in the lung and salivary gland which may have contributed to the lower abundance of iga protein in these tissues. as expected ighg was the most highly expressed immunoglobulin mrna in the lymph node, spleen and pbmc. interestingly, while ighg mrna was not highly expressed in the mucosal tissues, lc-ms/ms revealed igg was the dominant immunoglobulin in these tissues and secretions. this result suggests the majority of igg protein within the mucosal tissues and secretions is not locally produced and is more likely transported to these sites. in humans, the transportation and regulation of igg across the mucosal epithelium is driven by the neonatal fc receptor [51] . the importance of mucosal igg in the defence against epithelium associated pathogens has been demonstrated previously and is now recognised as playing a key role in mucosal immunity along with iga [52] . the relative abundance of igg over iga within the milk of p. alecto may have implications for the transfer of material immunity. in humans the majority of igg is transferred to the fetus via the placenta and only trace quantities of igg are found in lacteal secretions [53, 54] . other species, such as cows, sheep and horses do not transfer igg across the placenta and the majority of igg, and a smaller proportion of iga, is provided to the newborn through the colostrum and milk [53, 54] . the data presented herein would suggest p. alecto, unlike humans and rabbits, transfers the majority of igg through the lacteal secretions rather than through the placenta. selective iga deficiency is a common condition in humans affecting approximately 1 in 600 individuals [55] . in most cases individuals deficient in iga show no clinical symptoms. however, severe allergies, autoimmune and gastrointestinal disorders have been reported in some patients [55] . studies in iga knockout mice samples were run on separate gels and bands were excised from the region predicted to comprise the immunoglobulin heavy chains . gel images are presented in figure s2 . [56] . interestingly, however, the iga deficient mice produced higher quantities of systemic igg. similar findings have also been reported in humans [57, 58] . indeed, significantly higher levels of igm and igg were found in the saliva of iga deficient individuals compared to normal healthy controls [58] . in light of these studies, we hypothesise that the dominance of igg in the mucosal secretions of p. alecto may be an evolutionary compensation for an inherent lack of iga. further research is required to determine what functional relevance this would have on the immune response. 2-de resolution of p. alecto serum demonstrated the existence of multiple igg h isoforms. the immunoblot reactive spot train spanned a wide pi range of 5 to 9. this range is in agreement with the predicted pi range of 15 separate ighg mrna previously sequenced (range of 6.3 to 8.3) [28] . a doublet of slightly different molecular weights for the igg h chain was also observed after 1-de immunoblotting. most mammalian species contain multiple igg subclasses and therefore it is not surprising that p. alecto contained multiple subclasses of igg. indeed, butler et al. [29] reported the presence of multiple ighg subclass transcripts in a number of bat species including myotis lucifugus, eptiscus fuscus and cynopterus sphinx. in contrast to igg h , a much smaller number of reactive igm h isoforms were detected in serum following 2-de western blotting. however, following deglycosylation with pnga-sef, two bands of igm h were observed in p. alecto. this result was not observed in human igm. this finding suggests the possible existence of a subclass of igm h in p. alecto that is absent from humans. to our knowledge the existence of multiple igm subclasses has not been previously described in bats. considering that igm provides a first line of defence against microbial invasion, diversification of this immunoglobulin class may confer a distinct immunological advantage to the species. indeed, different igm subclasses may confer an enhanced and diverse classical complement pathway which, to some extent, may compensate for the lack of serum iga in p. alecto. further studies are required to elucidate the functional significance of putative igm subclasses within p. alecto. p. alecto igg from serum and plasma binds to both protein a and protein g but has significantly greater binding affinity to protein g. protein g recognises most igg subclasses in many species [59] whilst protein a binds only certain igg subclasses and in some species does not bind igg (e.g. rat and goat). this finding may reflect the presence of different p. alecto igg subclasses with different binding affinity to protein a. p. alecto igg h antiserum cross reacted strongly against two other bats (p. conspiculatus and r. megaphylus) and horse igg h . a close evolutionary relationship between chiroptera bats and horse has been reported on a number of occasions and may have contributed to this cross reactivity [60, 61] . the fact that p. alecto igm h antiserum did not cross react with horse suggests this immunoglobulin class has diverged more rapidly since the separation of horse and bats compared to igg. divergence of igm may have contributed to the rise of different igm subclasses as described above. given that serum iga is drastically reduced in p. alecto, diversification of igm may have functional significance for the early antibody response. the novel method involving the use of immobilised anti-fabspecific antibody described herein allows for the simultaneous purification of immunoglobulin classes from serum and production of class-specific antiserum without prior knowledge of the species' immunoglobulin repertoire. igg and igm were purified from p. alecto serum, and rabbit antiserum specific to both molecules were produced. while igg and igm were successfully isolated from serum, no visible enrichment of serum iga was achieved. trace quantities of iga were detected only by mass spectrometry in serum. iga was also detected in the mucosal secretions of the small and large intestine lavages, milk and tears. the glycosylation pattern of p. alecto igg and igm appears typical of that described for other mammalian immunoglobulins. furthermore deglycosylation of igm h and 2-de immunoblotting of igg h revealed the possible existence of multiple igm h and igg h subclasses in p. alecto. taken together, this study suggests healthy p. alecto bats have considerably less serum iga than expected. diversification of igm and igg subclasses may be an evolutionary compensation for this lack of iga. further research is now required to investigate the functional relevance of this unique antibody repertoire, particularly in response to viral infection. the knowledge and tools developed here will facilitate this research. (table s2 ) (tif) table s1 protein identification by lc-ms/ms analysis of igm affinity purified fractions. the four fractions were separated by sec from p. alecto serum and plasma (see figure s1 ). (docx) table s2 protein identification by lc-ms/ms analysis of 19 excised bands. proteins were purified by jacalin affinity chromatography from igg depleted p. alecto serum (see figure s3d ). (docx) flying-foxes: fruit-and blossom-bats of australia isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus serologic evidence for the presence in pteropus bats of a paramyxovirus related to equine morbillivirus bats: important reservoir hosts of emerging viruses bats, emerging infectious diseases, and the rabies paradigm revisited bats as a continuing source of emerging infections in humans fatal encephalitis due to nipah virus among pig-farmers in malaysia nipah virus infection of pigs in peninsular malaysia henipaviruses:an updated review focusing on the pteropid reservoir and features of transmission cedar virus: a novel henipavirus isolated from australian bats henipavirus neutralising antibodies in an isolated island population of african fruit bats human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo experimental nipah virus infection in pteropid bats experimental inoculation of plants and animals with ebola virus transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats experimental hendra virus infection in pregnant guinea-pigs and fruit bats (pteropus poliocephalus) australian bat lyssavirus infection in a captive juvenile black flying fox pathogenesis studies with australian bat lyssavirus in grey-headed flying foxes (pteropus poliocephalus) iga and the iga fc receptor p (1974) presence of j-chain in human immunocytes containing various immunoglobulin classes role of j chain in secretory immunoglobulin formation dual function of human-iga antibodies -inhibition of phagocytosis in circulating neutrophils and enhancement of responses in il-8-stimulated cells suppression by iga of igg-mediated phagocytosis by human polymorphonuclear leukocytes human serum iga down-regulates the release of inflammatory cytokines (tumornecrosis-factor-alpha, interleukin-6) in human monocytes antiinflammatory properties of human serum iga: induction of il-1 receptor antagonist and fc alpha r (cd89)-mediated down-regulation of tumour necrosis factor-alpha (tnf-alpha) and il-6 in human monocytes the impact of glycosylation on the biological function and structure of human immunoglobulins immunoglobulin heavy chain diversity in pteropid bats: evidence for a diverse and highly specific antigen binding repertoire the two suborders of chiropterans have the canonical heavy-chain immunoglobulin (ig) gene repertoire of eutherian mammals severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats fruit bats as reservoirs of ebola virus antibody mediated immune response in the bat immune response in chiroptera to bacteriophage phix174 studies of arthropod-borne virus infections in chiroptera .4. immune response of big brown bat (eptesicus fuscus) maintained at various environmental temperatures to experimental japanese b encephalitis virus infection establishment, immortalisation and characterisation of pteropid bat cell lines vaccine-induced protection against gastrointestinal bacterial infections in the absence of secretory antibodies complement activation and protein adsorption by carbon nanotubes purification and some properties of streptococcal protein-g, protein-a novel igg binding reagent separation of human iga1 and iga2 using jacalin agarose chromatography isolation and detection of human iga using a streptococcal iga-binding peptide the staphylococcal superantigen-like protein 7 binds iga and complement c5 and inhibits iga-fc alpha ri binding and serum killing of bacteria molecular cloning : a laboratory manual a new adjuvant comparative 2d dige analysis of the depleted serum proteome for biomarker discovery mass spectrometric identification and characterisation of the nucleocapsid protein of menangle virus generation of tioman virus nucleocapsid-like particles in yeast saccharomyces cerevisiae molecular characterisation of toll-like receptors in the black flying fox pteropus alecto a novel iga-like immunoglobulin in the reptile eublepharis macularius discovery of a unique ig heavy-chain isotype (igt) in rainbow trout: implications for a distinctive b cell developmental pathway in teleost fish a third immunoglobulin class in amphibians neonatal fc receptor for igg regulates mucosal immune responses to luminal bacteria beyond iga: the mucosal immunoglobulin alphabet immunoglobulins and immunocytes in the mammary gland and its secretions perspectives on immunoglobulins in colostrum and milk iga deficiency mucosal immunity to influenza without iga: an iga knockout mouse model compensatory levels of salivary igm anti-streptococcus mutans antibodies in iga deficient patients immunoglobulin levels in saliva in individuals with selective iga deficiency -compensatory igm secretion and its correlation with hla and susceptibility to infections purification of igg using protein a or protein g pegasoferae, an unexpected mammalian clade revealed by tracking ancient retroposon insertions the immune gene repertoire of an important viral reservoir, the australian black flying fox the authors thank susanne wilson for the management of the bat colony and rabbits at aahl, and for assistance with sample collection. for serum and plasma donation we acknowledge dr kristen glenister from the australian red cross blood bank, dr hume field from queensland primary industries and fisheries, dr kaku yoshi from the national institute of infectious diseases (tokyo), jennifer barr and meagan gillespie from aahl. we are also grateful to mary tachedjian and dr kim blasdell for critical reading of this manuscript. key: cord-347661-q9lgliph authors: zevenhoven-dobbe, jessika c.; wassenaar, alfred l. m.; van der meer, yvonne; snijder, eric j. title: production of monospecific rabbit antisera recognizing nidovirus proteins date: 2007-11-28 journal: sarsand other coronaviruses doi: 10.1007/978-1-59745-181-9_16 sha: doc_id: 347661 cord_uid: q9lgliph the importance of monospecific antisera for the experimental analysis of viral proteins is undisputed. they make it possible to identify and analyze the target protein against a background of a large number of other proteins, either in whole fixed cells or in cell lysates. this chapter describes our experience with the production of such rabbit antisera directed against proteins of coronaviruses and other nidoviruses. the use as antigens of either synthetic peptides (coupled to a carrier protein) or proteins expressed in escherichia coli is described, and detailed protocols for immunization and preparation of test bleeds are provided. for screening of the immune response following immunization, detailed protocols for three commonly used techniques are described, all of which are based on the use of infected cells or cells expressing the protein of interest, side by side with appropriate controls. the in situ immunodetection of the target in fixed cells by immunofluorescence microscopy is described, as are protocols for techniques that can be applied to cell lysates containing the target protein (western blotting and immunoprecipitation). the latter techniques are performed in combination with polyacrylamide gel electrophoresis, thus allowing confirmation of the molecular weight of the target that is recognized by the antiserum. coronaviruses, and other nidoviruses such as arteriviruses, produce one of the largest sets of viral polypeptide species among the rna viruses. this feature is related to the polycistronic nature of their genome, which contains up to 12 open reading frames and, in particular, to the polyprotein strategy used to produce the large viral replicase/transcriptase, the complex set of nonstructural proteins that is responsible for replication and transcription of the nidovirus genome [see (1) and the references therein]. in coronaviruses (fig. 1) , the larger of the two replicase polyproteins (pp1ab) can be more than 7000 amino acids long and is processed into 15 or 16 cleavage products, now commonly referred to as nonstructural protein (nsp) 1 to 16 (2,3) . in arteriviruses, despite their much smaller replicase size (pp1ab is "only" ∼3100 to 3900 amino acids), the complexity is not very different and replicase processing yields 13 or 14 cleavage products. the regulated autoproteolysis of nidovirus replicase pp1a and pp1ab is driven by two to four proteinases that reside in the orf1a-encoded part of the replicase. cleavage sites for coronavirus and arterivirus proteinases have been identified for several prototypic nidoviruses through a combination of theoretical and experimental methods and can now be confidently predicted for other members of these virus groups. since an additional 4 to 12 proteins are expressed from subgenomic mrnas produced from the 3 -end of the viral genome ( fig. 1) , including the structural polypeptides responsible for virion formation, the proteome of the average nidovirus consists of between 20 and 30 protein species. it should be noted that intermediates of replicase polyprotein processing, which have been described for various nidoviruses, are not taken into consideration in this count and are likely to even further increase the repertoire of viral polypeptides produced in infected cells. replicase precursors and processing intermediates in fact complicate certain types of analyses, such as those that rely on microscopy, since antibodies will usually not discriminate among precursors, intermediates, and processing end products. in the infected cell, many nidovirus proteins are targeted to specific locations (fig. 2) , some of them even to multiple specific locations. over a period of about 15 years, our laboratory has been involved in the production and characterization of polyclonal rabbit antisera directed against specific structural and, in particular, nonstructural proteins of nidoviruses (4-8). our experience in this area is summarized in this chapter. the usefulness and importance of monospecific antibodies for the experimental analysis of almost any protein in biology is undisputed. in particular, when depicted is the cytoplasmic replication cycle, which starts with virus entry and release of the genome into the cytoplasm. subsequently, the genome is translated to produce replicase polyproteins pp1a and pp1ab, which are cleaved to yield 16 nonstructural proteins (2). a complex for viral rna synthesis is assembled on cytoplasmic double-membrane vesicles (dmvs). this complex is involved in genome replication and the synthesis of a nested set of subgenomic mrnas, which are required to express the genes in the 3 -proximal third of the genome. translation of the smallest subgenomic mrna yields the viral nucleocapsid protein (n) that assembles into new nucleocapsid (nc) structures together with newly generated genome rna. other subgenomic mrnas encode viral envelope proteins that are (largely co-translationally) inserted into membranes of the host cell ' the target protein has to be studied against a background of a large number of other proteins, either in whole fixed cells or in cell lysates, specific antibodies can provide a rapid and reliable method for discriminating signal from noise. three commonly used antibody-based detection techniques, which are also important for screening of the immune response during antiserum production, are discussed below: • in situ immunodetection of the target in fixed cells, either by immunofluorescence (if) microscopy or immunoelectron microscopy (iem). immunofluorescence microscopy images of vero-e6 cells infected with sars-coronavirus illustrating the expression of four important viral genes and the different subcellular localization of their products. nonstructural protein 3 (nsp3) is one of the replicase subunits expressed from the genomic rna, revealing the localization of the membrane-bound viral replication complex. spike protein s localizes to different compartments of the exocytic pathway. membrane protein m accumulates in the golgi complex, in particular early in infection. nucleocapsid protein n is a largely cytoplasmic protein. • detection of the target by western blotting (wb), i.e., electrophoresis of a cell lysate containing the target and transfer of the (denatured) proteins to a solid carrier such as nitrocellulose or polyvinylidene fluoride (pvdf) membranes. • immunoprecipitation (ip) of the target from a cell lysate, often used in combination with radioactive labeling of protein synthesis in the cells for a specific time prior to cell lysis. although in several techniques experimental data obtained with monoclonal antibodies can be superior, in particular because of reduced background signal, the generation and screening of hybridoma cell lines requires a considerable investment, in terms of both labor and capital. furthermore, in the context of specific research questions, polyclonal antisera may sometimes even be preferred over monoclonal antibodies since they contain a mixture of immunoglobulin molecules, derived from different b-cell lines in the immunized animal, often recognizing multiple epitopes of the target protein. moreover, if desired, the chances of cross-reactivity of the antiserum with related proteins, e.g., from different strains of the same virus or from closely related other virus species, are considerably better for polyclonal antisera, particularly when they have been raised using a larger and/or conserved part of the target protein. obviously, the primary goal during polyclonal antiserum production in laboratory animals is to obtain a reasonable volume of a high-affinity antiserum. for rabbits, if necessary, bleeds of ∼15-20 ml can be obtained at 4-week intervals, and the final bleed can yield up to 50 ml of serum from about 80 ml of whole blood. since monoclonal antibodies are routinely produced in mice, the use of other species such as rabbits also creates the possibility of convenient in situ double-labeling experiments (see below). in such experiments, using speciesspecific secondary antibodies, a polyclonal rabbit antiserum against the target of choice and, for instance, a mouse monoclonal antibody recognizing a cellular protein can be used in combination to detect both proteins in the same specimen simultaneously. 7 the (obvious) standard prerequisites for the production of a polyclonal rabbit antiserum are: (i) an antigen, (ii) a (pathogen-free) rabbit to be immunized, (iii) a test method to detect the response, and (iv) a permit for animal experiments. the last relates to institutional and/or governmental regulations controlling animal use, which differ from country to country and are not discussed here in detail. important considerations are the choice of adjuvant, method and site of administration of the antigen, sedation of animals, maximum volume of test bleeds, and various safety precautions regarding animals and personnel. two types of antigens have been used in our studies: synthetic peptides and proteins expressed in and purified from escherichia coli. the production of antigens in e. coli are not covered in any detail in this chapter and the reader is referred to the extensive literature on this topic published elsewhere (and in chapter 13 in this volume). e. coli allows for the cheap production of large amounts of antigen, but clearly such antigens have to be purified prior to use for immunization. this can be facilitated, e.g., by the use of a variety of tags (such as fusion to glutathione-s-transferase, maltose-binding protein, or the commonly used hexahistidine tag) for which convenient affinity resins are available. reasonably pure antigens (>80% pure) are required and in the case of larger tags one should consider removing the tag proteolytically to ensure that the immune response will be directed against the target rather than against its tag. if the e. coli expression product turns out to be insoluble, which will usually interfere with its straightforward affinity purification, it may still be possible to purify a reasonably pure protein sample from inclusion bodies by repeated extraction of this material with increasing concentrations of urea (or guanidium isothiocyanate). as long as small amounts of this material can be used (i.e., the protein concentration is sufficiently high), after washing of the protein pellets with pbs, such urea-containing samples can be used for mixing with the adjuvant and immunization without the need for dialysis. synthetic peptides offer the advantage of being pure, and with certainty they contain exclusively the amino acid sequence of the selected target. since they normally cover only 10 to 25 residues of this target, their sequence has to be selected with care (see below). it is not straightforward to confidently predict antigenic peptides from an amino acid sequence. several programs to support this activity can be found on the internet and algorithms for this purpose are included in most dna/protein analysis software (9-11). if the structure of the target is known, peptides located on its surface are to be preferred. entirely polar and helical peptides should be avoided. in our experience, peptides located at (or close to) the n-or cterminus of the target also have a high probability of being immunogenic. this was true in particular for peptides derived from the nidovirus replicase polyproteins, which were usually selected on the basis of known or predicted cleavage sites (although based on relatively small numbers; success rate with terminal peptides was around 75% and with internal peptides only around 25%). the peptides to be synthesized usually are 10-25 amino acids long. peptides that are very hydrophobic may be more difficult to handle (e.g., poor solubility prior to coupling). to protect peptides against host proteases and thus increase their stability, the c-terminal carboxyl group can be replaced with an amide group during synthesis. (this may not be advisable when using peptides that normally form the c-terminus of a protein.) to facilitate coupling to bsa used as the carrier protein (see below), one or two lysine residues can be added to one side of the peptide (to the n-terminus when targeting the c-terminus of a protein, and vice versa). synthetic peptides are available from a variety of commercial or in-house sources. in our institute, peptides are synthesized by solid-phase strategies on an automated multiple peptide synthesizer (syroii, multisyntech, witten, germany). the purity of the peptides is determined by analytical reversed-phase hplc and should be at least 80%. the identity and homogeneity of the peptides is confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and analytical reversed-phase chromatography. freeze-dried peptides, dissolved peptides, or coupled peptides in solution are best stored at -85 • c. synthetic peptides have to be coupled to a carrier protein to enhance their immunogenicity. bovine serum albumin (bsa) and keyhole limpet hemocyanin (klh) are the two most commonly used carrier proteins. we have always relied on bsa (a very soluble and stable plasma protein) and a coupling reaction using glutaraldehyde, which links the amino groups of carrier and synthetic peptide (in lysine residues and at the amino terminus of the peptide). for example, crosslinking at lysine residues can be represented as: note that the above calculations are based on a number of assumptions and should be considered as a rule of thumb. clearly, since the sequence is known, the mr of the peptide can be calculated more precisely. however, since we usually add extra lysines to the peptide there will be different ways in which it can be coupled to the carrier and a variety of complexes will be formed (bsa "trees" with a varying number of peptide "branches" scattered all over the backbone), presenting the epitope in many different ways. 3 . to the peptide-bsa mixture, add pbs to give a final volume of 670 l; then (carefully, slowly, and while mixing) add 330 l of a freshly prepared 0.3% glutaraldehyde solution (equaling approximately 10 mol). 4. incubate the coupling reaction on a roller device at room temperature for 1 h to allow cross-linking of peptide and carrier protein. 5. add 200 l of 1-m glycine in order to quench the remaining free glutaraldehyde and continue rolling for 1 h. 6. dialyze the coupling reaction overnight against pbs to remove small molecules such as uncoupled peptide, glycine, and glycine-glutaraldehyde. dialyze two or three times against at least 500 volumes of pbs at 4 • c. the final step can be overnight. usually, after dialysis, the concentration of peptide-bsa complex is around 4 mg/ml and the solution is ready for direct use as an antigen in immunization. when using synthetic peptides or proteins purified from e. coli, adjuvants containing immunostimulatory molecules are applied to enhance the immune response to the antigen. freund's adjuvants have been used in our studies; freund's complete adjuvant (fca) for the initial immunization and freund's incomplete adjuvant (fica) for subsequent booster immunizations. fca (but not fica) contains heat-inactivated mycobacterium tuberculosis or mycobacterium butyricum (or extracts thereof), which stimulate both cellular and humoral immunity. the water-in-oil emulsion that is the basis for fca guarantees the slow release of the antigen from the site of immunization, but it should be noted that the mineral oil component is quite toxic and induces granulomatous reactions. mix fca/fica with the antigen-containing solution to form a "toothpaste-like" stable emulsion. see below for details. in our standard protocol, ∼200 g of antigen (peptide-bsa complex) is used for the primary immunization, and the same amount for subsequent booster reactions. 1 . before use, mix the fca, e.g., gently on a roller device to get the bacteria into suspension. for the primary immunization, the antigen (200-250 g) is diluted with pbs to give a final volume of 0.8 ml pbs, which is mixed with 0.4 ml of fca. transfer this mixture to a 3-ml syringe (luer-lock). use a p1000 micropipette; put the tip in the opening of the syringe, and slowly pull the plunger back. in the same manner, fill a second syringe with 0.4 ml of fca and 0.4 ml of air (to promote the formation of the emulsion). 2. connect the two syringes with a three-way stopcock (fig. 3) . mix the contents of the two syringes until a thick, white emulsion is obtained. this means that the suspension is converted into an oily mass that includes the water. this emulsion, when properly prepared, is stable and will not disperse when a droplet is put into pbs. the emulsion will ensure the slow release of the antigen into the animal. 3. disconnect the syringes and use the antigen suspension the same day. optionally, the syringes can be stored for later use, overnight at 4 • c or for longer periods at -80 • c. 4. for booster immunizations fica is used instead of fca. material for two boosts can be prepared in one action by doubling the volumes above. filled syringes can be stored at -80 • c for prolonged periods of time. young adult new zealand white rabbits (preferably females, weighing between 2 and 3 kg) have been used routinely in our studies (fig. 3) . the young age of the animals is thought to be particularly important to ensure a strong primary response to the antigen. although it is often advised to use two animals per antigen, in our experience-with just a few exceptions-both animals generally give the same (positive or negative) result, although titers of the antiserum and background signal(s) may vary between such duplicates. still, if the number of animals available or housing capacity is limited, and also for financial and ethical reasons, it may be wiser to use a single animal per antigen and-in the event of failure-return to such an antigen in a subsequent round of immunizations. the rabbits are injected subcutaneously at three or four places on their back (fig. 3) . given the strong inflammatory response induced by fca, the animals are monitored closely following the primary immunization. the interval between the primary immunization and the first booster, and between subsequent boosters, is about 4 weeks. it is essential to obtain a preimmunization bleed on (or before) the day of the primary immunization, which will be used to assess the response against the antigen (see below). the standard schedule for immunization and bleeding would look like this: bleeds should be limited to 15% of the animal's total blood volume and a 4-week recovery period should be allowed. as a "rule of thumb," the blood volume of rabbits is about 56 ml/kg of body weight-assuming the animal is mature, healthy, and adequately nursed. in our experience, a (good) response is unlikely (but not impossible. . .) if there is no positive signal after two or three boosts. although there have been some exceptions to this rule, it is advisable-for financial reasons as well as ethical considerations-to terminate the experiment after ∼70 days and, if necessary, try again with an additional animal or an alternative antigen later on. 1. collect 15-20 ml of blood (usually from an ear vein of the rabbit; fig. 3 ) in a 50-ml centrifuge tube and allow complete clotting of the blood in a water bath at 37 • c for 1 h. 2. use a pasteur pipette with a melted tip to gently liberate the clot from the wall of the tube. be sure to cause minimal lysis of red blood cells, to ensure a clear serum sample later on. subsequently, leave the clot to shrink overnight at 4 • c (fig. 3) . 3. centrifuge the tube at 1300 × g for 10 min to further reduce the volume of the clot. 4. carefully, with a pipette, remove as much serum as possible from the tube without touching/damaging the clot; in case of doubt, use a second collection tube for the last few ml (also an additional spin might help), which may be less clear and might be saved as a backup sample only. 5. prepare some smaller and some larger aliquots of the serum and store at -20 • c or -80 • c. sera can be kept for many years, but repeated freezing and thawing should be avoided. also, it is advisable to use tubes with a screw cap lid containing a rubber seal, which will minimize evaporation during prolonged storage. store a small sample (0.5 to 1 ml) at 4 • c for testing. 6. working stocks of antisera (in screw cap tubes) can be kept at 4 • c for many months; if desirable, 0.05% (final concentration) of sodium azide (highly toxic!) can be added as a preservative, but in our experience this is not necessary if sera are kept cool and handled with care. it is advisable to spin antisera (in particular the less clear ones) for 1 min at full speed in a microcentrifuge prior to use. the native proteins that are the targets for antiserum production are usually highly structured molecules and obviously the reactivity of the antiserum will be influenced by the conformation of the antigen used for immunization. in particular, when synthetic peptides are used the structural resemblance between antigen and target may be limited. this probably explains why-in our experiencepeptides derived from the termini of the target protein, which are often less structured than the protein core, generally work better than internal peptides. although an elisa approach, based on the antigen used for immunization, can be employed for an initial screening of the immune response in the animal, this result may be relatively meaningless when it comes to the question of whether the antiserum will ultimately react with the viral protein target. one will in fact be measuring the response against the combination of peptide and carrier protein or, in the case of immunization with proteins expressed in and purified from e. coli, against the target and contaminants present in the antigen. all the immunizations we carried out with bsa-coupled peptides produced sera that reacted positively in an elisa using plates coated with the uncoupled peptide, but less than half of those turned out to be useful in if, wb, or ip. therefore, it is advisable to screen immediately using samples containing the native, full-length viral protein target. moreover, during this screening process, the conformation of the target is an issue. when screening formaldehyde-fixed whole cells by microscopy techniques the target is fixed but structured. during wb analysis proteins are subjected to denaturing conditions and fixed to the membrane used for blotting, whereas ips are done in solution and can be performed under both native and denaturing conditions. in fact, the level of denaturation in ip experiments can be easily influenced by varying the percentage of sds in the ip buffer. often, ips with antisera raised using synthetic peptides work better in the presence of relatively high (0.25-0.5%) concentrations of sds, probably because the epitope in the denatured target resembles the antigen used for immunization more closely under these conditions. an additional significant advantage is the fact that the higher sds concentrations will strongly reduce the background ip signal and result in a much cleaner analysis (see below). in our experience, antisera that work well in if assays usually also work well in wb and ip. conversely, sera that are negative in if may still be highly reactive in wb and/or denaturing ip. consequently, it is important to test new antisera in at least two of these three assays. in our daily practice, we have usually relied on if for preliminary screening and wb for confirmation. there is an additional reason to confirm the if results by subsequent wb or ip analysis: in if assays antisera can sometimes show a strong reaction with cellular proteins (fig. 4) , either-at low dilutions-because of the lack of a specific response against the target or-at higher dilutions-because of an unexpected cross-reactivity cov nsp3 (4) , which was very specific in vero-e6 cells (see fig. 5 ), resulting in a strong, punctate nuclear labeling in bhk-21 cells that expressed the ace-2 receptor for sars-cov. (d) sars-cov-infected cells labeled several weeks after fixation and embedded using prolong mounting fluid. an aspecific nucleolar background labeling was observed, especially in the green range of the fluorescence spectrum. the specific, much brighter signal is derived from an anti-nsp3 labeling using a rabbit antiserum that was directly coupled to alexa fluor-488 (4). with cellular proteins. wb or ip analysis provides important information about the molecular mass of the target that is recognized, and may thus prevent premature conclusions about the specificity of the antiserum. the correct assessment of specificity is also aided by the inclusion of two important controls: the preimmunization serum (which should obviously not show the same signal) and mock-infected control cells (which might reveal that it is in fact a cellular target that is being recognized). when performing initial screening with if assays, it is practical to use cell cultures that have been infected at an moi of 0.5-1 and therefore contain a mixture of virus-infected and mock-infected cells in the same specimen. semiconfluent cells seeded on glass cover slips (10-mm-diameter) are the preferred specimens for initial testing. the cells can either be infected with the target virus or transfected with a vector expressing the target protein. obviously, cells should be fixed at a time point when the target protein is convincingly expressed. the most reactive rabbit antisera that we have produced can be used in if assays at dilutions of between 1:1000 and 1:5000. for initial testing, however, dilutions on the order of 1:100 to 1:500 are advised. 1 . wash the cells once with pbs and fix the cells at room temperature with 3% paraformaldehyde in pbs for at least 30 min (or overnight). 2. wash the cells once with pbs containing 10 mm glycine (coverslips in sealed dishes can be stored at 4 • c in pbs for many weeks). 3. using sharp tweezers, carefully transfer coverslips to the wells of a prelabeled 24well cluster containing pbs-glycine. be sure to remember, every time you handle the coverslips, on which side there are cells. while in the cluster, cells should always be facing upward. 4. permeabilize the cells at room temperature for 10 min in pbs containing 0.1% triton x-100. 5. in 5 min, wash the cells three times with pbs-glycine and leave them in the last wash step. 6. dilute the primary antiserum (initial dilutions, e.g., 1:100 and 1:500) in pbs containing 5% fcs; 50 l per coverslip (10-mm-diameter) will be needed. 7. cut a large piece of parafilm, place it in a larger dish, label the position of the various samples, and place 50-l drops of the antiserum dilutions on the parafilm. one by one, take the coverslips from the 24-well cluster, remove excess pbs by touching a tissue to the side of the coverslips, and place the coverslips on the drops with the cells facing the antiserum. 8. incubate for 30-60 min at room temperature (or 37 • c); make sure the samples do not dry out during this incubation (e.g., by placing a wet tissue in the dish). 9. return the coverslips to the 24-well cluster; in 20 min wash three times with pbsglycine and leave them in the last wash step. 10. prepare a dilution of the fluorescently labeled secondary antibody. optimal dilutions of conjugates should be determined separately using a well-defined primary antiserum. again 50 l per coverslip will be needed. optionally, 1 g/ml of hoechst 33258 can be added to this dilution for staining of nuclear dna. 11. incubate and wash as described under steps 7 to 9. 12. take the coverslips from the 24-well cluster. embed the specimens onto glass microscopy slides in a mounting fluid [e.g., mowiol or prolong (molecular probes)]. avoid air bubbles in the mounting fluid by slowly and carefully sliding the coverslip on a small drop (∼5 l) of mounting fluid. 13. store the specimens in the dark at 4 • c. the mounting fluid should harden at least overnight before high magnification lenses and immersion oil are used. 14. analyze the specimens in a fluorescence microscope using the filter sets required for the label attached to the secondary antibody. double-labeling experiments can be done to compare the localization of two (or more) proteins of interest in the same cell by combining a rabbit antiserum recognizing one protein and, e.g., a mouse monoclonal antibody recognizing a second target. obviously, the two primary antisera have to be detected with suitable conjugates carrying different fluorescent labels, preferably with wellseparated emission spectra. following initial optimization (testing of different dilutions, balancing of the two signals, and controls for specificity of primary and secondary antibodies), one can usually carry out the labeling using a twostep approach. first, specimens are simultaneously incubated in a combined dilution of the two primary antibodies and, subsequently, after extensive washing, in a combined dilution of the two conjugates. in case of background and/or specificity problems, however, it may be necessary to perform the labeling in four consecutive steps. in if assays, double labeling with two antisera from the same species is only possible when one of the two is directly coupled to a fluorescent group. we have recently been successful (fig. 5) (4) in purifying the ig fraction from small volumes (2-3 ml) of rabbit antisera using a commercially available protein a column and have subsequently conjugated these antibodies to alexa fluor-488. the if assay then consisted of three incubation steps: (i) incubation with the uncoupled rabbit antiserum, (ii) incubation with the anti-rabbit conjugate recognizing the first antibody, and (iii) incubation with the alexa fluor-488-coupled second rabbit antiserum. the order of these steps is very important to avoid binding of the anti-rabbit conjugate to the directly labeled second rabbit antiserum. during western blotting (wb) analysis samples containing the protein of interest are separated according to size in acrylamide gels and transferred to a membrane, which is subsequently incubated with the antiserum. protein bands reacting with the antiserum are detected using a secondary antibody and accompanying assay (a variety of enzyme-linked or fluorescent conjugates is available; here we use a peroxidase-coupled secondary antibody). as in the case of if assays, lysates to be tested can be derived from cells that are either infected or transfected with an appropriate expression vector. for infection lysates, we use a high multiplicity of infection to avoid a mixture of infected and uninfected cells in the sample. cells are lysed in protein lysis buffer, which leave the nuclei intact and allow their removal by centrifugation. for initial testing, an amount of lysate equaling ∼5 × 10 5 cells per 5 cm gel width (or 10-15 slots) can be used. alternatively, purified protein (e.g., expressed in e. coli) can be used (100-500 ng is fig. 6 . example of a standard test of a rabbit antiserum in western blot and immunoprecipitation. the antiserum used here was raised against a domain in the c-terminal region of pp1a of the arterivirus eav (8) and recognizes a large set of processing intermediates and end products, which are indicated by arrowheads: (a) western blot analysis: eav-and mock-infected cell lysates were prepared at 8 h postinfection, run on an sds-polyacrylamide gel, blotted to pvdf membrane, and incubated with the postimmune serum at a 1:1000 dilution. detection was with an anti-rabbit igg hrpo conjugate and a chemiluminescence assay. [reproduced from (13).] (b) immunoprecipitation: eav-infected cells were labeled with 35 s-methionine/cysteine for 3 h, from 5-8 h postinfection and lysates were used for ip using 5 l of antiserum per sample. following the ip, samples were run on an sds-polyacrylamide gel and signal was detected using autoradiography. as specificity controls, the preimmune serum was tested on eav-infected cells and the postimmune serum was tested on mock-infected cells. furthermore, for the left panel the binding of the antiserum to the antigen was done in the presence of 0.1% sds, whereas 0.5% sds was used for the right panel. the comparison illustrates how higher sds percentages can reduce the background signal and improve the overall picture. usually sufficient). most antisera can be used at a 1000-fold dilution or higher, but we advise starting the testing with a 1:500 dilution (fig. 6a) . protein-antibody complexes are then purified by having them bind to beads carrying the ig-binding proteins a or g on their surface, which are subsequently spun down and washed repeatedly to remove unbound proteins. the simplest form of protein a carrying beads are formalin-fixed staphylococcus aureus cells (e.g., pansorbin; calbiochem), but alternatively protein a or g coupled to sepharose can be used. usually, metabolically labeled protein lysates (e.g., 35 s-methionine-labeled) are used for immunoprecipitation studies and samples are run on sds-page gels that can then be used for autoradiography. in contrast to wb analysis, immunoprecipitation relies on recognition of the target in solution. depending on the conditions used, proteins can either be close to their native confirmation, partially denatured, or completely denatured (e.g., in the presence of high concentrations of sds). thus, the conformation of the antigen used for immunization may influence the results. in our experience, linear antigens such as synthetic peptides may yield antisera that work considerably better in immunoprecipitation assays carried out under stringent denaturing conditions (e.g., 0.5-1% sds), which will often also reduce the background in the assay (fig. 6b) . nevertheless, the optimal concentration of sds should be determined for every antigen-antiserum pair since the optimum may be as low as 0.1% sds, e.g., when using antibodies recognizing mainly (or exclusively) conformational epitopes. furthermore, the amount of antiserum in the immunoprecipitation assay is important. for initial screening of rabbit antisera we usually test 1 and 5 l of the antiserum in a 300 l total ip volume. coverslips with infected cells or cells expressing the protein of interest. 2. fixative: 3% paraformaldehyde in pbs pbs: see section 2.1, step2 pbs with 5% fetal calf serum (fcs) pbs with 10 mm glycine protein lysis buffer (20 mm tris-hcl amersham biosciences, hybond p, #rpn303f) 10x western blot transfer buffer (wtb): 250 mm tris, 1.92 m glycine pbs: see section 2.1, step 2 pbs-tm with 1% bsa) anti-rabbit igg horseradish peroxidase conjugate, e.g., swine-anti-rabbit igg hrpo amersham biosciences, ecl plus western blotting detection kit ip buffer (20 mm tris-hcl weak wash buffer a (20 mm tris-hcl ph 7.6; 150 mm nacl laemmli sample buffer (lsb): 50 mm tris-hcl, ph 6.8; 100 mm dtt; 10% glycerol formalin-fixed staphylococcus aureus cells; calbiochem #507858) or protein a/g sepharose beads wash the cells with cold pbs and-to a 10-cm 2 dish with 1-2 × 10 6 cells-add 300 l of cold protein lysis buffer transfer the lysate to a labeled microfuge tube and spin down the nuclei for 2 min at full speed in a microcentrifuge transfer the supernatant to the new tube, leave the pellet (nuclei, often barely visible) behind and add 1/100 the lysate can now be used for wb or stored in the -20 • c/-80 • c freezer prepare an sds-page gel (or minigel) of a suitable acrylamide percentage (depending on the size of the protein of interest) and run: (i) the lysate containing the protein of interest, (ii) a control lysate (mock-infected or untransfected cells), and (iii) a molecular weight marker. several sets of these samples can be run on one gel to try different antiserum dilutions, or wider slots can be used and strips cut with the right samples after blotting with a pencil mark one side of the membrane; this is the side of the membrane that will face the gel during blotting and, subsequently, the solutions during incubation. prewet the pvdf membrane in methanol for 5 min; never touch the pvdf membrane with bare fingers; always use gloves! dilute 10x wtb to give 1x wtb and incubate the membrane in 1x wtb for 10 min take the gel from between the glass plates and briefly wash it in 1x wtb build the electroblot stack-from cathode to anode-with the following layers: three sheets of whatman paper presoaked in wtb (and optionally one sheet of whatman paper soaked in wtb with 0.1% sds), the equilibrated gel, the equilibrated and marked membrane :500) and the preimmune serum as a control (1:500) in pbs-tmb and incubate the blots for 1 h at room temperature while swirling dilute the peroxidase-conjugated secondary antibody (swine-anti-rabbit igg hrpo) in pbs-tmb and incubate the blots for 1 h at room temperature while swirling prepare the solutions for the chemiluminescence assay according to the manufacturer's instructions gently "semidry" the blot with some whatman paper (let the fluid run off; do not really dry it!) put the chemiluminescence solution on the plastic foil and incubate the blot with the "protein side down" for 5 min remove the excess of fluid with some whatman paper and wrap the blot in plastic foil expose an x-ray film to the blot for 1-2 min, develop the film, and check if a longer exposure is required. to avoid overexposure of the film, it may be wise to wait about 30 min before making the first exposure take 50 l of 35 s-labeled cell lysate in protein lysis buffer (12) (equaling about 2-4 × 10 5 cells; obviously the expression level of the target protein is to be considered as well); add 250 l of ip buffer and the antiserum (1 or 5 l). incubate these ∼300-l samples overnight wash the pansorbin cells (25 l for each sample) by mixing with an equal volume of ip buffer, spin down for 20 sec at full speed in a microcentrifuge, and resuspend the pansorbin cell pellet in the original volume (again add 25 l of washed pansorbin cells to each immunoprecipitation and incubate at 4 • c for more than 1 h while swirling spin down for 1 min at full speed in a microcentrifuge, remove the supernatant, and add 500 l of weak wash buffer a; vortex until the pellet is completely resuspended. (optionally, this step can be repeated once to get a cleaner result spin down for 1 min at full speed in a microcentrifuge again and repeat the same washing procedure with weak wash buffer b resuspend the pellet in 30 l of lsb by pipetting up and down before loading the samples on an sds-page gel, denature at 96 • c for 3 min. (for some proteins, e.g., very hydrophobic ones, it may be better not to heat the samples and just leave them in lsb for 15 min or longer spin the pansorbin cells down for 2 min at full speed in a microcentrifuge; do not resuspend the pellet! transfer the supernatant to a new tube and load part of this sample onto an sds-page gel following sds-page, process the gel for autoradiography or phosphorimager analysis according to standard protocols and expose for 1-7 days not all bleeds of the same rabbit have the same concentration of antibody and thus the dilutions to be used can vary mounting solutions can sometimes have strange side effects, especially when the cells are not freshly fixed. for example, the use of prolong gold mounting fluid can generate a green nonspecific nucleolar signal when microscopy slides used for embedding are not clean, wipe them first with a tissue with 70% ethanol, next with water, and then with a dry paper towel/tissue. when ethanol is not removed, a (sometimes bright) orange background may result occasionally, antisera judged to be negative in if using paraformaldehyde-fixed cells can become positive using methanol-fixed cells (or the other way round) however, methanol fixation is less gentle and generally the morphology of the cell is less well preserved. fixation is performed for 10 min at -20 • c (using ice-cold 100% methanol or 95% methanol/5% acetic acid) and cells are subsequently transferred to and kept in pbs-glycine. methanol immediately dissolves all cellular membranes topley and wilson's microbiology and microbial infections: virology volume virus-encoded proteinases and proteolytic processing in the nidovirales unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex nuclear localization of nonstructural protein 1 and nucleocapsid protein of equine arteritis virus the putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral rna synthesis processing of the equine arteritis virus replicase orf1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains proteolytic processing of the replicase orf1a protein of equine arteritis virus prediction of protein antigenic determinants from amino-acid-sequences the antigenic index-a novel algorithm for predicting antigenic determinants semiempirical method for prediction of antigenic determinants on protein antigens structural proteins of equine arteritis virus proteolytic maturation of replicase polyprotein pp1a by the nsp4 main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp7 the authors would like to thank jan wouter drijfhout and willemien benckhuijsen (lumc department of immunohaematology) for advice and assistance on peptide design and synthesis. we are grateful to the staff of the lumc animal facility for almost 15 years of pleasant collaboration and reliable housing and handling of the rabbits used for antiserum production. this work was supported (in part) by the european commission int the context of the activities of the euro-asian sars-dtv network (sp22-ct-2004-511064). key: cord-258624-041cf99j authors: ahmad, sajjad; navid, afifa; farid, rabia; abbas, ghulam; ahmad, faisal; zaman, naila; parvaiz, nousheen; azam, syed sikander title: design of a novel multi epitope-based vaccine for pandemic coronavirus disease (covid-19) by vaccinomics and probable prevention strategy against avenging zoonotics date: 2020-05-23 journal: eur j pharm sci doi: 10.1016/j.ejps.2020.105387 sha: doc_id: 258624 cord_uid: 041cf99j the emergence and rapid expansion of the coronavirus disease (covid-19) require the development of effective countermeasures especially a vaccine to provide active acquired immunity against the virus. this study presented a comprehensive vaccinomics approach applied to the complete protein data published so far in the national center for biotechnological information (ncbi) coronavirus data hub. we identified non-structural protein 8 (nsp8), 3c-like proteinase, and spike glycoprotein as potential targets for immune responses to covid-19. epitopes prediction illustrated both b-cell and t-cell epitopes associated with the mentioned proteins. the shared b and t-cell epitopes: drdaamqrk and qarsedkra of nsp8, edmlnpnyedl and eftpfdvvr of 3c-like proteinase, and vnnsyecdipi of the spike glycoprotein are regions of high potential interest and have a high likelihood of being recognized by the human immune system. the vaccine construct of the epitopes shows stimulation of robust primary immune responses and high level of interferon gamma. also, the construct has the best conformation with respect to the tested innate immune receptors involving vigorous molecular mechanics and solvation energy. designing of vaccination strategies that target immune response focusing on these conserved epitopes could generate immunity that not only provide cross protection across betacoronaviruses but additionally resistant to virus evolution. a recent outbreak of pneumonia in wuhan, china, is associated with betacoronavirus of group 2b from family coronaviridae and the order nidovirales [1] [2] . the viruses are positive-sense rna, enveloped and non-segmented [2] . this coronavirus disease (covid-19) is known as a third human zoonosis of the 21 st century and is caused by a new strain not previously identified in humans [1] . the coronaviruses causing minor infections of the respiratory tract in humans are nl63, oc43, hcov-229e, and hku1 while, the lethal coronavirus infections that emerged in this century are the middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov) and the recent sars-cov 2 or covid-19 [2] . the source of the covid-19 is still not confirmed but some evidence suggests that the source may be in the seafood market of huanan in wuhan, china [3] [4] . the center for disease control and prevention (cdc) reported that the recent covid-19 is caused by betacoronavirus just like the previous two outbreaks of coronaviruses; mers and sars, the source of which is camels and bats, respectively [5] . the first transmission of cov from animals to humans was notified in 2002 causing sars-cov with a 10% mortality rate [6] . it was suggested that the virus needs some intermediate reservoir to infect humans efficiently as confirmed later by a thorough investigation revealing palm civets and raccoon dogs of the wet market carried sars-cov viral rna and might act like intermediate reservoirs [3] . the covid-19 rna virus carries a high mutation rate and ability to transfer from person to person as compared to other coronaviruses. according to the world health organization (who), till 23rd february 2020, the covid-19 affected a total of 78,811 individuals across globally of which, 77,042 were reported in china while 1769 were reported in other countries. the death toll in china is 2445 and 17 deaths have been reported in the rest of the world. according to the reports till 2nd january 2020, 41 patients have been admitted to the hospital of which most of the patients were men and 66% of them had exposure to the huanan seafood market and the median age of patients was 49 years [2] . the health-care workers are also diagnosed with the infection including those working in similar wards [6] . the time taken by covid-19 to infect other individuals is similar to that of sars. it is estimated that on average each infected person infects 2-3 persons and this occurrence increases two-folds, every 6.4 days [7] . it was observed that people with mild infection are more actively spread the infection [8] . due to critical nature of the outbreak, the virus was sequenced on urgent basis and the first sequence was available on 10 th january, 2020 online at virological.org. it was noted that covid-19 has much resemblance to sars-cov at the genomic level [4] . symptoms of covid-19 include fever, dry cough, shortness of breath and dyspnea, sore throat and leukopenia. to date no vaccine covid-19 is available and is need of an hour to develop a vaccine to prevent further spread of the disease. to this end, immuno-informatics can be applied to a complete protein data set of the virus for deep antigen analysis and thus can save time and cost for designing a vaccine against covid-19. this will ease the early development of a vaccine and proposed design can be subjected immediately to experimental trials. the stepwise flow of the methodology followed to design a vaccine against covid-19 is illustrated in fig.1. fig.1 . designed workflow for in silico vaccine engineering against covid-19. the complete dataset of proteins available in (ncbi) [9] coronavirus data hub (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/virus?seqtype_s=nucleotide&viruslineage_ss =wuhan%20seafood%20market%20pneumonia%20virus,%20taxid:2697049&utm_campaign=w uhan_ncov&utm_source=insights&utm_medium=referral) was retrieved and subjected to screening phase to identify potential vaccine candidates. first, host non-similar proteins of the pathogen were filtered that show no homology to the human host (taxonomic id: 9606). proteins having sequence e score < 1.0 e -5 , bit score > 100, and sequence identity ≤ 30 % were selected [10] . again, a blastp search against the mouse (mus musculus, taxonomy id: 10090) was performed using host non-similar proteins keeping the input parameters e score cut-off 0.005, bit score > 100, and identity < 30% [11] . the screened mouse non-similar proteins were then subjected to tmhmm 2.0 [12] and hmmtop 2.0 [13] for observing the number of transmembrane helices. proteins having less than two transmembrane helices were subjected to spaan [14] for predicting the adhesive proteins as they have the potential to facilitate attachment to the host tissues [15] . selected vaccine candidates were then used in blastp tool to align the selected adhesive protein candidates with the probiotic bacteria proteome including three lactobacillus species: l. rhamnosus (tax id: 47715), l. johnsonii (tax id: 33959), and lactobacillus casei (tax id: 1582) to avoid accidental inhibition of the useful gut bacteria [11] . selected vaccine candidates were then subjected to the immune epitope database (iedb) bepipred linear epitope prediction 2.0 [16] [17] [18] . the threshold of 0.5 was used for the prediction of linear b-cells epitopes which were then utilized in t-cell epitopes mapping to identify subsequences with the potential to bind reference set of major histocompatibility complex (mhc) class i and ii alleles [16] . epitopes were ranked according to their percentile score, the ones with the low percentile were considered as high affinity binders. the selected b-cell derived t-cell epitopes were then subjected to mhcpred 2.0 [19] analysis for interpreting their binding affinity potential. the cut-off criterion was set to ic 50 values < 100 nm for drb*0101 [20] . following this, the virulentpred [21] and vaxijen 2.0 [22] were utilized to validate the virulence and antigenicity of the selected epitopes, respectively. allertop 2.0 [23] was applied to remove the allergic epitopes. clc main workbench was employed to check the conservation of non-allergic epitopes required for designing an effective broad-spectrum vaccine. overlapping immunodominant epitopes were used to construct a multi-epitope peptide (mep) which is considered a promising strategy to stop viral infections [24] . one of the key issues with the design of peptide vaccine is its weak immunogenicity that can be resolved by designing a mep with appropriate adjuvants [25] . in the current study, mep was designed using aay linkers to combine screened multiple epitopes [26] . to the n-terminal of mep, an adjuvant in the form of b subunit of cholera toxin was linked to the mep thus creating a multi-epitope peptide vaccine construct (mepvc) [27] . the tertiary structure of the mepvc was created through a software called 3dpro of scratch protein predictor [28] , i-tasser [29] , and swiss-model [30] . the best model was further loop modelled using galaxyloop [31] and refined using galaxyrefine [32] of galaxyweb. to improve the construct's stability, disulfide bonds were introduced in the structure [33] using design 2.0 [34] . the sequence of the mepvc was translated in reverse and then optimized for codon usage according to the escherichia coli, which will end up in the increased expression of the mepvc sequence cloned in the mentioned expression system [35] . the entire activity was accomplished using java codon adaptation tool (jcat) server [36] . in order to assess the expression of sequence that have been cloned, the gc content and codon adaptation index (cai) were measured. the value of 1 cai is contemplated ideal [37, 38] whereas the appropriate gc content should be fluctuated between 30-70% due to favorable transcriptional and translational efficiencies [27] . there were other input factors carefully calculated to prevent rho-independent transcription termination, the binding sites of the prokaryotic ribosome, and the cleavage sites of restriction enzyme. as the final step of this phase, the cloning of the engineered construct was carried out into pet-28a (+) expression vector using snapgene (https://www.snapgene.com/). protparam tool [39] was applied for analyzing the physical and chemical properties of mepvc such as amino acid composition, estimated half-life, instability index, extinction coefficient, theoretical pl, atomic composition, molecular weight, and grand average of hydropathicity (gravy) to assist experimental studies. instability index is one of the key parameters that is significantly considered as it helps in discarding the unstable protein candidates (protein instability index > 40). in this step, the vaccine construct underwent immune response profiling and immunogenicity classification, which was done using the c-immsim server [40] . in order to predict the immune epitopes, a position-specific scoring matrix (pssm) employed by c-immsim was used. whereas, the different machine learning procedures were used to forecast the immune connections. this server is concurrently used to execute an immune simulation for 3 compartments such as bone marrow, tertiary lymph nodes, and thymus [41] . default simulation parameters were used which are as follows: random seed (12345), simulation steps (100), simulation volume (10), host hla selection (a mhc class i a0101 allele, b mhc class i b0702, dr mhc class ii drb1_0101 allele), and time step of injection was set to 1. the technique of molecular docking was utilized to predict conformation of the mepvc with respect to a suitable innate immune receptor. this analysis plays a significant role to determine the high-affinity contacts amid the vaccine construct and the immune receptor. the pdb id: 4g8a and tlr3 pdb id: 2a0z were retrieved from the protein data bank (pdb) for the tlr4 and tlr3, respectively. blind docking employed to calculate the regular pose of the vaccine construct with the mentioned receptors using an online patchdock server [42] . the resulting structures were further refined using the fast interaction refinement in molecular docking (firedock) [43] . the top ranked complex with the minimum global energy was selected and used to analyse the binding pose and intermolecular connections using ucsf chimera 1.13.1 [44] , discovery studio (ds) visualizer 17.2.0 [45] .16349, and visual molecular dynamics (vmd) 1.9.3 software [46] . in order to gain insights into the dynamics of the vaccine construct with the receptors, molecular dynamics (md) simulations have been carried out. the simulation analysis was also important to validate the exposure of epitopes towards the host structure for identification and handling of a substantial outcome. the md simulations took place in three different phases: system preparation, pre-processing and production [47] with an assistant model building with energy refinement (amber) 16 [48] . the antechamber program [49] was used to build the libraries and parameters for the tlr4 and vaccine construct. the tip3p solvation box (size 12 å) was inserted to solvate the construct. to study the intermolecular interactions, force field, ff14sb [50] was used whereas, the system was neutralized by the addition of na + counter ions. in the second phase of md simulations, the energy minimization of the complexes was carried out. each complex was minimized using the following steps: the energy minimization of hydrogen atoms (500 cycles), energy minimization of water box (1000 cycles, control of 200 kcal/mol -å 2 on rest of the system), minimization of the whole atoms of the system (1000 cycles with the restraint of 5 kcal/mol -å 2 on cα atoms), and the rest of the system was subjected to non-heavy atoms minimization with 300 cycles and restraint of 100 kcal/mol -å 2 . in the next step, the system was gradually heated from 0 k to 300 k with the time step of 2 femtoseconds and restraints of 5 kcal/mol -å 2 on cα atoms. in order to sustain the temperature of the system, langevin dynamics [51] with the gamma value of 1.0 was castoff. the shake algorithm [52] was used to put constraints on the hydrogen bonds of the system for heating. in the next step, systems were equilibrated for 100 ps with a time step of 2 fs followed by pressure equilibrium, which was attained using the npt ensemble with restraints of 5 kcal/mol -å 2 on cα atoms. the same step was extended for 50 ps with the 1 scale down on restraints on carbon atoms. however, the step of system equilibration was carried out for a time scale of 1 nanosecond followed by the production run of 100 ns with a time scale of 2 fs. for the production run, the berendsen algorithm [53] with the nvt ensemble cast off with a cut-off of 8.0 ǻ. simulation trajectories were calculated to investigate the strength of a complex via the cpptraj module [54] of amber. however, the visualization of simulation trajectories was done with ucsf chimera [55] , ds visualizer [45] and vmd [46] . in order to estimate the mmpbsa binding free energies for the receptors and multi-epitope peptide vaccine construct, the mmpbsa.py module [56] of amber16 was castoff. the program generated the input files for the complex, receptor and mepvc molecule using the ante-mmpbsa.py module. to compute the variance between the solvated and un-solvated phases, 100 frames of simulation trajectories were picked and analyzed [57] . for the precise values of binding free energies, the two different conformations were matched to the binding energies of significant residues. to estimate the free binding energy of the anticipated complex, δg bind, solv was resolved using the three equations given below: the net free binding energy was then decomposition into each residue to highlight the interacting and stable residues. the ncbi most recently dedicated a coronavirus disease data hub containing all nucleotide and protein sequences information published from across the world. in this study, we aimed at in silico prioritization potential vaccine candidates and designing a chimeric peptide vaccine for covid-19 based on all available protein sequences in the data hub. several bioinformatic and immunoinformatics techniques are employed with the aim to assist experimentalists in vaccine development against the virus. prioritization of potential vaccine candidates could help in minimizing time, labor cost and resources for developing and optimizing the success of getting an effective vaccine against the pathogen. in total, 193 protein entries were retrieved (stable 1 ) and analyzed first for sequence homology with the human host proteome. this was significant to evaluate as homology between virus protein (s) to be used in vaccine designing and the host is likely to cause strong autoimmune reactions in the host [58] . this check identified two proteins: (orf1a polyprotein (accession id, yp_009725295) and nsp3 (accession id, yp_009725299) as host homologous thus discarded from further evaluations. experimental studies of the human vaccines are usually done in mice because of the many practical advantages they provide compared to vaccine research in higher animal models [22] . the appropriateness of mice as an animal model for vaccine research is defined by its ability to reproduce relevant human physiology [59] . considering this, a homology check was devoted to the pipeline and applied to the filtered human non-similar proteins (191 in number) to ensure the selection of non-similar mice proteins. this assessment resultant into shortlisting of orf1ab polyprotein (accession ids, qhq82463, qhd43415, qhr63279, qhr63259, qhq71962, qho62106, qhq71972, qhr63249, qho60603, qho62111, qhn73794, qhr84448, qhr63269, qhn73809, qhr63289, and qho62876) and nsp3 (accession id, yp_009724389) as mice similar proteins. the mice nonsimilar proteins will aid in discouraging false positive results during in vivo experimentations and accurate interpretation of immune protective efficiency of the prioritized vaccine candidates against the virus [22, 60] . next, enumeration of transmembrane helices in the pooled 174 mice non-similar proteins was accomplished. this transmembrane topology characterization is deemed vital in relatedness to the afterward experimental expression studies of the proteins. protein with transmembrane helices less than 2 in numbers are often considered as best vaccine candidates as multiple helices make recombinant proteins purification and expression difficult in vaccine development [20] . this result 33 proteins spanning across five types, and include: orf3a polyprotein (accession id, qhq71974, qhr84450, qhd43417, qhq71964, qhq82465, qho60595, yp_009724391, qhn73811, qhn73796, qho62878), nsp4 (accession id, yp_009725300), membrane glycoprotein (accession id, qhd43419, qhq71966, qhq71976, qhq82467, yp_009724393, qho60597, qhn73813, qhn73798, qho62880, qhr84452), membrane protein (accession id, qhr63283, qhr63263, qhr63273, qhr63293, qhr63253), matrix protein (accession id, qho62109, qho62114), and nonstructural protein ns3 (accession id, qhr63251, qhr63281, qhr63261, qhr63271, qhr63291) containing multiple helices therefore not proceeded further. the creation of adhesin-based vaccines is considered an attractive and effective strategy and is being explored as a solution to number of infectious pathogens [61] . the idea behind exploiting adhesin for a vaccine is based on the promising preclinical findings. the aim is to confer protective immunity via two main mechanisms: (i) opsonization driven by opsonising antibodies that is capable of binding the target antigen as an immunological tag leading to activation of other components of the host immune system for enhance recognition of the pathogen and subsequent complement system activity and virus killing by phagocytosis, (ii) neutralization driven by adhesin-specific antibodies that block virus binding ability to host tissues. the adhesion probability computation revealed 26 protein to have adhesion probability value greater than a threshold as tabulated in stable 2 and can be ideal putative vaccine candidates against covid-19. the adhesin probability of protein ranges from 0.593 to 0.796 (mean, 0.645).antigenicity of proteins was predicted to reflect their ability of binding to products of adaptive immunity: antibodies or t-cell receptors. in total, 7 proteins: nsp8, nsp9, nsp10, 3c-like proteinase, spike glycoprotein, surface glycoprotein, and orf1ab polyprotein were recognized as antigenic and scored higher than the threshold. coronavirus nsp8 suggested having diverse activities, including template-dependent rna polymerase activities, canonical rna-dependent rna polymerases, cofactor function of nsp8 for nsp12-mediated rna-dependent rna polymerase activity, and metal ion-dependent rna 3′ polyadenylation activities [62] . nsp9 is a non-structural protein 9, key to coronavirus replication and is a single-stranded rna-binding protein [63] . nsp10 is a critical cofactor that switches on multiple enzymes in replication cycle. it is known to interact with nsp14 and nsp16 subunits activating their respective 3′-5′ exoribonuclease and 2′-o-methyltransferase functions [64] . the 3c-like proteinase is main cysteine protease and is nonstructural protein number 5 (nsp5) and essential in mediating cleavage of nsp4 to nsp16 [65] . the trimeric transmembrane coronavirus spike glycoprotein initiates infectious cycle by binding to a specific receptor on the host membrane followed by viral fusion [66] . the surface glycoprotein was analyzed to be different in a sequence patch (leu3-phe11) at the start of the spike glycoprotein. the orf1ab is replicase polyprotein cleaved by papain-like protease and 3c-like protease at specific cleavage sites to yield 15 to 16 non-structural proteins (nsps) [67] . the final numbers of potential vaccine candidates obtained in this step by step subtraction phase are presented in fig.2. identification of epitopes in given antigens is vital for a number of practical reasons, including understanding etiology of a disease, monitoring of immune system, development of diagnostic assays, and epitope-based vaccines designing [68] . from vaccine designing point of view, host adaptive immunity is highly specific and is able to recognize and destroy the invading pathogen [69] . additionally, adaptive immunity is able to remember the pathogens, creating long-lasting pathogen-specific protective memory enabling stronger attacks against the pathogen reencountered on successive times [70] . this arm of host immune system is driven by lymphocytes of two types: b and t-cells responsible for the humoral and cell-mediated immunity, respectively [71] . both cells recognize pathogen molecular components called antigens. the antigens interact with specific receptors present on the surface of b and t-cells. the activation of both these cells required antigen recognition by these receptors, in addition, to the second activation signals from the innate system. the vaccine candidates prioritized in the first phase were deeply investigated for b-cell epitopes. different lengths of linear b-cell epitopes were predicted for each protein and only recurrent epitopes simultaneously predicted by different servers were selected for chimeric vaccine designing. the b-cell epitopes predicted for the vaccine candidates were in the following order: nine for nsp8 and 3c-like proteinase, five for nsp9, eight for nsp10, 34 for spike glycoprotein and surface glycoprotein, and four for orf1ab polyprotein| partial. these b-cells epitopes are recognized as solvent-exposed antigens through b-cell receptors (bcr) and upon activation, b-cells secrete antibodies. antibodies have different functions including neutralizing pathogens, toxins and labeling pathogens for destruction [72] . the proteins were also analyzed for t-cell epitopes through very stringent criteria of p-value less than 0.005. the epitopes were of different lengths and interact with several different alleles of mhc-i and mhc-ii. for nsp8, 94 epitopes predicted whereas nsp9, nsp10, 3c-like proteinase, spike glycoprotein, surface glycoprotein and orf1ab polyprotein| partial protein were mapped for 61, 76, 153, 657, 651, and 44, respectively t-cell epitopes. these epitopes are presented on the surface through specific receptors known as t-cell receptor (tcr) allowing recognition of these antigens when displayed by antigen-presenting cells bound to mhc molecules [73] . epitopes presented by mhc i are recognized by cd8 (cytotoxic t lymphocytes) [74] whereas those presented by mhc ii are recognized by cd4 t-cells [75] . the cd4 t-cells later become helper t-cells that amplify the immune responses against the pathogen. comparative analysis of the predicted epitopes was further carried out to select epitopes that are common to b-cell, cd4 t-cell and cd8 t-cell alleles in order to design a specific, effective, and strong vaccine. on this basis, three epitopes from nsp8 (drdaamqrk, qarsedkra, eqavangdsev), none for nsp9 and orf1ab polyprotein| partial protein, four for nsp10 (gcscdqlrep, ylasggqpit, ylasggqpi, tvtpeanmdqesfg), three for 3c-like proteinase (edmlnpnyedl, kynyepltqdhv, eftpfdvvr), five for spike glycoprotein (rvystgsnvfq, vnnsyecdipi, ladagfikqygdclg, gqskrvdfc, rnfyepqiittd) and surface glycoprotein (rvystgsnvfq, vnnsyecdipi, ladagfikqygdclg, gqskrvdfc, rnfyepqiittd). the epitopes were then reevaluated in antigenicity check to make sure their binding potential of binding to immune cells. for nsp8, drdaamqrk and qarsedkra were found antigenic with score higher than the default threshold of 0.4 whereas the third epitope eqavangdsev was found non-antigenic hence removed. all the four epitopes of nsp9 revealed non-antigenic therefore not processed further. in case of 3c-like proteinase, kynyepltqdhv was found non-antigenic whereas edmlnpnyedl and eftpfdvvr were antigenic therefore considered in afterward analysis. the spike glycoprotein contains epitopes vnnsyecdipi, and rnfyepqiittd as antigenic whereas none of the surface glycoprotein provided epitopes were antigenic. following, the affinity of the filtered antigenic epitopes for the most prevalent drb*0101 allele in humans was evaluated through ic 50 value and those with value < 100 nm were classified as high affinity binders. all the pooled antigenic epitopes were found to have great ability of binding to the mentioned allele. similarly, these epitopes were evaluated in allergenicity and virulent potential check and only virulent and nonallergen were selected. virulent check was significant in ensuring selection of epitopes mediating infectious pathways in the host. the final selected epitopes that cleared all these checks are tabulated in table 1 . a mep was constructed first comprising epitopes finalized in the previous phase. mep based vaccines are considered an ideal approach to prevent and treat viral infections [24] . the epitopes shown in table 1 were linked to each other through flexible aay linkers as such it allows efficient separation required for the effective working of each epitope. once the mep was designed, to its n-terminus an adjuvant of cholera toxin subunit b (ctb) was added [76] . the schematic representation of the mepvc is shown in fig.3 . ctb is nontoxic part of cholera toxin and is considered an accelerator in protective immunity and a break in auto-immunity. it shows high affinity for monosialotetrahexosylganglioside displayed on variety of cell types, including gut epithelial cells, antigen-presenting cells (dendritic and macrophages) and b-cells [77] . ctb is a preferred choice as an adjuvant because of its ability of self-expression in variety of organisms and can be coupled to antigens through several approaches involving chemical manipulation and genetic fusion resulting in strong immunological responses against the antigens to which it is attached. the vaccine construct was then used in a comparative 3d structure prediction to ensure confidentiality in selection of the most suitable model for the construct with minimum structural errors. several different physicochemical properties of the mepvc were deduced from its sequence. the vaccine construct is 189 amino acids long with total number of 2984 atoms. the molecular weight of the construct is ideal i.e. 21.36 kd as small size construct is easy to handle and purify during experimental evaluation. the construct has instability index value of 35.43, signifying its high stability. the aliphatic index computed for the construct is 79.68, reflecting high thermostability. the estimated half-life in mammals, yeast, and escherichia coli is 30 hours, > 20 hours, and > 10 hours, respectively. the grand average of hydropathicity (gravy) score is -0.315 which highlights hydrophilic nature of the construct. the theoretical pi is 6.10 pointing to construct slightly acidic nature. the secondary structure elements of the vaccine construct can be divided into the following order: alpha helix (56.08 %), 3 10 helix (0 %), pi helix (0 %), beta bridge (0 %), extended strand (16.40 %), beta-turn (7.94 %), bend region (0 %), and random coil (19.58 %). compared to the itasser, phyre2, and swiss-model, the 3dpro predicted structure was determined as the most suitable structure based on the complete modeling of the given length of the amino acid sequence. loop modeling was done at leu29-gln37, ser51-gln70, glu72-gln77, glu57-ser76, val103-thr113, and glu167-asp186. the model was refined to minimum rmsd of 1.734 å and molprobity score of 1.134 that is quite low compared to the original structure score of 3.312, reflecting good quality of the modelled structure. similarly, the clash score in contrast to the original structure is 94.7 times lower demonstrating steric clash free structure. the galaxy energy of the structure is very stable (-3784.31) and ramachandran favored distribution increased from 93.6 to 95.7 %. the top 10 refined models of the mepvc are tabulated in table 2 . the 3d models of the vaccine construct after loop modelling and refinement is presented in fig.4a . the overall z-score of the modelled structure is -4 and the score is within the range of same size proteins in the pdb illustrating good quality as depicted in fig.4b . refinement of the structure ramachandran plot demonstrated the construct to contain 93.2 % of its residues in the most favored regions, while 5.7%, 0.0%, 1.1% residues are in additional allowed region, generously allowed region, and disallowed regions, respectively (fig.4c) . the overall average g-factor of the construct is 0.05. ramachandran plot for the mepvc illustrating distribution of torsion angles (blue squares) comparative to the core (shown in red) and allowed (shown in brown) regions. residues in the generously allowed region are shown in dark yellow whereas disallowed regions are depicted in pale yellow. enhancing protein stability is important in many biomedical applications and is an appealing approach to emulate nature stabilizing molecular interactions [78] . the covalent disulfide bonds provide substantial stability to target proteins and disulfide engineering had achieved considerable success in broad range of applications [33] . in total, 11 pairs of residues were selected for the purpose of disulfide engineering. these include met1-ser81, lys5-ala59, val12-asp28, ser16-asp28, thr40-ser47, val73-gln77, ala119-ala133, met122-ala133, ala126-asp129, leu156-leu163, and asn159-asp162. the average chi3 and energy value for the pairs is 12.54 (max, 108.65 and min, -110.64) and 3.12 (max, 4.39 and min, 1.14). the disulfide engineered mepvc structure is presented in fig.5 . in the follow up experimental studies, the maximum expression of mepvc is highly desirable [35] . one requirement for that is the codon usage of mepvc that must be adapted according to the expression system, for instance, here we used e. coli k12 as a mepvc expression system. the codon adaptation index (cai) and gc content revealed for the improved sequence are highly satisfactory with value of 0.96 and 48.85, respectively strongly indicating high mepvc expression. the mepvc then enclosed on both sites by 6x histidine tag to ease its purification process and inserted at appropriate sites of pet28a(+) vector as shown in fig.6. fig.6 . in silico restriction cloning of the mepvc into pet28a(+) vector. the insert is shown in red. molecular interactions and binding conformation of the designed mepvc with tlr3 and tlr4 innate immune receptors were deciphered via a protein-peptide docking approach. both tlr3 and tlr4 belong to toll-like receptor family of pattern recognition receptor and function to activate intracellular signaling nf-κb pathway and production of inflammatory cytokines responsible for the development of effective innate immunity [79, 80] . these receptors recognize viral associated molecular patterns and induce the production of interferon leading to activation of strong host defense responses. also, the specific adaptive immunity takes time to establish against antigens therefore it's important to evaluate mepvc affinity for the innate immune receptors. in case of mrpvc-tlr3 complex, the patch dock predicted 10 best solutions sorted based on the docking geometric shape complementarity score (stable 3) . a high score implies enhanced affinity of the interacting molecules and best docked conformations of the molecules with respect to each other. solution 3 was visualized for docked conformations and intermolecular forces responsible for such high affinity of the molecules. the selection was based on the firedock analysis (stable 4 ) which is an efficient package for refinement and reassigning procedure of docking scores to rigid body docking solutions. the global binding energy of solution 3 is better i.e. -6.39 kj/mol compared to the rest of predicted solutions. the contribution to the total score form attractive van der waals (vdw) energy is -12.12 kj/mol, repulsive (vdw) energy (4.79 kj/mol), hydrogen bond (hb) energy (-1.63 kj/mol), and atomic contact energy (ace) (5.95 kj/mol). the mepvc, within 3 å, was noticed to posed right in the center of the tlr3 receptor (fig.7) interacting via hydrogen and hydrophobic bonds with his156, asp180, lys201, glu203, ser206, phe227, asp229, asp230, ser254, ser256, asp257, ser282, tyr283, asp284, asp285, glu301, tyr302, phe304, glu306, tyr307, arg325, glu358, his359, lys382, tyr383, p408, his410, iso411, gly431, his432, glu434, pro408, asp457, phe459, gln483, and glu533. similarly, among 10 predicted mepvc-tlr4 complexes (stable 5 ), solution 7 (stable 6 ) was affirmed as best with total global energy of -10.39 kj/mol whereas the rest of complexes were noticed as highly unstable with score in positive. the attractive vdw, repulsive vdw, hb and ace contribution to the global energy is -35.75 kj/mol, 22.04 kj/mol, -5.51 kj/mol, and 12.61 kj/mol, respectively. visual analysis of the complex revealed binding of the mepvc at the interface of chains b and d (fig.8) . the mepvc is surrounded by chain b residues: pro23, glu24, ser25, asp44, lys47, asp50, asp51, arg67, arg87, glu89, pro113, gln115, asp137, his159, asp160, ser184, and lys186 and chain d residues: lys20, phe64, and asp114. the dynamic simulations of the human immune system in response to the designed vaccine construct were deciphered through c-immsim server [40] . the vaccine construct upon administration revealed to generate robust primary immune responses. as can be seen in fig.9a that combine igm and igg antibodies has a titer scale close to 10,000/ml followed by igm antibody (> 6000 antibody titer per ml). the combined igg1 and igg2 and igg1 were seen to generate high titer scale of around 6300/ml, 3100/ml, and respectively. the igg2 antibody response revealed to be low throughout post vaccine administration period. the dimerized soluble cytokine ifn-g produced against the antigen is > 400000 ng/ml (fig.9b ). in silico simulation of the host immune system using mepvs as an antigen. a. antibodies titer (a) and cytokines and interleukins (b) in response to the antigen. the stability and dynamics of the designed vaccine construct ensemble docked to innate immune receptors were disclosed through 100-ns of md production run and interpreted through the root mean square deviation (rmsd) [81] , root mean square fluctuation (rmsf) [82] , the radius of gyration (rg) [83] and beta factor (β-factor) [84] assays as depicted in fig.10 . the average cα atomic distance over 10,000 frames of tlr3-mepvc and tlr4-mepvc was decoded through rmsd assay. an average rmsd of 3.44 å (maximum, 6.30 å) and 3.36 å (maximum, 5.22 å) were estimated for tlr3-mepvc and tlr4-mepvc, respectively. the tlr3 and tlr4 receptors are observed more compact than the mepvc and as a result, continuous movements of the vaccine construct through its length are noticed at the exposed regions though rooted stable at the docked position. this seems to be responsible for bringing small structural deviation as probed by rmsd. visual inspection of the trajectories illustrated no major global and local secondary structure conformation changes in the receptor tlr3 and tlr4 structure. the mepvc movements with respect to the tlr3 are shown at different snapshots as illustrated in fig.11 . the mepvc seems responsible for the deviations in the receptor tlr4 at positions glu1-ser79, hie540-asn614, gln1144-gln1209, gln1347-pro1371, and lys1489-lys1491. continuous flexibility of the loops of mepvc exposed region is observed and is highly flexible responsible for the movements of the mepvc at the docked site though rooted stably (fig.12) . hydrogen bonding results when a hydrogen atom attached to a highly electronegative atom is attracted by another electronegative atom [85] . in a biological system, these hydrogen bonds are vital in determining specificity and directionality fundamental in molecular recognition [86] . the patterns of hydrogen bonds for both complexes were illustrated in each frame within 3 å in order to probe the strength of intermolecular association across the simulation period. the maximum number of hydrogen bonds of mepvc with tlr3 and tlr4 are 11 and 12, respectively demonstrating the high strength of interactions. the number of hydrogen bonds for both complexes are shown in fig.13. fig.13 . the number of hydrogen bonds formed during simulation between mepvc and tlr3 and tlr4 receptors. in a protein molecule, salt bridges are formed between charged side chains of amino acids at neutral ph. the residues mainly involved in these interactions include negative full electron charge glutamine and aspartate and opposite positive full electron charge arginine and lysine [87] . the presence of salt bridges between the interacting molecules is a clear sign of strengthening interaction stability. for tlr3-mepvc complex high numbers of salt bridges were estimated within 3.2 å between receptor glu8, glu276, arg306, glu333 with mepvc lys3, lys8, glu83, arg16, respectively. in case of tlr4-mepvc complex, receptor residues arg646, asp268, arg1396 are involved in salt bridging with asp146, arg175 and glu184 of the mepvc, respectively. the binding free energies of both tlr3-mepvc and tlr4-mepvc complexes were computed using continuum solvation mmpbsa and its complementary mmgbsa. the binding free energies of the vaccine construct for the receptors were estimated considering molecular mechanics energies as well as solvation energies. the net binding free energy for both complexes revealed to be very high illustrating the high interacting affinity of the molecules. for tlr3-mepvc, the total free energy of binding is -41.4273 kcal/mol in mmgbsa while in mmpbsa it is -84.4908 kcal/mol ( after performing the immune system simulation to mepvc and a thorough peptide dynamics analysis, a novel mepvc is proposed as a probable solution to the widely spread viral coronaviruses outbreak. meanwhile, this seems necessary to have a deep down lesson as covid-19 viral outbreak is propagating to the human species on this part of the universe called earth. as we are already living under a serious threat of antibiotic and antimicrobial resistance [88] which if ignored can cause havoc and the current spread of covid-19 is a clear example of even a viral resistance coming in line. referring to the introduction section where the probable cause of this viral attack is discussed, this is mandatory to mention that there is retaliation from animals and animal-associated viruses or bacteria observed against homo sapiens quite frequently in the recent past [89] . this not only alarms the way of consuming animals in our food in the form of livestock but at the same time explains the advancing defense system of various species around. either it is horizontal gene transfer among bacteria or transient nature of viral transmission throughout the world as one way or the other this is a form of advanced defense of organisms. it is meant to state that even before and after the cognitive revolution, homo sapiens cannot be exempted from biological laws. while considering under the realm of these biological laws this is much expected from all other species to have the right of keeping and exercising a defense mechanism [90] . instead of spending on wars among humans there is a dire need to be equipped for all wars to come against human species under the disguise of either environmental hazards or antimicrobial resistance. even viruses are finding new ways of infecting hosts. under this perspective the role of avian has been observed as much dominating in the recent past [91] . after having insights from comparative advancing mechanism we mention here and thereafter the preliminary existence of a "theory of retaliation" on the basis of currently available facts and observations. this theory of retaliation will play its role in coming future. the continuous attacks in the form of outbreaks by them have raised many challenges and threats to human beings. a recent mechanism study by bazel et al reported h5n1 influenza viruses is posing threat to human and animal health and is currently unclear what restricts these interspecies jumps on the host side. it is further signified that pb1-f2 (a short viral peptide) assists h5n1 bird influenza viruses to overcome a human restriction factor of the viral polymerase complex hax-1. it is also evidenced that a functional pb1-f2 aids in direct transmission of viruses from birds to humans [92] . additionally, there is already a mechanism existing for the antimicrobial defense of avian eggs which illustrates their efficacy in defense mechanisms [93] . furthermore, discovery of avian antimicrobial peptides, classified as bdefensins, present in chicken and turkey found active against bacteria, fungi, and yeast. this is another example of advance mechanism showed by avian and the possibility of a common ancestral gene between avian and other mammalian peptides seems obvious [94] . who alarmed quite often that influenza viruses with a vast silent reservoir in aquatic birds are impossible to eradicate while avian influenza is proved to be a threat to human health [95] . even having the phylogenetic identity between sars-cov-2 and sars-cov, some clinical characteristics differentiate sars-cov-2 from sars-cov, mers-cov, and seasonal influenza infections [2, 96] . this leads to the fact that viruses are attacking humans with different clinical features every time. the complex relationships between the human and animal species never faced a halt in evolving giving rise to numerous infectious pathogens. whatsoever is the case, the dramatic impact of infectious diseases affecting the modern human population worldwide is evident and unexpected rise of coronavirus infections raised to 100000 while writing this research and getting beyond the normal control (https://www.nature.com/articles/d41586-020-00154-w). after all we belong to the kingdom animalia with even ten on ten embarrassing similarities to chimpanzees [97] . probable prevention in this context is to somehow avoid the excruciating utilization and consumption of other mammals as edibles from homo sapiens and stop becoming a reason for the extinction of certain species. avenging from these species from time to time by utilizing the microbes associated with them against human has become obvious and vaccine design and discovery is of utmost importance. this is crucial to strategize a preventive control not only for symptomatic relief but in general taking steps to prevent hunting and strategy required to maintain a balanced ecosystem where specifically avian will not dwell under threat by sapiens. the advent of various new mechanisms of viral survival has remained sapiens perplexed and a broader strategy is required to circumvent this problem. in this study, we used available immunoinformatics approaches for the purpose to prioritize potential vaccine candidates against covid-19 considering their ease of use in experimental investigations. bearing in mind, the wide immunological applications of peptide vaccines only highly antigenic, virulent, conserved and non-allergic epitopes targeted by both b and t-cells were disclosed. a multi-epitope peptide was constructed and an appropriate adjuvant was added to allow suitable delivery and efficient immune processing of the epitopes. these promising computational findings might deliver preliminary epitopes set for a vaccine against the covid-19 notwithstanding the experimental testing in appropriate animal models to unravel real effectiveness. meanwhile, probable prevention discussed in this study for homo sapiens is to avoid becoming a reason for the extinction of various species either by hunting and/or over utilizing other mammals as this may be a reason for resistance both from microbes and other animal species of this kingdom. there must be strategic studies keeping in view the advancements in defense mechanism of avian and avian related microbes avenging humans. preventive use of animals or avian in human diet and avoiding hunting can be preventive options self-defense in this connection and/or maintenance of a balanced ecosystem should be reinvestigated supplementary files s-table 1. complete protein data set of covid-19 at ncbi coronavirus data hub adhesive proteins shortlisted for covid-19 top 10 patchdock solutions of the mepvc-tlr3 complexes s-table 4. firedock refinement of tlr3-mepvc complexes top 10 patchdock solutions of the mepvc-tlr4 complexes s-table 6. firedock refinement of tlr4-mepvc complexes authors are highly grateful to the pakistan-united states science and technology cooperation program (grant no. pak-us/2017/360) for granting financial assistance a novel coronavirus from patients with pneumonia in china clinical features of patients infected with 2019 novel coronavirus in return of the coronavirus: 2019-ncov the continuing 2019-ncov epidemic threat of novel coronaviruses to global health &#x2014; the latest 2019 novel coronavirus outbreak in wuhan, china novel coronavirus (2019-ncov) situation summary | cdc a novel coronavirus outbreak of global health concern sciencedaily: your source for the latest research news database resources of the national center for biotechnology information exoproteome and secretome derived broad spectrum novel drug and vaccine candidates in 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and chimpanzee neural progenitors during cerebral cortex development key: cord-339091-3xk2w0d2 authors: flower, darren r; macdonald, isabel k; ramakrishnan, kamna; davies, matthew n; doytchinova, irini a title: computer aided selection of candidate vaccine antigens date: 2010-11-03 journal: immunome res doi: 10.1186/1745-7580-6-s2-s1 sha: doc_id: 339091 cord_uid: 3xk2w0d2 immunoinformatics is an emergent branch of informatics science that long ago pullulated from the tree of knowledge that is bioinformatics. it is a discipline which applies informatic techniques to problems of the immune system. to a great extent, immunoinformatics is typified by epitope prediction methods. it has found disappointingly limited use in the design and discovery of new vaccines, which is an area where proper computational support is generally lacking. most extant vaccines are not based around isolated epitopes but rather correspond to chemically-treated or attenuated whole pathogens or correspond to individual proteins extract from whole pathogens or correspond to complex carbohydrate. in this chapter we attempt to review what progress there has been in an as-yet-underexplored area of immunoinformatics: the computational discovery of whole protein antigens. the effective development of antigen prediction methods would significantly reduce the laboratory resource required to identify pathogenic proteins as candidate subunit vaccines. we begin our review by placing antigen prediction firmly into context, exploring the role of reverse vaccinology in the design and discovery of vaccines. we also highlight several competing yet ultimately complementary methodological approaches: sub-cellular location prediction, identifying antigens using sequence similarity, and the use of sophisticated statistical approaches for predicting the probability of antigen characteristics. we end by exploring how a systems immunomics approach to the prediction of immunogenicity would prove helpful in the prediction of antigens. vaccines are agentseither molecular or supramolecular -which can stimulate protective immunity against microbial pathogens and the diseases they cause. protective immunity is a specific and enhanced adaptive immune response to subsequent re-infection or, when luck holds, infection by related organisms. such augmented immunity is mediated by the exacerbation of immune memory, which militates against the effects of infectious organisms. the word vaccine itself is derived from vacca (latin for cow). [1] [2] [3] . it is a thing of near universal agreement that mass vaccination -synergising as it does with the herd immunity it helps engender -is the most effective and efficacious prophylactic treatment currently available for contagious disease. humankind is commonly affected by over seventy infectious diseases, many of which are or will be targets for vaccines. there are in excess of fifty licensed vaccines, half of which are deemed to be in common use. most vaccines prevent childhood infections or are used by travellers to tropical or subtropical regions; only a minority combat disease in third-world countries. as recently as the late 1960s, there were over 10 million cases of smallpox spread through 31 countries, with about two million deaths a year, yet now smallpox is wholly and totally eradicated. poliomyelitis or polio is the other key global disease close to eradication. in 1991, the pan american health organization eliminated polio from the western hemisphere. in the first world, the annual death rate arising for contagious diseases such as polio, diphtheria, or measles is less than one in a thousand. the global polio eradication program has now greatly reduced the prevalence of polio in the rest of the world. only 784 cases of polio were reported in 2003. nevertheless, polio remains endemic in nigeria, afghanistan, pakistan, and india. despite such outrageous and egregious success, many major issues persist. no licensed vaccines exist for hiv and malaria, two of the world health organization (who)'s three big global killers, and there are no realistic hopes for such vaccines appearing in the short to medium term. bacille calmette guérin (bcg), the only vaccine licensed currently for the third major world disease, tuberculosis, has only limited efficacy [4] . add to this the 35 new, previously unknown infectious diseases identified in the past 25 years: hiv, marburg's disease, sars, dengue, west nile, and over 190 human infections with the potentially pandemic h5n1 influenza. it is commonly believed that new contagious diseases will continue to emerge in the 21st century. the world of the 21st century is threatened by parasitic diseases and emerging zoonotic infections; antibiotic-resistant bacteria; and bioterrorism [2] the vaccine arena has long been neglected, partly as a consequence of the extraordinary success just adumbrated, but activity within it is now feverish and febrile [5, 6] . dozens of vaccine candidates have passed through phase ii clinical trials, and during the past decade, vaccines in late development have numbered over 150. unlike antibiotics, resistance to vaccines is negligible. in the same way that vaccines target many kinds of disease, themselves caused by microbial pathogens of all types, so there are many types of fundamentally distinct vaccine. see figure 1 . these include attenuated or inactivated whole pathogens, subunit vaccines, peptide vaccines, and vaccines based on carbohydrates, amongst others. historically, the most successful and prevalent types vaccines have been those based on attenuatedthat is "weaken" or noninfective -whole pathogen vaccines, for example bcg for tb or sabin's polio vaccine. safety concerns have fomented other vaccine strategies to develop, which focus on antigens and latterly epitopes as the intrinsic component of single or composite vaccines. hepatitis b vaccine is an antigen -or subunitbased vaccine. while many epitope-based vaccines have now entered clinical trials, they are yet to fulfil their potential, medically or commercially. antigens, immunogenicity, and subunit vaccines subunit vaccines are typically but not exclusively protein molecules and their discovery is often based around a somewhat haphazard, essentially empirical search for antigenic or immunogenic protein antigens. the word antigen has several meanings in immunology and thus in vaccinology. the oxford english dictionary defines an antigen as a "…foreign substance which, when injected into an organism, stimulates antibody production." they trace its first use to 1908, and by the middle of the century the word had found its way into common technical dictionaries. an immunogen is on the other hand: "any substance capable of eliciting an immune response", although the word had other, earlier definitions. its first use in its modern rendering is traced to 1959, but was doubtless in use before then. figure 1 a schema summarising the components of a generic vaccine. the immunogenic component will typical be an attenuated or chemically-inactivated microbe, a protein antigen, or a poly-epitope or conjugate. this component will be combined with an appropriate delivery mechanism and an adjuvant. delivery mechanisms and adjuvants overlap in their ability to increase the immunogenicity of a weaklyactive vaccine. dictionary definitions seldom capture the scientific meaning of scientific terms particularly well. so, to be more precise, an immunogen, that is a moiety exhibiting immunogenicity, is any substance able to elicit a specific immune response, while an antigen -a moiety exhibiting antigenicity -is a substance which is recognized by an existing immune response, and its associated underlying molecular moieties such as t cells or antibodies, in a recall response. immunogenicity is the most important and interesting property for vaccine design and discovery. it is this property that allows a molecular moiety to induce a significant immune response. for the purposes of this review, our use of the terms antigen and immunogen shall be restricted to a single meaning; that of a protein, specifically one from a pathogenic microorganism, that evokes a measurable immune response. currently, the prophylactic responses of almost all effective vaccineswith the exception of bcg which is mediated by the quadrille of interacting antigen presenting cells (apcs), t-cells, and other phagocytotic cells that characterise cellular immunityare mediated by humoral immunity and antibodies. the pathogenesis of most diseases for which vaccines are not currently available are typically if not exclusively mediated by cellular immune mechanisms. thus, when seeking to identify immune responses for untreated disease, the immunogenicity sought need not be mediated by antibodies; mediation by cellular mechanisms or a combination of humoral and cellular mechanisms is equally valid. in what follows, we will endeavour to explore the availability of informatic tools and techniques for the identification of candidate subunit vaccines. even today, in the early years of the 21 st century, many experimental scientists still retain an atavistic distrust of any and all computational approaches. the very suggestion that algorithms can help them, saving effort and time is an anathema to them and their way of thinking, undermining all that they hold dear. they are deeply distrustful of the reliability of computer methods, preferring what they perceive as the infallible reliability of the experiment. not that they would admit to views so philosophically -and practicallynaive; but deep down this is how they think and how they feel. yet things have changed and changed dramatically, and things will continue so to change. slowly but surely, reverse vaccinology is becoming a discernibly more prevalent means of identifying subunit vaccines; and slowly but surely the contribution made by computation to the practice of reverse vaccinology is becoming ever more significant and ever more obvious [7] . see figure 2 . conventional laboratory-based, experimental microbiological approaches to antigen identification typically start with the cultivation of the target pathogenic micro-organism under laboratory conditions, then dissecting them into their component proteins, assaying these in some cascade of in vitro and in vivo assays, leading ultimately to the identification of proteins which display requisite protective immunity. it would indeed be wonderful if the identification of candidate vaccines was really and consistently this simple and systematic? unfortunately, it is often so much more complex, confusing, and confounding. it is not always possible to cultivate a particular pathogen outside of the host organism. many proteins are only expressed transiently during the course of infection. nor are all proteins easily expressed in sufficient quantities in vitro. thus many potential candidate vaccines may be missed. reverse vaccinology [7] [8] [9] [10] is, by contrast, able to analyze a genome to identify potential antigens. initially, the pathogenic genome is scanned for "open reading frames" or orfs. once all orfs have been identified, proteins are selected on the basis that they will be accessible to immune system surveillance, usually using some form of informatic-based prediction methodology or, more likely, set of methdologies. reverse vaccinology was established by a group studying neisseria meningitidis, which is responsible for sepsis and meningococcal meningitis. vaccines are available for all serotypes except subgroup b. potential orfs in n. meningitidis genome were identified [11] [12] ; 570 proteins were identified, 350 expressed in vitro, and 85 were surface exposed. seven proteins conferred immunity over a broad range of strains. another good example is streptococcus pneumoniae, a major cause of sepsis, pneumonia, and meningitis [13] [14] . 130 potential orfs were identified, 108 of which could be expressed; six proteins were found to induce protection. another example is porphyromonas gingivalis, a gram-negative anaerobic bacterium found in subgingival plaques present in chronic adult inflammatory gum disease periodontitis. of the 370 orfs identified in the genome [15] , 120 protein sequences were predicted to be open to surveillance by the immune system, 40 were positive for one or more sera. when used to vaccinate mice, two antigens exhibited clear protection. this very brief survey highlights the potential for vaccine discovery using this approach. however, the number of potential antigens is still high, particularly when we explore all potential difficulties in expressing and characterising them. both experimental and computational methodologies may thus omit potentially important antigenic proteins from analysis, albeit for different reasons, and both require continual optimisation. in particular, bioinformatics support for preclinical vaccine discovery has yet to flourish; but, as interest in vaccines waxes, this situation is changing: expanding from the nursery to at least the kindergarten. there are two main forms of support for vaccine discovery provided by bioinformatics. the first is technically indistinguishable from support for more general target discovery, and includes genomic annotation for both host and pathogen sequences and immunotranscriptomics, the application of microarray analysis to the immune system. the other type kind of support is focussed on immunoinformatics: it addresses problems such as the accurate prediction of epitopes. currently, prediction of t cell epitopes is essentially confined to predictions of varying accuracy of peptide binding to major histocompatibility complex. binding of peptides to class i mhc are reasonably accurate, at least for well characterised alleles [16] . however, several comparative studies have shown recently that the prediction of class ii mhc binding prediction t-cell epitopes is typically poor [17] [18] [19] , and likewise for structure-driven prediction of mhc-mediated epitopes [20] [21] . similarly, both structure[22] and data-driven [23] prediction of antibody-mediated epitopes is suboptimal. while the prediction of b cell epitopes remains primitive, or depends on an oft elusive knowledge of protein structure, methods for t cell epitopes prediction displaying not inconsiderable algorithmic sophistication have been and continue to be developed. despite this, and for different reasons, reliable and consistent prediction remains somewhat elusive; this regrettable decision will doubtless continue so for some time. ultimately, all methods, however sophisticated, are severely circumscribed by the data used to propagate and parameterise them. no data-driven method can go beyond the data used to train it; all methods are likewise much superior in their ability to interpolate than their ability to extrapolate. it is only by compiling extensive, high-quality data that we can aspire to create excellent models of general applicability. what we require are sets of data that are complete, thorough, and properly thought through. such sets would be able to explore both efficiently and effectively the complex and confounding multi-dimensional phase space of properties evinced by the molecules we target. iedb [24] [25] [26] [27] [28] [29] is a step-change in this direction. iedb is the dominant force in current immunoinformatics and stands as one of the principal achievements of the field. the number and quality of epitopes within it are an enormous improvement over all that has gone before. alex sette and his co-workers are to be congratulated on their achievement, but, like all dominant things, iedb has tended to choke most other efforts in the field, and even iedb has its limitations. data is usually multi-dimensional, and each dimension will typically be correlated, to a greater or lesser extent, with each other dimension. together, these many dimensions chart a space: a space of structural variation or variation of properties. if the data we use is itself of sufficient quality and provides a good enough coverage figure 2 whole antigen discovery. taking a reverse vaccinology dynamic, the process of discovering candidate subunit vaccines begins with a microbial genome, perhaps newly sequence, progresses through an extensive computational stage, ultimately to deliver a shortlist of antigens which can be validated through subsequent laboratory examination. the computational stage can be empirical in nature; this is typified by the statistical approach embodied in vaxijen [98] . or this stage can be bioinformatic; this involves predicting subcellular location and expression levels and the like. or, this stage can take the form of a complex mathematical model which uses immunoinformatic models combined with mathematical methods, such as metabolic control theory [116] , to predict cell-surface epitope populations. of the space, then straightforward methods drawn from, say, computer science -of which there are indeed very many -are now of satisfactory accuracy to build models of high predictive accuracy. as we have said, there is an on-going need for the quality, quantity, and availability of data to improve and increase. prediction is enthral to its underlying data. bias within the data places strict limitations upon the interpretability and generality of models from which it is derived. in general, for mhc-peptide binding experiments, the sequences of peptides studied are very biased in terms of amino acid composition, often favouring hydrophobic sequences. this arises, in part, from preselection processes that result in self-reinforcement. binding motifs are often used to reduce the experimental burden of epitope discovery. very sparse sequence patterns are matched and the corresponding subset of peptides tested, with an enormous reduction in sequence diversity. irrespective of their poor performance in prediction, several other problems exist, albeit different for t-and b-cell epitope prediction. for t-cell prediction, the key issue is the availability of data. it has recently been shown that t-cell epitopes, which were previously thought to be short peptides of 8-10 amino acids, can be up to 16 amino acids or perhaps even more. the existence of these longmer epitopes has greatly expanded the repertoire of peptides open to inspection by t-cells [30] [31][32] [33] . problematic as this seems, the situation is made worse by the fundamental logistic constraints of sampling within the specificity exhibited by a single allele. the number of possible sequences that a nonameric peptide might possess is in the region of 512 billion; considering that a single model is built from at most a few hundred peptides, the sampling undertaken is infinitesimally small. similarly, the 3000+ different mhc alleles known to exist in the global human population indicates the extraordinary potential for distinct peptide specificities within the potential patient cohort. for b-cell prediction, explaining the poor performance of b-cell epitope prediction algorithms may point to a fundamental misinterpretation of extant epitope data. pepscan is perhaps the most abundant data available currently but may not be what it seems. experimentally derived epitopes are identified by being assayed against pre-existing antibodies with affinity for whole antigens. however, if such "epitopes" are mapped on to their parent antigen structure they are randomly located throughout the protein and do not equate to clear surface patches, as one might expect if they simply mimic discontinuous epitopes identified by crystallography; in situ these antigenic regions can be completely buried, and thus inaccessible to antibody binding, rather than exposed. if we compare the conformation of antibodybound peptides with those from the intact antigens, they are usually quite different. however, b-cell epitopes in intact antigen and in whole antigen-antibody complexes are similar. it may be that the preformed antibody recognizes denatured antigen in vivo or that the isolated peptide adopts a conformation able to mimic the surface features of a discontinuous epitope. moreover, there is also evidence that the responsiveness of the immune system to pathogenic proteins is only poorly correlated with the possession of t cell epitopes, and that many potential epitopes have been deleted in proteins regularly accessible to immune surveillance, perhaps as an evolutionary counter measure in the war between host and pathogen [34] . taken together, these factors suggest that methods which rely solely on the possession of epitopes are unlikely to be fully effective when tasked with identifying antigens or immunogens. this conjecture is confirmed by what information there is, which indicates that there is little simple correspondence between antigens selected on this basis and experimentally verified antigenic or protective proteins. thus we must seek alternative methods for selecting, and prioritising within that selection, proteins likely to be antigenic and protective. we shall below examine three key approaches: subcellular location prediction, sequence similarity, and empirical statistical approaches, typified by vaxijen. for a protein to be accessible to surveillance by the immune system, it is often assumed to be physically external to the microbial organism or at least present on its surface rather than being sequestered away far from the roving eye of the immune system. for bacteria this means it must be secreted or located on -or in -the outer membrane surface. in this context, immune surveillance is undertaken by a variety of cellular and molecular actors, but chiefly by neutralising antibodies. thus the goal of identifying antigens through the use of subcellular location prediction is principally to identify surface proteins accessible to binding by neutralizing antibodies. unlike the relatively straightforward and well-studied task of identifying orfs, selecting the subcellular location of proteins can be challenging to the point of confusion. nonetheless, and notwithstanding the obvious caveats inherent in this strategy, this has become a favoured approach taken by many when tasked with selecting vaccine candidates. we should remember, however, that antigens are not epitopes, and epitopes are not antigens. so possession of t-cell and b-cell epitopes is in certain senses independent of sub-cellular location, and they may in principle be possessed by any protein. as we have said, there is evidence for t-cell epitope depletion in pathogenic proteins [35] ; or it may be that just picking immune-accessible proteins naturally favours proteins rich in antibody and helper t-cell epitopes, as opposed to cytotoxic t-cell epitopes, at least for membrane proteins. as data accumulates on the specific responses made to differently located accessible proteins we can build these recondite subtleties into prediction strategies. there are two basic kinds of prediction method: manual construction of rules of what determines subcellular location and the application of data-driven machine learning methods, which determine factors that discriminate between proteins from different known locations. accuracy differs markedly between different methods and different compartments, mostly due to a paucity of data. data used to discriminate between compartments include the amino acid composition of the whole protein; sequence-derived features of the protein, such as hydrophobic regions; the presence of certain specific motifs; or a combination thereof. databases, such as prodom [36] , pfam [37] , and pro-site [38] , contain within them the capacity to identify sequence motifs characteristic of certain protein families; this can in turn help us to predict if a protein belongs to an family of proteins which are typically extracellular. however, gross sequence similarity alone is neither a necessary nor a sufficient condition for a protein to share a common sub-cellular location. very similar sequences may be located quite differently, while lack of similarity is no guarantee that proteins will not be co-located. moreover, many proteins can and do exist in several locations within the cell, and often evince different if related functions in these different locations [39] . likewise, different organisms, and specifically eukaryotes versus prokaryotes, have quite different locations, both in number and in physical nature. the number of such compartments used in prediction studies varies considerably, and few papers address the full rich complexity of cell biology. for example, a common schema reduces eukaryotic cells to 4 compartments (cytoplasmic, extracellular, mitochondrial, and nuclear) and prokaryotic to 3 compartments (cytoplasmic, extracellular, and periplasmic), though some papers use over 10 compartments for eukaryotes. in fact, 10 compartments is an under-estimate, and the richness and the complexity of sub-cellular location is daunting, since any attempt to predict it must account for transient and permanent location, multiple locations, and both membrane organelles and multi-protein complexes as potential locales. among binary approaches, arguably the best method is signalp, which employs neural networks and predicts n-terminal secretion signals cleaved by signal peptidase-i-and their associated cleavage site [40] [41] [42] . the signal predicted is the type ii signal peptide common to both eukaryotic and prokaryotic organisms, for which there is a wealth of data, in terms of both quality and quantity. a recent enhancement to signalp is the implementation of a hidden markov model (hmm) which seeks to discriminate uncleaved signal anchors from cleaved signal peptides. one of the limitations of signalp is overprediction, as it cannot reliably discriminate between several very similar yet distinct signal sequences, regularly predicting membrane proteins and lipopteins as type ii signals. many other kinds of signal sequence exist. a number of methods have been developed to predict lipoproteins, for example. the prediction of proteins that are translocated via the tat-dependent pathway is also important but has yet to be addressed in any depth. many programs and servers have been devised and developed able to predict the subcellular location of gene products and proteins. psort is a well-known, well-used example; it is knowledge-based, multicategory prediction method, composed of several programs [43] [44] [45] [46] . psort i predicts 17 different subcellular compartments, while psort ii predicts 10 locations. there are several specialized versions of psort. ipsort deals specifically with secreted, mitochondrial, and chloroplast locations. psort-b only predicts bacterial sub-cellular locations. it reports precision values of 96.5% and recall values of 74.8%. another effective program is hensbc [47] . this constructs a hierarchical ensemble of classifiers by applying a series of if-then rules. hensbc is able to assign proteins to one of four different types (cytoplasmic, mitochondrial, nuclear, or extracellular) with approximately 80% accuracy for gram-negative bacterial proteins. another program is subloc [48] , a client/server suite which offers an interface for the prediction of prokaryotic subcellular location in three compartments. another still is gpos-ploc [49] , which fuses many basic classifiers, engineered using the optimized evidence-theoretic k-nearest neighbours rule. other programs include tatp 1.0 [50] , lipop 1.0 [51] , and phobius [52] . this list goes ever on and on. a comparison of a subset of these programs, which used a test set of 272 mycobacterial proteins [53] , indicated that subcellular localization methods generally had high predictive specificity and recognised true negatives reliably. we have also developed a range of programs specifically aimed at addressing the prediction of subcellular location within bacteria. building on a set of algorithms able to predict membrane proteins, tat secretion, and lipoproteins [54] [55] [56] [57] [58] [59] [60] , a set of bayesian networks, which were able to make individual predictions for specific subcellular locations was implemented in three pipelines with different architectures: a parallel implementation with a confidence level-based decision engine and two serial implementations with a hierarchical decision structure [61] . these two hierarchies were rooted differently, one by prediction between membrane types and the other by soluble versus membrane prediction. the parallel pipeline outperformed the serial pipeline, but took twice as long to execute. the soluble-rooted serial pipeline outperformed the membrane-rooted predictor. assessment using genomic test sets was more equivocal, as many more predictions are made by the parallel pipeline, yet the serial pipeline identifies 22 more of the 74 proteins of known location. this work is indicative of a clear direction that the development of future subcellular prediction strategies might take. currently, the richness of subcellular structures is yet to be integrated into location prediction. many cellular organelles are not included in such studies, nor are proteins with multiple locations, nor are the different routes by which proteins can reach the same compartment. the list of such caveats continues. clearly, if we can develop high specificity predictors for each such compartment, then combining them appropriately [61] , must be the way forward. there is then a need for a more sophisticated phase of supervised learning, where better and more complete annotation is built into any approaches taken. many difficulties remain however, most notably the lack of validated, high quality datasets where the location of proteins has been established unambiguously. this paucity is particularly acute for certain important but less well-studied kinds of secreted protein, such as those secreted by the type iii secretion system. while databases such as dbsubloc [62] have been developed, their utility is open to question. so too is the extraction of datasets directly from swiss-prot. here the inherent uncertainty in some of the available annotation can be confounding or at least frustrating. likewise, much more work on the prediction of the subcellular location of viral proteins is required; while some studies have been undertaken [63] [64] , the complexity of their interactions with their host organism must be factored into future analyses. generally, we can make the observation that ignoring this complexity is, in trying to simplify matters, more likely to render any analysis simplistic. fortunately, subcellular location prediction, useful though it can be, and quibbles and cavils notwithstanding, is not the only method available to us. there are other weapons in our arsenal, many with an equal provenance and of equal utility. one of the most obvious ways to identify new potential antigens in newly sequenced microbial genomes is through similarity searching. assuming that we know the sequence of one or more extant antigens, we can make use sequence searching programs of various complexity and sophistication, such as blast or fasta, to identify similar sequences in the target genome. this set of selected candidate antigens can then be prioritised for further theoretical and ultimately experimental validation. obviously, all the same caveats that exist for any sequence search hold here also: which thresholds are appropriate? are apparently high-scoring matches real or an artefact of search methodology? this process also presupposes that enough known antigens are available so that such searches can be comprehensive and thus effective. such compilation is the role played by the database. in the last decade, factory-scale experimentation allied to extensive literature mining has generated many functional immunology databases. databases, such as syf-peithi [65, 66] , which focus on properties of cellular immunology, and look primarily at data relevant to mhc processing, presentation, and t-cell recognition have existed since the mid 1990s. arguably, the best such database is the hiv molecular immunology database [67] , although clearly the depth of the database is at the expense of breadth and generality. it archives cd4+ and cd8+ t-cell and b-cell epitopes derived from the virus. it also includes detailed biological information regarding the response to the epitope, including its impact on long term survival, common escape mutations, whether an epitope is recognized in early infection, and curated alignments summarizing the epitope's global variability. other recent databases include mhcbn [68, 69] , which contains 18,790 mhc-binding peptides, 3,227 mhc nonbinding peptides, 1,053 tap binders and nonbinders, and 6,548 t-cell epitopes. epimhc [70] is a relational database of naturally occurring mhc-binding peptides and t-cell epitopes. presently, the database includes 4867 distinct peptide sequences from various sources, including 84 tumor antigens. two databases in particular, warrant special attention, albeit for different reasons. they are antijen [71] , formerly known as jenpep [72, 73] , and iedb [24] . antijen seeks to integrate a wider range data than is archived by other databases. implemented as a relational postgresql database, antijen is sourced from the primary literature and contains over 24,000 entries; it includes quantitative kinetic, thermodynamic, functional, and cellular data within the context of immunology and vaccinology. as well as t-cell and mhc binding data, antijen holds over 3,500 entries for linear and discontinuous b-cell epitopes, and includes measurements of peptide interactions with tap transporter and peptide-mhc complex interactions with t-cell receptors (tcr), as well as immunological protein-protein interactions. iedb is a database lavishly funded by the nih, which addresses issues of biodefence. as we have said, it is on a much larger scale than any other similar database, and benefits significantly from the input of 13 dedicated epitope sequencing projects. these exist, in part at least, to populate the database. iedb has largely eclipsed other efforts in functional immune databases. however, it does not, as a priority, address antigenicity at the whole protein level. at this point it is worth discussing the distinction between functional annotation and the objective discussed here. generally speaking, the function that a protein performs within the context of its organism of origin is irrelevant to its status as an antigen. here the ubiquity and multiple meanings of the word antigen are of little if any help. a protective antigen is a protein which is recognised and recalled by the host. this characteristic does not seem to be linked to the fact that a protein is an enzyme or a dna binding protein, nor does logic require such a link. thus identifying function is not a necessary condition for a protein to be an antigen, though the unequivocal identification of certain functions, such as being a virulence factor for example, greatly increases the probability that it will be such. concerning antigens, however, these databases, although replete with information concerning individual b cell epitopes, t cell epitopes, and major histocompatibility complex (mhc) binding peptides, remain otherwise partial and incomplete. their focus is on the epitope, not the antigen. there are many antigens for which specific epitope or mhc binding information is not currently available, yet many such antigens are known experimentally to induce either or both innate or adaptive immune responses. fortunately for the future of vaccine design and discovery specific antigen-orientatedrather than epitope-orientated -databases are now becoming available. arguably, the clearest and most unequivocal example of an antigenic protein is the so-called virulence factor (vf). these proteins are able to undertake the colonization of a host organism and/or induce disease. they are the front-line weapons in the pathogenic arsenal. analysis of known pathogenic species, such as vibrio cholerae or streptococcus pyogenes, has enabled the recognition of recurrent "systems" of vfs and toxins that may total 40 or more distinct proteins. these may exist as discrete pathogenicity islands or be spread more widely in the genome. traditionally, classification of vfs has categorised them as belonging to several thematic groups: adherence/colonization factors, invasions, exotoxins, transporters, iron-binding siderophores, and miscellaneous cell surface factors. a broader definition groups vfs into three classes: (1) "true" virulence factors; (2) vfs associated with the expression and regulation of class 1 vf genes; and (3) vfs required for the colonization of the host [74] . a number of databases that archive vfs have been reported. the virulence factor database (vfdb; url: http://www.mgc.ac.cn/vfs/) contains 16 characterized bacterial genomes with an emphasis on functional and structural biology and can be searched using text, blast, or functional queries [75, 76] . tvfac (los alamos national laboratory toxin and virulence factor database; url: http://www.tvfac.lanl.gov/) contains genetic information on over 250 organisms and separate records for thousands of virulence genes and associated factors. the fish pathogen database (url: http://www. fishpathogens.eu/vhsv/index.php), set up by the bacteriology and fish diseases laboratory, has identified over 500 virulence genes using fish as a model system. pathogens studied include aeromonas hydrophila, edwardsiella tarda, and many vibrio species. candida albicans virulence factor (candivf) is a small species-specific database that contains vfs, which may be searched using blast or a hla-dr hotspot prediction server [77] . phi-base is a noteworthy development as it integrates vfs into a cohesive whole a variety of pathogens of plants and animals [78] . obviously, antigens need not be vfs, they need only be accessible to immune surveillance and need not be directly or indirectly involved in infectivity. because of this, other types of database are required, able to capture and contain a wider tranche of relevant data. in the recent past, another database has been developed: anti-gendb [79] contains a compilation of over 500 antigens drawn from the literature and other immunological resources. it marks a new beginning in immunoinformatics, signalling a switch away from the peptide epitope and toward the whole protein antigen. these antigens come from 44 important pathogenic species. in antigendb, a database entry contains information regarding the sequence, structure, origin, etc. of an antigen with additional information such as b and t-cell epitopes, mhc binding, function, gene-expression and post translational modifications, where available. anti-gendb also provides links to major internal and external databases. antigendb will be updated on a rolling basis, with the regular addition of antigens from other organisms. this database will form the core of future attempts to predict antigens both by sequence similarity and using more recondite analysis. at this point it is worth mentioning the issue of thresholds. clearly, when one runs a sequence search, using blast for example, one might generate huge lists of near-identical proteins or get no hits at all; and, for that matter, we could also obtain almost any intermediate result. the issue is to judge which result is useful and which is not. this typically equates to setting a threshold, above which we anticipate usefulness and below which we might expect a lack of utility. setting such thresholds is however no easy task. they are dependent on the nature of the family in question. for the lipocalin family [80] [81] [82] , for example, hits are still valid in terms of structure and function at levels that would simply be noise for most other protein families. thus thresholds are family dependent, as well as problem dependent. empirically-selected cut-offs are thus the order of the day, but much thought and experimentation is needed in order to select appropriate values. as well as seeking similarity to known antigens, there is another, quite prevalent, idea that is deserving of comment: that the immunogenicity of protein is determined solely by its lack of similarity to the host. what we search for is some quantitatively-meaningful measure of the "foreignness" of a protein which correlates highly with its immanent immunogenicity. in this context, what we mean by the word "foreignness" is the evolutionary distance between the hosta man or a cow or mouse -and the candidate antigen, or the organism that produced it, or both. some consider this to be the prime factor determining the potential immunogenicity of a protein [83, 84] . clearly, since we are dealing with proteins and carbohydrates and the like, this evolutionary distance must be specified in terms of their molecular structures, or more likely their sequences, rather than in terms of morphological features. the potential importance of such a concept is supported by the observation that immune systems are actively educated to lack reactivity when presented with self-proteins [85] , a processoften called immune tolerancewhich is generated via epitope-specific mechanisms including clonal anergy, receptor editing, and clonal deletion [86] [87] . but how can we progress beyond this rather inexactly-specified philosophical standpoint to something which is practically useful when we select vaccine candidates? a potentially more useful way to express this conjecture is that the likelihood that a protein is immunogenic is solely a function of that protein's dissimilarity to the whole host proteome at the sequence level. or, to be more precise, how close in terms of sequence similarity is the candidate to the closest or most similar member of the host proteome. most sequence software is well suited to solving this problem, since it is precisely this problem that they were written to address. more difficult is assessing average dissimilarity to the whole proteome, a problem compounded when we use the similarity of overlapping peptide fragments rather than looking at the similarity at the level of global sequence alignment. in terms of choosing the length of such fragments, the epitope would seem to be the most logical choice, since this immunological quantum is the moiety most likely to be recognised during the immune response. yet even at the level of the epitope, a peptide of say 10 amino acids, even one mismatch in an otherwise perfect match can be significant, since such sequence differences, comprising a single amino acid, may exacerbate or abrogate neutralizing antibodies binding to a particular antigenic protein. moreover, the crossreactivity of a single high-affinity monoclonal antibody is rather different in nature to the cross-reactivity of large set of less affine polyclonal antibodies, and so too their ability to tolerate amino acid mismatches. it will also vary between individuals, since the immunization history of each organism will dictate to a large extent the recognition of epitopes. our understanding of epitopes can inform our understanding of mismatch tolerance, since the affinity of t-cell epitopes is more dependent on the possession of suitable anchor residues than it is on the possession of non-anchor residues. having said, the dogma of anchordependent affinity was long ago debunked, since all residues make some kind of contribute to affinity, entropic or enthalpic, although generally it is right to say that socalled anchors do make more significant contributions. our understanding of the structural-basis that determines the affinity of antibody-mediated epitopes is much less assured and complete, and the underlying thermodynamic causes of affinity, if strict causes they are, typically only become clear when high resolution structural data combines with measured thermodynamic metrics. likewise, when one looks not at a representative individual, but at the whole population, then the deletion of a single protein, within one host versus another, can render candidates previously valid and immunogenic as suddenly neither. again, these are difficult issues; as yet, they remain unresolved. given the hypothesis that immunogenicity is in some sense mediated by the level of similarity between a pathogenic protein and the host proteome, we have, in as yet-unpublished work, sought to bench-mark sequence similarity analysis as a means of quantifying the differences between populations of antigens and non-antigen. to that end, we identified sets of 100 known antigenic and 100 non-antigenic protein sequences derived from a variety of sources: allergens, bacteria, fungi, parasites, tumours and viruses. these were compared to the human and mouse genomes using standard sequence similarity searching protocols. whole pathogen proteomes were also aligned to these host proteomes. most antigenic and non-antigenic sequences were observed to be non-redundant; this implies a lack of clear homologues between pathogens and the human or mouse proteomes, although a number of parasite antigens were found to have a much higher level of similarity. these proteins comprised heat shock proteins, catalases, and enzymes involved in hydrolysis. these protein families are structurally conserved, though they might display significant functionality diversity. we also used statistical approaches such as the nonparametric mann-whitney test to assess if comparisons between the two populations were significant. the statistical null hypothesis was accepted in most cases, signifying that the effect presumably resulted solely from chance. the mann-whitney test supported the observations from sequence similarity analysis. we were unable to determine a threshold or cut-off based on the hypothesis of non-redundancy to the host's proteome. these results suggest that this is not in itself a solution to the identification of antigens. a variant based on fragments may be more successful, and this is clearly an issue crying out for further, deeper research. there are, of course, many other ways to approaches identifying antigenic proteins. one notable way, is looking for the horizontal transfer of so-called pathogenicity islands, clusters of pathogenic proteins acquired by transfer between micro-organisms. detection of such islands, which are typically large gene clusters with an atypical yet characteristic g + c content, can in turn lead to the identification of antigenic proteins [88] [89] [90] [91] . analysis at the nucleic acid level rather than at the protein level can facilitate the discovery of virulence proteins, perhaps using similar approaches to that used to identify cpg islands [92] [93] [94] [95] . however, rather than look at nucleic acid sequences, or at protein sequences directly, a new approach, based upon alignment-free techniques, has been developed which shows significant potential; we examine this next. most in silico approaches to predicting antigens make use of bioinformatics tools of one kind or another. such tools can identify membrane proteins, signal peptides, or lipoproteins with some success, yet most algorithms still rely on sequence alignment to identify characteristic sequence relationships or motifs characteristic of antigens. this may prove problematic in several ways. some proteins formed through divergent or convergent evolution lack obvious sequence similarity, although they may share similar structures and/or biological properties [96, 97] . in such a situation, alignment-based approaches may produce ambiguous results or fail spectacularly. many methods presuppose a direct sequence relationship such as that which can be revealed by simple sequence search techniques, such as blast. this is not always the case. immunogenicity, as a property, may instead be encoded within the sequence and structure of a protein in such a cryptic and subtle manner as to go beyond the conventional limitations imposed when one seeks direct identification by sequence alignment protocols. likewise, the discovery of truly novel antigens will be frustrated by their lack of similarity to antigens of known provenance. as a departure from alignment-dependent techniques, which dominate immunoinformatics as much as they do bioinformatics, we have implemented an approach which seeks to discriminate between candidate vaccines and nonantigens, using an alignment-free sequence representation [98, 99] . rather than concentrate on epitope and nonepitope regions, the method uses data on immunoprotective antigens derived from pathogenic sources to derive statistical models for predicting wholeprotein antigenicity. in attempting to overcome the limitations imposed by alignment-dependent sequence similarity methods, we have implemented a novel alignment-independent method for antigen identification based on auto cross covariance (acc) transformation of protein sequences into uniform equal-length vectors. the acc transform is a protein sequence mining method originally devised by wold et al. [100, 101] , which has found much application to the quantitative structure-activity relationships (qsar) study of peptides and also for the classification of proteins [102] [103] [104] [105] [106] [107] [108] [109] . the principal properties of the amino acids were represented by z descriptors [110] [111] [112] , which describe amino acid hydrophobicity, molecular size and polarity. the acc transformation accounts for neighbour effects, i.e. the lack of independence between different sequence positions. initially, we sought to develop models able to distinguish immunoprotective proteins from the general microbial proteome. we applied acc pre-processing to sets of known bacterial, viral and tumour antigens and developed alignment-independent models for identifying antigens. in a separate paper, extra models were added which address fungal and parasite antigens. for bacterial, viral and tumour antigens, models had prediction accuracies in the 70% to 89% range [98, 99] [113] . for the parasite and fungal antigens, models had good predictive ability with 78% to 97% accuracy under internal cross validation in 7 groups. under external validation, they gave 69% sensitivity ranking the true immunoprotective proteins in the first 25% of their proteomes. the models were implemented in a server for the prediction of protective antigens and subunit vaccines, which we have christened vaxijen [98] (url: http:// www.darrenflower.info/vaxijen). the accuracy values noted above are indicative of an approach that is more accurate than has been seen before; for example, for b-cell epitope prediction. vaxijen is an imperfect beginning; future research will yield significantly more insight as the number of known protective antigens increases [79] . there are several bioinformatics problems unique to immunology: the foremost and greatest is the challenge of accurately predicting immunogenicity. the successful computational prediction of immunogenicity may manifest itself at one extreme in the identification of a t-cell or b-cell epitope. this is mediated by straightforward if not uncomplicated molecular recognition events, which can be understood through properly exploring relationships between fundamental physical propertieshydrogen bonding and lipophilicity for exampleand apparent biological activity. establishing such structureactivity or property-activity relationships is a considerable concern of current immunoinformatics. at the other extreme, we encounter an altogether more complex phenomenon, where we see immunogenicity made manifest in the accurate estimation of antigenicity at the whole protein level. this is a system property; that is a property of the whole immune system rather than an individual molecular recognition event. at present, this is very much a secondary aspect of modern day immunoinformatics. the task of predicting whole protein immunogenicity is probably several orders of magnitude more complex and demanding than say predicting the binding of peptides to an mhc or even of predicting the binding of a pmhc complex to a tcr. this is not to say that such calculations are in any way trivial or lacking difficulty, but rather than that such complex prediction exercises are themselves subsumed in the task of predicting immunogencity at the system level. in our view, and in the view of other commentators, the clinical manifestation of the immunogenicity of a vaccine antigen, as opposed to the immunogenicity of an isolated epitope, arises as the very complex amalgam of many intertwining intrinsic and extrinsic factors, operating at various length-scales and at various different rates. such factors include host-side properties and pathogen-side properties, as well as protein-side properties that arise more or less exclusively from the protein itself. see figure 3 . so-called host-side properties are properties intrinsic to the immune system of the host. they include the possession of b-cell epitopes or t-cell epitopes, as recognised by the adaptive immune system, or the possession of pathogen-associated molecular patterns or pamps, which are recognised by pattern recognition receptors, such as toll-like receptors (tlrs), part of the innate immune system. pathogen-side properties are properties intrinsic to the pathogen as a whole organism. they include the expression level evinced by an antigen, the time-course of its expression and secretion, and also its subcellular location. protein-side properties include the state of aggregation exhibited by a candidate vaccine and any post-translational danger signals that the protein might possess. some of these properties have been discussed before. thus one would expect, at least naively, that a bona fide vaccine antigen would be both highly expressed and available for immune surveillance, as well as possessing epitopes that the host can recognise, where as a protein which is not immunogenic would lack such characteristics. identifying and predicting this diverse tranche of properties is a problem and thus a challenge. indeed, each component part is a challenge unto itself. consider the prediction of a t cell epitope: the best understood and most accurate of immunoinformatic prediction problems. such an epitope needs to be processed properly into a population of different peptides, and for the processed peptides to exist in a reasonable amount, and then be bound by an mhc with reasonable affinity, before being recognized appropriately within the context of a pmhc complex by a tcr. this then is a complex and contingent process, similar to a generic markov process, comprised of many stages which are themselves dependent on preceding steps. in terms of both mechanism and what steps-within-steps there may be, many of these steps are less than adequately understood. however, each step remains amenable to statistical evaluation. all stages are inherently predictable given an appropriate accumulation of relevant data. the situation is very similar but not identical for biopharmaceuticals. in this case, we can dispense with the pathogen and replace it with several classes of extrinsic factors which include amongst others product manufacture, patient health, and medication strategy. acute illness, particularly systemic illness, or latent bacterial or viral infections such hcv, can have a most profound effect on the manifestation of immunogenicity. likewise, a patient's genetics may alter profoundly the immune reaction to biopharmaceuticals, most obviously, the mhc haplotype will affect host t-cell responses. the nerve program is an expert system for vaccine antigen discovery, which addresses in a practical way some of the issues of prioritisation discussed above. the program has been developed to help automate the process of reverse vaccinology [114] . the identification and prioritising of potential orfs using nerve comprises 6 stages. firstly, the prediction of subcellular localization; then whether the protein is an adhesin; followed by the identification of transmembrane domains; then the protein is compared with both the human and pathogen proteomes; after which the protein is finally assigned a suggestive function. vaccine candidates are thus filtered and ranked, and a list of proteins is produced graded by precedence and the probability that it will be an immunogenic antigen. but nerve and other extant programs, such as dynavacs [115] , tasked with such a confounding task necessarily fall far short of what is needed or indeed what is possible. there needs to be a concerted and farreaching effort to address this issue. the most direct line of attack is to address first the subcellular location issue, as we discussed above. likewise, we can deconstruct the antigen presentation pathway, building models for each step and then integrating them into a fully functional model. we can develop empirically-based, statistically-grounded approaches -of which vaxijen [98, 99, 113] is merely the vanguardthat have their basis in emerging antigen databases. yet all of this is just the beginning. we need to factor in b-cell and antibody mediated issues using structural data. we need to properly assess the role of aggregation, of expression levels, of post-translational danger signals and the possession of pathogen-derived pamps, as well as the ability of large molecule and small molecule adjuvants to raise the intrinsic immunogenicity of candidates to useful levels. we should note in passing that many hostderived molecules are also labelled pamps; these include fibronectin, hsp70, and heparan sulphate, amongst others. however, these molecules are endogenous activators of innate immunity and not relevant to this discussion, though they may figure in auto-immune scenarios. vaccines are a good thing; putting caveats and cavils to one side, no one sensible really doubts the truth of this statement. vaccines have proved their worth time and again, but for them to continue to prove their worth we must find new ways of making them. almost all present-day vaccines are mediated by antibodies, and the majority target viral diseases. unfortunately, we are now running short of target diseases which fit this restrictive bill. many of the pathogens responsible for current recalcitrant and emergent diseases are, and are set to prove, much more demanding and difficult to target. in many senses, the low hanging fruit has been cut down and now the fruit we most desire is well out of reach. many diseases, including the who's big three diseases: hiv, tb, and malaria, are much more complicated functionally and/or structurally, and cellular, as opposed to humoral, immunology is tasked with defence against these dark arts. peptide vaccines and those based on apcs are important new if as yet unspectacular directions for research in vaccine discovery, yet modern strategies for vaccine figure 3 factors underlying immunogenicity as elaborated in the text, the phenomenon of immunogenicity can be explored through the diversity of underlying individual factors contributing to the instigation of the immune response. these factors can be assigned to the host (epitope recognition), the pathogen (location and expression level), and also factors intrinsic to the protein antigen itself, such as the possession of post-translational danger signals. development hinge primarily upon the effective search for vaccine antigens, at least for the proactive search for vaccines targeting long-standing but untreated diseases rather than the reactive response to so-called pandemics. such antigens, once discovered, will, in time, and with careful manipulation and an appropriate delivery system and/or appropriate adjuvant, become first candidate subunit vaccines and, after proper clinical evaluation, the vaccines of tomorrow. there are, as we have outlined, many competing ideas, thoughts, and concepts that can help us in our quest. certain of these hypothesis we have had cause to outline, are indubitably persuasive, even compellingly convincing, yet in execution many such methods fall far short of our desires. no single approach, however promising, is able to deliver on its promise. the reason for this failure is comparatively simple if uncompromisingly unsatisfying; as we are dealing with over simplified abstractions, we cannot hope to capture what is necessary for prediction by looking in a relatively superficial way at a single contributory factor, since protein immunogenicity arises from many, many factors. this is not a bioinformatics problem that is easily solved; instead it is like protein secondary structure prediction which has resisted all attempts over many decades, so obscure and recondite and far removed from direct experience a problem as it is. factors mediating protein immunogenicity are many and include host-side properties -possession of b or t cell epitopes for example -and pathogen-side propertiesprotein expression levels and sub-cellular location -as well as its aggregation state and the possession of posttranslational danger signals. a candidate vaccine should be highly expressed, available for immune surveillance, and possess epitopes that the host recognises. predicting such diverse properties remains challenging, though several contributing factors can be reliably predicted. yet, no one factor is itself enough. what we need is an integrative, systems biology approach to the problem. as the poet maya angelou so neatly puts it: "we all should know that diversity makes for a rich tapestry, and we must understand that all the threads of the tapestry are equal in value no matter what their colour." no one method is universally applicable and successful; rather we need to integrate several equally-valid, equally-partial methods and draw from their synergy, useful, helpful data. this is the as yet unattained goal; yet as these are issues of such importance even a partial solution would be prize enough. computer-aided biotechnology: 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their application to a structure activity relationship for oxytocin peptide analogs peptide binding to the hla-drb1 supertype: a proteochemometrics analysis proteochemometrics mapping of the interaction space for retroviral proteases and their substrates proteochemometrics analysis of substrate interactions with dengue virus ns3 proteases generalized modeling of enzyme-ligand interactions using proteochemometrics and local protein substructures rough set-based proteochemometrics modeling of gprotein-coupled receptor-ligand interactions improved approach for proteochemometrics modeling: application to organic compound-amine g protein-coupled receptor interactions melanocortin receptors: ligands and proteochemometrics modeling proteochemometrics modeling of the interaction of amine g-protein coupled receptors with a diverse set of ligands peptide quantitative structure-activity-relationships, a multivariate approach multivariate parametrization of 55 coded and non-coded amino-acids new chemical descriptors relevant for the design of biologically active peptides. a multivariate characterization of 87 amino acids bioinformatic approach for identifying parasite and fungal candidate subunit vaccines. the open vaccine journal nerve: new enhanced reverse vaccinology environment dynavacs: an integrative tool for optimized dna vaccine design fell da: enzymes, metabolites and fluxes computer aided selection of candidate vaccine antigens we should like to thank the referees for their helpful an insightful comments. this article has been published as part of immunome research volume 6 supplement 2, 2010: computational vaccinology: state-of-the-art assessments. the full contents of the supplement are available online at http://www.immunome-research.com/supplements/6/s2. the authors declare no competing financial interests. key: cord-356064-q56jnhss authors: bartel, sebastian; doellinger, joerg; darsow, kai; bourquain, daniel; buchholz, rainer; nitsche, andreas; lange, harald a title: proteome analysis of vaccinia virus ihd-w-infected hek 293 cells with 2-dimensional gel electrophoresis and maldi-psd-tof ms of on solid phase support n-terminally sulfonated peptides date: 2011-08-01 journal: virol j doi: 10.1186/1743-422x-8-380 sha: doc_id: 356064 cord_uid: q56jnhss background: despite the successful eradication of smallpox by the who-led vaccination programme, pox virus infections remain a considerable health threat. the possible use of smallpox as a bioterrorism agent as well as the continuous occurrence of zoonotic pox virus infections document the relevance to deepen the understanding for virus host interactions. since the permissiveness of pox infections is independent of hosts surface receptors, but correlates with the ability of the virus to infiltrate the antiviral host response, it directly depends on the hosts proteome set. in this report the proteome of hek293 cells infected with vaccinia virus strain ihd-w was analyzed by 2-dimensional gel electrophoresis and maldi-psd-tof ms in a bottom-up approach. results: the cellular and viral proteomes of vacv ihd-w infected hek293 cells, uv-inactivated vacv ihd-w-treated as well as non-infected cells were compared. derivatization of peptides with 4-sulfophenyl isothiocyanate (spitc) carried out on ziptipμ-c18 columns enabled protein identification via the peptides' primary sequence, providing improved s/n ratios as well as signal intensities of the psd spectra. the expression of more than 24 human proteins was modulated by the viral infection. effects of uv-inactivated and infectious viruses on the hosts' proteome concerning energy metabolism and proteins associated with gene expression and protein-biosynthesis were quite similar. these effects might therefore be attributed to virus entry and virion proteins. however, the modulation of proteins involved in apoptosis was clearly correlated to infectious viruses. conclusions: the proteome analysis of infected cells provides insight into apoptosis modulation, regulation of cellular gene expression and the regulation of energy metabolism. the confidence of protein identifications was clearly improved by the peptides' derivatization with spitc on a solid phase support. some of the identified proteins have not been described in the context of poxvirus infections before and need to be further characterised to identify their meaning for apoptosis modulation and pathogenesis. despite remarkable progress in the control and treatment of infectious diseases, the problem of emerging and re-emerging pathogens is likely to be one of the main issues of medical science and public health in the twenty-first century [1] . in this respect viral diseases are of particular concern, because advances in the field of antiviral drugs have lagged behind those regarding bactericidal drugs and antibiotics. it was shown by the emergence of the severe acute respiratory syndrome (sars) that new members of neglected virus families can cross into humans from unsuspected reservoirs, making rapid advances in our understanding of virushost dynamics necessary [2] . in this regard poxviruses are of particular importance. despite the success of the who-led smallpox eradication programme, other related human-pathogenic poxviruses remain a considerable threat. in particular, the 2003 outbreak of human monkeypox virus in the united states of america pointed out the imminence posed by zoonotic pox infections originating from animal-borne viruses [3] . moreover, an increasing number of human cowpox virus infections, especially affecting younger people lacking smallpox vaccination, have been reported in europe in recent years [4] . in addition to the threat of zoonoses, the relevance of a deeper understanding of the interactions of poxviruses with their host cells is pointed out by considering the classification of smallpox as a category a bioterrorism agent by the centers for disease control and prevention (cdc), especially since no acceptable treatment is available and the immunity in the population is declining [5] . in contrast to many other viruses, the ability of poxviruses to productively infect a given cell is not determined at the level of specific host receptors, but is regulated downstream of virus adsorption to the cell surface and virus entry by intracellular processes which for the most part are unexplored [6] [7] [8] [9] [10] . since the permissiveness of orthopoxviruses is therefore dependent on the hosts proteome set, the identification of cellular proteins affected by the protein products of the so-called viral host-range genes with proteomic approaches will be a starting point for further functional protein analysis. the characterization of the vaccinia virus (vacv) virions' proteome has enlightened the protein equipment of the virus at the entry stage of infection [11] . furthermore, virus-host dynamics have been analysed by yeast two-hybrid (y2h) studies to identify interaction partners [12] . no closer look at the dynamic changes of the human proteome during a vacv infection has been taken yet on a global scale. in this report an infection of human embryonic kidney (hek) 293 cells with well-studied vacv was chosen as a model system. uninfected hek 293 cells serve as a control, while vacv-infected hek 293 cells inactivated with uv enable the study of the influence of the virions' proteins on the cellular proteome. the investigation of infection effects on the expression of the cellular proteome was carried out in the late phase of virus replication. an offline-coupling of two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis (sds-page) and matrix assisted laser desorption/ionisation time of flight mass spectrometry (maldi-tof ms) was used in a bottom-up approach. in-gel trypsinated proteins from relevant spots of the 2-d gels are unambiguously identified via mascot database search of the peptide mass fingerprint (pmf) recorded in positive ion reflectron mode of the maldi-tof ms. identification rate was improved by obtaining additional sequence data of selected peptides in post source decay (psd) mode, whereas a considerably advanced data quality was achieved by n-terminally derivatising the peptides on a ziptipμ-c18 solid phase support with 4-sulfophenyl isothiocyanate (spitc) prior to ms measurement. psd spectra of spitc derivatized peptides improve protein identification efficiency improved protein identification by combined mascot search of pmf and psd spectra is shown exemplarily in figure 1 for the spot c-5, human prohibitin (phb). database search of the underivatised pmf has not led to unambiguous protein identification. the best hits held protein scores between 55 and 57 at a threshold value for significance of 64. among these, prohibitin is ranked third with a score of 56. after spitc derivatization, the ten most intensive m/z values of the derivatised pmf were selected by an ion gate for psd fragmentation. the amino acid sequence of selected peptides can be distinguished considering the series of y-fragments. for the peptide of m/z 1365.0 the sequence fdagelitqr (figure 1c ), for the peptide of m/z 1401.1 the sequence dlqnvnitlr (data not shown) and for the peptide of m/z 1160.3 the sequence iftsi[ge]dyder (data not shown) was confirmed. sequencing enables the mapping of a peptide to a certain protein within the confidence interval of its m/z value and increases its mascot score. if the sequenced peptides do not represent homologue sections in the human proteome even one peptide sequence can be significant. in case of prohibitin each of the three peptides alone was sufficient for unambiguous protein identification. the combined search of pmf and psd spectra of spitc-derivatised peptides identifies spot c-5 as being human phb with a mascot ion score of 237 at a threshold for significance of 34. the peptide mass fingerprint of tryptic digested protein standard serum albumin (bovine) before and after spitc derivatization is shown in figure 2 . the intensity is normalised on m/z value of 1479.9 of underivatised pmf spectra. the spitc derivatization introduces a mass shift of 215 da to the peptides and has strongly decreased their intensities. many peptides could no longer be detected after derivatization, which negatively affects the sensitivity of protein identification. the underivatised protein standard was unambiguously identified with a mascotscore of 129 and a significant threshold of 61, but could no longer be identified from the pmf after spitc derivatization. the influence of the n-terminal sulfonation on the quality of psd spectra is shown exemplarily in figure 2c and 2d for the m/z value of 1480.4 of 10 pmol tryptic digested bsa. the intensity is normalised on the precursor ion of the underivatised psd spectra. despite the reduction of the precursor ions' intensity of 95%, the signal to noise (s/n) ratio of the peaks in the psd spectra as well as the frequency of fragmentation were clearly increased by the n-terminal sulfonation. the improved coverage of the precursor ion y-series raised the mascot ion score of the peptide with the mass of 1480.4 da from 10 to 88. the sulfonation of primary amines was done in aqueous solution at slightly alkaline ph value (ph = 8.6). purification of peptides was carried out with ziptipμ-c18 columns according to the principle of reversed phase chromatography. generally, spitc derivatization of peptides can be performed in solution prior to column binding as well as solid phase supported. both sulfonation methods were modified compared to the literature with respect to signal intensity in order to enable protein identification with maximum sensitivity [15] [16] . exemplarily, the resulting pmf spectra for in-solution trypsinated bsa has been compared with the solid phase supported approach concerning signal intensity, which is shown in figure 2e /f. the signal to noise ratios (s/n) of m/z values used for identification were about 2 to 4 times higher for peptides derivatised on solid phase support than for the ones sulfonated in solution. the overall signal of pmf spectra in the mass range between 1000 and 2500 m/z is about 7 times larger for the standard derivatised on c18 columns. the improved s/n ratio and the increased signal intensity of the solid phase supported approach in psd mode enlarges the coverage of the precursor ions' y-series and simplifies the automation of peak picking. the mascot score of the database search of ten accumulated psd spectra was 176 for the solid phase supported approach and 139 for in solution sulfonation at a threshold value for significance of 33. protein identification via maldi-psd-tof ms spectra of on solid phase support spitc derivatized peptides the recording of psd spectra from m/z values of peptide mass fingerprints provides information about the peptides' primary structure and enables the unambiguous identification of proteins from few peptides with significant mascot ion scores by an analysis of fragmentation patterns. the difficulties of protein identification with maldi-psd-tof ms spectra are low s/n ratios of fragments, incomplete sequence coverage and complex fragmentation patterns as a cause of random charge stabilisation. the protonation of peptides during the maldi process is mediated by carboxyl groups of the matrix and mainly stabilised at primary amine groups of the n-termini and the lysine residues. the stabilised charge induces different fragmentations of the same ion. the derivatization of peptides with spitc binds a negatively charged sulfonic acid to the peptides' n-terminus [13] . this negative charge neutralises the protonation of c-terminal fragments which are no longer detectable in mass spectrometry. primarily, the psd spectra of spitc-derivatised peptides show fragments of the y-series, whose appearance among the n-terminal fragments is promoted by the large electron density around the peptide bond. the derivatization with spitc enables the detection of complete y-series with good s/n ratios and offers the elucidation of the peptides' primary sequence. the unambiguous mapping of peptides with completely known primary sequences to proteins registered in databases is remarkably simplified and is expressed in increased mascot scores [14] . however, the application of spitc derivatization for protein identification from complex mixtures is limited by the reduction of the peptides' signal intensities following n-terminal sulfonation. this degradation of sensitivity could be decreased by an optimization of the reaction conditions. for increased sensitivity the n-terminal sulfonation of peptides bound on c18 solid phase support is preferable compared to an in solution approach since signal intensity is increased and quality of psd spectra is raised. the comparison of the proteome analysis of vacv ihd-w-infected hek293 cells with non-infected control cells 9 h post infection has enabled the identification of 24 human proteins, whose expression has been regulated by infection, as well as of 3 viral proteins. since the genome of vacv ihd-w is not available, the identification of viral proteins occurred via sequence homologies of the vacv strains western reserve and copenhagen. afterwards peptide sequences were derived from spectra of 13 spots representing proteins regulated in their expression, whose database search gained no result. single-base substitutions among different vacv strains, which led to amino acid replacement, might have resulted in different peptide mass fingerprints. psd spectra are in correlation to the confidence interval of the m/z values of the precursor ion. therefore, protein identification via mascot search is disabled by mass shifts of precursor ions resulting from an amino acid replacement. de novo sequencing of viral proteins could not be assured because of the polyproteonality of 2d-gel spots. the unambiguously identified differences in the expression profile of hek 293 cells resulting from vacv ihd-w infection are discussed in the following sections. the infection of hek293 cells with active and inactivated vaccinia virus ihd-w significantly affects the cells' energy metabolism. the expression of fructosebisphosphate aldolase a (a-1), which acts as a central enzyme in the glycolysis by catalysing the retro-aldol cleavage of fructose-1,6-bisphosphate into dihydroxyacetone phosphate and glycerinaldehyde, is increased by both types of infection [15] . also the enhanced expression of the enzyme malate dehydrogenase (a-2) is directly associated with a boost in the energy metabolism, which serves the viruses' need for energy for dna replication. this enzyme is an integral part of the citric acid cycle by catalysing the oxidation of malate to fumarate [16] . the resulting reduction equivalent nadh/h + transfers electrons to the electron transport chain which builds up a proton gradient at the inner mitochondrial membrane, resulting in atp-synthesis catalysed by the h + -transporting two-sector atpase (a-3, a-4) [17] . a modification of the α-chain precursor of the h + -transporting two-sector atpase (a-3, a-4), resulting in spot migration, is triggered by both types of infection and has been detected as well as an overexpression of the mtdna stabilising atpase family aaa domain containing 3a (a-6) protein, whose existence has just been evidenced at transcript level [18] . another energy metabolism-related protein whose expression is up-regulated by the vacv ihd-w infection is the bsubunit of the electron transfer flavoprotein (a-5) which acts as a specific electron acceptor for several dehydrogenases, e. g. malate dehydrogenase (a-2), and transfers them to the respiratory chain [19] . all changes detected in the human proteome profile caused by the infection which are related to energy metabolism indicate an enhancement of the metabolic rate of the glycolysis as well as of the oxidative phosphorylation in order to fulfil the viruses' energy need for replication. the expression of the eukaryotic translation initiation factor 4h (eif-4h) (b-3) is up-regulated by infectious and inactivated vacv ihd-w. the protein eif-4h stimulates protein biosynthesis, atp hydrolysis and helicase activity of eif-4a. enzymatic investigations show that the affinity of eif4a to rna is increased two-fold and helicase activity four-fold by an interaction with [20] . the regulation of eif-4h may contribute to an enhancement in protein biosynthesis which is required by the virus' need for protein expression. further on an increased expression of nuclear protein hcc-1 (cip29) (b-4) is denoted in both types of infected hek293 cells. this protein possesses a binding domain for ss and dsdna and is postulated to be part of the ribonuclein complex. interactions with the rna-helicases ddx39 and bat 1 are verified which proves the influence of hcc-1 on the transcription of dna. the overexpression of hcc-1 in hek293 cells is known to decrease the cells' growth rate [21, 22] . several members of the spliceosome, heterogeneous nuclear ribonucleoprotein b1 (b-5), novel protein similar to small nuclear ribonucleo-protein polypeptide a (b-6), heterogeneous nuclear ribonucleoprotein m (b-7), are modulated in both types of vacv ihd-w-infected hek2093 cells. this heterogeneous protein complex splices introns of the hnrna. since the poxvirus genome has no introns and splicing of mrna is therefore obsolete, viral effectors may control the cells' protein biosynthesis by interfering with the cellular hnrna processing [23, 24] . far upstream element-binding protein 1 (fubp1) (b-8) stimulates the expression of transcription factor c-myc by binding onto far upstream element (fuse) upstream of the c-myc promoter [25] . the protein is a known link between the apoptosis cascade and the c-myc oncogene, it further possesses helicase activity for dsdna. experiments with transfected cells show that a high fubp1 expression increases the expression level of c-myc and in this way protects the cell from apoptosis, while caspase-mediated cleavage of fubp1 induces apoptosis [26] . both infectious and inactivated vacv ihd-w enhance the expression of fubp1. ewing sarcoma breakpoint region 1 (b-9) is a multifunctional protein with essential functions in gene expression, signal transduction, and mrna transport and processing. mutations in the protein-expressing gene lead to the development of ewing sarcomas and other tumours. the protein expression is suppressed after an infection with uv-inactivated as well as with active viruses [27] . prefoldin subunit 1 (b-10) is part of a heterohexamer chaperon (prefoldin) which binds cytosolic chaperonin (c.cpn) and transports target proteins to the hexamer. it is also involved in the folding of nascent peptides. gene deletions of prefoldin result in a dysfunction of the actin-tubulin cytoskeleton [28] . prefoldin subunit 1 is exclusively identified in cells infected with uv-inactivated vacv. the described alterations caused by the infection are results of the fact that the virus is taking control of the cells' gene expression as well as the protein biosynthesis and so aims for effective expression of viral proteins and the control of the cellular response. several viruses in general and poxviruses in particular are known to modulate the hosts' apoptosis pathways in order to avoid the antiviral defence mechanism at a cellular level [29] . this results in an altered expression profile of apoptosis-relevant genes caused by viral gene products. during vacv transcription double-stranded rna (dsrna) is synthesised, since the virus possesses overlapping genes on both strands of its dna. the presence of dsrna activates the dsrna-dependent serine/ threonine kinase (pkr) which phosphorylates the α-subunit of the translation initiation factor eif-2 which is essential for protein biosynthesis. this phosphorylation inhibits the translation of mrna and thereby induces apoptosis [30] [31] [32] [33] . an altered modification on the γ-subunit of the translation initiation factor eif-2 (b-1, b-2) caused by the infection was detected by a spot migration in the 2-de gel the relevance of which has not yet been determined in the literature. the dsrna-dependent pkr is usually inhibited by nucleophosmin 1 (c-7) which is ubiquitously expressed in human cells and translocates between nucleus and cytoplasm [34] . the downregulation of nucleophosmin 1 (c-7), which can not be detected in cells infected with active vacv ihd-w, suggests that it is a cellular response to the infection for the purpose of anti-viral defence. this immune response is undercut by the product of the vacv e3l gene, the putative double-stranded rna binding protein (d-1) which binds dsrna and inhibits apoptosis induction by preventing pkr from phosporylating the γ-subunit of the translation initiation factor eif-2 [35, 36] . the e3l gene product has been identified in hek 293 cells infected with active vacv ihd-w, which is in correlation to proteome analysis of vacv virions that indicate that the putative double-stranded rna binding protein (d-1) is not present in the virion but is expressed in the early replication phase. the present proteome analysis has led to the identification of several further apoptosis-relevant proteins. the expression of two isoforms of prohibitin (c-5, c-6) is decreased after infection with active vacv ihd-w. prohibitin regulates cell division, inhibits dna synthesis and sensibilises cells for apoptosis by destabilising the mitochondrial membrane [37] . beyond that, prohibitin enhances the transcription of p53 [38] . the infection of hek293 cells with active and inactivated vacv ihd-w inhibits the expression of the protein guanine nucleotide-binding protein subunit beta 2-like 1 (c-1) which is an acceptor for activated protein kinase c (pkc). pkc is then bound to the cytoskeleton and conveyed to its target proteins, the marcks proteins. the receptor is also engaged in the regulation of the src kinase which can inhibit apoptosis by a phosphorylation of caspase-8 [39, 40] . the reduced expression of the proteasome subunit alpha type-7-like (c-2) can be seen as a further cellular defence strategy. the proteasome subunit alpha type-7like (c-2) is part of the proteasome complex which cleaves peptides proteolytically at the amino acids arg, phe, tyr, leu and glu [41] . the inhibition of the proteasome in tumour cell lines induces apoptosis. the phosphatidylethanolamine-binding protein (c-3, c-4) is modified by an infection with active and inactivated vacv ihd-w, which results in an altered pi value. the protein binds to phosphatidylethanolamine which is presented at the surface of apoptotic cells, inhibits the rafkinase, prevents the activation of the map-kinase-cascade and the antiapoptotic transcription factor nf-b [42, 43] . it is to be postulated that the protein modification is associated with an altered physiological activity. in this comparative proteome analysis the 14 kda cell fusion protein (d-2) is identified in hek293 cells infected with active vacv ihd-w. its presence is predicted according to swissprot [44] . the variola homologue of the a27l gene is known to play an import role in virus penetration by fusing the outermost of the golgi-derived membranes enveloping the virus with the cells' plasma membrane. as expected, the major core protein p4a (d-3) of vaccinia virions is further identified in both infected cell culture approaches [45] . peroxiredoxin-2 (e-1) is an antioxidative enzyme which reduces hydrogen-and alkyl-peroxides and supports the antiviral activity of cd8(+) t-cells [46] . in contrast to the negative control, protein expression is enhanced by an infection with active and uv-inactivated viruses. transgelin-2 (sm22-alpha homologue) (e-2) has not been functionally characterised yet. the protein possesses an actin-binding domain and is overexpressed in cells infected with both infectious and inactivated vacv ihd-w. it seems as if human transgelin is packed into vaccinia virions and transferred from cell to cell for an unknown reason [47] . it can be hypothesised that it may serve as an anchor for the virus to move along the cytoskeleton. sorting nexin 3 (e-3) has a binding domain (px domain) for phospholipids and transports proteins within cells from organelles to membranes [48] . the protein expression is enhanced after infection of hek293 cells with active and uv-inactivated vacv ihd-w. profilin (e-4) binds actin and participates in the build-up of the cytoskeleton. as a response of extracellular signals, high concentrations of profilin inhibit the actin-polymerization which is enhanced by low profilin concentrations [49] . both types of infection increase expression of human profilin, while vacv possesses a profilin homologue by itself. chemical assisted fragmentation (caf) of peptides by nterminally sulfonation with spitc improves confidence in protein identification from psd spectra. ziptipμ-c18 columns solid phase-supported spitc derivatization is preferable compared to an in-solution approach, since s/n ratios as well as signal intensities are increased, and so the sensitivity for recording high quality psd spectra is enhanced. proteome analysis of vacv ihd-w-infected hek293 cells in the late replication phase shows the upregulation of the energy metabolism and alteration of gene expression regulation. further on the modulation of apoptosis which counteracts the cellular anti-viral response is demonstrated by the regulation of several proteins from different signalling pathways. interestingly the effects of uv-inactivated viruses and infectious viruses on the hosts' proteome are quite similar. there is no difference between the identified alterations of the energy metabolism and just one differentially expressed protein associated with gene expression and protein biosynthesis (b-10). therefore the up-regulation of the energy metabolism and alterations in the regulation of the cellular gene expression are results of virus entry and virions' proteins. however, the modulation of apoptosis is clearly affected by effects that correlate with infectious viruses. the absence of nucleophosmin in cells infected with infectious viruses, for example, may be the response on viral dsrna resulting from transcription of viral dna, as the competing apoptosis modulator putative double-stranded rna binding protein (d-1) is not a part of the virion. in part, the identified proteins have not yet been described in the context of poxvirus infections and need to be further characterised using virological methods to identify their meaning for apoptosis modulation and pathogenesis. all of the electrophoresis apparatus (ipgphor ii, multiphor ii chamber electrophorese, eps 3500 power supply) were purchased from amersham pharmacia biotech (uppsala, sweden). ipg drystrips and pharmalyte ph 3 virus stock solution containing 11.5 × 10 7 pfu was centrifuged for 1 h at 21,000 × g and 4°c. the virus pellet was resuspended in 1 ml phosphate buffered saline (pbs), transferred into a 6-well plate and inactivated by uv irradiation of 0.05 w/m 2 in a stratalinker 2400. success of inactivation was checked by immunofluorescence staining of infected cells. the two-dimensional polyacrylamide gel electrophoresis with immobilised ph gradient was performed according to the procedure by görg et al [50] . initially, 100 μg dried protein was solubilised in 400 μl rehydration solution in the second dimension the proteins were separated in a 12-%-polyacrylamide gel (250 × 200 × 1 mm 3 ) at 4 w, 600 v and 30 ma for 14 h at 12°c in a horizontal system. the size marker was rotimark ® 10-150 kda (roth, karlsruhe, germany) and a tris-glycine buffered system according to laemmli (1970) was used [51] . after electrophoresis, the proteins were silver stained with the proteo-silver plus silver stain kit (sigma-aldrich) according to the manufacturer's instructions. differences in the protein expression profile of the 2-d gels were identified by use of the image analysis software melanie 7.0 (swiss institute for bioinformatics). a quantitative analysis was disclaimed since silver staining is not an end point method. a match set was created from scanned gel images in order to compare protein spots across gels. the gel containing the best resolution and most spots was chosen as the master gel. landmark spots were defined and manual editing of the matching procedure was performed to improve the automated matching results. finally, the mismatched spots representing differences in protein expression were manually checked and selected for mass spectrometric analysis. differences in the protein expression profile of the 2-d gels were identified by use of the image analysis software melanie 7.0 (swiss institute for bioinformatics, lausanne, switzerland, http://www.expasy.org/melanie). a quantitative analysis was disclaimed since silver staining is not an end-point method. spots representing differences in protein expression were excised as dice with 1 mm edge length each, which were destained with the proteosilver™ plus silver stain kit (sigma-aldrich) according to the manufacturer's instructions. gel dice were then dried in a speedvac after dehydration in 100 μl acn. the dried dice were swollen in 10 μl reaction buffer (25 mm ammonium bicarbonate [nh 4 hco 3 ] ph 8.0) containing 500 ng trypsin (trypsin profile igd kit, sigma-aldrich) for 15 min at 4°c, before another 40 μl reaction buffer was added. the tryptic digest was incubated overnight at 37°c. the proteolytic peptide containing supernatant was transferred into a new reaction tube. peptides remaining in the gel dice were extracted by adding 20 μl 1:1 acn/0.1%tfa for 30 min. pooled peptides were evaporated to dryness in a speedvac for further analysis. reduction of disulfide bonds was done by addition of 2 μl 2 m dithiotreitol (dtt) to 200 μl 0.1 μg/μl bsa in 25 mm nh 4 hco 3 ph 8.0 at 60°c for 30 min. subsequently free thiol groups were carboxyamidomethylated by adding 6 μl 1 m iaa at 25°c for 30 min in the dark. excessive iodoacetamide (iaa) was converted with 1 μl 2 m dtt at 60°c for 15 min before the proteolytic digest was induced by addition of 1 μg trypsin (1:20 w/w) and incubated for 16 h at 37°c. for spitc derivatization the desired amount of protein was transferred to a microcentrifuge tube and evaporated to dryness in a speedvac. n-terminal peptide derivatization with 4-sulfophenyl isothiocyanate on ziptipμ-c18 columns dried peptides were resolved in 5 μl 0.1% tfa, 0.5 μl of which were mixed with 0.5 μl matrix (10 mg α-cyano-4hydroxycinnamicacid [chca] in 1 ml acn/0.1% tfa [1:1 v/v]) on a stainless steel carrier and were air-dried. prior to the recording of peptide mass fingerprints, the spots were washed with 1.5 μl 10 mm biammonium citrate three times. the remaining peptide solution was bound to a ziptipμ-c18 (millipore) pipette tip which was wetted with 10 μl acn/0.1% tfa (1:1 v/v) and washed twice with 10 μl 0.1% tfa. subsequently the ziptipμ-c18 was loaded with 10 μl 5 mg/ml 4-sulfophenyl isothiocyanate (spitc) in 20 mm nahco 3 ph 8.6 and incubated at 55°c for 1 h in a circulating air oven. derivatised peptides were washed five times with 0.1% tfa and three times with 10 mm biammonium citrate prior to elution with 1 μl acn/0.1% tfa (1:1 v/v) directly onto the maldi target. 0.5 μl matrix (10 mg chca in 1 ml acn/0.1% tfa [1:1 v/v]) was added and the spot was air-dried. dried peptides were resolved in 10 μl derivatization solution (10 mg/ml 4-sulfophenyl isothiocyanate [spitc] in 20 mm nahco 3 ph 8.6) and incubated at 60°c for 30 min. purification, elution and co-crystallization of derivatised peptides was done by use of ziptipμ-c18 (millipore) pipette tips as described above. the mass spectrometric investigations have been carried out using an axima-cfr plus (kratos analytical, manchester, uk). peptide mass fingerprints were recorded in positive ion reflectron mode with a setting of the laser intensity of 2.2 μj and a maximum laser-repetition rate of 5.0 hz at a pulse extraction of 1500 m/z (delay-time). each recorded spectrum was the sum range of 200 profiles, accumulated from five laser shots. ten m/z values greater than 1200 were automatically selected with descending intensity by the launchpad software (kratos analytical) for post source decay (psd) measurement. 100 profiles accumulated from ten laser shots were accumulated for one psd spectrum which was recorded in positive ion reflectron mode with a laser intensity of 4.0 μj and a maximal laser-repetition rate of 5.0 hz at a pulse extraction of 1500 m/z (delay-time). an external 5-points calibration was performed by using appropriate peptide standard (calibration mix [proteomix] 500-3500 da [laserbio labs]) prior to each measurement. a combined data file of m/z values of the pmf and psd spectra was sent to the expasy server (swiss institute for bioinformatics) and synchronised with the swissprot database for protein identification. peaks from the raw data were picked using mascotdistiller (matrix science, boston, usa) with a minimal signal/noise of 3 and a peak width of 0.3 m/z values were annotated with a mass tolerance of +/-0.3 da in ms 1 and +/-0.7 da in psd mode. carbamidomethylated cystein residues and spitc derivatised n-termini were set as fixed modifications, oxidised methionine residues as a variable modification. the challenge of emerging and reemerging infectious diseases rapid response research to emerging infectious diseases: lessons from sars human monkeypox: an emerging zoonosis cowpox virus infection: an emerging health threat potential biological weapons threats dissociation of progeny vaccinia virus from the cell membrane is regulated by a viral envelope glycoprotein: effect of a point mutation in the lectin homology domain of the a34r gene a27l protein mediates vaccinia virus interaction with cell surface heparan sulfate cell surface proteoglycans are necessary for a27l protein-mediated cell fusion: identification of the n-terminal bartel et al region of a27l protein as the glycosaminoglycan-binding domain vaccinia virus envelope d8l protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells vaccinia virus envelope h3l protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo pox proteomics: mass spectrometry analysis and identification of vaccinia virion proteins analysis of vaccinia virushost protein-protein interactions: validations of yeast two-hybrid screenings de novo sequencing of tryptic peptides sulfonated by 4-sulfophenyl isothiocyanate for unambiguous protein identification using post-source decay matrix-assisted laser desorption/ionization mass spectrometry improved protein identification efficiency by mass spectrometry using n-terminal chemical derivatization of peptides from angiostrongylus costaricensis, a nematode with unknown genome determination of aldolase in animal tissues the isoenzymes of malate dehydrogenase and their regulation in saccharomyces cerevisiae ecto-f1fo atp synthase/ f1 atpase: metabolic and immunological functions mutations and polymorphisms of the gene encoding the beta-subunit of the electron transfer flavoprotein in three patients with glutaric acidemia type ii further: biochemical and kinetic characterization of human eukaryotic initiation factor 4h hcc-1 is a novel component of the nuclear matrix with growth inhibitory function growth inhibitory effect of hcc-1/cip29 is associated with induction of apoptosis, not just with g2/m arrest structure and expression of the gene (hnrpa2b1) encoding the human hnrnp protein a2/b1 purification and characterization of native spliceosomes suitable for three-dimensional structural analysis a sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new dna-binding motif far upstream element-binding protein-1, a novel caspase substrate, acts as a cross-talker between apoptosis and the c-myc oncogene ewing sarcoma fusion protein ewsr1/ fli1 interacts with ewsr1 leading to mitotic defects in zebrafish embryos and human cell lines prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin rice ap: interferon mediated, double-stranded rna-dependent proteine kinase is inhibited in extracts from vaccinia virus-infected cells the dsrna proteine kinase pkr: virus and cell control induction of apoptosis by the dsrna-dependent protein kinase (pkr): mechanism of action characterization of a specific kinase inhibitory factor produced by vaccinia virus which inhibits the interferon induced proteine kinase nucleophosmin interacts with and inhibits the catalytic function of eukaryotic initiation factor 2 kinase pkr the e3l gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded rna-dependent protein kinase vaccinia virus (strain ioc) putative double-stranded rna binding protein prohibitins control cell proliferation and apoptosis by regulating opa1-dependent cristae morphogenesis in mitochondria prohibitin induces the transcriptional activity of p53 and is exported from the nucleus upon apoptotic signaling essential role of pkc[delta] in apoptosis induction of mouse thymocytes src kinase phosphorylates caspase-8 on tyr380: a novel mechanism of apoptosis suppression the mgc project team: the status, quality, and expansion of the nih fulllength cdna project: the mammalian gene collection (mgc) exposure of phosphatidylethanolamine on the surface of apoptotic cells raf-1 kinase inhibitor protein: structure, function, regulation of cell signaling, and pivotal role in apoptosis uniprotkb/swiss-prot p20535 (vfus_vaccc) integrated into uniprotkb/ swiss-prot the complete dna sequence of vaccinia virus plasma and red blood cell protein maps: update 1993 comparative proteomics of human monkeypox and vaccinia intracellular mature and extracellular enveloped virions snx3 regulates endosomal function through its px-domain-mediated interaction with ptdins(3)p distinct biochemical characteristics of the two human profilin isoforms the current state of two-dimensional electrophoresis with immobilized ph gradients cleavage of structural proteins during the assembly of the head of bacteriophage t4 proteome analysis of vaccinia virus ihd-w-infected hek 293 cells with 2-dimensional gel electrophoresis and maldi-psd-tof ms of on solid phase support n-terminally sulfonated peptides author details 1 friedrich-alexander university erlangen-nuremberg, institute of bioprocess engineering, henkestraße 91, 91052 erlangen, germany. 2 robert koch-institute, center for biological security 1, nordufer 20, 13353 berlin, germany.authors' contributions jd carried out the two-dimensional gel electrophoresis, image analysis as well as the mass spectrometric measurements. sb established the method for fragmentation of spitc derivatized peptides and executed the database searches for protein identification. the manuscript was drafted by sb and jd in equal parts. the cell culture part of this study was performed by db, whereas kd supported the image analysis and database searches. rb financed the analytical part of the study. hl coordinated the study and gave relevant contributions to the technical background. an conceived the study, and participated in its design. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-337067-j8ebslif authors: mades, andreas; gotthardt, katherina; awe, karin; stieler, jens; döring, tatjana; füser, sabine; prange, reinhild title: role of human sec63 in modulating the steady-state levels of multi-spanning membrane proteins date: 2012-11-15 journal: plos one doi: 10.1371/journal.pone.0049243 sha: doc_id: 337067 cord_uid: j8ebslif the sec61 translocon of the endoplasmic reticulum (er) membrane forms an aqueous pore, allowing polypeptides to be transferred across or integrated into membranes. protein translocation into the er can occur coand posttranslationally. in yeast, posttranslational translocation involves the heptameric translocase complex including its sec62p and sec63p subunits. the mammalian er membrane contains orthologs of yeast sec62p and sec63p, but their function is poorly understood. here, we analyzed the effects of excess and deficit sec63 on various er cargoes using human cell culture systems. the overexpression of sec63 reduces the steady-state levels of viral and cellular multi-spanning membrane proteins in a cotranslational mode, while soluble and single-spanning er reporters are not affected. consistent with this, the knock-down of sec63 increases the steady-state pools of polytopic er proteins, suggesting a substrate-specific and regulatory function of sec63 in er import. overexpressed sec63 exerts its down-regulating activity on polytopic protein levels independent of its sec62-interacting motif, indicating that it may not act in conjunction with sec62 in human cells. the specific action of sec63 is further sustained by our observations that the up-regulation of either sec62 or two other er proteins with lumenal j domains, like erdj1 and erdj4, does not compromise the steady-state level of a multi-spanning membrane reporter. a j domain-specific mutation of sec63, proposed to weaken its interaction with the er resident bip chaperone, reduces the down-regulating capacity of excess sec63, suggesting an involvement of bip in this process. together, these results suggest that sec63 may perform a substrate-selective quantity control function during cotranslational er import. the er is a major site of synthesis for both secretory and membrane integrated proteins. generally, nascent polypeptides are translocated cotranslationally across or into the er membrane at sites termed translocons. the translocon minimally comprises one sec61a-b-c heterotrimer and associated proteins forming an aqueous conduit that aligns with the ribosome during translocation. in cotranslational translocation, signal sequences or transmembrane (tm) domains within a nascent polypeptide are recognized by the signal-recognition particle (srp). the srpbound ribosome nascent chain complex then binds to the membrane via the srp receptor, and after srp release the translating ribosome interacts with the translocon, thereby enabling the growing polypeptide to be inserted directly into the translocon channel. soluble proteins, such as those ultimately secreted from the cell or localized inside the er lumen, cross the membrane completely, while membrane proteins exit laterally into the lipid phase, a step referred to as membrane integration [1, 2, 3] . the events accompanying the gating of the lateral exit portal of the translocon are currently less resolved. numerous components near the mammalian sec61 core translocon complex have been described that could contribute to the overall efficiency of translocation. these include the spc (signal peptidase complex) [4] , ost (oligosaccharyl transferase complex) [5] , tram (translocation chain-associated membrane protein) [6, 7] , trap (translocon-associated protein) [8] , ramp4 (ribosome-associated membrane protein 4) [6] , er proteins with j domains inside the er lumen such as erdj1 and erdj2/sec63 [9, 10, 11] , the sec63-interacting sec62 protein [10] , and others. the functional roles of the translocon-associated sec62 and sec63 proteins in protein biogenesis at the er have been well established in the yeast saccaromyces cerevisiae, where nascent chains can be translocated either co-or posttranslationally [1, 2, 3] . both translocation pathways depend on the protein-conducting sec61 channel, but differ in their targeting mechanisms. in the cotranslational mode of translocation, nascent precursors are targeted in an srp-dependent manner. in the posttranslational mode of translocation, the sec61 channel partners with another membrane protein complex, consisting of the essential sec62p and sec63p proteins, and the non-essential sec71p and sec72p proteins, that together mediate targeting and translocation of completed polypeptide chains. the posttranslational pathway is used predominantly by precursors with low hydrophobic signal peptides [1, 2, 12] . in the mammalian er, the cotranslational mode of protein translocation dominates. even so, orthologs of the yeast sec62p and sec63p, referred to as sec62 and sec63, are found in stoichiometric amounts compared with sec61a and in association with the sec61 complex [10, 13] . sec62 is a double-spanning membrane protein with a cytosolic domain that associates with ribosomes, suggesting a cotranslational function of this protein in mammals [14] . sec62 also teams up with sec63, an integral membrane protein of the hsp40 co-chaperone family [10, 14] . similar to its yeast ortholog, mammalian sec63 contains three tm domains with a lumenal j domain that functionally interacts with the er resident bip chaperone (kar2p in yeast) [10, 12, 15, 16] . the conservation of structural and biological features of sec62 and sec63 from yeast to humans strongly suggests that these proteins are likewise involved in protein transport into the mammalian er, but their precise function is less known. loss of function mutations in the human sec63 are not lethal but are linked to polycystic liver disease, indicating that it may contribute to er import and/or folding of proteins involved in biliary cell growth [1, 17] . in the case of human sec62, its overexpression was found to be associated with sporadic colorectal cancer and prostate cancer [1] . the participation of sec62/sec63 in the biogenesis of particular er substrates is also suggested by the finding that both proteins can transiently associate with newly synthesized polypeptides during er membrane integration in cell-free systems [18] . our previous work has been focused on the biogenesis of three related envelope proteins of the hepatitis b virus (hbv) that are synthesized as multi-spanning proteins at the er membrane. while the small s and middle m envelope proteins are integrated into the er membrane in a typical cotranslational process, the large l envelope protein forms a dual topology via a process involving cotranslational membrane integration and subsequent posttranslational translocation of its pres subdomain into the er [19, 20, 21] . similar to posttranslational translocation processes in yeast, the posttranslational pres translocation of l requires bip [22, 23] , a known interaction partner of sec63 [10] . we took this as a rationale to study the function of human sec63 and its partner proteins in cell culture systems. against expectation, we find that sec63 does not play a role in the special topogenesis of the hbv l protein (k.a and r.p., unpublished observation), but is rather more generally involved in the control of the steady-state levels of polytopic membrane proteins. overexpressed sec63, but not sec62, reduces the steady-state level of the polytopic hbv.s envelope protein to study the role of the human sec62 and sec63 proteins, expression vectors encoding n-terminally myc-tagged versions of these proteins were constructed and transiently transfected into huh-7 human liver carcinoma cells. upon deconvolution immunofluorescence and phase contrast microscopy, the ectopically expressed sec62 and sec63 constructs yielded an er-like staining and partially colocalized with calnexin, a membraneanchored er chaperone (fig. 1a) . the constructs were next transfected together with a c-terminally hemagglutinin (ha)tagged version of the s envelope protein of hbv (hbv.s). hbv.s is cotranslationally integrated into the er membrane, directed by the action of an uncleaved signal-anchor and a stop-transfer sequence encoded within its first and second tm segments [24] . the hydrophobic c-terminal half of hbv.s is predicted to further span the membrane two times thereby resulting in a fourfold membrane-spanning protein. after transient coexpression of the constructs, cell lysates were prepared with the non-denaturing detergent np-40 and analyzed by myc-and ha-specific western blotting. as shown in fig. 1b , sec62 and sec63 were efficiently synthesized and appeared in about 46 and 87 kda forms, respectively, in accord with their calculated molecular masses. hbv.s was obtained in its characteristic doublet of a 24 kda nonglycosylated (p24) and a 27 kda glycosylated (gp27) form as a consequence of partial n-linked glycosylation [25] . intriguingly, the intracellular level of hbv.s was significantly lower when ectopically expressed sec63 was present. in contrast, excess sec62 reproducibly did not affect the level of hbv.s (fig. 1b) . similar results were obtained with cell lysates prepared with the denaturing detergent sds (fig. 1b) , indicating that the observed down-regulation of hbv.s by excess sec63 was not merely due to changes in the solubility profile. since the overexpression of sec63 might be harmful for the cells, cytotoxicity assays of cellular supernatants were performed which did not reveal significant indications of cell damage (fig. 1b) . because sec62 and sec63 have been shown to associate with each other [13, 14] , the upregulation of sec63 could evoke an imbalance of the complex partner. therefore, the fate of hbv.s was analyzed in cells overexpressing both sec62 and sec63 proteins. under these conditions, the levels of sec62 and sec63 were lower as compared to their individual expression. one explanation may be that sec63 also reduced the steady-state level of the double-spanning sec62 protein at the er. alternatively, this could be the consequence of a lower degree of transfection caused by the higher amount of plasmid dna used for triple transfection. nonetheless, even in consideration of a lower transfection rate, hbv.s was downregulated ( fig. 1b) , implicating that the steady-state level of sec62 is less important. in vitro-binding studies have identified the negatively charged c-terminus of sec63 as the interaction domain for basic oligopeptide motifs within the n-terminus of sec62 [14] . to examine whether the sec62/sec63 interaction might play a role in the function of sec63, we analyzed a c-terminally truncated sec63 mutant lacking large parts of the cytosolic domain including the sec62 binding site. this sec63dc mutant similarly down-regulated hbv.s (fig. 1b) , thus arguing against an essential role of sec62 in the observed action of sec63. following er translocation, the hbv.s protein is able to selfassemble into subviral envelope particles (svps) that bud into intralumenal cisternae of post-er/pre-medial-golgi compartments and exit the cell by the constitutive pathway of secretion [25, 26] . to investigate whether the intracellular reduction of hbv.s by overexpressed sec63 was simply due to an enhanced svp export, lysates and supernatants of cotransfected cells were examined by an hbv.s-specific elisa. this analysis, however, clearly revealed that excess sec63 reduced both the intra-and extracellular pool of hbv.s. in contrast, overexpressed sec62 did not interfere with the synthesis and secretion of hbv.s (fig. 1c ). overexpressed sec63 reduces the steady-state level of the polytopic hbv.s envelope protein in a dosedependent manner to ascertain the degree of sec63 overexpression, total sec63 levels were examined in control-versus sec63-transfected huh-7 cells by using a polyclonal sec63-specific antiserum for western blotting. in order to ensure the true identification of endogeneous sec63, cells were depleted for sec63 by treatment with specific sirna duplexes. as shown in fig. 2a , endogeneous sec63 was absent in lysates of sisec63-treated cells. due to the myc-tag, exogeneous sec63 displayed a slower electrophoretic mobility that allowed its distinction from the endogenous isoform ( fig. 2a) . upon serial dilution of the sec63-transfected lysate and quantification, we calculated that exogeneous sec63 was overexpressed about 3.2-fold. an accompanying titration experiment showed that sec63 down-regulated hbv.s in a dose-dependent manner (fig. 2b) . thereby, the most prominent decline in the hbv.s steady state level appeared when the concentration of the transfected sec63 dna was raised above 1.5 mg/10 6 cells (fig. 2b ). to rule out the possibility that up-regulated sec63 had effects on mrna production or stability, the hbv.s-specific transcript levels were measured by quantitative reverse transcription pcr. although we noticed a modest decline of the hbv.sspecific mrna in sec63-overexpressing cells (fig. 2c ), this drop is unlikely to account for the impact of sec63 on the steady-state hbv.s pool. to specify the role of excess sec63, we next analyzed the fate of an hbv.s mutant lacking the first tm segment (hbv.sdtm1). this mutant still enters the er pathway but fails to assemble into intralumenal secretory svps [27] . upon coexpression with sec63, the intracellular amount of hbv.sdtm1 was drastically decreased (fig. 3a) , similar to the situation of wild-type (wt) hbv.s. thus, excess sec63 depleted hbv.s proteins irrespective of their secretory phenotypes and therefore likely acts prior to svp assembly and secretion. to sustain this finding, the outcome of further hbv envelope proteins was studied. the l envelope protein of hbv (hbv.l) is unable to assemble into secretory svps due to its dual transmembrane topology [21, 25] . this deficit can be abrogated by the addition of a foreign signal sequence to the nterminus of hbv.l that blocks the formation of the dual topology and enables secretion [28] . the overexpression of sec63 decreased the non-glycosylated and glycosylated forms of both the wt hbv.l and hbv.ile9::l signal fusion proteins (fig. 3a ). this indicates that excess sec63 compromises the production of hbv envelope proteins independently of their particular secretory behavior. during er translocation, the hbv envelope proteins are modified by n-linked glycosylation conducted by the translocon-associated ost complex [5, 29] . because the overexpression of sec63 could impair the structural integrity of the ost complex concomitant with translocational constraints, we studied a glycosylation-defective hbv.l mutant (hbv.ldn309). due to the mutation of the n-glycan acceptor site, this mutant appeared only in the nonglycosylated p39 form (fig. 3a) . hbv.ldn309 was also downregulated by excess sec63, thus largely excluding a participation of the n-linked glycosylation machinery in this process. since hbv is a hepatotropic virus, we routinely used huh-7 cells for viral expression studies. some liver cells, however, have been implicated in sec63 abnormalities, as mutations in the sec63 gene contribute to the development of autosomal dominant polycystic liver disease [1, 17] . therefore, we examined the effect of excess sec63 in hek293t human embryonic kidney cells in parallel. as exemplified for the hbv.l protein, overexpressed sec63 reduced the intracellular hbv.l level to almost the same extent as compared to huh-7 cells (fig. 3b ), arguing against cell type-specific effects. in combining these data, we deduced that excess sec63 diminished the steady-state pools of the hbv envelope proteins synthesized at the er. however, there remained the possibility that the observed effects were simply due to an exceptionally high degree of cotransfection of the sec63 and hbv constructs, thereby provoking intracellular imbalances, unspecific protein interferences, and/or other effects. to address this issue, we used a bicistronic plasmid vector carrying expression units for ha-tagged hbv.l under the control of the human metallothionein iia (hmtiia) promoter and for ha-tagged hip (heat shock cognate protein 70 [hsc70] interacting protein) under the control of the cytomegalovirus (cmv) promoter [22] . human hip is a cytosolic cochaperone involved in the regulation of hsc70 chaperone activity. this construct offered the chance to monitor the effect of excess sec63 in the same cells and the same blot. upon cotransfection of hbv.l/hip with sec63, the level of hbv.l was again reduced, while the amount of cytosolic full-length hip was only marginally affected (fig. 3c ). for unknown reasons, we noted, however, that hip-specific degradation products were sensitive to excess sec63. to get insights into the underlying mechanism(s), we investigated the time frame of the action of up-regulated sec63. although the data presented so far favor a cotranslational mode of sec63 activity, posttranslational impacts of excess sec63, such as a disruption of the integrity of the er membrane, could not be ruled out. therefore, cells transiently expressing the hbv.s construct for two days were retransfected with sec63 and analyzed after an additional day. for comparison, cells were simultaneously cotransfected with hbv.s and sec63. as shown in fig. 4a , overexpressed sec63 did not affect a preexisting hbv.s pool. to exclude the possibility that the lack of sec63 action in the ''posttransfection'' experiment was due to constraints in the retransfection, we analyzed the rate of cotransfection by immunofluorescence. although the intensity of the hbv.s-specific signals was substantially lower as compared to cells expressing hbv.s alone, the amplification of the signals enabled the detection of transfected cells. upon quantification, 49614% or 4267% of transfected cells were determined to be co-or posttransfected with sec63, respectively (n = 5) (fig. 4a ). together, these data support a role for sec63 at an early stage of hbv.s protein synthesis, possibly occurring during translocation. nuclei and organelle structures in the cell periphery are shown in the right panels. bar, 10 mm. b. cells were transfected with ha-tagged hbv.s together with empty plasmid dna (control), myc-tagged sec62, sec63, or sec63dc at a 1:3 dna ratio (4 mg total dna), respectively. cotransfections of hbv.s with sec62 plus sec63 were done at a 1:3:3 dna ratio (7 mg total dna), respectively. three days after transfection, cellular lysates were prepared with np-40 (np-40) and analyzed by ha-or myc-specific western blotting (wb) to demonstrate expression of hbv.s (bottom) or sec62, sec63 and sec63dc (top). to confirm equal gel loading, the lysates were probed by anti-b-actin wb (middle top). numbers to the right refer to molecular weight standards in kd, while the non-glycosyated (p24) and glycosylated (gp27) forms of hbv.s (s) are indicated on the left (middle bottom). relative hbv.s expression values were determined by densitometric analysis and demonstrated below in the graph in percent amount relative to control cells. error bars indicate the standard deviation from three separate experiments. the analyses of sds lysates (sds) of correspondingly cotransfected cells are illustrated in the bottom panel. to probe for cell cytotoxicity, supernatants of cotransfected cells were assessed for ldh activity. c. cells were cotransfected with hbv.s and sec62 or sec63 exactly as in a. lysates (cell) and supernatants (medium) were examined by an s-specific elisa, and hbv.s levels are demonstrated in percent amount 6 sd relative to control cells (n = 3). doi:10.1371/journal.pone.0049243.g001 in yeast, sec63p and its kar2p partner protein have been indicated to be involved in the export of misfolded proteins to the cytosol during er-associated degradation (erad) by the proteasome [30, 31] . for the mammalian sec63, such a role is ill-defined. to examine whether the up-regulation of sec63 reduced the hbv.s level via an enforced export out of the er, transfected cells were treated with the proteasome inhibitor epoxomicin. the efficacy of the drug during the applied assay conditions was indicated by the appearance of c-terminally degraded sec63 derivatives that accumulated in epoxomicin-but not in mocktreated cells (fig. 4b) . importantly, the inhibition of the erad pathway did not abolish the ability of excess sec63 to downfigure 2 . overexpressing of sec63 reduces the steady-state hbv.s level in a dose-dependent manner without grossly affecting transcription. a. huh-7 cells were either treated with control sirna (sicon) or sirna duplexes targeting sec63 (sisec63) or were transfected with control dna (control) or myc-tagged sec63 (sec63; 3 mg dna). np-40 cell lysates were prepared after three days and probed by anti-sec63 wb. for calculation of the degree of sec63 overexpression, the sec63-transfected lysate was serially diluted prior to gel loading as indicated above the lanes (% load). b. cells were transfected with hbv.s (1 mg dna) together with increasing amounts of sec63 dna, as indicated above the lanes (in mg). total dna used for transfections was adjusted with empty plasmid dna (control). extracts were prepared with sds lysis buffer two days after transfection and assayed by myc-(top), b-actin-(middle), and ha-specific (bottom) wb. c. cells were cotransfected with hbv.s and control or sec63 plasmids as in regulate hbv.s (fig. 4b) , suggesting that the reduction of the hbv.s steady-state level is not mediated by proteasomal degradation. to account for this unexpected finding, different explanations are conceivable. first, non-proteasomal proteases, like amino-and carboxypeptidases, may contribute to the destruction of improperly translocated hbv.s chains. such proteases, acting in the cytosol and the er/secretory pathway, have been reported to participate in endogenous antigen processing [32] . second, excess sec63 may halt hbv.s translocation, thereby promoting cotranslational destruction prior to completion of protein synthesis. in doing so, full-length, cterminally tagged hbv.s chains would not be detectable. and third, autophagosomes/lysosomes may be involved in the disposal of hbv.s. this pathway has been described in cells permanently replicating hbv [33] . although being clients of sec63, the hbv envelope proteins do not directly interact with sec63 as measured in a split-ubiquitin yeast two-hybrid assay (data not shown). this prompted us to examine whether or not sec63 may play a more general role in the production of er proteins. to this aim, we employed a panel of different translocational reporter proteins. for soluble er marker proteins, hemopexin (hxp) and a yellow fluorescent er fusion protein (yfp.er) were used that are cotranslationally translocated across the er membrane by virtue of their n-terminal cleaved signal peptides [34] . while yfp.er is retained within the er lumen via its c-terminal kdel retention signal, hxp is secreted from the cells. as shown in fig. 5a , both proteins were efficiently synthesized in transfected huh-7 cells. because hxp is a substrate to n-and o-linked glycosylation [34] , it appeared in multiple forms with different electrophoretic mobilities. upon overexpression of sec63, the intracellular levels of both reporters as well as the glycosylation pattern of hxp were largely unaffected, indicating that excess sec63 did neither affect er translocation nor the steady-state levels of these soluble proteins. identical results were obtained when er membrane proteins with a single membrane-spanning segment were analyzed. here, we took use of either a golgi.yfp fusion construct encoding yfp with a nterminal membrane anchoring signal peptide of b1,4-galactosyltransferase or the g glycoprotein of vesicular stomatitis virus (vsv.g). vsv.g harbors a cleavable signal peptide and a single tm segment in its c-terminus and is a substrate to n-linked glycosylation [35, 36] . coexpression studies demonstrated that sec63 neither down-regulated golgi.yfp nor vsv.g (fig. 5b) . it also did not affect the glycosylation pattern of vsv.g. since these data are in seemingly conflict with the results obtained for the hbv envelope proteins, we next assessed the behavior of multispanning membrane proteins synthesized in the presence of upregulated sec63. the m envelope protein of the mouse hepatitis coronavirus (mhv.m) is a triple-spanning protein with its first and second tm segments serving as a signal-anchor and stop-transfer signal, respectively [37] . beside viral proteins, human cd63/ lamp-3 (lysosomal-associated membrane protein 3), a member of the tetraspanin family, was included as a cellular multi-pass reporter [38] . ha-tagged versions of mhv.m and cd63 were cotransfected with sec63 and cell extracts were probed by western blotting. consistent with published data [37, 38] , the mhv.m and cd63 proteins run as broad smear in sds-polyacrylamide gels likely due to glycosylation (fig. 5c) . importantly, overexpressed sec63 almost completely depleted each of the multi-pass reporters (fig. 5c) , as it is the case for the hbv envelope proteins. to probe whether the non-glycosylated yfp.er and golgi.yfp reporters were truly translocated into the er of sec63-overexpressing cells, trypsin protection experiments were performed. microsomes of cotransfected cells were prepared and either left untreated or treated with trypsin in the absence or presence of detergent. for both reporters, protection against trypsin was observed in intact microsomes (fig. 6a) . on disruption of microsomes with detergent, trypsin completely converted the proteins to tryptic fragments thereby leading to the disappearance of full-length yfp.er and golgi.yfp. we noted, however, that neither construct was fully protected from proteolysis in the absence of detergent (fig. 6a ), likely as a consequence of some leakage of the microsomes. nonetheless, the amounts of protected yfp.er and golgi.yfp chains were almost identical in controland sec63-overexpressing cells. thus, up-regulated sec63 does not interfere with the translocation of these substrates. as measured by quantitative reverse transcription pcr of representative reporter genes, up-regulated sec63 does also not impair the synthesis and stability of yfp.er-and vsv.g-specific mrnas (fig. 6b ). in the case of cd63, transcript levels were diminished in the presence of exogenous sec63 (fig. 6b ). this may in part, but not entirely contribute to the observed decline of the cd63 protein level (see fig. 5c ). in order to determine the step(s) at which sec63 is acting, we analyzed whether up-regulated sec63 affected the translation or cotranslational insertion of the multi-pass cd63 reporter. for this, we used two constructs encoding the cytosolic green fluorescent protein (gfp) or gfp with a c-terminal fusion of cd63 (gfp.cd63). both constructs were in identical vector backbones and under the control of identical transcriptional elements. upon cotransfection of either of the two constructs with control or sec63 dna, their common gfp-tag enabled immunodetection on the same gel. like the cd63 wt protein (see fig. 5c ), the gfp.cd63 fusion protein appeared in multiple bands likely as a result of glycosylation, implicating that it had entered the er/secretory pathway (fig. 7) . up-regulated sec63 diminished the yield of the gfp.cd63 membrane reporter, while it had little effects on the cytosolic gfp protein (fig. 7) . since the transcripts of the two reporters were generated from the same promoter/polyadenylation elements and hence should be similar, we inferred that excess sec63 appeared to affect the cotranslational insertion rather than the translation of polytopic proteins. collectively, these data suggest that excess sec63 interferes selectively with the formation of multi-spanning er proteins. therefore, we next analyzed er cargoes in sec63-deficient cells. for depletion of sec63, classic sirna duplexes (sisec63) as well as sirna molecules prepared by endoribonuclease digestion (esi-sec63) were transfected into huh-7 cells prior to transfection with the hbv.s construct. western blot analysis of cell lysates showed that both sirna species depleted sec63 by about 80% as compared to control sirna-treated cells (fig. 8a ). when lysates were probed for hbv.s, the knock-down of sec63 resulted in significant higher intracellular p24/gp27 levels as compared to control-treated cells. of note, the intracellular increase of hbv.s was not due to a block in svp secretion, since the extracellular amount of hbv.s was also elevated in sec63-depleted cells (data not shown). these results indicate that sec63 is per se not required for hbv.s production; rather, its limitation promotes hbv.s synthesis and/or stability. to corroborate these results, other er reporter proteins were studied under conditions of deficit sec63. as representative markers, the soluble hxp protein, the single membrane-spanning vsv.g protein, and the multiple-spanning proteins hbv.l and mhv.m were investigated. depletion efficacy of sec63 and intracellular steady-state levels of the marker proteins were monitored by specific immunoblotting. as shown in fig. 8b , the biogenesis of hxp and vsv.g were unaffected by the knock-down of sec63. conversely, the amounts of each of the multi-spanning reporters were increased in sec63-depleted cells. these observations suggest that human sec63 is not essential for cotranslational er translocation, but regulates the translocation efficiency of certain precursor polypeptides. beside sec63 (i.e., erdj2), six other resident er proteins with j domains inside the er lumen have been described in mammals [11] . erdj1 is a further translocon-associated erdj protein and constitutes a single-pass type i membrane protein. unlike sec63, it has the ability to associate with translating ribosomes, likely to modulate translation [9] . erdj4 is a soluble er protein and plays a role in the degradation of erad substrates, but has not been found in close contact with the sec61 translocon [39] . to analyze whether an up-regulation of these erdj proteins might also affect the level of a multi-spanning membrane protein, flag-tagged versions of erdj1 and erdj4 were cotransfected with hbv.s. flag-specific western blotting confirmed the ectopic expression of erdj1 and erdj4 in 63 and 25 kda forms, respectively, consistent with their theoretical molecular masses (fig. 9) . importantly, when lysates were probed for hbv.s, no significant change in the p24/gp27 level was noticeable (fig. 9) . thus, in contrast to sec63, the overexpression of neither erdj1 nor erdj4 compromises the production and/or life span of hbv.s. in the next set of experiments, we focused on the sec63/bip interplay. the yeast and mammalian sec63 proteins have been shown to interact with the er lumenal kar2p/bip chaperone in a productive manner [10, 12, 15] . these interactions require their lumenally exposed j domains containing the highly conserved his-pro-asp (hpd) motif [11, 40, 41] . because mutations of this sequence abolish the functional interaction between j domains figure 5 . overexpressing of sec63 reduces steady-state levels of polytopic proteins. er reporter constructs, including soluble (a), singlepass membrane (b), and multi-pass membrane (c) proteins were used for cotransfection studies as indicated above the panels. each reporter was transfected with control dna or myc-tagged sec63 at a 1:3 dna ratio, respectively, into huh-7 cells. three days later, cell lysates were subjected to specific immunoblotting. ectopic expression of sec63 was assessed with anti-myc antibodies, while anti-b-actin antibodies were used to demonstrate identical gel loading. hxp, mhv.m, and cd63 were probed with anti-ha antibodies, yfp.er and golgi.yfp were detected with anti-gfp antibodies, and vsv.g was verified with anti-g-specific antibodies. reporter expression values were determined by densitometry and demonstrated in percent amount 6 sd relative to control cells (n = 3). doi:10.1371/journal.pone.0049243.g005 and their chaperone partners [40, 41] , we reasoned to study a sec63 mutant devoid of the hpd motif (sec63dhpd). the wt and mutant sec63 proteins were coexpressed with hbv.s and cell extracts were examined by specific immunoblotting. the sec63dhpd mutant likewise reduced hbv.s, albeit its downregulating activity was reproducibly less pronounced as compared to wt sec63 (fig. 10a) . similar results were obtained with the polytopic cd63 reporter (fig. 10a) . these results suggest that the stimulation of the atpase activity of bip by the j domain of sec63 may not be mandatory for the role of sec63 in polytopic membrane homeostasis. however, they do not thoroughly exclude that sec63 may function in concert with bip. to dissect whether sec63 per se or the associated bip chaperone is responsible for hbv.s protein reduction, the reporter was studied in cells overexpressing sec63 and bip either individually or together. the up-regulation of bip neither reduced the level of hbv.s nor reverted the inhibitory action of excess sec63 on hbv.s (fig. 10b) . together, these data suggest that sec63 is primarily responsible for the control of membrane protein levels, possibly with aid of bip. this work provides support for a role of human sec63 in protein biogenesis at the er. by analyzing the effects of excess and deficit sec63 in human cell lines, we observed an inverse correlation between the cell content of sec63 and the level of particular er substrates. as reporters, we used soluble, single-and multispanning membrane proteins of viral, bacterial, and mammalian origin and observed that the overexpression of sec63 specifically reduces the steady-state levels of polytopic membrane proteins, while the limitation of sec63 has reciprocal effects. the data of the kinetic experiment support a role for sec63 at an early stage of multi-spanning membrane protein synthesis, and we favor a model where this occurs during translocation. overall, our data implicate that sec63 has a regulatory function during cotranslational insertion of multi-pass membrane proteins that is, however, not essential. its action as a translocational controller may explain why the loss of sec63 function associated with polycystic liver disease (pcld) is not lethal in humans [1, 17] . with regard to our finding that the depletion of sec63 augments the biogenesis of polytopic proteins, dysfunctional sec63 may evoke an unbalanced physiological situation in pcld patients, thereby eventually affecting proteins that are involved in the control of biliary cell growth and proliferation. previous studies have shown that the yeast and mammalian sec62 and sec63 proteins interact with each other [13, 14, 42] , suggesting a concerted action of these proteins. while this is true for posttranslational protein translocation into the yeast er [2, 39] , overlapping roles for the mammalian proteins have not been documented. rather, in vitro biochemical analyses of dog and bovine pancreatic microsomes revealed that only small fractions of sec62 and sec63 are associated with each other, while the majority of these proteins were not found in a complex [10, 13] . cross-linking experiments in cell-free systems also argue for figure 8 . silencing of sec63 increases the steady-state levels of polytopic proteins. a. huh-7 cells were treated with control sirna (sicon) or sirna duplexes (sisec63) and esirna molecules (esisec63) targeting sec63. two days later, cells were transfected with hbv.s and lysed after 48 h. lysates were subjected to sec63-specific wb to examine depletion. for hbv.s detection, anti-ha wb was used. to confirm equal loading of lysates, bactin levels were assessed. below the panels, the cell-associated hbv.s levels are demonstrated in percent amount 6 sd relative to control cells (n = 2). b. cells were transfected with control or sec63-specific sirnas as above, prior to transfection with the indicated er reporters. hxp was used as a soluble marker, vsv.g represented a single membrane-pass protein, while hbv.l and mhv.m served as multi-spanning membrane reporters. cell lysates were subjected to immunoblotting using anti-sec63 (top), anti-b-actin (middle), or anti-ha/vsv.g antibodies (bottom). reporter expression values were determined by densitometry and demonstrated in percent amount relative to control cells. doi:10.1371/journal.pone.0049243.g008 individual functions of mammalian sec62 and sec63 proteins, as sec63 was found in transient proximity to newly synthesized membrane proteins at early stages of membrane integration, while sec62 was detected at a later stage [18] . consistent with this, we show that the overexpression of sec62 did not limit hbv.s membrane protein expression. moreover, up-regulated sec63 exerts its inhibitory activity independent of the sec62 protein content of the cells and independent of its sec62-interacting motif, indicating that a stoichiometric complex formation between sec63 and sec62 may not be mandatory for function. a sec62independent action of sec63 is also used by the parasite trypanosoma brucei that lacks a sec62 ortholog, but engages sec63 for co-and posttranslational er protein import [43] . the substrate-specificity of the sec63 action is intriguing, as it selectively targets multi-spanning membrane proteins. a currently unresolved issue in membrane protein folding is how and when tms are released from sec61 once translocation is terminated. one view, supported by lipid cross-linking studies is that tms are dislocated from the translocon and move into the bilayer by passive thermodynamic partitioning through a lateral cleft in the sec61a subunit. hence, the driving force for the lateral exit of tms can be correlated to their intrinsic hydrophobicity. numerous studies have shown that such a model is likely to apply for the membrane integration of single-spanning membrane proteins [44, 45, 46] . the biogenesis of polytopic membrane proteins, however, presents distinct challenges to the er translocon and demands additional modulation. experimental data suggested that up to four tms of one polypeptide could be present in the translocation channel at the same time concomitant with widening of the pore [47, 48, 49] . moreover, tms appear to progress through different proteinaceous environments prior to their integration into the lipid bilayer either one by one, as pairs or even groups, suggesting the participation of additional factors beside the translocon core complex [50, 51, 52] . because mammalian sec63 is adjacent to sec61 [10] and because it selectively influences the synthesis of polytopic membrane proteins, it is tempting to speculate that sec63 may affect the structure and functionality of those translocons handling polytopic proteins. in this view, sec63 (i) may control expansion of the translocon to accommodate multiple tms, (ii) may act as a lateral gate keeper controlling the opening and closure of the exit portal towards the lipid layer, and/or (iii) may function as a chaperone or an escort for tms awaiting lipid integration. a regulatory function of sec63 may also explain why it is dispensable for cotranslational translocation into reconstituted proteoliposomes [6] . since excess sec63 inhibits the biogenesis of polytopic proteins, while its deficit has inverse effects, sec63 has features of a ''negative'' regulator. the concept of translocational regulation by substrate-selective trans-acting factors has been well documented [53] . for example, the translocon-associated tram and trap proteins can interact directly with some nascent chains and stimulate translocation in a signal sequence-dependent manner, but neither protein is absolutely required because at least some substrates can be translocated in their absence [7, 8, 54] . translocon-associated er lumenal factors, like the soluble bip chaperone or the membrane-embedded ost complex, appear to influence translocation of nascent chains by mechanisms including trapping, modification, and/or folding, thereby facilitating quality control of er substrate entry [5, 16] . for the translocon-associated sec63 protein, we suggest a quantity control function in such that it may regulate the amount of polytopic protein integration in order to prevent er membrane overload. at first glance, our rna interference data are in conflict with two recent works, studying the loss of sec63 function in vitro and in vivo. by using a mutant mouse model for pcld, fedeles et al. [55] demonstrated that sec63 differently affected the polycystic kidney disease gene products, polycystin-1 and polycystin-2. both proteins are synthesized as multi-spanning proteins at the er whereupon polycystin-1 uses a cleavable signal peptide, while polycystin-2 employs a non-cleavable signal [56, 57] . in sec63 knock-out cells, the biogenesis of polycystin-1 was strongly impaired, while polycystin-2 was only moderately affected [55] . by studying seven different lumenal, single-and multi-spanning cotranslational er reporters with and without cleavable signal peptides, lang et al. [58] showed that some proteins need the help of sec63, while others do not. the authors suggest that the sec63dependancy may be in part related to the properties of cleavable and non-cleavable signal peptides [58] . except for the hbv.i-le9::l reporter that contains an artificial, presumably uncleaved signal peptide (r. prange, unpublished observation), all polytopic cargos used in this work contain non-cleavable signal peptides. hence, apart from its quantity control function sec63 appears to play an additional ''active'' role in cotranslational protein import depending on the character of the signal peptide. a greater panel of proteins will have to be analyzed to deduce the exact function of sec63. the j domains of yeast and mammalian sec63 proteins have been demonstrated to functionally interact with the kar2p/bip chaperone which plays a role in many functions of the er, including lumenal gating of the translocon, assisting protein folding, and targeting misfolded proteins for proteasomal degradation [16] . thus, the up-regulation of sec63 could lead to trapping of bip concomitant with defects in er translocation and homeostasis. however, we do not favor this interpretation, because the overexpression of two other bip-interacting erdjs, erdj1 and erdj4, did not impair the hbv.s steady-state protein level. also, the simultaneous overexpression of sec63 and bip did not revert the inhibitory effect of excess sec63 on the hbv.s cargo. nonetheless, the question remains whether sec63 acts in conjunction with bip. in case of the yeast sec63p, mutations of the hallmark hpd motif of its j domain abolish the functional interaction with kar2p, i.e., lead to a failure to stimulate the atpase activity of the chaperone [15, 40] . the overexpression of a corresponding mutant of the human sec63 compromised the steady-state levels of polytopic reporters but half the amount as compared to the wt protein. one interpretation of this finding may be that bip is not absolutely required for the sec63 action. alternatively, the hpd mutation may weaken but may not completely prevent the sec63/bip interaction. in favor of the latter interpretation are observations that j domain proteins can form stable complexes with hsp70 chaperones in both the adpand atp-bound states [41, 59] . notably, a recent analysis of the multi-spanning cystic fibrosis transmembrane conductance regulator has shown that the release of tms from the sec61 translocon and their membrane integration is atp-dependent [60] , although -thus far -there are no known core translocon components that contain atpase activity. possibly, the sec63/bip participation may provide the missing link. human sec62, sec63, erdj1, erdj4, and bip were obtained as full-length cdna clones from imagenes. for n-terminal tagging with the myc epitope, sec62 and sec63 were cloned into pcmv-myc (bd biosciences, clontech). the sec63dhpd and sec63dc mutants were created by mutagenesis using the quikchangeh ii xl site-directed mutagenesis kit (stratagene). sec63dhpd carries alanine substitutions of the amino acids (aa) hpd at positions 132 to 134, while sec63dc contains a premature stop codon generated at aa position 444 thereby deleting the cterminal domain (aa 444-760) of sec63. erdj1, erdj4, and bip were inserted into p3xflag-cmv-14 (sigma-aldrich) thereby giving raise to c-terminally flag-tagged constructs. all constructs were verified by sequencing and cloning details are available on request. mammalian expression vectors carrying the wt or mutant hbv envelope genes have been described previously [22, 28] . briefly, pni2.s.ha (hbv.s) and pni2.l.ha (hbv.l) encode the s or the l gene, respectively, with a c-terminally fused ha epitope under the transcriptional control of the hmtiia promoter. the hbv.sdtm1 mutant lacks the first tm segment of the s protein, while the hbv.ile9::l mutant carries the l gene with a foreign n-terminal signal sequence derived from human interleukin-9 [27, 28] . the bicistronic plasmid pcdna/ha.hip-l (hbv.l/hip) encodes the ha-tagged l gene and the ha-tagged human hip gene under the control of the hmtiia or cmv promoter, respectively [22] . the m envelope gene of mhv strain a59, present in plasmid ptz19r-m [37] , was a gift from p. j. m. rottier (utrecht university, the netherlands) and was subcloned into pcmv.ha (bd biosciences, clontech) for c-terminal tagging with the ha epitope (mhv.m). plasmid pci-vsvg expresses the g glycoprotein of vsv under control of the cmv promoter [35] and was obtained from g. nolan (stanford university school of medicine, usa) as ''addgene plasmid 1733''. plasmid peyfp-er encodes yfp with an n-terminal er targeting sequence of calreticulin and a c-terminal er retrieval sequence (bd biosciences, clontech). plasmid peyfp-golgi carries a fusion gene consisting of yfp preceded by a n-terminal 81 aa sequence of human b1,4-galactosyltransferase (bd biosciences, clontech). the construct for the human ha-tagged hemopexin (hxp) has been described [61] . vectors encoding human cd63 with an nterminal ha-tag or n-terminal egfp fusion under control of the cmv promoter were gifts from g. spoden (university mainz, germany). for egfp tagging, the open reading frame of cd63 encoding aa 2-236 was fused in frame to aa 236 of egfp (cd63.gfp) present in plasmid pegfp-c1 (bd biosciences, clontech). to inhibit expression of sec63, sirna duplexes targeting the nucleotide positions 1942 to 1960 of sec63 (caa-gaatggtggtggcttt) or esirna were used (sigma-aldrich). as a control, a nonsense sirna with no known homology to mammalian genes was used (quiagen). commercially available antibodies were as follows: mouse antib-actin (sigma-aldrich), rabbit anti-calnexin (santa cruz biotechnology), mouse anti-flag (sigma-aldrich), mouse anti-gfp (bd biosciences, clontech), mouse anti-ha (babco), rat anti-ha (roche), mouse anti-myc (babco), rabbit antibody against human sec63 (sigma-aldrich), and mouse anti-vsv-g (sigma-aldrich). peroxidase-labeled, secondary antibodies were obtained from dianova, and fluorophor-labeled antibodies were purchased from molecular probes. to probe for protein expression, cells were lysed with either the non-denaturing detergent nonidet p-40 (np-40) or the denaturing reagent sodium dodecylsulfate (sds). np-40 lysates were prepared by incubating the cells with tris-buffered saline (50 mm tris-hcl ph 7.5/150 mm nacl) containing 0.5% np-40 for 20 min on ice. thereafter, lysates were centrifuged for 5 min at 13 000 g and 4uc. for lysis with sds, the cells were scraped from the plates using 1 6 laemmli buffer, and cell suspensions were boiled for 10 min prior to centrifugation. cell extracts were subjected to sds-page and western blotting analyses using standard procedures. immunoreactivity was determined by enhanced chemiluminescence (western lightningh plus-ecl, perkinelmer) and recorded on amersham hyperfilm ecl (ge healthcare). the densitometric analysis of western blot band intensities was based on linear, 8-bit, gray scale, transmitted light tiff scans of the exposed films and was performed by using imagequanth software (molecular dynamics). in parallel, cell culture media was harvested, cleared by centrifugation, and assayed for the secretion/release of proteins. the expression and secretion of the hbv.s envelope protein was measured using the murex hbsag version 3 elisa (abbott). to evaluate the presence of damage and toxicity of transfected cells, lactate dehydrogenase (ldh) activity was determined in culture media using a colorimetric quantification assay (roche). microsomes were prepared from homogenized cells and subjected to a trypsin protection assay as described [22] . briefly, microsomes were proteolyzed with trypsin in the presence or absence of 0.5% np-40 for 60 min on ice. after inactivation of trypsin with aprotinin, solubilized samples were subjected to sds-page and immunoblotting. for immunostaining, cells grown on cover-slips were fixed and permeabilized with ice-cold methanol containing 2 mm egta. cells were blocked in pbs containing 2% animal serum, incubated with the indicated primary antibodies for 1 h at 37uc, rinsed with pbs, and then incubated with alexafluor-tagged secondary antibodies for 1 h at 37uc. dna was stained with hoechst 33342 (sigma-aldrich). z-stack images were acquired separately for each channel using a zeiss axiovert 200 m microscope equipped with a plan-apochromat 1006 (1.4 na) and a zeiss axiocam digital camera and were optically deconvoluted using the software supplied by zeiss. phase contrast images were obtained with the same microscope using phase contrast optics. for quantitative immunofluorescence analyses, images of randomly selected cells (about 100 cells per coverslip; n = 5) were collected at 1006 magnification with identical settings. the mrna of cotransfected huh-7 cells was isolated using the dynabeadsh mrna direct tm kit (life technologies) accord-ing to the mini-scale guidelines. to eliminate trace amounts of genomic and plasmid dna, the mrna was treated with 5 u dnase i (roche applied science) at 30uc for 20 minutes. reverse transcription was performed with the transcriptor universal cdna master kit (roche applied science) and the cdna was diluted 1:3. the mrna levels were determined by a taqmanchemistry based quantitative real-time pcr as absolute quantification using the standard curve method. therefore, serial dilutions of plasmid dna containing the respective target dna (range 1610 6 to 1610 21 fg/ml) were amplified in parallel. the pcr was performed with a 7300 real-time pcr system and the sequence detection software 4.0 (life technologies). each pcr reaction contained 4 ml aqua bidest, 0.4 ml of each primer (100 mm), 0.2 ml of each taqman probe (100 mm), 12.5 ml taqmanh gene expression master mix (life technologies) and 7.5 ml cdna. primers and probes are listed in table 1 . the pcr reaction was initiated by a single step at 50uc for 2 minutes and 95uc for 10 minutes followed by 38 cycles at 95uc for 15 seconds and at 60uc for 60 seconds. amplifications of samples and standard curves were performed in duplicate. the calculated data were converted into percentages whereupon the mean value of the control samples was set to 100%. protein transport into the endoplasmic reticulum: mechanisms and pathologies protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes protein translocons: multifunctional mediators of protein translocation across membranes the beta subunit of the sec61 complex facilitates cotranslational protein transport and interacts with the signal peptidase during translocation an evolving view of the eukaryotic oligosaccharyltransferase protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane signal sequencedependent function of the tram protein during early phases of protein transport across the endoplasmic reticulum membrane substrate-specific function of the translocon-associated protein complex during translocation across the er membrane erj1p has a basic role in protein biogenesis at the endoplasmic reticulum homologs of the yeast sec complex subunits sec62p and sec63p are abundant proteins in dog pancreas microsomes life and death of a bip substrate bip and sec63p are required for both co-and posttranslational protein translocation into the yeast endoplasmic reticulum mammalian sec61 is associated with sec62 and sec63 evolutionary gain of function for the er membrane protein sec62 from yeast to humans interaction of bip with the j-domain of the sec63p component of the endoplasmic reticulum protein translocation complex the er function bip is a master regulator of er function mutations in sec63 cause autosomal dominant polycystic liver disease tailanchored and signal-anchored proteins utilize overlapping pathways during membrane insertion post-translational alterations in transmembrane topology of the hepatitis b virus large envelope protein a dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis b viral morphogenesis novel transmembrane topology of the hepatitis b virus envelope proteins chaperone action in the posttranslational topological reorientation of the hepatitis b virus large envelope protein: implications for translocational regulation mammalian bip controls posttranslational er translocation of the hepatitis b virus large envelope protein hepatitis b surface antigen: an unusual secreted protein initially synthesized as a transmembrane polypeptide hepatitis b virus morphogenesis hepatitis b surface antigen assembles in a post-er, pre-golgi compartment deletions in the hepatitis b virus small envelope protein: effect on assembly and secretion of surface antigen particles hepatitis b virus large envelope protein interacts with gamma2-adaptin, a clathrin adaptor-related protein posttranslational n-glycosylation of the hepatitis b virus large envelope protein mutant analysis links the translocon and bip to retrograde protein transport for er degradation one step at a time: endoplasmic reticulumassociated degradation multiple proteases process viral antigens for presentation by mhc class i molecules to cd8(+) t lymphocytes activation of erad pathway by human hepatitis b virus modulates viral and subviral particle production hemopexin: structure, function, and regulation rapid production of retroviruses for efficient gene delivery to mammalian cells using 293t cellbased systems a single n-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus g protein to the cell surface membrane assembly of the triple-spanning coronavirus m protein. individual transmembrane domains show preferred orientation the tetraspanin cd63 regulates escrt-independent and -dependent endosomal sorting during melanogenesis erdj4 protein is a soluble endoplasmic reticulum (er) dnaj family protein that interacts with erassociated degradation machinery the lumenal domain of sec63p stimulates the atpase activity of bip and mediates bip recruitment to the translocon in saccharomyces cerevisiae interaction of murine bip/ grp78 with the dnaj homologue mtj1 structural studies and the assembly of the heptameric post-translational translocon complex role of protein translocation pathways across the endoplasmic reticulum in trypanosoma brucei the proteinconducting channel in the membrane of the endoplasmic reticulum is open laterally toward the lipid bilayer the sec61p complex mediates the integration of a membrane protein by allowing lipid partitioning of the transmembrane domain x-ray structure of a protein-conducting channel sequential triage of transmembrane segments by sec61alpha during biogenesis of a native multispanning membrane protein two translocating hydrophilic segments of a nascent chain span the er membrane during multispanning protein topogenesis the hydrophobic core of the sec61 translocon defines the hydrophobicity threshold for membrane integration different transmembrane domains associate with distinct endoplasmic reticulum components during membrane integration of a polytopic protein cotranslational protein integration into the er membrane is mediated by the binding of nascent chains to translocon proteins forced transmembrane orientation of hydrophilic polypeptide segments in multispanning membrane proteins the concept of translocational regulation a protein of the endoplasmic reticulum involved early in polypeptide translocation a genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation pkd2, a gene for polycystic kidney disease that encodes an integral membrane protein epitope-tagged pkhd1 tracks the processing, secretion, and localization of fibrocystin differential effects of sec61alpha-, sec62-and sec63-depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells role of the j-domain in the cooperation of hsp40 with hsp70 sequencespecific retention and regulated integration of a nascent membrane protein by the endoplasmic reticulum sec61 translocon gammaadaptin, a novel ubiquitin-interacting adaptor, and nedd4 ubiquitin ligase control hepatitis b virus maturation we thank g. nolan, p. j. m. rottier, and g. spoden for generously providing plasmid dna constructs. key: cord-332811-kjgah8ts authors: lee, do hyun; jeon, young-soo; park, choi-kyu; kim, seungjoon; lee, du sik; lee, changhee title: immunoprophylactic effect of chicken egg yolk antibody (igy) against a recombinant s1 domain of the porcine epidemic diarrhea virus spike protein in piglets date: 2015-06-23 journal: arch virol doi: 10.1007/s00705-015-2494-z sha: doc_id: 332811 cord_uid: kjgah8ts porcine epidemic diarrhea virus (pedv) is a highly contagious enteric pathogen of swine causing high mortality rates in piglets. pedv outbreaks have occurred continuously in most swine-producing asian countries and have recently emerged in the united states, leading to large economic losses for both the asian and us pig industries. the spike (s) protein of pedv consists of the s1 and s2 domains, responsible for virus binding and fusion, respectively. the involvement of the s1 domain in specific high-affinity interactions with the cellular receptor and induction of neutralizing antibodies in the natural host makes it a logical target for the development of effective vaccines and therapeutics against pedv. passive immunization by oral administration of egg yolk antibodies (igy) obtained from immunized chickens provides an alternative source of specific antibodies for the prevention and treatment of pedv in newborn piglets. in this study, we produced an igy against the pedv s1 protein and investigated its immunoprophylactic effect in neonatal piglets. a codon-optimized pedv s1 gene consisting of amino acid residues 25–749 was synthesized and used to establish a stable porcine cell line constitutively expressing a recombinant pedv s1 protein containing the chicken immunoglobulin fc fragment at its c-terminus. the purified recombinant s1 protein was found to mediate potent immune responses in immunized hens. we next tested the ability of oral passive immunization with anti-pedv s1 igy to protect piglets against pedv. specific chicken igy against the s1 protein was orally administered to neonatal piglets, and their responses subsequent to a virulent pedv challenge were monitored. the results showed that oral administration of anti-pedv s1 igy efficiently protects neonatal piglets against pedv, suggesting its potential as a prophylactic or therapeutic agent against acute pedv infection. porcine epidemic diarrhea (ped) is characterized by acute enteritis and watery diarrhea followed by severe dehydration, leading to high mortality rates in neonatal piglets [5, 29, 33] . the disease was initially recognized in england in 1971 [27] , and the causative agent, ped virus (pedv), was not identified until 1978 [28] . ped epidemics were first reported in asia in 1982, and ped has since continued to threaten swine health, causing substantial economic losses for the asian swine industry [3, 11, 21, 30, 38] . in early 2013, sudden ped outbreaks occurred in the united states sweeping through the pork industry across the country [24, 35] . subsequently, starting in late 2013, large-scale outbreaks of ped rapidly recurred in korea, taiwan, and japan; a us-strain-like pedv was found to be responsible for the recent outbreaks in those countries, raising global concerns regarding control measures for ped prevention [17] [18] [19] 22] . pedv is a large enveloped virus possessing a singlestranded, positive-sense rna genome of approximately 28 kb with a 5 0 cap and a 3 0 polyadenylated tail, belonging to the genus alphacoronavirus within the family coronaviridae of the order nidovirales [28, 33] . the spike (s) protein of pedv is a type i membrane glycoprotein consisting of 1,383 to 1,386 amino acids (aa), depending on the strain, that can be divided into the s1 (aa 1-735) and s2 (736-end) domains based on homology to the s proteins of other coronaviruses [6, 9, 15, 36] . similar to other coronavirus s proteins, the pedv s protein plays a critical role by interacting with the cellular receptor to mediate viral entry and inducing neutralizing antibodies in the natural host [1, 2] . more precisely, the s1 domain has been reported to contain the receptor-binding region and the main neutralizing epitopes [16, 37] . furthermore, the s1 region has been established as a suitable target for determining genetic relatedness among pedv isolates and for developing differential diagnostic assays and effective vaccines [4, 7, 15, 26] . in korea, both modified live and killed vaccines against pedv have been developed for disease control and use in the domestic market. however, the continued occurrence of ped nationwide has caused enormous financial losses for the korean pork industry, raising questions regarding the efficacy of these commercial vaccines. this phenomenon appears to be due to genetic and antigenic differences between the vaccine strain and the strains prevalent in the field [15, 26] . thus, the lack of effective vaccines increases the need for next-generation field-virus-based vaccines or control measures against pedv infection. passive immunization by oral administration of specific antibodies represents an attractive approach against gastrointestinal pathogens in both humans and animals [23] . egg yolk immunoglobulin (igy) from immunized chickens has been demonstrated to be a convenient large-scale source for specific antibodies; it has also been shown to be safe and effective against pedv in newborn piglets [12] . the aim of the present study was to produce an igy against the pedv s1 protein and assess its immunoprophylactic properties in neonatal piglets. a codon-optimized pedv s1 gene was used to establish a stable porcine cell line constitutively expressing a recombinant s1 protein. the recombinant s1 protein was capable of inducing efficient cytokine and antibody responses in immunized hens. moreover, oral passive immunization using chicken igy raised against the pedv s1 protein was found to control and prevent ped post-challenge in suckling piglets. cells, virus, and antibodies hek-293t cells (crl-1573) were purchased from the american type culture collection (atcc; manassas, va, usa) and cultured in dulbecco's modified eagle medium (dmem) with high glucose (invitrogen, carlsbad, ca, usa) supplemented with 10 % fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solution (1009; invitrogen). pk-15 cells were grown in rpmi 1640 medium (invitrogen) containing 10 % fbs and antibiotic-antimycotic solution. vero cells were cultured in alpha minimum essential medium (a-mem; invitrogen) with 10 % fbs and antibiotic-antimycotic solution. the cells were maintained at 37°c in a humidified 5 % co 2 atmosphere. the sm98-1 pedv vaccine strain was obtained from the korean animal and plant quarantine agency and propagated in vero cells as described previously [8] . challenge pedv was prepared from intestinal contents obtained from a field case that was found to be free of other common etiologic agents of neonatal porcine diarrheal diseases, as described previously [26] . briefly, a 4-day-old suckling piglet was inoculated orally with a small-intestine homogenate containing the field virus. small-intestine tissues were collected and homogenized in a 10 % suspension with a-mem using a magna lyser (roche diagnostics, mannheim, germany) by three repetitions of 15 s at 7,000 rpm, and tissue suspensions were clarified by centrifugation for 10 min at 4,5009g (hanil centrifuge fleta5, incheon, south korea). the clarified supernatant was filtered through a 0.22-lm-pore-size syringe filter (millipore, billerica, ma, usa), aliquoted, and stored at -80°c until use as the crude challenge virus. horseradish peroxidase (hrp) or fluorescein isothiocyanate (fitc)-conjugated secondary antibodies were purchased from jackson immunoresearch laboratories (west grove, pa, usa), and alexa fluor 488-conjugated secondary antibody was obtained from molecular probes (carlsbad, ca, usa). the pedv s-protein-specific monoclonal antibody (mab) was a kind gift from sang-geon yeo (kyungpook national university, daegu, south korea), and the nucleocapsid (n)-protein-specific mab was obtained from choongang vaccine laboratory (cavac; daejeon, south korea). dna manipulation and cloning were performed according to standard procedures [34] . escherichia coli strain dh5a (rbc bioscience, taiwan) was used as the host for general cloning. the pfb-neo-pedv-rs1-ig plasmid encoding a full-length, codon-optimized pedv s1 gene (aa 24-735) was described previously [26] . the pcdna3.1/chicken fc plasmid encoding the fc domain of chicken igg was a kind gift from hyung-kwan jang (chonbuk national university, jeonju, south korea). the chicken fc (cfc) fragment from pcdna3.1/chicken fc was subcloned into pfb-neo-pedv-rs1-ig by replacing the fc domain of human igg1 to construct the gene expression plasmid, pfb-neo-pedv-rs1-cfc, which produces a chicken fc-tagged fusion protein, rs1-cfc. the constructed plasmid was verified by nucleotide sequencing. generation of a stable pk-15 cell line expressing pedv rs1-cfc the retrovirus gene transfer system (agilent technologies, santa clara, ca, usa) was used to generate a cell line constitutively expressing the recombinant pedv rs1-cfc gene or an empty retroviral vector as described elsewhere [15, 25] . antibiotic-resistant continuous cell clones were examined by rt-pcr to verify the presence of the fulllength rs1-cfc gene, and the positive clones (pk-rs1-cfc) were then amplified for subsequent analysis. pk-rs1-cfc cells were grown on microscope coverslips placed in 6-well tissue culture plates. at 48 h post-seeding, the cells were fixed with 4 % paraformaldehyde for 10 min at room temperature (rt) and permeabilized with 0.2 % triton x-100 in phosphate-buffered saline (pbs) at rt for 10 min. the cells were subsequently blocked with 1 % bovine serum albumin (amresco, solon, oh, usa) in pbs for 30 min at rt and then incubated with a goat antichicken igg antibody conjugated to fitc. finally, the cells were counterstained with 4 0 ,6-diamidino-2-phenylindole (dapi; sigma, st. louis, mo, usa), and cell staining was visualized using a leica dm il led fluorescence microscope (leica, wetzlar, germany). pk-rs1-cfc cells were grown in 100-mm-diameter tissue culture dishes to 90 % confluency in serum-free medium (optipro sfm; invitrogen). at 72 h post-seeding, the protein-containing cell culture supernatants were harvested, and soluble proteins were immunoprecipitated with chicken igy precipitating resin (genscript, piscataway, nj, usa) according to the manufacturer's protocol in the presence of protease inhibitors at 4°c for 16 h. the beads were collected by centrifugation at 5,0009g (eppendorf centrifuge 5415r, hamburg, germany) for 5 min at 4°c and washed three times with 0.5 m nacl in pbs. the samples were subsequently eluted with 50 mm sodium citrate/50 mm glycine (ph 2.0) and neutralized with 1 m tris-hcl (ph 8.0). the purified proteins were concentrated with amicon ultracentrifugal filters 100k (millipore). protein concentration was measured using a pierce bca protein assay (thermo scientific, rockford, il, usa), and the final products were analyzed by western blotting to confirm target protein purification. the protein-containing cell culture supernatants or purified proteins as described above were mixed with nupage 49 lds sample buffer (invitrogen) and boiled at 70°c for 10 min. the proteins were separated on a nupage 4-12 % gradient bis-tris gel (invitrogen) under reducing conditions and stained using simplyblue safestain (invitrogen) according to the manufacturer's instructions or electrotransferred onto immobilon-p polyvinylidene fluoride (pvdf) membranes (millipore). the membranes were then blocked with 3% powdered skim milk (bd biosciences, belford, ma, usa) in tbs (10 mm tris-hcl [ph 8.0], 150 mm nacl) with 0.05 % tween-20 (tbst) at 4°c for 2 h and then reacted directly with the goat anti-chicken igg hrp-conjugated secondary antibody or with anti-pedv s mab followed by the corresponding hrp-labeled secondary antibody at a 1:2,000 dilution for 2 h at 4°c. finally, the proteins were visualized using enhanced chemiluminescence reagents (ge healthcare, piscataway, nj, usa) according to the manufacturer's protocol. twenty 10-week-old white leghorn hens were randomly allocated into three groups and immunized by intramuscular injection into the breast muscle with either the binary ethylenimine (bei)-inactivated sm98-1 pedv vaccine strain, 200 lg of the purified pedv rs1-cfc protein resuspended in pbs, or pbs with a mineral oil-based adjuvant (montanide isa 70 vg; seppic, puteaux, france) ( table 1 ). the hens were then boosted three times with the same immunogens emulsified with adjuvant at 3-week intervals. blood samples were collected prior to immunization, at each boost, and three weeks subsequent to the eggs collected from immunized hens were washed with diluted sodium hypochlorite solution (yuhanclorox, seoul, south korea), disinfected with 70 % ethanol, and used for egg yolk fractionation. the separation of the yolk from the albumen and chalaza was performed as described previously [13] . the egg yolk material was mixed with distilled water (dw) at a 1:7 (v/v) ratio immediately followed by the addition of strong acid electrolytic water (ph \ 2.0) at 0.02 % of the total volume. samples were thoroughly whisked using a hand blender for approximately 10 min. the egg yolk mixture was kept at 4°c for 24 h, and supernatants were then separated and clarified by centrifugation for 20 min at 3,0009g (hanil centrifuge fleta5) to thoroughly remove lipids. the supernatants containing igy was collected, lyophilized using a spray dryer and stored at 4°c for further experiments. peripheral blood mononuclear cells (pbmcs) were isolated from whole blood using a standard gradient centrifugation purification protocol with histopaque 1077 (sigma) according to the manufacturer's instructions. total rna was extracted from the pbmcs of hens using trizol reagent (invitrogen) and treated with recombinant dnase i (takara, otsu, japan) according to the manufacturer's protocols. the concentrations of the extracted rna were measured with a nanovue spectrophotometer (ge healthcare). quantitative real-time rt-pcr was done using a thermal cycler dice real time system (takara) with a one step sybr primescript rt-pcr kit (takara) and the following gene-specific primer sets as described previously [20, 31, 32] : chicken ifn-a forward, 5 0 -atcctgct gctcacgctccttct-3 0 ; chicken ifn-a reverse, 5 0 -gg tgttgctggtgtccaggatg-3 0 ; chicken ifn-b forward, 5 0 -gcctccagctccttcagaatacg-3 0 ; chicken ifn-b reverse, 5 0 -ctggatctggttgaggaggctgt-3 0 ; chicken ifn-c forward, 5 0 -agctgacggtggacc-tattatt-3 0 ; chicken ifn-c reverse, 5 0 -ggctttgcgc tggattc-3 0 ; chicken il-6 forward, 5 0 -caaggtgac ggaggaggac-3 0 ; chicken il-6 reverse, 5 0 -tggcgag gagggatttct-3 0 ; chicken il-8 forward, 5 0 -ccaag-cacacctctcttcca-3 0 ; chicken il-8 reverse, 5 0 -gcaaggtaggacgctggtaa-3 0 ; chicken tnf-a forward, 5 0 -gaagcagcgtttgggagt-3 0 ; chicken tnf-a reverse, 5 0 -gttgtgggacagggtagg-3 0 ; chicken glyceraldehyde-3-phosphate dehydrogenase (gapdh) forward, 5 0 -ggtggtgctaagcgtgttat-3 0 ; chicken gapdh reverse, 5 0 -acctctgtcatctctccaca-3 0 . the steady-state mrna levels of each cytokine gene were normalized against the level of chicken gapdh mrna, and the relative quantity (rq) of mrna accumulation was evaluated using the 2 -ddct method. the relative fold change in the expression of each gene was then calculated by comparing pre-immune and immunized sera. pedv-specific neutralizing antibodies in the serum and igy samples collected from hens in all groups were determined using a serum neutralization test in 96-well microtiter plates with the sm98-1 pedv vaccine strain as previously described [14] . briefly, individual virus stocks were diluted in serum-free a-mem to a concentration of 200 plaque-forming units in a volume of 50 ll. the diluted virus was then mixed with 50 ll of a twofold serial dilution of individual inactivated sera or igy solution dissolved in dw (100 mg/ml) in 96-well plates, and the mixture was incubated at 37°c for 1 h. next, approximately 1 9 10 4 vero cells in 100 ll of a-mem with 10 % fbs were added to each well, and the mixture was further maintained at 37°c in a 5 % co 2 incubator for 3 to 4 days. the neutralization titer was calculated as the reciprocal of the highest serum dilution that inhibited virus-specific cytopathic effects in all duplicate wells. the in vivo swine experiments described here were performed at the improah animal facility under the guidelines established by the institutional animal care and use committee. a total of 18 newborn piglets were obtained from seronegative pregnant sows at a commercial pig farm with no known prior ped outbreak or vaccination with pedv. all animals were determined to be free of antibodies to pedv, as well as to transmissible gastroenteritis virus and porcine reproductive and respiratory syndrome virus. no other animals aside from those included in the study were housed at the facility for the duration of the experiment. the design for the present pig experiment is outlined in table 2 . piglets were randomly divided into four groups: group 1, administration of igy against pedv; group 2, administration of igy against pedv rs1-cfc; group 3, administration of igy against placebo; group 4, control. the groups were housed in separate rooms, and no physical contact was allowed between the groups. all neonatal piglets except for animals in the control group were inoculated orally with 1 ml of the small-intestine homogenate containing 10 4 tcid 50 /ml pedv field virus determined using real-time rt-pcr as described previously [10] . following challenge exposure, piglets were administered orally with 2 ml of the corresponding igy solution dissolved in dw (250 mg/ml) at 1 and 2 days post-inoculation (dpi). clinical signs of diarrhea and the mortality in challenged piglets were monitored daily throughout the study. stool samples from all groups were collected daily with 16-inch, cotton-tipped swabs and subjected to rt-pcr using an i-tgev/pedv detection kit (intron biotechnology, seongnam, south korea) to detect the presence of pedv. fecal shedding of pedv was measured by quantitative real-time rt-pcr as described above, and the results were analyzed using the system software as described previously [32] . all piglets from the challenged and control groups were euthanized at 5 dpi for post-mortem examination. smallintestinal tissue specimens collected from each piglet (\3 mm thick) were fixed with 10 % formalin for 24 h at rt and then embedded in paraffin according to standard laboratory procedures. the formalin-fixed paraffin-embedded tissues were cut into 5-to 8-lm-thick sections on a microtome, floated in a 40°c water bath containing dw, and transferred onto glass slides. the tissues were then deparaffinized in xylene for 20 min and washed in decreasing concentrations of ethanol (100 %, 95 %, 85 %, 70 %, and 50 %) for 3 min each. the deparaffinized intestinal tissue sections were stained with hematoxylin and eosin (h&e; sigma) to observe histopathological lesions of pedv infection and subjected to ifa using the pedv n-specific mab and goat anti-mouse igg secondary antibody conjugated to alexa fluor 488 as described above. the student's t-test was used for all statistical analyses and p-values of less than 0.05 were considered statistically significant. generation of stable porcine-origin cell lines expressing the full-length, codon-optimized s1 protein previously, we synthesized a full-length, codon-optimized s1 gene and confirmed that codon optimization greatly enhanced the expression level of s1 upon transient transfection [26] . in this study, the codon-optimized s1 gene was used for stable transfection to produce preparative amounts of the s1 protein. to accomplish this, sublines of pk-15 cells were established that stably expressed the recombinant codon-optimized s1 under the control of a retroviral ltr promoter. ten generated cell clones were initially collected and subjected to rt-pcr and western blot analysis to examine s1 expression at the mrna and protein level, respectively (data not shown). based on the results of the western blot analysis, one pk-rs1-cfc cell clone that consistently expressed the highest level of s1 was chosen for subsequent studies. to characterize the pk-rs1-cfc cells, intracellular and extracellular expression levels of s1 were examined by immunofluorescence and western blotting. as shown in fig. 1a , specific cell staining was clearly evident when pk-rs1-cfc cells were reacted with the anti-chicken igg antibody, confirming a consistent high expression level of the s1 protein. western blot analysis of cell culture supernatants revealed that the pk-rs1-cfc cells stably expressed and cumulatively secreted high levels of the approximately 180-kda s1. the recombinant s1 protein expressed in the supernatants of stable pk-rs1-cfc cells was purified using chicken igy precipitation resin beads. the purified s1 protein was detectable at a high level by simplyblue staining and was confirmed by immunoblotting with antichicken igg antibody (fig. 1b) . using our purification and concentration procedures, we were able to purify an average yield of 30 lg of s1 protein per 6 ml of pk-rsi-cfc cell culture supernatant cultivated in a 100-mm tissue culture dish for 72 h. in addition, the overall growth kinetics of s1 gene-expressing pk-15 cells were found to be similar to those of the parental pk-15 cells, indicating that s1 expression has no effect on cell growth (data not shown). to produce igy, hens assigned to the three groups were immunized intramuscularly, as outlined in table 1 . blood samples were collected before immunization (pre-immune), at each boost, and 3 weeks after the final boost. pbmcs were prepared from blood samples obtained during the first ifn-a, ifn-b , ifn-c, il-6, and il-8, was significantly altered by immu-nization of inactivated pedv or s1-cfc compared to levels in the placebo-inoculated group (fig. 2) . these data indicate that the immunogens used in this study efficiently stimulated immune responses in chickens. serum samples collected at each collection time were subjected to a serum neutralization test against the pedv vaccine strain. non-immunized hens showed only minimal neutralizing antibody titers, whereas immunized hens exhibited gradually increasing neutralizing antibody titers (fig. 3a) . furthermore, hens immunized with inactivated pedv (group 1) and s1-cfc protein antigen (group 2) at 3-week intervals produced similar neutralizing antibody titers ranging from 1:32 to 1:64 subsequent to the final immunization. in addition, egg yolk samples from each group were found to contain neutralizing antibody titers comparable to those in the corresponding serum samples, indicating the presence of neutralizing igy antibodies (fig. 3b) . to evaluate the immunoprophylactic efficacy of anti-pedv s1 igy, neonatal 4-to 5-day-old pedv-seronegative piglets were divided into four groups and challenged orally with wild-type pedv followed by the respective antibody treatment at days 1 and 2 post-challenge. animals in group 4 were not challenged with pedv and given igy orally from non-immunized chickens at the same time points as the other groups. clinical observations of mortality and diarrhea in challenged piglets are summarized in table 3 . none of the piglets from group 4 died or developed any clinical signs of diarrhea. additionally, fecal shedding of pedv was also detected in the rectal swabs of these animals for the duration of the study (fig. 4) . in contrast, all piglets from the challenged groups (1-3), regardless of igy administration, exhibited diarrhea that began at 1 or 2 days post-challenge and lasted throughout the challenge experiment. in group 3, one piglet died; the remaining piglets experienced severe watery diarrhea and shed pedv in their feces, with a mean cycle threshold (ct) value of 16.9 (range 14.8-18.6) during the study (fig. 4) . although none of the piglets from groups 1 and 2 treated with igy died during the challenge experiment, the number of piglets exhibiting diarrhea post-challenge varied depending on the group. all piglets from these groups showed mild-to-severe diarrhea lasting for the entire experiment but recovered from the diarrhea by the end of the study. furthermore, fecal shedding of pedv from these piglets was significantly lower than in group 3 piglets; pedv rna was detected in the fecal swab samples of all pigs in groups 1 and 2, with mean ct values of 24.1 (range fig. 1 constitutive expression of the recombinant s1 protein in pk-rs1-cfc cells. (a) immunofluorescence assay for the rs1 protein. pk-15 or pk-rs1-cfc cells grown in a 6-well tissue culture plate were fixed with 4 % formaldehyde at 48 h post-seeding and incubated with anti-chicken igg antibody (top panels). the cells were then counterstained with dapi (bottom panels) and examined using a fluorescent microscope at 4009 magnification. (b) purification of the rs1 protein. the recombinant s1 protein was purified from serum-free medium of pk-rs1-cfc cells grown in a 100-mm tissue culture dish. the cell culture supernatant and the purified rs1 protein were resolved on a 4-12 % gradient bis-tris gel and stained with simplyblue solution (left panel) or electrotransferred onto a pvdf membrane (right panel). the membrane was blotted with a chicken igg-specific antibody 23.1-24.8) and 21.9 (range 20.0-26.5), respectively (fig. 4) . however, despite the similar levels of diarrhea, the group 2 piglets shed slightly higher amounts of pedv in their feces compared to those in group 1. gross intestinal lesions consistent with viral enteritis, including thin-walled and fluid-content-dilated small intestines, were typically observed in all group 3 piglets; less-severe lesions were observed in piglets given either igy (data not shown). likewise, the majority of enterocytes over the entire villi in the control piglets with normal igy treatment (group 3) were affected by pedv, showing moderate-to-severe villous atrophy and destruction, while piglets from groups 1 and 2 exhibited mild intestinal lesions comparable to those in the non-challenged group, and viral antigens were only detected in their small intestines (figs. 5 and 6 ). however, anti-pedv s1 igy treatment (group 2) was more efficacious than treatment with igy raised against the whole virus (group 1) in reducing the overall severity of macroscopic and microscopic intestinal lesions in the piglets. collectively, all immunoprophylactic methods used in this study were capable of protecting neonatal piglets against mortality and disease severity after challenge with a virulent pedv. vaccination against pedv is one of the most important and effective prevention measures by passively transferring specific neutralizing antibodies present in vaccinated sows to their litters through colostrum and milk. despite the availability of commercial attenuated and inactivated vaccines in korea, pedv continues to plague the domestic pork industry, raising issues regarding their protective efficacy. recently, severe outbreaks of pedv have reemerged in korea and have been estimated to affect over 40 % of pig farms, causing serious economic impact on producers and customers [17, 18] . our previous studies suggested that antigenic and genetic variations between the vaccine virus and field pedvs may be the cause of the incomplete efficacy or failure of vaccination, which appears to be responsible for the periodic outbreaks in domestic herds [15, 26] . furthermore, a comparison of the amino acid sequence of the s1 domain of the pedv s protein, which is associated with viral binding to host cell receptors and contains neutralizing epitopes [16, 37] , has shown a difference of over 10 % between the vaccine strain and field isolates, suggesting the need for nexteffect of igy against the pedv s1 protein 2203 generation vaccine development [15, 17] . in addition to vaccination, artificial passive immunization using igy has been used commercially as an alternative method for controlling ped by providing supportive immunity to neonatal piglets exposed to acute infection in korea. however, opinions differ among swine practitioners and producers regarding the efficacy of this immunoprophylactic strategy. a similar debate regarding the commercial use of igy may arise since the vaccine strain is also used to immunize chickens for the production of anti-pedv igy in korea. we have previously demonstrated that the s1 protein of pedv could be considered a potential candidate antigen for vaccination [26] . in the present study, the s1 protein of the field pedv was used as an immunogen to inoculate hens, thereby producing anti-pedv s1 igy. we first aimed to stably express the full-length, codon-optimized s1 gene of pedv in porcine-origin cells and to evaluate the immunogenicity and efficacy of igy against the recombinant s1 protein. subsequently, we were able to successfully generate a stable pk cell line continuously producing large amounts of the codon-optimized s1 protein tagged with cfc. following the purification and concentration processes, approximately 30-40 lg of the recombinant s1-cfc protein could be consistently harvested from the culture supernatants of pk-rs1-cfc cells grown in a 100-mm culture dish. since humoral immunity is an important indicator for evaluating the effect of the s1-based immunogen used in this study, we immunized chickens with the s1-cfc antigen prepared from the cell culture supernatants of pk-rs1-cfc cells and investigated whether they developed cytokine and antibody responses. all of the proinflammatory cytokines tested, except for tnf-a, were stimulated in chickens immunized with either the whole virus or the s1-cfc antigen. of these, the expression levels of il-6 and il-8 genes were distinctly enhanced in the s1-cfc-inoculated group compared with the inactivated pedv-inoculated group. furthermore, the chicken sera raised against s1-cfc contained higher levels of neutralizing antibody than those raised against the sm98-1 vaccine virus. however, the final levels of anti-pedv igy were lower in the eggs of the s1-cfc-immunized chickens compared to those of the sm98-1-inoculated group. these inconsistent results may be attributed to the use of the heterogeneous sm98-1 virus in the serum neutralization assay, which possesses a high degree of genetic variation in field isolates. nevertheless, our data indicated that the recombinant s1 protein efficiently elicits immune responses in chickens. subsequently, we tested the prophylactic efficacy of each igy in suckling piglets post-challenge-exposure. our data showed that, regardless of the igy administered, all neonatal piglets treated with igy survived after challenge with virulent pedv, suggesting that the anti-pedv s1 igy provides effective passive immunity to prevent mortality comparable to whole-virus-based igy. despite the lower levels of virus shedding in the feces of animals treated with anti-pedv igy, the anti-s1 igy-based strategy resulted in more-efficient protection than the anti-pedv igy procedure, as determined by the duration and severity of diarrhea and histopathological lesions. these results indicated that the administration of anti-s1 igy could significantly reduce the mortality and clinical signs of piglets, suggesting that tissue specimens were collected from the small intestines of piglets from each group at the time of necropsy. the formalin-fixed and paraffin-embedded tissue sections were deparaffinized and stained with hematoxylin and eosin. the sections were examined using a light microscope at 409 magnification. the inset images are enlarged versions of parts of the picture effect of igy against the pedv s1 protein 2205 application of anti-s1 igy is capable of partially blocking the virus from invading the small intestine. a more promising approach would be to use the entire field virus or its full-length s protein to produce igy instead of using only the s1 domain, since the s protein contains multiple functional domains and neutralizing epitopes to efficiently stimulate non-susceptible hosts. for this purpose, the production and application of igy against a korean field isolate are currently under investigation. in conclusion, to the best of our knowledge, this is the first study to evaluate the immunoprophylactic effect of igy against the recombinant s1 protein of the field virus in a pig model. the results presented here indicate that the recombinant s1 protein can elicit cytokine and antibody responses and induce neutralizing antibodies in chickens. furthermore, challenge experiments revealed that administration of anti-s1 igy efficiently protected suckling piglets against field pedv by providing passive immunity. pedv infection causes high mortality rates (90-100 % in neonatal piglets under 7 days of age), and epidemiological observations indicate the rapid spread of the disease. in this circumstance, the preliminary results of the present study showed the potential of anti-pedv s1 igy application as a considerable measure against pedv. further experiments to optimize production procedures will be required to achieve higher titers of igy; field studies on farms will be needed to better evaluate the efficacy of anti-s1 igy. these studies will provide additional practical information for the future use of this alternative igy method as a supplement to passive immunity against ped and other economically important viral diseases. fig. 6 detection of pedv in small intestine tissues of piglets. tissue specimens were prepared from small intestine of piglets from each group at the time of necropsy. the formalin-fixed and paraffinembedded tissue sections were deparaffinized and subjected to immunofluorescence staining with an anti-pedv n antibody. the sections were then counterstained with dapi and examined using a fluorescence 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in immortalized porcine alveolar macrophages human telomerase reverse transcriptase-immortalized porcine monomyeloid cell lines for the production of porcine reproductive and respiratory syndrome virus diseases of swine molecular cloning: a laboratory manual emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences proteolytic cleavage of peplomeric glycoprotein e2 of mhv yields two 90k subunits and activates cell fusion spike protein region (aa 636789) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan acknowledgments this research was supported by technology development program for bio-industry, ministry for agriculture, food and rural affairs, republic of korea (311007-05-1-hd120). conflict of interest the authors declare that they have no conflict of interest.ethical approval all procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted. key: cord-329403-jzrlywfe authors: teo, su hui catherine; wu, jian-ping; mok, chee-keng; tan, yee-joo title: a ns1-binding monoclonal antibody interacts with two residues that are highly conserved in seasonal as well as newly emerged influenza a virus date: 2019-03-06 journal: pathog dis doi: 10.1093/femspd/ftz012 sha: doc_id: 329403 cord_uid: jzrlywfe the non-structural protein 1 (ns1) of influenza a virus (iav) is a multifunctional protein that antagonizes host antiviral responses, modulating virus pathogenesis. as such, it serves as a good target for research and diagnostic assay development. in this study, we have generated a novel monoclonal antibody (mab) 19h9 and epitope mapping revealed that two residues, p85 and y89, of ns1 are essential for interacting with this mab. furthermore, residues p85 and y89 are found to be highly conserved across different iav subtypes, namely seasonal h1n1 and h3n2, as well as the highly pathogenic h5n1 and h5n6 avian strains. indeed, mab 19h9 exhibits broad cross-reactivity with iav strains of different subtypes. the binding of mab 19h9 to residue y89 was further confirmed by the abrogation of interaction between ns1 and p85β. additionally, mab 19h9 also detected ns1 proteins expressed in iav-infected cells, showing ns1 intracellular localization in the cytoplasm and nucleolus. to our knowledge, mab 19h9 is the first murine mab to bind at the juxtaposition between the n-terminal rna-binding domain and c-terminal effector domain of ns1. it could serve as a useful research tool for studying the conformational plasticity and dynamic changes in ns1. influenza a virus (iav) belongs to the orthomyxoviridae family and is composed of eight single-stranded, negative-sense rna segments. of these, the ns segment is the smallest and it encodes for the non-structural protein 1 (ns1) and nuclear export protein (nep, also known as ns2). ns1 protein of iav is a potent antagonist of the cellular antiviral interferon (ifn) response. although it is not incorporated into the virus, it exerts multiple functions through the interaction with a large number of cellular components either in the cytoplasm or the nucleus. ns1 is able to inactivate the host antiviral interferon responses through a variety of mechanisms to facilitate replication during infection (kochs, garcia-sastre and martinez-sobrido 2007) . although strain dependent, the general functions of ns1 include inhibiting the antiviral effects of 2'-5'-oligo (a) synthetase (oas)/rnase l and protein kinase r (pkr) (li et al. 2006 ); activating phosphoinositide 3kinase (pi3k) (hale et al. 2006) ; inhibiting host gene expression by interacting with components of nuclear transport (melen et al. 2007 ) and blocking mrna export (fortes, beloso and ortin 1994) . moreover, ns1 is also involved in suppressing adaptive immunity by attenuating human dendritic cell maturation and the capacity of dendritic cells to induce t-cell response (fernandez-sesma et al. 2006) . the ns1 protein is composed of 230-237 amino acids depending on the specific strain (hale et al. 2008) . ns1 has two wellcharacterized functional domains, namely, n-terminal rnabinding domain (rbd) and c-terminal effector domain (ed). the rbd is composed of the first 73 amino acids of ns1, followed by the linker region (lr) at residues 74-88, and residues 88-202 constitute the ed (hale 2014) . ns1(rbd) contains the residues critical for the double stranded rna (dsrna) binding. ns1(ed) is responsible for interaction with various cellular proteins including pkr (lu et al. 1995) , the 30-kda subunit of cleavage polyadenylation specificity factor 30 (cpsf30) (nemeroff et al. 1998; noah, twu and krug 2003) , and some pdz domain ligand proteins (jackson et al. 2008) . given that ns1 is highly expressed in infected cells and plays multiple functions, we have previously produced monoclonal antibodies (mabs) binding to ns1 as tools for research and diagnostic assay development. two of them were found to bind to ns1(rbd) and are able to bind different subtypes of iav (tan et al. 2010) . in this study, we further generated and characterized another mab, named as 19h9, which binds to two highly conserved residues p85 and y89 in ns1. a549, 293t and mdck cells were purchased from american type culture collection (manassas, va, usa). a549 cells were cultured in minimum essential medium (gibco). 293t and mdck cells were cultured in dulbecco's modified eagle's medium (dmem) (invitrogen). both media were supplemented with 10% fetal bovine serum (hyclone), penicillin (10 000 units/ml)streptomycin (10 mg/ml) solution (sigma-aldrich). all cell lines were maintained at 37 • c with 5% co 2 . the ns1 fragment (residues 85-215) of a/puerto rico/8/1934(h1n1) was pcr amplified from a full-length ns1 gene (accession no.: abd77680.1) and cloned into the pgex-6p-1 vector (ge healthcare). the gst-tagged wild-type and mutant ns1(ed) proteins were expressed and purified as described previously (tan et al. 2010) . the purified fusion proteins were dialyzed against phosphate-buffered saline (pbs) and the final concentration was determined using the coomassie plus protein assay reagent (thermo scientific). the purified proteins were concentrated to 3 mg/ml in a centrprep-10 (amicon) and stored in −20 • c for subsequent assays. purified gst-ns1-85-215aa protein was used to immunize mice and generate hybridomas as described previously (oh et al. 2010) . mab isotype was determined using the isostrip mouse monoclonal antibody isotyping kit (roche). the mab was purified using a hitrap protein g hp affinity column (ge healthcare) and was stored at −80 • c. the purity of the mab was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic (sds-page) analysis. the concentration of the purified mab was determined using the coomassie plus protein assay reagent (thermo scientific). for immunofluorescence analysis, mdck cells on glass coverslips were fixed at 24 hr post-infection (hpi) in either ice-cold methanol for 10 min at −20 • c or in 4% paraformaldehyde for 10 min at room temperature (rt) followed by permeabilization with 0.5% triton x-100 (sigma) for 5 min. fixed cells were then blocked with pbs containing 10% fetal bovine serum for 30 min at rt. cells were immunolabeled for 1 hr at rt with mab 19h9 or mouse anti-nucleoprotein (np; millipore) and 45 min with alexa fluor 488-conjugated goat anti-mouse igg antibody (invitrogen) together with dapi (sigma), and mounting in prolong r gold antifade mountant (molecular probes). images were acquired with olympus ckx53 microscope using olympus lcach n 20x/0.40 ipc objective lens and olympus dp27 color camera with olympus cellsens software. each channel was collected separately, with images at 1024 × 1024 pixels. 293t cells were seeded onto 6-well plates 24 hr prior to the transient transfection using lipofectamine 2000 reagent (invitrogen) according to the manufacturer's protocol. ns1-expressing plasmids of different subtypes (h1n1, h3n2, h5n1 and h5n6) were used in this study. in addition, a series of n and c-terminal truncations and amino acid substitution mutants of h1n1-ns1 were generated by pcr using high fidelity polymerase (roche) and cloned into pxj40-myc expression vector. these mutant proteins were expressed by transient transfection. about 48 hr posttransfection, cells were harvested, spun down by centrifugation and washed with cold pbs twice. cells were then resuspended in ripa buffer [50 mm tris-hcl (ph 8.0), 150 mm nacl, 0.5% np40, 0.5% deoxycholic acid, 0.005% sds and 1 mm phenylmethylsulfonyl fluoride] and subjected to freeze-thaw cycle 5 times. clear supernatant containing the protein of interest was obtained by spinning down the cell lysate at 13 000 rpm at 4 • c to remove the cell debris and further analyzed by western blot analysis. commercial antibodies against p85β (santa cruz), gst (santa cruz), gapdh (santa cruz), myc (santa cruz) and np (millipore) were used. a home-made rabbit anti-ns1 polyclonal antibody (wu et al. 2016 ) as well as the mab 19h9 generated above were also used. a total of 25 μg of total cell lysates were resolved using electrophoresis on sds-page gels and transferred onto a nitrocellulose membrane (bio-rad). the membrane was blocked in 5% skimmed milk in tris-buffered saline with 0.05% tween 20 (tbst) for 1 hr at rt and incubated with primary antibodies at 4 • c overnight. after the membrane was washed with tbst, it was incubated with a hrp-conjugated secondary antibody (pierce) at rt for 1 hr. the membrane was then washed and full-length ns1 transfected cell lysates were subjected to ip with mab 19h9 and protein a agarose beads followed by detection of immunoprecipitated proteins using rabbit anti-myc antibody in western blot. masses of molecular weight markers are indicated on the left in kda. (c), mdck cells were mock-or infected with rgpr8 h1n1 at an moi of 1 for 24 hr, fixed and processed for immunofluorescence. the subcellular localization of ns1 and np was detected by mab 19h9 and mouse anti-np mab, respectively. images were taken at magnification of 20x. with tbst again and detected with enhanced chemiluminescence substrate (pierce) using chemidoc tm mp imaging system (bio-rad). for loading controls, the membrane was re-probed with rabbit anti-gapdh (santa cruz biotechnology) antibody overnight at 4 • c, followed by secondary antibody and addition of enhanced chemiluminescence substrate (pierce) for protein detection. for ip, mab 19h9 was used to pull down full-length ns1, ns1(rbd) and ns1(ed) in the cell lysates for 1 hr at 4 • c, followed by the addition of protein a beads (roche) and then incubation at 4 • c overnight. the beads were washed with ripa buffer 3 times before subjected to western blot analysis. rabbit anti-myc antibody was used as primary antibody to detect the presence of ns1 protein and goat anti-rabbit hrp-conjugated antibody (pierce) was used as secondary antibody, followed by the recombinant viruses were generated with phw2000 reverse genetic system as described previously (hoffmann et al. 2000) . residue 85 of ns1 was changed from p to a and residue 89 was changed from y to f or a by pcr mutagenesis and the resulting dna were inserted into phw2000 vector. this plasmid was co-transfected with another seven phw2000 plasmids encoding the other pr8 genomic rnas into 293t cells. two days post-transfection, culture supernatant was collected and used to infect mdck cells. when cytopathic effect was visibly detected, culture supernatant was collected and used to infect naïve mdck cells. individual plaque was then amplified and viral titer was determined by plaque assay. about 90% confluent mdck cells were adsorbed with serially diluted supernatants containing viruses for 1 hr at 37 • c. the medium was discarded and the cells were rinsed using pbs. the cells were overlaid with 2 ml of dmem supplemented by 0.3% agar and 2 μg/ml tpck-trypsin (thermo scientific). after incubation at 37 • c for 2 days, the cells were fixed using 10% formalin for 1 hr and stained using 0.1% crystal violet solution. about 5 μg gst-tagged ns1(ed) fusion proteins were mixed gently with 40 μl of 50% slurry of glutathione sepharose 4b beads for 2 hr at 4 • c. beads were washed three times with binding buffer [20 mm tris (ph 7.4), 0.1 mm edta and 100 mm nacl] followed by incubation with 100 μg total lysates containing overexpressed p85β protein together with a serial dilution of mab 19h9 or control antibody at 4 • c overnight. the control antibody is a murine mab 1a9 which binds to the spike protein of severe acute respiratory syndrome coronavirus (lip et al. 2006; ng et al. 2014 ). the p85β protein was overexpressed in 293t cells via transient transfection as described above and the p85β expression plasmid (pcmv6-xl6-p85β) was purchased (origene). subsequently, beads were sedimented by centrifugation, washed three times with the washing buffer [0.5% np40, 0.1 mm edta, 20 mm tris, ph 7.4, 300 mm nacl]. laemmli sample buffer was added to the beads which were boiled to release all bound proteins. western blot analysis was subsequently performed to detect for p85β and ns1(ed) proteins. densitometric analysis of band intensities for western blots were performed using imagej. two-tailed student's t-test was applied to evaluate the statistical significance of differences measured from the data sets obtained in three independent experiments. p < 0.05 was considered statistically significant. the generation of mab 19h9 was carried out using the recombinant gst-tagged ns1(ed) protein of a/puerto rico/8/1934(h1n1) virus, which was expressed in and purified from e. coli. mab were incubated with 100 μg 293t cell lysates transiently transfected to overexpress p85β protein together with indicated amount of control mab 1a9 (lane 3) or mab 19h9 (lanes 4-7) at 4 • c for 2 hr. after washing, bound p85β, gst and gst-ns1(ed) were probed by p85β and gst antibodies, respectively. (b), the relative p85β-ns1 interaction in the absence of 19h9 was arbitrarily set at 100%, which was used to normalize the relative binding in the presence of indicated concentrations of mab 19h9 or control mab 1a9. 19h9 was able to recognize the ns1(ed) upon initial screening using elisa (data not shown). the isotype of mab 19h9 was determined to be igg2a, using isostrip mouse monoclonal antibody isotyping kit (roche) according to the manufacturer's protocol. the myc-tagged full-length ns1, ns1(rbd) and ns1(ed) proteins were expressed in 293t cells via transient transfection. as shown by western blot analysis (fig. 1a, bottom panel) , mab 19h9 only detected full-length ns1 and ns1(ed), suggesting its specificity to ed. the expression level of ns1 proteins was verified using an anti-myc antibody (fig. 1a, top panel) . since western blot analysis only detected denatured form of proteins, immunoprecipitation assay was also performed to determine whether mab 19h9 could recognize ns1 under native condition. as shown in fig. 1b , mab 19h9 immunoprecipitated full-length ns1 and ns1(ed) proteins in native state, while no binding was detected with ns1(rbd). thus, mab 19h9 is able to bind to both denatured and native forms of ns1 with specificity to the ed. furthermore, for spatial analysis of ns1 expressed in h1n1infected cells using mab 19h9, mdck cells were mock-or infected with a/puerto rico/8/1934/(h1n1) (rgpr8) generated by reverse genetics at a multiplicity of infection (moi) of 1 for 24 hr, fixed and processed for imaging analysis. infection was determined by immunolabeling for another viral protein, np and the expression of np was observed in all the cells exhibiting nuclear and cytoplasmic localization. figure 1c demonstrates that mab 19h9 binds to ns1 expressed in h1n1-infected cells. in addition, ns1 displayed cytoplasmic as well as nucleolar localization. this results are consistent with previous data (greenspan, palese and krystal 1988; garaigorta, falcon and ortin 2005; melen et al. 2007; tsai et al. 2017 ) on the subcellular localization of ns1 as virus strains (newby, sabin and pekosz 2007) and duration of infection (melen et al. 2007) can determine ns1 localization pattern. thus, mab 19h9 could serve as a useful tool for studying ns1 cellular localization, especially in relation to its functions in the cytoplasm and nucleolus. to delineate the epitope of ns1 recognized by mab 19h9, a series of ns1 truncation mutants consisting of residues 1-84, 1-100, 91-230 and 85-230 were generated and their expression levels in transiently transfected 293t cells were verified using an anti-myc antibody ( fig. 2a, upper panel) . western blot results showed that the minimal epitope region lies in the amino acids 85-90 ( 85 pasryl 90 ) as mab 19h9 could only bind ns1 fragments containing this motif ( fig. 2a, lower panel) . next, alanine scanning was used to determine the relative contribution of each of these residues to the interaction with mab 19h9. ns1(ed) mutants containing a single alanine substitution (at residue 85, 87, 88, 89, 90, in the case of residues 86 and 89, a86v mutant and a more conservative y89f mutant were also constructed) were constructed and tested for the binding of mab 19h9. the expression levels of these mutants in transiently transfected 293t cells were verified using an anti-myc antibody (fig. 2b, upper panel) . as illustrated in fig. 2b (lower panel) , the binding of mab 19h9 to alanine mutants at position 85 or 89 was totally abolished, while the binding to the more conservative y89f mutant was significantly reduced. on the other hand, single substitution at residues 86, 87, 88 and 90 did not affect binding of mab 19h9. fig. 2 shows that residues p85 and y89 of ns1 are essential for the binding of mab 19h9. amino acid sequence alignment revealed that these two residues are highly conserved among the seasonal h1n1 and h3n2, as well as highly pathogenic h5n1 and h5n6 avian iav strains (fig. 3a) . in order to investigate whether mab 19h9 interacts with the two residues conserved across different subtypes, ns1 of h1n1 and h3n2, as well as h5n1 and h5n6 was expressed in 293t cells via transient transfection. indeed, mab 19h9 was able to detect ns1 of different subtypes (fig. 3b) . thus, mab 19h9 not only binds to ns1 of the homologous pr8 virus but also cross-reacts with ns1 of different iav subtypes. besides testing the binding of mab 19h9 to ns1 protein overexpressed in 293t cells, recombinant viruses with various ns1 mutations (rgpr8-ns1-p85a, rgpr8-ns1-y89a and rgpr8-ns1-y89f) were generated so that binding of mab 19h9 to ns1 expressed in infected cells can be assessed. the replication potential of these recombinant viruses was first assessed by comparing their multi-step growth kinetics at 12, 24, 36 and 48 hpi in a549 cells infected with the viruses at an moi of 0.01. as shown in fig. 4a , all mutant viruses displayed marginally slower growth kinetics as compared to the wild-type virus, however, the difference was not statistically different. in the single-step growth kinetics assay, 293t cells were infected with wild-type and mutant viruses at an moi of 1, harvested at 12 and 24 hpi and cell lysates were subjected to western blot analysis to assess the level of viral protein synthesis. as shown in fig. 4b , single mutation at either residue p85 or y89 of ns1 did not affect the rate of viral protein synthesis in single-cycle replication, since the expression levels of viral np in mutant viruses were comparable to that of the wild-type virus at both time points. similarly, the level of ns1, as determined by using a rabbit anti-ns1 polyclonal antibody (wu et al. 2016) , was also comparable for all four viruses. however, mab 19h9 only bound strongly to rgpr8-ns1-wt virus and did not bind the three mutant viruses. consistent with the western blot results obtained using recombinantly expressed ns1(ed) fragments, these results suggested that residues p85 and y89 are required for mab 19h9 binding to viral ns1 expressed in infected cells during viral infection. it has been shown that ns1 binds specifically and directly to the p85β regulatory subunit of pi3k to activate the pi3k/akt pathway, which in turn supports efficient virus replication (ehrhardt et al. 2007) . structural study has revealed that residue y89 of ns1 is positioned in the binding interface of ns1-p85β complex and the conservative substitution at this residue (y89f) disrupts this interaction (hale et al. 2010) . since y89 is also critical for the binding of mab 19h9 to ns1, an in vitro gst pull-down competition assay was performed to investigate the effect of mab 19h9 on the interaction between ns1 and p85β. bacterially expressed and purified gst (negative control) and gst-ns1(ed) proteins were incubated with glutathione sepharose 4b beads followed by incubation, in the presence of mab 19h9 or control antibody, with total cell lysates containing p85β protein expressed by transient transfection. the amount of p85β protein bound to gst or gst-ns1(ed) was detected by western blot using antibodies specific to the p85β subunit. as shown in fig. 5a , p85β specifically bound to ns1(ed) instead of gst, since gst alone failed to pull down the p85β protein. furthermore, mab 19h9 inhibited the interaction between p85β and ns1(ed) in a dose-dependent manner (fig. 5a) , implying that the binding of mab 19h9 and p85β to ns1(ed) is competitive. quantification by densitometry showed that there was a significant reduction in the interaction between p85β and gst-ns1(ed) when mab 19h9 was used at 0.07 nm and 0.13 nm (fig. 5b) . on the other hand, the presence of 0.13 nm of control antibody (mab 1a9) showed no effect on the interaction between p85β and gst-ns1(ed). this result shows that mab 19h9 binds to residue y89 of ns1 and blocks its interaction with p85β. as mabs targeting ns1 can be used for research and diagnostic assay development, multiple hybridomas have been generated to produce anti-ns1 antibodies. for those mabs with epitope mapped, all of them have been shown to bind to motifs in either ns1(rbd) or ns1(ed) (tan et al. 2010; he et al. 2013; rahim et al. 2013; wen et al. 2015; sun et al. 2016) . in this study, we have generated a novel mab 19h9 and found that it interacts with two residues, p85 and y89, of ns1 that are highly conserved in seasonal as well as newly emerged highly pathogenic avian iav strains. consistently, mab 19h9 did not bind to rgpr8 mutant viruses carrying substitution at either of these positions. to our knowledge, mab 19h9 is the first murine mab binding at the juxtaposition between the rbd and ed of ns1. it could serve as a useful research tool for studying the conformational plasticity and dynamic changes in ns1. interestingly, a fully human single chain antibody fragment (huscfv clone 10) was isolated from a human scfv phage display library and found to bind to residues 75-90 of ns1 (a/duck/thailand/144/2005(h5n1)) (yodsheewan et al. 2013) . the overlap between the epitopes of mab 19h9 and huscfv clone 10 suggests that the juxtaposition between the rbd and ed of ns1 is immunogenic. the residue y89 is part of the ed of ns1 and previous study has demonstrated that it is important for ns1's interaction with the p85β subunit of the pi3k complex (hale et al. 2010 ). indeed, this study has shown that the interaction between ns1(ed) and p85β was abrogated by the binding of mab 19h9 to y89 of ns1. on the other hand, p85 is found in the lr between the rbd and ed. while the exact role of the lr in viral replication has not been defined, five amino acids deletion (residues 80-84) is found to be implicated in the high virulence of the h5n1 strains like a/vietnam/1203/2004/(h5n1) (long et al. 2008 ). in addition, structural analysis showed that the ns1(ed) dimer in the full-length ns1 of a/vietnam/1203/2004/(h5n1) exhibited significant difference from its structure when solved as an isolated domain (bornholdt and prasad 2008) . structural analysis of the full length h6n6-ns1 and a mutant h6n6-ns1 80-84 further revealed that the lr influences the orientation of ns1(ed) with respect to ns1(rbd) and thus plays an important role in determining the quaternary structure of ns1 (carrillo et al. 2014) . the lr could be the basis for strain-specific functions associated with ns1 protein during iav infection, possibly by determining the protein's correct subcellular localization. using mab 19h9, ns1 displayed cytoplasmic as well as nucleolar localization. as such, it could be used to study ns1 temporal distribution, and its interactions with host nucleolar or cytoplasmic proteins, during the course of virus infection. the interaction of mab 19h9 with the lr of ns1 could potentially be useful in investigating the less well defined functions of this region. x-ray structure of ns1 from a highly pathogenic h5n1 influenza virus the influenza a virus protein ns1 displays structural polymorphism influenza a 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the ns1 protein four c-terminal residues modulate pathogenicity multiple antiinterferon actions of the influenza a virus ns1 protein binding of the influenza a virus ns1 protein to pkr mediates the inhibition of its activation by either pact or double-stranded rna monoclonal antibodies targeting the hr2 domain and the region immediately upstream of the hr2 of the s protein neutralize in vitro infection of severe acute respiratory syndrome coronavirus virulence of h5n1 avian influenza virus enhanced by a 15-nucleotide deletion in the viral nonstructural gene binding of the influenza virus ns1 protein to double-stranded rna inhibits the activation of the protein kinase that phosphorylates the elf-2 translation initiation factor nuclear and nucleolar targeting of influenza a virus ns1 protein: striking differences between different virus subtypes the primary function of rna binding by the influenza a virus ns1 protein in infected cells: inhibiting the 2'-5' oligo (a) synthetase/rnase l pathway influenza virus ns1 protein interacts with the cellular 30 kda subunit of cpsf and inhibits 3'end formation of cellular pre-mrnas the rna binding domain of influenza a virus ns1 protein affects secretion of tumor necrosis factor alpha, interleukin-6, and interferon in primary murine tracheal epithelial cells substitution at aspartic acid 1128 in the sars coronavirus spike glycoprotein mediates escape from a s2 domain-targeting neutralizing monoclonal antibody cellular antiviral responses against influenza a virus are countered at the posttranscriptional level by the viral ns1a protein via its binding to a cellular protein required for the 3' end processing of cellular pre-mrnas an antibody against a novel and conserved epitope in the hemagglutinin 1 subunit neutralizes numerous h5n1 influenza viruses generation and characterization of a new panel of broadly reactive anti-ns1 mabs for detection of influenza a virus identification of a highly conserved epitope on avian influenza virus non-structural protein 1 using a peptide microarray a new panel of ns1 antibodies for easy detection and titration of influenza a virus the novel mitochondria localization of influenza a virus ns1 visualized by flash labeling identification of a novel linear epitope on the ns1 protein of avian influenza virus biochemical and structural characterization of the interface mediating interaction between the influenza a virus non-structural protein-1 and a monoclonal antibody human monoclonal scfv specific to ns1 protein inhibits replication of influenza viruses across types and subtypes key: cord-355913-fhvt1ht1 authors: burrell, christopher j.; howard, colin r.; murphy, frederick a. title: virus replication date: 2016-11-11 journal: fenner and white's medical virology doi: 10.1016/b978-0-12-375156-0.00004-7 sha: doc_id: 355913 cord_uid: fhvt1ht1 understanding the molecular events accompanying virus replication is essential for the proper understanding and control of all virus diseases. the virus replication cycle generates new viral genomes and proteins in sufficient quantities to ensure propagation of the viral genome; this requires that the extracellular viral genome is protected from enzymatic degradation and can be introduced into further target cells for further rounds of replication. the initial recognition between virus and host is more complex than originally supposed and may involve more than one cellular receptor. a critical first intracellular step is the generation of viral mrna by one of a limited number of strategies first described by david baltimore. lacking ribosomes, viruses have no means of producing protein and are reliant on the host cell for protein synthesis. viral proteins are often modified by host cell glycosylation during or after virus assembly. temporal regulation of intracellular events is critical in all but the very simplest of viruses, and some form of suppression of the host innate immune response is common to nearly all human viruses. infected cells often produce non-infectious particles with incomplete genomes, and these defective interfering particles may play a role in pathogenesis. understanding these processes will open up a range of targets for the development of novel therapies. understanding the molecular events accompanying virus replication has been a major focus of experimental virology, and is essential for the proper understanding and control of all virus diseases. the biological "purpose" of any replication cycle is the generation of new viral genomes and proteins in sufficient quantities to ensure propagation of the viral genome (a clear example of the "selfish gene"); this requires that the extracellular viral genome is protected from enzymatic degradation and can be introduced into further target cells for further rounds of replication. much has become known about the initial stages of attachment, and more detailed study shows that the initial recognition between virus and host is more complex than originally supposed. temporal regulation of intracellular events is critical in all but the very simplest of viruses, with some form of suppression of the host innate immune response being common to nearly all human viruses. there are examples where the innate immune response is even used to enhance virus spread to cells otherwise unavailable to the virus. over the next few years, the boundaries between virus-directed events and cellular processes that control specialized cell functions are likely to be even more complex; nevertheless understanding these processes will open up a range of targets for the development of novel antiviral therapies and immunotherapy. this chapter presents an overview of the subject, indicating similarities and differences in the replication strategies adopted by viruses of each family that contains human pathogens. major features and replication requirements of human viruses are shown in table 4 .1. more detailed information about the replication of individual viruses can be found in the relevant chapters of part ii of this book. before the development of in vitro culture techniques, viruses of humans could only be propagated in experimental animals. thus much of our knowledge about virus growth and replication relied in the first instance on the study of bacterial viruses. in 1931 it was shown that vaccinia virus and herpes simplex virus could be grown on the chorioallantoic membrane of embryonated chicken eggs. this system became routine for the study of many mammalian viruses as virtually all virus families contain viruses that can be cultured in this way. although cell culture has long since replaced the use of eggs for this purpose, the technology is still in use for the preparation of influenza virus stocks and the manufacture of influenza vaccines. the development of in vitro cell culture systems was a watershed development in virology: not only did it become possible to dissect the intracellular events accompanying virus replication in a manner similar to that of the study of bacteriophages in bacterial cells, it also provided a means of quantifying the amount of infectious virus in samples and virus stocks. artificial medium was developed to maintain cell viability independent of the source species: these cells could be in the form of organ cultures, explant cultures, primary cell culture monolayers, or monolayer cell cultures immortalized into cell lines. organ cultures maintain the three-dimensional structure of the tissue of origin and can be useful for short-term experiments that depend upon preserving fully differentiated cells. for example, tracheal epithelial cells attached to the cartilage matrix of the trachea during culture played a critical role in the isolation of many human respiratory viruses. the preparation of primary cell cultures uses proteases such as trypsin or collagenase to separate individual cells of a tissue such as fetal kidney or lung, and the individual cells then attach to a cell culture substrate where a limited number of divisions will occur. the limited life span of these cells requires repeated preparation of new cultures from source tissue, clearly presenting problems with reproducibility. in contrast, continuous propagation of cells is possible with two types of long-term culture: 1. "semicontinuous," "diploid" cell strains, for example, human lung or foreskin fibroblasts , in which cells eventually senesce after 20 to 30 divisions (the "hayflick number") due to progressive shortening of telomeres, and 2. continuous immortalized cell lines, for example, hela cells, bhk-21 cells. these are derived either from tumors or from primary cells that have undergone a spontaneous transformation event during cell culture, and can undergo an almost infinite number of cell divisions, thus generating consistency although often accompanied by a loss of differentiated cell functions. for example, hela cells were originally isolated in cell culture from a patient who died of cervical cancer chapter 4 in 1951, but the cells remain an important system for culturing viruses to this day, and in fact proliferate so successfully that hela cells can become contaminants of cell cultures from other sources. recognition of the presence of a virus was dependent initially on observing cultures at regular intervals under the light microscope for signs of morphological change or cell death, compared to uninoculated cell cultures acting as controls. the specific appearance of the cytopathic effect (cpe) is often diagnostic for a certain family of viruses, for example herpesviruses can cause a distinct cytopathology often accompanied by the fusion of dying cells. however, reliance on cytopathology can give false results especially if the sample is contaminated with bacterial toxins or other adventitious agents not related to the pathological process under study. using such systems, the basic mechanisms of transcription, translation, and nucleic acid replication have been characterized for all the major families of vertebrate viruses and the strategies of gene expression and regulation have been clarified. every family of viruses employs unique replication strategies. one important unifying and simplifying concept, as originally proposed by david baltimore in 1971, was to assign all viruses to one of six classes based on their genome composition and the pathway used to produce mrnas for translation using host cell ribosomes ( fig. 4.1 ). this also reflects the parasitic property of all viruses in requiring a host cell for the synthesis of viral proteins. most studies of the replication of viruses of humans have used cultured mammalian cells, grown either in suspension or as a monolayer adherent to a plastic surface. classic studies of this kind defined the one-step growth curve, in which all cells in a culture are infected simultaneously by using a high multiplicity of infection, and the increase in infectious virus over time is measured by sequential sampling and titration of infectious virus ( fig. 4.2) . it is important to understand that, with synchronous infection of the whole cell population, the events observed in the total culture can be used to study events in a single cell. the time to achieve maximal yield of virus ranges from 6 hours (poliovirus) to more than 24 hours (adenovirus), compared with a generation time for e. coli in broth culture of 15 to 20 minutes. virus released into the medium can be titrated separately from virus that remains cell-associated. shortly after infection, the inoculated virus "disappears"-infectious particles cannot be detected, even if the cells are disrupted. this eclipse period continues until the first progeny virions are detected some hours later. as infection progresses, non-enveloped viruses mature within the cell and may be detected as infectious, intracellular virions for some hours before being released by cell lysis. many enveloped viruses, on the other hand, mature by budding from the plasma baltimore, d., 1971 . expression of animal virus genomes. bacteriol. rev. 35, 235-241. then, modified from flint, s.j., et al., 2016 membrane of the host cell and are released into the medium. virus release via cell lysis is abrupt, whereas release via budding may continue over a protracted period of time. the eclipse period generally ranges from 2 to 12 hours for viruses of different families. early studies, relying on quantitative electron microscopy and the assay of infectious virions, provided an overview of the replication cycle (attachment, penetration, maturation, and release), but with little detail as to the processes involved. investigation of the expression and replication of the viral genome became possible after the introduction of biochemical methods for the analysis of viral nucleic acids and proteins. furthermore, imaging methods are also available nowadays that enable the tracking of viral and cellular proteins as the viral replication cycle proceeds. in order to initiate infection, virions must first bind to cells. binding occurs between ligands on the surface of the virion (viral attachment proteins) and receptors on the plasma membrane of the cell (table 4 .2). this initial interaction between virus and host cell is sometimes complex and there is frequently a lack of correlation between attachment studies in cultured cells versus intact hosts. for example, several viral envelope glycoproteins of herpesviruses may serve as attachment proteins and several cellular receptors may be involved, in sequential order, first achieving a loose attachment via one receptor, then irreversibly binding via a second receptor and ligand. exploitation of more than one cellular ligand provides redundancy and also may assist herpesviruses in invading both epithelial and neural tissues to support latent/recrudescent infection cycles. while there is a degree of specificity about the recognition of particular cellular receptors by particular viruses, quite different viruses (e.g., orthomyxoviruses and paramyxo viruses) may utilize the same receptor and, conversely, viruses in the same family or genus may use different receptors. viruses have thus evolved by making opportunistic use of a wide variety of host cell surface proteins as receptors-in most cases viruses employ as receptors host cell proteins that are crucial for fundamental cellular functions, many of which are strongly conserved over evolutionary time. the receptor-ligand interaction between human cells and human immunodeficiency virus 1 (hiv-1) illustrates the complexity of the interaction between virus and host proteins. attachment initially involves cd4 molecules on the surface of target cells, notably macrophages and t helper lymphocytes, via the viral gp120 envelope glycoprotein. this binding induces a conformational change exposing a highaffinity chemokine receptor-binding site on gp120, which binds one of several chemokine receptors on the cell surface. this latter binding in turn exposes a fusogenic domain of the viral transmembrane protein gp41. direct contact between the fusogenic domain of gp41 and the cell membrane brings about the fusion of the viral envelope with the plasma membrane of the cell, permitting the viral nucleocapsid to enter the cell cytoplasm (see fig. 23 .6). the cellular receptor for most orthomyxoviruses is the terminal sialic acid on an oligosaccharide side-chain of a glycoprotein (or glycolipid) exposed at the cell surface: the viral ligand is in a cleft at the distal tip of each monomer of the trimeric viral hemagglutinin glycoprotein (see chapter 3: virion structure and composition). viruses can adapt to new hosts through modification of the relevant attachment ligand. for example, influenza a viruses originate in aquatic birds and recognize sialic acid residues on the surface of cells along the respiratory tract. however, influenza a viruses of birds have a greater affinity for sialic acid linked to the penultimate galactose moiety through α2-3 bonding, whereas those of humans have a greater affinity for sialic figure 4.2 one-step growth curve of an enveloped and a non-enveloped virus. attachment and penetration are followed by an eclipse period of 2 to 12 hours during which cell-associated infectivity cannot be detected. this is followed by a period of several hours when viral maturation occurs. virions of non-enveloped viruses (e.g., adenoviruses-top) are often released late, when the cell lyses. release of enveloped virions (e.g., the togavirus western equine encephalitis virus) occurs concurrently with maturation by budding from the plasma membrane. adapted from flint, s.j., et al., 2009. principles of virology, third ed. asm press, washington, dc. acid linked via an α2-6 bond; this reflects differences in the relative abundance of the two linkages in the different host species. there is considerable subtlety in these interactions, and as with many human and animal viruses a single amino acid change within a receptor site may manifest as a significant change in host range and epidemiology. proinflammatory reactions in the host may also affect the expression of receptors and thereby indirectly modify interactions between virus and potential host cells. for example, human adenovirus 5 recognizes both the coxsackievirus and adenovirus receptor (car) and αvβ3/5 integrin in order to infect cells, yet neither is normally exposed on the apical surfaces of cells until macrophages respond to infection and secrete il-8. this in turn moves the car and αvβ3/5 integrin receptors away from the tight junctions of polarized cells toward the apical cell surface and thus become exposed and available for virus attachment. the majority of mammalian cells are continuously engaged in receptor-mediated endocytosis for the uptake of macromolecules via specific receptors. many enveloped and non-enveloped viruses use this essential cell function to initiate penetration and uncoating ( fig. 4 .3). virion attachment to those receptors clustered at clathrin-coated pits is followed by endocytosis into clathrin-coated vesicles. vesicles enter the cytoplasm and, after removal of the clathrin coat, fuse with endosomes (acidic prelysosomal vacuoles). acidification within the vesicle triggers changes in virion proteins and surface structures. for example, the acidic ph within the endosome, induces the hemagglutinin molecules of influenza viruses to undergo a conformational change. this enables fusion to occur between the viral envelope and the endosomal membrane and results in the release of the viral nucleocapsid into the cytoplasm. many other non-enveloped and enveloped viruses undergo comparable changes. fusion at neutral ph is an alternative mechanism. the f (fusion) glycoprotein of paramyxoviruses causes the viral envelope to fuse directly with the plasma membrane of the cell, even at ph 7. this allows the nucleocapsid to be released directly into the cytoplasm. a number of other important enveloped viruses for example filoviruses, have the ability to fuse with the host cell plasma membrane, thereby allowing entry of viral nucleic acid into the cytosol. a third entry mechanism ("direct entry") is used by some non-enveloped viruses (e.g., poliovirus, adenovirus). this involves binding of virus to receptors at the endosomal membrane, which induces conformational changes in the viral capsid; these changes then expose regions that react with the endosomal membrane to induce channels for transport of the genome across the plasma membrane ( fig. 4.4) . the receptors induce conformational changes in the virion (the 160s infectious virion becomes a 135s particle), with the loss of vp4 and the externalization of the n-terminus of vp1. the 135s particles are then internalized by a clathrin-and caveolin-independent, but actin-and tyrosine kinase-dependent, mechanism. the release of the viral genome takes place only after internalization from an endocytotic compartment localized within 100 to 200 nm of the plasma membrane. upon release of the rna genome, the empty capsid (80s) is transported away along microtubules. reproduced from brandenburg, b., et al., 2007, imaging poliovirus in order for viral genes to become available for transcription it is necessary that virions are at least partially uncoated. in the case of enveloped rna viruses entering by the process of fusion, the nucleocapsid is discharged directly into the cytoplasm and transcription commences from viral nucleic acid while still associated with the nucleocapsid protein(s). with the non-enveloped icosahedral reoviruses, only certain capsid proteins are removed and the viral genome expresses all its functions without being released from the virion core. for most other viruses, however, uncoating proceeds to completion, otherwise genome duplication cannot proceed. for some viruses, uncoating takes place in the nucleus where later stages of replication occur. replication of viral dna different mechanisms of dna replication are employed by each family of dna viruses ( fig. 4 .5). these involve either synthesis of daughter strands via a replication fork (papillomaviruses, polyomaviruses, herpesviruses), or via strand displacement (adenoviruses, parvoviruses, poxviruses). since cellular dna polymerases cannot initiate synthesis of a new dna strand but can only extend synthesis beginning from a short (e.g., rna) primer, one end of newly synthesized viral dna molecules might be expected to retain a short single-stranded region. various dna viruses have evolved different strategies for circumventing this problem. viruses of some families have a circular dna genome, others have a linear genome with complementary termini that serve as dna primers, yet others have a protein primer covalently attached to the 5ʹ-terminus of each dna strand. several virus-coded enzyme activities are generally required for replication of viral dna: a helicase (with atpase activity) to unwind the double helix; a helix-destabilizing protein to keep the two separated strands apart until each has been copied; a dna polymerase to copy each strand from the origin of replication in a 5ʹ-to 3ʹ-direction; an rnaase to degrade the rna primer after it has served its purpose; and a dna ligase to join the okazaki fragments together. often a single large enzyme performs two or more of these activities. the genomes of papillomaviruses and polyomaviruses structurally and functionally resemble cellular dna in binding to cellular histones. the viral genome utilizes host dna polymerase α-primase to synthesize the rna primer for genome replication. among the polyomaviruses an early viral antigen, large t, binds to the regulatory sequence-and in the case of papillomaviruses e1 and e2-thereby initiating dna replication. replication of these circular double-stranded dna molecules commences from a unique palindromic sequence and proceeds simultaneously with replication forks in both directions. both continuous and discontinuous dna synthesis occur (of leading and lagging strands respectively) at the two growing forks as for the replication of mammalian dna. the discontinuous synthesis of the lagging strand involves repeated synthesis of short oligoribonucleotide primers, which in turn initiate short nascent strands of dna (okazaki fragments), each is then covalently joined by a dna ligase to form one of the growing strands. the replication of adenoviruses is distinct from that of other dna viruses. the adenovirus dna genome is linear, the 5ʹ-end of each strand being identical to the other (terminally repeated inverted sequences) and covalently linked to a protein, the precursor of which serves as the figure 4.5 two mechanisms of synthesis of dna virus genomes. a replication fork is used by papillomaviruses, polyomaviruses, and herpesviruses. this involves initiation from an rna primer; synthesis along the leading strand is continuous, but discontinuous along the lagging strand with formation of okazaki fragments which are subsequently ligated. with circular dna genomes, synthesis proceeds bidirectionally from the site of origin. alternatively, the dna genomes of adenoviruses, parvoviruses, and poxviruses replicate by strand displacement, using as primers either protein (adenoviruses) or dna hairpins (parvoviruses and poxviruses). reproduced from flint, s.j., et al., 2009. principles of virology, third ed. asm press, washington, dc, with permission. primer for viral dna synthesis. dna replication proceeds from both ends, continuously but asynchronously, in a 5ʹ-to 3ʹ-direction, using a virus-coded dna polymerase. it does not require the synthesis of okazaki fragments. herpesviruses code for many or all of the proteins required for dna replication, including a dna polymerase, a helicase, a primase, a single-stranded dna binding protein, and a protein recognizing the origin of replication. poxviruses, which replicate entirely within the cytoplasm, are selfsufficient in dna replication machinery. hepadnaviruses, similar to retroviruses, utilize positive-sense single-stranded rna transcripts as intermediates for the production of progeny dna by a process of reverse transcription. singlestranded dna parvoviruses use 3ʹ-palindromic sequences to form a double-stranded hairpin structure to provide a primer for cellular dna polymerase binding. transcription of rna from an rna template is a phenomenon unique to viruses (fig. 4.6) , and requires an rna-dependent rna polymerase, a virus-coded enzyme not found in uninfected cells. the replication of viral rna requires first the synthesis of complementary rna: this in turn serves as a template for the synthesis of further copies of viral rna. for viruses where the viral rna is of negative-sense (orthomyxoviruses, paramyxoviruses, rhabdoviruses, filoviruses, bornavirus, arenaviruses, and bunyaviruses), the complementary rna is of positive-sense and the rna poly merase involved performs the same function as the virion-associated transcriptase used for the primary transcription of mrnas. most transcripts from such negative-sense viral rnas are subgenomic mrna molecules, but some full-length positive-sense strands must also be made, in order to serve as templates for full-length progeny viral rna synthesis (replication). for some viruses there is good evidence that the rna polymerases used for transcription and replication are distinct, in others the same enzyme performs both functions. in the case of the positive-sense rna viruses (picornaviruses, caliciviruses, togaviruses, flaviviruses, coronaviruses, and arteriviruses), the complementary rna is negative-sense, and its sole purpose is to provide a template for synthesis of more positive-sense rna. several viral rna molecules can be transcribed simultaneously from a single complementary rna template, each rna transcript being the product of a separately bound polymerase molecule. the resulting structure, known as the replicative intermediate, is therefore partially double-stranded with single-stranded tails. some rna-dependent rna polymerases can initiate rna synthesis de novo, while others require a primer with a free 3ʹ-oh group to which further nucleotides can be added. initiation of the replication of picornavirus and calicivirus rna, similar to that of adenovirus dna, requires a bound protein as primer, rather than an oligonucleotide. this small protein is covalently attached to the 5ʹ-terminus of nascent positive and negative rna strands in addition to virion rna, but not to molecules used as mrna. little is known about what determines whether a given picornavirus positive-sense rna molecule will be directed (1) to a replication complex (a structure bound to smooth endoplasmic reticulum), where it serves as template for transcription by rna-dependent rna polymerase into negative-sense rna, or (2) to a ribosome, where it serves as mrna for translation into protein, or (3) to a procapsid, with which it associates to form a virion. other rna polymerases require an oligonucleotide 5ʹ cap structure, which may be synthesized by viral enzymes or derived from cellular mrnas by a process of "capsnatching" (e.g., chapter 25: orthomyxoviruses). retroviruses have a genome consisting of positive-sense single-stranded rna. unlike other rna viruses, retroviruses replicate via a dna intermediate. the virion-associated reverse transcriptase, using a transfer rna (trna) molecule as a primer, makes a single-stranded dna copy, while the same enzyme functions concurrently as a ribonuclease and removes the parental rna molecule from most of the dna:rna hybrid, except for several short rna stretches known as poly-purine tracts. the poly-purine tracts then act as primers for copying the negative-sense single-stranded dna strand to form a linear double-stranded dna that contains an additional sequence known as the long terminal repeat (ltr) at each end. this double-stranded dna then is processed by the viral integrase and integrates into cellular chromosomal dna. from this point on, transcription of viral rna occurs from the integrated (proviral) dna (see chapter 23: retroviruses). distinguishing the various strategies used by viruses for the production of viral proteins is fundamental to an understanding of virus replication (fig. 4.1) . a summary of the properties of viral proteins is given in table 4 .3. for many viruses, the production of viral proteins immediately after entry of the viral genome is a critical step. these early proteins act to subvert the molecular machinery of the host, to allow the later production of new virus particles and to inhibit the activation of the host innate immunity that would otherwise prevent virus replication and spread to adjacent cells and tissues. for most families of dna viruses, transcription and dna replication take place in the cell nucleus, using cellular rna polymerase ii and other cellular enzymes. for viruses such as poxviruses and herpesviruses it is possible to identify a significant number of genes by gene deletion (more than 40%) that are not essential for the replication of the virus in cultured cells. of course, in the strict economy of viral genomes it is likely that most or all of such genes are important for virus survival in nature. the situation is quite different for rna viruses as these are unique having genetic information coded as rna. thus an effort needs to be made to understand the distinct replication and expression strategies, particularly as these have a bearing on understanding virus pathogenesis. rna viruses with different types of genomes (single-stranded or double-stranded, positive-or negative-sense, monopartite or segmented) have necessarily evolved different routes to the production of mrna. since eukaryotic cells do not contain an rna-dependent rna polymerase, all rna viruses need to code for a unique rna-dependent rna polymerase. in the case of positive-sense single-stranded rna viruses, the incoming rna genome can bind directly to ribosomes and be translated in full or in part without the need for any prior transcription; all other forms of incoming viral rna must first be transcribed to produce mrna, in order to begin the process of expression of the infecting viral genome. thus, both negative-sense single-stranded rna viruses and double-stranded rna viruses need to carry an rnadependent rna polymerase in the virion, originating from the preceding round of infection. in the case of dna viruses dependent upon the nucleus for replication, cellular dna-dependent rna polymerase ii performs this function, while double-stranded dna viruses that replicate in the cytoplasm carry a virus-encoded dna-dependent rna polymerase. many, but not all, viruses express different genes at different stages of the replication cycle. early viral genes are first transcribed into rna, which may then be processed in a number of ways, including splicing. the early geneproducts translated from this mrna are of three main types: proteins that shut down cellular nucleic acid and protein synthesis, proteins that regulate the expression of the viral genome, and enzymes required for the replication of the viral nucleic acid. following viral nucleic acid replication, late viral genes are transcribed. the late proteins are principally viral structural proteins used for assembly of new virions; some of these are subject to post-translational modifications before use, and are often made in considerable excess. structural proteins for the coating of nascent viral genomes are required in multiple copies for every new nucleic acid molecule destined for encapsidation. for this reason, many viral strategies have evolved so that new copies of the viral genome can act as templates for the further specific transcription and translation of structural proteins required for new virus particles. the existence of overlapping reading frames, multiple splicing patterns of rna transcripts, post-translational cleavage of polyproteins, etc., mean that it is too simplistic to assume one particular gene codes for a particular protein. it is more appropriate to refer to a transcription unit, defined as a region of the genome beginning with the transcription initiation site and extending to the transcription termination site (including all introns and exons in between), the expression of which falls under the control of a particular promoter. some viral proteins serve as regulatory proteins, modulating the transcription or translation of cellular genes or down-regulating early viral genes. the large dna viruses also encode numerous additional proteins, called virokines, which do not regulate the viral replication cycle per se, but influence the host response to infection. included among these are viral protein homologues of cellular cytokines. in 1978 walter fiers and his colleagues presented the first complete description of the genome of an animal virus, namely that of the polyomavirus sv40 (see fig. 20 .2). analysis of the circular double-stranded dna molecule and its transcription revealed some remarkable insights, many of which are now applicable to other double-stranded dna viruses. first, early and late genes are transcribed in opposite directions, from different strands of the dna. second, certain genes overlap, the protein products generated having common amino acid sequences. third, some regions of the viral dna may be read in different reading frames (orfs), so that quite distinct amino acid sequences are translated from the same nucleotide sequence. fourth, certain long stretches of the viral dna consist of transcribed introns that are not translated into protein as these are spliced out of the primary rna transcript (see fig. 20 .2). adenoviruses exemplify some of the mechanisms that regulate the expression of viral genomes at the level of transcription. there are several adenovirus transcription units; at different stages of the viral replication cycle, "preearly," "early," "intermediate," and "late" transcription units are transcribed in a precise temporal sequence. a product of the early region e1a induces transcription from the other early regions including e1b, but following viral dna replication there is a 50-fold increase in the rate of transcription from the major late promoter relative to early promoters such as e1b, and a decrease in e1a mrna levels. a second control operates at the point of termination of transcription. early in the replication cycle transcripts terminate at a particular point in the genome, while later in infection these are read through the point of termination to produce a range of longer transcripts with different polyadenylation sites and proteins of different functions. this is but one of the many examples of the economy of viral genomes in coding for complex functions using minimal sequences of nucleic acid. for rna viruses, the regulation of transcription is, on the whole, not as complex as is the case for dna viruses. in particular, the temporal separation into early genes transcribed before the replication of viral nucleic acid, and late genes thereafter, is not nearly so well defined. in the simplest examples (e.g., picornaviruses), a full-length polycistronic mrna is translated and the resulting polyprotein is subsequently cleaved to yield equimolar amounts of all protein products. togaviruses synthesize excess amounts of structural proteins from a separate subgenomic rna. yet other mechanisms of regulation have evolved among viruses with non-segmented negative-sense rna genomes. once the nucleocapsid is released into the cytoplasm of an infected cell, the rna polymerase initiates transcription from the 3ʹ-end of the genome. with paramyxoviruses, discrete genes along the viral rna are each separated by a consensus sequence that includes termination and start signals as well as short intergenic sequences of u residues enabling the transcriptase to generate a long poly(a) tail for each mrna by a process of reiterative copying (also known as stuttering). each discrete mrna is released from the template while the enzyme continues to transcribe the next gene in sequence. as the polymerase moves from 3ʹ to 5ʹ along the template, decreasing amounts of each mrna are sometimes made due to decreasing efficiency of the transcription process-thus, gene order becomes an efficient way of modulating the relative amount of each protein synthesized. paramyxovirus transcription also involves a process known as rna editing. the p gene codes for two proteins, p and v, which share a common n-terminal amino acid sequence but differ completely in the c-terminal sequences as a result of a shift in the reading frame brought about by the insertion of two uncoded g residues into the rna transcript by transcriptase stuttering. eukaryotic cells are not equipped to translate polycistronic mrna into several individual species of protein, as mammalian cells cannot normally reinitiate translation partway along an rna molecule. dna viruses overcome this limitation by using cellular mechanisms for cleavage (and sometimes splicing) of viral polycistronic rna transcripts to yield monocistronic mrna molecules. rna viruses, most of which replicate in the cytoplasm, do not have access to the rna processing and splicing enzymes of the host cell nucleus, but have developed a remarkable diversity of solutions to the difficulty of punctuating a large genome to produce multiple individual gene products. some (e.g., orthomyxoviruses) have developed a segmented rna genome in which each gene is, in general, expressed and duplicated as a separate molecule. others (e.g., paramyxoviruses) have evolved a polycistronic genome but produce monocistronic rna transcripts by termination and reinitiation of transcription. yet other rna viruses (e.g., coronaviruses) make use of a nested set of overlapping rna transcripts, each of which is translated into a single gene product. finally, some (e.g., picornaviruses) have a polycistronic genomic rna, which is translated into a polyprotein that is later cleaved proteolytically to yield the final products. with certain viruses, polycistronic mrna can be translated directly to produce several gene-products as a result of initiation, or reinitiation, of translation at internal aug start codons. internal initiation of translation is facilitated by an upstream rna motif known as an internal ribosomal entry site (ires), first discovered in 1988 in poliovirus and encephalomyocarditis virus rnas (another picornavirus). where initiation of translation at an internal aug takes place, a frameshift can also occur. another mechanism, known as ribosomal frameshifting, occurs fortuitously when a ribosome happens to slip one nucleotide back and forth along an rna template. this phenomenon is exploited by retroviruses, where frameshift read through leads to about 5% of gag protein molecules being extended as a gag-pol polyprotein. thus, taken together with the phenomenon of rna splicing and rna editing described above, it can be seen that there are several mechanisms of exploiting overlapping reading frames to maximize the limited coding potential of viruses with comparatively small genomes. there is considerable interest in the untranslated regions of viral genomes, particularly the numerous conserved (consensus) sequences or motifs that represent responsive elements. many of the latter have a critical role to play in the regulation of transcription. for example, each transcription unit of a viral genome has near its 3ʹ-end an mrna transcription initiation site (start site), designated as nucleotide +1. within the hundred or so nucleotides upstream of the start site is the promoter, which upregulates the transcription of a particular gene or series of genes. upstream or downstream from the start site there may be a long sequence with several, in some cases repeated, elements known as enhancers that amplify transcription even further. these regulatory regions are activated by the binding of either viral or cellular dna-reactive proteins. several such proteins may bind to adjacent responsive elements to form an interactive structure, or otherwise interact to facilitate attachment of the viral rna polymerase. viral regulatory genes encoding such regulatory proteins may act in trans as well as in cis, that is, these proteins may trans-activate genes residing on a physically separate molecule of nucleic acid. a description of the role of the regulatory genes of human immunodeficiency virus 1 (hiv-1) serves to illustrate the sophistication of such regulatory mechanisms. a dna copy of the viral genome is integrated into a chromosome of a resting t cell, and remains latent until a t cell mitogen or a cytokine induces synthesis of the cellular nf-κb family of dna-binding proteins. nf-κb then binds to the enhancer present in the integrated provirus, thereby triggering transcription of the hiv-1 regulatory genes. one of these, tat, found in all lentiviruses, codes for a protein specific for a responsive element, tar, within the provirus, greatly augmenting (trans-activating) the transcription of all viral genes (including tat itself). a positive feedback loop is thereby established that stimulates the production of hiv rna transcripts. moreover, by interacting with tar present in all viral mrnas as well as in the proviral dna, tat also enhances translation. the hiv-1 regulatory protein rev plays a different role to modulate gene expression by regulating the nuclear export of viral mrnas. although the control of lentivirus transcription may be unusually complex compared to many rna viruses, these viruses contain only 9 genes, compared with up to 200 in the case of some dna viruses. primary rna transcripts from eukaryotic dna are subject to a series of post-transcriptional alterations in the nucleus prior to export to the cytoplasm as mrna. first, a cap, consisting of 7-methylguanosine (m 7 gppp), is added to the 5ʹ-terminus of the primary transcript; this cap structure facilitates the formation of a stable complex with the host 40s ribosomal subunit, necessary for the initiation of translation. second, a sequence of 50 to 200 adenylate residues is added to the 3ʹ-terminus. this poly(a) tail acts as a recognition signal for processing and the transport of mrna from the nucleus to the cytoplasm, and assists in the stabilization of mrna against cytoplasmic degradation by ubiquitin. third, a methyl group is added at the sixth position to about 1% of the adenylate residues throughout the rna (methylation). fourth, introns are removed from the primary transcript and the exons are linked together in a process known as splicing, an important mechanism for regulating gene expression in nuclear dna viruses. a given rna transcript can have two or more splicing sites and, moreover, be spliced in several alternative ways to produce a variety of mrna species coding for distinct proteins; both the preferred poly(a) site and the splicing pattern may change in a regulated fashion as infection proceeds. for example, the hiv-1 protein rev assists the nuclear export of unspliced or singly spliced (intron-containing) viral mrna; thus, early in the replication cycle, the mrna present in the cytoplasm is largely doubly spliced, while later in the cycle after rev accumulates, a temporal switch in mrna species occurs. special mention should be made of an extraordinary phenomenon known as cap-snatching. the transcriptase of influenza virus, which also carries endonuclease activity, steals the 5ʹ-methylated caps from newly synthesized cellular rna transcripts in the nucleus and uses these host cell rna sequences as primers for initiating transcription from the viral genome. the rate of degradation of mrna provides another possible level of regulation. not only do different mrna species have different half-lives but the half-life of a given mrna species may change as the replication cycle progresses. capped, polyadenylated, and processed monocistronic viral mrnas bind to ribosomes and are translated into protein in the same fashion as cellular mrnas. the sequence of events has been closely studied in reovirusinfected cells. each monocistronic mrna molecule binds via its capped 5ʹ-terminus to the 40s ribosomal subunit and this then moves along the mrna molecule until reaching the initiation codon. the 60s ribosomal subunit also binds, together with methionyl-transfer rna and various initiation factors, after which translation proceeds. most viral proteins undergo various sorts of post-translational modification such as phosphorylation (for nucleic acid binding), fatty acid acylation (for membrane insertion), glycosylation, myristylation, or proteolytic cleavage. newly synthesized viral proteins must also be transported to the various sites in the cell where they are needed, for example, into the nucleus in the case of viruses where the nucleus is the major site of replication. the sorting signals that direct intracellular trafficking are only now beginning to be understood, as are the polypeptide chain-binding proteins (molecular chaperones) that regulate folding, translocation, and assembly of oligomers of viral as well as cellular proteins. the polycistronic viral rna is translated directly into a single polyprotein in the case of the positive-sense picornaviruses and flaviviruses. this large molecule carries protease activity that cleaves the polyprotein at defined recognition sites into smaller proteins. the first cleavage steps are carried out while the polyprotein is still associated with the ribosome. some of the larger intermediates exist only fleetingly; others are functional for a short period but are subsequently cleaved by additional virus-coded proteases to smaller proteins with alternative functions. post-translational cleavage occurs in several other rna virus families, for example, togaviruses and caliciviruses, in which polyproteins corresponding to large parts of the genome are cleaved. some viruses encode several different proteases. most are either trypsin-like (serine or cysteine proteases), pepsin-like (aspartyl proteases), or papain-like (thiol proteases). cellular proteases, present in organelles such as the golgi complex or transport vesicles, are also vital to the maturation and assembly of many viruses. for example, cleavage of the hemagglutinin glycoprotein of orthomyxoviruses or the fusion glycoprotein of paramyxoviruses is essential for virion infectivity. viruses frequently exploit those cellular pathways normally used for the synthesis of host cell secretory glycoproteins. the amino terminus of viral envelope proteins contains a sequence of 15 to 30 hydrophobic amino acids, known as a signal sequence, which facilitates binding of the growing polypeptide chain to a receptor site on the cytoplasmic side of the rough endoplasmic reticulum and its passage through the lipid bilayer to the lumenal side. oligosaccharides are then added in n-linkage to certain asparagine residues of the nascent polypeptide by en bloc transfer of a mannoserich core of preformed oligosaccharides, and glucose residues are removed by glycosidases (called trimming). the viral glycoprotein is then transported from the rough endoplasmic reticulum to the golgi complex. here the core carbohydrate is further modified by the removal of several mannose residues and the addition of further n-acetylglucosamine, galactose, and the terminal sugars, sialic acid, or fucose. the completed side-chains are a mixture of simple oligosaccharides (also called high mannose oligosaccharides) and complex oligosaccharides which are usually n-linked (to asparagine) or less commonly o-linked (to serine or threonine). a coated vesicle then transports the completed glycoprotein to the cellular membrane from which the particular virus buds. the precise composition of the oligosaccharides is determined, not only by the amino acid sequence and tertiary structure of the proteins concerned, but more importantly by the particular cellular glycosyl transferases active in the type of cell in which the virus happens to be growing. all non-enveloped viruses of vertebrates have an icosahedral structure. the structural proteins of simple icosahedral viruses associate spontaneously to form structural units (called capsomers when considered morphologically), which then self-assemble to form capsids into which viral nucleic acid is inserted, often accompanied by conformational changes to the nascent capsid structure. completion of the virion may also involve proteolytic cleavage of one or more species of capsid protein. the mechanism of packaging viral nucleic acid into a pre-assembled empty procapsid has been well elucidated for adenoviruses. a particular protein binds to a nucleotide sequence at one end of the viral dna known as the packaging sequence; this enables the dna to enter the procapsid bound to basic core proteins, after which some of the capsid proteins are cleaved to produce the mature virion. most non-enveloped viruses accumulate within the cytoplasm or the nucleus and are released only when the cell eventually lyses (fig. 4.7) . [rer] or golgi), then pass through the cytoplasm in smooth-walled vesicles and are released by exocytosis. reproduced from maclachlan, n.j., dubovi, e.j., 2011. veterinary virology, fourth ed. academic press, london (fig. 2.12) , with permission. all mammalian viruses with helical nucleocapsids, as well as some with icosahedral nucleocapsids (e.g., herpesviruses, togaviruses, and retroviruses) mature by acquiring an envelope by budding through cellular membranes. enveloped viruses may bud from the plasma membrane, from internal cytoplasmic membranes, or from the nuclear membrane; viruses that acquire an envelope within the cell are then transported within vesicles to the cell surface. budding usually occurs through patches of membrane that contain viral glycoprotein(s) inserted into the lipid bilayer of the membrane. this occurs by lateral displacement of cellular proteins from that patch of membrane ( fig. 4.8) . the cleaved single molecules of viral glycoprotein associate into oligomers to form typical rod-shaped or clubshaped peplomers with a hydrophilic domain projecting from the external surface of the membrane, a hydrophobic trans-membrane anchor domain, and a short hydrophilic cytoplasmic domain projecting slightly into the cytoplasm. in the case of icosahedral viruses, for example togaviruses, each protein molecule of the nucleocapsid binds directly to the cytoplasmic domain of the membrane glycoprotein oligomer, thus molding the envelope around the nucleocapsid. in the more usual case of viruses with helical nucleocapsids, a matrix protein attaches to the cytoplasmic domain of the glycoprotein peplomer, and the nucleocapsid protein recognizes the matrix protein to initiate budding. release of each enveloped virion does not breach the integrity of the plasma membrane, hence many thousands of virus particles can be shed over a period of several hours or days without significant cell damage. many but not all viruses that bud from the plasma membrane are only slowly cytopathic: and non-cytopathic may be associated with persistent infections. epithelial cells display polarity, that is they have an apical surface facing the outside world and a basolateral surface facing the interior of the body, the two separated by lateral cell-cell tight junctions. these surfaces are chemically and physiologically distinct. viruses that are shed to the exterior (e.g., influenza viruses) tend to bud from the apical plasma membrane, whereas others (e.g., lentiviruses, such as human immunodeficiency virus) bud through the basolateral membrane (fig. 4.9) . in type i budding, as with the alphaviruses, both the envelope glycoproteins and the internal capsid are essential. if altered or chimeric envelope proteins reach the plasma membrane, these do not support budding, indicating that the quaternary structures of the envelope heterodimers and capsid are involved in driving the process. type ii budding, for example gag-dependent budding of many retroviruses, requires only the internal capsid or matrix proteins. type iii budding can be driven solely by the envelope proteins. finally type iv budding is driven by viral matrix proteins but requires additional components; for example, internal matrix proteins alone can drive the budding of rhabdoviruses or orthomyxoviruses. but the process is inefficient or results in deformed particles unless envelope glycoproteins or the internal ribonucleoprotein are also present. reproduced from flint, s.j., et al., 2009. principles of virology, third ed. asm press, washington, dc (fig. 13.15) , with permission. figure 4.9 polarity of virus release from infected cells. viruses that bud from apical surfaces can be shed in respiratory or genital secretions or intestinal contents. viruses budding from basal surfaces are available for systemic spread via viremia or lymphatics. some viruses, for example, flaviviruses, bunyaviruses, and coronaviruses, take a more circuitous route in exiting the cell. viruses that do not bud are usually released by cell lysis. reproduced from maclachlan, n.j., dubovi, e.j., 2011. veterinary virology, fourth ed. academic press, london (figure 2.11), with permission. flaviviruses, coronaviruses, arteriviruses, and bunyaviruses mature by budding through either membranes of the golgi complex or the rough endoplasmic reticulum; vesicles containing the virus then migrate to fuse at the plasma membrane thereby releasing the virions by exocytosis (fig. 4.8) . uniquely, the envelope of the herpesviruses is acquired by budding through the inner lamella of the nuclear membrane; the enveloped virions then pass directly from the space between the two lamellae of the nuclear membrane to the exterior of the cell via the cisternae of the endoplasmic reticulum. satellite viruses are subviral particles that contain a dna or rna genome coding for a capsid protein, but that are absolutely dependent upon the presence of another virus for replication. the vast majority are found among plants but a few have an impact on medical virology and public health. replication of the dependoviruses (family parvoviridae, genus dependovirus-adeno-associated viruses, now used as vectors for delivering heterologous genes of interest, e.g., vectored vaccines) for example, is dependent upon co-infection with an adenovirus-the adeno-associated virus produces coat protein but not the enzymes necessary for genome replication. satellite viruses share little or no nucleotide sequence homology with the helper virus, yet can sometimes interfere with the replication of the helper virus. viroids are small, rod-like single-stranded rna molecules with a high degree of secondary structure, approximately 250 to 450 bases in size, all sharing a common structural feature of a conserved central genomic region essential for replication. the rna forms a hammerhead structure with the enzymatic properties of a ribozyme, an autocatalytic, self-cleaving molecule. this ribozyme function is used to cleave multimeric rna structures produced during the course of replication (chapter 22: hepadnaviruses and hepatitis delta). most are plant pathogens, remarkable in not coding for any protein. first described by theodore diener, it has been suggested that viroids may represent crucial intermediate steps in the evolution of rna-based life forms from inorganic precursors. hepatitis delta virus (hdv) is a unique example of a defective virus that requires co-infection with hepatitis b virus to provide its outer hbsag-containing envelope. the hdv genome is a branched or rod-like, single-stranded rna molecule similar to plant viroids in conformation, but unlike other viroids it codes for a single protein, the delta antigen. this nuclear phosphoprotein exists in two forms generated by rna editing: the shorter form (195 amino acids) is required for hdv genome replication whereas the larger form (214 amino acids) is necessary for assembly and release from infected liver cells. the hdv genome is thought to be replicated by the host cell rna polymerase ii enzyme via a rolling circle mechanism to produce a consecutive series of concatemers of first, antisense rna and then, rna of genome sense. these are immediately cleaved by the ribozyme domain in the rna genome to generate new viral genomes: this is the only known example of a mammalian virus utilizing a ribozyme for genome replication (see fig. 22 .9). a sample of any given virus inevitably contains a population of closely related genome sequences ("quasi-species"), replicating simultaneously at different and varying rates according to selection pressures. the molecular mechanisms involved in generating diversity include nucleotide substitution, insertion, or deletion; sequence duplication or deletion; genetic recombination; and genetic reassortment for viruses with segmented genomes. the error rate in nucleotide copying is approximately 1 in 10 5 nucleotides for dna viruses, and 1 in 10 4 nucleotides for rna viruses. this quasi-species population is then constantly varying its composition according to the most recent selection applied, providing the raw material for antigenic drift and for adaptation of the virus population to new hosts. the selection properties that are desirable in vivo include the ability to replicate to high levels, the ability to be transmitted from host to host, evasion of the immune response, and resistance to host antiviral mechanisms and/or antiviral therapy. not all of these properties are as necessary for successful replication in cell culture as they are in vivo, and virus stocks prepared in vitro are likely to include a different set of variants from those produced in vivo, say in experimental animal tissues. serial passaging of wild-type virus through cell culture or a foreign host species has been a long used and successful way to generate virus strains with lowered virulence for the original host, to be used as "modified live-virus" vaccines. many different viral genes may be involved in such adaptations; for example, mutations in viral polymerases may affect replicative ability or resistance to antiviral drugs, changes in antigenic epitopes can lead to immune escape, changes in receptor ligands that affect receptor preference can alter many aspects of pathogenesis, and so on. examples are discussed in chapter 15: emerging virus diseases. advances in our knowledge of virus replication, pathogenesis, diagnosis, vaccine production, and many other areas would not have been possible without the tools to quantitate how much virus there is in a given sample or preparation. with the greater use of antiviral drugs, most notably in blood-borne diseases such as hiv, hepatitis b virus, and hepatitis c virus, the stage of disease and effectiveness of treatment are now routinely assessed by quantifying the level of virus (viral load) in the blood. there are two approaches to measuring the titer of virus, either biological or physical. physical assays measure actual virus particles, and include electron microscopy, hemagglutination, and serological assays for the amount of viral antigens; biological assays measure some viral function, for example, infectivity, reverse transcriptase activity. tests performed on the same sample with different techniques will in some cases give significantly different results, and thus it is important to understand the reason for these differences. the difference between the amount of virus detected using a physical assay such as particle counting by electron microscopy and a biological assay, for example a plaque assay (fig. 4.10) , is often referred to as the particle to plaque-forming unit (pfu) ratio. in virtually all instances, the number of particles exceeds the number determined in a biological assay, often with ratios greater than 1000:1. this is due to a number of reasons; first, not all virions may be capable of replication due to intrinsic defects, for example, because only a partial or defective genome is present, or the particle is empty, faulty capsid assembly, or because of lethal mutations in the genome; second, virions may have undergone environmental inactivation; third, the choice of cells for virus isolation and growth may not mimic the optimum intracellular environment of the natural target organ; fourth, the interaction between a fully viable virus particle and a permissive cell may not always successfully initiate infection and an excess of particles may be necessary to achieve a statistical chance of success. perhaps no other procedure has contributed as much to virology as the development of the plaque assay. the test was originally developed a century ago by felix d'herelle in his initial studies of bacteriophages and was subsequently adapted to mammalian viruses in 1953 by renato dulbecco and marguerite vogt. the assay is elegantly simple: serial 10-fold dilutions of a virus sample are made in a cell culture medium. these diluted samples are then added to preformed monolayer cells and incubated under agar or a carboxymethyl cellulose layer to prevent the spread of secreted virus. after 24 to 48 hours (or longer, according to the virus of interest), plaques of necrotic cells become visible under the light microscope. at an appropriate time, fixation and staining, for example, with crystal violet, reveals clearly visible holes in the monolayer, each corresponding to a focus of necrotic cells initiated by one viable virus particle. serial dilution of the virus preparation facilitates the counting of discrete plaques so that, knowing the dilution, volume tested, and the plaque count, the concentration (titer) in the original sample can be determined. immunohistochemical staining procedures using specific antisera can be used as an alternative for visualizing plaques formed by non-cytopathic viruses. with the development of real-time pcr assays, the concentration of viral nucleic acid in a test sample can now be measured in virtually any context. by comparison with copy number controls, the concentration of nucleic acid in the treated sample can be determined. this type of assay does not detect empty capsids (those that do not contain viral nucleic acid), and, importantly, it does not necessarily relate to the infectivity of the sample. chapter 10: laboratory diagnosis of virus diseases, describes a further range of assays that are used in a diagnostic context, most of which can be also used both quantitatively and for research applications. this chapter has hitherto described how, during virus replication, non-infectious particles that lack the full genetic information essential for infectivity, are commonly produced-analogous to the assembly of defective motor figure 4.10 plaque assay for virus quantitation. cell monolayers are grown, typically in petri dishes, and when confluent the virus inoculum is allowed to adsorb to the monolayer in a small volume. the monolayers are overlaid with agarose to restrict diffusion of virus particles, and incubated to allow virus replication beginning with single cells and then spreading as more cells become successively involved. the vital stain neutral red is then added; this is taken up by viable cells, staining the monolayer red and leaving clear holes. plaque sizes and rates of development depend on the replication cycle and yield of the individual virus (see text). to determine the virus titer of the starting inoculum, serial 10-fold dilutions are inoculated on to duplicate monolayers. for those monolayers where discrete plaques can be distinguished (typically 10 to 50 plaques per dish), plaque numbers are counted and the original virus titer calculated by multiplying this plaque number by the dilution factor. by harvesting virus from a single plaque, a stock of genetically homogeneous virus can be obtained ("plaque-purifying"). spontaneous mutants in a virus inoculum may be recognized by plaques of abnormal size or morphology, and can thus be harvested and separated for further study. vehicles from a car assembly plant. one special example involves defective interfering (di) particles; these (1) lack the complete genome of the wild-type virus (deletion mutants), but (2) can still replicate in a new cell if the cell is also co-infected by wild-type virus which provides the missing function (by a process known as complementation). such defective particles may then also (3) interfere with the ongoing replication of the wild-type helper virus, possibly due to possessing a competitive replication advantage over wild-type virus. the production of di particles is encouraged by infection at high multiplicity, where the chances of co-infection with full-length and defective viruses occurring in the same cell are increased. in fact, this phenomenon was first observed by preben and herdis von magnus in 1944 with influenza virus; they found that very high multiplicities led to poor virus replication and the production of "interfering" virus stocks. a cyclical pattern may be observed, where di particles increase in number as long as the culture has sufficient wild-type helper virus replication; however, as di particles then inhibit the numbers of wild-type virus, the whole virus population diminishes, fresh rounds of wildtype virus replication can then occur and so a milieu for a return to di replication is built up again. despite several suggestions, it is not clear to what extent di particles play a role in the pathogenesis of an ongoing infection in a host, for example, in modulating recovery from an acute infection or promoting chronic infection. timing is everything: finetuned molecular machines orchestrate paramyxovirus entry principles of virology hiv-1 assembly, release and maturation viral membrane fusion. virology 479-480 poliovirus cell entry: common themes in viral cell entry pathways 2013. fields virology how viruses hijack the erad tuning machinery the rna synthesis machinery of negative-stranded viruses key: cord-350558-qfdp4ov9 authors: shaban, mohammed samer; müller, christin; mayr-buro, christin; weiser, hendrik; albert, benadict vincent; weber, axel; linne, uwe; hain, torsten; babayev, ilya; karl, nadja; hofmann, nina; becker, stephan; herold, susanne; schmitz, m. lienhard; ziebuhr, john; kracht, michael title: inhibiting coronavirus replication in cultured cells by chemical er stress date: 2020-08-26 journal: biorxiv doi: 10.1101/2020.08.26.266304 sha: doc_id: 350558 cord_uid: qfdp4ov9 coronaviruses (covs) are important human pathogens for which no specific treatment is available. here, we provide evidence that pharmacological reprogramming of er stress pathways can be exploited to suppress cov replication. we found that the er stress inducer thapsigargin efficiently inhibits coronavirus (hcov-229e, mers-cov, sars-cov-2) replication in different cell types, (partially) restores the virus-induced translational shut-down, and counteracts the cov-mediated downregulation of ire1α and the er chaperone bip. proteome-wide data sets revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including herpud1, an essential factor of er quality control, and er-associated protein degradation complexes. the data show that thapsigargin hits a central mechanism required for cov replication, suggesting that thapsigargin (or derivatives thereof) may be developed into broad-spectrum anti-cov drugs. one sentence summary / running title suppression of coronavirus replication through thapsigargin-regulated er stress, erqc / erad and metabolic pathways the er is critically involved in surveying the quality and fidelity of membrane and secreted protein 40 synthesis, as well as the folding, assembly, transport and degradation of these proteins (wang & 41 kaufman, 2016). the accumulation of unfolded or misfolded proteins in the er lumen leads to er 42 stress and upr activation, thereby slowing down protein synthesis and increasing the folding capacity 43 of the er (karagoz et al, 2019) . as a result, cellular protein homeostasis can be restored and the cell 44 survives. if this compensatory mechanism fails, er stress pathways can also switch function and will 45 eventually induce oxidative stress and cell death (hetz & papa, 2018; wang & kaufman, 2016). 46 47 the system relies on three er membrane-inserted sensors, including the protein kinase r (pkr)-like 48 er kinase (perk), inositol-requiring protein 1α (ire1α) and cyclic amp-dependent transcription 49 factor 6α (atf6α). perk and ire1α are ser/thr kinases whose conserved n termini are oriented 50 towards the er lumen (wu et al, 2014) . in non-stressed cells, the highly abundant major er 51 chaperone and er stress sensor binding-immunoglobulin protein bip (also called 78 kda glucose-52 regulated protein, gpr78; heat shock protein family a member 5, hspa5) binds to perk and ire1α, 53 which keeps these two proteins in an inactive monomeric state ( the activation of er stress by infectious agents has been widely observed. however, with few 68 exceptions, it remains to be studied how this response is shaped in a microbe-specific manner and 69 whether or not these responses are beneficial or detrimental to the host (grootjans et al, 2016). 70 moreover, there is a lack of knowledge on cov-mediated (de)regulation of er stress components at 71 the protein level. the latter is important because covs, in common with many rna viruses, are 72 known to cause a global shutdown of host protein synthesis (hilton et al, 1986). 73 74 here, we report that cov infection activates upr signaling and induces er stress components at the 75 mrna level but suppresses them at the protein level. strikingly, the well-known chemical activator of 76 the upr, thapsigargin, exerts a profound antiviral effect in the lower nanomolar range on three 77 different covs in different cell types. a detailed proteomics analysis reveals multiple thapsigargin-78 regulated pathways and a network of proteins that are suppressed by cov but (re)activated by 79 chemically stressed infected cells. these results reveal new insight into central factors required for 80 cov replication and open new avenues for targeted cov antivirals. 81 82 results to investigate how covs modulate er stress components at the mrna compared to the protein level, 84 we determined the expression levels of 166 components of the er stress pathway kegg 04141 85 "protein processing in endoplasmic reticulum" in human huh7 liver cells, a commonly used cellular 86 model for cov replication, in response to infections with hcov-229e and mers-cov, respectively. 87 for untreated huh7 cells, we obtained mrna (by rna-seq) and protein (by lc-ms/ms) expression 88 data for 119 components which revealed a positive correlation between mrna and protein 89 abundancies (fig.1a, upper graph) . however, in cell lysates obtained at 24 h post infection (p.i.), this 90 effect was largely lost (fig. 1a , middle and lower graph). pearson correlation matrix confirmed a 91 progressive loss of correlation between mrna levels and protein levels for this pathway over a time 92 course from 3 h to 24 h p.i. (fig. 1b) . thus, out of 37 (for hcov-229e) or 56 (for mers-cov) er 93 stress factors that were found to be regulated at the mrna level, only a few remained (down)regulated 94 at the protein level at late time points (fig. 1c, fig. s1 ). 95 to determine the functional consequences of this opposing regulation at the mrna and protein levels 96 in cov-infected cells, we focused on hcov-229e and assessed key regulatory features of the er 97 stress pathway as shown in fig. 2a . as a reference, we included samples from cells exposed to 98 thapsigargin, a compound that has been widely used to study prototypically activated er stress 99 mechanistically (bertolotti et al., 2000; oslowski & urano, 2011) . this setup included experiments, in 100 which thapsigargin and virus were added simultaneously to the cell culture medium (followed by a 101 further incubation for 24 h) or thapsigargin was added to the cells at 8 h p.i. for 16 h (fig. 2b) . the presence of thapsigargin in the growth medium resulted in a major drop in viral titers by more 104 than 150-fold (from 9.18*10 6 to 5.7*10 4 pfu / ml) which was paralleled by reduced amounts of viral 105 rna isolated from thapsigargin-treated, hcov-229e-infected infected cells at 24 h p.i. (fig. 2c) . immunofluorescence analysis of hcov-229e-infected cells treated with thapsigargin confirmed the 107 impaired formation of functional viral replication/transcription complexes (rtcs) as shown by the 108 reduced levels of both double-stranded rna (an intermediate of viral rna replication) and 109 nonstructural protein (nsp) 8 (an essential part of the viral rtc) (fig. 2d) . a strong suppression of viral replication was also demonstrated by the reduced protein levels observed 112 for the nucleocapsid (n) protein (a major coronavirus structural protein) as well as nonstructural 113 proteins (nsp) 8 and 12, both of which representing essential components of the viral replication 114 complex (snijder et al, 2016) (fig. 2e, f) . in all cases, the antiviral effect of thapsigargin remained 115 readily detectable when the compound was added at 8 h p.i, suggesting that it does not prevent viral 116 entry but rather suppresses intracellular pathways required for efficient rna replication and/or 117 particle formation and release or activates unknown antiviral effector systems (fig. 2c-f) . 118 119 next, we investigated er stress signaling under these conditions. both virus and thapsigargin were 120 confirmed to activate the perk branch of er stress (fig. 2e, f) , as shown by the retarded mobility of 121 perk in sds gels (indicating multisite phosphorylation) and by phosphorylation of the perk 122 substrate eif2α at ser51 (fig. 2e, f) . unlike thapsigargin treatment, hcov-229e infection led to a 123 weak but significant decrease of perk (mean 71±15 %) and eif2α (mean 67±13 %) levels compared 124 to the controls. infection also caused an approximately twofold (mean 42±22 %) reduction in bip 125 expression (fig. 2e, f) . in contrast, long-term thapsigargin treatment (for 16 h or 24 h) caused a 3-4-126 fold increase in bip expression, also in hcov-229e-infected cells, thus reversing the suppression by 127 viral infection (fig. 2e, f) . similarly, thapsigargin treatment for 16 h or 24 h caused a 1.5-2-fold 128 increase in ire1α expression (but not phosphorylation), again also in infected cells (fig. 2e, f) . in 129 this set of experiments, atf3 proved to be the only protein that was induced by virus alone (fig. 2e, 130 f), while the expression levels of atf4 remained largely unchanged (fig. 2e, f) . 131 132 these data show that both cov infection and chemicals like thapsigargin activate er stress through 133 the same proximal perk pathway, although they affect downstream cellular outcomes differentially. 134 the restoration of bip and ire1α levels by long-term thapsigargin treatment further suggests that the 135 cov-induced block of inducible host factors is not irreversible and can be reprogrammed by a 136 (presumably protective) thapsigargin-mediated response. our comparative analyses of viral replication 137 and host response lead us to conclude that chemically and virus-induced forms of er stress, although 138 proceeding through the same core perk pathway, do not simply potentiate each other but rather 139 (somewhat counterintuitively) counteract each other. 140 141 to explore a potential pharmacological exploitation of this effect, we assessed the cytotoxicity of the 142 combined thapsigargin treatment and virus infection, because both conditions are known to promote 143 cell death. 144 at 24 h p.i., cell viability of hcov-229e-infected huh7 cells was only marginally reduced (mean 145 90.02±12.32 %) (fig. 2g, upper graph) . after 24 h of incubation, thapsigargin decreased cell 146 viability in a dose-dependent manner with a cc50 of 10.7 µm in line with previous reports (fig. 2g, 147 middle graph, fig. 2h ) (sehgal et al, 2017; tombal et al, 2000) . the combination of thapsigargin 148 and hcov-229e infection did not cause additional cytotoxicity as shown by a nearly identical cc50 of 149 9.7 µm (lower graph, fig. 2g, fig. 2h ). at 1 µm thapsigargin, i.e., a concentration shown to 150 completely abolish viral protein translation and replication (see above), the cell viability of cells 151 infected with hcov-229e and treated with thapsigargin was 76.6±7.9 %, suggesting that thapsigargin 152 exerts its antiviral effects at concentrations well below its cytotoxic concentrations (fig. 2g, h) . to further characterize the metabolic state of the cells under the conditions used in these experiments, 155 we investigated protein de novo synthesis. newly produced proteins were quantified by in vivo 156 puromycinylation tagging of nascent protein chains followed by immunoblotting using anti-puromycin 157 antibodies. hcov-229e was found to shut down protein biosynthesis by 90.3±5.4% while 1 h of 158 thapsigargin treatment led to a shutdown by 94.3±4.3% (fig. 2i) . however, in infected cells, the 159 simultaneous or delayed addition of thapsigargin restored (or rescued) protein biosynthesis to 160 approximately 50 % of the level observed in untreated cells (fig. 2i) . these data demonstrate that, 161 although both viral infection and thapsigargin treatment (individually) induce er stress and cause a 162 translational shut-down, their combination shows no additive harmful effects to the cells. on the 163 contrary, their combination appears to have opposing effects that result in a partial restoration of the 164 cellular metabolic capacity while retaining a profound antiviral effect. 165 166 we next assessed if these effects were cell-type or virus-specific. in line with the results described to characterize the underlying molecular mechanisms responsible for the observed antiviral effects of 177 thapsigargin, we focused on two highly pathogenic coronaviruses, mers-cov and sars-cov, for 178 which, to our knowledge, no side-by-side comparison of proteomic changes has been reported to date. 179 the large-scale proteomic study included (i) untreated cells and cells that were (ii) infected with 180 mers-cov, (iii) sars-cov-2, (iv) treated with thapsigargin, (v and vi) infected with one of these 181 viruses in the presence of thapsigargin. we used label-free quantification of six replicates per sample 182 to determine the expression levels of > 5000 proteins from total cell extracts. 183 184 in a systematic approach, we identified differentially expressed proteins (deps) based on pairwise 185 comparisons of proteins obtained from untreated cells, virus-infected cells or thapsigargin-treated cells 186 using a p value of -log10 ≥ 1.3 as cut-off. as visualized by volcano plot representations, mers-cov 187 infection suppressed 413 (at 12 h p.i.) and 1171 proteins (at 24 h p.i.), respectively, and increased the 188 levels of 150 proteins (at 12 h p.i.) and 508 proteins (at 24 h p.i.), respectively (fig. 4a, b) , while 189 sars-cov-2 suppressed the expression of 232 proteins at 12 h p.i. and 141 proteins at 24 h p.i. and 190 increased the expression of 184 proteins at 12 h p.i. and 56 proteins at 24 h p.i. (fig. 4c, d) . we then devised a bioinformatics strategy to identify patterns of co-regulated or unique pathways and 205 link deregulated protein sets identified in these data to specific (known) biological functions. as 206 shown schematically in up-/or downregulated deps we found that many of the most highly enriched categories are related to 218 rna, dna, metabolic functions and localization (fig. s5a) . we then combined the 400 pathway 219 categories and searched this list for identical or unique go terms in response to mers-cov, sars-220 cov-2 or thapsigargin. by filtering 227 pathways (out of 400) with enrichment p values ≤ -log10 3 we 221 found 27 pathway categories shared by both viruses and by thapsigargin, which are mostly related to 222 rna, folding, stress and localization (fig. 4f, fig. s5b ). 61 pathway categories unique to 223 thapsigargin almost exclusively represented metabolic and biosynthetic pathways as shown for the top 224 20 overrepresented pathways containing up-or downregulated deps, suggesting that thapsigargin on 225 its own, unlike cov infection, initiates a broad metabolic response (fig. 4f, fig. s5b ). this raised the question of whether the thapsigargin effects were retained in infected cells or, 228 alternatively, drug-sensitive pathway patterns were reprogrammed (or masked) by the virus infection. 229 to address this point, we pooled all pathways enriched under virus+thapsigargin conditions and 230 compared them to virus infection or thapsigargin alone. 53% (132 out 248) pathway terms were shared 231 by these three conditions reflecting multiple stress-related, catabolic and rna regulatory processes 232 (fig. 4g, h) . 21 pathway terms were unique to the virus+thapsigargin situation. they primarily 233 mapped to specific splicing, signaling (torc, rhoa, arf3) and transport/localization pathways 234 (fig. 4g, h) . the 34 categories shared by virus+thapsigargin and thapsigargin conditions but were not 235 detectable in cells infected with virus (only) recapitulate the thapsigargin-regulated metabolic 236 pathways (pyruvate, aldehyde, carbohydrate, amino/nucleotide sugar, amino acid and glutamine 237 metabolism, tca cycle, erad pathway, n-linked glycosylation) (fig. 4g, h) . for several of these 238 pathways (e.g. erad, heat stress, carbohydrate metabolism), some deps were induced while others 239 were repressed, indicating remodeling of pathway functions at the protein level (fig. 4g, h) . the 53 240 pathway terms that were absent in the virus+thapsigargin group of terms (groups 27, 9, 17 of the venn 241 diagram shown in fig. 4g ) represent a distinct set of terms, mostly related to nucleotide and dna-242 related processes, such as dna repair, dna unwinding, chromatin silencing (fig. 4g, h) . in 243 summary, the functional analysis of deps at the level of differentially enriched pathway categories 244 shows that the antiviral effects of thapsigargin strongly correlate with the activation / suppression of a 245 range of metabolic programs. 246 247 the enriched pathway terms provided important overarching information on shared and unique 248 biological processes but not necessarily encompassed identical sets of deps as exemplified by the ten 249 pathways shown in fig. 4i . we therefore refined our analysis to the individual component level to 250 identify proteins with similar regulation between both viruses across both cell types. the proteomes of 251 huh7 and vero e6 cells overlap by 57 % (fig. 5a) . in this group, only 43 identical proteins were 252 found to be deregulated by both mers-cov and sars-cov-2 (fig. 5b, it becomes apparent that the majority of proteins are regulated in the same direction by thapsigargin 256 alone; demonstrating that thapsigargin largely overrides any virus-induced modulation of host 257 processes (fig. 5c ). 258 259 in the absence of thapsigargin, the virus infection generally has little or opposite effects on the levels 260 of the 108 proteins, as exemplified by the suppression seen for bip (hspa5) or herpud1 (fig. 5c, 261 highlighted in green). the 108 induced factors map to pathways involving copi-mediated 262 anterograde transport, er stress, organelle organization and apoptosis (fig. 5d) . across their pathway 263 annotations, 59 out of the 108 proteins were reported to strongly interact, thus probably being involved 264 in protein:protein networks that coordinate activities of the enriched pathways (fig. 5e, left graph) . 265 likewise, the 61 repressed proteins map to specific (though different) pathways, such as fatty acid 266 degradation or viral life cycle (fig. 5d) . 26 components can be allocated to a few small protein 267 interaction networks (fig. 5e, right graph) . 268 269 we then validated mass spectrometry data by immunoblotting, confirming the induction of herpud1 270 in thapsigargin-treated cells infected individually with each of the three covs (fig. 5f , g). we also 271 confirmed an additional hit belonging to the enriched pathways go:0006520 (cellular amino acid 272 metabolic process) and go:0034976 (response to endoplasmic reticulum stress, as shown in fig. 273 4g, h), cystathionine-γ-lyase (cth), a regulator of glutathione homeostasis and cell survival (lee et 274 al, 2014), as a further independent example for the fidelity and robustness of the proteomics data (fig. 275 5f, g). 276 277 the highly inducible herpud1 protein has an essential scaffolding function for the organization of searching our proteomics data for further erad factors we were able to retrieve a total of 34 (for 284 mers-cov) and 20 (for sars-cov-2) proteins of the canonical erqc and erad pathways for 285 which a differential expression was observed in virus-infected cells treated with thapsigargin (fig. 286 5h) . mapping of these data on the kegg 04141 pathway suggests that thapsigargin enhances or 287 restores these mechanisms at key nodes of erqc and erad in coronavirus-infected cells (fig. s6) . 288 289 we also intersected the 108+59 proteins jointly regulated by thapsigargin in mers-cov and sars-290 cov-2 infected cells with data from a recent genome-wide sgrna screen that reported new erad 291 factors required for protein degradation (leto et al, 2019) . this analysis identified 31 additional 292 thapsigargin-regulated factors that may further support antiviral erad, including uba6 and znf622, 293 which were recently described either as negative regulators of dna virus infections or of autophagy, 294 the latter process playing diverse roles during cov infection (fig. 5i) in conclusion, these data show that thapsigargin forces the (re)expression of a dedicated network of 298 proteins with roles in er stress, erqc, erad, and a range of metabolic pathways. collectively, these 299 changes at the protein level confer an "antiviral state" and profoundly suppress cov replication as 300 summarized schematically in fig. 5j . discussion 303 in this study, we report a potent inhibitory effect of the chemical thapsigargin on the replication of 304 three human covs in three different cell types. clearly, the mechanistic basis for these effects remains to be identified in additional studies. the 353 proteomic data show that thapsigargin affects multiple pathways beyond the core er stress response. 354 they also indicate that it will not be trivial to identify the essential targets that mediate thapsigargin's 355 antiviral effects. our data provide a rich resource for further drug target analysis, also in conjunction 356 with the few deep protein sequencing studies available for sars-cov-2 (but not mers-cov) 357 ( in the absence of effective therapeutic and prophylactic strategies (antivirals and vaccines) to combat 362 coronaviruses, and in view of the current sars-cov-2 pandemic, we report these observations to 363 invite other laboratories to embark on a broader investigation of this potential therapeutic avenue. 364 given that thapsigargin concentrations in the lower nanomolar range were shown to abolish cov 365 replication in cultured cells, even when added later in infection (8 h translational shut-down and are secreted in a cell-type specific manner (fig. s7) . some of these 380 cytokines may contribute to the cytokine storm observed in some covid-19 patients (mehta et al, 381 2020). while thapsigargin had no effect on il-8, il-6, cxcl2 and ccl20 in cell culture (fig. s7) , a 382 single bolus of the compound was shown to efficiently reduce the translation of pro-inflammatory 383 cytokines in preclinical models of sepsis (wei et al, 2019) . thus, an additional benefit of thapsigargin 384 treatment may arise from dampening overshooting tissue inflammation in covid-19 patients. in 385 summary, the study provides several lines of evidence that thapsigargin hits a central mechanism of 386 cov replication, which may be exploited to develop novel therapeutic strategies. this compound or 387 derivatives with improved specificity, pharmacokinetics and safety profiles may also turn out to be 388 valuable to mitigate the consequences of potential future cov epidemics more effectively. infection. sci pepstatin 874 a, pmsf and microcystin were dissolved in ethanol and leupeptin in water. other reagents were from 875 sigma-aldrich or thermo fisher scientific anti perk (abcam, #ab65142), anti bip (cell signaling, 879 #3177), anti eif2α (cell signaling #9722), anti p(s51)-eif2α (cell signaling #9721), anti p anti ire1α #sc-188), anti herpud1 antibody (abnova, 882 #h00009709-a01), anti cth antibody (cruz, #sc-374249), anti hcov-229e n protein ((ingenasa, 883 batch 250609), mouse anti hcov-229e nsp12 (gift from carsten grötzinger), rabbit anti hcov-229e 884 nsp8 (ziebuhr & siddell, 1999), anti mers-cov n protein (sinobiological, #100213-rp02), rabbit 885 anti sars-cov n protein cross-reacting with sars-cov-2 n protein (gift from friedemann weber), 886 anti sars-cov-2 n protein (rockland, #200-401-a50), anti puromycin the following secondary antibodies were used: dako p0447; polyclonal goat anti-mouse 889 immunoglobulins/hrp, dako p0448; polyclonal goat anti-rabbit immunoglobulins/hrp, cy3-coupled 890 anti rabbit (rb) igg (dk, merck millipore, #ap182c), dylight 488-coupled anti mouse (ms) igg (dk, immunoblotting was performed essentially as described (hoffmann et al after blocking with 5% 923 dried milk in tris-hcl-buffered saline/0.05% tween (tbst) for 1 h, membranes were incubated for 924 12-24 h with primary antibodies, washed in tbst and incubated for 1-2 h with the peroxidase-925 coupled secondary antibody. proteins were detected by using enhanced chemiluminescence (ecl) 926 systems from millipore or ge healthcare mrna expression analysis by rt-qpcr 930 0.5 -1 µg of total rna was prepared by column purification (machereynagel) and transcribed into 931 cdna using moloney murine leukemia virus reverse transcriptase #ep0441) in a total volume of 10 or 20 µl. 1 or 2 µl of this reaction 933 mixture was used to amplify cdnas using fisher scientific) for gusb (81 bp, hs99999908_m1), il6 (95 bp il8 (101 bp, hs0017 4103_m1), cxcl2 (68 bp, hs00236966_m1), and ccl20 (81 936 bp, hs00171125_m1), as well as taqman fast universal pcr master mix (applied biosystems/ 937 all pcrs 942 were performed in duplicate on an abi 7500 real-time pcr instrument. the cycle threshold value (ct) 943 for each individual pcr product was calculated by the instrument's software, and the ct values 944 obtained for inflammatory/target mrnas were normalized by subtracting the ct values obtained for 945 gusb. the resulting δct values were also used to calculate relative changes of mrna expression as 946 ratio (r) of mrna expression of treated after 2x 952 washing, cells were fixed with 4% paraformaldehyde in pbs (santa cruz, #281692) for 5 min, washed 953 3x 10 min with hank's bss (pan, #p04-32505), blocked with 10% normal donkey serum (jackson 954 immunoresearch, #017-000-121) for 20 min and incubated with primary and secondary antibodies 955 diluted in hank's bss containing 0.005% saponin (sigma-aldrich, #s4521-10g) for 2 h at room 956 temperature. following 3 washing steps with hank's bss containing 0.005% saponin in brief, 1.2 x 10 4 huh7 or 1 x 10 4 mrc-5 968 cells were seeded in 96-well plates for 24 hours and thereafter treated with dmso, thapsigargin, virus 969 alone or virus plus thapsigargin for 24 hours as indicated in the figure legends. then, the medium was 970 replaced by 100 µl complete cell culture medium including 20 µl celltiter 96® aqueous one solution 971 reagent according to the manufacturer's recommendations. cells were further incubated for 0.5 -1 972 hour at 33° c. then, absorbance values were measured at 490 nm after 24 h, 200 µl mtt mix (dmem supplemented with 10% fcs 976 containing 250µg/ml tetrazolium bromide, sigma) was added to each well. next, cells were incubated 977 for 90-120 min at 37°c and fixed using 3.7% pfa in pbs. the tetrazolium crystals were dissolved by 978 adding 200 µl/well isopropanol and the absorbance at 490 nm was measured using an elisa reader 979 (biotek) elisa 984 sandwich elisas from r&d systems (duoset elisa for human il-8 (dy208) the cell culture supernatants were harvested, centrifuged at 15,000 × g at 4°c for 15 s 988 and stored at -80°c. 100 µl of the supernatants were either used undiluted or were diluted in cell 989 culture medium as follows or mock infection) using two biological replicates resulting in 32 rna-seq data sets. 998 rna was sequenced (with rrna depletion) using illumina reagents and an illumina hiseq 4000 999 instrument (single read, 150 bases) cysteines were alkylated with iodoacetamide and 8 m urea buffer was exchanged 1015 to 50 mm ammonium-bicarbonate buffer with a ph of 8.0. samples were digested within the filter 1016 devices by the addition of sequencing grade modified trypsin (serva) and incubation at 37 °c over-1017 night. thereafter, the filter-units were transferred to fresh tubes. peptides were eluted by the addition 1018 of 50 µl 0.5 m nacl solution and centrifugation (14.000 x g for 10 min) finally, peptides were dissolved in 25 µl water with 5% acetonitrile and 0.1% 1022 formic acid. the mass spectrometric analysis of the samples was performed using a timstof pro mass 1023 spectrometer (bruker daltonic). a nanoelute hplc system sample loading was performed at a constant pressure 1027 of 800 bar. separation of the tryptic peptides was achieved at 50°c column temperature with the 1028 following gradient of water/0.1% formic acid (solvent a) and acetonitrile/0.1% formic acid (solvent 1029 b) at a flow rate of 400 nl/min: linear increase from 2%b to 17%b within 60 minutes, followed by a 1030 linear gradient to 25%b within 30 minutes and linear increase to 37% solvent b in additional 10 1031 minutes. finally b was increased to 95% within 10 minutes and hold for additional 10 minutes. the 1032 built-in "dda pasef-standard_1.1sec_cycletime" method developed by bruker daltonics was used 1033 for mass spectrometric measurement. data analysis was performed using maxquant with the 1034 andromeda search engine and uniprot databases were used for annotating and assigning protein 1035 identifiers the normalized expression values assigned 1041 to uninfected huh7 cells were derived from a total of 59 mock samples representing multiple 1042 technical repeats of two biological samples generated at each of the 3 h, 6 h, 12 h, 24 h time points in 1043 order to generate a common reference sample for the mean protein expression found in uninfected / 1044 untreated huh7 cells. this mean reference was used to calculate all ratio values. from the entire data 1045 set, only protein intensity values for 166 uniprot ids assigned to kegg 04141 were extracted, 1046 quantile normalized and further analyzed using the software tools described below. 1047 for the data shown in fig. 4 and 5, raw data from 96 lc-ms/ms runs (representing two independent 1048 experiments and three technical replicates per sample) were mapped to homo sapiens all data sets were processed by 1052 maxquant version 1.6.10.43 (raw data submission was done with version 1.6.17.0) (tyanova et al., 1053 2016a) including the match between runs option enabled resulting in the identification of ids 1056 assigned to contaminants and reverse sequences were omitted. for calculation of ratio values between 1057 conditions, the 2 x 6 replicates from each condition were assigned to one analysis group. 1058 differentially expressed proteins were identified from log2 transformed normalized protein intensity 1059 values by volcano plot analysis using perseus functions. all subsequent filtering steps and heatmap 1060 representations were performed in excel 2016 as described in the figure legends pearson correlations) were 1070 calculated using sigmaplot 11 thirty minutes 902 prior to harvest, the medium was supplemented with 3 µm puromycin (invivogen, #ant-pr-1). then, 903 cells were lysed as described above. after immunoblotting (see below), membranes were stained with 904coomassie brilliant blue and then hybridized with an anti puromycin antibody (kerafast, #eq0001) to 905 detect puromycinylated polypetides. 906total cell lysates of mers-cov-and sars-cov-2-infected cells used for immunoblotting or mass 907 spectrometry were prepared as follows. cells were scraped in ice-cold pbs and pelleted at 500 x g for 908 5 min at 4°. cell pellets were washed in ice-cold pbs and stored in liquid n2 (or lysed and processed 909 immediately). after thawing, cell pellets (corresponding to ≈ 300.000 cells seeded in 60 mm dishes at 910 the start of the experiment) were resuspended in 90 µl of ice-cold ca 2+ / mg 2+ -free pbs and 911 transferred to fresh tubes. after addition of 10 µl of 10% sds, samples were heated at 100 °c for 10 912 min and centrifuged at 600 x g for 1 minute at room temperature. supernatants were transferred to a 913 fresh tube and heated again at 100°c for 10 minutes and centrifuged at 600 x g for 1 minute at room 914 temperature. protein concentrations were determined with the detergent compatible bradford assay kit 915 (pierce™, #23246) using a 150-fold dilution. aliquots corresponding to 20-25 µg protein (per lane) 916were mixed with 4 x sds sample buffer (roti ® load, roth, #k929) and stored at -20 °c prior to 917 sds-page, or loaded immediately. cell lysates were subjected to sds-page on 8-10% gels. the 918pageruler™ prestained protein ladder (thermo scientific, #26616) was used as mr marker. 919 key: cord-334592-54dofkxh authors: levine, beth; mizushima, noboru; virgin, herbert w. title: autophagy in immunity and inflammation date: 2011-01-20 journal: nature doi: 10.1038/nature09782 sha: doc_id: 334592 cord_uid: 54dofkxh autophagy is an essential, homeostatic process by which cells break down their own components. perhaps the most primordial function of this lysosomal degradation pathway is adaptation to nutrient deprivation. however, in complex multicellular organisms, the core molecular machinery of autophagy — the 'autophagy proteins' — orchestrates diverse aspects of cellular and organismal responses to other dangerous stimuli such as infection. recent developments reveal a crucial role for the autophagy pathway and proteins in immunity and inflammation. they balance the beneficial and detrimental effects of immunity and inflammation, and thereby may protect against infectious, autoimmune and inflammatory diseases. t here is only one known mechanism that eukaryotic cells possess to dispose of intracellular organelles and protein aggregates that are too large to be degraded by the proteasome. it is therefore not surprising that this mechanism -the lysosomal degradation pathway known as autophagy -is also used to degrade microorganisms (such as viruses, bacteria and protozoa) that invade intracellularly 1, 2 . indeed, the mutation of autophagy genes increases susceptibility to infection by intracellular pathogens in organisms ranging from plants to flies to worms to mice, and possibly to humans. perhaps less apparent, but teleologically as intuitive, the autophagy pathway or unique functions of autophagy proteins also have a central role in controlling other diverse aspects of immunity in multicellular organisms. the autophagy machinery is thought to have evolved as a stress response that allows unicellular eukaryotic organisms to survive during harsh conditions, probably by regulating energy homeostasis and/ or by protein and organelle quality control. the same machinery might therefore be expected to diversify functionally in complex metazoan organisms, so as to regulate new layers of defences used by multicellular organisms to confront different forms of stress. a plethora of genetic, biochemistry, cell biology, systems biology and genomic studies have recently converged to support this notion. the autophagy machinery interfaces with most cellular stress-response pathways 3 , including those involved in controlling immune responses and inflammation. this interface is not only at the level of the autophagy pathway, but also entails direct interactions between autophagy proteins and immune signalling molecules 4 . there is a complex reciprocal relationship between the autophagy pathway/proteins and immunity and inflammation; the autophagy proteins function in both the induction and suppression of immune and inflammatory responses, and immune and inflammatory signals function in both the induction and suppression of autophagy. moreover, similar to cancer, neurodegenerative diseases and ageing 5 , defects in autophagy -through autophagy gene mutation and/or microbial antagonism -may underlie the pathogenesis of many infectious diseases and inflammatory syndromes. in this review, we describe recent advances in our evolving comprehension of the interface between autophagy, immunity and inflammation. we discuss how emerging concepts about the functions of the autophagy pathway and the autophagy proteins may reshape our understanding of immunity and disease. this set of proteins not only orchestrates the lysosomal degradation of unwanted cargo, but also exerts intricate effects on the control of immunity and inflammation. thus, the autophagy pathway and autophagy proteins may function as a central fulcrum that balances the beneficial and harmful effects of the host response to infection and other immunological stimuli. autophagy is a general term for pathways by which cytoplasmic material, including soluble macromolecules and organelles, is delivered to lysosomes for degradation 6 . there are at least three different types of autophagy, including macroautophagy, chaperone-mediated autophagy and microautophagy. macroautophagy, usually referred to simply as autophagy, is the subject of this review (fig. 1 ). in this pathway, a portion of cytoplasm (usually 0.5-1 μm in diameter) is engulfed by an isolation membrane, or 'phagophore' , resulting in the formation of a double-membrane structure known as the autophagosome. the outer membrane of the autophagosome fuses with the lysosome to become an autolysosome, leading to the degradation of autophagosomal contents by lysosomal enzymes. autophagosomes can also fuse with endosomes or multivesicular bodies and major histocompatibility complex (mhc)class-ii-loading compartments 7 . autolysosomes become larger as more autophagosomes and lysosomes fuse, but at a termination phase lysosomes are tubulated and fragmented for renewal 8 . the membrane dynamics of autophagosome formation involve complex processes, which are not completely understood. nonetheless, the molecular dissection of autophagy membrane dynamics, stimulated by the discovery of atg (autophagy-related) genes in yeast 9 , has shed considerable light on this topic (table 1) . several recent studies suggest that the endoplasmic reticulum (er) is crucial for autophagosome formation. the er cisternae often associate with developing autophagosomes, and electron tomography analysis has demonstrated direct connections between the er and autophagosomal membranes 10, 11 . moreover, the function of several key autophagy proteins seems to be at the level of the er (fig. 1) . autophagy is induced by nutrient starvation through the inhibition of mammalian target of rapamycin (mtor), resulting in translocation of the mtor substrate complex (ulk1/2, atg13, fip200 (also known as rb1cc1) and atg101) from the cytosol to certain domains of the er or closely attached structures 12, 13 . this leads to the recruitment of the class iii phosphatidylinositol-3-oh kinase (pi(3)k) complex, which includes at least vps34 (also known as pik3c3), vps15 (pik3r4 and p150), beclin 1 and atg14, to the er 13, 14 . the pi(3)k complex produces phosphatidylinositol-3-phosphate (ptdins(3)p), which recruits effectors such as double fyve-containing protein 1 (dfcp1) and wdrepeat domain phosphoinositide-interacting (wipi) family proteins. dfcp1 is diffusely present on the er or the golgi, but translocates to the autophagosome formation site in a ptdins(3)p-dependent manner to generate er-associated ω-like structures termed omegasomes 15 . among the four wipi isoforms, wipi2 is the major form in most cell types and functions downstream of dfcp1, and it may promote the development of omegasomes into isolation membranes or autophagosomes 16 . an er-associated multispanning membrane protein, vmp1, is also important for autophagosome formation. although vmp1 interacts with beclin 1 and is present at the autophagosome formation site at an early stage, it seems to function at a late stage in autophagy 13 overview of the autophagy pathway. the top right box shows a model of our current understanding of the molecular events involved in membrane initiation, elongation and completion of the autophagosome. the major membrane source is thought to be the endoplasmic reticulum (er), although several other membrane sources, such as mitochondria and the plasma or nuclear membrane, may contribute. after induction of autophagy, the ulk1 complex (ulk1-atg13-fip200-atg101) (downstream of the inhibitory mtor signalling complex) translocates to the er and transiently associates with vmp1, resulting in activation of the er-localized autophagy-specific class iii phosphatidylinositol-3-oh kinase (pi (3) this is perhaps consistent with other evidence that beclin 1-class iii pi(3)k complexes may function in autophagosomal maturation (in addition to vesicle nucleation), a process that can be regulated by other beclin-1-interacting partners such as rubicon (table 1) . at the final step of autophagosome formation, elongation of the isolation membrane and/or completion of enclosure require two ubiquitin-like conjugates. the first is the atg12-atg5 conjugate, which is produced by the atg7 (e1-like) and atg10 (e2-like) enzymes, and functions as a dimeric complex together with atg16l1 (ref. 19 ) . the second is the phosphatidylethanolamine (pe)-conjugated atg8 homologues -lc3, gate16 and gabarap -which are produced by the atg7 and atg3 (e2-like) enzymes 9, 20 . although the proteins involved in autophagosome membrane formation have been characterized as discrete complexes (table 1) , several potential interconnections between components of the different complexes were identified by a recent proteomics study 21 . such interconnections may function in autophagosome membrane formation or other distinct cellular functions. for example, the atg12-atg3 conjugate is implicated in mitochondrial homeostasis but not in autophagosome membrane formation 22 . in addition to the er, other membranes may be involved in autophagosome formation (fig. 1) . atg9, another multispanning membrane protein, is essential for autophagy 23 and traffics between the trans-golgi network, endosomes and autophagosome precursors 24 . studies suggest that mitochondria, the plasma membrane and the nuclear membrane could also be membrane sources for autophagosome formation [25] [26] [27] . however, the lack of detection of specific protein markers for these structures on the autophagosomal membrane leaves the decades-old question of the membrane source of the autophagosome unanswered. it is possible that cells may use different membrane sources to form the autophagosome in different contexts, thereby permitting specialization of membrane dynamics in a manner that allows divergent autophagyinducing signals to stimulate the capture of spatially distinct cargo. autophagy was originally considered to be a non-selective bulk degradation process, but it is now clear that autophagosomes can degrade substrates in a selective manner 28 . in addition to endogenous substrates, autophagy degrades intracellular pathogens in a selective form of autophagy, termed xenophagy. similar to bulk autophagy (such as that induced by nutrient deprivation) and other forms of selective autophagy (such as degradation of damaged mitochondria, peroxisomes, aggregate-prone proteins or damaged er), the precise membrane dynamics and specificity determinants of xenophagy are not fully understood. nonetheless, considerable advances have been made, and interesting similarities and differences are beginning to emerge between cellular recognition and degradation of self versus foreign microbial components through autophagy-like pathways (figs 1 and 2) . the vacuoles used for the engulfment of intracytoplasmic bacteria are similar to autophagosomes, and their formation requires the core autophagy machinery. but one apparent difference is the vacuole size; for example, the diameter of group a streptococcus-containing autophagosome-like vacuoles (gcav) can be as big as 10 μm. these large gcavs are generated by the rab7-dependent fusion of small isolation membranes 29 . by contrast, the formation of starvation-induced autophagosomes requires rab7 later in the autophagy process, at the autophagosome-lysosome fusion step. a more complex question is how autophagosomes (or components of the autophagy pathway) capture pathogens that are inside vacuolar compartments (fig. 2) . there are at least four general pathways that may be used for autophagy-protein-dependent targeting of bacteria to the lysosome. these include autophagy-protein-facilitated fusion of bacteria-containing phagosomes with lysosomes, the envelopment of bacteria-containing phagosomes or endosomes by autophagosomal membranes, the fusion of bacteria-containing phagosomes or endosomes with autophagosomes, or the xenophagic capture of bacteria that have escaped inside the cytoplasm. in some cases, the route of autophagy-dependent targeting to the lysosome has been well defined, such as for group a streptococcus that escapes from endosomes 30 . for several bacteria, however, the precise route is unclear. many studies define bacterial autophagy as the co-localization of bacteria and lc3, but we now know that lc3 can decorate membranous compartments other than autophagosomes (including phagosomes). several lines of evidence suggest that autophagy proteins function more broadly, not only in classical macroautophagy, but also in the process of phagolysosomal maturation during antigen presentation and microbial invasion. autophagy proteins are required for the fusion of phagosomes that contain toll-like receptor (tlr)-ligand-enveloped particles with lysosomes in macrophages 31 , and for the fusion of phagosomes that contain tlr-agonist-associated apoptotic cell antigens with lysosomes in dendritic cells during mhc class ii antigen presentation 32 . the self ligand and cell-surface receptor slam functions as a microbial sensor that recruits the beclin 1-class iii pi(3)k complex to phagosomes containing gram-negative bacteria, facilitating phagolysosomal fusion and activation of the antibacterial nadph oxidase (nox2) complex 33 . in addition, the engagement of tlr or fcγ receptors during phagocytosis recruits lc3 (and atg12) to the phagosome through nox2dependent generation of reactive oxygen species (ros) 34 . thus, in bacterial infections, a paradigm is emerging in which the coordinated regulation of microbial sensing, phagolysosomal maturation and antibacterial activity involves the recruitment of autophagy proteins to the phagosome. as a corollary, an interesting speculation is that impaired recruitment of autophagy proteins to the phagosome may contribute to the pathogenesis of chronic granulomatous disease, a genetic disorder caused by mutations in the nox2 gene (also known as cybb) and characterized by recurrent bacterial and fungal infections and inflammatory complications, such as inflammatory bowel disease. another autophagosome-independent function of autophagy proteins in pathogen destruction has been described in interferon-γ (ifn-γ)treated macrophages infected with the parasite toxoplasma gondii. the parasite-derived membrane, termed the parasitophorous vacuole, undergoes destruction through a mechanism that involves atg5-dependent recruitment of the immunity-related gtpase proteins to the parasitophorous vacuole 35, 36 , leading to the death of the parasite in the infected cell 35, 37 . together, these studies suggest that autophagy proteins have diverse roles in membrane dynamics to benefit the host in the removal of invading pathogens ( fig. 2 ), through xenophagy, phagolysosomal maturation, the recruitment of molecules that damage pathogen-derived membranes, and presumably, many other as yet undiscovered mechanisms. the mechanisms that cells use to target intracellular bacteria (and probably viruses) to autophagosomal compartments are notably similar to those used for selective autophagy of endogenous cargo. cellular cargo is commonly targeted to autophagosomes by interactions between a molecular tag (such as polyubiquitin), adaptor proteins such as p62 (also known as sqstm1 or sequestome 1) or nbr1 (which recognize these tags and contain an lc3-interacting region (lir) characterized by a wxxl or wxxi motif), and lc3 (ref. 28 ). these adaptor molecules enable autophagy to target designated cargo selectively to nascent lc3-positive isolation membranes. as reviewed elsewhere 38 , a similar mechanism involving ubiquitin and p62 seems to be involved in the targeting of intracellular bacteria, such as salmonella enterica serotype typhimurium (s. typhimurium), shigella flexneri and listeria monocytogenes, to autophagosomes. after escape into the cytoplasm or in vacuolar membrane compartments damaged by type iii secretion system (t3ss) effectors, bacteria or bacteria-containing compartments, respectively, may become coated with ubiquitin and associate with p62 and nascent lc3-positive isolation membranes. the autophagosomal targeting of salmonella also requires another cellular factor, ndp52 (nuclear dot protein 52), an autophagy adaptor protein that, like p62, contains an lir and ubiquitin-binding domains and restricts intracellular bacterial replication. a ubiquitinindependent pathway (that does not involve p62 or ndp52) could also function in targeting damaged salmonella-containing vacuoles (scvs) to the autophagosome. in this pathway, a lipid second messenger, diacylglycerol, acts as a signal for the co-localization of scvs with lc3-positive autophagosomes by a mechanism that involves protein kinase c and its downstream targets, jnk and nadph oxidase 39 . the autophagic targeting of a cytoplasmic positive-strand rna virus, sindbis virus, also occurs in a ubiquitin-independent manner, but involves the interaction of p62 with the viral capsid protein 40 . thus, diverse molecular strategies, including ubiquitin-dependent and -independent mechanisms, may be used to target microbes inside the cytoplasm or vacuolar compartments to the autophagosome. beyond targeting intracellular pathogens for degradation, p62 may have further beneficial effects in infected host cells. for example, shigella vacuolar membrane remnants generated by bacterial t3ss-dependent membrane damage are targeted by polyubiquitination, p62 and lc3 for autophagosomal degradation 41 (fig. 2) . these membrane remnants also accumulate numerous molecules involved in sensing and transduction of pathogen-associated molecular pattern (pamp) and danger-associated molecular pattern (damp) signals, and there is an increase in nuclear factor-κb (nf-κb)-dependent cytokine production, ros production and necrotic cell death in autophagy-deficient cells. thus, the ubiquitin-p62-dependent autophagic targeting of pathogen-damaged membranes could help to control detrimental downstream inflammatory signalling during bacterial invasion into host cells. another emerging concept is that selective autophagy of viral proteins, similar to selective autophagy of aggregate-prone toxic cellular proteins, may protect post-mitotic cells such as neurons against cell death. for example, in sindbis-virus-infected mice with neuron-specific inactivation of atg5, there is an accumulation of sindbis virus antigens (without increased levels of infectious virus), increased neuronal cell death and increased animal mortality 40 . moreover, p62 is required for starvation and ifn-γ-induced targeting of fau (and perhaps other ubiquitylated protein complexes) to mycobacteria-containing phagosomes, resulting in the generation of antimycobacterial fau-derived peptides 42 .the role of p62 in innate immunity is probably evolutionarily ancient, as the drosophila p62 orthologue ref(2)p was originally identified in a screen for modifiers of sigma virus replication 43 . we speculate that p62, as well as the other known lc3-interacting adaptor proteins (nbr1 and ndp52), may represent the tip of the iceberg in terms of cellular adaptor proteins that bind to ubiquitin (or other molecular tags) and target microbial substrates and cytosolic material to autophagosomes to coordinate innate immune responses. a recent proteomics study showed that the mammalian atg8 family, which includes lc3, gate16 and gabarap, has 67 high-confidence interactions with other cellular proteins 21 . some of these new atg8-familymember-interacting partners may have an as yet undiscovered role in innate immunity. another open question is whether the known proteins involved in selective autophagy of mitochondria (called mitophagy), such as nix (also known as bnip3l) and parkin 44 , also function in microbial autophagy. the autophagy pathway and/or autophagy proteins have a crucial role in resistance to bacterial, viral and protozoan infection in metazoan organisms. the genetic deletion or knockdown of autophagy genes to the proteins shown in the cargo-recognition box in fig. 1 ; however, as yet undiscovered adaptors may be involved in pathogen recognition, and pathogen targeting may involve ubiquitin-dependent or -independent mechanisms. protects plants from viral, fungal and bacterial infection by preventing the uncontrolled spread of programmed cell death during the plant innate immune or hypersensitive response 45 . in other organisms, autophagy proteins function in a cell-autonomous manner to control infection by intracellular pathogens. in drosophila, autophagy gene mutation increases susceptibility to viral (vesicular stomatitis virus) 46 and bacterial (l. monocytogenes) 47 infection. in dictyostelium and caenorhabditis elegans, autophagy gene mutation increases susceptibility to lethal s. typhimurium infection 48 . in mice, knockout of atg5 in macrophages and neutrophils increases susceptibility to infection with l. monocytogenes and the protozoan t. gondii 35 , and neuron-specific atg5 knockout increases susceptibility to central nervous system sindbis virus infection 40 . as noted in the next section, the autophagy pathway and proteins may also have 'proviral' or 'probacterial' effects in in vitro studies; however, in vivo evidence for such effects is so far lacking. the mechanisms by which autophagy genes mediate in vivo resistance to infection are not fully understood, but are likely to involve a combination of xenophagy, other autophagy-protein-dependent effects on microbial replication or survival, activation of innate and adaptive immune responses, and/or alterations in pathogen-induced cell death (fig. 3 ). an important question is whether this function of autophagy in broad resistance to infection with intracellular pathogens extends to humans. recent human genetic studies provide some clues. the immunityrelated gtpase human irgm, which regulates autophagy-dependent clearance of mycobacteria in vitro 49 , was identified as a genetic risk locus for tuberculosis in a west african population 50 . numerous studies have shown a crucial role for autophagy in defence against mycobacterial infection in human cells 1 , and a genome-wide analysis of host genes that regulate mycobacterium tuberculosis replication demonstrated that a predominance of factors were autophagy regulators 51 . thus, it is possible that autophagy has a central role in resistance to one of the most important global infectious diseases -tuberculosis. mutations in nod2, which encodes an intracellular pathogen-recognition receptor (nucleotide-binding oligomerization-domain-containing protein 2) that functions in bacterial autophagy 52, 53 , are also associated with susceptibility to infection with another mycobacterial agent, mycobacterium leprae, the aetiological agent of leprosy 54 . an exciting future venture will be to confirm whether irgm, nod2 and other autophagy-related genes are involved in resistance to infection with mycobacteria and other infections in further human populations and, if so, whether this resistance is mediated by autophagy. microbes undergo strong selective pressure to develop strategies to block host defence mechanisms; the number of such strategies is a surrogate measure of the importance of the host defence mechanism in immunity. as reviewed elsewhere 1,55 , viruses and intracellular bacteria have evolved several ways to adapt to host autophagy. they can antagonize autophagy initiation or autophagosomal maturation, evade autophagic recognition, or use components of the autophagy pathway to facilitate their own replication or intracellular survival. an emerging theme is that microbial antagonism of autophagy not only blocks the xenophagic degradation of intracellular pathogens, but also blocks the functions of autophagy in innate and adaptive immunity. a relatively unexplored yet crucially important frontier is how microbial antagonism may contribute more broadly to the role of microbes in diseases characterized by defective autophagy, such as cancer, neurodegenerative diseases, ageing and, potentially, autoimmune and inflammatory diseases. viral strategies to shut off autophagy include the blockade of positive upstream regulators of autophagy (such as the ifn-inducible rna-activated eif2α protein kinase (pkr) signalling pathway), the activation of negative upstream regulators of autophagy (such as the nutrient-sensing tor kinase signalling pathway) or direct antagonism of the autophagy machinery 55 . the overlapping functions of the eif2α kinase signalling pathway in stress-induced general translational arrest, transcriptional activation of stress-response genes and stress-induced autophagy enable viruses to disarm several facets of the cellular stress response to infection by one mechanism -that is, antagonism of eif2α kinase signalling. the mtor signalling pathway has a central role in the control of cell growth and metabolism, and interestingly, many of the viruses that activate mtor are oncogenic (for example, epstein-barr virus, kaposi's sarcoma-associated herpesvirus, hepatitis b virus and retroviruses). this suggests another type of pluripotent viral weapon -one that can promote oncogenesis by simultaneously inactivating autophagy and promoting cell growth through tor activation. hiv envelope protein-dependent activation of mtor signalling is also proposed to be a mechanism for hiv evasion of innate and adaptive immune responses in dendritic cells, including the degradation of incoming virions by lysosomes, blockade of hiv transfer to cd4 + t cells, stimulation of tlr4 and tlr8 ligand responses, and presentation of hiv gag antigen to cd4 + t cells 56 . it will be important to determine whether these effects of hiv-mediated mtor activation and autophagy inhibition contribute to impaired dendritic cell function during hiv infection in vivo. several viral proteins target the core autophagy protein beclin 1. autophagosome initiation is blocked by interactions between beclin 1 and the herpes simplex virus 1 (hsv-1) neurovirulence factor icp34.5 or the oncogenic γ-herpesvirus-encoded viral bcl2-like proteins, whereas autophagosome maturation is blocked by interactions between beclin 1 and the hiv accessory protein nef or the influenza virus matrix protein 2 (ref. 55 ). the interactions between beclin 1, hsv-1 icp34.5 and viral bcl2 are probably physiologically important in vivo; a mutant hsv-1 virus lacking the beclin-1-binding domain of icp34.5 is attenuated in mouse models of encephalitis (presumably through its failure to control xenophagy and innate immunity) 57 and of corneal disease (through its failure to control adaptive immunity) 58 . moreover, a mouse γ-herpesvirus that encodes a mutant viral bcl2 unable to bind to beclin 1 demonstrates impaired ability to maintain chronic infection 59 review insight beclin 1 is preferentially targeted by viral virulence proteins because of its central role in autophagosome formation, or more likely, whether we are just beginning to identify pairs of viral proteins and their autophagy pathway targets. in support of the latter, viral flice-like inhibitors encoded by kaposi's sarcoma-associated herpesvirus and molluscum contagiosum virus suppress autophagy by interacting with the atg3 e2-like enzyme, thereby preventing it from binding and processing lc3 (ref. 60) . bacteria possess diverse strategies to avoid degradation by autophagolysosomal pathways. as reviewed elsewhere 1,38 , many bacteria that reside in phagosomes or other vacuolar compartments have methods to inhibit lysosomal fusion or maturation, which, in the case of mycobacteria, can be partially overcome by treatments that stimulate autophagy. another possible mechanism for bacterial evasion of autophagy has emerged from a genome-wide screen to identify host factors that regulate the intracellular survival of m. tuberculosis 51 . according to bioinformatics analyses, m. tuberculosis infection of human macrophage-like cells activates cellular pathways that inhibit autophagy. intracellular bacteria that escape into the cytoplasm, such as s. flexneri and l. monocytogenes, use strategies to camouflage themselves to avoid autophagic recognition. the shigella t3ss effector icsb competitively binds to virg, thereby preventing the interaction between atg5 and virg, a bacterial surface protein required for actin-based motility and shigella targeting to autophagosomes 38 . the listeria protein acta interacts with cytosolic actin polymerization machinery (arp2/3, vasp and actin), which blocks bacterial association with ubiquitin, p62 recruitment and autophagic targeting 61 . the precise mechanism of this block is unknown, but it has been proposed that the acta-dependent recruitment of host cell cytoskeletal proteins may enable the bacterium to disguise itself as a host cell organelle 61 . this concept sheds light on the autophagy pathway in a fundamental aspect of immunology -the basis for discrimination between self and non-self. microbes have evolved not only to antagonize autophagy (as a cellular defence mechanism that threatens their survival), but also to exploit its components and functions in membrane trafficking for their own self-serving purposes 1,55 . an early concept in the field is that autophagosomes may serve as a protected niche for intracellular bacteria (provided fusion with acidic compartments is blocked) and/or serve as a source of nutrients for intracellular pathogens (which would require intact autophagolysosomal fusion) 1 . trafficking of the intracellular bacteria yersinia pseudotuberculosis to acidic compartments was recently shown to be enhanced by genetic inhibition of autophagy 62 . this seemingly contradicts other evidence that the autophagy proteins promote phagosomal maturation, but is consistent with the concept that autophagosomes function as a protected intracellular niche for bacteria. the role of the autophagy machinery in promoting and/or inhibiting vacuolar acidification -and the counter effects of microbes that reside in vacuolar compartments -needs to be explored further. the function of autophagy proteins in membrane formation and/ or trafficking is exploited by numerous viruses, including poliovirus, rota virus, hiv, coronaviruses, dengue virus, and the hepatitis b and c viruses 55 . autophagosomes (defined as lc3-positive membranes, see caveat below) may act as a scaffold for intracellular membrane-associated replication of certain cytoplasmic rna viruses 55 . autophagy may assist in hiv biogenesis, because the processing of the hiv envelope precursor protein gag and extracellular viral release are enhanced by the autophagy machinery 63 . similarly, autophagy proteins are required for maximal levels of poliovirus egress 55 . another newly defined proviral function of autophagy is its role in productive hepatitis c virus replication; several different autophagy proteins (such as beclin 1, atg4b, atg5, atg7 and atg12) assist in the translation of incoming, but not progeny, viral rna 64 . atg7 and class iii pi(3)k activity also enhance hepatitis b virus dna replication 65 . the mechanisms by which autophagy proteins facilitate the replication and/or egress of certain viruses are not yet understood. some observations may relate to the role of autophagy proteins in remodelling the er (vis-à-vis viral replication) or the role of autophagosomes in fusing with multivesicular bodies (vis-à-vis viral egress). it is possible that autophagy proteins function to provide membrane for viral replication complexes or translation machinery. this may be true for viruses such as hepatitis c virus, for which genetic knockdown of several different autophagy genes decreases productive replication 64 . however, for other viruses such as coronaviruses, the biogenesis of double-membrane, er-derived vesicles used for replication proceeds through a pathway that involves the nonlipidated form of lc3 (lc3-i) but not the general autophagy machinery 66 . thus, caution must be exercised in interpreting the significance of the co-localization (or biochemical interaction) of viral proteins and lc3, as lc3 may have autophagy-independent roles in membrane dynamics. the central importance of autophagy in immunity is further underscored by the multitude of immune-related signalling molecules that regulate autophagy. as reviewed in detail elsewhere [2] [3] [4] 38 , autophagy is induced by different families of pathogen-recognition receptors (such as tlrs, nod-like receptors and the double-stranded rna-binding protein pkr), damps (such as atp, ros and misfolded proteins), pathogen receptors (such as cd46), ifn-γ and downstream immunityrelated gtpases, and dap kinase, jnk, cd40, tumour necrosis factor-α (tnf-α), inhibitor of nf-κb (ikk) and nf-κb ( fig. 1) . high mobility group box (hmgb) proteins have also been shown to function as both universal sensors of nucleic acids in innate immune signalling 67 and inducers of autophagy 68 . autophagy is inhibited by bcl2, nf-κb, t helper 2 (t h 2) cytokines and the canonical nutrient-sensing insulin-akt-tor pathway. inactivation of this nutrient-sensing pathway may contribute to vesicular stomatitis virus stimulation of autophagy in drosophila 46 , and autophagy activation in c. elegans with loss-of-function mutations in this pathway may mediate pathogen resistance in long-lived mutant nematodes 48 . thus, both 'immune-specific' and more general nutrient-response signals control autophagy in response to infection. studies with vitamin d3 have uncovered a possible link between nutrition, innate immunity and the control of autophagy during mycobacterial infection. low vitamin d3 levels are associated with increased susceptibility to tuberculosis. vitamin d3 generates an antimycobacterial peptide, cathelicidin, and induces autophagy and mycobacterial killing in human monocytes through cathelicidin-dependent effects 69 . although cathelicidin is required for vitamin-d3-dependent transcriptional upregulation of autophagy genes such as becn1 and atg5, and vitamin d3 enhances the recruitment of cathelicidin to autophagosomes, it is not yet clear how cathelicidin promotes autophagy. nonetheless, these observations may begin to provide some insight into the century-old nobel prize award (niels ryberg finsen, 1903) for the use of ultraviolet-light therapy (which generates active vitamin d3) in the treatment of diseases such as tuberculosis. in most instances, the mechanisms of autophagy control by immunerelated signalling molecules are not understood. however, there are some examples of specific interactions between immune signals and autophagy proteins that may be relevant to these mechanisms. for example, the interaction between beclin 1 and bcl2 (which inhibits its activity) is thought to be disrupted by the tlr adaptors myd88 and trif, as well as by hmgb1, which bind to beclin 1 and displace bcl2 (refs 3, 68) . two intracellular sensors responsible for inducing autophagy in response to bacterial infection, nod1 and nod2, interact with atg16l1 and recruit it to the plasma membrane, resulting in enhanced association of invasive bacteria (s. flexneri) with lc3 (ref. 53 ). interestingly, a nod2 mutation associated with crohn's disease impairs atg16l1 plasma membrane recruitment and bacterial co-localization with lc3 (ref. 53 ). the identification of other possible protein-protein interactions between core autophagy proteins and immune signals by a large proteomics screen 21 has the potential to foster further advances in understanding how different immune signals regulate the autophagy machinery. for example, tectonin proteins with multivalent 2 0 j a n u a r y 2 0 1 1 | v o l 4 6 9 | n a t u r e | 3 2 9 review insight β-propeller folds are known to function in pathogen recognition and innate immunity in invertebrates 70 . thus, the interactions between previously uncharacterized human proteins of this tectonin family, tecpr1 and tecpr2, with the atg5-atg12-atg16l1 complex and atg8 family members, respectively 21 , may contribute to pathogen-induced autophagy stimulation or selective autophagic targeting of pathogens in mammals. further links between immune signalling molecules and autophagy regulation were suggested by a genome-wide short interfering rna screen 71 . the analysis identified 219 genes that suppressed basal autophagy, largely in a mammalian tor complex 1 (mtorc1)independent fashion. these included several cytokines such as clcf1, lif, igf1, fgf2 and the chemokine sdf1 (also known as cxcl12), as well as cellular signalling molecules regulated by cytokines such as stat3. these findings raise the possibility that cytokines may have a broader role in the control of autophagy than previously thought. moreover, because these cytokine signalling pathways are important in immune cells, another central question is to what extent cytokinemediated regulation of autophagy governs immune cell function. given the general function of autophagy in cellular homeostasis 5 , and the more specific functions in regulating immune and inflammatory signalling (discussed in 'regulation of immune signalling by autophagy proteins'), cytokine-mediated changes in autophagy levels in immune cells may have a central role in immunity and inflammation. autophagy proteins function in adaptive immunity, including in the development and homeostasis of the immune system and in antigen presentation (table 1 and fig. 3) . the knockout of different autophagy genes in specific lymphocyte populations in mice has shown a crucial role for autophagy proteins in the maintenance of normal numbers of b1a b cells, cd4 + t cells, cd8 + t cells and fetal haematopoietic stem cells 2, 44, 72 . in t cells, in which mitochondrial numbers are developmentally regulated during the transition from thymocyte to mature circulating t cell, the developmental defect in autophagy-deficient cells may be related to the defective clearance of mitochondria 44 . another crucial function of autophagy in the development and homeostasis of the immune system is the elimination of autoreactive t cells in the thymus 44 . high levels of autophagy are present in thymic epithelial cells, in which autophagy participates in the delivery of self-antigens to mhc class ii loading compartments. genetic disruption of atg5 in thymic epithelial cells leads to the altered selection of certain mhc class ii restricted t-cell specificities and autoimmunity 73 . beyond these functions in lymph ocyte survival and thymic negative selection, autophagy may exert other functions in lymphocyte differentiation, perhaps, in part, indirectly through effects on cytokine expression (see the next section). it is not yet known whether autophagy is involved in the development and/or homeostasis of immune cell populations other than lymphocytes and haematopoietic stem cells. autophagy proteins may participate in different facets of antigen presentation, including the delivery of endogenous antigens for mhc class ii presentation to cd4 + t cells 74, 75 , the enhancement of antigen donor cell cross-presentation to cd8 + t cells 75 , dendritic cell crosspresentation of phagocytosed antigens to cd4 + t cells 32 and, in one report, mhc class i presentation of intracellular antigens to cd8 + t cells 27 . the discovery that autophagosomes could deliver endogenous antigens to mhc class ii loading compartments sheds light on one of the central mysteries of antigen presentation -how the immune system elicits cd4 + t-cell responses to antigens that originate in all parts of the cell. the autophagic delivery of endogenously synthesized antigens for mhc class ii presentation has been demonstrated in vitro for certain viral antigens 75 , and probably explains the essential role of atg5 in vivo in negative thymic selection 73 . however, the relative importance of this pathway in antigen presentation during infection in vivo is not yet known. there is nonetheless interest in exploiting this pathway for optimizing vaccine-elicited cd4 + t-cell responses, by either pre-treating dendritic cells with autophagy-inducing agents in cell-based vaccine strategies or fusing antigens with lc3 to enhance their targeting to autophagosomes 1 . of note, autophagy proteins are required for antigen cross-presentation during infection in vivo 32 . dendritic-cell-specific deletion of atg5 in mice results in defects in priming cd4 + t-cell responses after hsv and listeria infections, and mice succumb more rapidly to lethal disease after intravaginal hsv infection. atg5-deficient dendritic cells have normal migration, innate responses, endocytic and phagocytic activity and cross-presentation of peptides on mhc class i molecules. however, they exhibit defects in phagosome-to-lysosome fusion and in cross-presentation by mhc class ii molecules of phagocytosed antigens containing tlr ligand. thus, the interior of the phagosome, like that of the autophagosome, is a cellular compartment that autophagyprotein-dependent antigen presentation accesses to generate peptides for presentation to cd4 + t cells. a potential evolutionary advantage of this autophagy-protein-dependent cross-presentation is that, by delegating antigen presentation duties to uninfected dendritic cells, the host can bypass the blockade of antigen presentation that may result from microbial antagonism of autophagy in infected cells. in response to infection, the host must activate those arms of the innate and adaptive immune system (including autophagy-dependent functions; fig. 3 ) that help to control infection while, in parallel, triggering specific responses that limit detrimental, uncontrolled immune activation and inflammation. an exciting new frontier in autophagy research is the growing recognition of the function of autophagy proteins in achieving this balance (fig. 4) . autophagy proteins function in both the activation and inactivation of innate immune signalling 4 . the autophagy pathway activates type i ifn production in plasmacytoid dendritic cells by delivering viral nucleic acids to endosomal tlrs 76 . by contrast, autophagy proteins negatively regulate rig-i-like receptor (rlr)-mediated induction of type i ifn production through the autophagic elimination of damaged mitochondria (and reduction of ros) 77 , and by the binding of atg5-atg12 to caspase recruitment domains of rlr signalling molecules 78 . moreover, the autophagy protein atg9a, but not atg7, negatively regulates the activation of sting, a transmembrane protein that is required for efficient activation of type i ifn and pro-inflammatory cytokine production in response to stimulatory dna 23 . thus, it seems that autophagy proteins can negatively regulate ifn production by both autophagy-dependent and -independent mechanisms. with respect to the latter, different autophagy proteins may be specialized to target different innate immune signalling molecules. the autophagy pathway and/or proteins also have a crucial role in the control of inflammatory signalling. a major effect is on the regulation of inflammatory transcriptional responses. increased levels of the adaptor protein p62, which accumulates in autophagy-deficient cells, activate the pro-inflammatory transcription factor nf-κb through a mechanism involving traf6 oligomerization 79 . the accumulation of p62 in atg7-deficient hepatocytes results in enhanced activity of the stress-responsive transcription factor nrf2 and nrf2-dependent liver injury 80 . in addition, paneth cells (intestinal immune epithelial cells) from mice hypomorphic for atg16l1 (atg16l1 hm ) show enhanced transcription of pro-inflammatory cytokines and adipokines 81 . a second important effect of autophagy proteins on inflammatory signalling is at the level of the inflammasome. this complex contains nod-like receptor cryopyrin proteins, the adaptor protein asc and caspase 1, and is activated by cellular infection or other stress to promote the maturation of pro-inflammatory cytokines such as interleukin-1β (il-1β) and . atg16l1-or atg7deficient mouse macrophages produce increased levels of mature il-1β and il-18 after tlr4 stimulation by endotoxin 82 . in addition, mouse chimaeras engrafted with atg16l1 −/− fetal liver haematopoietic progenitors have increased serum concentrations of il-1β and il-18 3 3 0 | n a t u r e | v o l 4 6 9 | 2 0 j a n u a r y 2 0 1 1 after treatment with dextran sodium sulphate (dss), which contributes to increased dss-induced colitis 82 . the mechanism(s) by which autophagy proteins negatively regulate inflammasome activation are not yet understood. mutually nonexclusive possibilities include direct interactions between autophagy proteins and inflammasome components, indirect inhibition of inflammasome activity through autophagic suppression of ros accumulation, or autophagic degradation of danger signals that activate the inflammasome. in line with the latter model, the autophagic degradation of amyotrophic-lateral-sclerosis-linked mutant superoxide dismutase has been proposed to limit caspase 1 activation and il-1β production 83 . in addition to regulating inflammatory signalling, the autophagy pathway may prevent tissue inflammation through its role in apoptotic corpse clearance. the efficient clearance of apoptotic corpses during development and tissue homeostasis prevents secondary necrosis, which releases danger signals (damps) that trigger inflammation. autophagy genes are essential for the heterophagic clearance of dying apoptotic cells during developmental programmed cell death (by the generation of atp-dependent engulfment signals) 84 , and the retinas and lungs of embryonic mice lacking atg5 have a defect in apoptotic corpse engulfment that is associated with infiltration of inflammatory cells 84 . on the basis of growing evidence that autophagy proteins function in tlrmediated phagolysosomal pathways, it is possible that autophagy also functions in phagocytes to facilitate apoptotic corpse clearance. thus, in tissues such as the intestine, in which physiological regeneration involves continuous shedding or apoptosis of epithelial cells, autophagydependent functions in dying cells and/or phagocytic cells may promote efficient corpse clearance, thereby limiting inflammation. perturbations in autophagy-protein-dependent functions in immunity may contribute not only to increased susceptibility to infection, but also to chronic inflammatory diseases and autoimmune diseases. the only well-characterized link thus far is between mutations in autophagy regulators and crohn's disease, a chronic inflammatory disorder of the small intestine, in which a breakdown in clearance or recognition of commensal bacteria, as well as altered mucosal barrier function and cytokine production, is thought to lead to intestinal inflammation (fig. 5) . other emerging links include the autoimmune disease systemic lupus erythematosus (sle), inflammation-associated metabolic diseases such as obesity and diabetes, and inflammation associated with cystic fibrosis lung disease (fig. 4) . the role of autophagy proteins in crohn's disease was not suspected until genome-wide association studies identified three crohn's disease susceptibility genes, irgm, nod2 and atg16l1, that are involved in autophagy 85 . the irgm risk allele contains a deletion in the promoter region of the gene that may be associated with changes in irgm protein expression and may contribute to crohn's disease, given irgm's role in autophagy-dependent control of bacterial infection 49 . however, this hypothesis has not yet been tested. the three major crohn's-diseaseassociated nod2 variants (a frameshift mutant and two missense mutants) may be loss-of-function mutants, with impaired muramyl dipeptide (mdp)-induced inflammatory signalling 86 . how the loss of function of a pro-inflammatory signal mechanistically contributes to an inflammatory disorder has been unclear, but the recently discovered links between nod2 and autophagy may solve this conundrum. in primary immature human dendritic cells, nod2 is required for mdpinduced autophagy, a process that is essential for the mhc class ii presentation of bacterial antigens to cd4 + t cells and for bacterial targeting to lysosomes 52 . dendritic cells expressing crohn's disease nod2 risk variants are defective in both of these functions 52 . thus, in patients with crohn's disease and nod2 risk variants, aberrant autophagy-dependent bacterial clearance and immune priming could act as a trigger for intestinal inflammation. a mechanistic link may also exist between atg16l1 mutation and crohn's disease pathogenesis. similar to findings with nod2 variants, dendritic cells from patients with the crohn's-disease-associated atg16l1(t300a) risk variant are defective in presenting bacterial antigen to cd4 + t cells 52 . however, it is not yet known how the t300a mutation affects the function of the mammalian atg16l1 protein. this mutation resides in the carboxy-terminal wd-repeat domain that is absent in yeast atg16 and is dispensable for autophagy. although some studies have suggested that the atg16l1(t300a) variant has reduced autophagic clearance of enteric pathogens such as adherent-invasive escherichia coli 87 or s. typhimurium 88 , it remains controversial whether the risk versus protective alleles of atg16l1 have differences in stability or antibacterial autophagic activity 89 . despite the uncertain nature of the effects of the t300a mutation on atg16l1 function, atg16l1 mutation (null or hypomorphic alleles) in mice results in abnormalities that are relevant to crohn's disease pathogenesis. as noted earlier, loss of atg16l1 function in mice results in enhanced tlr-agonist-induced pro-inflammatory cytokine production by macrophages 82 , enhanced dss-induced colitis 82, 90 and altered inflammatory gene transcriptional profiles in paneth cells 81, 90 . in addition, the paneth cells of mice expressing low atg16l1 levels (atg16l1 hm ) show defects in the packaging and extrusion of antimicrobial granules into the gut lumen; paneth cells from patients with crohn's disease and the atg16l1(t300a) risk variant show similar defects 81 . this suggests that, in addition to the overlapping functions of nod2 and atg16l1 in a common bacterial-sensing pathway that promotes bacterial antigen presentation, atg16l1 may have unique protective functions, including paneth cell antimicrobial peptide release and the negative regulation of pro-inflammatory cytokine production. to connect the striking phenotypes in atg16l1-mutant mice and the pathogenesis of crohn's disease in humans with the atg16l1(t300a) risk allele, the precise effects of the t300a mutation on atg16l1 protein function need to be uncovered. a new dimension in understanding the multifactorial basis of chronic inflammatory diseases such as crohn's disease has emerged from the discovery that a virus trigger is required to observe intestinal figure 4 | autophagy/autophagy proteins act to achieve a balance between activation and inactivation of innate immune signalling. a general model in which the levels of autophagy and autophagy proteins control disease in response to stressors. normal autophagy protein function (green) contributes to balanced inflammatory and metabolic responses, resulting in protection against disease. altered autophagy protein function (red) results in maladaptive inflammatory and metabolic responses, increased inflammation and more severe disease. abnormalities in atg16l1 hm mice 90 . in mice raised in a pathogen-free facility, only atg16l1 hm mice (and not wild-type mice) infected with a virus found in routine conventional animal facilities, a murine norovirus, showed abnormal paneth cell granule secretion, paneth cell proinflammatory gene-expression profiles, and intestinal inflammation in response to dss treatment 90 . this mucosal inflammation depended on the presence of the microbiome and pro-inflammatory cytokines, as it was reversed by antibiotic treatment or by tnf-α or ifn-γ inhibition. thus, variations in a host autophagy gene, exposure to a specific virus and the microbiome can act together to trigger intestinal inflammation in mice that is similar to that in patients with crohn's disease. although environmental factors, including the gut microbiome, have long been suspected to contribute to crohn's disease in genetically susceptible individuals, formal proof of this concept was lacking, and viruses were a previously unsuspected trigger. another implication of this work is the concept that autophagy proteins, through their diverse roles in immunity and the control of inflammation, may serve as a central rheostat that prevents inflammatory diseases triggered by environmental stress (fig. 4 ). an important unanswered question is whether perturbations in autophagy may also result in inflammatory autoimmune disease. genome-wide association studies have linked several single nucleotide polymorphisms (snps) in atg5 to sle susceptibility [91] [92] [93] . sle is a multifactorial, heterogeneous disease characterized by autoimmune responses against self-antigens generated from dying cells. although the effects of these snps on atg5 expression and function are not known, the lack of atg5-dependent negative thymic selection generates autoimmunity and multi-organ inflammation in mice 73 . loss of other atg5dependent effects, including regulation of ifn and pro-inflammatory cytokine secretion 77,78 , clearance of dying cells 84 figure 5 | the link between mutations in autophagy regulators and the chronic inflammatory disorder crohn's disease. an overview of the many possible mechanisms by which defects in autophagy and autophagy protein function may contribute to the pathogenesis of a type of inflammatory bowel disease, crohn's disease. a micrograph of a human small intestine from a patient with crohn's disease is shown (centre), demonstrating the severe transmural inflammation that is characteristic of this disease. the postulated mechanisms by which defects in autophagy protein function might contribute to the development or perpetuation of intestinal inflammation are based on studies in vitro and animal models. there is no direct evidence that autophagy defects contribute to human crohn's disease, although mutations in three autophagy-related genes, atg16l1, nod2 and irgm, are known to enhance risk of the disease. e, epithelium; igm, immunoglobulin m; l, lumen; la, lymphoid aggregates; tm, thickened muscle. scale bar, 200 µm. antigen presentation 32 , might also contribute to the autoimmunity and inflammation associated with sle. thus, a link between atg5 mutation (or mutation of other autophagy genes) and sle pathogenesis is biologically plausible, although not yet proven. defects in autophagy may contribute to inflammation-associated metabolic diseases such as diabetes and obesity, which are both linked to insulin resistance. the metabolic inflammasome -a complex composed of signalling molecules such as pkr, eif2α, jnk, irs and ikkmay act as a link between er stress and more global stress responses, inclu ding inflammation and metabolic dysfunction (as observed in insulin resistance and obesity) 94 . although most components of the metabolic inflammasome promote autophagy, the induction of autophagy by this signalling complex would be expected to serve as a negativefeedback mechanism that limits er stress and disease progression. consistent with this postulated protective effect of autophagy, hepatic suppression of the autophagy gene atg7 in mice results in increased er stress and insulin resistance 95 , and mice deficient in the autophagy adaptor protein p62 develop mature-onset obesity and insulin resistance 96 . furthermore, obesity is associated with the accumulation and activation of macrophages and subsets of t cells in adipose tissue and the production of cytokines such as tnf-α and il-6 (ref. 97) . thus, the failure of autophagy-dependent control of er stress, immune cell homeostasis, immune cell activation and/or pro-inflammatory cytokine secretion may contribute to inflammation-associated responses that underlie the pathogenesis of metabolic diseases. another potential link between autophagy deficiency and chronic inflammation is in cystic fibrosis 98 , a life-threatening genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (cftr). mutations in cftr lead to autophagy inhibition in lung epithelial cells through a mechanism that may involve ros-mediated sequestration of the beclin 1-class iii pi(3)k complex in perinuclear aggregates (redirecting it from its site of autophagy action at the er). restoration of beclin 1 and autophagy in cystic fibrosis epithelial cells rescues the disease phenotype, and antioxidants reverse the airway inflammation in a cystic fibrosis mouse model by a mechanism postulated to involve autophagy. the first series of studies demonstrating that the autophagy machinery is used to attack invading intracellular bacteria was published in 2004 (refs 30, 99, 100) . although autophagy had been observed at the ultrastructural level in cells infected with intracellular bacteria and viruses decades earlier, these studies were a seminal advance. for the first time, pharmacological and genetic manipulation of autophagy, which built on the discoveries of the yeast screens that identified the autophagy machinery, challenged the very notion of autophagy as an 'auto'-(self), 'phagy' (eating) pathway. indeed, we learned that the same genes that are used to orchestrate the degradation of self-constituents, either for nutritional/energy homeostasis or cellular damage control, are also used to orchestrate the degradation of foreign invaders, termed xenophagy. in the past few years, research in the field has uncovered new layers of complexity and functional diversity in terms of how this set of genesoriginally characterized in the context of macroautophagy -may function to protect multicellular organisms against not only the threats of infection but also the threats of the host's own response to infection. the autophagy machinery does much more than form autophagosomes to engulf microbes -it somehow allows microbes in phagosomes and vacuoles to be targeted to the lysosome; it enables crucial cells in the immune system to develop properly and perform some of their 'normal' functions (such as produce ifn, secrete antimicrobial peptides or present antigens to stimulate adaptive immunity); and it ensures that these responses do not become out of control by functioning in central immunological tolerance and the negative regulation of innate and inflammatory signalling. thus, recent advances may not only modify our understanding of immunity (in terms of understanding new roles of the autophagy machinery in immune regulation) but also reshape our understanding of the pathogenesis of inflammatory diseases (in terms of understanding how perturbations in autophagy protein function may contribute to such diseases). clearly, our understanding of the molecular mechanisms of the plethora of functions of autophagy proteins in immune-related processes is still quite primitive. we speculate that, similar to the way in which the initial genetic screens in yeast transformed autophagy research, current proteomic and genomic screens have the potential to transform research on autophagy and immunity. such a transformation would include facilitating a much deeper understanding of the molecular mechanisms of the existing known immunological functions of autophagy through the use of the tools of modern systems biology to understand autophagy protein-protein interaction and signalling regulatory networks on a broad scale. perhaps more exciting is the possibility that such a transformation will uncover new ways in which this ancient self-defence machinery can function in immunity. ■ autophagy, immunity, and microbial adaptations autophagy genes in immunity autophagy and the integrated stress response regulation of innate immune responses by autophagyrelated proteins autophagy in the pathogenesis of disease methods in mammalian autophagy research this paper provides a concise and critical review of current methods to monitor and modulate autophagy in mammalian cells antigen-loading compartments for major histocompatibility complex class ii molecules continuously receive input from autophagosomes termination of autophagy and reformation of lysosomes regulated by mtor dynamics and diversity in autophagy mechanisms: lessons from yeast a subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation 3d tomography reveals connections between the phagophore and endoplasmic reticulum the role of the atg1/ulk1 complex in autophagy regulation characterization of autophagosome formation site by a hierarchical analysis of mammalian atg proteins two beclin 1-binding proteins, atg14l and rubicon, reciprocally regulate autophagy at different stages autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum mammalian atg18 (wipi2) localizes to omegasome-anchored phagophores and positively regulates lc3 lipidation the pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells c. elegans screen identifies autophagy genes specific to multicellular organisms the atg16l complex specifies the site of lc3 lipidation for membrane biogenesis in autophagy lc3 and gate-16/gabarap subfamilies are both essential yet act differently in autophagosome biogenesis network organization of the human autophagy system atg12 conjugation to atg3 regulates mitochondrial homeostasis and cell death atg9a controls dsdna-driven dynamic translocation of sting and the innate immune response new insights into the function of atg9 mitochondria supply membranes for autophagosome biogenesis during starvation plasma membrane contributes to the formation of pre-autophagosomal structures autophagy enhances the presentation of endogenous viral antigens on mhc class i molecules during hsv-1 infection selective autophagy: ubiquitin-mediated recognition and beyond an initial step of gas-containing autophagosome-like vacuoles formation requires rab7 autophagy defends cells against invading group a streptococcus together with reference 99, provides the first evidence that autophagy has a key role in bacterial infection. atg5 is shown to be essential for controlling the replication of group a streptococci that escape into the cytoplasm toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis in vivo requirement for atg5 in antigen presentation by dendritic cells this study shows that the autophagy machinery is necessary for dendritic cells to process and present extracellular microbial antigens for mhc class ii presentation in vivo, which protects mice against lethal viral infection slam is a microbial sensor that regulates bacterial phagosome functions in macrophages activation of antibacterial autophagy by nadph oxidases autophagosome-independent essential function for the autophagy protein atg5 in cellular immunity to intracellular pathogens coordinated loading of irg resistance gtpases on to the toxoplasma gondii parasitophorous vacuole disruption of the toxoplasma gondii parasitophorous vacuole by ifnγ-inducible immunity-related gtpases (irg proteins) triggers necrotic cell death autophagy and innate immunity: triggering, targeting and tuning a diacylglycerol-dependent signaling pathway contributes to regulation of antibacterial autophagy autophagy protects against sindbis virus infection of the central nervous system shigella phagocytic vacuolar membrane remnants participate in the cellular response to pathogen invasion and are regulated by autophagy delivery of cytosolic components by autophagic adaptor protein p62 endows autophagosomes with unique antimicrobial properties contrôle génique de la multiplication du virus de la sensibilité héréditaire au co 2 chez drosophila melanogaster. caryologia (suppl autophagy in mammalian development and differentiation autophagy regulates programmed cell death during the plant innate immune response autophagy is an essential component of drosophila immunity against vesicular stomatitis virus autophagic control of listeria through intracellular innate immune recognition in drosophila autophagy genes protect against salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance human irgm induces autophagy to eliminate intracellular mycobacteria autophagy gene variant irgm -261t contributes to protection from tuberculosis caused by mycobacterium tuberculosis but not by m. africanum strains genome-wide analysis of the host intracellular network that regulates survival of mycobacterium tuberculosis nod2 stimulation induces autophagy in dendritic cells influencing bacterial handling and antigen presentation nod1 and nod2 direct autophagy by recruiting atg16l1 to the plasma membrane at the site of bacterial entry genomewide association study of leprosy viruses and the autophagy machinery human immunodeficiency virus-1 inhibition of immunoamphisomes in dendritic cells impairs early innate and adaptive immune responses hsv-1 icp34.5 confers neurovirulence by targeting the beclin 1 autophagy protein interaction of icp34.5 with beclin 1 modulates herpes simplex virus type 1 pathogenesis through control of cd4 + t cell responses viral bcl-2-mediated evasion of autophagy aids chronic infection of γherpesvirus 68 flip-mediated autophagy regulation in cell death control listeria monocytogenes acta-mediated escape from autophagic recognition autophagosomes can support yersinia pseudotuberculosis replication in macrophages autophagy pathway intersects with hiv-1 biosynthesis and regulates viral yields in macrophages the autophagy machinery is required to initiate hepatitis c virus replication this study shows that many autophagy proteins are essential for the initial stages of hepatitis c viral rna translation, illustrating that autophagy proteins may be subverted to enhance the replication of intracellular pathogens the early autophagic pathway is activated by hepatitis b virus and required for viral dna replication coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication hmgb proteins function as universal sentinels for nucleic-acidmediated innate immune responses endogenous hmgb1 regulates autophagy vitamin d3 induces autophagy in human monocytes/ macrophages via cathelicidin a novel human tectonin protein with multivalent β-propeller folds interacts with ficolin and binds bacterial lps a genome-wide sirna screen reveals multiple mtorc1 independent signaling pathways regulating autophagy under normal nutritional conditions fip200 is required for the cell-autonomous maintenance of fetal hematopoietic stem cells autophagy in thymic epithelium shapes the t-cell repertoire and is essential for tolerance this study provided the first evidence that the autophagy machinery functions in mhc class ii antigen presentation in vivo, specifically in shaping the cd4 + t-cell repertoire during negative thymic selection, and thereby preventing autoimmunity and multi-organ inflammation endogenous mhc class ii processing of a viral nuclear antigen after autophagy this study provided the first evidence that the autophagy machinery can deliver endogenously synthesized antigens for presentation on mhc class ii molecules to cd4 + t cells antigen processing via autophagy-not only for mhc class ii presentation anymore? autophagydependent viral recognition by plasmacytoid dendritic cells absence of autophagy results in reactive oxygen speciesdependent amplification of rlr signaling the atg5-atg12 conjugate associates with innate antiviral immune responses m. t. p62 at the crossroads of autophagy the selective autophagy substrate p62 activates the stress responsive transcription factor nrf2 through inactivation of keap1 a key role for autophagy and the autophagy gene atg16l1 in mouse and human intestinal paneth cells this study demonstrates that autophagy proteins negatively control endotoxin-induced inflammasome activation. references 81 and 82 also show that autophagy gene deficiency increases susceptibility to experimentally induced inflammatory bowel disease mutant superoxide dismutase 1-induced il-1β accelerates als pathogenesis autophagy gene-dependent clearance of apoptotic cells during embryonic development this paper shows that autophagy genes are necessary for apoptotic cells to generate engulfment signals required for successful apoptotic corpse clearance and the prevention of tissue inflammation genome-wide association defines more than 30 distinct susceptibility loci for crohn's disease nod2-mediated autophagy and crohn disease crohn's disease-associated adherent-invasive e. coli are selectively favoured by impaired autophagy to replicate intracellularly impaired autophagy of an intracellular pathogen induced by a crohn's disease associated atg16l1 variant differential involvement of atg16l1 in crohn disease and canonical autophagy: analysis of the organization of the atg16l1 complex in fibroblasts this study shows that both hypomorphic expression of an autophagy protein and a viral-infection trigger are necessary for experimentally induced inflammatory bowel disease, suggesting that the interaction between host defects in autophagy and environmental stressors such as infection may be crucial for the pathogenesis of certain inflammatory diseases genome-wide association scan in women with systemic lupus erythematosus identifies susceptibility variants in itgam, pxk, kiaa1542 and other loci a large-scale replication study identifies tnip1, prdm1, jazf1, uhrf1bp1 and il10 as risk loci for systemic lupus erythematosus genome-wide association study in a chinese han population identifies nine new susceptibility loci for systemic lupus erythematosus double-stranded rna-dependent protein kinase links pathogen sensing with stress and metabolic homeostasis defective hepatic autophagy in obesity promotes er stress and causes insulin resistance mature-onset obesity and insulin resistance in mice deficient in the signaling adapter p62 inflammation and metabolic disorders defective cftr induces aggresome formation and lung inflammation in cystic fibrosis through ros-mediated autophagy inhibition autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages autophagy induction by ifn-γ, rapamycin or starvation results in the conversion of mycobacterial phagosomes into phagolysosomes, thereby enhancing mycobacterial killing escape of intracellular shigella from autophagy the work in the authors' laboratories was supported by national institutes of health (nih) grants ro1 ca109618 and u54 ai057156 (b.l.); by grants-in-aid for scientific research from the ministry of education, culture, sports, science and technology, japan, and by the takeda science foundation (n.m.); and by nih grants ro1 ai054483, u54 ai057160, ro1 ai084887 and ro1 ca096511 and the broad medical foundation (h.w.v.). we thank t. stappenbeck for discussions, and a. diehl and m. harstein for scientific illustration. we apologize to those authors whose work could not be cited owing to space limitations. key: cord-334220-sqvfr31q authors: messina, francesco; giombini, emanuela; montaldo, chiara; sharma, ashish arunkumar; piacentini, mauro; zoccoli, antonio; sekaly, rafick-pierre; locatelli, franco; zumla, alimuddin; maeurer, markus; capobianchi, maria r.; lauria, francesco nicola; ippolito, giuseppe title: looking for pathways related to covid-19 phenotypes: confirmation of pathogenic mechanisms by sars-cov-2 host interactome date: 2020-11-03 journal: biorxiv doi: 10.1101/2020.11.03.366666 sha: doc_id: 334220 cord_uid: sqvfr31q in the last months, many studies have clearly described several mechanisms of sars-cov-2 infection at cell and tissue level. host conditions and comorbidities were identified as risk factors for severe and fatal disease courses, but the mechanisms of interaction between host and sars-cov-2 determining the grade of covid19 severity, are still unknown. we provide a network analysis on protein–protein interactions (ppi) between viral and host proteins to better identify host biological responses, induced by both whole proteome of sars-cov-2 and specific viral proteins. a host-virus interactome was inferred on published ppi, using an explorative algorithm (random walk with restart) triggered by all the 28 proteins of sars-cov-2, or each single viral protein one-by-one. the functional analysis for all proteins, linked to many aspects of covid-19 pathogenesis, allows to identify the subcellular districts, where sars-cov-2 proteins seem to be distributed, while in each interactome built around one single viral protein, a different response was described, underlining as orf8 and orf3a modulated cardiovascular diseases and pro-inflammatory pathways, respectively. finally, an explorative network-based approach was applied to bradykinin storm, highlighting a possible direct action of orf3a and ns7b to enhancing this condition. this network-based model for sars-cov-2 infection could be a framework for pathogenic evaluation of specific clinical outcomes. we identified possible host responses induced by specific proteins of sars-cov-2, underlining the important role of specific viral accessory proteins in pathogenic phenotypes of severe covid-19 patients. whilst covid-19 predominantly affects the respiratory system, it is a multisystem disease, with a wide spectrum of clinical presentations from asymptomatic, mild and moderate, to severe, fulminant disease (1) . host conditions and comorbidities such as high age, obesity, diabetes, hypertension, organ damages, inflammation and coagulation dysfunctionality, were identified as risk factors for severe and fatal disease courses (2) , but the mechanisms of interaction between host and sars-cov-2 that activate pathological pathways and determine severity, are still unknown. in last months, many studies clearly described several mechanisms of sars-cov-2 infection at cell and tissue level. it was observed that the replication of sars-cov-2, as well as all +rna viruses, occurs in the cytoplasm of the host cell, inducing a membrane rearrangement of rough endoplasmic reticulum (er) membranes into double-membrane vesicles (3, 4) . moreover, nsp8 along with nsp7 and nsp12, which yield the rna polymerase activity of nsp8, are assembled into the replicase-transcriptase complex, that begins to generate anti-sense (-) genomic rna molecules, templates for positive-sense genome (+) and mrna transcripts (5, 6) . during virus entry, a well-known process is the cleavage of s protein by furin on the cell membrane, which lead to split s protein into two subunits, s1 and s2, which the last can interact with ace2 (7, 8) . however, the sars-cov-2-host interaction is not restricted to local infection, but it triggers a systemic reaction, including the activation of the bradykinin storm, as described in many severe covid-19 patients (9) . indeed, sars-cov-2 infection causes from one side a decrease of ace level in the lung cells and, on the other side, an increase of ace2 level, leading to increase bradykinin level (9) . furthermore, microvascular injuries, due to systemic inflammatory response and endothelial dysfunction, were frequently found in severe covid-19 patients (10) . increased circulating d-dimer concentrations, reflecting pulmonary vascular bed thrombosis with fibrinolysis, and elevated cardiac enzyme concentrations, linked to emergent ventricular stress induced by pulmonary hypertension, were associated to early features of severe pulmonary intravascular coagulopathy related to covid-19 (11) . moreover, myocardial injuries were found to be linked to the risk of fatal outcome in covid-19 patients (12) (13) (14) . in 43% severe patients (21% of all covid-19 patients) the myocardial injury was observed and a severe patient had a 4.74-fold increase in the risk of myocardial injury than non-severe patients (15) . although microvascular injuries and microthrombi formation frequently occurred in severe covid-19 patients, the role of sars-cov-2 in this phenotype is not completely explained yet. recently, results of a meta-analysis study on interleukin-6 concentrations in patients with severe or critical covid-19, pointed out that the systemic inflammatory profile of covid-19 is distinct from that of non-covid-19 ards, sepsis, and car t cell-induced cytokine release syndrome, and it might be involved in organ dysfunction in covid-19, such as endovasculitis, direct viral injury and viral-induced immunosuppression (16) . although many aspects of covid-19 pathogenesis and mechanisms of sars-cov-2 have been investigated, only few papers describe the interactions among sars-cov-2 proteins by wet experiments. physical associations in human cells, between sars-cov-2 proteins and human proteins, were found by affinitypurification mass spectrometry (ap-ms), identifying 332 high-confidence sars-cov-2-human proteinprotein interactions (ppis), highlighting an activation of innate immune pathways (17) . then, the influence of sars-cov-2 on transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line, was described through a multi-omic approach. such a multilevel representation highlighted autophagy mechanisms regulation by orf3a and nsp6, the modulation of innate immunity by m, orf3a and ns7b, and the integrin-tgf-β-egfr-rtk signalling perturbation by orf8 of sars-cov-2 (18). virus-host interactome by computational approach has been applied to covid-19 for drug repurposing (17) , allowing the identification of new drug targets (19) and contributing to explain clinical manifestations (20, 21) . the structural information on sars-cov-2 proteins and their interactions with human proteins and other viral proteins, allowed to better understand the mechanisms of sars-cov-2 infection, also comparing it with sars-cov (22) . the interactome based on ppi and gene expression data, have been applied also to uncover the molecular origins of phenotypes of other complex diseases (24, 25) , in order to better define the mechanisms of covid-19 pathogenesis. the understanding of all mechanisms of sars-cov-2 infection also passes by overall visualization of biological reactions and pathways involved in covid-19 and h-cov infections. it can carry out on a disease map, such as covid-19 disease maps, containing many diagrams about host molecular response during the infection (26) (27) (28) . in this context, defining the host response induced by specific viral proteins would be of great importance and can guide the identification of functional viral targets, helping to better define the pathologic phenotypes of the infection. here, we carry out a network analysis on ppi to better identify host biological response induced by sars-cov-2. furthermore, the interactome analysis was applied to design network of sars-cov-2 -host proteins that could lead to a bradykinin storm. we used three different applications to identify interacting proteins: 200 proteins that interact closely with 28 sars-cov-2 proteins, 50 protein interactomes around each sars-cov-2 protein and 200 protein associated with kng1, the pre-cursor of bradykinin (bk). in all three models, pathways associated with proteins were identified, including dna damage/repair, tgf-β signalling, complement cascades ( figure 1 ). to define the massive effect of sars-cov-2 infection on the host cell, an interactome between the entire set of sars-cov-2 proteins and host was carried out. observing the colours' distribution, it is possible to distinguish three different areas, corresponding to as many subcellular districts, where sars-cov-2 proteins seem to be distributed (figure 2 a). in fact, n, nsp1, nsp4 and nsp15 are posed around nuclear proteins, while s, along with nsp5, nsp10, nsp12, nsp13, nsp14, nsp16, orf1a and orf9b, were among cytosolic and membrane proteins, at the bounder of the interactome. protein m and many accessory and not-structural proteins (ns7b, nsp6, nsp7, orf3a, orf7a, orf8, orf10 and orf14) are around mitochondrial and endoplasmic proteins. we described the high values of betweenness centrality and degree for 9 host proteins: two proteins multiorganelle amyloid-beta precursor protein (app) and dual-specificity protein phosphatase (pten); the membrane protein sodium/potassium-transporting atpase subunit alpha-1 (atp1a1); two cytoplasmic proteins, 14-3-3 protein theta (ywhaq) and ubiquitin (ubc); two proteins of endoplasmic reticulum and golgi apparatus, sarcoplasmic/endoplasmic reticulum calcium atpase 2 (atp2a2) and unconventional myosin-vi (myo6); the mitochondrial atp synthase subunit alpha (atp5f1a); one nuclear receptor for export of rnas, exportin-1 (xpo1), suggesting their main role in this infection (sfigure 1 , stable 1 ). in order to further dissect the interactions with the entire proteome of sars-cov-2, enrichment analysis was carried out with wikipathways and kyoto encyclopaedia of genes and genomes (kegg) databases. wikipathways gene enrichment analysis revealed biological pathways of dna replication, ubiquitination and proteasome, with high significance (fdr <0.01%). in addition, kegg pathway enrichment analysis revealed dna and rna replication pathways as the most significant pathways (fdr <0.01%), as well as signalling pathways and viral infection pathways (figure 2 b) . to define the effect of specific viral proteins on host response, interactomes were built, choosing one specific viral protein as seed, imposing to find the 50 closest proteins. these analyses allowed to produce 28 restricted interactomes, which defined the strictest biological interactions associated to seed proteins both among human proteins and other viral proteins. in sfigure for kegg database the gene enrichment analysis on interactomes of ns7b, orf1a, orf3a and orf8 showed pathway clusters highly significant and consistent with possible pathogenic mechanisms, such as the activation of the complement and of the coagulative cascade, (29) and the tgf-β-dominated immune response (30) . in particular, the ns7b interactome revealed snare interactions in vesicular transport pathway, describing the mechanisms of intracellular vesicle trafficking and secretion, as the most significant pathway among all interactome (fdr< 0. 00001%). the orf1a interactome revealed a cluster of pathways, composed by as the virus-host interactome has been efficient to describe the response to specific viral proteins, this method was applied to reconstruct the possible involvement of sars-cov-2 proteins in triggering the bradykinin storm during the infection. in this case, kininogen-1 (kng1), precursor of bradykinin made by proteolysis cleavage, was considered as seed for rwr, imposing 200 closest proteins as limit to stop the algorithm. the resulting interactome showed ns7b, orf3a, orf8 and s as proximal to the seed protein, suggesting their role in the modulation of biological processes around kininogen-1. indeed, it would provide a direct influence of in this study, we built a functional interactome between sars-cov-2 and human proteome through rwr algorithm, to identify biological mechanisms and cell responses during sars-cov-2 infection and to propose a simulated model of infection contributing to a better understanding of covid-19 pathogenesis. districts. the high likelihood of our model to in vivo real mechanisms strengths the representative power of the interactome and the explorative algorithm to simulate biological processes. moreover, the proximity among specific viral proteins into the network, such as nsp7, nsp8, nsp9, nsp10, nsp12, nsp13, nsp14 involved in the viral rna replication, can be a good way to investigate their possible function in viral infection. the closeness among s, nsp5, nsp16, orf9b in the network is difficult to interpret, but s, nsp5 and orf9b, along with n, had significant positive responses of igg antibody in sera of covid-19 patients (31) . in sars-cov infected cell culture, the location of nsp2 in the cytoplasm and to some extent in the nucleus, as well as orf3a, orf7b, orf6 and m in er, seems to be consistent with their location among nuclear and cytoplasmatic, and er proteins respectively (32). the involvement of proteasome and ubiquitination pathways, along with rna replication, represent the principal pathways activated for assembly and replication of sars-cov-2 (18) . in fact, strong increases in rna-modifying proteins were revealed in cell culture after infection with sars-cov-2 (33) . the ubiquitin proteasome system deletes viral proteins to control the infection, but the virus can use them for its propagation (34) . the interactomes, built around a single protein of sars-cov-2, allowed to draw effects on cell, involving specific pathways. vesicular transport mechanism by snare interactions, identified for ns7b, is used by pathogens to penetrate host cells through their membranes and in particular in sars-cov (35, 36) . to study the pathogenesis of covid-19 in systemic context, bradykinin storm was investigated by the interactome approach. we showed ns7b and orf3a interacting with ece1, which can inactivate bk (40) . patients and reinforce the role of orf3a to enhance the bradykinin dysregulation. these host-virus interactions could enhance a better viral fitness during the infection, as suggested by the interaction already observed with sars-cov between ns7b in ece1(18). bradykinin storm in covid-19: the locking of ece1 activity due to ns7b and especially orf3a interaction, could reduce the activation of the vasoconstrictor endotelin-1, and amplify the vasodilatation effect of bk. the ece1 gene is much more expressed in all the tissues than ace2 and ace, especially in the lungs, where ece1 is 128 fold and 3.8 fold expressed compared to ace2 and ace, respectively (41) . similar to the angiotensin peptides, bk is produced from an inactive pre-protein kininogen-1 through the activation by the serine protease kallikrein (42) . furthermore, the excess of bk can lead to vasodilatation, hypotension and hypokalaemia (9, 43) , which is associated with arrhythmia (44, 45) . all these clinical conditions have been widely reported in covid-19 patients (14, 46, 47) . it is also notable that many of the other symptoms reported for covid-19 (myalgia, fatigue, nausea, vomiting, diarrhoea, anorexia, headaches, decreased cognitive function) are remarkably similar to other hyper-bk-conditions that lead to vascular hyperpermeabilization (48) . moreover, the activation of bk is strongly linked to coagulation: active f12 activates plasma kallikrein (pk), that liberates bradykinin by cleavage (49) . the direct interactions between there are many experimental platforms for deriving such physical interactions, such as affinity purification mass-spectrometry (ap-ms) and yeast-two-hybrid (y2h), which enable the accurate identification of interactions with a relatively long time. the scenario reported in this study refers to few experimental data available on public databases and could be different respect to real phenotypes of covid-19 patients. the pathways' analysis did not consider tissue and cell type diversity. finally, the low threshold established for the number of nodes found by rwr (200) limited the reconstruction of the entire pathways. however, this was a software-imposed threshold. although such network-based approach showed great potential in identifying mechanisms not yet observed, experimental tests will be necessary to confirm what we have described. we developed a network-based model for sars-cov-2 infection, which could be a framework for pathogenic evaluation of specific clinical outcomes. here, the ppi interactomes were used to identify mechanistic processes of the viral molecular machinery during sars-cov-2 infection, for viral survival and replication within the host. with this knowledge, proteins interactions crucial for pathogenesis could be discerned. we identified different host response induced by specific proteins of sars-cov-2, underlining the important role of orf3a and orf8 in phenotypes of severe covid-19 patients. the interactome approach applied to identification of biological reactions around kininogen-1, allowed to view ns7b and orf3a interactions with ece1, which might have a role to enhance bradykinin storm. this network-based model of sars-cov-2 -host interaction could guide to develop novel treatments against specific viral proteins, such as monoclonal antibodies. the virus-host interactome was made by merging sars-cov-2 -host protein-protein interaction (ppi) data from intact (51), with data from human ppi databases, such as biogrid, innatedb-all, imex, intact, matrixdb, mbinfo, mint, reactome, reactome-fis, uniprot, virhostnet, biodata, obtained by r packages psicquic and biomart (52, 53) . for sars-cov-2 -host interaction experimentally obtained, 1407 protein interactors and 2305 interactions were download from intact at 12 september 2020. 28 sars-cov-2 proteins are used for all the analyses: e, m, n, s, nsp1, nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, orf1a, orf3a, orf6, orf7a, ns7b, orf8, orf9b, orf10, orf14. to obtain functional information about ppi ex vitro, to simulate biological reactions in infected cells and to explore cell response against viral infection, we employed a random walk with restart (rwr) algorithm, a state-of-the-art guilt-by-association approach (54) . this algorithm allows to establish a proximity network from a given protein (seed), to study its functions, based on the premise that nodes related to similar functions tend to lie close to each other in the network. for this study, two types of interactome were performed: the whole sars-cov-2 -host interactome and an interactome per single sars-cov-2 protein. in the first imputation, every protein of sars-cov-2 proteome was used as seeds, with the limit of 200 closest host's proteins to every sar-cov-2 protein, every protein of sars-cov-2 proteome was chosen as seeds for random walk with restart, imposing the limit of 200 closest host proteins to every sars-cov-2 proteins. this analysis allowed to design sars-cov-2-host interactome, where the different colours correspond to the localization of the proteins within the cell. for the second kind of interactome, one sars-cov-2 per time was used as seed, lowering to closest 50 proteins in order to define the induced biological response as better as possible. for each node, a score was computed as a measure of proximity to the seed protein (23) . in total, a large ppi interaction database was assembled, including 13334 nodes and 73584 interactions. graphical representations of networks were performed by gephi 0. 9. 2 (55) . to identify hub protein in the sars-cov-2 -host interactome, the values of betweenness centrality and degree were plotted. betweenness centrality score measure how a specific node is in-between other nodes and then can be considered a hub, while the degree of node corresponds to number of connections. pathways of proteins involved in host response were tested by gene enrichment analysis on kyoto encyclopaedia of genes and genomes (kegg) human pathways and wikipathways databases (56) . to allow gathering of results for every running, the r package enrichr was used, an r interface to web-based tool 'enrichr' for analysing gene sets (57) . the enrichr analysis was performed using these statistical parameters: p-value (fisher exact test), q-value (adjusted p-value for false discovery rate, fdr). results for kegg and wikipathways were considered significant with a revised p-value < 0. 05. to infer pathways involved in single viral interactome, gene enrichment analyses for each 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host protein target of chloroquine and melatonin for immunoregulation in covid-19: a network-based metaanalysis a network medicine approach to investigation and population-based validation of disease manifestations and drug repurposing for covid-19. chemrxiv : the preprint server for chemistry 10 identifying human interactors of sars-cov-2 proteins and drug targets for covid-19 using network-based label propagation structural genomics of sars-cov-2 indicates evolutionary conserved functional regions of viral proteins covid-19: viral-host interactome analyzed by network based-approach model to study pathogenesis of sars-cov-2 infection network medicine: a network-based approach to human disease uncovering disease-disease relationships through the incomplete interactome covid-19 disease map, building a computational repository of sars-cov-2 virus-host interaction mechanisms systems medicine disease maps: community-driven comprehensive representation of disease mechanisms covid-19 disease 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complement associated microvascular injury and thrombosis in the pathogenesis of severe covid-19 infection: a report of five cases endothelin-converting enzyme-1 regulates endosomal sorting of calcitonin receptor-like receptor and beta-arrestins the gtex consortium (2020) the gtex consortium atlas of genetic regulatory effects across human tissues the contact activation and kallikrein/kinin systems: pathophysiologic and physiologic activities bradykinin stimulates renal na(+) and k(+) excretion by inhibiting the k(+) channel (kir4.1) in the distal convoluted tubule hypokalemia and sudden cardiac death hypokalemia-induced arrhythmias and heart failure: new insights and implications for therapy. frontiers in physiology clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china kallikrein-kinin blockade in patients with covid-19 to prevent acute respiratory distress syndrome bradykinin: inflammatory product of the coagulation system kinin b1 receptors as a therapeutic target for inflammation intact: an open source molecular interaction database psicquic and psiscore: accessing and scoring molecular interactions biomart--biological queries made easy random walk with restart on multiplex and heterogeneous biological networks gephi: an open source software for exploring and manipulating networks wikipathways: a multifaceted pathway database bridging metabolomics to other omics research enrichr: a comprehensive gene set enrichment analysis web server 2016 update figures and tables figure 1. workflow to describe sars-cov-2 host interactome analysis based on ppi data figure 2. a) ppi interactome, based on human ppi and sars-cov-2-host interactions, with top 200 closest proteins identified by rwr, using together 28 proteins of sars-cov-2 as seeds for only one rwr run. different colours of node and edges represent different locations in the cell wikipathways gene enrichment analyses for 200 proteins identified by rwr algorithm using together 28 proteins of sars-cov-2 heatmap reporting top 5% smallest p values of gene enrichment analysis on list of proteins, obtained by every interactome of single viral seed. the values were reported as -log10 (p value). a) p values of gene enrichment based on kegg 2019. b) p values of gene enrichment based on wikipathways the nodes in red are human proteins, while the nodes in green are virus proteins. b) kegg human pathway and wikipathways gene enrichment analyses for 200 proteins closest to kng-1. c) ppi interactome, where proteins belonged to human complement system wp2806 (modes in purple) and complement and coagulation cascades (nodes in blue) pathways were highlighted. the proteins belonged to both pathways were marked in light blue 1: table with human and viral proteins identified by rwr in all sars-cov-2 proteins interactome are reported, along with statistical parameters and biological information table of gene enrichment analysis obtained by kegg 2019 database all interactions selected by rwr in interactome for single seed viral protein. detailed information about interactors, interaction and database were reported plot betweenness centrality vs. degree of 199 human proteins, retained in all proteins of sars-cov-2 -host interactome e) as seed. the nodes in red are human proteins interactomes based on human ppi and sars-cov-2-host interactions, with top 50 closest proteins identified by rwr, using accessory proteins (orf1a, orf3a, orf6, orf7a, ns7b, orf8, orf9b, orf10, orf14) as seed. the nodes in red are human proteins s-figure 4. interactomes based on human ppi and sars-cov-2-host interactions, with top 50 closest proteins identified by rwr, using accessory proteins key: cord-354050-kcn67stj authors: shi, guoli; schwartz, olivier; compton, alex a. title: more than meets the i: the diverse antiviral and cellular functions of interferon-induced transmembrane proteins date: 2017-11-21 journal: retrovirology doi: 10.1186/s12977-017-0377-y sha: doc_id: 354050 cord_uid: kcn67stj the first responders of human antiviral immunity are components of the intrinsic immune response that reside within each and every one of our cells. this cell-autonomous arsenal consists of nucleic acid sensors and antiviral effectors strategically placed by evolution to detect and restrict invading viruses. while some factors are present at baseline to allow for constant surveillance of the cell interior, others are upregulated by cytokines (such as interferons) that signal a viral infection underway in neighboring cells. in this review, we highlight the multiple roles played by the interferon-induced transmembrane (ifitm) proteins during viral infection, with focuses on ifitm3 and hiv-1. moreover, we discuss the cellular pathways in which ifitm proteins are intertwined and the various functions they have been ascribed outside the context of infection. while appreciated as broadly-acting, potent restriction factors that prevent virus infection and pathogenesis in cell culture and in vivo, questions remain regarding their precise mode of action and importance in certain viral contexts. continued efforts to study ifitm protein function will further cement their status as critical host determinants of virus susceptibility and prioritize them in the development of new antiviral therapies. the ifitm protein family is encoded by five genes in humans, including the immune-related ifitm1, ifitm2, and ifitm3, as well as ifitm5 and ifitm10 which have no characterized roles in immunity [1] . today, ifitm genes are present in many vertebrate animal species yet they likely emerged in early unicellular eukaryotes via horizontal gene transmission from a bacterium [2] . since then, species-specific gene expansions have given rise to unique ifitm gene repertoires that vary at the level of sequence and copy number [3] [4] [5] . in addition to the canonical ifitm gene locus on chromosome 11 in humans, there are a number of ifitm-like genes dispersed throughout our genome for which a functional understanding is lacking [2] . despite what their name implies, only the immune-related ifitm genes are interferon-inducible, and furthermore, moderate to high levels of expression may be seen in several tissue types even in the absence of interferon. the immunological and clinical importance of ifitm proteins to innate immunity is tied to their unique ability to inhibit the earliest step of the virus life cycle: entry into cells. as a result, they prevent not only viral replication but also the sequelae of virus-associated disease, such as cytopathicity (cell death) and inflammation. initially revealed to be endogenous inhibitors of influenza a virus (iav), west nile virus (wnv), and dengue virus, today it is recognized that a growing list of viruses are sensitive to ifitm-mediated restriction [6, 7] . the use of retroviral "pseudotypes, " in which different viral envelope glycoproteins are swapped into the same retrovirus capsid core, demonstrated that the route of cellular entry is a major determinant of restriction and identified strains exhibiting resistance. in general, those that require a ph-dependent triggering of viral fusion machinery in endosomes are the most affected when ifitm proteins, in particular ifitm3, are overexpressed or silenced in cells [8] [9] [10] . these findings suggested that ifitm-mediated antiviral activity manifests at the entry stage. the subcellular localization of ifitm proteins is important to our understanding of how virus entry is inhibited. they are regularly detected in endosomes, lysosomes, autophagosomes, and the plasma membrane, and the extent of which can vary by cell type and by ifitm family member. their presence at various cellular compartments is the result of dynamic protein trafficking that begins with de novo synthesis in the endoplasmic reticulum and ends with degradation in lysosomes [11] . multiple pieces of evidence suggest that ifitm3 takes on a type ii transmembrane protein topology, with a cytosolic aminoterminus, a luminal/extracellular carboxy-terminus, and two hydrophobic domains: one intramembrane domain (hd1) and one transmembrane domain (hd2) [12] [13] [14] . interestingly, there is also support for alternative membrane topologies in which the termini orientations are reversed [13] . the hydrophobic domains of ifitm members ensure movement between membrane-enclosed vesicles in the biosynthetic-secretory pathway, and posttranslational lipidification with an s-palmitoyl group promotes durable membrane associations [15, 16] . after passage through the endoplasmic reticulum, golgi complex, and plasma membrane, endocytic sorting motifs in the amino-terminus of ifitm2 and ifitm3 allow internalization into endosomes, and this positioning is crucial for antiviral activity [5, 17, 18] . late endosomes, multivesicular bodies, and lysosomes form hybrid organelles in cells and are nearly indistinguishable by conventional methods, and hence the subcellular localization of ifitm3 is often described as endolysosomal. however, some of the overlap with lysosomes must be attributed to the degradative pathway controlling ifitm3 protein turnover, which is mediated by the e3 ubiquitin ligase nedd4 [19] . furthermore, recent reports highlighting roles for ifitm3 in autophagy, a catabolic process involving the degradation of cellular cargo in lysosomes, may explain the apparent association with autophagosomes [20] . in summary, while endolysosomes appear to be the key compartment whereby ifitm3 restricts virus entry, its detection in other organelles may be indicative of uncharacterized cellular functions that are only now being investigated. as residents of cellular membranes, an attractive explanation for the effects of ifitm proteins on virus entry involves direct modification of membrane rigidity and curvature, but other indirect mechanisms have also been proposed (fig. 1 membrane proteins can alter the shape and fluidity of lipid bilayers, and furthermore, that virus-cell fusion is affected by these factors [21] . most of what we know about the antiviral activities of ifitm proteins results from work using iav and vesicular stomatitis virus (vsv), which perform ph-dependent fusion reactions in endosomes to gain access to the cell interior [22] . early findings using fluorescent microscopy and flow cytometry of single cells implicated ifitm3 as an obstacle to virus-cell fusion. when cells overexpressing ifitm3 are challenged with iav, productive infection is inhibited [8] . upon close examination, virions undergo attachment and internalization into cells before becoming cleared from the cell interior [9] . parallel experiments further showed that ifitm3 may expand the size and acidity of the endolysomal compartment itself. in effect, ifitm3 appears to trap endocytosed virions inside vesicles slated for destruction in a degradative pathway [6] . it remains to be determined whether ifitm3-containing structures represent a distinct, hostile subset of endolysosomes that are not conducive to virus-cell fusion. indeed, an article reported that iav sensitivity to ifitm3 is determined by the acidic threshold at which viral hemagglutinin (ha) triggers virus-cell fusion in endosomes. that is, ha variants which drive fusion at higher ph (less acidic) are more resistant to the block by ifitm3, suggesting that evasion of more acidic, late endosomes (where ifitm3 resides in most cell types) enables infection [23] . this assertion is supported further by the observation that vsv infection, which requires virus-cell fusion in early endosomes, is less affected by the presence of ifitm3 [8, 22, 24] . the effects of ifitm proteins on the makeup of the cell interior may present important clues about its principal mechanism of action [9] . simple experiments examining cell-cell fusion, in which ifitm proteins and viral envelope proteins are co-expressed, have been instrumental in testing different mechanistic possibilities. initial reports showed that ifitm members inhibit cell-cell fusion mediated by three classes of viral fusion proteins (some more than others) in a way that did not affect envelope expression itself [25] . this finding suggests that ifitm proteins may affect the physical aspects of membranes in a way that prevents fusion by diverse viral fusion proteins. while informative, an important caveat is that cell-cell fusion experiments in tissue culture do not accurately reproduce the true nature of virus-cell encounters, since ifitm and viral fusion proteins may not be expressed at physiologically relevant levels or at correct subcellular sites. attempts at identifying the precise step of the fusion sequence inhibited by ifitm suggested a block prior to hemifusion, the point at which lipid mixing begins between leaflets of two juxtaposed membranes [25] . however, other techniques including virus particle tracking detected lipid mixing between cell and virus in the presence of ifitm but no viral escape into the cytoplasm, suggesting that dilation of the fusion pore is restricted [26] . the basis for these observations may lie with increased lipid density or increased positive curvature of ifitm-containing membranes [25] . another piece of evidence in support of ifitm proteins as membrane remodelers is the finding that amphotericin b, an antifungal compound known to enhance membrane fluidity, counteracts the activity of ifitm3 to render cells permissive to infection [27] . furthermore, the discovery of an amphipathic helix within the first hydrophobic domain of ifitm3 was found to be crucial to the inhibition of virus entry [14] . this previously unappreciated structure is positioned adjacent to palmitoylated cysteine residues involved in membrane targeting and is also important for the inhibition of cell-cell fusion. based on previously recognized functions of other proteins endowed with an amphipathic helix, ifitm proteins may sense membrane changes occurring during virus-driven hemifusion and prevent fusion pore dilation. these latest findings implicate the first hydrophobic domain (hd1) of ifitm3 as the functional "arm" responsible for its antiviral activity. however, this contrasts with a previous finding that proposed a role for hd2 in cholesterol augmentation in endosomes [28] . residues within hd2 of ifitm3 were reported to interact with the vesicle-associated membrane protein-associated a (vapa), previously linked to cholesterol trafficking. while the effects of cholesterol on membrane fluidity and virus-cell fusion are well-characterized, a number of studies have failed to draw a mechanistic link between ifitm3 activity and cholesterol levels [26, 27, 29, 30] . therefore, effects of ifitm3 on lipid trafficking may not underlie virus inhibition, yet may be indicative of presently uncharacterized activities in the cell. interestingly, vapa and the related protein vapb can enhance the replication of some viruses, most likely through the manipulation of the lipid composition of cell membranes used for virus replication [31, 32] . overall, the study of pathogenic rna viruses demonstrating a high degree of sensitivity to ifitm-mediated restriction has provided some mechanistic insight behind the block they impose to virus entry. nevertheless, the demonstration that certain viruses are resistant to, or even benefit from, the ifitm proteins indicates that antiviral activity may be achieved through the coordination of other cellular proteins. ifitm proteins, especially ifitm1, are known to reside in the plasma membrane and to interact with transmembrane proteins, such as tetraspanins. hepatitis c virus (hcv) utilizes cd81 and occludin as co-receptors, which are components of cellular tight junctions and also happen to interact with ifitm1 [33] . overexpression of ifitm1 leads to inhibition of hcv entry into cells as well as disruption of tight junction complexes, suggesting that these two effects are functionally linked [34] . since cd81 and other tetraspanin proteins can modulate fusion events in multiple virus infections [35] [36] [37] [38] , the ability for ifitm proteins to alter the location or clustering of membrane protein complexes may be central to their antiviral activity. an 'indirect' mode of action is in agreement with the observation that cellular entry of the coronavirus hcov-oc43 is promoted by ifitm proteins [39] . this exceptional case may be explained by the fact that different viruses take advantage of distinct cellular receptors and thus distinct entry routes that are differentially impacted by the presence of ifitm proteins. a transmembrane metalloprotease known as zmp-ste24 (also known as face1) has recently emerged as a downstream effector of ifitm3 [40] . the authors posit that ifitm3 traffics zmpste24 to the sites of virus fusion at endosomes via a direct protein-protein interaction. importantly, in zmpste24 knockout cells, ifitm3 overexpression no longer provides protection from virus challenge. no mechanistic information is available for the antiviral activities of zmpste24 and thus future studies must address the specific determinants of how it binds to ifitm3. it is possible that domains of ifitm3 previously shown to be essential for antiviral function play roles in modulating the location or function of this and other cellular factors, resulting in an organized block to virus entry. the path to identifying the roles played by ifitm proteins during hiv-1 infection was less straightforward for multiple reasons. first, while the impact of ifitm proteins on viruses that require endocytosis and ph-dependent fusion is clearly appreciated, the role for endocytosis in hiv-1 cellular entry has been widely debated [41] . second, hiv-1 exhibits the capacity to spread between cells in a process known as cell-to-cell transmission, which likely allows escape from immune barriers [42] . despite these challenges, the study of ifitm and hiv-1 was mutually instructive: a new antiviral function was described for the former and the ports of cellular entry were defined for the latter. in the first formal description of the antiviral properties of ifitm family members, ifitm3 silencing had little to no effect on hiv-1 infection in hela-cd4 cells, thus grouping it with another retrovirus (amphotropic murine leukemia virus) deemed to be resistant [8] . also, in the paper that identified tetherin/bst-2 as the target of viral accessory protein vpu, which promotes hiv-1 release from cells, it was shown that ifitm proteins exhibited no impact on hiv-1 egress [43] . however, two highthroughput screens studying the activities of interferon stimulated genes (isgs) provided evidence that ifitm proteins impact hiv-1 replication when silenced or overexpressed [44, 45] . lu et al. provided the first in-depth functional demonstration of ifitm-mediated hiv-1 restriction at the level of entry in t cells, and this work was later extended to other lentiviruses of non-human primates [46] . in addition, experiments allowing ongoing replication in tissue culture revealed that the antiviral properties of ifitm proteins may not be limited to the inhibition of virus entry (see next section). as the primary determinant for virus-cell attachment and the subsequent fusion reaction, the viral envelope glycoprotein (env) was suspected to play an important role in whether or not hiv-1 and related lentiviruses are subject to inhibition by ifitm proteins. a report taking advantage of retroviral env pseudotyping provided key insight into viral factors that govern susceptibility and resistance. the authors showed that one can decrease sensitivity to ifitm proteins by increasing amounts of lentiviral env incorporated into virions [47] . however, they pointed out that the capsid core (vector) is also a determinant, indicating that the structure of the viruslike particle and/or its coordination with env also affects the degree of restriction. another study corroborated the importance of env with regards to inhibition by ifitm proteins, this time via examination of patient-derived hiv-1 clones known as transmitted/founder (tf) strains [48] . these viral variants represent a close approximation of the viral sequence that seeds infection in a newlyinfected individual, and they tend to utilize a specific co-receptor on the cell surface known as ccr5. here, it was revealed that the sensitivity of hiv-1 entry to ifitmmediated restriction depends on coreceptor usage and the subcellular localization of ifitm in the host cell. cxcr4-tropic hiv-1 strains were shown to exhibit sensitivity to ifitm2 and ifitm3, which are mostly localized to endolysosomes, while ccr5-tropic strains were sensitive to ifitm1 at the plasma membrane. this differential outcome suggests that hiv-1 fusion may occur in endosomes or at the plasma membrane depending on which virus coreceptor is engaged at the cell surface. tf strains are relatively resistant to ifitm-mediated restriction, yet matched viral clones derived 6 months following initial infection exhibited a gain of sensitivity to ifitm2 and ifitm3 [49] . this finding suggests that founder viruses enter cells at the plasma membrane, while viruses isolated at later stages of infection might increasingly rely on endosomal entry. however, ifitm2 and ifitm3 also transit to the plasma membrane before endocytosis, and thus the varying sensitivity of hiv-1 strains may result from fusion at different plasma membrane microdomains. furthermore, the authors show that the site of hiv-1 entry, as inferred by sensitivity to ifitm proteins, may also depend on cell surface levels of cd4. another report reinforced the link between ifitm and hiv-1 entry by demonstrating that cxcr4-tropic, but not ccr5-tropic hiv-1, is hypersensitive to a splice variant of ifitm2 lacking the amino terminus. notably, this ifitm variant is especially abundant in primary human cells (cd4+ t cells and monocytes) that serve as targets for hiv-1 infection in vivo [50] . together, these recent data suggest that ifitm2 and ifitm3 may be major selective pressures responsible for the use of ccr5 during primary hiv-1 infection. in addition to restricting virus entry, recent findings indicate that ifitm proteins perform antiviral functions impacting late stages of the hiv-1 life cycle. in contrast to previous strategies in which infections were launched using only cell-free virus preparations, the use of cell coculture experiments using infected cells (donors) and uninfected cells (targets) revealed new antiviral functions [51] . we found that ifitm overexpression in targets had little to no consequence for hiv-1 transmission and spread in this context, while, surprisingly, overexpression in donor cells led to potent decreases [51] . further experiments showed that the block to virus spread is attributed to an inhibition of virion infectivity, with ifitm3 exhibiting the most potent restriction. virus-cell fusions assays showed that hiv-1 virions produced in the presence of ifitm3 are less fusogenic when incubated with fresh target cells, and assessment of virion content indicated that ifitm3 incorporates into the viral lipid bilayer. this antiviral activity is enhanced upon expression of an ifitm3 mutant that is defective for endocytosis, indicating that restriction of hiv-1 virion infectivity is performed at the plasma membrane [5] . two other teams reported similar findings on this phenomenon in hiv-1 producing cells, with one adding a layer of mechanistic insight involving hiv-1 env glycoprotein [52] [53] [54] . here, the production of hiv-1 particles in the presence of ifitm3 via co-transfection of 293t cells resulted in defects in env maturation and decreases in virion-associated gp120, the infectious form of env [53] . the authors posited that ifitm3 interferes with env via a proteinprotein interaction in virus-producing cells. of note, this relationship between antiviral protein and env appears to hold true upon examination of non-human primate ifitm3 and their lentiviral counterparts [54] . nonetheless, there exist certain discrepancies that cloud the potential importance of this observation. first, ifitm members inhibit hiv-1 infectivity to varying degrees (ifitm3 > ifitm2 > ifitm1) [51] yet all three proteins inhibit certain env proteins to a similar extent [55] . second, ifitm3 overexpression in t cells reduces virus infectivity but has no detectable impact on hiv-1 env levels in infected cells or purified virions [51] , and a lack of effect has been reported by others using diverse experimental systems [5, 48, 56] . third, we now know that the negative "imprinting" of virions by ifitm3 occurs with various dna and rna viruses, apparently in the absence of envelope glycoprotein perturbation [56] . therefore, follow-up experiments will require the study of endogenous ifitm3 in various cell types as well as the effects of type-i interferon on hiv-1 env maturation and virion incorporation. overall, it is unclear whether modification of env is directly responsible for the inhibition of hiv-1 virion infectivity, nor is it known whether the virion incorporation of ifitm3 is critical for this effect. it is possible that both play mechanistic roles which are not mutually exclusive. of note, enriched plasma membrane localization of ifitm3 enhances both anti-hiv-1 activity as well as ifitm3 incorporation into virions, suggesting a functional link [5] . nonetheless, the recent identification of env variants that are resistant to the ifitm3-mediated restriction of virion infectivity confirms this viral protein as an important determinant. it was shown that a particular ccr5-tropic strain (ad8) of env is unaffected by ifitm3 overexpression in virus-producing cells, exhibiting no defects in virion infectivity. this resistance phenotype maps to the v3 loop of gp120 and is transferrable to other env isolates [57] . another feature of hiv-1 strains that are most sensitive to the ifitm3-imposed block to virus fusogenicity is the use of env proteins with high sensitivity to both soluble cd4 and the neutralizing antibody 17b, which recognizes a cd4-induced epitope. therefore, virus susceptibility to the infectivity defect caused by ifitm3 is linked to conformational changes in the cd4-binding site of env, although the stability or clustering of virion-incorporated env trimers may also be involved. based on what we know about the changes to cellular membrane fluidity caused by ifitm proteins, it is possible that similar disturbances in the viral lipid bilayer containing env are involved in the restriction of virion infectivity. another important observation made in these studies is that resistance to one antiviral function of ifitm3 is associated with resistance to another, since the ad8 strain exhibits relatively less sensitivity to the effects of ifitm3 in both target cells and producer cells, and tf strains are completely resistant to both modes of restriction [48, 56] . thus, ifitm3 performs at least two antiviral activities against hiv-1 for which there may be mechanistic overlap. for reasons that are not yet clear, cd4 engagement by env is an important determinant for whether virus is restricted at the level of target cells and producer cells [48, 55] . it is possible that cd4 binding and the conformational changes in env that follow (which dictate interactions with co-receptors) affect the route and kinetics of hiv-1 entry into cells [58] , and in turn, affect sensitivity to ifitm-mediated antiviral activities. globally, the multiple constraints that ifitm proteins place on hiv-1 env suggest that these cellular factors contribute to the genetic bottleneck that selects for interferon-resistant virus at the earliest stages of hiv-1 infection in vivo [59] . the study of diverse viruses has been central to discoveries involving the ifitm protein family. the physiological importance of ifitm activity has been clearly demonstrated for those viruses that exhibit the greatest sensitivity in cell culture experiments, such as iav. however, additional functions were found using viruses that are actually resistant to the block at cellular entry. in both cases, the use of transgenic mice deficient for the murine ifitm locus was central to establishing the in vivo significance of ifitm proteins in diseased and healthy states. downregulation of ifitm3 in target cells can lead to increased cytopathicity upon virus challenge in vitro, which has been attributed to its role as a protective barrier preventing virus entry and subsequent replication [60, 61] . nonetheless, in vivo experiments using mouseadapted virus strains have been critically important to understanding the full spectrum of downstream consequences resulting from ifitm deletion, ranging from cell death to the manifestation of virus-associated disease propagated throughout the host organism. for example, accelerated disease progression and mortality are observed in ifitm3-deficient mice challenged with iav, suggesting that ifitm3 confers a survival advantage to cells exposed to virus [62, 63] . indeed, the expression of ifitm3 may preserve the integrity and function of cells that would otherwise be destroyed by uninterrupted viral replication [64] . it was reported that elevated levels of ifitm3 protein in memory cd8+ t lymphocytes, which kill virus-infected cells and serve as important targets themselves for iav infection in the lung, promotes their survival during infection and enables long-term defense against future viral exposures [65] . in a murine model of chikungunya virus (chikv), mice lacking ifitm3 sustained greater joint swelling which was correlated to increased viral burden and pro-inflammatory cytokine production [66] . ifitm3-deficient mice were also found to be more vulnerable to lethality in the context of wnv. while wnv disease is associated with neurotropism, ifitm3 limited pathogenesis by suppressing viremia first in peripheral organs [67] . thus, by acting as the first line of defense in cells exposed to invading viruses, ifitm proteins prevent a plethora of adverse events that can lead to disease and death. yet unexpected antiviral activities associated with ifitm3 were revealed during murine cytomegalovirus (cmv) infection. in this case, ifitm3 does not limit virus entry into cells (the protein was previously shown to facilitate cmv virion morphogenesis [68] ). rather, cmv infection in ifitm3-deficient mice led to much higher production of the pro-inflammatory cytokine interleukin-6 (il-6) [69] . the result was dysregulation of cellular immunity and impaired control of virus replication, suggesting that murine ifitm3 plays a part in regulating cytokine production important for resolution of virus infection. another example in which ifitm3 could be found performing a non-canonical activity was in the setting of sendai virus (sev) infection. this murine paramyxovirus has been shown to be insensitive to the ifitm3-mediated block to virus entry [70] . notwithstanding, it was shown that ifitm3 regulates interferonbeta production triggered by sev infection in human cell lines [71] . that is, overexpression of ifitm3 inhibited production of the cytokine while knockdown had the opposite effect. the authors propose that ifitm3 associates with the transcription factor driving interferon-beta gene expression, irf3, and accelerates its turnover in autophagosomes. since interferon-beta itself induces the expression of ifitm3, this implies a role in negative feedback of the interferon pathway [71] . this finding warrants testing in an in vivo murine model, but it seems likely that the effects of ifitm proteins on interferon signaling will yield much interest from researchers and clinicians in the field of microbial pathogenesis. naturally occurring variation in ifitm genes has provided an additional genetic platform for the study of pathogenic virus infections important to human public health. population-level associations have been drawn between single-nucleotide polymorphisms (snp) in ifitm3 and severe outcomes following iav infection, corroborating an important in vivo function but lacking a mechanistic explanation. the most cited example is rs12252-c, a nonsynonymous mutation in the first coding exon of ifitm3, which was predicted to affect mrna splicing and to produce protein with an amino-terminal truncation [63] . this minor variant was found to be enriched in a group of individuals hospitalized following iav infection, and it is found at relatively high frequency among asian ethnic groups. however, the reasons for the disease association has remained elusive, as the truncated isoform of ifitm3 predicted to result from the snp had not been detected in cells or individuals. it has now been reported that cell lines derived from individuals who are homozygous for rs12252-c express a mrna transcript encoding full-length ifitm3, whereas the shorter isoform was not found [50] . over the course of several years, a slew of publications has either confirmed or refuted the genetic association between rs12252-c and iav infection outcome. a recent and comprehensive study now links an additional variant in the ifitm3 locus to severe influenzaassociated illness in three independent cohorts, albeit the snp identified is distinct from rs12252-c. known as rs34481144-a, it is found in the 5' untranslated region and is linked to lower ifitm3 protein levels in cells [72] . experimental evidence showed that the snp controls gene promoter activity via decreased binding of transcription factor irf3 and increased binding of ctcf, which promote and repress ifitm3 transcription, respectively. in agreement with the previous finding that ifitm3 preserves antiviral cd8 + t cells [65] , individuals harboring rs34481144-a contained reduced numbers of these cells in lung airways during infection [72] . the evolutionary pressures responsible for the maintenance of this 'defective' allele in humans are unclear, but its existence may allude to the involvement of ifitm3 in cellular processes requiring fine-tuned protein expression. before it was realized that ifitm proteins perform broad-spectrum antiviral activities, they were implicated in pathways important to embryonic development and cancer (exhaustively reviewed in [73] ). a crucial contribution to developmental processes seems unlikely, since transgenic mice in which the murine ifitm locus is knocked out exhibit no obvious abnormalities apart from being fat, suggesting a metabolic irregularity [74] . there is ample evidence, on the other hand, for both positive and negative regulation of ifitm expression during tumorigenesis. while detailed descriptions were previously lacking, recent developments have provided a mechanistic grounding to many observations linking ifitm proteins to cell proliferation, adhesion, and migration. in addition to the interferon signaling pathways, ifitm gene expression is controlled by a number of cascades involved in cellular homeostasis (fig. 2) . for example, growth factor receptors lead to downstream upregulation of ifitm2 upon triggering by insulin-like growth factor-1 (igf1), which relies on signaling via phosphatidylinositol-3-kinase (pi3k) and akt kinase. in gastric cancer cohorts, the upregulation of ifitm2 is associated with accelerated disease progression and shorter survival time [75] . silencing of ifitm2 in gastric cancer cells decreased cell proliferation, migration, and metastasis, while ifitm2 depletion in a mouse model resulted in dramatic decreases in tumor size [75] . ifitm3 has also been functionally implicated in this cancer type, as its knockdown suppressed tumor cell migration, invasion and proliferation capacity [76] . there is also evidence that ifitm3 acts indirectly to affect these cellular properties through regulation of other cellular proteins, such as osteopontin [77] . recently, the involvement of ifitm3 in cancer was further extended to include spatial regulation of the src oncoprotein. the trafficking of src between focal adhesions and the cell interior, which is regulated by activating molecule in beclin1-regulated autophagy (ambra1) and focal adhesion kinase (fak), is important for cell migration and metastasis. ambra1 redirects src towards autophagosomes to disfavor substrate attachment and favor cell movement in an ifitm3-dependent manner [78] (fig. 2) . collectively, the twin antiviral proteins ifitm2 and ifitm3 may serve as biomarkers for tumorigenic phenotypes as well as targets for anticancer interventions. it remains to be determined which domains of ifitm proteins are involved in the control of cellular properties and if they overlap with those known to be involved in antiviral immunity. for example, just as the subcellular localization of ifitm proteins is important to their antiviral activities [11] , it may also affect their involvement in cellular housekeeping functions. as a proof of principle, ifitm2 can act as a cell surface receptor for a secreted form of bag3, which promotes pro-survival signaling through the pi3k and p38 mapk pathways [79] . several reports highlighting ifitm-mediated impacts on basic cellular processes provide further opportunities to test how these proteins inhibit virus infections. a focus on functional roles of endogenous, rather than overexpressed, ifitm proteins has been crucial to this end. a prime example is the observation that deletion of the ifitm locus in murine cells led to interruption of clathrin-mediated endocytosis and loss of acidity within endosomes, suggesting that endogenous ifitm proteins might positively regulate these processes in vivo [80] . this possibility is further supported by work in astrocytes showing that knockdown of ifitm3 inhibits clathrindependent endocytosis [81] . as previously mentioned, the ifitm-deficient mice exhibit an age-related obesity associated with defects in leptin signaling, which could be explained by disturbances in ligand-receptor internalization [74] . interestingly, the ifitm proteins are also involved with the endocytosis-associated protein caveolin-1 (cav-1), with consequences for cell signaling events [82, 83] . together, these data hint that impacts on endocytic trafficking may also contribute to the mechanisms by which ifitm proteins inhibit virus entry. while the antiviral effects of ifitm proteins are generally assumed to manifest at the stage of virus-cell fusion, these data suggest that an effect on virus internalization must be carefully considered on a case-by-case basis. for example, since knockdown or knockout of ifitm3 leads to decreases in clathrin-mediated endocytosis and increases in virus entry, it is possible that ifitm3 promotes the trafficking of incoming virions into endocytic pathways that are acidified, degradatory and/or otherwise non-productive. since ifitm proteins share a high degree of sequence homology (especially ifitm2 and ifitm3, which differ by only 13 amino acids), future efforts must assess the specific or redundant role for each ifitm family member when describing new activities. only then can the functional utility of each be properly understood, be it during virus infection or basal cellular homeostasis. furthermore, it is important to test whether interferon signaling serves as a 'switch' to promote antiviral functions over housekeeping ones. we expect that boosting efforts towards understanding the host factors interacting with ifitm proteins will expose novel purposes in cells, which may, in turn, reveal additional ways by which these proteins interfere with virus infections. cell-based experiments have identified many binding partners of ifitm proteins, but in most cases, it is unclear whether cytokine regulation; ifitm3 is a negative regulator of the interferon response because it accelerates the turnover of irf3 in autophagosomes. it also suppresses the production of il-6, a pro-inflammatory cytokine which, itself, can also induce the expression of ifitm genes. ifitm2, in contrast, promotes the upregulation of il-6 by acting as a cell surface receptor for secreted bag3. as bag3 is also a well-characterized chaperone for selective autophagy, it will be of interest to determine if ifitm2 also participates with bag3 in autophagy-related processes. cell migration and invasion; ifitm3 is a central component of a multi-protein interaction involving src, fak, and ambra1, which is important for regulating cell adhesion and movement. ifitm3 assists in the subcellular trafficking of src between focal adhesion points and autophagosomes. cell growth, proliferation, and cell cycle regulation; interferon signaling is known to negatively regulate cell division and growth via stat signaling, with ifitm proteins serving as downstream effectors. ifitm1 interacts with caveolin-1 (cav-1) to inhibit erk/mapk signaling, a pathway which stimulates cell proliferation when active. ifitm1 also stabilizes p53, a tumor suppressor with anti-proliferative functions these interactions are direct or specific. interrogation of potential interactors in transgenic mice will help clarify their precise role and enable prioritization of different hypotheses. in general, increased experimental crosstalk between virology and cell biology will provide much needed opportunities for discovery and will facilitate the development of host-directed therapies targeting ifitm proteins in the setting of infection and cancer. the broad-spectrum antiviral functions of ifit and ifitm proteins the dispanins: a novel gene family of ancient origin that contains 14 human members evolution of vertebrate interferon inducible transmembrane proteins evolutionary dynamics of the interferon-induced transmembrane gene family in vertebrates natural mutations in ifitm3 modulate post-translational regulation and toggle antiviral specificity ifitms restrict the replication of multiple pathogenic viruses ifitm proteins-cellular inhibitors of viral entry the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus ifitm3 inhibits influenza a virus infection by preventing cytosolic entry distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus regulation of the trafficking and antiviral activity of ifitm3 by post-translational modifications combined approaches of epr and nmr illustrate only one transmembrane helix in the human ifitm3 interferon-induced transmembrane protein 3 is a type ii transmembrane protein ifitm3 requires an amphipathic helix for antiviral activity palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm3 the palmitoyltransferase zdhhc20 enhances interferoninduced transmembrane protein 3 (ifitm3) palmitoylation and antiviral activity phosphorylation of the antiviral protein interferon-inducible transmembrane protein 3 (ifitm3) dually regulates its endocytosis and ubiquitination identification of an endocytic signal essential for the antiviral action of ifitm3 e3 ubiquitin ligase nedd4 promotes influenza virus infection by decreasing levels of the antiviral protein ifitm3 a balancing act between ifitm3 and irf3 membrane curvature and its generation by bar proteins acid-dependent viral entry ph optimum of hemagglutinin-mediated membrane fusion determines sensitivity of influenza a viruses to the interferon-induced antiviral state and ifitms host cell factors and functions involved in vesicular stomatitis virus entry ifitm proteins restrict viral membrane hemifusion ifitm3 restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion amphotericin b increases influenza a virus infection by preventing ifitm3-mediated restriction the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterol-independent mechanisms the interferon-induced transmembrane proteins, ifitm1, ifitm2, and ifitm3 inhibit hepatitis c virus entry rhinovirus uses a phosphatidylinositol 4-phosphate/cholesterol counter-current for the formation of replication compartments at the er-golgi interface noroviruses co-opt the function of host proteins vapa and vapb for replication via a phenylalaninephenylalanine-acidic-tract-motif mimic in nonstructural viral protein ns1/2 cd81 (tapa-1): a molecule involved in signal transduction and cell adhesion in the immune system ifitm1 is a tight junction protein that inhibits hepatitis c virus entry the role of tetraspanins in fusion evidence showing that tetraspanins inhibit hiv-1-induced cell-cell fusion at a post-hemifusion stage tetraspanins cd9 and cd81 function to prevent the fusion of mononuclear phagocytes formation of syncytia is repressed by tetraspanins in human immunodeficiency virus type 1-producing cells interferon induction of ifitm proteins promotes infection by human coronavirus oc43 zmpste24 defends against influenza and other pathogenic viruses on the whereabouts of hiv-1 cellular entry and its fusion ports unique features of hiv-1 spread through t cell virological synapses tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu the ifitm proteins inhibit hiv-1 infection a diverse range of gene products are effectors of the type i interferon antiviral response primate lentiviruses are differentially inhibited by interferon-induced transmembrane proteins virion background and efficiency of virion incorporation determine susceptibility of simian immunodeficiency virus env-driven viral entry to inhibition by ifitm proteins resistance of transmitted founder hiv-1 to ifitm-mediated restriction coupling of replication and assembly in flaviviruses î�20 ifitm2 differentially restricts x4 and r5 hiv-1 ifitm proteins incorporated into hiv-1 virions impair viral fusion and spread ifitm proteins are incorporated onto hiv-1 virion particles and negatively imprint their infectivity ifitm proteins restrict hiv-1 infection by antagonizing the envelope glycoprotein nonhuman primate ifitm proteins are potent inhibitors of hiv and siv the v3 loop of hiv-1 env determines viral susceptibility to ifitm3 impairment of viral infectivity interference with the production of infectious viral particles and bimodal inhibition of replication are broadly conserved antiviral properties of ifitms the v3-loop of hiv-1 env determines viral susceptibility to ifitm3 impairment of viral infectivity delineating cd4 dependency of hiv-1: adaptation to infect low level cd4 expressing target cells widens cellular tropism but severely impacts on envelope functionality resistance to type 1 interferons is a major determinant of hiv-1 transmission fitness the ifitms inhibit zika virus replication zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells ifitm3 limits the severity of acute influenza in mice ifitm3 restricts the morbidity and mortality associated with influenza respiratory dc use ifitm3 to avoid direct viral infection and safeguard virus-specific cd8 + t cell priming enhanced survival of lung tissue-resident memory cd8 + t cells during infection with influenza virus due to selective expression of ifitm3 the interferonstimulated gene ifitm3 restricts infection and pathogenesis of arthritogenic and encephalitic alphaviruses the interferon-stimulated gene ifitm3 restricts west nile virus infection and pathogenesis human cytomegalovirus exploits interferon-induced transmembrane proteins to facilitate morphogenesis of the virion assembly compartment the antiviral restriction factor ifn-induced transmembrane protein 3 prevents cytokine-driven cmv pathogenesis palmitoylation on conserved and nonconserved cysteines of murine ifitm1 regulates its stability and anti-influenza a virus activity ifitm3 inhibits virus-triggered induction of type i interferon by mediating submit your next manuscript to biomed central and we will help you at every step: autophagosome-dependent degradation of irf3 snp-mediated disruption of ctcf binding at the ifitm3 promoter is associated with risk of severe influenza in humans the small interferon-induced transmembrane genes and proteins age-related onset of obesity corresponds with metabolic dysregulation and altered microglia morphology in mice deficient for ifitm proteins igf1/igf1r/stat3 signaling-inducible ifitm2 promotes gastric cancer growth and metastasis mechanism and biological significance of the overexpression of ifitm3 in gastric cancer interferon-induced transmembrane 3 binds osteopontin in vitro: expressed in vivo ifitm3 reduced opn expression ambra1 spatially regulates src activity and src/fak-mediated cancer cell invasion via trafficking networks bag3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages interferon-inducible transmembrane proteins of the innate immune response act as membrane organizers by influencing clathrin and v-atpase localization and function astroglial ifitm3 mediates neuronal impairments following neonatal immune challenge in mice binding of ifitm1 enhances the inhibiting effect of caveolin-1 on erk activation ifitm1 promotes the metastasis of human colorectal cancer via cav-1 we thank the late mark wainberg for his support and for his efforts at the retrovirology journal. os and aac conceived the manuscript. gs os and aac wrote and edited the manuscript. all authors read and approved the final manuscript. the authors hereby state that no competing interests exist. not applicable. all authors consent to the publication of this manuscript. not applicable. work in the lab of aac is supported by the intramural research program of the nih, national cancer institute, center for cancer research. work in the lab of os is supported by grants from the anrs, the vaccine research institute, the "chikv-viro-immuno" anr-14-ce14-0015-01 and "timtamden" anr-14-ce14-0029 projects, the labex ibeid program anr-10-ihub-0002, a gilead hiv cure grant and institut pasteur. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-023026-2r84ndzv authors: nan title: posters date: 2013-06-14 journal: glia doi: 10.1002/glia.22530 sha: doc_id: 23026 cord_uid: 2r84ndzv nan university of colorado school of medicine, aurora, united states oligodendrocyte progenitor cells (opcs) migrate long distances before differentiating and myelinating axons. it is well established that both short and long-range soluble guidance factors, as well as contact with the surrounding extracellular matrix (ecm) and neighboring cells, can modulate opc migration and differentiation. however, the influence of cell-matrix interactions on opc responses to guidance molecules is less clear. previous in vitro studies of opc migration in response to semaphorins and netrin-1 yielded inconsistent results, but these studies were performed on a variety of ecm substrates, i.e., explants, collagen, poly-d-lysine. in the current study, we systematically studied opc migration in response to guidance factors on several different ecm substrates, to better understand the influence of ecm interactions on opc responses to chemotropic molecules. understanding the regulation of opc migration and differentiation has important implications for the treatment of demyelinating diseases such as multiple sclerosis (ms). for example, semaphorins 3a and 3f are expressed in active, but not chronic, ms lesions, and in demyelination/remyelination studies in animal models, semaphorins influence opc migration into lesions, affecting the rate at which remyelination occurs. in addition, aberrant expression of ecm ligands occurs in and around ms lesions. we performed live imaging experiments of opc migration on different ecm substrates in response to gradients of chemotropic molecules. these studies are combined with ihc, co-ip, western blot and rt-pcr analyses to determine the effects of ecm substrates on opc migration and signaling pathway responses to chemotropic molecules. our preliminarily results suggest that ecm substrate can regulate both the direction and rate of opc migration. semaphorin 3a is repulsive to opcs on laminin 1, while on fibronectin opc migration remains uniform in the presence of semaphorin 3a. since laminin 1 is the ligand for a6b1 integrin heterodimers, while opc migration on fibronectin is mediated by avb3 integrins, the repulsive effect of semaphorin 3a appears likely to function through b1 integrin. thus, signaling through the neuropilin-plexin receptor complex that underlies the repulsive effects of semaphorin 3a on opc migration may be modulated by interactions with b1 integrins. x. bai university of saarland, homburg, germany secondary injury processes after acute brain trauma involve activation of different cell types like astrocytes, oligodendrocytes and microglia cells. a complex and yet not completely understood sequence of cellular responses initiate functional recovery after the neurodegeneration process. by using double-transgenic mice gcpb (gfap-egfp 3 plp-dsred1) mice, we were able to identify a particular type of activated glia expressing astro-and oligodendroglial properties simultaneously (ao cells) that transiently appeared after three types of acute cortical injuries (stab wound injury, pial vessel disruption and middle cerebral artery occlusion). ao cells could be labeled by oligodendrocyte precursor cell (opc) markers olig2 and sox10, but not for markers of mature astrocytes or neurons. two-photon live imaging of gcpb mice revealed that ao cells originated from plp-dsred-positive oligodendrocyte lineage cells. electrophysiological inspection of ao cells in acutely isolated brain slices revealed the expression of delayed rectifying, voltage gated k 1 currents, a typical property of ng2 glia. therefore, we classified ao cells as opcs. to follow the fate of ao cells which disappeared about 15 days after the injury, we generated gfap-n-cre 3 plp-c-cre 3 rosa26eyfp triple transgenic (crec) mice. under non-injury conditions, few recombined cells were observed. however, numerous recombined cells appeared after 1 week of stab wound injury adjacent to the lesion site, and lasted over 8 weeks. we found that 55-70% of recombined cells were gfap-positive, 18-29% of them were pdgfra-positive and 19-30% were positive for the oligodendrocyte marker gstp. in line with that, about 46% of newly generated gfap-positive astrocytes were observed from ng2-creert2 x rosa26tdtomato mice after 1 week of stab wound injury. these results provide a strong evidence for opcs giving rise to astrocytes after acute cortical injuries. to further understand the molecular mechanism, we performed intra-cerebral injection of bone morphogenetic protein 4 (bmp4, known to promote astrocyte, but blocking oligodendrocyte differentiation) into gcpb and crec mice. bmp4, but not leukemia inhibitory factor (lif), significantly increased the number of ao cells as well as astrocytes in the respective mice. in conclusion, ng2/opcs display strong potential to differentiate into astrocytes after acute cortical injury. for instance, sox9 and sox10 have a crucial role in opc specification and differentiation, respectively. using microarray analysis of oligodendrocyte lineage cells, we previously identified sox17 as a major regulator of oligodendrocyte development (sohn et al., 2006) . in opc cultures, sox17 expression was maximal at developmental corresponding to cell cycle exit and onset of differentiation. these findings, suggest that sox17 has critical functions in the controlling of opc cell cycle exit and differentiation. in order to decipher the functional role of sox17 in oligodendrocyte development, we generated a tet-sox17:sox10 rtta double transgenic mouse line. this mouse strain allows a sox17 overexpression specifically in sox101 cells in a doxycycline dependant manner (tet-on system). in the cns of dox-treated transgenic animals, we showed that sox17 overexpression was specifically targeted in sox101 oligodendroglial cells. in the developing cns, sox17 overexpression was targeted in pdgfra1/nkx2.21 opcs and in olig21/cc11 differentiated oligodendrocytes. to assess the effect of sox17 gain-of-function on oligodendrocyte development and myelination, we analysed opc proliferation, apoptosis and differentiation, after doxycycline induction from e12.5 to p15. in the embryonic spinal cord, we showed that overexpression of sox17 did not modify cell proliferation or the number of pdgfra1 opcs. these data strongly suggest that sox17 is not involved in early steps of oligodendrocyte development. interestingly, we found that sox17 gain-of-function drastically reduces the density of cc11 mature oligodendrocytes at postnatal stages, correlating with a delay in myelination in transgenic mouse spinal cords. altogether, our data reveal critical functions of sox17 in oligodendrocyte lineage progression and myelination. supports: arsep and nmss (usa). m.f is a phd fellow of the french mesr (ed3c). m. rocha, p. fernandes, a.c. manhães, p.c. barradas, f. tenorio universidade do estado rio de janeiro, instituto de biologia, rio de janeiro, brazil nutrient restriction during the perinatal period exerts a profound influence on brain development. previous studies using an undernutrition protocol (0% protein diet) during the first half of the lactation period in rats showed that the offspring presented an altered feeding pattern, which reflected a metabolic imprinting effect on feeding behavior. there is a delay in the pattern of npy expression in the arcuateparaventricular (arc-pvn) pathway, reflecting an effect on the development of the hypothalamic circuitry, leading to metabolic imprinting. here we studied the effects of undernutrition on glial differentiation using the same model. we used rats from 5 to 30 postnatal (p) days of age whose dams were either fed a 0% protein diet (dg) or a normoprotein diet (cg) from p1 to p10. we assessed ki67, vimentin, gfap, ed-1 and cnpase distribution in hypothalamic nuclei using immunohistochemistry. our results showed impairment in cell proliferation, more evident in the pvn and in the lateral hypothalamus (lh), where dg animals showed a lower number of ki-67positive (ki671) cells at p5 when compared to cg. however, at p10 and p15, the number of ki671 cells was significantly increased in dg. from p20 onwards, staining intensity remained relatively stable and similar in all groups. dg animals showed a delay in astroglial differentiation, presenting a decrease in gfap expression and a peak of vimentin expression at p10 and p15 accompanied by an increase in vimentin/ki671 cells. an increase in the number of ed1 positive cells next to the third ventricle was also shown at the same ages in dg. cnpase staining was very faint in most nuclei and did not show any obvious difference in dg animals. our results suggest that glial differentiation is delayed in dg, which may contribute to the changes in hypothalamic circuitry that causes metabolic imprinting. recent studies have demonstrated that neural precursor cells (npcs) residing in the adult subventricular zone also exhibit the capacity to generate new oligodendrocytes following experimental demyelination. the relative capacities of these two cell types to contribute to oligodendrogenesis in this context remain largely unexplored. we have adopted transgenic approaches that enable either the specific labeling of npcs or oligodendrocytes and the interrogation of their subsequent fate within the corpus callosi of mice subjected to central demyelination induced by cuprizone. adult nestin-creer t2 : r26r-eyfp mice were administered tamoxifen (0.3g/kg/day) for 4 days by oral gavage, inducing the permanent expression of yellow fluorescent protein (yfp) in npcs and their progeny. mice were subsequently fed 0.2% cuprizone for 6 weeks followed by 6 weeks recovery on normal food to enable the analysis of remyelination. expression of yfp and other cellular markers was examined immunohistochemically to determine the fate and migratory potential of npcs in the remyelinating corpus callosum (cc). rostrocaudal analyses of cc in the cuprizonechallenged mice demonstrated that approximately 60% of yfp 1 npcs commit to an oligodendroglial fate. there was robust recruitment of npc-derived oligodendroglial cells, with a 14-fold increase in yfp 1 sox10 1 cells compared to unchallenged mice. most of these cells were mature oligodendrocytes (yfp 1 cc1 1 ) and their density in the remyelinating cc was highest adjacent to the dorsolateral corner of svz and decreased proportionally with distance in the mediolateral axis. on the other hand, fate mapping of opcs using pdgfracreer t2 : r26r-eyfp mice revealed that oligodendrocytes derived from parenchymal opcs were distributed in a converse manner to svz-derived oligodendrocytes. in addition, we found that, independent of the cellular source, many oligodendrocytes repopulated the corpus callosum as linear arrays of clonally derived cells, in a manner that recapitulated the postnatal development of these structures. these results demonstrate that lineage determination and maturation of oligodendroglia from npcs occurs efficiently in situ. we also reveal that remyelination occurs in a coordinated way, most likely secondary to interactions between a restricted pool of axons and a sentinel opc induced to proliferate in situ. play an important role in developmental oligodendrogenesis and myelination and mounting evidence from rodent models of demyelination suggest that they also promote remyelination. the therapeutic application of thyroid hormone to demyelinating disorders is limited by the potential for cardiac side effects mediated by thyroid hormone receptor (thr) a signaling. here we report that gc-1, a thyromimetic with selective thrb1 action promotes in vitro oligodendrogenesis from both rodent and human oligodendrocyte progenitor cells. in addition, we used pdgfar-creer;rosa26-eyfp double-transgenic mice to examine the effect of gc-1 on the fate of oligodendrocyte progenitor cells and find that treatment with gc-1 during developmental myelination promotes oligodendrogenesis in vivo. these results indicate that a b receptor selective thyromimetic can enhance ol differentiation in vitro and during developmental myelination and warrants further study in demyelinating models. the olfactory epithelial layer contains multipotent horizontal basal cells (hbcs) that differentiate into olfactory sensory neurons. we generated olfactory sphere (os) cells in cultures that were derived from adult rat olfactory mucosa. fluorescence-activated cell sorting and immunofluorescence analyses showed that os cells were derived from hbcs. os cells underwent neuronal and glial differentiation in vitro. to examine multipotent differentiation in vivo, os cells were transplanted into injured rat spinal cords. the transplanted cells integrated into host tissue and underwent glial differentiation. our data showed the stem cell properties of hbcs. oligodendrocyte progenitor cells (opcs) comprise the main cycling cell population of the cns parenchyma during the early postnatal period and at adult stages. however, the molecular mechanisms implicated in opc divisions are still by large obscure. with the aims to unveil i) whether opc divisions exploit the same molecular machinery of neuronal precursors, and ii) whether distinct opc subsets exist with different cell division machineries, we focused on a mutant mouse where citron-kinase, a crucial regulator of cytokinesis in neuronal precursors, is germinally ablated (cit-k ko). these mice have severe cns developmental abnormalities, reduction of selected neuronal populations due to apoptosis triggered by defective divisions, display ataxia, epileptic seizures and die by the third postnatal week. interestingly, they were reported to have myelin defects, suggesting that the cit-ko affects oligodendroglial cells. notably, already early after birth we found a 2-fold decrease in opc density in both the cortical grey (gm) and white matter (wm), compared to wild-type mice (wt). here, most opcs were hypertrophic and multinucleated, indicating a cytokinesis failure after nuclear division. at later ages, opcs progressively disappeared from the cortical gm, while in both wm and ventral areas of the telencephalon (i.e. striatum, hypothalamus), uni-and multi-nucleated opcs could be detected, although at lower densities compared to wt. these data suggests opc heterogeneity in both cit-k requirement for cytokinesis and susceptibility to death. however, even where normal uninucleated opcs persisted, we could not find pre-myelinating or myelinating cells, in line with additional differentiation defects. to discriminate the contribution of cell-autonomous vs. environmental factors in differentiation impairment, we performed homochronic grafts of wt fluorescently tagged (gfp1) cells into perinatal cit-ko mice. strikingly, gfp1 opcs invaded the whole cit-k ko brain and actively divided. however, they hardly ever differentiated into more mature cells at difference with cells grafted in the wt, indicating that altered environmental signals contribute to myelination defects in cit-k ko. in conclusion, our results show that cit-k is involved also in glial cell divisions and suggest distinct cit-k requirement and proneness to death of dorsal and ventral opcs. additionally, cit-k ablation results in pervasive nervous tissue alterations affecting opc maturation. further studies will clarify the molecular mechanisms underlying these alterations. knowledge of how these cells act in repair is lacking, partly due to a limited understanding of their behaviour in normal developmental processes. radial glia cells are a unique population of neural progenitors that have recently been suggested to function in neurogenesis in the cerebral cortex. however, many questions still remain regarding their specific role in the developing spinal cord. using an organotypic spinal cord slice culture model and two-photon microscopy we directly visualized radial glial cell behaviour in the developing spinal cord. 400 mm slices of rat embryonic spinal cord (e12-e16) were cultured in order to preserve the 3d cytoarchitecture of the cellular environment. using electroporation, slices were transfected with a brain-lipid binding protein (blbp) driven gfp expression plasmid, which specifically labels endogenous radial glial cells. slice cultures were then imaged using two-photon time-lapse microscopy, allowing for high spatially and temporally resolved 4d imaging. we were able to follow individual gfp expressing radial glial cells over time (24 hours to 6 days) and characterized precise morphological changes. at early developmental ages, gfp expressing cells initially exhibited a radial morphology with the soma located in the ventricular zone and processes extending out to the pial surface. over time, many cells developed a multipolar morphology and migrated away from the lumen, primarily using somal translocation. using immunohistochemistry we identified populations of transfected cells which also expressed gfap, representing the differentiation of blbp-expressing radial glia cells into astrocytes. we also found that radial glial cells differentiate into oligodendrocytes (olig2 and cnpase positive) but found little evidence for gfp co-localization with neuronal markers (neun, dcx, biiitubulin). using this model we can directly observe complex developmental behaviours of specific cell populations and elucidate the relevant genetic regulation and molecular mechanisms that govern spinal cord development. by understanding these intricate events, we will gain insight into how a simple tube of undifferentiated neuroepithelial cells generates different cell populations that will eventually develop into the adult spinal cord. methods: we used the new cre-expressing mouse strain cx 3 cr1 creer to express a diphtheria toxin receptor (dtr) specifically on microglia/macrophages. in these mice, administration of tamoxifen leads to cre-mediated excision of a loxp flanked transcriptional stop element in both macrophages and microglia, thus allowing for the expression of the dtr as well as an yfp reporter. due to their fast turnover macrophages are replaced by unaffected precursors whereas microglia persist in the modified stage. these mice were used for in vivo and ex vivo analysis of microglia by facs and histology. results: our optimized protocol with two tamoxifen injections at the age of 2 weeks resulted in 80-90% of reporter-positive microglia in adulthood, whereas all peripheral macrophages were reporter-negative. with additional three successive dt injections we could achieve a microglia ablation efficiency of 80%. interestingly, histological as well as facs analysis revealed 50% of microglia repopulation already 4 days after depletion. two weeks after depletion microglia numbers were even higher compared to unaffected controls. on day 6, we found a cd45.2 high ly6c 1 population, indicating replenishment by peripheral monocytes. in bm chimera experiments, where peripheral cells were labeled with a congenic marker, we obtained striking evidence showing that almost all of the repopulating cells are of peripheral origin. conclusions: after depletion microglia were rapidly repopulated, which emphasizes their critical role in brain homeostasis and function. even though ms has an onset in young patients, it persists throughout life and progresses along adulthood and aging. therefore, mscs from aged individuals should be tested for their pro-oligodendrogenic activity before moving into the clinical trials. this is insofar of clinical relevance, since the brain's capacity for self-repair and remyelination strongly declines with aging. thus, the aim of this study was to determine whether mscs from aged individuals maintain their known prooligodendrogenic activity. mscs were obtained from bone marrow of young (2 months) as well as old (17-20 months) rats and analyzed their stem cell properties as well as their pro-oligodendrogenic activity. as expected, mscs derived from aged bone marrow presented diminished stem cell properties and differentiation capacity. to test the msc prooligodendrogenic activity, npcs were incubated with msc conditioned medium. surprisingly, soluble factors derived from old rats mscs maintain their ability to induce an increase of mbp-expressing oligodendrocytes on npcs. moreover, aged mscs conserve their oligodendrogenic activity also on npcs derived from old donors (20 months). these results indicate that mscs keep their oligodendrogenic activity regardless of age and, therefore, this study provides a rationale for clinical trials of autologous msc transplantation in ms therapy. 6 munich cluster for systems neurology (synergy), munich, germany astrocytes fulfill essential functions under physiological conditions and are involved in various beneficial and adverse functions after brain injury (reviewed by sofroniew 2009). in order to improve functional recovery after injury, it is essential to delineate the beneficial from adverse effects. however, little is yet to know which extent distinct sets of astrocytes perform distinct functions or whether all astrocytes behave in a uniform manner. live imaging of astrocytes after stab wound injury in the adult cerebral cortex in vivo revealed a striking heterogeneity of astrocyte behaviour with distinct subsets extending long processes towards the site of injury, others proliferating and yet other subtypes undergoing only hypertrophy. most strikingly, the vast majority of astrocytes proliferating were located with their somata directly at blood vessels (capillaries or post-capillary vessels). this close proximity was confirmed at ultrastructural level and identified as juxtavascular positions, sometimes adjacent to pericytes located in the perivascular space. this novel population of reactive astrocytes at juxtavascular positions is of particular interest, as the capacity of astrocytes to reactivate proliferation ( cell stem cell, in press). therefore we examined whether this population of juxtavascular astrocytes would also preferentially proliferate in even more severe injury conditions, such as 1 hour of middle cerebral artery occlusion (mcao). the proliferation of juxtavasular astrocyte was analyzed immunohistochemically in fixed brain sections co-stained for s100b/gfap/ki67/cd31 at different time points after injury. at 3 days post injury, 18% of all astrocytes were actively dividing (ki671). strikingly the majority of these was again located at juxtavascular positions (68%), suggesting a general bias of astrocytes at this position to proliferate after injury, while astrocytes further distant from blood vessels virtually fail to divide. we shall further present data from conditional knock-out mice with reduced proliferative activity of juxtavascular astrocytes in order to elucidate the function of this highly specific astrocyte population and their proliferative reaction after injury. a.d. rivera, a. butt university of portsmouth, portsmouth, united kingdom the generation of oligodendrocytes (ols) from their precursors (opcs) occurs throughout adult life and is particularly important following demyelinating insults, such as occurs in multiple sclerosis (ms). glycogen synthase kinase 3-b (gsk3b) acts as a constitutively active negative regulator of multiple pathways that regulate proliferation, survival and differentiation of opcs. lithium is a potent inhibitor of gsk-3 and is used for the treatment of bipolar disorder, but in addition is neuroprotective in a wide range of diseases, including stroke, amyotrophic lateral sclerosis, and alzheimer's disease. lithium inhibits gsk-3 by binding directly to the enzyme's magnesium-sensitive site and indirectly by enhancing its phosphorylation. notably, by inhibiting gsk3b, lithium acts as a wnt mimetic to promote b-catenin-dependent transcriptional events, including enhancement of genes involved in apoptotic inhibition. here, we have examined the effects of lithium on oligodendrocytes in the adult mouse optic nerve, a typical cns white matter tract; all procedures were in accordance with the animals scientific procedures act (1986) . transgenic mice aged postnatal day (p) in the developing and adult cns multipotent neural stem cells reside in distinct niches. specific carbohydrates and glycoproteins are expressed in these niche microenvironments that are important regulators of cell fate determination and stem cell maintenance. in previous studies we described lewisx (lex) glycan as a specific marker of neural stem precursor cells (nspcs) in developing brain and showed it to be involved in nspcs migration and proliferation. using lex glycosylation as a biomarker we aimed to identify the lex carrier glycoproteins which could have a functional relevance for cns development. in addition to already known lex carriers we have found lrp1 as new major lex carrier and for the first time showed it's expression in nspcs and developing brain. lrp1 is essential for the normal neuronal function in adult cns, whereas the role of lrp1 in development is still unclear, mainly due to the lethality of the lrp1 knock-out in mice. in the current study we investigated the basic properties of lrp1 knock-out nspcs created by means of cre-loxp mediated recombination. the elimination of lrp1 in vitro was induced by the addition of cell permeable cre-recombinase to nspcs derived from embryonic brain of lrp1flox/flox mice. the functional status of lrp1-deficient cells was subsequently studied using proliferation, migration and differentiation assays. lrp1 knock-out cells maintained as neurospheres, retained the ability to migrate and differentiate. interestingly, lrp1 knock-out nspcs generated 3-times less opcs in comparison to lrp1wt/wt cells. this suggests that lrp1 is involved in the control of the oligodendroglial differentiation. astrocytes are multifaceted cells in the adult central nervous system and react to pathology of the adult brain with a finely graded continuum of morphological and behavioral changes. given that astrocytes in healthy adult brain rarely divide outside the stem cell niches, but activate both proliferation as well as a stem cell potential after injury (for review see robel et al., 2011) , it is important to understand their exact mode of cell division. indeed, a defining hallmark of a stem cell is the capacity to self-renew -often by asymmetric cell division. therefore we set out to image reactive astrocyte divisions in vitro by developing a primary culture system for reactive astrocytes obtained from the somatosensory cortex of adult mouse at 5 days after stab wound injury. continuous live imaging and single cell tracking (costa et al., 2008 (costa et al., , 2011 allowed us observing the mode of cell division mode as well the cell fate of their immediate progeny. results of our study reveal (i) that reactive astrocytes can undergo multiple rounds of cell division, (ii) the shh signaling has a profound influence on this behavior, (iii) reactive astrocytes divide with distinct modes of cell division, which can be influenced by the local microenvironment. taken together, this new culture system allows close examination of signals on the mode and multitude of reactive astrocyte cell divisions and provides a great tool to identify signals derived from the extracellular matrix, other cell types or as diffusible signals. interestingly, even without injury temporary depletion of proliferating opcs leads to behavioral deficits which will be described. to identify the molecular mechanisms by which ng2 1 cells may react and influence other cells after brain injury, we isolated sox10-gfp 1 cells by facs either from the intact or the lesioned cerebral cortex at three days after stab wound injury. comparative genome-wide expression profiling revealed exciting candidates for opc function in the physiological and pathological brain. oligodendrocytes are the myelin forming cells of the central nervous system (cns) that enable fast salutatory signalling transduction and mediate trophic support and protection of axons. oligodendrocytes originate from a population of precursors cells (oligodendrocyte precursor cells, opcs) that are formed at distinct developmental stages. the biological process leading to opc differentiation involves a cascade of molecular events that eventually constitute the complex and multifaceted cellular program that determines the generation of mature oligodendrocytes. to identify the earliest detectable transcriptional correlates of differentiation we used a microarray approach investigating changes in gene expression occurring in primary rat opcs within the first 12 hours after purification. amongst the most regulated factors that were detected was hairless (hr), a nuclear receptor co-repressor that has been associated with thyroid hormone signalling in the neonatal cns. validation of the microarray results by rt-qpcr, western blot and immunochemistry confirmed a rapid increase of hr expression during opc differentiation in media containing thyroid hormone. in the absence of thyroid hormone, hr expression was suppressed and associated with impaired opc differentiation. similarly, rnai mediated gene silencing of hr induced an impairment of opc differentiation demonstrating an important functional role of hr in the differentiation program. thyroid hormones regulate hr expression via the thyroid hormone receptors thra and thrb as rnai-mediated gene silencing of each of the receptors resulted in a decrease in hr expression. hr is known to negatively regulate rar-related orphan receptor alpha (rora) and vitamin d receptor (vdr). however, rnai-mediated gene silencing of rora and vdr did not change the course of opc differentiation. interestingly, an interaction between hr and histone de-acetylases (hdacs), of which hdac1 and 2 are known regulators of opc differentiation, has been noted in the past. using an in situ protein-protein interaction assay we were able to demonstrate a direct interaction of hr with hdac1 and hdac2 in differentiating opcs. moreover, chip qpcr analysis revealed hr-hdac1 recruitment to the opc differentiation inhibitor hes5. taken together these data show that thyroid hormone promotes opc differentiation via hr by modulating hdac1 and 2 function. as iodine deficiency results in hypothyriodism our results give an example of a developmental disease that is triggered by environmental factors (the absence of iodine) causing epigenetic dysregulation. c. stagni, a.d. rivera, a. butt university of portsmouth, portsmouth, united kingdom astrocytes respond to all forms of cns insults by reactive astrogliosis, which involves changes in their molecular expression and morphology, and in most severe cases glial scar formation. reactive astrogliosis is potentially neuroprotective, but can also act as chemical and physical barrier to axon growth and repair. however, mechanisms underlying this process are still poorly understood. the enzyme glycogen synthase kinase 3-b (gsk-3b) is a key regulator of many signalling pathways involved in cell proliferation, survival and differentiation, including wnt signalling. lithium is a potent inhibitor of gsk-3 and acts as a wnt mimetic to promote b-catenin-dependent transcriptional events. wnt signalling is altered following cns injury and in a range of neurodegenerative diseases, such as stroke, amyotrophic lateral sclerosis, and alzheimer's disease. notably, lithium is neuroprotective in these same diseases. here, we have examined the effects of wnt3a and lithium on astrocytes of the adult mouse optic nerve, a typical cns white matter tract; all procedures were in accordance with the animals scientific procedures act (1986). we used transgenic mice aged postnatal day (p) 35 inwhich gfap drives the expression of egfp. optic nerves (ons) were isolated with the retina intact and maintained in organotypic culture for 3 days in vitro (div), in control medium, or medium containing lithium chloride, or wnt3a. both agents resulted in a significant increase in the number of astrocytes, compared to controls (p < 0.05, unrelated t-tests), but lithium and wnts3a had markedly divergent effects on astrocyte morphology. in the presence of lithium, astrocytes became highly polarised, extending long thin processes that traversed the nerve perpendicularly to the long axis. in contrast, wnt3a treatment resulted in the generation of morphologically simple astrocytes, with short, fine branching processes that extended radially from a centrally located cell body. genome wide microarray analysis indicated wnt3a and lithium differentially altered chondroitin sulphate proteoglycans (cspgs), a family of extracellular matrix molecules highly expressed at cns injury sites. further experiments are required to define the molecular phenotypes of the novel astrocytes generated by these treatments, but the results provide evidence that wnt signalling regulates astrogenesis in situ and is therefore a potential molecular target to modulate astrogliosis. the contrasting effects of lithium suggest it is acting via other pathways, which require further study. studies of mouse schwann cell culture. the proliferation of scs is regulated by axonal signals and several growth factors. growth factors that are essential for rodent sc survival in culture include heregulin-b1 and forskolin. in the case of rat scs, these factors are also sufficient to allow a robust proliferation. in contrast, the proliferation of mousederived scs in culture requires the presence of other growth factors. we studied effect of several factors known to be expressed in scs after nerve injury on mouse sc proliferation. these included fibroblast growth factor type 2 (fgf), pituitary extract (pe), epidermal growth factor, platelet-derived growth factor bb, and transforming growth factor b1. we found that heregulin-b1 and forskolin are essential for mouse sc to survive in culture, fgf and pe were the best combination of growth factors promoting the mouse sc proliferation, and that neuronal axons provide effective mitogenic environment for mouse scs. the expression of the large myelin-associated glycoprotein in pns was studied in scs cultured alone and in cocultures of scs and drgns. when schwann cells are alone without axons, expression of the cytoplasmic domain of l-mag is seen in the nucleus of schwann cells. when schwann cells make contact with neuronal axons, the location of the expression shifts out of the nucleus to the perinuclear and cytoplasmic regions; the extracellular domain is not visible. until the schwann cells initiate the myelination programme. these results support the hypothesis that l-mag might have role in the regulation of the myelination programme in the pns. it has been postulated that trophic support provided by either stem cells or precursors is actuallyone of the most beneficial effects in the cell-based therapies. to address this issue, a comparison between the neuroprotective properties of opcs and the stem cells derived from warton jelly was carried out in the co-culture system with ischemically injured organotypic hippocampal slices. those two cell types were selected in respect of the advancement in their differentiation process. while the mesenchymal stem cells residing in warthon jelly are still pluripotent, the opcs are already the glia-committed precursors. to evaluate their neuroprotective properties, we set -up an ex vivo co-culture system of either stem cells or opcs isolated from neonatal rat brain with organotypic hippocampal slices subjected to a short oxygenglucose deprivation (ogd). the cell death, as well as the number of the newborn cells has been quantified in slices co-cultured for one week. a microarray analysis of a broad spectrum of trophic factors and cytokines expressed by opcs was performed for the purpose of finding the factor(s) contributing to the observed effect. three of them were selected for the subsequent blocking experiments with specificl antibodies to verify their influence on the injured tissue. the results obtained in the study prove that both cell types secrete soluble factors and are potent in exerting the neuroprotective effect in a paracrine-like manner, promoting cell survival and proliferation in damaged hippocampal slices. in conclusion, the observed advantageous qualities of stem cells and oligodendroglia-biased progenitors significantly contribute to anticipating a clinical improvement in cell replacement therapies. ng2 is a type i transmembrane glycoprotein and also known as chondroitin sulphate proteoglycan 4 (cspg4). in the central nervous system ng2-expressing cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes in the developing white matter. for temporally-controlled gene targeting of ng2 glia in vivo, we generated a mouse line in which the open reading frame of the tamoxifen-inducible form of cre recombinase (creert2) was inserted into the ng2 locus by homologous recombination. here, we investigated the differentiation potential of ng2 glia at different developmental stages of the forebrain. cre recombinase activity was induced at embryonic day 17.5, postnatal day 0 (p0), p3, p8, p30 and p60 by intraperitoneal tamoxifen injections into novel tgh(ng2-creert2) mice crossbred to rosa26-tdtomato and rosa26-eyfp reporter lines that helped to identify ng2 glia and its progeny. recombination and cell identification were determined 10 to 60 days after the first tamoxifen injection. induction of recombination at embryonic stages revealed that embryonic ng21 cells mainly generated more ng2 glia and oligodendrocytes, however, a significant number of astrocytes could be detected as well. recombined cells were predominantly restricted to the ventral brain. in contrast, after tamoxifen injections at p0 and p3 recombined cells were widely found in all brain regions, thereby suggesting the presence of a distinct subpopulation of ng21 cells after birth. interestingly, only very few recombined astrocytes could be found, which are probably derived from few remaining embryonic ng2 glia. starting from p8, ng2 glia stopped generating astrocytes, with the progeny largely restricted to the oligodendrocyte lineage. however, consistently, we found recombined neun1 cells with the morphology of neurons in the ventral cortex after administration of tamoxifen at p8. even more of such cells were present in the whole cortex when cre activity was induced in adult mice. the morphology, the presence of neun immunoreactivity and our electrophysiological characterization that demonstrated bona fide action potentials clearly identify these cells as functional neurons. our results suggest that ng21 cells display a broad differentiation potential that appears to be highly age-dependent during development. brdu-assay) and the number of surviving cells (by counting dapi-stained nuclei). after we had observed, that the choice of the basal medium (neurobasal, rpmi or dmem) exerts a pronounced effect on opc proliferation we here investigated the effect of cell density on opc proliferation. starting from a small seeding density (10.000 cells/cm 2 ) a tenfold increase in seeding density only resulted in a fourfold augmentation of the density of surviving cells after 7 days in culture. using plating densities of 5.000; 20.000 and 80.000 cells/cm 2 we observed, that an increase in seeding density led to a decrease in the proliferation rate, as tested with a brduassay and monitored after 3 days in culture. similarly, the percentage of a2b5-positive, immature, cells was lower, the higher the cell density. we then tested whether this finding is due to proliferation inhibiting factors secreted in cultures with high seeding densities and whether the remaining microglia population could be involved. to this aim we seeded opcs at low density (5.000 cells/cm 2 ) and cultivated them with the medium supernatant of cultures with high density (80.000 cells/ cm 2 ). furthermore we seeded opcs in low density (5.000 cells/cm 2 ) and co-cultivated them with an added density of microglia as counted in cultures of high density (80.000 cells/cm 2 ). the resulting cell counts after 3 days showed, that the percentage of brdu positive cells after cultivation with the supernatant was significantly reduced compared with control cultures, although the number of a2b5-positive cells was not influenced by this treatment. the addition of microglia to the cultures lead to a strong reduction of surviving cells, even though the percentage of brdu and a2b5 positive cells were not altered. so apoptosis, necrosis or phagocytosis might be responsible for the reduction of surviving cells. our findings suggest that the reduction of surviving opcs in high density cultures is due to secreted factors inhibiting opc proliferation and that microglia could be involved in this effect. in neuropathological studies, it is important to detect microglial cells; however, a specific microglia marker has yet to be determined. [methods] in this study, we examined and compared the usefulness of various microglia markers, including human glucose transporter-5 (glut-5), mhc class ii, cd68, and iba-1. [results] all of the microglia markers consistently stained for microglia in paraffin sections fixed with formalin. cd68 staining of microglia in the gray matter was not as well defined as the other microglia markers. moreover, cd68 did not clearly stain the microglial processes. with regard to mhc class ii, it was difficult to observe the microglial processes in the white matter. in contrast, both glut-5 and iba-1 clearly stained for microglia in the gray and white matter, and provided detailed staining of microglial processes. although it has previously been shown that perivascular cells are negative for glut-5, we found perivascular macrophages to be glut-5 positive. [conclusions] nevertheless, glut-5 and iba-1 may be considered as markers suitable for routine histopathological staining procedures. neurons and glia of the enteric nervous system (ens) originate from a pool of sox101 progenitors. in addition to being a scaffold for enteric neurons, enteric glial cells (egcs) have been suggested to function in mucosal integrity, neuroprotection, adult neurogenesis, neuro-immune interactions and synaptic transmission. currently, diverse glial populations are known to reside within the ens. however, whether, specific functions are carried out by specialized glial subtypes, with characteristic morphologies and molecular markers and within specific locations in the gut remains elusive. to address this question we used a repertoire of genetic tools, immunostaining and imaging techniques. for high single-cell resolution study of egcs, we mosaic analysis with double markers (madm) in conjunction with sox10-cre. we analyzed adult gut tissue to characterize the different glial populations based on morphology, position in the ens and their relationship with the intestinal epithelial and vascular system. moreover, we used reporter mice (sox10-icreer t2 ; r26r-yfp and gfap-icreer t2 ; r26r-yfp) to quantify the expression of three glial markers -gfap, s100b and sox10 in myenteric plexus (mp) preparations of adult mice. our investigation on madm-mp preparations, revealed three subtypes of enteric glia in the mp of adult mice. type i and type ii glia, present in the primary plexus of mouse ens have been previously defined. in addition, we characterized a third subtype in the extraganglionic space of the mp of adult mice, at present termed as type iii-extraganglionic glia. furthermore, our analyses using 3d-imaging, on madm-gut sections allowed us to characterize the relationship of enteric glia located within the mucosa, with the intestinal epithelium and vascular system of the villi. our marker expression analysis showed that while the majority of glial cells co-expressed gfap and s100b, a large fraction of glia within the myenteric ganglia was positive for either s100b ($30%) or gfap ($10%). we also found that s100b 1 /gfapglia were abundant outside the ganglia. most glial cells co-expressed sox10 with gfap and s100b yet a significant proportion ($10%) of mainly extra-ganglionic glia (type iii) expressed only sox10. surprisingly, $5% of the cells that were s100band sox10displayed high gfap expression. use of gfap-icreer t2 ; r26r-yfp mice showed that about $30% of yfp-labelled cells did not express gfap, implying dynamic regulation of gfap expression. overall, our high resolution studies reveal extensive heterogeneity of enteric glial cells in terms of morphology, position and expression of molecular markers suggesting differential functional roles. our future experiments will address whether different physiological roles of egcs can be assigned to specific subtypes. university of washington, seattle, united states m€ uller glia are the major type of glia in the retina, spanning its entire thickness. it is known that bmp signaling promotes astroglial differentiation in cortex, but its role in m€ uller glial development in the retina has not been studied. the purpose of this study was to investigate the role of bmp-smad1/5/8 signaling in m€ uller glial development. c57bl/6 mouse retinas were collected at various ages between postnatal day 0 (p0) and p21 in order to analyze the expression and activation of smad1/5/8. smad1/5/8 was transiently but robustly activated in the inner nuclear layer, including developing m€ uller glia between p5-p8. the timing of this transient activation of smad1/5/8 corresponds with the timing of m€ uller glial differentiation and maturation. to test the hypothesis that activation of bmp-smad1/5/8 signaling is essential for m€ uller glial development, retinas were explanted at p3 or p6 and treated with bmp4 or a bmp inhibitor dorsomorphin (dm) for 5 days in order to activate or inhibit smad1/5/8, respectively. when p3 and p6 retinal explants were treated with dm for 5 days in vitro, there was a significant reduction in m€ uller glial markers (cralbp, gs, sox9, and sox2). smad1/5/8 activation and cralbp expression were also significantly attenuated in vivo 1 day after intraocular injection of a bmp inhibitor dm at p5. our data suggest that activation of bmp-smad1/5/8 signaling is necessary for the development of m€ uller glia. this work was supported by 1r01ey021482 to tar. huazhong university of science and technology, wuhan, china rho-associated kinase (rock) has been identified as an important regulator of proliferation and cell cycle progression in a number of cell types. although its effects on astrocyte proliferation have not been well characterized, rock has been reported to play important roles in gap junction formation, morphology, and migration of astrocytes. in the present study, our aim was to investigate the effect of rock inhibition by [(1)-(r)trans-4-(1-aminoethyl)-n-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] (y27632) on proliferation and dna synthesis in cultured astrocytes from rat spinal cord and the possible mechanism involved. western blots showed that treatment of astrocytes with y27632 increased their expression of cyclin d1, cdk4, and cyclin e, thereby causing cell cycle progression. furthermore, y27632-induced astrocyte proliferation was mediated through the extracellular-signalregulated kinase signaling cascade. these results indicate the importance of rock in astrocyte proliferation. here we used cell transplantation experiments to determine to which extent this differential behavior is regulated by environmental signals differing between the wm and gm, or intrinsic fate determinants restricted to one or the other cell population. these experiments indicate that the wm promotes differentiation of opcs to mature oligodendrocytes, as the rate of differentiation of gm cells increased when exposed to this environment. interestingly, cells derived from the wm retained their higher differentiation rate also in a less supportive environment like the gm. these results reveal not only important environmental differences for opc maturation, but also support the concept of intrinsic heterogeneity among adult opcs, persisting even when exposed to more inhibitory environments. thus, this work provides the basis to identify molecular pathways regulated by distinct niche/environmental signals and involved in the heterogeneity of adult opcs. multiple sclerosis (ms) is a chronic inflammatory and neurodegenerative demyelinating disease of the central nervous system (cns) characterized by inflammation, which leads to formation of demyelinating areas due to loss of oligodendrocytes, astrogliosis and, finally, axonal degeneration. different studies have suggested that 3 0 -5 0 -cyclic adenosine monophosphate (camp) levels might play an important role in neuroprotection and neuroinflammatory response so the modulation of this nucleotide intracellular levels might control the neuroinflammatory pathological process and, consequently, to delay the progression of ms. intracellular camp levels depend on its synthesis by adenylyl cyclases, and on its degradation by cyclic nucleotide 3 0 ,5 0 -phosphodiesterases (pdes). specific inhibitors for the different isoforms of pdes family, particularly camp-specific pde, emerge as putative treatments of this kind of disease. pde7 has emerged as a new therapeutic target, not only for a variety of immunological and immunodeficiency conditions to alleviate chronic inflammation, but also for several neurodegenerative disorders, including ms, in which both immune system and cns are implicated. in the present work, we have detected the expression of pde7 in oligodendrocyte precursor cells (opcs) isolated from cerebral cortex of p0 and p15 mice and from adult human samples derived from neurosurgery of epilepsy. opcs are abundant in the mice and human cns during development but they also represent 5-7% of the total number of cells in the adult cns, where they serve as a source of new oligodendrocytes to replace those which die. however, in ms these endogenous opcs are not able to effectively replace dead oligodendrocytes. in vitro, we have demonstrated that two newly-developed pde7 selective inhibitors (tc 3.6 and vp 1.15) favour opc survival and differentiation towards mature oligodendrocytes, being both more effective than the commercial pde7 inhibitor brl-50481. erk intracellular pathway has been identified as a key in these cellular processes related with the camp/pka/creb pathway. moreover, we have observed that both specific inhibitors (vp1.15 and tc3.6) increase the differentiation of human opcs. these findings, combined with the already known anti-inflammatory effect carried out by these pde7 inhibitors, point them as potential therapeutic agents to treat ms. the knowledge of factors that affect the biology of murine opcs and even more, human opcs, are critical for possible remyelination in this kind of pathologies. their role in cns remyelination has not been fully investigated. previous studies using genetic fate mapping techniques revealed their recruitment into injured spinal cord and differentiation into astrocytes and oligodendrocytes. in this study, we used aninducible cre-floxed-stop-rosa26-yfp mediated lineage tracing strategy to characterize the fate of foxj1 expressing ependymal cells during remyelination of toxin-induced demyelination. we found that in unlesioned spinal cord, foxj1 labelled ependymal cells. however, it also labeled a population of cells in the spinal roots with immnochemical features of fibroblasts. following lysolecithin-induced demyelination in the dorsal funiculus, there were few yfp expressing cells in the lesion area at 5 and 14 days post lesion (dpl) indicating that the ependymal cells were not recruited in demyelinated area. however, in ventral white matter lesions, which were adjacent to damaged nerve roots, many cells were detected expressing yfp. the distribution of yfp positive cells overlapped that of remyelinating schwann cells and a number of yfp expressing cells colocalised with mature schwann cell marker periaxin. these data revealed a foxj1 expressing population in peripheral nerve which migrates into demyelinated lesions andcontributes to cns remyelinaiton. the generation of myelinating cells from multipotential neural stem cells in the cns requires the initiation of specific gene expression programs in oligodendrocytes (ols). we reasoned that micrornas (mirnas) could play an important role in this process by regulating genes crucial for ol development. here we identified mir-7a as one of the highly enriched mirnas in oligodendrocyte precursor cells (opcs), overexpression of which in either neural progenitor cells (npcs) or embryonic mouse cortex promoted the generation of ol lineage cells. blocking the function of mir-7a in differentiating npcs led to a reduction in ol number and an expansion of neuronal populations simultaneously. we also found that overexpression of this mirna in purified opc cultures promoted cell proliferation and inhibited further maturation. in addition, mir-7a might exert the effects just mentioned partially by directly repressing proneuronal differentiation factors including pax6 and neurod4, or prool genes involved in oligodendrocyte maturation. these results suggest that mirna pathway is essential in determining cell fate commitment for ols and thus providing a new strategy for modulating this process in ol loss diseases. a. guzman de la fuente 1 , j. huang 1 new sheaths can be regenerated following the recruitment of adult neural progenitor cells (opc) into areas of injury and their differentiation into myelinating oligodendrocytes. although this regenerative process (called remyelination) occurs efficiently in the early stages of multiple sclerosis, its efficiency gradually declines leaving axons demyelinated and vulnerable to degeneration. several signalling pathways are involved in regulating opc differentiation and subsequent myelination. understanding their mechanisms is necessary to design regenerative therapies to overcome remyelination failure. we have identified rxrc as a key positive regulator of opc differentiation and remyelination (huang et al., 2011). rxrc is a nuclear receptor forms a heterodimer with other nuclear receptors to activate its downstream signalling cascades. here we describe rxrc binding partners expressed in both opc and oligodendrocytes, and the binding of vdr, rarb and pparc to rxrc within oligodendrocyte lineage cells by coimmunoprecipitation. vdr-rxrc heterodimer is necessary for the oligodendrocyte differentiation. treatment of oligodendrocytes with vdr antagonist inhibits opc differentiation and abrogates the effect of 9-cis retinoic acid, an rxrc agonist, in opc differentiation. our data suggests a model in which rxrc is an essential anchor for different nuclear receptors, including vdr, with a shifting and complex rxrc signaling in oligodendrocyte lineage cells. cleavage of type i membrane proteins by a-and c-secretases to yield biologically active fragments is best exemplified for the evolutionarily conserved protein notch. here we show that the protein ng2, a type i membrane protein which has homologues in mouse (ng2) and drosophila (kontiki) and is expressed by multiple immature cell types including glia, is processed in a similar fashion to notch. in both mouse and drosophila this generates a major 290 kd shedded ectodomain, a c-terminal fragment (ctf) and a small intracellular domain (icd). the ng2 icd is present after overexpression in cells of biochemical isolates of nuclear fractions, and can be visualized in the nucleus by immunofluorescence of cultured murine glial cells. in glial nuclei of developing drosophila larva, a striking intranuclear staining of the kontiki icd is observed. levels of expression of ng2 mrna and protein in mouse glial cells are regulated by the neurotransmitter glutamate and overexpression of cleaved fragments modulates protein expression in murine glial cells. these results demonstrate that cleavage of ng2 takes place in mouse and drosophila glia and that this generates fragments with signaling properties. modulation of ng2 expression by glutamate provides a way for neuronal activity to regulate ng2 signalling. our goal is to understand the glial signaling that controls inflammatory responses in the central nervous system (cns). this glial response can play both a detrimental and beneficial role. toll like receptor (tlr) signaling is implicated in responses to pathogens or endogenous signals. tlr signaling mediates immune response by inducing cytokines, including type i interferons (ifn). ifn-beta, a member of the type i ifn family, is a first-line therapeutic for multiple sclerosis. type i ifns signal through a common receptor, ifnar. the aim of the present study was to investigate the in vivo response of microglia and astrocytes to cns administration of tlr ligand/agonist, and to examine whether this response involves type i ifn signaling. mice were administered tlr ligands/agonists by injection to the cisterna magna. we analyzed leukocyte entry to the cns by flow cytometry, and their localization by immunostaining. the induction of ifn-beta was examined by in vivo imaging of an ifn-beta reporter mouse. astrocytes and microglia were sorted by facs and gene expression was measured using quantitative real time-pcr. injection of ligands/agonists for tlr2, 3 and 4 resulted in infiltration of cd451 leukocytes after 18 hrs, most strongly by tlr2. immunostaining showed parenchymal localization of infiltrating cells in cerebellum. facs sorted astrocytes expressed equivalent levels of tlr3 mrna to microglia but lower levels of tlr2 or 4. astrocytes were induced by tlr3 signaling to express interferon regulatory factor 7, which regulates the induction of type i ifn. the induction of ifnbeta in cns in response to tlr3 signaling was verified in ifn-beta reporter mice. tlr2, 3 and 4 signaling led to increased levels of mrna for glial cxcl10. together these results suggest the involvement of type i ifn signaling. however, unlike cxcl10 gene expression, that was dependent on ifnar signaling, tlr2-induced leukocyte infiltration was not affected in ifnar deficient mice. these studies point to a role for tlr signaling in the innate glial response that regulates cns inflammation. microglial phagocytosis plays a key role in neuroprotective and neurodegenerative responses of the innate immune system in the brain. here we investigated the regulatory function of the signaling protein phosphoinositide 3-kinasec (pi3kc) in phagocytosis of bacteria and zymosan particles by mouse brain microglia in vitro and in vivo. using genetic and pharmacological approaches our data revealed pi3kc as an essential mediator of microglial phagocytosis. unexpectedly, microglia expressing lipid kinase deficient mutant pi3kc exhibited similar phagocytosis as wild-type cells. detailed analysis disclosed pi3kc dependent stimulation of camp phosphodiesterase as a crucial mediator of phagocytosis. both stimulation of protein kinase a and epac were shown to convey inhibitory effects of camp on phagocytosis. together our findings indicate pi3kc-dependent suppression of camp signaling as an indispensible regulatory element of microglial phagocytosis. v. kovaleva, e. berezhnaya, m. rudkovskii, a. uzdensky southern federal university, rostov-on-don, russian federation photodynamic therapy (pdt) is currently used in oncology, particularly, in treatment of brain tumors. the possible role of no-mediated signaling in photodynamic injury and protection of neurons and surrounding glial cells (gc) was studied. crayfish stretch receptor consisting of a single neuron enveloped by gc was photosensitized with alumophthalocyanine photosens (10 nm, 30 min incubation) and irradiated with laser diode (670 nm, 0.4 w/cm2). application of no generators notably 10 mm sodium nitroprusside and 10 mm nonoate reduced pdtinduced necrosis of gc and showed the same tendency in neuronal necrosis. nonoate significantly increased pdt-induced apoptosis of gc. inhibitor of neuronal no-synthase l-name (1mm) significantly increased the percentage of necrotic glial cells after pdt but did not influence neuronal necrosis. this confirmed the anti-necrotic effect of no in glial cells and involvement of neuronal no synthase in protection of glial cells. 1 mm l-name and 1 mm l-nna (another inhibitor of neuronal no-synthase) as well as 50 lm s-methhylisothioharnstoff sulfat (inhibitor of inducible no synthase) protected gc from pdtinduced apoptosis. therefore no may be involved in pdt-induced apoptosis of glial cells. inhibition of no-activated protein kinase g with 10 lm kt5823 decreased the percentage of necrotic glial cells but not neurons. therefore protein kinase g appeared to be involved in pdtinduced necrosis of gc, possibly independently on no. kt5823 also increased the level of apoptosis of gc indicating the anti-apoptotic role of protein kinase g. thus no is involved in regulation of pdt-induced necrosis of neurons and glial cells as well as in apoptosis of glial cells. g. tyzack, t. cymes, n. lau, a. lakatos university of cambridge, cambridge, united kingdom background and question. emerging evidence suggests that signalling by damaged neurones leads to reactive astrocyte response that may have a fundamental impact on synaptic reorganisation. soluble factors, such as cytokines, have been described to induce such astrocyte activation. however, rapid contact dependent signalling between damaged neurones and astrocytes is also a possibility. in light of recent findings of ephb1 upregulation on injured neurons closely surrounded by reactive astrocytes, we hypothesized that ephb1 can signal to ephrin-b1 expressing astrocytes. methods. to test this in simplified in vitro assays, purified astrocyte cultures were treated with clustered-ephb1-fc, and the characteristic features of glial activation, such as cytoskeletal protein expression, stat3 phosphorylation, and p-stat3 nuclear transfer were monitored. results. we have demonstrated that 1) astrocytes express ephrin-b1, 2) ephb1 triggers a two-fold increase in gfap and ezrin expression, 3) ephb1 induces both stat3 phosphorylation and nuclear transfer by a three-fold value, and 4) all effects were significantly diminished by knocking down ephrin-b1 in astrocytes. conclusions. our data show that ephb1 induces astrocyte activation by triggering ephrinb1-mediated reverse signalling via stat3 activation. this work therefore suggests a potential novel route in neuron-astrocyte communication after injury. a further characterisation of this signalling pathway may provide a better understanding of mechanisms underlying glial activation and its role in plasticity. n. kramann 1 , r. pf€ ortner 1 , u.-k. hanisch 1 , k. hagemeier 2 , t. kuhlmann 2 , w. br€ uck 1 , c. wegner 1 1 university medical center g€ ottingen, department of neuropathology, g€ ottingen, germany 2 university hospital m€ unster, institute of neuropathology, m€ unster, germany introduction: laquinimod (laq) is an oral well-tolerated molecule that has been shown to reduce brain atrophy, disability progression and relapse rate in patients with relapsing-remitting multiple sclerosis. recent experimental data indicate that laq minimizes demyelination, inflammation and axonal damage in mice with cuprizone challenge. aim: since astrocytic nfjb activation plays a crucial role in cuprizone-induced demyelination, we investigated the effects of laq on cns cells in vitro and in vivo. methods: in vitro experiments used primary cells to test effects of laq on oligodendroglial survival as well as on cytokine secretion and nfjb activation in astrocytes and microglia. primary mouse astrocytes and microglia were pre-treated with 0, 0.25 and 2.5 mm laq and stimulated by pro-inflammatory cytokines. a dual-luciferase reporter assay was used to measure nfjb activity. for in vivo experiments, 10-week-old male c57bl/6j mice were challenged with 0.25% cuprizone and treated daily with laq (25 mg/ kg) or water. to assess astrocytic and microglial nfkb activation in vivo, nuclear translocation of nfjb p65 was assessed in astrocytes and microglia by double immunofluorescence with antibodies against p65 and iba1 or gfap. results: in vitro, astrocytic but not microglial nfjb activation was markedly reduced by laq as evidenced by nfjb reporter assay. in astrocytes, pre-treatment with 0.25 and 2.5 mm laq significantly reduced the induced nfjb activity after tnfa stimulation compared to stimulated controls. pre-treatment with 2.5 mm laq also significantly reduced nfjb activation after stimulation with the combination of il-1b and ifnc compared to untreated stimulated controls. oligodendroglial viability and survival were not affected by laq treatment. to confirm the in vivo relevance of these findings, we also examined astrocytic and microglial p65 translocation in mice with and without laq treatment after cuprizone challenge. the proportion of astrocytes with nuclear p65 immunoreactivity was significantly reduced in laqtreated mice (14.0% 6 0.9%) compared to untreated controls (25.8% 6 1.1%). microglia did not display marked translocation of nfjb p65 after cuprizone challenge. conclusion: our data indicate that laq prevents cuprizoneinduced demyelination by attenuating astrocytic nfjb activation. in vitro, laq reduced the astrocytic nfjb activation by up to 46% as evidenced by nfjb reporter assay. similar quantitative findings were obtained in vivo when laq treatment also led to a 46% reduction of astrocytes with nfjb activation, as evidenced by nuclear translocation of p65. these findings suggest that targeting the astrocytic nfjb pathway might have therapeutic effects in demyelinating cns disorders. . we asked whether brain-derived neurotrophic factor (bdnf), known to be highly expressed in a circadian manner in scn, might be involved in the mechanisms governing these astroglial rearrangements. using an analog-sensitive kinase allele murine model (trkb f616a ) and semi-quantitative electron microscopy, we found that the pharmacological blockade of the tropomyosin-related kinase receptor type b (trkb), the high affinity receptor of bdnf, abolished the day/night changes in glial coverage of vip dendrites. the bdnf/trkb signaling pathway therefore exerts a permissive role on the ultrastructural rearrangements that occur in scn across the day/night cycle. in contrast, the extent of glial coverage of non-vip dendrites was not different at daytime and nighttime in trkb f616a mice submitted to trkb inactivation or not receiving any pharmacological treatment. considering the well-established role of bdnf in the clock's photic synchronization process, these dataprovide strong support to the view that the daily astroglial rearrangements in scn could be involved in the photic entrainment of circadian rhythms. above all, they highly suggest that they are necessary for the modulatory action of bdnf on the scn responses to light. features of this form of neurodegenerative ataxia, including a striking reduction in lifespan when polyq atrophins are expressed in the glia. a sensible hypothesis is that affected glia will act non-autonomously on neurons as in other neurodegenerative pathologies. this is an emerging field that yields new and important therapeutic possibilities. we now wish to elucidate the mechanisms through which degenerating glia impairs organism functions, and identify genes involved cell-autonomously and non-autonomously in generating this aspect of the pathology. first, we investigated the impact of glial polyq atrophin on lifespan. using different glia marker, we have demonstrated that the expression of polyq atrophin in glia, or specifically in astrocyte-like cortex glia, has a dramatic effect on drosophila lifespan while brood brain barrier glia do not have any phenotype. we will then investigate the effect of other subtypes of glia looking at lifespan and motility to identify the relevance of each subtype in drpla. second, to identify new regulators of gliopathology we will conduct unbiased genetic screens for mutations in neurons that modify the lifespan of drpla flies, expressing polyq atrophin within the glia. our studies will be paramount in deciphering the complex glia-neurons interactions in ataxias and may be beneficial for several ataxias. they will open up new avenues for therapies aimed at targeting glianeuron interactions during disease progression. in alzheimer's disease, microglial clearance of beta-amyloid (ab) is considered neuroprotective. whether ab uptake regulates microglial cell responsiveness, and whether macrophages participate, remains unclear. here we investigated whether in vivo phagocytosis of endogenously-produced ab disturbs the turnover of microglia and macrophages by changing rates of cellular proliferation and apoptosis. using app swe /ps1 de9 tg mice, we show that plaque-associated microglia and cd11b 1 cd45 high macrophages bind and ingest endogenously-produced ab in vivo. plaqueassociated microglia and cd45 1 leukocyte-like cells were also observed in brains from patients with alzheimer's disease. phagocytic microglia and macrophages had a reduced rate of proliferation compared to abcells. instead, high proportions of apoptotic annexin v 1 microglia and macrophages were found in aged app/ps1 tg mice. phagocytic microglia and macrophages had greater caspase activity and increased p53 expression after in vivo uptake of ab, than cells that did not phagocytose ab. in vitro, we found that ab uptake induced caspase activation in microglia, whereas blocking ab uptake triggered apoptosis-induced cell death in macrophages. our data demonstrate that ab uptake impairs microglial/ macrophage viability and responsiveness. amyloid clearance may fail as phagocytic microglia and macrophages undergo ab-induced apoptosis. f. michetti, s. ceccariglia, a. d'altocolle, f. pizzolante, m. barba, a. delf a, c. gangitano universit a cattolica del s. cuore, rome, italy trimethyltin (tmt) is a neurotoxicant producing neuronal degeneration in the mammalian central nervous system , especially in the hippocampus (for reviews, 1,2). magnetic resonance imaging (mri) investigation in tmt-treated rats has evidenced dilation of lateral ventricles, possibly correlated to alterations in blood brain barrier permeability. we have investigated in the hippocampus and cortex of rats the expression of aquaporin 4 (aqp4), a glial water channel protein which is regarded to play a role in brain oedematous conditions, to explore the molecular mechanisms involved in the phenomenon. tmt-treated aqp4 expression was tested both by real-time pcr and western blotting analysis in hippocampus and cortex homogenates. to confirm molecular results and visualize the aqp4 cell distribution, double-label immunofluorescence for aqp4 and gfap was performed. real-time pcr and western blotting data show a significant upregulation of aqp4 starting from 14 days of tmt treatment in the hippocampus and, at a lower degree, in the cortex. accordingly, the immunofluorescence shows an intense astrogliosis and aqp4 immunoreactivity diffusely pronounced in the hippocampal and cortex areas starting from 14 days after intoxication. aqp4 immunolabelling was localized in astrocytic end-feet encircling the blood vessels, as expected. the study of the rhodamine b fluorescent tracer, intraperitoneally administered, also revealed an intense vascular reaction, characterized by hypertrophic vessels with abnormal course and dimensions in the brain of tmt-treated rats, indicating a vascular involvement in the tmt-induced neurodegenerative processes. aqp4 over-expression and the concurrent astrogliosis occurring in the brain of tmt-treated rats might be candidated to play a role in alterations of vascular permeability and brain oedema formation evidenced by mri studies. amyotrophic lateral sclerosis (als) is a neurodegenerative disease affecting predominantly motoneurons but with a particular pathological role of non-neuronal cells. previous research suggested that increased concentration of extracellular potassium could lead to motoneuron dysfunction. the na 1 /k 1 -atpase should regulate the homeostasis of potassium ions and thus help maintain regular neuronal activity. this homeostasis may be regulated by na 1 /k 1 -atpase in both neurons and astrocytes. in order to reveal the possible role of the na 1 /k 1 -atpase in als pathology, we explored the expression of the alpha1 catalytic subunit of na 1 /k 1 -atpase in neurons and astrocytes from different brain regions of the sod1 g93a rat model of als. labeling immunofluorescently the alpha1 isoform of the na 1 /k 1 -atpase revealed a decreased signal in the trigeminal nucleus and cerebellum of the als rat. double immunofluorecence with neun for neurons showed that the reduced alpha1 was on the neuronal surface in the trigeminal nucleus. in addition, the alpha1 ring-like staining of cerebellar granule neurons was lower in als as compared to the wild type rat. in comparison to neurons, astrocytes from the wild type rat were characterized with a lower expression of alpha1. in the examined brain regions we did not observe a prominent difference in alpha1 expression between astrocytes of wild type and als rats. based on the findings of this study the prominent reduction of na 1 /k 1 -atpase level observed in neurons and not in astrocytes could contribute to further understaning of impaired ion homeostasis affecting proper neuronal physiology in als. the role of glial na 1 /k 1 -atpase needs to be further studied with markers of other alpha subunit isoforms. although commonly used as nutritional supplements, excessive intake of bcaas might favour the establishment of neurotoxic conditions as indicated by the severe neurological symptoms characterising inherited disorders of bcaa catabolism such as maple syrup urine disease (msud). recent evidence indicates that bcaas induce excitotoxicity through mechanisms that require the presence of astrocytes. in the present study, we evaluated the effects of bcaas on microglia, the main immune cells of the brain. as an experimental model we used primary microglial cells harvested from mixed glial cultures that had been kept in normal or high bcaa medium (h-bcaa). we show that h-bcaa microglial cells exhibit a peculiar phenotype characterized by a partial skewing toward the m2 state, with enhanced il-10 expression and phagocytic activity but also increased free radical generation and decreased neuroprotective functions. we suggest that such an intermediate m1/m2 phenotype might result in a less efficient microglial response, which would promote the establishment of a low grade chronic inflammation and increase the likelihood of neurodegeneration. although based on in vitro evidence, our study adds on to an increasing literature indicating that the increasing use of dietary integrators might deserve consideration for the possible drawbacks. in addition to excitotoxicity, the altered immune profile of microglia might represent a further mechanism by which bcaas might turn into toxicants and facilitate neurodegeneration. the proteolipid protein gene (plp1), located on the x-chromosome, undergoes alternative splicing to produce the most abundant proteins of central nervous system myelin: plp and dm20. related to the extent of myelin defect, mutations in the plp1 gene in humans are associated with a spectrum of x-linked disorders, from the severe pelizaeus-merzbacher disease (pmd), to the mild form spastic paraplegia type 2 (spg2). phenotype-genotype correlation exists, large duplications of plp1 gene are predominantly found in pmd and null mutations in spg2. plp is almost 100% conserved across mammalian species, and the large spectrum of phenotype severity is also found plp transgenic mice. experiments on these models have contributed to our understanding in the pathophysiology of plp related disorders. in one hand, mice with extra copies of the plp1 gene (1plptg mice) have neurological symptoms and cns pathology similar to those found in pmd patients while mice with plp1 gene inactivation (plp null mice) share similarities with spg2 patients. then, these two mouse models represent very useful tools to evaluate the efficacy of a therapeutic strategy for the severe and the mild form of the plp related disorders on the neuropathological and behavioral aspects. olesoxime (tro19622), a cholesterol-like small molecule, has been shown to rescue motor neurons from cell death and to promote axonal regeneration both in vitro and in vivo. more recently, olesoxime has also been shown to promote central remyelination by accelerating oligodendrocyte maturation both in vitro and in vivo. altogether, these results strongly suggest that olesoxime may be a potential drug candidate for the treatment of white matter neurodegenerative disorders and notably to plp related disorders. here we treated 1plptg mice with olesoxime for 2 months starting at birth, as well as plp null mice from 3 to 12 months of age (presymptomatic treatment) or from 9 to 15 months of age (symptomatic treatment). behavioral and neuropathological analysis revealed that olesoxime failed to correct the development of symptoms in mice modeling the severe form of plp related disorders. on the contrary, treatment of plp null mice with olesoxime resulted in a 3-month delay in the onset of abnormal behaviors related to anxiety and in the slowing of central nerve conduction. these beneficial effects are observed only when the drug is administered before disease onset. treatment with olesoxime also reduces the effect of aging on rotarod performance decline in both wt and plp null old mice. altogether these results suggest that olesoxime could be of interest as a treatment to delay the onset of symptoms in plp related disorders provided that patients suffer from a mild form of the disease and are treated before the establishment of symptoms. objectives: glial cells play an important role in amyloid-beta (aß) clearance, however little is known about the cellular routes involved in internalization of different aß aggregation forms by adult human microglia and astrocytes. therefore, we evaluated the uptake mechanism of aß 1-42 by primary human adult microglia and astrocytes isolated from brain tissue of non-demented control and ad cases. methods: cells were preincubated for 1 hour with different inhibitors, before exposure to fluorescence labelled (fam) oligomeric and fibrillar forms of synthetic aß. the number of amyloid positive cells was quantified by flow cytometry. inhibitors of various pathways tested, include cytochalasin b (inhibits actin polymerization; general endocytosis inhibitor), nocadozole (inhibits tubulin depolymerization; general endocytosis inhibitor) and fucoidan (scavenger receptor inhibitor). results: cytochalasin b inhibited aß fibril uptake (90% reduction, p < 0.01) and to a lesser extent oligomer (50% reduction, p < 0.05) uptake in microglia, whereas no effect was seen on aß uptake by astrocytes. in contrast, nocodazole was found to inhibit aß fibril uptake (54% reduction, p < 0.05) and oligomer (77% reduction, p < 0.01) uptake in astrocytes, whereas no effect was seen on aß uptake by the microglia. interestingly, fucoidan prevented aß uptake in both astrocytes (oligomers:86% (p < 0.01); fibrils: 79% (p 5 0.01) and microglia (oligomers:67% (p < 0.001); fibrils: 79% (p < 0.05)). conclusions: these data indicate that both human astrocytes and microglia use scavenger receptors to internalize aß oligomers and fibrils. whether different types of scavenger receptors are involved in aß uptake by astrocytes and microglia will be further investigated. tel-aviv university, tel aviv, israel alzheimer's disease (ad) accounts for the majority of cases of dementia worldwide, affecting over 50% of the population over 85. the disease is characterized by a progressive memory loss, cognitive deterioration, behavioral disorders, deposit of beta-amyloid (ab) peptides, neurofibrillary tangles, reactive astrocytosis and activation of microglia cells. mutations in the genes that encode app (amyloid precursor protein) and components of the proteases that generate amyloid beta cause familial forms of ad. microglia, the resident immune cells of the central nervous system (cns) were suggested to play both beneficial and harmful roles in the course of the disease. therefore, modulating the microglial response is being considered as a promising approach for the treatment of this disease. we have recently shown using in vitro and in vivo systems that the ectoenzyme cd38, regulates microglial activation. in view of the significant role of activated microglia in ad and the important role played by cd38 in microglial activation, we hypothesized that cd38 deficiency would affect the course of the disease. in order to test this hypothesis, we implicated the app swe ps1de9 transgenic mouse model for ad, and compared the cognitive performance (as evident by behavioral studies) of aged app swe ps1de9 mice, to app swe ps1de9 cd38 -/mice. in addition, abload, accumulation of microglia and astrocytes was assessed in the brains of these mice. our preliminary results show that cd38 deficiency has a neuroprotective role in this ad mouse model as indicated by improvement in cognitive performance and dramatic reduction in ab load. oxidative stress induced by reactive oxygen species (ros) is associated with various pathological conditions, including aging, neurodegenerative disorders, traumatic and ischemic insults. the mechanism by which the gap junction protein connexin43 (cx43) contributes to oxidative stress-induced cell death or the "rescue" of the dying cells is still unclear. cx43 is highly expressed in astrocytes. we have previously shown that astrocytic cx43 is critical for neuroprotection during ischemic insults in vivo. to investigate the protective effect of cx43 in oxidative stress pathways, we induced oxidative stress in wild-type and cx43 knockout astrocytes using hydrogen peroxide (h 2 o 2 ). cx43 knockout astrocytes exhibited increased h 2 o 2 -induced cell death as measured by the loss of membrane integrity using the dye rhodamine b dextran, supporting a cell protective effect of cx43; this effect was not observed in wild-type astrocytes. to examine the involvement of cx43 in h 2 o 2 -induced cell death, we studied the expression and distribution of cx43 in response to h 2 o 2 in wild-type astrocytes. h 2 o 2 treatment altered the expression level and phosphorylation of cx43. this was accompanied with changes in cx43 distribution and gap junction plaque formation. hemichannel activity was measured using dye uptake. h 2 o 2 (0.7 mm) increased dye uptake, an effect that was blocked by gap26 (syntethic mimetic peptide, 200 mm) or carbenoxolone (100 mm), two connexin hemichannels blockers. however, h 2 o 2 treatment did not result in any measureable gap junction activity in cx43 knockout cells as measured by a scrape-loading dye transfer assay. these findings suggests that gap junction activity contributes to cx43-mediated protection in response to ros. one of the receptors found on microglia is the triggering receptor expressed on myeloid cells 2 (trem2), which signals intracellularly via an adapter molecule referred to as tyro protein tyrosine kinase-binding protein (tyrobp, known also as dap12). in humans, loss of function of either trem2 or tyrobp leads to nasu-hakola disease, which is characterized by neuroinflammation and bone cysts. by using a trem2 knock-out (ko) mouse line, we provided further insights on the trem2 function in neurodegenerative diseases. first, we observed mild signs of dopaminergic neurodegeneration and a slight increase in microglial iba1-immunoreactivity in different brain regions of 3 months trem2 ko mice compared with the wild type (wt) control animals. however, the transcription levels of inflammatory cytokines (tnfa, il1b) or inducible nitric oxide synthase (inos) were unaffected in mice of different ages (3, 9 or 12 months). to shorten the time required for neurodegeneration to occur, we have injected the mice intraperitoneally with lipopolysaccharides (lps) on four consecutive days and analyzed the dopaminergic neurodegeneration in the substantia nigra after a three weeks period. while the quantification of dopaminergic neurons in the substantia nigra showed a slight decrease in number for wt animals, a higher loss was recorded in the trem2 ko mice after systemic lps challenge. concomitantly, significant increase of microglia activation was detected in the lpstreated trem2 ko mice compared with lps-treated wt animals. our results are pointing towards a neuroprotective effect of the microglial trem2 receptor under chronic and systemic inflammatory conditions. increasing evidence suggest that brain energy metabolism is severely altered in multiple sclerosis patients, which may contribute to the process of neurodegeneration. imaging studies reveal a disturbed cerebral perfusion and glucose uptake and metabolism in ms patients. moreover mitochondrial dysfunction is recognized to play a crucial role in ms pathology. to date little is known about the distribution of glucose transporters (gluts), key molecules for the cellular uptake of glucose, in human brain and their role in the pathogenesis of ms. therefore, the aim of this study was to provide a complete overview of the distribution and expression levels of glucose transporters in control and ms brain. our data so far show that mrna levels of glut1, 3 and 4 as well as their regulators hypoxia inducible factor (hif)-1a, hif-2a and peroxisome proliferator-activated receptor gamma coactivator (pgc)-1a, are increased in normal appearing white matter (nawm) of ms patients compared to controls. immunohistochemical analysis revealed that brain endothelial cells not only express glut1 but also glut3 and glut4. glut 1, 3 and 4 are present in resting microglia and highly expressed in activated microglia and macrophages in active ms lesions. interestingly reactive astrocytes expressed increased levels of glut1 and glut4 compared to control, in addition glut3 positive astrocyes were observed in ms brain. axons expressed high levels of glut3 in active ms lesions, which was evidently reduced in chronic lesions. strikingly, all glucose transporters studied were down regulated in chronic ms lesions. together, these data emphasize the extent to which glucose metabolism appears to be affected in all stages of ms. a better understanding of these metabolic changes, especially in the nawm and chronic stage of the disease, is essential to develop strategies to improve neuronal survival and to gain more insight in the mechanisms underlying lesion formation. pericytes are vascular mural cells which are enriched within the capillary basement membranes of the brain. recent studies have provided evidence that pericytes regulate the blood-brain barrier (bbb) and modulate cerebral blood flow. neurovascular dysfunction and bbb damage are hallmarks of alzheimer's disease (ad), but the role of pericytes in ad pathogenesis is still unclear. we have examined post-mortem samples of middle frontal gyrus in collaboration with the university of cambridge after ethical approval by the local irb. ten cases with neither history of neurological disorder nor evidence of neuropathology were compared with 10 ad cases provided by the neurological foundation of the new zealand human brain bank. we performed immunostainings for laminin and plateletderived growth factor receptor (pdgfr)-b which mark vessels and pericytes, respectively. we also stained for ab 1-42 for evidence of amyloid pathology. quantification of pericyte and vessel density was performed using stereological methods, which are considered as gold standard in morphometric quantification. the spaceballs method was used to measure the density of capillaries. pericyte density was assessed using the optical fractionator. the age of the subjects did not differ between the control and ad groups (76,2 6 4,0 vs. 75,4 6 5,5 years). the cortex of ad cases contained more capillary vessels than controls (mean 6 sd: 308 6 40 vs. 365 6 41 mm/mm 3 ; p < 0,01; fig. 1a ). the capillary density increased in correlation to the total plaque load (r 5 0,58; p < 0,001). in contrast, we could not observe a significant difference in the number of pericytes per mm capillary length between control and ad cases (8,56 6 1,21 vs. 8,17 6 0,94 cells/mm; p 5 0,44; fig. 1b ). relative to tissue volume, the cortex of ad individuals showed a tendency to contain more pericytes than controls (2624 6 406 vs. 2985 6 588 cells/mm 3 ; p 5 0,13; fig. 1c ). this is the first study using state-of-the-art techniques to investigate the relationship between capillaries and pericytes in ad brains. impaired clearance of b amyloid across the pericyte-regulated bbb and deficient microvascular regulation of blood flow may contribute causally to ad pathogenesis. in consequence, loss of pericytes could contribute to disease progression. however, our results show that ad cortical pathology is characterized by an increase in the density of capillary vessels without deficit in pericyte coverage. thus, they do not substantiate the notion that pericyte loss is a significative feature of ad. m. noack, j. leyk, c. richter-landsberg carl von ossietzky universit€ at oldenburg, oldenburg, germany histone deacetylase 6 (hdac6), a member of the class ii hdacs, is a unique cytoplasmic a-tubulin deacetylase that interacts with the microtubule (mt)-associated protein tau. in healthy human brain six tau isoforms are expressed generated by alternative mrna splicing. these contain either three (3r-tau) or four (4r-tau) microtubule binding repeats regulating mt assembly and stability. tau hyperphosphorylation impairs its mt-binding activity. tau deposits in nerve cells and glia are the characteristic hallmark of a number of neurodegenerative diseases termed tauopathies. in progressive supranuclear palsy and corticobasal degeneration, pathogenic glial cell inclusions are observable in oligodendrocytes, the myelin forming cells of the cns. hdac6 plays an important role in the degradation and accumulation of misfolded proteins by promoting transport of aggregated proteins to the aggresome, localized at the microtubule-organizing center where protein aggregates are deposited and processed by autophagy. the present study was undertaken to elucidate the functional significance of hdac6 in oligodendrocytes and its role in pathological protein aggregate formation. towards this cultured rat brain oligodendrocytes and oln-t40 cells, an oligodendroglial cell line expressing the longest human tau isoform, were used. treatment of primary oligodendrocytes with tubastatin a (tst), a selective inhibitor of the tubulin deacetylation activity of hdac6, caused a significant increase in the level of acetylated tubulin, as determined by indirect immunofluorescence and immunoblot procedure. mts appeared more bundled and stabilized. inhibition of hdac6 attenuated the phosphorylation of tau at the phf-1-epitope (serine 396/ 404) and its enhancement at the 12e8-epitope (serine 262, located within the microtubule binding repeat region 1). furthermore, altered protein levels of tau isoforms containing either three or four mt binding repeats were observed. while 3r-isoforms were reduced, 4r-isoforms were increased in primary oligodendrocytes suggesting an impact on microtubule binding ability. hdac6 knockdown in oln-t40 cells revealed no alteration in tau phosphorylation at phf-1-epitope, but also augmented phosphorylation at the 12e8-epitope. cells were morphologically damaged and mt organization was disturbed. the data indicate that hdca6 modulates tau expression and phosphorylation, its inhibition leads to the accumulation of 4r-tau which is also prominent in tauopathies with oligodendroglial pathology. to test the hypothesis, that these cells are part of a possible compensatory mechanism to cope with the 6-ohda-induced loss of dopaminergic neurons, we studied the expression of tyrosine hydroxylase (th), the rate-limiting enzyme in the catecholamine synthesis pathway, in the cortex of 6-ohda-lesioned animals. performing immuncytochemistry at 4d after the lesion we demonstrated a 21-fold increase in the number of th-positive (th1somata in rat cortex following 6-ohda injection) compared to sham-lesioned animals. th1somata in the parietal cortex were restricted to the layers ii-v with most of the cells being bipolar. combining th immuncytochemistry with classical nissl stain yielded complete congruency. a small fraction of th1cells co-expressed calretinin thus pointing to an interneuron affiliation. virtually none of the th1cells expressed other markers of interneurons (calcium binding proteins, neuropeptides) or the glial markers gfap and nestin. in contrast, we found a co-localization of th with markers of glial progenitor cells (sox2 and s100b) and with psa-ncam, which has been shown to be expressed in immature, but not recently generated cortical neurons in cortical layer ii of the cat (varea et al.; front. neurosci. 5: 17 2011). taken together, these findings indicate that 6-ohda lesions might induce a glial de-differentiation followed by a fate re-direction towards neuronal committed cells. future studies have to be performed to confirm this and to find out if the th1cells are capable of dopamine synthesis. objective: using the mouse optic nerve (mon) wm model, we tested whether hydrogen in drinking water reduced functional wm ischemic injury, and if it reduced cellular lipid peroxidation and mitochondrial dysfunction including mitochondrial and nuclear dna oxidation. methods: functional integrity of mon was determined by quantitatively monitoring the area of mon compound action potential (cap) before, during and after a standardized 60 min period of oxygen and glucose deprivation (ogd), the ischemia equivalent ex vivo. nuclear 8-oxoguanine (nu8-oxog) was used as a marker of oxidative dna damage. results: a 60 min period of ogd caused prompt loss of the cap, followed by an average 20% recovery. in mice that had received hydrogen-containing drinking water for 7-10 days, the cap area did not disappear during ischemia and recovered to a significantly greater extent during reperfusion (normal oxygen and glucose levels). immunostaining of axonal neurofilament by smi-31 showed significant protection in mons from mice drinking hydrogen-water. accumulation of nu8-oxog was observed mainly in oligodendrocytes after ogd. the levels of 8-oxog after ogd were significantly reduced in optic nerves from hydrogen-water drinking mice. the 'protection' induced by 7-10 days of hydrogen-water drinking lasted several days. conclusions: our results show that several days of hydrogen exposure reduced the extent of cns wm irreversible injury associated with ogd. the importance of these observations is that oligodendrocytes may play an important role in the protective effect against ischemic injury. these observations raise intriguing therapeutic options. amyloid-b (ab) deposits in the brain extracellular space (ecs) and neurofibrillary tangles are typical features of alzheimer's disease (ad). both affections are expressed in triple transgenic (3xtg-ad) mice, making them a useful model for studying the pathophysiology of ad. in these mice, significant changes in astroglial morphology appear in the limbic brain structures [1, 2] , which may affect the diffusion of neuroactive substances through the ecs. using the real-time iontophoretic method, we determined the ecs volume fraction a (a 5 ecs volume / total tissue volume) and the geometrical factor tortuosity k (k 2 5 free / apparent diffusion coefficient) in the ca1 region of the hippocampus, dentate gyrus (dg) and prelimbic cortex (plc) in brain slices obtained from 10-month-old (10m) and 20month-old (20m) 3xtg-ad mice and age-matched wild-type (wt) controls. to study potential diffusion anisotropy, the measurements were performed along 3 orthogonal axes: medio-lateral, rostro-caudal and ventro-dorsal. astroglial morphology and the presence of ab were assessed using immunohistochemical staining for gfap and ab. we found no anisotropy in the ca1 region, dg, or plc, thus the data for each structure along the three orthogonal axes were pooled. the ecs diffusion parameters measured in the ca1 are shown in table 1. in the ca1 of 10m mice, there was no significant difference in the ecs diffusion parameters between wt and 3xtg-ad mice. in 20m mice, a decreased by 9% in wt but increased by 27% in 3xtg-ad mice in comparison with the values found in the younger animals. during aging, there was also a significant decrease in k in wt but not in the 3xtg-ad mice. a similar pattern of changes in the ecs diffusion parameters during aging was also observed in the dg and plc. unlike in the ca1, we found significant differences in young animals: a was lower in the dg and k was higher in the dg and plc of 3xtg-ad mice than in wt mice. in 20m 3xtg-ad mice, ab deposits accompanied with astroglial atrophy or hypertrophy were observed. we suggest that the amyloid load in 3xtg-ad mice and the subsequent prevailing astroglial atrophy affect the normal aging process by inducing an increase in the ecs volume during aging instead of the normal decrease. this leads to a larger ecs space in aged 3xtg-ad mice than in age-matched wt mice, which may alter the efficacy of extrasynaptic as well as synaptic transmission and contribute to the impaired cognitive functions in ad. the study was supported by the grants: gacr p304/11/0184, gacr p304/12/g069. significant differences between 10m and 20m mice of the same group are marked with *, differences between wt and 3xtg-ad mice of the same age are marked with 1. sequence or any treatment. however growing evidences are in favor of the involvement, besides neurons, of several partners such as glia and muscles. to better characterize the time course of pathological events in an animal model that recapitulates human als symptoms, we investigated functional and cellular characteristics of hsod1 g93a mice. we have evaluated locomotor function of hsod1 g93a mice through dynamic walking patterns and spontaneous motor activity analysis. we detected early functional deficits that redefine symptoms onset at 60 days of age, i.e. 20 days earlier than previously described. moreover, sequential combination of these approaches allows monitoring of motor activity up to disease end stage. to tentatively correlate early functional deficit with cellular alterations we have used flow cytometry and immunohistochemistry approaches to characterize neuromuscular junctions, astrocytes and microglia. we show that (1) decrease in neuromuscular junction's number correlates with motor impairment, (2) astrocytes number is not altered at pre-and early-symptomatic ages but intraspinal repartition is modified at symptoms onset, and (3) microglia modifications precede disease onset. at pre-symptomatic age, we show a decrease in microglia number whereas at onset of the disease two distinct microglia sub-populations emerge. we are now establishing the transcriptomic profile of microglia over the course of the disease. in conclusion, precise motor analysis updates the onset of the disease in hsod1 g93a mice and allows locomotor monitoring until the end stage of the disease. early functional deficits coincide with alterations of neuromuscular junctions. importantly, we identify different sets of changes in microglia before disease onset as well as at early-symptomatic stage. this finding not only brings a new sequence of cellular events in the natural history of the disease, but it may also provide clues in the search for biomarkers of the disease, and potential therapeutic targets. in order to gain more knowledge about the disease pathophysiology, two mouse models for vwm are developed in our lab. co-cultures between astrocytes (wt and vwm) and wt oligodendrocyte progenitor cells (opcs) are done to investigate the effect of astrocytes on opc maturation. furthermore, induced pluripotent stem cells (ipscs) from both vwm and wt mice are compared in their glial differentiation efficiency in vitro and in vivo. the astrocyte-opc co-cultures show that vwm astrocytes inhibit opc maturation. this suggest that astrocytes might have a crucial role in the development of the oligodendrocyte and myelin pathology in vwm. currently our research focuses on rescuing the maturation inhibition in vitro, which can aid the development of stem cell therapy for vwm. particularly in response to b-amyloid. using the appps1 alzheimer's disease mouse model, we observed the production of the common interleukin (il)212 and il-23 subunit p40 by microglia. genetic ablation of various il-12/il-23 signaling molecules, of which deficiency in p40 had the strongest effect, resulted in a drastic decrease in cerebral amyloid burden. although deletion of il-12/il-23 signaling from the radiation-resistant glial compartment of the brain was most efficient in mitigating cerebral amyloidosis, peripheral administration of a neutralizing p40-specific antibody likewise resulted in reduction of cerebral b-amyloid in appps1 mice. furthermore, intracerebroventricular delivery of antibodies to p40 significantly reduced soluble ab species and reversed cognitive deficits in aged appps1 mice. our results suggest that inhibition of il-12/il-23 signaling reduces cerebral amyloidosis and cognitive dysfunction, and may pose a novel potential pharmacological target to combat alzheimer's disease. charit e -universit€ atsmedizin berlin, berlin, germany question: microglia are attracted to and surround b-amyloid deposits, the main hallmark of alzheimer's disease (ad), suggesting a role for these cells in disease pathogenesis. however, recent in vivo data using the cd11b-hsvtk system for microglial depletion suggests that resident microglia are not sufficiently capable of restricting and clearing bamyloid plaques. to underpin the cd11b-hsvtk system and shed more light into the biological mechanistics of microglial depletion mediated by ganciclovir (gcv), we aim to intravitally monitor the depletion process. methods: we are using cd11b-hsvtk transgenic mice crossed to fractalkine-gfp 1/mice harboring green fluorescent microglia. a chronic cranial window is implanted onto the head of the offsprings followed by depletion of microglia and subsequent in vivo two-photon (2p) imaging. as 2p imaging is able to visualize the upper 300 mm of the cortex, a new topical depletion approach of cd11b-hsvtk 1 microglia was established. here, gcv is applied directly onto the brain surface in the area of the cranial window through a catheter. results: manipulation of the brain skull by installation of the cranial window and placing a catheter for topic gcv application resulted in strong depletion of microglia up to 85% in fractalkine-gfp 1/mice crossed to cd11b-hsvtk mice. conclusions: taken together, we established a minimal invasive technique for visualization of the microglia depletion process in cd11b-hsvtk mice using intravital 2p microscopy. these tools will allow the underpinning of the cd11b-hsvtk system as well as the study of relevant biological questions involving resident and peripheral derived microglia in ad. (icrea) , barcelona, spain x-linked adrenoleukodystrophy (x-ald) is a metabolic genetic disorder of the central nervous system characterized by demyelination in brain and/or axonopathy in the spinal cords, adrenal insufficiency and accumulation of very long-chain fatty acids (vlcfa) in plasma and tissues. the disease is caused by inactivation of the abcd1 transporter with the consequence production of oxidative stress mediators and damage. here we aimed to determine: i) the existence of endoplasmic reticulum (er) stress and the ensuing unfolded-protein response (upr) in brains and fibroblasts from x-ald patients, and in the x-ald mouse model (the abcd1 null mouse); and ii) whether the early and rampant oxidative stress formerly identified accounts for the er stress. indeed, here we report signs of er stress and upr response in human and mouse tissues. a common feature is the activation of atf6 and lower amounts of ire1, two er transducers constituting the core signature of upr in x-ald. chaperone grp78 and grp94 expression, by contrast, differ between variants and stages of the disease. we highlight how chaperones are down-regulated in x-ald mice at 12 months of age, pointing to selective depletion of upr-related factors overtime. treatment of the mouse model with a combination of antioxidants reversed the initial activation of transducers and chaperones, indicating that oxidative stress caused er stress. altogether, we postulate that oxidative-stress elicited er stress occurs in x-ald, and that a defective upr may contribute to axonopathy progression. thus, potentiation of the upr may be a therapeutic avenue in x-ald. representing opc-like cells with the potential to differentiate upon growth factor withdrawal and addition of triiodothyronine (t3) into premyelinating oligodendrocytes. upon differentiation, significant fewer cg4_wt-a-syn cells express the myelin basic protein (mbp; see figure a) as a marker for terminal differentiation and additionally, mrna levels were remarkably reduced compared to control cells (see figure b ). as mrna levels of the myelin protein proteolipid protein 1 (plp1) as well as the major myelin-gene regulatory factor (mrf) are also reduced in differentiated cg4_wt-a-syn cells (see figure b) we hypothesized that the process of differentiation is delayed upon a-syn expression. using various differentiation-promoting agents targeting distinct maturation-associated pathways we aimed to dissect the a-synmediated effect on differentiation in more detail and evaluate potential disease-modifying approaches for this devastating neurodegenerative disorder. methods: two nutritionally distinct groups of male newborn ratswell-nourished (w; n 5 26) and malnourished (m; n 5 33) were treated from the 5th to the 24th day of postnatal life with daily intraperitoneal injections of saline (sal) or cona (sigma-aldrich) at doses of 1 mg/kg (group w-l1 and m-l1) or 10 mg/kg (groups w-l10 and m-l10). two groups of "naive" (non-injected) pups were used as additional controls. when the pups reached adult age (90-120 days), under ip anesthesia (1 g/kg urethane 1 40 mg/kg chloralose) they were submitted to the csd recording (ecog and slow potential change) in two points of the parietal cortical surface. csd was triggered every 20 minutes by applying 2% kcl for 1 min at a point in the frontal cortex. this study was approved by the ethics committee of our university, case no.: 23076.031272/2010-53. results: compared to w-sal controls (mean 6 sd velocity in mm/ min 5 3.41 6 0.10), m-sal rats presented significantly higher velocities (4.26 6 0.16; p < 0.05). in both nutritional conditions l1 and l10 treated groups presented significantly lower csd velocities (3.12 6 0.14 and 2.82 6 0.18 for the w-l1 and w-l10, respectively, and 3.74 6 0.13 and 3.25 6 0.16 for the m-l1 and m-l10). this effect was dose-dependent, and was greater in the m-condition. the na€ ıve-and salgroups had similar csd velocities. conclusions: the lectin cona administered early in life to rats decelerated csd in a dose-dependent manner at adulthood, suggesting a long-lasting effect. the largest relative reduction was observed in the m group, suggesting modulation of the effect by the nutritional status. the involvement of glial cells in this effect is discussed. the endocannabinoid system is of growing interest as a therapeutic target in neurological diseases. the two major endocannabinoids 2arachidonoylglycerol (2-ag) and n-arachidonoyl ethanolamine (anandamide, aea), are the endogenous ligands for the cannabinoid receptors 1 (cb 1 ) and 2 (cb 2 ). cb 1 is mainly expressed in the brain and associated with neuroprotective effects, whereas cb 2 is primarily found in hematopoietic cells and associated with anti-inflammatory effects. in neurodegenerative diseases, e. g. amyotrophic lateral sclerosis (als) and parkinson's disease (pd), an increased cb 2 expression on microglia has been reported and is therefore an interesting therapeutic target. furthermore, an increase of endocannabinoid levels occurs in the affected neuronal tissues. stimulating this disease-related increase in endocannabinoids might thus be of therapeutic interest. in our project, we evaluated the novel, highly selective inhibitor kml29 of an enzyme central to the degradation of the endocannabinoid 2-ag, the monoacylglycerol lipase [1] . an in vitro monoacylglycerol lipase inhibitor screening assay for the comparison of the utilized inhibitor with others was performed. additionally, behavioral experiments including body temperature measurement, pain threshold measurement and motor activity analysis in healthy control mice were performed. furthermore, endocannabinoid system baseline levels of healthy control mice vs. sod1 (superoxide dismutase 1) mice as an als mouse model were compared at the gene expression and protein level. in vitro inhibition of the monoacylglycerol lipase by the recently published compound kml29 resulted in lower ic 50 values and therefore indicated a higher potency of kml29 when compared to the other monoacylglycerol lipase inhibitors jzl184 and cpc12 applied in the assay. in vivo administration of the reported monoacylglycerol lipase inhibitor kml29 resulted in significant decrease in body temperature, increase in pain threshold in b6sjlf1/j mice as well as in impairments in the nocturnal motor activity of c57bl/6j mice when compared to vehicle-treated mice. in summary, our data show a comparatively high potency in vitro and significant dose-dependent effects on physiological parameters in vivo of the monoacylglycerol lipase inhibitor kml29 and therefore lay the foundation for a future therapeutic study targeting neuroinflammation in mouse models of als and pd. glaz/apod protects against oxidative stress and promotes axon regeneration after injury, but its mechanism of action is unknown. we hypothesize that glaz/apod modulates membrane dynamics and oxidation. to contrast this hypothesis we study glaz protective role on the polyq-based spinocerebellar ataxia type i (sca1) model in drosophila. human polyq-ataxin1 expression in fly photoreceptors triggers glaz up-regulation. overexpressing glaz in photoreceptors or in retinal support cells rescues neurodegeneration. when expressed in retinal support cells, the glaz rescue is dependent on lipocalin-receptor expression by photoreceptor neurons, and the glaz-gfp fusion protein co-localizes with photoreceptor markers, suggesting the existence of receptor-mediated endocytosis of glaz. upon neurodegeneration, oxidative stress and induction of autophagy coexist. to test whether the glaz beneficial effects are mediated by the modulation of autophagy we quantified atg8a expression and monitored the accumulation of ubiquitinated proteins and p62. overexpression of glaz reduces atg8a induction, but concurrently reduces the accumulation of ubiquitinated proteins and p62. upon autophagy stimulation by rapamycin treatment, the levels of sca1-dependent accumulation of ubiquitinated proteins and p62 are further reduced by glaz. in addition, glaz decreases the sca1-dependent induction of gsts1, a sca1 genetic modifier contributing to the clearance of lipid peroxides. our data support that glaz enters the degenerating neurons by a receptor-mediated mechanism and promotes the resolution of autophagy, increasing its flow and helping to clear polyq-induced protein aggregates. moreover, the beneficial effects of glaz are linked to lipid peroxide clearance, either directly or through receptor-mediated modulation of protective gene networks. micinn(bfu2011-23978); jcyl(va180a11-2); fund. rodr ıguez-pascual. we investigated the effect of three different intensities of running exercises on the survival of sod1 g93a mice. at the early-symptomatic stage (p60), males were isolated and randomly assigned to 5 conditions: 2 sedentary groups ("sedentary" and "sedentary treadmill" placed on the inert treadmill), and 3 different training intensity groups (5 cm/s, 10 cm/s and 21 cm/s; 15 min/day, 5 days/week). we first demonstrated that an appropriate "control" of the environment is of the utmost importance since comparison of the two sedentary groups evidenced an 11.6% increase in survival in the "sedentary treadmill" group. moreover, we showed by immunohistochemistry that this increased lifespan is accompanied with motoneurons survival and increased glial reactivity in the spinal cord. in a second step, we showed that when compared with the proper control, all three runningbased training did not modify lifespan of the animals, but resulted in glial and motoneuronal changes. conclusions/significance: we demonstrate that increase in survival induced by a slight daily modification of the environment is associated with motoneurons preservation and strong glial modifications in the lumbar spinal cord of sod1 g93a . using the appropriate control, we then demonstrate that all running intensities have no effect on the survival of als mice but induce cellular modifications. our results highlight the critical importance of the control of the environment in als studies and may explain discrepancy in the literature regarding the effect of exercise in als. alzheimer's disease is the most prevalent neurodegenerative disorder, characterized by occurrence of senile plaques, neurofibrillary tangles and aberrant function of classical neurotransmitters and peptide messengers, such as neuropeptides and growth factors. in the last years a role of astrocytes in mediating and amplifying the progression of neurodegenerative disorders has been proposed. moreover, a growing body of evidence has shown that astrocytes can release non-peptide and peptide transmitters to influence neuronal development, function and plasticity. here we analyzed the impact of the main component of senile plaques, the amyloid-b (ab), on two key components of peptide vesicles, carboxypeptidase e (cpe) and secretogranin iii (sgiii), in astrocytes in vitro and in vivo. we consistently located cpe and sgiii proteins in cultured and in situ astrocytes. traffic and secretion of astroglial cpe and sgiii were analyzed under basal and stimulated conditions in primary cultures. we found that exposure of astrocytes to ab1-42 markedly impairs expression, traffic and secretion of glial cpe and sgiii. protein levels were decreased in the media, but increased into the cells, by ab1-42 incubations. the effect of ab on cpe and sgiii in glial cells was also investigated in vivo using the amyloid-forming transgenic mice appswe/ps1de9. an aberrant accumulation of cpe and sgiii was found in numerous activated-astrocytes surrounding senile plaques through all the cns of aged transgenic mice. taken together, the present study shows that ab dramatically alters expression, traffic and secretion of cpe and sgiii. because cpe and sgiii are essential in the process and targeting of neuropeptides and growth factors, an involvement of the astroglial secretory pathway in the pathology of alzheimer's disease is suggested. glia cytokine that is produced subsequent to h-i, that collaborates with other cytokines to stimulate the production of astrocytes from subventricular zone glial progenitors. the specific goal of this study was to evaluate gliogenesis after h-i when the tgfb1 receptor alk5 is antagonized in the vanucci p6 h-i rat model. by q-pcr, the relative amount of tgfb1 mrna peaked 7 days after h-i. therefore, sb-505124, and alk5 antagonist, was given intraperitoneally (i.p.) 7 days after the injury to a group of rats while another group received pbs. animals were sacrificed at different time points and brain samples processed for western blot analysis. validating the importance of alk-5 signaling, sb505124 reduced the levels of phosphorylated smad 2/3 that increased after h-i. in another study, sb-50512 was administered i.p. from p10 to p15 and animals sacrificed at p20 for immunohistochemistry for gfap, iba-1, gstpi and mbp. gfap and iba-1 positive cells were dramatically increased in the injured striatum and corpus callosum after h-i and mbp staining was decreased. by contrast, both the extent of injury and the degree of reactive gliosis was decreased by sb-505124 treatment. altogether, our results indicate that sb-505124 inhibits alk5 signaling in the damaged brain. furthermore, sb-505124 administration decreases the extent of microgliosis and astrogliosis while preserving myelination in the damaged brain after neonatal h-i. supported by nih r01 hd052064 and a grant from the leducq foundation awarded to swl. [2] . despite efforts to improve air quality, dep persists as a problem and the share of diesel engined passenger cars in western europe is increasing steadily. honey bees are an important pollinator of food crops and wild flowering plants. they not only contribute to food security by providing pollination services of an enormous economic value but also play an important ecological role. over the last five years, bee keepers worldwide have reported a decline in honey bee populations. the reasons for this decline remain unknown, but it is thought to be multi-factorial [3] . honey bee colonies are confronted with a number of stressors, for example infection with the mite varroa destructor or exposure to pesticides. to forage successfully honey bees rely on their learning and memory abilities. our research aims to establish whether dep might be a factor contributing to declines in honey bee populations through impairment of healthy cns function. methods: we have investigated the impact of dep exposure on the learning abilities of the honey bee using the proboscis extension reflex and classical conditioning. western blotting and histology were used to determine regional expression levels of stress proteins in the brain in response to an acute diesel exhaust exposure. we are examining the morphological appearance of glial cells to investigate the impact of dep in the honey bee cns of dep exposed, and control, forager bees. results and conclusions: we hypothesize that dep acts as a stressor in the brain of the honey bee causing changes in glial cells that are detectable using morphological and molecular techniques -similar to microglial activation or immunological responses of astroglia in the mammalian brain. this work will further our understanding of how dep acts on the brain and how it might impact on the health of the animal. current results indicate that diesel exhaust pollution impacts on the neurobiology of the honey bee and this may contribute to decline by impairing cns functions. hepatic encephalopathy (he) is a serious neurological disorder caused by liver failure. the prime candidate responsible for he pathology is an increased ammonium concentration; and following acute liver failure, ammonium can reach levels of up to 5 mm in the brain. astrocytes are a major target of ammonium toxicity and earlier studies suggested that this toxicity includes a disturbance in intracellular calcium signaling. in the present study, we analyzed the effect of acute hyperammonia on the intracellular calcium concentration of astrocytes in tissue slices of mouse brain, using quantitative intracellular calcium imaging with the indicator dye fura-2. astrocytes were identified by staining with the vital dye sr101; cerebellar bergmann glial cells were identified based on their morphology. in all brain regions studied, the hippocampal ca1 area, somatosensoric cortex, and cerebellar cortex, bath perfusion with 5 mm ammonium for 30 min induced a small, but persistent elevation in intracellular calcium, averaging about 50 nm. in addition, a fast and transient increase by on average 100 nm that lasted about 10 minutes was seen in a small percentage of astrocytes in hippocampal and cortical slices at the onset of ammonium perfusion. this transient increase was never observed in cerebellar bergmann glial cells. both the transient, as well as the plateau phase of ammonium-induced calcium changes were unaltered in the presence of ttx, indicating that they are independent from action potential generation by neurons. furthermore, ammonium-induced calcium increases largely persisted upon removal of extracellular calcium, indicating that they are caused by release from intracellular stores. taken together, our experiments demonstrate that ammonium evokes complex changes in the intracellular calcium concentration of astrocytes in the intact tissue, which differ both between cells in one preparation as well as between different brain regions. because of the central role of astrocyte calcium in gliotransmission, the observed ammonium-induced calcium dysbalance might result in disturbed neuronglia interaction and contribute to the pathology of he. it has been shown in a number of animal models that microglia in the degenerating brain are primed to show exaggerated cytokine responses to subsequent stimulation with toll-like receptors agonists such as lps and poly i:c. it is not clear whether the degenerating brain shows similarly exaggerated responses to pro-inflammatory cytokines. in the current study we hypothesised that glial cells in the hippocampus of animals with chronic neurodegenerative disease (me7 prion disease) would display abnormal responses to central cytokine challenges. in normal animals it has previously been established that intracerebral cytokine challenges elicit specific pathways, i.e. il-1b a cxcl-1 a neutrophil recruitment or tnfa a ccl-2 a monocyte recruitment. unilateral 1ll doses of tnfa (300ng/ll), il-1b (10ng/ll) or saline were administered intrahippocampally via pulled glass microcapillary, in normal (nbh) and me7 mice. these animals were terminally perfused for formalin fixation and paraffin embedding at 2, 24 and 72 hours post challenge. at 2 hours post challenge me7 microglia produced il-1b following either il-1b or tnfa challenge while nbh microglia did not. furthermore, there was very robust nuclear localisation of the nfkb subunit p65 in the astrocyte population and this was associated with very marked astrocytic synthesis of the chemokines cxcl-1 and ccl-2 in response to both cytokine challenges in me7 animals. conversely, very limited expression of these chemokines was apparent in nbh animals similarly challenged. thus astrocytes are the primary chemokine synthesizing brain cell and in the prion-diseased brain are they primed to produce exaggerated chemokine responses to acute stimulation with pro-inflammatory cytokines. this abnormal pattern of chemokine expression is predicted to have consequences for leukocyte infiltration and the ramifications of an altered cellular infiltration pathway for the degenerating brain will be discussed. these findings suggested that the presence of mutated htt in astrocytes alters glial glutamate transport capacity early in the disease process and may contribute to excitotoxicity. to decipher whether astrocytic mutated htt expression was directly associated with msns dysfunction, we investigated the functional properties of individual msns in proximity of mutated htt expressing astrocytes in acute slices. results from whole-cell patch clamp technique showed that msns surrounded (0 -100 microns distance) by astrocytes expressing mutated htt vs. astrocytes expressing normal htt did not differ with regard to passive membrane properties (input resistance, resting membrane potential), action potential firing rates nor spontaneous excitatory postsynaptic current properties (averaged amplitude and frequency). thus, astrocytic expression of mutated htt does not lead to readily apparent functional consequences in msns in these conditions. our data show that 11 month old tg mice displayed a significant increase of ab-plaques in the brain, compared to wt mice. furthermore, we show that the a1c subunit colocalizes with 90% of the abpositive plaques. the cellular expression of a1c was predominantly found in activated gfap1 astroglia around plaques. qpcr profiles of the hippocampus revealed expression of the majority of calcium channel subunits, which was stable during aging. the auxiliary beta4 subunit was expressed in these mice but did not co-localize with a1c1 astroglia around plaques. in cultures of primary astrocytes, ab(42) slightly enhanced the a1c mrna expression after 3 days. we are currently underway to characterize this expression in more detail. in summary, our data provide evidence for a largely stable expression of most ltcc subunits in alzheimer tg mice during aging. finally, the expression of a1c but not beta4 is upregulated in activated astrocytes located around ab-plaques and ab(42) may directly induce astroglial ca v 1.2 calcium channels. this study was supported by the sonderforschungsbereich sfb f4405-b19 and f4406-b19 of the austrian science funds. increasing evidence indicate that both physical and cognitive stimulation induce plastic changes in the brain, such as increase in synaptic and spine densities or neurogenesis, and counteract b amyloid (ab) pathology recovering cognitive function. in the present study we, for the first time, demonstrate the effects of psychostimulation on glial fibrillary acid protein (gfap) architecture in the dentate gyrus (dg) of 3xtg-ad, a well-established mouse model of ad. starting from 3 months of age, 3xtg-ad and their corresponding controls (non-tg) were housed either in the presence of a running wheel (run) or in the enriched environment (enr) for 9 months. as reported previously (olabarria et al., 2010), in standard housing, 3xtg-ad animals showed marked atrophy of gfap-positive profiles, evidenced by significant reduction in gfap surface area, by 39%, and volume, by 42%, compared with non-tg mice. however, physical and cognitive stimulation affected astrocyte morphology by increasing their cytoskeleton surface area and volume, both in non-tg and in 3xtg-ad mice. the surface area of gfap-ir astrocytes increased by 145% and 154% in 3xtg-ad mice housed in run and enr, respectively, compared with transgenic mice kept in standard housing. in addition, the cell volume of gfap-ir astrocytes was higher by 169% and 199% in 3xtg-ad mice housed in run and enr, respectively. the hypertrophy was also evidenced by an increase in the surface area and the volume of astroglial somata and processes in both non-tg and 3xtg-ad mice. further correlation analysis between 3xtg-ad and their corresponding control mice housed under the same conditions, revealed that run and enr not only reversed the genotype-induced astrocytic atrophy, but even induced a hypertrophy in 3xtg-ad mice, with gfap positive profiles rising to the levels of non-tg mice. thus our study indicates that long-term exercise and enriched environment restore and improve astroglial plasticity in the context of adlike pathology, which may recover cognitive functions by compensating disease-associated degeneration of neuronal-glial network. methods: intraperitoneal administration of wa at a dosage of 4mg/ kg of body weight was initiated from postnatal day 40 (p40) till end stage in sod1g93a mice, from 9 months till end stage in sod1g37r mice and from 6 months of age in tdp43a315t mice. results: wa was able to improve the survival by more than a week in sod1g93a and by two weeks in sod1g37r familial mouse model. in addition wa administration also conferred significant neuroprotection in tdp43a315t transgenic mice model. beneficial effects of wa in sod1g93a mice model was accompanied by reduction in loss of motor neurons and also by reduction in level of misfolded sod1 protein in the spinal cord as detected by immunoprecipitation with antibody specific to misfolded sod1. wa was found to be an inducer of heat shock protein 27 (hsp-27), which could possibly explain reduced level of misfolded sod1 and increased neuroprotection in wa treated mice. moreover real-time imaging with the use of biophotonic sod1g93a transgenic mice carrying luc (luciferase) and gfp (green fluorescent protein) reporter genes under the control of the murine gap-43 promoter revealed that wa was able to reduce neuronal stress at post symptomatic stage. conclusion: taken together, our results suggest that wa may represent a promising therapeutic drug for treatment of als. the temporal lobe epilepsy (tle) is the most common form of epilepsy that originates from the hippocampus and then propagates to other limbic areas such as the amygdala and entorhinal cortex. the pathological feature associated with tle is hippocampal sclerosis which is characterized by atrophy, induration, gliosis and loss of neurons in ca1, ca3 and the dentate hilar regions. some animal models, albeit do not exactly match the complex etiologies identified in humans, are found to recapitulate most of the pathological features observed in tle. there is evidence that administration of kainic acid can cause seizures in the ca3 region of the hippocampus that can lead to loss of neurons and astrogliosis characteristic of tle, but the underlying mechanisms associated with the degeneration of neurons remain unclear. since lysosomal enzymes, cathepsins b and d, can have important roles in the loss of neurons in a variety of experimental conditions, we evaluated their potential roles along with the insulin-like growth factor-ii (igf-ii) receptor, which is involved in the intracellular transport of these enzymes, in the kainic acid treated rats. our results clearly showed that systemic administration of kainic acid evoked severe loss of neurons along with hypertrophy of astrocytes and microglia in the hippocampal region of the adult rat brain. the levels and expression of cathepsins b and d as well as igf-ii receptor increased progressively with time in the hippocampus of kainic acid treated rats compared to control rats. the activity of both cathepsins b and d was also found to be enhanced in the hippocampus of treated rats. our double labelling studies revealed that expression of both cathepsins were initially increased in the pyramidal neurons and then decreased with time. this was accompanied by the expression of immunoreactive igf-ii receptors as well as cathepsins b and d in a subset of gfap-labelled activated astrocytes and iba1-labelled microglia in kainic acid treated rats. these results, taken together, suggest that enhanced levels/expression and activity of lysosomal enzymes may have a role in the loss of neurons observed in kainic acid treated rats. in addition, increased ng2 glia immunoreactivity and myelin loss were found specifically in the gray matter of patients' motor cortex and spinal cord ventral horn. furthermore, oligodendroglial specific monocarboxylate transporter 1 expression is decreased not only in als mouse models but also in patients. given that oligodendrocytes not only facilitate saltatory conduction of action potentials by myelinating axons, but also support neuronal functions metabolically through monocarboxylate transporter 1 (mct1), the injury to oligodendroglia in als may have pathogenic consequences. methods. to further investigate whether oligodendrocytes play a role in als development, we selectively removed mutant human sod1 (g37r) from postnatal ng21 cells in pdgfar-creer;loxsod1(g37r) mice. results. the administration of 4-hydorxytamoxifen (4ht) caused significant decrease in the expression of mutant human sod1 transgene in ng2 glia as determined by quantitative pcr analysis. we found that excision of mutant hsod1 from ng2 glia dramatically delayed disease onset and early disease, and significantly prolonged animal survival. in addition, at disease onset stage, activated astroglial and microglial responses were delayed in the animals received 4ht treatment. moreover, the removal of mutant sod1 helped to preserve mct1 expression at disease onset. conclusions. these data indicate that expression of mutant sod1 in ng21 cells and their oligodendrocyte progeny has a deleterious effect on motor neuron survival, and suggest that a key negative consequence of mutant sod1 expression in oligodendrocytes is to diminish their capacity to provide metabolic support to neurons. recent studies suggest that innate immunity might be also involved in the pathogenesis of neurodegenerative diseases, cerebral ischemia and brain injury. although the recent works demonstrated that adaptive immune system is involved in the motor neuron disease process, the role of innate immune system in motor neuron disease was not fully investigated. to assess the contribution of innate immunity in the pathogenesis of amyotrophic lateral sclerosis (als), the gene expression profile of lumbar spinal cord from symptomatic mutant sod1 mice was obtained by microarray approach and subsequent pathway analysis indicated the involvement of innate immune pathway. next, to test the role of innate immunity in the pathogenesis of als, sod1 g93a als model mice were mated with myd88 and trif (tir domain-containing adaptor inducing ifnb) deficient mice. myd88 and trif are the essential adaptor proteins for toll-like receptor mediated signaling pathway. as compared with sod1 g93a mice, myd88/trif double-deficient and trif-deficient sod1 g93a mice exhibited the substantially shorter survival times with accelerated disease progression. the disease duration was shortened by 50% in trif-deficient sod1 g93a mice. in contrast, elimination of myd88 in sod1 g93a mice showed marginal effect in survival time. in addition, the expression levels of pro-inflammatory chemokines, ccl5 and cxcl-10 were significantly suppressed in the spinal cord of trifdeficient sod1 g93a mice, as compared with sod1 g93a mice. to determine the cell type in which trif-dependent pathway contributes to the production of these chemokines, we examined the expression of poster abstracts s75 glia chemokines in lps-stimulated primary microglia or astrocyte derived from trif-deficient or myd88-deficient mice. trif-dependent induction of these chemokines was observed only in lps-stimulated microglia. moreover, we found that infiltration of t-lymphocytes and other immune cells were significantly decreased in the spinal cord of symptomatic trif-deficient sod1 g93a mice. these results suggest that the basal level of trif-dependent innate immune activation of microglia is beneficial to slow disease progression of als models through the maintenance of pro-inflammatory chemokines and the infiltration of the immune cells to the spinal cords. the detailed analyses to clarify the role of these infiltrating immune cells are underway. alzheimer's disease (ad), the most common age-dependent neurodegenerative disorder, causes a chronically progressive decline in cognitive functions. there is growing evidence that glial changes are early involved in this pathology. pdapp mouse, a well-defined model of ad, accumulates toxic soluble and deposited ab, derived from proteolytic processing of the amyloid precursor protein (app) and develops adlike synaptic deficits and cognitive impairment. on the other hand, autophagy has been associated to the neurodegenerative process, in particular with the clearance of aggregation-prone proteins like ab. the amyloid plaques, mainly located in cortex and hippocampus, are closely surrounded by reactive and hypertrophic gfap1 astrocytes. the aim of this work was to evaluate the potential astrocyte autophagic activity during the progression of ad in pdapp mice from 5 to 20 months (m) of age. lc3ii/i ratio was studied by western blot. amyloid deposit load, stained with congo red, exhibited a continuing rise according to age in transgenic mice. the markers gfap and lc3 were analyzed by immunofluorescence and confocal microscopy on hippocampal sections showing an increasing colocalization that reached the top at 14 m, where 52.5 6 9.9% of plaque associated gfap cells were lc31. at 20 m, this proportion was lower. conversely, the subpopulation of astrocytes located far from ab deposits were gfap1/lc3-, besides a decreased cell volume compared with control mice astrocytes. additionally, the density of astroglial cells in the stratum radiatum diminished with aging (5 compared to 14 m, total gfap1 cells, p < 0.05) along with higher plaque load, in a more prominently manner than in control mice. our results clearly show that 1) astroglia is strongly affected during ad progression: two different morphologic subpopulations -close to or far from deposits-are distinguished in the hippocampus. aging correlates with a glial density reduction; 2) a gradual increase of autophagic activity in plaque associated-astrocytes is verified by the first time, suggesting a contribution to ab clearance until 14 m in pdapp mice, with a significant decay after this age. it also shows widespread neuron loss and gliosis in the brain. interestingly, in cstb-/-mice pronounced microglial activation has been detected in selected brain areas already in presymptomatic mice, preceding astrocytosis and neuronal death. in this study, we examine the microglial contribution to the neuronal dysfunction and death in epm1. we aim to characterize the functional properties of cstb-/-microglia and their effects on the survival of cstb-/-neurons. our results show that the cstb mrna level in cultured wild type mouse microglia analyzed by real time quantitative pcr is high in relation to primary astrocytes or neurons. a gene-expression profiling of microglia from cstb-/-mice and from wild type mice has been obtained by using the affymetrix mouse exon 1.0 st array and reveals a down-regulation of interferon-regulated pathways in cstb-/-microglia. the stimulation of primary wild type mouse microglia with the endotoxin lipopolysaccharide (lps) up-regulates cstb mrna expression measured by real time quantitative pcr. cstb-/-microglia show an altered inflammatory response to the stimulation with lps by increased secretion of chemokines demonstrated by cytokine array analyses. moreover, the release of nitric oxide from cstb-/-microglia measured by griess assay is elevated. our data indicate an altered response of primary cstb-/-microglia to inflammatory stimuli, which may contribute to neurodegeneration in epm1. the data provide a basis for further detailed studies on the pathophysiology and therapy of this devastating disease. activation of astrocytes and microglia surrounding amyloid extracellular plaques in the brain is a hallmark of alzheimer's disease (ad). increasing evidence suggests that chronic neuroinflammation and elevated levels of several cytokines play important roles in ad development. beside astro-and microgliosis, demyelination and oligodendrogenesis can be observed in human patients as well as in murine models of ad. oligodendrocyte progenitor cells, also termed ng2 cells because they express the neuron/glial antigen 2 (ng2) proteoglycan, comprise about 5% of total cell number of the adult brain and are the main proliferating cells present. ng2 cells receive gabaergic and glutamatergic synaptic input and can secrete pro-and antiinflammatory substances upon activation by microglia-derived cytokines. overall, ng2 cells are highly reactive cells and one of the first cells which respond to changes in the cns. however, their role during ad pathology is still uncompletely characterized. using immunohistochemistry, we have analyzed their expression pattern in a transgenic murine model of ad (coexpressing a mutated form of the amyloid precursor protein (app) and the presenilin1(ps1) gene). we focused on the spatial arrangement of ng2 cells in the hippocampus of transgenic mice at 3, 7 and 10 months of age and age-matched controls (n 5 4/ time point). our goal is to provide an anatomical and structural basis for elucidating the role of ng2 cells in ad. the first results indicate an uniform distribution of ng2 cells in the hippocampus of app/ps1 mice, with an accumulation at the sites of plaque deposits in close association with microglia. ng2 cells are normaly absent from the pyramidal and granular cell layers. however, when plaques deposits were found at these principal cell layers, ng2 cells could be seen surrounding them, suggesting an active migration. changes in ng2 cell phenotype were noted from 3 months of age on. like microglia, ng2 cells assume an activated morphology, as in a more "bushy" appearance. in numerous plaques, ng2 immunoreactivity was strongly increased, mostly in the intermingled processes that encircle the core of the plaques. whether these activated ng2 cells proliferate and differentiate into oligodendrocytes or communicate with activated microglia in the plaque microenvironment remains to be investigated. further, levels of the glutamatergic ampa receptor expression will be evaluated. ultimately we aim at understanding how ng2 cells and microglia interact in ad, which could be usefull in the development of novel therapies. accumulating evidence supports an increasing role of astrocytes in the initiation or progression of a variety of neuropathological conditions. in alzheimer's disease (ad), numerous data indicate that astrocyte properties are modified with potential deleterious effects on neurons. accordingly, we have described changes in the expression of astroglial connexins, the gap junction channel and hemichannel forming proteins, in the vicinity of amyloid-b (ab) plaques in brains from ad patients and murine models of ad. also ab peptide was shown to trigger astroglial cx43 hemichannel activation in culture and in acute slices leading to neuronal degeneration. hence, we have investigated hemichannel function of astroglial connexins in 8-9 months old app swe /ps1 de9 mice that exhibit ab plaques in the cortex and hippocampus. ethidium bromide (etbr) uptake performed in acute brain hemisphere slices was used as an index of hemichannel activation and was quantified in astrocytes identified by gfap immunostaining. in app swe /ps1 de9 mice, compared to wildtype mice of the same age, an increased uptake of etbr was detected in the overall population of astrocytes. this uptake was blocked by carbenoxolone and lanthanum ions, indicating connexin hemichannel involvement. such activation may be linked to the increase in resting astroglial [ca 21 ] i described in this mouse model. interestingly, etbr uptake was higher in reactive astrocytes contacting ab plaques than in non-reactive astrocytes located far from (! 50 mm) these deposits. we are currently investigating their respective pharmacological profiles. moreover, in app swe /ps1 de9 mice knock-out for cx30 generated in our facility, etbr uptake was comparable to that observed in app swe /ps1 de9 mice, suggesting that cx43 is the major contributor to the hemichannel activity observed. altogether, these results indicate that neuroglial interactions could be affected by astroglial hemichannel activation and could account for neuronal alterations or death observed in neurodegenerative diseases. since it is well established that amyloid plaques are associated with activated astrocytes we asked whether concentrations of the astrocyte-derived proteins glial fibrillary acidic protein (gfap) and s100b in human csf might serve as additional biomarkers. to functionally link the role of astrocytes to mechanisms involved in memory formation, we asked whether the amount of gfap-positive astrocytes correlates to the level of ltp in the hippocampus of aged ad transgenic mice. we used elisa kits for the quantitative analysis of gfap (ibl-international, hamburg, germany) and s100b (ibl). in a mixed disease cohort (n 5 52 diseased patients, n 5 16 controls), we correlated standard biomarkers (abeta and tau-protein) and gliaderived proteins. furthermore we compared mean levels between groups of ad patients (n 5 36) and healthy control patients (n 5 35) and correlated levels of astroglial proteins in the csf and cognitive performance (mmst values). in transgenic (app/ps1 transgenic mice), aged (10-15 months) mice (n 5 10), we performed ltp experiments at the schaffer collateral synapses in the ca1 region of the hippocampus. the number of gfap-positive astrocytes was quantified contralaterally. results: in the mixed cohort, we found a significant correlation of t-tau and gfap levels in csf (r 5 0.261, pbetween the s100b levels and the patients cognitive performance as measured by the mmst values (r 5 -0,42) as well as between the gfap levels and the mmst values (r 5 -0,38). when correlating the number of astrocytes in the ca1 region to the level of ltp, 30 min after induction, we found a significant correlation of the number of astrocytes per mm 3 to the level of ltp (r 5 0,648, p conclusions: the astrocyte-derived proteins gfap and s100b can be reliably detected in human csf. measurement of astroglial biomarkers might allow an improved pathobiological staging of ad, also since astrocytes appear to play a functional role in memory formation in the context of an ad-like pathology in mice. retinitis pigmentosa is a group of inherited disorders affecting photoreceptors or retinal pigment epithelium (rpe) that leads to progressive loss of vision. the pde6b rd1 mouse model is a very well-known animal model for retinal degeneration, in which rods carry a mutation in b subunit of rod cgmp-phosphodiesterease. glial cell line-derived neurotrophic factor (gdnf) has already been shown to rescue morphology as well as function of rod cells in pde6b rd1 mouse. this effect was indirect, through stimulation of m€ uller glial cells (rmg). to better understand the neuroprotective effect of gdnf, primary retinal rmg were stimulated in vitro with gdnf and the secreted proteome was analyzed by proteome profiler arrays. among others, cyr61/ccn1, a member of ccn family, was found strongly induced upon rmg stimulation with gdnf. when applied directly to medium, cyr61 significantly reduced photoreceptor death in organotypic ex vivo cultures of pde6b rd1 retinas. in order to identify the target cells of cyr61 we treated, arpe19 and mio-m1 cell lines with cyr61, and observed an increase in phosphorylation of akt and erk1/2 signaling molecules. these results suggest that stimulation of rpe and rmg cells may have a protective influence on photoreceptor survival in organotypic ex vivo conditions. we postulate cyr61 as a novel potential candidate for future therapeutic approaches in neurodegenerative retinal disorders. since astrocytes react to multiple physiological and pathological stimuli in the microenvironment of the brain, they could be mediators for gene x environment interactions in the pathogenesis of psychiatric disorders. altered gene/protein expression and regressive changes have been reported for astrocytes in post-mortem studies of psychiatric disorders, i.e. schizophrenia (scz), bipolar disorder (bp) and autism spectrum disorders (asd). by contrast, no genetic link to astrocytes has emerged from the extensive genomic analysis of psychiatric disorders. genetic findings mostly relate to neurons for disorders rooted in neurodevelopment (asd, scz). it is timely to perform a formal analysis of genomic information emerging for psychiatric disorders for a contribution of genes expressed by astrocytes. methods: we generated a meta-list of genes highly expressed in rodent astrocytes using published gene lists and literature mining. datasets were prepared for candidate genes and genes from gwas in attention deficit/hyperactivity disorder (adhd), asd, bp and scz, by using literature and databases (szgene, sfari base, dbgap). datasets for copy number variations (cnvs) associated with neurodevelopmental delay were also assembled. then the overlap between the meta-list of genes highly expressed in astrocytes and gene datasets for psychiatric disease was determined. overlapping genes were pooled and subjected to david bioinformatics analysis. results: the meta-list of genes highly expressed in astrocytes contained n 5 2,680 genes (13.4% of the genome). datasets for risk genes in psychiatric disorders showed random overlap with the meta-list (adhd 18%; asd 13%; bp 12%; scz 13%). cnvs related to neurodevelopment were not enriched for astrocytic genes (8%). when the overlapping genes were pooled from all disorders, a small set of shared astrocytic genes emerged (n 5 23). cntnap2, nrxn1 and sdc2 were linked by david analysis (p 5 0.013). conclusions: this correlative analysis makes it unlikely that genes highly expressed in astrocytes make a major contribution to the genetic risk in four major psychiatric disorders. no genetic link was found between astrocytes and neurodevelopmental delay. changes in gene/ protein expression and pathology in astrocytes in the adult brain in scz and bp may be explained by chronic neuronal dysfunction, chronic medication or acute changes. the neuronal ceroid lipofuscinoses (ncls, batten disease) are a group of autosomal recessively inherited lysosomal storage disorders affecting children and young adults, each of which is caused by a mutation in a different gene. a common feature across all ncls is the early, localised glial activation that occurs long before the onset of neuron loss. this glial activation appears to be an accurate predictor of which neuron populations are vulnerable and will subsequently die. both astrocytes and microglia express the ncl gene products and it is likely that normal glial cell function may be compromised. given the close functional relationship between neurons and glial cells it is possible that glial activation and/or dysfunction may affect neuronal health. we have begun to explore the biology of astrocytes and microglia in the juvenile form of ncl using primary cultures from cln3 -/mice. defects in both astrocytes and microglia, including altered protein secretion profiles and impaired morphological and proliferative responses were apparent. co-culturing with mutant microglia and astrocytes influenced the survival and morphology of wild type neurons, with more profound effects upon cln3 -/neurons. intriguingly, these effects were largely reversed by substituting wild type glia, which rescue mutant neurons. a pilot study has provided similar evidence for glial dysfunction in infantile ncl, including and altered protein secretion and response to stimulation. these changes appear to be different from those observed in juvenile ncl and we are in the process of exploring whether the glial cell phenotypes discovered so far, and their impact upon neurons are specific to the different forms of the disease. the accumulation of aggregated proteins in nerve cells and glia underlie the pathogenesis of many neurodegenerative diseases. glial pathology is characteristically observed in tauopathies, such as corticobasal degeneration and progressive supranuclear palsy, and alexander disease, a primary disease of astrocytes caused by mutations in the gfap (glial fibrillary acidic protein) gene. irreversibly damaged proteins can be degraded by the proteasomal system. for proteasomal degradation they are covalently linked to ubiquitin in a three-step enzymatic pathway (e1, ubiquitin-activating enzyme; e2, ubiquitin-conjugating enzyme; and e3 ubiquitin ligases). the polyubiquitin chain needs to be removed before the translocation of the substrates to the lumen of the proteasome. this is achieved by deubiquitinating enzymes (dubs), which oppose the functions of e3-ligases and assist in targeting substrates to specific pathways. to assess whether impairment of dubs may contribute to age-related neurodegenerative diseases and the regulation of cell death and survival, we have subjected primary cultures of rat brain astrocytes to pr-619, a dub inhibitor with broad specificity. immunoblot analysis demonstrates that after a 18h treatment, pr-619 caused the induction of heat shock proteins, hsp70 and hsp32, and an increase in p62, which is considered a cargo receptor for ubiquitinated proteins and a common constituent of ubiquitinated protein inclusions. as analyzed by indirect immunofluorescence, protein aggregates positively stained by antibodies against ubiquitin and p62 assembled in the perinuclear region at the microtubule organizing center (mtoc). these aggregates were surrounded by a cage-like network of gfap intermediate filaments, thus resembling aggresomes. using mitotracker and lysotracker staining procedures, the fluorescent images demonstrate that after dub inhibition lysosomes and mitochondria are recruited to the mtoc. this process was dependent on an intact microtubule network, since it was interrupted after treatment with nocodazole, a microtubule destabilization drug. furthermore, pr-619 caused mitochondrial fragmentation which may be a stress response to enable the removal of damaged mitochondria by mitophagy. in conclusion, dub inhibition impairs the cellular architecture, leads to protein aggregate formation and mitochondrial alterations in astrocytes. our data sustain the hypothesis that an imbalance in dub activities may contribute to the pathogenesis of neurodegenerative diseases. shown that the collapsin response mediator protein 2 (crmp-2), which play a significant physiological role in neuronal cell bodies and axons within the cns, is phosphorylated during the neurodegenerative phase of the inflammatory disease. methods: we investigated the limitation of axonal degeneration by transducing retinal ganglion neurons with a phosphorylation mutant of crmp-2 utilising an intraocular adeno-associated virus 2 (aav2) delivery system in eae-induced mice. the other group of mice injected with aav2 consisting of the green flourescent protein reporter only (aav2-gfp) with eae was used as a control (n 5 8 per each group). results: we showed substantial preservation of axons of the optic nerve in the eae-induced mice injected with the aav2 carrying the crmp-2 phospho-mutant compared ($10-fold difference) with those mice injected with aav2-gfp. the aav2-gfp transduced axons showed significant degeneration during the peak stage of eae. conclusion: our data suggest that phosphorylation of crmp-2 may be a central mechanism that governs axonal degeneration during inflammatory demyelination of the cns (as occurs in ms) and inhibition of phosphorylation of crmp-2 may be of therapeutic potential for the progressive phase of the disease in ms patients. y. shinozaki, s. koizumi univ. of yamanashi, yamanashi, japan atp, a major gliotransmitter integrating neuron-glia networks, is known to be released or leaked from injured cells. in the cns, atp dramatically changes glial phenotypesand could affect pathogenesis of brain disorders. however, whether such glial responses facilitate or rather inhibit the diseases is still under debate. to address this issue,we studied the neuroprotective role of atp/purinergic signaling using in vivo stab injury model on mouse cerebral cortex. without injury or the contralateral side of the injured brain exhibited no fluoro-jade(fj)-positive neuronal death, cd45 1 infiltrating leukocytes or gfap 1 reactive astrocytes. three days after injury, fj signals, cd45 1 cells and reactive astrocytes were increased in the ipsilateral side of the brain. the cd45 1 cells were enclosed by reactive astrocytes-formed glial scar. administration of an atp-degrading enzyme apyrase, a broad p2 receptor antagonist ppads, and a p2y 1 receptor antagonist mrs2179 significantly reduced the stabincreased fj signals. immunohistochemical analysis revealed p2y 1 receptor was expressed in gfap 1 astrocytes. p2y 1 receptor knockout (p2y 1 ko) mice also exhibited reduced fj signals and infiltration of cd45 1 cells. they exhibited higher level of gfap expression, more remarkable hypertrophy of astrocytes and tighter glial scar formation than wild type mice did. the accelerated glial scar formation reduced cd45 1 cell number and inhibition of astrocytes by fluorocitrate increased cd45 1 cell number. although glial scar formation was accelerated, no enhanced proliferation was observed. we then analyzed the mechanism of enhanced glial scar formation and neuroprotection using in vitro scratch-wound model. in cultured astrocytes, the scratch induced a transient increase in extracellular atp, which was followed by decrease in extracellular atp mainly due to an increase in ectoatpase activity. suppression of purinergic signaling by either apyrase, ppads or mrs2179 enhanced scratchevoked astrocytic migration rather than proliferation. neither activation nor inhibition of p2y 1 receptors in cultured cortical neurons affected the scratch-induced damages. our data suggest that the decrease in atp/ p2y 1 receptor-mediated signal should be a trigger that transforms astrocytes into migratory phenotype, which forms tighter glial scar structure thereby suppressing neuronal death. background: oligodendrocyte damage and loss are key features of multiple sclerosis (ms) pathology and oligodendrocytes appear to be particularly vulnerable to ros. in vitro studies showed that ros induce cell death and prevent the differentiation of oligodendrocyte precursor cells (opcs) into mature myelin-producing oligodendrocytes. hence, a potential therapeutic strategy to protect these cells from ros-mediated damage is urgently needed. here we investigated the efficacy of several compounds that are able to boost antioxidant enzyme production, including monomethyl fumarate (mmf), tert-butylhydroquinone (tbhq), sulforaphane (sfn) and protandim. these compounds are thought to exert their protective function via activation of the nuclear-factor-e2-related factor-2 (nrf2) transcriptional pathway, which is involved in the production of antioxidant enzymes necessary for oxidative stress defense. methods: primary rat oligodendrocytes were treated with different concentrations of mmf, tbhq, sfn and protandim. expression of antioxidant enzymes were analyzed by pcr and western blot analyses. to study the beneficial effects of the different nrf2 activators, oligodendrocytes were first incubated with nrf2 activators and subsequently exposed to various concentrations of hydrogen peroxide. oligodendrocyte cell survival was measured by a live/dead cell viability assay. results: sfn, mmf and protandim are well-tolerated and induce nrf2driven antioxidant enzyme production in oligodendrocytes. protandim was the most potent compound with regard to antioxidant enzyme induction and protected oligodendrocytes against ros-induced cytotoxicity. conclusions: our findings indicate that several nrf2 activators are able to induce antioxidant enzyme production in oligodendrocytes. interestingly, protandim, a dietary supplement consisting of herbal ingredients, was the most potent protector of primary rat oligodendrocytes. in future experiments we will determine whether protandim can also promote the differentiation of opcs under oxidative stress and test the clinical efficacy of protandim in an experimental animal model for ms. the arising of the "tripartite synapse" concept and the discovery of the astrocytic excitability have been highlighting astrocytes as active elements concerning information flow in the brain. however, the impact of such remarkable features in complex brain function is still under-explored mostly due to the difficulty of studying neuron-astrocyte interactions in vivo. the aim of this work was to use an in vivo model of astrocytic dysfunction and investigate the impact of this treatment in complex cognitive functions. the rationale consisted in studying behavioral performance that relies on the prefrontal cortex, such as behavioral flexibility and working memory in an animal model of astrocytic dysfunction. for that purpose, we used a pharmacological model in which wistar-han rats were subjected to bilateral intracranial injections of aminoadipate in the prelimbic portion of the medial prefrontal cortex to cause astrocyte depletion specifically in this region, mimicking pathological states such as depression, in which marked decreases of gfappositive cells are observed. this animal model was tested for its cognitive abilities by the attentional set-shifting task (asst) and water maze-based tests. a clear impairment of cognitive function was observed in the animals treated with aminoadipate. a detailed morphological and histological analysis showed that along with the astrocytic lesion, neurons were also affected at the site of lesion. this seems to contribute to the cognitive decline observed in this model and may explain similar observations under pathological states. a. sternotte, e. hermans universit e catholique de louvain, brussels, belgium amyotrophic lateral sclerosis (als) is an adult disease characterized by a selective loss of motor neurons, resulting in progressive paralysis and death within 3-5 years. in several inherited cases, the disease is caused by point mutations in the gene encoding for superoxide dismutase 1 (sod1) which promotes its aggregation, leading to biochemical damages, including mitochondrial dysfunction. alteration of mitochondrial membrane potential and/or respiratory chain leads to atp depletion and these metabolic dysfunctions could contribute to motor neuron death in patients and animal models of als. in motor-neurons, the combination of altered axonal transport with mitochondrial dysfunction likely leads to considerable decrease in atp levels at distant neuromuscular junctions. commonly know as the fuel gauge of mammalian cells, amp-activated protein kinase (ampk) is a key enzyme in the control of cellular atp and energy homeostasis. in this study, the implication of ampk in the progression of als was specifically investigated by measuring the expression and activity of this enzyme in the spinal cord of mice overexpressing the mutated human sod1 (hsod1 g93a ), a commonly used experimental model of als. no consistent modification of the enzyme was detected in spinal cord samples throughout the progression of als. indeed, such experiment did not discriminate between cell types present in the tissue. hence, a more detailed characterization of ampk in cultured astrocytes derived from als animals revealed a robust induction of the enzyme activity, which correlates with decreased atp levels in these cells. in a second part of our study, we have investigated the consequences of ablating ampk in the mouse model of als by breeding ampk knock out mice with hsod1 g93a mice. while we did not detect any difference in the lifespan of ampk(-/-)/hsod1 g93a mice as compared to hsod1 g93a mice, we observed substantial alterations in the progression of the disease in selected behavioural tests. in particular, analysis of the gait (catwalk) which enables early detection of muscle weakness revealed that the onset was delayed by up to 30 days while the progression of the disease after onset was accelerated. further studies will be conducted to establish the importance of atp-in the disease, and to validate this enzyme as a putative pharmacological target in als and in other neurodegenerative diseases involving mitochondrial dysfunction. institute of neuroscience, sibs, cas, shanghai, china ng2 glia is the fourth type of neuroglia in vertebrate nervous system, which also known as oligodendrocyte progenitor cell (opc) in a developmental point of view. in adult brain, ng2 glia has a small cell body with highly branched extensions and represents a new type of glial cell differing from other glial cell types such as astrocyte, microglia and mature oligodendrocytes. published studies by others have shown that ng2 glia is able to promptly respond to brain injury and activated in several neurodegenerative disease animal models including 6-ohdainduced rat parkinson's disease model. however, the pattern and function of these activated ng2 glia remain largely unknown. here, we performed a time-course study of ng2 glia activation in mptp mouse model of parkinson's disease using immunohistochemistry. it was found that ng2 glia was activated as early as 24 hours after the last mptp injection in the substantia nigra (sn) of mptp-treated mice. this temporal character is very similar to those of microglia, indicating ng2 glia respond to mptp challenges very quickly. the activation of ng2 glia became stronger over time and reached the peak at about 5 days after the final mptp injection. then, the extent of ng2 glia activation declined gradually and diminished 10 days after mptp injection. interestingly, we found that prominent ng2 glia activation could only be observed in the substantia nigra. the dorsolateral striatum which is the projection target of nigral dopaminergic neurons, however, was devoid of ng2 glia activation in all the time-points examined. in summary, we characterized the ng2 glia activation in mptp mouse model of parkinson's disease. these data suggest that activated ng2 glia may play a role in the degenerative process of dopaminergic neurons in pd. hallmarks of cns inflammation, including microglial and astrocyte activation, are a feature of post-mortem tissue from amyotrophic lateral sclerosis (als) patients and in transgenic mice overexpressing mutant superoxide dismutase-1 (sod1 g93a ). administration of glucocorticoids does not significantly alter disease progression, but this may reflect poor cns delivery. here, we sought to discover whether cns-targeted liposomally-packaged glucocorticoid would inhibit the cns inflammatory response and reduce motor neuron loss. sod1 g93a mice were treated with saline, free methylprednisolone (mp, 4mg/kg/week) or glutathione pegylated liposomal mp (2b3-201, 4mg/kg/week) and compared to saline treated wild-type animals. animals were treated weekly with intravenous injections for 9 weeks from 60 days of age. animal weights and motor behaviour were monitored during this period. at the end of the experimental paradigm (116 days) mice were imaged using t 2 -weighted mri for brainstem pathology, and brain and spinal cord tissue was collected for histological analysis. all sod1 g93a animals showed a significant decrease in motor performance compared to baseline from $100 days. sod1 g93a animals showed a significant increase in signal intensity on t 2 weighted mr images, which may reflect the combination of neuronal vacuolation and glial activation in these motor nuclei. treatment with 2b3-201, but not free mp, significantly reduced t 2 hyperintensity, which correlated with significantly reduced histopathological manifestations in the brainstem motor nuclei, but not the spinal cord. interestingly, there was a significant reduction in astrogliosis but not microglial activation following treatment with 2b3-201. the results of this work indicated that the cns-targeted anti-inflammatory agent 2b3-201 has therapeutic potential in als. the toxic effects of new antiretroviral drugs on the central nervous system (cns) are unclear. because these drugs penetrate the brain even at low concentrations, it becomes crucial to determine the doses which can be toxic for the cns resident cells. moreover, after the recent introduction into clinical practice, it is unclear whether the efficacy of the antiretroviral drugs of new generation may also derive from their ability to exert extravirological effects on factors responsible for the development of hiv brain injury, e.g. matrix metalloproteinases (mmps). objective: to investigate on the toxicity of four different antiretroviral drugs and their ability to modulate the expression of gelatinase b (mmp-9) in astrocyte cultures. methods: primary cultures of rat astrocytes were activated by exposure to 10 mg/ml lipopolysaccaride (lps) (positive control) and simultaneously treated for 20 h with increasing doses (1-5-10-25. 50 mm) of: efavirenz (efv); darunavir (drv); maraviroc (mvc) or raltegravir (ral). mmp-9 mrna expression was assessed by rt-pcr. quantitative determination of mmp-9 expression was done by computerized scanning densitometry. single drug toxicity was assessed by the mtt test. each drug was considered toxic at the concentration able to induce a percentage of cell survival above 60%. results: the treatment with antiretroviral drugs inhibited mmp-9 mrna expression in lps-activated astrocytes in a dose-dependent manner. in particular, a statistically significant inhibition of mmp-9 expression was observed when astrocytes were treated with 25mm efv (54% of inhibition) or with 50 mm ral (43% of inhibition). as assessed by the mtt test, the toxicity of the antiretrovirals ranges from 10 and 50 mm. in particular, efv was toxic for astrocytes at the concentration of 25 mm, mvc at 10 mm, while drv and ral were toxic at the concentration of 50mm. conclusions: the present results indicate that efv and ral directly inhibit mmp-9 expression in lps-activated astrocytes with mechanisms that are independent from their antiviral activity. the toxic doses of antiretrovirals are much higher than those found in the csf of hiv-positive patients. our results highlight some beneficial/deleterious extra-viral effects of the antiretroviral drugs that may be useful to improve the development of new therapeutic strategies for the management of hiv infection. we used a parahippocampal kainate injection mouse model to induce mtle-hs and to study early expression patterns of kir4.1 and apq4 after se. mice were sacrificed 24 h, 72 h and 7d after kainate injection. immunhistochemistry of the hippocampus was performed to assess kir4.1 and aqp4 expression, and gfap staining was used for identification of astrocytes. gfap expression was increased compared to saline injected controls, suggesting that kainate injection induces astrogliosis. comparison of kir4.1 and aqp4 staining showed a similar change in the expression pattern. our data indicate a drop in kir4.1 and aqp4 expression within 24 hrs after se followed by an upregulation within the first week. altered expression of kir4.1 and aqp4 could contribute to dysregulation of potassium and water homeostasis and play a role in the early phase of epileptogenesis. c. l€ o€ ov, a. erlandsson uppsala university, neuroscience/neurosurgery, uppsala, sweden we have previously shown that astrocytes effectively engulf dead cells after trauma both in vitro and in vivo, but that they store the ingested material rather than degrade it. compared to macrophages, which degrade engulfed, dead cells within hours, our data show that astrocytes store the ingested material for weeks before the degradation is completed. to further study the routes of degradation and the possibilities to speed up this process we have used a cell culture model where uvtreated, dead cells are added to stem cell-derived astrocytes. by labeling the dead cells with the ph-sensitive dye phrodo prior to the engulfment, we demonstrate that the ph in the astrocytic phago-lysosomes is higher than in professional phagocytes. interestingly, lamp1 and lamp2, which are involved in the phago-lysosome fusion, are both highly expressed in the astrocytes, particularly around the ingested material. dendritic cells are known to express the inhibitory protein rab27a that slow down the degradation in order to preserve antigen for presentation. rab27a prolongs the actin coating around the phagosomes, which physically inhibit the phago-lysosome fusion, and interacts with nox-2, which leads to an increased consumption of protons. western blot analysis shows a high expression of rab27a in our cell cultures which may explain the slow degradation. moreover, dead cells in astrocytes are surrounded by actin rings for a long period of time after the ingestion. to counteract this, the astrocytes were treated with 0.1 or 1 mm of the actin inhibitor latrunculin b. the number of actin rings were significantly lower in the cultures treated with 1 mm of latrunculin b compared to controls, but the intensity of phrodo was unaltered indicating that the prolonged degradation may be due to a higher ph in the lysosomes rather than a delayed actin coating. next we will investigate the effect of rab27a expression on the degradation process by performing sirna experiments. one of the promising strategies for the treatment of spinal cord injury is the transplantation of glial cells obtained from the olfactory system; olfactory ensheathing cells (oecs). effective proliferation and migration of oecs are essential for optimizing clinical applications and there is a need for identification of small molecules that regulate glial cell biology. curcumin is a natural polyphenol compound found in the spice turmeric, which is known for its neuro-protective properties and has been reported to have an effect on neurogenesis and nerve regeneration. however, the effect of curcumin on oecs has not been determined. we have examined the effect of curcumin on oecs at the cellular level using fluorescence and timelapse microscopy. oecs were purified from s100b-dsred transgenic mice in which oecs express the fluorescent protein dsred. different treatments: (i) control medium, (ii) curcumin (0.5 to 20 mm), (iii) g5 commercial growth factor, or (iv) a combination of g5 and curcumin were used to determine the effect of curcumin on oecs proliferation and migration. cell proliferation was quantified at two different times by cell counting and mts proliferation assay. timelapse microscopy was used to visualize changes in cell morphology and cell migration. cell branching, number and area of lamellipodia and speed of migration per cell were measured for each treatment. we found that lower concentrations of curcumin (0.5 and 1 mm) increase oec proliferation. as well, combination of g5 and curcumin showed the highest proliferative effect on oecs suggesting a possible synergistic relation between g5 and curcumin. live cell imaging analysis showed that curcumin increased the number and area of lamellipodia resulting in faster migration of oecs. these results suggest that curcumin can regulate the proliferation, morphology and migration of oecs which could improve the therapeutic use of oecs for spinal cord injury repair. methods: the dams were divided in four groups and had received 50 ml of nonalcoholic or alcoholic beer solution -control, vehicle (nonalcoholic solution), etoh 5% (nonalcoholic solution 1 5%v.v ethanol) or etoh 10% (nonalcoholic solution 1 10%v.v ethanol) during gestational day 1 up to weaning (postnatal day 22). male offspring (30 -35 days old) were sacrificed and hippocampal acute slices were prepared. we analyzed gfap content, s100b and glutamate uptake as astrocyte parameters. we tested the animals in the plus-maze discriminative avoidance task to check anxiety and memory. results: gfap content decreased after pnee for etoh 5% and etoh 10% treatment (f 3,59 5 17.75, p < 0.0001). interestingly, we found that only the etoh 10% treatment showed an increase of hippocampal s100b content (t 5 2.38, p 5 0.02) and in the csf (f 3,18 5 3.249, p 5 0.0462), but no difference in secretion. glutamate uptake was significantly decreased for etoh 10% group (f 3,20 5 4.069, p 5 0.0208). to perform the plus-maze discriminative avoidance task we selected pups from control, vehicle and etoh 10% treatment on pnd 30. two-way anova revealed significant effects of treatment (f 2,46 5 4.32, p 5 0.019), arm type (f 1,46 5 103.9, p < 0.0001) and treatment x arm type interaction (f 2,46 5 6.66, p < 0.01). in the test session, only a significant prenatal treatment group effect (f 2,46 5 6.44 p < 0.01). bonferroni's post-hoc test revealed that only control group presented significantly less time in the enclosed aversive arm than nonaversive one. conclusions: in this study we showed that pnee with moderate doses could alter the structure and functional astrocytes balance. thus, high levels of s100b are indicated in some neurodegenerative disorders. taken this data together we could demonstrate that moderate ethanol exposure can be harmful to fetal brain. aging has been associated to neuroinflammation in the central nervous system, however it is not known whether microglial changes induced by aging are affected by early effects, such as litter size and sedentary life style. in addition, the lateral septum has been recognized by its complementary role in the memory processing for tasks of object recognition for identity and spatial location. in the present report, we investigated whether aging cognitive decline and microglial morphological changes in the lateral septal region are influenced by changing litter size early in life and by a sedentary life style. to assess these questions, wistar rats suckled in litters of either six or 12 pups per dam were raised sedentarily in groups of 2 up to 3, from the 21 st post-natal day onwards. at 4 (young adult) or 23 (aged) months-old, half of the sedentary rats underwent progressive daily treadmill exercise for five weeks, while the others remained sedentary. after performing the tests of recognition for spatial localization and object identity all of the animals were sacrificed and their brains were processed for selective microglia/macrophages immunolabeling with anti-iba-1 antibodies. a representative sample of the immunolabeled cells in the lateral septum was analyzed after three-dimensional reconstruction with neurolucida software (microbright field inc.) and morphological features of each cell were quantified by neuroexplorer (microbright field inc.). it was found that sedentary life style of wistar rats maintained in standard laboratory cages is associated with spatial memory deficits in both mature and aged subjects no matter the litter size, and that exercise decreased these effects in aged subjects raised in small but not in large litters. on the other hand, all sedentary aged rats, despite of the litter size, presented impairment of object recognition memory for identity, and exercise decreased this effect in animals from both large and small litter size groups. microglial morphological analysis revealed that cell soma area, perimeter and branches volume seem to be more intensely affected by aging and that these changes are mainly associated with animals raised in large litters. furthermore, it was observed important shrinkage and thickening of the microglial branches in aged individuals on a higher proportion in the sedentary group suckled in large litters. taken together the results suggest that litter size and sedentary life style may affect object recognition and spatial memories in both adult and aged rats and that exercise minimizes these effects on animals suckled in small but not larger litters. in addition, we suggest that these changes are related to distinct effects on the soma and branching patterns of septal microglia from young and aged subjects. huntington disease (hd) is a dominant inherited neurodegenerative disorder caused by an unstable expansion of a cag repeat within the gene encoding for huntingtin protein (htt). this mutation induces formation of a poly-glutamine tract in the mutant htt (mhtt) protein that prones its aggregation into the cells. hd is characterized by an initial and massive degeneration of the medium spiny neurons in the striatum with later neuronal loss in cortex, globus pallidus, and other structures leading to motor and cognitive symptoms. alterations of energy metabolism contribute to hd pathogenesis but the mechanisms responsible for these alterations are not well understood. a recent pet study provided evidence for a selective impairment of striatal glycolytic and not oxidative metabolism of pre-symptomatic hd patients. in the brain, glycolysis is predominantly an astrocytic metabolic process, whereas oxidative metabolism is primarily neuronal raising the question that metabolic defects in hd could originates from astrocytes. the purpose of our study was to characterize (1) the metabolic alterations in vivo in a transgenic mouse model of hd (bachd mice) and (2) the respective roles of astrocytes and neurons in metabolic defects occurring in hd using an in vitro strategy. bachd mice express the human full length htt protein with 97 glutamine repetitions under the human htt promoter into a bacterial artificial chromosome. bachd mice present a slow disease progression with motor deficits occurring at 6 months and progressive neuronal aggregates from 12 months, reflecting human pathology. first, we performed [14c]-2-deoxyglucose autoradiography experiments in vivo to assess energy metabolism in 14 months bachd (n 5 6) and wt (n 5 6) mice. we performed a 3d analysis of the whole brain glucose uptake without any regional a priori. using statistical parametric mapping (spm), a voxel-wise statistical analysis approach, we showed that compared to age-matched wt mice, 14 months bachd mice present hypometabolism in the striatum, like pre symptomatic hd patients, and also in the hippocampus. hypermetabolism was also detected in the hypothalamus. second, we performed an in vitro study to dissect out the cellular mechanisms responsible for these metabolic changes. by using cell insert, we realized neurons/astrocytes co-culture without any physical contact. this culture system allowed us to grow neurons in presence of wt or bachd astrocytes. we showed that both bachd and wt neurons have a decreased [14c]-2-deoxyglucose uptake when cultured in presence of bachd astrocytes but not wt astrocytes. these results collectively suggest that energy defects in bachd mice are due to noncell autonomous mechanisms by which astrocytes expressing mhtt induce neuronal metabolic dysfunction. naag and naa are abundant metabolites in the brain which can be utilised by oligodendrocytes to make myelin. but elevated levels of naag and naa are associated with myelin loss in the leukodystophies canavan and pelizaeus-merzbacher-like disease, whereas lower levels of naa are observed in brain tissue of ms patients. currently, the physiological function of these two metabolites as well as their role in white matter pathology is unknown. naag and naa can also act as signalling molecules as they can affect glutamate receptors. oligodendrocyte precursor cells (opcs) express nmda receptors and activation of these receptors may be important for their differentiation and efficient myelination. in disease opcs are recruited to demyelinating lesions where they proliferate and differentiate into new myelinating oligodendrocytes. we therefore investigated the effects of both physiological and pathological concentrations of naa and naag on opc biology in vitro. we used calcium imaging to see whether naag or naa can induce intracellular calcium changes in opcs as they are known to act on neuronal nmda receptors. 1mm naa or 1mm naag evoked intracellular calcium changes in opcs (df[340/380] 44.6 6 0.7 3 10 23 and 9.0 6 3 3 10 23 respectively), but only one third of the cells responded which corresponds to the proportion of nmda responsive cells in the cultures. however, not all opcs that responded to nmda did also respond to naag or naa. therefore, it seems that the naa and naag evoked calcium raise is via an nmda receptor independent pathway. naag and naa evoked a small but significant increase in intracellular calcium in opcs, which even after prolonged application of pathological levels of naa and naag did not affect the cells' viability as propidium iodide staining of opcs showed no significant difference in viability. over 90% of the opcs survived 4hrs application of 1mm naa and 1mm naag. naag and naa are produced and released by neurons but it is not known how opcs take them up from the environment and how it affects them. we therefore addressed which possible transporters opcs express and the effect naa and naag have on opcs' proliferation and differentiation. it is unlikely that naa and naag even at high concentrations have a detrimental effect on opcs. perhaps these high concentrations of naa and naag found in canavan and pelizaeus-merzbacher-like disease indicate rather opcs' deficiency to transport and utilise naa and naag for energy and myelin formation. vanishing white matter (vwm) disease is a severe leukoencephalopathy, classically starting in childhood. the disease is characterized by neurological symptoms, including uncoordinated movements and balance disturbances which are slowly progressive. episodes of rapid deterioration are triggered by febrile infections and minor head trauma, which can lead to coma. a treatment is unavailable and eventually all patients die within a few years upon diagnosis. the disease is caused by mutations in any of the five genes encoding the subunits of the eukaryotic translation initiation factor 2b (eif2b). although this protein has an important function in all cells, mainly the cells in the brain white matter (astrocytes and oligodendrocytes) are affected by these mutations. pathologically the brain shows cystic degeneration with meager gliosis, dysmorphic astrocytes, lack of myelin and an increased number of oligodendrocyte and astrocyte progenitor cells. we have developed two mouse models for vwm to further unravel the disease mechanisms at molecular level and to develop and test possible treatments. the 2b4 mouse line is homozygous for a mutation in the eif2b4 gene, encoding the eif2bd subunit, and the 2b5 mouse line is homozygous for a mutation in the eif2b5 gene, encoding the eif2be subunit. both mutations have been described in human patients and are associated with a severe form of vwm. preliminary data show that these mice models recapitulate the clinical and neuropathological phenotype observed in vwm patients. pathologically, the central nervous system white matter is vacuolated and shows lack of myelin while astrocytes are dysmorphic and immature. these transgenic mice appear to be a representative model for vwm. microglial cell lines were differentiated from ips cells and represent in vitro models for human microglia. rt-pcr and flow cytometry analysis of ipsdm showed gene transcription and protein expression of cd33 respectively. the protein expression was increased by breaking the cis-interacting sialic acids on the microglial glycocalyx using sialidases. cd33-fc fusion protein was designed and purified via the fc tag to study binding patterns of cd33 to various cell types expressing different glycocalyx structures. additionally, ipsdm exhibiting either an over expression or knockdown of cd33 will be used to determine protein functions. furthermore, preliminary immunohistochemical analysis of control brain tissue compared to ad patients showed increased cd33 protein expression in cd68 positive cells, in the latter. in summary, data show that cd33 is expressed by human microglial cell line enabling further characteristic and functional analysis of the protein. furthermore, preliminary analysis on primary brain tissue corroborates the association of cd33 -expressed on microglia -to lateonset ad. the neurotrophins are essential for peripheral nervous system development and myelination. we have previously demonstrated that the neurotrophin bdnf exerts contrasting influences upon myelinationacting through neuronal p75 ntr to enhance myelination, but inhibiting it via neuronal trkb. recently we have generated a small peptide called cyclo-dpakkr that structurally mimics the region of bdnf that binds p75 ntr . here we aim to investigate whether utilising cyclo-dpakkr to selectively target p75 ntr is an approach that could exert a unified promyelinating response. like bdnf, cyclo-dpakkr promoted myelination of ngf-dependent neurons in vitro, an effect dependent on the neuronal expression of p75 ntr . importantly, cyclo-dpakkr significantly enhanced the myelination of bdnf-dependent neurons in vitro, whereas bdnf inhibited it. local injection of cyclo-dpakkr adjacent to the neonatal sciatic nerve in vivo significantly enhanced myelin protein expression and increased the number of myelinated axons. we found that injection of cyclo-dpakkr also significantly upregulated the expression of neuregulin 1 type-iii, a key factor required to induce peripheral myelination. thus, our data demonstrate that cyclo-dpakkr promotes peripheral myelination in vitro and in vivo. to investigate whether cyclo-dpakkr promoted the remyelination of peripheral neurons, we utilized the ean, a rodent model of peripheral demyelinating neuropathy. our data show that administration of cyclo-dpakkr significantly delayed the disease onset and reduced clinical severity in ean. this improved disease phenotype is supported by reduced demyelination, as cyclo-dpakkr substantially reduced the extent of myelin damage in myelinated peripheral nerves subjected to ean. collectively, our data demonstrate that using cyclo-dpakkr to selectively target p75 ntr promotes peripheral myelin development, and is effective at ameliorating ean disease and protecting against myelin damage. our findings suggest that selective targeting of p75 ntr is a strategy worthy of further investigation for the treatment of peripheral demyelinating diseases introduction huntington's disease (hd) is a neurodegenerative disease characterised by huntingtin protein misfolding and the intra-cellular accumulation of mutant huntingtin (mhtt). accumulation of mhtt is most pronounced in neurons and is thought to underpin neuronal dysfunction. oligodendrocytes have been shown to accumulate mhtt aggregates, which may contribute to cellular dysfunction in these cells. evidence from diffusion tensor mri (dt-mri) studies shows that white-matter changes are amongst the earliest pre-symptomatic changes observed in hd. these findings have been taken to implicate axonal and or glial aberrations prior to disease onset. methods we have used a variety of techniques, which include, emulsion in situ hybridisation, whole brain sub-fractionation, immunohistochemistry, meso scale cytokine assay, and western blots. results our investigations using the r6/2 mouse model show that mhtt aggregates result in a specific down-regulation of the small heat shock protein (shsp) hspb5 (alpha-b-crystallin). localisation of hspb5 by whole brain sub-fractionation shows that hspb5 is enriched in the myelin fraction. emulsion in situ hybridisation further shows localises hspb5 in oligodendrocytes. detergent extraction of myelin fractions show differential hspb5 partitioning between soluble and insoluble fractions. cumulatively, these findings suggest that hspb5 is constitutively expressed in oligodendrocytes and is present in two distinct pools. recent studies implicate hspb5 as being a negative downregulator of inflammation. as such down-regulation of the shsp in r6/ 2 mice may promote increased oligodendrocyte vulnerability to mhtt cytotoxicity. to investigate the role of hspb5 as a negative regulator of inflammation, we obtained transgenic hspb5 knockout mice. the knockout mice are viable and show very little phenotypic difference against littermate controls. we have analysed the inflammatory profiles of these mice using the meso scale. to validate our findings in the r6/2 mice, we are currently investigating human tissue from distinct vonsattel stages of disease, to see if hspb5 downregulation is also observed in the human disease. conclusion as hspb5 has several critical cellular roles, its downregulation may compromise oligodendrocyte structure and function, which ultimately has negative consequences for neurons. it is therefore worthwhile to investigate it's specific role in the cns. the myelin sheath is attached to the axon through multiprotein complexes that form the axon-glial interactions. they consist of cell adhesion molecules and voltage gated ion channels and divide the axon into distinct domains: the node of ranvier, the paranode, the juxtaparanode and the internode. this molecular organization is crucial since recent studies identified distinct perinodal proteins as autoantigens in ms patients: nodal and paranodal neurofascin isoforms and juxtaparanodal tag-1/contactin-2. tag-1 is a cell adhesion molecule expressed both by axons and glial cells. in the adult nervous system, tag-1 organizes the juxtaparanodal domain of the fiber, where it interacts with caspr2 and the potassium channels (vgkcs). question: in this study we have analyzed the alterations of the juxtaparanodal proteins caspr2, tag-1 and vgkcs in relation to paranodal caspr and nodal sodium channels upon the onset and progression of experimental autoimmune encephalomyelitis (eae) and in the cuprizone model of toxic demyelination. methods: we immunohistochemically and biochemically analyzed both models of cns demyelination. results-conclusions: our results show a higher paranodal susceptibility to disruption compared to juxtaparanodes in eae. as eae progresses there is subsequent compensatory efforts for myelinated fiber restoration that include paranodal protein re-clustering and increased heminodal formation, without subsequent juxtaparanodal protein aggregation. in contrast, juxtaparanodal organization was observed only when remyelination occured in the cuprizone model of toxic demyelination. supported by: imbb-forth scholarship, manassaki scholarship (university of crete), the project "myelintag" (593) is implemented under the "aristeia" action of the "operational programme edu-cation and lifelong learning" and is co-funded by the european social fund (esf) and national resources. multiple sclerosis (ms) is a chronic disease of the human central nervous system that is characterized by focal lesions with inflammation, infiltration of immune cells, demyelination and axonal damage. activation of cannabinoid cb 1 /cb 2 receptors is considered a potential therapeutic strategy for the treatment of ms, based on the evidence that exogenous cannabinoid agonists exert neuroprotective and immunosuppressive effects in experimental models of the disease. nevertheless, the therapeutic use of synthetic and/or plant-derived agonists acting on brain cannabinoid receptors is limited by the possible adverse responses related to memory and learning impairment. an alternative approach that could avoid this limitation consists of enhancing the concentration of the main endocannabinoids (aea, 2-ag) by increasing their synthesis or decreasing their degradation. the main objective of this study was to analyze the effects of jzl184, a selective inhibitor of 2-ag hydrolyzing enzyme monoacylglycerol lipase (magl), in the chronic eae model of ms. mice were treated daily with jzl184 (8 mg/ kg and 32 mg/kg) or vehicle from onset of the motor symptoms to the end of the experiment. comparison of the motor score curves indicated that both doses of jzl184 ameliorated the deficits observed in vehicletreated mice during the disease course. nevertheless, the beneficial effect of the 32 mg/kg dose was no longer evident in mice scored at 40 dpi. chronic treatment with 8 mg/kg jzl184 reduced the conduction latency of the corticospinal tract measured at the end of the experiment, as well as the number and size of inflammatory lesions in spinal cord, whereas chronic administration of the high dose of the magl inhibitor had no effect on these parameters. importantly, treatment with the 32 mg/kg dose of jzl184 reduced the coupling ability of cannabinoid receptors to g i/o proteins, measured by [ 35 s]gtpcs autoradiography, in all the brain areas analyzed. finally, incubation with jzl184 prevented cytotoxicity by activation of ampa-type glutamate receptors in oligodendrocyte precursors in vitro. this protective effect of the jzl184 was blocked by the cb 1 receptor antagonist am281. our findings point to the involvement of cb 1 receptors in the in vivo therapeutic effects of jzl184, and suggest that chronic administration of magl inhibitors may be a promising strategy for the treatment demyelinating disorders in which oligodendrocyte excitotoxicity plays an important role as mechanism of white matter damage. under disease conditions, connexins can also release these signaling molecules into the extracellular milieu through hemichannels. since astrocytes are involved in the disease progression of als, we are exploring if abnormal gj activity can serve as a potential mechanism for the reported astrocyte-mediated toxicity. we used the sod1 g93a mutation as a als mouse model to address the role of connexins in als. we observed that at the end stage of the disease, spinal cords of sod1 g93a mice exhibited a significant increase in expression of connexin 43 protein compared to their spinal cords of control mice. this increase in cx43 co-localizes primarily to astrocytes in the gray matter of the spinal cord. importantly, human post-mortem tissue from als patients compared to control patients exhibited a significant increase in cx43 rna and protein levels in the motor cortex and a trend for increase in cx43 in the cervical spinal cord. this is an important finding in pursuing cx43 as a potential candidate contributing to astrocyte-mediated toxicity in als. in addition, when astrocytes were isolated from wt and sod1 g93a mice, endogenous levels of cx43 rna and protein were elevated in sod1 g93a mice compared to the wt derived astrocytes. we are currently investigating if functional properties of astrocytes from sod1 g93a mice are altered in addition to biochemical changes. in vitro studies using cx43 specific blockers to assess neuroprotection are also being conducted in co-cultures of neurons and astrocytes. these findings will be novel and an important step towards harnessing therapeutic strategies for als. the pdz protein omi/htra2 is localized in the intermembrane space of mitochondria. it is involved in mitochondrial homeostasis and may act as a chaperone. mutations in the serine protease domain of omi/ htra2 (mnd2 mice), leads to massive loss of striatal neurons and neuromuscular disorders in mice confirming the protective function within mitochondria. upon cellular stress, omi/htra2 is translocated from mitochondria into the cytosol when the mitochondrial outer membrane is permeabilised. in the cytosol, omi/htra2 blocks the action of the inhibitors of apoptosis proteins (iaps) by competitive binding or by degradation via the protease activity. this leads to caspase activation and apoptosis induction. omi/htra2 also regulates apoptosis in a caspaseindependent manner via its protease activity; recent studies have defined multiple substrates of the serine protease. in a yeast twohybrid screen we identified the ng2 proteoglycan as a binding partner of omi/htra2. ng2 is expressed by oligodendrocyte progenitor cells (opc). in the presence of hydrogen peroxide, which is a model of cellular oxidative stress, omi/htra2 is released from mitochondria and can bind to ng2 in opcs. binding of ng2 to omi/htra2 via the pdz domain may thus be a way to sequester or regulate the serine protease. oligodendrocyte-lineage cells are known to be particularly sensitive to cellular stress and white matter diseases of new borns is a major pathology in situations where premature infants are exposed to unphysiological oxygen concentrations. it has been recently shown that glioma cells often express the ng2 protein which may play a protective role. our results indicate that the interaction between omi and ng2 may help protect opcs from cellular stress. microglia are the immunocompetent cells of the cns that continuously survey the extracellular milieu for foreign antigens and play an instrumental role in brain inflammation. in contrast, astrocytes extend highly ramified processes between neurons and synapses and play a vital role in regulating brain homeostasis, synaptic function and plasticity. interestingly, both microglia and astrocytes adopt a specialized 'activated' or 'reactive' state when exposed to adverse brain conditions. the changes in their molecular profiles include the release a diversity of proteins such as pro-and anti-inflammatory cytokines and extracellular matrix molecules that could impact each other's behavior and regulate the properties of nearby neurons. in alzheimer's disease (ad), activated microglia and reactive astrocytes are detected around plaques in mouse models of ad and human ad tissues. however, exactly how these glial types contribute to the onset or progression of ad still remains an open question. to gain a better understanding of the bidirectional communication and response of microglia and astrocytes in the development of ad-like symptomatology, we investigated the properties of these cells types in the crnd8 transgenic ad mouse model by applying confocal imaging and western blot analysis. crnd8 is an early-onset model of ad-like symptomatology showing ab deposits present at 2 months of age and neuritic pathology by 5 months. our results show complex spatio-temporal changes in the interaction of microglia and astrocytes around ab plaques during the progression of the disease in both cortex and hippocampus. we found that subtle rearrangements of microglial morphology and astrocyte reactivity were detected as early as 1 month when ab plaque deposition is absent. by 2-3 months, low numbers of activated microglia and reactive astrocytes begin to surround small ab deposits. the establishment of complex 'reactive glial domains (rgds)' can be observed at 4 months, when amoeboid microglia fully encompass large ab plaques and become surrounded by reactive astrocytes forming an elaborate outer shell-like structure. intriguingly, after 6 months, the domains become progressively disrupted due to the reorganization and recruitment of additional microglia and astrocytes. we also observed gradated inflammatory phenotypes of glial cells. these are first restricted to rgds in the early and middle stages of ad but spread locally in late stages when disorganized rgds are observed. interestingly, neuronal dystrophy was correlated with the severity of the inflammation. we conclude that communication between microglia and astrocytes may be a key process in the ability of the brain to surmount a reparative response to early neurodegenerative conditions but may be disrupted or become overwhelmed in later stages of ad. supported by alzheimer society of canada (db) and by cihr (kkm and rq). poster topic 04 extracellular matrix and cell adhesion molecules opcs exist in mature rodent and human central nervous system (cns; around 5-7% of the total cells) and constitute an interesting source regenerative therapies in demyelinating diseases, like multiple sclerosis (ms). different molecules are involved in opc morphofunctional development during embryogenesis and postnatal stages, and some of them are upregulated in ms lesions, suggesting their involvement in the pathogenesis of the disease. this is de case of anosmin-1, an extracellular matrix glycoprotein coded by the kal1 gene and responsible for the x-linked form of kallmann syndrome. the best known mechanism of action of anosmin-1 seems to be mediated through the interaction of this protein with fibroblast growth factor receptor 1 (fgfr1) and the modulation of the activation of this receptor by fgf2. this protein participates in the adhesion, migration and differentiation of various cell types in the cns; among others, anosmin-1 promotes the adhesion of neurons, neurite outgrowth, axonal guidance and branching of different cns projection neurons, as well as having a role in the migration of different types of neuronal precursors, immortalized gnrh-producing neurons and embryonic opcs. in addition, previous results of our group also suggest a role of anosmin-1 in demyelinated lesions from ms patients and point to the feasible pharmacological and genetic manipulation of the fgf2/fgfr-1/anosmin-1 system on endogenous and/or exogenous opcs in demyelinating lesions. in the present work we studied the functional implications of anosmin-1 and fgf-2 on neurobiology (cell death, proliferation, motility, migration) of postnatal/adult murine opcs isolated from cerebral cortex (p0, p15, p60) and of opcs isolated from adult human biopsies, using both in vivo (immunohistochemistry) and in vitro (chemotaxis chambers, migration, brdu uptake, tunel, immunocytochemistry) techniques. these results would be useful for the design of effective neuroreparative therapies in ms and other demyelinating diseases. funded we generated ipsc lines from fibroblasts isolated from pitx3-gfp knock-in mice which allowed facs-purification of mature midbrain da neurons upon in vitro differentiation based on the expression of the mature da marker pitx3. subsequently, mouse pitx3-gfp knock-in ips cells were transduced with lentiviral vectors containing genes encoding for hl1 and for stx, the enzyme that add psa onto ncam. next they were differentiated in vitro into da neurons. the effects of hl1 and psa-ncam on differentiation, survival and outgrowth of ipsc-derived da neuron were analyzed in vitro and in vivo. indeed, we were able to obtain a pure suspension of mature ipscderived da neurons upon in vitro differentiation and fac sorting. transfection of pitx3-gfp knock-in ips cells with hl1 or stx did not affect the pluripotent potential of these cells and did not interfere with the process of dopaminergic differentiation itself. we continued to establish the beneficial effect of l1 and psa-ncam expression on survival and outgrowth of ipsc-derived da neurons in vitro and after grafting in the striatum of parkinsonian rats. vascular endothelial growth factor (vegf) is a major regulator of neurovascular remodeling following brain injury. while much have been learnt about vegf functions on target endothelial cells, little is known about its intracellular trafficking, mode of secretion and matrix interactions in "source" cells, i.e. mainly reactive astrocytes. here, we generated a vegf::gfp fusion protein to follow the distribution of vegf165 during its trafficking in primary astrocytes and cos7 cells. we found that vegf::gfp forms dimers and gets glycosylated similarly to wild type, and as expected, the molecule follows the endoplasmic reticulum-golgi pathway. however, its post-golgi trafficking suggests a unique route with features that do not conform the classical constitutive secretory pathway: the secretion of vegf165 occurs even at 19 c and shows a ca21-and pkc-induced increase. we investigated the distribution of vegf in polarized primary astrocytes in an in vitro scratch wound assay. in these cells, vegf::gfp appeared to follow a vectorial distribution and accumulated behind the leading edge. the accumulation appeared to be on the extracellular surface, moreover, ultrastructural immunogold labeling revealed that extracellular vegf::gfp remains associated with discrete areas of the cell membrane and is accumulated in caveolae as well as in microvesicular shedding elements. this particular localization corresponds to fibrillar adhesions (fb), where vegf::gfp is co-localized with fibronectin. we also observed that vegf::gfp is targeted to adherens junctions between astrocytes, however, at these sites vegf::gfp was not co-localized with fibronectin. finally, we show in frap experiments that integrin turnover is decreased in vegf associated fbs, which raises the possibility of an autocrine/paracrine regulation of astrocytic functions. together, these findings have strong implications for understanding focal coordination of angiogenesis and vascular remodeling by astroglia derived vegf. after invading the embryonic cns, microglia migrate extensively to occupy their final positions. however, the cellular and molecular mechanisms of microglial migration are unknown. therefore, this study aims to elucidate the microglial migration behavior, cellular interaction partners and ecm-integrin interactions essential for migration in the developing neocortex. methods: immunohistochemical stainings and immunogold labeling for transmission electron microscopy were carried out on fixed brain tissue of c57bl/6 cx3cr1 1/egfp mice embryos (embryonic day (e) 10.5-17.5). multicolor facs analyses were performed on homogenized age-pooled cx3cr1 1/egfp embryonic cortici. microglial migration was recorded in cx3cr1 1/egfp -hgfap-cfp acute brain slices using multiphoton time-lapse excitation and analyzed using mtrackj in imagej. results: laminin (lm) is expressed as punctuate dots near the pia and in the ventricular zone at e10.5 and disappears at e15.5 to remain only in blood vessels and the pia. fibronectin (fn) is present as aggregates from e10.5 until e17.5. both ecm proteins follow the course of radial cells. preliminary data indicate a transient upregulation of the lm receptor (lmr) on e15.5 and e16.5. in addition, the percentage of microglia expressing the fn receptor (fnr) tends to decrease during development. ex vivo time-lapse recordings show that embryonic microglia are dynamic cells that migrate in random patterns. these cells seem to make brief contacts with the processes of radial glia. conclusions: embryonic microglia express laminin and fibronectin receptors and possibly change the expression level during development. they are dynamic cells that migrate in random patterns, and possibly make occasional contact with radial glia. knowledge about ecm-integrin interactions during microglial migration could reveal targets for intervention in pathological settings, such as multiple sclerosis and alzheimer's disease. multiple sclerosis (ms) is a chronic demyelinating disease of the central nervous system in which astroglial cells become hypertrophic, produce various extracellular matrix (ecm) proteins which become aggregated when secreted. these aggregated ecm proteins contribute to a non-permissive environment for repair. tissue transglutaminase (tg2) expression is enhanced in astrocytes in ms lesions. this enzyme is well-known for protein cross-linking capacities. moreover, tg2 can act as a co-receptor for b-integrins to bind to ecm proteins, thereby mediating cell adhesion processes. in this study we questioned whether astrocyte-derived tg2 affects ecm protein production and/or aggregation, and if astrocyte-derived tg2 mediates cell adhesion onto various ecm proteins. primary rat astrocytes were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype tg2. alternatively, primary rat astrocytes were transduced with a lentiviral vector expressing scrambled shrna or tg2 shrna to knockdown endogenous rat tg2. the rat primary astrocytes overexpressing human tg2 showed an increased production of fibronectin and collagen v. conversely, knocking-down tg2 showed a decrease in fibronectin and collagen production. interestingly, overexpression of tg2 resulted in enhanced aggregation of extracellular laminin. also, human astrocytoma cells (u373) were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype tg2 and allowed to adhere onto ecmcoated wells. astrocytoma cells overexpressing human tg2 showed increased adhesion onto laminin-, and fibronectin-coated wells. we conclude that astrocyte-derived tg2 affects production/aggregation of fibronectin and laminin. moreover, the astrocyte-derived tg2mediated elevated production/aggregation of fibronectin and laminin coincides with more adherence of astrocytes onto these ecm proteins. we hypothesize that tg2-mediated enhanced production/aggregation of fibronectin and laminin is related to increased astrocyte adhesion which together could contribute to the non-permissive environment for repair in the central nervous system of ms patients. a. tripathi, z. parikh, p. pillai the m.s. university of baroda, vadodara, india background: oligodendrocytes originate as progenitor cells (opcs) in discrete areas of the developing brain which undergoes a complex and precisely timed program of proliferation, migration, differentiation, and myelination in cns. during migration it interacts with its surrounding environment through integrins. purpose: to evaluate the interactions between integrins and growth factors (gfrs) that promote the molecular events such as proliferation, cytoskeletal reorganization and migration in oligodendrocytes leading to oligodendrocyte mediated myelination. methods: cortical cells were prepared from p0 rat cerebral cortex following standard protocols. immunocytochemistry (icc), western blot (wb), immunoprecipitation (ip), migration assay and real-time rt-pcr analysis were performed. for dissecting out the roles of different intracellular signalling proteins in the regulation of events downstream of receptor activation, use of pharmacological inhibitors was carried out. results: tnfa, pdgfa and estradiol significantly increased cell proliferation and cell processes. data also suggest differential effects of extracellular matrix (ecm) on signalling component activation. significant increase in erk expression was found following gfrs-integrin interactions. ecm proteins bind to integrin family members and mediate bidirectional signalling between the cellular interior and the external environment through the activation of focal adhesion complexes. conclusion: integrin switching, oligodendrocyte proliferation, cytoskeletal reorganization and differentiation profile is highly influenced by ecm and gfrs. integrin governs the signalling cascades underlying oligodendrocyte differentiation and myelination. this study was performed in strict accordance with the recommendations for the care and use of laboratory animals. all the protocols were duly approved by the institutional ethical committee, the m.s. university of baroda. topography, roughness and mechanical properties of micro environment are crucial parameters influencing cell adhesion/motility, morphology and mechanics as well as the development of cells e.g. stem/progenitor cells, and neuronal cells [1] [2] [3] [4] [5] . atomic force microscopy is a powerful tool not only to study the morphology in terms of high resolution imaging and roughness measurements, but also to map mechanical and adhesive properties of the sample/cells and tissues. combining these remarkable abilities with advanced optical microscopy allows for extensive characterization of biomaterials and tissue slices [6] [7] [8] . however, commercially existing technical solutions are either time consuming, or only limited practicable for soft, sticky, or fragile samples. therefore, we developed a new force curve based afm mode -quantitative imaging (qi tm ). the novel qi tip movement algorithm prevents lateral forces and controls the vertical forces for nondestructive imaging. to demonstrate the performance and flexibility qi tm mode we investigated topography, adhesion properties, and young's modulus local distribution of living dorsal root ganglion cells. a specialized platform -cellhesion v r -has been developed to run single cell force spectroscopy (scfs) with a need for long-range cellsurface and cell-cell binding experiments with up to 100 microns pulling length. using single cell force spectroscopy (scfc), cell-cell adhesion can be quantified [e.g. 3], and the contribution of different components e.g. from the extra cellular matrix, can be assessed [9] . a new technical solution will be demonstrated if a cantilever attached cell can be transferred through the liquid-air interface. this approach allows to measure the adhesion of the same individual single cell on different materials within different dishes as it will presented for cho cell adhesion on plastic as well as albumin coated surfaces. we want to present the progress in automatic data processing and display to investigate topography, young's modulus and local adhesion phenomena on transparent and non-transparent biomaterials like titanium, peptide functionalized surface or cell layers, and tissue slices. l. vargova 1 , m. cicanic 1,2 , e. sykova 1,2 1 charles university, 2nd faculty of medicine, prague, czech republic 2 institute of experimental medicine, v.v.i., department of neuroscience, prague, czech republic bral-2 is a link protein that is localized in distinct brain regions, where it stabilizes the perineuronal nets of the extracellular matrix (ecm). our previous studies showed that qualitative and quantitative ecm changes, might evoke changes in the diffusion properties of the extracellular space (ecs), thus affecting the movement of neuroactive substances in the cns. in the current study, we determined by the real-time iontophoretic method the extracellular space volume fraction a (a 5 ecs volume / total tissue volume) and the geometrical factor tortuosity k (k 2 5 free / apparent diffusion coefficient) in coronal brain slices obtained from the sensorimotor cortex and the ventral posteromedial thalamic nucleus of sixteen-month-old bral-2 deficient mice (bral-2 -/-), age-matched bral-2 1/1 controls and young adult (5-month-old) bral-2 1/1 controls. the diffusion parameters in the cortex of young adult bral-2 1/1 mice were: a 5 0.21 6 0.01 (mean 6 s. e. m.) and k 5 1.48 6 0.02, n 5 6, n 5 5 (n -number of slices, n -number of animals). in the thalamus, there was a smaller a and a larger k (0.16 6 0.01 and 1.53 6 0.01, respectively, n 5 26, n 5 10). in young adult mice, we found no significant differences in the ecs diffusion parameters between bral2 positive and negative mice in either the cortex or the thalamus. in the cortex of aged bral-21/1 mice we found a smaller a (0.18 6 0.01, n 5 10, n 5 7) than in the cortex of young adult mice. during aging, there was no significant difference in the thalamic diffusion properties in wild-type animals, but in the thalamus of aged bral-2 -/-mice, a significantly decreased (0.13 6 0.01, n 5 28, n 5 11) in comparison to the younger bral2-/-animals as well as to age-matched controls. immunohistochemical analysis of the ecm in the ventral posteromedial thalamic nucleus revealed no positivity for bral2 protein in either group of bral2-/-mice. in the youger mice, bral deficiency was associated with a small attenuation in the positivity of brevican and another link protein, ctrl1; however, in aged bral2-/-animals, brevican and ctrl1 positivity was increased in comparison with the age-matched controls. in contrast to the cortex, we found no decrease in a in the thalamus of aged wt animals, suggesting that the process of aging may differ between the cortex and the thalamus. a decrease in the ecs volume fraction in the thalamus of bral-2 -/-aged mice may result from a disruption of the perineuronal nets and rearrangements of the molecular assembly, which seem to differ in young adult and aged animals. here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of camp pathways. a bulk of astroglial transcripts were differentially regulated by camp signaling. camp analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. moreover, genes typically activated in reactive cells, such as immunological mediators and scar components, were repressed by camp. gene set enrichment analysis and evaluation in situ of gene expression in astrocytes in different states showed that camp signaling conferred a mature and in vivo-like transcriptional profile to cultured astrocytes. these results indicate that camp signaling is a key pathway restricting developmental and activation features of astrocytes and promoting their maturation. a positive modulation of camp signaling is suggested to suppress the mechanisms of activation driven by pathological situations and to promote the physiologically normal state of differentiated astrocytes. question: microglia, the innate immune system of the central nervous system, are crucial to maintain homeostasis by cleaning debris and attacking stressors. in the ageing brain, increased numbers of microglia with ameboid morphology and increased inflammatory cytokine levels are reported. the altered microglial phenotype in the ageing brain is characterized by hyperresponsive to immune stimuli, which is called immune priming. immune priming could be a potential cause of age-associated neurodegeneration. the aim of this study is to characterize the immune pathways and biological processes that are altered in ageing-induced immune primed microglia and to find genes potential drivers of this process. design/methods: the effect of ageing on microglia has been addressed in naturally aged mice (24 months old) and in ercc1 transgenic mouse model in which accelerated ageing is induced by dna damage accumulation. from each of these models a pure population of microglia was isolated. microarray technology was used to obtain information about the genome wide gene expression profile. weighted gene co-expression network analysis (wgcna), uses correlational structures to find clusters of genes to make optimal use of a dataset in biological interpretation. results: using weighted gene co-expression network analysis, a very robust microglial immune priming gene module was discovered. in this module, there is a significant enrichment of the complement system, antigen presentation, interferon type-1 signaling and phagocytosis-machinery. the average expression of this module is stronger up-regulated in ercc1 transgenic than in normally physiologically aged mice, supporting the notion that ercc1 is characterized by excessive immune priming. conclusion: microglial priming which occurs in normal ageing or in accelerated ageing models is marked by increased expression of several immune signaling pathways. methods: we investigated three single nucleotide polymorphisms (snps) (rs1042713 (arg16gly), rs1042714 (gln27gly) and rs1042711) and five haplotypes in adrb2 to explore whether these polymorphisms were associated with ms progression in 436 well phenotyped ms patients. genotyping was done using illumina bead microarray and missing genotypes were imputed using beagle. results: a trend towards a decreased frequency of rs1042714c allele (27gln) was in people with progressive ms than in people with relapsing remitting ms. this was seen in both sp-ms (p (uncorrected) 5 0.0072) and in pp-ms (v 2 5 6.85, p (uncorrected) 5 0.0325). however, those p values did not reach statistical significance after correction for multiple testing. neither haplotypes nor snps were significantly associated with altered msss, age of onset, time between fist and second relapse, processing speed (sdt) or brain atrophy. conclusion: we found no evidence that polymorphisms in adrb2 influence severity in multiple sclerosis. here we examine the expression of these brain-specific pgc-1a isoforms in neurons and glia cells. we characterize the abundance of the different isoforms of pgc-1a in primary murine cell cultures of neurons, glia cells, including oligodendrocytes, astrocytes and microglia under control conditions and metabolic stress. we show that pgc-1a is differentially expressed in the different cell types, e.g. neurons and oligodendrocytes express much higher levels of the novel brain-specific isoforms of pgc-1a. the different expression levels might reflect the different metabolic needs of the different cell types. s. marques, g. castelo-branco karolinska institutet, stockholm, sweden oligodendrocytes (ol) are neuroepithelium-derived cells that insulate neuronal axons through myelin-containing membranes, essential for axonal integrity and impulse transmission. defects in this process are present in several diseases, including the highly debilitating multiple sclerosis (ms). the process of remyelination can be promoted by ol precursor cells (opc) endogenously and occurs during a short window of time at earlier stages of ms, ultimately failing as the disease evolve. the understanding of development and differentiation regulation of ol is essential to clarify the reasons behind the defective myelination during pathologies and to open the pave to new remyelinating cell therapies. opcs start to be specified early during embryogenesis but regardless of the origin, their terminal differentiation and functional maturation occurs only at post-natal stages. in this project, we are characterizing the transcriptome of opcs during development, which will allow us to better characterize the heterogeneity of these cells and point out what might be the key factors involved in transition between states. recent studies suggest that non-coding rnas can interact with proteins complexes containing chromatin modifying enzymes and transcription factors, regulating their activity, acting as scaffolds and/or recruiting them to specific loci in the genome. therefore, we are investigating their role in opcs by rna interference and how they might modulate the function of key transcription factors. neural stem cells (nscs) represent a potentially valuable resource when considering repair strategies for cns damage. unlike their embryonic counterparts, adult neural stem cells (anscs) in the two neurogenic niches have an astrocyte-like identity, raising the question of what regulates this ansc state. some early postnatal astrocytes in the parenchyma also retain some nsc-like characteristics and in vitro display self-renewal and multipotency, though these properties are lost following in vivo maturation. interestingly, following injury, adult astrocytes can become reactive and reacquire nsc-like properties, whilst others can generate new neurons through forced expression of specific transcription factors. we are aiming to elucidate the gene regulatory networks underlying specific nsc and astrocyte states and transitions between them using transcriptomic and epigenomic tools alongside computational methods in different nsc and astrocyte populations. we will describe our latest findings from an in vitro system that aims to model immature versus mature astrocytes derived from a common progenitor. from microarray data, we have identified candidate genes and pathways involved in regulating astrocyte potential versus differentiation, including a role for specific pro-inflammatory signalling. we will also describe the associated epigenetic signatures that accompany cell state and discuss the implications of our latest findings. microglia are the cns immune-related cells and associated with the pathogenesis of several types of neurological diseases. following peripheral nerve injury (pni), spinal microglia become activated and have crucial roles in generating neuropathic pain. we have previously shown that interferon regulatory factor-8 (irf8), upregulated in spinal microglia after pni, regulates gene expressions that switch to a reactive phenotype. here we identified irf5 as a crucial factor of reactive microglia that works cooperatively with irf8. we found that pni increased expression of irf5 in the spinal cord, the expression of which was restricted to microglia. forced expression of irf8 in cultured microglia markedly increased expression of irf5 in a manner that depended on its ability to bind dna. furthermore, irf8-deficient mice failed to increase the expression of irf5 in spinal microglia after pni, indicating irf8-dependent expression of irf5 in microglia in vivo. interestingly, upregulation of p2x4 receptor (p2x4r; an atp-gated channel crucial for producing neuropathic pain) by forced expression of irf8 in cultured microglia was markedly suppressed by knockdown of irf5 expression. in a model of neuropathic pain, suppressing upregulated irf5 protein by spinal administration of sirna targeting irf5 alleviated pain hypersensitivity. altogether, the irf8-irf5 axis drives a program of gene expression that promotes p2x4r hi microglia gating neuropathic pain. astrocytes are the most abundant glial cell type. they express a range of neurotransmitter receptors localized along their processes that cover synapses and constitute "microdomains" for signalling pathways. to investigate astroglial purinergic p2y1 and glutamatergic nmda receptors we generated knockout mice in which gene recombination of "floxed" receptors was controlled by an astrocyte-specific and tamoxifen-inducible cre recombinase (glast-creert2). here, we present data on the dna recombination efficiencies of glast-creert2 x floxed p2y1 mice and glast-creert2 x floxed glun1 mice in different brain regions (cerebellum, hippocampus, brainstem, optic nerve and cortex). recombination is determined by quantitative real-time pcr of genomic dna using primers across the loxp sequences flanking exon 1 of the p2ry1 gene and exon 11-21 of the grin1 gene, respectively. dna recombination was induced in young adult mice by intraperitoneal tamoxifen injection for three consecutive days and analyzed three weeks later. primers were designed such that reduction of the pcr signal reveals increasing recombination (i.e. gene excision and knockout) compared to control. the degree of reduction in the investigated brain regions is expected to be similar in both mouse lines; allowing conclusions (1) about the reliability of the glast-creert2/loxp system in general (i.e. recombination efficiency at different chromosomal locations), and (2) gives a percentage of glastpositive cells (predominantly astrocytes) in the given brain area. first data demonstrate 30% recombination in the cortex, which is well in line with the percentage of astrocytes in this brain region. these results could be confirmed by using a reporter mouse line expressing the red fluorescent protein tdtomato (r26-tdtomato) and using immunohistochemistry with astrocyte markers. in conclusion, the combination of quantitative real-time pcr analysis of gene recombination and cell-specific cre mouse lines represents a reliable approach to reveal the percentage of a given cell type in a given tissue region. the transcription factor creb is protective in injury and neurodegeneration, but the contribution of neuronal vs astrocytes is unknown. we have generated a mouse with targeted over-expression of a constitutively active creb (vp16-creb) in astrocytes by crossing teto-vp16 and tta-gfap mice. vp16-creb/gfap mice show expression of vp16 in astrocytes -as detected by immunohistochemistry -restricted to cerebellum, hippocampus and outer layers of cortex. we determined the role of astrocytic creb in the outcome of brain injury by subjecting wild type (wt) and vp16-creb/gfap (tg) mice to a focal cryolesion (c) in the frontal cortex. at 3 days post-lesion, wt mice presented a necrotic region filled with macrophages (labeled with lectin) surrounded by a rim of tissue with robust gliosis (labeled with gfap). tg mice showed reduced macrophage infiltration as compared with wt mice. a dna array analysis of the damaged zone (agilent) has revealed differentially expressed genes in paired comparisons. wtc/wt: 13,984; wtc/tgc: 5,821; tgc-t:913; tg/wt:0 (statistical linear model in limma., p values computed by empirical bayes moderated t-statistics at 0.05). there were 159 significantly enriched kegg pathways regulated by the cryolesion, according to gsea and hypergeometric tests set at p < 0.05. vp16-creb had an effect on 127 of the pathways. upon increasing the analysis stringency by setting the cut-off at p < 0.0001, the number of significantly enriched kegg pathways regulated by vp16-creb was reduced to 16. we selected 10 of these pathways related to 5 functions: energy metabolism, inflammation, structural reorganization, genomic stability and apoptosis, which we define as the core signature of the vp16-creb-elicited change. the differential expression of representative genes related to this signature was validated by real-time pcr. thus, vp16-creb rescued the decreased expression of enzymes regulating energy metabolism, while decreasing the expression of pathways related to apoptosis, extracellular matrix and inflammation. the latter is consistent with the decreased macrophage infiltration detected by immunohistochemistry in tg mice. we posit that vp16-creb protects the brain against bionergetic failure and secondary damage due to macrophage infiltration, while rendering the tissue more conducive to neurite regeneration by reducing extracellular matrix components. the study supports the notion of astrocytes as therapeutic targets in brain injury. furthermore, ca 21 /cn/nfat dependent mmp3 expression was confirmed in pure astrocyte cultures derived from neural stem cells (ast-nsc), demonstrating that the induced mmp3 expression occurs in astrocytes, and not microglial cells. in an in vivo stab-wound model of brain injury, mmp3 expression was detected in nfatc3-positive scarforming astrocytes. since [ca 21 ] i increase is an early event in most brain injuries, these data support an important role for ca 21 /cn/ nfat-induced astrocyte mmp3 expression in the early neuroinflammatory response. understanding the molecular pathways involved in this regulation could provide novel therapeutic targets and approaches to promoting recovery of the injured brain. the posterior hypothalamus (ph) is crucial for maintaining wakefulness whereas the anterior hypothalamus (ah) constitutes a sleep-promoting region. consequently, the energy needs of ah and ph should probably change throughout the sleep-wake cycle. the glycogen mobilization altogether with the astrocyte-neuron lactate shuttle (anls) likely constitutes two major mechanisms by which astrocytes ensure the neurometabolic coupling. therefore, we hypothesized that astrocytes of ah and ph may display differences in genes expression involved in these mechanisms during the sleep-wake cycle. to investigate this, we measured the gene expression levels in astrocytes obtained from transgenic mice expressing the fluorescent protein egfp under the control of the astrocyte-specific gene gfap promoter. mice were sacrificed at four time, zt0, zt6, zt12 and zt18 (zt0 corresponding to light-on). after brain dissection, "punches" of ah and ph were micro-dissected from slices. cell suspensions were obtained from a pool of 3 samples by enzymatic digestion and cell trituration. then, gfp-positive cells, which were highly enriched in astrocytes, were sorted by facs. total rna was extracted from these cells and gene expression levels were assayed by qrt-pcr and normalized by cyclophylin mrna levels. seven genes related to anls were tested: the alpha 2 sub-unit of the na-k atpase (atp1a2), the two sub-unit of the lactate dehydrogenase (ldha and ldhb), the glutamate transporters (glast and glt1) and two monocarboxylate transporters (mct1 and mct4). levels of mrna encoding genes related to glycogen metabolism such as ptg, the glycogen synthase (gs) and the glycogen phosphorylase (gphos) were also measured results showed that the levels of mrna encoding ldha, mct1 and mct4 expressed significant circadian variations which can reach 200% for mct4 at zt0, compared to zt6 levels in ah. the glt1 mrna levels as well as mct1 and mct4 mrna levels also displayed significant circadian variations in ph. levels of expression of genes encoding the gphos, gs and ptg did not display major regional circadian variation. this study shows that astrocytes displayed some transcriptional regulation in hypothalamic areas involved in the sleep-wake cycle. the difference in the pattern of expression of anls related genes in ah and ph, such as glt1, suggests that neuro-metabolic coupling capacity of each structure might be different and might play a functional role in the sleep-wake regulation. , are a heterogeneous cell population that is despite its functional importance still not well defined. while astrocytes are no longer just considered the supporting cells in the cns and are recognized to play a significant functional role in e.g. synapse formation, regulation of the blood brain barrier and synaptic transmission, little is known whether specific subtypes of astrocytes fulfill these functional tasks differently. studies in our laboratory described brain region specific differences in the expression of astrocytic glutamate transporter 1 (glt-1) with significantly increased levels of glt-1 in the spinal cord compared to brain. the mechanisms of glutamate transporter regulation are not well defined, and to date little is known about the factors that are responsible for regulating protein expression and transporter activity and whether this regulation is specific for certain subtypes of astrocytes. to study glt-1 regulation in more detail, we generated a family of transgenic mice using increasing lengths of the 5 0 non-coding region of the glt-1 gene controlling expression of tdtomato. these mice were crossed with full length bac-glt-1-egfp mice to compare astrocytic expression of the reporter genes. interestingly, only a subpopulation of gfp-positive astrocytes was found to have strong tdtomato fluorescence signal, when we crossed an 8.3 kb eaat2 promoter fragment mouse with the bac-glt-1 mouse, suggesting the existence of astroglial subtypes that are differentially regulated for eaat2 expression. such differences might account for variance in local glutamate-dependent excitotoxicity to motor neurons. to this end, we purified both tdtomato1/gfp1 and tdtomato-/gfp1 astrocytes from multiple cns regions by fluorescence-assisted cell sorting (facs) and analyzed their transcriptomic profiles using microarray analysis. a list of candidate genes, mainly transcription factors and cell membrane molecules, was complied from this analysis and validated by both quantitative rt-pcr and immunofluorescence. pax6 and kir4.1 were found to be highly expressed in astrocytes, consistent with previous findings. more importantly, kir4.1 mrna was noted to be selectively enriched in cortical tdtomato1/gfp1 cells, compared to gfp1 only cells. it suggests kir4.1 may serve as an astroglial subtype marker. . we have hypothesized that microglial c/ebpb is a potential target to attenuate the neurotoxic effects of neuroinflammation. in order to test this hypothesis in vivo, c/ebpb knockout mice are not suitable because they show multiple phenotypic alterations as a result of the widespread expression of c/ ebpb. mice with specific c/ebpb deletion in microglia would be a better model. unlike gfap promoter for astrocytes, there is not a gold standard promoter for cre expression in microglia. cd11b, cx3cr1 or lysozime m (lysm), among other promoters, have been used to this end. we have generated lysm-cre/c/ebpb fl/fl mice with two main aims: 1) to validate the use of lysm as a suitable promoter for microglial gene deletion by cre-lox recombination 2) to analyze the effects of the absence of microglial c/ebpb in vitro and in vivo lysm-cre/c/ebpb fl/fl mice which were fertile and viable and did not show the female infertility and high perinatal mortality described in c/ ebpb knockout mice. western blot experiments revealed the presence of c/ebpb in protein extracts from wild-type but not from lysm-cre/c/ ebpb fl/fl microglial cultures. immunocytochemistry experiments confirmed this observation. thus, c/ebpb immunoreactivity was present in all cells in wild-type microglial cultures and it was absent in all microglial cells in lysm-cre/c/ebpb fl/fl microglial cultures. the microglial specificity of lysm-cre recombination was demonstrated in primary mixed glial cultures. whereas in wild-type primary mixed glial cultures c/ebpb immunoreactivity was observed both in astrocytes and microglia, in lysm-cre/c/ebpb fl/fl mixed glial cultures it was detected in astrocytes, but never in microglia,. to assess the functional outcome of microglial c/ebpb deficiency, we analyzed lps1ifncinduced no production and we observed a marked decrease in lysm-cre/c/ebpb fl/fl microglial cultures. this decrease was also observed in mixed glial cultures in fitting with the microglial origin of no production in this model. in summary, these findings show that lysm-cre promoter causes recombination in 100% of microglial cells in primary culture and it is therefore suitable for microglial-specific gene deletion in vitro. experiments are in progress to determine the degree and specifity of microglial c/ebpb deletion in vivo in these mice. supported by grants pi10/378 and pi12/00709, instituto de salud carlos iii, spain. to study the functional role of cpeb3, transgenic mice overexpressing cpeb3 in astrocytes were generated. cpeb3 overexpression without the endogenous 3 0 untranslated region (utr) led to the downregulation of astrocytic connexins (cx43 & cx30). consistently, in the hippocampus of cpeb3 overexpressing mice, intercellular gap junction coupling was strongly impaired. cpeb3 overexpression also led to downregulation of glutamate transporter 1 (glt-1) and glutamine synthetase (gs), key players involved in extracellular glutamate clearance. we have now raised mice overexpressing cpeb3 with the endogenous 3 0 utr. in these mice, we observed the downregulation of cx43, cx30, glt-1 and gs. since interastrocytic coupling and astrocytic glt-1 and gs activities are downregulated in epilepsy patients with hippocampal sclerosis, we hypothesize that upregulation of cpebs in astrocytes may contribute to the pathogenesis of epilepsy by causing astrocyte dysfunction. to study the influence of the p53 gene in the structural and quantitative characteristics of astrocytes in the mice retina methods: adult mice of the c57bl/6 strain (12 months old) were distributed into two groups: 1) mice with two extra copies of p53 ("super p53"; n 5 6) and 2) wild-type p53 age-matched control, as the control group (wt; n 5 6). retinas were immunohistochemically processed with gfap. gfap1 astrocytes were quantified. results: in comparison con wt: i) retinal-astrocyte distribution followed established patterns; however, morphological changes were seen through the retinas in relation to p53 availability: astrocytes were more robust and secondary processes were more evident; ii) astroglial density was significantly higher in the sp53 retinas, both in the whole-retina (p < 0,01 student's t-test) and in the intermediate and peripheral concentric areas of the retina (p < 0.05 student's t-test). conclusion: the astrocyte changes in the sp53 retinas might improve the resistance of the retinal cells against oxidative stress and its downstream signalling pathways. however, neuronal damage caused due to excessive microglial activation is a well known phenomenon in neurodegenerative diseases. mirnas are novel regulators of gene function, controlling several biological processes. recent reports show altered mirna expression in immune-mediated pathologies, suggesting that mirnas have roles in modulating immune responses. thus, the present study was initiated to identify micrornas and their target mrnas which can modulate microglial inflammatory responses. a pilot study was designed to understand the role of mirna-200b in modulating microglia-mediated immune response as this was found to be localized in the microglia. recently, mirna 200b has been shown to target several proteins including c-jun, the substrate of jnk mapk (mitogen-activated protein kinase) which mediates the release of proinflammatory cytokines in activated microglia. qrt-pcr revealed the decrease of mir-200b expression level in activated bv2 microglia. loss-of-function and gain-of-function studies confirmed c-jun to be the target of mir-200b in microglia. overexpression of mir-200b in activated microglia resulted in a decrease in c-jun and jnk expression and activity, thereby dysregulating the mapk-jnk pathway and proinflammatory cytokines. further, treatment of neuronal cells, mn9d with conditioned medium obtained from activated microglial cells, resulted in increased inflammatorymediated cell death upon knockdown of mir-200b. overexpression of mirna-200b also reduced the phagocytic ability in activated microglia. taken together, these results demonstrate the important role of mir-200b in modulating the mapk pathway via c-jun which in turn affects different aspects of the inflammatory process accompanying microglia activation including cytokine response, no production, phagocytosis and neuronal cell death. thus, mir-200b may prove to be a useful target for developing therapeutic strategies to control microglial mediated inflammation in neurodegenerative diseases. further in order to understand the global mirna changes contributing to microglial activation, a global mirna microarray was carried out using control, lps-activated and amyloid b-activated primary microglia. this screen identified several families of mirnas differentially expressed between the different treatment groups enabling us to analyze the mirna signature in activated microglia. national university of singapore, singapore, singapore an inflammatory process in the brain as a result of an injury or infection is mediated by the microglia, the prime immune effector cells of the central nervous system (cns). microglia have also been shown to display chronic inflammatory responses leading to neurodegenerative disorders. small ubiquitin-like modifier-1 (sumo-1) is a post-translational protein modifier, which has been shown to be associated with nuclear factor kappa b (nfjb)-mediated inflammatory pathway. in this study, we showed the expression of sumo-1 in microglia and characterized the roles of sumo-1 in activated microglia. we found that sumo-1 is specifically expressed in the amoeboid microglial cells of the early postnatal rat brain. secondly, lipopolysaccharide-mediated activation of microglia led to a significant increase in the expression as well the nuclear translocation of sumo-1 in microglia in vitro. in order to establish a function for sumo-1 in activated microglia, we sought to silence its expression using sirna-mediated gene knockdown. sumo-1 knockdown in lps-treated bv-2 microglia in vitro subsequently showed the decreased nuclear translocation of nfjb and expression of tumour necrosis factor-alpha (tnf-a) level. further, we performed an in silico screening to identify the targets of sumo-1 in the nfjb pathway. our study thus, suggests that sumo-1 regulates nfjb translocation into the nucleus for the transcription of pro-inflammatory genes including tnf-a in activated microglia. microglia are the main resident immunological cells the cns. in the healthy brain, microglial cells are in a surveillance state whereas upon rupture of cns homeostasis they enter activated states. in ischemic stroke, activated microglia is one of the most important cellular components of post-stroke neuroinflammation, which mainly occurs in and around the area of the infarct. microglia activation is characterized by morphological alterations, functional and transcriptional remodeling, which account for the acquisition of immune phenotypes. purinergic receptors are known to play important roles in microglia activation, and p2x4r have been suggested as potential therapeutic target to limit microglia-mediated inflammatory responses associated with brain diseases. in the present study, we have developed an experimental approach based on laser micro-capture to isolate microglia from the penumbra area at different post-lesion times (from 4h to 7 days post-lesion). the repertoire of genes expressed by microglial cells was determined through cdna microarray analysis. with this approach we have been able to determine clusters of genes that are co-regulated thus revealing the time course of microglia activation in this model. in addition, we have compared the transcriptional remodeling of microglia in wild-type and p2x4-deficient mice. our results suggest that wild-type and p2x4r ko mice present specific transcriptional profiles, which only partially overlapped. thus our data suggest that p2x4r play significant roles in the regulation of microglial cells functions. z. chen 1 , m. ghosh 2 , r. sattler 1 , m. robinson 2 , j. rothstein 1 1 johns hopkins university, baltimore, united states 2 university of pennsylvania, philadelphia, united states question: astrocytes, the most abundant cell type in the central nervous system (cns), are a heterogeneous cell population that is despite its functional importance still not well defined. while astrocytes are no longer just considered the supporting cells in the cns and are recognized to play a significant functional role in e.g. synapse formation, regulation of the blood brain barrier and synaptic transmission, little is known whether specific subtypes of astrocytes fulfill these functional tasks differently. studies in our laboratory described brain region specific differences in the expression of astrocytic glutamate transporter 1 (glt-1) with significantly increased levels of glt-1 in the spinal cord compared to brain. the mechanisms of glutamate transporter regulation are not well defined, and to date little is known about the factors that are responsible for regulating protein expression and transporter activity and whether this regulation is specific for certain subtypes of astrocytes. methods: to study glt-1 regulation in more detail, we generated a family of transgenic mice using increasing lengths of the 5 0 non-coding region of the glt-1 gene controlling expression of tdtomato. these mice were crossed with full length bac-glt-1-egfp mice to compare astrocytic expression of the reporter genes. results: interestingly, only a subpopulation of gfp-positive astrocytes was found to have strong tdtomato fluorescence signal, when we crossed an 8.3 kb eaat2 promoter fragment mouse with the bac-glt-1 mouse, suggesting the existence of astroglial subtypes that are differentially regulated for eaat2 expression. such differences might account for variance in local glutamate-dependent excitotoxicity to motor neurons. to this end, we purified both tdtomato1/gfp1 and tdtomato-/gfp1 astrocytes from multiple cns regions by fluorescence-assisted cell sorting (facs) and analyzed their transcriptomic profiles using microarray analysis. a list of candidate genes, mainly transcription factors and cell membrane molecules, was complied from this analysis and validated by both quantitative rt-pcr and immunofluorescence. pax6 and kir4.1 were found to be highly expressed in astrocytes, consistent with previous findings. more importantly, kir4.1 mrna was noted to be selectively enriched in cortical tdtomato1/ gfp1 cells, compared to gfp1 only cells. it suggests kir4.1 may serve as an astroglial subtype marker. conclusions: our data clearly suggest the existence of molecular subtypes of astrocytes. factor expressed by activated astrocytes and microglia. studies with non-glial cells have shown that c/ebpd is able to regulate the expression of proinflammatory genes. however, the role of c/ebpd in neuroinflammation has not been established. we have here analyzed the effects of c/ebpd absence on glial activation in vitro and in vivo. in primary mixed glial and microglial-enriched cultures the expression of the pro-inflammatory genes nos-2, cox-2 and il-6 induced by lps1ifnc, but not by lps alone, was attenuated in the absence of c/ebpd. chip experiments revealed binding of c/ebpd to the promoters of these genes in activated, but not in control, mixed glial cultures. in neuronal-microglial co-cultures the neurotoxicity elicited by lps1ifnc-activated microglia was completely abolished when microglial cells were devoid of c/ebpd suggesting that this transcription factor plays a key regulatory role of the expression of potentially detrimental proinflammatory genes by activated microglia. to analyze the role of c/ebpd in neuroinflammation in vivo mice were treated systemically with lps (100 ug/mouse; i.p.). this treatment induced a marked increase in c/ebpd mrna and protein brain levels. double immunofluorescence experiments showed the presence of c/ebpd in astrocytes and microglia in lps-treated mouse brain. in c/ ebpd -/-mice, systemic lps-induced brain expression of nos-2, tnfa, il-1b and il-6 was attenuated. finally, in mouse experimental autoimmune encephalitis (eae) c/ebpd was upregulated in the spinal cord and the severity of eae symptoms was reduced in c/ebpd -/-mice. altogether these findings demonstrate that c/ebpd plays a major role in the regulation of proinflammatory gene expression in neuroinflammation and point to microglial c/ebpd inhibition as a novel strategy to reduce the detrimental effects of neuroinflammation. supported by pi10/378 and pi12/709 from instituto de salud carlos iii, spain gene targeting strategies have become a powerful technology for elucidating mammalian gene function. the recently generated knockout (ko)-first strategy produces a knockout at the rna processing level and also allows for the generation of conditional ko alleles by combining flp/frt and cre/loxp systems, thereby providing high flexibility in gene manipulation. however, this multipurpose ko-first cassette might produce hypomorphic rather than complete knockouts if the rna processing module is bypassed. moreover, the generation of a conditional phenotype is also dependent on specific activity of cre recombinase. here, we report the use of an efficient molecular biological approach to test pannexin1 (panx1) mrna expression in global and conditional panx1 ko mice derived from the ko-first mouse line, panx1 tm1a(komp)wtsi . using qrt-pcr, we demonstrate that tissues from wild-type mice show a range of panx1 mrna expression levels, with highest expression in trigeminal ganglia, bladder and spleen. unexpectedly, we found that in mice homozygous for the ko-first allele, panx1 mrna expression is not abolished but reduced by 70% compared to that of wild-type tissues. thus, panx1 ko-first mice present a hypomorphic phenotype. crosses of panx1 ko-first with flp deleter mice generated panx1 f/f mice. further crosses of the later mice with mgfap-cre or nfh-cre mice were used to generate astrocyte-and neuronal-specific panx1 deletions, respectively. a high incidence of ectopic cre expression was found in offspring of both types of conditional panx1 ko mice. our study demonstrates that panx1 expression levels in the global and conditional panx1 ko mice derived from ko-first mouse lines must be carefully characterized to ensure modulation of panx1 gene expression. the precise quantitation of panx1 expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of panx1 under physiological and pathological conditions. agarwal, e. hughes, d. bergles johns hopkins university, baltimore, united states astrocytes elevate intracellular ca21 in response to stimulation of metabotropic receptors. these ca21 transients are linked to processes as diverse as synaptic plasticity and functional hyperemia, but the relevance of this signaling remains uncertain. although most studies of ca21 signaling in astrocytes have focused on somatic events, anatomical and functional studies indicate that ca21 signaling in astrocytes may be compartmentalized into functionally isolated "microdomains." to facilitate analysis of ca21 signaling, we generated transgenic mice in which the cytosolic and membrane anchored variant of genetically encoded calcium indicator gcamp3, referred as cgcamp3 and mgcamp3 respectively, can be expressed conditionally in a cell specific manner. a ubiquitous cag promoter is used to control cgcamp3 and mgcamp3 expression, and a "stopper" sequence flanked by loxp sites was placed upstream of the coding sequence, preventing expression until cre excises this dna. these constructs were targeted to rosa26 locus to ensure widespread expression. to evaluate whether cgcamp3 and mgcamp3 were expressed at levels sufficient to resolve ca21 transients, we bred r26-lsl-cgcamp3 and r26-lsl-mgcamp3 mice to glast-creer mice. double transgenic offspring were injected with tamoxifen at 3 weeks of age and analyzed 2-3 weeks later. histology revealed that cgcamp3 and mgcamp3 were expressed in astrocytes throughout the brain. astrocytes in acute cortical slices exhibited large amplitude, spontaneous increases in both cgcamp3 and mgcamp3 fluorescence. the signal to noise ratio of the fluorescence intensity was greatly improved in astyrocytes expressing mgcamp3, partially due to the ability of mgcamp3 to better resolve near membrane ca21 flux. ca21 transients were typically restricted to small regions of an individual astrocyte, consistent with ca21 elevations within microdomains. spontaneous ca21 transients were occasionally observed in the soma of astrocytes expressing cgcamp3 but not mgcamp3, and were not coincident with activity in the processes, suggesting that these regions are functionally uncoupled. to determine if gcamp3 expression is sufficient to visualize ca21 signaling in vivo, we made cranial windows over the somatosensory cortex and performed 2-photon imaging. microdomain ca21 transients were prominent in cortical astrocytes, indicating that these mice provide new opportunities for understanding the significance of this form of signaling. in the trigeminal ganglion (tg), satellite glial cells (sgcs) surround neurons and are able to release substances that may affect sensory transmission through the tg and contribute to mechanisms underlying craniofacial pain. the aims of this study were 1) to investigate whether sgcs could be provoked to release glu in vitro and 2) to study the in vivo response properties of trigeminal afferent fibres upon artificial elevation of glu concentration in the tg. methods: confocal microscopy was used to assess glu content in and the expression of excitatory amino acid transporter 1 (eaat1) and eaat2 by sgcs of the tg. glu release was investigated in vitro, where sgcs were isolated from the tg of 9 adult male sprague-dawley (sd) rats and treated with control medium or medium containing 10 mm kcl together with 0, 0.1, 1, 10 mm of the eaat1/2 inhibitor tfb-tboa. in vivo electrophysiology experiments were conducted in 6 additional anaesthetized adult male sd rats to determine the effect of repeated intraganglionic injections of glu (500 mm, 3 ml 3 2, 30 min apart) on the response properties of 6 tg neurons that innervated either the temporalis or masseter muscle. cumulative afferent discharge was calculated as the sum of action potentials (ap) 10 min post injection subtracted from the sum of ap 10 min prior to injection. an electronic von-frey hair was used to measure afferent mechanical threshold (mt) at 1 min intervals for 10 min prior to the first and again 20 min after the second microinjection of glu. results: most sgcs were found to be glu positive and express eaat1 and eaat2. treatment with 10 mm kcl resulted in a $2-fold increase in glu concentration, compared to control (10.6 6 1.1 vs. 4.8 6 0.1 mm, respectively; p 5 0.038). treatment with 1 mm tfb-tboa significantly reduced the glu concentration to 5.8 6 1.4 mm (p 5 0.047), which was further lowered to 3.0 6 0.8 mm when 10 mm was applied (p 5 0.001). cumulative afferent discharge evoked in tg neurons by the second injection of glu (20 6 9 ap) was not significantly different from that evoked by the initial injection (21 6 10 ap; p 5 0.92). glu injections significantly reduced muscle afferent mt (baseline: 35 6 19 g) by 30 6 3% (p 5 0.03). these results indicate that glu can be released from sgcs through eaats, and that increased glu concentrations in the tg excite neurons and induce a state of peripheral sensitization. this may hypothetically be a mechanism, whereby activated tg sgcs could contribute to e.g. migraine headache. in an accompanying poster, we present data demonstrating that in cultured neurons l-lactate induces plasticity-related genes expression through a nmda-dependent mechanism (i.e. arc, zif268 and c-fos). here, we describe the effect of l-lactate on neuronal excitability by performing electrophysiological recordings of cultured cortical neurons. we observed that application of l-lactate (10 mm) triggers an inward current with an amplitude of 20.55 1/-0.1 na and slow kinetics, with a peak reached at 189 1/-55s (called ilac). the membrane current decreases gradually to reach a plateau, with a mean inward current of 20.13 1/-0.04 na persisting up to 780s (13 minutes) after addition of l-lactate (called ipyr). in order to assess l-lactate specificity on the generation of these currents l-pyruvate, another monocarboxylate, as well as d-lactate, the non-metabolized enantiomer of l-lactate) were tested. in contrast to l-lactate, l-pyruvate (10 mm) induced a low-amplitude sustained current (20.07 1/-0.03 na) similar to the late-phase slow current evoked by l-lactate (i.e ipyr), but no current similar to ilac. dlactate on its side did not elicit any specific current. pharmacological characterization of ilac and ipyr currents demonstrate that ilac is generated by nmda receptors (as it is blocked by mk801) and relies on active nmda receptors (as it is blocked by either glutamate or glycine binding sites inhibitors). in contrast, generation of ipyr by both l-lactate and lpyruvate is prevented by diazoxide, a katp opener, whereas ilac was unaffected by the treatment. in order to determine the importance of katp for plasticity-related gene expression, the katp closer glibenclamide (200 um) was applied to neurons and mrna gene expression of arc and zif268 quantified. results obtained demonstrate that arc and zif268 mrna expression are unaffected by glibenclamide. as a whole this set of data demonstrates that l-lactate generates two types of inward currents in neurons i.e. nmda-dependent (ilac) or katp-dependent (ipyr). moreover, they highlight the nmda-dependent ilac current as the key mediator, in contrast to katp, of l-lactate-induced plasticity gene expression. this is further sustained by the observation that l-pyruvate does not reproduce the effect of l-lactate on gene expression (see accompanying poster). this set of results provides novel insights into the mechanisms of action of l-lactate as an inducer of plasticity gene expression, and an important signaling molecule for neuronal plasticity. the reliance on drg neuron co-culture for sclcs fate commitment stands as a major hurdle for the therapeutic application of this finding. thus, we aim to unravel the mechanisms underlying sclcs fate commitment in order to develop a drg neuron-free condition for generating fate committed schwann cells. we hypothesised that the switch is brought about by the membrane bound neuregulin (nrg), nrg 1 type iii, but not the other soluble isoforms of neuregulin. as our results show that sclcs treated with soluble neuregulin did not show significant changes in morphology nor marker expression when compared with untreated sclcs. nrg 1 type iii expression was observed on purified drg neurons by immunocytochemistry, western blot analysis as well as reverse transcription pcr for the mrna. mammalian expression constructs for nrg 1 type iii were made by transfection into human embryonic kidney cells, hek 293t, and mouse embryonic fibroblasts (mef) with the aim of generating surrogate cell types for co-culture with sclcs to pursue cell-specific effects of nrg 1 type iii on differentiation of sclcs. the findings promise a way for generating fate committed schwann cells for autologous transplantation for recovery after nerve injury. (2) the co 2 /hco 3 buffer system. the latter being an open buffer system, because biomembranes are usually permeable to co 2 , and, in addition, most cells express hco 3 transporting membrane proteins. the enzyme family of carbonic anhydrases (cas) catalyses the reversible reaction from co 2 and h 2 o to hco 3 and h 1 , and thereby modulates the intracellular h 1 buffer dynamics.we have performed calibrated in situ live-cell imaging with the proton-sensitive fluophore bcecf in glial cells and neurons of acute cerebellar slices of wild-type and knockout mice. the studies were used to quantify the intracellular buffer capacity and to dissect the contribution of the intracellular caii and the extracelluar caiv by comparing intracellular h 1 shifts in glial cells and neurons from wild-type mice and mice deficient in their caii or caiv gene. we found that only one proton in 400000 is unbound and thereby chemically active, and that about 50% of this buffer capacity is mediated by the co 2 /hco 3 buffer system and the other 50% by intrinsic buffers. the rate of co 2 -induced change in intracellular h 1 concentration was increased by intracellular caii in both glial cells and neurons. the extracellular caiv on the other hand affected primarily neurons, but also showed unexpected intracellular activity (schneider et al. 2013, pnas 110). the rates of acidification and alkalinisation in wild-type and knockout mice were significantly reduced in the presence of the ca blocker 6-ethoxy-2-benzothiazolsulfonamid (10 mm). we confirmed ca protein expression by western blot and could also show that the expression of the remaining ca is not upregulated. these results provide new insights into cellular proton-coupled processes in acute neural tissue, which shows some new complex roles of carbonic anhydrases in shaping proton dynamics in cells and tissues. objective: it is becoming increasingly evident that inflammatory mechanisms promote neuronal hyper-synchronization and epileptogenesis following brain insults. our recent studies identified serum albumin as a key player in the inflammatory cascade leading to epilepsy, at least partially through the activation of tgf-b signaling pathway in astrocytes. in this study we set out to explore the mechanisms by which tgf-b1 induces glial activation and epileptogenesis. methods: primary cultures of microglia and astrocytes were treated with tgf-b1 and gene expression analysis along with protein quantification were performed by quantitative pcr and elisa. electrophysiological response to il-6 was obtained in acute brain slices (field potential recordings) and in-vivo (sub-dural corticoencephalography) following to a continuous administration of il-6 into the lateral ventricle for 7 days. results: in glia cultures tgf-b1 induced early and rapid up-regulation of il-6 at both mrna and protein levels whereas the upregulation of other pro-inflammatory cytokines such as il-1b and tnf-a was milder and at a later time point. notably, smad2/3-dependent tgf-b1 signaling induced the expression of il-6 primarily in astrocytes and to a significantly lesser extent in microglia. il-6 was sufficient to induce epileptiform activity in acute brain slices and recurrent seizures within 3-4 days a following continuous intracerebroventricular injection of il-6. conclusions: we thus suggest astrocytic release of il-6 as a potential key mechanism in epileptogenesis. okayama univ., dept. of brain science, okayama, japan dopamine transporter (dat) is expressed not only in catecholaminergic neurons but also in astrocytes. we previously showed that repeated l-dopa treatment markedly induced expression of dat and apparent dopamine (da)-immunoreactivity in the reactive astrocytes in the striatum of animal models of parkinson's disease. it is thought that astrocytes can uptake both l-dopa and da via neutral amino acid transporter lat and dat, respectively. therefore, uptake and metabolism of l-dopa and da in the striatal astrocytes may influence their availability in dopaminergic system of parkinsonian patients. to clarify uptake and metabolism of l-dopa and da in striatal astrocytes, the contents of l-dopa, da and their metabolites were measured after the l-dopa/da treatment using primary cultured astrocytes. first, we revealed the expression of neutral amino acid transporter lat and aromatic amino acid decarboxylase (aadc) in the striatal astrocytes. the level of l-dopa in astrocytes was markedly increased after 4-hr l-dopa exposure, but da was not detected in the astrocytes 4 or 8 hr after the treatment. on the other hand, the da treatment for 4 hr increased levels of da and its metabolites, especially dopac. these results indicate that uptaken da into astrocytes is rapidly metabolized and that uptaken l-dopa never be converted to da in astrocytes. furthermore, level of uptaken l-dopa in cultured striatal astrocytes was rapidly decreased after removing extracellular l-dopa. taken together, the present results suggest that striatal astrocytes act as a reservoir of l-dopa to uptake or release l-dopa depending on the extracellular l-dopa concentration, but have less ability to convert l-dopa to dopamine. s. limmer, c. kl€ ambt universit€ at m€ unster, m€ unster, germany neuronal function consumes a large amount of energy and thus the brain requires a constant but regulated supply of metabolites. in invertebrates, such as drosophila, the nervous system is floating in the hemolymph and all metabolites that enter the brain have to be transported across the blood brain barrier (bbb). as in primitive vertebrates, the drosophila bbb is generated by glia. a layer of so-called subperineurial glial cells completely encapsulates the nervous system and thus, all metabolites reach the neurons en route through glia. the main energy supply of the inner organs of drosophila is provided by trehalose that is found in high concentrations in the hemolymph. trehalose is a nonreducing disaccharide, in which two dglucose units are linked via a a,a-1,1-glycosidic bond. the uptake of trehalose is mediated by two trehalose transporters, which are members of the slc2a gene family. the subperineurial glial cells then either secrete c 6 sugars to the neurons -or alternatively supply energy to neurons by secreting c 3 metabolites as described for the bee retina. to discriminate between these possibilities we have followed a number of different approaches. the relevance of trehalose transporters in the different cell types has been determined by mutant analysis and rnai studies. furthermore, we have studied the role of all other putative sugar and monocarboxylate transporters in glial cells by rnai knockdown. to determine whether c 6 or c 3 carbohydrates are secreted by glial cells we suppressed the expression of core enzymes regulating glycolysis or components of the respiratory chain in either glial cells or neurons. moreover, we have ablated mitochondria specifically in glia or in neurons through expression of a restriction enzyme targeted to mitochondria. we will summarize our results and discuss approaches towards identifying neuronal signals that control glial carbohydrate secretion during energy homeostasis of the brain. microglia have long been known to respond to virtually any insult to the central nervous system. they quickly migrate to the site of the insult, and play many important roles, such as barricading the injury site, phagocytosing debris, and releasing cytokines. the role of microglia in the normal brain, however, has only recently begun to be appreciated. with the advent of in vivo imaging, microglia have been shown to be constantly surveying their environment with rapid extensions and retractions of their processes. subsequent studies have shown that these 'resting' microglia (now known as 'surveying microglia') are indeed active throughout development and adulthood. during development, microglia have been shown to be involved in synaptic monitoring and pruning as well as synaptic stripping after injury. thus far, many studies have focused on the role of microglia at the synapse. however, we report here, for the first time, that in the cortex of normal adult rodents, a small percentage of microglia are specifically associated with the axon initial segment (ais). the ais is characterized by a high density of voltage-gated ion channels and plays an important role in initiation of the action potential and maintenance of neuronal polarity. this interaction seems to be limited to the 'surveying' phenotype of microglia and much less frequent in 'activated' microglia. this overlap of processes appears early in development and continues throughout adulthood although the function of microglia at the ais is still currently unknown. x. liu 1 , j.-m. petit 2,3 , p.j. magistretti 2,3 , c. giaume 1 1 cirb, collège de france, paris, france 2 lndc, brain mind institute, epfl, lausanne, switzerland 3 centre de neurosciences psychiatriques, chuv, prilly, switzerland astrocytes act as modulators of neuronal activity through mechanisms such as neurotransmitter uptake, 'gliotransmitters' release, and metabolic supply. recently, astrocytes were shown to modulate sleep by vesicular release of atp. besides the cellular mechanism, we are interested in the network behavior of astrocytes in sleep-wake cycle. indeed, astrocytes form networks of communicating cells via gap junction channels constituted by connexins (cxs). recently, we have demonstrated that astroglial networking is regulated by neuronal activity and that neuronal activity is affected by deletion of the two main astroglial cxs (cx43, cx30). to investigate how astroglial networks behave in sleep-wake cycle, we used pharmacological treatments and sleep deprivation to manipulate sleep-wake status of mice and determined whether astroglial networks would change in response. astroglial networks in acute cortical slices were revealed by "dye coupling", i.e. by loading of a patch-clamped astrocyte with sulforhodamine b that diffuses into neighboring astrocytes through gap junction channels. also, cxs expression at mrna and protein levels was measured as an index of connections among astrocytes. the results are as follows. first, i.p. injection of modafinil, a potent wakefulness-promoting drug, increased both mrna and protein expression of cx 30 in the mouse cortex. superfusion of modafinil also enhanced dye coupling among astrocytes in acute coronal slices of the mouse somatosensory cortex. this effect was abolished by ttx treatment, which demonstrates neuronal activity is necessary for the effect of modafinil. in contrast, c-hydroxybutyric acid (ghb), a sleep-promoting agent, and propofol, a general anesthetic, decreased astroglial coupling in cortical slices. interestingly, the effect of ghb was not affected by ttx, suggesting a different pathway of action from modafinil. next, we used a "gentle" sleep deprivation model to sleep deprive mice for 6 hours from the onset of the light period. as a result, mrna of cx30, but not cx43, was increased in both the cortex and hippocampus. finally, dye coupling in cortical astrocytes was enhanced in mice after sleep deprivation compared to mice after normal sleep. this increase in dye coupling, however, was not observed in slices from cx30 knock out mice after the same sleep deprivation treatment. altogether these results indicate that astroglial networks are bidirectionally regulated by perturbations in sleep-wake cycle and that cx 30 is the major cx sensitive to such perturbations. in neurons, synaptic and extrasynaptic gaba a receptors (gaba a rs) differ in their subunit composition, conferring them distinct functional and pharmacological properties. oligodendrocyte precursor cells, also called ng2 cells, are contacted by bona fide neuronal gabaergic synapses. however, we recently showed a synaptic to extrasynaptic switch in the mode of transmission between gabaergic interneurons and ng2 cells during postnatal development of the somatosensory cortex. we therefore hypothesized that the postnatal switch of gabaergic transmission in ng2 cells is accompanied by changes in the expression of gaba a r subunits. to test for this hypothesis, we stimulated neuronal fibers to evoke gaba a r-mediated responses in ng2 cells recorded in acute slices of the somatosensory cortex of ng2-dsred transgenic mice. the effect of zolpidem and a5ia on evoked gabaergic responses reveals the predominance of functional a1-and a5-containing gaba a receptors, respectively, at interneuron-ng2 cell synapses early in development. however, the expression level of a5 decreases when responses rely exclusively in extrasynaptic transmission at more mature developmental stages. more importantly, specific pharmacology for the c2 subunit, a crucial molecular component for the clustering of gaba a receptors at postsynaptic sites, demonstrated a down-regulation of this subunit in ng2 cells prior to the complete loss of gabaergic synaptic activity. in keeping with the synaptic nature of the c2 subunit in neurons, this molecular change in ng2 cells correlates with the switch from synaptic to extrasynaptic transmission. to corroborate these functional data, we performed single cell multiplex rt-pcr of a1-5, b1-3, c1-3 and d subunit mrnas in ng2 cells of the second and fourth postnatal weeks. despite the large heterogeneity of subunit mrna expression at both developmental stages, we found that the number of cells expressing c2 mrnas dramatically decreases in the fourth postnatal week. in conclusion, the expression loss of the c2 subunit is an important molecular determinant impacting the change of transmission modes between interneurons and ng2 cells during cortical development. financial support: frc, anr blanche, arsep, dfg (sfb/tr3) and eu (fp7-202167 neuroglia). university of rochester, medical center, rochester, united states synaptic plasticity is a critical process throughout the lifespan for achieving and maintaining normal and efficient operation of the nervous system. while much is known about the functional and structural changes that occur at the synapse during synaptic plasticity, the mechanisms implementing these changes are poorly understood. despite being classically characterized as immune cells, microglia have recently been shown to play a role in normal brain function by restructuring and removing synapses. given the novelty of this role, few details are known about how microglia behave to implement such a role during periods of plasticity. to characterize the changes in microglial behavior during ocular dominance plasticity in the developing visual cortex, we examined microglial morphology in fixed brain sections as well as in vivo using two-photon microscopy. for analysis of microglia in fixed sections, c57/bl6 mice were monocularly deprived for either 12 hours, 1, 2, 4, or 7 days during the visual critical period (p23-p30). fixed coronal sections were stained with iba1, a specific marker for microglia, imaged on a confocal microscope and analyzed using imagej. microglial density decreased rapidly (within 12 hours) and remained low for the 7 day deprivation period. characterization of microglial morphology revealed an increase in the complexity of the microglial process arbor after 12 hours, which steadily decreased back to baseline by 7 days. this suggests that microglia do undergo a rapid change in morphology during plastic events that is reminiscent but distinct from the traditional activation pattern observed during pathological events. for analysis of microglia in vivo, heterozygous cx3cr1/gfp (jung 2000) mice were monocularly deprived directly preceding the first imaging session, the binocular segment of primary visual cortex was imaged, and the same cortical area was re-imaged 2 and 4 days later. while no change in microglial density was observed, analysis of microglial process motility showed a decrease in motility after periods of monocular deprivation. these results, combined with the observed morphological changes, show that microglia undergo behavioral changes after a period of monocular deprivation that are inconsistent with pathological activation, suggesting that microglia take on different roles and activities during normal brain function. g. baltazar, j. oliveira, t. roxo, c. fonseca faculty of health sciences, cics-ubi, covilhã, portugal neuroinflammation is a pathological hallmark in patients and experimental models of parkinson's disease. both present the classical features of inflammation, with evidence of an uncontrolled process. moreover, microglia may become activated early in the disease process and remain primed, responding strongly to subsequent stimuli, and thereby enhancing inflammation-induced oxidative stress and cytokinedependent toxicity in vulnerable neuronal populations. we have previously demonstrated that glial cell line-derived neurotrophic factor (gdnf), released by ventral midbrain astrocyte cultures, potently prevents microglial activation induced by a pro-inflammatory agent. therefore, gdnf is an important mediator in the astrocytemicroglia crosstalk keeping microglia in a resting state. however, in the brain, microglia and astrocytes are not alone and other cell types, e.g. neurons, may interfere and influence this astrocytic control of inflammation. therefore, it is important to investigate if the presence of neurons alters gdnf-mediated astrocytic control of microglial activity. in this work we aimed at investigating whether the presence of neurons, injured or not, changes the ability of astrocyte-derived gdnf to prevent microglial activation. for that purpose, we used primary cultures of ventral midbrain astrocytes and microglia, as well as neuronastrocyte mixed cultures from the same brain region. neuron-astrocyte cocultures were exposed to vehicle (control) or to the da neurotoxin mpp 1 . the media conditioned by control and mpp 1 -challenged neuron-astrocyte mixed cultures, or by astrocyte cultures, was collected and transferred to ventral midbrain microglia cultures, which were then exposed to the pro-inflammatory agent lipopolysaccharide. the effect of conditioned media obtained from the different cell culture systems on microglial activity was determined by analyzing the production of nitric oxide by the griess reaction and of the phagocytic activity by determining the engulfment of fluorescent microspheres by fluorescence microscopy. university of lausanne, lausanne, switzerland lactate is produced by astrocytes and used by neurons as metabolic substrate during activity. recent evidence indicates that lactate may not only serve as energy substrate but also as modulator of glutamatergic or gabaergic neurotransmission. we tested this hypothesis using primary cultures of cortical neurons obtained from wild type and gad67-gfp knock-in mice (c57bl/6). neuronal excitability was monitored by electrophysiological recordings and calcium imaging using the fluorescent probe fluo-4 am. spontaneous action potentials and rhythmic calcium transients were present in more than 50% of cultured neurons, which were either gabaergic or glutamatergic cells. in the presence of 5mm glucose, l-lactate application reversibly diminished calcium transient frequency in a concentration-dependent manner in both cell types (glutamatergic neurons ic50 4.23 6 1.9mm and gabaergic neurons ic50 4.18 6 2.8mm). to test whether lactate effects were dependent on metabolism, we applied the closely related substrate pyruvate (5mm) or switched to different glucose concentrations (0.5 or 10mm). none of these conditions altered the calcium transient frequency. in contrast, the application of d-lactate, thought not to be metabolized by neurons, decreased the spiking activity in the same concentration range as l-lactate (ic50 4.58 6 1.2mm). we determined that d-lactate was taken up by neurons, however more than two-fold less efficiently than l-lactate. these results suggest that the mechanisms of lactate sensitivity are not purely metabolic. since d-lactate has the same potency as l-lactate but it is less internalized, it is likely that lactate acts as an extracellular ligand that triggers the reduction of neuronal activity. this hypothesis is currently under investigation. this modulating effect of lactate represents a novel role for the compound that has to be taken into account in the context of the interactions between astrocytes and neurons. ben-gurion university, ber-sheva, israel microglia integrate within the neural tissue with a distinct ramified morphology through which they scan the surrounding neuronal network, a process which appears to contribute to the integrity, maintenance and functioning of the brain. since microglia are long-lived, they are subjected to senescence processes which may severely compromise their function with age. here we used a digital tool for the quantitative morphometric characterization of fine cortical microglia structures in mice. we thus followed the morphological changes microglia underwent with aging and with the progression of alzheimer's-like disease. compared with microglia in young mice, microglia in old mice are less ramified and possess less branches and fine processes, a phenomenon which was associated with increased expression of pro-inflammatory genes. notably, a similar microglial pathology appeared 6-12 months earlier in mouse models of alzheimer's disease (ad). we thus demonstrate that in addition to promoting neurotoxic inflammation, amyloid plaques attract the microglia and modify their structure. this, in turn, causes a severe microglial process deficiency, which possibly results in compromised neuronal function and repair. blood-borne glucose is the main energy substrate for the brain under physiological conditions. however, the question remains unresolved if the two main cell types in the brain -neurons and astrocytes -consume glucose upon their individual needs or if glucose is mainly taken up by one cell group and glycolytically degraded to energy-rich substrates (e.g. lactate), which are then shuttled to the other cell group. a novel genetically encoded fret glucose sensor (bittner et al., 2010, 2011) together with two-photon microscopy now provides for the first time the opportunity to determine intracellular glucose concentrations in single cells in the intact organism. cortical microinjections of an adenoviral vector (aav9 and aav6) coding for the sensor flii 12 pglu600md6 with the promoter sgfap for astrocytes and synapsin for neurons were performed in mice and chronic windows were implanted above the somatosensory cortex. after two weeks, specific expression in astrocytes and neurons was observed. the ratiometric fret signal in both cell types increased after intraperitoneal glucose injection and decreased after insulin application demonstrating the functionality of the construct in vivo. the astrocytic sensor displayed a large linear range at blood glucose levels ranging from 3 to 20 mmol/l. in a second set of experiments, we examined the dynamic behavior of cellular glucose concentrations upon increased brain activity. during electrical hindpaw stimulation 21% of examined astrocytes showed a 0.5% decrease of the fret signal, whereas neuronal glucose levels did not exhibit activity-dependent fluctuations. to be able to draw conclusions about metabolic rates of substrate oxidation we recently started with experiments where cellular glucose uptake was transiently inhibited using different blockers of glucose transport. drugs were applied systemically and intracortically. we could demonstrate this principle on first measurements on astrocytes. glucose fret sensors allow measurements of the dynamics of glucose concentrations of single neurons and astrocytes in the intact organism. measurements of cellular glucose transients during transport blockade hold the potential to compare glycolytic rates of neurons and astrocytes. this tool may substantially contribute to uncover the complex organization of brain energy metabolism. neuron-glial communication in the cns is fundamentally important for many brain processes including synaptic transmission and plasticity. perisynaptic astrocytic processes (pap) contact excitatory synapses, forming tripartite structures with neurons. in the hippocampus, the morphology of pap has been shown to remodel rapidly and continuously but the mechanisms and roles of this form of structural plasticity remain unknown. this study investigated the physiological mechanisms driving pap movements and their role during long-term potentiation (ltp). pap and spines contacts were labeled by viral gene delivery of farnesylated fluorescent proteins in hippocampal slice cultures and imaged by confocal microscopy. electron-microscopy of infected slices confirmed that pap-synapse morphology is preserved in organotypic cultures. pap movements adjacent to dendritic spines were evaluated with an index of motility (mi). increasing neuronal activity by schaffer collaterals (sc) stimulation elevated pap mi and this was prevented by both ttx and mglur inhibition, suggesting that neuronal activity modulates pap movements. we next investigated whether intracellular calcium (ca 21 i ) in astrocytes influenced pap motility. bapta-am bulk-loading specifically chelated ca 21 i in astrocytes and reduced pap movements. when exogenous gq-coupled receptors mrga1 or mrgc11 were specifically targeted to astrocytes, their agonist fmrfa induced ca 21 i increases and accelerated pap motility. moreover, fmrfa delivery at the synaptic level by two-photons flash photolysis was sufficient to elevate pap mi. we then investigated pap dynamics in relation to ltp. application of theta-burst sc stimulation initially increased pap motility, but then resulted 30min later in a decrease of pap motility. interestingly, the reduction of pap movements correlated with both spine enlargement and increased pap's spine coverage. to assess the consequences of these changes, we then specifically triggered elevation of pap movements at the synaptic level by flash photolysis. spine stability analysis 24 hour after uncaging revealed that spines in contact with activated paps were more stable than others. this study suggests that excitatory synapses control the motility of surrounding paps through the triggering of neurotransmitter-evoked astrocytic ca 21 i elevations. increased pap motility seems to be necessary to elevate their coverage of the synapse during ltp, leading to higher synapse stability. astrocyte reactivity occurs in response to many pathological brain situations. reactive astrocytes display morphological and functional changes that could result in concentration variations of some brain metabolites localized both in astrocytes and in neurons. such changes could be detected by magnetic resonance spectroscopy (mrs), allowing non-invasive monitoring of astrocyte reactivity and thereby neuronal dysfunction in vivo. one of the major peak detected by mrs in the brain corresponds to neuronal metabolite n-acetyl-aspartate (naa). although its function is unclear, it is considered as a biomarker for neurodegeneration and a decrease in its concentration is interpreted as neuronal death or dysfunction. however, there is not clear evidence that other cell types, specifically astroglia, could not contribute to change in naa levels in the brain. in this study, we used a rat model of astrocyte activation by stereotaxic injections of lentiviral vectors encoding for either the cytokine ciliary neurotrophic factor (lenti-cntf) into the right striatum or betagalactosidase (lenti-lacz) into the left striatum, as a control. mrs data were acquired on a 7t magnet from two voxels placed over each injected striatum. a diffusion-weighted laser sequence with echo time te 5 40 ms was used. concentrations were measured using lcmodel software for total n-acetyl-aspartate (naa 1 naag), myo-inositol (ins), total choline (cho), glutamate and taurine relative to total creatine. lenti-cntf injection promotes a sustained, extensive and selective activation of astrocytes, as evidenced by overexpression of gfap and vimentin and cellular hypertrophy (fig.1) . importantly, neurons displayed unaltered morphological, molecular and electrophysiological features, as described previously (escartin et al., 2006; beurrier et al., 2010). mrs evidences changes in metabolite concentration: in the lenti-cntf injected striatum, levels of the astrocyte metabolites ins and cho were increased, whereas levels of the neuronal metabolite naa and glutamate were decreased. increased levels of ins and cho are generally found associated with astrocyte reactivity, however, the decrease in naa is unexpected given that neurons are not dysfunctional with cntf. to understand how astrocyte reactivity can influence naa levels, we are currently characterizing the molecular changes occurring in the naa metabolism using quantitative pcr, biochemistry, histology and hplc. our data suggest that reactive astrocytes alone result in alterations of metabolite concentrations detectable by nmr. although the mechanisms by which activated astrocytes regulate neuronal metabolism remain to be elucidated, our work challenges the mrs dogma that decreased neuronal metabolites can be used as a biomarker for neurodegeneration. microglial phagocytosis is a vital phenomenon for the clearance of damaged and death cells or infectious agents in a context of brain injury or infection, respectively. in addition, microglia can boost synaptic plasticity by the phagocytosis of axon terminals and dendritic spines. therefore, it is crucial to better understand the mechanisms involved in microglia clearance in order to devise new strategies to promote an efficient brain repair. recently, we showed that histamine modulates microglia motility and cytokines release. in this work, we aimed to investigate the role of this molecule and its receptors in microgliainduced inflammation by evaluating microglial phagocytic activity and reactive oxygen species (ros) production. for that purpose, an iggopsonized latex bead assay was performed in n9 murine microglial cell lines exposed to lipopolysaccharide (lps) or histamine 1, 10 and 100 mm. we showed that histamine significantly stimulated phagocytosis of opsonized latex beads via h1 receptor activation. this effect was accompanied by the rearrangement of cytoskeleton analyzed by phaloidin and acetylated tubulin expression and cellular distribution. the phagocytic activity was significantly reduced after pretreatment with apocynin, a putative napdh oxidase (nox) inhibitor. all nox isoforms were expressed by microglial cells, but only the expression of nox1 was increased by treatment of histamine. rac1, an important subunit for the activation of nox1, was also activated by histamine. in addition, histamine induced ros production via h1 and h4 receptor activation. in conclusion, histamine plays an important role in the regulation of microglial phagocytosis, which is mediated by nox1 and rac1 activation. multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system characterized by demyelination and axonal damage, involving both white (wm) and gray matter (gm) areas. furthermore, cortical pathology correlates with ms progression and cognitive dysfunction. we previously found altered expression of gap junction (gj) proteins connexin32 (cx32) and cx47 in oligodendrocytes and cx43 in astrocytes in wm lesions and normal appearing wm (nawm) in brain samples from ms patients, and in a mouse model of experimental autoimmune encephalomyelitis (eae), implicating uncoupling of myelinating cells as an important aspect of ms pathology. here we studied cx32 and cx47 and their astrocytic partners cx30 and cx43 in and around cortical lesions and the normal appearing cortex (nacx) in ms samples and non-ms controls. we examined their expression by real-time pcr, quantitative immunoblot and immunohistochemichal analysis. compared to non-ms controls, expression levels of cx32 and cx47 mrna were reduced within lesions whereas cx30 and cx43 were increased. in nacx, all four connexins were upregulated with cx47 being significantly increased. immunoblot analysis of ms cortex revealed reduced cx32 and increased cx43 levels, while cx30 and cx47 showed no significant change. immunohistochemical analysis showed severely reduced cx47 gj plaques within lesions with gradual increase toward nacx. in contrast to nawm, cx47 gj plaques were not increased in nacx. both astrocytic gj proteins were increased in ms, but in distinct patterns: cx30 gj plaque counts were higher within lesions and perilesional areas. cx43 was increased in ms cortex compared to non-ms controls, but in contrast to nawm, cx43 gj numbers were higher in nacx than in lesions. similar alterations were found in the eae mice, in which cx32 and cx47 gj counts were reduced whereas cx43 and cx30 gjs were increased. our results indicate that oligodentrocytic as well as astrocytic gjs are affected not only in ms wm but also in the gm. loss of oligodendrocyte gjs and in parallel increase of astrocytic gjs reflecting astrogliosis occurs throughout the ms brain and may negatively affect the prospects of repair and remyelination, worsening disease progression. acknowledgements: this project was funded by the cyprus research promotion foundation (grants access/0308/11 and health/bios/ 0308/01 to kak) and by cyprus telethon (2010-14 grant to kak). post-mortem human brain tissues were kindly provided by the uk multiple sclerosis society tissue bank, imperial college london, funded by the uk multiple sclerosis society. the mainly astroglialocated enzyme glutamine synthetase is required to synthesize glutamine from the re-uptake of either glutamate or gaba. thereby, this enzyme significantly contributes to the control of brain glutamate and gaba levels. there is good evidence that both neurotransmitter systems are dysregulated in depression. hence, we decided to study the cellular expression of this enzyme in subjects with major depression and bipolar disorder. material and methods: we investigated the numerical density of glutamine synthetase immunoreactive astroglial cells in different brain areas (prefrontal cortex, anterior insula) of 14 subjects with major depression, 15 subjects with bipolar disorder and 19 matched control cases. results and discussion: we found that compared with controls in cases with major depression there was a significant reduction of the density of immunostained glial cells in all brain regions studied, whereas bipolar cases showed a normal expression of the enzyme. our findings point to a profound disturbance of the glutamateglutamine-gaba cycle in major depression, which is in good agreement with neuroimaging observations and molecular biologic data of others. m.-c. marx, d. billups, b. billups university of cambridge, cambridge, united kingdom perisynaptic astrocytes are thought to play a key role in the recycling pathway of glutamate by supplying the precursor glutamine to the presynaptic terminal. using fluorescent ph i measurements of astrocytes and direct patch-clamp recordings from the calyx of held synapse in rat brain slices, we have investigated the transport mechanisms that may mediate this release of glutamine from astrocytes in situ and assessed the role of glutamine in maintaining glutamatergic neurotransmission. glutamine transporter currents were elicited by puff application of 10 mm glutamine from a nearby pipette. for imaging astrocytic ph i , hpts was included in the patch pipette. system a (sa) transporter function was probed using the substrate inhibitors meaib, alanine and serine, while system n (sn) function was isolated using its unique ability to transport li 1 in place of na 1 . histidine was used as a substrate inhibitor of both sa and sn. glutamine-induced membrane currents i gln (220.0 6 2.4 pa, n 5 21) were blocked by bath applied alanine (10 mm) and meaib (20 mm), as well as substitution of external na 1 with li 1 . fluorescent ph i measurement revealed an alkalinisation during puff application of glutamine. while bath application of histidine (20 mm) abolished the ph change, meaib and serine (20 mm) only attenuated it. most of the ph change (63 6 11%; n 5 7) was insensitive to the substitution of na 1 with li 1 . in presynaptic terminals, puff application of glutamate did not induce a membrane current indicating a lack of direct glutamate uptake; however, puff application of glutamine resulted in a i gln which was eliminated by the removal of external na 1 and by meaib. miniature epscs recorded from postsynaptic neurons (238.8 6 3.5 pa) were inhibited by meaib (228.6 6 3.4 pa; n 5 5) following turnover of the presynaptic glutamate pool. single epscs were unaffected by meaib indicating no direct effect on postsynaptic ampa receptors. together, these data identify two glutamine transport systems within astrocytes. properties of the glial i gln indicate expression of sa while ph imaging reveals a non-electrogenic glutamine transporter with properties identifying it as sn. sn is known to be readily reversible under physiological conditions and we propose that this mediates the astrocytic glutamine release mechanism within the glutamate-glutamine cycle. furthermore, system a mediates glutamine transport in glutamatergic nerve terminals and is important in maintaining the supply of glutamate for excitatory neurotransmission during periods of high frequency stimulation. a. briens, m. schwalm, f. docagne, d. vivien inserm u919, caen, france astrocytes are key players in the brain, cooperating with neurons to modulate crucial processes. in particular, they are able to uptake and release neurotransmitters and neuromodulators to control their synaptic level. in our study, we hypothesize that astrocytes can regulate some effects of the serine protease plasmin in the cns through a mechanism of uptake. conversion of the zymogen plasminogen to the serine protease plasmin by the activators (tpa or upa) is the basis of fibrinolysis in the vasculature. but cerebral functions for the plasminogen activator/plasmin system have recently arisen. it has been shown that plasmin was essential to activate the brain derived neurotrophic factor (bdnf) in its mature form, promoting long-term hippocampal plasticity. in alzheimer's disease, plasmin plays a protective role by participating in beta-amyloid clearance. plasmin can also lead to neuronal injury by degrading extra-cellular matrix proteins. in animal models of multiple sclerosis, plasmin can also help removing intraparenchymal deposits of fibrin, thus limiting axonal damage. these data suggest that a system of regulation of plasmin effects in the brain is necessary. in this study, we showed that astrocytes can actively and specifically uptake plasminogen and plasmin in a time-dependent and a dose-dependent manner. this mechanism involves clathrin pits formation in astrocytes and the lysine-binding site of plasminogen/plasmin. plasminogen and plasmin, following endocytosis are found in early endosomes, late endosomes, lysosomes and recycling endosomes. finally, we noticed that astrocytes uptake plasmin more efficiently than plasminogen. this study brings out a mechanism of glial uptake of plasminogen and plasmin. this could control their extra-cellular levels and regulate their effects within the brain. further investigations should determine whether this mechanism can modulate plasminogen activation and synaptic plasmin activity. besides, plasminogen activators (tpa) and cerebral substrates of plasmin (pro-bdnf) are also taken up by astrocytes. a link could thus exist between these processes to regulate the amount of synaptic mature bdnf. this would reveal a cross-talk between neurons, releasing precursors in the synapse (pro-bdnf and plasminogen) and astrocytes, regulating their activation and their clearance. understanding and modulating plasmin uptake could be very important in pathological conditions where the plasminogen/plasmin system is involved, such as alzheimer's disease, multiple sclerosis or stroke. healthy brain aging is characterized by neuronal loss and cognitive decline being inflammation a major causative factor. microglial activation is considered to be a major driver for the neuropathological findings in alzheimer's disease (ad). we evaluated whether young vs. aged microglia responded differently to ab soluble oligomers (abo) and to conditioned media from abo-treated hippocampal neurons. microglia from 1 day cd1 pups were incubated for 24 h at 2 (young) and 16 (old) days in vitro (div) with abo (ab1-42 at 50 and 1000 nm). in parallel, e16 mice hippocampal neurons were treated with abo at 4 (young) and 18 (old) div for 24 h, and the incubation media used as conditioned media to treat microglia, at respective div, for further 24h. cell migration (boyden chamber), extracellular content in glutamate (commercial kit), and activity of matrix metalloproteinases (mmp)22 and 29 (gelatin zymography) were assessed. an increased migration towards abo and atp was observed in young microglia but almost undetectable in aged cells (p < 0.01). interestingly, microglia exposure to conditioned media from 1000 nm abotreated neurons markedly decreased both young and old microglia migration, while treatment with media from 50 nm abo-treated neurons enhanced migration, mainly in the young microglia (p < 0.05). concerning glutamate, aged cells released less than young ones upon abo treatment (p < 0.01). in addition, when exposed to neuronal conditioned media, only the young microglia were able to exert a neuroprotective role by reducing the media glutamate content. curiously, while abo promoted a microglia release of mmp-9, independently of cell age, it only induced mmp-2 secretion in old cells in an abo concentrationdependent manner (p < 0.05 for 1000 nm abo). in contrast, conditioned media from abo-treated neurons elicited mmp-2 release only from young microglia. together, our data point to a loss of microglia function towards abo with ageing and are unique in suggesting that senescent microglia may release high levels of mmp-2 in ad. in addition, our results indicate that abo-treated neurons enhance the release of mmp-2 even by young microglia. whether this finding may contribute to enhance inflammatory stress and the onset of ad will be the aim of further studies. human immunodefeciency virus-1 (hiv-1) productively infects microglia within the central nervous system (cns). viral replication within microglia and the resulting immune response and neurotoxicity leads to significant cognitive impairments not limited to dementia, neurocognitive abnormalities, and memory deficits. combination antiretroviral therapy reduces the cognitive impairments associated with hiv-1, but the regulatory hiv-1 tat protein is still produced in the cns during treatment, even in the absence of productive infection. our laboratory analyzes the effect of tat on the murine cns by stereotactically injected into cortex, which mimics in part the neuroinflammation and neurodegeneration characteristic of hiv-1 associated neurocognitive disorders (hands). additionally, in vitro tat-treated bv-2 microglial cells exhibit activation associated with increased cytokine expression and phagocytosis. mixed lineage kinase 3 (mlk3) has recently been implicated in the tat-mediated activation of microglia. mlk3 activity is increased by the presence of tat protein, which results in microglial activation and increased production of inflammatory cytokines. conversely, the brain penetrable, small molecule, new chemical entity urmc-099 inhibits the mlk3 pathway. we hypothesize that urmc-099 attenuates tat-induced microglial activation as measured by microglia process length, phagocytosis assay and cytokine expression. in vitro application of urmc-099 decreased tatinduced production of ccl2 and il-6, microglia process length, and phagocytosis of charged and uncharged beads in bv-2 microglia cells. these data, in aggregate, strongly support a role for the mlk3 pathway in neuroinflammation and suggest that inhibition of this pathway with urmc-099 may have significant anti-inflammatory effects in an inflammatory cns milieu. in conclusion, this work will advance our understanding of mlk3 activity and may provide a viable drug therapy for hands. the contributions of glial cells to the modulation of adult synaptic plasticity are becoming increasingly more appreciated. astrocytes in the supraoptic nucleus (son) of the hypothalamus demonstrate prominent temporary structural changes during physiological events such as dehydration and lactation. such structural remodeling is characterized by a reduction in the astrocytic coverage of oxytocin neurons and their synapses. these astrocyte morphological changes also contribute to the observed functional changes in synaptic activity during dehydration and lactation. in order to elucidate the underlying mechanisms regulating the astrocytic contributions to synaptic plasticity we have established an astrocyte protein expression profile for structural changes in the son during lactation and hyperosmotic challenge. for this, son from virgin, lactating, and hyperosmotic rats were collected from acute hypothalamic slices and analyzed by mass spectrometry to identify proteins differentially regulated by the changes in synaptic structure induced by lactation and hyper-osmolarity. our mass spectrometry data analysis has placed particular focus on regulation of astrocyte-specific proteins and the roles of these proteins in molecular pathways known to be involved in son plasticity, such as cytoskeleton remodeling, cell adhesion, and cell signaling. gene ontology analysis revealed over representation of pathways known to be highly active in astrocytes including metabolism and antioxidant activity. promising target proteins for future functional studies will be discussed. here we elucidate a novel merlin isoform 2 (merlin-iso2)-specific function in maintaining axonal integrity and propose that reduced axonal nf2 gene dosage leads to nf2-associated polyneuropathy. we identify a merlin-iso2-dependent complex corresponding to activation of the gtpase rhoa, enabling downstream rho-associated kinase to promote neurofilament heavy chain phosphorylation. in vivo, specifically merlin-iso2 knockout mice exhibit impaired locomotor capacities, delayed sensory reactions, and electrophysiological signs of axonal neuropathy. sciatic nerves from these mice and sural nerve biopsies from nf2 patients show reduced nf-h phosphorylation, decreased interfilament spacings and irregular-shaped axons. a. catenaccio, p. silva, f. court pontificia universidad cat olica de chile, santiago, chile axonal degeneration is an active process involved in a variety of neurodegenerative conditions triggered by diverse stimuli. this degenerative process can cause permanent loss of function, so it represents a focus for neuroprotective strategies. we have recently shown that axonal degeneration triggered by distinct mechanical and toxic damage is dependent on the activation of the mitochondrial permeability transition pore (mptp), which probably represents a central execution program of axonal degeneration, upon which several pathways converge (barrientos et. al., journal of neuroscience 2011). nevertheless, the participation of other cellular types in this degenerative process has been largely overlooked. after axonal damage in the peripheral nervous system (pns), schwann cells enveloping axons have been regarded as responsible for clearing degenerating axonal debris and to mount an inflammatory response for tissue clearance by invading macrophages. nevertheless, a possible function of glial cells in modulating axonal degeneration has not been addressed so far. here we explored the possibility that glial cells actively participates not only in the clearance of degenerating axons, but directly in the axonal degeneration program during wallerian degeneration. we found that axonal injury activates an early response of schwann cells that results in the fragmentation of their associated axons by actin-rich cytoplasmic domains of schwann cells known as schmidt-lanterman incisures (sli). this first step of schwann cell fragmentation of axons is dependent on the erk signaling pathway, wich control the dedifferentiation program initiated in schwann cells after nerve injury (napoli et. al., neuron 2012). in the axonal compartment, injury triggers a parallel cell autonomous degenerating program dependent on mitochondrial mptp activation, which is cell non-autonomously assisted by schwann cells through the activation of a cell extrinsic pathway of axonal destruction by tnf-alpha secretion. we showed for first that time that axonal degeneration in the pns proceeds by axonal autonomous mechanisms, as well as cell non-autonomous ones dependent on scs. therefore, effective targets for neuroprotection should consider not only the axonal compartment, but also the associated glial cell. glutamate is the major excitatory neurotransmitter in the brain. its concentration is the synaptic area is highly regulated in order to maintain point-to-point transmission and to prevent excitotoxicity associated with chronic activation of glutamate receptors. as there is no endogenous extracellular enzyme to degrade glutamate, the clearance of glutamate is ensured by transporters located on the surface of astrocytes. among these surface transporters glt-1 ensures almost 90% of glutamate uptake at synapses. it is widely accepted that many receptors and transporters on the surface of neurons and glia display surface trafficking properties and are not simply static proteins. the basic surface trafficking properties of glutamate receptors at the synapse have been studied in depth and have allowed us to understand many important trafficking events that occur during synaptic plasticity. the action of glt-1 is fundamentally important in synaptic communication. surface trafficking of this transporter may play a pivotal role in the spatial and temporal properties of glutamate diffusion at the synapse. however, it is still unknown whether glt-1 is dynamically regulated on the surface of astrocytes. thus we set out to uncover the surface trafficking of glt-1 in astrocytes by using a combination of high-resolution imaging, such as single particle (quantum dot [qd]) tracking, molecular biology and electrophysiological approaches. here we have demonstrated that glt-1 is indeed subject to surface trafficking on astrocytes. using pharmacological approaches we have shown that this diffusion is subject to regulation by the activity of the transporter itself as well as neuronal activity. furthermore, we have demonstrated that the speed of glt-1 diffusion is greatly reduced at excitatory synapses. this leads us to the conclusion that glt-1 surface diffusion is highly regulated at the synapse where it can effectively carry out its role as the principal glutamate transporter at the synapse. finally, to elucidate the physiological role of glt-1 surface diffusion, we have carried out electrophysiological experiments in which glt-1 trafficking is effectively reduced using a cross-link (x-link) protocol. we observed that a reduction in the surface trafficking of glt-1 resulted in an increase of neuronal excitability. thus glt-1 surface trafficking on astrocytes has an implicit role in regulating basal neuronal activity through glutamate uptake at the synapse. o. chever, e. dossi, n. rouach collège de france, paris, france astrocytes play crucial roles in brain physiology by dynamic interactions with neurons. they form plastic and extensive networks mediated by gap junction channels. it has recently been shown that astroglial networks limit neuronal network activity. however, it is currently unclear how astroglial networks influence neuronal excitability and population activity. to investigate how astroglial assembly regulates neuronal network activity, we performed electrophysiological recordings in hippocampal slices (ca3 and ca1 areas) from mice with disconnected astrocytes, in which both astroglial gap junction forming proteins, connexin 30 (cx30) and connexin 43 (cx43), are knocked out (gfap-cre cx30 -/-cx43 fl/fl , dko). synchronized excitatory discharges were recorded in an acute pharmacological model of epileptic-like activity. in this model, spontaneous bursting discharges are characterized by a sharp and large amplitude shift in field potential, generated by a major depolarization and firing of all putative neurons. pyramidal cells depolarized up to 40 mv during seconds and such depolarization is followed by a long-lasting undershoot of the membrane potential. we found that the frequency of bursting activity in dko hippocampal slices is drastically increased. however, the duration of neuronal depolarizing bursts is severely reduced. furthermore, pyramidal neurons are more depolarized due to an increase of synaptic bombardment. this suggests that increased synaptic background promoting resting membrane potential depolarization facilitates triggering of neuronal bursts, but compromises the strength of synchronized events. to investigate local dynamics of bursts generation, we performed multielectrodes arrays recordings in the ca3 area of the hippocampus. in slices from dko mice, bursting activity in the ca3 region is generated within a more restricted area, indicating that the number of neurons to be recruited is highly decreased. altogether, these results indicate that gap junction-mediated astroglial networks strengthen the coordination of neurons during synchronized events. acutely loaded hypoxia increases ventilation, and increased ventilation does not immediately return to the pre-hypoxic level, but persists for a while after resuming room air inhalation. this phenomenon is referred to as short term potentiation or plasticity of breathing, and is thought to contribute to the stabilization of ventilation. although extensive studies have been conducted to clarify the cellular mechanism of respiratory plasticity focusing on neurons and synapses, its precise mechanism remains unknown. on the basis of the recent discovery that astrocytes are actively involved in neural plasticity in various brain regions, we hypothesized that the post-hypoxic potentiation of breathing is mediated by astrocytes. we used unanaesthetized unrestrained adult mice. ventilatory parameters (respiratory frequency, tidal volume and minute ventilation) were measured by whole body plethysmography. to test the hypoxic response, mice breathed room air, hypoxic gas (7% o 2 , 93% n 2 ), and again room air. we also analyzed the hypercapnic response that was conducted under a hyperoxic condition to eliminate the influence of the carotid body. to test the hypercapnic response, mice breathed pure o 2 , hyperoxic hypercapnic gas (95% o 2 , 5% co 2 ), and again pure o 2 . after recording of the hypoxic and hypercapnic responses with their recovery time courses, arundic acid (ono-2506) was injected intraperitoneally. arundic acid is a compound that suppresses the function of astrocytes through the inhibition of s100b production in astrocytes. arundic acid did not affect ventilatory augmentation induced by either hypoxia or hypercapnia. however, arundic acid suppressed the short term potentiation induced by hypoxia (but not by hypercapnia) in a dose-dependent fashion. high dose arundic acid completely eliminated hypoxia-induced short term potentiation. these results suggest that the post-hypoxic potentiation of breathing is mediated by astrocytes with the involvement of s100b. the mechanism of hypercapnia-induced potentiation is different from that of hypoxia. we propose a model to explain the cellular mechanism of post-hypoxic potentiation: hypoxia stimulates the respiratory neuronal network in the brainstem through the excitation of the carotid body. neurotransmitters spilled from excited neuronal synapses activate nearby astrocytes. the activation of astrocytes is sustained, and these astrocytes persistently excite respiratory neuronal network by releasing gliotransmitters. monosynaptic c-fibre evoked excitatory postsynaptic currents (epscs) were recorded in lamina i neurons using whole-cell patchclamp in rat transverse spinal cord dorsal-root slice preparations. spontaneous epscs were recorded simultaneously. in all experiments bicuculline and strychnine were added to the bath solution to block gaba a -and glycine-mediated synaptic currents. the superfusion of spinal cord slices in vitro with fkn results in a rapid facilitation of evoked epscs in spinal lamina i neurons receiving monosynaptic input from primary afferent c-fibres. both the microglial cell inhibitor minocycline and a fkn neutralising antibody, completely prevented fkn-induced changes in synaptic transmission. in addition, the inclusion of the ca 21 chelator bapta or the nmda receptor antagonist mk801 in the pipette solution completely prevented fkn-induced enhancement of synaptic strength, suggesting a post-synaptic ca 21 -dependent mechanism of ltp induction. analysis of paired-pulse ratio (ppr) indicated that fkn-induced facilitation of synaptic strength is accompanied by a decrease in ppr suggesting a pre-synaptic mechanism of ltp expression. in support of this finding, fkn increases the number of spontaneous epscs recorded from lamina i neurons. these data indicate that stimulation of microglial cells in order to induce a pro-nociceptive activity state is sufficient for facilitation of synaptic strength within the dorsal horn. both pre-and post-synaptic mechanisms contribute to fkn-induced facilitation of synaptic strength. this work was funded by a wellcome trust flexible travel fellowship to akc. the organization and connectivity of the developing neocortex remains largely unresolved. the establishment of neuronal microcircuits is a complex process that partly depends on the diversity of neuronal cell types and their ultimate role in the mature brain. this complexity is increased by the existence of bona fide synaptic connections between neurons and oligodendrocyte precursor cells, also called ng2 cells, which properties and function remains largely unknown. to unravel the physiological characteristics of gabaergic synapses between interneurons and ng2 cells, we performed paired recordings in the developing somatosensory cortex. experiments were carried out during the second postnatal week in vgat-venus;ng2-dsred double transgenic mice that allowed us to identify interneurons (venus 1 ) and opcs (dsred 1 ) in acute slices. our results demonstrated that ng2 cells are synaptically contacted by fast-spiking (fsi) and non-fast-spiking (nfsi) interneurons. however, a predominant input was received by fsi, a class of interneuron that constitutes only a minor population in the second postnatal week. on the contrary, only a small proportion of the abundant nfsi were connected to ng2 cells. all the connections showed paired-pulse depression, low probability of response, and preliminary analyses suggest that each interneuron form a single synaptic contact onto ng2 cells. paired recordings, extensively used to examine the synaptic properties of neuronal microcircuits, may open a new perspective to understand in detail the mechanisms of gaba release and signaling between interneurons and ng2 cells in the healthy and pathological developing brain. first, we characterized a microglial cell line obtained from cd1 mouse cortex (n9), in order to clarify the mechanisms involved in the activation pattern of these cells. for that, we incubated n9 microglia with 300 ng/ml of lipopolysaccharide (lps) for 24 h. morphology (anti-iba1 staining), phagocytic ability (fluorescent latex beads) and chemotaxis to atp (boyden chamber) were evaluated. we observed that n9 microglia is ramified in control conditions and after lps treatment they change to an amoeboid morphology, which is accompanied by increased phagocytosis and decreased migratory ability, indicating that lps induces activation of n9 cell line. next, we investigated the role of the released factors from a mn-like cell line expressing human sod1 with g93a mutation (nsc-34/ hsod1g93a) on microglia functions. nsc-34 expressing human sod1 wt were used as control. thus, n9 microglia were incubated for 4 and 24 h with nsc-34 conditioned media that had been collected at 1 and 4 days of differentiation (div) and reactivity tests were performed as above. although after 4 h of incubation no significant changes were observed, data evidenced that incubation for 24 h with nsc-34/ hsod1g93a conditioned media collected at 4 div changed the bipolar morphology of the n9 microglia into an active amoeboid state, decreased phagocytic ability (22%, p < 0.05) and induced apoptosis (50%) (fig.1) . moreover, microglia have shown to not be attracted by the released factors from degenerating mn, as determined by the chemotaxis assay. together, our results evidence that released factors from nsc-34/ hsod1g93a degenerating cells although inducing microglia morphological activation trigger a reduction in cell phagocytic ability and loss of viability, suggesting a role for microglia along als progression. organotypic explants culture has a pivotal role in studying the complex structure of neuronal tissue. although embryonic tissue explants and slices are used to obtain long term culture, most applications such as drug testing require adult tissue for reliable results. in this study, we show that nanostructured metal-oxide arrays can be employed to yield long-term organotypic culture of adult neuronal tissue explants as demonstrated for the adult guinea pig retina and adult murine brain slices. even after 14 days of culture, the thickness and the layered structure of the retina were well maintained. quantitatively, the number of nuclei within the inner and outer nuclear layers was comparable to freshly isolated retinae and no significant change in the densities of the nuclei within the layers was observed. the outer plexiform layer, as the site of synaptic connection between photoreceptors and neurons, was still well preserved. moreover, the thickness of the photoreceptor layer was conserved, indicating that the inner and the outer segments of the photoreceptors survived well during two weeks culture. this is hardly observed in long-term cultures since the outer segments were cultured without retinal pigment epithelium. concerning the adult murine brain slices from neo-cortex, after 9 days of culture, the neurofilaments and cell nuclei were still well preserved and the neuronal network displayed the typical arrangement of long axons. however, preservation of the tissue depends strongly on the geometry of the nanostructured array surface such as roughness and spacing, whereby organotypic brain slice culture requires different geometries than retina culture. since organotypic culture of adult tissue could only be obtained for about 7 days with conventional techniques, our new substrate material could be the breakthrough for in vitro studies on tissue regeneration, neuroplasticity, as well as drug testing. the intrinsic optical signal (ios) is widely used for mapping neuronal activity in afferent activated brain areas. ios evoked by afferent stimulation in vitro is generally attributed to glial cell swelling via activation of na 1 /k 1 /clcotransporter that follows postsynaptic activation. despite the phenomenon is known for decades, the relative contribution of different cell types and the underlying molecular mechanisms have not been disclosed in detail. our goal was to explore the glial and neuronal correlates underlying in vitro ios genesis. we characterized ios initiated by schaffer collateral stimulation in the rat hippocampal slice using simultaneous high-frequency imaging and field potential recordings. we used a 464-element photodiode-array device (pda) that enables ios detection with 0.6 ms time resolution, making it achievable to align optical and electrophysiological signals. ios was primarily observed in the proximal region of the stratum radiatum and stratum pyramidale of the hippocampus. ios was decreased by blockade of neuronal activity by voltage-gated na 1 channel inhibitor tetrodotoxin and was significantly enhanced by suppressing inhibitory signaling with the gaba a antagonist picrotoxin. we found that ios was predominantly initiated by postsynaptic glutamate receptor activation and progressed by the activation of astroglial glutamate transporters and mg 21 -independent astroglial nmda receptors. in agreement with previous studies, furosemide, the blocker of both the neuronal k 1 / clcotransporter kcc2 and the glial na 1 /k 1 /clcotransporter nkcc1 decreased the ios amplitude. to decide which cotransporter is responsible for this effect of furosemide, we applied selective blockers of nkcc1 and kcc2, bumetanide and (1)[r]-dioa, respectively. we evidenced, that nkcc1 did not contribute to the ios generation, in contrast to previous suggestions. enhancement and slight inhibition of ios through anion and volume-regulated anion channels, respectively, were also depicted. major players of ios mechanisms disclosed by high-frequency ios imaging imply that spatiotemporal ios reflects glutamatergic neuronal activation and astroglial response, as observed within the hippocampus. our model may help to better interpret in vivo ios and support diagnosis in the future. question: a poor x3 polyunsaturated fatty acids (x3 pufa) status, favored by the low x3/x6 ratio in western diets, seems to contribute to cognitive decline in the elderly, but mechanistic evidence is lacking. we therefore explored the impact of x3 status on the evolution of glutamatergic transmission, astroglial regulation and neurogenesis in the hippocampus during ageing in rats. these processes are involved in memory formation and their dysregulation participates to the agerelated brain damage leading to cognitive decline. methods: we have compared 6 groups of rats aged 6 to 22 months fed x3-deficient, x3/x6-balanced, or x3 (fish oil) supplemented diets: young x3 balanced (yb), deficient (yd) or supplemented (ys), and old x3 balanced (ob), deficient (od) or supplemented (os) rats. we have evaluated synaptic efficacy and plasticity (electrophysiological recording), astroglial regulations (glutamate uptake and gfap expression), neuronal markers (glutamate transporters and receptors), neurogenesis (proliferation of neuronal precursors in the sub granular zone), and analyzed brain fatty acids composition. results: dietary modulation of x3 intakes efficiently modified the incorporation of docosahexaenoic acid (dha, the main x3 in cell membranes) in brain (250% deficient vs balanced, 110% supplemented vs balanced). ageing induced a 35% reduction of synaptic efficacy due to decreased pre-synaptic glutamate release, and a 30% decrease in the astroglial glutamate uptake associated to a marked astrogliosis (1100% gfap). x3 deficiency further decreased these hallmarks of ageing (od vs ob rats: 235% synaptic efficacy, 215% glutamate uptake, 130% gfap). on the opposite, x3 supplementation increased synaptic efficacy (125% os vs od) and seems to abolish astrogliosis (os vs ys : no change in gfap). neurogenesis was altered by x3 deficiency but not by supplementation. conclusion: our results characterize some specific age-related alterations of the glutamatergic synapse in the hippocampus that are aggravated by a dietary deficit in x3 and attenuated by x3 supplementation. methods: sgcs were isolated from the trigeminal ganglion (tg) of adult male wistar rats (n 5 9). animals were deeply anaesthetized and both tg were extracted and digested first in 5 mg/ml collagenase for 15 min and then in 0.125% trypsin containing dnase for 10 min. in initial experiments, isolated sgcs were treated with control medium or medium containing 16.5, 33, or 66 mm cis for 8h. in the subsequent experiments, sgcs were pretreated for 24h with control medium or medium containing 100 mm ibu or 1 mm skf. afterwards, sgcs were stimulated for 8 hours with medium containing 66 mm cis. pge 2 concentration was determined by elisa. results: cis caused a concentration-dependent increase in pge 2 release from sgcs. treatment with 33 mm cis significantly increased pge 2 concentration to 354.8 6 87.3 pg/ml, compared with control 211.3 6 39.0 pg/ml (p 5 0.018). pge 2 concentration was further increased to 464.1 6 77.0 pg/ml (p 5 0.002) following 66 mm cis. pretreatment with ibu significantly lowered the pge 2 concentration compared with cis-stimulated sgcs (197. 5 6 85.2 pg/ml vs. 415.8 6 143.8 pg/ml, respectively; p 5 0.05). a more pronounced inhibitory effect was observed following skf pretreatment, where it reduced the pge 2 concentration to 19.7 6 1.8 pg/ml (p 5 0.003, compared with cis-stimulated sgcs). conclusion: these findings suggest that cis contributes to an inflammatory response in the tg by provoking release of pge 2 from sgcs, which may lead to development or maintenance of peripheral sensitization involved in cipn. the data also suggest that treatment with agents that target pge 2 release cascade may prove beneficial for preventing development or maintenance of cipn. functional alterations in synaptic contacts have often been described as a hallmark of major depressive disorder (mdd). antidepressants (ads) have been shown to restore neuronal circuits through an enhancement of synaptogenesis in some regions of the brain. nevertheless, the underlying mechanisms are still unclear. glia cells have been since long acknowledged as active partners of neurons in orchestrating molecular signals crucial for the proper arrangement of neuronal circuits in the developing and adult brain. therefore, understanding how both neurons and astrocytes respond to antidepressants (ad) is of high interest. using rat c6 glioma cells and primary cultures of astrocytes and neurons, we showed a time-dependent cell-autonomous modulation of the erk/mapk signalling pathway after acute treatment with various classes of ads in both cell types. specifically, glia cells responded to ads with the simultaneous and fast activation (after 10 min treatment) of both erk1/2, in contrast to treatment with antipsychotics or mood stabilizers. this activation induced 48 hrs later an increased release of gdnf, a factor involved in synapse formation and axonal wiring, which was mapk-dependent. on the contrary, hippocampal neurons showed a reduction in erk activity that correlated with neuronal activity inhibition after short term (10 min) ads administration, as demonstrated by quantification of c-fos expression after kcl stimulation. interestingly, this inhibitory effect was reversible after long term (48 hrs) ads treatment. to further identify whether gdnf released from astrocytes treated with ads might be responsible for long term changes in neuronal synapses, we examined how ads influenced synaptic densities in neurons alone or co-cultured with astrocytes. strikingly, we found that the number of synapses was reduced at 48hrs after ad treatments, but only in the presence of intact astrocytes and not in cultures of neurons alone or neurons treated with astrocyte-conditioned media. this effect was reversed after 120 hrs treatment and rescued by the soluble form of the specific gdnf receptor gfralpha1, but not by gdnf alone. moreover, live imaging of astrocytes showed dramatic morphologic changes of their processes in response to ads that might underlie the observed synaptic remodelling. our analysis of changes occurring at the neuronglia interface upon ad treatments might elucidate novel mechanisms of ads action that may open the avenue for unravelling the role of astrocytes in psychiatric diseases and implement pharmacologic treatment regimens for mdd patients. here we extend these findings showing that the neurotrophin nt-3, which also belongs to the putative factors secreted from glial cells after t3-stimulation, also increases the navd in neuron enriched cultures. the effects of nt-3 and fgf-2 are, however, not additive. antibodies against nt-3 potentiated the effects of fgf-2, suggesting that by converging signal cascades nt-3 could reduce the effect of fgf-2. in neuron-glia mixed cultures antibodies against nt-3 enhanced the t3-induced up-regulation of navd, suggesting, that a co-secretion of nt-3 from glial cells could limit the effects of fgf-2. in a second series of experiments we investigated, whether the well-known effect of t3 on the expression of na 1 /k 1 -atpases, thought to contribute to the effect of thyroid hormone on metabolism, is also influenced by glial cells. we found, that the effect of t3 on 3 [h]ouabain-binding and on the expression of na 1 / k 1 -atpase alpha2 subunits detected by western-blots was highest in neuron-mixed cultures, suggesting that a neuron-glia interaction is also involved in the t3 effects on na 1 /k 1 -atpase expression. in the healthy brain microglia engage in bi-directional interactions with neurons and other brain cells, which is crucial for maintaining normal physiology and cognitive function of the brain. disturbances in homeostasis and cell-cell communication may predispose to age-related dysfunction and neurodegenerative disease. for decades it has been speculated that microglia exhibit phenotypic diversity allowing specialisation to a variety of brain microenvironments. previous observations of regional variations in microglial densities and morphologies support the existence of microglial heterogeneity. however it is clear that a more comprehensive understanding, in particular of the molecular basis of microglial phenotypic and functional diversity on a global scale, is required. the limitation of recent gene expression studies is the use of mixed whole brain or dissected mixed cell extracts which preclude the detection of cell-specific gene expression profiles. thus, our aim was to validate an efficient extraction process for microglia, ideally minimising the impact on the resting state. the method developed is based on existing procedures and was refined to ensure consistency and to maximise cell yield and purity. the extraction of highly pure microglia as a single cell type was achieved by a two-step isolation technique using density-gradient and magnetic bead separation. purity between 90-95% was verified by flow cytometry and immunohistochemistry. quantitative pcr also demonstrated expression of specific microglial genes indicating that isolated cells are microglia. cytokine assays suggest that no or only minimal activation of cells was induced by the isolation. hence, the microglia retained their ability to respond to immunogenic stimuli and produce il-1b as a key inflammatory cytokine. overall, the purification procedure was shown to achieve consistent high purities and retain crucial functional properties. thus, the extracted microglia are likely to adequately represent the real in vivo state and enable the study of microglial phenotypes and functionality in the healthy and ageing whole brain and accordingly brain regions. early diversity experiments showed region-specific microglia phenotypes in the brain of healthy and immune challenged animals as well as differences between unchallenged and challenged animals. question: activation of nmda receptors increase the respiratory frequency in mammals. astrocytes are in intimate contact with neurons, specially in glutamatergic synapses, and are able to sense neuronal activity, react to, and even influence nearby neurons. furthermore, they can sense changes in h 1 or pco 2 , and in response to these stimuli, they can release molecules like atp and d-serine, which will activate neuronal circuits. then d-serine, an agonist of the glycine site of the nmda receptor, can affect the respiratory rhythm during hypercapnia. our aim was to study the probable role of d-serine in the modulation of the respiratory rhythm in mice neonates. methods: fictive respiration was recorded with suction electrodes from c4-c5 ventral roots in "en bloc" (brainstem-spinal cord) preparations from p0-p3 cf1 mice. superfusion was done with artificial cerebrospinal fluid equilibrated with o2:co2 5 95%: 5%, (ph 7.4,28 c) containing different d-serine concentrations (0.1-100 mm) in presence or absence of d-amino acid oxidase (daao), which degrades d-serine. in addition, hypercarbic acidosis (switching equilibration from 5 to 10% co2, changing ph from 7.4 to 7.2) was done in absence or presence of daao. results: application of d-serine increased the frequency of the respiratory rhythm in a concentration-dependent way. application of dao attenuated the effects of d-serine application. likewise, daao reduced slightly the effect of hypercarbia on the respiratory rhythm. conclusions: our preliminary experiments indicate that d-serine is a potent agent increasing the respiratory rhythm frequency in in vitro neonatal preparations, and likely, in association with atp, is mediating the respiratory response to hypercarbia. there, we showed that mac-2 positive microglia accumulate within the motoneuron area and phagocyted apoptotic bodies at the onset of motoneuron developmental cell death (from embryonic day 12.5-e12.5). motoneuron developmental cell death and microglia accumulation in the motoneuron area increased at e13.5, decreased at e14.5 and disappeared at e15.5. since it was supposed that microglia promote developmental cell death in the developing central nervous system, at least in vitro, we determined to what extent it was also the case in the embryonic spinal cord in vivo. to address this issue, we analysed in vivo the progression of developmental neuronal death in the spinal cord of transgenic mouse embryos lacking microglia (pu.1-ko mice). as opposed to what is observed in the sc of wild type mouse embryo, we found that activated caspase-3 staining was detectable before e12.5 in pu.1-ko mouse embryos. in pu.1-ko mouse embryos, activated caspase-3 staining was not restricted to the motoneuron area, as several positive cells are also present in the dorsal region of the spinal cord and in the progenitor zone. at e13.5, activated caspase-3 staining was 10 times greater in the spinal cord ventral area of pu.1-ko mouse embryos when compared to wild type littermates. moreover, and opposed to what occurs in wild type animals, caspase-3 staining in the motoneuron area of pu.1-ko mouse embryos increased at e14.5 and persisted at e15.5, being likely partly due to the accumulation of apoptotic bodies in the absence of microglia. our results suggest that, in addition to their phagocytic role, embryonic microglia are likely to protect neuron from death at the onset of developmental cell death and to promote survival of progenitor-like cells in the embryonic sc in vivo. accordingly, microglia could have an opposite effect on cell survival in the embryonic sc when compared to postnatal snc developmental stages and pathological conditions in the adult. we recently demonstrated a key role of the a-secretase tace, also known as adam17, in peripheral nervous system (pns) myelination by tace-mediated cleavage and subsequent inhibition of neuregulin1 (nrg1) type iii activity. unlike the pns in which nrg1 type iii is an essential instructive signal for myelination, oligodendrocyte (ol) development and myelination in the central nervous system (cns) are likely controlled by several growth factors some of which undergo cleavage by secretases. to study the role of tace on opc development, immunopanned a2b51 opcs were cultured in proliferating or differentiating medium and treated with a soluble form of tace (rhtace). when ols were scored according to their morphology, we observed enhanced opc differentiation upon addition of rhtace to proliferating opcs. addition of rhtace to differentiating opcs also increased the number of ols with a complex morphology and already presenting membrane sheets, suggesting that tace might promote opc differentiation. to further investigate the role of neuronal tace in cns, we used an in vitro ol myelinating coculture system. when cocultured with tace null drg neurons, rat wild type opcs never myelinate, suggesting that neuronal tace might control opc differentiation. in agreement, preliminary ultrastructural analyses of transgenic mice lacking tace in cns neurons showed hypomyelination and signs of myelin degeneration. accordingly, conditional transgenic mice lacking tace in ol are normally myelinated. these studies suggest that in the cns the a-secretase tace promotes opc differentiation and might regulate cns myelination. a better understanding of the role of tace will provide novel insights into the mechanism regulating cns myelination. in the excitatory network glutamate is released in the synaptic cleft during synaptic activity. glutamate clearance by astrocytes is mainly performed by na 1 -dependent glutamate transporters. the na 1 load during glutamate uptake evokes an activation of the na 1 /k 1 atpase, which dramatically increases energy demands in astrocytes. astrocytes are also involved in the regulation of extracellular potassium (k e 1 ) which significantly increases during the repolarization of excitatory neurons. in this study we evaluated how these fundamental functions of astrocytes coexist and if they interact. we investigated the impact of altering k e 1 concentrations on glutamate transporter activity measuring intracellular sodium (na i 1 ) using the na 1 sensitive fluorescent dye asante sodium green loaded in cultured astrocytes. we observed that glutamate uptake caused a reversible rise in na i 1 , which was tightly modulated in amplitude, slope and recovery rate by k e 1 levels. in order to evaluate the impact of physiologically relevant k e 1 fluctuations on the kinetics of the glutamate transporter the na 1 /k 1 atpase was inhibited. therefore we found that low k e 1 evoked a significantly faster influx of na 1 , whereas high k e 1 markedly slowed down glutamate capture, indicating that k e 1 directly modulates the kinetics of glutamate transporters. we then investigated the energy demands of astrocytes caused by k e 1 alterations. atp hydrolysis was indirectly measured through the changes in intracellular free mg 21 using the mg 21 sensitive fluorescent probe magnesium green. we found that low k e 1 led to higher energy demands of astrocytes during glutamate uptake. overall these results indicate that k e 1 acts as a negative feedback on glutamate uptake in astrocytes. the physiological consequences of these findings have to be considered in the context of k 1 buffering and of maintenance of neurotransmitter and energy homeostasis. recovery was significantly improved by nf fractions, as well as tubulin (tub), added after lpc removal: compared to cultures treated with lpc alone the number of olp (a2b51 cells), and their proliferation increased significantly, as well as the number of differentiated (cnp1) and mature (mbp1) ol. moreover, nf and tub protected ol from lpc toxicity when added at the time of lpc treatment: they increased olp proliferation, as well as the number of cnp1 and mbp1 ol significantly, compared to cultures treated only with lpc. on the contrary irrelevant proteins (actin, and skin proteins) were ineffective, demonstrating the specificity of the cytoskeleton proteins effects. importantly, nf and tub increase olp survival and proliferation when challenged with lpc, without delaying differentiation and maturation. we hypothesize that release of nf and tub following axonal damage in ms could participate in the regulation of remyelination through this process. this putative phenomenon might be complexified by alterations of axon protein expression, or of proteolysis, known to occur in ms models. purpose: microglial proliferation is commonly accepted as a hallmark of glial activation. an automatic method that allows assessing the number of microglial cells to analyze the proliferative microglial behavior in a laser-induced ocular hypertension (oht) model has been developed. methods: adult albino swiss mice were organized in two groups: naive (age-matched control; n 5 6) and lasered (n 5 6). retinal wholemounts were immunostained with anti-iba1 and images of iba-11 microglias were recorded with a fluorescence microscope. new algorithms of segmentation and control of distances were developed in matlab to obtain the number of iba-1 microglial cells. automatic results were compared with those obtain by direct human observation of the samples. results: the automatic method detected the number of iba-11 cells in the inner and outer plexiform layers of the retina in both, na€ ıve and oht-retinas. the number of cells present in the samples was not an obstacle for the program to run properly. the time required for counting iba-11 cells decreased from the human guide to the program based counting method from days to one hour. results show a strong correlation between both automatic and manual method (pearson correlation test, r 5 0.979; p 5 0.000 and r 5 0.942; p 5 0.000 for outer and inner plexiform layer respectively) indicating the consistency of the automatic counting. conclusions: a new, reliable and quick algorithm was developed with matlab to obtain the number of iba-11 microglial cells and also cellular density maps through retinal wholemounts in both na€ ıve and other proliferative conditions (i.e. glaucoma). due to this new automatic method, a bigger set of images or samples could be included in future studies to analyze the behavior of microglial cells. we are interested to understand whether scs in the peripheral nerves of mammals express functional ionotropic glutamate receptors, and whether scs use these receptors for communication with axons. to start addressing this question, we established a preparation of mouse live sciatic nerve slices employing "know-how" of live brain slices technique. using this preparation, we performed whole-cell patch-clamp recordings of scs at different stages of development (embryonic and early postnatal). we included a fluorescent dye into the pipette solution in order to perform post-recording morphological analysis of single scs. first, we recorded current responses of scs to a series of depolarizing voltage steps, aiming to test whether all scs in the nerve are electrophysiologically akin. we found that mouse scs differ in their passive membrane properties and expression of voltage-gated k1 channels: depending on the age, 3 to 4 cell types could be distinguished. post-recording morphological characterization of the recorded cells showed that scs in the mouse sciatic nerve differ in their size, as well as in the number and length of their processes. next, we used fast pressureinduced application of glutamate to investigate whether scs possess ionotropic glutamate receptors, and whether electrophysiologically distinct sc types differ in their expression of glutamate receptors. application of 1 mm glutamate caused an inward current in at least two types of scs, and preliminary analysis revealed a peak current amplitude in the range of 14-250 pa (n 5 18). glutamate-induced currents in scs were blocked by gyki53655, indicating that mouse scs carry ionotropic glutamate receptors of ampa/kainate type. currently we are studying which subunits of ampa receptors are present in scs, and how these receptors get activated in situ. although cx32 is expressed by myelinating schwann cells in the peripheral nerves, patients with cmt1x develop relatively early axonal degeneration, which correlates best with chronic disability. we found in previous studies of gjb1-null mice, a model of cmt1x, that the diameter of myelinated axons was progressively reduced at the age of 2 months compared to wild type (wt) littermates, resulting from progressive dephosphorylation of axonal neurofilaments and increased packing density. fast axonal transport was slower in distal axons of mutant compared to wt animals, with impaired mobility of synaptic vesicleassociated proteins and accumulation of beta-amyloid precursor protein and tau. how cx32-formed gjs are involved in the regulation of axonal cytoskeleton dynamics remains unclear. methods-here we investigated the signaling mechanisms regulating axonal cytoskeleton focusing on major kinases and phosphatases involved in neurofilament phosphorylation by immunohistochemistry and immunoblot. in addition we examined the localization of axonal mitochondrial, which also depend on axonal transport. we focused on nerve pathology in 2 month-old gjb1-null mice, when demyelination and inflammation is minimal, in order to identify the direct effects of gj loss on the axon. results-our results show that erk1/2 kinase and pp2a phosphatase were localized in non-compact myelin areas, including the schmidt-lantermann incisures and paranodes, where gjs are normally formed by cx32. another kinase, cdk5, was more diffusely localized along the axon. biochemical analysis showed altered amounts of erk1/ 2, pp2a and cdk5 as well as their upstream activators in gjb1-null nerves. the pp2a inhibitor phapi was also localized in non-compact myelin areas and significantly increased in gjb1-null nerves. assessment of axonal mitochondria by porin immunostaining showed significantly increased density in gjb1-null nerves, consistent with slower axonal transport. conclusion-our findings clarify some mechanisms of early axonal pathology that are independent of demyelination in this mouse model of cmt1x. several components of the signaling pathway regulating axonal cytoskeleton and axonal transport appear to be functionally related to gjs at the non-compact myelin sheath. loss of myelin gjs results in impaired regulation of axonal cytoskeleton, energy supply, and axonal transport. we have recently discovered that oligodendrocytes secrete exosomes containing a distinct set of proteins as well as mrna and microrna. intriguingly, oligodendroglial exosome release is stimulated by the neurotransmitter glutamate indicating that neuronal electrical activity controls glial exosome release. in this study we examined the role of exosomes in neuron-glia communication and its implications in glial support. results: to analyze the transfer of oligodendroglial exosomes to neurons, we exposed cultured cortical neurons to fluorescently labeled oligodendroglial exosomes. indeed, cultured cortical neurons internalized and accumulated oligodendroglial exosomes in the neuronal cell soma in a time-dependent manner. addition of endocytosis inhibitors or expression of dominant negative dynamin interfered with neuronal exosome internalization indicating that exosome uptake is mediated by clathrin-dependent endocytosis. furthermore, neuronal internalization of exosomes resulted in functional retrieval of exosomal cargo in vitro and in vivo upon stereotactic injection of exosomes. to investigate the influence of oligodendroglial exosomes on neuronal gene expression we performed a microarray screen of neuronal mrnas after exosome exposure. interesting candidates were further validated by qrt-pcr. conclusion: taken together, our results represent a proof of principle of exosome transmission from oligodendrocytes to neurons suggesting a new route of horizontal transfer in the cns. astrocytes are essential for the regulation of neuronal excitability, synaptic plasticity, and the vascular coupling of brain metabolism. unlike neurons, they are electrically silent, but produce a complex repertoire of ca 21 events that coordinate their major functions. however, the canonical cellular mechanism for ca 21 integration in astrocytes is unknown. using cultured astocytes and astrocytes in brain slices expressing ca 21 sensor, gcamp2, we report a candidate principle for ca 21 integration in single astrocytes. analysis of the spatiotemporal dynamics of ca 21 signaling in cultured astrocytes demonstrated that ca 21 event distribution can be described by a power law. we show that metabotropic glutamate receptor activation or changes in extracellular ca 21 regulate the power law exponent. these findings demonstrate scale-free ca 21 dynamics in astrocytes that can provide a potential computational theory for brain operation and metabolism. the biogenesis of the axon-myelin unit is controlled by reciprocal interactions between oligodendrocytes and neurons, which continue throughout life. oligodendrocytes secrete endosome-derived microvesicles termed exosomes, implicated in intercellular communication. these exosomes carry a specific set of proteins as well as rna species. here, we show that the neurotransmitter glutamate stimulates exosome secretion from oligodendrocytes by provoking ca 21 entry through oligodendroglial nmda and ampa receptors. furthermore, active neurons evoke exosome release from oligodendrocytes, indicating that neuronal activity controls oligodendroglial exosome release. in turn, neurons internalize oligodendroglial exosomes and functionally retrieve the exosomal cargo. thus, oligodendrocytes may influence the neuronal metabolism by an exosome-dependent transfer of bioactive substances to neurons. axonal degeneration resulting from lack of glial support occurs in plp and cnp deficient mice. intriguingly, both proteins are components of oligodendroglial exosomes. a proteomic approach revealed that amount and composition of exosomes derived from plp -/and cnp -/oligodendrocytes are altered, supporting the hypothesis that disturbed intercellular transfer of substances by exosomes may contribute to axonal degeneration in these mouse models. a. shahraz, j. kopatz, h. neumann university of bonn, bonn, germany microglial cells are the resident macrophages of the brain, which are derived from the embryonic haematopoiesis of the yolk sac. recently, we established a protocol for in vitro differentiation of pluripotent stem cells into microglial precursors by mimicking the embryonic haematopoiesis in a neural microenvironment. we now succeeded to produce microglia from human induced pluripotent stem (ips) cells. these human microglial cells were responsive to amyloid-b by increasing reactive oxygen species (ros) production. in addition, we produced primitive neural stem cells (pnsc) from ips cells that differentiated with suitable growth factors towards neurons. in human microglia-neuron co-culture, amyloid-b treatment resulted in shorter neural branches length compare to the control group. furthermore, results showed that the inhibitory human lineage specific receptor sialic acid binding immunoglobulin-like lectin-11 (siglec-11) was expressed on human microglial lines. we are now studying the neuroprotective role of siglec-11 in the amyloid-b-mediated microglia-neuron co-culture model. by expressing the human-lineage specific receptors, these microglial cells will be a suitable model to investigate these receptors in a human microglianeurons co-culture system.x purpose: organotypic retinal culture is a useful tool to perform preclinical drug testing, in which cellular interactions as well as tissue threedimensionality are preserved. the present study aimed to provide a detailed characterization of the morphologic changes of macro and microglial cells in this culture system. methods: retinas were isolated from 7 days old crl: cd(sd) rats with the retinal pigment epithelium attached as described previously (arango-gonzalez et al. 2010). explants were cultured for 4, 10 and 14 days (div4, div10 and div14). glial cell populations were characterized by immunostaining cryosections and wholemounts of age-matched control and cultured retinas using specific antibodies against gfap, vimentin and cd11-b. results: in div4 and div10 cultures, gfap-positive astrocytes were more robust and not homogeneously distributed as observed in vivo: in some retinal areas the astrocytic network was thicker and in other regions astrocytes were sparsely distributed. at div14 only few thinned gfap-positive astrocytes remained. gfap immunoreactivity of m€ uller cells was up-regulated in culture. however, no major difference was observed in vimentin staining between both, in vivo and in vitro retinas. m€ uller cells extended processes from the inner to the outer limiting membrane were observed for all examined retinas. microglial cells stained with cd11-b at div4 and div10 had somas more robust than in vivo and cell processes were thicker and retracted. same cells at div14 exhibited mainly a rounded morphology. conclusions: progressive morphological changes were observed in glial cell populations in retina explants. these changes include reactive macrogliosis, rearranged astrocytic distribution and microglial activation. since glial response is a hallmark of several retinal diseases, our organotypic retinal culture is a valuable resource for future investigations in retinal degenerative processes and therapy. it is widely recognized that grouped housing in enriched environment (enr) impacts the animals' brain structure and function. for instance, rats reared in enr exhibit enhanced neurogenesis in the dentate gyrus, improved hippocampus-dependent spatial memory task performance, and spine density increases of ca3 and ca1 pyramidal neurons. we found that that enr housing for four weeks after weaning induces an enhancement in gamma band local field potential oscillation power and an increase in spine density in the right ca1 stratum radiatum of long-evans rats. the mean spine size, as assessed by serial measurements of postsynaptic density areas, did not change substantially by laterality or rearing condition. one standing question is how glial processes are organized in enr treated rats. unlike cerebellar parallel fiber synapses, the astrocytic coverage of a hippocampal synapse varies from synapse to synapse. our preliminary electron microscopic observation suggests that right ca1 str. radiatum excitatory synapses tend to have larger degrees of astrocytic coverage in enr treated rats than singly caged rats.we are currently making morphometric measurements to quantitatively assess the peri-synaptic glial changes. it is becoming more and more evident that proper functioning of the neuron-astrocyte-microglia triad is fundamental for the functional organization of the brain, and thus it is of the utmost importance to better characterize their interactions in physiological and pathological processes. we recently demonstrated, in rat models of normal brain aging and lps-induced acute inflammation, that astrocytes and microglia actively collaborate in the clearance of apoptotic neurons and neuronal debris associated with programmed cell death. here we studied the interactions between neurons, microglia and astrocytes within the ca1 region of the hippocampus after bilateral common carotid artery occlusion (bccao) in the rat, a valid model of chronic cerebral hypoperfusion which leads to persistent ischemic conditions and ultimately to neuronal death. male wistar rats were subjected to permanent bccao. a group of rats was infused into the jugular vein with dipyridamole (7 days, 4 mg/kg/day by an osmotic minipump). sham-operated animals were used as controls. three months after bccao, immunohistochemical studies were performed on brain coronal slices, focussing on the hippocampal ca1 region. using an antibody against glial fibrillary acidic protein (gfap) no astrogliosis was detected. we found a significant increase in the number of total microglia, visualized using the iba1 antibody, in bccao-treated rats in comparison to the sham group (1 18%, p < 0.01, one way anova and newman-keuls) and this effect was completely reverted by dipyridamole (p < 0.01, one way anova and newman-keuls). as exemplified in figure 1 , in the ca1 and str. radiatum of bccao-treated rats, many neurons (anti-neun antibody, red) showing signs of degeneration were closely apposed to and infiltrated by astrocyte branches (anti-gfap antibody, green) which appeared to be bisecting the cell body into cellular debris, and microglia cells (anti-iba1, antibody, blue) were actively phagocytosing the damaged neurons. this finding is consistent with the scavenging activity of microglia upon dying neurons or debris, a possible mechanism that prevents further injury to neighboring neurons. it will be interesting to investigate which intercellular communication mechanisms allow the recruitment and activation of different glial cells in a well-organized reciprocal interaction to scavenge the damaged neurons and to verify the mechanisms of the protective effects of dipyridamole found in this chronic cerebral ischemic model. astrocytes are glial cells which provide important metabolic support to neurons, actively tune synaptic activity and influence brain microcirculation [1] . one of the key processes which sustain astrocyte communication with neighbouring cells is regulated exocytosis mediating the release of gliotransmitters (peptides, amino acids, atp), delivery of membrane transporters, channels and other molecules to the plasma membrane. the exocytic gliotransmitter release is thought to be associated with snare complexes. these consist of four a-helices where one of these is contributed by syntaxin-1, one by synaptobrevin-2 (sb2) and two by snap-23 in astrocytes. out of these, sb2 is a trans-membrane protein present on the secretory vesicle [2] . however, the subvesicular nano-architecture and the number of sb2 molecules per vesicle are unclear. moreover, it is also unknown how many sb2 molecules are involved in the fusion between the vesicle and the plasma membrane in astrocytes. to study single vesicles and their association with sb2 in live astrocytes, we generated a ph-sensitive indicator yellow synapto-phluorin (ysph) as a marker for sb2 and as a functional readout for monitoring the properties of fusion pores, which are formed following the merger between the vesicle and plasma membranes. in an acidic environment ysph is non-fluorescent and becomes fluorescent upon alkalinisation, permitting the study of fusion of vesicles with the membrane during gliotransmitter release [3] . our preliminary results, obtained by confocal fluorescence microscopy (with the resolution limit of about 200 nm) and by a super-resolution microscopic technique structured illumination microscopy (sim; with the resolution limit of about 120 nm) show that the new probe (i.e. ysph) efficiently reports the fusion pore establishment in astrocytes and the configuration of sb2 molecules in a vesicle. the number of schwann cells is fitted to the axonal length in the peripheral nerves. this setting is lost when tumorigenic stimuli induce uncontrolled schwann cell proliferation, generating tumours such us neurofibromas and schwannomas. schwann cells proliferate as well during wallerian degeneration. in both cases proliferation is finally arrested. we show that in neurofibroma, the induction of jmjd3 removes trimethyl groups on lysine-27 of histone-h3 and epigenetically activates the ink4a/arf-locus, forcing schwann cells towards replicative senescence. remarkably, loss of function of this mechanism allows unrestricted proliferation, inducing malignant transformation of neurofibromas. interestingly, our data suggest that in injured nerves, schwann cells epigenetically activate the same locus and go as well into the senescence program. indeed, when this pathway is genetically blocked, cell proliferation results increased after nerve injury. we postulate that the ink4a/arf-locus is expressed as part of a physiological response that prevents uncontrolled proliferation of the de-differentiated schwann cells generated during nerve regeneration, a response that is also activated to avoid overproliferation after tumorigenic stimuli in the peripheral nervous system. university of rochester medical center, rochester, united states synaptic plasticity is critical for normal neurodevelopment and proper circuit function in the adult nervous system. recent evidence suggests that microglia, classically studied in neuroinflammation, play critical roles in neurodevelopment and synaptic plasticity. however, the mechanisms driving these roles are not well understood. to start elucidating the molecular players involved in microglial communication with neurons during plasticity, we decided to first explore the role of purinergic signaling in this process. while purinergic signaling has been implicated in microglial behaviors, studies have primarily focused on neuroinflammatory roles. however, noninflamed microglia highly express the purinergic receptor, p2y12, which has known functions in microglial chemotaxis in early inflammation and can stimulate the release of cytokines implicated in synaptic plasticity. thus, we posited that purinergic signaling could contribute to the microglial motility that underlies synapse surveillance in the non-inflamed brain. this in turn may be critical for microglial roles in synaptic refinement during development. to explore this possibility, we took advantage of the p2y12 knock-out (ko) mouse and examined the morphology and motility of microglia in the visual cortex as compared to microglia in wildtype mice. first, we used immunohistochemistry for the microglia specific ionized calcium-binding adapter (iba-1) protein to stain microglia in fixed sections. we then used confocal imaging and scholl analysis to investigate the complexity of the microglial processes. we also used 2-photon microscopy to monitor microglial motility in p2y12 ko mice by crossing them with cx3cr1-gfp knock-in mice that allow visualization of microglia including fine processes in vivo. our preliminary evidence suggests that p2y12 disruption alters basal microglial morphology and behavior making microglia less complex and altering their motility patterns. we are now investigating whether microglia-synapse interactions, as well as functional plasticity elicited by visual experience are altered in p2y12 ko mice. our studies will expand our understanding of emerging roles for microglia in neurodevelopment and will pioneer new non-pathological roles for microglial purinergic signaling. a. dvorzhak, a. wojtowicz, r. grantyn charit e -university medicine, berlin, germany in neurodegenerative diseases, the afflicted brain is both an important object of study and an opportunity to characterize a given cellular interaction from a pathophysiological perspective. this dual approach is particularly advantageous when human disease is based on a monogenetic defect and an appropriate animal model becomes available for detailed investigation, as in case of z_q175_ki (q175), a new knock-in mouse expressing a mutant form of murine huntingtin. electrophysiological recordings of gabaergic unitary ipscs from striatal output neurons (sons) in sagittal slices from wild-type and homozygous q175 challenged the current viewpoint that gabaergic transmission is enhanced in the hd striatum. quantal analysis in combination with high frequency stimulation and paired pulse tests revealed that synaptic gaba release is in fact tonically suppressed resulting in disinhibition of striatal output activity (dvorzhak et al j physiol 2013). the underlying mechanism involves a retrograde endocannabinoid signalling pathway linking postsynaptic mglur5 with presynaptic cb1 and gaba release. in addition to this deficit, z-q175_ki homozygotes exhibited a significant reduction of tonic inhibition via extrasynaptic gaba(a) receptors. using pharmacological tools to alter the ratio of ambient glutamate and gaba concentrations made clear that the hd-related depression of synaptic and extrasynaptic gabaergic actions depends on the state of both the astrocytic glutamate transporter glt-1 and the gaba transporter gat-3. in support of h eja and colleagues, our imaging experiments suggest that in normal striatal astrocytes gat-3 is in a releasing mode and driven by the activity of glt-1. however, the latter activity is much lower in the hd striatum. sbfi recordings of glt-1-related na transients and patch clamp recordings of glt-1 transporter currents revealed a marked reduction (less than 50% of wt level) in sr-labelled astrocytes from symptomatic hd mice. together, our results suggest that the deficiency of astrocytic glutamate uptake might represent a pathophysiological key mechanism underlying the observed disinhibition in the striatum of mice affected by huntington's disease. nodes of ranvier are highly enriched in voltage-gated sodium channels (nav), allowing rapid action potential propagation on myelinated axons. cell adhesion molecules (neurofascin-186 (nfasc186) and contactin) and scaffold proteins (ankyring and bivspectrin), which are also enriched at the nodes, have a critical role in assembly and/or stabilization of na v clusters. oligodendrocytes have been described to induce nav channel clustering at nodes in the central nervous system (cns); however, the nature of the signal(s) involved in nodal formation remains mostly unknown in the cns. to gain insight into cns node assembly, we developed hippocampal neuronal cultures from e18 rat embryos. during the first week, as expected, na v channel accumulation was detected at the axon initial segment (ais), co-localized with ankyring and nfasc186, whereas expression along the axon was diffuse and hardly visible. in contrast, at 14 days in vitro (div), regularly spaced clusters of na v channels, ankyring and nfasc186 were detected along the axon. the percentage of hippocampal axons with clusters increased from 4.1 6 3.1% at 14 div, to 15.8 6 3.5% and 17.167.1% at 17 and 21 div, respectively (mean 6 sd). importantly, these clusters were formed in the absence of myelin and no accumulation of the paranodal protein caspr was detected at this stage. to study the role of extrinsic versus intrinsic pro-aggregating factors, we analyzed nearly pure hippocampal neuronal cultures. these purified neurons still formed ais, while only few axons formed na v clusters. interestingly, when these purified neurons were co-cultured with oligodendrocytes or with conditioned medium from oligodendrocytes, the percentage of neurons with nascent nodes was greatly enhanced. taken together, these results confirm that some glial soluble factor(s) might be involved in na v channels clustering, as previously shown for retinal neurons (kaplan et al., 2001). furthermore, nascent nodes were detected on gabaergic neurons, but not on glutamatergic hippocampal neurons, thus arguing also for the role of intrinsic neuronal factors in their establishment. using hippocampal sections from gad67-gfp mice, we showed that some gabaergic interneurons were myelinated in vivo. moreover, we observed that nascent node formation prior to myelination also takes place in vivo, strengthening physiological relevance. the electrophysiological properties of neurons with nascent nodes will be studied. microglia in the degenerating or aging brain are primed by aspects of pathology to produce exaggerated pro-inflammatory responses to subsequent central and systemic inflammatory insults. however, the mechanisms by which microglia become primed, rather than adopting an m1 phenotype, are not clear. triggers for priming may include altered expression of complement, recognition of amyloid or neuronal/synaptic debris for phagocytosis, decreased engagement of microglial regulatory molecules such as cd200r, trem2 or fractalkine receptor. there is also evidence that loss of basal neurotransmitter tone may contribute to this state. microglia are known to express the nicotinic cholinergic a 7 receptor, which has been shown to influence macrophage and microglial reactivity. in the current study we performed limited lesions of the basal forebrain cholinergic system using the murine-p75-saporin immunotoxin (mu-p75-sap), to investigate the degree to which loss of cholinergic tone predisposes the microglia to subsequent inflammatory stimulation and to assess the consequences of this for cognitive function in the vulnerable brain. intracerebroventricular injection of mu-p75-sap (0.08 lg) depleted cholinergic neurons in the basal forebrain and decreased cholinergic innervation of the hippocampus, but left performance on hippocampal-dependent reference and working memory tasks relatively intact. however, decreased cholinergic innervation of the hippocampus conferred increased susceptibility to cognitive deficits induced by systemic lps (100 lg/kg) 40 days after lesioning, but microglia were not primed 40 days after 20% lesions. to investigate the regional, temporal and neurochemical basis of this we induced more severe lesions of the basal forebrain (using 1.2 lg mu-p-75 sap) and assessed microglial priming at 14 or 40 days post-lesion. these data demonstrate that microglia are primed by low dose challenge at 14 but not 40 days and, when more robust lesions are induced, priming remains even at 40 days. these responses are observed at the site of injury (medial septum) but more profoundly at the site of denervation, the hippocampus. indicating that neuronal injury and, particularly, loss of cholinergic tone have a profound effect on the reactivity of the microglia to subsequent inflammatory challenge. these data expand our knowledge of microglial priming and have clear implications for the interaction of cholinergic tone and inflammation in cognitive dysfunction such as dementia and delirium in which cholinergic dysfunction is implicated. ). however, glucose may also be metabolized to glycogen or oxidized to co 2 , so it is not obvious that the stimulation of glut1 and hexokinase leads to lactate release. the objective of this work was to investigate whether lactate may be released by cultured mouse astrocytes within the time frame of the interstitial lactate rise observed in vivo. a first approach with an enzymatic assay showed a significant increase in extracellular lactate after 1 minute of exposure to 12 mm k 1 or 50 lm glutamate. to obtain better time resolution we developed a "lactate sniffer", a hek293 cell that expresses the fret lactate nanosensor laconic (san mart ın et al., plos one 2013). seeded on top of an astrocytic monolayer, the sniffer cell responded within 10 seconds of exposure to elevated k 1 12 mm and a similar response was elicited by 3 mm ba 21 . the response to k 1 and ba 21 supports a role for the na 1 /bicarbonate co-transporter nbce1, previously shown to mediate the stimulation of astrocytic glycolysis (ruminot et al., j. neurosci., 2011). the sniffer cell detected an equally fast increase in extracellular lactate when the culture was exposed to 50 lm glutamate. previous work had shown that glutamate does not stimulate glucose consumption in the short-term (bittner et al., j. neurosci. 2011), and therefore the rapid release of lactate came as a surprise. this phenomenon may relate to glycogen mobilization or perhaps to inhibition of astrocytic oxygen consumption (azarias et al., j. neurosci. 2011). k 1 , ba 21 and glutamate did not produce detectable effects on the sniffer cell in the absence of astrocytes. the rapid release of lactate by astrocytes exposed to k 1 and glutamate supports a role for these cells in the fast increase in interstitial lactate concentration that accompanies synaptic activity. ] i ) are regularly occurring as a result of uptake of glutamate from the synaptic space. glutamate uptake occurs via the glutamate/na 1 co-transporters glast and glt-1, where one glutamate is accompanied by 3 na 1 and 1 h 1 in exchange for 1 k 1 . the salt pump, na,k-atpase (nka), is responsible for the maintenance of the trans-membrane na 1 gradient by exporting 3 na 1 and importing 2 k 1 for each atp. nka is dose-dependently inhibited by ouabain. astrocytes express two isoforms of the catalytic nka a subunit, the ubiquitous a1 and in the cns the glia-specific a2. the isoforms differ with regard to na 1 affinity, which is lower for a2 and with regard to ouabain affinity, which is higher for a2 than for a1. although a2 mutations give rise to neurological symptoms -familial hemiplegic migraine type 2, the relative roles of the a1 and a2 isoforms in astrocytes are still incompletely understood. we hypothesized that the low na affinity of a2 makes it more suitable to cope with transient large increases in na i . to test this, we compared the effect of 200 mm glutamate for 10 min on real time changes of [na 1 ] i in primary astrocytes, which, following transient transfection, predominantly expressed either the a1 or the a2 isoform. in a1 cells glutamate caused a significantly larger increase in [na 1 ] i and longer recovery time to base line [na 1 ] i than in cells expressing a2. glutamate uptake dependence on either a isoform was determined by aspartate uptake in astrocytes expressing endogenous a1 and a2. in the presence of ouabain concentrations that selectively inhibit a2, we found a modest (1.9mm) increase in [na 1 ] i , accompanied by a disproportionately large decrease (24%) in glutamate uptake. it has been suggested that astrocyte glutamate transporters are clustered in microdomains where they may interact with nka. in gst pull down assays on rat brain we found that both glt-1 and glast interacted with the 1st intracellular loop of both a1 and a2, but the interaction was significantly stronger for the a2 isoform. no interaction was found with other segments of the a molecule. the data indicate that the nka a2 isoform, which has a more restricted expression than a1, plays a specific role for the interaction with the glutamate transporters and for sodium homeostasis in astrocytes. this specificity may be attributed to low sodium affinity of a2 and to the relatively high capacity of a2 to interact with the astrocyte glutamate transporters. ozone, a major component of air pollution, has considerable impact on public health. besides its well described inflammatory and dysfunction effects on the respiratory tract, there is accumulating evidence indicating that ozone exposure also affects brain functions. however, the mechanisms through which ozone exerts toxic effects on the cns remain poorly understood. we previously showed that in addition to lung inflammation,ozone exposure caused a neuronal activation in the dorsolateral regions of the rat nucleus tractus solitarius (nts, a sensory nucleus involved in visceral information processing) overlapping terminal fields of lung primary afferent running in the vagus nerves to investigate this hypothesis, we used electron microscopy and immunoblot techniques. in ozone-exposed animals, the astrocytic coverage of nts glutamatergic synapses is increased while the astrocyte volume fraction and the astrocyte membrane densities are not significantly increased. moreover, the expression of specific astrocyte markers gfap, s100beta and ezrin did not change in ozone-exposed animals. altogether, our results indicate that ozone inhalation induces glial plasticity, which is restricted to the peri-synaptic coverage. . we recorded pp-gc synaptic activity after eae induction via adoptive transfert (at-eae) and found that mepsc frequency was increased compared to control animals. moreover synaptic alteration in at-eae mice depended on astrocytic tnfr1 because the effect was absent in tnfr1-/-mice but reappeared upon tnfr1 re-expression in astrocytes. nr2b receptors are also necessary because in vivo ifenprodil injection prevented synaptic alteration. overall, our study reveals a specific mechanism responsible for hippocampal synaptic dysfunction possibly relevant for cognitive impairment in ms. research supported by snsf grant 31003a-140999 and nccr "synapsy" to av. we patched ng2 cells in the hippocampal ca1 region of ng2-dsred mice (8-20 days old) and investigated in current clamp mode how sodium and potassium channels affected synaptic depolarizations. epsps and ipsps were evoked from a resting membrane potential of 285mv by injecting mock epsc or ipsc waveforms derived from miniature currents. epsps which depolarized ng2 cells to more than 245 mv were increasingly shortened by vgcs. at 216 6 1 mv, corresponding to a quantal content of 640, the half width of epsps was shortened to 47 6 3% while the amplitude was hardly altered. application of ttx, 4-ap and tea showed that most of the observed shaping of epsps was primarily due to activation of a-type k1 current rectifying the depolarizing input. na1 currents only slightly ($10%) amplified epsp input, but this effect was outcompeted by the dampening effect contributed by a-type current ($15%). tea was almost without effect on epsps. due to their slower time course ipsps were very differently shaped by vgcs: while recruitment of vgcs by mock ipsps started at a similar threshold voltage, the maximal suppression of ipsp amplitude was much stronger. when depolarizing ng2 cells to 235 6 1 mv, corresponding to a quantal content of 235, ipsp amplitudes were suppressed to 52 6 4% and the duration of ipsps was not shortened but increased to 193 6 5%. a-type k1 current was the primary contributor for rectifying as well. interestingly, if a-type k1 current was blocked, ipsps with a quantal content of 105 unmasked a small ttx-sensitive spikelike depolarization of 20 6 2 mv (duration: 7 6 1 ms). for comparison, while synaptic responses to simultaneous release of 100 glutamatergic vesicles were hardly altered by vgcs, the synaptic response of 100 gabaergic vesicles was substantially low-pass filtered by a-type potassium currents. altogether, our results suggest that vgcs in ng2 cells normalize the synaptic strength of excitatory and inhibitory inputs and support their differentiation by ng2 cells through further emphasizing their pre-existing kinetic differences. to explore the involvement of this pathway in pain and analyze its impact separately in sensory neurons and sgcs, we tested pharmacological inhibitors and transgenic mice in an orofacial pain model. transient inflammation in the submandibular region of mice was induced using complete freund's adjuvant (cfa) and tactile sensitivity was quantified using von frey filaments. although inflammation was resolved by 28 days after injection, tactile hypersensitivity persisted in wildtype mice for at least 53 days, consistent with chronic post-inflammatory pain. tactile hypersensitivity in mice was reversed by systemic injection of the panx1/gap junction blockers mefloquine or carbenoxolone at 7 days (peak inflammation) and at 28 days after cfa-injection, suggesting the contribution of these channels to tactile hypersensitivity. in dissociated trigeminal ganglia (tg), which innervate the submandibular skin, bzatp-induced yopro uptake into neurons and glia was prevented by mefloquine, demonstrating the functional presence of the p2x 7 r-panx1 complex in tg. moreover, tg of cfa-injected mice showed more atp release and immunostaining of tg revealed higher expression of panx1 at 1 week after cfa injection compared to controls. development of hypersensitivity was prevented in p2x 7 r-null and in panx1-null mice, and was attenuated in mice with glia-specific deletion of panx1 (gfapcre-panx1f/f) but not with neuron-specific deletion (mnfhcre-panx1f/f), emphasizing the importance of glial panx1 signaling in pain. because p2x 7 r and panx1 both have a key role in the immune response by activating the inflammasome, we are currently dissecting the relative importance of neuron-glial communication compared to inflammatory responses in the development and maintenance of orofacial pain. overall, our results show that the p2x 7 r-panx1 complex likely plays a major role in signaling events contributing to tactile hypersensitivity. synaptic plasticity is central to the process of learning and memory and is accompanied by strengthening of existing synapses and/or formation of new synapses. numerous studies have investigated the molecular mechanisms of learning and memory with neurons as the primary interest. given the tight coupling between synaptic activity and brain metabolism, it seems reasonable to assume that synaptic plasticity changes occurring at the synaptic level may also occur at the metabolic level in order to keep up with the augmented demand at the synapse. in this study we investigated the importance of genes involved in brain energy metabolism, and in particular those involved in neuronglia metabolic coupling during fear-motivated inhibitory avoidance learning. using ( 14 c) 2-deoxyglucose (2-dg) technique we first defined the brain areas that are involved in this learning process. context-dependent avoidance behavior was tested in c57bl/6 mice using the step-through inhibitory avoidance paradigm (ia). regional brain metabolic activity, as measured by the 2dg uptake, was quantified in several brain regions. brain metabolic mapping revealed increased glucose utilization in hippocampus, amygdala [(basolateral complex (bla) and the central nucleus (cea)], and anterior cingulate cortex and mammillary bodies. quantitative mrna levels were assessed in dorsal hippocampal tissue at different time points following ia learning. results obtained demonstrate that learning modulates the expression pattern of genes involved in brain energy metabolism, i.e. glycogen synthesis and degradation, pyruvate metabolism, the pentose phosphate shunt and the astrocyte-neuron lactate shuttle (anls) in a time dependent manner. we found a late-phase of enhanced dorsal hippocampal gene expression, particularly for anls related genes, including monocarboxylate transporter 1 (mct1). furthermore, we found that mice deficient in mct1 transporter display impaired long term memory in the inhibitory avoidance and spatial learning in morris water maze. these observations indicate that metabolic adaptation shown by neuron-glia metabolic coupling might be a relevant phenomenon following learning in conjunction with synaptic plasticity. in order to systematically as well as functionally analyse rmg reactivity to retinal degeneration and thus explore the rmg-derived signalling molecules in depth, we aim at comprehensively profiling proteomewide cellular responses to stimuli. we thus developed a proteomics approach screening specifically for cell surface and membrane proteins as well as secreted protein expression profiles and in addition monitor quantitative changes induced by prototype inflammatory inducer lipopolysaccharide lps. this workflow utilises sugar residues of cell surface proteins on intact rmg which are mildly oxidized and then covalently coupled to biotin. biotinylated proteins are affinity purified and glycosylated peptides are specifically released by pngasef followed by protein identification and quantification by label-free lc-msms. this workflow resulted in the identification of more than 500 proteins on cell surfaces complemented by more than 700 proteins identified in the rmg secretome. bioinformatic analysis allocates 75% of the cell surface proteins to be truly membrane or extracellular matrix proteins. this comprehensive cell surfaceome includes transmembrane receptors, transporters, adhesion molecules, signalling molecules and proteases, including 18 cd markers, 18 integrins, 41 solute carriers, two ephrins, five ephrin receptors and six plexins. treatment of cells with lps results in a highly reproducible significant shift of cell surface proteome with upregulation of 36 proteins and downregulation of 13 proteins. among those lps-induced cell surface expression changes are proteins that suggest an active role of these glial cells in inflammatory processes. the specific changes will be discussed in detail. cell surface biotinylation on glycosyl-residues in combination with label-free lc-msms is a sensitive and reproducible method to profile cell surface proteomics and sheds light on the biological properties of cells. background: neuronal activity alters calcium ion (ca 21 ) dynamics in astrocytes, but the physiologic relevance of these changes is controversial. to examine this issue further, we generated an inducible transgenic mouse model in which the expression of an inositol 1,4,5-trisphosphate absorbent, "ip 3 sponge", attenuates astrocytic ca 21 signaling. results: attenuated ca 21 activity correlated with reduced astrocytic coverage of asymmetric synapses in the hippocampal ca1 region in these animals. the decreased astrocytic 'protection' of the synapses facilitated glutamate 'spillover', which was reflected by prolonged glutamate transporter currents in stratum radiatum astrocytes and enhanced n-methyl-d-aspartate receptor currents in ca1 pyramidal neurons in response to burst stimulation. these mice also exhibited behavioral impairments in spatial reference memory and remote contextual fear memory, in which hippocampal circuits are involved. conclusions: our findings suggest that ip 3 -mediated astrocytic ca 21 signaling correlates with the formation of functional tripartite synapses in the hippocampus. they are the only non-neuronal cell type in the brain that receives synaptic input from neurons. ng2-cells exhibit a variety of ca 21 -signalling pathways, which might be triggered by pre-synaptic neuronal activity. however, no global increase in the intracellular ca 21 -concentration ([ca 21 ] i ) was detected in ng2-cells employing the minimal stimulation technique at neuron-glial synapses. therefore we assume that post-synaptic ca 21 -microdomains might be activated under physiological conditions. these ca 21 -signals are locally restricted to the plasma membrane. membrane bound ca 21 -sensors with a high signal-to-noise ratio, such as lck-gcamp3, are best suited for optimal visualisation of ca 21 -microdomains. here, we set out to express lck-gcamp3 in ng2cells together with the cytosolic reporter dye dsred. we generated transgenic mice which express a bidirectional promoter encoding both proteins under the control of a tet-off system. this strategy allows an exclusive expression of the transgenes only in the presence of a tet-responsive transcriptional activator (tta). the construct was successfully cloned and expressed in hek293 cells. as expected, lck-gcamp3 was located only at the inner plasma membrane while dsred was expressed all-over the cytosol. functionality of the ca 21 -sensor was tested in vitro. elevation of [ca 21 ] i reliably increased the fluorescence intensity of lck-gcamp3. after zygote injection seven positive founder animals could be identified and will be crossbred with a ng2-tta mouse line. taken together, the presented construct appears suitable for functional imaging of ca 21 -microdomains in post-synaptic ng2-cells. according to the "lactate shuttle" hypothesis, glycogen stored in astrocytes can be converted to l-lactate and exported to neurones as preferred energy substrate. we use optogenetics to investigate signalling between astrocytes and noradrenergic (naergic) neurones of the locus coeruleus (lc). to selectively activate g s -protein-mediated signalling in astrocytes, we generated a viral vector to express a chimera of rhodopsin and b 2 -adrenoceptor (optob 2 ar). we confirmed that this construct activates camp-mediated signalling in astrocytes. in cultured astrocytes, excitation of optob2ar resulted in acidification as detected using snarf-5 indicator, and this acidification could be blocked by pre-incubation with an inhibitor of glycogen breakdown 1,4-dideoxy-1,4-imino-d-arabinitol (dab), suggesting that the acidification was due to build-up of l-lactate. fast scan cyclic voltammetry (fcv) was used to measure noradrenaline (na) release in organotypic slices cut at the level of the lc in which astrocytes were expressing optob 2 ar. light stimulation of astrocytes powerfully induced release of na which could be prevented by pre-incubation with dab or application of d-lactate. bath application of l-lactate (!100 mm) in absence of optogenetic stimulation triggered the release of na. in patch clamp experiments, l-lactate depolarised lc neurones and evoked vigorous firing of action potentials. these experiments suggest that activation of g s -protein-coupled receptors in astrocytes could potentiate the release of na from lc neurones via l-lactate. the cellular and molecular mechanisms of this signalling mechanism are currently under investigation. supported by the british heart foundation. rgcs. in addition to this, the organization of astrocytes in the retina of a rat model of glaucoma was found to be significantly different to those of a healthy retina. these alterations in glial cell morphology may reflect changes in their relationship with rgcs and could signify profound changes in their secretome, which may influence rgc survival. in this study we investigated labeling of astrocytes by sr101 in acute slices from the ventro-lateral medulla and the hippocampus of transgenic mice expressing egfp under the control of an astrocyte-specific promoter. while sr101 efficiently labeled egfp-expressing astrocytes in hippocampus, we found that sr101-staining was very weak in the ventro-lateral medulla and rather unspecific. thus, sr101 is not a reliable marker for brainstem astrocytes. although carbenoxolone decreased the labeling of astrocytes in the hippocampus significantly, mefloquine which blocks pannexin and connexin hemichannels, was unable to prevent sr101 uptake in hippocampal astrocytes. time-lapse 2-photon imaging revealed that in hippocampus both astrocytes and neurons showed temporary sr101-loading. in astrocytes the rise of sr101 fluorescence was slower as compared to egfp-negative cells and sr101 was quickly removed from non-astrocytic cells during washout, while it was retained in astrocytes. in brainstem astrocytes, however, only a very weak and transient sr101-labeling was observed. to test if sr101 is actively removed from astrocytes in the brainstem, we applied mk-571 to block the multi-drug resistance transporters mrp-1. we did not observe any increase of sr101-labeling in brainstem astrocytes. in contrast, astrocytic sr101 labeling was significantly reduced by substrates of organic anion transport, probenecid, estron-3sulfate and dehydroepiandrosterone sulfate suggesting that sr101 is actively transported into hippocampal astrocytes by an organic anion transporting polypeptide (oatp). additionally, the data suggest that astrocytes modulate extracellular concentration of neurosteroids in the hippocampus. as an alternative approach we have used scanning electron microscopy (sem) to obtain high-resolution back scattered images (bsi) of serial ultrathin sections over a wide-area. in tem the size of the specimen is limited to less than 1 mm 2 , whereas in sem it can be expanded to 20-30 mm 2 , allowing us to mount and examine more than 100 serial ultrathin sections on the same stage, collecting 3d information about many nodal structures simultaneously. here we demonstrate a variety of nodal architectures assembled from bsi-sem (su8010,hitachi). in our preliminary study using this technique, the pattern of cellular processes approaching the perinodal axolemma is quite different among axons even in only four nodes examined in optic nerve. in the present study, we analyzed perinodal elements at 25 whole nodes in rat optic nerves and reconfirmed this variety of perinodal glial elements include the followings: 1) perinodal space with extracellular matrix (20-100%); 2) closely abutting astrocytic processes (0-80%) ; 3) unidentified glial cell, presumable ng21 glial processes (0-50%), but could not find 4) pre-synapse-like pseudo-neuronal processes, which was observed at the perinode in the cns grey matter (cerebral cortex) in the previous study. additionally, we found unexpected structures, small teardrop-like protrusion(s) of axolemma at 18 out of 25 nodes examined (72%). in 16 nodes out of these 18 nodes (88.9%), glial elements encircled or densely contacted with the teardrop protrusions. a typical case of 3d reconstructed image is shown in the figure. nodal axon (ax, red), unidentified glial process (yellow; ug, presumable ng2 cell) and teardrop-like protrusion (asterisks) are demonstrated. outer part of the protrusion from the position indicated two arrows is encircled by the process of ug cell. based on these observations, we propose that some glial cells, including astrocytes and/or ng2 cells, might contribute to remove wasted membranous elements from the axon at the node of ranvier, which is a new type of neuron-glial interaction. to understand what makes this ssc network more synchronous and excitable, we performed a morphological study of neurons and astrocytes by estimating densities of neun and s100-positive cells respectively. although we found similar densities of neurons and astrocytes between gaers and non-epileptic controls, the thickness of the ssc was 30% smaller in gaers. western blotting quantification of gfap, another astrocytic marker, was significantly higher at p15-p17, as compared with non-epileptic control. moreover, gfap-immunolabeling suggested that these astrocytes were reactive. we hypothesized that these modifications are linked with an alteration of astrocytic and/or neuronal physiological calcium excitability that could lead to the occurrence of epileptic seizures. to better evaluate the role of astrocytes and neurons networks during the development of ssc between p15 and p25, we used two-photon microscopy imaging coupled with eeg recording in animal under anesthesia and neuroleptanalgesia. we first analyzed neuropile, representing ascendant projection of deeper cortical layers, astrocytes and neurons calcium activities. preliminary results obtained from animals under isoflurane anesthesia showed similar neuropile activities. astrocytic and neuronal activities were too weak to highlight differences between gaers and non-epileptic control. this is likely due to the mode of anesthesia known to strongly decrease astrocyte calcium signaling and neuronal synchronization. current experiments are therefore performed using neuroleptanalgesia know to allow the occurrence of swd. the expected data should lead to a better understanding of neuron-astrocyte interactions during brain development and the mechanism underlying epileptogenesis in a genetic model of epilepsy. purpose: to study the effects of laser-induced ocular hypertension (oht) in the astrocytes of contralateral eyes two weeks after lasering. methods: adult swiss mice were divided into two groups: na€ ıve (n 5 6) and lasered (n 5 6). retinal whole-mounts were immunostained with antibodies against gfap. the gfap-labelled retinal area (gfap-ra) and the number of astrocytes were quantified. results: in comparison with na€ ıve: i) astrocytes were more robust in contralateral eyes. in oht-eyes, the astrocyte population was not homogeneous, given that astrocytes displaying only primary processes coexisted with astrocytes in which primary and secondary processes could be recognized, the former having less intense gfap-ir (p<0.001). the mean percentage of astrocytes in which only primary processes could be detected was 37.8%; ii) gfap-ra was increased in contralateral (p<0.05) and decreased in oht-eyes (p <0.001); iii) the mean intensity of gfap-ir was higher in oht-eyes (p<0.01), and the percentage of the retinal area occupied by gfap1 cells with higher intensity levels was increased in contralateral (p 5 0.05) and in oht-eyes (p<0.01); iv) the astrocyte number did not differ significantly among the eyes analyzed. however, in oht-eyes the number of astrocytes in which primary and secondary processes could be observed were decreased (p<0.01). conclusions: two weeks of laser-induced oht caused changes in the gfap-labelled retinal area but not in astrocyte number in both, contralateral and oht-eyes. on the basis of the astroglial changes detected in the present work, the use of the contralateral eye as an internal control in experimental model unilateral oht should be reconsidered. astrocytes show a high level of functional and morphological heterogeneity and are involved in many aspects of neural function, e.g., formation of the blood-brain barrier, regulation of ion homeostasis, synaptogenesis, and synaptic plasticity. despite the diversity of this cell type, the various astrocyte subpopulations are not well characterized, which is mainly due to the lack of markers that would allow for classification of astrocyte subsets. our study aimed at phenotyping of astrocyte subpopulations based on the expression of cell surface markers, which are associated with certain functions. we used two novel monoclonal antibodies, acsa-1 and acsa-2 (acsa: astrocyte cell surface antigen), directed against extracellular epitopes of astrocyte-specific cell surface markers to identify astrocyte subtypes. the acsa-1 antibody was generated by immunization of glast1 knockout mice and specifically detects the astrocyte-specific l-glutamate/l-aspartate transporter glast (eaat1, slc1a3), whereas the acsa-2 antibody results from an immunization of rats with astrocytes isolated from gfap-egfp transgenic mice. a mass spectrometric approach for the ligand-based identification of the acsa-2 antigen pointed to a number of candidates, which have to be validated by further experiments. the antibodies acsa-1 and acsa-2 were carefully analyzed by costaining experiments with commonly used intracellular astrocyte markers. we found that both antibodies specifically detect astrocytes in the developing and adult central nervous system. in contrast to antibodies against intracellular markers, such as gfap, s100ß, or glutamine synthetase, acsa-1 and acsa-2 can be used to detect and isolate living astrocytes, which enables further analysis and culture of the separated cells. flow cytometric analysis of acsa-1 and acsa-2 antigen expression on cells from different brain regions, as well as immunohistochemical analysis, revealed differences in the expression patterns. this allowed us to define different astrocyte subtypes especially in the cerebellum and olfactory bulb of the neonatal mouse brain. future work will focus on the identification of additional astrocytespecific cell surface proteins for a comprehensive classification of astrocyte subtypes based on cell surface marker expression or expression patterns. the reciprocal communication between neuronal and glial cells represents the key component of the immunosurveillance system of the brain. it has been proposed that the neuro-glial interaction is impaired in human alzheimer's disease. in this study we focus on neuronal "on" and "off" signalling molecules in the transgenic rat model for alzheimer's disease. using enzyme-linked immunosorbent assay we determined the level of cd47, cx3cl1 ("off" signalling molecules) and mmp3, trem2 ("on" signalling molecules) in brain homogenates. our results demonstrated significantly up-regulated level of cd47 (p < 0,001) in our ad rat transgenic animal model. on the other hand, quantification of mmp3 level revealed significant decrease of mmp3 expression (p < 0,001) in tested transgenic animals. previous studies showed that cd47 and mmp3 are predominantly expressed on neuronal cells in cns where cd47 functions normally as a marker of "self" to protect intact body component or "don't eat me" molecule which protect cd47-expresing cells from phagocytosis. our results indicate that overexpression of cd47 on neuronal cells expressing pathologically modified truncated tau protein can be used as a neuroprotective mechanism potentiating spread and accumulation of pathological modified tau protein in neurons affected by ad pathology which in final stage support progression of the developing disease. in conclusion, we showed, that pathologically modified tau protein can modulate specific "on and off" signalling molecules and thus affects neuron-glia interaction in ad. field-excitatory post-synaptic potentials (fepsp) were recorded from the ca1 area of hippocampal slices prepared from wistar rats (3-5 weeks old) as before (fontinha et al., 2008) . after obtaining a stable value of the fepsp slope for at least 30 min, ltp was induced by delivery of 3 trains of 3 pulses at 100hz, each train being separated by 200 ms. ltp magnitude (% increase of fepsp) was evaluated 50-60 min after induction. ltp magnitude in control conditions was 16 6 5.9%, whereas in a second independent pathway of the same slices but in the presence of bdnf (20ng/ml) it was 39 6 4.7% (n 5 7; p <0.05). ltp was completely abolished when hippocampal slices were superfused with the gliotoxin fluorocitrate (fc, 200 mm), which selectively reduces the astrocytic metabolism decreasing intracellular ca 21 signalling and the release of gliotransmitters. interestingly, in presence of fc, the facilitatory action bdnf (20 ng/ml) upon ltp was completely prevented. noteworthy, when fc (200 mm) treated slices were superfused with the selective adenosine a 2a receptor agonist, cgs21680 (30nm), the facilitatory action of bdnf (20 ng/ml) upon ltp was rescued (n 5 5; p < 0.05), so that dltp caused by bdnf (ltp in the presence of bdnf -ltp in the absence of bdnf in two independent pathways of the same slices) in fc 1 cgs 21680 treated slices (19%) was similar to that observed in control slices (23% in the absence of fc and cgs 21680). the results suggest that the facilitatory action of bdnf upon ltp is controlled by astrocytes, and that this role of astrocytes results from their contribution to the extracellular accumulation of adenosine allowing a 2a receptor activation to gate plasticity actions of bdnf. several studies demonstrated the ability of astrocytes to sense, respond to and regulate neuronal function. among the many functions of glial proteins, glutamate (glu) and gaba transporters play important roles in balancing excitatory and inhibitory signals in the brain. here we show that astrocytes regulate the tonic inhibition of neurons by the concerted action of glu and gaba transporters, thereby protecting both neurons and glial cells from overactivation. we demonstrate that the uptake of glutamate triggers the reverse function of glial gat-2/3 transporters by elevating the intracellular na 1 concentration in astrocytes. the released gaba significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. we also describe the source of the releasing gaba that is synthesized by an alternative pathway from polyamines. moreover, in the low-[mg 21 ] model of epilepsy, we show that blockade of the glial glu/gaba exchange mechanism increases the duration of seizure-like events and also results in increased activity of astrocytes, demonstrating the neuroprotective impact of the mechanism. finally, we show that the released glial gaba modulates the power of gamma range oscillation in vivo, suggesting that the glu/gaba exchange mechanism is also functioning in the intact hippocampus under physiological conditions. revealing this novel molecular mechanism by which astrocytes provide an adjustable, in situ negative feedback on the excitability of neurons is expected to broaden our understanding about the regulation of neuronal activity by astrocytes and may open up new targets for the treatments of pathological conditions, such as epilepsy or ischemia. chronic stress is well recognised to decrease the number of gfap1 astrocytes within the prefrontal cortex (pfc). recent research, however, has suggested that our understanding of how stress alters astrocytes may be incomplete. specifically, chronic stress has been shown to induce a unique form of microglial remodelling, but it is not yet clear whether astrocytes also undergo similar structural modifications. such alterations may be significant given the role of astrocytes in modulating synaptic function. accordingly, in the current study we have examined changes in astrocyte morphology following exposure to chronic stress in adult rats, using three-dimensional digital reconstructions of astrocytes. our analysis indicated that chronic stress produced profound atrophy of astrocyte process length, branching and volume. we additionally examined changes in astrocyte-specific s100b, which is both a putative astrocyte marker, and a protein whose expression is associated with astrocyte distress. while we found that s100b levels were increased by stress, this increase was not correlated with atrophy. we further established that while chronic stress was associated with a decrease in astrocyte numbers when gfap labelling was used as a marker, we could find no evidence of a decrease in the total number of cells, based on nissl staining, or in the number of s100b1 cells. this finding suggests that chronic stress may not actually reduce astrocyte numbers and may instead selectively decrease gfap expression. together, these results provide a significantly more elaborate picture of how chronic stress alters the pfc. the drosophila nervous system is ensheathed by several types of glial cells, one of which, namely the subperineurial glia, forms pleated septate junctions (psj) to build a tight blood-brain barrier (bbb). hence, nutrients from the surrounding hemolymph must cross these glial cells to reach the neurons. the amount of nutrients entering the nervous system is likely to be tightly controlled through neuron-glia communication to ensure optimal metabolic supply while avoiding disturbance of the extracellular homeostasis. we aim to elucidate signals involved in this regulatory interaction. to this end, an rnai-based screen in glia of adult flies is performed using the gal4/gal80 ts system. we analyze the intake of blue-dyed food by photometric measurement. since impairment of the metabolic supply of the brain should severely affect the organism, compensatory changes in feeding behavior are expected. for instance, knockdown of nutrient transporters specifically in glia would then induce excessive feeding in normally nurtured individuals. the first set of rnai lines screened includes genes that have previously been found to be essential in glia for survival or locomotion of the animal. furthermore it comprises metabolic enzymes, all carbohydrate transporters, neuropeptides and their corresponding receptors. candidate genes will be further characterized regarding their role in controlling the energy homeostasis of the nervous system. the discovery that activation of non-neuronal cns microglia plays a causal role in spinal processing of nociceptive signaling has shed new light on the processes underlying neuropathic pain facilitation. however, there remains much uncertainty as to the necessary contribution of microglia to enhanced pain states. we aim to define the particular role of microglia for the initiation of neuropathic pain and by answering this question also learn if the function of peripheral myeloid cells is distinct or redundant in this process. methods: to specifically investigate spinal microglia and peripheral macrophages in the pathogenesis of neuropathic pain, we model chronic pain by performing partial ligature of the sciatic nerve in cd11b-hsvtk mice engrafted with gfp bone marrow (gfp>cd11b-hsvtk). cd11b-hsvtk 1/mice allow the exchange with peripherally-derived, gfp 1 macrophages upon central depletion of endogenous cd11b 1 microglia. following this depletion/ repopulation paradigm, behavioral analyses of mechanical and thermal allodynia are conducted. results: we established a selective tool to exchange cns parenchymal microglia with peripheral gfp 1 myeloid cells. in chronic pain tests for mechanical and thermal hyperalgesia, gfp>cd11b-hsvtk 1/mice show considerable decreases in paw withdrawal thresholds in response to mechanical, but not thermal stimuli ipsilateral to the injury, indicating distinct roles of microglia and macrophages in the facilitation of thermal hyperalgesia. conclusions: we identified a differential contribution of resident spinal microglia vs. peripheral myeloid cells in the development of neuropathic pain in gfp>cd11b-hsvtk chimeras. future studies aim to examine the exact mechanisms underlying the distinction between these two populations. a deeper understanding of the processes involved in the compartmentation of brain energy metabolism is of major interest. not only to enwrap pathomechanisms of brain diseases associated with metabolic deficits but also for a more accurate interpretation of functional neuroimaging signals which often use metabolic signals as surrogate marker for brain activity (e.g. fdg pet). large amount of knowledge in the field has been obtained from small animal neuroimaging studies. but due to their invasiveness they often need anesthesia. anesthetics heavily alter physiological read-outs. the aim of this study was 1.) to test if metabolic imaging using two-photon microscopy (2pm) and genetically encoded glucose sensors is feasible in the awake, head-fixed mouse and 2.) to examine the effects of isoflurane anesthesia on the signals. in two mice, a recombinant adeno-associated virus as a carrier for the genetically encoded glucose sensor flii 12 pglu600md6 was injected into the whisker barrel cortex. by using an astrocyte-specific promoter (short gfap) astrocytic expression was ensured. in addition, a chronic window and a head post were implanted. behavioral training started a few days after surgery. animals had to learn to tolerate head fixation without spontaneous motor activity. within a single trial animals were trained not to move for 10 seconds (imaging period) to receive a water reward. animals were subjected to water restriction during the behavioral training. animals were trained for three weeks before 2pm imaging started. animals learned to tolerate head fixation up to 90 minutes allowing acquisition of up to 400 trials per session. upon oral glucose administration (per single water reward $1 mg glucose was administered) fret signal started to increase within 5 minutes and went up to a maximum of 8%. surprisingly, isoflurane anesthesia led to an even higher increase of the signal (mean increase of 12%) compared to the awake state. in some experiments the barrel cortex was activated by vibrotactile single whisker deflections generated by a piezo bending actuator. fret signal did not show a difference between baseline and stimulated condition (1.133 6 0.3 vs. 1.136 6 0.29). in conclusion, the presented experiments demonstrate for the first time the feasibility of awake 2pm imaging for metabolic measurements on a single cell level. the signal increase upon peroral glucose administration unambiguously supports the functionality of the sensor. isoflurane exhibits a large effect on intracellular astrocytic glucose concentration distinctly revealing the need for experiments in awake animals for unbiased results. future experiments will now be expanded to neuronal glucose sensors to enable comparisons between astrocytes and neurons. our previous studies indicate that neuronal electrical activity controls glial exosome release resulting in subsequent neuronal uptake and functional retrieval of the exosomal content. proteomic analysis of oligodendroglial exosomes revealed a list of candidates with potential neurotrophic action in neurons. to elucidate the impact of oligodendroglial exosomes on the neuronal metabolism, we analyzed the metabolic activity of neurons after co-culture with oligodendrocytes or direct treatment with exosomes. methods: neurons were subjected to different stress paradigms such as oxidative stress, hypoxia, and nutrient deprivation. neuronal vitality was assessed by mtt assay and the mitochondrial membrane potential was visualized by staining with mitocapture. results: neurons grown under optimal conditions were not affected by the presence of exosomes. intriguingly, when neurons were subjected to stress (oxidative stress, nutrient deprivation, oxygen-glucose deprivation) their metabolic activity was significantly increased in the presence of exosomes. when challenged with oxidative stress prior to exosome treatment, neurons were not able to recover. mitocapture staining demonstrated that oligodendroglial exosomes prevent the breakdown of the mitochondrial membrane in nutrient-deprived neurons. conclusions: our results indicate that exosome supply is protective for neurons but is unlikely to support their recovery. we suggest that oligodendroglial exosomes carry neuroprotective substances, which protect neurons from stress. interestingly, this effect of lactate on synaptic plasticity mechanisms was not fully mimicked by glucose, suggesting that the effect was not only related to supporting the potential increase in energy demands related to plasticity, but that l-lactate could act as a signaling molecule for the regulation of expression of plasticity-related genes. we have followed up on these observations and show that l-lactate significantly stimulates mrna expression of key immediate early genes (iegs), in a time-and concentration-dependent manner, in cultured neurons. following one hour of treatment with 20 mm l-lactate, arc, zif268 and c-fos mrna levels were increased by 5.2, 3.7 and 8.2 folds, respectively. in addition, the increased iegs mrna expression levels induced by l-lactate are correlated at the protein level with respectively 5.5, 4.0 and 3.2 fold increase compared to control values at the same time point. these effects are specific for llactate, since d-lactate (non-metabolized enantiomer of l-lactate), l-pyruvate and d-glucose (at equicaloric concentrations) have no effect on gene expression. interestingly, we observed that, in similar culture conditions, l-lactate-induced iegs expression is only observed in neurons but not in astrocytes, implying a cell specific effect. characterization of the underlying molecular mechanisms of l-lactate action on iegs expression demonstrate the involvement of nmda receptors. finally, we obtained evidence that such regulatory mechanisms of ieg expression also operate in vivo as direct intracortical injections of l-lactate (10 mm) into the somatosensory-motor cortex areas resulted, within 1 hour of application, in a significant stimulation of arc, zif268 and c-fos expression (by 61 6 13%, 46 6 8% and 60 6 12%, respectively) as compared to d-lactate (10 mm)-injected contralaterally areas. taken together these findings demonstrate that l-lactate acts as a direct signaling molecule which regulates neuronal plasticity-related iegs expression both in vitro and in vivo. interestingly, the underlying mechanism of action of l-lactate involves nmda receptors.this set of observations therefore reveals a novel signaling role of lactate which may represent a likely mechanism to account for the role of astrocyticderived l-lactate on ltm formation. neuronal activity in the brain is associated with a transient increase in the extracellular k 1 concentration. the excess k 1 is removed from the extracellular space, primarily by the surrounding glial cells, leading to an intracellular accumulation of k 1 via mechanisms not fully identified and/or quantified. post-stimulus recovery of [k 1 ] o has been proposed to be dependent on kir4.1-mediated spatial buffering and/or to na 1 /k 1 -atpase activity. to resolve the molecular mechanisms involved in k 1 clearance, we initially determined the contribution from the different k 1 -transporting mechanisms present in primary culture of rat astrocytes and their k 0.5 for k 1 . the na 1 /k 1 /2clcotransporter, nkcc1, increased its activity within a physiological concentration range of k 1 , while the na 1 / k 1 -atpase saturated at much lower concentrations. consequently, a concentration-dependent switch occurs between the two mechanisms of k 1 uptake in cultured astrocytes. in addition, nkcc1 was capable of mediating robust k 1 -induced astrocytic cell swelling. thus, nkcc1 could potentially act as a molecular mechanism responsible for clearance of the stimulus-evoked rise in [k 1 ] o and the associated shrinkage of the extracellular space. to determine the contribution of each of the three molecular mechanisms to k 1 -clearance in native brain tissue, we used rat hippocampal brain slices and employed ion-sensitive microelectrodes in association with high-frequency electrical stimulation as well as focal appliances of k 1 by ionophoresis. inhibition of kir4.1 (100 um bacl 2 ) or nkcc1 (10 um bumetanide) failed to show a significant effect on the rate of k 1 removal from the extracellular space. in contrast, inhibition of the a2 and a3 isoforms of the na 1 /k 1 -atpase significantly delayed post-stimulus recovery of [k 1 ] o and this delay was further potentiated by additional inhibition of the a1 isoform. the na 1 /k 1 atpase emerged as the primary factor responsible for stimulus-evoked k 1 -clearance with no evidence in favor of kir4.1 and nkcc1 involvement. further characterization of the different na 1 /k 1 -atpase isoforms, by heterologous expression in xenopus laevis oocytes, revealed that the a1 isoform reached its maximal activity already at resting k 1 concentrations, hinting at a "housekeeping" function. the a2 subunit displayed voltage-sensitivity and increased turnover rate along physiologically relevant increments in extracellular k 1 concentration. these a2-related features may render this astrocyte-specific subunit variant specifically geared for post-stimulus recovery of [ as a model of ischemic injury, and sham-operated mice were used as controls. we isolated gfp 1 cells from the dorsal part of the lv of sham-operated-and post-ischemic brains and employed a neurosphereforming assay in order to estimate their ability to proliferate and selfrenew. furthermore, we followed their immunocytochemical and electrophysiological properties during in vitro differentiation and compared the differentiation potential of gfp 1 cells isolated from the controls to those isolated from post-ischemic brains. the gfp 1 cells isolated from the dorsal part of the lv of controls formed neurospheres and differentiated only into a glial phenotype. the gfp 1 cells isolated from the dorsal part of the lv of post-ischemic brains were able to form neurospheres as well; however, besides their differentiation into a glial phenotype, they also gave rise to cells with the properties of neuronal precursors. we also performed in situ immunohistochemical/electrophysiological analyses of gfp 1 cells in the adult brain of controls and those after mcao. in situ analyses revealed that gfp 1 cells expressed the phenotype of adult nscs or neuroblasts in controls or following ischemia and that their number was increased in both hemispheres following mcao. compared to controls, we found that the number of gfp/doublecortin-positive cells significantly increased in the dorsal part of the lv as well as the number of gfp 1 cells in the olfactory bulb, where they probably differentiated into calretinin 1 interneurons. our results indicate that gfp 1 cells with an active mdach1 gene in the dorsal part of the lv play an important role in the increased production of neuroblasts after injury and that this process is also enhanced in the contralateral hemisphere. collectively, our results reveal the involvement of the mdach1 gene in adult neurogenesis. cells expressing this gene exhibit the properties of adult nscs or neuroblasts and respond to mcao by enhanced neurogenesis. experimental cerebral ischemia leads to activation of microglia and astrocytes, which subsequently release pro-and anti-inflammatory factors. furthermore, during the earliest periods of neuronal damage, large quantities of dopamine are released, which may exacerbate the vulnerability of the lesioned tissue. on the other hand, delayed treatment with levodopa has been proven beneficial in stroke patients, suggesting a more complex effect of dopamine. recent studies on astrocytic involvement in neuroinflammation have shown a remarkable modulation of the inflammatory response over the dopamine d2 receptor (d2r). after stroke, reactive astrocytes in the astroglial scar were found to be immunopositive for d2r. in this study, we report the expression of d2r in activated microglia, both after experimental stroke in vivo and after oxygen-glucose deprivation (ogd), an in vitro model of ischemia. using immunohistological stains, we analyzed the expression of d2r in iba1 positive cells 1, 3, 5, 7, and 14 days after middle cerebral artery occlusion (mcao; n 5 2-4 mice/time point). for each animal, >4 pictures were taken from the ischemic core as well as from the contralateral, non-lesioned cortex from 3 sections. a total of >300 (control) and >500 (ischemic core) iba11 cells were analyzed for each time point. in the ischemic core, $25% of cells immunopositive for iba1 expressed d2r in contrast to <1% in control areas. a strong depletion of d2r immunoreactivity was observed in the ischemic striatum, accompanied by an increase in d2r expression in the adjoining cortex and an elevated presence and activation of microglia. for in vitro experiments, we used fluorescence activated cell sorting to isolate cd45 low /cd111 microglial cells. no significant d2r expression at mrna level could be detected by rt-pcr in this population, while we were able to detect d2r in non-microglial cells. interestingly, cultured primary mouse microglia expressed low quantities of d2r, as revealed by western blotting and rt-pcr, suggesting that culturing is sufficient to induce d2r in microglial cells. upon in vitro activation of primary microglia through 60 minutes of ogd, we found a 1.5 fold increase in d2r expression after 24 hours (n 5 4). furthermore, treatment of primary microglia with the selective d2r agonist pramipexole showed a dose-dependent tendency to increase lipopolysaccharideinduced tnfa release (n 5 5). our studies indicate that activated microglia express a functional d2r, which may contribute to the pro-inflammatory reaction of microglia after ischemia. white matter (wm) is injured in most strokes and axonal injury and dysfunction contribute to disability associated with clinical deficits. in young wm, the damage from ischemic injury involves the sequence of energy depletion (ionic pathway), excessive glutamate release (excitotoxicity), generation of reactive oxygen species and oxidative stress (oxidative pathway). in older wm the injury is mediated by ca21-independent excitotoxicity due to an earlier and more robust glutamate release. because excitotoxicity leads to oxidative stress in wm we investigated whether blocking nitric oxide synthase (nos) activity before or after a period of oxygen glucose deprivation (ogd) promoted axon function in an age-dependent manner. acutely isolated optic nerves from young and old (1 and 12 month) swiss webster (sw) or c57bl/6 (bl6) mice were used to ascertain quantitative measurements of wm function and structure. to support a biological basis for nos inhibitor nitro-l-arginine methyl ester (l-name) action in themonpreparation, we evaluated the expression and localization of brain nos (bnos) using immunohistochemistry in conjunction with confocal imaging. the expression of bnos co-localized with gfap (1) astrocyte nuclei, cytoplasm, end-feet as well as nf-200 (1) axons. the pattern of bnos expression paralleled astrocyte morphology with age and became more punctate in appearance. evoked compound action potentials (caps) recovered to 21.8 6 2.8% (n 5 18) after 60 min of oxygen glucose deprivation (ogd) in young bl6 mons. pretreatment of mons with l-name (200 mm) promotedcaprecovery to 69.4 6 11.3% (n 5 8) compared to ogd while caps recovered to 39.9 6 4.4% (n 5 11) when l-name was applied after the end of ogd. pretreatment of mons from 1 or 12 month old sw with l-name improvedcaprecovery to 49.6 6 3.7% (n 5 8, vs ogd 21.3 6 3.7%, n 5 12) or to 51.1 6 9.3% (n 5 7, vs ogd 5.7 61.7%, n 5 8) respectively. l-name application after the end of ogd failed to promote aging axon function (8.8 6 3.5%, n 5 6). changes in nos activity help unveil agedependent oxidative injury mechanisms in ischemic white matter. question: cerebral endothelial cells have been reported to exert a protective effect against brain damage. does transplantation of healthy endothelial cells have a beneficial effect on the outcome of ischemic brain damage? methods: we injected endothelin-1 (et-1) into the rat internal capsule to induce lacunar infarction. seven days after et-1 injection, microvascular endothelial cells (mvecs) prepared from adult rat cerebral cortices were transplanted into the internal capsule. meningeal cells prepared from adult rat cerebra or 0.2% bovine serum albumin-hank's balanced salt solution were injected as controls. two weeks later, the footprint test and histochemical analysis were performed. results: we found that mvec transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (p <0.01) and induced remyelination (p<0.01) compared with the control groups. also the inflammatory response was repressed by mvec transplantation, judging from fewer ed-1-positive activated microglial cells in the mvec-transplanted group than in the other groups. conclusions: elucidation of the mechanisms by which mvecs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia. objective: though lactate is a known neuroprotective agent, its mechanism of action is not known. we hypothesized that lactate can provide neuroprotection by activating trek channels. the effect of lactate on the electrophysiology of trek channels was studied both in an in vitro brain slice model, after blocking the activity of other ion channels and in a heterologous expression system. results: whole cell patch clamp experiments, on ca1 stratum radiatum astrocytes, in acute hippocampal slices, significantly increased trek channel activity by 23.3 6 6.4% and hyperpolarized their resting membrane potential by 5 6 0.6 mv, on bath application of 30 mm lactate. comparison of rectification index at negative potentials between control and 30 mm lactate treatment showed no significant difference, suggesting that the conductance activated by lactate is outward rectifying. lactate-evoked increase in trek channel activity was reversibly inhibited by 200 mm quinine, a potent inhibitor of trek channels. further, lactate was unable to increase trek channel activity after disruption of intracellular lactate uptake with 2 mm a-cyano-4-hydroxy cinnamate, a blocker of monocarboxylate transporter. this was confirmed by inside-out patch clamp experiments on human trek1 channel expressed in hek293 cells. conclusion: the experimental observations suggest uptake of lactate by astrocytes to activate trek channels intracellularly. regenerative responses occurring after different types of postnatal brain injury often involve expansion of an endogenous neural progenitor cell pool, but the molecular mechanisms underlying this process are still poorly understood. we studied expansion of the glial progenitor cell pool in a mouse model of neonatal hypoxia (hx) that reproduces the hallmarks of perinatal brain injury in infants, including diffused white matter injury (dwmi). we have previously shown that hx causes proliferation of wm oligodendrocyte progenitor cells (opcs), and that this process is regulated by the cyclin-dependent kinase 2 (cdk2) pathway. our analysis in wm in vivo and in cultured cells demonstrated: i) higher expression of cdk2, cycline, prb806/811 and e2f1 in wm opcs after hx; ii) activation of the cdk2 pathway after hx, and iii) reduced hx-induced opc proliferation in cdk2-null mutant mice. here, we studied the upstream molecular mechanisms that regulate activity of the cdk2 signaling pathway resulting in opc proliferation after hx. we show that sirtuin 1 (sirt1) histone deacetylase activity is a major regulator of cdk2 signaling and of the regenerative response of opcs after hx. under hypoxic conditions, sirt1 is upregulated in wm, and interacts with both rb and cdk2. patterns of posttranslational modifications of cdk2 or sirt1 proteins after silencing either of these genes in cultured cells from normoxic and hypoxic wm indicate that cdk2 is a substrate for deacetylation by sirt1, and that sirt1 is phosphorylated by cdk2. also, sirt1 causes rb deacetylation resulting in dissociation of e2f1 from the rb/e2f1 complex, which ultimately leads to higher opc proliferation. knockdown of sirt1 in hypoxic wm cell cultures suppresses opc proliferation. finally, inhibition of sirt1 activity by sirtinol accelerates opc differentiation to mature oligodendrocytes, and reduces the number of opcs. we are currently analyzing the effects of hx on opc proliferation and differentiation in the wm of sirt1-null mice. our results provide evidence that sirt1 is an essential regulator of the cdk2-dependent regenerative response observed in wm opcs after hx, and that inhibition of sirt1 activity facilitates oligodendrocyte regeneration from opcs, which might ultimately promote wm recovery after hx. supported by nih r01ns045702, p01ns062686 and iddrc p30hd40677. gonadal steroid hormones reveal a potential neuroprotective role in acute brain ischemia. in rat in vivo studies using the transient occlusion of the middle cerebral artery as ischemic stroke model, we have shown that 17ß-estradiol and progesterone reduce the infarct area by more than 70% given 1h after the onset of stroke, prevent neuronal death and behavioral deficits. an intriguing aspect of this study was that the application of steroid hormones reduced the number of microglia and microglia-related markers in the penumbra during the first 24 h after the onset of stroke. besides microglia, penumbral astroglia coevally appeared as cellular target for steroid hormones by reducing the expression of proinflammatory-and molecules necessary for the attraction and activation of microglia and lymphocytes. this suggests that protective steroid hormones influence glia cell cross-talk and activity during early damaging conditions in the lesioned brain site. by using an in vitro hypoxic approach, we have now demonstrated that microglia express steroid hormone receptors and directly respond to ischemic conditions and steroid hormone treatment by changing expression and secretion of inflammatory compounds, trans-signaling factors and by modulating phagocytotic activity. this clearly shows that microglia is an important direct target for steroid hormones but also indirectly influenced via cross-talk by adjacent astrocytes at the damaged brain site. this generally points at an extensive bidirectional crosstalk between both glial cell types to convey steroid-mediated neuroprotection in ischemic brain tissue. in summary, the balance of inflammatory astrogliamicroglia interactions within the injured brain tissue during an early phase of ischemic destruction is a pivotal step for tissue repair. cannabinoids are considered as key regulators in the pathology of various diseases, including ischemic stroke. we previously demonstrated that post-ischemic treatment of two naturally occurring phenylpropanoids, trans-and cis-hinokiresinols (hrs) significantly reduced ischemic injury in rats subjected to middle cerebral artery occlusion (mcao) . in studies to screen their cellular targets, we found that hrs selectively bind to both type 1 and type 2 cannabinoid receptors (cb1r and cb2r). in the cb1r reporter gene assay, both hrs demonstrated antagonistic activity. in cb2r-overexpressing u 2 os cell lines, both hrs increased forskolininduced camp accumulation, suggesting their inverse agonism. since camp induces expression of heme oxyagenase-1 (ho-1), a well-known antioxidant stress protein, we further investigated the effect of hinokiresinols on ho-1 expression in mixed cortical neuron/glia cultures. trans-hr increased astroglial expression of ho-1 greater than cis-hr did. a selective ho-1 inhibitor, tin protoporphyrin ix significantly reduces the neuroprotective effect of trans-hr in cortical cultures exposed to oxygenglucose deprivation. in addition, both hrs reduced the number of ed-1immunopositive cells (i.e., active microglia and macrophages) in ischemic lesions. also, both hrs significantly reduced microglial migration induced by an endocannabinoid, 2-arachidonoylglycerol. the present study suggests that hrs reduce ischemic injury via regulation of glial cbrs. , the main anaplerotic enzyme in the brain, is predominantly located in astrocytes. in the adult brain, ischemia is known to reduce pyruvate carboxylation. moreover, reperfusion after ischemia leads to production of reactive oxygen species (ros). the pentose phosphate pathway (ppp) is, by making nadph necessary for the regeneration of reduced glutathione, a major pathway in the protection against ros. however, astrocytic and neuronal metabolic function in the neonatal brain after hypoxic-ischemic brain injury (hi) remains to be explored. methods: hi was induced in 7-day-old rats by unilateral severing of the carotid artery followed by 2 hours recovery and subsequent exposure to hypoxia (8%o 2 ) for 90 min. 30 min after end of hypoxia (the early reperfusion phase), rats were injected with [1,2-13 c]glucose and decapitated 30 min later. one group was sham-operated, and not exposed to hypoxia, thus reflecting normal metabolism in the neonate. extracts of ipsilateral hemispheres from hi and sham animals were analysed with 1 h-and 13 c-nmr spectroscopy. results: the sham animals had similar glutamine content, but lower amounts of 13 c labelled glutamine compared to corresponding values from adult rats, reflecting a generally lower rate of glucose metabolism in the neonatal astrocytes. however, approximately equal amounts of 13 c labelled isotopomers of glutamine were derived from pc and pyruvate dehydrogenase (pdh), resulting in a relatively high pc/pdh-ratio compared the same ratio in adults. following hi and reperfusion, a reduction in glucose metabolism and mitochondrial metabolism was seen by increased glucose and reduced incorporation of 13 c labelling in glutamate, glutamine and aspartate via pc and pdh. however, the pc/pdh-ratio in glutamine was similar in hi and sham. total mean values of glutamate, glutamine and aspartate were decreased, but only the latter to a significant degree. labelling in lactate via the ppp was reduced following hi. conclusion: the low labelling of lactate via the ppp indicates that the flux through the ppp was in fact reduced in the early reperfusion phase after hi. moreover, mitochondrial metabolism of pyruvate from glucose was reduced in both astrocytes and neurons. impaired anaplerosis in astrocytes was confirmed by lower amounts of aspartate. however, the proportionally similar decrease in 13 c labelling in glutamate and glutamine via pc, and the maintained pc/pdh-ratio implies that astrocytes continue to provide metabolic support for the neurons during the early reperfusion phase. ret receptor tyrosine kinase is the signaling component of the receptor complex for the family ligands of the glial cell line-derived neurotrophic factor (gdnf). ret is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons and it is necessary for the postnatal maintenance of dopaminergic neurons. ret expression has been as well demonstrated on microglia and several evidence indicate that gdnf regulates not only neuronal survival and maturation but also certain functions of microglia in the brain. here we demonstrated that isolectin ib4, commonly used as a microglial marker in the brain, binds to the glycosylated extracellular domain of ret both on the surface of living nih3t3 fibroblasts cells stably transfected with ret than in adult rat brain sections as revealed by immunoblotting. further, confocal immunofluorescence analysis demonstrated a clear overlap staining between pret and ib4 labeling in primary microglia cultures as well as in adult rat sections obtained from control or postischemic brain after permanent middle artery occlusion (pmcao). interestingly, ib4 staining identified activated or amoeboid retexpressing microglia under ischemic conditions. collectively, our data indicate ret receptor as one of the ib4-reactive glycoconjugate accounting for the ib4 stain in microglia under physiological and ischemic conditions. astrocytes can adapt to lower oxygen tensions by enhancing their glycolytic rate of energy production. in this case, adequate expression of monocarboxylate transporters (mcts) may be essential to sustain prominent lactate efflux and prevent glycolysis inhibition. here we show that oxygen level is an important factor controlling mct4 expression in primary cultures of mouse cortical astrocytes. indeed, while mct4 is clearly expressed by astrocytes in vivo, its basal in vitro expression in primary cultures of mouse cortical astrocytes is undetectable under classical culture conditions (95% air, 5%co 2 ). exposing astrocytes to more physiological brain oxygen tensions (hypoxic chamber flushed with 1%o 2 /5%co 2 /94%n 2 , leading to a concentration of dissolved oxygen in the culture medium of 40-60mmhg) restored mct4 expression. mct4 mrna expression became detectable after 12 hours under lower oxygen tension and was still present after 10 days with a maximum at 36 hours of incubation. this effect was paralleled by the appearance of the mct4 protein which reached a plateau between 48 hours and 96 hours, with a further enhancement at 7 and 10 days of incubation. this induction was specific for mct4 since mct1 expression was not altered. lactate release was significantly enhanced after 48 hours of incubation and further increased up to 10 days compared to astrocytes maintained under atmospheric conditions. treatment with dimethyloxalylglycine (dmog), a compound that mimics hypoxia by stabilizing the hypoxia-inducible factor 1 alpha (hif-1a) , produced a similar effect. both mct4 mrna and protein induction reached their maxima when astrocytes were treated with a concentration of dmog ranging between 1 to 2.5mm, which is also the range for greatest hif-1a expression. again, no effect was detectable on mct1 expression. transfecting astrocyte cultures with a sirna against hif-1a significantly reduced the effect of lower oxygen tension on mct4 expression. our results suggest that 1) under physiological conditions, changes in local oxygenation might regulate the capacity of astrocytes to supply lactate to neighboring cells via notably alteration in mct4 expression 2) under pathological conditions, mct4 may be upregulated as a consequence of hypoxia and contribute to neuroprotection by favouring the release and accumulation of lactate in the extracellular space. indeed, this process might give rise to the beneficial effect of lactate for neurons observed during the recovery from hypoxic/ischemic conditions in vitro and in vivo. early onset dementia and. the loss-of-function of trem2 or its coreceptor dap12 is responsible for the recessively inherited nasu-hakola disease (also known as plosl). the brains of plosl affected patients show strong microglial activation in the cerebral white matter. reports demonstrate that trem2-mediated phagocytic function of microglia is required for debris clearance and cns tissue homeostasis. perinatal brain injury is the underlying etiology for a host of developmental disabilities that includes spastic motor deficits and cognitive, behavioral and learning difficulties. hence, our aim is to characterize the expression of trem2 in the control of neuroinflammation following hypoxia/ ischemia (hi) in the newborn brain. we performed hi brain damage in postnatal day 7 (p7) c57/bl6 mice by permanent left carotid occlusion and litters were exposed to 8% of oxygen balanced with nitrogen for 50 minutes in a hypoxic chamber with controlled humidity and temperature maintained at 37 c. pups were then returned to their dam until sacrifice. the age-matched controls and samples from 3 hours to 7 days after hypoxia were collected and processed for immunohistochemistry. in control animals, trem2 expression was observed in the corpus callosum and in the subventricular zone at p7 that almost disappeared at p10. following hi, an increase in trem2 staining was observed in the corpus callosum, hippocampus, caudate-putamen, fimbria, cortex and thalamus in the ipsilateral (il) hemisphere, following a similar regional pattern of microglia activation. the increased expression of trem2 was observed from 24 hours to 7 days post-hypoxia. trem2 colocalized mainly with microglia markers, such as iba-1 and cd68. oligodendrocyte expression of trem2 was also analyzed. in conclusion, hi produced an increase in trem2 expression on the il damaged regions. these results suggest that the modulation of trem2 might be a possible target for damage control in neonatal brain. increasing evidence shows that astrocytes play a critical role in neuronal protection during an ischemic insult. in the vertebrate retina, most of astrocytic functions are executed by m€ uller glial cells, the major macroglial cell type in this tissue. the aim of this study was to evaluate the role of m€ uller glia in the onset and progression of retinal ganglion cell (rgc) degeneration after acute ischemic injury in the mouse eye in vivo. here, we used the selective gliotoxin fluorocitrate (fc) in order to transiently impair m€ uller glial metabolism at different time points during and after induction of an acute retinal ischemia/reperfusion by means of elevated intraocular pressure (iop). the effect of fc (3 mm) on metabolism was determined by measuring retinal glutamine levels, aconitase activity and mitochondrial membrane depolarization after intravitreal injection of the gliotoxin. retinal ischemia was unilaterally induced by elevating iop for 45 min. fc was intravitreally injected 4 h before onset of ischemia and after 6, 12 or 24h of reperfusion. saline was injected in the contralateral eye and served as control. surviving rgcs were quantified by means of confocal scanning microscopy and automated cell counting-based analysis on retinal wholemounts 7 days after lesion. fc treatment reduced glutamine levels to 35-38% as compared to control 4-6 h after injection. inhibition was completely reversed 24 h after injection. aconitase activity was reduced to $30% from control levels 4 h after fc injection. mitochondrial membrane potential was reduced by 50-70% as indicated by reduced intensity of the specific stain mitotracker red cmxros. one week after ischemia, only 40% of rgcs survived as compared to unlesioned controls. impairment of m€ uller glial metabolism during ischemia further reduced rgcs by 15% as compared to ischemia alone. fc treatment did not significantly modify rgcs survival 6 and 12 h after lesion. results support the notion for a critical neuroprotective role of m€ uller glial cells during ischemia. in the acute phase following ischemia, however, m€ uller glia inhibition does not further affect rgcs survival. further research is required to establish the time point for this switch in m€ uller glial function and the underlying mechanisms. extract has been widely investigated in animal models and clinical studies for various diseases including ischemic stroke. its major metabolite, cordycepin, has been reported to act as a selective a3 adenosine receptor agonist and to protect neurons against ischemic injury. however, their underlying mechanisms remain unclear. in the present study, we report that the standardized c. militaris extract, wib-801c, which contains cordycepin 8% of total dry weight of the extract, markedly reduced ischemic injury by inhibiting post-ischemic inflammatory responses. postischemic treatment with wib-801c (orally administered twice at 3 and 8 h after onset of mcao) significantly reduced infarct size and edema in rats subjected to transient middle cerebral artery occlusion (mcao, 1.5 h) and subsequent reperfusion (22 h). wib-801c also significantly improved integrity of glial cells in ischemic lesions, as evidenced by reduced white matter degeneration and loss of blood-brain barrier. importantly, wib-801c significantly ameliorated neurological deficits in not only transient but also in permanent stroke models. moreover, wib-801csignificantly improved long-term survival of mcao rats over 30 days. as we previously reported with selective a3 receptor agonists introduction: cd200 & cd200r are immune inhibitory molecules that have been shown to be involved in inducing immune tolerance and in contributing to immune privileged status of the cns. the developing brain exhibits distinct morphological as well as physiological characteristics determining a peculiar response to injury showing an aggravated susceptibility to excitotoxicity and pro-inflammatory cytokines, along with an exacerbated inflammatory response. previous studies from our group have described the expression of cd200-cd200r in brain during development showing a distinct pattern of expression in the cortex and the hippocampus. hence, the aim of this study is phenotypic characterization of cd200r1 microglia/macrophages and cd2001 neuronal cells following hypoxia/ischemia (h/i) in neonatal mice brain. wild-type c57/bl6 mice postnatal (p) day 1,3,5,7,10,14,21 and adult were used for developmental studies. p7 mice was used for h/i injury that was administered using vannucci model modified for neonatal mice (8% o 2 , 55 min) and samples were collected 3h, 12h, 24h, 48h, 72h & 7 days after hypoxia for immunofluorescence staining. results: phenotypic characterization of cd200r1 microglia/macrophages showed them to express markers of pro-or anti-inflammatory phenotype in a temporal fashion post lesion. they expressed m2 phenotype as observed by colocalization with cd2061 cells throughout the time points studied but a subpopulation of cd200r1 cells exhibited m1 phenotype as exhibited by mhcii1, cd861 cells from 24-48 hrs post lesion. calretinin (cr) used for the characterization of cd2001 neurons showed that most cr1 neurons were cd2001 especially the interneurons in the innermolecular layer of hippocampus, layer i of the neocortex and the hilus at most age groups studied. after h/i, cd200 immunolabeling was increased in the hippocampal fissure until 7 days post-lesion. this followed a change in cd200r1 microglial cells described above. to conclude, a balance between m1/m2 phenotype of cd200r1 microglia/macrophages and expression of cd200 by cr1 cells are involved in evolution of h/i induced brain injury in neonatal mice. objective: the cytokine interleukin-1 (il-1) and its naturally occurring receptor antagonist (il-1ra) play a key role in determining neuronal cell death and survival in focal cerebral ischemia. the objective of this study was to determine the cellular production of il-1/il-1ra, and to test the neuroprotective potential of post-surgically injected il-1ra overexpressing bone marrow (bm) cells in a mouse model of focal cerebral ischemia. materials and methods: c57bl/6 mice were injected i.v. with bm cells isolated from sil-1ra overexpressing mice, 30 min after permanent middle cerebral artery occlusion (pmcao). physiological parameters and behaviour were recorded post-surgically, infarct sizes were estimated, and microglial-leukocyte expression of il-1/il-1ra was analyzed by flowcytometry, in situ hybridization and immunohistochemistry. results: we identify microglia, and not recruited leukocytes as the major producers of il-1ra after pmcao in mice, and we show by using il-1ra knock out mice that microglial-produced il-1ra is neuroprotective. we report that the sil-1ra produced by the post-surgically injected bm cells, potentiates the neuroprotective effect of microglialderived il-1ra, at both 24 h and 5 days, which is consistent with behavioural improvement at 5 days, and detection of recruited bm cells in the ischemic area 1.5 h after pmcao. interestingly, we also find that the sil-1ra overexpressing bm cells stimulate microglial production of il-1ra 6 h after pmcao, at which time both il-1a and il-1b is upregulated. conclusion: our results provide proof of principle that increasing the production of il-1ra by recruited bm cells can counteract the effect of il-1a/b, increase neuronal survival and improve motor function alone or through induction of microglial-produced il-1ra after pmcao in mice. bogomoletz institute of physiology, kyiv, ukraine short-term oxygen-glucose deprivation (ogd) is known to activate excitatory glutamatergic synapses, induce long-term potentiation and result in neuronal and synaptical ultrastructural remodelling in organitypic cultured hippocampal slices (ochs). in this work we studied changes in glial synaptic coverage following 30 min ogd episode using 3d reconstruction of ochs confocal and electron microscopic images. propidium iodide (pi) and mitotracker orange cmtmros (mto) were used to estimate cell viability and their mitochondrial potential. astroglial and microglial cells were identified using immunohistochemical staining with anti-gfap and anti-iba-1 antibodies. the study demonstrated that astroglial and microglial cells retain viability (there was no significant increase in the amount of pi-labbelled cells). 3d reconstruction revealed significant increase in excitatory synapses glial coverage as early as 1 hour following ogd accompanied by a significant increase in number of astroglial and microglial processes. the deprivation also resulted in gradual increase of mitochondrial potential from 4 hr to 24 hr in both astroglial and microglial cells. it was previously shown that pyramidal neurons under the same conditions lose mitochondrial potential and became pi-positive after 4 and 24 reoxygenetion, indicating damage of cytoplasmic membrane, the effect prevented by application of nmda receptor antagonist d-ap5. thus, unlike neurons astroglial and microglial cells are ogd-resistant and demonstrate marked mitochondrial activation -a process that might be involved in maintaining neuronal function during oxygen-glucose deficiency. using a microfluidic high throughput qpcr platform, we measured the expression of 47 selected genes encoding astrocytic and polydendrocytic markers, ion channels, transporters and receptors that participate in maintaining k 1 and glutamate homeostasis in each of 292 individual gfap/egfp-positive glial cells. in this study we show how the expression of these selected genes changes during development (postnatal days 10, 20, 30 and 50) as well as 3, 7 and 14 days after middle cerebral artery occlusion (mcao). self-organizing maps and principal component analyses divided the cells according to their similarity in gene expression into three subpopulations of astrocytes present within the first 10-50 days of postnatal development (p10-p50). the first subpopulation comprises immature glia mainly from p10, characterized by the high transcriptional activity of all of the studied genes, including polydendrocytic markers. the second subpopulation is dominated by cells from p20 and displayed low transcript levels of all of the studied genes. the third subpopulation represents mature astrocytes mainly from p30 and p50. three, seven and fourteen days after ischemia (d3, d7, d14), additional astrocytic subpopulations appear: resting astroglia (mainly from p50 and d3), transcriptionally active early reactive astroglia (mainly from d7) expressing the mrna of polydendrocytic markers, and, presumably, permanent reactive astroglia (solely from d14). following focal cerebral ischemia, reactive astrocytes undergo pronounced changes in their expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and markers of reactive astrocytes and polydendrocytes. from the data obtained from single cell pcr analysis, we calculated the spearman correlation coefficients between pairs of genes, which revealed interesting positive gene expression correlations with polydendrocytic markers (gria2-4, grik1-5, grin3a, kcnj16, kcnk2 and kcnk10 genes). this study was further supplemented by an immunohistochemical analysis of nmda receptor subunits and pdgfar, which confirmed that changes in gene expressions correlate with immunodetected proteins. ga cr 13-02154s, astf 110-2011, gauk 604212. reactive astrogliosis is often regarded as universal type of astroglial response to various forms of injuries. among its most characteristic features one may include: glial fibrillary acidic protein (gfap) up-regulation, cellular hypertrophy and proliferation and gliotic scar formation. an important element of glial cells response is their ability of de-differentiation, which is related with their re-gaining of immunological and molecular properties characteristic for earlier developmental forms. the origin of cells proliferating in response to various types of brain injury is still a subject of debate. among others it is related to the pathomechanism of lesion, affected brain structure and developmental stage of the nervous system. aim: in our study we aimed to assess the differences of de-differentiation and proliferation potential of astroglia localized in the cerebral cortex and striatum to the transient focal brain ischemia. material and methods: transient focal brain ischemia was evoked in 20 adult male wistar rats by placement of the monofilament surgical thread into the internal carotid artery and occlusion of the middle cerebral artery for 1h. the postoperative survival period was up to 6 weeks. immunocytochemical double-staining for astrocytic marker (gfap), developmental (pax6) and proliferative (ki-67) markers was performed and subsequent qualitative and quantitative study was done by means of confocal microscopy. results: double-labeled gfap/pax6 and gfap/ki67 reactive astroglial cells were revealed in the cerebral cortex and striatum of the ischemic region, starting from 24h after initiating the ischemia. the apparent difference in the intensity of morphological changes, quantity of de-differentiating and proliferating astroglial cells was observed between ischemic cerebral cortex and striatum. intensity of astroglial response and proliferative potential were much higher in the striatum than in the cerebral cortex. summary and conclusion: apparent difference in proliferative response between the cerebral cortex and striatum may be consequence of metabolic and molecular differences between astroglial cells populating two mentioned above structures. it may be also consequence of uneven decrease of cerebral blood flow and resulting different metabolic conditions influencing astroglial function in various fragments of ischemic area. reassuming, the differentiated potential of astroglial proliferative response to ischemic changes in different brain regions must be taken into account while analyzing clinical consequences of cerebral infarcts of different localization. triggering receptor expressed on myeloid cells-2 (trem2) is a microglial surface receptor involved in phagocytosis. clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. the role of trem2 following stroke is currently unclear. as an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). quantitative pcr (qpcr) revealed a greatly increased transcription of trem2 after stroke ($10fold). we subsequently analyzed the expression of proinflammatory cytokines, chemokines and their receptors in trem2knockout (trem2-ko) mice via qpcr. microglial activation (cd68, iba1) and cd3-positive t-cell invasion were analyzed via qpcr and immunohistochemistry. functional consequences of trem2 knockout were assessed by infarct volumetry. the acute inflammatory response (12 h reperfusion) was very similar between trem2-ko mice and their littermate controls. however, in the sub-acute phase (7 d reperfusion) following stroke, trem2-ko mice showed a decreased transcription of pro-inflammatory cytokines tnfa, il-1a and il-1b, associated with a reduced microglial activity (cd68, iba1). furthermore, trem2-ko mice showed a reduced transcription of chemokines ccl2 (mcp1), ccl3 (mip1a) and the chemokine receptor cx3cr1, followed by a diminished invasion of cd3-positive t-cells. no effect on the lesion size was observed. conclusions although we initially expected an exaggerated pro-inflammatory response following ablation of trem2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in trem2-ko mice. we therefore conclude that trem2 appears to sustain a distinct inflammatory response after stroke. metabotropic glutamate receptors (mglurs) are seven transmembrane domain g-protein-coupled receptors for l-glutamate, the main excitatory neurotransmitter in the mammalian brain. in addition, glutamatemediated excitotoxicity plays a central role in mediating neuron and oligodendrocyte death during ischemic episodes. in vitro, developing oligodendrocytes (ols) in particular have been shown to be highly vulnerable to excitotoxicity and oxidative stress. notably, activation of group 1 mglurs has been shown to attenuate ol excitotoxicity in vitro and in brain slices has been shown to reduce ischemia-mediated impairment of astrocytes. here, we have examined the functional expression of mglurs and their role in acute ischemic injury in developing cns white matter of the mouse optic nerve. calcium imaging experiments were performed in optic nerves from p9-13 wild-type mice. optic nerves were isolated intact, loaded with fluo-4 am and visualized using a zeiss lsm 5 pascal confocal microscope; images were collected in optical z-sections and changes in fluorescence intensity were measured. the application of mglur agonists acpd and dhpg showed that group i mglur mediate a rise in glial [ca 21 ] i . the action of the group i mglur antagonist aida versus acpd indicated that the rise in [ca 21 ] i also involves group ii (and possibly group iii) mglur. immunohistochemistry confirmed glial expression of group i subunit mglur5 and group ii subunits mglur2 and mglur3 in the optic nerve, although further analysis of cellular localization is required. to examine the role of mglurs in white matter ischemia, optic nerves from p8-9 mice in which fluorescent reporters are driven by astrocyte (gfap-egfp) or oligodendrocyte (sox10-egfp) genes were isolated intact into artificial cerebrospinal fluid (acsf) and subjected to oxygen glucose deprivation (ogd) for 1 hour. activation of group i or group ii mglur with the agonists acpd, dhpg or ly379268 protected astrocytes and oligodendrocytes from ischemic cell death. further experiments are required to determine the mechanisms involved, but these findings demonstrate functional expression of mglur in optic nerve glia and indicate that activation of mglur is a possible therapeutic strategy to protect against glial damage in response to ischemia. the cerebral cortex is one of the most often affected areas after stroke, but there are no studies comparing the outcome in different cortical regions after focal ischemia. the aim of this investigation was to evaluate the patterns of glial activation, tissue degeneration and neuronal loss in different survival times following cortical ischemia. focal ischemia was induced by stereotaxic microinjections of endothelin-1 (et-1) into the somatosensory, motor and association cortices of adult rats (n 5 45). the control animals were injected with the same volume of sterile saline (n 5 27). the animals were perfused at 1, 3 and 7 days after ischemia. gross histopathology was evaluated using cresyl violet staining. 20 lm sections were submitted to immunohistochemistry for astrocytes (anti-gfap), activated microglia/macrophages (anti-ed1) and microglia (anti-iba1 and ed1). tissue loss and glial activation were more intense in the somatosensory cortex than in other cortical areas. the motor cortex was the second more affected area. the association cortex was the less damaged area, which was confirmed by quantitative analysis (anova-tukey). the results suggest that an ischemic lesion of the same intensity induces a differential pattern of tissue loss and neuroinflammation, depending on the cortical area, and that the primary sensory and motor areas are more susceptible to ischemia than association areas. tlr4 is required for ipc-induced neuroprotection. in order to elucidate the mechanisms by which microglial tlr4 contributes to ipc, we carried out cell-targeted genomic analyses specifically on microglia exposed to either ischemic conditions in vitro or ipc in vivo. for our in vitro model, we exposed wt and tlr4 -/mouse primary microglia to hypoxic/hypoglycemic conditions for 24 hours. for our in vivo model, we carried out transient middle cerebral artery occlusion (mcao) or sham surgery as our ipc pulse on wt and tlr4 -/adult male mice. three days later mice were sacrificed and microglia acutely isolated from ipsilateral cortex using magnetic affinity chromatography cell separation and ex vivo flow cytometry. microarray analysis was carried out on rna from both in vitro and in vivo microglia. results from both datasets identified robust expression of type 1 and/or type 3 interferon (ifn)-stimulated genes (isgs) as the predominant transcriptomal feature of ischemia-exposed microglia. in vitro, we found that 25 out of the 60 (40%) individual genes that were significantly up-regulated >2fold by hypoxia/hypoglycemia in wt, but not tlr4 -/-, microglia were isgs including mx1, oas2, irf7 and ccl5. promoter analysis demonstrated that the top four most active transcription factors were isgf3, irf2, irf1 and irf7; all ifn regulatory factors (irfs). a similar pattern emerged from the in vivo dataset with multiple irfs again among the most activated transcription factors in sorted microglia from ipcexposed mice. however, unlike the isg response in vitro, the isg response from the in vivo dataset was not tlr4-dependent. the ifn family of cytokines is recognized as a key component of the innate immune response to infection and other types of injury. the role of microglial ifn-signaling in ipc is unknown. these results suggest that broad activation of isgs may be a key feature of the microglial response to ischemic conditions. astrocytes respond to central nervous system (cns) injury by the formation of a glial scar that develops within few days after insult and is characterized by astrocyte proliferation and cellular hypertrophy. reactive astrocytes change their immunohistochemical profile, such as increasing expression of gfap, nestin and vimentin; however, they also markedly alter the expression of ion channels, receptors and transporters, which participate in the maintenance of ionic-, water-and neurotransmitter homeostasis, such as k 1 channels, aquaporins or glutamate transporters. here, we aimed to characterize the gene expression profile and functional properties of reactive astrocytes 5 weeks after global cerebral ischemia (gci) or focal cerebral ischemia (fci) with a specific focus on k 1 channels or non-specific cationic channels. gci was induced in adult rats by bilateral, 15-minute common carotid artery occlusion combined with low oxygen, while fci was induced in adult egfp/gfap mice by permanent middle cerebral artery occlusion. using the patch-clamp, we investigated the membrane properties of astrocytes in situ 5 weeks after gci in the rat hippocampal ca1 region and 5 weeks after fci in the mouse cortex. regardless of the type of ischemic injury, astrocytes from both cns regions depolarized their membrane potential by $14 mv 5 weeks after ischemia. when compared to astrocytes from the non-injured ca1 region or the cortex, the post-ischemic astrocytes displayed large hyperpolarizationactivated time-and voltage-dependent, non-inactivating inward currents, the current density of which increased 3-fold in response to gci or fci. in addition, these non-specific cationic channels were sensitive to zd7288 and to a low extracellular na 1 concentration, suggesting that they may belong to the family of hyperpolarization-activated cyclic nucleotide-gated (hcn) channels. we have taken advantage of fluorescently labeled astrocytes in egfp/gfap mice and isolated egfp 1 cells by facs from non-injured as well as ischemic regions and performed gene expression profiling using single-cell rt-qpcr. our data revealed that cortical astrocytes after fci express, besides the increased expression of gfap, gfapd, aqp1 and 9, extremely high levels of hcn1-3 transcripts that encode hcn 1-3 channels. moreover, our immunohistochemical analyses confirmed the presence of hcn1-3 in reactive astrocytes, especially hcn1, 2 and 3 channels. in summary, our results show that reactive astrocytes from post-ischemic tissue express hcn channels and that their activity might significantly contribute to na 1 influx, possibly maintaining atpase function and/or contributing to intracellular na 1 -dependent events, such as glutamate transporter or na 1 /ca 21 exchanger reversal. ga cr 13-02154s, p304/12/g069 and gauk 383711. the optimal operation of electrogenic astrocytic transporters and exchangers for some well-defined astrocyte brain homeostatic functions depends on the presence of k 1 channels in the cell membranes and the hyperpolarized membrane potential. our previous study showed that astrocytes functionally express two-pore domain k 1 channel trek-1, which helps to set the negative resting membrane potential. however, the roles of trek-1 on astrocytic function under normal and ischemic conditions remain unclear. in this study, we investigated the effects of trek-1 activity on astrocytic function and neuronal death under ischemic conditions. in an in vitro hypoxia model with astrocytes culture, trek-1 immunoreactivity was up-regulated after hypoxia. suppression of trek-1 activity inhibited the glutamate clearance capability, enhanced the inflammatory secretion of astrocytes derived s100b and led to increased neuronal apoptosis after ischemic insult. after middle cerebral artery occlusion induced focal ischemia in rats, the trek-1 expression was significantly increased which correlated with reactive astrogliosis. application of lineonic acid, a trek-1 agonist which could potentiate the astrocytic conductance and hyperpolarization membrane potential, up-regulated the expression of astrocytic glutamate transporter glt-1, inhibited the micro-glial inflammatory response and attenuated neuronal damage. our results suggest that trek-1 activity is involved in astrocytic function and neuronal survival. this would provide evidence showing astrocytic trek-1 involvement in ischemia pathology which may serve as a potential therapeutic target in stroke. oligodendrocytes are the myelinating cells of the central nervous system (cns). they are responsible for ensheathing myelin layers around axons, which contributes to improve conduction of neuronal impulses and additionally provides axonal support. oligodendrocytes are generated by oligodendrocyte precursor cells (opcs), a self-renewing and proliferating adult stem/precursor cell population in the cns. this process occurs not only during developmental myelination but also mediates cns remyelination, a regenerative process that restores myelin sheaths to demyelinated axons. although energy metabolism in the oligodendrocyte lineage is thought to be very active to support the lipid production specific to this cell type, it is still poorly characterized. as a first step towards a detailed characterization of the energy metabolism associated with specific lineage stages we investigated how glucose -the main cerebral energy substrate -is metabolized at an immature opc stage and in differentiated oligodendrocytes. primary cultures of opcs and oligodendrocytes were incubated with medium containing [1,6-13 c]glucose for 4h and metabolites in both cell extracts and culture medium were collected and analysed for excess % 13 c enrichment using gas-chromatography-mass spectrometry (gc-ms) as well as for total amounts using hplc. the significant 13 c-enrichment in citrate, malate, and aspartate in cell extracts indicate that oligodendrocytes oxidize glucose-derived metabolites extensively in the tricarboxylic acid (tca) cycle. moreover, they release 13 c-labelled aspartate and glutamate to the extracellular medium, although a significant consumption of aspartate, glutamate and glutamine from the medium was also observed. enrichment of extracellular lactate and increased alanine synthesis in oligodendrocytes as compared to opcs suggests that the rate of aerobic glycolysis is increased in immature as compared to differentiated cells. in conclusion, these data confirm that both opcs and oligodendrocytes are highly metabolically active and, in addition to extensively oxidizing glucose, synthesize and release distinct metabolites. we anticipate that further studies on this subject will contribute to a better understanding of the physiological role of oligodendrocytes in the cns and the role of glycolysis and mitochondrial metabolism during their maturation. schwann cells in the peripheral and oligodendrocytes in the central nervous system require a tremendous amount of lipids to be capable of producing all cell membranes forming the myelin structure. furthermore, when compared to other cells, myelin membrane presents an extremely high lipid content ($80% of dry weight) coupled to an unique lipid composition. the importance of myelin lipid composition towards myelin formation and maintenance is highlighted by the frequent appearance of myelin defects in lipid-metabolism human disorders. one of the main represented class of lipids in myelin are phospholipids, of which fatty acids (fas) represents the building blocks. most cells preferentially uptake circulating free fas. however, cells under increased membrane production, e.g. highly proliferating cells, can endogenously produce them through the enzyme fatty acid synthase (fasn), which synthesize palmitate. in these cells, fasn is transcriptionally augmented via the mtorc1 pathway. thus, we hypothesized that due to their increased lipid demand myelinating cells would require fasn activity towards myelin production. the functional role of fasn in the metabolic control of myelin membrane lipid composition and myelination is unidentified. thus, we addressed it experimentally by conditionally deleting fasn in myelinating cells in vivo. we show that endogenous fas biosynthesis in myelinating cells is essential to maintain myelin membrane lipid homeostasis. furthermore, maintaining a correct lipid homeostasis is critical for myelination. thus, we analyzed whether fasn is required to allow palmitoylation of myelin proteins and their targeting to the myelin membrane. in addition, we show that interfering with myelin fas homeostasis results in the activation of systemic compensating metabolic loops promoting fas uptake, although not sufficient to preserve efficient myelination. taken together our data demonstrate that in myelinating cells fas homeostasis is under the synergistic control of endogenous metabolic fas production via fasn and systemic functional regulation of free fas uptake. most importantly, maintaining a correct homeostasis of fa-derived lipids in myelin is critical for myelination. homeostasis and signaling, respectively (1). in mammals, two cytosolic and two mainly mitochondrial located glutaredoxins have been identified (2). we already described an essential role for the formation of a functional neuronal network during embryonic development of one of these proteins, the vertebrate specific glutaredoxin 2 (3). here, we will focus on the contribution of glutaredoxin 2 on basic cellular functions of oligodendrocytes, the myelinating cells of the central nervous system, under physiological and pathophysiological situations, e.g. multiple sclerosis, which is characterized by inflammatory axonal demyelination (4). oligodendrocyte progenitors and mature oligodendrocytes are very sensitive against oxidative stress induced by release of nitric oxide by activated microglia (5) . using a2b5 positive glia restricted progenitor cells as well as organotypic cerebellar slice cultures, we found that increased levels of glutaredoxin 2 protect against cell death induced by either activated microglia or s-nitrosoglutathione, a physiological nitric oxide donor. under physiological conditions elevated levels of glutaredoxin 2 increased migration of a2b5 positive cells nearly three fold. western blot analyses, quantitative real time pcr, as well as immunocytochemistry revealed that glutaredoxin 2 inhibited further differentiation of ng2 positive oligodendrocyte progenitor cells. in summary, our data indicate the importance of specific thiol redox signaling during cellular protection and function in this type of glia cells. current multiple sclerosis therapeutics act as immunomodulators and/ or immunosuppressors. this strategy is largely ineffective at preventing progression of the disease. significant benefits to multiple sclerosis patients might be seen with a therapeutic agent which induces remyelination, however, to date no such agents have been developed. using in vitro and in vivo models we have identified nefiracetam as a potential remyelinating therapeutic. to study remyelination in vitro, hippocampal organotypic cultures were exposed to lysophospatidylcholine (lpc) for 18hrs to induce demyelination before being allowed to recover. cultures were treated with nefiracetam for 24hrs of this recovery period. in all cases immunofluorescent labelling of myelin basic protein (mbp) was used as a measure of myelin. nefiracetam was shown to increase mbp expression, relative to untreated controls, accelerating recovery from lpc. two different models were used to investigate the efficacy of nefiracetam in vivo; an inflammatory model, experimental autoimmune encephalomyelitis (eae), and a toxin induced model of demyelination, achieved through the use of cuprizone chow. nefiracetam treatment did not alleviate the symptoms of eae. however, nefiracetam did restore the amount of myelin in cuprizone exposed animals, compared to saline-treated controls. in combination, these observations suggest that nefiracetam is capable of accelerating remyelination through a mechanism of action which is independent of immunomodulation. we conclude that nefiracetam encourages remyelination both in vitro and in vivo. these pro-myelin properties identify nefiracetam as a potential therapeutic for demyelinating disorders. university of naples "federico", neuroscience, naples, italy changes in intracellular [ca 21 ] i levels have been shown to influence the developmental processes that accompany the transition of human oligodendrocyte precursor cells (opcs) into mature myelinating oligodendrocytes and are required for the initiation of myelination and remyelination processes. in the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (ncx) family members ncx1, ncx2, and ncx3, play a role in oligodendrocyte maturation. functional studies, as well as mrna and protein expression analyses, revealed that ncx1 and ncx3, but not ncx2, were divergently modulated during opc differentiation into oligodendrocyte phenotype. in fact, while ncx1 was down-regulated, ncx3 was strongly up-regulated during the oligodendrocyte development. the importance of calcium signaling mediated by ncx3 during oligodendrocyte maturation was supported by several findings. indeed, whereas the knocking down of the ncx3 isoform in opcs prevented the up-regulation of the myelin protein markers cnpase and mbp, its overexpression induced an up-regulation of cnpase and mbp. furthermore, ncx3 knock-out mice exhibited not only a reduced size of spinal cord but also a marked hypomyelination, as revealed by the decrease in mbp expression and by the accompanying increase in opcs number. collectively, our findings indicate that calcium signaling mediated by ncx3 plays a crucial role in oligodendrocyte maturation and myelin formation. to explore the role of kif13b in myelination in vivo, we generated conditional knock-out mice with kif13b specific ablation in either schwann cells or oligodendrocytes. we found that kif13bfl/fl p0cre mouse nerves are hypomyelinated with reduced myelin thickness, thus confirming in vitro data. in the pns, axonal neuregulin (nrg) 1 type iii is one of the main signaling promoting myelination and remyelination after injury. nrg1 type iii binds to erbb2/erbb3 receptors in schwann cells, which signal through a plethora of downstream signaling pathways also including the pi3k/akt complex. the pi3k/akt signaling pathway is attenuated by the pten phosphatase, which dephosphorylates ptdins(3,4,5)p 3 (s. goebbels et al., 2010). dlg1 is believed to interact with pten and potentiate its ability to dephosphorylate ptdins(3,4,5)p 3 , thus negatively modulating the akt-mtor pathway (l. cotter et al, 2010). as kif13b interacts with dlg1 in schwann cells, we investigated the pi3k/akt pathway in kif13b-null nerves and surprisingly, we found that dlg1 expression levels are increased whereas akt phosphorylation is decreased in mutant nerves. our findings suggest that kif13b negatively regulates dlg1 activity and acts as a promoter of myelination in the pns. on the contrary, when kif13b is lost specifically in oligodendrocytes, myelin sheaths are thicker. at the molecular level, the hypermyelination is consistent with increased akt phosphorylation. we are currently investigating the molecular mechanisms at the basis of the kif13b/dlg1 interaction in both schwann cells and oligodendrocytes. we have identified the nootropic nefiracetam as a potential novel remyelinating therapeutic using in vivo and in vitro models of demyelination. in this study we investigate the mechanism of action of nefiracetam in the context of remyelination as regulated by wnt/b-catenin signalling and spontaneous activity. to investigate whether nefiracetam acts by modulating wnt signalling, organotypic hippocampal cultures were exposed to nefiracetam or the wnt/b-catenin inhibitor cardamonin. both increased immunofluorescent staining for the opc marker ng2. myelin formation, as assessed by mbp staining, was enhanced by nefiracetam but was not affected by cardamonin. thus, modifying wnt signalling appears to impact opc development without effecting differentiation, suggesting the pro-myelinating effects of nefiracetam involve an additional mechanism. we next investigated the effect of nefircatam on opc differentiation using purified opc cultures. nefiracetam increased the ol:opc ratio, as assessed by immunofluorescent staining for mbp and ng2 respectively, suggesting nefiracetam is capable of promoting ol maturation through a direct action on opcs. finally, we investigated the effect of nefiracetam on spontaneous activity. organotypic hippocampal cultures were exposed to lysophospatidylcholine (lpc) for 18hrs to induce demyelination before being allowed to recover. cultures were treated with nefiracetam for 24hrs of the recovery period. spontaneous calcium activity was then measured using fluo4-am. cultures not exposed to lpc displayed low levels of activity which were unaffected by nefiracetam. lpc exposure caused an increase in activity which was enhanced by nefiracetam. additionally, in these cultures, lpc decreased mbp staining compared to control while nefiracetam post-lpc caused a return to control levels. these data suggest increased activity may be an adaptive response related to myelin repair which is enhanced by nefiracetam. together, these findings suggest a complex mechanism of action for nefiracetam as a remyelinating agent involving modulation of wnt signalling, spontaneous activity and opc differentiation. in an assay in which cortical oligodendrocytes ensheath dorsal root ganglion cells 3 we found that nrg and nmda receptors interact to regulate myelination. without nrg, blocking nmda receptors had no effect on myelination. adding nrg increased myelination at all timepoints analysed (2-5 weeks), suggesting that nrg did not merely accelerate myelination. strikingly, nrg led to the majority of myelination becoming dependent on activation of nmda receptors and action potential activity. nrg's effect was associated with a 4-fold increase in nmda receptor current in oligodendrocyte lineage cells. thus, nrg switches myelination from a default programme, which is independent of neuronal activity, to a mechanism that is regulated by glutamate released from active axons. the effects of nrg were associated with an increase in the phosphorylation of akt and the transcription factor creb, but not associated with changes in the phosphorylation of erk. these data reveal a function for oligodendrocyte nmda receptors. the absence of nrg in multiple sclerosis lesions, and enhanced remyelination by added nrg, suggest a role for neuregulin/nmda receptor dependent remyelination after pathology. low-density lipoprotein receptor-related proteins (lrps) are endocytic receptors that internalize several ligands and are involved in lipoprotein homeostasis in adult brain. lrp-2, also known as megalin, is involved in the development of the central nervous system (cns) and in the transport of molecules across the blood brain barrier (bbb). in a previous work, our group revealed that megalin is selectively involved in sonic hedgehog-mediated effects on oligodendrocyte precursor cell migration and proliferation during the development of the cns. although there are some reports describing the presence of lrps in acute lesions of multiple sclerosis (ms) patients and pointing to an important role of shh in the pathogenesis of this disease, there are not data about the expression of megalin in ms tissue. in the present study, we analyse the distribution and the cellular characterisation of megalin in samples of human brain from ms patients to elucidate the role of this receptor in the pathogenesis of demyelination. changes in megalin distribution parallel the different ms histopathological lesions: we detected megalin in active demyelinating lesions and in the periplaque of chronic-active lesions, areas where remyelination spontaneously occurs. on the contrary, megalin was absent in the demyelinated region of chronic lesions. in addition, we observed the up-regulation of megalin in a subpopulation of perivascular astrocytes within the normal appearing grey matter (nagm) of ms patients. moreover, megalin was up-regulated in blood vessels within active lesions, in the periplaque of chronic-active lesions and in the nagm. in parallel, we also analysed the bbb profile to determine structural and functional alterations. altogether, we suggest that megalin is an important receptor expressed in diverse areas of the cns of ms patients representing different aspects of ms development: i) a potential target to improve remyelination; ii) an indicator of a functional rather than a structural disruption of the bbb. therapeutic strategies to modulate specific lrps would be useful to effectively treat ms. bdnf is known to promote central nervous system myelination both in vitro and in vivo. adopting in vitro myelination assays, we identified that bdnf acts through oligodendroglial-expressed trkb receptors to promote myelination. this was verified in vivo, as deletion of trkb in mature oligodendrocytes (in trkb fl/fl mbp-cre mice) resulted in a hypomyelinating phenotype during development. question: here we investigate the consequence of trkb deletion in oligodendrocyte progenitor cells (in trkb fl/fl cnpase-cre mice) and the signalling pathways downstream of trkb that promote myelination. methods and results: trkb fl/fl cnpase-cre mice were born in mendelian ratios and were phenotypically indistinguishable from littermate control mice. surprisingly, analysis of myelinated axonal tracts in the spinal cord and optic nerve revealed normal myelin development. in addition, the number of cells in the oligodendroglial lineage was the same as control mice during postnatal development. these data indicate that the timing of trkb deletion in the oligodendroglial lineage is critical, as deletion in opcs results in no significant phenotype, whereas deletion in mature oligodendrocytes results in a hypomyelinating phenotype. these data suggest that opcs are able to compensate for the loss of trkb and myelinate normally in the absence of bdnf signalling. our in vitro data have shown that the promyelinating influence of bdnf strongly correlates with erk1/2 activation. here we show that overexpression of erk2 in opcs exerts a more significant promyelinating effect than erk1. as erk1/2 are known to exert some of their effects through direct transcription factor phosphorylation, we have undertaken an in silico analysis of myelin-specific transcription factors and identified several that contain potential erk1/2 binding domains and phosphorylation sites. co-immunoprecipitation experiments show an interaction between erk1/2 and these transcription factors. investigation into the nature of these interactions and their functional consequences are ongoing. conclusions: this work suggests a novel role for erk1/2 signalling within oligodendrocytes that regulates cns myelination, possibly via activation of myelin-specific transcription factors. as erk1/2 is activated by multiple receptors, other receptors could potentially compensate for the loss of trkb in opcs, resulting in a normally myelinated cns in vivo. myelin is vulnerable to impairment by genetic, toxic, and immunological triggers, but the molecular basis for many aspects of myelin pathology has remained unknown. to identify molecules required for the structural integrity of central nervous system myelin we have systematically analyzed its protein composition by quantitative proteome analysis. based on the high abundance of filament-forming septins in myelin, we hypothesized that septin filaments may constitute a cytoskeletal membrane cortex in myelin. we find that septin filaments localize to the non-compact adaxonal myelin compartment, which is considered relevant for the metabolic maintenance and the turnover of myelin. for functional analysis, we generated mice lacking the most abundant septin of myelin, sept8, from myelinating glia. our results indicate that sept2, sept4, sept7 and sept9 multimerize with sept8 and that this interaction is essential for their incorporation into myelin. myelin lacking septin filaments display pathogenic outfoldings emerging from adaxonal myelin. we propose that this phenotype reflects that myelin septins provide long-term structural integrity to the adaxonal compartment of cns myelin. here, we used primary opc cultures to investigate the mechanisms underlying gpr17 endogenous regulation. this is quite important since gpr17 is upregulated at injury sites in both a demyelinating in vivo model (boda et al., glia, 2011) and in multiple sclerosis (ms) patients (chen et al., nat neurosci 2009), which, could, in turn, cause defective myelination. in line with these findings, we have recently shown that gpr17 forced over-expression at late differentiation stages, obtained by transfecting cultured opcs with a gfp-gpr17 fusion vector, indeed impairs terminal cell maturation. this suggests that the receptor needs to be down-regulated/ desensitized to allow opc terminal maturation. physiologically, gpr17 downregulation may occur through agonist-induced receptor phosphorylation via g-protein coupled receptor kinases (grks) (daniele et al., j pharm exp ther, 2011; frantangeli et al., j biol chem, 2013), which are, in turn, controlled by mtor kinases, and are altered in ms. our in vitro data show that rapamycin, an inhibitor of mtor kinase, reduces grk2 levels, with parallel increases in gpr17 expression and strong impairment of opc maturation. globally, these data suggest that dysregulation of these interconnected pathways leading to aberrant gpr17 overexpression may prematurely block opc maturation. analysis of gpr17 in vivo in ms models will confirm if the receptor is dysregulated during disease development and will help finding ways to re-establish myelin repair in demyelinating diseases. loss of central nervous system myelin, and the failure of remyelination by oligodendrocytes, contributes to the functional impairment that characterizes neurodegenerative disorders and demyelinating diseases such as multiple sclerosis (ms). since incomplete remyelination will irreversibly damage axonal connections, treatments effectively promoting (re)myelination are pivotal in precluding progression of disease. although the reasons for remyelination failure are likely multifactorial, one of the major impediments to successful remyelination is the altered lesion microenvironment. our previous findings suggest that fibronectin aggregates, as an environmental factor, contribute to remyelination failure. here, we aim at elucidating whether exogenous gangliosides, i.e., cell surface lipids with a potential to modulate fibronectin-cell surface interactions, could overcome fibronectin-mediated inhibition of myelination. of the gangliosides tested, only addition of gd1a, perturbed adherence of oligodendrocyte progenitor cells to fibronectin, but not to poly-l-lysine, without affecting cell viability. gd1a slightly increased growth factor-mediated proliferation on fibronectin, but in the presence of the lipid migration of oligodendrocyte progenitor cells on fibronectin was reduced. furthermore, addition of gd1a increased myelin-like membrane formation on (aggregated) fibronectin, whereas gm1 reduced oligodendrocyte differentiation, as reflected by a decrease in the number of mbp positive cells. most interestingly, gd1a counteracted the myelination inhibiting effect of aggregated fibronectin in co-cultures of dorsal root ganglion neurons and oligodendrocytes. also, gd1a enhanced remyelination in demyelinated cerebellar slice cultures that contained fibronectin aggregates. taken together, gd1a is able to overcome the inhibiting effect of aggregated fibronectin in remyelination. given the persistent presence of these aggregates in ms lesions, gd1a might therefore act as a potentially novel molecular tool to selectively modulate the impeding signaling environment, apparently detrimental to remyelination. its mechanism of action and exploration of gd1a's potential in the treatment of ms, are currently investigated. in the peripheral nervous system (pns) it is secreted by schwann cells and fibroblasts and its expression is strongly induced upon injury. we are interested in the function of apod in the molecular events that take place after a pns injury. we have studied in vivo the process of wallerian degeneration following sciatic nerve crush and we have assayed ex vivo the phagocytosis of labelled myelin from wt and apod-ko mice by flow cytometry. the analysis of cellular processes and protein or mrna expression of a group of signalling molecules induced by injury shows that the lack of apod results in an exacerbated mcp-1 and tnfa-dependent macrophage recruitment. at the injury site, free aa decreases in the wt. lack of apod results in higher basal levels and a stronger injurytriggered depletion of aa. control by apod of the availability of aa to produce the lipid mediators involved in macrophage recruitment is therefore a key mechanism that conditions both wallerian degeneration and injury resolution later on. on the other hand, the phagocytosis of apod-ko cns myelin is less efficient than the phagocytosis of wt myelin. these results indicate that there are also genotype-dependent differences in myelin composition and/or in the interaction between myelin and macrophages. an electron microscopy analysis of crude myelin preparations reveals that the cns myelin from apod-ko mice has abnormal periodicity and shows defective myelin compaction. lipid analysis of apod-ko myelin shows altered phospholipid composition, particularly in phosphoinositid species. a study of the function of apod in the myelination process is currently underway, our results demonstrate that apod function is relevant for both, myelin membrane properties that influence myelin-macrophage interactions, and the control of the lipid-mediated signalling events controlling the extent of macrophage recruitment. adhesion g protein-coupled receptors (ad-gpcrs) are a subgroup of gpcrs that facilitate cell-cell and cell-matrix interaction. structurally, they differ from other gpcrs by the presence of an extremely larger extracellular n-terminal region that is separated from the 7-transmembrane region by a gpcr autoproteolysis-inducing (gain) domain that in most ad-gpcrs facilitates an auto-catalytic process to produce an nand c-terminal fragments during the maturation process. gpr56 is a member of the ad-gpcr family and its mutations are responsible for a human brain malformation known as bilateral frontoparietal polymicrogyria (bfpp). individuals with bfpp present with changes in the white matter tracts in the form of hypomyelination, in addition to cortical malformation. here, we show that gpr56 knockout mice and zebrafish exhibit a reduction of myelin-specific protein expression, fewer myelinated axons and decreased number of mature oligodendrocytes in the central nervous system (cns). during cortical development, gpr56 functions together with its ligand collagen iii to regulate neuronal migration and cortical lamination. however, it is not clear whether collagen iii is also the ligand of gpr56 during cns myelination. gpr56 has a very long and poorly characterized n-terminal fragment, allowing for the possibility of multiple binding partners that mediate cell-extracellular matrix interactions. indeed, gpr56 also binds to tissue transglutaminase (tg2) in melanoma cells. we have begun to study the possible role of collagen iii and tg2 in oligodendrocyte development and cns myelination. the likelihood of these two proteins as being the ligand(s) of gpr56 during oligodendroglial development will be discussed as will our ongoing work to define the role of gpr56 in cns myelination. proper control of cell cycle, proliferation and differentiation is fundamental to regulate oligodendrocyte (ol) development and recruitment of oligodendrocyte precursors (opc) after brain damage. failure in this process results in severe dysmyelinating disorders and defective brain repair. the molecular machinery that couples differentiation to proliferation withdrawal plays therefore a critical role in brain development and repair and is regulated by multiple extracellular cues including extracellular matrix (ecm) and growth factor derived signals. however, how these signals are integrated into ol to control cell cycle progression and differentiation is only partially understood. we are investigating the role of jun activating binding protein 1 (jab1), an intracellular signalling molecule, shuttling between nucleus and cytoplasm, involved in the control of cell proliferation, survival and gene transcription. recent evidence showed that jab1 function is regulated by ecm and growth factors. we demonstrated that jab1 is expressed in glial cells in peripheral and central nervous system (cns), and conditional inactivation of jab1 in schwann cells results in dysmyelinating neuropathy. here we present preliminary data showing that jab1 plays a role in cns development. mice with conditional inactivation of jab1 in ol, by cnp-cre tansgene, show cns hypomyelination, including corpus callosum, optic nerve and spinal cord. concomitant fiber degeneration was observed. our preliminary data suggest that jab1 expression in ol plays a role in cns myelination and possibility axonal survival. c. gonsior, j. trotter university of mainz, mainz, germany myelin basic protein (mbp), a major component of central nervous system (cns) myelin, is essential for oligodendroglial function and myelination in the cns and its expression is tightly regulated in time and space. the mrna of mbp is sorted to cytoplasmic rna granules and transported up to the distal processes of oligodendrocytes in a translationally silenced state. we and others could show that, dependent on axonal signals, activation of the non-receptor tyrosine kinase fyn leads to a release of translational inhibition allowing synthesis of mbp protein at the axon-glial contact site. a key factor in this pathway is the fyn target and rna-binding protein heterogeneous nuclear ribonucleoprotein (hnrnp) a2, that recognizes the a2 response element (a2re) in the mbp 3 0 utr and orchestrates the dynamics of the mbp mrna granule. moreover, we identified hnrnp f as an additional target of fyn kinase present in these ribonucleoprotein complexes and contributing to posttranscriptional regulation of mbp. cell stress conditions, as present in various disease states such as multiple sclerosis, may result in the formation of cytoplasmic rna-containing stress granules (sgs), potentially influencing the fate of myelin transcripts including their translation.mbp mrna was shown to be sorted to these cytoplasmic foci. we here identified the hnrnps a2 and f to be associated with oligodendroglial sgs. de-regulation of such factors involved in mbp mrna metabolism could affect stress dependent synthesis of mbp and thus (re-)myelination and myelin maintenance. we performed the experiments in biozzi abh mice known to develop a chronic relapsing-remitting form of eae following immunization with myelin oligodendrocyte glycoprotein. our findings suggest that in demyelinating lesions, myelin layers are pulled off from the myelin sheath and form bulb-like structures that we call "myelinosomes". we can detect the formation of such structures in acute and in chronic stages in the animal model, as well in biopsies from multiple sclerosis patients. we are currently investigating the molecular interactions that underlie the formation of myelinosomes to gather further insight into the mechanisms that underlie immue-mediated demyelination. adenosine is a potent neuromodulator which can be released from most cells and its extracellular concentration increases dramatically after brain injury. it acts on four g protein-coupled receptors, a 1 , a 2a , a 2b and a 3, and their activation is involved in myelination as well as in apoptosis linked to neurodegenerative diseases. in this study, we initially found that rat oligodendrocytes in vitro express all four adenosine receptor subtypes, adenosine transporters ent1 and ent2 and the adenosine degrading enzymes adenosine deaminase and adenosine kinase. activation of adenosine receptors caused mitochondrial dysfunction and oligodendroglial apoptosis. notably, selective activation of adenosine receptors revealed that a 3 receptors are the main subtype mediating oligodendrocyte death. thus, a 3 receptor agonist 2-ci-ib-meca caused a robust increase in ros levels, mitochondrial membrane depolarization and caspase-dependent apoptosis. in turn, selective a 3 receptor activation induced mapk modifications compatible with cellular damage, which was prevented by the selective antagonist mrs1220. the modulation of g protein and adenylate cyclase suggested that 2-ci-ib-meca acts via camp-pka. moreover, selective activation of a 3 receptor triggered intracellular calcium mobilization via plc pathway. finally, we used cerebellar organotypic slices and optic nerve ex vivo to investigate adenosinemediated oligodendroglial death in more integral preparations. consistent with data in dissociated cultures, a 3 receptor activation induced a significant damage in the optic nerve as well as in the cerebellum white matter, an effect that was prevented by caffeine, an adenosine receptor blocker. together, these results indicate that adenosine can trigger oligodendrocyte death via activation of a 3 receptors, and suggest that this mechanism may contribute to the etiology of demyelinating diseases. question-one of the commonest forms of inherited neuropathy, xlinked charcot marie-tooth disease (cmt1x) leads to progressive distal muscle weakness and sensory loss. the disease is caused by a large number of mutations in the gjb1 gene encoding the gap junction protein connexin32 (cx32). cx32 is expressed by schwann cells in peripheral nerves and forms important channels of communication between the layers of the non-compact myelin sheath and is necessary for the homeostasis and survival of the myelinating cells and the axon. transgenic disease models generated in our lab as well as clinical-genetic analysis of large number of patients have demonstrated that most cmt1x mutations result in loss of cx32 function and failure to form gap junctions. cx32 ko mice offer a well characterized and relevant model of the disease to study future therapies for cmt1x, for which gene replacement may be an effective treatment strategy. methods-for this purpose, we have generated a novel 3 rd generation lentiviral vector to allow gene delivery to peripheral nerves. in this vector, the cx32 open reading frame is placed under the control of cell-specific promoter for schwann cells, the rat mpz/p0 promoter. the expression cassette also includes a green fluorescent protein (egfp) as a marker. we have successfully produced lentiviral vector particles and delivered them to the sciatic nerves of 2-month old wild type (wt) and cx32 ko mice, immediately distal to the sciatic notch. the expression of egfp on wt background and of cx32 on ko background was determined by western blot analysis and immunohistochemistry of teased nerve fibers at different time points after injection. results-our initial studies in wt mice (n 5 15) show that more than 60% of schwann cells from different nerve fascicles expressed egfp as far as 5 mm distal to the injection site. expression started a week after the intraneural injection and remained stable for up to 3 months. when the lentiviral construct was injected into the sciatic nerves of cx32 ko mice (n 5 5), virally delivered cx32 was expressed and was properly localized at the non-compact myelin areas including the paranodal regions and incisures of myelinated fibers, where gap junctions are normally formed. poster abstracts s139 glia conclusions-thus, we have generated lentiviral vector that leads to efficient gene expression specifically in schwann cells. these results indicate that gene replacement therapy is feasible and may lead to correction of the gap junction loss in myelinating cells of the pns. perinatal inflammation is intensively discussed to have a long-term impact on various cell populations and on the development of autoimmune diseases such as multiple sclerosis (ms) in adulthood. in order to determine long-term effects of perinatal inflammation on the course of demyelination in adulthood we mimic a bacterial inflammation by exposure to lipopolysaccharide (lps) of either pregnant or newborn mice. demyelination as a "second hit" was induced by feeding adult mice with the oligodendrocyte toxin cuprizone (bis-cyclohexanone oxaldihydrazone). a single prenatal lps injection at e13.5 did not affect the course of demyelination in adulthood. in contrast, serial postnatal lps injections lead to a delayed demyelination and a reduced number of activated microglia in the corpus callosum, suggesting a long-lasting effect of perinatal inflammation on the function of microglia. surprisingly, mice exposed to lps after birth showed an enhancement of early remyelination accompanied by an increased number of newly differentiated oligodendrocytes. furthermore, independent of cuprizone administration the perinatal lps injections seem to induce a long-term impact on the blood-brain barrier (bbb) by decreasing the number of claudin-5 positive vessels. in summary, perinatal inflammation mimicked by the administration of lps has long-lasting effects on the bbb, microglia activation, and remyelination. question: epigenetic control is crucial for the differentiation of a variety of cells including oligodendrocytes, the myelinating glial cells of the central nervous system. however, studies about the implication of epigenetic factors in peripheral nervous system maturation are just emerging and we were wondering whether methylation of histones is involved in this process. methods: we describe the function of ezh2 in primary schwann cells and drg cocultures using immunohistochemistry, transfection and lentiviral transduction, shrna, qrt-pcr analysis, morphological analysis, western blotting and chromatin immunoprecipitation. results: here, we demonstrate for the first time the impact of a histone methyltransferase, encoded by the enhancer of zeste homolog 2 (ezh2) gene, on schwann cell differentiation. in sciatic nerves, ezh2 expression was found in schwann cells and to peak perinatally. suppression of ezh2 expression in cultured primary rat schwann cells reduced the length of cell processes. these morphological changes were accompanied by widespread alterations in the gene expression pattern, including downregulation of myelin genes and induction of p57kip2, which we have recently identified as an intrinsic inhibitory regulator of schwann cell maturation. in addition, we show that ezh2 suppression in dorsal root ganglion cocultures interferes with in vitro myelination. chromatin immunoprecipitation analysis revealed binding of ezh2 at the p57kip2 promoter and reduction of histone h3k27 trimethylation upon gene suppression. ezh2 suppression-dependent effects on morphology and myelin genes could be reversed by concomitant suppression of p57kip2, indicating that p57kip2 is a downstream effector of ezh2. furthermore, we describe hes5 as transcriptional repressor of myelin genes in schwann cells, which was induced upon ezh2 suppression and downregulated in p57kip2-suppressed schwann cells. conclusions: we have identified a molecular link between histone methylation and control of schwann cell differentiation and demonstrate that this epigenetic mechanism is crucial for glial differentiation to proceed. previous studies, performed primarily in cell culture, suggest that electrical activity may regulate multiple stages of oligodendrocyte development including proliferation, migration, initial wrapping and myelination. in this study, we have examined the requirement for electrical activity in oligodendrocyte development in vivo using zebrafish as a model system. results: we find that electrical activity is not required for proliferation, migration, or the initiation of axon wrapping. rather, our data indicate a specific role for electrical activity in oligodendrocyte differentiation by regulating a subset of myelin genes, including myelin basic protein (mbp). silencing electrical activity with tetrodotoxin reduced mrna expression of mbp, as assessed by rna in situ hybridization and quantitative rt-pcr. in contrast, myelin protein zero, claudin k, claudin 11a, ugt8, and 36k levels were not regulated by electrical activity. ongoing experiments are aimed to uncover the mechanism of activity-dependent mbp mrna expression during oligodendrocyte differentiation. conclusions: taken together, these data indicate that electrical activity can modulate myelin gene expression in vivo but do not support the notion that electrical activity drives key axon-glia messengers that are required for the initial stages of myelination. (fancy et al., 2011) . eight-week-old lgals3-/-and wild type (wt) mice were fed a diet containing 0.2% cpz w/w during 6 weeks, after which cpz was withdrawn in order to evaluate remyelination 2 weeks after. according to our results, cpz-induced demyelination in lgals3-/mice showed an exacerbated astrocytic and microglial response as compared to wt littermates. electron microscopy showed a significant lack of myelinated axons in lgals3-/-mice as compared to controls. furthermore, the few myelinated axons present were nearly 50% less myelinated than those of controls and were found to be collapsed. remarkably, the remyelination process seemed to be faster in lgals3-/-mice than in wt. remyelinated lgals3-/-mice showed a higher myelin basic protein (mbp) recovery rate as compared to their controls. flow cytometry assays showed a sharper microglial response in lgals3-/-mice, which was supported by an exacerbated number of cd11b1 and cd451 cells. however, electron microscopy images from remyelinated lgals3-/-animals showed, again, collapsed axons with a defective myelin wrap, as compared to wt mice showing normal axons without any relevant myelin wrap disruption. behavioral performance observed during cpz treatment recovery correlates with alterations in the morphological studies, which show that neither lgals3-/-nor wt mice reach basal myelination levels. we have shown that new oligodendrocytes (ols) continue to be generated in healthy adult mice after 8 months of age, even in the optic nerves, which are considered to be completely myelinated already [1] . this raises the question of whether the adult-born ols and myelin are engaged in remodeling existing myelin (e.g. intercalating among the existing myelin sheaths), or replacing ols that die in use. to ask whether ols die and are replaced during healthy adulthood we generated transgenic mice that express icreer t2 under the transcriptional control of the myelin basic protein promoter (mbp6-icreer t2 ). the $6kb mbp upstream fragment that we used is reported to direct transcription to ols but not to either ol precursors (ops) or schwann cells [2] . we confirmed that our mbp transgene targets cre recombination specifically to differentiated ols by crossing to ros26-yfp conditional reporters; when tamoxifen was administered to double-transgenic offspring, essentially all yfp 1 cells in the cns were also cc1 1 . when mbp6-icreer t2 is crossed to tau-mgfp conditional reporter mice (mgfp is membrane-tethered) we can visualize differentiated ols and their myelin internodes in white and grey matter, allowing us to follow the fates of myelinating ols for extended periods of time after tamoxifen labeling. following tamoxifen administration to mbp6-icre t2 : tau-mgfp mice on postnatal day 60 (p60) or p120, a large proportion of ols became gfp-labelled in the corpus callosum, optic nerve and spinal cord. examining these mice at increasing times post-tamoxifen will reveal if and how the number and fraction of mgfp-labeled ols/ internodes changes with age -whether there is net loss or gain of ols or whether new ol synthesis is balanced by the rate of ol death (i.e. there is ol turnover). our preliminary results indicate that new ol synthesis exceeds ol death, so the function of adult ol genesis is not simply to replace dying ols. this study aims to identify protein networks that participate in peripheral myelin formation and maintenance. we use a cell culture system, where c57bl/6j mouse embryos (e13.5) are used for the isolation of dorsal root ganglia neuron explants. explants are cultured in matrigel, and endogenous schwann cells appear in the culture within a few days. myelin formation is induced by adding myelination-promoting substances to the culture medium. immunocytochemistry is used to evaluate the myelination efficiency and the developmental timetable of myelination in culture. electron microscopy is used to study structural features of myelin in culture and to compare its ultrastructural features with those of myelin in situ. rna microarray analysis is performed to study gene expression changes during myelin development in culture. the rna expression data indicates the changes in the rna expression levels, but it does not give information about the amount of final protein product, their posttranslational modifications or stability. we combine the rna expression analysis with a proteomics approach. proteins are extracted for 2-dimensional electrophoresis analysis at the same time points, and protein spots exhibiting time-dependent changes in their expression levels are analyzed by mass spectrometry. we show that mouse dorsal root ganglion explant cultures produce substantial amounts of mature myelin in 3 weeks after the induction of myelination. ultrastructural analysis shows that myelin in our cell culture system has the same structural characteristics as those observed in peripheral nerves in situ. rna microarray analysis reveals changes in myelin-, nervous system-and schwann cell-related genes. the most significant changes in gene expression occur at the induction of myelination. at the protein level, peaks of specific proteins can be observed at the first and the last time points, which complement the results obtained using microarray data. we have developed a reproducible and efficient method to study molecular changes in myelination at the proteomic and genomic levels. this approach will be exploited further for the characterization of the molecular networks involved in the myelination process. results: our findings show that primary rat schwann cells respond to ivig stimulation with altered cell morphologies accompanied by an accelerated growth of cellular protrusions in early stages of the differentiation process. stimulation with ivig was also found to reduce cellular proliferation rates without affecting cell survival. furthermore, we observed that ivig treatment transiently boosts myelin gene expression of immature cells, whereas myelin gene expression of differentiating schwann cells is promoted on a long-term scale. importantly, myelin gene expression responses cannot be detected upon application of igg1 control antibodies (herceptin, synagis, avastin). in addition, we could demonstrate that primary rat schwann cells express the cd64 fc receptor and that in differentiating schwann cells cd64 levels were significantly increased. on the other hand, we could also reveal a specific binding of the human ivig on the schwann cell surface. conclusions: we therefore conclude that schwann cells are not only able to respond to but also specifically bind immunoglobulins and that ivig stimulation can promote their differentiation. here, we demonstrate that cnp deficient mice exhibit alterations in mechanical and thermal sensation. histological analyses of cnp deficient mutants reveal loss of sensory axons when quantified in the dorsal root and the predominantly sensory saphenous nerve. in contrast ventral roots remain unaltered. moreover, electron microscopical assessments of saphenous nerves display a hypermyelination of small to mid-sized caliber axons and an overrepresentation of the smallest fibers. the myelin pathology of sensory axons is reflected by alterations of sensory nerve conduction velocities. thus, our data demonstrate that, contrary to findings in the central nervous system, cnp has an impact on myelination and is especially important for the integrity of small to mid-sized caliber axons within the sensory peripheral nervous system. to unravel the pathogenetic mechanism of the s63del mutation, we generated transgenic mice that express p0s63del protein and manifest a demyelinating neuropathy similar to cmt1b. p0s63del is a misfolded protein retained in the er, where it activates a chronic unfolded protein response (upr). the upr is a cellular response to er stress, aimed to reduce the load of abnormal proteins by attenuating protein translation, increasing the er folding capacity and stimulating protein degradation. among the degradation strategies upregulated by the upr is the er-associated-degradation (erad) pathway. in erad, proteins destined for degradation are retrotranslocated from the er to the cytosol, ubiquitylated and degraded by the proteasome. microarray analysis of s63del nerves revealed that several erad genes, such as derlins, are strongly upregulated and derlin-3 expression returns towards normal in association with the amelioration of demyelination produced by ablation of chop, indicating a possible involvement of this pathway in cmt1b neuropathy. derlins elicit particular interest for exerting a protective role in several er stress-mediated diseases. in s63del nerves, derlins co-immunoprecipitate with p0s63del protein and, finally, derlin-1 overexpression reduces the level of er stress in cos7 cells expressing p0s63del protein, suggesting a positive role in the modulation of p0s63del protein retrotranslocation. these observations indicate that derlins may be part of the schwann cell adaptive response that attempts to cope with chronic er stress by modulating p0s63del clearance. we are now in the process of investigating the role of derlins, in the pathophysiology of pns myelination using in vitro, ex vivo and in vivo approaches. during the myelination process, the oligodendrocyte extends processes to and wraps around multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. this is one unique example of how cells in the central nervous system establish asymmetry. transport of mrna and local regulation of protein synthesis represent one mechanism by which such cellular asymmetry can be generated and the different requirements for myelin sheath at each axo-glia interaction can be controlled. the mrna of a key myelin sheath protein, myelin basic protein (mbp), is known to be transported into the oligodendrocyte processes, and a tight temporal and spatial control of mbp mrna translation is thought to be required for normal myelination. the molecular basis for the tight control of mrna transport and translation is the assembly of large mrna-protein complexes. prior work has identified a number of proteins that interact with the mbp mrna, including hnrnp-a2, hnrnp-k and hnrnp-e1. however, it is currently unknown how these protein functions together to prevent translation during transport. it is also unknown how targeting of the mbp mrna to the myelin sheets occurs. to delineate the precise role of the individual binding protein in regulating mbp mrna translation, we have identified regulatory elements within the 3 0 utr of the mbp mrna involved in translational inhibition, and we have identified binding sites in the mrna for hnrnp-k and hnrnp-e1. furthermore, we have analyzed the effect of sirna-mediated knockdown and overexpression of these proteins in primary oligodendrocytes. together, our results allow us to propose a model, in which the individual mrna binding proteins are assigned distinct roles for correct spatial and temporal expression of mbp. remarkably, this manipulation was sufficient to achieve both the expansion of oligodendrocyte precursor cells (opc) and the de novo myelination of a large fraction of enlarged parallel fiber axons in the cerebellar molecular layer, independent of axonal neuregulin-1 expression. by laser-guided microdissection and gene expression profiling of cerebellar gc, we identified neuronal factors that promote opc proliferation and myelin synthesis in vitro. these findings suggest that oligodendrocytes and their precursors respond to multiple 'instructive' signals expressed by cns neurons in vivo, but only on axons with diameters >0.2 lm that became 'permissive' for myelination. l. marziali, p. franco, j. pasquini university of buenos aires, caba, argentina promyelinating effects of both apo-transferrin (atf) and thyroid hormone (th) on rat brain are well documented. th effects are mediated by nuclear receptors which act as transcription factors and regulate different cell processes. previous results from our laboratory showed that th promotes the commitment of oligodendrocyte progenitors (opcs) to mature myelinating oligodendrocytes (olg). on the other hand, atf is able to promote the commitment of neural stem cells (nsc) to cells of the oligodendroglial linage and favors olg maturation after an intracranial injection. our previous demonstration that th administration is able to up-regulate tf mrna expression leads us to hypothesize that both factors converge in the control of oligodendrogenesis. to test if the combined effects of both atf and th are required for proper myelination in the rat brain, studies were done at p10 and p20 for each experimental condition. a hyperthyroid state was rendered by daily subcutaneous th administration from the day of birth (hyper). hypothyroidism was achieved by giving propylthiouracil to the mother from gestational day 18 to the end of the experiment (hypo). half of the hypo pups received an intracranial injection of atf at postnatal day 3 (hypo1atf). a set of euthyroid animals received an intracranial injection of atf at postnatal day 3 (atf). control groups received an intracranial injection or subcutaneous saline solution (c). at p10, hyper animals showed an up-regulation of 55% in tf mrna expression and 87% in protein levels as compared to controls. hypo showed a decrease of 25% in tf mrna levels. this effect was not affected by exogenous administration of atf. at p20, hyper showed no differences in the expression of tf mrna or protein levels as compared to controls. hypo showed a decrease of 25% in tf mrna and a 40% decrease in protein levels. this effect was not affected by exogenous administration of atf. no differences were observed in the expression of insulin growth factor i (igf-i) or igf-i receptor between groups at p10 or p20. immunohistochemical analyses were performed at p20 in all groups usingspecific markers anti caii, anti mbp, anti rip and pdgfra. our conclusion is that, in a hypothyroid state, tf is not able to produce olg and myelin maturation at the corpus callosum, which indicates that th is necessary for atf action. however, the finding that hyperthyroid animals showed a significant increase in tf mrna strongly suggests that tf could be involved in th effects. new experiments are carrying out in order to knock down the tf gen to probe this last piece of evidence. h. yigit, l. meyer, c. gonsior, p. hoch-kraft, j. trotter johannes gutenberg university, mainz, germany myelination is the prerequisite for fast saltatory conductance of action potentials. in the central nervous system, the myelin sheath is produced by oligodendrocytes which extend processes and wrap the axons. this structure is subsequently compacted by the help of certain proteins. of major importance here is the myelin basic protein (mbp) which interacts with the cytoplasmic leaflet of the plasma membrane. mbp is a highly basic protein and binds to the negatively charged plasma membrane with high affinity. it has 6 isoforms in mice produced by alternative splicing. the mostly studied 4 isoforms are 14 kd, 17 kd, 18,5 kd and 21,5 kd. interestingly, exon-ii containing 17 kd and 21,5 kd isoforms are actively transported into the nucleus while 14 kd and 18,5 kd isoforms are rather localized to the distal part of the plasma membrane. transport to the nucleus depends on temperature and energy. recently, it was reported that exon-ii contains a non-canonical py nuclear localization signal. as a cytoplasmic protein, mbp is essential for proper myelination. furthemore mbp is supposed to have multiple additional functions, e.g. in calcium homeostasis and cytoskeleton dynamics. it was also shown that the expression level of mbp exon-ii containing is changed in schizophrenia patients. selectively transport of exon-ii containing isoforms to the nucleus raises the question of its nuclear functions which have not been elucidated yet. this study aims to unravel the role of exon-ii containing mbps in the nucleus. gene expression profiling of the microdissected svz regions revealed that wnt-responsive genes were enriched in the postnatal dorsal svz compared to the lateral svz. we performed inhibition of gsk3b by intraventricular infusion of ara to initiate wnt-signalling. several approaches confirmed activation of the wnt-signalling pathway in the dorsal svz as well as within ops. thus, gene expression profiles of wnt-target genes of the dorsal svz indicated that axin2, lef1, fzdl1 and tcf4, but not those other pathways, were dramatically up-regulated. furthermore, nuclear b-catenin was transiently up-regulated in the dorsal svz including in a large population of sox10-egfp expressing cells following pharmacological wnt-activation. gene expression profiling and immunostainings revealed that the densities of nscs, cycling progenitors and subsequent op differentiation in the dorsal svz were augmented. in parallel, we genetically manipulated wnt-signalling in stem and progenitor cells of the dorsal wall of the lateral ventricle. targeted dorsal svz electroporation of cre-gfp plasmids in floxed stabilised b-catenin (gain-of-function) and floxed b-catenin (loss-of-function) transgenic mouse lines were performed and resulted in a phenocopy of the results observed following ara-intraventricular infusion. in summary, our findings illustrate that wnt-signalling endogenously persists in the dorsal svz after birth. pharmacological as well as genetic interventions reveal an important role of this signalling pathway in the regulation of op genesis during the period of myelination. in the cerebellum the generation of neurons and glia and the relationships between these two lineages are poorly understood. all the cerebellar neuronal phenotypes derive from two distinct embryonic germinal neuroepithelia that are restricted regarding the neuronal cell fate: the rombic lip (rl) gives rise to the glutamatergic neurons, whereas the ventricular zone (vz) generates all gabaergic neurons. in particular, different types of inhibitory interneurons are generated during the postnatal life from a common pool of progenitors expressing the transcription factor pax2. on the other hand, the glial development is relatively unexplored. part of the oligodendrocytes derives from extracerebellar sources, whereas some of them derive from endogenous precursors. astrocytes, instead, may derive from progenitors that delaminate from the vz and continue to divide in the prospective white matter (pwm) up to postnatal life. given the vn origin of both astrocytes and interneurons, we wondered whether a lineage relationship exists between these two cell populations and whether they share the same bipotent progenitor. in order to address these issues, we performed a genetic fate mapping study of glastcreert2 mice. we showed that both cerebellar gabaergic interneurons and astrocytes derive from proliferating glast 1 precursors. in particular, we found that these progenitors are able to produce interneurons at earlier developmental phases, whereas after p6 they appear restricted to the glial lineage. proliferating glast 1 cells that may produce interneurons comprise bergmann glia and pwm progenitors. however, when recombination was specifically induced in bergmann glia, we found that these cells were not neurogenic. therefore, we concluded that glast 1 progenitors reside in the pwm. to get further insight in the behaviour of these precursors, we injected in the pwm a gfp-containing lentiviral vector with a specific tropism for astroglial cells. some days after, infected cells generated pax2 1 -interneurons that were able to mature into interneurons of different cortical layers. moreover, in vitro and in vivo clonal analysis of pwm derivatives indicates that the pwm is neurogenic and both astrocytes and interneurons may derive from a common progenitor. evidence is accumulating that neurogenesis in the subventricular zone (svz) is boosted after trauma or ischemia, also through the interaction with surrounding parenchyma or niche cells. nevertheless, the number of newborn neurons that survive and integrate in the damaged areas is negligible, suggesting a non-permissive environment. thus, understanding the complex signaling network guiding neuroblast generation/survival could help identifying strategies to limit negative inputs and promote regeneration. extracellular nucleotides (ents) are among the hypothetical modulators of svz cell functions, especially under pathological conditions where their concentrations raise several folds and they contribute to reactive astrogliosis. few literature data have recently pointed for a role of the p2y 1 receptor subtype (one of the 8 known g protein-coupled nucleotide receptors) in controlling the proliferation and differentiative potential of svz cells. thus, we tested the ability of adpbs, a stable p2y 1 agonist, to modulate stem cell properties in the adult brain, with a focus on the possible modulatory effects exerted by reactive astrocytes. the administration of adpbs in the lateral ventricle of adult mice led to reactive astrogliosis in the surrounding brain parenchima, and to a massive reaction of gfapexpressing precursors and astrocytes in the svz. also proliferation was increased, paralleled by a significant expansion of the population of mash11 transit-amplifying cells and of doublecortin1 neuroblasts. thanks to the conditional glast::creert2 yfp mouse model, we also demonstrated that adpbs promoted the proliferation of glast-expressing progenitors in the neurogenic niche, and sustained their progression towards the generation of rapidly dividing transit-amplifying cells. in vitro the nucleotide analog increased the proliferation of svz cells grown as neurospheres, and their differentiation towards neurons, fully confirming in vivo data. interestingly, a significant enhancement in neurosphere generation was detected when svz cells were initially grown in the supernatant of astrocytes exposed to adpbs, and then shifted to normal medium. this suggests that adpbs stimulates the release of yet-to-be identified astrocytic mediator(s) whose removal from the culture medium boosted proliferation of svz cells. our results further strengthen the notion that the purinergic system is a key regulator of the neurogenic potential of svz cells, both directly and through the involvement of reactive astrocytes. cambridge stem cell institute, cambridge, united kingdom oligodendrocytes, one of the principal glial cell-types of the central nervous system, are critical for neuronal function and represent the primary target of many neurological diseases, including multiple sclerosis and leukodystrophies. despite the recent progress in stem cell research and the possibility to generate various neural cell types in vitro, the derivation of oligodendrocytes from human pluripotent stem cells remains inefficient and unreliable. we employed a new experimental approach termed "direct cellular reprogrammng" which was already succesfully applied for the generation of different types of neurons and neural stem cells. aided by bioinformatic filtering we have generated a pool of seventeen candidate reprogramming factors that could potentially convert fibroblasts directly into oligodendrocyte lineage cells. combinatorial testing of the candidate factors in overexpression experiments has revealed a combination of just two transcription factors that was sufficient to convert mouse embryonic fibroblasts directly into o4-expressing oligodendrocyte precursors. supplementation of additional factors and multicistronic gene delivery strategies render the reprogramming process more efficient and widen the applicability of this protocol. cellular reprogramming offers a new tool for the generation of oligodendrocyte precursors. this approach, that circumvents the protracted specification and differentiation process inherent to the oligodendrocyte lineage will foster the quest for a culture system of human oligodendrocyte lineage cells. transplanted are derived from progenitors within the intestine. to gain further insight in possible stem cell niches the compelling evidence that mouse enteric glia can also be neuronal precursors was related to human tissue. human colon from infants with hirschsprung's disease was investigated with a panel of glial and stem cell markers. tissue samples from infants with hirschsprung's disease were investigated with glial and stem cell markers along the gut axis. the tissue samples from ganglionic, aganglionic and transient segments were immuonstained either for s100/nestin, s100/p75, gfap/nestin and gfap/p75. in all samples investigated, nestin positive ganglia could be found, even in the distal parts with a severe hypo-or aganglionosis. beside nestin positive cells in all segments, there were also different expression pattern glial markers within the ganglia, indicating that distinct phenotypes of glia cells could be found. neural and glial precursor cells are present in the ganglionic as well as in the hypoganglionic segments of hirschsprung's colon, suggesting that these cells might be suitable for ncsc generation and further transplantation. the subventricular zone (svz) of the lateral ventricle, the largest adult stem cell niche, has a striking pinwheel organization specific to regions of adult neurogenesis. the pinwheel's core contains the apical endings of b1 cells and in its periphery two types of ependymal cells: multiciliated (e1) and biciliated (e2) cells. previous studies showed that svz cells are reactivated in response to demyelination. however, the mechanisms inducing svz reactivation in response to inflammatory demyelination such as in multiple sclerosis (ms) are poorly understood. we hypothesize that b1 and e, which are in direct contact with csf through cilia, may play a crucial role in initiation of svz reactivation. in the present study, we examined the svz reactivation in mice in which we targeted an ms-like pathology to the forebrain white matter (t-eae). animals were immunized with subclinical doses of myelin oligodendrocyte protein and after 20 days, received a stereotaxic injection of a cytokine cocktail consisting of tumour necrosis factor (tnfa) and interferon (ifn c) into the corpus callosum. to obtain an en face view of the ventricular surface, we dissected whole mounts of lateral ventricle of control and eae animals at 3, 7, 14 and 28 days post-eae induction (dpi). we investigated by immunohistochemistry whether and how the pinwheel architecture of the svz is modified and ependymal cells are reactivated during disease course of eae. in svz niche of t-eae animals we found that the number of b1 cells increased and peaked at 7 dpi compared to control. the number of e2 cells and pinwheels decreased and then reached control level at 7 dpi. while the number of e1 cells remained steady, their surface area increased at 3dpi and then returned to normal size over time. in addition, the number of their ciliary basal bodies increased during eae time courses, and e1 cells highly expressed gfap in eae animals compared to controls. thus, the findings of our ongoing research suggest that neuroinflammation induces some changes in cell number and morphology of svz niche as well as reactivation of ependymal cells, which altogether may contribute to svz reactivation mechanisms. further study will provide detailed information on the homotypic and heterotypic interactions between pinwheel cells in response to t-eae. to test whether hypothalamic tanycytes act as bona fide neural stem/ progenitor cells in vivo, we analysed their immumoprofile and proliferative potential, and lineage-traced them in vivo. for this, we exploited our earlier findings that in the adult hypothalamus, fibroblast growth factor 10 (fgf10) is expressed exclusively by tanycytes (hajihosseini et al. 2008 mcn 37: 857-68) , a radial glial-cell like population that lines the floor and ventral walls of the third ventricle. using a line of fgf10-lacz reporter mice, we found that fgf101 beta tanycytes express a panel of neural/stem progenitor markers such as nestin, musashi1, blbp and sox2. fgf101 tanycytes also incorporated brdu under cumulative brdu-labelling paradigms, and formed neurospheres in vitro. to directly test the potential of fgf101ve tanycytes, we lineage-traced them at p28 and p70 by activating the constitutive expression of the marker gene, tomato-dsred, in these cells and their descendants. this was achieved by applying tamoxifen to fgf10-creer-t2::rosa26r-tomato double transgenic mice. after short survival time-points, tomato was detected exclusively in tanycytes, whilst with longer survival time-point, tomato1 cells emerged in the neighbouring parenhcyma. the majority of these were neun1 neurons with fine arborizations. we noted that the newly-generated neurons become associated mainly with hypothalamic nuclei that regulate appetite and energy-balance. moreover, they respond to appetite/energy balance regulating signals such as acute food deprivation and leptin administration. our findings establish fgf101 beta-tanycytes as a putative population of neural stem/ progenitor cells that generate appetite/energy balance regulating neurons in the postnatal and adult hypothalamus. we used an inducible transgenic mouse line (hgfap-creert2) to conditionally label and fate map putative gfap(1) nscs populations in the adult svz and spinal cord (sc), and compare their self-renewal and multipotential properties. we evidence that a population of gfap 1 cells that share the morphology and antigenic properties of svz-nscs reside in the dorsal and ventral aspects of the central canal (cc) throughout the spinal cord. these cells are non-proliferative in the intact spinal cord, but incorporate edu, an s-phase marker following spinal cord injury. multipotent yfp-expressing neurospheres (i.e. which derive from recombined gfap-expressing cells) were successfully obtained from both the intact and injured spinal cord, confirming their early gfap origin. notably, only spheres isolated from the injured spinal cord revealed long-term self-renewal properties resembling those of svz neurospheres. altogether, this work highlights discrepancies in the identity of nscs that reside in distinct regions of the adult cns. our observations however reconcile divergent views on the location and identity of spinal cord-nscs by showing a population of multipotent gfap 1 progenitors with limited self-renewal properties co-existing alongside with multipotent, self-renewing ependymal cells within the spinal cord central canal. during development neural precursors (nps) both divide, to expand the cell population, and produce many different kinds of neurons and glia. this balance appears to be regulated by par complex proteins, which polarize neural precursors and can thereby direct daughter cells for different fates. how par complex proteins are regulated to appropriately polarize nps remains unknown. in recent years, regulation of gene function by micrornas has emerged as an important mechanism during development. using bioinformatics we identified the polarity gene pard3 as a candidate target for microrna219 (mir-219). mir-219 is specifically expressed in the developing central nervous system (cns) of vertebrates and mir-219-deficient zebrafish embryos have a deficit of oligodendrocytes, the myelinating glial cells of the cns. because a disruption in polarity could affect the types of cell divisions that nps undergo, thus altering the balance of cell types that arise, we hypothesize that neural precursor maintenance is regulated by modulation of polarity cues through mir-219. by using an in vitro reporter assay we found that mir-219 can downregulate expression of a luciferase gene fused to the pard3 3 0 utr. reduction of pard3 function in zebrafish embryos suppressed the oligodendrocyte phenotype resulting from loss of mir-219 function and injection of a morpholino oligonucleotide designed to block binding of mir-219 to its pard3 target sequence phenocopied mir-219 knock down. together, these data provide strong evidence that pard3 is a functionally relevant in vivo target of mir-219. to further investigate the role of mir-219 function we tested expression of np, radial glial and neuronal markers in mir-219-deficient embryos. the number of cells expressing sox2, a marker of nps and neural stem cells, was significantly increased upon mir-219 knockdown. concomitantly, mir-219-deficient embryos had a dramatic deficit of radial glia and late born neurons. these data provide evidence for a new mechanism of np regulation, in which mir-219 regulates pard3 levels, thereby regulating the transition of dividing neural precursors to differentiated neurons and glia. in demyelinating diseases such as multiple sclerosis myelin repair activities based on recruitment, activation and differentiation of resident progenitor and stem cells can be observed. however, the overall degree of successful remyelination is limited. it is therefore of considerable interest to understand oligodendroglial precursor cell (opc) homeostasis and maturation processes in order to develop remyelination therapies. mesenchymal stem cells (msc) were shown to exert positive immunomodulatory effects, to reduce demyelination, to increase neuroprotection and to promote adult neural stem cell differentiation towards the oligodendroglial lineage. we here addressed whether msc secreted factors can influence primary opcs in a myelin non-permissive environment. methods: to this end we analyzed cellular morphologies, expression and regulation of key genes/proteins involved in oligodendroglial cell fate and maturation upon incubation with mesenchymal stem cell conditioned medium. results: this demonstrated that msc derived soluble factors promote and accelerate oligodendroglial differentiation, even under astrocytic endorsing conditions. accelerated maturation featured elevated levels of 2 0 , 3 0 -cyclic nucleotide 3 0 -phosphodiesterase and myelin basic protein expression, reduced glial fibrillary acidic protein expression and was accompanied by downregulation of prominent inhibitory differentiation factors such as id2 and id4. conclusions: we thus conclude that besides the previously established immunomodulatory and neuroprotective roles of mscs these cells can also positively influence oligodendrogenesis in the adult central nervous system. in this study, we evaluated the effects of epo and the epo splicing variant (vepo) on murine neurogenesis ex vivo. for this purpose, we analyzed the survival of cultured neural stem cells (nscs) either exposed transiently to recombinant protein, or transduced with lentiviral vectors to achieve stable protein expression. in comparison to non-exposed controls, recombinant epo and vepo enhanced survival of nscs ex vivo (epo: 150 6 25%; vepo: 250 6 60% survival). nscs transduced with pcl20-mscv-epo-and pcl20-mscv-vepo showed also higher viability ex vivo compared to nontransduced nscs (epo: 182 6 4% vepo: 131 6 4% survival). furthermore, pcl20-mscv-epo, but not pcl20-mscv-vepo, increased the proportion of differentiated neurons when compared to non-transduced controls. thus, stable expressed vepo may have rather an effect on nsc survival than on nsc differentiation. our results demonstrate that vepos are neuroprotective in vitro. vepo may serve as therapeutic protein for treatment of neurodegenerative disease associated with impaired neurogenesis such as huntington disease. in the subependymal zone (sez) cytogenic niche of the adult mouse brain neural stem cells drive the continuous generation of new cells, mostly of olfactory bulb interneurons, but also of cells of the oligodendroglial lineage. previous reports have shown that newly-born cells exit the sez and migrate to the adjacent corpus callosum (cc) where they differentiate into oligodendroglial cells. however, the real contribution of these niche-derived oligodendrocyte progenitor cells and oligodendrocytes (nopcs and noligos) to the oligodendroglial population of the cc has not been assessed so far. in this study we used the hgfap-cre ert2 3 rosa26-eyfp double transgenic mice in order to label specifically the progeny of sez-located adult neural stem cells. we found that cells expressing markers of opcs, such as pdgfra and olig2, are constantly generated in the sez and migrate to the proximal fraction of the cc. interestingly though, these cells do not remain in the cc for more than 15 days with their contribution to the total population of oligodedroglial cells remaining stably below 2% even in the ageing brain. moreover, immunostaining for markers of cell cycle revealed that a higher fraction of nopcs undergoes mitosis, when compared with local opcs; hence, they constitute almost 25% of proliferating opcs of the cc. notably, during their transient presence in the cc, nopcs express markers of maturing oligodendrocytes and are incorporated in established local cell structures. when the cc is challenged with a focal demyelinating insult the local and the niche-derived oligodendroglial machineries exhibit differential cell kinetics, nopcs increase their contribution to the total pool of oligodendroglial cells; however, their presence remains transient. in the human and mouse retina m€ uller glia (mg) are well known to undergo gliosis in all major types of retinal diseases -which sometimes may even lead to scar formation due to proliferative gliosis. some studies suggest that in the mouse retina mg derived neuronal regeneration can be stimulated, but only to a very limited extent. here, we started to find out, if conditional immortalization might stimulate mg derived proliferative gliosis and /or neuronal regeneration. others and we reported that in the juvenile mouse retina, after retinogenesis is finished, some m€ uller glia shift from a quiescent differentiated state into a proliferative state upon damage of retinal explants ex vivo. we now observed that this process is differentially inducible by mitogens like egf (epidermal growth factor). edu (s-phase marker) pulse chase experiments revealed a tremendous increase in mg proliferation within the first two days, a peak of edu positive cells at day 4 (14 122 sem, n 5 4) and a massive decrease until day 6 (4 120.4 sem, n 5 4) per 400 mm of a central retina section. next, we used transgenic mice with tightly and temporally controlled expression of the proto-oncogene sv40 large t-antigen (csv40lt). it is well reported that csv40lt binds several proteins including the tumor suppressor p53 and retinoblastoma and bypasses cell cycle checkpoints. induction of the csv40lt for 6 days ex vivo led to an overall increase in proliferation compared to control. the number of edu1 cells was 8.5 fold increased (csv40lt: 23 126 sem, n 5 4; control: 3 120.16 sem, n 5 4; p our results so far suggest that induction of csv40lt not only overcomes the proliferative restriction of m€ uller glia but also maintains its progeny in the cell cycle over extended period of time. surprisingly, major parts of the generated cell progeny formed gliotic cell clusters, which were all located within the boundaries of the retina. further, we present data of successful m€ uller glia isolation at high purity by fac-sorting. in our current and future work we study the m€ uller glia and its derived progeny to find out the underlying mechanisms that enable neuronal regeneration and prevent gliotic scar formation. in mammals, regeneration capacity of the central nervous system (cns) is considered too low to recover the neurological functions after traumatic injury. in fishes and amphibians, however, the spontaneous regeneration is vigorous enough to complete the anatomical reconstruction and functional recovery even after severe damage of cns, in which the ependymal cells play the central role to exhibit proliferation, epithelial-mesodermal transition, cell migration, support of regenerating axons and neurogenesis to contribute to recovery from injury. these positive roles of the ependymal cells observed in fish and amphibian damaged cns may be good candidates for treating mammalian cns injury. in this study, we attempted to control the activity of the ependymal cells in the adult rodent spinal cord by virus-mediated gene transfer for imitating the reactions that are observed in the ependymal cells of fish and amphibian damaged cns. we introduced the gene known to be expressed in both the ependymal cells and neural stem cells (nscs) and found the proliferation of ependymal cells. in this case, the ependymal cells expressed glial fibrillary acific protein suggesting that this gene regulates proliferation and differentiation into astrocytes. in addition, introduction of another gene, also known to be expressed in the ependymal cells and nscs, caused cell proliferation without differentiation tendency. these findings indicate that the cell fate of the ependymal cells in the adult rodent spinal cord can be modulated by artificial intervention. future studies will clarify whether cell fate modification in the ependymal cells can contribute to anatomical reconstruction and functional recovery in the adult mammalian damaged spinal cord. methods neural stem cells are isolated from different parts of the human gastrointestinal tract, and also from different compartments (muscle, submucous and mucous layer) using enzymatical digestion approaches. generated neurospheres were screened for neuronal, glial, neural stem cell and pluripotency markers, prior and after differentiation. additional experiments were performed where neurospheres were either co-cultured with tissue blocks from different parts of the brain, or transplanted into brain slices from the rat. results neurospheres could be generated from all areas investigated and differentiated on glas coverslips. nestin and musashi-1 could be found in all neurospheres. moreover sox-10, nanog and oct-4 could be found in the differentiating cultures. after differentiation tubulin and pgp 9.5 positive neurons could be found. transplantation experiments showed that cells from the transplanted spheres migrated into the brain slices and differentiated into neurons or glial cells. neurospheres co-cultured with tissue blocks from cortex, cerebellum or hippocampus showed a significant higher outgrowth than neurospheres cultured without any brain tissue. conclusions in general, the ens is a suitable tissue for the isolation of neural stem cells in the individual patient and might so be an appropriate source for autologous neural stem cells. neurogenesis. at the end of embryogenesis, oligodendrocytes and astrocytes appear during the process of gliogenesis. although in the last decades diverse extrinsic and intrinsic factors involved in these developmental processes were identified, our understanding of detailed mechanisms is still comparatively low. in order to increase the knowledge about the cellular and molecular mechanisms that underlie central nervous system development, transfection of nscs showed to be a promising method to understand the function of specific genes and proteins. to overcome the well-known difficulties to transfect cells of the central nervous system, we improved the electroporation conditions to achieve high efficiency transfection and survival rates for embryonic and adult nscs isolated from mouse forebrain structures. one day after electroporation transfection and viability rates of around 80% were achieved in murine nscs (bertram et al. j neurosci meth 2012). due to our interest in differentiation and maturation of oligodendrocytes we are currently improving the transfection conditions of oligodendrocyte precursor cells (opcs), since a sufficient protocol for the transfection of opcs derived from neonatal rat cortices is currently not established. the opcs are isolated from rat cortical tissue by a shaking protocol and transfected with the 4d-nucleofector (lonza). when we used the same pulse protocol that successfully works on nscs, oligodendrocytes were sensitive to the transfection process, and many cells died or did not undergo normal differentiation. this demonstrates that other pulses are needed to electroporate opcs. in cooperation with lonza we tested several recommended pulses to increase the transfection and survival rates of opcs. a dramatic increase of the viability rate has already been achieved. in further experiments the pulse protocol will be improved to obtain transfection rates above 20%. by establishing this transfection protocol we want to accomplish transfection and viability rates comparable to murine nscs. this improved protocol for the transfection of sensitive opcs will allow for reliable studies of gene function by over-expression or knockdown experiments, and will enhance our understanding of cell biological mechanisms important for oligodendrocyte development. recent studies demonstrate that astroglial cells isolated from non-neurogenic brain regions have the potential to be reprogrammed into functional neurons through forced expression of transcription factors known to instruct neurogenesis. based on our previous studies on the potential of the neurogenic gene cend1 in directing neural stem/precursor cells (nsc) to exit the cell cycle and acquire a neuronal phenotype, in parallel with evidence demonstrating activation of cend1 expression by genes of the neurogenin family, we explored the combined effect of cend1 and neurogenin-2 on their reprogramming potential on postnatal cortical astrocytes. to this end, forced expression of either cend1, neurogenin-2 or both, resulted in an important increase of two morphologically distinct subpopulations of gfapcells with elongated morphology, that strongly expressed the radial glial marker glutamate transporter glt-1. further characterization revealed that a subpopulation of these cells differentiates towards the neuronal lineage, as they were exhibiting a differentiated neuronal morphology and expressed b-iii tubulin, as well as neuronal subtype-specific markers, including gaba and th. further analysis using long time live cell imaging and brdu cumulative labeling revealed that the majority of cend1-transduced astrocytes undergo 1-2 cell divisions before differentiating to gabaergic neurons, whereas most ngn2 1 astrocytes directly transdifferentiate giving rise to th 1 neurons. additionally, only in the doubletransduced cultures, cend1 1 /ngn2 1 astrocytes seemed to take a step back forming colonies of small round glast 1 /nestin 1 cells, detected 24h following transduction. a day later, these colonies grew as three-dimensional spheres of high proliferative potential attached to the culture dish. when 'astrospheres' were isolated and cultured under nsc conditions, they grew as neurospheres and were propagated for over ten passages. importantly, as soon as 'astrospheres' were cultured in the absence of growth factors, they differentiated into neurons, astrocytes and oligodendrocytes, implying that they possess neural stem cell properties. at the moment we are performing electrophysiology recordings to assess the functionality of cend1derived neurons in vitro, while studies are in progress to explore the potential role of cend1 and ngn2 on astrocytic reprogramming in vivo following cortical brain injury. average membrane potential (v m ) was 277 mv and input resistance (ir) was 92 mx, while dcx 1 or map2 1 cells expressing fast activating outwardly rectifying k 1 currents (ka), and delayed outwardly rectifying k 1 currents (kdr) were defined by a v m of 272 mv and high values of ir (1943 mx). ng2 1 cells with a complex current pattern expressed inwardly rectifying k 1 (kir) currents, in addition to kdr and ka currents, and their v m was 276 mv and ir was 383 mx. the electrophysiological analysis revealed that the inhibition of the wnt signaling pathway significantly increased the incidence of cells with a complex current pattern, while the number of cells with the marked expression of outwardly rectifying k 1 currents declined. wnt signaling inhibition also resulted in increased kir and decreased ka current amplitudes. 4oht-treated cells were also hyperpolarized when compared to controls. immunocytochemistry using antibodies against map2 and dcx showed that neuronal progenitors in 4oht-treated cultures were less developed, having fewer and less branched processes. in summary, our data imply that the canonical wnt pathway in the neonatal svz promotes nsc differentiation into cells with neuronal characteristics. gacr p303/12/0855; p304/12/g069. adult neurogenesis plays a critical role in the overall health of, and repair processes occurring within the nervous system. while a great deal of information has been gained about the elements of the neurogenic niche and the factors that mediate neurogenesis, many unanswered questions remain. specifically, studies suggest that microglia have a significant impact on adult neurogenesis, but no unifying theory exists to explain their exact role in this process. in order to determine if microglia play a pivotal role in adult hippocampal neurogenesis and to examine the mechanisms through which microglia exert their effects on the hippocampal neurogenic niche, we employed a transgenic mouse model that allows for the selective ablation of microglia in the central nervous system (cns), the cd11b-herpes simplex virus thymidine kinase (cd11b-hsvtk) mouse. using this system, we removed microglia from the adult hippocampal neurogenic niche and evaluated the effects of this manipulation on neurogenesis and hippocampal homeostasis, including immunohistochemical analyses of stem cell survival and proliferation, as well as analyses of neuroblast development, migration and electrophysiological activity. taken together, these studies help to provide a better understanding of the factors governing adult neurogenesis and further our knowledge about the increasingly diverse role of microglia in the cns. the current assumption is that ependymal damage, caused by viral infections may result in reactive gliosis in the subventricular zone (svz), leading to sgns. however, as the svz is currently recognized as one of the neurogenic niches in the adult human brain, and svz astrocytes have been identified as neural stem cells (nscs), the assumption of sgns being reactive astrocytes has to be reconsidered. therefore, we have immunophenotyped the cell types present in these structures in the brains of individuals of various ages. in addition, we assessed nodule incidence in the svz in cases infected with hiv, where cells in the svz are likely to be to be affected. we found sgns in almost 90% of all cases, independent of age or hiv infection. we determined that cells in the sgns express adult nsc markers, rather than markers for reactive astrocytes. we hypothesized that direct contact of the nscs with cerebrospinal fluid (csf) may induce nodule formation. indeed, we could show that human ventricular csf stimulated the proliferation of human nscs in culture. from our data, we conclude that sgns most likely are formed upon ependymal damage in response to exposure to csf and represent local expansions of the svz. . in experimental murine model of demyelination, the repair capacity is robust and sufficient, even if it might decrease with aging. one major therapeutic goal in ms is to favour endogenous myelin repair, to prevent neurodegeneration and disability progression. using a transcriptomic approach on pure populations of opcs, we try to identify mechanisms specifically involved in opcs activation. we set up a method to isolate a purified population of opcs, by fluorescent-activated cell sorting from pdgfar::gfp mouse brains. we created the gene expression profile of neonatal opcs, adult opcs and adult oligodendrocytes (ols) in control conditions and of adult opcs in cuprizone-induced demyelinating conditions. we analyzed the transcriptomic profile of adult opcs, neo-natal opcs and ols, and identified more than 1000 differentially expressed genes. after demyelination, we identified more than 2000 adult-opc-specific genes that are either up-or down-regulated compared to control conditions. preliminary analysis suggests that adult opcs are more "mature" than neonatal opcs and partially revert to a more immature profile after demyelination. bio-informatic analysis is ongoing, with the perspective of identifying key candidates for myelin repair capacity, then develop functional experiments to validate their involvement in remyelination. in parallel, we plan to compare the transcriptomic profile of adult opcs from aged versus young mice, to get insight into age-related decrease of repair efficacy. question: after damage, astrocytes are activated and exert specific roles for central nervous system (cns) repair. among these, astrocytes within the injury site may represent a novel source of cells with stem cell potential. in this study, we used sedimentation field-flow fractionation (sdfff) method to rapidly sort well preserved astrocyte subpopulations and we identified stem cell properties within one of these subpopulations. methods: cells were collected from newborn rat cortex, separated mechanically and subjected to sdfff. cell fractions obtained were analyzed by immunocytofluorescence using antibodies against specific epitopes: glial fibrillary acidic protein (gfap), o4, b iii tubulin, and cd68, labelling respectively astrocytes, oligodendrocytes, neurons, and microglial cells. then, proteomic analysis was performed on astrocyte subpopulations and their behaviour in culture was analyzed, particularly under culture conditions usually allowing neurosphere development. results: three main fractions (f1, f2, and f3) were isolated and compared with the total eluted population (tp). after one week in vitro, tp and f2 derived cell cultures contained respectively 74.5% and 11.9% of gfap expressing astrocytes while this percentage was extremely high in f1 and f3 derived cell cultures (95.6% and 98.0% respectively). f3 derived cells displayed higher migratory capacities than those of f1. proteomic analysis of f1 and f3 derived cells indicated a high expression of vimentin in f3 derived cells. in the presence of epidermal growth factor (egf), f3 derived cells formed neurospheres containing cells expressing gfap and nestin (figure) that, when placed in appropriate conditions, were able to generate the three major cell types of the cns (neurons, astrocytes and oligendrocytes). moreover, colony-forming cell assay in a collagen-containing semi-solid matrix allowed in the presence of egf the formation of large colonies. in addition, 7 days after cortical lesion in adult rats, cells presenting similar stem properties were obtained after sdfff cell sorting. conclusions. our study demonstrates that sdfff enables the isolation of almost pure astrocyte subpopulations after one week in vitro. in addition, after sdfff cell sorting, we isolated an astrocyte subpopulation expressing vimentin and displaying the self-renewal and multipotency properties of neural stem cells. our data support that sdfff is an efficient tool to explore the different astrocyte subpopulations of the cns, particularly those involved in cns repair. experimental studies have shown that loss of myelin results in axonal loss and disability. so, finding of an expandable and autologous source of myelin-forming cells to enhance remyelination is required. induced pluripotent stem cell-derived neural progenitor cells (ipsc-npcs) have been developed recently. the remyelination potential and safety of these cells still remained to be well-addressed. the main goal of this study is to characterize mouse ips-npcs in vitro and in vivo after transplantation. we used embryonic mouse neural progenitor cells (mnpcs) as control and characterized mouse ips-npcs for their expression of major markers of immature or mature cells by immunostaining. rt-pcr was performed to analyze expression of nestin, olig2, b3-tubulin, olig1, sox10, and nkx2.2 mrnas. furthermore, to investigate the fate of these cells in vivo, we injected the lysolecithin to induce focal demyelination in the spinal cord of adult nude or shiverer mice. we transplanted cells in the lesion site 2 days after demyelination. animals were sacrificed 2 days, 1, 2, 6 and 8 weeks post transplantation to assess survival and differentiation of the grafted cells. our preliminary results showed that mips-npcs similar to mnpcs were nestin1, ki671, olig21, b3-tubulin1 but o4-and map5-. mips-npcs, were more proliferative than mnpc. moreover, mips-npc expressed all mrna transcripts except sox10. a first line of mips-npcs, was transplanted in the newborn shiverer:rag forebrain or the demyelinated spinal cord of immunosuppressed shiverer mice with a constant failure in terms of cell survival beyond 2 days of transplantation, highlighting the fragility of some of the reprogrammed cell lines. cells of a second line of mips-npcs were transplanted in nude mice to be sacrificed at 1, 2 and 6 weeks. an additional serie of transplantation was performed in the demyelinated shi:rag spinal cord. data at 1 and 2 weeks indicate excellent survival and integration of the mips-npc at both time points. some of the grafted cells expressed gfap, olig2 and ki67 (few only) and so far, no tumors were observed at these timepoints. immunohistochemistry with other markers and analysis of 6 weeks post transplantation animals, are in progress. our preliminary results show that although mips-npcs have very similar immature phenotype of mnpcs in vitro, they may have different survival capacities in vivo. future studies will reveal whether transplanted cells which showed short-term survival after grafting are capable of differentiation into myelin-forming cells. . however these putative stem cells have only been partly characterized in vitro and their therapeutic potential for cns repair has not been addressed. we cultured adult mouse drg cells in sphere-forming conditions. in parallel, we derived spheres from adult spinal cord of the same animals and compared their properties. this last tissue contains well-described stem/progenitor cells whose repair capacities have already been proven for spinal cord injury but not for primary demyelination. in vitro, regardless of their origins, all tissues generated self-renewable spheres up to 10 passages. rt-pcr and immunocytochemistry revealed that sphere-forming cells actively proliferate and express neural stem cells markers. drg spheres were multipotent: as they differentiated into schwann cells, neurons and myofibroblasts, whereas spinal cord spheres differentiated into astrocytes, neurons and oligodendrocytes. we then compared the integration, differentiation potential and remyelination capacity of primary sphereforming cells isolated from actin-gfp transgenic mice after their engraftment in two distinct environments: the adult nude lpc-demyelinated spinal cord and the developing newborn shiverer forebrain. both cell types survived but with distinct integration and differentiation patterns. in the mbp deficient mutant, spinal cord cells exhibit a substantial tropism toward white matter tracts and differentiated mainly into myelinating oligodendrocytes while drg cells were largely distributed in the parenchyma in close association with blood vessels and differentiated in vascular smooth muscle cells. surprisingly, after focal demyelination, in the adult spinal cord, drg cells migrated extensively towards the lesion and differentiated into remyelinating schwann cells whereas adult spinal cord cells migrated less efficiently, and gave rise to oligodendrocytes and a large population of astrocytes. our study reveals that both cell types present different but promising potential for cns remyelination. funding by ela foudation, dim nerf, arsep foundation. ischemia-induced progenitor cell proliferation is a prominent example of the adult mammalian brain's ability to regenerate injured tissue resulting from pathophysiological processes. a key locus of regeneration is the neurogenic niche located in the subgranular zone of the dentate gyrus (sgz). within the sgz, radial glia-like progenitor cells generate neural precursor cells which migrate into the granule cell layer (gcl) of the dentate gyrus, mature, and integrate into the hippocampal synaptic network. in order to better understand and ultimately exploit the cell signaling mechanisms that regulate ischemia-induced proliferation in the sgz, we tested the role of the p42/44 mitogen-activated protein kinase (mapk) cascade effector ribosomal s6 kinase (rsk) in this process. using the endothelin-1 ischemia model in c57bl/6 mice, we report that intrahippocampal cerebral ischemia triggered rsk activation in sgz progenitor cells. further, microinjection of a rsk inhibitor significantly reduced ischemia-induced sgz progenitor cell proliferation. using the neurosphere assay, we also show that sgz-and subventricular zone (svz)-derived adult neural stem cells (nsc) exhibit a significant reduction in proliferation in the presence of rsk and mapk inhibitors. taken together, these data indicate that rsk functions as a cell-autonomous regulator of ischemia-induced progenitor cell proliferation. here we demonstrated that the inpcs not only possess npc-specific marker genes, but also have qualitites of primary brain-derived npcs (wt-npcs), including tripotent differentiation potential, mature neuron differentiation capability and synapse formation. moreover, the mature neurons derived from inpcs exhibit significant physiological properties, such as potassium channel activity and generation of action potential-like spikes. importantly, besides survival, the transplanted inpcs exhibit the migratory property responding to inflammatory stimulation in vivo. these results suggest that directly reprogrammed inpcs closely resemble wt-npcs, which may suggest an alternative strategy to overcome the restricted proliferative and lineage potential of induced neurons (incs) and broaden applications of cell therapy in the treatment of neurodegenerative disorders. the pathogenesis of neurodegenerative disorders, including human immunodeficiency virus (hiv)21 associated neurocognitive disorders (hand), is exacerbated by an imbalance between metalloproteinases (mmps) and their inhibitors, tissue inhibitors of metalloproteinases (timps). as the timps exhibit diverse non-classical functions including anti-apoptotic effects, the induction of timp-1 in neuroinflammatory conditions likely serves multiple roles in addition to modulating mmp activity. our work demonstrates differential timp-1 expression in acute versus chronic activation of astrocytes and hiv-1 associated dementia (had) brain tissues. the timp-1 promoter harbors five consensus ccaat boxes. ccaat enhancer binding protein (c/ebp)b levels were elevated in brain specimens from hiv-1 patients and this transcription factor regulated astrocyte timp-1 expression. hiv-relevant stimuli increased c/ebpb expression in human astrocytes and localized the factor to the nucleus. overexpressing c/ebpb in astrocytes increased timp-1 promoter activity, mrna and protein expression, while knockdown of c/ebpb decreased timp-1 mrna and protein expression. erk1/2 activation is critical for il-1b-mediated astrocyte timp-1 expression and p38k activation contributes to il-1b-induced astrocyte timp-1 and c/ebpb expression. these data suggest that erk1/2 signals downstream of c/ ebpb to facilitate il-1b-induced astrocyte timp-1 expression. the role of astrocyte-timp-1 as a neurotrophic factor was examined. interestingly, cotreatment with timp-1 protected neurons from apoptosis and reversed neuronal morphological changes induced by staurosporine (sts) and hiv-1 ada virus. further, the anti-apoptotic effect was specific to timp-1 and partially independent of mmp-inhibition. additionally, timp-1 modulated the bcl-2 family of proteins and inhibited opening of mitochondrial permeability transition pores induced by hiv-1 or sts. together, these findings describe a novel function, regulatory mechanism and direct role of astrocyte-timp-1 in neuroprotection suggesting its therapeutic potential in had and possibly in other neurodegenerative diseases. mounting evidence and our pervious study has also confirmed that the axon outgrowth inhibition receptor ngr was also expressed on microglia, and regulated cell adhesion and migration behavior in vitro. however, microglia has been proposed to play a pivotal role in the neuroinflammation through expressing a range of proinflammatory enzymes and proinflammatory cytokines under pathological stimulus. these findings initiate the possibility of nogo/ngr signal on mediating neural inflammation processes during cns injury beside neurite outgrowth inhibition. in the present study, we found that nogo-p4, a 25-aa core inhibitory peptide sequence of nogo-66, was able to induce microglia expressing cox-2, inos, as well as releasing proinflammatory cytokines including il-1b, no and pge2, which could been reversed by nep1-40 or pi-plc treatment. microglia stimulated with nogo-p4 increased the phosphorylation lever of signal transducer and activator of transcription 3 (stat3). the activation of stat3 further mediated the promotion of nogo-p4 on microglia expression proinflammatory factors. furthermore, administration nep1-40 was capable to attenuate the inflammation response in a mouse spinal cord compression injury model by reducing the expression of several proinflammatory mediators, as well as attenuating the activation of stat3. taken together, our results demonstrated, for the first time, that nogo-66 may directly take part in cns inflammation process by influencing microglia expressing proinflammatory factors, which was related to ngr /stat3 signal pathway. these results imply that the interaction of nogo-66 with ngr expressed on microglia may participate in diverse cns diseases related to neural inflammation, including trauma, stroke, and neurodegeneration diseases, etc. our results indicate that ccl-1 is one of the key molecules in pathogenesis and ccl-1/ccr-8 signaling system can be a potential target for drug development in the treatment for neuropathic pain. demyelination plays a central role in the pathophysiology of multiple sclerosis (ms) not only because it disrupts nerve conduction, but also because of effects that compromise axonal survival. there is a growing consensus that any effective treatment for ms must include a component that restores or enhances remyelination by endogenous oligodendrocyte progenitor cells (opc). however achieving this goal requires a far better understanding of why remyelination fails in the first place. we now report that fgf9 is selectively up regulated in demyelinating ms lesions and acts to inhibit (re)myelination in vitro. to explore the mechanism involved we compared the activity of fgf9 to that of fgf2, another member of the fgf family with well characterised effects on opc differentiation and myelination. fgf2 acts directly on opc to stimulate proliferation, while at the same restricting their differentiation into mature oligodendrocytes. in contrast, fgf9 is neither an opc mitogen, nor is it able to inhibit opc differentiation directly. however, opc differentiation and myelination in myelinating cultures were inhibited by supernatants harvested from fgf9 treated astrocytes, even in the presence of an excess of fgf9 neutralising antibody. microarray studies were performed to identify astrocyte derived factors responsible for this inhibitory effect. this approach identified several potential candidates that were significantly up regulated by fgf9 in astrocytes. these included lif, a member of the gp130/il6 cytokine family that is generally described as having pro-myelinating effects, although our own studies indicate when present in excess relative to other gp130/il6 family members it will inhibit myelination in vitro. our experiments demonstrate that fgf9-mediated signal transduction in astrocytes disrupts their ability to maintain a growth factor milieu that can support myelination. been shown that inflammation is primarily mediated by macrophages that are recruited to the tissue. similarly, a recent study demonstrated reactive microgliosis in the hypothalamus of mice fed a high-fat diet. in the cns, however, the relevance of microglial activation in response to a high fat diet remains unclear. we aim to determine if microglia activation occurring in response to overnutrition is a cause or consequence of dysregulation of energy homeostasis and obesity. to determine if the absence of microglia (and therefore microglia-mediated inflammation) may influence the metabolic response to a high fat diet, we used a transgenic mouse model, the cd11b-hsvtk mouse (tk), which allows for an inducible, specific ablation of microglia in response to treatment with the nucleoside analog, gancilovir (gcv). mice were treated with gcv for 4 weeks during which time they were maintained on a diet high in fat (60%) or a respective control diet. body weight, food intake, locomotor activity and energy expenditure were measured and compared to wildtype mice. analyses revealed alterations in the metabolic phenotype of mice fed both hfd and control diet in the absence of microglia cells. our findings point towards a physiological role of microglia in energy homeostasis, the study of which will be the focus of further experiments. interleukin-10 (il-10), a cytokine classically related with anti-inflammatory and protective functions in the central nervous system (cns), has been reported to be produced by astrocytes and microglia in different neurodegenerative and neuroinflammatory conditions. in order to study the specific role of il-10 in the cns, we have created a new transgenic mouse with astrocyte-targeted production of il-10 (gfap-il10tg). the aim of this study was to evaluate if production of this cytokine in the cns has any effect on the phenotype of glial cells. for this purpose, coronal cryostat free-floating sections from the brain of both adult transgenic mice and their corresponding wild-type (wt) littermates, were processed for the study of astrocytes using gfap immunohistochemistry and microglia using antibodies against iba1 and several markers commonly related to the activated phenotype of these microglial cells, such as cd16/32 (fc receptor), f4/80, cd11b, cd206, cd150 and mhc-ii. our results showed that astrocyte-targeted production of il-10 induces an increase in the area covered by gfap immunolabelling. microglial cells in these gfap-il10tg animals displayed also morphological changes in specific areas of the brain, including the hippocampus, cortex and cerebellum, characterized by coarser processes and a notable enlargement of the cell body. distinctively, in the hippocampus, microglial cells adopted an elongated morphology parallel to the dendrites of pyramidal neurons. in addition, in the aforementioned areas, microglia exhibited a statistically significant increase in iba1, cd16/32, f4/80 and cd11b markers and "de novo" expression of cd150, whereas no detectable levels of either cd206 or mhc-ii was found. in conclusion, these results indicate that in specific areas, glial cells are influenced by the presence of astrocyte-targeted il-10. further studies are necessary to evaluate whether after injury these transgenic animals will present changes in the microglial and astrocyte activation that can influence the evolution of the lesion. networks. brain-resident macrophages, microglial cells were considered ''resting,'' but it has recently become evident that they actively sense neuronal microenvironment with their motile and ramified processes. they develop specific activities (cytotoxic, neuroprotective or phagocytic) depending on brain molecular context. for example, they contribute directly to the development of neuronal networks by eliminating or maintaining synapses in an activity-dependent manner. morphofunctional plasticity of microglial cells is finely tuned in order to keep brain homeostasis and to ensure such developmental activity. like macrophages at the periphery, microglial cells indeed adopt m1 or m2 phenotypes, distinguishable through their pattern of cytokine expression and of specific cell surface markers, a m1 phenotype being more pro-inflammatory while a m2 phenotype is more related to phagocytosis. linoleic acid (la, 12:2n-6) and alpha-linolenic acid (ala, 18:3n-3) are essential fatty acids that cannot be synthesized de novo by mammals and have to be provided through the diet. they are precursors of arachidonic acid (aa; 20:4n-6) and docosahexaenoic acid (dha; 22:6n-3) respectively which are key structural components of brain cell membrane phospholipids and precursors of bioactive lipid messengers involved in the regulation of inflammation and microglial activity. most aa and dha accumulate in the brain during the perinatal period via placenta and milk. we previously demonstrated that depleting the diet in n-3 pufas from the first day of gestation alters some neuronal functions of the offspring at adulthood. we here hypothesized that this is due to an alteration of microglial mrophofunctional activity in the developing brain. to test this hypothesis, mice were submitted to a diet deficient or not in ala throughout gestation and lactation. microglial morphology, phenotypes and functions were determined in the brain of pups at post-natal day 21 (pnd21). we show that dha levels is decreased in membrane phospholipids of microglia. in addition, microglial cells from mice fed with an ala deficient diet present a drastic increase of the cd36 marker expression combined to a dysregulated cytokine/chemokine expression profile and an altered phagocytic activity. all together, our results show that dietary n-3 pufas deficiency is likely to impair brain innate immune system activity at pnd21. the outcome of such microglial impairment on brain function is discussed. in recent years, one of the most promising therapeutic means for ad was thought to be immunization with specific antibodies against ab. several therapeutic antibodies were developed, however, most of the clinical trials were canceled due to unexpected deaths and neuroinflammatory responses in some patients. the causes and mechanisms of such responses are currently only partially understood. we have recently raised monoclonal antibodies against oligomeric forms of ab and were investigating whether these antibodies would prevent the neurotoxicity of ab oligomers in primary neuronal-glial cerebellar granule cell (cgc) cultures. antibodies were not directly toxic to cgcs when applied at concentrations relevant to concentrations reported to be in blood serum. however, surprisingly, the antibodies when in complexes with ab oligomers or firbrils dramatically increased the neurotoxicity of ab. similar effects were also observed with antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein vp1, human metapneumovirus nucleocapsid protein and measles virus nucleocapsid protein strongly potentiated the neurotoxicity of their antigens. the neurotoxicity of antibody-oligomeric antigen complexes was abolished by removal of the fc region from the monoclonal antibodies or by the removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. in conclusion, we find that immune complexes formed by ab oligomers or other oligomeric antigens and their specific monoclonal antibodies can cause neuronal death in primary neuronal-glial cultures via fc-dependent microglial activation. the results suggest that therapies resulting in antibodies to oligomeric ab or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation. additionally, if endogenous antibodies contribute to neuronal loss in ad or viral encephalitis, suppression of microglial activation may be therapeutic. antidepressants have often been recommended for potential treatment of chronic pain, especially for neuropathic pain caused by nerve damage. it is known that nerve injury-induced activation of microglia and astroglia can be a cause for the development of neuropathic pain. recent data indicate that abnormal neuronal activity mediated by glia activation may influence the level of neuroimmune signaling molecules in chronic pain. however, it remains unclear whether the mechanism underlying antidepressant effects is related to glia activation. this study investigated the potential pain-relieving properties of milnacipran and venlafaxine, selective serotonin/noradrenaline reuptake inhibitors (snri), in neuropathic pain and attempted to resolve how these antidepressants affect the factors synthesized by the activated glia in neuropathy. chronic constriction injury (cci) was performed in wistar rats by loose ligation of the sciatic nerve. after a single intraperitoneal (i.p.) injection of antidepressants, the responses to mechanical and thermal stimuli in neuropathic rats were evaluated 7 days after cci by the von frey test and the cold plate test, respectively. to confirm the influence of antidepressants on microglia and astroglia, western blot analysis of iba-1 (microglia marker) and gfap (astroglia marker) in the spinal cord and dorsal root ganglions (drg) was performed. the experiments were carried out according to the iasp and local bioethics committee rules. our findings confirmed that the administration of milnacipran (20 mg/kg) and venlafaxine (10 mg/kg) elicited antiallodynic and antihyperalgesic effects in cci-exposed rats. western blot analysis showed that milnacipran elevated the level of iba-1 protein in the spinal cord (but not in drg) in cci-exposed rats. venlafaxine decreased the cci-induced iba-1 protein level in the spinal cord and in drg. we observed no changes in gfap in the spinal cord and drg after milnacipran and venlafaxine administration. these results provide a substantial evidence that both used antidepressants belonging to snri attenuated allodynia and hyperalgesia in an animal model of neuropathic pain, although their effect on microglial cells and astrocytes was different, namely, milnacipran increased the microglia activation, in contrast to venlafaxine. this effect can be explained by comparing the effects of these drugs during their repeated application, and relevant experiments are currently in progress. myeloid cells can respond to intra-and extracellular danger/stress signals by inflammasome-mediated activation. this process consists of two steps: toll-like receptor-induced production of pro-il-1b followed by inflammasome-mediated cleavage and secretion of bioactive il-1b. inflammasome-mediated activation is strictly regulated by expression of regulatory proteins and by inhibitory processes like autophagy. recent studies describe that microglia are markedly different from other myeloid cells. they originate from a separate progenitor, are chronically exposed to the neural microenvironment and their activation may be regulated differently. the objective of this study was to determine the expression profile of inflammasome components in primary microglia and to compare inflammasome-mediated microglial activation and its regulation with other myeloid cells. primary microglia, bone marrow-and blood-derived macrophages (bmdms) of adult rhesus macaques of the same donor were profiled for all nod-like receptors (nlrs), inflammasome-associated adaptor proteins, caspases, and regulatory proteins by qpcr. inflammasome function and kinetics were assessed by exposing lps-primed microglia to atp, silica or msu (danger-associated molecular patterns: damps) followed by measuring the transcription of pro-il-1b-encoding mrna and the secretion of il-1b. primary microglia expressed nlrs (nalp1-3, nod1/3/4, aim2, ipaf, naip), adaptor proteins (asc), caspases (1/3-5/7/8), and regulatory proteins (a20), and the expression profile closely resembled that of bmdms. priming of microglia with lps induced high levels of pro-il-1b-encoding mrna, but il-1b protein was only secreted in response to subsequent stimulation with various damps. interestingly, in lpsprimed bmdms the window for inflammasome-mediated activation was 4-8 hours, while lps-primed microglia remained sensitive for inflammasome-mediated activation for at least 20 hours. in conclusion, primary microglia express multiple inflammasome components closely resembling the expression profile of bmdms and they can be induced to form functional inflammasomes. importantly, primed microglia remain sensitive to inflammasome-mediated activation for much longer than other macrophages. we will present data on whether this reflects a difference in negative regulation of the inflammasome, or differences in autophagy or apoptosis sensitivity. objective: the susceptibility of the aging brain to neurodegenerative diseases may in part be attributed to the intrinsic senescence of the microglia. cellular aging or senescence is linked to telomere shortening/dysfunction. evidence is accumulating that aging microglia show morphological changes, referred to as microglial dystrophy, which may be accompanied by accumulation of the iron-binding protein, ferritin. here, we investigated the effect of telomere shortening on microglial morphology, numbers, and activation state in telomerase deficient (terc -/ -) mice, a model of premature aging due to shortened telomeres. methods: male terc 1/1 mice and 3 rd generation terc -/mice, which show a clear aging phenotype, were perfusion-fixed with 4% paraformaldehyde. brains were processed into 60 lm thick horizontal sections. every fourth section was used for stereological estimation of cd11b 1 microglia in the molecular layer of the dentate gyrus by the optical fractionator method. sections were also stained for the microglial markers iba-1 to monitor microglial morphology, cd45 and cd68 to study microglial activation, and ferritin to study microglial aging. further sections were stained for the apoptosis marker cleaved poly (adp-ribose) polymerase (cparp). results: unlike resting microglia in littermate terc 1/1 mice, which display thin, finely branched processes, microglia in terc -/mice exhibited partial retraction and hypertrophy of processes. microglial cd45 and cd68 immunoreactivity were similar in terc 1/1 and terc -/mice. absolute numbers of mac-1 1 microglia were significantly reduced in young and old terc -/versus terc 1/1 mice. an increased microglial density in old terc -/versus terc 1/1 mice could be attributed to a volume reduction of the dentate gyrus. we also observed an increased number of cparp 1 cells in old terc -/versus terc 1/1 mice. this increase was most prominent in the sub-granular zone, an active site of neurogenesis, but also occurred in the molecular layer of the dentate gyrus and might reflect microglial apoptosis. we observed no difference in ferritin staining in terc -/versus terc 1/1 mice. conclusion: our findings suggest that telomere shortening impairs microglial capacity for self-renewal. surprisingly, changes in microglial morphology in terc -/mice occurred without differences in cd45 and cd68 expression. ongoing studies will show if increased numbers of microglia in terc -/mice co-express cparp or ferritin as evidence of increased microglial aging. tetrahydrocannabivarin (d9thcv) and cannabidivarin (cbdv). we have developed a series of new cannabinoid quinones, among them the cbg quinone (vce-003) that shows pparg and cb2 receptors agonism. in addition we have found that vce-003 activates the nrf2/are pathway in neuronal cell lines. in the present study, we investigated the therapeutic potential of vce-003 in the experimental autoimmune encephalomyelitis (eae) model of multiple sclerosis (ms) by immunization con mog 33-55 . vce-003 (5mg/kg ip, daily) was administered to susceptible c57/bl6 mice at the onset of symptomatology. clinical score and weights of mice were daily recorded until the day of sacrifice (28 days post-immunization). vce-003 treatment delayed the onset of disease and ameliorated the symptomatology. histological analysis of spinal cord of eae mice treated with vce-003 showed decreased microglia reactivity and reduced cellular infiltrates, in particular cd4 1 t lymphocytes. double labeling with neurofilament and the myelin protein, rip indicated that vce-003 diminished the axonal damage. demyelination was evaluated by luxol fast blue labeling. changes in the expression of several cytokines, adhesion molecules and nrf2-dependent genes were determined by qrt-pcr. the implication of pparg and cb2 receptors in the beneficial effects of vce-003 in the eae model of ms is being investigated by using specific receptors antagonists. taken together our results support the potential of vce-003 for the treatment of ms and other chronic inflammatory diseases. we found that the lipid molecule lactosylceramide (laccer), is upregulated in ms patients. to better understand the role of laccer in ms, we used an animal model of spms, and found that laccer synthase (b4galt6) is upregulated during disease progression by astrocytes, and that its inhibition halts the progressive phase of the disease, attenuating cytokine and chemokine production by the astrocytes, and reducing the recruitment of inflammatory ly6c high monocytes to the cns. moreover, it also skewed the microglia and monocytes towards a m2 phenotype, and shifted the astrocyte phenotype to support re-myelination and axonal growth. in vitro experiments, using primary cells demonstrated that the inhibition of laccer synthesis directly inhibits cytokines and chemokines production in astrocytes. importantly, similar results were also obtained with human astrocytes, reinforcing the biological importance of our findings. in summary, we identified a novel lipid-signaling pathway that promotes the activation of local cns immunity and neurodegeneration in experimental spms and is a potential therapeutic target for spms. converging clinical studies report an increased prevalence of comorbid neuropsychiatric symptoms, particularly major depressive disorders, in a number of conditions (e.g., aging, obesity) and diseases (e.g., atherosclerosis, congestive heart failure, rheumatoid arthritis) sharing inflammation as a common denominator. by using an experimental approach in mice exposed to innate immune system (iss) stimulation, we demonstrated that induction of depressive-like behavior is mediated by cytokine-induced brain activation of a tryptophan-catabolizing enzyme, the indoleamine 2,3-dioxygenase (ido). in the metabolic syndrome (mets), a widely accepted concept that identifies a cluster of individual risk factors for type 2 diabetes and cardiovascular disease (including obesity, metabolic dysregulations and basal low-grade inflammation), neuropsychiatric symptoms emerge as significant factors for aggravation of the disease and related outcomes. recently, we demonstrated in a model of mets, the diabetic and obese db/db mice that cognitive alterations and increased anxiety-like behavior are related to hippocampal inflammation. on the other hand, depressive-like behavior is not affected by basal inflammation displayed by db/db mice. given the data linking increased depressive-like behavior and ido activation by cytokines in conditions of iss stimulation, the question arises as to whether depressive-like behavior is increased in those conditions in db/db mice. to answer this question, we measured in db/db mice and in their healthy db/1 littermates the effect of a lipopolysaccharide (lps) challenge (5 mg/mouse, ip) on behavioral reactivity in a depression test (the forced swim test, fst), plasma levels and hippocampus expression of inflammatory cytokines and related neuronal targets, and brain ido activity. plasma levels and hippocampus expression of il-1b, il-6, tnfa and il-10 are similarly increased 2h after lps in both db/1 and db/db mice. as expected, brain ido activity and duration of immobility in the fst are increased in lps-treated db/1 mice 24h after lps. on the contrary, induction of brain ido activity is significantly blunted in db/ db mice compared to their db/1 counterparts and no increase of depressive-like behavior is observed. moreover, some data suggest an impairment of the neuroimmune interactions in db/db mice. we need now to understand how obesity and related neurobiological alterations impair ido activation by cytokines and their consequences in terms of vulnerability to infections in mets. in this study, we tested it in a collagenase-induced mouse intracerebral hemorrhage (ich) model using tlr2 knock-out (ko) mice. to induce ich, collagenase or blood was injected into the right caudate putamen in 8-10 week old male mice. tlr2 expression was upregulated in the ipsilateral hemorrhagic tissues of the collagenase-injected mice. brain injury volume and neurological deficits following ich were reduced in the tlr2 ko mice as compared to the wild type (wt) mice. heterologous blood-transfer experiments show that tlr2 signaling in the brain-resident cells, but not leukocytes, contributes to the injury. in our study to elucidate underlying mechanisms, we found that damage in the blood-brain barrier (bbb) integrity following the ich was attenuated in the tlr2 ko mice compared to the wt mice, which may be due to reduced mmp-9 activation in brain astrocyte. the reduced bbb damages accompanies with reduced neutrophil infiltration and proinflammatory gene expression the injured brain parenchyma, which may account for the attenuated brain damage in the tlr2 ko mice after ich. conclusively, these data demonstrate that tlr2 contributes to brain injury following ich by compromising bbb through activating mmp-9 in brain. the pathological relevance of this autoantibody response is unknown, but it is generally assumed that it exacerbates demyelination via activation of complement and/or cell meditated effector mechanisms. however in a majority of cases the mog-specific autoantibody titre is far lower than that required to induce widespread demyelination and exacerbate disease severity in animal models of ms. to explore the possibility mog-specific antibodies may play other more subtle roles in disease pathogenesis we investigated possible effects in myelinating cultures derived from embryonic rat spinal cord; a model system that allows us to explore antibody-dependent effects in the absence of exogenous complement and effector cells. myelinating cultures were treated continuously with monoclonal antibodies specific for either mog (clone z2, igg2a), sulphatide (clone o4, igm), proteolipid protein (plp) (clone o10, igm), or an appropriate isotype control from 18 days in vitro onwards. in the absence of an exogenous source of complement none of these antibodies induced demyelination, but by 24 div had all had a significant inhibitory effect on myelination compared to cultures treated with appropriate isotype control antibodies. to investigate possible mechanisms contributing to this inhibitory effect qpcr arrays were used to determine if this complement-independent effect on myelination was associated changes in expression of immune mediators. unexpectedly we found recognition of antigens exposed at the surface of the myelin sheath induced a rapid increase in expression of three chemokines known to be involved in recruitment of effector t cells into the cns. within 24 hours of adding antibody to the cultures expression of ccl5, ccl20 and cxcl11 increased by at least three orders of magnitude and then declined to baseline over the following five days despite continuous presence of antibody. this transient increase in mrna transcripts for these cytokines resulted in sustained protein synthesis and secretion of biological active products as demonstrated by analysis of culture supernatants. these results challenge the traditional view that myelin-specific autoantibodies contribute to the pathogenesis of diseases such as ms and adem by virtue of their ability to initiate immune-mediated demyelination. we now demonstrate that even in the absence of recruited immune effector mechanisms myelin-specific autoantibodies not only inhibit myelination but trigger secretion of chemokines predicted to trigger or exacerbate inflammation within the cns. the blood-brain barrier (bbb), comprising specialized brain endothelial cells, is crucial in maintaining a controlled environment within the central nervous system (cns) to safeguard normal neuronal function. in multiple sclerosis (ms) the function of the bbb is disturbed, leading to uncontrolled influx of metabolites and immune cells and neuroinflammation. therefore, restoring the function of the bbb may be a novel therapeutic tool to halt disease progression or new lesion formation. we previously showed the importance of the vitamin a metabolite retinoic acid (ra) in bbb function during cns development 1 , where fetal astrocyte-derived ra is needed to induce bbb function during cns embryogenesis. considering the possible involvement of developmental pathways that could regenerate the disrupted bbb, we investigated the possible role for ra in the protection or reinstatement of disrupted bbb function. we show that ra counteracts the deleterious effects of inflammatory cytokines such as tumour necrosis factor (tnf)-a and interferon (ifn)c on bbb function in vitro. moreover, ra diminishes the induction of inflammation-related genes in human brain endothelial cells, and induces general immune quiescence in resting brain endothelium. our preliminary data as well as a recently described study in the cuprizone animal model for demyelination suggest that ra synthesis in the cns is increased under inflammatory conditions. the impact of cns-derived ra on the inflamed bbb, as well as the cellular source is currently under investigation. insight into the ra-synthetic pathway and its protective effect on the bbb may lead to the development of novel therapies which are aimed to restore bbb function and reduce the inflammatory cascade in ms lesions. tissue transglutaminase (tg2) is a multifunctional enzyme whose expression and activity is enhanced during inflammatory processes. we previously observed that the expression of tg2 is increased in monocytes in lesions in post-mortem material of ms patients. moreover, tg2 activity and expression is enhanced in chronic-relapsing experimental autoimmune encephalomyelitis (cr-eae) in rats. in this same ms model, pharmacological inhibition of tg2 activity dramatically reduced clinical symptoms and attenuated the influx of monocytes. in the present study we question whether tg2 plays a role in mouse eae, as transgenic mice have to be used for subsequent in vivo imaging of leukocytes/monocytes during eae. methods and results: eae was induced in tg2 -/mice and littermate wildtype mice (c57bl/6) using mog 35-55 . during the early phase of disease (day 14-15), the clinical symptoms observed were significantly less severe in tg2 -/compared to the wildtype mice. moreover, the maximal clinical score was significantly lower in the knockout mice. there was no difference in the day of onset of disease symptoms between the two groups. secondly, we aimed at visualizing leukocytes/monocytes in the spinal cord of lysm-gfp and cd11c-yfp mice suffering from mog 35-55induced eae using intravital video microscopy. a permanent window was fixed on top of the spinal cord of mice to allow reimaging of the same field of view in the same mouse over the disease course. in a pilot experiment we observed numerous gfp positive cells in the white matter around the imaged blood vessel in the spinal cord of a lysm-gfp eae mouse. although equally present, this was less prominent in a cd11c-yfp mouse suffering from eae. subsequent immunohistochemical analysis revealed that various cell types had infiltrated the spinal of cord of both mice. outlook: to further address the role of tg2 in the infiltration of leukocytes/monocytes into the spinal cord of eae mice in vivo, specific inhibitors for tg2 activity will be administered to the transgenic mice and leukocytes/monocytes will be visualized using intravital video microscopy. z. muneer 1 , c. wiesinger 1 , g. regelsberger 2 , j. berger 1 , s. forss-petter 1 1 medical universty of vienna, center for brain research, vienna, austria 2 medical university of vienna, institute of neurology, vienna, austria x-linked adrenoleukodystrophy (x-ald) is the most frequently occurring peroxisomal neurodegenerative disorder and is often associated with cerebral demyelination and inflammation. x-ald is caused by mutations in the atp-binding cassette sub-family d member 1 (abcd1) gene encoding adrenoleukodystrophy protein (aldp), which is a transporter for coa esters of very long-chain fatty acids (vlcfa) across the peroxisomal membrane. adrenoleukodystrophy related protein (aldrp), another member of the abcd sub-family, encoded by abcd2, is the closest homologue of abcd1. upon over-expression, abcd2 has been shown to compensate for abcd1 deficiency in vitro and in vivo. several lines of evidence imply an important role of microglia/macrophages in the disease progression. here we used mouse peritoneal macrophages (mpmu) to correlate the gene expression levels of candidate modifiers with x-ald associated defects, like accumulation of vlcfa and decreased peroxisomal b-oxidation, in abcd1 and abcd2 single ko mutants as well as in abcd1/abcd2 double deficient (doko) mice. by quantitative rt-pcr analysis, in mpmu from wild type mice the abcd2 mrna was present at about half the level of the abcd1 mrna. abcd1 ko mpmu showed elevated accumulation of vlcfa (c26:0) compared to the mpmu from wild type mice as measured by gc-ms. the vlcfa levels of abcd2 ko mpmu were similar to wild type amounts. interestingly, there was much higher accumulation of vlcfa in the mpmu from doko mice. there were no differences in the mrna expression level of the elovl1 gene, encoding the enzyme catalyzing the elongation step of vlcfa biosynthesis. peroxisomal boxidation activity was decreased in abcd1 ko mpmu compared to wild type level. this defect was strongly enhanced in mpmu from abcd1/ abcd2 doko mice. these results show that the increased accumulation of vlcfa in mpmu from doko mice as compared to abcd1 ko mice was due to a more severe defect in peroxisomal b-oxidation in doko mpmu. we conclude that a substantial expression of abcd2 mrna prevents a more severe metabolic phenotype in abcd1 deficient mouse peritoneal macrophages. this study also supports the validity of abcd2 up-regulation as a potential therapeutic target in x-ald. j. claude, b. linnartz-gerlach, h. neumann reconstructive neurobiology, bonn, germany microglia have innate immune receptors recognizing pathogens and disease-associated molecular patterns but also molecules that could sense the intact tissue. a subfamily of these receptors is the inhibitory signaling sialic acid-binding immunoglobulin-like lectin (siglec) group including siglec-e that has an immunoreceptor tyrosine-based inhibitory motif (itim) in the cytoplasmic tail to suppress activatory microglial signals. in this study, we used primary and stem cell-derived microglia that were modified by lentiviral vectors. here we show that siglec-e is expressed on microglia and is up-regulated following interferon-c (ifnc) treatment. we performed lentiviral knock-down and overexpression of siglec-e. lentiviral overexpression of siglec-e decreased, while knock-down increased the phagocytosis of neural debris and its associated reactive oxygen burst. the extracellular domain of siglec-e linked to a fc-part of immunoglobulin bound to the sialic acid residues of the neuronal glycocalyx. therefore, we co-cultured these modified microglia with primary hippocampal neurons. overexpression and knock-down of siglec-e showed an increase and decrease in relative neurite-length, respectively. the neuroprotective effect of siglec-e was abrogated after removal of the sialic acid residues on the neuronal glycocalyx. treatment with the anti-oxidant trolox abolished the neurotoxic effect of the siglec-e knock-down on neurite length. in summary, our data suggests an immunomodulatory function of siglec-e on microglia which leads to a neuroprotective phenotype by decreasing the production of reactive oxygen species and a reduced phagocytosis rate of neural debris. a. nadjar, c. madore, a. sere, a. aubert, s. lay e university of bordeaux 2, bordeaux, france many epidemiological studies have linked maternal exposure to infections during pregnancy to later development of cognitive disorders in the descendants. these alterations are likely to originate from a generalized neuroinflammation in the fetal brain following activation of the maternal immune system. this neuroinflammatory response relies on microglial cells activation. these latter normally contribute to the development of neuronal networks especially by eliminating/maintaining synapses, a phenomenon also known as synaptic stripping, in optimal conditions of brain development. neonatal inflammation may alter the synaptic stripping capacity of microglial cells. thus, targeting this persistent inflammatory microglial activation could represent an original and promising strategy to improve cognitive performances in the descendants. omega-3 are known as immunomodulators and target microglial cells. we thus hypothesized that enriching the diet with omega-3 from the first day of gestation may impair the development of neurological deficits at adulthood, through limitation of microglial inflammatory response in favor of its synaptic stripping activity. to characterize the beneficial effects of omega-3 on microglial activity, pregnant mice were fed with an omega-3-deficient diet, or a balanced omega-3/ omega-6 diet or supplemented with omega-3 and received a peripheral injection of either lps or saline at e18 of gestation. using the golgi method, we first evaluated the morphology of dendrites and quantified the dendritic spines density as a measure of neuronal networks maturation in the hippocampus. we found that a prenatal exposure to lps increased the number of immature spines and this was reversed by an omega-3 supplemented diet. to correlate these data with alterations of microglia-neurons interactions, we took advantage of the cx3cr1-gfp mice and injected them with a neuronspecific lentivirus containing dsred protein, in the hippocampus, 7 days prior to two-photon experiments. we focused on microglia general dynamic and microglia-neuron physical interactions and found an alteration in microglial behavior and microglia-neurons interactions in pups from lps-treated females. these alterations were reversed by an omega-3 supplemented diet. overall, our data show that alterations of microglia-neurons interactions in the developing brain may explain the neurological disorders observed in the descendants of females with prenatal inflammation. these deleterious effects may be prevented by nutritional strategies. , which is characterized by a relapsing phase with inflammatory cell infiltrates and a remitting period, where patients partially recover. among the different cell types involved in the necessary immunomodulation to allow the relapsing-to-remitting transition, the role of myeloid-derived suppressor cells (mdscs) gains importance. mdscs form a heterogenic population of immature myeloid cells that is able to suppress the inflammatory response. this cells act, among other mechanisms, through arginase-i (arg-i) activity on tcells. a previous study of our group showed that arg-i 1 -mdscs transiently enter the spinal cord of eae mice and takes part in the immune response control by inducing t cell apoptosis around the peak of the clinical score. therefore, changes in the mdsc population during eae should modify the evolution of the disease. the retinoid acid family molecules are used for the treatment of different leukaemia due to their role as mdsc differentiation factors into diverse cell populations, abolishing t cell immunosuppression. am80, a synthetic analogue of the retinoic acid with a higher bioavailability and less side effects than the natural ones, has controversial actions on eae depending on its administration period. in this work, we administered am80 specifically in the critical moment for immune modulation (around the maximum clinical score). drug administration affected mdsc population and clearly worsened the eae course. our results point to endogenous strengthening of mdsc population as a new and promising therapeutic strategy to treat ms by speeding up the transition from the relapsing to the remitting period. sildenafil induces cgmp accumulation by pde5 inhibition. we have demonstrated that sildenafil decreases microgliosis and astrogliosis and proinflammatory cytokines expression in a demyelination model. however, little is known about mechanisms of sildenafil neuroprotection. since nfjb plays an important role in the regulation of glial activation, we examine the hypothesis that nfkb is part of the mechanism underlying the sildenafil neuroprotective effects. five male mice (c57bl/6), 6 weeks-old, were used per group. the groups received for four weeks: 0.2% cuprizone (cpz) mixed into the chow; cpz into the chow plus sildenafil (viagra v r , pfizer, 25 mg/kg) in the drinking water, starting concomitantly (sild-t0) or fifteen days (sild-t15) after initiation of cpz; controls received pure chow/water. cerebella were processed for western blotting and immunofluorescence (if). results showed that cpz increased gfap expression compared to control (astrocytes activation). sild-t0 (but not sild-t15) significantly decreased gfap compared to cpz group. in controls, microglia showed resting phenotype, with thin and branched processes positive to iba1/nfjbp65 (inactive fraction) double labeling. cpz treatment decreased inactive-nfjb expression, indicating that this transcription factor was activated. in agreement, ikba (nfjb inactivating protein) was also decreased. if showed increase of iba-1 expression, indicating microglia activation. sild-t0 strongly increased the nfjbp65 and its inhibitory protein, ikba, suggesting inactivation of nfjb. if showed decreasing in iba-1 labeling, suggesting inactive microglia. the delayed treatment (sild-t15) did not decrease iba-1 or increase inactive-nfjb and ikba expression. in conclusion, treatment with sildenafil concomitant with cpz exposure prevents micro-and astrogliosis in mice, possibly through ikba-nfjb signaling pathway. sildenafil may be suitable to improve neuroinflammatory/neurodegenerative diseases treatment. financial support: cnpq, capes, facepe, fapesp. mt-1&2 levels have been found to be increased in several human neurodegenerative diseases including alzheimer's disease (ad). moreover, mt112 are also upregulated in different ad mouse models, for example tg2576 mice, which show a significant up-regulation of these proteins in the vicinity of the amyloid plaques. in the present study we generated a double transgenic mouse line that develops ad-like pathology in addition to having an overexpresion of mt1, in order to determine the role of mts in different aspects of ad pathology. the results show that the overexpression of mt1 does affect the mortality rate of the tg2576 and control mice in a gender-dependent manner and partially reverses the behavioural phenotype of young (4-5 months) tg2576 mice, reducing the exploratory activity and improving the learning process; in contrast, mt1 does not cause relevant changes in deambulations and anxiety. on the other hand, the amyloid cascade and neuroinflammation is increased in the hippocampus of old (15-16 months) apptgmt mice, but the lower level of gliosis in the hippocampus of young male apptgmt mice suggests that the overexpression of mt1 is capable of reducing inflamatory response well before amyloid plaques are formed but not afterwards. further molecular and immunohistochemistry analyses are underway to give further insight into the role of mts in amyloidosis and neuroinflammation in this ad mouse line. exposure to prenatal inflammation is a risk factor for neurodevelopmental and neurobehavioural abnormalities that manifest in later life in disorders such as autism, schizophrenia and seizure development. the amygdaloid complex is composed of more than 10 nuclei with subdivisions that have different cytoarchitectonic, chemoarchitectonic, and projection characteristics and that are responsible for the processing of higher functions such as memory consolidation and emotional conditioning. it is also known that the basolateral nucleus of the amygdala is implicated in seizure propagation and initiation. seizure onset later in life may be linked to abnormal development of the amygdalaoid complex. neuropeptide y (npy) and its receptors are known to be involved in a number of important functions such as feeding behaviours, anxiety and seizure modulation. npy y1 and y2 receptors are the most abundant in the brain and are expressed at different stages of development. during foetal development different brain regions are populated by neurones and glia at different times and the interface between mother and foetus plays a crucial role in the development process. lipopolysaccharide (lps) induced maternal inflammation is a known animal model for studying the inflammatory effects on the developing foetus. in this study we are investigating the effects of an acute prenatal systemic insult using the lps model on amygdala morphology and structure. embryonic day 12 (e12) c57bl6j pregnant mice receive an intraperionteal injection of lps (50 mg/kg) or 0.9% sterile saline. embryos at e12, e16, e18 and pups at p1, p7 p14 and p40 are taken and used to study the developing hippocampus and amygdala. for each age and treatment group foetal brains, maternal brains, maternal serum and placentas are collected. incidence of preterm birth, resorption rate, litter size and weights are also assessed. immunohistochemistry, western blot and rt-pcr are then used to assess the expression of neuronal and glial markers in the amygdala and surrounding limbic structures. specifically alterations in expression, activation and distribution of npy and its receptors, microglia and astrocytes in the developing amygdala and hippocampus at different stages following lps treatment are being investigated. it is hoped that this study will further enhance our understanding of how the maternal environment influences brain development and, if disturbed at a critical time in the development of the amygdala, may predispose individuals to amygdala related disorders in later life. in vivo imaging of transgenic fluorescent mice in the cns by twophoton laser-scanning microscopy (2p-lsm) has become a powerful tool in neuroscience. for in vivo imaging of the cortex a craniotomy of the skull has to be made. in addition to anaesthesia and analgesia treatment, we occasionally apply anti-inflammatory drugs to prevent immunological activity that can obscure the images. as anti-inflammatory drug we used carprofen ((rs)22-(6-chloro-9h-carbazol-2-yl)propanoic acid), a known cyclooxygenase-2 (cox-2) inhibitor. adult cx3cr1egfp mice in which microglia are labeled by expression of the green fluorescent protein egfp were treated with a single dose of carprofen or with vehicle 12 hours before the craniotomy. in all mice we exposed the right somato-sensory cortex, and quantified the microglial response to a laser-induced micro-lesion. this micro-lesion was caused by increasing the power of the laser for 1 second in the middle of the region of interest. results -conclusions unexpectedly, we observed that carprofen reduced the microglial process motility significantly. the speed with which the processes approached the lesion dropped from 0.5 mm per minute in the untreated mice to 0.2 mm per minute in the treated mice. a molecular understanding of microglia response in inflammatory processes and how anti-inflammatory drugs modify normal microglia response will provide a strong impact in developing treatment strategies for diseases with strong inflammatory components. resting and activated (lipopolysaccharide, lps) cultured cstb-/-and control microglia were analyzed for cytokine production, chemotaxis and phagocytosis. microglia extracted directly from the mouse brains were studied for expression of mhc ii and m1-m2 polarization using flow cytometry. mouse brain cortex (p14) was analysed for m1-m2 microglial phenotypes and the presence of other immunological cells from the periphery. moreover, we have checked myeloid cell population in bone marrow and spleen of p14 animals as well as cytokines' level in blood serum. our results from cultured microglia show that secretion of proinflammatory chemokines is elevated by cstb-/-microglia. also, cstb-/microglia are chemotactically more active compared to the controls whereas their phagocytic activity is decreased. cstb-deficient microglia show decreased expression of mhc ii on the cell surface indicating reduced antigen presentation. activated cstb-/-microglia directly extracted from the brain has predominantly proinflammatory m1 phenotype. cstb-/-mice display inflammatory changes in the peripheral tissues at their early postnatal period. we have registered high concentrations of proinflammatory chemokines and cytokines in blood serum of p14 cstb-/-pups, but no difference in amount of neutrophils and lymphocytes between brains of wild-type and knockout mice. also, in bone marrow and spleen amount of granulocytes is enhanced in cstb-/mice during early postnatal period as well as level of granulocyte macrophage colony-stimulating factor in their blood serum. our results suggest a role for cystatin b in regulation of immune response and that cstb-deficiency is associated with early inflammatory processes both in the brain and the peripheral tissues. therefore, we consider that epm1 should be treated as a disorder combining neuropathological and immunological features. the contribution of glial cells in the pathophysiology of epilepsy is increasingly valued. furthermore, clinical and experimental evidence suggests a direct relationship between epileptic activity and cns inflammation, which is characterized by accumulation, activation and proliferation of microglia and astrocytes. concomitant glia-mediated mechanisms of action of several aeds have been proposed. however, their direct effects on glial cells, especially microglia, have jet been hardly investigated. we aimed to investigate the influence of commonly used aeds on the glial viability and microglial in-/ activation state in a physiological and inflammatory modified in-vitro astroglia/ microglia coculture model. methods:primary astrocytic cultures were prepared from brains of postnatal (p0-p2) wistar rats and cocultured with a physiological amount of 5% (m5), as well as 30% (m30) microglia in order to mimic inflammatory conditions. cocultures were treated for 24 hours with valproic acid (vpa), carbamazepine (cbz), phenytoin (phe) and gabapentine (gbt) with a concentration of 10, 25, 50 and 100 mg/ml. viability and proliferation was measured using the tetrazolium (mtt) assay. the microglial activation state was determined by immunocytochemical labeling using a monoclonal antibody to the ed1 marker. results:m5 and m30 cocultures showed a dose-dependent, significant reduction in glial viability after incubation with phe and cbz. furthermore, low doses of vpa led to highly significant microglial activation in m5 cocultures. however, cbz significantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia in the inflammatory modified m30 cocultures. conclusion: cns inflammation is characterized by a disturbance of glial cell functions. strong microglial activation, a typical hallmark of inflammation, was induced by vpa in m5 and continued in m30 cocultures. with regard to the direct relation between cns inflammation and seizures, vpa seems to be unsuitable to reduce inflammatory condition. the reverse effect was achieved after cbz. we noticed significant microglial inactivation, after incubation of the m30 cocultures. as it has been demonstrated for levetiracetam before, we assume a beneficial therapeutic effect of anti-epileptic drugs (aed) with an antiinflammatory glial potential in epileptic patients with persistent inflammation. microglial cell activation due to homeostatic imbalances of external and/or internal etiology implies, among others, secretion of proinflammatory cytokines. the fact that microglial activation, inflammation and neurodegeneration often coexist, suggests that proinflammatory cytokines might induce and/or enhance neuronal damage. we investigated the neurotoxic impact of microglial activation in organotypic hippocampal slice cultures by exposing them to the toll-like receptor 4 ligand, lipopolysaccharide (lps), for 72 hours. microglial activation was characterized by unbiased estimation of their number (stereology) and quantification of soma and branching morphology (neurolucida v r -based cell reconstructions). moreover, proinflammatory cytokine and nitrite levels in the culture supernatant were estimated by elisa and griess. the neurotoxic impact was assessed by morphology (nissl staining, fluoro-jade v r b) and extracellular electrophysiological recordings of spontaneous and evoked neuronal activity in the hippocampal ca3 subregion. lps exposure induced microglial population expansion, morphological changes, such as process thickening and somatic enlargement, as well as substantial secretion of proinflammatory cytokines (tnfa, il6) and nitrite accumulation in the culture medium. however, these changes did not coincide with neurodegeneration and were associated with only minor effects on neuronal function, as demonstrated by the amplitude of evoked neuronal responses and short-term plasticity properties. we conclude that, in contrast to what has been observed in vivo, chronic microglial activation by lps is not sufficient to drive neuronal death and dysfunction in organotypic hippocampal slice cultures. the neonatal brain is particularly susceptible to oxidative stress. our group has previously shown that following hypoxic-ischemic injury, hydrogen peroxide (h 2 o 2 ) levels rise significantly particularly in the neonatal brain and are sustained for up to 24 hours. this rapidly accumulated h 2 o 2 is detrimental in the iron-rich immature brain as it can lead to the generation of dangerous free radicals that can cause extensive injury. to date, there is limited literature on the effects of increased h 2 o 2 levels, particularly on microglial cells, which have been extensively indicated in the mediation of ensuing injury. here we describe the effects of a continuous exposure of microglia to h 2 o 2 , as generated using the glucose oxidase-catalase (gox-cat) system. this system allows us to generate and continuously maintain for up to 24h pathophysiological levels of h 2 o 2 >1 um. microglial cultures were derived from the p1 mouse brain and exposed to either bolus concentrations of h 2 o 2 [1-100 mm] or varying concentrations of gox-cat for varying lengths of time. conditioned medium was collected from cells at 4, 18 and 24h of treatment and analyzed for secreted cytokine levels using the 25-plex cytokine microbead array kit. treated cell extracts were processed for protein and fixed cells were labeled with m1 and m2a phenotype markers. continuous exposure to very low levels of h 2 o 2 can produce 10-20-fold higher (over control) pro-inflammatory cytokine protein levels of il5, il6, il12p40, il12p70, il15, il17 and ifng and at least a 100-fold higher level of il1a, il1b, il7 and tnfa by 18h. anti-inflammatory cytokine (il4, il10 and il13) and chemokine (rantes, g-csf and gm-csf) protein levels were also increased by 18h. interestingly, no prominent cytokine responses were seen with bolus treatment at any of the time points studied. low, continuous h 2 o 2 promoted a predominantly m2a microglial phenotype by 24h. we conclude that studying continuous exposure effects will help delineate the various specific effects h 2 o 2 can have on microglial cells. these specific effects can then be used to clarify when and how microglial cell responses can be beneficial or detrimental following injury in the immature brain. we have previously shown that natural (15-deoxy-d 12,14 prostaglandin j 2 , 15d) and synthetic (pioglitazone) agonists of peroxisome proliferator activated receptor-c (ppar) potentiate intrinsic cellular mechanisms protecting oligodendrocyte (ol) progenitors (ops) from oxidative insults and promote their differentiation to ols. in addition, ppar-c agonists potentiate mitochondrial activities, as the mitochondrial respiratory chain activity and the regulation of cytoplasmic ca21 waves, which are known to be crucial for ol differentiation. in the present study we sought to investigate whether ppar-c agonists can protect ol cultures from conditions causing mitochondrial stress. first, to specifically induce a mitochondrial impairment, we used the complex i inhibitor rotenone. as expected rotenone, at concentration not affecting cell viability, significantly inhibited ol differentiation, as indicated by the reduced number of cells expressing specific markers of differentiation (o 4 and o 1 ). in ppar-c agonist-treated ols the inhibitory effects of rotenone were significantly attenuated, suggesting a protective effect of the agonists against the mitochondrial toxin. we next examined a condition mimicking inflammatory stress by challenging op cultures with tnf-a, an inflammatory cytokine known to retard the differentiating program of ops. in parallel with the expected reduction of the percentage of o 4 and o 1 positive cells, the cytokine induced a significant reduction of mitochondrial membrane potential (mmp), suggesting an impairment of the mitochondrial functions. the simultaneous treatment with tnf-a and ppar-c agonists (15d or pioglitazone) significantly reverted both tnf-a dependent reduction of ol differentiation and mmp. at the molecular level, we found that in op cultures, ppar-c agonists increased the expression of the uncoupling protein-2 (ucp-2), a mitochondrial protein known to contribute to the protection of mitochondria against oxidative stress. these findings suggest that ppar-c agonists protect ols and promote myelination through several mechanisms, including those involving mitochondrial functions. our studies support the therapeutic potential of ppar-c agonists in brain diseases in which mitochondrial alteration, oxidative stress and demyelination occur and point to the need to better understand the role of ppar-c and its agonists in ol biology. it is characterized by lesions of demyelination and inflammation, which can be classified according to the activation state of the microglia/macrophages. well before any myelin and blood-brain barrier damage clusters of activated microglia are noticeable. these clusters are considered to be the first stage of lesion formation and therefore called 'pre-active lesions. however, they do not always develop into demyelinating lesions. here we postulate that stressed oligodendrocytes play an crucial role in pre-active lesion formation, by producing factors that will attract and activate microglia to either a more pro-inflammatory (m1) or antiinflammatory (m2) activation status. this can contribute to the amount of damage to the environment and further lesion formation. this study sets out to identify the best method to mimic this early lesion formation in vitro. first, human primary isolated microglia are stimulated to either a m1 or m2 phenotype and characterized by marker expression and cytokine profile. oligodendrocytes are isolated out of the human brain. medium of (stressed) oligodendrocytes will be used as attractant as well as activator for microglia. by immunohistochemical staining the activation status of microglia in pre-active lesions will be determined. preliminary data reveal that human microglia are able to be skewed towards a pro-and anti-inflammatory phenotype. first experiments of primary human oligodendrocyte cultures, showed pure oligodendrocytes. thus far our data support our hypothesis, however further research is required to make our conclusions more robust. question: although cell transplantation is increasingly suggested to be beneficial for the treatment of various neurodegenerative diseases, the therapeutic application of such intervention is currently hindered by the limited knowledge regarding central nervous system (cns) transplantation immunology. in this study, we aimed to investigate the early post transplantation innate immune events following grafting of autologous mesenchymal stromal cells (msc) in the cns of immune competent mice. methods and results: first, the survival of grafted luciferase/egfpexpressing msc (msc-luc/egfp) was demonstrated to be stable from on day 3 post implantation using in vivo bioluminescence imaging (bli), which was further confirmed by quantitative histological analysis of msc-luc/egfp graft survival. additional histological analyses at week 1 and week 2 post grafting revealed the appearance of (i) graftsurrounding/-invading iba11 microglia and (ii) graft-surrounding gfap1 astrocytes, as compared to day 0 post grafting. while the density of graft-surrounding astrocytes and microglia did not change between week 1 and week 2 post grafting, the density of graft-invading microglia significantly decreased between week 1 and week 2 post implantation. however, despite the observed decrease in microglial density within the graft site, additional phenotypic analysis of graftinvading microglia, based on cd11b-and mhcii-expression, revealed > 50% of graft-invading microglia at week 2 post implantation to display an activated status. although microglial expression of cd11b and mhcii is already suggestive for a pro-inflammatory m1-oriented phenotype, the latter was further confirmed by: (i) the expression of nos2 by microglia within the graft site, and (ii) the absence of arginase 1-expression, an enzyme known to suppress no activity in m2oriented microglia, on graft-surrounding and -invading microglia. conclusions: in summary, we here provide a detailed phenotypic analysis of post transplantation innate immune events in the cns of mice, and warrant that such intervention is associated with an m1-oriented microglia response and severe astrogliosis. it is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (cns). cd300f is a novel immunoreceptor that dampens inflammatory reactions in experimental autoimmune encephalitis (eae) and diverse allergy models. we have analyzed the function of cd300f immunoreceptor in an nmda excitotoxicity model and in a traumatic brain injury model (tbi). for this purpose, we determined the pattern of expression of both cd300f and its putative ligand(s) in the cns, as well as the neuroprotective role of cd300f after both acute brain injuries. first, we used a human and rat cd300f-ig soluble protein to show by confocal microscopy the presence of endogenous ligand(s) for this receptor mainly in cns white matter oligodendrocytes and on the surface of oligodendrocytes, fibrous astrocytes and neurons in vitro. interestingly, when we analyzed the in vitro expression pattern of rcd300f in brain cells by q-pcr and immunohistochemistry, in addition to the expected expression in microglial cells, we detected expression of cd300f in oligodendrocytes and neurons. in vivo, after a tbi, cd300f is expressed at early timepoints (3d) in small round macrophages, and at later time-points (14d) in neurons of the penumbra. q-pcr studies showed significant upregulation of the mrna for rcd300f at 7 and 14d after tbi. interestingly, a delayed (4 hours after the lesion) intraparenchymal injections of a non-viral modular recombinant gene therapy vector termed nlsct overexpressing hcd300f induced a decrease in the lesion volume 3d after nmda injection or tbi when compared to control gfp over-expression. in addition, the over-expression of hcd300f decreased the long-term sensitive functional deficits observed by the "sticky tape" test. in order to validate these results with the endogenous molecule, we cloned the rat ortholog of cd300f protein. on-going positron emission tomography (pet) studies using 2-[ 18 f]-fluoro-2-desoxiglucosa ( 18 f-fdg) are being used to evaluate the long-term functional recovery after tbi. the overexpression of rcd300f receptor had a comparable neuroprotective effect as the human molecule after nmda injection. these data suggest that cd300f may be important in neuron-glia inflammatory and trophic interactions. in addition, our results suggest that delayed cd300f overexpression by means of a modular recombinant gene therapy may constitute an interesting therapeutic strategy for acute brain injuries. depending on the type of signals produced after an inflammatory event, microglia develops specific activities, including cytotoxic, neuroprotective or phagocytic activities in order to come back to brain homeostasis. as described for macrophages, microglia can adopt m1 (proinflammatory) or m2 (phagocytic) phenotypes distinguishable through their pattern of factors expression and specific cell surface markers. interestingly, polyunsaturated fatty acids (pufas) are precursors of bioactive lipid messengers involved in the regulation of inflammation and in particular, docosahexanoic acid (dha, 22:6n-3) an n-3 pufa, presents anti-inflammatory properties. the aim of our study was thus to investigate the effect of an increase of n-3 pufas on the inflammatory response and cognitive abilities after a peripheral immune challenge. to increase n-3 pufas, we took advantage of transgenic mice carrying the fat-1 gene from the roundworm caenorhabditis elegans. this fat-1 transgenic mouse is capable of producing n-3 fatty acids from the n-6 type endogenously, eliminating confounding factors of the diet. this conversion leads to abundant n-3 fatty acids with reduced levels of n-6 fatty acids in tissues, including the brain. to induce a neuroinflammation, we injected mice intraperitoneally with lipopolysaccharide (lps), components of gram negative bacteria walls. we first studied the microglial phenotype 24 h after lps injection and found that microglia in fat-1 mice presented a significant increase of m2 phenotype markers. we then measured cytokine expression in the hippocampus after a lps challenge and found a significant decrease in il-1b mrna in fat-1mice compared to wildtype littermates. in terms of hippocampal memories abilities, only control mice presented a deficit in the y-maze task whereas fat-1 mice were able to perform the test correctly. our results indicate that 24 h after a lps injection, fat-1 mice presented a decreased of the inflammatory response and normal hippocampal memory abilities compared to wild-type littermates. glia integrity and homeostasis as they have prominent role in blood brain barrier formation and maintenance, as well as in active regulation of an immune response. however, astrocytes are affected in neuroinflammation, when their protective roles might be suppressed and when they might be shifted towards pro-inflammatory phenotype. experimental autoimmune encephalomyelitis (eae) is a widely used model of autoimmune neuroinflammation. chemokine cxcl12 produced by astrocytes and endothelial cells has profound anti-inflammatory and anti-encephalitogenic role in eae. nitric oxide (no) produced by inducible no synthase (inos) within the cns is considered as disease promoting, while interleukin (il)-10 as protective in eae. the aim of this work was to investigate effects of no and il-10 on cxcl12 gene expression in astrocytes. methods: astrocytes were stimulated with supernatant collected from concanavalin a-stimulated splenocytes (conasn) and simultaneously exposed to no donor, sodium-nitroprusside (snp) or anti-il-10 antibody. also, astrocytes were stimulated with ifn-c1il-17 in the absence or presence of il-10. peritoneal macrophages isolated from healthy rats were co-cultivated with astrocytes. after 24h incubation period cxcl12 gene expression in astrocytes was measured by rt-qpcr. phosphorylation of p38 mapk and nf-jb was assessed by immunoblot in astrocytes exposed to snp. results: we found that no released from snp or macrophages significantly reduced cxcl12 gene expression in astrocytes. this reduction was mediated by inhibition of p38 mapk activation, but not of nf-jb signaling. on the contrary, il-10 stimulated and anti-il-10 antibody suppressed cxcl12 gene expression in astrocytes. conclusions: these results imply that expression of cxcl12 in astrocytes is inversely regulated by no and il-10 in the central nervous system affected by inflammation. having in mind, profound regulatory role of cxcl12 in neuroinflammation, these data contribute to our understanding of pro-and anti-inflammatory nature of no and il-10, respectively. in the presence of pathological insults, they acquire reactive phenotypes aimed at re-establishing brain homeostasis and minimizing neuronal damage. however, reactive microglia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect. consequently, the progression and resolution of microglial activation have to be tightly controlled to avoid detrimental secondary effects. signals arising from neuronal cells play an important role in the activation state of microglial cells. among them, inhibitory mechanisms, such as neuronal cd200 and microglial cd200r1 interaction, keep the proinflammatory phenotype of microglia under control. alterations in the expression of cd200 and cd200r1 have been described in pathological conditions, and the modulation of cd200-cd200r signalling could be an interesting target to be considered in therapeutic approaches against neuroinflammation occurring in neurodegenerative diseases. little is known on the molecular mechanisms involved in the regulation of cd200 and cd200r1 expression. the aim of the present work is to study the possible modulation of cd200 and cd200r1 by ppar-c agonists, and the involvement of cd200-cd200r1 in the anti-inflammatory and neuroprotective effects of ppar-c activation. we have used mouse microglial, mixed glial and neuronal cell cultures, as well as neuron-microglia co-cultures. cd200r1 expression was detected in microglial cells, and it was decreased in response to pro-inflammatory stimuli such as lps/ifn-c. the ppar-c agonist 15-deoxy-d 12, 14 -prostaglandin j 2 (15d-pgj 2 ) abrogated the inflammatory response in lps/ ifn-c-treated microglial cells and prevented cd200r1 expression inhibition. cd200 expression was detected in neuronal cultures and, at a lesser extent, in astrocytes in mixed glial cultures. lps/ifn-c-treatment did not modify cd200 expression in neuronal cultures, but it induced an increase in mixed glial cultures. this increase was inhibited by 15d-pgj 2 pre-treatment. 15d-pgj2 also protected against lps/ifnc-induced neurotoxicity in neuron-microglia co-cultures, but this effect was abolished when cd200-cd200r1 interaction was interrupted using an anti-cd200r1 blocking antibody. these results show that ppar-c agonists modulate cd200 and cd200r1 expression in reactive glial cells, and that cd200-cd200r1 interaction is necessary for the neuroprotective effect of ppar-c agonists. to gain insight into the contribution of the cwbc pathway to remyelination in ms, we analyzed the expression pattern of tcf7l2, a downstream target of the cwbc pathway and coactivatior of b-catenin in ms lesions. tcf7l2 was expressed in ol and astrocytes in a subset of early ms lesions, but was also observed in tissue samples from patients with inflammatory, non-demyelinating diseases. in contrast, in chronic lesions, no expression of tcf7l2 was detected. by wb we found slightly increased b-catenin levels in ms lesions compared to normal appearing white matter. aspirin (ass), a non-steroidal anti-inflammatory drug, attenuates bcatenin signaling activity by inhibition of protein phosphatase 2a (pp2a) via increased phosphorylation. therefore, we determined the effect of ass on ol differentiation and myelin gene expression (mge). as a control for the cwbc pathway activation or inhibition the selective cwbc pathway inhibitor (icg-001) was chosen. icg-001 binds specifically to cbp and causes a disruption of the b-catenin/cbp interaction. exposure of ol to 0.3 mm icg-001 for 48 hours increased mge and had a positive effect on ol differentiation. in contrast, ass had no positive effect on process formation and did not promote mge in primary murine ol. our results suggest that icg-001, but not ass increases the expression of myelin genes. further experiments are required to determine the functional role of the cwbc pathway for ol differentiation and remyelination in ol and ms. (braak et al., 2003) . furthermore, neuroinflammation, i.e. activation of microglial cells in the substantia nigra (sn), has been widely implicated in pd progression (mcgeer et al., 1988) . in the present study, we question whether microglial activation occurs in brain regions beside the sn which are affected in pd patients. methods: to this end, we studied microglial activation and protein pathology in the ob and hc of clinically and neuropathologically verified pd patients (braak 4-6) and control subjects (braak 0). human post-mortem formaldehyde-fixed material of pd patients included sn (n 5 13), ob (n 5 9) and hc (n 5 12), and material from age-matched control subjects without neurological deficits included sn (n 5 10),ob(n 5 6), and hc (n 5 8). results: the presence of a-syn pathology concentrated in the anterior olfactory nucleus of the ob and in the ca1-2 region of the hc. furthermore, using cd68 as a microglial marker, we observed a significant increase in the number of immunopositive microglial cells, with an activated, amoeboid morphology, in the sn as well as in the ob and hc of pd patients compared to control subjects. co-localization studies indicated that these activated microglia were found in the proximity of a-syn inclusion bodies and neurites, but did not co-localize suggesting that the microglial cells do not actively phagocytose a-syn. conclusion: we conclude that microglial activation occurs in other brain regions beside the substantia nigra of pd patients. the functional role of the microglial cells in those brain regions and their contribution to pd pathology remains to be established. in the present study, we question whether ccl2 and cx3cl1 and its receptors are present in hippocampal lesions of ms patients. to this end, semi-quantitative rt-pcr was performed on cdna transcribed from rna isolated from hippocampi of ms patients and control subjects. moreover, immunohistochemical analysis of ccl2, cx3cl1 and its receptors ccr2 and cx3cr1 was performed on post-mortem, formalin-fixed hippocampal lesions (plp -) from ms patients and on hippocampal material (plp 1 ) from control subjects. ccl2 and ccr2 mrna was significantly enhanced in demyelinated ms hippocampal homogenates compared to non-demyelinated and control hippocampal homogenates. cx3cr1 mrna was significantly increased in demyelinated ms hippocampal homogenates compared tot non-demyelinated hippocampal homogenates, but cx3cl1 mrna levels showed no difference between groups. ccl2, ccr2, cx3cl1 and cx3cr1 immunoreactivity was present mainly in white matter of control and ms hippocampi and was clearly enhanced in hippocampal grey (cornu amonis) and white (stratum lacunosum, stratum radiatum, alveus) matter lesions. the intensity of the ccl2 and cx3cl1 immunoreactivity was most pronounced when active lesions were present. co-localization studies indicated that ccl2 and cx3cl1 are present in astrocytes, whereas cx3cr1 and ccr2 are present in or on microglial cells. we conclude that the chemokines ccl2 and cx3cl1 and their respective receptors are enhanced in hippocampal ms lesions. as no clear influx of leukocytes is observed in the grey matter lesions, we propose that astrocyte-derived ccl2 and cx3cl1, via interaction with their receptors, affect microglial activation and/or function thereby contributing to neuronal dysfunction in the hippocampus as seen in ms patients. , is a 17 kda cytokine-inducible protein, produced by activated macrophages during chronic transplant rejection and in inflammatory reactions. in central nervous system (cns), iba1 is a sensitive marker associated to activated microglia and is upregulated following neuronal death or brain lesions. iba1-like factors have been described in several metazoan and share a well conserved amino acid primary structure throughout evolution suggesting a common, functional role. the medicinal leech hirudo medicinalis is able to regenerate its cns after injury, leading to a complete functional repair. similarly to vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. we investigated the expression of hirudo iba1 to track the activation state of leech microglial cells involved in nerve repair events. results: we recently identified a gene, named hmiba1, coding a 17.5 kda protein showing high similarity with vertebrate iba1 factor. quantitative rt-pcr analyses showed that hmiba1 is constitutively expressed in cultured nerve chains. a weak down regulation was observed in the days following experimental injury. gene transcripts rise back to basal level one week later. cultured nerve chains stimulated with atp shows a significant increase of hmiba1 transcript 6 hours after treatment. immunoblot analysis, performed with anti-hmiba1 polyclonal antibodies, revealed an immunopositive band at the predicted size. the presence of hmiba1 protein in na€ ıve and experimentally challenged tissues was evaluated by immunohistochemistry. the protein is constitutively present in spread, stellar shaped microglial cells, distributed in connective fibers and in segmental ganglia. a few hours after experimental injury of cns, the amount of immunopositive microglial cells increases at the lesion site and at the cut end of nerve fibers until. the amount of hmiba11 cells in connectives rapidly increases in atp treated nerve chains. this augmentation is visible in ganglia microglia and in connective fibers, where cells located between axon fibers display an elongated and stretched shape. conclusion: hmiba1 is a good marker of activated microglia. like in vertebrates, the atp induces its expression in leech cns. also if the functional role of hmiba1 has to be further elucidated, this molecule appears as a good activation marker of microglia and an interesting tool to study and follow the activity of such cells during nerve repair in leech. hypertension is the single most important risk factor for cardiovascular disease. despite significant advancements, 20-30% of all hypertensives remain resistant to all current available pharmacotherapy. these patients exhibit elevated sympathetic drive, increased norepinephrine spillover, and dampened parasympathetic drive. the dysfunctional autonomic nervous system of these resistant patients indicates a neurogenic origin for their pharmacotherapy resistance. previous studies have proposed that neuroinflammation in the autonomic brain regions plays an important role in neurogenic hypertension. this coupled with the emerging interest in microglia led us to hypothesize that activation of microglial cells in the brain could be a critical event in the initiation and establishment of hypertension pathophysiology. we have previously established that chronic low dose angiotensin ii (ang ii) induced hypertension involves activation of microglia, and increase in proinflammatory cytokines and reactive oxygen species (ros) in cardioregulatory brain areas, such as the paraventricular nucleus of the hypothalamus (pvn). intracerebroventricular (icv) delivery of minocycline, an anti-inflammatory antibiotic that inhibits microglia activation, decreased activated microglia, inhibited increase in cytokines and ros, and attenuated hypertension. in addition, inhibition of brain mitochondrial ros attenuated hypertension, microglial activation, and the overexpression of brain pro-inflammatory cytokines. a time-course experiment demonstrated that there was a significant increase in activated microglia before the increase in blood pressure was detectable by radiotelemetry. furthermore, the origin of microglia in established hypertension appears to be both from resident and bone marrow derivation. these observations indicate that the activation of microglia is a critical player in the development of neurogenic hypertension. they suggest that activation of microglia in cardioregulatory regions of the brain is an early occurrence that may initiates a cascade of signaling events leading to increase sympathetic nerve activity and initiation of hypertension. this research is supported by nih grant (r37 hl 33610 to mkr). our results demonstrate significant inflammatory activation of the neurons and their sgc not only in drg associated but also non-associated with injured nerve. a distinct expression of cytokines and their receptors was identified in sgc surrounding largesized drg neurons. significant inflammatory reactions of sgc were found in all drg of pac-treated rats. moreover, inflammatory activation sgc was also observed in drg of sham-operated rats indicating other kinds of trigger than traumatic nerve injury. the nerve injury and pac-treatment also triggered socs3 expression in sgc to control stat3 activation. inflammatory activation of sgc significantly contributes to ectopic activation of the drg neurons not only associated with injured nerve, and is involved in npp induction. aging is associated with reduced function and degenerative changes of the central nervous system (cns). increasing evidence suggests that changes in microglia cells, i.e. the resident macrophages of the cns, contribute to the age-related deterioration of the cns. the most prominent age-related change of microglia concerns enhanced sensitivity to proinflammatory stimuli of microglia in mice, rats and primates, called priming. we have addressed this issue in ercc1 mutant mice, ercc1 d/mice, a progeroid, dna repair deficient mouse model that displays features of accelerated aging in multiple tissues including the cns. in aged ercc1 d/mice, microglia showed increased proliferation and enhanced immune function including expression of cytokines and antigen presentation molecules, increased phagocytosis and production of reactive oxygen species (ros). this microglial functionality was found to be surprisingly hyper-responsive to immune stimuli indicative of microglia priming. transcriptome analysis revealed an expression pattern that characterizes microglia priming, featuring genes associated with increased antigen pattern recognition and antigen presentation and phagocytic-, adhesion-and chemotactic ability. in mice where the ercc1related dna repair deficit was targeted to forebrain neurons, microglia priming was restricted to forebrain areas, suggesting that the dna damage-induced changes in neurons provided the signals leading to microglial immune priming. acidosis is a clinical consequence of many major diseases. non-infectious diseases are increasing in prevalence and many have been shown to have an inflammatory component, which in the absence of infection, will be sterile. the nlrp3 inflammasome, a multimeric protein complex which induces processing of il-1b into its active form, is a well established mediator of sterile inflammation and is known to drive the worsening of brain injury in numerous experimental disease paradigms. we sought to investigate whether acidic conditions, typical of a disease environment, affected il-1b processing in primary mouse glial cells. mixed glial cultures were grown from wild type and nlrp3 knockout mice. culture media was reduced to ph6.2 and il-1b release was measured following addition of activators of the nlrp3 inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, atp) with or without pre-treatment with caspase-1 or cathepsin d inhibitors (yvad-cho or pepstatin a respectively). subsequently, il-1b release was measured following addition of lactic acid with or without pre-treatment with the above inhibitors. at ph6.2, activators of the nlrp3 inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, atp) induced the release of il-1b from mixed glial cultures. the il-1b released at this low ph was 20kda in size in addition to the mature 17kda il-1b. this 20kda il-1b release was maintained in nlrp3 deficient cells and was not significantly altered by pre-treatment with the caspase-1 inhibitor yvad-cho. lactic acid, itself released during disease and a common cause of acidosis, also induced the release of 20kda il-1b from mouse glial cultures. as with the nlrp3 activators under acidic conditions, the lactic acid-induced 20kda il-1b release was maintained in nlrp3 knock-out cultures and with pre-treatment with yvad-cho. pre-treatment with the cathepsin d inhibitor pepstatin a, however, significantly reduced the 20kda il-1b released with the nlrp3 activators and lactic acid. here we show that under disease relevant conditions (low ph), 20kda il-1b is released from mouse glial cultures and this 20kda il-1b is independent of the classical nlrp3 inflammasome/caspase-1 pathway and likely mediated by cathepsin d. further investigation of this caspase-1-independent il-1b pathway may in future provide novel targets for the treatment of inflammatory disease. the phoneutria nigriventer (ctenidae-araneaeomorpha) spider venom (pnv) causes reactive gliosis, neuroinflammation and blood-brain barrier (bbb) impairment. nitric oxide(no)-soluble guanylate cyclase(sgc)-cgmp has been implicated in pnv-induced cavernosal relaxation. we investigate whether no-sgc-cgmp signaling acts on astrocytes/microglia activation and neuroinflammation induced by pnv. male wistar rats (6-8-week-old) were pre-treated (i.p.) with sgc inhibitor (odq, 10 mg/kg), nnos inhibitor (7-nitroindazole-7ni, 40 mg/kg), no donor (nitroprussiate-ntp, 3 mg/kg) or pde5 inhibitor (sildenafil, 10 mg/kg). after 30 minutes, the venom (0.85 mg/kg) was injected in the tail vein (i.v.). saline (i.v.), pnv alone or dmso (i.p) followed by pnv were used as controls. one hour after injection, cerebella were processed for immunofluorescence or western blotting. pnv increased gfap, iba-1, ifn-c and sgc, compared to saline control, indicating astrocytes and microglia activation, neuroinflammation and sgc-cgmp involvement. the sgc inhibition by odq intensified the pnv effects, increasing even more gfap, iba-1 and ifn-c levels. this indicates that sgc-cgmp production can be inhibited by venom. in agreement, the cgmp accumulation by sildenafil inhibited the pnv effects, decreasing gfap, iba-1, ifn-c and sgc. the increase of sgc by venom could result from a feedback mechanism, when sgc activity is inhibited and its expression is increased. 7ni and ntp pre-treatment did not show difference compared to venom alone, suggesting that no per se is not involved in the inflammatory effects of venom. however, it is not discarded no and sgs-cgmp coupling in the mediation of pnv effects. we suggest that pnv induces glial reaction and neuroinflammation by sgc-cgmp inhibition. the study gives evidence that sgc-cgmp, coupled or not to no signaling, mediates pnv cerebellar effects including the bbb impairment. pnv can be a useful tool for studies on the mechanisms involved in glial regulation. cnpq/fapesp/faepex support. j. engele, m. puchert, v. € odemis university of leipzig, leipzig, germany it is currently believed that cxcl12 predominantly signals through cxcr4. in addition, it is assumed that the alternate cxcl12 receptor, cxcr7, represents a non-classical g protein-coupled receptor which primarily acts as a modulator of the function of cxcr4. discrepant from this view, we demonstrated recently that in primary rodent astrocytes and human glioma cell lines, sdf-1 exclusively signals through cxcr7 by a g protein-dependent mechanism. we now provide evidence that cxcr4 is essential for cxcl11/i-tac signalling in primary rodent astrocytes and some human glioma cells. treatment of cultured rat astrocytes with cxcl11 for 10 min resulted in the dose-dependent activation (phosphorylation) of erk1/2 and akt with maximum activation in the presence of 100 ng/ml of the chemokine. cxcl-11-dependent activation of both signalling molecules persisted in astrocytes in which expression of the established receptors for cxcl11, cxcr3 and cxcr7, were inhibited by rnai. however, cxcl11-dependent activation of erk and akt was abrogated following sirna-mediated inhibition of cxcr4. likewise, cxcl11 failed to activate erk and akt in cortical astrocytes cultured from a cxcr4-/-transgenic mouse line. moreover, similar to primary astrocytes cxcl11 activated erk and akt in the human glioma cell line, a767. again activation of both signalling molecules was abrogated following rnai-mediated inhibition of cxcr4. cxcl11-dependent activation of erk and akt further remained undetectable in a non cxcr4-expressing subpopulation of a767 cells, previously isolated by flow cytometry. together, these findings unravel a unique processing of cxcl11 and cxcl12 signalling in primary astrocytes which is preserved at least in some malignant astroglial cells. long-lasting activation of gfap-positive astrocytes occurs in brain tissue exposed to injuries that promote epilepsy development. studies in experimental models of epilepsy showed that activated astrocytes release various molecules, such as proinflammatory cytokines and danger signals, that play a role in seizures generation and recurrence. these activated cells also loose their ability to buffer extracellular k 1 and glutamate. this set of evidence suggests, therefore, that astrocytes may contribute to epileptogenesis (i.e. the post-injury phase prodromal to generation of spontaneous seizures), thus representing a putative biomarker of epilepsy. to test this hypothesis, we set up an in vivo longitudinal study using 1 h-magnetic resonance spectroscopy (mrs) to measure the hippocampal levels of metabolites that could reflect the extent and the duration of astrocytes activation after an epileptogenic brain injury. status epilepticus (se), which provokes epilepsy, was induced by pilocarpine in adult male rats. 1 h-mrs measurements were done in the hippocampus every 24 h for 7 d post-se, thus encompassing the epileptogenesis phase, and in chronic epileptic rats using a 7 tesla bruker biospec. spectra were processed and analysed using jmrui and tarquin freeware softwares. we studied changes in myo-inositol (mins) and glutathione (gsh), which reflect astrocytes activation. we found a progressive (2-fold, pwhich was maintained in epileptic rats. immunohistochemical analysis (ihc) of s100ß and gfap confirmed the concomitant activation of astrocytes in separate timematched groups of rats. gsh levels during epileptogenesis showed a negative correlation with the frequency of spontaneous seizures which developed after se. a negative correlation was also found between gsh and mins levels during epileptogenesis and the extent of neurodegeneration in hippocampus of epileptic rats. ihc done in epileptic rats at the end of the mrs experiments, showed that hippocampal s100ß levels positively correlated with spontaneous seizure frequency. since this is a soluble protein, further investigations of its csf and blood levels are warrented. our mrs findings show that gsh levels during epileptogenesis could serve as a predictive biomarker of the ensuing seizure frequency (thus of epilepsy severity) and, together with mins levels, also predict the extent of neuronal cell loss which develops following se in epileptic tissue. notably, ihc-detected s100ß levels in the epileptic tissue also reflect seizure frequency. these findings highlight the potential use of serial 1 h-mrs analysis of astrocyte activation for predicting the severity of epilepsy and the extent of neuropathology in the clinical setting. interleukin-6 (il-6) is a highly plurifunctional cytokine, with many pleitropic actions, considered one of the main cytokines controlling the immune system and coordinating it with the nervous and endocrine systems. il-6 is produced in multiple cell types in the cns, and in turn many cells do respond to it. it is therefore important to ascertain which the contribution of each cell type is in the overall role of il-6 during both physiological and pathological conditions. astrocytes are major responders to il-6 as well as one of the main cns producers of il-6. for this work we used astrocytary il-6 ko (ast-il6 ko) mice, which we already proved to have an important role in physiological conditions (like body weight control and exploratory/ locomotion behavior), in order to test astrocytary il-6 role during a neuroinflammation situation. for this purpose, we induced either an extensively used animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (eae), or a traumatic brain injury (cryolesion) in our mice. regarding eae, results indicate that lack of astrocytary il-6 delays clinical course of eae and ameliorate eae symptomatology in ast-il6 ko respect littermate controls in a gender-dependent way. further immunohistochemistry analyses confirm a decreased number of cellular and lymphocytes infiltrates and lesser demyelination, angiogenesis and gliosis in spinal cord of ast-il6 ko animals. regarding traumatic injury, astrocytary lack of il-6 facilitates microgliosis, lymphocytes infiltration and a faster decrease of the injured area. all these results are likely to bring some answers to astrocytesecreted il-6 involvement in neuroinflammation pathways and eae pathogenesis. interleukin-10 (il-10) is a counterregulatory cytokine that plays an important role in controlling inflammatory and immune reactions. in the central nervous system (cns), production of il-10 has been demonstrated in activated astrocytes and microglia after different types of injuries. the specific role played by il-10 inmodulating glial responses is however still unclear. hence, the objective of this study was to evaluate the effects of local astrocyte-targeted production of il-10 on glial reactivity using the axonal anterograde degeneration paradigm. for this purpose, unilateral perforant pathway transection (ppt) was performed on adult gfap-il10 transgenic (tg) animals and their corresponding wild types (wt) littermates. at 3 and 7 days post-lesion (dpl), animals were intracardially perfused with 4% of paraformaldehyde, brains frozen and parallel free-floating sections processed for glial analysis using immunohistochemistry for iba-1 (microglia) and gfap (astrocytes). our results showed that in comparison with wt animals, microglial cells in the dennervated dentate gyrus molecular layer of gfap-il10tg showed differential reactivity changes. after lesion, astrocytic reactivity presented a higher hypertrophy in gfap-il10tg animals when compared with their corresponding wt littermates. in conclusion, this study demonstrated that local production of il-10 inthe cns can modify the glial response associated with ppt. further studies are warranted to evaluate if these alterations in microglial and astrocytic reactivity have any repercussions for the evolution of the lesion and the associated axonal sprouting. astrocyte and microglia become reactive under many brain pathological conditions, making this process of neuroinflammation a surrogate marker of neuronal dysfunction. several studies have reported that reactive microglia overexpress the translocator protein 18kda (tspo, formerly known as peripheral benzodiazepine receptor). positron emission tomography (pet) using radioligands of tspo is thus considered as a potent technique to detect reactive microglia in situ. however, it is still controversial whether tspo pet imaging is also able to monitor reactive astrocytes in situ. this is an important question as reactive astrocytes and reactive microglia play very different roles in brain physiology and could impact disease progression in opposite ways. to address this question, we used a model of selective astrocyte activation through unilateral lentiviral gene transfer of the cytokine ciliary neurotrophic factor (lenti-cntf) in the rat striatum. cntf induced an extensive activation of astrocytes, which overexpressed gfap and became hypertrophic. microglia, on the contrary, displayed a minimal increase in the expression of markers of reactivity. cntf-activated astrocytes overexpressed tspo at the mrna and protein levels. pet imaging experiments demonstrated a significant and specific binding of two tspo radioligands [ 18 f]dpa-714 and [ 11 c]ssr180575 in the lenti-cntf-injected striatum. we show that reactive astrocytes can be monitored by tspo-pet imaging in the rat brain. this technique is thus well suited to monitor reactive microglia as well as reactive astrocytes, the two cell types involved in neuroinflammation, but would not allow their discrimination in situ. chronicity might establish a neuroinflammatory ganglion profile with inflammatory cells contributing to the hypersensitive phenotype. we first investigated whether, in trigeminal sensory ganglia, cytokines such as tnfa might contribute to a local inflammatory phenotype of a transgenic mouse model of familial hemiplegic migraine type-1 (fhm-1, cacna1a r192q knock-in mice). with respect to wild-type, r192q ki trigeminal ganglia were enriched in activated macrophages and expressed higher mrna levels of il1b, il6, il10 and tnfa cytokines and the mcp-1. functional consequences of crosstalk between macrophages and sensory neurons were studied in primary ganglia cultures, where larger release of soluble factors and larger currents mediated by pain-transducing atp-gated p2x3 receptors were found. consistently, we observed that, following lps injection, tnfa expression and macrophage occurrence were significantly higher in r192q knock-in ganglia with respect to wild-type ganglia. our data suggest that, complex cellular and molecular environment of sensory ganglia could support a new tissue phenotype compatible with a neuroinflammatory profile. we propose that, in selected patients, this condition might contribute to pain pathophysiology through release of soluble mediators, including tnfa and atp that may modulate the crosstalk between sensory neurons and resident glia, underlying the sensitisation process. cultures of astrocyte/microglia are stimulated with ifn-gamma and then infected with tachyzoites of neospora caninum they release nitric oxide (no) that controls parasite proliferation. in order of elucidating the immune response of cns against this parasite, this study investigated the participation of inducible nitric oxide synthase (inos) in the control of its proliferation in co-cultures of neuron-glia obtained from rat brain. the cells were stimulated with ifn-gamma (100 iu/ml-24h) then supplemented with l-ng-nitroarginine methyl ester/l-name, an inhibitor of inos (1.5 mm/ml -60 min) before infection with tachyzoites of n. caninum (1:1 parasite:cell). after 72h, in the cultures supplemented with l-name, it was verified, a reduction of tachyzoites number in 55.7% (control cells) and 59.8% (ifn-y stimulated cells). however, in infected co-cultures, it was not observed any release of no, even when cells where modulated by ifn-g. additionally, in cultures infected and treated with l-name, the levels of il-10 were increased in six fold (non stimulated) and nine fold (ifn-g stimulated). these data suggest that no should not participate as a mediator of n.caninum control in neuron/glia co-culture but, the regulatory pattern of immune response must play a role in its down modulation regulating the inflammatory mediators released during the cns infection, perhaps to protect neurons. methods: adult albino swiss mice were organized in two groups: naive (age-matched control; n 5 6) and lasered (n 5 6). anti-iba1 was used in retinal whole-mounts immunolabelled to analyze the distribution, morphology, density and arbor area of iba11 microglial cells. results: in na€ ıve, contralateral and lasered eyes, iba11 microglias were distributed throughout the retina in the: subretinal space (ss), outer plexiform layer (opl), inner plexiform layer (ipl), nerve-fibre layer (nfl) and ganglion-cell layer (gcl). in na€ ıve eyes, iba11 microglias: i) had rounded bodies with fewer and shorter processes in the ss; ii) exhibited a branched morphology emanating from small cell bodies in the opl and ipl; iii) were less ramified and were related with the retinal vessels in the nfl and in the gcl. by contrast, the morphological features exhibited by iba11 cells in contralateral and oht-eyes differed from na€ ıve: i) somas were more robust; ii) processes were thicker and more branched in contralateral eyes and thicker and retracted in oht-eyes. in oht-eyes and contralateral untreated eyes the iba11 microglial number was increased in comparison with na€ ıve (p < 0.001 and p < 0.05 respectively, t-test). in comparison to na€ ıve retinas the iba-11 cell arbor in the opl and ipl decreased in both oht-eyes (p < 0.001 and p < 0.001 respectively, t-test) and in the contralateral untreated eyes (p < 0.05 and p < 0.001 respectively, t-test). conclusions: two weeks of laser induced-oht triggered microglial retinal changes suggestive of activation in the contralateral untreated and oht-eyes. the microglial activation in contralateral eyes could be related to the immune response. this behaviour in contralateral untreated eyes, lead us to suggest that the use of contralateral eyes as internal controls in experimental unilateral oht, should be reconsidered. interleukin-6 (il-6) is a pleiotropic cytokine involved in inflammatory and non-inflammatory responses. among the latter, it participates in the regulation of body weight and metabolism. il-6-deficient mice develop mature onset obesity and have higher blood glucose, as well as impaired glucose tolerance and elevated blood leptin levels, suggesting that il-6 participates in suppressing adiposity in mice. moreover, transgenic mice with astrocyte-targeted production of il-6 challenged with a high-fat diet are resistant to high-fat diet-induced obesity, highlighting the role of centrally produced il-6 in the regulation of body weight. in this context, we hypothesize that mice lacking il-6 produced by astrocytes (ast-il6ko) will be more prone to develop obesity. we therefore challenged ast-il6ko mice (obtained with the cre-lox technology) with a high fat diet (58% kcal from fat) for 17 weeks and compared their body weight and food intake with those of mice fed a standard diet (18% kcal from fat). on weeks 14 and 15, the metabolic status was evaluated by an insulin tolerance test (itt) and an oral glucose tolerance test (ogtt), respectively. regarding body weight gain, we see a clear increase in ast-il6ko females on a high-fat diet in comparison to their controls with no apparent difference in food intake, which is also already apparent in the control diet. in males a similar tendency is observed. part of this difference can be attributed to the heavier subcutaneous white adipose tissue depots (relative to body weight) in ast-il6ko mice (fig 1) . insulin and oral glucose tolerance are compromised in mice fed a high-fat diet, but no differences between genotypes are observed. taken together, these results indicate that centrally produced il-6 has indeed a major role in the regulation of body weight, affecting subcutaneous white adipose tissue, but without significantly altering peripheral glucose metabolism. we are currently working on assessing hypothalamic neuropeptides with in situ hybridization to study the effects of astrocytic il-6 deficiency at the central level. minocycline is an agent with pleiotropic properties that targets multiple proteins and cellular processes associated with development of neuropathic pain, including inhibition of the injury-induced glia activation. the aim of our study was to examine the effects of minocycline on the injury-induced changes in immune factors and on morphine effectiveness in a rat model of neuropathic pain. chronic constriction injury (cci) to the sciatic nerve in rats was performed according to bennett and xie (1988) and 2 behavioral tests were conducted to measure allodynia (von frey test) and hyperalgesia (cold plate test). minocycline was administered intraperitoneally 16h and 1h before cci and then twice daily for 7 days. the studies were performed using competitive rt-pcr in the spinal cord and drg from control, cci-exposed and minocycline-treated cciexposed rats. morphine was administrated i.t. (chronic catheterization according to yaksh and rudy, 1976) and ip 1h after the last minocycline injection. the experiments were carried out according to iasp rules (zimmermann, 1983) . repeated administration of minocycline attenuated allodynia and hyperalgesia when measured 7 days after cci. minocycline downregulated the spinal and drg level of several immune factors: c1q, mmp-9, mmp-2, il-1beta, il-6, il-18, nos1 and nos2 which were increased in consequence of the injury. moreover, chronic administration of minocycline improved the response to i.t. and i.p. morphine injections. our results demonstrate that minocycline reduces the level of pro-inflammatory factors and increases the effectiveness of morphine, which may have clinical significance for enhancing analgesic effects of opioids in neuropathic pain. to date, there is no standardized simple model available to investigate the biology of human microglia. the aim of this study was to establish a new in vitro microglia model using blood-derived precursor cells. for that purpose, human peripheral blood monocytes were cultured in serum free medium in the presence of a mixture of cytokines and chemokines (m-csf, gm-csf, ngf and ccl2) to generate monocyte-derived microglia (m-mg). monocyte-derived dendritic cells (m-dc) were also generated as a control population using gm-csf and il-4. the human microglia cell line hmc3 was used as control. m-mg were clearly different in morphology, phenotype and function from m-dc, but shared many properties with hmc3 cells. m-mg acquired a ramified morphology with primary and secondary processes, comparable to hmc3. they expressed very low levels of cd45, cd14 and hla-dr, cd11b and cd11c; but a distinct pattern of chemokine receptors, including ccr1, ccr2, ccr3, ccr4, ccr5, cxcr1, cxcr3, cx3cr1. similar to hmc3, under non-activated condition, the m-mg secreted of il-8 and il-6. in comparison with m-dc, m-mg displayed lower t-lymphocyte stimulatory capacity, as well as lower phagocytosis activity. in summary, we have established a new protocol for the generation of human monocyte-derived microglia, which is is feasible, well standardized and reliable, as it uses well defined culture medium and recombinant cytokines, but no serum or conditioned medium. this model will certainly be very helpful for future studies investigating the biology and pathology of human microglia. w. schaafsma university of groningen, neuroscience, section medical physiology, groningen, netherlands background: microglia are the innate immune cells of the cns. like other tissue macrophages, they can activate and commit to distinct reactive phenotypes in response to tissue damage and infections. however, the microglial response to tissue damage may change over age and by past experience. these changes in activation patterns may be caused by epigenetic modifications which in turn can result in changes in gene expression of, for example inflammatory cytokines. a well studied condition with an altered responsiveness of innate immune cells is 'endotoxin tolerance' (et). innate immune cells that are pre-exposed to endotoxins are dampened in their inflammatory response upon re-exposure to these endotoxins. while et is well described for peripheral macrophages/monocytes, very little is known about et in relation to microglia. objective: in our experiments we set out to investigate the presence of et in microglia and if an 'epigenetic' memory is involved. design/methods: in our in vitro experiments, microglia cells were stimulated with lps (100 ng/ml). naive microglia only received one lps stimulation at the end of culture, pre-stimulated microglia received an 24h pre-stimulation of lps (100 ng/ml) and a second stimulation 5 days later. in vivo, male c57bl/6 mice received an i.p. injection with either pbs or lps (1 mg/kg) and 4 weeks later again an i.p. injection with pbs or lps. transcription levels and secretion of il1-b and tnf-a were assessed by use of quantitative pcr and elisa. in order to answer if there is a long lasting epigenetic memory involved in this tolerance we used chromatin immune-precipitation (chip), to investigate changes in covalent histon tail modifications associated with either permissive or repressed chromatin. results : in vitro, we show a 'tolerant' phenotype in microglia which receive a second lps stimulation after 5 days. a reduced expression level of pro-inflammatory genes il1-b and tnf-a and reduced secretion of tnf-a were observed in response to a second lps challenge. furthermore, with in vivo experiments we showed that mice which received a second lps injection after 4 weeks, showed a dampened response in their microglia inflammatory reaction at the level of il1-b and tnf-a gene transcription. in both in vitro and in vivo experiments we showed a corresponding pattern for histon tail modifications in the enrichment of activation marks h3k4me3 and ach3 at the il1-b and tnf-a promotors. we are the first to show a long lasting 'tolerant' phenotype in mouse microglia in vitro and in vivo and the involvement of epigenetic changes at the level of histon modifications. morphological criteria and dual immunofluorescent labelling confirmed expression of er stress protein in neurons, astrocytes, microglia or oligodendrocytes. an intriguing finding was localisation of crt to the rim of oro-positive myelin fragments and the 'patchy' nature of crt staining seen when tissue was dual-labelled with crt and gfap or iba1. these results are the first demonstration of significantly higher levels of crt in rodent eae. chop and p-eif2a data has also not been reported in rat eae. this study highlights the potential importance of er stress in inflammatory demyelination. the authors acknowledge support from the irish research council and from the foundation office of nui, galway. (3). considering the responsible signaling pathways regulating adult neurogenesis (4), we observed differential regulation of wnt members in the hippocampus on transcriptional level, e.g. wnt dependent transcription factors (axin2, tcf4) and ligands (wnt3, 5a, 8b and 11), as well as on translational level represented by the wnt signaling cascade (active-bcatenin, phospo-glycogen synthase kinase 3b) during acute and chronic states of disease. by using in vitro studies in primary hippocampal cultures, we further show the role of transforming growth factorbb1 (tgfb1), a key cytokine early involved during eae (5), in regulation of wnt ligand expression and wnt signaling activity. furthermore we are able to visualize the connection between tgfb1 and wnt signaling by using the axin2-lacz mouse, a wnt reporter mouse, for functional and histological investigation of the hippocampus. taken together our results suggest a cross talk of inflammation and wnt signaling for dysregulated hippocampal neurogenesis. interleukin-6 (il-6) is a cytokine with major regulating effects of the inflammatory response. moreover, il-6 is a neuropoietin that has neurotrophic effects related to neuronal survival and protection. to establish the importance of il-6 produced only in the central nervous system, we have generated mice producing il-6 essentially only in the brain by crossing gfap-il6 mice (transgenic mice with astrocyte-targeted production of il-6) with il-6 ko mice (il-6-deficient). we studied the inflammatory response in a traumatic brain injury model, cryolesion, after 3 and 10 days post-lesion gfap-il6-il6 ko mice, comparing them with appropriate controls. in basal conditions wt and il-6 ko mice showed a similar phenotype. this was also the case for gfap-il6 and gfap-il6-il-6 ko mice, which showed prominent astrogliosis, microgliosis, increased recruitment of t lymphocytes and vascularisation compared with the other two groups. in response to cryolesion of the cortex an increased astrogliosis, microgliosis, recruitment of t lymphocytes and vascularisation was observed. il-6 deficiency produced an altered inflammatory response, in a time-and gender-dependent manner. this study with il-6 ko and gfap-il6 transgenic mice indicates that during an acute neuropathological insult such as traumatic brain injury il-6, from either the brain or the periphery, has an important role on the inflammatory response. increasing evidence suggests that iron accumulation in the brain might contribute to neurodegeneration. iron is a potential source of free radicals as it can catalyze the production of hydroxyl radicals under oxidative conditions. this can lead to amplification of tissue injury caused by oxidative damage. we thus characterized iron storage within the central nervous system (cns) of animal models of different neurodegenerative diseases. we examined animals with acute inflammation mediated by cd8 or cd4 positive t cells and animals suffering from t cell and antibody mediated chronic inflammation due to active immunization. further, we studied lps induced lesions which represent cns disease caused by the innate immune system. similarly, we characterized oxidative damage in the different models for inflammation. we did not find evidence for the presence of oxidized phospholipids or oxidized dna in the experimental lesions, which is in contrast to our results of ms lesions. none of these models showed iron accumulation in glial cells comparable to what is seen in ms tissue. however, some iron positive microglia and perivascular macrophages were observed in acute and chronic models. in preliminary experiments we found evidence that the presence of iron exacerbates h 2 o 2 induced cell death in purified glial cells in vitro. as a next step, we wanted to study a possible amplification of oxidative damage and neurodegeneration by iron in a more elaborate system. for this purpose we treated myelinating spinal cord cultures with iron chloride (fecl3) or ferritin. we observed iron loading in microglia in both experimental setups. moreover we found a selective decrease of microglia in iron chloride treated cultures but not after ferritin application. we did not find iron loaded oligodendrocytes in this in vitro model. iron accumulation is only minimal compared to the human brain in all the tested animal models. these observations necessitate the search for additional animal models mimicking the human situation more closely. objectives: microglia are a brain resident population of immune cells, involved in homeostatic surveillance of the brain parenchyma, with high importance in the regulation of inflammatory processes after injury. there are unresolved questions regarding microglia origin, and their similarities to peripheral macrophage populations. our objective was to compare the capacity of microglia and bone marrow derived macrophages (bmdms) to adopt different phenotypes, and how this influenced cell death after brain injury. methods: we compared the phenotype of bmdms and microglia (both bv2 microglial cell line and primary microglia) after treatment with well known polarising agents. lipopolysaccharide (lps) was used as an inducer of the classical m1 phenotype and il-4 was used to induce an alternative m2 phenotype. microglia or bmdms, of different phenotypes, were added to hippocampal organotypic slices (hosc) subjected to oxygen glucose deprivation (ogd) as an in vitro model of brain injury. results: bmdms and microglia (bv2 and primary microglia) both adopt m1 or m2 phenotypes after treatment with lps or il-4 respectively. however, addition of polarised microglia or bmdms onto hosc resulted in different outcomes. addition of activated bmdms, of either m1 or m2 phenotypes, led to cell death in control hosc and increased death when combined with ogd. on the other hand, addition of bv2-microglia did not induce any cell death in control hosc, and were protective after ogd, except for m1microglia which were toxic. endogenous microglia within the hosc were also able to adopt different phenotypes and exert neuroprotection after il-4 treatment. these ex vivo data correlated with the in vivo situation where we observed increases in microglial activation early after experimental stroke, although the vast majority of these activated cells did not express markers of the m1 phenotype. conclusions: this study highlights functional differences between macrophages and microglia, especially in response to brain injury. although microglia, like peripheral macrophages, can adopt different demyelination, thereby creating an unfavorable environment for remyelination. exploring efficient ways to prevent or overcome tlr-induced fibronectin aggregation by astrocytes might pave the way for new approaches in remyelination-targeted therapeutic strategies in ms. to study the tlr2 response in-vivo, sod1 g93a mice were crossed with the tlr2-luc-gfp reporter mice, a transgenic mouse bearing the dual reporter system luciferase and green fluorescent protein under the transcriptional control of the murine tlr2 promoter. double transgenic mice and littermates controls were monitored longitudinally using in-vivo biophotonic/bioluminescent imaging to measure microglial activation over the course of the disease. surprisingly, this analysis did not reveal an increase of activation in sod1 g93a mice as the disease progressed, with only weak signal coming from olfactory bulb and brain area. however, quantification of this signal suggested a lower level of tlr2 activation in the sod1 g93a mice compared to wild-type littermates. to further study the microglial impairment observed in the sod1 g93a mice, these transgenic mice were given lps with i.p. injections at 5 mg/kg and were followed for 48h by bioluminescence imaging, both at a pre-symptomatic age (60 days) and closer to paralysis (115 days). the level of tlr2 activation was lower in the sod1 g93a mice than the wild-type littermates, both at 60 days and 115 days. immunostaining of olfactory bulbs reveal the same phenomenon, which is less iba1 and less tlr2-positive cells in the sod1 g93a tissue. mrna analysis by in-situ hybridization confirms a similar pattern of microglial response. in a parallel study, primary microglial cell culture, derived from adult mice (60days) were similarly stimulated with lps to further understand the altered sod1 mutant microglia response. here again, sod1 cells appears less responsive to lps stimulation as observed in our mice studies. these combined results suggest that sod1 g93a microglial cells might have a pre-symptomatic suboptimal immune response. the role of reactive glia in the etiology and progress of neurological diseases is still unknown. reactive glia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect, highlighting the relevance of a strict control of the progression and resolution of glial activation. under some circumstances glial activation does not resolve, but it exacerbates or becomes chronic resulting in detrimental secondary effects. it is necessary to develop strategies to halt the negative outcome of glial activation in these situations, by controlling the pro-inflammatory phenotype of reactive glial cells and potentiating their beneficial effects. we studied the temporal pattern of inflammatory reaction in spinal cord and brain regions in the experimental autoimmune encephalomyelitis (eae) model of multiple sclerosis. special attention was paid to the involvement of members of the c/ebp family of transcription factors, which regulate the expression of pro-inflammatory genes in reactive glia, and cd200, cd200r1 and trem-2, membrane-associated inhibitors of the pro-inflammatory response in resting/surveillant microglia. mice were scored for signs of eae for up to 28 days postimmunization (dpi). eae symptoms were present from 10 dpi, reaching score peak at 14 dpi. in the spinal cord, we observed a sustained increase in c/ebpa and a transient increase in c/ebpb and c/ebpd expression peaking at 14 dpi. cd200 expression decreased at 9 dpi, remaining below control values at 28 dpi. cd200r1 and trem-2 expression increased at 14 dpi, thereafter this effect progressively attenuated. no alterations were observed in the brain. an acute increase in the expression of pro-inflammatory genes (il-1b, il-6, tnf-a, inos, cox2) occurred in the spinal cord at 14 dpi. our results suggest that cd200 expression decreases before onset of eae symptoms resulting in downregulation of the microglia inhibitory signal mediated by cd200-cd200r1, which would facilitate the proinflammatory response observed after onset of the eae symptoms. the increase in cd200r1 expression observed after the onset of eae symptoms suggests a concomitant intend of microglia/macrophages to resolve the inflammatory response. the transient increase in c/ebpb and c/ebpd expression could be related to the acute increase in proinflammatory gene expression, while the sustained increases of c/ebpa expression and trem-2 could be involved in the resolution of the inflammatory response. recently, we showed a beneficial effect of myelin reactive t cells on oligodendrocyte precursor cell differentiation in zones of axonal degeneration in the hippocampal dentate gyrus, which mirrors grey matter injury in multiple sclerosis. this effect was associated with a marked expression of t-cell cytokines, such as interferon-c and interleukin-17, enhanced microglial clearance of myelin debris, and an enhanced sprouting of calretinergic axons. to gain better insight in which cytokines and signaling pathways are elevated in response to the t cell enhanced regenerative responses, we performed a mrna microarray study, in which the transcript profile in hippocampi from mice with adoptively transferred proteolipid protein specific t cells combined with an axonal lesion were compared to the transcript profile from mice with axonal lesion. thereby we identified 1446 transcripts, which were differently expressed. we identified the interleukin-1 (il-1) signaling pathway as a potential target by performing pathway analysis using david and amigo databases. we discovered that il-1a, il-1b and il-1 receptor antagonist (ra) mrna as well as the il-1 signaling pathway were highly upregulated in response to myelin reactive t cells. in order to determine if il-1b mrna was translated into protein, we performed immunohistochemical stainings on tissues from mice with 2 days post lesion survival. expression of il-1b was observed in the molecular layer of t cell infiltrated, but not in t cell na€ ıve mice with axonal lesion, suggesting that myelin reactive t cells stimulate il-1b protein expression. ongoing studies will determine the expression of il-1a and il-1ra protein and determine the cellular expression of the il-1 signaling pathway in t-cell infiltrated versus non-t cell infiltrated mice with axonal lesion. understanding the regulation of the expression of il-1a, il-1b and il-1ra in t cell compared to non-t cell infiltrated cns may lead to a better understanding of the functional consequences of inflammatory responses in the cns. when io were challenged with kainate in the presence of conditioned medium (cm) from astrocytes, susceptibility to kainate-induced cell death was reduced, but cm from l-ap4-treated astrocytes reverted kainate toxicity. in contrast, treatment with cm from control or lpsactivated microglia, either untreated or pre-exposed to l-ap4, was not able to affect kainate-induced io cell death. to establish whether mglur4 activation could modulate the antigen presenting activity of microglia, the bv2 microglia cell line was used. treatment with l-ap4 (50 lm/48 h) did not affect changes in morphology induced by activation with lps (0.1 lg/ml/48 h), but significantly reduced the percentage of cells expressing mhc class ii, as detected by flow cytometry. these data suggest that during neuroinflammation, activation of mglur4 directly on io or through astrocytes and microglia may contribute to a protective effect resulting in survival of the oligodendrocyte lineage. mirnas are small non-coding rna molecules that modulate gene expression at a post-transcriptional level and their role in the regulation of biological processes makes them an emerging class of therapeutic targets. dysregulation of mirna networks has been linked to neurodegeneration and immune dysfunctions and has become a research focus in the context of alzheimer's disease (ad). in this work, we demonstrate that mir-155 expression is increased in the brain of 33trg ad transgenic mice, prior to senile plaque formation, and co-localized with intraneuronal app accumulation in the cortex and hippocampus. in view of the pro-inflammatory functions of mir-155 and aiming at clarifying its role in ad, we evaluated the levels of mir-155 in ab-stressed astrocytes and microglia cultures, two brain-related cell types involved in neuroinflammation. we show that mir-155 expression levels are increased in astrocytes and microglia following activation with ab fibrils but not with ab oligomers. these findings correlate with the observed decrease in socs-1 expression, a mir-155 validated target, and with the increase in the production of tnf-a, il-1b and il-6 by these cells. importantly, we show that mir-155 expression is regulated by c-jun, since silencing of this transcription factor led to the reduction of mir-155 levels following astrocyte and microglia exposure to ab fibrils or lps. c-jun was also found to be upregulated in the 33trg ad transgenic model since early ages, which can help to explain the observed early increase in mir-155 expression. overall, our results demonstrate the important role of mir-155 in ad and show that silencing of c-jun in glial cells may constitute an interesting and promising anti-inflammatory therapeutic strategy towards this disease, by targeting mir-155-mediated inflammatory responses. here we demonstrate that tgfb is an important endogenous factor promoting quiescence of microglia in vitro. inhibition of microglial tgfb signalling resulted in a microglia polarisation towards the classical activation state. moreover, tgfb is able to significantly decrease ifnc-induced classical activation of primary microglia and to enhance il4-induced alternative microglia activation. we further provide evidence that il4-mediated alternative microglia activation is dependent on active tgfb signalling. these results demonstrate the essential functions of tgfb as an important regulator of microglia activation states and further suggest that tgfb might be an interesting therapeutical molecule to modulate microglial functions during development and progression of neurodegenerative diseases. we found that young mice lost weight only in the first 24h after infection, whereas aged mice exhibited a biphasic pattern of weight loss, with the second phase of weight loss beginning 1 week after infection and continuing for approximately 2 1 =2 weeks. aged mice also demonstrated a co-ordination and balance deficit in the static rod test 1 week after infection compared to uninfected or 3 week post infection aged mice, whereas young mice were unimpaired at any timepoint. s. typhimurium infection caused a significant, progressive reduction in open field rearing activity at 1 week and 3 weeks after infection in both young and aged mice. a similar but not significant trend was apparent in novel object performance. forced swim test data showed a trend towards depressive behaviour at 1 week after infection, but not at 3 weeks. analysis of pro-inflammatory mediators in the hippocampus did not indicate significantly upregulated il-1b, tnf-a, cox-1 or cox-2 transcript at any timepoint, suggesting that microglia might not play a significant role in mediating these behavioural effects, or that the changes in these molecules were too subtle to reliably detect using qpcr in the hippocampus. the effects of ageing on s. typhimurium induced weight and co-ordination changes may be a result of regional differences in the sensitivity of microglia to peripheral infection within the cns or increased sensitivity to activation of neuronal circuits involved in weight loss or co-ordination with age. we have studied microglial cells with anti-iba1 and anti-cd68 immunohistochemistry in the brains of 18 subjects with pretangle neuropathology ranging from 4 to 51 years of age. regions examined included the locus coeruleus and surrounding brainstem areas, entorhinal cortex, hippocampal formation, and other parts of the mesial temporal lobe. ten of the subjects (median age 34) showed severe neuroinflammation secondary to infectious disease (hiv, sepsis, toxoplasmosis) and cns trauma, while 8 subjects (median age 32.5) died from non-infectious conditions and lacked neuroinflammatory changes (controls). we also examined two 52-year-old subjects with down syndrome both of which showed braak stage vi neurofibrillary degeneration, where one had comorbid sepsis and the other one did not. pretangle neuropathology was staged 1a and 1b in all 18 subjects, i.e. they exhibited primarily subcortical tau pathology in the locus coeruleus and only sporadic lesions in the transentorhinal cortex. our findings show that rampant microglial activation in the ten subjects with infections and trauma was coincident with the same level of tau pathology as seen in the 8 control subjects, demonstrating that neuroinflammation does not initiate or exacerbate neurofibrillary degeneration. in addition, our findings show that the extent of neurofibrillary pathology seen in down syndrome is unrelated to neuroinflammation since the down subject without comorbid sepsis revealed a complete absence of microglial activation. overall we conclude that neuroinflammation is not causally involved in the development of neurofibrillary degeneration which is most characteristically associated with alzheimer's disease dementia. it has been suggested that peripheral infection/inflammation may play a role in the onset or development of not only neurodegenerative diseases but also depression, autism and chronic fatigue syndrome through inflammatory responses in the brain. to investigate the role of microglia in this hypothesis we used a model of neuroinflammation induced by an intraperitoneal (i.p.) injection of the toll-like receptor 3 (tlr3) agonist, polyinosinic:polycytidylic acid (poly i:c) in rats. as we reported previously an injection of poly i:c (i.p) decreased the daily amounts of spontaneous running wheel activity to $60% of the preinjection levels until day 7. microglia were morphologically activated in the prefrontal cortex (pfc) until 72 hrs after the injection of poly i:c. pretreatment with minocycline for the 3 consecutive days (40 mg/kg/ day) blocked the poly i:c-induced decrease in the running wheel activity as well as microglial activation. quantitative analysis of mrna levels demonstrated that interferon-a (ifn-a) increased in the pfc on both days 1 and 7. we found in the present study that poly i:c injection induced increases in mrnas relevant to tlr-signaling such as tlr3, interferon regulatory factor 3 (irf3) and irf7 in the pfc. furthermore, direct application of poly i:c to the primary cultured microglia also induced an enhanced expression of ifn-a, tlr3, irf3 and irf7. it has been reported that permeability of blood-brain barrier is increased after poly i:c injection (wang et al., 2004) . therefore, it is possible that the peripheral poly i:c enters into the brain to induce neuroinflammation by activating microglia. interestingly, intracerebroventricular (i.c.v.) injection of primary cultured microglia activated by poly i:c, but not by lipopolysaccharide, effectively produced a significant decrease in spontaneous activity in rats. taken together, these findings suggest that microglial activation is important for poly i:cinduced neuroinflammation through inducing tlr3-related genes. roles of each gene in the behavioral changes will be further studied. results: microglia produced nanogram levels of pgrn (10 fold higher in microglia than in astrocytes), and pgrn release from microglia was suppressed by the tlr ligands or il-1/ifnc, but increased by il-4 or il-13. unexpectedly, while astrocytes stimulated with proinflammatory factors released large amounts of slpi, none were detected in microglial cultures. we also identified mmp-12 as a pgrn proteolytic enzyme, and slpi as an inhibitor of mmp-12-induced pgrn proteolysis in human microglia. conclusions: our results establish microglia as a significant source of pgrn. mmp-12 and slpi are modulators of microglial pgrn proteolysis. negative and positive regulation of microglial pgrn release by the proinflammatory/th1 and the th2 stimuli, respectively, suggests a fundamentally different aspect of pgrn regulation compared to other known microglial activation products. microglial pgrn appears to function as an endogenous modulator of innate immune responses. at pathological condition such as injury or ischemia to the central nervous system (cns), microglial cells and astrocytes become activate and inflammation occurs like proliferate, migrate and secrete some proinflammatory cytokines, il-1ß, tnf-a or il-6. however, it is unclear that the reason why inflammation is induced in the cns, and whether or not it is necessary for the brain during recovery from pathological situation. to know the molecular mechanisms for inflammation occurs in activated glial cells, we made a stab wound mouse model to the brain. we performed microarray analysis for rna extracted from the brain tissue around stab wound site compared among day after 0, 1, 3 and 7. it revealed that most of the genes from top 20 genes at high expression level around lesion site were concerned in immunological or inflammatory functions. we successfully identified and focused on to osteopontin (opn), which is an inducer of pro-inflammatory cytokine production expressed not only in reactive microglial cells but also in reactive astrocytes around the injured cns. furthermore, we also found receptors against opn functioning in the injured brain. however, functional role of opn in reactive astrocytes is not yet clarified. to analyze the central role of opn in reactive astrocytes, we examined primary culture of astrocytes from opn-deficient (opn/ko) mice and found that the morphology of these cells was unusual. by the stimulation with lipopolysaccharide to the primary culture of astrocytes from opn/ko mice, some of the pro-inflammatory cytokine expression was altered. moreover, reactivity of astrocytes caused by stab wound to the brain was examined using analogue of nucleic acid, bromo-deoxy-uridine (brdu), and clarified that it was decreased in opn/ko mice. from these results, we conclude that opn might play a major role in the activation of astrocytes under inflammation occurred to the cns. here we determined the influence of a hfd on eae. we show that feeding mice with a hfd exacerbates eae severity through the induction of the brain renin angiotensin system (ras), resulting in an increased immune cell infiltration into the cns, oxidative stress and th1 cytokines. moreover, peripheral inflammation was boosted upon a hfd due to a ras independent mechanism, indicating that a hfd exacerbates neuroinflammation in various manners. these data suggest that nutritional modulation may have an important influence on the course of ms and other neuroinflammatory conditions. the brain's immune privilege has been attributed to a lack of dendritic cells (dc) within its parenchyma and the adjacent meninges implying maintenance of antigens rather than their presentation in lymphoid organs (lo). using cd11c-gfp mice, we have recently reported the existence of a cd11c/iba-1/cd11b 1 -population in the juxtavascular parenchyma which extent their processes into the glia limitans, an ideal position for antigen-presentation. therefore, we phenotypically compared them to the cd11c 1 /cd45 1 population (all isolated with the same protocol used for brain cd11c 1 cells) from lung, liver and spleen in healthy mice using 7-color flow cytometry. we found unique, sitespecific expression patterns of f4/80, cd80, cd86, cx3cr1, ccr2, flt3 and mhc-ii. as described before, two different cd45 1 -populations (cd45 high and cd45 int ) in the brain can be separated, whereas liver, lung and spleen exhibit a more homogeneous cd45 high population. a higher percentage of the brain's cd45 1 /cd11c 1 -cells were f4/ 80-positive compared to spleen, liver and lung, but expressed it at lower levels. within the cd45 int /cd11c 1 -population most cells expressed the microglia marker cx3cr1 and ccr2 low (marker for inflammatory, motile cells). most importantly, compared to spleen and liver, cd45 int /cd11c 1 -cells from the brain almost completely lacked mhc-ii expression and cd45 high /cd11c 1 -cells from the brain have a lower percentage of mhc-ii 1 -cells. since the cd45 int cells are widely regarded as the resident, intraparenchymal microglial population and cd45 high as dcs and perivascular macrophages, our data confirm the view that a small subpopulation of microglia (cx3cr1 high , f4/ 80 1 ,ccr2 low ) share established immune phenotypical characteristics of dcs (cd11c 1 ). additionally we showed that intraparenchymal cd11c 1 microglia are unique in their low expression of mhc-ii. their weak expression of cd80 is in line with a tolerogenic phenotype. methods: we performed a series of experiments, where we examined the expression of pro-inflammatory factors in resident microglia and infiltrating macrophages after fluorescence-activated cell sorting (facs) at different eae stages. in order to properly discriminate the two cell types we used a set of markers where resident microglia were defined as cd11b 1 cd45 int ly6c -, and infiltrating macrophages cd11b 1 cd45 hi ly6c 1 . using this protocol we have analyzed the rna expression levels of mhcii (cd74, h2-aa), co-stimulatory molecules (cd80, cd86, cd40, cd274), pro-inflammatory genes (il-1b, tnf-a) and inflammasome-regulated cytokines in these immune cells with quantitative pcr. followed by analysis of mhcii, cd80 and cd40 at the protein level with flow cytometry. results: our results indicate that infiltrating macrophages have a pronounced activated (cd74 and h2-aa) and pro-inflammatory phenotype (il-1b and tnf-a) at rna level. this is supported with an increase of the expression of mhcii and the key co-stimulatory molecules cd80 and cd40 at rna as well as protein level. in contrast, microglia display a decrease in the expression of the co-stimulatory molecules cd80 and cd86 at the rna level and do not up-regulate expression of il-1b and tnf-a during the progression of eae. conclusion: our data suggest that upon eae, infiltrating macrophages display an inflammatory profile whereas microglia are only mildly immune activated. background: age-related hearing loss (presbycusis) affects half of people by the age of 75. it has a large impact on quality of life and is associated with accelerated cognitive decline. however, presbycusis is not an inevitable process of aging, suggesting that its progression could be minimised. hearing relies on sound vibrations from the middle ear to elicit movement of the basilar membrane in the cochlea, resulting in excitation of hair cells. spiral ganglion neurons relay the inner hair cell signals to the central auditory system via the vestibulocochlear nerve. sounds are integrated in the central auditory pathway and then processed by cognitive areas of the brain. degeneration of cochlear structures, including hair cells and spiral ganglion cells occurs in presbycusis. in the central auditory system there are alterations in neuronal plasticity and neurodegeneration may occur. chronic inflammation has previously been correlated with agerelated hearing loss in humans, implicating immune cells in the progression of presbycusis. microglia within the central nervous system are susceptible to priming by neurodegeneration or aging, both factors in presbycusis. it is feasible that microglia in the cochlea, an organ with similar immune privilege as the brain, could also become primed. priming increases the chance of microglia becoming activated by subsequent inflammatory events, such as 'flu, which may cause them to contribute to neurodegeneration in the cochlea and central auditory system. hypothesis: our working hypothesis is that as age-related hearing loss progresses, microglia in the cochlea and central auditory pathway will become primed. we predict that systemic inflammation will cause primed microglia to be activated to a pro-inflammatory damaging phenotype, which will increase the rate of cochlea and auditory pathway degeneration and hence hearing loss progression. methods: c57bl/6j mice are genetically predisposed to develop age-related hearing loss. young and middle-aged mice will be challenged with lps or saline (control) and microglial phenotype assessed in the cochlea and central auditory system. neurodegeneration will be evaluated at corresponding points in the cochlea and auditory pathway using molecular techniques. conclusion: if our hypothesis is proved this would suggest that rigorous control of infection in older individuals would reduce progression of hearing loss by minimising degeneration in the auditory system. this receptor triggers the response via both myd88-and trif-dependent signalling pathways, leading to production of cyto-and chemokines and thereby alarming and attracting the immune cells of the periphery to invade the brain. however, tlr4 is only fully functional in complex with several co/receptors, such as cd14. even though cd14 has been traditionally considered simply as a lps affinity provider, recent data indicate that cd14 is also necessary for the endocytosis of tlr4, which links tlr4 to intracellular signalling via the adapter protein trif. our latest data reveal the critical role of this receptor for the profile as well as the magnitude of microglial cyto/chemokine production in response to diverse lps variants. cd14 increases the sensitivity towards lps in a cell type-specific manner, making microglia far more sensitive to lps than bone marrow and peritoneal macrophages. while striatal applications of low lps doses reveal less incoming neutrophils into a brain of cd14ko mice, as compared to wildtypes, high doses lps lead to an excessive neutrophil infiltration. this phenomenon is well correlating with the observations in vitro, where cd14 absence in microglia stimulated with high doses lps causes an excessive production of selected chemokines, with the highest impact on the neutrophil chemoatractant cxcl1. these regulatory activities of cd14 require its membrane insertion and prolonged functionality, pointing to an involvement of signalling. in order to reveal candidates for such signalling mechanisms, we tested the role of syk and plc. even though these enzymes were described to play a role in cd14-dependent signalling in dendritic cells, they have no contribution in microglia, further pointing to a cell typespecific organization of the cd14/tlr4 complex in microglia. importantly, we identified receptor systems that impose influences on cd14 expression itself, which would thereby determine the extent and impact of a cd14-mediated regulation of tlr4 functions. supported by the dfg (for1336). in neurodegenerative disease misfolded proteins and cues released by dying cells can attract immune cells by chemotaxis and alter their phenotype. to better understand and differentiate between the effect of these cues, we aim to elucidate the spatiotemporal immunological events in the brain in vivo. zebrafish are an excellent model system to study leukocytes in vivo, and we previously showed by live imaging how engulfment occurs in the developing zebrafish brain (van ham et al., curr biol 2012). here we use genetically targeted cell ablation to specifically induce cell death of target brain cells. we subsequently track dying cells and immune cells by single and multiphoton 4d imaging using fluorescent reporter genes. to analyse immunological infiltrate and resident cells in an unbiased fashion, we use large-scale electron microscopy (em) of complete zebrafish cross-sections in parallel as a complementary approach. we find that within a day after onset of cell death multiple types of phagocytic cells, including microglia, accumulate in areas where cell death occurs, engulfing dying cells. granulocytes, do not migrate towards these dying cells and are not found inside the brain at these stages. we also find total numbers of immune cells, microglia in particular, are increased. in addition to microglia, we find phagocytic peripheral cells to be involved in clearance of dying cells. our findings provide an initial in vivo characterization of the immune response to programmed cell death in the brain, indicating involvement of resident and peripheral immune cells. we show spatiotemporal recruitment of these different immune cell types, and proliferation of microglia. the latter together with the recruitment of peripheral leukocytes are likely triggered to increase capacity to dispose of dying cells efficiently. our results indicate zebrafish provides a model to tease apart basic dynamic properties of immune maintenance of the diseased brain in vivo. our current studies focus on identifying the sequence of immunological events in resolving brain injury in vivo and how this is controlled, to ultimately help identify which of these aspects are harmful during brain damage or promote tissue recovery. in humans, lif and osm both signal through the lif receptor (lifr), however osm can also activate its specific receptor, osmr. lifr signaling promotes the survival of glial cells and neurons, and is thought to limit the development of pathogenic t helper 17 cells while promoting differentiation of protective regulatory t cells. osmr signaling is also neuroprotective, however the effects on immune cells are not yet elucidated. in this study, the immunomodulatory effects of lifr and osmr signaling in humans are investigated. we determined which immune cells express the receptors for lif and osm in healthy donors and compared this to the expression levels in ms patients. we found that in blood of healthy donors approximately half of the monocytes express the lifr and osmr, while 5-10% of the t cells and b cells express the receptors. more importantly, in untreated ms patients higher numbers of t cells and b cells express both the lifr and osmr as compared to healthy controls and treated ms patients. available treatments suppress the immune system, resulting in less activated t and b cells, which could explain the lower receptor expression. indeed, we measured the receptor expression on t cells and b cells after activation and demonstrated this strongly induces expression of both receptors. currently, we are investigating the effect of lif and osm on proliferation, cytokine production and antigen presentation of the different immune cell subsets. as blood circulating immune subsets of ms patients have a higher expression of lif and osm receptors, patients show increased susceptibility for modulation by these cytokines. thus, their immunoregulating properties together with the protection of glial cells and neurons indicates that these cytokines are promising candidates for the treatment of ms and other neuroinflammatory diseases. interleukin-6 (il-6) and interleukin-10 (il-10) are key cytokines with an important role in the regulation of the inflammatory and immune responses. in the central nervous system (cns), increased expression of both il-6 and il-10 occurs in a wide range of pathological conditions. meanwhile il-6 is recognized as a cytokine with a dual role acting as a pro-inflammatory or anti-inflammatory signal inducing glial activation, il-10 is well known by its ability to counteract the inflammatory and immune reactions. the objective of the present study was to evaluate the effects of local production of either il-6 or il-10 on the microglial response and the neuronal degeneration induced by facial nerve axotomy, a sterile neuronal injury model. to accomplish that, we used two transgenic mice, gfap-il6tg and gfap-il10tg, which express either il-6 or il-10 under the gfap promoter on astrocytes, i.e. a local production within the cns. unilateral facial nerve 1mm resection was performed, one set of transgenic and wild-type (wt) axotomized animals were sacrificed at 3, 7, 14, 21 and 28 days post injury (dpi) and cryostat free-floating sections were processed for immunohistochemical analysis. another set of animals were sacrificed at 21dpi to study neuronal survival. our results showed that, at 21 days after facial nerve axotomy, in comparison with the usual neuronal death observed in wt, selective cns il-6 production had a detrimental effect on neuronal survival, whereas, il-10 production was able to reduce neuronal death. these effects on neuronal survival correlated with changes in the expression of different molecules associated with microglial activation such as iba1, cd11b, cd16/32, mhc class ii, and some integrins like osteopontin and its receptors (cd44 and a5), along the different time points after injury. remarkably, when compared with their wt littermates, cd11b expression was higher on gfap-il10tg mice, whereas remains lower on gfap-il6tg animals along all the time-points analyzed. similarly, cd44 expression, which increases after axotomy in wt mice, was higher on gfap-il10tg mice at 14 dpi and 28 dpi, whereas no induction of this molecule was detected on the gfap-il6tg axotomized animals. in conclusion, our results indicate that astrocyte-targeted il-6 and il-10 production has a direct impact on neuronal survival, glial activation and integrin mediated signaling, producing a specific outcome of facial nerve axotomy. methods: we used bv-2 cells, a murine microglial cell line. in a first experiment aimed at determining the time course of the induction of expression of the lipoxygenases and receptors, they were incubated with lps. in a second experiment aimed at determining the effects of the resolvins and lipoxin on the production of il-10, il-1b, il-6 and tnfalpha, they were firstly incubated with lxa4, rvd1 and rve1 and then incubated with lps. results: our results indicated that lps only enhanced the expression of alx, the receptor for rve1 and lxa4 ( i2) (p < 0.05). the expression of the 15-lipoxygenase was also affected by lps (p < 0.01) and varied during the time course, reaching a maximum after 6h of incubation (p < 0.05). the production of the proinflammatory cytokines decreased after 18h of incubation: il-1b with rvd1 (100 nm, p < 0.001) and rve1 (10 nm, p < 0.01); il-6 with rve1 (1 and 10 nm, p < 0.05 and p < 0.001); tnfalpha with rve1 (1 and 10 nm, p < 0.001 and p < 0.01). the production of the anti-inflammatory cytokine il-10 increased only when cells were incubated with lxa4 after 6h (1 and 10 nm, p < 0.01). conclusions: these results suggested a potential role of pufa-derivates in the resolution of inflammation. methods: the expression of a panel of m1 and m2 markers on human monocyte derived m1 and m2 macrophages was analyzed using flow cytometry. this revealed that cd40 and mannose receptor (mr) were the most distinctive markers for m1 and m2 macrophages, respectively. we next examined the activation status of macrophages/microglia in ms and control tissue using a panel of m1 and m2 markers. results: our data show that m1 markers, were abundantly expressed by microglia in normal appearing white matter and by activated microglia/macrophages throughout active demyelinating ms lesions. m2 markers, mr and cd163, were expressed by myelin-laden macrophages in active lesions and perivascular macrophages. double stainings using anti-cd40 and anti-mr revealed that 70% of the results and conclusions: the aim of the present study was to evaluate the specific binding of [ 3 h]pk11195, a compound targeting tspo, to human tissue sections from patients with ms, and to correlate these findings with presence of tspo1 microglia in different types of lesions. specific binding of [ 3 h]pk11195 was evaluated using autoradiography, and histological analysis was performed on tissue sections to evaluate lesion type, tspo expression and presence of microglia. there was a clear correlation between level of specific binding of [ 3 h]pk11195 and lesion type, such that there was an increased binding to tissue with active or chronic-active lesions compared to control tissue or tissue with chronic lesions. this was concomitant with an increased tspo1 expression and increased presence of microglia in tissue sections with active or chronic-active lesions. in conclusion, specific binding of [ 3 h]pk11195 is correlated with lesion type, tspo expression and presence of microglia in tissue sections from ms patients. being astrocytes key regulators of microglial cell activation. during inflammatory response, glial cells secrete cytokines such as tnfa, il1b and tgfb, as well as nitric oxide (no), and activate phagocytosis, chemotaxis, increased expression of receptors that bind cytokines and inflammatory ligands. among ligand receptors are induced pattern recognition receptors such as scavenger receptor (srs), including class a receptor (sra), expressed by astrocytes and microglia. we will evaluate if sra has active roles in ab clearance and inflammatory activation of glia. methods: we evaluated the role played by sra in glial cell activation, and the regulatory capacity of astrocytes upon microglia in vitro, assessing production of no griess assay and cytokines by elisa; phagocytosis and activation of activity identity markers of signaling pathways by western blot. results: in primary glial cultures from wild type (wt) and knockout mice (ko) for sra, it was found that lps-stimulated sra ko astrocytes release 50% more no than sra wt astrocytes. these differences were associated with an extended activation of erk signaling pathway. in contrast, evaluation of lps-induced cytokines production, only wt astrocytes released il1b, whereas production of tnfa and tgfb reached similar levels in wt and sra ko animals. the abolition of il1b release by sra ko astrocytes appeared to be associated to the inhibition of activation of jnk and p38 signaling, and to the delayed activation of ijb/nfjb pathway in response to lps. microglia from sra ko mice also showed several differences in response to lps stimulation compared with their wt counterparts. they failed to show activation-associated morphological changes and were unable to secrete il1b, although they showed an increased release of no. in terms of activation pattern, sra ko microglia showed increased expression of mhc-ii, indicating that sra could be involved in a mechanism inhibiting mhc-ii expression that has not been previously documented. conclusions: our results suggest that sra participates in the activation of specific inflammatory responses by astrocytes and microglia, and the activation of signaling pathways involved in the production of soluble molecules, being also needed for astrocytes in order to be able to modulate microglia activation. microglia are the resident macrophages of the brain and the first responders to disturbances of brain homeostasis. they exist in one of two broadly defined states, a rather inaptly named "resting state" in which they exhibit a ramified morphology and, upon tissue disturbance, an "activated state" in which they exhibit an amoeboid morphology. the characterization as "resting" is in stark contrast with the frantic morphological changes that ramified microglia undergo in brain slices and in vivo, constantly extending and retracting processes. while the purinergic receptor p2y12 has been identified as a key receptor via which microglial processes are guided to a source of atp or to the site of an injury, the signals and pathways guiding microglial processes in the healthy tissue remain elusive. here, by imaging microglia in acute hippocampal brain slices, we assessed the baseline and targeted motility of microglial processes in the presence of different pharmacological agents targeting purinergic signaling. we found that while bath application of a p2y12 specific blocker alone (psb-0739 at 500 nm) or of a p2y1 specific blocker alone (mrs2179 at 25 mm) did not affect the baseline motility of microglial processes, bath application of a p2y1/12/13 blocker (mrs2211 at 25 mm) strongly reduced the baseline motility of microglia, leading them to retract most of their processes. moreover, a strong rebound of motility was observed after washing out mrs2211 that did not lead to microglial activation within the time window of the experiment (up to 90 minutes). in experiments measuring the chemotactic response of microglia to a pipette containing 1 mm of atp inserted into the slice, we found that chemotaxis was completely abolished by psb-0739 (bath applied at 2 mm) but not by mrs2211 (20 mm). finally, no chemotaxis was observed in response to a pipette containing udp-glucose (5 mm), an agonist of the p2y14 receptor. these results confirm that p2y12 alone controls the targeted chemotactic response of microglial processes to atp, with no discernable involvement of p2y1, 13 or 14, but suggest that p2y1, 12 and 13 might collectively play a role in controlling the baseline motility of microglial processes in the healthy tissue. how the different p2y receptors interact with each other to control microglial movements remains to be determined. supported by an eu marie curie fellowship, the wellcome trust and the erc. the ubiquitin-proteasome-system (ups) plays a central role in degradation and clearance of short lived regulatory as well as misfolded or damaged proteins. upon stimulation by interferons (ifn) the catalytic subunits b1, b2 and b5 of the 20s proteasomes (s-proteasomes) can be replaced with b1i/lmp2, b2i/mecl-1 and b5i/lmp7 subunits, giving rise to the catalytically active immuno-proteasomes (i-proteasomes), respectively. since i-proteasomes play an important role in clearance of oxidant-damaged proteins that preferentially accumulate upon inflammatory stimulation, and many neurodegenerative diseases, including alzheimer's disease (ad) and parkinson's disease (pd) are associated with a chronic inflammatory reaction, we tested the impact of i-proteasome deficiency on the pathogenesis and progression of ad and pd. while appps1 mice, a model for ad associated extracellular plaque pathology, exhibited increased levels of i-proteasome subunits in the brain, genetic ablation of the b5i/lmp7 subunit, which significantly impairs i-proteasome function, resulted in a reduction of cerebral plaque burden, indicating a disease exacerbating role of iproteasomes in ad. in contrast, transgenic alpha-synuclein (tgasn) mice, a mouse model for intracellular alpha-synuclein pathology associated with pd, crossed to b5i/lmp7 subunit deficient mice, highlight an increase of aggregated alpha-synuclein accompanied by worsening of pathology in tgasn mice lacking functional i-proteasomes, suggesting a protective role of i-proteasomes for intracellular amyloid pathologies. future studies will aim at resolving the mechanistic underpinnings of the seemingly opposing effects of i-proteasome deficiency on the pathogenesis and progression of extracellular and intracellular protein aggregation to aid better understanding and guide novel therapeutic opportunities for neurodegenerative diseases. in the most severe form x-linked adrenoleukodystrophy (x-ald) is a fatal neurodegenerative disorder with inflammatory demyelination of the brain caused by a deficiency of the adrenoleukodystrophy protein (aldp), encoded by the abcd1 gene. aldp, a member of the abc transporter subfamily d, is located in the peroxisomal membrane and transports very long-chain fatty acids (vlcfa) as coa-esters for degradation into the peroxisome. currently, the only curative therapies are allogeneic hematopoietic cell transplantation (hct) or genetically corrected autologous hematopoietic cd34 1 stem cell therapy (hsct). the success probably relies on the settlement of microglia and perivascular macrophages derived from the myelo-monocytic donor cells in the brain of the patient. therefore, we explored the phenotypes of the major immune cell types derived from the cd34 1 stem cell. immune cells from peripheral blood of controls and x-ald patients were isolated by magnetic activated cell sorting (macs). purity of cell isolations was verified by flow cytometry. in purified cells qrt-pcr analysis was used to determine mrna levels of the abcd transporters and gc-ms was used for the quantification of vlcfa levels, a diagnostic marker. the three peroxisomal abc transporters were differentially expressed in cd34 1 derived cell types of healthy controls: abcd1 and abcd2 mrna were inversely expressed in all cell types, whereas abcd3 was equally distributed. abcd2, the closest homolog of abcd1 could be expected to compensate for the loss of abcd1 function. however, the expression pattern of abcd2 was unchanged in x-ald patients; accordingly, cell types lacking abcd2 mrna (e.g. monocytes) displayed the most severe biochemical phenotype concerning vlcfa catabolism, whereas cells with substantial abcd2 expression (e.g. t-lymphocytes) were barely affected. thus, not all investigated immune cells present an intrinsic metabolic defect. therefore, we propose that the beneficial effect of hct and hsct may rely on the replacement of those cells lacking sufficient abcd2 expression in abcd1 deficiency. in addition, these findings support the concept that abcd2 is a target gene for pharmacological induction, as an alternative treatment strategy, to rescue vlcfa metabolism and possibly halt the inflammation in x-ald patients. interleukin-33 (il-33) is a cytokine that has important functions in inflammatory and autoimmune diseases. little is known, however, about il-33 in the brain. we analyzed expression of il-33 in the mouse brain during embryonic and postnatal development. being undetected until late embryogenesis, il-33 was highly expressed during the first two weeks of postnatal life, after which expression gradually decreased and was then absent from the normal adult brain. astrocytes and oligodendrocyte precursors expressed il-33, but not neural progenitors or neurons. the vast majority of il-33 positive cells displayed nuclear staining. apart from its function as a pro-inflammatory cytokine, a role for nuclear il-33, potentially in transcriptional regulation, is also emerging. the important role of neuro-inflammation in traumatic brain injury is being increasingly recognized, and local cytokine production can mediate the inflammatory response. because of its potential for attracting inflammatory cells we analyzed il-33 expression in a cortical contusion infarction model (cci). we found that il-33 expression was induced by injury, with a peak of expression three days after cci, and at six days the levels declined. il-33 positive cells showed an overlap with astrocytic and oligodendrocytic lineage markers, but not with neurons, neural progenitors, endothelial cells or microglia/macrophages. a similar time course for il-33 expression in human brain trauma was found, using cerebral microdialysis, which allows continuous sampling of the parenchymal concentrations of molecules over several days in vivo. the peak of released il-33 for the patient analyzed was 4 days post trauma. il-33 binds to a heterodimeric receptor composed of st2 and il-1racp. following injury of st2 knockout mice we found a general reduction of inflammatory cells at the site of injury, compared to wild type animals, and there were fewer microglia at the trauma site of mice lacking il-33 receptors. our data show that il-33 expression is under tight regulation in the normal brain, but is induced by traumatic injury where is important for attracting inflammatory cells. the small heat shock protein alpha b-crystallin (cryab) is expressed by oligodendrocytes (ol) and astrocytes at several stages of multiple sclerosis (ms) lesions. cryab has been shown to be protective and therapeutic in the eae model of ms, possibly due to its anti-apoptotic functions and regulation of inflammatory pathways. in this study, we further investigated the role of cryab in de-and remyelination in vivo, applying the cuprizone model of demyelination to cryab -/mice. surprisingly, we found that cuprizone-induced lesions are significantly smaller, less severe and less inflammatory in cryab -/mice, compared to wild type (wt) controls. staining for microglia and astrocytes revealed that astrocytes are the main inflammatory cell type that is affected by cryab expression: lesions in cryab -/mice display less reactive astrogliosis than wt controls. conversely, although less severe, lesions in cryab -/mice also contain fewer oligodendrocyte progenitor cells (opc) than in wt mice. in addition, our data suggest that remyelination is less efficient in cryab -/mice than in wt controls and that remyelinated areas in cryab -/mice contain fewer ol than remyelinated areas in wt mice. together, we hypothesize that cryab, due to its anti-apoptotic actions, mediates protection in both astrocytes and ol, enabling on one hand reactive astrogliosis, which contributes to demyelination, and on the other hand ol survival, facilitating remyelination. additional in vitro experiments support this hypothesis, revealing that cryab -/astrocytes are less reactive than wt astrocytes and that sirna-mediated knockdown of cryab affects ol survival. interestingly, analyzing phosphorylation patterns of cryab in cuprizone lesions revealed that cryab is differentially phosphorylated in astrocytes and ol. furthermore, we found that cryab is differentially phosphorylated in astrocytes in active demyelinating ms lesions, indicating that phosphorylation of the protein might underlie its (pathogenic) role in active demyelination. these paradoxical findings not only suggest a role for cryab as a potential target in a regenerative approach for ms treatment, but also point to a possible pivotal role for reactive astrogliosis in cuprizoneinduced demyelination and, more importantly, in early ms pathology. in a comprehensive immunohistochemical survey, we compared cortical ms lesions to those of inflammatory (tuberculous meningitis, rasmussen's encephalitis, b-cell lymphoma, and meningitis) and neurodegenerative (alzheimer's disease) diseases. although complex and fulminant immune responses were seen in many disease cases, primary demyelination was only detected in ms. in search of ms-specific molecular mechanisms, we performed whole-genome microarrays on micro-dissected archival formalin-fixed paraffin-embedded material. apart from cortical ms lesions, we also included brain tissue from patients suffering from tuberculous meningitis (inflammatory control) and alzheimer's disease (neurodegenerative control). additionally, control cases without brain pathology were included. using a restrictive cut-off, we identified 301 genes differentially expressed in ms lesions. more than 80% of these candidate genes could be assigned to t-cell mediated inflammation, microglia activation, oxidative stress, tissue injury, dna damage/repair as well as remyelination and regenerative processes. subsequent neuropathological analysis confirmed that significantly more oxidatively damaged neurons (stained positive for oxidized phospholipids) were present in cortical ms lesions than in any other examined disease. tunel stainings for the detection of dna strand breaks showed that neurons and oligodendrocytes with tunel-positive nuclei were most abundant in ms lesions containing zones of active demyelination and tissue injury. interestingly, immunohistochemical stainings for radical producing enzymes revealed that oxidative stress-mediated tissue damage in cortical ms lesions seems to be driven by nadph oxidases rather than by nitric oxide synthases. taken together, we were able to show that ms-specific primary demyelination is driven by mechanisms of tissue injury that differ from any other investigated inflammatory and neurodegenerative disease. among these mechanisms, oxidative tissue injury seems to play a major role. f. labombarda 1 , s. gonzalez 1 , i. jure 2 , a. de nicola 1 1 ibyme/conicet/uba, buenos aires, argentina 2 ibyme/coni-cet, buenos aires, argentina reactive gliosis and inflammatory mediators are implicated in demyelination and secondary damage after spinal cord injury (sci). we have previously reported that after sci, short-term progesterone treatment (3 days) stimulates oligodendrocyte precursor cells proliferation and decreases reactive gliosis, whereas chronic treatment (21 days) differenciates oligodendrocytes precursor cells into mature oligodendrocytes and enhances remyelination. presently, we further studied whether progesterone was able to modulate the inflammatory reaction and cytokine production by astrocytes and microglial cells. thus, the timecourse mrna expression of pro-inflammatory cytokines (il1b, tnfa and il-6) and pro-inflammatory enzymes (cox-2 and inos) was studied by pcr in real time. results showed that the highest increase in cytokine and pro-inflammatory enzymes mrna production occurred in rat spinal cord 6 h after sci. progesterone treatment significantly decreased the early rise of proinflammatory mediators mrnas at 6h. as progesterone action in spinal cord involves multiple mechanisms, the role played by the classical progesterone receptor (pr) on the progesterone inhibitory effects on cytokines, was assessed in prko mice. in agreement with data obtained in the rat model, sci strongly stimulated tnfa, il1b and il-6 mrna levels in spinal cord of both wild type and prko mice. however, whereas progesterone treatment inhibited the mrnas of cytokines in wild type mice 6h after sci, it was ineffective in prko mice, involving pr in the inhibition of cytokines production. finally, we measured astrocyte and microglial cells density by immunohistochemsitry after 6 h of progesterone treatment. the steroid administration significantly decreases gfap1 and ox-421 cells, which correlated with cytokine and pro-inflammatory enzymes inhibition. we conclude that progesterone attenuates reactive gliosis and inflammatory reaction, probably reducing the secondary spinal cord damage and favouring remyelination. in the steady state they are rarely observed in the cns parenchyma. however, during cns inflammation they cross the blood-brain barrier where together with microglia (cns-resident apc) they can present antigen to infiltrating t cells. the goal of this study was to compare two subpopulations of microglia: cd11c-and cd11c1 with brain dc in terms of promoting different t cell responses. two subpopulations of microglia: (cd11c-cd45dim cd11b1) and (cd11c1 cd45dim cd11b1) as well as (cd11c1 cd45hi) bdc have been sorted from the cns of c57bl/6 mice subjected to eae. sorted cells were used either for ex vivo proliferation and cytokine assays or for qrt-pcr. our results show that bdc and cd11c1 microglia are similar with regard to ability to induce proliferative t cell response from both primed and na€ ıve t cells. nevertheless, they differ in their cytokine profile and ability to promote cytokine release by t cells. our findings suggest that different subtypes of apc in the cns promote different immune responses. this work has been supported by lundbeckfonden. melanocortin 4 receptor (mc4r) is predominantly expressed in the brain and it is the only mcr expressed in astrocytes. our previous results showed that a-melanocyte-stimulating hormone (a-msh) the anti-inflammatory action is mediated by mc4r in astrocytes and in the hypothalamus of male rats and that these effects may lead to neuroprotection. we have already demonstrated that ndp-msh (an a-msh analogue) increased brain-derived neurotrophic factor (bdnf) expression through the camp-pka-creb pathway in astrocytes. in the present study we examined the participation of mitogen activated protein kinases (mapk) and phosphatidylinosotol-3 kinase (pi3k)-akt pathways in mc4r signaling in astrocytes. we also investigated the effect of a-msh on bdnf expression in vivo in brains of male rats.we preincubated cultured rat primary astrocytes with mapk (p38, jnk and erk) and pi3k-pdk1-akt inhibitors 15 min before addition of 1 mm ndp-msh for 1 h. we found that ndp-msh-stimulating effect on bdnf expression assayed by qrt-pcr was abolished only in the presence of erk and pi3k inhibitors. accordingly, levels of phospho-erk1/2 determined by western blot were increased by ndp-msh whereas phospho-akt levels were not modified at 30 min. ndp-mshinduced erk1/2 activation was decreased by adenylate cyclase and pi3k inhibitors but not by a pka inhibitor, suggesting a camp and pi3k involvement in this effect. we also investigated if a-msh was able to induced bdnf expression in vivo. for that purpose we injected male rats with a-msh (0.5 mg/kg, ip) or vehicle (saline) once and sacrificed them after 3 h. rats were also injected twice daily and for two days and killed 48 h after the first injection. we observed that bdnf mrna levels increased after a-msh treatment at 3h in the hypothalamus whereas in the cortex bdnf expression was not modified. at 48 h bdnf expression was not modified by a-msh. we showed by immunohistochemistry that mc4r and bdnf co-localizes with neurons and astrocytes in the brain. since melanocortins have anti-inflammatory and neuroprotective effects in the brain, the mechanisms described in astroglia help understand mc4r action. our results indicate that melanocortins induce bdnf expression in astrocytes through erk and pi3k, suggesting that this effect could be involved in the neuroprotective actions of melanocortins. the fact that bdnf expression also increases in vivo in the hypothalamus reinforce this idea. methods: we analyzed microglia activation and the role of nmda receptors in this process using the microglia cell line bv2 and cortical slice cultures. the in vivo analysis was performed in two models: first, in odtr mice that express the diphtheria toxin receptor specifically in oligodendrocytes (odcs) which allows induced killing of odcs (locatelli et al., 2012) and second in mice that were immunized with mog/ cfa to induce eae. results: the proinflammatory cytokine il-17a plays a pivotal role in the pathogenesis of ms and eae. we showed il-17a activates microglia in vitro. therefore, we activated the microglia cell line bv2 and cortical slice cultures with il-17a to and subsequently treated the cultures with the nmda receptor antagonists mk801 and ap5 to block nmda receptor signaling. this treatment inhibited il-17a-mediated activation of microglia and in particular ros production, proliferation and migration. additionally, we detected increased secretion of il-6 and g-csf. furthermore, il-17a enhanced activation of the nmda receptor through phosphorylation leading to higher influx of calcium upon ligand binding. to further analyze nmda receptor-depended microglia activation we used two different mouse models that provoke different mechanisms of microglia activation. whereas in eae a strong inflammatory process activates microglia, in odtr mice, activation occurs due to induced odc death. interestingly, in the context of eae microglia activation was clearly diminished when mice were treated with mk801 whereas inhibition of the nmda receptor had no effect on microglia activation in the odtr model. in line with the in vivo data we show that inhibition of microglia activation takes place only when the cells were stimulated with il-17a and not upon stimulation with lps. conclusions: our findings show that nmda receptors are not involved in general microglia activation but rather play a role in microglia activation in an inflammatory context with specific cytokines such as il-17a. aims of the study: to investigate whether cx3cl1-cx3cr1 signaling contributes to microglia migratio00ctivation and subsequent astrogliosis following msc transplantation in the cns. methods: first, in order to allow localization of grafted msc in vivo, wt c57bl/6 msc were transduced with a lentiviral vector encoding the blue fluorescent protein (bfp). next, bfp1 msc were injected into the cns (striatum) of cx3cr1 1/-(n 5 10) and cx3cr1 -/-(n 5 7) transgenic mice. these mice have respectively one or both copies of the cx3cr1 gene replaced by the egfp reporter gene. at 10 days posttransplantation, histological analyses were performed to determine iba11 microglia and s100b1 astrocytes within and surrounding the bfp1 msc graft site. astrogliosis was determined based on staining for gfap. cx3cr1 expression was evaluated based on egfp expression. all histological evaluations were quantified using tissuequest and/or imagej cell analysis software. results: (i) bfp1 msc graft survival is observed in both cx3cr1 -/and cx3cr1 1/mice; (ii) microglia migration towards grafted msc occurs independent of functional cx3cr1-cx3cl1 signaling; (iii) down-regulation of egfp gene-expression (and thus also cx3cr1 gene expression) is observed in both cx3cr1 -/and cx3cr1 1/mice on msc graft-invading microglia, but not on msc graft-surrounding microglia; (iv) the absence of functional cx3cr1-cx3cl1 signaling in cx3cr1 -/mice results in significantly less astrogliosis around the msc graft site, as compared to the msc graft site in cx3cr1 1/mice. conclusions: here we identified cx3cr1-cx3cl1 signaling as a potential target to modulate and/or control astroglial scarring following (stem) cell grafting in the cns. the latter is of significant importance as grafted cells can only functionally interact with (injured) brain tissue in the absence of a graft-surrounding astroglial scar. this accumulation is known to be essential for the usual sprouting of injured axons and leads to a functional nerve repair. because of the insignificant infiltration of macrophages in the injured leech cns, we investigate the chemotactic mechanisms of the recruitment of only resident microglial cells in order to explore in fine the crosstalk between damaged neurons and activated microglia leading to the leech cns repair. methods: in vitro and ex vivo chemotactic assays were performed by using recombinant form of hmc1q on microglial cells which were pre-incubated or not with anti-cc1qr or anti-gc1qr antibodies. then, affinity purification analyses using biotinylated c1q were performed from leech microglial cell protein extracts. finally, immunohistochemistry analyses were located the production of newly characterized c1q receptors in microglial cells. results: we demonstrated that rhmc1q contributes to the recruitment of some leech microglial cells. chemotaxis assays showed that the blocking of respective receptors, homologous to human gc1qr and cc1qr, inhibits the hmc1q-dependent recruitment. importantly, affinity purification analyses demonstrated the interaction between both receptors and hmc1q. finally, those receptors were located on distinct microglial subsets. conclusion: this work is used to specify the activation processes of only resident microglial cells. the results show the importance of hmc1q in the microglial accumulation leading to the nerve repair. in h. medicinalis, hmc1q activity is driven through two different receptors which are not present on the same cells suggesting that hmc1q activates two distinct microglial cell subpopulations. the question whether blood-borne immune cells are infiltrating brain areas afflicted with neurodegeneration in ad and other tauopathies yielded contradictory results. some authors showed that the chronic neuroinflammation in ad was provided almost exclusively by resident cns cells without any apparent influx of leukocytes from the blood while others reported that hematopoietic cells can enter the brain in alzheimer's disease and may contribute to an increased inflammatory burden. in order to address this issue we have used two independent transgenic lines expressing human misfolded truncated tau in two different genetic background (wistar and shr). we have shown that tau neurodegeneration can induce inflammatory responses mediated by reactive microglia in both transgenic lines, however the microglial responses showed a striking difference between the lines. we have identified two significantly different inflammatory responses to the same inducer. moreover, we found that the genetic background significantly modified the molecular cascade influencing leukocyte influx into the brain. in both transgenic lines, the majority of the blood cells entering the brain belonged to antigen presenting cell family -monocytes and dendritic cells. at the proteomic level we found striking differences between the lines. while in the wistar transgenic line we observed significant increase of cytokine-induced neutrophil chemoattractant-1, in shr transgenic line, tissue metalloproteinase 3 was significantly reduced. these findings suggest that blood cell infiltration of the brain affected by tau neurodegeneration is modified by genetic background. this inter-individual variability should be taken into the consideration in the development of novel drugs with anti-inflammatory properties. acknowledgement: this work was supported by axon neuroscience and research grants vega 2/0161/11, 2/0205/11, 2/0193/11, apvv 0200-11, apvv 0206-11 and structural fund 26240220046. charit e -universit€ atsmedizin, berlin, germany toll-like receptors (tlrs) are key molecules of the innate and adaptive immune response in vertebrates. the original protein toll in drosophila melanogaster regulates both host defense and morphogenesis during development. tlrs recognize host-and pathogen-derived stimuli. single-stranded rna is sensed by tlr7 localized to the endolysosomal compartment of immune cells. we found that both extracellular viral rna and host-derived micro-rnas induce cell-autonomous and microglia-mediated neuronal cell death through the endosomal receptor tlr7 in vitro and in vivo. however, the exact intracellular signalling cascade and the cellular mechanisms through which tlr7 leads to tissue injury in this context remained unclear. unc93b1 is a molecule specifically involved in trafficking of nucleotide-sensing tlrs, such as tlr7, in immune cells of both humans and mice. unc93b1 physically interacts with this receptor in the endoplasmic reticulum (er), and the function of the membrane protein unc93b1 is to deliver the nucleotidesensing receptors from the er to endolysosomes. making use of real-time pcr, immunocytochemistry, and histochemistry we systematically examined the expression of unc93b1 in the murine cns. whereas a distinct expression for this molecule was observed in microglia and astrocytes, expression of unc93b1 in cultured neurons was negligible. however, expression of this molecule was detected in cortical neurons of brain sections. moreover, unc93b1 was strongly regulated during different embryonic, postnatal, and adult stages of the developing mouse brain. neurons of various brain regions were identified as the main cell type expressing unc93b1 in the developing brain. taken together, our data reveal a specific expression pattern of unc93b1 in the cns, in particular in a developmental context, and lay foundation for further investigation of the pathophysiological significance of this molecule for both injurious and developmental processes in the central nervous system of vertebrates. has benefited from a number of studies that precisely depicted different types of plaques in white or grey matter areas. also, both inflammation and diffuse axonal loss in the normal appearing white matter (nawm) were extensively described. however, little attention was given to the so-called periplaque, which is usually defined as a partially demyelinated ribbon of tissue surrounding the plaque. whether periplaques correspond to expanding lesions, sites of ongoing remyelination or areas of tract degeneration remains uncertain. methods: in this context, our study aimed to bring quantitative insights to the neuropathology of ms periplaques in the spinal cord of primary or secondary progressive ms patients. a neuropathological quantitative assessment of inflammation, axonal loss and myelin loss was performed concurrently in the periplaques, plaques and normal-appearing white matter (nawm) of cervical spinal cord samples from 16 patients with progressive ms. results: periplaques formed large areas of incomplete myelin loss that extended distant away from the border of plaques. axonal loss was quantitatively similar in plaques and periplaques but signs of axonal dystrophy were predominantly observed in plaques. surprisingly, axons that remained myelinated in periplaques presented a thicker myelin sheath than myelinated axons of the nawm. inflammation in the periplaque was mainly characterized by an accumulation of macrophages/microglia that were closely apposed to myelin sheaths but exerted poor phagocytic activity. finally, we found that neuropathological features of periplaques were overall disconnected from that of plaques with regard to size, shape, inflammation and axonal integrity. conclusions: our work indicates that in ms spinal cords, periplaques correspond to demyelinating rather than remyelinating lesions. it further suggests that in progressive forms of ms, periplaques are likely to impact significantly on neurological disability. finally, we propose that tract degeneration might not be the only cause of periplaque extension and that slowly-expanding demyelination, temporally remote from plaque formation, might occur in periplaques. molecular results will be presented that support these hypotheses. cell-adhesion between endothelial cells at the blood-brain barrier (bbb) makes the endothelium impermeable to blood-derivatives and immune cells. to establish and maintain this barrier during development, adulthood, and during disease, brain endo-thelial cells must develop and sustain these strong adhesive contacts, through expression of tight junction molecules. however, we do not know whether netrin supports inter-endothelial cell adhesion at the blood-brain barrier. given this, we hypothesize that netrin tightens the bbb during development, adulthood, and protects it during disease. methods: to test this, we used both human adult primary brainderived endothelial cells and newborn netrin-1 knockout mice and evaluated netrin's effect on inter-endothelial cell adhesion and barrier permeability. we also assessed netrins' therapeutic potential to maintain the barrier and limit immune cell infiltration into the central nervous system (cns) during experimental autoimmune encephalomyelitis (eae). results: our results demonstrate that brain endothelial cells express netrins. they help to form a tighter bbb during development. they also maintain and protect the adult barrier by increasing the expression of endothelial junction molecules, thus promoting inter-endothelial adhesion and reducing protein leakage across the barrier. netrins also reduce bbb breakdown and diminish initial myeloid cell infiltration into the brain and spinal cord during eae, which delays disease onset and ameliorates disease severity. discussion: we conclude that netrins enhance bbb stability, and protects the bbb during neuroinflammatory disease. microglial cells were shown to play an important role in many diseases of the central nervous system, not least due to their capability to become activated upon signs of brain injury. they migrate to lesion sites, proliferate, release cytokines and chemokines, and phagocytose cells or cellular debris. therefore, the microglial cytoskeleton has to be highly rearrangeable. one major element of the contractile system in nonmuscle cells is nonmuscle myosin ii (nm ii). as the role of nm ii in glial cells is poorly characterized, it is an aim of this project to study nm ii-dependent functions in microglia. we found that inhibition of nm ii by blebbistatin, which is a highly selective inhibitor of nm ii adenosine phosphatase, prevents any morphological shaping in freshly plated microglia and leads to functional deficits during migration and phagocytosis of fluorescence-labeled beads. using confocal microscopy, we could find diffuse expression of nm ii in resting microglia in vitro, whereas activation with bacterial lipopolysaccharide (lps) led to perinuclear accumulation of nm ii protein. in an activated state, microglial cells release pro-inflammatory cytokines and reactive oxygen species; interestingly, in the presence of blebbistatin the release of nitric oxide (no) as well as rearrangement of nm ii was prevented. taken together, these results illustrate a key role for nm ii in microglial shaping and functioning. ongoing studies will improve our knowledge on spatial and functional aspects of the actomyosin complex during demyelinating processes and open targets for development of therapeutic strategies towards remyelination in the cns. prolonged activation of the nucleus basalis of meynert (nbm), the primary source of cholinergic projection to the cerebral cortex has been reported to cause significant increases in cerebral cortical blood flow (cbf) in rodents. while the nbm also gives rise to gabaergic and glutamatergic projections to the cerebral cortex, the nbm-driven increase of cbf has been described to be dependent in part on muscarinic acetylcholine receptors. lately, several groups reported that astrocytes modulate local cbf via intracellular ca 21 signaling. considering that cortical astrocytes express machrs and in vivo activation of the nbm leads to machr-dependent ca 21 surges in astrocytes, cholinergic modulation of cbf via astrocytic ca 21 surges is conceivable. we report here that a single train stimulation of the nbm (stnbm; 100 hz, 0.5 s, 200 ma) induces a biphasic (a rapid increase, followed by an overshooting slower decrease that goes back to baseline within a minute) laser doppler flowmetry (ldf) response in the somatosensory cortex. moreover, the stnbm-induced ldf response was sensitive to the machr antagonist atropine. stnbm with a weaker stimulation intensity (50 ma) induces a transient decrease. surprisingly, we find that ip3r2 knockout mice, which lack cytosolic ca 21 surges in astrocytes, show similar ldf responses to stnbm. a.-c. boulay, c. giaume, m. cohen-salmon cirb, paris, france astrocytic endfeet are specialized processes, which enwrap blood vessels and express a large molecular repertoire dedicated to the physiology of the vascular system. one of the most striking properties of astrocyte endfeet is their enrichment in the gap junction proteins connexin (cx) 43 and cx30 allowing for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. however, the role of these proteins at the gliovascular interface is not fully understood. we have recently demonstrated that astroglial perivascular cxs expression starts after birth during bloodbrain barrier maturation and controls its integrity (ezan et al. cx30 d/d purified vessels showed essentially a strong upregulation (fold change > 12) of sgcg. this gene encodes gamma-sarcoglycan, a membrane glycoprotein associated with the dystrophin-dystroglycan complex which is essential for muscle integrity and is impaired in duchenne muscular dystrophy (petrof et al., 1993) . at the gliovascular interface, the dystrophin-dystroglycan complex anchors astrocyte endfeet to the basal lamina (nico et al., 2010) , and is crucial to the bbb integrity. however, expression of sgcg and its role in the brain has until now only been poorly documented. moreover, we found an upregulation of s100a8 and s100a9 (fold change 6,3 and 5,5, respectively) encoding the calgranulin a and b, two proteins present in neutrophils and monocytes, and involved in their migration to inflammatory sites, attachment to endothelial cells and extravasation (ryckman et al., 2003) . altogether, our results suggest that cx30 might modify the dystrophin-dystroglycan complex thereby influencing blood-brain barrier properties, and control the attachement as well as the transmigration of leukocytes through the endothelium, thus contributing to the regulation of inflammatory processes in the brain. the blood-brain barrier is supported by the perivascular glia. a crucial event during the gliogenesis is the replacement of radial glia by astrocytes. immunohistochemical staining of nestin visualized the first gliovascular connections after e16, and by e20 the radial glial endfeet cover the vessels completely. by e20 numerous nestin immunoreactive astrocyte-like cells participated in it. in parallel, nestin immunoreactivity also appeared in the wall of the vessels. by about p10 both radial glia and nestin immunopositive astrocytes disappeared and gave place to astrocytes immunoreactive to s100 protein and glutamine synthetase, markers of mature astrocytes. in the radial glia these markers were not found, and they colocalized with nestin only scarcely. the s100 and glutamine synthetase immunoreactive cells appeared at first in the middle zone of the pallium about p2, and their population gradually extended to both the pial surface and the deep cortical zones, colonizing the whole thickness by p8, in parallel with the disappearance of nestin immunoreactive glial elements, either radial or astrocytic ones. at p6 gfap immunoreactive cells were still confined to the most superficial and deepest cortical zones, so showed no colocalization with s100 and glutamine synthetase, unlike that found after p8. whether this glial 'takeover' results in a transitory increase of leakage of the blood-brain barrier, it remains to be answered. it seems however, to underly that cerebrovascular fibronectin and laminin immunoreactivities disappear at first from the middle zone of the developing cortex and only later from the superficial and deep ones, between p2 and p10. according to krum and rosenstein (1991) disappearance of these immunoreactivities refers to the fusion of the glial and vascular basal laminae, the maturation of the definite gliovascular connections. surrounded by astroglial scars that inhibit such growth. little is known about how astrocytes assemble to form this scar, and a deeper understanding of the cellular and molecular mechanisms that control this process is needed if this goal is to be achieved. in vitro data suggest that certain bioactive molecules can alter astrocyte morphology into a more growth supporting state. other molecules have been shown to promote axon growth. clinical application of many of these molecules would require neurosurgical delivery directly into the spinal cord. injectable biomaterials have the potential to serve as depots and scaffolds for in vivo delivery of bioactive molecules. we are developing di-block co-polypeptide hydrogels (dch) as fully synthetic biomaterials that could safely and easily be injected into specific sites after sci to deliver molecules in order (i) to understand better, and (ii) to manipulate, the mechanisms that control glial scar formation. we have previously shown that dch are biocompatible after injection into brain or spinal cord and selfassemble into structures with finely controllable properties. our current work shows that dch depots can deliver diffusible bioactive growth factors that influence local neurons or astrocytes in predictable ways and form gradients that are effective up to several mm away from depots. we are now testing the ability of dch depots to deliver specific growth factors that will manipulate scar-forming cells when dch are injected at clinically realistic sub-acute times after sci. to investigate the molecular phenotype of esdm, we performed a whole transcriptome analysis of esdm in comparison to primary cultured microglia, flow cytometry-sorted microglia, different myeloid cells, t cells, astrocyte-and neuron-enriched cultures. while being distinct from t cells, neurons and astrocytes, esdm were closest related to primary cultured microglia followed by flow cytometry-sorted microglia and other myeloid cells. esdm and primary cultured microglia showed a strong overlap in their transcriptome by only having 143 gene transcripts differentially expressed. most of these differentially expressed genes were immune-related genes that were up-regulated in primary cultured microglia. this indicates that esdm were in a less activated state rather reflecting the in vivo biology of these cells. flow cytometry analysis of cell surface markers selected from the microarray confirmed the close relationship between esdm and primary cultured microglia. esdm resemble primary microglia not only in respect to surface marker expression and functional properties, but also display a molecular phenotype similar to primary microglia. our data suggest that esdm are a reliable tool for the replacement of primary microglia. spinal cord injury (sci) is a devastating condition which usually leads to paralysis. this outcome of injury in most cases is permanent due to the cascade of immunological factors that create an anti-repair environment around the site of injury and due to the limited capacity of central nervous system (cns) axons to regenerate, creates a daunting challenge to establish an effective therapy. from many years of research it is apparent that a combination of therapies would be the best course of action for treatment of sci; this however greatly increase the animals cohorts and time to establish the most effective course of treatment. for this reason we have developed an in vitro model of spinal cord injury as a moderate throughput screen, which is in accordance with the 3r's. this culture consist of dissociated mixed rat embryonic spinal cord cells plated onto a monolayer of astrocytes this is essential for the spinal cord cells, obtained from rats of embryonic day 15, which then develop into a carpet of neurites that over time become myelinated forming internodes of myelin interspaced with nodes of ranvier. this mixed cns culture was utilized using a scalpel blade to axotomise the axons and a persistently neurite-free area is created which is accompanied by many features of sci, including demyelination and reduced neurite density adjacent to the lesion and the presence of and reactive astrocytes. these finding are congruent with changes seen in vivo after spinal injury. data will be presented regarding the effects from proven and novel pharmacological agents on neurite outgrowth and myelination looking to understand the mechanism in which they act. as well as modifications of the culture system to incorporate methods to align axons and isolate cell bodies from their processes for a more detailed analysis of regeneration. the role of increased activity of astrocyte networks in structural synaptic plasticity following remote axonal injury is unexplored. we set out to investigate the efficiency of synaptic rearrangements in models where astrocyte reactivity is selectively diminished by the inhibition of the main cytokine pathway in transgenic mice (tg) in comparison to that observed in wild type mice (wt). first, synapse densities were compared in the facial nuclei two weeks after unilateral facial nerve transection by synaptotagmin-1/psd-95 immunolabelling and ultrastructural analysis. we found that the recovery of synapse density was decreased by 40% in tg mice compared to wt mice, and that the reduction in synaptic density correlates with a relative loss of neuronal coverage by reactive astrocyte end-feet in the same conditions. specifically, we observed a 30% reduction in excitatory synapse density in the tg mice. second, we developed an organotypic entorinho-hippocampal slice culture system in which astrocyte activation and synaptic plasticity could be more closely monitored in real time. this was assisted by two-photon microscopy, using glial ca 21 -imaging and dendritic spine motility analysis after perforant pathway transection. in summary, we demonstrated that full astrocyte activation is necessary for partial structural recovery of synapses after axonal injury. our work, using simplified models, may help experimental approaches aimed at optimizing adaptive plasticity in cns injuries. pathological loss of myelin in diseases like multiple sclerosis (ms) is usually followed by a phenomenon of remyelination, in which oligodendrocytes synthesize new myelin sheaths to envelope exposed around axons in the adult central nervous system (cns). the importance of remyelination arises from the fact that this process not only restores saltatory conduction, but also protects axons from different insults and thus limits clinical disability in demyelinating diseases. in this scenario, the mammalian subventricular zone (svz) has garnered attention as a potential source of replacement cells after injury. this zone harbours stem cells and supports long-distance migration, and is activated in ms patients to promote gliogenesis. although ng2 1 precursor cells are the first to react to demyelination and show the highest proliferation rate in comparison with other cells, the relative contribution of the svz with respect to the inflammatory demyelination induced by the theiler's virus infection and oligodendrogenesis has never been addressed. in the present study, we have investigated the behavior of the svz in theiler's murine encephalomyelitis virus -induced demyelinating disease (tmev-idd). we report that in this viral model for ms, there is a preclinical phase of the disease with demyelination in the corpus callosum that is followed later by an attempt of remyelination. this phase is accompanied by an activation of the svz with no apparent activation of ng2 1 precursors, and a strong and clear staingalectins (gals) are a family of soluble lectins that, when binding to b-galactosides, form multivalent complexes with glycoconjugates of the cellular surface and induce a modulation of intracellular signaling pathways for differentiation and survival. recently, galectin-1 has been described as a microglial deactivator which prevents neurodegeneration and promotes neuroprotection in an eae model. however, its action at neuronal level is poorly understood. spinal cord injury (sci) also remains a major challenge to neurological research, as several factors such as extracellular matrix molecules inhibit axonal regeneration. among them, semaphorine 3a (sema3a) contributes to the inhibition of axonal regeneration by acting on microtubules and actin cytoskeleton. neuropilin-1 (nrp-1) is a neuronal receptor whose binding of sema3a generates repulsion of axonal growth. in addition, nrp-1 has been described as a target of gal-1 in non-neuronal systems. the aim of this work was to study the role of gal-1 intraumatic sci, considering that sema3a expression is "turned on" in sci and prevents axonal regeneration via nrp-1. gal-1 knockout mice (lgals1 -/-) were submitted to a full traumatic sci at thoracic level (t9-t10) and treated with different concentrations of recombinant gal-1. we demonstrated that lgals1 -/mice treated with gal-1 have a dose-dependent functional recovery of motility as early as 6 days post-treatment, which is structurally associated to neuronal repopulation and high axonal regeneration. these effects were only observed in animals treated with gal-1 with dimerizing capacity, but not in mice treated with a mutant gal-1, only present in a monomeric form. in addition, we observed that microglial response was "turned off" in gal-1-treated mice. moreover, exogenous dimeric gal-1 bound to the surface of injured motoneurons and promoted the spread of nrp-1. 3d confocal microscopy reconstruction showed gal-1 and nrp-1 spatial proximity. to confirm this result, we carried out immunoprecipitation assays, which allowed us to determine that only the dimeric form of gal-1 interacted with nrp-1 and induced a decrease in the levels of sema-3a bound to nrp-1. our results show that gal-1-based treatments combine its neuronal effect on nrp-1 with its deactivating effect on microglia, which leads to a better restorative process after medullar lesion. the findings presented here support the potential of dimeric gal-1 as a therapeutic agent for human sci patients. theophylline and caffeine. it depresses activation of microglial cells and astrocytes which is associated with neuronal damage during inflammation and hipoxia. it is largely known that ethidium bromide (eb) injection into the cns induces local oligodendroglial and astrocytic death, resulting in primary demyelination, blood-brain barrier disruption and schwann cell invasion, and thus serving as an experimental demyelinating model in animals. the aim of this study was to evaluate if prop had the capacity of affecting glial cell behaviour during the process of demyelination and remyelination following gliotoxic injury with eb. wistar rats were divided into 2 groups: i-rats injected with 10 microlitres of 0.1% eb into the cisterna pontina and treated with prop; ii-rats injected with eb and not treated with the xanthine. prop (agener) treatment was done using 12.5 mg/kg/ day by intraperitonial route during all experimental period. the rats were euthanized from 7 to 31 days after eb injection and brainstem sections were collected and processed for light and transmission electron microscopy studies. results from both groups were compared by using a semi-quantitative method developed for documenting in semithin sections the extent and nature of remyelination in gliotoxic lesions. in general terms, by 7-11 days following gliotoxic lesion, the center of the lesion was expanded and filled with huge amounts of myelin debris among foamy macrophages and demyelinated axons. no astrocytic processes were found in this site and some lymphocytes were seen in the neuropil and around blood vessels. the most prominent feature of the 15 th day was the initial association at peripheral locations between naked axons and remyelinating cells. schwann cells were associated with one or multiple demyelinated axons or already forming thin myelin lamellae around single axons in astrocytic-free areas. oligodendrocytes began to form thin myelin sheaths in areas completely or partially filled with astrocytic prolongations. by 21-31 days, it became evident that rats treated with prop presented a small improve in the repair process when compared to untreated animals, the latter presenting greater amounts of myelin-derived membranes in the central area and a lesser extension of oligodendrocyte remyelination, but without any apparent difference regarding to schwann cells. by 31 days results showed that prop administration following eb injection seemed to stimulate oligodendroglial remyelination (mean remyelination scores of 3. peripheral axotomy results in a disconnection of the neuronal cell body from the target organ. a complex rearrangement of the nerve microenvironment, the so called wallerian degeneration, takes place distally to the lesion and involves schwann cell proliferation as well as macrophage infiltration. the process results in the formation of the bands of b€ ungner which guide the axonal sprouts throughout the nerve distal stump. although this process has been extensively studied, a thorough understanding of the three-dimensional nerve tissue reorganization is not completely known. such knowledge may in turn lead to more effective strategies aiming at the repair of peripheral nerves following transection or crushing. in this sense, the tetrahydrofuran (thf)-based tissue-clearing technique has been adapted for sciatic nerves of mice and used for subsequent observation to 2p-lsm. transgenic mice that express fluorescent proteins in neurons, astrocytes, schwann cells and microglia/macrophages as well as double and triple hybrids were used. additionally, by immunohistochemical analysis, it was possible to observe the structure of the nerve after injury by the expression of neurofilament and s100b, in non fluorescent mice. also, increased non neural cell responsiveness after injury was studied with anti-p75 ntr immunostaining. transmission electron microscopy allowed the evaluation of the ultrastructure of the nerve microenvironment, as well as the regenerative process by 3, 7 and 14 days after injury. microscopic examination of cleared tissues of transgenic mice through 2p-lsm consisted in comparing the different cell type interaction before and after injury, with particular interest on the schwann cells, macrophages and growing axons. question: mass spectrometry (ms) has become nowadays an essential tool for the detection and identification of biomolecules in various contexts of biological problematic. ms can be performed from all types of biological materials ranging from biological fluids to tissues. end of 90's it was shown that with certain instrumentation such as those equipped with a maldi ion source it was possible to record ms spectra directly in situ from tissue sections. more, point to point automated acquisition of maldi ms spectra from a whole tissue section was shown to allow for reconstruction of the 2d images of biomolecules contained in the section distribution through ion density maps (caprioli rm et al., anal. chem. 1997). methods: maldi ms imaging (maldi msi) is a molecular imaging modality that presents several advantages including possible imaging of hundreds of biomolecules in one step acquisition and the untargeted character of the method allowing to study molecules without any prerequisite knowledge or use of probes. results: over the past decade maldi msi has demonstrated its potentiality in terms of applications for biology and clinics (franck j et al., mol. cell. proteomics. 2009). maldi msi can be use to study both endogenous molecules namely metabolites, lipids, peptides and proteins or exogenous ones such as drugs or xeniobiotics and was applied to various problematic. understanding of physiological or physiopathological processes requires knowledge on the identity and localization of molecules within a tissue. maldi msi reseals a clear potential to answer this objective and give a unique contribution. indeed, more recently maldi msi has gained new strategies for identification of molecules in situ on tissues. in particular, many efforts were given to develop confident identification of proteins from tissue sections. conclusion: here we will review this novel technology and discuss some of its current and potential contributions for neurosciences application (wisztorski m et al., dev. neurobiol. 2008). this will be illustrated by presentation of the investigation of invertebrate neuroimmune response as well as vertebrate neuropathological disorders using either fresh frozen or formalin fixed paraffin embedded tissues. the sphingosine 1-phosphate (s1p) signaling pathway is known to influence astroglial reactivity in experimental autoimmune encephalomyelitis and the synthetic s1p analog fty720 has been shown to provide neuroprotection in experimental models of acute stroke. however, the effects of a manipulation of s1p-s1p1 signalling at later time points after experimental stroke have not yet been investigated. we examined whether a relatively late initiation of a fty720 treatment has a positive effect on long-term neurological outcome with a focus on reactive astrogliosis, synapses and neurotrophic factors. we induced photothrombotic stroke (pt) in adult c57bl/6j mice and allowed them to recover for three days. starting on post-stroke day 3, mice were treated with fty720 (1 mg/kg b.i.d.) for 5 days. behavioral outcome was observed until day 31 after photothrombosis and periinfarct cortical tissue was analyzed using tandem mass-spectrometry, taqman v r analysis and immunofluorescence with especial emphasis on markers of astroglial reactivity. fty720 treatment results in a significantly better functional outcome persisting up to day 31 after pt. this is accompanied by a significant decrease in gfap-immunoreactivity (-ir) and an increase in psdsize. however no change in gfap-mrna, gs-ir or cs56-ir was observed. while fty720-treatment leads to a decrease in s1p concentration, it increases vegf-mrna in the periinfarct cortex. the initiation of fty720 treatment in the convalescence period has a positive impact on long-term functional outcome, probably mediated through reduced astrogliosis, a modulation in synaptic morphology and an increased expression of neurotrophic factors. due to the restrictions in regenerative capacity of the brain the tissue most severely damaged after traumatic brain injury (tbi) will end up as a fluid filled cavity. presently, it is an impossibility to effectively restore lost brain tissue, but ongoing research on the stimulation of endogenous neural stem cells has increased the hope to viably and functionally repopulate the injured parenchyma. however, it is crucial that possible therapies can show a long-term effect on both regeneration and functional recovery to be of clinical interest. in this study the enhanced induction of neurogenesis in rats after experimental tbi was evaluated three or six weeks after injury. severe focal tbi was performed using the controlled cortical impact model after which a combination therapy of intracerebroventricular administration of epidermal growth factor (egf) for seven days followed by deposition of extracellular matrix scaffold, containing vascular endothelial growth factor, into the cortical cavity. the treatment was devised to accomplish an optimal effect on the stem cell regeneration. the animals with six weeks survival time were functionally evaluated using the morris water maze (mwm). before sacrifice the animals were injected with bromodeoxyuridine (brdu) to identify newly generate cells. sections were made and immunohistochemically double stained for brdu and cell type markers for neurons, astrocytes and blood vessels. the sections were micrographed and analyzed for hemispheric tissue loss as well as number of new astrocytes, neurons and blood vessels. three weeks after injury there was a significant treatment effect as shown by an increase in neuronal and astrocytic regeneration. however, after six weeks there was no difference in the number of newly generated neurons and astrocytes. evaluation of tissue loss and spatial learning in the mwm corroborated that the treatment had no effect at the later time point. the results clearly show the importance of longterm studies in rodents to ensure that a promising effect on tissue regeneration and functional outcome is not merely temporary. astrocyte activation to extracellular matrix (ecm) -producing cells is inhibitory to regeneration in cns injury or disease. here we show that the cleaved neurotrophin receptor p75 ntr controls transforming growth factor (tgf)-beta-mediated astrocyte activation via regulation of nucleocytoplasmic shuttling of smad2. loss of p75 ntr completely rescues tgf-beta-induced hydrocephaly, astrocyte activation, and ecm production in gfap-tgf-beta transgenic mice. nuclear fractionation of tgfbeta-treated p75 ntr knock out astrocytes revealed reduced nuclear phosphorylated smad2 than their wt counterparts. in accordance to p75 ntr depletion, inhibition of tgf-beta-induced gamma-secretasemediated p75 ntr cleavage also reduces nuclear accumulation of smad2 and the secretion of proteoglycans that inhibit neurite outgrowth. taken together, our results identify regulated intramembrane cleavage of p75 ntr as a mechanism for astrocyte activation resulting in the regulation of tgf-beta signaling and scar formation in the diseased brain. the reaction of glial cells toward brain injury comprises a proliferative response with some reactive astrocytes even reacquiring stem cell potential as indicated by forming long-term self-renewing and mutlipotent neurospheres (for review: robel et al., 2011). here we addressed first the question to which extent the type of injury selectively affects the proliferative response of ng2 glia and astrocytes and to which extent the proliferative reaction of astrocytes may be linked to the stem cell response. interestingly, while both astrocytes and ng2 glia mounted a profound proliferative response after acute invasive injury, such as stab wound and mcao, non-invasive injury models, such as amyloid plaque deposition or widespread neuronal death could not activate the proliferation of these macroglial cells, while microglia proliferated actively in all these lesion models. intriguingly, the proliferative response of astrocytes correlated to the activation of their potential to form self-renewing, multipotent neurospheres with a steep decline with age. we then set-out to determine the signals regulation reactive astrocyte proliferation and neurosphere formation after invasive injury and demonstrate that invasive lesions result in entrance of sonic hedgehog (shh) into the brain from extraneural sources, such as the cerebro-spinal fluid, and activate astrocyte proliferation and neurosphere formation. this response can be blocked by systemic injection of the shh-signaling antagonist cyclopamine as well as inducible astrocyte-specific deletion of the receptor smoothened by glast creert2 . interestingly, shh-agonists can boost the proliferative response of astrocytes in vivo and their subsequent neurosphere-forming capacity, thereby providing a new approach how to activate this response in conditions where it is limited, as e.g. in age or noninvasive injury conditions. taken together, our work highlighted for the first time differences in the proliferative and stem cell response of reactive astrocytes in different injury paradigms and identified the shh signaling pathway as a key signal in this response. tumor necrosis factor (tnf) is a pleotrophic cytokine involved in normal brain function and inflammation, apoptosis, and other responses that occur following injury and disease. there is a considerable amount of confusion as to whether or not tnf activation is beneficial or detrimental following injury to the cns. genetic studies in mice have suggested that inflammation in disease models involves soluble tnf (soltnf) and that maintenance of innate immune function involves transmembrane tnf (tmtnf). these findings suggest that the outcome of selective pharmacologic inhibition of tnf may depend on whether the pharmacologic intervention is targeted towards soltnf or tmtnf. therefore, we took advantage of a dominant-negative inhibitor of soltnf, xpro195, that selectively inhibits soltnf and compared the effect of this inhibitor to a more widely used igg1 fc-tnf receptor (tnfr) 2 fusion protein etanercept, an inhibitor of both soltnf and tmtnf, that has been a successful treatment strategy for several autoimmune diseases. however, etanercept has been shown to display severe side effects by increasing the risk of infections. female c57bl/6 mice were subjected to a spinal cord injury at t9 level. mice were divided into three groups, receiving etanercept, xpro1595, or saline. the drug was delivered directly onto the lesion site using alzet mini osmotic pumps for 3 days starting immediately after introduction of the lesion. mice were allowed to survive 35 days. functional evaluation was performed using the basso mouse scale, rung walk, open field test and hargreaves test. in contrast to etanercept, xpro1595 significantly ameliorated recovery of hind limb function (evaluated by motor recovery score) compared to saline when administered continuously to the lesioned cord, whereas s.c. injections every 3 days for 8 weeks following sci had no effect. our study suggests that selective inhibition of soltnf is beneficial following sci when administered directly to the contused spinal cord and raises the possibility that selective inhibition of soltnf may represent a new therapeutic strategy for the treatment of sci without compromising the innate immune response. here, we show that expression of oncostatin m (osm) is upregulated after spinal cord injury (sci). to reveal the relevance of increased osm signaling in the pathophysiology of sci, osm was applied locally after hemisection. acute osm-treatment significantly improved locomotor recovery after mild and severe sci. delayed osm-treatment in the chronic phase was ineffective. improved recovery in osm-treated mice was associated with a reduced lesion size, less astrogliosis and diminished neuroinflammation. the underlying mechanism involves neuroprotective effects of osm. furthermore, osm promoted nerve fiber regeneration both in vitro and in vivo. thus, local production of osm after sci is an endogenous response that limits cns lesion propagation and promotes recovery. the university of western australia, crawley, australia following neurotrauma, cells beyond the initial trauma site undergo secondary degeneration, with excess ca 21 a likely trigger for loss of neurons, compact myelin and function. treatment of secondary degeneration by limiting excess ca 21 entry into cells using inhibitors of specific ca 21 channels showed promise in preclinical studies, but clinical trials were disappointing and combinatorial approaches are acknowledged as necessary. we assessed efficacy of every possible combination of three ca 21 channel inhibitors at reducing secondary degeneration 3 months after partial optic nerve (on) transection in rat. we used lomerizine to inhibit voltage gated ca 21 channels (for 3 months); oxidised adenosine-triphosphate (oxatp) to inhibit purinergic p2x 7 receptors and/ or 2-[7-(1h-imidazol-1-yl)26-nitro-2,3-dioxo-1,2,3,4-tetrahydro quinoxalin-1-yl]acetic acid (inq) to inhibit ca 21 permeable a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (ampa) receptors (both for the first 2 weeks after injury). only the three ca 21 channel inhibitors delivered in combination completely preserved visual function to levels not different from normal animals. in order to determine the mechanism/s by which the three inhibitors delivered in combination preserved function, we assessed neuroprotection, nerve swelling, myelin compaction and node/paranode complexes. preservation of retinal ganglion cell (rgc) somata and axons is unlikely to have accounted for the full recovery of function as only partial neuroprotection of rgcs vulnerable to secondary degeneration was achieved. a range of the ca 21 channel inhibitor combinations prevented swelling of optic nerve vulnerable to secondary degeneration, with no one agent selectively effective. each of the treatments involving lomerizine significantly increased the proportion of axons with normal compact myelin and decreased the proportion of axons with partially decompacted myelin, indicating the importance of sustained control of ca 21 flux via voltage gated ca 21 channels for prevention of chronic myelin decompaction. nevertheless, limiting decompaction of myelin or formation of atypical node/ paranode complexes was not sufficient for preservation of function in our model. it is likely that the additional prevention of the lengthening of the paranodal gap that was only achieved by treatment with the three ca 21 channel inhibitors in combination was an important effect that contributed to the associated preservation of visual function by this combinatorial treatment strategy. to generate myelinating oligodendrocytes opcs undergo a distinct series of morphological and molecular changes. these are regulated by a combination of extrinsic and intrinsic factors, which have only been partially been revealed so far. in the present study we identify a novel signaling mechanism as potent negative regulator of opc maturation. ephrin b3, a member of the b-class family of ephrins, inhibits opc process formation and blocks the formation of mature oligodendrocytes in vitro. the presence of ephrinb3 results in activation rhoa and pkca signalling and deactivation of fak kinase in opcs in vitro. moreover, epha4 rtk, a receptor responsive to ephrinb3 in opcs, is also expressed by opcs in demyelinating lesions. infusion of recombinant ephrin b3 into demyelinating lesions results in a failure of remyelination caused by an inhibition of opc maturation. in contrast, antibody-mediated masking of ephrin b3 epitopes in vitro as well as in demyelinating lesions of aged rats promotes opc differentiation and accelerates myelin regeneration. our results demonstrate an important role of ephrin b3 for the process of remyelination. after peripheral nerve injury, axons rapidly degenerate and start regrowing only after a few days. full functional recovery, however, is rare. a progressive loss of intact axons also determines the clinical phenotype of chronic demyelinating peripheral neuropathies, such as charcot marie tooth disease type 1a (cmt1a). cmt1a is the commonest inherited neuropathy, caused by a duplication of the peripheral myelin protein 22kda (pmp22) gene. we demonstrate that the transgenic neuronal overexpression of the growth factor neuregulin-1 type i enhances axonal regeneration and functional recovery after experimental acute peripheral nerve injury. similarly, neuregulin-1 type i overexpression preserves axonal loss and improves nerve function in a mouse model for cmt1a. importantly, neuregulin-1 type i induced axonal preservation in cmt1a mice was independent of myelination, as myelin sheath thickness was not altered. we conclude that axonal recovery and preservation relies on common supportive factors and that paracrine neuregulin-1 type i constitutes a beneficial signal in peripheral nerve disorders. g. szab o university of tuebingen, tuebingen, germany question: the enormous capacity for repair of the cns after lesion is one of the most amazing property of lower vertebrates. in the mammalian central nervous system (cns) the astrocytes reveal the so-called orthogonal arrays of particles (oaps); the clusterization of the aqp4 molecules at the astrocytic endfeet. in lower vertebrates these structures are lacking. in the case of the amphibia -anolis carolinensis-the caudal spinal cord has the capability for regeneration after loss of the tail, an ability which might be in relation with the presence of tight junctions (tj) between the glial cells like the olfactory ensheating cells act in mammalian olfactory system through the lifespan. the growing axons of the fish optic nerve are embraced by astrocytes which are also interconnected by tjs. tj connected glial cells could contribute to a growth-supportive microenvironment which might reinforce our hypothesis namely the mutual exclusion of aqp4 occurrence in astrocytes and regenerative growth of nerve fiber. methods: we performed immunocyto-and immunohystochemical staining, western blot, freeze-fracture electron microscopy and pcr on cultured aqp4 knockout and wildtype astrocytes and also on formalin fixed paraffin embedded tissue sections regarding junctional proteins, such as occludin, claudin 1,3,5. result: in the light of our experiments, there is no mutual exclusiveness between the presence of aqp4 and tight junctions in astrocytes, at least in mammals. further investigations need to be completed. glutamate and electrical activity influence opc differentiation and myelination in normal development. opcs receive synaptic input from unmyelinated axons, possibly to initiate myelination. both opcs and mature oligodendrocytes respond to glutamate via ampa and nmda receptors. activation of glutamate receptors in vitro increase opc migration but decreases proliferation. here we examine the role of glutamate signalling in remyelination following experimental demyelination in the adult rodent cns. we voltage-clamped opcs in brain-slices of adult rat cerebellar peduncle containing focal areas of primary demyelination and post-identified these by ng2-immunolabelling. recruited opcs mainly expressed ampa receptors at the peak of the opcs proliferation (5-10 days postlesion), as over 90% of the glutamate evoked current was blocked with nbqx (ampa/kainate receptor antagonist) and unaffected by ap5 (nmda receptor antagonist). the demyelinated axons continued to propagate action potentials with latencies similar to those in unmyelinated axons during development. critically, the demyelinated axons established synapses with opcs expressing voltage-gated sodium currents. these synaptic inputs had identical decay times to those recorded during developmental myelination. pharmacological inhibition of neuronal activity or glutamate signalling decreased remyelination efficiency by impairing differentiation. these results indicate that 1) glutamate currents are mainly mediated via ampa and kainate receptors during the opc recruitment 2) opcs establish synaptic contact with demyelinated axons and 3) the neuronal activity is essential for remyelination. together, these data suggest that demyelinated axons remain active and communicate with opcs through glutamate release during the regenerative process to guide their remyelination. there has been much recent interest in the use of intraocular stem cell transplantation to slow or reverse visual loss in glaucoma. however, migration and integration of stem cells into the retina is limited by reactive gliosis, a major barrier to retinal engraftment. our aim was to identify glial modulators of low toxicity and measure their potential to facilitate stem cell entry into the host retina. an explant organotypic culture system was used as a stem cell transplantation model and screening tool for candidate drugs. gfp1 mouse mesenchymal stem cells (mmscs) were co-cultured on the surface of the explants for 4 days.the microglial contribution to mscinduced reactive gliosis was investigated by assessing microglial activation and proliferation using immunohistochemistry for f4/80 and edu. distribution of chondroitin sulphate proteoglycans (cspgs) and their role in the barrier was also explored by immunostaining for chondroitin sulphate (cs-56) and by chondroitinase abc(chabc) treatment. stat3 inhibitor vi and jak1 inhibitor were used to suppress glial fibrillary acidic protein (gfap) expression. the efficacy of each drug to overcome the barrier was evaluated by immunohistochemistry for the glial markers gfap. mmsc integration was assessed by simultaneous immunostaining for gfp. cell viability after drug treatment was investigated by measuring the preservation of retinal thickness and by immunostaining for the neuronal marker neun and for caspase3. co-culture of retinal explants with mmscs caused a 1.5 6 0.4 fold increase in gfap expression. microglial activation and proliferation was not markedly affected by the presence of grafted mmscs on retinal surface. cspgs, highly expressed in the outer retina but only moderately in the inner retina, do not contribute substantially to the barrierto inner retinal engraftment. in contrast, interfering with the jak/stat pathway successfully reduced gfap expression by 60% 6 10% compared to control and facilitated mmscs integration into the host tissue. no toxic effect on cell viability was detected. on the contrary, over the 4 days ex-vivo culture, stat3 inhibitor vi treatment resulted in a neuroprotective effect on the outer nuclear layer, whose thickness (50.84 6 4.4 mm) is better preserved compared to control (38.4 6 9.0 mm) the results show that microglia and cspgs do not play a dominant role in the barrier to retinal engraftment, but the jak/stat pathway represents a promising pharmacological target.targeting modulators of glia reactivity to promote stem cell integration in the host tissue may facilitate stem cell therapy in glaucoma and other neurodegenerative diseases. oecs possess many characteristics favorable for the repair of sci including the secretion of growth and survival factors, the expression of cell adhesion molecules and the ability to integrate with astrocytes. oecs have also been reported to promote exceptional outgrowth of severed axons across the inhibitory environment of the lesion scar. the unique combination of regenerative properties suggests oecs would be beneficial for the repair of ventral spinal root avulsion injuries. after ventral root avulsion anterior horn neurons often fail to regenerate axons back into the surgically re-implanted nerve root resulting in poor functional recovery. to evaluate the efficacy of oec transplants for the repair of ventral root avulsions, the ability of oecs to promote neurite outgrowth from spinal cord neurons was investigated in a spinal cord organotypic co-culture. the integration of oecs with the spinal cord tissue was also examined by confocal fluorescence microscopy. oecs were harvested from adult sprauge dawley (sd) rats, cultured for 14 days and transfected with gfp. slices of cervical spinal cord were prepared from p8 sd pups and cultured on collagen coated membrane inserts. each slice was surrounded by a collagen gel with or without the addition of gfp oecs and glial derived neurotrophic factor (gdnf). our results indicate that oecs alone and in combination with gdnf have a positive effect on neurite outgrowth and morphology. the aim of this study was to obtain an hydrogel for the controlled delivery of vascular endothelial growth factor (vegf-a165). hydrogels are a class of liquid-gel biomaterials with high water content, typically composed of cross-linked, three-dimensional hydrophilic water-soluble polymer networks. the advantages of injectable hydrogel systems are: biocompatibility, biodegradability, adjustable shape, low cost and similarity to the extracellular matrix; moreover, they can be used as growth factor and cell delivery systems for tissue engineering applications. agarose/gelatin (a/gl) hydrogel was cross-linked with genepin. rheological measurements show that a/gl hydrogel can be injected through a syringe into the inner cavity of a nerve guide. in vitro assays performed to evaluate adhesion, proliferation, viability and migrationusing a fibroblast cell line (nih3t3), neonatal olfactory bulb ensheathing cells (nobec) and a schwann cell line (rt4-d6p2t)show that hydrogel allows cell adhesion, proliferation, viability and migration. vegf-a165 is a potent angiogenic factor and angiogenesis has long been recognized as an important and necessary step during tissue repair. vegf is known to have a positive effect on schwann cell proliferation and migration, neuron survival and outgrowth of regenerating nerve fibers. the hydrogel preparation protocol was optimized to be functionally efficient at physiological conditions, allowing the loading of vegf without the risk of its denaturation. different amounts of vegf (50-100-200 ng/ml) were incorporated in the a/gl hydrogel and the releasing rate in vitro up to 65 days was quantified by elisa . released vegf bioactivity was validated in human umbilical vein endothelial cells (huvec) and in nobec by evaluating phosphorylation of vegf-receptor 2 (vegfr2), akt and erk1-2. moreover, dorsal root ganglia explants were cultured on hydrogel containing different amounts of vegf, and an increased neurite outgrowth was quantitatively assessed. these results demonstrate that vegf can be successfully incorporated and bioactive released from a/gl hydrogel inducing vegfr2 activation and neurite outgrowth. in vivo test are ongoing on adult rats: one centimetre lesions on medial nerve were performed, followed by implantation of porous polye-caprolactone tubes filled with a/gl hydrogel containing vegf-a165. functional tests and histological analysis will be carried out to evaluate peripheral nerve regeneration. .spinal cord injury (sci) often leads to death of oligodendrocytes and considerable demyelination. demyelination of axons compromises signal conduction, and contributes to the functional deficits following sci. enhancing remyelination may serve to restore conduction and likely prevents axonal degeneration. here we focussed on a potential therapeutic intervention to promote remyelination, the transplantation of human oligodendrocytes precursor cells (opc)s capable of differentiating into mature oligodendrocytes and remyelinating denuded axons. human platelet derived growth factor (pdgf)-responsive neural precursor (prp) cells were isolated from the fetal forebrain and had been previously shown to differentiate into mature oligodendrocytes with a higher efficiency in vitro than traditional egf/fgf responsive neural stem cells (chojnacki and weiss 2004; deleyrolle et al. 2006 ). therefore, we tested the potential of human prps to differentiate into new oligodendrocytes and remyelinate axons in a rodent thoracic contusion spinal cord injury model. one week following injury, human prps were transplanted rostral and caudal to the lesion site. subsequent histological examination at two, seventeen and forty-two days post-transplantation demonstrated that human prps survived and integrated well into host tissue, particularly when nk cells were depleted. transplanted human prp's labelled with the mature oligodendrocyte marker apc, and 45% of the cells colabelled with the oligodendroglial marker olig2. astrocyte differentiation was also observed, suggesting human prps function as a multipotent glial progenitor following sci. human-specific immunoreactive processes were seen in close association with axons expressing the myelin protein mbp, providing evidence of remyelination by the transplanted cells. overall, these findings indicate that human prps are a source of new oligodendrocytes capable of producing myelin in response to spinal cord injury. remyelination is a regenerative process during which demyelinated axons are enwrapped by newly formed myelin sheaths. in the central nervous system (cns), this process takes place in two stages: first, during the recruitment stage, oligodendrocyte precursor cells (opcs) divide, migrate and engage the demyelinated axons. then, during the differentiation stage, opcs differentiate to myelin-forming oligodendrocytes. under pathological conditions such as multiple sclerosis (ms), remyelination often fails and as a consequence chronic demyelinated areas accumulate in ms. post mortem evidence suggests that opc differentiation is often impaired in ms patients. however, the reason why opc differentiation fails in ms is not completely understood. it has been proposed that ms lesions contain factors that act as specific inhibitors of opc differentiation. amongst them, myelin associated inhibitors play an important role. we have demonstrated that the inhibitory effect of myelin protein extracts (mpe) is mediated by activation of signalling cascades including pkca-marcks in vitro. furthermore, modulation of pkca has been shown to rescue opc differentiation in the presence of inhibitory mpe. here we demonstrate that tamoxifen, a drug that is used in various clinical settings because of its pkc-inhibitory activity, is able to promote opc differentiation in vitro. in a series of in vivo experiments, we tested the hypothesis that inhibition of pkca by tamoxifen promotes cns remyelination. demyelination was induced in the caudal cerebellar peduncle (ccp) of young-adult sprague-dawley rats by the administration of ethidium bromide. animals received daily doses of tamoxifen. although tamoxifen treatment did not induce changes in the number of opcs expressing immature markers (ng2 and pdgfr-a), a significant increase in the number of cells expressing mature marker of oligodendrocytes (cc1/olig2 and plp) was detected. furthermore, analysis of resin-embedded tissue demonstrated that tamoxifen significantly promotes remyelination in the cns when compared to controls (mann-whitney's test, p < 0.05). although further investigations are required to elucidate the mechanisms by which tamoxifen exerts cns remyelination, our results demonstrate that administration of tamoxifen represents a potent and clinically accessible strategy to promote endogenous myelin regeneration. induced pluripotent stem (ips) cell technology now allows development of autologous oligodendrocyte precursor cells for putative remyelination cell therapy for multiple sclerosis (ms). we have successfully developed mouse and human ips cells and converted these into oligodendrocyte precursor cells (opcs) that can differentiate into fully mature oligodendrocytes. furthermore, we have shown that (intracerebral) implantation of ips-derived opcs in mouse ms models enhances remyelination.before implantation-induced remyelination therapy with autologous ips-derived opc can proceed to a clinical trial for remyelination cell therapy in ms patients, we have to provide solid evidence for long term efficacy and to establish the safety thus ultimately excluding contamination of teratogenic (pluripotent) stem cells. these issues are best examined in a nonhuman primate model. the biomedical primate research center (bprc), rijswijk has developed a welldocumented experimental autoimmune encephalomyelitis (eae) model in marmoset monkeys that most closely approximates the pathology of ms in humans. in this animal model we will verify that intrathecally administered ips-derived opcs migrate to and remyelinate eae lesions and determine their long-term fate.we have developed an efficient protocol to induce marmoset ips cells from skin fibroblasts. for that we have used a lentiviral polycistronic construct, containing the four (human) reprogramming genes oct4, klf4, sox2 and cmyc, that is excisable using cre-recombinase. the pluripotent nature of the marmoset ips cells was established based on the endogenous expression of pluripotency transcription factors and the ability to differentiate in-vitro (via embryoid bodies) and in-vivo (subcutaneous injection for teratoma formation) into all the different tissues of the three germ layers. presently we are developing protocols for the differentiation of these marmoset ips cells into an oligodendrocyte cell lineage. published protocols have been modified for production of opcs for research purposes, and produced cells are used for characterization of opcs. ultimately the aim is to define an opc population with optimal myelination capacity. results and discussion: currently, human oligodendrocytes and opcs can be produced from pluripotent stem cells. these cells are routinely characterized in protein expression level, and methods for more comprehensive characterization are under development. produced opcs can be purified with fluorescence-activated cell sorting based on ng2 protein expression in order to study if this cell population is heterogeneous in terms of myelination capacity. conclusions: oligodendrocytes and opcs can be produced from human pluripotent stem cells. studies are ongoing to determine the molecular characteristics and myelination capacity of the differentiated and purified opc-population. remyelination, the regenerative process in which myelin is restored to demyelinated axons, is carried by oligodendrocyte precursor cells (opcs). in a previous study we have shown that oligodendrocyte precursor cells are multipotent and they can differentiate into oligodendrocytes as well as to schwann cells and occasionally astrocytes in the remyelinating lesions. we observed that schwann cells derived from opcs in the central nervous system occupied almost exclusively tissue around blood vessels in astrocyte deficient areas. therefore, we postulated the occurrence of specific niches or microenvironments that modulate opc fate. the aim of present study was to identify the factors and their downstream effectors that significantly discriminate vascular and nonvascular niche and determine the fate of oligodendrocyte precursor cells. we microdissected tissue from vascular and non-vascular regions of lesion areas at 6, 10 and 14 days after bilateral stereotaxic injection of ethidium bromide into the brain white matter of adult rats and performed microarrays to profile the whole transcriptome of both niches separately. this approach allowed us to distinguish between mrnas that are enriched within two separate environmental niches within the same lesion area. the study identified 138 differentially expressed transcripts. comparative bioinformatic analysis of global gene expression combined with signalling transduction pathway structure and genes function analysis revealed specific candidate signaling pathways differentially expressed in local peri-vascular niche compared to nonvascular one. results of rt-pcr-based validation of microarray data have proven that expression gradient of bmp4, bmp7, their antagonist sostdc1, as well as wnt2b, wnt6, dhh and scube1 act as the major regulators of opcs fate in the vascular niche. moreover, we observed that blood vessels undergo substantial regeneration in the course of remyelination and postulated the important role of blood vessels reconstruction in creating the specific microenvironment of neurovascular niche during tissue regeneration. our analysis shown that unique properties of tissue around blood vessels differ from the rest of the lesion which can be one of the factors favoring opcs alternative differentiation in this area of lesion. . however, key aspects of dynamic behavior, such as cell migration or process orientation and motility, can only be monitored by live imaging. to elucidate these key aspects of opc behavior after injury, we used repetitive in vivo two-photon laser scanning microscopy (2plsm) to follow ng2 1 -cells after smaller and larger stab wound injuries in the mouse somatosensory cortex. we therefore used the bac transgenic mouse line sox10 icreert2 (simon et al., 2012) crossed to the inducible reporter line cag-gfp (nakamura et al., 2006) selectively labeling cells of the oligodendrocyte lineage. gfp 1 opcs were imaged through a cranial window at the day of injury and at different time points thereafter. identification of the same cells was possible based on blood vessels labeled by rhodamine dextran as stable landmarks. live imaging revealed that the majority of opcs reacts within 2 days after injury with hypertrophy (enlarged soma and processes), polarization (elongation and concentration of processes towards the injury site), directed migration towards the injury site (defined as movement of the cell body for at least 10 mm) and proliferation (defined by a single cell being replaced by 2 or even more daughter cells). only a small proportion of cells within $500 mm of the injury did not react in any detectable manner. we noted that polarization and hypertrophy occur rather fast after inflicting the injury, while proliferation peaks 4 days after injury. taken together, the live observation of opcs reacting to stab wound injury supports the concept of opc heterogeneity and reveals new insights into the functional role of these cells after injury: the fast process orientation towards the injury site implies a contribution to wound closure and their substantial proliferation sometimes for several rounds amplifies the number of ng2 1 -cells surrounding the injury site with implications for scar formation. the aim of this study was to create a non-invasive tool for cell visualization and cell population tracking that can be used in long term studies. methods: this study was performed using human pluripotent stem cell lines derived in institute of biomedical technology (former regea), university of tampere, finland. cells were differentiated to neural cells using neural differentiation protocol described earlier by lappalainen and colleagues. fluorescent probes used in this study were 1) 5chloromethylfluorescein diacetate (celltracker green cmfda, ct) and 2) 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindodicarbocyanine perchlorate (did). cell viability and proliferation of the labeled populations was briefly studied. also, the labeled cells were characterized using immunocytochemistry and neuronal functionality was studied using micro electrode array (mea). results: during labeling optimization a wide range of different labeling parameters was tested for both probes to obtain long term labeling. most suitable parameters for human neural cells were 10 mm ct with 72 hours incubation time and 10 mm did with 2 hours incubation time. with these concentrations the labeling was visible up to 4 weeks and had no statistical effect on cell viability. ct labeling was not affecting to cell proliferation but with did slight decrease in proliferation was seen in long term culture. labeled populations contained both map-2 positive neurons and gfap-positive astrocytes. also, the ct or did labeled population and their mixed-cultures expressed normal spontaneous network activity when cultured on mea-platform. conclusions: this study indicated that fluorescent probes ct and did are suitable for long-term labeling of human derived neural cultures in vitro. the labeling had no negative effects on cell behavior with the exception of did labeled cells having slight decrease on cell proliferation. importantly, the labeled neural networks were electrically functional. axonal regeneration after spinal cord injury (sci) is limited mainly due to the presence of an inhibitory microenvironment, especially reactive astrogliosis and glial scar formation. overcoming this inhibitory barrier of reactive astrocytes and inhibitory substances making the microenvironment of the damaged neurons permissive for axonal regrowth might be crucial for cns repair. single-dose irradiation has been proved to be neuroprotective in cns trauma through elimination of reactive astrocytes, however, the irradiation conditions with singledose protocols are not in clinical use. in the present study, we aimed to investigate whether low-dose-fractionated irradiation, which is currently being used widely for clinical treatment of several tumor types, could regulate cell cycle elements of reactive astrocytes just like its use in cancers, inhibit glial scar formation, attenuate astrogliosis-mediated inhibition of axonal regeneration and facilitate recovery of motor function after spinal cord hemi-section in beagle dogs. as expected, our results demonstrated that low-dose-fractionated irradiation reduced reactive astrogliosis, attenuated astrogliosis-associated neural injuries, ameliorated microenvironment of axonal regeneration, and benefited recovery of the animals subjected to spinal cord trauma. accordingly, irradiation might be a promising therapeutic strategy targeting at excessive astrogliosis for sci in the near future. mesenchymal stem cell transplantation has been proven to have beneficial effects in various degenerative diseases, including demyelinating models. both in our lab as well as others have shown that they express a number of trophic factors that are capable of inducing remyelination, mainly by activating nearby oligodendrocyte progenitors towards mature oligodendrocytes that in turn remyelinate the damaged area. however, this effect was only observed locally, in the area surrounding the graft, thus in order to achieve general remyelination in various brain structures simultaneously, we decided to perform bone marrow-derived mesenchymal stem cell injections into the lateral ventricles. in this manner, the cells may attach to various areas such as the hippocampus, corpus callosum and fornix, among others, all of which are demyelinated. previous to the graft, the cells were incubated with iron nanoparticles. this way, it is possible to track in vivo the grafted cells by magnetic resonance imaging (mri). as a result, the cells were observed at different time points (0-15-30-60-90 days) by mri. also, the demyelinated areas can also be visualized and myelin quantified using image analysis software. this allows the quantification myelin density in the same individual mice at different moments before and after transplantation. j. muenzel 1,2 , c. becker 2 , t. becker 2 , a. williams 1 1 university of edinburgh, centre for regenerative medicine, edinburgh, united kingdom 2 university of edinburgh, centre for neuroregeneration, edinburgh, united kingdom central nervous system (cns) remyelination is important for the restoration and protection of proper nervous system function in demyelinating diseases such as multiple sclerosis but is poorly understood, and inefficient in humans. in contrast, zebrafish are able to regenerate many tissues very efficiently. as zebrafish are being increasingly used to study myelination, we aimed to develop and characterise an adult zebrafish model of cns de-and remyelination, to answer the question if remyelination is also very efficient, to describe the biology and to identify similarities and differences with mammalian remyelination. this then may allow us to better understand why zebrafish have a high regenerative capacity compared to rodents and humans. previous studies in teleost fishes have used optic nerve crush as a paradigm of myelin injury to investigate myelin repair. this, however, involves the de novo myelination of regenerated axons, rather than deand remyelination of the same axons. lysophosphatidylcholine (lpc) is a detergent-like toxin, which is widely used in rodent models to demyelinate axons. we administer lpc to the optic nerve of adult zebrafish and observe less myelin by both immunoreactivity and electron microscopy at the lesion site at 8 days post lesion (dpl) and microglia activation along the optic pathway. immunoreactivity and electron microscopy suggest complete regeneration of myelin at 28 dpl. we cannot identify any axonal injury in this model. in young zebrafish (aged 4-6 months), the myelin thickness of remyelinated fibres shows no difference to the pre-lesion state, which is different to mammals, where the myelin thickness is reduced. however, in old fish (aged 18 1 months), although the axon diameter is the same as pre-lesion and in young animals, , remyelinated fibres have thinner myelin, suggesting that the regenerative capacity of zebrafish declines with age. we believe that this new zebrafish model of cns remyelination can be added to the suite of current models to better understand the remyelination process, why it fails, and to test for compounds to improve it, with the added benefit that zebrafish are rapid breeders, transgenesis is easy and there is a high potential for live imaging. although often described as a hard-wired component of the vertebrate body, the nervous system is a plastic and considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity of its core components. unlike the cns, the pns is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that underlie this phenomenon. peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. they are ensheathed by myelinating or non-myelintaing schwann cell glia, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. this structure forms from centrally-derived glial cells and serves as a protective barrier that is essential for nerve function. following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris, regenerate and re-innervate targets. schwann cells have been shown to play an important role in this process by trans-differentiating, proliferating, clearing debris, and guiding re-growing axons, but less is known about the potential role of perineurial glia. to investigate the role of perineurial cells in pns regeneration, we have developed an injury response assay that uses a micropoint laser to create injuries along the motor nerves in live transgenic zebrafish. time-lapse imaging of injured nerves reveals that perineurial glia rapidly respond to nerve injury and extend processes toward the injury zone. this is in contrast to schwann cells, which we observe orienting towards the distal stump where they engulf and clear axonal debris. these data demonstrate that perineurial glia respond immediately to motor nerve injuries in a manner distinct from schwann cells, and future work is aimed at defining the molecular mechanisms that mediate the cellular responses of perineurial glia and schwann cells, as well as determining if developmental paradigms are recapitulated in these glial populations during the regenerative process. we have previously shown that in schwann cells the transcription factor c-jun acts as a master regulator of wallerian degeneration and is required for successful repair following nerve injury (arthur-farraj et al., methods. we here investigate on the release of the gliotransmitter glutamate from superfused isolated purified glial processes (gliosomes) obtained from adult rat cerebellum. intracellular ca 21 levels were measured by a microfluorimetric technique. immunocytochemical confocal and western blot analysis were also carried on cerebellar gliosomes. results. confocal and western blot analysis confirmed that cerebellar gfap-positive gliosomes are a purified preparation of glia processes. more then 80% of gliosomes were positive for brain-specific lipid binding protein (blbp), indicating that they derived from bergmann cells. by measuring the release of glutamate we obtained evidence for the presence of glutamate release-facilitatory ampa receptors on the bergmann glial processes. in fact, ampa evoked [ 3 h]d-aspartate or endogenous glutamate release which was abolished in ca 21 -free medium; effectiveness of the selective inhibitor naphthylacetyl spermine indicated the involvement of ca 21 -permeable, glua2-lacking ampa receptors. ca 21 imaging confirmed the presence of ca 21 -permeable, glua2lacking ampa receptors on bergmann glia processes. activation of the glua2-lacking ampa receptors triggered vesicular exocytotic glutamate release: inhibition of vesicular loading by the vesicular glutamate transporter (vglut) inhibitors rose bengal or trypan blue, or by the h 1 -atpase inhibitor bafilomycin a1 prevented the ampa-evoked glutamate release (see figure) . confocal analysis confirmed that blbpand gfap-positive processes expressed vglut1 and vglut2. conclusions. we here report evidence that: 1. functional processes of bergmann cells are recovered in purified preparation of adult rat cerebellar gliosomes; 2. ampa receptors located on bergmann glia processes show features of ca 21 permeable, glua2-lacking ampa receptors; 3. activation of the ampa receptors evokes ca 21 entry in isolated bergmann glia processes and ca 21 -dependent vesicular glutamate release from the processes. activation of ca 21 -permeable, glua2lacking ampa receptors coupled to vesicular glutamate release might represent a mechanism by which bergmann glia processes modulate synaptic transmission at parallel fibers/purkinje cell synapses. the formation of brain edema, which accompanies ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to extracellular k 1 elevation and neurotransmitter accumulation in the extracellular space. an increased uptake of these osmotically active substances, predominantly provided by astrocytes, is accompanied by intracellular water accumulation via aquaporin-4 (aqp4). previously, it has been shown that the removal of perivascular aqp4 via the deletion of alpha-syntrophin, a protein responsible for anchoring aqp4 on the astrocytic membrane, delays edema formation and k 1 clearance (amiry-moghaddam et al.,2003, pnas 11;100(23):13615-20). therefore, we aimed to elucidate the impact of alpha-syntrophin deletion on astrocyte volume changes in the cortex during pathological states, such as hypoosmotic stress or oxygen-glucose deprivation (ogd), using three-dimensional (3d) confocal morphometry in situ. in addition, single cell rt-qpcr profiling was carried out to reveal possible differences in the expression profiles of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. in order to visualize individual astrocytes that lack alpha-syntrophin, double transgenic mice were generated by crossbreeding gfap/egfp mice ( . 3d-confocal morphometry revealed that alpha-syntrophin deletion results in significantly smaller/slower astrocyte swelling when induced by 20 min hypoosmotic stress (210 mosm), 30 min ogd or by high extracellular k 1 (50 mm), while alphasyntrophin deletion had no effect on the astrocytic shrinkage evoked by hyperosmotic stress (350 mosm). the volume recovery of cortical astrocytes from gfap/egfp/alpha-syntrophin knockout mice was significantly slower following their exposure to hypo-or hyperosmotic stress, whereas no differences were found in astrocyte volume recovery following ogd or after their exposure to high extracellular k 1 . compared to the cortical astrocytes of gfap/egfp mice, single cell rt-qpcr analyses revealed that astrocytes from gfap/egfp/alpha-syntrophin knockout mice express higher mrna levels for two-pore domain k 1 channels (twik-1, task-2, task-3), inwardly or outwardly rectifying k 1 channels (kir3.1, kir5.1, kv1.3, kv1.6) and chloride channels (clc1,clc4), while mrna expression for the glutamate transporter glt-1 is lower. in summary, the deletion of alpha-syntrophin slowed down astrocyte swelling during hypoosmotic stress, ogd or high k 1 ; however, it also resulted in alterations in astrocytic gene expression profiles. supported by grants ga cr 13-02154s and p304/12/g069 from the gacr. microglia undergo a process of activation in any type of pathology which is controlled by many factors such as cytokines, chemokines or growth factors, but also by neurotransmitters. we found that a subpopulation (16%) of freshly isolated microglial cells from the adult brain respond to the muscarinic acetylcholine receptor agonist carbachol with a ca 21 response, indicating the expression of functional receptors. the carbachol-sensitive population increased in microglia/ brain macrophages isolated from the tissue of mouse models for stroke (57%) and alzheimer's disease (26%), but not for glioma and multiple sclerosis. microglia cultured from adult and neonatal brain contained a similar carbachol-sensitive sub-population (17% and 10%) which was increased by treatment with interferon-c to 60% within 12 hours, and was sensitive to blockers of protein synthesis. carbachol was a chemoattractant for microglia and decreased phagocytosis activity, indicating that microglia are a functionally heterogeneous population. t13-05a astrocytes and s1p receptors the family of sphingosine 1-phosphate receptors (s1prs) are g protein-coupled comprising five subtypes (s1p1r-s1p5r). these receptors are expressed in cells of the immune, cardiovascular, and central nervous systems (cns), in addition to others. s1prs play important roles in celular proliferation, differentiation, survival and migration. recently, the immunomodulatory drug, gilenya v r has been approved as the first oral therapy for multiple sclerosis (ms), after proving efficacious in clinical trials. the active ingredient of gilenya v r is the phosphorylated compound fty720 (pfty720), which is a potent agonist on all s1prs, except s1p2rs. pfty720 has been suggested to work as a 'functional antagonist' causing s1p1r internalisation in lymphocytes, thus limiting t cell auto-immunity. in addition to regulating the immune system, the lipophilic nature of the pro-drug fty720 allows it to readily cross the blood-brain-barrier (bbb) where it may also activate s1prs expressed on both neurons and glia. here, the role of s1prs in the cns was investigated, by specifically investigating their role in astrocyte function. a range of methods were used to examine the effects of s1pr activation and their roles in astrocytes, including: (i) s1p1r subtype trafficking, (ii) transient and continued intracelular signalling, (iii) astrocyte cell migration, (iv) release of pro-inflammatory cytokines, (v) oligodendrocyte cell differentiation and survival and (vi) myelination state in brain slice cultures. the data showed that activation of the s1p1r subtype leads to (i) its internalisation and (ii) continued signalling in astrocytes, and that s1p1rs were found to (iii) promote astrocyte migration, (iv) limit release of pro-inflammatory cytokines, (v) promote oligodendrocyte survival and (vi) limit demyelination. these studies demonstrate a role for s1p1rs in regulating astrocyte function and suggest their use a drug targets for neuroinflammtory and neurodegenerative disorders. in particular, both astrocytes and oligodendrocytes express kir4.1, which are essential for setting their strongly negative rmp, as well as the maintenance of [k 1 ] o following neuronal activity. notably, strong expression of the kir7.1 subtype was determined during a whole genome microarray analysis of the mouse optic nerve, a typical cns white matter that contains mainly astrocytes and oligodendrocytes. the kir7.1 subunit is known to be involved in potassium transport in epithelial cells and has been identified in cerebellar purkinje neurons, but has not been reported in glia. here, we examined functional expression of kir7.1 in mouse brain and optic nerve. rt-pcr and western blot confirmed expression of kir7.1 mrna and protein expression in the brain and optic nerve. positive kir7.1 immunostaining was observed in astrocytes and oligodendrocytes in brain sections and in optic nerve explant cultures and the results indicated a developmental increase in kir7.1 expression. in astrocytes, kir7.1 was localised to perivascular end-feet, supporting a potential role in k 1 regulation. in oligodendrocytes, kir7.1 were localised to cell somata, suggesting a developmental role in setting their rmp. in addition, oxygen and glucose deprivation (ogd) experiments were performed on isolated intact optic nerves from mice aged p10 and examined for the effects of the kir7.1 channel blocker vu590. after 60 min ogd, blockade of kir7.1 resulted in increased cell death of optic nerve glia, measured using propidium iodide (pi) labelling, from 80 6 30 in controls to 189 6 49 in vu590 (n 5 4 nerves per group, 6 fov per nerve; p < 0.001, anova). our results demonstrate expression of kir7.1 in glial cells, and indicate they are important in protecting glial cells from ischemic damage. is an eight transmembrane domain protein highly expressed in brain. human mutations in mlc1 lead to the rare genetic disorder "megalencephalic leukoencephalopathy with subcortical cyst" (mlc). characteristic for the disease are the onset of macrocephaly within the first year of life, and a slowly progressive loss of motor skills with epilepsy and cognitive decline. at the cellular level, countless fluid-filled vacuoles occur within myelin sheaths surrounding axons and in astrocytic endfeet. in cell culture models, mlc1 mutations are associated with defects in chloride currents and cell volume regulation. thus far, the expression and function of mlc1 in intact murine and human brain are still controversial. to understand the role and developmental expression of mcl1 in the brain, we have developed a mlc1 mutant null mouse model carrying an egfp reporter gene under the mlc1 promotor. we now show the exclusive expression of mlc1 in astrocytes throughout the brain with specifically higher expression around blood vessels, at the sub-ventricular zone and at the glia limitans. the functional loss of mlc1 in mutant mice recapitulates in part the human mlc disease. ko mice show macrocephaly with high brain wet weight. water filled vacuoles develop in the white matter of the cerebrum and in large fiber tracts of the brainstem. by contrast, the heterozygous loss of mlc1 has no consequences on myelin structural integrity. both heterozygous and homozygous mlc1 ko mice have dysmorphic peri-vascular and peri-ventricular astrocytes. in contrast to humans, ko mice have no motor deficits, but are hyperactive and show an anxiety-like behavior. in the mlc1 ko mouse, a dysfunction of an astrocytic protein causes loss of myelin structural integrity leading to vacuolating myelinopathy. therefore, the mlc mutant mouse could be a key model to study the astrocytic involvement in brain water and ion homeostasis. gaba activated slowly desensitizing responses in ng2 cells which were mimicked by muscimol and inhibited by bicuculline. to elucidate the subunit composition of the receptors we tested its pharmacological properties. co-application of pentobarbital, benzodiazipines and zolpidem all significantly increased the gaba responses. the presence of small tonic currents indicated the presence of extrasynaptic gaba a receptors. to further analyze the subunit expression, single cell transcript analysis was performed subsequent to functional characterization of ng2 cells. the subunits a1, a2, b3, c1 and c2 were most abundantly expressed, matching properties resulting from pharmacological characterization. importantly, lack of the c2 subunit conferred a high zn 21 sensitivity to the gaba a receptors of ng2 cells. to determine the effect of gaba a receptor activation on membrane potential, perforated patch recordings were performed. in the current-clamp mode, gaba depolarized the cells to about 230 mv, indicating a higher intracellular clconcentration (about 50 mm) than previously reported. gaba-induced depolarization in ng2 cells might trigger ca 21 influx through voltage-activated ca 21 channels. schwann cells (sc) play important roles in the development and regeneration of the peripheral nervous system (pns) following injury. several molecules such as neurosteroids and neurotransmitters have been suggested as potential pharmacological targets in regulating sc physiology and regenerative potential. nevertheless, the slow growth rate and difficulties in harvesting limit sc applications in regenerative medicine. adipose-derived stem cells (asc) can be differentiated into a sc-like phenotype (dasc) sharing morphological and functional properties in the present study, we analysed changes in glutamate transporter expression and function following a mechanical lesion in organotypic slice cultures of the mouse hippocampus using immunohistochemistry, western blots and dynamic imaging. the lesion was positioned perpendicular to the stratum pyramidale in the ca1 area and comprised the entire hippocampus proper. after three-six days, a glial scar had formed along the lesion site. activated astrocytes in close proximity (100-150 mm) to the lesion ("scar cells") directed long, palisading gfap-positive processes towards the lesion, had significantly swollen somata and lost their ability to take up sr101. furthermore, some exhibited distinct clustering of glast and glt-1 immunoreactivity. scar cells showed greatly diminished increases in intracellular sodium in response to application of d-aspartate, an agonist for glutamate transporters. astrocytes in the periphery to the lesion, in contrast, maintained their ability to take up sr101 and showed only slight upregulation of gfap, as well as less swollen cell bodies. cells in the periphery displayed only marginal changes in glutamate transporter immunoreactivity and unaltered amplitudes of sodium changes in response to d-aspartate. taken together, our data show that mild astrogliosis in the periphery of a mechanical lesion is not accompanied by a significant change in glial glutamate uptake capacity. at the scar itself, a strong clustering of glutamate transporters is observed that apparently goes along with a severe functional reduction in glutamate uptake. transport of base equivalent (hco 3 -) across the membrane is a crucial process to maintain the intracellular and extracellular proton concentrations within a narrow physiological range. the electrogenic sodium-bicarbonate cotransporter (nbce1, slc4 a4) is a major base transporter in eukaryotes and expressed in many tissues, especially in epithelial cells. nbce1 may transport significant amounts of bicarbonate into and out of the cell. in the brain, nbce1 is predominantly expressed in glial cells and operates with a transport stoichiometry of 1na:2hco 3 -. we have studied the bicarbonate sensitivity of nbce1, in primary cultured mouse cortical astrocytes (c57bl6/n) using live cell fluorescence imaging with confocal microscope and bcecf-am as a proton sensitive fluorescence probe. we have also used the primary cortical astrocyte culture from nbce1-ko mouse to compare the nbce1-mediated processes in wt astrocyte culture. bicarbonate dose response protocols suggest that nbce1 has a very high affinity for bicarbonate (k m <1 mm). due to this high affinity for bicarbonate, nbce1 can sensitize even residual bicarbonate concentrations of 150-300 lm present in nominally bicarbonate-free extracellular solutions (buffered with hepes). the removal of residual bicarbonate by saturating extracellular buffer (hepes) with 100% o 2 reduced these nbce1-mediated intracellular proton changes significantly. in astrocytes of nbce1-ko mice, cytosolic h 1 changes could not be detected at hco 3 concentrations lower than 3 mm. it has been reported that astrocytic nbce1 activity mediates the activation of astrocytic glycolysis by a mechanism dependent of intracellular bicarbonate and ph (ruminot et al 2011 j neurosci 40: 64-71). using a genetically encoded fret-based glucose nanosensor, we show that glucose metabolism can be activated at low millimolar bicarbonate concentration, which was largely absent in astrocytes from nbce1-ko. our results demonstrate the highest bicarbonate sensitivity yet described in animal cells, mediated by nbce1 in cortical astrocytes. nbce1 may function as a bicarbonate sensor in these cells, e.g. for modulating energy metabolism. supported by the deutsche forschungsgemeinschaft de 231/24-1. inward currents which were significantly blocked by the l-type calcium channel antagonist nimodipine. to better investigate the role of l-type calcium channels in ng2 glia at different developmental stages of the cns, we take advantage of the inducible cre-lox system by breeding homozygous cav1.2 floxed mice and cav1.3 flexed mice with ng2-creert2 knock-in mice. we are currently investigating how the removal of cav1.2/1.3 alters proliferation, migration and survival of ng2 glia. a particular focus lies on the analysis of behavioral abnormalities generated by cav1.2/1.3-deficient ng2 glia. chronic neuropathic pain is a frequent consequence of spinal cord injury (sci). yet despite recent advances, upstream releasing mechanisms and effective therapeutic options remain elusive. previous studies have demonstrated that sci results in excessive atp release to the peritraumatic regions and that purinergic signaling, among glial cells, likely plays an essential role in facilitating inflammatory responses and nociceptive sensitization. we sought to assess the role of connexin 43 (cx43) as a mediator of cns inflammation and chronic pain. to determine the extent of cx43 involvement in chronic pain, a weight-drop sci was performed on transgenic mice with cx43/cx30 deletions. sci induced robust and persistent neuropathic pain including heat hyperalgesia and mechanical allodynia in wild-type control mice, which developed after 4 weeks and was maintained after 8 weeks. notably, sciinduced heat hyperalgesia and mechanical allodynia were prevented in transgenic mice with cx43/cx30 deletions, but fully developed in transgenic mice with only cx30 deletion. sci-induced gliosis, detected as upregulation of glial fibrillary acidic protein in the spinal cord astrocytes at different stages of the injury, was also reduced in the knockout mice with cx43/cx30 deletions, when compared with littermate controls. in comparison, a standard regimen of post-sci treatment of minocycline attenuated neuropathic pain to a significantly lesser degree than cx43 deletion. these findings suggest cx43 is critically linked to the development of central neuropathic pain following acute sci. since cx43/cx30 is expressed by astrocytes, these findings also support an important role of astrocytes in the development of chronic pain. mast cells (mcs) are immune cells that reside in normal brain tissue. they store in granules an array of pro-inflammatory mediators, which are rapidly released to the extracellular milieu upon activation through an extracellular ca 21 -dependent mechanism. neurotoxic amyloid peptides are known to induce mcs degranulation but the membrane transduction mechanism remains unknown. here, the possible role of pannexin 1 hemichannel (panx1 hc) known to be permeable to ca 21 was studied. objective: since ca 21 influx is essential for mcs degranulation, we evaluated if the neurotoxic amyloid fragment ab 25-35 peptide promotes mcs degranulation via activation of panx1 hcs. methods: primary na€ ıve mcs were differentiated from bone marrow precursors of wild type (wt) and panx1 knock out (panx1 -/-) c57bl/6 mice using il-3 present in wehi3 conditioned medium. the presence of panxs 1, 2 and 3 mrnas presence was evaluated by rt-pcr analyses. the hc activity was assessed using the 4',6-diamidino-2-phenylindole (dapi) uptake assays and measurements of membrane current using whole cell patch clamp while intracellular ca 21 signal was evaluated using fura-2am. degranulation was assessed by quantification of extracellular histamine as well as release of toluidine blue (tb) from tb preloaded mcs from wt and panx1 -/mice. results: only panx1 mrna was detected in wt mcs. stimulation with ab 25-35 but not ab 35-25 peptide induced histamine and tb release. it also enhanced dapi uptake and total membrane current in wt mcs. these responses were drastically reduced in wt mcs pretreated with 10 mm carbenoxolone and 200 mm 10 panx1, blockers of panx hcs and were absent in panx1 -/-mcs. in wt mcs treated with ab 25-35 increase the intracellular ca 21 signal and was drastically prevented by 10 panx1, absence of extracellular divalent cations and in panx1 -/-mcs. conclusion: these findings indicate that mcs express functional panx1 hcs, which are essential for ca 21 influx required for the degranulation response induced by ab 25-35 . thus, inhibition of panx1 hcs might avoid spreading of mc-dependent inflammatory mediators released during alzheimer's disease progression. gliomas are the most frequent primitive cns tumors and are thought to derive from astrocytes or from neural progenitors/stem cells. however, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. tgf alpha is frequently over-expressed in the early stages of glioma progression. sharif & al (oncogene. 2007 (19):2695-706) previously demonstrated that prolonged exposure of normal astrocytes to tgf alpha is sufficient to trigger their reversion to a neural progenitor-like state. when astrocytes de-differentiated with tgf alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. our study aims to identify and characterize the protein signature of those in vitro transformed cells in an attempt to understand their neoplastic behavior and the effect of transformation on metabolic processes. this involves a global proteomic analysis using the 2d-dige methodology and a study of the metabolism of these cells (carbon source, lactate, glucose and glutamine use, ros metabolism…) by 1hand 13c-nmr nmr quantification of metabolites. such approaches are expected to provide information allowing understanding the metabolic reprogrammation that occurred during transformation. the comparative 2d-dige proteomic analysis of normal and transformed astrocytes shows that during transformation, the cells increase their expression of glycolytic enzymes, thus acquiring the ability to use aerobic glycolysis (warburg effect). moreover, the transformed cells reduce their capacity for tricarboxylic acid oxidation and for neurotransmitters (glutamate and gaba) metabolism. ingenuity pathway analysis indicates major effects on carbohydrates, amino acids and nucleotides metabolic components. using enzymatic activity measurements and the detection of protein isoforms by 2d-western blot and zymography, we document a change in expression and activity of various isoenzymes that may be responsible for those metabolic reprogrammations. r.e. k€ alin, a. jarczewski, f. apel, r. monk, s. kraft, j. radke, f.l. heppner charit e -universit€ atsmedizin berlin, neuropathology, berlin, germany question: glioma are the most frequent malignant primary tumors of the brain. glioma survival and growth is determined by the interaction with brain parenchyma, which includes intense tumor angiogenesis. this process is supported by glioma-invading myeloid (gim) cells, namely brain resident microglia or brain-invading macrophages. depletion of gim cells leads to a significant decrease in glioma volume. in a previous study, we established the novel neuroendocrine hormone apelin as an angiogenic factor and described its upregulation in human glioblastoma multiforme. our aim is thus to study the role of apelin signaling in tumor angiogenesis but also its possible effect on brain monocytes for the regulation of glioma growth. methods: to investigate apelin signaling during gliomagenesis we took advantage of an orthotopic mouse model for tumor formation. intracerebral xenotransplantation of the human glioma cell line u87mg, expressing lentiviral control or apelin shrna, allows us to study the specific contribution of glioma-derived apelin to gliomagenesis. to further dissect a putative immune function of apelin, we used the isogenic mouse glioma cell line gl261 for intracerebral implantation into immunocompetent mice lacking (apelin-ko) or overexpressing apelin (apelin-tg). results: loss-of-apelin expression in u87mg cells resulted in a reduced glioma volume and attenuated the formation of the xenograft vasculature. gl261 implants in apelin-ko mice produced a similar phenotype. in addition, invasion of glm cells was significantly reduced. interestingly, expression analysis showed an upregulation of the apelin receptor apj in brain myeloid cells making them competent to respond to glioma-derived apelin. conclusions: our findings demonstrate that apelin plays an important role in tumor angiogenesis and glioma growth. we show here that both, glioma cell-derived apelin and apelin expressed by the tumor neovasculature are contributing to tumor angiogenesis, gim invasion and glioma growth. we anticipate that our study will provide insights whether apelin signaling may serve as a future novel target for antiangiogenic tumor therapy. tumor infiltrating microglia/macrophages (tims) constitute the largest population of infiltrating cells in glioblastoma (gbm), the most aggressive brain tumor. data from the clinic and from experimental work performed in murine and human models indicate that tims play a significant role in gbm biology as they support proliferation, migration and invasion of tumor cells. evidence is amounting that tumor cells actually polarize tims towards this m2-like, tumor-supportive phenotype. we showed that toll-like receptor 3 ligand reverses this m2-into a m1-like phenotype. pre-activated, m1-like polarized tims incubated with spheroids of gbm cells reduced migration, killed and phagocytosed tumor cells over a 15 day-period, indicating a sustained m1 activation of tims in absence of exogenously added stimuli. in order to analyse in more detail tims-gbm cells interactions, we have undertaken two approaches. we use spheroids of cells (tumor with/without tims) embedded in collagen matrix, in which tims can be implanted, as an experimental model. this three-dimensional in-vitro system is well suited for a qualitative and quantitative monitoring of cell proliferation, death, migration and invasion. data experimentally generated are then implemented in a mathematical model that is, to our knowledge, the first one proposed to take into account tumor cells and tims in order to simulate gbm progressive behaviour. in a first step, the capacity of spheroids made of murine glioma cells to invade collagen matrices was evaluated in absence and presence of microglia. invasion was monitored by photography of the spheroids and images were processed with photoshop cs5 software. in order to achieve a precise determination of invasion, we chose to measure the diameter of the core plus the invasive rim as a read out of expansion rate. untreated microglia promoted growth and invasion of tumor cells. data generated through the proposed in-vitro system were comparable to and reproduced the insilico simulations obtained with the mathematical model, hence validating our mathematical and experimental approaches. we currently evaluate the rate of proliferation and death of tumor cells in various settings that modulate tims polarization, using flow cytometry and confocal (live) imaging. data contributed by these two approaches should facilitate the delineation of a predictive model for tumor progression in a tims-enriched microenvironment with possible therapeutic fallouts. sialic-acid-binding immunoglobulin-like lectins (siglecs) are type 1 membrane proteins displaying an amino-terminal immunoglobulin-like variable (v-set ig-like) domain that binds sialic acid and variable numbers of immunoglobulin-like constant region type 2 (c2-set ig-like) domains. siglec-h is a recently identified mouse-specific cd33-related siglec that signals via the itam linked adaptor protein dap12. so far the function of siglec-h and its ligand are unknown. in this project, we demonstrate gene transcription and protein expression of siglec-h by mouse microglia after interferon-c (ifn-c) treatment or polarization into a m1-subtype. targeting of beads to siglec-h by specific antibodies triggered the phagocytic uptake of the beads by the microglia. a siglec-h fc fusion protein generated by our laboratory selectively bound to cells with an altered glycocalyx, particularly glioma cells, but not to normal control cells. m1-polarized microglia phagocytosed glioma cells and also limited their proliferation in a co-culture system. interestingly, no signs of apoptosis were observed before phagocytosis of the glioma cells by siglec-h expressing microglia. phagocytosis of glioma cells was reduced after shrna mediated siglec-h knock down in microglia. furthermore, microglial clearance of glioma in vitro was dependent on the interaction of siglec-h with its corresponding adaptor protein dap12. our data show that m1-polarized microglial cells can engulf glioma cells via a dap12-mediated and siglec-h dependent uptake. glioblastomas (gbm) are the most common brain tumors in humans. although advances in the chemotherapeutic management of glioblastoma have been made, almost 80% of the patients die within the first 2 years after diagnosis. on the basis of these observations, the identification of new molecules that can counteract the gbm growth and invasiveness appears relevant. previously, we have demostrated that m2 muscarinic receptor agonist arecaidine inhibited cell proliferation in a time and dose-dependent manner and induced a severe apoptosis both in two glioma stable cell lines (u251 and u87) and in primary cultures derived from human biopsies. in order to clarify the mechanisms causing the decreased cell proliferation, we have evaluated the ability of m2 receptor activation to counteract the notch-1 and egfr pathways. the analysis by real time pcr and western blot have demonstrated that, in both cell lines, m2 receptor caused a decreased expression of egfr and notch-1 and its ligands (e.g. delta-1, jagged-1 and 22), suggesting that the decreased cell proliferation may dependent on altered expression and activity of these pathways. although facs analysis have showed that arecaidine induced an arrest of cell cycle progression, we observed, in both cell lines, a significant increase of dividing cells already after 24 hours of treatment. in particular, the number of metaphases increased significantly, while the percentage of anaphases diminished. furthermore we observed in both cell lines, a dis-regulation of mitotic spindle assembly, misalignment of chromosomes and the presence of multipolar spindles. moreover, due to prolonged activation of the promethaphase/methaphase mitotic checkpoint, chromosomes appeared more condensed in cells treated with arecaidine. facs analysis and immunocitochemistry using anti-histone c-h2ax, a marker of double strand breaks, have also demonstrated that arecaidine treatment induced dna damage. on the other hand, m2 agonist induced also oxidative stress, followed by an increased expression of the enzyme superoxide dismutase and sirtuins (sirt1 and sirt2). in conclusion, the data obtained suggest that the activation of m2 receptor induces cytostatic and cytotoxic effects in glioblastoma cell lines, opening new therapeutic perspectives for this receptor in glioblastoma therapy. univdersidade federal do rio de janeiro, rio de janeiro, brazil glioblastoma (gbm) is one of the most aggressive human cancers. despite current advances in multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome for patients with high grade glioma remains fatal. there is now a growing awareness that the main limitations in understanding and successfully treating gbm might be bypassed by the identification of a distinct cell type that has defining properties of somatic stem cells, as well as cancer-initiating capacitybrain tumor stem cells, which could represent a therapeutic target. in addition, experimental studies have demonstrated that the combination of antiangiogenic therapy, based on the disruption of tumor blood vessels, with conventional chemotherapy generates encouraging results. emerging reports have also shown that microglial cells can be used as therapeutic vectors to transport genes and/or substances to the tumor site, which opens up new perspectives for the development of gbm therapies targeting microglial cells. finally, recent studies have shown that natural toxins can be conjugated to drugs that bind to overexpressed receptors in cancer cells, generating targeted-toxins to selectively kill cancer cells. further, extensive effort is being dedicated to characterize the molecular basis of gbm resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. we show that a non-cytotoxic concentration of equinatoxin ii (eqtx-ii), a pore-forming toxin from the sea anemone actinia equina, potentiates the cytotoxicity induced by temozolomide (tmz), a first-line gbm treatment, and to etoposide (vp-16), a second-or third-line gbm treatment. we also suggest that this effect is selective to gbm cells and occurs via pi3k/akt pathway inhibition. finally, magnetic resonance imaging (mri) revealed that a non-cytotoxic concentration of eqtx-ii potentiates the vp-16-induced inhibition of gbm growth in vivo. these combination therapies constitute a new and potentially valuable tool for gbm treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy. a growing body of evidence indicates that glioblastoma stem-like cells (gscs) play a central role in glioblastoma development and resistance to current therapies. we recently described a cluster of micro-rnas, the mir-302-367, which induces the exit of gsc from their stem state and suppresses their tumorigenic properties in an irreversible manner (fareh et al, 2012). we postulated that such a drastic change in the cell phenotype should be accompanied with metabolic alterations that could be instrumental in the loss of functional properties of gscs induced by the micro-rna cluster. metabolome profiling by mass-spectrometry of gscs and gsc-mir-302-367 pointed to changes in the gaba synthesis pathway (see abstract el-habr et al). remarkably, exposure of na€ ıve-gsc to metabolites of the gaba synthesis pathway found to be overproduced in gsc-mir-302-367 reproduced at least in part the effects of mir-302-367, including inhibition of clonality, down-regulated expression of selfrenewal markers, and loss of self-renewal properties. the metabolites studied acted by interfering with progression of the cell cycle and the nuclear localization of transcription factors crucial for maintenance of gsc stem-like properties. studies assaying the effects of the metabolites on gsc tumor-initiating properties are under way. these results demonstrate that metabolic regulations can participate in the control of gsc properties, and opens novel paths in therapeutic targeting of glioblastoma. *lgd and eae contributed equally. glioblastomas are the most frequent and aggressive form of adult primary brain tumors. glioblastoma stem cells (gscs) are thought to play key roles in the development and resistance of the tumor to existing radio-and chemotherapies. our previous results showed that the cluster of micrornas, mirna-302-367, induces loss of gsc stem-like and tumor-inducing properties (fareh et al. 2012). to further understand the molecular pathways controlling the peculiar properties of gscs, we sought for metabolic changes likely to accompany the drastic change in cell phenotype induced by mir-302-367 expression. we measured the intracellular and secreted levels of 271 metabolites by mass spectrometry. reconstitution of the metabolomes revealed significant and coordinated changes in components of the krebs cycle, and the glutamine/glutamate neuropeptide metabolic pathway that suggested altered turnover of the gaba synthesis pathway. exon array hybridization and western blot analysis, revealed changes in expression of several enzymes of the gaba synthesis pathway in mir-302-367-gscs. of note, none of the analyzed enzyme transcripts appeared as a direct target of mir-302-367. our results could be integrated in a complex multistep schema compatible with enhanced turnover of the gaba synthesis pathways, resulting in decreased cell gaba levels and increased levels of gaba by-products, as observed in mir-302-367 gscs. further studies showed that these metabolic changes participate in the induced loss of gsc properties (see abstract by dubois et al). * eae and lgd contributed equally colonization of the brain by carcinoma cells is barely understood, in particular when considering interactions with the host tissue. we recently demonstrated that microglia assist carcinoma cell invasion. current findings indicate that this is part of a danger response of the entire brain tissue. in the brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia as well as astrocytes comparable to that seen at the interface of human cerebral carcinoma metastases. this response tries to protect the brain from intrusion of benign epithelial cells by inducing apoptosis. however, this is ineffective against malignant cells which did not undergo apoptosis and actually exploited this reaction to invade instead. gene expression and functional analyses revealed that c-x-c chemokine receptor type 4 (cxcr4) and wnt signaling are involved in this process. furthermore, cxcr4 regulates the recruitment of microglia towards brain injury in a zebrafish model and was expressed in human stroke patients, suggesting a conserved role of cxcr4 in various danger reactions. we propose that glia-assisted malignant invasion takes advantage of the physiological two-stage glial defense reaction. carcinoma cells escape the second step of cell killing and misuse the damage response to colonize the brain. university of tuebingen, tuebingen, germany aquaporin-4 (aqp4), the main water channel of the brain, is highly expressed in animal glioma and human glioblastoma in situ. in contrast, most cultivated glioma cell lines do not express aqp4, and primary cell cultures of human glioblastoma lose it during the first passages. accordingly, in two glioma cell lines of the rat (c6 cells and rg2 cells) and in sma mouse glioma cell lines we found no aqp4 expression. this let us consider the possibility that aqp4 expression depends on brain microenvironment. aqp4 negative rat glioma cells were implanted into rat brain. within two weeks, a tumor developed. aqp4 staining of the tumor cells was positive. however, if the identical cells were implanted into the rat's flank, they did not express aqp4. in contrast to the normal brain, where aqp4 staining is polarized in the astrocytic endfoot membranes, the aqp4 staining in c6 and rg2 tumors was distributed over the whole glioma cell as in human glioblastoma. we conclude that the micro-environment is crucial for aqp4expression in brain and brain-tumor. glioblastomas (gbm) are infiltrative and highly vascularised brain tumors containing a subpopulation of multipotent stem-like cells. here we questioned whether notch pathway activation in these cells influenced the extent of tumoral vascularisation and dissemination. we used two cd133 1 sox2 1 gbm stem-like cultures generating highly infiltrative but poorly vascularised tumors upon intracranial grafts. the use of a hes5 promoter reporter construct indicated that the notch pathway was only active in a small fraction of cells. sustained notch activation in these cultures, using an activated form of the notch-1 receptor (nicd), induced a profound alteration of their morphology, phenotype and properties. a severe decline of their migration and proliferation rates was observed in vitro and in vivo. intracranial and subcutaneous transplantation revealed that nicd triggered an extensive vascularisation of tumoral cells indicated by the presence of wellformed vessels with large lumens. close interactions between gbm cells and host endothelial cells were readily observed but no evidence for endothelial transdifferentiation was found either in vitro or in vivo. microarray and proteomic analyses showed that nicd induced the expression of vascular adhesion proteins (icam-1, vcam-1, itga9), proangiogenic cytokines (plgf, il-8, hb-egf), metalloproteinase (mmp-9), a-smooth muscle actin and dll4, a notch ligand essential for vascularisation. this was associated with a remarkable transcription factor switch whereby cells became klf9 1 snai2 1 while ascl1, olig2, and sox2 were reduced or lost. thus in addition to its well-described role in vascular development and remodelling, the notch pathway is also central in controlling proliferation, migration and vascularisation of gbm-stem like cells. t04-04b extracellular space diffusion parameters in the mouse thalamus in bral2 knock-out mice retinal wholemounts were immunostained with antibodies against gfap, iba-1 and mhc-ii. results: in the na€ ıve retinas, weak constitutive mhc-ii expression was scarcely found in some iba-11 microglial cells and rarely in gfap1 astrocytes. only a small dendritiform subpopulation of iba-11 cells, located in the juxtapapillary area and in the marginal region of the retina, had a strong mhc-ii immunoreaction. in comparison with na€ ıve both, in contralateral and in oht-eyes: i) gfap was upregulated in m€ uller cells and microglia was activated; ii) mhc-ii was upregulated on macroglia and microglia. in microglia, it was similarly expressed in contralateral and ohteyes. by contrast, in macroglia, mhc-ii upregulation was observed mainly in astrocytes in contralateral eyes and in m€ uller cells in oht-eyes conclusions: both, contralateral and oht-eyes had macro-and microglial retinal changes in mhc-ii expression after two weeks of laser-induced oht approximately 70% of the nmo patients harbor antibodies against the water-channel aquaporin-4, aqp4-igg (seropositive nmo), expressed mainly by perivascular astrocytes. however, some patients diagnosed with nmo don't have aqp4-igg (seronegative nmo). the aim of this work is to compare in vitro the neuroinflammatory profile of igg from seropositive (igg-nmo1) and seronegative (igg-nmo-) nmo patients. pool of purified igg from igg-nmo1 and igg-nmo-patients fulfilling diagnostic criteria of 2006, and from multiple sclerosis (igg-ms) patients and healthy controls (igg-hc) were include in the study. mixed glial cell cultures of astrocytes (40%), oligodendrocytes (40%) and microglia (15%) were obtained from spinal cord of postnatal c57bl6/j mice. after 21 days in vitro, cells were treated with the igg pools for 12 hours for rt-pcr, and 24 hours for western blot and immunocytofluorescence (if) igg-nmo1 caused a stronger upregulation of c/ebpb mrna and nos2 protein than igg-ms and igg-nmo-. all these data show that igg-nmo1 and igg-nmo-induce a different proinflammatory glial activation. the proinflammatory pattern of igg-nmo-is quite similar to that induced by igg-ms patients. these findings raise the question if igg-nmo-could be a different disease. such as gdnf, artemin, bdnf and sonic hedgehog, adhesion molecules including p75ntr, n-cadherin and n-cam and the transcription factor olig1. c-jun also controls the structure of the regeneration tracks. in schwann cell c-jun null mice, the initial rate of axonal regeneration after injury is impeded and long term recovery of function measured by footprint analysis, toe pinching , von frey hairs, and the hargreaves test is not seen 10 weeks after injury. there is widespread death of both large and small drg neurons and although spinal cord motor neurons survive there is widespread failure of both sensory and motor neurons to reinnervate target muscles united states mutations in the neurofibromatosis 2 (nf2) gene cause neurofibromatosis type 2 (nf2) characterized by formation of multiple schwannomas and meningiomas. nf2 encodes a tumor suppressor protein called merlin. loss of function of merlin is associated with an increased level of active rac and p21-activated kinases (pak). the lim domain kinases (limk1 and 2) are rac-pak substrates and modulate actin dynamics and cytoskeletal organization by phosphorylating cofilin, an actin severing and depolymerizing agent acknowledgements: this work was supported by grant ncn 2011/03/b/ nz4/00042 and statutory funds. phenotypes, their functions are fundamentally different in a model of brain injury. these data provide new insights into the protective role of microglia after brain injury. . although the pathogenesis is still not clear, activation of the immune system at some point of the disease process plays a crucial role. it is known that myelin-specific autoreactive t-cells and monocytes cross the blood-brain barrier (bbb) and migrate into the cns. within the cns, these cells secrete proinflammatory cytokines which stimulate local glial cells to produce inflammatory factors, including reactive oxygen species that contribute to demyelination and ultimately axonal loss. the destruction of the bbb and the influx of monocytes and t-cells from the circulation are key events in the progression of ms pathology. tissue transglutaminase (tg2) is a multifunctional enzyme that has been shown to play a role in monocyte/macrophage adhesion and migration onto extra cellular matrix proteins in vitro. this binding occurs via interaction with bintegrins. previously, we observed the appearance of tg2 in monocytes in active human post-mortem ms lesions in the cns.in the present study we question whether tg2 expression is regulated in primary human monocytes by inflammatory mediators and whether it is modulated in ms patients. methods: to this end, tg2 in monocytes was detected by semiquantitative rt-pcr.results and conclusions: our preliminary results show that activation of primary human monocytes with lps or lps1ifnc results in a time-dependent increase in tg2 mrna whereas the expression of factor xiiia (another member of the transglutaminase family) remains stable over time. moreover, the tg2 mrna level is higher in unstimulated monocytes derived from ms patients compared to control subjects while factor xiiia mrna level remains unaffected.these initial data are promising with respect to a possible role for monocyte-derived tg2 being involved in adhesion to and migration across the bbb under inflammatory conditions (e.g. multiple sclerosis). this hypothesis has not been proven yet. in addition, by increasing the number of patients, a consistent difference in tg2 expression in monocytes between ms patients and control subjects may point to tg2 as a novel biomarker for disease. an important aspect of chronic neurodegenerative diseases, such as alzheimer's, parkinson's, huntington's and prion disease, is the generation of an innate inflammatory response within the central nervous system (cns). microglial and astroglial cells play a key role in the development and maintenance of this inflammatory response, showing enhanced proliferation and morphological activation. using a laboratory model of chronic neurodegeneration (me7 murine model of prion disease), we studied the time-course and regulation of microglial proliferation. our results show that resident microglial cells have an increased proliferation rate during the development of the disease, leading to a significant increase in the population, without a contribution from circulating cells. microglial proliferation is differentially regulated in diverse regions of the cns, pointing to a heterogeneous development of the pathology. we have identified novel molecular regulators of the proliferative response, and addressed the significance of the contribution of microglial cells to the pathological course of the disease by modifying their proliferation. we also found a correlation of our results with the scenario present in chronic human neurodegenerative conditions variant creutzfeldt-jakob disease (vcjd) and alzheimer's disease. our results demonstrate that microglial proliferation is an important feature of the evolution of chronic neurodegenerative disease, with direct implications for understanding the contribution of the cns innate immune response to disease progression. here, we examined the underlying mechanism of fibronectin aggregation and address whether inflammatory mediators, such as toll like receptor (tlr) agonists or pro-inflammatory cytokines induce fibronectin aggregation by astrocytes. our findings reveal that only tlr3 and 24 agonists, including the endogenous tlr3 agonist stathmin, increased stable fibronectin aggregation. interestingly, only white matter-derived astrocytes displayed fibronectin aggregation whereas in cortical astrocyte cultures fibronectin remained unaffected, suggesting regional differences in the functional response of astrocytes. furthermore, in rat cerebellar slice cultures, the tlr3 agonists only induce aggregation of fibronectin after lysolecitin-induced demyelination. in this manner, efficient remyelination is impeded and, consequentially, progressive neuronal decline occurs.taken together, tlr3 and 24 agonists promote fibronectin aggregation by primary rat astrocytes. since tlr3 on astrocytes and stathmin are both upregulated in ms lesions, stathmin, as an endogenous tlr3 agonist, might play a role in inducing fibronectin aggregation after c-jun reprograms schwann cells of injured nerves to generate a repair cell essential for regeneration. neuron. 75(4): 633-47). in this study we identify a novel role for c-jun in the activation of notch signalling in the denervated schwann cell. we find that c-jun is required to activate notch signalling, leading to upregulation of the bhlh protein hes1. hes1 then plays two functions in the denervated cell, promoting myelin breakdown and acting as part of a negative feedback loop to reduce c-jun levels. as a result of this, ablating notch signalling specifically in schwann cells acts to increase c-jun levels. we show that this upregulation of schwann cell c-jun accelerates axon outgrowth, target re-innervation and remyelination by generating a cell, which promotes faster than normal functional recovery. these results identify novel functional links between the c-jun and notch signalling pathways. they also show that not only is schwann cell c-jun necessary for successful nerve regeneration, but that nerve repair can be improved by enhancing normal c-jun signalling. it is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (cns). after injury of the peripheral nervous system (pns), schwann cells and macrophages phagocyte myelin debris in wallerian degeneration in order to regenerate axons to distal targets. the immunoreceptor cd300f is normally expressed in the myeloid line and in nervous system in microglia, oligodendrocytes and neurons under certain conditions. however, little is known about the cd300f ligands. by using a cd300f-fc fusion protein we have analyzed the expression of the cd300f ligands in the pns. moreover, we have also analyzed the possible role of the cd300f immunoreceptor in peripheral nerve regeneration by blocking the interaction between cd300f and its ligands with the same fusion protein. thy1-yfp-h mice sciatic nerves were injected with cd300f-fc, control migg2a or pbs immediately before a crush injury. the results show that cd300f ligand is expressed in schwann cells. moreover, after a sciatic nerve injury, animals injected with the cd300f-fc protein show a lower number of yfp-positive fibers growing into the tibial nerve after 10 days post-lesion (dpl) than control groups. moreover, the cd300f-fc group shows a higher number of macrophages and cd206-positive cells at 10 dpl when compared to control groups. we do not see these differences in axon regeneration and macrophage infiltration after 28 dpl. we have also evaluated the mrna expression of the pro-inflammatory cytokine il-1b at 24 hours after crush and injection of the fusion protein. q-pcr shows an up-regulation of the mrna in control groups and a lower mrna expression level in the cd300f-fc protein group. together these results show that the pair cd300f receptor and ligand is implicated in some aspects of wallerian degeneration and nerve regeneration such as the modulation of both the influx and phenotype of macrophages. in response to the central nervous system (cns) injury, hematopoetic cells migrate to the lesion where they can potentially contribute to tissue regeneration. following cns injury, these cells can secrete a plethora of immunomodulating cytokines and/or pro-regenerative growth factors as well as phagocyte proinflammatory tissue debris. nevertheless, the role of primitive hematopoetic stem/progenitor cells (hspc) -derived from bone marrow -to support cns regeneration has not been studied in detail. therefore, the aim of the present study was to examine characteristics of hspc in vitro as well as to test proregenerative potential of these cells to support cns repair in vivo.highly pure population of primitive hematopoetic stem/progenitor cells was isolated from the bone marrow and the cns with fluorescence activated cell sorting (facs). the number of hspc in the cns lesion was significantly increased compared to intact cns tissue. sorted-bone marrow hspc were co-cultured with primary astrocytes to examine their phenotype according to the presence of specific antigens and gene expression as well as to test their phagocytosis potential. in the presence of astrocytes, bone marrow-derived hspc differentiated in vitro into microglia-like cells expressing specific myeloid/microglia markers and showing high ability to ingest fluorescent microbeads. the in vivo potential of hpsc for supporting regeneration was examined by their transplantation into the cns lesion.results of our study demonstrated that primitive hematopoetic stem/progenitor cells are the source of microglia-like cells which can support regeneration in the central nervous system. when the brain or spinal cord is injured, glial cells in the damaged area undergo complex changes resulting in the formation of the glial scar. whether the scar is beneficial and detrimental to recovery remains controversial. the signals that initiate the formation of the glial scar are unknown. because both canonical and non-canonical wnts are increased after spinal cord injury (sci), we examined the role of canonical wnt signaling in the glial reactions to cns injury. to disrupt b-catenin-dependent wnt signaling specifically in opcs, we created transgenic mice carrying an opc-specific conditionally deleted bcatenin gene. after moderate contusion injuries to the thoracic spinal cord of control mice, opcs proliferate and accumulate in the penumbra region surrounding the injury epicenter. in the b-catenin-depleted mice, there is reduced proliferation and accumulation of opcs after sci, reduced accumulation of activated microglia/macrophages and reduced astrocyte hypertrophy. using a crushed optic nerve model, we show that these reduced glial reactions create an environment that is permissive for axonal regeneration. these results suggest that canonical wnt signaling in glia after cns injury is necessary for the formation of the glial scar and they identify wnt signaling as a new therapeutic target for promoting axon regeneration. the crucial role of central nervous system (cns) glial cells in the integrity and physiology of neuronal networks, is well known. however, little is known about glial cells in the peripheral nervous system (pns). one of the main glial cell types in the pns is the perineuronal satellite glial cells (sgcs), that surround drg neurons in an envelope-like fashion. despite their abundance in peripheral nerves, having stem cells properties and playing a role in neuropathic pain, there is limited information on their physiology. although sgcs have similarities to astrocytes in terms of purinergic-and gap junction-mediated signaling, their membrane properties and ionotrophic glutamate receptor expression are more or less unknown. our aim was therefore to characterize the membrane properties and glutamatergic ion channel expression of sgcs. we performed patchclamp experiments on sgcs wrapped around sensory neurons in the rat dorsal root ganglion (drg) in vitro. whole-cell recordings revealed that sgcs are slightly hyperpolarized in comparison to the neurons they ensheath, with a resting membrane potential of approximately $80mv and a high input resistance (&gt;1gx). moreover, upon membrane depolarization no detectable voltage-gated na 1 , ca 21 or k 1 currents were detected. extracellular application of glutamate agonist, kainic acid, n-methyl-d-aspartic acid (nmda) and 2-amino-3-(3hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (ampa) during the recording, did not evoke any response from the cells, indicating their lack of ionotropic glutamatergic receptor (iglur) expression. double immunocytochemistry on lucifer yellow (ly) filled scgs revealed that they express the scip/oct6 transcription factor, which is also expressed by promyelinating schwann cells.drg ganglion sgcs differ from cns perineuronal cells as they lack glutamatergic receptors, but are similar to astrocytes as they have no voltage-gated ion channels, are gap-junctionally coupled and express glutamine synthetase. thus, we are currently addressing if sgcs respond to neuronal activity in a similar manner to astrocytes in the cns with the use of calcium imaging. these studies will provide further insights into the physiological role of sgcs in the pns. multiple sclerosis (ms), a chronic neuroinflammatory disease with presumed autoimmune etiology, is the most common demyelinating disorder of the central nervous system among young adults. progressive neurological impairment of the patients are associated with accumulation of characteristic brain lesions characterized by inflammation, demyelination, gliosis and axonal damage. demyelination and loss of oligodendrocytes contributes to axonal loss and permanent neurological deficits. remyelination occurs but is limited; one contributing factor is the incapability of oligodendrocyte precursor cells (opc) to differentiate and form new myelin sheaths. k 1 channels are not only known to predominantly set and maintain the membrane potential but also to be important regulators of various cell functions like proliferation and maturation. specific regulation of these cellular functions is based on high channel diversity and their ability to form heteromers as well as alternate mrna splicing and posttranslational modifications. so far the expression and function of potassium channels in cells of the oligodendroglial lineage has not been clearly identified. previous studies indicate that an unspecific blockade of k 1 channels in opc suppresses maturation as well as proliferation. moreover, for one distinct channel (k ir 4.1) a functional relevance has been shown, as deficiency of kir4.1 leads to altered opc maturation and hypomyelination in mice. here, we elucidate the expression and functional relevance of k 1 channels in oligodendrocytes. our first electrophysiological recordings from primary murine opcs revealed the presence of different k 1 channels with both inward and outward rectification. in future experiments, we aim at identifying k 1 channel subtypes that specifically regulate opc cell functions and might influence cell maturation under neuroinflammatory conditions. thereby, we want to offer new insights in the functional role of k 1 channels in opcs/oligodendrocytes and contribute to novel therapeutic strategies for the treatment of ms. aqp4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. aqp4 has been described as an important entry and exit site for water during formation of brain edema and regulation of aqp4 is therefore of broad interest. phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. protein kinase (pk)-dependent phosphorylation of ser 111 has been reported to increase the water permeability of aqp4 expressed in an astrocytic cell line. this possibility was, however, questioned based on the crystal structure of the human aqp4. our study aimed to resolve if ser 111 was indeed a site involved in phosphorylation-mediated gating of aqp4. the water permeability of aqp4-expressing xenopus oocytes was not altered by a range of activators and inhibitors of pkg and pka. mutation of ser 111 to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of aqp4. pkg activation had no effect on the water permeability of aqp4 in primary cultures of rat astrocytes. molecular dynamics simulations of a phosphorylation of aqp4.ser 111 recorded no phosphorylation-induced change in water permeability. a phospho-specific antibody, exclusively recognizing aqp4 when phosphorylated on ser 111 , failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. thus, our data indicate a lack of phosphorylation of ser 111 and of phosphorylation-dependent gating of aqp4. undocked connexons may form open hemichannels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules with masses lower than 1 kda. a range of physiologically relevant molecules, smaller than the assumed molecular cut-off of 1 kda, have therefore been proposed to permeate connexin 43 (cx43) in its hemichannel configuration. however, the permeability profile of cx43 hemichannels remains unresolved as does the molecular substrate for hemichannel activity in astrocytes. exposure of cx43-expressing xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. in spite thereof, a range of smaller biological molecules, such as water, glutamate, lactate, glucose, and atp, did not gain detectable access through the pore. in contrast, permeability of glutamate, glucose and atp was observed in oocytes expressing the other major astrocytic connexin, cx30. exposure to low divalent cation solutions also induced a robust membrane conductance in cx30-expressing oocytes but none in cx43-expressing oocytes. expression of cx43 in c6 glioma cells failed to induce hemichannel activity. our results thus call for caution when assigning molecular identity to astrocytic channel activity and when interpreting hemichannel-mediated dye-uptake as equal to permeability of physiologically relevant molecules. atp-gated p2x4 receptor channels expressed in spinal microglia actively participate in central sensitization, making their functional regulation a key process in chronic pain pathologies. p2y6 metabotropic g q -coupled receptors, also expressed in microglia, are involved in the initial response to nerve injury, triggering phagocytosis upon activation by udp. it has been reported recently that expression of both p2x4 and p2y6 is upregulated in activated microglia following nerve injury. we show here, in resting as well as lps-activated primary microglia, that p2y6 decreases p2x4-mediated calcium entry and inhibits the dilation of p2x4 channels into a large-conductance pore measured with a yo-pro-1 uptake assay. furthermore, p2y6 activation modulates the atp-dependent migration of microglia, a process likely involved in their shift from migratory to phagocytic phenotype.reconstituting the p2x4-p2y6 interaction in recombinant systems shows that p2y6 activation decreases p2x4 current amplitude, activation and desensitization rates, and reduces p2x4 channel permeability to the large cation nmdg 1 . phospholipase c-mediated hydrolysis of the phosphoinositide pi(4,5)p 2 , a necessary cofactor for p2x4 channel function, underlies this inhibitory crosstalk. as extracellular levels of both atp and udp are increased in the spinal cord following nerve injury, the control of p2x4 activity by p2y6 might play a critical role in regulating neuropathic pain-inducing microglial responses. with sc, thus representing a valid sc alternative. we have previously shown that dasc express c-aminobutyric-acid (gaba) receptors (i.e. gaba-a and gaba-b receptors), which modulate their proliferation and neurotrophic potential, although little is known about the role of other neurotransmitter systems in asc.in this study we investigated the expression of purinergic receptors in dasc. using rt-pcr, western blot analyses and immunohistochemistry we have demonstrated that asc express p2x 3 , p2x 4 and p2x 7 purinoceptors. interestingly, differentiation of asc towards glial phenotype was accompanied by up-regulation of p2x 4 and p2x 7 receptors. using ca 21imaging techniques, we have shown that stimulation of purinoceptors with adenosine-5 0 -triphosphate (atp) results in intracellular ca 21 signals, indication functional activity of these receptors.. moreover, we have shown that the increase of intracellular ca 21 leads to sc death, an effect that can be prevented using specific p2x 4 or p2x 7 antagonists.altogether, these results show, for the first time, the presence of functional purinergic receptors in sc-like derived from asc and their link with critical physiological processes such as cellular death and survival. the presence of these novel pharmacological targets in dasc might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches using dasc for peripheral nerve repair. university of freiburg, freiburg, germany intracellular ph homeostasis is a vital function shared by all cells and is mainly regulated by the coordinated action of several acid-base transporters. in the brain, ph homeostasis is of particular importance since changes of extracellular ph (pho) are events associated with both physiological conditions and pathological states in brain function. transient pho changes usually accompany physiological processes, including neuronal activity, and astrocytes respond to a rise in extracellular k 1 with plasma membrane depolarization and intracellular alkalinization. on the other hand, loss of brain ph homeostasis can lead to severe pathological conditions and significant changes of pho have been observed during seizures and spreading depression.in the present study, we sought to investigate regulation mechanisms of the electrogenic sodium/bicarbonate cotranspoter 1, nbce1, and of the vacuolar h 1 -atpase (v-atpase) in primary hippocampal astrocytes following extracellular acid-base changes, following neuronal activity, and using the 4-aminopyridine (4-ap) model of epilepsy in vitro.we show that extracellular acidosis, but not extracellular alkalosis, increased the number of activated astrocytes. our data also show differential regulation of acid-base transporters in the hippocampal glial cells during extracellular acid-base changes. intracellular ph measurements revealed a crucial role for v-atpase in regulation of intracellular ph, a process accompanied by increased membrane expression of the transporter, as assessed by immunofluorescence and surface biotinylation. nbce1-b is the nh2-terminal variant of nbce1 predominantly expressed in glial cells and is transcriptionally regulated followed neuronal activity or treatment of the cells with 4-ap.these data propose transcriptional and post-translational modification of acid-base transporters as putative regulatory mechanisms in astrocytes to cope with changes of extracellular ph serving the maintenance of intracellular ph homeostasis. these astroglial networks fulfil a variety of functions in the brain, e.g. potassium buffering and metabolite transport. in this study we compared glial networks in different brain regions to investigate site specific effects on network formation. we combined electrophysiology and immunohistochemstry with semi-quantitative rt-pcr and western blot analysis to investigate connexin expression, gap junction coupling and antigen profiles. experiments were performed in wild type and transgenic mice with glia specific fluorescence labelling as well as in cx30ko mice. astrocytes were investigated between postnatal days 12-60. gap junction networks in the ca1 region of the hippocampus and the ventrobasal thalamus show abundant coupling in hgfap-egfp mice. intriguingly, we found significant coupling between oligodendrocytes and astrocytes in the thalamus, while in the hippocampus panglial coupling was less abundant. other glial cells did not participate in the networks. we also found that a fluorescent glucose analog, 2-nbdg, propagates through the thalamic panglial network. the function of these panglial networks remains largely unclear.in heterozygous cx43-ecfpki mice, deletion of one allele of the major hippocampal cx43 significantly reduced the number of coupled astrocytes only in the hippocampus, while the thalamic networks remained unchanged. sr101 labelling of astrocytes and subsequent 2p microscopy identified a significant subset of thalamic sr1011 cells lacking cx43-ecfp expression. sr101 did not label oligodendrocytes as analysed in plp-gfp mice. semi-quantitative rt-pcr and western blot analysis revealed stronger expression of cx30 in thalamic nuclei while cx43 levels were higher in the hippocampus. this indicates a minor role for cx43 in gap junction coupling of astrocytes in the thalamus. consistent with these findings, the thalamus of cx30ko mice displayed a strong decrease in astrocytic cell coupling compared to wild type littermates.together, these results indicate that thalamic astrocytes differ in various aspects from their counterpart in other brain regions and support the emerging concept of astrocyte heterogeneity. the mechanism of secondary damage spread after brain trauma remains unresolved. in this work, we redirected the attention to astrocytic communication pathways. using an in vitro trauma model that consists of a scratch injury applied to a rat astrocyte monolayer, we found a significant induction of connexin43 hemichannel activity, demonstrated by ethidium uptake, in regions distal from the injury ($17 mm away and maximal $1 h after injury). this response was abolished by two connexin hemichannel blockers, la 31 and the peptide gap26. in addition, the trauma-induced increase in hemichannel activity was prevented by inhibition of purinergic p2 receptors. the scratch-induced increase in hemichannel activity was absent in astrocytes from cx43 knockout mice. this activity took place with a singular spatial distribution, since cells located at $17 mm away from the scratch gained connexin hemichannel activity. however, the functional state of gap junction channels (dye coupling) was not significantly affected in the same locations. the connexin43 hemichannel activity was also enhanced by the acute extracellular application of 60 mm k 1 . the increase in hemichannel activity was correlated with an increment in apoptotic cells, measured by immunofluorescence to annexin v at 24 h post-trauma, which was totally prevented by gap26 peptide. these findings could open a new approach to prevent or reduce the secondary cell damage due to brain trauma. l. schlosser, a. scheller, f. kirchhoff institute of physiology, saarland university, homburg, germany current research suggests that astrocytes represent heterogeneous neuroglial populations in different regions of the brain. this is particularly evident when the expression pattern of various transmitter receptors is analyzed. hippocampal astrocytes, for example, do not express ampa receptors, while cerebellar bergmann glia is loaded. functional analysis of glial receptors requires dedicated efforts for each distinct brain region and developmental age. unfortunately, the molecular identification using antibody approaches is often hampered by either poor antibody quality or abundant receptor expression on adjacent neuronal membranes. therefore, we are using cre/lox-mediated gene ablation for proper functional analysis.here, we are focusing on the characterization of astrocyte-specific gene deletion of the gaba b1 receptor. metabotropic gaba b receptors consist of two subunits gaba b1 and gaba b2 forming a heteromeric receptor complex of which gaba b1 is essential. in contrast to neurons, activation of astroglial gaba b receptors leads to transient rises of intracellular ca 21 . the functional impact of these glial gaba b -receptors is largely unknown.to address the gaba b receptor function in astrocytes we took advantage of the cre/lox system and crossbred glast-creert2 knockin mice with floxed gaba b1 receptor mice. first immunohistochemical analysis reveals a selective deletion of gaba b1 on a majority of astroglial processes. functional studies addressing the role of these receptors in astroglial ca 21 signaling are in progress. purinergic signaling is the most diverse system in astrocytes to communicate with other glial cells and neurons. astrocytes express a variety of p2x (ionotropic) and p2y (metabotropic) receptors. especially in case of brain injury, atp levels and receptor expression on astrocytes are increased. atp is immediately degraded to adp, amp and adenosine. these nucleotides/nucleosides act back on the preferred purinoreceptor expressed on glial and neuronal cells. in the last decade researchers tried to evaluate the functional impact of astrocytic purinoreceptors on the complex information flux at the tripartite synapse. a prominent role has been suggested for the p2y1 receptor subtype. we generated conditional mouse mutants to investigate the function of p2y1 receptors in astrocytes in vivo and crossbred mice carrying the floxed p2y1 gene with mice that express the inducible dna recombinase (creert2) under the control of the glast (l-glutamate/ l-aspartate transporter) locus. ablation of the p2y1 receptor is induced in development and adulthood by intraperitoneal tamoxifen injections. successful recombination of the targeted p2y1 receptor is evaluated by qrt-pcr on genomic dna and mrna usually 21 days after tamoxifen application. at the genomic level we find $25% and $50% recombination in astrocytes of the cerebellum and hippocampus, respectively. similar recombination frequencies are observed in young (p11) or adult mice (6-10 weeks). when looking at the transcript level, the mrna is reduced to 57% in the cerebellum of adult mice and to 76% in the hippocampus. in young mice we determined a reduction to 40% and 42% mrna expression in the cerebellum and hippocampus, respectively. given the high level of p2y1 expression on other cells of the brain these findings indicate a successful astrocyte-specific knockout. further fluorescence-activated cell sorting (facs) of recombined cells expressing the red fluorescent protein td-tomato will be used to verify the knockout. the potential function of these astroglial receptors in neuronal networks will be as well investigated using histological (em and light microscopy), twophoton ca 21 imaging in situ and in vivo and behavioral approaches. we recently showed that they invade the cortex at 11.5 days of embryonic age (e11.5). they first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. the absence of the expression of mac-2 and mhc ii suggest these cells have a na€ ıve/quiscent phenotype during embryonic development of the cortex. besides immunohistochemical markers is the presence of different k 1 channels on microglia also an indication of their activation stage. however most studies have been conducted on postnatal and adult microglial cells. therefore we aimed at determining the electrophysiological activation phenotype of these embryonic microglia.at the age of e13.5 and e15.5, microglial cells display a small inward rectifying k 1 current and this independent of their location in the embryonic cerebral cortex and their cell morphology. these cells also express functional p2x receptors, which based on the profile of the response are most probably p2x7 receptors. time-lapse analysis showed that embryonic microglia are highly dynamic cells. suggesting that these cells are already very active during fetal development. rapid extracellular removal of glutamate, the major excitatory neurotransmitter in the cns, is essential for normal brain function. this task is primarily accomplished by the action of the sodium-dependent, high-affinity transporters glast and glt-1 (rodent analogous of eaat1 and eaat2), which are mainly expressed by astrocytes. impairment or failure of glast and glt-1 plays an important role in many pathological conditions. question: pelizaeus-merzbacher-like disease (pmld) is a hypomyelinating leukodystrophy caused by mutations in the gjc2 gene encoding the gap junction (gj) protein connexin47 (cx47). cx47 is expressed in oligodendrocytes and forms most gj channels to astrocytes and to other oligodendrocytes. since pmld-associated gjc2 mutations cause loss of cx47 function, gene replacement strategies may be promising for developing future treatments. a mouse model for plmd, the cx32/ cx47 double knockout (ko), is characterized by early onset of severe leukodystrophy at 4 weeks of age leading to death by 6 weeks, offering the possibility to test therapies. the aim of the present study was to generate a lentiviral vector to allow gene delivery specifically to oligodendrocytes, and to examine the transduction efficacy, distribution, duration and levels of egfp reporter gene and cx47 expression throughout the cns, in order to establish a method for oligodendrocyte-targeted gene therapy.methods: the expression cassette with the cx47 gene along with the ires-egfp as a reporter gene under the control of oligodendrocyte-specific cnp promoter was cloned into the lentiviral vector pcclsin.ppt.hpgk.gfp.pre. mock vectors lacking the cx47 gene were also generated as controls. viral particles were produced to high titers and purified, before injection. lentiviral vectors were delivered into the brain of wild type (wt) and cx47ko mice at postnatal day 1 (p1), intraventricularly and in the stratum radiatum of the dorsal hippocampus. egfp and cx47 expression was assessed using immunochemistry and immunoblot analysis at different time points post injection.results: we found widespread expression of virally delivered egfp in different brain regions colocalizing with oligodendrocyte markers (cc1 and olig2). the expression of egfp was detected from p15 until 3 months post-injection. on average 20.3 6 2.56% of oligodendrocytes were egfp positive, with highest rates in the subventricular zone (29.3 6 6.31%, n 5 6) and olfactory bulb (19.9 6 4.36%, n 5 8) and lower rates in the cortex (16.1 6 2.60%, n 5 6) and corpus callosum (12.3 6 3.40%, n 5 3). in cx47ko brain expression of virally delivered cx47 was also found after p21 with formation of gj plaques in a subpopulation of oligodendrocytes.conclusions: our results show that neonatal lentiviral gene delivery may result in stable and widespread cns expression targeted to oligodendrocytes by using cell specific promoters. thus, gene therapy approaches using lentiviral vectors may be feasible for future treatment of leukodystrophy and should be further studied. recent reports identified mrna coding for different cav subtypes like 1.2 and 1.3 in hippocampal ng2 glia. using acute brain slices prepared from developing and mature ng2-eyfp mice, we also found that all ng2 glia upon depolarization in different brain regions elicited a. grimaldi, g. d'alessandro, c. lauro, m. catalano, c. limatola sapienza university of rome, rome, italy glioblastoma (gbm) is one of the most aggressive tumor of the central nervous system, being characterized by a great invasiveness and a very low survival rate of patients. this work wants to better define the role of tumor microenvironment in the modulation of tumor invasiveness. it is known that tumor migration and invasiveness can be modulated by the chemokine cxcl12 and its receptor cxcr4. the expression of both these proteins has been demonstrated in various human gbm cell lines and it has been shown that, blocking this pathway, tumor cells greatly reduce their invasive ability. we have recently demonstrated that an important molecule directly implicated in tumor migration is the intermediate conductance calcium-activated potassium channel (kca3.1). given the expression of these channels also in other cell types infiltrating the tumor mass, like microglia, we investigated the effect of kca3.1 inhibition on microglia-glioblastoma interaction. preliminary data indicate that kca3.1 activity on microglia is involved in the movement of these cells toward the tumor mass. a growing body of evidence indicates that glioblastoma stem-like cells (gscs) play a central role in human glioblastoma development and resistance to current therapies. we recently described a cluster of micro-rnas, the mir-302-367, which induces the exit of gscs from their stem state and suppresses their tumorigenic properties in an irreversible manner (fareh et al, 2012) . to elucidate the gene networks that govern gsc exit from their stem state, we used chip-seq (chromatin immunoprecipitation followed by next generation dna sequencing), to identify gene loci showing enrichment of the active (h3k4me3) and repressive (h3k27me3) epigenetic marks in gsc-mir-302-367 as compared to na€ ıve gscs. functional significance of the chip-seq results was evaluated with confrontation of the chip data with the transcriptomes of the corresponding cells derived from exon array hybridization. western blot analysis showed that global h3k4me3 and h3k27me3 expression levels were similar in gsc and gsc-mir-302-367. chip-seq analysis revealed similar gross number of gene loci associated with h3k4me3 or h3k27me3 (13000 and 5000 genes, respectively) in either cell type. interestingly, 16% (2381 genes) of the analyzed genes exhibited a change in either h3k4me3 or h3k27me3, or both in mir-302-367-gscs as compared to na€ ıve gscs. these changes resulted into a transcript variation in 14% of the cases (332/2381) considering as significant an increase or a decrease of at least 2-fold and the 77% of these genes translated into congruent alterations in the corresponding transcript levels.analysis of the functional significance of the changes observed using functional annotation databases identified novel gene networks, likely to participate in the regulation of gsc properties. studies under way focus on members of these networks specifically activated in mir-302-367 gscs that might alter gsc dialog with their microenvironment. malignant gliomas are the most frequent primary tumors of the brain with poor clinical prognosis. infiltrating peripheral macrophages and resident microglia contribute significantly to the tumor mass. we have previously shown that microglia as the intrinsic immune competent brain cells promote glioma expansion by up-regulating metalloprotease mt1-mmp through toll-like receptor (tlr) and its adaptor protein myd88. in this study we identified tlr2, as the main tlr controlling mt1-mmp expression and pro-tumorigenic signaling in microglia. glioma-derived soluble factors and synthetic tlr specific ligands induced mt1-mmp expression in microglia from wt mice but not tlr2-/-mice. by using the organotypic brain slice model, we found that tumor expansion depended on both parenchymal tlr2 expression and the presence of microglia. tlr2 is also highly expressed in human gliomas and inversely correlates with patient survival. in search for an endogenous tlr2 ligand released from glioma cells, we screened glioma conditioned medium (gcm) by mass spectrometry for endogenous tlr2 agonists produced by glioma cells and found versican, an extracellular matrix proteoglycan and a reported ligand of tlr2. to examine if versican is the factor mediating the glioma-microglia interaction, we silenced versican expression in gl261 cells. primary microglia were then stimulated with gcm from versican sirna and non-target sirna transfected gl261 cells and microglial mt1-mmp expression was analyzed by real-time pcr. an almost 3-fold up-regulation in microglial mt1-mmp expression was observed using gcm from non-target transfectants while the expression was reduced with gcm from versican knock-down gl261 transfectants. our results show that tlr2 activation is an essential part of the signaling cascade employed by glioma cells to convert microglia into a pro-tumorigenic phenotype and tlr2 thus may be a novel target for glioma therapies. two ipsdms were used to determine their responses in the presence of glioblastoma cells. using lc-ms/ms method, proteomic profiles were generated and compared between ipsdms exposed to human glioblastoma cells and ipsdms exposed to normal human astrocytes as control.results: immunolabeling of the two ipsdm cell lines showed specific expression of microglial markers such as iba-1, cd11b, and cd68. the comparative proteomic analyses indicated that microglia exposed to glioblastoma cells showed differential protein expressions relevant for cytoskeletal activity, energy production, and cell survival compared to control.conclusion: this is the first study showing the proteomes of human induced pluripotent stem cell-derived microglia exposed to glioblastoma cells, and the findings could provide further insight of the interaction between microglia and glioblastoma. key: cord-346314-o9fjpqaj authors: jarboui, mohamed ali; bidoia, carlo; woods, elena; roe, barbara; wynne, kieran; elia, giuliano; hall, william w.; gautier, virginie w. title: nucleolar protein trafficking in response to hiv-1 tat: rewiring the nucleolus date: 2012-11-15 journal: plos one doi: 10.1371/journal.pone.0048702 sha: doc_id: 346314 cord_uid: o9fjpqaj the trans-activator tat protein is a viral regulatory protein essential for hiv-1 replication. tat trafficks to the nucleoplasm and the nucleolus. the nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rrna and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. importantly, transient nucleolar trafficking of both tat and hiv-1 viral transcripts are critical in hiv-1 replication, however, the role(s) of the nucleolus in hiv-1 replication remains unclear. to better understand how the interaction of tat with the nucleolar machinery contributes to hiv-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of jurkat t-cells stably expressing hiv-1 tat fused to a tap tag. using an organellar proteomic approach based on mass spectrometry, coupled with stable isotope labelling in cell culture (silac), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in jurkat t-cell nucleolus upon tat expression. numerous proteins exhibiting a fold change were well characterised tat interactors and/or known to be critical for hiv-1 replication. this suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by tat provide an additional layer of control for regulating cellular machinery involved in hiv-1 pathogenesis. pathway analysis and network reconstruction revealed that tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, t-cell signaling pathways and genome integrity. we present here the first differential profiling of the nucleolar proteome of t-cells expressing hiv-1 tat. we discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust hiv-1 production. the nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (nors) formed by clusters of hundreds of rdna gene repeats organised in tandem head-to-tail repeat [1, 2] . a membrane-less organelle originally described as the ''ribosome factory'', the nucleolus is dedicated to rna-polymerase-i-directed rdna transcription, rrna processing mediated by small nucleolar ribonucleoproteins (sornps) and ribosome assembly. ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60s) and small (40s) ribosomal subunits, which are subsequently exported to the cytoplasm. this fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] . the nucleolus is the site of additional rna processing, including mrna export and degradation, the maturation of uridine-rich small nuclear rnps (u snrnps), which form the core of the spliceosome, biogenesis of t-rna and micrornas (mirnas) [4] . the nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] . the concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions. of importance, the nucleolus is also the target of viruses including hiv-1, hcmv, hsv and kshv, as part of their replication strategy [2, 17] . proteomics studies analysing the nucleoli of cells infected with human respiratory syncytial virus (hrsv), influenza a virus, avian coronavirus infectious bronchitis virus (ibv) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . interestingly, both hiv-1 regulatory proteins tat and rev localise to the nucleoplasm and nucleolus. both their sequences encompass a nucleolar localisation signal (nols) overlapping with their nuclear localisation signal (nls), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . furthermore, tat and rev interact with the nucleolar antigen b23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . nevertheless, a recent study described that in contrast to jurkat t-cells and other transformed cell lines where tat is associated with the nucleus and nucleolus, in primary t-cells tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . while the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing tat and rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . importantly, two major studies by machienzi et al. have revealed important functional links between hiv-1 replication and the nucleolus [49, 50] . first, they could inhibit hiv-1 replication and tat transactivation function employing a tar decoy specifically directed to the nucleolus. furthermore, using a similar approach, with an anti-hiv-1 hammerhead ribozyme fused to the u16 small nucleolar rna and therefore targeted to the nucleolus, they could dramatically suppress hiv-1 replication. collectively, these findings strongly suggest that hiv-1 transcripts and tat nucleolar trafficking are critical for hiv-1 replication. however the nature of these contributions remains to be elucidated. in this report, we systematically analysed the nucleolar proteome perturbations occurring in jurkat t-cells constitutively expressing hiv-1 tat, using a quantitative mass spectrometry approach. following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon tat expression, we focussed on the tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, dna replication and repair and metabolism and discuss their potential involvement in hiv-1 pathogenesis. in this study, we investigated the quantitative changes in the nucleolar proteome of jurkat t cells constitutively expressing hiv-1 tat (86aa) versus their tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (silac) technology, followed by esi tandem mass spectrometry and implemented the experimental approach described in figure 1a . first, using retroviral gene delivery, we transduced hiv-1 tat fused to a tandem affinity purification (tap) tag (consisting of two protein g and a streptavidin binding peptide) or tap tag alone (control vector) in jurkat leukemia t cell clone e6-1 and sorted the transduced cells (gfp positive) by facs. this resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( figure 1b) . the functionality of tap-tat was confirmed by transfecting jurkat tap-tat and tap cells with a luciferase reporter gene vector under the control of the hiv-1 ltr (pgl3-ltr) [36] . tap-tat up regulated gene expression from the hiv-1 ltr by up to 28 fold compared to control ( figure 1c ). to further address the functionality of tat fused to tap, we compared jurkat tap-tat with jurkat-tat, a cell line stably expressing untagged tat [51] . both cell line exhibited comparable hiv-1 ltr activity following transfection with pgl3-ltr ( figure s1 ). next, tat expression and subcellular localization was verified by subcellular fractionation followed by wb analysis ( figure 1e ). tap-tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. these observations were further validated by immunofluorescence microscopy ( figure 1e ). of note, jurkat-tat presented similar patterns for tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by wb analysis (figure s2 and s3). we next compared the growth rate and proliferation of the jurkat tap and tap-tat cell lines (materials and methods s1), which were equivalent ( figure s4a ). similarly, facs analysis confirmed that the relative populations in g1, s, and g2/m were similar for jurkat tap-tat and tap cells ( figure s4b ). we labeled jurkat tap-tat and jurkat tap cells with light (r0k0) and heavy (r6k6) isotope containing arginine and lysine, respectively. following five passages in their respective silac medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( figure 1a ) [52] . each step of the procedure was closely monitored by microscopic examination. to assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by western blot analysis ( figure 1d ). nucleolin (110 kda) and fibrillarin (fbl) (34 kda), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. this distribution pattern for nucleolin appears to be specific for jurkat t-cells as show previously [52, 53] . the nuclear protein parp-1 (poly adpribose polymerase 1) (113 kda) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. alpha-tubulin (50 kda) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination. subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. comparative quantitative proteomic analysis was performed using maxquant to analyse the ratios in isotopes for each peptide identified. a total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. the fully annotated list of the quantified nucleolar proteins is available in table s1 and the raw data from the mass spectrometry analysis was deposited in the tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. we annotated the quantified proteins using the toppgene suite tools [54] and extracted gene ontology (go) and interpro annotations [55] . the analysis of go biological processes ( figure 1f ) revealed that the best-represented biological processes included transcription (24%), rna processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of jurkat t-cell nucleolar proteome [52] . subcellular distribution analysis ( figure 1f ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. the subcellular distribution was similar to our previous analysis of the jurkat t-cell nucleolar proteome [52] . table s1 . the distribution of protein ratios are represented in figure 1g as log 2 (abundance change). the silac ratios indicate changes in protein abundance in the nucleolar fraction of jurkat tap-tat cells in comparison with jurkat tap cells. the distribution of the quantified proteins followed a gaussian distribution ( figure 1g ). a total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon tat expression (table 1) . of these, 30 proteins were enriched, whereas 19 proteins were depleted. cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (npm1/ b23), c23, fbl, nucleolar protein p120 (nol1), and nucleolar protein 5a (nol5a). the distinct ratios of protein changes upon tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus. we performed wb analysis to validate the silac-based results obtained by our quantitative proteomic approach ( figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon tat expression, including 9 enriched (hsp90b, stat3, prb, ck2a, ck2a', hsp90a, transportin, zap70, ddx3), and 3 depleted (ilf3, bop1, and ssrp1) proteins. in addition, we also tested by wb analysis, protein abundance not affected by tat expression (importin beta, fbl, b23, c23). these results highlight the concordance in the trend of the corresponding silac ratios, despite some differences in the quantitative ranges. of note, using wb, we could observe a change of intensity for protein with a silac fold change as low as 1.25-fold. of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. on the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in table 1 . alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. therefore, we next focused on both enriched or depleted individual proteins with activities associated with hiv-1 or tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes. we initially focused on signaling proteins interacting with tat and/or associated hiv-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by tat expression. phospho-protein phosphatases. phospho-protein phosphatase pp1 and pp2a are essential serine/threonine phosphatases [56, 57] . importantly, pp1 accounts for 80% of the ser/thr phosphatase activity within the nucleolus. in our study, pp1 was found to be potentially enriched by 1.52-fold in the nucleolus of jurkat cells expressing tat, which supports previous studies describing the nuclear and nucleolar targeting of pp1a by hiv-1 tat and how pp1 upregulates hiv-1 transcription [58, 59, 60, 61, 62] . pp1 c was also identified as part of the in vitro nuclear interactome [63] . similarly, ppp2ca, the pp2a catalytic subunit (1.29-fold) and its regulatory subunit pp2r1a (1.27-fold) were similarly enriched upon tat expression. interestingly, tat association with the pp2a subunit promoters results in the overexpression and up regulation of pp2a activity in lymphocytes [64, 65] . furthermore, pp2a contributes to the regulation of hiv-1 transcription and replication [61, 66] . retinoblastoma protein. the tumour suppressor gene prb protein displayed a 1.4-fold change in the nucleolus upon tat expression [67] . furthermore, wb analysis confirmed the distinct translocation of prb from the nucleoplasm to the nucleolus by tat ( figure 2 ). depending on the cell type, prb can be hyperphosphorylated or hypophosphorylated upon tat expression and can negatively or positively regulate tat-mediated transcription respectively [68, 69, 70] . interestingly, the hyperphosphorylation of prb triggers in its translocation into the nucleolus [71] . phosphorylation of prb is also associated with an increase in ribosomal biogenesis and cell growth [72] . stat3. the transcription factor signal transducer and activator of transcription 3 (stat3) was significantly enriched (1.86-fold) in the nucleolar fraction by tat constitutive expression. furthermore, wb analysis indicated that tat expression could promote the relocalisation of stat3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( figure 2) . interestingly, previous studies have demonstrated tat-mediated activation of stat3 signaling, as shown by its phosphorylation status [73] . interestingly, stat3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] . ybx1. ybx1, the dna/rna binding multifunctional protein was enriched by 1.38-fold in the nucleolus of jurkat cells upon tat expression. interestingly, ybx1 interacts with tat and tar and modulates hiv-1 gene expression [63, 75] . zap70. the protein tyrosine kinase zap70 (zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of jurkat cells expressing tat [76] . furthermore, wb analysis revealed that tat expression could promote the relocalisation of zap70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( figure 2 ). of note, zap70 is part of the in vitro nuclear tat interactome [63] . matrin 3. the inner nuclear matrix protein, matrin 3 (matr3), presented a 1.39-fold change in the nucleolus of jurkat cells expressing tat. it localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . matrin 3 has been identified as part of the in vitro hiv-1 tat nuclear interactome [63] . two recent studies have described matrin 3 as part of ribonucleoprotein complexes also including hiv-1 rev and (rev response element) rre-containing hiv-1 rna, and promoting hiv-1 post-transcriptional regulation [78, 79, 80] . casp10. the pro-apototic signaling molecule, caspase 10 (casp10), was significantly depleted from the nucleolus of jurkat-tat cells (0.82-fold) [81] . importantly, tat expression downregulates casp10 expression and activity in jurkat cells [82] . adar1. adenosine deaminase acting on rna (adar1), which converts adenosines to inosines in double-stranded rna, was significantly depleted from the nucleolus of jurkat-tat cells (0.78-fold). interestingly, adar1 over-expression up-regulates hiv-1 replication via an rna editing mechanism [83, 84, 85, 86, 87, 88] . furthermore, adar1 belongs to the in vitro hiv-1 tat nuclear interactome [63] . to underline the structural and functional relationships of the nucleolar proteins affected by hiv-1 tat, we constructed a network representation of our dataset. we employed cytoscape version 2.6.3 [89] and using the mimi plugin [90] to map previously characterised interactions, extracted from protein interaction databases (bind, dip, hprd, ccsb, reactome, intact and mint). this resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( figure 3a ). centrality analysis revealed a threshold of 23.7 interactions per protein. topology analysis using the centiscape plugin [91] showed that the node degree distribution follows a power law ( figure s5 ), characteristic of a scale-free network. importantly, when we analysed the clustering coefficient distribution ( figure s6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around hiv-1 tat. furthermore, node degree connection analysis of our network identified hiv-1 tat as the most connected protein ( figure s6 ). specifically, the topology analysis indicated that the values for tat centralities were the highest (node degree, stress, radiality, closeness, betweeness and centroid), characterising tat as the main hub protein of the nucleolar network. indeed, a total of 146 proteins have been previously described to interact with tat ( figure 3b , table s2 ). these proteins are involved in a wide range of cellular processes including chromosomal organization, dna and rna processing and cell cycle control. importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold). in parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( figure 4) . ribosomal biogenesis. we initially focused on ribosome biogenesis, the primary function of the nucleolus. we could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by tat expression (figure 4 ). while some ribosomal proteins remained unaffected, tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except rpl35a, for which tat expression caused a marked decrease at the nucleolar level (0.29-fold). similarly, several proteins involved in rrna processing exhibited an overall increase in nucleolar accumulation upon tat expression. these include human canonical members of the l7ae family together with members participating in box c/d, h/aca and u3 snornps ( figure 4) . conversely, bop1, a component of the pebow (pescadillo bop1 wdr12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of jurkat tap-tat cells (0.81-fold) and this was confirmed by wb analysis (figure 2 ) [93] . nevertheless, the other pebow complex components, pes1 (0.94-fold) and wdr12 (1.1fold), were not affected by tat expression. of note, we did not detect change in the abundance of protein participating in rdna transcription such as rnapoli, ubf. spliceosome. we identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . these proteins include the small nuclear ribonucleoproteins u1, u2 and u5, sm d1, d2, d3, f and b, and the heterogeneous nuclear ribonucleoproteins. our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of hiv-1 tat in jurkat t-cells (figure 3 and 4) . the only three proteins that were significantly depleted from the nucleolus upon expression of hiv-1 tat were rbmx (0.89-fold), hnrnpa2b1 (0.84-fold) and snrpa (0.81-fold). several investigations showed expression alteration in cellular splicing factors in hiv-1 infected cells [95, 96] . molecular chaperones. we have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of t-cells upon tat expression (figure 3 and 4) , many of which were previously characterised as part of the tat nuclear interactome [63] . several heat-shock proteins including dnajs, specific hsp90, hsp70 and hsp40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of jurkat cells expressing tat ( figure 4 ). as shown by wb, while hsp90a and b are mostly cytoplasmic, tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (figure 2) . similarly, heat-shock can cause the hsp90 and hsp70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . in a recent study, fassati's group has shown that hsp90 is present at the hiv-1 promoter and may directly regulate viral gene expression [102] . we also observed the coordinated increased abundance of class i (groel and groes) and class ii (chaperonin containing tcp-1 (ctt)) chaperonin molecules (figure 3 and 4) upon tat expression. ubiquitin-proteasome pathway. the ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . the 26s proteasome is composed of the 20s core particle (cp) and the 19s regulatory particle (rp). alternatively, cp can associate with the 11s rp to form the immunoproteasome. all the quantified proteins in our study are part of the 19s regulatory complex and include psmd2 (1.5-fold), psmd3 (1.32-fold), psmd11 (1.25-fold) and psmd13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of tat (figure 4) . interestingly, tat interacts with distinct subunits of the proteasome system, including the 19s, 20s and 11s subunits. the consequences of these interactions include the competition of tat with 11s rp or 19s rp for binding to the 20s cp, which resulted in the inhibition of the 20s peptidase activity [106, 107, 108, 109, 110, 111] . furthermore, tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic t-lymphocytes [112] . importantly, a recent study demonstrated that in the absence of tat, proteasome components are associated to the hiv-1 promoter and proteasome activity limits transcription [113] . addition of tat promoted the dissociation of the 19s subunit from the 20s proteasome, followed by the distinct enrichment of the 19s-like complex in nuclear extracts together with the tat-mediated recruitment of the 19s subunits to the hiv-1 promoter, which facilitated its transcriptional elongation [113] . we also quantified uba1 (1.36-fold), the e3 ubiquitin-protein ligase uhrf1 (1.13-fold), ubc (1-fold) and two ubiquitinspecific-peptidases, usp30 (1.28-fold) and usp20 (0.06-fold) (figure 4) . dna replication and repair. upon hiv-1 tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with dna replication and repairs pathways (figure 4) . tat induced the coordinated enrichment of the miniature chromosome maintenance mcm2-7 complex (from 1.23-to 3.30fold, respectively) [114] . mcm7, 6 and 3 were identified as part of the in vitro nuclear interactome of hiv-1 tat [63] . the structural maintenance of chromosomes 2, smc2, was enriched (1.35-fold) in the nucleolar fraction by tat expression. smc2 was identified as part of the in vitro nuclear interactome of hiv-1 tat [63] . while replication factor c1 (rfc1) and rfc2 (1.31-and 1.28-fold respectively) displayed an increased fold change and rfc5/3 were not affected, rfc4 was severely depleted (0.69-fold) from the nucleolar fraction upon tat expression [115] . rfc1 and rfc2 were identified as part of the in vitro nuclear interactome of hiv-1 tat [63] . tat induced the enrichment of xrcc6 (1.27-fold) and xrcc5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous dna end joining (nhej) [116] . xrcc6 associates with viral preintegration complexes containing hiv-1 integrase and also interact with tat and tar [117, 118, 119] . furthermore, in a ribozyme-based screen, xrcc5 (ku80) knockdown decreased both retroviral integration and tatmediated transcription [120] . as part of the base excision repair (ber), we have identified a major apurinic/apyrimidinic endonuclease 1 (apex1) (1.29-fold) . importantly, in a sirna screen targeting dna repair factors, apex1 knockdown was found to inhibit hiv-1 infection by more 60% [121] . the high mobility group (hmg) protein, hmga1 (1.30-fold), was enriched in the nucleolus following tat expression [122] . hmga1 interact with hiv-1 integrase and is part of the hiv-1 pre-integration complex [123, 124] . importantly, hmga1 has been identified in a proteomic screen, as a cellular cofactor interacting with the hiv-1 59leader [125] . metabolism. our proteomic data suggest that tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (figure 4 and figure s7 ). notably, in t cells expressing tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase b (ldhb) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (gapdh) (1.17-fold) and phosphoglyceric acid mutase (pgam1) (0.89-fold) ( figure 4 and figure s7 ). briefly, gpi catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. subsequently, pfkp catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. at the end of the glycolytic pathway, pkm2, in its tetrameric form, is known to generate atp and pyruvate, while ldhb diverts the majority of the pyruvate to lactate production and regeneration of nad+ in support to continued glycolysis, a phenomenon described for proliferative tcells [126] . of note, in highly proliferating cells, pkm2 can be found in its dimeric form and its activity is altered. this upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. as part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (g6pd) (2.11-fold), which branches of the glycolysis pathway to generate nadph, ribose-5phosphate an important precursor for the synthesis of nucleotides. consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. more specifically, impdh2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (prps2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (ctps) (1.74-fold) which catalyses the conversion of utp to ctp and the ribonucleotide reductase large subunit (rrm1) (1.56-fold). in parralel, we noted the increased abundance of the phosphoserine aminotransferase psat1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro. the host-virus interface is a fundamental aspect in defining the molecular pathogenesis of hiv-1 [127, 128, 129, 130, 131, 132, 133] . indeed, with its limited repertoire of viral proteins, hiv-1 relies extensively on the host cell machinery for its replication. several recent studies have capitalized on the recent advances in the ''omics'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . hiv-1 tat is essential for viral replication and orchestrates hiv-1 gene expression. the viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . we and others have employed system-level approaches to investigate tat interplay with the host cell machinery, which have characterised hiv-1 tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . here, we have investigated the nucleolar proteins trafficking in response to hiv-1 tat expression in t-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by tat in the fine-tuning of protein availability and function. we have developed for this study, a cellular model using jurkat t-cells stably expressing tat fused in its n-ternminal to tap-tag. jurkat t-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. importantly, they have been widely employed to evaluate tat-mediated pathogenesis using system-wide approaches and to analyse t-cell key cellular signaling pathways and functions [144, 150, 151, 152] . indeed, we have found them particularly suited for prolongued in vitro culture in silac medium and subsequent isolation of their nucleolus followed by ms analysis, which requires up to 85 millions of cells. we fused tat to the tap tag to enable future downstream applications such as tandem affinity purification or chromatin ip analysis. importantly, we have confirm that n-terminal tap-tag did not interfere with tat function nor its localisation in jurkat cells, when compared to untagged-tat. of note, tat subcellular distribution can vary according to the cell type employed. while tat is known to accumulate in the nucleus and nucleolus in jurkat cells and other transformed cell lines, in primary t-cells, tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . these differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by kuusisto et al. [39] . furthermore, stauber and pavlakis have suggested that tat nucleolar localisation could be the results of tat overexpression [31] . here, we have selected and employed a polyclonal population of jurkat t-cells expressing tat at different levels. we propose that this heterogeneity in tat expression levels might reflect tat stochastic expression described during viral replication [153] . using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. the extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with hiv-1 pathogenesis. hiv-1 tat is expressed early following hiv-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. in this context and based on our results, we propose that tat could participate in shaping the intracellular environment and metabolic profile of t cells to favor host biosynthetic activities supporting robust virions production. indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. with the notable exeption of rpl35a nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (nhp2l1, rlp14, rpl17, rpl27, rps2, rpl13). furthermore, this effect appears to be specific to hiv-1 tat since transcription inhibition by actinomycin d resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. in parralel, we observed the concordant modulation of pathways regulating protein homeostasis. we noted the significant nucleolar accumulation of multiple molecular chaperones including the hsps, the tcp-1 complex, and canx/calr molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . these observations further support previous studies describibing the modulation of the proteasomal activity by tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . we also observed the concomitant depletion of casp10 in the nucleolus of jurkat tap-tat. it has been suggested that casp10 could be targeted to the nucleolus to inhibit protein synthesis [154] . interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . as viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability. coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. similarly, these pathways are elevated in proliferative t-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the warburg effect [156, 157, 158, 159] . there, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the tca cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl coa and nadph for redox homeostasis. consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including imph2, a rate limiting enzyme known to control the pool of gtp. similarly, we noted the nucleolar enrichment of psat1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . collectively, we propose that by controlling protein homeostasis and metabolic pathways, tat could meet both the energetic and biosynthetic demand of hiv-1 productive infection. of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . furthermore glycolytic enzymes, such as pkm2, ldh, phosphoglycerate kinase, gapdh, and aldolase, also have been reported to display nuclear localization and bind to dna [162] . more specifically, pkm2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] . hiv-1 tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote tcr signaling [164] . we have observed the nucleolar enrichment by tat of key proximal or downstream components of t-cell signaling pathways, including zap70, ilf3 and stat3, which play crucial roles in t-cell development and activation. we had previously identified them as t-cell specific components of the nucleolus, and if studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . our results further support that tat could contribute to the dysregulation of tcr-derived signals and that the nucleolus could represent an important spatial link for tcr signaling molecules. we observed the coordinated nucleolar enrichment of key components of the dna replication, recombination and repair pathways by tat. these include xrcc5 and xrcc6, hmga1, apex1, mcm2-7, smc2, rfc1 and rfc2, while rfc4 was found to be significantly depleted. interestingly, these cofactors have been associated with the efficiency of retroviral dna integration into the host dna or the integrity of integrated provirus [166] . whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined. the mechanisms of tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with tat, since subcellular fractionation combined with wb analysis showed that the pattern and extent of subcellular redistribution between proteins varied. we could observe cases where tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (ddx3, tnpo1). alternatively, tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (prb). additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon tat expression (stat3, zap70 and hsp90). finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (ssrp1). it remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. remarkably, we identified several known hiv-1 tat partners involved in hiv-1 pathogenesis, which suggests that tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. this is exemplified by the following enriched proteins, prb, pp1 and stat3, for which phosphorylation is induced by tat. importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by tat. moreover, our data indicates that serine/threonine kinases (ck2 a') and phosphatases (pp1) were significantly enriched in the nucleolar fractions of jurkat tap-tat. these enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. in addition, tat significantly decreased the levels of sumo-2 in the nucleolus. similarly, sumo-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . given the potential importance of post-translational modifications, including phosphorylation in the tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach. the control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. ribosomal proteins are degraded by the ubiquitin-proteasome pathway to ensure their abundance matches up with rrna transcription levels. conversely, heat shock proteins hsp90s protect them from degradation. interestingly, our data showing that tat modulation the abundance proteins associated with the ubiquitin-proteasome and heat-shock pathway. this could contribute to the observed enrichment of ribosomal proteins by tat. nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of tat-mediated prevention of their export to the cytoplasm. interestingly, using a different cellular system, a drosophila melanogaster tat transgenic strain, ponti et al, analysed the effects of tat on ribosome biogenesis, following 3 days heat shock treatment to induce tat expression under the control of the hsp70 promoter [167] . following tat expression, they observed a defect in pre-rrna processing associated with a decrease in the level of 80s ribosomes [167] . nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours. while previous system-level studies have monitored the effects of hiv-1 tat expression on t cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to hiv-1 tat expression. using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to tat constitutive expression. our findings underscore that tat expressing t-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. importantly, we noted the functional relationship of nucleolar proteins of our dataset with hiv-1 pathogenesis and hiv-1 tat in particular. this further increases our confidence in our experimental strategy and suggests a role for tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. ultimatly, our study provides new insights on the importance of tat in the cross talk between nucleolar functions and viral pathogenesis. importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with hiv-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention. jurkat t-cells, clone e6.1 (atcc), jurkat ntap-tat and jurkat ntap were maintained in rpmi-1640 medium supplemented with 10% (v/v) foetal bovine serum (gibco, eu approved), and antibiotics. phoenix-gp cells (g.p. nolan; www.stanford.edu/ group/nolan/), were maintained in dmem medium supplemented with 10% (v/v) foetal bovine serum (gibco, eu approved). cells were counted using scepter tm 2.0 cell counter (millipore). the sequence of hiv-1 tat (hiv-1 hxb2, 86 amino acids) was sub-cloned into pentr 2b vector (invitrogen, a10463). using the gateway technology (invitrogen), we introduced the hiv-1 tat sequence into the plasmid pcemm-ntap(gs)-gw [168] . phoenix cells (g.p. nolan; www.stanford.edu/group/ nolan/), were transfected using fugene 6 (roche) with 5 mg of the plasmid ntap-tat or ntap and 3 mg of the pmdg-vsvg. viral supernatants were collected after 48 h, filtered and used to transduce the jurkat cell lines. the construct is termed ntap-tat, the empty vector was termed ntap. using retroviral gene delivery, we stably transduced jurkat cells (clone e6.1 (atcc)). the positive clones named jurkat ntap-tat and jurkat ntap were sorted to enrich the population of cells expressing gfp using the bc moflo xdp cell sorter (beckman coulter). sub-cellular fractions (10 mg) were resolved by sds-page and transferred onto biotrace pvdf membranes (pall corporation). the following primary antibodies were used: a-tubulin (sc 5286), c23 (sc 6013), and fibrillarin (sc 25397) were from santa cruz biotechnology, and parp (am30) from calbiochem, mouse anti-zap 70 (05-253, millipore), rabbit anti-stat3 (06-596, millipore), rabbit anti-ilf3 (ab92355, abcam), rabbit anti-hsp90 beta (ab32568, abcam), mouse anti-adar1 (ab88574, abcam), rabbit anti-hdac1 (ab19845, abcam), rabbit anti-ssrp1 (ab21584, abcam) rabbit anti-bop1 (ab86982, abcam), mouse anti-kpnb1 (ab10303, abcam), rabbit anti-hiv-1 tat (ab43014, abcam), rabbit anti-ck2a (ab10466, abcam), rabbit anti-ddx3x (ab37160, abcam), mouse anti-tnpo1 (ab2811, abcam), mouse anti-hsp90a (ca1023, merck), and rabbit-anti rb1 (sc-102, santa cruz).the following secondary antibodies were used ecl: anti-mouse igg and ecl anti-rabbit igg (ge healthcare), and donkey anti-goat igg (sc 2020) (santa cruz biotechnology). for silac analysis silac-rpmi r0k0 and silac-rpmi r6k6 (dundee cells) media supplemented with 10% dialyzed fbs (gibco, 26400-036) were used. the jurkat cells expressing ntap-tat and ntap were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. cells viability was checked with trypan blue (0.4% solution, sigma) and further confirmed using pi staining and facs analysis. cells were mixed to the ratio 1:1 to obtain 140610 6 cells. nucleoli were isolated from the mixed cell population as previously described in jarboui et al., [165] . nucleolar extracts (100 mg) were resuspended in 50 mm ammonium bicarbonate and in solution trypsin digested as previously described in jarboui et al. [165] . sample was run on a thermo scientific ltq orbitrap xl mass spectrometer connected to an eksigent nano lc.1dplus chromatography system incorporating an auto-sampler. sample was loaded onto a biobasic c18 picofrittm column (100 mm length, 75 mm id) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nl min-1. the mass spectrometer was operated in positive ion mode with a capillary temperature of 200uc, a capillary voltage of 46v, a tube lens voltage of 140v and with a potential of 1800 v applied to the frit. all data was acquired with the mass spectrometer operating in automatic data dependent switching mode. a high resolution ms scan was performed using the orbitrap to select the 5 most intense ions prior to ms/ms analysis using the ion trap. the incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''heavy'' medium as described in [169] . our analysis showed that we had an incorporation efficiency .95% (data not shown). the ms/ms spectra were searched for peptides identification and quantification using the maxquant software [170] (version 1.1.1.36), the human ipi database (version 3.83) and the andromeda search engine associated to maxquant [171] . standard settings were used for maxquant with the acetyl (protein n-term) as variable modification and carbamidomethyl (cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 da, respectively. each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. proteins were identified with the minimum of one peptide with a false discovery rate less than 1%. gene ontology, kegg pathway and pfam terms were extracted from uniprot entries using perseus, a software from the maxquant data analysis package (http://www.maxquant.org ), and the toppgene suite tools [54] . the jurkat ntap-tat and jurkat ntap were transfected using the amaxa electroporation system (amaxa biosystem) with the pgl3 (pgl3-ltr) (promega) as recommended by amaxa biosystem. dual-luciferase assays (promega) were performed according to the manufacturer's instructions. luciferase activity was measured and normalized against the total amount of proteins as quantified by the bca protein quantification kit (pierce, thermo scientific). to preserve their original shape, we performed immunostaining of jurkat cells in suspension. cells were fixed in 2% pfa for 10 min at rt, permeabilised in 0.5% triton x-100 for 15 min at rt and blocked with 5% fcs. cells were incubated with the rabbit hiv-1 tat antibody (ab43014, abcam) followed by the secondary antibody anti-rabbit alexa fluor 647 (a-21246, invitrogen). cells were allowed to attach to cell-tak (bd) coated silanised slides (daocytomation), and stained with dapi. images were captured with a carl zeiss confocal microscope equipped with a plan-apochromat 63x/1.4 oil dic objective. the proteomics raw data file from the mass spectrometry analysis was deposited to the tranche repository(https:// proteomecommons.org/tranche/) [172] . the file can be accessed and downloaded using the following hash key: (r3o5sv5z6hvwqrbndhp21txfetludwyxvwmifu-h6e1kmgaraucsq4dlncxeuvfohdezledcg4x5y8resb6-mua6wm1kiaaaaaaaab/w = = ). materials and methods s1 description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis. (docx) the epigenetics of rrna genes: from molecular to chromosome biology nucleolus: the fascinating nuclear body the nucleolus: a paradigm for cell proliferation and aging. brazilian journal of medical and biological research = revista brasileira de pesquisas medicas e biologicas/sociedade brasileira de biofisica in search of nonribosomal nucleolar protein function and regulation deciphering the human nucleolar proteome functional proteomic analysis of human nucleolus directed proteomic analysis of the human nucleolus nopdb: nucleolar proteome database-2008 update nucleolar proteome dynamics nopdb: nucleolar proteome database quantitative nucleolar proteomics reveals nuclear re-organization during stress-induced senescence in mouse fibroblast -dependent subcellular proteome localization following dna damage the nucleolus takes control of protein trafficking under cellular stress quantitative proteomics and dynamic imaging of the nucleolus reveals distinct responses to uv and ionizing radiation a quantitative proteomics analysis of subcellular proteome localization and changes induced by dna damage the nucleolus: reviewing oldies to have new understandings involvement of the nucleolus in replication of human viruses nucleolar proteomics and viral infection nucleophosmin phosphorylation by v-cyclin-cdk6 controls kshv latency elucidation of the avian nucleolar proteome by quantitative proteomics using silac and changes in cells infected with the coronavirus infectious bronchitis virus quantitative proteomics using silac coupled to lc-ms/ms reveals changes in the nucleolar proteome in influenza a virus-infected cells quantitative proteomic analysis of a549 cells infected with human respiratory syncytial virus quantitative proteomics using stable isotope labeling with amino acids in cell culture reveals changes in the cytoplasmic, nuclear, and nucleolar proteomes in vero cells infected with the coronavirus infectious bronchitis virus proteomics analysis of the nucleolus in adenovirus-infected cells viral nucleolar localisation signals determine dynamic trafficking within the nucleolus protein b23 is an important human factor for the nucleolar localization of the human immunodeficiency virus protein tat spatial association of hiv-1 tat protein and the nucleolar transport protein b23 in stably transfected jurkat t-cells specific complex of human immunodeficiency virus type 1 rev and nucleolar b23 proteins: dissociation by the rev response element effects of a highly basic region of human immunodeficiency virus tat protein on nucleolar localization a region of basic amino-acid cluster in hiv-1 tat protein is essential for trans-acting activity and nucleolar localization intracellular trafficking and interactions of the hiv-1 tat protein phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of hiv-1 tat by infected t-cells the ins and outs of hiv-1 tat tuning the transport properties of hiv-1 tat arginine-rich motif in living cells in vivo study of hiv-1 tat arginine-rich motif unveils its transport properties direct interaction of the human i-mfa domain-containing protein tat results in cytoplasmic sequestration and control of tat activity the arginine-rich domains present in human immunodeficiency virus type 1 tat and rev function as direct importin betadependent nuclear localization signals the hiv-1 tat nuclear localization sequence confers novel nuclear import properties global enhancement of nuclear localization-dependent nuclear transport in transformed cells intermolecular masking of the hiv-1 rev nls by the cellular protein hic: novel insights into the regulation of rev nuclear import nuclear factor 90, a cellular dsrna binding protein inhibits the hiv rev-export function functional analysis of the interaction of the human immunodeficiency virus type 1 rev nuclear export signal with its cofactors the ins and outs of hiv rev the hiv-1 rev protein the specificity of the crm1-rev nuclear export signal interaction is mediated by rangtp requirement of ddx3 dead box rna helicase for hiv-1 rev-rre export function mechanisms of receptor-mediated nuclear import and nuclear export the nuclear pore component nup358 promotes transportin-dependent nuclear import a nucleolar tar decoy inhibitor of hiv-1 replication ribozyme-mediated inhibition of hiv 1 suggests nucleolar trafficking of hiv-1 rna constitutive expression of hiv-1 tat protein in human jurkat t cells using a bk virus vector gautier vw proteomic profiling of the human t-cell nucleolus nucleolin undergoes partial n-and o-glycosylations in the extranuclear cell compartment toppgene suite for gene list enrichment analysis and candidate gene prioritization interpro: the integrative protein signature database mitotic phosphatases: no longer silent partners emerging roles of nuclear protein phosphatases nuclear targeting of protein phosphatase-1 by hiv-1 tat protein expression of a protein phosphatase 1 inhibitor, cdnipp1, increases cdk9 threonine 186 phosphorylation and inhibits hiv-1 transcription regulation of hiv-1 transcription by protein phosphatase 1 dephosphorylation of cdk9 by protein phosphatase 2a and protein phosphatase-1 in tat-activated hiv-1 transcription nuclear protein phosphatase-1 regulates hiv-1 transcription vitro nuclear interactome of the hiv-1 tat protein association of tat with promoters of pten and pp2a subunits is key to transcriptional activation of apoptotic pathways in hiv-infected cd4+ t cells pp2a targeting by viral proteins: a widespread biological strategy from dna/rna tumor viruses to hiv-1 protein phosphatase 2a enhances activation of human immunodeficiency virus type 1 by phorbol myristate acetate rb1, development, and cancer human immunodeficiency virus type 1 tat protein modulates cell cycle and apoptosis in epstein-barr virus-immortalized b cells hiv-1 tat elongates the g1 phase and indirectly promotes hiv-1 gene expression in cells of glial origin retinoblastoma gene inhibits transactivation of hiv-ltr linked gene expression upon co-transfection in he la cells nucleolar size and activity are related to prb and p53 status in human breast cancer nucleolus, ribosomes, and cancer intracellular tat of human immunodeficiency virus type 1 activates lytic cycle replication of kaposi's sarcoma-associated herpesvirus: role of jak/stat signaling getting the message across, stat! design principles of a molecular signaling circuit interaction of yb-1 with human immunodeficiency virus type 1 tat and tar rna modulates viral promoter activity the structure, regulation, and function of zap-70 bipartite nuclear localization signal of matrin 3 is essential for vertebrate cells matrin 3 and hiv rev regulation of mrna characterization of the hiv-1 rna associated proteome identifies matrin 3 as a nuclear cofactor of rev function matrin 3 is a co-factor for hiv-1 rev in regulating post-transcriptional viral gene expression playing the disc: turning on trail death receptor-mediated apoptosis in cancer hiv-1 tat protein concomitantly down-regulates apical caspase-10 and up-regulates c-flip in lymphoid t cells: a potential molecular mechanism to escape trail cytotoxicity enhancement of replication of rna viruses by adar1 via rna editing and inhibition of rna-activated protein kinase multiple levels of pkr inhibition during hiv-1 replication adar2 editing enzyme is a novel human immunodeficiency virus-1 proviral factor alternate rrna secondary structures as regulators of translation adar1 interacts with pkr during human immunodeficiency virus infection of lymphocytes and contributes to viral replication double-stranded rna adenosine deaminases enhance expression of human immunodeficiency virus type 1 proteins cytoscape: a software environment for integrated models of biomolecular interaction networks michigan molecular interactions (mimi): putting the jigsaw puzzle together analyzing biological network parameters with centiscape network biology: understanding the cell's functional organization interdependence of pes1, bop1, and wdr12 controls nucleolar localization and assembly of the pebow complex required for maturation of the 60s ribosomal subunit comprehensive proteomic analysis of the human spliceosome the expression of the essential nuclear splicing factor sc35 is altered by human immunodeficiency virus infection hiv-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages nucleolar targeting of the chaperone hsc70 is regulated by stress, cell signaling, and a composite targeting signal which is controlled by autoinhibition stress inhibits nucleocytoplasmic shuttling of heat shock protein hsc70 heat-induced nuclear accumulation of hsc70s is regulated by phosphorylation and inhibited in confluent cells intracellular localization of the 90 kda heat shock protein (hsp90alpha) determined by expression of a egfp-hsp90alpha-fusion protein in unstressed and heat stressed 3t3 cells localization of heat shock proteins in mouse male germ cells: an immunoelectron microscopical study gyrase b inhibitor impairs hiv-1 replication by targeting hsp90 and the capsid protein ubiquitin-like and ubiquitin-associated domain proteins: significance in proteasomal degradation ubiquitin and ubiquitin-like proteins in the nucleolus: multitasking tools for a ribosome factory analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins the rtp site shared by the hiv-1 tat protein and the 11s regulator subunit alpha is crucial for their effects on proteasome function including antigen processing hiv-1 tat inhibits the 20 s proteasome and its 11 s regulator-mediated activation peptide sequencing identifies mss1, a modulator of hiv tat-mediated transactivation, as subunit 7 of the 26 s protease new human gene encoding a positive modulator of hiv tat-mediated transactivation a cdna for a protein that interacts with the human immunodeficiency virus tat transactivator human immunodeficiency virus-1 tat protein interacts with distinct proteasomal alpha and beta subunits hiv-1 tat protein modulates the generation of cytotoxic t cell epitopes by modifying proteasome composition and enzymatic activity the proteasome regulates hiv-1 transcription by both proteolytic and nonproteolytic mechanisms oncogenic activity of mcm7 transforming cluster unzipped and loaded: the role of dna helicases and rfc clamp-loading complexes in sister chromatid cohesion the xrcc genes: expanding roles in dna double-strand break repair role of the non-homologous dna end joining pathway in the early steps of retroviral infection lupus autoantigen ku protein binds hiv-1 tar rna in vitro effect of ku80 depletion on the preintegrative steps of hiv-1 replication in human cells identification of cellular cofactors for human immunodeficiency virus replication via a ribozyme-based genomics approach sirna screening of a targeted library of dna repair factors in hiv infection reveals a role for base excision repair in hiv integration the dynamics of hmg proteinchromatin interactions in living cells retroviral cdna integration: stimulation by hmg i family proteins hiv-1 cdna integration: requirement of hmg i(y) protein for function of preintegration complexes in vitro activity of the human immunodeficiency virus type 1 cell cycle-dependent internal ribosomal entry site is modulated by ires trans-acting factors fuel feeds function: energy metabolism and the t-cell response human immunodeficiency virus type 1, human protein interaction database at ncbi patterns of hiv-1 protein interaction identify perturbed host-cellular subsystems the biological context of hiv-1 host interactions reveals subtle insights into a system hijack hivhost interactions: a map of viral perturbation of the host system cataloguing the hiv type 1 human protein interaction network retroviral proteomics and interactomes: intricate balances of cell survival and viral replication dual role of host cell factors in hiv-1 replication: restriction and enhancement of the viral cycle decoding the multifaceted hiv-1 virus-host interactome functions of tat: the versatile protein of human immunodeficiency virus type 1 chromatin dynamics associated with hiv-1 tat-activated transcription modifications in host cell cytoskeleton structure and function mediated by intracellular hiv-1 tat protein are greatly dependent on the second coding exon pathway analysis in hek 293t cells overexpressing hiv-1 tat and nucleocapsid expression profiles and pathway analysis in hek 293 t cells overexpressing hiv-1 tat and nucleocapsid using cdna microarray proliferative activity of extracellular hiv-1 tat protein in human epithelial cells: expression profile of pathogenetically relevant genes proteomics analysis of human astrocytes expressing the hiv protein tat hiv-1 tat reprograms immature dendritic cells to express chemoattractants for activated t cells and macrophages gene expression profile of hiv-1 tat expressing cells: a close interplay between proliferative and differentiation signals modifications in the human t cell proteome induced by intracellular hiv-1 tat protein expression cell-type-specific proteome and interactome: using hiv-1 tat as a test case differentially expressed genes in hiv-1 tat-expressing cd4(+) t-cell line hiv-1 tat assembles a multifunctional transcription elongation complex and stably associates with the 7sk snrnp the histone chaperone protein nucleosome assembly protein-1 (hnap-1) binds hiv-1 tat and promotes viral transcription genome-wide binding map of the hiv-1 tat protein to the human genome jurkat t cells and development of the t-cell receptor signalling paradigm global survey of human t leukemic cells by integrating proteomics and transcriptomics profiling a genome-wide short hairpin rna screening of jurkat t-cells for human proteins contributing to productive hiv-1 replication stochastic gene expression in a lentiviral positive-feedback loop: hiv-1 tat fluctuations drive phenotypic diversity dedd and dedd2 associate with caspase-8/10 and signal cell death chaperones and multitasking proteins in the nucleolus: networking together for survival? evidence for an alternative glycolytic pathway in rapidly proliferating cells glutamine uptake and metabolism are coordinately regulated by erk/mapk during t lymphocyte activation on the origin of cancer cells understanding the warburg effect: the metabolic requirements of cell proliferation characterization of human phosphoserine aminotransferase involved in the phosphorylated pathway of lserine biosynthesis proteomics profiling of nuclear proteins for kidney fibroblasts suggests hypoxia, meiosis, and cancer may meet in the nucleus glycolytic enzymes as dna binding proteins emerging roles of pkm2 in cell metabolism and cancer progression viral modulation of t-cell receptor signaling proteomic profiling of the human t-cell nucleolus the role of unintegrated dna in hiv infection the hiv tat protein affects processing of ribosomal rna precursor an efficient tandem affinity purification procedure for interaction proteomics in mammalian cells use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification andromeda: a peptide search engine integrated into the maxquant environment proteomecommons.org collaborative annotation and project management resource integrated with the tranche repository the authors wish to acknowledge access to and use of the ucd conway mass spectrometry resource instrumentation at the mass spectrometry resource (msr), conway institute for biomolecular and biomedical research, university college dublin is gratefully acknowledged. the authors wish to acknowledge prof. dimitri scholz director of biological imaging, conway institute, ucd, for his assistance with image acquisition and analysis. key: cord-354547-eomm1sl5 authors: wang, jibin; fang, shouguo; xiao, han; chen, bo; tam, james p.; liu, ding xiang title: interaction of the coronavirus infectious bronchitis virus membrane protein with β-actin and its implication in virion assembly and budding date: 2009-03-16 journal: plos one doi: 10.1371/journal.pone.0004908 sha: doc_id: 354547 cord_uid: eomm1sl5 coronavirus m protein is an essential component of virion and plays pivotal roles in virion assembly, budding and maturation. the m protein is integrated into the viral envelope with three transmembrane domains flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. in this study, we showed co-purification of the m protein from coronavirus infectious bronchitis virus (ibv) with actin. to understand the cellular factors that may be involved in virion assembly, budding and maturation processes, ibv m was used as the bait in a yeast two-hybrid screen, resulting in the identification of β-actin as a potentially interacting partner. this interaction was subsequently confirmed by coimmunoprecipitation and immunofluorescence microscopy in mammalian cells, and mutation of amino acids a159 and k160 on the m protein abolished the interaction. introduction of the a159-k160 mutation into an infectious ibv clone system blocks the infectivity of the clone, although viral rna replication and subgenomic mrna transcription were actively detected. disruption of actin filaments with cell-permeable agent cytochalasin d at early stages of the infection cycle led to the detection of viral protein synthesis in infected cells but not release of virus particles to the cultured media. however, the same treatment at late stages of the infection cycle did not affect the release of virus particles to the media, suggesting that disruption of the actin filaments might block virion assembly and budding, but not release of the virus particles. this study reveals an essential function of actin in the replication cycle of coronavirus. enveloped viruses acquire their envelope by budding from the host cell. in this process, viral envelope proteins gather at a special membranous structure and cooperate with other viral components to induce budding [1] . for example, some viruses including human immunodeficiency virus bud from the plasma membrane and release the virion from host cells by pinching-off. others are budding at intracellular membranes [2, 3] . in this way, virions are wrapped within intracellular membrane-bound compartments, such as the endoplasmic reticulum (er) and golgi apparatus, and the newly budded viruses exit the cell by using the cellular secretory pathway [2] . however, the detailed mechanisms of viral assembly and budding, especially the host factors that are involved in these processes, are yet to be revealed for many viruses. in this study, we report that interaction between coronavirus membrane protein (m) and actin with functional implication in facilitating virion assembly and budding. coronavirus is an enveloped virus with a large, positivestranded rna genome of about 27 to 31 kilobases in length. the avian coronavirus infectious bronchitis virus (ibv) belongs to the third group of coronaviruses genus. like many other coronavriuses, ibv virion is built from four structural proteins, including the nucleocapsid (n) protein with which the genomic rna is packed, the spike (s) protein that forms the prominent coronavirus spikes, the m protein which is the most abundant component of coronavirus, and the envelope (e) protein which is a minor but yet critical component in virion assembly [4] . some group ii coronaviruses also encode an additional structural protein, the hemagglutinin-esterase (he). coronaviruses are known to assemble and bud at membranes of the intermediate compartment (ic) , locating between the er and golgi complex [5] . the m protein is a type iii membrane protein and a key player in coronavirus assembly. it spans the membrane bilayer three times, leaving a short amino-terminal domain on the virion exterior surface (or exposed luminally in intracellular membranes) and a large carboxy-terminal tail in the virion interior (or in the plasma) [6] . lateral interactions between m proteins are thought to mediate the formation of the virion envelope [7] . when expressed alone, m protein accumulates in the golgi complex in the form of homomultimeric complexes [8, 9] . however, in combination with the e protein, m is retained in the budding compartment and incorporated into virus-like particles (vlps) with similarity in size and shape to authentic virions, demonstrating that the m and e proteins are the minimal requirements for envelope formation for most coronaviruses, [10] . the m protein appears to interact with s and he proteins, and the s-m-he protein complexes can be detected in cells infected with the bovine coronavirus [6] . the m protein was also shown to interact with the mouse hepatitis virus (mhv) nucleocapsid consisting of the genomic-size mrna 1 and n protein in a pre-golgi compartment, probably at the er membrane. it may interact directly with the genomic rna through the packaging signal, initiating the m-nucleocapsid interaction [11] . there is also a detectably direct interaction between m and n proteins in the nucleocapsid, which may further stabilize the m-genomic rna interaction [11] . actin is the most abundant protein in a typical eukaryotic cell, accounting for about 15% in some cell types [12] . the protein is highly conserved, differing by no more than 5% between species as diverse as algae and humans. it polymerizes in a helical fashion to form actin filaments (or microfilaments) that form the cytoskeleton, a three-dimensional network inside a eukaryotic cell. actin filaments provide mechanical support for the cell, determine the cell shape, enable cell movements (through lamellipodia, filopodia, or pseudopodia), and participate in certain cell junctions, in cytoplasmic streaming and in cell contraction during cytokinesis [13] . in the present study, actin was shown to be co-purified with the ibv particles and was identified as a potential interacting protein of the ibv m protein. the interaction was subsequently confirmed by coimmunoprecipitation and immunofluorescent staining. mutation of amino acids a159 and k160 in the m protein abolished the interaction. introduction of the a159-k160 mutation into an infectious ibv clone system showed no infectious virus could be recovered, although viral rna replication and subgenomic mrna transcription were detected. furthermore, treatment of the infected cells with cell-permeable agent cytochalasin d at early, but not late, stages of the replication cycle showed no release of the virion to the cultured media, suggesting that disruption of actin filaments might block virion assembly and budding. yeast two-hybrid screening yeast two-hybrid screening was performed with the pretransformed matchmaker human bone marrow cdna library (clontech) according to the instructions of the manufacturer. in brief, the bait plasmid was transformed into yeast strain ah109, and transformants containing the bait plasmid were mated with the pretransformed cdna library. candidates were initially selected on sd-ade/-his/-leu/-trp plates, and plasmid dna was isolated from positive clones and sequenced according to the instructions of the manufacturer. h1299 and vero cells were cultured in rpmi-1640 and complete dulbecco's modified eagle's medium (invitrogen), respectively, supplemented with 10% new born calf serum (sterile) and 1% penicillin/streptomycin (invitrogen) and maintained at 37uc in humidified 5% co 2. constructs containing plasmid dna under the control of a t7 promoter were transiently expressed in mammalian cells using a vaccinia virus-t7 system. briefly, 90% monolayers of h1299 cells were infected with 10 plaque forming units (pfu)/cell of the recombinant vaccinia virus (vtf-3), which express the t7 rna polymerase gene, for 2 hours at 37uc prior to transfection. the plasmid dna was transfected into vtf-3 infected cells using lipofectamine 2000 reagent according to the instructions of the manufacturer (invitrogen). hela cells transfected with appropriate plasmids were lysed with tntg lysis buffer (30 mm tris-hcl ph 7.4, 150 mm nacl, 1% np40, and 10% glycerol) in the presence of 16protease inhibitor mixture (sigma) at 24 hours post-transfection. total cell lysates were immunoprecipitated with appropriate antibodies for 2 hours at 4uc and further incubated for 2 hours at 4uc after adding buffer-balanced protein a agarose beads. the beads were washed three times and subjected to electrophoresis on sds-12% polyacrylamide gel. ibv m protein was transiently expressed in h1299 cells grown on 4-well chamber slides (iwaki). after rinsing with phosphatebuffered saline (pbs), cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.2% triton x-100, followed by incubating with specific antibodies diluted in fluorescence dilution buffer (pbs with 5% newborn calf serum) at room temperature for 2 hours. cells were then washed with pbs, incubated with fitc-conjugated anti-rabbit secondary antibodies (dako) in fluorescence dilution buffer at 4uc for 1 hour and with alexa fluor 488 phalloidin (molecular probe) at rt for 20 minutes before mounting. confocal microscopy was performed on a zeiss microscope. samples were lysed with 26sds loading buffer and subjected to 10% sds-page. proteins were transferred to pvdf membrane (bio-rad) by using a semi-dry transfer cell (bio-rad, trans-blot sd), and blocked overnight at 4uc with 10% nonfat milk in pbs-t. the membranes were probed with specific primary antibodies followed by anti-mouse or anti-rabbit secondary antibodies conjugated with harderadish peroxidase (sigma). membranebound antibodies were detected with the enhanced chemiluminescence (ecl) detection reagents (amersham, uk). the virus was layered onto 20% tne-buffered sucrose solution (tne buffer: 50 mm tris-hcl, ph 7.4, 100 mm nacl, 1 mm edta) and centrifuged at 175,0006g for 3 hours at 4uc (cushion). the resulting virus pellets were resuspended in tne buffer, layered onto 10-50% linear sucrose gradient prepared with tne buffer, and centrifuged at 175,0006g for 18 hours at 4uc. aliquots of fractions starting from the top of the gradient were analyzed by sds-page. in vitro assembly and transcription of full-length cdna clones were carried as previously described [14] . briefly, plasmids were digested with either bsmbi (fragment a) or bsai (fragments b, c, d and e). bands corresponding to each of the fragments were cut from the gels and purified with qiaquick gel extraction kit (qiagen inc.). all the fragments were ligated with t4 dna ligase at 16uc overnight. the final ligation products were extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol, and detected by electrophoresis on 0.8% agarose gels. full-length transcripts and n transcripts (using a linearized pkt0-ibvn containing ibv n gene and the 39-utr region as templates) were generated in vitro using the mmessage mmachine t7 kit (ambion, austin, tex) according to the manufacturer's instructions. vero cells were grown to 90% confluence, trypsinized, washed twice with cold pbs, and resuspended in pbs (14) . rna transcripts were added to 400 ml of vero cell suspension in an electroporation cuvette, and electroporated with one pulse at 450 v, 50 mf with a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in 1% fbscontaining dmem in a 60 mm dish and further cultured in dmem without fbs. reverse transcription, rt-pcr, and real-time pcr viral rna was reverse-transcribed to cdna using superscript iii (invitrogen) with modification to the protocol as follows: random hexamers (300 ng) and total rna (5 mg) were incubated for 10 minutes at 70uc. the remaining reagents were added according to the manufacturer's recommendation and the reaction was incubated at 55uc for 1 hour followed by 20 minutes at 70uc to inactivate the rt. for rt-pcr, a forward primer in the leader sequence and a reverse primer were used to generate a product by pcr. quantitative real time rt-pcr was conducted using smart cycler ii (cepheid) with sybr green (cepheid) to detect subgenomic cdna with primers (7.5 pm) optimized to detect a product spanning the leader sequence to the 59 end of m gene or genomic cdna with primers (7.5 pm). the cdna from the rt reaction of each virus was diluted 1:10, and 1 ml was used for each pcr, with a total reaction volume of 25 ml. m gene was cloned by digesting pibvm with ncoi and ecori, and inserted into ecori/ncoi digested pgbkt7 to generate pgbkt7-m. the pcr fragment for actin was amplified by using primers 59-cgcggatccatggatgatgatatcgccgcg-39 and 59-ccgctcgaggaagcatttgcggyggacgat-39. the pcr fragment was digested with bamhi and xhoi and ligated into bamhi and xhoi digested pxj-myc to generate pxj-myc-actin. the deletion constructs md1, md2, md3, md4 and md5 were made by two rounds of pcr as described previously [15] . the primer used for md1 is 59-aactgcagttaagc-aagccactgaccctc-39. the primers used for md2 are 59-tcttttgtaggttataagtgtgaaccagac-39 and 59-gtctggttcacactta taacctacaaaaga-39. the primer for md3 is 59-aactgcagccgcttt ggtcaccag-39. the primers for md4 are 59-tgtgagggtccagac-cacttg-39 and 59-caagtggtctggaccctcaca-3. the primers for md5 are 59-ggtcagtggc tttgtgaa-ccagac-39 and 59-gtctggttcacaaagccactgacc-3. all constructs were confirmed by automated nucleotide sequencing. in a previous study, it was observed that a cellular protein with migration properties on sds-page similar to actin was consistently co-purified with highly purified ibv particles [16] . however, as no suitable antibodies against actin were readily available at the time, the identity of this protein was not established. this issue was revisited in this study. confluent monolayers of vero cells were infected with ibv at a multiplicity of infection of approximately 2 pfu/cell. the supernatants were collected at 18 hours post-infection and centrifuged at 4,000 rpm for 20 minutes to remove cell debris. the virus particles were spun down by ultracentrifugation through a 2 ml sucrose cushion (20%), and purified using a 10-50% sucrose gradient. following ultracentrifugation, 11 fractions were collected from top to bottom, and the presence of viral proteins was checked by western blot with anti-n antibodies, showing that the ibv n protein was detected in fractions 7-11 with majority of the protein located in fractions 8-10 ( fig. 1, top panel) . analysis of the same fractions by western blot with anti-actin antibodies showed that actin appeared in the same fractions as n protein, with the majority of the protein detected in fractions 8-10 ( fig. 1 , middle panel). fractionation of total lysates from cells without ibv infection under the same conditions showed that actin was mainly detected in fractions 3-4 ( fig. 1, bottom panel) . these results demonstrate that actin could indeed be co-purified with the virus particles. however, it is currently uncertain if actin could be incorporated into the virions. in an attempt to search for ibv proteins that may interact with b-actin, ibv structural and nonstructural proteins were used as baits to screen a human cdna library in yeast two-hybrid screening. among all the ibv proteins used, only the c-terminal cytoplamsic portion of the ibv m protein was able to interact with b-actin. to confirm the interaction by co-immunoprecipitation, the fulllength cdna for b-actin was amplified by rt-pcr from hela cells, cloned into an expression vector with a c-myc tag at its nterminus (myc-actin), and co-transfected into hela cells with the ibv m. analysis of cells expressing the myc-tagged actin either on its own or together with the m protein by western blot with anti-myc monoclonal antibody showed the detection of the myctagged actin (fig. 2, lanes 1 and 3) . similarly, analysis of cells expressing the m protein either on its own or together with the myc-tagged actin by western blot with anti-m polyclonal antibodies showed the detection of the full-length glycosylated (two upper bands) and unglycosylated (25 kda) forms of the m protein (fig. 2, lanes 5 and 6) . the same cell lysates were then subjected to immunoprecipitation with anti-myc antibody. western blot analysis of the precipitates with the same anti-myc antibody showed the detection of the myc-tagged actin expressed either on its own or together with the m protein (fig. 2, lanes 7 and 9) . western blot analysis of the same precipitates with anti-m antibodies showed the detection of the m protein only in cells coexpressing the two proteins (fig. 2, lane 12) . no detection of the m protein was found in cells expressing either the m protein or mycactin alone (fig. 2, lanes 10 and 11) . these results confirm that the ibv m protein could indeed interact with actin. to map the region in the m protein responsible for the interaction with actin by yeast two-hybrid screening, five deletion and one mutation constructs were made (fig. 3a) . in figure 3a , md1 contains the amino acid sequence from 104 to 159 (with deletion of amino acids 160 to 225); md2 contains the deletion of amino acids 104-159; md3 contains the amino acid sequence from 104 to 192 (with deletion of amino acids 193 to 225); md4 contains the deletion of amino acids 155-162; and md5 contains the deletion of amino acids 159-160 (fig. 3a) . the mutant construct mm1 contains the mutation of amino acids 159 and 160 from alanine and lysine to proline and glutamic acid, respectively (fig. 3a) . introduction of individual deletion and mutant constructs into the yeast stain ah109 together with pact-actin showed growth of wild type and md3 constructs on sd-trp/-leu/-his/ -ade selective plates (fig. 3b) . however, none of the other deletion and mutant constructs could grow on the same plate (fig. 3b) . western blot analysis showed similar expression levels of wild type and mutant constructs in yeast (data not shown). as md5 contains the minimal deletion of two amino acids, this study maps the actinbinding site on the m protein to the region containing amino acids a159 and k160. co-immunoprecipitation was then carried out by expressing the myc-tag wild type m (c-terminal domain from amino acid 104 to 225, myc-m) and myc-mm1 in cells. western blot analysis with anti-myc monoclonal antibody showed the expression of myc-m and myc-mm1 at a similar level (fig. 3c, upper panel, lanes 1 and 2) . the same cell lysates were then subjected to immunoprecipitation with anti-actin antibody, and western blot analysis of the precipitates with anti-myc antibody showed the presence of myc-m but not myc-mm1 (fig. 3c, lower panel, lanes 1 and 2) . the detection of myc-m in the immunoprecipitates was greatly enhanced by co-expression of myc-m and actin (fig. 3b, lane 4) . colocalization of the m protein with actin in cells treated with cytochalasin d as a type iii membrane protein with three transmembrane domains, the ibv m protein is mainly localized to the golgi apparatus in virus-infected cells and in cells expressing the m protein [8] . cytochalasin d is a cell-permeable fungal toxin. it can bind to the barbed end of actin filaments and inhibit both the association and dissociation of actin subunits, resulting in the disruption of actin filaments and inhibition of actin polymerization. to test the subcellular localization of the m protein in cells treated with cytochalasin d, h1299 cells were transfected with m construct and 12.5 mg/ml of cytochalasin d was added to the cells at 4 hours post-transfection. cells were permeabilized with 0.2% triton x-100 and stained with anti-m antibodies at 48 hours posttransfection. the same cells were also stained with alexa fluor 488 phalloidin, a specific dye for actin (molecular probe). staining of cells treated with dmso and alexa fluor 488 phalloidin showed a normal three-dimensional network (fig. 4, upper panels) . phalloidin showed that the regular cell actin filaments were destroyed (fig. 4, lower panels) , displaying random distribution of actin dots in cells without the m protein expression. in cells expressing the m protein, both m protein and actin were found mainly in the golgi area with fairly well overlapping of the two staining patterns (fig. 4, lower panels) . effects of deletion and mutation of amino acid residues a159 and k160 on the replication, budding and release of ibv two approaches were used to test the effect of deletion and mutation of amino acid residues a159 and k160 in the m protein on the replication, budding and release of ibv. first, the a159-k160 deletion and mutation were introduced into a full-length ibv infectious clone, and the effect of this minimal deletion and mutation on the replication of viral rna and the recovery of infectious ibv was analyzed. introduction of full-length transcripts derived from wild type (wt), the a159-k160 deletion construct (md5) and a159-p/k160-e mutation construct (mm1) into vero cells by electroporation showed efficient recovery of infectious ibv from cells transfected with wild type transcripts. however, it was consistently observed that no infectious virus could be recovered from cells transfected with either md5 or mm1 transcripts. as no infectious virus was recovered from cells transfected with md5 and mm1 transcripts, rt-pcr amplification of the negative strand rna was performed to check if rna replication occurred in the transfected cells. the primer pair (59-14931 gcttatc-cact agtacatc 14949 -39 and 59-15578 cttctcgcacttc-tgcactagca 15600 -39) was chosen to amplify the negative strand ibv sequence from nucleotides 14931 to 15600 by the rt-pcr. if replication of viral rna occurred, a 670 bp pcr fragment would be expected. as shown in fig. 5a , the 670 bp rt-pcr fragments were obtained from cells transfected with both wild type and mutant transcripts at 24 and 72 hours posttransfection, respectively (upper panel, lanes 2-7). interestingly, the amounts of the fragment detected from cells transfected with md5 and mm1 transcripts were increased at 72 hours posttransfection (fig. 5a , upper panel, lanes 3-4 and 6-7), demonstrating the replication of the transfected mutant transcripts. no rt-pcr fragment was detected in cells without transfection (fig. 5a, upper panel, lane 1) . rt-pcr amplification of subgenomic mrnas was then carried out to check if subgenomic mrna synthesis could occur in cells transfected with the mutant transcripts at 24 and 72 hours postelectroporation. the forward primer (59-acttaagataga-tatta a-39) used in this reaction corresponds to the leader sequence from nucleotides 26-46 in the genomic rna and the downstream primer (59-ctctggatccaataacctac-39) covers the ibv sequence from nucleotides 24784 to 24803. if transcription of subgenomic mrnas occurs, a 415 bp pcr product corresponding to the 59-terminal region of the subgenomic mrna4 would be detected. as shown in fig. 5a , a dominant 415 bp was observed in cells electroporated with wild type full-length transcripts at two time points (lower panel, lanes 2 and 5). in cells transfected with md5 and mm1 transcripts, a much weaker band was detected in cells transfected with the two mutant transcripts at 24 hours post-transfection (fig. 5a, lower panel, lanes 3-4) . similarly to the results described above, the amounts of the subgenomic rna 4 fragment detected from cells transfected with md5 and mm1 transcripts were increased approximately 3 to 5 fold, respectively, at 72 hours posttransfection based on real time pcr assay (fig. 5a, lower panel, lanes 3-4 and 6-7) . as a negative control, no detection of the amplified fragments was obtained from a mixture of rna templates containing the in vitro transcribed rna and total rna extracted from mock infected cells (fig. 5a, lower panel, lane 1) . in the second approach, plasmids containing wild type m (pibvm) and the a159-k160 deletion m sequences (pmd5) were transfected into h1299 cells using the vaccinia/t7 recombinant antibodies, respectively (fig. 5b) . in cells transfected with both wild type and md5 constructs with or without ibv infection, detection of similar amounts of m protein was observed (fig. 5b, top two panels, lanes 1 and 2) , suggesting that both constructs were expressed at similar efficiencies. however, much more n protein was detected in the infected cells transfected with wild type m construct than that in cells transfected with the md5 mutant (fig. 5b, middle panel, lanes 1 and 2) . as a loading control, similar amounts of actin were detected in cells transfected with both constructs (fig. 5b, bottom panel) . these results confirm that expression of the a159-k160 deletion m protein significantly reduces the infectivity of ibv, suggesting that expression of this mutant protein could inhibit the production of infectious virus in a dominant-negative manner. to rule out the possibility that the deletion and mutation may render detrimental effect on the overall structure of the m protein and its interaction with other viral proteins that are essential for virion assembly, interaction of the mm1 mutant with the e protein was tested by co-immunoprecipitation. as shown in fig. 5c , western blot analysis with anti-ibv e protein antibodies showed the presence of e protein in cells expressing the protein on its own or together with wild type or mm1 mutant protein (top panel). immunoprecipitation of the same lysates with anti-m antibodies and subsequent analysis of the precipitates by western blot with anti-m antibodies led to the detection of wild type or the mm1 mutant proteins in cells expressing m forms either alone or together with the e protein (fig. 5c, middle panel) . western blot analysis of the same precipitates with anti-ibv e antibodies, however, showed the presence of e protein only if it was coexpressed with either wild type or the mm1 mutant protein (fig. 5c, bottom panel) . these results confirm that the a159-p/ k160-e mutations did not affect the interaction between ibv m and e proteins. to define more precisely the stage of the viral replication cycle that is facilitated by the interaction between m protein and actin, the effects of disrupting actin filaments by cytochalasin d on the replication, budding and release of ibv were tested by detailed time-course experiments. confluent monolayers of vero cells in six-well plates were infected with ibv at a multiplicity of infection of approximately 3 pfu/cell. at 0, 4, 8, 12 and 16 hours postinfection, either 12.5 mg/ml of cytochalasin d or equal volume of dmso was added to the cells. the supernatants and cell pellets were separately harvested at 24 hours post-infection and the presence of ibv n protein was checked by western blot with anti-n antibodies. typical n protein profiles, including the full-length and posttranslationally modified forms of the protein, were detected in the total cell lysates (fig. 6, top panel) . in cells treated with cytochalasin d, slightly less amounts of the n protein were detected when cytochalasin d was added to the cells at 0, 4, 8 and 12 hours post-infection, respectively (fig. 6, top panel, lanes 1-4) . no obvious difference in the expression of n protein was seen when cytochalasin d was added at 16 hours post-infection (fig. 6 , top panel, lane 5). western blot analysis of actin was included as a loading control (fig 6, middle panel) . analysis of the n protein in the clarified supernatants showed the detection of a single n protein species that represents the n protein incorporated into the virion particles in cells treated with dmso alone (fig. 6, bottom panel, lanes 6-10) . in supernatants collected from cells treated with cytochalasin d, much reduced amounts of the n protein were detected in cells treated with the reagent at 12 and 16 hours postinfection, respectively (fig. 6, bottom panel, lanes 4 and 5) . no n protein was detected in supernatants harvested from cells treated with cytochalasin d at 0, 4 and 8 hour post-infection, respectively (fig. 6, bottom panel, lanes 1-3) . coronavirus m protein plays essential roles in virion assembly and budding [18, 19] . the protein itself cannot bud. however, with the help of e protein, vlps may be formed at the ic membranes. as coronaviruses do not contain a matrix protein that underlines the membrane, the m protein may serve as a matrixlike protein. it is proposed that the formation of coronavirus envelope is dominated by lateral interaction between m molecules that form a two-dimensional lattice in intracellular membranes [20] . the m-m interaction is essential but not sufficient for coronavirus envelope assembly. free energy is needed to generate and stabilize membrane curvature, suggesting that interaction of m protein with host proteins would be a must [21] . to support this speculation, the results present in this study demonstrate that interaction of the ibv m protein with b-actin is essential for virion assembly and budding. the interacting region in the m protein was pinpointed by deletion and mutation studies. as md3 is able to interact with actin, but md1 and md2 could not, it was reasoned that the domain responsible for this interaction may be located in the region covering amino acids 159-160. this region was chosen for further deletion and mutational analysis, confirming that it is indeed involved in the interaction with actin. however, the two amino acids are not conserved in other coronavirus m proteins. at present, we do not know whether interaction between coronavirus m protein and actin is a general phenomenon for all coronaviruses, or is just ibv-specific. available evidence suggests that the host cytoskeleton, especially actin, is involved in the budding process of several animal viruses [22] . actin has been identified in many enveloped viruses, such as rous sarcoma virus, mouse mammary tumor virus, sendai virus and measles virus. in the case of measles virus, actin was originally thought as a cellular contaminant, but was later demonstrated to be an internal component of the virus [23] [24] [25] . actin was found in the purified paramyxovirus particles and actin filaments were seen in association with the budding virions by electron microscopy [24] [25] [26] . polymerized actin was also found in hiv preparations [27, 28] . the functional implication for the incorporation of actin into these virus particles remains unclear. one possibility is that actin polymerization serves as an additional force for membrane bending during budding [1, 29] . as an illustrative example, actin was shown to control the movement of measles virus envelope proteins on the surface of infected cells [30] . interaction of actin with viral components was also reported in several other viral systems. one example is the interaction of the nuclocapsids of murine mammary tumor virus with actin, which was required for extruding the virus particles from virus-infected cells [31] . the m protein of newcastle disease virus could interact with actin in vitro [1, 32] . the sars-cov n protein could aggregate elongation factor 1a through direct interaction, leading to the inhibition of protein translation and cytokinesis by blocking f-actin bundling [33] . it was also reported that the sars-cov n protein could induce apoptosis and actin reorganization in mammalian cells under stress conditions [34] . the results shown in this study demonstrate that actin could be co-purified with ibv virions and that the ibv m protein is associated with actin, suggesting that actin is likely to be incorporated into ibv virions through its interaction with the m protein. however, more conclusive data, such as immunogold labeling of highly purified virions, are currently lacking. it is interesting that ibv replication cycle was disturbed in cells treated with cytochalasin d. cytochalsin d could prevent actin from polymerization, leading to malfunction of actin. when actin was disrupted, much less ibv virions were released from the cells into the culture medium, demonstrating that actin plays a very important role in the ibv replication cycle. two possible mechanisms were considered. first, actin may be involved in the formation of the m lattice that may promote and stabilize its curving, resulting in the enhancement of some late processes, such as virion budding and release. second, the mhv m protein was shown to be recycled from the golgi complex back to early compartments, such as the er and ic, which are functionally important in coronavirus infected cells [21] . therefore, interaction of actin with m protein may help the retrograde transport of m protein from golgi apparatus to some early compartments and provide more m protein for virion assembly and budding. the actin-binding proteins are grouped into 60 distinct classes based on primary structures [35] . the sequence of the actinbinding sites ranges from 10-30 residues, which shows no obvious homology among different classes [36] . for example, aldolase, a glycolytic enzyme, binds actin filaments to concentrate enzyme and substrate [37] . the sequence 32-adestgsiakrlqsig-tente-52 of aldolase has been identified as the actin-binding motif [38] . furthermore, carcinoembryonic cell adhesion molecule 1 (ceacam1) was found to bind f-actin. the actin binding site is flhfgktgssgplq, which is not similar to other existing actin-binding sequences [39] . coronavirus m protein is an essential and predominant component of the virions. it plays pivotal roles in virion assembly and budding by interaction with other viral components. firstly, the monomers of m could interact with each other particularly via the transmembrane domains [21] . besides, the m-n interaction region was narrowed to the 16 residues adjacent to the carboxy terminus of tgev m protein [40] . however, the specific amino acids on m protein responsible for m-s and m-e interactions are still enigmatic. in the present study, deletion or mutation of amino acids a159 and k160, which are essential for the interaction of m protein with actin, is detrimental to the recovery of ibv from an infectious ibv clone due to a defect in late stages of the ibv replication cycle. although, the deletion or mutation of amino acids a159 and k160 could also interact with another structural protein e, we cannot exclude the possibility that this region is essential for maintaining the integrity of the m protein and involved in the interactions with other viral components. nevertheless, it lends support to the conclusion that interaction of the ibv m protein with actin is essential for the completion of the ibv infection cycle. more evidence is derived from the study using cytochalasin d. similar amount of viral proteins were detected in the total cell lysates but were not detected in supernatants when cytochalasin d was added early in the viral replication cycle suggests that actin filaments are essential for some early events, such as virion assembly and budding, during the assembly and maturation processes of ibv. as ibv proteins can be efficiently detected in the supernatants when cytochalasin d was added at 12 and 16 hours post-infection, respectively, it suggests that actin filaments are unlikely involved in the release of the ibv particles. instead, our data support that actin filaments may be involved in the virion assembly and budding process of the coronavirus replication cycles. as coronavirus m protein could bind to the viral genomic rna through packaging signal [41] and rna replication in the cytoplasm would rely on the support of actin [42] , the interaction of m protein with actin may serve as a platform to promote the binding of m protein to viral rna as well as viral rna replication. alternatively, coronavirus m protein could bind to the n protein-rna complex to stabilize the nucleocapsid [11] and extra energy would be needed in this process [21] . the interaction of m protein with actin may serve as a power station to provide energy for completion of the viral replication cycle. virus maturation by budding cell biology of viruses that assemble along the biosynthetic pathway cargos and genes: insights into vesicular transport from inherited human disease coronavirus particle assembly: primary structure requirements of the membrane protein characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step the coronavirus membrane glycoprotein protein interactions during coronavirus assembly coronavirus m proteins accumulate in the golgi complex beyond the site of virion 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and contributes to core stability nucleocapsid-independent specific viral rna packaging via viral envelope protein and viral rna signal cellular factors in the transcription and replication of viral rna genomes: a parallel to dna-dependent rna transcription key: cord-347710-ff64y6ef authors: wan, qianya; song, dan; li, huangcan; he, ming-liang title: stress proteins: the biological functions in virus infection, present and challenges for target-based antiviral drug development date: 2020-07-13 journal: signal transduct target ther doi: 10.1038/s41392-020-00233-4 sha: doc_id: 347710 cord_uid: ff64y6ef stress proteins (sps) including heat-shock proteins (hsps), rna chaperones, and er associated stress proteins are molecular chaperones essential for cellular homeostasis. the major functions of hsps include chaperoning misfolded or unfolded polypeptides, protecting cells from toxic stress, and presenting immune and inflammatory cytokines. regarded as a double-edged sword, hsps also cooperate with numerous viruses and cancer cells to promote their survival. rna chaperones are a group of heterogeneous nuclear ribonucleoproteins (hnrnps), which are essential factors for manipulating both the functions and metabolisms of pre-mrnas/hnrnas transcribed by rna polymerase ii. hnrnps involve in a large number of cellular processes, including chromatin remodelling, transcription regulation, rnp assembly and stabilization, rna export, virus replication, histone-like nucleoid structuring, and even intracellular immunity. dysregulation of stress proteins is associated with many human diseases including human cancer, cardiovascular diseases, neurodegenerative diseases (e.g., parkinson’s diseases, alzheimer disease), stroke and infectious diseases. in this review, we summarized the biologic function of stress proteins, and current progress on their mechanisms related to virus reproduction and diseases caused by virus infections. as sps also attract a great interest as potential antiviral targets (e.g., covid-19), we also discuss the present progress and challenges in this area of hsp-based drug development, as well as with compounds already under clinical evaluation. stress proteins (sps) are a diverse group of proteins that are synthesized at increased levels when cells are exposed to either intracellular or extracellular stressful stimuli. they exhibit protective effects against stresses. stress proteins include heat shock proteins (hsps), rna chaperone protein (rnps), and proteins mainly function in the endoplasmic reticulum (er): peptidyl-propyl isomerases, protein disulfide isomerases (pdis) and the lectin-binding chaperone system. 1 sps are ubiquitously expressed in all kind of cells, triggering signal cascades for neutralizing and eradicating the stresses occurring both intracellularly (e.g., pathogen invasion) and extracellularly (e.g., starvation, stimulation by cytokines/chemokines or hormones). responses triggered by sps can either activate pathways to promote cell survival or initiate cell death (i.e., apoptosis, necrosis, pyroptosis or autophagic cell death) for eliminating the damaged cells to protect a particular organ/ tissue under given conditions. it is widely noted that the dysregulation of stress proteins is associated with a variety of human diseases, including cardiovascular diseases, neurodegenerative diseases (e.g., parkinson's diseases, alzheimer disease), stroke, human cancers and infectious diseases. in this review, we focus on their functions and update findings involved in infectious diseases, particularly, the diseases caused by viral infections. heat shock proteins in 1962, an italian geneticist ritossa inadvertently elevated the incubation temperature of drosophila larvae and discovered an increased gene transcription of unknown proteins. he nominated these protein as hsps. 2 further studies have revealed a large number of hsps, which form a big family and are ubiquitously expressed in cells. based on the molecular weight, hsps are classified into different families, including hsp100s, hsp90s, hsp70s, hsp60s, hsp40s, and some small hsps . [3] [4] [5] hsps belong to the largest family of chaperones. the hsp expression is rapidly induced when cells meet physiological or environmental attacks such as starvation, high temperature, hypoxia or hyperoxia, pathogen invasion, malnutrition and exposure to chemicals or uv, etc. they form a network to promote or stabilize the correct folding of substrate protein to gain its functional/active conformation (fig. 1 ), although they may not associate with the substrate protein in the final structure. 3, 6 hsps are important factors in regulating cell survival, differentiation and cell death. accumulating evidence shows that some hsps participate in not only innate cellular immunity but also antigen presentation in adaptive immune response. 7, 8 hsps also serve as potential biomarkers for some diseases. it has been shown that the increase of hsp70 in plasma is associated with heart failure, 9 and the elevated hsp27 level in human peripheral blood mononuclear cells is related with coronary artery disease (cad). 10 hsp90s. hsp90, an abundant chaperone in all eukaryotic cells, controls a variety of critical signalling pathways in eukaryotic cells. 5, 6, 11 hsp90 is an atp-dependent chaperone with different isoforms, including 1) hsp90α (hsp90aa1, or hspc1), hsp90α-a2 (hspaa2, or hspc2) and hsp90β (hspab1, or hspc3) locate in cytoplasm. 2) glucose regulated protein grp94 (hspc4 or gp96) locates in the er. 3) trap1 (hspc5) locates in mitochondria. 12 among them, hsp90α and hsp90β account for the greatest proportion in humans. hsp90 contains three regions: an atpasedependent hydrolytic domain in the n-terminal region, a middle linker region, and a dimerization domain in the c-terminal region. 5 like other hsps, hsp90 binds non-native substrate peptide to prevent its aggregation and degradation. when hsp90 binds atp, a transient dimerization of the n-terminal domain allows the substrate peptide binding to hsp90. then the atp hydrolysis and energy release lead to a conformational change of the n-terminal domain that facilitates the correct folding of the substrate petite ( fig. 1) . 5, 6 besides, hsp90 is also involved in telomere maintenance, apoptosis, and cell cycle progression, etc. 6, 13 it is well known that hsp90 not only interacts and contributes to rna polymerase assembly and nuclear import of some (−) ssrna viruses (e.g., pb2 of influenza virus), but plays crucial roles in the folding process of viral capsid proteins and virion assemblies as well. 14 cochaperones of hsp90. cochaperones of hsp90 regulate hsp90 functions at many aspects. cdc37 (also called p50) delivers kinase to hsp90 and inhibits its atpase activity; carboxyl terminus of hsp70-interacting protein (chip) functions as e3 ubiquitin ligase; hsc70/hsp90-organizing protein (hop, also called sti1) inhibits dimerization of the n-terminal domain; and the activator of hsp90 atpase 1 (aha) and p23 participates the maturation of substrate peptides. 11, 15 hsp70s. hsp70 is a subfamily of hsps' superfamily with~70 kda molecular weight. it accounts for the majority of molecular chaperones in cells. 11 the members of the hsp70 family mainly include: (1) hsp72 (hspa1a), hsp70-2 (hspa2), hsp70b' (hspa6) and hsc70 (hspa8) are commonly located in the cytosol; (2) grp75 (hspa9) is located in mitochondria; (3) grp78 (hspa5) is associated with the er. 16 hsp70 consists of two domains: a 44-kda nucleotide-binding domain (nbd) which can be divided into four subdomains (ia, ib, iia, and iib) in the n-terminal region and a 28-kda substrate-binding domain (sbd) composed of c-terminal α-helical (sbdα) and n-terminal β-sheet (sbdβ) subdomains in the c-terminal region. 17, 18 as a critical component of cellular protein surveillance, the atp-dependent molecular chaperone protects cells from damage caused by stress and takes part in a number of folding processes, including folding of newly synthesized polypeptides, recognition and refolding of misfolded or aggregated proteins, solubilization or degradation of proteins, transporting proteins, assembly or disassembly of oligomeric protein complexes, and the regulation of certain natively folded proteins. 5, 13, 19, 20 the functions of hsp70 are not limited to host protein folding. its functions are considerable during viral infection. the members of hsp70s exhibit quite different roles in the course of virus life cycle. for example, hsp72, hsp70b' and hsc70 participate in the hcv viral entry, virion assembly and translation of the viral genome. grp78 in er is associated with the homeostasis of viral proteins and prevents the overload of viral proteins in host cells. in hepatocytes, the elevated grp78 stimulates innate immunity to restrict or eliminate hepatitis b virus (b) replication. 21 grp75 interacts with the ns5a protein of hcv in mitochondria. 22 accumulating evidence shows that hsp70 interacts with viral components of human cytomegalovirus, rabies virus, respiratory syncytial virus, human papillomavirus, herpes simplex virus. chaperone cycle of hsp70. the chaperone cycle is mediated by the n-terminal nbd, which regulates the binding of hsp70 with substrates through switch of two states. the first state is the atpbound state with low affinity for substrate binding, i.e., a high association and dissociation rate of the substrate peptides to the sbd. the second state is atp hydrolysis that switches to the adpbound and nucleotide-free state. at this state, the substrate exchange rates are low while the affinity to substrates is high. the chaperone activity of hsp70 mostly relies on atp hydrolysis. the basal atpase of hsp70 is normally low unless it is stimulated by the substrate peptide itself. it takes 20-30 min for a molecule of atp to be hydrolysed per hsp70 molecule at 30°c. as a result, it is necessary for some cochaperones to encounter with hsp70 atp to induce atp hydrolysis and help the increase of the affinity for substrate peptides. 19, 23 cochaperones of hsp70. the most crucial cochaperones of hsp70 are members of the j-domain proteins (jdps) family and nucleotide exchange factors (nefs) family. previous studies focused on the function of hsp70 machinery and led to the development of a "canonical model" for its mode of action. the model contains two steps. first, the unfolded peptide substrates bind jdps of the hsp40 family; then the substrates are delivered to hsp70 that stimulate the hsp70's atpase activity. simultaneously, jdps prevent the aggregation of unfolded proteins. second, nefs work as substrate release factors that assure the substrate to fold into the correct and active conformation. in this way, the cochaperones strongly facilitate the function of hsp70. therefore, hsp70 generally does not work individually but cooperates with cochaperones. 23, 24 hsp60s. hsp60s are large cylindrical oligomers with two back-toback rings. 19 the non-native proteins of the central cavity in each ring are folded into the native protein through an atp-dependent process. 25, 26 hsp60s are classified into two subfamilies. group i is mainly in prokaryotes, while group ii appears in eukaryotic cytosol and some archaea. 27, 28 the most well-studied one in group i is the groel-groes chaperonin system in the cytosol of bacteria. groel is an about 57 kda protein with two rings arranged back-to-back; and 7 subunits form a tetradecamer structure. groes is the cochaperone of groel. 11 the two rings-tetradecamer structure appears in two fig. 1 the general chaperone cycle of heat shock proteins. initially, unfolded client protein bound to the hsp70-hsp40 chaperones interacts with the hsp90. atp binding to hsp90 induces the client proteins transfer from hsp70 to hsp90. later, the conformation of hsp70-hsp40 chaperones will be released. finally, the hydrolysis of atp induces additional conformation changes leading to the client protein release forms include asymmetric (1 groel: 1 groes) and symmetric (1 groel: 2 groes) complexes, which are described as "bullet" 14, 29, 30 and "american football" 31 shaped respectively. the groel-groes chaperonin undergoes allosteric regulation dependent on atp and which completes the protein folding function. the polypeptide binds to the hydrophobic sites of one of the seven subunits of groel and changes conformation upon atp binging and hydrolysis. with the help of cochaperone groes, the polypeptide finishes its folding process. 32 in contrast to the groel-groes system, the mammalian homolog hsp60/hsp10 system is less studied. hsp60 is thought to be imported into the mitochondria and converted into its mature form with a molecular size of 58 kda. [33] [34] [35] hsp60 exhibits important roles in facilitating protein folding, transportation and proteostasis in mitochondria. 36, 37 group ii chaperonins include the archaeal thermosome and eukaryotic cct (chaperonin-containing tcp1, or called tric), which are oligomers with eight to nine subunits with molecular weight 57-61 kda in each ring. compared to group i, group ii chaperonins show different allosteric movements by atp binding. 11, 19 hsp60 family is known to participate in viral life cycle at various stages from viral attachment to the replication of the viral genome. hsp60s are essential for host cell immunity regulation. some viral proteins require hsp60 for its translocation within host cells. pb2 is a subunit of influenza a viral rna polymerase, which mostly locates in the nucleus but also appears in mitochondria. 38 when the virus infects the host cells, pb2 is responsible for maintaining the function of mitochondria. pb2 interacts with mitochondrial antiviral signaling protein (mavs) to downregulate intracellular immune response by decreasing the level of ifnβ so that the invading virus can easily escape from the defence of host cells. 39 hsp60 takes the role of transporting pb2 from cytosol to mitochondria in the host cells. besides, hsp60 shows great regulatory functions on innate immunity by inducing proinflammatory cytokine release, such as tnf-a, il-6, and il-1b, etc. 40 small heat shock proteins. small heat shock proteins (shsps) are a group of small proteins with a low molecular weight ranging from 15 to 40 kda. 4 there are 10 members in the shsp family and some are ubiquitous including hsp27 (hspb1), hspb5 (αb-crystallin, or αbc), hsp20 (hspb6), and hsp22 (hspb8), while the others are tissue-specific including hspb2 (myotonic dystrophy protein kinase, or mkbp), hspb3, hspb4 (αa-crystallin, or αac), hspb7 (cvhsp), hspb9, and hspb10 (sperm outer dense fiber protein, or odf). 41 shsps can exist as monomers, dimers or even large multimeric complexes in the cells. 42 the structure of shsps is different from the other hsps due to their less conserved sequences. the basic structure of shsps is a conserved α-crystallin domain (acd) flanked by two non-conserved domains including the n-terminal sequence (nts) and the c-terminal sequence (cts). among these domains, acd becomes the characteristic of different shsps. 43, 44 shsps play crucial roles in several physiological processes regarding stress tolerance, apoptosis, aging, and longevity. [45] [46] [47] [48] the phosphorylation together with the n-terminal wdpf motif helps shsps to form homo-or hetero-oligomers. 49, 50 the oligomerization is the hallmark of shsps for supporting their quite different activities. phosphorylation favors small oligomer formation while dephosphorylation provokes a shift toward large oligomer formation. 51 oligomerization dynamics is crucial for chaperone activity because it gives rise to the possibility to format different homo-and hetero-oligomers, each with different binding properties to chaperone a broad range of substrates. 41, 52 for instance, the phosphorylated species are required for actin dynamics. small phosphorylated dimers/tetramers bind f-actin to regulate actin polymerization. 53 among different shsps, hsp27 has been broadly studied. hsp27 exists in all tissues though it is known to mainly express in cardiac, skeletal and smooth muscles. 54 its importance has been demonstrated in cell differentiation, cell survival, cellular innate immunity, viral protein translation, and intracellular virus transport, etc. [55] [56] [57] same as the other shsps, hsp27 shares a highly conserved α-crystallin domain. 41, 58, 59 hsp27 is capable of oligomerization and phosphorylation. there are three serine residues 15, 78, and 82 can be phosphorylated by different kinases including p90rsk, pkg, mapkap kinases, etc. 55 the phosphorylation of hsp27 is a reversible process. the dephosphorylation contributes to the formation of large size oligomers. 60, 61 hsp27 can not only form large homo oligomers up to 800 kda; but it can also cooperate with other shsps (e.g., hsp20) to form heteromeric structures. 56, 58 hsp27 is upregulated and activated upon infections. 62, 63 the elevated hsp27 activity promotes either cytoskeletal stability or cell motility, 64, 65 and prevents apoptosis. 66 hsp27 is required for il-1-induced expression of the pro-inflammatory mediators il-6, il-8, and cyclooxygenase-2. [67] [68] [69] hsp27 is also linked to various signalling pathways involved in the development, differentiation, and cell growth. 70, 71 the long-term and high-level expression of hsp27 stimulated by variety of stresses (such as hbv or ebv infections) enhances carcinogenesis, cell survival, stemness of cancer cells, cancer metastasis, tumour formation and drug resistance. transcriptional regulation of the hsps. heat shock factors (hsfs) display great contributions in regulating the transcriptional activation of hsp genes. in all invertebrate animals, only hsf1 is responsible for the transcriptional activation. in vertebrates, four members of hsf family (hsf1-4) regulate hsp expression. 72 among them, hsf1 is the most critical one. the fibroblasts from hsf1 −/− mice undergo apoptosis upon heat stress because of no hsp transcription. 73 upon stress conditions, the originally monomeric hsf1 in the cytoplasm could trimerize and translocate into the nuclei to promote the hsp expression by binding on the heat shock elements (hse) in the promoter region. 74 protein disulfide isomerase protein disulfide isomerase (pdi) is a multifunctional oxidoreductase and chaperone that catalyses the formation, isomerization and reduction of disulfide bonds in the endoplasmic reticulum (er). during disulfide bond formation, cysteine residues at the cghc active site of pdi accept two electrons from the cysteine residues in polypeptide substrates, leading to the reduction of pdi and oxidation of the substrate. then pdi transfers the electrons to an acceptor to start another cycle of disulfide bond formation. 75 in addition to pdi's catalytic function as a thiol-disulfide isomerase, it also exhibits molecular chaperone properties for glycosylated protein quality control. 76 erp57 (pdia3, grp58) is possibly the most thoroughly studied pdi family member that shares a similar structure consisting of four domains (namely a-b-b'-a') and possesses two localization sequence-an er retention signal (qdel), and a nuclear localization signal (kpkkkkk). unlike other pdi family members that directly bind the substrates for their reductase or isomerase activities, the b domains of erp57 have a high affinity to associate with calreticulin (crt) and calnexin (cnx), which would help to recognize and recruit polypeptide segments of the glycoproteins. 77 if the protein is not correctly folded, udpglucose:glycoprotein glucosyltransferase (uggt) would be recruited to reglycosylate the proteins, allowing them to be recognized and re-associated by erp57/crt/cnx complex. 76, 78, 79 considering the essential roles of pdis in the oxidative folding and chaperone-mediated protein quality control, they are now linked to a growing range of diseases including those are caused by virus infection. proteins that interact non-specifically with rna and resolve the non-functional inhibitory structures are usually referred to as rna chaperones, which have distinct roles without common sequences or motifs. 80, 81 they participate in a large number of cellular processes, including chromatin remodelling, transcription regulation, rnp assembly and stabilization, rna export, histone-like nucleoid structuring, intracellular immunity, and viral rna replication and translation. rna molecules mostly rely on welldefined 3d structures to fulfill their functions. however, the process of rna folding is very complicated. 82 the multitude of possible rna base-pairings together with the high stability of rna duplexes would give rise to a large number of alternative secondary and tertiary structures that are thermodynamically as stable as the functional, native structure. 83 rna chaperones promote rna folding by accelerating the escape from kinetic folding traps and prevent rnas from being trapped in nonfunctional conformations. [84] [85] [86] so far, no protein has been characterized whose primary function is to resolve nonspecifically misfolded rnas in cells. 80, 81 hnrnps are a group of heterogeneous nuclear ribonucleoproteins. they are essential factors for manipulating both the functions and metabolisms of pre-mrnas/hnrnas transcribed by rna polymerase ii. more than 20 hnrnps have been identified to date. hnrnps contain common rna binding motifs like arginine glycine boxes (rgg boxes), rna recognition motifs (rrms), hnrnp k homology (kh)-domains and zinc finger (zf)-domains (khzf domain). 87 well-defined functions of this family include transcription regulation, pre-mrna splicing, 3′-end formation, mrna packaging, rna transport, translational regulation, rna silencing, dna repair, and telomere biogenesis. they also have the ability to shuttle between nucleus and cytoplasm, therefore could transiently help to form rnp complexes in nucleus and also participate in rna metabolism in cytoplasm. 88 a large collection of hnrnps are involved in virus activities, most of which were first identified using viral rna-protein binding assays, followed by functional assays. 89 the importance of stress proteins one of the main functions of stress proteins is to maintain cellular homeostasis. under pressure, stress proteins are hyperactive to release the pressure. hsp27, hsp70 and hsp90 accumulate to a very high level in quite a few types of cancer cells. [90] [91] [92] although the mechanism underlying the increase has not been fully understood, it suggests that the fast increased hsps respond to the folding stresses. with tumor progression, the accumulating oncogenic proteins need powerful protein folding ability. under long-term stresses, hsps participate or promote carcinogenesis, cell survival, anti-apoptosis, angiogenesis, cancer cell stemness, invasion and metastasis. 93 however, hsps and rna chaperones are downregulated in nearly all age-related neurodegenerative diseases including alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, and several polyglutamine diseases such as huntington's disease and different forms of spinocerebellar ataxias 94, 95 (fig. 2) . downregulated rna chaperones lead to disorder of rna metabolism; 94 while the attenuated hsps result in a failure of the protein quality control (pqc) system to adequately handle the folding or timely degradation of proteins in neurologic disease. 95 both mechanisms cause protein aggregates, the hallmark of age-related neurodegenerative diseases. in this review, instead of paying much attention to these topics, we would only focus on the main biological functions and target values of stress proteins in human diseases caused by pathogen infections, particularly by virus infections because the welldeveloped antibiotics have already controlled the bacterial infection very well since 1950s. viruses infection causes a variety of diseases that are highly associated with dysregulation of stress proteins (fig. 2 ), e.g., respiratory symptoms, 96, 97 gastroenteritis, [98] [99] [100] [101] hemorrhagic fevers. 102, 103 critically, it has been demonstrated that neurodegenerative diseases as well as neurobehavioral disorders are the consequences of loss of neurons and axons in the central nervous system with ageing. it is evidenced that these diseases are possibly caused by chronic neuropathic viral infections. 104 stress proteins are involved in many steps of virus infection process, including virus entry, uncoating, replication, gene expression, as well as virus assembly and releasing as steps 1-5 shown (fig. 3 ) some viruses replicate in the nucleus, while stress proteins take part in the virus protein and/or rna nuclear import/export processes as setp a-c shown (fig. 3 ). the summary of the relationship between stress proteins, virus infection as well as host cell response is listed in table 1 . in this section, we would review and present new findings on hsp90 family proteins in the life cycles of different viruses including rna virus, dna virus and retrovirus. in addition, we also discuss their functions in virus-induced cellular response. the function of hsp90 family in rna virus infection virus entry. in the case of rna virus, hsp90 is critical for the entry of enterovirus a71 (ev-a71), 107 japanese encephalitis virus (jev) and dengue virus (denv). 108 ev-a71 entry is significantly blocked when cells are pre-treated with hsp90 inhibitors or hsp90-specific sirnas. 107, 109 since both denv virus and jev belong to the flavivirus, the entry of the these two viruses differently utilize hsp90 with the support of hsp70s in both neuroblastoma and microglial cells. 95, 108 denv depends much more on hsp90 to enter into cells as compared with jev since anti-hsp90 antibodies or hsp90 inhibitors block the majority of denv entry 110 but only a small portion of jev entry. 111 additionally, both hsp90 and hsp70 are associated with membrane lipid rafts in response to denv infection. 110 however, in denv infected liver cells (hepg2), neither hsp90 nor hsp70 works as the receptor to enable denv internalization, 110 indicating alternative entry mechanisms in different cell types. it is likely that the receptor functions of hsp90 and hsp70 are replaced by other unknow molecules. virus replication. hsp90 protein facilitates virus replication in several aspects. first, hsp90 works as a classic chaperone protein to stabilize virus proteins. hsp90 stabilizes paramyxoviruses polymerase and l protein, as well as assists virus replication. inhibition of hsp90 could hamper virus replication and shorten the half-life of l protein in vesicular stomatitis virus (vsv), human parainfluenza viruses-2 (hpiv-2), human parainfluenza viruses-3 (hpiv-3), simian virus 41 (sv41), or respiratory syncytial virus (rsv) infection cells. 112, 113 similarly, hsp90 is shown to maintain the stability of chikungunya virus (chikv) non-structural proteins (nsps) including nsp3 (a protein essential for rna synthesis) and nsp4 (rna-dependent rna polymerase, rdrp); 114 and the protease of hcv nonstructural protein ns3. 115 second, hsp90 modulates virus polymerase activity to enhance virus replication. taking hcv as an example, hsp90 indirectly modulates the hcv polymerase ns5 activity by maintaining the stability of kinase phosphoinositide dependent kinase l (pdk1), an upstream kinase of ns5 phosphorylation kinase prk2. 116 besides, mediated by fkbp8, hsp90 forms a complex with ns5 and directly regulates ns5 activity. 117 hsp90 inhibitors suppress viral replication by disrupting the hsp90-ns5 complex formation. 117 third, hsp90 manipulates the proper location of virus polymerases. during influenza virus infection, hsp90 interacts with the viral rdrp subunits polymerase basic protein-1 (pb1) and -2 (pb2) to form a complex, and then co-translocates into nucleus. 14, 118 in this process, hsp90 maintains the stability of pb1 and pb2. after entry into nucleus, hsp90 dissociates from the hsp90/pb1/pb2 complex and forms a new functional complex with polymerase acidic protein (pa). 119 extending study shows that the state of hsp90 acetylation is strictly regulated by histone deacetylases 6/8 while some viruses (such as influenza, sv40, hbv etc) also enter into nucleus of host cells. they may undergo other steps in their life cycle which are labelled as a-c: virus nucleus import, nucleus export, and virus rna processing. during the process of virus infection, hsp90, hsp70, hsp60, hsp40, hsp27, and pdis participate in the virus entry and uncoating steps. hsp90, hsp70, hsp60, hsp40, hsp27 and rna chaperones take part in the virus replication step. hsp70, hsp40 and rna chaperones are required in virus gene expression step. hsp90, hsp70 and hsp40 assist virus assembly. hsp70 and rna chaperones contribute to the virus release. while hsp90 is also important in virus nucleus import and export. hsp70 plays a role in the virus nucleus import. and rna chaperones play a major role in virus rna processing, including replication, initiation of translation, stabilization and decay 485, 486 (hdac6/8) in the influenza rdrp nuclear import stage. hdac6/8 inhibitors efficiently limit the polymerase nuclear import and suppress virus replication. 120 in the course of influenza infections, hsp90 expression is stimulated through mtor/p70s6k signalling. 121 our recent studies show that hsp90 also exhibits significant importance on ev-a71 replication through pim1 signalling (unpublished). it has been shown that ev-a71 infection elevated both the mrna and protein levels of pim1. 122 the elevated pim promoted ev-a71 replication while knockdown of pim1 decreased ev-a71 replication. knockdown of hsp90β decreases 60% of virus replication 12h post infection (p.i.), while the secreted virions decrease by approximately 80%, indicating the crucial roles of hsp90β in both virus replication and secretion (fig. 4a-c) . other researchers reported that hsp90β is responsible for ev-a71 assembly which may be the reason that hsp90β attenuates the secretion of ev-a71 virions in our study. 107, 109, 122 notably, hsp90β contributes to virus replication more than that of secretion. our data also shows that knockdown or knockout of hsp90β decreased the proteins expression level of ev-a71 structure protein (fig. 4d , e). and hsp90 inhibitors 17-aag, geldanamycin (ga) and ver50588 all dramatically inhibit ev-a71 protein expression (fig. 4f ). among them, ver50588 display the strongest inhibitory effect which has not been reported before. we predicted that hsp90β is a potential target of pim1 (fig. 4g ). to address this hypothesis, we conducted experiments by overexpression pim1 and knockdown of pim1. accordingly, the phosphorylated status of hsp90β is increased and decreased (fig. 4h,i) . more importantly, knockout of hsp90β by crispr/cas9mediated gene editing almost completely abolishes the effects of pim1 signaling on ev-a71 replication (fig. 4j, k) . viral protein maturation, virion assembly, and release. during the viral protein expression and maturation, hsp90 works as a classic chaperone to monitor the proper folding of viral proteins. hsp90 modulates the maturation of hcv nonstructural protein 2/3 (ns2/ 3) kinase. 123 hcv ns2/3 is cleaved into two separate proteins right after translation, a key step of ns2/3 protein maturation. hsp90 strictly regulates the proper folding of newly synthesized ns2/3 protein. 123 hsp90 and its co-chaperone p23 form a complex to assist the proper folding of capsid precursor polyprotein p1 of poliovirus, rhinovirus, and coxsackievirus; 124 while the inhibitor ga reduces the maturation of p1, leading to immature p1 degradation in proteasome. 124 during the virion assembly, hsp90 interacts with capsid vp1 protein of noroviruses and the termini of the murine norovirus 1 genome. 124, 125 this interaction not only stabilizes vp1, but ensures the viral genome to be encapsulated into capsids as well. 124 hsp90 interacts with and stabilizes influenza neuraminidase (na), a major surface glycoprotein involving in virion release. 126 more importantly, it emphasizes the hsp90-na complex formation on promoting cell survival, leading to more virus production. 126 the function of hsp90 family in dna virus infection virus entry. the entry of dna viruses includes steps of crossing over cell membrane and nuclear import. hsp90 is mainly shown the ability to assist the nuclear import of virus. the nuclear transport of many viruses depends on the microtubules (mt) and mt-dependent molecular motor dynein/dynactin complex. 127 virus strictly modulates the status of tubulin acetylation, a critical event for the transportation of viral components. [128] [129] [130] in hsvinfected cells, hsp90 co-localizes with acetylated tubulin and capsid protein vp5. 131 hsp90 inhibitors disrupt its binding to the acetylated tubulin, thereby inhibiting the nuclear transport of hsv capsid protein. 131 during hbv infection, the glucocorticoid receptor shows a strong possibility to enhance the nuclear import of hbv. 132 hsp90 facilitates glucocorticoid receptor redistribution from the cytoplasm to the nucleus. 133 virus replication. during dna retroviral replication, hsp90 mainly contributes to regulating and maintaining the reversetranscriptase (rt) activity. taking hepatitis b virus (hbv) as an example, the reverse transcription is an essential step to generate viral genomic dna in type vii viruses. the beginning of reverse transcription is the recognition and interaction of rt with an rna signal (the packaging signal, ε) on the pre-genomic rna. 134 it was identified that hsp90 is an essential host factor that facilitates duck hepatitis b virus (dhbv) replication by interacting with viral rt. 135 treating with hsp90 inhibitors or monoclonal antibodies (mab) sufficiently block rt-ε binding. 135 hu et al. demonstrated that rt-ε interaction depends on hsp90's atp hydrolysis activity. 136 two independent regions in the terminal protein (tp) and the rt domains of polymerase separately bind with hsp90 at the n-terminal and c-terminal fragments, and both domains are essential for ribonucleoprotein (rnp) and protein priming. 137, 138 although a model is established to show how hsp90 bridges the two separate rt domains of polymerase together to enable the formation of an rnp complex with the hbv rna; 137 there are still some fundamental questions to be addressed. firstly, whether the hsp90 chaperones or hsp70 chaperones are essential for the rt-ε interaction. stahl et al. believed that hsp70 chaperones are much more important for the rt-ε interaction. they proposed that hsp90β another hsp90 family member, is shown a critical regulator in stabilizing and activating rt, allowing its preferential binding to the pregenomic rna during hbv replication. 143 the replication of most dna viruses occurs in the nucleus, where virions form in replication centres. therefore, the proper location of viral proteins is quite important for virus replication. hsp90 is also found to regulate the location of virus dna polymerase in virus-infected cells. after treated with hsp90 inhibitors, hsv polymerases is mislocalized from the nucleus to the cytoplasm and subsequently degraded in a proteasomedependent manner. 144, 145 similar to hsv, the nuclear translocation of dna polymerase of ebv also requires hsp90. 146, 147 during the polymerase transportation, the polymerase catalytic subunit balf5 forms a complex with bmrf1 in the assistance of hsp90β. hsp90 inhibitors effectively block the translocation of viral dna polymerase. 146, 147 virus gene expression. hsp90 is important for virus gene expression both at the transcription and translation levels. the transcription of hsv immediate-early α (ieα) genes is initiated by the transcription factor complex, which is composed of octamerbinding transcription factor 1 (oct-1), host cell factor 1 (hcf-1) and viral protein 16 (vp16). 148, 149 in the complex, vp16 is the major virus-encoded transcriptional activator that controls the efficiency and level of viral gene transcription. in the transcription process, hsp90α is shown to maintain the stability of vp16 by keeping it from degradation in a macroautophagy-mediated manner. 150 similarly, hsp90 also regulates the transcription of human cytomegalovirus (hcmv) immediate-early genes through activating akt and nf-κb signalling pathways, which are critical for major immediate early genes transcription. 151, 152 at the translation level, hsp90 promotes the translation of conserved herpesvirus protein kinases (chpks), including herpes simplex virus type 1 and 2 (hsv-1, hsv-2), varicella-zoster virus (vzv), ebv, kshv. 153 chpks play important roles in multiple processes, including gene expression, 154-156 viral dna replication, [156] [157] [158] capsid nuclear egress, 159, 160 and dna damage responses. 161, 162 the translation of ebv nuclear antigen 1 (ebna1) protein is also manipulated by hsp90. 163, 164 ebna1 is critical for cellular transformation, tumorigenesis, and the maintenance of viral episomes. [165] [166] [167] the ebna1 translation is strictly regulated by hsp90 through the gly-ala repeat domain to keep ebna1 at a relatively low level. 168 it has been demonstrated that hsp90 inhibitors block the translation of ebna1; and mutation of gly-ala repeat domain abrogates the inhibition of ebna1 translation. 163, 164 hsp90 does not interact with ebna1directly, a bridge protein may be involved in this process. inhibiting ebna1 expression strongly suppresses both ebv-induced primary b cell transformation in vitro and lymphoproliferative disease in scid mice in vivo. 163, 164 virus assembly. only a few papers reported the function of hsp90 in dna virus assembly. the activated hsp90 is needed for hbv assembly. 169, 170 hsp90 enhances the affinity of core protein dimers for capsid formation and prevents capsid dissociation. 169 besides, the reactive oxygen species (ros)-promoted hbv capsid assembly also requires an active form of hsp90. 170 virus-induced tumorigenesis. as described before, ebv is the causative regent of several tumors, including burkitt's lymphoma 171 and nasopharyngeal carcinoma (npc). 172 the latent membrane protein 1 (lmp1) is regarded as an oncoprotein that promotes tumor metastasis and invasiveness through inducing the expression of matrix metalloproteinase 9 (mmp9), mimicking the tumor necrosis factor receptor (tnfr) superfamily proteins, and activating the nf-kb, mapk, pi3k/akt and jak/stat signal transduction pathways. 173, 174 hsp90 seems positively promoting cell growth in ebv-positive nasopharyngeal carcinoma cells and ebv-infected t and nk cells. 175, 176 hsp90 inhibitors, at13387 and biib021, potently inhibit cell growth and induce apoptosis by impeding lmp1 function through activating its downstream signaling pathways described above. 175, 176 immunity modulation. we have discussed how hsp90 is hijacked by dna viruses to promote viral replication above. under certain conditions, hsp90 exhibits antiviral activities by promoting cell immunity. in the acute infection stage, hsp90 is induced to express in the cell surface of ebv-infected b cells. it has a strong ability to expand gamma delta t cells (γδ t cells) population. 177 the γδ t cells have potent antiviral ability in the acute phage when a host is infected by hiv, influenza, sendai, coxsackie, vaccinia, vsv, or hsv-1. the γδ t cells work as both early sentinels of the immune system by providing immediate protection and as bridging elements between innate immunity and adaptive immunity. 178 since hsp90 can work as an immune sensor and assist antigen presentation, it may function in the same way in ebv infection. 177, [179] [180] [181] [182] the function of hsp90 family on cell transformation during retrovirus infection several hsps functions as oncoproteins to promote cellular transformation. hsp90 participates in the htlv-1-induced cellular transformation. the tax protein of htlv-1 controls viral replication and induces t lymphocyte transformation. 183 nf-κb pathway is one of the main targets essential for this process; 184 while hsp90 acts as an important partner of tax by binding with and maintaining its stability in nucleus. 153, 185 hsp90 inhibitors (e.g.,17-dmag) or hsp90-depletion by sirnas cause tax degradation in proteasome, inhibition of nf-κb signalling, and activation of the long terminal repeat (ltr) of htlv-1. 153, 185 oral treatment with fig. 4 pim1 signaling promotes ev-a71 replication through hsp90β phosphorylation(unpublished data). a rd cells were treated with scramble/hsp90β sirna for 24h, the effects of hsp90β knockdown were determined by rt-qpcr assay. the results are shown as the means, and ±error indicates the standard deviation (sd). data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed student's t-test. b, c rd cells were treated with scramble /hsp90β sirna for 4h, then infected with ev-a71 at moi of 1 for indicated time. the intracellular (b) and extracellular (c) viral rna levels were detected by rt-qpcr assay. the results are shown as the means, and ±error indicates the standard deviation (sd). data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed student's t-test. d, e knockdown of hsp90β by sirna or knockout by cripsr/cas9 mediated gene editing in rd cells, and then cells were infected with ev-a71 at moi of 1 for indicated time. the protein level of ev-a71 was determined by western blot. f rd cells were treated with hsp90 inhibitors at different concentrations and infected with ev-a71 at moi of 0.01 for 48h. the protein level of ev-a71 was determined by western blot. g pim1protein interaction network was predicted using online genemania program. h pim 1 was overexpressed in 293t cells and cell lysate was collected for immunoprecipitation assay. the phosphorylation status of hsp90 was detected by western blot. i pim1 was overexpressed/ knocked down in 293t cells. and cell lysate was collected for native page analysis. j wt rd cells and hsp90β knockout (hsp90β-ko) cells were pretreated with 2 μm pim1 inhibitor sgi-1776 for 2 h, then the cells were infected cells with ev-a71 at moi of 0.01 for 48h. intracellular viral rna level was determined by rt-qpcr. the results are shown as the means, and ±error indicates the standard deviation (sd). data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed student's t-test. k pim1 was overexpressed in wt/hsp90β -knockout rd cells, and infected with ev-a71 at moi of 1 for 12 h. the protein level of ev-a71 was determined by western blot hsp90 inhibitor 17-dmag significantly suppresses the aggressive infiltration into multiple organs in atl mice. 153, 185 functions of hsp70 family in rna virus infection virus entry. viruses in different families (e.g., picornaviridae, flaviviridae, and reoviridae) take advantage of hsp70 family proteins for their entry into host cells. in the case of picornaviridae, for example, the coxsackievirus a9 (cav-9) uses hsp70 homolog grp78 for its entry. 186 it shows that antibodies against grp78 block 50% of virus binding. integrin αvβ3 is another famous virus receptor. 187 when cells are simultaneously treated with grp78 and integrin αvβ3 antibodies, virus binding is blocked completely. therefore, grp78 functions as a co-receptor of cav-9. besides, grp78 can interact with major histocompatibility complex (mhc) i molecules on the host cell membrane after infection of cav-9. mhc i molecules help virus internalization into mammalian cells. 186 in the course of ev-a71 infection, hsp70 is dramatically upregulated and interacts with ev-a71 on the cell surface. hsp70 antibody significantly inhibits virus binding to the cell surface. 188 besides enteroviruses, many viruses of flaviviridae family also require hsp70 to entry into host cells. by affinity chromatography assay, hsp70 is discovered to form a complex with hsp90 and denv receptor that facilitates viral entry. 110, 189 hsp70 interacts with dnev envelope protein (e protein) and plays a significant role in virus attachment. 190 similarly, antibodies against hsp70 and hsp90 significantly inhibit denv infection. 110, 189 the same mechanism is also observed in jev infection. 191 hsp70 is enriched in the lipid raft and colocalized with the e protein in jev-infected huh7 cells. 192 the depletion of cholesterol disrupts the enrichment and colocalization of the e protein and hsp70 to a raft membrane. eventually, it decreases jev entry without any effects on virus attachment. 192 these results suggest that hsp70 works as a receptor of jev; and lipid rafts serve as an organizing centre to facilitate jev entry. at the late stage of jev entry, hsc70 (isoform d) is upregulated in c6/36 cells upon jev infection. however, it seems that hsc70 is not required for virus attachment to the cell membrane but needed for virus penetration into the host cells. it is suggested that hsc70 holds an intense involvement in clathrinmediated endocytosis at the late stage of viral entry, which helps jev to penetrate into host cells. 193 recently, it is reported that grp78 is also required for jev both in the attachment and entry steps. 194 antibody targeting the n-terminal of grp78 significantly prevents virus attaching to host cells whereas antibody targeting the c-terminus fails to block the attachment. knockdown of grp78 also inhibits jev internalization. the colocalization and interaction between grp78 and jev envelope protein provide solid evidence to show the importance of grp78 in the process of virus attachment and entry. 194 interestingly, grp78 is secreted out of host cells after jev infection, and the secreted grp78 cooperates with jev to promote virus infection. 195 recent studies show that grp78 is a receptor of sars-cov, mers-cov, and sars-cov-2 viruses. 196 zikv infection is positively regulated by hsp70 at multiple stages. 197 hsp70 inhibitors impair virus entry, rna replication, and capsid assembly of different zikv strains in diverse cell lines. 190, 197 rotavirus infection also needs the assistance of membrane-resident hsc70. 198 hsc70-specific monoclonal antibodies inhibit virus internalization and infection without effect on virus attachment. 198 further evidence shows that the whole virus particle and a short domain (or a peptide) in the cterminal region of vp5 is sufficient to bind to hsc70. 199 the atpase domain of hsc70 is proved to be necessary for its interaction with vp5 and induction of virion conformation change for the entry. 200 virus replication. hsp70 family proteins participate viral replication by employing different mechanisms. first, hsp70 family proteins facilitate the formation of virus replication complex and/ or maintain the stability of complex proteins. in some cases, hsp70 family proteins directly interact with viral polymerase to enhance viral replication. for example, during the mumps virus (muv) infection, the expression level of hsp72 is increased. the c-terminal region of hsp72 interacts with the n-terminal region of p protein, which is an essential component of rdrp complex. knockdown of hsp72 results in accumulated ubiquitinated p protein as well as increased cell apoptosis. 201 besides, hsp70 is also reported to regulate l protein, another muv polymerase component. hsp70 cooperates with hsp90 to regulate l protein levels. hsp90 inhibitor, 17-aag, reduces the l protein level through promoting degradation via the c terminus of hsp70interacting protein (chip) -mediated proteasomal pathway. hsp70 inhibitor ver155008 together with 17-aag enhances l protein degradation. therefore, hsp90 and hsp70 together regulate the stability of l protein and ensure the proper virus replication complex (vrc) formation. 202 in the case of canine distemper virus (cdv) infection, the increased hsp70 results in an elevated expression of light nucleocapsid (nc-l) variant, which displays polymerase activity. 203, 204 a more direct evidence is that hsp70 facilitates viral rna production in cell-free transcriptional assays. 204 furthermore, it is demonstrated that hsp70 interacts with and regulates nc polymerase activity dependent on the hsp70 atp activity; because hsp70 antibody significantly inhibits nc polymerase activity and supplementation of purified recombinant hsp70 enhances both the basal and stress-induced nc polymerase activity. 205 the other members of hsp70s also regulate vrc formation. hsp72 physically interacts with several replication proteins of flavivirus including ns5a, ns3, and ns5b (rdrp). for example, hsp72 participates the vrc formation of hcv. 206 downregulating hsp72 leads to a decreased number of vrc in hcv-infected cells, while overexpression of hsp72 raises the number of vrc. 206 hsc70 is associated with vrc by binding on the 3' polyu/uc motif of hcv rna genome. 207 hcv accumulation and virion production are significantly suppressed when cells are treated with hsp70 or hsc70 inhibitors. 208 similar result is reported in the case of rsv infection. ectopic expression of rsv nucleocapsid protein (n protein) and phosphoprotein (p protein) are detected to interact with hsp70 in 293t cells. 209 the n protein is responsible for interacting with the viral rna, and p protein interacts with n protein and with the rdrp l to form the nucleocapsid. in rsvinfected cells, hsp70 redistributes into lipid-raft membranes and colocalizes with virus n protein and lipid raft marker gm1. 210 although hsp70 inhibitors suppress rsv polymerase activity; 210 it only disrupts viral gene expression but do not affect rna polymerization. 211 therefore, more detailed studies are needed to understand the functions of hsp70 in modulating rsv gene expression and replication. in term of ebola virus (ebov) replication, the mechanism of hsp70 involved is much more complicated. by using immunoprecipitation and mass spectrometry assays, it has been identified that the n protein interacts with hsp70, nef, bag2, and the hsp70 co-chaperone dnaja2. 212 the n protein recognizes and binds to the viral rna genome to establish a steady n protein-rna complex structure (rnp). this complex further interacts with viral proteins vp30, vp35 and rdrp l, to finally form vrc. here, hsp70 functions to maintain the stability of n protein and helps to facilitate vrc formation. 212, 213 in addition, hsp70 is also copurified with l polymerase in insect cells. 214 besides, hsc70 interacts with the terminal non-coding regions of the ebov genome. disruption of the interaction by mutating the binding site potently inhibits the minigenome replication of ebov. 215 another mechanism that hsp70 employs to support virus replication is to modulate nuclear import of polymerase or nuclear capsid. some rna viruses also replicate in the nuclear such as cdv and influenza virus. therefore, nuclear transportation becomes a critical step for their replication. upon cdv infection, hsp70 is shown a strong contribution to viral replication by interacting with and promoting the translocation of the nucleocapsid particles from the cytosol to nucleus. 216 similarly, during influenza infection, hsp70 interacts with pb2 or pb1 monomers and pb2/ pb1 heterodimer in hela and hek293t cells, and sequentially translocates into the nucleus with pb2 monomers or pb2/pb1 heterodimers. 217 if hsp70 and pb2/pb1 polymerases are retained in the cytosol, the polymerase activity reduces dramatically. the shuttling of hsp70 between nuclear and cytoplasmic compartments underlies the modulatory effect of hsp70 on influenza virus replication. 217 virus gene expression. besides viral entry and replication, hsp70 also contributes to viral protein translation. positive singlestranded rna viruses (e.g., sars-cov-2, hcv, zika, ev-a71, etc) use the internal ribosome entry site (ires) to initiate the translation of their own proteins but inhibit the host cellular cap-dependent translation through regulation or cleavage of eukaryotic translation initiation /elongation factors (eifs/eefs). in the case of coxsackievirus b3 (cva b3) infection, hsp70 is upregulated to enhance the initiation and elongation of viral translation. 218 in the translation initiation step, hsp70 upregulates ires-acting factor lupus autoantigen protein expression and activates eif4e binding protein 1 (eif4ebp1), a cap-dependent translation suppressor. in the elongation step, hsp70 activates the akt-mammalian target of rapamycin complex 1 (mtorc1) signal cascade, leading to activation of eef2 via kinase p70s6k-and cdc2-mediated phosphorylation and inactivation of eef2 kinase (ef2k). 218 hsc70 enhances the ires activity in ev-a71 infected cells. hsc70 interacts with 2a protease of ev-a71 to enhance eif4g cleavage that impairs host cell cap-dependent translation but enhances viral ires-mediated translation. 219 therefore, hsc70 may serve as an antiviral target against ev-a71 and hcv infections. some viruses do not have ires sequence, and virus replication produces plenty of dsrnas which trigger the activation of protein kinase-rna-activated (pkr)-eif2a signalling cascade that shuts down global translation in cells and releases stresses. 220, 221 to circumvent the pkr-mediated block to viral proliferation, influenza a virus induces the cellular tetratricopeptide repeat (tpr)domains containing jdp protein, p58ipk. influenza virus downregulates pkr in an hsp70-dependent way. [222] [223] [224] [225] [226] in uninfected and unstressed cells, p58ipk activity is clogged with hdj1 by forming a complex. 225, 226 during influenza a infection, the amount of hdj1 in p58ipk-hdj1 complex decreases to an undetectable level. these findings suggest that the activation of p58ipk appears to be a sequel to the hsp70-mediated release of hdj1 from the p58ipk-hdj1 complex, which allows the monomeric p58ipk to inhibit pkr. 225 in the hsp40 chaperone part, we would discuss more details about the regulation of pkr signaling by influenza virus. virion assembly. hsp70 is reported to assist some viruses' assembly. during morphogenesis of the double-stranded rna reovirus, hsp70 contributes to the assembly of trimeric sigma 1 protein, which is responsible for the interaction with host cell receptor. 227 while the n-terminal segment of the sigma 1 protein folds and trimerizes cotranslationally in an hsp70-independent manner, a post-translational fold of the c-terminal globular domain is dependent on hsp70. in this process, hsp70 binds cotranslationally to the region downstream the n-terminal αhelical coiled-coil, which presumably helps to inhibit unwanted interaction and misfolding. trimerization of the c-terminal domain of the sigma 1 protein is coupled to the atp-dependent release of hsp70 from the ribosome. 227 besides, in hela cells infected by poliovirus or coxsackievirus b1, hsp70 is detected to interact with the capsid precursor p1. 228 in the complex, p1 is mainly newly synthesized and has a longer half-life than that of total p1. the hsp70-p1 complex is regarded as an assembly intermediate of picornaviruses. 228 interestingly, some researchers demonstrate that hsp70 inhibits influenza virus replication by blocking nuclear export of viral ribonucleoprotein complex (vrnp), and subsequent viral morphogenesis via disassociating m1 from vrnp. 229, 230 virus release. the evidence of hsps on virus releasing is limited. both hsp70 and hsc70 can interact with ns5a protein; although they play different roles in hcv infection. 231 silencing of hsp70 decreases viral protein expression, but the virus protein level is not affected. 232, 233 instead, interfering hsc70 reduces extracellular virion production. 232, 233 moreover, hsc70 is embedded in the viral capsid. and co-localization between hsc70 and core and e2 structural proteins of hcv has been found in lipid droplets. therefore, hsp70 and hsc70 may regulate hcv infection release at different steps. the function of hsp70 family proteins in dna virus infection virus entry and genome releasing. instead of virus attachment and entry into the host cells, here we talked about virus entry into the cytosol from er. the cytosol entry from er is a key step in sv40 infections. hsc70 is reported to be essential in this step. hsc70 interacts with and is regulated by sgta. 234 further studies show that hsp70 superfamily member hsp105 forms a subunit in the hsc70-sgta complex to facilitate sv40 cytosol entry. 235 in addition, grp78/bip also plays a role in sv40 cytosol entry. grp78 interacts with sv40 capsid protein in a dnajb11-dependent manner to help sv40 disassemble and enter into the cytosol. 236 hsp70 is also needed for the genome release of some dna viruses. such a process is described in adenovirus infection. after the release of the virion from endocytic vesicles into the cytoplasm, hsp70 and hsc70 immediately attach to the hexon protein, one of the major adenovirus coat proteins. 237 hsp70/ hsc70 and its co-chaperone bag3 interact with the penton base protein, the viral capsid constituent responsible for virus internalization. 238, 239 the intact nucleocapsid is transported to nucleus through the typical nls-dependent nuclear import machinery. 240 the nucleocapsid anchors to the nuclear pore through its hexon protein by interacting with components of the pore complex. then viral dna is transferred into the nucleus in a hsp70-dependent manner but leaving hexon outside the nucleus because the purified hexon, instead of viral dna, enter the nucleus in a hsp70-independent manner. 240 a possible explanation is that the intact nucleocapsid is too large to pass through the nuclear pore complex, while the disassembly of nucleocapsid facilitates the entry of viral dna into nucleus. however, more solid evidence is needed for such an explanation. 238 other examples for the contribution of hsc70 in viral genome release in host nucleus are hsv and polyomavirus. in hsv-infected cells, the translocation of hsc70 from the cytosol to nucleus is triggered by the immediate-early viral protein icp0. hsc70 is colocalized with the components of the 26s proteasome and virus ul6 portal protein, which provides the conduit for dna entry and exit from the capsid. ul6 is highly ubiquitinated in the nucleus, indicating that hsc70 may be responsible for the correct folding and degradation of ul6 in the ubiquitin-proteasome pathway, though there was no direct evidence that ul6 is a substrate of hsc70. 241 it is also believed that hsc70 contributes to polyomavirus genome nuclear import through its interaction with all viral capsid proteins vp1, vp2 and vp3 both in vitro and in vivo. hsc70 translocates from the cytosol to the nucleus accompanied by the translocation of capsid proteins upon infection with polyomavirus. 254 gene expression and protein maturation. most viruses manipulate the cellular transcription and translation machinery and shut off host protein synthesis, so that they can take advantage of these machineries and recruit initiation and elongation factors for the expression of viral proteins. some host factors exploited by viruses interact closely with components of hsp70 complex. therefore, the chaperone system is highly important for viral gene expression. several transcription initiation factors are well known to physically interact with the hsp70 co-chaperone bag1 in vitro. bag1 stimulates general transcription activity in an hsp70dependent manner. [242] [243] [244] the stimulation of global transcription is detected in cells upon infections by either human polyomavirus, john cunningham virus (jcv) or hcmv. 245, 246 however, the detailed molecular mechanism of the general transcriptional activation by viruses is to be further investigated. the typical example of hsp70 system in regulating the maturation of viral proteins is shown in hbv large envelope protein (lhbsag). [247] [248] [249] the mature lhbsag has a unique dual transmembrane topology. initially, the c-terminal of lhbsag cotranslationally resides in er, while the n-terminus is resident in the cytosol and later is translocated into er for post-tranreported that the expression of grp78 is stimulatedslation. to ensure the correct topology, hsp70 system strictly regulates the post translocation of n-terminal of lhbsag. during the translation, hsc70 interacts with the n-terminal of lhbsag at amino acids 63 to 107 and suppresses lhbsag translocation into er. 248, 249 hsc70 cochaperones hip and bag1 also regulate the activity of hsc70 in an antagonistic way. 250, 251 overexpressed hip promotes hsc70 activity resulting in more cytosol retention of lhbsag n-terminal; 250 while bag1 overexpression could inhibit hsc70 activity to promote nuclear translocation. 251 in the post-translation process, grp78 binds with lhbsag and hsc70 to facilitate the er translocation of hbv large surface antigen. 247, 248, 252, 253 the function of grp78 is regulated by both positive regulator er-localized dnaj-domain-containing protein 4 (erdj4) and negative regulator bip-associated protein (bap). 253 increased bap destabilizes lhbsag/bip complex. 253 together, hsp70 chaperone system is crucial in modulating the sophisticated topogenesis of hbv envelope protein. 247 virus assembly. a lot of dna viruses normally assemble in the nuclei of infected cells. taking polyomavirus as an example, all capsid proteins are synthesized in the cytosol, whereas subsequent assembly of virions only takes place in the nucleus. during polyomavirus infection, the constitutive form of hsp70 and hsc70 are coimmunoprecipitated with all three viral capsid proteins (vp1, vp2, and vp3). hsp70 is translocated from cytoplasm to the nucleus in the late stage of infection, coincident with localization change of the viral capsid proteins. 254 in vitro studies show that hsp70 functions to keep proper assembly of polyomavirus. 255 the prokaryotic hsp70 chaperone dnak also interacts with recombinant vp1 at the c-terminal domain where it links pentamers in an assembled capsid. 255 when dnak binds to vp1, it inhibits vp1 assembly, which is induced by calcium in vitro. however, combining the hsp70 chaperone system including dnak, dnaj and grpe with vp1 together is sufficient to assemble vp1 into uniform capsids in the presence of atp alone without calcium. 255 the function of hsp70 family in retrovirus infection human t lymphotropic virus type 1 (htlv-1) is a well-investigated example of retrovirus that interacts with hsp70 proteins. during htlv-1 infection, syncytium formation is a key factor for cell-tocell virus transfer. the syncytium formation is subject to close cellto-cell interactions. 256, 257 cell membrane-resident hsc70 is necessary in this process as hsc70-specific monoclonal antibodies eliminates the syncytium formation and htlv-1 infectivity. the same outcome presents when cell is treated with a peptide derived from the htlv-1 glycoprotein gp46, which binds to hsc70. 258, 259 interestingly, although hsc70 enhances the syncytium formation, it has no effect on virus entry. 259 hsp70 also plays an important role in the post-entry steps. during human immunodeficiency virus type 1 (hiv-1) infection, viral protein r (vpr) stimulates interaction between the viral preintegration complex and karyopherin-alpha to facilitate viral nuclear import. hsp70 functionally overlaps with vpr in this process. 260 when vpr is deficient, hsp70 could rescue virus nuclear import by interacting with karyopherin-alpha at the n-terminal that also binds vpr. interestingly, some researchers argue for the antiviral role of hsp70 because hsp70 and vpr share the same substrate. it seems that hsp70 would compete and inhibit vpr function. since hiv-1 needs vpr to manipulate cell cycle and apoptosis, hsp70 neutralizes the function of vpr in hiv-1 infection. 261, 262 recently, hiv is observed to package hsp70 as part of virion core. the virion-incorporated hsp70 atpase activity and correct conformation of hsp70-virion are essential for hiv infection, since inhibition of hsp70 atpase activity interrupts the hsp70-virion core association and diminishes virus infectivity. 263 the effects of hsp70s on host cells upon dna virus infection cellular transformation. dna viruses those do not encode polymerase in their genome are dependent on host dna replication machinery. to replicate in quiescent cells, the virus has to reinitiate cell cycle thereby transforming the host cells. some mechanisms have evolved in enabling viruses to overcome the restriction points of cell cycle. the best-investigated example is sv40. the large and small t antigen (tag) are central for the sv40 transformation ability. at their n terminus, both types of tag contain the j-domain, the signature motif of an hsp70 cochaperone. mutation or deletion of the j-domain disrupts the functional association of tag with hsp70s. the ability of tag to transform mammalian cells is subsequently obliterated. 264 also, tag sequesters the retinoblastoma family proteins (prb, p107, and p130) and liberates members of the e2f family of transcription factors in a hsc70-atp hydrolysis-dependent manner. 265, 266 the free e2f family proteins subsequently trigger the expression of the s-phase genes leading to dna replication. 267 the above observations are interpreted in the following four steps. firstly, the large tag combines with prb-e2f complex. subsequently, the prb-e2f-tag complex associates with hsc70 in the presence of atp as hsc70 atp-bound form presents high substrate association rate to facilitate tag-hsc70 complex formation. the third step is that the j-domain of tag in the tag-hsc70 complex stimulates atp hydrolysis. this process is dependent on the active site of hsc70 and does not occur at the t-antigen binding site. meanwhile, hsc70 transfers to the high-affinity conformation allowing the trap of the substrate protein prb or e2f. in the last step, hsc70 induces the conformation of the substrate protein in the complex which assists the dissociation of prb and e2f. after adp dissociation and rebinding of atp to hsc70, e2f and the prb-tag complex is released from the hsc70-substrate complex. [268] [269] [270] [271] a second strategy of tag to transactivate e2f transcription is independent of the disruption of the prb-e2f complex that also involves the jdomain and hsp70 protein. [271] [272] [273] [274] tag functions like bag1 to assiste the assembly of a transcription initiation complex on the respective promoters in the presence of hsc70. alternatively, tag could induce hsc70 to disassemble an inhibitory silencer complex fig. 5 model for the participation of hsc70 in the tag induced disassembling of prband e2f. firstly, tag combines with prb-e2f complex to facilitate t-antigen-hsc70 complex formation. then tag-hsc70 complex stimulates atp hydrolysis, and hsc70 transfers to the high-affinity conformation allowing the trap of the substrate protein prb or e2f. finally, hsc70 induces the conformation of the substrate protein in the complex which assists the dissociation of prb and e2f to initiate cell cycle reprogram or to assist with remodelling the chromatin (fig. 5) . hpv and adenovirus have similar transforming activities by disrupting prb-e2f complexes. although neither e7 (hpv) nor e1a (adenovirus) protein contains a j-domain, both proteins could transform cells in a way similar to that described for sv40 tag. e7 interacts with tumor suppressor htid-1. 275 the c terminus of e7, which mediates the interaction with htid-1, is essential for the physical disruption of the prb-e2f complex though it is not necessary for direct interaction with prb. 276, 277 these observations suggest that the interaction with htid-1 is involved in the disruption of the prb-e2f complex, providing e7 with the jdomain necessary to recruit hsc70 for the complex dissociation in analogy to the function of sv40 tag. alternatively, the binding of e7 to htid-1 could transform cells through inhibition of the assumed tumor suppressor function of htid-1. e1a directly interacts with hsc70 to disrupt the prb-e2f complex. 278 in conclusion, most double-stranded dna viruses depend on hsp70 chaperones for the reprogramming of the host cells to reenter the cell cycle. cell survival and apoptosis. since hsp70 systems are essential modulators for cell survival under stress conditions, the induction of hsp70 protein facilitates virus infection by keeping the cell alive until mature viruses are ready to leave. this is the main reason why the disruption of apoptotic pathway is a quite common phenomenon in viral infections. [279] [280] [281] in the early stage of viral life cycle, viral reproduction is simply vulnerable to cell death. naturally, viruses are evolved in manipulating cellular apoptosis. in ebv-infected cells, nuclear oncoprotein ebna3a helps hsp70 nuclear translocation and hsp70 chaperone complex formation to immortalize b cells through inhibiting apoptosis. 282 in contrast, apoptosis is beneficial for virus spreading when virions are finally assembled. [283] [284] [285] [286] [287] [288] [289] [290] virions are found in apoptotic bodies and subsequently engulfed by phagocytic cells. it has been suggested that virus can infect neighbour cells without being detected by the host immune system. 291, 292 on the other hand, the decrease of hsp70 mrna level may lead to the timed induction of apoptosis at the late stage of adenovirus infections. innate immunity. hsp70 has been previously described to influence innate immunity and inflammatory responses. 293 it has been believed that hepatocyte is devoid of innate immunities. ma et al. reported that the expression of grp78 is stimulated by hbv replication. the elevated grp78 protein in turn activated innate immune response by induction of ifnβ expression. 294 further studies showed that hsp70 greatly contributes to cellular innate immunity in response to either virus or bacterial infections. in the course of bacterial infections, the elevated intracellular levels of hsp70 protect cells from lps-mediated inflammation. through its interaction with tnf receptor associated factor 6 (traf6), hsp70 can inhibit its ubiquitination and thereby block the activation of transcription factor nf-kb. 295, 296 weiss et al. has presented more details on this mechanism. hsp70 binds with ikk, leading to disturbing the function and stability of nf-κb/ikbα/ikk complex and further impairing ikbα phosphorylation. 297 the disturbance of this complex also affects ikbα proteasomal degradation and nuclear translocation of nf-κb complex. 298 the inhibition of nf-κb signalling has great therapeutic significance because it can prevent massive tissue damage medicated by the excessive inflammation response. a more recent study provides another approach for hsp70 to regulate inflammation. pierre et al. described an nf-κb independent pathway that hsp70 could impact nlrp3/asc inflammasome formation through its association with nlrp3. 299 aside from the anti-inflammation function of intracellular hsp70, the secreted extracellular hsp70 binds to dendritic cells and macrophages before being recognized by its binding elements, most of which are innate immune receptors. 293, 300 toll-like receptors (tlrs) detect virus invasion and immediately trigger intracellular innate antiviral response. 301 they belong to type i integral membrane glycoproteins of il-1 receptor (il-1r) superfamily. 302 it has been suggested that tlr2/tlr4 involved in the initiation of innate immunity by extracellular hsp70. hsp70 utilizes both tlr2 and tlr4 to transduce its proinflammatory signal in a cd14-dependent manner to promote proinflammatory cytokine production via myd88/irak/nf-κb axis signalling cascade. 8, 301 another study clearly demonstrated the function of tlr4 and its direct interaction with hsp70. 303 after ligand binding, tlrs dimerize and undergo a conformational change required for recruiting downstream signaling molecules, including the adaptor molecule myeloid differentiation primary-response protein 88 (myd88), il-1r-associated kinases (iraks), tgfβ-activated kinase (tak1), tak1-binding protein 1 (tab1), tab2 and tnf-receptor-associated factor 6 (traf6). [304] [305] [306] [307] many other innate immunity-related signaling pathways would then be activated, for example, phosphorylation of nf-κb via tak1/ikk activation. mapks p38, jnk, and erk pathway are also activated, then subsequently activate creb and ap-1 transcription factors. both ap-1 and nf-κb activate proinflammatory cytokine expression, including tnfα, il-6, il-1β, and a number of other cytokines and chemokines. 308-311 the effects of hsp60s on host cells upon rna virus infection immunity modulation. few studies show the function of hsp60s in the life cycle of rna virus; however, the function of hsp60 in regulating host immunity has been widely studied. [312] [313] [314] [315] [316] the majority of studies show that hsp60 works as an activator of immune response. in the case of jev infection, hsp60 facilitates virus-induced inflammation by promoting il-1β production via increasing nlrp3 inflammasome activity and nfκb phosphorylation. 317 however, under certain conditions, viruses also utilize hsp60 to evade host cell immune response. the genome-wide rna interference (rnai) screen identifies the interaction of hsp60 with pb2 of influenza virus. 318 hsp60 helps pb2 translocation from the cytosol into mitochondria. 38, 39 then the mitochondrial pb2 interacts with and modulates the activity of mitochondrial antiviral signaling protein (mavs) to suppress interferon β (ifnβ) production. 38 therefore, hsp60 determines the effect of pb2 on both mitochondrial stability and the level of ifn-β production. 38, 39 besides, denv infection also elevates hsp60 expression. silencing of hsp60 results in an increase of ifn-α production and decrease of virus reproduction in macrophages. 319 however, the detailed mechanism remains elusive. apoptosis regulation. viruses use different mechanisms to modulate apoptosis. in the case of hcv infection, ros production is regarded as the major contributor of hcc although it also promotes cell apoptosis. 320 study shows that the n-terminal domain of hcv core protein can induce ros production by interacting with hsp60 and inhibiting the normal function of hsp60 in releasing protein stress. 321, 322 while another rna virus, rotavirus sa11, tries its best to delay the early apoptosis through modulating hsp60 stability. 323 hsp60 helps to translocate nsp4 protein of rotavirus into mitochondria from cytosol and induces apoptosis. 323 to yield more viruses, rotavirus infection increases the hsp60 phosphorylation at tyr 228 by activated src kinase that leads to the ubiquitin-mediated proteasomal degradation. 323 even though virus postpones apoptosis via hsp60, the main function of hsp60 is to refold proteins in mitochondria. 323, 324 the function of hsp60 family in dna virus infection virus replication. in the previous sections, we have discussed that hsp90 is important for rt-ε rna complex formation in hbv infection. however, it has been shown that hsp60 directly regulates rt activity before the rt-ε rna complex formation. 325, 326 hbv replication is markedly suppressed when hsp60 is knocked down by specific sirnas. 325 hsp60 transiently interacts with rt to activate rt in an atp-dependent manner. 326 upon rt activation, hsp60 immediately dissociates from the hsp60/rt complex without being encapsidated into viral nucleocapsid. 326 more detailed research shows that at least one of two rt fragments, residues 1-199 of terminal protein (tp) domain and 680-842 of rnase h (rh), is necessary for hsp60 binding. 327 the tp domain is also responsible for the binding of hsp90 in the rt-ε rna priming step. 138 apoptosis regulation. similar to hcv infection, hbv infection also induces strong apoptosis which is thought mainly contributed by hbx protein. 328 studies show that overexpression of hsp60 facilitates hbx-induced apoptosis. 329, 330 the interaction of hbx and hsp60 has been observed and confirmed by different methods including affinity purification, mass spectrometry and co-immunoprecipitation. 329 hsp60 binds on a small domain (residues 88-117) of hbx. 329 furthermore, hsp60 also forms a complex with hbx and hsp70 in the mitochondria. 330 however, the mechanism of how hsp60 enhances the apoptosis remains unclear during hbv infections. immunity modulation. hsp60 is involved in both the innate and adaptive immune response. 314 here, we focus on how hbv harnesses hsp60 to evade host immune response. numerous studies show that hbv employs active means to escape innate immune response and induce immunosuppression. 331 among these strategies, hbv infection increases the population of cd4 + cd25 + t regulatory cells (tregs) which can produce an amount of il-10 and tgf-β. 332 il-10 is also called cytokine synthesis inhibitory factor (csif) displaying anti-inflammatory properties. 333 it influences both the first and the second line of immune defence. 334 hbv infection increases serum shsp60 level and makes use of hsp60 to activate cd4 + cd25 + regulatory t via tlr2/myd88/il10 signaling. 335 the function of hsp60 family in retrovirus infection although the role of hsp60 in dna/rna virus infection process has been widely studied; only limited knowledge has been obtained in retrovirus infection. hsp60 is encapsidated into hiv particles, 336 but we have no idea about its function. the integrase catalyzes the integration of hiv-1 pro-viral dna in the host genome. 337 a small portion of hsp60 colocalizes and interacts with the viral integrase (in). 338 hsp60-hsp10 complex maintains the integrase at an active form and stimulates its activity in an atp-dependent manner. 338 the integration is a critical step for successful infection of hiv-1. the function of hsp40 family in rna virus infection virus replication. hsp40s regulate rna viral replication by modulating polymerase activity, replication complex and nuclear transportation. in jev-infected cells, hsp40/dnaj homolog hdj2 interacts and colocalizes with ns5 protein, an rdrp essential for viral rna genome replication. 339 overexpressed hdj2 promotes jev replication significantly. however, how hdj2 modulates ns5 activity remains elusive. 339 influenza virus also takes advantage of hsp40 to promote replication by assisting vrc to relocate into nucleus. the replication of influenza virus occurs in the nucleus of host cells; therefore, nuclear trafficking of viral ribonucleoprotein (vrnp) complex is required. the vrnp is composed of viral rna (vrna), polymerase heterotrimer (pa, pb1, pb2) and nucleoprotein (np). 340 np has a key function of interacting with importins through its nuclear localization signals. 341, 342 hsp40s have two strategies to help vrnp transport into nucleus. first, hsp40/ dnajb1 interacts with np at the early stage of infection and ensures efficient association between np and importin alpha. 343 this interaction is mediated by the j domain of hsp40 and the nterminal region of np. 343 another strategy is that hsp40/dnaja1 binds with the pb2 and pa polymerase subunits, then cotranslocates into nucleus with pb1-pa complex. 344 besides, hsp40/dnaja1 enhances viral rna synthesis both in vivo and in vitro. 344 different from dnajb1, dnaja1 mainly depends on its c-terminal substrate-binding domain instead of typical j domain to manage viral rna synthesis. 344 in the replication process, hsp70 is reported to enhance polymerase nuclear translocation. 217 thus it is proposed that dnaja1 cooperates with hsp70 and assist rna polymerase nuclear import. 217, 343 virus gene expression. viral rna replication produces plenty of double-stranded rna (dsrna) molecules. host cell detects these dsrna and activates interferon-induced protein kinase (pkr) to restrict viral replication by phosphorylating eukaryotic initiation factor eif2α and preventing protein synthesis. 220, 221, 345 however, in order to escape from the antiviral response of host cells, the influenza virus smartly blocks the activation of pkr/eif-2α. influenza virus ns1 directly binds the n-terminal of pkr and inhibits pkr activation. 225 besides, np protein also interacts with hsp40, leading to dissociation of hsp40 and p58ipk. 346 it is reported that type iii hsp40/p58ipk is an inhibitor of pkr, [222] [223] [224] 226, 347 while hsp40 is the inhibitor of p58ipk. 225, 348 therefore, the dissociation of hsp40 results in activation of p58ipk. subsequently, p58ipk inhibits the activity of pkr/eif-2α. as a result, influenza virus releases the inhibition of protein synthesis. however, in the late stage of infection, influenza virus m2, hsp40 and p58ipk form a stable complex that would lead to pkr activation, er-stress-induced cell death and virion release. 349 protein maturation. flavivirus genome encodes a large polyprotein which is later cleaved into several mature structures and nonstructure proteins. the mature proteins then form vrcs. at the beginning, hsp40 family protein dnajc14 participates in the vrc formation of flavivirus. during yellow fever virus (yfv) infection, dnajc14 is recruited to non-structural protein clustering sites with ns3 and ns5 to form vrc. 350 however, either knockdown or overexpression of dnajc14 inhibits yfv and hcv replication. 350, 351 later, it has been demonstrated that dnajc14 overexpression affects yfv polyprotein processing and alters vrc assembly. overexpression of dnajc14 alters the cleavage sites of ns3/4a and ns4a/2k and gives rise to abnormal ns3 to ns3-4a ratios, suggesting that the chaperone activity of dnajc14 modulates ns3/ 4a/2k cleavage that ensures appropriate expression level of ns3 and ns4a. the inhibition of vrc formation upon ectopic expressing dnajc14 is caused by chaperone dysregulation. 351, 352 immunity modulation. hsp40s sometimes act to help the virus evade host immunity, while sometimes it exhibits antiviral activity by increasing host immune response under certain conditions. it is reported that the expression of dnajb1/hsp40 and hsp70 is induced by polyi:c stimulation. 353 hsp40 cooperates with hsp70 to suppress the mda5/mavs pathway though interacting with mda5 and inhibiting mda5 multimer formation. 353 however, dnaja3 shows its ability to suppress virus replication. during hfdv infection, vp1 is able to suppress the type i interferon signaling via suppression of phosphorylation, dimerization, and nuclear translocation of irf3. 354 however, dnaja3 induces lysosomal degradation of vp1 protein. therefore, dnaja3 indirectly stimulates the immune response of host cells. 354 the function of hsp40 family in dna virus infection virus replication. evidence suggests that hsp40s regulate the initiation of dna virus replication. in the case of hpv replication, it starts with the recognition of protein e2 on the origin (ori) sequence and recruitments of replication initiator e1, which displays atpase and helicase activities. [355] [356] [357] hsp40 (hdj1 and hdj2) and hsp70 enhance e1 binding on the ori independently and additively. hsp40 directly binds with e1 and remains in the e1ori complex, whereas hsp70 transiently interacts with e1 in an atp-dependent manner. 355 subsequent study reveals an additional role of hdj2 in facilitating e1 helicase function by replacing e2 in the e1/e2/ori complex. 358 the stable association of e2 to ori flanking the e1 binding site may act as a dna clamp to prevent dna unwinding. similarly, hsp40 (htid1) also has similar functions to hdj1 and hdj2 with independent chaperone activity. hsp40 interacts with hsv-1 replication initiator protein and helicase protein ul9, thereby promotes their binding to the replication origin. 359, 360 this provides another example of the involvement of j-proteins in the replication process of eukaryotic dna viruses. except for enhancing hbv replication, a possible negative role of hsp40 has also been reported. the core protein is a key component of viral capsid and essential for virion assembly, while hbx is a multifunctional virulence factor implicated in viral replication and hepatocarcinogenesis in human. a yeast 2-hybrid approach is used to identify interactions between the core protein and two hsp40s, hdj1 and htid1. 361, 362 individual expression of each hsp40 in hepatocytes transfected with a replicationcompetent hbv construct shows an inhibitory effect on both viral replication and capsid formation. further studies reveal that both core and hbx proteins are destabilized by co-expression with hdj1 or htid1; because they are targeted for enhanced proteolytic degradation. except inhibiting hbv replication, hdj1 also facilitates proteasome-mediated degradation of hbx. 361 virus induced cellular transformation. as mentioned above, some viruses can induce cell transformation and tumorigenesis. here, we discuss the role of hsp40 proteins on virus-induced cellular transformation. hsp40 may have a negative role in hbv-induced cell transformation. it has been demonstrated that hbx is the major factor for induction of hepatocyte transformation. [363] [364] [365] [366] nevertheless, overexpression of hsp40s (hdj1 and htid1) significantly enhance the proteasome-mediated degradation of hbx. another study suggests that htid1 interacts with e7 oncoprotein and promotes the cellular transformation by hpv16; 275 because the binding sequence of e7 shares high similarity to the tag oncoproteins of sv40. nuclear entry of viral pre-integration complex. hiv can efficiently infect nondividing cells. 367 this requires active transport of the viral pre-integration complex (pic) into the nucleus without breaking down nuclear membrane. some components of pic implicated in regulating nuclear import include the central dna flap and viral proteins in, ma, and vpr of hiv-1 (or vpx of hiv-2). 288,368-372 hsp40/dnajb6 interacts and enhances the nuclear localization of vpx as well as promotes the nuclear import of viral pic. 373 similarly, dnajb6 also promotes vpr nuclear localization during hiv-1 infection; 374, 375 particularly, the long isoform of dnajb6 is extremely important in this process. 375 the expression level of dnajb6 s/l isoform is regulated by the polyadenylation factor cstf64. high level of cstf64 favors dnajb6-s isoform production, whereas a low level of cstf64 enhances dnajb6-l isoform production. 374 regulation of viral gene expression. a notable example of hsp40 involvement in regulating retrovirus gene expression is its importance in enhancing the gene expression during hiv-1 infection. 376 nef protein, an important viral protein associated with pathogenesis and disease progression, stimulates hsp40 expression by enhancing hsp40 promoter activity via hsf1 transcription factor. [377] [378] [379] hsp40 and nef co-translocate into nucleus where they become a part of cdk9-associated transcription complex to enhance long terminal repeat (ltr) mediated gene expression. 376, 380 the binding of hsf1 on hiv-1 ltr promoter induces viral gene expression directly. 380 interestingly, hsp70 seems to act contrary to hsp40, which also presents in the nef-hsp40 complex. different from hsp40, hsp70 suppresses viral replication and gene expression; while hsp40 rescues the hsp70downreguled viral gene expression. hsp70 inhibits cdk9 phosphorylation, an essential event for high-affinity binding of hiv-1 transactivator of transcription-positive transcription elongation factor b complex for transactivating response rna. 381 it is also reported that some other hsp40 family proteins negatively regulate hiv replication. hsp40a1, b1, b6 and c5 (but not c3) are able to limit hiv-1 production, while they have no effect on viral gene expression upon infection by adenovirus, hsv-1 or vaccinia virus. the conserved dnaj domain is suggested to be responsible for the inhibiting hiv-1 reproduction. the hsp70/ hsp40 complex specifically recognizes and inhibits the rev translation or accelerates its degradation, leading to inhibiting viral gene expression. 382 small heat shock proteins are the most upregulated proteins identified in host cells under stress conditions, for example, when cells are exposed to elevated ros level, abnormally high temperature, or pathogen invasions. 383 in most cases, shsps are responsible for recognizing misfolded proteins and transferring them to other atp-dependent chaperones for proper folding, or proteasomes or autophagosomes for degradation. 49, 384 hsp27 is one of the most ubiquitously expressed shsps with the highest level in skeletal, smooth, and cardiac muscles. 59, 385 like all other shsps, hsp27 shares a highly conserved α-crystallin domain, or socalled c-terminal domain. it contains 6-8 β-strands, forming 2 β-sheets as intermolecular interaction sites. 41, 59 because of the importance of hsp27, in this section, we mainly focus on the function of hsp27 in virus infection. hsp27 has been shown particular importance in viral infections. rajaiya et al. suggested that the association of hsp27 with p38 or nfκb/p65 plays key roles in controlling the expression of proinflammatory mediators in virus-infected cells. 64 fukagawa et al. suggested that the phosphorylated hsp27 is upregulated through the pi3k/akt pathway upon ebv infection. 386 we would discuss the special roles of hsp27 in different viral infections in the following sections. the function of hsp27 in rna virus infection hsp27 is upregulated during ev-a71 infection by proteomics analysis. knockout of hsp27 results in the suppression of virus replication and protein expression level, while their restoration appears after hsp27 is restored. also, hsp27 enhances viral ires activity by promoting 2a pro -mediated eif4g cleavage. 57 interestingly, the nuclear translocation of hsp27 from cytosol is reversely correlated with the relocalization of rna chaperone hnrnpa1 from nucleus to the cytosol for initiating viral protein translation upon ev-a71 infection. however, knockout of hsp27 blocks hnrnpa1 cytosol relocolization, indicating a fundamental role of hsp27 in regulating the import/export of nuclear proteins during virus infection (fig. 6) . 383 hsp27 is rapid upregulated at the early stage (4 hours post infection) of coronavirus infection, 387 suggesting an important role in virus early replication and possibly a good target for treating sars-cov-2 infection. swine fever virus (csfv) is a member of the family flaviviridae. hsp27 is found to bind with ns5a, which is a non-structural protein in response to viral replication and assembly. hsp27 depletion enhances the virus replication while the replication is suppressed by ectopic expression of hsp27 through activating nf-κb signaling in pk-15 cells. 388 porcine epidemic diarrhoea virus (pedv) infection causes high fatality in swine. the hsp27 is obviously upregulated in pedv infected marc-145 cells. 389 however, the virus titer is declined by ectopic expression of hsp27. hsp27 could activate nf-κb signalling and enhance the transcription of ifnβ and downstream interferon stimulated genes (isgs). 389 the function of hsp27 in dna virus infection pro-inflammatory cytokines play crucial roles in early antiviral infections. adenovirus infection causes a wide variety of diseases. 390 the internalization of adenovirus leads to the activation of erk1/2, which could in turn trans-activate nf-κb and ap-1 to induce pro-inflammatory cytokine il-8 expression in different experimental systems. [391] [392] [393] a study shows that downregulation of hsp27 leads to an increased release of il-8 from keratinocytes stimulated with uv or tnfα. the increase of il-8 is suppressed by nf-κb inhibitor and correlated with an enhanced iκb-α degradation and phosphorylated iκb-α accumulation in hsp27-depleted cells. in addition, hsp27 is shown to be associated with the iκb kinase (ikk) complex. because the synthesis of prostanoid pge2 and il-8 is regulated by nf-κb, it could be a likely mechanism of hsp27 in modulating inflammatory cytokine production. 70 further studies also show that hsp27 is of particular importance in the cyclooxygenase-2 and il-6 expression via activating p38 mapk/mk2 signalling and the consequential stabilization of cyclooxygenase-2 and il-6 mrnas. 68 aside from its involvement in pro-inflammatory response regulation, researches have also found the antioxidant role of hsp27 in regulating stress caused by ros. 49 it is commonly agreed that hsp27 regulates enzyme activities by upholding glutathione in reduced form, such as glutathione reductase and glucose-6phosphate dehydrogenase. 394 more recent evidence correlates the level of shsps with the intracellular content of iron, a catalyzer of hydroxyl radical generation. hsp27 also exhibits an important role in oxidized protein degradation machinery. 395, 396 however, there is also controversial evidence indicating the involvement of hsp27 in accumulation of oxidized proteins that benefits herpesvirus replication. experimental models are set up using two distantly related herpesviruses rhesus rhadinovirus (rrv) and hsv-1. they are close relative of kaposi's sarcoma-associated herpesvirus (kshv). the oxidized proteins are accumulated during these viral infections. results show the removal of only a part of oxidized proteins in a proteasome-dependent manner, while some others resisting degradation. 397 oxidized proteins resisting proteolysis become sequestered in foci within the nucleus and coincided with hsp27-enriched foci; although they are not associated with virus-induced chaperone enriched domains (vice). furthermore, the accumulation of oxidized proteins is more pronounced in hsp27-depleted cells. 398 one possible explanation is that hsp27 buffers the toxic effects of those defective proteins undergoing proteolysis through aggregation in the nucleus. the roles of hsp27 are most likely not mutually exclusive during virus infection. hsp27 also contributes to virus replication. porcine circovirus type 2 (pcv2) is a single-stranded dna virus that causes the postweaning multisystemic wasting syndrome (pmws) in pigs. the phosphorylated hsp27 is upregulated in the nucleus in pcv2infected pk-15 cells. hsp27 inhibitors such as sb203580 suppress pcv2 replication. the same result appears upon hsp27 knockdown. in contrast, ectopic expression of hsp27 promotes viral replication. 399 moreover, the phosphorylation of hsp27 is also upregulated in ebv-positive cells, as well as the phosphorylated (activated) akt levels. when ebv-positive cells are treated with pi3k inhibitors, the phosphorylated hsp27 level is decreased, suggesting that the phosphorylation of hsp27 is upregulated through the pi3k/akt signalling pathway upon ebv infection. 386 however, studies also show that hsp27 can both positively and negatively regulate the virus replication depending on the virus/ cell types. tong et al. reported that hsp27 works as an antiviral protein against hbv replication through enhancing ifn production in hepatocytes. the hsp27 expression level is increased in both hbv-infected human liver tissues and hbv-producing hepg2.2.15 cells. 400 the function of pdis on virus entry and uncoating the entry of some viruses into eukaryotic cells is governed by redox-regulated processes. one example is newcastle disease virus (ndv), a bird virus in the family of paramyxoviruses. this negative-sense, single-stranded rna paramyxovirus gains entry to its host cell through large conformational changes in its fusogenic f-protein, which involves thiol/disulfide exchange. 401 overexpression of pdi and erdj5 (a pdi family reductase with an extra j domain) leads to an increase of viral membrane fusion, indicating a route whereby virus can take advantage of the pdi family to gain access to host cells. 402 fig. 6 the controversial function of hsp27 during virus infection. hsp27 would enhance 2a protein-mediated eif4g cleavage, which would in turn promote ires function and viral genome replication. it correlates with hnrnpa1 translocation from host cell nuclei to cytosol, and the consequential virus protein translation. hsp27 is also able to suppress apoptosis via caspase 3 inhibition. on the other hand, hsp27 could help activating ikk complex, modulating nf-κb and ap-1 to induce the expression of pro-inflammatory cytokines reduces β1 and β3 integrins allowing denv entry. 403, 404 also, thiol blockers and pdi inhibitors decrease the entry of rotavirus in ma104 cells, indicating the involvement of thiols for infectivity. 405 the entry of hiv is regulated by pdis. the envelope of hiv becomes unhinged by pdi for entry. ryser et al. firstly reported that cleavage of two disulfide bonds in the gp120 surface component of the hiv-1 envelope is required for virion entry into cd4 + cells. in this process, the pdi on cell surface is responsible for this effect. 406 pdi inhibitors sufficiently prevent the reduction and block the cleavage of surface-bound disulfide conjugate, thereby prevent infection at the level of hiv-1 entry. 407 now, there are numerous studies on how envelope binds on host cells. results from different groups have demonstrated that gp120 moves laterally along the membrane surface until it collides with a patch of pdi in a domain of the membrane that distinguishes from a typical lipid raft. pdi reduces two disulfide bonds in gp120, producing conformation changes that likely stabilize the binding of gp120 to cd4 and expose the v3 loop for subsequent binding to the chemokine coreceptor. following this, gp41 undergoes rearrangement into its fusogenic intermediates and entry occurs. 408, 409 during the entry, galectin-9 binds pdi to regulate the redox environment on the cell surface and enhance hiv entry. 410 taken together, the reports from these three groups have profound implications for our understanding of the hiv virion surface structure and viral entry. the other examples are the nonenveloped polyomavirus (py). py particles contain a layer of coat protein vp1. this single protein, arranged as 72 pentamers, forms the shell surrounding the viral genome. 411 after internalization, py penetrates the er membrane to gain access to the cytosol and then the nucleus for viral genome transcription and replication. pdis isomerize or reduce the virus disulfide bonds to generate a membrane transportcompetent intermediate for host cell membrane penetration. 412 for example, sv40 entry involves caveolar/lipid raft-mediated endocytosis, vesicular transport to er and translocation into the cytosol. erp57 isomerizes the interchain disulfides connecting vp1 for virus uncoating. 413 the further study demonstrates that erp57 and pdi operate in concert with erp29 to unfold the vp1 c-terminal arm. pdi and erp72 reduce py, while erp57 principally isomerizes the virus in vitro. mutagenesis study subsequently identified that the residues c 11 and c 15 of vp1 are important for infection, suggesting a role for these residues during isomerization. 414 pdis regulate viral protein translation a little evidence shows that pdis are involved in viral protein translation. some positive single-stranded rna (+ssrna) virus depends on ires-mediated translation for viral protein synthesis. ev-a71 infection is potently inhibited by an active compound oblongifolin m (om), which is isolated from herb garcinia oblongifolia. further studies show that om suppresses the viral ires-mediated translation of polypeptide via suppressing erp57, and ectopic expression of erp57 increases the ires activity and partially rescues the decreased viral replication caused by om treatment. 415 the detailed mechanism how erp57 downregulates ires activity would be further investigated. several studies have demonstrated the implication of redox balance disruption in the establishment of viral infection and the progression of virus-induced diseases. and accumulated ros in turn may modulate the viral replication and cellular response that also contribute to viral pathogenesis. 416, 417 virus-induced oxidative stress has been reported during hiv, 418 influenza virus, 419 hbv, 420 hcv, 421 encephalomyocarditis virus (emcv), 422 respiratory syncytial virus (rsv), 423 and jev 424 infections. considering its thioredoxin-like sites, erp57 has been thought a main player in the mechanisms of cell protection against oxidative stress, regardless of its subcellular location. redox proteomics analysis of hpv positive tissues shows that the expression level of erp57 and gst is positively correlated with tissue redox status, suggesting its potential association with viral-induced oxidative dna and protein damages. 425 er stress is another consequence caused by virus activities. viral infection would lead to exploitation of the er membrane, accumulation of misfolded proteins, and imbalance of calcium concentration. influenza a virus (iav), hbv, jev, denv, and zika virus all hijack host cell process to enhance viral pathogenesis, such as facilitating viral folding and trafficking, affecting receptor interaction, and modulating host immune responses. [426] [427] [428] therefore, as a major factor in er stress response, erp57 would be critical for viral protein glycosylation and maturation, which may in turn affect virus release and infection (fig. 7) . the life cycle of most rna viruses is completed in the cytoplasm of host cells. to complete the life cycle, virus is often able to induce redistribution of many host cell proteins; particularly, those proteins involved in rna metabolism and functional regulation of viral rnas. hnrnps, such as hnrnp a1, 429 hnrnp c1/c2, 430 hnrnp d 431 and hnrnp i, 432 are mainly resident in the nuclear but quite often shuttle to cytoplasm for stabilizing the viral rna and initiating cap-independent translation. viruses employ various means to redistribute hnrnps. for instance, the ebov inhibits the nuclear import of hnrnp c1/c2. vp24 binds to npi-1 subfamily karyopherin-alpha nuclear import proteins at the c-terminal region (amino acids and prevents their interaction with tyrosine-phosphorylated stat1 (pstat1) and hnrnp c1/c2. the inhibition results in cytoplasm retention of pstat1 and hnrnp c1/c2. 430 435 however, the detailed mechanism of how 2a pro and core protein induce the redistribution remains unknown. virus replication. it has been well documented that hnrnps greatly contribute to the virus replication. for example, hnrnp i/ ptb and hnrnp c participate replication of different viruses, e.g., coronavirus, 436 hcv 437 and poliovirus. 438 the role of hnrnp i/ptbs in viral rna synthesis seems to vary among different viruses. ptbs bind ucuaa pentanucleotide repeats at ires and 3′-utr. ptbs modulate coronavirus rna synthesis. 436 however, the function of hnrnp i/ptb is to some degree complicated in hcv rna replication. it selectively interacts with the 3′ end of the hcv genomic rna. the upstream sl2 and sl3 stem-loop structures are essential for hnrnp-i/ptb i binding whereas the most 3′-terminal stem-loop sl1 is not needed. hnrnp i/ptb i and hnrnp c specifically bind the pyrimidine-rich region of in the 3′ntr of hcv rna genome. 437 possibly, hnrnp i/ptb i is recruited for helping to initiate viral negative-strand (−) rna genome replication, to stabilize rna genome, and/or to regulate the encapsidation of genomic rna. 439, 440 up to date, the detailed function of hnrnp i/ptb i binding on 3′utr has not been elucidated. some studies showed that ptbs have an inhibitory effect on the synthesis of hcv rna genome, 441 while aizaki et al. reported that ptbs are required for efficient hcv rna replication. 442 it has been postulated that they suppress the initiation of viral genome rna replication but enhance ires-mediated translation or facilitate the replication-translation switch. it is shown that hur can compete hnrnp i/ptb i binding on 3′ utr of the viral rna to facilitate la binding to the 3′ utr; while la protein is critical for hcv genome replication. [443] [444] [445] although hnrnp c has many similarities with hnrnp i and both bind with the 3′utr of the hcv rna genome to facilitate viral rna replication; 437 more details demonstrate that only hnrnp c binds the 3′-ends of viral rnas with both negative and positive polarities. 446 besides, hnrnp c also binds at the 5′-end on negative-strand rna of poliovirus to facilitate the synthesis of positive-strand rna; 438 while mir-555 decreases the expression of hnrnp c thereby inhibits poliovirus replication. 447, 448 virus rna splicing. the main function of hnrnps is modulating rna process and metabolism, including splicing, stabilization and transportation. it has been documented that hnrnp k helps the rna splicing of iav. iav is a major human pathogen with a genome comprised of eight negative-stranded rna segments. two viral rna segments, ns1 and m, undergo alternative splicing and yield several proteins including ns1, ns2, m1, and m2 proteins. two of the influenza virus rna segments generate spliced products: ns segment codes for non-structural protein ns1 and nuclear export protein nep/ns2; m segment encodes the matrix protein (m1) and ion channel (m2). ns1-bp properly splices the viral m1 mrna segment to yield the m2 mrna without affecting the splicing of m4 mrna or ns mrna segments. in this process, hnrnp k works as a mediator to bridge the interaction of ns1-bp (binding protein) and m mrna. lack of neither ns1-bp nor hnrnp k ensures the proper splicing of m mrna. 449, 450 further studies show that ns1-bp and hnrnp k bind m mrna downstream of the m2 5′ splice site (5′ ss). ns1-bp binds most proximal to the 5′ss, partially overlapping the u1 snrnp binding site, while hnrnp k binds further downstream and promotes u1 snrnp recruitment. mutation of either or both the hnrnp k and ns1-bp-binding sites results in m segment mis-splicing and attenuated iav replication. 451 virus translation. virus rna translation often harnesses host cellular proteins. most hnrnps positively regulate virus translation. positive-sense single-stranded rna viruses normally contain an ires sequence that drives viral protein translation independence of the cellular cap-dependent translation. hnrnps bind with the ires of different viruses to assist their translation. during hcv virus infection, hnrnp i, hnrnp l, hnrnp d, [452] [453] [454] hnrnp a1 and hnrnp k 455 all participate the translation of hcv viral protein by binding with 5′utr sequence. hnrnp i (ptb3), is one of the polypyrimidine tract-binding proteins (ptbs) that has been reported to bind viral rna recurrently. they could bind the ires of picornaviruses, including cardio-aphthovirus group (including emcv, ev-a71) and entero-rhinovirus group (poliovirus and hrv-2). 89, 456 ptbs also bind hcv utr regions, 441 calicivirus rna 457 and coronavirus rnas. 436 for picornavirus and hcv, it is proposed that ptbs could help ires folding into a translation-competent structure. 458 although unlike canonical transient interactions shared by rna chaperones, it is not clear whether ptbs are eliminated after the ires has folded properly. 459 ptb binds the 5' utr of hcv rna genome to initiate translation. in translation assays, ptb antibody efficiently blocks the ires-mediated translation in vitro. 460 three rrm motifs of ptb monomer directly bind with ires of fmdv to stabilize ires structure and enhance eif4g entry to ires. 459 hnrnp l is capable of binding the 3′ border of the ires. [461] [462] [463] hnrnp l binds with single stranded-hcv rna when preannealing singlestranded rna with mir-122. 463 hnrnp d, also referred to as aurich element rna-binding protein 1 (auf1), shuttles between the cytosol and nucleus. it is found that hnrnp d could also interact with the stem-loop ii of hcv 5′ utr, and its overexpression enhances hcv ires-dependent translation. 452 pim1 inhibitors induce hnrnp d relocalization from nucleus to cytosol so that it binds the ires and inhibit ev-a71 protein translation. 122 similarly, hnrnp k interacts with sl1 located in the 5′ -utr of hcv genome where is bound with mir-122 binding site. 464, 465 mir-122 is required for hcv replication. it binds at a conserved sequence in the 5′ -utr and increases the stability of hcv rna. [466] [467] [468] it is also reported that residues 25-91, a hydrophilic region near the n terminus of hcv core protein, binds to proline-rich domains of hnrnp k and negatively regulates the effect on human thymidine kinase transcription. 469 however, its function on viral replication is not well addressed. while hnrnp a1 binds with both 5′-and 3′-utr of hcv rna, they form a complex with ns5b and septin 6 to assist viral protein translation. the c-terminal of hnrnp a1 and n-terminal of septin-6 are required in the translation process. since hnrnp a1 has many homologous such as hnrnp a/b, hnrnp a2/b1, and hnrnp a2; all of them may substitute for hnrnp a1 in regulating ires-mediated translation. 470 the enteroviruses' ires sequence also interacts with hnrnps. hnrnp a1 specifically binds on the 5′-utr of ev-a71 and enhances ires-dependent translation. 471 apigenin, a dietary flavonoid, interacts with hnrnps and interferes with their rna editing activity. 472 the binding of hnrnp a1 with viral rna is significantly blocked when the cells are infected with ev-a71 upon treating with apigenin, leading to marked suppression of iresmediated translation. it is noted that hnrnp a1 redistribution is not affected in this experiment. recent studies show that hnrnp a1 cytoplasmic translocation is strictly regulated by some signalling or stress proteins, such as mink/p38 mapk pathway 473 and hsp27. 57 p38 inhibitors or hsp27 knockout dramatically block hnrnp a1 translocating from nucleus into cytosol, leading to inhibition of virus replication. 57, 473 except for promoting viral protein translation by binding with the viral ires sequence, hnrnps can directly bind with virus proteins to facilitate virus replication. upon cvb3 infection, 3c pro binds and cleaves hnrnp m to facilitate virus replication. 474 however, hnrnp m does not affect ires activity and rna stability. 475 hnrnp k is required for denv replication. 476 the core protein of dnev interacts with hnrnp k to release the inhibitory effects of hnrnp k on transcriptional activator c/ebpb. 477, 478 other studies demonstrate that hnrnp c1/c2 interact with viral rna and ns1 protein of dnev and facilitate virus reproduction. [479] [480] [481] [482] hnrnp d binds on both 5′-and 3′-utr of enteroviruses and inhibits translation without affecting rna decay. 434 on the other hand, virus also applies strategy to release the inhibitory effects of hnrnp d via protease 3cd cleavage. 434 the cytoplasm translocation of hnrnp d is dependent on the expression of 2a pro and viral rna replication. 433, 434 hnrnp d also inhibits nucleocapsid expression by interacting with an au-rich sequence (nt 41 to 60) in the 3′ utr of niv. 483 virus rna export. hnrnp a2/b1 interacts with ns1 of influenza virus, leading to decrease of the viral ns1 rna/protein levels as well as ns1 rna nuclear export. 484 rna polyadenylation. the gag gene of rous sarcoma virus contains a cis-acting negative regulator of splicing (nrs) element. the general function of nrs is usually manifested by binding serine/arginine-rich (sr) protein hnrnp h and u1/u11 snrnps, resulting in inhibition of splicing by acting as a decoy 5′ splice site. evidenced by in vitro polyadenylation analysis, a new function of nrs element is revealed that it is required for 3′ ltr polyadenylation. in this process, hnrnp h binds on nrs element and promotes polyadenylation; however, mutation of the binding sites of u1 and u11 snrnps to the nrs does not affect polyadenylation. 485, 486 an opposite result shows that polyadenylation stimulatory activity of nrs is dependent of u1 and/or u11 snrnp as well as sr proteins; while hnrnp h seems not participating in the splicing control or rous sarcoma virus polyadenylation. 487 the functions of rna chaperones in regulating viral activities of dna viruses virus replication. the transient lytic dna replication of hcmv relies on the cis-acting element orilyt, six viral-encoded core proteins, the proposed dna replication initiator protein ul84, ie2, irs1 and the gene products from the ul112/113 loci. here it is reported that hnrnp k is sufficient to interact with ul84 protein of hcmv thereby promotes viral replication. the interaction is further enhanced by another two virus proteins ul44 and ie2. 488 gene expression. hnrnps regulate dna virus gene expression at both transcriptional and post-transcription levels either positively or negatively. at the transcription level, hnrnp d0b and hnrnp a/b are capable of binding with cis-acting aagtatgca core element of hpv18 late promoter to suppress the late genes l1 and l2, which are components of virus capsid proteins. 425, 489 hnrnp k binds enhancer ii (enii) to promote hbv replication. ectopic expression of hnrnp k augments hbv replication; while hnrnp k knockdown significantly decreases hbv viral load. 490 further study reveals that apobec3b forms a super complex with hnrnp k that alters the enii binding activity via conformational change, therefore suppresses the s gene promoter activity. 491 in the course of hsv infection, the immediate early protein ie63 (icp27), hnrnp k and casein kinase 2 (ck2) together form a big complex. ck2 is capable of phosphorylating hnrnp k and icp27. the phosphorylated icp27 is responsible for its nucleocytoplasmic translocation and interaction with hnrnp k. up to date, the function of the complex formation is not well elucidated. 492, 493 it may affect icp27 to recruit the cellular rna polymerase ii for the transcription of certain late genes. [494] [495] [496] at the post-transcription level, hnrnps regulate the polyadenylation, splicing, and translation during dna virus infections. hpv genome can be divided into an early region and a late region, followed by the proximal early (pae) and the distal late (pal) polyadenylation signals, respectively. 497, 498 the virion production mainly depends on the differentiation-dependent induction of l1 and l2 late genes. it has been shown that hnrnp h downregulates the late gene l2 at an early stage by interacting with the multiple ggg motifs located 174 nucleotides downstream of the early polyadenylation signal pae. the hnrnp h binding promotes polyadenylation at early polyadenylation signal and inhibits the l2 mrna production, since l2 mrna production need read-through into the late region and polyadenylation of the late transcripts at the pal. 499 while at the late infection stage, hnrnp h is captured by l1 to release the inhibitory effect on l2. 499, 500 this process is called late gene autoregulation which enables rapid viral capsid protein production. 500 another example for hnrnps modulating the polyadenylation is that sm polymerase (pol) mrna of ebv early protein is cleaved and polyadenylated inefficiently. 501 under certain conditions, ebv early protein sm may harness hnrnp a1 and hnrnp c to help the processing of polymerase mrna. viral rna splicing is also modulated by hnrnps. hnrnp a1 negatively regulates the expression of hpv late genes by affecting rna splicing. it directly binds with the late regulatory element (lre) in differentiated hpv16-infected cells. 502 hnrnp a1 binds with splicing silencer element to suppress the use of the 3′ splice site located immediately upstream of aug at late 1 (l1) mrna. hnrnp a1 inhibits the splicing of late mrnas through the splicing silencer sequence and prevents the premature expression of l1 gene. [503] [504] [505] on the other hand, there is another 17 nucleotides resident immediately downstream of the splice site which counteracts the effect of hnrnp a1-binding splicing silencers. 506 hnrnp i also interferes with the splicing inhibitory elements locating at the upstream and downstream of major late 5′ splice site sd3632, thereby activates the late gene expressions. 507 at the translation level, hnrnp i and hnrnp k inhibit the translation of hpv late gene l2 via binding on a specific cis-acting element in the 3′ end of l2 mrna; 508 whereas the inhibition of l2 translation can be disrupted by c-src-mediated phosphorylation of hnrnp k at multiple tyrosine residues. 509 cellular transformation. hnrnps also exhibit some other functions during virus infections. auf1 works as a major component of c promoter binding factor 2 (cbf2) to interact with ebna2 and mediate ebna2 targeting to the latency c promoter (cp) of ebv, thereby inducing b-cell immortalization and viral latency in humans. 510 auf1 also binds with the eber1 noncoding rna of ebv. in ebv-positive cells, eber1 is abundant; therefore it may compete with auf1-interacting targets in the host cells. 511 both ebna2 and eber1 are proposed to facilitate cell transformation. immunity modulation. aside from affecting virus life cycle directly, hnrnps are able to regulate viral activities through modulating immune response. during hsv-1 infection, hnrnp a/b form a complex with viral dna, followed by homodimerization and demethylation. these events result in translocating the complex to cytosol and activating natural immunity through type i interferon signalling. moreover, the complex promotes n6methyladenosine modification and cgas-sting-related mrnas translocation upon infection by dna viruses, further enhancing the immune response for virus elimination. 512 the function of rna chaperones in regulating the viral activities of retroviruses as we have mentioned above, hnrnp a1 can bind to rna/proteins and participate intracellular nucleo-cytoplasmic transportation, 513 as well as alternative splicing of mrna in eukaryotes. during retrovirus infection, hnrnps participate in viral rna transcription, 514 splicing, 515-517 translocation 516 and translation. transcription. in hiv-infected cells, viral nef protein is required for high-level viral replication. 518 it is reported that nef, eed, kinase lck and npkc subfamily (pkcδ/θ) form a complex nakc (nef-associated kinase complex) responsible for promoting viral replication. 519 later on, it is found that hnrnp k is also a partner of the complex. it bridges the interaction of nef, eed and the kinases. the hnrnp k-nucleated complex activates erk1/2 and results in suppressing hiv promoter, enabling suboptimal amounts of tat and transcription factors (e.g., nf-kb) for initiation of transcription. 514 post-transcription. hiv-1 takes advantage of alternative splicing to generate doses of messenger rnas to encode the various viral proteins. it is known that over 40 message rnas are created by alternative splicing from a single pre-mrna. 520,521 alteration of splicing patterns dramatically affects the infectivity and pathogenesis of hiv-1. 521, 522 the splicing of hiv mrna is mainly controlled at the early stages of spliceosome assembly on pre-mrna by a stepwise association of the small nuclear ribonucleoprotein particles (snrnps) u1, u2, and u5·u4/u6. the early steps include the recognition of the 5′ splice site and the branch point sequence by u1 and u2 snrnps, respectively. 523 rs splicing factors, which contain a domain rich in arginines and serines (rs domain), assist these steps. u2af, one of the splicing factors which consists of two subunits u2af65 and u2af35, interacts with the polypyrimidine tract and the 3′ splice site, respectively. 524 then the interaction mediates the association of u2 snrnp with the branch point sequence. [525] [526] [527] sr splicing factors are essential for virtually every step of spliceosome assembly, including early recognition of splice sites, recruitment of basic splicing factors to the pre-mrna, and formation of bridging contacts with other rs domain-containing splicing factors. 528 prior to forming spliceosomes, the pre-mrna is packed with hnrnps. 529 it has been documented that pre-mrnas bind with different subsets of hnrnps, indicating sequence specificity at some degrees. a direct role of hnrnps in constitutive splicing has not been observed; although it seems that the binding of hnrnps exhibits an unspecific nature. it is widely believed that hnrnps employ crucial functions of modelling pre-mrna structure and initiating recognition of splice sites. 530 the cis-acting sequence elements of cellular and viral pre-mrnas undergoing alternative splicing regulate the process either positively or negatively. they bind trans-splicing factor machinery together and form splicing complex. the majority of trans-acting factors are either hnrnps or sr family proteins. these proteins also regulate alternative rna splicing either positively or negatively. sr proteins often regulate splicing in a positive way while some hnrnps mainly do the job in a negative way. 528, 530 alternative incomplete splicing of hiv-1 genomic mrna produces more than 40 unique viral mrna species within an hiv-1-infected cell through highly accurate regulation by cis exon splicing silence elements (ess) and trans hnrnps. the production of tat, rev and vpr proteins is highly controlled because they play key roles in hiv-1 multiplication. ess2 is the first identified ess in the hiv-1 genome that locates downstream of 3′ ss a3 within exon 4 and specifically represses tat mrna splicing. 531 ess2 is mapped to a 10 nt core sequence cuagacuaga. 532 ess3, which locates at exon 7, represses splicing at 3′ ss a7. 533 fine structure mutagenesis indicated that ess3 is bipartite; and each sub-elements [agaucc (ess3a) and uuag (ess3b)] inhibits splicing independently. 534 a third ess (auaguuaguccuagg, essv), which locates downstream of 3' ss a2 in exon 3 within the vif coding sequence, represses splicing at 3′ ss a2. 535 to modulate the expression of tat protein, several hnrnps participate the splicing process by interacting with these ess, intron splicing silencer (iss) elements directly or interfering enhancer splicing element (ese) activity. for example, hnrnp a1 and hnrnp k synergistically bind on ess2 and inhibit the utilization of a3 splicing site. 536, 537 the uag triplets in ess2 is required for hnrnp a1 binding, and several hnrnp a1-binding sites are also found at sls3. the c-terminal gly domain of hnrnp a1 is important for the binding. sr proteins sc35 and srp40, the strong activators of site a3, have similar binding sites to hnrnp a1. hence, they may compete with each other to bind ess2 and ese2. 536 another study shows that hnrnp h displays an inhibitory effect on splicing. hnrnp h binds to ess2 and competes with u2af35 for binding to the exon sequence flanking 3′-splice site a3. this binding results in the inhibition of splicing at the 3′-splice site a3. 538 hnrnp a1 also inhibit tat splicing by binding with iss, which is independent of exon splicing silencer (ess2) and blocks the entry of u2 snrnp but not u2af65. [539] [540] [541] the up1 domain of hnrnp a1 is responsible for the iss binding, while the rgg domain of hnrnp a1 is not needed for the alternative splicing activity. 542, 543 in addition to ess2 and iss elements that are regulated by hnrnp a1, blocking the binding of sr protein sc35 on ese is another strategy for the virus to inhibit the tat expression at the early infection stage. hnrnp a/b proteins bind the ese locating at tat exon 2 to repress splicing, whereas sc35 binds the ese to activate splicing. the binding sites of hnrnp a1 and sc35 are overlapping within the juxtaposed ese/ess9. it seems that hnrnp a1 inhibits the splicing of the upstream intron by binding to the ess and directly masking the sc35 binding site. 544, 545 similarly, hnrnp a1 is also reported to compete with asf/sf2 at ese3/(gaa)3. therefore, the ratio of asf/sf2 to hnrnp a1 determines the utilization of ese3/(gaa)3 for activation or repression at site a7. 515, 541, 546 the expression of other hiv proteins is also regulated by hnrnps via modulating splicing. hnrnp a/b proteins inhibit the splicing at 3′ splice site a2 which is used to generate vpr mrna. hnrnp a/b proteins bind with essv with a consensus sequence pyuag. the splicing at splice site a2 is increased when the three pyuag motifs within essv are mutated, leading to increased vpr mrna levels and reduced skipping of the noncoding exon flanked by a2 and d3. 547 others reported that hnrnp d also binds at essv and functions as an inhibitor of splicing. 548 the binding of hnrnp a/b proteins at essv blocks u2af65 binding to the ppt of the repressed 3′ splice site and inhibits the splicing efficiency of 3′ splice site. 549 interestingly, hnrnp h is found to positively regulate the exon 6d splicing of hiv mrna. it interacts with the sequence cgga and enables u1 snrnp assembly onto exon 6d. 550, 551 further study shows that hnrnp h family proteins function as a splicing enhancer through enhancing the atp-dependent spliceosomal complex formation. 552 rna translocation. after transcription and splicing, lots of spliced and unspliced rna molecules of hiv exist in the nucleus. translocation of rna into the cytoplasm is dependent on the rev protein. on one hand, some hnrnps facilitate rna translocation. in hiv-1 genome, two sequences similar to the hnrnp a2 response element (a2re) function as cis-acting rna trafficking sequence that binds to the trans-acting trafficking factor hnrnp a2. the binding mediates a specific rna trafficking pathway characterized extensively in oligodendrocytes. a2re-1 locates within the major homology region of gag gene; while a2re-2 locates at a region overlapped between vpr and tat coding sequence. hnrnp a2 binds to both a2res in vitro to form a complex, which is necessary for rna transportation in oligodendrocytes in vivo. a2re-mediated rna transport requires both microtubule and hnrnp a2. if gag and vpr rnas containing a2re-1 and a2re-2 respectively are differentially labelled, it is observed that they are co-assembled into the same rna trafficking granules and cotransported to the periphery of the cell. although the tat rna contains a2re-2, it is not transported as efficiently as vpr rna. [553] [554] [555] hnrnp d also has a similar function in assisting rna translocation. 556 on the other hand, hnrnp a2b1, hnrnp c and hnrnp u can retain the hiv-1 genomic rna in nucleus. depletion of hnrnp a2b1 results in cytoplasmic redistribution of the virus rna genome in the absence of rev. 557, 558 an n-terminal fragment of the hnrnp u specifically targets the 3′ long terminal repeat (3′ltr) and blocks the cytoplasmic accumulation of mrnas, thereby affecting hiv gene expression. 559 besides hiv, the regulatory protein orf57 of kaposi's sarcomaassociated herpesvirus (kshv) also interacts with hnrnp k. ck2 can phosphorylate orf57 and promote orf57-hnrnp k interaction. the orf57-hnrnpk-ck2 complex may be important for rna export of kshv since orf57 is responsible for the nuclear export of viral mrnas. 560,561 hnrnp a1 interferes with the binding of rex to rexresponse element (xre). the rex protein of htlv-1 mediates the nuclear export of unspliced and incompletely spliced viral mrnas. this process partly depends on the binding of rex to rex-regulatory sequences xre. 562 viral protein translation. hnrnp a1 is induced to redistribute into the cytoplasm in the late infection stage of hiv and enhances the ires-mediated translation. 563 hnrnp d helps the redistribution of hiv mrna into the cytoplasm, and p45 and p42 isoforms increase viral gag protein synthesis while p40 and p37 suppressed this process. 556 hnrnp i interacts with a novel ires within a latently expressed gene (vcyclin) of kshv and enhances vflip expression. 564 the network of hsps and their co-chaperones is essential for cells to maintain protein homeostasis, including nascent protein folding, protein translocation across membranes, and protein complex formation. many stress stimuli can disrupt protein homeostasis such as thermal stress, nutrient starvation, chemical toxicity, oxidative stress, hypoxia, inflammation, and virus infection. [565] [566] [567] [568] stresses can lead to protein misfolding and aggregation that need to be resolved quickly to prevent cell and tissue damage. 568 hsps and their partners may facilitate protein degradation when cells cannot cope with massive damaged proteins. many lines of evidence suggest hsps as major factors for protein surveillance to protect host cells against virus infections. it was observed that hsp70/hsp90 expression increased dramatically in hsv-infected cells. 569 overexpression of hsp70 inhibits the translocation of viral capsid into nucleoli during flavivirus infections. although the interaction has not yet been investigated in natural infection, the in vitro findings have suggested that hsp70 may act as a negative regulator of viral capsid protein to protect host cells against wnv infection by abolishing cytotoxic effects induced by the viral capsid. 570 studies from ours and other groups show that almost all hsp subfamily members (hsp90s, hsc70, hsp70, grp78, erp57, hsp27) are highly responsible to enterovirus infection and play key roles in all stages of virus life cycle. not surprisingly, demonstrated that all most all hsps (hsp90s, hsp70s, hsp60s, hsp40s and hsp27) participate coronavirus infection, 145, 196, 387, 571 suggesting good target for anti-covid-19 drug development. as discussed in above sections, hsps exhibit immunomodulatory effects on both innate and adaptive immune responses. [572] [573] [574] hsps are capable of regulating not only intracellular innate immunity, but extracellular innate and adaptive immunity as well. hsps released from tumor cells can bind to surface receptors on antigen presenting cells (apcs) and elicit tumor-specific killers by means of antigen crosspresentation. 575, 576 for example, hsp27 positively modulates nf-κb phosphorylation, increases ifnβ transcription and downstream antiviral interferon-induced genes (isgs). pedv infection downregulates hsp27 expression to escape host antiviral surveillance. 389 additionally, extracellular hsps can also regulate cytokine production by dendritic cells (dcs), underscoring a connection between innate and adaptive immune responses modulated by hsps. 577 the purified or recombinant hsp70 can stimulate the production of pro-inflammatory cytokines and c-c chemokines in monocytes, macrophages and dcs, and upregulate mhc and costimulatory molecules in dcs. 578 binding of extracellular hsp72 to human monocytes and dendritic cells can induce the production of the pro-inflammatory cytokines including tnf-α, il-1β, il-6 and il-12 and ifn-γ. 579 in the process of above hspinduced cytokine production, hsps act as internal stimulus of the cd14/tlr (tlr2 and tlr4) complex signal transduction pathways that further activates nf-κb and mapks signalling. 580, 581 although the main functions of hsps are to protect cells upon stresses, they are often hijacked by many viruses to achieve successful infections. recent study has demonstrated that denv ns3 protein acts as a viral suppressor of rna silencing by interacting with hsc70, thereby interrupting host antiviral system by rnai pathway and subsequently enhancing denv replication. 582 ev-a71 takes advantage of hsps (hsp90, hsc70, erp57, and hsp27) to enhance 2a pro -mediated eif4g cleavage that is favour viral protein translation and blocks host protein translation. 57, 219, 415 viruses also initiate er stress in host cells after infection. they have to manipulate upr activation leading to cell survival, rather than inflammation induction, autophagy and apoptosis. denv modulates upr activation in a sequential manner to prolong the viral life cycle by allowing cellular adaptation to cope with the infection-induced er stress. upr is transiently induced by perk pathway resulting in phosphorylation of eif2α and subsequently translational attenuation in the early phase of infection. this transient event allows viral protein synthesis and accumulation that finally trigger upr by ire1-xbp1 (x-box binding protein 1) axis in the mid-phase of infection. this results in the increased expression of grp78 to facilitate protein folding and also the increased expression of gadd34 (growth arrest and dna damage 34), which dephosphorylates eif2ɑ, and thus allowing protein translation to be continued. the increased grp78 also prevents cellular apoptosis-mediated by chop (pro-apoptotic protein during stress persistence). finally, the increased viral proteins transiently trigger atf6 arm of upr to provide the active spliced xbp1 for sustaining upr activation in the late phase. 583 jev infection requires hsp70s in particular stages of its life cycle for survival and establishment of infection, including viral entry, replication, 194 and maturation. 195 cell-surface hsp70 directly interacts with jev envelope protein. 192 antibodies against hsp70 or 90 significantly block virus entry. 111 it is also observed that hsp70 colocalizes and directly interacts with jev replicase complex. in addition, hsp70 also interacts with ns3, ns5 and viral dsrna that stabilizes vrc formation during jev infection. 194, 584 er stress and antiviral in mammalian cells, the er stress is sensed and mediated by three er transmembrane receptors: pancreatic er kinase (pkr)-like er kinase (perk), inositol-requiring enzyme 1 (ire1) and activating transcription factor 6 (atf6). in resting cells, these three sensors are maintained in inactive states via interactions with the er resident chaperone grp78. when unfolded or misfolded proteins accumulate in the er lumen, grp78 dissociates from these three transducers, resulting in activation and initiation of the upr. viruses also take advantage of or revolt er stress response by different means for favour its life cycle at specific stage(s) of infection. the er stress response constitutes a cellular process triggered by a variety of conditions disturbing protein folding in the er. eukaryotic cells have developed an evolutionarily conserved adaptive mechanism, i.e., the er stress and upr, aiming to clear unfolded/misfolded, and excessive amount of protein production; thereby restoring er homeostasis. in cases er stress cannot be resolved, upr would be triggered and lead to cell death. er stress and upr could be observed in a large amount of virus infections, as most of viruses require host er machinery for viral protein synthesis and modification. virus infection usually causes er stress. when large amount of misfolded/unfolded proteins or excessively expressed viral proteins accumulate inside er lumen, er stress response is triggered as indicated by fast elevated expression of er chaperone proteins, including grp78/ bip, grp94, calnexin and calreticulin. it is well documented that er stress response is triggered by a number in some cases, er stress plays a role in virus pathogenesis. for instance, jev, bvdv, tulv, severe acute respiratory syndrome coronavirus (sars-cov) and wnv have all been shown to induce apoptosis through upr. [585] [586] [587] [588] [589] oxidative stress is mediated by er stress during hcv infection. 21 certain viruses modulate er stress response or preferentially activate different pathways of upr. hcv infection activates the atf6 pathway while blocks the ire1 pathway. instead, hbv infection stimulates both atf6 and ire1 signalling but has no effects on perk signalling although both virus infect the same kind of hepatocytes. 590-592 hsv-1 develops a virulence factor enabling dephosphorylation of eif2α, one of the downstream effectors of the perk pathway. 593 some consequences of upr are beneficial for viral life cycle. for example, atf6-induced expression of chaperone proteins may help viral protein folding and prevent protein aggregation. the perk-eif2α-activated atf4 may help re-establish cell metabolism and resume protein translation. the ire1-xbp1 pathway may facilitate virus replication by enhancing er protein-folding ability and er membrane biosynthesis. 594 the activated atf6 promotes the replication of asfv and lymphocytic choromeningitis virus (lcmv). 595,596 denv envelope protein directly interacts with grp78, which provides a scaffold for its association with two other chaperones calnexin and calreticulin. this complex significantly improves the proper folding and stability of denv e protein, thereby leading to increase of virus production. 597 other than denv, grp78 is also demonstrated to promote hcmv, jev and rgnnv infections. 195, 598, 599 the ire1-xbp1 pathway is activated by iav to facilitate its replication. 600 however, other upr outcomes are detrimental for virus replication. the perk-eif2α-mediated global translation attenuation is known as an antiviral response to restrict viral replication, such as infection by denv or wnv. 583, 601 the activated pkr phosphorylates eif2α at the ribosomal interface, which in turn causes a general inhibition of protein synthesis and blockage of vsv replication. 602 the ire1-xbp1(s)-mediated er-associated protein degradation (erad) pathway reduces intracellular hbv particles by degrading its envelope proteins. 603 another approach of er stress contributing to virus pathogenesis goes through modulating host cell immune responses. vsv, hcv and sars-cov are able to inhibit the type i ifn signaling pathway by activating the perk signalling, which leads to the phosphorylation-dependent ubiquitination and subsequent degradation of ifnar1, thereby promoting immune evasion and virus pathogenesis. 604, 605 wnv has also been reported to induce er stress and inhibit type i ifn signaling pathway for escaping from the host immune response. 601 us11 protein of hcmv activates upr to facilitate the degradation of class i major histocompatibility complex (mhc1), leading to immune evasion. 606 moreover, er stress is responsible for viral pathogenesis by interconnecting with the inflammatory responses. for example, hcv induces inflammatory responses by activating ire1, which interacts with traf2 to phosphorylate jnk, leading to activation of inflammation mediators. 607 ns4b and ns5a proteins of hcv activate nf-κb via er stress-elicited calcium depletion and ros production. 604 the x protein (hbx) of hbv induces the expression of cyclo-oxygenase 2 (cos2), a key mediator of inflammation, through perk-eif2α-activated atf4. 608 rna chaperons and human diseases caused by viral infections hnrnps impact mrna metabolism including transcript synthesis, processing and degradation as well as translation. 432 the function of hnrnps is determined or modulated by cellular localization. the mechanisms that regulate the nucleo-cytoplasmic shuttling are, therefore, of extreme importance. most hnrnps possess a conventional nuclear localization signal (nls) and are predominantly present in the nucleus during steady state. they are able to translocate in the cytosol upon post-translational stimulation or by the recruitment of other hnrnps. 609 post-translational modifications like methylation, phosphorylation, ubiquitination and sumoylation are reported to affect biological activity and subcellular localization of hnrnps. 610 as stated in the above sections, virus always employs different strategies to redistribute hnrnps in host cells. 430, 431, 433, 434 how dysregulation of hnrnps causing human diseases has gained an increasing interest. the expression level of hnrnps is altered in many types of diseases, including varieties of human cancers and neurodegenerative diseases (e.g., spinal muscular atrophy (sma), amyotrophic lateral sclerosis (als), alzheimer's disease (ad), and fronto-temporal lobe dementia). in this section, we mainly focus on the relationship between hnrnps and diseases caused by virus infections. enteroviruses are common pathogens that cause human diseases worldwide. although most enteroviral infections are subclinical, they may cause a wide spectrum of diseases including mild upper respiratory illness (common cold), febrile rash (hand, foot, and mouth disease and herpangina), aseptic meningitis, heart failture, pleurodynia, encephalitis, acute flaccid paralysis (paralytic poliomyelitis) and neonatal sepsis-like disease. 293, 611 besides, several studies showed that enterovirus sequences could be detected in neuronal cell bodies of the spinal cord of 60-88% amyotrophic lateral sclerosis (als) patients, suggesting that persistent enterovirus infection may have a relationship with als. 295, 297, 299 enterovirus infections are supposed to cause or increase the risk of als; because the replication and translation of poliovirus, ev-a71, and coxsackievirus redistribute numerous hnrnps (such as hnrnp a1) into the cytoplasm, the same localization during als pathogenesis. for example, hnrnp k, hnrnp a1, hnrnp m and hnrnp d shuttle into cytoplasm to assist virus replication or translation. 300, 455, 472, [612] [613] [614] dislocated hnrnp a1 and loss of splicing function have been regarded as a toxic mechanism in als pathogenesis. 301 hsv infection is one of the most common causes of infectious disease in humans. 302 hsv infection often causes watery blisters in the skin or mucous membranes of the mouth, lips, nose, or genitals. 615 as neurotropic and neuroinvasive viruses, hsv-1 and -2 persist in the infected individuals through hiding in the neuron cells from immune surveillance. when the carrier's immunity compromised, hsv is reactivated that causes new sores. more seriously, accumulating evidence shows that hsv infection is associated with the pathogenesis of alzheimer's disease. 105, 616, 617 during hsv infection, hnrnp k positively promotes hsv replication; 618 while hnrnp a2/b1 negatively regulates hsv replication by triggering immunity response. 512 hbv, hcv, ebv, hpv and hiv are oncogenic viruses that account for over 20% of human cancers. hcv or hbv infection leads to a wide spectrum of liver disease ranging from acute hepatitis (including fulminant hepatic failure) to chronic hepatitis, cirrhosis, and hepatocellular carcinoma (hcc). 619,620 hdv can only infect people who are already infected with hbv. the coinfection of hdv and hbv increases the risk of liver cirrhosis and liver cancer. 621, 622 except for damaging the liver, 10% of hbv infected patients also have other symptoms outside the liver, such as serumsickness-like syndrome, membranous glomerulonephritis, acute necrotizing vasculitis (polyarteritis nodosa) and papular acrodermatitis of childhood (gianotti-crosti syndrome). 623 multiple steps of hcv reproduction need the presence of hnrnps. the rna replication of hcv needs the presence of hnrnp i, hnrnp c; 437, 442, 446 while virus rna translation requires hnrnp a1, hnrnp d, hnrnp i, hnrnp k and hnrnp l. 452, 460, [462] [463] [464] [465] besides, hnrnp a2, hnrnp l, hnrnp u and hnrnp k are highly expressed in hcc tissue. they promote liver cancer progression. 8, 304, 305, 624 ebv has been implicated in several diseases, including infectious mononucleosis, 625 burkitt's lymphoma, 626 hodgkin's lymphoma, 627 nasopharyngeal carcinoma, 628 multiple sclerosis 629 and lymphomatoid granulomatosis. 630 other diseases associated with ebv infections include gianotti-crosti syndrome, erythema multiforme, acute genital ulcers, oral hairy leukoplakia, 631 and disorders related to α-synuclein aggregation (e.g. multiple system atrophy, dementia with lewy bodies, and parkinson's disease). 632 during ebv infection, hnrnp a1 and hnrnp c assist the polyadenylation of ebv rna. hnrnp d takes part in cellular transformation-induced by ebv. 510 hpvs are a group of dna viruses with more than 150 members that infect cutaneous and mucosal epithelia. the acute infection of hpv causes benign cutaneous lesions such as non-genital and genital warts, or flat cervical condylomas. 633 about 15 hpv subtypes that infect genital tract are capable of inducing malignant tumors, most commonly in the cervix. 308,309 these cancer-associated hpvs are grouped into high-risk types, while those not associated with cervical cancer are regarded as low-risk types. 307 most infections by low-risk type hpvs are asymptomatic, except for infection by hpv6 and hpv11 that cause most cases of genital warts (condyloma acuminatum). 309 hpv-related cancers are the result of long-term persistent infection with high-risk types. hpv16 and hpv18 are the most common carcinogenic types which account for a larger proportion of cervical cancers, squamous cell cancers and adenocarcinomas in all regions. 311 hpvs are also implicated in the development of a variable proportion of vulvar, vaginal, penile, and oropharyngeal cancers, anal cancer, 634 head and neck cancers, 635-637 lung cancer and skin cancers. hnrnp a1 is involved in splicing of hpv rna; while hnrnp h plays a role in the polyadenylation of hpv. hnrnp k and hnrnp i are required for viral protein translation. besides acquired immunodeficiency syndrome (aids), hiv infection is also able to cause human cancers because hiv infection impairs immunity system. people with aids are not only more easily infected with bacteria, viruses, fungi, and parasites; 638 but at high risk for developing various viral-induced cancers as well, including burkitt's lymphoma, kaposi's sarcoma, primary central nervous system lymphoma, and cervical cancer. 639 515, [532] [533] [534] [535] [536] [537] [544] [545] [546] [547] [548] [549] [550] [551] [552] [553] [554] [555] [556] [557] [558] [559] 564 potential therapeutic value by targeting stress proteins interest in targeting stress proteins in various diseases is increasing overtime. in this part, we will discuss the potential therapeutic value by targeting stress proteins. first, targeting stress proteins have a wide spectrum of antiviral ability. for example, hsp90 inhibitors, which are originally developed for anticancer, have been demonstrated to possess antiviral activity in cultured cells against poliovirus, rhinovirus, ev-a71, 107,109 coxsackievirus, 124 rsv, 112, 113 vsv, paramyxoviruses (hpiv2, hpiv3, sv5, sv41), influenza virus, 126 chikv, 114 hcv, 115, 124 and hsv, 131, 144, 145, 148, 149 hbv, 135, 169 ebv, 146, 147, 163, 164 hcmv 151, 152 and htlv. 153, 185 depleting hsp60 by sirna functionally suppresses infections by influenza virus, 38,39 denv, 319 hbv, 325, 329, 331 hcv, 321, 322 rotavirus, 323 and hiv. 338 targeting hnrnp a1 is able to inhibit the reproduction of hiv, [539] [540] [541] 563 hpv, 503-505 hcv, 471 ev-a71 472 and htlv-1. 562 inhibition of hnrnp c could be a strategy to combat viral infections by hcv, 438 [461] [462] [463] 475 therefore, stress proteins are particularly attractive as antivirals targets for those lacking therapies and newly emerging viral diseases. second, under disease situations, the cellular need of stress proteins is usually stronger than that in normal conditions which enable specific selectivity of those drugs targeting stress proteins. for example, mutant p53 relies much more on hsp90 function than wild type p53. hsp90 inhibitor gm can easily disturb the association of mutant p53 with hsp90, resulting in mutant p53 degradation while not affecting wild type p53. 310 additionally, stress proteins derived from stressed cells display higher affinity to clients and inhibitors. tumor cell-derived hsp90 exhibits a 100fold higher binding affinity for 17-aag than that from normal cells, since tumor cell-derived hsp90 complexes with activating cochaperones p23 and hop exhibit increased atpase activity and possess higher affinity to hsp90 inhibitors. in contrast, hsp90 in normal cells exists as an uncomplexed species with low atpase activity and low affinity to hsp90 inhibitors. 294 further study demonstrates that the difference is caused by a distinctive portion of hsp90 forming complexes in cancer cells with oncogenic partners, such as bcr-abl-hsp90 complex in mutant b-raf-hsp90 in skmel28 melanoma cells, k562 chronic myeloid leukemia cells, her3-hsp90 and raf1-hsp90 complex in mda-mb-468 breast cancer cells. hsp90 inhibitor pu-h71 selectively binds to specific hsp90-oncoprotein networks in these cancer cells. 570 these characters of cancer cells enable stress proteins to exhibit stronger biological activity and to be discriminated by their inhibition under stress as compared with normal conditions. similarly, inhibitors of stress proteins show high prospect for antiviral. hsp70 inhibitor jc40 inhibits pan-flavivirus (denv2 and denv4) replication in mddcs, with negligible toxicity to host cells. 640 these experiments highlight the feasibility of using hsp inhibitors therapeutically in humans. third, it is not observed drug resistance using hsp inhibitors for antiviral. for instance, hsp90 inhibitors are refractory to develop drug resistance. this is clearly demonstrated in rsv infection. 113 when rsv is repeatedly treated with hsp90 inhibitors, no drug resistance was observed either in extensive passaging of the virus in cultured cells or in mice undergoing long-term treatment with hsp90 inhibitors. similar result is also observed in denv infections. 640 treatment of denv up to 10 passages with hsp70 inhibitor jc40 does not cause any drug resistance. the lack of viral drug resistance to hsp90 and hsp70 inhibitors suggests that such an antiviral approach may be particularly useful for treating chronic viral infections in which drug resistance is most frequently observed. the three features of stress proteins inhibitors make them extremely powerful antiviral agents, suggesting great application potential for treating the diseases caused by virus infections, such as covid-19. challenges and perspectives: target-based drug development targeting hsp90 for antivirus drug development. hsp90 is thought to be the most abundant and evolutionarily conserved heat shock protein. a unique pocket in n-terminal region of hsp90 is required for binding with atp and co-chaperones. 641 hsp90 forms a flexible dimer by interaction of c-terminal domains. the formation and dissociation of compact dimers involving nterminal domains are important for the molecular chaperone activity. 642 the middle domain of hsp90 tends to recruit and facilitate unfolded client proteins to assemble. the c-terminal domain of hsp90 possesses a highly conserved peptide sequence for interacting with co-chaperones. 643 over 20 co-chaperones selectively interact with hsp90 to regulate atpase activity and recruit client proteins to assemble a big complex under certain conditions. [642] [643] [644] [645] it has been shown that more than 300 client proteins require hsp90 and co-chaperones for folding and maturation. 646 the mechanism of selectivity may rely on the direct interaction of co-chaperones with specific clients. therefore, most hsp90 inhibitors achieve their inhibitory effects by suppressing the atpase activity or disrupting the interaction between hsp90 and its co-chaperones. expression of hsp90 and its client proteins are increased during viral infection and in most cancer cells. hsp90 as an effective anticancer drug target has already grabbed attention; and a series of hsp90 inhibitors as potential drugs have been intensively investigated in the laboratories, preclinical and clinical scenarios. the successful use of hsp90 inhibitors in cancer therapy makes it much easier to apply them for treating virus infections. as described above, we have comprehensively summarized the functions of hsp90 and its client proteins in a diversity of virus reproduction processes. the potential of hsp90 inhibitors has been well demonstrated to protect cultured cells against infections by ev-a71, 107,109 poliovirus, rhinovirus, coxsackievirus, 124 paramyxoviruses (hpiv2, hpiv3, sv5, sv41),vsv, rsv, 112,113 influenza virus, 126 chikv, 114 hcv, 115,124 hsv, 131,144,145,148,149 hbv, 135,169 ebv, 146, 147, 163, 164 hcmv, 151, 152 and htlv. 153, 185 notably, administration of hsp90 inhibitors to infected animals exhibits little toxicity while potently suppresses the replication of poliovirus, 107,109 ebv, 163, 164 hbv, 135 chikv 114 and hcv. 647 these experiments highlight the feasibility of using these inhibitors therapeutically in clinic. here we would briefly enumerate the present hsp90 inhibitors and their potential for antiviral therapies. most hsp90 inhibitors hamper hsp90 function by competitively binding to the atp binding site of hsp90, blocking the interaction with co-chaperones, or modulating acetylation. by doing so, we also try to address the possibility of repurposing hsp90 inhibitors as candidate antiviral drugs ( table 2) . inhibitors targeting hsp90 atpase activity. some hsp90 inhibitors competitively bind to the atp pocket in the n-terminus of hsp90, leading to blocking of atp hydrolysis and the closure of nterminus of hsp90 dimer. in addition, another atp binding site has been found in the c-terminus of hsp90. 648 recently, some natural products and their derivatives have been reported to competitively bind to the atp pocket in the c-terminus of hsp90. generally, inhibitors of hsp90 atpase are classified into three types: (i) ansamycins, (ii) non-ansamycins and (iii) those block hsp90 c-terminal atpase activity. ansamycins are antibiotics that possess the benzoquinone as structure core. these antibiotics include geldanamycin (ga), herbimycin a, and the macbecins. they inhibit hsp90 activity and degrade its client proteins. 649 ga competitively binds to the atp pocket in the n-terminus and inhibits the atpase activity. 649 it has shown potent antiviral activity against coronavirus infection in the culture cells, 145 indicating a good potential for treating covid-19. tanespimycin (17-allylamino-17-demethoxygeldanamycin, 17-aag) is an analogue of ga. 650, 651 alvespimycin (17-dimethylaminoethylamino-17demethoxygeldanamycin, 17-dmag) is an analogue of 17-aag, with high solubility in water. in addition, retaspimycin hydrochloride (ipi-504) is a more water-soluble derivative of 17-aag. 652 non-ansamycin inhibitors of hsp90 include luminespib, 653 biib021, 654 ganetespib, 655 onalespib, 656 snx-5422, 657 etc. another type of hsp90 inhibitors binds to the c-terminal atp pocket rather than n-terminal atp pocket. novobiocin blocks the c-terminal atpase activity. 658, 659 in in vitro and in vivo assays, treatment with novobiocin strongly reduces the expression of hsp90-dependent client proteins, such as raf-1 and p60v-src. a natural rotenoid, deguelin, is suggested to bind to the atp pocket in the c-terminus without affecting the atp pocket in the nterminus. 660 treating with deguelin leads to reduced hsp90 clients, such as cdk4, akt and mek1/2. 661 like deguelin, epigallocatechin gallate (ecgc) is reported to bind to the atp pocket in the c-terminus of hsp90 without affecting the nterminal atp pocket. 662 compared with inhibition of the nterminal atp pocket of hsp90, inhibitors targeting the c-terminal atp pocket lead to a stability of hsp70 and a negative instability of client proteins of hsp90. however, the potential of these cterminal inhibitors and their molecular mechanisms remain elusive. the structural features of the c-terminal domain of hsp90 need to be further analysed. such analysis may provide important hints on how to design effective hsp90 inhibitors without hsr induction. inhibitors of hsp90's co-chaperones. as the functions of the hsp90 chaperone machinery are highly dependent on the associated co-chaperones, it is possible to selectively inhibit downstream signaling of hsp90 by disrupting certain protein-protein interactions. inhibitors targeting the interactions may present an alternative approach to prevent the toxicity induced by other hsp90 inhibitors. as a result, great efforts are put into disrupting the interaction between hsp90 and its cochaperones and are rewarded recently in this area. here we briefly present the advance in hsp90-cdc37 complex and hsp90-hop-hsp70 ternary complex. human cdc37 is a well-studied co-chaperone of hsp90. the middle domain and c-terminus of cdc37 are critical for the interaction with hsp90. currently, most of the reported agents for manipulating the hsp90-cdc37 interaction are natural products and their derivatives. these agents include celastrol, aferin a, sulforaphane, kongensin a and platycodin d. they are capable of dissociating the hsp90-cdc37 complex. [663] [664] [665] [666] [667] in addition, a peptide (pep-1, ac-khfgmlrrwdd-nh2) is developed and shows strong inhibitory effects on the formation of hsp90-cdc37 complex. 668 another well-known hsp90/co-chaperone complex is hsp90-hop-hsp70 ternary complex, which facilitates the transfer of unfolded client proteins from hsp70 to hsp90. notably, six active compounds have been identified after screening the ncgc compound library. 669 these compounds possess similar structural cores and have no effect on hsp70 expression. studies on hsp90-co-chaperone inhibitors are limited. great effort should be paid on the efficacy and toxicity for further drug design and clinical studies. hsp90 inhibitors blocking deacetylation of hsp90. the chaperone function of hsp90 is also controlled by posttranslational modification (ptm). the well-studied ptms in hsp90 are acetylation and deacetylation in the m-domain of hsp90. vorinostat (suberoylanilide hydroxamic acid, saha) dissociates her2/erbb2 from hsp90 via acetylation of hsp90 that leads to degradation of her2/erbb2. 670 laq824 induces hsp90 acetylation that reduces hsp90 client protein levels. 671 romidepsin is involved in the dissociation of mutant p53 and raf-1 from hsp90 via acetylation of hsp90. 672 in addition, the nuclear import of iav polymerase needs the deacetylation of hsp90 which is strictly regulated by histone deacetylases 6/8 (hdac6/8). 120 hdac6/8 inhibitors efficiently limit polymerase nuclear import and suppress virus replication. 120 therefore, hsp90 inhibitors that block hsp90 deacetylation would potentially be used to treat influenza a virus infection. their potential usage for combating other viruses (such as sars-cov-2) needs to be extensively explored. novel class of hsp90 inhibitors for hsp90 cleavage. recently, hsp90 cleavage has been observed under various stimuli such as uv irradiation, 673 ascorbate/menadione, 674 hdac inhibitors, 675 proteasome inhibitors 676 etc. therefore, hsp90 cleavage is considered as another mechanism. hsp90 cleavage induced by these inhibitors can be classified into two types: enzymatic cleavage and non-enzymatic cleavage. 674, 676 the enzymatic cleavage produces a 55 kda fragment of hsp90 via caspase 10 activation, while the non-enzymatic cleavage utilizes reactive oxygen species (ros) to chemically degrade hsp90 to an~70 kda fragment. additionally, some substances have been reported to present hsp90 cleavage activity, but the mechanism remains unclear. targeting hsp70s for drug development: one kind of antiviral drugs are currently designed based on different properties of hsps. considering hsp70 and hsc70 are potential targets for hcv, they load the dcs with hsp70 for a prolonged potent antigen-and tumor-specific t cell responses directed against multiple epitopes. 677 hsp70 inhibitors (such as quercetin, ver155008 and jc40) also show great potential for treating flavivirus and enterovirus infections. 202, 208, 640 inhibitors blocking the interaction between grp78 and spike protein of coronavirus would be a useful approach for combating the infection by sars-cov, mers-cov, and sars-cov-2 infections. 196, 387, 571 however, the efficacy and toxicity of these inhibitors are not yet well investigated in clinic studies. targeting hsp60s for drug development: as described above, hsp60s play critical roles in different diseases including autoimmune diseases, human cancers and virus infection-induced diseases. studies show that silencing of hsp60s by sirna significantly decrease influenza virus, 38,39 denv 319 and hbv 325, 331 reproduction by activating immunity response; and is capable of releasing hcv 321,322 , rotavirus, 323 hbv 329 and hiv 338 infectioninduced pathogenesis. therefore, small molecule regents targeting hsp60s are potentially useful as therapeutics in these diseases. 678, 679 currently, the known hsp60 inhibitors are either from natural products or synthetic compounds (table 3) . mechanistically, they can be grouped as two types. type i inhibitors block atp binding and hydrolysis. type ii inhibitors covalently interact with certain cysteine residues of hsp60. however, the detailed binding sites have not been identified. the natural products of hsp60 inhibitors include mizoribine, epolactaene, myrtucommulone a, stephacidin b. mizoribine is an imidazole nucleoside antibiotics isolated from eupenicillium brefeldianum. 680 it directly binds to hsp60 and inhibits the chaperone activity of the hsp60-hsp10 complex. 33 epolactaene is isolated from the fungal strain penicillium sp. bm 1689-p. 681 its derivate tert-butyl ester etb has similar activity as epolactaene. 682 they inhibit hsp60-hsp10's chaperoning activity by covalent and selective bound to hsp60 at residue of cys442, close to the atp binding pocket. 683, 684 interestingly, etb does not inhibit the atpase activity of hsp60, 685 suggesting that the covalent interaction between etb and cys442 may allosterically modulate hsp60-hsp10's chaperoning activity without interfering with its atpase activity. myrtucommulone a (mc) binds to hsp60 and inhibits the refolding activity of the hsp60-hsp10 complex. 686 mc is a non-prenylated acylphloroglucinol with multiple reported bioactivities, including antibacterial, 687,688 antioxidant, 689 antiinflammatory, 690, 691 and anti-tumor properties. 692, 693 in addition to these natural products, several other natural products are also reported to interact with hsp60 without direct proof. stephacidin b is isolated from aspergillus ochraceus wc76466 while avrainvillamide is isolated from aspergillus sp. cnc358. 694, 695 interestingly, dimeric stephacidin b is converted into monomeric avrainvillamide in tissue culture media which implies avrainvillamide is the actual active species. 696 pull down assay only shows hsp60 may be a putative target. 696 further validation studies have yet to be performed. besides the natural products identified above as potential hsp60 inhibitors, quite a few synthetic molecules have also been discovered to be able to modulate hsp60 activity. o-carboranylphenoxyacetanilide is firstly identified as an hif-1α inhibitor. 697 later it is found that o-carboranylphenoxyacetanilide selectively and directly binds to hsp60 and inhibits hsp60-hsp10's atpase activity and refolding activity. 685 hsp60 can interact with hif-1α, suggesting that inhibition of hif-1α activity by carboranylphenoxyacetanilide is possibly through targeting hsp60. 685 the other class of synthetic compounds identified to inhibit hsp60 is gold porphyrins. 698 it directly binds to hsp60 and inhibits the refolding activity of the hsp60-hsp10 complex. although several hsp60 inhibitors potently suppress virus reproduction, a lot of work remains to be done to verify if they can serve as drugs for treating diseases caused by virus infections. targeting hnrnps for antivirus drug development. drugs targeting rna chaperones may have a wide spectrum of antivirus capability. hnrnp a1 participates the reproduction of hiv, [539] [540] [541] 563 hpv, 503-505 hcv, 471 ev-a71 472 and htlv-1. 562 inhibition of hnrnp c suppresses the replication of hcv, 438 514, 536, 537 however, the available regents developed targeting hnrnps as antiviral drugs are limited. to date, only three agents targeting hnrnps have been reported. apigenin is a flavonoid abundant in fruits and vegetables. it targets hnrnp a2 and blocks hnrnp a2 dimerization thereafter affects the alternative splicing of key mrnas. 699 apigenin efficiently inhibits the interaction of hnrnp a1 and a2 with ev-a71 rna thereby inhibits ev-a71 rna translation. 472 quercetin is also a flavonoid abundantly present in plants. it binds to the c-terminal region of hnrnp a1, impairing the ability of hnrnp a1 to shuttle between the nucleus and cytoplasm and ultimately resulting in its cytoplasmic retention. 700 in addition, quercetin down-regulates hnrnp a1 expression. 701 the antiviral ability of quercetin has been verified in some viruses such as inhibiting influenza entry, 702 ev-a71, 703 and hcv, 208 jev and denv reproduction. 704 however, in these researches, they explain the antivirus mechanism of quercetin is by inhibiting hsp70 expression 208 or inhibiting virus proteases 703 without mentioning the function of hnrnp a1. another compound vpc-80051 targets rna binding motif of hnrnp a1 and alters hnrnp a1 splicing activity. 705 its antiviral ability remains to be further investigated. targeting er stress pathways for drug development: during the past few years, the enlarging role of er stress-mediated pathways is becoming quite an attractive topic for broad-spectrum antiviral therapy, especially in the field of virus reproduction and pathogenesis, considerable progress has also been made for potential antiviral agents development. among pathways involved in er stress, the three upr pathways are the most thoroughly studied, thus antiviral targets related with these pathways are considered as the first class. extensive investigation for antiviral development has been put int to the perk-eif2α pathway. a small chemical compound named salubrinal, identified by boyce and his colleagues, specifically inhibits the formation of pp1/gadd34 complex as well as suppresses hsv γ134.5-mediated eif2α dephosphorylation. it could also significantly reduce hsv replication. 706 their research suggested that salubrinal was able to attenuate pp1/gadd34-dependent eif2α dephosphorylation, which would in turn inhibit denv replication. 707 a glucose analog 2-deoxy-d-glucose is responsible for the activation and phosphorylation of eif2α, leading to suppression of herpesvirus (kshv) replication. this agent is a potential candidate for anti-herpesvirus, and hopefully is tested in clinical therapies. 708 other than eif2α, tremendous efforts have also been made in developing drugs to target ire1-xbp1 pathway. 3,5-dibromosalicyladehyde is an endoribonuclease that specifically interacts with ire1 and blocks the downstream iav replication. 600 wp1130 is another ire1-xbp1 inhibitor with a broad-spectrum antiviral effects against multiple viruses, namely mnv-1, lcv and so on. 709 er stress-mediated apoptosis signaling is another noteworthy pathway for drug development and selection. rana catesbeiana ribonuclease (rc-rnase) is reported to be efficient in suppression of jev replication via activation of caspase-3, caspase-8 and caspase-9 pathways. 710 dubble-stranded rna activated caspase oligomerizer (draco) is reported to have broad anti-viral effects in clinical therapy. draco selectively activates apoptosis in cells infected with dsdna viruses but has no harmful effect on normal healthy cells. over 15 different kind of viruses can be eradicated by draco, including hiv and dengue virus. 711 drugs targeting other signal transducers are also investigated, for example jnk, jak, bcl-2 and chop. a promising agent vaticanol b can protect host cells from er stress-induced apoptosis. 712, 713 some researchers choose to focus on viral proteins that could trigger er-stress, including non-envelope glycoproteins of sfv, precursor glycoprotein of lcmv, glycoproteins tulv, several nonstructural protein of sars-cov, multiple non-structural proteins of flavivirus family, envelope protein of pedv, e1, e2 and nonstructral protein of hcv, surface protein of mhv, icp0 glycoprotein b of hsv1, structural protein 4 of chikv. viral proteins that are responsible for upr activation and apoptosis induction are given special attentions such as hcv (e1, e2, core), hcmv (pul37x1, pul38) and sv5 v protein. great advances have been made in this area, current progress includes, clemizol for hcv ns4b, vaccine vectors for hsv-1 γ134, and norakin for iav hemagglutinin a. 714, 715 due to their potential side effects on normal cell functions, the usage of inhibitors targeting stress proteins are very cautious in clinic settings; and the gone with wind infection manner of some virus (e.g., sars-cov) limits the opportunity of clinic studies, giving rise to additional challenges for developing stress protein-based drugs against the common and special infectious diseases such as severe acute respiratory symptom (sars) caused by coronavirus. we thank prof. robert weiss at cornell university college of veterinary medicine for critical reading and comments in preparation of this manuscript. the work was partially supported by grants from the science technology and innovation committee of shenzhen municipality [jcyj20170818100531426, jcyj20180507181627057]; grants from national science foundation of china in and out of the er: protein folding, quality control, degradation, and related human diseases a. new puffing pattern induced by temperature shock and dnp in drosophila molecular chaperone functions of heat-shock proteins the growing world of small heat shock proteins: from structure to functions 70 and 90, anti-apoptotic proteins, in clinical cancer therapy molecular chaperones in protein 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rna polymerase degradation following hsp90 inhibition in a range of negative strand viruses hsp90 inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus chikungunya virus nsp3 & nsp4 interacts with hsp-90 to promote virus replication: hsp-90 inhibitors reduce chikv infection and inflammation in vivo heat-shock protein 90 is essential for stabilization of the hepatitis c virus nonstructural protein ns3 destabilization of pdk1 by hsp90 inactivation suppresses hepatitis c virus replication through inhibition of prk2-mediated viral rna polymerase phosphorylation hepatitis c virus rna replication is regulated by fkbp8 and hsp90 identification of hsp90 as a stimulatory host factor involved in influenza virus rna synthesis hsp90 inhibitors reduce influenza virus replication in cell culture mc1568 inhibits hdac6/8 activity and influenza a virus replication in lung epithelial cells: role of hsp90 acetylation autophagy is involved in regulating influenza a virus rna and protein synthesis associated with both modulation of hsp90 induction and mtor/p70s6k signaling pathway pim1 impacts enterovirus a71 replication and represents a potential target in antiviral therapy host cell factor requirement for hepatitis c virus enzyme maturation evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance molecular chaperone hsp90 is a therapeutic target for noroviruses influenza a virus neuraminidase protein interacts with hsp90, to stabilize itself and enhance cell survival viral transport and the cytoskeleton the hepatitis e virus open reading frame 3 product interacts with microtubules and interferes with their dynamics the ins and outs of tubulin acetylation: more than just a post-translational modification? regulation of microtubule assembly and stability by the transactivator of transcription protein of jembrana disease virus heat-shock protein 90 promotes nuclear transport of herpes simplex virus 1 capsid protein by interacting with acetylated tubulin glucocorticoid stimulates hepatitis b viral gene expression in cultured human hepatoma cells geldanamycin, a heat shock protein 90-binding benzoquinone ansamycin, inhibits steroid-dependent translocation of the glucocorticoid receptor from the cytoplasm to the nucleus hepatitis b virus replication hsp90 is required for the activity of a hepatitis b virus reverse transcriptase requirement of heat shock protein 90 for human hepatitis b virus reverse transcriptase function in vitro reconstitution of a functional duck hepatitis b virus reverse transcriptase: posttranslational activation by hsp90 hbv polymerase interacts independently with nterminal and c-terminal fragments of hsp90beta chaperone activation of the hepadnaviral reverse transcriptase for template rna binding is established by the hsp70 and stimulated by the hsp90 system efficient hsp90-independent in vitro activation by hsc70 and hsp40 of duck hepatitis b virus reverse transcriptase, an assumed hsp90 client protein hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids hsp90 makes the human hbv pol competent for in vitro priming rather than maintaining the human hbv pol/ pregenomic rna complex expression of stable hepatitis b viral polymerase associated with grp94 in e. coli herpes simplex virus type 1 dna polymerase requires the mammalian chaperone hsp90 for proper localization to the nucleus geldanamycin, a ligand of heat shock protein 90, inhibits the replication of herpes simplex virus type 1 in vitro hsp90 inhibitors: a potential treatment for latent ebv infection? nuclear transport of epstein-barr virus dna polymerase is dependent on the bmrf1 polymerase processivity factor and molecular chaperone hsp90 the herpes simplex virus vp16-induced complex: the makings of a regulatory switch herpes simplex virus disrupts nf-kappab regulation by blocking its recruitment on the ikappabalpha promoter and directing the factor on viral genes heat-shock protein 90α is involved in maintaining the stability of vp16 and vp16-mediated transactivation of α genes from herpes simplex virus-1 geldanamycin, a potent and specific inhibitor of hsp90, inhibits gene expression and replication of human cytomegalovirus curcumin inhibits human cytomegalovirus by downregulating heat shock protein 90 a novel hsp90 inhibitor, 17-dmag, induces tax down-regulation and its oral administration to atl-model mice intervenes against the infiltration property of the atl-like lymphocytes and provides extended survival period processing of the herpes simplex virus regulatory protein alpha 22 mediated by the ul13 protein kinase determines the accumulation of a subset of alpha and gamma mrnas and proteins in infected cells icp22 and the ul13 protein kinase are both required for herpes simplex virus-induced modification of the large subunit of rna polymerase ii epstein-barr virus-encoded protein kinase (bglf4) is involved in production of infectious virus the human cytomegalovirus ul44 protein is a substrate for the ul97 protein kinase distinct and separate roles for herpesvirus-conserved ul97 kinase in cytomegalovirus dna synthesis and encapsidation the human cytomegalovirus ul97 protein kinase, an antiviral drug target, is required at the stage of nuclear egress viral mimicry of cdc2/cyclin-dependent kinase 1 mediates disruption of nuclear lamina during human cytomegalovirus nuclear egress conserved herpesvirus kinases target the dna damage response pathway and tip60 histone acetyltransferase to promote virus replication sumo binding by the epstein-barr virus protein kinase bglf4 is crucial for bglf4 function hsp90 inhibitors block outgrowth of ebv-infected malignant cells in vitro and in vivo through an ebna1-dependent mechanism ganetespib, an hsp90 inhibitor, kills epstein-barr virus (ebv)-infected b and t cells and reduces the percentage of ebv-infected cells in the blood ebp2 plays a key role in epstein-barr virus mitotic segregation and is regulated by aurora family kinases epstein-barr virus provides a survival factor to burkitt's lymphomas structure of the p53 binding domain of hausp/usp7 bound to epstein-barr nuclear antigen 1 implications for ebv-mediated immortalization self-inhibition of synthesis and antigen presentation by epstein-barr virus-encoded ebna1 heat shock protein 90 facilitates formation of the hbv capsid via interacting with the hbv core protein dimers reactive oxygen species promote heat shock protein 90-mediated hbv capsid assembly virus particles in cultured lymphoblasts from burkitt's lymphoma epstein-barr virus in the pathogenesis of npc all three domains of the epstein-barr virus-encoded latent membrane protein lmp-1 are required for transformation of rat-1 fibroblasts epstein-barr virus induces invasion and metastasis factors a novel hsp90 inhibitor at13387 induces senescence in ebvpositive nasopharyngeal carcinoma cells and suppresses tumor formation the heat shock protein 90 inhibitor biib021 suppresses the growth of t and natural killer cell lymphomas heat shock protein 90 expression in epstein-barr virus-infected b cells promotes gammadelta t-cell proliferation in vitro tcrgammadelta cells and viruses heat shock protein 90 (hsp90) contributes to cytosolic translocation of extracellular antigen for cross-presentation by dendritic cells efficient cross-presentation by heat shock protein 90-peptide complex-loaded dendritic cells via an endosomal pathway nlr, the nucleotide-binding domain leucine-rich repeat containing gene family the hsp90-sgt1 chaperone complex for nlr immune sensors molecular mechanisms of cellular transformation by htlv-1 tax activation of nf-kappab by htlv-i and implications for cell transformation hsp90 protects the human t-cell leukemia virus type 1 (htlv-1) tax oncoprotein from proteasomal degradation to support nf-κb activation and htlv-1 replication grp78, a coreceptor for coxsackievirus a9, interacts with major histocompatibility complex class i molecules which mediate virus internalization integrin alpha v beta 6 is an rgd-dependent receptor for coxsackievirus a9 heat shock protein 70 as a supplementary receptor facilitates enterovirus 71 infections in vitro surface proteins of c6/36 cells involved in dengue virus 4 binding and entry zika virus dependence on host hsp70 provides a protective strategy against infection and disease heat shock protein 70 on neuro2a cells is a putative receptor for japanese encephalitis virus association of heat-shock protein 70 with lipid rafts is required for japanese encephalitis virus infection in huh7 cells heat shock cognate protein 70 isoform d is required for clathrin-dependent endocytosis of japanese encephalitis virus in c6/36 cells grp78 is an important host factor for japanese encephalitis virus entry and replication in mammalian cells japanese encephalitis virus co-opts the er-stress response protein grp78 for viral infectivity natural products may interfere with sars-cov-2 attachment to the host cell heat shock protein 70 (hsp70) mediates zika virus entry, replication, and egress from host cells heat shock cognate protein 70 is involved in rotavirus cell entry interaction of rotaviruses with hsc70 during cell entry is mediated by vp5 the peptide-binding and atpase domains of recombinant hsc70 are required to interact with rotavirus and reduce its infectivity heat shock protein 70 regulates degradation of the mumps virus phosphoprotein via the ubiquitin-proteasome pathway heat shock protein 90 ensures efficient mumps virus replication by assisting withfang viral polymerase complex formation constitutive overexpression of the major inducible 70 kda heat shock protein mediates large plaque formation by measles virus enhanced production of morbillivirus gene-specific rnas following induction of the cellular stress response in stable persistent infection the highly inducible member of the 70 kda family of heat shock proteins increases canine distemper virus polymerase activity heat shock protein 72 is associated with the hepatitis c virus replicase complex and enhances viral rna replication tylophorine analogs allosterically regulates heat shock cognate protein 70 and inhibits hepatitis c virus replication the heat shock protein inhibitor quercetin attenuates hepatitis c virus production human respiratory syncytial virus n, p and m protein interactions in hek-293t cells evidence for an association between heat shock protein 70 and the respiratory syncytial virus polymerase complex within lipid-raft membranes during virus infection interactome analysis of the human respiratory syncytial virus rna polymerase complex identifies protein chaperones as important cofactors that promote l-protein stability and rna synthesis elucidation of the cellular interactome of ebola virus nucleoprotein and identification of therapeutic targets an rna polymerase ii-driven ebola virus minigenome system as an advanced tool for antiviral drug screening recombinant rna-dependent rna polymerase complex of ebola virus a small stem-loop structure of the ebola virus trailer is essential for replication and interacts with heat-shock protein a8 cellular stress response induces selective intranuclear trafficking and accumulation of morbillivirus major core protein heat shock protein 70 modulates influenza a virus polymerase activity heat shock protein 70 promotes coxsackievirus b3 translation initiation and elongation via akt-mtorc1 pathway depending on activation of p70s6k and cdc2 hsc70 regulates the ires activity and serves as an antiviral target of enterovirus a71 infection the double-stranded rna-dependent protein kinase is also activated by heparin repression of the pkr protein kinase by the hepatitis c virus ns5a protein: a potential mechanism of interferon resistance the 58,000-dalton cellular inhibitor of the interferon-induced double-stranded rna-activated protein kinase (pkr) is a member of the tetratricopeptide repeat family of proteins purification and partial characterization of a cellular inhibitor of the interferon-induced protein kinase of mr 68,000 from influenza virus-infected cells characterization and regulation of the 58,000-dalton cellular inhibitor of the interferon-induced, dsrna-activated protein kinase the molecular chaperone hsp40 regulates the activity of p58ipk, the cellular inhibitor of pkr the cellular inhibitor of the pkr protein kinase, p58(ipk), is an influenza virus-activated co-chaperone that modulates heat shock protein 70 activity c-terminal trimerization, but not n-terminal trimerization, of the reovirus cell attachment protein is a posttranslational and hsp70/atp-dependent process association of heat shock protein 70 with enterovirus capsid precursor p1 in infected human cells heat shock protein 70 is related to thermal inhibition of nuclear export of the influenza virus ribonucleoprotein complex heat shock protein 70 inhibits the activity of influenza a virus ribonucleoprotein and blocks the replication of virus in vitro and in vivo polo-like kinase 1 is involved in hepatitis c virus replication by hyperphosphorylating ns5a the ns5a-binding heat shock proteins hsc70 and hsp70 play distinct roles in the hepatitis c viral life cycle structural characterization of the hsp70 interaction domain of the hepatitis c viral protein ns5a sgta-dependent regulation of hsc70 promotes cytosol entry of simian virus 40 from the endoplasmic reticulum a non-enveloped virus hijacks host disaggregation machinery to translocate across the endoplasmic reticulum membrane bip and multiple dnaj molecular chaperones in the endoplasmic reticulum are required for efficient simian virus 40 infection adenovirus capsid proteins interact with hsp70 proteins after penetration in human or rodent cells novel partner proteins of adenovirus penton co-chaperone bag3 and adenovirus penton base protein partnership nuclear import of adenovirus dna in vitro involves the nuclear protein import pathway and hsc70 nuclear sequestration of cellular chaperone and proteasomal machinery during herpes simplex virus type 1 infection transcriptional stimulation by the dna binding protein hap46/bag-1m involves hsp70/hsc70 molecular chaperones transcriptional activation by the human hsp70-associating protein hap50 the hsp70-associating protein hap46 binds to dna and stimulates transcription bag-1, a novel bcl-2-interacting protein, activates expression of human jc virus bag-1m, an isoform of bcl-2-interacting protein bag-1, enhances gene expression driven by cmv promoter chaperone action in the posttranslational topological reorientation of the hepatitis b virus large envelope protein: implications for translocational regulation sequence-specific repression of cotranslational translocation of the hepatitis b virus envelope proteins coincides with binding of heat shock protein hsc70 chaperones involved in hepatitis b virus morphogenesis bag-1m accelerates nucleotide release for human hsc70 and hsp70 and can act concentration-dependent as positive and negative cofactor hsp70 interacting protein hip does not affect glucocorticoid receptor folding by the hsp90-based chaperone machinery except to oppose the effect of bag-1 molecular chaperone grp78/bip interacts with the large surface protein of hepatitis b virus in vitro and in vivo mammalian bip controls posttranslational er translocation of the hepatitis b virus large envelope protein in vivo and in vitro association of hsc70 with polyomavirus capsid proteins chaperone-mediated in vitro assembly of polyomavirus capsids productive infection and cell-free transmission of human t-cell leukemia virus in a nonlymphoid cell line detection of lymphocytes producing a human retrovirus associated with adult t-cell leukemia by syncytia induction assay 71-kilodalton heat shock cognate protein acts as a cellular receptor for syncytium formation induced by human t-cell lymphotropic virus type 1 heat shock cognate protein 70 is a cell fusion-enhancing factor but not an entry factor for human t-cell lymphotropic virus type i heat-shock protein 70 can replace viral protein r of hiv-1 during nuclear import of the viral preintegration complex heat-shock proteins reverse the g2 arrest caused by hiv-1 viral protein r heat shock protein 70 protects cells from cell cycle arrest and apoptosis induced by human immunodeficiency virus type 1 viral protein r atpgammas disrupts human immunodeficiency virus type 1 virion core integrity the amino-terminal transforming region of simian virus 40 large t and small t antigens functions as a j domain the molecular chaperone activity of simian virus 40 large t antigen is required to disrupt rb-e2f family complexes by an atp-dependent mechanism atp-dependent simian virus 40 tantigen-hsc70 complex formation the regulation of e2f by prb-family proteins mechanism of regulation of hsp70 chaperones by dnaj cochaperones investigation of the interaction between dnak and dnaj by surface plasmon resonance spectroscopy priming polyvalent immunity by dna vaccines expressing chimeric antigens with a stress protein-capturing, viral j-domain loss of p19(arf) eliminates the requirement for the prb-binding motif in simian virus 40 large t antigenmediated transformation novel mechanisms of e2f induction by bk virus large-t antigen: requirement of both the prbbinding and the j domains j domain-independent regulation of the rb family by polyomavirus large t antigen the j domain of simian virus 40 large t antigen is required to functionally inactivate rb family proteins a novel human dnaj protein, htid-1, a homolog of the drosophila tumor suppressor protein tid56, can interact with the human papillomavirus type 16 e7 oncoprotein identification of a novel retinoblastoma gene product binding site on human papillomavirus type 16 e7 protein the human papillomavirus e7 oncoprotein 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expression alters proteasomal degradation of ikappab kinase in experimental acute respiratory distress syndrome hsp70 is a negative regulator of nlrp3 inflammasome activation functional domains of hsp70 stimulate generation of cytokines and chemokines, maturation of dendritic cells and adjuvanticity hsp70 peptidembearing and peptide-negative preparations act as chaperokines toll-like receptor signalling toll-like receptor 4 (tlr4) is essential for hsp70-like protein 1 (hsp70l1) to activate dendritic cells and induce th1 response toll-like receptor signaling and its inducible proteins inflammation in gastric cancer: interplay of the cox-2/prostaglandin e2 and toll-like receptor/myd88 pathways tak1-ecsit-traf6 complex plays a key role in the tlr4 signal to activate nf-kappab cutting edge: traf6 mediates tlr/il-1r signaling-induced nontranscriptional priming of the nlrp3 inflammasome heme oxygenase-1 regulates dendritic cell function through modulation of p38 mapk-creb/atf1 signaling activated apoptotic cells induce dendritic cell maturation via engagement of toll-like receptor 4 (tlr4), dendritic cell-specific intercellular adhesion molecule 3 (icam-3)-grabbing nonintegrin (dc-sign), and beta2 integrins il-1β induction of muc5ac gene expression is mediated by creb and nf-κb and repressed by dexamethasone induction of proinflammatory response in prostate cancer epithelial cells by activated macrophages autoimmune and inflammatory mechanisms in atherosclerosis heat shock proteins as ligands of toll-like receptors the hsp60 immune system network heat shock protein and innate immunity heat shock proteins: mediators of atherosclerotic development hsp60 critically regulates endogenous il-1β production in activated microglia by stimulating nlrp3 inflammasome pathway genome-wide rnai screen identifies human host factors crucial for influenza virus replication rna interference mediated silencing of hsp60 gene in human monocytic myeloma cell line u937 revealed decreased dengue virus multiplication hepatitis c virus: from oxygen free radicals to hepatocellular carcinoma mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis c virus core protein interaction of hepatitis c virus core protein with hsp60 triggers the production of reactive oxygen species and enhances tnf-alpha-mediated apoptosis tyrosine phosphorylation modulates mitochondrial chaperonin hsp60 and delays rotavirus nsp4-mediated apoptotic signaling in host cells cooperation between heat shock proteins in organizing of proteins spatial structure antisense oligodeoxynucleotides targeted against molecular chaperonin hsp60 block human hepatitis b virus replication human hepatitis b virus polymerase interacts with the molecular chaperonin hsp60 binding site analysis of human hbv pol for molecular chaperonin, hsp60 hepatitis b virus x protein: a multifunctional viral regulator interaction of the hepatitis b virus x protein (hbx) with heat shock protein 60 enhances hbx-mediated apoptosis hbx protein of hepatitis b virus (hbv) can form complex with mitochondrial hsp60 and hsp70 innate immune responses in hepatitis b virus (hbv) infection circulating and liver resident cd4 + cd25 + regulatory t cells actively influence the antiviral immune response and disease progression in patients with hepatitis b interferon effects on interleukin-10 secretion mononuclear cell response to interleukin-10 is normal in multiple sclerosis patients il-10-producing regulatory b cells (b10 cells) in autoimmune disease hepatitis b virus replication could enhance regulatory t cell activity by producing soluble heat shock protein 60 from hepatocytes an hsp60 related protein is associated with purified hiv and siv molecular mechanisms in retrovirus dna integration functional interactions of human immunodeficiency virus type 1 integrase with human and yeast hsp60 dnaj homolog hdj2 facilitates japanese encephalitis virus replication structure of the ribonucleoprotein of influenza virus a classical bipartite nuclear localization signal on thogoto and influenza a virus nucleoproteins an unconventional nls is critical for the nuclear import of the influenza a virus nucleoprotein and ribonucleoprotein human heat shock protein 40 (hsp40/dnajb1) promotes influenza a virus replication by assisting nuclear import of viral ribonucleoproteins dnaja1/hsp40 is co-opted by influenza a virus to enhance its viral rna polymerase activity molecular cloning and characterization of the human doublestranded rna-activated protein kinase induced by interferon influenza a virus nucleoprotein exploits hsp40 to inhibit pkr activation p58ipk, a novel endoplasmic reticulum stress-inducible protein and potential negative regulator of eif2alpha signaling biochemical and genetic evidence for complex formation between the influenza a virus ns1 protein and the interferon-induced pkr protein kinase interaction of hsp40 with influenza virus m2 protein: implications for pkr signaling pathway flavivirus replication complex assembly revealed by dnajc14 functional mapping identification and characterization of the host protein dnajc14 as a broadly active flavivirus replication modulator chaperone-assisted protein folding is critical for yellow fever virus ns3/4a cleavage and replication dnajb1/hsp40 suppresses melanoma differentiation-associated gene 5-mitochondrial antiviral signaling protein function in conjunction with hsp70 cellular dnaja3, a novel vp1-interacting protein, inhibits footand-mouth disease virus replication by inducing lysosomal degradation of vp1 and attenuating its antagonistic role in the beta interferon signaling pathway human hsp70 and hsp40 chaperone proteins facilitate human papillomavirus-11 e1 protein binding to the origin and stimulate cell-free dna replication targeting the e1 replication protein to the papillomavirus origin of replication by complex formation with the e2 transactivator cell-free replication of the human papillomavirus dna with homologous viral e1 and e2 proteins and human cell extracts chaperone proteins abrogate inhibition of the human papillomavirus (hpv) e1 replicative helicase by the hpv e2 protein the human dnaj protein, htid-1, enhances binding of a multimer of the herpes simplex virus type 1 ul9 protein to oris, an origin of viral dna replication activation of the herpes simplex virus type-1 origin-binding protein (ul9) by heat shock proteins turnover of hepatitis b virus x protein is facilitated by hdj1, a human hsp40/dnaj protein negative regulation of hepatitis b virus replication by cellular hsp40/dnaj proteins through destabilization of viral core and x proteins hepatitis b virus x protein in the pathogenesis of hepatitis b virusinduced hepatocellular carcinoma high-level expression of hepatitis b virus hbx gene and hepatocarcinogenesis in transgenic mice x protein of hepatitis b virus modulates cytokine and growth factor related signal transduction pathways during the course of viral infections and hepatocarcinogenesis hepatitis b. x antigen and p53 are associated in vitro and in liver tissues from patients with primary hepatocellular carcinoma retroviral infection of non-dividing cells: old and new perspectives hiv-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway a nuclear localization signal within hiv-1 matrix protein that governs infection of non-dividing cells hiv-1 genome nuclear import is mediated by a central dna flap functional analysis of the simian immunodeficiency virus vpx protein: identification of packaging determinants and a novel nuclear targeting domain simian immunodeficiency virus vpx is imported into the nucleus via importin alphadependent and -independent pathways hsp40 facilitates nuclear import of the human immunodeficiency virus type 2 vpx-mediated preintegration complex interference of dnajb6/mrj isoform switch by morpholino inhibits replication of hiv-1 and rsv large isoform of mammalian relative of dnaj is a major determinant of human susceptibility to hiv-1 infection heat shock protein 40 is necessary for human immunodeficiency virus-1 nef-mediated enhancement of viral gene expression and replication hiv-1 nef and host cell protein kinases nef triggers a transcriptional program in t cells imitating single-signal t cell activation and inducing hiv virulence mediators the plasma membrane as a combat zone in the hiv battlefield cellular heat shock factor 1 positively regulates human immunodeficiency virus-1 gene expression and replication by two distinct pathways reciprocal regulation of human immunodeficiency virus-1 gene expression and replication by heat shock proteins 40 and 70 novel role of hsp40/dnaj in the regulation of hiv-1 replication heat shock proteins as cellular lifeguards structural and functional diversities between members of the human hspb, hsph, hspa, and dnaj chaperone families muscle develops a dpecific form of small heat shock protein complex composed of mkbp/hspb2 and hspb3 during myogenic differentiation epstein-barr virus upregulates phosphorylated heat shock protein 27 kda in carcinoma cells using the phosphoinositide 3-kinase/akt pathway proteomics analysis of differentially expressed proteins in chicken trachea and kidney after infection with the highly virulent and attenuated coronavirus infectious bronchitis virus in vivo cellular hsp27 interacts with classical swine fever virus ns5a protein and negatively regulates viral replication by the nf-κb signaling pathway down-regulating heat shock protein 27 is involved in porcine epidemic diarrhea virus escaping from host antiviral mechanism the heat-shock response adenovirus type 7 induces interleukin-8 production via activation of extracellular regulated kinase 1/2 nf-κb and the map kinases/ap-1 pathways are both involved in interleukin-6 and interleukin-8 expression in fibroblast-like synoviocytes stimulated by protein i/ii, a modulin from oral streptococci signaling pathways for glycated human serum albumin-induced il-8 and mcp-1 secretion in human rpe cells mammalian small stress proteins protect against oxidative stress through their ability to increase glucose-6-phosphate dehydrogenase activity and by maintaining optimal cellular detoxifying machinery heat shock proteins: essential proteins for apoptosis regulation apoptosis versus cell differentiation: role of heat shock proteins hsp90, hsp70 and hsp27 small heat shock proteins hsp27 (hspb1), αb-crystallin (hspb5) and hsp22 (hspb8) as regulators of cell death modification and reorganization of the cytoprotective cellular chaperone hsp27 during herpes simplex virus type 1 infection heat shock protein 27 is involved in pcv2 infection in pk-15 cells hspb1 is an intracellular antiviral factor against hepatitis b virus role of thiol/disulfide exchange in newcastle disease virus entry overexpression of thiol/disulfide isomerases enhances membrane fusion directed by the newcastle disease virus fusion protein endothelial cell surface expression of protein disulfide isomerase activates beta1 and beta3 integrins and facilitates dengue virus infection protein disulfide isomerase mediates dengue virus entry in association with lipid rafts inhibiting rotavirus infection by membrane-impermeant thiol/disulfide exchange blockers and antibodies against protein disulfide isomerase inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent hiv-1 entry the catalytic activity of protein disulfide isomerase is involved in human immunodeficiency virus envelope-mediated membrane fusion after cd4 cell binding protein-disulfide isomerase-mediated reduction of two disulfide bonds of hiv envelope glycoprotein 120 occurs post-cxcr4 binding and is required for fusion galectin-9 binding to cell surface protein disulfide isomerase regulates the redox environment to enhance t-cell migration and hiv entry structure of murine polyomavirus complexed with an oligosaccharide receptor fragment how viruses use the endoplasmic reticulum for entry, replication, and assembly simian virus 40 depends on er protein folding and quality control factors for entry into host cells a pdi family network acts distinctly and coordinately with erp29 to facilitate polyomavirus infection oblongifolin m, an active compound isolated from a chinese medical herb garcinia oblongifolia, potently inhibits enterovirus 71 reproduction through downregulation of erp57 cellular self-defense: how cellautonomous immunity protects against pathogens role of free radicals in viral pathogenesis and mutation ex vivo induction of apoptosis in lymphocytes is mediated by oxidative stress: role for lymphocyte loss in hiv infection. free radic role of oxidants in influenza virus-induced gene expression investigation of oxidative stress and antioxidant defense in patients with hepatitis b virus infection and the effect of interferon-alpha plus lamivudine combination therapy on oxidative stress hepatitis c virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ros) production oxidative damage to neurons caused by the induction of microglial nadph oxidase in encephalomyocarditis virus infection cellular oxidative stress response controls the antiviral and apoptotic programs in dengue virus-infected dendritic cells japanese encephalitis virus stimulates superoxide dismutase activity in rat glial cultures oxidative stress in hpv-driven viral carcinogenesis: redox proteomics analysis of hpv-16 dysplastic and neoplastic tissues folding and oligomerization of influenza hemagglutinin in the er and the intermediate compartment n-glycosylation of the premembrane protein of japanese encephalitis virus is critical for folding of the envelope protein and assembly of viruslike particles hepatitis c virus glycoprotein folding: disulfide bond formation and association with calnexin an rna chaperone activity of non-specific rna binding proteins in hammerhead ribozyme catalysis the ebola virus vp24 protein prevents hnrnp c1/c2 binding to karyopherin α1 and partially alters its nuclear import hnrnps relocalize to the cytoplasm following infection with vesicular stomatitis virus hnrnp proteins and the biogenesis of mrna direct and indirect effects on viral translation and rna replication are required for auf1 restriction of enterovirus infections in human cells cellular mrna decay protein auf1 negatively regulates enterovirus and human rhinovirus infections heterogeneous nuclear ribonucleoprotein a2 participates in the replication of japanese encephalitis virus through an interaction with viral proteins and rna viral and cellular proteins involved in coronavirus replication hnrnp c and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3'ntr of the hcv rna genome mechanistic consequences of hnrnp c binding to both rna termini of poliovirus negative-strand rna intermediates characterization of multimeric complexes formed by the human ptb1 protein on rna heterogeneous nuclear ribonucleoprotein i (hnrnp-i/ptb) selectively binds the conserved 3′ terminus of hepatitis c viral rna role of la autoantigen and polypyrimidine tract-binding protein in hcv replication polypyrimidine-tract-binding protein is a component of the hcv rna replication complex and necessary for rna synthesis hur displaces polypyrimidine tract binding protein to facilitate la binding to the 3′ untranslated region and enhances hepatitis c virus replication the la antigen binds 5′ noncoding region of the hepatitis c virus rna in the context of the initiator aug codon and stimulates internal ribosome entry site-mediated translation hepatitis c virus internal ribosome entry site-mediated translation is stimulated by specific interaction of independent regions of human la autoantigen hur, a protein implicated in oncogene and growth factor mrna decay, binds to the 3′ ends of hepatitis c virus rna of both polarities delayed kinetics of poliovirus rna synthesis in a human cell line with reduced levels of hnrnp c proteins microrna-555 has potent antiviral properties against poliovirus cellular rna binding proteins ns1-bp and hnrnp k regulate influenza a virus rna splicing ns1-binding protein (ns1-bp): a novel human protein that interacts with the influenza a virus nonstructural ns1 protein is relocalized in the nuclei of infected cells co-regulatory activity of hnrnp k and ns1-bp in influenza and human mrna splicing rna-binding protein hnrnp d modulates internal ribosome entry site-dependent translation of hepatitis c virus rna two distinct binding modes of a protein cofactor with its target rna mechanism for nucleic acid chaperone activity of hiv-1 nucleocapsid protein revealed by single molecule stretching heterogeneous nuclear ribonuclear protein k interacts with the enterovirus 71 5′ untranslated region and participates in virus replication the polypyrimidine tract binding protein is required for efficient picornavirus gene expression and propagation feline calicivirus replication: requirement for polypyrimidine tract-binding protein is temperature-dependent internal initiation of translation from the human rhinovirus-2 internal ribosome entry site requires the binding of unr to two distinct sites on the 5′ untranslated region evidence for an rna chaperone function of polypyrimidine tractbinding protein in picornavirus translation interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis c virus rna genome and its functional requirement in internal initiation of translation hnrnp l is required for the translation mediated by hcv ires heterogeneous nuclear ribonucleoprotein l interacts with the 3 border of the internal ribosomal entry site of hepatitis c virus hnrnp l and nf90 interact with hepatitis c virus 5′-terminal untranslated rna and promote efficient replication a human proteome microarray identifies that the heterogeneous nuclear ribonucleoprotein k (hnrnp k) recognizes the 5′ terminal sequence of the hepatitis c virus rna heterogeneous ribonucleoprotein k (hnrnp k) binds mir-122, a mature liver-specific microrna required for hepatitis c virus replication modulation of hepatitis c virus rna abundance by a liver-specific microrna global epidemiology and genotype distribution of the hepatitis c virus infection position-dependent function for a tandem microrna mir-122-binding site located in the hepatitis c virus rna genome hepatitis c virus core protein interacts with heterogeneous nuclear ribonucleoprotein k an rna-binding protein, hnrnp a1, and a scaffold protein, septin 6, facilitate hepatitis c virus replication hnrnp a1 interacts with the 5' untranslated regions of enterovirus 71 and sindbis virus rna and is required for viral replication apigenin inhibits enterovirus-71 infection by disrupting viral rna association with trans-acting factors the role of misshapen nck-related kinase (mink), a novel ste20 family kinase, in the ires-mediated protein translation of human enterovirus 71 n-terminomics tails identifies host cell substrates of poliovirus and coxsackievirus b3 3c proteinases that modulate virus infection heterogeneous nuclear ribonucleoprotein m facilitates enterovirus infection the heterogeneous nuclear ribonucleoprotein k (hnrnp k) is a host factor required for dengue virus and junín virus multiplication the heterogeneous nuclear ribonucleoprotein k (hnrnp k) interacts with dengue virus core protein identification of heterogeneous nuclear ribonucleoprotein k (hnrnp k) as a repressor of c/ebpmediated gene activation role of human heterogeneous nuclear ribonucleoprotein c1/c2 in dengue virus replication vimentin interacts with heterogeneous nuclear ribonucleoproteins and dengue nonstructural protein 1 and is important for viral replication and release identification of human hnrnp c1/c2 as a dengue virus ns1-interacting protein heterogeneous nuclear ribonucleoprotein k supports vesicular stomatitis virus replication by regulating cell survival and cellular gene expression downregulation of nipah virus n mrna occurs through interaction between its 3′ untranslated region and hnrnp d hnrnp a2/b1 interacts with influenza a viral protein ns1 and inhibits virus replication potentially through suppressing ns1 rna/ protein levels and ns1 mrna nuclear export the negative regulator of splicing element of rous sarcoma virus promotes polyadenylation a cellular protein, hnrnp h, binds to the negative regulator of splicing element from rous sarcoma virus efficient polyadenylation of rous sarcoma virus rna requires the negative regulator of splicing element analysis of the interactions of viral and cellular factors with human cytomegalovirus lytic origin of replication viral dna replication orientation and hnrnps regulate transcription of the human papillomavirus 18 late promoter host heterogeneous ribonucleoprotein k (hnrnp k) as a potential target to suppress hepatitis b virus replication cytidine deaminase apobec3b interacts with heterogeneous nuclear ribonucleoprotein k and suppresses hepatitis b virus expression the multifunctional herpes simplex virus ie63 protein interacts with heterogeneous ribonucleoprotein k and with casein kinase 2 ck2 protein kinase is stimulated and redistributed by functional herpes simplex virus icp27 protein rna polymerase ii holoenzyme modifications accompany transcription reprogramming in herpes simplex virus type 1-infected cells association of herpes simplex virus type 1 icp8 and icp27 proteins with cellular rna polymerase ii holoenzyme herpes simplex virus 1 icp27 is required for transcription of two viral late (gamma 2) genes in infected cells human papillomaviruses: a compilation and analysis of nucleic acid and amino acid sequences a downstream polyadenylation element in human papillomavirus type 16 l2 encodes multiple ggg motifs and interacts with hnrnp h specific interaction between hnrnp h and hpv16 l1 proteins: implications for late gene auto-regulation enabling rapid viral capsid protein production the epstein-barr virus (ebv) sm protein enhances pre-mrna processing of the ebv dna polymerase transcript the alternative splicing factor hnrnp a1 is up-regulated during virus-infected epithelial cell differentiation and binds the human papillomavirus type 16 late regulatory element a 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hfip1, cstf-64, hnrnp c1/c2, and polypyrimidine tract binding protein identification of an hnrnp a1-dependent splicing silencer in the human papillomavirus type 16 l1 coding region that prevents premature expression of the late l1 gene inhibition of hpv-16 l1 expression from l1 cdnas correlates with the presence of hnrnp a1 binding sites in the l1 coding region identification of a 17-nucleotide splicing enhancer in hpv-16 l1 that counteracts the effect of multiple hnrnp a1-binding splicing silencers polypyrimidine tract binding protein induces human papillomavirus type 16 late gene expression by interfering with splicing inhibitory elements at the major late 5' splice site, sd3632 translational inhibition in vitro of human papillomavirus type 16 l2 mrna mediated through interaction with heterogenous ribonucleoprotein k and poly(rc)-binding proteins 1 and 2 c-src-mediated phosphorylation of hnrnp k drives translational activation of specifically silenced mrnas regulation of the epstein-barr virus c promoter by auf1 and the cyclic amp/protein kinase a signaling pathway auf1/hnrnp d is a novel protein partner of the eber1 noncoding rna of epstein-barr virus nuclear hnrnpa2b1 initiates and amplifies the innate immune response to dna viruses hnrnp a1 selectively interacts through its gly-rich domain with different rna-binding proteins hiv nef enhances tat-mediated viral transcription through a hnrnp-k-nucleated signaling complex cooperative-binding and splicing-repressive properties of hnrnp a1 trafficking of hiv-1 rna is mediated by heterogeneous nuclear ribonucleoprotein a2 expression and impacts on viral assembly actin-associated hnrnp proteins as transacting factors in the control of mrna transport and localization genomic structure of an attenuated quasi species of hiv-1 from a blood transfusion donor and recipients novel (n)pkc kinases phosphorylate nef for increased hiv transcription, replication and perinuclear targeting cloning and functional analysis of multiply spliced mrna species of human immunodeficiency virus type 1 alternative splicing of human immunodeficiency virus type 1 mrna modulates viral protein expression, replication, and infectivity a naturally arising mutation of a potential silencer of exon splicing in human immunodeficiency virus type 1 induces dominant aberrant splicing and arrests virus production exon recognition in vertebrate splicing intron recognition comes of age identification, purification, and biochemical characterization of u2 small nuclear ribonucleoprotein auxiliary factor interaction of u2af65 rs region with pre-mrna of branch point and promotion base pairing with u2 snrna a potential role for u2af-sap 155 interactions in recruiting u2 snrnp to the branch site sorting out the complexity of sr protein functions hnrnp complexes: composition, structure, and function alternative pre-mrna splicing: the logic of combinatorial control balanced splicing at the tat-specific hiv-1 3'ss a3 is critical for hiv-1 replication splicing efficiency of human immunodeficiency virus type 1 tat rna is determined by both a suboptimal 3' splice site and a 10 nucleotide exon splicing silencer element located within tat exon 2 the tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3′ splice site behind the scenes of hiv-1 replication: alternative splicing as the dependency factor on the quiet an exonic splicing silencer downstream of the 3' splice site a2 is required for efficient human immunodeficiency virus type 1 replication biochemical and nmr study on the competition between proteins sc35, srp40, and heterogeneous nuclear ribonucleoprotein a1 at the hiv tat exon 2 splicing site rna biology identification of protein partners of the human immunodeficiency virus 1 tat/rev exon 3 leads to the discovery of a new hiv-1 splicing regulator, protein hnrnp k a second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of tat mrna and binds protein hnrnp h control of hiv-1 env rna splicing and transport: investigating the role of hnrnp a1 in exon splicing silencer (ess3a) function the hnrnp a1 protein regulates hiv-1 tat splicing via a novel intron silencer element hnrnp a1 controls hiv-1 mrna splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure solution structure of the hiv-1 intron splicing silencer and its interactions with the up1 domain of heterogeneous nuclear ribonucleoprotein (hnrnp) a1 a truncated hnrnp a1 isoform, lacking the rgg-box rna binding domain, can efficiently regulate hiv-1 splicing and replication sc35 and heterogeneous nuclear ribonucleoprotein a/b proteins bind to a juxtaposed exonic splicing enhancer/exonic splicing silencer element to regulate hiv-1 tat exon 2 splicing hnrnp a1 recruited to an exon in vivo can function as an exon splicing silencer a janus splicing regulatory element modulates hiv-1 tat and rev mrna production by coordination of hnrnp a1 cooperative binding rna splicing at human immunodeficiency virus type 1 3' splice site a2 is regulated by binding of hnrnp a/b proteins to an exonic splicing silencer element differential hnrnp d isoform incorporation may confer plasticity to the essv-mediated repressive state across hiv-1 exon 3 human immunodeficiency virus type 1 hnrnp a/b-dependent exonic splicing silencer essv antagonizes binding of u2af65 to viral polypyrimidine tracts sr proteins and hnrnp h regulate the splicing of the hiv tev-specific exon 6d the secondary structure of the human immunodeficiency virus type 1 transcript modulates viral splicing and infectivity members of the heterogeneous nuclear ribonucleoprotein h family activate splicing of an hiv-1 splicing substrate by promoting formation of atp-dependent spliceosomal complexes rna trafficking signals in human immunodeficiency virus type 1 trafficking of hiv-1 rna is mediated by heterogeneous nuclear ribonucleoprotein a2 expression and impacts on viral assembly a late role for the association of hnrnp a2 with the hiv-1 hnrnp a2 response elements in genomic rna, gag, and vpr localization differential effects of hnrnp d/auf1 isoforms on hiv-1 gene expression depletion of hnrnp a2/b1 overrides the nuclear retention of the hiv-1 genomic rna mapping of determinants required for the function of the hiv-1 env nuclear retention sequence inhibition of hiv-1 gene expression by a fragment of hnrnp u nuclear mrna export: insights from virology protein kinase ck2 phosphorylation regulates the interaction of kaposi's sarcoma-associated herpesvirus regulatory protein orf57 with its multifunctional partner hnrnp k heterogeneous nuclear ribonucleoprotein a1 interferes with the binding of the human t cell leukemia virus type 1 rex regulatory protein to its response element human immunodeficiency virus type 1 (hiv-1) induces the cytoplasmic retention of heterogeneous nuclear ribonucleoprotein a1 by disrupting nuclear import: implications for hiv-1 gene expression a polypyrimidine tract facilitates the expression of kaposi's sarcoma-associated herpesvirus vflip through an internal ribosome entry site heat shock protein hsp72 plays an essential role in her2-induced mammary tumorigenesis. oncogene the role of heat shock protein 70 in mediating agedependent mortality in sepsis peptides and aptamers targeting hsp70: a novel approach for anticancer chemotherapy activation of hsp70 reduces neurotoxicity by promoting polyglutamine protein degradation monitering heat shock protein 70 and heat shock protein 90 during herpes simplex virus type 1 infection hsp70 functions as a negative regulator of west nile virus capsid protein through direct interaction mitochondrial hsp70, hsp40, and hsp60 bind to the 3′ untranslated region of the murine hepatitis virus genome immunomodulatory activity of extracellular hsp70 mediated via paired receptors siglec-5 and siglec-14 extracellular cell stress proteins as biomarkers of human disease molecular chaperones and protein-folding catalysts as intercellular signaling regulators in immunity and inflammation cross-presentation of the oncofetal tumor antigen 5t4 from irradiated prostate cancer cells-a key role for heat-shock protein 70 and receptor cd91 membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen stress for maintaining memory: hsp70 as a mobile messenger for innate and adaptive immunity novel heat shock protein hsp70l1 activates dendritic cells and acts as a th1 polarizing adjuvant chaperokine activity of heat shock proteins hsp70 as endogenous stimulus of the toll/interleukin-1 receptor signal pathway chlamydial heat shock protein 60 activates macrophages and endothelial cells through toll-like receptor 4 and md2 in a myd88-dependent pathway association of hadha with human rna silencing machinery dengue virus modulates the unfolded protein response in a time-dependent manner heat shock protein 70 is associated with replicase complex of japanese encephalitis virus and positively regulates viral genome replication japanese encephalitis virus activates autophagy through xbp1 and atf6 er stress sensors in neuronal cells bovine viral diarrhea virus infection induces autophagy in mdbk cells the expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis the emerging roles of viroporins in er stress response and autophagy induction during virus infection stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy hepatitis c virus suppresses the ire1-xbp1 pathway of the unfolded protein response glucose-regulated protein 78 is an intracellular antiviral factor against hepatitis b virus hepatitis b virus x protein (hbx) activates atf6 and ire1-xbp1 pathways of unfolded protein response herpes simplex virus 1 infection activates the endoplasmic reticulum resident kinase perk and mediates eif-2alpha dephosphorylation by the gamma(1)34.5 protein membrane biogenesis and the unfolded protein response the atf6 branch of unfolded protein response and apoptosis are activated to promote african swine fever virus infection role of the host cell's unfolded protein response in arenavirus infection interaction of dengue virus envelope protein with endoplasmic reticulum-resident chaperones facilitates dengue virus production human cytomegalovirus specifically controls the levels of the endoplasmic reticulum chaperone bip/grp78, which is required for virion assembly betanodavirus up-regulates chaperone grp78 via er stress: roles of grp78 in viral replication and host mitochondria-mediated cell death influenza a viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (ire1) stress pathway west nile virus differentially modulates the unfolded protein response to facilitate replication and immune evasion resistance to vesicular stomatitis virus infection requires a functional cross talk between the eukaryotic translation initiation factor 2α kinases perk and pkr activation of erad pathway by human hepatitis b virus modulates viral and subviral particle production hepatitis c virus ns4b induces unfolded protein response and endoplasmic reticulum overload response-dependent nf-κb activation the sars coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor human cytomegalovirus protein us11 provokes an unfolded protein response that may facilitate the degradation of class i major histocompatibility complex products hcv causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response endoplasmic reticulum stress induced by hepatitis b virus x protein enhances cyclo-oxygenase 2 expression via activating transcription factor 4 functional diversity of the hnrnps: past, present and perspectives heterogeneous nuclear ribonucleoproteins (hnrnps) in cellular processes: focus on hnrnp e1's multifunctional regulatory roles acute and chronic disease caused by enteroviruses hnrnp a1 alters the structure of a conserved enterovirus ires domain to stimulate viral translation heterogeneous nuclear ribonuclear protein k interacts with the enterovirus 71 59 untranslated region and participates in virus replication molecular mechanism of action of hnrnp k and rtn3 in the replication of enterovirus 71. chin sherris medical microbiology, 6e anti-herpetic medications and reduced risk of dementia in patients with herpes simplex virus infections-a nationwide, population-based cohort study in taiwan antivirals reduce the formation of key alzheimer's disease molecules in cell cultures acutely infected with herpes simplex virus type 1 the heterogeneous nuclear ribonucleoprotein k is important for herpes simplex virus-1 propagation hepatitis b virus epidemiology and natural history of hcv infection pathobiology of chronic hepatitis virus infection and hepatocellular carcinoma (hcc). tal hepatitis delta virus polyarteritis nodosa and extrahepatic manifestations of hbv infection: the case against autoimmune intervention in pathogenesis autoantibody recognition of an n-terminal epitope of hnrnp l marks the risk for developing hbv-related hepatocellular carcinoma benign lymphadenopathies epstein-barr virus-associated lymphoproliferative disorders: experimental and clinical developments epstein-barr virus-associated hodgkin's lymphoma human papillomavirus and epstein-barr virus in nasopharyngeal carcinoma in a low-incidence population epstein-barr virus infection and multiple sclerosis: a review lymphomatoid granulomatosis: a practical review for pathologists dealing with this rare pulmonary lymphoproliferative process epstein-barr virus and skin manifestations in childhood monoclonal antibodies against epstein-barr virus cross-react with alpha-synuclein in human brain human papillomavirus (hpv) type distribution and serological response to hpv type 6 virus-like particles in patients with genital warts the global health burden of infection-associated cancers in the year 2002 trends in human papillomavirus-associated cancers-united states epidemiology and natural history of human papillomavirus infections in the female genital tract case-control study of human papillomavirus and oropharyngeal cancer review of human immunodeficiency virus type 1-related opportunistic infections in sub-saharan africa the treatment of patients with hiv defining hsp70 subnetworks in dengue virus replication reveals key vulnerability in flavivirus infection the hsp90 mosaic: a picture emerges the chaperone hsp90: changing partners for demanding clients structure of tpr domain-peptide complexes: critical elements in the assembly of the hsp70-hsp90 multichaperone machine hsp90 and co-chaperones twist the functions of diverse client proteins the 'active life' of hsp90 complexes structure and functional relationships of hsp90 cellular growth kinetics distinguish a cyclophilin inhibitor from an hsp90 inhibitor as a selective inhibitor of hepatitis c virus novobiocin and additional inhibitors of the hsp90 cterminal nucleotide-binding pocket the amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an atp/adp switch domain that regulates hsp90 conformation 17-allylamino-17-demethoxygeldanamycin induces downregulation of critical hsp90 protein clients and results in cell cycle arrest and apoptosis of human urinary bladder cancer cells inhibition of signal transduction by the hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis antitumor efficacy of ipi-504, a selective heat shock protein 90 inhibitor against human epidermal growth factor receptor 2-positive human xenograft models as a single agent and in combination with trastuzumab or lapatinib nvp-auy922: a novel heat shock protein 90 inhibitor active against xenograft tumor growth, angiogenesis, and metastasis biib021, an orally available, fully synthetic small-molecule inhibitor of the heat shock protein hsp90 ganetespib, a unique triazolone-containing hsp90 inhibitor, exhibits potent antitumor activity and a superior safety profile for cancer therapy the heat shock protein 90 inhibitor, at13387, displays a long duration of action in vitro and in vivo in non-small cell lung cancer snx2112, a synthetic heat shock protein 90 inhibitor, has potent antitumor activity against her kinase-dependent cancers novobiocin and related coumarins and depletion of heat shock protein 90-dependent signaling proteins the heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized atpbinding domain in the carboxyl terminus of the chaperone design, synthesis, and biological evaluation of novel deguelinbased heat shock protein 90 (hsp90) inhibitors targeting proliferation and angiogenesis structural basis for depletion of heat shock protein 90 client proteins by deguelin epigallocatechin-3-gallate is a novel hsp90 inhibitor novel hsp90 inhibitor platycodin d disrupts hsp90/cdc37 complex and enhances the anticancer effect of mtor inhibitor sulforaphane inhibits pancreatic cancer 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inhibitor clemizole is highly synergistic with hcv protease inhibitors spontaneous and engineered compensatory hsv mutants that counteract the host antiviral pkr response open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source [81671995]; the general research grant of hong kong [11100215] ; and strategic funds from city university of hong kong. competing interests: the authors declare no competing interests.commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-335310-61wibso4 authors: chen, hui-wen; huang, chen-yu; lin, shu-yi; fang, zih-syun; hsu, chen-hsuan; lin, jung-chen; chen, yuan-i; yao, bing-yu; hu, che-ming j. title: synthetic virus-like particles prepared via protein corona formation enable effective vaccination in an avian model of coronavirus infection date: 2016-08-15 journal: biomaterials doi: 10.1016/j.biomaterials.2016.08.018 sha: doc_id: 335310 cord_uid: 61wibso4 the ongoing battle against current and rising viral infectious threats has prompted increasing effort in the development of vaccine technology. a major thrust in vaccine research focuses on developing formulations with virus-like features towards enhancing antigen presentation and immune processing. herein, a facile approach to formulate synthetic virus-like particles (svlps) is demonstrated by exploiting the phenomenon of protein corona formation induced by the high-energy surfaces of synthetic nanoparticles. using an avian coronavirus spike protein as a model antigen, svlps were prepared by incubating 100 nm gold nanoparticles in a solution containing an optimized concentration of viral proteins. following removal of free proteins, antigen-laden particles were recovered and showed morphological semblance to natural viral particles under nanoparticle tracking analysis and transmission electron microscopy. as compared to inoculation with free proteins, vaccination with the svlps showed enhanced lymphatic antigen delivery, stronger antibody titers, increased splenic t-cell response, and reduced infection-associated symptoms in an avian model of coronavirus infection. comparison to a commercial whole inactivated virus vaccine also showed evidence of superior antiviral protection by the svlps. the study demonstrates a simple yet robust method in bridging viral antigens with synthetic nanoparticles for improved vaccine application; it has practical implications in the management of human viral infections as well as in animal agriculture. vaccine is historically the most effective countermeasure against infectious threats, as agents resembling pathogens are administered to mount an immune response against specific targets. amidst continuing and emerging viral threats, vaccine technology continues to advance with the aim of effectively promoting antiviral immune responses, and a major development effort lies in retaining or integrating virus-like features in vaccine formulations for improved immune processing. several morphological and antigenic characteristics of viral particles have been demonstrated to promote immune potentiation. for example, particles at the nanoscale have been shown to have better lymphatic transport as compared to smaller subunit antigens [1, 2] . in addition, the display of multiple antigens on a single particle facilitates more effective antigen presentation to immune cells [1] . as compared to traditional vaccine formulations, vaccines preserving virus-like features have shown superior capability in eliciting immune responses [3e5] . these results and observations have also prompted material scientists to apply synthetic nanomaterials towards mimicking viral features for vaccine development [6e9] . given their high radii of curvature, synthetic nanoparticles frequently possess high surface energies that induce adsorption of biomolecules in a phenomenon known as protein corona formation. in protein-rich media, strong nanoparticle/protein association occurs spontaneously as a means to passivate surface energies, and the resulting particles are encased in a protein layer that dictates the particles' interactions with the environment [10, 11] . while protein corona formation is gaining increasing scientific interest owing to its implications in biomedical applications [10, 12, 13] , we herein demonstrate harnessing this phenomenon can be beneficial towards mimicking viral features for vaccine applications. we show that synthetic virus-like particles (svlps) with close semblance to native virions in physicochemical properties and antigen display can be facilely prepared through spontaneous antigen-particle association in optimized incubation conditions. using 100 nm gold nanoparticles (aunp), a biologically inert material commonly used for biomedical research [14e16] , and a spike glycoprotein derived from an avian infectious bronchitis virus (ibv), a single-stranded positive-sense rna virus that belongs to the family coronaviridae [17] , we controlled the incubation condition to prepare spike glycoprotein-laden svlps (fig. 1) . the morphological features and antigen display by the svlps were compared to native ibv viral particles using nanoparticle tracking analysis and immunogold staining. in addition, vaccination potency between the svlps and free spike glycoproteins was compared in an avian model of coronavirus infection. a commercial whole inactivated virus (wiv) formulation that is the current standard vaccine for ibv management was examined in parallel. coronaviruses are a major viral family of which the most publicized examples include the pathogens behind severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) [18] . in animals, ibv is a prime example of coronavirus that infects the respiratory and urogenital tracts of chickens, posing a serious economic threat as one of the most important pathogens in the poultry industry. the ibv spike glycoprotein, which forms the large, pental-shaped spikes on the surface of the virion, is chosen as the antigen candidate as it is implicated as a determinant of virus pathogenicity. among coronaviruses, spike glycoproteins possess a variety of biological functions, including triggering cell attachment, inducing cell-cell fusion, and binding to cellular receptors [19, 20] . as spike glycoproteins are the primary targets in ongoing vaccine development efforts for coronavirus vaccinations, the present study has broad implications across both human and animal disease management [21, 22] . s. frugiperda sf9 (atcc crl-1711) insect cells were cultured in grace's insect cell medium (invitrogen, carlsbad, ca) and supplemented with 10% fbs (thermo fisher, rockford, il) and 1% p/ s/a antibiotics (biological industries, beit-haemek, israel) at 27 c. 100 nm gold nanoparticle (aunp) solution was purchased from sigma-aldrich (st. louis, mo). avian coronavirus ibv strain 2575/98 was propagated in 10-dayold specific-pathogen-free (spf) chicken embryos via the allantoic route as previously described [23] . the virus titers of ibvs were determined with the method of reed and muench [24] in spf chicken embryos and expressed as 50% embryo infectious dose (eid 50 ) [25] . the virus-containing allantoic fluid was concentrated and purified using sucrose gradient solution as previously described to derive the native virions [23] . full spike (s) protein of avian coronavirus ibv was cloned and expressed using the bac-to-bac baculovirus expression system (invitrogen). briefly, a recombinant plasmid was constructed by inserting full spike protein gene of ibv strain 2575/98 (accession no. dq646405) [26] into the pfastbac-1 vector using the following primer set: ibv-s-bamhi-f: 5 0 -ttggg atccg atgtt ggtga agtca c-3 0 ; ibv-s-sali-f: 5 0 -cttgt cgaca ttaaa cagac ttttt aggt-3 0 . the recombinant pfastbac-1 shuttle vector was then transposed to the bacmid in e. coli strain dh10bac, and recombinant bacmid was purified using the hipure plasmid midiprep kit (invitrogen). sf9 cells were used for transfection with the recombinant bacmid, and recombinant baculoviruses were then harvested in the supernatant and designated rbac-2575s. recombinant spike proteins (r2575s) were harvested from sf9 cells infected with rbac-2575s (multiplicity of infection ¼ 1). sf9 cells were washed and lysed with the i-per insect cell protein extraction reagent (thermo fisher). recombinant proteins were purified using the glycoprotein isolation kit, cona (thermo fisher) according to the manufacturer's instructions. after purification, r2575s protein was stored in 10% sucrose at à20 c. citrate-buffered 100 nm gold nanoparticles were washed repeatedly in water to remove the citrate stabilizer, and the resulting pellet was resuspended in 10% sucrose. protein solutions ranging in concentrations between 100 mg/ml to 3 mg/ml of purified spike proteins were then mixed with 1 â 10 11 /ml of gold nanoparticles (determined by nanoparticle tracking analysis) in 10% sucrose. the mixtures were bath sonicated for 1 min followed by incubation in an ice bath for 30 min. the nanoparticles were then removed from unbound spike proteins via centrifugation at 1500g for 3 min. following 3 centrifugal washes with 10% sucrose, pelleted nanoparticles were mixed with 1x pbs and sonicated in a bath sonicator for 30 s. dispersible, stabilized svlps were retrieved and their protein content was quantified using a bca protein assay (thermo fisher) with 25 ml of 1 â 10 11 particles/ml following the manufacturer's protocol. visualization of unstable nanoparticles and colloidally stable svlps was performed using a 200 kv high resolution transmission electron microscope (fei tecnai tf20). particle stability was assessed by monitoring the size of svlps for 7 days. particle size, polydispersity index (pdi), and concentrations were measured by nanoparticle tracking analysis using nanosight ns-500 (malvern, uk) at a concentration of 1 â 10 8 particles/ml based on the manufacturer's instructions. particle size and zeta potential were also measured by dynamic light scattering using zetasizer nano zs at a concentration of 1 â 10 10 particles/ml (malvern, uk) based on the manufacturer's instructions. antigen display was examined using freshly prepared svlps. antigen retention was examined by mixing svlps in protein-poor (pbs) or in protein-rich (10% bsa) conditions for varying periods of time. at 0, 3, 10, and 24 h marks, svlps were pelleted from their respective solutions. the particles were then processed using a previously published protocol with sds-page loading buffer for protein removal and quantification [27] . ibv spike proteins eluted from the svlp were analyzed in 6% discontinuous sds-page under non-reducing condition. protein gel was then transferred onto a 0.45 mm nitrocellulose membrane (bio-rad). after transfer, the membrane was soaked in blocking buffer (5% skim milk in pbs) at room temperature for 1 h and probed with anti-s monoclonal antibody (mab) for another 1 h. after three washes, the membrane was incubated with peroxidase-conjugated goat anti-mouse igg (h þ l) (jackson immunoresearch laboratories, west grove, pa) in blocking buffer at room temperature for 1 h. after three washes, the protein blots were detected with either tmb membrane peroxidase substrate (kpl) or enhanced chemiluminescence (ecl) substrate (pierce). band intensities were analyzed via imaging analysis using imagej. presence of ibv spike proteins on the svlps was further verified by immunogold staining, and purified ibv 2575/98 virions were used as a control. briefly, 3 ml of svlp or virion samples were deposited onto a glow-discharged carbon-coated grid for 2 min. the virion sample was fixed with 4% paraformaldehyde for 5 min. after 3 washes with pbs, the samples were blocked with 1% bsa for 15 min. the samples were then incubated with anti-s mab for 1 h. after pbs washes, the samples were incubated with 6 nm goldconjugated goat anti-mouse igg (jackson immunoresearch laboratories) for another 1 h. after pbs washes, native virions were further stained with 1% uranyl acetate for 15 s. all experiments were performed at room temperate. particles were visualized under a 200 kv high resolution transmission electron microscope (fei tecnai tf20). the care and use of animals were approved by the institute animal care and use committee, national taiwan university (approval no. ntu-102-el-89). all animal experiments were carried out in accordance with the approved guidelines. 8-week old balb/c mice were injected with 50 ml of pbs, free protein formulation, or svlps containing 2 mg of viral antigens via the intrafootpad route. after 24 h, the mice were sacrificed and the popliteal lymph nodes were harvested (n ¼ 6). cryosections (6 mm) were made and fixed for 10 min in acetone, followed by 8 min in 1% paraformaldehyde. sections were blocked by 5% normal goat serum (invitrogen) in pbs for 10 min and stained with anti-s mab for 4 h at room temperature. after washes, sections were further incubated with fitc-conjugated anti-mouse igg (jackson immu-noresearch laboratories) for 1 h at room temperature. nuclei were counterstained with dapi (invitrogen). fluorescence signal was observed under a fluorescence microscope (leica dmi8), and quantified via imaging analysis using imagej. 8-week old balb/c mice were injected intramuscularly in the thigh with 100 ml of formulations containing pbs, free protein, or svlps (10 mg of viral antigens) mixed with the complete freund's adjuvant. mice blood was collected on day 14 and 21 for antibody titer quantification (n ¼ 4e5 per group). three-week-old spf chickens were obtained from jd-spf biotech (miaoli, taiwan). chickens were randomly divided into four different experimental groups (n ¼ 4e6 per group) receiving pbs, free protein (r2575s), whole inactivated virus (wiv) vaccine (merial laboratories, lyon, france), or svlps. briefly, free protein or svlps (10 mg of viral antigen in 100 ml) were emulsified with the complete freund's adjuvant and administered via an intramuscular route. the commercially available wiv vaccine (oily-adjuvanted) was administered to chickens according to the manufacturer's recommendation (0.3 ml per chick). chicken sera and tears were collected on day 0 (before immunization), 14, and 21 post-immunization. all chickens were intranasally challenged with ibv 2575/98 live virus (10 6 eid 50 ) on day 21, and were observed for disease signs for 7 days. chickens were sacrificed on day 28. for serum iga and igg virus-specific elisa, 100 ng of purified ibv 2575/98 virions was diluted with coating buffer (15 mm na 2 co 3 and 35 mm nahco 3 , ph 9.6) and coated onto flatbottomed microtiter plates (nunc) at room temperature overnight. the wells were washed with pbst (0.1% tween 80 in pbs) three times and blocked with blocking reagent (5% skim milk in pbst) at 37 c for 1 h. after washes, 100 ml of chicken serum was added and incubated at room temperature for 1 h. following three washes, 100 ml of peroxidase-conjugated goat anti-chicken igy (h þ l) or iga (jackson immunoresearch) in blocking buffer was added into each well and incubated at room temperature for 1 h. after three washes, 100 ml of sureblue reserve tmb microwell peroxidase substrate (kpl) was added to each well and incubated in the dark at room temperature for 10 min. the reaction was stopped by adding 100 ml of tmb stop solution (kpl). the od was measured at 450 nm using an automated plate reader (thermo fisher). for total tear iga quantification, elisa was performed with chicken iga elisa kit (ab157691, abcam) according to the manufacturer's protocol. on day 28 post immunization, chicken spleens were minced and passed through a 70-mm cell strainer (corning) to obtain single-cell suspensions. red blood cells (rbcs) were lysed using an rbc lysis buffer (ebiosciences), and cells were resuspended in rpmi 1640 medium (gibco, grand island, ny) containing 10% fbs. viable cells were determined by trypan blue staining. 10 6 splenocytes were plated in 96-well u-bottom plates (corning), and were stimulated with 1 mg of purified ibv 2575/98 virions in the presence of brefeldin a (golgiplug, bd biosciences) for 6 h at 37 c. for the quantification of cytokine expression, the stimulated splenocytes were lysed, and total rna was isolated by trizol (invitrogen) according to the manufacturer's manual. real-time rt-pcr was performed using iscript (bio-rad) and iq sybr green supermix kit (bio-rad) with previously described primers for chicken ifn-g and gapdh [28] . melting curve analysis following real-time pcr was conducted to verify the specificity for each primer set. all obtained ct values were normalized to gapdh. the relative expression of chicken ifn-g (fold change of naive control) was determined by a 2 àddct method [29] . disease signs of chickens were recorded on a daily basis after virus challenge. the clinical score index of ibv infection was interpreted according to a previously described method [30] . the clinical signs were evaluated as: 0 ¼ no clinical signs; 1 ¼ lacrimation, slight shaking, watering feces or tracheal rales; 2 ¼ lacrimation, presence of nasal exudate, depression, water feces, apparent sneezing or cough; 3 ¼ high degree of lacrimation, nasal exudate, and severe watery feces; 4 ¼ death. after necropsy, gross lesions at the tracheas and kidneys were recorded. chicken kidneys were further harvested and homogenized in tryptose phosphate broth (bd biosciences). viral load in kidneys was assessed by quantitative rt-pcr described below. 2.11. viral rna quantification rna in chicken kidneys was extracted using trizol (invitrogen) according to the manufacturer's manual. for viral load assessment, quantitative rt-pcr was performed with iscript (bio-rad) and iq sybr green supermix kit (bio-rad) using previously described primer sets that target the s protein gene of ibv (rc2u and rc3l) [31] and chicken 28s rrna [32] . quantitative rt-pcr experiments were performed in duplicates. data was expressed as arbitrary units. data was analyzed by anova followed by dunnett's multiple comparison tests using graphpad prism (graphpad software, san diego, ca). p values smaller than 0.05 were considered significant. following aunp incubation in solutions of different protein concentrations, the resulting nanoparticles were pelleted from free proteins and re-dispersed through sonication in pbs. consistent with previous studies on nanoparticle/protein interactions [33] , it was observed that higher protein concentrations yielded particles with increased colloidal stability as evidenced by the disappearance of a discernable pellet and a purple solution characteristic of aunp suspensions ( fig. 2a) . svlps prepared from the 3 mg/ml protein suspension were readily dispersible and manifest as distinct, nonclustered nanoparticles under transmission electron microscopy (fig. 2b) , indicating passivation of the high particle surface energy upon sufficient protein coating. in contrast, particle preparations with lower protein content (1000 mg/ml) yielded clustered aunps. to further characterize svlps, we assessed aunps, svlps, and native ibv virions (fig. 2c) using nanoparticle tracking analysis, which examines particle samples on a particle-by-particle basis via tracking of scattered laser light from individual particles [34] . between aunps and svlps, we observed an overall reduction in the light scattering intensity. given that aunps are known to scatter light at an extraordinary efficiency, the intensity reduction in svlps can be attributed to successful protein coating, which restricts light passage to the aunp surfaces. likewise, native virions have the lowest light scattering under the analysis as they are comprised entirely of organic materials. the result demonstrates the feasibility of studying the evolution of nanoparticle protein corona formation using nanoparticle tracking analysis, which reveals changes in light scattering and size simultaneously. upon examining the size distributions of the different particles, svlps showed a broader distribution as compared to the sharply distributed 100 nm aunps. protein corona formation increased the nanoparticle size from 100.6 nm (pdi ¼ 0.012) to 139.2 nm (pdi ¼ 0.073) and increased the zeta potential from à23.2 mv to à16.7 mv (fig. 2c,d) . in comparison to native ibv virions, which have an average diameter of 147.3 nm (pdi ¼ 0.081) and a zeta potential of à16.6 mv, the svlps are similar in overall physicochemical properties. examination of particle stability showed that the svlps remained stable in pbs over a 7-day period with its size ranging from 136.7 nm (pdi ¼ 0.071) to 140.2 nm (pdi ¼ 0.091) (fig. 2e) . analysis of antigen display with freshly prepared svlps showed that 1 â 10 11 aunps retained 23.5 ± 2.2 mg of spike proteins, corresponding to approximately 900 ibv spike proteins per particle. western blotting using analysis revealed a sharp protein band of approximately 160 kda (fig. 2f) , which is characteristic of the viral antigen [17] . transmission electron microscopy and immunogold staining further highlight the similarity between svlps and native ibv virions. it was observed that immunogold clustered around the svlps, mirroring the staining pattern on the native virions (fig. 2g) . these observations demonstrate the close semblance between the svlps and native virions regarding their physicochemical properties and antigen display. examination of antigen retention in protein-poor (1x pbs) and protein-rich (10% bsa in 1x pbs) conditions also shed light on the characteristics of the protein corona around the svlps. in pbs, particle-bound antigen level remained steady over a span of 24 h, yielding similar ibv spike protein band intensities across the different incubation samples (fig. 2h) . a rapid drop-off in particlebound spike protein was observed upon incubation in 10% bsa. immediate retrieval of svlps from the bsa solution resulted in~65% reduction in the spike protein level, and at the 24 h mark,~25% of the initial antigen remained on the svlps. this observation suggests the formation of two distinctive corona layers distinguishable by their interaction dynamics with surrounding biomolecules, reflecting the presence of both a reversible "soft corona" and an irreversible "hard corona" that have been frequently observed in prior nanoparticle studies [35e37]. the results indicate that approximately 200e250 ibv spike proteins are stably bound to each svlps. these proteins are expected to remain in the particulate form in complex biological environments upon in vivo administration. to examine antigen delivery and lymphatic transport by the svlps as compared to free spike proteins, svlp formulation was administered to mice through a footpad injection. popliteal lymph nodes, which are the draining lymph nodes by the footpads, were subsequently collected and sectioned for immunofluorescence assay. ibv spike protein-specific immunofluorescence staining showed a significantly enhanced antigen delivery by the svlps as compared to the free protein formulation, resulting in an increased number of fluorescent punctates (green) in the lymph node sections (fig. 3a) . imaging analysis on multiple lymph node sections showed that the svlp formulation increased lymphatic delivery by approximately 6 fold (fig. 3b) . the observation of increased delivery attests to the strong protein/particle binding in the "hard corona" layer as the particle carrier is capable of facilitating antigen transport in vivo. the enhanced lymph node localization of the svlps is consistent with prior observations on nanoparticles and virus-like particles [2] . owing to their nanoscale morphology and physicochemical properties, these nanoparticles are known to facilitate free lymphatic drainage via convective transport [38, 39] as well as cell-mediated lymphatic delivery via increased cellular uptake [2] . immunogenicity of the svlps was also examined following intramuscular inoculation in mice. anti-ibv igg serum titers were compared between mice vaccinated with svlps and with free ibv spike proteins (fig. 3c) , and it was observed that the svlps elicited significantly higher igg levels, demonstrating improved vaccination potency over the free protein formulation. the improved immunogenicity can be explained in part by the enhanced antigen delivery to the lymph node, where a high number of antigen presenting cells reside. in addition, the particulate nature of the svlps likely also favors other immune activation mechanisms, such as improved cellular uptake, enhanced complement activation [38] and presentation by follicular dendritic cells [40] . these nanoparticle-specific immunological features make the svlps a promising vaccine candidate for disease management. to evaluate the svlps' effectiveness against viral infections, we vaccinated spf chickens with free ibv spike proteins or svlps (10 mg of total viral antigens) via the intramuscular route. as an additional reference, a commercial wiv vaccine for ibv was administered based on the manufacturer's suggested dosage. following vaccination, blood and tear were collected for analysis and a live ibv challenge was performed (fig. 4a) . elisa analysis showed that the svlps were superior in generating both igg and iga titers as compared to the free protein formulation and the wiv vaccine (fig. 4b,c) . the total iga in the tears of the vaccinated chickens were also quantified. despite that intramuscular vaccination is generally known to be non-ideal for promoting mucosal immunity [41] , elevation of tear iga level was observed for all three vaccine formulations (fig. 4d) . it is expected that mucosal vaccination in future studies may further increase tear iga levels and better highlight the differences among the formulations in eliciting mucosal immunity. besides humoral immunity, cellular immunity, a major component of effective antiviral immune responses [42] , was analyzed using splenocytes extracted on day 28. the svlp sample showed a significant increase in the ifn-g mrna level as compared to the control, free protein, and the wiv vaccine samples (fig. 4e) , demonstrating superior promotion of antigen-specific cellular immunity. we further examined the effect of the different vaccinations in protecting against a viral challenge. clinical scores evaluated based on stamina, posture, and voice show that the svlp group had the lowest overall symptoms, on par with animals vaccinated with the wiv formulation (fig. 5a,b) . in comparison, vaccination with the free protein formulation was less effective and highly variable in moderating the disease symptoms. on day 28, necropsies were performed to examine the tracheas and kidneys, which are characteristic sites for infections by ibv [43] . as indicated in the gross lesion photos, the best antiviral protection was observed in the svlp-immunized group, whereas organs from the free protein group and the wiv vaccine group showed observable mucus secretion and petechiae in tracheas (fig. 5d, upper panel, arrowed) and swollen lesions and hemorrhages in kidneys (fig. 5d, lower panel, arrowed) . the prophylactic effect of the svlp vaccination was further demonstrated by examining the viral load in kidneys. analysis by quantitative rt-pcr showed that immunization with svlps more consistently reduced the viral content, resulting in the lowest relative viral mrna expression across the animal samples (fig. 5c) . the results further corroborate the protective effect by the svlp vaccination, which enhanced both humoral and cellular immunity for increased protection against the viral challenge. coronavirus spike proteins are the primary antigenic signatures on coronaviruses as they contribute to the characteristic crown-like morphology underlining this virus family. as these proteins comprise the outermost layer of coronaviruses, the spike proteins have a pivotal role in viral pathogenesis and are recognized as the primary target for vaccine preparations [44] . present vaccination strategies for coronaviruses include recombinant viruses and viruslike particles, and there is a continuing effort in developing new strategies for improving vaccine potency and safety [22] . to the best of our knowledge, incorporating coronavirus spike protein with synthetic nanoparticles has not been previously explored. by exploiting the high surface energies of synthetic nanoparticles, spontaneous assembly of svlps covered with ibv spike proteins were demonstrated. the strong particle/antigen association resulted in virus-sized particulates displaying ibv spike proteins, and the svlps elicited strong immune protection against a live ibv challenge. the enhanced immunopotentiation by the particle carrier is consistent with previous studies and echoes the curious observation that gold nanoparticles not only promote humoral but also cellular immune responses upon association with antigens [14, 15] . as the increased cellular immune response suggests that the nanoparticles may play a role beyond a passive antigen carrier, future studies examining the impact of nanomaterials and nanoparticle surface energies on immunological interactions are warranted. it should be noted that the phenomenon of protein corona formation is an evolving field of study in which scientists continue to examine nanomaterials in biological medium with increasing complexity [45e47]. subtle changes on the environment and on nanoparticle properties can have dramatic and unpredictable impact on the overall corona identity with significant biological implications. to demonstrate a practical utility for the protein corona phenomenon, the present study adopts a reductionist approach in examining protein-particle interactions. aunps are incubated in a highly controlled condition with proteins of a singular species to form svlps with virus-mimetic features, and the dynamics of such association are expected to vary with different biomolecules and nanomaterials [48] . in general, inorganic nanoparticles promote stronger protein adsorption as compared to organic nanoparticles as inorganic nanoparticles tend to have higher surface energies. decreasing particle size also tends to increase biomolecule interactions as it increases radii of curvature of nanoparticle surfaces. other forces, such as electrostatic interactions, van der waals forces and covalent interactions all play intertwining roles in governing the nano-bio interface, and factors including nanoparticle functionalizations, buffer conditions, and biomolecule species have significant impact on the corona formation [48] . nonetheless, in a controlled and optimized condition, the phenomenon may be exploited to facilely prepare formulations with defined characteristics and favorable biological performance. the present work takes advantage of this spontaneous interaction between nanomaterials and biomolecules towards improving vaccine development. this strategy may find practical applications in disease management against coronaviruses as well as other infectious threats. in summary, we demonstrate by incubating viral antigens with synthetic nanoparticles in optimized conditions, spontaneous formation of protein corona induces the assembly of virus-like nanostructures with viral antigens encasing the particulate core. results from the present study validate the successful preparation of svlps via nanoparticles' innate tendency to induce protein coating. in comparison to typical virus-like particle preparations, the present strategy offers practical advantages owing to its simple and facile process. amidst the growing health threats of coronavirus infections as well as the ongoing economic impact of ibv infections, virus-like particles are garnering increasing scientific interest as vaccine candidates owing to their improved efficacy in comparison to subunit antigens [49, 50] . in the present study, vaccination with the svlps resulted in enhanced humoral and cellular immune responses, improving protection against an avian model of coronavirus infection as compared to free protein antigens and a commercial wiv vaccine. strong immunity against the viral challenge following svlp vaccination was evidenced by multiple criteria, including improved physical symptoms, reduced organ lesions, and decreased overall viral load. the enhanced immunopotentiation by the svlps is attributable at least in part to increased lymphatic delivery and multivalent antigen display. given the robustness and versatility of the approach, it can be envisioned the technique can be broadly applied for different vaccine development. enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand t-fh cells and promote germinal center induction vaccine delivery: a matter of size, geometry, kinetics and molecular patterns development of a new hydrogen peroxidebased vaccine platform virus-like particles as immunogens self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h1n1 antibodies interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses nanoparticulate sting agonists are potent lymph node-targeted vaccine adjuvants ph-degradable imidazoquinoline-ligated nanogels for lymph nodefocused immune activation cancer cell membrane-coated nanoparticles for anticancer vaccination and drug delivery rapid formation of plasma protein corona critically affects nanoparticle pathophysiology the effect of nanoparticle size, shape, and surface chemistry on biological systems protein adsorption is required for stealth effect of poly(ethylene glycol)-and poly(phosphoester)-coated nanocarriers spontaneous protein adsorption on graphene oxide nanosheets allowing efficient intracellular vaccine protein delivery modulating antibacterial immunity via bacterial membrane-coated nanoparticles gold nanoparticles as a vaccine platform: influence of size and shape on immunological responses in vitro and in vivo gold nanoparticles in delivery applications coronavirus ibv: structural characterization of the spike protein from sars to mers: 10 years of research on highly pathogenic human coronaviruses the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis a truncated receptorbinding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice a type-specific blocking elisa for the detection of infectious bronchitis virus antibody a simple method of estimating fifty per cent endpoints a laboratory manual for the isolation and identification of avian pathogens identification of taiwan and china-like recombinant avian infectious bronchitis viruses in taiwan quantitative profiling of the protein coronas that form around nanoparticles comparison of the expression of cytokine genes in the bursal tissues of the chickens following challenge with infectious bursal disease viruses of varying virulence analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method in vivo evaluation of the pathogenicity of field isolates of infectious bronchitis virus s1 and n gene analysis of avian infectious bronchitis viruses in taiwan chicken interferon alpha pretreatment reduces virus replication of pandemic h1n1 and h5n9 avian influenza viruses in lung cell cultures from different avian species formation mechanism for stable hybrid clusters of proteins and nanoparticles critical evaluation of nanoparticle tracking analysis (nta) by nanosight for the measurement of nanoparticles and protein aggregates biomolecular coronas provide the biological identity of nanosized materials reversible versus irreversible binding of transferrin to polystyrene nanoparticles: soft and hard corona the evolution of the protein corona around nanoparticles: a test study exploiting lymphatic transport and complement activation in nanoparticle vaccines a sensitive in vivo model for quantifying interstitial convective transport of injected macromolecules and nanoparticles follicular dendritic cells: dynamic antigen libraries what role does the route of immunization play in the generation of protective immunity against mucosal pathogens? contributions of humoral and cellular immunity to vaccine-induced protection in humans coronavirus avian infectious bronchitis virus the spike protein of sars-covea target for vaccine and therapeutic development effects of the presence or absence of a protein corona on silica nanoparticle uptake and impact on cells protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles understanding and controlling the interaction of nanomaterials with proteins in a physiological environment understanding biophysicochemical interactions at the nano-bio interface mers-cov viruslike particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques assembly and immunogenicity of coronavirus-like particles carrying infectious bronchitis virus m and s proteins the study was supported by the ministry of science and technology (103-2321-b-002-066, 104-2321-b-002-023, 105-2321 -b-001-055) and national taiwan university (104r7320). key: cord-355327-d3gcfepx authors: fan, samuel w; george, richard a; haworth, naomi l; feng, lina l; liu, jason y; wouters, merridee a title: conformational changes in redox pairs of protein structures date: 2009-08-01 journal: protein science doi: 10.1002/pro.175 sha: doc_id: 355327 cord_uid: d3gcfepx disulfides are conventionally viewed as structurally stabilizing elements in proteins but emerging evidence suggests two disulfide subproteomes exist. one group mediates the well known role of structural stabilization. a second redox-active group are best known for their catalytic functions but are increasingly being recognized for their roles in regulation of protein function. redox-active disulfides are, by their very nature, more susceptible to reduction than structural disulfides; and conversely, the cys pairs that form them are more susceptible to oxidation. in this study, we searched for potentially redox-active cys pairs by scanning the protein data bank for structures of proteins in alternate redox states. the pdb contains over 1134 unique redox pairs of proteins, many of which exhibit conformational differences between alternate redox states. several classes of structural changes were observed, proteins that exhibit: disulfide oxidation following expulsion of metals such as zinc; major reorganisation of the polypeptide backbone in association with disulfide redox-activity; order/disorder transitions; and changes in quaternary structure. based on evidence gathered supporting disulfide redox activity, we propose disulfides present in alternate redox states are likely to have physiologically relevant redox activity. emerging evidence supports the concept of two distinct types of disulfides in protein structures which have different functional roles. 1 the best known group act as structural stabilizers in proteins, significantly lowering the gibb's free energy of the protein chain by crosslinking it, resulting in proteins that are more resistant to denaturation. the second group are redoxactive and best known for their catalytic role in proteins such as thiol-disulfide oxidoreductases. an increasing role for the redox-active group in protein redox regulation is emerging. 1, 2 these disulfides act as redox-sensitive switches and are best viewed as posttranslational modifications akin to phosphorylation. it has now been established that oxidative regulation: important in processes such as the cell cycle, apoptosis, and response to oxidative stress; is an important control mechanism in the cytosol. transient oxidation mediates control of enzymes, signaling pathways, as well as transcriptional and translational control. however, these characterized cytosolic proteins likely only represent the tip of the iceberg. increasingly, reduction of disulfides is also emerging as a key regulator of protein function in oxidizing environments. [3] [4] [5] importantly, ageing and several major diseases including neurodegenerative diseases such as alzheimer's disease, type ii diabetes, cancer, and cardiovascular disease have been associated with abnormal redox conditions. structural and redox-active disulfides can be distinguished by their redox potentials. disulfide redox potentials measured in thiol-disulfide oxidoreductases range from à120 to à270 mv. [6] [7] [8] [9] for disulfides serving structural purposes, the redox potential can be as low as à470 mv. 10 computational methods for distinguishing the two groups would be a boon. reactivity of a redox-active cys is modulated by its immediate protein environment. in addition to the effects of the electrostatic environment, physical stress applied to a disulfide bond both through stretching and twisting enhances its reactivity. these forces can be applied to a protein dynamically 11 or may be captured during the protein folding process, rendering the disulfide poised to act upon exposure to variations in redox conditions. 12 because of their enhanced reactivity, redoxactive disulfides are also more susceptible to cleavage/ oxidation under nonphysiologic conditions. for instance, redox-active disulfides are prone to cleavage by synchrotron radiation during the process of x-ray structure determination. [13] [14] [15] other factors such as the ph of crystallization or the presence of mutations that change the electrostatic environment near a disulfide may also influence reactivity. here, we mined the protein data bank (pdb) 16 for highly similar proteins that have been solved in multiple redox states, that is, disulfide-bonded in one structure and reduced in another. we present evidence that most of the disulfides present in our redox pairs dataset are likely to be physiologically redox-active. many of the proteins have known thiol-based redox activity, while others have a high likelihood of thiol-based redox activity based on their involvement with redox processes. we also observed and classified types of conformational changes which occur during redox transitions. the redox pairs dataset contains 18,003 pairs of protein structures consisting of 4333 protein chains, of which 1238 (28.57%) are high-resolution structures (<2.2 å). many structures are of the same protein solved in different redox environments. a filtered dataset consists of 322 unique protein groups clustered at 95% sequence identity. further evidence of disulfide redox activity was sought based on experimental data in the literature, association of protein function with known redox activity, and implication of the disulfide in redox activity by independent computational prediction methods. for 41 proteins, direct experimental evidence of redox activity for the particular disulfide was available (des-ignated by ''k'' in table s1 , supporting information). the proteins belong to several major groups known to have redox activity. 11 unique redox pairs are associated with aerobic metabolism in eukaryotes or carbon fixation in plants (in groups d and e of table s1); a further five are associated with maturation of proteins of these pathways (in groups e and g). four redox pairs are associated with ion channel activity (group a): two of these, the chloride channel clic1 and the detoxification enzyme arsenate reductase, have known redox activity 17, 18 ; while a third, aquaporin, was recently implicated as a pore for the reactive oxygen species hydrogen peroxide, as part of the redox signaling process. 19 seven redox pairs are associated with the cell cycle, a process known to be redox regulated 20 (group b). formation of disulfides involving the catalytic cys in the phosphatases pr-1 and cdc25b regulate their resident protein's function. 21 , 22 41 redox pairs (group d) are involved in transcriptional control, replication, and dna synthesis including the oxidative stress response transcription factor oxyr 23 and ribonucleotide reductases from several species in multiple redox states. 24 12 redox pairs generated or destroyed reactive oxygen species (groups f and y). the latter comprised three different peroxiredoxins 25 as well as superoxide dismutases from several species. 26 four different amine oxidases generate peroxide while inducible nitric oxide synthase generates no. other large groups consist of proteins devoted to cell entry 27 figure 1 . to further assess the likelihood that the presence of proteins in the pdb with disulfides in both redox states is an indicator of physiological disulfide redox activity, we looked for gene ontology (go) terms overrepresented in the redox pairs dataset compared to the entire pdb. go is a dynamic controlled vocabulary of over 16,000 terms used to describe molecular function, process and location of action of a protein in a generic cell. 28 the analysis revealed many go terms consistent with redox function, including cell redox homeostasis and multiple pathways of cysteine biosynthesis (supporting information table 2 ). interestingly, several of the enriched go terms are linked to proteins mediating cell entry, including bacterial toxins and viral entry proteins. reduction of disulfides, often accompanied by irreversible conformational changes, have previously been implicated as a general mechanism in cell entry proteins of diverse types. 27 experimental evidence directly supporting such a general process exists for hiv gp120, newcastle disease virus, sars coronavirus and chlamydia. [29] [30] [31] [32] thus the go terms support redox roles for proteins in the redox pair dataset. disulfides were tested computationally for likely redox activity by screening the dataset for oxidized structures where the disulfide had particular properties previously associated with redox activity. flagged disulfides were those (a) with high torsional energies; (b) found in known redox-active sequence motifs; or (c) which disobeyed known rules of protein stereochemistry. 12 studies of disulfide-bonds in model proteins have shown that those bonds with high torsional energies are more easily cleaved than disulfide-bonds with lower stored energy. [33] [34] [35] [36] 148 disulfides in 139 proteins have torsional energies >15 kj mol à1 : a con-servative threshold associated with redox activity. 27 these are designated by ''h'' in supporting information table 1 . redox-pair disulfides were also found in sequence motifs previously shown to be associated with redox activity. 67 proteins, designated by ''m'' in table s1 , contain known redox-active motifs such as the cxxc motif: a redox-active disulfide motif commonly found in the thioredoxin fold, but which has also arisen convergently in other folds. a further 65 proteins, designated by ''f'' in table s1 , contain disulfides in protein structures that disobey known rules of protein stereochemistry. 37, 38 these disulfides, which table 1 . key to groups: a, ion channel, ion pump modifier; b, protein folding, isomerization; c, cell cycle; d, calvin cycle; e, photosynthesis, respiration, transport, maturation of proteins involved in these processes; f, destruction of oxygen radicals; g, redox metal homeostasis; h, amino acid metabolism; i, sulfur metabolism; j, nucleotide metabolism/homeostasis; k, isoprenoid biosynthesis; l, rna-interacting; m, proteases; n, dna repair; o, actin-related; p, carrier proteins; q, other metabolism; r, nadp þ -dependent oxidoreductase; s, other oxidoreductase; t, signaling; u, development; v, cell growth pathways; w, enzyme inhibitors; x, redox homeostasis; y, ros generating; z, apoptosis; a, protein fate; b, cell entry; c, cell adhesion; d, dna-interacting proteins; e, translation; f, immunity; g, carbohydrate metabolism; h, nad þ -dependent oxidoreductases; i, ca handling; j, methyltransferase; k, other. fan et al. we refer to as ''forbidden'' disulfides, conform to canonical structural motifs. deformation of the resident structure enhances disulfide reactivity by inducing stress in the protein chain. 37, 38 forbidden disulfides are proposed to have a functional role through redox activity. 12 in summary, a total of 226 proteins in the redox pair dataset contain disulfides predicted to be redox-active by a second independent bioinformatic analysis method. nonetheless, it is possible that some proteins with alternate cys-pair redox states are non-native, that is, not encountered under normal, or abnormal, physiological conditions. we reasoned that proteins demonstrated selective reduction of disulfides were more likely to represent physiological states. accordingly, for proteins with more than one disulfide, the dataset was partitioned into pairs where all the disulfides were reduced and those where some disulfides were selectively reduced while others remained intact, to further test the likelihood of involvement in physiological redox processes. the enriched go terms for both the partially and fully reduced sets contained terms previously associated with thiol-based redox regulation indicating both the fully and partially reduced sets have likely redox activity (supporting information table 2). having established the redox-pair dataset is enriched in proteins involved in redox-regulated processes, we studied conformational changes between redox pairs of proteins. for many proteins in the data set there were negligible conformational differences between the sg atoms of each cys residue of the reduced and oxidized states (see fig. 2 ). for this group, other factors may be required for, or preclude, conformational changes in these proteins. crystal packing forces, for example, may prevent a subsequent conformational change after cleavage of the disulfide by synchrotron radiation. for another group, the small distance changes of 2-4 å observed are most likely due to rotation of one or both cys sidechains toward each other to form the sas bond. some of the proteins exhibiting these small conformational changes are oxidoreductases with catalytic thiols such as thioredoxin, thioredoxin reductase, and protein disulfide isomerase. for proteins which exhibit major conformational changes, four classes were identified based on the nature of the change. these groups were: proteins that oxidize disulfides following expulsion of metals such as zn; proteins that exhibited major reorganization or ''morphing'' of portions of the polypeptide backbone in association with disulfide redox-activity; proteins that exhibited order/disorder transitions; and proteins that exhibited changes in quaternary structure. redox regulation of proteins that form disulfides following expulsion of zn is an emerging area of signaling. 40 for example, reversible redox regulation of the zn-binding site has recently been proposed for small tim proteins of the mitochondrial intermembrane space (ims). 41 small tim proteins have an essential role in the transport of proteins into the ims. these proteins can exist in both a reduced zn-bound state figure 2 . change in disulfide separation in protein redox pairs. excluding interchain disulfides, around 21% of redox structure pairs have a change of over 1 å in the sulfur atom separation between the oxidized and reduced structures. maximal intrachain differences of up to 18.8 å were apparent in 1-deoxy-d-xylulose-5-phosphate reductoisomerase (dxp reductoisomerase) and the rna sulfuration enzyme trmu. 39 and an oxidized disulfide-bridged state. both states likely represent physiological forms of the proteins which are adopted in response to changing redox conditions in the ims. 41 in addition, expelled zn acts as a second messenger to effect further cellular responses. proteins that form disulfides following expulsion of metals such as zn were identified by searching for ligation of metals by cys in reduced structures. a total of 73 redox pairs in 53 proteins were found (table i , z in s2). proteins identified include transcriptional regulators such as smad and anti-trap; transcriptional silencers such as sirt2; and the enzymes of sulfur metabolism, bhmt, mete, and meth. also notable were a number of rna-interacting proteins including amino-acid-trna synthetases, rna/dna polymerases, ribonucleotide reductase and the ribosome. in addition to zn, a few other bound metal atoms were represented including fe, ni, cu. there were several examples of inorganic iron-sulfur (fe-s) clusters associated with redox-active disulfides including: xanthine dehydrogenase, a major source of ros in the failing heart; and components of respiratory complexes (table i) . other proteins containing fe-s clusters contained other redox-active disulfides that were not associated directly with the fe-s cluster. all proteins containing fe-s clusters (designated with ''c'' for cofactor) are listed in table i regardless of whether the fe-s cluster is directly associated with a labile dithiol pair or not. crystallographers typically differentiate two types of zn sites: catalytic zn sites, which in their simplest, mononuclear form have three amino-acid sidechains and one water molecule as ligands; and structural zn sites, which have four amino acid ligands. from a redox point of view, a second variable -lability, is important. some structural zn sites expel zn upon variation of physiological redox conditions, whereas others bind zn tightly, and would not be expected to release zn under physiological conditions. we refer to this first redox-regulated group as ''labile'' zn sites; and the second, purely structural group, as ''inert.' ' we wished to determine whether zn sites associated with redox pair disulfides detected in the study were labile or inert. the number of zn ligands and their identity are key indicators of zn site lability. for catalytic mononuclear zn sites, three amino acids, which are often his, ligate the zn. in binuclear zn sites there are usually six ligands, and the carboxylated residues asp and glu predominate over his. 56, 57 cys is more commonly found as a ligand in structural zn sites which generally have four ligands. on consideration of ligand type alone, s 4 sites -those with four cys ligands, are the most labile type of structural zn site, and are almost as reactive as a free thiol. 58 most of the structural zn sites identified in this study were of the s 4 type (table i) , suggesting they are labile. four catalytic zn sites were also found in the dataset. these catalytic zn sites are highly unusual because of their utilization of cys as a ligand instead of the more usual his, glu, or asp. two of the catalytic sites are within bhmt and meth: enzymes of sulfur metabolism, which is redox-regulated in plants. 2 selection of sulfur ligands for zn in these enzymes may confer an additional level of redox control on sulfur metabolism. as an additional control, we checked whether metal atoms were expelled from oxidized structures. a metal atom is not present in over a third of the oxidized structures. for another structure, aspartate transcarbamylase (1tug), the metal site was only partially occupied in the oxidized chain. for the remaining structures where the metal atom is retained in the oxidized structure, the shortened sg-sg distance may be caused by a heterogeneous mix of oxidized and reduced, metal-bound structures in the ensemble. we reasoned that if the metal was expelled from some members of the ensemble, or partially expelled (still in the site but held, for example, by only two of four potential ligating cys), then the b-factor should be higher than for chains where no shortened sg-sg distances are observed. although b-factors cannot be compared between different structures, for some of the redox pairs, different chains of the same structure in different disulfide redox states could be compared. our analysis confirmed that the b-factor of the metal was higher in the oxidized chains for 80% of structures (see fig. 3 ). a similar result was obtained for proteins with fe-s clusters. there were no instances of expulsion of the fe-s cluster from the oxidized structure but oxidation of fe-ligating cys residues to form disulfides was generally associated with a higher b-factor of the relevant fe atom than fe atoms where the ligating cys remained reduced. in contrast, in reduced structures all fe atoms of the fe-s cluster had similar b-factors. further inspection of proteins with likely redoxactive zn sites reveals that two forbidden disulfide motifs, the jump strand disulfide (jsd) and a type of b-diagonal disulfide (bdd), 12 are repetitively associated with redox-active zn-binding sites in the dataset. oxidized structures that contain a bdd include isoleucyl trna synthetase (1ile 180-184, 502-504), the apoptosis-related proteins birc8 and survivin (1xb1, 300-303;1f3h, 57-60), aspartate transcarbamylase (1r0b, 138-141) and the dna repair enzyme recr (1vdd, 57-60). oxidized structures that contain a jsd include the amino-acyl trna synthetases for leu and ile (1obh, 439-484; 1jzs, 461-502), the enzymes of sulfur metabolism meth and bhmt (1q7m, 207-273; 1lt7, 217-300) and gtp hydrolase gtphi (1gtp, . a large group of proteins in the dataset were observed to undergo plastic deformations involving large scale rearrangements of the polypeptide backbone. we refer to these conformational changes as morphing transitions. these transitions, which typically involve unraveling portions of secondary structure, are illustrated in figure 4 and in animations in the supporting information. the well known redox-sensitive protein oxyr belongs to this group. in oxyr, a protein of 305 residues, significant refolding of the c-terminus occurs involving residues 196-221. 23 in the reduced structure, residues 200-204 form a short 3 10 helix flanked by regions of coil. upon oxidation, residues 196-221 adopt a different fold with residues 189-193 and 213-218 forming two parallel b-strands joined by a loop with a disulfide-linkage between cys199 and cys208. in the reduced oxyr structure, the two cys that form the disulfide are separated by almost 13 å. cys 199 in oxyr is known to undergo alternate modifications such as glutathionylation. 60 because of the spectacular nature of the transition between the reduced and oxidized structures, and the existence of an alternate mode of redox regulation, the physiological relevance of the disulfide-linked structure has been called into question. 60 to determine whether the oxidized structure of oxyr is indeed an oddity, or if other proteins exhibit similar conformational changes, we systematically searched for morphing proteins in the dataset by scanning for protein structure pairs that exhibited large differences of the backbone torsional angles over at least seven contiguous residues. changes in pseudo-dihedral angles calculated using four consecutive ca atoms exceeding a summed threshold of 1000 degrees over seven consecutive residues were used to detect changes in secondary structure. twenty-two parents were found in the fully reduced set and four in the partially reduced set (designated m in supporting information table 1 ). proteins showing the largest conformational changes are in table ii . these proteins include the cl à channel clic1: a protein that forms ion channels by inserting itself into membranes in response to oxidation; the detoxification enzyme arsenate reductase, which undergoes multiple conformational changes associated with a disulfide relay as part there is partial oxidation of the metal site, the b-factor of the metal should be higher because it has fewer ligands and hence more conformational freedom. this is true for most structures (bars above the axis). standard errors are depicted where they could be calculated. key to structures: clostridium acetobutylicum thymidine kinase 1-1xx6; e. coli aspartate transcarbamylase 2-1r0b, 3-1tug; thermotoga maritima sir2 4-2h59; homo sapiens sir2 5-1j8f; equus caballus alcohol dehydrogenase 6-1axg; bacillus subtilis tryptophan rna-binding attenuator protein-inhibitory protein (antitrap) 7-2bx9; severe acute respiratory syndrome coronavirus nonstructural protein 10 8-2g9t, 9-2ga6; homo sapiens estrogen receptor 10-1hcq; deinococcus radiodurans recombinational repair protein 11-1vdd; norovirus 3c-like protease 12-1wqs; moorella thermoacetica bifunctional carbon monoxide dehydrogenase/acetyl-coa synthase 13 17, 61 and cyclic phosphodiesterase, a trna-splicing enzyme where the disulfide modulates access to the active site. 59 like oxyr, several of these proteins exhibit large changes in ca separations of the two cys between alternate redox states (table ii) . thus large physical separation of the reduced cys pair seems to be a common feature of many of these proteins and does not preclude disulfide formation. another recognizable group of conformational changes involves order/disorder transitions. in these transitions, alternate redox states of the protein correlated with differing amounts of disorder in the protein structure, as evidenced by missing electron density. a total of 282 redox pair structures in 27 parents exhibited order/disorder transitions correlated with the redox state of the disulfide (table iii, fig. 5 ). proteins were classified into four groups based on the location of disulfide-forming cys residues with respect to the disordered region in the protein chain. proteins in group a had a single cys residue in a disordered region, with the other cys residing on a more stable region of structure. in group b, both cys residues reside on a single disordered region. in group c, both residues lie on disordered regions which are not contiguous in the sequence. in group d, disulfide reduc-tion is associated with multiple regions of disorder in the reduced protein chain. disorder-to-order transitions have previously been observed upon binding of ligands. 73 acquisition of order upon binding of ligands concomitant with disulfide formation was apparent for the group b oxidoreductase gdhb, where the disulfide straddled part of the pqq binding site, 74 and the group a rna sulfuration enzyme ectrmu, where the disulfide straddles the trna-binding site. however, the reverse was true for the thermotoga maritima trna-processing enzyme, psi55s, where significant disorder of the protein chain concomitant with disulfide reduction occurred upon binding of the trna substrate fragment. 75 the introduction of disorder in this case may facilitate further co-operative binding of the rna and protein after the initial docking step. 75 analyses of the composition of disordered sequences have detected amino acid biases from global frequencies. in particular, aromatic amino acids and cys are depleted in disordered sequences, whereas lys is enriched. 76, 77 the disordered sequences in the redox pair dataset are also depleted in aromatics, in agreement with more general studies of disordered sequences, but are not depleted in cys, or enriched in lys; and thus do not conform entirely to previously detected sequence patterns. redox pair disordered sequences are instead enriched in oxygen-bearing sidechains such as asp, glu, ser and thr. some changes in secondary structure are also apparent during the order/disorder transitions. in the oxidized state, both cys residues generally reside on coil in their proteins, but for a small number of pairs the oxidized form has a secondary structure that ''melts'' upon reduction of the disulfide. examples include vitamin d-binding protein 78, 79 and the e. coli oxidoreductase ghdb, 74, 80 where helices are lost upon reduction; and the bacteriophage cell entry protein prd1 81 where a small b-sheet disappears upon reduction. the amount of disorder in the chains is also of interest. for most reduced structures, the disorder introduced upon reduction of the disulfide affects less than 10% of the polypeptide chain. however, three of the four group d proteins exhibited large regions of disorder in excess of 20% of the involved protein chain. interestingly, all were associated with a second more-ordered monomer that was oxidized. for example, the thermotoga maritima trna-processing enzyme, psi55s undergoes a spectacular loss of secondary structure in one monomer upon binding of rna. 75 it is possible that the fully reduced state of these protein dimers is uncrystallisable and that only partially and fully oxidized oligomers are sufficiently ordered to enable structure solution. we also investigated whether formation of interchain disulfide bonds altered the quaternary state of proteins. different redox states of the interchain disulfides are associated with two general types of quaternary interaction: those where the number of oligomers differs between the reduced and oxidized state; and those where the number of subunits involved in the oligomer stays constant, but the oligomerization interface changes. some redox pairs exhibit a mixture of the two types of quaternary change. for example, disulfide-bonded dimers of the macrophage sialoadhesin 67 in the more ordered oxidized structure: a disulfide is formed between cys 297 and cys 311 which straddles a key phosphorylation site at thr 309. formation of the disulfide is proposed to inhibit kinase activity by recruiting the cognate phosphatase. 70 the t-loop was recently identified as a target for selective cancer drugs. 66 b: a larger order/disorder transition associated with expulsion of zn from the oxidized structure of leucyl-trna synthetase. 71, 72 in the more ordered reduced structure, several new secondary structures condense enabling rigid co-ordination of the zn. zn is represented by red spheres in the reduced structure. oxidized structures are shown on the left, reduced structures on the right. fan et al. form an interface distinct from that found in trimers formed upon binding of sialosides, 82 and may negatively regulate ligand binding. twenty-nine redox pair protein clusters with intermolecular disulfide bonds exhibit changes in quaternary structure upon oxidation/reduction. redox regulation of the quaternary state has previously been associated with cooperative behavior in proteins. for example, the hoxb5 protein binds dna in vitro when either oxidized or reduced but formation of an intramolecular disulfide between hoxb5 monomers is necessary for co-operative binding. 83 other well-known examples are oxyr 23 and p53. 84 several other proteins in the dataset exhibit co-operative behavior. cytoglobin is a member of the vertebrate haemoglobin family residing in the nucleus and cytosol that is upregulated figure 6 . quaternary changes between different redox states. a: changes in the number of subunits in the multimer between different redox states of chloroplast gapdh. the a subunit of spinach chloroplast gapdh in a reduced, monomeric form (pdb 2hki) is shown on the right and the oxidized homotetramer in complex with nad þ (pdb 1nbo) is shown on the left. in higher plants, photosynthetic gapdh exists in vivo mainly as heteromeric isoforms, a 2 b 2 and a 8 b 8 being the most abundant, interconvertible conformations. the b subunit is homologous to the a subunit but has an additional nonhomologous cterminal extension. stable homotetramers of a subunits (a 4 -gapdh) are found at low levels in chloroplast preparations and may be the only form of photosynthetic gapdh present in unicellular green algae and cyanobacteria. calvin cycle enzymes are known, as a group, to be redox regulated. 85 the activity of the ab isoform of chloroplast gapdh is redox regulated by formation of a disulfide between c-terminal thiols in the nonhomologous part of the b chain in light, and its reduction in the dark. 86 the interchain disulfide between a subunits may be important for redox control of the a 4 isoform, which is known to form complexes with the small chloroplast protein cp12 and phosphoribulosekinase in the dark. 87 the disulfide-forming cys 200 (1nbo numbering) is not present in the b subunit of chloroplast gapdh which is transcribed from a different gene, or in cytosolic gapdhs. b: changes in the oligomeric interface in cytoglobin, a member of the vertebrate haemoglobin family residing in the nucleus and cytosol that is upregulated under conditions of hypoxia and certain types of stress. intermolecular disulfide bonds in cytoglobin modulate the quaternary structure and may regulate the ligand-binding properties. 89 both the reduced and oxidized states are dimers. under conditions of hypoxia and certain types of stress (see fig. 6 ). it has recently been implicated as a tumor suppressor. 88 intermolecular disulfide bonds in cyto-globin modulate the quaternary structure and may regulate the ligand-binding properties 89 (table iv) . the homologous lamphrey haemoglobin is in an equilibrium italicized prefixes are species abbreviations. protein names in roman font. ao, aminooxidase; bcp, bacterial comigratory protein peroxiredoxin; bmp2, bone morphogenetic protein 2; dna pol iii k, dna polymerase iii k; ef-ts, elongation factor ts; gapdh, glyceraldehyde-3-phosphate dehydrogenase; gef, guanine nucleotide exchange factor; gga1, golgi-localized c adaptin; grp1, guanine nucleotide exchange factor and integrin binding protein homolog grp1; grx, glutathione reductase; pcbp2, poly(c)-binding protein-2; phsam domain, polyhomeotic-proximal chromatin protein sterile alpha motif domain; pnmt, phenylethanolamine n-methyltransferase; rubiscol, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit; sam-s-adenosyl-l-methionine; vegf, vascular endothelial growth factor; ae, aeropyrum pernix; af, archaeoglobus fulgidus; bpv, bovine papilloma virus; cr, crithidia fasciculata, dm, drosophila melangaster; fmdv, foot and mouth disease virus; hpv, human papilloma virus; hs, homo sapiens; me, methanobacterium thermoautotrophicum; mm, mus musculus; mp, mycoplasma pneumoniae; nt, nicotiana tabacum; ps, pisum sativum (pea); rn, rattus norvegus; sp, spinacia oleracea; td, themiste dyscritum; tt, thermus thermophilus. a 2nd cys in different monomer. state between a low o 2 affinity oligomer and a high-affinity monomer which contributes to its co-operativity. there was also a small group of transmembrane receptors which form covalent dimers in a neck region close to the membrane. these include the low-density oxidized ldl receptor, and the asialoglycoprotein receptor. covalent dimer formation may promote further quaternary changes such as higher oligomer formation in response to bound ligands as has been recently demonstrated for the metabotropic glutamate receptors. 96 relevance to disease importantly, ageing and several major diseases including neurodegenerative diseases such as alzheimer's disease, type ii diabetes, cancer and cardiovascular disease have been associated with abnormal redox conditions. inadvertent triggering of redox-active disulfide switches is a likely contributor to these phenotypes. this view is supported by the association of several redox pairs in the dataset with disease. sod1 (also known as cuzn-sod) is an enzyme mutated in familial lateral sclerosis (fals), an age-dependent degenerative disorder of motor neurons in the spinal cord, brain stem and brain. sod1 is largely localized to the cytosol but is also found in the intermembrane space of mitochondria. sod1 is activated by formation of the disulfide and insertion of cu by the copper carrier ccs. both of these proteins are represented in the redox pair dataset (tables i and s2 ). the conserved disulfide in sod1 is essential for activity and unusual because it remains oxidized in the cytosol. although the disulfide is intrachain, it is thought to play a critical role in stabilizing the sod1 homodimer. formation of the disulfide is part of the sod1 maturation process which is regulated by physiological oxidative stress. the three reduced proteins in the dataset are three distinct mutants associated with familial lateral sclerosis (fals): g37r, h46r, and i113t. the exclusive association of the reduced proteins with the disease mutants suggests redox imbalance of this disulfide is implicated in the disease. this hypothesis is supported by the finding that 14 fals mutants have the common property of being more susceptible to disulfide reduction than wild-type protein. 97 toxicity associated with disulfide-linked multimerization of sod1 mutants has recently been implicated in the disease mechanism. 98 several types of conformational change were observed in the redox pair dataset suggesting there may be common structural modes of redox regulation. proteins exhibited disulfide oxidation following expulsion of metals such as zn, morphing, order/disorder transitions, and changes in quaternary structure. in an individual protein more than one mode may be employed. for example in leu-trna synthetase, part of the protein chain becomes disordered upon zn expulsion but this is not the case for all proteins with labile zn. redox regulation of proteins that form disulfides following expulsion of zn is an emerging area of signaling. well known and characterized examples including hsp33 which is activated by zn expulsion as part of the oxidative stress response 99 ; and the small tim proteins which are kept in a transport-competent state in the cytosol by conjugation with zn. 41 upon delivery to mitochondria, zn is expelled from small tim proteins which are activated by disulfide formation. zn is not expelled from all protein zn-binding sites with equal facility, and is probably not expelled from some sites at all under physiologic conditions. for structural zn sites an analogous situation may exist to the bipartite distribution of redox-active and structural disulfides in proteins, with labile zn sites being redox-active and inert zn sites corresponding to the purely structural group. key to zn expulsion under oxidizing conditions is the presence of oxidation-prone cys as a zn ligand. sites with more cys are likely to be more facile. based on the number and nature of ligands, the zn sites detected in the redox pair dataset likely belong to the redox-active group. the repeated association of forbidden disulfides of the bdd type with labile zn binding sites is particularly interesting because of the recent characterization of the oxidative process that leads to activation of the bacterial chaperone hsp33. 99 the presence of the zn center keeps hsp33 monomeric and functionally inactive. upon exposure to elevated temperatures, the cterminal domain swings away from the protein's center of mass, exposing the zn centre. the n-terminal thiols in the zn site, cys 232 and cys 234, are preferentially oxidatively modified. the remaining two cys, 269 and 272, form a disulfide of the bdd type, releasing zn and unfolding the c-terminal domain. unfolding the c-terminus removes a steric barrier to dimerization, activating the protein's chaperone function. the function of disulfide formation between cys 269 and 272 in the zn binding site has not been fully delineated. potential benefits are protection from other oxidation modifications and restriction of the conformational freedom of the protein chain allowing easier refolding upon return of the protein's milieu to normal conditions. however, these benefits apply to any disulfide and the repeated appearance of the bdd in labile zn sites of proteins of the redox pair dataset suggests a more specific function, possibly recognition by a foldase or metallochaperone. we hypothesize the forbidden disulfide would become stressed upon condensation of the b-sheet in its vicinity, 12 possibly allowing easier re-insertion of zn. without any detailed experiments as a guide, the significance of the other repeated forbidden disulfide motif, the jsd, is not clear, but the same arguments relating to its forbidden disulfide nature would apply. the jsd-containing proteins meth and bhmt are known to be reversibly inactivated by disulfide formation. 100 in vitro, bhmt can be reactivated by dialysis against dtt and zn, 101 but the specific method of reactivation in vivo has not been characterized. for most structures exhibiting order/disorder transitions in the dataset, the reduced structure is associated with more disorder than the oxidized structure. energetically, formation of a disulfide bond between cys residues would have a favorable entropic contribution to the stability of the chain. a notable exception is leu-trna synthetase where the more ordered reduced structure binds zn (fig. 5(b) ). this may be generally true for order/disorder transitions associated with labile zn sites: the zn-bound state is entropically favored over a single disulfide because the polypeptide chain is constrained at three or four points instead of two. 40 a subset of redox pair proteins exhibiting order/ disorder transitions correlated with disulfide redox status contain regions of disorder in excess of 20% of the protein chain. all the proteins in this group exist as dimers with the other monomer being more ordered. the disordered monomers in these class d structures may correspond to the molten globule state. for these proteins, the molten globule state adopted may be physiologically relevant to their function. molten globules are collapsed forms of the protein chain which have some native-like secondary structure but a dynamic tertiary structure as seen by far and near circular dichroism spectroscopy, respectively. the radius of gyration of proteins in the molten globule state is $10% larger than the native state but precise tertiary structures at atomic resolution have not yet been obtained for commonly studied molten globule proteins such as lactoglobulin a. however, one of the proteins in group d: platelet factor 4, which like hepatocyte growth factor, binds heparin and regulates angiogenesis, has been shown to adopt the molten globule state upon reduction of its disulfides. 102 it seems likely proteins in group d adopt a molten globule state in vivo but can also adopt more ordered states upon binding of appropriate ligands. several of the proteins bind large polymers such as rna and heparin and the order/disorder transitions may mediate co-operative behavior between monomers. general studies of disordered sequences suggest there may be several different types of disordered sequence, three of which have been previously detected using an automatic classifier. 103 the oxygen-rich sequences of the disordered regions of proteins of the redox pair dataset seem to represent a novel type of disordered sequence not previously recognized. oxygen-rich sequences may confer bistable switch-like properties on the sequence: an oxygen-rich environment stabilizes the disulfide in the oxidized state by increasing its pka 104 ; while the subset of negatively charged asp and glu residues in oxygen-rich sequences may contribute to disorder in the reduced state via likecharge repulsion. disordered regions are known to undergo post-translational modifications including acetylation, glycosylation, methylation, and phosphorylation which may regulate the order/disorder transition. 73 redox-activity of resident cys residues should be added to this list of post-translational modifications. perhaps the most astonishing conformational changes associated with disulfide reduction are the morphing transitions. the current paradigm, due to anfinsen, is that the primary sequence of a protein encodes a unique three-dimensional structure. various discoveries that have challenged this notion include the spectacular ph-induced conformational change in influenza a haemagglutinin (ha), 105 molecular chaperones, and the previously discussed disordered sequences. all of these discoveries have been difficult to reconcile with the concept that protein sequences adopt a unique minimally frustrated fold. ha is believed to be kinetically trapped in a higher energy fold by an n-terminal domain which is subsequently cleaved, allowing it to cascade into a minimally frustrated fold. the conformations of disordered sequences appear to be principally governed by entropic factors: weak sequence preferences for a variety of conformational substates or modes appear to be co-selected by ligands. morphing structures appear to be a new type of challenge to the concept of a unique minimally frustrated fold. for morphing sequences, the structure appears to be dependent on the environment. unlike influenza a ha, where kinetic trapping during the folding process clearly plays a part in the structure adopting two different folds, for morphing sequences the two folds are energetic minima for the sequence in a particular environment. as such structural changes are reversible and dependent on environment. we were previously aware of two instances where subdomain morphing of proteins has been associated with reversible disulfide reduction: a redox-controlled structural reorganization of the ion channel clic1 proposed to regulate its insertion into membranes, 18 and sequential oxidation of the transcription factor oxyr in response to oxidative stress which modulates its quaternary structure and dna-binding properties. 23 here we showed that morphing is a common type of major structural change associated with disulfide reduction. the most spectacular morphing transition is that of clic1, where an entire subdomain of the protein rearranges in response to different redox conditions. several redox pairs were found exhibiting large morphing conformational transitions of similar magnitude to oxyr, supporting the notion that these types of transitions may be reasonably common. large separations of the cys residues in the reduced state are a typical feature of these proteins, and do not preclude disulfide linkages forming. possibly attack by a helper molecule, such as glutathione, may be required as an intermediate in disulfide formation to bridge the intervening distance. large physical separation of partner cys residues may represent an example of negative morphing transitions are not limited to changes in disulfide redox status. other morphing transitions not associated with disulfide redox state have been noted in mad2 and lymphotactin. [106] [107] [108] ph and redox are probably only two of multiple environmental changes associated with structural morphing. the relative importance of these and other environmental changes in protein morphing remain to be determined. morphing transitions may also have some relevance to evolutionary bridging states. in 1995, based on structures of arc repressor, sauer proposed the existence of evolutionary bridging states. a substructure in wild-type arc repressor adopts a b-strand conformation which forms a two-stranded b-sheet with the adjacent monomer. in the n11l mutant of arc repressor, the substructure was shown to fluctuate between the native state and a helical state on a millisecond time scale. a double mutant known as ''switch arc'' (n11l, l12n) was shown to be stabilized in the alternate helical conformation. sauer proposed the fluctuating n11l mutant represented an evolutionary bridge between two stable low energy folds. proteins that can adopt two different folds from the same sequence could evolve two different functions upon gene duplication and mutation hence acting as an evolutionary bridge between two different folds. 109 it is clear morphing proteins could also act as bridge states: deselection of one state over the other by point mutation may lead to loss of the ability to morph in the daughters resulting in proteins with different but related folds. although we have concentrated solely on disulfide reduction in this study, several anecdotal examples of disulfide isomerization should be mentioned in the context of the aforementioned discussion. a second example of an evolutionary bridging state was proposed based on a study of cnidarian collagen. the nterminal domain of minicollagen 1 has 44% sequence identity with the c-terminal domain, but the structures show a different disulfide bonding pattern and fold. 110 at this stage it is not clear if the two forms are interconvertible or represent true one-state endpoints evolved from an evolutionary bridging state. a different example where major structural organization is regulated by disulfide isomerization is the activation of p1 lysozyme. 111 this conformational change is irreversible and associated with release of a hydrophobic transmembrane sequence by membrane depolarization, suggesting the latent state may be a kinetically trapped state rather than a true morphing protein. our study mined pairs of protein structures which contain disulfides in both reduced and oxidized states and represents the first systematic compilation of such a dataset. quaternary changes, morphing and expulsion of transition metals, particularly zn, have previously been associated with redox-related changes in proteins and here we showed these conformational changes are more common than anecdotal examples suggest. to our knowledge order/disorder transitions have not previously been linked with redox changes but also seem to be a common type of conformational change. like morphing transitions, changing redox conditions appear to be only one of many mechanisms regulating order/disorder transitions. the oxygen-rich sequences of the disordered regions of proteins of the redox pair dataset seem to be a novel type of disordered sequence not previously recognized. finally, two forbidden disulfide motifs are associated with redoxregulated zn binding here for the first time. the physiological significance of redox-controlled structural changes is only beginning to be appreciated. previously, all oxidative changes were thought to be irreversible and damaging. it is now known that redox signaling pathways can modulate homeostatic and adaptive responses. however, continued stimulation of adaptive responses may lead to maladaptive responses. the redox pair dataset is a valuable resource for the investigation of physiological and pathological processes associated with disulfide redox activity. the pdb 16 was mined for pairs of structures with cys in different redox states. first, all disulfide-containing structures were identified by querying their dssp records, 112 as ssbond records in the pdb header were found to be unreliable. for each disulfide the atomic residue positions were mapped to the sequence position. the connectivity of the disulfides in the sequence was then used to assign a unique identifier, d mn score [eq. (1)], to each protein. 113 the d mn score allows direct comparison of the disulfide connectivity between structures as structures with the same disulfide bonding will have the same d mn score. where i and j are the sequence positions of disulfidebonded cys. all protein sequences in the pdb were clustered at 95% sequence identity using blastclust. 114 the 95% threshold was chose to capture the same protein with some natural and engineered mutations allowed. some highly conserved orthologs from closely related species were also captured in the same cluster, for example, the nf-jb orthologs in human and mouse. proteins within each group were compared to detect subgroups with different d mn scores-indicating alternative disulfide connectivity. for fully oxidized proteins, a set of ''pseudo-reduced'' d mn s were calculated by iteratively ignoring one or more disulfides. the set of pseudoreduced d mn s for the oxidized protein were matched against d mn s for the reduced proteins in the dataset to select redox pairs. as an additional control, proteins were split into two groups based on whether some or all disulfide bonds in the protein were reduced. proteins in which cys residues were engineered into proteins were filtered from the list. analysis of gene ontology term enrichment go terms that are over and under-represented in the oxidized proteins in the redox-pairs dataset compared to the pdb as a whole were identified using fisher's exact test, in the same way as applied in tools that analyze gene-expression data. 115 go annotations for the entire pdb were taken from the goa resource, downloaded from the ebi. 28 to ensure a fair comparison, the go annotations of a nonredundant set of the oxidized structures (95% sequence identity) were compared to go annotations of a nonredundant pdb data set (95% sequence identity). to improve overall annotation coverage, a representative pdb structure is assigned all the go terms belonging to the pdbs within its cluster group. because go is a hierarchical data structure, go terms are set to a single level in the hierarchy to enable a fair comparison. here we used go level three. at this level there is a compromise between information quality and the number of annotations available. 116 where a go term is below this level we move up the hierarchy until we reach the third level go term(s). go terms above level three cannot be used. a p-value was calculated representing the probability that the observed number of each go term (n) could have resulted from randomly selecting this go term in the pdb as a whole (n). fisher's exact test was used in order to approximate this p-value. go terms that are most specific to the redox pairs set will have p-values near zero, and those that are under-represented will be near one. protein conformational changes were examined using a variety of complementary approaches. we used profit (www.bioinf.org.uk/software/profit) to search for large conformational changes such as hinge movements. profit superimposes two structures using ca atoms and was used to calculate rmsd and distances between residues. large movements were indicated by the change in distance between the sulfur groups of the cys residues in a disulfide. because rmsd is averaged over the entire molecule, large changes confined to a subdomain of protein may not be obvious using rmsd. large conformational changes between structure-pairs corresponding to rearrangements of the polypeptide backbone were also identified using an implementation of a method by flocco and mowbray. 117 for pairs of structures, the method compares the change in a pseudo-torsional angles defined by four consecutive ca-atoms. conformational changes were identified by running a smoothing window (five residues long) over the torsional-difference plot. stretches of contiguous residues with seven or more pseudo-dihedral angles that exhibited individual movements in excess of 20 were initially filtered from the dataset. these conformational changes were further classified by comparing dssp annotations, solvent accessibility and secondary structure, between the protein pairs. proteins with conformational changes where the summed changes in the pseudo-torsional angles exceeded a threshold of 1000 degrees with accompanying secondary structure changes were classified as morphing. the presence or absence of multiple chains in the pdb structure could cause errors when calculating changes in solvent accessibility; therefore, dssp was run on individual chains only. all solvent accessibility measures were normalized using values derived by chothia. 118 each amino acid was assigned to one of two states; core or exposed. core residues are those with a solvent accessibility of less than 10% and exposed residues are those with a solvent accessibility equal to 10% or more. secondary structures were assigned to one of three states: helix, strand and coil. regulation of the protein disulfide proteome by mitochondria in 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trypanosoma brucei -photoreduction of the redox disulfide using synchrotron radiation and evidence for a conformational switch implicated in function oxidized and synchrotron cleaved structures of the disulfide redox center in the n-terminal domain of salmonella typhimurium ahpf specific chemical and structural damage to proteins produced by synchrotron radiation the protein data bank all intermediates of the arsenate reductase mechanism, including an intramolecular dynamic disulfide cascade the intracellular chloride ion channel protein clic1 undergoes a redox-controlled structural transition membrane transport of hydrogen peroxide a combined in vitro/bioinformatic investigation of redox regulatory mechanisms governing cell cycle progression structure and biochemical properties of prl-1, a phosphatase implicated in cell growth, differentiation, and tumor invasion structural mechanism of oxidative regulation of the phosphatase cdc25b via an intramolecular disulfide bond structural 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signaling crystal structure of the atx1 metallochaperone protein at 1.02 å resolution engineering, characterization and phage display of hepatitis c virus ns3 protease and ns4a cofactor peptide as a single-chain protein mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding crystal structures of zinc-free and -bound heme domain of human inducible nitric-oxide synthase: implications for dimer stability and comparison with endothelial nitric-oxide synthase reversible interconversion of two forms of a valyl-trna synthetase-containing protein complex thioredoxin-linked processes in cyanobacteria are as numerous as in chloroplasts, but targets are different mutational analysis of the redox-sensitive transcriptional regulator oxyr: regions important for oxidation and transcriptional activation chloroplast transcription at different light intensities. glutathione-mediated phosphorylation of the major rna polymerase involved in redoxregulated 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structures the authors thank dr. kieran scott for interesting discussions. disulfide torsional energy calculations were performed at the nci national facility. key: cord-337158-0iw2kcaf authors: tiernan, hannah; byrne, bernadette; kazarian, sergei g. title: atr-ftir spectroscopy and spectroscopic imaging for the analysis of biopharmaceuticals date: 2020-06-22 journal: spectrochim acta a mol biomol spectrosc doi: 10.1016/j.saa.2020.118636 sha: doc_id: 337158 cord_uid: 0iw2kcaf attenuated total reflection fourier transform infrared (atr-ftir) spectroscopy is a label-free, non-destructive technique that can be applied to a vast range of biological applications, from imaging cancer tissues and live cells, to determining protein content and secondary structure composition. this review summarises the recent advances in applications of atr-ftir spectroscopy to biopharmaceuticals, the application of this technique to biosimilars, and the current uses of ftir spectroscopy in biopharmaceutical production. we discuss the use of atr-ftir spectroscopic imaging to investigate biopharmaceuticals, and finally, give an outlook on the possible future developments and applications of atr-ftir spectroscopy and spectroscopic imaging to this field. throughout the review comparisons will be made between ftir spectroscopy and alternative analytical techniques, and areas will be identified where ftir spectroscopy could perhaps offer a better alternative in future studies. this review focuses on the most recent advances in the field of using atr-ftir spectroscopy and spectroscopic imaging to characterise and evaluate biopharmaceuticals, both in an industrial and an academic research based environment. biopharmaceuticals, also commonly known as biologics, are drugs derived from biological organisms. they are utilised in treating or preventing a range of diseases such as arthritis, diabetes, and some cancers. [4] [5] biopharmaceuticals have a huge diversity of function due to their variation in chemical composition and their conformational flexibility. biopharmaceuticals are highly selective and have a low nonspecific toxicity, but their complexity promotes significant challenges in production. this is due to their multiple routes of administration to patient (e.g. oral, pulmonary, transdermal), and variation in pharmacokinetic parameters, such as half-life (t 1/2 ), protein binding, and bioavailability. their comparatively large size means it is unusual for biopharmaceuticals, even small antibody fragments, to cross the blood brain barrier (bbb). 9 although this can limit their application to brain disorders, it also means there is reduced risk of unwanted side effects. monoclonal antibodies (mabs) (fig 1) are the most common biopharmaceuticals, and account for more than half of total biopharmaceutical product sales. their major benefits are their ability to identify and bind to cell surface targets with very high specificity, and their capacity to be isolated effectively through changes in salt concentration and ph. 10 alterations in the environment of biopharmaceuticals are common in production, such as the implementation of extreme changes in ph during cleaning-in-place (cip) protocols, which results in effective separation of mabs due to their intrinsic resistance to ph changes. 11 the hydrophobic interaction chromatography (hic) step, j o u r n a l p r e -p r o o f 2 also works through altering the environment of the protein, the sample is loaded onto the column at high salt concentrations, and is eluted at lower salt concentrations. 12 the complexity of biopharmaceutical manufacture is two-fold. firstly, these large biopharmaceutical drugs are unpredictable due to their sensitivity and propensity to aggregate. secondly, their manufacture is challenging due to the development and optimisation of the industrial multi-level production pathway. current techniques in this pathway such as mass spectrometry (ms) can be expensive to run, and others such as nuclear magnetic resonance (nmr) have stringent sample pre-requisites for optimum results, which include but are not limited to, a ph range of 4-7, the use of a deuterated solvent, and the absence of excipients. this complex biopharmaceutical production pathway therefore requires a technique which can be applied to multiple uses, is inexpensive to run, and requires minimal to no sample preparation. ftir spectroscopy is a label-free, non-destructive technique, which can be used to analyse biological systems, as previously outlined in a number of comprehensive review papers. [13] [14] ftir spectroscopy can be used to observe key characteristics of biopharmaceuticals such as denaturation and aggregation, prediction of metabolites, concentration, and to quantify levels of phosphorylation and glycosylation. these characteristics of ftir spectroscopy are critical to providing insights into biotherapeutic behaviour and stability, and can also be used to optimise the production process through acting as a quality control measure. ftir spectroscopic imaging uses a focal plane array (fpa) detector instead of a traditional single element detector. this typically consists of 64 x 64 or 128 x 128 pixels, and collects an interactive image containing thousands of spectra. ftir spectroscopy and spectroscopic imaging have a variety of setups and approaches, offering the capability to investigate biopharmaceutical structural integrity, which is paramount to producing a reliable and safe product. the applications of ftir spectroscopy and spectroscopic imaging to the manufacture of biopharmaceuticals are outlined and critically discussed in the following review. in recent years there has been rising demand for effective biopharmaceutical drugs; in 2019, 8 of the 48 drugs approved by the fda were for antibodies or antibody-drug conjugates. 15 however, the fast commercialisation of biopharmaceuticals in comparison to small molecule drugs remains a challenge, partly due to their relative chemical and physical instabilities. analytical techniques are therefore required for characterisation of biopharmaceuticals, to ensure the effective monitoring of their stability and efficacy throughout production and delivery to the patient. this characterisation commonly involves the use of techniques such as liquid chromatography tandem mass spectrometry (lc-ms-ms) to monitor changes in mass, 16 high pressure liquid chromatography (hplc) to determine impurity profiles of samples, 17 and infrared spectroscopy (ir) to identify impurities and compare biosimilars to the original, 'reference drugs'. 8 most studies where ftir spectroscopic analysis has been utilised to investigate biopharmaceuticals are focussed on mabs as opposed to other biopharmaceuticals due to their highly specific, comparatively stable nature. 10, [18] [19] this specificity is determined by the chemical composition, physical forces, and molecular structure at the fragment antigen-binding (fab) region (fig 1) . the umbrella term of mabs also includes derivative antibodies, such as bispecific antibodies (bsabs), antibody-drug conjugates, radiolabelled antibody conjugates, antigen-binding fragment fab, and fc-fusion proteins. 20 some of the more unusual biopharmaceuticals such as fc fusion proteins, pegylated proteins and antibody drug conjugates (adcs), viruses, and virus like particles (vlps), pose unique problems and challenges which require stringent monitoring of their higher order structures. for example, during production, vlp-based vaccines encounter problems due to their lack of a viral genome (essential for the formation of a virus) causing instability in downstream processing. 21 the high demand for mabs is well-founded, and despite the challenges associated with industrial grade production of homogeneous and efficacious product, they have been used in a number of novel applications. [22] [23] research has suggested mabs, when combined with bbb peptide shuttles, could penetrate the bbb, and deliver an effective treatment for brain metastases. 9 this could occur through the use of antibody fragments to reduce molecular weight, or the incorporation of cell penetrating peptides (cpps) into j o u r n a l p r e -p r o o f 3 mabs to deliver large cargoes across cell membranes, and even the bbb. 24 another created an affordable immunoassay using 4c9c9 and 4c9e11 mabs in a sandwich-type umelisa® assay to identify cystic fibrosis in new-borns, specifically for cuba and other latin american countries. 22 irrespective of the benefits, producing biopharmaceutical drugs has some significant drawbacks. production requires substantial costs in terms of time, monetary investment, and protocol optimisation. an unfortunate consequence of this is a high cost to the patients. one course of biotherapeutics for arthritis costs around usd $20,000 annually, 25 and for crohn's disease it can exceed usd $44,000 annually. 26 although this cost can be reduced by the use of biosimilars, it is still essential that efficiency in the process is retained through the minimisation of loss of product. the structural and functional integrity of the drug product must also be preserved throughout the varied conditions required for production, including isolation and storage, and delivery to the patient. 27 selected biopharmaceuticals such as therapeutic antibodies and antibody fragments are prone to significant aggregation and misfolding during production and delivery. 28 to make both production and delivery more effective, it is essential to be able to observe, and fully understand the mechanisms by which aggregation and/ or unfolding occur. certain points have been identified in the production pathway where therapeutic proteins are more prone to aggregation or unfolding. these points are generally where the proteins are placed under stress conditions such as thermal, 29 oxidative, 30 and mechanical stress, 31 repetitive freeze thaw conditions, 29 and/ or interface agitation. 32 it is therefore ideal that data is collected at each biopharmaceutical process stage, and is utilised to make a real-time decision in order to produce high quality products. diversity in production pathways mean a variety of monitoring techniques are employed at each phase to ensure safety and efficacy of final product. in-line, on-line, and off-line measurements (fig 2) , summarised by holzer et al 33 all have their merits, but in-line measurements such as nir spectroscopy 34 and atr-ftir spectroscopy can be combined with other techniques to offer real-time decision making. 35 for example in-line atr-ftir and nir spectroscopy can be incorporated into a feedback loop to reduce the presence of contaminants or aggregates, therefore reducing the time and monetary costs of production. ftir spectroscopy offers fast data acquisition with minimal sample preparation, and has limited sample volume requirements. there are abundant review articles outlining ftir spectroscopy and spectroscopic imaging, 13, 36 and the successful application of ftir spectroscopy to proteins in general. 37 38 as these review papers discuss, the capabilities of ftir spectroscopy can be enhanced with different accessories to cater to specific requirements, including micro and macro imaging, atr, transflection, and transmission modes. atr-ftir spectroscopy measures samples at a depth of penetration of 0.5-5 µm from the surface of internal reflection element (ire). despite the fact that ftir spectroscopy in transmission measures the whole thickness of a sample, measured thickness for aqueous solutions is generally limited to 6 µm due to the strong absorbance of water. 39 sample preparation using this transmission mode of measurement can also be laborious. therefore, the most commonly used technique when studying solutions of proteins is atr-ftir spectroscopy (fig 3) , which usually employs a diamond, ge, si or znse ire, and collects information from the layer of the sample adjacent to the surface of the ire. recent research has identified that igg aggregates distribute unevenly close to the surface of the znse ire using a high-throughput atr-ftir spectroscopic imaging approach. 6 thus, appropriate spectral processing must be undertaken when current techniques used in biopharmaceutical processing are typically suited to only monitoring a few sample characteristics simultaneously, require lengthy sample preparation, or are costly. for example, mass spectrometry (ms), is usually focussed at the molecular level to monitor post translational modifications (ptms), and for the identification of numerous proteins in a protein mixture, but it is costly to run. 40 uv spectroscopy is utilised in a variety of set ups to elucidate protein-protein interactions through monitoring changes in uv absorbance using delta absorbance (δabs), but is used primarily for protein concentration measurements. [41] [42] [43] a recently revived technique, analytical ultracentrifugation, obtains high resolution data about molecule size of the sample in liquid state, allowing information to be collected about the aggregation status of the protein. 44 although these techniques can all be applied to investigate protein structure and function, and to determine the feasibility of biopharmaceuticals in the production pathway, they all have their downsides. ftir spectroscopy addresses most, if not all of these concerns, through its high throughput capabilities, inexpensive and easy sample preparation, and its range of technical applications. although multifaceted and useful, ftir spectroscopy requires appropriate spectral processing to ensure reliability and to optimise results. this is carried out straight after data collection and is primarily used to remove or reduce unwanted signals in spectra. incorrect usage of these processing steps can have a serious impact on the reliability of data. [45] [46] post data collection spectral processing such as second derivative analysis is commonly used for quantification of proteins, and can combat the slightly changing backgrounds between different samples, as well as to accentuate spectral features. [47] [48] [49] [50] researchers have proposed slightly altered techniques such as simultaneous fitting of absorption and second derivative spectra, 51 and have conducted comparability studies on the most effective second derivative techniques. 52 the correct usage of spectral processing techniques is essential in order to exploit the full power of atr-ftir spectroscopy in biopharmaceutical monitoring and processing. the choice and composition of biopharmaceutical buffers is also paramount to ensuring bio stability, 53 and has a large effect on spectral quality, therefore sufficient corrections must be made to final spectra in order to account for this, and to obtain the most reliable information. atr-ftir spectroscopic set up demonstrating ir light being directed from the spectrometer to the accessory mirrors, towards the ire, where it will interact with the sample on the surface of the ire to a penetration depth of 0.5 -5 µm, and will then be directed to the fpa detector j o u r n a l p r e -p r o o f 5 it is well known that protein secondary structural changes can be detected using ftir spectroscopy. 29, [54] [55] [56] amide spectral bands are most commonly used for this protein characterisation, particularly the amide i (1650 cm -1 ) and amide ii (1545 cm -1 ) bands. 47, [57] [58] the amide i band between 1600-1700 cm -1 mostly consists of c=o stretching vibrations and c-n groups, and its peak position is determined by the backbone conformation and hydrogen bonding pattern, whilst the amide ii band between 1510-1580 cm -1 consists primarily of n-h bending, but also c-n, and c-c stretching vibrations. 59 due to the strong overlap of the bending mode water band (1635 cm -1 ) with amide i, it has been proposed that other bands such as amide iii (1300-1200 cm -1 ), could be used to discern secondary structure. 49 this could be beneficial due to the lack of overlap with the bending mode water band, and the supposed improved resolving nature of individual bands. [60] [61] however, these claims are disputed by goormaghtigh et al, who suggest that amide iii bands are irrelevant when you have obtained information from the 1545 cm -1 amide ii band. 49 others imply that the amide iii band is significantly affected by side chains, and will be altered by the amino acid composition. 62 therefore, the majority of research still uses amide i and ii bands for effective analysis and structure elucidation. ftir spectroscopy, combined with recent advances in data processing and chemometrics, is an ideal tool to investigate the stability and suitability of biopharmaceuticals. if proteins are of a high concentration (>30 mg/ml), which is typical in biopharmaceutical production, amide bands can more easily be seen, however lower concentrations also yield good amide i and ii spectra (fig 4) . 63 macro atr-ftir spectroscopy is commonly used due to the ease of sample preparation and data collection. it is therefore well suited as an in-line technique to the analysis of biopharmaceuticals both during and post production, for example, the monitoring of protein concentration and secondary structure, as well as providing insights into ptms (fig 5) . atr-ftir spectroscopy has many uses in biopharmaceutical processing, some examples being the effective characterisation of biomaterials, 64 the monitoring of monoclonal antibody purification, 3 and biopharmaceutical bioactivity under stress conditions. 65 other uses include the investigation of structural stability of biopharmaceuticals such as bevacizumab®, 65 humatrope®, 66 and humalog®. 67 atr-ftir spectroscopy has been used to study protein adsorption on znse ire, 68 and in-column affinity chromatography purification of monoclonal antibodies, where atr-ftir spectroscopy was used to demonstrate changes in protein content at various stages of purification, such as after culture fluid binding (fig 6) . 3 6 atr-ftir spectroscopy is able to monitor a wide range of ptms. biochemical variability can include deamidation, phosphorylation, methylation, and acetylation, 69 but glycosylation is the most common ptm in biopharmaceuticals, and has a significant impact on their efficacy and safety. 70 ptms are still commonly analysed in biopharmaceutical processing using ms, 71 and more recently, nmr, 72 but these methods are respectively expensive to run and better suited to low concentration samples. one recent paper has successfully characterised glycosylation in biotherapeutic monoclonal antibodies with atr-ftir spectroscopy using 0.5 µl of dried igg on a diamond ire, and the multivariate tools, principal component analysis (pca), and multivariate analysis of variance (manova). here, the authors obtained valuable information from only the infrared spectrum of the glycoprotein, with no labelling or separation required to determine an accurate fingerprint of the glycosylation profile ( fig 5) . 2 previous reports have shown contrasting relationships between protein synthesis rates and glycosylation of different proteins, 73 however, glycosylation of the final product is certainly influenced by the host cell line, the cell culture and the purification process. 70 goldsztein et al have used ftir spectroscopy for identifying phosphorylation of gastric atpase. this research managed to observe the binding of a single phosphate on atpase using a combination of atr-ftir spectroscopy with biospecific interaction analysis (bia)-atr biosensors, which allows the surface of the ire to adsorb to a biomembrane. 74 atr-ftir spectroscopy has been used to investigate secondary structure of denatured bovine serum albumin (bsa) alone and in a mixture with a native protein solid, which could be applied to investigate the solid-state formulation development process more thoroughly. 75 atr-ftir spectroscopy has also been used extensively to study proteins, in particular biopharmaceuticals, and exciting research has shown the potential applications of it to investigate and monitor changes effectively in ptms and secondary structures. due to the extensive applications of ftir spectroscopy, it is surprising that it is still underutilised in biopharmaceutical production, however the wealth of data and robustness of this technique should ensure it is included in future innovative analyses. a biosimilar can be defined as a biopharmaceutical which has no clinically significant differences from the reference product in terms of quality, safety, and efficacy. 76 the european medicines agency (ema) was the first to approve a biosimilar (somatropin) in 2006, and the first monoclonal antibody biosimilar (infliximab) in 2013, whereas the first biosimilar (zarxio) was not approved by the fda until 2015. 77 biosimilars are driving cost effective manufacture of biopharmaceuticals as a whole, and as the patents on high income yielding original biopharmaceuticals come to an end, a number of which will expire this year, 78 there is increasing demand for the development and manufacture of these biosimilars. j o u r n a l p r e -p r o o f 7 although they are not expected to be identical, maximum similarity between reference product and biosimilar is desired, and any differences must be justified to meet safety and efficacy requirements. the lack of public information on reference biologics makes biosimilars difficult to produce. in contrast to small molecule drugs, when biosimilars are reproduced there will be changes in the manufacturing process due to their complex structure and lengthy processing parameters which might affect the chemical properties, efficacy, and stability of the product. two of the most prominent causes for concern in the production of biosimilars are their variable potency and immunogenicity. 79 there is also conflicting research into the effects of switching patient prescription drugs from original biopharmaceuticals to biosimilars, a potentially problematic practice. 80-81 however, one review paper believes these concerns are unfounded, and that the possible costs outweigh the benefits, 82 and another reviewed 90 studies and found little differences reported between reference drugs and biosimilars. 83 this proposed interchangeability was thought to be problematic due to the difficulties in ensuring the product does not invoke different immune responses than the reference product. recently, the fda has approved the introduction of 'interchangeable products', which require additional information outlined by the biologics price competition and innovation act. these interchangeable products are tested to ensure they produce the same clinical result as the reference product, and these drugs can therefore be dispensed by a pharmacy, even with a prescription for the reference product, and without the need for a separate prescription from the healthcare prescriber. 84 when properly utilised, ftir spectroscopy can replace some other, more traditional methods. 85 although circular dichroism (cd) can be used to estimate secondary structure, it is unsuitable for highly concentrated samples, or samples containing other highly absorbing molecules such as excipients. 63 in contrast, ftir spectroscopy, particularly atr-ftir 8 spectroscopy, caters to these requirements. for example, atr-ftir spectroscopy was used to assure that the secondary structure of the biosimilar ristova® was similar to that of the original biologic. 86 in addition, using ftir spectroscopy, quality control to a high standard has been carried out on humalog®, an approved biosimilar, to investigate stability at varying temperatures. 67 it is common to use ftir spectroscopy for secondary structure elucidation, however tertiary structures which are frequently explored using either fluorescence spectroscopy 87 or nmr, 88 can also be characterised, and more quickly, by using ftir spectroscopy. it is well documented that ftir spectroscopy has been frequently used to clarify higher order, tertiary, structures of biosimilars. 19, 57, 89 however one conflicting study compared a reference biopharmaceutical with its respective biosimilar using ftir spectroscopy, and did not observe differences between the original drug and the biosimilar. though they simply compared the amide i and amide ii band shape with no further spectral analysis, which could have conclusively identified any differences. 90 methionine oxidation is a significant degradation pathway in biopharmaceuticals, and has an important impact on biosimilar stability and efficacy. this is exacerbated by the presence of phosphate buffer, 91 and is typically investigated using liquid chromatography with mass spectrometry (lc-ms), although this is time consuming. ftir spectroscopy has been used to successfully quantify oxidation, 92 and measure subtle spectral changes. this study demonstrated the power of ftir spectroscopy to provide detailed analysis of biosimilars, and other interchangeable products compared to the original licensed biotherapeutic. process analytical technology (pat), has been used since 2004 to enhance the understanding and control of processes throughout product development and manufacturing, 93 and has been previously outlined in a comprehensive review. 94 in biopharmaceutical production, processes are largely based on historical data, it is therefore important that analytical methods such as atr-ftir spectroscopy are employed in-line to ensure rapid responses to processing requirements. research has shown that a reduction in the costs of products available to market, possibly through the use of efficient, expedited processes, would result in increased investment in r&d. 95 previously most sampling techniques were off-line, meaning samples were extracted and measured with a time-delay, resulting in inefficiencies. ftir spectroscopy offers noninvasive, in-line, and instantaneous measuring of numerous critical processing parameters (cpps). for example, grosshans et al used in-line ftir spectroscopy, along with partial least squares (pls) analysis, as an effective process analytical tool (pat) for preparative protein chromatography, they found it to be useful to distinguish and selectively identify proteins based on their secondary structure. 96 traditional manufacturing of biopharmaceuticals is typically divided into two sections: upstream processing (usp), which includes cell culture and harvesting, and downstream processing (dsp), consisting of multiple steps of chromatography, filtration and diafiltration, and finally product formulation (fig 7) . upstream techniques in processing of biopharmaceuticals experience high levels of monitoring and feedback when compared to downstream techniques. 97-99 for exa mple , inline appli catio ns of atr-ftir spec trosc opy inclu de upst rea m proc ess mon itoring of glucose and ethanol in different cell lines, observing changes in α-glucose spectra with time, and the possibility of simplified calibration techniques using a spectral library. 100-101 ftir spectroscopy is also commonly combined with a flow through atr capability to monitor ethanol fermentation. 102 ftir spectroscopy can also be utilised in a wide range of downstream applications in biopharmaceutical production such as purification, where the sample is placed under high stress levels due to fluctuations in shear and ph. ftir spectroscopy has been successfully used for an in-line estimation of the degree of peglycation of eluting sample from a chromatography column in near real-time. 103 ftir spectroscopy was applied in-line to the bioreactor cell culture process, and correlated with offline measurements, for real time monitoring of bioreactor mab igg3 cell culture process dynamics. the concentration profiles of four key cell culture metabolites (glucose, glutamine, lactate and ammonia) were predicted using multivariate calibration models. 18 mab binding propensity during the protein a capture step has been monitored using ftir spectroscopy. 3 when mabs are in the purification step of manufacture, protein a resin experiences a decrease in binding capacity and product recovery. ftir spectroscopy can therefore be used to monitor changes in resin composition via a continuous feedback loop to eventually make purification more effective. some recent research argues that fluorescence is better for this downstream application, due to its ability to monitor changes in situ. 104 however others have already shown this is not only possible, but more facile, with atr-ftir spectroscopy. 105 lin et al suggests that ftir spectroscopy can be used effectively for monoclonal antibody comparability studies, but for higher order structure identification only, such as secondary or tertiary structure, or for product characterisation. they came to this conclusion through the comparison of size exclusion and ion exchange chromatographies (sec and iec) with cd and ftir using mabs samples of varying phs and concentrations, 1 and by carrying out second derivative analysis of proteins (fig 8) . another paper used ftir spectroscopy for secondary structure classification of mabs subjected to environmental stresses. 19 here, ftir spectroscopy was interestingly used to prove the apparently negligible effect small alterations in tertiary structure had on overall long-term stability and bioactivity. 106 despite this research indicating the use of ftir spectroscopy solely for higher order structure identification, it has been shown conclusively that ftir spectroscopy could be applied to investigating the primary structure of biopharmaceuticals, such as ptm changes. 74 mabs are the most common class of biopharmaceuticals, and are mostly favoured due to their reduced immunogenicity owing to the humanisation of murine mabs, which greatly improves their in vivo tolerability. 107 immunogenicity can also be affected by genetics, impaired immunity of patient, and administration of and impurities within a drug. 108 typically mabs are produced using the upstream processes of cell culture; centrifugation, and depth and membrane filtration, and the downstream processes of protein a chromatography; low ph viral activation, cation exchange chromatography, anion exchange chromatography, viral filtration, and ultrafiltration/ diafiltration. 109 mab production supported by the inline capabilities of atr-ftir spectroscopy complement the trend of moving from batch to continuous processes for biopharmaceutical processing in order to reduce costs, and improve quality of drug and flexibility of processes. 110 aside from detecting protein presence, ftir spectroscopy can also detect impurities in solution, such as detergents, buffer residues, and protein contaminants in manufacturing (fig 9) , not detectable by reversed phase hplc electrospray ionisation ms (rp-hplc-esi-ms) or sds-page. in fact it is predicted triton x-100, the detergent seen in fig 9(b) , could not be seen by any other method, 8, 111 a significant advantage when compared to cd. this detection of impurities is possible through the comparison of sample ftir spectra with standard ftir spectra. another recent study has developed a reusable immuno-infrared sensor, which uses a functionalised germanium atr crystal to analyse human blood and csf fluid, and subsequently detect biomarkers for alzheimer's disease (ad). this modified technique reduces time and cost of identification, and could be applied to identify impurities in biopharmaceutical processing. 112 some reviews have proposed other techniques such as size exclusion chromatography (sec) and mass spectrometry (ms) for this application, 113 however, these alternative techniques are more time consuming and costly. one paper limited the application of ftir spectroscopy in biopharmaceutical production to only identifying higher order structural changes such as aggregate levels in purification steps. 114 in this review paper we have presented the full potential of ftir spectroscopy to identify changes of biopharmaceuticals under a range of conditions, and at primary, secondary, and tertiary structural levels. ftir spectroscopic imaging is especially useful for studying complex and heterogeneous samples, as it produces a chemical image of the sample or system being investigated, and offers the opportunity to collect dynamic images. macro atr-ftir spectroscopic imaging consists of a large sample compartment containing an atr accessory attached to a spectrometer, whilst micro atr-ftir spectroscopic imaging involves the use of an infrared microscope with an atr objective. the first paper to study aggregation of igg by macro atr-ftir imaging investigated secondary structural changes of 1 mg/ml igg4 solution under thermal stress and found igg4 is more stable at high ph or at low or high salt concentrations. 115 the use of macro atr-ftir spectroscopic imaging and depth profiling is particularly interesting as it could be used to visualise biopharmaceutical therapeutic release systems. macro atr-ftir spectroscopic imaging of biomaterials has increased in popularity in recent years. research using this technique has demonstrated the ability of a germanium atr ire crystal to monitor distribution within materials, and the presence and states of live cells, 116 and it has also been used to investigate cancerous tissue samples and bio fluids. 117 as previously seen, macro atr-ftir spectroscopic imaging can be applied to depth profiling studies (fig 10) . 6 micro atr-ftir spectroscopic imaging has previously been used to investigate protein crystallisation, 118 and aggregates in live cells, 119 as well as to probe protein hydration in living cells. 120 terahertz time-domain spectroscopy (thz-tds) in the far ir region was used to investigate bsa protein solution at phs ranging from 2.5-10, this paper found different conformations of bsa have varying impact on the dynamics of surrounding water molecules. 121 micro atr-ftir imaging has recently been used for depth profiling of prostate cancer tissue in the z-direction, at varying angles of incidence, to study embedded components using a created 3d model. 122 micro ftir spectroscopic imaging has also been applied to determine the structure of protein-based silk fibroin/polyethylene oxide (sf/peo) polymer blends, 7 where conformation transitions of random coil and/ or α-helical conformation were shown in the sf-rich domains, to β-sheet conformation in peorich matrix (fig 11) . this research used ftir and x-ray microscopy spectroscopic imaging (stxm) as complementary techniques, and found they are useful to study phase behaviour and molecular conformation of protein based polymers. this could be applied to the biopharmaceutical industry through the use of therapeutic delivery systems. 123 macro atr-ftir spectroscopic imaging has been used to monitor affinity chromatography purification of monoclonal antibodies, 3 a particularly useful investigatory method which looks at the binding capacity of resin. this is important due to the high financial cost of replacing protein a resin. 124 macro atr-ftir spectroscopic imaging has been successfully applied to cleaning procedures of a fouling layer on membranes such as polypropylene (pp), and polytetrafluoroethylene (ptfe), 125 the small penetration depth of atr is well suited to this application due to the thin nature of membranes. it is predicted that the membrane filtration market will be worth $8.6 billion usd by 2024, 126 therefore the identification of ftir as a valid monitoring technique of membranes could have significant positive impacts on the acceleration of this market due to the ease of data collection. microfluidics and ftir spectroscopic imaging have previously been combined to investigate pharmaceuticals (or small molecule drugs) such as ibuprofen. 127 in this study ibuprofen was studied at neutral and acidic conditions, and the structural change from compacted powder to crystalline form was characterised. macro atr-ftir imaging allowed the phase change of different areas in the tablets to be observed simultaneously. another application for macro atr-ftir spectroscopic imaging is the monitoring and quantification of lysozyme within water droplets studied under dynamic flow. 128 building on this research, protein solutions under flow and atr-ftir spectroscopic imaging could be applied to j o u r n a l p r e -p r o o f 12 biopharmaceutical processing. one review has found that microfluidics, in tandem with chromatography and ms is effective in characterising the stability of therapeutic proteins. 129 these experiments could be adapted to work with macro atr-ftir spectroscopic imaging, and provide an alternative low cost, fast acquisition time, and high throughput tool. this review has highlighted the use of atr-ftir spectroscopy for the investigation of efficient production of biopharmaceuticals. although atr-ftir spectroscopy is already in situ at a number of points in the production pathway, there is real potential for it to be utilised more effectively. atr-ftir spectroscopy will shorten measurement times 6 (compared to other analytical techniques such as hplc), and increase the effectiveness of measurements through improved signal to noise ratio, and the increased number of parameters and characteristics of the biopharmaceuticals which can be observed from a single spectral measurement. macro atr-ftir spectroscopic imaging has yet to fulfil its potential in biopharmaceutical analysis. in the future it could be used to study biopharmaceuticals under flowing conditions, in a scaled down approach. an example of this would be the study of live cells in the first stages of usp, and the study of ptms, along with higher order structures, in a single measurement. macro atr-ftir spectroscopic imaging could also be applied to investigate the level and distribution of aggregation in dsp, which would be ideal as an in-line technique, reducing the margin of error that comes with off-line measurements. the true power of biopharmaceuticals, and monoclonal antibodies in particular, is being realised in the worldwide battle against sars-cov-2. some companies are using single cell sequencing to structure the library of antibodies from sars-cov-2 patients, and then using polyclonal therapies for treatment. 130 another company is harnessing the power of monoclonal antibodies to develop a therapy for treatment, and are considering various other drugs which could be repurposed for covid-19 treatment, such as those which treat rheumatoid arthritis or some cancers. 131 indeed some researchers think they have already found an effective treatment using monoclonal antibodies. 132 the fast development of monoclonal antibody therapies has previously been effective, for example in the ebola epidemic in west africa. despite the disadvantages of monoclonal antibodies, such as transport and delivery difficulties, they offer a promising solution in the weeks, months, and years to come. 133 if these proposed monoclonal antibodies therapies are taken to production, atr-ftir spectroscopy will almost certainly have a role to play in stability testing, but the capabilities of atr-ftir spectroscopy and spectroscopic imaging reach much further. it is hoped that future research will utilise these techniques to the limit of their capabilities, to improve efficiency and sample yield. assessing the utility of circular dichroism and ftir spectroscopy in monoclonal-antibody comparability studies ftir spectroscopy as an analytical tool to compare glycosylation in therapeutic monoclonal antibodies in-column atr-ftir spectroscopy to monitor affinity chromatography purification of monoclonal antibodies overlapping effects of new monoclonal antibodies for severe asthma monoclonal antibodies in cancer therapy insight into heterogeneous distribution of protein aggregates at the surface layer using attenuated total reflection-fourier transform infrared spectroscopic imaging structural determination of protein-based 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enabling cell culture process analytical technology the therapeutic monoclonal antibody market advances and challenges in enveloped virus-like particle (vlp)-based vaccines generation and characterization of murine monoclonal antibodies against immunoreactive trypsinogen for newborn screening of cystic fibrosis potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody cell-penetrating peptides selectively cross the blood-brain barrier in vivo very large scale monoclonal antibody purification: the case for conventional unit operations a high-throughput method for membrane protein solubility screening: the ultracentrifugation dispersity sedimentation assay structural properties of monoclonal antibody aggregates induced by freeze-thawing and thermal stress forced degradation testing as complementary tool for biosimilarity assessment effects of solution conditions, processing parameters, and container materials on aggregation of a monoclonal antibody during freezethawing process control and monitoring for continuous production of long-length fiber optic near-infrared (nir) spectroscopy probes for on-line quality control of processed land animal proteins model-based prediction of purity and quantity during a chromatographic capture of fibroblast growth factor 2 attenuated total reflection fourier-transform infrared (atr-ftir) imaging of tissues and live cells qualification of ftir spectroscopic method for protein secondary structural analysis ethanol or/and captopril-induced precipitation and secondary conformational changes of human serum albumin analysis of protein glycosylation and phosphorylation using liquid phase separation, protein microarray technology, and mass spectrometry assays for determination of protein concentration ultraviolet spectroscopy and its pharmaceutical applications-a brief review an application of ultraviolet spectroscopy to study interactions in proteins solutions at high concentrations large-scale human metabolomics studies: a strategy for data (pre-) processing and validation breaking with trends in pre-processing? trac-trend anal chem systematic ftir spectroscopy study of the secondary structure changes in human serum albumin under various denaturation conditions advanced statistical and numerical methods for spectroscopic characterization of protein structural evolution extracting biological information with computational analysis of fourier-transform infrared (ftir) biospectroscopy datasets: current practices to future perspectives simultaneous fitting of absorption spectra and their second derivatives for an improved analysis of protein infrared spectra comparability of protein therapeutics: quantitative comparison of second-derivative amide i infrared spectra the importance of buffers in downstream processing examination of the secondary structure of proteins by deconvolved ftir spectra protein secondary structures in water from second-derivative amide i infrared spectra1 spectroscopic ftir and nmr study of the interactions of sugars with proteins obtaining information about protein secondary structures in aqueous solution using fourier transform ir spectroscopy proteolytically-induced changes of secondary structural protein conformation of bovine serum albumin monitored by fourier transform infrared (ft-ir) and uv-circular dichroism spectroscopy conformational analysis of protein secondary structure during spray-drying of fourier transform infrared analysis of amide iii bands of proteins for the secondary structure estimation a distinct utility of the amide iii infrared band for secondary structure estimation of aqueous protein solutions using partial least squares methods ftir analysis of proteins and protein-membrane interactions infrared absorbance spectroscopy of aqueous proteins: comparison of transmission and atr data collection and analysis for secondary structure fitting application of ftir method for the assessment of immobilization of active substances in the matrix of biophysical study of bevacizumab structure and bioactivity under thermal and ph-stresses application of atr-ftir spectroscopy to the study of thermally induced changes in secondary structure of protein molecules in solid state molecular monitoring of different commercial insulins using ftir-atr spectroscopy for pharmaceutical quality control proteomic analysis of post-translational modifications challenges of glycosylation analysis and control: an integrated approach to producing optimal and consistent therapeutic drugs current trends in the analysis of post-translational modifications characterizing post-translational modifications and their effects on protein conformation using nmr spectroscopy protein glycosylation control in mammalian cell culture: past precedents and contemporary prospects gastric atpase phosphorylation/dephosphorylation monitored by new ftir based bia-atr biosensors attenuated total reflection fourier transform infrared (atr ft-ir) spectroscopy as an analytical method to investigate the secondary structure of a model protein embedded in solid lipid matrices ten years of biosimilars in europe: development and evolution of the regulatory pathways these drug patents are expected to expire in 2020 biosimilarity versus manufacturing change: two distinct concepts key concepts and critical issues on epoetin and filgrastim biosimilars. a position paper from the italian society of hematology, italian society of experimental hematology, and italian group for bone marrow transplantation switching reference medicines to biosimilars: a systematic literature review of clinical outcomes fda approves amjevita, a biosimilar to humira assessment of structural and functional similarity of biosimilar products: rituximab as a case study how to measure and analyze tryptophan fluorescence in membranes properly, and why bother? multivariate analysis of two-dimensional c methyl nmr spectra of monoclonal antibody therapeutics to facilitate assessment of higher order structure structural similarity, characterization of poly ethylene glycol linkage and identification of product related variants in biosimilar pegfilgrastim metal-catalyzed photooxidation of histidine in human growth hormone detection and identification of the vibrational markers for the quantification of methionine oxidation in therapeutic proteins advances in downstream processing of biologics -spectroscopy: an emerging process analytical technology analysis of manufacturing costs in pharmaceutical companies in-line fourier-transform infrared spectroscopy as a versatile process analytical technology for preparative protein chromatography antigen generation and display in therapeutic antibody drug discovery --a neglected but critical player on-line fermentation monitoring by mid-infrared spectroscopy simplified fourier-transform mid-infrared spectroscopy calibration based on a spectra library for the on-line monitoring of bioprocesses an on-line approach to monitor ethanol fermentation using ftir spectroscopy fourier-transform infrared spectroscopy as a process analytical technology for near real time in-line estimation of the degree of pegylation in chromatography analytical tools for monitoring changes in physical and chemical properties of chromatography resin upon reuse cleaning-in-place of immunoaffinity resins monitored by in situ atr-ftir spectroscopy technical decision making with higher order structure data: impact of a formulation change on the higher order structure and stability of a mab the immunogenicity of humanized and fully human antibodies immunogenicity in protein and peptide based-therapeutics: an overview challenges and opportunities in biopharmaceutical manufacturing control integrated control of continuous (bio)pharmaceutical manufacturing applications of ftir in identification of foreign materials for biopharmaceutical clinical manufacturing reversible immuno-infrared sensor for the detection of alzheimer's disease related biomarkers analytical tools for characterizing biopharmaceuticals and the implications for biosimilars at-line mid infrared spectroscopy for monitoring downstream processing unit operations high-throughput thermal stability analysis of a monoclonal antibody by attenuated total reflection ft-ir spectroscopic imaging spectroscopic imaging of biomaterials and biological systems with ftir microscopy or with quantum cascade lasers using fourier transform ir spectroscopy to analyze biological materials micro atr ftir imaging of hanging drop protein crystallisation a ftir microspectroscopy study of the structural and biochemical perturbations induced by natively folded and aggregated transthyretin in hl-1 protein hydration in living cells probed by fourier transform infrared (ft-ir) spectroscopic imaging a study of the effect of a protein on the structure of water in solution using terahertz time-domain spectroscopy three-dimensional depth profiling of prostate tissue by micro atr-ftir spectroscopic imaging with variable angles of incidence synthetic polymers for biopharmaceutical delivery membrane-based technologies in the pharmaceutical industry and continuous production of polymer-coated crystals/particles membrane fouling from ammonia recovery analyzed by atr-ftir imaging pharmaceutical membrane filtration market by product (filter, systems), technique (microfiltration, ultrafiltration), material attenuated total reflection-fourier transform infrared spectroscopic imaging of pharmaceuticals in microfluidic devices generation of chemical movies: ft-ir spectroscopic imaging of segmented flows microfluidic approaches for the characterization of therapeutic proteins how monoclonal antibodies might prove useful against the coronavirus. npr we would like to thank bbsrc and bristol myers squibb for provision of a case studentship to miss h. tiernan key: cord-346916-jj4l9ydl authors: girardi, erika; pfeffer, sebastien; baumert, thomas f.; majzoub, karim title: roadblocks and fast tracks: how rna binding proteins affect the viral rna journey in the cell date: 2020-08-23 journal: semin cell dev biol doi: 10.1016/j.semcdb.2020.08.006 sha: doc_id: 346916 cord_uid: jj4l9ydl as obligate intracellular parasites with limited coding capacity, rna viruses rely on host cells to complete their multiplication cycle. viral rnas (vrnas) are central to infection. they carry all the necessary information for a virus to synthesize its proteins, replicate and spread and could also play essential non-coding roles. regardless of its origin or tropism, vrna has by definition evolved in the presence of host rna binding proteins (rbps), which resulted in intricate and complicated interactions with these factors. while on one hand some host rbps recognize vrna as non-self and mobilize host antiviral defenses, vrna must also co-opt other host rbps to promote viral infection. focusing on pathogenic rna viruses, we will review important scenarios of rbp-vrna interactions during which host rbps recognize, modify or degrade vrnas. we will then focus on how vrna hijacks the largest ribonucleoprotein complex (rnp) in the cell, the ribosome, to selectively promote the synthesis of its proteins. we will finally reflect on how novel technologies are helping in deepening our understanding of vrna-host rbps interactions, which can be ultimately leveraged to combat everlasting viral threats. viruses are obligate intracellular parasites that strictly rely on host cells to translate and amplify their genomes [1] [2] [3] . viral rnas (vrnas) play a central role during infection as they bear all the necessary information for a virus to express its proteins, replicate and spread. unlike dna viruses where the messenger rna must be first transcribed for the synthesis of viral proteins, rna viruses use their rna molecules both for protein synthesis and as replication templates. viruses encode an array of vrna-binding proteins (vrbps), essential for different steps in the viral lifecycle, comprising translation, synthesis, packaging of the viral genome and cell-to-cell spread. importantly, vrna is never really naked in the cellular milieu. apart from its interactions with vrbps, it has evolved to co-opt specific sets of host encoded rbps. this is illustrated by the variety of described strategies that vrnas use to hijack key cellular machineries and evade the host's defense arsenal. unlike vrbps that promote infection, host encoded rbps can either have pro-or antiviral functions. indeed, a large body of work produced during the last decades describe the involvement of host encoded rbps in almost every known stage of vrna lifecycle. certain host rbps, ordinarily functioning in cellular tasks, are repurposed by vrnas to guide them through the viral lifecycle, including genome translation, synthesis, modification, localization and packaging. on the other hand, many host rbps have evolved dedicated antiviral functions, ranging from the recognition of the invading vrna to the restriction of viral replication. the ability of vrna to either subvert or get antagonized by cellular rbps determine the outcome of a viral infection. indeed, vrna-rbps interactions could dictate the permissiveness of certain cell types to infection, host range, tissue tropism, viral evolution, efficiency of viral replication, pathology of infection and immune clearance [4] . vrnas interact with numerous and very diverse rbps during infection. this is illustrated by the rich literature on the subject that has yielded many discoveries in the last decades. for example, the study of intimate vrna-rbp interactions shaped our understanding of how rna interference (rnai) functions as the main antiviral defense system in plants and invertebrates, and how bacteria defend themselves against invading phages through the crispr system [5, 6] . herein, we review the mechanisms at play when rna viruses infect their animal hosts, taking a vrna-centric view. focusing on viruses that are important human pathogens, we will describe how cellular rbps act on early steps after viral entry, driving mechanisms of vrna recognition, degradation and modification to limit or to promote viral replication and spread. we will further describe a number of crucial mechanisms during which vrna hijacks the ribosome to preferentially translate the viral program. finally, we will reflect on how new emerging techniques are allowing to get a better grasp on the diversity of host rbps-vrna interactions. elucidating the mechanisms by which rbp-vrna interactions influence viral infection, is advancing our understanding of cell biology by revealing unforeseen biological knowledge and may also provide new targets for host-directed antiviral therapies [7] . indeed, recent corona, ebola and zika virus outbreaks remind us that new innovative antiviral approaches are clearly needed, particularly for emerging rna viruses with important epidemic potential [8] [9] [10] . despite the fact that the molecular composition of rna is universal throughout life kingdoms, host immune pathways are able to differentiate and recognize non-self nucleic acids based on specific features, structures and modifications. moreover, despite the molecular mimicry set by rna viruses to resemble cellular mrnas and escape host recognition, the viral nucleic acid still needs to embark on a long journey through a hostile cell environment and must overcome the obstacles put in place by the host antiviral system in order to be translated and replicated. cellular innate immunity comprises a rather sophisticated set of host factors that discriminate molecular alterations with a high specificity and restrict rna virus infection through direct or indirect activity on the vrna. in vertebrate animals, a variety of cellular sensors have evolved to detect foreign rna sensing to activate the innate immune response, which involves transcriptional and post-transcriptional activation of genes of interferon-stimulated genes (isgs). pattern recognition receptors (prrs) are host-encoded proteins that sense molecular features of vrna molecules which are generally absent in the majority of cellular rnas. multiple molecular receptors are involved in the recognition of these so-called pathogen-associated molecular patterns (pamps) [11, 12] . a major molecular signature and weakness of all rna viruses is the long double stranded (ds) rna molecules that are generated during replication. the sensing of dsrna is believed to be sequence-independent and rather depends on the distinct molecular features of the viral dsrna structure mostly absent in uninfected host transcriptome [13] . among the first cellular proteins that detect the invading virus are the toll-like receptors (tlrs) (fig. 1) , which are transmembrane glycoproteins that are constitutively expressed or pathogen-induced, located on the plasma membrane or intracellular endosomes which are preferential entry routes for vrnas (reviewed in [14, 15] ). tlrs share strong similarities in their structures and organization. their n-terminal region recognizes ligands thanks to its extracellular or luminal domains and is connected to the c-terminal cytoplasmic domains (ctds) by a single membrane-spanning domain. among the 10 tlrs identified in humans so far, tlr7 and tlr8 recognize ssrnas and tlr3 recognizes dsrnas [12, 16, 17] . in tlr3, the n-terminal region recognizes the ligand, dsrna, in the lumen of endosomes whereas the c-terminal signalling domain resides in the cytoplasm, and upon ligand recognition, tlr3 initiates the signalling process that culminates in transcriptional induction of specific immune genes. tlr3 was believed to be a major sentinel against viral infections since it responds to a common by-product of viral replication ( table 1) . for instance, encephalomyocarditis virus (emcv) infection leads to a tlr3-dependent innate stress response, which is involved in mediating protection against virus-induced myocardial injury [18] . similarly, tlr3 recognizes dengue virus (denv), limiting its replication [19] , while zika virus (zikv)-mediated tlr3 activation was shown to deplete neural progenitor cells [20] . indeed, the protective versus pathogenic role of tlr3 in viral pathogenesis is debated [21] . another group of molecular sentinels that senses foreign rna is represented by the ifn-induced intracellular cytosolic receptors, named retinoid acid-inducible gene i (rig-i)-like receptors (rlrs), which comprise dexd/h-box helicases such as rig-i (or ddx58), melanoma differentiation-associated protein 5 (mda5 or ifih1) and laboratory of genetics and physiology 2 (lgp2 or dhx58) [22] (fig. 1 , table 1 ). all rlrs possess a central dexd/h-box rna helicase domain and a ctd which are necessary for detection of foreign rnas. rig-i and mda5 have as well two n-terminal caspase activation and recruitment domains (cards), which mediate downstream signal transduction, while lgp2 lacks them. although rig-i and mda5 share similar structural composition, their ability to detect vrna is not redundant but specific [22] ( table 1) . rig-i monitors the 5 ′ ends of rna molecules via its ctd and helicase domain in order to initiate cytokine production in response to a wide range of viruses. triphosphate (ppp) or diphosphate (pp) at the uncapped 5 ′ -end of rna molecules with partial base-pairing to a complementary strand of rna molecules are recognized and required for vrna is sensed by different families of receptors (e.g. rlrs, tlrs, pkr). vrna is edited by adars and methylated by host mettl proteins (m 6 a) or viral methyl transferases (2 ′ -o-me). vrna can also be degraded by 5 ′ -3 ′ or 3 ′ -5 ′ exonucleases (e.g. xrn1 and rna exosome respectively) or by endonucleases (e.g. dicer). rig-i activation [23] [24] [25] [26] . the typical structure of the panhandle of negative-strand viral genomes confers full rig-i ligand activity. rig-i recognizes many single stranded negative rna virus families, such as paramyxoviridae, rhabdoviridae, orthomyxoviridae, bunyaviridae and filoviridae [27] [28] [29] . rig-i also detects some positive-sense rna viruses such as dengue and zika viruses of the flaviviridae family, by recognition of nascent viral genomes containing 5 ′ -ppp groups prior to capping [30] . mda5 senses both rna length and secondary structure by recognizing uninterrupted rna duplexes longer than a few hundred base pairs [31] and forming filaments around dsrna to initiate signalling [32] . mda5 is required for immunity against several classes of viruses, including single-stranded positive rna viruses such as picornaviruses [28, 33, 34] , flaviviruses [35] and coronaviruses [36] (table 1) . the third rlr family member lgp2 lacks the amino-terminal tandem cards and thus lacks signal-transducing activity. several studies indicate a disparate regulatory role for lgp2 in either triggering or dampening the innate immune signalling pathways following rna virus infection [37] . on one hand, lgp2 can function as a feedback inhibitor of rig-i by sequestrating double-stranded but not single-stranded rnas. lgp2 negatively regulates sendai virus (sv)-induced irf-3 or nf-κb signaling acting as a natural inhibitor of antiviral responses, and as a repressor of rig-i signaling [38] . however, lgp2 has a positive effect on mda5-mediated antiviral response. lgp2-deficient mice exhibited a defect in type i ifn production in response to infection by the encephalomyocarditis virus, the replication of which activates mda5-dependent innate immune response [39] . moreover, a recent study indicated that lgp2 can inhibit antiviral rnai in mammals by competing with and preventing dicer-mediated processing of dsrnas into sirnas both in vitro and in cells [40] . interestingly, other dexd/h-box helicases, such as ddx17, ddx21, ddx6 or ddx56, are also emerging as important sentinels that influence viral sensing in multiple ways. these helicases have been shown to bind vrnas, acting either as antiviral effectors or contributing to viral replication [41] . the presence and replication of viral nucleic acids in vertebrate cells can also induce the activation of other antiviral enzymes that are both sensors and effectors ( table 1) . these also recognize viral signatures; however, their main function is not necessarily to only induce a transcriptional immune response, but rather to directly attack vrna by degrading it or inhibiting its translation. for this reason, these are not usually referred to as receptors [12, 42, 43] . among them, double-stranded rna (dsrna) activated protein kinase r (pkr; also known as eif2ak2) or adenosine deaminase and 2 ′ -5 ′ -oligoadenylate synthetase class of enzymes (oass), can bind dsrna in a sequence independent manner, recognizing the structure as a prr (fig. 1 first, pkr is a serine-threonine kinase constitutively expressed in all tissues at a basal level as an inactive monomer. viral dsrna binding to the two n-terminal rna binding motifs induces a conformational change that allows atp binding to the c-terminal kinase domain. pkr can be activated by dsrna from reoviruses [44] but can also recognize dsrna from replication intermediates of + rna viruses [45] ] (table 1) . moreover, it has been shown that highly structured rna elements from retroviruses such as human immunodeficiency virus (hiv) transactivation-response region (tar) rna hairpins [46] or dsrna formed by the antiparallel mrna transcripts of some dna viruses can also activate pkr [47] . upon dsrna binding pkr dimerizes and autophosphorylates threonine residues in its activation domains, thereby stabilizing the dimers and increasing kinase activity [48] . this in turn leads to a series of events that inhibit viral translation and replication, as will be detailed later on in this review. interestingly, pkr is an interferon stimulated gene (isg) itself and its transcription can be up-regulated upon type i interferon production by virus-infected cells, stimulating its accumulation in neighboring cells. pkr activation following infection of interferon-primed neighboring cells inhibits global protein synthesis and reduces viral spread, making this activation a key player in the innate response to viruses [49] . therefore, pkr can be considered a major molecular sentinel of antiviral innate immunity, recognizing a diverse set of stress-inducing stimuli and mounting an appropriate (fig. 3) . the second known class of dsrna sensors and effectors is represented by the 2 ′ ,5 ′ -oligoadenylate synthetases (oases) (fig. 1, table 1 ), which also act as prrs for the detection of many rna viruses [54] . the four human oas genes, oas1, oas2, oas3 and oasl (oas-like), are constitutively expressed at low levels in the cytoplasm and are activated in response to viral dsrna. as an example, oas1 protein accumulates in the cytoplasm as an inactive monomer [55] and following activation by viral dsrna, it forms a tetramer that synthesizes 2 ′ ,5 ′ -oligoadenylates (2-5 as). these 2-5as activate the ribonuclease l (rnasel) to cleave cellular and viral rnas and supress viral replication (see below). overall, the functional redundancy, provided by multiple host proteins that recognize different features of vrnas, is a successful strategy for the host to fight and contain the infection [56] . similar to pkr, the oas-rnasel axis can be antagonized by some rna viruses. for example, certain corona and rotaviruses encode proteins belonging to the phosphodiesterase family, able to cleave 2-5 a molecules, thereby preventing activation of rnasel [57] . in concert with innate immune sensors linked to ifn activation, mammalian cells can also depend on intrinsic factors that participate in the direct antiviral response to non-self nucleic acids. accumulating evidence suggests that the cellular rna surveillance pathway restricts viral infection by modulating vrna stability [58] . the presence of specific molecular features on the vrna and the binding of trans-acting antiviral host rbps can drive vrna through the host mrna quality control pathways (fig. 1 ). for instance, in mammalian cells, the non-sense mediated decay (nmd) pathway has recently been shown to function in the restriction of positive-sense (+) rna viruses such as alphaviruses in mammalian cells [59] . in fact, alphavirus rna genome resembles cellular mrnas and is translated into non-structural viral proteins (nsps) but has an untranslated 3 ′ utr, much longer than typical cellular mrnas, which is detected and degraded by the nmd pathway [59] . the 5 ′ to 3 ′ rna decay machinery can also directly target vrna for degradation in the cytoplasm. in this pathway, the monomethyl guanosine (m7g) cap structure is cleaved from mrnas by decapping enzymes exposing the 5 ′ monophosphorylated product to progressive 5 ′ →3 ′ exoribonucleolytic degradation by xrn1. the hepatitis c virus (hcv) genomic rna, which lacks a 5 ′ cap and is therefore susceptible to xrn-1 mediated decay, uses an unusual mechanism to overcome this pathway [60] . binding of the liver-abundant mir-122 to the 5 ′ utr of hcv in association with argonaute-2 (ago2) protein stabilizes the vrna and slows xrn1 decay of the viral genome in infected cells [60, 61] ( fig. 1) . other flaviviridae are able to take advantage of their susceptibility to xrn-1 mediated vrna decay. dengue, west nile, yellow fever or zika vrnas contain a highly structured 300-700 bp-long non-coding rna in their 3 ′ end. an incomplete 5 ′ -3 ′ degradation of the viral genome by the cellular xrn1 exonuclease produces the so called small flaviviral rna (sfrna). during rna degradation, xrn1 is stalled at the 3 ′ utr extremity of flaviviral vrnas, more precisely at stem-loops/pseudoknots, causing the accumulation of different species of sfrna. although, sfrna does not seem to have a direct role in viral replication per se, it contributes to viral pathogenicity in vivo partly by interfering or evading innate immune responses, by sequestering cellular rbps and inhibiting their endogenous functions [62] [63] [64] [65] [66] [67] [68] [69] [70] . interestingly, sfrna seems to function in a species-specific manner. while it facilitates replication and pathogenesis by inhibiting ifn-signalling in mammalian hosts, a recent report shows that zikv sfrna possess an anti-apoptotic function that helps the virus to disseminate and reach saliva in mosquito hosts, thus promoting productive infection and transmission [71] . it has also been proposed that sfrna could be a suppressor of rnai [72] . in the 3 ′ decay pathway, deadenylated mrna degradation is mediated by a cytoplasmic multisubunit 3 ′ -to-5 ′ exoribonuclease complex, namely the rna exosome ( fig. 1 ). this degradation pathway is emerging as a crucial effector for recognition and degradation of specific vrnas [73] . the exosome interacts with a variety of rbps, some of which are exported to the cytoplasm in response to viral infection and serve as adaptors to specifically target particular rnas. several antiviral rbps have been found to interact with the exosome, suggesting that their mechanism of action may involve exosomal degradation. for instance, the conserved rna helicase ddx17 translocates from the nucleus to the cytoplasm upon rift valley fever virus (rvfv) infection, binds a specific stem-loop structure in the vrna and recruits the exosome to degrade the vrna [74] . another example, is the zinc-finger antiviral protein (zap), which binds vrnas containing a zap response element (zre) and induces rna degradation via interaction of its n-terminal domain with host decay machinery mediated [75] (fig. 1 ). zap is constitutively expressed in many cell types and it is induced upon pathogen-sensing and in response to interferon. zap exhibits broad antiviral activity against a broad spectrum of viruses, such as alphaviruses, enteroviruses, filoviruses, influenza and porcine reproductive and respiratory virus [42] but also retroviruses [76] . for instance, zap binds regions of hiv-1 viral rna containing cpgs and induce degradation of viral rnas via interaction with the essential cellular cofactor khnyn [77] . moreover, zap antiviral activity has been linked to its subcellular localization in stress granules, which are induced upon infection by sindbis virus (sinv) for example [78] . interestingly, trim25, an e3 ubiquitin ligase with no previous known role in rna binding is emerging as a regulator of many antiviral factors, including zap [79, 80] . other ribonucleases can exhibit broad-spectrum antiviral effects through viral rna binding and degradation. the ribonuclease mcpip1 is involved in the suppression of japanese encephalitis virus (jev) and denv, but also other positive-sense rna viruses, such as sinv, hcv and emcv, and negative-sense rna virus, such as influenza virus [81, 82] . finally, two conserved endoribonucleases have been shown to play a major antiviral role in different animal species, namely rnase l and dicer ( fig. 1 , table 1 ). rnase l is composed of three major domains: an n-terminal regulatory ankyrin repeat domain (ard), a protein kinase (pk)-like domain, and a c-terminal ribonuclease domain (rnase). in the absence of 2-5 a, the ard represses the rnase domain. binding of 2-5as, generated by oases, to the ard alters the conformation of rnase l, thereby exposing protein-protein interaction domains and releasing the rnase domain from internal inhibitory sequence, triggering its dimerization and activation. rnase l cleaves single-stranded rnas at the uu/ua dinucleotides, which are relatively rare in the coding regions of cellular mrnas compared to viral rnas [55] . in the case of hcv infection, rnase l degradation of vrna produces small rna cleavage products with 3 ′ -monophosphate (3 ′ -p) and 5 ′ -hydroxyl (5 ′ -oh) at their termini, which fold back into potent pamps recognized by rig-i, amplifying the innate immune responses [83] . interestingly, some hcv genotypes escape rnasel cleavage by accumulating silent mutations at ua and uu dinucleotides, which are preferentially targeted by the nuclease [84] . the rnase iii endoribonuclease dicer is active against different rna viruses in both vertebrate and invertebrate animals (table 1 ) [6, [85] [86] [87] [88] [89] . rna interference (rnai) is a primordial form of antiviral immunity mediated by small rnas and is the major immune defence in insects, plants and nematodes species [12, 90] . in the antiviral rnai pathway, dicer is able to bind and cleave viral dsrnas into ~21 nt-long small interfering rnas (sirnas). the sirnas are subsequently loaded into the argonaute-containing rna induced silencing complex (risc) and program risc to target vrna in a sequence-specific manner, inducing vrna degradation or translational arrest. although evidence of dicer activity against certain rna viruses have been reported in undifferentiated mouse stem cells [88] , and in some other specific context [91, 92] , the physiological antiviral role of rnai and its interplay with the interferon response in vertebrate animals is still an area of active debate [93] [94] [95] . in recent years, it has been revealed that the rna code is much more complex than the primary rna sequence. in particular, covalent rna base and sugar modifications recently emerged as an additional layer in the regulation of gene expression. such modifications can change the rna structure and its interaction with other rnas or proteins, thereby regulating important steps in rna metabolism including splicing, rna stability and translation. these rna modifications are also utilized by host cells as a way to distinguish self-rnas from vrnas [96] [97] [98] . it is therefore not surprising that viruses evolved strategies to modify their own genomes and transcripts or hijack dedicated cellular enzymes responsible for these modifications. here we briefly describe three rna modifications known to be present on vrnas (adenosine-to-inosine editing, n 6 -methyladenosine (m 6 a) and 2 ′ -o-methylation) [88] [89] [90] . we expect that other rna modifications may have the ability to also influence vrna stability, translation and immune potential. deamination of adenosines to inosines (a-to-i editing) is a covalent rna modification occurring on dsrna structures, by the adenosine deaminase acting on rna (adar) enzymes (fig. 1, table 1 ). among the three mammalian adars, adar1 is expressed in its constitutive nuclear form adar1p110 and the ifn-inducible cytoplasmic one, adar1p150. both enzymes consist of a c-terminal deaminase domain, three consecutive dsrna binding motifs and one or two copies of n-terminal z-dna binding domains [99] . the consequence of the rna modification by adar1 is two-fold: first, if editing takes place in coding sequences, inosines will be misinterpreted by the translation machinery and will be read as guanosines. second, a-to-i conversion have the capacity to destabilize dsrna structures by changing the watson-crick base pairing and impairing recognition by cytoplasmic antiviral receptors, such as rlrs, pkr and oas1. for these reasons, adar proteins play a pivotal role in masking self rnas against innate immune detection by selective labelling [100] . given the opposite effects of adar1 and antiviral sensors in the control of innate immune responses to endogenous duplex rna, the ato-i editing contribution in antiviral defence remains unclear. a bias toward a-to-g mutations dependent on adar has been described for a wide range of viruses [101] . this could potentially lead to the synthesis of dysfunctional viral proteins and destabilize key rna structures such as dsrna replication intermediates. although several studies have reported high a-to-i editing levels indicative of a potential mutagenic and antiviral effect, adar activity seems to be rather proviral due to its dampening effects on the innate immune system [101] . one clear example where adar plays a proviral role is the case of hepatitis d virus (hdv). hdv contains only one orf responsible of expressing the hdv delta antigen (hdag), essential for hdv replication and spread. two forms of hdag are expressed: a short form s-hdag and one that is 19 amino acid longer (l-hdag). hdv needs both to be expressed in order to replicate and spread and this depends on adar1. in fact, adar1 recognizes a specific structure on the rod-like rna of hdv antigenomic rna and converts adenosine to inosine. replication of this mutated rna leads to the substitution of guanosine in its genome, specifically leading to the replacement of an amber codon (uag) for the small delta antigen by a tryptophan codon (ugg) which produces a protein that is 19 amino acid longer. the synthesis of this longer protein is crucial for hdv spread, therefore adar1 is a proviral host rbp essential for hdv infection [102] . in mammalian cells, n 6 -methyladenosine (m 6 a) is the most prevalent and dynamically modulated internal mrna modification. such a modification is reversible via the activity of two key enzyme classes: methyltransferases or "writers" (e.g. methyltransferase like 3 (mettl3) and 14 (mettl14)) and demethylases or "erasers" (e.g. (fto) and alkbh5) (fig. 1, table 1 ). the m 6 a writers and erasers can alter the cellular rna fate by destabilizing local rna structures or by affecting protein binding to modulate host rna stability or translation efficiency. in particular, the m 6 a readers such as yth domain-containing family proteins (ythdf1-ythdf3) play crucial roles [103] . recent reports show that m 6 a could be a regulator of the immune system, playing a role in the discrimination between endogenous and exogenous rnas [104] . indeed, several studies have identified m 6 a modifications on a variety of vrnas and this enhances global viral gene expression by increasing rna stability and translation [96, 105] . interestingly, mapping of m 6 a on the rna genomes of flaviviridae, including dengue, zika, yellow fever, and west nile virus, identified conserved regions modified by m [6] a, suggesting that this modification is a conserved regulatory mark across flaviviridae genomes [106] . another case where m 6 a appears to function in innate immune discrimination between self and non-self rnas is given by the human metapneumovirus (hmpv) [107] . m 6 a methylation of vrna promotes hmpv replication and gene expression. in the absence of m 6 a residues on the vrna, viral infection is compromised due to a rig-i-dependent increase of type i interferon [107, 108] . this notion is reinforced by the recent report of the effect of the loss of the m 6 a writer mettl3 in the mouse hematopoietic system. indeed, mettl3 knock-out results in the upregulation of mda5-rig-i, pkr and oas-rnase l pathways due to the aberrant accumulation of endogenous dsrna [109] . therefore, m6a modification appears to prevent formation of certain dsrna, so it would be interesting in future work to check whether this has implications during viral infection. the 2 ′ o-methylation (2 ′ o-me) modification is a highly abundant chemical modification found in the rna of virtually all eukaryotic species, whereby a methyl group is added to the 2 ′ -oh of the ribose ring by cellular 2 ′ -o-methyltransferases (2 ′ o-mtase) (fig. 1 ). 2 ′ o-me is enriched in the first and second transcribed nucleotides next to the n7 methylated 5 ′ cap structure of higher eukaryote mrnas. while n-7methylation is important for stability and translation of the mrna, the 2 ′ -o-methylation has been shown to antagonize the innate immune response by allowing endogenous mrnas to escape recognition by immune sensors [97, 110] . notably, 2 ′ -o-me of the first (cap 1) and often the second (cap 2) nucleotide abolishes interaction of host mrnas with rig-i and mda5 [97] . the functional importance of 5 ′ -structures and modifications in mrnas is supported by the fact that many cytoplasmic rna viruses (including picornaviruses, flaviviruses and coronaviruses) have evolved alternative 5 ′ elements. these include small viral proteins linked to the 5 ′ end of genomic rna, or encode functions associated with the formation of a 5 ′ cap that are homologous to those found in eukaryotic cells. many viruses have evolved mechanisms to generate their own cap structures with methyl ribose in 2 ′ -o position in addition to the one at the n7 position of the capped guanine to evade innate immune recognition. for instance, the coronavirus 2 ′ -o-methyltransferase activity associated with the highly conserved viral nonstructural protein 16 (nsp16) is necessary to inhibit mda5 recognition of vrna and activation of ifn pathway [111] . 2 ′ -o methylation of the 5 ′ cap of vrna also subverts mammalian antiviral responses by evading restriction by interferon-induced proteins with tetratricopeptide repeats (ifits). ifits are cytoplasmic antiviral proteins which differ in specificity against rna structures. among them, ifit1 binds 5 ′ ppp rnas [112] and non-2 ′ -o methylated viral capped transcripts [113, 114] to inhibit their translation by out-competing the cellular translation initiation apparatus [115] (table 1) . members of the flaviviruses and coronaviruses are able to evade ifit1 sensing by adding a 2 ′ o-methyl cap structure to their own mrna via viral proteins [116, 117] . interestingly, alphaviruses whose genomic rna has a 5 ′ cap lacking 2 ′ -o methylation can avoid ifit1 immune restriction and efficiently replicate thanks to a secondary structure within their 5 ′ untranslated region (utr) which alters ifit1 binding and function [118] . once it has managed to escape host cell sensing and degradation pathways, vrna must compete with endogenous mrnas for accessing the same protein synthesis machinery. rna viruses have evolved several mechanisms to bypass the highly regulated translation cycle, particularly at the initiation phase and most of these mechanisms rely on peculiar vrna-host rbps interactions. importantly, changes in abundance, modification and activity of translation factors could regulate both vrna and host mrna translation [50] . therefore, rna viruses often employ strategies that favor the selective translation of their genomes. they can accomplish that by targeting, modifying or cleaving host translation factors and/or by usurping the identity of endogenous mrnas to have a privileged access to the ribosome [119] . canonical cellular mrnas are capped at their 5 ′ end cotranscriptionally. the cap structure, consisting of a 7-methylguanosine (m 7 g) linked to the 5 ′ nucleoside of the mrna chain through a 5 ′ -5 ′ triphosphate bridge, is crucial for mrna stability and translatability. specifically, the cap protects mrna from 5 ′ -3 ′ exonucleases and ensures recognition by the cap-binding complex eukaryotic translation initiation factors (eif4f) [120] (fig. 2) . interestingly, vrna caps can be stolen ("cap-snatching") from cellular mrnas or synthesized using either a host-or virus-encoded capping apparatus, and these capping assemblies exhibit a wide diversity in organization, structure and mechanism [121] . recognition of the cap by the eif4f subunit eif4e is thought to prepare mrnas for recruitment of the 43s complex containing multiple eifs, the initiator methionine trna (met-trna i met ) as a ternary complex with eif2 and gtp (eif2-tc), and the small 40s ribosomal subunit (fig. 2) . the polyadenylated 3 ′ mrna end is recognized by a poly(a)binding protein (pabp), which is bridged to the 5 ′ end via pabp interactions with the eif4g subunit of eif4f end. the assembled 43s complex scans the 5 ′ utr of the mrna to locate the initiator start codon (aug) (fig. 2) . after aug recognition ribosomal 60s subunits joining trigger eifs release to form the 80s ribosome where elongation can proceed for synthesis of the polypeptide chain. translation is one of the most energy-expensive processes in the cell and is therefore tightly regulated. cells rapidly reprogram gene expression when subjected to stress to conserve energy and minimize damage [122] . the arrest of bulk protein synthesis is a classical reaction cells employ during viral infection and it is often characterized by a specific inhibition of translation initiation. in the never-ending arms-race, many viruses have however evolved mechanisms to overcome the cellular stress they induce. generally, translation initiation is inhibited either by the disruption of the formation of the eif4f complex or by the inhibition of eif2-tc recycling [122] (fig. 3) . one major cellular regulator of eif4f complex formation is the mechanistic target of rapamycin (mtor) pathway and will not be discussed here [122] [123] [124] . the second major mechanism that causes translational arrest is the disruption of eif2-tc recycling, caused by phosphorylation of the α subunit of eif2. mammalian cells encode four known eif2α kinases among which pkr (table 1 ) (see above). the other three eif2α kinases: haeme-regulated inhibitor (hri), general control non-derepressible protein 2 (gcn2) and pkr-like endoplasmic reticulum (er) kinase (perk), respond to various cellular stresses including heat shock, amino acid deprivation, and er stress [122] (fig. 3) . these cellular stress states can be indirect indicators of viral infections. for instance, flaviviruses like dengue and zika viruses, which replicate in close association to er-membranes, induce er stress that results in perk activation and eif2α phosphorylation early during viral infection [125, 126] (fig. 3) . in some cases, gcn2 can also be activated by vrna to arrest translation. for example, gcn2 can recognize two regions of sindbis virus (sinv) genomic rna and inhibit vrna replication by blocking early viral translation. strikingly, mice lacking gcn2 are extremely susceptible to intranasal sinv infections, demonstrating high virus titers in the brain compared to similarly infected control animals [127] . alphaviruses like sinv take advantage of eif2 inactivation and can be totally insensitive to translational arrest by eif2 phosphorylation. subgenomic mrnas of sinv and another alphavirus, semliki forest virus (sfv), initiate translation in the presence of high levels of phosphorylated eif2α, utilizing a highly stable rna hairpin loop downstream of the aug initiator codon that stalls the ribosomes on the correct site to initiate translation of their mrnas, thus bypassing the requirement for a functional eif2 [128] . as mentioned earlier, dsrna is detected by and activates pkr for the rapid inhibition of translation initiation [129] . following activation by dsrna binding, dimeriziation and auto-phosphorylation, pkr phosphorylates eif2α which locks it onto its guanine nucleotide exchange factor eif2b, thereby preventing the regeneration of the eif2-tc that is critical for canonical translation initiation [122] (fig. 3) . although initially identified because of its ability to regulate translation in response to dsrna, pkr can also be activated by other cellular stresses such as oxidative stress, cytokines or following the stimulation of tlrs [130, 131] . furthermore, upon activation, pkr not only inhibits translation initiation, but also modulates a plethora of different signal transduction pathways, including the activation of p38 mitogen-activated protein kinase (mapk), c-jun n-terminal kinase (jnk), signal transducer and activator of transcription 1 and 3 (stat1 and 3) and the tumor suppressor p53 [132] . pkr can indirectly activate the nuclear factor κb (nfκb), which has among other functions an important role in type i ifn induction [132] . selective translation is a common strategy used by rna viruses to maintain intact their protein synthesis during global translation shutoff. we will briefly describe mechanisms used by viruses to promote the selective translation of their genomes during global protein shutdown. sequence, structure and chemical modifications present in the viral genome can determine the identity of the interacting rbps serving as translation factors. selective translation can therefore be achieved when vrna shunts the lengthy and elaborate multi-step mechanisms that yield assembled and productive 80s ribosomes to gain a privileged access to the ribosome. other strategies, such as targeting and degrading translation factors, ribosomal rnas or messenger rnas might also confer a selective advantage for vrnas over endogenous ones. we will discuss mechanisms that target the translation initiation step, nevertheless it is important to note that viruses are also able to trick capped rna is recognized by eif4f complex recruiting the 43s complex to the 5 ′ utr of mrna. this is followed by scanning until a start codon in encountered, followed by the formation of 80s ribosomes before elongation. lower panel: depicted are different modes of ires-dependent translation (i, ii, iii and iv). note the minimal requirement for initiation factors depending on ires class. these can either act on endogenous mrnas, eif4f, eif2, eif3 or the ribosomal subunits. in red are listed the four known cellular eif2alpha kinases: gcn2, hri, perk and pkr. translation elongation or termination steps [133] . while some positive-sense rna viruses like flavi or coronaviruses possess a cap-structure on the 5 ′ end of their vrna, some others like caliciviruses or picornaviruses (e.g. poliovirus or rhinovirus) decorate their 5ʹend with a covalently bound viral protein, vpg. in some cases, vpg proteins linked to the 5 ′ end of positive-strand rna genomes (e.g. feline calicivirus (fcv) or human norovirus) plays the role of a cap structure, recruiting ribosomes through interaction with eif3 [134] . in fact, modifications at the 5 ′ end of vrnas largely dictate the translation initiation mode that could either be cap -dependent or -independent. coronavirus mrnas are thought to undergo a classical cap-dependent translation initiation mechanism. a small compound that prevents the formation of the eif4f complex, a hallmark of cap-dependent translation, by blocking the interaction between eif4e and eif4g is able to significantly decrease human coronavirus 229e replication and infectious virus titers [135] . to favor the translation of their vrnas over endogenous mrnas, severe acute respiratory syndrome coronaviruses (sars-cov) non-structural protein 1 (nsp1) is able to efficiently and selectively inhibit host mrna translation by binding 40s subunits [136] (fig. 3) and promote host mrna degradation. for example, sars-cov-1 nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas, however viral mrnas seem to be resistant to nsp1-induced rna cleavage [137] . one mechanism permitting this selectivity have been shown for middle east respiratory syndrome coronavirus (mers-cov) where nsp1 selectively target mrnas transcribed in the nucleus and spares mrnas of cytoplasmic origin, notably vrnas [138] . flaviviruses are also thought to undergo a canonical cap-dependent initiation of translation requiring both the eif4f complex and pabp [139] [140] [141] . interestingly, in contrast to most cellular mrnas, flavivirus vrna lacks a 3 ′ poly-a tail; however, pabp is still able to associate internally to denv 3 ′ utr upstream of a conserved 3 ′ stem-loop, pabp/3 ′ utr interaction mimicking the role of mrna poly-a tail and presumably stimulating translation initiation 141]. both utr regions (5 ′ and 3 ′ ) in flaviviruses possesses secondary structures shown to stimulate viral translation [139, 142, 143] . interestingly, under cellular conditions where translation factors are limiting or eif4e is inhibited, denv has been shown to alternate between canonical cap-dependent translation initiation and a noncanonical mechanism that does not require a functional m [7] g cap [144] . although the mechanism by which flaviviruses undergo cap-independent translation initiation is not completely clear, it has been recently suggested that denv and zikv 5 ′ untranslated regions could harbor internal ribosomal entry site (ires) functions [145] . in fact, ires-dependent initiation of translation is the other major mechanism used by many viruses to initiate their translation, dispensing them from cap-dependence. ires sequences are specialized cis-acting rna elements present in viral 5 ′ utrs capable of bypassing the extensive requirement of translation initiation factors to recruit ribosomal complexes and drive protein synthesis. iress therefore confer a considerable competitive advantage to viral mrnas, freeing them from host regulatory constraints and sustaining viral protein synthesis when canonical cap-dependent translation is impaired [119] (fig. 2) . there are four known types of viral iress (i, ii, iii and iv). although iress perform a similar function, there are no known universal ires sequences. in fact, ires elements present in the genome of different families of rna viruses lack overall conserved features [146, 147] .the classification of viral iress in four types stems from their structural organization, their respective dependence on sets of translation initiation factors, and whether they use scanning or instead directly recruit ribosomes to the start codon [148] (fig. 2) . type i and ii ireses were first discovered in picornaviruses, such as poliovirus (type i) and encephalomyocarditis virus (type ii). these two ires classes interact with eif4g c-terminal region which binds to eif3 and eif4a [149] . both require eif5b, eif2 and met-trnai and are stimulated by the activity of eif1, eif1a and eif4b [150] (fig. 2) . type iii iress, exemplified those of pestiviruses and hepatitis c virus ires, require even less translation initiation factors to recruit 40s ribosomes. in fact, the hcv ires bypasses the requirement for eif4f and the scanning step of translation initiation. hcv ires is able to directly interact with eif3 and the ribosomal 40s subunit, placing the start codon present in the vrna in the ribosomal p-site [151] (fig. 2) . finally, type iv iress are amongst the most remarkable as they completely obviate the need for canonical initiation factors. these iress are common in the dicistroviridae family of viruses such as cricket paralysis virus (crpv) or drosophila c virus (dcv), which infect insects. these viruses contain a single positive-sense rna genome that encodes two non-overlapping open reading frames separated by a short intergenic region (igr). the igr region acts as an ires to initiate translation by recruiting 80s ribosomes in the absence of initiator trna i met or any canonical initiation factors, from a gcu alanine codon located in the a-site of the ribosome [152, 153] (fig. 2) . indeed, the igr rna structure mimics a trna, thus tricking the ribosome into translation initiation without the need of the eif2-gtp-met-trna complex. using shortcuts for a privileged access to the translation machinery by tricking the ribosome is not the only strategy rna viruses employ to selectively favor the synthesis of viral proteins. another effective tactic is targeting and inhibiting the translation of cellular mrnas, which frees up a large pool of ribosomes, making them readily available for vrna translation. this is generally achieved by targeting host rbps, specifically translation initiation factors or ribosomal proteins. viruses that use ires-dependent translation for example, actively provoke the shut-off of host cap-dependent translation initiation, by targeting specific initiation factors. for instance, enteroviruses including rhino and polioviruses, encode proteases that cleave eif4g, impeding the formation of the eif4f complex and therefore cap-dependent host mrna translation [154] (fig. 3) . other picornaviruses, such as emcv suppresses cap-dependent translation by activating the translational repressor 4ebp1 [155] . hypophosphorylated 4ebp1 binds eif4e preventing, eif4e-eif4g interaction, thus eif4f assembly. in fact, 4ebp hypophosphorylation is a strategy common to many viruses to inhibit host mrna translation (e.g., crpv, vsv) [156] [157] [158] (fig. 3) . targeting eif4f-associated pabp proteins is also used by some viruses to influence host translation. for example, rotavirus nsp3 interacts with eif4g and displaces pabp, therefore inhibiting host translation [159] . likewise, enterovirus and calicivirus proteases cleave pabp, and the rubella virus capsid protein binds pabp to suppress cellular mrna translation [119, 154, 159] . other translation initiation factors like eif3 can also be targeted by viral proteins. eif3-binding proteins from measles and rabies viruses inhibit host protein synthesis [160, 161] , whereas foot-and-mouth disease virus protease degrades eif3a and eif3b subunits [162, 119] . also, the coronavirus spike protein has been shown to interact with eif3f influencing translational control [163] (fig. 3) . although initiation factors are the privileged target of viral proteins to shut-off host mrna translation, some examples of viruses targeting translation elongation factors are also reported. for instance, it has been shown that ef1a and eef2 are respectively inactivated by sars-cov-1 n protein and avian reovirus p17 [164, 165] . in many of these examples, how vrna translation is able to proceed upon inactivation of these essential initiation or elongation factors remains unclear. viral rbps can directly target the ribosome to promote translation of their genomes and viral rna may also directly interact with ribosomal proteins to preferentially translate their genomes. denv ns1 protein interacts with rpl18 to promote viral protein synthesis [166] . conversely, vrna can rely on specific nonessential ribosomal proteins, dispensable for translating most host mrnas. moloney leukemia virus and sinv stop codon readthrough and frameshifting efficiency, have been shown to depend on rpl4 [167] . vsv cap-dependent mrna translation have been shown to be affected by rpl40 presence [168] (fig. 3) . also, some ires functions, like those found in polio, hcv or dicistroviridae, have been shown to be regulated by small ribosomal proteins such as rack1 and rps25 [169] [170] [171] [172] [173] (fig. 3) . interestingly, rack1 seem to be a privileged target of poxviruses to promote selective translation of viral mrnas [174] . finally, rbps that are not necessary translation factors or ribosomal can also positively influence viral translation. for example, pelo has been shown to be a host factor needed for efficient translation of viral capsids of drosophila c virus (dcv) [175] . vigilin and rrbp1, two endoplasmic reticulum localized rbps have been shown to bind denv and zikv vrnas, promoting their translation, stability and replication [176] . during the last decades, our knowledge of the intricate interactions of cellular rbps with vrnas have been a direct result of technical advances in modern molecular biology. when it comes to the study of rbps that are viral sensors or antiviral effectors, many major discoveries in how tlrs, rlrs, dicer or other rbps recognize and/or antagonize vrna, were made thanks to in vivo genetic screening technologies and innovations [177] . both, hypothesis-driven reverse genetics approaches, and forward genetic techniques have been able to yield susceptibility or resistance phenotypes, revealing genes with unanticipated and important roles in recognizing and/or targeting vrnas. the involvement of tlr 3, 9, mda5 and rig-i in the innate antiviral response wouldn't have been possible without in vivo genetic manipulation and screening of mouse models [178] [179] [180] . another classical example where genetics played a fundamental role in discovering important antiviral host rbps, is the study of the rnai pathway components. argonaute and dicer proteins' involvement in antiviral defenses across several animal and plant species was only possible thanks to genetic manipulation and screening techniques in model organisms [85, [181] [182] [183] . importantly, biochemical and structural biology approaches including x-ray crystallography and cryo-electron microscopy (cryo-em) guided these discoveries and were able to determine structurally how sensor and effector rbps interact with vrna [184] [185] [186] [187] [188] . likewise, recent technical innovations and improvements in rna isolation, manipulation and sequencing have enabled detection of new rna species and rna chemical modifications (e.g. m 6 a, 2 ′ -o-me) in vrnas, revealing an underappreciated role of these species and modifications in viral lifecycles [96, 189, 190] . thus, the use of oxidative treatment on the rna prior to its sequencing helped to unravel the existence of 2 ′ -o-methylated viral small rnas species that accumulate during sinv infection [191] . more recently, the use of single cell rna sequencing allowed to determine at an unprecedented resolution the accumulation of viral transcripts in a dynamic manner [192] . while this has so far been used to study dna viruses' transcription, it might also prove useful to study replication of rna viruses. we are still lacking a global picture of rna modifications that are present in viral rnas, and more importantly, we need to determine their exact contribution to the infectious cycle. to this end, it is crucial to develop and validate good antibodies targeting modified ribonucleotides, or to turn to emerging sequencing technologies, such as the oxford nanopore one, that allows direct rna sequencing. this strategy has recently allowed the direct detection of m 6 a by sequencing of cellular rnas [193, 194] . similarly, the last decades of research brought very detailed insights into how vrnas co-opt host ribosomes. this is partly due to the history of research in the mrna translation field. the ribosome being the most conserved and largest known rnp complex in living organisms, deciphering mrna translation has been an endeavor pursued since the birth of modern molecular biology [195] . important knowledge in our understanding of mrna translation were made in the past thanks to technical innovations and improvements. for example, in vitro translation systems, reconstituted from rabbit reticulocytes, were instrumental to understand many aspects of endogenous mrna translational control [195] . interestingly, thanks to such in vitro systems, we know since the 1970 ′ s the inhibitory role of viral dsrna or eif2 phosphorylation on translation initiation [196, 197] . in vitro translation systems coupled to reporter assays were also crucial later on to decipher the different mechanisms by which ires-dependent translation proceeds [50, 152, 198] . more recently, cryo-em approaches were able to paint a more detailed picture of how different ireses can interact and co-opt host ribosomes [150, 199, 200] . the rapid development and improvement of cryo-em techniques is expected to generate further knowledge, surpassing ires-ribosome interactions. indeed, cryo-em techniques have been lately used to unravel the structural basis of flaviviral sfrna production for example [63] . most recently, cryo-em structures of sars-cov2 nsp1 with human 40s ribosomal subunit revealed that nsp1 c-terminus binds to and obstructs the mrna entry tunnel, explaining how this protein is able to shutdown endogenous mrna translation [201] . another very recent cryo-em study was able to identify residues of nsp1 crucial for mediating translation inhibition [202] . in the future, these approaches promise to aid structure-based drug design not only against sars-cov2 but also other emerging rna viruses [201] . it is finally important to note that other novel and innovative techniques developed to study translation such as single-molecule approaches [203] or ribosome profiling by deep sequencing [204] are revealing the complexity of vrna interaction with host ribosomes. for instance, single-molecule studies of ires-mediated translation have revealed insights into the dynamics of hcv and crpv ires interaction with ribosomes and clarified decades of biochemical research, providing an outline of the conformational and compositional trajectory of the ribosome during initiation [151] . likewise, ribosome profiling revealed strategies that some rna viruses use to induce programmed ribosomal frameshifting (prf) that would have not been discovered otherwise. frameshifting events are 'programmed' because they occur at specific sequences, stimulated by cis-acting elements generally present in the vrna at rates that are orders of magnitude more frequent than nonprogrammed events [205] . cardioviruses such as emcv and its relative theiler's murine encephalomyelitis virus (tmev) induce prf but are atypical due to the absence of an appropriately positioned stimulatory rna structure in their genomes and a failure to reconstitute prf in vitro, outside of the context of virus infection. remarkably, thanks to ribosome profiling, a recent study shows that emcv frameshifting is trans-activated by viral protein 2a. as a result, the frameshifting efficiency increases from 0 to 70 % (one of the highest known in a mammalian system) over the course of infection, temporally regulating the expression levels of the viral structural and enzymatic proteins [206] . in the future, ribosome profiling techniques are expected to reveal more secrets of vrna translation, especially for viruses that annexes organelles like the er to complete their translation [207] . each time a technological leap is achieved the scope of what we know about vrnas interaction with cellular rbps has expanded. two global strategies have been used to study vrna-rbp interactions; protein-centric or rna-centric methods. protein-centric methods start with a known rbp of interest and characterize its interaction with rna. these approaches involve uv-crosslinking followed by antibody purification of the rbp of interest and identification of bound rnas, often by deep sequencing, broadly termed hits-clip (cross-linking immunoprecipitation followed by high-throughput sequencing) [208] . these methods have been used in the past in the context of viral infections to characterize the rna species that bind either known host rbps [176, [209] [210] [211] or of viral proteins [212, 213] . we will not focus further on these methods here but readers can refer to excellent reviews on the subject in the context of viral infections [214] [215] [216] . rna-centric approaches are more recent and focus on purifying an rna of interest to identify its associated proteins. a large number of the so-called "unconventional rbps" have been identified as associated to rnas thanks to these rna-centric approaches. for example, rna interactome capture (rna-ic), have largely expanded the repertoire of known rbps [217] . rna-ic entails ultraviolet crosslinking of rbps to rna in live cells, followed by collective capture of rnps with polyadenylated (poly(a)) rna on oligo(dt) beads and identification of proteins by quantitative mass spectrometry (q-ms) [217, 218] . these methods discovered new rbps that generally lack canonical rna-binding domains and are conserved across species, suggesting the existence of previously unidentified modes of rna binding and new biological functions for protein-rna interactions [217] . this method was applied to study cellular rna-interactome during sinv infection and was able to identify over 200 rbps that interacted differentially with host rna upon viral infection, amongst which, rnas encoding proviral and restriction factors [219] . although rna-ic is able to identify rbps that directly interact with rnas, one limitation of this method is its reliance on oligo(dt) to pull-down poly(a) containing rnas. another novel method, comprehensive identification of rna binding proteins-mass spectrometry" (chirp-ms), addresses this limitation by using 20-mer oligonucleotide probes complimentary to the rna of interest. chirp-ms uses formaldehyde (fa) cross-linking prior to pull-down with biotinylated probes, able to hybridize with an rna of interest. unlike uv-crosslinking, the use of fa for crosslinking doesn't only identify direct rna binders but also their associated protein complexes. cross-linked rnp complexes are then captured with streptavidin beads prior to proteomic analysis [220] . originally developed to identify rbps associated with long non-coding rnas (lncrnas) [221] , chirp-ms was recently applied to identify rbps associated with denv and zikv vrnas [176] . chirp-ms identified viral non-structural proteins ns3 and ns5 as highly enriched with denv and zikv vrna. furthermore, this method identified 464 high confidence hits from the human proteome that were specifically and reproducibly associated with the denv or zikv vrna [222] . many of those proteins were localized to the er, in line with the known biology of flaviviral rna, being translated and replicated at specific er sites [223] . importantly, when viral infection is difficult to synchronize, chirp-ms is unable to differentiate at which step in the viral lifecycle vrna-rbps associations occur (e.g. entry, translation, replication, assembly). a recent approach addresses partly this limitation and is able to capture interactions with just the pre-replicated viral rna genome [224] . viral cross-linking and solid-phase purification (vir-clasp) relies on infection of unlabeled host cells with 4-thiouridine (4su)-labeled viral genomes [224] . irradiation of infected cells with 365 nm light generates covalent cross-links between 4su-labeled viral genomes and interacting host or viral proteins. solid-phase capture of these complexes with solid-phase reversible immobilization (spri) beads leads to purification of total rna but only proteins covalently crosslinked to the 4su-labeled viral rna. this purification precedes (lc-ms/ms) identification of the crosslinked proteins [224] . using vir-clasp, the study identifies early interactomes of different viral genomes (e.g. chikv, iav, zikv, vsv). the authors of the study find that incoming chikv vrna binds both proviral and antiviral factors. while on one hand, chikv incoming vrna hijacks the lipid-modifying enzyme fatty acid synthase (fasn) for pro-viral activity, it is on the other hand n6-methyladenosine modified, binding to ythdf1 which suppresses its replication [224] . the aforementioned rna-centric methods (rna-ic, chirp-ms, vir-clasp) all use crosslinking (uv or fa) to identify in vivo interactions by purifying the rna under denaturing conditions that remove noncovalent interactions, and subsequently extracting only the cross-linked proteins for identification [225] . other approaches, that do not require crosslinking of rnps, use proximity proteomics to identify rbp-rna interactions in live cells. these approaches rely on 'promiscuous' biotin ligases that are able to label preferentially proteins that are within 20 nm of distance with biotin [226, 227] . originally developed to identify protein-protein interactions, this approach was recently adapted to identify rna-rbps interactions. the rna-protein interaction detection (rapid) method allows to use this spatial detection constraint to detect rbps bound to rna by tagging an rna of interest with a boxb aptamer to recruit a fusion protein of λ-n and a promiscuous biotin ligase [228, 225] . the biotin sprayer binds the boxb motif through its λ-n domain and labels proteins proximal to its bound rna [225] . rapid-ms was successfully used to identify rbps that bind zikv utr sequences. interestingly, the identified rbps show enriched expression in neural tissue, which is in line with the neurotropic nature of zikv [228] . rapid pointed to a known rna-binding protein, qki, that is highly expressed in neural progenitor cells (npcs) and whose depletion reduces zikv rna levels by 90 % [228] . importantly, because these rna-centric methods rely on rna sequence specificity they can be transposed and applied to identify rbps associated with virtually any rna virus. after these proof-of-principle studies, all of these novel rna-centric approaches are expected to yield important fundamental knowledge about vrna interactions with host rbps, if applied to emerging or re-emerging rna viruses with epidemic potential such as ebola or sars-cov2 [229, 230] . rna-protein interactions are central to most cellular processes. it is thus not surprising that rna viruses and cellular genes evolved intricate vrna-rbp interactions that determine many aspects of the viral life cycle [4] . from the host cell perspective, the affinity of its rbps to the invading vrna, could be a double-edged sword. many cellular genes have evolved to specifically recognize and/or target vrnas and alert the immune system to mobilize cellular defenses. this is illustrated by the relative conservation of many vrna sensors and anti-vrna effectors across species, such as components of the rnai pathway or vrna sensors like rlrs and tlrs [12] . likewise, many viral genes and rna structures have specifically evolved to co-opt fundamental host cell machineries, without which the virus in unable to replicate. this is exemplified by the convergent evolution of many rna elements in the viral genomes, with roles in hijacking ribosomes and the cellular machinery in general [133] . in fact, structured rna elements, such as iress are shared amongst viruses from very different families, ranging from plant to animal viruses [133, 148, 231] . also, viral enzymes that can interfere with endogenous mrna lifecycles favoring vrnas translation, are also found across a wide range of viral families [119, 156] . during the last decades, since the rise of modern molecular biology we are witnessing a remarkable and accelerated development of tools and technologies that are enabling to delve deeper into fundamental cellular mechanisms. as mentioned in this review, each time a technical innovation emerged, our understanding of vrna-rbp interactions expanded. for example, proteins identified by the novel aforementioned rna-centric methods (e.g. chirp-ms, vir-clasp, rna-ic) are highly enriched with classical rbps as expected. however, hundreds of "unorthodox" rbps have also been found to bind vrnas [219, 222] . some of those unorthodox' rbps have already known cellular functions and weren't expected to have an influence on viral lifecycles by binding vrnas. for example, cellular transport motors like dyneins have been shown to have an rna binding activity that might be responsible of rnas trafficking, uncoating, reverse transcription and in some cases viral replication factory assembly [232] . another eloquent example comes from the interaction of er-resident multimolecular complexes, such as sec61 translocon complex or the oligosaccharyltransferase (ost) complex, with the lifecycle of flaviviruses [3, 233] . although these complexes have known and established roles in protein translocation and glycosylation in the er, new unexpected rna-related functions of these complexes are just emerging [234] . for example, flavivirus vrnas that are translated and replicate in close proximity to er membranes, seem to require "non-canonical" functions of the ost complex [3] . this might involve tethering viral rna genomes to the er for efficient translation and replication [176, 207, 232, 234] . future work will clarify which unconventional rbps are truly important for the viral infectious cycle. in the meantime, these systemwide approaches are uncovering new host rbps with unexpected new roles during viral lifecycles. understanding mechanistically the relevance of these newly discovered interactions is crucial in the future. alongside careful and rigorous biochemistry, this can be tackled with new complimentary cutting-edge methods (e.g. clip-seq, ribosome profiling) [204, 206, 210, 222] to determine the footprints of rbps on viral and host rnas. other new approaches to visualize, track, target or detect rnas (e.g. crispr-cas13), if applied to vrnas, will clarify the role of the newly discovered host rbp-vrna interactions [235] [236] [237] . finally, there are many reasons to look at the future of the field with optimism. indeed, continuing to dissect the intimate relationship between vrnas and host rbps with new tools, is expected to both expand our fundamental understanding of virology and cell biology, and to guide the development of much needed novel antiviral approaches. the authors report no declarations of interest. host factors in positive-strand rna virus genome replication human host factors required for influenza virus replication genetic dissection of flaviviridae host factors through genome-scale crispr screens diverse roles of host rna binding proteins in rna virus replication crispr-cas immunity in prokaryotes antiviral 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antiviral immunity in human neural progenitors and brain organoids the authors would like to thank dr. alex g. johnson for his thorough and critical reading of the manuscript. the authors apologize to colleagues whose work could not be cited due to space limitations. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.semcdb.2020.08.006. key: cord-342756-rgm9ffpk authors: senger, mario roberto; evangelista, tereza cristina santos; dantas, rafael ferreira; santana, marcos vinicius da silva; gonçalves, luiz carlos saramago; de souza neto, lauro ribeiro; ferreira, sabrina baptista; silva-junior, floriano paes title: covid-19: molecular targets, drug repurposing and new avenues for drug discovery date: 2020-10-02 journal: mem inst oswaldo cruz doi: 10.1590/0074-02760200254 sha: doc_id: 342756 cord_uid: rgm9ffpk coronavirus disease 2019 (covid-19) caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a highly contagious infection that may break the healthcare system of several countries. here, we aimed at presenting a critical view of ongoing drug repurposing efforts for covid-19 as well as discussing opportunities for development of new treatments based on current knowledge of the mechanism of infection and potential targets within. finally, we also discuss patent protection issues, cost effectiveness and scalability of synthetic routes for some of the most studied repurposing candidates since these are key aspects to meet global demand for covid-19 treatment. at the moment, the prevention aimed at reducing transmission in the community is the best alternative, indicating that enhanced public health interventions, including social distancing, use of masks and movement restrictions should be implemented to bring the covid-19 pandemic under control. aggressive isolation measures including travel restriction in china, have successfully led to a progressive reduction of covid-19 cases. (8) kissler and colleagues, simulated the relaxation of the protective measures using a post-pandemic mathematical model. (9) the authors demonstrated that social isolation will be necessary at the best scenario until 2021, and reaffirmed the need and importance of maintaining social isolation. they also suggested that in the absence of such restrictions, the pandemic could last until 2024. thus, in this context, the search for new medicines available to combat this disease is urgent. coronaviruses are members of the family coronaviridae that present crown-like spikes on their surface visualised by electron microscopy. the subfamily coronavirinae contains the four genera alpha, beta, gamma, and deltacoronavirus. coronaviruses infect birds (gamma and deltacoronaviruses) and several mammalian species (mainly alpha and betacoronaviruses), including humans. (10, 11) coronaviruses have been isolated from diverse species, including mammals like bats, rodents, bovines, swine, felines, pangolins, horses and others. (12, 13) human coronavirus was first identified in 1966 by tyrrell and bynoe. (14) nowadays the seven coronaviruses that can infect humans (hcovs) are classified in alpha coronavirus (229e, nl63) or beta coronavirus [oc43, hku1, middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov) and more recently the sars-cov-2]. 229e, nl63, oc43, and hku1 can cause upper and lower respiratory tract infection in adults and children. after the 2000s, two epidemic covs have arisen in humans: the sars-cov and the mers-cov which were reported in 2003 and 2012, respectively. (15, 16) in 2019, after the first report of novel pneumonia in wuhan, china, the seventh hcov was described. it also causes severe acute respiratory syndrome (therefore called sars-cov-2) and spreads very quickly worldwide . coronavirus are single-stranded positive-sense rna viruses and their genome size is approximately 30 kb, which encodes some important structural proteins. (17) the spike (s) glycoprotein is a well characterised protein that mediates coronavirus entry into host cells via fusion of the viral and cellular membranes through a pre to post fusion conformation transition. (18) the s protein s1-s2 subunits bind to cellular receptors that vary according to the coronavirus species: angiotensin-converting enzyme 2 (ace2) in sars-cov, sars-cov-2 and hcov-nl63; and dipeptidyl peptidase 4 (dpp4) and aminopeptidase n (apn) in mers or others alphacoronaviruses like tgev (porcine transmissible gastroenteritis coronavirus) and porcine respiratory coronavirus (prch). (18, 19, 20) other structural proteins are mandatory to assemble the complete viral particle like nucleocapsid protein (n), membrane protein (m) and the envelope protein (e). furthermore, they can be involved in other processes like morphogenesis, envelope formation, budding or pathogenesis. (17, 21, 22) by genomic sequencing analysis of other coronavirus strains and sars-cov-2, andersen and collaborators demonstrated that sars-cov-2 has mutations resulting in six different amino acids at the receptor-binding domain (rbd) that appears to be optimised for binding to the human receptor ace2. (23) they also showed that the gene encoding the spike protein has an insertion of 12 nucleotides giving it a polybasic (furin) cleavage site at the s1-s2. in this way, the high-affinity of the sars-cov-2 spike protein to the human ace2 is a consequence of natural selection on a human or human-like ace2. they suggest some possibilities to explain that: emergence in an animal host before zoonotic transfer; natural selection in humans following zoonotic transfer; or natural selection during the passage. (23) other researchers did a phylogenetic analysis of 160 genomes of sars-cov-2. (24) they showed 3 important variations in the composition of amino acids that allowed them to classify into different groups. group a has two subclusters that are distinguished by the synonymous mutation t29095c. while b is derived from a by two mutations t8782c and c28144t, type c differs from its parent type b by mutation of g26144t. the a and c types are found more often outside east asia, in europeans and americans. while the b type is the most common type in east asia. (24) in december 2019, covid-19 was initially reported as a new viral pneumonia, due to the clinical characteristics of the large number of cases that emerged in wuhan, china. (25, 26) sars-cov-2 typically causes respiratory sickness, the major clinical characteristics ob-served in infected patients are high fever, dry cough and dyspnea (shortness of breath or difficulty in breathing). minor symptoms include headache, diarrhea, nausea, vomiting, loss of smell and taste. (27) this clinical condition can progress to moderate or severe pneumonia. (28) in this case, first there is an accumulation of macrophages in alveoli, followed by release of cytokines and accumulation of fluids. neutrophils can also be recruited by the immune system leading to the destruction of type i and type ii alveolar epithelial cells causing a collapse of the alveoli function and consequently the acute respiratory distress syndrome (ards). in the severe condition, with an increase of the inflammation, the protein rich fluid from lungs enter in the bloodstream causing the systemic inflammatory syndrome (sirs). (29, 30) these complicating factors can lead to a multi-organ failure and septic shock, causing patient death. furthermore, some pre-existing conditions can enhance the risk to develop the severe form of the disease, including age over 60 years and a history of chronic diseases like chronic lung disease, asthma, heart diseases, immunosuppressed patients, cancer, diabetes or chronic kidney disease. (31, 32, 33, 34, 35) finally, the vast majority of people have mild symptoms or are asymptomatic, which is a big problem because they can also transmit the virus to the non-infected population. (36) drug repositioning, repurposing, reprofiling or retasking is the evaluation of existing drugs for new therapeutic purposes. (37) a candidate drug (investigational or approved) for repurposing efforts already has a known safety and toxicity profile, based on at least successful phase i or phase ii clinical trials. (38) considering the whole process, costs of bringing a repurposed drug to the market have been estimated to be ten times lower and the time is shortened by around a half, compared with a new drug. (38) even though the clinical phase iii and regulatory aspects remain similar for developing a new drug, drug repurposing possesses many advantages over developing a new drug from scratch: the reduced time and financial investment for development, the lower risk of failure and a consolidated pharmaceutical supply chain for production and distribution to the patients that effectively need treatment. (39) emerging or reemerging viruses pose major public health concerns globally. (40) for several pathogenic viruses, considerable efforts have focused on vaccine development and other therapies, like transfusion of convalescent plasma. (41, 42) however, during pandemics infected individuals need urgently to be treated on a large scale. a medicine armamentarium for the covid-19 outbreak is needed immediately and drug repurposing could be one of the best strategies to deal with this pandemic. (43, 44) computational and experimental approaches can be used, alone or combined, to achieve a more holistic point of view and increased chance of success in drug repurposing. in the following topic, we will review sars-cov-2 structure and mechanism of infection in order to discuss molecular targets from the virus or its human host that are being considered for drug repurposing and perhaps future development of new drugs. ongoing drug repurposing efforts will be described in more details later in this article, along with some clinical trials that have been carried out so far for covid-19 treatment. finally, as treatment availability is of utmost importance when dealing with a pandemic, we bring a discussion on patent protection and ease of large-scale production of some of the drugs that are more advanced in clinical studies. sars-cov-2: structure, mechanism of infection and drug targets sars-cov-2 structure -electron microscopy imaging of sars-cov-2 virions indicates that they have a spherical or pleomorphic shape, with diameters ranging from 60 to 140 nm, showing prominent spikes of 9-12 nm in their surfaces that resemble a solar corona, hence the name "coronavirus". (45, 46, 47, 48) sars-cov-2 is an enveloped virus with a single-stranded positive sense (5'-3') rna (+ssrna) (~ 30 kb) containing a 5'cap structure and a 3'-poly-a tail. (49, 50) its genomic rna (grna) has a variable number of open reading frames (orfs) that are predicted to encode 16 non-structural (nsp), 4 structural and several accessory proteins ( fig. 1 ). (26, 51, 52, 53, 54) orf1a and orf1b represent more than 2/3 of the whole length of grna, and encode two polyproteins: pp1a (440-500 kda) and pp1ab (740-810 kda). (53, 55) the polyprotein pp1a is translated from orf1a while pp1ab from orf1a/orf1b using a -1 ribosomal frameshift mechanism that occurs near the 3' end of orf1a which allows continued translation of orf1b. (53) together, pp1a and pp1ab originate all nsps (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) , such as m pro (nsp5) protease and rdrp (nsp12) rna polymerase, which form viral replicase/transcriptase complexes (rtcs), and are encapsulated in double-layered vesicles originated from the endoplasmic reticulum (er). (56, 57, 58) the orfs near the 3' end of the grna encode the structural and accessory proteins of sars-covs. (58) the first ones have a crucial role in the assembly of viral particles and virus invasion. (56, 58) the main structural proteins are named: spike (s), envelope (e), nucleocapsid (n) and membrane (m) proteins. most of them reside on the virion surface (s, e, m proteins) while n proteins are found in the core of the particle bound to grna. (59) s proteins are essential for virus attachment and entry into the host cells, tissue tropism and pathogenesis. (58, 60) e proteins exert several roles in virus infection, such as helping in virus assembly and release from infected cells, creating ion channels in cell membranes and suppressing host stress response. (58, 61, 62) n proteins interact with grna to form the ribonucleoprotein. (56, 62) m proteins have a role in virion assembly and in determining the shape of the envelope. they also bind to all other structural proteins promoting, for instance, the stabilisation of n protein-rna complexes. (56, 63) sars-cov-2 mechanism of infection -at present, the mechanisms that underlie sars-cov-2 infection have not been directly described. nonetheless, they seem to be similar to those proposed for other coronaviruses. (58) in one proposal, virus infection starts with the binding of its s proteins to host receptor ace2, a membrane protein largely expressed in the lung and small intestine cells (fig. 2 ). (44, 59, 64) after attachment, s protein is cleaved by host proteases initiating the fusion of virus and cell membranes that culminates in viral grna release into the cytoplasm. this event is proposed to occur through two distinct ways: via plasma membrane (early pathway) or via endosomes (late pathway). in the early pathway, s protein is cleaved by host plasma membrane proteases (e. g., tmprss2) while in the late pathway by endosomal proteases (e. g., cathepsin l). the route taken by the virus to enter the cell appears to be dependent on the availability of these proteases. (59, 64, 65) once in the cytoplasm, grna is readily translated into viral polyproteins (pp1a/pp1ab), which are cleaved into the individual nsps that compose the rtcs (fig. 2) . these complexes recognise transcriptional regulator sequences in grna and begin to transcribe a series of subgenomic rnas that encode structural and accessory proteins, otherwise the whole grna is replicated. (57, 58, 59) upon translation, s, e and m structural proteins are driven to the er-golgi intermediate compartment (ergic) where s proteins go through post-translational modifications (e. g., proteolysis, n-glycosylation). in parallel, a copy of the grna and n proteins bind in the cytoplasm to form the nucleocapsid and move into the ergic. in this compartment, nucleocapsid and the other (1) virus infection initiates with the binding of virus s proteins to the ace2 cellular receptors. after attachment, the virus may enter the cell through two distinct mechanisms: early and late pathways. (2) in the early pathway the genomic ssrna (grna) is liberated into the cytoplasm after the fusion between viral and cell cytoplasmic membranes, an event triggered by membrane proteases (e. g., tmprss2). (3) the grna is immediately translated into two polyproteins that undergo proteolytic cleavage giving rise to all nonstructural proteins (nsps). (4) the nsps form the replication-transcription complexes (rtcs) where the grna (blue ribbon) is replicated and the subgenomic rnas (red ribbon) are transcribed. (5) the subgenomic rnas are translated into viral structural and accessory proteins in the cytosol. (6) upon translation, e, m and s structural proteins are inserted into er and follow the secretory pathway to the er-golgi intermediate compartment (ergic). (7) meanwhile, a copy of the grna binds to n proteins in the cytoplasm forming the nucleocapsid, which is transported to the ergic. (8) virion assembly in the ergic. (9) the new virion travels through the cytoplasm inside a vesicle and leaves the cell by exocytosis. (10) alternatively, in the late pathway, the virus can undergo endocytosis to initiate the infection. (11) the virion membrane merges with the endosome membrane after s protein proteolysis by endosomal proteases (e. g., cathepsin l), allowing the grna to be released into the cytoplasm. from this point on, the cycle follows the same pathway described in 3-9 steps. the red arrows indicate the general replication pathway and the blue arrows indicate the grna movement through the cycle. some viral and host proteins have been explored as potential targets for drug repurposing. some of these drug candidates are shown with their site of action indicated in the cycle. (44, 59, 66, 67) viral proteins are assembled into a virion which travels through the cytoplasm inside a vesicle and leaves the cell by exocytosis. (48, 56, 59) candidate drug targets -in the search for a treatment for covid-19, several viral and host molecular proteins have been explored as potential drug targets. overall, they participate in key events of the virus infection cycle, such as cell entry and replication, as well in host metabolic pathways and immune response. in the following topics we will address in more details some of these targets. drugs under investigation for blocking the main steps of the sars-cov-2 virus replication cycle are indicated in fig. 2 . virus targets -during sars-cov-2 grna translation, two proteases, namely m pro and pl pro , act in concert to cleave and release from pp1a/pp1ab the 16 nsps that compose the rtc. (58) therefore, these proteases are essential for virus replication and represent useful targets for therapeutic intervention. recently, sars-cov-2 m pro and pl pro had their 3d structures published -pdb 6lu7, 2.16 å and pdb 6w9c, 2.70 å, respectivelywhich make them particularly useful for computational structure-based drug design methods. (68) 3-chymotrypsin-like protein (synonyms: coronavirus main protease, m pro , 3cl pro ) of sars-cov-2 is a 33.8 kda homodimeric protein (306 aa) that belongs to the cysteine protease class (possibly from c30 family and pa clan). (68, 69) this enzyme catalyses the hydrolysis of peptide bonds of polyproteins (possibly e.c. 3.4.22.69) at sites whose amino acid sequences generally follow the pattern leu-gln* (ser, ala, gly) (*marks the cleavage site). (70, 71) m pro is part of pp1a/pp1ab polyproteins (nsp5). (57) during polyproteins translation, m pro suffers autolyt-ic cleavage and is released from its polyprotein precursor, reaching a mature state that cleaves pp1a/pp1ab at no less than 11 sites downstream of the nsp4 coding region. (57, 68, 70, 72) each protomer of sars-cov-2 m pro is divided into three domains: chymotrypsin and picornavirus 3c protease-like i and ii domains, composed by antiparallel β-barrel structures, and domain iii which contains five α-helices arranged into an antiparallel globular cluster responsible for protease dimerisation (fig. 3a) . domains ii and iii are connected by a long loop, where lies a cleft that serves as a substrate binding site and where catalysis occurs using the cys 145 -his 41 dyad (fig. 3b ). (68, 70, 73) m pro has been widely explored in drug discovery campaigns using experimental and/or computational approaches. (68, 73, 74, 75, 76, 77) moreover, m pro has no human homologue, which reduces the chances of toxic effects of a given inhibitor. (68) potential sars-cov-2 m pro inhibitors include fda-approved antivirals, such as inhibitors of hiv-1 [e. g., lopinavir (1) /ritonavir (2) ] and hcv [e.g., boceprevir (3)] proteases, as well as antineoplastic [e.g. carmofur (4)] and antibacterial [e.g., doxycycline (5)] drugs. (73, 78, 79, 80, 81, 82) chemical structures for compounds 1-14 are shown in fig. 4 . nsp3 is the largest protein encoded by covs (~200 kda). in sars-covs it has 16 domains which include a papain-like proteolytic enzyme from the cysteine protease class (c16 family and ca clan for sars-cov). (57, 69, 83) pl pro is composed of a catalytic domain, an extended right-handed thumb-palm-finger structure with a cys-his-asp catalytic triad, and a ubiquitin-like domain (ubl) (fig. 5a ). the catalytic cys is located in the thumb subdomain while his and asp in the palm subdomain (fig. 5b ). pl pro catalyses the hydrolysis reaction of peptide bonds of pp1a/pp1ab at three sites (nsp1/nsp2, nsp2/nsp3 and nsp3/nsp4) that share the xlxgg* pattern (*represents the cleavage site). (57, 71, 83, 84) this activity is responsible for nsp3 release from polyproteins. pl pro also recognises and hydrolyses ubiquitin and isg15 from cellular proteins. (83, 84) the deubiquitination and deisgylation activities are proposed to modulate the post-translational modifications of signaling molecules that trigger innate immune response of the host. (85) these functions are pivotal for virus infection justifying the search for sars-covs pl pro inhibitors. (84, 86, 87, 88, 89, 90) putative inhibitors of sars-cov-2 pl pro include fda-approved drugs such as fostamatinib disodium (6) (a tyrosine kinase inhibitor used in the treatment of chronic immune thrombocytopenia) and natural products [e.g., platycodin d (7)]. (89, 91) rna-dependent rna polymerase (rdrp, nsp12) is an essential protein responsible for rna synthesis during viral rna transcription and replication cycles. (89) as described for sars-cov, the rna polymerase activity of sar-cov-2 rdrp (804 aa) also seems to require the binding of nsp7 and nsp8 cofactors to enhance rdrp binding and processivity. (92, 93) the overall rdrp structure has a "right hand" rdrp domain, composed by three subdomains (palm, fingers and thumb), and a nidovirus-unique n-terminal extension domain that forms a nidovirus rdrp-associated nucleotidyltransferase (niran) structure. these domains are connected by an interface domain. additionally, this protein also has a n-terminal β-hairpin (fig. 6a) . the active site of rdrp contains conserved polymerase motifs located in the palm subdomain and its configuration is similar to other rna polymerases. (94) rdrp substrates, rna template/primer and nucleotide triphosphate (ntps), access the catalytic centre through the template and ntp entry paths, respectively, while the product-template hybrid is released through the rna exit path. (92, 94) sars-cov-2 rdrp shares 96% identity in amino acid sequence with sars-cov protein. (93) moreover, the accessory proteins also have a high degree of amino acid sequence identity between these viruses: 98.1 % for nsp7 and 97.5 % for nsp8 -sequences obtained from pdb 7bv2 (2.5 å) and pdb 6nur (3.1 å) entries. (92, 95) therefore, it is reasonable to suggest that sars-cov rdrp inhibitors may also bind to the homologous enzyme from sars-cov-2, as demonstrated for remdesivir (8) (fig. 6b) , an adenosine triphosphate analog. (92, 93, 96) other potential inhibitors include clinically available drugs, such as favipiravir (9), a purine nucleic acid analog used in the treatment of influenza, and ribavirin (10), a synthetic guanosine nucleoside indicated for the treatment of hepatitis c virus (hcv) infection. (97, 98) the crucial role of rdrp and the lack of a host homolog turns this enzyme into a valuable target for anti-covs agents. (93) helicase (nsp13) is a multifunctional protein (predicted to have 596 aa in sars-cov-2) essential for sars-covs rna replication and proliferation. (74, 99) sars-cov helicase belongs to helicase superfamily 1 (sf1) and can unwind double-stranded dnas/rnas (helicase activity) in the 5'-3' direction, an energyconsuming process that is driven by the hydrolysis of nucleosides triphosphate (ntpase activity). (57, 100, 101) its structure contains five domains arranged in a triangular pyramid-shape: the triangular base is composed by two "reca-like" (1a and 2a) and 1b domains whereas the zinc binding domain (zbd), in the n-terminal, and the stalk domain are oriented towards the apex. the zbd and 1b domains are connected by the stalk domain ( fig. 7) . (101) the same domain arrangement is predicted to occur in sars-cov-2 enzyme. (74) sars-cov helicase has been explored as a potential target for drugs. (99, 100, 102) recently, some efforts have also been made to predict inhibitors for sars-cov-2 helicase. (74, 103, 104, 105) they include drugs used in the clinics to treat acquired immunodeficiency syndrome (aids), such as vapreotide (11) (somatostatin analog) and atazanavir (12) (hiv protease inhibitor), as well anti-hcv protease inhibitors [e.g., daclatasvir (13) ] and bismuth salts [e.g., bismuth potassium citrate (14) , a compound used in clinical treatment of gastrointestinal diseases]. (103, 104, 106) sars-covs replication also requires other enzymatic activities including (guanine-n7)-methyltransferase (n7-mtase), 2'-o-methyltransferase (2'-omtase) and exoribonuclease (exon). (57, 107, 108) the n7-mtase domain at the c-terminus of nsp14 is responsible for adding a methyl group in the n7 position of rna 5'-guanosine forming the 5'-cap structure of viral rnas. (108) additionally, nsp14 also has a catalytic domain in its nterminus with 3'-5' exon activity, which participates in rna proofreading mechanism (fig. 8 ). (57) 2'-omtase (nsp16) catalyses the methylation reaction at the ribose 2'-o position of the first and second nucleotide of the mrnas and it is one of the sars-cov-2 proteins whose 3d structure has already been resolved (pdb: 6w61, 2.0 å) (fig. 9 ). (109) both nsp14 and 16 use nsp10 as a cofactor to enhance their 3'-5' exon and 2'-omtase activities, respectively. (110) together, these proteins are responsible for inducing rna modifications that are essential for its stability and translation as well to avoid the activation of host immune response. (57, 107, 109) thus, nsp14 and 16 have been considered as potential targets for anti-sars agents that may affect their functions in a direct or indirect way (e.g., by blocking nsp 10 binding). (108,109,110,111,112 ,113,114,115) some compounds have been regarded as potential inhibitors of sars-cov-2 methyl transferases, such as adenine dinucleoside s-adenosylmethionine analogs [e.g., dinucleoside 13 (15) ], predicted for sars-cov n7-mtase activity, and some clinically available drugs [e.g., raltegravir (16) -a hiv integrase inhibitor], predicted for sars-cov-2 2'-omtase activity. (112, 115) chemical structures for compounds 15-29 are shown in fig. 10 . spike protein (s) is a viral type i transmembrane glycoprotein (~180-200 kda) responsible for covs interaction and invasion of host cells. (59, 64) this protein is synthesised as a monomer and suffers post-translational modifications in the er before becoming a trimeric glycosylated protein. (59, 116) its structure is divided into three main domains: an extracellular, a transmembrane and a short intracellular domain (fig. 11 ). (117) the former protrudes from the surface of the virus particle creating a crown-like halo and contains two functional subunits: s1 (bulbous shape), which binds to cellular receptors (e.g., ace2), and s2 (stalk shape) that promotes the fusion between cell and virus membranes. (59) in turn, s1 subunit has two domains: a n-terminal and a c-terminal domain. the latter serves as a rbd for sars-covs being responsible for ace2 recognition and binding. (59, 64, 117) apart from s1 and s2, sars-cov-2 s protein also has a ganglioside-binding subdomain at the tip of n-terminal domain, which may allow this protein to interact with gangliosides on cells' surface. in theory, this domain could facilitate virus attachment to the cell and facilitate the contact with ace2, being a potential site for drug interference. (118) fig. 9: ribbon representation and vdw surface of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) 2'-o-methyltransferase 3d structure (pdb 6w61, 2.0 å, nsp16, pink) in complex with its nsp10 cofactor (light blue). the zinc and chloride ions are represented as gray and green spheres, respectively. image created using maestro, release 2020-1 (maestro, schrödinger, llc, new york, ny, 2020). covs s proteins require proteolytic priming or cleavage to become fusion competent. (59) this is achieved by host proteases (e.g., tmprss2, cathepsin l, furins) which cleave the s2 subunit of s protein at two main positions: s1/s2 interface and s2'. the latter is located immediately upstream of the fusion peptide (fp), the fusion functional element of s2. contrary to sars-cov, sars-cov-2 s2 subunit also has an additional cleavage site for furin proteases in the s1/s2 region, something that also occurs in mers-cov. (60) it is still unclear its role in sars-cov-2 infection, but it is speculated that the ubiquitous expression of furins may increase cell and tissue tropism of sars-cov-2 in comparison to sars-cov, as well altering its transmissibility and pathogenicity. (60, 117) taken together, host proteases represent potential targets for anti-sars-cov-2 drugs, and thus some of them will be discussed in the following topics. (60, 64, 119) covs e proteins are small integral membrane polypeptides (76 -109 aa) encoded by subgenomic rnas. in sars-cov, they are found in virions, but also in large amounts in the ergic where they participate in virus budding and trafficking. their structures are divided into three main domains: n-terminal, transmembrane (tmd) and c-terminal domain. tmd is able to form pentameric α-helical bundles creating ion conductive pores in membranes. (120, 121, 122) the ion channel (ic) activity of e protein has been proposed to alter ion homeostasis, as well induce inflammatory response, which may lead to pulmonary damage. (123, 124) thus, the use of inhibitors of ic activity may represent a possible therapeutic strategy for covs-related diseases, including covid-19. ( this can be exemplified by the fda-approved drugs gliclazide (17), a sulfonylurea administered to non-insulin-dependent diabetes mellitus patients, and memantine (18) , an n-methyl-d-aspartate receptor antagonist used in the management of alzheimer's disease, which have shown ic inhibitory activity in bacteria expressing sars-cov-2 e proteins. (126) host cell targets -a key step in sars-covs infection is the attachment of s protein to host angiotensinconverting enzyme 2 (ace2), a membrane-bound carboxypeptidase (family: m2, clan: ma). (25, 65, 69) this enzyme catalyses the hydrolysis of angiotensin i and angiotensin ii into angiotensin-(1-9) and angiotensin-(1-7) peptides, respectively. (127) ace2 is expressed in the upper respiratory system, type i and ii alveolar epithelial cells in the lungs, heart, endothelial cells, kidney tubular epithelium and other tissues. (128) its role as a functional receptor of sars-cov-2 s protein in host cells makes this protein a potential drug target to treat covid-19. there are several therapeutic strategies targeting ace2 which include: vaccines based on s protein, exogenous administration of a soluble form of ace2, and administration of small molecules [e.g., arbidol (19) , an anti-influenza drug] or antibodies (e.g., sti-1499, an anti-spike antibody) to block the interaction of ace2 with s protein. (66, 129, 130) additionally, compounds could also have an anti-sars-covs activity by disturbing ace2 glycosylation, as proposed for chloroquine (20) . (131) renin-angiotensin-aldosterone system (raas) inhibitors, such as ace inhibitors [ace-i, e.g., captopril (21) ] and angiotensin receptor blockers [arb, e.g., losartan (22) ], are commonly used in clinics to treat hypertension and cardiovascular/renal diseases. (132, 133) recently, some researchers have debated over the administration of these drugs to patients with known or suspected covid-19. (133, 134, 135, 136) the main reason for this discussion is that raas inhibitors may induce ace2 expression, which, in theory, could increase the severity of the infection. (133, 136) nonetheless, evidence suggests that these drugs may protect patients from lung injury by suppressing angiotensin ii signaling mediated by angiotensin receptor 1 (at1r). (135, 136, 137) further investigation is still required to determine whether ace-i and arb have a beneficial or deleterious role in covid-19 treatment. (134) in order to become fusion-competent, sar-covs s proteins must be cleaved by host proteases. in sars-cov-2, this process appears to be mediated primarily by tmprss2 in the plasma membrane. (65) tmprss2 (transmembrane protease, serine 2) is a transmembrane protein (predicted to have 492 aa) from the serine protease class (family: s1 clan: pa). (69, 138) it is highly expressed in the epithelial cells of the prostate, and relatively less in lungs, colon, liver, kidney, and pancreas. its physiological function is still unclear. (138) tmprss2 has a major role in sars-cov-2 cell entry and replication, and thus represents an interesting therapeutic target since its inhibitors could potentially block virus infection in its initial stages. (65, 138) potential tmprss2 blockers include some serine protease inhibitors [e.g., camostat mesylate (23)], commercially available compounds [e. g., zinc64606047 (24), 3-[[5-(3-fluorophenyl)pyrimidin-2-yl]amino~(n)-(4-methyl phenyl)benzamide]] and natural products [e.g., withanone (25) ]. (65, 139, 140, 141) in the absence of exogenous or membrane-bound proteases (e g. tmprss2) to promote sars-covs infection on cells' surface, the virus can also be internalised via endocytosis, also known as "late pathway". (59) this process comprises several factors that operate in a sequential and partially overlapping fashion. (142) endocytosis initiates with the recruitment of endocytic coat proteins (e. g., clathrins) from the cytosol to gather on the inner leaflet of the plasma membrane. then, protein-coated pits bud into vesicles originated from plasma membranes and fuse with each other or with pre-existing early endosomes. (142, 143) gradually, early endosomes mature to late endosomes which have an acid environment (approx. ph 5.5), activating endosomal cathepsin l cysteine proteases (family: c1, clan: ca). (59, 69, 143) these enzymes are responsible for triggering the fusion activity of s protein and subsequent insertion of viral sars-cov grna into the cytosol, especially in cells with lower expression of tmprss2. (64, 65) some key elements of endocytosis have been explored as potential targets for anti-sars-cov drugs, such as agents that increase endosomal ph (avoiding cathepsin l activation) [e.g., chloroquine (20) and hydroxychloroquine (26) ] and cathepsin l inhibitors [e.g., teicoplanin (27) , a glycopeptide antibiotic]. (49, 131, 144) cytokines are short-lived small size proteins (15-20 kda) that exert an important role in autocrine, paracrine, and endocrine signaling controlling the development and function of several immune and nonimmune cells. (145, 146) they are classified in families based on certain properties of their receptor complexes, such as their structure, specificity and composition. (145) one example is interleukin (il)-6 family which includes several cytokines, such as il-6, that use the common signaling receptor subunit glycoprotein 130 kda (gp130). il-6 signaling requires the binding of il-6 to il-6 receptor (il-6r) which consists of a soluble il-6 binding domain (cd126) and membrane-bound gp130. il-6 acts as the main inducer of fever, inflammation and of hepatic acute phase proteins. therefore, il-6/il-6r antagonists have a therapeutic effect in inflammatory diseases. (145, 146) their use in covid-19 treatment may be beneficial to avoid the "cytokines storm" syndrome that some patients with the severe form of the disease may develop. (147) this deleterious event is caused by an amplified immune response and cytokine release that can damage the organs, including the lungs. (66) tocilizumab and sarilumab are two examples of humanised monoclonal antibodies that act as il-6r antagonists which are currently under clinical trials for covid-19 treatment. (148) other targets -apart from sars-covs infection, some relevant molecular targets of other viral diseases and host metabolism have also been investigated in covid-19 drug discovery, such as viral neuraminidases and the dpp4 cell receptor. neuraminidases (na) or sialidases, are glycoside hydrolases (family: gh34) largely found attached to the envelope of influenza viruses. (149, 150) they catalyse the hydrolysis of glycosidic bonds between sialic acid and adjacent sugar residues (ec 3.2.1.18) in glycoproteins, glycolipids and oligosaccharides. (149) na is mainly responsible for cleaving sialic acid acids from cell receptors and on carbohydrate side chains of nascent virion, facilitating its release from infected cells. (150) its critical role in virus infection and proliferation has been exploited by na inhibitors [e.g., oseltamivir (28) ] which are administered in clinics to combat influenza infections. apparently, this therapeutic strategy was employed at the beginning of co-vid-19 outbreak during the peak of influenza season in china when the etiologic agent of this disease was yet unknown. so far, there is no evidence that na inhibitors may have a role in covid-19 management. (66) dipeptidyl peptidase-4 (ddp4), also known as adenosine deaminase complexing protein 2 or t-cell activation antigen cd26, is a type ii transmembrane glycoprotein of 766 aa largely expressed in many tissues (e.g. lungs and immune cells). (151, 152) ddp4 belongs to serine protease group (family: s9, clan: sc) and catalyses the hydrolysis of n-terminal dipeptide, xaa-yaa-|-zaa-, (preferentially when yaa is a proline, as long as zaa is neither proline or hydroxyproline), from a variety of peptide substrates (ec 3.4.14.5), such as chemokines, neuropeptides, vasoactive peptides and growth factors. (69, 71, 152) therefore, it participates in many physiologic and pathological processes, including glucose/insulin metabolism and immune/inflammatory response. (153) in addition, dpp4 acts as the functional cell receptor of mers-cov s protein, helping virus entry into the cell. theoretically, this role may also be played in sars-cov-2 infection, as predicted by computational analysis. (154) recently, there has been a discussion about the use of ddp4 inhibitors, such as sitagliptin (29) (anti-diabetic drug), in the treatment of covid-19. (152, 153, 155, 156) in principle, these inhibitors could be beneficial for type 2 diabetes patients (or even without diabetes) with covid-19 since it could potentially block virus cell entry/replication, as well suppress inflammatory response. (152, 155) however, there is not enough evidence yet to prove this hypothesis. (152, 153, 156) computational approaches -the sars-cov-2 pandemic is creating a fertile ground for computational approaches to identify viable therapeutic options in the short-term, with the number of published studies growing rapidly. (157, 158) classical target-and ligand-based strategies (e.g., molecular docking and similarity analysis), are being applied to fda-approved drugs and compounds in clinical trials to explore possible interactions with viral proteins. (75, 159, 160) artificial intelligence (ai) methods are also being extensively applied to large molecule databases to find existing drugs that could be repurposed based on previously reported antiviral activities. (103, 161, 162, 163) ai approaches can even explore hidden connections between drugs, human and viral targets to find novel bioactivities and even combinations of drugs. (52, 161, 164) these studies emphasize how important computational methods are in modern drug discovery to analyse the huge amount of biomedical data available. table i summarises a selection of studies that suggested potential drugs for repurposing. below we will describe the main computational methods and results. smith and smith performed a large virtual high-throughput screening to identify drugs, natural products and metabolites that could disrupt the interaction between sars-cov-2 s protein and human ace2 receptor. the authors used a supercomputer called summit to conduct the simulations, which included structural modeling of the s protein:ace2 complex, generation of an ensemble of conformations for the complex and molecular docking. (160) the ensemble generation was a crucial step in this work, allowing the authors to explore different conformations of the complex and identify drugs that could interact with binding sites not easily identified in static conformations. using the gromacs molecular dynamics simulation suite, six different clusters of conformations were generated by restrained temperature replica-exchange molecular dynamics simulation (t-remd) and submitted to docking with autodock vina using the sweet-lead compound collection. (170, 171, 172) the authors explored two strategies, consisting of disrupting the s protein: ace2 complex and preventing its formation by docking on the isolated proteins. from this analysis, four compounds [pemirolast (30) , nitrofurantoin (31), isoniazid pyruvate (32) and eriodictyol (33) ] displayed potential to disrupt the s protein: ace2 interaction, and three natural compounds [cepharanthine (34) , ergoloid (35) and hypericin (36) ] showed potential to block host recognition. (160) chemical structures for compounds 30-49 are shown in fig. 12 . ge and coworkers constructed a virus-related knowledge graph (or network) by combining seven networks with information about target-drug and protein-protein interactions, molecule similarity and sequence similarity of human and viral sources to predict drugs targeting sars-cov-2. (164) well known biological and interaction databases were employed, such as drugbank, chembl, bindingdb, and uniprot . (173, 174, 175, 176) the final knowledge graph was assembled by merging the nodes and edges of the seven networks, where each node represent drugs or targets, while the edges between them are the identified relations, such as similarity (i.e., molecular and sequence similarities) and drug-target or target-target interactions. (164) a graph convolutional network (gcn) was used to learn and extract the hidden information on the nodes and edges of the knowledge-graph, allowing the authors to access novel drug-target and target-target interactions and find molecules that could be repurposed to sars-cov-2. (164) gcn's are powerful neural networks that can access the rich information within the nodes of a graph and return insights about possible relations (e.g., potential drugs and targets interactions), representing a valuable tool in the modern drug discovery scenario, where massive biological data are available in databases such as drugbank and chembl. (177, 178) the final knowledge-graph was then mined to gather an initial list of drugs, which was further refined by extracting previous evidence of antiviral activity from pubmed using a deep learning method called biomedical entity relation extraction (bere) and manual inspection. (179) in a subsequent step, the authors elaborated a final list of drug candidates using transcriptome analysis of ten sars-cov patients. the poly-adp-ribose polymerase 1 (parp1) inhibitor, mefuparib hydrochloride (cvl218) (37) , currently in phase i clinical trials, was identified as a potential drug for repurposing. in vitro and in vivo validation showed promising inhibition and safety profiles. concretely, compared to arbidol (19) , one of the standard treatments for covid-19 in china, cvl218 showed higher antiviral efficacy and higher concentration in lungs of rats. (164) the authors also found that cvl218 significantly inhibited the production of il-6 induced by the cpg oligodeoxynucleotide 1826 (cpg-odn1826) in peripheral blood mononuclear cells (pmbc) indicating it might be an alternative to treat the inflammation caused by sars-cov-2. furthermore, cvl218 showed favorable pharmacokinetics and toxicity profiles in rats and monkey models. (164) this integration of artificial intelligence with wet-lab experiments highlights the importance of in silico methods to mine baricitinib (39) rheumatoid arthritis human ap2-associated protein kinase 1 (aak1) knowledge-graph a pilot trial indicated that baricitinib (39) improved clinical parameters (e.g., cough and dyspnea) in a small group of patients. c (161) toremifene (40) (41) and mercaptopurine (42) ] and angiotensin receptor blockers [irbesartan (43) ]. d (52) a: cepharanthin (34) has been shown to decrease the expression levels of s-protein in mrc-5 cells infected with human coronavirus oc43 (hcov-oc43). (165) flavonoids have been described as inhibitors of m pro and ligands of the s-protein of sars-cov, which could give insights on possible antiviral activities of eriodictyol (33), hypericin (36) and other flavonoids on sars-cov-2. (166, 167) b: a recent study showed that atazanavir (12) inhibited m pro in vitro with greater potency than lopinavir (1). (168) c: despite the small size (12 patients) and open label, non-randomised format, baricitinib (39) showed potential safety when used in combination with lopinavir (1) / ritonavir (2). (169) as large amounts of data from databases and accelerate the discovery of potential drugs for the covid-19 pandemic. beck et al. used a natural language processing (nlp)-based approach to estimate the binding affinity of 3,410 fda-approved drugs against potential targets of sars-cov-2, including 3cl pro , s protein, rdrp, helicase, endornase, 3'-to-5'-exonuclease and 2'-o-ribose methyltransferase. (103) the premise of their approach is analogous to understanding text in different languages, for instance by learning the semantic relationships of words to execute a task, such as predicting the most probable word in a text or the sentiment expressed by it. (180, 181, 182) their model, called molecular transformerdrug target interaction (mt-dti) was trained on 1 billion smiles strings and the fasta sequence of target proteins, which bypass the need for 3d structures (e.g., x-ray) of protein-target complexes. (183) the pre-training approach allows the model to be used for other related tasks without the need to train from scratch, which is especially important when not enough data is available, which is the case for sars-cov-2 inhibitors. in addition, this approach is able to transfer more general features learned by the model, making it extremely flexible to deal with related tasks. (184, 185) therefore, the task consisted of training the model to understand the chemical structure of small compounds and protein targets. the authors found that atazanavir (12), remdesivir (8) and efavirenz (38) are potential inhibitors of m pro , while atazanavir (12) also yielded nanomolar predicted binding affinity for rdrp, helicase, endornase, 3'-to-5'-exonuclease and 2'-o-ribose methyltransferase. (103) it is important to highlight that although these drugs were predicted as nanomolar inhibitors, experimental confirmation is essential to validate the computational analysis. researchers at the uk-based company benevolen-tai (https://www.benevolent.com/) analysed medical data from different sources to create a knowledge graph of biomedical information, showing possible drug-target interactions on its nodes and edges (similar to the previously mentioned approach adopted by ge et al.) to explore new strategies for sars-cov-2. (161, 164) among the drugs identified by mining the hidden information within the graph, there were 378 inhibitors of the p2associated protein kinase 1 (aak1), a regulator of viral endocytosis in at2 alveolar epithelial cells. (161) inhibitors of aak1 could then potentially block viral entry into alveolar cells. however, the authors argued that only one of these drugs, baricitinib (39), a janus kinase inhibitor, has an acceptable safety profile. in addition, the low therapeutic dosing of baricitinib (39) (2mg or 4mg daily) makes it a promising drug for repurposing. (161) baricitinib (39) will be further discussed below. zhou et al. developed a network-based approach to identify potential repurposable drugs based on the interactions between their human targets and coronavirusassociated proteins. (52) the premise is that protein targets that are associated with viral infections are part of a subnetwork of the human protein-protein interaction network. the targets of a repurposable drug should be in or close in proximity to this subnetwork. the authors identified many potential targets, such as poly [adp-ribose] polymerase 1 (parp-1), glycogen synthase kinase 3 (gsk-3), and also biological pathways that could be explored, such as nf-kappa b signaling. (52) the most interesting drugs selected by the authors are from a diverse set, including toremifene (40) (selective estrogen receptor modulator), sirolimus (41) (immunosuppressant), mercaptopurine (42) (antineoplastic) and irbesartan (43) (antihypertensive). an interesting finding was that the selected molecules and targets have reported links to viral infection and some have been used for treatment of coronavirus-related disease, such as emodin (44) in sars-cov, and mercaptopurine (42) and toremifene (40) in sars-cov and mers-cov. (52) in addition to identifying repurposable drugs, possible synergistic combinations between them were also suggested by the network, such as sirolimus (41) and dactinomycin (45) , and mercaptopurine (42) and melatonin (46) . (52) in vitro and in vivo studies -genome and replication kinetics of sars-cov, mers-cov and sars-cov-2 viruses are very similar, suggesting that these diseases could be treated by a single drug that modulates the activity of a common target or mechanism among them. (52, 186) so far, no clinically available antiviral drugs have been developed for any of these three viruses. (187) this fact demonstrates that we did not learn a suitable strategy for treatment from these two prior diseases, and we are not yet prepared to deal with this new coronavirus epidemic, regarding therapeutic options. (188) experimental target validation (e.g., by genetic and proteomic approaches), cellular in vitro and in vivo animal models of sars-cov-2 infection, are pivotal for the discovery of molecular pathways involved in pathogenesis, supporting the discovery of new drugs. (189, 190) sars-cov-2 genome encodes less than 30 proteins that can be explored for generating new avenues for further research. heterologous viral protein expression could be used in a myriad of structural and functional targetbased biochemical studies. (191) these assays do not need to be performed in biosafety level 3 laboratory, democratising the early research on searching for a new drug candidate for covid-19 treatment. (192) genome-wide expression studies, nucleotide sequences, protein structures and associated sequences are available online providing a foundation for preclinical drug discovery and development research against sars-cov-2 (available from: https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/). it is good to point out that many drugs show in vitro effect (e.g., target and/or cellular assays) that may not last in vivo, in animal models, including humans. an ideal animal model of covid-19 should reflect the clinical signs, viral replication and pathology reported in humans. (193) in vivo animal infection experiments with sars-cov-2 require infrastructure of a biosafety level 3 laboratory, restricting scientific research to well-equipped research groups. animal models are indispensable, because they could identify toxic hits or molecules that could enhance covid-19 pathogenicity. table ii lists some compounds that have the potential to be clinically tested and that will be discussed in more details below in this topic. remdesivir (8) , a nucleotide analog pro-drug developed by gilead sciences inc. to fight ebola, acts on viral replication by inhibiting rna polymerase. (96) this molecule was also active against sars and mers viruses. (200, 201, 202) wang et al. demonstrated reduced viral copy numbers in cell culture supernatant of vero e6 cells infected with ncov-2019betacov/wuhan/wiv04/20192, in the presence of varying concentrations of remdesivir (8) . (194) this compound blocked virus infection at lowmicromolar concentration (ec 50 = 0.77 μm) and showed a high selectivity index (> 100). the authors also disclosed that remdesivir (8) inhibited virus infection efficiently in human liver cancer huh-7 cells. chloroquine (cq; 20) and its less toxic derivative, hydroxychloroquine (hcq; 26) , are used to treat malaria and lupus/rheumatoid arthritis, respectively. both drugs have already been described as possible antivirals. (195, 203, 204) cq (20) and derivatives probably act by decreasing acidity in endosomes and lysosomes, intervening on glycosylation of cellular virus receptors and modulating host immunological activity. (118, 205) studies in cultures of vero e6 cells have suggested that hcq (26) can affect the virus sars-cov-2 in both entry and post entry stages at host cells. (194) this molecule presented an ec 50 (196) the authors also performed in vitro physiologically based pharmacokinetic models for both drugs, separately. interestingly, when comparing toxicity in five animal models, mcchesney et al. demonstrated that hcq (26) is two to three times less toxic than cq (20) itself. (207) it is also good to point out that chloroquine (20) has shown in vitro activity against many different viruses, but no significant beneficial effect on animal models. (208) the association ritonavir (2)/lopinavir (1) is used together with other antiretrovirals for the treatment of human immunodeficiency virus since the beginning of the century. (209) ritonavir (2) is a potent cyp3a inhibitor therefore inhibiting the metabolism of lopinavir (1), increasing its plasma levels. both are reported as peptidomimetic molecules that inhibit hiv-1 protease activity in a competitive manner. (210) the m pro plays a major role in homeostasis of viral polyproteins essential for viral function and replication, being considered a validated drug target. (211) this combination may be useful against (194) chloroquine (20) (194, 195, 196) ritonavir (2) + lopinavir (1) lopinavir (1) nd (197) nitazoxanide (47) broad-spectrum antiparasitic and antiviral still needs confirmation 2.1 μm 16.8 (194) ivermectin (48) broad-spectrum antiparasitic still needs confirmation 2 μm nd (198) teicoplanin (27) antibiotic for gram-positive bacteria still needs confirmation 1.7 μm nd (199) sars-cov-2 virus by acting on its main protease, an enzyme essential in processing polyproteins translated from viral rna. (212) choy et al. demonstrated that it inhibits sars-cov-2 replication in vero e6 cells with ec 50 value of 26.6 μm. (197) these results corroborate with the study of de wilde et al. that demonstrated antiviral in vitro effect of lopinavir (1), but not ritonavir (2), against sars-cov, mers-cov, and hcov-229e, with mean ec 50 varying from 6.6 to 17.1 μm. (213) other compounds were also tested against sars-cov-2 in vitro. nitazoxanide (47), a broad-spectrum antiparasitic and antiviral, inhibits a low-micromolar range with an ec 50 of 2.1 μm but has a selective index of only 16.8. (194) ivermectin (48), another broad spectrum antiparasitic agent also demonstrated in vitro anti-sars-cov-2 activity. its ec 50 was determined to be 2 μm when added to vero-hslam cells 2 h post-infection and measuring viral rna at 48h post-infection. (198) teicoplanin (27) , an antibiotic used against gram-positive bacterial infections, was found to be active in vitro against sars-cov-2 with an ec 50 value of 1.7 μm, but in vivo efficacy still needs to be determined. (199) a robust preclinical drug discovery pipeline comprising in vitro, and in vivo models of sars-cov-2 infection is particularly important to identify new antivirals for human covid-19 treatment. this pipeline can munition clinical studies with molecules that have a higher chance to become a drug, decreasing attrition rates. clinical studies -over 1452 clinical trials have been unveiled, until the publication of this manuscript, focusing on covid-19 treatment interventions (available from: https://clinicaltrials.gov/ct2/results?cond=covid-19). with a growing number of patients suffering from acute severe respiratory symptoms and hospital capacities reaching its limit, therapeutic options are urgently essential to avoid human mass deaths. drug repurposing approaches of approved (with safe and effectiveness proven) and unapproved molecules, that were promising in pre-clinical and early stages of clinical studies of sars and mers, are in vogue. with this premise, world health organization (who) organised a simplified dynamic platform comparing the effectiveness of treatment strategies around the globe. this clinical trial design, called solidarity, can shrink by 80% the time of clinical studies, compared with the "gold standard" double-blind, placebo-controlled trials. this strategy could overcome the uncertainty of multiple small trials that do not produce a solid base necessary to establish the relative success of arising probable treatments. on the other hand, this shorter time required reflects the compassionate characteristic of the studies, that cannot rule out placebo effects and patient severe adverse effects, including death. (214) currently, who is focusing on four most promising therapies: remdesivir (8); cq (20) or hcq (26) ; ritonavir (2)/lopinavir (1); ritonavir (2) /lopinavir (1) plus interferon-beta, an immune response modulator. (44) a selection of studies with these drugs, alone or in combination, are summarised in table iii. for other studies, the reader can refer to kupferschmidt and cohen, which summarised several clinical studies on drug repurposing for covid-19 reported so far. (44) it is worth noting that many clinical studies are beginning to be carried out in different parts of the world and their number is increasing rapidly day by day. remdesivir (8), a nucleoside analogue that acts by inhibiting rna synthesis, is already in clinical studies for treatment of covid-19. (96) this prodrug was already investigated in clinical trial testing for ebola virus with no effect but showed efficiency against different types of coronaviruses in cell culture and animal models. (200, 221) from the drugs in the solidarity trial, remdesivir (8) has the best potential to be used in clinics, having a low toxicity profile to humans. (44) this molecule was used compassionately in the first covid-19 patient diagnosed in the united states by intravenous treatment and improved patient clinical condition. (222) grein et al. reported a study in a cohort of patients hospitalised for severe covid-19 who were treated with compassionateuse remdesivir (8) , clinical improvement was observed in 68% of the patients (36 of 53). (223) there are two clinical trials at phase iii, being designed to evaluate the efficacy and safety of parenteral remdesivir (8) in mild/moderate (nct04252664) and severe (nct04257656) hospitalised adults with covid-19. (224) these trials are evaluating intravenous remdesivir (8) at a dose of 200 mg on the first day plus 100 mg once daily, for an additional 9 consecutive days. the randomised, double-blind, placebocontrolled, multicentre trials were recently suspended (nct04252664) or terminated (nct04257656) because no eligible patients for enrollment were found, due pandemic control in china. (215) the study nct04257656 showed that remdesivir (8) was not associated with statistically significant clinical benefits in severe forms of covid-19. however, the authors observed a numerical reduction in time to clinical improvement in those treated earlier, but still requires confirmation. another study, performed by beigel et al. (with the same dosage and treatment period than the previous works cited above) enrolled 1063 patients with lower respiratory tract infection, demonstrated that individuals that were treated with remdesivir (8) had a shorter time to recovery than placebo group (11 days compared with 15 days). (216) even with a small scientific ballast, due to limited information about safety and effectiveness of using remdesivir (8), u.s. food and drug administration (fda) approved its emergency use on severe covid-19. (225) the approval was based on review of the topline data on two studies that are recruiting patients, (nct04280705) and (nct04292899). other clinical studies are being conducted to address the effect of remdesivir (8) on co-vid-19 treatment (available from: https://clinicaltrials. gov/ct2/results?cond=covid-19&term=remdesivir&cn try=&state=&city=&dist=). other phase iii clinical trials evaluated for co-vid-19 treatment included cq (20)/hcq (26) . these studies were endorsed by promising in vitro data that suggested an impairment of viral replication by acting on endosome-mediated viral entry or later phases of viral replication. (194, 226) both molecules have a well-studied toxicity profile being used for more than half-century as antimalarials, but in some cases can induce heart side (8) was not associated with clinical benefits in severe forms. however, an observed numerical reduction in time to clinical improvement in patients treated earlier, but still requires confirmation (215) ; nct04257656 (terminated by epidemic control in china with no eligible patients) remdesivir (8) (216) ; nct04280705 chloroquine (20) multicentre > 100 patients 500 mg per day, for 10 days compilation of various clinical studies that are in course, authors conclude that the dosage used could be sufficient (217) phase iib double-blind, randomised 81 patients 600 mg twice daily for 10 days and 450 mg twice on the first day, once daily for more 4 days higher dosage of chloroquine (20) has toxic effects and increased lethality, with any clinical benefit (218) hydroxychloroquine (26) non-randomised, non-double-blind, non-placebo-controlled 36 patients 600 mg of hydroxychloroquine (26) daily for 10 days hydroxychloroquine (26) significantly reduces viral load despite small sample size (219) ritonavir ( effects. (227) these drugs have already been examined against at least five other viruses with good initial in vitro results, but without significant effects in animal models and humans. (205) clinical trials to measure the effectiveness of cq (20) at 500 mg of chloroquine diphosphate (corresponding to 300 mg of chloroquine (20) base) per day, for ten consecutive days on the treatment of covid-19 were performed. (217) based on results for a very limited number of patients (100) , it was concluded that cq (20) presented apparent efficacy and acceptable safety against cov-id-19 associated pneumonia. borba et al. performed a double-blind, randomised clinical trial designed to assess the safety of cq (20) in two dosages: 600 mg twice daily for 10 days with an initial dose of 450 mg twice daily on the first day, decreasing to once daily for four more days. (218) the study suggests that the higher dosage should not be recommended for critically ill patients with covid-19 because of its potential side effects. it is important to note that both doses studied were above the brazilian recommended dose. a controversial study performed by gautret et al. with a small sample size demonstrated that hcq (26) treatment daily at 600 mg significantly decreases viral load in covid-19 patients and its effect is reinforced by azithromycin (az; 49) combination. (219) until this moment there is clinical robustness to support az (49) therapy in covid-19. (228) mehra et al. performed a registry analysis of 96,032 hospitalised patients regarding the use of cq (20) or hcq (26, combined or not with a macrolide) for treatment of covid-19. (229) the observational study verified that with covid-19 requiring hospitalisation a regimen containing cq (20) or hcq (26) had no evidence of benefit, but instead was associated with an increase in the risk of ventricular arrhythmias and a greater hazard for in-hospital death with covid-19. these findings suggest that these drug regimens should not be used outside of clinical trials and urgent confirmation from randomised clinical trials is needed. after the publication, the paper was retracted, which influenced who to return the studies on cq (20) and hcq (26) . (230) recently, on 17 june 2020, who stopped the hcq (26) arm of the solidarity trials again based on the recovery trials (available from: https://www.recoverytrial.net/). in this study 1542 patients were randomised to hcq (26) and compared with 3132 patients randomised to usual care alone. there was no significant difference in any of the parameters evaluated: the primary endpoint of 28-day mortality, hazard ratio and hospital stay duration or other issues. other study reported that hcq (26) did not prevent covid-19 when used as postexposure prophylaxis within 4 days after moderate or high-risk exposure. (231) robust data from well-designed control randomised clinical trials with large number of patients is still expected for concluding on the efficacy of hydroxychloroquine (26) . ritonavir (2) and lopinavir (1) were developed to target hiv-1 protease and postulated to inhibit the 3-chymotrypsin-like protease of sars and mers, being associated with improved clinical outcomes. (232) the combination has beneficial effects in marmoset monkeys infected with the mers-cov virus. (233) (1)/(2) may have clinical efficacy against sars-cov-2, as seen in the response against sars-cov. (234) in vitro sensitivity and satisfactory response in a preliminary non-randomised clinical trial of sars-cov to (1)/(2) have already been reported, encouraging its testing in sars-cov-2. (235) a total of 199 patients confirmed severe sars-cov-2 infection were randomly designated to be given either (1)/(2) (400 mg/100 mg) twice a day for 14 consecutive days plus standard care or standard care alone. (220) this first trial was not promising, no benefit was observed on clinical improvement, mortality and viral loads on severe covid-19 patients. other clinical trials are being carried out and should decide whether these drugs are useful for covid-19 treatment or not. ritonavir (2)/lopinavir (1) plus interferon-beta, a cytokine involved in inflammatory modulation, is the last bet of the solidarity megatrial. sheahan et al. demonstrated that this combination improves pulmonary function but does not reduce virus replication or severe lung pathology in mice model of mers-cov. (236) this combination also showed promising results in mers-cov infection in a nonhuman primate model. (233) the study (nct04276688) administered ritonavir (2)/lopinavir (1) 400 mg + 100 mg/ml twice daily for 14 days and interferon beta-1b 0.25 mg subcutaneous, every alternate day for 14 days. another study (nct04343768) will perform a randomised trial to verify the effects of interferon beta 1a, compared to interferon beta 1b and the base therapeutic treatment in moderate to severe covid-19. currently, there is a lot of research around the world examining drugs that can be used for the treatment of covid-19. unfortunately, until this moment no therapeutic options are promptly effectively available. most of the studies have a small number of patients with variable dose and/or duration, fact that hinders a comparison. to successfully undertake the covid-19 pandemic with new medicines, synchronised clinical trials with randomised, double-blind, placebo-controlled are still needed. infection prevention by social distancing and supportive medical care, are the only strategies to deal with this disease until this moment. the goal of this session is to consider potential obstacles for effective adoption of a repurposed drug to widespread treatment of covid-19. both patent protection and scalability of manufacturing processes are key aspects to consider while choosing the best therapeutic option to adopt when dealing with a pandemic. a patent can be defined as a government license that confers the owner the exclusivity over a new invention or medication. with the patent, the inventor is able to exclude others from making and selling the invention for a determined period of time. when it comes to medication, the market exclusivity granted by the patent generates enormous economic profit for the patent holder, once the company will have the monopoly over the product for around 20 years. once the patent term finishes, generic companies can start producing and selling the drug, increasing the market competition over the product. a strategy adopted by some companies is to maximise the term of patent of successful products, extending market exclusivity, for instance, increasing economic rewards with the invention. in order to extend patent terms, the companies can apply for new formulations, new routes of administration, new uses of the drug among other strategies. (237, 238) this is very important when considering drug repurposing, once those strategies can make it harder to patent a new method of use for the drug, compromising the legal rights of the new repurposed indication. as a consequence, the expected profit associated with repurposing can be severely affected. (39) remdesivir (8) a good example of maximising the term of patent is the drug remdesivir (8) , which was developed by the american pharmaceutical company gilead sciences, inc. in 2011. nowadays, (8) has shown promising results as a suitable repurposed drug in the treatment of the covid-19 disease. indeed, there are many ongoing clinical trials with this drug in cov-id-19 patients. (239, 240) due to the good results concerning remdesivir (8) , in january 2020, the wuhan institute of virology applied for a patent on gilead's remdesivir (8) for the treatment of coronavirus, in an attempt to protect china's economic and medical interests. however, remdesivir (8) has already 133 patents related to the coronavirus filed by gilead science in 43 countries around the globe since 2011. in fact, the company has a robust patent portfolio that includes structures of remdesivir-related compounds (family 1), the manufacturing method of the drug (family 2), the use of remdesivir (8) in treating coronaviridae infection (family 3), among other patents that claims the use of (8) for the treatment of a series of viral infections (families 3 and 4) (fig. 13) . (241) practically, this means that if the (8) is proved to be efficient for the treatment of covid-19, gilead science is the only pharmaceutical company owing the market exclusivity of the drug, at least until 2031. in fact, the company has already started to produce larger amounts of the drug and they already made improvements to optimise the drug manufacturing timeline. now, the company can produce the drug in 6 months, half the time that they used to need to get the final product. in addition, the company has been donating remdesivir (8) to ongoing clinical trials, a gesture that will not only help the trial patients, but gilead itself. (242) baricitinib (39) -in 2009, a patent assigned to incyte corporation disclosed the preparation of several active compounds as jak inhibitors, including baricitinib (39). in the same year, lilly and incyte made an agreement allowing lilly co. to manufacture and commercialise the medicine worldwide, making it lilly co. the only supplier around the globe. (243) there are two patents that protect the drug, one concerning its synthetic pathway and another disclosing the use of (39) in the treatment of rheumatoid arthritis. both of them will expire in nine years, which could open opportunities for generic drug companies to produce baricitinib (39) . nowadays, (39) has been investigated as a drug that could be used in the treatment of covid 19 patients, since its anti-inflammatory activity could minimise inflammatory compli-cations in covid 19 patients. according to the platform clinicaltrials.gov, there are 14 ongoing clinical trials to evaluate the efficacy and safety of baricitinib (39) in the treatment of covid 19. (244) if the drug succeeds, there will be a need for larger and faster production and distribution of the medicine worldwide. there are two patents disclosing a synthetic method for the preparation of (39). the first one is from 2009 and owned by incyte co. and the latest is from 2016, by lilly co. (245, 246) the main difference between them is how the central pyrazole ring is installed in the molecule. in the patent from 2009, baricitinib (39) is obtained by a convergent synthesis. the synthetic pathway starts with the protection in position 7 of 4-chloro-7h-pyrrolo[2,3d]pyrimidine (50) using 2-(trimethylsilyl)ethoxymethyl chloride (51) , affording intermediate (52) . next step is a suzuki-miyaura reaction, coupling the fused ring system to a 4-pyrazoleboronic acid pinacol ester (53), giving key intermediate (54) . parallelly, a couple of steps starting from 2-(chloromethyl)oxirane (55) lead to 1-boc-3-azetidinone (58) that reacts with diethyl cyanomethylphosphonate (59), affording key intermediate (60) . next, the key intermediates (54) and (60) react in the presence of dbu, giving (61) . then, steps involving hydrolysis of the boc, sulfonation and pyrrolopyrimidine deprotection afford (39) with an overall yield of 21 % (fig. 14) . (246) the synthetic pathway described in the patent from 2016 has only six steps in a linear approach, as a consequence, the product is obtained in a higher overall yield when compared to the synthetic pathway discussed above. intermediate (67) is obtained from azetidine-3-ol (64) in a couple of steps, including a sulfonation, an oxidation and the installment of the cyanomethylene moiety (fig. 15 ). furthermore, it is not necessary to protect any position in this sequence. additionally, the oxidation step can be performed under flow conditions. then, intermediate (67) is reacted with ester (53), affording (68) . a suzuki-miyaura reaction involving 7-boc-4-chloro-7hpyrrolo [2,3-d] pyrimidine (69) is applied, allowing the formation of the bound between the azetidinylpyrazole group and the pyrrolo[2,3-d]pyrimidine system. last step is a hydrolysis of the boc, affording baricitinib (39) with an overall yield of 50 % (fig. 15 ). (245) off-patent drugs -fortunately, a number of drugs that have been tested as possible agents in the covid-19 treatment are off-patent, meaning that generic drug companies already manufacture and commercialise the medicine, making it easier for drug repurposing. (39) the off-patent drugs that have been tested in covid-19 clinical trials include cq (20) , hcq (26), ritonavir (2) and lopinavir (1) . (239, 240) a recent research article published by hill and collaborators estimated the minimum cost of production associated with these drugs, showing that all the treatments under evaluation in current clinical trials are cheap to manufacture. however, list prices can be over 100 times higher than the costs to produce the drug. (247) if any of those drugs become approved for the treatment of covid-19, its demand will increase dramatically. therefore, it is important to consider the challenges associated with scaling up the production to meet the demand. for instance, there are few regulatory approved production facilities, and drug manufacturers rely on low-cost suppliers of raw materials in india and china, which might be scarce during a pandemic. consequently, it would be difficult to ensure short term global availability of the treatment, since the production depends on different countries. (248) those conclusions are very helpful in showing the importance of optimising the way of manufacturing the candidate drugs presented above. for this reason, recent and optimised synthetic pathways found in patents and articles for some ritonavir (2) and lopinavir (1) are discussed below, as cq (20) and hcq (26) have now been abandoned. finally, we will briefly discuss the ease of manufacturing in large scale each of the drugs we had the synthesis reviewed here. ritonavir (2) -the first disclosure of ritonavir (2) is presented in a patent from 1994, assigned to abbott laboratories. even though the patent consists of a range of markush structures, among which is found (2), its synthesis is not presented. (249) the synthetic pathway to obtain (2) was disclosed for the first time in a second-generation patent in 1995, by the same company. (250, 251) some drawbacks related to the synthesis presented by abbott include the employment of expensive condensing agents, as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (edc) and poor reaction yields on the first steps of the synthetic pathway, making this strategy too expensive and not suitable for scale-up production. considering the above deficiencies, a recent chinese patent concerning the synthesis of ritonavir (2) has been released. the synthetic pathway described in the patent starts with a nucleophilic addition-elimination reaction between the starting material (2-isopropylthiazol-4-yl)-nitro-methylamine (71) and n-[(2,2,2-trichloroethoxy)carbonyl]-l-valine (72), generating intermediate (73) . the intermediate is mixed with p-toluenesulfonyl chloride and triethylamine to activate the acid function, followed by the addition of reagent (74) in a one-pot procedure. the condensation allows the formation of intermediate (75) , which is submitted to acidic conditions for a boc deprotection, followed by another nucleophilic additionelimination reaction with reagent (76), thus obtaining the final product ritonavir (2) (fig. 16) . (252) when comparing both patents, it is possible to highlight important advantages present in the latest one, including the use of cheap and easily available reagents, for instance, the use of ptoluenesulfonyl chloride as an amide condensing agent, the synthetic pathway has a high yield (79% overall), low cost and it is ease to scale-up the production. lopinavir (1) -the first synthetic methods related to the synthesis of lopinavir (1) are present in a patent from 1996 owned by abbott laboratories. (249) (1) has 4 chiral centres, and the synthetic strategies reported by abbott are similar, involving the synthesis of a key intermediate amino alcohol unit, that is then connected to the appropriated side chains. some drawbacks that can be pointed out in the synthesis include the use of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (edc) as a condensing agent and the weak base 1-hydroxybenzotriazole, both expensive reagents, making the synthesis not suitable from the viewpoint of cost and industrial ap-plication. in addition, one of the methods described by abbott includes the synthesis of an acid chloride as an intermediate, which is very unstable, and it can be easily decomposed by humidity, making the synthesis not suitable for industrial production. the most recent patent related to the synthesis of lopinavir (1) is dated 2018 and it is from the chinese company shanghai desano pharmaceuticals co. ltd. (253) the patent is described as technical, with the aim of improving the synthesis of (1) presented in the patent from 1996, by abbott laboratories. the patent describes the synthesis of lopinavir (1) by a one-pot procedure starting with the formation of an acid chloride from (2s)-(1tetrahydropyramid-2-one)-3-methylbutanoic acid (77) followed by addition of a weak base and the reactant (78) to the reaction system. the amine function of (78) allows a nucleophilic addition-elimination reaction to occur, affording (1) after treatment with nahco 3 (fig. 17 ). therefore, it is possible to synthesise lopinavir (1) in high yield (90%) in an optimised method, which involves a few reagents, mild conditions and is performed in a one-pot procedure. synthetic scalability and cost-effectiveness -the synthetic pathways described above show how much improvement has been done concerning the preparation of these drugs in a more cost-effective way. furthermore, when comparing the drugs discussed above, it is worth pointing out which one could be the easiest one to produce when analysing the factors that influence the final cost of a synthetic pathway, such as chiral centres, cost of starting materials, number of steps and overall yield. regarding chiral centres, ritonavir (2) and lopinavir (1) have four chiral centres each, and they are not sold as racemate mixtures. moreover, to synthesise those compounds the chiral centres do not come from natural molecules, but have to be synthetically installed, which makes the chiral starting materials more expensive and enantiomeric excess has to be accessed more carefully at the end of the synthetic pathway. as a consequence, it can take longer and more expensive to obtain the final product. on the other hand, baricitinib (39) does not have any chiral centres, making the synthetic pathway easier and cheaper to perform. concerning the number of steps and overall yield, baricitinib (39) has the highest number of steps and lower overall yield when compared to ritonavir (2) and lopinavir (1) . however, one of the steps in the synthesis of baricitinib (39) can be performed under flow conditions, which is very interesting from the industrial point of view. therefore, when taking into account the most recent synthetic pathways proposed for these drugs, the synthesis of baricitinib (39) is the most cost-effective of the three of them. in conclusion -sars-cov-2 infection is a lifethreatening disease with such a high transmission rate that can surpass even the most well-structured health systems in developed high-income countries. while vaccines are the ideal solution for preventing the spread of infectious diseases like covid-19 their development cycle have intrinsic challenges and safety checking steps that require several months or years to complete their development. a similar situation is found for developing new drugs for covid-19 (as with any other disease), because of the long process involving discovery, validation and safety evaluation of new chemical entities for use in human health. in this scenario, repositioning drugs already in clinical use for treatment of covid-19 is an important shortcut, as we have discussed. data are accumulating about the molecular pathology of covid-19 as well as structural information on the viral and host proteins involved in the infection mechanism. this knowledge is essential for allowing researchers to have new insights on approved or investigational drugs that can be repurposed to treat sars-cov-2 infection. as reviewed here, over 10 targets distributed amongst distinct steps of the sars-cov-2 replication cycle or host cell mediators are currently being investigated. here, we highlighted several up-to-date examples of potential repurposable drug candidates proposed from either computational approaches or experimental trials (or their combination) at different levels of validation and stages of development. noteworthy, ai methods hold a great promise in finding occult links between drugs, human and viral targets to find novel bioactivities and even combinations of drugs. remdesivir (8) , cq (20)/hcq (26) (alone or in association with other drugs) and the association lopinavir (1)/ritonavir (2) have been the focus of most clinical studies. so far, results have been mostly disappointing with these trials, stressing the importance of carefully performed studies in patients to attest that biological activities observed in vitro actually be translated into clinical efficacy and safety. at the moment, even with limited information about safety and effectiveness, remdesivir is the only drug approved by the fda for emergency use on severe covid-19. as discussed here, a major concern with drugs for effectively fighting the pandemic is the drug's industrial production cost and its impact on final treatment cost. gilead, remdesivir's owner company, has recently signed non-exclusive voluntary licensing agreements with five generic pharmaceutical 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out a winner fierce pharma gilead turbocharges production of covid-19 hopeful remdesivir lilly and incyte announce collaboration for development and commercialization of oral anti-inflammatory and autoimmune therapies search results: baricitinib | covid-19 processes and intermediates for the preparation of {1(ethylsulfonyl)-3 inventors; incyte corporation assignee 2009. azetidine and cyclobutane derivatives as jak inhibitors. united states patent us 2009/0233903 a1 minimum costs to manufacture new treatments for covid-19 dozens of coronavirus drugs are in development -what happens next? inventors; abbott laboratories assignee 1995. process for the preparations of a substituted 2,5-diamino-3-hydroxyhexane discovery of ritonavir, a potent inhibitor of hiv protease with high oral bioavailability and clinical efficacy zhou z, inventors; guizhou yongnuo feite bio pharmacy co ltd assignee 2019. preparation method of ritonavir. chinese patent cn 109369562a inventors; yancheng desano pharmaceutical co ltd assignee 2018. method used for preparing lopinavir using one-pot method. chinese patent cn 108218791a all authors contributed to writing, reviewing and editing the manuscript. mrs, tcse and rfd contributed equally to this manuscript; fps-jr performed the manuscript conceptualisation and final review. the authors declare no conflict of interest concerning this manuscript. key: cord-354950-kmpbdvof authors: demurtas, olivia c.; massa, silvia; illiano, elena; de martinis, domenico; chan, paul k. s.; di bonito, paola; franconi, rosella title: antigen production in plant to tackle infectious diseases flare up: the case of sars date: 2016-02-05 journal: front plant sci doi: 10.3389/fpls.2016.00054 sha: doc_id: 354950 cord_uid: kmpbdvof severe acute respiratory syndrome (sars) is a dangerous infection with pandemic potential. it emerged in 2002 and its aetiological agent, the sars coronavirus (sars-cov), crossed the species barrier to infect humans, showing high morbidity and mortality rates. no vaccines are currently licensed for sars-cov and important efforts have been performed during the first outbreak to develop diagnostic tools. here we demonstrate the transient expression in nicotiana benthamiana of two important antigenic determinants of the sars-cov, the nucleocapsid protein (n) and the membrane protein (m) using a virus-derived vector or agro-infiltration, respectively. for the m protein, this is the first description of production in plants, while for plant-derived n protein we demonstrate that it is recognized by sera of patients from the sars outbreak in hong kong in 2003. the availability of recombinant n and m proteins from plants opens the way to further evaluation of their potential utility for the development of diagnostic and protection/therapy tools to be quickly manufactured, at low cost and with minimal risk, to face potential new highly infectious sars-cov outbreaks. severe acute respiratory syndrome (sars) is a dangerous infection with pandemic potential. it emerged in 2002 and its aetiological agent, the sars coronavirus (sars-cov), crossed the species barrier to infect humans, showing high morbidity and mortality rates. no vaccines are currently licensed for sars-cov and important efforts have been performed during the first outbreak to develop diagnostic tools. here we demonstrate the transient expression in nicotiana benthamiana of two important antigenic determinants of the sars-cov, the nucleocapsid protein (n) and the membrane protein (m) using a virus-derived vector or agro-infiltration, respectively. for the m protein, this is the first description of production in plants, while for plantderived n protein we demonstrate that it is recognized by sera of patients from the sars outbreak in hong kong in 2003. the availability of recombinant n and m proteins from plants opens the way to further evaluation of their potential utility for the development of diagnostic and protection/therapy tools to be quickly manufactured, at low cost and with minimal risk, to face potential new highly infectious sars-cov outbreaks. recombinant proteins expressed in plants have emerged as a novel branch of the biopharmaceutical industry and hold great potential to produce different types of therapeutic proteins at low cost and with reduced risks of contamination with human and animal pathogens (moustafa et al., 2015; paul et al., 2015; sack et al., 2015) . transient expression of target proteins can be easily achieved by plant viruses or by agroinfiltration (gleba et al., 2007) , saving the time spent in the generation of transgenic plants, often allowing higher protein yield due to the absence of chromosomal integration and consequently of position effects (komarova et al., 2010) . transient expression can also be used as a means for preliminary evaluation of correct expression before starting the generation of transgenic plants, or related platforms, such as plant cell cultures or microalgae (franconi et al., 2010) . antigen preparation plays a crucial role in the development of a diagnostic test, and plants represent an ideal biofactory system. the approach could be extended to other cases when a pathogen cannot be grown in the lab or is highly virulent and needs a methodology for fast and affordable production. indeed, virus-specific and 'orphan' vaccine candidates and therapeutics represent one of the most interesting applications of the plant-based technology, especially when it is necessary to produce 'rapid response' vaccines such as those directed against bioterrorism agents and diseases with pandemic potential, like influenza. the capacity for such a response has already been demonstrated by the (four) companies involved in the us in the production of 100 million doses of influenza vaccine a month (rybicki, 2014) . this opens the way for the use of this technology for other diseases such as, to name a few, ebola (henao-restrepo et al., 2015; heymann et al., 2015) , avian flu (su et al., 2015) , mers (al-tawfiq and memish, 2015; keener, 2015) and other viruses where the principles of the so-called "one health initiative" (strategies to control diseases across species) are important (http://www.onehealthinitiative.com/). the fact that the epidemiology of these diseases is associated to sudden and sometimes unforeseen contagious burst, results in an on/off attention about, in terms of research, prevention and pharma industry efforts (barber, 2014; lamattina, 2014) . for this reason, tools for a quick and relatively easy scale-up may provide a solution to such emergencies (streatfield et al., 2015) . among emerging and re-emerging diseases, severe acute respiratory syndrome (sars) appeared in china in november 2002. the epidemic spread to 29 countries over 5 continents, leading to more than 8000 infected patients globally (world health organization [who] , 2006) with a fatality rate of 9.6% (suresh et al., 2008) . the aetiological agent of the syndrome, the coronavirus sars-cov, was rapidly identified (drosten et al., 2003; marra et al., 2003; rota et al., 2003) . the end of the sars outbreak was declared by world health organization [who] (2003) in july, thanks to strong containment measures. however, several local outbreaks were subsequently reported in china, as a consequence of accidental laboratory contaminations or infections after contact with animals infected with sars-cov strains significantly different from those predominating in the 2002-2003 outbreak (peiris et al., 2004) . while no effective therapy is currently available, considerable efforts have been made to develop vaccines and drugs to prevent sars-cov infection, since a sars epidemic may recur at any time in the future (gimenez et al., 2009; chan and chan, 2013) . moreover, because of the highly contagious nature of the disease, and since sars-cov has been defined a potential biological weapon (centers for disease control and prevention [cdc] , 2012), it is still important to develop effective sars-cov sensitive diagnostic tools (suresh et al., 2008) . the large sars-cov genome, a polyadenylated rna of 29,727 nucleotides, encodes four major viral structural components, the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins, and 16 non-structural proteins (bartlam et al., 2007) . the structural n protein is the most abundant protein in the sars-cov virion. it is a highly basic protein of 422 amino acids (46 kda) of the helical nucleocapsid, playing an important role in viral pathogenesis, replication, rna binding, cell cytokinesis and proliferation (surjit and lal, 2008) . n protein has been recognized as the preferred target for detection of sars-cov infection by reverse transcription-polymerase chain reaction (rt-pcr; suresh et al., 2008) . in addition, the who guidelines for sars diagnosis, developed during the outbreak in 2003, suggested the use of n-based elisa for specific igg detection as confirmatory test of sars-cov infection (world health organization [who] , 2003 sars: laboratory diagnostic tests) due to the ability of the host to mount an early antibody response against the n protein (che et al., 2004) . furthermore, since the n protein is able to induce a long-term cell-mediated immune response in animal models, it represents a potential vaccine candidate as well (roper and rehm, 2009) . to date, the production of recombinant n protein has been achieved in a variety of heterologous expression systems, including plants, (zheng et al., 2009) , providing proofs of concept for its use in vaccine formulations (roper and rehm, 2009 ). however, the immune response in animal models (both natural and nonnatural sars-cov hosts) might be not useful to predict the human immune response. the m protein is the most abundant protein in the sars-cov viral envelope. it is functionally involved in the assembly and budding of virions from the cell. m protein forms homo-oligomers and interacts with s, e, and n proteins (hogue and machamer, 2008) . it consists of 221 amino acids (25 kda), with a short glycosylated n-terminal domain, three membrane-spanning domains and a long immunogenic c-terminal cytoplasmic domain. it has been reported that rabbit antiserum raised against recombinant m protein produced in yeast has a potent neutralizing activity in vitro (pang et al., 2004) . antibodies to the m protein were detectable in convalescent sars patients and b-cell epitopes of the m protein have been identified (he et al., 2005) . it has also been shown that m acts as a dominant immunogen for ctl response in humans (liu et al., 2010) . moreover, it has been demonstrated that sars-cov m-specific memory cd4+ and cd8+ t cells were persistent in the peripheral blood of recovered sars patients more than 1 year after infection . in a study, where different dna vaccines were used, m generated the strongest t-cell response in an animal model, and recovered sars patients had a long-lasting cd4+ and cd8+ memory for the m antigen (roper and rehm, 2009) . these data suggest that further research should be directed toward evaluating the potential efficacy of the m antigen for vaccine and diagnostic tools development. in this study, we demonstrate the feasibility of using plant transient expression systems (potato virus x [pvx]-mediated infection and agroinfiltration) to produce two sars-cov antigens, the n and m proteins, as useful tools to face sars-cov infection. in particular, we demonstrated that the sars-cov n protein produced in nicotiana benthamiana is recognized by the specific antibodies of convalescent sars patients. moreover, the expression of the sars-cov m protein was achieved for the first time in plant. the approach to rapidly get crucial antigens by transient expression in plants, potentially attains to other infectious, either emerging or re-emerging, diseases (e.g., mers, avian flu, ebola), that share with sars the features of rapid outbreak burst and need to rapidly produce diagnostic or therapeutic tools. escherichia coli xl1 blue strain was used as a host for cloning and protein expression. cells were grown in luria-bertani medium at 37 • c with shaking at 250 rpm. agrobacterium tumefaciens gv3101 and c58c1 strains were used to transiently express the m protein in n. benthamiana and were grown in yeb medium (5 g/l beef extract, 1 g/l yeast extract, 5 g/l peptone, 5 g/l sucrose, 2 mm mgso 4 ) at 28 • c with shaking at 250 rpm. when necessary, ampicillin (100 µg/ml) or kanamycin (25 µg/ml) was added to the culture medium. hek-293 cells were cultivated as a monolayer in dmem medium with 10% fetal bovin serum (fbs) and 50 µg/ml gentamicin at 37 • c with 5% of co 2 and relative humidity of 94%. the nucleocapsid (n, genbank protein id. aap33707.1) and membrane (m, genbank protein id. aap33701.1) full-length genes of the human sars-cov frankfurt i isolate, acc. no. ay291315 were cloned into pcr2.1-topo ta cloning vector (invitrogen; carattoli et al., 2005) . the inserted fragments were cut out by digestion with bamhi-noti and sub-cloned into the pqe-30 (qiagen) prokaryotic expression vector . the full-length n gene (1269-bp long) was amplified by pcr on the template plasmid pqe30-n with pfu polymerase with the forward primer 5 -ggccatcgatgaattcggatccatcatgagaggatcgc atcacatcc-3 (clai restriction site: underlined, ecori site: italic, initiation translation codon: bold) and the reverse primer 5 -gacttgtcgacgcggccgctta tgcctgagttgaatcagcag-3 (sali site: underlined, noti site: italic, stop codon: bold). for mammalian cells expression, the pcr product was cut out by digestion with ecori/noti and inserted into the pvax1 vector (invitrogen). for plant expression, the pcr product was cut out with clai/sali and cloned into the ppvx201 plant vector (baulcombe et al., 1995) . in this vector, the full-length viral cdna of the potato virus x (pvx) is inserted between the constitutive 35s promoter of the cauliflower mosaic virus (camv 35s) and the transcription terminator (nos-term) of the nopaline synthase gene of a. tumefaciens, necessary for the regulation of the viral genome expression upon infection with plasmid dna. characteristic components of the viral expression vector are the following: viral replicase gene (rdrp); triple gene block encoding protein for cell-to-cell movement (m1-3); viral coat protein gene necessary for encapsidation of viral rna (cp); coat protein sub-genomic promoter (sgp; figure 1a) . the full-length m gene (666-bp long) from the pcr2.1-topo ta vector was amplified in two subsequent steps. first, the forward primer 5 -ggccatcgatgaattcggatccatcatggcagacaacgg tactattac-3 (clai restriction site: underlined, ecori site: italic, initiation translation codon: bold) and the reverse primer 5 -gtgatggtgatga tgctcgagtgcctgtactagcaaagcaatatt-3 (end of the his 6 tag: italic, end of the m gene: bold) were used to add the his 6 tag at the c-terminus. subsequently, the same forward primer and the reverse primer 5 -gacttgtcgacgcggccgctcaatggtgatggtgatg atgctcg-3 (sali site: underlined, noti site: italic, stop translation codon: bold) were used in order to add restriction sites useful for cloning. for simplicity, we report the name of the recombinant genes n-his 6 and m-his 6 as n and m genes. the purified pcr products were cloned into the eco rv-linearized pbluescript sk(+; pbs) cloning vector (stratagene; pbs-n and pbs-m), sequenced for authenticity and sub-cloned by clai-sali in the ppvx201 plant vector (ppvx-n and ppvx-m) or by ecori/noti in the pvax1 vector (pvax-n and pvax-m). after xl1 blue cells transformation, ppvx-n and ppvx-m plasmid dnas were purified (maxiprep kit, qiagen) for plant infection, while pvax-n and pvax-m plasmids were purified with endotoxin-free purification kits (plasmid maxi kit lps-free, qiagen) for mammalian hek-293 cells transfection. the m gene was also sub-cloned by xbai/sali from the pbs-m construct into the pbi-121 plant binary vector (clontech laboratories, acc. no. af485783), obtaining the pbi-m construct. in this construct, the m gene is inserted between the camv 35s promoter and the nos terminator. characteristic components of the vector are: right and left borders (rb, lb), gene for kanamycin resistance (npt ii) under the transcription promoter and terminator of the nopaline synthase gene of a. tumefaciens ( figure 6a) . the pbi-m construct, obtained by substitution of the gus gene with the m gene, was used to transform the gv3101 and c58c1 strains of a. tumefaciens. the expression and purification of the n and m proteins were done according to standard protocols (the qiaexpressionist, qiagen). since it was previously described that the m protein is toxic in bacteria (carattoli et al., 2005) , culture growth was performed at suboptimal temperature of 30 • c. in these conditions, we isolated a clone able to express the m protein in e. coli. characterization revealed that it corresponded to a spontaneously mutated m gene, coding for a protein carrying the three substitutions k 13 > r, f 36 > l, and i 160 > v (m rlv ). the m rlv and the n proteins were purified by ni-nta affinity chromatography in denaturing conditions. for the n protein, when purified in native conditions, imidazole concentration in the wash buffer was 30 mm. quantification of the n protein purified in denaturing and native condition was performed by coomassie stained sds-page comparing the intensity of the band at 50 kda to known amount of bsa using a chemidoc imagelab system with imagelab 4.0 software (bio-rad). hek-293 cell line was transfected in six wells plates with the pvax-n or pvax-m constructs according to standard protocols (effectene transfection reagent, qiagen). 24 h post-transfection (pt) some wells were added with 12.5 µm of the proteasome inhibitor mg-132. 48 h pt both samples, treated or not with mg-132, were harvested and prepared for protein expression analysis (immunoblotting and immunofluorescence). after three washes with cold phosphate-buffered saline (pbs: 21 mm na 2 hpo 4 , 2.1 mm nah 2 po 4 , 150 mm nacl, ph 7.2) cells were centrifuged at 800 rpm for 5 , recovered and re-suspended in sds-loading buffer (10% glycerol, 60 mm tris-hcl ph 6.8, 0.025% bromophenol blue, 2% sds, 3% 2-mercaptoethanol) and boiled for 10 min. for immunofluorescence analysis, hek 293 cells were grown on multi-chamber glass slides and transfected at 40% of confluency. 24 h post transfection cells were washed three times with pbs, fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% triton x-100 in pbs. samples were blocked with 5% no-fat dry milk in pbs. for detection of the n and m proteins, cells were incubated with specific polyclonal antibodies (pab) validated in immuno-cytochemical assay on sars-cov infected and previously described (carattoli et al., 2005) . for n protein we used a mouse anti-n pab, (obtained after immunization of animals with the purified his 6 -n protein produced in e. coli) at 1:800 dilution. for the m protein we used a mouse anti-m pab (obtained by immunizing mice with the recombinant cytoplasmic domain, amino acids 138-222, of the m protein produced in e. coli) at 1:400 dilution. cells were then incubated with a 1:300 dilution of an anti-mouse biotinylated secondary antibody (ge healthcare) and a 1:50 dilution of streptavidin-fitc conjugated (sigma-aldrich, gmbh, steinheim, germany). nuclei were counterstained with dapi (2 µg/ml) in pbs. slides were examined using a fluorescence "axiolab zeiss" microscope (oberkochen, germany) interfaced with a coolsnap ccd camera (roper scient ., princeton, nj, usa). two leaves of n. benthamiana plants (4 week-old) were dusted with carborundum powder and inoculated with 10 µg of the plasmids ppvx-n, ppvx-m, or ppvx201 (empty vector, negative control) diluted in 100 µl bi-distilled water. plants were also treated with 100 µl bi-distilled water (mock-infected) for monitoring viral infection symptoms. plants were grown under 16 h daylight at 22 • c into a containment greenhouse (bio-safety level 2) and observed daily for pvx infection signs. inoculated and symptomatic leaves were harvested and stored at -80 • c until use. four week-old n. benthamiana plants were infiltrated with a. tumefaciens c58c1 and gv3101 cultures harboring the pbi-m construct that had been grown and induced as described (kapila et al., 1997) . plants were subsequently grown as described and the leaf disks collected 3, 4, 5, 6, 7, and 10 days post-infiltration (dpi) were homogenized with an ultraturrax in three volumes (w/v) of sds-loading buffer and boiled for 3 min. to analyze n and m protein accumulation in plant tissues, soluble proteins were extracted from plant material using different buffers, depending on their hydrophobic properties. crude plant extracts were prepared by grinding the tissue to a fine powder in liquid nitrogen. the powder was re-suspended and homogenized with an ultraturrax in three volumes (w/v) of pbs or, alternatively for the m protein, in one volume (w/v) of gb buffer (100 mm tris-hcl ph 8.1, 10% glycerol, 400 mm sucrose, 5 mm mgcl 2 , 10 mm kcl, 10 mm 2-mercaptoethanol) containing protease inhibitors ("complete, edta-free, " roche diagnostics, gmbh, mannheim, germany). tissue homogenates were centrifuged at 4 • c, 12,000 × g, for 10 min. the supernatant was transferred to a fresh tube and kept on ice (or at 4 • c) until use and total soluble protein (tsp) content was estimated by the bradford assay (bio-rad inc., segrate, italy). pellets were resuspended in appropriates volumes of sds loading buffer, and prepared as described in the paragraph below, constituting the insoluble fraction of leaf extracts. homogenized tissues of ppvx-n infected plants were also used to inoculate n. benthamiana plants to propagate the infectious recombinant pvx particles until the third round of infection. a small-scale purification of plant-produced n protein was performed. in brief, lyophilized leaf material was ground in liquid nitrogen, re-suspended and homogenized with an ultraturrax in 8 m urea, 100 mm nah 2 po 4 , 1 mm tris, ph 8.0, and incubated for 1 h at rt with gentle shaking. leaf material was finally lysed on ice by sonication at 10 hz output (10 s each) for three times. cell debris collected by centrifugation was discarded. the recovered supernatant was filtered (0.45 µm) and incubated for 2 h with the ni-nta resin. plant n protein purification was performed by decreasing the ph (the qiaexpressionist). protein elution was obtained at ph 4.5. for immunoblotting analysis of the n protein, plant extracts containing 20 µg tsp and purified protein were boiled for 3 min in sds-loading buffer. immunoblotting detection of the m protein was performed on plant extracts incubated at 40 • c for 20' in sds-loading buffer. samples were separated by 12% sds-page and transferred onto pvdf membranes (immobilon-p, millipore). after membrane blocking with 5% non-fat dry milk in pbs (mpbs) over night (o/n) at 4 • c, membranes were incubated for 2 h at room temperature (rt) either with an anti-his 6 monoclonal antibody (mab; h1029-clone his-1, sigma) or with specific polyclonal antibodies previously described (carattoli et al., 2005) . for n protein detection, we used a rabbit anti-n pab, (obtained after immunization of animals with the purified his 6 -n protein produced in e. coli) at 1:3000 dilution. immune complexes were revealed by 1 h incubation at rt with an antirabbit biotinylated secondary antibody (b8895, sigma), at 1:5000 dilution, followed by horseradish peroxidase (hrp)-conjugated streptavidin (rpn1231, ge healthcare) at 1:2000 dilution. for m protein detection, membranes were incubated with the mouse anti-m pab at 1:5000 dilution, followed by incubation with an anti-mouse hrp-conjugated secondary antibody at 1:10000 dilution (na931, ge healthcare). for both proteins, the bound antibody was detected using the ecl plus system ("enhanced chemi-luminescence", ge healthcare). the amount of n protein in the plant extracts was estimated by a quantitative triple antibody sandwich (tas) elisa. symptomatic systemic leaves, deriving from 15 plants infected with ppvx-n, were collected and pooled. three independent extractions were performed and analyzed. microtiter plates were coated with 100 µl/well of rabbit anti-n pab (diluted 1:3000 in pbs) for 3 h at 37 • c followed by coating with 100 µl plant extract with a normalized tsp content (50 µg) for 16 h at 4 • c. wells were then blocked with 150 µl/well of 5% mpbs at 37 • c for 3 h. the captured n protein was detected by incubating at 37 • c for 2 h with 100 µl/well of a 1:1500 dilution in 2% mpbs of a mouse anti-n pab (obtained after immunization of animals with the purified his 6 -n protein produced in e. coli, carattoli et al., 2005) followed by incubation with 1:10000 dilution of hrp-conjugated goat anti-mouse igg antibody. after each step, wells were washed three times with pbs + 0.1% tween 20 and one time with pbs. enzymatic activity was measured by adding 100 µl/well v/v h 2 o 2 /abts [2 , 2 -azino bis-(3-etilbenzotiazolin) sulphuric acid] (kpl inc., gaithersburg, md, usa) at rt in darkness condition. the absorbance of the samples was read after 30 at 405 nm on an elisa microtiter plate reader. known amounts of e. coli-purified n protein (0.5, 2, 5, 20, 50, 100, and 150 ng) were used as a standard. in order to give a better estimation of n protein yields in plant, the standard was diluted in n. benthamiana extract. a 'multi-strip' western blot assay was chosen to evaluate the antigenicity of the plant produced n protein. it is an immunoblotting procedure modified from kiyatkin and aksamitiene (2009) , in which the sample, loaded in a single-well polyacrylamide gel (a standard gel where a single preparative well is cast), is blotted onto a membrane that is then cut into strips of the same width, each containing same amount of protein. each strip is then incubated with different antibodies or sera and developed by a colorimetric assay (see ciufolini et al., 1999; di bonito et al., 2002) . in our experiments, single-well 12% sds-page gels were loaded with one of the following preparations: (i) e. coli purified n protein (7.5 µg); (ii) plant-purified n protein (3.7 µg); (iii) plant extract from ppvx-n symptomatic leaves containing about 3.7 µg of n protein and 2 mg tsp; (iv) plant extract from ppvx201 symptomatic leaves containing about 2 mg tsp. samples (iii) and (iv) were prepared as following: the powder ground from ppvx-n or ppvx201 symptomatic leaves was resuspended and homogenized by an ultraturrax in one volume (w/v) of tn buffer (25 mm tris-hcl ph 7.2, 150 mm nacl) containing protease inhibitors. tissue homogenates were centrifuged at 4 • c, 15000 × g, for 15 . tsp was calculated by bradford assay and precipitated by trichloroacetic acid (tca) at 25% final concentration. after 30' incubation in ice, samples were centrifuged, pellets were washed with cold 80% acetone, dried, re-suspended in sds loading buffer. gels were blotted using a semi-dry system (biorad) onto pvdf membranes. after blocking with 5% mpbs, membranes were dried at rt and cut in 15 strips, 0.5 cm width. each strip of samples (ii) and (iii) contained approximately 250 ng of the n protein (as plantpurified protein or in plant extract, respectively). as controls, strips containing 500 ng of e. coli-produced n protein [sample (i)], were prepared, as well as strips of plant extracts without the n protein [extract from ppvx201 symptomatic leaves, sample (iv)]. before incubation with human sera, one strip from either plant-derived or e. coli-expressed n protein was incubated with the mouse anti-n pab, as previously described (carattoli et al., 2005) , to confirm protein presence and to check that the amount of protein in the strips was sufficient for colorimetric detection. the strips were incubated with pools of sars sera collected during the sars outbreak in hong kong in 2003 (chan et al., 2005 ; 86 sars patients pooled in seven groups) at 1:100 dilution in 3% mpbs, o/n at 4 • c. blood samples were collected with informed written consent. the study was approved by the institutional human research ethics committee (the joint chinese university of hong kong-new territories east cluster clinical research ethics committee). as non-sars controls, strips were incubated with three pools of sera from 30 patients affected by unrelated respiratory diseases (carattoli et al., 2005) . antigen-antibodies complexes were revealed by a rabbit anti-human igg (h + l) hrpconjugate (southern biotechnology associates, inc, cat.no.6145-05) diluted 1:5000 in 3% mpbs. after 1 h of incubation at rt colorimetric reaction on the strips was induced by adding 3,3 -diaminobenzidine tetrahydrochloride substrate, dab (sigma d-5637) and hydrogen peroxide. to produce the recombinant sars-cov n protein, n. benthamiana plants (4 weeks old) were infected with the ppvx-n dna plasmid harboring the n gene ( figure 1a) . as a control, plants were either mock infected or infected with the wild type ppvx201 vector. while mock infected plants showed no symptoms, typical symptoms (mainly chlorotic spots) generally appeared on the inoculated leaves of plants infected with ppvx-n or ppvx201 vectors 4-5 days post inoculation (dpi). the infection spread systemically to apical leaves, where symptoms appeared 7-10 dpi. to examine whether the n protein accumulated in infected plants, soluble protein extracts were analyzed by elisa and immunoblotting. interestingly, after infection with the ppvx-n plasmid, protein expression corresponded to symptoms in systemic leaves in all plants analyzed (about 100). this result indicates that the construct is stable and that the recombinant virus pvx-n can spread systemically within the inoculated plant. immunoblotting of soluble protein extracts from plants infected with ppvx-n reveals a single band of about 50 kda in systemic leaves (figure 1b) , suggesting the absence of proteolysis. on the contrary, when the n protein is expressed in e. coli, additional bands of lower molecular mass are present ( figure 1b) . these bands probably correspond to protein degradation, in accordance with previous work demonstrating the intrinsic instability and/or autolysis of this protein when expressed in bacteria (mark et al., 2008) . furthermore, ppvx-n-derived extracts, stored at -20 • c for 2 months and then thawed (figure 1b) , as well as extracts obtained from freeze-dried ppvx-n-infected leaves showed an intact n protein (data not shown). immunoblotting of mammalian cells transfected with the pvax-n plasmid also revealed the presence of a single band of approximately 50 kda corresponding to the n protein (figure 2a) . immunofluorescence revealed a cytoplasmic localization of the n protein ( figure 2b) . plant extracts deriving from pvx-n-infected leaves were also used for subsequent inoculations. n protein expression was confirmed at least until the third cycle of re-infection (supplementary figure s1) , further demonstrating the stability of the construct and of the recombinant virion. the amount of recombinant n protein expressed in leaves, as measured by tas-elisa, was approximately 3-4 µg/g fresh leaf weight, corresponding to 0.2% tsp (figure 3) . although the n protein accumulated mainly in the soluble fraction in all the expression systems used (plant, bacteria and mammalian cells), for purification the best recovery of the n protein from e. coli was obtained in denaturing conditions (3 mg of protein/liter of culture under denaturing conditions versus 0.4 mg protein/liter of culture under native conditions, figure 4a ). therefore, we decided to perform n protein purification in denaturing conditions also from plant tissue. we performed a small-scale purification by loading plant extracts derived from freeze-dried leaves (obtained from a pool including primary-infected and re-infected systemic leaves) on a ni-nta affinity purification column. in this way, we obtained yields of about 1 µg of purified n protein/g of fresh leaf weight ( figure 4b ). the elisa and immunoblot analysis gave a first indication of antigenic features of the plant-derived n protein. in fact, in such analysis the n protein was specifically recognized by rabbit and mouse anti-his 6 -n hyper-immune sera that had previously been shown to recognize the n protein in sars-cov infected cells (carattoli et al., 2005) . to further characterize the n protein expressed in plant, we analyzed its reactivity with sera from sars patients, collected during the sars outbreak in hong kong in 2003. these sars sera were previously screened by an elisa assay with an e. coliexpressed n protein (di bonito and chan, data not shown). here, to evaluate sars-positive sera reactivity with n plant expressed protein, we used a 'multi-strip' western blot assay. in a first experiment, we evaluated the reactivity of the purified n protein with a pool of 5 sars sera. as shown in figure 5a , both preparations of purified n protein, from e. coli and from plant, are recognized by sars sera as well as by the mouse anti-n pab (positive control). then, we validated the results obtained for the plant-purified n protein analyzing its reactivity with other six groups of sars sera deriving from 86 patients and with three groups of sera from 30 patients affected by non-sars respiratory diseases ( figure 5b) . in this experiment, we observed that the n protein purified from plant is specifically recognized by all the groups of sars sera analyzed, while no reactivity was observed with sera from patients affected by other respiratory diseases. we also tested the reactivity of sars and non-sars sera with the unpurified n protein (soluble extract of ppvx-n symptomatic leaves) and with the extract from plant infected with the ppvx201 empty vector. a light reactivity with sars sera was observed only for the unpurified n protein, while no reactivity was observed for the same preparation when using non-sars sera (supplementary figure s2) . importantly, the ppvx201 leaf extract did not react with any human sera (supplementary figure s2) . to our knowledge, this is the first report of the antigenic properties of a plant-derived n protein as revealed by direct serology using sars patient sera, suggesting its possible use for the development of sars diagnostic assays. we investigated the ability of plant expression systems to cope with the synthesis of m protein. we started our studies on m protein expression in n. benthamiana plants (4-week old) by infection with the ppvx-m plasmid harboring the m gene as described for the n protein. also in this case typical symptoms generally appeared 4-5 dpi on inoculated leaves and 7-10 dpi on systemic leaves. however, m protein production was reported in just 2 plants out of 100 analyzed at detectable levels (data not shown). hence, we investigated the possibility to obtain the m protein by agroinfiltration. n. benthamiana leaves were infiltrated with a. tumefaciens suspensions (strains c58 and gv3101) harboring the pbi binary vector containing the m gene (pbi-m vector, figure 6a ). with this simple technology, and without the use of any post-transcriptional gene silencing suppressors, we were able to detect sars-cov m protein production in plant. the time course experiment revealed that the m protein is expressed mainly at 4 and 5 dpi (data not shown). for immunoblotting analysis, the m protein was extracted with an appropriate buffer (gb buffer) and, since it was previously reported that boiling causes m protein aggregation and precipitation (lee et al., 2005) , samples boiling was avoided. interestingly, the m protein accumulated almost totally in the soluble fraction of the plant extract ( figure 6b ). as described in the previous paragraph for the n protein, to better characterize the plant-derived m protein we worked, at the same time, on m protein expression in bacteria and mammalian cells. due to toxic properties of m protein for e. coli (carattoli et al., 2005) , we performed colony selection and m protein expression under sub-optimal growth conditions (30 • c). in this way, the m protein, with a mass of about 25 kda was purified by ni-nta affinity chromatography in denaturing conditions, obtaining yields of about 100 µg/l culture (data not shown). dna sequence analysis revealed the presence in the m gene of three spontaneous point mutations: r 13 > k in the n-terminal domain, l 36 > f in the first transmembrane domain and v 160 > i in the cytoplasmic domain (m rlv ). as the plant-derived recombinant m protein, the m rlv was also specifically recognized by the mouse anti-m pab ( figure 6c ) that had previously validated by immunofluorescence antibody assay (ifa) in sars cov infected vero cells (carattoli et al., 2005) . although we were not able to express the m protein with its original aa sequence in bacteria, the m rlv protein was useful as standard for the plant-produced m protein characterization. immunoblotting revealed that, contrary to prokaryotic cells, the plant system allowed the expression of the full-length m protein, especially by the use of the agrobacterium c58c1 strain ( figure 6b) . the m protein yield, calculated by immunoblotting using the quantified m rlv protein purified from e. coli as standard, was estimated to be 0.1-0.15% tsp. the mobility of the plant-derived m protein in sds-page was reduced compared to the mutated bacterial form (figure 6c) , suggesting a higher molecular mass of plant-expressed compared to the e. coliexpressed m protein. no attempts to purify the m protein from plant were done due to the expression estimates. the m protein was also expressed in mammalian cells. immunofluorescence on m-transfected hek-293 cells using the mouse anti-m pab revealed that m protein is primarily localized in the plasma membrane (figure 7) . similar results were previously reported by tseng and collaborators (tseng et al., 2010) , who described a perinuclear and plasma membrane localization of the m protein expressed in various cell lines. however, we did not observe m protein expression by immunoblotting, either in transfected cells or in the culture medium (not shown), probably because of its low expression level, as we reported in n. benthamiana plants by pvx-mediated infection. the importance to develop effective therapeutic and preventive strategies, to be readily applied to new emergent pathogens is established by the two novel coronaviruses that have emerged in humans in the twenty-first century: severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). both viruses cause acute respiratory distress syndrome and are associated with high mortality rates. there are no clinically approved vaccines or antiviral drugs available for either of these infections, thus their development represents a research priority (graham et al., 2013) . severe acute respiratory syndrome coronavirus was the first massive infectious disease outbreak and it still has the potential to cause large-scale epidemics in the future. the key to preventing and controlling a future outbreak of sars is to develop rapid and specific diagnostic methods so that suspected patients can be correctly assessed. moreover, effective and safe treatment/vaccination will be extremely important in minimizing the damage of a new pandemic. enormous efforts have been undertaken to these purposes. the four major diagnostic methods available for sars include viral rna detection by rt-pcr, virus induced antibodies by immunofluorescence assay (ifa), or by enzyme linked immunosorbent assay (elisa) of nucleocapsid protein (n) and inoculation of patient specimens in cell culture (world health organization [who] , 2003 sars: laboratory diagnostic tests). the sars-cov n protein, expressed at early stage of infection and triggering a strong antibody response by the host, is considered to be the best diagnostic target (surjit and lal, 2008) . plasmon resonance-based biosensors (huang et al., 2009; park et al., 2009 ) and nanowire/carbon nanotube transistors (ishikawa et al., 2009) have been developed for the detection of sars-cov n protein in patient sera. such sensors offer real-time detection of nanomolar concentrations of the protein. nevertheless, sars tests should also have other useful features such as cost-effectiveness and ease of operation. moreover, combinations of antigens may be necessary to provide a definitive diagnosis of sars in humans and susceptible animals (roper and rehm, 2009) . the production of recombinant n protein has been achieved in a variety of heterologous expression systems. a synthetic gene with optimized codons has been expressed in e. coli at high yield (das and suresh, 2006) but it was demonstrated that bacterially expressed n protein produces false seropositivity owing to interference of bacterially derived antigens (leung et al., 2006; yip et al., 2007; surjit and lal, 2008) or cross-reacts with antisera of human coronaviruses (hcov-oc43 and hcov-229e)-infected patients (woo et al., 2004 ). an interesting study correlated the phosphorylation state of the n protein with its antigenicity and specificity of antibodies recognition (shin et al., 2007) . these data underline the importance of producing the recombinant protein in eukaryotic platforms such as insect cells, yeast, or plants to set up more efficient and specific diagnostic tests. to date, it was demonstrated that the n protein produced in insect cells may be useful for the development of highly sensitive and specific assays to determine sars infection (shin et al., 2007) . later, the n protein was transiently expressed in plant by agroinfiltration, and its antigenicity was demonstrated in mice (zheng et al., 2009 ) giving a proof of concept of its use in vaccine formulations. previously, the s1 domain of sars-cov s protein had been stably expressed in tomato and low-nicotine tobacco plants obtaining about 0.1% tsp (pogrebnyak et al., 2005) . the plant-derived antigen was able to induce systemic and mucosal immune responses in mice. while previous works have primarily focused on the s and n proteins, there is growing evidence of the potential of the m antigen both as an effective vaccine and diagnostic candidate. thus, the obtainment of the recombinant full-length m protein in an eukaryotic expression system also represents a good way to develop an effective and safe sars-cov vaccine. in addition to the knowledge that the m protein elicits a strong humoral responses, and that a specific humoral and cellular immune response can be obtained by co-expressing s, m and e (lu et al., 2007) , it has been demonstrated that the m protein also contains t cell epitopes (liu et al., 2010) . the availability of recombinant m protein, in combination with other viral proteins might overcome the concern about the sensitivity and the specificity of n nucleoprotein-based assay, as described when using the n and the s proteins together (woo et al., 2004; haynes et al., 2007; gimenez et al., 2009) . this would help the development of more efficient reagents to detect antibodies in the infected human host. here, we propose the use of plants as an alternative system to produce the n and m antigens that could be useful to formulate new vaccines and diagnostic assays against sars. since plant transformation and regeneration of stable transformants require considerable time, we used transient expression systems (pvx and agroinfiltration) to evaluate the ability of the plant expression system to cope with the synthesis of the sars-cov m and n proteins. the n and m full-length genes of the human sars-cov frankfurt i isolate were cloned, without codon optimization, into different expression vectors. for the sars-cov n protein, we assessed the successful ectopic expression in n. benthamiana plants by ppvx-mediated infection (figure 1) . we were able to obtain the n protein in systemic leaves in most primaryinfected plants as well as in 100% re-infected plants. these results demonstrate the stability of the construct, a condition not easily obtained especially when large sequences are inserted in the ppvx-derived expression cassette (the n gene is about 1300 bp). in fact, several studies report that the use of 'first generation' plant viral vectors (like the ppvx series) for the expression of proteins in plants can lead to insert elimination by natural selection over replication cycles as early as the first infection passage with a positive correlation between insert length and elimination rate (avesani et al., 2007) . unlike the observed prokaryotic expression pattern, no proteolysis products were detected in immunoblotting by using polyclonal sera in ppvx-n-derived extracts, fresh or stored at -20 • c for 2 months ( figure 1b) . these data demonstrate the stability of the recombinant n protein when transiently expressed in n. benthamiana. the same stability was observed when the n protein was expressed in mammalian cells, even in the absence of proteasome inhibitor (figure 2a) . the purified plant-produced n protein is specifically recognized by sera from chinese sars patients of the 2003 outbreak, and not from patients affected by unrelated respiratory diseases (figure 5) . this result suggests that the plant-expressed sars-n protein is suitable in sars diagnosis. it is interesting to note that, when using crude plant extracts, sars human sera reveal a weak band, corresponding to the molecular weight of the n protein, without any cross-reaction with other components of the plant extract (supplementary figure s2 ). taken together, the results indicate that plant-derived n protein is specifically detected by antibodies of sars patients, using an assay where the antigen-antibody complex is revealed by a colorimetric method, less sensitive but more specific and suitable for hospital clinical laboratories. it should be noted that the only information available to date about plant-derived sars-cov n protein antigenicity were from zheng et al. (2009) who immunized mice and evaluated the impact on the regulation of cytokines and on the elicited igg subclasses. our study is the first demonstration by direct serology that plant-derived n protein is able to reveal human n-specific antibodies present in sera of sars patients, thus providing an adequate instrument to develop a rapid, low-cost, immune-based diagnostic assay to be used as an alternative or in association to molecular diagnosis. for the m protein, we obtained a spontaneously mutated m rlv protein in e. coli. this allowed to overcome toxicity of the wild type protein when expressed in bacteria (carattoli et al., 2005) and to purify the m rlv protein that was then used as positive control in our experiments. as the sars-cov m protein is difficult to express in recombinant form, the exact structure and function of the protein are not fully elucidated (neuman et al., 2011; tseng et al., 2013; siu et al., 2014) . unlike prokaryotic cells, the plant system allowed the expression of the full-length m protein by using a. tumefaciens (in particular the strain c58c1), demonstrating for the first time the possibility to express the sars-cov m protein in plants, without the need of codon optimization or addition of any further modifications. on the contrary, by pvx-mediated infection we observed very low expression levels for the m protein, only in 2 plants out of 100 analyzed. we can speculate that membrane m protein, that already proved to be toxic in e. coli, may be difficult to express by using a 'living vector' like pvx due to interference with virus replication and/or expression/assembly of viral components, while it is tolerated by the plant when expressed by agro-infiltration. compared to the mutated m rlv protein produced in e. coli, the m protein produced in plant shows a reduced electrophoretic mobility, suggesting a higher molecular mass ( figure 6c) . the reason for this difference remains to be elucidated, but it could be due to modified residues in the m rlv protein or by the presence of glycosylation in the m protein produced in the eukaryotic system (the native protein is n-glycosylated at the fourth residue). the plant-produced m protein yields were not sufficient to perform the test with human sera. thus, for plant-derived m protein characterization, efforts should be made in order to enhance expression yields. this includes the use of secondgeneration viral vectors (gleba et al., 2007) or other implemented platforms that have been developed in the last years (moustafa et al., 2015; paul et al., 2015; sack et al., 2015) . our results add further insights to the characterization of the n and m proteins and provide a proof of principle for using plants as a robust, rapid and flexible production system for protein reagents suitable to face potential recurring sars-cov outbreaks. rf, pdb concept and design of the research, analysis and interpretation of data, writing and revising the article, final approval of the version to be published. ocd, sm design of the research, acquisition of data, analysis and interpretation of data, drafting and writing the article. ei acquisition of data, analysis and interpretation of data. ddm writing and revising the article critically for important intellectual content. pksc design and revising the article critically for important intellectual content and final approval of the version to be published. the work was partially supported by the italian 'ministero della salute, ' grant 'progetto speciale lotta alla sars.' an update on middle east respiratory syndrome: 2 years later stability of potato virus x expression vectors is related to insert size: implications for replication models and risk assessment who pillories drug industry on failure to develop ebola vaccine structural proteomics of the sars coronavirus: a model response to emerging infectious diseases jellyfish green fluorescent protein as a reporter for virus infections recombinant protein-based elisa 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nucleocapsid protein expressed in insect cells: the effect of phosphorylation on immunoreactivity and specificity suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain plant-produced candidate countermeasures against emerging and reemerging infections and bioterror agents the epidemiology, evolution and recent outbreaks of avian influenza viruses in china: a review molecular targets for diagnostics and therapeutics of severe acute respiratory syndrome the sars-cov nucleocapsid protein: a protein with multifarious activities identifying sars-cov membrane protein amino acid residues linked to viruslike particle assembly self-assembly of severe acute respiratory syndrome coronavirus membrane protein longitudinal profile of immunoglobulin g (igg), igm, and iga antibodies against the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in patients with pneumonia due to the sars coronavirus severe acute respiratory syndrome (sars): laboratory diagnostic tests library cataloguing in publication data persistent memory cd 4+ and cd 8+ t-cell responses in recovered severe acute respiratory syndrome (sars) patients to sars coronavirus m antigen naturally occurring anti-escherichia coli protein antibodies in the sera of healthy humans cause analytical interference in a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for serodiagnosis of severe acute respiratory syndrome boosted expression of the sars-cov nucleocapsid protein in tobacco and its immunogenicity in mice we thank dr. alessandra carattoli from 'istituto superiore di sanità' for coordinating the sars project. we also thank orsola bitti for technical assistance. the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016. 00054 key: cord-355924-8sk9al0n authors: allam, loubna; ghrifi, fatima; mohammed, hakmi; el hafidi, naima; el jaoudi, rachid; el harti, jaouad; lmimouni, badreddine; belyamani, lahcen; ibrahimi, azeddine title: targeting the grp78-dependant sars-cov-2 cell entry by peptides and small molecules date: 2020-10-21 journal: bioinform biol insights doi: 10.1177/1177932220965505 sha: doc_id: 355924 cord_uid: 8sk9al0n the global burden of infections and the rapid spread of viral diseases show the need for new approaches in the prevention and development of effective therapies. to this end, we aimed to explore novel inhibitor compounds that can stop replication or decrease the viral load of the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2), for which there is currently no approved treatment. besides using the angiotensin-converting enzyme (ace2) receptor as a main gate, the cov-2 can bind to the glucose-regulating protein 78 (grp78) receptor to get into the cells to start an infection. here, we report potential inhibitors comprising small molecules and peptides that could interfere with the interaction of sars-cov-2 and its target cells by blocking the recognition of the grp78 cellular receptor by the viral spike protein. these inhibitors were discovered through an approach of in silico screening of available databases of bioactive peptides and polyphenolic compounds and the analysis of their docking modes. this process led to the selection of 9 compounds with optimal binding affinities to the target sites. the peptides (satpdb18674, satpdb18446, satpdb12488, satpdb14438, and satpdb28899) act on regions iii and iv of the viral spike protein and on its binding sites in grp78. however, 4 polyphenols such as epigallocatechin gallate (egcg), homoeriodictyol, isorhamnetin, and curcumin interact, in addition to the spike protein and its binding sites in grp78, with the atpase domain of grp78. our work demonstrates that there are at least 2 approaches to block the spread of sars-cov-2 by preventing its fusion with the host cells via grp78. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a new virus strain of the coronaviridae family. coronaviruses (covs) are enveloped viruses with a singlestranded positive-sense rna containing approximately 30 000 base pairs (bps). 1 covs are involved in many diseases affecting the upper respiratory tract, gastrointestinal system, and central nervous system in humans and animals. 1 two groups of proteins characterize covs: structural proteins, including spike (s), envelope (e), membrane (m) and nucleocapsid (n), [2] [3] [4] and nonstructural proteins, such as proteases (nsp3 and nsp5) and rdrp (nsp12). 5, 6 the cov s glycoprotein is a precursor protein composed of 1300 amino acids, located on the outer envelope of the virion. it plays an essential role in the attachment, fusion, and entry of the virus into the host cells. particularly, s protein mediates the fusion of the viral and the cellular membranes 7 by binding to different surface receptors of the host cells via its receptor-binding domain (rbd). the transmembrane (s) spike trimeric glycoprotein of the virus consists of 2 functional subunits: s1 and s2. the first, which made up of 4 domains (s1 a, s1 b, s1 c, and s1 d), contributes to the attachment of the virus to the host cell receptor. then, s2 coordinates the fusion of the 2 membranes. 8 this fusion activates proteases and allows the proteolytic cleavage of the s protein. 9 the latter causes conformational changes to prime the s2 subunit for the fusion of viral and cell membranes. 8 thus, rbd is the key component by which the virus is translocated inside the cells through its interactions with the angiotensin-converting enzyme (ace2), 10, 11 dipeptidyl peptidase-4 (dpp4) and or glucose-regulating protein 78 (grp78). 12 the sars-cov-2 rbd contains 13 disulfide bonds that correspond to 13 cyclic regions considered to be similar to the cyclic form of pep42. 13 these regions can interact with the grp78 cell surface. the grp78 or binding immunoglobulin protein (bip) is a member of the heat shock protein family. 14 it is traditionally regarded as a major chaperone of the endoplasmic reticulum (er) that facilitate protein folding and assembly, protein quality control, ca 2+ binding, and regulating er stress signaling. 15 when grp78 moves to the surface of the cell, it can interact with many ligands or other proteins like a multifunctional receptor. it is therefore involved in inflammatory and autoimmune diseases [16] [17] [18] and overexpressed in various human cancers, including prostate cancer, breast cancer, lung cancer, melanoma, and ovarian cancer. [19] [20] [21] in addition to its role in the proliferation, invasion, and metastasis of many cancer cells, grp78 also has a sensitivity to the recognition of viruses through its substrate-binding domain (sbd) 6 and is involved in the assembly of their envelope proteins. [22] [23] [24] [25] recently, grp78 has been recognized as an attachment factor for the middle east respiratory syndrome coronavirus (mers-cov), which improves the entry of the virus in the presence of dpp4, 12 and as one of the sars-cov entry receptors in the human cell. 1 most research carried out, or measures taken, are reposed on the idea that ace2 is the primary receptor of sars-cov-2. 10, 11 providing grp78 as a second receptor in the presence of special physiological conditions when the expression of grp78 is high, [16] [17] [18] [19] [20] [21] this could potentially be more favorable to infection with sars-cov-2. thus, the presence of ace2 and grp78 at a high quantity could classify these additional categories to very high-risk types. as there are no efficacy tools to fight against this disease, it is therefore essential to find out how to protect persons with chronic illnesses (cancer, diabetes, and high blood pressure). these people are at greater risk of developing an aggressive form of sars-cov-2 infection. like sars-cov and mers-cov, sars-cov-2 is a member of betacoronavirus 5,26 which also causes a severe respiratory tract infection with a higher mortality rate. 12, 27 sequence and structure analysis of s proteins from different cov strains showed that the specificity of interaction between s proteins and their receptors is the main determinant of the host tropism of these viruses. 28 the main cellular receptors for viral s proteins include ace2 and dpp4, as well as other molecules that may be involved in the interaction between the virus and the host cell. 29, 30 it has been suggested that grp78 may also act as another receptor that assists sars-cov-2 to penetrate the host cells. 13 this same proposition has confirmed by aguiar et al, 31 who concluded that the grp78-binding site overlaps with the ace2-binding site, although the residues involved in the interactions may be a little different. for this purpose, a targeted analysis of the expression of candidate genes involved in sars-cov-2 infection confirmed the presence of the grp78 protein in vitro in epithelial cells of the human respiratory tract and lung tissue. 31 this analysis suggests that for ace2 to be an integral receptor for sars-cov-2, there are probably other mechanisms dynamically regulating the expression of ace2 in the respiratory mucosa in the context of infection with sars-cov-2 32 and/or possibly other co-receptors. these can contribute to the functioning of ace2 and tmprss2 in the binding and fusion of sars-cov-2 in this cell type. 31 in the same context, palmeira et al 33 have verified that the level of expression of the grp78 gene is high in the blood of patients with sars-cov-2 pneumonia. the entry of the virus is a key step that can be targeted by a possible therapeutic strategy opting for the prediction of a molecule capable of causing simultaneous inhibition of the 2 proteins. 34, 35 based on the knowledge gained about the sars-cov-2 virus, 2 axes of investigation were explored in this study to prevent the translocation of the virus inside host cells. the first was the inhibition of grp78 recognition by sars-cov-2, by targeting both the nucleotide-binding domain (nbd) and sbd of grp78 with atp-competitive small molecules and/or peptides. the second consisted of the inhibition of the viral s protein of sars-cov-2 at the grp78-binding site. finally, we identified 9 compounds that could act as potential blockers of sars-cov-2 cell entry through grp78 for further consideration as possible therapies against this virus. a library of 100 active phytochemicals (polyphenols) [36] [37] [38] [39] was generated from pubchem database (pubchem.ncbi.nlm.nih. gov). biologically active peptides were obtained from the structurally annotated therapeutic peptides database (satpdb) (https://doi.org/10.1093/nar/gkv1114). two binding domains characterize the structure of grp78. the first is the nbd that houses atp, whereas the second (sbd) receives the substrate (peptide or protein) in the form of an excluded segment or partially folded protein. 40 the inhibition of the atp-binding site interrupts the functional cycle of the protein by modifying its conformation, which may lead to the inhibition of viral penetration. atp docking was conducted on grp78 (pdb id:5f1x) structure to identify the key residues of its atpase site. 41 the residues (asp-34, thr-38, tyr-39, ile-61, glu-201, asp-224, phe-258, gly-228, gly-249, asp-250, gly-364, ser-365, ile-368, and asp-391) were used as references for the search space determination. similarly, it was considered advisable to prevent the spike from being attached to the grp78 by directly targeting their interaction site. the grp78 residues involved in spike binding are ile-426, thr-428, val-429, val-432, thr-434, phe-451, ser-452, val-457, and ile-459 as was previously described. 14 according to the study by ibrahim et al, 13 grp78 binds to the sars-cov-2 spike in 4 regions, in which regions iii allam et al 3 (c391-c525) and iv (c480-c488) showed stronger affinities for the spike. consequently, it was proposed in our study to target these 2 regions to prevent any possibility of spike binding to the grp78 receptor. structures of the grp78 (pdb id: 5e84) and sars-cov-2 spike (pdb id: 6lzg) were downloaded from the rcsb pdb database (http://www.rcsb.org/pdb/home.do) in pdb format. the small-molecule sdf files (molecule.sdf ) downloaded in the previous step were converted to pdb format using open babel. 42 all inhibitors and receptors have been optimized and converted to pdbqt format using the standard protocol of the autodock tools. 43 molecular docking of small molecules (atp and inhibitors) was performed using autodock vina 1.1.2. docking of atp on grp78 was conducted to determine the key residues of its atpase site. docking search space was defined on each targeted site of grp78 and spike proteins as explained previously. the cluspro server 44, 45 was used for peptide-protein docking of the selected peptides. the resulting peptide-protein complexes were ranked by cluster size and visual inspection. small molecules and peptides were selected for each protein site based on their estimated binding energy and the protein residues with which they are to interact. visual analysis of docking poses and image rendering was performed using pymol version 2.3 (schrodinger, llc). the global burden of infections and the rapid spread of viral diseases point to the need for new preventive approaches and more effective therapeutic strategies. therefore, particular priority is being given to the emerging sars-cov-2, which is considered a pandemic under current circumstances. in this direction, our study focused on the repositioning of approved drugs as well as the investigation of other bioactive compounds that may prevent the penetration of sars-cov-2 into host cells by targeting the region of grp78 that is required for the interaction with the spike protein of the virus. two factors have guided our compound selection process. the first is the identification of peptides that can block the interaction of the sars-cov-2 spike and grp78, and the second is the screening of small molecules suitable for docking inside grp78's atp-binding pocket to inhibit its activity and thus its interaction with the spike. the effect of the selected phytochemicals and peptides on sars-cov-2 spike protein and grp78 was elucidated by molecular docking analysis. the promising candidates were selected based on their binding mode and affinity against the 2 targets. peptide-protein docking performed between the peptides and their designated targets. the peptides docked against the spike protein and its binding sites in grp78. results from their binding modes analysis were favorable, indicating that the selected compounds could potentially inhibit or prevent the entry of the virus. the sequences of the selected peptides are presented in table 1 . the peptide satpdb18674 selected to target the spike protein was mainly bound to region iv (c480-c488), whereas satpdb12488 and satpdb28899 showed more affinity to region iii (c391-c525) ( figure 1 ). thus, this could directly interfere with the spike/grp78 interaction as both regions are necessary for the spike to recognize the grp78. peptides reported as inhibitors of grp78 (satpdb18674, satpdb18446, satpdb12488, satpdb14438, and satpdb28899) were mainly bound to the region i426-i459 ( figure 2 ). this region of grp78 appears to be critical for the spike to attach to the host cell. these results revealed that the peptides satpdb18674, sat-pdb12488, and satpdb28899 had strong affinities simultaneously with the spike and with both regions of grp78 (regions iii and iv). we therefore hypothesize that these peptides can prevent the entry of the virus by binding either to regions iii and iv of the spike or to their binding site in grp78. the peptide satpdb18674 was particularly bound to the key residues ( figure 3 ) at the grp78 target site (val-429, analysis of the molecular interactions of the peptides stabilizes the target protein by interacting with the residues of their different binding pockets. several hydrogen bonds have been observed, ensuring strong bonds with the spike binding site residues (regions iii and iv) and with those of grp78. the residues (ile-13, ser-12, and thr-9) of satpdb14438 interacted with grp78 residues thr-434, val-429, and thr-458. finally, the peptide satpdb28899 residues asp-20, leu-19, ser-16, and thr-15 interacted with val-429 and thr-458 from the same region. the 5 compounds established multiple hydrogen bonds with the residues of the grp78 substrate-binding pocket. interactions with v429 and s452 were observed in the 5 peptides, except for satpdb28899 (v429 only). satpdb12488, satpdb14438, and satpdb18446 interacted with thr-434; satpdb14438 and satpdb28899 interacted with gln-449; satpdb14438 and satpdb28899 interacted with thr-458; and satpdb12488 interacted with lys-447 and phe-451. grp indicates glucose-regulating protein. table 2 illustrates the details of the interactions between the proteins and peptides selected. the screening of polyphenols was performed against the spike protein, its binding site in grp78, and the atpase domain of grp78. postdocking analysis showed favorable binding modes, indicating that the compounds epigallocatechin gallate (egcg), homoeriodictyol, isorhamnetin, and curcumin could bind simultaneously to the 3 sites. the 4 polyphenols have established multiple hydrogen bonds with the residues of the grp78 pocket ( figure 4) . also, these compounds showed high affinity toward the regions iii and iv of the spike protein ( figure 5 ). the docking results of the compounds egcg, homoeriodictyol, isorhamnetin, and curcumin in the atp-binding site of grp78 showed their favorable positioning inside the receptor's cavity, mimicking atp in its mode of interaction with the atpase domain of grp78 ( figure 6 ) with binding affinities higher than or close to that of atp. therefore, these compounds could potentially interfere with the virus entry to the host cells. finally, as covs acquire the ability to bind to multiple receptors, and cross the interspecies barrier, this work could provide new insights in the development of effective therapies against sars-cov-2 infections. the set of interactions between the small molecules and the active sites of the 2 targets (atpase domain, grp78/spike interaction sites) is summarized in table 3 . the best affinity was attributed to egcg at all the interaction sites. binding to egcg regularly folds the protein while atp binding unfolds the protein. a slightly unfolded form is the native functional form of grp78. thus, due to the link of the egcg and the movements with sbd, grp78 will not be able to perform its appropriate functions given the changes in conformation. this may be one of the likely mechanisms by which the egcg inhibitor acts on the full-length grp78 protein. the several researches show that polyphenols as an antiviral and antiinflammatory agent that can be helpful for both prevention and treatment of new emerging cov. 46 however, well-designed clinical trials are needed to demonstrate the potential efficacy of curcumin against sars-cov-2 infection and its ensuing complications. 47 current drugs have limited efficacy in the treatment of sars-cov-2, given the high number of deaths caused by this virus. designing new drugs that target specific activities of this virus or stop the stages of its infection cycle is a crucial step. due to the diverse behavior of sars-cov-2 depending on the population type, the production of an appropriate vaccine against this disease is difficult, hence the need for new therapeutic strategies, 48 proposing compounds affecting different stages of the virus's life cycle, and strategies that target the entry of the virus on the ace2 side and others on the sars protease side. 49 our study will complement these strategies by targeting the different sites involved in the sars/grp78 interaction (figure 7) . the neglect of the latter causes a high risk of infections. the set of molecules (4 polyphenols and 5 bioactive peptide) was selected in this study to inhibit the sars-cov-2 spike/grp78 interaction. satpdb18674 and egcg gave the best results for all the targeted sites for the 2 proteins. our results, therefore, meet an urgent need, given the absence of an effective drug available. as demonstrated in this combined approach, screening and reassigning bioactive molecules to prevent the entry of this virus on the spike/grp78 side can provide an accelerated approach to identify and develop new treatments for sars-cov-2 infection. inhibition of the interaction between the spike protein sars-cov-2 and the receptor by blocking the grp78 is a strategy interesting to identify drugs that decrease the rate of viral infection. 13 several studies have given confirmatory evidence to the presence of the grp78 protein in patients infected with sars-cov-2. [31] [32] [33] in addition, grp78 is part of receptors that allow the virus to enter and facilitate the initial infection of host cells, 31 the reason why it has become a molecular target to treat covid-19. these results, as well as those of the aforementioned studies, allow us to suppose that the grp78 protein could be a receptor for sars-cov-2 and to propose molecules capable of hindering this interaction. reducing the level of expression or inhibition of grp78 by small interfering rnas or by sirnas, respectively, blocked the entry of japanese encephalitis virus 50 replication. 51 a study recently showed by virtual screening that there are molecules that can in silico inhibit the grp78 protein and prevent virus binding. 33 interestingly, our results also suggest a compound capable to simultaneously inhibit the 3 binding sites that allow entry of sars-cov-2 via grp78. the peptides proposed in this work targets 2 regions of the s protein (iii and iv) and 1 of grp78. this interaction could interfere with or competitively bind to the s protein s sites of sars-cov and grp78. these results suggest a novel inhibitor mechanism distinct from the anti-sars-cov peptide that could disrupt the virus-host cell interaction. we propose, therefore, to combine 2 or 3 of these peptides before or after infection with the virus. inhibition of atpase activity of the grp78 nbd 52 is another potential inhibitory mechanism of sars-cov-2 infection. studies have previously reported that after competitive inhibition at the atp-binding site of grp78, the sbd of the latter adopts a conformation having a low affinity for the substrates, thus blocking their transport. 53 binding of various inhibitors to the atp-binding site established by crystallographic studies 54 allows altering the function of grp78 and, therefore, theoretically avoids infection with sars-cov-2. pretreatment of grp78 with the small molecule egcg inhibited the ebola virus infection, 51 what supports our results obtained where egcg is suggested to have an inhibitory effect on grp78 with high affinity (-10.5 kcal/mol). interestingly, a recent study showed by molecular docking that certain natural products (estrogen and phytoestrogens) could interfere with the attachment of sars-cov-2 to stressed cells. 55 as for the natural products (egcg, homoeriodictyol, isorhamnetin, and curcumin) selected in our study, they could also interfere with this attachment. there are several proofs on the antiviral potential of herbal compounds. 56 pharmacological investigations have revealed many pharmacological activities for certain phytochemicals as the egcg, and curcumin. reducing the charge and inhibiting the expression of viral core proteins are the main effects of polyphenols. 57, 58 these effects are of great interest for the development of synthetic drugs against sars-cov-2-specific polyphenols. the inhibitory effect of curcumin on sars-cov replication has been demonstrated by wen et al 59 . likewise, the impact of curcumin on cells before or after a viral infection reach the infectivity of certain viruses. 60 recently, mooring studies revealed that curcumin could potentially inhibit ace2 to remove covid-19 entry in the cell (potential effect). these results and that shown in our work are proof that confirms the potential role of curcumin as a promising antiviral agent. grp78 has a strong affinity for hydrophilic or hydrophobic regions, such as those of the sars-cov-2 spike. 13 as grp78 can act as a co-receptor for sars-cov-2, 61,62 compounds with a high affinity could hinder this interaction, by competing molecules have established at least 6 hydrogen bonds with these residues in grp78. the best affinity was attributed to egcg (-10.2 kcal/mol), higher than that of atp (-9.6 kcal/mol). all molecules interact through hydrogen bonds with gly-228, phe-258, asp-224 residues; egcg established 3 other h bonds with gly-364, ser-365, and ile-368. homoeriodictyol, isorhamnetin, and curcumine interact through hydrophobic bonds with thr-38 and thr-39. egcg indicates epigallocatechin gallate; grp78, glucose-regulating protein 78. with the viral spike 55 ; this specificity increases the probability of the compounds selected in our study to rival with the spike and grp78, thus preventing the entry of sars-cov-2 and the resulting infection. drug repositioning is an effective and rapid strategy for providing therapeutic solutions to covid-19. in silico approaches can be very useful in identifying new indications for approved drugs whose pharmacokinetic data are already known, allowing them to move rapidly to the final phases of clinical trials. in this work, we identified 4 phytochemicals (polyphenols) that could potentially inhibit the interaction of sars-cov-2 spike protein with grp78, in addition to 5 peptides (sat-pdb18674, satpdb18446, satpdb12488, satpdb14438, and sat-pdb28899) that target simultaneously the spike protein and its binding region in grp78. the satpdb18674 and egcg gave the best results for all of the targeted sites for the 2 proteins. as these results appear very promising, further bioassays are needed to confirm the inhibitory activity of these compounds against sars-cov-2 infection. figure 7 . the strategy description targeting the different sites involved in the sars/grp78 interaction. chemical molecules have been extracted from compound libraries (pubchem and satpdb). screening of these molecules (100 polyphenols and 40 peptides) was carried out by the docking method by autodock vina and cluspro server. this method offers a precise and efficient anchoring tool, which is based on empirical notation functions. it is based on the average binding energy scores in autodock vina, 43 and the root-mean-square deviation (rmsd) in cluspro to generate clusters with the most probable models of the complex. selection of structures based on energy minimization. grp78 indicates glucose-regulating protein 78. coronavirus spike proteins in viral entry and pathogenesis response of bacterial and fungal soil communities to chinese fir (cunninghamia lanceolate) long-monoculture plantations term vaccines for the prevention against the threat of mers-cov prospects for a mers-cov spike vaccine quantitative structure-activity relationship and molecular docking revealed a potency of anti-hepatitis c virus drugs against human corona viruses grp78: a cell's response to stress structure, function, and evolution of coronavirus spike proteins towards treatment planning of covid-19: rationale and hypothesis for the use of multiple immunosuppressive agents: anti-antibodies, immunoglobulins, and corticosteroids the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs coronaviruses post-sars: update on replication and pathogenesis middle east respiratory syndrome coronavirus and bat coronavirus hku9 both 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specific cell subsets across tissues preliminary virtual screening studies to identify grp78 inhibitors which may interfere with sars-cov-2 infection alteration and analyses of viral entry with library-derived peptides molecular screening and docking analysis of lmtk3and akt1 combined inhibitors effects of polyphenol compounds on influenza a virus replication and definition of their mechanism of action the antimicrobial and antiviral activity of polyphenols from almond (prunus dulcis l.) skin in silico inhibition studies of axl kinase by curcumin and its natural derivatives curcumin-synthetic analogs library screening by docking and quantitative structure-activity relationship studies for axl tyrosine kinase inhibition in cancers probing the atp site of grp78 with nucleotide triphosphate analogs three-dimensional structure prediction of the human lmtk3 catalytic domain in dyg-in conformation open babel: an open chemical toolbox autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading the cluspro web server for protein-protein docking cluspro peptidock: efficient global docking of peptide recognition motifs using fft natural product-derived phytochemicals as potential agents against coronaviruses: a review potential effects of curcumin in the treatment of covid-19 infection coronavirus disease 2019 (covid-19): a perspective from china repurposing of known anti-virals as potential inhibitors for sars-cov-2 main protease using molecular docking analysis grp78 is an important host factor for japanese encephalitis virus entry and replication in mammalian cells hspa5 is an essential host factor for ebola virus infection â��)â��epigallocatechin gallate overcomes resistance to etoposide-induced cell death by targeting the molecular chaperone glucose-regulated protein 78 glucose-regulated protein 78 substratebinding domain alters its conformation upon egcg inhibitor binding to nucleotide-binding domain: molecular dynamics studies adenosine-derived inhibitors of 78 kda glucose regulated protein (grp78) atpase: insights into isoform selectivity natural products may interfere with sars-cov-2 attachment to the host cell anti-infective properties of the golden spice curcumin identification of potent covid-19 main protease (mpro) inhibitors from natural polyphenols: an in silico strategy unveils a hope against corona an in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus inhibition of enveloped viruses infectivity by curcumin grp78, a coreceptor for coxsackievirus a9, interacts with major histocompatibility complex class i molecules which mediate virus internalization japanese encephalitis virus co-opts the er-stress response protein grp78 for viral infectivity key: cord-354030-8tfg881h authors: dong, rong; chu, zhugang; yu, fuxun; zha, yan title: contriving multi-epitope subunit of vaccine for covid-19: immunoinformatics approaches date: 2020-07-28 journal: front immunol doi: 10.3389/fimmu.2020.01784 sha: doc_id: 354030 cord_uid: 8tfg881h covid-19 has recently become the most serious threat to public health, and its prevalence has been increasing at an alarming rate. the incubation period for the virus is ~1–14 days and all age groups may be susceptible to a fatality rate of about 5.9%. covid-19 is caused by a novel single-stranded, positive (+) sense rna beta coronavirus. the development of a vaccine for sars-cov-2 is an urgent need worldwide. immunoinformatics approaches are both cost-effective and convenient, as in silico predictions can reduce the number of experiments needed. in this study, with the aid of immunoinformatics tools, we tried to design a multi-epitope vaccine that can be used for the prevention and treatment of covid-19. the epitopes were computed by using b cells, cytotoxic t lymphocytes (ctl), and helper t lymphocytes (htl) base on the proteins of sars-cov-2. a vaccine was devised by fusing together the b cell, htl, and ctl epitopes with linkers. to enhance the immunogenicity, the β-defensin (45 mer) amino acid sequence, and pan-hla dr binding epitopes (13aa) were adjoined to the n-terminal of the vaccine with the help of the eaaak linker. to enable the intracellular delivery of the modeled vaccine, a tat sequence (11aa) was appended to c-terminal. linkers play vital roles in producing an extended conformation (flexibility), protein folding, and separation of functional domains, and therefore, make the protein structure more stable. the secondary and three-dimensional (3d) structure of the final vaccine was then predicted. furthermore, the complex between the final vaccine and immune receptors (toll-like receptor-3 (tlr-3), major histocompatibility complex (mhc-i), and mhc-ii) were evaluated by molecular docking. lastly, to confirm the expression of the designed vaccine, the mrna of the vaccine was enhanced with the aid of the java codon adaptation tool, and the secondary structure was generated from mfold. then we performed in silico cloning. the final vaccine requires experimental validation to determine its safety and efficacy in controlling sars-cov-2 infections. in december 2019, covid-19, caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was first discovered in china and has rapidly spread across the world. as of 12:00 noon on june 4, a total of 6,392,319 confirmed cases of covid-19 have been reported globally, including 383,318 deaths. the prevalence of the disease has been increasing at an alarming rate. there were 1,849,852 cases in the united states, 555,383 in brazil, 431,715 in russia, 281,270 in the united kingdom, and 3,275,736 in a number of other countries (1) . the incubation period for the virus is ∼1-14 days, and all age groups are susceptible to a fatality rate of about 5.9%. the most common clinical manifestations are low-grade fever, dry cough, fatigue, and gastrointestinal symptoms (2) . about half of all patients with covid-19 develop shortness of breath, and severe cases may rapidly develop sars, septic shock, difficult-to-correct metabolic acidosis, and coagulation disorders (3) . covid-19 may also affect other organs, most commonly the heart and kidneys (4) (5) (6) . some patients may have mild symptoms, without fever, and may recover after 1-4 weeks (7) . other patients may show signs of serious illness and some may die; however, most patients show favorable progress (8) . male individuals with the disease and aged patients have the worst prognosis. in children, the disease is relatively mild (9) . covid-19 is caused by a novel single-stranded, positive (+) sense rna beta coronavirus, which is a pathogen of the coronaviridae family, named sars-cov-2 (10) . the full-length genome sequences revealed that sars-cov-2 has the greatest genetic similarity to bat coronavirus, ∼45-90% similarity to severe acute respiratory syndromerelated coronavirus (sarsr-cov), and a smaller similarity of 20-60% to the middle east respiratory syndrome-related coronavirus (mers-cov) (10) . thus, a bat might be the original host of sars-cov-2, but the intermediate host remains undiscovered (10) . the genes of sars-cov-2 encode structural proteins and non-structural proteins. four structural proteins are absolutely vital for viral assembly and invasion of sars-cov-2. spike protein homotrimers constitute the spikes on the viral surface, and these spikes are responsible for attachment to host cells by binding to their receptors (10) . the m protein has three transmembrane domains, which determine the shape of the virion, facilitate membrane curvature, and bind to the nucleocapsid. the e protein plays an important role in virion assembly and release, as well as involved in viral pathogenesis. the n protein has two different domains, both of which bind to the viral rna genome via totally different mechanisms. in addition, some reports have shown that non-structural proteins are essential for the replication of coronaviruses (10) . vaccination is a vital tool for the control and elimination of the virus, and the development of a vaccine for sars-cov-2 remains an urgent need (11) . traditional methods of vaccine development are time-consuming and very labor-intensive (12). the realm of immunoinformatics tools considers the mechanism of the host immune response to yield additional methodologies in the design of vaccine against diseases are cost-effective and convenient, as in silico predictions can reduce the number of experiments needed (13, 14) . dozens of studies have generated epitope-based peptide vaccine of sars-cov-2. baruah and bose (15) used immunoinformatics tools to discover cytotoxic t lymphocyte (ctl) and b cell epitopes for the spike protein of sars-cov-2. then, abraham et al. developed a multi-epitope vaccine that was designed using immunoinformatics tools that potentially trigger both cd4 + and cd8 + t-cell immune responses (16) . although there are many vaccines generated by immunoinformatics tools, most of these are based on spike protein. the spike protein is responsible for attachment to host cells by binding to angiotensin-converting enzyme 2 (ace2) (17) . a vaccine based on the spike protein could induce antibodies to block sars-cov binding and fusion or neutralize virus infection (18) . but there are still many obstacles, spike protein-based sars vaccine may induce harmful immune responses that cause liver damage of the vaccinated animals (19) . other virus proteins are considered as the candidates for designing vaccine with protective and less harmful immune responses (20) . vaccine-based on structural and non-structural proteins of the virus is revealed potential vaccine inducing protective immune responses (20, 21) . pandey et al. reported the more scientifically rigorous strategy of multi-epitope subunits based on multiple proteins against parasitic and viral diseases, such as malaria, visceral leishmaniasis, and hiv (22) (23) (24) . in this present, we employed immunoinformatics to predict multiple immunogenic proteins from the sars-cov-2 proteome and thereby design a multi-epitope vaccine. these proteins included non-structural and structural sequences of sars-cov-2, their reference sequences were retrieved from the national center for biotechnology information (ncbi) database. the proteins of the sars-cov-2 have been reported and reference could get from ncbi (25, 26) . the reference sequences of sars-cov-2 proteins were retrieved from ncbi protein database (https://www.ncbi.nlm.nih.gov/protein) and accession numbers in table 1 , then we stored the reference sequences as a fasta data type. the proteins with <100 amino acid sequences which are too short to predict epitopes were excluded, the remaining proteins were used for further analysis. vaxijen is the first server for alignment-independent prediction of protective antigens, which overcome the limitations of alignment-dependent methods (27) . to identify the potential antigenicity of sars-cov-2 proteins, an online prediction server, vaxijen v2.0 (http://www.ddg-pharmfac.net/vaxijen/ vaxijen/vaxijen.html) was used to predict the antigenic values of each protein (28) . this identification was applied according to the default parameters of the server. proteins having antigenicity were sorted according to an antigenic score of ≥ 0.5 (threshold for this model is 0.5) and were selected for further structural modeling (27) . there are no available experimental structures of sars-cov-2 proteins, phyre 2 provide model regions trough a new ab initio folding simulation with no detectable homology (29) . the sars-cov-2 proteins were modeled by phyre 2 server (http://www.sbg. bio.ic.ac.uk/phyre2/). because the sars-cov-2 proteins with no detectable homology protein to finish the modeling, we chose the intensive search and output the accurate alignment by the alignment of hidden markov models. modrefiner was used by the galaxyrefine server (http:// galaxy.seoklab.org /cgi-bin/submit.cgi?type=refine) (30) . the structure assessment was performed by the swiss-model workspace (https://swissmodel.expasy.org/assess) (31) . the three dimensional (3d) models were used for the conformational (discontinuous) b-cell epitope predictions while the sequences were utilized in linear b-cell and t-cell epitope predictions. netctl-1.2 is demonstrated to have a high predictive performance (32) . the netctl 1.2 server (http://www.cbs. dtu.dk/services/netctl/) was applied to predict ctl epitopes for the sars-cov-2 at the threshold value of 0.75 with high sensitivity and specificity (32) . to cover ∼90% of the world's population, three supertypes (a2, a3, and b7) were selected based on artificial neural networks, to predict mhc class i binding epitopes (33) . the best candidates for the sars-cov-2 vaccine construction were sorted for further prediction, based on a half-maximal inhibitory concentration (ic50) < 500 nm and an integrated score. the ic50 < 500 nm represents epitope has a high affinity to receptor. the integrated score indicated the transporter of antigenic peptides (tap) transport efficiency, class i binding, and proteasomal cleavage prediction (34) (35) (36) . then the specific treg epitopes were screened and excluded by the epitoolkit (https://epivax.com/). for mhc class ii t cell epitope predictions, the immune epitope database server predicted binders based on the percentile rank or mhc binding affinity (37) . the immune epitope database server (iedb; http://tools.iedb.org/mhcii/) was used to predict helper t lymphocyte (htl) epitopes (37) . we chose the combinatorial approach which recommended by iedb to predict htl epitopes. the combinatorial approach combined nn-align, smm-align, comblib, sturniolo, and netmhciipan methods (38) (39) (40) (41) (42) . the 17 alleles of the human leukocyte antigen (hla) were selected for the prediction at α and β chains, separately (43) . for final construction, epitopes were selected based on their scores (low scores indicated favorable binding), the release of interferongamma (ifn-γ), induction of emergent properties, and the ic50 < 500 nm. the ifn-γ cytokine makes a major contribution to antiviral mechanisms. it excites both native and specific immune responses by activating macrophages and natural killer cells (44) . further, ifn-γ augments the response of mhc to antigens. the ifn-γ epitope server (http://crdd.osdd.net/raghava/ifnepitope/ scan.php) was used to recognize ifn-γ epitopes (45) . we entered the htl epitopes with low scores into the ifn-γ epitope server. positive ifn-γ induction was predicted based on the support vector machine (svm) hybrid approach. the final htl epitopes were determined based on ifn-γ induction and mhc class ii binding, both of which facilitate the stimulation of t-helper cells (46) . the abcpred (http://crdd.osdd.net/raghava/abcpred/) and bepipred linear epitope prediction (http://tools.iedb.org/bcell/ result/) servers were utilized to predict linear b cell epitopes. the abcpred server is based on an artificial neural network (ann) (47, 48) . the linear b cell epitopes of the sars-cov-2 protein were predicted at a threshold of 0.5. the bepipred linear epitope prediction server is based on seven methods: (49) (50) (51) (52) (53) (54) . we used these seven methods separately to predict the average threshold. the overlap between abcpred and bepipred severs was selected to determine the candidate epitopes for the sars-cov-2 vaccine construction (55) . unlike t-cell epitopes that are linear continuous stretches of residues, b-cell epitopes are generally conformational (discontinuous) (56) . in this study, the ellipro servers (http://tools.iedb.org/ellipro/) were applied to predict the conformational b-cells epitopes (57) . the server predicts epitopes based on pi (protrusion index) value. the epitope with pi = 0.9 would include 90% of residues with 10% being outside the ellipsoid, discontinues b-cells epitopes with the top pi value was selected for vaccine designing (57) . to develop the final vaccine, epitopes determined by various immunoinformatics software were linked together with the aid of separate linkers. the ctl epitopes were linked by the aay linker, htl epitopes by the gpgpg linker, and b cells were linked by the kk linker (48, 58, 59) . to increase the vaccine immunogenicity, the β-defensin (45 mer) amino acid sequence was adjoined to the n-terminal of the vaccine with the help of the eaaak linker (60). the β-defensin peptides provoke innate immunity cells and recruit naive t cells through the chemokine receptor-6 (ccr-6) (61). the pan-hla dr binding epitopes (13aa) as well as added to the n-terminal of the vaccine with the aid of the same linker (59) . the pan-hla dr binding epitopes in vaccine construct facilitating binding to many different types of mouse and human mhc-ii alleles to induce cd4-helper cell responses (59) . to enable the intracellular delivery of the modeled vaccine, a tat sequence (11aa) was appended to c-terminal (62) . linkers (ayy, kk, and gpgpg) play vital roles in producing an extended conformation (flexibility), protein folding, and separation of functional domains, and therefore, make the protein structure more stable (59). the allergenic proteins induce a harmful immune response, allergenicity of the vaccine should be non-allergic (63) . the non-allergic character of the vaccine sequence was evaluated by the algpred server (http://www.imtech.res.in/raghava/algpred/) (63) . we predicted allergenicity of vaccine sequences choosing a hybrid approach (svmc+ige epitope+arps blast+mast) with the highest accuracy and sensitivity (63) . the vaxijen v2.0 server (http://www.ddgpharmfac.net/ vaxijen/vaxijen/vaxijen.html) was applied to evaluate the antigenicity of the vaccine (27) . the antigenicity prediction method was solely based on the physicochemical properties of proteins without recourse to sequence alignment. the precision rate of the server ranged from 70 to 89%. to determine immune response profile of this multi-epitope vaccine, computational immune simulations were performed by the c-immsim online server at (http://kraken.iac.rm.cnr.it/ c-immsim/) (64) . the c-immsim utilizes the celada-seiden model for describing both humoral and cellular profiles of a mammalian immune system against designed vaccine. as per the literature, three injections were administrated at different intervals of 1 month. the simulation was performed with default parameters. the vaccine sequence was administered 4 weeks apart. the simulation volume was 1,000, simulation steps was 1,000, random seed was 12,345, and the vaccine injection with no lps (64). the protparam tool (http://web.expasy.org/protparam/) was used to evaluate the physicochemical properties of the final vaccine protein (65) . the physicochemical properties included the number of amino acids, molecular weight, theoretical isoelectric point (pi), amino acid composition, atomic composition, formula, extinction coefficients, estimated half-life, instability index, aliphatic index, and grand average of hydropathicity (gravy) (66) . the molecular weight and theoretical pi were computed by user-entered sequences. the amino acid and atomic compositions were self-explanatory. the extinction coefficient of a protein was based on information about its amino acid composition. the instability index of a protein indirectly indicated the stability of the protein. if the computed instability index of protein was <40, it was regarded as a stable protein, while values >40 were regarded as unstable. in vivo half-life evaluation of proteins was based on the principle of the "n-end rule." furthermore, gravy is a measurement of the hydrophobic nature of the protein, which is calculated by determining the total hydropathy of all amino acids divided by the number of amino acid residues in the protein. to avoid inducing pathogenic priming and autoimmunity, the sequence homology of the final vaccine to human protein was screened by blastp online server (https://blast.ncbi.nlm.nih. gov/blast.cgi) (67) . an ideal vaccine should have non-sequence to human proteins. the designed vaccine was a reconstructed protein with no detectable homology (29) . phyre2 incorporates an ab initio folding simulation to model regions of proteins with no detectable homology. the phyre 2 server (http://www.sbg.bio. ic.ac. uk/phyre2/) was used to predict the three-dimensional structure of the designed vaccine. the server generates a fulllength 3d model of a protein sequence by employing both multiple template modeling and simplified ab initio folding simulation (29) . to enhance the overall and partial structural quality of the protein, the output 3d structure of the final vaccine from the phyre 2 server was further refined by the galaxyrefine server (http://galaxy.seoklab.org/cgi-bin/submit.cgi?type=refine) (30) . the galaxyrefine server predicted five refined models of our developed vaccine construct, in which model 1 was made by the structural perturbation based simply on the clusters of the side chains; whereas, models 2-5 were generated by deeper perturbations of loops and secondary structural elements (30) . for the assessment of the tertiary structure of the final vaccine protein, a ramachandran plot was performed by the swiss-model workspace (https://swissmodel.expasy.org/ assess) (31) . the ramachandran plot illuminates favored regions for backbone dihedral angles against amino acid residues in protein structure (31) . the structure assessment page shows the most relevant scores provided by molprobity and help we easily identify where residues of low quality lie in their model or structure (31) . then, prosa-web (https://prosa.services.came. sbg.ac.at/prosa.php) was employed in the final vaccine protein structure validation. a positive z-score commonly means an erroneous or erratic section found in the generated 3d protein model (68) . to revealing the binding affinity between the vaccine construct and antigenic recognition receptors of toll-like receptor-3 (tlr3, 2a0z) and major histocompatibility complex (mhc-i, 4wuu, and mhc-ii, 3c5j) present on the surface of immune cells (69) . docking analysis was performed using the cluspro server (https://cluspro.bu.edu/login.php?redir/queue. php). tlr3 act as receptors for antigenic recognition. the cluspro server computed the models based on electrostatic interactions and desolvation energy (69) . to reconfirm the binding affinity of the designed vaccine construct between these receptors, the patchdock server (https://bioinfo3d.cs.tau.ac.il/ patchdock/) was used for docking (70) . the server predicted the potential complex with the help of three algorithm-molecular shape representations, surface patch matching, filtering, and scoring (70) . after the acquisition of the output from the patchdock server, the complexes were refined by the firedock algorithm, which predicted the optimal complex with the aid of energy functions (70) . the pdb file of vaccine protein and receptor complex (tlr3, mhc-i, and mhc-ii) were used to start the molecular dynamic (md) simulations. the complexes were placed in a octahedron box of water molecules represented by the threepoint charge spc model, whose boundary is at least 10 å from any protein atoms. the solvated protein was subsequently neutralized by chloridions. covalent bonds involving hydrogen atoms were constrained using the lincs algorithm, and longrange electrostatic interactions were treated with particle-mesh ewald employing a real-space cutoff of 10 å. the system was first briefly minimized with backbone atoms restrained to the initial coordinates to remove close contacts, and the restrained system was gradually heated to 300 k under constant volume conditions in 100 ps. each system was equilibrated for 1 ns using the constant isothermal-isobaric ensemble at 1 atm and 300 k without any restraints. the parrinello-rahman barostat and a v-rescale thermostat were used with an integration time step of 2 fs. production run md simulations were performed for 10 ns with coordinates recorded every 10 ps. all simulations were performed using gromacs 2018.2 along with the gromos96 54a7 force field (16, 24) . for the purpose of cloning, codon adaptation of the designed vaccine was performed for analyzing the codon usage by the prokaryotic organism (escherichia coli, e. coli). the java codon adaptation tool (http://www.jcat.de/) was used to optimize codon (71) . then the secondary structure of mrna was predicted by mfold (http://unafold.rna.albany.edu/? q=mfold) (72) . for raising the expression efficiency of the final vaccine protein, the e. coli k12 strain was chosen. for the valid translation of the vaccine gene, we proofread and avoided rho-independent transcription termination, prokaryote ribosome binding site, and cleavage site of restriction enzymes. restriction endonuclease sites xhoi and bamhi were appended to n and c terminals of vaccine, respectively. then, it was inserted into the pet28a (+) vector between the xhoi and bamhi. the flow chart of the designed work is shown in figure 1 . the strategy of vaccine construction is presented in figure 1 . the proteome of sars-cov-2 was retrieved, which comprised 27 proteins. the reference sequences of those proteins were retrieved in the fasta format and their details are presented in table 1 . five proteins with <100 amino acid sequences are too short to predict epitopes (orf6 protein, orf10 protein, orf7b protein, nsp11, and envelope protein) were excluded. in order to develop a subunit vaccine, it is critical to identify candidate proteins that are important for inducing a protective immune response (27) . the remaining 22 proteins sequence were relayed to the vaxijen server to determine their antigenicity based on the antigenic scores ( table 1) . proteins with antigenic scores >0.5 were selected for further analysis (28) . nine proteins, namely orf7a protein, orf8 protein, nsp9, nsp6, nsp3, endornase, orf3a protein, membrane glycoprotein, and nucleocapsid phosphoprotein were finally selected for further epitope prediction. there is no available experimental structures of these nine proteins, we predicted homology models for the nine proteins applying the normal mode of phyre2 online server. the most suitable templates for the nine proteins were identified to be the pbd entries ( table s1 ). all of the modeled structures were showed over 90% residues in the ramachandran favored region figure s1 and table s2 . the prediction of ctl epitopes (9 mer) was performed by the netctl server. the binder sites were determined based on three supertypes (a2, a3, and b7), with a 95% coverage rate of the world's population. nine proteins were selected based on antigenicity. one epitope of each supertype was selected based on the highest score and an ic50 value < 500 nm. then the specific treg-inducing epitopes were excluded by epitoolkit. a total of 18 epitopes were selected from nine proteins as the candidates for the construction of the vaccine ( table 2) . the htl epitopes (15 mer) were evaluated for three hla supertypes: hla-dr (drb1 * 01:01, drb1 * 07:01, drb1 * 09:01, drb3 * 01:01, drb4 * 01:01); hla-dq (dqa1 * 01:01/dqb1 * 05:01, dqa1 * 01:02/dqb1 * 06:02, dqa1 * 03:01/dqb1 * 0:02, dqa1 * 04:01/dqb1 * 04:02, dqa1 * 05:01/dqb1 * 02:01, dqa1 * 05:01/dqb1 * 03:0 1); and hla-dp (dpa1 * 01/dpb1 * 04:01, dpa1 * 01:03/dpb1 * 02:01, dpa1 * 02:01 /dpb1 * 01:01, dpa1 * 02:01/dpb1 * 05:01, dpa1 * 03:01/dpb1 * 04:02). we sorted the top epitopes with the lowest scores (low scores indicated the highest binding capability) from three supertypes. the best candidate was then selected based on positive ifn-γ induction and an ic50 < 500 nm. then the specific treg-inducing epitopes were excluded by epitoolkit. thus, a total of 14 epitopes were selected for vaccine design ( table 3) . we used the abcpred and bepipred servers to identify the line b cell candidate epitopes. all predicted epitopes from both servers were compared, and only the overlapping epitopes were selected for the development of the vaccine. the line epitopes identified by abcpred had prediction scores ranging from 0.52 to 0.93, and line epitopes identified by bepipred had prediction scores ranging from 0.5 to 1. among these line epitopes, only 12 (16 mer) were found to be common or partly common in both servers ( table 4) . these 12 line epitopes were selected for vaccine construction ( table 4) . the non-continuous b cell epitopes were predicted by the ellipro severs, a total number of 27 non-continuous b cell epitopes were generated from ellipro. amino acid residues, sequence location, the number of residues, and the pi scores of the predicted conformational epitopes are shown in table 5 and the graphical depiction of these epitopes can be seen in figure s2 . twenty-four epitopes were excluded because it added the allergenicity of vaccine, three epitopes were marked red and selected for vaccine construction. the best candidate epitopes were used for the construction of the vaccine. a total of 18 ctl epitopes, 14 htl epitopes, 12 the half-maximal inhibitory concentration (ic50) value was > 500 nm, which ensured a higher binding capability of the selected epitopes to mhc molecules. linear, and three non-continuous b cell epitopes were fused together with the aid of linker sequences. the ctl epitopes were linked by ayy (the aay liner helps the epitopes produce suitable sites for binding to tap transporter and enhances epitope presentation), the htl epitopes were combined with the aid of gpgpg (the gpgpg linker stimulate htl responses and conserve conformational dependent immunogenicity of helpers as well as antibody epitopes), and b cell epitopes were merged with the aid of kk. the final to enhance vaccine immunogenicity, the human β-defensin-3 sequence (45aa) and pan-hla dr binding epitopes (the pan-hla dr binding epitopes in vaccine construct facilitating binding to many different types of mouse and human mhc-ii alleles to induce cd4-helper cell responses.) was added to the nterminal of the vaccine with the aid of the eaak linker. to enable the intracellular delivery of the modeled vaccine, a tat sequence (11aa) was appended to c-terminal. the vaccine was developed to be 864 amino acids in length ( figure s3) . the sequence homology of final vaccine protein to human protein sequence shown that there were no significant alignments ( figure s4 ). the allergenic character of the vaccine was determined by the algpred server and was based on the hybrid approach (svmc + ige epitope + arps blast + mast) with a 93.5% coverage. the vaccine was non-allergen with 84% accuracy and 82.78% sensitivity at threshold value was −0.2. similarly, the antigenic the half-maximal inhibitory concentration (ic50) value was < 500 nm, which ensured a higher binding capability of selected epitopes to mhc molecules. nature of the vaccine construct was evaluated and showed that the protein was a favorable antigen with a global prediction score of a protective antigen of 0.5308 (probable antigen). the default threshold value for antigenicity was 0.4 in the virus model. moreover, the vaccine constructs contained 864 amino acids, and its molecular weight was 95.4 kda. the theoretical pi was predicted to be 9.71. the vaccine contained 63 negatively charged residues and 125 positively charged residues. the vaccine construct was composed of 13,541 atoms, and its chemical the immune response profile in silico immune simulation the immune stimulation of the final vaccine was performed using c-immsim online server, which gives the immune profiles of the designed vaccine. the proliferation in the secondary and tertian immune response were identified by igg1 + igg2 and igm, as well as, the decreasing of the antigen count igg + igm showed the proliferated (figure 2a) . the stimulation result revealed the development of immune response after immunization. b cell population was highly stimulated upon immunization ( figure 2b) . similarly, the cytotoxic and helper t cell levels were proliferated that suggested the development of secondary and tertian immune response (figures 2c,d) . during the exposure time, it was also observed that the production of ifn-γafter immunization ( figure 2e) . these results were significant for the immune response against sars-cov-2. hence, the tertiary structure of the full-length vaccine sequence was predicted by phyre 2, and it was applied for refinement and further analysis. twenty-five templates were employing modeling as figure s5 shown. there were three templates from human defensin which were we added in to enhance the immunogenicity, others from virus ( figure s5) . the immune epitopes were not structural homology to human proteins that could avoid inducing autoimmune. the secondary structure of the predicted model contained 18% alpha-helix, 21%tm helices 44% beta-sheets, and 27% disordered figure s6 . to optimize the 3d structure of the modeled protein, the initial model was refined in the galaxyrefine server. the galaxyrefine server-generated five models based on the rootmean-square deviation (rmsd) and molprobity algorithm. the details of the five models are shown in table s3 . model 1 with the top ramachandran favored, therefore selected for docking purposes (figure 3) . a model with more residues in the ramachandran favored region, less in outliers region and rotamer region was considered as a more ideal one. the initial model generated from phyre 2 server and refine model from galaxyrefine were evaluated with the aid of the swiss-model workspace. the initial model was 63.46% of residues in the ramachandran favored region, 19.49% in the ramachandran outliers region, and only 10.22% in the rotamer region (figure 4 ). the refine model was 89.1% of residues in the ramachandran favored region, 2.09% in the ramachandran outliers region, and only 0.15% in the rotamer region (figure 3) . other favorable parameters of the refined model were as follows: gdt score of 0.9922, rmsd value of 0.260, molprobability of 2.049, clash score of 8.9, and poor rotamers totaling 0.3 ( table s1 ). the quality and potential errors in the final vaccine 3d model were verified by prosa-web. the z-score indicates overall model quality, the model with a lower z-score was considered as the higher quality one. the z-score of the initial model was −2.81, refine model is −3.64 (figure 5 ). to further evaluate the binding affinity between the developed vaccine construct and the relative antigenic receptors (tlr3, mhc-i, and mhc-ii), molecular docking was performed. the server yielded 44 candidate models with different binding energies. twenty-nine model complexes of tlr3 and covid-19 vaccine were determined, from which just one complex with the lower binding energy score of −1156.2 was selected to show ( table 6 and figure 6) . a total of 29 model complexes of mhc-i and the covid-19 vaccine were discovered, and the lowest binding energy score was −1346.8 ( table 6 and figure 6) . a total of 29 complex models of mhc-ii and the covid-19 vaccine were predicted, among which, one model complex with the lowest binding energy score of −1309.1 was chosen to show ( table 6 and figure 6) . further, the vaccine construct was evaluated using the patchdock server, which identified different models and produced a score table. the top 10 complexes identified were refined by the firedock algorithm. among those top 10 models, the model with the lowest binding energy was further selected to show in this paper. the refinement outcomes of tlr3 and the vaccine complex was solution number 1 with global energy of −38.40, attractive van der waals energy (vdw) of −26.02, repulsive (vdw) of 8.62, and atomic contact energy of −11.06 ( table 6 and figure 6 ). the complex of mhc-i and the vaccine was ranked number nine, with global energy of −22.97, attractive vdw of −26.84, repulsive vdw of 12.82, and atomic , g26, t27, t28, l30, k32, e33, p34, c35, s36, s37, g38, p45, h47, p48, l49, a50, d51, n52, k53, c58, c67, p68, d69, g70, v71, r80, s81, v82, s83, p84, k85, l86, f87, i88, r89, e91, e92, e95, t936, c937, g938, q939, q940, q941, t942, t943, l944, k945, g946, k962, k963, g964, v965, q966, i967, p968, c969,t970, c971, g972, k973, q974, a975, t976, k977, y978, l979, v980, q981, q982, e983, s984, p985, f986 50 0.719 12 k839, p841, q842, v843, n844, g845, l846, t847, w851, a852, d853, n854,n855, c856, l956, s957, a991, p992, p993, a994, q995, y996, e997, l998, k999, h1000, g1001, t1002, f1003, t1004, e1008, y1009, t1010, g1011, n1012, y1013, q1014, c1015, g1016, h1017, k1019, t1022, s1023, k1024, e1025, t1026, l1027, y1028, c1029, i1030, d1031, g1032, a1033, l1034, l1035, t1036, k1037, s1038, s1039, e1040, y1041, k1042, g1043, p1044, i1045 65 0.648 13 d806, d807, t808, l809, v811, e812, f814 7 0.62 14 k1051, e1052, n1053 3 0.602 endornase 15 s1, l2, e3, n4, v5, a6, f7, n8, v9, v10, n11, k12, g13, h14, f15, d16, g17, q18, q19, g20, e21, v22, p23, v24, s25, i26, i27, n28, n29, t30, v31, y32, t33, k34, v35, d36, g37, v38, d39, v40, e41, l42, e44, n45, k46, t47, t48, l49, p50, v51, n52 51 0.755 16 e145, g146, s147, v148, k149, g150, l151, g169, e170, a171, v172, k173 12 0.707 17 l200, p205, s207, m209, i211, d212, l214, e215, l216, a217, m218, d219, e220, f221, i222, e223, r224, y225, l227, e228, g229, y230, a231, f232, e233, h234, i235, y237, g238, d239, f240, s241, h242, s243, q244, l245, g246, k256, r257, f258, k259, e260, s261, p262, e264, f279, t281, d282, a283, q284, t285, g286, s287, s288, k289, c290, k307, s308, q309, d310, l311, s312, v313, v314, s315, k316, v317, m330, l331, w332, c333, k334, d335, g336, h337, v338, e339 77 0.699 18 t98, i99, g100, c102, s103, m104, t105, d106, i107, a108, k109, k110, p111, t112, e113, t114, i115, c116, a117, p118, l119, t120, g125, r126, v127, d128, g129, v131, d132, l133, f134, r135, n136, a137, r138, n139, k181, v182, d183, g184 (table 6 and figure 6 ). to accomplish the estimate of the stability of the vaccinereceptor complex, we performed the simulation of the docked complexes (vaccine and tlr-3, mhc-i, and mhc-ii) with the help of gromacs. then, various analysis like energy minimization, pressure assessment, temperature, and potential energy calculations were performed. the temperature and pressure of the simulation system during the production run was around 300 k and 1 atmosphere, respectively, indicating a stable system and successful md run. the temperature and pressure of the three simulation systems (vaccine and tlr-3, mhc-i, and mhc-ii complexes) during the production run were around 300 k and 1 atmosphere, respectively, indicating the stable systems and successful md run (figures 7a-f) . the complex root mean square deviation (rmsd) plot represents the structural fluctuation of the overall structure of the complex of vaccine and immune receptor. the rmsd of vaccine-tlr3 complex has large fluctuation during 0-6 ns simulation. after 6 ns, the rmsd value was kept around 1.25 nm, indicating that the conformation of this complex was stable ( figure 7g) . otherwise, the rmsd of vaccine-mhc-i and -mhc-ii complexes has large fluctuation during 0-4 ns simulation. after 4 ns, the rmsd value were kept around 1 nm, indicating that the conformation of the two complexes were stable (figures 7h,i) has low rmsf value, indicating these residues has low structural flexibility. by contrast, residue 0-200 and 600-800 has relatively higher rmsf value, indicating the larger flexibility during those regions (figures 7j-l) . to fuse the final vaccine to an expression vector, codon conversion of the vaccine protein was performed by the java codon adaptation tool. restriction site xhoi and bam hi were added to n and c terminals of the codon sequence, then was inserted into the pet28a (+) vector between the xhoi and bamhi (figure 8) . the rna secondary structure using the mfold program was generated foldings contain 4,381 base pairs out of 2.3% in the energy dot plot. mfold predicted an identical secondary structure of 4,381 bp formed by nucleotide fragments (figure s7 ). sars-cov-2 is characterized by high infectivity and high transmission speed; thus, a prophylactic vaccine is needed (11) . the availability and advantages of the multi-peptide vaccine developed by immunoinformatics methods have been confirmed by previous studies (73, 74) . ojha et al. used the immunoinformatics methods to develop a multiepitope subunit vaccine to epstein-barr virus-associated malignancy (73) . in recent studies, genomics and proteomics information of sars-cov-2 have been retrieved, stored, and utilized (75, 76) . in the present research, we tried to develop a multi-epitope subunit prophylactic vaccine of sars-cov-2, with the help of immunoinformatics tools. a line of research have tried to develop the vaccine of sars-cov-2 by immunoinformatics tools. baruah and bose (15) used immunoinformatics tools to discover cytotoxic t lymphocyte (ctl) and b cell epitopes for the spike protein of sars-cov-2. then, abraham et al. developed a multi-epitope vaccine that was designed using immunoinformatics tools that potentially trigger both cd4+ and cd8+ t-cell immune responses (16) . most of those research just focus on the spike protein-based vaccine. a vaccine based on the spike protein could induce antibodies to block sars-cov-2 binding and fusion or neutralize virus infection (18) , as well as induce harmful immune responses that cause liver damage (19) . other proteins should be ideal candidates for designing vaccines. in the present report, we selected nine proteins with positive antigenicity for further epitope prediction. all proteins from sars-cov-2 with <100 amino acid sequences were excluded, and the antigenic nature of the remaining proteins was evaluated. this method can facilitate the discovery of potential antigens of sars-cov-2 when the precise immunity mechanisms are unknown. to design an effective vaccine, we selected the sars-cov-2 protein through the above-mentioned methods for epitope prediction. in recently, asaf et al. reported that identify multiple epitopes for cd4 + 12 and cd8 + t cells based on muti-protein (77) . their protein list was the same as this in our research. in asaf 's report, they just predicted the t cell epitopes, non-b cell, b cell peptide was not predicted (77) . the b cell epitopes are antigenic determinants from the antigen that are recognized by the b cell surface membrane receptor and evoke the production of specific antibodies. the persistent challenge in immunological prediction tools is the prediction of epitopes to a higher level of accuracy (78) . to determine accurate linear b cell epitopes from the antigenic proteins, we used two bioinformatics tools based on different algorithms of prediction. we identified nine overlapping linear b cell epitope candidates from two different bioinformatics tools. this method was superior to the prediction of epitopes from a single tool (78) . moreover, we also have predicted the noncontinue b-cell epitopes. the b cell immune response is preferred in the design of a vaccine. however, t cells may also elicit a strong immunoreaction. the vaccine that activates both ctls and htls should be more effective than a vaccine that only targets ctl responses (79) . to generate a more effective vaccine, we predicted both ctl epitopes and htl epitopes. the t cell epitopes were decomposed fragments from the antigen presented by the mhc molecules of t cells and stimulated the production of effector t cells, immunological memory t cells, and ifn-γ. the cellmediated immune response induced by ctls plays a vital role in the defense against viral infections through the recognition of intracellular viral pathogens by mhc class i molecules. in the present report, mhc-i binding epitopes were predicted by choosing a2, a3, and b7 alleles, which cover ∼95% of world's population. we selected 18 ctl epitopes. the htls play a vital role in the antiviral immune response by producing ifn-γ. moreover, htls are able to induce and maintain ctl responses. furthermore, 14 htls epitopes were chosen based on both the binding capability and ifn-γ induction. bhattacharya et al. also used the spike protein sequence predicted for mhc-i and mhc-ii epitopes of sars-cov-2, but not predicted capability of producing ifn-γ (80). the t cell epitopes enhanced ifn-γ inducing capability, which evokes both the native and specific immune responses by activating macrophages and natural killer cells, and augmenting the response of the mhc to the antigen (81, 82) . in this study, the immunogenic epitopes from b cells, ctls, and htls were chosen to develop a more valid, reliable, and effective vaccine against sars-cov-2. a multiepitope approach was used by splicing together epitopes with the aid of their respective linkers. to improve the immunogenicity of this multiepitope vaccine, an adjuvant β-defensin and pan-hla dr binding epitopes (13aa) were fused to the n-terminal with the aid of an eaaak linker, then a tat sequence (11aa) was appended to c-terminal with the added of kk. the final vaccine constituted 864 amino acids. the allergenicity, antigenicity, and stability of the designed vaccine constructs were then evaluated. the tertiary structure of the generated vaccine was predicted by using the phyre 2 server and then refined by the galaxyrefine server. the binding affinity of complexes of the developed vaccine and receptors, in which tlr-3, mhc-i, and mhc-ii (present on the surface of the immune cell) were confirmed by the cluspro server was based on molecular docking. furthermore, to ensure the translation efficiency of the designed vaccine in a specific expression system, the mrna of the vaccine was enhanced with the aid of the java codon adaptation tool. the restriction enzyme cutting sites of xho? and bamh? were then appended to the n and c terminals, respectively. the vaccine sequence was subsequently cloned in pet28a (+), the expression vector. further experimental validation of the safety and efficacy of the designed vaccine for sars-cov-2 is warranted. all datasets presented in this study are included in the article. rd and zc performed the experiments. rd and yz wrote the paper. yz and fy edited the final version. all authors participated in the experimental design, data analysis, and agreed with the final version of the paper. available online at clinical characteristics of 140 patients infected with sars-cov-2 in wuhan severe acute respiratory syndrome coronavirus 2 from patient with 2019 novel coronavirus disease, united states a preliminary study of the relationship between 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mining, and immunoinformatics in vaccinology: where are we? role of allograft inflammatory factor-1 in the pathogenesis of diseases we extend the sincerest appreciation to nhc key laboratory of pulmonary immunological diseases, and guizhou provincial people's hospital, for their technical assistance. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.01784/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 dong, chu, yu and zha. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-022888-dnsdg04n authors: nan title: poster sessions date: 2009-08-19 journal: eur j immunol doi: 10.1002/eji.200990224 sha: doc_id: 22888 cord_uid: dnsdg04n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx3, the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx3 facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th1-oriented adaptive response. actually, ptx3-deficient mice are highly susceptible to aspergillosis and develop th2 skewed responses; moreover, ptx3-deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx3, which does not show direct activity on fungal cells. finally, ptx3 alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx3-mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx3 amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx3-opsonised conidia, cd11b activation, internalization, recruitment to the phagocytic cup and cd11b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd11b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx3, fcgriia/cd32 mediates inside-out activation of cd11b and consequently phagocytosis of c3b-opsonised conidia. in vivo phagocytosis experiments performed with c1q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx3) and the cellular arm (fcgrs, cr3). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx3 is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx3 binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx3-/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd14, immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx3. we found high concentration of ptx3 in human colostrum (47.62 ± 13.5 ng/ml at day 1 post-delivery) compare to the one found in human serum ( x 2 ng/ml). the presence of ptx3 in human colostrum seems to be due to the secretion of ptx3 by human mammary gland since we report the production of ptx3 by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx3 production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx3 production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx3 supplying by breast feeding: (i) soluble ptx3 in colostrum (ii) hmc that can secrete ptx3 upon stimulation in the specific tissue, (iii) an increase of ptx3 production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx3 may participate to the beneficial role of breast feeding on the newborn health. a. m. baru 1 , j. stephani 2 , h. wagner 2 , t. sparwasser 1 1 twincore, institute for infection immunology, hannover, germany, 2 technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of 10 receptors in humans (tlr 1-10) and 12 in mice (tlr 1-9, 11-13). as transmembrane receptors, tlrs are expressed on the cell surface (tlr 1, 2, 4, 5, 6, (10) (11) (12) (13) and at endosomal membranes (tlr 3, 7, 8 and 9) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about 5 years later to this, toll-like receptor 9 (tlr9) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr9 as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr9 (hutlr9) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr9 (mutlr9) is also expressed on conventional dendritic cells (cdcs). consequently, tlr9 ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr9 (henceforth called as hut9 mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr9 knock-out background. we expect that hut9-mutlr -/mice mimic the human specific expression pattern of tlr9, i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr9. by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr9 ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c1q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin-12 is a heterodimeric cytokine consisting of the two subunits p35 and p40. the main inducers of il-12p40 are microbial components activating toll-like receptors with the magnitude of il-12p40 induction depending on the specific tlr engaged. differential induction of il-12p40 upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il-12p40 due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il-12p40 promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il-12p40 promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone 3 and 4 acetylation in specific regions of the proximal promoter region. acetylation of h4 showed a specific distribution pattern and occured mainly in regulatory elements within the il-12p40 promoter, whereas acetylation of h3 took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue 4 on h3 turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue 1 , w. ellmeier 1 1 medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi 1 , m. buxadé 1 , j. minguillón 1 , r. berga 1 , j. aramburu 1 , c. lópez-rodríguez 1 1 universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat5 is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat5 participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat5 is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat5, the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat5 could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat5 is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat5 is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat5. our work indicates that nfat5 is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr3, tlr4, tlr7 and tlr8 and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p50 and p65 nfxb subunits. interestingly, we observed that tlr3 stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr4, tlr7 and tlr8 stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl-2 expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr3, tlr4, tlr7 and tlr8. finally, in vivo tlr7 stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr4, tlr7 or tlr8 triggering can directly favor tumor development whereas tlr3 signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms-354825) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd8 + and cd4 + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr 4 ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd4 + and cd8 + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd4 + and cd8 + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: 1. innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr 4 stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . 2. adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni 1 , r. oatuni 1 , m. collini 2 , m. caccia 2 , p. castagnoli 3 , g. chirico 2 , f. granucci 1 1 university of milano-bicocca, biotechnology and bioscience, milan, italy, 2 university of milano-bicocca, physics, milan, italy, 3 singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin-2 (il-2) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il-2 production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca2+/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca2+ influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr4 engagement, depending instead exclusively on cd14. we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase-2 (cox-2) that, by generating prostaglandins (pgs), such as pge2, regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd14. given the essential involvement of cd14 in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd14 represents a major step towards the development of potential treatments with modes of action involving interference with cd14 functions. we have examined the interaction of cd55, a 80-kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd14. human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd14 antibody coupled to biotin visualised by qd-525-streptavidin and anti-cd55 antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd55 and cd14, suggesting a new functional role of cd55 as a member of a multimeric lps receptor complex. l. lundvall 1 , r.r. schumann 1 1 charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase-1 induction leads to an increased release of mature il-1b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d39) was established for assessing il-1b induction during this disease. the murine raw 264.7 cell line, the human astroglial u-87 mg and the murine microglial cell-line bv-2 were stimulated with the tlr4 ligand lps, the tlr2 ligand pam 2 cys, the nod2 ligand mdp, or the nod1 ligand c12-ie-dap, and, as control, atp alone or in combination. we assessed il-1b by elisa and caspase-1 and pro-il-1b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il-1b induction pathway. s. pneumoniae (d39) infected mice showed a significant increase in il-1b release after 24 hours. in vitro, an increase in il-1b levels after costimulation with lps or pam 2 cys, and mdp or c12-ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il-1b by tlr-and nlr-ligands was observed in raw cells, bv-2 cells, u-87 cells and primary astrocytes. active caspase-1 (p10) was induced by mdp or c12-ie-dap, corresponding with high il-1b responses when lps or pam 2 cys was added. sirna experiments show that a knock-down of nod2 leads to a diminished il-1b release after lps-and mdp-stimulation. the precursor forms of il-1b and caspase-1 seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il-1b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il-1b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il-1b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg 1 1 toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd3 and cd28 activation model system -tlr4 presence on cd4+ cells is found in mouse t cells, human t cells and jurkat cell lines. following cd3/cd28 activation for 48 hours we have identified a small but distinct populationof tlr4+ cells. further characterization indicates these cells to be cd4+cd25+ cells. further characterization of the expression and functional acitvity of the tlr4+ t cells indicates co-expression of tlr4 with md-2 indicating a functional tlr4 receptor. in addition lps activiation did not lead to upregulation of tlr4 expression in t-cells. the data indicate that tcr activation leads to tlr4 expression. the expression appears to be associated with cd4+cd25+ cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann 1 , d. kaul 1 , c. krüger 1 , f. zipp 1 , r. nitsch 2 , s. lehnardt 1 1 charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, 2 charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr7 plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna40) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr7 (and human tlr8) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr7 is expressed in these cells. incubation of microglia with all three of the above mentioned tlr7 ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il1-b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr7 knock out (ko) microglia by realbecause neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin-1 was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin-1 is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin-1-transfected plb985 cells, coronin-1 overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd11b labeling. concerning apoptosis, increased coronin-1 expression in dmf-differentiated plb985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin-1 significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or bid truncation suggesting that coronin-1 interfered with mitochondria-related events. to validate the prosurvival role of coronin-1 in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin-1 immunolabeling. we concluded that coronin-1 could constitute a potential target in resolving inflammation. p.-n. hsu 1 1 national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw264.7 macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p38 map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf-6 sirna and traf-6 decoy peptide, indicating this pathway is traf-6 dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b7-h1 and b7-dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il-6 was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il-2 and ifn-g which could not be overcome by restimulation with acd3. this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd4 t cells into t helper 17 (th17) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th17 differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th17 cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il-1 receptor-related protein st2 could be implicated in lps tolerance. the natural ligand of st2 was recently identified as interleukin-33 (il-33), a new member of the il-1 family. in this study, we investigated whether il-33 triggering of st2 was able to induce lps desensitization of mouse macrophages. we found that il-33 actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il-33 enhancing effect of lps response is mediated by the st2 receptor since it is not found in st2 ko mice. the biochemical consequences of il-33 pretreatment of mouse macrophages were investigated. our results show that il-33 increases the expression of the lps receptor components myeloid differentiation protein-2 (md2), cd14 and tlr-4 and the myeloid differentiation factor 88 (myd88) adaptor molecule. in addition, il-33 pretreatment of macrophages enhances the cytokine response to tlr-2 but not to tlr-3 ligands. thus, il-33 treatment preferentially affects the myd88-dependent pathway activated by the tlr. c. klotz 1 , b. lenz 1 , r. lucius 1 , s. hartmann 1 1 humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., 2008) . the cystatin effect was blocked by the application of anti-il-10 receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin-10 (il-10). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il-10 in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il-10 production after avcystatin stimulation. application of specific inhibitors revealed that the il-10 induction was p38 and erk dependent, and inhibitor titration indicated a higher sensitivity towards p38. western blotting experiments confirmed the phosphorylation of p38 and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il-10 is also regulated by the phosphatidylinositol-3-kinase (pi3k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il-10 and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose-7-phosphate (s-7p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose-5p and xylulose-5p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p38/jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s-7p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts 1 , y. morias 1 , p. de baetselier 1 , a. beschin 1 1 vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m1) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m1 activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m1 activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m1 in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd88, ifng, il-10, ccr2 and nf-kb to the development and/or recruitment of pathogenic m1 in the liver was investigated using knock-out mice or by delivering il-10 in infected mice. results: we established that cd11b+ly6c+cd11c+ tnf and inos producing dcs (tip-dcs) represent the major m1 liver subpopulation. tip-dcs differentiated in an ifng/myd88-dependent manner from cd11b+ly6c+ inflammatory monocytes in the liver of infected mice. ccr2 promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr2 ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il-10 enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il-10 in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p50 was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p50 and il-10 play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m1, in particular tip-dcs. the inflammatory activity of liver m1 is controlled by il-10 and/or p50 nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov 1 , j. driesen 1 , z. abdullah 2 , a. niño castro 1 , t. chakraborty 3 , m. krönke 4 , o. utermöhlen 4 , c. wickenhauser 5 , j.l. schultze 1 1 limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, 2 institute of molecular medicine and experimental immunology, bonn, germany, 3 institute of medical microbiology, university of giessen, giessen, germany, 4 institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, 5 institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine 2,3-dioxygenase, cyclooxygenase-2 and cd25 and production of prostaglandin e2 (pge 2 ) and interleukin 10. all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd25, expressed by regulatory myeloid cells, acts as an il-2 scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge 2 or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd25 + ido + cox-2 + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin 12 (il-12) and il-18 in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il-12 and il-18 or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il-12 and il-18. in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il-12 and il-18. therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel 1 , m. rodriguez gomez 2 , m. niedermeier 2 , y. talke 2 , n. göbel 2 , k. schmidbauer 2 , m. mack 2 1 unversity hospital regensburg, internal medicine ii, regensburg, germany, 2 university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il-4 and il-6. activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to 24h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp-1 cells infected with m. bovis bcg, the avirulent m. tuberculosis h37ra strain and the virulent m. tuberculosis h37rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva 1 , l.d. miteva 1 , s.a. stanilova 1 1 trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il-23 is a heterodimeric cytokine composed of a p19 subunit associated with the il-12/23p40 subunit. like il-12, il-23 is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il-12p40-related cytokines controls the appearance of normal th 1 and pathological th 17 mediated immune responses. in this study, we examined the dynamics of inducible il-12p40 and il-23p19 mrna expression and protein production in purified human monocytes and how jnk and p38 mapks inhibitors influenced il-12p40 and il-23 production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il-23p19 gene expression was higher than those observed for il-12p40 gene at all time-points. the level of il-23p19 mrna increased after 6 th h and reaching a maximum level at 9 th h (43.4 fold for c3bgp and 22.7 fold for lps). c3bgp and lps triggered il-12p40 gene transcription were almost equal at the 3 rd h (4.4 and 4.1 fold) and at 9 th h (7.8 and 7.9 fold, respectively) after stimulation. the higher level of il-12p40 gene expression was detected at 6 th h in lps compared to c3bgp stimulated monocytes (8.1 vs. 4.9 fold). however, il-12p40 and il-23 protein production was increased in the highest level after c3bgp stimulation. the inhibition of p38 led to the statistical significant augmentation of c3bgp stimulated il-12p40 production. the inhibition of the same map kinase enhanced lps stimulated il-12p40 production without significant difference. the inhibition of jnk and p38 mapks significantly decreased c3bgp stimulated il-23 production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c3bgp and lps to induce the expression of il-12p40 and il-23p19 mrnas in purified human monocytes. we showed that inhibition of p38 mapk down regulated il-23 and upregulated il-12p40 production in stimulated monocytes. we concluded that in human monocytes p38 map kinase activation has an opposite effect on the il-12p40 and il-23p19 expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd83, cd86 and cd40, and secrete il-12, thus acquiring the ability to induce proliferation and th1 polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd1c, bdca-3, and cd16. by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd16+ subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd16+ dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd16+ dc, including their survival and their ability to produce il-12p70. besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il-6, il-8 and mcp-1 involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg 1 , s. wolke 2 , a. brakhage 2 , m. gunzer 1 1 otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, 2 hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in 2004 nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and 2-photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to 3 hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor 7 (irf7), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr9. in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr9 to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens 1 , s. vander beken 1 , p. bogaert 1 , j. grooten 1 1 ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th1-driven immunological counterpart of the th2-driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd45 bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th1-and th2-driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd45.1 + ram remained constant in cell number for the first 2 days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day 4 of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker 1 , a. stojanovic 1 , a. cerwenka 1 1 german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr-1 + cd11b + f4/80 + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas 1 , f. marchesi 1 , m. fabbri 1 , s. schiarea 2 , c. chiabrando 2 , a. mantovani 1,3 , p. allavena 1 1 istituto clinico humanitas, rozzano, italy, 2 istituto mario negri, milano, italy, 3 università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m1 and m2. m1 macrophages act as a first line of defence against pathogens whereas m2 cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about 50 % of the monocytes differentiated after 5 days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd16, cd68 and low levels of mhc class ii. tc-macro produced il-10, il-6, tnf but not il-12, even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl2, cxcl8, ccl17 and cxcl10. the transcriptional profile of tc-macro revealed that several genes in line with an m2 polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g 100.000 kda). il-3 and il-6 were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl-2h3 rat mast cell line. we demonstrate that gr nuclear translocation begins within 5 minutes and completes after 30 minutes in dx treated rbl-2h3 cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within 5 minutes as non-genomic. studying gc-caused apoptosis, rbl-2h3 cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl-2h3 cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. 5 minutes of dx treatment inhibited ca 2+ -signaling in antigen stimulated rbl-2h3 cells in the concentration range of 100nm -10mm. moreover, 5 minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl-2h3 cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box 1 (hmgb1) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb1 is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin (1) . extracellular hmgb1 can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb1 exerts antibacterial functions in human adenoid and testis (2) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity (3). we asked whether hmgb1 from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for 40 or 120 minutes with 25 nm phorbol ester (pma), 100 ng/ml interleukin 8 (il-8), or 100 ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h2a-histone h2b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb1 in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb1 is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb1 localizes on nets. we hypothesize that net-bound hmgb1 might exert a direct antimicrobial function, or that nets might concentrate hmgb1 locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation (37°c, 3 h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor-1 induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr-1 and anti-vegfr-2 antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il-18. altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd1 molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd1a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd1a and cholesterol-dependent lipid rafts impact on cd1a surface expression and cd1a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd1-restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd1 group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd1a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd1 molecules in multiple sclerosis. we first analyzed cd1 expression on monocytes from 16 ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd1a was not expressed on ms patient monocytes, while the other members of the cd1 family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd1 molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd1 molecules in this context. c. ohnmacht 1 , d. voehringer 1 1 ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il-4 reporter (4get) mice as well as in vivo depletion of basophils are used to uncover their role during type 2 immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about 60h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th2 cells. these results demonstrate that basophils play a crucial role as effector cells in type 2 immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il-1beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water (10 mg and 100 mg daily) for 30 days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il-6 and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il-6 and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant 143038). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type-1 macrophages, mph1), and during resolution of inflammation (type-2 macrophages, mph2). methods: mph1 / 2 were generated by culturing cd14 + human monocytes for 6 days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph1 / 2 of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph1 / 2 were stimulated with lps and secreted il-6, il-8, il-10, il-12p40 and tnf were quantified by elisa. results: mph2 are relatively efficient phagocytes for apoptotic neutrophils whereas mph1 are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph1 and mph2 which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il-6, il-12 and tnf production is suppressed while il-10 secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp70 in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of 90 years old and older (long-livers). intracellular ros and hsp70 levels were registered by flow cytofluorimetry upon labeling with 2',7'-dichlorofluorescin diacetate (invitrogen) and anti-hsp70 antibody (brm-22, sigma), respectively. intracellular level of hsp70 was also estimated in neutrophils after heat shock (hs) performed at 43°c for 10 min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at 1-15 min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for 1 min at 42°c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp70 (hsp70 basal ) and ros level, both intracellular and extracellular. at the same time increased hsp70 level immediately after hs (hsp70 (0 min)) correlated negatively with intracellular ros (initial and after hs). the hsp70 increase value (hsp70 (0 min) -hsp70 basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp70 increase was registered in 60 min after hs (hsp70 (60 min) -hsp70 basal ). thus it was found that within this age group the alteration in hsp70 induced by hs in neutrophils but not basal hsp70 itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant #3303. d. goyeneche-patiño 1 , z. orinska 1 , f. mirghomizadeh 1 , s. bulfone-paus 1 1 forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd8 + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type 1 ifn. two novel genes, receptor transporter protein (rtp4) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp4 and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd88 and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd8 -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during 8 and 48 h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp4 and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine 2000. stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp4 in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd88 knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr 3 and 9, as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp4 and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd88 and trif and the pathways that they represent are not relevant in the expression of rtp4 and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano 1 , e. erba 2 , r. frapolli 2 , m. d'incalci 2 , a. anselmo 1 , s. pesce 1 , p. allavena 1 , a. mantovani 1, 3 1 humanitas clinical institute, rozzano, italy, 2 mario negri institute, milan, italy, 3 institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et-743) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl2, cxcl8 and the inflammatory protein pentraxin3 (ptx3) either at transcriptional and protein level, especially after tnfa/il1b stimulation. down-regulation of ccl2, cxcl8, ptx3 and also of il6 and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd31+ vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin-4 (il-4) is a key cytokine of the t helper 2 cell response. il-4 has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il-4 production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il-4 producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day 3-6 after birth, a small population of il-4 producing cells was observed in the absence of any stimuli. the il-4 + population was not detectable at 6 or 12 weeks of age. other cytokine producing cells (ifn-g, il-10) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il-4 + cell population. phenotyping of the neonatal il-4 + cells showed that they were ige + /mhcii -/cd4cells. the occurrence of cd4 + il-4 producing cells after pma stimulation increased slowly with age and did not reach adult levels by 12 weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il-4 at day 5 after birth. neonatal pbmc collected before colostrum uptake did not produce il-4 in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il-4 responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il-4 response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel 1 , d. voehringer 1 1 ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th1 cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat6-dependent manner when stimulated with the th2 cytokines il-4 or il-13. alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat6-dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat6-deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat6 in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat6 expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat6 expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat6-deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il-4 exposed macrophages from wild-type and stat6-deficient mice. candidate genes will be expressed using retroviral transfections of stat6-deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of 38 pleural effusions, including 15 malignant and 23 nonmalignant tumors. the following human malignant cell lines were tested: a549, ht29, hct116, sw60, mcf7, mda-mb231, jurkat and hl60. results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m1, and the tams isolated from the malignant effusions similar to m2. among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms4a4a and ms4a6a: highest trascriptional level after 18h of stimulation with 10-6m dexamethasone. ms4a murine genes are differently expressed respect to the human counterpart and only the homologs of ms4a6a (ms4a6b, 6c and 6d) have a similar regulation. finally, egfp-tagged ms4a4a, ms4a6a, and ms4a7 expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms4a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms4a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein-1 (mcp-1)/ccl2 and the gpcr ligand fmlp or the tlr4 agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il-1b or ifn-g, significantly and dose-dependently synergized with ccl2 in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin-8/cxcl8, but not of the ccl2 receptor ccr2 in monocytic cells. in turn, the induced cxcl8 synergized with ccl2 for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr1/cxcr2, because cxcl8 receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl2 intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, 2004) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for 20 min at 37°c with gentle shaking, followed by spinning down of pmns for 5 min, at 4°c and 500g. the supernatant was sedimented at 15000g for 10 min, 4°c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd56 has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd56 low cd33+ blood monocytes (mo) in healthy individuals and the increase in cd16+cd56+ blood mo in patients with inflammation has been reported. the functional activity of human cd56+ blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd56 expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd33 and activation markers such as hla-dr and trem-1. percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a 12.3 fold increase (p x 0.0001) in the total number of circulating cd56 low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd33 and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd56+ blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd33+ blood mo expressed increased levels of cd56 on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem-1+) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd56 on cd14 high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m1-and m2-polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m1 and m2 macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m1 type and were cd14 negative but cd80 and mhcii positive and produced high levels of il-8, tnfalpha and il-6 following lps stimulation. culture in the presence of mcsf resulted in induction of the m2 phenotype. these cells were cd14 positive with intermediate expression of cd80 and mhcii expression and produced high levels of il-10, il-6 and il-8 following lps stimulation. interestingly, already baseline il-8 production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m1-and m2-polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr4 ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl60 cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma (50 ng/ml) treatment for 48h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl60 cells expressed antigen receptors cd14, tlr2, tlr4 and cd68 , and displayed enhanced phagocytic activity and production of ros. expression of cd13, cd33 and cd15 was also maintained however the cells were hla-dr and cd1a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl60 cells to differentiate into macrophages in response to pma. hl60 may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta 1 , n. ferrer 2 , a. gómez 2 , j. gonzalo 2 , a. arbués 2 , a. anel 1 , c. martín 2 , j. pardo 1 , apoptosis, immunity and cancer 1 university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, 2 university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so2, a m. tuberculosis phop mutant strain that was shown (perez et al 2001) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al 2006) . in the present study, we compare the time course and phenotype of cell death induced by so2, bcg and wild type m. tuberculosis on the murine macrophage cell line j774 and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase-3 activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl2x4 deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type 1 receptor for vip (vipr1) gene is highly conserved through species and, in humans, is highly polymorphic. vipr1 has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from 53 blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr1 in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr1 after 3, 6, 9 and 12 h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g 50 %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from 53 donors that had been typed for the relevant vipr1 snps. the down-regulation of vipr1 correlates with the presence of a t at rs896 mapping in the 3'-end of the gene (p= 0.004). the vipr1 protein level was decreased about 40 % in monocytes of 3 subjects typed as t/t at rs896 whereas 3 subjects typed as c/c at rs896 maintained a high level of expression after 9h of lps treatment. the data show that different haplotypes of the vipr1 gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr1 vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska 1 , t. vavrochova 1 , d. filipp 1 , immunobiology 1 institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e7.5-13.5) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd14 in the early mouse embryo (me). facs analysis of cell suspension prepared from 10.5 day me showed that about 0.7-1 % of cells were positive for cd11b. these cells exclusively were also positive for cd14, tlr2, and cd45 antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days 7,5-13,5. reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e7,5), embryo-derived mhcii + macrophages start to appear in the embryo around day 13. multicolor facs analysis of cd11b, cd45, cd14, f4/80, tlr2, tlr4, c-kit and mhcii surface markers revealed differential expression of tlr2 and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd11b + tlr2 + cells isolated from the e10,5 embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il12/il23-dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies 16 presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and 11 have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in 9 out of these 11 patients mutations in several ifng pathway genes, other candidate genes are being investigated for the 2 other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp1. we did focus on the study of 2 genes which are considered as important pathogen recognition receptors of the innate immune system: tlr2 is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il-10 cytokine. using a case/household-contact cohort we did investigate polymorphisms of these 2 genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to 70-80 % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, 2009. hsp70 are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp70 have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp70 on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp70 (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp70 were used. exogenous hsp70 was used in concentration 1-10 ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio 1:1 and 2:1 directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp70 on their surface. results demonstrating effect of exogenous hsp70 on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp70 on amplitude of respiratory burst. the cells expressing surface hsp70 impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp70 might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp70 to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited 28 pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd14/cd86 and cd14/cd163 double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd14/86 subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd14/163 subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m2 polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević 1 , i. pilipović 1 , s. stanojević 1 , k. mitić 1 , k. radojević 1 , v. pešić 2 , g. leposavić 1,2 1 institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, 2 faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h 2 o 2 ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to 14-day-long propranolol treatment and measured both no and h 2 o 2 production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b 2 -and a 1 -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h 2 o 2 production. an increase in h 2 o 2 production in the presence of the a 1 -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h 2 o 2 production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b 2 -and a 1 -adrenoceptors on peritoneal macrophages (a stimulatory effect on b 2 -adrenoceptors and a suppressive effect on a 1 -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd14 ++ cd16 -('classical') and cd14 + cd16 + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd14 + cd16 + monocytes display higher tlr2 and -4 expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd14 ++ cd16monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr2 stimulation. methods: cord blood (n=8) and peripheral-blood (n=8) mononuclear cells were stimulated in vitro for 24hrs with peptidoglycan and subsequently analysed for cd14 and cd16 and intracellular il-12p70 and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il-12p70, both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il-12p70 was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il-12p70 (% positive cells and geomfi) was significantly higher for cd14 + cd16 + cells than for cd14 ++ cd16cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd14 ++ cd16cells were positive for tnf to a significantly higher extent than the cd14 + cd16 + cells. in particular the tnf response to tlr2 stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd14 ++ cd16monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a 16-year-old boy who admitted with complaints of seizures during the previous 2 months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g (50 mgr/m2/day, subcutaneously every other day) was started. after 2 months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for 3 months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf1 gene concerns the well-known gt deletion in the second exon of ncf1 gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = 45) subjected to moderate brain injury were randomized, and received either 1.5 mg/kg ubiquitin or vehicle (placebo) intravenously within 5 min after cci. levels of tnf-a, il-1b, il-6, il-10 and il-1 receptor antagonist were analyzed in brain tissue using real time rt-pcr at 4 and 72 hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at 24h and 7 days. data were analyzed with the mann-whitney u test and a two-tailed p x 0 .05 was considered significant. all cytokines were highly up-regulated 4 hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il-10 were significantly lower in the ubiquitin treated animals, whereas the levels of il-6 and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day 7. the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight (38) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while 18 were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p=0.02), tpp (p=0.00), osi (p = 0.00), mda (p = 0.00) were significantly raised but taa (p = 0.01) was significantly reduced in ptb patients compared with controls. the levels of mda (p = 0.04), neopterin (p=0.00) and tpp (p=0.00) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p=0.00), mda (p=0.00), neopterin (p=0.02), osi (p=0.00) were significantly reduced while taa (p=0.01) was significantly raised in c+m after 4 weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after 4 weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas 1 , e.m. cunha 1 , m.j. oliveira 1 1 icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles (20 nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to 5 minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage (70 %) of macrophages contained the metal particles than neutrophils (55 %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap-70 is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap-70 regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., 2006; sivalenka and jessberger, 2004) . swap-70-/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., 2002; sivalenka and jessberger, 2004; sivalenka et al., 2008) . crucial regulators of these processes are members of the rho family of small gtpases such as rac1 and rhoa. swap-70 interacts with rac1 in vitro and preferentially binds the active gtp-bound rac1. swap-70 supports the increase of active rac1 in vitro by a yet to be defined mechanism (shinohara et al., 2002) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap-70 and rac1. it was found that fulllength swap-70 preferentially binds to constitutively active rac1 (rac1q61l) but not to its dominant negative form (rac1t17n). binding assays with swap-70 truncated mutants showed interaction of swap-70's n-terminus with gtpgs rac1 or rac1 depleted of guanine nucleotide, whereas swap-70 central or c-terminal regions do not bind to any form of rac1. preliminary competitive-binding assays with overlapping 18mer peptides, spanning the entire swap-70 sequence, mapped the rac1 binding site near the n-terminus of swap-70. full-length swap-70 site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap-70 with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap-70. v. c. barbosa 1 , c. d. polli 1 , m.c. roque-barreira 1 , m.c. jamur 1 , c. oliver 1 , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl-2h3 cells were sensitized with ige anti-tnp and stimulated with dnp 48 -hsa or artinm. artinm binding to rbl-2h3 cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp-1 and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp-1 release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand 1 (psgl-1), b1 and b7-integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd44 (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il-1b, il-18 and tnf-a. herpes simplex virus 1 (hsv-1) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv1 infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il-18 and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv1. methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) (10ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv-1 for 18 h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot 2.0 software. results: in the first itraq experiment over 300 human proteins were identified in the hsv1 infected cell supernatants. from these proteins 119 had at least 3 fold increase after poly(i:c) + hsv1 infection compared to the uninfected cells. hsv1 infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of 2.7 fold increase in the protein amount in the poly(i:c) + hsv1 infected cell supernatant and a 2.6 fold increase in the hsv1 infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand 10, il-6, tnf-a induced protein 6, complement factor b, galectin-1 and mxa. at present, further experiments are on-going for more detailed analysis of the hsv1 infected macrophage secretome. h. p. prakash 1,2 1 german cancer research centre, translational immunology, heidelberg, germany, 2 max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein-1 (ciap-1) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap-1 and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal-3 (25 mg/ml) binding to monocytes, in the presence or absence of 10mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal3, laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal3 or rpmi and monocytes (1x10 5 ) were added into each insert. when necessary, hrgal3 was pre-incubated with 10mm lactose or sacarose. mcp-1 (100ng/ml) was used as positive control. we observed that hrgal-3 binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal-3 is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal-3, we observed that the association between these glycoproteins and hrgal-3 resulted in a 60 % increase in the number of migrating cells. both n-and c-terminal domains of hrgal-3 are involved in the association between laminin or fibronectin and hrgal-3, since the presence of lactose resulted in 50 % and 20 % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal-3 induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for 3 weeks in the presence of bmp4,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd14+cd45+ cells. the sorted cd14+cd45+ cells were further cultured for 7 -10 days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd14, m-csf receptor cd115 and the scavenger-receptor cd36, we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd14 and cd16 (fcgammariii) : the cd14lowcd16-and cd14+ cd16+. trancscriptional, phenotypic and functional assays suggest the alternative (m2) polarization of cd14+cd16+ embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m2 -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi 1 , p. kropf 1 1 imperial college london, immunology department, london, united kingdom the balance between t helper (th) 1 and th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th1 or th2 responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd4 + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg9vd2 t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f1-atpase) is expressed on many cell types and is a possible specific ligand for the vg9vd2 tcr. the present study aims at understanding the role of f1-atpase in antigen regognition. using video microscopy calcium imaging in single vg9vd2 t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f1-atpase. purified f1-atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg9vd2 cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f1-atpase. thus the f1-atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg9vd2 t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f1-atpase. by using purified f1-atpase and peptides derived from vg9vd2 tcr sequences, interaction sites between f1-atpase and tcr were identified on both ligands. based on these findings a generalized model for vg9vd2 t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg2d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg2d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg2d activation in vivo. by transiently overexpressing the nkg2d ligand rae-1-beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma5vdelta1 gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg2d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg2d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg2d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae1 induction. the primary systemic th2 response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg2d receptor suggest that different ones may play unique roles. a novel nkg2d-ligand, h60c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae-1 induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg2d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd1d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th1 (ifn-g, tnf) and th2 (il-4, il-13) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type 1 diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd4 -nk1.1 -inkt cells producing high levels of the pro-inflammatory cytokine il-17 has been identified (inkt17 cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt17 cells in type 1 diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il-17 as compared to c57bl/6 mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt17 cells present in nod mice were mainly cd4 -nk1.1 -, express the ror-g transcription factor and il-23 receptor, both molecules being usually associated with th17 commitment. we are currently analyzing, using co-transfer experiments, whether these inkt17 cells play a beneficial, a deleterious, or any role in the development of type 1 diabetes in nod mice. j. s. dodd 1 , r. muir 1 , s.s. affendi 1 , p.j. openshaw 1 1 imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd1d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd1d-deficient mice with poor nkt cell responses have inefficient induction of cd8 t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th2 immunity (increasing il-5 and il-10), promoting pulmonary eosinophilia and ablating cd8 t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd8 t cell activity (as measured by cd25 expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d4) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d7) weight loss by a cd8 t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd4 + ab t cells, are regarded as immature or t h 2 biased. vg9 + vd2 + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg9 + vd2 + t cells towards these activators. because il-23 is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg9 + vd2 + t cells. herein, we observed that zoledronate induced neonatal vg9 + vd2 + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h 1-like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h 2-like cytokines such as il-4 and il-5 were not induced. addition of il-23 to zoledronate selectively costimulated ifn-g production from neonatal vg9 + vd2 + t cells. furthermore, zoledronate/il-23 treatment resulted in neonatal vg9 + vd2 + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il-23 (il-23r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il-23 resulted in a further increase of t-bet expression in neonatal vg9 + vd2 + t cells. these changes in the expression of il-23r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il-23 treatment. of note, in contrast to adult peripheral blood vg9 + vd2 + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg9 + vd2 + t cells. altogether, these observations show that neonatal vg9 + vd2 + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il-23 is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e3 ligases such as hhv8 encoded k3, k5 or mhv68 encoded mk3. these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e3 ligases manipulate them. we discovered that both the cellular march and viral e3 ligases ubiquitinate cd1 molecules. however, whereas viral molecules inhibit cd1-antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd1 molecules. in contrast mhc class ii was only targeted by cellular and not by viral e3 ligases. furthermore cd1 molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e3 ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd2 negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht29 colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd2 neg clones in vivo, we generated hypodermal ht29 tumors in immunodeficient mice. concomitant injections of vd2 neg clones, in contrast to vd2 + cells, prevented the development of ht29 tumors. vd2 neg clones expressed chemokine c-c motif receptor 3 (ccr3) and migrated in vitro in response to chemokines secreted by ht29 cells, among which were the ccr3 ligands macrophage inflammatory protein (mip)-1d and monocyte chemoattractant protein (mcp)-4. more importantly, a systemic intraperitoneal (i. p.) treatment with vd2 neg clones delayed the growth of ht29 subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr3 antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd2 neg clones were not able to inhibit the growth of a431 hypodermal tumors. our findings suggest that cmv-specific vd2 neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht29 cells expressing the luciferase and realized orthotopic injection of ht29-luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint1, currently the only known determinant of a canonical iel compartment, that is selectively required for vg5vd1 + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint1 expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint1 function; for example, we demonstrate that the constitutive expression of wild-type skint1 fully restores detc development in a skint1 mutant mouse, but does not rescue normal detc function. thus, skint1 provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd1d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd19 + cd21 hi cd23 lo cd11c -) and splenic conventional dendritic cells (cdcs) (cd11c hi cd8a +/-cd11b +/-b220 -) from wt and cd1d -/mice as apcs for nkt cells from va14-ja18 transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd1d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd1d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il-4 by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il-4 producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h 2 response, whereas cdc-primed nkt cells rather favor a t h 1 response. objectives: il-18 is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il-18 give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd1d -/-) mice. we set out to investigate the activated b cells in il-18 injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il-18 (2 mg) for 10 days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day 14 was evaluated by flow cytometry and immunohistology. results: mice injected with il-18 developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd138 + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il-18 was increased in inkt cell deficient (cd1d -/-) mice, in contrast to published data. an increased response to il-18 was also observed in ja281 -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il-18 induced antibody responses. further characterization of the recruitment of b cells in il-18 injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il-18 induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd1d, are prone to autoantibody production and often involved in early immune responses. the il-18 induced antibody response in mzb deficient (cd19 -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il-18 induced antibody responses. we conclude that the role for inkt cells in il-18 induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c57bl/6 mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha 18-/-and cd1d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il-4. moreover, ifn-g production required the presentation of ehlppg by cd1d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il-12. similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd16 in cmv-infected individuals. cd16 is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that 71.9 ± 15.9 % of gamma-delta t cells from 21 cmv-infected ktr expressed cd16, when compared with only 19.8 ± 16.7% in 11 non cmv-infected ktr (p x 0.0005). similarly, 51.9 ± 25.7 % of gamma-delta t cells from 13 cmv-seropositive blood donors expressed cd16 compared to 27.1 ± 25.1 % in 15 cmv-seronegative donors (p x 0.01). cd16+ gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd16-dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il-12 and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd16 mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd107a on cd16+ gamma-delta t cell lines. cd16 is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd16+ gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a431 skin carcinoma cell line pre-incubated either with rituximab (anti-cd20) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd16-dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k5mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il-6, ifn-g, tnf-a, osm, ccl2, cxcl8, cxcl10, and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd40, cd86, hla-dr, and dc-sign, and lost cd14, ccr2, ccr5, and cxcr4. addition of further microbial stimuli (lps, peptidoglycan) induced ccr7 and enabled these inflammatory dcs to trigger antigenspecific cd4 + effector ab t cells expressing ifn-g and/or il-17. importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg9/vd2 t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg9/vd2 t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg9/vd2 t cells stimulated with hmb-pp in the presence of il-21 express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il-21 regulate expression of the b cell attracting chemokine cxcl13/bca-1, its receptor cxcr5, and co-stimulatory molecules involved in b cell help. purified peripheral vg9/vd2 t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to 4 days with and without hmb-pp, in the absence or presence of il-2 or il-21, or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl13 protein were detected in co-culture supernatants only when both il-21 and hmb-pp were provided, implying an il-21-dependent and tcr-dependent expression. vg9/vd2 t cells were confirmed as producers of cxcl13 by flow cytometry and immunofluorescence. under the same conditions, activated vg9/vd2 t cells expressed cd27, cd28, cd40l, cd70, icos and ox40. in contrast, neither cxcr5 nor ccr7 changed markedly by il-21 stimulation of peripheral vg9/vd2 t cells. conclusion: our findings confirm on the protein level that stimulation of vg9/vd2 t cells with hmb-pp and il-21 induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto 1 , m. emoto 1 1 gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)-12. although downmodulation of surface t cell receptor (tcr)/nkr-p1c (nk1.1) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il-12 stimulation. to determine whether failure to detect inkt cells after il-12 stimulation is caused by dissociation/internalization of tcr and/or nkr-p1c or by block of de-novo synthesis of these molecules, and to examine the role of il-12 in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p1c by inkt cells after stimulation with a-galcer or il-12, and influence of il-12 neutralization on down-modulation of stcr/snkr-p1c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il-12 neutralization. whereas s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of il-12, s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p1c remained unaffected by a-galcer or il-12 treatment, despite the down-modulation of ctcr and/or cnkr-p1c protein expression. in contrast, ctcr + cnkr-p1c + stcr -snkr-p1c -inkt cells and cnkr-p1c + snkr-p1c -inkt cells were detectable after in-vitro stimulation with a-galcer and il-12, respectively. our results indicate that tcr and nkr-p1c expression by inkt cells is differentially regulated by signaling through tcr and il-12r. they also suggest that il-12 participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p1c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg9-gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region 3 d1 (cdr3d1) and cdr3d2 repertoire, with a striking enrichment in a specific germline-encoded cdr3d1 sequence. differentiated gd t cells and the enriched cdr3d1 sequence were detected as early as after 21 weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd1-jd1 and vgi-jg1.3/2.3 rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd2-jd1 and vg9-jg1.2 selection was seen in pb tcrgd cells. detailed analysis of the cdr3 motifs revealed selection determinants in both vg9-jg1.2 (canonical length and cdr3 motif) and vd2-jd1 (minimal cdr3 length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚4) and tcrgd cb cells (6-7) to tcrgd pb cells (˚10 or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type 1 diabetes (t1d). we have previously shown that transgenic expression of a cd1d-restricted, va3.2-vb9 tcr in nod mice lead to an increase in cd1d-restricted type ii nkt cells (24abnkt cells), and prevention of the development of t1d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by 24abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc2.5 cd4+ t cells, in the presence or absence of selected cells from 24abnkt cell transgenic mice. results: in 24ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, 24abnkt cells expressed a high level of cxcr3 and a low level of ccr7 and cd62l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd4+ bdc2.5 t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from 24ab transgenic mice with bdc2.5 cd4+ cells resulted in the prevention of diabetes development. the protection from disease required a minor cd4+ subset of 24ab+ nkt cells, but was independent of cd25+ t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd1d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs44, induces in vitro inkt cell expansion and ifng and il-4 secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th2 profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd1d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il-10 with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr1 cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th17-type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th17 immune response is characterized by the secretion of il-17a and il-17f. the il-17 locus encodes the highly conserved il-17a and il-17f cytokines that are syntenic in 44kb distance to each other. besides cd4 + th17 and nkt cells, approximately 50 % of the il17a producers are gd t-cells. like cd4 + th17 cells, il-17 producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il-17 production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th17 cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il-17 or ifn-g. combined with the well known classification of il-17 producing gd t-cells along the markers cd27 and cd122, our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th17-type immune responses in vivo. in this context, the potential redundancy of il-17a and il-17f may complicate the analysis. so far, most studies were carried out with il-17a single-deficient or il-17f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il-17a and il-17f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg9vgd2 t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b1 and il-15 differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd2 tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp3) and, similarly to ab tregs, suppress the proliferation of anti-cd3/anti-cd28 stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd2 tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il-17 was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd11b in the liver following a-galcer treatment, numbers of cells lacking cd11b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma9/vdelta2 t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma9/vdelta2 t cells expansion upon stimulation with zoledronic acid (za) and interleukin-2 (il-2). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by 1) the bioinformatic analysis of gene expression profiling data 2) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in 43 patients (49 %) (responders, r), whereas 44 patients (51 %) were non-responders (nr). vgamma9/vdelta2 t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt2, whereas temra of r patients had an higher expression of the costimulatory molecule nkg2d. the proliferative response of vgamma9/vdelta2 t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, 82 % of r patients were m, whereas 77 % of um patients were nr (p x 0.001). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c57bl/6 mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il-12 p40 (clone r2-10f6; atcc) or anti-cd-1d (clone 1b1) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at 18 hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il-12p40, a component of both il-12 and il-23; pretreatment with anti-cd1d mab had no effect. il-12rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il-12 was not a critical factor. this suggestion is supported by the increased expression of il-23p19 and il-12p40 (but not il-12p35) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il-23 production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb1) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd27 expression. gd 27+ cells secrete interferon-g, while gd 27cells are capable of producing il-17. this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd27-defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine 123. cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to 40 % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚2% of gd 27+ cells but not at all in gd 27cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, 40-60 % of gd 27+ cells were positive for pgp activity, although their gd 27counterparts remained largely negative (p x 0.01). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd27. as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd 27+ and gd 27cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma9vdelta2 t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma9vdelta2 t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma9vdelta2 t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma9vdelta2 t cells from healthy donors were stimulated with different compounds (zol, ipp) for 24 hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma9vdelta2 t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma9vdelta2 t cells. moreover, mcp-2 depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma9vdelta2-mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma9vdelta2 tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma9vdelta2 t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg9vd2 t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg9vd2 t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg9vd2t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg9vd2 t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg9vd2 t cells and their effects on the viability of glioma cells, we expanded in vitro vg9vd2t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg9vd2t cell lines to kill three different glioma cell lines (t70, u251, u373) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg9vd2t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg9vd2 t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg9vd2 t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg9vd2 t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately 15 % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e7 protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd1d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e7-induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e7-expressing transplanted skin is dependent on interactions between populations of cd1d-expressing cd11c+/f480+ myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e7 graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il-2 and a-galcer. expression of cd1a, cd1d and the costimulatory molecules cd86 and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il-4) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd1d expression on the cell surface in comparison with control cells. in contrast cd1a expression was decreased. cd86 and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd1d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd1a, cd1b and cd1c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il-4 producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd1d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, 0.5-10 % of circulating lymphocytes express a vg9vd2 t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg9-enriched genes compared to conventional mhc-restricted cd8 + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf4 & crtam in gd and ab t cells respectively. because igsf4 binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd8 + ab t cells express crtam, and that engagement of igsf4 on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf4/crtam. we therefore sought to answer: 1. what is the function of igsf4/crtam on gd t cells? 2. how is the igsf4-crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf4 enrichment on resting gd t cells, with expression also detected on˚10 % of ab t cells. the properties of those cells are being examined. however, igsf4 generally correlates with markers of activation/antigen experience such as cd45ro. thus, igsf4 cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg9 + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf4 and crtam within 48 hours. however, engagement of igsf4 by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf4 may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg2d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf4 interactions are sufficient for cd8 t cells to kill gd t cells and/or vice versa. instead, crtam-igsf4 interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd3z is a 16 kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately 3-6 % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd2 tcr variable segment associated with the vg9 segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg9vd2 t cells without antigen presentation. in vitro stimulated vg9vd2 t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg9vd2 t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg9vd2 t cell anergy observed in hiv+ patients. to this aim, cd3z expression and ifn-g production by vg9vd2 t cells from hiv+ and hiv-subjects were analyzed. we show that vg9vd2 t cells from hiv-infected patients expressed lower level of cd3z compared with healthy donors. a direct correlation between cd3z expression and ifn-g production capability by vg9vd2 t cell was found. however, pkc activation by pma is able to restore cd3z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg9vd2 t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner 1 , m. swamy 1 , s.l. clarke 1 , a. hayday 1 1 king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd8aa or cd8 -cd4cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to 15 days. the cells are initially activated by plate-coated acd3 antibody and a cytokine cocktail and maintained further in medium containing low levels of il-2. after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg2d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il-6) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after 6 hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd94 in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg2 molecules in heterodimer with cd94, we screened the presence of these receptors on t cell subsets in bd. the expression of nkg2 a/c/d molecules on gd and cd8+ t cells were analyzed in 17 active and 9 inactive patients with bd and 21 healthy controls. expression of nkg2 molecules was evaluated on cd8+, gd t and cd56+ nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls (4.4 vs. 2.8 %, p=0.001). in addition to the increase of gd t cells, increased expression of activating nkg2c molecules was also observed on gd t cells (20 % vs. 11 %, p= 0.01). nkg2a expression on gd t cells was found to be higher than nkg2c expression in patients and controls; but nkg2a expression on the t cells was not statistically different in both groups (33.5 vs. 40 %). nkg2d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd8+ cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th1 and th2 cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg9/vd2+ lymphocytes. we have screened a panel of 26 lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg9/vd2+ cells, and selected two susceptible ("target") cell lines (over 60 % death in the assay) and two vg9/vd2+ resistant ("non-target") cell lines (under 20 % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs 697 (non-target), and validated the results by rt-qpcr quantification. results: we identified 37 commonly up-regulated and 50 commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp1, ifitm1 and prame, for example, are up-regulated, whereas cd274 and clec2d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg9/vd2+ target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd1d, in the rat. mice and rats have very similar cd1d and inkt tcr genes, with the exception of the va14 gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd1d revealed a very similar pattern of cd1d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il-4 production was 10-100 fold lower in the best responder rat strain (f344) compared to mouse (c57/bl6). since nkrp1a (rat homologue of mouse nk1.1) and tcr are not appropriate markers for rat inkt, cd1d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd1d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd1d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd1d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd1d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g9d2 t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g9d2 t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g9d2 t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g9d2 t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp-1. a significant (p x 0.01) reduction in intracellular bacterial numbers was observed (n=8) in the presence of pbmcs cultured with picostim+il-2 in comparison with pbmcs cultured with il-2 or media alone. picostim+il-2 caused significant expansion and activation of gd t cells following culture of pbmcs for 10-14 days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers 100-fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il-2, reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma9/vdelta2 (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than 5 % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: 1) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; 2) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); 3) to establish whether the same issues could be solved using a simplified protocol of dc generation. 1) dc were generated from cd14 + cells of healthy donors/mm patients; immaturedc on day 6 were induced to fully mature by incubation for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za. after 7 days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; 2) idc generated from cd14 + cells of hla-a*0201 + healthy donors/patients were pulsed with sv-peptide and stimulated for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za; after 2 rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd8 + t cells was determined by svpentamers staining; 3) the same experiments were performed both with dc generated following a standard protocol and a 48h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [6-3 h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd4 + cd25 + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin-2 on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp3 negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd1 subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg2d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd8+ have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n=8) and inactive (bdna) (n=20), versus healthy controls (hc) (n=30) and patients with recurrent oral ulcerations (ru) (n=14). to determine gd cytokine profile and surface markers treg-related in bd (n=9) and hc (n=11). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta1, vdelta2, cd8alpha, cd8beta, nkg2d, nkg2a and cd103. -intracellular expression of ctla-4, and foxp3. -intracellular expression of il-2, il-4, ifngamma, il-10 and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta2+ cells were significantly increased in ru. vdelta1+ and gdcd8+ lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg2d was slightly increased in gd from bda. -nkg2a expression by gdcd8+ was not different in bd versus hc. -most of gdcd8+ presented cd8alpha-alpha homodimers in bd and hc and were negative for cd103, foxp3 and ctla-4. gdcd8+ and gdcd8-subsets were (in bd and hc): -high ifngamma-producers without differences. -low il-2-producers: il2+ cells were lower in gdcd8+ than in gdcd8-. -low il-10-producers: il10+ cells were lower in gdcd8+ than in gdcd8-. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd8+ than in gdcd8--very low producers of il4 in most of cases. the hallmark in bd was the increase of gdcd8/vdelta1+. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg2a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd8+, except a lower percentage of il-2+ cells than in the gdcd8-subset. gdcd8+ from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd56 in humans. a subgroup, the invariant nkt cells (inkt), expresses the va24vb11 tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd3 + cd56 + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from 10 healthy umbilical cord blood samples and stimulated with ifn-g (50 ng/ml), anti-cd3 (25 ng/ml) and il-2 (500 ui/ml). these cells were cultured for 21 days and the expanded cd3 + cd56 + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd3 + c56 + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x 0,05 was considered significant. results: we could significantly expand cord blood cd3 + cd56 + nkt cells from 0,87±0,57 % to achieve an enrichment of 46,89±13,31 % (p=0,0002). table 1 shows the percentage (mean±sd,n=10) of phenotypic markers in cd3 + cd56 + cells at baseline (day 0) and after 21 days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb4 and vb8 families (p x 0,001). conclusion: our results show that cord blood-derived nkt cells are mainly cd8 + and cd94 + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb4 and vb8 families in these cells. l. marischen 1 , d. wesch 1 , p. rosenstiel 2 , a. till 2 , d. kabelitz 1 1 institute of immunology, kiel, germany, 2 institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod2. peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod2 gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp1 19 and pvama1 proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day 5 culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd69 + and cd25 + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day 3 after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd107a + (lysosomal associated membrane proteins: lamp-1) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th1 and th2 cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th1-like and th2-like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n=43) and healthy controls (n=13) were determined by flow cytometry using anti-cd3 and monoclonal antibody specific for the cdr3 loop of the invariant tcr a chain of inkt cells (clone:6b11). furthermore, after pma/ionomycin stimulation for 4 hours, intracellular ifng and il-4 cytokines were detected in cd4+cd8-, cd4-cd8-(dn), cd4-cd8+ and cd4+cd8+ subsets of inkt cells by five colour flow cytometry in patients with ad (n=10) and healthy controls (n=10). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x 0.01) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x 0.01). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r=0.726 and p x 0.001) and healthy controls (r=0.693 and p x 0.001). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il-4 level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x 0.05). the frequency, the number of inkt cells and the cytokine producing capacity of the cd4/cd8 inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il-4 that promotes the th2 differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting 37.5%, 16.5%, 12.1%, 7.9%, 7.9% and 7.5% of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells (1.8 %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd4 + nkt and cd4 -cd8 -nkt cells. nk1.1 + nkt cells may release large amounts of il-2, il-4, ifn-g and il-10 after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il-2 had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il-2. there was a significantly increase in the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd69 molecule existed in 68.95 % of the cells from large-scale selection. the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cell subsets in the giant lymphocytes were enhanced to 84.0 and 38.9 folds, respectively. under a light microscope at x400 magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of 5 fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than 1.0 and they are non-adherent cells. the differentiation pathway of the seb-activating cd8 + and tcrvb8 + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd8 + nkt cell and tcrvb8 + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl3 which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo-1am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h2begfp reporter mice. stimulation with anti-gd clone gl3 or anti-cd3 clone 2c11 elicited activation of gd t cells suggesting that tcr gd and cd3 molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl3 in vitro produced ccl4, ifng and tnfa. therefore, we were interested whether the ccl4 production of gd iiel influenced the homing of ccr5 cells such as lamina propria (lp) cd4 + foxp3 + cells (tregs). to test this, wt mice were i. p. treated with gl3 mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr5 + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr5 + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr5 expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il-4 production in g4 reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il-4 gene. in the absence of any manipulation gfp was expressed from the il-4 locus in populations of immature thymic nkt cells (predominantly cd4+cd44lotcrhi cells on a balb/c background, and cd4+cd44lonk1.1-on a c57bl/6 background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il-4 production was induced predominantly in cd4+ nkt cell subsets of the liver and spleen, and after i. n. administration, in cd4+ nkt cells of the airways. spontaneous and a-galcer-induced expression from the il-4 locus occurred in the absence of stat6 signalling, and did not require initial exposure to il-4 protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin-17 (il-17) plays an important role in neutrophil recruitment. herein, we investigated the role of il-17 receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c57bl/6 (wt) and il-17 receptor gene-deficient (il-17r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated 6 hours after surgery. the ability of il-17 mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il-17r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il-17 induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il-17r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il-17receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il-17 showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il-17 also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il-17 receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin-1 receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il-1r associated kinases to activate nfxb and p38 mitogen-activated protein kinase. the il-33-induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il-33 in different mast cell subsets. methods: different mast cells subsets (hmc-1, human cbmcs and murine bmmcs) were stimulated with il-33. the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk1/2, pakt, pnfxb, p38 and pjnk). additionally, we studied the signal transduction of il-33 in il-33r transfected hek293t cells. results: we found, that a tir family member, il-33r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il-33-induced cytokine production depends on c-kit transactivation. il-33r and il-1r accessory protein (il-1racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il-1r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il-33-induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il-33 in mast cells in absence of iger activation. (1) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase-3 activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease-1 (ho-1) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho-1 expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin 1 and 2. recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin 2 in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap1 small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap1 activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr2 shows a bimodal kinetic, with the first peak at 30''/1' and the second at 5' after stimulation. rna interference-mediated depletion of beta-arrestin 2 specifically inhibits the occurence of the second wave of rap1 activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin 2 is involved in rap1 activation and that the oscillations in the formation of rap1-gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c3g (rap1gef) and spa1 (rap1gap): preliminary results suggest that spa-1 has probably a role in the early activation peak. since this oscillatory chemokine-induced rap1 activation is present on other myeloid cell lines (hl60, 32d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin 2, localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh3 containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of 153 different sh3 domains that revealed over 20 putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)-3 and il-5 stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il-3 and il-5 receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il-3 , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn 328 are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: (1) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna-146 expression is induced by activation of the toll-like/interleukin-1 receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna-146a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna-146a transfected cells showed significantly reduced levels of the active proapoptotic caspases 8 and 3 (casp8/3). in line with this, mirna-146a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna-146a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna-146a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna-146a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il-15, il-7r a-chain and il-15r a -chain genes in hiv disease progression. methods: we studied 70 antiretroviral treated patients (progressors) and 71 long term non progressors (ltnp). we analyzed 2 snps in the il-15 gene, 5 snps in the il-7r gene and 3 snps in the il-15r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il-15r aa 182 and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il-7r and il-15r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd4 and cd8 t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art1, and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art1 in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art1 transcripts by rt-pcr analysis in human blood leukocytes. soluble art1, released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek293 cells with art1 and tnf resulted in modification of tnf at at least 2 distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r32, a site that has previously been implicated in binding to tnfr2. binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit225 than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art1 resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik 1 , t.v.poroshina 1 1 institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: 23 patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and 11 control men were assessed for slpi, tnf-alpha, il-8 and free tgf-b1 in ejaculate by elisas using stat-fax 303 plus. a 3 to 5 day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at -20 degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients (5127.571 ± 971.731 pg/ml, p x 0.001) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid (125.49 ± 17.9 pg/ml; p x 0.05). the il-8 concentration was elevated in all patients (366.7± 49.5 pg/ml; p x 0.05) in seminal fluid. free tgf-b1 was present in normal seminal plasma in high concentrations (346.4 ± 29.2 pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b1 concentrations were 36.2 ± 6.2 pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il-8 and free tgf-b1 are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi 1 , e. a. elgaaeid 1 1 faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th1 or th2 cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod2/card15 protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod2/card15 gene in cd. we performed a cases /controls study upon 75 cd patients and 90 healthy controls. this study suggests that in northen tunisian population, 3020insc mutation in nod2/card15 gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il-10 polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il-10 cytokine genetic profile in patients with ibd. we examined the contribution of il-10 gene promoter polymorphisms (-627 and -1117) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card15/nod2 gene mutations in cd presentation and location. in tunisian population, the 3020insc insertion in nod2/card15 gene is a marker of susceptibility to cd, while the a allele at position -627 in the il-10 promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il-10 gene polymorphisms and card15/nod2 gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il-2-dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il-2-driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il-2 molecule by ganglioside. but gangliosides apparently can also form complexes with il-2r; such complexes influence on the signal transduction through il-2r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il-2r subunits. in our work we use il-2-dependent cytotoxic t-cell murine line ctll-2. two different approaches for study of possible interaction types between exogenous gangliosides and il-2r subunits were applied: antibody staining of il-2r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with 125 i-dcp-gm1 followed by immunoprecipitation of il-2r subunits. the fluorescence intensity of the antibody-labeled il-2r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by 29.5 % after cells incubation with ganglioside gm1, and by 10.7 % after incubation with gm2. labeling of the cells with antibodies to the il-2r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm1 (2.8 %) or with gm2 (5.9 %). to determine the mode of this impressive masking influence of ganglioside gm1, photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm1 with subunits of il-2r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il-2r, but not for il-2r a-subunit. these results demonstrate that exogenous ganglioside gm1 can interact with a-and bsubunits of il-2r in different modes. interaction of il-2r b-subunit with ganglioside gm1 requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa 1 , j. bukur 1 , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak2 and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak2. results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak2 expression due to a gene deletion on chromosome 9, whereas the expression and functionality of other ifn-g signal transduction components like stat1 and jak1 were not affected. jak2 blockade by two jak2-specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak2 leading to a lack of jak2 expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak2 inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of 506 controls of the spanish population. methods: two il18 polymorphisms located on the il-18 promoter region, snps -607 a/c (rs1946518) and -137 g/c (rs187238) were genotyped using taqman snp genotyping assays. the functional il-18 gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il-18 -607 p=0,318. il18 -137 p=0,740) but may contribute to disease onset and aggressiveness: the genotype il-18 -607 cc genotype, which is associated with higher il-18 production, was significantly associated with higher tumor size (p=0,001), grade (p=0,030), t (p=0,001), m (p=0,012) and stage (p=0,002). the influence of the il-18 -103 gg genotype was less relevant, however was correlated with higher tumor size (p=0,036), grade (p=0,017), t (p=0,026) and stage (p=0,011). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il-18 polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il-18 gene (il-18 -607 and -137) may be associated with an worse prognosis of rcc. high levels of il-18 production can play an important role in grow, invasion and metastasis of renal cancer. interleukin-18 (il-18) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il-18 is its ability to induce the production of ifn-gamma in presence of il-12. moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l0. then it seems that il-18 has a crucial role in immunity against brucella infection. since the expression of il-18 can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il-18 gene polymorph isms and brucellosis. methods: a total of 188 patients with brucellosis and 77 healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il-18 polymorphisms at positions -656, -607, 137, +113, +127 and codon 35/3 using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il-18 polymorphisms at position -137 g/+113t/+127c (correlated with high production of il-18), codon 35/3c and -656g/-607c (correlated with higher production of il-18) were significantly higher in the healthy controls than the patients (p=0.000022, p=0.00185 and p=0.0441, respectively). discussion: as data revealed genotypes that have correlation with higher production of il-18 are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il-18 at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor 2 (ifngr2) mouse mutant. we have generated a mouse line carrying a conditional ifngr2 allele using the 2 loxp 2-flp recognition target (frt) approach. a targeting vector with 2 loxp sites flanking exons 4-6 and 2 frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat1 following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat1 activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd4 cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr2 on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez 1 , r. carretero 2 , p. saenz-lopez 2 , j. cantón 2 , j. carretero 2 , f. ruiz-cabello 2 , l.m. torres 1 , cts-143 1 hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, 2 hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and 130 normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca)12 allele of ifgn is significant more frequent in healthy control than in cin patient (p=0,030) in contrast homozygosis for (ca)12 allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p=0,030 and p=0,045 respectively). conclusions: our study suggest that ifng (ca)12 allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin-15 (il-15), a th1-related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il-15 single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: 190 patients with brucellosis and 78 healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il-15 (c267t, g367a, c13687a and a14035t) polymorph isms using pcr-rflp. results: at position 267, the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p=0.025 and p=0.042, respectively). at position 13687, the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p=0.050, p=0.015). discussion: as shown the frequency of cc genotype and c allele at position 267 were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position 13687 is lower in patients than the healthy controls and the frequency of c allele at position 13687 is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf-7 and skbr-3, as well as their ability to respond to several cytokines and to the g-1 gpr30 agonist. the mcf-7 and skbr-3 human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il-2ra, il-2rg, il-3/il-5/gm-csfr, il-4/il-13r, il-5ra, il-6ra, il-6r (gp130), il-7ra, il-10r, tnfr i, tnfr ii, ifngra, cxcr1, cxcr2, cxcr3, cxcr4, cxcr5. cells were incubated with il-10, ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr30 agonist. cytosolic ca 2+ concentrations [ca 2+ ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr-3 cells were found positive for a larger number of receptors than the mcf-7 cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf-7 cells with il-10 reduced the calcium response to g-1, while pretreatment with ifn-g and tnf-a potentiated the calcium response to g-1. tnf-b had no effect on mcf-7 g-1 stimulation. no direct effect on basal [ca 2+ ] i stimulation could be noticed when administering the cytokines alone. in skbr-3 cells, pretreatment with il-10 or tnf-b had no effect on basal [ca 2+ ] i and did not significantly alter the calcium response to g-1, while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g-1. pretreatment with tnf-a produced calcium oscillations and reduced the response to g-1. conclusions: mcf-7 and skbr-3 cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr30 stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd8a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd8a -pdcs and cdcs were infected with mcmv, whereas after 24h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd8 t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag1-deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag1 and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd8 t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg 1668, respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd8a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt3-l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f4/80 + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd8 t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr7 and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd11c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd8 t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd4 t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd4 t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd8 t cells in response to an adova infection due to an impaired cd4 t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than 500 passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il-12. to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp33 and vacv-gp33 expressing the gp33 epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p14 t cell receptor recognizing the gp33 epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p14ifnar -/mice. upon adoptive co-transfer of p14ifnar -/and p14ifnar wt/wt t cells and subsequent challenge with mva-gp33, a massive expansion of p14ifnar wt/wt t cells was observed, whereas p14ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp33 induced a rather similar expansion of ifnar competent and ifnar deficient p14 t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h5n1-specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h5n1 induced an exceptionally nf-kb dependent antiviral response. irf3 is essential part of this interferon-response of human endothelia. furthermore, we identified hmga1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h5n1. finally, nfatc4 was found to be a transcriptional regulator for specifically h5n1-induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h5n1 which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns1, delta-ns1 influenza virus (a recombinant influenza virus lacking the ns1 gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns1 during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting 2 days post-infection, reached its maximum at day 4, and triggered t cell priming in vivo. a direct comparison of delta-ns1 virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns1 protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns1 protein. thus, we propose that the virally encoded ns1 protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin 18 is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin 18 is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that 14-3-3 proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of 14-3-3 target proteins. thus, these results suggest that 14-3-3 proteins have a regulatory role in host defence against viral infections. i. wessels 1 , d. fleischer 1 , l. rink 1 , p. uciechowski 1 1 institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)-1b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il-1b expression is not elucidated, yet. it is known that the il-1b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il-1b when stimulated. b-cells which are il-1b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il-1b promoter and the impact of methylation on il-1b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl-60 cells were differentiated into monocytic cells after dihydroxyvitamine d 3 treatment. the monocytic phenotype was confirmed by flow cytometry. the il-1b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with 5-aza-2-deoxycytodine (aza) and changes in il-1b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl-60 cells, differentiated cells displayed upregulation of cd14 antigen and acquired the ability to express il-1b. by comparing the accessibilities of il-1b promoter we detected that the il-1b promoter was not accessible in undifferentiated hl-60 cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl-60 cells, demonstrating that the chromatin remodeling of the il-1b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il-1b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il-1b expression were found. our data indicate that the il-1b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il-1b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg 1 , g. wetzel 1 , h. arnold 1 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p65 family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il-1b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp2, 3, 4, 5 and 6 mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd88 was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. (2)). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd8 + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. 2008 aug 1;181 (3)). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. 2008 dec; 82 (24) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il-1b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory 4t1 breast cancer cells over expressing il-1b (4t1/il-1b) tumor cells, but rarely in untransfected 4t1 cells. secretion of il-1b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg2d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il-4ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used 130 healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array 5.0 having p x 10 -4 in two independent cohorts each consisting of 200 blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for 24h and we measured the levels of il-6, il-8, il-10, tnf-alfa and il1-ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed 150 snps with p x 1,0*10 -4 . to identify/replicate the association of cytokine production for these 150 we reanalysed these on a 200 cohort. a combined analysis revealed 10 snps with p x 9,1*10 -5 .these results are not genome wide significant. the 10 snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find 10 nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp60 have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp60 on adipocytes. for the first time we could show a hsp60-stimulated release of the proinflammatory cytokines il-6, cxcl1 and mcp-1 in a time-and concentration-dependent manner from murine 3t3-l1 adipocytes. analyses of hsp60-signalling in these adipocytes revealed that members of the mapk-family (erk1/2, p38) and the transcription factor nfkb are involved in hsp60-mediated induction of the mediators il-6, cxcl1 and mcp-1. binding-studies with fluorescence-labelled hsp60 demonstrated that the interaction of hsp60 with adipocytes exhibits basic features of a receptor-mediated binding. hsp60-binding to adipocytes was saturable and reached its maximum at 3.5 mm. binding was inhibitable only by the unlabelled ligand (52 %), but not by unrelated proteins, thereby proving the specificity of hsp60-binding. further analyses to characterize hsp60-receptor structures on adipocytes revealed the presence of toll-like receptor (tlr)4 on adipocytes. tlr4 has been found to be expressed on macrophages and to interact with hsp60, therefore suggesting tlr4 as a potential receptor candidate for hsp60 on adipocytes. in order to identify the responsible binding-epitope of hsp60 we investigated the effect of specific antibodies directed against different epitopes of the hsp60-molecule. incubation with antibodies directed against the n-terminus of hsp60 (aa1-200; 5-25 mg/ml) were capable of inhibiting the hsp60-binding to adipocytes (47-80 %) indicating that the n-terminal region of hsp60 is involved in receptor binding. our experiments demonstrate that hsp60 stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp60-mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf3. we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf3 knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with 3 % of fetal calf serum at 37°c and 5 % co2. bone marrow nonadherent cells were removed after 24 h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd10, cd29, cd73, cd21 and stro-1 and were negative for cd45. intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost 100 % of msc. interleukin-2, ifn-gamma and tnf (th1 cytokines) increased msc contractility, whereas il-10 (a th2 cytokine) decreased msc contractility. by immunofluorescence, we observed that il-2, ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il-10 decreased that incorporation. our results suggest that th1 and th2 cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca 2+ -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as 40 nm and 400 nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents (10 mm) at the 24-h culture interval reached the values of approximately 3 ng/ml and 2 ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of 2-6 h in rat pecs. the 24-h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p38 and erk1/2. it was not suppressed by the calcium chelating agents bapta-am and tmb-8. the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr 305/07/0061. pin1 is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin1 substrates and targeting sites. recent data show that pin1 interacts with apo-bec3g (a3g). the pin1/a3g interaction results in a reduced a3g expression and a diminished a3g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin1 gene (-842 g/c and -667 t/c) modulate pin1 expression; in particular, the -842 gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, 28; 69-74, 2007) . the -842 c/g and -667 t/c polymorphisms in the promoter of pin1 gene as well as pin1 protein levels were analyzed in 30 exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; 40 hiv-infected patients (hiv) and 40 healthy controls (hc). the genotype and allele distributions of the -842 snp was skewed in esn (genotype: p= 0.008; allele: p= 0.013). in particular esn showed a significantly lower frequency of the -842 gg genotype compared to hiv and hc (p=0.017 and p=0.019, respectively) and consequently a lower g allele frequency (p=0.026 and p=0.028, respectively). no significant differences were found for the -667 snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, 9-(r)[2-(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, 9-[2-(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. secretion of cytokines was determined after the 5-h culture by elisa. production of no was assayed at the interval of 24 h using griess reagent. approximately 300 compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. 9-[2-(phosphonomethoxy)ethlyl]-2,6-diaminopurine derivatives, c) 9-[2-hydroxy-3-(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) 9-heteroalkyl substituted 2-amino-6-guanidinopurines, and f) 2-amino-3-(purin-9-yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately 30 compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip-1a and cytokines tnf-a and il-10. although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n 6 -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as 2 to 5 mm. the remarkably enhanced secretion of chemokines was reached within 2-4 h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant 1m0508. host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and 10 million units of ifn-a treatment three times a week for 6 months was initiated. before and after treatment: percentages of the il-2 and ifn-g in cd4+ t cells were assessed to determine intracellular t helper cell 1 (th1) type cytokine expression. similarly, percentages of intracellular il-2 and ifn-g were detected to verify cytotoxic t cell 1 (tc1) type cytokine expression in cd8+ t cells. percentage of th2 and tc2 type cytokine expression, (il-4 and il-13) were determined in cd4+ and cd8+ t cells, respectively. six (50 %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th2 cells with respect to healthy controls before treatment. tc percentages, both tc1 and tc2, were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il-4 expression was higher and the percentages of th1 type cells were significantly low. il-13 expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency (29,8 %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects (6,6 % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt 1 , f. juengerkes 1 , b. schumak 1 , g. gielen 1 , j. kalff 2 , p. knolle 1 , b. holzmann 3 , a. limmer 1 1 university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, 2 university hospital bonn, department of surgery, bonn, germany, 3 department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il-12, the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh 1 1 rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il-17 and il-23 in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, 45 h. pylori-infected du patients (23 patients were positive for anti-caga antibody and 22 patients were negative for anti-caga antibody), 30 h. pylori-infected as carriers (15 subjects were positive for anti-caga antibody and 15 subjects were negative for anti-caga antibody) and 15 healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il-17 and il-23 was measured by elisa method. the mean serum levels of il-17 in total du patients (9.28 pg/ml ± 5.48) was significantly higher than those observed in total as subjects (5.19 pg/ml ± 3.75, p x 0.001) and healthy control group (3.55 pg/ml ± 3.76, p x 0.0001). in du group, it was found that the mean serum levels of il-17 in subjects with positive test for anti-caga (10.84 pg/ml ± 5.79) was significantly higher than those observed in subjects with negative test for anti-caga (7.65 pg/ml ± 4.74; p x 0.05). the mean serum levels of il-23 in du (8.66 pg/ml ± 8.41) and as groups (7.25 pg/ml ± 5.66) was significantly higher than those found in uninfected control group (3.64 pg/ml ± 3.36, p x 0.02 and p x 0.03, respectively). however, no significant difference was observed for mean serum levels of il-23 between du and as groups. moreover, in both du and as groups the mean serum levels of il-23 was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il-17 and il-23 in h. pylori-infected subjects as compared with control group. in du group the expression of il-17 influenced by the bacterial caga factor. a. aral 1,2 , a. atak 1 1 gazi university faculty of medicine, department of immunology, ankara, turkey, 2 gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il-6 levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il-6 elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il-6 levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the 48th hour and reached to maximum levels at the 1st week and decreased again at the 3rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the 1st week and started to decrease at the 3rd week. il-6 reached to its peak at the 3rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic 1 , m. jurisic 2 1 university of kragujevac, school of medicine, kragujevac, serbia, 2 university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in 43 radicular inflamed cysts and 15 odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd3, cd20 and cd68. tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x 0.05) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. 1) drawings blood from 20 patients and 20 healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. 2) detection of the gene expression of prolactin and tlr-2 in cd14+ peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p 0.05) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr-2 and peripheral prolactin expression in cd14+ monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u937 culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table-2 ). when tb household contacts and healthy controls were compared, cfp10 and esat6 seemed to be more useful than tst in tb contacts for displaying ltb (table-2) . although cfp10 spot numbers were much more than esat6 spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: 0,069)( table-3 ). both esat6 and cfp10 spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g 20 analytes immediately prior to liver transplantation and at sequential time points up to 90 days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip-10, il-6 and il-10. notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type 1 ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/1/68 (h3n2) (hk) differs from its putative avian precursor by 7 amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk-5aa-i62r, n81d, k92n, s193n, g144a, human (2-6); rhk-r2-l226q, s228g and rhk-7aa-i62r, n81d, k92n, s193n, g144a, l226q, s228g, avian (2-3)). among these variants, the double mutant rhk-r2 and the seven mutant (rhk-7aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l226q and s228g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about 50 pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip-10 and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r2 and rhk-7aa as compared to rhk and rhk-5aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il-8, shedded adhesion molecules (cd25, vcam-1, icam-1), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk-5aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of 98 newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il-10, and neutrophilic cd64 levels, procalcitonin and il-10 were measured by elisa technique while, neutrophilic cd64 by single colour flowcytometric technique. of these 49 "infected" infants, 16 had positive blood culture (subgroup ia: culture-positive sepsis), and 33 infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another 49 newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il-10, cd64, and crp. il-10 had the highest sensitivity and specificity, 92 % and 84 %, respectively, using cutoff n 17.3 pg/ml. for pct, the highest sensitivity and specificity, 65 % and 60 %, respectively, were at a cutoff value of n 36.4 pg/ml. neutrophilic cd64 had maximal sensitivity and specificity of 92 % and 71 %, respectively, at cutoff value of 2.6 %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il-10 and neutrophilic cd64, which together provided sensitivity and specificity of 95 % and 83 %, respectively, and npv 86 %. the combination of il-10 and crp had high sensitivity and moderate specificity, 93 % and 79 %, respectively. conclusions: il-10 and neutrophilic cd64 levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp3, naip5 or ipaf and the adaptor asc are involved in caspase-1 activation in response to bacterial infection, triggering the processing and secretion of il-1b and il-18. recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi3k, vps34 and can be inhibited with the pi3k inhibitors wortmannin and 3 methyladenine (3ma). in contrast, activation of akt, via class i pi3k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il-1b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with 3ma, wortmannin or an akt inhibitor. supernatants were analysed for il-1b by elisa. results: 3ma enhanced il-1b secretion by bmdc treated with the tlr3 and tlr4 ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il-1b secretion was greatly reduced in bmdc from nalp3 -/mice compared to wild type c57/bl6 controls. treatment with the akt inhibitor had no effect on lps-induced il-1b secretion by bmdc. tlr-dependent secretion of il-1a was also enhanced by treatment with 3ma. conclusions: these data demonstrate that il-1b secretion by bmdc in response to treatment with pi3k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp3 inflammasome. this response is limited to tlr3 and tlr4 agonists. inhibition of akt had no effect on lps-induced il-1b production, suggesting that the effect of wortmannin and 3ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi3k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr2 signaling by the inflammatory lipid mediator sphingosine 1-phosphate (s1p) through receptors 1 and 2 in human monocytes-macrophages, which could explain some of the s1p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s1p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s1p, and later analyzed by flow cytometry. a pharmacological analysis of the s1p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam-1 expression was increased following lps and s1p concomitant stimulation in both venous and arterial cells, suggesting that tlr4 and s1p receptors cooperate in the expression of icam-1. conversely, no cooperation was observed when tlr2 ligands were used. in order to elucidate which s1p receptor subtype was involved in the increase of icam-1 expression, we used a pharmacological approach with s1p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam-1 after lps and s1p challenge was significantly reduced by blocking s1p receptor 3 and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s1p receptors 2 and 3, which suggest that s1p receptor 1 might be involved in the effect. conclusions: altogether these data demonstrate that tlr4 and s1p receptors can interact to increase adhesion molecules such as icam-1 in human endothelial cells, and the s1p receptor subtype involved in the effect differs between arterial and venous cells. 15 with ssc without pah) and a pool of 12 sera of healthy controls (hc) were tested. results: in 1 dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with 7-10, 4-8 and 2-5 protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in 2 dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized 145±48, 127±26, 130±25 and 150 protein spots respectively. twenty one protein spots were recognized by more than 80 % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein 1 and peroxyredoxin 6 and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: 27 patients suffering from different diseases were enrolled in our study. 16 patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas 11 of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam-1 and vcam-1 were assesed by an enzyme immunoassay (elisa). results: 11 out of the 27 patients (40,7 %) had elevated levels of the intercellular adhesion molecule. 6 out of the 16 patients suffering from bone diseases (37,5%) had raised values (mean value 400 ng/ml) whereas 5 patients out of the 11 suffering from soft tissue diseases and diabetes (45,5 %) had raised values (mean value 735,5 ng/ml). reference value for icam-1 was 130-299 ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients (40 %) having increasd levels of icam-1. high icam-1 levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes (45,5 %) than in patients with bone diseases (37.5 %). mean values were found 735,5 ng/ml and 400 ng/ml accordingly. those findings verify the positive correlation between icam-1 and inflammatory diseases and tissue damage but not for vcam-1. colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: 16 cases of ulcerative colitis (urc), 16 adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, 33 tubular or tubulo-villous adenomas with low grade dysplasia, and 33 infiltrating adenocarcinomas. immunohistoobjectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd18 in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind 20 patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). 40 mg of pravastatina oral 2 hours they were administered before the procedure (group study, n=10) or placebo (group placebo, n=10), and control (group control, n=10). samples of outlying veined blood were extracted to the 24 hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd18 in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: 3 types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in 3 degrees: degree 0. without expression. degree 1. weak; degree 2. moderate; degree 3. intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree 0-1. the placebo group: mixed pattern, degree 1-2. group study (40 mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree 2-3. the percentage of cells that expressed cd 18 was greater in the group study (40 mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd18 answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam10 and adam17. we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam17 as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam17. these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam17 activity in the tissue. in the presence of the adam17 inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam17 as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam17-mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf-460/ vegf+405 is associated with rcc risk ( p= 0,017), metastases ( p=0,043), nuclear grade ( p=0,05), tumor stage ( p=0,029), and tumor size (p=0,04). on the other hand, the polymorphism vegf -2578 a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol 3,4,5-trisphosphate. therefore, pten is one of the main antagonists of the pi3-kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il-6 as well as il-8 levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il-6 as well as il-12 and il-23 mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th-17 t cells, we measured th-17 cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il-17 and a strong reduction of il-22 mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il-10 exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il-1ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il-10. based on our previous observation that support a direct role of il-10-activated stat3 in the enhancement of il-1ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il-1ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il-10. quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il-1ra promoter. crosslinked nuclear lysates were immunoprecipitated 30 and 60 min after il-10 addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il-1ra promoter. chip assays showed that the pol ii recruitment to the il-1ra promoter induced by lps is significantly increased by il-10, further strengthening the concept that the rapid enhancement of lpsinduced il-1ra gene expression by il-10 initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat3 immunoprecipitated dna showed statistically significant levels of stat3 binding to the il-1ra promoter only in cells stimulated with lps in the presence of il-10. surprisingly, anti-p65 and anti-p50 chip assays revealed enrichment of both p65 and p50 recruitment to the il-1ra promoter when il-10 was added to lps-stimulated cells, suggesting that il-10 enhances the recruitment of nf-kb to the il-1ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il-10-treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il-1ra promoter site is dependent on il-10-activated stat3, since it is greatly reduced when stat3 activation by il-10 is impaired. the molecular mechanism through which il-10-activated stat3 promotes the recruitment of nf-kb to the il-1ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il-15 acts as a specific negative transcriptional regulator of mouse mast cell protease-2 (mmcp-2). we examined the mechanisms underlying the repression of mmcp-2 gene expression. our data show that the "repressor" effects of il-15 on mmcp-2 promoter activity are still operating on the mmcp-2 591 bp long minimal promoter. moreover, il-15 deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy1. furthermore, chromatin immunoprecipitation revealed that il-15 promoted specific reciprocal recruitment of c/ebpb but not yy1 to the mmcp-2 promoter. finally, il-15 deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp-2. thus, we proposed that the expression of mmcp-2 and possibly other immunoregulatory genes may be regulated by il-15 through epigenetic modification and by balancing the content and binding of c/ ebpb and yy1 in mast cells. i. nagy 1 , k. filkor 1 , a. vörös 1 , l. kemény 2 , a. szász 1 1 bzaka, baygen, szeged, hungary, 2 university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir-203, mir-146a and mir-155, which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir-203 expression; in contrast, pgn re-stimulation had no further effect on mir-146a and mir-155 expression. next, we investigated the correlation between the expression of mir-203 and its two known direct targets: regulatory protein p63 and suppressor of cytokine signalling-3 (socs-3). although the gene-expression profile of neither p63 nor socs-3 changed, we found that the expression of mir-203 reversibly correlates with both p63 and socs-3 protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir-203 prior to pgn-treatment completely abolished both p63 and socs-3 down-regulation, revealing the involvement of mir-203 in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir-203 expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb1+/-had a lower incidence and burden of benign papillomas when compared to tgfb1+/+ animals. however, more scc developed in the tgfb1+/-mice. after acute and chronic promotion, tgfb1+/-skin showed a reduced proliferative response with no increase in epidermal tgfb1 or nuclear p-smad2 compared to tgfb1+/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb1 gene dosage. further, pharmacological inhibition of alk5 suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb1 are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb1+/-skin, tgfb1+/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb1+/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb1+/+ but not tgfb1+/-keratinocytes, indicating that tgfb1 switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb1 +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb1 acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of 132 patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp-1) and fibroblast (mrc-5) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of 132 ra patients and 82 controls the levels of survivin correlated to urokinase (upa) (r=0.46), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that 30/132 ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc-5 and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol 1,4,5-trisphosphate 3-kinase type b (itpkb) phosphorylates inositol (1, 4, 5) trisphosphate (ins(1,4,5)p 3 ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca 2+ responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h1 and h2 receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il-1b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il-1b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase-1 to generate the mature secreted active form. caspase-1 is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of 425 genes involved in splicing with an average 5-fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified 30 genes that significantly affect the production of il-1b by thp-1 cells after a 24h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il-1b secretion. tissue transglutaminase (tg2) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg2 in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg2 expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg2 can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg2 not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg2 inhibitor, in a transgenic mouse model cf and in the taz10 transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz10 tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg2 in generating inflammation in two very different pathologies. this work underlines the critical role of tg2 in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge 2 stimulated a-dc. preliminary results indicate no accumulation of hif-1alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif-1alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p38 but not erk1/2 or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt5a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il-12 and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt5a -but not wnt3a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt5a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt3a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp130 on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp130 in te. after infection with t. gondii, mice lacking neuronal gp130 (synapsin-cre gp130 fl/fl ) died significantly earlier in the chronic phase of infection than control gp130 fl/fl mice. death of synapsin-cre cre gp130 fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp130 fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp130-deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp130 expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp130 fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il-1b, il-6 and il-8 have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il-4, il-10 and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb1 further may generate more effective therapies. as tnfa mrna 3' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb1 suppresses tnfa protein production by upregulating the rnabinding protein fxr1, which can bind to tnfa mrna and inhibit translation. methods: using raw 264.7 cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb1, we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 3'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb1 and luciferase expression was quantified. cells treated with lps and tgfb1 were also examined for fxr1 expression using pcr and western blot. following fxr1 knockdown using sirna, the influence of tgfb1 and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb1. using the luciferase-tnf-3'utr vector we show that tgfb1 targets the 3'utr of tnfa. furthermore, tgfb1 and il-10 both upregulate fxr1 mrna and protein; and treatment with tgfb1 and lps can synergistically upregulate mrna expression, more than tgfb1 alone. following sirna inhibition of fxr1, tgfb1 can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp-1 and mmp-3 secretion from saecs, nhbes and fibroblasts to a peak of 2.5 +/-0.5 ng/ml, at 72 hours. interleukin-17 augmented comtb-stimulated up-regulation of mmp-1 and mmp-3 secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin-17 down-regulated mmp-9 secretion from saecs by 50 %. interleukin-22 up-regulated mmp-1 and mmp-3 secretion from fibroblasts but not from saecs. timp1 secretion from saecs was enhanced by interleukin-17 but there was no effect of interleukin-22. mmp up-regulation by interleukin-17 and comtb was inhibited by the pi3kinase inhibitor ly294002 and on western analysis akt (protein kinase b) was phosphorylated at 30 minutes. chemical inhibition of the p110d isoform of pi3kinase with ic87114 abrogated the il-17 and comtb driven secretion of mmp-3 from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome 10) accentuated mmp-3 secretion. these inhibitory effects were confirmed with sirna. mmp-3 up-regulation was secondary to increased gene expression with promoter activity peaking 24h after stimulation. in summary, interleukin-17 and interleukin-22 drive transcription dependent mmp-1 and mmp-3 secretion from airway epithelial cells and fibroblasts. interleukin-17 also increases timp but down-regulates mmp-9 gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi3kinase pathway is central in interleukin-17 driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto 1 , l.s. moreira 2 , e. gonzalez-rey 2 , m. delgado 1 1 institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, 2 university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within 15 years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper 1 response. cholesterol metabolism is regulated by factors such as pparg1 (proliferator activated receptor g), srb1 (a class b scavenger receptor), cd36 or abca1, that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th1 immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg1, srb1, cd36, and abca1. we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to 60 % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il-6 in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il-6 and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il-6, tnf-a, il-1b, il-8 and il-12, and the anti-inflammatory cytokine il-10, in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il-23 was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il-6 and il-10, and in some cases, of il-23. the maximal plasma levels of il-6 and il-10 were found at 24 hours and of il-23 between 48-72 hours. modest plasma levels of il-8 were also observed, with maximal production at various time points (4-24 hours). by contrast, production of tnf-a, il-1b and il-12 did not occur to a significant extent, while production of il-17 occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il-6, il-8, il-23 and il-10 also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il-6, il-10, il-8 and il-23, i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving 165 patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il-6, activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il-33 is a novel il-1 cytokine family member that is expressed as an intracellular precursor (pro-il-33) and is thought to be cleaved by caspase-1 to yield a mature bioactive form of the molecule (mat-il-33). to date however, evidence of cell-associated proteolytic processing and caspase-1 dependent secretion of mat-il-33 has not been reported. here we show that pro-il-33 but not mat-il-33 is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il-33 to the il-33r and also il-33r-dependent bioactivity of pro-il-33 on mast cells. we propose a previously unrecognized role for pro-il-33 as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il-23 p19 mrna expression in myeloid cells, and markedly increase secretion of il-23, but not il-12, by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il-23 promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop10. chromatin immunoprecipitation (chip) assays using anti-chop10 and isotype control mab were performed using nuclear lysates from u937 cells and il-23 promoter dna measured by qpcr. chop10 binding on the il-23 promoter was detected following stimulation of u937 cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il-23 promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop10 in il-23 gene transcription, u937 cells expressing shrna's specific for chop10 or non-specific gene target were tested for their ability to express il-23 following tlr and er stress stimulation. u937 expressing three independent shrna targets for chop10 exhibited significant reductions in il-23p19 mrna (up to 87 % reduction of the response to lps+tp) compared to u937 expressing a control shrna. chop10 shrna expression did not affect the expression of other lpsresponsive genes, including il-1, il-8, ccl3 and sod2. to identify if er stress induction of il-23 mediated by chop10 expression plays a role in a more physiological setting, we examined the role of chop10 in the induction of il-23p19 gene expression following chlamydia trachomatis (ct) infection. infection of u937 cells with live but not g-irradiated ct induced expression of er stress response genes, including chop10. u937 infected with live ct exhibited increased il-23p19 mrna expression compared to u937 infected with nonviable bacteria. chop10 silencing significantly reduced the ability of live ct to induce il-23p19mrna, confirming the important role of chop10 in this response. these data suggest that er stress induction of chop10 could contribute significantly to the pathogenesis of diseases in which il-23 plays an major role, through induction of il-17 and il-22 producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd45b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd45b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd45b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd45b maintained a sustained activation of p38 kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd45b-deficient mice showed only transient p38 activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd45b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd5 and cd69 were elevated on gadd45b-deficient thymocytes. thus, we provide evidence that gadd45b and a resulting persistent activation of p38 constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo 1 1 institut pasteur, paris, france, 2 monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il-7 is recognized as an essential factor for thymopoiesis, the nature of the thymic il-7 niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il-7 promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il7 transcripts (il-7 hi cells). il-7 hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl19, ccl25, cxcl12) and cytokines (il15) that are critical for normal thymopoiesis. in the adult thymus, il-7 hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il-7 hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il-7 levels. conversely, the frequency of il-7 hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il-7-expression by tecs. > together, our temporal-spatial analysis of il-7-expressing cells in the thymus suggests that thymic il-7 levels are dynamically regulated under distinct physiological conditions. this novel il-7 reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il-7 expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l5 and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl2-tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl2 and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl2-tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl2-tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg2 plasma cells and serum igg2 levels were˚5-10 fold increased. importantly, serum from young slc-tg;em-bcl2-tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in 2/3 and 6/6 cases, respectively. these values were increased when compared with control groups: 1/6 in fcgriib -/and 0/6 in bcl2-tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd27 + cd135 + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd117 hi cells, representing multipotent progenitors, or cd127 + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb2 and ephb3 expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b1 and/or ephrin b2, the ligands of ephb2 and ephb3 receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb1 or ephrinb2 genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb1/b2 double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd45+ cells in the cortex, increased proportion of k5+k8+ cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb1 and ephrinb2 in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink 1 , d. vanhecke 1 1 university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op9-dl1 cell culture system (1) . using this in vitro assay we obtain large numbers of human cycd3 + and cd4 + cd8 + double positive thymocytes starting from umbilical cord blood (ucb) derived cd34 + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il-7 and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd34 + cells upon notch signaling is cd7 followed by cd127. t cell specification is accompanied by the induction of cd1a and loss of cd34 on cd7 + cd127 + cells. these cd34 -cd1a + cd7 + cells become dependent on continuous il7 and notch signaling for sustained survival and further differentiation into cd4 + cd8ab + dp thymocytes. we found that flt3l is not essential for the differentiation of cd4 + cd8 + human thymocytes but that addition of exogenous flt3l in the co-cultures increases the number of cd34 + precursors and consequently result in higher yields of developing cd4 + cd8ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd3 + cd4 + cd8ab + dp subset in this in vitro assay suggesting that op9 stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn3 to dn4 stage, where bdnf and its receptor p75 are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga 1 , c. lópez-rodríguez 1 1 pompeu fabra university, barcelona, spain nfat5 is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat5 are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat5 participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat5 deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd8 + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat5-null mice are unable to mount cd4+-and cd8 + -immune responses. data from our laboratory indicate that nfat5-null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat5 during the development of t lymphocytes, we developed mouse models that delete nfat5 at early (lck-cre + /nfat5 flox/flox ) or late (cd4-cre + /nfat5 flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat5 is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat5 at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p53, is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues 58-88 of the proline-rich domain of p53. methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p53 null (p53-/-) and wild type (p53+/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p53-/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p53 has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p53 is not important for preventing thymic t-cell tumors. s. myrczek 1 , r. pardi 2 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, 2 vita-salute san raffaele university school of medicine, milano, italy jab1 is the catalytic subunit of the highly conserved cop9 signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab1 regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab1 regulates the activity of ap1 transciption factors. to date jab1 is thought to be essential for every cell type as jab1 knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab1. to investigate the function of jab1 in b cells we established a mouse strain deficient for jab1 selectively in b cells. mice with floxed alleles of jab1 kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb1-locus (m. reth, freiburg). ablation of jab1 expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b1 and b2 cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl2 under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab1-deficient, bcl2-transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab1 in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab1 when apoptosis is prevented. t. nitta 1 , s. murata 2 , k. tanaka 3 , y. takahama 1 1 university of tokushima, tokushima, japan, 2 university of tokyo, tokyo, japan, 3 rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd8 t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd8 t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd8 t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th1 and th2 antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd4+ t cells or functional deficits that selectively interfere with th1 or germinal centre responses. in this talk i will present data from some of the first 12 strains that have been identified including the first 5 strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn1 c-kit high (etp), dn2 and dn3 thymocyte populations was hybridised to affymetrix mouse 430a-2.0 genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors (85 out of 623 genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included 64 signal transducers (out of 590 genes) such as acvr1, bmpr1, fzd7, chemokine receptors cx3cr1, cxcr6 and integrins a2, a4, a9, ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata-3, tcf-1, notch-1, rag-1, rag-2 and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn2 stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn3 population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd8aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks 3 out of 4 of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e14 thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by 7-10 days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day 18 of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd34 + cd1ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged 1 month to 3 years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd34 + cd1precursors with recombinant wnt3a (100 ng/ml) or with licl (10 mm) for 12hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab (8e7) under conditions of phosphatase activity inhibition. wnt3a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il-15 and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st2 cells suplemented with il-7 and flt3l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt3a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt3a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il-7 and flt3l and modify the transcription factor profiles of cd34 + cd1thymocytes mainly increasing hes-1 and id3 expression levels. human th17 clones and circulating th17 cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th1 and th2 clones or circulating th1oriented t cells, respectively. accordingly, human th17 cells exhibited lower expression of clusterin, and higher bcl-2 expression and reduced apoptosis in the presence of tgf-beta, in comparison with th1 cells. umbilical cord blood naï ve cd161(+)cd4(+) t cells, which contain the precursors of human th17 cells, differentiated into il-17a-producing cells only in response to il-1beta plus il-23, even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd161(+) t cells but it increased the relative proportions of cd161(+) t cells differentiating into th17 cells in response to il-1beta plus il-23, whereas under the same conditions it inhibited both t-bet expression and th1 development. these data suggest that tgf-beta is not critical for the differentiation of human th17 cells, but indirectly favors their expansion because th17 cells are poorly susceptible to its suppressive effects. m. irla 1 , w. reith 1 1 university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd4(+) or cd8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd4(+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd40 on mtecs by cd40l induced on the positively selected cd4(+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd4(+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu 1 , i. ravens 1 , g. bernhardt 1 1 hannover medical school, institute of immunology, hannover, germany cd155 is originally identified as human poliovirus receptor (pvr) and as rodent tage4, which is overexpressed in rodent colon carcinoma. cd155 is also known as necl-5, a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd155 expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd155 is overexpressed in transformed cells and promotes the cell cycle. thus, cd155 seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd4 and cd8; the dn subset is further subdivided into four stages (dn1-4) by differential expression of cd44 and cd25. thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd4+ sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd8 lineage. here we show that the frequency of terminally matured cd8+ sp cells but not that of cd4+ sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd155, a selective deficiency of mature cd8+ sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd155 is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd8+ sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd8+ t cells. in cd155 deficient animals, a shift in the tcr repertoire displayed by the pool of cd8+ sp cells was found demonstrating that cd155 is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. 2005 , 202, 1599 . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht 33342, propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e9 (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd71 + ter119 + erythroid and cd45 + cd11b + myeloid cells are simultaneously cycling (s/g2/m) in the post-gastrulation mouse embryo (e10-12). the peak of lymphohematopoietic cell proliferation occurs at e13 in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence 24-48 hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e11-12 that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd40-cd154 interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd40, we hypothesised a role for cd40-cd154 interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd40 expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd154 message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd154-/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd154 on reconstitution of b cell numbers following depletion. we show that cd40 is expressed by pro-b cells, and these cells proliferate in response to cd40 signalling in vitro. pcr identified a source of cd154, negative for cd3eta, in the bm of wt mice showing this cd154 is not provided by activated re-circulating t cells. we have shown that when cd154 -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn1 (cd4 -/cd8 -/cd44 + /cd25 -) to the dp (cd4 + /cd8 + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn1 stage on. in the bone marrow we found yfp + /b220 + and yfp + /b220populations. thus these pta expression analyses show closely similar pattern to those observed with hucd25 preta-reporter transgenic mice (gounari f. et al. 2002 , martin et al. 2003 . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. 3, 489-496 (2002 ) martin c. h. et al., nat. immunol. 4, 866-873 (2003 . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz 1 , g. klein 1 1 zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm-332 which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x 70kda.mmp-19, a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm-332. in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp-19. by western blotting the zymogen and the activated form of mmp-19 can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm-332 and mmp-19 can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp-19 which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp-19, timp-2 and timp-3, are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp-19 plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of 15-20 % compared to only 5 % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd4/cd8 expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd4+cd8+ double positive (dp) tcrab low cells entering selection, and their cd4+cd8+ dp tcrab-immediate precedents followed by underrepresentation of the selected cd4+cd8+ dp tcrab high and the most mature cd4-cd8+ and, particularly, cd4+cd8-single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd4-cd8-double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd4+cd8-/cd4-cd8+ sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a 1 -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a 1 -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a 1 -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd4/ cd8/tcrab), in adult wistar rats subjected to 14-day-long treatment with a 1 -ar blocker urapidil (0.20mg/kg body weight/day s. c.). the a 1 -ar immunoreactivity was found in both thymocytes (mainly less mature cd3and cd3 low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed1-postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd4/ cd8/tcrab expression were observed. these changes comprised of an increase in the percentage of cd4+8+ tcrabthymocytes, which was accompanied by the reduction in that of cd4+8+ tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd4+8-tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd4-8+ tcrab high was reduced. in addition, the percentage of cd4+ t regulatory and cd161+tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a 1 -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a 1 -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around 4 weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c57bl/6 mice infected with m. avium (10 6 cfus, iv) were sacrificed at different time points after infection (5, 16 and 25 weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il-10) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at 16 weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at 24 weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il-10 in this organ. in the spleen, ifn-g reaches a peak of expression earlier (5 weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm29935 and ai067906 from nih and grant i-823 from the robert a. welch foundation to wtg, and by grants hl067256 and hl61897 from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. 2001) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c57bl/6 x balb/c)f1 mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f1 mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski 1 , s. lang 1 , m. stein 1 , t. winkler 1 1 friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h3ac) or methylation of h3 on lysine 4 (h3k4me2/3). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h3ac and h3k4me2/3) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar #3) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 5'rlm race at the ivar #3 element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo2 locus, is thought to be accessible only during the double-negative (dn) 1 and 2 thymocyte stages based on mrna expression, implying that translocations between lmo2 and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx1 (hox11), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed 1) to evaluate lmo2 and tlx1 breakpoint-site accessibility during thymocyte development; 2) to determine in which stage of development there is an increased chance for lmo2 or tlx1 translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo2 accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo2 and tlx1 loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn1 and dn2 development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn1, dn2, dn3 and immature single positive (isp) stages for both lmo2 and tlx1. conclusion: our findings show that both the lmo2 and tlx1 loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn1 and dn2 stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that 1 to 3 successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e10-12), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag2 -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under 6 months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd4 cell depletion. however, cd8 depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd8 t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd8 cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd8 t cells has important implications for vaccine development against neonatal infections. (2008) showed that irf8 knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf8 as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf8 in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf8 were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs17444416. conclusions: although recent findings indicated that irf8 function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf8 that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez 1 , t. boehm 1 1 max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn1 transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l1 expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd4 and cd8 t cells from tuberculosis patients highly express pd-1 when compared to healthy uninfected individuals. in addition, analysis of pd-1 expression in lung biopsies from tuberculosis patients revealed that pd-1 is expressed on cd4 and cd8 t cells confined to lung granulomatous lesions. finally, blocking of the pd-1/pd-l1 axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd-1/pd-l1 pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p100 to p52. among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt 1 aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly 1 wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th17 and th1 cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd4 + t cells, nfatc1 and c2 are predominantly expressed. nfatc1 is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc1/a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc1/c. as demonstrated by y2h screen and co-ips, nfatc1/c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc1/c -but not the unsumoylated nfatc1/a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc1 target gene interleukin-2. other lymphokines like ifng and il13 are reversely regulated. interestingly, ntreg cells which do not express il2 exerted only nfatc1/c, but no nfatc1/a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc1 from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc1 function. therefore, especially ntreg cells and anergized cd4 + t cells might be regulated by the long sumoylatable isoform nfatc1/c. lnk/sh2b3 and aps/sh2b2, two members of the lnk/sh2b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b-1 cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il-7 signalling in pre-b cells overexpression of lnk dramatically inhibits il-7-dependent growth demostrating that lnk negatively regulates il-7 pathways. furthermore, we showed that il-7 stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e3 ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav1. to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd3 complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd3 complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd4 + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh2 or sh3 domains known to mediate protein-protein interactions are key players in these processes. sly2 (sh3 domain protein expressed in lymphocytes 2) was identified as a putative adaptor protein containing a sh3 and a sam domain as well as a bipartite nls. sly2 belongs to a family of three molecules: sly1, sly2 and sash1.in humans, the sly2 gene is located on chromosome 21, in mice on chromosome 16. sly2 is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly2 protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly2 we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin3-associated polypeptide p30 (sap30) as a putative interaction partner of sly2. sap30 is a conserved member of the sin3a-hdac corepressor complex that contains histone deacetylase 1 (hdac1) and histone deacetylase 2 (hdac2) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected 293t cells. in addition, we could show a direct interaction between sly2 and hdac1. to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly2 increases the activity of hdac1 in whole cell lysates and, more precisely, in nuclear extracts of 293t cells. the interaction of sly2 with sap30 and hdac1 indicates a transcriptional function of this protein. within the sin3a-hdac corepressor complex sly2 might act as a switch for the activity of hdac1. cd46-cyt1 and cyt2 are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd19 (b cell marker) fused to the transmembrane and intracellular domain of cd46-cyt1 or cyt2 in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt1 coengagement controlled il-10 secretion, while cyt2 coligation inhibited ifng production. moreover, our preliminary data suggest that cd46-cyt2 inhibits the phosphorylation of several molecules known to be activated by cd46 stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd46 cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh2 domain and three sh3 domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut78 cells with gst fusion proteins containing full length nck, the three sh3 domains or the individual sh3 and sh2 domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp76 and cd3epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam68) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip55 once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g4, results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g4) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g4) degranulation as measured by cd107a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi3k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose-6-phosphate receptor which exhibits structural and functional similarity to the vps10p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose-6-phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla-4 (sctla-4) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla-4 in immune responses. using a specific anti-human soluble ctla-4 monoclonal antibody, jmw-3b3 that selectively binds the soluble isoform but not membrane bound ctla-4, or cleaved fragments of it, we demonstrate that sctla-4 plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla-4, secreting increased amounts of cytokines including interferon-g, il-17 and tnf-a, but lower amounts of il-10. soluble ctla-4 was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla-4 induced secretion of the immunoregulatory cytokine il-10 by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine 2,3 dioxygenase enzyme cascade was also initiated by sctla-4. it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla-4 it is crucial to t cell inhibition. membrane-bound ctla-4 exists as a homo-dimer on t cells but sctla-4 is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b7 ligands on antigen presenting cells. a third important observation from this study is that sctla-4 exists both in serum and culture supernatants as a natural 46kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla-4, concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il-10 dependent regulation is most critical, boosting sctla-4 secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin-1/efhd-2, in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin-1/efhd2 is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin-1/efhd-2 in the immature murine b cell line wehi231 enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin-1/efhd-2 impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g1 cell cycle arrest. to understand how swiprosin-1/efhd2 enhances pro-apoptotic bcr signals, we analyzed whether swiprosin-1/efhd2 is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin-1/efhd2 enhanced bcr-induced calcium flux in wehi231 cells, whereas shrna-mediated down-regulation of swiprosin-1/ efhd2 impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin-1/efhd2 interacts with phospholipase cg2 (plcg2) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg2 and swiprosin-1/efhd2 was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin-1/efhd2 silenced wehi231 cells with swiprosin-1/efhd-2 was inhibited by the syk inhibitor bay 61-3606. in analogy, swiprosin-1/efhd2 regulated syk activity positively. moreover, swiprosin-1/efhd2 re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg2 and of syk tyrosine residue 352, which is involved in syk activation. finally, reconstitution of swiprosin-1/efhd2 knock-down cells with swiprosin-1/efhd2 mutants revealed that the n-terminal putative sh3-binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi231 cells. interestingly, swiprosin-1/efhd2 re-expression in swiprosin-1/efhd2-silenced cells induced already in unstimulated cells raft partitioning of syk, plcg2 and the bcr, which was reversed after 2 min of bcr stimulation. in summary, swiprosin-1/efhd2 is an accelerator of proximal bcr signalling and acts through syk and plcg2 by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e3 ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc1 tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd28 co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p14 mice die within hours after a second challenge with p33 peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e3 ligase dead mice can spontaneously reject tc1 tumors. conclusion: cbl-b e3 ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e3 ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e3 ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd8 t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd8 + cd45ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip10 and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal 1) and co-stimulatory signals (signal 2), provided by beads coated with anti-cd3/cd28 antibodies. gene expression patterns were compared for cells stimulated with anti-cd3/cd28 beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip10, itac). the enhanced expression of granzyme-b, ifn-g, trail and ip10 were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd3-coated p815 cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal-3 to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd3 + foxp3 + and cd4 + cd25 high t cells. moreover, t cell receptor activation with combined anti-cd3 and anti-cd28 stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk-506, but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv-1 infection leads to immune dysfunction owing to a successive loss of the cd4 + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv-1 virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd4 and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk1/2. this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk1/2 directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap-1, nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk1/2 is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal 1 , t. brdicka 1 , v. horejsi 1 1 institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd45 are key regulators of src-family kinases in leukocytes. while cd45 is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh2 domain of csk binds phosphotyrosine 317 of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd25 and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd69 upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd25-csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd8 t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd8 t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie-2kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd8 t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd8 t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd8 t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd8 t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa-1 integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git2 in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi 1 , s. parusso 1 , b. frossi 1 , g. gri 1 , c. pucillo 1 1 university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il-6 -/-mcs and anti-il-6 receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il-6 derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd40 on naï ve b cells and the interaction of cd40 on b cell surface and cd40l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd40l e cd40 on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves 1 , n. bercovici 1 , a. caignard 1 1 inserm u567, paris, france inhibitory killer ig-like receptors (kir2dl1-2/3) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd8 + t cell subsets. these receptors suppress cd8+ t cell activation through recruitment of the src homology 2 domain-containing protein tyrosine phosphatase 1 (shp-1). to further analyse the yet largely unclear role of inhibitory kir receptors on cd4+t cells, kir2dl1 transfectants were obtained from a cd4 + t cell line and primary cells. the transfection of cd4 + t cells with kir2dl1 dramatically increased the t cell receptor (tcr)-induced production of il-2 independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir2dl1-itim phosphorylation, shp-2 recruitment, zap-70 and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp-2 and p-pkc-v but not of shp-1. in contrast, the kir2dl1/hla-cw4 interaction led to a strong synaptic accumulation of kir2dl1 and the recruitment of shp-1/2, inhibiting tcr-induced il-2 production. kir2dl1 may induce two opposite signaling outputs in cd4 + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir2dl1 receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg1 is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg1 still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg1 antibody during these conditions. the observed igg1 switching behaviour mimics that of b cells responding to lps and il-4, but is mediated by a different, stat6-independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana 1 , c. schwindling 1 , m. pasche 2 , c. junker 1 , c. kummerow 1 , u. becherer 2 , e.c. schwarz 1 , j. rettig 2 , m. hoth 1 1 saarland university, biophysics, homburg, germany, 2 saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai1 channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than 150 nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd4 + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd4 + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of 21 days. under tolerogenic conditions (ova alone), cd4 + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, 4-1bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd4 + t cell expansion and contraction kinetics in the early phase of the t cell response (days 1-6). in the late phase of the primary response (days 7-21), under immunizing conditions, the large majority of transgenic cd4 + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il-2, ifn-g, and il-17a, and only few ova-specific foxp3 + regulatory t cells ( x 10 %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd4 + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells (30 %) was substantially increased. on day 14, both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells (50 %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd4 + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il-17a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd8 t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd8 tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin-2, interferon-g and macrophage inflammatory protein-1 by cd8 tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd8 tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd8 tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog 35-55 protein. our results provide direct evidence that mc contribute to cd8-specific priming in eae and show that the tc proliferation failure is specific for cd8 tc from mog 35-55 -immunized w/w sh mice. the role of mc-cd8 tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd8 tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd8 tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd8 tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd28 engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd28 costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd3complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla-4, but not via any conventional motifs in this region. overexpression of trim augments ctla-4 surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla-4 localisation, mainly restricted to the tgn. ctla-4 vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla-4 trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla-4 expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla-4 transport to the cell surface. it is imperative to reveal the mechanisms by which ctla-4 is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd1 molecules, nkt cells are activated and release cytokines, including ifn-g, il-2 and il-4. nkt cells are efficiently recruited to the liver via cxcr6-dependent chemotaxis toward cxcl16 and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd8 t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd8 t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd8 t cell tolerisation via interaction with lsec. to this end we analysed cd1d expression on lsec and their ability to activate nkt cells by presentation of the cd1d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd1d, as agalcerpresenting-lsec were capable to induce tnf-a, il-2, il-4 and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd54 and b7-h1 on lsec. as naï ve cd8 t cell tolerisation by lsec critically depends on b7-h1, we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb2 in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg9, jg1.2, and vd2 gene products. they recognize nonpeptide antigens like (e)-4hydroxy-3-methylbut-2-enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd-1. one of the inhibitory receptors, pd-1, is a member of cd28/ctla-4 family and contains a single ig v-like domain in its extracellular region. pd-1 can bind to two b7 homologue molecules, pd-l1 and pd-l2. it has been reported that interaction of pd-1 with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd-1 is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with 2-methyl-3-butenyl-1-pyrophosphate plus il-2 to obtain gd t cells. pd-l1+ and pd-l1-human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l1 mabs, the pd-l1 extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd-1 in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd-1/pd-l1 interaction. results: gd t cells expressed pd-1 upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l1. we first examined whether or not the engagement of pd-1 receptor could modulate the cytotoxic activity of gd t cells. pd-l1-expressing tumor cells tempered cytotoxic activity of pd-1+ gd t cells, and cytokine production such as tnf-a was down-regulated by pd-1 engagement. in addition, inclusion of anti-pd-l1 mab reversed cytotoxic activity and cytokine production when pd-l1-expressing tumor cells were challenged by pd-1-expressing gd t cells. conclusion: pd-1 delivers inhibitory signals in gd t cells upon engagement with pd-l1. peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il-2 production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e3 ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e3 ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd4 + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova 323-339 peptide)-specific t cells from do11.10 tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e3 ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e3 ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler 1 , p. aichele 1 1 immh, university freiburg, immunology, freiburg, germany interleukin 12 (il-12) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il-12 has a direct influence on cd 8 + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal 1) and costimulation (signal 2). we analysed direct il-12 signaling to cd8 t -12 signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd8 t cells lacking il-12 signaling failed to up-regulate klrg1 and to down-regulate cd127 in the context of listeria but not viral infections. thus direct il-12 signaling to cd8 t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd8 + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd8 + t cells are tightly controlled in their effector functions by cd152 (ctla-4). we demonstrate that signals induced by cd152 reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd8 + t cells. for this novel function cd152 specifically represses the transcription factor eomes, but not t-bet. a cd152 mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd152-mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd8 + t cells differentiated in the absence of cd152 signaling could be demonstrated in vivo. the novel insights that cd152-mediated signal transduction in vivo indeed alters cd8 + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd8 + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p2 receptors. p2x 1-7 receptors open to non-selective ion channels, whereas p2y1, 2, 4, 6, 11-14 are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p2 receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr9 but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p2x7 antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca(2+)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek 1 , a. brouckova 1 , d. filipp 1 1 institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih3t3 cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y505flck. comparative 2-d gel analyses followed by ms/maldi identified rack1 as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih3t3 cells of ha-tagged rack1 with either a wild type lck or constitutively active y505flck revealed a significantly enhanced complex formation between y505flck and rack1 compared to that of wtlck. ectopic expression of y505flck with its domain-inactivating mutations showed that lck-rack1 interaction depends on functional sh2, sh3 and the c-terminal tail sequence of lck. lck-rack1 interaction is readily detectable also in primary cd4 + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack1 with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack1 co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack1 in the context of lck translocation to lr is further strengthen by the observation that rack1 is associated with elements of cytoskeleton. these results are the first to characterize rack1 as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin 21 (il-21) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd19+cd20+cd27-cd38-igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th2 response to alumova inducing large early germinal centres and massive plasma cell formation with more than 75 % of these switching to igg1. the plasma cells up-regulate cxcr4, but not cxcr3, a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th1-associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚5%), igg2a (˚30 %), igg2b (˚30 %) or igg1 (˚30 %). in addition to cxcr4, some 70 % of these plasma cells strongly express cxcr3. the induction of cxcr3 in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg2a switching during th1 responses. this is functionally significant for oti-dependent cxcr3 expression, as well as induction of switching to igg2a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg2b. t-bet is known to be induced in b cells exposed to ifng or tlr9 stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd88. objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd272) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd4+ t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd4+ t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla-4 pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa-1 and cd2 in the psmac of untransformed human t-cells. in marked contrast, tcr/cd3 accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl-1 regulates interclonal t cell competition during acute and chronic immune activation. we found p53-independent noxa gene induction and mcl-1 downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl-1, which was delayed in noxa -/cells. using ot-1 cells and altered peptide ligands we observed that the level of mcl-1 downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl-1 -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np366) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl-1 axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen 9 (ebag9) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag9 confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag9, which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g2-adaptin in t cells. both interactions suggested an involvement of ebag9 in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag9-/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag9-deficient ctls. these data imply a role for ebag9 in regulating the formation of mature ctl granules and identify ebag9 as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag9 defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag9-related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd8 t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd80/86 and il-12. in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd3/acd28 or pma/ionomycin could not significantly activate cd4 and cd8 t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca 2+ influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena 1 , s. carrasco 1 , i. merida 1 1 centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp1-ras-erk signal intensity is critical to determine the final cell outcome. rasgrp1 is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. 1. analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. 2. develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd8 cells expressing gfp-c1 domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c1 domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h4, cd278), a cd28-like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th2 biased responses and high production of the anti-inflammatory cytokine il-10, and was essential to the development of germinal centres. however, icos can also help in the il-21-dependent differentiation of inflammatory th17 cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd28 a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p85 subunit of class ia pi-3 kinases. these can complex with one of the three 110 kda catalytic subunits (p110a, p110b, and p110d) expressed by leukocytes that generate pip 3 affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi3k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi3 kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi3-k regulatory (p85a, p85b, p53a) and catalytic (p110a, p110b, and p110d) pi3-kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p85a g p53a g g p85b and p110a g p110d g g p110b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi3-kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd3-induced secretion of il-10 and il-4. during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap450, a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap450 construct that acts as a dominant negative, or sirna knockdown of endogenous akap450 expression in t cells prevents the correct organization of cd3z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa-1 localization was also analyzed to assess p-smac architecture and, interestingly, confocal 3d reconstruction revealed that lfa-1 ring was not clear in the akap450-disrupted cells. moreover, akap450 was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap450 decrease the phosphorylation of molecules such as lat, plcg1 and pkcv. these defective activation events as reflected in a reduction of il-2 production. together, our results underscore a key role for akap450 in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin85/cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh3 domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca2+ mobilization and cell activation involving the pi3k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc1d10c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr 1) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as 2) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. 3) we then studied the phenotype of carabin kd (shrna expressing) a20 b cells after bcr stimulation. 1) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t1 tot2 b cells and to follicular mature b cells. 2) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. 3) bcr simulation, but not lps stimulation, of carabin kd a20 b cells shows an acceleration of ras target erk1/2 phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk1/2 pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor 3 (tlr3) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd4 + t cells express tlr3 and respond to the well characterized synthetic tlr3 ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd4 + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr3. tlr3 stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd4 + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor 3 (irf3) as revealed by realtime-rcr analysis of ifn b and irf7, whose transcription depends on the activity of irf3. combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr3 signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd4 + t cells. this study was supported by dfg spp 1110 "innate immunity" (ka 502/8-3). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd3e cytoplasmic domain (cd3e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd3e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd3e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd3e cd and a fluorescent membrane dye (r18). with this assay, we show that the cd3e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd3e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd3e cd to lipid bicells. membrane binding by the cd3e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia 1 , y. ge 1 , u. quitsch 1 , p. beckhove 1 1 german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd4+ t cell help is believed to contribute to optimal cd8+ memory expansion via cd40l on cd4+ t cells binding cd40 on dendritic cells. however, a few reports suggest that cd40l-cd40 engagement may mediate direct cell-cell contacts between cd4+ and cd8+ t cells. in this study, we investigated the importance of cd4-cd8 co-operation and cd40l-cd40 interactions for t em proliferation. methods: we isolated human cd4+ and cd8+ t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd4+ and cd8+ populations were activated in vitro using anti-cd3/cd28 beads. proliferation was measured by [ 3 h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd40 or cd40l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd4+ or cd8+ t em cells, demonstrating that optimal t em expansion requires direct cd4-cd8 interactions. surprisingly, not only cd8+ but also cd4+ t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd4+ t em proliferation depends on signals from cd8+ t em cells. activation induced the expression of cd40 on both populations and cd40l on subsets of cd4+ and cd8+ t cells. blocking of cd40l on cd8+ t em cells impaired significantly cd4+ t em proliferation, which confirms that the improved expansive potential of cd4+ t em cells in mixed populations depends on cd40l co-stimulation by the cd8 t em subset. conclusions: our data demonstrate for the first time that activated cd8+ t em cells deliver help to the cd4+ t em subset via cd40l-cd40 signalling and may play an important role for cd4+ t em expansion upon stimulation. the t cell surface glycoprotein cd5, a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd5 localization upon t cell:apc conjugation. we have questioned which domains of cd5 mediate the localization within the is, and for this we have expressed cd5 mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd5 mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd5 by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd5 depends on sequences within the cytoplasmic domain, as a cd5 deletion mutant lacking most of the cytoplasmic tail, cd5.k384 stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd5 translocation was mapped within amino acids glu 418 and his 449 since the cd5.h449 stop mutant, just short of 22 aa is still able to translocate to the is, whereas cd5.e418 stop , that lacks 2 important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd5 translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu 418 -his 449 ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna-2 is missing, but lmp-1 is still expressed. using a hl derived cell line, we have shown that the cytokine il-4 can induce lmp-1 expression in vitro and can replace ebna-2. we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is 2, 3 or 4. a high affinity stat6 binding site is spaced by 4 nucleotides. we found three potential stat binding sites in the lmp-1 promoter, which we named lrs, tr and edl1. they were spaced by 4, 3 and 2 nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il-4-treated or non-treated kmh2-ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat6 binding site, or lrs-stat6. a stat6 complex binding to the gl-epsilon promoter and lrs-stat6 was induced by il-4. the specificity of the stat6 complex was shown by supershift experiments with anti-stat6, but not anti-stat5 antibodies. when gl-epsilon or lrs-stat6 was used as cold competitors in a 100-fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat6 contains a functional stat6 binding site. oligonucleotides, corresponding to lrs in which the stat6 site had been mutated, could not compete for stat6 binding. interestingly, the unlabeled lrs-tr with 3 nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by 2 nucleotides (lrs-edl1), it could not compete. thus, expression the transforming protein lmp-1 can be induced directly by the t cell derived cytokine il-4 in a stat6 dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp-1 and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd8 t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji 2007 (ji . 179:2250 . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd4 t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd4+ t cells primarily affect the proportion of cd4+ t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd40, cd80 and cd86 on parameters of cd4+ t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd40, a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd40l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd40 leads to an enhancement of ig and cytokine production. the current dogma postulates that these 2 signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd40l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd40l (rmcd40l) can increase proliferation induced by tlr3 (poly ic) and tlr4 (lps) agonists. by contrast, we never observed any synergy between rmcd40l and tlr1/2 (pam3csk4) or tlr2/6 (pam2csk4) agonists. to go further in the study of cd40l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd40l, named mini-cd40ls, based on a c 3 -symmetry core holding cd40-binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd40ls bind to immobilized human cd40 and compete with the binding of cd40l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd40l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd40l, mini-cd40ls synergize tlr4 (lps), tlr3 (poly ic) and tlr7/8 (r848) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd40ls and tlr 1/2 (pam3csk4), tlr 2/6 (pam2csk4) and tlr9 (odn 2395) agonists. synergy between cd40l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd40l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd8 + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd8 + t cell tolerance. we have defined a phenotypic profile for cd8 + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd25 and cd69, and high levels of expression of cd62l and ly6c. whereas, cd8 + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd25 and cd69, and loss of cd62l and ly6c expression. ly6c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly6c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly6c, expressed on naïve cd8 + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd8 + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for 3 weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to 60 days after infection. results: daily injection of paraoxon induced g 50 % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining 2 weeks of treatment. mice exposed to paraoxon exhibited g 80 % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with 80 % of mice surviving the infection compared to 20 % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th1 and th17 cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide 3-kinases (pi3k) constitute a family of enzymes that generate 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi3k are divided into two types: class ia p85/p110 heterodimers, which are activated by tyr kinases, and the class ib p110g (p110gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p110g deletion affects tcr-induced t cell stimulation. mice lacking p110g show a partial defect in t cell differentiation, activation and survival. p110g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd4+/cd8+ t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi3kg-specific inhibitor causes a reduction in the number of cd4+ memory t cells that mediate renal injury. similarly, pi3kg deletion in p65 pi3k transgenic mice also reduces the numbers of cd4+ memory t cells. there is therefore evidence that pi3kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi3kg regulates this process is not well understood. we studied the specific role of p110g in t cell activation. methods: we studied whether the tcr activates p110g and the consequences of interfering with p110g expression or function on t cell activation. results: we found that after tcr engagement, p110g interacts with and forms a complex with ga q/11 , lck and zap70. tcr stimulation activates p110g, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p110g controls rac1 activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p110g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p110g-/-t cells. our observations clarify the activation mechanism and mode of action of p110g in the control of t cell activation, confirming a crucial role for p110g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a4b2 and a7, expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c57bl/6j mice. they were stained with fluorescently labeled igm-, cd40-or cd23specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by 3 h-thymidine incorporation upon stimulation with anti-cd40 and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a7 nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a4 or b2 nachr subunits inhibited binding of igm-and cd23-specific antibodies but facilitated that of cd40-specific antibody. in contrast, antibody against a7 subunit prevented binding of anti-cd40 but not of anti-igm or anti-cd23 suggesting that a7 nachrs are located close to cd40, while a4b2 ones are close to bcr/cd23. consequently, anti-cd40-induced b lymphocyte proliferation was increased by mla much stronger than by a4b2-specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine-3. in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd40-mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a7 nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd4 + t h -lymphocytes to antigen presenting cells or of cd8 + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim1 and trpc3), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about 96 %. down-regulation of stim1 was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd4 + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, 2008) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be 300-600 pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein 70 (hsp70) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd8 + cytotoxic t-lymphocyte (ctl) and cd4 + t-helper cell (th1) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp70 with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k562 cells as well as targetindependent cytotoxicity. cd4 + cells exhibited a strong increase in proliferation after stimulation with hsp70, with rates of up to 29 %. in the presence of target cells, a 35-fold up-regulation of granzyme b mrna was observed after stimulation of cd4 + t-helper cells with hsp70 in combination with il-7, -12 and -15. the target cell-independent secretion of granzyme b by cd4 + cells was greatly augmented after stimulation with hsp70 plus il-2 or il-7, -12 and -15. in this study, we have shown that hsp70 is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd4 + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd3 + and cd8 + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp70 on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd40, which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd150 receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk1 overexpression in a model system was achieved by transfection. pjnk1/2 expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd150 on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk2, but not jnk1 activation. cd40 ligation on primary tonsillar b cells also resulted in jnk2 activation. however, bcr crosslinking did not affect the level of jnk1/2 phosphorylation. cd150-mediated jnk2 activation was independent from sh2d1a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd150 and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase 1 (hpk1) was associated with cd150 in primary b cells as well as in b cell lines. cd150-hpk1 association was independent from cd150 tyrosine phosphorylation and sh2d1a expression. overexpression of hpk1 in a model system significantly enhanced cd150mediated jnk2 phosphorylation. it is known that tnf family receptors such as cd30, cd40, rank trigger survival signals in hrs cells. we observed the expression of pjnk1/2 in hrs cells of primary classical hl. cd150 could be involved in sustained jnk2 activation in primary hrs cells, and this may reflect the role of cd150 receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk2 is activated via cd150 in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk1 is involved in cd150-mediated jnk2 activation. objectives: cd5 has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd5 upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd5 are involved in c-cbl phosphorylation and association. methods: el4 thymoma cell line was stably transfected with wild-type human cd5 or hcd5 cytoplasmic tail mutants: cd5.k384stop (maintaining only a pseudo itim); cd5.h449stop (lacking the distal s and y in the carboxy-terminal region); cd5. ¿ e418-l444stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y700 in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd3 in combination or not with anti-human cd5 biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py700) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x 0.05). in murine thymocytes, co-crosslinking of cd3 with cd5 induces an increase in c-cbl phosphorylation compared to cd3 alone. analysis of the el-4 transfectants showed that mutants cd5.k384stop and cd5.h449stop lost the ability to costimulate cd3-mediated phosphorylation of c-cbl. in contrast, cd5. ¿ e418-l444stop mutant, was able to efficiently costimulate cd3-mediated c-cbl phosphorylation, similarly to the hcd5wt. our results indicate that the absence of the pseudo itam in cd5 does not interfere with c-cbl phosphorylation in response to cd3 plus cd5 crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd5 appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd5 cytosplasmic tail, but rather, may indirectly associate with cd5 through the interaction with other sh2-sh3 domain-containing molecules, that may be recruited to cd5 through its carboxy-terminal region. l. kolly 1 , s. narayan 1 , j. tschopp 2 , a. so 1 , n. busso 1 1 chuv, rheumatology, lausanne, switzerland, 2 unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase-1 into inflammasomes and thus plays a key role in regulating capase-1-dependent il-1b and il-18 production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd4 + and cd8 + t cells were activated in vitro through anti-cd3 stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd3 ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd3 activated asc -/-t cells predominantly displayed a more th 2 phenotype, producing more il-10 (199 vs. 692 pg/ml; asc +/+ vs. asc -/-t cells respectively; p=0.0074) and less ifn-g (15,831 vs. 6 ,921 pg/ml; asc +/+ vs. asc -/-t cells respectively; p = 0.0021). when asc +/+ and asc -/-t cells were purified into cd4 + and cd8 + t cell fractions and activated individually using anti-cd3, no inhibition in proliferation was observed amongst activated asc -/-cd4 + and cd8 + t cells. interestingly, the activated asc -/-cd4 + t cell fraction produced significantly more il-10 when compared to activated asc -/-cd8 + t cells and asc +/+ cd4 + and cd8 + t cells (asc -/-cd4 + t cells = 380 pg/ml il-10; asc -/-cd8 + t cells = undetectable il-10; asc +/+ cd4 + t cells = 11 pg/ml il-10; asc +/+ cd8 + t cells = undetectable il-10). cd4 + and cd8 + t cell mixing experiments revealed that asc -/-cd4 + t cells are able to inhibit the proliferative ability of asc -/-cd8 + t cells, asc +/+ cd4 + and cd8 + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd4 + t cells. collectively, these results demonstrate that the absence of asc drives cd4 + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd4 + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge 1 and pge 2 suppress some t-cell functions including proliferation, activation and cytokine production. pge 2 signals through four types of gpcrs called the ep receptors. at low concentrations, pge 2 is believed to be necessary for t cell function, whereas at higher concentrations, pge 2 inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge 2 and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge 2 diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd4 + t cells with ep receptor antagonists was found to impair cell surface expression of cd71, cd69, cd25 and ox40 but not cd44. suppression of t cell proliferation by pge 2 has already been widely studied. however, blocking ep receptors in cd4 + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd4 + t cells to the chemokine sdf-1b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd44 + cd4 + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd4 + t cells. our results also suggest that considering pge2-mediated camp signaling in cd4 + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd4 + human t-cells was examined. oxidation affects several ca 2+ signalling pathways by altering the activity of ip 3 receptors, trp channels and store operated ca 2+ channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca 2+ signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd147 (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd147 knockout mouse possess enhanced mixed lymphocyte reactions and cd147 monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd147 signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd147 in jurkat t cells augments the secretion of the t cell growth-factor interleukin-2 (il-2) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il-2 promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd147, we identified the immunomodulatory sub-domain of cd147. supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd40 and il4 (simulates t cell-dependent activation). microarray assays identified about 104 mirnas in unstimulated b cells. 35 of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor-4 (irf-4). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf-4 transcripts differs from that observed for irf-4 protein abundance after b cell stimulation. further analysis identified the irf-4 transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk592 and the dfg forschergruppe for832. objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on 11 different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey 1 , f. hauck 2 , i. berberich 1 , f. berberich-siebelt 2 , gk 520 -immunomodulation 1 institute for virology and immunobiology, university of wuerzburg, würzburg, germany, 2 university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd4+ t lymphocytes, the transcription factor is predominantly expressed in t helper 2 (th2) compared to t helper 1 (th1) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g1 phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp-1 encoded by prdm1 is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp-1 is expressed in differentiated effector t cells where it is higher in th2 than th1 cells. the regulation of the blimp1 expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm1 promoter and activates blimp-1 expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp-1 in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp-1 in b cells using cre recombinase. moreover we found a new putative blimp-1 isoform lacking exon 2. currently, we analyze the expression of c/ebpb and blimp-1 in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening 53 cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd21low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd4 + t cells but not transitional b cells or cd8 + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein-1a (mip-1a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy2 revealed constitutively high background levels in cd21low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas 1 , v. courtois 1 , k. de luca 1 , r. sodoyer 1 1 sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il-2, il-4 or il-6 could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il-2, il-4 and il-6. furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a-136-0033-01835). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen-4 (ctla-4) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla-4 during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il3) in the tissue and the released eggs and first stage larvae (l1) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd3 and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla-4 during the infection, a neutralysing antibody (a-ctla-4; 4f10) was administered intraperitoneally (300 mg) two hours before subcutaneous infection with s. ratti il3. the in vivo neutralisation of ctla-4-signalling by applying a-ctla-4 during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th2 cytokines, such as il-4 and il-5 and a reduction of the proinflammatory cytokines ifn-g and il-17. the investigation of the humoral response showed a remarked increase of the igg1-titer in the serum during secondary infection in mice that had been treated with a-ctla-4 during primary infection. furthermore, the blockade of ctla-4 resulted in a diminished worm burden as indicated by reduced release of l1 in the faeces. these results suggest that the blockade of ctla-4 during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th2 response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg1 during secondary infection also reflects the induction of a potent th2 response. objectives: cd36 is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd36 is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd36 on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd36 expression. cd36 knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd36 ko compared to heterozygous mice. since reduced levels of cd36 are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd36 ko mice. after one injection of apoptotic cells, cd36 ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd36 on b-cells are involved in setting this threshold. conclusion: our data suggest that cd36 is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd152 acts as a major check-point of immune responses, but the mechanism by which cd152 controls peripheral t cell responses is unknown. the consequences of cd152 signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd152 in th1 cells reduced migration towards ccl4, cxcl12 and ccl19. crosslinking of cd152 together with cd3 and cd28 stimulation on activated th1 cells increased expression of the chemokine receptors ccr5 and ccr7, which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd152 expression and cd152-mediated migration-enhancing signals. importantly, migration of cd152 positive th1 lymphocytes in in vivo experiments increased, as compared to cd152 negative counterparts, showing that indeed cd152 orchestrates specific migration of selected th1 cells to sites of inflammation and antigenic challenge in vivo. these data show that cd152 signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd152 adds to the already significant role of cd152 in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd152 signaling on the longevity of cd28 null t cells. using a sensitive staining method for cd152, we show that human cd4 + cd28 null and cd8 + cd28 null t cells rapidly express surface cd152. serological inactivation of cd152 using specific fab fragments or blockade of cd152 ligands using ctla4ig in cd4 + cd28 null and cd8 + cd28 null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd152 crosslinking on activated cd28 null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd152 is mediated by pi3'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd152 act directly on activated cd28 null t lymphocytes and, due to its exclusive expression as a receptor for cd80/cd86 on cd28 null t cells, prevention of cd152 mediated signaling is likely a major target mechanism taking place during therapy with ctla4ig. objectives: cd45 is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd45 -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd148 (ptprj, dep-1) which acts as a positive regulator of sfk in cd45 -/-b cells and macrophages and can compensate for cd45 deficiency in these cells. indeed, combined deficiency of cd45 and cd148 in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd148 and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd148 -/mice were reported so far. however, in human t cells the role of cd148 may be different since naive human t cells express cd148 at a level comparable to b cells. using cd45 -/-/cd148 -/human t cell line (jurkat-derived js-7 cells) we tested the ability of cd148 to complement cd45 deficiency in t cells. we used retroviral transduction to express human cd45 or cd148 in js-7 cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd148 to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js-7 cells. expression of wild type cd45 or cd148 in js-7 cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd69 upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js-7 cells expressing cd148 when compared to control cells. finally, cd148 substrate trapping mutant expressed in js-7 cells interacted with lck in vivo suggesting that lck is a direct substrate of cd148 in js-7 cells. the results suggest a level of redundancy between cd45 and cd148 in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam1.6) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr 505 and tyr 394 we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native 2d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca2+ release-activated ca2+) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa-1 (leukocyte function-associated antigen 1) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa-1 prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al.2006) we describe a non-viral vector based approach (vockerodt et al. 2008) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in 9 independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd40 signaling cascade was conducted. after activating this particular signaling cascade (cd40) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. 2005 ). a rat thymic epithelial cell (tec) line (r-tnc.1) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc.1 cell line in vitro. it was found that a number of adhesion molecules, such as cd2, cd4, cd8, cd11a, cd18, cd54, cd90 was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc.1 line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il-2 activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc.1 cells. we found that both the adhesion (30 min and 3h) of activated thymoytes were partially blocked by mab to cd2 and cd8 molecules (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells). early adhesion was inhibited by mab to cd90, abtcr, mhc class i molecule (ifn-g stimulated r-tnc.1 cells) and cd4 molecule (ifn-g unstimulated r-tnc.1 cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc.1 cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay (6h), namely mab to cd2, cd4, cd8, cd90 molecule (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc.1 cells). our results also suggest the involvement of cd11a/cd18 dependent -cd54 independent pathway in adhesion and cd11a/cd18 dependent -cd54 dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc.1 cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin-27 (il-27), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il-27 favours naï ve cd4 t cell differentiation into th1 cells to the detriment of th17 or th2 differentiation. the il-27 receptor (il-27r) is a heterodimer composed of tccr, which confers ligand specificity, and gp130, a signal transducing chain that is utilized by several other cytokines. il-27 has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd8 t cells, but the potential impact of il-27 on human cd8 t cells has not been elucidated. our goal is to investigate the impact of il-27 on human cd8 t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il-27r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd8 than cd4 t cells expressing the complete surface il-27r (gp130+tccr). however, we detected high amounts of intracellular tccr in both, cd4 and cd8 t cells, but only polyclonal activation (anti-cd3) of cd8 t cells led to an actual increase of il-27r surface expression. purified cd8 t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il-27 activated stat1 and stat3 signalling with rapid kinetics in both cd8 and cd4 t cells, indicating the capacity of il-27 to signal through these molecules. addition of il-27 to anti-cd3 activated cd8 t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il-27 on human cd8 t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd-1/pd-l1 pathway is associated with production of the immunoregulatory cytokine il-10, the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l1 and pd1, as well as myelin basic protein (mbp)-stimulated il-10 production, pakt inhibition, and apoptosis (annexin v), in 50 ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); 24 had a diagnosis of stable disease (sms). results showed that: 1) pd-l1 -expressing cd14+ and cd19+ cells are reduced in ams compared to sms individuals (p=0.04); and 2) pd1 expression is increased in cd4+ t cells of sms individuals and is comparable on cd8+ t cells of ams and sms patients. this is associated with a significant decrease in il-10 production by mbp-stimulated cd14+ and cd19+ cells of ams patients (p=0.03). additionally, cd8+ anexin v+ (av+) cells were diminished and cd8+ pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd4+av+ and cd4+ pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l1/pd-1 pathway seen in ams patients result in a reduced mbp-specific il-10 production by cd14+ and cd19+ cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd8+ t. the pd1/pdl1 pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti-4-1bb in cd8 cells. this difference could be due to down regulation of cd28 by activated lymphocytes and possible preferential response of cd8 cells to anti-4-1bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin7 (stx7) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx7. rna interference technique was also used to down regulate stx7 expression in primary human ctls. results: we identified stx7 in ctls by pcr and immunocytochemistry. stx7 accumulates at the is after ctl/target cell contact. when stx7 function was blocked by overexpression of a dominant negative stx7 mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx7 is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the 20 years history of mouse t h 1 and t h 2 subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h 0, t h 1 or t h 2 phenotypes, based on their cytokine production (il-2, ifng or il-4). a comparative analysis of t-bet, ifng and il-4 mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca 2+response, membrane raft expression/organization, k + -and ca 2+ -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h 1 g t h 0 g t h 2, although the membrane cholesterol level (detected with anti-cholesterol ab, ac8) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h 1 cells upon activation than in t h 2 cells. t h 1 cells produced a more sustained calcium response with higher amplitude than t h 2 cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav1.2 and kv1.3 ion channels, major functional determinants of the sustained calcium influx. t h 2 cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd19-dynabeads. cd19 + isolated cells were facs sorted into cd19 + cd27naïve b or cd19 + cd27 + memory b cells. dna synthesis was measured by 3h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd19 + cd27naï ve b cells, of which bmp-6 and -7 were most efficient (40 % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd19 + cd27 + memory b cells by 40 -50 %. to induce differentiation, both naï ve and memory b cells were stimulated with cd40l and il-21. this increased the production of igm, iga and igg 10 -100-fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad1/5/8 in cd19 + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd40l/il-21-induced production of igs in mature human b cells. s. gutenberger 1 , k. warnatz 1 1 university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases 1 and 2 (erk 1/2). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd86 upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of 20 hd and 25 cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk1/2 phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk1/2. to increase the signal intracellular phosphatases were inhibited by h2o2. as markers of activation and initiation of proliferation, cd86 and ki67 expression were measured after 2 days of in vitro stimulation. k. theil 1 , p. aichele 1 1 immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd8 t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p14 cd8 t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b6-recipient mice and compared their expansion with wild-type (wt) p14 t cells after viral infection. we could demonstrate a severe impairment in the capacity of p14 t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p14 t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd8 t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p14 t cells into h8 mice. h8 mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp33-41. therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h8 mice are infected with lcmv8.7. this is a lcmv variant that has got a point mutation in gp33-41 and consequently cannot be recognized by the p14 t cells. s. frischbutter 1 , r. baumgrass 1 1 deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap1 which are important for expression of cytokines such as il-2, ifng and il17. it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl-10 was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl-10 phosphorylation but rather an inhibition of bcl-10 dephosphorylation. furthermore, calcineurin and bcl-10 co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl-10/malt1 signaling complex and dephosphorylates bcl-10 and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop 1 , j. lamoureux 1 , c. fortin 1 1 université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: 25 healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla-4 has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla-4 expression was higher after stimulation in t cells of elderly. there was differences between cd4 and cd8 t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd95l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh3 and ww domain proteins. since fasl surface expression is regulated by adam10-mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl2a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh3 domain proteins that potentially interact with the riped fasl prd, we used a sh3 domain phage display library containing all 288 sh3 domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx9 family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conseoptimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b7 family members on antigen-presenting cells with cd28 on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd28 co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound12, with classical gcs regarding their suppressive effect on cd28-costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd3 and anti-cd28, and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd28-costimulated human memory/effector cd4+ t cells by compound12 than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound12 was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound12 and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound12 than prednisolone. when evaluating possible mechanism for the increased activity of compound12 in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound12 which was not prevented by the partial gc receptor antagonist, ru-486, in vitro. moreover, in vivo we observed less induction of il-1beta and tnf-alpha by pre-treatment with compound12 than with prednisolone. our data indicate that the non-steroidal segra, compound12, may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b6 mice elicits robust cd8+ t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il-2+ cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa 224 -specific cells also express tnfa, but only about half of the ifng+ np 366 -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd8+ t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd8+ t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova 257-264 peptide shows a close correlation with division in vivo. early after antigen encounter (0-2 divisions) the vast majority of cells express only tnfa. after 3-4 divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions (4-6 divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd4+ t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd8+ t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd8+ t cells, expansion plays a very important role in the regulation of cd8+ t cell effector function. in addition to its chemo-attractant function, sdf-1a (stromal-cell derived factor-1a, cxcl12) has been described to costimulate cd4 + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd4 + t cells was increased by immobilized sdf-1a to a level similar to that obtained with the costimulatory molecule cd28. as visualized by real time confocal microscopy, t cells entering in contact with sdf-1a formed a tether and displayed an active scanning activity. since sdf-1a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd3 mabs, it is conceivable that the sdf-1a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf-1a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf-1a in the context of cd4 + t activation by antigen-presenting cells secreting sdf-1a. this study should help us to better define how sdf-1a modulates cd4 + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd4 + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd3 and anti-cd28 antibodies and cape for 2 days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by 3h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il-5 production and lymphoproliferation in cd4 + t cells stimulated by anti-cd3/cd28. cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd4 t cells express a variety of molecules on their surface, such as mhc-class ii, cd80, cd86, cd70, whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd4 t cells might induce t cell proliferation and differentiation from cd4 resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd4 t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after 5 days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd4 memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd25, cd30, and cd69. analysis of the cytokine profile of these cells revealed the differentiation of il-10-and ifn-g-double-producing cells in response to contact with th1 effector cells, and il-4-producing cells in response to contact with th2 effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd3/cd28 stimulation. whereas neutralization of ifn-g or il-4 during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd70, cd80, and cd86 could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to 64-85 %. conclusion: interaction of cd4 memory t cells with activated t cells resulted in the production of the cytokines il-4, il-10, and ifn-g. given the immunomodulatory capacity of il-4 and il-10, these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir 1 , s. thompson 1 , j.j. murphy 1 1 king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl1 leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il-2 and il-5 and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp36l1 by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp36l1 has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl1 differentiation was observed to be associated with downregulation of zfp36l1 protein. in an attempt to determine whether zfp36l1 downregulation was directly linked to bcl1 differentiation, a zfp36l1 shrna expressing lentivirus (psicor) was employed to knockdown zfp36l1 expression. this reagent downregulated zfp36l1 expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp36l1 shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp36l1 downregulation in promoting bcl1 plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of 32 patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all 32 isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd95 (fas, apo-1) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd3/cd28-triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo1 mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd95l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il-2 and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk1/2 phosphorylation, the expression of various activation markers, the il-2 production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb415) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb1,3galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd4+ and cd8+ t cells. in addition, mitogenic stimulus increase 3-fold the all binding to cd4+ t cells. previous studies in human pbmc showed that all binds to human cd4+ t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd4+ t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd3-dependent activation of purified cd4+ t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd25 and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd25 expression, but enhanced the anti-cd3-dependent proliferation and cd25 expression of purified cd4+ t cells. analisis of calcium influx showed that all enhanced anti-cd3 dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd4+ t cells by up-regulating t cell activation mediated by anti-cd3 stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in214609) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd3 complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd95 receptor. it has been previously demonstrated that cd95 ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp-76, slap-130 and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il-1r, il-2r, il-3r, il-7r, il-12r, il-18r, cd27, cd28, cdw137( 4-1bb), cd 95(fas) and cd178 (fasl) on hpbls in different cultured time, i. e. 0d, 1d, 3d, 5d, 7d, 9d, 11d and 14d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. 1. the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. 1) the expressions of membrane immune molecules before cultured. 2) expressions of the membrane molecules on hpbls during culturing. 5% fbs rpmi 1640 group (1640 group), il-2 group, pha group... (1) mtt assay. (2) proliferative times and growth curves of hpbls... 1. during cultured in vitro, there are expression changes of the il-1rs (il-1ra, il-2ra, il-2rg, il-3r, il-7r, il-12r, il-18r), co-stimulatory molecules (cd27, cd28, 4-1bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in 1640 group, il-2 group and pha group, but the rests are different. 2. our data also suggest that the hpbls cultured in cd3mcab+cd28mcab+il2+il1a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. 3. celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun 1 , r. tauber 1 1 zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p56lck and the ras/rac2 signalling pathway (1) followed by mitogen-activated protein kinases (2) and c-jun n-terminal kinase (1), which leads to an enhanced binding of l-selectin to soluble ligands (3). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues (4). here we show that the protein phosphatase 2a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp2a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp2a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer 1 , e. glasmacher 1 1 helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago1-4 genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm1 and rck or expression of dominant-negative gw182. interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd4 + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd8 + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd8 + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd8 + t-cell activation and differentiation. naïve cd62l hi cd44 lo cd8 + t cells were sorted and stimulated by anti-cd3 and anti-cd28 antibodies. results: firstly, we show that the activation and differentiation of cd8 + t cells require il-2 provided by activated cd4 + t cells at the initial priming stage after stimulation. secondly, this critical il-2 signal is delivered through il2rbg of cd8 + cells and is independent of il-2ra. besides promoting cell proliferation, il-2 stimulation increases the amount of ifng and granzyme b produced by cd8 + t cells. conclusion: therefore, our studies demonstrate that a full cd8 + t-cell response is elicited by a critical temporal function of il-2 released from cd4 + t cells, providing mechanistic insights into the regulation of cd8 + t cell activation and differentiation. most antigenic peptides recognized by cd8 t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a3168-176 is a tumor antigenic peptide presented by hla-a1 and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a3168-176 we setup an in vitro digestion assay using a 20-mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a3168-176 by tumor cells. by electroporating hla-a1 cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart-1/melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart-1/melan-a 26-35 specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart-1/melan-a 26-35 ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart-1 expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart-1/melan-a 26-35 epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart-1/melan-a 26-35 ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd8 + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd4 t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev 207: 293-313, 2005) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h2-m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity 11: 453-462, 1999) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide 84-103, whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from 84-103 peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the 84-103 epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, 2008), we found, after an initial induction at 8 hrs p. i., a strong inhibition of tapasin transcription at 24 hrs p. i. furthermore, also reduction of tap1 and tap2 transcription was observed contrasting to the elevated levels of erp57 and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß1, ß2 and ß5, which are replaced by their ifng-inducible counterparts ß1i, ß2i and ß5i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß5i is replaced by a thymus-specific subunit ß5t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß1i, ß2i, ß5i and ß5 in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß1,ß2 and ß5i (single intermediate proteasome), and the other comprises ß1i, ß2 and ß5i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent 10-20 % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about 50% of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a*0201) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a*0201 binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a*0201 from tapasin, but only ttp-ha dissociated hla-a*0201 from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl-721.220-a2 showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a*0201 from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a*0201. m. basler 1,2 , c. lauer 2 , m. groettrup 1,2 1 biotechnology institute thurgau, kreuzlingen, switzerland, 2 university of constance, division of immunology, department of biology, konstanz, germany two lmp7-dependent antigens have been described that relied on the 'structural presence' of lmp7 in the proteasome but not on the activity of lmp7. here we have investigated processing of the h-2d b -restricted uty 246-254 epitope of the male minor antigen uty reported to be lmp7-dependent. using splenocytes from lmp7 -/-, lmp2 -/and mecl-1 -/mice we found that the uty 246-254 epitope requires lmp7 and lmp2 but not mecl-1. curiously, a selective lmp2 inhibitor did not interfere with uty 246-254 presentation. objective: we investigated why the deletion but not the inhibition of lmp2 interferes with uty 246-254 presentation. we hypothesized that the 'structural' requirement for lmp2 is based on replacement of the caspase-like activity of b1 in the proteasome. methods: it was determined if t1a mutants of lmp2 and/or b1 can rescue the uty 246-254 epitope. we used a b1-selective inhibitor to determine if the inhibition of the caspase-like activity of b1 preserves the epitope. finally we determined by mass spectrometry if the uty 246-254 epitope embedded within a 25mer precursor peptide is differentially cleaved by lmp2-deficient and proficient immunoproteasomes in vitro. results: we found that t1a mutants of lmp2 and b1 rescue presentation of uty [246] [247] [248] [249] [250] [251] [252] [253] [254] . also inhibition of cells with a b1-selective inhibitor preserves uty [246] [247] [248] [249] [250] [251] [252] [253] [254] presentation. an aspartate in position 7 of the uty 246-254 sequence wmhhnmdli is preferentially used as a cleavage site by lmp2-deficient but not half as frequently by lmp2-proficient immunoproteasomes. the generation of the uty 246-254 epitope relies on the replacement of the caspase-like activity of b1 by lmp2 because the b1 activity destroys the uty 246-254 epitope. this is the first example for the 'structural' requirement of lmp2 for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp2 and perhaps also lmp7 and mecl-1 exert their function in antigen processing. a. linnemann 1 , a. musiol 2 , r. lindner 1 1 hannover medical school, cell biology, hannover, germany, 2 hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf6-regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf6) were overexpressed in 3t3 fibroblasts. we show that antibody-mediated oligomerization of mhc i in 3t3 fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf6: endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf6-regulated to a novel, arf6-independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv40 virus and since sv40 binding triggers mhc i oligomerization, this novel pathway may be involved in sv40 uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of 57 rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab3b, 3c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta2-microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab3b and rab3c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab3b or 3c positive vesicles. furthermore, the rab3b, 3c positive compartment were colocalizd with a fraction of rab27a at a juxtaposition of phagosomes. together these data demonstrate that rab3b and rab3c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over 90 % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over 80 viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd8+ t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf2a and bglf5 have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf1, has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens 2009). this represents a novel function for a virally-encoded gpcr. bilf1 is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf1 displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf1 is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf1 can hinder the recognition of virally-infected cells by cytotoxic cd8+ t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe 131 natural ligands, including two peptides derived from semenogelin-1, a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg1 is transcribed in thymus from both male and female individuals. finally, we detected the semg1 mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta1, beta2, beta5) and immuno-subunits (beta1i, beta2i, beta5i). deficiency in beta5i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta5i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta5i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta5i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta5i-deficient cells could be restored by treatment with d3t, which increases the amount of proteasomes independent of beta5i via induction of mixed proteasomes containing beta1i, beta2i and beta5. consequently, not the lack of the specific proteolytic activity of beta5i or immunoproteasomes, but the reduced proteasome quantity in beta5i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr2, 4 and 5, which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd4 + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd4 + t cells. the presence of exogenous tlr3 and tlr8 ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james 1 , i. bailey 1 , t. elliott 1 1 university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct26 colon carcinoma. this response is long lasting and mediated by both cd4 and cd8 t cells. importantly, the treg depleted ct26 specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a20, c26, bcl1, renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw11, which is h2-d d restricted. analysis of the generation of gsw11 in ct26 revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct26 cells substantially increased the amount of gsw11 present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct26 and immunised balb/c mice. greater than 80 % of mice injected with eraap kd ct26 were found to reject the tumour. analysis of t cell responses revealed the presence of gsw11-specific t cells, however, these responses were small (0.5-1 %). this compared to a much larger response to ct26 (˚5 %). preliminary results indicate that the majority of the t cell responses (non-gsw11-specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [1] . the di-allelic hla-a2 restricted minor histocompatibility antigen ha-1 locus codes for the highly immunogenic ha-1 his and the non-immunogenic ha-1 arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha-1 arg immunogenicity was the estimated numbers of cell surface presented copies i. e. 80/cell for ha-1 his and less than 5/ cell for ha-1 arg [2] . as ha-1 his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha-1 his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha-1 mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha-1 allelic peptides, we investigated the impact of the ha-1 his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha-1 his and ha-1 arg peptides were performed. moreover, the crystal structures of hla-a2 in complex with either ha-1 his , ha-1 arg or a ha-1 variant with a citrulline residue at position 3 were determined in order to obtain atomic level insights into the conformation of the hla-a2/ha-1 peptide complexes. our results exclude a role for antigen processing in preventing ha-1 arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha-1 arg allele in the hla-a2 peptide binding groove [3] . they provide a rationale for the lack of ha-1 arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd8 t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd8 t cell response. on the other hand, immunosubunit expression is not essential for development of cd8 t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp7 (ib5) and mecl-1 (ib2) develop a variety of autoimmune responses, including a latent form of t1d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd8 but not cd4 t cells. a cd8 t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose-6-phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd8 t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd8 t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, 162 autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr3 and dr4. analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type 1 (hsv-1) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd4 + and cd8 + t cells in hsv-1 immunity have yet to be clearly elucidated. to better understand the role of hsv-1-specific cd4 + t cells in immune control we have identified a 13 amino acid epitope derived from glycoprotein d of hsv-1. following flank infection, gd-specific cd4 + t cells were first detected in the draining brachial and axillary lymph nodes (ln) 5-days post-infection (pi), peaking at day 7 and declining thereafter. gd-specific cd4 + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day 6 pi and peaked at day 9. while hsv-specific t cells were first observed in the draining ln at day 5 pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as 2 days pi, with peak activity 4 days pi before declining to background by day 7. however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd4 + t cells was observed up to 23 days post-infection in the brachial ln. ex vivo analyses suggest that only cd11c + cells were involved in gd antigen presentation at days 2, 5 and 15 post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd8a + dcs can present the gd antigen to cd4 + t cells at day 2 pi, whereas by day 5 pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv-1 infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap1 and erap2 unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap1/erap2 in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp57. upon assembly of the heavy chain with b 2 m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp57. both crt and erp57 are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp57 to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp57 or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp57 must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp57 point mutant that fails to bind to cnx or crt was just as effective as wild type erp57 in normalizing rates of disulfide formation. we conclude that erp57 does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp57 point mutant, class i heavy chains, crt and the tapasin-erp57 disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp57 and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp57 have no effect on plc stability or class i surface expression, suggesting that erp57 plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies 5-11 % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb1*0405/dqb1*0401 haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr*0405 transgenic ab0 nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd4+ and cd8+ t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr*0405 transgenic mice (cd4+ t cell competent) develop aip even unprovoked, similar to ab0 nod mice (cd4+ t cell deficient), while hla-dr*0401, hla-dq8 or hla-dr*0405/dq8 (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr*0405 fails to protect from aip, likely due to defects in the induction of cd4+ regulatory t cells. our results identify hla-dr*0405 as a prominent risk factor for aip on the hla-drb1*0405/dqb1*0401 haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan 1 , c. britten 1 , h. overkleeft 2 , g. van der marel 2 , k. melief 1 , d. filippov 2 , f. ossendorp 1 1 leiden university medical center, section tumorimmunology, leiden, netherlands, 2 leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd8 and cd4 t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi 1,2 , x. hu 1,2 , a. kawanatachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping 8-mer and 10-mer epitopes (rypltfgwcf (nef138-10) and rypltfgw (nef138-8)) were found to be presented by hla-a*2402 and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y2f, y2w, t5c, f6l, w8r, f10r, f10y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef138-10 (wild type) and its four mostly common immune escape mutants (y2f, t5c, y2f&t5c, f6l), and also nef138-8 (wild type). we found that there was little difference between the nef138-10 (wild type) and nef138-10 (y2f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y2 or f2. interestingly the central bulge region of the peptide was becoming very flexible for the nef138-10 (t5c) and nef138-10 (y2f&t5c), which may affect the binding of peptide and the recognition of t cell receptor. for nef138-10 (f6l), the side chain of l6 was more flexible compared to the nef138-10 (wild type). alignment of the nef138-10 and nef138-8 showed that the nef138-8 became flat and the side chain of f6 was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef138-10 was featured, while the peptide nef138-8 was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef138-10 was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd8+ and cd4+ t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a2 restricted cmvpp65 derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a 2-3 log reduction of activation efficiency. this pattern was seen for 5/9 epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to 2-5 log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b7 restricted cmvpp65 rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t2 cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv 9-mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd8+ t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp100 and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp110, and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor-5 (fgf-5) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of 40 amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf-5 peptide was produced in vitro after incubation of proteasomes with a 49-amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf-5 spliced peptide by cells transfected with mutant constructs encoding fgf-5 proteins where the intervening segment was shortened from 40 amino acids to 30, 20 or 8 residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp100 peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b*4402, are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b*4405, appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b*4402 and b*4405 are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us3 protein. to address this hypothesis, us3 was stably coexpressed in b lymphoblastoid cell lines expressing hla-b*4402 or hla-b*4405. in the presence of us3, the surface expression of hla-b*4402 was substantially reduced whereas hla-b*4405 expression was relatively unaffected. although us3 was able to form complexes with both hla class i allotypes, only hla-b*4402 was retained intracellularly in an immature form whereas hla-b*4405 was transported to the cell surface. accordingly, in the presence of us3, hla-b*4405, but not hla-b*4402, constitutively presented a hla-b44 restricted alloantigen to reporter t cells, suggesting that us3 binds hla-b*4405 without interfering with peptide loading. us3 has been reported by others to bind the plc but surprisingly we have not detected such us3-plc complexes in our system. rather, in the presence of us3 we identified a pool of class i molecules distinct from the plc and only present in us3 expressing cells, implying that us3 may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa 1 , b. seliger 1 1 martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il12p70. since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for 24h with the gold standard of maturation (tnfa, il1b, il6 and pge2) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase-3 (lap3) had a more than 20-fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap1 and erap2 and of the immunoproteasome subunits lmp2 and lmp10 was enhanced between 2 and 5-fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr4 ligand mpla in comparison to the tlr-3 and 7/8 ligand polyi:c and r848. these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd4 + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il-4 induced surface expression of mature mhc class ii molecules and cd86. when ifn-g/il-4 primed pcmc were used as antigen presenting cells for cd4 + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd69 up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type 1-diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b15-23 to specific cd8+ t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd8 + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th1 immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd8+ t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd8+ tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd8+ t cell clones, faure, 2009, eur j immunol 39 (2): 380-90). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd8+ t cells after their in vitro differentiation into dc, 6 days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv-1 genome contains arfs within gag, pol and env genes encoding for a panel of hla-b*07 restricted epitopes. qprsdthvf (q9vf/5d) is one such epitope but its parental epitope qprsnthvf (q9vf/5n) has a significant higher frequency among hiv-1 isolates. strikingly, q9vf/5d-or q9vf/5n-specific ctls recognize apcs infected with hiv strains encoding for q9vf/5d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad8 ) encoding for q9vf/5n do not activate ctl responses raising the possibility that q9vf/5n epitope is not presented by infected cells. we asked whether introducing mutations within q9vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q9vf/5n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q9vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q9vf mhc-i presentation. we performed in vitro proteasomal digestions of 28mer peptides encompassing q9vf/5d or q9vf/5n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q9vf/5n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q9vf/5n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q9vf/5d or q9vf/5n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q9vf/5d peptide. we cloned and sequenced hiv-1 genomes from the three donors. surprisingly, out of 20 hiv proviral genomes isolated from pbmcs of q9vf/5d reactive donors, we could not find any virus bearing the q9vf/5d sequence. the isolated hiv sequences either encoded for q9vf/5n or had a stop codon within the epitope. in contrast, viruses encoding for q9vf/5d were isolated from pbmcs of the q9vf/ 5d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv-1 seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch 1 , y. wang 1 , d.c. tscharke 1 1 the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd8 + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd8 + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd8 + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h-2 bxd f 1 mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f 1 progeny, with some peptides being almost equally immunogenic in f 1 and inbred mice, while others elicit responses that are reduced by more than 90 % in f 1 mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f 1 mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f 1 mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd8 + t cells with a restricted vb usage in f 1 mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr2 agonist, s-[2,3-bispalmitoyiloxy-(2r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp-2), as an adjuvant for cross-priming against cellular and soluble antigens. malp-2 has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd8 + and cd8 -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr2/6 results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap1 expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap1) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to 90 % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, 50 and 10 % as compared to control rma cells, were injected s. c. in the flank of c57bl/6 syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to 45 days after injection. all mice injected with rma clone with a 50 % level of eraap expression developed a tumor and died within 23 days after injection. surprisingly, any animal injected with rma clone with a 10 % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd4 + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd4+ t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor (207 bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd20 therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor (2.5 bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg4 levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb1*0701-restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with 20-mer synthetic fviii peptides to stain epitope-specific cd4+ cells.cd4+ t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a2 domain: fviii 405-424 , fviii 421-440 and fviii 653-672 , as well as the c1 domain peptide fviii 2093-2112 , but not any c2 domain peptides. the c1 domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost.85:123-33, 2005 ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni 1 , s. albini 1 , m.z. limongi 1 , l. cifaldi 1 , d. fruci 1 1 ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p50 nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap1 and erap2, we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap1, erap2 and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap1 and erap2. this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p65 nf-kb subunit, we demonstrated that nf-kb is involved in erap1 and erap2 expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap1 and erap2 and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap1, erap2 and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch 1 , s. tenzer 1 , h. schild 1 , e. von stebut-borschitz 1 1 uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c57bl/6 mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th1 immunity, whereas ifng secretion of both cd4 + th1 and cd8 + tc1 cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il-12-dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd8 + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd8 + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm7 (lmp7 -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c57bl/6 mice. in addition, ex vivo co-cultures with infected dc from either lmp7 -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc1 cell ifng secretion and the dc restimulatory capacity of cd8 + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd8 + t cells, isolated from low dose infected c57bl/6 wildtype or lmp7 -/mice, were not detected. in summary, our data indicate that despite the fact that cd8 responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd8 + t cells against l. major is independent of the lmp7 subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a20, generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd4 + t cell epitope (84-103) from the g1 domain of aggrecan 10,000 times more efficiently than non-specific b cells and over 100 times more efficiently than the macrophage line j774. however, despite this highly efficient aggrecan capture, processing and presentation of the 84-103 epitope took at least 5 hours, comparable to the time required for presentation of aggrecan by j774. treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the 84-103 epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions 114, 115 and 116 at the peptide binding groove. position 116 also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b27 is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b27 subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b27 folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b*2705, peptide loading is relatively independent of this chaperone. we analyzed the effect of b27 subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b27 subtypes. the association of b27 heavy chain with tapasin was analyzed in c1r cells transfected with hla-b27 subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta-1, which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b27 peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me1 monoclonal antibody that recognizes b27properly folded b27/peptide complexes. the formation of fully assembled b27 molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc10, which recognizes mhc class i free heavy chains (hc), or with me1. maturation was analyzed by pulse-chase analysis, immunoprecipitation with me1 and treatment with endoglycosidase h (endo h). hla-b27 polymorphic positions other than 116, both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b27 subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd4 + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd8 + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c57bl/6 mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately 15 % of vacv global presentation in the context of h-2 b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd8+ and cd8-dc, which differ in their mhci antigen presentation capacities. cd8+ dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd8-dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd8+ and cd8-dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd8+ and cd8-dc was determined. surprisingly, cd8+ dc exhibit rapid surface mhci turnover compared to cd8-dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd8+ and cd8-dc were stabilized and no longer underwent rapid turnover. this suggests that cd8+ and cd8-dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd8+ and cd8-dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h-2kb loaded with the ova-derived peptide, siinfekl. cd8+ and cd8-dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd8+ dc, but not the cd8-dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd8+, and not cd8-dc. this data further validates the role for cd8+ dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp-1 cytosol using gelatinase b/mmp-9 as a model enzyme. in the first dimension, ion exchange chromatography separated the thp-1 proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp-9. in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp-9 on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp-1 cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp-9-cleaved or intact thp-1 cytosol. proliferation was assessed by measuring incorporated 3 hthymidine. results: 100-200 mmp-9 candidate substrates were isolated, of which 69 were identified, revealing many novel mmp-9 (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about 2/3 of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp-9 decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e3-19k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue 56 in hla-a11 and residue 93 in e3-19k are highly critical for association of both proteins. this defines a putative interaction surface between e3-19k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e3-19k and the class i antigen presentation pathway. moreover, because soluble e3-19k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap1 and tap2. tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap1 and tap2 were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap1 on tap2 was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap1 mutants suggested that the lack of tap1 protein expression is associated with a strong reduction of tap2 protein levels, which could be restored by tap1 gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap1 on tap2 different shrna plasmids specifically targeting tap1 or tap2, respectively, were stably transfected into constitutively tap1 and tap2 expressing hacat keratinocytes and colo794 melanoma cells. in both cell types the shrna-mediated tap1 and tap2 inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap1 caused not only an almost complete loss of tap1, but also a strong decrease of tap2 protein expression. in contrast, the tap2 knock down exhibited no influence on the tap1 expression. specific inhibition of the proteasome prevented the degradation of tap2 in the tap1 knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap1 on tap2 protein expression is not restricted to rare tap1 mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of 27 healthy volunteers was exposed twice to 1.5 minimal erythema dose of uv. blister roofs were then collected before and 1, 4 and 10 days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd14 + cd36 + and of a macrophagic cd14 -cd36 + cell subsets that emerge 1 and 4 days post exposition, respectively. more importantly whereas classical cd1a hi cd207 + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd1a low cd207and cd1a low cd207 + that emerged 1 and 4 days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd86 and hladr. finally, 10 days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey 1 , e. reeves 1 , t. elliott 1 , e. james 1 1 university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd4+ cd25+ regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct26, stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct26-derived cross-protective antigen, gsw11, which was found to be encoded within the ectotropic murine leukaemia virus (emv-1) envelope protein, gp70. this protein has previously been shown to encode ct26-specific cd8 and cd4 antigens, implicating it as a 'hot-spot' for ct26 tumour antigens. interestingly, we have identified a truncated version of gp70 which may be responsible for generation of gsw11. expression studies have revealed increased gp70 expression in ct26 compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw11) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p1 pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p4, p6 and p9 tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp2 molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp85-99, bound to hla-dp2 with high affinity (10-30 nm). binding studies of mbp85-99 derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f91a, f92a and k93a) were affected, and only by a 20-50 fold reduction in affinity for hla-dp2. the observation that none of the substitutions resulted in a complete loss of binding to hla-dp2 indicates that 1) hla-dp2 binding to peptides does not depend on a large hydrophobic residue accomodated in p1, or 2) mbp85-99 can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp2 molecule binds the immunodominant epitope in ms, mbp85-99, possibly in more than one register. additional studies are required to resolve the hla-dp2 peptide binding properties, and to determine whether expression of hla-dp2 affects the disease course in ms patients. results: depletion of mncs for cd14+ cells abrogated the tg-induced cytokine production and proliferation of cd4+ t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g 98 % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd4+ t cell proliferation was significantly reduced in cd14/cd19-depleted mnc cultures, as compared to cultures depleted for cd14+ monocytes alone. the same applied to the production of il-2, il-6 and tnf-a. production of ifn-g and il-10 was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa 1,2 , p. pala 1 , g. miiro 3 , j. todd 3 , p. kaleebu 1,2 , n. imami 2 , f. gotch 2,3 1 mrc uganda, basic science, entebbe, uganda, 2 imperial college, london, united kingdom, 3 mrc uganda, entebbe, uganda background: hiv-1-specific t-cell responses are preserved in hiv-1 infected individuals with non-progressing hiv-1 disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv-1 for g 8 years, maintaining cd4+ t-cell counts g 500, and with minimal cd4+ decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: 17 art naï ve hiv-1 infected patients from the entebbe cohort in uganda were recruited and stratified by cd4+ t-cell count, cd4+ decline slopes, and time of enrolment, into 2 groups -10 ltnp and 7 rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd4 t-cell count, and ifn-g, il-2 and il-4 elispot responses to pools of 22 to 34 peptides (18-mers overlapping by 10aa). peptides were based on consensus sequences of gag and nef from hiv-1 clades a1, a2 and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il-4 responses were significantly higher in the ltnp than in rp (p=0.02, ifn-g responses to gaga2 pool 1; p=0.04, il-4 responses to gaga1 pool 2). il-2 responses were low and not significantly different between ltnp and rp. there was a positive correlation between il-4 responses to gaga1 pool 2 and cd4 t-cell counts (r 2 =0.502, p=0.04), but no correlation between either il-4 or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv-1 specific responses were higher in ltnps than in rps confirming previous results. non-specific il-4 responses were high possibly reflecting baseline th2 responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her2/neuand her2/neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using 7-aad for dna analysis and specific antibodies directed against the proliferation marker ki-67, the m-phase specific phistone h3 as well as for the h-2l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her2/neuversus her2/neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g 0 /g 1 -, s-and g 2 /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g 0 /g 1 -phase, which continuously raised and peaked within the s-phase. however, h-2l d surface antigen expression was not altered in her2/neucells during cell cycle progression. in contrast to her2/neufibroblasts the her2/neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h-2l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd207) and ccr6 were cultured from cd34+ stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd34+ derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il-4-dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mø). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to 45 min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mø than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mø have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, 1999) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of 30 healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, 1992, 1999) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x 0,0001), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd4+, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, 2252 patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for 2207 of them. for 538 of these patients hla-drb1 data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p=0.02). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb-1a preparations, whereas no differences were shown for ifnb-1b. an association between hla-drb1*11 and nab-negativity was detected (p=0.028). the known associations between hla-drb1*15 and csf-positive ms and hla-drb1*04 and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb1 potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb-1a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb-1b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart-1 and gp100 more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni 1 , s. lorenzi 1 , f. mattei 1 , f. spadaro 1 , l. gabriele 1 1 istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd8 t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd8a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd8 t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd8 t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg7 thymoma line, we show that type i ifn promote the ability of cd8a + dc to capture apoptotic eg7 cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd8a + dc and the persistence of apoptotic eg7-cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg7-derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd8a + dc. as a result, eg7-loaded dc become competent at inducing ot-i cd8 t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd8a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. (2). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd8+ dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam3csk4, and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd8+ dc but not cd8-dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm1, which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a549 human lung cancer cell line. methods: dm1 induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a549 cells. we examined the morphological change using optical microscope. and gfp-lc3 punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm1 was showed high cytotoxicity on various cancer cell line, especially a549 cells. dm1 treated a549 cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc3 and beclin-1 protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc3 was increased concentration and time-dependently. beclin-1 also was increased. and then, we examined gfp-lc3 punctation on a549/ gfp-lc3 stable cells using confocal. a549 cells were formed gfp punctuation after dm 1 treatment. to confirm these data, we measured cell death ratio using 3-methyladenine (3-ma), an autophagocytosis inhibitor. after 3-ma treatment, dm1 induced cell death was decreased comparing with dm1 treatment. conclusion: dm1 did not induce apoptotic cell death. but dm1 showed several characteristics of autophagic cell death. taken together, dm1 induced autophagocytosis on a549 cells. these finding indicated that therapeutic potential of dm1 by triggering autophagic cell death. s. b. rasmussen 1 , s. r. paludan 1 1 university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr)9, which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr9 initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr9 recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv-1 infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv-1, we found that tlr9 dependent ifn regulatory factor 3 activation was abrogated. in addition, inhibition by 3-methyladenine of phosphoinositol 3-kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr9 dependent recognition. in the ongoing project we are examining how hsv-1 triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko 1 , i. berberich 1 , gk 520 -immunomodulation 1 universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl-2 family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a1 and its human homologue bfl-1 are anti-apoptotic members of the bcl-2 family. a1 is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a1. unless the c-terminal ends of other bcl-2 proteins the tail of a1 does not function as a strong membrane anchor. nevertheless, the last amino acids of a1 are important for the protein. in fact, the c-terminus of a1 serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a1 undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl-2 factor bimel results in increased stability of a1. this is due to reduced ubiquitination of a1 after binding of bimel. we conclude that the cterminus of a1/bfl-1 serves as a docking site for e3 ubiquitin ligase(s) that control the stability of a1 by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e3 ubiquitin ligase that interacted with a1 in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs-1 in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs-1 is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs-1 significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs-1 was manifest not only by the suppression of jaks acting upstream of p38 mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak 1/2 activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs1 which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs-1 overexpressing cells. the results strongly suggest that socs-1 acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl12/sdf-1a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl12 effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u-87, and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p53 and fas-l expression was carried out by western-blot. results: cxcl12 induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p53 pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p53-ser46 levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after 24h of tnf-a treatment. conclusions: cxcl12 induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p53function/levels. s. y. demiroglu 1 , r. dressel 1 1 universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp70 is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp70 on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp70 does not protect against cell death mediated by ctl. acute overexpression of hsp70 can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res 63: 8212) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp70 on granzyme b-induced apoptosis. methods: hsp70 was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp70 induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type 5. results: hsp70 did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp70 in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g1 peak measurements (p=0.01). in contrast, a partial protection of ge-tet cells was observed after acute hsp70 overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp70 does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp70 effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood 102:1788) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk1034 and dr394/2-3. the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr1 and tnfr2, both belonging to the tnf receptor superfamily. tnfr1-mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr2-associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr2, resulting in depletion of traf2 from the cytoplasm. here we confirm that tnfr2 induced depletion of cytosolic traf2 by the use of tnfr1-and tnfr2-specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf2 in the alternative nfkb pathway, we show that tnfr2 induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr2 and membrane tnf. j. c. morales 1 , d. ruiz-magaña 1 , d. carranza 1 , c. ruiz-ruiz 1 1 universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase-8. in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r2 and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp36l1 is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp36l1 may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl-2 protein is an important cell survival protein at different stages of b cell development. bcl-2 mrna also contains are regions that could possibly be targeted by zfp36l1 protein. in the present study, we have tested the ability of zfp36l1 protein to bind to a bcl-2 mrna are probe and to degrade bcl-2 mrna. recombinant bacterially expressed zfp36l1 protein was shown to bind specifically to a bcl-2 are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp36l1 also provided evidence that endogenous zfp36l1 in b cells could bind to the bcl-2 are. in order to examine whether zfp36l1 binding to bcl-2 are resulted in bcl-2 mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp36l1 knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl-2 mrna was expressed in both wild-type and knockout mefs. the half-life of bcl-2 mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp36l1 does play a role in degradation of bcl-2 mrna. overall, our data are consistent with a role for zfp36l1 in degradation of bcl-2 mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp1. since lmp1 is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp1 function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi3-kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te1) and lymphocytic (nc5) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes1 or tes2 (transforming effectors site) derived from the c-terminal intracellular part of lmp1, in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp1's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase-8. using this nc5 tumorigenic model in scid mice, we showed that induction of the dn lmp1-tes2 prevent development of tumours and mouse death. these dominant negative derived from lmp1 could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs1 and socs3 differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs1 exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p38mapk activities, while socs3 promoted apoptosis with an increase in p38 activities. in contrast, both socs1 and socs3 display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs1 or socs3 induced a slight decrease in g1 or s phase cells and a prominent increase in g2/m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g1/s transition and g2/m arrest induced by socs1 or socs3. socs1 promoted dna damage-induced p21 induction and g2/m cyclin b expression, while socs3 induced decrease in g1 cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein 90 (hsp90) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp90 has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp90 can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp90 is released during necrotic cell death and proinflammatory effects of extracellular hsp90 have been observed. thus, we asked whether apoptozing cells cleave hsp90 during apoptosis or how hsp90 is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp90 protein content. we observed that hsp90 is degraded during apoptosis resulting in the formation of a fragment of about 50 kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp90 (hsp90 alpha and hsp90 beta) we could show that the 55 kda fragment is formed after degradation of the alpha isoform of hsp90. further, we were able to show, that hsp90 cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp90 and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp90 was inhibited or if exogenous hsp90 was added. these results demonstrate that cleavage of hsp90 represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc3-i is processed to lc3-ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf-7 gfp-lc3 cells were therefore incubated in starvation medium for 3 hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl-2/beclin-1 control of autophagy. recently, jnk was shown to be necessary for beclin-1 upregulation, and jnk-mediated phosphorylation of bcl-2 is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf-7 cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol 3-angelate (pep005), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep005 provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb4 turned their response to pep005 from an increased to decreased rate of apoptosis. furthermore, our data show that pep005 inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl-1 and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p75, trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch11 and analysed by flow cytometry. expression of the caspase 8 inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch11 treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase 8 in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase 8 by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases 9 and 3. diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase-8 abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor 1 (tnf-r1), and tumor necrosis factor (tnf)-a receptor 2 (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid 1-o-octadecyl-2-o-methyl-rac-glycero-3-phosphocholine (et-18-och 3 ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd95 independent of its ligand fasl/cd95l. fas/cd95 is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin-1, an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl2-family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment (5-fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo 201, sw1116, lovo, caco-2, ht-29) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki-67, pcna, p53, bcl-2) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in 88 patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl-2, bax, caspase 3, 6, 9, nfkb, parp1/89kda, tnfr1/cd120a and cd95/fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl-2, bax, nfkb, parp1 was significantly lower and expression of caspases, tnfr1 as well as percentage of cd95 cells significantly higher as compared with control group. caspase 3 expression was significantly higher as compared with nfkb, parp1 and tnfr1. in comparison to the standard nutrition preoperative immunonutrition increased bcl-2 and nfkb expressions and decreased caspases and parp1 expressions. in addition, we found a significant down-regulation of bcl-2 expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega-3 fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x 0.01). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g1 phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x 0.007). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with 54 healthy volunteers receiving either la1 (10 9 cfu/day) or placebo, during 57 days prior to uv (2 × 1.5 med). blister roofs, liquid and skin biopsies were collected 1, 4 and 10 days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p53). while a similar decrease of lc for both groups was observed on day 1 after uv exposure compared to placebo, la-1 group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd1a low cd207 -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il-10 and a tendency to inhibit il-6 was observed in la-1 group compared to placebo. on day 4, la-1 group presented a greater recruitment of early lc precursors and a trend to increase cd1a low cd207 + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il-6, tnfa, il-8, and il-10) was observed in la1 group compared to placebo. we show that la1 limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la1 supplementation for photoprotection at a lower dose (1 log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn1 is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn1 appears to be expressed in all epithelial cells of the early thymic rudiment starting around e11.5. previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn1 in a nude (foxn1-deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn1. if true, it should be possible to target this cell type by use of foxn1-promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either 19 or 82 glutamine residues under the control of foxn1 promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn1 promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei 1,2 , y. toriumi 3 , k. kimura 4 1 european university viadrina, frankfurt (oder), germany, 2 shimane institute of health science, izumo, japan, 3 shimane university faculty of medicine, department of pediatrics, izumo, japan, 4 japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a 10-minute-rest period, followed by a 15-minute yoga exercise called asana, a 15-minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a 20-minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp2). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r=0.83, p x 0.02), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r=0.77, p x 0.05) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd4 (r=0.96, p=0.0001). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd4, within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper 2 response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd4 + cd25 + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox40l expressed on mcs and the constitutive expression of ox40 (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox40l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca 2+ responses and degranulation. the cross-talk between t reg cells and mcs through ox40-ox40l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th1 or th2 effector cells and the induction of antigen specific foxp3+ regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th2 cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th2 and treg subset, gata3 and foxp3 respectively, counter regulate each other (mantel y et al., 2008) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp3+ tregs and high gata3+ th2 cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost 100 million allergic patients are sensitized to the major birch pollen allergen bet v 1, which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v 1 recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v 1 molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g 90 %) of patients' ige binding to bet v 1 was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa 30-59 (p2) and aa 74-104 (p6) of bet v 1. cross-reactive ige epitopes between bet v 1 and related pollen allergens and plant food allergens involved primarily p2. p2 and p6 are not adjacent peptides in the bet v 1 sequence but define a surface-exposed patch on the three-dimensional structure of bet v 1. as determined by surface plasmon resonance, monoclonal antibody mab2 specific for p2 and mab12 specific for p6 showed high affinity, i. e., dissociation constants, k(d) = 8.35e-11 m and k(d) = 1.05e-9 m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p2 and anti-p6 antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v 1 allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th1 cells as well as tc1 cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th2 immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th2-mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th1/tc1 cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd4 + and cd8 + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd4 (gk1.5) or anti-cd8 (53.6.72) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr-1 monoclonal antibody rb6-8c5 or monoclonal antibody nimp-r14. one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd8 + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd4 + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd8 + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd8 + t cells, cd4 + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd4 + or cd8 + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th1 and tc1 cells operative in the elicitation of airway inflammation. conclusions: robust type 1 immune responses, although highly effective in the counter-regulation of local th2-mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h292. the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p 1, we were able to show that the major allergen of phleum pratense, phl p 1, although sharing molecular similarities with der p 1, does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p 1. chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il-6 and il-8 from nci-h292 cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p 1. in contrast to hdm extract grass pollen allergens induce the release of mcp-1 from respiratory epithelial cells, as well as moderate levels of il-12. detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr22) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, 3 distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): 7.2±5.7) and of il-13 (mean±sd: 1157±909 pg/ml), and reduced il-10 (mean±sd of il-10-spot forming units/2x10 5 cells (sfu): 912±510), compared to healthy subjects (3.4±2.7 si, p x 0.05; 355±396 pg/ml, p x 0.05; 1272±623 sfu, for proliferation, il-13 and il-10, respectively); children with non-ige mediated cma had a significant reduction of il-13 (192±362 pg/ml), compared to ige patients (p x 0.0004) and an increased, although not statistically significant, production of ifn-g (33.7±54.6 sfu) compared to control (9.5±9.5 sfu) and to ige-allergic patients (0.6±0.8 sfu). finally, tolerant patients showed reduced il-13 (636±1048 pg/ ml, p x 0.05) and proliferation (3.9±3.5 si), compared to acute ige-cma children. interestingly, the high level of il-10 observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il-4 was undetectable in all patients even blocking the il4-receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th2-like response with il-13 and proliferation is dominant in ige-mediated cma patients; by contrast a th1-skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang 1 , b. chua 1 , f.c. lew 1 , a. ho 1 , k. wong 1 , k.l. wong 1 , d. m. kemeny 1 1 national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd4 th2 t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th2 cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd8 t cells inhibits the induction of the th2 response. here we have investigated the effect of cd8 t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd8 t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of 3 airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd8 + t cells (36.7 %±4.1 % to 17.6 %±2.7 %). when ifn-gamma -/-ot-i cd8 t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced (39.6 %±5.1 %), suggesting an important role for cd8 t cell ifn-gamma. cd11c + cd103 + cd11blung dcs from cd8 transferred mice secreted higher levels (500pg/ml) of il-12p70 following ex-vivo stimulation as compared with animals that were not given cd8 t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd8 t cells can subsequently divert the local lung environment to one that favors th1 immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in 8 % of patients with hiv infection. this reaction is strongly associated with hla-b*5701 and appears to be mediated by cd8+ t cells producing inflammatory cytokines. we show that cd8+t cell responses can be primed in vitro, in normal blood donors who are hla-b*5701+, but not in non-b*5701+ donors. cd8 t cells, but not cd4 t cells, are expanded by abacavir pulsed autologous apc over a13-day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b*5701. similar responses were detected in abacavirhypersensitive hlab*5701+ patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b*5701 since they were undetectable using apc expressing closely related hla-b57 or b58 allotypes. responses to apc expressing mutants of hla-b*5701 demonstrated a crucial role for residue 116. isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b*5701 triggering cd8 t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b*5701 may be relevant to the role of other disease-associated class i allotypes such as hla-b27 and b51. a. jenckel 1 , s. bulfone-paus 1 , n. föger 1 1 research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin1a (coro1a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro1a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro1a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro1a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro1a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro1a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro1a during mast cell activation. cell fractionation experiments demonstrated that the association of coro1a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro1a phosphorylation. a functional correlation between coro1a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro1a. thus, coro1a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin1a (coro1a) deficient mice have demonstrated that coro1a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin1b (coro1b) is a closely related homolog of coro1a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro1b deficient mice and crossed them with coro1a deficient mice to obtain coro1a/coro1b double deficient mice. analysis of t lymphocytes from coro1a/coro1b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro1a/coro1b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro1a/1b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro1a/1b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn1 (also known as hacs1 or sly2) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology 3 (sh3) domain and a predicted bipartite nuclear localization signal. the samsn1 gene is located on mouse chromosome 16 and encodes a well conserved protein with 364 amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn1 in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn1 expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn1 in all tested hematopoietic cell types. the highest expression level of samsn1 mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd4+ and cd8+ t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly1 (hacs2) and sly3 (sash1) -were expressed only at a very low level in mast cells. the high level of samsn1 mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn1 in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn1 deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn1 deficient mast cells. in additional studies we are now investigating the effects of samsn1 deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn1 in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg1 antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., 2008). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg1 abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st8siai-v; st6galnac i-iv, st3gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg1 abs. results: we observed that the expression of st3gal i, iii and v coding genes was similar in both hybridomas, while the st3gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg1. in addiction, the expression levels of st8sia and st6galnac genes in the hybridoma producer of anaphylactic igg1 were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg1. conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg1 abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h 2 cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after 2 times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il-5 and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il-5 and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf1 mediates the differentiation into th1 cells by activating multiple genes which are independently crucial for the development of naive t cells into th1 cells. because irf1 is expressed in many different tissues, it can be considered as a master switch factor for th1 cell differentiation. methods: here, we tested mice deficient in irf1 in the murine acute asthma model to evaluate its importance in this th2 cell-mediated disease. the protocol setup was the following: 3 sensitizations s. c. with ova, followed by 3 challenges via ova inhalation and adoptively transferred wildtype cd8+ t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf1 deficient mice with the help of adoptively transferred cd8+ t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by 3.5 fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. 1) were also significantly higher in irf1 deficient mice. conclusion: interferon regulatory factor 1 plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd8+ t cells. mechanistically, a potential counterregulation of asthma by th1 cells is not available in irf1 knockout mice. together with our previous report that irf1 represents a susceptibility gene for allergy in the human, our data highlight irf1 as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c57bl/6 mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for 24hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk1-receptor and ppt1 mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd11c expressing apc substets characterized by cd4 and cd8 expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th-1 cytokine profile and increased levels of il-2 mrna. further the number of cd25 + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: 1) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase3, 2) asthma and ee are prototypic th2 diseases in which the cytokines il-4 and il-13 play a principal role through the activation of the transcription factor stat6, and 3) we have recently demonstrated that some chloromethyl ketones can downregulate stat6 by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat6, we analyzed its effect in the activation of stat6 by il-4 and il-13. we found that treatment of cells with ppis inhibited the ability of il-4 and il-13 to signal stat6 activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th2 mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied 12 asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd4 and cd25 high (treg)), that might inhibit the development of a th2 response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for 48 hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd69 or cd25 at 48 hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h4r. the level of intracellular ccl2 production in human lc was reduced after stimulation with h4r agonists and basal production could be restored when h4r was blocked with the specific antagonist jnj7777120. moreover histamine and a h4r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h4r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h4r in allergic skin diseases and encourage further exploration of the h4r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g1p3, an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g1p3 was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g1p3 which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd20 antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th2 to chronic th1-predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received 4 weekly intravenous infusions of rituximab at a dose 375 mg/m 2 body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g 5,000; g 6,000; g 19,000 mg/dl, respectively). all patients underwent prior treatment with omalizumab for 6 months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the 2 nd week after initiation of rituximab therapy up to 1 year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up 1 year), ige levels decreased from 5,512 to 1,500 mg/dl. the other two patients are in the 3 and 1-months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd20 antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) 1 and th2 cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either 1 %dncb, 25 %tma or to vehicle alone for 0.5-6h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il-2 (mean=131pg/ml, n=12, p x 0.001), il-17 (mean=69pg/ml, n=12, p x 0.001) and il-1a (683pg/ml, n=12, p x 0.001) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of 50ng/ear of murine recombinant il-2 or of the known regulator of lc migration; il-1b. interleukin-1b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after 4 (32 %) and 16h (31 %) (n=6, p x 0.001). in contrast, il-2 or control injections were without effect. however, il-2 administration caused an increase in cutaneous il-17 production (347pg/ml, n=12, p x 0.001) compared with control injection and naï ve tissue (19 and 63 pg/ml, n=12, ns). in addition, systemic treatment with anti-il-2 antibody failed to impact on lc migration provoked by dncb (33 % reduction; n=6, p x 0.001). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il-1a (987pg/ml, n=10, p x 0.001) and il-17 (248pg/ml, n=6, p x 0.01) expression was reduced to baseline levels by anti-il-2 treatment. these data demonstrate that il-2 is not involved in the regulation of lc migration, unlike il-1b and tnf-a. however, il-2 is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il-2 may influence lc and dermal dc maturation, via the expression of il-1a and il-17. allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey 2005), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p38-mapk-pathway related protein (p38prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p38 mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct-2005 -018681, www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, 15 % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il-1 receptor antagonist (il-1ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il-1b and of il-1-like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il-1-related genes by real-time pcr on mrna from blood cd14+ monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il-18, il-18bp, and caspase-1, both before and after therapy with kineret. il-1b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il-1b, tnfa, il-12) nor anti-inflammatory cytokines (il-10, tgfb) were detected. as expected, il-1ra was only detectable after therapy. il-18 was detectable in schnitzler's sera at higher levels than in controls (23.0 vs. 10.3 pm) and decreased after therapy (13.2 pm). the circulating il-18 inhibitor il-18bp was lower than in controls and not affected by therapy. thus, free il-18 levels were increased in schnitzler's patients as compared to controls (17.6 pm vs. 7.2 pm in controls) and decreased after therapy (9.9 pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il-1-related cytokines (il-1b, il-18), nor of the il-1/il-18-converting enzyme caspase-1 in blood monocytes. however, the high circulating levels of il-18 suggest an increased activity of caspase-1, as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around 20 % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr3 signal via myd88 to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd88 signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c57bl/6 myd88-deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd88-/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg1-titers in ova-treated myd88-/-mice compared to wildtype mice, although the nacl-treated myd88-/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over 15,000 species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies (6 mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = 4) or from non-infested contra-lateral sites. expression of mip-1a, igf-1, mcp-1 and ip-10 genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd3, cd4, cd8, cd21, mhc class ii, and p46 than that of holsteins. lymphocytes expressing wc1 and cd21 antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x 0.05). infested skin of nelores contained significantly more message for mip-1a, igf-1, mcp-1 and ip-10 than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th2 response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd3+ t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il-1 plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa-1 in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam-1/lfa-1 binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th1 than a th2 local response pattern by virtue of il-1 secretion and icam-1/lfa-1 interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th2 like micromilieu in acute towards a th1 dominated milieu in chronic lesions. the genotyping of ccl4l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi07/0329). psoriasis is an inflammatory dermatosis with 2 % prevalence among caucasians. hla-cw6 allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in -308 and -238 positions. strong association was found between polymorphisms in the -238 region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b27, and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to 40 years of age) were selected. hla-a -b -c -dr -dq alleles and tnf-238 and -308 snp were differentiated by pcr/ssp. analyzing the total group, 21 patients (30.5 %) were male, 48 (69.5 %) were female. in the male group, the mean age at disease onset was 16,3 years. severe forms were seen in this group (psoriatic arthritis in 4 cases and erythroderma in 2). seven patients (33,3 %) had a favorable evolution of the disease, but 14 (66,4 %) developed extensive psoriasis, covering over 30 % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw*06 allele was positive in 10 cases (47,62 %), tnf 238 g/a genotype was found in 5 (23,8 %) and tnf 308 g/a in 4 (19,1 %). in the female group, the mean age at disease onset was 14,6 years, one case of psoriatic arthritis and one of erythroderma. twenty-nine (60,4 %) had a favorable evolution of the disease and 19 (39,6 %) an unfavorable evolution. cw*06 allele was positive in 26 cases (54,2 %), tnf 238 g/a genotype was presented in 16 (33,3%) and tnf 308 g/a in 8 (16,6 %). severe disease was seen in male patients. there was no difference in frequency of cw*06 allele between male and female groups, but there was a tendency of significant difference in tnf 238 g/a genotype. we found that c57bl/6 mice were more susceptible than balb/c and dba/2 mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il-6 and mrp8/14, among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: 136 cases, which were studied retrospectively, included children (84 boys and 52 girls), aged 3-14 years, who had an anaphylactic reaction, out of the 915 that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food (44 %-particularly sea food and dried fruit), drugs (25 %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites (9 %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema (76 %), and gastrointestinal symptoms (37 %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g 100 was found in 26 out of the 47 severest cases (55,4 %), in which the adequate control was held, while in their vast majority (45 out of 47) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in 53,7 % (73 cases) there was a hereditary family history of atopy, while in 52 children (38 %) there was also a personal history of asthma. finally, at a great percentage (69 %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that 1)the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. 2) in particular, in many cases it is proved that there is a personal but also a family history of atopy. 3) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd86 we could observe dose dependent upregulation of programmed death ligand 1 (pdl-1) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a 60 year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since 35 years. in march 2006 a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters (120 mg) every second day. in april 2008 the patient presented himself in the consult with multiple livid papules with a diameter of 3 mm in the area of the auricle. the histological examination showed an hhv-8 positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th1 type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs1, socs3 and foxp3 in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n:4), the patients who showed recurrence or progression after treatment (non-responders, n:4) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n:7) were evaluated for socs1, socs3 ve foxp3 mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd3, cd4, cd25, foxp3, cd4 + cd25 high , cd4 + foxp3 + . expression of socs3 and foxp-3 mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs1 was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd25, foxp3, cd4+cd25 high , cd4 + foxp3 + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs1, socs3 and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs1 and socs3 molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of 62 silicosis patients, with mild (21), moderate (23) and severe (18) silicosis, 92 coal workers, exposed to inorganic dust containing fcs (exposed), and 43 healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than 0.05 was considered significant.neopterin level was significantly elevated in exposed (3,2±0,8ng/ml) compared to controls (1,8±1,3ng/ml; p=0,0001). moreover, the neopterin level in exposed was similar to silicosis patients (2,9±1,6ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls (81,9±22,7au vs 64,3±16,7au p=0,0001) and to silicosis patients (69,6±20,0au p=0,002). in contrast, igmcic was significantly elevated in silicosis than in exposed (70,2±18,8au vs 62,1±24,2au; p=0,03).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p=0,001 and 0,02 respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a*01, b*08, drb1*03 (these of good prognosis) *12/*13/*14/*15) or tbc (drb1*02/*05/*14/*16 and associations with dqa1*02/*03/*05) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a*01, b*08 and drb1*03 (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a*01, drb1*14. as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd4 + , cd25 ++ , foxp3 + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein 60 (hhsp60). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd4 + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il-17, il-6, il-8, tgf-b and ifn-g production prevailed, pointing to a th17/th1 weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp60. importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th17/th1 cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp60 might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices (1) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a 384 well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c3-complement, myeloid related protein 14 and two new proteins, integrin-ß4 and fibrinogen. data from more than 100 patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than 10 % and 16 %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least 3 mice per group with distinct cytokine pattern: th1 (c57bl/10, c57bl/6) and th2 (balb/c). muscular injury was performed by injection of bupivacaine. both th1-dominant strains presented more areas with regenerating myofibers and macrophages at 4 dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd3 bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at 4 dpi. balb/c mice showed increased collagen expression and decrease of mmp-9 activity associated with more mrna for tgf-b1. this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th1 cytokines) or myonecrosis and collagen deposition (th2 cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß1-adrenergic receptor (anti-ß1ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß1ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß1ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß1ec ii -specific rat monoclonal antibody (clone 13f6), and showed by elisa that it binds to the linearized ß1ec ii peptide. additionally, we confirmed with flow cytometry that 13f6 also binds the ß1ec ii in its native conformation, i. e. directly labeled circular ß1ec ii (dyl649-ß1ec ii ) peptide. moreover, we demonstrated activation of the ß1-adrenoreceptor by 13f6 using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß1ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß1ec ii -specific peptide-based therapy, by intravenously applying a circular ß1ec ii peptide in the dcm lewis rat model to neutralize the anti-ß1ec ii antibodies. while the peptide therapy strongly reduced the anti-ß1ec ii titers in the serum by up to 80 % and consecutively lead to clinical remission, elispot assays for the detection of ß1ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß1ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß1ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b3 or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca 614-629 epitope elicits autoreactive cd4 + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca 614-629 peptide. in a first step, hybridoma cells were generated by fusing bw5147 tcra -cd8lymphoma cells with myhca 614-629 -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca 614-629 -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle 1 , a. schwarting 1 , p.r. galle 1 1 university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase 3 (pr3), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr3 in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr3-positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il-8. pr3-mrna from the murine cancer cell line wehi-274 was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr3-promoter in the second intron of the mouse pr3 gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr3 transcript and its promoter indicates that the murine pr3 may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th1 mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd1d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th2 cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n=13) or pbs (n=11) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr4 activation, in the presence/ absence of och. subsequently we transferred 1.5x10 6 mdcs (n=11) or och-primed mdcs (n=11) (3 times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a 70.6 % reduction in plaque size compared to mice treated with mdcs (p x 0.05). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant 23.7 % (p x 0.05) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a 2 to 3-fold increase in these cells was detected 3 days after the last treatment with och-primed dcs (p x 0.05). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il-10. discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th2 phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c57bl/6 background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed 350 igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c57bl/6 and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr3 regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg2c were isolated only from fcgriib deficient mice, but not from c57bl/6 control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box 1 protein (hmgb1), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb1. we investigated if hmgb1-containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb1 remains associated with ncs released from late apoptotic cells in vitro. hmgb1-ncs complexes were detected also in the blood of patients with sle. hmgb1 containing ncs from apoptotic cells induced secretion of il-b, il-6, il-10, and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd88 and toll-like receptor 2. neither hmgb1-free ncs from living cells nor from apoptotic hmgb1-or hmgb1/2-deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb1 activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il-10 release by macrophages. immunizations with hmgb1-containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr2-dependent manner. in conclusion, hmgb1 in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher 1 , k.p. hoefig 1 , e. kremmer 1 , v. heissmeyer 1 stretches and helps in the selection of the correct splicing borders. a allele of (r61h) creates a strong binding site for a splicing enhancer protein srp40 according to bioinformatics. our findings indicate that, the putative branch point, r61h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank1. finally, we believe that bank1 delta 2 protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta 2 protein may contribute to the observed reduction in sle susceptibility. s. beermann 1 , r. seifert 1 , d. neumann 1 1 hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h 1 receptor (h 1 r), h 2 r, h 3 r, and h 4 r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd3 antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or 4-methylhistamine, a h 4 r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and 4-methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c57bl/6 mice induced by either a-cd3 antibodies or cpg-odn. this histamine effect was completely inhibited by the h 2 r-specific antagonist famotidine, while h 4 r-, h 1 r-, and h 3 /h 4 r-selective antagonists had no or only moderate effects. interestingly, the h 4 r-selective reagent jnj7777120, which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd3 induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h 4 r, jnj7777120 may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h 2 r and, to a much lesser extend, the h 4 r. using this assay system, cells obtained from control c57bl/6 mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega-3 fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of 20 ng/ml resolvin e1(rve1) on the release of tgfb, il-6 and il-17 in the culture of peripheral mononuclear cells (5x10 6 /ml) stimulated by phorbol ester (pma) (1nm), and the combination of pma and ionomycine (3 mg/ml) for 72 hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from 7 healthy subjects and from 10 patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = 0.010) and sjögren's (p=0.017) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve1 significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p=0.041) or pma+ionomycin (p=0.021), however rve1 was ineffective at reducing tgfb release in the sle and ss patients. rve1 caused a non-significant decrease in il-6 release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il-17 was not significantly modified by rve1 in any of the groups tested. the release of tgfb by 20 ng/ml of rve1 can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve1 tested, il-6 and il-17 release were not significantly affected in healthy or autoimmune patients. omega-3 fatty acid derived rve1 may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis 1 , a.k. tsirogianni 1 , m. herrmann 2 , h. m. moutsopoulos 1 , m.n. manoussakis 1 1 university of athens, dpt pathophysiology, athens, greece, 2 university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included 29 with primary ss (american-european criteria 2002) and 14 with sle (acr criteria 1997). age-and sex-matched healthy blood donors to the ss and sle groups (13 donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, 2007) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma (5 patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd68-staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p=0.0001). in ss patients, defective mb-phagocytosis involved only monocytes (p x 0.0001) and significantly correlated with the presence of extraglandular manifestations (p=0.02). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x 0.0001) and of ss (p=0.001). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: 30 healthy persons have been included in our study and 71 sle patients (66 women and 5 men) with various clinical signs (44 persons had 1 st degree of disease activity, 27 persons -2 nd degree of pathological process activity). 18 women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b 2glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in 36,6 %, aphl of igg class -in 45,1 % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x 2 =6,4; p x 0,02). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x 2 =3,9; p x 0,05). the increased levels of anti-ada were revealed in 11 of 18 women with hnp, and the combination of anti-ada and aphl (9/18) was found out more often than isolated anti-ada (2/18, x 2 =6,5; p x 0,02) or isolated aphl (3/18, x 2 =4,5; p x 0,05). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc1 and mdc2, and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc1, mdc2 and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from 31 pss patients fulfilling the american european consensus group criteria (aecc) and 28 gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p=0,0002) and mdc2 (p x 0,0001) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd5+ b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin 21 (il-21) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd5+ b cells and increased il-21 levels. we therefore hypothesized that il-21 may have similar biological effects on cd5+ b cells in autoimmune diseases. here we demonstrate that the amount of il-21 in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to 14 % of cd5+ b cells in sle individuals expressed grb. in-vitro experiments revealed that il-21 was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor 9. importantly, il-21 significantly decreased the cd5+/cd5-b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd5+ b cell death. these results suggest that il-21-induced grb may play a regulatory role for cd5+ b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il-21 and grb levels in sle and showing that il-21 reduces the cd5+/ cd5-b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il-21 may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il-21 in sle and other autoimmune diseases. r. de palma 1 , e. d'aiuto 1 , s. vettori 1 , g. abbate 1 , g. valentini 1 1 second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd4+ t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il-1beta, il-6 and il-8, cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed 169 igg antibodies from single facs purified cd19+cd27+cd38+cd138+ bone marrow plasma cells of 3 patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran 1 , e. moazemi godarzi 1 , e. kamali sarvestani 1 , e. aflaki 1 1 shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin 6 (il-6) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il-6 gene at positions -572 g/c and -174 g/c have been described that are key regulators of il-6 gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il-6 gene polymorphism at -174 position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the -572 position significantly differed in sle patients and controls. at this position gg genotype was observed in 77.9 % of patients compared to 68.9 % in the control group (p x 0.014). the frequency of -572 g allele in patients (87.3 %) was also higher than in controls (83.2 %) (p=0.034). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. -174 polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x 0.04). at -572 polymorphism, a significant difference with regard to photosensitivity in male patients (p=0.04) was found. in conclusion, results of this study showed that -572 polymorphism plays an important role in susceptibility to sle and that -174 polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained 161 patients with ibd (89 with cd, 41 with uc, 31 with gluten sensitive enteropathy, gse) and 33 healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of 7 asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd4-ve cd8b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd4(+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling-1 (rgs-1) protein, we have now found substantial over-expression (10-100 fold) of rgs-1 in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs-1 decreases primary t cell responses to cxcl12 and ccl19, strongly implying that it may regulate t cell localisation. thus, rgs-1 may be a novel target for modulating t cells in ibd, consistent with which snps in rgs-1 have been associated with both coeliac disease and type 1 diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c57bl/6j mice were divided in control (c) and experimental (e) groups. c1-c3 received peanut protein immunization, animals of the control groups c4 were sham immunized, and control group c5 received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a 30 day peanut containing cd, the experimental group was divided into 5 groups (e1-e5). ova feeding began 7 days prior cd (e1) on day 0 (e2), 7 (e3), 14 (e4) and 21 (e5) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e1) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio 2 as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase 1 and secretion of bioactive il1-b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il1-b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying 5 % destrane sulphate sodium (dss) in the drinking water for 8 days to wildtype mice. when ultrafine nanoparticles were administered on day 6 and 7 by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il10 -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl 1 , n. schneiderhan-marra 1 , m. blum 2 , g. stein 2 , m. schmolz 2 , t. joos 1 1 nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, 2 edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [1] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco-2 cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of 24-well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h 1-associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h 2-like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il-17 production and t h 17 polarization and decreases recruitment of cd4 + cd25 + foxp3 + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd11c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd11c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h 17 and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen 1,2 , g. van assche 3 , p. rutgeerts 3 , a. liston 1 , j. l. ceuppens 2 1 k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, 2 k. u. leuven, experimental immunology lab, leuven, belgium, 3 k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp-1 allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced 3 types of colitis dss (th1/th17), tnbs (th1) and oxazolone (th2) in hp ko mice. neutralizing anti-il-17 mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th17/th1 cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. 1) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; 2) in dss but not in oxa colitis mice, il-17, ifn-g, tgf-b and il-6 were significantly increased (p x 0.01, dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x 0.05, ko vs wt). in tnbs colitis, we found elevated il-12 and ifn-g (p x 0.01, tnbs vs control). although not significant, il-17 was also somewhat upregulated; 3) in dss colitis we observed that il-23 enhanced differentiated th17 cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il-6 and il-21, more mln-t cells from hpko mice differentiated into th17 cells; 4) anti-il-17 mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il-17 showed reduced il-6, il-17 and ifn-g in both mln and lp (p x 0.05, anti-il-17 vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il-17, supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann 1 , g. talabér 1 , g. süt" o 2 , p. németh 1 , t. berki 1 1 university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, 2 university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that 2.2 million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd4+cd25hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th1 and th2 cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd4+ and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd4+ inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells (74.5 ± 3.5 %, mean ± sem), while cd4+ inkt cells ratio was moderate (25.4 ± 3.5 %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: 38.0 ± 7.1 %; cd: 34.0 ± 5.2 %, mean ± sem), while the percentage of cd4+ inkt cells was elevated (uc: 62.0 ± 7.1 %; cd: 65.9 ± 5.2 %, mean ± sem) in both disease groups. proportions of foxp3+ treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b1 gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b1 adenovirus (tgf-b1-exo). the t cell inhibitory function of tgf-b1-exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b1-exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b1-exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd4 + foxp3 + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b1-exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b1-exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd4 + foxp3 + regulatory t cells (treg) revealed that the relative numbers of cd4 + foxp3 + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b1 gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd4 + foxp3 + treg. tgf-b1-exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd8+ t-cells was similar in the four vaccinees as observed by ifng, il-2 production-profiles. however, the killing capacity of expanded cd8+ t-cells was distinct as observed by the competence to kill ns3-peptide presenting transfectants in vitro. as depicted in figure 1 , cd8+ t-cells cells from both vac1 (cleared ) and vac2 (chronic) produced il-2 and ifng after stimulation with ns3-peptide59. however, specific killing of the peptide loaded transfectants was only observed in vac 1, who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure 1 ] killing of ns3 peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd8+ t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd8 t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns3 from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr4-expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns3 might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns3 or eda-ns3 under the control of cmv promoter were prepared. recombinant ns3 and the fusion protein eda-ns3 were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a2 molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd40 agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns3 rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns3. results: immunization of mice with the plasmids expressing eda-ns3, but not ns3 alone, induced strong t cell responses against the main hla-a2 restricted cytotoxic t cell determinants from ns3. the recombinant eda-ns3 fusion protein, but not ns3, was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp-1 monocyte cell line. immunization of hhd mice with eda-ns3 fusion protein induced both cd4+ and cd8+ t cell responses against ns3 and, when immunized with poly(i:c) and anti-cd40 antibodies, was able to down-regulate the intrahepatic expression of hcv-ns3 rna. the recombinant eda-ns3 fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even 25 years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e2 protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd207+cd1a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd8+ responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to 3 years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd1a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd8+ t cells by mature dcs transduced with melan-a-recombinant human coronavirus 229e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin 2 (il2) and interleukin 15 (il15) will be involved, and fms-like tyrosin kinase 3 ligand (flt3l) will be expressed to modulate dendritic cells. il2 is known to enhance early t cell expansion and limits t cell overshoot, whereaes il15 guarantees survival of high affinity t cells during memory phase. on the other hand flt3l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik 1 , ag waisman 1 uniklinik mainz, 1. med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd8 t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il-10 production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd8 t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response 7 days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il-10 produced by b cells by conditional deletion of the il-10 gene in these cells. we found that b cell secreted il-10 has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb490 to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from 8 healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for 10 days in the presence of il-2 and il-7 with exposure to overlapping peptide pools of latent ebna-1 and lytic bzlf-1 antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd3, cd4, cd8, ccr7, cd45ra, il-2, tnfa, ifng and cd107a. the majority of ex vivo ebv-reactive cd4+ t cells as well as ebna-1-reactive cd8+ t cells were il-2 and tnfa-producing memory cells, the later being more frequent in bone marrow (cd4+, median, ebna-1: bm 0.69 %;pb 0.12 %; bzlf-1: bm 0.37 %;pb 0.01 %, p=0.039). after in vitro expansion a major subset of ebv-specific cd4+ and cd8+t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd4+ and cd8+ t cells retained, however, a tnfa single, tnfa/il-2 or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd4+ and cd8+ t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr7/cd45ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il-2 delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd4 + and cd8 + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd154 or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd4 + and cd8 + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd8 + and cd4 + t cells in peripheral blood with a hexon protein-spanning pool of synthetic 15-mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to 29-90 % in the t cell lines and the absolute numbers of both hexon-specific cd4+ and cd8+ t cells were 2 to 3 log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd8 + and cd4 + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype 5 (ad5) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad5 vaccine vectors expressing whv core protein was first examinated in c57bl6 mice. groups of mice were immunized with a dna prime-ad5 boost regimen or with dna and ad5 alone. ad5 was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd8+ t cell responses against peptide pools 1 and 3 in spleens of dna and dna-ad5 immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd8+ t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad5 very weak cd8+ t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad5 level of antibodies did not change. those antibodies were only igg2a subclass what indicates th1 t helper type of response. ad5-immunized mice had over 3-fold lower level of anti-whc: both igg2a and igg1 subtypes were detected. the weak response induced by ad5 may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m2e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal 2007) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m2e serum ab titers against peptide and native m2e. this correlated with a large number of m2-specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h1n1), which contains a variant m2e-sequence different in 3 amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x31 (h3n2) and the highly pathogenic pr/8 (h1n1) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m2e as a potential "universal" influenza vaccine. this research is supported by a nih t32 fellowship ca09171-32, the nih grant ai 46457 and a grant from the commonwealth of pa. l. yu 1 1 zhejiang university, zhangzhou, china interleukin-18 (il-18) is a cytokine produced by stimulated mononuclear macrophage system. in this report, 18-day-old chicken embryos were vaccined with the plasmid dna (pci-chil-18) encoding chicken interleukin-18 and the copy numbers of chil-18 in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil-18 in 18-day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil-18 could accelerate high concentrations of chil-18 in nonage peripheral blood, accelerate high expression of chil-18 in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil-18 could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil-18 (p x 0.05). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of 40nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd8 cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on 12 volunteers and a phase i study on 24 volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd8 cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv-1 neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) 120 and the transmembrane protein gp41. two sites on gp120 and gp41 are attractive targets for vaccine design: the epitope in the third hypervariable region (v3) is recognized by the human monoclonal antibody 447-52d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies 4e10 and 2f5. in order to elicit anti-hiv-1 neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp120-v3 region or the gp41-mper. the vlps are based on the acyltransferase component (e2 chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e2 chain self-assembles into a 24 nm protein scaffold resembling a vlp and that contains 60 copies of e2. efficient display and refolding of the v3 and mper regions in e2 vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the 5' of the gene coding for e2. the priming and boosting with a combination of vlps and specific hiv-1 envelope dna were used to immunize mice and rabbits. results: the v3-e2 and mper-e2 vlps were purified as stable 60mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of 447-52d, 4e10 and 2f5 antibodies to hiv-e2 monomers was confirmed by western blot. we obtained high titers of hiv-1 gp140-specific antibodies in mice immunized with a combination of vlps plus dna (hiv-1 sf162 gp160). these antibodies generated a low (20 %-27 %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e2 vlps plus dna elicited a low titer of hiv-1 gp140 specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp160 plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e2 vlps are able to display antigenic determinants of hiv and to induce high titers of hiv-1-specific antibodies. the e2 vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h5n1 whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h5n1 influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [1] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h5n1 whole virus wild-type vaccine, could induce an immune response and protect mice against h5n1 influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h5n1 antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h5n1 virus. the protective efficacy of the trivalent vaccine derived mainly from the h1n1 component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h5n1 virus, suggesting that antibodies are the main contributor to protection. h1n1 specific serum did not inhibit neuraminidase activity of h5n1 virus suggesting that protection was not mediated by neuraminidase n1-specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h5n1 whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h5n1 vaccine enhanced anti-h5n1 antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h5n1 vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h5n1 candidate vaccine. [1] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to 6 years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of 335 children in the first grade of primary school in the area of central and west macedonia. there were 234 greek and 101 foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following 1) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to 100%. 2) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). 3) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. 4) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type 1 diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om-85, a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd28 -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om-85. cytokine secretion, activation and proliferation of b cells and foxp3+ tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr2, tlr4 or the myd88 adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om-85. two synthetic tlr4 agonists used as adjuvant in human (om-174-dp and om-197-mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om-85-induced protection of diabetes required natural tregs, as nod-cd28 -/mice were not protected. remarkably, om-85 activated b cells and not t cells, promoting their proliferation and il-10 secretion, two phenomena that were tlr4-and myd88-dependent. om-174-dp and om-197-mp-ac two synthetic murine tlr4 agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd4 + cd25 + foxp3 + t cells and the proliferation of il-10 secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr4 signaling in the protective effect of om-85 on development of diabetes and show that two other tlr4 agonists induce proliferation of b cells and their secretion of il-10 as well as stimulation of regulatory cd4 + cd25 + foxp3 + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit 1 , a. legat 1 , s. thomas 1 , m. van mechelen 2 , p. hermand 2 , m. goldman 1 1 institute for medical immunology/université libre de bruxelles, gosselies, belgium, 2 glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide 4-phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor 4 (tlr4). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd14 (scd14) for its tlr4 ligand activity, we investigated the involvement of both forms of cd14 in the responses elicited by crx-527, a prototypical agp. first, we found that crx-527 efficiently induces nf-kb and irf3 activation in hek cells transfected with tlr4 and md-2 genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd14. likewise, crx-527 efficiently induces the synthesis of nf-kb and irf-3 dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd14 prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd14 respond to crx-527 but not to lps in serum-free medium. the addition of the soluble form of cd14 did not modify the levels of il12 and tnf produced by crx-527 stimulated dc but increased the levels of interferon-b (ifn-b). when scd14 was added to hek cells expressing tlr4/md-2, nf-kb activity was not modified but irf3 activity was increased in a dose-dependent manner in response to crx-527. we will further compare the responses induced by crx-527 in wild-type and cd14 deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h-2d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled 14 and 28 days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin-15 gene polymorph isms (c267t, g367a, c13687a and a14035t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [1] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n=14) have higher amount of both nonactivated (subtype infg+/cd69-, p=0.02) and activated (subtype infg+/cd69+, p=0.028) cbmc, producing ifn-g, as compared with newborns from urban mothers (n=79) exposure to pets and the risk of allergic symptoms during the first 2 years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc09/16 to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity (12-14 %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: 5 months -3 years (n=22), 4 -7 years (n=15) and 8 -12 (n=6) tb has the highest diagnostic value in children n 4 years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp-10 antigen is more sensitive for detection of tb-specific t cells compared to esat-6 antigen. 4. in children with tst 5-14 mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr2 and tlr4 in the development of spontaneous lupus disease by creating tlr2 or tlr4 deficient c57bl/6 lpr/lpr mice. methods: tlr2 and tlr4 deficient lupus prone mice have been generated by crossing c57/bl6-tlr2 -/-or c57/bl6-tlr4 -/-mice with c57/bl6 lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr2 or tlr4 deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr2 or tlr4 in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr4 deficient mice suggesting an important role of tlr4 in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr4 also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr4 pc14/13 expression of full length mcl-1 and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl-1, an anti-apoptotic protein. a splice variant of mcl-1 arises by removal of exon 2 and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl-1 (mcl-1l) and its splice variant (mcl-1s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl-1. method: neutrophils were isolated from children (diagnosed x 17 years) with jsle (n=14) and non-inflammatory conditions (control, n=14) and incubated with control serum, jsle serum alone or with jsle serum plus 20pg/ml gm-csf. quantitative real time pcr was used to assess mcl-1l and mcl-1s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl-1s to mcl-1l was also higher in jsle patients compared to controls (p x 0.05). the addition of gm-csf to jsle serum was associated with an increase in mcl-1l (1.66 ± 0.31) and a decrease in mcl-1s (2.56 ± 1.1) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl-1 compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl-1s, and less anti-apoptotic full length mcl-1 cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd4 + cyld ex7/8 t cells were transferred into rag1 -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex7/8 cd4 + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex7/8 mice are capable to control inflammatory responses. for this purpose cd4 + cd25 + cells were co-transferred with naïve wt cd4 + t cells into rag1 -/-recipients. interestingly, rag1 -/-recipients of cyld ex7/8 t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc17/16 the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a*03 in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb1*13, *01 and *0103 alleles, and a decreased allele frequency of drb1*15 in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb1*0103 is associated with the development of severe disease and positive association of cd with drb3*0301 and drb1*13. indeed, drb1*15 was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb1*07 frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb1*04 in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb1*15 were strongly increased with respect to cd and controls. however, the frequency of drb1*04 was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb1*15 is associated with uc in european, north american, japanese and korean populations methods: a total of 176 children were studied, (92 boys and 84 girls), up to 17 years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, 51 positive cases of children were found (29 %) with active infection : 28 boys (14 x 5 years of age, 12 5-10 years of age and 2 g 10 years of age) and 23 girls (10 x 5 years of age, 6 5-10 years of age and 7 g 10 years of age). pharyngitis was present in 47 children (92,2%), 39 had fever (76,5%) and 48 had lymphadenitis (94 %). the lab tests revealed leukocytosis up to 20.000 leukocytes in 29 cases (56,9 %) and leukocytosis g 20.000 in 9 cases (17,6 %). the most frequent complication documented was streptococcal superinfection in 13 children (25,5 %) and thrombocytopenia in 8 children (15,7 %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and 3) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il-13ra2, previously believed to be a decoy for il-13 only, is able to transmit a signal via il-13. our results support this and may suggest that il-13/ il-13ra2 signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il-4ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il-13 or il-13ra2 and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd4 + cd25 -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il-6 and il-1b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd4 + cd25 -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi-98, l. acidophilus ncfm tm and b. bifidum bi-504) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi-98 strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il-6 or il-1b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 1 1 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed 21 pediatric cd patients (13 active, 8 remission), 24 pediatric uc patients (17 active, 7 remission), and 37 age-matched non-ibd controls. nkg2d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x 0.05 was considered significant. results: nkg2d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd3+cd56+ nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg2d ligands on circulating monocytes is also observed. the dramatic increase of nkg2d+ lymphocytes, and the strong upregulation of nkg2d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg2d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc17/26 peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp3+ t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd4+ t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp7 knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß5i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp7 knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp7 knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp7 knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than 200 million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec-205 antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa 1-191) or hcv-ns3 (aa 1027-1218) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns3 and core we successfully optimized culture and protein purification conditions. briefly, ns3 was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec-205 to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec-205/ hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd01/2 mva-nef vaccination induces polyfunctional cd4 t-cells and increases the proliferative capacity of cd8 t-cells in hiv-1 infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv-1 protein nef (mva-nef) was safe in hiv-1 infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd4 t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il-2 and mip-1b, the expression of the activation marker cd154 and the differentiation marker cd45ra in nef-specific cd4 and cd8 t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il-2 and mip-1b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd4 t-cell response and a significant increase of polyfunctional nef-specific cd4 t-cells, simultaneously expressing ifn-g, il-2 and cd154. using the standard ics no increase of nef-specific cd8 t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd8 t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il-2 and cd154 expressing cd4 t-cells and the increase of proliferating cd8 t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd4 t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd8 t-cells in hiv-1 infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv-1 mediated membrane fusion and p24-detected replication. db81 was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with 25mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd4 + and cd8 + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns4 (1677-1756 aa) and rns5a (2061-2302 aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/2j mice were immunized intraperitoneally 2 times at a month interval with different doses (0.1 -2 mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns4 protein. immunization with rns5a-immunomax conjugate and rns5 in cfa (1.4 mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns5a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns5a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd01/21 degree of cross-genotype reactivity of hcv-specific cd8 t cells directed against ns3 the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd8 t cell response targeting hcv genotype 1 (gt1) and genotype 3 (gt3) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: 53 subjects (17 with gt1, 22 with gt3 and 14 anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns3 from gt1 or gt3. individual reactive peptides and the degree of cross-reactivity between the gt1 and gt3 variants were determined by ics. complete ns3 is sequenced from all viremic patients pd01/22 anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes 1, 4, 6 or 9 suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes 1, 4 or 9, but not 6 led to a significant reduction in viral loads. decreased splenic viral load after ifna1 treatment correlated with an expansion of activated fv-specific cd8 + t cells and nk cells in the spleen, whereas in ifna4-and ifna9-treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna2, 5 and 11 are under investigation pd01/23 elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd8 t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd8 t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd8 t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a*0201-or k b -restricted cd8 t cell responses by these dna vaccines differed. k b /ova 257-264 -and k b /s 190-197 -specific cd8 t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd4 + and cd8 + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least 16 healthy, randomly chosen blood donors were cultured for12 days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. 48 out of 72 tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than 50 % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of 5 peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, 3 hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd8 + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd8 + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il-12-exposed cd8 + t cells showed at least five-fold increase of survival rate in vivo than il-2-exposed cd8 + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd8 + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il-12-exposed cd8 + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd8 + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il-12 and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x 0.01) . there was a significant elevation of t ada and ada1 activities in iga deficient patients as compared to healthy individuals (p x 0.01) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd01/61 hiv-1 sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after 2-6 weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in 12 out of 17 patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, 10-day-old ross 308 broiler chickens divided randomly into 3 groups, 2 experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/79 strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/79 and h120 strains, via the drinking water at 10 days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, 7&14 days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd4 t cells from 25 patients with active tb and 28 patients with successfully treated tb were analysed for simultaneous expression of ifng and il2 at the single cell level using multi-colour flow-cytometry after 6 hours stimulation with ppd. moreover, cells were stimulated with esat-6 and cfp-10 receiver operator characteristics analysis revealed that a percentage of ifng /il2 dual positive cells x 56 % served as an accurate marker for active tb patients (specificity 100 %, sensitivity 65 %), while frequencies g 56 % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd03/7 necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in 1993 and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately 10 100 000 inhabitants) is 5.33 to 100 000. among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, 12 out of 14 children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently 6.12 to 100 000 inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of 1986 to 1993 led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd03/8 novel analogues of thalidomide inhibit cd80 expression and production of tnf-a, il-12, ifn-g, cxcl-9 this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta1-dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta1r7k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd80 and cd86. these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il-1a and il-1b, while il-6 levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il-1b production. using bmdc from nlrp3 -/-mice we examined il-1b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il-1b production is dependent on nlrp3. collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho-1 expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from 20 % in wild type (hmox1 +/+ ) mice to 87 % in ho 1 deficient (hmox1 -/-) mice. hmox1 -/-but not hmox1 +/+ mice developed end-stage multiorgan failure. mortality of hmox1 -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h 2 o 2 or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb1. similarly, circulating and cytoplasmic hmgb1 was increased in hmox1 -/-relative to hmox1 +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b-1,2-linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at 4-6 years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and 2-4 weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were 7.7 and 9.4 iu/ml and those of dtwp-pasteur were 8.2 and 8.6 iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were 30.2 eu/ml for dtwp-local and 47.9 eu/ml for dtwp-pasteur vaccines, respectively (p x 0.001). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd05/18 united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b:2 , a gram-negative coccobacillus. jrmt12, an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of 10 8 cfu of jrmt12. a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b:2 on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for 1 h before adding concanavalin-a (cona) pd06 -vaccination and immunotherapy against parasitic diseases pd06/1 evaluation of simian adenoviral vector adch63 expressing msp-1 as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype 5 (adhu5) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch63 is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp-1 and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm128 while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho-1 expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox1 locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho-1 is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp63 expression was confirmed by sds-page and elisa using monoclonal antibody against gp63. discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a5-180recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a5-180recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least 6 month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at -20°c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and 2 seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r=0.216, p=0.641) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il-17 also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il-17 on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il-17 to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il-17 and il-17-related inhibition of cfu-e growth were dependent on p38 mapk activity, since the p38 mapk inhibitor, sb203580, markedly downregulated il-17-induced activation of nos and reversed the growth inhibitory effects of il-17 on cfu-e. the in vivo stimulating effect of il-17 on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il-17 effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il-17 acts on bone marrow cells and also revealed a link between the il-17, no and hematopoiesis. further studies on il-17-mediated induction of both inos and enos methods: a total of 1785 blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of 68 samples (3,8 %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x 100 iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had 100 iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of 10 iu/l anti-hbs in all the blood units, which also goes for our study pd14/20 neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen 1,2 1 national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine 297 can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa-1a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa-1a iggs we monitored 12 children (9 boys and 3 girls) in ages from 3.2 to 17.2 years with average age of 8.9 years. in 11 of them all was diagnosed for the first time. 1 subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest (11 subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc3h1 gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. 2005, nature 435, 452-8). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 3'utr of icos, in which a 47bp sequence, containing a possible mir-101 binding site, was sufficient (yu et al. 2007, nature, 450, 299-303). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. 2000, j biol chem 275, 33655-62). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia-1-and ago2-deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r01 ai 42269. we have recently reported that 6-hydroxyl-1-methylindole-3-acetonitrile (6-hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw 264.7 macrophages. in this report, we investigated the effect of 6-hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin-8 (il-8), monocyte chemotactic protein-1 (mcp-1) in ht-29 human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed 6-hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated 6-hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. 6-hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p65. in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of 6-hma significantly reduced the mrna levels of il-8 and mcp-1 stimulated by tumor necrosis factor-a (tnf-a) in the ht-29 cells. pretreatment of 6-hma also significantly blocked the ixba degradation and nf-xb p65 nuclear translocation stimulated by tnf-a in the ht-29 cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of 6-hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, 6-hma could be new potential therapeutic agent for inflammatory bowel disease.cd4 serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd4 igg immune response in hiv-1-exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp120-binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd4 domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp120, reacted with the 2 external domains of soluble human cd4, in the absence of the target cells expressing the co-receptor ccr5. the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd4-gp120 complex and trigger the membrane fusion between effector (gp120 expressing) cells and target (ccr5 expressing) cells. thus, in the absence of ccr5 we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv-1 infections. a conventional protocol for the generation of monoclonal antibodies was used. db-81 (igg1, x), one of the anti-cd4 antibodies obtained, recognized preferentially cd4 complexed to gp120, as compared to cd4 alone, not competed for the gp120 binding site on cd4 and was specific for the second extracellular domain of cd4. g. röder 1 , l. geironson 2 , a. darabi 3 , m. harndahl 1 , c. schafer-nielsen 4 , k. skjödt 5 , s. buus 1 , k. paulsson 2 1 copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, 2 lund university, immunology bmc d14, lund, sweden, 3 lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, 4 schafer-n, copenhagen, denmark, 5 department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first 87 n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a*0201. to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn 1-87 /80 , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn /80 was demonstrated to be located on tapasin [40] [41] [42] [43] [44] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first 87 n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd8+ t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region 301-498 of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b*2705-restricted np-flu epitope (aa 383-391) was replaced by hla-b27 or hla-a2-restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard 51 cr release assay and through the ifn-g production. results: using hla-b27 or hla-a2 restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b27 molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a2 molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a2 restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b27 as a remarkable hla-class i molecule that, differently from hla-a2, can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b27 molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv-1)-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv1. consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p17-gag and p24 region was determined in vitro. 25mer peptides were digested with i20s and c20 proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv-1 p17-and p24-gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers 1 , g. koster 1 , o. landsverk 1 , f. skjeldal 1 , o. bakke 1 1 university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi3 kinases and thus ptdins(3)p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier 1 , l. visvabharathy 1 , j. fu 1 1 university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e3-19k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e3-19k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type 2 e3-19k and class i molecules.results: we showed that e3-19k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a11 molecules, with the mature form being more tightly associated. we also provided evidence that e3-19k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e3-19k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e3-19k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il-12, il-6, tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands 2 and 4, but also with the t-celldependent stimulus cd40l (cd154) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd40, cd83, and cd86 was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p38 and erk-1/2. strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd4+ t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th2 (il-4+) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th1 (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th1 / th2 balance and to identify potential t cell target antigens (ags), we analyzed circulating cd4+ t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il-4 expression in peripheral blood cd3+cd4+ lymphocytes were measured in 9 ccs patients and 7 healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il-4/ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il-4 and lower ifn-g intracellular expression were detected in cd4+ t cells from css patients, as compared to hs (il-4: 1.3±0.3 % vs. 0.50±0.21 %, p x 0.05; ifn-g: 14.2±4.5 % vs. 27.0±4.8 %, p x 0.025). similar results were obtained by elisa (il-4: 0.39±0.16 vs. 0.30±0.07 pg/ml, p x 0.05; ifn-g: 31.0±14.3 vs. 79.0±15.0 pg/ml, p x 0.05). elispot counts of hexavalent vaccine-stimulated cd4+ cells were positive for il-4 in 4/5 (80 %) css patients and in 5/5 (100 %) hs, and for ifn-g in 2/9 (22 %) css patients and 7/7 (100 %) hs. mpo stimulation determined significant ifn-g release in 5/8 (62 %) css patients, but not in hs (0/5) no il-4 response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd4+ cells from css patients show a th2-polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd4+ t cells, and responses towards it are instead th1-polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd4+ t cells in css. a. voigt 1 , e. opitz 1 , k. savvatis 2 , k. klingel 3 , k. stangl 1 , u. kuckelkorn 1 , p.-m. kloetzel 1 1 charité -universitätsmedizin berlin campus mitte, berlin, germany, 2 charite -campus benjamin franklin, berlin, germany, 3 universität tübingen, tübingen, germanymurine models of coxsackievirus b3 (cvb3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp2 of the stress-induced ip, were infected with 1x10e5 pfu cvb3 nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp7 as early as day 4 p. i. in lmp2-deficient mice. however, lmp2-deficiency was linked to less severe myocarditis at day 8 p. i. (he stain of cardiac tissue sections: wt 1.95 ± 0.20 vs. lmp2-deficiency 0.71 ± 0.06 (grade of myocarditis; scale 0-4; p x 0.001). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x 0.05), there was no difference in lmp2-deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb3 infection in lmp2-deficient mice. likewise, diastolic function was preserved in lmp2-deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp2-deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp2-immunosubunit function in regulatory processes of viral replication. absence of lmp2 confers host protection in enterovirus myocarditis. h. w. liao 1 , j. xu 1 , j.q. huang 1 1 sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype 2 (den-2) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr1 and tnfr1-2 were constantly very low whereas trailr2-4 decreased after den-2 infection. fasl was expressed at similar levels on huvecs throughout den-2 infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase 8, caspase 3 were also observed by western blot after by den-2 infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for 7 days into wis rat paw web tissue in saline containing 7.5x10 7 cells. group ii. s.epidermis was injected as in group 1 after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day 8.they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors 2,23 and 3,91 respectively (p x 0.05). immunohistochemical pictures showed increase in percentage of ox62 (migrating dendritic cell), mhc ii, his48 (granulocytes), ox7 (stem cells) and cd54 (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed1 (macrophages) and ox62 cells. popliteal lymph nodes contained evidently less of ox62, his48 and mhcii cells than in group i (p x 0.05). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega-3 fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega-3 fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega-3 fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega -3 fatty acids consumption. the results indicate that omega-3 fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega-3 fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega-3 fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density 1.24 and 202,000g, top 20 % layer contained lipoproteins only and 20-50 % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising 1 % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin-1. objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e2f2 mutation, and other affecting b cell apoptosis control, such as bcl-2 over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c57bl/6 (b6) genetic background. e2f2-/-bcl-2tg were obtained backcrossing e2f2-/-and e2f2+/+hbcl-2tg mice. e2f2-/-bcl-2tg, e2f2-/-, e2f2+/+hbcl-2tg and control mice (e2f2+/+) were followed up to 18mo-old. serologic studies: serum samples obtained at 3, 9 and 15 month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of 3, 9 and 15 mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h3]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl-2tg in b lymphocytes of e2f2-/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e2f2-/-bcl-2tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e2f2-/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e2f2 or the overexpression of a bcl-2 tg in the b6 genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor 9 (tlr9) abolishes the generation of antinucleosome igg2a and igg2b autoantibodies but exacerbates lupus disease. however, the tlr9-dependent tolerance mechanism is unknown. here we show that loss of tlr9 in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th1 and th17 t cells. transfer of a synthesized monoclonal polyreactive igm to tlr9 deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr9-dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n=115) (vitali et al. 2002) . ninety-seven of 115 (84 %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g 1 (fs+), while biopsies from 18/115 (16 %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in 27/115 (23 %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x 0.05). interleukin (il) 17, il-1ra, il-1beta, il-12p40, il-15, macrophage inflammatory protein (mip) 1alpha, mip-1beta, eotaxin, interferon (ifn) alpha, and il-4 levels were significantly increased in the gc+ patients (n=27) compared with the gc-patients (n=70). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il-4 found in these patients (gao et al. 2006) . increased titers of th17-associated cytokines, il-17, il-1beta and the il-23 subunit il-12p40, may indicate a higher activity of these cells in gc+ patients (nguyen et al. 2008) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il-6. the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg2. production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g 50 %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd88-dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd88-dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/-1 baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il-8. objectives: increased levels of il-18, an innate and inflammatory cytokine of the il-1 family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il-18 and other genes of the il-1/il-1r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from 48 patients and 32 healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il-18 and il-18bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il-18 are higher than in controls. serum il-18 correlates with disease activity and decreases upon remission. monocyte expression of the receptor il-18rb is increased and correlates with disease severity, while expression of tir8/sigirr (a down-regulatory receptor of the il-1r/il-18r family) is reduced. in mrl lpr/lpr mice, expression of il-18, caspase-1 and il-18rb genes precedes disease onset in lymph-nodes. in other organs, changes in il-1-related genes (il-33 and tigirr-1 up-regulation, tir8/sigirr down-regulation) occur after disease onset. free il-18 levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il-18 levels correlate with autoimmune lupus both in mice and humans. free il-18 may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il-18rb is a marker of pathology, suggesting increased il-18-dependent activation. both in mouse organs and human monocytes, tir8/sigirr expression decreases with disease, suggesting impaired control of il-18r activation. thus, il-18 may be involved in autoimmune lupus pathology, and il-18-related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f4 (amino acid (aa) 451-593) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f4 was recognized by all patients. fragment f1 (aa 5-30) was recognized by 9 of 34 dcssc patients. fragment f8 (aa 350-400) was recognized by 4 of 8 sle patients. analysis of clinical data revealed a significant difference between the f1 negative and f1 positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f1 and f8 could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd4 + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd4 + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril-2. by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd4 + t cells enriched in tregs were co-cultured with activated cd4 + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd4 + t cells anergy was also evaluated based on cbl-b, grail and foxp3 mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd4 + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd4 + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b-1 cells through targeting p110d by shrnas strategy. methods: we used the drugs, ly294002 and wortmannin, pan-specific inhibitors against pi3ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p110d. we then introduced either pan-specific inhibitors against all pi3ks or p110d-targeting shrnas into an sle-prone animal model, nzb/w f1 mice, for therapeutic purposes. the results suggested that pi3ks are not only important for the development of b-1 cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p110d. either pan-specific inhibitors against pi3ks or p110d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f1 mice. one inhibitor, ly294002, and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi3ks are critical for the maintenance of b-1 cell populations might shed light on future treating other diseases associated with b-1 cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool 1 , m. alarcón-riquelme 1 , s. kozyrev 1 1 institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank1 gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs10516487, r61h). we identified that bank 1 gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon 2 (delta 2). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank1 is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank1 isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs17266594), which is in complete ld with r61h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia1, which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a 57-year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti-2gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last 1year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow 1 1 cnrs 9021, strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed 18 patients, with minor clinical and/or biological manifestations of their disease, for at least 6 monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below 2. b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd19 surface protein for all patients. this cd19 lower expression is associated with cd45 lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf2 expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf2 lacking an exon in the c2-domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf2 in t-cells. smurf2 expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf2 expression was upregulated by tgf-beta stimulation in t-cells and smurf2 was markedly upregulated in tumor infiltrating cd4+ lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day 42, smurf2 transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf2 showed an upregulation of the tgfbrii and an activation of smad2 and 3 as compared to wild-type t-lymphocytes, which were previously described as typical smurf2 targets for degradation. in addition the transfection of smurf2 and a caga-luc plasmid into cos-cells for smad3-promotor studies yielded the same effect as shown by upregulation of the smad3 activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf2 splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf2 has beneficial effects on mucosal inflammation and tumor development. smurf2 thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat3 has important functions in cytokine signalling in a variety of tissues. however, the role of stat3 in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat3 activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat3 activation in dss colitis is dependent on il-22 rather than il-6. il-22 was secreted by colonic cd11c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat3 activity were highly susceptible to experimental colitis, indicating that epithelial stat3 regulates gut homeostasis. stat3 iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat3 regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il-22 and epithelial stat3 was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat3 activation regulates immune homeostasis in the gut by promoting il-22-dependent mucosal wound healing. stat3 seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd40 and tlr4 expression at day 3 and day 7 after infection. mycobacterial infection did not result in differential tlr2 expression as compared to uninfected cells. cd40 is involved in stimulating th1 pro-inflammatory responses, although map may interfere with cd40 signalling (1) . tlr4 signalling elicits anti-inflammatory responses, which can contribute to bacterial replication (2) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd40 and tlr4 receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd8 t cells by exogenous antigen leads to colitis. methods: eight million naïve cd8 + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h2-kb, were transferred i. v. into b6 mice. at day 0 and 4, mice were treated intra-rectally (i. r.) with 50 % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day 2 after the injection of ot-i cells. the phenotype of effector cells was evaluated at day 5 by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd8, the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd8 + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd8 + t cells. our study demonstrates that the local activation of antigen-specific cd8 t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b6 mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of 535 patients, (248 male and 287 female), with age average 69,7 years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x 2 and t-test programmes. results: in 372 patients (69,5 %,with age average 62,7 years of age) no antibodies were detected. on the contrary, in the remaining 163, 52 male and 111 female, (30,5 %, with age average 76,4 years of age), antibodies were detected. out of them, in 106 cases the results were strong positive (32 male and 74 female) and weak positive in 57 cases (20 male and 37 female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: 1) helicobacter pylori infection is relatively common in the general population (30,5%).2) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women).3) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova 1 , t. ulmannova 1 , k. stechova 1 , k. tesarova-flajsmanova 1 , v. stavikova 1 , j. nevoral 1 1 charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of 20 mothers whose infants were diagnosed with allergic colitis and 20 mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis (2-27 weeks, average 16.8 weeks of infant's age) or at the age of 12 weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il-4, il-6, il-10, il-17, il-23, ifn-gamma, tgf-beta1, egf and eotaxin. man-whitney u test was used for statistical analysis, p x 0.05 was considered statistically significant.results: il-10 as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x 0.001) in breast milk of mothers whose infants were suffering from allergic colitis (range 0-8.45 pg/ml, mean 2.1 pg/ml) than in control group (range 0-3.41 pg/ml, mean 0.35 pg/ml). higher concentrations of il-4 and lower concentration of tgf-beta1 were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. 8310-5. background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il-8 (murine homologs kc and mip-2) and its receptor cxcr2 are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated 12 h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at 2, 4, 16 and 22 h post-transfer. anti-kc antibody or its isotype control was administered at 20mg/mouse one hour before transfer followed by whole body and organ imaging 4 hours post-transfer. results: facs analysis revealed 80 % neutrophil purity, 35 % of which were cxcr2 + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, 4 h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than 10 15 bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model (1.5 % dss in drinking water for 1 week, followed by pure drinking water for 1 week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, 2 % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell 2004), treatment of commensal-depleted mice with the tlr4 ligand lps in drinking water protected against the lethality of 2 % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of 1.5 % they may become pathogenic and drive an intestinal inflammation. at 2 % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by 1.5 % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type 2 immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il-13 production which is an important pathological factor. neutralizing il-4 or il-13 prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th2 cytokines in colitis remain undefined, we used mice deficient in il-4/il-13 or the key receptor through which they signal, il-4ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il-4ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il-4/il-13 double deficient mice which were protected from colitis. removing il-13 production from il-4ra -/mice, by using il-4ra/il-13 double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il-13 mediates susceptibility in an il-4ra independent manor. recent evidence pc17/33 introduction: the activation of cd4 + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th1-like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th1 cytokines, such as il-5,-6 and il-13. however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc2 was found in uc and cd colonic tissue compared to control specimen. transmitted to the th2-mediated oxazolone-induced colitis model, nfatc2-production is significantly increased in both diseases, too. nfatc2 deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc2 deficient mice compared to control mice, which can be observed by tunel assays, caspase3 and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl-2 and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il-6, ifn-gamma, il-13 and il-17 by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc2 regulates il-23/il-17 in an indirect way. last, administration of il-6 blocked the protective effects of the nfatc2 deficiency in experimental colitis, suggesting that nfatc through il-6 signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc2 in colitis by controlling mucosal t cell activation in an il-6 dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd4cd45rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd4cd45rb high t cells also regulatory cd4cd45rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at 3 different time points during colitis progression. after 3 weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at 9 weeks. microrna was isolated from colons of mice in different stages of colitis progression (3, 6 and 9 weeks) and control mice that do not induce colitis (n=3 for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from 11 micrornas that demonstrated an induction during the development of disease we selected 4 micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd4cd45rb high transfer model. objective: the purpose of this clinical trial (id: nct00287677 of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for 6 months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in 3 groups: group a (n=8) receiving haart+ gh (for 6 months) + tt+hva/b vaccines (at month 6 post gh adminsitration); group b (n= 6) receiving haart+gh but not vaccines; and group c (hiv control group, n=7) with haart+vaccines (at month 6) but without gh. all patients are followed up 6 months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after 24 weeks of administaring hormone the absolute numbers of cd4 incresase from 562 ± 93 to 704 ± 112 cells per mm 3 (mean and sem; p x 0.0025). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd4 absolute numbers from 550±97 to 470±103 cells per mm 3 (p x 0.02). viral load remained undetectable in all patients. despite the increase in cd4 counts the percentage of recent thymic emigrants (as assessed by the expression of cd31) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv (80,000-160,000), hepatitis c virus (hcv; 2.3-4.7 million) and hepatitis b virus (hbv; 8-16 million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv-1 infected patients with g 350 cd4 + t-cell counts and plasma hiv-rna levels of x 1.7 log 10 copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from 4 timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g 50 and when the responses were g 2 times the standard deviation above the mean of replicate negative controls (mock electroporated dc). 16/17 patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in 4/16 patients before and 11/16 patients after vaccination. for rev, 7/16 patients showed a pre-existing rev specific response and 14/16 patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already 11/16 patients responding before and 16/16 patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in 13 out of 16 patients before vaccination and 16/16 patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus 71 (ev71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev71 is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev71 inactivated virus. methods: mice were immunized intraperitoneally with 10 mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after 0, 2, and 4 weeks. blood samples were collected at 0, 21, 35, and 45 days. the total serum anti-ev71 igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il-5 from splenocytes was also measured. results: immunization with ev71/ps-g showed that the anti-ev71 igg levels were significantly increased compared with ev71 alone or ev71/cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev71/ps-g group compared to ev71 alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev71/ps-g-immunized mice compared to those of ev71 or ev71/cfa/ifa-immunized mice, indicating a th1 cells response elicited by heat-inactivated ev71 vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd8+ t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd8+ t-cell responses against hcv-ns3, studying induction of il-2, ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il-2 and ifng production by cd8+ t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv-1-recognizing cd8 + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv-1 replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd8 + t cells by electroporation, and chose tcrs which were able to recognize the hla-a2 restricted hivpol-peptide iv9, or the hivgag-peptide sl9. results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il-2, tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd25, after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd4 + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd8 + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd8 + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt3l pretreated balb/c mice were incubated for 3 hrs with hcv ns5-coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g 80 % dcs and was used for immunization. dc expression of the maturation markers cd40, cd80 and cd86 was determined before and after ingestion of ns5-coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns5 expressing sp2/0 cells). results: in immunized animals, the ctl activity was increased 3-fold compared to mock immunized mice. accordingly, tumor challenge with ns5 expressing tumor cells showed a significant reduction in tumor growth. the number of cd4 + ifn-g + cells was increased g 3-fold and the number of cd4 + il-2 + increased g 5fold in the dc-ns5-bead immunization group. these results paralleled the proliferative response of splenocytes to ns5 protein obtained from immunized animals with the most significant response in the cd4+ population of dc-ns5-bead immunized animals. the use of ns5 coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th1 biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec-205 antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec-205 induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec-205 promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec-205 antibody can be developed. we screened the anti-dec-205 sequence computationally for putative hla dr4-restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec-205 antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec-205 as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih 1r21ai078800 a live oral vaccine based on human adenovirus (ad)4 has proved safe and effective in us military recruits for nearly 50 years. in these experiments, we have investigated whether replication-deficient ad4 can be an efficient potential vaccine carrier for oral vaccination. ad5 vectors were used throughout to provide a benchmark for efficacy. we generated novel ad4 and ad5 vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad4 than ad5. ad5 routinely infected and provided transgene expression in˚10 % of human cd4+ and cd8+ t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to 25 -30 % of cd8+ t cells present, showing that ad5 infected a surprisingly large proportion of t cells. in comparison, ad4 provided egfp expression in x 2 % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad4 and ad5 vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad4 and ad5 induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day 3 after dosing, and transgene expression being reduced below detectable levels by day 8. interestingly, when delivered together ad4 and ad5 vectors targeted the same subset of cells. together, these data show that ad4 is a viable alternative to ad5-based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies (20 recently commercialized; g 300 in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the 667 mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that 667-treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g 8 months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, 2005; gros et al, 2008; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd8 + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp60) of rhdv at two different locations: 1) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp-2), and 2) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp-306). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd8 + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp-2) was more immunogenic than insertion at position 306 for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp-2 at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr-2 ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd8+ t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd8+ t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np 147-155 specific cd8+ t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr-2 targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l929cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during 7 days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd2+, cd4+, cd8+, cd16+ t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by 1,5-2-fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns4 and ns5a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h5n1 influenza virus inhibits the ifn-g synthesis (mibayashi et al., 2007) and causes a decrease in cd4 + and cd8 + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., 2000) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a 10 amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions 129-131 (tat'kov c. i. et al., 2000) . deltaferon was i. p. administered to male non-inbred mice in doses of 2-40*10 4 iu once or twice at an interval of 48 hours, alone or in combination with double-stranded yeast rna preparation (5 mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., 1983) . results: when deltaferon was administered in doses of 2-20*10 4 iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of 2*10 4 iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon (2*10 4 iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd8 responses (tetramer+ hiv-1 gag p24 cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd8 responses after pla-p24 immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p24 were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas 1 , c. prego 2 , s. vicente 2 , m.j. alonso 2 , a. gonzález-fernández 1 1 university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, 2 university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer 188, with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with 1 or 2 immunizations to balb/c female mice (4 weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day 15 to 260 post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease (100 miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigenbackground: infection with human immunodeficiency virus type 1 (hiv-1) is characterized by dysfunction of hiv-1-specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p24 antigen from hiv-1 and the tlr3 ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p24 microcapsules containing p24 and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for 10 days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p24 peptides. intracellular staining of interferon-gamma (ifn-g), interleukin-2 (il-2) and cd107a after p24 stimulation was also performed. results: mddc from hiv(-) subjects, incubated for 24 hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p24 containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il-2, upon p24 peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd16b between our model cytokine il-2 and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p30gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il-2 dependent proliferation assays of il-2 variants. results: murine il-2 attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il-2 dependent cell line ht-2. we found that the membrane anchors comprising one and four ig-like domains (il-2::1iggpi and il-2::4iggpi) resulted in severely reduced vlp production by 293 producer cells and despite of an increased targeting of il-2::4iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il-2 fused to the minimal gpi-anchor acceptor sequence of hcd16b (il-2::gpi). il-2::2iggpi, however, showed comparable particle production and biological activity in vitro when compared to il-2::gpi. furthermore, il-2 fused to 2ig::gpi showed an increased capacity to co-stimulate primary p14 tcr transgenic t-cells specific for lcmv-gp 33-41 in the context of h2-d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. 2ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il-2::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f1816-b13 of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant 812079 & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild 1 , interdisciplinary transplant laboratory 1 charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects 2-25 % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of 2-3 months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with 6 different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from 2,5 month to 16 days. t cell lines are composed of cd8 and cd4 cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker 1 , m. assenmacher 1 , a. richter 1 1 miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp65-specific cd4 + and cd8 + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd45ro -cd25 -) cd4 + and cd8 + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp65 peptide pool and cd3-depleted autologous pbmc as feeder cells in the presence of il-2, il-7, and il-15. already 9-13 days after primary activation pp65 495-503 /a2-tetramer + cd8 + t cells were detectable for 3 hla-a2 + blood donors. to analyze cd8 + t cells having other specificities than for the peptide pp65 495-503 as well as probably primed cd4 + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to 3.9% of the cd8 + t cells and up to 3.8% of the cd4 + t cells after restimulation with pp65 peptide pool, but not with either irrelevant ie-1 peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for 20 days led to a 10 -100 fold expansion of pp65-specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd154 and cd137 only if restimulated with the pp65 peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il-2, indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd4 + and cd8 + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf 1 , k. mozdzanowska 1 , l. otvos 2 , j. erikson 1 1 the wistar institute, philadelphia, united states, 2 temple university, philadelphia, united statesthe influenza virus a matrix protein 2 ectodomain (m2e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m2e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine 2003; virology journal 2007), we generated a novel peptide and investigated its efficacy in inducing an anti-m2e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd8 + t cells, have been considered. clinical data confirm a crucial role for antiviral cd8 + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a*0201 and hla-b*0702 previously. considering other frequent hla alleles cmv specific cd8 + t cells were monitored longitudinally in 30 hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd8 + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd8 + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a*2402/pp65 (341-349) (7.6x10 6 cells/l) and hla-b*3501/pp65 (123-131) (4.4x10 6 cells/l) specific cd8+ t cells are significantly lower than those for hla-a*0101/pp50 (245-253) (17.2x10 6 cells/l) and hla-a*0201/pp65 (495-503) (13.2x10 6 cells/l) specific cd8 + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. (2,5 -50 mcg) . no adverse effects were indicated during trials (up to 25 month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after 4th immunization with 50 mcg of vichrepol.no differences were detected in cd4+ and cd8+ t cell counts and cd4+/cd8+ ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp70 or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp70 or gp70 alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb6f1 hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb6f1 mice with adenoviral nanoparticles expressing fusion proteins containing gp70 resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd4 + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd4 + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf01 exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted 90-99 % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf01 did not show any increase in their levels of ifn-gamma. conclusion: caf01 enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from 13 children aged 1-2 years old (6 boys and 7 girls) -group 1, and 11 children (6 boys and 5 girls) 6-7 years old -group 2 were isolated on a gradient of density before vaccination ( or revaccination) with priorix, 1 week, and 2 months after and incubated with cfse. then 2 million/ ml pbl were incubated in rpmi-1640 supplemented with 10 % fcs (the negative control), at presence of 5 mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing 5 % cî 2 at 37°c within 7 day. intensity of a fluorescence estimated on fl1 by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control 90 % pbl in both groups did not enter mitosis. in the positive control 90 % of cells have passed one and more mitoses. in group 1 measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in 2 months 15-25 % of lymphocytes demonstrated antigen-specific proliferation. in group 2, on the contrary, before the vaccination the most part of cells (75-80 %) has not entered division, but 20-25% of cells have passed 2 and more mitoses. in a week specific lymphocyte proliferation decreased and in 2 months it was increased up to 40-50 %. production of the interleukin (il) 6, ifn-g, tnf-a, il-4, il-1 was more informative than il-5, il-7, il-8, il-12. measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp120 cd4-binding site (cd4bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv-1 neutralizing polyclonal iggs directed against the cd4bs. the mabs were shown to react with an anti-cd4bs human neutralizing mab (b12), to elicit antibodies that recognize the gp120 molecule and an anti-hiv-1 neutralizing response in rabbits, confirming them as cd4bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp120 antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd4bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd4bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp120 titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b12 in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p1 and p2) reacted in elisa only with the cd4bs-directed igg fraction. the clones were also recognized in elisa by b12. p1 and p2-immunized rabbit sera showed a strong anti-gp120 titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb2 strain glycoproteins. in particular, 3/5 rabbits in the p1 group and 1/5 in the p2 group showed an 80 % hiv neutralization at dilutions ranging from 1:20 to 1:150. the immunoenzymatic assay used, allowed to detect a p1 and p2 reactivity in hiv-positive sera and was able to detect a b12 concentration equal to 10 ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines (17d and 17dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a 23-year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following 17d-204 vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd16, cd32 and cd64) along with increased levels of nkt-cells (cd3 + cd16 +/-cd56 +/-/cd3 + ratio) and activated t-cells (cd4 + and cd8 + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il-6, il-17, il-4, il-5 and il-10) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il-4 + ), cd8 + t-cells (il-4 + and il-5 + ) and b-lymphocytes (tnf-a + , il-4 + and il-10 + ). the analysis of cd4 + t-cells revealed a complex profile with increased frequency of il-12 + and ifn-g + and decreased percentage of tnf-a + , il-4 + and il-5 + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester 1 , h.-g. rammensee 1 , s. stevanović 1 1 eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd8 t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd8 t cell epitopes for the frequent mhc class i alleles a*01, a*02, and a*24 from the proteins pii (hexon), pviii, and e1a of adv strains ad2 and ad5 by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a 12-day recall stimulation of pbmcs from at least 16 healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd8 t cells. we could identify 27 new peptides eliciting ifn-g responses, several of which were confirmed as novel cd8 t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle (5 ara criteria) was diagnosed in a 40-year-old african female patient with hiv-1 (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd4 t-cell count x 200 cells/mm 3 . initiation of 500 mg mmf bid was associated with biological and clinical remission of sle and cd4 t-cell increase. no opportunistic infections or cancers were noted during a 3-year follow-up and the patient remained always naive to art. hiv-1-specific cd4 and cd8 t-cell responses were analyzed after 18 months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il-2 production following stimulation with a panel of 192 hiv-1-derived optimal epitopes (9/10-mers) covering various hiv regions and a pool of 105 hiv-1-derived peptides (15-mers overlapping by 11 aminoacids) encompassing the entire gag protein. all peptides are derived from hiv-1 consensus strain iiib. results: highly polyfunctional hiv-1 specific cd4 and cd8 t-cell responses against gag were detected. 11 epitope-specific cd8 t-cell responses were identified: except for one response restricted by hla a*23 and another one by hla cw*07, all the others were restricted by hla-b alleles and mostly by b*58 (n = 5). seven out of 11 responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il-2+) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv-1 specific cd4 and cd8 t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv-1 t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of 10 6 dl50/ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf-1 of 12-16 g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the 7 th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt-46767, cofaa and cgpi-20090305. . state of vaccine-induced measles immunity was determined by means of elisa in 1-3, 4-6 and 7-9 years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in 4-6 years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p 0.05) measles immunity and antibody level much lower (p p 0.05) than among children with mt conversion. in 7-9 years the comparison group kept decreased (p p 0.05) measles immunity, the majority (92±5.54 %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p 0.05) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p 0.05), the majority (83.3±4.1 %) of persons lost protected antibody level; among children with mt conversion in 1-3 years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p 0.05) to low values; among children with hyperergic mt igg level decreased (p p 0.05) and reached low (in 1-3 years), minimal protected (in 4-6 years) and lower than protected (in 7-9 years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il-17 producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein 65 (dnahsp65). methods: balb/c female mice were infected by intra-tracheal route with 10 5 h37rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp65 genetic vaccine was done at days 30, 40, 50 and 60 post-infection. each dose consisted of 100 micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd4+, cd8+ and gamma-delta t cells from lungs were determined 10 and 50 days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x 0.05 were considered significant (t test). results: at day 10 after the end of the immunotherapy, dnahsp65 treated mice exhibit increased numbers of absolute cd8+ and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il-17 producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp65 treated mice showed more ifn-gamma producing cells in both cd8+ and cd4+ cell populations. at day 50 after the end of the therapy, the main observation in mice which received dnahsp65 treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd4+ and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd8+ cells, together with a more frequency of gamma-delta t cells producing il-17. finally, the immunotherapeutic effects of dnahsp65 vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd8+ cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il-17, are the main effects of dnahsp65 immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il-17 and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as 60 loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of 3-5 log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput (96-well format), requires only 100 ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in 5 different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that 5 candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls 71 % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all 5 m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards 4 of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to 100 % in pb, 75 % in rx and 93 % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd40l, a co-stimulatory molecule preferentially expressed on activated cd4+ t cells, is the ligand of cd40. cd40-cd40l interaction induces the production of il-12 and the initiation of a th1-type immune response. several studies show that cd40l is required for the activation of macrophages and the maturation of dcs. moreover, cd40l enhances the capacity of cd8+ t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd40l. we have constructed the recombinant bcg strain expressing cd40l (rbcg2) by electroporation of bcg with pgfm11/signalsequenceag85b-cd40lec and an another recombinant strain with empty vector pgfm11 (rbcg1) as a control. the expression of cd40l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th1 type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd40l 2 weeks after vaccination but not at 4 and 8 weeks. rbcg2 seems to be more protective against paratuberculosis than rbcg1, but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd40l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il-1a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x 0.05) of tnfa, ifng and il-1a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x 0.05) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd40 mab and ag85a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd40 binding on cd40 transfected l929 fibroblasts. the conjugates were tested in vivo in wild type and cd4 + cell-depleted mice for the induction of specific anti-ag85a serum antibodies. splenocytes were challenged ex vivo with ag85a and were examined for their ability to produce th1-related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il-2. we developed a method to successfully crosslink anti-cd40 mab to ag85a. serum antibodies against ag85a were detected after immunisation with this conjugate vaccine in both wild type and cd4 + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag85a alone. production of two other th1-related cytokines, tnfa and il2, was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd40 conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd4 + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer 1 , r. burger 2 1 robert koch-institute, cellular immunology, berlin, germany, 2 robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd4-positive and cd8-positive t-lymphocytes. cd8-positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd8+ t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for 5 days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to 30 % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd4+ t-lymphocytes in vivo. similarly, the cd4-positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd4+ and the cd8+ subpopulation depended on the presence of apc. stimulation oft cd8+ cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd8-positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target 6 (esat-6) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, 83 iranian and afghan adults (38 patients with sputum smear and culture positive tuberculosis, 24 recovered patients during 6 months after full course of chemical treatment and 21 healthy individuals) were recruited to quantify the frequency of esat-6 and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of 4 spot forming unit ( g 20 spots per million), we found detectable response to esat-6 in almost 80 % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and 77 % of these individuals had detectable esat-6 specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in 14 %, 57% and 62 % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th1 immune response, against esat-6, in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish 1331 in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal 1 , p.k. upadhyay 1 1 national institute of immunology, pdc-1, delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl 2 solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c57bl/6 mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: 2.4 % alginate, 0.012 % pva concentration gives particles with size of 2-10 micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with 5 % trehelose and 3.5 % pvp (poly vinyl pyrollidone). there was 6 fold increase in proliferation index of spleenocytes and releases 600 pico-gram of interferon gamma after 4 week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by 10 % increase in cd86 and 10.5 % in ccr7 expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b100 is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b100s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b100s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b100s. by cell cycle analysis we determined that b100s is able to induce apoptosis in up to 80 % of jurkat and molt-4 t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b100s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b100s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase 9 is the first to be cleaved, followed by caspases 3 and 8, thus suggesting that b100s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b100s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b100s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd40, cd80 and cd86, and of the inflammatory cytokines il-6, il-23 and mcp-1. using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd4 t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th2 and th1/th17 skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd4 t cell responses. microcapsule based vaccination resulted in a marked induction of il-17 secreting th17 cells, without inducing strong th1 (cfa) or th2 (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg1, igg2c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th17 responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd4 + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd80, cd86 and cd40 and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il-6 and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr4-dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr4-defective c3h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il-1b secretion by dc, through its effects on caspase-1 processing, which is also tlr4-independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr4 signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc2461 (st11) or lactobacillus rhamnosus ncc4007 (lpr), each at 5x10 8 cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week 1 to week 7 of life. fresh feces were collected at 3, 5 and 7 weeks of life for determination of iga levels (assessed by elisa). pups were bled 2 and 4 weeks after immunization for determination of measles-specific igg1 and igg2a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st11-fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from 200 kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at 3:1 ration and washed 3 times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw264.7 by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of 24 extracts from 10 euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc6/propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that 15 up to 24 euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd3+ cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on 35 th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il-5 and ige serum levels were increased on animals from group i when compared to control.the enhancement on th2 immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd8+ ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd4+t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg2a and igg2b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren 1,2 , n. almqvist 2 , a. lönnqvist 1 , s. östman 1 , c. rask 1 , e. telemo 2 , a.e. wold 1 1 university of göteborg, department of clinical bacteriology, göteborg, sweden, 2 university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; 0.8 mg/ml) in the drinking water for 5 days and, 3 days later, 50 mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin-5 and interleukin-13. examination of gut sections from sea treated donor mice revealed increased density of cd8a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp21, proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna 1826, a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp21, or groel together with cpg-dna 1826 reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp21 together with cpg-dna 1826. the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp21 microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp21 microspheres induced protective ifng-secreting cd4 + t-cells and raised pulmonary pmp21-specific iga levels in vivo. also, pmp21 microspheres caused lower il-6 serum levels upon administration than the injection of pmp21 together with cpg-dna 1826, indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in 44 cases of s. aureus bacteraemia in iv drug users and 44 cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised 44 iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen 2006).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day 0) and four weeks thereafter (day 28). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter 2007) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st59 (spa-type t172, agr 1, and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p=0.01).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st59 strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st59 strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at 4 and 9 years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in 15 and 7 ds children, respectively. samples were taken before and 3-4 weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg 1 -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at 4 years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg 1 , igg 2 and igg 4 ). at 9 years, ds children had lower postvaccination geometric mean igg 4 anti-tt-titers only. post-vaccination igg 1 -anti-tt avidity levels were decreased in 8/15 and 4/7 ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. 2 kda; 30, 6 kda; 23, 9 kda; 19, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] 6 kda and 18, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 7 kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of 100 families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an 8-month period. sera were obtained from healthy subjects from the same community (2 months to 88 years of age). the highest incidence of salmonella-associated diarrhea, 74/1000, occurred in children under 5 years of age. the lowest incidence, 10/1000, was observed in the population aged 10 to 59. whereas serum from individuals ranging 15-70 years of age showed maximum igg1, igg3 and iga anti-s. typhimurium titres, children less than 5 years-old did not show detectable igg1 and igg3 titres and had weak igg2, iga and igm antibody levels; only their igg4 levels were comparable to those detected in adults. moreover, the levels of igg2 and igg3 antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs 07-18, a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses (67%), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a-1,6linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after 7-fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková 1 , s. bystrický 1 1 institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o135 using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd19, expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age 18-55 years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg1, igg2 and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present 5 years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after 5 to 7 days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg2 response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers 1:3200). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones (1:800 vs. 1:3200). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was 5. 85 fold of normal control). subcutaneous immunization gave a weaker stimulation: 1. 52 fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv 0032-06 and vega 2/7029/27 grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a-1,6-linked d-mannopyranose units and many branches composed of a-1,2 a-1,3 and/or b-1,2-linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant (6mg oligosaccharide per one conjugate dose) two times in 14 days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day 14 after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs (250g-350g) were treated orally with 50 mg respivax five consecutive days. after the last application, on days 3, 7, 10, 14, 28 and 42 six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on 4mm thick serial sections, stained with hemalauneosin. the populations of cd8, cd4 and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day 3 subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd8 positive cells, which number reached maximum at the end of the second week. on day 14 b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd4 and cd8 positive cells. on days 28 and 42 the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta 1 , s. majumdar 1 1 bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein-10 (ip-10) and interleukin-8 (il-8) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip-10 mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase2 (inos2) expression and was linked to the mapk signaling pathway via antagonistic regulation of p38mapk and erk1/2. further, ip-10 was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th1 cytokines and no. thus this study strongly demonstrates that ip-10, like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th1/th2 cytokines mediated by cd8+ t cells and activating cd4+ t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x 0.05) following immunization and after challenge with leishmania major. il-4 values were increased in all groups, but there was no statistical difference between the groups(p g 0.05) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x 0.05) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x 0.05) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x 0.05). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at 3 week intervals. three weeks after booster injection, 5×10 5 stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg2a/igg1was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type 1 immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin 10 (il-10), and cellular expression of cd4 and cd25. results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd4+ lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with 1,600 infective n. brasiliensis larvae on day 2 post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day 6 post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg1 and igg2a). this was independent of level of me supply. feeding regime did not affect levels of the type-2 cytokines il-4 and il-13. conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp63 is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp63 of l.major strain mrho/ir/75/er. methods: l.major promastigotes were grown in rpmi1640 supplemented with 10 % fcs. l.major rna extraction and cdna synthesis were carried out. gp63 gene segment was amplified by specific primers and cloned into ptz57r to construct ptz57r/gp63. the presence of gp63 into ptz57r was confirmed by pcr. then, ptz57r/gp63 was sent to determine the sequence of its nucleotides. after that the gp63 gene segment was sub-cloned into pet32 a (+) expression vector and transformed into e.coli bl21 (de3) plyss and gp63 protein was expressed in presence of 1mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of 100 % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, 2007) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including 50 volunteers. 10 male volunteers (18 and 45 years) for the adult group, and 40 children of both sexes (5-9 years) were enrolled. subjects received virosomal formulations containing 50 mg of ama 49-c1 (pev301t), an apical membrane antigen-1 derived synthetic phospatidylethanolamine (pe)-peptide conjugate and 10 ug of uk39 (pev302t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days 0, and 90. results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling 29 % of the treated mice to survive till 21 days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only 0.04 % of the amount fed and it remained in circulation in the blood only for 30 minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to 0.4 % ( of the amount fed but it's circulation was sustained till 6 hrs post feeding. under such conditions when 200mgm of curcumin bound to 300mgm of chitosan nano particles were fed one time daily for 10 days post infection to plasmodium yoelii infected mice 100 % of mice were cured and survived atleast for 100 days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma9vdelta2 t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il-10 responses, to epitopes on the dominant rbc autoantigen, anion exchanger-1 (ae-1, or band 3) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of 1 methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla-4 + regulatory t cells or soluble forms of ctla-4, may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =-0.81) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd8 + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b 1 (r medium =-0.74) and il-10 (r medium =-0.65) in isolated splenic hscs ex vivo. analysis of effector cd4 + t cells in spleen showed decrease of t h 1 cells quantity and simultaneous t h 2 cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd4 + cd25 + ctla-4 + -and cd4 + cd25 -il-10 + il-4regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h 1, t h 2 and regulatory t cells were correlated (r th1 =-0.74, r th2 =0.86 and r th3 =0.80 and r tr1 =0.68) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th3 =0.84 and r tr1 =0.76) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b1-and il-10-produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p38, jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin-17, t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il-17, as compared to non-infected controls. the infection also altered the effect of il-17 on mapk activation by preventing its stimulating effect on p38 mapk. moreover, in s.obvelata-infected animals il-17 markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il-17 induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. 2.-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from 1 ⁄2 to 1/64 with a normal serum pool. 3.-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r 2 g 0.98. the analytical range was from 1 g/l to 70 g/l. the coefficient of variation (cv) was x 10 % for [mc] n 1 g/dl, and x 20 % for [mc] x 1 g/dl. this procedure was successfully implemented to quantify the mc in 1100 serum samples between march 2008 and february, 2009. among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: 1. we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. 2. the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from 7 day and reached plateau level after 30 day of teratocarcinoma insertion. moreover, cd34 + cd38cells showed in spleen and main lymph nodes from 15 day and achieved plateau level after 60 day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at 22 day and had a maximum pick at 60 day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than 1,5 times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b 1 , il-10, pge 2 and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number =0.81 and r activity =0.92) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd40-ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd40l is cleaved to soluble (s)cd40l. we sought to examine the levels of scd40l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded 5 atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd40l -along with other products -were assayed by quantitative elisa. levels of il8, cd62p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd40l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l . to examine if scd40l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il-6 secretion. the il-6 concentration was consistently below 5-10 pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il-6. the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il-6 production over control (p x 0.05) at d2 after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l (p x 0.05). pre-incubation of b cells with cd40-blocking antibodies substantially abrogated il-6 secretion, unlike isotype-matched control. the partial blocking of cd40 binding on cd40 + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd40l (under investigation) and indicates a sustained role for pc-derived scd40l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd40l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule-1 (dnam-1) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from 30 aml patients before specific anti-leukemia therapy and 47 healthy donors. all results were analyzed statistically using spss version 15. results: aml patients under 65 years showed a significant reduction in the expression of dnam-1, nkp30 and nkp46 compared with age-matched controls. both healthy individuals and aml patients older than 65 years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp44 expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam-1 on nk cells and its ligand cd112 on aml blasts has been found in aml patients under 65 years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam-1 expression on aml patients and confirm previous reports showing a significant decrease of nkp30 and nkp46 on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel 1 , e. gorska 1 , u. demkow 1 , m. wasik 1 1 medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved 10 children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for 2 or 48 hours with or without dexamethasone in concentration 10 -6 m. analysis of: p-gp surface expression, p-gp function (rhodamine 123 test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was 14,52 %±11,32. after 48 hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine 123 efflux, which characterized p-gp activity, was 2,62 %±3,15. after 2 hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine 123 (4,31 %±4,03, p=0,015). average phi in lymphoblasts was 7,78±0,19. acidification of cells incubated 2 hours with dexamethasone was seen in 10-25 % percentage of cells 17) . rest of lymphoblasts showed alkalization (phi -8,00).the percentage of lymphoblasts in early stage of apoptosis after 48 hours incubation with dexamethasone (annexin-v test) was higher than in control cells (19,34 % vs 14,13 %; p=0,01). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd49d/cd29 integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd29 on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd38 and anti-cd29 (clon huts21) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures (18 hours). results: cd29 active form was expressed in the majority of normal and tumoral bm pc from healthy subjects (67.3±6.6, n=9) and mm patients in the early stages of the disease (32.6±7.5, n=17). in these cells, huts21 epitope was clearly upregulated by mn 2+ . in contrast, circulating pc were almost all huts21 negative, and levels did not significantly augment when these cells were exposed to mn 2+ . moreover, not only pb but also bm malignant cells from pcl patients were also huts21 negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index (2.35±0.9 brdu+ cells, n=3) in comparison to pc from mm patients in the first stages of disease (1.3±0.2 brdu+ cells, n=5). these results suggest that the active form of cd29 must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either 10 % fetal bovine serum (fbs) with and without fgf2,or 10 % human platelet lysate (hpl), 5 %hpl, (10 % fbs + 10 % hpl), (10 % fbs + 5 % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day 18). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p53 tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p53 dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: 1) ten cases with fa diagnosed on the basis of dna breakage analysis, 2) ten cases with aaa, and 3) ten normal control cases. the presence of p53 dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p53 dna was demonstrated in bone marrow of 90 % of children with fa, compared to 10 % in children with aaa (p x 0.001), while, no p53 dna was seen in normal control. a positive correlation between dna breakage and presence of p53 dna was seen in bone marrow from fa (p x 0.02, r0.81). the presence of p53 tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord-334511-lx9608vy authors: emwas, abdul-hamid; szczepski, kacper; poulson, benjamin gabriel; chandra, kousik; mckay, ryan t.; dhahri, manel; alahmari, fatimah; jaremko, lukasz; lachowicz, joanna izabela; jaremko, mariusz title: nmr as a “gold standard” method in drug design and discovery date: 2020-10-09 journal: molecules doi: 10.3390/molecules25204597 sha: doc_id: 334511 cord_uid: lx9608vy studying disease models at the molecular level is vital for drug development in order to improve treatment and prevent a wide range of human pathologies. microbial infections are still a major challenge because pathogens rapidly and continually evolve developing drug resistance. cancer cells also change genetically, and current therapeutic techniques may be (or may become) ineffective in many cases. the pathology of many neurological diseases remains an enigma, and the exact etiology and underlying mechanisms are still largely unknown. viral infections spread and develop much more quickly than does the corresponding research needed to prevent and combat these infections; the present and most relevant outbreak of sars-cov-2, which originated in wuhan, china, illustrates the critical and immediate need to improve drug design and development techniques. modern day drug discovery is a time-consuming, expensive process. each new drug takes in excess of 10 years to develop and costs on average more than a billion us dollars. this demonstrates the need of a complete redesign or novel strategies. nuclear magnetic resonance (nmr) has played a critical role in drug discovery ever since its introduction several decades ago. in just three decades, nmr has become a “gold standard” platform technology in medical and pharmacology studies. in this review, we present the major applications of nmr spectroscopy in medical drug discovery and development. the basic concepts, theories, and applications of the most commonly used nmr techniques are presented. we also summarize the advantages and limitations of the primary nmr methods in drug development. the unexpected sars-cov-2/covid-19 outbreak, with over 34 million confirmed cases globally (oct. 2020) and the struggle for survival in the absence of a proven and efficient treatments, emphasizes molecules 2020, 25 the critical need to develop effective, novel, and rapid drug discovery methodologies. even though the pharmaceutical industry works constantly to discover and develop novel drugs, the process is still slow and expensive. the cost of introducing a new drug has increased steadily, with current cost estimates predicting that a future drug will cost in excess of $2.6 billion. the typical development cost is usually spread out over the course of 14 years [1] [2] [3] , making investment even more difficult (i.e., cost recovery delay). this high investment barrier for drug development is a result of numerous testing phases (scheme 1), with each phase requiring a statistically significant number of cases. although there are several other substantial costs to drug development, that discussion of experimental methods to reduce costs is beyond the scope of this review. emphasizes the critical need to develop effective, novel, and rapid drug discovery methodologies. even though the pharmaceutical industry works constantly to discover and develop novel drugs, the process is still slow and expensive. the cost of introducing a new drug has increased steadily, with current cost estimates predicting that a future drug will cost in excess of $2.6 billion. the typical development cost is usually spread out over the course of 14 years [1] [2] [3] , making investment even more difficult (i.e. cost recovery delay). this high investment barrier for drug development is a result of numerous testing phases (scheme 1), with each phase requiring a statistically significant number of cases. although there are several other substantial costs to drug development, that discussion of experimental methods to reduce costs is beyond the scope of this review. the emergence of a pandemic and the emergencies it creates worldwide understandably drive and motivate the rapid development and/or optimization of drugs. however, patient safety and subsequent earned public trust is a primary requirement. drug redirecting/repurposing (scheme 1) is an efficient short-cut method in disease treatment that utilizes existing tools, and combines artificial intelligence, machine learning algorithms, and experimental nmr techniques (i.e. "from bench to bedside"). this process must be relatively rapid and efficient to have any benefit to patients and the health-care system. scheme 1. schematic representation of drug discovery [4] . numerical data were reported from meigs et al. [3] . compared to mass spectrometry and high-performance liquid chromatography (hplc), nuclear magnetic resonance (nmr) is another powerful technique with several unique advantages [5] [6] [7] [8] . nmr is intrinsically quantitative, and it provides several different approaches that are routinely utilized to identify and structurally elucidate molecules of interest [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . in contrast to mass spectrometry, nmr is non-destructive, non-invasive, has extremely high reproducibility permitting in addition to the advantages provided by nmr, it is often used with complementary methods such as x-ray crystallography, hplc, and mass spectrometry [69] . an example of this is found in work by wyss et al. [36] , where they combined x-ray crystallography with nmr fragment-based screening to create the first inhibitor candidate for bace-1 in alzheimer's disease. bace-1 is a membrane-anchored aspartic acid protease and is responsible for the production of amyloid beta peptides in neurons related to the progression of alzheimer's disease [36, 70] . using nmr fragmentbased screening, wyss et al. identified isothiourea as binding to bace-1 and confirmed this observation with the x-ray crystal structure of the complex of a ligand-efficient isothiourea fragment. information obtained from these experiments aided in design optimization, resulting in the selection in addition to the advantages provided by nmr, it is often used with complementary methods such as x-ray crystallography, hplc, and mass spectrometry [69] . an example of this is found in work by wyss et al. [36] , where they combined x-ray crystallography with nmr fragment-based screening to create the first inhibitor candidate for bace-1 in alzheimer's disease. bace-1 is a membrane-anchored aspartic acid protease and is responsible for the production of amyloid beta peptides in neurons related to the progression of alzheimer's disease [36, 70] . using nmr fragment-based screening, wyss et al. identified isothiourea as binding to bace-1 and confirmed this observation with the x-ray crystal structure of the complex of a ligand-efficient isothiourea fragment. information obtained from these experiments aided in design optimization, resulting in the selection of iminopyrimidinones as bace-1 inhibitors [36] . this is a perfect example of using different complementary methods to maximize scientific outcome. however, in order to be efficient, one must know the advantages and disadvantages of each method. one of the major issues regarding nmr is the effective size restriction when measuring targets such as proteins above 40kda. recent progress has extended this mass limit; an example of this is the resolved structure of chaperone secb in complex with unstructured prophoa (pdb id 5jtl) with a total mass of 119kda using nmr [71] . in this review, we present practical guideline to use nmr techniques in drug design studies and provide examples of the successful use of nmr in drug-design. nmr is a versatile tool for studying biomolecules of all kinds and is a unique regarding the biophysical analysis of drugs [72] [73] [74] [75] . the basic feature of nmr lies in the fact that it inductively detects the larmor precession of individual nuclei (i.e., spins) which vary because of different atomic, electronic, and chemical environments (i.e., structural atomic relationships). initially, the sample is placed in a strong, static, and homogeneous magnetic field. because spins contain angular momentum, they exhibit larmor precessions around this static magnetic field. a net magnetization builds up over time as the spin population (represented by different energy levels) is minutely differential in the presence of the magnetic field. these levels are dictated by the spin quantum number and can be roughly thought of as different orientations with respect to the static field. subsequently, induced electromagnetic fields at radiofrequencies (called rf pulses) are applied transverse to the plane of the static magnetic field, and the net or bulk magnetization undergoes an effective rotation. the bulk coherence moves into the transverse plane and the subsequent coherently precessing magnetization vectors induce a detectable alternating voltage in the nmr receiver coil. this tiny alternating voltage is amplified and converted from an analog time domain signal to a frequency reading via fourier transformation. these signals are recorded in response to the induced radio-wave pulses ( figure 2 ) and are representative of the larmor frequencies that are converted into normalized values termed chemical shifts in order to be field independent. molecules 2020, 25, x for peer review 4 of 66 of iminopyrimidinones as bace-1 inhibitors [36] . this is a perfect example of using different complementary methods to maximize scientific outcome. however, in order to be efficient, one must know the advantages and disadvantages of each method. one of the major issues regarding nmr is the effective size restriction when measuring targets such as proteins above 40kda. recent progress has extended this mass limit; an example of this is the resolved structure of chaperone secb in complex with unstructured prophoa (pdb id 5jtl) with a total mass of 119kda using nmr [71] . in this review, we present practical guideline to use nmr techniques in drug design studies and provide examples of the successful use of nmr in drug-design. nmr is a versatile tool for studying biomolecules of all kinds and is a unique regarding the biophysical analysis of drugs [72] [73] [74] [75] . the basic feature of nmr lies in the fact that it inductively detects the larmor precession of individual nuclei (i.e. spins) which vary because of different atomic, electronic, and chemical environments (i.e. structural atomic relationships). initially, the sample is placed in a strong, static, and homogeneous magnetic field. because spins contain angular momentum, they exhibit larmor precessions around this static magnetic field. a net magnetization builds up over time as the spin population (represented by different energy levels) is minutely differential in the presence of the magnetic field. these levels are dictated by the spin quantum number and can be roughly thought of as different orientations with respect to the static field. subsequently, induced electromagnetic fields at radiofrequencies (called rf pulses) are applied transverse to the plane of the static magnetic field, and the net or bulk magnetization undergoes an effective rotation. the bulk coherence moves into the transverse plane and the subsequent coherently precessing magnetization vectors induce a detectable alternating voltage in the nmr receiver coil. this tiny alternating voltage is amplified and converted from an analog time domain signal to a frequency reading via fourier transformation. these signals are recorded in response to the induced radio-wave pulses ( figure 2 ) and are representative of the larmor frequencies that are converted into normalized values termed chemical shifts in order to be field independent. table 1 . h chemical shift assignments is shown as an example. the chemical structure and simulated 1 h-nmr spectrum were created using chemdraw 18.1. this is the final, representative spectroscopic signature of the chemical and magnetic environment of the atom, and it provides detailed atomic resolution information about the molecular structure [76] [77] [78] [79] . a wealth of information can be derived from the nmr signal made up components such as the chemical shift position, signal linewidth, and observed couplings/multiplet structure. the table 1 . h chemical shift assignments is shown as an example. the chemical structure and simulated 1 h-nmr spectrum were created using chemdraw 18.1. this is the final, representative spectroscopic signature of the chemical and magnetic environment of the atom, and it provides detailed atomic resolution information about the molecular structure [76] [77] [78] [79] . a wealth of information can be derived from the nmr signal made up components such as the chemical shift position, signal linewidth, and observed couplings/multiplet structure. the signal contains precise details about the chemical environment of the involved and interacting spins in the structure of the molecule, dynamics of the spins in various timescales, conformational exchange, etc. [80] [81] [82] . any change in the environment of the associated spin can be observed. these changes include molecules 2020, 25, 4597 5 of 63 molecular binding, interactions, and/or exchange between different conformations [20, [83] [84] [85] . thus, nmr has been used to study a wide range of functional molecules such as natural products [86] [87] [88] , saccharides [89, 90] , metabolites [91, 92] , dna [93, 94] , and proteins [95] , and its use as an analytical tool in drug design research has increased immensely in recent years (see figure 1 ). as nmr is non-destructive in nature, the same sample can be analyzed repeatedly. nmr can be performed first and then submitted to mass spectrometry (ms); however, the addition of common deuterated nmr solvents (such as d 2 o) can perturb ms results and should be avoided (e.g., tube-in-tube or by using non-deuterated solvent and running the nmr unlocked). in fact, high-performance liquid chromatography (hplc), ion-trap ms and nmr have been combined to detect the effects of drugs demonstration in urine and blood serum samples [69, 96, 97] . corcoran and spraul [98] emphasize that liquid chromatography (lc), ms, and nmr utilized in parallel give comprehensive structural data on molecules of novel drugs in development. in the following subsections we briefly describe nmr methods that have been used in drug design, and then discuss how nmr principles are used in drug discovery research. the one-dimensional (1d) experiment is by far the most common nmr experiment used for drug studies. the 1d acquisition takes the least amount of time, has one of the simplest hardware requirements, and therefore, in most cases, 1d-nmr is more attractive for high throughput studies. one dimensional nmr spectroscopy normally incorporates a preparation period, some form of induced excitation to form coherence, and lastly, a signal "read" detection period. the preparation period can be modified according to the needs of the experiment or the specifics of the sample. simple 1d nmr is capable of rapidly producing high-quality spectra of drugs and their targets while revealing how the drugs and targets may interact at the atomic level. 1d 1 h-nmr is extremely effective in drug design studies because it has a (relatively) high sensitivity, it is non-destructive, and because hydrogen atoms are extremely abundant in most molecules of interest. therefore the resulting spectra usually contains a large amount of relevant information and this wealth of data can be acquired in a relatively short period of time. the basic 1d 1 h-nmr, along with 1d 13 c-nmr, 1d 15 n-nmr, and 1d 31 p-nmr, and their respective uses in drug design/discovery are briefly discussed below. the 1 h hydrogen isotope is nmr visible, has the highest gyromagnetic ratio (apart from tritium) of all of nmr active nuclei, and is combined with a vast natural abundance in organic chemical compounds. this makes the 1d-1 h-nmr experiment the most commonly applied nmr approach. moreover, many software databases [99] [100] [101] [102] are well established for 1 h-nmr spectra therefore assisting with processing, analyzing, and identifying the detected molecules automatically. since almost all drug discovery and drug development studies are performed on samples dissolved in water, many different solvent suppression methods have been applied. the most common is presaturation [103, 104] . the key point of this method is to use a low power induced field at the specific frequency of water. this effectively averages out any coherence of the water resonance. the experiment is simple for common hardware to perform and easy to set up; however, presaturation has a substantial disadvantage in that signals resonating close to the solvent signal will show decreased intensity [103, 104] or may be lost entirely. this is due to the fact the even selective pulses or very low power pulses also excite some area around the water signal. also suppressed hydrogen from h 2 o in solution can exchange with atoms of interest in the molecule and effectively bleed the suppressive spin state to any neighboring atoms. the water signal itself is usually broad, so a wider area of suppression is not necessarily undesirable but affects more of the molecule(s) of interest. more recent water suppression techniques have been developed such as those based on a scheme known as excitation sculpting [105, 106] . the basic pulse sequence consists of a double pulsed field gradient echo (dpfge) in each of which a selective component pulse is flanked by two pulsed field gradients [107] . the particular elements differ for different applications. in the case of water suppression known as water suppression by gradient tailored excitation (watergate), this involves an initial encoding gradient along with the middle element; a combination of two selective 90 • rotations on the water along with a central non-selective 180 • excitation of all resonances [108] . this is predicated in that water experiences a 360 • rotation (effectively nothing) while all other spins experience 180 • rotation. the application of the second refocusing gradient does not rephase the water and therefore removes the signal. the reader is referred to the detailed literature [103, 104, 109] for further information. in principle, a water suppression element (or many elements combined) can be incorporated in any existing pulse sequence to enhance the performance, and it has been implemented in various 1d, 2d, and triple resonance 3d/4d experiments. although 1 h is the most sensitive nucleus for nmr yielding strong, sharp signals within a few minutes [110] , chemical shift dispersion of 1 h is quite narrow (only around 10 ppm). this has prompted the consideration of other nuclei such as 13 c, 15 n, or 31 p for resolution improvements. compared with 1 h, 13 c has a much higher chemical shift dispersion (~200 ppm), however the natural abundance of 13 c is low (1.1%). additionally, the gyromagnetic ratio is~4 times weaker than 1 h and therefore 13 c spectra are far more difficult to obtain especially for less concentrated samples. there are some polarization transfer techniques such as distortionless enhancement by polarization transfer (dept) or insensitive nuclei enhanced by polarization transfer (inept), which can enhance signal intensity by starting the magnetization on a higher sensitivity and abundance proton and then transferring magnetization to the less sensitive carbon nuclei for subsequent direct detection [111] , but this requires additional hardware and acquisition times. the use of 1d 13 c in drug design studies was illustrated by tsujimoto et al. [112] . the goal of the study was to examine if a metabolomics approach based on 1 h and 13 c offers significant improvements when comparing potential drugs. the authors prepared a total of 40 samples with five different citrus-type crude drugs (kijitsu, tohi, chimpi, kippi and seihi) and measured 1d 1 h and 1d 13 c for each sample. while 1 h-nmr spectra allowed the identification of three compounds (naringin, sucrose, and β-glucose), using 13 c-nmr allowed unambiguous identification of eight additional compounds (naringin, neohesperidin, αand β-glucose, sucrose, limonene, narirutin, and synephrine). the added signal resolution from 13 c-nmr spectra allowed researchers to obtain better structural information about the compounds than from 1 h-nmr spectra alone. in comparison to the previous example, 15 n has a lower shift dispersion (~100ppm) than 13 c, but higher than that of 1 h. here, the situation is unfortunately severely limited due to an even lower natural abundance (0.37%) and a gyromagnetic ratio~10 times smaller than 1 h. this means that 15 n's combined sensitivity is around 260,000 times lower than 1 h. as a result, isotopic enrichment of 15 n combined with 1 h-mediated enhancement via indirect detection is often needed in order to obtain a satisfactory 1d 15 n spectra. similar to 13 c, a few methods are available to overcome such low sensitivity. one of them focuses on tagging molecules with carboxyl groups using 15 n-ethanolamine and later detecting the signal using a 2d heteronuclear correlation nmr experiment [113] . currently, novel approaches such as "smart isotope labeling" have been developed [114] . also, the sofast (band-selective optimized flip angle short transient) technique can help but results in substantial hardware considerations/drawbacks and often increased concentrations, and/or dramatically longer experiments are still required [115] [116] [117] . promising methods are on the horizon. these methods include 15 n heteronuclear signal enhancement via signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (sabre-sheath); however, more work and research are required before such methods can be applied for biomedical purposes [118] . with a natural abundance of 100% and a gyromagnetic ratio of about 2.5 times smaller than 1 h, one may think that phosphorus could be broadly used for nmr experiments regarding the drug discovery and development. however, the application of 31 p is limited due to the fact that most of the molecules of interest simply do not contain a phosphorus atom. therefore 31 p-nmr is usually applicable for studies related to energy, phospholipid metabolism (atp, nadp), and/or characterization of changes in dna [94, 119, 120] . for example, overall et al. conducted an experiment in which they showed that 31 p solid-state nmr can be used for quantitative analysis of dna dynamics within live bacteria [94] . for that, the researchers first prepared untreated cultures of e. coli, and measured them using a hartmann-hahn 1 h to 31 p cross-polarization ( 31 p cp) experiment. afterwards, they measured e. coli treated with ampicillin and maculatin 1.1 (mac1.1) in a similar manner. spectra obtained from treated bacteria compared to those obtained from untreated bacteria showed alterations in the lineshape, reduced signal intensity at the spectrum's edges, and a shift in spectral density towards 0 ppm which indicated the increased dynamics of the phosphorus from nucleic acids [94] . over time, several innovations have been applied to expand the usage of 31 p. like in 13 c and 15 n labeling of specific biological compounds, incorporation of 31 p can also be used. in order to achieve that, 2-chloro-4,4,5,5-tetramethyldioxaphospholane (ctmdp) can be used for tagging lipids containing hydroxyl, aldehyde, and carboxyl groups that can later be detected with better resolution [121] . another fairly recent method enables toxicological screening of 31 p in living cells for several hours without affecting cell viability [122] . this specific method can be used to observe the changes in energy metabolism in real-time while enabling the evaluation of the effects of administered drugs. nmr experiments are not limited to one-dimensional direct acquisition; they can be extended to multidimensional methods including 2d, 3d, 4d, and even higher dimensionality. the focus of this section is common 2d nmr experiments that have been used in drug design and drug development. a brief description of correlation spectroscopy (cosy), total correlation spectroscopy (tocsy), and heteronuclear multiple bond correlation (hmbc), along with their uses in drug design and discovery will be presented. cosy is one of the simplest and most frequently used 2d nmr experiment [123] . it shows the homonuclear coupling of nuclei (i.e., 1 h-1 h) separated by up to several covalent bonds. the pulse sequence consists of a 90 • excitation pulse followed by a specific evolution time (t 1 ), a second pulse, and finally a measurement period (t 2, not to be confused with relaxation rates or times). the second pulse can be 90 • or 45 • or 135 • , depending upon the specific requirements, and respectively yield cosy [124] , cosy-45 or cosy-135 functionality (see [125] [126] [127] ). a two-dimensional fourier transform (ft) yields the final spectra and shows the frequencies for proton ( 1 h) or carbon (in the case of carbon detection) along both axes. there are two types of peaks; (i) diagonal peaks, which represent the peaks of the conventional 1d spectra, and (ii) cross-peaks, which have different values in the two frequency axes and are therefore off the diagonal. these off diagonal cross-peaks are the most important pieces of information as they mark correlations between pairs of nuclei due to through bond magnetization transfer. this helps in identifying which atoms are connected [128] , critical for structural elucidation of both known molecules and unknown molecules in solution [129] . by implementing phase-cycling [130, 131] , it is also possible to distinguish different types of coupling and yields further helpful information about the chemical structure of a molecule [132] . as an example, the use of the cosy experiment was presented in the work of zheng et al. [88] . the main goal of their work was to investigate potential biological differences and compare the pharmacological effects between danggui (an herbal drug used in traditional chinese medicine) and european danggui. the difference between the two experiments is that a tocsy experiment will show multiple cross-peaks including indirectly coupled nuclei (i.e., longer range via scalar coupling) throughout the j-coupled spin system of a chemical compound. the basic pulse sequence of the tocsy consists of excitation by a 90 • pulse, followed by a free variable evolution period which encodes the indirect dimension. this is normally followed by an isotropic mixing sequence to transfer magnetization between spins via the strong scalar coupling. the mixing generates in-phase magnetization throughout a spin coupled network of the associated nuclei during the mixing time. lastly, a direct detection is performed. a major advantage of the tocsy experiment is that it detects in-phase magnetization (i.e., pure absorptive line-shape) which is far easier to analyze compared to the anti-phase information in the phase sensitivity cosy-type experiment. the isotropic mixing is usually performed using a composite pulse scheme such as waltz, mlev or dipsi [133, 134] pulse train, and can be sandwiched between two z-filters [135] where isotropic mixing is performed on the longitudinal magnetization. the most obvious advantage of tocsy is that all cross-peaks of the same spin system can be observed for whole spin system at once. this is useful for identifying the complete network of spins and reducing the ambiguity of any spectral overlap. the tocsy experiment can be produced as 1d with a relatively shorter time and easier analysis compared to 2d but lacks the benefit of multi-dimensional resolution. the 2d tocsy is usually done to resolve spectra overlap [50] when first identifying molecules [136] [137] [138] . for example, jiang et al. used this to predict the response to gemcitabine-carboplatin (gc) chemotherapy in patients with metastatic breast cancer who were previously exposed to treatment with both anthracyclines and taxanes [137] . for that, researchers collected serum samples from 29 patients prior to treatment and measured them using 1d 1 h-nmr. additionally, they conducted 2d nmr experiments such as the 1 h, 1 h-cosy, 1 , 1 h-tocsy, 1 h, 13 c-hsqc, and 1 h, 13 c-hmbc to help assign serum metabolites. after receiving the treatment with gemcitabine-carboplatin, patients were divided into four groups based on the results from the computed tomography: complete response (cr), partial response (pr), stable disease (sd), or progressive disease (pd). after comparing nmr results prior to the treatment with the outcome of chemotherapy, the researchers observed lower baseline levels of serum format and acetate in breast cancer patients who progressed with the disease than in those who achieved a clinical benefit from therapy, indicating that those two biomarkers could be used to distinguish between patients who will benefit from gc treatment from those who do not [137] . 2d-heteronuclear single quantum coherence (hsqc) experiments are commonly used to help resolve spectral overlap [139] while providing 13 c information without the inherent sensitivity losses involved in 13 c direct detection (see below). hsqc shows the correlations between directly coupled nuclei [140] , e.g., 1 h-13 c or 1 h-15 n [140] . as such, an hsqc spectrum will show clean peaks for each unique proton directly connected to the heteronuclear nuclear atom of interest [140, 141] . in 1 h, 13 c/ 15 n hsqc experiments, the magnetization is transferred from the more sensitive nucleus (i: 1 h) to the less sensitive nucleus (s: 13 c/ 15 n) [142] [143] [144] (figure 3 ). this is especially useful when applying nmr spectroscopy to drug design, as most drugs are organic (i.e., contain carbon atoms), and the relative abundance of 13 c (1.1%) is quite low [143] . by transferring sensitivity from 1 h to 13 c, one can circumvent the long experimental time required for 1d 13 c experiments [143] . molecules 2020, 25, x for peer review 9 of 66 abundance of 13 c (1.1%) is quite low [143] . by transferring sensitivity from 1 h to 13 c, one can circumvent the long experimental time required for 1d 13 c experiments [143] . for example, de castro et al. [145] studied ptac2s and its related cytotoxicity to the cisplatinresistant epithelial ovarian carcinoma (eoc), skov-3 cells. in the study, they used nmr spectroscopy and multi-variate statistical analysis to observe how skov-3 cells reacted to treatment with ptac2s. in particular, they used 1 h, 13 c-hsqc along with 1 h-cosy and heteronuclear multiple bond correlation (hmbc), and the human metabolome database to assign the chemical shifts of the lipid metabolites present in the studied samples. interestingly, skov-3 cells treated with ptac2s produced more pyruvate than skov-3 cells treated with cisplatin. the authors also noticed an unexpected difference in lipid metabolite expression levels between the cells treated with ptac2s and those treated with cisplatin. these results provide a possible explanation for how ptac2s is able to overcome cisplatin resistance in skov-3 cells [145] . heteronuclear 2d experiments are useful for transferring magnetization from sensitive nuclei (i.e. 1 h) to less sensitive nuclei (i.e. 13 c) [146] thereby reducing the time needed for the acquisition of spectra [147] . heteronuclear single quantum coherence (hsqc) will only show one cross peak for each coupled pair [92, 128] of nuclei. this makes hsqcs useful for assigning the backbone of proteins [148] and in metabolites of complex biofluids [149] , whose 1d 1 h-nmr spectra can suffer from severe spectral overlap. the hmbc technique, while similar to hsqc, is an example of a heteronuclear 2d experiment that reveals correlations between nuclei separated by two or more chemical bonds while also suppressing one-bond correlations at the same time. this experiment combined with hsqc is often used to assign nmr spectra for studied molecules in drug design experiments [65, 66, 137, 145] . as an example, hmbc was used in a recent study by xu et al. [66] to investigate the changes in the metabolic profiles of rats treated with different dosages of the "renqingmangjue" pill, a traditional tibetan medicine. in this study, the rats were divided into four groups based on the amount of "renqingmangjue" administered: low dose group (ld)-83.33 mg/kg/day, middle dose group (md)-333.33 mg/kg/day, high dose group (hd)-1333.33 mg/kg/day and a control group (nc). after 15 days of consecutive administration, half of the rat population was used to collect samples such as serum, kidney, and liver tissue, while the other half underwent an additional 15 days of recovery before the same samples were acquired. the samples were measured using 1 h-nmr cpmg (an experiment used to suppress signals from larger molecules, see below) [150] [151] [152] along with 1 h, 1 h-cosy, 1 h, 13 c-hsqc, and 1 h, 13 c-hmbc used for molecules assignment. the obtained spectra showed that the "renqingmangjue" pill alters many metabolites, which are related to a variety of metabolic pathways including energy metabolism, amino acid metabolism, and lipid metabolism indicating potentially harmful effects on kidneys and liver. for example, de castro et al. [145] studied ptac2s and its related cytotoxicity to the cisplatin-resistant epithelial ovarian carcinoma (eoc), skov-3 cells. in the study, they used nmr spectroscopy and multi-variate statistical analysis to observe how skov-3 cells reacted to treatment with ptac2s. in particular, they used 1 h, 13 c-hsqc along with 1 h-cosy and heteronuclear multiple bond correlation (hmbc), and the human metabolome database to assign the chemical shifts of the lipid metabolites present in the studied samples. interestingly, skov-3 cells treated with ptac2s produced more pyruvate than skov-3 cells treated with cisplatin. the authors also noticed an unexpected difference in lipid metabolite expression levels between the cells treated with ptac2s and those treated with cisplatin. these results provide a possible explanation for how ptac2s is able to overcome cisplatin resistance in skov-3 cells [145] . heteronuclear 2d experiments are useful for transferring magnetization from sensitive nuclei (i.e., 1 h) to less sensitive nuclei (i.e., 13 c) [146] thereby reducing the time needed for the acquisition of spectra [147] . heteronuclear single quantum coherence (hsqc) will only show one cross peak for each coupled pair [92, 128] of nuclei. this makes hsqcs useful for assigning the backbone of proteins [148] and in metabolites of complex biofluids [149] , whose 1d 1 h-nmr spectra can suffer from severe spectral overlap. the hmbc technique, while similar to hsqc, is an example of a heteronuclear 2d experiment that reveals correlations between nuclei separated by two or more chemical bonds while also suppressing one-bond correlations at the same time. this experiment combined with hsqc is often used to assign nmr spectra for studied molecules in drug design experiments [65, 66, 137, 145] . as an example, hmbc was used in a recent study by xu et al. [66] to investigate the changes in the metabolic profiles of rats treated with different dosages of the "renqingmangjue" pill, a traditional tibetan medicine. in this study, the rats were divided into four groups based on the amount of "renqingmangjue" administered: low dose group (ld)-83.33 mg/kg/day, middle dose group (md)-333.33 mg/kg/day, high dose group (hd)-1333.33 mg/kg/day and a control group (nc). after 15 days of consecutive administration, half of the rat population was used to collect samples such as serum, kidney, and liver tissue, while the other half underwent an additional 15 days of recovery before the same samples were acquired. the samples were measured using 1 h-nmr cpmg (an experiment used to suppress signals from larger molecules, see below) [150] [151] [152] along with 1 h, 1 h-cosy, 1 h, 13 c-hsqc, and 1 h, 13 c-hmbc used for molecules assignment. the obtained spectra showed that the "renqingmangjue" pill alters many metabolites, which are related to a variety of metabolic pathways including energy metabolism, amino acid metabolism, and lipid metabolism indicating potentially harmful effects on kidneys and liver. relaxation in nmr is a phenomenon describing the time dependence involved in signal intensity after an induced rf (radiofrequency) pulse is applied [153] . after application of a 90 • rf pulse, the bulk magnetization will move to the transverse (xy) plane and will gradually return to its original equilibrium position along the longitudinal (z) axis [154] . this process is described in figure 4 , and is termed t 1 relaxation. the details are beyond the scope of the manuscript and interested readers are directed to [155] and references therein. relaxation times for nmr are even more complicated and exist in two categories: t 1 and t 2 . t 1 refers to the rate of longitudinal (or spin-lattice) z-axis relaxation as the system returns to equilibrium. a second component also contributes, i.e., t 2 relaxation and refers to the rate of transverse (or spin-spin) relaxation [154] which occurs in the xy plane. t 2 is independent of the longitudinal relaxation (t 1 ) and represents the loss of coherence in the precessing spins. therefore nmr relaxation spectroscopy can be based on t 1 and/or t 2 [156] , and is collectively referred to as "relaxation edited nmr" [157] . molecules 2020, 25, x for peer review 10 of 66 relaxation in nmr is a phenomenon describing the time dependence involved in signal intensity after an induced rf (radiofrequency) pulse is applied [153] . after application of a 90° rf pulse, the bulk magnetization will move to the transverse (xy) plane and will gradually return to its original equilibrium position along the longitudinal (z) axis [154] . this process is described in figure 4 , and is termed t1 relaxation. the details are beyond the scope of the manuscript and interested readers are directed to [155] and references therein. relaxation times for nmr are even more complicated and exist in two categories: t1 and t2. t1 refers to the rate of longitudinal (or spin-lattice) z-axis relaxation as the system returns to equilibrium. a second component also contributes, i.e. t2 relaxation and refers to the rate of transverse (or spin-spin) relaxation [154] which occurs in the xy plane. t2 is independent of the longitudinal relaxation (t1) and represents the loss of coherence in the precessing spins. therefore nmr relaxation spectroscopy can be based on t1 and/or t2 [156] , and is collectively referred to as "relaxation edited nmr" [157] . over time (ms to seconds, and in extreme cases, minutes [155] ), the bulk magnetization will decrease in the transverse plane, and increase in the longitudinal axis, returning to its original, equilibrium value. (a) represents t1 relaxation and (b) represents t2 relaxation. t1-based methods typically measure and compare the t1 times of the free and bound ligands. a common way to measure the t1 value of a small molecule is the inversion recovery experiment [158, 159] , although other experiments are also available such as ultrafast nmr t1 [160] and saturation inversion recovery [161] . in general, the shorter t1 the relaxation time the less intense the peak signal will be and the broader the signal linewidth [162] . the t1 values of free and bound ligand will differ depending on how strongly the ligand binds because molecular interactions with the target will influence the ligand's molecular motion, and hence, its longitudinal relaxation [156] . bound ligands will have smaller t1 values than in their free form because, overall, they will experience slower molecular motion upon interacting with a target [163] therefore behaving like a much larger molecule. they can (depending on molecule size) also display a negative noe difference spectrum (transferred noe) [164] , whereas non-binding ligands normally show small-positive noes [156] . for binding ligands to display negative noes, their t1 values must be comparatively longer than the 1/koff value of the target [156] . t 1 -based methods typically measure and compare the t 1 times of the free and bound ligands. a common way to measure the t 1 value of a small molecule is the inversion recovery experiment [158, 159] , although other experiments are also available such as ultrafast nmr t 1 [160] and saturation inversion recovery [161] . in general, the shorter t 1 the relaxation time the less intense the peak signal will be and the broader the signal linewidth [162] . the t 1 values of free and bound ligand will differ depending on how strongly the ligand binds because molecular interactions with the target will influence the ligand's molecular motion, and hence, its longitudinal relaxation [156] . bound ligands will have smaller t 1 values than in their free form because, overall, they will experience slower molecular motion upon interacting with a target [163] therefore behaving like a much larger molecule. they can (depending on molecule size) also display a negative noe difference spectrum (transferred noe) [164] , whereas non-binding ligands normally show small-positive noes [156] . for binding ligands to display negative noes, their t 1 values must be comparatively longer than the 1/k off value of the target [156] . t 1 relaxation times can be easily used to screen small molecules as ligands for dna [165] and serve as a basis for hts experiments [166] . an experiment related to drug design that utilized 1d and 2d relaxation edited nmr was done by hajduk et al. [167] in which he and others used 1d and 2d relaxation edited nmr techniques to detect ligands that bind to fk506 binding protein and stromelysin. one year earlier, liu et al. [157] used relaxation edited one-and two-dimensional 1 h-nmr spectroscopy to characterize biological fluids. tang et al. [168] extended this by applying relaxation edited nmr spectroscopy to improve the detection of metabolites in blood plasma. more recently, jaremko et al. commented on available models used to interpret 15 n protein relaxation data [169] , and even used deficient 15 n relaxation data to rapidly calculate the dynamics of proteins [170] . the t 2 relaxation experiment relies on so-called carr-purcell-meiboom-gill (cpmg) building blocks ( figure 5 ). molecules 2020, 25, x for peer review 11 of 66 t1 relaxation times can be easily used to screen small molecules as ligands for dna [165] and serve as a basis for hts experiments [166] . an experiment related to drug design that utilized 1d and 2d relaxation edited nmr was done by hajduk et al. [167] in which he and others used 1d and 2d relaxation edited nmr techniques to detect ligands that bind to fk506 binding protein and stromelysin. one year earlier, liu et al. [157] used relaxation edited one-and two-dimensional 1 h-nmr spectroscopy to characterize biological fluids. tang et al. [168] extended this by applying relaxation edited nmr spectroscopy to improve the detection of metabolites in blood plasma. more recently, jaremko et al. commented on available models used to interpret 15 n protein relaxation data [169] , and even used deficient 15 n relaxation data to rapidly calculate the dynamics of proteins [170] . the t2 relaxation experiment relies on so-called carr-purcell-meiboom-gill (cpmg) building blocks ( figure 5 ). figure 5 . cpmg pulse sequence. first, a 90° rf pulse is applied and results in transverse magnetization in the xy plane. then a 180°y pulse is applied to re-phase the magnetization vectors. after 180°y, the vectors who were faster during the dephasing are overtaken by the slower vectors, which results in re-phasing and generation of a spin-echo signal. this process is repeated several times. this pulse sequence is explained with the following steps: first, application of a 90° rf pulse creates a transverse (xy plane) magnetization. second, a spin-echo period (delay-180°-delay block) is responsible for mx/y magnetization decay. this period is repeated "n'' times (cpmg building blocks). it is essential to point out that every nmr experiment involving a large number of pulses (e. g. due to the repeating building blocks) is likely to be sensitive to hardware restrictions and small miscalibrations of the duration of the applied pulses. to attenuate the unwanted effects of miscalibrations, meiboom and gill modified the previously used carr-purcell sequence [171] by changing the phase of the applied 180° pulses from x to y [172] . this procedure can be used to measure t2 relaxation times of any type of nuclei. for instance, in the case of 13 c, all pulses and acquisitions are applied on 13 c channel, while broadband proton decoupling is applied during all pulse sequences. it works analogically for different nmr-active nuclei [173] . in a typical cpmg experiment, the effective transverse relaxation rate, r2,eff, is typically measured by fitting the signal decay as a function of a variable number of cpmg blocks [174] . the experimental half-height linewidth (d) of a given resonance signal is directly related to t2 * (also called as 'effective' or "observed'') by the following equation: (1) figure 5 . cpmg pulse sequence. first, a 90 • rf pulse is applied and results in transverse magnetization in the xy plane. then a 180 • y pulse is applied to re-phase the magnetization vectors. after 180 • y , the vectors who were faster during the dephasing are overtaken by the slower vectors, which results in re-phasing and generation of a spin-echo signal. this process is repeated several times. this pulse sequence is explained with the following steps: first, application of a 90 • rf pulse creates a transverse (xy plane) magnetization. second, a spin-echo period (delay-180 • -delay block) is responsible for mx/y magnetization decay. this period is repeated "n" times (cpmg building blocks). it is essential to point out that every nmr experiment involving a large number of pulses (e. g. due to the repeating building blocks) is likely to be sensitive to hardware restrictions and small miscalibrations of the duration of the applied pulses. to attenuate the unwanted effects of miscalibrations, meiboom and gill modified the previously used carr-purcell sequence [171] by changing the phase of the applied 180 • pulses from x to y [172] . this procedure can be used to measure t 2 relaxation times of any type of nuclei. for instance, in the case of 13 c, all pulses and acquisitions are applied on 13 c channel, while broadband proton decoupling is applied during all pulse sequences. it works analogically for different nmr-active nuclei [173] . in a typical cpmg experiment, the effective transverse relaxation rate, r 2,eff , is typically measured by fitting the signal decay as a function of a variable number of cpmg blocks [174] . the experimental half-height linewidth (d) of a given resonance signal is directly related to t 2 * (also called as 'effective' or "observed") by the following equation: t 2 represents the transverse relaxation times, and additional broadening comes from the magnetic field inhomogeneities (t 2 inh ), which must be taken into account. t 2 measurements of ligands are also useful for determining the binding nature of a small molecule. the t 2 values of small molecules are quite large compared to those of bigger molecules (i.e., proteins) mostly because macromolecules have more spin-spin diffusion [175] . bound ligands will, therefore, display shorter t 2 values than non-binding ligands because they interact with the target (i.e., protein), adopting similar vibrational and rotational energies to the target [176] . this interaction is represented by the resonance line broadening in the binding ligand's spectrum when a receptor is introduced into the sample [156] . given the sizable difference of t 2 values of binding and non-binding ligands, one can utilize 1d relaxation-edited experiments to distinguish the binding ligands from the non-binding ligands efficiently and effectively based on the differences in the t 2 values [167] . these and other related relaxation edited experiments prove useful in drug design. relaxation edited nmr spectroscopy takes advantage of an inherent atomic property (i.e., the return of bulk magnetization back to equilibrium [177] ), so no molecular enrichment (e.g., 15 n isotopic enrichment of protein targets) is required [167] . furthermore, the slow time scale of nmr relaxation allows the user to manipulate the external conditions (i.e., length and power of pulse) to increase the resolution of targets and potential drugs [155] in nmr drug design experiments. however, this slow timescale also sets the lower limit at which nmr drug design experiments can be performed [155] , meaning that any external manipulations cannot decrease experimental time below a certain threshold. this varies based on the drugs and targets used in the experiment. low drug solubility is also a challenge, as the ligands must be at a sufficiently high concentration to allow detection via nmr, although the use of organic solvents has helped to attenuate this effect in relaxation edited nmr spectroscopy [156] . for examples of experiments that use different nmr techniques mentioned above, see table 1 . emodin can affect the immune response and interrupt energy metabolism (citric acid cycle) along with glutathione synthesis, which can lead to oxidative stress. dox decreases atp production and induces oxidative stress in h9c2 cells. dex counteracts those changes, having a cardioprotective effect on h9c2 cell lines. [67] curcumin shows antihyperlipidemic effects on c57bl/6slac mice by partially restoring metabolic defects induced by a high-fat diet. affected metabolic pathways include the citric acid cycle, glycolysis and gluconeogenesis, ketogenesis of bcaa, synthesis of ketone bodies and cholesterol, and choline and fatty acid metabolism. formosanin c shows the ability to inhibit synthesis and methylation of dna as well as reducing the activity of the citric acid cycle and energy metabolism in the mitochondria of hepg2 cells. melamine melamine disrupts metabolism of glucose, nitrogen, and protein in the liver of wistar rat. 1 h-nmr cpmg [182] aristolochic acid (aa) aristolochic acid causes renal lesions and a disorder in tubular reabsorption in wistar rats. 1 evaluating response outcome for patients with rheumatoid arthritis, treated with rituximab. identification of metabolites changes between responders and non-responders such as succinate, taurine, lactate, pyruvate and aspartate. no distinction between metabolite profiles of serum from patients treated with levetiracetam, lamotrigine and topiramate. could not evaluate response of initial treatment of epilepsy. [184] metallaprism mainly affects lipid metabolism in a2780 (human ovarian cancer) and hek-293 (human embryonic kidney) cells, and increases gsh levels in all cell lines. in a2780cisr (cisplatin resistant a2780) cells, lipid biogenesis and glycosylation are affected by treatment with metallaprism. extract from centella asiatica promotes glycolysis, boosts the citric acid cycle and decreases gluconeogenesis and lipid metabolism in t2dm sprague-dawley rats. vr24 and vr27 improve glycerol metabolism, decrease betaine levels, and normalize the altered level of myoinositol in serum of dmh-induced crc (colorectal cancer) wistar rats. evaluating the efficacy (determined by the concentration of a drug able to reach the therapeutic site) of four drugs in cerebrospinal fluid of tuberculous meningitis patients. genipin can recover energy metabolism to normal levels, and regulate methylamine and amino acid metabolisms of diabetic sprague dawley rats. 1 h-noesy-1d [190] adriamycin (adr) identification of seven biomarkers: trimethylamine oxide (tmao), taurine, trimethylamine (tma), hippurate, trigonelline, citrate and 2-oxoglutarate that can predict tumor's (gastric adenocarcinoma) response to adr treatment in balb/c-nu/nu mice. 1 h-noesypresat-1d [191] danggui/european danggui fuzi causes a shift in energy metabolism (from aerobic respiration to anaerobic), induces membrane toxicity, and disrupts the balance of gut microbiota of wistar rats. administrating fuzi with gancao diminishes the toxic effects of fuzi. 1 h-nmr spectra enabled the identification of three compounds (naringin, sucrose, and β-glucose), and 13 c-nmr enabled the identification of eight compounds (naringin, neohesperidin, αand β-glucose, sucrose, limonene, narirutin, and synephrine). as stated, nmr spectroscopy can be fundamental in studying how drugs interact with their targets. this has been done mainly via the fragment based drug design (fbdd) approach, which has two sub-approaches: target-(i.e., protein) based, or ligand-(drug) based. target based screening monitors how the target responds to binding molecules in a method called structure activity relationship ("sar") by nmr. ligand (drug)-based screening methods provide ways to observe the binding/non-binding behavior of the drug in approaches such as saturation transfer difference (std) and other nuclear overhauser effect (noe) type methods, diffusion-based methods, relaxation-based methods (i.e., t 1 and t 2 ). target based screening, ligand (drug) based screening, and their respective methods, are discussed in detail below. nmr-based drug discovery can be broadly classified into two groups: chemical and biological (in-cell) categories. one of the principal methods of drug discovery using nmr spectroscopy is called fragment-based drug design (fbdd) [194] . in-cell nmr (biological) based drug discovery techniques will be discussed later in this review. fbdd was first reported in 1996 [195] and used throughout the late 1990s as evidenced by the use of keywords related to fbdd in papers published during this time [196] . the use of fbdd as a viable drug screening technique began to be widely adopted in the mid-2000s [197] . high throughput screening (hts) is another technique widely used in drug discovery [198] . hts analyzes molecules from a chemical library to see which ones are suitable leads [198] [199] [200] [201] (see figure 6) . fbdd techniques will screen against a carefully designed fragment library composed of a few thousand molecules (for details on the choice of compounds and design of fragment libraries, see [202, 203] ) and identified hits are further developed via fragment growing, fragment merging, or fragment linking [194] . for examples of drugs derived from the fbdd approach that are currently in clinical trials, refer to table 2 . at the time of writing, and to the best of our knowledge, there are three food and drug administration (fda)-approved drugs derived from the fbdd approach [221] , and over 30 are in clinical trials [222] . the first marketed drug derived via the fbdd approach is vemurafenib [223] . vemurafenib is also the first drug approved for treatment of braf-mutant cancer [224] , and is reported to exhibit significant clinical benefit for patients with metastatic melanoma [224] . venetoclax, a common drug used to treat patients with chronic lymphocytic leukemia [225] , is considered the second drug to be discovered using the fbdd approach [221] , and ribociclib, a cdk4 inhibitor, the third [221] . the names, structures, targets/applications, and clinical status of vemurafenib, venetoclax, ribociclib, and other drugs are listed in table 2 . hts has been productive in drug design [204, 205] , but the method is time and resource intensive [206] and expensive [206] because of the numerous molecules to be examined (~100 million) [207] . furthermore, the success rate is only estimated to be at~50% [204, 208] . unlike traditional hts, which can survey a large number of molecules ranging from a few hundred thousand to a few million [209] , fbdd usually surveys a few thousand molecules (~1000-15000) from libraries with greater chemical diversity [209, 210] . fbdd is a main-stream screening technique for drug discovery [207, 209, [211] [212] [213] [214] [215] [216] and nmr is standard for many fbdd studies [209] . additional methods and techniques such as spr, x-ray crystallography [209, [217] [218] [219] [220] etc. have also been used in fbdd studies, accompanied or unaccompanied by nmr experiments. for examples of fbdd derived drugs using methods besides nmr, refer to table 2 . at the time of writing, and to the best of our knowledge, there are three food and drug administration (fda)-approved drugs derived from the fbdd approach [221] , and over 30 are in clinical trials [222] . the first marketed drug derived via the fbdd approach is vemurafenib [223] . vemurafenib is also the first drug approved for treatment of braf-mutant cancer [224] , and is reported to exhibit significant clinical benefit for patients with metastatic melanoma [224] . venetoclax, a common drug used to treat patients with chronic lymphocytic leukemia [225] , is considered the second drug to be discovered using the fbdd approach [221] , and ribociclib, a cdk4 inhibitor, the third [221] . the names, structures, targets/applications, and clinical status of vemurafenib, venetoclax, ribociclib, and other drugs are listed in table 2 . nmr, x-ray crystallography, inhibition of cathepsin d as mentioned, nmr spectroscopy can be used in fbdd in two different ways: (1) target (or receptor) based screening, and (2) ligand-based screening. with the stated advantages and disadvantages, researchers must select based on their available compounds. target based screening typically utilizes the "sar by nmr" (structure-activity-relationship by nuclear magnetic resonance) approach [246] . sar is primarily used to identify and develop extremely tight-binding ligands [247] . the ligand to target binding is traditionally monitored via chemical shift changes [247] using a correlation spectroscopy such as 1 h-15 n hsqc starting with the target and no ligand present [248] . multiple spectra for the target are recorded in the presence and absence of ligands. the binding ligand will cause chemical shift perturbations in the target, and these perturbations are often easily visualized by overlaying the two spectra [247] . for example hajduk et al. investigated the binding interactions of 2-phenylimidazole with the fkbp protein as shown in figure 7 [249] . disadvantages, researchers must select based on their available compounds. target based screening typically utilizes the "sar by nmr" (structure-activity-relationship by nuclear magnetic resonance) approach [246] . sar is primarily used to identify and develop extremely tight-binding ligands [247] . the ligand to target binding is traditionally monitored via chemical shift changes [247] using a correlation spectroscopy such as 1 h-15 n hsqc starting with the target and no ligand present [248] . multiple spectra for the target are recorded in the presence and absence of ligands. the binding ligand will cause chemical shift perturbations in the target, and these perturbations are often easily visualized by overlaying the two spectra [247] . for example hajduk et al. investigated the binding interactions of 2-phenylimidazole with the fkbp protein as shown in figure 7 [249] . from the overlaid spectra, chemical shift changes are measured, and from the molecular location, extent, and rate of the chemical shift changes, the binding site and affinity of the ligand is calculated [250] . then, by following a procedure completely analogous to that of fbdd (see figure 6 ), a ligand developed from multiple fragments can be optimized for the binding site of interest, again by monitoring the changes in chemical shifts of the target. several examples of the successful applications of sar by nmr in drug design research are replete in the scientific literature [204, 251, 252] . sar by nmr spectroscopy allows researchers to observe directly ligand binding [247] in both solution state and solid-state spectra [253] , increasing the method's versatility [254] . it works particularly well for targeting proteins with adjacent "subpocket" binding sites [248] . furthermore, sar by nmr is cost-effective when combined with hts (high throughput screening) [255] . sar by nmr can also be used even when atomic peak assignments in spectra are unknown, though it is much more powerful when the resonance frequency of each atom is known [254] . the main limitation of sar by nmr, however, is its inability to distinguish between multiple binding modes (i.e. cleavage from the overlaid spectra, chemical shift changes are measured, and from the molecular location, extent, and rate of the chemical shift changes, the binding site and affinity of the ligand is calculated [250] . then, by following a procedure completely analogous to that of fbdd (see figure 6 ), a ligand developed from multiple fragments can be optimized for the binding site of interest, again by monitoring the changes in chemical shifts of the target. several examples of the successful applications of sar by nmr in drug design research are replete in the scientific literature [204, 251, 252] . sar by nmr spectroscopy allows researchers to observe directly ligand binding [247] in both solution state and solid-state spectra [253] , increasing the method's versatility [254] . it works particularly well for targeting proteins with adjacent "subpocket" binding sites [248] . furthermore, sar by nmr is cost-effective when combined with hts (high throughput screening) [255] . sar by nmr can also be used even when atomic peak assignments in spectra are unknown, though it is much more powerful when the resonance frequency of each atom is known [254] . the main limitation of sar by nmr, however, is its inability to distinguish between multiple binding modes (i.e., cleavage of covalent bonds or allosteric changes), and if multiple binding modes are present, it can be difficult to pinpoint the "true" binding site of the ligand solely using data obtained using sar by nmr [254] . ligand-based screening, the second approach of nmr in fbdd, has three main categories: 1) saturation transfer difference (std) and nuclear overhauser effect (noe) type methods, based on 2) diffusion methods, or 3) relaxation-based methods (i.e., t 1 and t 2 ). saturation transfer difference (std) nmr depends on the nuclear overhauser effect (noe), which is often used to enhance the sensitivity of less sensitive nuclei such as 13 c and 15 n [256,257] . this increase in sensitivity is possible because of dipolar coupling (i.e., through space interactions of separate nuclei) [257] . the increase in sensitivity is actually brought about by applying a long, low power radiofrequency pulse that selectively saturates the magnetization [256] of a specific chemical group (i.e., the methyl groups on a protein), which is then given time to transfer to another chemical group via the noe dipolar coupling within a few angstroms [258] . the transfer in magnetization is easily visualized on a nmr spectrum that takes the differences in the signal intensities from before and after the irradiation. this new spectrum is called a "difference spectrum", and it reveals what chemical groups interact with the irradiated signal [259] (see figure 8 ). of covalent bonds or allosteric changes), and if multiple binding modes are present, it can be difficult to pinpoint the "true" binding site of the ligand solely using data obtained using sar by nmr [254] . ligand-based screening, the second approach of nmr in fbdd, has three main categories: 1) saturation transfer difference (std) and nuclear overhauser effect (noe) type methods, based on 2) diffusion methods, or 3) relaxation-based methods (i.e. t1 and t2). saturation transfer difference (std) nmr depends on the nuclear overhauser effect (noe), which is often used to enhance the sensitivity of less sensitive nuclei such as 13 c and 15 n [256,257] . this increase in sensitivity is possible because of dipolar coupling (i.e. through space interactions of separate nuclei) [257] . the increase in sensitivity is actually brought about by applying a long, low power radiofrequency pulse that selectively saturates the magnetization [256] of a specific chemical group (i.e. the methyl groups on a protein), which is then given time to transfer to another chemical group via the noe dipolar coupling within a few angstroms [258] . the transfer in magnetization is easily visualized on a nmr spectrum that takes the differences in the signal intensities from before and after the irradiation. this new spectrum is called a "difference spectrum", and it reveals what chemical groups interact with the irradiated signal [259] (see figure 8 ). std nmr is an application of noe used to probe the binding of ligands to a specific site within the targeted proteins [256] . a generic example of detecting ligand binding via std is presented in figure 9a . the std nmr method follows the same concepts as a normal noe experiment: a spectrum of the ligand in the free, non-binding form is recorded, the ligand is allowed to bind to the protein, which has a functional group of interest (i.e. methyls) with a saturated signal from a previous selective radiofrequency pulse. the saturated signal travels to the ligand, increasing the intensity of a signal on the ligand spectrum and finally a difference spectrum is used to determine precisely which sections of the ligands bind. the difference in peak intensities proves the presence of ligand binding [260] . water-ligand observed through gradient spectroscopy (waterlogsy) is a second type of std (see figure 9b ). the main difference with normal std nmr is that water is the saturated signal [261] , and instead of observing lower peak intensities, peak inversions indicate the presence of ligand binding [209] . std nmr is an application of noe used to probe the binding of ligands to a specific site within the targeted proteins [256] . a generic example of detecting ligand binding via std is presented in figure 9a . the std nmr method follows the same concepts as a normal noe experiment: a spectrum of the ligand in the free, non-binding form is recorded, the ligand is allowed to bind to the protein, which has a functional group of interest (i.e., methyls) with a saturated signal from a previous selective radiofrequency pulse. the saturated signal travels to the ligand, increasing the intensity of a signal on the ligand spectrum and finally a difference spectrum is used to determine precisely which sections of the ligands bind. the difference in peak intensities proves the presence of ligand binding [260] . water-ligand observed through gradient spectroscopy (waterlogsy) is a second type of std (see figure 9b ). the main difference with normal std nmr is that water is the saturated signal [261] , and instead of observing lower peak intensities, peak inversions indicate the presence of ligand binding [209] . for std nmr to work properly, the ligand concentration must be in large excess (often 100-1000 fold) over the receptor so that effective saturation transfer can take place [260] . this means that for std nmr, and waterlogsy, only small amounts (µg) of protein are required to get results [261] [262] [263] . this is advantageous for researchers, as they can perform std nmr on a protein of interest, and preserve the rest of the unused sample for future/other experiments. also, the same sample can be used for multiple nmr measurements. std nmr facilitates the differentiation of binding ligands from non-binding ligands because the change in signal (as determined by the difference spectrum) is easy to measure and observe, as shown in figure 9 . waterlogsy has been extended to study ligand interactions with dna and rna [261] . for std nmr to work properly, the ligand concentration must be in large excess (often 100-1000 fold) over the receptor so that effective saturation transfer can take place [260] . this means that for std nmr, and waterlogsy, only small amounts (µ g) of protein are required to get results [261] [262] [263] . this is advantageous for researchers, as they can perform std nmr on a protein of interest, and preserve the rest of the unused sample for future/other experiments. also, the same sample can be used for multiple nmr measurements. std nmr facilitates the differentiation of binding ligands from non-binding ligands because the change in signal (as determined by the difference spectrum) is easy to measure and observe, as shown in figure 9 . waterlogsy has been extended to study ligand interactions with dna and rna [261] . there are additional noe-type experiments (trnoe, inpharma, salmon, etc.) used for drug design, and specific details regarding individual methods are found in the scientific literature [264] . with the pressing search for new antiviral drugs, any techniques for identifying and characterizing novel leads has become increasingly important. benie et al. [265] described the use of saturation transfer difference (std) nmr spectroscopy [262, [266] [267] [268] [269] [270] [271] to identify and characterize the binding of an antiviral compound to native human rhinovirus serotype 2 (hrv2). the experiments demonstrated that it is possible to subject targets of the size and complexity of whole viruses (for a model of an hrv2 particle cut open, cf. the table of contents) to std nmr experiments. the principles of std nmr have been known for many years [267, 268] but it was only recently that the potential of this technique for screening libraries for compounds with binding activity toward protein receptors has been realized [262, 266] . the technique also permitted the analysis of epitopes of ligands bound to receptor proteins. previous nmr studies of virus-ligand interactions used chemical shift titrations, which required very large quantities of the virus. this approach was unworkable when studying pathogenic viruses. benie et al. [265] demonstrated that solution state std methodology not only reduces the amount of virus required by approximately 2 orders of magnitude, but also allows for the identification and characterization of virus-ligand interactions with atomic resolution [272] . there are additional noe-type experiments (trnoe, inpharma, salmon, etc.) used for drug design, and specific details regarding individual methods are found in the scientific literature [264] . with the pressing search for new antiviral drugs, any techniques for identifying and characterizing novel leads has become increasingly important. benie et al. [265] described the use of saturation transfer difference (std) nmr spectroscopy [262, [266] [267] [268] [269] [270] [271] to identify and characterize the binding of an antiviral compound to native human rhinovirus serotype 2 (hrv2). the experiments demonstrated that it is possible to subject targets of the size and complexity of whole viruses (for a model of an hrv2 particle cut open, cf. the table of contents) to std nmr experiments. the principles of std nmr have been known for many years [267, 268] but it was only recently that the potential of this technique for screening libraries for compounds with binding activity toward protein receptors has been realized [262, 266] . the technique also permitted the analysis of epitopes of ligands bound to receptor proteins. previous nmr studies of virus-ligand interactions used chemical shift titrations, which required very large quantities of the virus. this approach was unworkable when studying pathogenic viruses. benie et al. [265] demonstrated that solution state std methodology not only reduces the amount of virus required by approximately 2 orders of magnitude, but also allows for the identification and characterization of virus-ligand interactions with atomic resolution [272] . the very large size of viruses makes them particularly attractive for studies by std nmr, as they inherently yield large line widths allowing for easy irradiation of the virus without affecting the ligand protons. furthermore, because of the larger correlation time of a virus in comparison to an average-sized protein, spin diffusion, and thus saturation transfer, is very efficient. the large line width has additional benefits not just for std-based nmr methods but also for transfer noesy spectra, as protons from the virus capsid are invisible in the nmr spectra (for an example of a transfer noesy spectrum, see [265] ). moreover, competitive std titration experiments can be used to determine the k d value of a ligand [271] . analysis of the std spectra using the group epitope mapping method [271] allows for the determination of the binding epitope. std nmr methods can considerably speed up the determination of the binding epitope for potential antiviral lead compounds. simple std nmr experiments provide substantial information on the binding of ligands to native viruses and require very small amounts of the virus with measurement times in the range of tens of minutes. this allows for a high throughput of ligand samples without significant consumption of viral material because it remains unaffected by the experiments and is easily separated from the low molecular weight ligands by ultra-filtration subsequently. in addition to the detection of binding, a complete mapping of the ligand-binding epitope can be achieved [265] . noroviruses (nv) are non-enveloped, single-stranded, positive-sense rna viruses that are the major cause of epidemic outbreaks of gastroenteritis worldwide [273] [274] [275] . the viral coat consists of a single protein, vp1, which assembles into a capsid with overall icosahedral symmetry [276] [277] [278] . attachment of human noroviruses to histo-blood group antigens (hbgas) is thought to be critical for the infection process [279] . the protruding domains of the vp1 proteins, called p-domains, harbor highly conserved binding sites for hbgas. std nmr-based epitope mapping was used [262, 271] to identify structural features of different core types critical for the binding of synthetic a-and b-tetrasaccharides [280] to virus-like particles (vlps) of a highly homologous gii.4 strain (ast6139). std nmr experiments provide a robust and straightforward technique for obtaining ligand binding epitopes at atomic resolution. comparing binding epitopes of related ligands then delivers critical information about structural requirements for ligand recognition. conversely, comparison of binding epitopes of a given ligand binding to wild type, and to mutant proteins reveals the importance of individual amino acids for binding. std nmr experiments with l-fuc and b-trisaccharide in the presence of wild type and mutant vlps yield virtually identical binding epitopes and suggest that these two mutations do not significantly alter hbga recognition. the std nmr approach to characterize binding of hbga ligands to noroviruses has employed vlps as targets and thus taken advantage of the large size of vlps yielding excellent signal-to-noise ratios of the corresponding std nmr spectra, as demonstrated previously [281] . the application of the transferred noe (tr-noe) effect was first demonstrated by bothner-by [282] . the tr-noe is the nuclear overhauser effect between ligand spins, which are in chemical exchange between the bound and unbound form with the protein or receptor. ligands, which are a mixture of target molecules, are small in size (below 500-1000 da). since they are usually low molecular weight molecules, they exhibit much shorter correlation times when compared to the receptor and have slow noe build-ups with no spin diffusion. this is the reason they show small positive noes in the free form. when binding to a protein receptor, the situation changes, where the ligand acquires large correlation times in the bound state with rapid noe build-up. then they show spin diffusion and a strong negative noe, which is termed the transferred noe. signals arising from the protein are usually not observed for large proteins as they are generally kept low in concentration, with ligands in a high excess concentration. in addition, most of the time protein signals are suppressed by their very short t 2 period. it is worthwhile to mention that ligands that are in fast exchange between the bound and the free form (dissociation constants ranging from µm to mm) get enough bound time to transfer the negative noe from the protein complex to the population of the free molecules, yet usually retain the chemical shift of the free molecule along with the relaxation characteristics. in order to observe tr-noes, the following condition have to be fulfilled: where n and ∂ represent the number of molecules and the cross-relaxation rate, respectively. the subscript b and f represent the bound and free form, respectively. therefore, to observe the tr-noes, a high excess concentration of ligands over protein is maintained. on the other hand, if the ligand concentration is kept too high, the excess free ligand in solution will exhibit positive noe, which can result in a significant reduction of the tr-noesy enhancements due to negative noe developed by the very small concentration of bound ligand. hence, the preparation of the sample becomes tricky and an optimum ratio between 10-30 to 1 is maintained while considering the dissociation constant values. the binding of a ligand to a receptor protein can easily be identified by observing the sign and size of the noes. there are some distinct experimental features for the discrimination between tr-noes from the bound state and noes of the ligand in free states like the build-up rate. for tr-noes, this is in the range of 50 to 100 ms, whereas for small ligands it is much longer. there have been various instances of experimental implementations to quickly determine the binding activity of ligand libraries. one example was to find the ligand molecule among a library of 10 similar structure polysaccharides that is bioactive in binding with recombinant e-selectin [283] . this is a protein present in an igg chimera with a molecular weight of about 220 kda. in this case, two 2d noesy spectra were recorded. the noesy spectra for the ligand library was measured at several temperatures and it was found that most of the 10 compounds exhibited the weak positive noes at 310 k, which was then chosen to differentiate between trnoes showing large negative values. the trnoesy spectra of the ligand library in the presence of protein was recorded at different ratios, such as 5:1, 8:1, 12:1, 15:1, and 20:1, at 310 k. in all the ratios, trnoes were observed; however, the ratio of 15:1 represented the best-case scenario. the inpharma method (see figure 10 ) was designed to determine the relative orientation between two competitive ligands in the receptor-binding pocket through the observation of inter-ligand noe between the two ligands. it is a tr-noe in nature as it is mediated by the bound conformation of the competing ligands and in exchange with the receptor protein. the first example was competitive binding and observation of inter-ligand noe between baccatin iii and epothilone a in the presence of tubulin, which acts as a receptor [284] . since the observation is on the ligand site, it provides unique advantages. the detailed conformation of a ligand-protein complex can be addressed by conventional nmr. however, it is time-consuming and demands full solving of the structure and there is also a size limitation. from that aspect, ligand-based methods are more useful. the only limiting fact is that it should fulfill all the conditions of tr-noe explained previously in terms of dissociation constant (k d ), fast exchange regime, and proper ligand to protein ratio. then, information on the ligand structure can be derived from tr-noe build up as a function of mixing time. this can be readily explained using the originally proposed schematics [284] . the noesy spectrum of a mixture of the two ligands a and b in the presence of the common receptor (t) is recorded. under the situation that each of a and b exhibit competitive binding in a fast exchange regime with the receptor t, intermolecular tr-noe peaks between the two ligands a and b can then be observed in the noesy spectrum due to extensive spin diffusion. during the noesy mixing time, the first proton of ligand a (h a ) binds to receptor t, which results in transfers of magnetization from h a to h t . subsequently, the complex at dissociates as they fulfill the dissociation constant range, which creates the opportunity for ligand b to bind to the receptor t at the same binding site. this results in the transfer of the magnetization of h t , which had been originally coming from h a , to h b . as a result, an inter-molecular correlation h a -h b can be seen, and this inter-molecular noe will be a function of mixing time as described above. the detailed analysis of such intermolecular noe peaks helps in assessing the relative orientation of each ligand in the binding pocket. diffusion is the random, translational motion of molecules in solution as a consequence of their thermal energy [285] . this type of motion is often referred to as "brownian motion", a motion that describes molecular movement induced by random collisions between the molecules [286] . in the presence of a concentration gradient, molecules will naturally move from places of higher concentration to places of lower concentration [287] after a period of time, t, as shown in figure 11 . fick's law can be used to model this type of movement [288] . the distribution of the diffusing molecules is accurately represented by a gaussian curve, a normal distribution centered at a single point, which gradually "flattens" as t approaches infinity [213] . the extent to which a molecule diffuses is directly related to its shape, size, and mass [285] . in homogeneous isotropic solutions, the root mean square distance (zrms) traveled by a molecule is given by following equation [289, 290] : where d is the diffusion coefficient of the molecule, and t is the diffusion time. making the assumption that the molecules are solid rigid spheres, the value of d can be calculated according to the famous einstein-stokes equation (equation (2)): where kb is the boltzmann's constant (1.3807 × 10 −23 j/k), t is the absolute temperature, η is the solution viscosity, and rs is the hydrodynamic radius of the molecule [290] . equation (1) and equation (2), however, are not universally applicable; they only apply to molecules that are freely diffusing in isotropic, homogeneous solutions, and importantly that can be accurately described as hard, rigid spheres [285] . different molecular geometries and additional modes of diffusion (i.e. restricted and anisotropic) require more advanced mathematics and theory [291, 292] , but the essential concepts of diffusion remain the same. diffusion is the random, translational motion of molecules in solution as a consequence of their thermal energy [285] . this type of motion is often referred to as "brownian motion", a motion that describes molecular movement induced by random collisions between the molecules [286] . in the presence of a concentration gradient, molecules will naturally move from places of higher concentration to places of lower concentration [287] after a period of time, t, as shown in figure 11 . fick's law can be used to model this type of movement [288] . the distribution of the diffusing molecules is accurately represented by a gaussian curve, a normal distribution centered at a single point, which gradually "flattens" as t approaches infinity [213] . the extent to which a molecule diffuses is directly related to its shape, size, and mass [285] . in homogeneous isotropic solutions, the root mean square distance (z rms ) traveled by a molecule is given by following equation [289, 290] : z rms = (2dt) 1 2 where d is the diffusion coefficient of the molecule, and t is the diffusion time. making the assumption that the molecules are solid rigid spheres, the value of d can be calculated according to the famous einstein-stokes equation (equation (2)): where k b is the boltzmann's constant (1.3807 × 10 −23 j/k), t is the absolute temperature, η is the solution viscosity, and r s is the hydrodynamic radius of the molecule [290] . equation (1) and equation (2), however, are not universally applicable; they only apply to molecules that are freely diffusing in isotropic, homogeneous solutions, and importantly that can be accurately described as hard, rigid spheres [285] . different molecular geometries and additional modes of diffusion (i.e., restricted and anisotropic) require more advanced mathematics and theory [291, 292] , but the essential concepts of diffusion remain the same. molecules 2020, 25, x for peer review 27 of 66 figure 11 . visual representation of how molecules diffuse in solution from a high concentration to a low concentration across an arbitrarily defined point (x = 0) after some period of time (t = ∞) [289] . the red arrow indicates the direction of translational motion as the molecules (green) move from an area of higher concentration (left) to an area of lower concentration (right) [287] . the earliest pulse sequence used to measure diffusion in nmr spectroscopy is the gradient spin echo sequence (se), developed by stejskal et al. [293] . the se pulse sequence is shown in figure 12 . the se pulse sequence uses a gradient (g) of the externally applied magnetic field, (pulsed field gradient), the first after the 90 o pulse, and the other after the 180 o refocusing pulse. the first gradient pulse (g1) labels or gradient-encodes the nmr-active nuclei based on their physical position in the sample tube. if the molecules diffuse during the time period they are not in the correct position to experience the second gradient which re-focuses the spins. this is detected via nmr as a signal intensity decrease. after a diffusion time (∆), the second gradient pulse is applied to decode the spatial labeling of nmr-active nuclei, obtaining a well-defined spectra of diffusing molecules in solution [294] . additional nmr sequences are available for diffusion experiments [295] , and are detailed in more comprehensive reviews dealing with the subject [296, 297] . the signal intensity of the diffusing molecules depends on three factors, as described by equation where i is the observed intensity, i0 the reference intensity (unattenuated signal intensity), d is, of course, the diffusion coefficient referred to earlier, γ is the gyromagnetic ratio of the observed nucleus, g is the strength of the gradient, δ the length of the gradient, and ∆ the diffusion time [294] . from equation (3), it is easy to see that the signal intensity decreases exponentially with time, so it is vital to optimize the values of g, δ, and ∆ for diffusion nmr measurements [294] . the earliest pulse sequence used to measure diffusion in nmr spectroscopy is the gradient spin echo sequence (se), developed by stejskal et al. [293] . the se pulse sequence is shown in figure 12 . the se pulse sequence uses a gradient (g) of the externally applied magnetic field, (pulsed field gradient), the first after the 90 • pulse, and the other after the 180 • refocusing pulse. the first gradient pulse (g 1 ) labels or gradient-encodes the nmr-active nuclei based on their physical position in the sample tube. if the molecules diffuse during the time period they are not in the correct position to experience the second gradient which re-focuses the spins. this is detected via nmr as a signal intensity decrease. after a diffusion time (∆), the second gradient pulse is applied to decode the spatial labeling of nmr-active nuclei, obtaining a well-defined spectra of diffusing molecules in solution [294] . additional nmr sequences are available for diffusion experiments [295] , and are detailed in more comprehensive reviews dealing with the subject [296, 297] . molecules 2020, 25, x for peer review 29 of 66 figure 12 . spin echo (se) sequence, as discovered by stejskal et al. [293] . g1 is the first gradient pulse applied after the first 90° pulse, and g2 is the second gradient pulse applied after the first 180° pulse. δ and ∆ are the gradient length, and diffusion time, respectively. in silico (virtual) screening is now a standard technique in drug design and discovery [310] that has been in use since at least 1991 [311] , though the exact origin of the phrase "in silico" is not clear [312] . the nearly ubiquitous use of virtual screening is due to its efficiency in searching massive chemical databases in order to generate lead molecules [313] that inhibit protein-protein interactions [314] , and its ability to help identity ligand (drug) binding sites on the target of interest [310] to lend insight to the mechanisms of action for lead compounds [315, 316] . virtual screening is often accompanied by in vitro or in vivo techniques for pharmacology drug research [312] , to increase drug throughput, helping to reduce the time and cost of developing novel drug candidates [317] . virtual screening has also been used to identify candidates for anti-viral drugs [318] and anticancer drugs [319] . several chemical databases are available both for public and academic use [320] . virtual screening is properly identified as a high-throughput screening (hts) technique [321] , though using its full capacity as an hts technique is not required for most purposes. virtual screening requires a minimum of two inputs, (1) a three-dimensional model of the ligand (drug), and (2) a three-dimensional model of the receptor (protein) [322] , the latter generated from the atomic studies of proteins via x-ray crystallography or nmr spectroscopy [323] . virtual screening is not a truly "stand-alone" technique and has often been combined with additional biophysical techniques besides nmr spectroscopy and/or x-ray crystallography [324] , such as differential scanning fluorimetry [325] , fluorescence polarization, and surface plasmon resonance [324] . in this section, we briefly introduce how virtual screening has been combined with nmr spectroscopy, and how they are complementary approaches to each other in drug design. the complete details of how [293] . g 1 is the first gradient pulse applied after the first 90 • pulse, and g 2 is the second gradient pulse applied after the first 180 • pulse. δ and ∆ are the gradient length, and diffusion time, respectively. the signal intensity of the diffusing molecules depends on three factors, as described by equation (3) [294] : where i is the observed intensity, i 0 the reference intensity (unattenuated signal intensity), d is, of course, the diffusion coefficient referred to earlier, γ is the gyromagnetic ratio of the observed nucleus, g is the strength of the gradient, δ the length of the gradient, and ∆ the diffusion time [294] . from equation (3), it is easy to see that the signal intensity decreases exponentially with time, so it is vital to optimize the values of g, δ, and ∆ for diffusion nmr measurements [294] . the drug design approach based on diffusion nmr is basically a screening technique used to differentiate the binding ligands (drug) from non-binding components [264] . ligands able to bind should have significantly different diffusion coefficients (d) compared to non-binding ligands [297] , i.e., the diffusion coefficients of binding ligands will be smaller than those of non-binding ligands [264] . thus, diffusion-based nmr is a way of effectively "filtering" and identifying which ligands are binding [264] . diffusion-based nmr spectroscopy has advantages in ligand based screening applied to drug discovery. for example, diffusion ordered spectroscopy (dosy) does not require prior separation/purification of the ligand/target solution [298] . diffusion based nmr allows simultaneous determination of diffusion coefficients in multicomponent systems containing large molecules (i.e., proteins) and possible binding partners (i.e., small drug compounds) [285] , and no special labeling or contrasting agents are required, though their use is not exclusively inhibited (for an example of the use of labeled compounds in diffusion nmr spectroscopy, see [299] ). a problem occurs when there is significant chemical shift overlap between the binding molecule signals and the target. this situation makes it hard to distinguish the nmr signals [300] , and the calculations typically assign an intermediate value to the diffusion rate (i.e., one gets a smear). multidimensional diffusion nmr pulse sequences are available [301] , which may help resolve spectral overlap in 1d experiments [300] . another issue is that molecules in chemical databases may have generally low solubility [302, 303] . low solubility decreases the overall signal intensity and therefore makes accurately measuring diffusion experiments far more difficult [304] . there are many examples demonstrating the successful application of diffusion nmr in examining drugs of pharmaceutical interest [305] , and ligand-target interactions [167] . hajduk et al. [167] exploited the changes in diffusion rates to detect ligands that bind to the fk506 binding protein and the catalytic domain of stromelysin. nishimura et al. [306] utilized dosy, in combination with noesy to determine the orientation of two guest molecules, p-ethoxyiodobenzene and 2-iodo-6-methoxynaphthalene, within a host composed of a tetrakis(4-hydroxyphenyl)-cavitand and a tetra(4-pyridyl)-cavitand. furthermore, matthias et al. [307] used 1 h molecular diffusion and 19 f spin diffusion to probe the drug loading properties of the rf-peg hydrogel for 5-fluorouracil (fu) and 1,3-dimethyl-5-fluorouracil (dmfu), two anticancer drugs. dosy can be combined with saturation transfer difference (std, discussed earlier in this review) to yield new insights about ligand-target interactions. kramer et al. [308] combined std with dosy to analyze a mixture composed of wheat germ agglutinin and two derivatives of n-acetyl glucosamine (ligands). using this new technique they were able to obtain high quality spectra of the components in the mixture. tanoli et al. [309] also combined std and dosy to explore the interactions of smaller molecules with bovine serum albumin. these are just a few examples to show that diffusion nmr spectroscopy has played, and will continue to play, a prominent role in drug design. in silico (virtual) screening is now a standard technique in drug design and discovery [310] that has been in use since at least 1991 [311] , though the exact origin of the phrase "in silico" is not clear [312] . the nearly ubiquitous use of virtual screening is due to its efficiency in searching massive chemical databases in order to generate lead molecules [313] that inhibit protein-protein interactions [314] , and its ability to help identity ligand (drug) binding sites on the target of interest [310] to lend insight to the mechanisms of action for lead compounds [315, 316] . virtual screening is often accompanied by in vitro or in vivo techniques for pharmacology drug research [312] , to increase drug throughput, helping to reduce the time and cost of developing novel drug candidates [317] . virtual screening has also been used to identify candidates for anti-viral drugs [318] and anticancer drugs [319] . several chemical databases are available both for public and academic use [320] . virtual screening is properly identified as a high-throughput screening (hts) technique [321] , though using its full capacity as an hts technique is not required for most purposes. virtual screening requires a minimum of two inputs, (1) a three-dimensional model of the ligand (drug), and (2) a three-dimensional model of the receptor (protein) [322] , the latter generated from the atomic studies of proteins via x-ray crystallography or nmr spectroscopy [323] . virtual screening is not a truly "stand-alone" technique and has often been combined with additional biophysical techniques besides nmr spectroscopy and/or x-ray crystallography [324] , such as differential scanning fluorimetry [325] , fluorescence polarization, and surface plasmon resonance [324] . in this section, we briefly introduce how virtual screening has been combined with nmr spectroscopy, and how they are complementary approaches to each other in drug design. the complete details of how virtual screening works, and how it applies to drug design outside of its combination with nmr is well documented in additional reviews [310, 322, [326] [327] [328] [329] [330] . a prime example of the complementarity between nmr screening and virtual docking is found in the work of chen et al. [331] , in which the authors sought to target the a 2a adenosine receptor (a 2a ar) protein, a drug target for the treatment of parkinson's disease [332] . they used virtual screening and an nmr-based screening method against the same 500 molecules in a fragment library so they could compare the results of both methods. the virtual screen successfully predicted (based on calculated binding affinities) four out of the five orthosteric ligands discovered by nmr that were within the top 5% of the fragment library, showing that the two separate methods can give similar and reliable results. later on, chen et al. discovered that virtual screening picked up three additional fragments that remained undetected by the nmr-based method, and were, in fact, a 2a ar ligands; this shows that though neither method is flawless, they are still perfectly complementary approaches for drug design [322, 331] . in another scientific work that integrated nmr with virtual screening, di lello et al. [333] found small molecular inhibitors of the enzyme ubiquitin specific protease 7 (usp7), a key regulator of the tumor suppressor protein, p53 [334] . a fragment screen by nmr revealed a series of small molecules that bind in the active site of usp7 near the catalytic cysteine (amino acid 223). a ligand-based virtual screen utilizing the fastrocs program identified~30 hit molecules, several of which were further characterized by 1 h-15 n trosy chemical shift perturbation and line broadening to probe the binding site of the active hits. di lello. also tested the active compounds against eol-1 cells to verify the hits as identified by virtual screening and further characterized by nmr, showing that the active compounds do indeed inhibit usp7 activity. through additional study of the active molecules and further optimization of their structures, they eventually discovered a series of ligands that bind in the "palm" region of the catalytic domain of usp7, inhibiting its catalytic activity [333] . this study clearly demonstrates that nmr screening-based techniques can be combined with virtual screening to find viable drugs for targets of interest. additional examples of the successful integration of nmr and virtual screening as applied to protein targets are also found in the literature, further demonstrating the practicality and complementarity of virtual screening and nmr [329, [335] [336] [337] . for example, li et al. [338] used virtual screening filtered by nmr to identify and characterize non-metal chelating metallo-β-lactamase (mbl) inhibitors, and in particular, verona integron-encoded mbl (vim)-2, when previously there were no clinically significant inhibitors of mbl, since mbl enzymes hydrolyse many, if not all, β-lactam antibacterials compounds specifically designed to inhibit their activity [339] . furthermore, shan et al. [340] and bertini et al. [337] both used virtual screening and nmr, in their respective studies. through the combined use of nmr and virtual screening, shan et al. was able to identify, design, and synthesize novel pdz domain inhibitors, which are proteins implicated in tumorigenesis [340] . bertini et al. was able to combine nmr to study the interaction of ligands with metalloproteinases, using known inhibitors of metalloproteinases as a starting point [337] . while hsqc noesy nmr data provided structural and spatial constraints for the proposed 3d models, virtual screening was used to refine the models, and to probe the ligand-protein interaction. in each case (i.e., ligand-protein interaction), bertini et al. was able to obtain a well-defined ligand conformation in the protein binding site, thus offering a viable alternative to other approaches described in the literature [337] . clearly, combining virtual screening with nmr-based methods is advantageous in studying how ligands (drugs) bind and interact with targets (proteins) of interest. paramagnetic nmr (pnmr) can also play a prominent role in drug discovery [341] , as pnmr can provide key structural information in situations where crystal structures cannot due to the weak binding of ligands [341] . pnmr can be used to quantify the binding between ligands and large biomolecules such as proteins, dna, and rna [342] . pnmr depends on the presence of a group (called the paramagnetic center) with an unpaired electron [343] , and since many naturally occurring biomolecules and organic compounds lack a paramagnetic center, one such as caged lanthanide (clanp) [344] , must be introduced artificially [341] . once the paramagnetic center (often a metal ion) is present, paramagnetic effects can be used to measure the distance and the relative orientation (i.e., angle) between molecules [345] . this information is crucial when it comes to determining how ligands and substrates bind. thus, pnmr is quite a useful technique for drug discovery when a paramagnetic center is present. the most relevant consequence of pnmr for drug discovery is paramagnetic relaxation enhancement (pre), although there are a number of studies demonstrating the use of pseudocontact shift (pcs) effect in drug discovery research [341] . paramagnetic relaxation enhancement (pre) is proportional to the inverse sixth power of the distance between the paramagnetic center and the nucleus of interest (i.e., 1 h), although it does not reveal anything about relative orientation [341] . pre can give quantitative information in the range of 10-25 angstroms [346] . several researchers have taken advantage of this outstanding property to study the structural and dynamic properties of complex biomolecular machineries in their native environment [347] . for example, iwahara et al. (2003) demonstrated that a protein's binding polarity to dna can be determined by pre, using edta-derivatized deoxythymidine (dt-edta) with a chelated metal ion (such as cu 2+ or mn 2+ ) as a probe. dt-edta with a chelated metal ion is a convenient choice, as it can be inserted into any position of a synthesized oligonucleotide. with data derived from the pre effect, one can easily determine the polarity of the protein (or drug) binding to dna [348] . several researchers have investigated dna as a drug target [349] , and the study of iwahara et al. clearly demonstrates, and even indicates, that pre can potentially be used to study the interactions between a drug and dna [348] , provided that a paramagnetic center such as dt-edta or a metal ion is present. brasuń et al. [350] also used pre derived distances between a paramagnetic center and a nucleus of interest. they replaced the cys-s-s-cys bridge found in oxytocin and vasopressin with the his-cu 2+ -his motif to investigate if doing so would alter the stability of oxytocin and vasopressin. they determined the distances between the cu 2+ ion and 1h nuclei (possible because of pre), and used these values to generate three-dimensional models of the his-cu 2+ -his motifs in both oxytocin and vasopressin. in doing so, they indicated that such an approach using pre can help in designing new biologically active compounds [350] , and hence in drug discovery research, as many drug discovery studies require a reliable models for the successful generations of hit-lead molecules, especially in the case of in silico docking [351] . this study again proves the usefulness of pre, and therefore, pnmr, in drug discovery research. in two additional studies, huang et al. [352, 353] used pre in their individual studies of protein binding and protein dynamics, respectively. in the huang et al. case [352] , these authors used pre to establish a model of the binding between the g-actin protein, and thymosin β4, an actinbinding protein. using pre determined constraints (distances) and 1 h-15 n hsqc, they were able to establish a well-converging docking structure of the g-actin/thymonsin β4 complex [352] . on the other hand huang et al. [353] did not measure protein binding, but studied the conformational changes and dynamics of select large membrane proteins utilizing 19 f-nmr spectroscopy, and ni 2+ as the paramagnetic center. through a series of extensive experiments, they showed that conformational exchange rates of membrane proteins can be determined from measurements of the metal-enhanced longitudinal relaxation (i.e., pre) of the 19 f nuclei [353] , thus yielding additional information (i.e., protein conformation dynamics) that could be utilized in drug discovery projects targeting proteins (i.e., understanding how the protein changes shape based on its environment can be used to find potential binding sites for drug candidates). all these examples prove that pnmr is powerful approach in drug discovery research, given that pre can aid in generating trustworthy models of interacting molecules, and that it can help researchers understand better how the molecules interact in the first place. since the late 1970s solid state nmr (ssnmr) has demonstrated its usefulness in complex biomolecular systems such as collagen or lipid bilayers [354] . however, over the past years ssnmr has gained attention in the field of drug design and is slowly becoming a commonly used technique as its proving to be a powerful tool for structural analysis of membrane proteins and amyloid fibrils [354] [355] [356] . ssnmr is becoming a more attractive alternative for several different reasons. one of them is the fact that it enables the characterization of a chemical compound in a solid-state form such as in a tablet/pill [356] [357] [358] . moreover, ssnmr is not only restricted to analyzing the chemical structure but it can also provide insight into the physical properties of a compound such as polymorphism (different crystalline structures of the same compound), disorder (crystal defects and amorphous solids in the compound) or the presence of cocrystals (multicomponent crystal made of a compound and one or more small organic molecules) [356, 357] . ssnmr can also be used to quantify the amount of crystalline against the amount of amorphous material in the sample to establish phase purity (the amount of desired phase separated from other, undesirable phase) [356] [357] [358] . ssnmr differs from liquid state nmr by the presence of anisotropic interactions. in liquids nmr these effects are averaged to zero as a consequence of rapid molecular tumbling. in solid state however, the molecules are not tumbling rapidly and the residual effects of anisotropic (orientation depended) interactions such as anisotropic chemical shift, magnetic dipolar coupling, and quadrupolar coupling could be observed in the form of broad peaks, with could be much wider than the chemical shift range of the nucleus [355, 358, 359] . as a results, there has been a constant effort to improve the sensitivity and resolution of solid state nmr spectra, which increased the potential of ssnmr in future applications [360] . one of the methods that works for nuclei with spin value of i = 1/2 is called magic-angle spinning (mas). it increases the resolution by rapidly rotating the sample around a fixed (or so-called magic) angle of 54.736 • [360] . this method can be combined with decoupling, to remove the dipolar couplings between spins. this is done by applying radiofrequency pulses or cross-polarization (cp) transfer of magnetization from abundant and sensitive nuclei such as 1 h to less sensitive such as 13 c [328, 333] . a broader comparison between ssnmr and liquid state nmr is provided in [361] . as mentioned before, ssnmr can provide information about membranes and membrane proteins. for this reason, ssnmr can be used to detect interactions of ligands with receptors embedded to the membrane which enables the mapping of binding site of a receptor by utilizing cp-mas (cross-polarization magic-angle spinning) nmr and site specific mutagenesis [355] . ssnmr can provide the conformation of ligands bound to the receptor which can then be used to optimize future drug in terms of better affinity and efficiency [355] . since ssnmr is also applicable to amyloid research, it can be used for probing polypeptide structures of amyloid and intermolecular contacts between fibrils. the potential is for the design a drug that will inhibit the process of aggregation of proteins and peptides [355] . lastly, since ssnmr gains insight into physical properties of a chemical compound it can be used for control of the process of formulation and processing of a drug to help assess the purity of a compound [358] . an example of ssnmr application related to drug design is the work of callari et al., who monitored the effect of drug loading on the properties of micelles [362] . polymer micelles are widely used as nano-carries for drug delivery, but so far the effects of drug loading on the morphology of a drug carrier had not been thoroughly investigated [362] . they created a model consisting of a fructose hydrophilic block and a pmaa block (micelle), to which a different amount of platinum complex was anchored. the results from this experiment showed that micelles loaded with a higher amount of platinum complex had reduced cellular uptake, release, and cytotoxicity. the micelles with a lower load (ll) of platinum complex were more effective at targeting cancer cells (of cell lines mda-mb-231 (breast cancer) and a549 (lung cancer) than the micelles with a higher load (hl) of the platinum complex. this is evidenced by the lower ic 50 (half maximal inhibitory concentration) values of the ll micelles as compared to the hl micelles. both of those results could be related to the micellar structure and their potential for interaction between the sugar moieties and the cell wall [362] . another example of practical application of ssnmr is the work of lee and colleagues [363] in which they investigated the structure of a designed zinc-binding amyloid fibril that catalyzed ester hydrolysis. metals ions such as zinc where found to affect the process of protein aggregation which resulted in arise of amyloid like structures. therefore, understanding the processes of aggregation and the factors related to them is crucial for creation of new drugs for amyloid related diseases [364] . in the experiment lee et al. used ac-ihvhlqi-conh 2 peptide (referred as hhq) to form fibrils with varying zn 2+ :hhq molar ratios. the results showed that zn 2+ -bound hhq fibrils form parallel-in-register form of packing β-strand in each sheet and his residues are coordinated to zn 2+ via nδ1, while half of the his residues are also coordinated to zn 2+ via nε2. additionally, zn 2+ binds in a 1:1 metal ion/peptide ratio. after further analysis using structural bioinformatics, it was concluded that each zinc ion was coordinated by three histidine nitrogens from two adjacent strands. half of all histidines bridged to zn 2+ ions forming a metal-imidazolate chain [363] . a "hit" is a molecule identified from a screening technique (hts, fbdd, etc) as having a desirable effect (i.e., decreased cellular growth, high affinity score) on a target [365, 366] . however, the question of whether the activity is related to actual binding to the target, or to interference with one of the components of the assay readout mechanism, is uncertain. thus, a validation step is required. hit-validation is therefore the process of confirming, or validating, that the molecule(s) identified previously have on target activity and selectivity [367, 368] . one of the highest-impacts of nmr on drug discovery is the use as a hit-validation tool. though the hit-validation or confirmation of drugs is mostly limited to the solution state [369] , this aspect of nmr truly is a "gold standard" technique in drug discovery. nmr by itself is a powerful tool for drug validation as in the case of sharma et al. (2012) [370] who sought to identify potential drug-like inhibitors against l-aspartate α-decarboxylase (adc) an enzyme responsible for the decarboxylation of l-aspartate in order to generate β-alanine and carbon dioxide [371] , in mycobacterium tuberculosis. they began with known inhibitors of adc, and developed a protocol to measure the enzymatic activity of adc. upon addition of adc to a solution of l-aspartate, l-aspartate gradually disappeared because adc was converted to l-aspartate to β-alanine; therefore the peak intensity of l-aspartate decreased, and the peak intensity of β-alanine increased in the presence of adc (no inhibitor drug present). using this newly developed nmr-based protocol allowed direct measurement of adc enzymatic activity, and sharma et al. were able to confirm the enzymatic inhibiting activity of seven previously discovered inhibitors of adc [370] . this study demonstrated that nmr can be an effective validation tool for known drugs and for new drugs generated by a screening approach. nmr is also able to remove false positives that emerge from biochemical screens [372] . for example, an aptly named technique called a la assay to detect reactive molecules by nuclear magnetic resonance (alarm nmr) is able to eliminate false positives from hts methods [373] , and in the presence of a test compound or mixture, measures dithiothreitol (dtt)-dependent 13 c chemical shift changes of the human la antigen [373] . dahlin et al. provided an updated protocol of alarm nmr to aid researchers in the production of the 13 c-labeled la antigen reporter protein, in testing compounds with the la protein, and in the analysis of obtained nmr spectra. using alarm nmr prioritized hits identified from hts screening [374] . an example of alarm nmr is found in the work of dahlin et al., where they used this technique to test molecules that were assumed to be inhibitors of histone acetyltransferase (hat) inhibitors, and from their studies, actually discovered that 65% (15 out of 23) of the most commonly reported hat inhibitors were actually faulty. they were actually nonselective interference compounds, not necessarily specific to the inhibition of hat [375] . thus, alarm nmr (and nmr in general) served as a useful validation method, especially for unvalidated hits identified from biochemical screens [372] or other screening techniques. the last example highlights the need for cross validation, or the combination of two or more techniques to verify identified chemical hits. of course, nmr is not the sole technique used for drug validation. most often, nmr drug validation is coupled with additional methods [367] such as surface plasmon resonance (spr) [376, 377] x-ray crystallography [377] [378] [379] , isothermal calorimetry (itc) [379] , uv-vis and/or fluorescence spectroscopy [380] . the work of goudreau et al. is an excellent example of combining nmr with another biophysical technique, in this case x-ray crystallography, for drug validation [378] . a series of benzodiazepine inhibitors of human immunodeficiency virus 1 (hiv-1) was identified using an in vitro capsid assembly assay, and further characterized by 19 f-nmr. analysis of the chemical shift perturbation and line broadening effect on the 19 f-nmr spectra of the benzodiazepine inhibitors revealed the specificity and reversibility of the binding inhibitors. the same set of 19 f-nmr spectra were used to identify the n-terminal domain of the capsid as the binding site of the benzodiazepine inhibitors. the specific amino acids involved in the binding of the benzodiazepine inhibitors were identified from the chemical shift perturbation of 1 h, 15 n-trosy nmr spectra. later, use of x-ray co-crystallography confirmed binding locations of the benzodiazepine inhibitors and their binding modes, which was useful for further development and optimization of the benzodiazepine inhibitors [378] . the work of goudreau et al. therefore showed how nmr could be used as a co-validation technique with another biophysical method [378] . nmr can be also coupled with multiple biophysical techniques to validate a molecule's ability to inhibit protein-protein interactions (ppis) [367] . an example of the combination of nmr with spr and x-ray crystallography can be found in the work of fry et al., where the authors sought to understand how the nutlin molecule inhibits mdm2-p53, a protein-protein interaction that has been an important cancer therapy target for several years [381] [382] [383] . fry et al. [377] gradually deconstructed rg7112, the first nutlin molecule to enter clinical trials [384] , into 11 fragments so they could study the inhibitory effect of rg7112 on the mdm2-p53 interaction by spr, nmr, and x-ray crystallography. spr was used to determine the k d values of the rg7112 fragments and confirmed that rg7112 and some of its fragments do bind to mdm2, inhibiting the mdm2-p53 interaction. 1 h, 15 n-hsqc nmr chemical shift perturbation was also used to assess and verify binding identified by spr. of the six fragments of rg7112 confirmed by 1 h, 15 n-hsqc nmr as binding to mdm2, spr showed binding for five of them; thus, the two separate techniques were in good agreement with each other. the fragments of rg7112 that were confirmed to bind by both spr and 1 h, 15 n-hsqc nmr were further studied with x-ray crystallography, which can tell precisely where and how the molecules bind to the protein. using co-crystallization, fry et al. were able to obtain structures for several of the verified binding fragments in complex with mdm2 and were able to visualize the binding of the fragments to the mdm2 protein [377] . nmr is obviously a powerful drug binding validation tool, but it becomes much more powerful when coupled with additional biophysical techniques, as seen in the work of fry et al. [377] . dias et al. [379] took a similar approach as fry et al. [377] in that they took known inhibitors of a protein-protein interaction, and dissected them into individual fragments to assess a protein's drug-ability. the interaction studied was that between the proteins von hippel-lindau (vhl), and the alpha subunit of hypoxia-inducible factor 1 (hif-1α). twelve compounds (known inhibitors and derived fragments) were developed using a crystal structure of hif-1α peptide bound to the stable multiprotein complex pvhl-elongin c:elongin b (vcb). each of these compounds was screened using three separate nmr techniques, saturation transfer difference (std), carr-purcell-meiboom-gill (cpmg) relaxation experiments, and waterlogsy (to assess drug binding and to predict drug binding mode. each compound that was unambiguously detected (i.e., the molecule was identified as successfully binding by at least two of the three nmr methods of std, cpmg, and waterlogsy) was subjected to further analysis by itc and x-ray crystallography. itc was used to determine the dissociation constants of binding molecules, and x-ray crystallography was used to confirm the binding mode predicted by the nmr studies. generally speaking, the designed fragments had similar ligands efficacies compared to the parent molecules but had much higher dissociation constants (k d values), meaning that the fragments bound less tightly than the original parent molecule [379] . with this example, it is possible to see the strength of using nmr as its own hit-validation tool (i.e., three different nmr techniques were used for screening compounds [379] ), and yet, the follow-up of nmr studies with itc and x-ray crystallography was useful in providing a basis for assessing the drug-ability of a protein-protein interaction [385] [386] [387] [388] . thus, it is clear to see that nmr is a prominent method of hit-validation in drug discovery research, especially in combination with other biophysical techniques. substantial progress has been made in the nmr field over the past 5-10 years, and various methods were established to determine the drug-target complexes. most of them utilize either noe or chemical shift perturbations (csp) although in silico models/programs, using nmr-derivate data also exist. one of the methods called difference of inversion recovery rate with and without target irradiation (direction) is used to map pharmacophores and can be an alternative to std experiments. this method uses the difference between longitudinal relaxation rates of ligand protons with-and with-out irradiation of the protons of the target protein. the direction approach, however cannot be used for slowly exchanging (strong binding) ligands. the practical approach of this method was demonstrated on the experiment when analyzed the interactions between p38 mapk (p38 a mitogen-activated protein kinase) and its inhibitor-sb203580 [389, 390] . the results from this experiment showed that protons h1, h4, h5, and h6 of sb203580, are in close neighborhood with the protons of p38 mapk when compared with h2, h3, and methyl protons. it indicates that two aromatic rings (a pyridine ring and fluorophenyl ring) of sb203580 interact tightly with p38 mapk. the results were later confirmed with proton density map of each ligand's proton, based on the crystal structure of sb203580-p38 mapk complex [391] . moreover, the same authors already created a new and improved protein-ligand docking method by combining the direction obtained nmr data with docking software. [392] . a second method that can be used to map pharmacophores is called inter-ligand nuclear overhauser effect (iloe). this 2d nmr experiment detects when two ligands bind simultaneously to adjacent sites on a protein surface although both of the ligands do not have to bind to the same binding pocket (opposite to inpharma, see above) [5, 393] . a negative ligand−ligand noe signal will be created when ligands bind in close proximity to each other whereas ligands that do not bind will show no noes, or at most very weak positive ones [372, 394] . iloe also enables determination of the ligand orientations with respect to one another [393] . as in the case of inpharma, iloe can be utilized even in the absence of a 3d protein structure and used with large proteins. additionally, iloe differs from inpharma in mixing times-for iloe the mixing times are typically in the range of 600-800 ms [345] . application of iloe was first shown on glycolate+nad + in the presence of porcine heart lactatedehydrogenase, and by glucose-6-phosphate+nadph in the presence of l. mesenteroides glucose-6-phosphatedehydrogenase and from that time it has been widely used [393, 395, 396] . a third method called structural information using overhauser effects and selective labeling (sos-nmr), relies of std experiments performed on ligand complexes with different protein samples that have been fully deuterated excluding a specific type of amino acid. in other words, the data obtained by sos-nmr gives insight into the ligand-binding amino acid composition and when taken into consideration the 3d structure of targeted protein can be used to establish the structure of protein-ligand complex. this approach has been demonstrated using two complexes-fkbp complexed to 2-(3 -pyridyl)-benzimidazole and mura complexed to uridine diphosphaten-acetylglucosamine (udp-glcnac). the results showed that for fkbp and mura, only four and three amino acids (fkbp: ile, val, leu, met; mura: trp, phe, his) were needed to be selectively protonated in perdeuterated samples to establish the ligand-binding site. additionally, on average only 6 amino acids were required for accurate identification of ligand-binding surface. according to authors sos-nmr can greatly improve the early stages of the drug discovery process [397] . moreover, combining sos-nmr with other methods can even further increase chances for a positive outcome of an experiment [398] . a completely different approach to this topic was taken by chen et al. [399, 400] . instead of using isotope labeling, chen's group decided to use a tert-butyl group contained within ligand-1 to obtain structural information about the protein-ligand complex [400] . the tert-butyl group formed an intense singlet in 1.0 to 1.5 ppm range thanks to rapid methyl rotation and methyl reorientation within that group. when compared with the protein's 1 h-nmr signal, the tert-butyl signal tended to be much narrower and resulted in easy detection without the need for isotopic enrichment even in protein complexes of high molecular mass such as bacillus stearothermophilus dnab hexamer (320 kda) [399] . additionally, the tert-butyl group produces intense noesy cross peaks that can be observed even in the situations where normally cross-peaks of the proteins are barely detectable. this is partially because the signal corresponded to nine protons within tert-butyl group. those aspects enable measurements of pseudo-contact shifts generated by paramagnetic tags attached to the protein. as a result, it allows positioning of the ligand on the protein. an example of this approach, is dengue virus ns2b-ns3 protease from serotype 2 (referred as denpro) in complexed with ligand containing a tert-butyl group. the result of this experiment showed noes between the tert-butyl group of ligand-1 and residue val155 from denpro [400] . solvent accessibility, ligand binding, and mapping of ligand orientation by nmr spectroscopy (salmon) is another method based on the data obtained via nuclear overhauser effect. this method utilizes waterlogsy [401] to probe for solvent accessibility to the ligand and determine the orientation of the ligand by analyzing signal changes in waterlogsy spectra (positive signal from unbound ligand vs. negative for protein-bound ligands). this method was first used to determine the orientation of prodrug called tretazicar ((5-(aziridin-1-yl)-2,4-dinitrobenzamide) known as cb1954 in nqo2 (quinone oxidoreductase 2) binding site. previous attempts had been made to obtain the orientation of tretazicar bounded to nqo2, however the results obtained from x-ray crystallography were inconclusive as two orientations of tretazicar could be possible. the information obtained via salmon showed that the side chain of asparagine at position 161 formed a hydrogen bond with 2-nitrogroup of tretazicar, and that the aziridine moiety of tretazicar pointed toward the solvent [401] . another variant of waterlogsy method called logsy utilizes the titration slopes as a measure of solvent accessibility. the titration slopes are created by a constant increase of protein concentrations. this method also provides more insight into the process of ligand solvation by checking the influence of protein concentration onto the process. this approach was used on the bromodomain 1 of protein 4 (brd4-bd1) by mapping epitopes of two ligands interacting with brd4-bd1 and predicting ligands position. the results showed that the triazolopyridazine moiety of both ligands was implanted into the binding pocket of the brd4. additionally, the results from logsy titration showed that methyl-group 1 of ligand 1, aromatic proton 8 of ligand 2 and aromatic proton 8 of ligand 1 exhibit strong water noe. this information enabled researchers to utilize a chemical replacement strategy (substitute bound water molecules by suitable functional groups) for aromatic proton 8 in a series of ligands containing the triazolopyridazine ring. those protons were replaced with an amino or aminomethyl groups and as a result, the binding affinity of those ligands increased 100-fold. finally, the results obtained from x-ray crystallography for ligands with such modifications allowed to find the binding mode of the triazolopyridazine ring of ligand 1 (with methyl group pointing internally) and the substituted amino group was found to create hydrogen bond to the side chain of asn140 of brd4-bd1 [402] . the most recent approach called nuclear magnetic resonance molecular replacement (nmr 2 ) utilizes spatial data obtained through solution-state nmr in order to locate the binding pocket of a complex structure. for that, it uses a receptor model, e.g., a x-ray structure of a homolog, to conduct an analysis and at the same time excluding the need for protein resonance assignment. to conduct an experiment using such an approach requires a few steps. first, either the protein or ligand used in the complex must be uniformly 13 c and 15 n labeled. then, an experiment to assign the ligand is needed such as 2d 13 c, 1 h-hmqc or 13 c, 1 h-hmbc. the next step is the evaluation of ligand intra-and ligand-protein intermolecular distances through noe cross peaks obtained from f 1 -15 n, 13 c-filtered 1 h, 1 h-noesy. lastly, choosing a proper input structure is required which can be either x-ray or nmr structures in apo form, with another bound ligand, or a homolog to the protein of interest. then the nmr 2 program analyzes for all possible partial assignments (such as methyl groups of a protein) and calculates the complex structures for all options [403, 404] . this method was already successfully used to resolve complex structures in case of slow and fast exchange ligands [403] [404] [405] [406] . in silico methods combined with nmr derived information can also be used to determine accurate drug-target complexes. 1 3.6.9. samplex another program that can utilize csp called smoothed automatic mapping of protein from listed extremes (samplex) can help to determine the interaction surface of proteins complexes. samplex takes the chemical shifts of the protein of interests in both the free and bound state and corresponding 3d structure of a protein in the free state. the programs returns a confidence value for each residue to be in a perturbed or unperturbed state (0.05 as being in a perturbed state, −0.05 as remaining in their unperturbed state). this approach was tested on five examples, one of which was subtilisin bpn' (serine protease) complexed with its inhibitor-chymotrypsin inhibitor 2. the results showed that residue 2, and residues 56-62 of chymotrypsin inhibitor-2 were perturbed and residue 63 was in an ambiguous state. to compare, the x-ray crystallography data showed residues 50 and 54-61 to be involved in the interaction. for subtilisin bpn' the program predicted residues 33, 97, 99-109, 126-128, 141, 154-156, 167-171 and 218-219 to perturbed and residues 65, 98 and 220 to be in ambiguous state. that information was also confronted with the x-ray crystallography data which shown residues 99-104, 125-128, 154-157, 167, 218-221 to be perturbed [408] . the interactions between targets (proteins) and ligands (small molecules) can be analyzed independently of the biological systems by using 'cell-based' nmr drug design approaches. three basic approaches [409] are as follows: (1) compound-detected in-cell nmr, (2) target-detected in-cell nmr, and (3) reporter-detected in-cell nmr. these methods, with the exception of compound detected in-cell nmr, differ according to the isotopically labeled structure (protein, cell structure, etc.), which enables nmr detection. a cartoon representation of each of these methods is given in figure 13 . as we have attempted to emphasize and demonstrate, nmr has a powerful and unique role in drug design. nmr provides detailed structural information about a molecule along with kinetic information over extended time periods, i.e. not just a snapshot [24, 25] . moreover, nmr is quantitative and highly reproducible, allowing applications in diverse fields such as relaxometry, combinatorial chemistry, fluxomics, and targeted analysis [91] . nmr can be combined with other analytical techniques, such as mass spectrometry "in tandem" analysis of the molecules of interest [436] [437] [438] . 1 h 1d-nmr is used particularly in the analysis of metabolites, while the strength and std nmr is a technique that lies within the compound-detected in-cell nmr method but does not require isotope-labelling of the studied compound. however isotopic labelling of the compound may be used to enhance the quality of the spectra. in the target-detected in-cell nmr only the target of interest is isotopically labeled (i.e., 15 n labeled protein). for instance, target proteins can be isotopically labeled during cell growth in isotopically enriched ( 13 c, 15 n, or both 15 n/ 13 c) media [410] . the cell type and the labeling method may vary across experiments. different cell types, including bacteria [411] , oocytes [412] , yeast cells [413] , mammalian cells [414] , hela cells [415] and even insect cells [416] have been reported in the literature. the fact that in-cell nmr applies to more than one cell type testifies of the versatility and potential application of this technique. in terms of labeling, 15 n is one of the most commonly used approaches [417] when the targets of interest are proteins. recently, 19 f labeling has been reported as a useful probe for protein-ligand interactions [418] . it was shown that 19 f can reveal information about the dynamics of protein-ligand interactions [419] . methyl groups [420] have also been used as probes for proteins and complexes in vivo [420] , proving that labeling specific chemical groups instead of the entire biomolecule (i.e., protein) is feasible. in-cell nmr extends beyond proteins, and has been applied successfully to dna [93, 421] and rna molecules [422, 423] . telomeric repeats have also been studied using target detected in-cell nmr [424] . the reporter-detected in-cell nmr technique isotopically labels neither the ligand nor the target, but rather a receptor that indirectly measures the effects of ligand-target binding [409] . the "reporter" varies according to the experimental context. for instance, dose et al. [425] used acetylation-and deacetylation-based assays to monitor the activity of histone deacetylase and acetyl-transferase. thongwichian et al. [426] used peptide-based reporters to identify active kinases and phosphatases in cellular conditions. lastly, doura et al. [427] designed a 19 f probe that operates in biological conditions in order to study the adherence and dynamics of proteins found in human blood. in many viruses and phages, scaffolding proteins (sps) are required to ensure the correct organization of coat proteins (cps) and other minor capsid proteins into a precursor structure, called a procapsid [428, 429] . although sps are critical for viral assembly and therefore potential therapeutic targets their structural properties (with only a few exceptions [430, 431] ) are poorly understood. the size limitation of nmr can be used advantageously as a filter to identify disordered segments even in very large supramolecular protein complexes. in this way, nmr can provide a unique perspective on the dynamic and disordered elements of macromolecules not accessible by other techniques. the procapsid encapsulation experiments described by whitehead et al. [432] were conceptually analogous to in-cell nmr experiments [433] [434] [435] in which signals from small proteins, or flexible segments of proteins, can be observed when they are incorporated inside living cells, as long as the isotope-labeled proteins of interest do not interact strongly with other large cellular components [433] [434] [435] . the so called "in-virus" nmr strategy applied by whitehead et al. [432] could be more generally used to study the dynamic properties of macromolecules encapsulated into virus particles, including cargo molecules encased in viral capsids for nanotechnology applications. additionally, such studies could assess the level of interaction of cargo molecules with the virus and probe the release properties of cargo nmr [432] . as we have attempted to emphasize and demonstrate, nmr has a powerful and unique role in drug design. nmr provides detailed structural information about a molecule along with kinetic information over extended time periods, i.e., not just a snapshot [24, 25] . moreover, nmr is quantitative and highly reproducible, allowing applications in diverse fields such as relaxometry, combinatorial chemistry, fluxomics, and targeted analysis [91] . nmr can be combined with other analytical techniques, such as mass spectrometry "in tandem" analysis of the molecules of interest [436] [437] [438] . 1 h 1d-nmr is used particularly in the analysis of metabolites, while the strength and intensity of the recorded signals is directly proportional to the concentration of the sample [44, 439, 440] . 1 h 1d-nmr can also be used to follow "real-time" analysis of different molecules [441] . in the 1d 1 h-nmr experiment, there are no polarization transfer techniques required (the 1 h atom is already highly sensitive) and covers a spread of interesting nuclei in the molecule(s) being studied [91] . assuming that the sample can be stored stably for extended periods of time, the non-destructive nature of nmr permits the re-use on the sample for different experiments [50, 442] . aiding in the reproducibility of nmr [91] , sample-recycling offers a significant advantage of nmr [443, 444] . while always important in drug design, sensitivity and resolution of nmr are two major factors that need special consideration. since both factors improve with increasing magnetic field strength, we have seen a spike in the demand for ultra-high-field nmr spectrometers. recently, 28.2 t (i.e., 1.2 ghz for 1 h) magnets have become commercially available, and with recent advances in magnet technology, such as liquid helium recycling and magnetic field shielding [91] , nmr has begun to offer far better resolution and higher sensitivity while reducing the substantial costs of maintaining the instruments compared to past decades. other steps have been taken to enhance the sensitivity of nmr including: the development of cryoprobes increasing the signal to noise ratio 3 to 4 times, and micro-coil probes that not only increased sensitivity but also reduced the amount of sample required for the measurements [445] . from another perspective, one can further optimize the process of obtaining spectra by using different methods of measurements. one of these, called sofast, helps to reduce the delay between scans resulting in lowering acquisition time for 2d experiments such as hmqc utilizing 1 h, 15 n or 1 h, 13 c [117,446,447] . the basic principle of this method is to use selective 1 h pulses that will excite only a small portion of the available nuclei pool, while the unperturbed spins provide a magnetization "heat sink" thus improving the spin-lattice relaxation (t 1 ) rate via dipolar interactions [446] . these methods can be highly efficient when studying drug binding and molecular interactions [117] . another method, called ultrafast 2d nmr, enables obtaining a 2d spectra within a single scan but with the associated cost of reduced sensitivity [117, 448] . the principle of this method is to divide the sample into n number of fractions, and apply the appropriate incremental aspect to each fraction, while recording them all simultaneously within the one scan [448] . this method has been applied to many metabolomic studies [177, 447, 449] as well as in the analysis of natural products [450] . the difficulty is that the effective concentration of the sample is lowered by the fractionation level. the more slices the sample is split into, the greater the reduction of combined signal that is obtained. lastly, a method termed non-uniform sampling (nus) may provide an advantage by reducing the total time for measurement while maintaining the same resolution of spectra [117] . nus effectively skips over parts of the total dataset, collecting only around 20 to 30% of the total. usually sections containing higher concentrations of signal (over noise) are emphasized in the selection scheme, known as non-linear data acquisition. these methods reconstruct the complete data subset by applying various algorithms such as multidimensional decomposition (mdd), which essentially separates the sets of multidimensional data into one dimensional problems that are much easier to solve given the common process of signal overlapping in multidimensional nmr spectra [117] . other algorithms such as compressed sensing (cs), maximum entropy method (max ent) and iterative soft threshold (ist) each have their own advantages but all focus on decreasing the time needed for collection of spectra [117, 451] . the cost is in signal to noise, as no gain is ever absolutely free. the inherent advantages of nmr do not eliminate the disadvantages (table 3) , which are namely being limited for some nuclei, and inherent insensitivity for many types of experiments. nmr can provide high quality resolution and sensitivity for some experiments [91, 452] but the application can be challenging. when the experiments become multidimensional, there is often a tradeoff between the improved higher resolution and/or the resulting sensitivity and/or the amount of time an experiment takes, i.e., high-quality spectra for multidimensional experiments take much longer than their simple 1d counterparts [117] (scheme 2). one must always choose between resolution, sensitivity, and the amount time; as you can only ever have two out of the three. advantages disadvantages ability to identify simple chemical compounds. high quality resolution and sensitivity for many 1d experiments. less time consuming compared to 2d nmr. compared to 2d nmr less details can be obtained for more complex molecules. for nuclei other than 1 h and 19 f, relative sensitivity is fairly low-requires extra labeling to obtain better spectra. ability to identify complex molecules and observe different interactions between the nuclei, e.g., correlations between all spins in one spin system using tocsy experiment. requires long times to obtain a proper spectra (up to days). greatly reduces time to obtain 2d spectra. reduced sensitivity. significantly reduces acquisition time of hmqc. relatively low resolution lowers the time of measurement while keeping the same level of resolution. requires use of reconstruction algorithms since missing data points can lead to artifacts in spectra. multidimensional data are "broken" to one dimensional, which are easier to analyze. ability to resolve overlapping resonances. data must be (approximately) symmetrical (lorentzian shape) to obtain good spectra. good reconstruction of weaker peaks. large computational costs, low performance on noisy data. significant reduction in acquisition time. nominally lorentzian peak shapes may be distorted, and peak intensities may be altered. greatly reduced time to obtain nmr spectra. requires a grid of uniformly sampled data points. often makes stronger binding ligands from weakly binding fragments. less time and resource intensive. can be used only for small fragments of compound of interest. direct observation of target binding to ligand. several types of nmr experiments are possible. inability to distinguish binding modes, difficult to gage the "true" binding site of ligand to protein. only requires a small amount of sample. highly reproducible. allows direct observations of ligand binding. only works for ligands with low binding affinity (fast chemical exchange). inability to distinguish binding modes. in vivo studies are possible, can focus on specific cell parts. special labeling techniques may be required. spectra may be more challenging to interpret. can model protein drug interactions, helps speed up and reduce cost of drug delivery protein models need to be validated through experimental approach. can observe proteins interacting with metal ions, long observation distance (10-25 angstroms) between paramagnetic left and nearby atoms. paramagnetic left required in the system. enables the characterization of a chemical compound in a solid-state form such as a tablet/pill. provides insight into the physical properties of a compound. significant broadening of the spectral lineshapes due to anisotropic spin interactions. noticeable difference in spectra of binding and non-binding ligands. sets the lower limit of time for which experiments can be performed. unfortunately, in the real world, when working with nmr spectroscopy, we are mainly forced to choose between high or low precision data (scheme 2) with fixed available instrument times. scheme 2 can be useful, when choosing nmr technique and/or method/approaches in drug studies. it shows the general representation of different nmr techniques and method/approaches in costtime matrix, bearing in mind that the exact position in the matrix can be influenced by the environmental conditions, pulse sequence and sample preparation (e.g. concentration). regarding, nmr methods/approaches, the exact position depends on the choice of proper nmr technique. for the purposes of scheme 2, the 2d techniques were chosen as described in the practical examples. nevertheless, with relatively short times and at low cost, we can acquire numerous data sets and therefore the low precision can be partially compensated for by statistical analysis. fortunately, significant efforts have been undertaken to reduce the amount of time it takes to record multidimensional spectra, especially for 2d nmr, and still obtain high quality spectra (table 3) . novel pulse sequences have been developed to decouple nuclei in pure-shift nmr [117] . dynamic nuclear polarization (dnp) can induce hyper-polarization in atoms ( 13 c, 15 n) with an inherently low sensitivity [453, 454] . parahydrogen-induced polarization (phip) and signal amplification by reversible exchange (sabre) are other polarization techniques used to increase the sensitivity of inherently insensitive nuclei [455] . unfortunately, in the real world, when working with nmr spectroscopy, we are mainly forced to choose between high or low precision data (scheme 2) with fixed available instrument times. scheme 2 can be useful, when choosing nmr technique and/or method/approaches in drug studies. it shows the general representation of different nmr techniques and method/approaches in cost-time matrix, bearing in mind that the exact position in the matrix can be influenced by the environmental conditions, pulse sequence and sample preparation (e.g., concentration). regarding, nmr methods/approaches, the exact position depends on the choice of proper nmr technique. for the purposes of scheme 2, the 2d techniques were chosen as described in the practical examples. nevertheless, with relatively short times and at low cost, we can acquire numerous data sets and therefore the low precision can be partially compensated for by statistical analysis. fortunately, significant efforts have been undertaken to reduce the amount of time it takes to record multidimensional spectra, especially for 2d nmr, and still obtain high quality spectra (table 3) . novel pulse sequences have been developed to decouple nuclei in pure-shift nmr [117] . dynamic nuclear polarization (dnp) can induce hyper-polarization in atoms ( 13 c, 15 n) with an inherently low sensitivity [453, 454] . parahydrogen-induced polarization (phip) and signal amplification by reversible exchange (sabre) are other polarization techniques used to increase the sensitivity of inherently insensitive nuclei [455] . nmr has become a "gold standard" method in drug design due to its speed, simplicity, and reproducibility. the standardized ppm scale allows one to compare all nmr results and gather them in databases for the common use of researchers. although sample labeling was limiting in the beginning, it has now become a strength of nmr that permits the observation of big molecules and/or biomolecular processes "through the door lock". nmr is the only analytical technique that permits qualitative and quantitative analysis without previous sample purification or separation. high precision nmr data still requires long experiment times and has elevated costs; however, these will no doubt be alleviated in the future. estimating the cost of new drug development: is it really $802 million? health aff. (millwood) the price of innovation: new estimates of drug development costs animal testing and its alternatives-the most important omics is economics what is the question? nmr in drug design evaluation of ligand-based nmr screening methods to characterize small molecule binding to hiv-1 glycoprotein-41 ramped-up nmr: multiplexed nmr-based screening for drug discovery extraction, characterization, and anticoagulant activity of a sulfated polysaccharide from bursatella leachii viscera ultra-low thermal conductivity in na/sb chalcobismuthates: synthesis, crystal structures, optical properties and 23na nmr spectroscopy layered copper thioaluminate k2cu3als4: synthesis, crystal structure, characterization and solid-state 27al and 39k nmr studies identification and characterization of sse15206, a microtubule depolymerizing agent that overcomes multidrug resistance amide versus amine ratio in the discrimination layer of reverse osmosis membrane by solid state 15n nmr and dnp nmr current and future perspectives on the structural identification of small molecules in biological systems nmr experiments for the rapid identification of p=o···h-x type hydrogen bonds in nucleic acids separation and structural elucidation of a novel analogue of vardenafil included as an adulterant in a dietary supplement by liquid chromatography-electrospray ionization mass spectrometry, infrared spectroscopy and nuclear magnetic resonance spectroscopy three-dimensional nmr spectroscopy of fluorinated pharmaceutical solids under ultrafast magic angle spinning essential parameters for structural analysis and dereplication by 1h-nmr spectroscopy uplc-hrms and nmr applied in the evaluation of solid-phase extraction methods as a rational strategy of dereplication of phyllanthus spp. aiming at the discovery of cytotoxic metabolites a light responsive two-component supramolecular hydrogel: a sensitive platform for the fabrication of humidity sensors nuclear magnetic resonance study of nanoscale ionic materials variable-temperature nmr and conformational analysis of oenothein, b nmr at low and ultralow temperatures 1h-nmr study of the effect of temperature through reversible unfolding on the heme pocket molecular structure and magnetic properties of aplysia limacina cyano-metmyoglobin spectroscopic studies of the intermediates in the conversion of 1,4,11,12-tetrahydro-9,10-anthraquinone to 9,10-anthraquinone by reaction with oxygen under basic conditions heterogeneous intraand intermolecular hydroamination catalysts -(phosphonomethoxy)ethyl]adenine (pmea) and of its n 1, n 3, and n 7 deaza derivatives with copper(ii) in aqueous solution online reaction monitoring by single-scan 2d nmr under flow conditions solvent-and anion-dependent li+-o2-coupling strength and implications on the thermodynamics and kinetics of li-o2 batteries nmr approaches in structure-based lead discovery: recent developments and new frontiers for targeting multi-protein complexes screening protein-single stranded rna complexes by nmr spectroscopy for structure determination intramolecularly stapled amphipathic peptides via a boron-sugar interaction structure determination using solution nmr: is it worth the effort? nmr spectroscopy brings invisible protein states into focus fragment-based screening using x-ray crystallography and nmr spectroscopy utilizing nmr and epr spectroscopy to probe the role of copper in prion diseases combining nmr and x-ray crystallography in fragment-based drug discovery: discovery of highly potent and selective bace-1 inhibitors. in fragment-based drug discovery and x-ray crystallography real-time structure-based drug development-tutorial: triad's nmr-based structural determinations are smart chemistry method development and validation: quantitation of telmisartan bulk drug and its tablet formulation by 1h-nmr spectroscopy 31p and 15n solid-state nmr study for the development of a novel membrane protein drug-screening methodology functional analyses of target proteins for drug development by nmr band-selective excitation nmr spectroscopy and quantitative time-domain analysis using complete reduction to amplitude-frequency table (craft) to determine distribution coefficients during drug development aggregation ability of three phylogenetically distant anammox bacterial species host-dependent nitrogen recycling as a mechanism of symbiont control in aiptasia recommended strategies for spectral processing and post-processing of 1d 1h-nmr data of biofluids with a particular focus on urine anti-cancer agents in saudi arabian herbals revealed by automated high-content imaging synthesis and evaluation of modified chalcone based p53 stabilizing agents chapter three-paclitaxel synthesis and characterization of a homogeneous and silica supported homoleptic cationic tungsten(vi) methyl complex: application in olefin metathesis ft-icr mass spectrometry, and nmr spectroscopy study of heavy fuel oil recommendations and standardization of biomarker quantification using nmr-based metabolomics with particular focus on urinary analysis marine bacterial transparent exopolymer particles (tep) and tep precursors: characterization and ro fouling potential active site arginine controls the stereochemistry of hydride transfer in cyclohexanone monooxygenase synthesis, characterization and conformational study of new α,β-unsaturated acylhydrazones based on calix [4] arene backbone poly-γ-p-biphenylmethyl-glutamate as enantiodifferentiating alignment medium for nmr spectroscopy with temperature-tunable properties elucidation and partial nmr assignment of monosulfated maitotoxins from the caribbean elucidation of the structure of the 2-amino-3,5-dibromochalcone epoxides in solution and solid state synthesis, spectroscopy, and theoretical calculations of some 2-thiohydantoin derivatives as possible new fungicides identification of polyunsaturated triacylglycerols and cc location isomers in sacha inchi oil by photochemical reaction mass spectrometry combined with nuclear magnetic resonance spectroscopy coordination geometry determination of stannane compounds with phosphinoyldithioformate ligands using multinuclear nmr, sn mössbauer and dft methods clathrate structure determination by combining crystal structure prediction with computational and experimental 129xe nmr spectroscopy synthesis and biological activity of n-(arylsulfonyl) valine hydrazones and assistance of nmr spectroscopy for definitive 3d structure sández macho, i. use of 1h-nmr std, waterlogsy, and langmuir monolayer techniques for characterization of drug-zein protein complexes nmr spectroscopy techniques for screening and identifying ligand binding to protein receptors sequential 1h-nmr assignment of the complex of aponeocarzinostatin with ethidium bromide and investigation of protein-drug interactions in the chromophore binding site nmr-based metabolomic techniques identify the toxicity of emodin in hepg2 cells 1h-nmr-based metabolomics study of the toxicological effects in rats induced by new insights about doxorubicin-induced toxicity to cardiomyoblast-derived h9c2 cells and dexrazoxane cytoprotective effect: contribution of in vitro 1h-nmr metabonomics nmr-based metabonomic study on the subacute toxicity of aristolochic acid in rats solid state nmr and bioequivalence comparison of the pharmacokinetic parameters of two formulations of clindamycin bace1 is the major β-secretase for generation of aβ peptides by neurons structural basis for the antifolding activity of a molecular chaperone chapter ten-binding site identification and structure determination of protein-ligand complexes by nmr: a semiautomated approach fragment-based drug design nmr in target driven drug discovery: why not? nmr in integrated biophysical drug discovery for ras: past, present, and future nmr in pharmaceutical discovery and development clinical magnetic resonance spectroscopy principles of pulse nmr spectroscopy spin dynamics: basics of nuclear magnetic resonance nmr spectroscopy explained: simplified theory, applications and examples for organic chemistry and structural biology solid-state nmr paramagnetic relaxation enhancement immersion depth studies in phospholipid bilayers protein dynamics from nmr an introduction to nmr-based approaches for measuring protein dynamics using nmr spectroscopy to investigate the role played by copper in prion diseases monitoring dendrimer conformational transition using 19f and 1h2o nmr retention of strong intramolecular hydrogen bonds in high polarity solvents in binaphthalene-benzamide derivatives: extensive nmr studies potential hepatoxicity risk of the shell of herpetospermum caudigerum wall in rats based on 1h-nmr metabonomics metabolomic analysis of anti-hypoxia and anti-anxiety effects of fu fang jin jing oral liquid integrative drug efficacy assessment of danggui and european danggui using nmr-based metabolomics ardá, a. nmr and molecular recognition of n-glycans: remote modifications of the saccharide chain modulate binding features obtaining 3d atomistic structure of saccharides from raman/roa/nmr spectroscopic techniques the strengths and weaknesses of nmr spectroscopy and mass spectrometry with particular focus on metabolomics research monitoring dna-ligand interactions in living human cells using nmr spectroscopy situ monitoring of bacteria under antimicrobial stress using 31p solid-state nmr nmr studies of protein structure and dynamics combined hplc, nmr spectroscopy, and ion-trap mass spectrometry with application to the detection and characterization of xenobiotic and endogenous metabolites in human urine bioequivalence assessment of two formulations of ibuprofen lc-nmr-ms in drug discovery asics: an automatic method for identification and quantification of metabolites in complex 1d 1h-nmr spectra metabolite identification via the madison metabolomics consortium database a gui r package for proficient automatic profiling of 1d 1h-nmr spectra of study datasets open source software for identifying and quantifying metabolites in nmr spectra chapter 3-experimental aspects of nmr spectroscopy practical aspects of n-dimensional data acquisition and processing water suppression that works. excitation sculpting using arbitrary waveforms and pulsed field gradients distortion-free homonuclear spectra of peptides and nucleic acids in water using excitation sculpting water suppression that works. excitation sculpting using arbitrary wave-forms and pulsed-field gradients gradient-tailored excitation for single-quantum nmr spectroscopy of aqueous solutions solvent signal suppression in nmr experiments on ax systems distortionless enhancement of nmr signals by polarization transfer 13c-nmr-based metabolic fingerprinting of citrus-type crude drugs chemoselective 15n tag for sensitive and high-resolution nuclear magnetic resonance profiling of the carboxyl-containing metabolome 15n-cholamine-a smart isotope tag for combining nmrand ms-based metabolite profiling fast reconstruction of non-uniform sampling multidimensional nmr spectroscopy via a deep neural network 15n and 13c-sofast-hmqc editing enhances 3d-noesy sensitivity in highly deuterated, selectively [1h,13c]-labeled proteins chapter 5-new advances in fast methods of 2d nmr experiments heterogeneous microtesla sabre enhancement of 15n nmr signals information from combined 1h and 31p nmr studies of cell extracts: differences in metabolism between drug-sensitive and drug-resistant mcf-7 human breast cancer cells assessment of pharmacological treatment of myocardial infarction by phosphorus-31 nmr with surface coils application of 31p nmr spectroscopy and chemical derivatization for metabolite profiling of lipophilic compounds in human serum development of a bioreactor system for cytotoxic evaluation of pharmacological compounds in living cells using nmr spectroscopy the organic set of nmr spectra. in 50 and more essential nmr experiments: a detailed guide pulse pair technique in high resolution nmr a reprint of the historical 1971 lecture notes on two-dimensional spectroscopy choosing the best pulse sequences, acquisition parameters, postacquisition processing strategies, and probes for natural product structure elucidation by nmr spectroscopy the phase transfer catalysed synthesis of isoflavone-o-glucosides determination of the relative signs of 2j(31p-31p) in complexes of tungsten(0) and molybdenum(0) using two-dimensional [31p-31p]-cosy-45 nuclear magnetic resonance chemical shift correlation two dimentional nmr knowns and unknowns in metabolomics identified by multidimensional nmr and hybrid ms/nmr methods product operators, coherence pathways, and phase cycling. part iii: phase cycling artifacts in two-dimensional nmr chapter 6-correlations through the chemical bond i: homonuclear shift correlation analytical optimization of active bandwidth and quality factor for tocsy experiments in nmr spectroscopy novel selective tocsy method enables nmr spectral elucidation of metabolomic mixtures elimination of zero-quantum interference in two-dimensional nmr spectra 1h-nmr and ms based metabolomics study of the intervention effect of curcumin on hyperlipidemia mice induced by high-fat diet pharmacometabonomics analysis reveals serum formate and acetate potentially associated with varying response to gemcitabine-carboplatin chemotherapy in metastatic breast cancer patients based metabolic profiling of cells in response to treatment with a hexacationic ruthenium metallaprism as potential anticancer drug the use of coupled hsqc spectra to aid in stereochemical assignments of molecules with severe proton spectral overlap chapter 2-nmr spectroscopy methods in metabolic phenotyping. in the handbook of metabolic phenotyping high-resolution nmr techniques in organic chemistry doubly compensated multiplicity-edited hsqc experiments utilizing broadband inversion pulses product operator formalism in nmr acac)(γ-acac)(dms)] treatment revealed by a metabolomic 1h-nmr study a comprehensive discussion of hsqc and hmqc pulse sequences evaluation of band-selective hsqc and hmbc: methodological validation on the cyclosporin cyclic peptide and application for poly(3-hydroxyalkanoate)s stereoregularity determination chapter 4-nmr molecular recognition studies for the elucidation of protein and nucleic acid structure and function metabominer-semi-automated identification of metabolites from 2d nmr spectra of complex biofluids an improved 15n relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins improved hsqc experiments for the observation of exchange broadened signals quantification of metabolites by nmr spectroscopy in the presence of chapter 5-relaxation and dynamic processes basic principles of nmr a complete introduction to modern nmr spectroscopy nmr in drug discovery high-resolution diffusion and relaxation edited one-and two-dimensional 1h-nmr spectroscopy of biological fluids measurement of spin relaxation in complex systems 15-multiple-pulse nmr experiments ultrafast nmr t1 relaxation measurements: probing molecular properties in real time saturation-inversion-recovery: a method for t1 measurement chapter 8-the physical basis of the nuclear magnetic resonance experiment. part ii: pulse and fourier-transform nmr spin-lattice relaxation time in drug discovery and design nmr methods to characterize protein-ligand interactions the interaction of dna with intercalating agents probed by sodium-23 nmr relaxation rates nmr screening techniques in drug discovery and drug design one-dimensional relaxation-and diffusion-edited nmr methods for screening compounds that bind to macromolecules use of relaxation-edited one-dimensional and two dimensional nuclear magnetic resonance spectroscopy to improve detection of small metabolites in blood plasma the quest for simplicity: remarks on the free-approach models fast evaluation of protein dynamics from deficient 15n relaxation data effects of diffusion on free precession in nuclear magnetic resonance experiments modified spin-echo method for measuring nuclear relaxation times measuring t1 and t2 relaxation rates an exact solution for r2, eff in cpmg experiments in the case of two site chemical exchange nmr-based screening in drug discovery extraction of t2 from nmr linewidths in simple spin systems by use of reference deconvolution quantitative metabolic profiling of nmr spectral signatures of branched chain amino acids in blood serum isoniazid-induced hepatotoxicity and neurotoxicity in rats investigated by 1h-nmr based metabolomics approach 1h-nmr-based metabolic profiling of naproxen-induced toxicity in rats 1h-nmr toxicometabolomics following cisplatin-induced nephrotoxicity in male rats the antitumor effect of formosanin c on hepg2 cell as revealed by 1h-nmr based metabolic profiling an nmr-based metabonomic investigation of the subacute effects of melamine in rats metabolomic profiling predicts outcome of rituximab therapy in rheumatoid arthritis response to drug treatment in newly diagnosed epilepsy: a pilot study of 1h-nmr-and ms-based metabonomic analysis metabolic alteration in obese diabetes rats upon treatment with centella asiatica extract novel 1,3,4-thiadiazoles inhibit colorectal cancer via blockade of il-6/cox-2 mediated jak2/stat3 signals as evidenced through data-based mathematical modeling plasma-metabolite-biomarkers for the therapeutic response in depressed patients by the traditional chinese medicine formula xiaoyaosan: a 1h-nmr-based metabolomics approach metabonomic profiles delineate the effect of traditional chinese medicine sini decoction on myocardial infarction in rats 1h-nmr spectral identification of medication in cerebrospinal fluid of pediatric meningitis 1h-nmr-based metabolomics approach to investigating the renal protective effects of genipin in diabetic rats potential metabolomic biomarkers for evaluation of adriamycin efficacy using a urinary 1h-nmr spectroscopy 1h-nmr-based serum metabolomics reveals erythromycin-induced liver toxicity in albino wistar rats nmr-based metabonomics study on the effect of gancao in the attenuation of toxicity in rats induced by fuzi nmr-fragment based virtual screening: a brief overview discovering high-affinity ligands for proteins: sar by nmr when fragments link: a bibliometric perspective on the development of fragment-based drug discovery current perspectives in fragment-based lead discovery (fbld) adaptation of high-throughput screening in drug discovery-toxicological screening tests high-throughput screening: the hits and leads of drug discovery-an overview review of applications of high-throughput sequencing in personalized medicine: barriers and facilitators of future progress in research and clinical application review article: high-throughput affinity-based technologies for small-molecule drug discovery design principles for fragment libraries: maximizing the value of learnings from pharma fragment-based drug discovery (fbdd) programs for use in academia how size matters: diversity for fragment library design high-throughput screening: update on practices and success industrial scale high-throughput screening delivers multiple fast acting macrofilaricides virtual high throughput screening using machine learning methods introduction to fragment-based drug discovery. in fragment-based drug discovery and x-ray crystallography the influence of lead discovery strategies on the properties of drug candidates biophysical screening in fragment-based drug design: a brief overview high-throughput screening and fragment-based design: general considerations for lead discovery and optimization nmr screening and hit validation in fragment based drug discovery structural biology in fragment-based drug design fragment-based drug discovery using nmr spectroscopy chapter nine-practical aspects of nmr-based fragment screening fragment-based drug design nmr-based screening: a powerful tool in fragment-based drug discovery nmr in drug discovery: a practical guide to identification and validation of ligands interacting with biological macromolecules applied biophysical methods in fragment-based drug discovery concepts and core principles of fragment-based drug design application of fragment-based drug discovery to versatile targets a three-stage biophysical screening cascade for fragment-based drug discovery protein crystallography and drug discovery: recollections of knowledge exchange between academia and industry twenty years on: the impact of fragments on drug discovery churcher, i. fragment-based drug discovery-from hit discovery to fda approval: lessons learned and future challenge the first drug approved for braf -mutant cancer venetoclax: evidence to date and clinical potential fragment-based drug design: strategic advances and lessons learned krimm, i. fragment linking strategies for structure-based drug design discovery and development of venetoclax, a selective antagonist of bcl-2 an inhibitor of bcl-2 family proteins induces regression of solid tumours x-ray and nmr structure of human bcl-x l, an inhibitor of programmed cell death ribociclib for the first-line treatment of advanced hormone receptor-positive breast cancer: a review of subgroup analyses from the monaleesa-2 trial the csf1 receptor inhibitor pexidartinib (plx3397) reduces tissue macrophage levels without affecting glucose homeostasis in mice structure-guided blockade of csf1r kinase in tenosynovial giant-cell tumor characterizing and overriding the structural mechanism of the quizartinib-resistant flt3 "gatekeeper" f691l mutation with plx3397 discovery and chemical development of verubecestat, a bace1 inhibitor for the treatment of alzheimer's disease discovery of the 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative verubecestat (mk-8931)-a β-site amyloid precursor protein cleaving enzyme 1 inhibitor for the treatment of alzheimer's disease fragment-based drug discovery applied to hsp90. discovery of two lead series with high ligand efficiency discovery of (2,4-dihydroxy-5-isopropylphenyl)-[5-(4-methylpiperazin -1-ylmethyl)-1,3-dihydroisoindol-2-yl]methanone (at13387), a novel inhibitor of the molecular chaperone hsp90 by fragment based drug design optimization of pyrrolamide topoisomerase ii inhibitors toward identification of an antibacterial clinical candidate (azd5099) using fragment-based approaches to identification of n-(4-piperidinyl)-4-(2,6-dichlorobenzoylamino)-1h-pyrazole-3-carboxamide (at7519), a novel cyclin dependent kinase inhibitor using fragment-based x-ray crystallography and structure based drug design cyclin-dependent kinase inhibitor at7519 as a potential drug for mycn-dependent neuroblastoma biological characterization of at7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines development of second-generation cdk2 inhibitors for the prevention of cisplatin-induced hearing loss practical fragments: fragments in the clinic: 2016 edition sar by nmr: putting the pieces together encyclopedia nmr-based approaches for the identification and optimization of inhibitors of protein-protein interactions nmr-based screening of proteins containing 13c-labeled methyl groups estimating protein−ligand binding affinity using high-throughput screening by nmr exploring the binding of peptidic west nile virus ns2b-ns3 protease inhibitors by nmr blocking the interactions between calcium-bound s100a12 protein and the v domain of rage using tranilast characterization of protein−ligand interactions by high-resolution solid-state nmr spectroscopy using chemical shift perturbation to characterise ligand binding combining automated peak tracking in sar by nmr with structure-based backbone assignment from 15n-noesy atia-tul-wahab chapter 6-nuclear overhauser effect accuracy in determining interproton distances using nuclear overhauser effect data from a flexible molecule chapter 10-more 1d and 2d nmr experiments: the nuclear overhauser effect-polarization transfer-spin lock experiments-3d nmr use of nmr saturation transfer difference spectroscopy to study ligand binding to membrane proteins waterlogsy as a method for primary nmr screening: practical aspects and range of applicability characterization of ligand binding by saturation transfer difference nmr spectroscopy detecting binding affinity to immobilized receptor proteins in compound libraries by hr-mas std nmr ligand based nmr methods for drug discovery virus-ligand interactions: identification and characterization of ligand binding by nmr spectroscopy method for detecting biologically active compounds from compound libraries evolutionary change of the heme c electronic structure: ferricytochrome c-551 from pseudomonas aeruginosa and horse heart ferricytochrome c nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase application of nmr based binding assays to identify key hydroxy groups for intermolecular recognition determining binding sites in protein-nucleic acid complexes by cross-saturation group epitope mapping by saturation transfer difference nmr to identify segments of a ligand in direct contact with a protein receptor synthetic derivatives of pyrrole and pyrrolidine suitable for the therapy of infections caused by rhinoviruses global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis economic impact of outbreaks of norovirus infection in hospitals norovirus-host interaction: implications for disease control and prevention x-ray crystallographic structure of the norwalk virus capsid structural insights into antigenic diversity and host specificity. proc. natl. acad. sci structural basis for the recognition of blood group trisaccharides by norovirus epitope mapping of histo blood group antigens bound to norovirus vlps using std nmr experiments reveals fine details of molecular recognition synthesis and nmr studies on the abo histo-blood group antigens: synthesis of type iii and iv structures and nmr characterization of type i-vi antigens nmr experiments reveal the molecular basis of receptor recognition by a calicivirus negative nuclear overhuaser effects as probes of macromolecular structure identification of an e-selectin antagonist in a substance mixture by transfer noe the inpharma method: protein-mediated interligand noes for pharmacophore mapping diffusion nmr spectroscopy in supramolecular and combinatorial chemistry: an old parameter-new insights beyond passive: chaotic transport in stirred fluids 3.5 passive transport chapter 7-momentum, heat and mass transfer in boundary layers diffusion nmr spectroscopy chapter 10-diffusion nmr spectroscopy the diffusion equation models for diffusion spin diffusion measurements: spin echoes in the presence of a time-dependent field gradient diffusion by nmr the stejskal-tanner equation generalized for any gradient shape-an overview of most pulse sequences measuring free diffusion diffusion ordered nuclear magnetic resonance spectroscopy: principles and applications measuring ligand-protein binding using nmr diffusion experiments chapter 6-dosy nmr for drug analysis diffusion-weighted nmr spectroscopy allows probing of 13c labeling of glutamate inside distinct metabolic compartments in the brain spatial encoding and spatial selection methods in high-resolution nmr spectroscopy spatially encoded 2d and 3d diffusion-ordered nmr spectroscopy enumeration of 166 billion organic small molecules in the chemical universe database gdb-17 exploring chemical space for drug discovery using the chemical universe database 19f dosy diffusion-nmr spectroscopy of fluoropolymers pulsed-field gradient nuclear magnetic resonance measurements (pfg nmr) for diffusion ordered spectroscopy (dosy) mapping guest-encapsulation properties of a self-assembled capsule by dynamic boronic ester bonds properties of small molecular drug loading and diffusion in a fluorinated peg hydrogel studied by 1h molecular diffusion nmr and 19f spin diffusion nmr a new nmr method to analyze multi-component enzyme/substrate systems the exploration of interaction studies of smaller size, mostly ignored yet intrinsically inestimable molecules towards bsa; an example of std and dosy nmr. open chem structure-based virtual screening for drug discovery: principles, applications and recent advances from data banks to data bases in silico pharmacology for drug discovery: methods for virtual ligand screening and profiling ultra-high-throughput structure-based virtual screening for small-molecule inhibitors of protein-protein interactions in silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen in silico target prediction for elucidating the mode of action of herbicides including prospective validation docking, virtual high throughput screening and in silico fragment-based drug design in silico virtual screening approaches for anti-viral drug discovery virtual screening strategies: recent advances in the identification and design of anti-cancer agents a review of ligand-based virtual screening web tools and screening algorithms in large molecular databases in the age of big data virtual high throughput screening (vhts)-a perspective application of nmr and molecular docking in structure-based drug discovery virtual ligand screening against comparative protein structure models biophysical screening for the discovery of small-molecule ligands the use of virtual screening and differential scanning fluorimetry for the rapid identification of fragments active against mek1 the holistic integration of virtual screening in drug discovery retrospect and prospect of virtual screening in drug discovery docking and scoring in virtual screening for drug discovery: methods and applications nmr and in silico screening acceleration of the drug discovery process: a combinatorial approach using nmr spectroscopy and virtual screening complementarity between in silico and biophysical screening approaches in fragment-based lead discovery against the a2a adenosine receptor adenosine a2a receptors in parkinson's disease treatment discovery of small-molecule inhibitors of ubiquitin specific protease 7 (usp7) using integrated nmr and in silico techniques molecular recognition of p53 and mdm2 by usp7/hausp design and characterization of libraries of molecular fragments for use in nmr screening against protein targets antihypertensive drug valsartan in solution and at the at1 receptor: conformational analysis, dynamic nmr spectroscopy, in silico docking, and molecular dynamics simulations combining in silico tools and nmr data to validate protein−ligand structural models: application to matrix metalloproteinases nmr-filtered virtual screening leads to non-metal chelating metallo-β-lactamase inhibitors metallo-β-lactamase inhibitors inspired on snapshots from the catalytic mechanism synthesis of potent dishevelled pdz domain inhibitors guided by virtual screening and nmr studies paramagnetic nmr in drug discovery paramagnetic nmr in drug discovery paramagnetic nmr in solution and the solid state clanp-an artificial paramagnetic centre to study proteins by nmr current nmr techniques for structure-based drug discovery requirements on paramagnetic relaxation enhancement data for membrane protein structure determination by nmr paramagnetic nmr as a new tool in structural biology edta-derivatized deoxythymidine as a tool for rapid determination of protein binding polarity to dna by intermolecular paramagnetic relaxation enhancement dna as a target for drug action the structural effects of the cys-s-s-cys bridge exchange by the his-cu(ii)-his motif studied on natural peptides-a promising tool for natural compounds-based design towards reproducible computational drug discovery utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin monitoring dynamics of large membrane proteins by 19f paramagnetic longitudinal relaxation: domain movement in a glutamate transporter homolog solid-state nmr spectroscopy on complex biomolecules solid-state nmr spectroscopy as a tool for drug design: from membrane-embedded targets to amyloid fibrils new approaches to the characterization of drug candidates by solid-state nmr saikiran solid state nuclear magnetic resonance spectroscopy-a review solid-state nmr spectroscopy in pharmaceutical research and analysis solid-state nuclear magnetic resonance spectroscopy: theory and pharmaceutical applications recent advances in solid-state nuclear magnetic resonance spectroscopy solid-state nmr in drug design and discovery for membrane-embedded targets the effect of drug loading on micelle properties: solid-state nmr as a tool to gain structural insight zinc-binding structure of a catalytic amyloid from solid-state nmr aggregation of biologically important peptides and proteins: inhibition or acceleration depending on protein and metal ion concentrations principles of early drug discovery hit discovery and hit-to-lead approaches chapter twelve-hit-to-lead: hit validation and assessment biophysics: for hts hit validation, chemical lead optimization, and beyond advances in nuclear magnetic resonance for drug discovery validation of drug-like inhibitors against mycobacterium tuberculosis l-aspartate α-decarboxylase using nuclear magnetic resonance expression of bacterial l-aspartate-alpha-decarboxylase in tobacco increases beta-alanine and pantothenate levels and improves thermotolerance methods for identification of false positives in biochemical screens alarm nmr: a rapid and robust experimental method to detect reactive false positives in biochemical screens alarm nmr for hts triage and chemical probe validation assay interference and off-target liabilities of reported histone acetyltransferase inhibitors a combination of 19f nmr and surface plasmon resonance for site-specific hit selection and validation of fragment molecules that bind to the atp-binding site of a kinase deconstruction of a nutlin: dissecting the binding determinants of a potent protein-protein interaction inhibitor monitoring binding of hiv-1 capsid assembly inhibitors using 19f ligand-and 15n protein-based nmr and x-ray crystallography: early hit validation of a benzodiazepine series is nmr fragment screening fine-tuned to assess druggability of protein-protein interactions de novo design of chiral organotin cancer drug candidates: validation of enantiopreferential binding to molecular target dna and 5 -gmp by uv-visible, fluorescence, 1h and 31p nmr inhibiting the p53-mdm2 interaction: an important target for cancer therapy inhibiting mdm2-p53 interaction suppresses tumor growth in patient-derived non-small cell lung cancer xenograft models targeting p53-mdm2-mdmx loop for cancer therapy. in mutant p53 and mdm2 in cancer mdm2 small-molecule antagonist rg7112 activates p53 signaling and regresses human tumors in preclinical cancer models small molecules, big targets: drug discovery faces the protein-protein interaction challenge exploring protein-protein interactions as drug targets for anti-cancer therapy with in silico workflows targeting protein-protein interactions for drug discovery protein-protein interaction modulators: advances, successes and remaining challenges sb 203580 is a specific inhibitor of a map kinase homologue which is stimulated by cellular stresses and interleukin-1 novel homologues of csbp/p38 map kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles an accurate pharmacophore mapping method by nmr spectroscopy protein-ligand docking guided by ligand pharmacophore-mapping experiment by nmr the inter-ligand overhauser effect: a powerful new nmr approach for mapping structural relationships of macromolecular ligands sar by iloes: an nmr-based approach to reverse chemical genetics targeting apoptosis via chemical design: inhibition of bid-induced cell death by small organic molecules epitope mapping and competitive binding of hsa drug site ii ligands by nmr diffusion measurements a saturation transfer nmr-based method for determining the structures of protein−ligand complexes how much nmr data is required to determine a protein-ligand complex structure? o-tert-butyltyrosine, an nmr tag for high-molecular-weight systems and measurements of submicromolar ligand binding affinities sensitive nmr approach for determining the binding mode of tightly binding ligand molecules to protein targets solvent accessibility, ligand binding, and mapping of ligand orientation by nmr spectroscopy direct nmr probing of hydration shells of protein ligand interfaces and its application to drug design protein-ligand structure determination with the nmr molecular replacement tool, nmr2 nmr-based determination of the 3d structure of the ligand-protein interaction site without protein resonance assignment the nmr2 method to determine rapidly the structure of the binding pocket of a protein-ligand complex with high accuracy protein-fragment complex structures derived by nmr molecular replacement using ligand-induced protein chemical shift perturbations to determine protein-ligand structures automatic mapping of perturbed and unperturbed regions of proteins and complexes applications of in-cell nmr in structural biology and drug discovery protein 19f nmr in escherichia coli 3d structure determination of a protein in living cells using paramagnetic nmr spectroscopy structure of proteins in eukaryotic compartments direct structural evidence of protein redox regulation obtained by in-cell nmr cell nmr spectroscopy in protein chemistry and drug discovery high-resolution heteronuclear multidimensional nmr of proteins in living insect cells using a baculovirus protein expression system isotope labeling for solution and solid-state nmr spectroscopy of membrane proteins. in isotope labeling in biomolecular nmr 19f-nmr in target-based drug discovery applications of 19f-nmr in fragment-based drug discovery methyl groups as probes for proteins and complexes in in-cell nmr experiments nmr-based investigations into target dna search processes of proteins isotope labeling strategies for nmr studies of rna the first successful observation of in-cell nmr signals of dna and rna in living human cells g-quadruplex dna and ligand interaction in living cells using nmr spectroscopy nmr profiling of histone deacetylase and acetyl-transferase activities in real time a multiplexed nmr-reporter approach to measure cellular kinase and phosphatase activities in real-time an adhesive 19f mri chemical probe allows signal off-to-on-type molecular sensing in a biological environment scaffolding proteins and their role in viral assembly mechanism of scaffolding-assisted viral assembly structure of the coat protein-binding domain of the scaffolding protein from a double-stranded dna virus11edited by m. summers structural assembly of the tailed bacteriophage φ29 nmr mapping of disordered segments from a viral scaffolding protein enclosed in a 23 mda procapsid hydrogen exchange of monomeric α-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in escherichia coli investigating macromolecules inside cultured and injected cells by in-cell nmr spectroscopy physicochemical properties of cells and their effects on intrinsically disordered proteins (idps) beyond the paradigm: combining mass spectrometry and nuclear magnetic resonance for metabolomics sample collection and preparation of biofluids and extracts for gas chromatography-mass spectrometry gas chromatography-mass spectrometry of biofluids and extracts application of nmr metabolomics to search for human disease biomarkers optimized metabolite extraction from blood serum for 1h nuclear magnetic resonance spectroscopy molecular thermodynamics using nuclear magnetic resonance (nmr) spectroscopy. inventions a review of nondestructive characterization of composites using nmr chapter 2-mass spectrometry dehydrodimerization of pterostilbene during electrospray ionization mass spectrometry cryogenic probe 13c nmr spectroscopy of urine for metabonomic studies sofast-hmqc experiments for recording two-dimensional deteronuclear correlation spectra of proteins within a few seconds sofast-hmqc-an efficient tool for metabolomics ultrafast 2d nmr: an emerging tool in analytical spectroscopy evaluation of fast 2d nmr for metabolomics real-time separation of natural products by ultrafast 2d nmr coupled to on-line hplc compressed sensing and the reconstruction of ultrafast 2d nmr data: principles and biomolecular applications application of ex situ dynamic nuclear polarization in studying small molecules dynamic nuclear polarization for sensitivity enhancement in modern solid-state nmr determinants for optimal enhancement in ex situ dnp experiments hyperpolarized nmr spectroscopy: d-dnp, phip, and sabre techniques this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to thank king abdullah university of science and technology (kaust) for financial support. the authors declare no conflict of interest. key: cord-268239-neb6xxlf authors: illiano, anna; pinto, gabriella; melchiorre, chiara; carpentieri, andrea; faraco, vincenza; amoresano, angela title: protein glycosylation investigated by mass spectrometry: an overview date: 2020-08-28 journal: cells doi: 10.3390/cells9091986 sha: doc_id: 268239 cord_uid: neb6xxlf the protein glycosylation is a post-translational modification of crucial importance for its involvement in molecular recognition, protein trafficking, regulation, and inflammation. indeed, abnormalities in protein glycosylation are correlated with several disease states such as cancer, inflammatory diseases, and congenial disorders. the understanding of cellular mechanisms through the elucidation of glycan composition encourages researchers to find analytical solutions for their detection. actually, the multiplicity and diversity of glycan structures bond to the proteins, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies make their detection much trickier than other kinds of biopolymers. an overview of the most prominent techniques based on mass spectrometry (ms) for protein glycosylation (glycoproteomics) studies is here presented. the tricks and pre-treatments of samples are discussed as a crucial step prodromal to the ms analysis to improve the glycan ionization efficiency. therefore, the different instrumental ms mode is also explored for the qualitative and quantitative analysis of glycopeptides and the glycans structural composition, thus contributing to the elucidation of biological mechanisms. glycosylation is a co-and post-translational modification that involves the covalent bonding of an oligosaccharide chain with a polypeptide chain [1] . glycans are secondary gene products generated by the coordinated action of many enzymes in the subcellular compartments of a cell. therefore, monosaccharide units can be coupled together in many different ways (adopting different conformations and binding positions) without following a specific pattern, unlike proteins or dna. mammalian glycans are synthesized in intricate biosynthetic pathways by assembling only ten monosaccharide units: fucose (fuc), galactose (gal), glucose (glc), n-acetylgalactosamine (galnac), n-acetylglucosamine (glcnac), glucuronic acid (glca), iduronic acid (idoa), mannose (man), sialic acid (sa), and xylose (xyl) [2] [3] [4] . nand o-linked glycosylation are the most common forms of glycosylated conjugate present in cells. to fully understand the origin of the diversity of glycan structures, it is necessary to deepen their biosynthetic transformations [5, 6] . the synthesis of n-glycans begins in the endoplasmic reticulum (er), where a lipid-bound oligosaccharide (which generally β1,2-xyl, α1,3-fuc, and lewis type a structures, are conserved. this evidence has also been found in higher plants and in mosses such as physcomitrella patens as reported by fitchette et al. 1999 ; wilson, zeleny, et al. 2001; viëtor et al. 2003 [20] [21] [22] . with regard to o-glycosylation, implicated in cell signaling, it is substantially different in plants. mucin-type o-glycans have not been detected on plant proteins as well as the glycosyltransferases necessary for the initiation and elongation of these o-glycans have not been found in plant genomes. studies on the glycoforms structures have been conducted in plants by taylor et al. 2012; tryfona et al. 2012 ; highlighting that a residue of gal can be transferred to ser residues on specific proteins and arabinose chains, and that structurally complex arabinogalactans are present on the hydroxyproline residues of the proteins belonging to the cell wall [23] [24] [25] . the need to include the fungi into the relative kingdom originated from several similarity with animalia one. actually, the highly mannose glycans like plants are characterized by extensive repeating α1-6-linked units branched by short chains of α1-2and α1-3-linked mannose structures [26] . the intense o-mannosylation characterized the o-glycosylated proteins displaying a higher variable in sugar components and the linkage type of glycans determining the multiple functions [27] . for instance, the catalytic activity of α-l-arabinofuranosidase of fungus pleurotus ostreatus relied in o-glycosylation of s160 residue crucial for enzyme structural stability [28] . the main aim of glycomics is to understand the structure, enzymatic and biological mechanisms of glycosylation as well as to compare biosystems under normal and pathological conditions [29] in order to figure out glycans alterations as possible biomarkers of a disease. the difficulties caused by microheterogeneity largely prevent a simple and direct approach to investigate the entire set of glycans from glycoproteins after their hydrolysis or by glycoconjugates, and over the years different protocols have been developed [30] [31] [32] [33] . an important aspect in glycoforms micro-heterogenicity molecular structures comprehension relies in their possible different pharmacological profiles [4] . saccharide motifs commonly found in mammalian are object of pharmaceutical studies for the synthesis of therapeutically significant glycoconjugates like glycosaminoglycans, glycoproteins, glycolipids, glycosylphosphatidylinositol-anchored proteins, glycosylated recombinant proteins, and the development of carbohydrate-based vaccines [34] and manufacturing glycoprotein pharmaceuticals [35, 36] . a new discipline born in 1980, glycobiology, has focused on the structural and functional characterization of glycoproteins and glycoconjugates. recently, glycobiology has evolved in glycomics regarding the investigation of the complete set of glycoconjugates and carbohydrates present in an organism. during the last five to ten years, many papers have been written on the role of glycosylation in different human diseases, whereas the highest contribution came out from the implication of glycosylation in cancer, followed by inflammation and involvement in the immune system ( figure 1 ). an overview of each of these aspects will be discussed to support the crucial role of glycosylation and the relevance of new ms technologies in glycomics for the identification and quantification of the glycopeptides and the relative glycans. immune system cells express glycoproteins and glycolipids associated with the cell surface able the number of papers published during the last five years seems to be time-dependent, suggesting that the interest for the glycosylation in any disease is enormously increasing (figure 1 ). what about the used technique along the time? even the number of papers providing the use of the nuclear magnetic resonance (nmr) technique is maintained linear along that time frame, although it resulted to be almost 5-fold lower than those dealing with the mass spectrometry (ms) approach (data not reported in the figure 1) . a number of papers as high as 4863 and 2570 during the period 2010-2020 and 2015-2020, respectively, makes mass spectrometry a helpful tool for glycomics and matrix-assisted laser desorption/ionization mass spectrometry (maldi-ms) or liquid chromatography tandem mass spectrometry (lc-ms/ms), equally contributing at the same aim (roughly 19% for both within last 5 years), whereas still a few papers referred to the targeted ms approaches) and even less to the lc-ms/ms in multiple reaction monitoring (mrm) ion mode ( figure 1 ). an overview of each of these aspects will be discussed to support the crucial role of glycosylation and the relevance of new ms technologies in glycomics for the identification and quantification of the glycopeptides and the relative glycans. immune system cells express glycoproteins and glycolipids associated with the cell surface able to detect environmental signals and modulate the adaptive immune response. a comprehensive review must deal with the antigenic role of glycoproteins, glycoconjugates, polysaccharides, or glycolipids as components of t-cell epitopes or toward their presentation by major histocompatibility complex (mhc) pathways on antigen-presenting cells [37] . glycopeptides can bind to mhc molecules and to specifically stimulate t-cells thanks to their glycosylated portion, suggesting the structural importance of carbohydrate moiety for the t-cell stimulation and of the amino acid sequence to be allocated within a closed binding groove of mhc-i or mhc-ii, in agreement with the length of peptide [38] . many immune receptors expressed on innate and adaptive immune cells recognize the glycans present on the surface as molecular epitopes associated with pathogens (examples include bacterial lipopolysaccharides and peptidoglycans) [39] . although protein glycosylation in prokaryotic organisms appears to be a rare event, regulated by a different machinery compared to eukaryotic cells, bacteria can express glycosylated protein [7] . glycoproteins displayed on their surface are involved in pathogenicity, antigenicity, host-pathogen interactions, and immunity evasion as well as having structural functions [40, 41] . for example, the alanine-proline-rich antigen (apa) glycoprotein, expressed on the cell surface of different mycobacteria species, induced glycan-specific t-cell response, whereas the non-glycosylated form of the same protein in escherichia coli showed reduced stimulation of the cd4 + t-cell system compared to the native antigen, giving evidence of the crucial involvement of glycosylation in t-cell activation by apa during infection [42] . a recent review greatly explored the different role of envelope glycoproteins along the virus pathobiology from immune evasion by glycan mimicry/shielding toward the recognition of glycans on host cell receptors up to induction of innate immune cell response mediated by complement activation [43] . other authors lingered on the spike (s) envelope protein of the currently emerging virus (cov) inducing severe acute respiratory syndrome (sars) to explain the crucial role of glycoprotein in infection initiation by binding receptor-binding domain of s protein to the cellular receptor ace2 and in the phase of viral envelope fusion with the host-cell membrane through the endosomal pathway [44] . actually, the s proteins of coronaviruses display a larger number of n-linked glycan sites (23-38) per protomer but a lower population of oligomannose-type glycans (30%) compared to the other viruses [45] . the increase of the number of glycosylation sites and the reduced density by oligomannose-type glycans seems to be based on an evolutive selection, reflecting a balance between immune evasion by epitope shielding and functionality by attachment to the host cell. although the s protein of sars-cov and sars-cov-2 showed~91% identity with a high number of glycosylation sites shared between these two viral strains, the exclusivity of new sites of n-glycosylation in sars-cov-2 allowed to speculate a different shielding and camouflage from the host defense system [46] . the study of the different mechanisms of recognition based on the identification of glycosidic components of the virus by the immune system are fundamental for the development of antiviral vaccines [44, 47, 48] . another important focus is the immune response by carbohydrates inducing the t cells to release cytokines in agreement to the activation mechanism [37] . in addition, known pro-inflammatory proteins, cytokines, can also induce direct changes to the n-glycosylation of endothelial cell membrane proteins, highlighting that glycosylation could contribute to the amplification of inflammatory vascular diseases [49] . a well-documented example is represented by the cd43 and cd45 glycoproteins abundantly expressed on the surface of b and t cells and which contain both o-glycans and n-glycans. during cell differentiation and activation, modulation of the glycosylation of these proteins is observed. this structural modification appears to be the molecular signal for the regulation of multiple t cell functions: cell migration, signaling of t cell receptors, cell survival, and apoptosis [50, 51] . aberrant glycosylation and/or alterations in serum protein glycosylation have been reported in many autoimmune and inflammatory diseases (e.g., rheumatoid arthritis, ra). the state of inflammation involves many physiological and biochemical systemic changes. many studies have found that the mutation of the glycoside structure linked to a polypeptide chain reflected the pathophysiological state of the cell producing the protein. therefore, knowledge of serum protein glycosylation can be an excellent starting point for the diagnosis and prognosis of many diseases [52, 53] . the most representative example of the involvement of mutations in the saccharide structure in the onset of autoimmune and/or inflammatory diseases is given by immunoglobulin g (igg). igg is a glycoprotein with a n glycosylation site conserved in the fc region and variable glycosylation (linked to o or n) in the fab region [54] . glycosylation of igg molecules is essential for its binding with all receptors through the maintenance of an open conformation of the two heavy chains, whereas deglycosylated igg antibodies are unable to mediate the inflammatory response triggered in vivo. a therapeutic application of gamma globulins intravenously indicates how these acts as anti-inflammatory [55] . among all inflammatory conditions, rheumatoid arthritis is that in which igg glycosylation has been studied mostly: decreased terminal sialylation and galactosylation of igg is resulted to be the common denominator of autoimmune disorders [56] . the defect in glycosylation probably involves a greater interaction with the rheumatoid factor (rf), an autoantibody, which could contribute to increase the activation of cytokines and therefore the inflammatory response of the subject [57, 58] . genetic defects in glycosylation (cdg) are often embryonic lethal, underlying the vital role of glycans in congenital defects affecting a single step in the formation of a glycoform or an entire pathway [59, 60] . in fact, the cdgs were categorized into two different classes: type i concerns anomalies in the formation of the oligosaccharide structure on the glycolipid precursor before the attachment on the asn residue of a protein; while type ii concerns anomalies in the control of the branched oligosaccharide n-linked structure present on the new glycoprotein [61] . cdg phenotypes can result from altered activation or transport of sugar precursors; and altered expression and/or activity of enzymes (glycosidases or glycosyltransferases) or proteins implicated in the golgi apparatus functioning. a clear illustration of congenital glycosylation disorder is the family of α-dystroglycanopathies whom frequently include alterations in the central nervous system [62] and ocular disease manifestations, in addition to muscular dystrophy, intellectual disability, developmental delay, hypotonia, macrocephaly, growth retardation, adducted thumbs, failure to thrive, cardiac anomalies, wrinkled skin, and early death. [39, 63] genetic alterations support the development of various pathologies, including neoplastic ones, but epigenetic changes in response to stimuli can significantly influence the genesis and neoplastic transformation. the dynamic redistribution of proteins between subcellular compartments in response to cellular functional state has been clearly described as a consequence of system perturbation underlying breast cancer [64, 65] . even the glycosylation pathways are driven by the cellular response to microenvironmental cues that activate oncogenic pathways reprogramming cancer cells along the entire disease evolution up to invasion and disease dissemination [66] . changes in protein glycosylation of both o-glycans (galnac-ser/thr) and n-glycans [67] can occur at the beginning as well as at end of cancer progression and metastasis. for instance, it has been shown that apparently minimal alterations in the structure of carbohydrates reflect the changes of cell surface components crucial for neoplastic transformations and the metastatic behavior of tumor cells [68, 69] . several reviews focus on the type of glycan alteration driving cancer hallmarks [66, 70, 71] : (1) glycan upregulation is a result of the cellular reprogramming likely due the metabolic shift from oxidative phosphorylation to aerobic glycolysis (warburg effect) [72] . the increase of cellular glucose not only contributes to more sustained glycolysis but also to the upregulation of the hexosamine biosynthetic pathway. in parallel, the increase of β1-6 highly branched n-glycans observed in cancer cells [73] is also due to upregulation of n-acetylglucosaminyltransferase v enzyme (gnt-v encoded by mgat5 gene). differently, gnt-iii enzyme (encoded by mgat3 gene) contrasts n-glycans elongation by the addition of a bisecting glcnac residue in a β1,4-linkage. the overexpression of branched-n-glycan structures has been shown to interfere with cell-cell adhesion mediated by cadherin-epithelium, promoting dissociation and invasion of cancer cells. (2) incomplete biosynthesis of glycans can be due to the impairment of the normal synthetic mechanism of complex glycans or misfolding of proteins, triggering the end of biosynthesis. truncated structures originated as a consequence of these events, often leading to the expression of tn and t antigens, more commonly occurring in early carcinogenesis [74] . (3) the de novo expression of specific glycoepitopes e.g., certain antigens (such as sialyl lewis a (sle a ) [siaα2,3galβ1,3(fucα1,4)glcnac] and sialyl lewis x (sle x ) [siaα2,3galβ1,4(fucα1,3)glcnac]) is observed in advanced stages of cancer [75, 76] . lewis antigens originate from branched-n-glycan structures elongated with poly-n-acetyllactosamine (repeats of galβ1,4 glcnacβ1,3) and further capped with fucose and sialic acid. these antigens are particularly enriched on the surface of cancer cells for high affinity to the carbohydrate-binding proteins, such as galectins and selectins. the interaction of glycans to both carbohydrate-binding proteins is a crucial event for neoplastic progression and the formation of cancer metastases [77] [78] [79] . an increased core fucosylation mediated by fucosyltransferase 8 is detected in metastatic melanoma [80] , whereas the fucosyltransferase 8 expression could facilitate invasion and tumor dissemination, in part due to a reduced cleavage of the cell adhesion molecule l1 [81] . in each of these situations, the types of cell surface glycans present on a given glycoprotein are dictated in part by the expression, location, and activity of glycosyltransferases in a given cell [82] . (4) other modifications on individual sugars, including o-acetylation of sialic acids and o-sulfation of galactose and n-acetylglucosamine residues can occur in cancer cells for modulating their growth and differentiation. (5) new motifs e.g., galβ1-4galβ1-can take place within the complex-type oligosaccharide chain as an expression of dramatic changes in their biosynthesis during oncogenic transformation. these new-formed glycans are capped with α2-3-linked sialic acid residues demonstrated to facilitate the migratory behavior and to increase the invasiveness of metastatic melanoma cells [83] . the final glycosylation product reflects the coordinated effort of many different enzymes, whose expression, localization, and post-translational modifications are significantly influenced from cellular response of metabolic reprogramming [2] . as a result, reagents that specifically interact with the glycan product are crucial to manage the potential changes in glycosylation as an effective diagnostic, prognostic, and even therapeutic target in routine clinical practice. although the examination of changes in glycosylation in cancer lesions poses unique challenges, recent developments in glycomics offer promising solutions and may reveal specific associations between altered glycosylation and neoplastic or diseases progression. technological breakthroughs in mass spectrometric analysis for specific glycan epitopes provide a more molecular approach to examine potential changes in glycosylation or to display a sufficient degree of alteration in glycosylation, as mixtures of commonly occurring glycosylation patterns associated with normal cells or tumor-associated signals [82, 84] . a very interesting study concerns follicular melanoma (fl), the most common indolent b cell lymphoma, which represents about 40% of all non-hodgkin lymphomas [85] . in this type of neoplasm, in about 85% of cases, variable domain glycans have been reported to be rich in high mannose structures [86, 87] . the discovery of these structures in almost all patients implies that they are useful or even essential for improving proliferation and survival [88] . in healthy donors, these high mannose structures have not been detected on cell surface, advancing the hypothesis that in fl glycans do not fully mature within the golgi complex due to enzyme inaccessibility [89, 90] . all these changes of glycosylated chains contribute to increased molecular heterogeneity of tumor cells compared with their non-transformed counterparts, which in turn can alter the glycan structure and function. therefore, the study of changes in the glycosylation mechanisms connected with the disease has become crucial to get information on the progression of cancer [91] . such investigations constitute a breeding ground in considering glycans as important markers in the early diagnosis, in the determination of the prognosis and in the stratification of the risk, as well as in serving as a target of specific drug therapies [70] . the bottom-up proteomics studies provide the use of simple protocols of in solution or in situ digestion to release peptides from proteins to be submitted to the ms analysis. this approach enables the detection of a wide plethora of peptides mainly free from any variable modifications, such as phosphorylation or glycosylation, for the higher ionization efficiency in comparison to the modified peptides. actually, an increase of the negative charge and the acidity of the phosphorylated or glycosylated peptides affects their ionization efficiency. this event clearly speculates that the ms signals of phosphorylated and glycosylated peptides undergo an effect of ion suppression due to the competition with a much higher number of non-modified peptide counterpart displaying a more intense ion current. many methods have been developed during the last decades to overcome the challenges associated to the glycoproteome analysis mainly based on mass spectrometry. the most widely used approach to characterize glycosylation involves the enzymatic or chemical cleavage of glycans from glycoprotein, followed by purification steps previous in the ms analysis. this method is limited exclusively to the glycoproteins with one glycosylation site, because it is unable to correlate glycan composition with the different glycosylation sites. this limitation can be overcome using another approach based on the characterization of intact glycopeptides released by glycoprotein proteolysis. thus, the glycan composition can be correctly correlated to the glycosylation site of specific glycopeptides by using this approach referred to as 'a glycosylation site-specific analysis'. the information obtained in a site-specific manner can be extremely important in correlating glycosylation profiles with specific glycosylation sites, which is useful in understanding structure-function relationships [92, 93] . numerous protocols are now effective for the highly sensitive characterization of broad glycans by ms analysis [94] . figure 2 summarizes the main stages of sample preparation combined with the ms analysis. the most used protocols for biological samples provide the enrichment and purification of glycopeptides by using different molecular mechanisms. to overcome ionization difficulties and to prevent the suppression of the glycopeptide signal from the non-modified peptides in complex mixtures, it is possible to combine several purification methods. one of the most common methods for purifying glycopeptides before ms analysis is reverse phase purification (rp) on the high-pressure liquid chromatography (hplc) system. this method provides a separation of the various glycopeptides based on their different amino acid sequence since the retention mechanism is governed by the hydrophobicity of peptide portion [95] . the techniques that could be combined with rp-hplc include purification strategies based on hydrazine resins, lectin affinity chromatography, carbohydrate-based gels such as cellulose or sepharose, and gel filtration, or size exclusion chromatography (figure 2) . a classical approach is based on the use of hydrazine-based resins to capture of n-linked glycoproteins. the oligosaccharides can be oxidized to the corresponding di-aldehydes and then immobilized on the solid hydrazide support. the resulting isolated glycoproteins are digested, and unmodified peptides washed out the resin. the glycopeptides are then enzymatically deglycosylated using pngase (peptide-n-glycosidase) f (or a), an enzyme cleaving the bond between glcnac and an asn residue converted to asp, and quantified by isotopic labeling [96] . the oxidative chemical coupling between the glycan and resin is also the own limitation due to structural modification of glycans [97] . additionally, chemical derivatization has several side reactions, therefore purification protocols based on lectin affinity chromatographic enrichment have recently been developed (figure 2) . a single lectin recognizes specific glycoforms, therefore lectins array could be used to capture several glycoproteins in a single step. multi-lectin affinity columns have been developed by combining different lectins, e.g., hancock and colleagues combine cona (concanavaline a), wga (wheat germ agglutinin) and jacalin for the analysis of serum glycoproteins [98] . these enrichment methodologies have been used for comparative studies of human serum in pathological and non-pathological samples in order to identify oligosaccharides as disease biomarkers [98] [99] [100] [101] [102] . alternative methods are based on the chemical and/or enzymatic hydrolysis of glycoproteins followed by multi-lectin affinity capture of the glycopeptides. this selective isolation of glycopeptides provides a great recovery of glycoforms because the steric hindrance by the protein portion seriously affects the interaction with lectin. other affinity purification techniques implemented in the last few years to purify glycopeptides are hydrophilic interaction chromatography (hilic) or normal phase chromatography, and porous graphitized carbon. the porous graphitized carbon technique is used to enrich glycopeptides with small peptide portions, since not enough selectivity is obtained with larger tryptic glycopeptides. hilic separation takes advantage of polar interactions between the hydroxy groups of glycans and the stationary phase. the efficient removal of the non-glycosylated counterpart takes place by using organic solvent washing followed by glycopeptide elution with an aqueous buffer as detailed in a study on the central nervous system glycoproteomics [103] . this method allows the glycopeptides separations based on their oligosaccharide portion, and its results are extremely useful when there is more than one glycosylation on the same peptide moiety. among the main enrichment techniques, the use of immunoaffinity columns plays an important role in the characterization of site-specific occupancy due to the natural affinity of glycan epitopes to the specific antibodies on the functional regions. more often, the immunoaffinity is combined to another mentioned above protocol to reduce the complexity and increase the recovery of n-glycoproteins, for instance, in human plasma [104] . the further advantages and disadvantages associated with each used strategy is extensively reviewed in previous papers [92, 93] . the analysis of glycans or glycopeptides has always been very challenging due to the limited quantities that are released from glycoproteins. since the structure of a glycan may depend on the expression, activity, and accessibility of the different biosynthetic enzymes, it is not possible to use recombinant dna technology in order to produce large quantities of glycans for structural and functional studies as it is for proteins. mass spectrometry could be a valuable tool for their characterization thanks to high sensitivity and selectivity. maldi-ms is an effective technique for n-glycan analysis of simple or complex matrices such as recently reported [105, 106] . amoresano et al. published a glycoproteomic characterization of human sera from healthy donors and patients affected by myocarditis for the identification of glycoproteins (even the least abundant), including the location of n-glycosylation sites and the profile of glycans present [107] . the strategy was simply based on the proteolytic digestion of serum proteins followed by a single enrichment step of glycopeptides by affinity chromatography using cona lectin. the glycopeptides were then deglycosylated by treatment with pngase-f and the free peptides analyzed by nano-lc/msms, which allowed both the identification of the individual proteins and the elucidation of their modification sites. profiles of oligosaccharides released by maldi-tof (time of flight) were also obtained. the glycans profile is obtained by maldi-tof analysis of the intact glycan mixture and the attribution of the different structures is carried out by checking the molecular weight and the knowledge of molecular pathways for the biosynthesis of oligosaccharides. however, this approach is useful in glycoforms profiling, but nevertheless it does not provide structural information such as sugar anomericity, neither on glycans site-specificity. to obtain this type of information, the combination of a profile by maldi-tof, with experiments of tandem mass spectrometry by post-source decay (psd) or collision-induced dissociation (cid), is generally required [108] . the lc-ms/ms of whole glycopeptides provide, instead, more information about the site-specificity of glycans. usually the cid fragmentation of the glycopeptides produces a wide fragmentation on the oligosaccharide portion (such as typical oxonium ion fragment at m/z 163 [105, 106] , and y-and b-type ions from the peptide moiety, therefore these ms/ms data are useful for assigning the glycan compositions (see figure 4 below). in an analogous way, neutral losses of saccharides such as hexose (162 da) , n-acetylhexosamine (203 da), fucose (146 da), n-acetylneuraminic acid (291 da) could be used to indicate the presence of glycopeptides in the mass spectra. other types of fragments, called cross-rings, may be useful in determining the glycosidic linkage. ms n experiments, on glycans moiety or directly on glycopeptides, are useful to characterize glycosidic structures present in glycoproteins as well as the type of branching, the sequence of the antennas, and the possible presence of modifying groups (e.g., sulfate, phosphate, acetyl groups, etc.). moreover, by selecting fragments (typically oxonium ions) of the most abundant glycopeptides, it is possible to set up a selective ion monitoring (sim) method for glycopeptides identification with high sensitivity in ion trap ms [107] , quadrupole-tof mass analyzers [108] . another fragmentation method used in the analysis of glycopeptides are electron-capture dissociation (ecd) and electron transfer dissociation (etd). in both techniques, the glycan portion does not undergo fragmentation while the peptide fragments provide both the z and c ions (see figure 4 below). ecd experiments are typically performed on ionic resonance instruments of the fourier transform cyclotron (ft-icr) while the etd can be performed in an ion trap mass spectrometer. finally, the combination of ion mobility-mass spectrometry (im-ms) to the fragmentation induced by cid has been successfully used for improving the glycoform separation; indeed, the higher charged states associated to branched glycans generate repulsive interactions more intense than those less branched, leading to an improved separation between the different glycoforms on the same glycosylation site [109] . many biomedical researches were based on the use of im-ms to determine the profile of glycans in glycoproteins, as some specific glycans can change with disease progression [110] . clemmer's group showed that im-ms was a promising technique to explore the glycan heterogeneity and glycan isomers [111] and to associate for the first time the ion mobility distributions of specific glycans to pathological conditions in liver cancer and cirrhosis patients [112] . structural characterization of complex carbohydrates is labor-intensive and time-consuming and requires orthogonal methods to identify: (i) the constituent monomers, (ii) their sequence including branching points, (iii) configuration of the glycosidic bond, (iv) position of the glycosidic bond, (iv) anomeric configurations [113, 114] . in recent years, the high-throughput ms analyses of glycoforms have made significant progress and are now commonly applied [115, 116] . although ms has developed to be one of the most powerful technologies for the analysis of structures (e.g., protein sequencing, structural characterization of small organic molecules), it rarely allows to differentiate isobaric monosaccharide residues and to get information on the monosaccharide linkage, as mentioned above. glycoproteins and their associated glycans quantification using ms techniques is at a nascent stage [117] . the improvements made in the sample preparation protocols and in the development of high-throughput platforms have led to an increase in the request of tools supporting data analysis in order to overcome the limitation of time expenditure [118] [119] [120] . to this aim, many software applications (open access and commercial) have emerged in recent years to offer new and promising capabilities as greatly reported in recent reviews [121] [122] [123] [124] . although these tools differ in the computational strategies, many of them have been designed to support specific workflows and/or limited data sets. as described by liang et al., all these tools share the same fundamentals: (i) determining the accurate mass in the precursor ms spectra; (ii) hypothesis of glycan composition and the peptide backbone sequence from the analysis of the lc-ms/ms spectra; (iii) interrogation of theoretical spectra contained in databases of proteins and glycans that matches the marker fragment ions for the peptide backbone and glycan, or attempt to de-novo sequencing the portion of the glycan; and (iv) data validity score [124, 125] . the use of such predictive software is particularly valuable for implementation of the lc-ms/ms method based on mrm, an ion mode widely recognized for the quantification accuracy of molecules. the main applications of mrm were limited to the metabolomics and proteomics, but their usefulness is also expanding in the field of glycoscience. mrm methods are very suitable for robust, fast, sensitive, and specific quantitative analysis of multiple target compounds, simultaneously detected also in the presence of other more abundant compounds (for instance, in complicated mixtures, such as biofluids). since mrm is a targeted approach, both the knowledge of the mass and charge state of the analyte and its fragmentation behavior in cid are essential for the choice of best transitions during the method development. numerous studies have focused on the fragmentation model of glycans and glycopeptides [126] [127] [128] , emphasizing that the typical fragmentation occurs on glycosidic bonds under low energy cid conditions normally used in triple quadrupole instruments. in these low energy conditions, the cross-ring cleavages are often low abundant, while intense fragments of glycan (oxonium ions) and those related to peptide fragmentation have been shown to be characteristic of each n-glycopeptides. several authors performed glycoprotein quantification directly on the enzymatic digest of the plasma without any enrichment step in glycopeptides by using specific characteristic fragment ions [129] [130] [131] . as an example, an mrm/ms method was developed by hong et al. to quantify immunoglobulins igg, iga, and igm and their glycoforms by using the most intense signals associated to oxonium ion fragments for the quantification. on the other hand, in the case of o-glycosylation, the major fragment of the o-glycosidic bond is typically the y0 fragment. these characteristic fragment ions are valuable to set up an mrm/ms method for glycoproteomics [132] [133] [134] [135] . characteristic oxonium ions, which represent hexose (m/z 163), hexnac (m/z 204), neuac (m/z 292), hexhexnac (m/z 366), hexhexnac-fuc (m/z 512), and hexhexnacneuac (m/z 657), with or without loss of water, were used as reporter ions [136] . moreover, due to the high variability of glycosylation, an important step in developing an mrm method for glycoproteins is the peptide and glycopeptide profiling (normally performed by lc-q-tof ms/ms) to evaluate the fragmentation behavior of the peptides and for validating the assignment of parent glycopeptide ion [137] . a glycosylation profile of standard (sigma) pituitary human follicle stimulating hormone (hfsh) was performed by lc-ms/ms analysis using q-exactive plus spectrometer by chiara guerrera and coworkers (paper under revision). an example of high resolution ms 2 fragmentation spectrum of hfsh alpha chian glycopeptide ([hexnac4hex5neuac1] glycosylation on asn52 site of l.vqkn 52 vtsestccvaksy.n peptide) was reported in figure 3a . the fragmentation pattern of high-energy collision dissociation (hcd) is an alternative type of fragmentation method characterized by higher activation energy and shorter activation time comparing to the traditional ion trap cid (figure 4) . hcd for peptides with posttranslational modifications (ptms) can provide both the sequence information (b-and y-type fragment ions) and the localization of the modification sites as it can identify cid-labile ptms. thus, high mass accuracy ms2 spectra have been successfully applied for ptms studies, as certain diagnostic ions specific for hcd could be recognized for ptms identification [138] . the ms/ms spectra offered a valid support for the choice of specific transitions to be used during the development of the quantitative mrm method. actually, once established, the glycosylation pattern of the glycopeptide of interest, the most intense signals were selected to set up the mrm method. the extracted ion currents (eic) associated to the different transitions for the same glycopeptide were recorded at the same retention time. this finding ensured the unequivocal identification above to contribute glycoprotein quantification. in figure 3b , is shown the eic best transitions of figure 3a glycopeptide. since the peptide's glycan is a di-antennary mono-sialylated structure, this glycopeptide will exist as a combination of two isoforms generated by the position of the sialic acid on one or the other antenna and characterized by slightly different retention times ( figure 3b) . therefore, transitions are representative of intense fragments ions that are unique for the peptide to be quantified, thus ensuring high sensitivity and reduced interference from other peptides [139, 140] . major complications can also arise in developing a glycopeptide quantification method because of the absence of exogenous glycopeptide standards and incomplete proteolytic digestion caused by steric hindrance due to the glycan chains [132, 141, 142] . represents nacetyl-glucosamine; represents mannose; represents galactose; represents c sialic acid. the fragmentation pattern of high-energy collision dissociation (hcd) is an alternative type of fragmentation method characterized by higher activation energy and shorter activation time comparing to the traditional ion trap cid (figure 4) . hcd for peptides with posttranslational modifications (ptms) can provide both the sequence information (b-and y-type fragment ions) and the localization of the modification sites as it can identify cid-labile ptms. thus, high mass accuracy ms2 spectra have been successfully applied for ptms studies, as certain diagnostic ions specific for hcd could be recognized for ptms identification [138] . represents nesents mannose; represents galactose; represents c sialic of high-energy collision dissociation (hcd) is an alternative type of rized by higher activation energy and shorter activation time n trap cid ( figure 4) . hcd for peptides with posttranslational e both the sequence information (b-and y-type fragment ions) and on sites as it can identify cid-labile ptms. thus, high mass accuracy lly applied for ptms studies, as certain diagnostic ions specific for ms identification [138] . represents n-acetyl-glucosamine; cells 2020, 9, x acetyl-glucosamine; represents mannose; represents galactose; repr acid. the fragmentation pattern of high-energy collision dissociation (hcd) is an fragmentation method characterized by higher activation energy and short comparing to the traditional ion trap cid (figure 4) . hcd for peptides with modifications (ptms) can provide both the sequence information (b-and y-type f the localization of the modification sites as it can identify cid-labile ptms. thus, h ms2 spectra have been successfully applied for ptms studies, as certain diagnos hcd could be recognized for ptms identification [138] . the fragmentation pattern of high-energy collision dissociation (hcd) is an alt fragmentation method characterized by higher activation energy and shorter comparing to the traditional ion trap cid (figure 4) . hcd for peptides with p modifications (ptms) can provide both the sequence information (b-and y-type frag the localization of the modification sites as it can identify cid-labile ptms. thus, hig ms2 spectra have been successfully applied for ptms studies, as certain diagnostic hcd could be recognized for ptms identification [138] . represents nnose; represents galactose; represents c sialic rgy collision dissociation (hcd) is an alternative type of higher activation energy and shorter activation time ( figure 4) . hcd for peptides with posttranslational sequence information (b-and y-type fragment ions) and t can identify cid-labile ptms. thus, high mass accuracy for ptms studies, as certain diagnostic ions specific for ication [138] . represents c sialic acid. as reported by lebrilla et al., the application of mrm in the field of glycomics can be divided into three main areas: quantification of glycoproteins, glycopeptides, and oligosaccharides, showing each of them intrinsic troubles. in order to quantify glycoproteins, the crucial step is the selection of glycopeptides to be monitored [143] . sample treatment protocols, as well as the choice of proteolytic enzymes, also largely influence the mrm analysis of both glycoproteins and glycopeptides [144, 145] . enzymatic hydrolysis is typically carried out with trypsin but, due to the uneven distribution of lysine and arginine residues in the amino acid sequences of proteins, this can generate high mw peptides not easily detectable by ms. numerous advantages in this field have been obtained by using immunoaffinity enrichment of peptides or proteins followed by mrm/srm-based quantification, achieving sensitivity suitable to the concentration range (ng/ml) at which low-abundance biomarkers are normally found [146] [147] [148] . furthermore, in the analysis of glycopeptides, doubly glycosylated peptides can be generated, thus complicating the resolution of the glycosylation profile. quantification strategies can be separated into label-based or label-free; standards with label or internal standards are often used in mass spectrometry to minimize the effects of ionization and to increase the accuracy of absolute and relative quantification. in particular, the type of labeling preferred in mrm analyzes is non-isobaric [149] . in order to overcome the limits of the large quadrupole inclusion windows (generally at least 0.5 da), an alanine labeled with d 6 has been used for the labeling of glycans, resulting in an ∆m of 6 da [150] or lysine and arginine for labeling at 13 c and 15 n of proteotypic peptides (∆m 8 and 10 da). anyway, in order to conduct a quantitative analysis of the glycoforms present in a mixture, it is essential to quantified non-glycosylated peptides in order to normalize the glycosylation profile to the total protein content [151] . figure 3a is used as example. the ms/ms spectra offered a valid support for the choice of specific transitions to be used during the development of the quantitative mrm method. actually, once established, the glycosylation pattern of the glycopeptide of interest, the most intense signals were selected to set up the mrm method. the extracted ion currents (eic) associated to the different transitions for the same glycopeptide were recorded at the same retention time. this finding ensured the unequivocal identification above to contribute glycoprotein quantification. in figure 3b , is shown the eic best transitions of figure 3a glycopeptide. since the peptide's glycan is a di-antennary mono-sialylated structure, this glycopeptide will exist as a combination of two isoforms generated by the position of the sialic acid on one or the other antenna and characterized by slightly different retention times ( figure 3b) . therefore, transitions are representative of intense fragments ions that are unique for the peptide to be quantified, thus ensuring high sensitivity and reduced interference from other peptides [139, 140] . major complications can also arise in developing a glycopeptide quantification method because of the absence of exogenous glycopeptide standards and incomplete proteolytic digestion caused by steric hindrance due to the glycan chains [132, 141, 142] . as reported by lebrilla et al., the application of mrm in the field of glycomics can be divided figure 3a is used as example. quantitative protein assays by using targeted mrm strategies have been developed to investigate protein concentrations in various biological fluids [152] [153] [154] e.g., in human plasma or serum, and other animal biofluids (e.g., bovine milk) [155] [156] [157] . some examples of glycoproteins quantification based on mass spectrometry in mrm mode are annotated below to support the high potential and versatility of approach even in rapid routine clinical screening. recently, a method for the quantification of total glycosylated and sialylated prostate-specific antigen (psa) was recently developed. periodate-oxidized psa tryptic glycopeptides are captured using immobilized hydrazide, released by pngase f, and quantified by mrm using a triple quadrupole lc-ms [158] . in a recent study by song et al. [159] , mrm assays were developed for the quantification in serum samples of fetuin and alpha1-acid glycoprotein glycopeptides. kurogochi et al. developed an mrm assay to identify and quantify 25 glycopeptides from 16 different glycoproteins found in mice serum [135] . moreover, ms in mrm mode has long been used for the quantitative determination of haptoglobin glycopeptides in the serum of psoriasis patients [160] or affected by pancreatic cancer [161] . lebrilla et al. applied the lc-ms/ms methodology to investigate the glycoprotein profile of human milk by selecting the best transitions precursor ion-product ions for each glycopeptide. the extensive literature on protein glycosylation reveals numerous examples in which these post-translational modifications play essential roles in the events of biological recognition, signaling and cell-cell communication. in fact, oligosaccharide structures perform crucial functions throughout the cell: in the cytosol, on the cell surface, in the secretory compartments, and in the extracellular space. among the many roles that glycans play on the cell surface, the importance of specific glycosylated forms of the protein domain to facilitate or modulate biological recognition events has been highlighted. therefore, the knowledge of the diversity of the structures of glycans becomes a further level of information content in the understanding of biological systems, laying the foundations for the greatest challenge of the near future, that is, identifying the critical contexts in which the functions of the glycans contribute to the biological regulation of the inside the surprisingly large array of heterogeneous glycan structures. as widely described in this review, recent advances in qualitative and quantitative analytical strategies based on the use of mass spectrometry provide the necessary breadth, depth, and sensitivity of the analysis to define the entire spectrum of the complexity of glycan in various biological contexts. the continuous high-performance adaptations of these methodologies enable the collection of essential structural datasets to reveal the mechanisms in the biosynthetic pathway regulation, to define unique glycan signatures for pathological states and to provide correlations between structure and biological functions. in this way, by increasing the ability to decode the numerous functions of glycoprotein or glycans in complex biological systems, the development of new therapeutic approaches, such as vaccines or targeted pharmacotherapies, is encouraged. although technological progress in the field of targeted mass spectrometry has made great strides in the recognition of specific glycan structures, future studies are necessary for the realization of standardized methodologies that can be used in common clinical and diagnostic practice. with the improvement and evolution of ms technology and sample preparation techniques, these types of test will play a more important role in the 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liver tissue by combination of multiple enzyme digestion and hydrazide chemistry characterization of glycopeptides from hiv-isf2 gp120 by liquid chromatography mass spectrometry targeted quantitative analysis of streptococcus pyogenes virulence factors by multiple reaction monitoring multiple enzymatic digestion for enhanced sequence coverage of proteins in complex proteomic mixtures using capillary lc with ion trap ms/ms consecutive proteolytic digestion in an enzyme reactor increases depth of proteomic and phosphoproteomic analysis quantification of low-abundance proteins in biological fluids. in serum/plasma proteomics an automated and multiplexed method for high throughput peptide immunoaffinity enrichment and multiple reaction monitoring mass spectrometry-based quantification of protein biomarkers peptide immunoaffinity enrichment coupled with mass spectrometry for peptide and protein quantification stable isotope labeling methods in protein profiling targeted glycomics by selected reaction monitoring for highly sensitive glycan compositional analysis applications of multiple reaction monitoring to clinical glycomics combinatorial peptide libraries facilitate development of multiple reaction monitoring assays for low-abundance proteins multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution product ion monitoring" assay for prostate-specific antigen in serum using a linear ion-trap qualitative and quantitative profiling of the bovine milk fat globule membrane proteome advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics multiple reaction monitoring-based determination of bovine α-lactalbumin in infant formulas and whey protein concentrates by ultra-high performance liquid chromatography-tandem mass spectrometry using tryptic signature peptides and synthetic peptide standards simultaneous analysis of glycosylated and sialylated prostate-specific antigen revealing differential distribution of glycosylated prostate-specific antigen isoforms in prostate cancer tissues multiple-reaction monitoring liquid chromatography mass spectrometry for monosaccharide compositional analysis of glycoproteins quantitative determination of haptoglobin glycoform variants in psoriasis site-specific analysis of n-glycans on haptoglobin in sera of patients with pancreatic cancer: a novel approach for the development of tumor markers selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants the sweet and sour of serological glycoprotein tumor biomarker quantification this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license author contributions: all authors have contributed to write and revise the review. all authors have read and agreed to the published version of the manuscript.funding: authors wish to thank european union (fse, pon ricerca e innovazione 2014-2020, "dottorati innovativi con caratterizzazione industriale"). this research received no external funding. we thank chiara guerrera for providing assistance for lc-ms/ms analysis by using q-exactive plus spectrometer. this publication was in partial fulfilment of the requirements for the degree of phd in chemical sciences−xxxiii cycle, university of naples federico ii. the authors declare no conflict of interest. key: cord-279432-aik5bo6o authors: digard, paul; blok, vivian c.; inglis, stephen c. title: complex formation between influenza virus polymerase proteins expressed in xenopus oocytes date: 1989-07-31 journal: virology doi: 10.1016/0042-6822(89)90523-0 sha: doc_id: 279432 cord_uid: aik5bo6o abstract all three influenza virus polymerase (p) proteins were expressed in xenopus oocytes from microinjected in vitro transcribed mrna analogs, with yields of up to 100 ng per oocyte. to examine the functional state of the xenopus-expressed p proteins, the polypeptides were tested for their ability to form stable complexes with each other. as seen in virus-infected cells, all three p proteins associated into an immunoprecipitable complex, suggesting that the system has considerable promise for the reconstruction of an active influenza rna polymerase. examination of the ability of paired combinations of the p proteins to associate indicated that pb1 contained independent binding sites for pb2 and pa, and so probably formed the backbone of the complex. sedimentation analysis of free and complexed p proteins indicated that pb1 and pb2 did not exist as free monomers, and that similarly, complexes of all three p proteins did not simply consist of one copy of each protein. the heterodisperse sedimentation rate seen for complexes of all three p proteins did not appear to result from their binding to rna, suggesting the incorporation of additional polypeptides polymerase complex. influenza virus gene expression depends on the coordinate transcription and replication of eight segments of negative-sense single-stranded rna. this is mediated by the three viral polymerase proteins pbl , pb2, and pa in association with the nucleoprotein (lnglis et a/., 1976) and possibly with other viral proteins or unidentified host cell factors. on infection of a permissive cell the ribonucleoprotein-polymerase complexes migrate to the nucleus where viral mrnas are transcribed (her-z et a/., 1981) . these mrnas are initiated by, and contain, 5'-capped rna fragments generated by a viral cap-dependent endonuclease from host cell mrnas (plotch eta/., 1979) . they are also polyadenylated, but lack sequences complementary to the extreme 5'-end of their corresponding genome rna template (hay et a/., 1977a) . at later times, dependent on the production of virus specific proteins, full-length, nonpolyadenylated, noncapped copies of the genomic rnas are made, which then serve as templates for production of more vrna (hay et a/., 197713) . the polymerase (p) proteins have been shown to exist and probably function as a complex both in virions (braam et al., 1983) and in the infected cell (detjen et a/., 1987; ak-' to whom requests for reprints should be addressed. kina et a/., 1987), but little is known about the role of the individual proteins, or the precise composition of the (presumably differing) complexes which catalyze the synthesis of the three types of virus rna. to address the structure-function relationship of the polymerase proteins a system is needed whereby individual virus proteins can be expressed in an appropriate environment in sufficient quantity to facilitate reconstitution of activity. it should then be possible to dissect the system to reveal the individual functions of particular proteins. our approach has been to seek expression of the polymerase-associated polypeptides through transcription of cloned dna into artificial mrna analogs using the bacteriophage sp6 rna polymerase, and translation of these mrnas in xenopus oocytes. such a system offers the advantage that proteins may be produced individually, or in any desired combination, simply by translating the appropriate mrna mixture. in addition, since the polypeptides can be produced simultaneously in an environment similar to that in the infected cell, the likelihood is that they will display appropriate physiological interactions. thus, the system offers considerable promise for the reconstitution of enzyme activity. in this report we describe the establishment of such a system and its preliminary characterization. in addition, as a first step toward reconstitution of enzyme ac-tivity, we show here that in the absence of any other virus specific polypeptides all three p proteins can interact to form complexes, and report preliminary characterization of these complexes. all enzymes were obtained from boehringer-mannheim, and all radiochemicals from amersham (england). nuclease-treated rabbit reticulocyte lysate was obtained from dr. t. hunt (cambridge). influenza strain a/pr8/34 was propagated in, and purified from, embryonated eggs as previously described (inglis et al., 1976) . infected cell lysates were prepared at 6 hr postinfection from confluent monolayers of chick embryo fibroblasts infected at a multiplicity of infection of around 10. transcription vectors for the three polymerase genes were constructed by the insertion of cdna copies of the relevant influenza a/pr8/34 segments (the generous gift of dr. p. palese) into a plasmid containing an sp6 rna polymerase promoter. the sp6 vector used, psp64-t (krieg and melton, 1984 ; obtained from dr. a. colman), also provides flanking 5'-and 3'-noncoding sequences from the xenopus ,&globin gene, which enhance the translation of foreign genes in oocytes (drummond et al., 1985) . copies of segments 1, 2, and 3 were excised from the plasmids paprlo1, papr206, and papr303 (young et a/., 1983) by digestion with the restriction enzymes barnhi, hindill, and ecori, respectively. segment 1 was inserted directly into the bg/ll site separating the 5'-and 3'-globin noncoding sequences in psp64-t, while segments 2 and 3 were end-filled by klenow fragment dna polymerase (boehringer) and blunt-end-ligated into a similarly endfilled psp64-t. the resulting plasmids containing the pbl , pb2, and pa genes, in positive orientation relative to the sp6 promoter, were designated pstl+, pst2+, and pst3+, respectively. all manipulations were carried out according to standard procedures (maniatis et a/., 1982) . in vitro transcription and translation was carried out as previously described (brierley et a/., 1987) . briefly, the three transcription plasmids were linearized downstream to the globin 3'-noncoding sequence by digestion with smal, and sp6 rna polymerase run-off transcripts were synthesized under conditions which re-sulted in the incorporation of a synthetic 5'-m'gpppg cap structure (new england bio-labs). the product rna was then phenol-extracted and checked for structural integrity by agarose gel electrophoresis (maniatis eta/., 1982) . for in vitro translation, messenger-dependent reticulocyte lysate (mdl; pelham and jackson, 1976) was programmed with mrna to a final concentration of around 0.1 pg/pl and incubated at 30" for 1 hr. oocytes were taken from the frog, maintained, and injected essentially according to standard procedures (colman, 1984) . each oocyte received a maximum of 50 ng of rna in a constant injection volume of 50 nl. at 2 hr postinjection, groups of eight oocytes per rna were transferred to modified barth's saline (mbs) containing [35s]methionine (sp act 1 150 cvmmol; amersham, england) at 1 .o mci/ml (10 &i/oocyte) and subsequently harvested by mechanical disruption into tkm buffer (20 mm tris-hci, ph 7.6, 100 mni kci, 5 mm mgcl>, 1 mm phenylmethylsulfonyl fluoride (pmsf), 50% glycerol; 10 plloocyte) at 5 hr postinjection. the resulting lysates were then clarified by microcentrifugation and stored at -20" prior to analysis. the production of antisera directed against the pb2 protein has already been described (brierley et a/., 1987) and a similar strategy was used to prepare antisera against pbl and pa. briefly, portions of p protein coding sequence were expressed in bacteria as c-terminal fusions with ,&galactosidase, using the pex series of plasmids (stanley and luzio, 1984) . these fusion proteins were then purified by gel elution and used to immunize rabbits. in this study, antibodies raised to amino acids 50-370 of pbl, 342-463 of pa, and 585-759 of pb2 (f5) were used. lmmunoprecipitation from oocyte lysates for immunoprecipitation, 10 ~1 of [35s]methionine-labeled oocyte lysate (containing the equivalent of one oocyte) was diluted to 100 ~1 with oocyte immunoprecipitation buffer, (oipb; 50 mm tris-hci, ph 7.6, 100 mm kci, 5 ml\/l mgci,, 1% triton x-l 00, 1% sodium deoxycholate, 0.1% sds, 1 mll/l pmsf) and left on ice for 30 min before the addition of 4 ~1 of rabbit antiserum. after a further 30 min on ice, 50 ~1 of a 50% suspension of protein a-sepharose (sigma) in oipb was added, and the tubes were rotated at 4" for 30 min. finally, the sepharose-bound material was collected by centrifugation, washed once with 1 ml of oipb, and pbid pb2d ipa abcdefghij fig. 1. expression of the influenza virus p proteins in xenopus oocytes, and their reactivity with the monospecific antipolymerase sera. lanes a, d. g, j-unprecipitated lysates: oocytes microinjected with pstl+. pst2+, pst3+, h20, respectively. lanes c, f, i-precipitated with a-pbl , or-pb2, a-pa, respectively. lanes b, e. h-precipitated with the corresponding preimmune bleeds. eluted in laemmli sample buffer (laemmli, 1970) . proteins contained in the supernatant were separated on a 10% polyacrylamide gel and detected by autoradiowwb immunological detection of nitrocellulose-bound proteins samples were subjected to polyacrylamide gel electrophoresis (page) (laemmli, 1970) and transferred to nitrocellulose according to standard procedures (towbin et a/., 1979). the nitrocellulose was blocked by incubation with a solution of 4% bsa in pbs for 1 hr at 37", followed by a 1 hr incubation at room temperature with the antiserum-diluted 1 in 200 in 4% bsa, 2% newborn calf serum in pbs. the blot was then rinsed with lo/o np-40 in pbs, before incubation with 1 &i of lz51labeled protein a for 1 hr. a final wash with 1% np-40 in pbs was carried out before the blot was air-dried and exposed to film. oocyte lysates (from 10 oocytes) were layered on top of linear 5-20% (w/v) sucrose gradients in 20 ml\/l tris-cl, ph 7.6, 100 mm kci, 5 mm mgc&, 0.1% np-40, 1 mm pmsf and centrifuged at 100,000 g,, for 12 hr. fractions were then collected, adjusted to 1% triton x-l 00, 1% sodium deoxycholate, 0.1% sds, and analyzed for their p protein content by immunoprecipitation and page. all gradients had bovine serum albumin (bsa; m, 68,000) and apoferritin (rji, 440,000) included as internal standards. to test the capacity of xenopus oocytes for expression of the influenza p proteins, artificial mrnas corresponding to each gene were transcribed in vitro using the sp6 rna polymerase and microinjected into the oocytes. after a 2 hr recovery period, the cells were labeled by incubation with [35s]methioninefor 3 hr, harvested, and analyzed by gel electrophoresis before and after immunoprecipitation with specific anti-p protein sera (fig. 1) . in the unprecipitated tracks, strong bands of the expected mobility could be seen [lane a, pstl+ transcript (pbl); lane d, pst2+ transcript (pb2); lane g, pst3+ transcript (pa)] and were not seen in control water-injected oocytes (lane j). in each case these polypeptides were specifically precipitated by the homologous antiserum (lanes c, f, i), but not by the corresponding preimmune sera (lanes b, e, h). the extent of radiolabeling of the p proteins in the above experiment suggested that their production was quite efficient. to obtain a quantitative estimate, the accumulation of the p proteins was examined over a period of time after microinjection of mrna, by western blotting with the directed antisera. the result of such an experiment for pbl is shown in fig. 2a . the antiserum specifically detected a single band in lysates from pst1 +-injected oocytes which comigrated both with [35s]methionine-labeled pbl (synthesized in mdl using pstl+ transcript) and with pbl from purified virions. the results indicated that the total amount of pbl increased up to 40 hr postinjection and then declined slightly. known amounts of purified virus were also included in the western blot as standards to quantify the amount of pbl produced. since the equivalent of half an oocyte was loaded in each track, it was estimated from densitometry scans that after 40 hr each oocyte had accumulated on average 100 ng of pbl [assuming that pbl comprises 1% w/w purified influenza virions (inglis et al., 1976) ]. similar analyses were carried out for the other p proteins (data not shown). the accumulation of pa was similar to that of pbl, but pb2 was produced less efficiently (approximately 10 ng per oocyte). this appears to be the result of protein turnover, since microinjected [35s]methionine-labeled pb2 (synthesized prior to injection in mdl programmed with pst2f transcript) was found to have a half-life of 3 hr, and since the accumulation of pb2 was similar to that of the other p proteins up to about 3 hr postinjection (data not shown). a further experiment was carried out to assess p protein production in oocytes relative to virus-infected cells; fig. 2b shows a western blot comparing the amount of pbl accumulated per half oocyte 36 hr after microinjection, with that present in an equivalent amount (in terms of total protein) of chick embryo fibroblast (cef) cells at 6 hr postinfection (approximately 10" cells). a more intense signal was observed from the pst1 +-injected oocyte track than that from the infected cell track. however, the rate of production in the infected cell must be higher given the difference in incubation times. nevertheless, the overall quantities seem comparable. an important initial step in the biogenesis of the influenza polymerase is likely to be the formation of a complex of the three p proteins; accordingly, the oocyte-expressed p proteins were examined for their ability to associate with each other. groups of a dozen oocytes were injected with solutions containing one, two, or all three of the mrna analogs. for these experiments, the rnas were mixed in equal quantities and single rnas were diluted in hz0 to keep the concentration of any one transcript constant throughout. the oocytes were then metabolically labeled and harvested 3 . analysis of the ability of oocyte-expressed p proteins to form complexes with each other. oocytes were injected wrth the combinations of pst transcripts shown, and radiolabeled lysates prepared as described under materials and methods. the lysates were then precipitated with lu-pbl (lanes l), a-pb2 (lanes 2) or a-pa (lanes 3). see text for full description, as before. a 3-hr labeling period was employed to avoid the problem of the instability of pb2, and allow the production of approximately equal amount of all three proteins. following harvest, the oocyte lysates were analyzed by immunoprecipitation and page (fig. 3) . the antisera can be seen to be truly monospecific in that none of the p proteins were significantly precipitated by the heterologous antisera when expressed in isolation (lanes a-i). however, each antiserum precipitated all three p proteins from oocytes injected with a mixture of all three transcripts (lanes j-l). this specific coprecipitation provides evidence that when cotranslated in an oocyte, the p proteins are present as a complex. given that all three p proteins would associate into a complex, it was of interest to determine which of the polypeptides were interacting with which. therefore, paired combinations of the mrna analogs were microinjected, and their products assayed for association as before by immunoprecipitation with specific antisera (fig. 3, lanes m-r) . pbl and pb2 (lanes m and n), and pbl and pa (lanes q and r) were found to coprecipitate, but not pb2 and pa (lanes o and p), thus indicating that pbl may act as the backbone of the complex, and also implying discrete binding sites on pbl and pb2 and pa. in order to characterize further the association of the three p proteins in oocytes, individual and complexed the absence of pb2 in the fourth fraction from the top of the gradient is the result of an error during the immunoprecipitation of that sample. p proteins were analyzed by velocity gradient sedimentation. the aims of this were twofold; first to confirm the physical existence of complexes, by showing an increased sedimentation rate for the coexpressed p proteins, and second, to examine the size of complex formed. lysates from oocytes microinjected with single or mixed p protein mrnas were fractionated on sucrose gradients, and then each fraction was assayed for its p protein content by immunoprecipitation. figure 4 shows the result of this experiment for individually expressed p proteins. pa migrated slightly faster than the bsa marker (as expected for an m, 82,000 protein), but surprisingly pbl and pb2, which are similar in size, sedimented as much larger and more heterogeneous bodies. the majority of the proteins sedimented in fractions corresponding to a size of around m, 250,000, but significant amounts of material were also present in fractions corresponding to much larger sizes. for pbl and pb2 then, it seemed likely that demonstration of a convincing mobility difference between the individual and complexed form would be difficult. however, such an experiment remained possible for pa, given its lower and more discrete sedimentation rate. accordingly, oocyte lysates containing either pa alone or pa in combination with pbl and pb2 were fractionated on a gradient, and the fractions immunoprecipitated with anti-pa serum. the results of this experiment are shown in fig. 5 . again, paalone migrated as a reasonably defined band near the top of the gradient (top panel). however, in the presence of the other two p proteins, it sedimented throughout the whole of the gradient (middle panel), suggesting its inclusion in complexes. in addition, the coprecipitation of pbl and pb2 with pa could also be seen, further confirming the existence of an interaction between the polypeptides. it is interesting to note, however, that the ratio of the three polypeptides present in the complexes was not constant throughout the gradient. in particular, the ratio of pbl and pb2 to pa increased in the faster sedimenting species, suggesting either that pa has more than one binding site for each of the basic p proteins, or perhaps more likely, that the former proteins can form complex structures linked through self-association. from the migration pattern of pa seen in complexes, the marked heterogeneity in sedimentation rate of pbl and pb2 appears to be extended to a complex of all three p proteins. in view of the fact that the p proteins must interact with rna, it seemed possible that the high sedimentation values obtained for individually expressed pbl and pb2 (fig. 4) and for complexes containing pbl and pb2 could have arisen from the polypeptides binding to rna present in the lysate. if this was the case, rnase treatment of the lysate prior to gradient fractionation should significantly decrease the sedimentation rate of the complexes. the bottom panel in fig. 5 shows the result of such rnase treatment; no difference in mobility between treated and untreated complexes could be seen. furthermore, rnase treatment of lysates containing individually expressed pb2 also failed to affect its sedimentation pattern (not shown), suggesting that the heterogeneous size distribution of the p protein complexes is not the result of association with rna. a major goal in the study of the influenza polymerase is the reconstitution of an active enzyme from cloned components. this would then allow detailed analysis of the biochemical reactions catalysed by the enzyme, while manipulation of the dna templates would facilitate structural and functional analysis of individual components. here, we have demonstrated the feasibility of producing all three influenza virus polymerase proteins for functional studies by the translation of in vitro transcribed mrna analogs in xenopus oocytes. high levels of expression were achieved for pbl and pa after prolonged incubation, reaching 100 ng per oocyte. however, pb2 appeared to be unstable, with a half-life of about 3 hr, which meant maximal expression was around 10 ng per oocyte. other groups have noted the instability of pb2 when expressed in isolation; pb2 expressed in nih 3t3 cells using an inducible bovine papilloma virus vector has a similar half-life of around 3 hr (braam-markson el al., 1985) . nevertheless, 10 ng of each individual polymerase protein is equivalent to the amount present in about 1 pg of purified virus, and virion transcriptase activity can easily be detected in reactions containing only 5-10 pg of purified virus (bishop et al., 1971) . therefore, we believe that the system offers considerable promise for the reconstitution of enzymatic activity. our results indicate that all three p proteins expressed in oocytes formed a complex. this parallels the situation seen in the infected cell, where similarly, all three proteins associate, and also confirms the observations of other workers that a complex of the p proteins can exist in virus-infected cells independently of virus rnps (detjen et al., 1987; akkina et al., 1987) . it is also the first direct demonstration that all three artficially expressed p proteins can reassociate into a complex, probably a vital preliminary step toward reconstitution of the influenza polymerase. in a previous report, where the influenza p proteins were expressed using baculovirus vectors (st. angelo eta/., 1987) only pbl and pb2 formed an immunoprecipitable complex. the authors suggested from this that the presence of another influenza gene product was needed for the incorporation of pa into a stable complex. in the light of the results presented here this seems unlikely; an alternative explanation is that amphibian cells provide a more suitable environment for the complex formation than do insect cells. for example some kind of posttranslational modification of the p proteins might be necessary for formation of a full complex and this could be more faithfully carried out in xenopus oocytes. the factors involved are likely to be quite subtle, because when the p proteins are cotranslated in mdl, no complex formation can be detected (not shown). akkina et al. (1987) reported that most of the p proteins found in the cytoplasm of virus-infected cells were not in the form of complexes. however, our work would suggest that a functional nucleus is unlikely to be necessary for complex formation, as the p proteins do not localize to the nucleus in xenopus oocytes, and furthermore, all three proteins associate normally in enucleated oocytes (not shown). expression of pairs of p proteins indicated that pb2 and pa can associate independently with pb 1, yet cannot form a complex directly with each other. thus pbl can be considered the "backbone" of the complex. this idea is consistent with its suggested central role as the protein responsible for elongation (braam et a/., 1983) , with the other two polypeptides as adjuncts fulfilling more peripheral functions such as substrate selection or cap-binding. gradient analysis of individually expressed p proteins indicated that pbl and pb2 did not exist as free monomers, but rather as heterogeneous populations of high-molecular-weight aggregates. it is not clear at present whether these structures represent self-aggregation, or the proteins binding to cellular components. this property of a large and heterogeneous sedimentation rate was also a feature of complexes of all three p proteins, as seen in the markedly different migration of pa when expressed alone or in the presence of the other two p proteins. most of the complexed material sedimented with an apparent molecular weight much greater than 250,000, the expected size for a simple trimolecular complex. again, this could reflect some kind of association with a cellular component, but it is unlikely to be the result of the complexes binding to rna present in the lysate, as rnase treatment prior to gradient analysis does not reduce their rate of sedimentation (fig. 5) or that of pb2 expressed alone (not shown). a second possibility is that the larger complexes contain extra copies of the p proteins. support for this idea comes from the observation that the faster sedimenting forms of the complex appear to contain an increased ratio of pbl and pb2 to pa, suggesting the existence of different stoichiometric forms of complex, varying in their relative content of pbl and pb2. these might arise from pa binding directly to more than one copy of each protein, but given the heterogeneous size distribution of individually expressed pbl and pb2 (and the lack of any observable direct interaction between pa and pb2), the existence of pa associated multimers of the basic p proteins seems a more likely explanation. it has also been suggested previously that the influenza polymerase is a multimeric structure (krystal et a/., 1986) . it is possible that pbl and pb2 behave in a manner analogous to that of sv-40 large t antigen, a large multifunctional protein which exists in different oligomeric forms with discrete biochemical activities (reviewed in rigby and lane, 1983) . in respect of the varying ratios of the p proteins found in the xenopus-expressed complexes, it is interesting to note that complexes detected in infected cells do not necessarily seem to consist of equimolar quantities of all three proteins. akkina et al. (1987) reported that complexes deficient in pb2 were present in the cytoplasm, and showed data to suggest the existence of rnp-associated complexes with a less than equimolar ratio of pa (our own unpublished observations support the latter observation). it is therefore possible that different functions of the influenza rna polymerase may be attributable to different forms of the complex. the successful reconstruction of the influenza polymerase complex provides an indication of the potential of the xenopus system for reconstruction of an active polymerase. as yet we have been unable to observe reassociation of the xenopus-expressed polymerase complex with vrna, the next step toward reassembly of the transcription complex, because vrna is rapidly degraded in oocytes (data not shown). however, it may be possible to circumvent this problem by partially purifying the complexes from an oocyte lysate before incubating them with the rna substrate, or alternatively, by providing natural or artificially assembled rnp structures. various other expression systems have been used to study the influenza polymerase. krystal et a/. (1986) constructed cell lines expressing all three p proteins and showed that these were able to functionally complement viruses bearing ts lesions in the p protein genes. however, no biochemical data were reported on this system, possibly because of the low levels of expression obtained. st. angelo eta/. (1987) expressed the p proteins in recombinant baculoviruses, but although the system offered considerable promise in terms of the levels of protein expressed, as discussed above, only an incomplete polymerase complex was formed. recently, it was demonstrated that an active influenza polymerase could be reassociated from the purified polypeptide components of disrupted virion rnps by renaturation with escherichia co/i thioredoxin (szewczyk et al., 1988) . although this is a significant result and provides an exciting system for the study of the polymerase, it is ultimately limited to the examination of wild-type proteins. our approach has the significant advantage that mutant polypeptides can be generated easily by manipulation of the dna protein coding sequence. characterization of such altered polypeptides will allow a more detailed analysis of the structure and function of the polymerase proteins. intracellular localisation of the viral polymerase proteins in cells infected with influenza virus and cells expressing pbl protein from cloned cdna transcription of the influenza ribonucleic acid genome by a virion polymerase. 1. optimal conditions for in vitro activity of the ribonucleic acid-dependent ribonucleic acid polymerase activity molecular model of a eukaryotic transcription complex: functions and movements of influenza p proteins during capped rna-primed transcription expression of a functional influenza viral cap-recognising protein by using a bovine papilloma virus vector an efficient ribosomal frame-shifting signal in the polymerase encoding region of the coronavirus ibv translation of eukaryotic messenger rna inxenopus oocytes the three influenza virus polymerase (p) proteins not associated with viral nucleocapsids in the infected cell are in the form of a complex the effect of capping and polyadenylation on the stability, movement and translation of synthetic messenger rnas in xenopus oocytes influenza virus messenger rnas are incomplete transcripts of the genome rnas transcription of the influenza virus genome influenza virus, an rna virus, synthesises its messenger rna in the nucleus of infected cells polypeptides specified by the influenza virus genome. 1. evidence for eight distinct gene products specified by fowl plague virus functional messenger rnas are produced by sp6 in vitro transcription of cloned cdnas expression of the three influenza virus polymerase proteins in a single cell allows growth complementation of viral mutants cleavage of structural proteins during the assembly of the head of bacteriophage t4 molecular cloning: a laboratory manual messenger rna translation in reticulocyte lysate transfer of 5-terminal cap of globin mrna to influenza viral complementary rna during transcription in vitro structure and function of simian virus 40 large t-antigen two of the three influenza viral polymerase proteins expressed by using baculovirus vectors form a complex in insect cells construction of a newfamilyof high efficiency expression vectors: identification of cdna clones coding for human liver proteins purification, thioredoxin renaturation, and enzymatic activity of the three subunits of the influenza a virus rna polymerase electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications cloning and expression of influenza virus genes we thank dr. p. palese for the gift of the influenza cdna clones, dr. a. colman for the plasmid psp64-t, and dr. c. dingwall for advice and oocytes. this work was supported in part by mrc grant ge62397 1 ca and a grant from the wellcome foundation. p. digard thanks the states of guernsey education authority for support. key: cord-265887-g5zhoyo9 authors: mukherjee, shruti; bhattacharyya, dipita; bhunia, anirban title: host-membrane interacting interface of the sars coronavirus envelope protein: immense functional potential of c-terminal domain date: 2020-08-11 journal: biophys chem doi: 10.1016/j.bpc.2020.106452 sha: doc_id: 265887 cord_uid: g5zhoyo9 the envelope (e) protein in sars coronavirus (cov) is a small structural protein, incorporated as part of the envelope. a major fraction of the protein has been known to be associated with the host membranes, particularly organelles related to intracellular trafficking, prompting cov packaging and propagation. studies have elucidated the central hydrophobic transmembrane domain of the e protein being responsible for much of the viroporin activity in favor of the virus. however, newer insights into the organizational principles at the membranous compartments within the host cells suggest further complexity of the system. the lesser hydrophobic carboxylic-terminal of the protein harbors interesting amino acid sequencessuggesting at the prevalence of membrane-directed amyloidogenic properties that remains mostly elusive. these highly conserved segments indicate at several potential membrane-associated functional roles that can redefine our comprehensive understanding of the protein. this should prompt further studies in designing and characterizing of effective targeted therapeutic measures. induce global metabolic changes on infected cells, leading to the rearrangement of the lipid dynamics to facilitate viral multiplication. (27) in some cases, these alterations produce the reorganization of intracellular membranes of the host cell, building an adequate microenvironment for viral replication. all these findings highlight the intimate connections between viruses and host lipid interactions. typical of the genus, the initial steps of sars-covs entry include the attachment of the virus particle to a cellular receptor, angiotensin-converting enzyme 2 (ace2-specific for sars-cov) located on the host plasma membrane surface. (28) the viral genome has to take entry into the host cell to reach the replication sites. different lipids found either on the plasma membrane and/or the endosomal membranes can contribute to these processes by enabling receptor clustering, virus internalization, or membrane fusion. the host spectrum/tropism of individual covs have been known to be primarily determined by the s protein. (29) the functional aspects of the s protein have been known to mediate receptor binding and virus-cell fusion that occur at either the interface of the plasma membrane or within the endosomal vesicles. (30) the mechanism of membrane fusion of sars-cov-1 mostly belongs to the type i fusion system, where s protein of sars-cov-1 comprises of a number of membrane binding regions in the s2 domain identified as fusogenic peptide or fps by several researchers. (31) (32) (33) recently bhattacharjya et. al. have shown that one of these fusion peptides adopts β-sheet conformation either in the presence of phosphatidylcholine or phosphatidylcholine/cholesterol comprising membrane models with the help of circular dichroism spectroscopy. (34) moreover, it has also been shown that the peptide undergoes oligomerization with increasing concentration of cholesterol in the membrane. in addition to receptor binding, the action of host cell-specific proteases can likely cleave the s protein, in turn, regulating the cov infection and host-or tissue-tropism. (35) once inside the host cellular environment, positive-stranded rna viruses such as cov compartmentalize their replication machinery efficiently within the host membranous compartments to evade detection by host pattern recognition receptors. (36) such compartments are formed for the benefit of the virus by taking advantage of cellular pathways and lipid-modifying enzymes. (27) after transcription within the rough endoplasmic reticulum (er), the viral membrane proteins associate with the endoplasmic reticulum-golgi intermediate compartment (or the ergic) before being folded and promoting the budding off of the viral particle by hijacking the cellular secretory mechanism.(37) a major portion of the viral e protein has been known to remain localized at the site of intracellular trafficking, viz. the ergic system, participating in cov assembly and budding. (38, 39) thus, much of the lipid components of the virus particles are comparable to these host organellar membranes being rich in phosphatidylcholines, phosphatidylethanolamines and a small fraction of phosphatidylinositols. (40) this has provided direct evidence that the host membranes can serve as crucial functional domains that are indispensable for viral propagation. thus, mechanistic insight into the functional interface of host-membranes is particularly essential to obtain a comprehensive understanding of the target conformers for effective therapeutic strategies. e protein of cov is an integral membrane protein about 76-109 residues long and has been known to be most dynamic at the membranous functional interface. (41) the primary structural elements of the protein make it particularly interesting that delve into its possible role in viral propagation. interestingly, despite the everevolving nature of the virion particles, the primary sequence of the protein remains mostly conserved. the sars-cov-2 e protein bears about 97% sequence similarity with that of the sars-cov. this alternatively directs into understanding the uniqueness of the sequence that is conserved to mediate some crucial functional roles of the protein (figure1). the primary amino acid sequence reveals a very short n-terminal hydrophilic sequence of about ten amino acids. (42, 43) this is followed by a stretch of hydrophobic sequence that serves as the transmembrane domain (tmd). this twenty-eight residue long stretch has been known for the membrane-induced topologies of the protein that have been extensively defined and studied. (44) the absence of a canonical cleaved signal sequence suggests equal likelihood for the protein to be a type ii (with its c-terminal targeted to the er lumen) or type iii (with its n-terminal targeted to the er lumen) membrane protein. therefore, the topology of the cov e protein is highly debated in the literature and remains largely inconclusive. table 1 summarizes the contradictory topology predicted for some of the widely studied cov strains. the predicted topologies of the sars cov-2 e protein using prediction servers have been represented in table 2 . however, further complicating matters, the topology models devised using prediction programs are mostly inconsistent with the previous experimental observations. two membrane-associated topologies have been demonstrated for e protein (figure2)-hairpin or transmembrane. (24, 45) in a report by arbely et al. (2004) , the protein was shown to adopt a highly unusual topology, comprising of a short transmembrane helical hairpin conformation that inverses about a previously unidentified pseudo-center of symmetry. (46) this hairpin structure could deform lipid bilayers and cause fragmentation. (46, 47) it has been suggested that post-translational modifications and several intermediate stages of maturation in the er and golgi environment can modulate the overall conformation of the molecule in response to the physiochemical properties of the membrane. (48) (49) (50) the specific lipid composition of the particular lipid bilayers of the er-golgi protein synthesis-packing machinery of the cell defines divergent physicochemical properties at the membranous interface. the lipid packing density has been known to underlie significant features of the resulting membrane-inducing characteristic local curvatures, surface charge, phase behavior, elasticity, hydrophobicity, degree of hydration, etc.(51) membrane phase behavior in a cell has been known to permit the transient concentration of specific proteins and lipids into dynamic nanoscopic domains. (52) the level of palmitoylation may moderate the relative proportion of the membrane-associated conformers of the protein that again, in turn, can modulate the local membrane curvatures, facilitating further favorable association of the protein conformers. (53) substitutions of the hydrophobic amino acid residues in the tmd of the e protein with charged amino acids have been shown to alter the migrating properties of the e protein in sds-page. (44) this, therefore, suggested changes in the overall conformation and membrane association of the mutant forms in comparison to the wild type e protein. the tmd is followed by a lesser hydrophobic c-terminus with a fraction incorporated as part of the envelope in the virion particle ( figure 1 ). the c-terminus, in particular, has been known to be associated with the redirection to the host er and golgi complex membranes that underlies some of the major functional domains of the protein. (54, 55) comprising of more than 50% of the entire protein sequence, this segment has been known to influence the topology in the host cell. there are several conflicting theories that either suggests a luminal localization of the c-terminal when associated with the ergic system or directed towards the cytoplasm. (38, 45) the several experimental and in-silico prediction studies have been mostly inconclusive with respect to its localization and hence functional domain in the different strains of cov. nevertheless, the amino acid sequence of the c-terminal harbors some of the answers to the many known functional attribute of the protein sequence. interestingly, it comprises of the conserved "fyxy" peptide sequence (where x= any amino acid) that can be correlated to its high amyloidogenic propensity. (15, 25) also, another highly conserved cysteine containing motif-"cxxc" can enable disulfide isomerization to prompt membrane-directed conformational changes. (56) apart from these highly conserved sequences throughout the genus, there are distinct potent glycosylation sites along the stretch that can serve as chaperone interacting motifs to help in the protein folding and/or aid in j o u r n a l p r e -p r o o f journal pre-proof trafficking along with the cellular machinery.(57) glycosylation of particular asparagine residues (asn 45, asn 48, asn 64, and asn 68) in the sars-cov has been shown to be crucial in maintaining the proteinoligomerization events associated with the host membranes. (58) conversely, maintaining the glycosylation events have been suggested to be critical to maintaining the broad functional roles of the protein. (59, 60) further terminally, there is a distinct phosphorylation domain comprising of the "s 67 sr 69 " motif. additionally, a pdzbinding domain (pbm) in the c-terminal plays an essential role in host cell modifications necessary for viral infection and pathogenesis (figure 1 ). (61, 62) mutation studies with the pbm have established its importance for viral infection, making it a significant factor of research for therapeutic and vaccine designing. (61, 62) 2. the e protein as a functional viroporin. the primary structural features of the e protein have striking similarities with the viroporin class of proteins. (63) (64) (65) viroporins ideally comprise of about 60-120 amino acids, with a distinct hydrophobic transmembrane domain. they are known for their interactions with membrane surfaces, often resulting in the expansion of the lipid bilayer. (65) viroporins participate in several viral functions, including the prompting of viral particle release from host cells. (66) although not essential for viral replication, viroporins modulate the host cellular machinery to make it favorable for viral propagation. they affect host cellular functions, including membrane vesicularization, glycoprotein tracking, and membrane permeability. the transmembrane domain could form hydrophilic pores in the membranes of virus-infected cells by oligomerization. (63, 67) these hydrophilic channels would allow low molecular weight hydrophilic molecules to cross the membrane barrier, leading to the disruption of membrane potential, collapse of ionic gradients, and release of essential compounds from the cell several studies with sars cov e protein have demonstrated the structural and functional similarities with viroporins. it has been demonstrated that the sars-cov e protein could enhance the membrane permeability of bacterial cells to o-nitrophenyl-β-d-galactopyranoside and hygromycin b, suggesting that the protein may function as a viroporin. (68) similar observations were reported on mouse hepatitis virus e protein. (64) the e protein, as a viroporin, is present in low copy numbers in the virus particle, but has been implicated in membrane scission being mostly associated with the ergic and cis golgi-consistent with a predominant role as a mediator of virus assembly and release at this location. (69) the lipid content at these locations may also enhance virus budding. (70) compartments are formed by manipulating the protein-synthesis, packaging, and distribution system of the cell involving the er-ergic-golgi complex. this helps the viral replication machinery to evade detection by host pattern recognition receptors ( figure 2 ). both viral and hijacked host proteins are used in this process, taking advantage of cellular pathways and lipid-modifying enzymes to the benefit of the virus, sequestering newly formed rnas away from host immune sensors. (75, 76) this eventually allows the virus to manipulate the normal secretory pathway of the host cellular machinery to transport and deliver the virus protein cargo at the site of final packaging, and eventual budding. (69) the virulence of the strain is, in fact, determined by the efficiency in their mechanism to concentrate the rna synthesis machinery and subsequent packaging within these specialized compartments that are, in turn, modulated by the associated structural proteins.(77) among the structural proteins, the e protein has been particularly intriguing either alone or in collaboration with the m protein. (71) interestingly, studies have shown that overexpression of the e protein is sufficient to induce the packaging and assembly of the cov particles upon efficient manipulation of the host cellular machinery. (78) rottier et al. suggested that the e protein has no genuine structural function in the virion envelope itself where it occupies frequent, regular positions within the lattice built by m protein. (79) however, its primary role lies in the functional interface of the host packaging and assembling membranes ( figure 3 ). the frequent but strategic positions within the lattice hint at a possible morphogenetic function to generate the required membrane curvature for the final viral particle formation ( figure 3 , inset). studies have endowed the c-terminal to be associated with the redirection of the e protein to the er, and golgi complex for assembly, budding, and intracellular trafficking of infectious virions.(55) interestingly, sequence analyses of the c-terminal residues suggest the prevalence of some degenerate er export signal sequences. these are closely comparable to the most common fxxxfxxxf(80) and the [r/k](x)[r/k](81) dibasic motifs that have been known to be responsible for the export of glycosyltransferases. the last 6-residue "r 103 dklys 108 " stretch from the ibv e protein terminal has been defined to serve as the er retention signal. (72) the site-directed mutagenesis of this composite lysine residue (k105) to glutamine resulted in the accumulation of e in the golgi apparatus. [56] maturing through the golgi complex, the e protein is expected to impart some very crucial functions with respect to vesicularization events, by inducing local curvatures at the site of membrane-directed accumulation ( figure 3 ). two principle mechanisms have been described for moving and delivering cargo proteins through the secretory pathway of the golgi complex, the cisternal maturation, and the formation of mega-vesicles. (82, 83) despite the extensive studies performed, experimental evidence for both the systems remains inadequate and far from being elusive. during cov infection, virions have been observed in large vesicle depots resembling megavesicles derived from golgi/ergic membranes, indicating that remodeling of the golgi complex may be crucial for virion trafficking. (84) understanding the structural transitions that the e protein undergoes, from after its synthesis to its association with the ergic membrane complex, would greatly facilitate the characterization of potential targets. we expect the protein to undergo stepwise dynamic structural transitions with specific roles bound to the membranous component. this gradual transition is dependent equally on both the respective membrane composition along with the conformational changes in the protein as it is translated, modified, and matures along with the er-golgi network system. previous studies performed by knoops as membrane fusion and fission to prompt viral propagation by hijacking the entire cellular machinery. however, progress in our in-depth understanding of this whole machinery has been stymied by the inability to track the functional interface of the protein at the atomic-level resolution in real-time. identifying these intermediate functional conformers could broaden our chances of finding the best target motif for therapeutic design. the cov e protein has been known for its possible pro-apoptotic viroporin activity majorly by mobilizing calcium ions, prompting an overall change in the ionic environment of the homeostatic cell. (86, 87) the virus particles, generally, hijack the cellular homeostatic machinery of the host cell by compromising the membrane integrity that facilitates its regulatory mechanisms within the environment of the host cells. it has been suggested that the e protein can directly disrupt membranes through the formation of ion channels upon membrane-directed oligomerization events mediated upon the insertion of the tmd. in this context, the tmd has received much interest owing to their membrane-spanning attribute. molecular dynamic simulation studies with synthetic peptides have revealed that the tmd could form ion channels by homo-oligomerization into stable dimers, trimers, and pentamers. 90) additionally, in silico prediction studies based on high-resolution nmr spectroscopic studies provided useful insight into understanding the probable conformers that define the tmd induced ion channels. hence, these studies directed the defining of therapeutics targeting the resultant ion channel pores. (91) the hollow structure of the oligomers allows the unregulated efflux of ions from the cellular compartments across the hydrophilic interior of the pore, modulating the membrane potential, altering the ionic environment of the lumen. alterations in ion concentration would promote translation of viral mrnas, given the translation of mrnas from many cytolytic animal viruses is reasonably resistant to high sodium concentrations. (63, 67) since the secretory pathway of the host cellular machinery is highly sensitive to perturbations in ionic strength, viral propagation can trigger a detrimental apoptotic cascade. unregulated ion channel formation upon e protein oligomerization in the host membrane is believed to be one of the primary processes underlying the viral pathophysiology.(92) as discussed previously, much of the studies have been focusing on the tmd for the high molecular weight conformers (multimers) of the protein upon host membrane interactions. (93, 94) however, interestingly, the immediate adjacent segment (towards the c-terminal) hint, at least in part, at a possible role in the membrane directed oligomerization propensity of the molecule. upon inspection into the c-terminal segment of the protein, we can see that it is marked by the presence of unique motifs that can prompt much of the membrane-directed conformational changes of the protein molecule. the primary sequence of the c-terminal region reveals possible functional characteristics of the protein that are lesser known in literature. interestingly, the sequence is distinctive of amyloidogenic proteins and peptides that have been studied in the literature for their membrane-directed indispensable functions. highresolution structural models of amyloid oligomers have been obtained in the context of membrane interactions in neurodegenerative diseases.(95) membrane-induced oligomerization prompts membrane compromise, often leading to vesicularization into smaller lipid aggregates. (96) (97) (98) studies with amyloid proteins have hinted at the possibilities of a change in the local curvature being induced by the membrane-associated high molecular weight conformers of the proteins (figure 3 , inset).(98) several reports have suggested specific lipid domains to serve as templates for protein anchorage. this is followed by rearrangement of the protein conformers resulting in aggregates that form "wedges" within the bilayer to facilitate curvature change. (99, 100) such protein aggregates are driven mostly by the hydrophobic interactions that relocate within the acyl chain region of the lipid bilayer. these interactions result in a change in the lipid packing density and hence the overall membrane integrity. (101) the structural similarity of the c-terminal segment of the e protein to amyloid proteomics indicates at analogous purposes that can be complementary to or function in parallel to its viroporin activities. the oligomerization properties of e protein have been defined previously.(25) e protein expressed in bacterial and mammalian cell systems under reducing conditions was shown to exist as monomers. however, upon change to non-reducing conditions, they formed homodimers and homotrimers. (68) the existing literature has focused on the phenomenon of tmd multimerization associated with the ion channel formation that lacks mechanistic details on the initiation. however, site-directed mutagenesis studies revealed that the two-cysteine residues, immediately adjacent to the tmd in the c-terminal, were essential for the oligomerization, leading to the induction of membrane permeability. the tmd is immediately followed by a "cxxc" redox motif, which is highly conserved among the coronaviridae family of viruses. this motif is very crucial for several reasons. the oxidized cxxc motif can maintain the structural topology of the transmembrane region as well as that of its contiguous cytoplasmic domain, inclusive of glycosylation sites, involved in signaling and protein-protein interaction.(102) a probable activation of a thiol in this motif can trigger the participation of the other cys residues in the formation of the inter-subunit disulfide bond. the activated thiol will then attack the disulfide and cause its isomerization into a disulfide isomer within the motif. this may lead to the refolding of the transmembrane region and may activate its fusogenic potential.(103, 104) interestingly, a similar cxxc motif is present as a part of the c-terminal of s protein sequence. bioinformatic studies have suggested at the possibilities of forming inter-protein disulphide bridges that might indicate at a crossover or cooperation between these two structural proteins in the viral membrane interactions. (41) the formation of a disulfide bond may also play a crucial role in the oligomerization of the e protein, forming stable dimers, trimers, and pentamers depending on its functional requirement.(105) thus even though the tmd spans the lipid bilayer, the cxxc motif could serve as an essential key to defining the membrane-associated oligomerization events-providing newer targets for preemptive therapeutic intervention. apart from the consequential involvement of this segment in the viroporin function, the amyloidogenic segment of the e proteins can function independently at the membranous interface. further downstream, the cterminal harbors the "fyxy" composite peptide sequence ( figure 4 ) that has been known for a high propensity of amyloid formation. (106, 107) interestingly, a self-assembling short peptide segment with the composite sequence has been studied to have a membrane-mediated function. bhunia and co-workers studied a 9-residue long peptide stretch (tk9, t 55 vyvysrvk 63 ) in sars cov e protein (figure 4 ) for its physical changes in the presence of both zwitterionic and negatively charged model membrane micelles.(15) the tk9 peptide segment was shown to adopt amyloid fibrillary structures in solution ( figure 4 ). (25) further studies with tk9 provided evidence for its aggregation propensity that closely resembles amyloid proteins. intriguingly, the composite 5-residue peptide motif-t 55 vyv 58 in tk9 was found to serve as the critical sequence motif that may be critical for the beta-sheet formation. (25) however, in vitro measures demonstrated the differential orientation of the tk9 peptide with respect to the membranous environment: correlating well with the structure and function at the membrane-protein interactions interface. this atomic-resolution nmr based structural elucidation in the presence of membrane mimics have improved understanding of the molecular mechanism of cov infection under physiological conditions. studies demonstrated the helical propensity of this particular segment from the c-terminal region (figure 4 ) of the protein to impart host specificity to the composite virus. the peptide's affinity was further j o u r n a l p r e -p r o o f journal pre-proof manifested by its pronounced membrane-integrity disruption ability towards the mammalian compared to the bacterial membrane mimic-implicated in the viral pathogenesis. tk9 penetrated deeper into the acyl region of the neutral micelles mimicking the mammalian membrane and its intracellular organelles as opposed to the superficial conformation when in association with the bacterial model membrane mimics (figure 4 ). this provided evidence for the host membrane-associated conformational change in the protein segments that is specific to a particular lipid composition (species specificity). this study alternatively offered useful insight into the fact that the composite lipid molecules play particularly important roles in the interaction dynamics and conformational uptake. these segment-based studies highlighted the exclusive function of the amyloidogenic segment of the e protein that acts independently of the tmd-associated oligomerization events. similarly, another short 9-residue peptide, nk9 (n 45 ivnvslvk 53 ) further upstream, was identified and characterized, being marked by sheet breaker-hydrophobic residues, like ile, val, and leu. (25) the experimental intervention into these segments can provide useful insight into the topology of the protein, its membrane-bound orientation, and eventual functional conformation uptake. these preliminary studies have provided useful links to the immense possibilities of the functional membrane-directed conformers of the e protein, which remains largely unexplored. nevertheless, epitope mapping for these interfaces can serve as critical targets for effective therapeutic intervention. preliminary sequence homology analyses performed to compare the e protein primary sequence of sars cov to that of the novel cov-2, revealed a minimal difference with the "fyxy" motif remaining conserved (figure 1 ). the first threonine and the second valine from the tk9 sequence have only been substituted by a serine and phenylalanine residues, respectively ( figure 4 ). this might indicate at an unperturbed oligomerization potency of the segment. this conserved amyloidogenic sequence indicates a very crucial functional role of the segment that contributes significantly to the virulence. the fact that hydrophobic interactions can provide stability in localization within the acyl-chain layer of the lipid bilayers leaves us with some obvious open questions. does the c-terminal induce the initial docking of the protein to the host membranes? do these hydrophobic interactions prompt the downstream oligomerization events to the formation of the membrane-associated multimers? alternatively, does the lipid-composition specificity of the c-terminal determine the conformational uptake and the eventual functional topology? these questions are vital for a comprehensive understanding of viral propagation. interestingly, hydrophobic protection of these segments within the acyl-chain region can serve as open templates for downstream aggregation. this can result in membrane-associated aggregates that, in analogy to the amyloidogenic proteins, can modulate the overall membrane integrity. thus, it is essential to study the interface of interaction between the e protein and the host membrane, either as whole or directed to particular functional peptide segments. one of the major functional aspects of cov e protein has been associated with virus assembly and its subsequent release for propagation in the host physiology. indirect immunofluorescence microscopy showed that e protein is localized to the golgi complex in cells transiently expressing ibv e.(108) when co-expressed with ibv m, both from cdna and in ibv infection, the two proteins are colocalized in golgi membranes, near the cov budding site.(109) thus, even though ibv e is present at low levels in virions, it is apparently expressed at high levels in infected cells near the site of virus assembly. the subcellular localization of the sars cov e protein was analyzed using the sera from immunized mice by elisa and immunofluorescence using cells j o u r n a l p r e -p r o o f infected with recombinant (rsars-cov) and viruses lacking the e gene (rsars-cov-∆e) as a negative control. (38) several parallel studies have endowed the c-terminal to be associated with the redirection of the e protein to the er, and golgi complex for the intracellular trafficking of infectious virions. (55) experimental studies have shown that the c-terminal domain of the e protein, in fact, plays a crucial role in virus budding. (110, 111) the deletion of the domain resulted in the free distribution of the mutant protein and a dysfunctional viral assembly. alternatively, mutations introduced into the cytoplasmic tail of mhv e protein by targeted rna recombination resulted in elongated virions, further indicating at the critical involvement of the domain in the budding mechanism. (72) the difference in the lipid composition along the secretory pathway might, in fact, have a direct role on the e protein topology. moving from the er to the golgi and finally the plasma membrane, there is a definite gradient in the concentration of specific lipid components that induce a unique physical property of the lipidbilayers in terms of thickness and rigidity. (40) the er presents a lower fraction of cholesterol or sphingolipids that allows maintenance of a relatively fluid lipid compartments for the remodeling associated with the protein association, sorting and accumulation. (112) this fluidity can have a direct role in the gradual compartmentalization of the er-associated membranes prompting viral particle formations. the host membrane-mediated oligomerization of the e protein may be responsible for much of the membrane compromise in favor of the viral propagation. the membrane-associated aggregates can compromise the overall lipid bilayer integrity. (113, 114) the hydrophobic interactions involving the lipid acyl regions can result in increased fluidity of the membrane surface, prompting phase separation of the lipid domains. biomolecular phase separation is suggested to drive the organization of cellular organelles involved in cellular packaging and compartmentalization.(115) differential distribution of the membrane proteins and their site of interaction with specific lipid rafts, help determine the local curvature and hence characterize propensity to bud off from the surface. (112, 116) the membranous compartments of er and the golgi complex presents with a fair fraction of zwitterionic and neutral lipids-i.e., phosphatidylcholine and phosphatidylethanolamines. (112) in vitro studies by khattari et al have demonstrated that the membrane-directed topology of the sars cov e protein imposes a direct constraint on the lipid-bilayer thickness and the acyl-chain ordering. (117) their studies suggested that the increasing protein concentrations in the organellar membrane along the secretory pathway (greater protein/lipid concentrations), the tmd of the sars cov e protein induces bilayer lipid arrangements. specific inter-molecular interactions with the lipid molecules and intra-chain hydrogen bonds and salt-bridge interactions allow the uptake of a topology that traverses across the membrane bilayer. however, the exact transmembrane topology of the protein was highly debated and remains elusive.(23) nevertheless, these intra-and intermolecular interactions can be considered to have the ability to rearrange and induce direct morphological changes in the host membranes. the final viral packaging requires assembling of the replicated rna strands and the other viral proteins before the final membrane budding. the putative transmembrane domains of the e protein can also serve a 'catalytic' function in the membrane packaging. (118) cross-talk between distant protein molecules can, in fact, serve as a "zipper" to close the 'neck' of the viral particle as it pinches off from the membrane in the terminal phase of budding. (8, 119) despite the immense possibilities, much of the progress in our understanding of the actual mechanism of action is still hypothetical and far from being elusive. it is, therefore, essential to study the interface of interaction between the e protein and the replication complex. particularly, the membrane-mediated aggregation, that compromises the local curvature, inducing the final scission. cov gained particular notoriety when the sars cov outbreak shook the world around 2002-2003. the aftermath leads to the identification of many newer family members. soon after that, studies were in full swing all around the globe to determine the mechanism of viral propagation. several parallel studies provided useful insight into the understanding of the structural and functional uniqueness of the viral proteins and rna in their search for finding the ideal target for therapeutic intervention. almost immediately after sars, the endemic spread of the mers-cov in 2012 reignited the urge to gain in-depth insights into the system all over the world for designing effective therapeutic measures. the recent outbreak of the novel covid-19 soon turned into a pandemic, threatening health standards globally. lu et al. first published the genomic characterization of covid-19 and also reported it to be significantly divergent from sars-cov, claiming it to be a new human-infecting βcoronavirus. (120) a comparative analysis of small membrane/ envelope protein of different coronaviruses could provide an interpretation of the molecular events that could advance these viruses from developing acute infections to one producing a pandemic. an analysis of the sequence conservation can also help in identifying the regions required in the typical functions of membrane interaction, oligomerization, localization in the host cell, and viral infection. such information is of immediate significance and would contribute to vaccine designing and facilitate the evaluation of vaccine candidate immunogenicity. additionally, this would help in reflecting the potential effects of mutational events as the virus is transmitted through the human population. recent studies have revealed that the homology with a cov strain isolated from pangolin was ~99%. this indicated that while sars-cov-2 have been known to evolve from the bat cov, pangolins might have helped as an intermediate host. (121) nevertheless, further research is required to justify these theories. we performed a blast (https://blast.ncbi.nlm.nih.gov/blast.cgi) search for the e protein and selected the sequences from the different genus of the coronaviridae family. homology analyses and sequence alignment were conducted using the mega software. the muscle program of the mega software was used to perform multiple sequence alignment, and a phylogenetic tree was created by using a maximum likelihood approach ( figure 5 ). (122) predictive tools such as tmhmm (123), memsat(124), phobius (125) , and the hydrophobic moment plot method(126) of eisenberg and co-workers allowed the calculation of the transmembrane regions. much has been defined to correlate the structural conformation of the tmd with its ion channel formation. however, sequence analyses of the c-terminal have revealed crucial information that has been out of the limelight for several years. despite the fact that the c-terminal has shown the potential to underlie much of the protein's dynamic functionality associated with membrane budding and scission, it has received much lesser attention. several secondary structure prediction programs predicted two β-strands within the sars-cov e protein tail sequence, including the n 45 ivnvslvk 53 and the t 55 vyvysrvk 63 . the predicted β-strands fit the criteria for forming a β-hairpin, which is a simple structural motif with two β-strands linked by a short loop of two to five amino acid residues. interestingly, a highly conserved proline residue, pro 54, resides between the predicted βstrands-responsible for the β-coil-β motif. (54) the antiparallel β-strands form hydrogen bonds to stabilize the hairpin structure. previous reports with the synthetic peptide fragment of e (i.e., i 46 -s 60 ), encompassing the predicted β-hairpin segment, was found to produce ∼100% β-structure and was completely resistant to hydrogen-deuterium exchange in fourier transform experiments. alternatively, the study showed that titration with the drug molecule, hma (5-(n, n-hexamethylene) amiloride) resulted in large perturbations of the val 49 and leu 65 residues, which are far apart in the sequence. the structural model proposed by jaume torres's group, therefore, suggested that these two residues were coming spatially close in the membrane-associated pentamer and might belong to different monomers. comparison with mutation-based data hypothesized this β structure to be in dynamic equilibrium with an α-helical intermediate form. in fact, a delicate balance between j o u r n a l p r e -p r o o f these two forms may alter the membrane-dynamics and subsequent processes in the infected cell (e.g., membrane scission, binding to protein partners, or e protein localization). disturbing this predicted β-strand region along with the substitution of the conserved proline residues had resulted in the disruption of the cytoplasmic golgi complex signal, consequently changing the subcellular localization of e protein. (55) taken together, the results support the hypothesis that the three-dimensional structure of e protein and not the primary sequence dictates much of its function. the sum of amino acid substitutions per site from between sequences is presented in figure 5 . analyses were performed using the poisson correction model. the estimated value of the shape parameter for the discrete gamma distribution is ~15.1, suggesting that the occurrence of the conserved amino acid residues among the coronavirus family e protein is not entirely random. however, upon close inspection with other e protein sequences of different species of the same genus, the value decreased to ~6.8. substitution patterns and rates were estimated under the jones-taylor-thornton (1992) model (+g). (127, 128) the model evolutionary rate differences among sites were obtained using a discrete gamma distribution (10 categories, [+g]). all the ambiguous positions were eliminated for each of the sequence pairs (pairwise deletion option). evolutionary analyses were conducted in mega x. (129) collectively, the data reveals that sites observed to be constant among sequences might have crucial biological significance correlated to their function. besides, deleterious mutations are much more likely to be found in population-level data. this indicates that sites that would predictably be considered as 'invariable' at the phylogenetic level might have transient polymorphisms at the population level. in the analyses of the e protein sequences from the cornaviridae family, altering the number of gamma categories did not have any noticeable effect on the estimate of the substitution rate. cov e protein has received lesser attention when compared to the other structural proteins (like s and m) majorly owing to a very low copy number in the viral envelope. (42, 130, 131) it is the smallest and yet has been the most puzzling of the major structural proteins. earlier deletion based studies had proven that the viral life cycle endures the absence of e protein, implying that other viral genes could counterbalance for its loss. (132) however, recent evidence collected with recombinant covs missing e protein, exhibit considerably lowered viral titers, crippled viral development, or produce progeny incapable of further propagation. (10, 11) several parallel studies have highlighted the crucial role played by the e and m proteins in the propagation of the viral genome into the host cell and virulence. remarkably, studies have reported that viruses produced by the deletion of the sars-cov e gene can be attenuated in at least three different animal models that conferred protection against sars-cov pathogenesis. (133, 134) but, the lack of complete information and a limited number of findings have prevented an understanding of the exact mechanism underlying the definite functional role of e protein in viral infection. nevertheless, these have provided the basis of understanding that the cov e might be involved in several aspects of the viral replication cycle, including host cell responses such as apoptosis, inflammation, and even autophagy in association with other nonstructural viral proteins.(135) soon after the identification, studies have been in full swing all around the globe to determine the mechanism of the viroporin action. several parallel studies from all over provided useful insight into understanding the structural and functional uniqueness of the viral protein in their search for finding the absolute target for therapeutic intervention. extensive studies performed in prof. torres's laboratory provide useful insight into the structural conformation of the ion channel pores created by e protein that seems to be a very attractive target for therapeutic intervention. (91) their studies have prompted the designing and characterization of specific inhibitors for these channels that would perturb the primary functional attribute of the protein. hexamethylene amiloride has been studied to have blocked this e protein-associated ion channel activity in the mammalian cells expressing sars-j o u r n a l p r e -p r o o f cov e protein. (136) however, viroporin proteins, like the sars-cov e protein, can exhibit a multitude of diverse functions unrelated to their ion-channel properties that are yet to be defined. the dynamic functional interfaces of the protein have been underestimated for its functionality in the propagation. this study can be correlated with earlier docking screening by gupta et al., where more than 4000 phytochemicals were evaluated on the same protein, and three of those, i.e., belachinal, macaflavanone e, and vibsanol b were found to be particularly promising. (130) based on the experimental evidences, it is worth mentioning that the two hits, i.e., glycyrrhizic acid and cepharanthine, are supporting their significant activity against the sars viruses. (130) more recently, anatoly chernyshev has established the potential of the sars-cov-2 e protein as a pharmaceutical target. (94, 137) these studies have defined 36 approved drugs, which theoretically could block the e protein-induced ion channel and eventually hinder the virus's life cycle. (138) however, these studies have been based on in silico approaches, and hence much of the drug candidates have to be subjected to further experimental investigations. considering the lengthy procedure of new drug development, the existing strategy of drug repurposing has turned into one of the preferred solutions for the immediate treatment of sars-cov-2 affected individuals. (139) long-term drug development objectives for the pharmaceutical industry incorporate the identification of inhibitors targeted at the replication or infection routes associated with sars-cov-2 or other allied coronavirus infections. (140) knowledge of the coronavirus structural proteins and their mechanisms of viral action can be crucial in defining alternative therapeutic targets. despite the extensive research efforts worldwide, a definite cure to target cov propagation is still elusive. effective therapeutic intervention into viral systems without compromising the host cell integrity requires an in-depth understanding of the specific functional interface. especially, with the ever-evolving viral components, as seen in the covid-19 specimen, immense multidimensional research is the need for the hour. among the structural proteins, the e protein have not been a convincing choice for therapeutic targeting, despite the evidence for a crucial viroporin activity. much of the knowledge has remained restricted to only its ion channel formation ability. although this might be serving as a significant functionality, it is definitely not the comprehensive representation of the immense potential that the protein is possibly capable of undertaking. we believe, in-depth understanding of its functional interface with the host-membranes can undermine newer possibilities and functional attributes that have not received the deserved attention so far. attempts to determine the crucial protein domains associated with the host membranes are innovative in terms of targeted therapeutic designing. several studies have been gradually exemplifying the protein's essential role in viral packaging and propagation. the conserved sequences in the c-terminal region directly prompt the understanding of evolutionarily crucial functional roles of this segment that is yet to be conclusively defined. the knowledge is incomplete, essential insight into the protein's functional interface should provide newer insight into understanding and structure-function correlation. this should enable the effective designing of targeted therapeutics. the entire portrayals of biologics with characters suitable for inhibiting several key cov proteins could serve as a basis for drug development. j o u r n a l p r e -p r o o f infected cell table 2 : list of predicted topologies of sars cov-2 envelope protein using in silico prediction servers. alternatively, the segment has been studied for the differential membrane-directed functioning. the peptide undergoes a helical conformation when in association with membranous environments. comparison between bacterial and mammalian model membrane mimicking systems showed the different orientation of the peptide. this suggested the specific functional role of this peptide segment and its membrane-directed structural change. preliminary studies with the ty5 peptide segment (underlined) had also shown the significance of the "fyxy" sequence, characteristic of amyloid proteins, and peptides. (15, 25) the figure was prepared with https://biorender.com. j o u r n a l p r e -p r o o f highlights:  the sars cov envelope protein is a small structural protein of the virus that has been suggested to have significant viroporin like activity.  majority of its function is mediated at the interface of host-membrane interactions j o u r n a l p r e -p r o o f  focus at the membrane-directed features of the protein provide useful insight into gaining mechanistic insight into its viroporin functions.  studies have elaborated the central hydrophobic transmembrane domain of e protein, known to affect ion-channel formation.  the c-terminal region of the protein show further potential host-membrane directed functional roles.  the highly conserved amyloidogenic amino acid stretches of the c-terminal suggest at significant contribution to cov propagation. sars-cov-2: an emerging coronavirus that causes a global threat world health organization declares global emergency: a review of the 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title: a potential treatment for covid-19 based on modal characteristics and dynamic responses analysis of 2019-ncov date: 2020-10-21 journal: nonlinear dyn doi: 10.1007/s11071-020-06019-1 sha: doc_id: 275124 cord_uid: 7l53bvp1 the 2019-ncov is ravaging the world, taking lots of lives, and it is emergent to find a solution to deal with this novel pneumonia. this paper provides a potential treatment for covid-19 utilizing resonance to destroy the infection ability of 2019-ncov. firstly, the geometry size of 2019-ncov is scaled up by 10,000 times. the additional mass is used to represent the effect of the fluid around a spike protein. the finite element analysis (fea) is used to study the modal characteristics of the tuned 2019-ncov model and mistuned 2019-ncov model in blood, respectively. based on fea, the lumped parameter mechanical model of 2019-ncov is established. then, the dynamic responses of mistuned 2019-ncov are investigated through harmonic response and dynamical analysis. finally, a potential method utilizing 360° sweep excitation to cure covid-19 is put forward. a novel coronavirus (2019-ncov) was identified as the causative virus linked with a cluster of cases of pneumonia detected in wuhan city by chinese authorities on 7 january [1] . due to the discovery of 2019-ncov, the virus has been diagnosed quickly [2, 3] . from december 2019, coronavirus disease 2019 (covid-19) has been ravaging the world and killed more than 181 thousand people [4] . medical scientists around the world are exerting themselves to develop vaccines, but vaccine researches are timeconsuming, and vaccines can lose efficiency when ribonucleic acid (rna) for this virus mutates. this paper tries to provide a potential method based on vibration to damage the infectivity of 2019-ncov and cure covid-19, and it will not fail if the virus mutates. 2019-ncov is a kind of coronavirus, which consists of virus sphere and spike proteins on the sphere surface. when 2019-ncov infect humans, they enter cells of humans by binding their spike proteins to receptors on the cell membrane, and then copy viruses in the cell. it is theoretically feasible to disable the 2019-ncov by destroying the spike protein structures through resonance, and then it is easy for the immune system to eliminate these viruses and recover. this paper combines finite element analysis (fea) and theoretical methods to study the modal characteristics and dynamic responses of 2019-ncov in the blood, the geometry size of 2019-ncov is about 200 nm, and for the convenience of fea, the geometry size is scaled up by 10,000 times to 2 mm. all other parameters of length dimension are magnified 10,000 times, so that we can get the accurate result by scaling down length dimension of results by 10,000 times. generally, it is necessary to utilize the nonlocal elasticity theory to consider the size effect of nanomaterials. however, based on studies given by peddieson et al. [5] , for a cantilever under concentrated force, its bending behavior utilizing the nonlocal elasticity theory is identical to the local result. in this paper, spike proteins structure on the novel coronavirus sphere surface is a cantilever structure. therefore, it is reasonable to apply traditional mechanical methods to conduct the analysis. the 3d 2019-ncov model is built in solidworks software as shown in fig. 1 with protein material, which is input to ansys workbench to do modal analysis. in the process of analyzing the modal characteristic of structures in the fluid, the additional mass is used to represent the effect of the fluid around the structure. according to fluid mechanics by mao and wu [6, 7] , the additional mass of the blood per unit length around a spike protein can be calculated as follows where q b is the density of the blood, r is the equivalent radius of the environment, and r is the radius of the spike protein. since r is infinite compared to r, m bp is simplified as m bp ¼ q b pr 2 . the parameters are set as the additional mass of the blood around a spike protein is m b ¼ m bp l 1 ¼ q b pr 2 l 1 ¼ 2:6 â 10 à22 kg in the real situation, while in the modal analysis it is added to the spike protein by an equivalent point mass 2:6 â 10 à10 kg. because of the relatively heavy mass of the sphere body compared to spike proteins, its modes are rigid body motion, which the sphere of covid-19 model is fixed in modal analysis. a tuned 2019-ncov model indicates that every spike protein is identical. the frequencies of the tuned 2019-ncov model are shown in table 1 . the first 200 frequencies are very close to each other, and they are all first-order bending vibrations. figure 2 is one of the corresponding mode shapes. these frequencies are supposed to be identical according to the vibration theory, and the discrepancies are caused by the intrinsic error of finite element analysis (fea). in the realistic situation under the electron microscope, the number of spike proteins varies from different novel coronavirus. even though the number of spike proteins in the same kind of novel coronavirus varies, it is hard to detect the exact number currently. in this paper, the 2019-ncov model is built based on estimation. in order to achieve mistuning, the circle of spike proteins picked to lengthen could be chosen randomly in principle. but for the sake of analysis, the circle of 30 spike proteins in our model is selected. a mistuned 2019-ncov model is obtained by lengthening one chosen circle, which has 30 spike proteins. at the same time, keeps the other circles unchanged. the frequencies of the mistuned 2019-ncov model are shown in table 2 . the first 60 frequencies are very close to each other, and they are all first-order bending vibration of the lengthened spike proteins. from 61st frequency, they are the first-order bending vibration of unchanged spike proteins. figure 3a , b is one of the corresponding mode shapes, respectively. the discrepancies of the first 60 frequencies are caused by the intrinsic error of fea. the spike proteins in realistic 2019-ncov are different from each other in geometry, so the frequencies of the realistic 2019-ncov model are a range of frequencies close to each other, and every frequency corresponds to the first-order bending vibration of one spike protein. 3 lumped parameter mechanical model of 2019-ncov according to the mode shape of single spike protein as shown in fig. 4 , the first-order bending mode shape is employed as the vibration form of spike proteins. based on studies given by weaver and timoshenko et al. [8] , the rayleigh method is utilized to calculate the first-order frequency to check this choice where p is circular frequency, q is the density, e is the young's modulus, i is the bending moment, y is the first-order mode function, a is the area of cross section, and l is the length. compared with the fea result, which is 198,340,000 hz, the relative error is 0.22%. this indicates a reasonable choice. based on the first-order bending vibration form, the lumped mechanical model is established as illustrated in fig. 5 . according to the equivalent frequency principle, spike proteins are simplified as mass-spring structures. each spike protein is identical in geometry except for its location. based on studies given by beer et al. [9] , the equivalent bending stiffness is calculated as k eq ¼ 3ei l 3 ¼ 1:41 â 10 à3 m/n, and the equivalent mass m eq is calculated as m eq ¼ 9:14 â 10 à22 kg by the following eq. (3) where t represents the kinetic energy, a 2 ðxþ ¼ p á ð5 â 10 à9 á x þ 1 â 10 à9 þ 2 is the area of cross section of the end part in a spike protein, v is the velocity at the indicates the location of the additional mass of the blood around a spike protein. according to studies given by weaver et al. [8] , the forced vibration theory of multiple degrees of freedom system is used to yield the natural frequency, which the natural frequency is calculated as f ¼ 1 2p ffiffiffiffiffi k eq m eq q ¼ 1:977â 10 8 hz. comparing with eq. (2), the relative error is 0.33%, which validates the accuracy of the model. the lumped parameter mechanical model of mistuned 2019-ncov is roughly the same with tuned 2019-ncov. but, there are small differences on equivalent stiffness and mass. 4 dynamic response analysis of mistuned 2019-ncov the harmonic response is conducted after the modal analysis of the mistuned 2019-ncov model, and acceleration excitation is set as 1 mm/s 2 , as shown in fig. 6 . the result of the harmonic response is shown in fig. 7 , where one of the lengthened spike proteins has the largest amplitude. the fea amplitude-frequency curves of a lengthened spike protein and an unchanged spike protein, whose h is p=8, are illustrated in figs. 8 and 9 , respectively. these results accord with the modal characteristics. the excitation f sin xt is imposed on the end of spike proteins of the mistuned 2019-ncov model. there are totally 32 circles, and each circle consists of spike proteins, which have the same angle h between their displacements and excitation. the viscosity of blood is considered as viscous damping. according to studies given by weaver et al. [8] , the vibration theory of multitude degrees of freedom model and the forced vibration equation of lumped parameter model are utilized to obtain the following equation and the steady-state response as follows fig. 6 acceleration excitation in the process of harmonic response analysis fig. 7 harmonic response analysis result where h i ¼ ðià1þp 16 , u i ¼ tan à1 r eqi x=k eq ð1 à k 2 i þ, h i is the angle between excitation and displacement, . it is easy to find that the vibration amplitude increases with cosh i j j. the spike proteins, whose displacements parallel with the excitation direction, have the biggest vibration amplitude. when 5p=16 h i 11p=16 and 21p=16 h i 27p=16, cos h i j j 0:56, and these vibration amplitudes are relatively small, which it can somehow achieve response concentration. according to studies given by weaver et al. [8] , the relationship between amplitude and frequency of the lumped parameter model is obtained as follows fig. 8 the fea amplitude-frequency curve of a lengthened spike protein fig. 9 the fea amplitude-frequency curve of an unchanged spike protein the amplitude-frequency curves of the spike proteins are analyzed in harmonic response, as shown in fig. 10 , where b 1max ¼ 3:56 â 10 à12 m, b 2max ¼ 7:01 â 10 à13 m. compared with the fea results in figs. 8 and 9, relative errors are 5.10% and 7.83%, respectively. the spike proteins of a realistic 2019-ncov are usually different from each other in geometry, and every spike protein has a different frequency, which there is small discrepancies. therefore, it would be possible to utilize an ultrasonic vibration exciter to 360°sweep, whose frequency range is set as the firstorder bending vibration frequencies 1.9 9 10 8 hz to 2.0 9 10 8 hz in order to excite every spike protein of 2019-ncov to resonate. 360°rotating sweep excitation could evoke resonance of spike proteins in turn and vibration concentration. given a proper excitation amplitude, it could destroy the spike protein structures and disable the infectivity of 2019-ncov. effects of ultrasonic resonance destruction of viral proteins are discussed further. since the bending strength of the spike protein is unknown, for the reliability of the analysis, it is set as the same as the bending strength of common steels, which is 300 mpa. if the stress in the spike protein is bigger than 300 mpa, it would crack at the base and lose its ability to invade human cells. then, the required destruction excitation amplitude is analyzed by fea, as shown in fig. 11 , which is calculated as 1.041 9 10 -5 mm and the equivalent acceleration is 4.164 m/s 2 . based on studies given by glaister [10] , this acceleration is safe for human body. based on studies given by randall et al. [11] , it is known that the range of resonant frequencies of standing human bodies was found to be from 9 to 16 hz. since the resonant frequencies of human bodies are below 20 hz, the vibration energy would hardly be absorbed by human body and it would not cause much harm and discomfort to patients. therefore, it is efficient and safe to utilize an ultrasonic vibration exciter to conduct 360 o rotating sweep excitation to human body if the vibration frequencies are set as 1.9 9 10 8 hz to 2.0 9 10 8 hz and the amplitude is set as larger than 1.041 9 10 -5 mm. in this paper, the modal characteristics and dynamic responses of 2019-ncov in the blood are investigated by fea and theoretical analysis. a possible treatment for covid-19 based on vibration is put forward. several findings are listed below. (1) the frequencies of the tuned 2019-ncov model are very close to each other, and they are all first-order bending vibrations. the frequencies of the realistic mistuned 2019-ncov model are a range of frequencies close to each other, and every frequency corresponds to the first-order bending vibration of one spike protein. (2) the first-order bending vibration is adopted as the vibration form, and the established lumped parameter mechanical model of 2019-ncov is in good agreement with the fea model. when applying an excitation on the 2019-ncov model, the vibration amplitude increases with cos h i j j, and it can somehow achieve response concentration. (4) in order to help cure the covid-19, a potential method utilizing 360 o sweep excitation to destroy the spike protein structures is put forward. when an ultrasonic vibration exciter is applied 360°rotating sweep excitation to human body, the vibration frequencies is set as 1.9 9 10 8 hz to 2.0 9 10 8 hz and the amplitude is selected as larger than 1.041 9 10 -5 mm, it is efficient and safe to disable the 2019-ncov from infecting human cells, which maybe help cure covid-19. world health organization. who statement regarding cluster of pneumonia cases in wuhan national health commission of the people's republic of china. interim protocol of diagnosis and treatment of 2019 novel coronavirus-associated pneumonia (the second version) a novel coronavirus from patients with pneumonia in china world health organization. novel coronavirus (2019-ncov) situation report-22 application of nonlocal continuum models to nanotechnology fluid mechanics fluid mechanics vibration problems in engineering mechanics of materials human tolerance to impact acceleration resonant frequencies of standing humans conflict of interest the authors declare that there is no conflict of interest regarding the publication of this paper. key: cord-023209-un2ysc2v authors: nan title: poster presentations date: 2008-10-07 journal: j pept sci doi: 10.1002/psc.1090 sha: doc_id: 23209 cord_uid: un2ysc2v nan the boron neutron capture therapy (bnct) based on the interaction between 10 b isotope and thermal neutron has been highly noted in recent years as one of promising techniques for treatment of cancers. p-( 10 b)borono-l-phenylalanine (l-10 bpa), in which boron atom is enriched with 10 b isotope, is now using clinically as an effi cient 10 b carrier for treatment of patients in particular with malignant brain tumor and melanoma (1) . in order to develop a practical method for the synthesis of l-10 bpa, we have recently examined the enantioselective method utilizing the negishi reaction based on the coupling of 4-( 10 b)boronoiodobenzene derivative with 3-iodo-l-alanine derivative (2) . from the standpoint of diagnosis of cancers, the magnetic resonance imaging (mri) is noted as one of common techniques. in particular, mri based on the measurement of 19 f atom is becoming a remarkable one. to create practical materials utilizing as not only the 10b carrier but also mri probe, we had already synthesized the compounds containing both 10 b and 19 f atoms in a single molecule such as [4-( 10 b)borono-2,6-difl uorophenyl]-dl-alanine [dl-10 bpa(2,6f 2 )] 3., [4-( 10 l-proline is conformationally unique among coded amino acids in that its torsion angle is blocked (-65 ± 10) by its characteristic fi vemembered pyrrolidine ring structure and the preceding torsion angle (tertiary amide) can undergo cis (0) <-> trans (180) isomerization much easier than the secondary amides of the usual peptide bonds. in addition, its torsion angle is commonly found either in the right-handed 3 10 -/helical region (-30 ÷ -50, or cis' conformation) or in the left-handed, semiextended, region [-150 ±10, or trans', or poly-(l-pro) n conformation]. methylation at the c -position of a pro residue was suggested to block the preceding tertiary amide ( ) torsion angle of the resulting ( me)pro to the trans disposition and to restrict the , surface to the single region where the 3 10 / -helices are found. we have synthesized a large set of n -blocked, ( me)pro-containing, dipeptide n'alkylamides having the general formulas p-d-( me)pro-xxx-nhipr and p-xxx-d-( me)pro-nhipr, where p is ac or boc and xxx is d-ala, l-ala, aib, gly, d-( me)pro, or l-( me)pro. the results of the present x-ray diffraction analysis clearly show that the region of the conformational map overwhelmingly preferred by ( me)pro is indeed that typical of 3 10 / -helices, but the semi-extended, type-ii poly(pro) n helical region can exceptionally be explored by this extremely sterically demanding c -tetrasubstituted -amino acid. in addition, the known high propensity for -turn formation of the pro residue is even enhanced in peptides based on its c -methylated derivative. to complete the picture of the preferred conformation of ( me)pro, the synthesis and crystal-state investigation of a series of terminallyprotected homo-peptides from d-( me)pro are currently in progress in our laboratory the interest in guanidine-containing compounds is due to their important role in biological recognition processes. five-member cyclic n-amidinoamino acids represent hybrid structures, combining both proline rigidity and high positive charge of arginine guanidine group. structurally similar n-amidino-proline, n-amidino-pyroglutamic acid and cyclocreatine (2imino-1-imidazolidine acetic acid) were chosen as objects of our study. the problem of their incorporation into peptide structure requires use of substituted derivatives or effective guanidylation technique. recently we have presented the synthesis of mts-protected n-amidinoproline. nevertheless, its application in n-termini modifi cation of peptides shows slow condensation rate in classical as well as solid phase synthesis for short peptides (2-5 residues) or completely fails in case of longer ones (10 residues). on the other hand, polymer supported guanidylation of proline by bis-boc-protected carboxamidine-1hbenzotriazole seems to be preferred route to n-amidino-proline containing peptides. three methods of n-amidino-pyroglutamic acid synthesis have been investigated and model dipeptide has been acquired on wang polymer by the cyclization of guanidine-glutamic acid. for selective boc-deprotection on wang resin the literature technique has been utilized. however, in this case esi-ms and nmr analyses evidence for side-reaction of triethylamine alkylation by polymer linker fragment. hplc analysis shows n-amidino-pyroglutamyl-phenylalanine stability at acidic and physiological ph but fast ring opening in water solution at ph 9. for cyclocreatine application in peptide synthesis we suggest the use of its p-toluenesulfonate having good solubility in dmf and showing effective coupling in cl-hobt/dic condensation. the examples of namidino-amino acids practical application in peptide synthesis will be presented. tryptophan is often a key pharmacophore which determines the affi nity of peptide ligands for their receptors. cyclic analogues of tryptophan which introduce local constraints and reduce the fl exibility of the indol moiety are very valuable tools to probe the bioactive conformation of the peptide ligands. one of the possibilities to freeze indol moiety of tryptophan is the synthesis of the additional 6-member ring by the formation of 1,2,3,4-tetrahydro--carbolines. we report the synthesis of 1,3-disubstituted 1,2,3,4-tetrahydro--carbolines via the pictet-spengler reaction. methyl ester of tryptophan or dipeptides with n-terminal trp (trp-ala-ome, trp-leu-ome) were used as arylethylamine substrates and -amino aldehydes derived from l and d-amino acids were used as carbonyl components. we determined that there were no differences of the stereoselectivity of the pictet-spengler reactions in the case of trp-ome or trp-dipeptides. we also investigated the conformation of the newly created 6-membered ring in cis and trans diastereomers. the roesy spectra were used to assign the more stable conformation for each isomer. the conformations of cis isomers derived from tryptophan and dipeptides were the same and substituents on c-1 and c-3 were in both cases equatorial. the conformation of trans isomers were depended on the carboxyl part of trp. for methyl ester of tryptophan the ester group was axial, whereas for dipeptides we observed the opposite conformation with equatorial substituent on c-3. our results show that the pictet-spengler reaction can be successfully performed for amino acids and peptides and the conformation of the newly created 6-membered ring depends on the substrates and the size of c-1 and c-3 substituents. helical-screw handedness of peptides composed of diastereoisomeric cyclic amino acids nagano 3 helical-screw handedness in proteins is believed to result from thecarbon chiral center of l--amino acids. recently we have reported that the helical-screw sense of oligopeptides can be controlled without a chiral center on the peptide-backbone but by chiral centers at the side chain. that is to say, we designed and synthesized an optically active cyclic , -disubstituted amino acid (s,s)-ac(5)c(dom), in which the -carbon atom is not a chiral center but chiral centers exist at the side chain. conformational analysis of the (s,s)-ac(5)c(dom) peptides revealed that the hexapetide formed left-handed 3 10 -helices both in solution and in the solid state, and the octapeptide assumed a left-handed -helix. herein we designed new two diastereoisomeric cyclic , -disubstituted amino acids; (1s,3s)-and (1r,3s)-1-amino-3-(methoxy)cyclopentanecarboxylic acid (ac(5)c(om)) having chiral centers both at the -carbon atom and at the side chain. the amino acids (1s,3s)-and (1r,3s)-ac(5)c(om) were synthesized starting from l-(-)-malic acid. that is to say, at fi rst, the malic acid was converted to diiodide compound, and bisalkylation of dimethyl malonate with the diiodide gave a cyclic diester. monohydrolysis of the diester, followed by curtius rearrangement produced separable mixtures of (1s,3s)-and (1r,3s)-ac(5)c(om). we prepared both diastereoisomeric (1s,3s)-and (1r,3s)-homooligomers by solution-phase methods, respectively, and studied their preferred secondary structures using 1 h nmr, ft-ir, cd and x-ray crystallographic analysis. zobel, ansgar; gaus, katharina; wollschläger, katrin; nieß, anke; juodaityte, jovita; sewald, norbert organic and bioorganic chemistry, bielefeld university, germany novel highly active and specifi c dna binding molecules have a considerable potential particularly with regard to applications in medical science. the major and minor grooves of the dna serve as recognition areas. it is of high interest for chemical biology to control dna-binding abilities of synthetic molecules. a possible modulator is light in combination with photoswitchable molecules. triostin a is a natural bisintercalator which belongs to the quinoxaline antibiotics originally isolated from streptomyces s-2-210. it consists of a bicyclic depsipeptide with n-methylated amino acids and a cystine bridge. its heteroaromatic quinoxaline moieties are able to intercalate gc-specifi cally into the dna inducing a change in conformation and therefore inhibit the transcription by blocking spezifi c enzymes. tandem is the n-unmethylated derivative of triostin a which binds at selectively to the dna due to the change in the hydrogen bonding pattern. the exchange of the cystine bridge of tandem with substituted azobenzene units leads to photoswitchable triostin a analogs. the synthesis of the depsipeptidic basic structure which contains the quinoxaline moieties is introduced as well as the coupling with azobenzene amino acids. the double cyclization step is accomplished under pseudo high dilution conditions. in order to differ between cisand trans-confi gurations of the molecule, nmr-and ir-spectroscopy was carried out as well as the photoswitchability of different analogues was tested by irradiation and rp-hplc analysis. for synthetic peptide vaccine prototype development: (pc) , which has an heteroatom in its structure and their bioconjugates obtained by microwave and carbodiimide methods were explained. for pc obtaining, the synthesis of the monomer, 1azabicyclo[4.2.0]octane, (c), was carried out by organic methods which then will be used in polymer synthesis [1] [2] . consequently, polymers having different molecular weights and their water soluble bioconjugates were synthesized and the characterization of these polymers and conjugates were done by different methods such as uv, atr ft-ir, sec with four detectors and zeta sizer. for the chemical modifi cation of pc, bromoacetic acid was used and a water soluble polyampholyte synthesis was achieved with the quaternization of polymer. atr ft-ir spectra of pc was recorded and molecular weights, polidispersity values of polymers, mark-houwink constants and molecular diameters of the polymers were measured with size-exclusion chromatography with four-detectors (light-scattering, refractive index, viscosity and uv). with zeta sizer, size-analysis of polymer chains were done and their zeta potentials were measured. it is determined that molecular weights of the polymers were affected by the change in the amount and the type of initiators used and also from the polymerization media. analysing of having biodegradable characteristics of the synthesized polymers has been being reviewed. after synthesis and modifi cation of pc, its conjugates with antigenic peptides like avian infl uenza hemagglutinin (ha 98-106) ypydvpdya and rgdsggc cell receptor peptide were obtained. their characterization was also performed. japan; 3 in alzheimer fs disease research, sparing water-solubility and uncontrolled self-assembly of amyloid peptide (a ) 1-42 are signifi cant obstacles to establish an experimental system that clarifi es a pathological mechanism of a 1-42. to solve these problems, we herein disclose water-soluble "click peptides" based on an o-acyl isopeptide method. these peptides contain an o-acyl instead of n-acyl residue at the gly 25 -ser 26 of a 1-42, and are converted to a 1-42 via an o-n intramolecular acyl migration triggered by phchange (ph-click) or photo-irradiation (photo-click). these peptides had remarkably higher water-solubility than a 1-42. in addition, these peptides clearly adopted monomer state with a random coil structure, which were verifi ed by various physicochemical assays. importantly, we could establish an in situ system predominantly comprised of monomer a 1-42 as a result that the monomer click peptide was converted to a 1-42 quickly and quantitatively in accordance with ph-change. both selfassembly and conformational change of the produced a 1-42 in situ were observed with time. similarly, the photo-triggered click peptide afforded a 1-42 by uv-irradiation. because the in situ production of intact a 1-42 from the click peptides could overcome the handling problems of a 1-42, this strategy would provide a reliable experimental system for investigating a pathological function of a 1-42 in alzheimer fs disease. the antioxidant effect of introducing phenylpropenoyl moiety in either in n-terminal group of analgesic oligopeptides or in c-terminal end of opioide active amino acid, modifi cated with polyamines have been evaluated. the series of hydroxycinnamoyl peptide and amino acid amides were synthesized by standard method used in peptide chemistry. obesity is a major public health problem associated with morbidity and mortality and continues to increase worldwide. the gastrointestinal tract and the pancreas release hormones regulating appetite and body weight. obestatin is a recently described 23 amino-acids peptide derived from preproghrelin. it was identifi ed by bioinformatic prediction (1) . obestatin decreases food intake and body weight gain, decelerates gastric emptying, promotes sleep in rat, inhibits water drinking. it has been described that obestatin effects on feeding behavior and an u-shaped dose-response relationship was found: low doses (0.01-3 nmol/kg) and high doses (1-3 nmol/kg) were ineffective. the 23 amino-acid carboxy-amidated human obestatin nalapropheaspvalglyilelysleuserglyvalglntyrglnglnhissergln alaleunh 2 and corresponding rat obestatin hpheasnalapropheasp valglyilelysleuserglyalaglntyrglnglnhisglyargalaleunh 2 were synthesized by different schemes. synthesis was developed on the basis of solid phase synthesis of protected peptide fragments followed by their assembly into the fi nal product. fragments 1 -8, 9 -13, 1 -13, 14 -20, 14 -22 were built on the acid-labile 2-chlorotrityl chloride resin using the orthogonal fmoc/tbu strategy. fragment 21-23 (hcl•hargalaleunh 2 ) was obtained in solution. the assembly of the full-length peptide was carried out by fragments coupling in solution or using solid phase method. the free peptide was obtained by treating the protected peptide with mixture of tfa: h 2 o:tis (95: 2.5: 2.5). the peptide was purifi ed with hplc and isolated by lyophilization with 95%+ purity according to analytical reversed-phase hplc. the mass of molecular ion determined by maldi-tof spectrometry was in fi ne agreement with calculated value. the inverstigation of biological actions of obestatins is in progress. straightforward synthesis of enantiopure tfm-amino acids from chiral cf 3 brigaud, thierry; chaume, grégory; huguenot, florent; caupène, caroline university of cergy-pontoise, france trifl uoromethylated amino acids (tfm aas) are current synthetic targets due to their unique properties and their synthesis in enantiopure form remains a challenge. we will report that chiral 2-trifl uoromethyl-1,3oxazolidines (fox) are highly versatile synthons for the stereoselective synthesis of various functionalized -trifl uoromethylamino compounds such as -amino nitriles , -and -amino acids, diamines and amino alcohols. 1, 2 moreover, trifl uoropyruvate-based oxazolidines proved to be very valuable building blocks for the stereoselective synthesis of both enantiomers of -trifl uoromethyl proline and -tfm-pyroglutamic acid, in enantiopure form. 3 moreover, the synthesis of the (s)--tfm--allylglycine and the novel (s)--tfm norvaline were achieved in a few steps from this starting material. we will also report a recent straightforward synthetic route to tfm-dihydroxyprolines in enantiopure form from trifl uoropyruvate-based chiral oxazolidines. chitinases catalyse the hydrolysis of chitin, the natural homopolymer of (1,4)-linked n-acetyl-d-glucosamine. chitin is a key structural component of the cell walls, exoskeletons, and eggshells of pathogenic fungi, insects, and nematodes, respectively, which all rely on the ability to hydrolyse chitin at specifi c points in their life cycles. chitinase inhibitors are now attracting considerable interest as novel fungicides and insecticides, as well as chemical tools to study human diseases as diverse as asthma and malaria. in this context, the cyclic pentapeptide natural products, argifi n and argadin, are two exciting inhibitors which pose some interesting synthetic challenges. the argifi n structure includes two sensitive -linked asp residues, as well as an unusual carbamoylated arg side chain, while argadin contains a unique aspsemialdehyde residue, that is cyclised to the peptide backbone to generate a potentially labile hemiaminal. we will describe improved routes to both compounds that allow us to avoid signifi cant side reactions such as aspartimide formation and homoserine-mediated backbone cleavage that are observed in our previously reported syntheses. [1, 2] this is achieved by carrying out the assembly and cyclisation of both peptides, including key side chain derivatisation steps, entirely on solid phase. the application of the strategies devised to the automated synthesis of argifi n and argadin and related natural products will be described. during the last two decades combinatorial chemistry has become an important tool for the quick synthesis of large numbers of small molecules used, for example, in the generation of the new lead structures for medicinal applications. additionally, it allows rapid access to diverse chemical libraries with novel structures and properties. small molecules libraries are generally prepared on solid support simplifying tedious purifi cation steps of the intermediates and facilitating the entire synthesis. piperazines and keto-piperazines are amongst the important backbones in today's drug discovery. the piperazine framework has been defi ned in medicinal chemistry as a "privileged scaffold". it is a molecular backbone with versatile affi nity properties representing a frequently-occurring binding motif, and providing potent and selective ligands for a wide range of biological targets. the high number of positive hits revealed in biological screens with the piperazine scaffold urged chemists to develop plenty of different synthetic methods that allow fast and effi cient building of these heterocyclic systems on solid support as well as homogenous chemistry. however, the majority of these methodologies is not stereospecifi c and the complex mixtures of the stereoisomers are generated. in order to preserve important chiral centers in potential hits we propose to use pipearzine backbone, initially prepared in optically pure form, bearing various tethers with orthogonally protected groups applicable via solid phase organic chemistry (spoc). we will describe a novel synthesis of keto and diketopiperazine building blocks. these chiral building blocks are applied in "around-the-scaffold" modifi cation strategy by spoc, introducing valuable physico-chemical properties in 3 independent diversity points. solid-phase strategies for constraining peptide structures can serve for elucidating the relationships between conformation and activity. for example, the systematic substitution of natural amino acids by their aza-amino acid counterparts has been accomplished using a fmoc strategy featuring couplings with aza-amino acid chlorides to provide insight into the biologically active conformers of the melanocortin receptor agonist ac-his-d-phe-arg-trp-nh2 as well as the calcitonin gene-related peptide antagonist [d31, p34, f35]cgrp27-37 (1) (2) (3) . employing cyclic sulfamidates, we have now developed fmoc strategies for the effective solid-phase introduction of alpha-and beta-amino gamma-lactam constraints into peptides. since their pioneering use in a somatostatin analog with about 9 fold greater activity (4), lactam restraints have been used to study a variety of relevant targets (5, 6) . our strategies now provide effective means for performing lactam scans of biologically active peptides as demonstrated using the growth hormone secretagogue ghrp-6, and an allosteric modulator peptide antagonist of the il-1 receptor. our presentation will focus on recent developments in the solid-phase chemistry for synthesizing such constrained peptide analogs and the relationships between peptide conformation and biology, elucidated by these novel methods. references: 1 barcelona science park, university of barcelona, 08028-barcelona, spain; 2 linaclotide is a 14-residue peptide currently undergoing phase ii clinical trials for the treatment of gastrointestinal diseases such as chronic constipation (cc). linaclotide, which can be administered orally, is an agonist of the guanylate cyclase type-c receptor found in the intestine. from a structural point of view, this small peptide presents a constrained structure with the presence of three disulfi de bridges between cys1-cys6, cys2-cys10, and cys5-cys13. h-cys( 1)-cys( 2)-glu-tyr-cys( 3)-cys( 1)-asn-pro-ala-cys( 2)-thr-gly-cys( 3)-tyr-oh in order to reach the large amounts required for a marketed peptide, an effi cient synthesis needs to be attained. to optimize the synthesis, its fundamental limitations need to be determined and addressed. in the case of linaclotide, the key points are related to the numerous cys (some of them consecutive) present in the peptide, for two reasons: the potential risk of racemization upon assembling the linear chain, and the misfolding of the three disulfi de bridges. for that, the concourse of different protecting groups and folding conditions as well as the analysis of the disulfi de bridges in the fi nal folded peptide has been studied and will be discussed in this presentation. balalaie, saeed 1 ; arabanian, armin 1 ; mohammadnejad, mahdieh 1 ; gross, juergen h. 2 1 university, iran (islamic rep.); 2 university, germany gnrh analogues have been used for the treatment of steroid-dependent tumors, such as prostate and breast cancers. the development of more potent gnrh analogues depended largely on the important made in the science of peptide chemistry. ugi-4mcr reaction is known for the synthesis of amide bond. we wish to report herein an effi cient method for the synthesis of some gnrh analogues based on ugi reaction using fourcomponent reaction of n and c-terminus peptides, aromatic aldehydes and isocyanides. this ugi-4mcr could describe to build up novel gnrh analogues deriving from triptorelin and gonadorelin. all of the products were purifi ed using preparative hplc and the structures were assigned according to maldi mass spectrometry data. peptide synthesis is based in a proper combination of protecting groups and in the right choice of the right coupling method. nowadays, almost all peptide bond formed are carried out in the presence of 1hydroxybenzotriazole (hobt) or its derivatives (hoat, cl-hobt). thus, hobt derivatives are used in combination with a carbodiimide or another coupling agent or built into a standalone reagent such as immonium ( thus, a replacement of hobt should be found for preparation of peptides for research purposes and, more important, for the production of peptide based apis. herein, several alternatives to hobt will be discussed taking into account the explosivity properties. furthermore, a new family of immonium salts, which incorporates an proton acceptor in its carbocation skeleton. the novel proton acceptor coupling reagent has shown superiority to the described previously. an oxygen in the carbocation moiety confers to the reagent more solubility, enhances coupling yields and decreases racemization, allowing the use of just one equivalent of base. examples on the use of the non-explosive replacement for hobt together with carbodiimides or built into phosphonium and immonium salts, with the proton acceptor, will be discussed. thiocarbamate-linked peptides by chemoselective peptide ligation using phenylthiocarbamate chemistry peptide chemical ligation chemistries, which allow the chemoselective coupling of unprotected peptide fragments, are useful tools for synthesizing native polypeptides or unnatural peptide-based macromolecules. native chemical ligation (ncl) (1), staudinger ligation (2) lead to the formation of a native peptide bond at the ligation site. other methods result in the formation of unnatural covalent bonds such as oxime (3), thioester (4) linkages. these methods are of great interest when native peptide bonds are not absolutely required. in this context, we examined the potential use of the phenylthiocarbamate group in ligation chemistry. the phenylthiocarbonyl group can be easily introduced into peptides on alpha or epsilon amino group using phenylthiochloroformate and standard solid-phase method (5) . it reacts chemoselectively with cysteinyl peptides to give an alkylthiocarbamate bond. s,n-shift of the alkylaminocarbonyl group from the cys side chain to the alpha-amino group did not occur. the method was used for linking two peptides chain through their n-termini, for the synthesis of a cyclic peptide or for the synthesis of di-or tetravalent multiple antigenic peptides. synthetic peptide derivatives which contain secondary amide moiety at c-terminus are inhibitors of serine and cysteine proteinases and thus could be effi ciently used as ligands in affi nity chromatography in order to isolate these enzymes. there is a number of chemical syntheses of amidous peptide derivatives in the literature, but the reaction yields are usually low and reaction conditions and purifi cation procedures are too complicated. it is also known that the application of enzymatic catalysis allows to obtain high reaction yields with simultaneous simplifi cation of synthetic and purifi cation procedures, and at the same time preserves the optical purity of target compound. subtilisin 72 sorbed on silochrome could be effi ciently used as catalyst of acylation of secondary amides by the esters of acylpeptides. subtilisin's wide substrate specifi city allows to carry out the syntheses with peptides which contain hydrophilic, hydrophobic, dicarbonic-and diamino-acids at c-terminus. piperidine, morpholine, indole, diethylamine and other secondary amines are used in the syntheses as the amino-components. the reaction yield equals about 60-80% in dependence of the nature of c-terminal amino acid of acylating peptide, pka of amine and the hydrophobicity of the fi nal compound. the distribution of the reaction product in liquid and solid reaction phases is investigated, the optimal reaction conditions for enzymatic acylation of secondary amines are established. all the compounds obtained are characterized by its nmr and mass spectra. the inhibition constants for some proteolytic enzymes with the compounds of research are defi ned. 1 unnatural amino acids are becoming increasingly important substrates in modern drug design, synthesis and discovery research. in particular, arylhistidines naturally occur in the active site of the heme-copper oxidases as well as in cytotoxic and antifungal marine peptides (1) . a method of choice for the preparation of unsymmetrical biaryl systems is the suzuki-miyaura cross-coupling of an aryl halide with an arylboronic acid. it has been shown that microwaves signifi cantly enhance this reaction leading to higher overall yields and purities as well as shorter reaction times. although a great variety of biarylic compounds have been prepared following this approach, so far, it has not been applied to the arylation of the histidine imidazole ring. initially, we studied a methodology for the synthesis of 5-arylhistidines via a microwave-assisted suzuki-miyaura cross-coupling reaction in solution (2) . taking into account the advantages of the synthesis on solid support, such as the avoidance of tedious work-up which are particularly valuable for palladium-catalyzed reactions, we studied the application of the above methodology on solid-phase. here, we report the suzuki-miyaura reaction between a 5-bromohistidine and an arylboronic acid on solid support. the reaction conditions were optimized by varying several parameters such as the solvent, the reagent concentrations and the reaction time. the optimized conditions were subsequently applied to the synthesis of peptides containing a 5-arylhistidine residue. this work constitutes the fi rst suzuki-miyaura coupling involving the imidazole ring of a histidine on solid support. microwave-assisted attachment of fmoc-amino acids to resins via triazine "superactive esters". the substitution level of the new functionalized resin was calculated following a standard protocol based on the spectroscopic measurement of the soluble chromophore piperidine-dibenzofulvene. coupling reactions to the resin proceeds at room temperature in 3 hours. moreover we carried out the time-consuming attachment of fmoc-amino acids to the resin by an automatic monomode microwave (mw) instrument (liberty, cem), in fact microwave is proposed as a valid alternative to enhance effi ciency of coupling reactions and it has been widely applied to spps. novel peptide-hetorocycle conjugates: derivatives of 3-(benzimidazol-5-yl)alanine (1) . considering the biological activity and complexing abilities of benzimidazoles we developed a direct solid-phase synthesis of benzimidazole-peptide conjugates, expecting these new compounds to express novel biological properties (2) . the peptide-heterocycle conjugates are obtained by on-resin reaction between aldehydes and peptides containing a specially designed -(3,4-diaminophenyl)alanine residue (3) . the stoichiometric amount of aldehyde leads to 2'-substituted 3-(1h-benzimidazol-5-yl)alaninecontaining peptides, the increase in aldehyde content results in 1`,2`disubstituted derivatives. the reaction with dialdehydes gives novel amino acid residues containing tricyclic systems, in the case of ophthalic aldehyde -the pyrido-[1,2-a] benzimidazole. the compatibility of our method with the fmoc solid phase peptide synthesis protocols was proven by the synthesis of analogues of immunosuppressory fragments of ubiquitin and hla-dq [4, 5] . the biological activity of the modifi ed oligopeptides was compared to that of original fragments 6. as well as to similar quinoxaline-peptide hybrids. the collision-induced dissociation of substituted benzimidazole-peptide conjugates leads to characteristic fragmentation ions, which may be used as a diagnostic tool in the fragmentation of the benzimidazole-peptide hybrids. cardona, valérie; oswald, benoit genzyme pharmaceuticals, switzerland dehydroamino acids are an important class of compounds due to their presence in many biologically active natural products including the antrimycins, tentoxin, phomopsin a, or the phosphatase inhibitors like microcystin and nodularin. in the last decade an increasing interest in -branched dehydroamino acids has been developed based on their importance as commodity chemicals and value as tools in structure relationship studies. the incorporation into a peptide of such unsaturated amino acids introduces an element of conformational rigidity, as well as changes in reactivity, allowing for the development of high affi nity ligands for receptors. a variety of methods exist for the synthesis of dehydroamino acids. some of these approaches rely on thermodynamic control to dictate the alkene geometry. if there is no strong thermodynamic preference, or if the desired product is not the thermodynamically favoured isomer, the existing synthetic methods are often ineffective. we describe here an effi cient stereoselective method for the synthesis of , -branched dehydroamino acids (iii) from -aryldehydroamino acids (i). the -aryldehydroamino acids (i) were prepared from commercially available z/boc-phosphonoglycine trimethylester and aldehydes using the schmidt protocol. the transformation of (i) into their -bromodehydroamino acid derivatives (ii) is performed by a hoerrner reaction in a very good yield giving the trans derivatives. suzuki cross-coupling on (ii) with a boronic derivative generated the high value building blocks (iii). a variety of side chains can be incorporated using this type of reaction. the stereochemistry of these compounds has been determined using noe enhancement experiments. we report here a scalable and high yielding synthesis of stereospecifi c , -aryldehydroamino acid derivatives. versatile methods for synthesizing acyl-tetramic acids peptide analogs. davidov, gali; mozes, tamar; khandadash, raz; byk, gerardo bar ilan university, israel acyl-tetramic acid derivatives have been identifi ed as potential antiviral and antibacterial compounds. in this work we have improved their synthesis starting from tetramic acid lactams using peptide coupling reagents such as bop under microwave heating for generating the carbon-carbon bond of the acyl-tetramate. using this procedure we have synthesized a number of new tetramic acid derivatives in solution and generated a series of acyl-tetramic acid building blocks that were introduced into peptides using conventional spps methods. additionally, we present here a new solid phase microwave assisted synthesis of acyltetramates on pre-synthesized peptides and amino acids. antibacterial and antiviral activity of the products will be presented. peptide sequence and include all side-chain groups. in practice this is diffi cult to achieve. the task of making a turn mimetic can be regarded from the perspective of the backbone dihedral angles -to make a beta turn mimetic the phi and psi angles of two consecutive residues have to be controlled. limiting the dihedral angle space is most effectively accomplished with a cyclic constraint -one or more may be used. in this case a single medium ring cyclisation from the (i) carbonyl to the (i+3) amine -in place of the classical hydrogen bond -forces the four backbone dihedral angles into a turn conformation. due to the polyfunctional nature of peptide systems the introduction of unnatural constraints can be synthetically challenging. in the present case a number of side reactions were encountered and had to be overcome. some were well known such as diketopiperazine formation and others involving transannular interactions were unexpected and gave rise to interesting byproducts. ultimately the side reactions were overcome by choice of cyclisation position and synthetic modifi cations. auto-assembling antimicrobial cyclic pseudopeptides including aza-3 -amino acids antibiotic resistance of pathogens against conventional antibiotics is increasing at a rate that far exceeds the pace of new development of drugs. so, antimicrobial peptides, both synthetic and from natural sources, have raised interest as potential useful drugs in the future. however, due to proteolytic degradation, peptides are not ideal candidates for pharmaceutical development. that is why numerous researches try to develop non natural peptidic analogues for enhancing metabolic stability, bioavailability, and biological absorption. in this class of peptidomimetics, pseudopeptides consisting exclusively or including aza 3 -amino acids have emerged as a promising new class of compounds that favour hydrogen bond formation and can enhance biological activities when compared to natural parent peptides (1) . we have designed "mixed" cylic pseudopeptides composed of -and aza3 -amino acids that target bacterial cell wall and induce the death of the pathogens. particularly, some of these cyclic pseudopeptides have broad spectrum antibactericidal activities on gram positive and gram negative bacteria with low minimum inhibitory concentrations (mic). on the other hand, this type of molecules is not haemolytic and cytotoxic at antimicrobial activity levels(2) now, we try to explain the mechanism of action of our pseudopeptides that act on the microbial membranes. with nmr studies we have demonstrate that in solution cycles auto-associate at high concentrations and we investigate their behaviour in presence of small unilamellar lipidic vesicules (suv) (3) . this phenomenon of auto association seems to be facilitated at the lipid interface. liu, hongqiang 1 ; pattabiraman, vijaya r. 2 ; vederas, john c. 1 1 university of alberta, canada; 2 lantibiotics are a class of antimicrobial peptides containing lanthionine (1) and/or -methyllanthionine (2) residues in cyclic moieties, and are currently used for food preservation. they also have potential as human therapeutics. lacticin 3147 is a two-peptide lantibiotic produced by l. lactis subsp. lactis dpc3147, and its components (a1 and a2) are active against a wide range of gram-positive bacteria by a synergistic mechanism in sub nm concentrations. however, the oxidation of the thioether of lanthionine (1) and -methyllanthionine (2) is a major source of lantibiotic instability and loss of activity. to investigate structure-activity relationships and determine whether sulfur can be replaced, an analogue of lacticin 3147 a2 in which oxygen atoms replace sulfur was synthesized by solid phase peptide synthesis. utilizing the stereochemicaly pure oxa-lanthionine (3) and oxa--methyllanthionine (4) with orthogonal protecting groups, the conformationally constrained tricyclic moiety in oxa-lacticin 3147 a2 was constructed. the results of biological evaluation of the oxidatively stable analogue will also be described. controlling -helical secondary structure of oligopeptides and its use as a chiral catalyst replacement of -hydrogen atom of -amino acids results in ,disubstituted amino acids. as an , -disubstituted amino acid, achiralaminoisobutyric acid (aib) is well-known, and widely used to construct helical secondary structures of oligopeptides. the helical secondary structures constructed by using aib usually show 3 10 -helices, but nothelices in the case of short oligopeptides. recently we designed and synthesized a chiral cylic , -disubstituted amino acid (s,s)-ac 5 c dom , in which the -carbon atom is not a chiral center but chiral centers existing at the side chain. homooctapeptide composed of (s,s)-ac 5 c dom formed a left-handed -helix both in solution and in the solid state. furthermore, in the case that the (s,s)-ac 5 c dom was incorporated into l-leuhexapeptide, the hexapeptide cbz-{l-leu-l-leu-(s,s)-ac 5 c dom } 2 -ome preferentially formed a right-handed -helix in the crystal state, whereas the hexapeptide cbz-{l-leu-l-leu-aib} 2 -ome formed a right-handed 3 10 -helix. the fi nding that the propensity of cyclic amino acid ac 5 c dom is to form -helix over 3 10 -helix, stimulated us to use the -helical oligomer containing the cyclic amino acid as an asymmetric catalyst. we prepared several l-leu-oligopeptides containing , -disubstituted amino acids, analyzed their preferred secondary structures, and studied a enantioselective reaction of prochiral substrate using -helical oligopeptides as a chiral catalyst. fbp28 domains: spps using pseudoproline and depsipeptide approaches, stability and structure of glutamine-rich analogs (nature biotech., 2008) . one peptide will soon be tested in clinical studies. the success depends on effi cient synthetic access to large amounts of the peptide. systematic modifi cations of the lead structure hbvpres/2-48 have to be performed resulting in a panel of peptide variants to be studied with respect to their applicability, pharmacokinetics and serum stability. we selected a series of peptides carrying deletions, point mutations, d-amino acid exchanges and sequence permutations. the syntheses of 40 derivates were performed by fmoc-solid-phase synthesis on a rink amide am resin using hbtu/dipea activation on an applied biosystems 433a peptide synthesizer. after completion of the peptide sequence, stearic acid was attached to the n-terminus. the products were deprotected and detached from the resin by tfa treatment and subsequently purifi ed by hplc. the stability of the peptides was determined in human serum. we were able to synthesize all hbvpres lipopeptides in acceptable yields (7-40%). preparative hplc was used to obtain intended compounds in high purity as determined by hplc and esi mass spectrometry. the serum stability studies revealed exceptionally long biological half-lives (t1/2 > 24 h for all lipopeptides) mainly due to the fatty acid residue. the peptides belong to a class of intrinsically unfolded peptides and are therefore not prone to aggregations within the synthesis. consequently solid phase peptide synthesis provides a suitable access to the peptides described. it can be speculated that the peptides are protected against degradation due to an association via their lipophilic end to serum proteins. sugar derivatives for self-assembling -cyclic peptides brea last years, numerous inorganic and organic nanotubes have been developed. self-assembling peptide nanotubes (spn) made from cyclic peptides have structural and functional properties that may be suitable for various applications in biology and material science. recently, our group have reported , -cyclic peptide ( , -cp) that forms highly stable homo-and/or heterodimers with partial hydrophobic cavities. in the present communication we will describe the design, synthesis and applications of a new class of self-assembling -cyclic peptides containing sugar derivatives that modify both the inner and the outer surfaces of the resulting supramolecular entities. peptido2.rotaxanes: can a tetramide macrocycle travel to a station by wrapping up around a helical peptide thread? we are currently expanding this fi eld by synthesizing and studying the properties of new sets of peptido2.rotaxanes. the initial set examined includes symmetrical, achiral compounds with a fmoc stopper at each terminus, threads built up with a central fumaric diamide station and two helical aib ( -aminoisobutyric acid) homo-peptides of different lengths (from 1 to 5 residues), and an aromatic tetramide macrocycle. these supramolecular systems have been characterized spectroscopically and two of them by x-ray diffraction as well. the fundamental interactions between macrocycle and thread (the same interactions offering the major contribution to the template-directed preparation of this family of molecules) are intercomponent h-bonds comprising the four amide groups in the ring and the two amide bonds in the fumaric derivative. the second, more complex, set of peptido2.rotaxanes examined is represented by non-symmetrical compounds involving a thread based on a central helical -(aib) 6 -peptide linker and two stations of opposite chirality (a -d-leu-gly-gly-tripeptide at the n-terminus and a fumaric diamide-l-leu moiety at the c-terminus). as stoppers and the macrocycle, we selected two diphenylacetyl groups and the usual aromatic tetramide, respectively. as spectroscopically assessed, the macrocycle initially positions on the fumaric diamide-l-leu station. subsequently, by using photons as stimuli to induce the fumaric<->maleic equilibrium, we were able to switch partially the relative macrocyclebinding affi nity in favor of the -d-leu-gly-gly-tripeptide station. this is the fi rst example of a rotaxane where the ring makes a journey to one of the stations by wrapping up around a helical peptide thread. synthetic novel n phenyl and bi-phenyl tetracarboxamides bis peptides. an approach to dna threading intercalators as expected potential pharmaceutical carriers for catatonic agents. naphthalene diimides were reported to bind to dna opposite grooves via the threading intercalation mode (1, 2) . on the other hand, peptides constitute an excellent class of molecules for rapid drug discovery and lead optimization. the title bis dipeptides may, consequently, offer signifi cant dna biological probes. potential anticancer drugs or drug carriers could thus be presumed. herein, the title bis peptides a and b were suggested and synthesized as new prototype candidates of such class compounds. pre-assembled and purifi ed peptides via the conventional methods of peptide synthesis are, subsequently, coupled to either 1,2,4,5-benzene tetra-carboxylic dianhydride or 1,4,5,8-naphthalene tetra-carboxylic dianhydride. the expected structures of obtained a and b as preliminary candidates were confi rmed via the chemical, chromatographic and spectroscopic methodologies. the chemistry of both a and b will be discussed. synthesis of other candidates, the corresponding biological, pharmacological and physicochemical investigations are under realization. x = phenyl or bi-phenyl ring [compound a and b respectivel 63 the fragment pth(1-34) is suffi cient to bind and activate the pth type i receptor (pth1r). the molecular mechanisms by which pth binds to and activates the pth1r have been extensively investigated. recent investigations focusing on the interaction of n-terminal modifi ed fragments pth(1-11)nh2 with pth1r showed that certain modifi cations can increase signalling potency and that enhancement of the -helicity in the pth(1-11) sequence yielded potent analogues of pth(1-11)nh2. the design of cyclic analogues represents a widely used strategy to increase peptide stability and potency. the structural constraint induced by cyclization reduces conformational fl exibility and may enhance potency, selectivity, stability and bioavailability as well as membrane barrier permeability. initially, the work was concentrated on conformational constrains in n-terminal such as the introduction of ctetra-substituted amino acids. global restrictions in the conformation of a peptide are possible by limiting the fl exibility of the peptide strand through cyclization. to this purpose, the amino acid side chains that are not involved in receptor recognition are connected together or with the peptide backbone. previous works on the role of side chains of aa determined through d-scan, analogues which maintained better -helical structure, contained d-gln in position 6 and 10. recently gardella and co-workers reported that an analogues of pth(1-11) cyclized between 6 and 10 residues exhibited almost the same activity of linear analogues. so, in this work two new potent cyclic analogues in position 6 and 10 were synthesized directly on spps, using side chains of lys and glu or ser. then they were biologically tested and analyzed by cd, according to our previous work. boudreau, marc; vederas, john university of alberta, canada the neopetrosiamides a and b are two diastereomeric tricyclic peptides that inhibit amoeboid invasion of human tumor cells. they were isolated by anderson and co-workers from the marine sponge neopetrosia sp. collected in papua new guinea, with their subsequent structure elucidation by the same group in 2005. the peptides are 28 residues in length and contain three disulfi de bonds, as well as the unusual amino acid methionine sulfoxide at position 4. the two peptides differ only by being epimeric at this sulfoxide functionality. we report the total synthesis of neopetrosiamides a and b as well as an analog wherein the methionine sulfoxide has been replaced by norleucine, the carbon analog of methionine. the strategy involved solid phase peptide synthesis to generate the linear species, and selective formation of the disulfi de bonds from orthogonally protected cysteine residues. synthesis of mimetics of the antibody 82d6a3: a new class of antitrombotics. in the lab for tromboses research of the kulak (belgium) an antitrombotic antibody (ab) called 82d6a3 has been characterized (1) . the ab inhibits the interaction between the plasma peptide von willebrand factor (vwf) and by injury exposed collagen, a prerequisite interaction in thrombusformation in arteries (high blood shear). it has been shown in vivo that the ab 82d6a3 has an antitrombotic effect without showing the bleeding complications that known antitrombotics exhibit. this makes the ab an attractive lead for antithrombotic drug design. by x-ray and mutagenesis studies, the paratope (active site) of the antibody has been determined. 6 amino acids, discontinuous in the primary structure of the antibody but a quasi linear unit in space, play an important role in the interaction. incorporation of the functional groups of the amino acids and fi xation of the secondary structure of the paratope will be the aim in the design of the paratope mimetics. with this rational design we will try to develop an orally available, synthetic paratope peptidomimetic with the same antitrombotic effect as the antibody. the development (solid phase and solution peptide synthesis) of the mimetics is a stepwise process. in each step conformational restrictions are introduced. in this we hope to divert away from the peptide and go to a drug like molecule. 1 peptide ligands for sh2-and ptp domains containing phosphotyrosine are of great interest to infl uence the activity of kinases, phosphatases and other functional proteins. backbone cyclization can help to stabilize these ligands against proteolytic degradation and to form their bioactive conformation. till now backbone cyclization was performed with bifunctional and in few cases with trifunctional amino acids but not with phosphotyrosine. the assembly of such peptides requires preformed building units. because the necessary reductive alkylation of phosphotyrosine derivatives leads only to very low yields we used n-functionalized pseudodipeptides with the unprotected phenolic hydroxyl group. for fragment condensation we synthesized building units of the common structures: fmoc-aaø[co-n(x)-(ch2)n-nh-alloc)tyr(oh)-oh and fmoc-aaø[co-n(ch2)m -cooall)tyr(oh)-oh. depending on the steric hindrancy of the n-terminal amino acid these pseudodipeptides were synthesized in solution or at sasrinresin. they were purifi ed by fl ash chromatography and analytically characterized by hplc, esi-ms and nmr. we tested our strategy on the synthesis of an octapeptide-ligand for the n-terminal sh2-domain of the phosphatase shp-1: glu-gly-leu-asn/abu-ptyr-nle-asp-leu-nh2. couplings of the dipeptide units were performed with pybop, stepwise coupling to the peptide fragments with unprotected phenolic hydroxyl group with pentafl uoro phenylesters. after fi nishing the assembly the obtained polymer bound octapeptides were consecutively cyclized, phosphorylated, removed from the resin and purifi ed by hplc. based on elucidated side reactions the synthetic strategy was optimized. the obtained backbone cyclic and phosphorylated ligands were tested for their infl uence on the phosphatase activity of shp-1. the found enzymatic activities are correlated to size, direction, hydrophobicity and conformational fl exibility of the lactam bridges. biology and medicine. in this context, we have devised a new family of compounds named « polyamide amino acids » (paas), constituted by a pna (peptide nucleic acids) backbone mimicking rna sugarphosphate backbone, onto which aminoacid residues are linked. therefore, these paas could constitute a new type of rna ligands, liable to specifi cally interact with an rna target through an original interaction mode. to assess the ability of paas to be potential rna binders, we fi rst prepared 8 tetra-paas (t1-t8) via solid-phase synthesis, starting from 4 paa monomers deriving from alanine, phenylalanine, lysine and arginine residues. interactions of the 8 tetramers with a hiv-1 tar rna fragment, taken as a target model, was investigated by fl uorescence spectroscopy and circular dichroism. to give insights about specifi city, binding affi nities to tar were also assessed using an excess of a trna mixture. results showed kd values varying from 0.08 to 2032 m, indicating the importance of the aminoacid side chains in the interaction. thermodynamic analyses revealed that even if electrostatic interactions play a part in the complex formation, the binding is enthalpy-driven, highligthing the importance of non-electrostatic interactions in the recognition process. these results are of special interest since rna/ ligand association specifi city is typically assumed to be due to shortrange non-electrostatic interactions. moreover, t1-t4 were shown to be specifi c to tar in the presence of an excess of trna. all together, these results are encouraging and they highlight the potential of paas as rna ligands. indeed, the use of only four paa monomers as building blocks leads to tetra-paas displaying both affi nity and specifi city for their rna target. studies on the solid phase synthesis and selective detection of peptide derived amadori products by mass spectrometry stefanowicz mass spectrometric analysis of glycation products of proteins and peptides attracts increasing attention (1) . peptide-based amadori products could be used as markers of diabetes mellitus, which makes them the subject of interest in clinical chemistry. recently, several procedures of siteselective synthesis of amadori-modifi ed peptides has been published [2, 3] . in this communication we will present the synthesis of a new, fully protected derivative of glycated lysine which was successfully applied as a building block for incorporation of the glycated lysine moiety into peptide chain according to the standard fmoc-solid phase synthesis protocol. we will also present a new and straightforward method of selective detection of peptide-derived amadori products by esi-ms basing on characteristic neutral losses in the sugar moiety. the proposed approach in contrast to the neutral loss scanning, which requires triple quadrupol mass spectrometer, can be performed on the instruments with higher resolution and sensitivity, like q-tof . new apa inhibitors interacting with the s1 subsite bring new insights in the apa substrate specifi city mediated by the calcium ion. 3 aminopeptidase (apa, ec 3.4.11.7) is a membrane-bound zinc metallopeptidase involved in the maturation of brain angiotensin iii, a peptide which exerts a tonic stimulatory action on blood pressure in hypertensive animals (1) . therefore, developing inhibitors of this enzyme should result in new antihypertensive agents with possible new application in the treatment of certain forms of hypertension (2) (3) . we and others have previously reported the design of such inhibitors (4) (5) . nevertheless, the improvement of the affi nities of the inhibitors is relatively impaired since the three dimensional (3d) structure of the enzyme is not available. with the aim of getting insight into inhibitors optimization, a 3d model of apa was constructed in our group as an alternative (6) . furthermore, it is well established that apa substrate specifi city and enzymatic activity are calcium dependent. in order to render this model more accurate and so on to identify the amino acids residues involved in calcium binding, we have designed new inhibitors able to explore the apa s1 subsite to better understand its specifi city. we will report the synthesis of these new inhibitors, as well as an inversion of the specifi city of apa s1 subsite in absence of calcium. these results and the identifi cation of the amino acids implicated in calcium binding will be discussed on the basis of site-directed mutagenesis studies. one of the designed inhibitors, ni 926 (ki = 70 nm) is to date the more potent inhibitor of apa activity measured without calcium. altogether, these data should allow the improvement of apa inhibitors by structure aided design. synthesis, resolution, and absolute confi guration of bpaib, a benzophenone-containing c -tetrasubstituted -amino acid photoreactive amino acids with benzophenone side chains, the prototype of which is bpa (4-benzoyl phenylalanine), have found numerous applications as photo-probes for covalent modifi cation of enzymes and receptors, as well as in intramolecular quenching by a nitroxide free radical in trichogin peptide analogs. however, the remarkable fl exibility of the bpa side chain may question the extrapolation of results of photo cross-linking experiments and photophysical data to protein mapping and intramolecular distances, respectively [m. saviano et al., chembiochem, 2004, 5, 541-544 and references cited therein]. to overcome this problem, we designed a new "constrained bpa" amino acid, bpaib, belonging to the sub-class of the ci <->ci cyclized, c -tetrasubstituted -amino acids (strong -turn and helix inducers in peptides). racemic boc-bpaib-oh was prepared by bis(alkylation) of ethyl isocyanoacetate under phasetransfer conditions with 1-benzoyl-3,4-(bis)bromomethyl benzene as alkylating agent, followed by acidic hydrolysis, n -boc protection, and saponifi cation of the ester function. resolution was achieved through poster abstracts the terminally-blocked dipeptide bz-bpaib-l-phe-nhchx with chromatographic separation of the diastereomers and acidic hydrolysis. x-ray diffraction analysis of a crystal of a z-bpaib-l-phe-nhchx diastereomer allowed the assignment of the absolute confi guration of the bpaib enantiomers. photo-crosslinking studies with this residue are currently in progress. n-methylation of n -acetylated, c -ethylated, fullyextended homo-peptides: synthetic and conformational aspects moretto peptides characterized by single or multiple n-methylated, ctrisubstituted (protein) -amino acids are one of the subject of increasing interest in medicinal chemistry. several naturally occurring peptides, remarkably stable to proteolytic attacks, are based on n-methylated peptides. n methylation of the -conh-function is a useful tool for discriminating solvent exposed from intramolecularly h-bonded secondary amide groups in peptides. we are currently extending this reaction to linear peptides based on c -tetrasusbtituted -amino acids. after having investigated synthesis and conformation of the n-methylated homo-peptides from the c -methylated, helicogenicaminoisobutyric acid and c -methylnorvaline residues [a. moretto et al., biopolymers (pept. sci.) 2006, 84, 553-565], in this work we examined the n-methylation reaction on homo-peptides from c , -diethylglycine (deg), known to overwhelmingly adopt the fully-extended, multiple c5, conformation. we studied the following peptide series: ac-(deg) n -n(et) 2 , with n = 1-5. under the experimental conditions used, only monomethylation (on the n-terminal, acetylated residue) takes place. our ft-ir absorption, nmr, and x-ray diffraction analyses support the view that the fully-extended conformation preferred by the original peptides is dramatically perturbed in all of the derivatives mono-methylated at position 1. computational study on secondary structure of oligopeptides containing chiral , -disubstituted -amino acids we have shown mcmm conformational search method and amber* force fi eld using macromodel is useful to predict secondary helical structures ( -helix, 3 10 -helix) of oligopeptides prepared from ,disubstituted -amino acids. moreover, we have studied conformational analysis of oligopeptides containing chiral , -disubstituted -amino acids to predict the helical screw sense of helical structures. we calculated , -disubstituted peptide using mcmm conformational search with various force fi elds (amber*, mmff, opls) . in the case of using amber* force fi eld the results were in agreement with those of x-ray and were most stable conformation evaluated by 3-21g level molecular orbital calculation. these results indicated that computational simulation using conformational search calculations with amber* force fi eld is most useful for conformational analysis of oligopeptides containing , -disubstituted -amino acids. a new colorimetric test for solid-phase amines and thiols. simple colorimetric tests still remain the most convenient tests for providing information on the course of solid-phase reactions. a colour test, which indicates the presence or absence of a functional group by visual detection, is a simple, practical tool to monitor the completeness of the reaction. although the variety of colour tests for amines, there is still a need for a reliable and sensitive test in some synthetic approaches. here, we wish to report a new simple, fast and sensitive colorimetric assay for the visual detection of solid-phase bound primary, secondary amines and solid-phase bound thiol groups using 1-alkyl-2-aryl-imidazo[1,2-a]pyrimidinium salts. in the search for a new synthetic approach to polysubstituted 2-aminoimidazoles (1), we have reported a new procedure, employing substituted 2-aminopyrimidines and -bromocarbonyl compounds. the intermediate imidazo [1,2a] pyrimidinium salts in the synthesis of substituted 2-aminoimidazoles appeared to give a strong colour when reacted with primary and secondary amines. due to the strong colour change of amino functionalised resins after reaction with the "desc" reagent (2), a protocol for the detection of resin bound primary and secondary amines has been developed. the "desc" test is a new, practically, simple, quick and reliable test for the visual detection of resin bound primary and secondary amines in a sensitive way. the "desc" reagent is accessible from the cheap starting materials and can be prepared in two steps. tetrafl uoroborates of chiral n-triazinylammonium salts were found stable and useful in enantioselective (ee up to 98%) peptide synthesis from racemic amino acids in solution. several chiral n-triazinylammonium tetrafl uoroborates were obtained as stable l and/or d selective coupling reagents (1) . broad range of common amines (strychnine, brucine, sparteine etc.) were found useful as a chiral auxiliary, opening access to amino acids of both required confi gurations. we have attempted to expand the scope of application of this family of reagents expecting their effi ciency in enantiodifferentiating reactions in solid phase peptide synthesis (spps). tetrafl uoroborates of chiral n-triazinylammonium salts were obtained by treatment 2-chloro-4,6-dimethoxy-1,3,5-triazine with tetrafl uoroborates of appropriate tertiary amines in the presence of sodium bicarbonate (2) . enantiodifferentiating reagents were used in synthesis on wang resin under classic condition for triazine based coupling reagents (3) . the most important advantage of application of chiral n-triazinylammonium tetrafl uoroborates is the predictability of confi guration and repeatability of enantiomeric purity of incorporated amino acid. even if only threefold excess of racemic carboxylic acid, enantiomeric purity of coupling products in spps reached ee 98-99. the demand for diversity of non-proteinogenic amino acids, willingly used as new building blocks, is severely restricted by laborious procedures leading to enantiomerically homogeneous individuals. typical sources such as isolation from natural products, biotechnological methodology, even the asymmetric syntheses posses a limited value because complex or tedious synthetic procedures or limited access to the pool of chiral auxiliaries. herein we present the novel, general approach allowing enantioselective incorporation of any enantiomer of amino acids directly from racemic substrate. according to our procedure, the chiral auxiliary is used only at the stage of enantioselective activation. the departure of chiral auxiliary yields in the traceless way, the activated derivative of n-protected amino acid identical with those which is obtained with the well known, classic, achiral triazine coupling reagent (1) . therefore, the traceless chiral coupling reagents can be successfully applied directly in the coupling stage without additional studies of their reactivity. several chiral n-triazinylammonium tetrafl uoroborates were obtained as stable l and/or d selective coupling reagents. broad range of common amines (strychnine, brucine, sparteine etc.) were found useful as a chiral auxiliary, opening access to amino acids of both required confi gurations. the most important advantage of application of chiral n-triazinylammonium tetrafl uoroborates is the repeatability and predictability of enantiomeric purity and confi guration of incorporated amino acid. freeman, noam s.; hurevich, mattan; gilon, chaim hebrew university jerusalem, israel azapeptides are peptide analogues in which one or more of thecarbons, bearing the side chain residues, has been replaced by a nitrogen atom. azaamino acid residues conserve the pharmacophores necessary for biological activity while inducing conformational changes and increased resistance to proteolitic degradation. these properties make azapeptides an attractive tool for structure-activity relationship studies and drug design. a general approach for solid phase synthesis of azapeptides has been developed based on the in-situ activation of n-2-(3,5dimethoxyphenyl)propan-2-yloxycarbonyl (ddz), n €™-substituted hydrazines, with phosgene, followed by introduction to n-terminus of resin-bound peptide. the ddz-aza-amino building units include aliphatic, aromatic and functionalized side chains, protected for synthesis by the fmoc strategy. solid-phase azapeptide synthesis is demonstrated including selective mild deprotection of ddz with mg(clo 4 ) 2 and coupling of the next amino acid with triphosgene. the mild ddz deprotection is also orthogonal with boc chemistry. we describe the synthesis of n €™-substituted ddz protected hydrazines which have wide applications in the synthesis of azapeptides as well as in general synthesis of substituted hydrazines and aza containing peptidomimetics. ddz protected hydrazines offer the possibility to construct novel and unique drug-candidate structures such as branched and cyclic azapeptides. fast improved synthesis of insulin-like peptides barlos the a-and b-chains of insulin-like peptides were synthesized by various methods. the best results were achieved by dividing the sequences of the a-chain into three protected fragments and that of the b-chain into two fragments. the fragments were prepared on 2-chlorotrityl resin and/ or 4-methoxybenzhydryl resin using fmoc-amino acids. condensation of the fragments was carried out either by solution phase or solid phase techniques. the joining of the a and b chains was also studied using either biomimetic or chemoselective oxidative folding methods. a new approach for amides and peptides chemical synthesis by means of phosphonic acid/alkylene oxide chemistry few different derivatives of l-phe were synthesized, using originally discovered method of phosphonic acid/alkylene oxide chemistry. this method provides the successful synthesis of variety amides and dipeptides without preliminary protection of alfa-nh2 group of lamino acids. the supposed mechanism displays the formation of nphospholane carboxyanhydride (1-oxy-3-aza-2-phospholane-5-one), or an o-hydroxypropyl h-phosphonate salt of an amide of l-phe. the nphospholane carboxyanhydride is protected at the n-terminus, as well as is activated at the c-terminus. this pathway favors the desired reaction with a variety of nucleophiles from the n-to c-terminus. this method also allows the synthesis of different esters of amino acids employing alcohols as nucleophiles previously described by us (1). formation of disulfi de bonds in synthetic peptides is one of the more challenging transformations to achieve in peptide chemistry, in view of possible formation of oligomeric by-products and other side reactions, as well as occasional solubility problems in aqueous oxidizing media. the recently introduced polymer-supported oxidant, clear-ox™, has proven to be a very valuable tool in the preparation of disulfi de-bridged peptides. [1, 2] oxidations using clear-ox were carried out at ph ranging from 4 to 7 in water/acetonitrile solutions, with concentrations 10-15 times higher than comparable solution oxidations. the latest progress in the preparation of various monocyclic and bicyclic peptides will be presented. the amyloid -peptide (a ) is believed to play a causal role in alzheimer's disease (ad). one of the hypotheses of a neurotoxicity is that it induces the generation of reactive oxygen species (ros) and hydrogen peroxide (h 2 o 2 ) formation by binding to metals such as copper and iron. it is hypothesized that the sole tyrosine residue plays an important role in a -mediated toxicity, due to its ability to form a dityrosine cross-link from tyrosyl radicals generated in the highly oxidative environment in the brain. hence, studies of the dityrosine cross-linked a peptide dimers would increase our understanding of the oxidative alteration and dimerisation of a in amyloid formation and ad related neurodegeneration. we are investigating the synthesis of the a peptide dimers containing the dityrosine cross-link. dityrosine, suitably protected for solid-phase peptide synthesis (spps), has been prepared from iodotyrosine using a miyaura borylation-suzuki coupling method. studies on the synthesis of dityrosine-linked peptide dimers through incorporation of dityrosine in spps are underway. initial model studies employing 2,6-diaminopimelic acid (dap) have validated this approach. preparation of the model daplinked a peptide dimers will be discussed, as well as progress towards the dityrosine-linked a peptide dimers. nepomniaschiy, natalia; brik, ashraf ben-gurion university of the negev, israel the staudinger reaction, discovered nearly a century ago, occurs between a phosphine and an azide to form an aza-ylide. this transformation was further explored and developed, leading to several reactions of highly synthetic importance. traceless staudinger ligation is one example of such modifi cations, in which an ester moiety is placed within the phosphine structure to capture the nucleophilic aza-ylide, by intramolecular cyclization, leading to a stable amide bond. while the aza-ylide intermediate is known to be stable in organic solvents, it tends to hydrolyze rapidly under aqueous media to furnish the primary amine product. in the traceless staudinger ligation, however, the reduction of the azide to amine is a competing side reaction, as it would reverse the capture step leading to two peptide fragments. we have found that such reduction step would be benefi cial if an electrophile and an azide (rather than the phosphine) are placed within the switch peptide system. investigating various reducing reagents led us to the discovery that tris(2-carboxyethyl) phosphine hydrochloride (tcep) is excellent azide reducing reagent. both the reduction and the acyl transfer steps are rapid and occur in a few minutes. applying the tcep-mediated triggering in switch peptides, derived from the a to understand the currently unclear processes of pathological folding, self-assembly, and aggregation of amyloid peptide will also be reported. fast fmoc-deprotection reagent for peptide synthesis diffi cult sequences have long been known in solid phase peptide synthesis, theses sequences can experience sever aggregation both during synthesis and under purifi cation. the aggregation is believed to depend mainly on -sheet formation or/ and hydrophobic properties of the peptide. now a days there are several strategies to circumvent theses problems, one of the most successful is to incorporate a backbone protective group as the hmb group, develop by johnson and co-worker(1), and thereby prevent aggregation due to -sheet formation. an additional charge will increase the solubility of peptides towards aqueous solutions and thereby facilitate the purifi cation by hplc (using acetonitrile and water system) and analysis (by maldi-tof mass spectrometer) of the peptide. here we present the nmec (n-methyl-n-[2-(methylamino)ethyl] carbamoyl) group(2) that is a orthogonal protective group for tyrosine side chain and the hydroxyl moiety of the hmb group. the nmec group is fmoc compatible and is protected during the synthesis with a boc group. the fi nal cleavage from the resin with tfa renders the amine of the nmec group protonated and will increase the solubility of the peptide during purifi cation and analysis. the nmec group can be easily removed with a mild alkaline treatment by a cyclization elimination reaction. the nmec group has been employed in the synthesis of diffi cult and hydrophobic peptides as the membrane spanning sequence of the calciton receptor, amyloid (25-35) and amyloid (1-42) with success. [3, 4] . for example, infl uenza a virus-related peptide (h-gilgfvftl-h) with a diffi cult sequence was synthesized using o-acyl isodipeptide unit. analysis of the crude peptide revealed high purity of the product with no by-product derived from the diffi cult sequence or epimerization. using ch 2 cl 2 as solvent in coupling the isodipeptide unit, a 1-42 was also synthesized with almost no major side reaction 5.. racemization-free segment condensation method was developed by employing n-segments possessing a c-terminal urethane-protected o-acyl ser/thr residues 6.. the synthesis of long peptides/proteins by racemization-free segment condensation has thus become possible at ser/thr residues and not only c-terminal gly/pro residues. growing interest to peptide substrates, inhibitors and other peptidomimetics required new highly effective synthetic techniques. the important goal of enzymatic peptide bond formation is the optical purity of the peptide, which facilitates the product isolation. thus using enzymatic condensation as a last step in peptidomimetics production is preferable. fixing of enzymes on/in suitable insoluble supports has many advantages: high operational stability, ease of separation, possibility of recycling and improved activity in low water media. chitosan, natural hydrophilic polysaccharide, was used as a matrix as it has distinct advantages over the other supports due to (a) its renewable nature -it is available in large quantities as a waste product from fi shing industry and (b) its excellent fi lm-forming ability, allowing attachment to reactor walls for advanced processing. serine proteases subtilisin and chymotrypsin, and cysteine protease papain were immobilized onto chitosan. the fi lms of biocatalysts were prepared by drying the mixed solution of chitosan and enzyme in acetate buffer ph 5.6. treatment with glutaraldehyde was found to give material with high stability and good mechanical properties. the obtained biocomposites showed high amidase activity against specifi c chromogenic peptide substrates and protein substrate azacasein. immobilized subtilisin and chymotrypsin also possess esterase activity against p-nitrophenyl acetate. the longterm storage in aqueous buffer and acetonitrile had a little effect on the hydrolytic activity of subtilisin-based biocomposite. the dependence of subtilisin/chitosan hydrolytic activity on temperature and ph was studied. obtained samples possessed high synthetic activity and were capable to catalyze peptide bond formation in dmfa/mecn mixture in reaction zaalome+fpna zaalfpna for subtilisin, zaaome+lpna zaalpna for papain. the product yield reached 60-100% in 24 h. acknowledgement: this work was supported by rfbr 06-03-33056 69 synthetic chemistry or on solid phase and b) the chemical ligation of the [cys12(acm)]-(1-25)-thioester with the [cys48(acm)]-(26-68) segment, both deprotected and hplc purifi ed. the required intermediate peptides were prepared by optimized fmoc-based methods either stepwise or by convergent synthesis. for the formation of the two disulfi de bridges a two step procedure was investigated, involving a dmso oxidation step to form the cys26-cys45 linkage, followed by iodine oxidation to form the cys12-cys48 bond. native chemical ligation at valine haase, christian; rohde, heike; seitz, oliver humboldt-universität zu berlin, germany native chemical ligation is perhaps the most useful technique for peptide segment coupling in water. (1) a c-terminal peptide thioester reacts with an n-terminal cysteine. the requirement for this rare amino acid represents the major bottle neck to the synthesis of native proteins. several strategies have been developed to overcome this limitation. in the extended ligation, n-terminally attached auxiliaries allow access to other ligation junctions by mimicking the cysteine-thiol moiety. the ligation-desulfurization approach employs -thiol amino acids for fragment coupling followed by sulphur removal. herein, cysteine acts as precursor of the abundant alanine(2) and phenylalanine can be obtained by using its -mercapto derivative. (3) . we demonstrate the application of penicillamine in the generation of valine junctions employing the ligation-desulfurization approach. the , -dimethylcysteine building block is commercially available with various protecting group patterns suitable for routine solid phase synthesis of peptides. the ligation at penicillamine proceeded surprisingly fast despite the steric demand at the thiol group. even leu-val ligation sites, which appear in hydrophobic peptide segments, are accessible. we also present an improved method for achieving metal-free desulfurization and show applications in the synthesis of valine-containing peptides.(4) references: 1. p. e. dawson the application of n-alkyl cysteine (nac)-assisted thioesterifi cation reaction to the synthesis of polypeptides by the thioester method azide as a protecting group for lysine side chains on the solid phase peptide synthesis oriented toward the peptide condensation by the thioester method peptide ligation chemistry has been developed based on the use of peptide thioester as a building block in the thioester method (1) and native chemical ligation (2) . we found that a cysteine-containing peptide is transformed into the corresponding s-peptide (peptide thioester) by the n to s acyl shift reaction (3). on the other hand, in 1985, zanotti et al. reported that a diketopiperazine thioester, cyclo(-cys(coch2ph)-pro-) (1) was formed when a dipeptide p-nitrophenyl ester, phch2co-cys(st-bu)-pro-onp (2), was treated under reductive aqueous conditions (4). thioester 1 would be formed via intramolecular n-s acyl shift reaction followed by diketopiperazine formation. based on these observations, we designed a cysteinyl prolyl ester (cpe) autoactivating unit for preparation of the peptide thioester and for the peptide ligation [5, 6] . a peptide containing the cpe unit, peptide-cpe 3, was transformed into a peptide thioester of diketopiperazine, cyclo(-cys(peptide)-pro-) 4, then thiol-thioester exchange reaction with an external thiol produced the peptide thioester 5 and a diketopiperazine, cyclo(-cys-pro-) (6) . the poster abstracts peptide-cpe 3 was also able to ligate with a cysteinyl peptide 7, via the thioester in one-pot, to give a polypeptide 8. (1) has enabled the synthesis of entire proteins including those carrying posttranslational modifi cations. one of the most frequently encountered modifi cation of eukaryotic secretory and cell surface proteins is the attachment of oligosaccharides to asparagine residues (n-glycosylation). despite many efforts in this fi eld the function of nglycosyation is poorly understood, which is mainly caused by the lack of pure glycoproteins. since purifi cation of natural glycoproteins is quite tedious due to heterogeneity in the sugar part, the total synthesis of homogeneous glycoproteins has become an attractive target (2) . we have addressed this topic by choosing rnase c as a model glycoprotein. instead of the oligomannosidic type rnase c is containing a complex type n-glycan. for synthetic reasons ligations had to be conducted in a sequential manner (3) by fi rst reacting the recombinant 40-124 cys-segment (4) with an n-terminally protected and glycosylated thioester 26-39 5.. selective deprotection of the ligation product 26-124 and ligation with synthetic thioester 1-25 in a one pot manner was accompanied by unexpected side reactions. these drawbacks were fi nally overcome leading to full-length glycosylated rnase c displaying enzymatic activity. a new pseudo-native ligation: multiple, successive azidealkyne cycloadditions. (1) the reaction has become a classics. as triazoles are stable to acid and basic hydrolysis, and reductive and oxidative conditions, the reaction has been widely used in chemistry and biochemistry, proving to be an useful tool in combinatorial, bioconjugate or medicinal chemistry. the reaction has been applied in the peptide fi eld as an easy way to synthesize cyclic, labelled and side chain modifi ed peptides including effi cient synthesis of pseudo-glycopeptides. conformational and structural studies prove that the triazole ring is an excellent trans-amide surrogate and thus, can be considered as a new pseudo-native linkage. (2) in order to explore the potential of the reaction as a way to successively assemble several peptide chains to generate mimics of proteins, we have elaborated a new strategy for consecutive triazole formation based on a semi-orthogonal alkyne protection scheme. (3) so far, we have improved our fi rst one-pot successive cycloaddition approach performing the fi rst three successive cuaac in mild conditions thanks to a highly selective deprotection of two different alkyne groups. the application of this strategy represents a new promising method for the chemoselective pseudo-native ligation dedicated to the synthesis of protein chimera or for the decoration of molecular templates. solid phase synthesis using lightly crosslinked polystyrene supports has proved to be a successful route to the manufacture of peptides. the methodology introduced by merrifi eld nearly fi fty years ago has remained largely unchanged. during the last twenty years or so, it has been argued that incorporating a hydrophilic polymer within the conventional hydrophobic polystyrene matrix is benefi cial. others have suggested that polyamide or polyether-based resins may prove to be superior to polystyrene. despite this, large-scale peptide manufacture has generally relied on traditional polystyrene supports. this is due in part to the diffi culty in producing composite supports economically in large volumes, but also due to the handling diffi culties that are often associated with very high swelling polar polymers. other problems arise from the fact that many of these types of resin are relatively low loading and so yields are greatly diminished. in this poster we offer a solution to these problems: a polystyrene derived support which is modifi ed to create economical, amphipathic resins suitable for large scale peptide synthesis. attachment of appropriate handles or linkers enables both peptide acids and peptide amides to be produced. the synthesis of a variety of peptides is demonstrated. peptide synthesis in water using boc-amino acids nanoparticles reactants. here, we studied in-water solution-phase method using water-dispersible nanoparticulate boc-amino acids, which are the most common building blocks but are diffi cult to use for peptide synthesis in water. water-dispersible boc-amino acid nanoparticles were prepared by pulverization using a planetary ball mill in the presence of peg, and the size of the resulting water-dispersible nanoparticles was determined by dynamic light scattering analysis. the scanning electron microscopy image of water-dispersible boc-phe-oh nanoparticles also revealed nanosize particles. we studied in-water coupling reaction using waterdispersible boc-amino acid nanoparticles, and leu-enkephalinamide was successfully synthesized in water according to the boc chemistry. an alternative way for conopeptide formation kádár, kinga 1 ; panyi, györgy 2 ; tóth, gábor k. 1 1 university of szeged, hungary; 2 university of debrecen, hungary the chemistry used to oxidize the free thiol bonds to the corresponding disulfi de bond in a controlled fashion remains a signifi cant challenge in spite of many advances in peptide chemistry. the primary reason of this lies in the diffi culties involved in the formation of multiple regioselective disulfi de bonds. in this work we focused on the elucidation of synthetic strategies for the preparation of multiple disulfi de containing peptide venoms regulating the ion channels of immune cells-anuroctoxin and tc32-, and a neuropeptide with regulatory functions-orexin a. for the synthesis of these naturally occuring cys-rich peptides we had chosen the oxidative folding being the most simple of the methods available. in the case of anuroctoxin we isolated the native form of the peptide toxin with high selectivity and we optimized the folding conditions, so within an hour the majority of the linear peptide folds in the cyclic form with all four disulfi de bonds in the correct form. the regioselectivity of the isolated isomer was verifi ed with biological measurements. for the synthesis of orexin a we optimized the folding conditions and, using a polymer-supported oxidant-the clear-ox resin-, within two hours we could isolate the correctly folded isomer as the major reaction product. the correct structure was proven by coelution of the isolated isomer and the commercially available orexin a. but oxidative folding does not always give the correctly folded isomer. the best proof for this is the tc32 scorpion toxin which albeit our tryings always gave the misfolded isomers if we used the linear, unprotected peptide. therefore a new chemical synthesis using different side-chain protection needs to be taken into consideration. the synthesis of orthogonally protected tc32 and its use for the preparation of the correctly folded peptide to be underway. poster abstracts have fundamental importance in biological recognition processes. one of the most challenging task among of them the rational preparation of the glycosylated peptides especially having oligosaccharide moieties. there are two main strategies for the synthesis of glycopeptides: the synthon and global (convergent) method. both of them can be implemented in liquid or solid-phase. since the glycosylation could appear on o and n atoms of the amino acid side-chain, due to the different reactivity of the glycosidic linkage different chemical strategies will necessitate. in this presentation we compare several chemical strategies for the preparation of two model peptides (leu-lys-asn*-gly-gly-pro, gly-val-glu-asp-ile-ser*-gly-leu-pro-ser-gly,*site of glycosylation). as glyco-part several mono, di and trisaccharide including chitobiose, galactosilxilose, mannosil-n-acetyl-glycosil-n-acetyl-glucosamine were used and several of the used strategies led to successful preparation of these glycoconjugates. site-specifi c pegylation of human igg1-fab using a rationally designed trypsin variant in the present contribution we report on a novel, highly selective biocatalytic method enabling c-terminal modifi cations of proteins with artifi cial functionalities under native state conditions. the approach is based on the fourfold trypsin variant k60e/n143h/e151h/d189k which was generated by site directed mutagenesis and initially specifi ed in terms of selectivity and activity using a peptide library of the general structure bz aax aa x bb x cc aag-oh. the trypsin variant was found to bear a restricted proteolytic activity towards the rare recognition sequence yrh with an occurance of less than 0.5% in native proteins. the specifi city is zinc ion dependent due to an artifi cial metal binding site in the s2´-subsite (1). placing of the recognition sequence at the cterminal region of respective proteins via standard mutagenesis protocols provides suitable precursor targets for site-specifi c modifi cation via a transamidation reaction. due to the great demand for polymer-modifi ed antibody fragments for pharmaceutical purposes we evaluated the function of the approach on example of the c-terminal pegylation (peg...polyethylene glycol) of the human igg1 fab fragment. the fragment itself was expressed with the recognition sequence and an additional strep-fusion enabling effi cient purifi cation. the derivatization of the antibody fragment with 40 kda peg via the trypsin variant (mw ~ 24 kda) proceeds effi ciently with a total yield of isolated modifi ed protein of 40%. interestingly, no undesired cleavage reactions could be detected leading to fully active modifi ed antibody fragment. references: 1. willett, w.s., brinen, l.s. et al. (1996) . "delocalizing trypsin specifi city with metal activation." biochemistry 35 (19): 5992-8. 2-chloro-4,6-bis-(2,2,2-trifl uoroethoxy)-1,3,5-triazine and n-4,6-(2,2,2-trifl uoroethoxy)-1,3,5-triazin-2yl)ammonium tetrafl uoroborates as highly effi cient coupling reagents jastrzabek, konrad; kolesinska, beata; kaminski, zbigniew, j. we expected that modifi cation of substituents in the triazine ring improve activity, stability and solubility of new triazine based coupling reagents. in order to increase activity we prepared 2-chloro-4,6-(2,2,2trifl uoroethoxy)-1,3,5-triazine by treatment of cyanuric chloride with 2,2,2-trifl uoroethanol. taking advantage of modular structure of triazine coupling reagents the entire family of n-(4,6-(2,2,2-trifl uoroethoxy-1,3,5-triazinyl-1)ammonium tetrafl uoroborates have been obtained. effi cient coupling reagents should be useful for amide bond formation between broad range of substrates, work in stoichiometric quantities, be soluble and stable in most of the solvents. it should function effi ciently in solution as well as in spps, to get high purity crude products and minimize racemization of the products. we found n-(4,6-(2,2,2trifl uoroethoxy-1,3,5-triazinyl-1)ammonium tetrafl uoroborates useful for activation of carboxylic components. the participation of triazine "superactive ester" as intermediate in the condensation has been proved in the model experiments. utility of reagents n-(4,6-(2,2,2trifl uoroethoxy-1,3,5-triazinyl-1)ammonium tetrafl uoroborates were confi rmed by peptide synthesis in solution in high yield. in our study we focused our attention on the performance (in terms of purity of the crude and extent of racemization) testing the solid phase synthesis of acp(65-74) and model peptides with aibaib fragment, which are a good example of diffi cult peptide sequence. purifi cation of chemically synthesised proteins by use of cleavable imac-based n -terminal protein protecting groups (tags) (1), there remains a challenge in terms of time and cost, for the separation of impurities from the chemically synthesised product in spps. this is particularly acute in the case of protein synthesis where the crude product mixture contains capped (acetylated) truncates of comparable size and structure to the protein product. such a common situation results in extensive, and expensive, purifi cation techniques such as large scale hplc. to this end we designed a hydrophobic tag, tbfmoc (2) , which incorporated the base-labile characteristics of the fmoc (3) group. the tbfmoc group causes retention on hydrophobic columns and hence separation from untagged impurities. although this was effective, we were of the opinion that a complementary tag was required which would have the characteristics of metal-complexation and cleavage by -elimination for subsequent removal of the tag from the protein product after imac. considerations implicit in the design of such an n -terminal protecting group, or tag, will be discussed , and applications to the chemical synthesis of chemokines will be dealt with in the presentation. in last few decades, acridines which are known as anticancer, antimicrobal and antiviral agents have been in the center of interest of scientists. the heterocyclic molecules demonstrate a noteworthy group of compounds which interact with biological targets e.g. topoisomerase i, ii [1, 2] . we wish to pay our attention to a new group of tuftsin-acridine conjugates. tuftsin offers a wide range of biological activities such as supporting of immune system, bactericidal, tumoricidal activity. however tuftsin is unstable in plasma which reduces its effi cacy. that is the reason for search of new analogues that were more resistance to proteolysis degradation (3). tuftsin-acridine conjugates were synthesized using a fmoc solid phase strategy. the elongation of peptide chain was based on twostep procedure: deprotection and coupling reaction with dic and the additive of hobt in dmf/dcm/nmp mixture. tuftsin analogues were modifi ed at -amino group of lysine via introduction of the simple amino acids to obtain isopeptide bond. tuftsin analogues were conjugated to the acridine molecule via fl exible linkers. the carboxylic group of linker was connected to n-terminal group of peptide-resin using standard method for amide bond formation. the coupling reaction was performed with peptide-resin and the 4-fold excess of 1-nitro-acridine derivative using tbtu, hobt in the presence of diea in dmf for 24h. simultaneous deprotection of peptide side chain and cleavage from resin was done with the tfa cocktail. final products were purifi ed by spe and characterized by elemental analysis, ms and 1 h-nmr spectroscopy. the effect of microwave irradiation under controlled temperature conditions on the solid-phase synthesis of peptides was investigated. for optimization studies a model peptide (h-gly-ile-leu-thr-val-ser-val-ala-val-oh) was selected which suffers from poor synthetic effi ciency under standard spps conditions. synthesis of the nonapeptide was performed using various combinations of solid supports (polysterene, tentagel, chemmatrix) and solvents employing fmoc/but orthogonal protection strategy. applying controlled microwave heating, the reaction times were signifi cantly reduced while maintaining a high purity of crude product with no racemization being observed. the optimized microwave synthesis method was succesfully applied for longer, aggregated peptides. microwave coupling and cleveage were accomplished in a dedicated reactor setup that allowed accurate internal reaction temperature measurment using a fi ber-optic probe system. comparison studies between microwave-and conventionally heated reactions will be presented. during the last few years microwave assisted peptide synthesis has became popular and it was reported to be useful in some cases. there is also an ongoing discussion about an additional "microwave effect". based on this background we have synthesized several model peptides on a microwave synthesizer and compared with the synthesis on conventional batch and continuous fl ow synthesizers with and without microwave irradiation. during this investigation we have also compared reaction rates and purity of the peptides synthesized under the infl uence of microwave irradiation or conventional heating on this different synthesizer systems by various conditions. in no case we can fi nd any additional postulated "microwave effect". the results of this investigation will be presented and discussed. conotoxins form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. the subgroup -conotoxins specifi cally and selectively bind to subtypes of nicotinic acetylcholine receptors (nachrs), which are targets for treatment of several neurological disorders. the aim of this work is to develop an improved method able to generate conotoxins in high yield and purity. this will overcome a key barrier currently preventing the effi cient synthesis of small focused libraries in order to investigate the structureactivity relationship (sars) of those peptides. the development of general synthetic strategies for the preparation of conotoxins and analogues are essential to effi ciently approach important questions within the area of neurobiology and for the development of novel drugs for treatment of various neurological diseases. a new and highly effi cient synthetic strategy for the synthesis of alpha-conotoxin-mii has been developed. this strategy combine solid phase synthesis with microwave assisted heating to produce the two disulfi de bonds peptide in high yield and purity. we fi rst report here the use of microwave assistance in order to form a disulfi de loop. this technique demonstrates the advantage of preparing the fi rst disulfi de bridge while the peptides are resin bound. this step is critical to provide the dicyclic native peptide, that was performed by a followed classical in solution oxidation by iodine strategy. an enhanced total procedure for the synthesis of ctxmii is recommended, which can be effi cient applied at several small disulfi de rich peptides of biological importance. solid phase peptide synthesis in aqueous environment using microwave assistance heating galanis, athanassios s. 1 since the fi rst reports on the use of microwave heating more than 20 years ago, microwave-assisted organic synthesis (maos) has become an important tool for rapid and effi cient synthesis of organic molecules. microwave assisted heating has been further applied to peptide synthesis in order to accelerate the rate of synthesis and to improve the yields for the synthesis of diffi cult sequences. as far as water as solvent is concerned, numerous recent publications report the combination of water as an environmentally benign solvent for chemical transformations with the use of microwave irradiation as an effi cient heating method. we report herein, the combination of the microwave-assisted heating and the use of water as solvent for solid phase peptide synthesis. a variety of common amino acids derivatives and coupling reagents have been studied in order to optimize coupling reactions in water by microwave-assisted heating. we also describe the total synthesis of a small peptide using water as solvent. the solid-phase synthesis using the environmental friendly aqueous medium dramatically reduces the cost of the synthesis and could be broadly applied in research or in industrial production of peptides. vanier, grace; collins, jr., michael; singh, sandeep; collins, jonathan cem corporation, united states the application of microwave energy for solid phase peptide synthesis (spps) represents a major breakthrough for overcoming incomplete and slow reactions typical of conventional spps. microwave energy has been applied successfully in a manual and automated approach for enhancing synthesis of peptides and peptidomimetics. common side reactions such as racemization and aspartamide formation have been studied and shown to be easily controllable with optimized methods that can be applied routinely. we will present the latest research on improving the synthesis of diffi cult peptides with microwave energy. synthetic antifreeze glycopeptides and analogues: synthesis, structural analysis, and functional studies norgren, anna s. 1 glycosylated peptides are involved in various biological processes such cell adhesion and differentiation. in contrast to mucin-type oglycan peptides which are present on the membrane of mammalian cells antifreeze glycopeptides (afgps) are a little investigated example of glycosylated peptides containing similar components. afgps usually consist of a varying number of repeating units of (ala-ala-thr) with minor sequence variations and the threonine hydroxyl oxygen glycosylated with the disaccharide -d-galactosyl-(1-3)--n-acetyl-d-galactosamine. antifreeze activity has been proven by different experimental observations like suppression of recrystallisation and ice nucleation, thermal hysteresis and change of the crystal habitus. although it is known that the n-acetyl group at the c2 position of the galactosamine, the -confi gured glycosidic bond to the threonine hydroxyl group and the -methyl group are essential, the adsorption mechanism is not yet understood. (1) . in contrast to fragment condensation, solid phase peptide synthesis gives the possibility to prepare one defi ned product and to introduce structure inducing amino acids such as proline. the disadvantage of spps with the bulky glycosylated amino acids is the low coupling effi ciency. this problem was overcome by using more active coupling reagents and microwave-enhanced methods during coupling leading to suffi cient coupling effi ciency without having to apply great excess of the glycosylated amino acid. after purifi cation the peptide structure was examined by cd and nmr in water and dmso at different temperatures. additionally, the substances were microphysically analysed according to their recrystalisation inhibition activity and their infl uence on the crystal habitus. microwave technology applied to spps has been recently proposed as valid support to the enhancement of coupling rates. we also used microwave energy in conjugation process between polyelectrolytepeptide bioconjugation with edc, hbtu. the use of peptide epitops of viruses particles in the composition of polymeric conjugates as vaccine has several potential advantages over whole viral or bacterial preparation. recently, our group report a novel approach to a totally synthetic vaccine which consists of fmdv (foot and mouth disease virus) vp1 peptides, prepared by covalent conjugation of peptide biomolecules with membrane active carbochain polyelectrolytes in the present study, peptide epitops of vp1 protein both 135-161(p1) amino acid residues (ser-lys-tyr-ser-thr-thr-gly-glu-arg-thr-arg-thr-arg-gly-asp-leu-gly-ala-leu-ala-ala-arg-val-ala-thr-gln-leu-pro-ala) and triptophan (trp) containing on the n terminus 135-161 amino acid residues (trp-135-161) (p2) were synthesized by using the microwave assisted solid-phase methods. synthesis of peptides were performed by microwave assisted spps. peptides characterized by lc-ms and purifi ed by rp-hplc. bioconjugation between polyelectrolytespeptide were synthesized by two different microwave assisted method. the fi rst one is classical carbodiimid activating method. the second one is hbtu activating method. the second method is a novel and effective method for bioconjugation process. peptides and polyelectrolytespeptide bioconjugates analysed and comparised by gpc system with four dedector (uv. refractive index, light scattering, viscosity). microwave-assisted solid-phase synthesis of peptide probes to detect specifi c biomarkers: shifting off limitations affecting conventional synthetic strategies biomarkers play a key role in development of diagnostic/prognostic tools. in fact, since their involvement in several diseases such as autoimmune diseases (i.e., multiple sclerosis, rheumatoid arthritis) and neurodegenerative diseases as alzheimer's disease, biomarkers represent a great promise for the development and set up of tuned therapies. for autoimmune diseases, we proposed the development of synthetic posttranslational modifi ed peptides as antigenic probes for characterization of specifi c and high affi nity autoantibodies as biomarkers. by an innovative "chemical reverse approach" we propose optimisation of antigenic probes by a statistically signifi cant screening of sera guided by autoantibodies circulating in patients' biological fl uids (1) . spps via the building block approach is the principal strategy leading to modifi ed peptides. high purity is a condicio sine qua non for effi cient biomarker detection. these syntheses can present several diffi culties as result of internal aggregation of resin-bound peptides during elongation steps, reducing reagents penetration, and signifi cantly decreasing reaction rates in both acylation and deprotection steps. such events strongly affect purity of the crude peptides and therefore, fi nal yield. we demonstrated that application of microwave (mw) energy in spps of modifi ed complex peptide probes exhibits several advantages improving coupling rates, possibly because of the decrease of chain aggregation during the synthesis (2) . we report the mw-assisted synthetic conditions of diffi cult peptides (i.e. glycosylated, citrullinated, multiple antigenic, amyloidogenic peptides, etc.) by which we strongly improved preparation of diagnostic/prognostic probes in terms of crude purity, fi nal yield, and time-consuming compared to conventional protocols. fidan, zerrin; volkmer, rudolf charité berlin, institute for med. immunology, germany the alpha-helical coiled-coil structure is a versatile protein-interactiondomain, in which the coiled-coil is composed of at least two righthanded amphipathic alpha-helices, which are coiled up around each other into a left handed supercoil. this widespread structural motif is involved in many biological procedures like transcription, scaffolding or signalling. in addition, the most characteristic and important identifying feature of a coiled-coil is the recurrence of a periodic heptad repeat sequence. coiled-coils are able to form complexes up to heptamers of different orientation. based on this signifi cant occurrence and also their structure coiled-coil peptides have the ability to function as molecular recognition molecules. our goal is to use self-associating coiled-coil sequences as molecular building blocks (lego brick). the ligation of desired functionalities on coiled-coil sequences opens up the opportunity to add different functionalities on artifi cial complex peptide molecules that stick together via coiled-coil moieties. this makes coiled-coil sequences to useful tools during complex oligopeptide assembly. we want to present novel chimeric oligopeptides which are build up by two segments, one responsible for association the other for functionality. as a model for the association segment several gcn4-leucine zipper mutants (1) and as functionality segments amongst others ww domains were used. both segments were linked by the native chemical ligation approach. we will present in detail the synthesis of a gcn4-leucine zipper mutant and also the following c-terminal thioester formation for the subsequent ligation step. furthermore, we will present the synthesis of the functional device, a ww-domain with a n-terminal cystein residue and also the native chemical ligation of both synthesized peptide fragments supported by hplc and mass-chromatography. dialysis-related amyloidosis, a disease arising from long-term dialysis is characterised by gradual accumulation of 2-microglobulin ( 2m) amyloid fi brils in bones and ligaments. although 2-m is known to form amyloid fi brils in vitro under acidic ph conditions, seeding of preformed amyloid fi brils or as an effect of ionic strength, the mechanism underlying aggregation of soluble 2-m into insoluble fi brils under physiological conditions is largely unknown. interestingly, eakin and co. recently showed that the chemical basis of the amyloidogenesis process is a backbone isomerisation of the conserved pro 32. our aim is to understand the molecular mechanism associated with 2-m amyloidogenesis using fourier transformed infrared (ftir) spectroscopy and site-directed-isotope labelling, a method in which the vibration of the labelled residue is shifted from its original position in the spectrum. we already demonstrated that ftir spectroscopy along with site directed-isotope-labelling is a promising approach to obtain information on the microenvironment of tyrosine side chain residues.2 to use this approach in the case of 2-m amyloidogenesis, we decided to synthesise an isotopically labelled 2-m at crucial residues such as pro 32 that should undergo drastic variations in their microenvironments during the misfolding. with its 99 residues, 2-m is over the limits of a reasonable synthesis using spps but the two suitably positioned cysteines in its sequence offer the possibility of a chemical synthesis using native chemical ligation. thus, we were able to realise the chemical synthesis of an isotopically labelled 2-m in a good yield using a three segments strategy with disconnections at the two cysteines and thiazolidine as a masked cysteine for the middle segment. the synthetic 2-m obtained was in full agreement with the characteristic 2-m ftir spectra. a novel cyanophycin synthetase from thermosynechococcus elongatus bp-1 catalyzes non-primer-dependent cyanophycin synthesis. cyanophycin (multi-l-arginyl-poly[l-aspartic acid]) is synthesized by cyanophycin synthetase (cpha). it was believed that cyanophycin synthetase requires asp, arg, atp, mg 2+ and primer (low-molecular mass cyanophycin) for cyanophycin synthesis and catalyzes the elongation of low-molecular mass cyanophycin. despite extensive studies of cyanophycin, the mechanism of primer supply is still unclear. in the present study, we searched for a cyanophycin synthetase that synthesizes cyanophycin from asp and arg without added primer in vitro. cyanophycin synthetase from thermosynechococcus elongatus bp-1 (tlr2170 protein) was produced by an escherichia coli geneexpression system as a c-terminal his-tagged protein. we found that tlr2170 protein synthesized cyanophycin without added primer. the tlr2170 protein had strict substrate specifi city and used only asp and arg as substrates. the optimal ph was 9.0, and mg 2+ or mn 2+ was essential for cyanophycin synthesis. atp could not be substituted by gtp, ctp, or ttp. the molecular mass of the tlr2170 protein as estimated by gelfi ltration chromatography was 400 } 9 kda. thus, the tlr2170 protein appeared to be a homo-tetramer of 100-kda subunits including the his-tag sequence. the tlr2170 protein had thermal stability and fully retained its activity after a 15-min incubation at 60 ‹c. additionally, we examined cyanophycin synthesis at 30 ‹c, 40 ‹c, 50 ‹c, and 60 ‹c. sdspolyacrylamide gel electrophoresis showed that the molecular mass of cyanophycin increased with increased reaction temperatures. synthesis of peptide thioester by fmoc chemistry through hydroxyl side chain anchoring barta , 2005) . azapeptides are usually synthesized on solid phase. a method, permitting the convergent synthesis of azapeptides starting from unprotected fragments, would offer the possibility to study aza amino acids effect in complex polypeptides or proteins. we have focussed our study on ligation reactions leading to the formation of an azagly residue at the ligation point, as gly is a frequent amino acid in peptides or proteins. the fi rst synthetic strategy relies on the reaction between a peptide thioester and a n-terminal azaglycine peptide. experimental conditions were found which permitted the chemoselective formation of azapeptide but with racemisation of the c-terminal amino acid of the thioester fragment. alternately, a synthetic strategy based on the chemistry of the phenylthiocarbonyl group, permitted the successful synthesis of azapeptide without racemisation. (2), native glycoforms are of therapeutic interest. our retrosynthetic strategy implies sequential native chemical ligation (3) and the split of il-6 into three fragments. an n-terminal fragment il-6 (1-42) thioester and the cysteine fragment il-6 (49-183) were expressed in e. coli. the carbohydrate carrying fragment il-6 (43-48) was synthesized via spps. to obtain the recombinant fragments specifi c termini were required. the n-terminal il-6 (1-42) was fused between two inteins and after expression and refolding from inclusion bodies the target peptide was released as a c-terminal thioester after dual intein cleavage. the cysteine fragment il-6 (49-183) was expressed as a single intein fusion also leading to inclusion bodies. this enabled the release of il-6 (49-183) with an n-terminal cysteine after intein cleavage of the refolded fusion protein. the central segment il-6 (43-48) contains a sugar residue attached to the n-glycosylation-site at asn44. this fragment features both a c-terminal thioester and an n-terminal cysteine protected as a thiazolidine. sequential ligations leading to the full length il-6 glycoprotein will be presented. synthetic chemistry syntheses of shorter fragments which are fi nally linked together (4) . this "small building block approach" should also simplify synthesis of modifi ed prion protein. in our plan of mouse prion (moprp) synthesis, we have employed consecutive chemical ligations. we have started with moprp(203-231) in which it is possible to study native chemical ligation between cysteine in the position of 213 and methionine in the position of 21(2) several moprp(203-212) peptide thioesters with aryl and alkyl thiols were prepared for further study of the native chemical ligation of moprp(213-231) and moprp(203-212). the optimal ligation conditions were found by a kinetic study which was carried out in a range of ph and with various thiols. infl uence of electron withdrawing and electron donating groups was studied in both aromatic and aliphatic thiols and it was found that it is possible to affect the ligation by choosing an appropriate thiol. ligation optimal conditions, kinetic study results, and suitability of various thiols for the ligation are discussed. low-cost industrial chemo-enzymatic synthesis of pharmaceutical, nutraceutical and diagnostic oligopeptides the production costs for oligopeptides and derivatives thereof by chemical synthesis are extremely high. therefore dsm pharmaceutical products embarked on a research programme on chemo-enzymatic peptide synthesis. important advantages of this technology are that no expensive stoichiometric coupling reagents nor side-chain protection are required and that no racemisation occurs. focus is on elongation in the n ¨c terminal direction using novel enzymatic c-protecting group interconversion methods. one of the preferred building blocks are amino acid amides, but selective deprotection of peptidic c-terminal amides is a challenge. we discovered 1 that using a peptide amidase 2 c-terminal peptide amides can be directly converted to methyl esters in almost pure methanol. thus, separate enzymatic hydrolysis and reactivation steps are no longer required. other versatile building blocks are amino acid t-butyl esters. we discovered that peptide c-terminal t-butyl esters can be transformed, using commercially available proteases, to activated alkyl esters such as methyl-and benzylesters which can be directly used in another protease-mediated coupling reaction. 3 furthermore, we found that using commercial enzymes side chain unprotected peptides with a free c-carboxyl terminus can be directly transformed to cterminal thioesters 4 and c-terminal aryl amides 5 in the presence of the corresponding thiol and aniline, respectively. finally, a real-life example of large-scale industrial chemo-enzymatic peptide production will be shown. it is known that chiral amino acids, as well as their dipeptides, may catalyze the asymmetric condensation of glycolaldehyde in water. on the basis of the particularly large erythrose enantiomeric excesses (ee) obtained when utilizing the chiral l-val-l-val catalyst and given the possibility of an abundant delivery of other types of amino acids to the early earth, we have studied the catalytic effect on this synthesis of the peptides based on c -methylated -amino acids, such as iva (isovaline or c -methyl, c -aminobutyric acid) and c -methylvaline, ( me)val, that are abundant in meteorites. results of the catalysis experiments showed the all c -methylated peptides to the tetramer level exhibit signifi cant chiral infl uence on the synthesis of tetroses and mimic the effect of the l-val-l-val catalyst in having a larger erythrose ee than threose ee, as well as in their confi guration relationship with the sugars (the product erythrose acquires ee of confi guration opposite to that of the catalyst in case of peptides, while it is the same for amino acids). interestingly, the largest ee (45% for erythrose) was obtained with the iva homo-tetrapeptide under mild conditions. the homo-dipeptides of both iva and ( me)val also produced a signifi cant ee (41% for erythrose) that appears to increase with time. because c -methylated amino acids are non-racemic in meteorites, do poster abstracts not racemize in aqueous environments, and are known to be (3 10 )-helix formers in peptides with as few as four residues, these results suggest that meteoritic, c -methylated, -amino acids may have contributed to molecular evolution upon delivery to the early earth by catalytically transferring their asymmetry to other prebiotic molecules. synthesis' and application of peptide adhesives for wound healing natural polymers with repeating peptide sequences, like spider silk and mussel glue have interesting properties that can be used for biomedical applications. however, the isolation from their natural sources is rather diffi cult and the desire to introduce modifi cations necessitates the development of novel methods to synthesize such polymers with repeating peptide sequences. recently, we have shown that the microwave-assisted copper(i)-catalyzed 1,3-dipolar cycloaddition can be used in the polymerization of the model dipeptide azido-phe-alapropargyl amide. by varying the reaction conditions we could either obtain small cyclic oligomers (4-20 aas) or linear polymers which consisted up to 300 aas residues (1). to broaden the scope of this polymerization reaction, two novel biodegradable monomers, azido-phe-ala-lys-propargyl amide and azido-phe-ala-glyc-lys-propargyl amide were polymerized. both monomers are water-soluble and contain recognition sites for the proteases trypsin and chymotrypsin; moreover, the tetrapeptide can be chemically hydrolyzed depending on the ph of the solution. the molecular weight of the polymers could be tailored between 4 -14 kda (33 to 100 aas residues). the enzymatic degradability of the polypeptides was monitored by a ninhydrin-based colorimetric assay and by maldi-tof. the results showed that the peptidic polytriazoles were smoothly degraded by trypsin and chymotrypsin. from these data we can conclude that the microwave-assisted copper(i)-catalyzed 1,3-dipolar cycloaddition reaction is an effective tool to synthesize biodegradable functional polymers which provides new opportunities to design (novel) biomedical materials. details of monomer synthesis, polymerization reactions and degradation studies will be presented. peptide synthesis on cellulose membranes, known as spot synthesis, is an ideal tool to determine biological interactions and/or key positions in proteins. in general we can differ in two kinds of synthetic peptides with different possibilities: over the past 15 years synthetic peptide library techniques have emerged as powerful approaches to determine t-cell epitopes and to specify peptide binding to mhc-i and/or mhc-ii molecules. the sequence-based approach is a straightforward method to directly identify t-cell antigens belonging to a specifi c target (e.g. a virus of interest) without the need of any further deconvolution steps. the entire protein sequence is presented as a set of overlapping peptides (peptide scan), which are subsequently screened for t-cell stimulation. several cd-8 t-cell epitopes in selected cmv proteins have been identifi ed using standard spps techniques. however, the peptide sequences synthesized by standard spot synthesis can only be cleaved without authentic c-termini (ß-alanine or glycine as c-terminal amino acid). here, we summarized three different established methods to generate cleavable peptides with authentic c-termini in adequate amounts, with suffi cient purity and within a justifi able time-scale. the standard spot synthesis to generate membrane bound peptides use glycine or ß-alanine membranes to map epitopes of b-cells, allergenic proteins, antibodies etc. furthermore, we have also established the "method of inverted peptides" to screen protein domains requiring peptidic ligands with free c-termini such as pdz-or 14.3.3 domains. another highlight of peptide libraries is the screen of posttranslational modifi cation in proteins by phosphorylation at serine, threonine, and tyrosine residues which plays a role in eukaryotic cell cycle regulation, cell differentiation, apoptosis, or cytoskeletal regulation. labeled peptide libraries, consisting of -helices, -loops and -sheets peptides, have been successfully prepared for use in peptide arrays that act as a protein-detection system and have been applied for proteinrecognition studies [1] [2] [3] . it is known that more than half the proteins in a mammalian cell are post-translationally modifi ed by such processes as phosphorylation, glycosylation, and acetylation. such modifi cations play an important role in various bio-recognition, for instance glycoproteins are involved in cell-adhesion, infection, and biological protection. hence the designed peptide library used for arrays has been expanded to include fatty acid attached peptides and glycopeptides. fatty acids were easily introduced by conventional fmoc-spps, while the synthesis of glyco-peptides was not easy. in the present paper we describe the effi cient construction of o-glycoside structured and labeled peptides by fmoc-spps using a building block strategy. glycosylated threonine has been used as a building block and was prepared in large amounts. glucose, mannose, galactose and lactose were acetylated and coupled to the hydroxyl group of fmoc-thr in the presence of bf3oet2. the resulting protected sugar-amino acids were manually assembled on to the peptidyl resin containing a fl uorescent dye using a peti-syzer® (hipep laboratories). after construction of the glycol-peptides the acetyl groups on the sugar were removed by hydrazine hydrate, and the peptides were then cleaved and purifi ed. the resulting glycopeptides were characterized by lcms. the present protocol has been used to prepare ca. one hundred glycopeptides that have been tested against various carbohydrate-binding proteins. parallel small scale peptide synthesis meets a fast, lowcost purifi cation method for the production of high quality peptide microarrays to analyze dna/protein interactions herein, a parallel synthesis in a small scale (0,1 mol) which takes place in 384-micro well plates is demonstrated. with regard to a fast but yet effi cient, low-cost purifi cation in a parallel manner a ''fmoc-on'' purifi cation method, which is integrated into the synthesis process, was developed. peptides were cleaved from the resin with their fmoc-group still on. the crude products were transferred by extraction into another 384-micro well plate fi lled with purifi cation material. purifi cation takes place due to the high affi nity of the terminal fmoc-group to this material. hplc measurement shows a high recovery (98%) and purity (90%). cleavage of the fmoc-group during the purifi cation process is also possible. here, hplc measurement demonstrated a recovery of 93% and a purity of 92%. a fully automated synthesis of more than 300 peptides in a 384-micro well plate combined with a parallel ''fmocon'' purifi cation is shown. the peptides were used for the production of microarrays to analyze dna/protein interactions. the "fmoc-on" purifi cation allows easy and cost-effi cient purifi cation of peptides for all kind of biological applications. is an antibiotic-macrolide related to a group of tylosin (tyl) which structure is based on 16-member lactone with carbohydrate substitutes attached. macrolides are well known translation inhibitors, they bind to ribosomal tunnel (rt) in a way that their lactone ring is located orthogonally to the long axis of the rt, covering most of its cleft; hence, the mechanism of protein synthesis inhibition by macrolides relies on the mechanical obstruction they provide to the passage of nascent polypeptide chain through the rt. recently we have designed and synthesized a number of peptide derivatives of macrolides where the peptide part modeled the growing chain, while the antibiotic served as an "anchor" for positioning the peptide at the specifi c site of rt. these derivatives are of interest both as antibacterial agents and as potential probes for investigation of the interactions of nascent peptide chain with the specifi c sites of the rt and their infl uence on the translation. now we report a new type of peptide -macrolide conjugates in which peptide binds by its -amino function through a spacer to the 4'-hydroxyl group of the mycaminose residue of des. two proline-containing peptides are chosen: gp and ggp. proline residues of these peptides are supposed to interact with the peptidyl transferase center region when the complexes of these 4'-peptidyldesmycosins with bacterial ribosomes are formed. the fi rst step of the synthesis was acetylation of 2'-, 2"-, 4"and 4"'-oh-groups of tyl by ac2o in pyridine following by hydrolysis in 1n sulphuric acid resulted in des with all protected oh-groups except 4'-oh-group of mycaminose. this free hydroxyl group was used for reaction with succinic anhydride leading to formation of reactive carboxyl group. the next step was a reaction between this carboxyl function with -amino groups of peptide methyl esters. the structures of new 4'-peptide derivatives of des were proved by ms maldi and nmr-spectroscopy. synthesis .0]octane, (c), was carried out by organic methods which then will be used in polymer synthesis [1] [2] . consequently, polymers having different molecular weights and their water soluble bioconjugates were synthesized and the characterization of these polymers and conjugates were done by different methods such as esi lc-ms, uv and atr ft-ir spectroscopy. for the chemical modifi cation of pc, bromoacetic acid was used and a water soluble polyampholyte synthesis was achieved with the quaternization of polymer. it is determined that molecular weights of the polymers were affected by the change in the amount and the type of initiators used and also from the polymerization media. analysing of having biodegradable characteristics of the synthesized polymers has been being reviewed. atr ft-ir spectra of pc and pc-hemagglutinine peptide was recorded. for peptide synthesis we have used microwave assisted solid phase peptide synthesis method. lc-ms was used to determinate of molecular weight of hemagglutinin. after we have obtained m/z values of peptide we used preparative hplc for purifi cation of hemagglutinin. after synthesis and modifi cation of pc, its covalent conjugates with hemagglutinin (ha 98-106) were obtained by carbodiimide activation. its characterization was also performed. the peptide coupling reagent fi eld has clearly evolved in the last decade from carbodiimides to onium (phosphonium and uronium) salts. the era of industrial coupling reagents began in 1955 with the introduction of dicyclohexylcarbodiimide (dcc), which at that time was already known and well studied, as a reagent for the formation of amide bond. unfortunately, carbodiimides did not comply with the concept of ultimate coupling reagents because its high reactivity provokes racemization and side reactions during the coupling reaction. at the beginning of the 70's, 1-hydroxybenzotriazole (hobt) was proposed as an additive to dcc to reduce racemization and from then on other benzotriazole derivatives such as 1-hydroxy-6-chlorobenzotriazole (cl-hobt) or 1-hydroxy-7azabenzotriazole (hoat) have also been used. the obt active esters are less reactive than the o-acylisourea, but are more stable and less prone to racemize. the use of the most reactive aminium salt, hatu , is inconvenient because of the price, which makes its use detrimental for industry. hctu/tctu, based on cl-hobt, are a good alternative to hbtu/tbtu, because of the presence of the chlorine atom that stabilizes the structure, hence, making these reagents less hazardous. herein, pyclock, the phosphonium salt of the cl-hobt is introduced p20100-001 n-methylation of biologically active peptides is of interest because it results in increased conformational integrity and stability against enzymatic degradation. furthermore, n-methylated peptides have a decreased ability to form h-bonds to water molecules and, consequently, a better ability to cross biological barriers. this is exemplifi ed by the naturally occurring peptide cyclosporine which is orally active. in an effort to improve the blood-brain barrier permeability of the cyclic enkephalin analogues h-dmt-c[d-cys-gly-phe-d(or l)-cys]nh 2 (dmt = 2',6'-dimethyltyrosine), we prepared analogues that were n-methylated at phe 4 and/or cys 5 . n-methylated cys derivatives were prepared either by direct methylation (ch 3 i)/nah) or by using oxazolidinone chemistry. single n-methylation of the two cyclic peptides at phe 4 or d(or l)-cys 5 produced four analogues that all showed very high mu and delta opioid agonist potencies in the guinea pig ileum and mouse vas deferens assays. the two analogues n-methylated at both phe 4 and d(or l)-cys 5 also retained high agonist activity at both receptors. results from molecular mechanics and molecular dynamics studies on the conformational behavior of these peptides will be presented. during the last years the number of deaths due to hemostatic impairments such as coronary angioplasts, coronary thromboembolisms, myocard heart attack, pulmonary embolism etc. has become equal to those caused by cancer formations. haemostasis is a key process whose correct functioning is an important defence mechanism of the human organism. it is a blood coagulation process activated in case of injury of the blood system. if it is functioning correctly vascular-motor and cell reactions are triggered and the blood coagulation cascade is activated. one of the most important enzymes in the blood coagulation cascade is factor xa. that's why its inhibitors are promising alternative against thrombotic disorders. in our previous work, we reported the synthesis of hybrid structure between isoform 2 and 3 of antistasin and the active sequences d-phe-pro-arg; d-arg-gly-arg; phe-ile-arg and tyr-ile-arg (1). beside the analogues with c-terminal cooh group, the peptide d-phe-pro-arg-pro-lys-arg-nh2 was synthesized. the biological activity of the last one was 60 times bigger than natural isoform 3 of ats and some times more active than all other synthesized analogues. in the current work we described synthesis and biological activity of c-terminal amide analogues of all early synthesized peptides in order to deduce the structure-activity relationship. the anticoagulant activity according to the aptt and ic50 was determined. the neo-angiogenesis is the formation of new blood vessels from a pre-existing vasculature which enables the supply of the nutrients and oxygen necessary for tumor growth. consequently, inhibiting the blood vessels sprouting in order to starve the tumour constitutes an attractive anti-tumoral therapy. the vascular endothelial growth factor or their receptors (vegfr-1 and 2) are key mediators of neo-angiogenesis and constitute nowadays validated targets for anti-angiogenic therapies (1) . despite this fact, strategies aiming to develop pure receptors antagonists and not inhibitors of the tyrosine kinase activity remain scarce. we have previously designed antagonists that interact with the extra-cellular domain of vegf receptor 1 and thus prevent vegf binding (2) . these antagonists were proved to be potent and able to interfere with the vegf signalling pathway. because these compounds were rationally designed by using the co-crystallized structure of vegf with the immunoglobulin like fragment 2 of vegfr-1, we raise the following question: do these antagonists really target the immunoglobulin like domain 2 of vegfr-1? in order to respond to this question but also with the future aim of optimizing our antagonist in a rational way we need to co-crystallize at least one of our antagonists with the d2 fragment of the vegfr-1. furthermore, since human vegfr-1 d2 is a potential target in the development of angiogenesis modulators, the availability of this protein domain, and mutants, should be useful in the screening and development of novel specifi c ligands. up to now, the 101-amino acid polypeptide chain of vegfr-1d2 has been only produced by gene expression (3) . here, we report the fi rst solid phase peptide synthesis of the vegfbinding domain of the vegf receptor 1 and the in vitro experiments which permitted to verify its biological activity. chung, nga n. 1 different types of turns are important elements of secondary structure in peptides and proteins. the most common ones are different kinds of -turns involving four consecutive amino acid residues. the dipeptide unit containing the second and the third residues of such turns exists in the cis-conformation. our cispeptide bond motif 4-aminopyroglutamic acid promotes the -turn type vi/vi', and can be treated as a hybrid of glycine and alanine. this feature prohibits its utilization as a replacement for any possible dipeptide unit. in order to convert our compound into a more valuable tool for probing the existence of a particular peptide bond in the cis-conformation, we have elaborated the synthesis of nmonoalkylated derivatives of 4-aminopyroglutamic residue through a reductive alkylation reaction performed on solid support. following this idea, we have obtained analogues with n-benzylated and n-(phydroxy)benzylated 4-aminopyroglutamic residues as scaffolds for the phe-ala and tyr-ala dipeptides units, respectively. only one of 12 enkephalin analogues, [n(bzl)-(r,r)-apy4-5]-enkephalin amide, possesses similar agonist potency as leu-enkephalin in the gpi assay, indicating that the c-terminal part of this peptide may assume the cisconformation upon binding to the receptor. this is the fi rst active opiod analogue containing a 4-aminopyroglutamic acid residue. supported by kbn (poland) grant 2 p05f 00129 and nih (u.s.) grant da-04443 mif-1 (l-prolyl-l-leucyl-glycine amide, plg) is a brain peptide, presented in hypothalamic tissue, as well as the c-terminal tripeptide of the neurohypophiseal hormone oxytocin and inhibits the release of melanocyte-stimulating hormone (msh) in some systems. recently, some chemical modifi cations of mif-1 to enhance opiate agonist/ antagonist actions as well as binding activity of analogues have been reported. on the other hand, the concept of structural modifi cation in peptide fragments to confer them specifi c properties is of current interest in the study and design of new bioactive targets. a well known example is the incorporation of unnatural amino acids into the molecule of natural biologically active peptides leading to analogues with signifi cant theoretical and practical importance. with this idea in mind we studied possibilities of introducing unnatural amino acids canaline (can), norcanaline (ncan), canavanine (cav), nor-canavanine (ncav) and slys into mif-moiety in order to achieve a better analgesic effect. to obtain the peptide mimetics, both fmoc-and boc-based spps approach were used. analgesic activity was determined by the paw-pressure (pp) and hp tests. the experiments were carried on male wistar rats. the changes in the mechanical nociceptive threshold of the rats were measured by the randall-selito paw pressure test using analgesimeter (ugo basile). it was found that substitution of leu in position 2 of mif-molecule by unnatural amino acids increased the pain threshold. the analgesic effect with cav-substitution was highest, whereas parent mif-1 showed only a minute increase of pain-threshold. the compound m6 had the strongest effect on learning and memory and the compound p6 showed the strongest and fastest analgesic effect. the compound m6 and p6 were also able to modify the effect of the model cns-drugs (hexobarbital and pentylenetetrazole). theoretically and experimentally determined octanol/water logp values of the compounds correlate with their cns-effects. m6 and p6 had higher logp than m3 and p3 and showed better antinociceptive and anticonvulsant activity in vivo. compounds possessed also chelating activity towards fe ions. the stronger chelating activity of the compounds with the 6-spacer clearly correlated with their better neuropharmacological activity in vivo (in comparison to the compounds with the 3-spacer). the position isomery may also contribute for the variations in their pharmacological activity. the limited number of the compounds does not allow derivation of well-defi ned structure-activity relationships, however, their 3d models show possibility for many low energy conformers with different atoms involved in formation of fe chelating complexes. a major challenge in opioid peptide chemistry is the synthesis of novel compounds mimicking the endogenous peptide ligands. these new peptidomimetics should be biologically active and more stable against enzymatic degradation than their parent ligands. one of the possibilities is the introduction of -amino acids into the peptides sequence. monosubstituted -amino acids ( 2 -or 3 -) due to their similarity in structure to -amino acids, moreover their tendency to give folded structures even in short peptides and to the stability towards mammalian peptidases may be very useful in the creation of the new potentially active compounds. we have developed simple and effi cient two step conversion of the cyanoacetate into fully protected 2 -amino acids. the procedure involves knoevenagel condensation of the methyl cyanoacetate and aromatic aldehydes (for aromatic path) or alkylation of the methyl cyanoacetate with various alkyl halides (for aliphatic path) at fi rst then reduction and boc-protection (performed in one pot) of the resulting fi rst stepproducts. we focused our attention on applying above method to the synthesis of different 2 -amino acids (as homologues of -amino acids) as elements for the synthesis and structure -activity relationship study of endomorphin analogues. small library of analogues has been created in which -amino acids in every position with exception of pro were substituted by their respective 2 -analogues. as templates, endomorphin-1 (tyr-pro-trp-phe-nh 2 ), endomorphin-2 (tyr-pro-phe-phe-nh 2 ) and its d-ala2-analogue (tapp) have been used. in this communication the pharmacological consequences of such modifi cation in endomorphins will be discussed. solid-phase synthesis and effects of amino acid and peptide analogues of non-protein amino acid canavanine on nociception non-protein amino acids have been widely used as components of peptides to enhance biological activity, proteolytic stability, and bioavailability. it is well known that unnatural amino acids with guanidine functionality exhibit diverse pharmacological effects when introduced in biologically active systems. our previous efforts were focused on the preparation and the characterization of unnatural amino acids, particularly those containing a basic functionality in the side chain. we have synthesized numerous unnatural amino acids, structural analogues of arginine and lysine, which demonstrated certain biological effects. recently, as part of our ongoing research focused on the search of novel arginine mimetics, we developed an effi cient approach for solid-phase synthesis of unnatural amino acids nor-canaline (ncan) and nor-canavanine (ncav). next we studied the possibilities of introducing ncan and ncav into the molecule of biologically active peptides. we also studied their antinociceptive effects using the paw pressure (pp) and hp tests. in the last few decades, considerable attention has been devoted on the potential role and activity of certain environmental pollutants (edcs) in increasing anomalies that involve the endocrine system of wild species and man. (1)the development of a fast high-throughput detection system for the quantitative analysis of edcs requires the development of a novel sensitive solid phase edcs extraction method. because of the high affi nity of edcs with the estrogen receptor, this has been greatly investigated. this study has leaded the recognition of the amino acids located in the ligand binding domain of the receptor which interact with edcs. use of a dipodal scaffold molecule and different combination of the crucial amino acids, will allow the generation of a library of small to medium size biomimetic receptors. in this communication, we will disclose our fi rst results in the preparation of a small library of biomimetic receptors, with and without the dipodal scaffold, to evaluate their affi nity towards edcs and the role played by the scaffold structure. romeralo tapia, rosa 1 ; van der eycken, johan 2 1 ghnet university, belgium; 2 ghent university, belgium endocrine disrupting chemicals (edc's) are an important class of pollutants, which have in common that they show affi nity for the hormone binding domain of the estrogen receptor, thus disturbing the endocrinal system. detection in waste water has not been succesful due to their low concentration. therefore, new sensitive screening methods are highly demanded. a possible solution is the use of estrogen receptor mimics for affi nity chromatography. to this end, scaffold 1 will be synthesized and used for building a tetrapodal peptidomimetic library. amino acids known to be important for the estrogen-receptor interaction will be preferably incorporated. first of all, we set out to prepare one dipodal peptide library member 6. the orthogonally protected scaffold 2 was synthesized in one single step starting from the commercially available 3amino-5-nitrobenzoic acid.2 using fmoc solid phase chemistry on wang resin as the solid support, this dipodal scaffold allowed the attachment of two different oligopeptide chains.employing this methodology the synthesis of a small library will be performed, and the affi nity of the different peptidomimetics for edc's will be investigated. on-resin microwaves-assisted ring closing metathesis for the synthesis of octreotide dicarba-analogues di . octreotide is an antitumoral agent used mainly as a carrier of radionuclides for cancer diagnosis and therapy. it shows the same disulphide bridge of the parent srif that is prone to be opened by oxidizing and reducing agents. this prompted us to search a more stable tether bridging the active motif of the cognate molecule. the premier reaction of rcm was performed in an oil bath under severe experimental conditions i. e. anhydrous argon atmosphere and long reaction times [3;4] . the microwaves assisted version was, instead, effi cient for the cyclopeptides yield and required very short reaction times. we also evaluated the effi cacy of different grubbs catalysts in the microwaves assisted rcm, operating in different conditions of temperature and time. in the search for potential antimicrobial drugs, we have been dealing with two microbial enzymatic systems: n á -succinyl-l-diaminopimelic acid desuccinylase (dape 1, 2 ) and n á -acetyl-l-ornithine deacetylase (arge 3, 4 ), which might be promising targets for potent and selective enzyme inhibitors based on the modifi cation of n á -succinyl-diaminopimelic acid (dap) and n á -acetyl-ornithine (orn). the inhibition of both the enzymes would possibly interrupt the pathways leading to development of bacteria due to a role of meso-dap as essential component of peptidoglycan based bacterial cell walls and orn, as well, being a component of biosynthetic pathway for arginine that can serve as a source of both the carbon and nitrogen in microorganisms. our effort was focused on the synthesis and characterization of two series of n á -substituted derivatives of dap and orn, potentially interfering with the hydrolytic action of dape and arge in the processes of bacterial growth. in this introductory study, the compounds prepared were also assayed against bacterial strains escherichia coli and bacillus subtilis and inhibitory activity of orn derivatives was found with regard to differences in the structure of n á -substituent. the fi rst report on practically effective bradykinin (bk) antagonists for b 2 receptors was published in 1984. the key to conversion of bradykinin into an antagonist was replacement of pro 7 with an aromatic d-amino acid; d-phe was fi rst used. however, our studies demonstrated that the d-amino acid residue at position 7 of the bradykinin antagonist, until recently considered to be necessary for b 2 antagonism, can be replaced by suitable l-amino acid or achiral residue or, together with the amino acid occupying position 8, by a sterically restricted dipeptide unit. having all this in mind we synthesized and bioassayed two new analogues of bradykinin. the peptides were designed by substitution of position 7 of bradykinin b 2 receptor antagonist ([d-arg 0 ,hyp 3 ,thi 5,8 ,d-phe 7 ]bk), previously described by stewart's group, with structural isomer of proline: 2 -iso-pro or its homologue: 3 -homo-pro. it's worth emphasizing that position 7 in bradykinin molecule is occupied by proline residue. our previous results demonstrated the importance of the position in the peptide chain into which the sterically restricted 1-aminocyclohexane-1-carboxylic acid residue (acc) was inserted. these fi ndings prompted us to investigate how introduction of l-pipecolic acid residue (l-pip) in position 7 or 8 of stewart's antagonist will affect pharmacological properties of resulting compounds. in comparison to the acc residue, the ring of l-pipecolic acid also consists of six atoms, but includes the nitrogen atom. bearing in mind that acylation of the n-terminus of several known b 2 blockers with a variety of bulky groups has consistently improved their antagonistic potency in the rat blood pressure assay, the aforementioned four analogues were also synthesized in the n-acylated form with 1-adamantaneacetic acid (aaa). the activity of eight new analogues was assessed in isolates rat uterus and in rat blood pressure test modifi cations of the myelin basic protein epitope mbp87-99 divert th2 to th1: immune responses in peripheral blood mononuclear cells (pbmc) from multiple sclerosis patients. postranslational modifi cations (citrullination, phosphorylation, deamidation, methylation, glycosylation) are common biological processes that alter specifi c parts of a protein after synthesis. nearly all known proteins undergo some form of postranslational modifi cation and almost all amino acids can be altered by one or more of these processes. the modifi ed protein thus, contains new or rare amino acids or new specifi c side groups that can have critical infl uence on the structure and function of the protein molecule. conversion of arginine to citrulline, an important postranslational modifi cation, was fi rst described by fearon. arginine residues in proteins can undergo this modifi cation and the resulting citrulline remains part of the protein in the position of arginine. citrulline is not a natural amino acid in proteins, and may induce immune responses. such responses have been recently implicated in the pathogenesis of autoimmune/infl ammatory diseases such as ms and rheumatoid arthritis 1. herein we investigated cytokine secretion in peripheral blood mononuclear cells (pbmc) of 7 ms patients and 7 controls, and attempted to correlate cytokine polarization with the nature of the antigenic stimulus. we synthesized peptide analogs that map to the myelin basic protein ( . analogs p4 and p5 resulted from the citrullination of the 91 and 97 arginine residues in epitopes p2 and p3. we then tested ms and control pbmc with various concentrations of the peptides and investigated cell proliferation by the brdu proliferation assay and cytokine secretion by elisa. we suggest that citrullination of self-antigens maybe an important step in triggering disease in susceptible individuals. incorporation of aza-3-amino acid into 26rfa(20-26), the endogenous ligand of gpr103 : structural analysis. aza-3-peptides, mixing -and aza-3-amino acids (the aza analogs of 3-amino acids), represent a novel and exciting type of peptidomimetics.1 in particular, we have shown that aza-3-amino acid induces a n-n or hydrazino turn, stabilized by an eight-membered-ring intramolecular hydrogen bond between the carbonyl acceptor group of the residue i-1 and the amide proton of the residue i+1. interestingly, this n-n turn promotes a well-defi ned -turn formation (hydrogen bond between the co of the residue i-2 and the hydrazidic proton of the aza-3-moeity) when an -amino acid is foregoing.2 26rfa, a novel neuropeptide of the rfamide superfamily, exhibits high affi nity for gpr103 and induces a potent orexigenic effect in mice. 3 in biomimetic environment, 26rfa encompasses an -helix between pro4 and arg17 residues and a canonical -turn centered on ser23. 26rfa(20-26), whose sequence is strictly conserved across species, is about 100 times less potent than 26rfa. this heptapeptide shows important distortions of the -turn that may be responsible for its weak potency. the aim of this study was to restore the -turn formation in 26rfa(20-26) (ggfsfrf-nh2) by the presence of an aza-3-amino acid. for this purpose, we have (i) synthesized the aza-3-counterpart of each residue of 26rfa(20-26), (ii) individually incorporate the surrogate into the heptapeptide and (iii) investigated the 3d structure under nmr restraints of the hybrid peptides. the results will be presented with a particular attention on the serine position. the analysis of the structure-antithrombotic activity correlation for peptide receptors of adhesive glycoproteins with the general formula arg-xaa-asp gribovskaya, olga; martinovich, vera; golubovich, vladimir institute of bioorganic chemistry, belarus structural analogues of the arg-gly-asp sequence with the general formula arg-xaa-asp, where xaa -ala, d-ala, -ala, -abu, -ak, pro, d-pro, asn-trp, were synthesized using classical methods of peptide chemistry in solution, the levels of their antithrombotic activity were determined and stable conformations were calculated using a pairwiseadditive approximation method. interatomic distances in the obtained conformers were calculated between various atoms in the functional groups, including carbons in the carboxylic groups, nitrogens in the -aminogroups, amino and imino nitrogens in the guanidine group, 16 distances in total. the analysis of correlation of the calculated interatomic distances and measured values of antithrombotic activity was carried out using statistical methods. we show that the distance between the amino nitrogen of the guanidine group of arginine and the -carboxyl carbon of aspartic acid determines the antithrombotic activity. it has the optimum value in the arg--ala-asp peptide, which was the most active among the synthesized analogues of the arg-gly-asp sequence (ic50 -10.6 mkm, adp, 1.5 mkm). microbial proteases with narrow specifi city as an instrument of peptide chemistry kotlova the problem of selective removal of short n-terminal peptides from the gene-engineered proteins is actual by many reasons. at fi rst it is connected with gene-engineering method of protein preparation in form of its precursors. the problem of n-terminal formyl-met removal from gene-engineered proteins, which are produced by bacillacae may be solved by introduction of specifi cally-cleaved insert after formyl-met. subsequent specifi c removal of such short n-terminal peptide results in mature protein. moreover, the analysis of cleaved short peptides could characterize the process of mature protein formation. we suggest microbial enzymes with narrow specifi city to solve the mentioned problem. for this aim we have isolated the trypsin-like enzyme and postproline-specifi c endopeptidase from aspergillus sp. and glutamyl-specifi c protease from b. intermedius. these enzymes have high specifi city approved by hydrolysis of short synthetic peptides and long polypeptides. for example, specifi city of postproline protease was established by hydrolysis of mellitin. the fi nal peptide mixture was analyzed by rp-hplc and mass-spectra. the use of enzymes with narrow specifi city is demonstrated in the analytic method of detection of formyl-met presence at the n-terminus of gene-engineered -interferon. trypsine hydrolysis of -interferon leads to more than 14 peptides are formed including the 12-membered n-terminal peptide. being hydrolyzed with glutamyl-specifi c enzyme -interferon is converted to 6 peptides, including 41-membered n-terminal peptide. application of postprolinespecifi c enzyme for -interferon hydrolysis leads to formation of 1 short 4-membered peptide and 4 long fragments. experimental data show that in case of analytic control of -interferon maturity the utilization of postproline-specifi c protease is preferable. in other cases the enzyme selection is ruled by the features of protein primary structure. bimodal action of cystatin related epididymal spermatogenic (cres) protein and its reactive site loop derived s-s bridge bicyclic peptides on proprotein convertase-4 (pc4) activity several precursor proteins found on sperm surface and reproductive tissues were proposed or confi rmed as substrates of pc4. these include adam proteins, growth factors proigf1 and 2 and hormonal protein pacap. lack of pc4 leads to impaired fertility in mice, suggesting its important role in reproduction. pc4 is thus considered an important target for development of nonhormonal contraceptive agents. pc4-inhibitors are expected to fi nd therapeutic, clinical and biochemical applications. a lot of interest has grown to develop specifi c pc4 inhibitors. recently natural inhibitor of serpin family have been described for pc1, 2, and furin, but not for pc4. however, a new serpin, cres, has been reported from epididymis fl uid, where pc4 may be found. so far, cres has only been shown to inhibit pc2, which is not present in reproductive tissues. we propose that cres may represent a natural regulator of pc4, based on localization and other studies. in this study we generated recombinant human cres protein (139 aa), and its reactive-site loop (rsl) derived acyclic and cyclic 43-mer peptides with various s-s linkage combinations. cres inhibited pc4 with ic50 ~50um, while rsl-peptides were found to be more potent with ic50 50-250nm, depending on the s-s linkage locations. furthermore, we noted that at lower concentrations, cres and its peptides fi rst enhanced pc4 activity before any inhibition occurred. this bimodal behavior may be due to cleavage. molecular modeling showed strong interactions between cres and pc4 via some of their key residues. overall, we noted that "rsl" is the most crucial element required for pc4 inhibition by cres. funds were from from cihr (ab). the de novo design of peptides and proteins has assumed considerable interest didehydroresidues, in particular alpha, in the recent years. alpha, beta-beta-didehydrophenylalanine (deltaphe) are being considered important conformational constraints inducing tools in de novo peptide design. deltaphe is a noncoded, achiral residue, an analog of the naturally occurring phenylalanine amino acid with a double bond between calpha and cbeta atoms. introduction of deltaphe in peptide sequences is known to induce conformational constraint, both in the peptide backbone as well as the side chain, and to provide the peptide with increased resistance to enzymatic degradation. in small peptides containing eta turn structure, and in peptides containing a single deltaphe, a type ii b more than one deltaphe residues, helical structures are stabilized. a number of deltaphe peptides varying in length, content and position of deltaphe residues have been found to contain 310 helices of both screw senses. following these design principles, we were able to design, synthesize and characterize super secondary structural motifs like helix-turn-helix, helical bundle and glycine zipper. we have extended this work to the de novo design of peptides with antibiotic and anti-fi brillization activity. a series of cationic peptides containing deltaphe residues have shown remarkable antibiotic activity and are being developed further. deltaphe containing peptides may have longer in vivo half life owing to their ability to resist enzymatic degradation. more recently, we have observed that small peptides containing deltaphe self-assemble in nanotubular and nanovesicular structures. these nanostructures have also been used to entrap small drug like molecules. we have fully characterized these systems and are exploring their potential as delivery agents in biological systems. the synthesis and design of peptidomimetic oligomers that adopt designed, compact conformations (foldamers) have become a challenging task in recent years. among them, -peptide foldamers exhibit rich diversity of secondary structures. [1, 2] a relationship has been established between the backbone chirality pattern and the prevailing secondary structure, which underlines the role of stereochemical control in the -peptide foldamer design.(3) an important challenge is to introduce proteinogenic side-chains to generate diverse anchor points on the molecular surface promoting their application in drug discovery. it has been proved that stable helices can be obtained when sequences were coupled with -amino acid enantiomeric pair motifs in a lego approach.(3) in this work, -amino acids and open chain functionalized -amino acids were inserted between the enantiomeric pair design element for 1 and 2, respectively. confi gurations in the backbone were designed to promote helix formation. nmr and ecd measurements augmented with ab initio calculations revealed that 1 forms a h9-12 helix and 2 forms a h14/16 helix. these helices are unprecedented in the literature. to prove that the stereochemical patterning is crucial in the folding, we perturbed the confi guration motifs of the backbones by swapping the sequence of the central -amino acids. the peptides with exchanged residue order could not fold into helices. these novel structures can be useful scaffolds for biomedical applications. introduction small length synthetic peptides, based on sp c-terminal fragment, increase the secretion of tnf-and prevent the proliferation of several cancer cell lines. we have already shown the antiproliferative activity of tri-and tetra-peptoids in breast and prostate cancer cells. the aim of this study was the synthesis of hexa-peptoids, analogs of sp c-terminal region, containing the residues d-trp and tic and the peptoid ones nhn(r)ch2co, nphe and nala in their sequence and their evaluation against cancer cells proliferation. methods and results all the syntheses were carried out stepwise using the fmoc/ but methodology on the solid support 2-cltr resin and dic/hobt as coupling reagent. all analogs were purifi ed (hplc) and identifi ed ( cyclotheonamides constitute a family of structurally related cyclic pentapeptides of marine origin that inhibit trypsin-like serine proteases. their binding mode has been elucidated by the x-ray structure of cyclotheonamide a in complex with trypsin. an extended peptide conformation which is stabilized by macrolactamization allows to address in a substrate-like manner beside the s1 pocket also the s1' and s2 pocket. the s1 ligand, (s)-3-amino-6-guanidino-2-oxo-hexanoic acid, interacts via its guanido function with asp 189 at the bottom of the s1 pocket. in addition, the ketone covalently modifi es the gammaoxygen of ser 195 by hemiketal formation (1) . recently, two novel cyclotheonamides have been isolated from a marine sponge of the genus ircinia. one of them, cyclotheonamide e4, is a potent inhibitor of human beta-tryptase (2) . in this study, cyclotheonamide e4 was modifi ed at two positions: (i) the s1 ligand was replaced by beta-homolysine or as betahomoarginine to obtain reversible acting tryptase inhibitors, and (ii) the alpha amino function of (s)-2,3-diamino propionic acid, which is not part of the cyclic backbone, was used as anchoring point for basic p3 residues to exploit interactions with the negatively charged glu 217 of tryptase. these analogs were synthesized by a combination of solid phase and solution phase chemistry 3.. synthetic details as well as the inhibitory profi le of these novel cyclotheonamide e4 analogs will be discussed. oostatic peptides containing d-amino acids: activity and degradation in the fl esh fl y neobellieria bullata bennettová deteriorating effect of c-terminus truncated 4p and 5p analogues [1, 2] of decapeptide h-tyr-asp-pro-ala-pro 6 -oh (3) on ovarian development (i.e. oostatic effect) of insect species diptera, orthoptera and hemiptera has stimulated an analysis of metabolic degradation of corresponding peptides after application to the insect body [4] [5] [6] [7] . radiolabeling in different positions of the peptide chain allowed determination of the degradation decisive steps -the fast splitting off the c-terminal pro from the 5p followed by successive cleavage of tyr and asp from the nterminus. after introduction of corresponding d-amino acids into peptide chain of 5p, we could see the same or even stronger oostatic effect of the analogues in comparison with parent peptide after application on the fl esh fl y neobellieria bullata. in the degradation assay, an elimination of enzymatic cleavage of peptide bonds pointing from the central labeled pro residue to either d-ala or d-asp residues in the neighbourhood was observed, resulting in total increase of the analogs stability. structure-activity relationships( sar) studies of camk ii inhibitors. in fact, in vitro it can phosphorylate up to 40 proteins, including enzymes, ion channels, transcription factors and a number of these proteins appear to be physiological substrates. for example, cam-kii is highly concentrated in the postsynaptic density of glutamatergic synapses where it phosphorylates and potentiates current through the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor ion channel (ampa-rs). in fact, this family is encoded by four genes ( , , and ), whereas the and isoforms are expressed in diverse tissues and , isoforms are most prominent in neural tissues. the identifi cation of camk ii inhibitors is important to better defi ne its physiological. in literature is reported a 79-aa brain-specifi c protein that and potently inhibited kinase and bound the catalytic domain of cam-kii activity with an ic50 of 50 nm. the inhibitory protein (cam-kiin), and a 27-residue peptide derived from it (camkiintide, krppklgqigrakrvvieddriddvlk), was highly selective for inhibition of cam-kii with little effect on cam-ki, cam-kiv, cam-kk, protein kinase a, or protein kinase c. cam-kiin interacted only with activated cam-kii (i.e., in the presence of ca2+/cam or after autophosphorylation). here we report a structure-activity relationships study using as template the camkiintide. we synthesised a peptide contains all 28 amino acids present in camkiin-tide, however, in random sequence (camkiin-tide scramble). then we operated a progressive deletion of amino acids from c-terminal and dall'n-terminal to detect minimum active sequence. moreover camkiintide and cankiintide scrumble was made cell-permeable by n-terminal addiotion of an antennapedia (rqikiwfqnrrmkwk). here we report the biological results of our study. ptprj: a receptor-type protein tyrosine phosphatase as a target of new peptides with antitumoral activity to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. it has been demonstrated that ptprj (also known as hptpeta, dep-1, cd148) is able to inhibit cell growth promoted by specifi c ptk some of which are over-expressed in certain types of cancer. moreover a role for ptprj was also assessed in vasculogenesis; infact its reduced activity in enhanced vegf-induced vegfr2 activity leads to increased cellular responses, supporting a ptprj-vegfr2 interaction. on these basis it is important the identifi cation of molecules with agonist activity which are able to stimulate the function of residual ptprj in malignant cells and stopping the proliferation and possibly trigger programmed cell death. using as a template a peptide, already identifi ed, with agonist activity against ptprj(h-[cys-his-his-asn-leu-thr-his-ala-cys]-oh), here we report a structure-activity study carried out through endocyclic modifi cations (ala-scan, d-substitutions, single residue deletions, substitutions of the disulfi de bridge) and the preliminary biological results of this set of compounds. faulty of chemistry, university of gdansk, poland; 2 faculty of chemistry, university of gdansk, poland isolated in 1999 from the sunfl ower seeds trypsin inhibitor sfti-1 is up to date the smallest naturally occurring peptidic proteinase inhibitor and therefore is an excellent starting structure to design peptidomimetic serine proteinase inhibitors. peptomeric library consisting of 360 monocyclic analogues of sfti-1 was designed and synthesized by the solid phase method with the intension of select chymotrypsin and cathepsin g inhibitors. all peptomers contained in positions 5 and 12 nbenzylglycine (nphe) that mimics proteinogenic phe. in the synthesized library different peptoid monomers were introduced in the segment 7-10. this is a turn region that makes an important contribution to structural integrity and rigidity of sfti-1. deconvolution of the library against both proteinases by the iterative method in solution revealed that the highest chymotrypsin inhibitory activity displayed analogue with n-[4-(2-aminoethyl)morpholyl]-glycine (naem) in position 8. this analogue was even more active than the one with pro in this position which is absolutely conserved in bownan-birk inhibitors. while, deconvolution carried out against cathepsin g indicated that analogue with npiperonylglycine (npip) in position 8 and 9 and with n-butylglycine (norleu) in position 10 presents the highest inhibitory activity. acknowledgements: this work was supported by ministry of science and higher education (grant no. 2889/h03/2008/34). 3 a platform for the design and optimization of new antimicrobial peptides effective against specifi c pathogens has been set up. the main features expected for these peptides are low environmental impact, broad spectrum of activity, reasonable bacterial selectivity, and low eukaryotic cytotoxicity. our approach also includes the use of a design of experiments protocol in order to fi nd peptide sequences that fi t these features. the obtained peptides would represent an alternative to currently used antibiotics or pesticides. this work focused on fi nding new control agents against economically important plant pathogenic bacteria such as erwinia amylovora, pseudomonas syringae and xanthomonas vesicatoria for which the available methods are not suffi ciently effective. nowadays, their control is mainly based on copper compounds and antibiotics. although antibiotics are highly effi cient, they are not authorized in several countries and resistance has been developed on plant pathogens. we have synthesized combinatorial libraries of cyclic decapeptides and linear undecapeptides. these libraries have been screened for antibacterial activity and eukaryotic cytotoxicity, and have led to the identifi cation of peptides with mic values of 1.6-12.5 microm. notably, cyclic peptides active against e. amylovora have been found, constituting the fi rst report of this type of peptides with activity towards this bacteria. the best peptides are bactericidal, display a low eukaryotic cytotoxicity at concentrations 30-120 times higher than the mics, and show a low susceptibility towards protease degradation. best peptides have been tested in vivo by evaluating their preventive effect of inhibition of p. syringae, x. vesicatoria and e. amylovora infections. the most active peptide is slightly less effective than streptomycin, currently used in fi eld. therefore, the best analogues can be considered as good candidates for the development of antibacterial agents for use in plant protection. selection of chromogenic and fl uorogenic substrates of neutrophil serine proteases using combinatorial chemistry approach human serine neutrophil and mastocytes proteases such as cathepsin g, neutrophil elastase, proteinase 3 and -tryptase are involved in several physiological processes. unwanted activity of those enzymes yields to severe pathological states like infl ammation, wegener granulomatosis or various types of cancer. therefore monitoring of the activity of these proteases is crucial for the proper therapeutical treatment. the simplest method for determination of protease activity is the use of synthetic substrates. they are also useful for characterization of the enzyme specifi city. in this work we report selection of chromogenic and fl uorogenic substrates of human serine neutrophil and mastocytes proteases applying combinatorial chemistry approach. peptide libraries were synthesized by the portioning-mixing method. deconvolution of synthesized libraries was performed using iterative approach in solution. 5-amino-2-nitro benzoic acid attached to the c-termini of synthesized peptides served as a chromophore released upon the interaction of peptide with enzyme. additional introduction of 7-methoxy-4-coumaryl acetic acid or 2-amino benzoic acid on á-amino groups of the chromogenic substrates converted them into fret displaying compounds. the most active substrates were subjected to further modifi cation applying non-proteinogenic amino acids. as a result, one of the most selective substrates of these proteases was obtained. we also attempted to construct a simple tools to detect activity of human serine neutrophil and mastocytes proteases by immobilization, through amide and peptoid based linkers, of selected substrates on solid phase. angiogenesis modulation: identifi cation of tetrameric tripeptide as inhibitor of vegfr-1 by the screening of peptide combinatiorial libraries 5 vascular endothelial growth factor receptor-1 (vegfr-1, flt-1) and their ligands are involved in complex biological processes associated to severe pathological conditions, like angiogenesis, infl ammation and metastasis formation (1). thus, the search for antagonists of flt-1 has recently gained a growing therapeutic interest. in order to identify new molecules able to selectively bind flt-1 and neutralize its activity, a screening of a combinatorial tetrameric tripeptide library built with nonnatural amino acids has been carried out by a competitive elisa-based assay. the library has been designed on a branched tetrameric structure in order to obtain molecules with a high recognition surface and, using 30 building blocks, a complexity of 27.000 different peptides has been achieved. peptide mixtures composing the library have been utilized as competitors of the flt-1/plgf (placental growth factor) interaction and the most active components have been isolated following an iterative deconvolution procedure. the selected most active hit shows a selective binding to flt-1 over kdr and inhibits in vitro its interaction with both plgf and vegf-a. the peptide is fully stable in biological environments, prevents flt-1 phosphorylation and blocks huvec capillary-like tube formation stimulated by plgf or vegf-a. in vivo the peptide inhibits the vegf-induced neoangiogenesis in chicken embryo chorioallantoic membrane assays and also stimulates cornea neovascularization (cnv) by displacing vegf from a complex with sflt-1. all data suggest that the compound has potential applications in diseases characterized by pathological angiogenesis, such as tumor growth and ischemic retinopathy. heinlein, christian; unverzagt, carlo bioorganische chemie, universität bayreuth, gebäude nw i, 95440 bayreuth, germany since the purifi cation of homogeneously glycosylated glycoproteins remains a diffi cult task the synthesis of entire glycoproteins is a fi eld of emerging interest (1) . especially the synthesis of the required protein fragments in high purity and good yields demands for effi cient methods. fragment condensation of protected peptides can reduce deletions in the crude product and thus facilitates purifi cation. condensation of peptide fragments in cspps (2) is however subject to epimerization upon cterminal carboxyl activation. to overcome this problem the use of cterminal gly or pro is usually performed. peptides containing c-terminal pseudoprolines can also be coupled without stereomutation because the serine or threonine residue has been reversibly protected as a prolinelike oxazolidine (3). this concept facilitates the synthesis of long peptides by epimerization-free fragment condensation at c-terminal ser/ thr residues in addition to gly/pro residues thus increasing the number of safe condensation sites. after solving several problems associated with the generation of peptide acids with c-terminal pseudoprolines the synthesis of the glycosylated rnase fragment was carried out as a racemization-free fragment condensation. tuftsin is liberated from fc-domain of the heavy chain of igg by two specifi c enzymes. being the tetrapeptide of biological origin is extremely important product because it can activate a few elements of immune system such as granulocyte and macrophage. tuftsin indicates not only immunological stimulating factor but also antibacterial, antivirial and antitumor properties. in spite of its wide range of activity, the peptide is unstable in plasma and it has become the aim of the formation of novel analogues more resistant to proteolysis degradation. the introduction of the additional residue at -amino group of lysine caused that new bond became stronger than peptide bond in central chain [1] [2] [3] . we synthesized linear tuftsin derivatives that were prepared on the solid phase using a fmoc/tbu procedure. the method of elongation of peptide chain was based on two-step procedure: deprotection and coupling step. segment coupling reaction was carried out with tbtu, hobt and in the presence of diea in dmf/dcm/nmp mixture. the introduction of the simple amino acid (ala, -ala, val, ile or gly) at -amino group of lysine let us obtain the isopeptide bond. the modifi cation was achived by introducing lysine residue, protected at -amino group with mtt. the selective removal of mtt group was caused by 2% tfa treatment. peptides were cleavage from resin and purifi ed. tuftsin derivatives were confi rmed by ms, amino acid analysis, elemental analysis and rp-hplc analysis. peptides were sent to assay their microbiological properties and the results will be described as a structure-activity relationship. the synthesis and structural study of iso-aß(1-42) the aß(1-42) peptide is diffi cult to synthesize, because of its high tendency for aggregation during the synthesis. both the couplings and the fmocremoval can be troublesome, due to the steric hindrance. the incorporation of the ester-bond disturbs the structure, thus ease the synthesis after the 25 th residue. if the peptide would be synthesized using boc-chemistry the removal of the -amino protecting group would be less problematic, because the 50% tfa/dcm mixture solubilizes well the peptide chain. thus a boc-strategy was devised for the synthesis of iso-aß(1-42). the purifi ed peptide was studied with cd-spectroscopy and dynamic light scattering. these structural studies revealed that the isopeptide has some ß-sheet content even in a ph 2 solution. it was realized also that small aggregates are present in the precursor peptide. both techniques mentioned above showed, that after altering the ph to 7.4, the ß-sheet content of the peptide increases and aggregation takes place. with the use of the isopeptide, aß oligomers and -with prolonged incubation -fi brillar structures can be formed for biological studies. continuing our program of syntheses of muramyl dipeptide (mdp) and nor-muramyl dipeptide (nor-mdp) conjugates as potential immunomodulators, we designed novel conjugates of mdp or nor-mdp with tuftsin derivatives containing isopeptide bond between -amino group of lysine and carboxylic group of simple amino acids such as alanine, glycine and valine. the synthesis of a greater number of conjugates will enable structure-activity relationship studies. tuftsin analogues containing isopeptide bond showed increased chemical resistance and activity in relation to tuftsin. the introduction of the additional residue at -amino group of lysine by nhco-formation caused that isopeptide bond became stronger than peptide bond in central chain. the protected pentapeptides (h-thr-lys(y)-pro-arg(no 2 )-obn, y= ala,gly,val) were synthesized by the conventional chemical procedure using mixed anhydride method. acylation of the thr amino group of partially protected pentapeptides by 1-benzyl-mdp or 1-benzyl-nor-mdp was performed using the mixed anhydride method with isobutyl chloroformate and n-methyl-morpholine (nmm) in dry dmf. the protected conjugates were isolated and purifi ed with a preparative tlc. the identities of the protected products were confi rmed by high resolution 1 h-nmr (500 mhz, cosy, tocsy, roesy, ghsqc, ghmbc) spectroscopy. the fi nal products were hydrogenated with h 2 / pd/c in 50% methanol-acetic acid and purifi ed with preparative tlc. the identities of the conjugates were confi rmed by tlc qualitative amino acid analysis, and elemental analyses. finally, the combined use of muramyl peptides with other immunomodulators, e.g. such as tuftsin, retro-tuftsin other chemotherapeutics is promising in the therapy of different infections, autoimmunological diseases and anticancer therapy. effi cient microwave-assisted synthesis of myelin epitopes mog35-55 and mog97-108 using cltr-cl resin fmoc/tbu methodology. unlike conventional heating, microwave energy directly activates any molecule with a dipole moment and allows for rapid heating at the molecular level. the protected peptides were synthesized using the cem liberty automated microwave peptide synthesizer in 18 and 13.5 hours respectively, with a 3-minute fmoc deprotection (25% piperidine solution in dmf) and 5-minute coupling reactions using dic/hobt in dmf solution. the maximum temperature reached during both the deprotection and coupling reactions was 80 °c except for the coupling of fmochis(trt)oh (50 °c). the fi nal crude products were of high purity as identifi ed by analytical rp-hplc. siah up-regulates the hif-1alpha hypoxic response pathway: siah binding peptides coupled to cpps inhibit siah activity and demonstrate proof of concept that siah is a viable anti-tumor drug target vegf, a secreted downstream component crucial for angiogenesis, has been successfully targeted clinically using antibody therapy (avastin). the hypoxic response, however, activates other pathways that stimulate tumor growth, suggesting that inhibitors of upstream components in the pathway may be useful to give a broader spectrum of inhibition. we have focused on the siah proteins that regulate hif-1ƒñ levels by ubiquitylation and degradation of the prolyl hydroxylases (phds) immediately upstream of hif-1ƒñ in the hypoxic response pathway. in a proof-of-principle study, we have shown that siah inhibition by expressed protein fragments (from a drosophila high affi nity interacting protein, phyllopod) can inhibit the stabilization of mammalian hif-1ƒñ during hypoxia and limit tumor growth in a mouse tumor model. a 23 amino acid peptide sequence (phyl) from the phyllopod protein has been identifi ed as the interaction site with siah. the aim of this work was to show that a small peptide could mirror the activity of the transfected recombinant phyllopod protein. to achieve this, phyl was covalently attached to cell penetrating peptides (cpps) and tested for its ability to inhibit hif-1ƒñ stabilization and hypoxic response in human u2os cells. both the tat sequence and penetratin were utilized as cpps and attachment was via disulfi de, maleimide or through a pro10 spacer sequence. the cpp¡vphyl constructs were found to have varying activities but a penetratin-pro10-phyl was found to be inhibitory in the u2os cell-line hif-1ƒñ stabilization assay. this result demonstrated proof of concept that siah inhibition could be attained by targeting a restricted and specifi c protein-protein interaction site. (1) . the principal event in the development of prion disease is the transformation of the normal cellular prion protein (prpc), which is highly helical in nature, into the pathogenic isoform prpsc which is insoluble and has an extensive ƒò sheet structure. the events surrounding this transformation are very poorly understood, but the mechanism of the detailed steps involved in this process is fundamental to the understanding of prion disease pathogenesis. in an endeavour to study the structure of polypeptide component of the n-terminal section of prpc, synthesis of a range of prp polypeptides were assembled on a cem liberty microwave synthesiser. these fragments ranged in length from 20 to 111 amino acids and span the protein sequence from position 1 to 144. standard cem synthetic coupling cycles were used, except when peptides were greater than 30 amino acids in length, whereby longer coupling cycles were employed. a number of purifi cation strategies were employed, such as c4 and c18 rp-hplc at either 25c or 60c and size exclusion chromatography. the purifi ed peptides were characterised by rp-hplc and esi-ms. in addition, they were analysed for secondary structure by cd spectrometry, and evaluated for cell toxicity and fi bril forming ability with tht. the synthesis of a 60mer peptide for investigating the mechanism of action of the hiv fusion inhibitor t20 however, the mechanism of action of t20 is still in debate. it is believed that peptides derived from gp41 chr region may share a common mechanism, by binding to gp41 nhr coiled coil and preventing formation of the fusogenic gp41 core-six helix bundle (6hb), thereby inhibiting fusion between the virus and target cell membrane. we have synthesized a 60-mer nhr peptide-n60-covering all the binding sites for any length of the chr-peptide. synthesis of the polypeptide was carried out using conventional as well as microwave assisted solid phase synthetic methods. details, including comparison of the synthetic approaches will be presented. using c34, another anti-hiv chr peptide containing the pocket-binding domain, as a control, we analyzed the activity of t20 to interact with n-60 to form 6hb and to inhibit the 6-hb formation between n-60 and c34 by cd spectroscopy and elisa. we found that t-20, unlike c34 could neither form a stable 6hb with n60, nor inhibit the 6-hb formation of the fusogenic 6hb core. our results thus suggest that t-20 and c-34 peptides inhibit hiv fusion by different mechanisms of action. the oligomerisation equilibrium of ts is modulated by a fi ne tuning; shifting this equilibrium towards the monomeric form would cause ts inactivity and low translation, overcoming resistance mechanisms encountered for (co)substrate-like inhibitors as the inactive monomer regulates its own expression. our group designed some small ligands to interfere with thymidylate synthase dimerisation, including short peptides taken from the interface sequence that represent the natural ligand for this region, mimicking the other subunit without forming a functional dimer. interesting biological data are arising from the activity tests but a rapid screening assay is necessary to prove they are really interfering with ts oligomerisation. fret is a spectroscopic phenomenon whose intensity depends on the distance between two fl uorophores; when this value varies due to conformational changes of the protein the probes are linked to, consequent fret variation can be used to sense the state of the protein(s) ( stromal derived factor-1 (cxcl12) is a 68 amino acid cxcchemokine with critical role in homing, migration and guiding of different cell types including hematopoietic progenitor cells (hpc), stem cells, tumour cells and neuronal cells during embryogenesis and in adults (1). these various functions in physiological as well as pathophysiological processes make this small protein interesting for regenerative medicine. to apply this chemoattractant in medicine, it is needed to form spatially and temporally controllable concentration gradients of active sdf-1 in response to a non-tissue damaging trigger like visible or near uv-light. after irradiation of an inactive prodrug dramatic change in conformation or lack of sterical hindrance should then lead to fully biological activity under physiological conditions within a few minutes. to prove the principle and assess its potential in photodynamic therapy of neuronal injuries a water-soluble, photosensitive sdf-1 analogue has been developed. for this the expressed protein ligation (epl) approach has been used, in which one segment has been recombinantly expressed and purifi ed using the impact ® -system to yield the corresponding peptide thioester (2) . the modifi ed peptide fragment has been chemically synthesized on solid phase using fmoc-strategy. the photocleavable moiety, the 6-nitroveratryloxycarbonyl (nvoc) protecting group, has been introduced at a side chain amino group. furthermore, the analogue has been characterised by physico-chemical methods and has also been tested in vitro on transfected cos-7 cells in order to determine its biological activity after activation. sdf-1 is a chemokine that plays a major role in traffi cking of hematopoietic stem cells (hsc). thus it enables the formation of bone marrow during embriogenesis and later in adult life it supports retention and homing of these cells in the bone marrow. furthermore it is involved in organogenesis and regeneration, respectively. 1 due to these promising features the subform sdf-1 could serve as a therapeutic target. for studies on the small protein concerning its molecular properties as well as its therapeutic potentials, it needs to be modifi ed chemically. in order to reach these goals, the n-terminus sdf-1 1-49 has been cloned and expressed recombinantly in e. coli er 2566 as a thioester, while the c-terminus sdf-1 50-68 has been synthesized via solid phase peptide synthesis. modifi cations are thereby introduced at the c-terminus at lys56. up to now carboxyfl uorescein has been coupled to the -amino group of the lysine residue. the two fragments then have been ligated via expressed protein ligation (epl), a subform of the native chemical ligation (ncl). 2 activity studies and fl uorescence microscopy on hek293 cells transfected with the sdf1-specifi c g-protein coupled receptor cxcr4 have been conducted. stueber, werner; lewandrowski, peter; frisch, juergen; weinschenk, toni; singh, harpreet immatics biotechnologies gmbh, germany ima901 is a multiple peptide vaccine for the treatment of renal cancer (rcc). the tumor-associated peptides (tumaps) contained in ima901 were identifi ed by immatics directly from primary renal cells (= primary rcc tumor tissue samples), selected regarding their over-expression in rcc and proven to be immunogenic using in vitro t-cell assays. ima901 consists of 10 individual peptides (10 tumaps) and nonactive ingredients which are used as excipients of the pharmaceutical presentation of ima901. all 10 peptides are synthesized by conventional fmoc chemistry. the sequences of the peptides will be presented and technical issues will be discussed. in the fi nal formulation of ima901 578 g of each peptide plus excipients are fi lled into glass vials and lyophilized. the challenges of the production of multi peptide drugs will be discussed. such challenges comprise the synthesis, the production of the formulation as well as the analyses of the fi nal presentation of ima901. results of the phase 1 trial in 28 vaccinated rcc patients showed that (1) ima901 was safe, (2) multiple t-cell responses to vaccinated peptides correlated with favourable clinical outcome and (3) patients with a lower percentage of regulatory t cells (tregs) were more likely to develop a vaccine-induced multiple t-cell response. novel peptides -msh analogs with high candidacidal activity in an attempt to improve the candidacidal activity of -msh and to better understand the peptide structure-antifungal activity relations, we designed and synthesized novel peptide analogs. because previous data suggested that the peptide [dnal-7, phe-12]--msh(6-13) has greater candidacidal activity than -msh and is the most potent of the analogs tested in the past, this compound has became our lead (1, 2) . from this lead compound we have synthesized a new library of peptides where we have replaced the glycine in position 10 with unconventional amino acids. here, we report new analogs with a strong antimicrobial and candidacidal activity. the obtained results are very encouraging in that they show the great potential of these peptides as a truly novel class of candidacidal compounds. derivative were modifi ed by the same amino acid to establish infl uence of the adjacent amino acid on antimicrobial activity of the compounds studied. the activity of all obtained peptides was screened against model gram-positive (bacillus subtilis) and gram-negative (escherichia coli) bacteria whereas antifungal activity was tested against yeast pichia pastoris. all tests were performed using antibiogram method whereas the minimal inhibitory concentrations were determined using two-fold serial dilution technique. huang, yen-hua 1 ; colgrave, michelle l. 2 the cyclotides are a family of naturally occurring macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a cystine knot motif, which impart extraordinary stability to this peptide family. a recent study demonstrated that the prototypic cyclotide kalata b1 possesses signifi cant activity against two economically important sheep nematodes haemonchus contortus and trichostrongylus colubriformis. an alanine scan of the molecule highlighted the residues critical for activity. in this work, we explore the relative importance of positively charged residues in different regions of the molecule to aid the understanding of the structural and biological basis of its nematocidal activity. a lysine scan has been conducted, in which each of the non-cys residues in this 29 amino acid peptide has been successively replaced with lysine and the suite of peptides have been assayed against the two sheep nematodes. substitution of residues in loop1, v10, g12, n15, t16, w23, v25, l2, p3, and v4 decreased or completely abolished the activity, suggesting that these residues are critical to the nematocidal activity of kalata b1. on the other hand, incorporation of a positive charge in positions g18, t20, t27, n29, and g1 signifi cantly enhanced the anthelmintic activity of the grafted peptides, up to fourfold, compared to native kalata b1. these increases in activity after lysine incorporation into the kb1 scaffold raise the possibility of being able to engineer greater anthelmintic activity into the peptides, further highlighting their potential as anthelmintic agents. ivanova, v.p. 1 interaction of cells with extracellular matrix (ecm) affects many aspects of cell behavior including growth, morphology, migration and differentiation. integrins are known to mediate cell adhesion to proteins of ecm. ligand-binding properties of integrins depend not only on composition of ecm ligands and level of integrin expression in cell, but also on spectrum and activity of soluble factors indirectly infl uencing cell-matrix contacts. interaction of cells with substratum may be divided into cell adhesion and spreading. following an initial cell attachment event, cell may or not spread depending on cell type and the nature of the molecular signals they receive. oligopeptides released from different proteins during their proteolysis in or out of cells may act as the such short-time existing signals. in the present study we investigated the effect of multiply repeated peptide fragment in different collagen types on cell spreading. murine embryonic fi broblasts (200000/ml) were allowed to adhere for 45 min at 37° c with or without peptide (in various concentrations) to plastic surface pre-coated or not with gelatin. it was shown that the synthetic peptide increased the number of spread cells and caused shape changes in cells spread on different substrata. a 30-min pretreatment of cells with the peptide (before conducting of cell spreading assay) resulted in inhibiting of cell spreading on gelatin. our results suggest that the peptide regulation of cell spreading could be related to its effect on re-distribution of integrin receptors in sites of cell contacts with substratum. bioactive peptides from cyanobacteria cyanobacteria are versatile source of small peptides from which vast majority are cyclic. cyanobacterial culture collection in university of helsinki contains over 1000 strains and this collection is used for screening of new bioactive compounds. cyclic heptapeptides, microcystins are the best known cyanobacterial peptide family. over 80 structural variants have been described from which most are strong hepatotoxins. our research group have participated to the determination of the structure of many novel microcystins and other cyclic and linear cyanobacterial peptides. in recent years we have found highly toxic and novel microcystins from lichen associated cyanobacteria (1) and from anabaena strains of baltic sea (2). in the structural analysis of new peptides from the known peptide families we have used liquid chromatography ion trap mass spectrometry which have proven to be very effective method and is in many cases the only method needed in the verifi cation of a new structure. with this lc-itms method we have found many new structural variants from anabaenopeptin, anabaenopeptilide and spumigin peptide families. in collaboration with many research groups we have found cyanobacterial compounds/extracts which inhibit protein kinase c activity, inhibit/activate boar sperm motility, disintegrate cell membranes or are antidotes for microcystin toxicity. structures of the compounds are under study except the microcystin antidote which structural analysis showed that it belongs to a rare peptide family containing imino bond in cyclic skeleton. the results of n-terminal modifi cation of arginine vasopressin with cis-1-amino-4-phenyl-cyclohexane carboxylic acid. the highly potent oxytocin receptor antagonists arginine vasopressin (avp) is a cyclic nonapeptide with multiple functions. the main peripheral physiological roles of avp are the regulation of water balance, the control of blood pressure, and the release of adrenocorticotropin hormone (acth). moreover, avp also exhibits to some extent typical oxytocin (ot, a closely related neurohypophyseal peptide) activities such as the galactogogic and the uterotonic effects. all peptides were tested for the pressor, antidiuretic and uterotonic in vitro activities in the rat. cis-apc 2 modifi cation at position 2 of avp is suffi cient to change the pharmacological profi le of the peptides. analogues i -iv were moderately potent antidiuretic agonists with prolonged action. in regard to the uterotonic activity, all peptides with cis-apc were highly potent antagonists, except compound i. it supports our earlier hypothesis that an amino acid residue in position 2 has signifi cant impact on pharmacological activities. the incorporation of unnatural non-proteinogenic -amino acids into peptides has emerged as a novel and promising approach in peptide modifi cation. the conformationally restricted amino acid derivatives are of particular interest. this thesis are also supported by our already 12-years research focused on the effects of steric restriction and the presence bulky substituent in the n-terminal part of avp molecule on biological properties of the resulting analogues. all peptides were tested for the pressor, antidiuretic and uterotonic in vitro activities in the rat. all the analogues were devoid of the pressor potency and exhibited only negligible antidiuretic activity. interestingly, in regard to the uterotonic activity, the new compounds exhibited moderate (i) or high (ii -iv) antioxytocic potency. it should be point out that single substitution e.g. replacement of tyr in avp molecule with igl results in moderately potent and highly selective antagonists of oxytocin (i). our new igl 2 substituted peptides proved that the presence of the sterically restricted amino acid residue may result in high active and selective antiuterotonic agents. these in our opinion interesting fi nding demonstrates the usefulness of our approach in the design of new highly active and selective analogues with desired pharmacological properties. arginine vasopressin (avp), a neurohypophyseal hormone and neuromodulator, is a cyclic nonapeptide with a disulfi de bridge between cys residues at positions 1 and 6. as a hormone avp exerts its biological effects upon binding to three receptor subtypes termed: v 1a , v 1b (v 3 ), and v 2 . furthermore, avp to some extent can interact with the oxytocin receptor (ot). biological activity of peptides is determined by their structure and conformation. conformational restriction of bioactive peptides is therefore a well-established strategy to change their pharmacological profi le. peptide fl exibility can be restricted by a local constraint imposed, e.g. by introducing amino acids with limited conformational freedom, that has an impact on specifi c orientations of the peptide backbone and the side chains. in this work, we decide to check the infl uence of the bulky ( design, synthesis and biological activities of temporin a and temporin l analogues. temporins a (ta) and l (tl) are antimicrobial peptides isolated from the skin of red european frog "rana temporaria". temporins are active against a broad spectrum of microrganism: ta (flpligrvlsgil-nh2) is preferentially active against gram-positive bacterial strains; tl (fvqwfskflgril-nh2) has the highest activity against fungi, and bacteria, including resistent gram-negative strains, but it shows haemolytic activity too. ta exerts its antimicrobial activity by its ability to form a transmembrane pore via a 'barrel-stave' mechanism or to form a 'carpet' on the membrane surface via the 'carpet-like' model. recently we investigated the preferential conformation of tl and ta in sds and dpc solutions which mimic bacterial and mammalian membranes, respectively. in sds, the peptides prefer a location at the micelle-water interface; in dpc, they prefer a perpendicular location to the micelle surface, with the n-terminus imbedded in the hydrophobic core. tl shows higher propensity, with respect to ta, in forminghelical structures in both membrane mimetic systems and the highest propensity to penetrate the micelles (1). on these results we designed and synthesized new ta and tl analogues and found interesting differences in their effi cacy against microbial species, and fi nding a new potent antimicrobial agent without haemolytic activity. arginine vasopressin (avp), a neurohypophyseal nonapeptide hormone [cycle1-6 (h-cys 1 -tyr 2 -phe 3 -gln 4 -asn 5 -cys 6 -pro 7 -arg 8 -gly 9 -nh 2 )], elicits a variety of responses both centrally and peripherally by acting on three distict g-protein coupled receptors: v 1a (vascular), v 1b (pituitary) and v 2 (renal). it also binds to the oxytocin (ot) receptor. in addition to its well-known antidiuretic activity, avp has also complex cardiovascular actions and adrenocorticotropic hormone (acth) releasing activity. binding of avp to the v 1a receptor subtype also stimulates glycogenolysis in the liver and promotes platelet aggregation. it is generally accepted that the conformation of the n-terminal part of neurohypophyseal hormones analogues is important for their pharmacological activity. in continuing our work aimed at the design of selective avp analogues, we synthesized twelve new analogues of avp containing mercapto propionic acid ( we also studied the effect of modifi ed c-terminal amide on biological potency of the new avp analogues. the analogues were synthesized by fmoc/bu t solid phase methodology and were tested for their rat uterotonic in vitro activity, rat pressor activity and antidiuretic activity using conscious rats. the modifi cations performed had a signifi cant impact on pharmacological activities of the analogues. acknowledgements: we thank the european social fund (esf), operational program for educational and vocational training ii (epeaek ii), and particularly the program pythagoras i, for funding the above work. it was also supported by the research project no. z40550506 of the academy of sciences of the czech republic. three-dimensional structure and mechanism of action of an antifungal peptide generated from hemocyanin cleavage in a penaeid shrimp an antifungal peptide, pvhct, which corresponds to the 23 amino acid c-terminal sequence of the shrimp respiratory protein hemocyanin, has been previously identifi ed in the plasma of the penaeid shrimp litopenaeus vannamei (1) . it is generated by proteolytic cleavage in response to a microbial challenge. similarly, a c-terminal fragment of hemocyanin displaying antimicrobial activity has been isolated from crayfi sh plasma (2) . these peptides are believed to contribute to the crustacean defence. the phenomenon of in vitro antimicrobial peptide generation from a respiratory pigment already observed with hemoglobin thus appears not to be restricted to mammals (3). pvhct displays a broad spectrum of antifungal activity with minimum inhibitory concentrations (mics) in the range 3-50 m (12.5 m against the shrimp pathogen fusarium oxysporum). its activity would be based on the inhibition of spore germination (1) . to contribute to the elucidation of the mechanism of pvhct antifungal activity, we determined its three-dimensional structure by circular dichroism (cd), nmr and molecular modelling and examined its effects on the f. oxysporum spore ultrastructure by transmission electron microscopy (tem). cd and nmr data indicate that pvhct is unfolded in an aqueous environment but adopts a similar helical structure in methanol solution and in dodecylphosphocholine (dpc) micelles used to mimic biological membranes. the structure consists of an amphipathic -helix spanning residues 8 to 18. tem shows that pvhct induces structural changes of the plasma membrane accompanied by a disorganization of the cytoplasm and a signifi cant decrease of lipid bodies. glucagon-like peptide-1 (glp-1), glucagon (gcg), and oxyntomodulin (oxm) are highly homologous peptide hormones derived from posttranslational processing of the preproglucagon gene. the sequence of oxm in particular, is identical to the sequence of gcg, with an 8amino acid extension at the c-terminus. pharmacological doses of oxm activate both the glp-1 receptor (glp1r) and the glucagon receptor (gcgr) albeit with lower affi nity compared to glp-1 and glucagon, respectively. in order to understand the origin of this dual specifi city, we synthesized a number of oxm analogs in which one or more of the native amino acids were replaced. first, a chimera was produced in which all the residues differing between glp-1 and gcg were grafted into the oxm native sequence, yielding an analog with the same pharmacologic profi le as glp-1. further studies showed that surprisingly, the switch from glp1r/gcgr co-agonism to glp1r-selective agonism could be obtained by a single amino acid substitutions at position 3 of oxm. in particular, the analog with gln3 substituted by glu (oxm-q3e) showed the same activity as native oxm on glp1r, but complete loss of activity on gcgr. oxm and oxm-q3e were compared in a hyperglycemic clamp study performed in diet-induced obese (dio) mice. due to the short half-life of the peptides, both were infused intravenously at ~16 g/kg/ min in chronically catheterized mice. the amount of exogenous glucose required to maintain the hyperglycemic level was 2-fold greater with the selective glp1r agonist oxm-q3e than with the glp1r/gcgr coagonist oxm, showing that abolishment of gcgr activity signifi cantly improves the glucose-lowering effect. we believe that these fi ndings could be useful for the development of a peptide therapeutic for the treatment of type 2 diabetes. hospitals across europe and north america have lately been plagued with infections caused by clostridium diffi cile, a diffi cult to treat bacterium due to its resistance to many commercially available antibiotics. recently, we isolated a two-component bacteriocin, thuricin, produced by bacillus thuringiensis that exhibits activity against c. diffi cile. we found that these peptides, called trn and trn , operate together in a synergistic fashion to inhibit bacterial growth. the peptide combination was found to be highly active against a wide range of c. diffi cile strains, including the virulent epidemic strain of the o27 ribotype, as well as most other clostridia and some bacilli and listeria species. maldi mass spectrometry revealed that both peptides have molecular weights that are lower than those predicted from their genetic sequences, indicating that they are post-translationally modifi ed. sequencing of the mature peptides through tandem mass spectrometry showed that each peptide has three modifi ed amino acid residues near its c-terminus. these residues were found to be two units lighter than their expected natural amino acid masses, suggesting a loss of two hydrogen atoms through dehydrogenation or oxidation. in order to confi rm these proposed modifi cations, we are investigating the production of 13 c-and 15 n-labeled trn and trn for structure elucidation by nmr. isolation of labeled peptides will be achieved by growing b. thuringiensis on defi ned media containing [u-13 c] glucose and ( 15 nh 4 ) 2 so 4 . subsequent nmr experiments will enable us to determine the three-dimensional solution structures of trn and trn , individually and bound together. by elucidating thuricin's structure, we aim to better understand its mechanism of action against c. diffi cile. the most potent peptidic human bradykinin (bk) b 2 receptor antagonist is hoe-140 (icatibant). in this study we present the synthesis of nine new analogues of hoe-140 substituted at position 7, 8 or 9 with chosen d-amino acid residues and/or nonproteinogenic amino acid residues, e.g. n-cyclohexylglycine (nchg), 1-aminocyclohexane-1-carboxylic acid (acc), octahydroindole-2-carboxylic acid (oic), piperidine-3-carboxylic acid (nip) and 4-phenylpiperidine-4-carboxylic acid (ppc). in the next nine peptides we combined the above mentioned modifi cations with the placement of 1-adamantane acetic acid (aaa) at position 0. all new analogues were tested on human umbilical vein for their antagonistic potency. only three compounds containing nchg 8 , nchg 9 or ppc 9 exhibited noticeable antagonistic activities on human bradykinin receptors thus still less potent then original hoe-140 sequence. each of them with aaa 0 showed lower activity then parent analogues. peptides: d-arg-arg-pro-hyp-gly-thi-ser-d-phe-nip-arg and aaa-d-arg-arg-pro-hyp-gly-thi-ser-d-phe-nip-arg, although strongly potent antagonists against bk-induced blood pressure lowering responses in rats, did not show noticeable antagonistic activity on bk-induced contraction of the isolated human umbilical vein, a well-established b 2 r bioassay system, suggesting a species dependent activity of the compounds. due to advances made in the peptide fi eld during the last few years, peptides as therapeutics have continued to gain more interest. the interest in peptide therapeutics generally comes from their high specifi city to targeted sites, their diversity and their usually low toxicity. pt-141, also known as bremelanotide, and mt-ii are potential future drugs and both are heptapeptides with a 23-membered cyclic monomeric lactam bridge. they are melanocortin receptor agonist and an analog of alpha-melanocyte stimulating hormone (a-msh). the pt-141 molecule has a c-terminal acid group while mt-ii has a c-terminal amide function. both neuropeptides have been tested in treating male sexual and erectile dysfunction as well as female sexual arousal disorder. pt-141, patented by palatin technologies, new jersey, usa, is the only known synthetic aphrodisiac and unlike other erectile enhancers like viagra, it does not act upon the vascular system. instead, it directly increases sexual desire and is used nasally as a spray. a scalable method for the synthesis of both peptides using fmoc-chemistry and the problems involved during the synthesis process will be discussed in details. tateaki, wakamiya; takahiro, nishimaru; kiyoe, mori; yoshihiro, yamaguchi kinki university, japan l-glutamate (glu) is known to be major excitatory neurotransmitter not only in the mammalian central nervous system but also in the ganglia of arthropods. binding of spider toxins to glutamate receptors (glurs) results in the inhibition of glu-mediated neurotransmission. in order to elucidate the mode of binding between glu and glurs, we focused on the visualization of glurs by complex formation with fl uorescentlabeled analogs of nptx-594 (1), a spider toxin with the structure of n 1 -(2,4-dihydroxyphenylacetyl-l-asparaginyl)-n 12 -l-lysyl-4,8-diaza-1,12-dodecanediamine [dhpa-asn-dada(12lys)]. in the present study, the modifi ed nptx-594, i.e., dhpa-asn-dada(12abg) (2) in which the lys residue of 1 was replaced with the n-(4-aminobutyl)glycine (abg) residue, was employed as a template compound to create the fl uorescentlabeled analogs of nptx-594, since the biological activity of 2 is three times higher than that of 1. we thus carried out the modifi cation of dhpa in the analog 2, i.e., 1) dhpa was replaced with the coumarintype acyl residues (type-1); 2) the phenylacetyl residues having alkyne side chains that can be converted into suitable fl uorophores based on the click chemistry (type-2); and 3) the coumarin-type acyl residues having the mercapto group to form disulfi de bond with the cys residue in glurs (type-3). this paper presents the synthesis and biological activity of various nptx-594 analogs to adopt as probes for visualization of glutamate receptors. an investigation of the functional requirements of apidaecin ib c-terminal fragment by means of peptoidpeptide hybrids by acting on one or more intracellular targets, without damaging the cytoplasmatic membrane. their particular killing mechanism and their low toxicity against mammalian cells make them attractive for the development of new antibiotics. structure-activity relationships studies have been carried out on short pro-arg rich antimicrobial peptides isolated from insects and the characterization of various natural isoforms of the 18-residues peptide apidaecin ib allowed to identify an evolutionary conserved region in the c-terminal part of the molecule. even a single point mutation in this region results in reduction or loss of antimicrobial activity. we recently described the synthesis of some apidaecin ib peptoid-peptide hybrids in which each arginine was replaced by the corresponding n-alkyl glycine residue (1). the afforded modifi cation made the resulting peptoid-peptide hybrids more resistant to proteolysis but moving the [narg]residue from the n-to the c-terminal end of the molecule progressively reduced the antibacterial activity. here we report the synthesis of a series of novel analogues containing a n-homoarginine or n-norarginine residue in position 4, 12 or 17. the effect of the size of the side-chain of the peptoid residue on the antimicrobial activity is also reported. in order to strengthen the peptide resistance to proteolysis and by considering that enzymic cleavage of the arg 17 -leu 18 peptide bond yields a fully inactive compound, we also prepared the [nleu 18 ]-apidaecin analogue. the conformational properties of the resulting peptoid-peptide hybrid, which is devoid of any antimicrobial activity, will be compared to those of apidaecin ib and the other [narg]peptoid-peptide hybrids. a wide variety of organisms produce antimicrobial peptides as part of their fi rst line of defense. however, antimicrobial activity is not the main function of some of these peptides. in the cuttlefi sh sepia offi cinalis we observed an antibacterial activity for the neuropeptide : h-alsgdaflrf-nh 2 (1). this decapeptide belonging to the fmrfamide family involved in regulation of reproduction and chromatophore function and is able to inhibit the growth of marine bacteria. circular dichroism studies have revealed for this amphiphilic peptide a helical structuration in sds micelles and in 50% tfe. to improve this antimicrobial activity, we fi rst introduce a lysine residue instead of aspartic residue, the antimicrobial activity of this new peptide has been improved by augmentation of the positive net charge (+1 to +3). moreover preliminary results have shown that the incorporation of aza-3 -amino acids analogues could create original hybrid pseudopeptide with superior activity . the natural peptide is not effi cient against staphylococcus aureus whereas the pseudopeptidic analogue revealed a minimum inhibiting concentration (mic) between 8-16 m. therefore some -amino acids were replaced by aza-3 -amino acids. we will show in this communication that depending on the substituted residue and on the structuration, these modifi cations could lead either to no activity or to a drastic enhancement of the antimicrobial activity, demonstrating that aza-3 -amino acids can facilitate the burying in lipidic bilayer of antimicrobial peptides that acts on bacterial membranes(2) references: isolation and structural characterization of capistruin, a lasso peptide predicted from the genome sequence of burkholderia thailandensis e264 the poor overall fragmentation behavior in ms n studies suggested a branched cyclic peptide with a rigid lasso structure. optimization of the fermentation conditions increased the production by 200-fold and subsequent nmr structural studies proved the lasso structure of the peptide that was named capistruin. heterologous production of the lasso peptide in e. coli showed that the identifi ed genes are suffi cient for the biosynthesis of capistruin, which exhibits antimicrobial activity against closely related burkholderia and pseudomonas strains. to our knowledge, this is the fi rst rational based identifi cation of a novel lasso peptide and the presented approach should be advantageous for the isolation of further lasso peptides in the future. endogenous antimicrobial peptides (amps) are the earliest molecular factors in the evolution of innate immunity. marine invertebrate animals have no acquired immunity with a system of antibodies diversifi cation. they are presumed to use an amps-based system as principal defense against potential pathogens. we have discovered a new family of small (21-residue) amps, termed arenicins, in coelomocytes of marine polychaeta lugworm arenicola marina. these amps exhibited activity against gram-positive, gram-negative bacteria and fungi. complete amino acid sequences were determined for each isoform. arenicins have one disulfi de bond (cys3-cys20). arenicins have no structure similarity to any previously identifi ed antimicrobial peptides. a novel 40-residue antimicrobial peptide, aurelin, exhibiting activity against gram-positive and gram-negative bacteria, was purifi ed from mesoglea of a scyphoid jellyfi sh aurelia aurita. complete amino acid sequence of aurelin was determined. aurelin has 6 cysteines forming three disulfi de bonds. the total rna was isolated from the lugworm coelomocytes and from the jellyfi sh mesoglea, rt-pcr and cloning were performed, and cdnas were sequenced. a 202-residue preproarenicin contains a putative signal peptide (25 amino acids) and a long prodomain. a 84residue preproaurelin contains a putative signal peptide (22 amino acids) and a propiece of the same size (22 amino acids). aurelin reveals partial similarity both with defensins and k+ channel blocking toxins of sea anemones and belongs to shkt domain family. overlapping of biological properties of marine animal amps and toxins along with their sequence homology might be a consequence of divergent evolution from a common ancestor. antimicrobial peptides from marine organisms could afford design of new antibiotics manifesting broad-spectrum antibacterial activity. cyclic enkephalin analogs containing two alkylurea units we shall present some structure-activity results for 8 enkephalin analogs, derived from the exhaustive combinations of d-lys and d-orn in position 2 with lys, orn, dab and dap in position 5, both positions coupled ¦ø-¦ø¡¯ by means of the urea bridge. accordingly, they all are restrained by 14-18-membered rings. in addition, we introduced the -nh-ethylurea unit instead of -nh2 of amide group in previously published analogs [1] [2] [3] . their in vitro activities were determined in the gpi and mvd assays. the peptides are more active than enkephalin in the gpi while have similar activities to the latter in the mvd assay. the effect of the introduction of ethylurea unit at the c-terminus on the activities is also discussed. chemical shifts of the peptides in water were fully assigned and their sequences confi rmed. several cross-peaks between the protons of amidoalkylurea unit and preceding residues have been observed in each case, suggesting that the unit may be involved in specifi c interactions between residues 4 and 5. understanding the structure/activity relationships of hepcidin iron is an essential element for nearly all living organisms and plays a key role in a range of processes including oxygen transport and storage, catalysis of redox reactions, production of metabolic intermediates and host-defence (1). until recently, little was known about the regulatory elements involved in the control of iron uptake and distribution within the body. the recently discovered peptide hepcidin has been shown to be a key regulator of iron metabolism within the body in response to a range of conditions, including infl ammation, hypoxia and anaemia (2) . this talk will focus on work towards elucidating structure/activity data for hepcidin with the aim of gaining a better undertstanding of the interaction between hepcidin and its receptor, ferroportin. we hope that this information will facilitate the design of synthetic agonists or antagonists of ferroportin to be used as potential drug leads. three-dimensional structures of investigated analogues were determined using two-dimensional nmr spectroscopy and molecular dynamics simulations with time-averaged restraints. the analysis of structural differences exhibited by different modifi cations provides the basis for understanding conformation -activity relationships and thereby the mechanism of interactions of the analogues with receptors. nmr structure of the micelle-bound 26rfa and 43rfa, two peptide ligands of the gpr103 receptor a novel rfamide peptide, named 26rfa and with no meaningful similarity with other members of this family, has been recently characterized. its precursor encompasses several potential cleavage sites and thus may generate various mature peptides including an nterminally extended form of 26rfa, termed 43rfa. both peptides act as endogenous ligands of the g-protein coupled receptor gpr103. this receptor has been recently implicated in the bone metabolism regulation the determination of the 3d structure of such peptides is essential for the elucidation of their structure/function relationships and for the design of potent agonists or antagonists. although structure elucidation of a ligand in the absence of the target receptor can deliver limited insight into the bioactive conformation, there is emerging evidence that interactions with the cell membrane is a key step required for receptor recognition. in this context, we have investigated the solution conformation of 26rfa and 43rfa by cd, nmr and molecular modelling in different media, in particular in one miming the "free" form of the molecule (methanol or mixture tfe/water) and in a cell membrane mimetic medium (dpc micelles). in an organic solvent, both peptides adopt the same conformation, i.e. an amphipathic alpha-helical structure, fl anked by two n-and c-terminal disordered regions. when bound to dpc micelles, the n-terminus remains fl exible and an helix is present at the same position as in the "free" form for both molecules. in contrast, the cterminal extremity becomes structured adopting an inverse gamma-turn conformation. these data represent the fi rst step for the rational design of new molecules that might be used in the treatment of osteoporosis. acknowledgements: supports were obtained from inserm and ifrmp 23. the nmr spectrometers are supported by grants of the conseil régional de haute-normandie. nmr and molecular modelling facilities are provided by the centre de ressources informatiques de haute-normandie. antimicrobial activity of analogues of a peptide isolated from venom glands of social wasps polistes major major inhabiting the dominican republic recently we have described isolation and biological activities of several new peptides from the venom glands of social wasps polistes major major found in dominican republic. we have also reported the synthesis of their analogues in order to investigate structure-activity relationship with respect to the antimicrobial and hemolytic activities (1). here we report the activities of a few further analogues of one of the peptides called pmm (h-ile-asn-trp-lys-lys-ile-ala-ser-ile-gly-lys-glu-val-leu-lys-ala-leu-nh2). the parent sequence or its truncated analogues were modifi ed on the n-terminus with 6-aminocaproic acid, glycolic acid, palmitoic acid, and 9-acridinyl and 9-(1,2,3,4-tetrahydro)acridinyl groups. the new analogues were tested for their antimicrobial activity (determination of minimal inhibitory concentration values -micusing broth dilution method) and hemolytic activity (determination of the ic50 value using suspension of rat erythrocytes). the palmitoylation unfortunately did not enhance antimicrobial activity against the tested microorganisms (bacillus subtilis, staphylococcus aureus, escherichia coli and pseudomonas aeruginosa). substitution of amino acids ser8 or glu12 subsequently for alanine, serine or lysine did not infl uence the activity of the peptides signifi cantly. gramicidin s, gs, c-(val-orn-leu-d-phe-pro)2, was isolated from bacillus brevis. it forms a two-stranded antiparallel -sheet fl anked by two ii' -turns. it was found that the distribution of hydrophobic and hydrophilic residues on the opposite sides of the sheet is a structural feature required for gs antimicrobial (am) activity. despite its wide gram+ and gram-antimicrobial activity gs is useless in therapy because of its high hemotoxicity in humans. it was found, however, that the analogues of gs-14 (gs with lys-leu inserted into each strand) got more am selective, when their amphipatic moments were perturbed by swapping adjacent lys leu/val or confi guration reversal at lys (1) . here, we report on effects of similar perturbations put on gs original c-decapeptide, using the following examples: c-(val-lys-leu-d-his-pro)2, 1, 1. as the mother compound, and its three analogs, viz. c-(val-d-lys-leu-d-his-pro-val-lys-leu-d-his-pro) -lys2 converted to d, c-(lys-val-leu-d-his-pro-val-lys-leu-d-his-pro) -val1-lys2 swapped, and c-(val-leu-lys-d-his-pro-val-lys-leu-d-his-pro) -lys2-leu3 swapped, 2-4; all having reduced ring-sequence symmetry. the peptides were synthesized by solid-phase methods using 9-fl uorenylmetoxycarbonyl (fmoc) methodology. having solved their structures by 2d-nmr and having tested their activities/selectivities, we confi rmed that only 1 had relatively favorable bio-profi le, as already published (1) cereulide, a foodborne peptide highly toxic towards the insulin producing beta-cells of the pancreas cereulide is a heat stable cyclic, lipophilic (log kow 5.96) peptide (1152 g mol/1) produced by certain strains of bacillus cereus, a bacterium connected to emetic food poisonings. it is insoluble in water, soluble in ethanol, methanol, dmso, food oils. cereulide exposure caused collapse of mitochondrial membrane potential in all tested human cells at low (ng/ml) exposure concentration (nk cells, t lymphocytes, caco2, calu3, neural paju, hela). toxicity is caused by its action as ion carrier with a selectivity of k + : na + = >1000 : 1. in various foods connected to human illness, concentrations of 0.01 to 3 g/g of cereulide were measured. in a case where the remains of a meal that had caused acute serious illness of two adult persons, were obtained for analysis, 1.3 g of cereulide was found /g of food. we undertook to explore the effects of purifi ed cereulide, and cereulide containing bacterial extracts, on porcine pancreatic islet cells in culture. foetal porcine islet cells were exposed to heat killed extracts from food-borne b. cereus strains producing or not producing cereulide and to purifi ed cereulide. effects were assayed using viability staining with fl uorochromes and cellular contents of dna and insulin. exposure to 1 ng/ml of purifi ed cereulide caused necrotic cell death of the islet cells impairing their insulin content within 2 days. cell extracts of cereulide positive b. cereus strains connected to food poisoning or isolated from food items were toxic, corresponding to their measured cereulide content. extracts of b. cereus strains producing or not producing the b. cereus diarrhoeal toxin, but not cereulide, were tolerated by the porcine islet cultures up to concentrations 1000 fold higher compared to extracts from strains containing cereulide, produced substance toxic towards porcine fetal langerhans islets and beta cells. application of non-sequential pharmacophore concept for design of antimicrobial peptide dendrimers unique structure of dendrimeric compounds consisting of a central core and several generations of branches provides opportunity of multiple practical solutions in the area of medicine. location of a high number of functional groups at the surface, allows to present multiple pharmacophoric units to the receptors, with immediate application in the design of a new generation drugs or vaccines, tools for studying autoimmune diseases or understanding gene delivery mechanism. here we present another possible application of dendrimeric compounds -preparation of drug molecules, which mimic active conformations of various macromolecular ligands. we focused on low molecular weight basic dendrimeric peptides, which mimic active conformations of recently discovered natural antimicrobial peptides. structurally, natural compounds are linear cationic peptides consisting of 10-50 amino acids that kill a broad spectrum of microbes destabilizing ordered structure of their cell membrane. it is generally accepted that positive charge and an induced amphipathic conformation are necessary for their antimicrobial activity. the project is related to multi-drug resistance of numerous bacteria against conventional antibiotics and involves de novo design of 1-2 generation dendrimeric peptides, which mimic sequencerelated active conformations of natural compounds (non-sequential pharmacophore concept). apparently, several groups of small peptide dendrimers were synthesized and structurally characterized (nmr, cd). they are potent antimicrobials, active against broad spectrum of species including mrsa and esbl strains. interactions between model membranes and peptide dendrimers of various structure will be discussed. seawater desalination is most commonly done today by reverse osmosis (ro) using thin-fi lm composite membranes. a major problem in ro desalination is biofouling, caused by adhesion and growth of bacteria to form biofi lm on the membrane surface. recently we proposed a new approach to reduce biofi lm formation on ro membranes that is based on immobilization of antimicrobial peptides (amps) onto the membrane surface. in this study we screen amps capable of reducing biofi lm growth under conditions simulating seawater desalination, and search for mode of binding to the membrane without affecting the peptides bioactivity. a specifi c bioassay was developed for screening peptides activity in high salinity conditions in order to evaluate the inhibition of biofi lm growth, based on growing biofi lmforming bacteria in a 96-wells microtiter plate. we prepared various amps known from the literature by solid phase peptide synthesis (spps) using fmoc-chemistry. the bactericidal activity of amps was examined in fresh water and compared to high salinity water. most amps lost their activity in high salinity conditions; yet, few peptides possessed their bactericidal activity and were used in subsequent experiments. searching for mode of linkage was performed by evaluating the bactericide activity of amps modifi ed with numerous types of linker molecules. based on literature studies that showed no decrease in bactericidal activity of amps upon n-terminal modifi cation, we prepared the corresponding peptides with modifi ed spacers on their amino-terminal and evaluated their antimicrobial activity in solution. indeed, we obtained gly 3 -spacers that retained activity, which were used subsequently as linkers to ro membranes. the mode of binding, as well as bactericide activity of the peptides and of the membranes will be presented and discussed. this study will lay the bases for a novel approach to decrease biofi lm formation on the surface of ro membranes during ro desalination. bioactive tripeptides ile-pro-pro and val-pro-pro protect endothelial function in vitro in normotensive and hypertensive rats milk drink containing casein-derived bioactive tripeptides isoleucylprolyl-proline (ipp) and valyl-prolyl-proline (vpp) has been shown to decrease blood pressure both in animal models and clinical studies. this effect can be attributed to the tripeptides. it has been suggested that one possible blood pressure lowering mechanism of the tripeptides could be angiotensin-converting enzyme (ace) inhibition. however, not all studies support this fi nding. the effect of tripeptides ipp and vpp on vascular function was investigated in vitro using rat mesenteric arteries. superior mesenteric arteries isolated from male wistar-kyoto and spontaneously hypertensive (sh) rats were incubated in krebs solution containing 1 mm of the peptide (either ipp or vpp) in +4 ºc for 48, 24, 12 or 1 h. after incubation mesenteric artery rings were mounted in an organ bath chamber and extensive vascular reactivity measurements were performed. acetylcholine-induced endothelium-dependent relaxation was better preserved (p < 0.05) in mesenteric arteries of both strains incubated with ipp or vpp compared to the control. clear differences were not observed in sodium nitroprusside-induced endotheliumindependent relaxation. the ace-inhibitory activity of ipp and vpp was studied by measuring the response to a single administration of angiotensin i and ii in organ chambers. proportioned to kcl-induced contraction, no clear reduction in angiotensin i -contraction was seen. thus, ace-inhibition may not be the main mechanism for the long-term effects of ipp and vpp in the protection of endothelial function. we suggest that the tripeptides do not affect smooth muscle but they protect endothelium during incubation indicated as preserved acetylcholine-induced endothelium-dependent relaxation. and directly and modulate other parts of host innate immunity. today, more than 800 cationic peptides have been identifi ed. they all have certain conserved physical features including a net positive charge, contain approximately 50% hydrophobic amino acids and have sizes ranging from 12 to 50 amino acids. however, virtually any -sheet, loop including -helix, type of secondary structure can arise including -turn and extended. the multitude of cationic peptide sources, structures and a spectra of activity is matched by a number of complex and controversial models attempting to describe and explain their modes of action. little is known about the sequence requirements of short host defense peptides like bactenecin (12mer). with help of our novel technique using a artifi cially created luminescence producing gram negative bacteria and peptide synthesis on cellulose we can investigate the sequence requirements of such peptides. hundreds of peptides are tested for their ability to kill pseudomonas aeruginosa. complete substitutional analyses of different bactenecin variants as well as a semirandom peptide library with about 2000 members were measured. the complete substitutional analysis will give us information about the importance of each single position whereas the peptide library will give us broader information which composition of amino acids results in an active antimicrobial peptide. the data will be analyzed using the quantitative structureactivity relationship approach (qsar) to identify sequence patterns that discriminate between superior activity cf. equivalently active and inactive. this will give us mechanistic cues for a better understanding of the mode of action of the short antimicrobial peptides. the results of these measurements and analyses will be discussed in detail. here we propose a simple and rapid fl ow cytometric method to assess internalization of the peptides in bacteria. the method is based on the use of fl uorescently-labeled peptides and of the extracellular quencher trypan blue to discriminate between a cell surface and cytoplasmic localization of the tested molecules. to his aim, we used bodipylabeled peptides showing different modes of action. these included some fragments of bac7, a proline-rich peptide known to penetrate bacterial and eukaryotic cells without membrane damage [1, 2] , and polymyxin b, a peptide antibiotic that binds to lps and to the cell membranes. by using this approach coupled to fl ow cytometric analysis, we showed that the fl uorescence intensity of e. coli and s. typhimurium cells treated with sub-inhibitory bodipy-bac7 concentrations did not decrease despite extensive washing and addition of the quencher trypan blue. in contrast, the fl uorescence of cells treated with bodipy-polymyxin b, as well as that of bacteria treated with a fl uorescein-labeled anti lps antibody, were promptly and almost totally quenched by addition of trypan blue, indicating their accessibility on the bacterial surface. these results confi rm the suitability of this method to rapidly infer the localization of labelled molecules in an accessible or inaccessible compartment of the treated bacterial cells. hp (2-20) is an antimicrobial peptide derived from the n-terminus of helicobacter pylori ribosomal protein l1 (rpl1). in our previous study, several analogues of hp (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) , with amino acid substitutions that increased or decreased net hydrophobicity, were designed and showed that an analogue, a3 designed by substituting gln and asp with trp at positions 17 and 19, respectively, caused increased antibacterial activity in minimal inhibition concentration (mic) and minimal bactericidal concentration (mbc) without having hemolytic activity. the peptide a3 acted also synergistically with known antibiotics including chloramphenicol against bacterial cells. fluorescence activated fl ow cytometry showed that a3-treated cells had higher fl uorescence intensity than untreated cells, similar to that of melittin-treated cells. the peptide a3 showed a strong antimicrobial activity against antibiotic-resistant pseudomonas aeruginosa from otitis media, clinically isolated mrsa and vrsa including biofi lm-forming bacteria in vitro and in vivo. the ototoxicity of a3 was studied in vivo by topical application to the middle ear in guinea pig model. twenty guinea pigs (5 groups, each group n=4) were each injected by transtympanic approach with 20ul of 8ug/ml,16ug/ml,32ug/ml, 128ug/ml of a3, and 0.4% gentamicin sulfate was instillated as a control. auditory brainstem responses (abr) to click were measured between 1st and 7th days after injection. histologic investigation of cochlea was performed by scanning electron microscope and light microscope. the results showed that topical application of a3 to the middle ear is well tolerated without cochlear damage. the present study, therefore, demonstrates that the usefulness of antimicrobial peptides for multi-drug resistant bacteria including as a new ototopical agent for crpa otitis media. structure-activity relationship study of kiss-10: identifi cation of an antagonist of gpr54 in the absence of ccda, ccdb inhibit the cell division and can kill bacteria by a mechanism that involves the dna gyrase. bacterial dna gyrase is unique among the type ii topoisomerase with ability to negatively supercoil dna. the enzyme consists of two subunits, a (gyra) and b (gyrb) and operates as an a 2 b 2 heterotetramer. the mechanism of the inhibition of the gyrase activity by ccdb is still an object of many debates, but is clear that r462 residue of gyra and the c-terminus of ccdb (w99-i101) play a crucial role in the gyrase-ccdb interactions. as an approach for a better understanding of this mechanism as well as for development of new gyrase inhibitors, we have synthesized peptide analogues of the ccdb protein and studied its activity by supercoiling assays and bacterial growth. five fragments (ccdb et1 , ccdb et2 , ccdb et3 , ccdb et4 and ccdb ss1 ) of the natural ccdb were designed and synthesized by spps. for the design, we considered the 13 residue c-terminal -helix (residues e87 to w99), the loop that connects two strands of the wing sheet (residues r40 to l50) and an n-terminal region that includes the fi rst of the fi vestranded antiparallel -sheet. all peptides, except ccdb et4 , showed inhibition of the supercoiling activity of the dna gyrase, especially ccdb et2 with a mic = 15 m. free peptides not showed antimicrobial activity, but when encapsulated in liposome (suv) were able to inhibit the bacterial growth in liquid culture medium. the growth inhibition in vitro for ccdb et2 was about 70% for gram negative bacteria. our fi ndings revealed a novel synthetic inhibitor of dna gyrase and ccdb et2 analogue is a good starting point for the development of a new and specifi c class of antibacterial agents based in the dna gyrase inhibition. acknowledgements: support: fapesp and cnpq synthesis and neuroprotective properties of short peptides consisting solely from glycine and proline residues now appears more and more data on biological activity of short peptides consisting solely from glycine and proline residues. it is suppose, that these peptides, named as "glyprolines" (gps), can be formed from collagen, åñì and related proteins. however an effect of these peptides on cns is not yet understood. the aims of this work were to improve a methodology of gps synthesis and to study cytoprotective properties of some new gps in culture of neuronal cells. gps with a common structure (gp)n, (pg)n, and (pgp)n (n=1-3) were synthesised by consecutive growing of peptide chain and fragment's condensation. synthesised peptides were characterised by hplc, mass-spectrometry, element analysis etc. cytoprotective activity of gps was assessed on an increase of survival of cultivated ðñ12 cells after í 2 î 2 -induced oxidative stress. it was shown, that from all tested peptides only (gp)n and pgp reveal cytoprotective activity. at the concentration 100 m these peptides reduced an amount of damaged cells 1.7-2.0 times in comparison to the control. thus preliminary data received show that some gps demonstrate a strong cytoprotective activity and therefore it is advisably to put them on further study on in vivo models. in preliminary investigations we found that all the peptides inhibited to a high degree the replication of hsv-1 in vero cells. moreover, these compounds did not show any cytotoxic activity against the vero cells. sexual dimorphism of hldf-6 peptide neuroprotective action in alzheimer's models in vivo and vitro we established that hldf-6 peptide, biologically active fragment of human leukemia differentiation factor (hldf), restores ability for learning, and also prevents loss of long-term memory and decrease in the exploratory behavior of wistar rat-males with experimental alzheimer's disease induced by intrahippocampal injection of betaamyloid peptide a (25-35) and ibotenic acid. the hldf-6 peptide was shown to render protective infl uence directly on primary culture of hippocampal and cerebellar neurones, isolated from newborn male brain, under conditions of the beta-amyloid toxicity. protective action of hldf-6 peptide on newborn rat-male neurons is connected with poster abstracts its ability to reduce 5-alpha reductase mrna expression in more than 30 times, blocking testosterone metabolism into dihydrotestosterone (dht) and thus interfering hyper activation of nmda receptors in ca1 areas of hippocampus. the protective action of hldf-6 peptide was investigated also and on female-rats with experimental alzheimer's disease. it was shown, that in contrast to males at which both forms of memory are broken: and long-term and working ones, in the case of females, injections of a (25-35) + ibotenic acid result in infringement of only working memory, at safety of long-term memory. introduction of hldf-6 peptide restored the broken working memory at females as effectively, as at males. however the mechanism of protective action of peptide on primary culture of hippocampal and cerebellar neurones, isolated from newborns female-rat brain, is connected not with the decrease in hyper activation of nmda receptors, but with abad (17 beta-hydroxysteroiddehydrohenase of the 10th type) mrna expression enhancement which is a target of beta-amyloid peptide action and blocking of progesterone conversion into 5 alpha-dihydroprogesteron due to 5-alpha-reductase mrna expression inhibition. in vitro and in vivo studies of p-19, an antimicrobial peptide active against multidrug resistant gram positive cocci we have designed octapeptide based on the sequence of sepecin b, an antibacterial protein of sacrophaga peregrina, effective against gram + bacteria. we systematically incorporated internal hydrophobic residues for a cationic, helical amphipathic structure effective for its high antimicrobial activity. it is also modifi ed c-terminally by a dehydro leucine residue effective for its structural stability. the synthesis of dehydro amino acid was done by solution phase and other amino acids were coupled by solid phase peptide synthesis method. anti-bacterial activity of the peptide was done by standard micro broth dilution technique against clinical isolates of gpc including methicillin resistant s. aureus (mrsa), methicillin sensitive s. aureus (mssa), hlar, group a and group b streptococci cultured from pus, wound and throat swab. all these isolates were known to be responsible for nosocomially acquired infections. s. aureus atcc 25023 was used as quality control strain. invivo effi cacy of this peptide was also tested in a mouse model with epidermal lesions caused by mrsa. the mic obtained for the peptide was 10 g/ml (average) for the tested strains. the peptide showed no hemolytic activity against human red blood cell. in the invivo studies the healing was induced early (after 72 hrs total healing was seen) in the experimental animals. this novel peptide has a potential to evolve as a therapeutic option in infections caused by resistant gpc. , an adhesive protein, is recognized by the activated platelet integrin á ééb 3 through the arg-gly-asp (rgd) sequence resulting to platelet aggregation and thrombus formation. inhibition of this process can be achieved by rgd peptide analogues that bind to á ééb 3 receptor. however, this class of inhibitors upon binding to receptor cause an outside-in signaling which induces a further activation of the platelet. in previous studies we presented cyclic (s,s)-cdc-containing compounds (ic 50~2 ìm) and á ééb derived sequences (y 313 mesradr 320 , á ééb 313-320 ) (ic 50~2 50ìm ) that exhibit a non-rgd-like inhibitory activity [1, 2] . this interesting aspect could be the basis for the design and development of a new class of anti-platelet agents that could overcame the drawback of platelet activation through the outside-in signaling. to this aim we designed, synthesized and tested for their inhibitory potency various á ééb 313-320 hybrid analogues incorporating the (s,s)-cdc-motif. cyclization reduces the allowed conformations, of both the backbone and the side chains, and possibly induces a favourable for the biological activity orientation of the charged side chains. the inhibition assays on the adp induced platelet aggregation revealed that incorporation of the (s,s)-cdc-motif considerably increases the inhibitory activity of the á ééb 313-320 analogue. the unique toxic effect of acrebol, a novel peptide from acremonium exuviarum a novel peptaibol, named acrebol, was isolated and purifi ed from the fungal strain bmb4 found in water-damaged wood-based indoor building material. the strain bmb4 was identifi ed as acremonium exuviarum based on morphology, its sequence, and cycloheximide resistance. ms/ms analysis showed that acrebol is a mixture of two almost identical peptaibols composed of 16-17 amino acid residues with masses 1726 and 1740 da, acephe-iva/val-gln-aib-ile-thr-leu-aib-pro-aib-gln-pro-aib and acephe-iva/val-gln-aib-ile-thr-leu-val-pro-aib-gln-pro-aib, respectively. the c-termini of the peptaibols was seroh however the sequence (mass of 216 da) between b13 and seroh could not be interpreted. both isoforms of acrebol had a strong toxic effect on boar spermatozoa, feline fetus lung cells, murine neuroblastoma, and mouse insulinoma min 62 cells. we found that, unlike other peptaibols, acrebol in toxic concentrations did not increase the ionic and solute permeability of membranes of isolated rat liver mitochondria. acrebol did not disturb the ionic homeostasis and osmotic balance of mitochondria and induced no release of apoptogenic proteins (cytochrome c) from the intermembrane space of mitochondria. acrebol strongly inhibited complex iii of the respiratory chain (ic 50 ~ 110 ng/ml), presumably, the outer quinone-binding center and, similarly to myxothiazol but in contrast to antimycin a, decreased the production of superoxide anion in the outer compartments of mitochondria. in the boar spermatozoa, acrebol, blocking the respiratory chain, caused the atp depletion due to the oligomycin-sensitive reversion of the reaction of atp synthesis, which resulted in the inhibition of progressive movement. acrebol induced necrosis-like death of mouse insulinoma min 62 cells whose energetic metabolism is strongly dependent on oxidative phosphorylation in mitochondria. thus, acrebol is a unique peptaibol with a specifi c pattern of the toxic effect. delta sleep inducing peptide (dsip), its analogues and deltaran®: biological activity and mode of action for the last decade we have been engaged in studies on the endogenous neuromodulator dsip (waggdasge) and a large group of its derivatives in respect of both their physiological activity and mechanism of action. a wide range of evidences confi rmed benefi cial effects of dsip and some active analogues under experimental stress models. dsip has emerged as promising and potentially effective therapeutic agent due to strong and unique adaptive and stress protective activity revealed during the study. dsip related drug deltaran® registered in russia has also showed the signifi cant effi ciency in animal test models and clinic. the peptides of dsip family often do not demonstrate any effects under normal and comfortable conditions or even cause slight prooxidative and stress promoting effects. these properties and established wide profi le of biological activities of dsip complicate the work on dsip mode of action. cellular and subcellular effects of this peptide still remain poorly studied. previously we investigated some biochemical events underlying the stress protective effi ciency of dsip and its derivatives. in continuation of these studies we have attempted to evaluate the putative dsip infl uence on classical cellular processes utilizing stressprotective heat shock proteins (hsps) and apoptosis. effect of dsip on the level of hsp70 expression in human erythroleukemia cell line k562 was detected.. we have found that dsip down regulates the increase of intracellular hsp70 level during incubation of cells in high density cell culture. according to our preliminary data dsip increased hsp70 level in murine t-cell line ctll-2 similar to -and ß-adrenergic receptor agonists. in murine thymocytes dsip increased both apoptosis and hsp70. we propose that registered effects of dsip are mediated through adrenergic receptors. cellular mechanisms of dsip and related peptides action are under way. identifi cation, chemical synthesis, and antimicrobial activity of tbd-1 -the fi rst -defensin isolated from reptiles antimicrobial peptides (amps) and proteins have been discovered in single and multicellular organisms indicating the importance of this peptide class for the innate immune system. amps are active against bacteria, fungi and viruses by affecting either the microbial cytoplasmic membrane, thus increasing its permeability or interacting with specifi c targets. defensins form one of the major subfamilies of amps, among which -and -defensins play a signifi cant role in bridging innate and adaptive immunity in mammals. the cationic -defensins are cysteine-rich and vary in length from 36 to 44 residues. the -stranded structure of -defensins is stabilized by a characteristic arrangement of three conserved disulfi de bonds between cysteines 1-5, 2-4, and 3-6. although -defensins lyse the bacterial membrane at higher concentrations it has been shown that they modulate also the immune system, e.g. being chemotactic for t-cells. we have isolated a novel 40mer -defensin called tbd-1 from leukocytes of the european pond turtle and deduced its complete sequence de-novo by combining different tandem mass spectrometry techniques (maldi, esi; cid and etd) and edman degradation. it was also possible to identify the disulfi de pattern in a tryptic digest by maldi-mass spectrometry. the deduced peptide sequence was afterwards confi rmed by solid phase peptide synthesis. thus two fragments were synthesized by standard fmoc/ t bu chemistry using three orthogonal cysteine protecting groups (trt, acm, tbu) to selectively form the right disulfi de bridges. after native chemical ligation of the two peptide fragments the fi rst two cysteines were oxidized on air followed by iodide oxidation for the second and dmso oxidation for the third disulfi de bridge. in antimicrobial activity assays, tbd-1 synthesized in -conformation was active against gram-positve, gram-negative bacteria, and fungi similar to the native peptide. trichogin ga iv (trga), an antimicrobial peptide of the lipopeptaibol family, continues to reveal peculiar properties since the time of its former identifi cation by rebuffat and coworkers. x-ray diffraction studies showed that trga is folded in a mixed 310/a-helix conformation, while nmr, cd and ir absorption experiments proved that this structure is predominantly populated in solution, although more disordered structures contribute to the conformational landscape of trga. we have recently shown by time-resolved experiments, that an equilibrium between helical conformers and more compact, folded conformers takes place in solution, with interesting transition dynamics in the microsecond time scale. in our conformational studies on trga we realized that the 3d geometry of the peptide chain reproduces the structural environment of the ion coordination site of calcium-binding proteins. this fi nding urged us to investigate the binding properties of trga with respect to ca(ii), gd(iii) and tb(iii), because lanthanide ions have been widely employed in biochemical studies as best substitutes of ca(ii). the binding of ca(ii), gd(iii) and tb(iii) to trga gives rise to a conformational transition, monitored by cd spectra at different ionpeptide molar concentration ratios. at high r values, the cd curves of all the ion-peptide complexes show a positive maximum at ~214nm, typical of type ii turns, suggesting the population of bent structures. the quasi-isodichroic point found for all systems between 198 and 202 nm, indicates that an equilibrium between extended helical and bent conformations actually takes place. fluorescence experiments on the tb(iii)/trga adduct have shown that, upon ion binding, the fl uorescence quantum yield of tb(iii) markedly increases, due to the release of water molecules from the ion coordination inner shell. molecular dynamics calculations on the peptide/ca(ii) adduct were also performed to obtain structural and dynamical information. antibiotic resistant bacterial strains represent a global health problem with a strong social and economic impact. thus, there is an urgent need for the development of antibiotics with novel mechanisms of action. castros group isolated and determined the sequence of the peptide hy-a1 (ifgailplalgalknlik) of skin secretion from the frog hypsiboas albopunctatus which showed antimicrobial activity. the aim of the present work was evaluated 4 analogues to supply information poster abstracts about the relationship structure-biological activity. the peptides were synthesized by spps using the fmoc chemical approach. the biological activities were assayed by measuring growth inhibition of two types of gram-positive bacteria and others two types of gram-negative. the synthesis and purifi cation of peptides by hplc was effi cient and a high purity level (96%) was obtained. the peptide containing trp in position 6 (for fl uorescent studies) replacing leu presented mic values comparable to wild type sequence: 32 um, 32 um, 8 um and 2 um for e. coli, p. aeruginosa, s. aureus and b. subtilis, respectively. two peptides with this modifi cation but containing at the n-terminal region one group acetyl or a residue of asp showed mic values of 128 um for e. coli and p. aeruginosa, although 4 um for gram-positive bacteria. different results were observed when the residue added was lys. in this case, the activity against whole bacteria was sustained or increased. conformational properties were investigated by cd techniques in water, tfe and in zwitterionic micelles (lpc). the cd experiments demonstrated that in water, the peptides have a random structure, but in tfe and lpc solutions they acquired an ordered structure, composed mainly by -helix. however, these data there is no relationship between the structure and activity against bacteria gram-positive. these results showed that the n-terminal region of the peptide hy-a1 develop key roles in its antibacterial action different types of bacteria. 4 pexiganan is an antimicrobial peptide remarkably effective against bacteria causing skin infections, and has been commercially developed as a topical cream to treat infected diabetic foot ulcers. [1] [2] [3] as peptide drug-based therapy often suffers with low drug stability in vivo, suitable delivery systems must be developed. with this purpose in mind, we have designed a pexiganan-chitosan conjugate to combine the exceptional bioadhesion and tissue regenerating abilities of chitosan [4] [5] [6] with the excellent antibiotic properties of pexiganan. we herein wish to report our fi rst results on the successful synthesis, ft-ir and amino acid analysis of a pexiganan-chitosan conjugate prepared by covalent attachment of a cys-containing pexiganan analogue to the chitosan's amino groups, by means of the heterobifunctional cross-linker sulfo-emcs.7. overexpression of e. coli oligopeptidase b confers resistance to the proline-rich antibacterial peptides scocchi, marco; de gobba, cristian; mattiuzzo, maura; gennaro, renato university of trieste, italy the proline-rich antimicrobial peptides (pramps) are a large group of cationic peptides isolated from mammals and insects, which show a spectrum of antibacterial activity limited to gram-negative bacteria and a remarkably low cytotoxicity towards eukaryotic cells. pramps are thought to act in a permeabilization-independent manner, via energydependent internalization into bacteria followed by recognition and inactivation of internal molecular targets. with the aim of investigating their mode of action, we have isolated a number of e. coli clones showing increased resistance to the bovine proline-rich peptide bac7 after transformation with a dna library from bac7-resistant mutants. among the recombinant plasmids responsible for resistance, some of them harboured the gene coding for the oligopeptidase b (opdb), a serine peptidase belonging to the prolyl oligopeptidase family (pop) broadly distributed among unicellular eukaryotes and gram-negative bacteria, which has emerged as an important virulence factor. opdb was cloned and expressed under the control of an inducible promoter and the transformants tested for susceptibility to a panel of antimicrobial peptides, including pramps. olipopeptidase activity of purifi ed opdb was then tested in vitro against the same peptides. the results indicate that the clones overexpressing opdb are more resistant to the pramps and that the degree of resistance correlates with the expression level of opdb. in addition, in vitro incubation of pramps with the purifi ed peptidase, followed by mass spectrometry analysis, showed that it promptly hydrolyzes all the peptides assayed to short, inactive fragments. these results suggest that opdb may contribute to cleavage and inactivation of antimicrobial peptides that are internalized into the target cells and support the notion that opdb is a novel virulence factor. guan, shuwen; huang, lei; li, pengfei; wang, liping; li, wei college of life science, jinlin university, china great progresses have been made in the research on identifying molecules of therapeutical potential for delaying aging. using caenorhabditis elegans, we had tested the effects on stress resistance and life span of treatment with dhhp-6 (deuterohaemin-alahisthrvalglulys), synthetic mimetics of the antioxidant peroxidases, which neutralizes peroxide. to further test the mechanisms of dhhp-6 on life span extension, exogenous protein sod and catalase levels were measured. we show that dhhp-6 is able to elevate in vivo sod and catalase activity levels after two days administration. treatment with exogenous dhhp-6 affected endogenous protein sod and catalase levels and elevated the expression of sod-3::gfp in the head and vulva. on the other hand, dhhp-6 can extend life span of c. elegans lacking sod-3 or ctl-2 gene expression by rnai. this suggested that the antioxidant enzyme in c. elegans may not the only target affected by dhhp-6. du, haidong; yan, yumei; wang, liping; li, wei college of life science, jinlin university, china high concentration of hydrogen peroxide (h2o2) induces nuclear dna fragmentation, lipid peroxidation and has been implicated in many diseases including heart failure, parkinson's disease, and cancer. in a systematic attempt to develop an effective scavenger of h2o2, we have successfully synthesized an artifi cial microperoxidase, deuterohaemin -alahisthrvalglulys (dhhp-6) as core catalytic center to which 6 amino acid peptide was covalently attached. dhhp-6 exhibited potent peroxidase activity, favorable membrane permeability and thermal stability.two experimental models of oxidative injury were established in order to investigate the anti-oxidative effect of dhhp-6: one was induced by hypoxia-reoxygenation in cultured heart-derived h9c2 cells bioactive peptides and another was caused by h2o2 in cultured neonatal rat ventricular myocytes. dhhp-6 could protect cells against oxidative injury by determining mtt cell proliferation assay, ldh leakage and ca2+-atpase activity. furthermore, dhhp-6 repressed the apoptosis gene expression, such as p21, hsp70 and heme oxygenase¢ñ, that proved dhhp-6 had protective effect in cultured neonatal rat ventricular myocytes . taken together, this small microperoxidase exhibited excellent ¡°druggable¡± properties, and could be a promising agent for ros-associated diseases with unmet needs. trichogin ga iv is the most extensively investigated member of the class of lipopeptaibols that are linear peptide antibiotics of fungal origin, characterized by the presence of a variable, but remarkable, number of aib residues, a fatty acyl group at the n-terminus, and a 1,2-amino alcohol at the c-terminus. several analogues of trichogin ga iv with amino acid substitutions or deletions were designed which allowed determination of the minimal inhibition concentration against gram-positive and gram-negative bacteria and various pathogenic fungal cells. the natural peptide exhibits a specifi c activity against s. aureus and only a marginal hemolytic effect. interestingly, trichogin ga iv is active also against several methicillinresistant s. aureus strains. studies on synthetic analogues demonstrated that substitution of the c-terminal leucinol by leu-ome, or substitution of one aib residues by the epr label toac do not perturb signifi cantly the biological activity of the peptide. on the other hand, removal of 3 or 7 n-terminal residues eliminated any antibacterial activity. finally, studies of proteolytic degradation on trichogin ga iv and analogues where the 3 aib residues are replaced by leu demonstrated that the presence of several non-coded aib residues endows the natural peptaibol with remarkable resistance to proteolysis. the present results indicate that trichogin ga iv is a promising lead compound for the development of new, selective and protease-resistant, antibacterial drugs. the siderophore microcin family: from the genetic systems to the antimicrobial peptides vassiliadis, gaëlle; peduzzi, jean; rebuffat, sylvie muséum national d'histoire naturelle-cnrs, france microcins are low molecular weight antimicrobial peptides secreted by enterobacteria and involved in microbial competitions within the intestinal tract. they are synthesized by the ribosomal pathway. we have isolated the fi rst siderophore peptide (1), a post-translationally modifi ed form of the chromosomally encoded microcin e492 (mcce492) from klebsiella pneumoniae, having a potent bactericidal activity mainly directed against escherichia coli. the post-translational modifi cation consists of a glycosylated catechol-type siderophore linked to the c-terminus. we have recently identifi ed the genes responsible for the acquisition of this modifi cation and proposed a model for its biosynthesis (2) . in order to identify novel siderophore peptides, we analyzed the genetic systems of several microcinogenic strains. based on the genetic organization of the microcin gene clusters, three microcins which had preparation of a close mimic of the n-terminal part of the c5a receptor (c5ar) by selective introduction of sulfated tyrosine residues (2) and chips in complex with our sulfated c5ar-model by multi-dimensional nmr techniques. based on these interaction data and the solution structure of the complex, chips-based c5ar inhibitors for use in anti-infl ammatory therapy might be designed. insulin-like peptide 5 (insl5) was fi rst identifi ed through a search of the expressed sequence tags (est) databases. primary sequence analysis showed it to be a prepropeptide that is predicted to be processed in vivo to yield a two-chain sequence (a and b) containing the insulin-like disulfi de crosslinks. the high affi nity interaction between insl5 and the receptor rxfp4 (gpcr142) coupled with their apparent co-evolution and partially overlapping tissue expression patterns strongly suggest that insl5 is an endogenous ligand for rxfp4. given that the primary function of insl5/rxfp4 pair remains unknown, an effective means of producing suffi cient quantities of this peptide and its analogues is needed in order to systematically investigate its structural and biological properties. a combination of solid phase peptide synthesis methods together with regioselective disulfi de bond formation were used to obtain insl5. both chains were identifi ed as being ¡ §diffi cult sequences¡¨ and were unusually resistant to standard synthesis protocols including those mediated by microwaves. the a-chain was also prone to signifi cant aspartimide formation. the b-chain, in particular, required the use of the strong tertiary amidine, dbu, for more effective nƒñ-deprotection during its assembly. following chain combination and sequential disulfi de bond formation, the resulting synthetic insl5 was obtained in good overall yield and shown to possess a similar secondary structure to human relaxin-3 (h3 relaxin). the peptide was able to inhibit camp activity in sk-n-mc cells expressing the human rxfp4 receptor with a similar activity to h3 relaxin. in contrast, it had no activity on the human rxfr3 receptor. novel cyclic bacteriocin-like peptides from strains of anabaena (cyanobacteria) leikoski (3) and microcystis aeruginosa (4) . the genome sequence of fi lamentous and diazotrophic cyanobacterium anabaena 90 revealed a putative gene cluster (acy) encoding a bacteriocin-like peptide. one of the acy genes encoded a prepeptide, which showed n-terminal homology to the known cyanobacterial prepeptides but the mature peptide product in anabaena 90 could not be predicted. we sequenced prepeptide genes from several anabaena strains to fi nd a prepeptide containing either cysteine or methionine, since sulphur containing amino acids enable the detection of the mature peptide product through 34s-labelling and lc-ms. the nucleotide sequence of the prepeptide genes revealed enormous variety in closely related anabaena strains. one strain, anabaena 844b contained a methionine in the prepeptide, and a cyclic heptapeptide product corresponding to the precursor was discovered in this strain. since the cleavage sites were found to be conserved in all anabaena strains the products could be predicted from the amino acid sequence and identifi ed by lc-ms. in addition, the biosynthesis of the decapeptide anacyclin in anabaena 90 was verifi ed by heterologous expression of the acy genes in e. coli and the structure of the peptide was confi rmed with a synthetic reference peptide. altogether, new cyclic peptides were found in 19 strains of anabaena with very little sequence conservation. cyanobacteria seem to be versatile producers of peptides using both ribosomal and non-ribosomal biosynthetic pathways. it is well established that n-terminal fl ank of corticotropin-releasing factor molecule is crucial for its biological activity. little, however, is known about in vivo effects of crf-derived peptides. this study was aimed to investigate possible actions of a tripeptide crf fragment 4-6 pro-pro-ile (ppi). tripeptide ppi was found to exert effects similar to fullsize crf molecule after central administration. the tripeptide induces behavioral activation in home cage, but inhibits behavioral activity under stressful conditions. ppi increases blood pressure and heart rate, blood glucose level and body temperature, decreases pain sensitivity, increases eeg amplitude and large doses of ppi induce seizures. ppi inhibits sexual motivation and performance in mating tests in males. crf antagonist -helical crf 9-41 abolishes ppi infl uence on circulatory system, glucose metabolism, thermoregulation and pain sensitivity. adrenalectomy does not interfere with hyperglycemic and hyperthermic actions of ppi, while pancreatectomy prevents hyperglycemic effect. nonselective beta-adrenergic blocker obsidan prevents hyperglycemic and decreases hyperthermic effects of the tripeptide and ganglionic blocker hexamethonium abolishes both effects. taken together effects of ppi correspond to stress-reactions, involving corticotropin-releasing factor and evidence that ppi is either 1) a part of a crf molecule active site, or 2) physiologically active crf derivative, realizing its effects through activation of crf receptors, or 3) an independent regulator, affecting crf-ergic neurons. acknowledgements: the work was supported by rfbr grant 06-04-49-620-à huggins, kelley; andersen, niels university, united states amyloid fi bril formation is associated with at least 17 human diseases; among these, the human-amylin(ham)-derived deposits in type ii diabetes were the discovery system and ham aggregation is one of the more thoroughly studied systems. to date, inhibitors of beta-aggregate and fi bril formation have been polyphenols, mutants of ham, or short peptide related to the ham(22-29) sequence, nfgailss. we now report that stable beta-hairpin scaffolds displaying trp and tyr residues are effective inhibitors, delaying the onset of both the cd changes associated with beta structure formation and the nucleation time and net enhancement of the fl uorescence observed with added thiofl avin-t (tht). under our test conditions (8 microm ham, 2% hfip in 5mm phosphate buffer, ph 7), ham begins to display an increase in beta structure by cd at 40 min with a constant maximal value from 85 -220 min. tht fl uorescence also indicates a circa 50 min onset time with a rapid (<20 min) rise to the full response. inhibition (delayed onset and reduction in the maximal fl uorescence enhancement) has been observed with a number of hairpins; those with two trp residues on a single face of the hairpin are more potent. of these, our best inhibitor to date, kkltvwipgkwitvsa (p = d-pro), increases the onset time more than 2-fold at equimolar concentrations. at 4 molar equiv., the onset time is greater than 320 min and tht fl uorescence levels out at < 40% of the control value. in analogy to the report by prof. ghosh (jacs 2006, p. 14456) that a beta-sheet protein with added tyr and trp residues inhibits fi bril formation by the alzheimer-related abeta peptide; we expect that designed beta-hairpin scaffolds will be more generally applicable and will afford new insights into the recognition phenomena of amyloidogenesis. baabur, hemda; ashkenasy, gonen ben gurion university, israel the main advantage in utilizing proteins for the development of sensors and receptors, over the more often used approach that exploits small molecules, is the ability to manipulate large recognition surfaces, which expose toward the bulk solution number of different functional groups. we search for stable artifi cial proteins that can be utilized as 'universal' recognition entities. to that end, modular chemical syntheses are exploited for the preparation of repeats-proteins. leucine rich repeat proteins (lrrs) are 20-29 residue sequence motifs present in several proteins with diverse functions. internalin b (inlb), a surface lrr protein of the human pathogen listeria monocytogenes, promotes invasion into various host cell types by inducing phagocytosis of the entire bacterium. consensus design uses statistical analysis of sequence alignments of families of homologous proteins for protein engineering. we show here that using consensus design, series of protein mutants that differ in the recognition surfaces are synthesized and will be probed for their ability to bind different natural and non-natural ligands. comparative structural studies of potent neuroprotective peptides of the humanin family although the rescue activity of hn peptides has been linked to a number of signaling pathways and receptors, their mechanism of action remains unknown. in this work cd and nmr data on the above peptides are presented and compared in an effort to defi ne structural characteristics related to their function. evaluation of our fi ndings in combination with existing structure-function relationship data for this class of peptides, brings forth fl exibility as an important structural feature that may facilitate interactions with functional counterparts of the neuroprotection pathway. the ability to adopt a partial helical conformation in the presence of low concentrations of tfe is another common structural feature that may be defi ning the interactions of these peptides in the environment of cell membranes. desleu-aga(c8r)hng17 and cl display a more complex behavior shifting from -helical to -sheet conformations depending on ph, peptide concentration, and % of tfe present in solution. this fact may be related to their high potency since in hn literature evidence for self-association ability has been linked with neuroprotection. our cd and nmr experimental data combined with theoretical modeling will hopefully provide important clues for the elucidation of the mechanism of action of the hn family of peptide the process of molecular recycling describes a dynamic mechanism by which individual amyloid fi brils are continuously dissolving and reforming (1) . the present work aims at the study of the dynamic properties of the -amyloid, a (1-42), amyloid fi brils. since the formation of a aggregates has been suggested as a key process in the pathology of alzheimer's disease, the study of the dynamic nature of a ( the coiled coil structure is widely distributed in natural proteins, such as transcription factors, receptor proteins and enzymes. this motif has been recently used by chemists for the design of functional synthetic proteins. specifi c coiled coil sequences can be utilized as templates for self replication processes (1, 2) . the stability and the activity of these replicators depend on the characteristics of the amino acid residues in the recognition interface. it has been suggested that it is possible to control protein structure and the replication process by external triggers such as light, and that the light can also be used to monitor the conformational changes and/or to follow the protein functionality(3). we show here our ability to control the folding stability of coiled coil peptides and their reversible or irreversible self replication effi ciency by light, and we demonstrate the possibility to exploit fret couples to facilitate in-situ monitoring of the folding and reactivity. we use 'caged' mutants with a photo-switchable molecule in the recognition interface of the peptidewhich disrupts its coiled coil structure -as inactive species. deprotection of the caged proteins is used as a mechanism to restore the self replication process. the ligation is followed by monitoring the changes in fl uorescence of either the donor or acceptor of the fret couple. we will describe synthesis and structural characterization of caged and cage-free peptides and measurements that show, as expected, that the cage free peptides are more stable as coiled coils and better catalyst for their own formation than the caged analogs. moreover, we discuss the reversible replication process and how we can shift and control its equilibrium staes. , a member of the rfamide peptide family, has been reported to have pronociceptive and analgesic activities as well as pro-and anti-opioid effects. these contradictory effects seem to result from npff capacity to bind two different gprotein coupled receptors (npff1 and npff2). although the exact role of npff1 and npff2 receptor is still unclear, this complex system appears to play an important role in pain regulation. structure-activity relationship (sar) studies using analogs of npff and derived peptides have shown that the c-terminal part of the molecule (i.e. pqrf-nh 2 ) is crucial for affi nity and activity. however, some of the essential features for ligand recognition by npff receptors are still missing to design highly selective molecules. in order to provide further insight into ligand recognition, we have investigated the solution conformation of npff in different media using circular dichroism and nmr spectroscopy. our results showed that (1) in the presence of methanol or trifl uoroethanol, turn-like elements are present in npff structure, and (2) are not yet established, although genetic and animal models have shown a causal role of amyloid -peptide (a ) in ad. however, recent debate has focused on whether amyloid fi brils or soluble oligomers of a are the main neurotoxic species contributing to neurodegeneration and dementia. one approach for preventing aggregation would be the conversion of the peptide conformation. prior investigations indicate that polymeric nanoparticles offer strong advantages in modulating the secondary structure of the peptide (1, 2) . these results have encouraged us to extend our work on polymeric nanostructures for conformational transformations contributing to the development of new therapies for these diseases. complexes of polyampholytes and dodecanoic or perfl uorododecanoic acid were prepared (2) resulting in nanoparticles with hydrodynamic diameters ranging from 3 to 5 nm. the fl uorinated nanoparticles induced -helix rich structures in a peptide, whereas their hydrogenated analogues were less effi cient leading in most cases to aggregation orsheet formation, as determined by circular dichroism spectroscopy. the degree of fl uorination, the hydrophilic balance and the charge density of the fl uoropolymers, as well as the size of the nanoparticles in aqueous solution, are decisive for the interactions. the impact of these structures on the a -induced toxicity in cultured neurons was studied. we report that the fl uorinated nanoparticles increased the a -mediated mtt (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazoliumbromide) reduction, which indicates a higher cell viability. the anti-apoptotic effect of the complexes was evaluated by determining the activation of caspase-3. this assay also confi rms the decrease of a -mediated cytotoxicity in the presence of fl uorinated biocompatible complexes. alternative strategy to investigate enzymatic activity using peptides containing toac spin probe (1), the present work extended the investigation of the specifi city of angiotensin i-converting enzyme (ace, ec 3.4.15.1), a dipeptidyl carboxypeptidase which cleaves the c-terminal dipeptide from angiotensin i (angi, drvyihpfhl) to produce the potent vasoconstrictor angiotensin ii peptide (angii). the use of paramagnetically labeled ai analogues attaching the toac (2,2,6,6tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) probe (2) advantageous allows the monitoring of their conformation and its enzymatic hydrolysis specifi city through the epr and fl uorescent methods, the latter due to the quenching effect induced by the stable free radical toac probe upon the tyr4 residue of angi. the study of toacattaching ai analogues at positions 0, 1, 3, 5, 8, 9 and 10 indicated that the fi rst four analogues are substrates for ace in the decreasing order 0 ~ 1 > 3 > 5, thus confi rming that greater the proximity of the unnatural probe to the cleavage site (8-9) of the sequence, the smaller are the substrate specifi city of analogues. otherwise the quenching effect of tyr4 fl uorescence by toac decreased with increasing distance between both residues, thus suggesting overall fl exible structures for most of analogues. these fi ndings were also corroborated in a combined cd and epr studies although some differences were detected among the derivatives either in the variation of ph or amount of the structuring tfe studies. finally, differences between epr spectral lineshapes of some labeled analogues and their corresponding cleavage products seems to allow a real time monitoring of the enzymatic reaction. the effect of the so called " -sheet breakers" (bsbs) on a 1-42 aggregates inhibition of aggregation of amyloid -peptide seems to be a critical step in the therapeutic approach to prevent amyloidosis in alzheimer's disease. the therm " -sheet breaker" (bsb) had been introduced by soto c. et. al. as a 5-residue peptide in 1998, that inhibits amyloidprotein fi brillogenesis. we synthetized several derivatives of the "soto peptide" as well as a big number of peptidomimetics. their mechanism of action has been studied by nmr spectroscopy, cd and transmission electron microscopy (tem). all of these methods showed that the soto peptide lpffd and the similar peptides can not prevent a aggregation. these compounds bind to the surface of a aggregates and decrease bioactive peptides the specifi c surface area of a which accessible for the cell-membranebound receptors, can lead to a decreased toxicity. the -sheet breaking effect does not work up to an a peptide-small peptide ratio of 1:10. as a consequence, these compounds are not -sheet breakers, only they modify the surface of a fi brils and rather speed up the formation of -sheet structure. designing trehalose-conjugated peptides for the inhibition of alzheimer's a oligomerization and neurotoxicity. interactions between cationic or anionic porphyrins and polypeptide templates with charges that are opposite to those of porphyrins have been extensively investigated for their possible applications in biomedicine and photodynamic therapy (pdt). the infl uence of porphyrin on the conformation of the peptide part of the complexes is studied in this work. non-covalent interactions of cationic tripeptide l-lysyl-l-alanyl-lalanine (kaa) with anionic meso-tetrakis(4-sulfonatophenyl)porph yrin (tpps) and its copper(ii) (without axial ligand), iron(iii) (one axial ligand), and manganese(iii) (two axial ligands) derivatives were investigated in aqueous solutions by vibrational (vcd) and electronic circular dichroism (ecd) spectroscopies. although both the cd spectroscopies are sensitive to conformation, particularly vcd is extremely sensitive to subtle conformational changes. the vcd spectra of pure kaa in the amide i' (c=o stretch vibration) region showed the spectral patterns typical for left-handed polyproline ii helical conformation (ppii). interaction of kaa with non-metallated tpps was accompanied by change of the amide i' vcd patterns -loss of vcd intensity and arising of a new negative band at ~1630 cm -1 -that was interpreted as a partial change of ppii into less compact conformation as extended helix or -sheet segment. in case of cu(ii)-and fe(iii)-tpps, the loss of the amide i' vcd intensity of kaa was observed only. for mn(iii)-tpps having two axial ligands, the vcd pattern was unchanged compared to the pure tripeptide indicating that this derivative is not able to change the conformation of kaa in peptide-porphyrin complex. suitable models of biologically important small-sized proteins -histons -located in the chromosomes of eucariotic cells. they are able to interact with negatively charged functional groups of many biologically important molecules as dna, polyuronic acids, and porphyrins and thus infl uence a broad range of biological functions. in this work, the solution conformation of synthetic oligotripeptides (llysyl-l-alanyl-l-alanine) n [n = 1, 2, 3] was investigated at the different temperatures and ph using combination of vibrational (vcd) and electronic circular dichroism (ecd) spectroscopies. vcd spectra of all the oligopeptides measured at room temperature show a negative couplet (positive to lower frequency) in amide i' region. this spectral pattern is indicative of left-handed polyproline ii helical conformation (ppii), which is stable at wide range of ph. the temperature dependence of the vcd spectra indicates that ppii conformation of all the oligopeptides remains stable even at 90°c, independently on length of oligopeptide chain. these results were confi rmed by temperature dependent ecd experiments, where the characteristic negative and positive bands at about 195 and 220 nm, respectively, were observed. taken together, vcd and ecd results suggest that ppii conformation of (lys-ala-ala) n sequence is the dominant conformation within the range of temperature from 20 to 90°c. all the results including spectral data analysis are discussed in detail. acknowledgment: the work was supported by the research grant 203/06/p371 from the grant agency of the czech republic. we thank miroslava žertová, msc (iocb prague) for the oligopeptide synthesis. -peptides are probably the most thoroughly investigated peptidomimetic oligomers. to extend the fi eld of -peptides towards the construction of possible new secondary structures, the replacement of the c and c atoms of the -amino acid with heteroatoms could be an attractive modifi cation, for example c -atom of -peptides by an nr moiety, leading to hydrazine peptides. in the literature, there are only a few studies about hydrazine peptides [1] [2] [3] , and hydrazine peptides with cyclic side-chain have not been studied yet. in order to determine the secondary structure preference of h-[(cis or trans)-acpc-2s-aza-acpc] 3 -nh 2 peptides (figure 1 ), their potential energy hypersurface were probed at the ab initio b3lyp/6-311g** level. the cis (1r,2s) formed 10/12-helices, while the opposite acpc enantiomer resulted 6 strand. the trans (1s,2s) formed 8-strand, while the opposite acpc enantiomer resulted 12-helix. the hybrid-peptides in question were synthetized on solid support, and their high-resolution 3d assignments were made by using nmr, which was supported by ecd, vcd, qls and tem methods. the hydrazino modifi cation resulted in better water solubility than that of the acpc homooligomers. this result is very important concerning the further biological applicability. mazur, adam; katarzy ska, joanna; zabrocki, janusz; jankowski, stefan technical university of lodz, poland cyclolinopeptide a, (cla, 1), a cyclic nonapeptide cyclo(-pro 1 -pro 2 -phe 3 -phe 4 -leu 5 -ile 6 -leu 8 -val 9 -), possesses strong immunossupresive and antimalarial activity as well as the ability to inhibit cholate uptake into hepatocytes [1, 2] . the mechanism of cyclolinopeptide a activity is similar to that of cyclosporine a. the object of our investigations are six cla analogues with pro 1 and pro 2 residues replaced by 2 -isoproline or 3 -homoproline. the immunosuppressive activity of these new cla analogues was evaluated on the basis of the mouse splenocyte proliferation assay 3.. nmr spectra analysis (chemical shifts assignment and structural constraints) was based on 1d and 2d nmr experiments at 700 mhz. long (300 ns) molecular dynamics calculations were carried out using gromacs program (gromos g53a6 force fi eld) for isomers of at least 30% content. in woolley, andrew university of toronto, canada thiol-reactive azobenzene-based cross-linkers provide a straightforward means for introducing a photo-isomerizable unit into a peptide or protein structure. we have shown that the conformational response of the peptide in such systems is critically dependent on the manner of attachment of the cross-linker as well as the cross-linker structure. cross-linkers can be introduced in which trans-to-cis photoisomerization leads of formation of helical structure, or conversely to loss of helical structure. these effects can be understood in a semi-quantitative manner by calculating the degree of mismatch between the steric requirements of the linker and the conformational ensemble of the peptide. kinetic studies reveal that in general photoisomerization of the linker is fast (several ps) whereas subsequent peptide folding or unfolding occurs over hundreds of microseconds. such cross-linkers are thus potentially useful phototriggers for studying protein folding processes, as well as for controlling the equilibrium stability of different conformational states. applications to photo-control of bioactive peptides and proteins will be discussed. short corticotropin-like peptides with stress-protective activity it was synthesized 87 linear è 9 cyclic corticotropin-like peptides, which contained from 13 to 2 amino acid residues. the ability of each of the synthesized peptides to inhibit the specifi c binding of tritium-labelled corticotropin (11-24) to the adrenal cortex membranes of rat in vitro was investigated. on the base of the obtained results 12 peptides with the highest inhibitory activity were selected for in vivo tests. the infl uence of the selected peptides on the level of 11-oxicorticosretoids and catecholamines in the adrenals and blood of rats in the experiments on acute hemorrhage and hypobaric hypoxia, cold and heat shock and low doses of g-radiation were studied. it was established that intravenous injection of 5 short peptides at the dose of 1 g/kg could correct disturbance of 11-oxycorticosretoids-catecholamines system in the adrenals and plasma of rats that were subjected to hemorrhagic shock and hypoxia. the rest of investigated peptides possessed lower stress-protective activity. it was also shown that under cold or heat shock, thrice-repeated intranasal injection of these peptides into rats at the dose of 10-20 g/animal abolished temperature induced changes in the level of 11-oxycorticosretoids and catecholamines in the adrenals as well as the content of free histamine and the activity of diaminoxydase in the myocardium. it has been shown that the stress-protective activity of corticotropin-like peptides is mediated by the corticotropin receptor in cortex of the adrenals. a study of immunobiological activity of the wrnwdyyk octapeptide the evaluation of the effect of japanese herbal in many cases, japanese herbal (kampo) medicines have been used in the empirical treatment of chronic hypofunction. however, the kampo medicines consist of several herbs, whose pharmacological mechanism is not clear. in western medical science, medical doctors usually treat patients according to disease diagnosis. in eastern medical science, treatment is based on diagnostics called gsho h, which is a unique concept in kampo medicines and quite different from diagnosis. the concept of gsho h is diffi cult for non-professionals to understand, furthermore, there are responders and non-responders when nonprofessionals prescribe kampo medicines. the concept of gsho h focuses on individuals, not diseases, therefore, it is diffi cult to gain a given effect for everyone by general clinical trials. in this study, we investigated the effects of prokinetic kampo medicines on plasma levels of gut-regulatory peptides (somatostatin, motilin and gastrin) and compared with that of dopamine receptor antagonists, the western prokinetics. the fi ve kampo medicines, including pinelliae tuber and zingiberis rhizoma, three dopamine receptor antagonists or placebo was orally administered. venous blood samples were taken before and till 240 min after administration. plasma peptide levels were measured using a sensitive enzyme immunoassay. the dopamine receptor antagonists and kampo medicines caused signifi cant increase of plasma gut-regulatory peptide levels compared with placebo group. in recent years, chronic hypofunction without mechanical problem, such as non-erosive refl ux disease and functional dyspepsia, is diffi cult to cure using western medicines. the preliminary study indicated the plasma somatostatin levels of patients with any symptoms are high and plasma motilin levels of them are low compared with those of healthy subjects. to evaluate kampo medicines using bioactive peptides as biomarkers, it may be possible to cure diseases that is diffi cult to treat by western medicines. bapst, jean-philippe; calame, martine; eberle, alex n.; tanner, heidi university hospital basel, switzerland radiolabeled -msh analogs are potential candidates for melanocortin-1 receptor (mc1-r)-mediated melanoma targeting. several short -msh peptides carrying a dota (1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid) metal chelator were designed and evaluated as potential diagnostic (e.g. with 111in, 67,68ga) or therapeutic (e.g. 90y, 67cu) radiopharmaceuticals. the analogs tested to date showed high affi nity for the mc1-r in vitro, excellent internalization into the tumor cells, as well as a good incorporation in tumor xenografts and a low uptake in normal tissues in vivo, except the kidneys where considerable uptake is observed. our current studies attempt to infl uence the pharmacokinetic parameters in order to address specifi c uptake (i.e. by melanoma) versus non-specifi c uptake (i.e. by the kidneys). as glycosylation had been shown to improve tumor-to-kidney ratios in the case of somatostatin and to reduce peptide re-uptake by the tubular system of the kidneys in general, we investigated glycosylated analogs of [nle4, asp5, d-phe7]--msh4-11 (napamide). carbohydrate moieties such as glucose, galactose and maltotriose were introduced at various positions on the msh peptide carrying the metal chelator dota for labeling with 111in. the peptides were evaluated in vitro in both murine and human cell lines for mc1-r binding and cellular localization, and in vivo in b16f1 tumor-bearing mice for tissue distribution. the tumor-to-kidney ratio for gal-napamide (4-48 h aucs) was superior to any of the previously published msh peptides. other glycopeptides showed very good binding affi nities but lower selectivity in vivo. in additional, a series of non-glycosylated dimeric derivatives, bearing one or two moieties of the chelator complex, were developed which displayed excellent receptor affi nity but tended to result in higher kidney accumulation. by contrast, at least one negatively charged dota-napamide showed excellent tumor-to-kidney ratios. there is a critical lack of validated early biomarkers for most conditions and diseases. early diagnosis does enable treatment of less severe disease states, the use of less invasive techniques and could potentially reduce the costs of healthcare systems. however, it is most likely that peptides in tissue or blood -or their modifi cations -can be specifi cally associated with different disease states. the discovery and verifi cation/ validation of such disease markers are two distinct workfl ows. during the discovery phase, a relatively small number of samples with a high number of potential biomarker candidates are screened. the highthroughput provided by the itraq™ reagent strategy allows for simultaneous analysis of such samples. once biomarker candidates have been identifi ed with initial statistical signifi cance, these have to be validated. this validation workfl ow involves analyzing a large number of samples with a relatively small number of candidates to establish the biological signifi cance of the biomarker candidates. rather than switching to immunological techniques for this validation step, we suggest a mass spectrometry based approach. this orthogonal strategy is a novel targeted, high throughput quantitative multiplexed multiple reaction monitoring (mrm) approach. the approach relies on assay development using a combination of mrms to target specifi c peptides identifi ed in discovery, followed by ms/ms to confi rm that the quantitative mrm signal results from the target peptide. in addition to being a quantitative method, this validation approach is extremely specifi c and sensitive. several published examples of the application of this mrm-based approach utilizing the actual or hypothetical physical properties of peptides in complex mixtures will be presented. infl uence of the charge on the in vivo behavior of radiolabeled bombesin analogues prostate and breast cancers are the second leading cause of cancer death in men and women, respectively. the side effects related to the treatments that are available today have a great infl uence on the patients' quality of life. therefore, the development of new diagnostic and therapeutic strategies may have an important impact in the outcome of these cancers. gastrin-releasing peptide (grp) receptors are present in high quantities in a variety of cancers, prostate and breast tumors among them. targeting of over-expressed grp receptors with radiolabeled bombesin (bbs) analogues would offer an interesting tool for tumor imaging and therapy, depending on the radionuclide used. some analogues of bbs, based on the fragment 7-14, were functionalized with the (n his)chelator for labeling with the 99m tc-and 188 re-tricarbonyl-core. despite an increased metabolic stability, these analogues showed very low tumor uptake. additional insertion of a ala-ala linker led to increased tumor uptake but still unfavorable in vivo properties. in order to further improve the biodistribution, novel polar linkers with different charge were introduced in the molecule. a positive charge resulted in increased kidney uptake, whereas one single negative charge led to a signifi cant increase in the tumor uptake and also signifi cantly higher tumor-totissue ratios. co-injection with natural bbs importantly inhibited the uptake in the tumors and receptor-expressing tissues, which confi rmed the specifi city of the in vivo uptake. moreover, imaging of the tumor xenografts by spect/ct was also much clearer with the analogues bearing one negative charge. additional negative charges, however, resulted in a loss of binding affi nity and internalization, and unfavorable biodistribution. in conclusion, bbs analogues with one single charge in the linker hold a greater potential for imaging and therapy of grp receptor-overexpressing tumors peptide-macrolide conjugates as novel instruments for protein biosynthesis study bacterial ribosomes are targets of numerous therapeutic agents. macrolide (ml) antibiotics prevent nascent polypeptide growth by blocking the ribosomal exit tunnel (rt). availability of high-resolution structures of complexes of ribosomal 50s subunits with ml enabled developing novel ideas concerning their inhibitory activity of translation. formerly we obtained peptide derivatives of ml in which the peptide part modelled a growing chain, while an antibiotic moiety served as an "anchor" for positioning the peptide in the rt. the goal of this study was to synthesise tryptophan-containing peptide derivatives of 5-omycaminosyltylonolide (omt) to monitor location of the specifi c trp binding site that modulate the activity of the peptidyl transferase centre of the ribosome. the peculiarity of compounds designed in this study lies in that of a trp containing peptide fragment, connected to the primary hydroxyl group in position c23 of omt, is oriented in the rt towards the hypothetical trp binding site. the following peptides were used: boc-l-trp-gly-oh, boc-l-trp-ala-oh, boc-l-trp-abu-oh, boc-l-trp-ape-oh. variable distance between trp residue and macrolide ring was achieved by introduction of glycine, -alanine, -aminobutyric acid and -aminovaleric acid as c-terminal amino acids of the dipeptides. the peptides were obtained from boc-l-trp-oh and ethyl esters of the corresponding amino acids by condensation with bop followed by saponifi cation of the peptide esters. the reactions between peptides and omt were produced using dcc and dmap as condensation agents. as a result peptide-macrolide derivatives were obtained. all compounds were purifi ed by column chromatography on silica gel and characterized by hplc, mass-spectrometry and nmr. it was shown by means of chemical probing that trp containing omt derivatives specifi cally bound to rt of the ribosome. moreover these compounds displayed an antibiotic activity in testes with several staphylococcus strains. zwanziger, denise 1 ; neundorf, ines 1 ; schatzschneider, ulrich 2 ; beck-sickinger, annette g. 1 1 university of leipzig, germany; 2 university of bochum, germany npy is a 36 amino acid peptide amide that belongs to the pancreatic polypeptide-hormone family (1) . it is the most abundant neuropeptide in the brain and triggers a number of central activities, such as regulation of food intake as well as stress induced reactions [2] [3] [4] . npy forms a selective interaction with at least three receptors, which are called y1, y2 and y5. in the past it was shown that y1-receptors are overexpressed in >90% of all breast tumors as well as in 100% of the derived metastases. normal breast tissue expresses y2-receptors, while the neoplastic tissue expresses y1-receptors 5.. as a result, the npy-y1-receptor system can be used for tumor targeting and therapy. in the fi rst step we synthesized the truncated and modifi ed npy analogue [pro30,nle31,bpa32,leu34 ]npy(28-36) (nle-norleucine; bpa-benzoyl-phenylalanine) by solid bioactive peptides phase peptide synthesis using fmoc/tbu strategy. the npy analogue was characterized with respect to in vitro binding affi nity and selectivity at y1-receptor expressing human breast cancer mcf-7 cells and the metabolic stability in human blood plasma, respectively. then we coupled different potential chelators to [pro30,nle31,bpa32,leu34]np y(28-36), which was n-terminally modifi ed by two ß-alanines as spacer between the peptide sequence and the chelator. next, we determined their ability of metal conjugation of diverse metals, as cu2+ and re3+. metal conjugated peptides were characterized by maldi-tof-ms (matrix assisted laser desorption/ionization mass spectrometry), rp-hplc (reversed phase high performance liquid chromatography) and ir-spectroscopy (infrared). after optimization of the metal conjugation fi rst binding affi nity studies were performed. 2 chain is mainly expressed in skeletal muscles and peripheral nerves, and interacts with cell surface receptors such as integrin, dystroglycan, and heparan sulfate proteoglycans. biological functions of the laminin 2 chain n-terminus including integrin binding and heparin/heparan sulfate binding were found previously. here, we focused on the n-terminal region of the mouse laminin 2 chain (position 1-1566) and screened the biologically active sequences using 142 peptides. the synthetic peptides were generally 12 amino acids in length and overlapped with neighboring peptides by 4 amino acids. cell attachment activity of the peptides was evaluated using a peptide-coated plastic plate assay and a peptide-conjugated sepharose bead assay using ht-1080 human fi brosarcoma cells. eleven peptides showed cell attachment activity on plate assay and fi ve peptides showed cell attachment activity on beads assay. previously we screened active sites on the laminin 1 chain and identifi ed several active sequences. when we compared with active sequences of the 1 and 2 chains, a2-31 (yydetvasrnlsln) and a2-112 (ggklkyaiyfea) are unique active peptides only within laminin 2 chain. these results suggest that the biological activity of a2-31 and a2-112 are 2 chain specifi c and are useful for investigating the 2 chain specifi c functions of laminin 2 chain. novel peptide biopharmaceuticals by using phage display technology štrukelj, borut; lunder, mojca; bratkoviè, tomaž university of ljubljana, faculty of pharmacy, slovenia libraries of random peptides displayed on the surface of bacteria, mammal cells or bacteriophages are an essential tool that enables systematic study of target molecule interactions. the identifi cation of ligands from large biological libraries by phage display has now been used for almost 15 years. in a last few years several improvements have led to numerous high affi nity peptide ligands that express various biological activities. phage-displayed peptide libraries have been used successfully to isolate peptide ligands directed to a functional site for which the natural ligand is or is not a protein or peptide. by using a modifi ed, in-house developed selection proctocol, we successfully selected several phage clones with high affi nity to pancreatic lipase, ghrelin and cysteine protease cathepsin k as target proteins. based on their deduced animoacid sequences, twenty heptapeptides with the highest affi nity to target proteins were synthesized and characterized for their capacity to inhibit enzyme or acceptor function. the most succesful peptide candidates inhibited pancreatic liapase with the apparent inhibition constant of 16 um, and cathepsin k with the apparent inhibition constant of 0,10 um. a set of 22 peptidomymetic compounds were sinthesyzed based on the aminoacid sequence of selected peptides and their inhibitory activity was determined. the most potent candidates were selected for further development of peptide biopharmaceuticals aganist obesity (inhibitors of pancreatic lipase and ghrelin) and in the prevention of osteoporosis (cathepsin k inhibitors). novel approach to non-specifi c elution in phage display using ultrasound phage display is used to select and optimize peptides or protein domains binding to virtually any protein and sometimes even non-protein targets. several rounds of screening are performed, until the increase in phage output, or binding assays performed with phage pools, indicate that the population of binding phages has been adequately enriched. in cases where ligands of particular target are not known or available, targetbound virions are released by non-specifi c elution for example with acidic buffer, or competitively with free target molecule, or by addition of bacterial host directly to the target-bound phages. we used streptavidin, immobilized by adsorption, as a model target protein for affi nity selection of peptides from phage display library. the effi ciencies of a number of well-known typically used non-specifi c elution strategies in selecting and retrieving a phage clone displaying the tripeptide biotin mimetic (hpq) from a streptavidin coated surface were compared. all the commonly used elution strategies have failed to elute and select high affi nity hpq-bearing phage clones bound to streptavidin. the failure was shown to be due to the inability of eluants to break the interaction of high affi nity clones with the target, which is thus likely to be the cause for failed selection with other targets also. to surmount this, we have introduced a new elution strategy, combining low ph elution buffer with sonication which, in addition to loosening the peptidetarget interaction, also serves to detach the target molecule from the immobilization surface. this ultrasound-based method enabled single step selection of a high affi nity peptide from a library of diversity greater than 10 9 , and thus represents a dramatic improvement in searching for novel, specifi c peptide ligands. apoptosis to various tumor cells but not in normal cells. using fmoc solid phase synthesis, cellulose membrane-bound octameric peptide library of trail scan was prepared and cell viability assay was directly performed on peptide disk with jurkat cells. six peptide sequences that could induce cell death were found, and particularly rnscwskd (trail227-234) peptide was shown the strongest effects. then, peptides of stronger effects were found through amino acid substitution, and the cnscwskd peptide induced >90% cell death in treated cells. features of apoptosis, such as dna fragmentation, activation of caspase, phosphatidylserine externalization, chromatin condensation, and competition with trail for binding to the death receptor (dr) 4 or dr5 were observed, suggesting that this peptide is a trail mimic. caspase-3 activation was observed in various tumor cells treated with this peptide as well as with trail, while no activation was observed in human normal fi broblasts. the cnscwskd peptide is a potential candidate for use in cancer therapy. the being an incretin mimetic, exendin-4 exerts the same action as glp-1 including the glucoregulatory and the insulinotropic effects through glp-1 receptor. exendin-4 may be preferable to glp-1 on the aspect of stability. however it was comprised with 39 amino acids and was not suitable for developing as an oral drug. recently, there was an upsurge in the development of exendin-4 mimetic as potential therapy for type 2 diabetes. in this study, a receptor-binding region on the surface of rinm5f cell was used as the target to screen peptide ligands for the receptor in a phage 12-mer peptide library. dna sequencing revealed a group of closely related peptides from the fourth round of selection. through the activity of decreasing blood glucose assay in vivo, the cell proliferation assay and capability against dppiv in vitro, one highest bioactivity peptide (qpsvgmkpsprh, ex-12pa) was acquired. the bioactivity of ex-12pa was almost identical with exendin-4 on the promoting cell proliferation in a dose-dependent manner. the decreasing blood glucose effect of ex-12pa was up to 45 min after administration. the stability against dppiv of ex-12pa maintained good performance that 35% parent peptide remained after 48h. in summary, ex-12pa as a shorter glp-1 receptor agonist mimicked the action of exendin-4 and built a good foundation for designing the oral diabetes drug. key words: exendin-4, mimetic, phage display peptide library, screen over the last decades we have witnessed the emergence of bacterial resistance to virtually all clinically important antibiotic types. therefore, continuous development of antibacterial agents with completely novel modes of action accompanied by rationalization of chemotherapeutic prescription is the key strategy to adopt in a long-run struggle against the growing problem of pathogen resistance. numerous indispensable antibiotics interfere with peptidoglycan cell wall biosynthesis making this unique metabolic pathway a well validated target for antimicrobials. while nearly all of these antibiotics inhibit late stages of murein synthesis occurring on the extracellular side of plasma membrane, initial cytoplasmic steps have not been extensively exploited as drug targets. we have performed affi nity selections from random linear and conformationally constrained (disulphide cyclized) peptide libraries displayed on bacteriophage particles against two essential bacterial enzymes murd and mure, involved in the cytoplasmic synthesis of peptidoglycan monomer precursor. selected peptides were found to inhibit respective targets in an in vitro assay with ic 50 values of 140 m to 1.5 mm. reported inhibitory peptides should be regarded as templates for design of low-molecular-weight peptidomimetics. resulting murd and mure inhibitors with improved potency and/or physicochemical characteristics (especially membrane permeability) have the potential to act as broad spectrum chemotherapeutics. fidan, zerrin; ljeskovica, nizama; portwich, michael; volkmer, rudolf charité-universitätsmedizin berlin, germany coiled coil (cc) sequence motifs are common structural motifs and versatile protein interaction modules. the cc is composed of at least two right-handed amphipathic a-helices that wrap around each other into a left-handed supercoil such that their hydrophobic surfaces are in contact. cc can associate up to heptamers, form homomeric and heteromeric complexes at different stoichiometries, and be aligned parallel and antiparallel. a characteristic of all coiled coils is the presence of heptad repeat sequences [abcdefg]i, where i denotes the heptad number. although cc motifs can be predicted with a high degree of confi dence, predicting the association states and topologies is still a great challenge. we will report on synthetic peptide arrays useful to study cc associations at the amino acid level. in contrast to rational design, which mostly depends on short model peptides, our approach relies on the full-length homodimeric gcn4 -and the heteromeric cjun/cfos coiled-coil domain formation. the infl uence of amino acid substitutions on association is tested without restrictions and presumptions and the stoichiometry is examined by biophysical methods. furthermore, we generate arrays comprising hundreds of (putative) cc sequences and subsequently probe them for association to a set of several native cc sequences. an interactome can be drawn for each of the investigated cc sequences, enabling one to interpret the biological functions. we will present: (i) association analyses of all single substitution variants of the gcn4-, cfos and cjun leucine zipper probed with the associated native leucine zipper; (ii) the exposure of a heteromeric cc-network deduced from gcn4 variants and the determination of the networkstoichiometry; (iii) the cc-interactom of several native coiled coils like the cc sequences of akap 18 , pqbp1 18, orai1 and others. almost two decades ago we used the spatial screening technology to optimize activity and receptor subtype selectivity among rgd recognizing integrins. this culminated in the avb3 selective superactive pentapeptide c(-rgdfv-) which gave rise to a number of peptidomimetic modifi cations. among them we studied all retro-, inverso-and retroinverso peptides of this structure. only one retro-inverso peptide exhibits full activity: the peptide c(-dgrvf-) strongly inhibits avb3/vitronectin binding (4 nm) but not aiibb3/fi brinogen binding (>10 000 nm). all investigated retro-sequences show very low affi nity for avb3. recently it was discovered that the ngr sequence in the fi bronectin domain fi5 after rearrangement into iso-asp-g-r exhibits high binding affi nity for integrin avb3. this surprising result stimulated us to investigate cyclic peptides containing the iso-asp-gly-arg sequence3.. after optimisation we obtained peptides which bind with high activity to integrin a5b1 and lower, but signifi cant activity for avb3. in the ongoing work we investigate if receptor modelling can help to understand these results. selectivity and activities for these two integrins have been obtained recently in our group using two different peptidomimetic scaffolds bioactive peptides (tyrosine based and diacylhydracine based) using a homology model of the integrin a5b1 for which no x-ray structure is known. binding of integrin ligands involves binding of a carboxyl group to the metal ion (ca or mn) in the so-called midas region of the integrin. so far all integrin ligands contain a carboxyl group and any attempt to substitute this group by an isoster failed. we recently found that hydroxamic acids (-conhoh) allow for the fi rst time a substitution of this carboxyl group. the activities and selectivities for integrin subtypes avb3, a5b1 and aiibb3 have been explored and give interesting results. development of gnrh-iii-antracycline conjugates as multifunctional drug delivery systems for targeted chemotherapy targeted chemotherapy based on the cell specifi c or overexpressed receptors on tumors might be an effi cient therapeutic approach for the treatment of cancer. expression of gonadotropin-releasing hormone (gnrh) receptor was identifi ed on different types of tumors.1 it has been shown that gnrh-iii (glp-his-trp-ser-his-asp-trp-lys-pro-gly-nh2) isolated from see lamprey has antiproliferative activity on numerous tumor cells, and signifi cantly less potency on releasing gonadotropin hormones (lh, fsh); therefore, it is a more selective antitumor agent than the human gnrh derivatives.2 in our work, gnrh-iii was used as targeting moiety for the preparation of multifunctional drug delivery systems for targeted cancer chemotherapy. daunomycin (dau) and doxorubicin (dox) as antineoplastic agents were attached to the side chain of 8lys of gnrh-iii through amide, oxime, hydrazone or ester bonds, either directly or by insertion of an enzyme cleavable tetrapeptide spacer. stability studies of the conjugates were performed in human serum, as well as in the presence of chymotrypsin. the effect of chemical structure of the conjugates on in vitro antitumor activity was studied using different cancer cell lines (mcf-7 human breast, ht-29 human colon and c26 murine colon cancer cells). oxime bond linked dau-gnrh-iii conjugate was selected for in vivo experiments using c26 colon tumor bearing mice. the conjugate showed similar antitumor activity as the free drug, but less toxic side effect and longer survival of the animals was determined in the case of the application of the conjugate. guillon, g. 1 (1) . it was also shown to be highly selective for the human v1b receptor (1) . we now report the synthesis and some pharmacological properties of three fl uorescent hv1br ligands (a,b,c), based on modifi cations of the lys8 residue in d[leu4,lys8]vp, with alexa 488 (a), alexa 647 (b) and antraniloyl (atn) (c). the fl uorescent peptides a, b and c exhibit the following affi nities for the hv1br: (a) ki = 1.2 nm, (b) ki =186 nm and (c) ki = 0.65 nm. peptides a and c exhibit moderate affi nities for the hotr and very weak affi nities for the hv1ar and for the hv2r. on v1b-transfected att20 cells, they activate plc coupling as evaluated by stimulation of ip3 levels. they also induced receptor internalization visualized by accumulation of fl uorescence in endosomial vesicles. one or more of these new fl uorescent hv1br ligands promise to be useful tools for studying human v1b receptor localization or traffi cking. distribution of prolyl oligopeptidase in the mouse wholebody sections and peripheral tissues prolyl oligopeptidase (pop) is a serine endopeptidase that hydrolyses proline-containing peptides shorter than 30-mer, including many bioactive peptides. the distribution of pop in the brain has been studied but little is known about the distribution of peripheral pop. we used immunohistochemistry to localize pop in mouse whole-body sections and at the cellular level in peripheral tissues. furthermore, we used a pop activity assay to reveal the associations between pop protein and its enzymatic activity. the highest pop protein densities were found in brain, kidney, testis and thymus, but in the liver the amounts of pop protein were small. there were remarkable differences between the distribution of pop protein and activity. the highest pop activities were found in the liver and testis while kidney had the lowest activity. in peripheral tissues, pop was present in various cell types both in the cytoplasm and nucleus of the cells, in contrast to the brain where no nuclear localization was detected. these fi ndings support the proposed role of pop in cell proliferation in peripheral tissues. the dissociation of the distribution of pop protein and its enzymatic activity points to nonhydrolytic functions of pop and to strict endogenous regulation of pop activity. the rapid cytoplasmic entry of cationic cell-penetrating peptides in the past few years, our understanding of the cellular import of cellpenetrating peptides has evolved rapidly. the initial concept of cell entry by direct permeation of the plasma membrane, was followed by endocytosis as a major route of import at least for most cellpenetrating peptide-cargo conjugates. more recently, we and others observed a rapid cytoplasmic delivery of fl uorescein-labeled analogs of the cationic cpps nonaarginine and tat-peptide at subtoxic lower poster abstracts micromolar concentrations (1). this import originates from spatially confi ned zones of the plasma membrane and depends on the presence of heparan sulfate proteoglycans. these molecules have been proposed to interact polyvalently with the guanidinium groups of the arginine residues. moreover, the threshold for the induction of this import could be lowered by inhibition of endocytic routes of entry. recently, it was reported that the absence of serum in the medium lowers the threshold for this uptake process (2) . in summary, these observations suggest that the concentration of peptide associated with the plasma membrane is a decisive trigger for this import process. currently, it is not known, to which degree this cytoplasmic entry of labeled conjugates at higher concentrations is based on similar molecular mechanisms as the direct permeation of unlabeled conjugates that has also been observed at lower concentrations (3). here we summarize the current knowledge on direct transport across the plasma membrane of cationic cpps and present new data on the molecular events involved in this uptake process. plasma membranes have numerous essential functions for maintaining cellular homeostasis. however, the membranes are also barriers to intracellular delivery of various therapeutic molecules. for improvement of their translocations, we developed a novel method using gala peptide/cationic lipid complexes. gala, a 30-residue amphipathic peptide with a repeat sequence of glutamic acid-alanine-leucine-alanine, was designed to mimic the function of viral fusion protein sequences that mediate escape of virus gene from acidic endosomes to cytosol (1) . when attached with bioactive cargoes, the gala peptide may thus serve as intracellular vector bearing effi cient endosomal escape function. however, because of negative charges from glutamic acids (7-residues) in the gala sequences, access of the peptide on negatively charged cell surface would be not so effi cient. to overcome this problem, cationic lipid was employed as an gadhesive h for pasting the gala peptide onto cell surface to accomplish effi cient cellular uptake. we examined the ability of gala peptide as a delivery vector using fitc as a model of membrane-impermeable low-molecular weight drugs. when fitc-gala (1 ƒêm) was administrated to hela cells, co-addition of cationic lipid, lipofectamine 2000 (lf2000), drastically increased the effi ciency in uptake of fitc-gala. in a time-dependent manner, the fitc-gala escaped from endosomes, and diffuse fl uorescent signals were observed in both cytosol and nucleus. also the gala/cationic lipid system was applied for the intracellular delivery of fitc-avidin protein (68 kda). when fitc-avidin was mixed with biotinylated-gala/ lf2000 complexes, fitc-avidin (250 nm) was internalized into cells effectively. in the absence of these complexes, internalization of fitcavidin was poor. these results suggest the usefulness of our approach for intracellular delivery using gala peptide and cationic lipid. the membrane repair response masks membrane disturbances caused by cell penetrating peptide uptake even though cell-penetrating peptides are able to deliver cargos of different sizes into cells, their uptake mechanism is still not fully understood and needs to be elucidated in order to improve their delivery effi ciency. recent studies have suggested that there might be a direct penetration of peptides in parallel with different forms of endocytosis. however, the direct penetration of hydrophilic peptides through the hydrophobic plasma membrane is highly controversial. three proteins involved in target cell apoptosis -perforin, granulysin and granzymes share many features common in uptake of cell-penetrating peptides e.g. they bind proteoglycans on the plasma membrane. the uptake of perforin activates the membrane repair response, a resealing mechanism triggered in cells with injured plasma membrane, due to extracellular calcium infl ux. upon activation of the membrane repair response, internal vesicles are mobilized to the site of the disrupted plasma membrane resealing it within seconds. in this study we present evidence that the membrane repair response is able to mask damages caused by cell-penetrating peptides when they internalize cells, thus preventing leakage of endogenous molecules out of the cell. the hypothalamic decapeptide gonadotropin-releasing hormone (gnrh-i; gnrh symmetric dimer derivatives ([ in vitro (cellular uptake and antiproliferative effect of the dimer derivatives) and in vivo (comparison of per os and intraperitoneal administration) antitumor effect of these symmetric dimers were investigated. the cellular uptake of dimer derivatives were studied by fl ow cytometry (bd lsr ii) on mcf-7(human breast cancer) and ht-29 (human colon carcinoma) cell lines using carboxifl uorescein labeled symmetric dimer derivative. the cells were treated with different concentration of synthetic dimers and after the treatment were analysed by fl ow cytometry in order to investigate their cellular uptake. the antiproliferative effect of symmetric gnrh-iii dimers were studied on mcf-7, ht-29 and t47-d (human breast cancer) cell lines. ht-29 xenograft was applied for studying in vivo antitumor effect of gnrh-iii dimers in different administration routes. the cellular uptake and the antiproliferative effect are cell type dependent as it can be found in the literature. we found that symmetric dimer derivatives of gnrh-iii signifi cantly decreased the tumor volume in vivo (40%). iib 3 receives intracellular signals (inside-out signaling) that allow cytoplasmic proteins to interact with the cytoplasmic domains of iib 3 subunits, resulting in platelet aggregation. the aim of this work is to inhibit platelet thrombus formation by specifi cally disrupting the insideout signalling pathway using synthetic peptides based on the cytoplasmic region of 3 subunit, residues 743-762. peptide analogues derived from 118 3 743-762 region ( 3 743-750, 3 743-750(ptyr747), 3 743-756, 3 755-762 and 3 749-756) were synthesized in their free, palmitoylated and/or tagged with the tat(48-60) signaling sequence and carboxyfl uoresceinlabeled in order to investigate their membrane permeability, as well as their inhibition potency on the platelet aggregation. from the biological assays in prp and washed platelets we concluded that the modifi ed peptides that carry either palmitoyl-group or the tat(48-60) signaling sequence penetrate platelet membrane and inhibit human platelet aggregation, in contrast to the corresponding free peptide analogues. acknowledgements: the gsrt (epan yb/88) and e.u. for the fi nancial support. ; aib = -aminoisobutyric acid) is a potent mitochondriotoxic analogue of mp that demonstrates a signifi cant intracellular co-localization with mitochondria. through co-operation with a protein of the mitochondrial permeability transition pore vdac, mitoparan specifi cally promotes apoptosis. in contrast, cytc 77-101 , a cryptic fragment of human cytochrome c, is a moderately potent apoptogenic cpp that demonstrates a strong propensity for colocalization within the endoplasmic reticulum. using a sychnologic modular design, we have synthesized and evaluated a broad range of chimeric constructs combining apoptogenic cpp (message) and peptidyl address motifs. the overriding objective of these studies was to enhance cytotoxicity and/or develop target-selective drug delivery vectors. address motifs that target both plasma membrane and nuclear envelope protein structures included i) the integrin-specifi c rgd sequence, ii) a fas ligand mimetic wewt, iii) heptagastrin (aygwmdf) that targets a novel membrane binding site on high grade astrocytoma and, iv) a mimetic of fg nucleoporins (nups). incorporation of peptidyl address motifs, by simple n-terminal acylation or via a fl exible aminohexanoic acid (ahx) linker, produced chimeric constructs with enhanced cytotoxic potencies. most signifi cantly, ld 50 values readily achievable in vivo were demonstrated by the modular apoptogenic cpp z-gly-rgdf-mitoparan, (ld 50 = 1.4 m) and ac-nfkfglss(ahx)cytc 77-101 (ld 50 = 1.0 m). in conclusion, targetspecifi c modular design of apoptogenic cpp is a promising strategy for the study and therapeutic induction of apoptosis. phage display screening of geminin-binding peptide and validation of its therapeutic use in tumors yoshida, kenichi meiji university, japan dna replication is controlled by the stepwise assembly of a pre-replicative complex (pre-rc). geminin is a component of the pre-rc and plays a role in preventing incorporation of the minichromosome maintenance protein complex into the pre-rc via binding and inhibiting cdt1 function. it may be possible to design low-molecular-weight chemicals that would bind to geminin to suppress the aberrant cellular proliferation in tumor cells. a random 12-mer peptide phage library was used for the selections. phage was selected by panning on immunotubes coated with recombinant geminin. positive clones were identifi ed by a screening phage produced from single colonies for specifi c binding to gst-geminin in elisa. fluorescent-labeled peptide linked to the sv40 nuclear localizing signal was synthesized by solid-phase synthesis, using fmoc chemistry. by detecting fl uorescein fl uorescence to mark specifi c transfected cells, we examined the effects of the peptide transfection on the synthesis of new dna in hct116 cells. by screening a random peptide phage library, we identifi ed a certain peptide sequence bound to geminin. using a series of mutant geminin, elisa test revealed the amino acid residues 31-111 are responsible for the peptide binding. we found fewer brdu positive cells following transfection of the geminin-binding peptide than following that of the control peptide. this study suggests that the chemical peptidomimetics of this peptide might form the basis for the development of drugs that could be used to prevent tumor progression. acknowledgements: this study was supported by kakenhi. it has been previously demonstrated that n3-(4-metoxyfumaroyl)-l-2,3diaminopropanoic acid (fmdp) is a strong inhibitor of glucosamine-6phosphate synthase, a potential target for antimicrobial chemotherapy. fmdp is transported into the cells when incorporated in a peptide chain and hydrolyzed by intracellular peptidase releasing free inhibitor as the "warhead" component, which can react with the target enzyme. the concept of utilizing of peptide transport system for delivery of toxic amino acids into the microbial cells is characterized by authors as "illicit transport". unfortunately, peptides are not stable in physiological fl uids, due to activity of peptidases. analogs with modifi ed amide backbone are more resistant towards enzymatic degradation. it has been shown that enzymatic hydrolysis of endothiopeptides is often signifi cantly slower than natural peptides. in our studies we synthesized nva[csnh]-fmdp, an analog of nva-fmdp, which is a strong antimicrobial peptide. because of the fact that the "warhead" component in this peptide contains an amide backbone, fi rstly we decided to synthesize free fmdp with thioamide backbone. the results of enzymatic kinetics studies showed that in this way modifi ed compound is much weaker inhibitor of glucosamine-6-phosphate synthase than fmdp. it was the reason why we planned and carried out a multi-step synthesis in order to obtain a potential more resistant towards enzymatic degradation, antimicrobial peptide containing a thioamide backbone only between nva and fmdp. activity of nva[csnh]-fmdp nva-fmdp against selected microorganism in medium containing blood serum was determined. neuroprotective effects of cortexin and cortagen in rats with focal brain ischemia and brain trauma there is a growing body of evidences that natural peptides and their smaller synthetic analogues have good outlooks for medical use. three compounds were studied in this research -cortexin, cortagen and nîleylcortagen. cortexin is a balanced combination of oligo-peptides isolated from calf/porcine cortex. the number of non-clinical and clinical studies confi rmed cortexin effi ciency as nootropic and neuroprotective drug. in spite of benefi cial results the natural source of the drug implies some diffi culties for it general use. to overcome this issue a number of putative cortexin synthetic analogues were developed. among them is tetrapeptide cortagen. in order to improve the peptide transport to the brain and lipophility the hydrophobic oleyl radical was added to the peptide chain. the aim of research was to study the effects of these peptides on recovery of conditional refl ex in active avoidance paradigm after heavy brain trauma and neurologic defi cit dynamics induced by focal brain ischemia in rats. 170 males albino rats were involved in experiment. closed craniocerebral trauma was modeled by weight-drop method. focal cerebral ischemia was induced by cortical phototrombosis. experimental therapy started in 0.5 hours after mechanical action and continued for 7 days. synthetic peptides were administrated i.p. once-a-day in dose of 10 -1000 mcg/kg. cortexin was used at the same schedule in dose of 150 mcg/kg. cortagen and cortexin administration in dose of 10 and 150 mcg/kg respectively resulted in pronounced neuroprotective effect in rats with focal cerebral ischemia. the observed effects were proved by limb-placing test. cortexin experimental therapy resulted in earlier restoration of conditional refl ex starting from the 3rd day after brain trauma. nîleylcortagen had weaker benefi cial effect with signifi cant improvement in conditioned response recovery by the 4th day. probiotics such as lactobacillus rhamnosus gg provide health benefi ts beyond their mere nutritive value, and are useful in the treatment and prevention of several diseases. isolation and use of the anti-apoptotic factor(s) would ultimately culminate in the development of novel therapeutic agents in enteropathy resulting from chemo-and radio-therapy in the treatment of cancer patients, which could in turn be administered in a consistent and pharmacologically sound manner. in present study, intestinal epithelial cells were isolated from wistrar rats (n=12), and were grown in dmem medium supplemented with 5% foetal bovine serum at 37°c in a humidifi ed 5% co 2 atmosphere. after 72 h, these cells were treated with bioactive peptides isolated from lgg (10-100 ìmol/ lit) for 2 h, and were incubated with camptothecin (20 ìmol/ lit) for 2 and 4 h, and cell viability was measured by mtt assay. caspase activity assay was carried out to determine caspase 3 and caspase 9 activity. moreover, cell lysates were also pretreated with caspase 3 inhibitor (200 ìm). dna fragmentation assay was carried out to determine the increase in dna fragmentation. bioactive peptides signifi cantly prevented camptothecin induced apoptosis in rat intestinal epithelial cells. the treatment of intestinal epithelial cells with camptothecin for 4 h resulted in fi ve-fold increase in dna fragmentation, and resulted in three-fold increase in caspase 3 activity, compared with controls. pre-treatment with bioactive peptides (10 ìmol/ lit) signifi cantly attenuated the camptothecin-induced dna fragmentation by 29% (p<0.001), and signifi cantly inhibited caspase 3 and caspase 9 activity by 35% and 39%, respectively, after 4 h of camptothecin exposure (p<0.001). hence, it was concluded that novel bioactive peptide from lgg were found to be potential anti-apoptotic agents and hence could be administered as therapeutic molecule for treatment of enteropathy resulting from chemo-and radiaton-therapy. cisplatin belongs to the most powerful and useful anticancer agents. it binds strongly to dna in regions containing several guanine units, forming pt-dna links within strands. through disrupting base-pairing guanine to cytosine cross-links lead to unwinding of the dna. as a result cisplatin works against both types of cells, destroying cancer and normal type ones. therefore more selective delivery system of platinum to cancer cells is still needed. based on the evidence of the presence of -and -opioid receptor types in carcinoma cells we proposed to use opioid peptides as selective carriers for delivering platinum ions to the cancer cells. we designed hybride molecules which combine two fragments. one part of the molecule contains the opioid pharmacophore and the other fragment is designed to form a complex with platinum ion. such molecule can serve not only as carrier for platinum, but also give a strong analgesic effect. as a result such hybride molecule should express analgesic properties and provide anticancer activity. we will present synthesis of a few opioid peptide-platinum(ii) complexes, the binding affi nity at the opioid receptors and effect on the proliferation of the human glioblastoma cells. peptides derived from the c-terminal heptad repeat region of the hiv fusogenic protein gp41 are potent inhibitors of viral infection, and one of them, t20 (enfuvirtide), is used for the treatment of therapy-experienced aids patients. the mechanism of action of these peptides is to interfere bioactive peptides with the fusion of the viral and target cell membranes, and it is known that hiv entry takes place in membrane microdomains ('lipid rafts') enriched in cholesterol and sphingolipids. therefore we explored the advantage of targeting a peptide fusion inhibitor to membranes by addition of a lipid moiety, specifi cally a cholesterol group. since derivatization with lipids is also known to improve the half-life of peptide therapeutics, we chose the antiviral peptide c34, which is more potent than t20 in cell-based assays, but unlike t20 has a very short half-life in vivo. we show here that attachment of cholesterol to c34 (c34-chol) dramatically increases its antiviral potency against a panel of hiv strains, including primary isolates: for strain hxb2, ic50 = 4 pm for c34-chol, versus 205 pm for c34, and 692 pm for enfuvirtide). consistent with the anticipated mechanism of action, c34-chol accumulates at the site of action, since washing of the target cells after incubation with the peptide, but prior to triggering fusion, increases ic50 only 5-fold, relative to 400-fold for c34. moreover, cholesterol must be strictly positioned at the c-terminus of c34, in line with the need of an antiparallel orientation of the c-peptide relative to n-peptide trimeric coiled-coil, present in the fusion-active conformation of gp41. in addition to boosting antiviral potency, derivatization with cholesterol has the expected benefi cial effect on the peptide pharmacokinetics. we believe that these fi ndings may be of general utility for viruses, which share with hiv the dependence on a type i fusogenic machinery. multiple sclerosis (ms) is an autoimmune demyelinating disease mediated primarily by cd4+ t cells of the th1 subset. the design of peptide mutants of disease-associated myelin epitopes to alter immune responses offers a promising avenue for the treatment of ms. we designed and synthesized a number of peptide analogues by mutating the principal tcr contact residue based on mbp83-99 epitope. the synthesis of the linear peptide agonist mbp83-99, as well as of the cyclic analogue was carried out by the fmoc/tbu methodology, utilizing the 2-chlorotrityl chloride resin. cyclization was achieved using obenzotriazol-1-yl-n,n,n',n'-tetramethyluronium tetrafl uoroborate (tbtu) and 1-hydroxy-7-azabenzotriazole, 2,4,6 collidine allowing fast reaction and high yield cyclization product. the purifi cation was achieved using hplc reversed-phase chromatography and the peptide purity was assessed by analytical hplc and by mass spectrometry (esi-ms). agonist and antagonist (linear and cyclic) peptides were conjugated to reduced mannan. immune responses were diverted from th1 to th2 in sjl/j mice and generated antibodies which did not cross react with native mbp protein. (1) . due to its high effi cacy and specifi city, hn represents a potential lead to new therapeutic approaches of ad. however, the molecular mechanism(s) of hn function(s) are not yet fully elucidated. wild-type hn and hn-derivatives were synthesized by spps and amino acid sequences and homogeneities of the rp-hplc purifi ed peptides ascertained by esi-and maldi-mass spectrometry (ms). the complex formation between hn and a (1-40) was studied by affi nity-chromatography and high resolution ms (fticr-ms), as well as by immunoanalytical (elisa) techniques. the binding sites between the two peptides were identifi ed by applying proteolytic epitope extraction/excision procedures [2, 3] integramide a, an effi cient inhibitor of the coupled reaction of hiv-1 integrase, is a 16-mer linear peptide characterized by 9 c -methylated -amino acids (5 iva, isovaline, and 4 aib, -aminoisobutyric acid, residues) that was isolated from fungal extracts of dendrodochium sp. the amino acid sequence was fully elucidated by the merck group a few years ago (s. b. singh et al., org. lett. 2003, 4, 1431-1434) . on the other hand, the chiral sequence was only partially determined. in particular, the precise stereochemistry of the iva 14 -iva 15 dipeptide (known to contain one d-and one l-residue) near the c-terminus was not reported. to solve this unsettled issue and to assess integramide a primary structure-bioactivity relationship we performed by solution methods the total chemical independent syntheses of both l-d and d-l 16-mer diastereomers and compared their properties with those of the natural inhibitor. for an unambiguous, complete stereochemical assignment of integramide a we relied heavily on hplc and nmr techniques. our results clearly indicate that the chirality sequence of the iva 14 -iva 15 dipeptide of the natural product is l-d. the two integramide a diastereomers were also evaluated as inhibitors of hiv-1 integrase in the coupled reaction of proviral dna into the host cell dna. . a prototype peptide, r11-29, spanning rantes residues 11 to 29, contains two hydrophobic clusters of fundamental biological importance connected by a nonessential hydrophilic linker. r11-29 has been rationally modifi ed to improve its ccr5 binding and its antiviral potency. nmr studies on the modifi ed peptides revealed important similarities and differences with the three-dimensional organization of rantes. although these peptides are consistently more active as dimers, an increase in anti-hiv-1 activity of the monomeric forms was observed in parallel with their molecular evolution. in addition, no tertiary interactions could be detected by nmr, indicating an autonomous folding of the monomers also in the context of dimeric peptides. the most potent peptide designed so far, rmax, shows anti-hiv-1 activity in the low nanomolar range (vangelista et al, in preparation). strikingly, a similar potency was observed in the same assay with t20, an hiv-1 gp41-derived peptide currently licensed for use in aids therapy. these results provide a rationale for the use of rantes-derived peptides as new candidates for treatment and prevention of hiv-1 infection. 10% of the population older than 65 years and 40% exceeding the age 80 years are affected by alzheimer's disease (ad).(1) a factor in the pathogenesis of ad is the cerebral deposition of amyloid fi brils as senile plaque. bace1 initiates the pathogenic processing of app by cleaving at the n-terminus and the resulting c99 membrane bound c-terminal peptide can then be hydrolyzed by -secretase to form apeptide (a 40 or a 42). bace1 is not only a promising target cause it is a key player in formation of a but also because bace1 knock-out mice are viable and free of the gross phenotypic changes. our group was recently able to shown that the phosphino dipeptide (pdp) isostere is a suitable replacement of the hydroxyl ethylene isostere in om00-3(2) resulting in pseudo peptidic inhibitors of about same potency. [2, 3] in our search for conformationally restrained pdp isostere bace1 inhibitors we speculated that the p1 and p3 cyclization would lock the active conformation of the cyclic linear pseudo peptidic inhibitor in the n-terminal region. first the detail analysis of the crystal structure of om00-3 bound to bace1(2), revealed that the ideal macrocyclus should consist of a 13-membered heterocycle. on this basis we developed a macrocyclic inhibitor with p1 and p3 cyclized side chains containing a pdp isostere with improved serum stability. specifi c tumor-targeting, via tumor-associated antigens (taa), selectively expressed or over expressed on tumor cells, is the goal of modern cancer therapy aimed at overcoming non-specifi c toxicity of most anticancer drugs. the expression of taa varies among different tumors and patients, resulting in highly variable response to tumor targeted therapies. therefore, diagnosis should provide information on the expression of the targeted antigen in each patient, thus allowing to predict possible effi ciency of a therapy mediated by targeting agents directed to the same tumor antigen. in this approach, the molecule used for tumor cell tracing should be as close as possible to that used for therapy. the use of peptides as tumor targeting agents was envisaged years ago with the fi nding that receptors for different endogenous regulatory peptides are over-expressed in several primary and metastatic human tumors, and can be used as tumor antigens (reubi jc. j nucl med 1995;36:1825-35). in previous works, we demonstrated that peptides synthesized in a branched form, result in molecules that are resistant to proteolytic activity and can retain (or even increase, through multivalent binding) peptide biological activity. a branched peptide that targets neurotensin (nt) receptors, known to be over-expressed in a number of tumors, including colon, pancreas and prostate carcinoma (reubi jc. endocr rev 2003;24:389-427) and was conjugated to effector units and proved to be stable and active in vivo (falciani c, et al. mol cancer ther. 2007;6:2441-8). here, we created new nt-based molecular tools conjugated to different fl uorophores and chemotherapeutics and demonstrated that branched peptides can be modulated either as tracers for measuring the presence of the specifi c target in primary tumor or metastasis on human surgical resections, or as specifi c drug-carriers, which use the same target to enter and eventually kill tumor cells in vitro and in vivo. aquaculture has become an increasingly important activity, as an immediate source of animal protein required for several countries growing population. vaccination of fi sh against bacterial and viral diseases is one of the best strategy for controlling certain infectious bioactive peptides diseases in aquaculture worldwide, thus decreasing the need for antibiotics, and increasing cost-effectiveness and net profi ts. infectious pancreatic necrosis virus (ipnv) is a pathogen of farm fi sh with a worldwide distribution. peptides derived from ipnv protein 2 (vp2), a major structural protein, could be used for development of effective vaccine against ipnv, according to a search of vp2 antigenicity we selected two peptide sequences, k 19 pyvrledetpqg 42 (vp2-19-42) and n 91 fslaeqpanetk 103 (vp2-91-103), that are expected to be highly immunogenic. these peptide sequences were synthesized in their n-terminus iodoacetylated form and conjugated to ac-(lys-aib-cys) 4 -nh 2 , a cell penetrating sequential carrier (cpsc). conjugation of four copies to cys side chains of the carrier was realised by the chemoselective ligation method. the resulted constructs were purifi ed by hplc and characterized by esi-ms. immunizations are in progress in order to test their immunoprotective potency. acknowledgements to the gsrt (greece-egypt project 266-å) for the fi nancial support. for more then two decades, the rgd sequence is known to bind integrins, e.g., 5 1, v 3, and iib 3 among many others. it is present in the natural ligands for these integrin receptors, as in fibronectin, vibronectin, or fibrinogen. recently, it was shown that fibronectin in which the rgd sequence has been mutated into an rge sequence (which is known not to be recognized by integrins) still has the ability to interact with its receptor. however, it forms fn fi brils only with a slightly different phenotype than wild-type fibronectin (1). a hypothetical model for these unexpected results was proposed by curnis et al. (2) . the ngr (asn-gly-arg) sequence, present at four positions in the fi bronectin molecule, is able to undergo a rearrangement to isodgr (isoasp-gly-arg), which shows activity on v 3 and -with less potency -on 5 1. the mechanism of asn-deamination is already known for a long time and has widely been considered to be a process of degradation, acting as a biochemical clock that limits protein lifetimes in vivo (3). curnis et al. were the fi rst to show that the deamination process increases protein function instead (2). these fi ndings stimulated us to create a library of cyclic peptides containing the isodgr sequence. a screening of this library showed various new peptides with high activity and selectivity towards the integrin receptor 5 1. the correlation between the intensities of muramyl peptides adjuvant effect and nod2 activation. it is well known that essential activity of muramyl peptides -minimal structures of bacterial cell wall -are adjuvanticity. the comparison of the adjuvant effect of these compounds with the ability to activate nf-kb pathway through nod2 was examined. the adjuvant activity of di, tetrasaccharide peptides and stearoyl containing derivatives has at least two peaks in dose-response curves and greater of them correlates with respective dose-response data for nf-kb stimulation through nod2. introduction of stearoyl moiety, with the aim of improving muramyl peptide interaction with the cell membrane and subsequent intracellular delivery, infl uenced the corresponding activities in vitro, but did not correlate with improved effects in vivo experiments. the comparison of the adjuvanticity in vivo and the nod2 activation in vitro revealed clear correlation between two responses. these fi ndings confi rm the view that nod2 pathway activation should account, at least in part, for the adjuvant effect of these compouds. the correlations of the nf-kb pathway induced by muramyl peptides with the aim of enhancing their adjuvant activity were investigated. the thymus gland plays an important role in overall immunomodulation. the studies in early 1960s by several authors have established that thymus is necessary for the normal development of immune response. it is thought to be responsible for the development and regulation of tcell immunity, acting through endocrine mechanism (1). it is known that thymus peptides play an important role in the development, maturation, differentiation, and activation of t-lymphocytes. investigation of the function and properties of this gland shows that the thymus contains pharmacologically active components with immunological properties. when the immune system is challenged, thymic peptides seem to regulate the expression of various cytokine and monokine receptors on t-cells and induce secretion of il-2, interferon alpha, and interferon gamma. up to date several thymic extracts have been isolated using different biochemical methods of extraction, homogenization and purifi cation. these are, usually, semipurifi ed aqueous and lipid calf thymus extracts. the goal of this study was to determine the biological activity of peptide components, isolated from the calf thymus. extract of calf thymus was prepared and fractioned into lipid and nonlipid (peptides) fractions. the nonlipid fraction was isolated and characterised by biuret, ir , nmr and hplc methods. analyses of ir and nmr spectra indicated the presence of charasteristic bands and peaks for peptides. results estimated from hplc analyse showed that molecular masses of isolated peptides were below 1500 daltons. biological activity was accesed by using proliferation of thymocites and splenocites in vitro in the presence of mitogen, and obtained results showed signifi cant imunomodulatory effect. keywords: thymus, peptides, immunomodulators, thymocites, splenocytes, proliferation according to contemporary knowledge about proteins, every protein can play the role of a precursor of biologically active peptides. bioactive peptides isolated from food proteins display various activities, for example: antihypertensive, antithrombotic, immunomodulating, opioid and antibacterial. the database of protein and bioactive peptide sequences designed in chair of food biochemistry (www.uwm.edu.pl/biochemia) contains information on proteins which are precursors of bioactive peptides. the database, biopep, enables an evaluation of food proteins according to the following criteria: the frequency of the occurrence of fragments with the given activity in a protein chain, a potential activity of protein fragments, and profi les of a potential biological protein activity i. e. the type and location of a bioactive fragment in the protein chain. the biopep database was used to determine the profi les of potential biological activity of food proteins and classify them into families and subfamilies. results that we obtained indicate that the greatest number of bioactive peptides can be released from milk proteins. we also analysed structural properties of bioactive fragments encrypted in protein chains which are predicted to be accessible for endopeptidases. the application of biopep and msblast software enabled us selection of a fragments with high degrees of identity to the celiac-toxic peptides in food proteins sequences. based on the biopep, we are able to design the processes of the release of bioactive peptides from protein sequence and with the use of mass spectrometry -to identify released peptides. the detailed results of authors in silico food proteins analysis will be presented. the computational methods and the above-mentioned tools can be applied for designing food with the special designed and desired properties (functional food) as well as production of nutraceuticals i. e. food with therapeutic properties. neurotensin analogs with high affi nity and selectivity at human neurotensin receptor 1. neurotensin (nt), pglu 1 -leu 2 -tyr 3 -glu 4 -asn 5 -lys 6 -pro 7 -arg 8 -arg 9 -pro 10 -tyr 11 -ile 12 -leu 13 -oh, exerts numerous physiological actions in the central nervous system and in the periphery. studies in animal models have suggested its involvement in the modulation of dopamine transmission, hypothermia, analgesia, locomotor activity, cardiovascular function, and others. at present, three receptors are known to bind nt; two of them are g-protein coupled-receptors (ntsr1 and ntsr2). the physiological effects of ntsr1 have been extensively studied but the functions associated with the nt binding to ntsr2 are less well defi ned. the c-terminal fragment of nt, arg 8 -arg 9 -pro 10 -tyr 11 -ile 12 -leu 13 -oh, has been long recognized as an essential and suffi cient segment for the effective interactions of nt with the receptors; a short peptide, arg 8 -arg 9 -pro 10 -tyr 11 -ile 12 -leu 13 -oh, designated nt(8-13), displays binding affi nity similar to that of the full-length nt at both ntsr1 and ntsr2. in this study, the role of arg 8 in the interactions of nt(8-13) with the human ntsr1 and ntsr2 was examined through ligand structure-function studies. the side chain of arg 8 was determined to not be critical for binding to hntsr1 but essential for binding to hntsr2. several analogs of nt(8-13) are reported which are high affi nity hntsr1 ligands (ic 50 = 0.1 to 3 nm) and more than 500-fold selective versus hntsr2. human umbilical vein endothelial cells (huvecs) form tubular networks when cultured on top of the matrigel basement membrane preparation, refl ecting the ability of the cells to form blood vessels. using this model, we have previously shown that psa (also known as klk3) inhibits endothelial cell tube formation(1), indicating reduced angiogenic potential. furthermore, we have shown that the antiangiogenic activity of psa is related to its enzymatic activity [1, 2] . we have developed peptides that stimulate the enzymatic activity of psa towards a small chromogenic substrate. these peptides also enhance the anti-angiogenic activity of psa both in vitro and in vivo. this supports our hypothesis that enhanced psa-activity by our peptides could be used to reduce tumor angiogenesis and, thus, to reduce tumor growth. the most extensively studied tads are those that contain acidic tads and one extremely important protein is the human tumour suppressor protein p53. given the presence of repetitive stretches of acidic amino acids tads are generally disordered in the free state and this has also been demonstrated for p53tad. using a combination of nmr spectroscopy, isothermal titration calorimetry and site-directed mutagenesis studies we have recently characterized the interaction of the p53tad with the pleckstrin homology (ph) domain of the tfb1/ p62 (yeast/human) subunit of tfiih. the nmr structure of the tfb1/ p53tad complex demonstrates that p53 forms a short alpha-helix in complex with tfb1 (1). this structure is an important step towards developing a sequence code for acidic tad binding to the ph domain of tfb1/p62. such detailed structural information is essential to design molecules that modulate transcription activators such as p53. we will present the structure of the p53/tfb1 complex, structural and biophysical characterization studies with peptides designed to mimic acidic tads and compete with p53 for binding to tfiih. integrins are a family of transmembrane cell surface receptors, which mediate cell-cell and cell-matrix adhesion. the binding of integrins to their natural ligands is the molecular basis of physiological processes such as cell adhesion, migration and signal transduction of cells, as well as of patho-physiological processes. thus, small molecules capable of interfering with this integrin-natural ligand binding process have pharmacological potential in the therapy of cancer and infl ammatory diseases. the amino acid sequence rgd, present on many of the natural ligands, is a prominent recognition motif of integrin ligands. synthetic rgd-containing peptides are an excellent starting point for the identifi cation, synthesis and development of selective integrin ligands. the affi nity and selectivity of the peptide ligands towards different integrins depend strongly on the secondary structure of the sequence and the overall three-dimensional shape. cyclization is frequently used as a method to reduce the accessible conformational space. additionally, the incorporation of non-natural conformationally constrained amino acids can greatly affect the secondary structure of the peptide, in such a way that the synthetic ligands prefer to adopt a particular conformation. the aim of this investigation are small cyclic peptides containing the rgd motif and constrained amino acids (such as -methylated amino acids, -amino acids or dehydroamino acids) that exhibit well-defi ned conformational properties. the present communication describes the synthesis of different cyclic rgd peptides with the general sequence c-(-arg-gly-asp-xaa-yaa-) and the evaluation of their activity as ligands for the v 3 and 5 1 integrins, present on human cells. acknowledgments: this work is supported by a marie curie intra-european fellowship from the 6th framework programme. in recent years there has been increasing interest in the design of chemical agents capable of inducing dna interstrand crosslinking (isc), which, shutting down the dna replication process, represents by far the most cytotoxic of all the alkylation events. quinone methides (qms) are interesting compounds that have been proposed as intermediates in a large number of chemical and biological processes. the asymmetry introduced by the presence of two electronically different substituents, carbonyl and methylidene, on the cyclohexadiene ring imparts a strong dipolar character to quinone methides not found in benzoquinones and quinodimethanes. qms have been successfully used to accomplish nucleoside alkylation and dna isc by photochemical and fl uorideinduced activation. apart from their involvement in biological processes, o-qms react with a variety of nucleophiles (michael addition), resulting in substituted hydroquinones. they also function as effi cient heterodienes in diels-alder reactions and undergo a variety of self dimerization reactions. here we present the synthesis of a new series of synthetic o-qms (bis-napthalene type) conjugated with rgd analogue peptides. the biological activity of these compounds is under investigation. aknowledgements: we thank the european social fund (esf), operational program for educational and vocational training ii (epeaek ii) and particularly the program pythagoras ii for funding the above work. platelets aggregation causes clotting in the blood vessels during the blood circulation due to several reasons. factor viii (fviii), a blood coagulation glycoprotein, is a key component of the blood coagulation system. fviii in its activated form (fviiia) acts as a cofactor to the serine protease fixa in the conversion of the zymogen fx to the active enzyme (fxa). the role of fviiia is to increase the catalytic effi ciency of fixa in the activation of factor x (fx). the target of this research is the synthesis of biologically active peptides, which are expected to inhibit selectively the maximisation of thrombin production depended on factor ix (fix) and accordingly the additional activation of platelets. these peptides are based on the regions in which the fviii interacts with fix. glycoprotein fviii is composed of three distinct domain types in the arrangement, a1-a2-b-a3-c1-c2. the sequence 558-565 of a2 subunit is the following: ser558 -val-asp-gln-arg-gly-asn-gln565 this work covers the synthesis and biological evaluation of linear and cyclic head to tail peptides, analogs of the loop sequence 558-565 of the a2 subunit, aiming at the inhibition of interaction of fviiia with fixa. substitutions have been taken place at asn564, which are related to the pharmaceutical groups at the side-chain, like asp(r)564. all the synthesized analogs are purifi ed (rp-hplc) and identifi ed (esi-ms). the synthesized peptides analogs were investigated for their inhibitory activity and tested for clotting defi ciency by measuring their activated partial thromboplastin time (aptt) in vitro. these results will be discussed in relation with their biological activity against the fviiia factor of blood coagulation. nerve growth factor (ngf) is an homodimeric protein that binds two different cellular receptors: 1) the transmembrane tyrosine kinase receptor trka, a member of tyrosine kinase family; and 2) p75, a member of the tumor-necrosis-factor receptor superfamily. both receptors are involved in the activation of different intracellular signal transduction cascades. small peptides retaining the most essential elements of ngf may be useful either as agonist or competitive antagonist in the treatment of several neurodegenerative desease and nerve injuries. the n-terminal fragment of ngf was previously demonstrated to be an important determinant for affi nity and specifi city in the binding to trka. crystal structure of ngf-trka complex, site-directed mutagenesis studies and substitution of individual amino acids, contributed to identify within the ngf molecule the most relevant domains for its biological activity in the loop-1 (29-35), loop-4 (92-98) and in the nterminal region (3-18). we synthesized twenty peptides mimicking the loop1 and 4 linked together with aminoacid-based spacers (n glycines) or with two unit of 8-ammino-3,6-dioxaottanoic acid with or without the n-terminal region. l1l1 and l4l4 homodimeric loops, moreover l1 and l4 were also synthesized, as single loop, and their biological properties were compared to l1l4 and n-l1l4 activity . the n-l1l4 (hpifhrgefsvadsvsvwvgdctdikgkctgacdgkqc) and l1l4 (ctdikgkctgacdgkqc) peptides showed a good ngf agonist activity at concentration as low as 3 um. both were able to induce differentiation of chick dorsal root ganglia (drg) and to stimulate the tyrosine phosphorylation of trka but not of trkb receptor. in addition the l1l4 peptide was able to induce the pc12 cells differentiation into sympathetic-like neurons. moreover the l1l4 peptide was shown to reduce neuropathic behaviour and restore neuronal function in a rat model of peripherical neuropathic pain, thereby suggesting a potential therapeutic role for this peptide. short proline-rich peptides interact with sh3 domains, exhibiting little or no secondary structure before their binding to the cognate proteintargets. under these conditions the binding process of a proline-rich peptide with the sh3 domain shows unfavorable binding entropy, likely resulting from a loss of rotational freedom on the formation of the ppii helix. with the aim of stabilizing the ppii helix conformation in sh3 binding motifs, in the previous years, we replaced the proline residues of the hpk1 proline-rich decapeptide, ppplppkpkf (p2), either with 4-r-(fp) or with 4-s-(fp) fl uoro-l-proline at different i, i+3 positions. the interactions of the fl uoroproline-peptides with the sh3 domain of cortactin protein were analyzed quantitatively by non-immobilized ligand interactions assay by circular dichroism (nilia-cd), whereas cd thermal transitions were measured to correlate their propensity to adopt ppii helix with their affi nity for sh3. results show that although the introduction of the fp residue stabilizes the ppii helix conformation of peptides in a position-dependent manner, the induction of a stable peptide conformation does not increase the ligand affi nity towards the sh3 domain of cortactin. to explore the effect of electron-withdrawing substituent in the pro residue, the fp residues of p2 were replaced by the natural amino acid 4-r-hydroxy-proline (hyp). unexpectedly, nilia-cd and cd thermal transitions results showed that the hyp-containing peptides exhibit a stable conformation in aqueous buffer and k d values lower than the corresponding fp peptide-analogues. in particular, hyp3 peptide containing hyp residues at i, i+3 and i+6 positions adopts the greater percentage of ppii helix conformation over the entire studied temperature range and shows a k d value comparable to that of the parent p2 peptide. we are now searching for inhibitory peptides able to reduce survivin function in breast cancer cells. we take advantage of the observation that survivin forms homodimers in vivo and have derived short peptides representing the dimerization domain. to enable intracellular expression and visualization of these short peptides, they will be fused to a fl uorescent carrier protein. alternatively, the peptides comprising the dimerization domain will be inserted into a scaffold protein. this allows the presentation of the peptides in a constrained and stabilized conformation. we will analyze the effects of lentiviral expression of these fusion peptides in cancer cells expressing high levels of survivin. the peptides will be analyzed for their ability to inhibit proliferation, cell-cycle progression and/or to induce apoptosis in cancer cells. cd11c/cd18 ( x 2) is a member of the leukocyte integrin (cd18 or 2 integrin) subfamily of adhesion molecules and it is expressed on monocytes, macrophages and granulocytes. its ligands include fi brinogen, ic3b, lps, collagen type i and denatured proteins as well as intercellular adhesion molecules icam-1 and icam-4. icam-4 is a red cell specifi c membrane glycoprotein that was fi rst described as anerythrocyte blood group antigen lw (landsteiner-wiener) and later it was found to belong to the family of intercellular adhesion molecules. the fi rst reported receptors of icam-4 were leukocyte integrins cd11a/cd18 and cd11b/cd18. later it has been reported to bind to several other integrins as well. latest reported interaction is to cd11c/cd18. the binding of icam-4 to integrins expressed in macrophages might clarify some of the controversy concerning the recognition and uptake of senescent red blood cells in spleen. another function of icam-4/ macrophage integrin interactions could be in retaining the maturing red cells in bone marrow until they are ready to be released in the circulation. adhesion between icam-4 and integrins have also been found to be important in different pathological situations and inhibition of these adhesion events could be of important therapeutic value. we have studied the interaction between icam-4 and cd11c/cd18 using peptides derived from both binding partners using pepspot method and synthetised peptides. the inhibitory or activating role of these peptides could beof clinical importance in pathological conditions where abnormal red cell adhesion is observed. the regulatory effects of relaxin in mammals realize via receptors of the serpentine type. in this work we studied the relaxin activating effect on the adenylyl cyclase (ac) activity in the rat tissues and muscle tissues of invertebrates using a peptide strategy. the strategy involved the synthesis of the peptides 619-629 and 615-629, as well as the palmitatemodifi ed peptide 619-629, all corresponding to the c-terminal region of the third intracellular loop (c-icl3) of the human type 1 relaxin receptor lgr7. the peptides 615-629 and 619-629-lys(palm) had a dose-dependent activating effect on the ac activity and gtp-binding in myocardium, brain and, to a smaller extent, skeletal muscles of rat. the 619-629-lys(palm) had a stronger ac effect than the longer peptide 615-629, because of the possibility to be anchored in the membrane by the hydrophobic radical and, hence, to interact with the g protein more effectively. in mollusks and earthworm muscles, the stimulatory effects of both peptides were weak. competitive inhibition of the stimulating effects of relaxin by the lgr7-derived peptides indicates that the relaxin signal in myocardium and brain is transmitted to ac via lgr7 receptor. in skeletal muscles of rat and muscle tissues of invertebrates, the inhibition of ac and gtp-binding stimulating effects of relaxin by peptides was almost indiscernible. it can be concluded that relaxin controls the ac of these tissues via the receptor different from the relaxin receptor lgr7. the stimulating effects of lgr7-derived peptides also decreased in brain and myocardium in the presence of ñterminal peptide 385-394 of mammalian g s -subunit and after cholera toxin treatment. thus, relaxin stimulates ac via lgr7 receptor and g s protein in myocardium and brain, and the coupling between receptor and g s protein is mediated by the interaction of receptor ñ-icl3 and ñ-terminal segment of g s . acknowledgements: supported by rfbi (grant 06-04-48809) and «russian science support foundation». normal tissue function depends on adequate supply of oxygen through blood vessels. angiogenesis is a fundamental process by which new blood vessels are formed and which is highly regulated in healthy individuals. however, many diseases are driven by unregulated angiogenesis. excessive angiogenesis is associated with cancer, rheumatoid arthritis, psoriasis, while insuffi cient angiogenesis results in ischemia or artherosclerosis. many new angiogenic modulators have been developed in the last years, mostly to inhibit angiogenesis but only few peptide-based angiogenic stimulators have been reported. there are data in the literature suggesting roles of small peptides or basichexa peptides as angiogenic modulators like ringseis et al. reporting effects on endothelial cell function (such as ec proliferation) and fazekas et al. describing effects for the latter. endostatin, an endogenous inhibitor of angiogenesis, among its proteolitic fragments contains both, an inhibitor and a stimulator of angiogenesis. we considered it possible that fragments of basic heptapeptide d-phe-cys(-)-tyr-d-trp-lys-cys(-)-thr-nh 2 (tt-232), a strong antitumor agent, could also act as angiogenic modulators. the heptapeptide was developed in our laboratory and has been recently in a clinical trial (phase ii). we synthesised, characterized and tested partially protected di-and tripeptide fragments of it such as boc-d-phe-cys(acm)-tyr-ome, d-phe-cys(acm)-tyr-ome, boc-tyr-d-trp-cyclohexilamide and h-tyr-d-trp-2-adamanthylamide. the biological activity of the compounds was tested in vitro using an immortalized kaposi sarcoma cell line to determine their pro-anti-angiogenic character. to test the angiogenic potential of the best compound, the aorta ring assay was used, which by using intact vascular explants reproduces more accurately the environment in which angiogenesis occurs. in this study we report the synthesis and angiogenesis modulating effects of the peptides. somatostatin is a neuropeptide that regulates several functions of the endocrine and exocrine systems. it also affects cell proliferation and neurogenic infl ammation through a family of g protein-coupled receptors. neurogenic infl ammation plays signifi cant role in the pathogenesis of numerous infl ammatory diseases (such as asthma, arthritis, allergy and migraine). inhibitory effect of somatostatin on infl ammation is well known but the pharmaceutical use of the native peptide is limited due to its broad spectrum of anti-secretory effects and short plasma half-life time. tt-232, a heptapeptide analogue of somatostatin (developed by our research group and it is in clinical phase ii trials) has selective antitumour and anti-infl ammatory effect without regulating other endocrine or exocrine processes. receptor-ligand binding experiments with various analogues of the somatostatin as well as tt-232 [d-phe-c(cys-tyr-d-trp-lys-cys)-thr-nh 2 ] verifi ed that the side chains of tyr, d-trp and lys are important pharmacophoric groups for somatostatin-like biological activity. linear, cyclic and branching derivatives were designed and synthesised using above amino acids to selectively inhibit infl ammatory actions. unnatural moieties also were applied to enhance their enzyme resistance. linear and cyclic peptides consist tyr and d-trp, while ring closed ones contain lys too. branching peptidomimetics have the same, fl exible core [tris(2aminoethyl)amine] and three protected or unprotected amino acids situated in equal positions. the biological activity of the compounds was evaluated by in vitro assay of substance p release and in vivo assay of plasma protein extravasation. the most potent agents strongly inhibited substance p release by 90 -95 % and one of them showed strong anti-infl ammatory activity when administered orally. the structure of the novel compounds and the relationship between their structure and biological activity will be discussed. urotensin ii (u-ii), a potent vasoconstrictor, is found in diverse species, including human. several biological studies indicate that u-ii is the most potent mammalian peptide vasoconstrictor reported to date, and it appears to be involved in the regulation of cardiovascular homeostasis and pathology. in order to elucidate the importance of trp residue for receptor interaction and biological activity recently we have designed, synthesized new analogues where trp7 was replaced with constrained analogues ltpi or dtpi (1,2,3,4-tetrahydronorharman-3-carboxylic acid). the tpi residue was replaced in both agonist p5u and antagonist urantide sequences. on these new ligands we performed biological and nmr conformational studies. the new ligands will be used in further biological investigations of the ut receptor. gnrh analogs as carriers for targeted suicide gene delivery suicide gene therapy represents one of the promising approaches to the cancer treatment. an application of herpes simplex virus thymidine kinase (hsvtk)/ganciclovir system possesses additional advantage due to bystander effect on neighboring cancer cells. overexpression of gnrh receptors in the case of most adenocarcinomas creates the basis of gnrh analogs use as carriers for targeted suicide gene delivery. we investigated different manners of gnrh molecule modifi cation; their infl uence on peptide/dna complex formation and its penetration into cancer cells. analogs, containing nls from large antigen of sv40 were synthesized using combination of boc-and fmoc-chemistry. depending on peptide structure (agonist or antagonist) nls was attached via position 6 or 1 of the natural molecule. the competition experiments demonstrated that internalization of peptide/ dna complex into hepg2 cells is mediated by specifi c receptor binding. moreover, nls/dna complexes, lacking gnrh moiety were unable penetrate cellular membrane. subsequent studies permit to identify the infl uence of analog structure on in vitro effi ciency of suicide gene therapy, followed by acyclovir treatment. it was shown that application of reference peptide gene delivery system damaged about 50% of tumor cells. use of cationic peptide conjugated with rgdf sequence provides high effi ciency of treatment, however can not ensure selective action on cancer cells. gnrh analogs completely suppress tumor growth in vitro due to specifi c interaction of peptide/dna complex with correspondent receptor. the effi ciency of suicide gene therapy depends on peptide structure and is in favor of agonists as compared to antagonists. preliminary data of experiments in vivo on laboratory animals demonstrated practical utility of tested peptides in the course of intravenous administration. thus, it was shown that gnrh analogs containing nls moiety represent promising candidates for the delivery of hsvtk gene into the cancer cells. the structural-functional study of putative grape (vitis vinifera) uncharacterized protein sequences was performed. we developed a special method of computer analysis (1) for this. this method has allowed to reveal new potentially active regulatory oligopeptide sequences yet not investigated experimentally. information on grape amino acid sequences of public databases [2, 3] , computer database erop-moscow (endogenous regulatory oligopeptides) 4. containing the information on structure and functions of known natural oligopeptides and specially created computer programs were used for this. protein amino acid sequences were compared with all known oligopeptide sequences in this method. as a result several tens of grape oligopeptide sequences were elucidated. the similarity of their sequences with the known oligopeptide structures of other biological species was the basis for the prediction their potential functional properties. it has been shown that grape contain putative regulatory oligopeptides possessing functions of antibacterial and antifungal agents, enzyme inhibitors, calmodulin binding structures, rapid alkalinization factors, etc. the primary structure similarity of grape sequences was found not only with plant species but with bacteria, fungi, and animals also. cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. their stability derives from a headto-tail cyclised backbone coupled with a cystine knot arrangement of the three-disulfi de bonds. taking advantage of this stable framework we developed novel vegf-a antagonists by grafting a peptide epitope involved in vegf-a antagonism onto the stable cyclotide framework. antagonists of this kind have potential therapeutic applications in diseases where angiogenesis is an important component of disease progression, including cancer and rheumatoid arthritis. a grafted analogue showed biological activity in an in vitro vegf-a antagonism assay at low micromolar concentration and importantly the in vitro stability of the linear epitope was markedly increased using this approach. in general, the stabilization of bioactive peptide epitopes is a signifi cant problem in medicinal chemistry and in the current study we have shown the cyclotide framework is ideally suited for such stabilization. cystine rich scaffolds are emerging as valuable templates in drug design and in the current study we have shown that the cyclotide scaffold, with the advantageous features of a knotted disulfi de core and a cyclic backbone, has signifi cant potential in stabilizing a wide range of bioactive peptide epitopes. gonadotropin releasing hormone (pglu-his-trp-ser-tyr-gly-leu-arg-pro-gly-nh2, gnrh) plays a signifi cant role in the controlling of gonadotropins and steroids hormones. a large number of linear gnrh analogues has been synthesized and tested for several medical uses. leuprolide acetate (pglu-his-trp-ser-tyr-(d)leu-leu-arg-pro-nhet, lpa) is a potent gnrh agonist and is used to treat a wide range of sex hormone related disorders, including prostatic cancer, endometriosis and precocious puberty. despite its widespread use, only limited information based on spectroscopic evidence regarding the solution conformation of leuprolide are known. moreover, non crystallographic data is available for the receptor of gnrh (g protein-coupled receptor). the aim of this study was to characterize the conformation of leuprolide and its modifi ed linear analogue (pglu-his-trp-ser-tyr(ome)-(d)leu-leu-arg-aze-nhet) in dmso solution (which simulates better the receptor environment) using nuclear magnetic resonance (nmr) and molecular modeling techniques. by using both nmr and molecular modeling we have characterized the secondary structural preferences of these gnrh analogues. structural determinants of binding to the mu-opioid receptor -an important target in analgesia -attracts great scientifi c attention. many natural and synthetic peptides and peptidomimetics were shown previously to bind to the mu-opioid receptor selectively but there is no consensus about what structure is responsible for such biological activity. no high resolution structure of this receptor is available and the binding site of ligands is not exactly known despite numerous site-directed mutagenesis studies. this suggests that the determination of structural aspects of mu-opioid activity should focus on the ligands. mu-opioid ligands with similar affi nity and selectivity should possess at least one common structural feature in which they differ from other ligands of different affi nity and selectivity. comparative structural analysis of such ligands, considering adequate representation of binding conditions may reveal key features of bioactivity. in this study ten mu-opioid receptor ligands, damgo, tyr-w-mif-1, morphiceptin, endomorphin-1 and 2 and their analogues, possessing different affi nity and selectivity were examined using molecular dynamics. conformational preference of these molecules was determined in aqueous and dmso media which were meant to model different possible binding environments. no structural trend, correlating with previously measured bioactivities was observed in aqueous media. in dmso it was found, that a preference for trans orientation of the tyr1 side chain and gauche (-) orientation of the third aromatic side chain, the free rotation of the phe4 side chain and a high propensity of bent backbone structure is favorable for high affi nity binding to the mu-opioid receptor, while deviations from these criteria results in variable loss of bioactivity. constellation of these four key conformational parameters may be a guiding principle in the future for the design novel mu-opioid receptor ligands. in 5 cui-catalyzed azide-alkyne 1,3-dipolar huisgen's cycloaddition -prototypic "click reaction"-is a recently developed synthetic procedure to obtain cyclopeptides carrying, as a rigid linking unit, the lactam bioisoster, 1,4-disubstituted [1, 2, 3] triazolyl ring (1). we have recently reported the synthesis and conformational analysis of the 1,4-disubstituted[1, 2, 3] triazolyl containing cyclopeptide derived from the sequence of the potent i-to-i+4 side chain-to-side chain lactam-containing antagonist of parathyroid hormone-related peptide (pthrp) 2.. the conformational properties of triazolyl-containg peptide were compared to those of the corresponding lactam analog. cd and nmr studies revealed that, despite a slight difference of the backbone arrangement, triazolyl containing cyclopeptide and lactam-containing cyclopeptide share a common orientation of the side chains (3). here we present the structural study of a new library of disubstituted[1, 2, 3] triazolyl containing cyclopeptides designed to obtain different cycle dimensions and different triazolyl-ring positioning. cd and nmr analysis in different solvent systems shows that both, the dimension of the cycle and the specifi c positioning of [1, 2, 3] triazolyl ring are critical to fully resemble the conformational properties of the potent lactamcontaining antagonist of parathyroid hormone-related peptide (pthrp). the somatostatin (srif) is a cyclic tetradecapeptide, which exerts inhibitory effects on the secretory processes in the endocrine and exocrine systems, and the cell proliferation through somatostatin receptors (sstr1-5). sstrs are distributed throughout human body, not only in normal cells but also in tumor cells. most of the somatostatin analogues developed for clinical use, such as octreotide which act longer than somatostatin are being used in the diagnosis and treatment of endocrine tumors. the use of these analogues as antitumor agents has been limited because of their antisecretory effects and poor oral bioavailability. tt-232 [d-phe-c(cys-tyr-d-trp-lys-cys)-thr-nh 2 ], was reported by keri et al. to have potent antiproliferative activity without antisecretory action 1.. based on the above, we aimed to design and synthesize somatostatin analogues with more potent antiproliferative activity and high oral bioavailability. we synthesized pyrazinone ring containing cyclic peptides (2) and linear peptides which are substituted at the c-terminus lys with hydrophobic and rigid groups. our focus was on the active sequence: tyr-d-trp-lys. we also examined their antiproliferative activity and found that boc/h-tyr-d-trp-1-adamantylamide exhibited the most potent antiproliferative activity higher than that of tt-232. furthermore, on the best analogues we studied dna fragmentation by facs analysis and cellular morphology. the results demonstrated that these somatostatin analogues induced cell death by apoptosis. 6 kobe gakuin universit, japan background and aims: endomorphin-2 (em-2: h-tyr-pro-phe-phe-nh 2 ) has high affi nity and selectivity for the mu opiod receptor (1). we focus on the pro residue, which imposes strong restraints on the conformation of the peptide chain or induces cis-trans isomerization of x-pro bonds. we substituted the pro with 2-azetidinecarboxylic acid (aze) or piperidinecarboxylic acids (pip) to increase or decrease the conformational fl exibility. in this paper, we deal with the synthesis of em-2 analogues containing aze and pip and the evaluation of the biological functions of the analogues on the opioid receptors. methods: the synthesis of peptides was achieved according to the procedure of okada y. et al. (2) the fi nal products were identifi ed by maldi-tof mass spectrometry and elemental analyses. the receptor binding activity of peptides was assessed by radio-ligand receptor binding assay using mu and delta opioid receptors from cos-7 cell membranes expressing each opioid receptors. for the evaluation of the biological function of peptides, the gpi and the mvd tests were performed. the new analogue of csf114(glc) 1., [pro 7 ,asn 8 (glc),thr 10 ]csf114 is characterized by a type i -turn around the minimal epitope asn(glc), and it was demonstrated to show the highest antibody affi nity in competitive elisa in multiple sclerosis (ms) patients' sera and thus it appears as a promising tool for the detection in patients' sera of specifi c autoantibodies. in previous studies, we synthesized and tested different fragments of this new glucosylated peptide, [pro 7 ,asn 8 (glc), thr 10 ]csf114, identifying the shortest sequence [pro 7 ,asn 8 (glc),thr 10 ] csf114(5-11) able to detect antibodies by elisa in ms patients' sera. this heptapeptide is characterized by thr in position 10 generating a characteristic n-glycosylation consensus sequence. according to the results obtained with the linear heptapeptide in ms, we applied the backbone cyclicization method to develop two backbone cyclic libraries of glycopeptides based on the sequence of the linear hepta active peptide. backbone cyclization is a method that allows obtaining cyclic peptides without changing the natural sequence or the chemical character of the amino acid residues, in order to enhance activity, stability to metabolic degradation, selectivity and bioavailability (2) . the fi rst library contains a gly building unit at the c-terminus and is connected to the n-terminus by linker of various lengths (n=2, 3, 4, 6) . in the second library, his 9 of the heptapeptide is replaced by gly building units with various alkyl chains (n=2, 3, 4, 6) [fmoc-n (n alloc(n-alkyl))gly-oh] that is connected to the n-terminus by linker of various lengths. twenty cycloglycopeptides were synthesized, and screened by competitive elisa s on ms patients' sera to select the most bioactive cyclic glycopeptide. peptide nucleic acids have become, arguably, one of the most interesting of dna mimics. owing to their high chemical stability and resistance towards nucleases and proteases, they are very attractive as antigene/ antisense agents, molecular biological tools and for genetic diagnosis. the lack of charge and polar groups in the backbone decrease their solubility in aqueous environment and their ability to cross cell membranes, reducing their performance in in vivo applications. in order to overcome these problems -to improve solubility, increase affi nity and specifi city of binding, a number of analogues were synthesized. this study describes the synthesis of pna-monomers on the base of non-protein amino acids analogues of basic amino acids lys and arg. nucleobases are the second residue we have selected. studies will include replacement for example of the uracil, with the fl uorinated analogue. growing evidences indicate that n-glycosylation is a co-translational modifi cation that, either native or aberrant, may play a fundamental role in a large number of biological events. in particular among postand co-traslational modifi cations glycosylation plays a crucial role in the immune system. in fact, almost most of all the key molecules involved in the immune response are glycoproteins. there are growing evidences of defects in glycosylation with diseases that assets to pathway oligosaccharides as code words. in previous studies, we demonstrated that the presence of a -dglucopyranosyl moiety on an asn residue at position 7 of csf114(glc) is fundamental for auto-ab recognition resulting the fi rst multiple antigenic synthetic probe (msap) able to detect autoantibodies in ms patients' sera. up to now we have investigated the carbohydrates infl uence and specifi city in ms antibody recognition introducing several glycosilated building blocks in the msap sequence (i.e. glc, man, glc glc, gal, glcnac on the side chain of ser, thr, asp, glu, hypro) 1.. due to microheterogeneity and the extremely high specifi city of carbohydrateprotein interaction we included in our library screening, the ribose. we report the synthesis of new asn-derivatives bearing on the side chain ribose rib and rib linked by an n-glycosidic bond and protected for spps.these building blocks introduced in the csf114 -turn scaffold lead to the new ribosylated peptides contributing to the library of glycopeptides to fi shing out families of autoantibodies specifi c for different autoimmune diseases. fraczyk, justyna; kujawska, nina; kaminski, zbigniew, j. we designed and prepared supramolecular structures formed from nlipidated oligopeptides immobilized in the regular pattern on the cellulose surface which are able to specifi c binding of ligand molecule. due to the conformational fl exibility of the fragments forming the supramolecular structure, the shape and prosperities the binding cavities are adjusted the most effectively to requirements of the guest molecules. the previous studies documented that process of binding guest molecules is highly selective, reversible and competitive. therefore, we supposed that under favorable circumstances the structures could operate as catalysts if suitable molecular fragment are included inside the binding pocket. in order to verify this hypothesis we prepared library of supramolecular hosts with catalytic triade: his asp(glu) ser, incorporated into the binding pocket 1.. for the fi rst generation library the rate of hydrolysis of p-nitophenyl esters of n-protected amino acids was measured by spectrophotometric determination of liberated p-nitrophenole in buffered, aqueous methanol and compared with appropriate data obtained in the absence of catalytic structures. the most active catalyst were selected from the library and their stability, selectivity and ability for re-use was studied. for the second generation of library the stereoselectivity of artifi cial esterase we present here a new technique for identifying very small quantity of peptide mixtures that are selected out from a peptide library in solution, by using multi-component fl uorescence labeling. the technique is basically a modifi cation of positional screening method associated with a 1:1 correspondence between the amino acid at the i-th position and the type of the fl uorescence lebel at the n-terminal. 2-dimensional fl uorescence spectroscopy was employed for identifying the fl uorescence labels in the mixture of peptides that bound to target cells or target proteins. the results of the new screening method will be presented for peptides that specifi cally bind to human cancer cells. show increased biological activity in a dimeric form1. in recent years there have been signifi cant efforts to obtain minimized versions of naturally occurring proteins such as dimeric dna binding proteins which retain their function. the gcn4 basic region peptides were connected trough a disulfi de bond to give a dimer which specifi cally bound the ap1-dna sequence. dimeric peptides and proteins were obtained also by non covalent interactions.2 in this work we propose a strategy for obtaining by expressed protein ligation (epl), one pot protein homodimers covalently connected at the c-terminus. the synthetic strategy was extended also to the synthesis of heterodimers. epl is a protein engineering tool for the chemo and region-selective modifi cation of proteins based on the use of intein containing constructs.3 in this work dimers were obtained by reacting a new bi-functional linker with carboxyl-activated polypeptides. we synthesized a linker containing two cysteines in a n-terminal-like position, separated by an ethylendiamine spacer, and obtained thioester proteins by intein mediated splicing reactions. this strategy affords chemically stable dimeric proteins. the linker can be easily modifi ed at need, changing the lenght and rigidity of the spacer between the cysteines. this strategy has potential in biochemical and bioorganic applications, for obtaining minimized and/ or modifi ed natural proteins and for joining two different proteins at the c-terminus position. this technique will be extended to the synthesis of dimeric proteins mimicking the transcription factor ap-1. 1 previous studies showed that csf114(glc), 2,3 a designed glycopeptide characterized by a -d-glucopyranosyl moiety, can detect and isolate specifi c autoabs in sera of a signifi cant number of ms patients. this synthetic ag could be considered a mimetic of aberrantly glycosylated myelin proteins triggering autoimmunity in ms. myelin oligodendrocyte glycoprotein (mog) is considered a putative autoag in ms. 4 our aim is to obtain mog properly glycosylated to characterize the molecular mechanisms of ab-mediated ms and to design new antigenic probes to detect autoabs as biomarkers. production of specifi c glycoproteins may benefi t from a chemical approach, such as expressed protein ligation (epl) a protein engineering strategy useful to introduce noncanonical amino acids and biological probes into proteins. 5 epl allows synthetic and recombinant polypeptides to be chemoselectively and regioselectively joined together. the recombinant rmog ed (1-97), obtained as c-terminal thioesther by protein splicing, will be ligated to the peptide fragment [gly 103 ,a sn 104 (glc)]mog ed (98-117) bearing a cys residue at the n-terminus. an alternative strategy exploits the selective reaction between a glucosyl iodoacetamide derivative and the cys free thiol of a protein. 6 a site directed mutation has been performed on rmog to introduce a cys residue at its native site of glycosylation. the semi-synthetic proteins will be tested by elisa using ms patients' sera. automation is an identifi ed goal in the peptides r&d at lonza. this approach would facilitate and accelerate peptide production. this is highly desired within peptides r&d, where the need exists for rapid synthesis of peptides to fulfi l iso and gmp projects requirements. automated peptide synthesisers are available on the market but many have limitations which make them inappropriate for lonza (e.g., low scale, coupling systems limitation, pre-activation procedure at low temperature). furthermore, there is no obvious standard instrument for scalable spps equipped with pat. ge healthcare provides scalable and complete solutions for automated solid-phase synthesis of oligonucleotides from small research amounts to full commercial production (1 mol-1mol) based on the äkta, oligopilot 400 and oligoprocess™ platforms. the unicorn™ software provides control and monitoring of the processes. ge healthcare synthesisers are designed around fl ow-through column reactors, giving faster kinetics and lower solvent and reagent peptide biotechnology and diagnostics consumption compared to batch synthesisers. based on experience with scale-up of oligonucleotides, ge healthcare and lonza are confi dent that the fl ow through-column technology can be scaled up for peptides to a cost effi cient process. this is a strong argument that the potential of the äkta platform as a peptide synthesiser should be explored. ge healthcare was approached by lonza to develop a peptide synthesiser based on the äkta platform instrument to compete with the state of the art in the peptide fi eld. the aim is to develop the fl uidics, programming and chemistry methods of the äkta system to lonza's needs for routine production of 0.4g to 2g of crude peptide, and later scale up to 2 kg. this synthesiser, controlled by the unicorntm software, has the capability to perform synthesis using on-line mixing or pre-activation at different times and temperatures of amino acids, coupling reagents, solvents and additives. results from peptides will be presented. neuropeptides are produced from precursor proteins by selective cleavage at specifi c sites. classical biosynthetic cleavage occurs at basic residues due to the activity of a small number of well-known proteases. however, with the discovery and characterization of new neuropeptides, a new non-classical pathway has been described with cleavage occurring at tryptophane, leucine and other residues. neuropeptide-processing peptidases involved in this new non-classical pathway are completely unknown but essential for correct processing of certain neuropeptides. therefore, we are interested in identifying proteases involved in the non-classical pathway using activity-based proteomics. for this purpose, we fi rst designed and synthesized some peptides based on the sequence the mature neuropeptides described in the literature but with an active functional group able to bind the active site of the proteases of interest. bound peptides were labelled with biotin or rhodamine by click chemistry, and the brain and pituitary proteome was then characterized by in-gel analysis and multidimensional nlc-ms/ms. several proteases that may be involved in neuropeptide processing have been identifi ed in this experiments. further studies concerning cloning, protein expression and activity assays will confi rm their role in biological tissues. the incidence of autoimmune diseases continues to increase. although an enormous research effort has been directed in understanding this increment, etiology of human autoimmunity remains enigmatic. primary biliary cirrhosis (pbc) is a chronic cholestatic liver disease characterized by destruction of bile ducts and the presence in the serum of antimitochondrial antibodies (ama positive in 90-95% of patients), directed against the e2, which is one of the three component enzymes of the 2-oxo acid dehydrogenase multienzyme complex family chiefl y pyruvate dehydrogenase complex (pdc-e2). environmentally induced co-or post-translational modifi cations of autoantigens are hypothesized to break immune tolerance leading to self reactivity in pbc 1.. in this context it is possible to take advantage of a unique technology that allows the monitoring of peptide epitope modifi cations that will lead to the identifi cation of altered autoantigens that the human host will recognize as foreign, similarly to what recently reported in multiple sclerosis (2) . our approach will lead to new diagnostic tools that can be used in earlier stage patients and possibly to monitor disease activity. herein we report the synthesis of lipoamide and glyco-peptides characterized by a -hairpin structure with the modifi cation on the tip of the -turn as peptidomimetic of native antigens involved in pbc. in order to identify the best synthetic antigens and to clarify the role of the -turn in exposing the modifi cation we compared the antibody recognition in pbc sera by elisa using the modifi ed peptides in comparison with the proposed autoepitope of pdc-e2 [pdh (44-63) ]. the synthetic antigens were able to detect by elisa autoantibodies in 30% of ama negative sera of pbc patients confi rming that ptm-peptides are useful diagnostic/prognostic tools. alzheimer fs disease is characterized by the abnormal accumulation of amyloid peptide (a ) into extracellular fi brillar deposits known as amyloid plaques. a can self-assemble into soluble oligomers, protofi brils, and amyloid fi brils, and all of these aggregated forms contain signifi cant -sheet structure. the core of a (14-23 residues) containing hydrophobic region is a key to promoting a aggregation. in the previous study, we constructed green fl uorescent protein (gfp) variants which have the core part of a sequence on the surface -sheets. it has been demonstrated that the gfp variants inhibit a aggregation 1.. in this study, we have utilized a small protein, insulin-like growth factor ‡u receptor domain 11 (igf2r-d11) as a scaffold, and a part of a sequence was incorporated into the -sheet surface of igf2r-d11. igf2r-kk and igf2r-ka were designed by substituting two a derived sequences for some amino acids in igf2r-d11 as parallel and anti-parallel -sheet models, respectively, of the a aggregates. these insoluble proteins expressed by e coli. were solubilized by denaturing buffer, and the denatured proteins were refolded in conditions permitting formation of native proteins. after refolding, the proteins were purifi ed as monomers by size exclusion chromatography. we have investigated the interaction between a and the designed protein variants by surface plasmon resonance studies. as a result, igf2r-kk bound tightly to a more than igf2r-ka. inhibitory activities of a fi brillization in the presence of igf2r-kk, igf2r-ka, or wild type igf2r-d11 were evaluated by thiofl avin t (tht) binding assay. it was demonstrated that igf2r-kk inhibited a fi brillization effectively. because of their high structural fl exibility. on the other hand, a 2stranded -structure is presumed to be more stable than a single strand as a fi bril forming intermediate. therefore, the aggregation mechanism of prion protein remains to be further studied on their precursor structures. recently, we have established a novel strategy to determine the amino acid sequence for amyloid formation, based on their essential interactions. a number of fi bril forming peptides at positions from 160 to 230 in the amino acid sequence were obtained by our calculation method. in order to confi rm whether aggregates of the candidate peptides consist of the 2-stranded -structure, a series of peptides consisting of g10 residues-turn-10 residues h were prepared. several candidate peptides, of which regions are 166-187, 168-189, 170-191, and 178-199 , showed the typical enhanced fl uorescence intensities in the thiofl avin t-binding assay, suggesting the amyloid formation. ir spectra of these peptides showed the typical bands corresponding to the -structure. in addition, the peptide (178-199) exhibited a shoulder band at 1654 cm -1 in ir spectrum, refl ecting a turn structure. these results revealed that the amyloids of the peptide (178-199) are constructed with the 2-stranded -structure, which may be formed by intra-molecular hydrogen bonds. in conclusion, the results obtained here indicate that the sequence from 178 to 199 could be a key region for the transition from a normal to an abnormal structural state. single-molecule force spectroscopy provides a powerful tool to investigate biomolecular interactions. a different approach measures forces required for breaking a bond in a differential format by comparison with a known reference bond of dsdna (1). here we apply this molecular force balance to the integrin v 3, which is over expressed in ovmz-6 cells. this protein interacts with ligands containing an -arg-gly-asp-(rgd) motive. ligands containing the rgd sequence were made by peptide synthesis and linked to a gcn4-peptide using a peg-spacer. for detection carboxyfl uoresceine was introduced to a lysine side chain between both peptides. the gcn4-peptide can be recognized by specifi c anti-body fragments (2) and can act as a reference bond. these force balances was linked to a pdms-surface, whereas the reference bond is attached to the surface and the rgd-peptide is accessible to the integrin. when the pdms-stamp gets in contact with the ovmz-cells, the interaction of ligand and receptor can occur. by applying a force at the stamp the force balance is stretched and the weaker bond brakes. investigation of the fl uorescence-level on stamp and cells enables the localization of the balance construct and thus an estimation of the bond force. first stamp-experiments on living cells showed that the gcn4anti body interactions are too strong to act as a reference system. since the binding forces of dna are well investigated by afm and small dna-molecules were already used as force sensor, dna can act as a more sensitive reference. thus in a further approach the rgd-peptide was linked to a biotinylated peg. this enables the application of short double-stranded dna as a reference bond via a fl uorescence-labelled streptavidin. using this system the force balance could be transferred to the v 3 integrines located in the cell membrane of ovmz-cells. nanogaps, which allow making electrical contact to structures on the nanoscale, are increasingly used for the preparation of biosensors. the positioning of the synthetic or biological species inside the nanogap must be controlled to reach optimal electrical or detection properties. (1) (2) (3) in this context, the chemical properties of the layer between the electrodes is of prime importance, since they will impose the imbibition of the nanogap and permit the formation of chemical bonds between the molecules of interest and the substrate. thin fi lms can be characterized by a variety of physical or chemical methods. however, the characterization of the chemical properties of nanogaps is complicated because of the small size of the substrate delimited by the electrodes. in this context, novel experimental tools are needed for probing rapidly the chemical reactivity of nanogaps. we show here for the fi rst time that a specifi c functional group in a 30-90 nm nanogap can be detected by combining peptidecapped gold nanoparticles and electrical detection.(4) a semicarbazide layer and semicarbazone chemoselective ligation was used in this proofof-concept study, which thus required the preparation of stable peptidecapped gold nanoparticles modifi ed by aldehyde groups and control gnps derivatized by amide groups. the chemoselective insertion of gold colloids into the nanogaps led to current increases from 2 to 4 orders of magnitude, in accord with the number of gold nanoparticles in the nanogaps detected by scanning electron microscopy. 3 we present the electrical detection of immunoglobulin g (iggs) from human serum using a nanogap-based biosensor. the detection method is based on the capture of iggs by a probe immobilized between gold nanoelectrodes of 30 to 90 nm spacing. the captured iggs are further reacted with secondary antibodies labelled with gold nanoparticles (gnps). insertion of gnps into the nanogap resulted in increasing the conductance through the nanogap. the use of a chip with ninety nanogaps enabled the calculation of a quality factor for the detection which, coupled with a non-linear regression analysis of the data, easily discriminated specifi c and differential capture of human antibodies by arrayed probes. we obtained a 500-fold higher quality factor with protein a compared to goat anti-murine antibodies. this method can be applied, through these proof-of-concept experiments, to the detection of protein-protein interactions in biological samples. in the past several decades, hundreds of peptides have been identifi ed which have specifi c biological activity and are highly potent in in vitro assays but lack the prerequisite pharmacokinetics to become effi cacious human therapeutics. we have developed a novel antibody-based platform technology that provides an improved pharmacokinetic profi le for biologically active peptides, resulting in a long duration of action. one such mimetibody™, cnto 528, is a novel erythropoietin (epo) receptor agonist. although cnto 528 bears no sequence homology to erythropoietin, it is a potent erythropoietin receptor agonist, rescuing epo dependent cells from apoptosis in vitro and stimulating erythropoiesis in vivo. studies were done in normal rats to explore the pharmacodynamics and pharmacokinetics of cnto 528 in normal rats and to demonstrate its effi cacy in rat models of anemia. in vitro, cnto 528 was approximately 10 fold less potent than rhepo in stimulating the growth of ut-7epo cells. despite this lower in vitro potency, when compared to rhepo and darbepoietin in normal rats, a single subcutaneous dose of cnto 528 resulted in a longer-lived reticulocytosis and longer-lived increase in hemoglobin. also, cnto 528 caused only minor changes in red cell distribution width (rdw) or mean cell volume (mcv) and led to the release of mature reticulocytes. we have also shown that cnto 528 was effi cacious in rat models of anemia and in a rat model of pure red cell aplasia. taken together, our data show that cnto 528 is a novel stimulant of erythropoiesis in rats. this platform has been applied to other biologically active peptides as well and has proven to be a robust platform for enhancing the pharmacokinetics of peptides that would otherwise be rapidly cleared. cell penetrating peptides (cpps) have been recognized as promising tools for the delivery of different therapeutic molecules. previous studies in our laboratory have shown that the s4(13)-pv peptide accumulates inside cells very effi ciently through a rapid, dose-dependent and nontoxic process. formulations based on the s4(13)-pv cell penetrating peptide presented great potential for the delivery of plasmid dna, which may prove useful for gene-based therapies. in the present work, we aim to 1) investigate the relevance of the dermaseptin-derived sequence and of the nuclear localization signal to the effi ciency of the overall process of plasmid dna delivery by the s4(13)-pv and related peptides; 2) compare the potential of the s4(13)-pv peptide to mediate plasmid dna delivery with that of the extensively studied tat cpp. a comparative analysis of the transfection effi ciency mediated by the systems based on the s4(13)-pv, reverse nls and scrambled peptides was performed in tsa and hela cells. in general, for both cell lines, the reverse nls peptide mediated transfection at effi ciencies comparable to those observed for the s4(13)-pv peptide. however, transfection mediated by the scrambled peptide was signifi cantly less effi cient than that obtained for the s4(13)-pv and reverse nls peptides. to compare the biological activity of the s4(13)-pv with that of the tat peptide, we transfected tsa cells with various s4(13)-pv-and tatbased formulations. our results have shown that both peptides enhanced the activity of cationic liposome-based systems. above a threshold peptide/cationic liposome/pdna charge ratio (10/1/1), the enhancing effect was independent of the peptide used, although for the lowest charge ratios, this effect seemed to be more relevant in the case of the s4(13)-pv peptide. higashi, nobuyuki; kawamura, yoko; koga, tomoyuki doshisha university, japan the aim of tissue engineering is to replace failed organs with new functional tissue and organs. to realize this, materials are needed, which can direct the growth of cells to generate new tissue. to control and direct cell behavior, a defi ned biomimetic environments is needed, which surrounds the cells and promotes specifi c cell interactions. the rgds sequence has been recognized as the cell attachment site of the natural extracellular matrix. the purpose of this study is to fabricate rgdscarrying nanoscaffords towards cell adhesion using a self-assembling technique. here we synthsize two types of rgds-based materials, one of which is an amphiphilic triblock peptide composed of leu and lys (rgds-l4k8l4) that has been revealed to self-assemble into ƒà-sheet nanofi ber under specifi c conditions 1., and another one is a rgdsended polystyrene (pst-rgds) that is a typical artifi cial polymer. these peptide and peptidomimetic were prepared by solid phase synthesis using fmoc-chemistry and by coupling fmoc-chemistry and atom-transfer radical polymerization (atrp) method, respectively. rgds-l4k8l4 was found to self-assemble into ƒà-sheet-based nanofi ber at ph 9.6, and by lowering ph the conformational change from ƒà-sheet to random coil structure was induced, giving no aggregation of the peptide. when mouse nih/3t3 cells were seeded on the rgds-l4k8l4 nanofi bercoated plate, successful cell adhesion and spreading were observed, but not for the rgds-l4k8l4 random coil-coated plate, indicating the importance of the rgds sequences densely and regularly located at the nanofi ber surface. the utility of pst-rgds nanofi lms will be discussed, in comparison with that of nanofi bers. the role of deiminated protein antigens in the diagnosis of rheumatoid arthritis autoantibodies directed against citrulline-containing peptide (acpa) have high specifi city of rheumatoid arthritis (ra). citrullinated proteins are formed by posttranslational modifi cation, namely by deimination of arginine residues in protein sequences by peptidylarginine deiminase enzymes (padi). autoantibodies to deiminated (citrullinated) proteins are the most specifi c serological markers of rheumatoid arthritis. rheumatoid arthritis symptoms develop gradually, and it is diffi cult to precisely date the beginning of the disease. these antibodies are detectable already years before the fi rst clinical symptoms of the disease. the aim of our study was to identify the epitopes of vimentin and fi laggrin derived-peptides targeted by ra specifi c antibodies to provide further information about the nature of the initial autoantigenic substance. we used conventional solid-phase peptide synthesis (fmoc strategy) carried out on "multipin ncp" (chiron mimotopes peptide system) non-cleavable kit. identifi cation of epitope structure of antigenic proteins represents one of the major applications of these technologies. the peptides were prepared in duplicates. citrullinated peptides and the nonmodifi ed counterparts containing arginine instead of citrulline residues were synthesized in order to compare their respective reactivities. in the "indirect" elisa experiments the presence of acpa was determined using serum samples of ra patients and healthy blood donors. this series of experiments effi ciently identifi es citrullinated epitopes of fi laggrin and vimentin as a potential antigenic target for ra specifi c antibodies. the determination of epitopes of these proteins could be important for the development of appropriate diagnostics in this most frequent human systemic autoimmune disease. at the present time the human lysosomal enzymes cathepsins b and l, cysteine proteinases of c1 family, attract great attention. they not only take part in protein degradation but also appear to play role in other important physiological processes, for example, antigen presentation, caspase-independent cell death and involved in different diseases such as osteosarcoma, acute pancreatitis, tumor invasion and metastasis. but selective detection of these enzymes is diffi cult because determination of their enzymatic activity is carried out using substrates also correspond to specifi city of trypsin-like enzymes. the chemo-enzymatic synthesis of selective substrates of cysteine proteinases of c1 family was developed. these compounds are chromogenic glp-phe-ala-pna (i), glp-val-ala-pna (ii) and fl uorogenic substrates abz-phe-ala-pna (iii), glp-phe-ala-amc (iv). peptides were obtained in preparative quantities and were characterized by the data of amino acids analysis, hplc, mass-spectrometry, spectrophotometry (i and ii) and fl uorimetry (iii and iv). the specifi c activities of cathepsins b, l and similar enzymes of plants papain, bromelain and fi cain were determined using synthesized substrates and commercial available substrates z-phe-arg-pna, z-arg-arg-pna and bzl-arg-pna. the specifi c activities of enzymes of other classes -serine (chymotrypsin, trypsin and subtilisin), aspartic (pepsin) and metalloproteinases (thermolysin) -were also defi ned with these substrates. it was shown, that glp-phe-ala-pna, glp-val-ala-pna and glp-phe-ala-amc are not detectable cleaved by enzymes of other classes. acknowledgements: this work has been supported by rfbr grant ¹ 06-03-33056à. to implement a new electrochemical biosensor for autoantibody detection in ms we used csf114(glc) analogues, properly modifi ed at n-terminus with ferrocenyl and ferrocenyl-thiophosphine derivatives, as "electrochemical probes" in cyclic voltammetry (2) . the electrochemical properties of ferrocene, coupled to thiophosphine ability to build simple monolayers on gold surfaces, allow peptides to be anchored on the working electrode used for detection. in particular, 4-fcphp(s)abu organometallic amino acid was specifi cally designed to be used directly in solid phase peptide synthesis. the organometallic moiety introduced in the new glycopeptides did not affect autoantibody recognition as demonstrated both in sp-elisa and in inhibition experiments. an electrochemical monitoring was able to detect interactions of the modifi ed glycopeptides with isolated antibodies from ms patients' sera. we demonstrated a detection sensitivity comparable to elisa method. therefore, the new electrochemical probes can be proposed to characterize autoantibodies as biomarkers of multiple sclerosis by a simple, rapid, and reproducible cyclic voltammetry-based diagnostic methodology. troponin is a structural protein complex, located on the thin fi lament of the contractile apparatus. it is composed of three protein subunits: troponin i (24kda), troponin c (18kda), and troponin t (37kda) and exists as isoforms specifi c to the cardiac and skeletal muscle cells, respectively. cardiac troponins are released in the peripheral blood during irreversible cardiac muscle damage in a time-specifi c manner. rapid troponin elisa assays based on the production of specifi c antibodies against the whole complex or individual subunits have been shown to possess suffi cient sensitivity and specifi city for use in the emergency departments. however, their usefulness sometimes is limited by various factors, such as the selection of epitopes for antibodies production, derived from cardiac troponins representing high homology against the skeletal isoforms, interfering blood factors etc. aiming to contribute in the fi eld of developing highly sensitive and specifi c reagents for the detection and isolation of cardiac troponins in the sera of patients with cardiovascular diseases, we selected epitopes derived from the cardiac isoforms for production of antibodies, mainly based on their predicted immunogenicity, the minimum homology against the skeletal isoforms and the lack of interferences of the produced antibodies with various blood factors. the selected sequences were conjugated to the tetrameric sequential oligopeptide carrier (soc 4 ), either by the classic solid phase step-by-step methodology or by chemoselective ligation reactions and the resulted conjugates were used as immunogens for releasing anti-troponin specifi c antibodies. the performed elisa experiments revealed the high affi nity and specifi city of the produced anti-troponin antibodies against the native protein. a chemical reverse approach for autoimmune diseases diagnosis alcaro an increasing number of individuals throughout the world is affected by autoimmune diseases, a large and diverse group of disorders that are categorized by tissue injury or pathology. although the incidence and prevalence of individual autoimmune diseases are not high, the population burdens of the disease are large and underestimated. thus, reliable diagnostic/prognostic tools are necessary for an early diagnosis and for monitoring disease activity. in this scenario, we proposed a 'chemical reverse approach' based on the use of patients' sera to screen synthetic modifi ed peptides. we showed that this approach could lead to the effective identifi cation of specifi c probes able to characterize highly specifi c autoantibodies as disease biomarkers of highly relevant autoimmune diseases such as multiple sclerosis and rheumatoid arthritis [1, 2] . toscana biomarkers is a r&d company involved in the application of the "chemical reverse approach" to different autoimmune conditions for the development of innovative diagnostic/prognostic tests. as a number of autoimmune diseases have been associated with posttranslational modifi cations, which alter the function and immunogenicity of protein/peptide antigens, we are synthesizing and screening focused peptide libraries based on post-translational modifi ed amino acid, conformational and minimal epitope diversity. lead compounds can be thus used as antigenic probes for specifi c recognition of autoantibodies as biomarkers of diseases in the set up of diagnostic/prognostic assays (3). autoimmune diseases are considered now as a plague. in fact some autoimmune diseases previously considered rare are actually increasing their frequency because of an earlier diagnosis (e.g. celiac disease). possibly also an environmental factor (bacterial and/or viral infection) should contribute to autoimmune diseases. the idea we have been investigating for years and more recently proposed by others, is that aberrant post-translational modifi cations, i.e. glycosylation, deimination etc., create neoantigens triggering autoantibodies. this could explain why proteins (both recombinant and isolated), components of target organs or tissues, are failing. anyway, it is evident that one single biomarker will never enable to reach successful diagnostic & prognostic tools. on the contrary, synthetic peptides specifi cally modifi ed (with sugars, citrulline, lipoyl moieties, etc.) are interesting tools to fi shing out of patients' sera these autoantibodies. we have recently reported that this can be effi ciently done following a "chemical reverse approach" 1.. we successfully applied this strategy in the development of the fi rst multiple sclerosis antigenic probe [msap] : an n-glucosylated peptide characterised by a -hairpin structure exposing at the best the minimal epitope asn( -glc) involved in antibody recognition (2) . a wider application of our sap is obtained by its citrullinatation and/or galactosylation (3) useful for rheumatoid arthritis or lipoylation for investigating primary biliary cirrhosis. in addition to being able to show the viral infection from a serum sample within a few minutes, at the point of care, the poc assays are inexpensive, easy-to-perform and do not require special equipment or laboratory. as antigen in the poc assay we used a 24-amino acid peptide in four branches of a lysine core, with the kyvtgin sequence in the middle. the peptide antigen was conjugated to gold particles and absorbed to a fabric ribbon and dried. in the test, a serum sample is added together with buffer, and the solution dissolves the conjugate. a positive result is obtained when the antibodies together with the gold conjugate bind to anti-human-igg on the nitrocellulose, and form a specifi c coloured line which can be detected in the test window. serum samples were collected from patients with acute b19 infection, and control samples were drawn many years after infection; additional control sera came from subjects devoid of b19 antibodies. the assay was shown to be stable in accelerated stability study. the conditions for industrial scale manufacturing were evaluated and the sensitivity and specifi city were addressed, whereby the assay proved to be highly specifi c for acute b19 infection. alzheimer's disease (ad) is a chronic neurodegenerative disease characterized by a progressive loss of memory and cognitive decline, for which the aggregation and plaque-formation by the -amyoild (a ) polypeptide has been identifi ed as a key event. recently, unpaired variable domains of llama single chain antibody (vhh) fragments against a have been found to exert considerable therapeutic potential for ad. vhh represents the smallest antigen-binding unit with a molecular size of ~15 kda, compared to ig-heavy and light chain variable domains, fab fragments and complete igg antibodies. human cystatin c (hcc) is a cystein protease inhibitor present in all human body fl uids which has a propensity to co-associate with a -plaques/fi brils, and has plaque-inhibitory properties. using proteolytic extraction and excision of the llama-vhh-a (1-40) immune complex (e.g., trypsin, glu-c protease) in combination with electrospray ionization (esi)-and maldi-mass spectrometry, the a -binding epitope was identifi ed at the middle-carboxyterminal domain of a , a (17-28). an analogous mass spectrometric approach was employed for the identifi cation of the binding epitopes of the hcc-a -complex, using immobilized hcc and a -affi nity matrices. an almost identical minimal epitope to that of the vhh-anti-a -antibody ( a (17-24) ) was found, which binds to a specifi c c-terminal domain of hcc, hcc(101-117) 1.. the identifi ed hcc epitope peptide was found to specifi cally inhibit a -oligomerization in vitro, in agreement with the a -epitope domain interefering with the a -aggregation. the identifi ed a and hcc epitopes represent new lead structures for designing neuroprotective inhibitors of the a -aggregation process, and for molecular ad diagnostics. using peptide arrays to reveal mechanisms of apoptosis aspp2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. the c-terminus of aspp2 contains ankyrin repeats and an sh3 domain (aspp2 ank-sh3 ), which mediate its interactions with apoptosis-related proteins such as p53 and bcl-2. we have used a combination of membrane-bound peptide arrays and biophysical methods to study the protein-protein interactions of aspp2 at the molecular level in order to reveal their possible role in apoptosis. using peptide arrays, we have mapped the binding interfaces of aspp2 with its partner proteins such as bcl-2, nf-b and other proteins that are involved in apoptosis. we then applied the peptide array results to study profoundly the interactions of aspp2 with proteins from the anti-apoptotic bcl-2 family. we found that aspp2 ank-sh3 binds to bcl-2, bcl-xl and bcl-w at two homologous sites in all three bcl proteins tested: (i) the conserved bh4 motif (ii) a binding site for pro-apoptotic regulators. quantitative biophysical analysis of the interaction with the free peptides revealed that the binding was selective, and the bh4 domain of bcl-2 binds tightest to aspp2. we propose a mechanism in which aspp2 induces apoptosis by inhibiting functional sites of the anti apoptotic bcl-2 proteins. the array screening results also served as a basis for docking studies that resulted in binding model for the complex between the full length protein bcl-2 and aspp2 ank-sh3 . we conclude that the use of combinatorial methods such as peptide arrays, combined with quantitative biophysical techniques, can signifi cantly contribute to better understanding of biological pathways. micron-sized monodispersed polystyrene for synthesis, tagging and biological screening of small molecules peptide arrays are useful tools to characterize antibodies, to determine sequence specifi cities of enzymes (e.g. kinases) or to fi nd interaction partners to given peptide sequences. one popular format for such arrays is a cellulose sheet with hundreds of synthetic peptides bound to it. these spot-arrays have been used successfully in a broad range of applications since their invention 15 years ago 1.. a drawback is the use of large reagent volumes and the limited throughput with only one copy of the library. celluspots™ represent a new method [2, 3] that allows the production of hundreds identical peptide arrays from a single synthesis run on individual membrane disks. the peptides are synthesized on a modifi ed cellulose support which is dissolved in a cleavage-mixture after the synthesis. resulting solutions of peptide-cellulose-conjugates are then spotted onto coated microscope slides by conventional spotting techniques. the identical arrays are useful tools for large, parallel sera screening projects. there is a great importance in biological macromolecules integration within electronic devices. this is since these molecules are expected to be suitable both as the active components of electronic devices, or as guides for bottom-up assembly of hybrid structures. the goal of this research is to study the assembly and electronic properties of novel devices that exploit synthetic protein molecules. here we show the fabrication of de-novo protein based diode-like devices. we have chosen coiled coil protein structures, which can adopt two conformational states that differ in their internal molecular dipole. the proteins have been equipped with surface binding groups, typically cysteine thiols, on both ends. dithiol bridges at one side facilitated self assembly on gold surfaces. protected cys residues were placed on the other end that after deprotection will allow successive binding processes in order to complete the device assembly process. characterization of the correct folding in solution has been achieved by hplc and cd. the dependence of the dipole in the dimeric protein has been shown using large scale kelvin probe and high resolution kelvin force microscopy (kfm) measurements. we believe that this work will contribute to the understanding of electronic processes in proteins and may serve as foundation for their exploitation in device confi gurations by making use of the ability to trigger conformational changes in order to control device activity. synthesis of new tetracyclic rgd peptides for specifi c tumor cell targeting. the integrin family of adhesion molecules participate in important cell-cell and cell-extracellular matrix interactions in a diverse range of biological processes. the avb3 integrin is overexpressed in several types of cancer cells and play an important role in angiogenesis as well as in tumour cell migration by interacting with vitronectin on the extracellular matrix mainly through the recognition of the tripeptide sequence rgd. the search for highly selective ligands to target the endothelial cell integrin avb3 is currently the focus of many research groups as they may represent new therapeutics or valuable diagnosis in a number of areas such as metastasis, angiogenesis, arthritis and retinopathy. to date, the cyclic (-rgdfx) peptides and related n-methylated analogues developed by kessler's group are among the most active and selective compounds for the avb3 integrin receptors. for instance, the use of c(-rgdf(nme)v-) is actually evaluated in several clinical trials. therefore, we designed a new class of cyclic tetrapeptides. rgdk peptides were fi rst synthesized by solid phase synthesis then cyclized in solution through a urea bond using n,n'-carbonyldiimidazole. using skmel28 and a549 cells, expressing and non-expressing avb3 respectively, we demonstrate that one of our peptide showed a better internalization than the reference peptide crgdfe. our strategy allowed the coupling of the best cyclic recognition peptide through its extracyclic acidic group to any molecular moieties containing an amino group that makes him a great cadidate for easy multimerization. along this line, different applications are currently developped in our group. design in this study, we described a simple method for aminocylation of unprotected saccharides with mildly activated amino acids esters as potential ace inhibitory activity. as a model reaction, we investigated esterifi cation of sucrose -" royal carbohydrate" with cyanomethyl ester of benzyloxycarbonyl-phenylalanine.the difference of reactivity between the eight hydroxyl groups is a key factor in chemical exploration. in polar solvent as dmf or dmso the primary positions might react faster than secondary ones if the reaction is essentially sensitive to steric interactions. on the other hand, the reaction might be more sensitive to the activity of the alcohol functions. in this respects, 2g-oh has been shown to be the most reactive among eight hydroxyl groups of sucrose. after removal of benzyloxycarbonyl group, the products possess groups which can accommolated in the hydrophobic s1 and s2 subsites of angiotensin i converting enzyme. the free amino group in the amino acid-carbohydrate esters can also serve as good ligands for zn2+ in the ace active site. carbohydrates possess both hydrophobic and hydrophilic groups in their structure and could also bind with enzyme subsites. the measurements of ic50 value of newly amino acyl esters of carbohydrates are in progress. synthetic polyelectrolytes (pe) have been widely used to modify proteins via complex formation and covalent attachment, increasing (or reducing) the immunoreactivity and/or immunogenecity of originally antigenic proteins and improving their in vivo stability with prolonged clearance times. such conjugates seem to be great importance for medicine and immunobiotechnology in particular with respect to drug delivery and vaccine innovation. synthetic peptides are promising candidate vaccines for the control of viral diseases. previous studies with foot-and-mouth disease virus (fmdv) have identifi ed fragments of isolated vp1 protein and synthetic peptides from vpi which stimulate antibody production, albeit of poor neutralizing activity, or are recognized by antivirus antibodies. fmdv is an attractive model with which to study the potential of peptide-based synthetic vaccines. in this study, we sought to evaluate the immunogenecity of a candidate containing 135-161 synthetic peptide epitops of vp1 capsid protein of fmdv. the immunogenic properties of the conjugates were also investigated and the relationship between immunogeneticity and structure formation in the solutions is analyzed. a new high immunogenic protein-polymer complex and conjugates with antibody production, processing relatively prolonged times was obtained. it was obtained that a single immunization of mice with pepeptide bioconjugates without classical adjuvant increased the primary and secondary peptide-specifi c immune response to fmdv. polyacrylic acid (paa) is a well known bioactive polymer (bioadhesive nano-and microparticles, ph-sensitive paa grafted poly(vinylidene fl uoride) membrane bags, ultrafi ne cellulose fi ber surfaces grafted with paa for enzyme immobilization, paa-polyvinylpyrrolidone biopolymeric systems for the treatment of the dry eye, etc. paa, their alkyl-esters and non-toxic copolymers with vinyl pyrrolidones are strong adjuvants for primary and secondary responses and that they are promising alternatives to the mineral oil-based adjuvants presently used in various veterinary vaccines. in this study, the interaction between peptide epitops of vp1 protein of foot-and mouth disease (fmdv) and polyacrylic acid (paa) will be discussed on the basis of the experimental results obtained by the size-exclusion chromatography (sec) with online quadruple detection system: uv absorption (uv), refractive index (ri), right angle light scattering (ls) and viscosity (vis) detectors and the binding coeffi cient, i.e., the binding ratio of peptide to polyacrylic acid. human igg-specifi c binding peptides to distinguish normal and abnormal conformers: applications for igg purifi cation and detection in recent years, human immunoglobulin g (igg) attracts attention as protein of the main format of the antibody medicine. in the purifi cation of igg, protein a originated from bacteria is frequently used as a affi nity ligand, but several problems such as the contamination of endotoxin and the deterioration of protein a by the repeated use were pointed-out in the use of protein a. on this account, the development of low molecular ligands and mimic peptides which can be used instead of protein a has been performed. we have searched for the peptides which specifi cally bind to human igg using a t7 random peptide phage library and, as a result, succeeded in isolation of two kinds of peptides called type i and type ii. type i peptide recognize normal structure of human igg and can be used for the purifi cation and the detection of igg. however, it not only binds to human igg but also to mouse and rabbit igg with comparatively high affi nity. on the other hand, type ii peptide is extremely specifi c only for human igg. a more important thing is that type ii peptide does not bind to igg in serum, but get possible to bind to igg antibody purifi ed by a protein a column. from this result, we elucidated the abnormal structure of igg is generated by acid condition used in the elution from a protein a column and type ii peptide recognizes this specifi c structure. in this presentation, we report the results that the type i peptides functioning as an affi nity ligand instead of protein a can be applied for the purifi cation system of the antibody by improvement by amino acid substitutions and chemical conversion. furthermore, we also report the generation condition of an abnormal conformer of igg structure by ph and the temperature and the effective removal of the generated specifi c conformer by the type ii peptideimmobilized column. acknowledgements: this research was partially supported by a grant of practical application research from japan science and technology agency. the isolation and detection of low abundant enzymes and proteins is one of the most challenging tasks in bioanalytical and pharmacological fi elds, especially if information about their state of activity is required. for this purpose tailored solutions for addressing the members of a protein family are required. peptide chemistry provides established methods to assemble building blocks to construct such molecular probes. we chose matrix metalloproteinases (mmp) for validation of such an approach. this protein family processes and degrades various extracellular matrix proteins and possesses a highly conserved catalytic site with a zinc ion in its centre. most of the known and potent inhibitors are peptidomimetics containing zinc chelating hydroxamate groups (e. g. marimastat). although it binds reversibly, it is potent and active against a wide range of metalloproteinases. it was chosen as synthetically available binding group to target the protein in its active state. it was modifi ed by peptide chemistry in order to introduce multiple functions. depending on the purpose, different reporter groups, photoreactive or cleavage sites were chosen. the probes were tested positively to inhibit various human recombinant mmps using activity assays. mmp were isolated using streptavidin coated magnetic beads and a biotinylated probe. furthermore a photoreactive group was introduced to enhance the binding to the target protein. a covalent interaction was achieved by irradiation of a probe protein mixture, isolation with magnetic beads and cleavage from the beads. pyclock superior coupling reagent for biosensors construction based on peptides coupling onto solid phase polymer atias, danit; abu-rabeah, khalil; marks, robert s. electrochemically biosensors are valuable analytical tools for the monitoring of various biological/ chemical molecule levels, and tremendous research effort has been put into the development of such accurate analytical devices. one crucial aspect in the fabrication of a biosensor is the deposition of biological macromolecules in high amounts with retention of their specifi c activity. among the various deposition methods of biomolecules, such as direct adsorption onto the surface, cross-linking, covalent attachment by carbodiimide chemistry is one of the few methods that allows a stable deposition into a biocompatible environment. hence the urgent need for proteins coupling with high yield to solid surface in the aim of biosensors construction. however the use of carbodiimide as peptide coupling agents was found to be limited in various aspects. thus, during the activation of hindered carboxylic components, such as those involved in bulky protein coupling reactions to solid phase fi bers pyclock was found to be very effi cient for slow coupling reactions and it can be used in excess to assure a complete activation of the carboxylic function. in this study 6-chloro-benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafl uorophosphate coupling reagent (pyclock) was shown to have better performance than 1-ethyl-[3-(dimethyl¬amino)propyl]-3ethylcarbodiimide (edac) and hydroxysulfosuccinimide (nhss) in peptides large sequence coupling to solid phase fi bers. cantel, sonia; valmalle, charlene; subra, gilles; enjalbal, christine; martinez, jean general approaches used in protein identifi cation and characterization involve consecutive purifi cation steps. then the desired protein extract is submitted to mass spectrometry analysis (lc-ms/ms) after enzymatic digestion. technical diffi culties are involved in determining the pmf of a protein particularly in relative low abundance. a typical protein will give rise to at least twenty to thirty peptides after trypsin digestion. not all of these peptides will appear in maldi analysis. one factor that is believed to cause incomplete detection is competition for protonation during the ionisation process inducing ion discrimination. we have recently developed a new technology allowing specifi c labeling of lysine residues in proteins and easy maldi-ms detection and identifi cation of labeled peptides following protein hydrolysis (1) . nhydroxysuccinimide ester of -cyano-4-hydroxycinnamic acid (chca) was used as a labeling reagent to increase maldi signal of lysinecontaining peptides in cytochrome c proteolytic mixture. this original approach enables to discriminate labeled peptides of interest among other abundant peptides. herein, we report the optimization process to investigate the limits of this tool. a defi ned receptor-binding domain (rbd) on the viral spike protein (s) mediates the attachment of sars-coronavirus to its cellular receptor, angiotensin-converting enzyme 2 (ace2). we have synthesized peptide libraries for identifi cation of rbd binding epitopes by surface plasmon resonance (spr) and saturation transfer difference (std) nmr spectroscopy. three dodecapeptides were identifi ed to have signifi cant affi nity to ace2; the best of them had a kd = 85 m . further refi nement yielded a hexapeptide from y438 to l443 (ykyryl) of s that bound ace2 at kd= 46 m. std nmr spectroscopy reveals close contacts of the aromatic tyrosine residues to the receptor. the peptide was also analyzed for antiviral activity using an in vitro assay that measures sars-cov infection of vero cells. at a concentration of 7 mm virus replication was reduced about 100 fold. no replication occurred at peptide concentrations above 10 mm. the peptide blocks the binding site on ace2 that is necessary for the virus to infect the cells. it can be used to design peptidomimetic compounds as entry inhibitors for sars-cov. to exclude other effects that were due to unspecifi c inhibition of viral replication, the peptide was tested during an alpha virus infection of vero cells. no signifi cant inhibition was observed. however, for the human corona virus nl63 that causes severe colds in humans and that uses the same receptor a clear inhibition could be observed comparable to the results obtained for sars-cov. additionally, we have synthesized a hexapeptide library with amino acid substitutions of the chemical lead ykyryl and measured their binding affi nity to ace2 via spr to analyze the importance of the individual amino acids. spr studies indicate an important role of r441. prostate cancer is one of the most common malignancies in men and is responsible for more deaths than any other cancer, except for lung cancer. in the last decade we have developed bradykinin antagonist peptides (b10234, b10238), peptide dimer (b9870, suim-(d-arg-arg-pro-hyp-gly-igl-ser-d-igl-oic-arg)2, suim: suberimidyl; hyp: trans-4-hydroxyproline; igl: -(2-indanyl)glycine; oic: 2s,3as,7asoctahydro-1h-indole-2-carboxylic acid) and four generations of our small molecule, bkm-570 which showed high inhibition against lung cancer in vitro and in vivo. some of the compounds also showed inhibition against prostate cancer. it is well known that prostate cancer metastasizes into bones. while prostate cancer itself has many treatment options, no drugs currently exist for the treatment of bone metastatic prostate cancer. we modifi ed our potent anti-cancer small molecules by incorporating an aminobisphosphonate group to target these compounds to bone. bkm-570, f5c-oc2y-atmp (f5c: 2,3,4,5,6pentafl uorocinnamoyl; oc2y: (o-2,6-dichlorobenzyl)-tyrosine; atmp: 4-amino-2,2,6,6-tetramethylpiperidine) is the fi rst generation of our small molecules. this compound consists of three parts: a, an acyl group; b, a tyrosine amino acid residue and c, an amide group. we introduced aminobisphosphonate or aminobisphosphonate derivatives at position c, and the new n-(bis-phosphonatoalkyl)amide small molecules had anti-cancer activity against prostate cancer metastases in mice. one of these compounds has been shown to be effective against the growth of pre-established human prostate tumors in mouse skeleton through the induction of apoptosis and blockade of survivin expression. the synthesis of these aminobisphosphonate small molecules, the structure relationship studies and the biological activity of these compounds in cultured human prostate cancer cells and animal models will be presented. hayouka novel, potent and selective angiotensin iv short analogues the hexapeptide angiotensin iv: h-val-tyr-ile-his-pro-phe-oh (ang iv) mediates a wide range of physiological actions, including control of blood fl ow and cognitive enhancement. it exerts its effects by binding to at4 receptors, which are widely distributed across tissues. the at4 receptor has been identifi ed as the insulin-regulated aminopeptidase or irap. it has been proposed that ang iv exerts its action by inhibiting the catalytic activity of this enzyme. we have reported that the -homo amino acid containing analog h-2 hval-tyr-ile-his-pro-3 phe-oh (al-11) is a potent, selective and stable ang iv antagonist, in which the 2 hval is responsible for stability and the 3 hphe for selectivity. 1 in this study we report the new ang iv short analogues. the incorporation of erythro-mephe 6 or tic 6 resulted in analogues which have great ability to inhibit the hydrolysis of leu-p-nitroanilide by irap or by ap-n. our data showed that the full ang iv sequence is not necessary for high potency. peptides with pro deletion containing tic or erythro-mephe were even more potent than full ang iv sequence. moreover a peptide design and synthesis of a non -peptide par1 thrombin receptor antagonist, using cyclohexane as template receptors that mediate thrombin action are attractive drug discovery targets because of their involvement in cardiovascular pathophysiology (dysregulation of platelet aggregation and endothelial cell function). the cellular actions of thrombin are, in large part, caused by the activation of proteinase-activated receptors (pars) 1, 3 and 4 (pharm. rev. 54:203). the serine proteinase thrombin cleaves and activates cellular par1 in many pathophysiological settings associated with hemostasis, tissue injury and the proliferation of vascular smooth muscle and tumor cells. in the present study, we synthesized a novel non-peptide par1 mimetic, based on a conformational analysis of the s 42 fllr46 tethered ligand sequence of par1 in order to inhibit the cellular actions of thrombin. the rational design, based on nmr constraints and molecular dymamics, led to compound 1 (fig.1) containing the spatially closed key pharmacophoric guanidyl and phenyl groups, attached to cyclohexane as a template. compound 1, inhibited both tfllr-amide (10 m) and thrombin (0.5 and 1 u/ml)-mediated calcium signaling in a cultured human hek cell assay (j pharm. exp ther. 288:358). aboye, teshome leta 1 ; clark, richard j 2 ; craik, david j. 2 ; göransson, ulf 1 1 biomedical centre, sweden; 2 institute for molecular bioscience, australia the cyclic cystine knot motif, as defi ned by the cyclotide peptide family, is an attractive scaffold for protein engineering (1). however, to date the utilization of this scaffold has been limited by the inability to synthesize members of the most diverse and biologically active subfamily, the bracelet cyclotides. here we describe the synthesis and fi rst direct oxidative folding of a bracelet cyclotide, cycloviolacin o2, and thus provide an effi cient method of exploring the most potent cyclic cystine knot peptides (2) . the linear chain of cycloviolacin o2 was assembled using fmoc solid phase peptide synthesis and cyclized by thioester-mediated native chemical ligation, and the inherent diffi culties of folding bracelet cyclotides were successfully overcome in a single step reaction. the folding pathway was characterized and included predominating fully oxidized intermediates that slowly converted to the native peptide structure. angiogenesis, the process of new blood vessel formation, is important in both physiologic and pathologic situations, and its inhibition can be useful in the fi ght against several diseases, such as tumor development, diabetic retinopathy, etc. one of the key factors in promoting angiogenesis is the vascular endothelial growth factor (vegf), which exerts its biological activity through interaction with specifi c receptors, vegfr-1 (flt-1), vegfr-2 (kdr, flk-1) and vegfr-3 (flt-4) . vegf is over-expressed in all examples of pathologic angiogenesis, and its effects on tumor growth are mainly mediated by kdr. our approach to novel anti-angiogenic drugs is centered on the search for inhibitors of vegf-kdr interaction. directed mutagenesis studies allowed the identifi cation of a surface of vegf recognized by kdr, with residues arg 82 , ile 83 , lys 84 , his 86 and glu 89 , located in a -hairpin of loop 3, identifi ed as essential for the interaction. most residues in this region are also located in the interface of interaction between vegf and some none-humanized phage-displayed antibodies with potent antiangiogenic activity, suggesting a binding to vegf similar to that of vegf receptors. starting from vegf 81-91 fragment (mrikphqgqhi), we have designed different hydrocarbon-bridged analogues to preserve the -hairpin native structure. this communication will describe the solid-phase synthesis of olefi n-bridged (c=c) peptides and their saturated (c-c) analogues. considering that the -turn of native vegf 81-91 is slightly distorted, due to the presence of an extra amino acid residue, in addition to the bridged undecapeptides, two series of decapeptide analogues have also been prepared, just by removing the residues glu 87 or gly 88 . the conformational behavior of linear and bridged-peptides has been analyzed by nmr (noe, 1 h and 13 c chemical shifts). the ability of these peptides to adopt the native vegf -hairpin structure will be compared with their anti-angiogenic activities. integrins constitute a family of heterodimeric, transmembrane cell adhesion receptors which connect cells to the scaffolding proteins of the extracellular matrix. the pioneering observation that integrins -especially v 3 and 5 1 -are hallmarks of metastatic cancer and seriously involved in the process of tumor angiogenesis turned them into attractive targets for cancer therapy. out of that, the inhibition of integrin function is a major challenge in medicinal chemistry. potent ligands are currently in different stages of clinical trials for the antiangiogenic therapy of cancer and age-related macula degeneration (amd). especially the subtype 5 1 has recently been drawn into the focus of research due to its genuine role in angiogenesis.(1) in here, we describe the rational design and the synthesis of high affi nity 5 1 binders and the optimization of their activity and selectivity against v 3 by means of extensive sar-studies and docking experiments.(2) starting from a tyrosine scaffold(3) we succeeded in getting compounds with affi nities in the low and even sub-nanomolar range and selectivities of 400 fold against v 3. the insights about the structure-activity-relationship gained from the tyrosine based ligands could then be successfully transferred to ligands bearing an aza-glycine(4) scaffold to yield 5 1 ligands with affi nities of even sub-nanomolar range and selektivites exceeding 10.000 fold. modeling studies and biological activities of a nonpeptide at 1 receptor angiotensin ii antagonist the octapeptide angiotensin ii (h-asp-arg-val-tyr-ile-his-pro-phe-oh) is the major factor of the renin-angiotensin system (ras) and plays a signifi cant role in the regulation of arterial blood pressure. in the present study, we have modeled a losartan analogue, the non-peptide angiotensin ii at 1 antagonist, 5-butyl-1-hydroxymethyl-1-{[2'-(1htetrazol-5-yl)biphenyl-4-yl]methyl} imidazole (v8). structure activity relationship (sar) and molecular modeling studies indicate close proximity of hydroxymethyl and tetrazole pharmacophoric groups of antagonist v8 and losartan. conformational analysis was performed using a grid scan search in order to derive all the possible conformations from which six energy local minima (syn and anti) were extracted after a cluster analysis. furthermore, these different conformations were superimposed with losartan, where a spatial correlation among the pharmacophoric groups is observed. antagonist v8 showed similar potency with losartan in our in vivo model with anesthetized rabbits and in vitro binding studies to at 1 receptor. at specifi c concentrations compound v8 showed higher affi nity compared to losartan indicating that reorientation of butyl and hydroxymethyl groups on imidazole template allows a better binding to at 1 receptor. 1h-benzotriazolium 1-[bis(dimethylamino)methylene]-5-chloro-,hexafl uorophosphate (1-),3-oxide (hctu) is a non-toxic, non-irritating and non-corrosive coupling reagent [1] [2] . seven biologically active peptides (ghrp-6, 65-74 acp, oxytocin, g-lhrh, c-peptide, hamylin 1-37 , and -amyloid 1-42 ) were synthesized with reaction times reduced to deprotection times of 3 minutes or less and coupling times of 5 minutes or less using hctu as the coupling reagent. no expensive coupling reagents or special techniques were used. total peptide synthesis times were dramatically reduced as much as 42.5 hr (1.8 days) without reducing the crude peptide purities. it was shown that hctu can be used as an affordable, effi cient coupling reagent for fast fmoc solidphase peptide synthesis. human sdf-1 contains sixty-eight amino acids and is a member of the chemokine family of peptides. this long peptide was synthesized step-wise using our quality control conditions in 51 hours. the reaction times were then reduced to deprotection times of 2 x 2 min and coupling times of 2 x 2.5 min, resulting in a total synthesis time of 22 hours. the effect of different resins, resin substitutions and deprotection reagents on the crude peptide purities were compared. a small portion of crude peptide was purifi ed using an rp-hplc column and the mass of the fi nal product was confi rmed with maldi-tof mass spectrometry. references: 1. steven l. kunkel and nuria godessart, chemokines in autoimmunity: from pathology to therapeutics, autoimmunity reviews, 1, 313-320 (2002). pedersen, søren l.; sørensen, kasper k.; jensen, knud j. despite the development of new coupling reagents and solid supports, spps is still often faced with diffi culties in the assembly of long or "diffi cult" sequences, e.g. due to aggregation and steric hindrance giving rise to incomplete reactions. the use of convenient and precise heating with microwaves for spps has gained in popularity as it for many syntheses has provided signifi cant improvement in terms of speed, purity, and yields, maybe especially in the synthesis of long and "diffi cult" peptides. thus, precise microwave heating has emerged as one new parameter for spps, in addition to coupling reagents, resins, solvents etc. we have previously reported on microwave heating to promote a range of solid-phase reactions in spps. here we present a new, fl exible semi-automated instrument for the application of precise microwave heating in solid-phase synthesis. it combines a slightly modifi ed biotage initiator microwave instrument, which is available in many laboratories, with a modifi ed semi-automated peptide synthesizer from multisyntech. a custom-made reaction vessel is placed permanently in the microwave oven, thus the reactor does not have to be moved between steps. mixing is achieved by nitrogen bubling. washing steps are automated, however the activated amino acid derivatives have to be added manually. first, we developed optimized protocols for short cycle times in semi-automated spps with general fmoc chemistry. we utilized a microwave-compatible temperature probe for exact temperature measurements during microwave heating. then we developed protocols for on-resin reductive amination for anchoring of the fi rst amino acids to a bal handle. finally, we used the new instrument and the optimized protocols to assist in the synthesis of a range of diffi cult and long sequences. we believe that these successful syntheses demonstrate that this semi-automated instrument and the methods developed for it, can be an effi cient starting point for spps with microwave heating. investigation of polyelectrolyte-antigenic peptide conjugates by fl uorescence spectroscopy in proteins or peptides, it is possible to localize the interaction between protein and pe at certain protein domains. we investigate covalent binding mechanism of synthetic peptides which include tryptophan residue in the peptide sequence with copolymers of acrylic acid and n-vinylpyrolidone (vp\aa) depending upon the weight concentration ratio of components by fl uorescence method. antigenic peptides are synthesized by microwave assisted solid phase peptide synthesis method. characterization of these peptides are performed with lc-ms. subsequently these crude peptides are purifi ed by preparative hplc system. peptide-polymer covalent conjugation are performed in organic and pbs media. covalent conjugation are carried out by carbodiimide method. carbodiimide is used for the activation of carbonile groups of synthetic polymer which is the binding area of peptides that includes free amino groups. from the analysis of peptide-pe conjugates, it is possible to discuss the mechanism of the conjugate formation and structure of forming particles. according to the fl uorescence analysis results, free peptide which containing trp residue, gives fl uorescence spectrum. however, after the conjugation reaction it is observed that products max decreased and it is characterized as blue shift of max. this indicates that when conjugates formed, trp is completely isolated from aqueous solution. antiangiogenic thrombospondin type 1 repeat analogs: synthesis, structure, and biological activity the thrombospondin type 1 repeat (tsr) has been shown to inhibit angiogenesis and tumor growth in a number of in vivo models of cancer, but the details of this activity, including structure-function relationships, are not well understood. we explore these tsr elements in further detail via structural and biological evaluation of a series of tsr analogs. the tsr domain has a characteristic fold consisting of a two-stranded -sheet and a third 'rippled' strand. three tryptophans on the 'rippled' strand form cation-stacking interactions with two arginines on the adjacent sheet, forming an extended cation-network. in addition its structural importance, it has been postulated that the surface formed by this interaction is a key site for protein-protein recognition. a synthetic approach to the generation of novel tsr analogs, utilizing spps and native chemical ligation, has allowed us to rationally introduce unnatural amino acids in an effort to probe the structural and functional landscape of this clinically relevant protein domain. we have synthesized analogs of the second tsr domain of human thrombospondin-1 (tsr2) designed to probe the importance of the cation-stack. the arginine residues have been replaced by ornithine and citrulline, and thermal denaturation experiments by circular dichroism have been used to estimate stability differences between the mutant tsr2 and the native. taken as a set, these thermodynamic measurements provide insight into the stability afforded by a cationinteraction in the context of a native protein fold. synthetic access to the tsr2 domain also provides the opportunity for the introduction of various modifi cations, including the introduction of aminooxy groups as chemical handles, pegylation for in vivo stability, and biotinylation to create an affi nity caputure reagent for identifi cation of protein-protein interaction partners. the tetradecapeptide bombesin (bbs) has a high affi nity for the gastrin-releasing peptide (grp) receptor. these receptors are overexpressed in human tumors such as breast and prostate cancers. therefore they can serve as targets for in vivo imaging and therapy of these tumors with 99m tc-and 188 re-radiolabeled bbs analogues, respectively. for the radiolabeling, a chelator is attached to the n-terminus of the bbs analogues. our group developed the (n his)ac chelator, which is easy to synthesize and has a very high affi nity for the 99m tc-and 188 retricarbonyl complexes. however, an important part of the injected radiolabeled bbs analogues accumulated in healthy organs, such as liver and kidneys. to improve the pharmacokinetic properties, a bbs analogue was glycated via the lys side chain of the spacer using the maillard reaction. unfortunately, during glycation, overalkylation on the secundary amine of the chelator was observed. therefore, two alternatives for this glycation were examined. the fi rst one was based on the chemoselective reaction between a hydroxylamine (incorporated into the spacer) and an aldehyde (glucose). the second alternative was the cu(i) catalyzed cycloaddition between an alkyne (incorporated into the spacer) and an azide (azido-glucose). these approaches for carbohydration circumvented the problem encountered during glycation via the maillard reaction. glycation by the cu(i) catalyzed cycloaddition was, by far, the easiest way to incorporate the glucose moiety. moreover, after 99m tc labeling, this analogue showed the best biodistribution and the best diagnostic properties of all glycated analogues. an alternate approach to improve the pharmacokinetics consisted of including different polar spacers such as ³hglu, ³hasp, ³hlys, ³hser and -nh(ch 2 ch 2 o) 2 co-between the (n his)ac chelator and the bombesin sequence. in particular the analogues with a negative charge ( ³hglu, ³hasp) in the spacer showed a favorable effect on the pharmacokinetics. determination of binding ratio of hydrofobic peptidepolymer conjugates by using fl uorescamine assay budama battal, yasemin; derman, serap; mansuroðlu, banu; mustafaeva, zeynep yildiz technical university, bioengineering department, turkey non-fl uorescent 4-phenylspiro-[furan-2(3h),1-phthalan]-3,3'-dione (fl uorescamine) reacts readily with primary amines in amino acids, peptides and proteins to form stable, highly fl uorescent compounds (fl uorophors). fluorescamine has been used to detect free amino groups on peptides or completion of coupling reactions in solid phase peptide synthesis and not only in aqueous solution but also in organic solvents and on solids. in this study, peptide epitops of vp1 capsid protein of foot-and-mouth-diseases virus 40-60 amino acid residues (val-lys-ile-asn-asn-thr-ser-pro-the-his-val-ile-asp-leu-met-gln-thr-his-gln-his-gly) were synthesized by sigma. we synthesized the covalent conjugate of 40-60 amino acid residues with copolymers of acrylic acids and nvinylpyrolidone (vp\aa) at different ratio of components (npeptide/ npolymer) and investigated the mechanism of condensation reaction by using different physicochemical analyses as hplc, fluorescence spectroscopy and fluorescamine assay. for determination of binding ratio of hidrofobic peptide-polymer congutages, fl uorescent measurements were performed in the presence of fl uorescamine at the wavelengts of excitation 390 nm and emission 475 nm. when conjugation reaction completed, the amount of free amino groups had decreased and observed that fluorescence intensity had decreased and the estimated degree of primary amino group after conjugation, calculated from fl uorescence spectrums. (4-phenylspiro-[furan-2(3h),1-phthalan]-3,3'-dione) which heterocyclic dione is a reagent for the detection of primary amines on peptides or completion of coupling reactions in the picomole range. its reaction with amines is almost instantaneous at room temperature in aqueous media, organic solvents ect . fluorescamine reacts with primary amines (in peptide, protein ect.) to form highly fl uorescent product (fl uorophors) whereas the reagent and its degradation products are nonfl uorescent (1). this is the basis of a fl uorescent protein assay [1, 2] . fluorescamine is used in many sensitive detection methods, e,g., characterization of poly-l-lysine (pll)/dna complexes post-modifi ed with a multivalent hydrophilic polymer (3), or synyhetic peptide-polymer conjugates. in this study, foot-and-mouth-diseases virus vp1 capsid protein's synthetic peptide epitope which containing tryptophane (trp), 135-161 (p1) amino acid residues (try-ser-lys-tyr-ser-thr-thr-gly-glu-arg-thr-arg-thr-arg-gly-asp-leu-gly-ala-leu-ala-ala-arg-val-ala-thr-gln-leu-pro-ala) were synthesized by using the solid-phase methods. we synthesized the covalent conjugate of copolymers of acrylic acids and n-vinylpyrolidone (vp\aa) with 135-161 amino acid residues at different npeptide/npolymer ratio and investigated the mechanism of condensation reaction by using fluorescence spectroscopy and fluorescamine assay. in the fl uorescamine assay the conjugate solution was measured on a pti qm-2003 steady state fluorescence spectrometer with an excitation wavelength of 390 nm and emission at 475 nm. after the conjugation reaction, the amount of free amino groups had decreased because of binding carboxyl group and observed that fluorescence intensity had decreased. determination of free amino group degree after conjugation, calculated from fl uorescence spectrums. newly developed high strength and chemically stable silica gel based preparative reversed phase packing materials kuriyama, naohiro; shoji, noriko; morishita, kiyoshi; omote, masakatsu ymc co., ltd., japan a new high strength silica gel and a bonding technology based on preparative bulk packing materials for hplc have been developed to provide improved recovery, selectivity, and longer life time for the preparative peptide separations. the novel preparative silica particle was successfully prepared by the new generation process, which allows the higher gel density than typical silica gel and the particle size distribution would be practically mono-dispersed character. for the effective reversed phase peptide separations, pore size and pore volume of these new particle were optimized depending on the molecular weight of peptides. to enhance chemical stability and selectivity under the typical peptide purifi cation conditions, the combination of chemical bonding method and functional group density was optimized for maximum performance. by repeated packing and unpacking of this synthesized gel with large dynamic axial compression column, it was demonstrated that no fi ne has appeared and no back pressure increasing has occurred comparing to commercially available packing materials. also cost effective peptide purifi cation with high loadability, productivity, and recovery was achieved with signifi cant small and large peptides. calcitonin gene-related peptide (cgrp) is a 37 amino acid neuropeptide produced by tissue-specifi c alternative mrna splicing of the calcitonin gene. the cgrp peptide signals through a seven transmembrane g protein-coupled receptor (gpcr) belonging to the secretin receptor family. co-expression of the calcitonin-like receptor (clr) with receptor activity modifying proteins-1 (ramp1) forms a mature cgrp receptor on the cell membrane surface. cgrp is widely distributed in the peripheral and central nervous systems. in the later, cgrp is expressed in trigeminal ganglia nerves and when it is released has potent dilator effects on cerebal and dural vessels. through this mechanism, cgrp is involved in the regulation of blood fl ow to the brain and painsensitive meninges. the pathology of migraine has been associated with the vasodilation effects of cgrp on cerebal circulation. it has been reported that the cgrp peptide consist of an alpha-helical n-terminal region, a fl exible center region, and two putative c-terminal beta-turns. truncation of the fi rst seven residues in cgrp results in antagonists of the cgrp1 receptor (ic50 4 nm), however, cgrp(8-37) is rapidly degraded in plasma. here, we report the iterative design and synthesis of new high affi nity cgrp antagonists with signifi cantly increased plasma stability, and higher affi nity for the human cgrp1 receptor. cordopatis, paul; pappa, eleni; zompra, aikaterini; magafa, vassiliki; diamantopoulou, zoi; lamari, fotini; katsoris, panagiotis university, greece mammalian gonadotropin releasing hormone (gnrh-i) and the sea lamprey gonadotropin releasing hormone type iii (gnrh-iii), belong to the class of conserved gonadotropin releasing hormone peptides. in addition to the classic hypophysiotropic action of gnrh-i, it has been shown that many malignant cells, such as prostate cancer cells, secrete gnrh-i and express the gnrh-i receptor/s. gnrh-iii has no endocrine activity in mammals even at high doses, but has been shown to suppress directly the growth of breast and prostatic cancer cells in vitro. in a continuation of our previous work and in order to study the effect of modifi cations in positions 4 and 6 of leuprolide, an agonist of gnrh-i, on prostate cancer cell proliferation, we synthesized ten new conformationally restricted analogues. d-leu6 of leuprolide was substituted by d-lys, d-or l-glu and ser4 by n-me-ser. we also synthesized fi ve new analogues of gnrh-iii and studied their effect on prostate cancer cell proliferation. asp in position 6 of gnrh-iii was substituted by asn and glu, pro9 was substituted by á-aminoisobutyric acid (aib) and trp3 and/or trp7 by d-trp. peptides were synthesized by the solid phase methodology and fmoc/but chemistry in high yields. results show that the inhibitory effect of gnrh-i analogues on the proliferation of human prostate cancer cells depends on the nature of the substituted amino acid in position 6. incorporation of d-trp in positions 3 and 7 of gnrh-iii preserved its antiproliferative activity, whereas all other modifi cations reduced its activity. mastoparan (mp) is an antimicrobial cationic tetradecapeptide with following primary structure inlkalaalakkil. this amphiphilic -helical peptide was originally isolated from the venom of wasp paravespula lewisii. however some mp analogues (tetradecapeptides with different amino acids sequence) have been also isolated from the venom of hornets, yellow jackets and paper or solitary wasps. they are rich in hydrophobic amino acids such as leu, ile, ala and commonly posses lys residues. mp shows a variety of biological activities such as inhibition of the growth of gram-positive bacteria, activation of mast cell degradation and histamine release, activation of phospholipase a2 and c or erythrocyte lysis. mp is also turned out to enhance the permeability of artifi cial and biological membranes and activate gtp-binding regulatory proteins by a mechanism analogous to that of g protein-coupled receptors. nowadays many mastoparan analogues have been synthesized. some of them contains in primary structure the mastoparan sequence but they loss some of the properties of mp, such as transportan (a cell penetrating peptide, consisting n-terminal fragment 1-12 of galanin and mastoparan at the c-terminus linked with lys residue) or its truncated analogue, transportan-10. in present study we have designed and synthesized several new chimeric mastoparan analogues composed of mp and other biologically active peptides (e.g. galanin, rnaiii inhibiting peptide) and containing natural or unnatural structures. next we examined each of these hybrid constructs as well as natural peptides for they antimicrobial activities. acknowledgement: this work was supported by the university of gdansk grand bw 8000-5-0133-8 cinnamic acid derivatives as esters, amides and glycosides are known to have antibacterial, antiviral, antiinfl ammatory, antiproliferative, immunostimulatory etc. properties. some of them (amide and ester analogues of caffeic acid with natural amino acid esters) exibit stronger antioxidative activity. in particular thiazole, oxazole and imidazole amino acids that may play a key role in biological activities of unusual peptides are also important intermediates for natural product synthesis and peptidomimetics. here we report the synthesis of novel cinnamic acid amides with oxazole containing glycyl-methyl ester as potential antioxidants. the amides were synthesized from sinapic, coumaric, ferulic acids and the corresponding glycyl-methyl ester hydrochloride form using n-ethyl-n-(3-dimethylaminopropyl) carbodiimide hydrochloride (edc) and 4-n,n-(dimethylamino)-pyridine (dmap). their antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl dpph (1,1diphenyl-2-pycrylhydrazyl) test. the venoms of marine cone snails contain a complex mixture of peptide neurotoxins known as conotoxins (1) . they are a diverse class of biomolecules that exhibit exquisite selectivity and potency for a wide range of pharmacological targets in the central nervous system, making them valuable tools for studying the mechanisms of neurotransmission and pain. despite their diversity, conotoxins have evolved from a relatively small number of rigid disulfi de bonded frameworks that give rise to a series of intervening loops of amino acids that project outwards from the framework and interact with the receptor binding site. receptor targets are generally determined by the shape of the framework, with the amino acids within the framework infl uencing receptor subtype specifi city. this work aims to use positional scan libraries (2) based on 4/3 -conotoxin frameworks to study the interactions of amino acids with the binding site of neuronal nicotinic acetylcholine receptors, and to discover new ligands that possess greater potency and selectivity for different subtypes these receptors. the disulfi de bond frameworks were formed by oxidizing each mixture in dilute aqueous buffer and their correct formation monitored by cd spectroscopy. an á7 nachr functional assay was used to screen each mixture to determine the activity of each mixture and the results of this assay were used to design a series of individual candidates for use in further structure activity relationship studies. melittin effect on the sensory neurons prelevated from double transgenic vs. normal mice melittin is a 62 aa peptide. it is the active compound of bee venom and a powerful stimulator of phospholipase a2. its antimicrobial effect was extensively studied in the literature, but little is known about its neuroactive properties. melittin increases the cell membrane permeability in excitable and non-excitable tissues, as a result of its interaction with the negatively charged phospholipids. our interest was focused on the algesic properties of melittin and on its potency against the electrophysiological parameters of sensory neurons from dorsal root ganglia. in our study, we have performed patch-clamp studies, by the whole-cell confi guration. primary cell cultures were obtained from double transgenic mice tcr-ha+/ins-ha+ with type i diabetes. this animal model is well-suited for the study of neuropathic pain and it enables us to test the analgesic effects of melittin (0.1-5 m). our recordings indicate that the active-peptide modifi es the algesic profi le of the sensory neurons, in particular by acting against the capsaicinactivated ionic currents. in addition, we have monitored the action of melittin on the i h currents, na + voltage -dependent currents, delayed rectifi er k + currents. a particular interest was focused on the recordings of ca 2+ voltage-dependent currents. the maximal effect was obtained at a dose of 1 m melittin, and at higher doses the active-peptide strongly disturbs the lipid membrane order. in conclusion, the algesic profi le of the sensory neurons from double transgenic mice is signifi cantly modifi ed by the neuroactive melittin in comparison with the profi le of neurons from balb/c mice. these electrophysiological data are important, and are very well corroborated with the increased thermal and mechanical hypersensitivity induced by melittin itself and the bee venom extract, that have been proved their effi ciency in behaviour tests . the toxic effects of amps tested on mammalian cell cultures the amps (antimicrobial peptides) is a class of molecules that belongs to the innate immunity. the ability of some of these peptides to penetrate the microorganism's external membrane and to induce their death suggests that this class of molecules may represent a new generation of antibiotic pharmaceuticals. in our work, we focused on the amps side effects on mammalian cells. the target was to characterize the toxicity of several amps evaluated on in vitro cellular cultures. the following amps were used in our experiments: melittin, magainin i and ii, cecropin a and mastoparan. the viability of cells in cultures was assessed by mts test on chinese hamster lung fi broblasts v79 and on human lymphocytes primary cultures. concentrations in the range of 0.1 -100 m were used. the ic50 value for each type of amp was evaluated from the viability versus concentration curves. haemolysis assays were also performed for these active-peptides. based on ic50 values, melittin has a stronger citotoxicity than magainin i and magainin ii. on the other hand, magainins appears to posses higher haemolytic properties than melittin. the less cytotoxic peptide seems to be mastoparan. these types of results are very useful to characterize the ability of amps to induce toxic effects in human body during the treatment. acknowledgements: financial support by the cnmp research grant pnii number 61-016/2007. the melanocyte-inhibiting factor (mif-1), which is the tripeptide pro-leu-gly-nh2, was isolated in the conventional way from bovine hypothalamus tissue. mif-1 represents a class of naturally occurring opiate antagonists with varying activities in independent situations. the purpose of the present study was to investigate the analgesic activity of mif-1 and its analogues modifi ed at position 2 with unnatural amino acids cav, slys, sleu, sile and snle. the experiments were carried out on male wistar rats (180-200 g). the changes in the mechanical nociceptive threshold were measured by the paw-pressure test using an analgesiameter (ugo basile). mif-1 and analogues (all in dose 1mg/ kg) were administered intraperitoneally (i.p.). mif-1 exhibits a weak analgesic effect and selective affi nity for the -opioid receptor. mifanalogues -mif-cav, mif-sleu, mif-ile and mif-nle were found to have a naloxone-reversible analgesic effects. pacap (pituitary adenylate cyclase-activating polypeptide) exists in two biological isoforms, i.e a 38-amino acid peptide and a shorter form of 27 residues corresponding to the n-terminal part of pacap38. previous structure-activity relationships studies revealed that the n-terminal segment is essential for the activation of the specifi c and selective type i pacap receptor (pac1). in order to clarify the molecular requirements for pac1 activation, we initiated structure-activity investigations of the n-terminal part of both pacap isoforms. in particular, an important library of analogs focusing on the fi rst seven residues (his1-ser2-asp3-gly4-ile5-phe6-thr7) was rationally developed and pharmacologically evaluated for their capacity to bind the pac1 receptor and to induce ca2+ mobilization in chinese hamster ovary (cho) cells stably expressing the human pac1 receptor. ala scan demonstrated that the carboxylic acid function of residue asp3 and the benzyl group of phe6 are essential feature for proper pac1 receptor activation. the inversion of chirality of residues ile5, phe6 and thr7 caused a loss of affi nity whereas the incorporation of d-amino acids in positions 1, 2 or 3 did not affect the binding properties of pacap. moreover, using a n-methyl scan, we showed that the n-terminal domain of pacap is not tolerant to back-bone modifi cations. however, replacement of ser2 by pro did not decrease signifi cantly the potency of the peptide while substitution of this same residue by d-pro totally inhibited the biological activity of pacap. furthermore, other structural constraints reduced the potency of pacap, suggesting that the n-terminal domain needs fl exibility to bind and activate the pac1 receptor. finally, residues 28-38 of c-terminally extended peptides played a favorable role for the binding affi nity towards the pac1 receptor as pacap38 analogs demonstrated highest binding affi nity compared to their pacap27 counterparts. neutrophil elastase-dependent synthetic host defence pro-peptides for the treatment of bacterial infections in cystic fi brosis patients chronic infection and infl ammation play a major role in the pathophysiology of lung disease in cystic fi brosis (cf). besides pseudomonas aeruginosa, there is increasing evidence that other multidrugresistant organisms, such as methicillin-resistant staphylococcus aureus (mrsa) are implicated in the activation of the infl ammatory response in cf patients. cationic host defence peptides, multifunctional mediators of the innate immune system, have been recognised as promising candidates for the development of novel antimicrobial agents (1). most of these macromolecules are produced as inactive prepropeptides and are proteolytically activated to release a c-terminal cationic peptide with antimicrobial activity. we thought to mimic this natural mechanism to target cf pathogens with synthetic host defence propeptides. saltresistant, all-d cationic antimicrobial peptides (2) were conjugated at their n-termini to a polyglutamic acid sequence to compensate the net positive charge of the parent peptide, through a linker selectively degraded by a disease-associated enzyme (neutrophil elastase). in vitro effects of the all-d p18 peptide candidate on planktonic and biofi lm forms of pseudomonas aeruginosa and staphylococcus aureus clinical isolates were assessed. reactivation studies of the propeptide were performed in presence of purifi ed neutrophil elastase and in physiologically relevant conditions, using bronchoalveolar lavage fl uids from cf patients. the nmr structures of the propeptide and active p18 sequences were also compared. these studies confi rmed that the propeptide remains inactive in the absence of neutrophil elastase and that the latter enzyme can potentiate its antimicrobial activity. synthesis and biological activity of a series of aza 3pseudopeptides related to 26rfa, the endogenous ligand of gpr103 26rfa, a novel neuropeptide of the rfamide family, is the natural ligand of the previously orphan receptor gpr103. both 26rfa and gpr103 mrnas are highly expressed in hypothalamic nuclei of rodents, and icv injection of 26rfa induces a potent orexigenic effect in mice. recently, it has been shown that gpr103-defi cient mice suffer from osteopenia. analysis of the 26rfa precursor reveals that it may generate several additionnal rfa-peptides including an n-terminally extended form (43rfa) and a truncated form (26rfa (20-26) ). 26rfa and 43rfa increase dose-dependently [ca 2+ ]i in gpr103-transfected cells while 26rfa(20-26) is about 100 times less potent than 26rfa. molecular modeling under nmr constraints of 26rfa shows that the n-terminal region encompasses an -helix and the c-terminal region adopts a -turn in dpc micelles. the c-terminal peptide 26rfa(20-26) exhibits major distortions of this turn that may be responsible for its weak potency. the aim of this work was to introduce an aza 3 residue in 26rfa(20-26) in order to stabilize the -turn. sequential aza 3-counterpart substitution of amino acids at positions 20 and 21 enhanced by 2 and 7 folds the potency of 26rfa(20-26), respectively. the aza 3-phe 22 analog was 2 times less potent than 26rfa(20-26). replacement of the native ser 23 by the aza 3 surrogate of (ho)homothr (aza 3-hth) to favor hydrogen bonding, generated an analog that was 2 folds more potent than 26rfa(20-26). in contrast, substitution of residues 24 to 26 led to analogs totally devoid of effect on calcium mobilization. as the -turn is centered on the ser 23 of 26rfa, we can assume that the aza 3-hth 23 moiety partially mimics the -turn in the 26rfa(20-26) sequence. these data constitute the fi rst step towards the development of new gpr103 analogs that could prove useful for the treatment of feeding disorders and/or osteoporosis. acknowledgements: supported by inserm (u413 and pnr-re) and the région haute-normandie. olm is recipient of a fellowship from mesr. it is well known that microtubule targeting agents (mtas) are used in clinical treatments as anticancer agents. recently, it has become clear that some mtas also function as gvascular disrupting agents (vdas), which induce tumor-selective vascular collapse. as one of such candidates, a natural cyclicdipeptide, phenylahistin (plh) exhibiting colchicine-like anti-microtubule activity, has been our focus. we have succeeded in synthesizing plh, and performed the structure activity relationship study of its derivatives. from the biological evaluations, according to these results, a highly potent derivative npi-2358 (ic50 = 15 nm, ht-29) was selected, which is now in phase i clinical trial as an anticancer drug in the us. furthermore, we have established the synthetic route of npi-2358 and its derivatives. about 100 analogs were prepared so far and screened by ht-29 citotoxicity assay. as a result, several highly potent derivatives ware developed. although npi-2358 and its derivatives are believed to be recognized around the colchicine binding site on tubulin, the three-dimensional structure of npi-2358 could not be superimposed over that of colchicine. in order to understand the precise binding mode of npi-2358, we developed a highly potent cytotoxic derivative, kpu-244 (ic50 = 3.9 nm, ht-29 cells) with a benzophenone structure. because benzophenone is recognized as a photo-reactive group, we synthesized biotin-tagged kpu-244 derivative as a photoaffi nity probe. since this probe has biological activity enough to function to tubulin, photoaffi nity labeling was performed to analyze the binding site. as a result, irradiation-time-dependent labeling towards tubulin was observed. the labeling was also dose-dependently inhibited by colchicines addition, suggesting that the probe specifi cally recognizes around the colchicine binding site on tubulin. chemical biology study for determining the precise binding site is now in progress. conformational analysis of the new temporin analogues: gln3ta and pro3tl. temporins are antimicrobial peptides (amp €™s) isolated from the skin of red european frog rana temporaria. they are active particularly against gram-positive bacteria, candida species, fungi. they have the ability to bind and permeate both artifi cial and biological membranes. we have recently investigated by spectroscopic means two members of this amp family temporin l (fvqwfskflgril-nh2) and temporin a (flpligrvlsgil-nh2) (1). based on the nmr results, we developed two new analogues of these peptides named pro3tl (fvpwfskflgril-nh2) and gln3ta (flqligrvlsgil-nh2). biological data indicate that pro3tl has a higher antimicrobial activity and a lower hemolytic activity than the native peptide tl. in contrast, gln3ta more hemolytic than the parent peptide ta. the conformational behavior of the new analogues was investigated in membrane mimetic environment (sds and dpc micelles) by spectroscopic and computational methods. diagnostic nmr parameters observed for glu3ta and pro3tl indicate a conformational propensity toward helical structure. for glu3ta, bturn structures are also observed along the n-terminal fragment of the peptide. we are currently studying the conformational behavior of the four peptides also by molecular dynamics simulation in explicit solvated dodecylphosphocholine and sodium dodecylsulfate systems. these simulations would provide a realistic picture of the interactions between the peptide and models of bacterial and mammalian membranes. where xaa was selected form a collection of cyclic and acyclic nonaromatic amino acids. the peptides were prepared by standard spps methods using either the fmoc or boc strategy and tested in vitro for their agonistic potency and effi cacy at the v1a and the related receptors. unlike avp, which has aromatic amino acids in positions 2 and 3, these analogues contain only non aromatic residues, but are still potent v1a agonists. compounds with cyclic aliphatic residues (e.g. cha) in position 2 were found to be generally more potent v1a agonists than the ones containing acyclic residues. the ala 2 analogue ([ala 2 ,ile 3 ,dab 8 ]vp) was totally inactive (no signifi cant v1ar agonism up to 1000 nm) as pharmacology and medical peptide chemistry reported previously for [ala 2 ]avp (2). the cha 2 containing peptides are slightly less potent as v1ar agonists than their phe 2 counterparts, but the overall in vitro pharmacological profi le of the two classes of compounds is similar. selected new analogues were also tested in vivo in a rat pressor model. the compounds were found to be effective in raising arterial blood pressure and appeared to be longer acting in vivo when compared to avp and related compounds, as demonstrated by slower disappearance of their pressor effect at equieffective doses. detailed experimental procedures, the results of biological testing, and sar discussion will be presented. the impact of lithium cations on the peptide bond lithium ions play an important role in some biological processes, e.g. in the treatment of neuronal dysfunctions and some metabolic pathways. however, the mechanisms for these actions are not known up to now. recently it has been shown that lithium cations infl uence the isomerisation state of peptide bonds which is essentially pronounced in the case of the amino acid proline. such an isomerisation goes hand in hand with conformational changes possibly resulting in altered folding and structuring. thus, lithium cations might essentially infl uence the biological function of peptides and proteins. to shed light on the underlying mechanism we carried out detailed studies on several model peptides. thus, the isomerisation as well as the kinetic properties of the peptides have been determined employing various techniques of nmr spectroscopy. besides, quantum chemical studies on li + -peptide complexes were performed to get insight into the structural aspects of the cation-peptide interaction. enhanced screening and understanding of hypolipidemic peptides assisted by informatics hypolipidemic peptides targeting the bile acid to inhibit cholesterol absorption in intestine has been indicated by several naturally obtained short peptides. these bile acid-binding peptides are known to disrupt the micellar formation of bile acid for capturing intestinal cholesterol, and lead the aggregates pass the intestine without absorbance. such effect also disrupts the hepatic circulation of bile acids, which accelerates the conversion of stored cholesterol into lacking bile acids. however, traditional extraction procedure of peptides form natural products is time-consuming. therefore, to enhance the screening of such functional short peptides, we have combined the array-based screening strategy with bioinformatics strategy, to design effective experiments by prediction of physicochemical peptide structures. as a result, we resulted in signifi cantly higher screening effi ciency and high bile acid affi nity compared to random screening. we hare report the introduction of one of supervised learning algorithms, fuzzy neural network, and unsupervised algorithm, hierarchical clustering scheme for such functional peptide screening. analogues of the kinin b1 receptor antagonist r-954 bearing n-terminal lipid moieties. adrenomedullin (am) is a 52-amino acid peptide that shares structural similarities with regulatory peptides belonging to the calcitonin family (calcitonin, cgrp and amylin). am is known to produce a potent vasodilation via the coupling to the calcitonin receptor-like receptor (crlr) associated with a chaperone protein, the receptor-activity modifying-protein (ramp). binding studies performed in mammalian tissues revealed that the largest distribution of am binding sites was found in the heart and lungs. in fact, the pulmonary blood vessels display a vast abundance of am binding sites that act as clearance receptors. pulmonary arterial hypertension (pah) is a condition associated with obliteration of pulmonary arterioles which carries a very poor prognosis, in part because of delayed diagnosis due to the lack of specifi c noninvasive diagnostic tools. likewise, there currently exists no molecular imaging agent to diagnose pulmonary embolism, a pathological condition that occurs when a blood clot blocks a lung artery. therefore, due to the high density and incidence of am receptors in the cardiorespiratory system, am could represent a key-target for diagnosis with radiolabeled am-related drugs. in this study, we looked at structureactivity relationships and synthesized various am fragments exhibiting high binding specifi city for am receptors but reduced biological activity. moreover, we introduced different chelating moieties into these am peptides to evaluate the possibility of labelling those molecules for imaging purposes. using cell binding assays, we observed that a cyclic moiety combined with the am(22-52) sequence is able to maintain a good affi nity for am receptors. furthermore, we showed that the incorporation of a 4-amino acid sequence into the peptide chain was the best chelating moiety that allows high effi ciency labelling with 99mtc. hence, our study strongly suggests that am derivatives are promising leads for lung specifi c imaging. 99m tc-labeled analogs with pansomatostatin properties may lead to clinically useful radiotracers and further search for analogs displaying improved biological profi le is warranted. and for the rat (r) sst 2 by competition binding assays in ar4-2j cell membranes with [ 125 i-tyr 3 ]octreotide as radioligand. after 99m tc-labeling, radiopeptide internalization was studied in ar4-2j cells. biodistribution of [ 99m tc]demotates was studied in healthy swiss albino mice and for [ 99m tc]demotate 1, 2, 3 and 6 in ar4-2j tumor bearing mice. results: demotate 1 showed the highest affi nity binding for the hsst 2 and the rsst 2 , while introduction of one asp linker(s) at the n-terminus diminished the affi nity for the hsst 2 and the rsst 2 and led to lower internalization rates. substitution of thr 8 by asp 8 in demotate 6 resulted in good affi nity for the rsst 2 and lower affi nity for the hsst 2 . single (d)phe 1 -substitution/5 or thr 8 substitution by asp 8 combined with asp introduction at the nterminus/7 led to inferior binding affi nity, poor internalization capacity and poor uptake of the respective radiopeptides in target-organs and, in the case of [ 99m tc]demotate 1, 2, 3 and 6, in ar4-2j tumors in mice. conclusion: introduction of asp residues in the original [ 99m tc]demotate 1 chain exerted a negative effect on receptor affi nity, internalization capacity and targeting of somatostatin binding sites in mice precluding their use as hydrophilic pharmacokinetic modifi ers. the family of secreted aspartic proteinases (saps), which are encoded by ten distinct genes, is an important virulence factor of the human pathogen candida albicans.(1) due to an increase in the number of candida strains that are resistant to the drugs currently used in therapy, these proteinases are highly promising new drug target candidates. based on the knowledge of sap2-substrate specifi cities(2)and x-ray structural studies of sap2 in complex with inhibitors,(3) we designed and synthesized a series of sap inhibitors by modifying the structure of the pentapeptide pepstatin a, which is a potent yet non-selective aspartic proteinase inhibitor. these inhibitors were synthesized manually via a spps methodology using a 2-cl-tritylchloride resin and fmoc-protected amino acids. in addition, the key residue of the designed peptide inhibitors, the -amino acid statine (sta), which was prepared according to a slightly modifi ed literature procedure,5. was further protected at its hydroxyl group as a silyl ether for the purpose of avoiding competitive side reactions during amino acid couplings. we prepared 9 new inhibitors by varying the pepstatin a structure at the p3, p2, or p2' position. all variants showed effi cient inhibition when screened against the isoenzymes sap1, sap3, and sap6, and some of them exhibit ic50 values similar to or even lower than that observed for pepstatin a. peptide library is a general technique for screening specifi c peptides that bind to a target protein. but, in a standard screening method, peptide libraries need to be fi xed on large carriers like phage, beads and so on. the large carriers would affect the binding between peptides and the target protein. in this work, we develop a new method for screening peptide libraries without carriers. we attached a variety of fl uorescent amino acids to peptide libraries and the peptides that bind to target proteins were identifi ed through 2-dimensional fl uorescence spectroscopy in aqueous solution. in this presentation, 36-different fl uorescent amino acids were synthesized or purchased. of those amino acids, we newly synthesized 30 fl uorescent amino acids. 2-d fl uorescence spectra of those amino acids were measured. they were different in fl uorescence excitation/emission wavelengths. excluding those of weak fl uorescence intensities, 15 fl uorescent amino acids are selected. those 15 fl uorescent amino acids are mixed in methanol/water (ph7.4) (=1/1(v/v)) and the 2-d fl uorescence spectrum was measured. then, concentrations of the component amino acids were evaluated by a linear least-squares method. the results suggested that all fl uorescent amino acids can be quantifi ed. the set of fl uorescent amino acids combined with the 2-d fl uorescence spectroscopy technique will be a powerful tool for screening peptides and other drug compounds without using any carriers. n-methyl phenylalanine-rich peptides as potential blood brain barrier shuttles malakoutikhah, morteza; teixidó, meritxell; giralt, ernest institute for research in biomedicine, barcelona, spain several peptide families containing n-methylated amino acids were designed and synthesized using solid-phase peptide synthesis (spps). the permeability of these compounds was studied by parallel artifi cial membrane permeability assay (pampa) and immobilized artifi cial membrane chromatography (iamc) so as to select the best peptides in terms of length, terminal groups and amino acid replacement to be used as carriers that pass through a model of blood-brain barrier (bbb) by passive diffusion for non-permeating agents. furthermore, their enzymatic stability in human serum and their cell viability were tested by mtt assay. these peptide families showed great stability and nontoxicity. the three best peptides were coupled to levodopa and assessed. these peptides transferred levodopa through an artifi cial membrane and transformed it from a non-passive permeating drug into a compound able to cross the membrane by passive diffusion. the structure-activity study of insulin-like peptide 3 (insl3) requires the design and synthesis of various analogues. in order to test these for their receptor binding affi nity, a high-throughput receptor binding assay is needed. we have therefore developed an effi cient solid phase synthesis protocol to prepare specifi cally mono-labeled human insulin-like peptide 3 (insl3) for the study of its interaction with its g-protein-coupled receptor, rxfp2. a commercially available chelator, diethylene triamine pentaacetic acid (dtpa), was coupled to the n-terminus of solid-phase bound insl3 a-chain and then a coordination complex between eu 3+ and dtpa chelator was formed. after combination of the purifi ed aand b-chains together with sequential formation of the three insulinlike disulfi de bonds, the labeled peptide was purifi ed at high yield using high-performance liquid chromatography with high ph buffer to prevent the liberation of europium from chelator. saturation binding assays were undertaken to determine the binding affi nity (pk d ) of labeled insl3 for rxfp2 in hek 293 stable cell line expressing rxfp2. the binding affi nity of dtpa-labeled insl3 (9.05 ± 0.03) was comparable to that of 125 i-labelled insl3 (9.59 ± 0.09). the effi cient solid-phase synthesis has provided a novel lanthanide-coordinated, dtpa-labeled insl3 with excellent sensitivity, stability and high specifi c activity and which is superior to the traditional 125 i-insl3. this labeled peptide can be used in a high-throughput screening of insl3 analogues in structure-activity studies. multimodal tumor imaging using mri and pet/spect provides comprehensive diagnostic information as it combines anatomical information (by mri) and functional information (by pet or spect). development of dota-derived imaging probes is advantageous for multimodal imaging as it accommodates many metal ions employed in different imaging modalities including gd(iii) for mri. however, high relaxivity and enhanced specifi c up-take at the targeted site is important for the gd-based mri contrast agents. the dota-based prochelators required for the multivalent vectorization of targeting ligands were synthesized by functionalizing cyclen or do2a-tertbutyl ester with modifi ed glutamic acid. multivalent conjugation of bombesin peptides, which specifi cally target tumors expressing gastrinreleasing peptide (grp) receptors, yielded the corresponding mono-, di-, and tetravalent bombesin analogues. the conjugates showed excellent chelating properties with gd(iii) (for mri applications) and also with 111 in, 177 lu and 68 ga (for radiopharmaceutical applications). the 111 in and 177 lu labeled divalent conjugates showed rapid internalization and slower externalization rate compared to their corresponding monovalent analogues as studied with prostate tumor cell lines. the gadolinium complexes of monovalent and divalent conjugates showed signifi cant relaxivities at 60 mhz ranging from 9.3 to 19.2 mm-1s-1 at 25°c. the values represent the 2 to 4 fold enhancement of relaxivity compare to clinically employed dotarem® (gd-dota). further, the relaxivities increased signifi cantly from monovalent to divalent conjugates. this is highly promising as we would expect much higher relaxivities in case of tetravalent conjugates, which are currently under investigation. in conclusion, the work demonstrates the development of high relaxivity multivalent bombesin analogues, which could be employed for the multimodal imaging such as pet/mri or spect/mri with improved tumor targeting capabilities. a new highly potent dota-conjugated bombesin antagonist for grpr-positive tumor targeted imaging peptide receptors are very promising targets for tumor imaging. the somatostatin receptors were successfully targeted with peptides labeled to -emitters ( 111 in, 67 ga and 99m tc) and positron emitters ( 18 f, 68 ga, 86 y) for diagnostic imaging. for tumor targeting, radiolabeled agonists were developed as they usually trigger the internalization of the peptidereceptor complex. we have recently shown that somatostatin-based radiolabeled antagonists may not only have a higher tumor uptake than equipotent agonists but also a longer lasting tumor uptake. among the most promising receptors to be targeted, the bombesin receptors are of great interest as they are overexpressed in major human tumors such as prostate and breast. the aim of this study was to develop a radiolabeled bombesin-based antagonist conjugated to the macrocyclic chelator dota which allows effi cient and stable labeling with a variety of two-and three-plus charged radiometals for imaging (pet, spect). we compared its in vitro pharmacologic properties and biodistribution in the pc-3 mouse model side-by-side with the potent agonist [ nat,111 in]-do3a-ch 2 co-g-4-aminobenzoyl-q-w-a-v-g-h-l-m-nh 2 (amba). in(iii)-amba was shown to be a potent agonist by ca 2+ fl ux and immunofl uorescence studies and in(iii)-rm1 was effi ciently antagonizing the activity of in(iii)-amba. the pharmacokinetics showed a distinct superiority of the radioantagonist with regard to the high tumor uptake as well as to all tumor to normal tissue ratios. [ 68 ga]-rm1 showed similar pharmacokinetic than [ 111 in]-rm1 with a lower initial kidney uptake and a faster wash out from the kidney. the pet/ ct scintigraphic studies of [ 68 ga]-rm1 in the animal model confi rm the signifi cant high tumor uptake and tumor to background ratio. as we found for somatostatin receptor-targeting radiopeptides,bombesin-based radioantagonists also appear to be superior to radioagonists for in vivo imaging and potentially also for targeted radiotherapy of grpr-positive tumors cell penetrating peptides (cpp) are a special class of peptides that possess the property to traverse the formidable barrier of the plasma membrane and deliver cargos into cells. using cpp as vectors and dna, mrna or proteins/enzymes as potential intracellular targets, a new generation of intracellular contrast agents (cas) can be developed. these agents have prospective use for molecular imaging (both optical and magnetic resonance imaging) by targeted labeling of cells. aiming to image the presence of specifi c mrnas or enzymes, two mrna targeting (contains a pna sequence antisense or non-sense to the target mrna of dsred) and one enzyme targeted (contains a unit cleavable by -galactosidase) cas were tested for their activity in the presence and absence of respective targets. the antisense targeting ca, their nonsense derivative and the enzyme targeted ca were taken up effi ciently into cells by an exclusively endosomal mechanism as observed by fl uorescence microscopy. cell free binding assays proved a specifi c interaction with a synthetic target for the antisense but not for non-sense ca. magnetic resonance studies showed a higher uptake in transgenic dsred expressing cells than the parent cells. however, no difference was observable for antisense versus non-sense ca in dsred cells, due to the vesicular entrapment which is preventing the specifi c interaction between ca and cytosolic target. since a comparable cellular distribution was visible for the enzyme targeted agent, a specifi c accumulation in -galactosidase containing cells is also unlikely. the results show that even though the designed cas were effi ciently taken up into cells, they can interact specifi cally with the target only if colocalization is achieved. however, a lack of specifi city is caused by the endosomal entrapment. further modifi cations are required to achieve the release from endosomes or a direct uptake into the cytosol. the infl uence of pegylation on the tumor accumulation of frop-1, a tumor specifi c peptide identifi ed by phage display the pool of natural peptides suitable for the tumor targeting is limited and therefore novel lead structures identifi ed by screening of phagedisplayed libraries are highly warranted. however, transfer of the novel peptide sequences to clinical application is often diffi cult. we had shown that the coupling of dota to frop-1, a peptide identifi ed in phage display libraries resulted in a fundamentally improved in vitro binding capacity. however, a biodistribution study revealed that the slow binding kinetics frop-dota (h-glu-asn-tyr-glu-leu-met-asp-leu-leu-ala-tyr-leu-lys(dota)-cys-nh2) allowed the excretion to forestall a signifi cant tumor accumulation. the aim of this study was to investigate whether the conjugation of peg to frop-dota results in a derivative with a prolonged residence time in the blood. frop-1 bearing a c-terminal dota residue to allow labeling with in-111 and a cysteine to attach a maleimido-modifi ed 2000 da peg oligomer was obtained by solid phase synthesis. the conjugate was purifi ed by size exclusion chromatography. several attempts to couple different peg derivatives on the solid support did not proceed satisfactorily and therefore the peg residue was attached in solution. the breast cancer cell line mcf-7 showed a relative low accumulation of the pegylated peptide in the cells. in contrast, biodistribution studies of the labeled conjugate in mice bearing human fro82-2 showed a time dependent increasing uptake of the pegylated peptide with a high retention (at 24 h p.i. 76% of the maximal activity concentration persisted in the tumor). the highest uptake values were determined at 120 min. p.i. reaching 2.3 %id/g tumor as compared to 0.06% %id/g observed for the non-pegylated derivative at 135 min p.i. apparently pegylation provides a substantially improved stabilization in the circulation which allows a stable tumor accumulation. in vivo spect/ct imaging of intracranial human glioblastoma xenografts with 111indium-labeled homing peptide. huhtala, tuulia* 1 ; enbäck, julia* 2 ; rautsi, outi 1 ; weisell, janne 1 ; laakkonen, pirjo* 2 ; närvänen, ale* 1 *equal contribution. 1 university of kuopio, finland; 2 university of helsinki, finland phage display libraries provide a powerful tool to identify new biologically active peptides that bind specifi cally to their target molecules. different tumors express a distinct range of molecular markers on their vasculature providing a possibility to use peptides for targeting. tumor-homing peptides offer an opportunity to target, image and destruct the target tissue. glioblastomas represent the most aggressive form of brain tumors. we have identifi ed a novel peptide, coop, using an ex vivo / in vivo phage display screen. this peptide homes specifi cally to the early stage astrocytoma model. in addition, the peptide homes to the u87 human glioblastoma xenografts (enbäck and laakkonen, unpublished data). we have studied the biodistribution and tumor homing properties of indiumlabelled peptide in mice bearing intracranial human u87 glioblastoma tumors using spect/ct imaging. coop peptide was conjugated with the dtpa (diethylenetriamine pentaacetic acid) via amino terminus and labeled with 111indium. imaging was performed in four different time points (15 minutes, 1 hour, 2 hours & 24 hours), and the organ and tissue specifi c radioactivity was measured after the scarifi cation of the animals. intravenously injected 111in-labeled coop peptide accumulated in the u87 braintumors. the amount of the peptide increased with time and a clear radioactive accumulation was seen in 1 /3 animals at 1 hour and in 5 / 6 animals at 2 hours after injection. due to the small size of the dtpa-peptide conjugate the majority of the molecules were secreted via the kidneys during the fi rst 15 minutes. at 2 h timepoint the tumorto-brain tissue ratio was 11,5. we report here that the coop peptide, which specifi cally homes to the brain tumor vasculature and tumor cells, strongly accumulated in vivo to the xenografted human glioblastoma tumors in mice. this is the fi rst time when a synthetic peptide has been used in spect imaging of brain tumors in animal mode. failure of the development of a novel peptide tracer for molecular imaging of cancer therapy response 343-9 (2008) have shown that fl uorescently labeled derivatives of the hvggssv motif accumulated in tumors undergoing therapy. this peptide motif had been identifi ed by the in vivo screening of phage-displayed peptide libraries. the ability of the peptide to differentiate between responding and nonresponding cancers after treatment could be utilized for the development of promising tracers to monitor cancer therapy. the goal of this study was to investigate whether radiolabeled derivatives of the hvggssv motif can be used for the noninvasive determination of therapy response in vivo. therefore three different conjugates were synthesized by solid phase synthesis using fmoc chemistry. the fi rst peptide was n-terminally modifi ed with the chelator dota (dota-ggghvggssv-conh2) to allow radiolabeling with metallic radioisotopes such as 111in or 68ga. a glycine linker was placed at the n-terminus to separate the peptide motif from the chelator. in order to obtain derivatives accessible to radioiodination a tyrosine was introduced at the n-terminus (h2n-yggghvggssv-conh2) of the second peptide. the third peptide was biotinylated at the n-terminal amino group and bound to radioiodinated streptavidin (sa-biotin-ggghvggssv-conh2). the biodistribution was monitored by scintigraphy in mice bearing a431-tumors treated with a vegf receptor-specifi c inhibitor (su5416). this study revealed that the 111in labeled hydrophilic dota conjugate shows a rapid renal clearance, the iodinated peptide accumulates mainly in the liver and kidneys. all of the peptide derivatives do not specifi cally accumulate in treated tumors after i.v. injection. in contrast to the promising results shown by han z. et al. our study shows that the hvggssv motif can not be easily adapted to radiolabeling approaches with clinical relevance. in conclusion, the peptide motif is not attractive for further clinical evaluation as new diagnostics for the imaging of cancer response. a novel approach to improve cellular delivery of 5-aminolaevulinic acid: new ala-containing peptide prodrugs for photodynamic therapy. photodynamic therapy (pdt) is a binary therapeutic modality which is currently under investigation for the treatment of several kinds of malignancies. it relies on the interaction of two individually harmless components: a photosensitiser and an external radiation. the interaction of the photosensitiser with light of the appropriate wavelength and molecular oxygen results in the generation of cytotoxic species, namely singlet oxygen and/or radicals, and localized destruction of tumours or infected tissue. in 5-aminolaevulinic acid photodynamic therapy (ala-pdt), exogenous administration of ala is employed to generate elevated intracellular levels of the natural photosensitiser protoporphyrin ix (ppix), via metabolism through the haem biosynthetic pathway. however this approach suffers from several drawbacks associated with ala's lack of stability at physiological ph and its highly hydrophilic nature, which prevent it from crossing biological membranes and hence limit tissue penetration. ala delivery with synthetic peptide prodrugs is a promising way to address these problems. in this work we report the synthesis and characterisation of a series of peptide prodrugs of general structure ac-xaa-ala-or, where xaa is an alpha amino acid, chosen to provide a prodrug with appropriate lipophilicity and water solubility. the uptake of the compounds and metabolism to ppix in pam212 keratinocytes, relative to ala is evaluated by fl uorescence spectroscopy, and further quantifi ed by recovery and chemical derivatisation of intact/ partially metabolised prodrugs. in a parallel study, we have also explored the possibility of coupling of ala and ala prodrugs to cell penetrating peptides (cpp). the conjugation of one or more molecules of ala to a cpp sequence represents an interesting approach to enhanced topical delivery of ala. preliminary results of these studies are reported herein. acknowledgements: thanks are due to biotechnology and biological sciences research council (grant bbd0127831) bioshuttle as a carrier for temozolomide transport into prostate cancer cells if metastatic prostate cancer gets resistant to antiandrogen therapy, there are few treatment options, because prostate cancer is not very sensitive to cytostatic agents. temozolomide (tmz) as an oral applicable chemotherapeutic substance has been proven to be effective and well tolerated with toxicity especially for brain tumors. unfortunately tmz was ineffi cient in the treatment of symptomatic progressive hormone-refractory prostate cancer. this may have different reasons like the short plasma half-life of tmz, a non adapted application schema and as a result, an insuffi cient bioavailability. to improve the specifi city, we built our so called tmz-bioshuttle-construct with cathepsin b (ctsb) mrna specifi city. this complex combines, a transmembrane transporter molecule connected via a disulfi de bridge to an antisensepeptide nucleic acid (pna) against a docking site in exon 1 of the ctsb mrna. furthermore this part is connected via a ctsb cleavable peptide substrate to a nuclear localization sequence coupled with tmz. inside the target-cell, the pna recognizes the cytoplasmic ctsb mrna and after annealing (the pna/rna hybride is not a substrate for rnase h) results in a cell-specifi c retention of the tmz in the cytosol especially of ctsb-expressing cells. then, after the cathepsin b-mediated cleavage in the cytoplasm, the nls-sequence is separated and activated for an ran/importin-mediated transport of the tmz-cargo into the nucleus of the target cells. this bioshuttle-mediated tmz transfer could be a step forward to a successful therapy with higher specifi city, avoiding adverse reactions and circumventing the previous therapy limiting situation. the intracellular delivery of protein segments and other bioactive molecules using membrane -permeable peptides has been investigated in multiple aspects. most of the currently recognized cell penetrating peptides (cpp) are of cationic nature and derived from viral, insect or mammalian proteins endowed with membrane translocation properties. these peptides enter the cell by a receptor independent mechanism, which is poorly understood and carry only one epitope. our target was to achieve a cell-penetrating carrier able to transport more than one bioactive molecule. to this aim we designed a cell penetrating sequential carrier (cpsc) formed by the repetitive -lys-aib-cysmoiety, which incorporates the cysteine residue for anchoring the bioactive molecules through a thioether bond. the lysine free side chain possesses the cationic nature of the construct while the á-amino isobutyric moiety could induce a 3 10 helicoid peptide backbone with amphipathic characteristics. to test the ability of the cpsc to penetrate the cell membrane we synthesized the carboxyfl uorescein -labelled cpsc, cf-[lys-aib-cys(-ch 2 conh 2 )] 4 -nh 2 , while the cf-tat-cys(-ch 2 conh 2 )-nh 2 analogue, which is a small basic peptide that has been shown to deliver a large variety of cargoes into the cells, was used as a positive control. the results indicated that cpsc had a homogeneous distribution into the cytoplasm suggesting that cpsc may provide a new powerful biological tool for drug transportation. acknowledgements: the gsrt and eu (pened 03åä629) for the fi nancial support. amphipathic pro-rich peptides as intracellular drug delivery systems pujals, silvia; fernandez-carneado, jimena; giralt, ernest institute for research in biomedicine, spain shared among many of the known cpps. in 2004 our laboratory described a novel group of cpps: amphipathic proline-rich peptides. the principal advantages of these compounds are non-cytotoxicity, nonviral origin and high solubility in aqueous media. the best candidate for cpp among the amphipathic pro-rich peptides was sap (sweet arrow peptide), (vrlppp) 3 .(1) derivatives with hydrophobic moieties, such as fatty acids or silaproline, have shown highly improved internalisation effi ciency; an all d-amino acid version of the cpp sap was shown to be completely protease resistant and was evaluated in a preliminary in vivo study. cd and tem studies regarding the self-assembly properties of this family of peptides highlight the possible role of aggregated species in the internalisation process. finally, these cpps have shown to be internalised via caveolae or lipid-rafts mediated endocytosis, which circumvents the lysosomal route of degradation. in order to test a challenging cargo for sap, gold nanoparticles were conjugated to sap. thereafter, the transport of au np by sap in hela cells has been studied by tem. while np alone are not internalised, np-c-(vrlppp) 3 are clearly uptaken. studies with qds (quantum dots) and egfp as sap cargo are currently being done in our laboratory. 1. pujals, sílvia; giralt, ernest. proline-rich, amphipathic cell-penetratingpeptides. addr (2008), 60, 473-484. novel cysteine-rich cell penetrating peptide: effi cient uptake and cytosolic localization introduction: crossing the plasma membrane is a prerequisite for intracellular targeted drug delivery. cell penetrating peptides are actively used as the delivery tool for intracellular delivery of various cargos. however, confi nement of biomolecules into endosomes limits their use for intracellular targeting. therefore, there is a need for vectors capable of transferring cargo molecules directly into the cytoplasm. herein, we focus on the development of a novel cpp (derived from polypeptide crotamine 1.) which shows an effi cient uptake at low concentrations ( 2.5 m) and cytosolic distribution along with vesicular uptake. methods: series of peptides were synthesized by fmoc strategy, introducing mutations in cro (27-39) (proposed cpp sequence in crotamine). all were n-terminally labeled with fl uorescein isothiocyanate for optical imaging. structure activity relationship (sar) studies were done by substitution and/or deletion of amino acid residues in the sequence observing the uptake behaviour by fl uorescence spectroscopy and microscopy. results: amongst 60 synthesized peptides, one of shorter length showed the best intracellular delivery and cytosolic distribution at lower concentration (2.5 m) when compared to other cpp. replacing or deleting cysteines had negative impact on internalization. results also displayed the involvement of tryptophans in cellular uptake indicating, along with cationic amino acids, the importance of each residue in this optimized sequence. conclusions: sar studies identifi ed a novel cell penetrating peptide showing, besides of endosomal uptake, also an effi cient delivery into the cytoplasm at low concentrations. thus, this peptide might prove useful for effi cient transmembrane delivery of agents directed to cytosolic targets. peptide nucleic acid (pna) is a dna mimic consisting of the four common bases of dna on a pseudopeptide backbone that makes it extremely stable in biological fl uids. antisense pna is targeted against mrna in cytoplasm in a sequence specifi c manner. however, the main hindrance to the effective use of pnas has been their relatively poor uptake by cells. endosomal release or direct uptake into cytosol of agents is mandatory for attaining mrna based targeting. there are reports on the cell penetrating peptide (cpp) based delivery system. it has also been reported that conjugates of cholesterol and sirnas facilitate cellular import (1) . the aim of this study was to synthesize different sequences of cholesterol coupled antisense pna and to compare its uptake characteristics with a cpp-pna conjugate. the synthesis of pna (anti-dsred pna (agcgcctgtacc), specifi cally targeted to mrna of dsred, a red fl uorescent protein) conjugated to cpp (d-tat) or cholesterol was performed in fully automated synthesizer (prelude, protein technologies, inc.) using continuous solid phase chemistry. to increase the solubility in water, linkers (aeea) and additional charged amino acids were coupled or the sequence of peptide, pna and cholesterol was changed. all compounds were labelled with fitc to confi rm the cellular uptake by fl uorescence microscopy and spectroscopy. cell uptake studies showed that the cpp bound pna was located predominantly in vesicles indicating an endosomal uptake mechanism and subsequent entrapment in vesicles. cholesterol bound pna was also effi ciently internalized. however, it was also located inside vesicles without detectable cytosolic distribution. pna-cholesterol has fewer synthetic steps than pna-cpp. however, it was also located inside vesicles restricting its applicability for mrna targeting. the effi cient uptake might make it a promising cellular delivery agent after further improvements. opioids are gold standards in pain treatments. unfortunately, fast development of tolerance and dependence creates limitations in application of these drugs in chronic pain treatment. over twenty years ago we have proposed development of multitarget medicines as a new avenue of drug discovery. identifi cation of numerous endogenous components that participate in the formation, transmission, modulation and perception of pain signals offers various strategies for the development of new analgesics. neurotensin is an endogenous neuropeptide that play important regulatory role of pain transmission. therefore, it was interesting to develop chimeric analogues that hybridize opioid and neurotensin active components. in such chimeric compounds the possible structural interference may signifi cantly infl uence on interaction of both components with target receptors and/or biological barriers transport systems. in this communication we present synthesis, pharmacological profi le and structural analysis of new potent chimeric compounds. acknowledgements: presented studies have been supported in part with eu grant normolife (lshc-ct-2006-037733) artifi cial ribonucleases based on short peptides. the synthesis of molecules that are capable of nonrandom rna cleavage has found a variety of important applications in molecular biology. for instance, such molecules are used as structural probes for nucleic acids in solution, or in rational design of novel anti-infectives, since rna is the genetic material of many pathogenic viruses. here we represent design and synthesis of peptide-like molecules mimicking the catalytic site of natural ribonucleases (a and t1): series f aa1 -aa2 -aa3 -phe -oalkyl; aa1 -glu/lys, aa2 -thr/ser/lys, aa3 -glu/ lys/thr/arg; series r glu -x -arg -gly -oalkyl; series k glu -x -lys -gly -oalkyl; -gly, -ala, 4-aminobutyric acid, 6aminohexanoic acid, p-aminobenzoic acid; series l aa3 -aa2 -aa1 -l1 (l2)-aa1 -aa2 -aa3 , l1 -4,9-dioxa-1,12-dodecanediamine, l2 -1,12-diaminododecane; aa1-aa3 -glu, lys, ser, arg. ability of artifi cial rnases to rna cleavage was shown in experiments with 96mer rna hiv-1. the effi cacy of rna cleavage depends on artrnases structure (for example, on location of negative and positive charged amino acids (glu, arg lys) in peptide). the effi cacy of the most active compounds was 60-98 % at 18 h incubation. anti-infl uenza activity in vitro of 12 such artrnases was investigated by inhibition of reproduction virus a/hong kong/1/68 (h3n2) in tissue cultures of chorioallantoic membranes (ccm) of 12-14 days age chick embryos. acknowledgements: this work was supported by rfbr (07-04-00990a, 08-04-90038-bel_a), pharmamed ruxo-008-n -06, the grant of novosibirsk region government for the best young scientists. (ziagen)-(1s,cis)-4-[2-amino-6-(cyclopropylamino)-9hpurin-9-yl]-2-cyclopentene -1-methanol is a synthetic carbocylcilic nucleoside analogue with inhibitory activity against hiv. serious and sometimes fatal hypersensitivity reactions have been associated with abacavir. a possible way to increase the side effects is by modifying the known antiviral drugs with various amino acids. the aim of this study was to design and to synthesize of new amino acids and peptide (gly, gly -gly) and thiazole containing (gly, gly -gly) esters prodrugs of abacavir and to explore their activity on the hiv. chemical stability of some purine analogues in the search of new prodrugs effective against herpes simplex virus, series of acyclovir-9-[(2-hydroxyethoxy)methyl]guanine (acv) esters with peptidomimetics, as well as abacavir-(1s,cis)-4-[2-amino-6-(cyclopropylamino)-9h-purin-9-yl]-2-cyclopentene-1-methanol derivatives have been synthesized and tested for antiviral activity. the chemical stability of some of them is studied at ph 1 and 7.4 and temperature of 37°c. a high-performance liquid-chromatografi c (hplc) method was developed for quantifi cation of the unchanged ester concentration. the promelittin represents a readymade cancer pro-toxin; the prodomain inhibits cytolytic activity and the sequence of the prodomain could be manipulated into a substrate for activating proteases. here we present the development of a novel promelittin pro-toxin that can be activated by the serine protease fi broblast activation protein (fap). fap is over expressed on the surface of reactive stromal fi broblasts present in the stroma of human epithelial tumors. fap is not expressed by tumor epithelial cells or by fi broblasts in other tissues, thus making fap a pantumor target for pro-toxin development. peptides containing truncated pro-domain sequences were tested on erythrocytes to determine the optimal prodomain length for inhibiting cytolytic activity. once the length was optimized, modifi ed promelittin peptides were generated that contained previously identifi ed fap substrate sequences in the prodomain. these peptides were digested with fap and tested in vitro against fap+ and fap-cell lines in order to determine their fap cleavage potential and toxicity. our lead promelittin peptide was found to be effi ciently activated by fap and selectively toxic to fap+ cell lines with an ic50 value in the low micromolar range that is similar to that of melittin. pharmacology and medical peptide chemistry region of highly hydrophobic and the antigenic part of hbsag by using microwave assisted solid phase peptide synthesis (spps). tryptophan at the n terminus of the sequence was added by our research group for fluorescence dedection analysis. this peptide was conjugated with synthetic polyanions. different conjugates comparised each other. the synthesized bioconjugates analysized by choromatographic and fl uorometric methods. the fi nal product peptide was characterized by lc-ms and purifi ed by rp-hplc. the bioconjugates were synthesized using different activation mechanisms and the different initial molar ratios (npeptide/npolymer = 1, 3, 5, 7, 9) of the polyanions and the peptide. also physicochemical properties of the bioconjugates were investigated. the structure and characterization of the synthesized conjugates was analyzed by various choromatographic and fl uorometric methods such as hplc, gpc and fluorescence spectrometer. the reaction of high temperature solid-state catalytic isotope exchange (hscie)(1) between insulin and spillover tritium was studied. hscie reaction with the solid mixture containing 1 mg recombinant human insulin was performed for 10 min at 120o c using 5% pd/baso4 catalyst. the product of the reaction was purifi ed by hplc on waters delta-pak c4 300a 3.9 x 150 mm column. tritium labeled insulin with specifi c radioactivity 40 ci/mmol and radiochemical purity of more than 98% was obtained. performic acid oxidation of cysteine residues and subsequent acid hydrolysis were used to analyze the distribution of tritium. it was demonstrated that specifi c radioactivity of à and b chains amounted to 8.2 and 31.8 ci/mmol respectively. all the amino acids contained tritium and its content in his residues of the b chain was equal to 45%. experiments for the evaluation of the biological activity of tritium labeled insulin were performed on cd-1 6-8-week-old awake male mice. activity of labeled insulin was compared with the activity of standard insulin. insulin was injected subcutaneously at a dose of 0.8 u/kg. twofold statistically signifi cant lowering of glucose level was observed 40 min after injection (4,9 + 0,4 and 4,3 + 0,3 mmol/l for standard and tritium labeled insulin, respectively). lowering of the glucose level in animals obtaining labeled insulin was the same, as in animals obtaining standard insulin. thus, hscie reaction of insulin with tritium doesn't change its hypoglycemic activity. cortexin is polypeptide (up to 10 kda) brain extract accepted for clinical use in several countries including russia due to its positive effects on memory, attention, and cortical processes. a synthetic peptide analog of cortexin, cortagen (ala-glu-asp-pro), was recently developed. the heptapeptide semax (met-glu-his-phe-pro-gly-pro) is an analogue of the acth(4-10) fragment, which is completely devoid of any hormonal activity associated with the full-length acth molecule. both cortexin and semax are currently effectively used for treatment of brain hypoxia and ischemia with comparable clinical benefi ts. however, the cellular and molecular mechanisms underlying the action in the brain are mainly unknown. in present work we studied effects of cortagen, cortexin, semax, its analogue pro-glu-pro (pgp) on glutamate-induced neuronal death and intracellular ca2+ homeostasis deterioration in cultured cerebellar granule cells. peptide drugs gain increased interest as a new supra-group of therapeutics between the classic-organic, small-molecule drugs and the large biotechnology-derived bio-drugs. they are used in different therapeutic areas like allergy, anti-infection, oncology, obesity, etc… due to their particular structure and biochemical origin, pharmaceutical development of a peptide drug poses special challenges which will be discussed here, exemplifi ed by own research as well as literature data. currently, there are about a dozen classical peptides described in pharmacopoeia, excluding the derived peptidomimetics like ace-inhibitors. these pharmacopoeial peptide-drugs, together with the approved marketing authorisations of new peptidic entities, are a good starting point to look at the desired characteristics. last, the regulatory developmental guidelines are in a general way seldom taken peptide drugs into account, leaving an interpretational gap between the two current main supragroups of therapeutics. after the initial active pharmaceutical ingredient (api) synthesis, analytical characterisation is aimed at integrity and purity evaluation of the api, which is also required for biomedical experiments. in this analytical characterisation, sample treatment issues like solubility and adsorption are considered as well. the chemical and plasma-metabolic stability, a critical parameter for peptides, is to be assessed to obtain kinetic and mechanistic information. functionality is tested in vitro using cell-and organ-based protocols, including ligand binding studies, as well as in vivo encompassing adme and target-organ confi rmation like brain. the pharmaceutical drugability information thus obtained allows further development decisions including required api modifi cations and proof-of-principle drug delivery formulations. lysine dendrimers and starburst copolymers as new carriers of anticancer drugs based on the complexes of platinum and gold. though the complexes of metals such as platinum and gold are very promising compounds for the treatment of different types of cancer, the low solubility complicates their application as medicines. in this work a series of lysine dendrimers of different structure and starburst copolymers of lysine and glutamic acid was studied as solubilizing carriers of anticancer drugs based on the complexes of platinum (cisplatin) and gold. cd analysis showed the small changes in the structure of the polymers upon metal binding. the esem study revealed that the presence of the amino groups on the surface of the carriers is crucial for the metal ligation. the highest content of metal bound was found in the samples with high amount of lysine residues. while the content of gold was rather low in all the samples (about 2%), the copolymer of lysine and glutamic acid with the ratio 2:1 as well as lysine dendrimer of fourth generation had 7 to 10% of platinum linked to the carrier. the absence of the bromine in the samples in the case of gold complex allowed to assume the covalent bond between metal and polymer. in the same time all the conjugates were highly soluble in water. the peculiarities of the synthesis and the anticancer activity in different cell lines will be discussed. acknowledgements: the work was supported by the "russian science support foundation". prospects for the development of synthetic peptide vaccines against hepatitis c no vaccinoprophylaxis of this disease or cardinal treatment means has been developed till now because of the problems produced by high genetic variability and heterogeneity of hepatitis c virus (hcv) isolates, lack of both hcv infection models and effi cient expression systems for the virus and its components. the problem of anti-hcv vaccine development can be solved via a principally new approach, the creation of artifi cial synthetic immunogenic constructs with the help of bioinformatics and high-throughput screening technologies. two approaches exist towards the development of anti-hcv peptide-based vaccines: one is to develop vaccines that stimulate cytotoxic t-cell response (peptides loading dendritic cells; the so-called "cell vaccines") and the second is to construct immunogenic peptides raising protective antibodies. however, the fi rst approach may lead to the massive hepatocyte death and the development of the heavy hepatic injury during hcv infection. in order to develop peptide immunogenic constructs able to raise antibodies against whole native hcv envelope proteins analysis of amino acid sequences of these proteins belonging to different hcv genetic variants has been performed. this analysis has allowed to choose the most conserved and presumably functionally important protein fragments. immunogenicity of these fragments in the whole protein molecules has been determined and interaction sites for one of the putative hcv receptor, heparansulfate, have been revealed with the help of peptide scanning. despite of the low immunogenicity of the most fragments inside the whole protein, antipeptide antibodies against them have been obtained via immunizing by conjugates of the peptides with certain carriers. two synthetic peptide immunogenic constructs have been developed that have raised antibodies against whole hcv envelope proteins. synthetic immunoactive fragments of endogenous proteins: selection and application for diagnostics and immunotherapy we have selected and synthesized the immunoactive fragments of four endogenous proteins: nucleophosmin, survivin, prion and alpha-7 subunit of acetylcholine receptor (achr). nucleophosmin and survivin are overexpressed in tumor cells and exhibit an antiapoptotic activity. their detection in tumor cells could be used for tumor differential diagnostics and selection of optimal chemotherapy scheme. accumulation of pathogenic isoform of prion protein in brain causes the neurodegenerative prion diseases and antiprion antibodies as known could be used for immunotherapy of prion disorders. alzheimer's disease (ad) development is accompanied by an accumulation ofamyloid peptides ( a) in brain and destruction of neurons . one of the ad development hypotheses assumed that a when binding to the alpha-7 achr forms a complex that penetrates into the cells, resulting in the neurons death. we have proposed that alpha-7 achr could be a new target in the ad therapy and antibodies against alpha-7 subunit could prevent its binding to a. selected synthetic fragments of these four proteins were able in a free nonconjugated to a protein carriers state induce antibody response in experimental animals. obtained rabbit antibodies against fragments of nucleophosmin and survivin were affi nity purifi ed. antibodies against nucleophosmin detected monoand oligomeric forms of this protein in immunoblotting of hela cells lysates. antibodies against survivin were able to detect this protein in immunohistochemical test of the breast cancer tumor samples. rabbit antiprion antibodies interfered with patigenic prion isoform in scn2a neuroblastoma cell culture. it was shown that synthetic alpha-7 achr fragments induced the antibodies formation in mice with symptoms of ad which penetrated the blood brain barrier, regenerated the spatial memory and normalized the beta-amyloid level in animal's brain. acknowledgements: supported by ras program mcb and rfbr grants 06-04-48710, 06-04-48388. investigation peptide-based cyclin a inhibitors: new tools to understand and to regulate the cell cyle-apoptosis signalling pathway the knowledge of the aetiology of cancer has increased considerably in the last two decades. the objectives have moved from unspecifi c cytotoxic chemotherapeutics to the identifi cation of small molecules (and antibodies) with a well defi ned molecular target. proliferation of eukaryotic cells is under control of a series of concerted molecular mechanisms defi ned as the cell division cycle whose progression is tightly governed by members of the cyclin-dependent kinase family. the protein-protein complexes formed between different cyclins and cdks (cdks) are central to cell cycle regulation. these complexes have been object of extensive research in cancer programs. considerable effort has been focused on the development of atp-competitive small molecule inhibitors of cdks. however, in general, these compounds have several alternative protein targets that compromise their demanded selectivity. cdk-2 was recently suggested to be dispensable for cell proliferation, although this does not appear to be the case for cyclin a, which therefore is defi ned as a more appropriate target for drug design. we have identifi ed an hexapeptide (nbi1) that inhibits the kinase activity of the cdk2-cyclin a complex through selective binding to cyclin a (1). the characterization of the inhibitory mechanism revealed that the hexapeptide does bind neither to the atp site nor to the cyclin recruitment site. a cell permeable derivative of the nbi1 peptide induces apoptosis and inhibits proliferation of tumor cell lines. we believe that the current structural and mechanisms of action studies on nbi1 will be useful for characterizing and controlling the important relation between cell cycle and apoptosis signalling pathways. the average size and size distributions of protein, polymer and peptideprotein or polymer-protein mixtures (complexes) and conjugates was investigated using photon correlation spectroscopy with a zetasizer nano zs instrument. the zeta potential measurements of this complex and conjugates were carried out in the folded capillary cell of the zetasizer nano zs instrument the same as used for dynamic light scattering measurements. in this study we investigate that size and zeta potential of synthetic peptide-carrier protein covalent conjugates. synthetic peptides are small antigenic molecules and not strong immunogens because of their small size for this reason, synthetic peptide must be coupling to a suitable carrier proteins or polymers (bsa, klh, ova) for increasing their immunogenity. most of the vaccine development studies are focused on the preparation of a good effective adjuvant. in this study we prepare carrier-protein (bovine serum albumin protein)-synthetic peptide conjugates whit carbodiimide method by using 1-ethyl -3-(3dimethylaminopropyl) carbodiimide hydrochloride (edc). for one protein molecule we studied with different ratios of peptides (npeptid/ nbsa) and synthesized the conjugates at these ratios. conjugates are purifi ed by using different column systems. purifi ed conjugates are characterized and the mechanism of the binding process was investigated comparatively with synthetic peptide, bsa and bsa-synthetic peptide physical mixture by using zeta-sizer nano zs depent on the time. an analysis of deletion mutants of the pld1 d4 domain defi nes short regions within the pld1 interacting with ped/pea15: implications for the development of peptides-specifi c antagonist. (1) . several studies have revealed that it regulates cellular functions by binding components of intracellular transduction pathways. recent reports also evidenced that it binds to phospholipase d (pld1) and enhances its stability, resulting in increased intracellular levels of diacylglycerol(2), deregulating protein kinase c signalling and generating resistance to insulin action on glucose transport (3) . thus, disrupting the interaction between these proteins by a cell-penetrating compound represents a novel strategy for improving insulin sensitivity in target cells. the expression of d4 domain, (the shortest ped-interacting region with pld1) in l6 skeletal muscle cells stably overexpressing ped, reduces the interaction of this protein with pld1 (4), suggesting that this region could bind ped preventing its interaction with the whole pld1 and restoring insulin action. aim of this work is the identifi cation of d4 crucial residues for ped interaction and the development of specifi c antagonists. we expressed three soluble truncated d4 domains, determining whether binds to ped like the d4 wild type, by carrying out dose-response elisa. only one of these regions exhibited an effi cacy similar to d4-wt and functional cellular data support this evidence. to further investigate the d4 residues involved in ped binding, we prepared a set of overlapping peptides covering the d4-region identifi ed. our results suggest that the n-terminus region of d4 encompassing residues 762-801 is involved in ped/pea15 recognition and, currently, cellular experiments on peptides are underway. ew-peptide family. one family, two drugs. two dipeptide analogs -l-glu-l-trp (1) and -d-glu-d-trp (2) -had been registered in russia as immunomodulator thymogen (1) and immunosuppressor thymodepressin (2). these two drugs possessed reciprocal activities in vivo [1, 2] . the individual peptides were initially separated by preparative hplc from the crude thymus homogenate and sequenced. dipeptide l-glu-l-trp was the most active in the majority of in vitro and in vivo tests. in the process of sar studies, the "signal" role of l-glu-l-trp in the immune response was discovered, as well as the critical role of manifestation of in vitro effects as well as the infl uence of precise chemical and optical structures on biological activity of different ewpeptide analogs will be discussed. the experiments were conducted on blood neutrophiles, monocytes, thymic epithelial cells, lymphocytes, thymocytes, endothelial cells of human blood vessels and newborn blood cord. we evaluated the peptide effects on the phagocyte activity of cells, on the expression of functionally important membrane molecules and cell activation markers, and on the capacity of cells for mutual adhesion. the dose dependence data of 1 and 2 on such activities as cytokine production, the processes of stimulation and suppression of apoptosis and proliferation of immunocompetent cells will be presented. ion exchange chromatography (iec) is widely used for analysis and purifi cation of biomolecules. we have newly developed polymer-based iec column, named ymc-biopro, specially designed for separation of proteins, peptides and nucleic acids. ymc-biopro iec columns are based on 5 micron @porous and non-porous hydrophilic polymer beads with low nonspecifi c adsorption, and they show higher binding capacity and higher recovery of biomolecules compared to conventional iec columns. the completely spherical and monodispersed beads, with optimal packing technology, provide high theoretical plate number and symmetrical peak shape. excellent resolution is achieved from the high column effi ciency coupled with the excellent selectivity of qa and sp ion exchangers. in this poster, we will show benefi ts of ymc-biopro iec columns and some example cases of superior separation of important biomolecules, such as monoclonal antibody and dna. the analysis of proteins and peptides by rp-hplc is important in the development of well-characterized biotechnology pharmaceuticals. since polypeptides interact only slightly with the stationary phase after desorption, short columns are suited for fast separations. also, by decreasing the particle size of hplc packings, column effi ciency is increased. we demonstrate the use of short hplc columns packed with 1.5um, large pore c18 silica for ultra-fast biomolecule analysis. due to the short column format, high fl ow rates and optimal separations are possible, with moderate backpressures (< 3000 psi), using a conventional high-pressure gradient, binary pump system. narrow-bore lc and lc-ms analyses were performed on a binary or a quaternary hplc system, with detection by uv or ms (ab/mds sciex q trap). the solvent system comprised of acn/water or n-propanol/water plus one or two ion pairing agents (formic acid, tfa, hfba) at 0.01 to 0.2%. a fl ow rate of 0.8 ml/min (1200 cm/h), 4x higher than typical for a 2.1 mm id column, allowed for ultra-fast one minute separations of proteins with broad physicochemical characteristics. closely related insulin variants (bovine, sheep, human) may be separated in under 1.5 minutes. while the typical hplc run time for the separation of hemoglobin chains on conventional columns ranges from 25 to 60 minutes, runs under two minutes may be realized with the large pore, sub-two micron column. accordingly, fi ve different hemoglobin samples (from different animal species) may be compared after a total of only 14 minutes (includes run and equilibration time). 10 highly reproducible runs of a synthetic peptide mixture can be completed in < 20 minutes, including equilibration with 20 column volumes between runs. intact igg in sheep serum may be separated from albumin in < 4 min. using a n-propanol/water solvent system containing 0.1% tfa. new peptides and triterpene derivatives as dimerization inhibitors of hiv-1 protease protein-oligopeptide fragmentomics zamyatnin the substantiation and defi nition of the term "fragmentomics" is given. within the framework of this scientifi c direction the theoretical structural-functional analysis of all possible fragments of protein molecule can be performed with the purpose of determination of its sites which could be potential sources of the regulatory oligopeptides. the data on the primary structure of proteins from public protein databases, information from the erop-moscow database (1) that contains data on the structures and functions of the natural oligopeptides, and special computer program complex were used for it. as a result the natural regulatory oligopeptides both representing exact structures of protein fragments and containing protein fragments were found. the method has allowed also to reveal new potentially active sites of protein amino acid sequence yet not investigated experimentally. it was shown that different fragments of the food protein molecules are involved in amino acid sequences of many natural antimicrobial oligopeptides, toxins, neuropeptides, and hormones. these results confi rmed deep relationship between the basic regulatory systems. in connection with the obtained data, the process of oligopeptide biogenesis, the possibility of natural formation of regulatory oligopeptides from different protein molecules, formation of exogenous oligopeptides pool, and correspondence of the obtained results with the conception of oligopeptide continuum are discussed. a possible practical importance of active fragments of proteins in regulatory processes of a living organism is noted. the binding of peptides containing tyrosine residue to -cyclodextrin cyclodextrins form inclusion complexes with various organic molecules. aromatic amino acid residues bind to -cyclodextrin with deep penetration of the cyclodextrin cavity. in case of oligopeptides binding depends on the peptide conformation. the formation ofcyclodextrin inclusion complexes with the tyrosine residues within three peptides was investigated using steady-state fl uorescence spectroscopy and molecular dynamic simulations. the free energy along the reaction pathway delineating the inclusion of tyrosine's aromatic ring into -cyclodextrin was computed using umbrella sampling molecular dynamic simulation. the association constant and the corresponding association free energy were derived by integrating the potential of mean force over a representative ordering parameter. the three peptides studied consist of eighteen amino acids and the tyrosine residues are located at the position 1, 2 or 4. selected sequences are fragments of notch receptors. (notch1=ykieavqsetveppppaq, notch2 =tlsyplvsvvsesltper, notch3=pyplrdvrgepleppeps). out of three peptides only notch3 binds strongly to -cyclodextrin with binding constant similar to that of actyrnhme. the binding of cyclodextrins with phenolic compounds involves nonspecifi c van der waals and hydrophobic interactions and depends on accessibility of tyrosine sidechain. role of carboxyl groups in the secondary structure and function of sturgeon gonadotropin free negatively charged carboxyl groups were selectively modifi ed (neutralized) in sturgeon (acipenser güldenstädti br.) gonadotropic hormone (gth) and subunits. 11 carboxyl groups, 3 in and 8 in subunit, were neutralized by the reaction with glycine ethyl ester. investigation of re-associated -dimers (recombinants) comprising one or both modifi ed subunits showed that specifi c hormonal activity was completely lost while immunoreactivity was lowered in comparison with that of the standard -dimer. cd-spectroscopy of the modifi ed subunits did not indicate considerable changes in their spatial structure. conclusion was made that free cooh-groups of gth are important as bearers of the negative charge necessary for the hormone activity on the level of the hormone-specifi c membrane receptors. wagner, nathaniel; dadon, zehavit; yishay, eliya; ashkenasy, gonen ben gurion university of the negev, israel living cells can process rapidly and simultaneously multiple extracellular input signals through the complex networks of evolutionary selected biomolecular interactions and chemical transformations. recent approaches to molecular computation have sought to mimic or exploit various aspects of biology. a number of studies have adapted nucleic acids and proteins to the design of molecular logic gates and computational systems, while other works have affected computation in living cells via biochemical pathway engineering. we described recently the graph structure and experimental analysis of a self-organized synthetic peptide network (1-3). the system was designed rationally to operate in neutral aqueous solutions based on sequence selective auto-and cross-catalytic template-directed coiled-coil peptide fragment condensation reactions. here we show that such de novo designed synthetic networks can also mimic some of the basic logic functions of the more complex biological networks. consequently, we describe experimental and simulation studies that highlight the possibility of segments of the networks to express all sixteen two-input boolean logic functions (4, 5) . many studies deal with the folding of double helices in nucleic acids. in contrast, much lesser attention is given to double helices in peptides. nevertheless, the investigation of peptides with alternating l-and d--amino acids, like gramicidin a, shows various double helix patterns with antiparallel and parallel arrangements of the strands. in this study, we want to give a systematic overview on the possibilities of double helix formation in -peptides on the basis of ab initio mo theory. according to general principles, double helices with characteristic intermolecular hydrogen bonding patterns were generated and verifi ed as minimum conformations at the hartree-fock level employing the 6-31g* basis set. the calculations show several types of antiparallel and parallel double helices with different stability, which is strongly infl uenced by backbone substituents. the dengue virus causes a spectrum of clinical symptoms, ranging from mild dengue fever to the severe forms of dengue hemorrhagic fever and dengue shock syndrome. there are four dengue virus serotypes (den1-4). the dengue virus ns3 protease is an attractive target for development therapeutics against dengue. the aim of the study was to investigate the prime side specifi city of dengue virus ns3 protease substrates using proteochemometrics, a new technology for drug target interaction analysis. a set of 48 internally quenched peptides were designed using statistical molecular design (smd), synthesized and assayed with proteases of four subtypes of dengue virus for michaelis (k m ) and cleavage rate (k cat ) constants. the obtained data were subjected to proteochemometrics analysis, concomitantly modeling all peptides on all the four dengue proteases, which yielded highly predictive models for both activities. the interpretation of the models suggested that considerably differing physico-chemical properties of amino acids (aa) contribute to k m and k cat activities. it was found that for k cat activity only p1' and p2' prime side residues played important role, while for k m all four prime side residues, p1'-p4', were important. high cleavage rate (characterized by k cat ) was obtained for peptides having small aa (ser, gly, ala) at the p1' position and small or fl exible aa at the p2' position. for high affi nity (low k m ), at the p1' position aa should be small and moderately hydrophilic, while acidic residue is highly unfavorable. for the p2' position, the most favorable was trp; however, this position allowed a broader diversity of aa. for the p3' position, cys was more benefi cial than the aa present in native cleavage sites of the dengue polyproteins. for the p4' position, the best affi nity would be obtained with ala, gly or cys. the models may be used to modify each p' substrate position to optimize separately substrate affi nity and cleavage rate for den1-4 proteases. identifi cation of novel bioactive peptide sequences from human proteins for the development of potential therapeutics many protein-protein interactions are facilitated by the binding of peptides recognition modules to short linear motifs within the target proteins's primary sequence. therefore, synthetic peptides based on parent sequences of human proteins containing functional motifs are useful tools to uncover protein signaling and interactions. to date, peptide studies typically derive sequences from a single identifi ed protein or (at the other extreme) screen random combinatorial peptides, often without knowledge of the signaling pathways targeted. the objective of the novel bioinformatic approach presented here was to determine whether rational design of oligopeptides specifi cally targeted to potentially signaling-rich juxtamembrane regions could identify modulators of human platelet function (1) . the aim of this project was to identify peptides that span residues strongly conserved in the corresponding proteins in other species (orthologues), but that differ from those in related human proteins (paralogues). synthetic selection rules to avoid unfavorable amino acid combinations and excessively hydrophobic peptides were devised to reduce the risk of by-products during synthesis, postsynthetic degradation and solubility problems. high-throughput in vitro platelet function assays of fatty acid-modifi ed cell-permeable peptides corresponding to these regions identifi ed many agonists and antagonists of platelet function. the combined bioinformatic and experimental screens of human protein subsequences can therefore be used to validate functions of candidate proteins and provide templates for the development of potential therapeutics. denessiouk, konstantin; denesyuk, alexander; johnson, mark s. åbo akademi university, finland many small molecule ligands (or parts thereof) with important roles in the biochemistry of the cell are recognized by different proteins with different cellular functions. these proteins do not necessarily share a common fold since their three-dimensional structures can differ completely such that they are considered to be unrelated proteins. surprisingly, we see on numerous occasions that similar ligands are often recognized in a very similar way by means of a common structural motif formed from main-chain elements of several consecutive amino acids within the ligand-binding site of the proteins. these short polypeptide segments can be found in many protein folds, and variations in their structure can often explain different elements of protein function. in particular, we have characterized the presence of a nucleotide binding motif present in more than 30 protein folds that function in part to recognize the adenine ring system in many essential biological ligands (e.g. atp, nad(p), fad, fmn, coa, etc.). furthermore, a study of unrelated folds among pyridoxal phosphate dependent enzymes led to the characterization of a c nn structural motif for protein recognition of phosphate ions common to 104 fold-representative protein structures that belong to 62 different folds. interestingly, amino acid side chains play little or no role in ligand recognition, explaining the evolutionary independence of such binding mechanisms (i.e. the motifs are found in unrelated folds), while possibly refl ecting the types of interactions that were characteristic of the earliest protein-ligand interactions during early stages of molecular evolution. bioinformatics meets medicine: structure-based peptide design as a basis for drug development here, we present different structure-based approaches on the design of peptides mimicking the whole surface, a specifi c region of a protein or even tumor markers. the potential of such methods stretches from the development of peptide-derived drug candidates to immunological screenings. mimicry of binding sites: we have developed superficial, a program that proposes peptide libraries representing the entire surface or just regions of proteins. these peptides can be synthesised, e.g. using the spot-synthesis technique, and tested for their ability to mimic linear as well as non-linear binding sites. as a proof of principle, a library of peptides representing the surface of lysozyme was generated starting from a crystal structure of a lysozyme-hyhel-5 complex. adjacent sequence segments were linked by spacers to conserve local conformations. disulfi de bonds were generated to tether the peptides. binding assays against the hyhel-5 antibody identifi ed several peptides representing the non-linear recognition site of the lysozyme. translation of a tumor marker into peptides: the tumor-associated carbohydrate antigen gd2 is an established target for immunotherapy in neuroblastoma. we proposed peptidic mimics of this ganglioside for active immunisation against the tumor marker gd2 using molecular modelling. the ability of these peptides to mimic the carbohydrate moiety was verifi ed with in silico docking experiments. in a next step they were successfully tested as mimotopes in a mouse model. design of switchable peptides: photo-switchable compounds are becoming increasingly popular for a series of biological applications. goal is the reversible photo-control of structure and function of biomolecules. a required death domain for the binding of proapoptotic proteins (e.g. bak) to the hydrophobic groove of anti-apoptotic proteins is the bh3 helix. inserting the photo-reactive compound hemithioindigo into this short peptide, stabilization towards proteolytic degradation is achieved. the identifi cation of peptides that bind to antibodies is an important step in characterizing antibody specifi city in order to study molecular recognition. numerous approaches have been developed to select peptides that bind with desired specifi city and affi nity to antibodies of interest. in our study peptide arrays produced by spot technology were used to develop and optimize binders to antibody derived by mutations from the anti-p24 (hiv-1) single chain fv antibody, cb4-1. three mutations were introduced in the complementarity determining region 3 of the light chain being in close proximity with epitope-homologous peptide. this mutated antibody had completely lost its affi nity for the epitope-homologous peptide. a substitutional analysis of epitopehomologous peptide was performed on cellulose, in which all positions of the peptide sequence were substituted by each of the 20 naturally occurring amino acids. the peptides representing the binding activity in spot membranes were synthesized using solid phase synthesis and their binding activity was confi rmed by fl uorescent polarization method. a substitutional analysis indicated that binding to mutated antibody could be restored and improved by combining favourable substitutions at the n-and c-terminal residues of the epitope-homologous peptide. this approach has allowed us to generate binders from non-binders to mutated antibody in high micromolar range. it is well known that physiological regulatory peptides that act as hormones and neurotransmitters are produced by the specifi c cleavage of their precursor proteins that per se have no biological functions. during these processes, many fragmented peptides are also produced from the same precursor proteins. it is expected that they may have various unexpected biological activities, but their biological functions have not been investigated in depth. the neutrophil-activating peptides we recently purifi ed turned out to be the peptides that are cleaved from mitochondrial proteins by proteolysis, suggesting that fragmented peptides produced by maturation and degradation of functional proteins may also have various biological functions [1, 2] . therefore, we named such functional cryptic peptides hidden in protein sequences cryptides, and those cryptides that are derived from mitochondrial proteins "mitocryptides" in particular (3). we then identifi ed many mitocryptides by the combined investigation with "dry" and "wet" experiments, i.e., the sequences of mitocryptides that activate g proteins were predicted based on the distribution of charged and hydrophobic residues and their activities were examined on granulocytic cells. receptors for these peptides were also characterized by the direct cross-linking experiments between peptides and their targeted proteins. moreover, it is demonstrated the presence of a novel signaling mechanism involving mitocryptides. the comprehensive identifi cation of cryptides is expected to lead to the elucidation of various cryptic signaling mechanisms. the 4-benzoylphenylalanine (bpa) residue is widely used as a photoaffi nity label for the study of intermolecular (peptide) ligand-protein (receptor) interactions, where it is thought to function by hydrogenabstraction from met residues followed by covalent c-c bond formation of the resulting radical pair. we are carrying out detailed studies of this reaction in a series of fi ve, structurally rigid, hexapeptides of general sequences boc-u x bu y mu z -ome and boc-u x mu y bmu z -ome, where b = l-bpa, u = aib, m = l-met, and u x +u y +u z = 4, with a view to determining the effects of spacer length (u y = 1-3) on the rate of the intramolecular excited state reaction and the chemical and 3d-structures of the resulting products. the triplet state lifetimes of the bpa residues in the compounds, determined in a deoxygenated, dilute, acetonitrile solution by laser fl ash photolysis, vary in the following order: ubu 2 mu, = 60 ns; umu 2 bu, = 190 ns; ubumu 2 , = 350 ns; ubmu 3 , = 430 ns; and ubu 3 m, = 920 ns. in addition to the information these data provide on the structural requirements for intramolecular excited state quenching in the molecules, they also serve to defi ne the conditions necessary for optimal intramolecular reaction in preparative photolysis experiments. accordingly, the products resulting from ubu 2 mu and ubumu 2 have been prepared in high yields, isolated, and structurally characterized by hplc, mass spectrometry, nmr, and cd techniques. for each of the two photoinduced macrocyclizations, two diastereomers, arising exclusively from the bpa diradical regioselective attack on the met s-methyl group, were found. gattin, zrinka; van gunsteren, wilfred eth, switzerland during the past decades, the dependence of the secondary structure of peptides on their amino-acid sequence has been the focus of numerous investigations. in addition to naturally occurring peptides based onamino acids, peptides based on -amino acids, have raised particular interest because of their potential for pharmaceutical use. these peptides often have high folding propensities and it has been found that peptides with as few as four residue may fold into a stable secondary structure. -peptides offer the possibility to investigate the folding-unfolding equilibrium in the context of very small systems, therefore they have also been the subject of a number of investigations based on atomistic molecular dynamics (md) simulations which revealed that the foldingunfolding equilibrium typically occurs on time scale of nanosecond to tens of nanoseconds. how the backbone bound heteroatoms (oh, nh 2 ,f) infl uence the structure of a peptide is little known by now, both experimentally as well as theoretically. we performed several md studies on the conformational behavior of centrally placed hala( -met) fl uoro and hydroxy analogues, hala( -f) and hala( -oh), as function of chirality and level of substitution to investigate the infl uence of these factors on secondary structure formation and stability. all -peptides were simulated in methanol solution at temperature of 340 k and a pressure of 1 atm using the gromos96 biomolecular simulation software and the gromos 45a3 force fi eld. the ensembles of trajectory structures were analyzed in terms of conformational space sampled by the peptide, folding behavior, structural properties such as hydrogen-bonding and in terms of the level of agreement with the available experimental nmr data, noe's and 3 j-coupling constants. the nitroxide spin-labelled 2,3 -cyclic amino acid poac was synthesized, resolved and its absolute confi guration assigned (k . wright et al., tetrahedron, 2008, 64, 4416-4426 ) in order to be used as a spin-probe to evaluate the 12-helicalpeptide secondary structure. a series of -hexapeptides, b o c -a c p c -p o a c -p o a c -a c p c -a c p c -a c p c -o m e , b o c -a c p c -p o a c -a c p c -p o a c -a c p c -a c p c -o m e , boc-acpc-poac-acpc-acpc-poac-acpc-ome, and b o c -a c p c -p o a c -a c p c -a c p c -a c p c -p o a c -o m e , based on the (3r,4r)-poac enantiomer, combined with (1s,2s)-acpc for confi gurational homogeneity of the amino acid components, was designed. in these hexapeptides two poac residues are incorporated at positions i, i+n (n = 1-4) to observe conformation-related spin-spin interactions. the peptides were synthesized by n-to -c chain elongation of n -boc protected peptide segments in solution. their conformational analyses, performed by cd, ft-ir absorption and esr spectroscopic techniques, will be also described. computational design has been successful in creating new proteins. we focus on the design of a -sandwich protein which is challenged by general problems as h-bonds between neighboring strands being dependend on a tightly packed hydrophobic core and aggregation. an improved version of our program propac was tested to repack the backbone of several natural protein structures with high reproducibility. starting with backbone coordinates we found amino acid sequences by packing amino acid side chains with defi ned conformations (rotamers) into the core of -sandwich proteins. in a fi rst approach we have assembled four synthetic -hairpins on a cyclic peptide template (tasp) to form a -sandwich with two identical four-stranded antiparallelsheets. improvement of surface residues reduced the aggregation of the proteins. spectroscopic analysis shows a fold with a free energy near -25 kj/mol and confi rms the -structure by cd and ftir. we improved the design to a partial asymmetric -sandwich assembled from three different -hairpins. the synthesized molecule was the fi rst -sandwich molecule showing a defi ned fold in 2d-nmr. this data and results from the symmetric betabellins (1) suggest that symmetrical sheets do not fold into defi ned structures. to synthesize completely asymmetric sheets we simplifi ed the synthesis of -sandwiches by directed coupling of two four-stranded antiparallel -sheets. the computed proteins were selected for tight packing of the hydrophobic core and analyzed for a stable fold during dynamic simulations. one of our goals is to fi nd a correlation between the experimentally determined protein stability and the parameters used for the selection of the residues in the hydrophobic core and at the surface. biocatalyst-catalyzed peptide synthesis using inverse substrates as acyl donor sekizaki previously we reported that the p-amidinophenyl esters behave as specifi c substrates for trypsin and trypsin-like enzymes. in these esters the site-specifi c group (charged amidinium) for the enzyme is included in the leaving group portion instead of being in the acyl moiety. such a substrate is termed an inverse substrate (1) . inverse substrates allow the specifi c introduction of an acyl group carrying a non-specifi c residue into the trypsin active site without recourse to a cationic acyl moiety characteristic of conventional substrates. we also showed in an earlier study that inverse substrates were applicable to enzymatic peptide synthesis. therefore, a general method for the preparation of a variety of inverse substrates would be valuable. we designed two series of new type inverse substrates, p-(amidinomethyl)phenyl and m-(amidinomethyl)phenyl esters derived from n-(tert-butyloxycarbonyl)aminoisobutylic acid, were prepared. we also analyzed the kinetic behavior of trypsin towards these synthetic esters (2) . they were found to be readily coupled with an acyl acceptor such as l-alanine p-nitroanilide to produce dipeptide. the optimum condition for the coupling reaction was studied by changing the organic solvent, ph, and acyl acceptor concentration. the optimum standard condition was selected as follow: acyl donor (inverse substrate), 1 mm; acyl acceptor (l-alanine p-nitroanilide), 20 mm; enzyme, 10 m; 50% dmso-mops (50 mm, ph 8.0, containing 20 mm cacl2); 25 ž. an -aminobutyric acid containing dipeptide was obtained in high yield. streptomyces griseus trypsin was a more effi cient catalyst than the bovine trypsin. it was found that the enzymatic hydrolysis of the resulting product was negligible. peptides derived from nk-2 selective for phosphatidylserine as novel cancer agents chemotherapeutic agents, commonly used as anti-cancer drugs, have severe side effects, also affecting healthy human cells. natural antimicrobial peptides, and their derivatives, have gained interest as potential anti-cancer agents. under normal conditions, due to the asymmetric distribution of plasma membrane lipids across the bilayer, mammalian cells comprise phosphatidylserine (ps) only in the inner leafl et. in the case of malignant transformation inner leafl et ps can move to the outer leafl et and act as a surface marker. the surface exposure of negatively charged ps on various tumour cells makes these cells susceptible to killing by cationic membranolytic peptides such as nk-2 (1).the aim of this study is to develop short peptide sequences still acting selectively towards ps exposed on cancer cells without damaging healthy cells. in order to use this new strategy with ps-specifi c peptides it is necessary to analyze the lipid composition of mammalian cancer and non cancer cell membranes. further as a basis for peptide activity studies the biophysical characteristics of cancer cell membranes and healthy counterparts with respect to lipid composition were determined by investigation of liposomal mimics composed of phosphatidylcholine and/or phosphatidylserine by dsc and x-ray. these model systems were also used to screen the activity of a series of peptides derived from nk-2. fluorescence spectroscopy was applied to test the release of fl uorescence marker molecules from liposomes composed of solely ps, pc or pc/ps mixtures in the presence of various concentrations of peptides. data revealed that some nk-2 derived peptides have a high affi nity towards ps causing signifi cant leakage of liposomal content, whereas healthy mammalian cell mimicking pc liposomes were not affected. optimized peptides resulting from these experiments will be used for in-vitro studies on prostate cancer cell lines. in most moths, the sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (pban), a 33-34 amino acid neuropeptide that is released from the subesophageal ganglion into the hemolymph in response to physiological and environmental cues. pban exerts its pheromonotropic effects by binding to pbanr, a member of the rhodopsin-like family of g protein-coupled receptors (gpcr) that is predominantly expressed in the pheromone-producing cells of the female pheromone gland. the structure-activity relationship studies for bombyx mori pban have revealed that the shortest peptide with pheromonotropic activity is the c-terminal pentapeptide-amide, pban(29-33)-nh 2 (fsprl-nh 2 ), and that the c-terminal amide group is required for the pheromonotropic activity of pban. in this study, we have analyzed the solution structures of the c-terminal decapeptides derived from bombyx mori pban with an amidated and a free c-termini by two-dimensional nmr. the pheromonotropically active decapeptide-amide, pban(24-33)-nh2 (srtryfsprl-nh2), and its inactive counterpart, pban(24-33)-oh (srtryfsprl-oh), were dissolved in three kinds of solvents: (1) 50 mm sodium phosphate buffer (ph 6.0)/100 mm nacl/0.02% nan3 in 90%(v/v) h2o/10%(v/ v) d2o (buffer a), (2) 500 mm dodecyl phosphocholine (dpc)-d38 in buffer a, and (3) 30%(v/v) 2,2,2-trifl uoroethanol (tfe)-d 3 in buffer a. the nmr data indicated that these decapeptides did not adopt specifi c conformations in buffer a, but they adopted specifi c conformations in the presence of dpc micelles or tfe. these peptides exhibited different chemical shifts for some protons in all the solvents used, and they took similar but different conformations in the presence of dpc micelles. the conformational difference between these peptides may refl ect their difference in pheromonotropic activity. structuring and membrane interactions of the human antimicrobial cathelicidin ll37 cathelicidins are a family of vertebrate host defence peptides with both a direct capacity to inactivate microbes and to modulate components of the innate and adaptive immune systems. they are characterized by a conserved pro-region carrying individual, highly variable antimicrobial sequences, that become active only after proteolytic release, and with quite different structural and aggregational features that lead to differential biological effects on prokaryotic or eukaryotic cells. ll-37 is the only human cathelicidin, and displays a broad-spectrum, medium sensitive antimicrobial activity in vitro that is accompanied by some cytotoxicty towards eukariotic cells at antimicrobial concentrations, and a strong capacity to modulate host immune and healing processes at lower concentrations. these activities likely all involve interaction with biological membranes at some point, and are related to its amphipathic, helical structure and strong tendency to aggregate in specifi c conditions. the latter is an evolved feature absent in some primate orthologues, whose activities appear more limited to a direct antimicrobial action. the structural and aggregational behaviours of ll37 and selected primate orthologues were systematically investigated by biophysical and biochemical methods. these included cd spectroscopy in different buffers, sds micelles or anionic or zwitterionic luvs, transmission ftir spectroscopy, dye release from liposomes, and atr-ftir spectroscopy on supported lipid monolayers, followed by atomic force microscopy to probe morphological effects on lipid order. data was also collected from antimicrobial, cell lysis and cytofl uorimetric assays, giving a more complete picture of the possible mode of action of these helical peptides, and highlighting the importance of biochemical parameters such as structuring and dimerization/oligomerization processes in the selective interaction with biological membranes, leading to cytotoxic or immunostimulatory effects. oligomeric structure of fowlicidin-1, an antimicrobial/ anti-endotoxic peptide from the family of cathelicidin, in lipopolysaccharide bilayer a recurring theme in the structure-function correlation studies of the cationic antimicrobial peptides (amps) is that the formation of oligomeric structures in lipid environments by amps. self-assembly of amps may result disruption of the membrane (outer/inner) structures of the microorganisms by forming pores or ion channels. however, high-resolution oligomeric structures of amps in lipid environments are diffi cult to obtain. to-date, oligomeric structure at atomic resolution of any antimicrobial peptide has not been reported. secondary structures of amps are usually obtained in perdeuterated sds or dpc micelles, as a mimic to the inner cytoplasmic membrane, by nmr spectroscopy. cathelicidins comprise a major family of host-defense antimicrobial peptides in vertebrates. these peptides are synthesized as a part of large precursor proteins containing a well conserved cathelin domain and an extremely variable, in terms of length and amino acid compositions, cterminal region. the c-terminal part of the cathelicidins is bestowed with antimicrobial and lps neutralizing activities. here, we report a tetrameric structure of a 22-residue active fragment of fowlicidin-1 or vk22, one of the fi ve cathelicidins found in chicken, in lipopolysaccharide by nmr spectroscopy. the tetrameric structure of the vk22 determined from trnoe is highly helical. a large number of trnoe cross-peaks were observed connecting residues far apart in the sequence. calculated structures reveal an anti-parallel arrangement of four helices. the interface of the tetramer appears to be rich in polar or charged residues delineating a plausible pore or channel. most of the non-polar residues are found to be exposed indicating plausible interactions with non-polar fatty acyl chains of lps or with membrane in general. the oligomeric structure of vk22 peptide may be useful to understand structure/activity relationship, in particular pore formation, by the helical antimicrobial peptides. the interaction of the antimicrobial peptide nk-2 with different lipid systems antimicrobial peptides are important components of the natural defense system of most living organisms against invading pathogens. these are relatively small (below 10kda), cationic and amphipathic peptides of variable length, sequence and structure. in this study, the antimicrobial peptide nk-2, which is a 27 amino-acid residue derivative of the cationic core region of nk-lysin, has been used. the used membrane mimetic model systems have different dimensionality: two-dimensional (monolayers at the air-liquid interface) as well as three-dimensional (vesicles, micelles) systems of different phospholipids. the aim of this study was to investigate the infl uence of peptide-lipid interactions on the lipid structure and vice versa on the secondary structure of the peptide. the peptide secondary structure was measured by cd (circular dichroism spectroscopy) in bulk and irras (infrared refl ection-absorption spectroscopy) at the air-liquid interface. the structure of lipid langmuir monolayers has been examined by numerous techniques such as pressure-area isotherm measurements, fl uorescence and brewster angle microscopy and x-ray techniques. charged as well as zwitterionic phospholipids have been used to study the adsorption of nk-2. the peptide adsorbs at the air-buffer interface due to its amphiphilic character. it also inserts into uncompressed phospholipid monolayers. the peptide reorients from random coil in bulk to -helix lying fl at at the interface. the incorporation of nk-2 into a dppg monolayer leads to a different orientation (either -helix with an oblique orientation or random coil) and to the fl uidization of the aliphatic chains. nk-2 infl uences also the structure of ordered dppg domains. to assess the location of the peptide nk-2 in the lipid matrix, specular x-ray refl ectivity studies were carried out. wadhwani, parvesh; reichert, johannes; buerck, jochen; ulrich, anne s. karlsruhe institute of technology, germany antimicrobial peptides (amps) constitute an essential part of innate immunity and act against an external microbial invasion where as cell-penetrating peptides (cpps) can carry a biologically functional molecule through the cell membrane and are relevant in drug delivery developments. fusogenic peptides (fps) are active on membraneinterfaces and are instrumental in fusion of membranes which is vital for various fertilization and viral processes. membrane fusion properties of short amps and cpps have been seldom tested and have therefore eluded the attention of most investigators, instead only their cytotoxicity is investigated. there is general lack of understanding if these peptides are fusogenically active. we have investigated the ability of amps and cpps to trigger membrane fusion. as an example, hiv-1-fusion peptide fp23 is used as benchmark to compare fusion activity of various amps and cpps. most fps are believed to be unstructured or conformationally fl exible ( -helix or -sheet); therefore the secondary structure of the peptides before and after the fusion reaction is investigated and correlated to their respective ability to execute membrane fusion. our results show that various amps and cpps which are capable to switch their secondary structure are also able to promote fusion to an extent that is even higher than the known hiv-1 fusion peptide fp23. these results will be presented in the poster. interaction of the minimal active peptide sequence of human growth hormone releasing factor with negatively charged liposomes zschörnig, olaf; thomas, lars; weigelt, heiko; köhler, guido university of leipzig, germany the most bioactive peptides such as hormones and neurotransmitters do not exist in an ordered structure in aqueous solution. the conformation of the peptide changes from this fl exible unordered structure into an inherent one, only when it reaches a biomembrane. that's why it is important to investigate the such peptides like growth hormonereleasing factor (ghrf) in the presence of lipid bilayers. human ghrf is an amidated peptide consisting of 44 amino acids residues. a lot of studies has show that the residues n-terminal part are the active core of the peptide. we studied the interaction of human ghrf (1-29) with phospholipid membranes. the interaction of ghrf (1-29) with the liposomes was investigated fl uorescence spectroscopy. to detect a fl uorescence signal the peptide were labelled at fi rst, 15th, 24th and 29th position with dansyl. the fl uorescence intensity of ghrf (1-29) at different lipid-peptide-ratios were analysed using a model, which includes the hydrophobic and the electrostatic interaction between the peptide and the phospholipid membrane. the hydrophobic binding constant of ghrf(1-29) and its effective charge were determined using this model. further the peptides position in the membrane were investigated using spin labelled phospholipids, which are able to quench fl uorescence over large wavelength arrays. the quenching effi ciency will decrease by increasing the distance between fl ourophore and spin probe. the use of 15 mol% tempo-pc, 5-doxyl-pc, 10-doxyl-pc and 16-doxyl-pc in the phospholipid composition of the liposomes allows the determination of the peptides membrane penetration depth. all results indicate, that the binding of ghrf (aa 1-29) to phospholipid membranes is increased strongly by increase of the surface charge density of the liposomes. the peptide is arranged parallel to the membrane surface and is localized in membrane-water-interface of the liposomes. folding propensity and biological activity of selected antimicrobial peptides bozzi, argante 1 ; di giulio, antonio 1 ; aschi, massimiliano 1 ; rinaldi, andrea c. 2 1 university of l'aquila, italy; 2 university of cagliari, italy the innate immunity of multicellular organisms relies in large part on the action of antimicrobial peptides (amps) to resist microbial invasion. crafted by evolution into an extremely diversifi ed array of sequences and folds, amps do share a common amphiphilic 3-d arrangement. this feature is directly linked with a common mechanism of action that predominantly (although not exclusively) develops upon interaction of peptides with cell membranes of target cells. it is generally agreed that amps are essentially unstructured in the aqueous phase and fold upon contact with the membrane, adopting an amphiphilic fold. this favours absorption of peptides onto lipid bilayer and their subsequent integration into the membrane with expansion of the outer leafl et, which in turn leads to membrane thinning and permeabilization. however, recent observation suggest that things might be more complicated than previously believed. indeed, md simulations coupled to cd spectroscopy, studies with model membranes, and antimicrobial assays, have shown that, at least for some peptides, a signifi cant correlation exists between the conformation adopted by the peptide in solution, i.e. before the interaction with membranes, and its antimicrobial activity. the linear peptides we have studied following this approach include two members of the frog skin-derived temporin family, namely temporin a and temporin l, and two members of amphibian bombinins h, i.e. bombinins h2 and h4. we observed that the presence of a partially folded structure in water solution may facilitate, both thermodynamically and kinetically, the peptide folding in the microbial membrane, and thus favour biological activity. looking for such built-in conformational characteristics could well help to rationalize the different spectrum and level of activity recorded for cationic alpha-helical amps on membraneenveloped targets, and assist the design of improved analogs and biomimetic synthetic peptides with antibiotic properties. investigation and computational modelling of antimicrobial peptides linser the uprising resistance of pathogenic bacteria against treatments with conventional antibiotics emerged an acute search for alternatives. one class of promising alternatives are naturally occurring antimicrobial peptides. we present a comparative study of computational modelling of peptide properties with structural characterization of the interaction and antibacterial or haemolytic activity of three peptides (nk-cs, nkcs-[lp] and nkcs-[aa]). we compared computational interaction models with measurements of antibacterial and haemolytic activity, small angle x-ray and surface plasmon resonance data, and structure predictions by circular dichroism (cd). all peptides were active against escherichia coli (gram negative) and staphylococcus carnosus (gram positive) bacterial cultures, but the haemolytic properties against human red blood cells were found to be poor and indicated the peptides' selectivity. cd studies of the peptide secondary structure confi rmed the prediction of peptide helicity. the antibacterial activity can be correlated with a change of the hexagonal phase transition temperature of 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (pope) as determined by small angle x-ray scattering (saxs). the calculated peptide membrane affi nity is not related in linear way with the antibacterial activity. the reason for this might be aggregation as shown by surface plasmon resonance. the inverse hexagonal phase transition temperature was increased by the peptides and this promotes a positive curvature of the membranes. we assume that this curvature fi nally leads to the disruption of the model membranes. in summary an over all helical structure, electrostatic and hydrophobic parameters as well as strong amphipaticity are good measures to describe antibacterial peptide interaction. kier, brandon; andersen, niels university, united states over the last decade, -hairpins have emerged as excellent model systems for probing the intrinsic stabilities of portions of -sheet structure and for studying the sequence dependence of the folding dynamics of -strand association and alignment. for many decades, it has been established that peptide helices can be substantially stabilized (3 -7 kj/mol) by starting the sequence with an n-cap (acetyl, asp or sede). no comparable strategy has appeared for reducing the end-fraying of -hairpins. fully folded models of hairpins have typically been constructed by conversion to a cyclic system: inserting another turn favoring sequence (e.g. d-pro-gly, pg) at the end of the hairpin strands. we now report a set of favorable through-space interactions that can serve to cap -hairpins. the discovery followed from nmr studies of ac-w-ixgk-wtg (x = p, n). these studies indicated a favorable face-to-edge (fte) w1/w6 interaction which also allows for a g8-hn to w6 indole ring h-bond and the sequestration of the thr hydroxyl by h-bonding to the acetyl. these interactions produce spectroscopic diagnostics: a cd exciton couplet together with extreme upfi eld shifts for he3 of the c-terminal trp (1.5 -2.1) and hn of the c-terminal gly (2.7 -3.6 ppm). these feature disappear when x = l-pro and the gly-hn shift returns to its coil value (as in ac-wtg) upon removal of the n-terminal ac. in its most favorable application, ch3ch2co-wipglwtgps, we were able to establish that dgu at 280k was 8.9 kj/mol (97.8% folded) by backbone hn h/ d exchange protection measures. we now report that these interactions are retained in longer hairpins. peptides of the general formula, ac-w-(zz)ningk(zz)n-wtg (n = 1, 2, or 3) display enhanced fold stability. in each case, the ac is essential to prevent end-fraying and to elicit the full set of chemical shift diagnostics of the " -cap". the unique structural features of polyproline peptides to adopt an extended, relatively rigid polyproline ii (ppii) backbone conformation in aqueous solution, has led to their widespread application as a "molecular ruler" in biological and biophysical investigations. in the left handed ppii helix the peptide bonds show the all-trans conformation resulting in an interterminal distance, which increases by 2.8 to 3.1 å per proline residue. the same backbone conformation has been identifi ed in important proline rich protein-protein recognition motifs involved in the regulation of physiological processes like transcription, cell growth, cytoskeleton rearrangement and postsynaptic signal transduction via modular sh3, ww or evh domains. to investigate the structural features of polyproline and proline rich sequences by fl uorescence resonance energy transfer (fret) and other spectroscopic methods, these peptides were labelled n-and c-terminally with different fl uorescent dyes. for synthesis of homopolymeric polyproline peptides with a sequence length of more than 20 amino acids a fragment condensation strategy has been applied to prevent the occurrence of shorter side products which can perturb distance measurement by fret. spectroscopic analysis of these peptides indicated that even for short polyproline sequences (< 5 residues) there is a remarkable deviation from the expected ppii conformation. fret measurements revealed that polyproline peptides show an increase of the mean interterminal distance of about 1.7 å per proline residue. single molecule fret measurements of polyproline peptides with a length of 21 amino acids point to a heterogeneous ensemble of different conformations caused by randomly distributed cis conformations resulting in diverse species with different interterminal distances. similarly the structural consequences of the integration of non-proline amino acids in a polyproline sequence have been investigated by fret. the unusual helix stability of a vegf mimetic peptide understanding helix stability and formation is a prerogative to elucidate mechanism of protein folding and design helix peptide with specifi c activity. peptide helix is a simple model system in which various contributions to helix formation can be dissected and understood qualitatively. many strategies have been pursued to design peptide helices and notable results have been achieved even with very short sequences, but mainly these methods rely on the use of non natural amino acids or introducing constraints. in this communication, we report the stability characterization, via cd, nmr and md studies, of a designed, -helical, 15-mer peptide, composed only of natural amino acids, which activates the vegf-dependent angiogenic response (1). this peptide shows an unusual thermal stability whose structural determinants have been determined. two factors, the n-terminal region and an hydrophobic interaction i, i+4, are found as playing a mayor role of this remarkable stability (2) . these results could have implication in the fi eld of protein folding and in the design of helical structured scaffolds for the realization of peptides to be applied in chemical biology. adiponectin, a cytokine secreted by adipose tissue, which has been shown to affect lipid and glucose metabolism, attracts interest because it is a target for therapeutics in the metabolic syndrome. the molecule has a tendency to associate to form various multimers. although this multimer formation could modulate its biological function, the details of this mechanism are still unclear. thus studies on this system from the aspect of molecular assembly are interesting. the molecule consists of three domains, i.e. a variable (v), a collagen-like (c) and a globular (g) domain. the structure of the g domain determined by x-ray crystallography showed that it exists as a trimer but the remaining c and v domains have not been well characterized. we synthesized various parts of the molecule, c, g, vc, cg, and full length vcg in e. coli. the cd spectra of cg and vcg showed a positive peak around 230nm which is the hallmark of collagen structure. this may be attributed to the repeats of typical collagen type triplet, x-y-gly. the temperature dependency of these cd spectra showed the three states conformational changes of these peptides. the lower transition, where the positive peak disappeared corresponds to the melting of collagen like structure and the higher one corresponds to that of globular structure of the g domain. the apparent molecular weights determined by ultra-centrifugal analysis showed that these peptides exist in trimeric form at the intermediate state. however, neither c nor vc showed such transitions. these results showed that, contrary to our expectations, the contribution of the triplet of x-y-gly is not the dominant one for stabilizing the trimeric state. in order to explore the stabilizing factor, we are carrying out various experiments, such as mutation analysis, titration of ca ++ , redox reaction of disulfi de bonds and so on. one result is that ca ++ was shown to be a factor for increasing the thermal stability of the trimer. are v57 mutants of amyloidogenic protein -human cystatin c more or less resistant to denaturation conditions? jankowska, elzbieta; orlikowska, marta; radulska, adrianna; szymanska, aneta university of gdansk / faculty of chemistry, poland cystatins are natural inhibitors of cysteine proteases -enzymes widely distributed in animals, plants and microorganisms. human cystatin c (hcc) has been also recognized as an amyloidogenic protein directly involved in formation of pathological fi brillar aggregates which deposit in the brain arteries of elderly persons causing cerebral amyloid angiopathy (1). our studies were performed to explore possibilities of preventing this lethal disease. the overall 3d architecture of monomeric human cystatin c is not known but its fold could be anticipated from the structure of the dimer which has been already determined (2) . the dimeric cystatin c is created through the exchange of 'subdomains' between two molecules (3d domain swapping) and consists of two identical subunits in great extent reconstituting the fold of the monomeric chicken analogue of hcc. it is possible that similar mechanism is involved in cystatin c oligomerization and fi brilization process. the most signifi cant structural changes during dimerization process are observed in the l1 loop (55-59, qivag). this loop occurred to be a hinge region in the 3d domain swapping event. with the aim to check implications of greater or decreased stability of this loop for dimerization and aggregation propensity of human cystatin c, we designed and construct hcc l1 mutants with val57 residue replaced by asp, asn or pro, respectively. the structural studies of these mutants and their thermal denaturation process have been performed by means of cd and ftir spectroscopies. results of these studies will be presented. azobenzene-mediated photomodulation of a collagen triple helix monitored by ir spectroscopy (1), our most recent efforts were addressed to the design and synthesis of a photoswitchable collagen triple helix. by replacing in suitable positions of the ac-(gly-pro-hyp) 7 -nh 2 a pro and hyp residue with (4s)mercaptoproline, respectively, a side chain-to-side chain crosslinking of the collagen peptide was afforded with a purposely designed azobenzene derivative. as expected from modeling studies, self-association of the modifi ed collagen model peptide into a stable triple helix was observed with the trans-azobenzene clamp, while its photoisomerization to the cis isomeric state leads to unfolding processes as well assessed by nmr structural analysis (2) . unfolding pathways can be studied by comparing ftir difference spectra induced by temperature or light. we found the photomodulation of the triple helix to be reversible and the effi ciency of the photoisomerization increased with temperature reaching a maximum value shortly below the melting point. ir spectroscopy was thus used to identify the optimal temperatures required for structure destabilization at suffi cient extents to enable unfolding by the weak driving force of the azobenzene clamp. the results confi rmed the correctness of the design and the ability of the azobenzene switch to photocontrol this complex tertiary structure, thus allowing for time-resolved monitoring of the triple-helix unfolding process. studies on various collagen model peptides (x-y-gly) 10 where x and y are often imino acids, pro or hyp r (4(r)-hydroxyproline), have shown that hyp r at the y position plays an important role in stabilizing the collagen triple helix. however substitution of non-natural 4(s)-hyp (hyp s ) at both positions decreases the thermal stability of triple helix as (pro-hyp r -gly)10 exists in a stable triple helical state, whereas (hyp s -pro-gly)10 and (pro-hyp s -gly)10 are in a single coil state. similar effects on the stabilities of triple helices are provided by the substitution of fl uoroproline. however there is an exception that (fpro s -pro-gly)10 takes a triple helix at 4 c whereas the counterpart, (hyp s -pro-gly)10, is in a single coil state. although the difference could be explained by the steric hindrance of hydroxyl group in s confi guration, it is still controversial how much this hindrance could obstruct the triple helix formation. therefore, even though apparently the tripeptide with the hyp s -pro-gly sequence is not capable of triple helix formation, we could expected that (hyp s -pro-gly) n forms a triple helix as n increases. the transition temperature of (pro-pro-gly) n was shown to have the chain length dependency. here, we synthesized (pro-pro-gly) n and (hyp s -pro-gly) n (n=10,15) and investigated their thermal stabilities. the cd spectra of (hyp s -pro-gly)15 showed the conformational transition from collagenlike triple helix to random coil with the melting temperature at 21 c. the apparent molecular weight, determined by the sedimentation equilibrium method, showed that (hyp s -pro-gly)15 exists as trimer at 4 c and as monomer at 37 c. the melting profi le was also clearly detected by dsc. @thus, it is concluded that the existence of hyp s at the x position is not essential to interfere the triple helix formation. we are on course to investigate the stabilizing mechanism of the collagen structure. the infl uence of o-glycosylation on the folding and stability of -helical coiled coil peptides glycosylated proteins have been shown to play a key role in many biological events like cell-cell communication, immune response, cell adhesion, intracellular targeting, protease resistance, and many other processes. recently, there is a growing interest in the effect of glycosylation on the secondary structure of proteins, because of the association with the so called conformational diseases that arise from the dysfunctional aggregation of proteins in not native conformations. in this study we used -helical coiled coil based peptides as model systems to investigate the effects of serine-linked -galactose on both the secondary structure and amyloid formation tendency. thehelical coiled coil structural motif consists of two to seven -helices which are wrapped around each other with a slight superhelical twist. its simplicity and regularity have made it an attractive system to explore fundamentals of protein folding.(1) the importance of the coiled coil motif is obvious with the amount of 5% of all amino acid residues in the protein data bank being part of a coiled coil motif. at fi rst we examined to which extent and at which positions a glycosylation is possible without destroying the coiled coil structure. therefore, we systematically incorporated one to six serine-linkedgalactose units into several solvent exposed positions of a 26 amino acid long coiled coil peptide. the hydrophobic dimerization face was not modifi ed. the preformed l-ser(ac 4 --d-gal)-oh building blocks were introduced by convenient solid-phase synthesis following the fmocstrategy. folding and stability of the glycopeptides were monitored by cd spectroscopy. the amyloid formation tendency was investigated by a tht fl uorescence assay. aging, associated with decreasing protein homeostasis (proteostasis) capacity and increasing oxidative stress, is a prominent risk factor for amyloid diseases. alzheimer fs disease (ad), which is one of the most common amyloid diseases, involves intra-and extracellular amyloid formation by the amyloid bata peptide (abeta). however, how abeta can aggregate in vivo even though its physiological concentration (pc) is much lower than its critical concentration (cc) for aggregation is a mystery. we have proposed that covalent modifi cation of abeta by small molecule oxidation products can explain how abeta can form amyloid at pc. the aldehyde-bearing cholesterol oxidation product 1(2), which can modify abeta by schiif-base formation, is an example of the products that could affect ad onset. however, signifi cant questions about the modifi cation of abeta by 1(2) persist, including: does modifi cation by 1(2) lower the cc of abeta aggregation into the pc range? and, is abeta modifi ed by 1 (2) able to aggregate at low concentrations on a biologically relevant time scale? in this study, these questions are answered by studying chemically synthesized analogs of abeta that are site-specifi cally modifi ed by 1(2) at asp1, lys16, or lys28. modifi cation at the different sites has a similar effect on the thermodynamic propensity for aggregation. in contrast, the effect of metabolite modifi cation on aggregation kinetics depended strongly on the modifi cation site. abeta modifi ed at lys16 formed amorphous aggregates fastest and at the lowest concentrations. in contrast, the appearance of thiofl avin-t positive aggregates at higher concentration was fastest for abeta modifi ed at asp(1). the infl uence of modifi cation site on the nature of the aggregates suggests that amorphous aggregation and fi brillization place different conformational demands on abeta. furthermore, these studies may partially explain how abeta can aggregate at nm pcs when the cc of unmodifi ed abeta is in the ƒêm range. code, christian; domanov, yegor; kinnunen, paavo k.j. antimicrobial peptides are ubiquitous in nature and consist of several families of cationic amphiphilic peptides. they partition into lipid membranes and promote the segregation of anionic phospholipids (2, 3) . we have shown several antimicrobial peptides, e.g. temporin b and l, indolicidin, and magainin 2 to activate secretory phospholipase a 2 (pla 2 ) (1, 4) and we concluded that this could represent synergistic action of these peptides/proteins in defense against microbes. the fact that the sequences of these amps are very different suggest rather non-specifi c mechanism to be involved. to pursue the latter in more detail we used förster-type resonance energy transfer (fret) between labeled pla 2 and temporin b. interestingly, fret coincides with concentration dependant activation of pla 2 hydrolysis of dipalmitoylphosphatidylcholine (dppc) liposomes. accordingly, temporin b and pla 2 interact forming a supermolecular complex terminating into amyloid-like fi brils (4). homo-oligomeric fi bers are formed on a slower time scale when pla 2 interacts alone on dppc liposomes (5) . a general mechanism of peptide induced pla 2 activation forming heterooligomeric cofi brils is suggested. human islet amyloid polypeptide forms lipid-encased amyloid fi brils on supported lipid membranes domanov, yegor; kinnunen, paavo university of helsinki, finland islet amyloid polypeptide (iapp) forms fi brillar amyloid deposits in the pancreatic islets of langerhans of the patients with type 2 diabetes mellitus and its misfolding and aggregation are thought to contribute to -cell death. increasing evidence suggests that iapp fi brillization is strongly infl uenced by lipid membranes and, vice versa, the membrane architecture and integrity is severely affected by amyloid growth. we performed direct fl uorescence microscopic observations of the morphological transformations accompanying iapp fi brillization on the surface of supported lipid membranes. within minutes of application in submicromolar concentrations, iapp caused extensive remodelling of the membrane including formation of defects, vesiculation, and tubulation. the effects of iapp concentration, ionic strength, and the presence of amyloid seeds on the bilayer perturbation and peptide aggregation were examined. growth of amyloid fi brils was visualized using fl uorescently labelled iapp or thiofl avin t staining. two-colour imaging of the peptide and membranes revealed that the fi brils were initially composed of the peptide only, and vesiculation occurred in the points where growing fi bres touched the lipid membrane. interestingly, after 2-5 hours of incubation iapp fi bres became "wrapped" by lipid membranes derived from the supported membrane. progressive increase in molecular level association between amyloid and membranes in the maturing fi bres was confi rmed by förster resonance energy transfer (fret) spectroscopy. the possible role of lipid wrapping in stabilization of iapp amyloid in vivo is discussed. fibrinogen-derived peptides that mediate cell adhesion: structure and activity studies interaction of human islet amyloid poly peptide with phospholipid membrane vesicles dannehl, claudia; zschörnig, olaf university of leipzig, germany amylin, also known as human islet amyloid polypeptide (hiapp), is a 37-residue peptide, suspected to play a major role in the malfunction of insulin secretion in diabetes mellitus type ii. co-secreted with insulin in the beta-cells, hiapp, in higher rates destroys the barrier function of the beta-cells, leading to a failure in insulin production. because of its amyloidogenity, aggregates of fi brils can be observed in the islands of langerhans to indicate its overexpression. we studied the physico chemical properties of hiapp by observing changes in its structure depending on time and the surrounding media using maldi-tof-ms, atr ft-ir-spectroscopy. to understand the process of penetration and toxicity to cells, we performed leakage measurements of carboxyfl uoresceine containing phospholipid large unilamellar vesicles by means of fl uorescence spectroscopy. moreover we determined membrane binding of dansyl-labeled hiapp. at physiological ph value, hiapp is positively charged and thus negative charges at the phospholipid membrane surface accelerate the process of peptide folding. being random coil as initial state, a mixture of anti-parallel betasheet and alpha-helices emerges in time. in the presence of negatively charged phospholipids, hiapp aggregates can be seen within a few minutes after titration. also in absence of any free charges, as seen in water, fi brils grow and after an incubation for 24 hours at 37°c, some alpha-helices are twisted and after two weeks, no random coil is detected anymore. titration of hiapp to carboxyfl uoresceine fi lled liposomes, showed different results concerning equilibrium time and maximal extent depending on age and preparation of the peptide. in particular the composition of the vesicles seems to determine their stability in the presence of hiapp. nanoparticle induced folding and fi bril formation of a coiled coil peptide nanoparticles present large surface areas and are capable to catalyze fi bril formation of peptides. (1) . one assumes that the nature of surface controls which of the peptides will interact with the nanoparticles and that an enhanced local peptide concentration reduces the lag-time for aggregation. in previous studies, we reported the design of a coiled coil peptide that can adopt a random coil, -helical structure, and -sheet folding in dependence of ph and peptide concentration. (2) here, we expose this peptide to au nanoparticles that are either negatively or positively charged. the interaction of peptide and nanoparticle was investigated by cd and uv/vis spectroscopy, gel electrophoresis, and transmission electron microscopy. we found that electrostatic interactions with au nanoparticles can affect the peptide folding resulting in more than one secondary structure, namely, a competition between the -helical and -sheet structures. the latter folding is very likely a consequence of the high local peptide concentration on the surface of nanoparticle that facilitates a -sheet fi brillation of peptide. moreover, several factors such as ph, peptide concentration, and size of the nanoparticle have a strong infl uence on the nanoparticle-mediated folding. these results provide valuable information on pharmaceutical applications of nanoparticles and will help to reduce possible adverse effects. antimicrobial peptides are synthesized by all living organisms as a part of their innate immune system. those molecules are endowed with a broad spectrum of activity against pathogens. they can be divided into two classes differing in the mechanism of killing: membrane disruptive antibiotics cause a dysfunction of the membrane and subsequent cell lysis; non-membrane disruptive peptides are focused on the intracellular targets. in both situations the initial stage consists in the interactions with the cytoplasmic membrane, in most cases without the exploitation of any receptors. a non-receptor type of interactions decreases the possibility of development of microbial resistance and makes peptides a very potent alternative to conventional antibiotics. in the face of the reduced effi ciency of traditional drugs there is an urgent need for the design of new therapeutic agents with the optimized activity. monte carlo simulations of peptide-membrane interactions can be one of the strategies. that method takes in the account biophysical properties of a membrane as well as structural and physicochemical nature of peptides. in our work we are focused on the development of new analogues of nkcs, a derivative of potent antibiotic peptide nk-2. in the present study the outcome of monte carlo simulations is compared to experimental results of antibacterial tests performed against escherichia coli. the insight into the molecular mechanism of peptides activity is obtained in vitro using saxs method and artifi cial systems mimicking a bacterial cytoplasmic membrane. the results indicate that monte carlo modelling is a good tool to predict the peptide -membrane interactions and can be very useful for the design of novel antibiotics. a 16-35 peptide: structural features in membrane mimicking systems amyloid (a ) peptides, the hallmark of alzheimer disease, depending upon conditions, undergo conformational transition from soluble monomers to the highly toxic -sheet oligomers which form the mature fi brils. conformational and biological analyses were carried out on several different a fragments, to understand the role of the single residues in the fi brillation process. a 25-35, is considered the shortest fragment exhibiting large -sheet aggregates and retaining the toxicity of the full-length peptide. we recently reported the conformational analysis of the synthetic a 25-35 under several solution conditions, and analyzed the modulation of the conformational behaviour of a 25-35 peptide, by interaction with nicotine-like molecules. a 16-35 is the 20-mer a peptide, composed of the a 25-35 fragment, and additional n-terminal residues, known to be endowed with fi bril disaggregating activity. a 16-35 encompasses part of hydrophobic (1-28) and hydrophilic (29-35) a -regions. due to the amphipathic character of this fragment, common to the full a 1-42 and a 1-40, a tossicological mechanism involving a -peptide-membrane interaction may be hypothesized. on these bases we decided to perform the structural investigation of the fragment a 16-35 in membrane mimicking systems including micelle and vesicles aggregates, differing for composition and structural complexity. high resolution three dimensional structure was solved by nmr spectroscopy in negatively charged sds and in dpc/ sds mixed micelles. fluorescence and epr spectroscopies were used to monitor the peptide lipid interaction. the data agree on the critical role played by the membrane composition on the stabilization of different conformers, potentially driving to different oligomeric and/or polimeric toxic species. machan, radek; jurkiewicz, piotr; benda, ales; hof, martin j.heyrovsky institute of physical chemistry, czech republic charge and hydrophobicity are important determinants of membrane activity of antimicrobial peptides. the model peptide lah 4 whose charge can be easily controlled by ph of the medium (1) is a very convenient tool to study the relationship between physical properties of peptide molecules and their membrane activity. it was shown that the changes in the charge of lah 4 molecules result in changes in their orientation with respect to phospholipid bilayers (1). in the present study, effects of lah 4 on the properties of phospholipid bilayers at different ph values are studied by several methods. its infl uence on the lateral mobility of lipids within an spb and on the stability of suspensions of large unilamellar vesicles were characterized by fl uorescence correlation spectroscopy, showing evidence of peptide induced aggregation of lipids at basic ph. changes of hydration and local viscosity within the bilayer following changes in orientation of peptide molecules were measured by solvent relaxation technique. the effect of phospholipid charge was also taken into account in each case. several bioactive peptides, such as antimicrobial, cell-penetrating or fusogenic peptides, exert their biological function by interacting with cellular membranes. therefore, structural data on the location of these molecules inside lipid bilayers are very important for a detailed understanding of their mechanism of action. fluorescence spectroscopic methods are particularly suited to the study of peptide-membrane association, but give only low-resolution information on peptide position in the lipid bilayer. molecular dynamics simulations, on the other hand, can provide a very detailed picture of the peptide-membrane interaction, but need to be validated by quantitative comparison with experimental data. we applied several fl uorescence approaches, together with md simulations, to the investigation of two antimicrobial peptides: the lipopeptaibol trichogin ga iv, and pmap-23, a member of the cathelicidin family. to perform the spectroscopic studies, a variety of peptide analogues containing a single fl uorophore were synthesized. fluorescence spectra, depth-dependent quenching experiments, and peptide-translocation assays were employed to determine the location of the two peptides inside lipid bilayers, in particular as a function of peptide/lipid ratio. molecular dynamics simulations were performed by a "minimum bias" approach, starting from a random mixture of water, lipid and peptide, and following the spontaneous self-assembling of the lipid bilayer. the fi nal membrane-bound 3d-structure is in quantitative agreement with the position of the fl uorescent labels determined by depth-dependent quenching experiments. for both peptides investigated, the atomic details of md simulations provide new insights on the mechanism of membrane destabilization. acknowledgements: with the support of the ministry of education, university and research, and of foreign affairs of italy. cavaco-paulo, artur university of minho, portugal surfactant proteins (sp-) are found in lungs of mammalians. most surfactant proteins have a carbohydrate binding domain (crd) and neck domain. crd have defense function in mammalian mucosa's, by eliminating microbes, due to strong binding to sugars in the cell walls. neck domains are found to order phospholipids allowing the exchange of oxygen between the alveoli and blood. x-ray structures indicate that neck domains are alpha-helixes, but circular dicroism indicates that in the presence of phospholipids, those peptides present a disordered structure. neck domains with 20 residues from natural sequences of sp-a, sp-b, sp-c, sp-d have been tested to be delivered in lipophilic media. only disordered structures would have the ability to cross the lipid barriers. the results obtained here indicated that neck domains possibly can be used as carriers to deliver molecules across skin and hair. fraternal twins ! -peptides and oligoureas are isosteric, isostructural foldamers endowed with yet distinct biomolecular recognition properties. in the fi eld of peptidomimetics, there has been a sustained interest towards the design of non-natural oligomeric backbones with new folding patterns. over the past 12 years, the amide linkage has become the quintessential motif to elaborate folding oligomers. aliphatic and aromatic oligoamides (peptoids, -, -peptides) have provided numerous helical-folded structures, many of which have shown interesting biological activities (1). interestingly, substituting urea for the ch 2 -co-nh units in the 4 -peptide backbone represent a spectacular case of isosteric and iso-structural replacement. detailed nmr studies of the resulting oligoureas revealed a helical fold very similar to that reported for the cognate 4 -peptides. defi nitive confi rmation of this isostructural relationship came with the recent x-ray structure determination of the canonical 2.5-helix of oligoureas. how such isosteric and isostructural oligoamide and oligourea backbones compare in biomolecular recognition events is an interesting question that we attempted to address in the present work. notably the two systems were compared for their antimicrobial activity, membrane interaction and disruption properties. both -peptides and oligoureas designed to mimic globally amphiphilic alpha-helical host-defense peptides have been synthesized and tested. the results showed a surprising dichotomy in bactericidal activity between the two isostructural systems and enlightened the unique antibacterial of amphiphilic oligourea helices. to question whether this functional difference results from differential membrane disruption activities, we have undertaken detailed physicochemical investigations using negatively charged phospholipid membranes as model systems. understanding of the cellular uptake of an amphipathic cell penetrating peptides complexed with sirna konate, karidia; divita, gilles; heitz, frédéric crbm umr5237 -cnrs, france the effi ciency of delivery of macromolecules into living cells is very important for therapeutic purposes. the discovery of a class of peptides, known as cell-penetrating-peptides (cpps), with the ability to mediate translocation of various cargoes both in vitro and in vivo has provided new perspectives in the fi eld of delivery. we have designed a new secondary amphipathic cpp, caddy, which can transfect sirna. many studies have tried to elucidate cpp internalization mechanisms and there is currently still much controversy between endocytosis phenomena and direct interaction with membranes. however, elucidating the interactions between peptides and lipid membranes is essential to understand how caddy delivers cargoes in cells. to highlight these interactions, we have applied biophysical method to phospholipids monolayer and bilayer as model plasmic membranes. regarding the increase of the phospholipids monolayer surface pressure in presence of caddy, it is obvious that this peptide have high affi nity with lipids. while cholesterol presence does not induce anything in peptide insertion on a phospholipids monolayer, the cargo and a widely studied partner of the internalization: heparan sulfate of the gag' s family, do not induce the same behaviour. the intrinsic probe of caddy, tryptophan residues, and the fitc probe bound to the cargo allowed to follow and identify interactions between these two entities by fl uorescence spectroscopy. adding a bilayer vesicular solution unsettle these interactions, tryptophan residues interact with phospholipids while fitc probe are less embarrassed by peptides. caddy alone in solution is not structured but cd measurements show a typical spectrum of helical conformation in presence of phospholipids vesicles. and when the peptide complex its cargo, cd spectrum show a contribution of the sirna relevant of a conformational change inside both partners interacting together. high membrane coverage as the basis of antimicrobial peptide activity the interaction of the antimicrobial peptides (amps) omiganan (h-ilrwpwwpwrrk-nh 2 ) and bp100 (h-kklfkkilkyl-nh 2 ) with model bilayers was characterized. the activity and selectivity of these peptides could be attributed to a strong preference towards anionic membrane model systems, which mimetize bacterial membranes. regarding the interactions with bacterial membrane models, there were marked differences in the interaction patterns, as well as in functional properties of the peptides at high peptide:lipid ratios. these differences occurred for both peptides, despite their being unrelated in sequence and in occurrence in nature. such events at high membrane coverage could represent the equivalent at the molecular scale of the conditions at which the antimicrobial activity of the peptides is triggered. although the lipid: peptide ratios at these transitions are lower than 10 phospholipids per peptide molecule, the plausibility of this hypothesis was demonstrated taking into account an estimate of the amount of lipid per bacterium, and the bacterial concentration in minimum inhibitory concentration (mic) assays. according to the partition constants obtained towards bacterial membrane models, these peptides are expected to reach, at the mic, precisely those high concentrations in the membrane. in addition, surface charge neutralization was shown to occur in these conditions. activity at high membrane coverage is thus likely not only for these peptides but also for any peptide displaying high membrane affi nity and micromolar mics, which is common amongst amps. biosensors based on artifi cial membrane system that permits the functional reconstitution of transmembrane receptors have been studied for the past decade. immobilized liposome encapsulates intracellular chemicals in the internal water cavity and provides a potential advantage over supported planar lipid membrane. to prepare a stable liposome adlayer, we have proposed an immobilizing method based on small-peptide modifi cation of solid surface. in the present study, we investigated kinetics of liposome adsorption on the surface-bound synthetic peptides which have several alanine, lysine and tryptophan residues as amphiphilic segment with a cysteine at terminus. the quartz crystal microbalance studies showed that the peptide sequences can be divided into two groups according to liposome-size dependence of langmuir adsorption constant. while the initial adsorption processes could be satisfactorily described by simple langmuir adsorption kinetics, the amounts of liposome adsorbed irreversibly were increased as time advances. the results from afm reveal that most of the liposomes are bound to peptidic surfaces as single fl attened particles without fusion together for several hours. we next performed the detection of ganglioside gm1-lectin interaction on the liposome surface. all the binding constants and binding amounts, as observed by qcm, were fairly consistent with each other and and showed the effectiveness of our approach. herce, henry rensselaer polytechnic institute, united states recently we have proposed theoretically an energy independent pathway for the uptake of cell penetrating peptides (cpps) that challenges fundamental concepts associated with protein membrane interactions, h. d. herce and a. e. garcia, pnas, 104, 20805 (2007) . this mechanism involves strong interactions between cpps peptides and the phosphate groups on both sides of the lipid bilayer, the insertion of charged side chains that nucleate the formation of a transient pore, followed by the translocation of cpps peptides by diffusing on the pore surface. this mechanism explains how key ingredients, such as the cooperativity among the peptides, the large positive charge, and specifi cally the arginine amino acids, contribute to the uptake. we will describe the details of this mechanism and present novel experimental results that directly validate the model. a comparative studies on lipid affi nity of cell penetrating peptides in presence or absence of cargo a growing number of natural and /or synthetic peptides with cell membrane penetrating capability have been identifi ed and described in the past years. these molecules have been considered as targeting structures for the delivery of bioactive compounds into various cell types. although the mechanism of uptake is still unclear, it is reasonable to assume that the relative contribute of each proposed mechanism could differ for the same peptide, depending on experimental protocol and cargo molecule composition. in this work we try to connect the capability to interact with model lipid membrane of cpp and their structural and chemical characteristics in order to obtain a biophysical classifi cation that predicts the behavior of cpp-cargo molecule in cell system. indeed, the interaction with cell membrane is one of the primary step in the interaction of cpp with cells, and consequently the studies on model membrane could become important for understanding peptide-membrane interaction on a molecular level, explaining how cpps may translocate a membrane without destroying it and how this interactions come into play in shuttling cpps via different routes with different effi ciency. we analyzed by fl uorescence spectroscopy the binding properties of six different cpps (kfgf, antp and tat derived peptides, and oligoarginine peptides containing 6, 8 or 10 residues) in absence or presence of the same cargo peptide (the [392-401]ptyr 396 fragment of hs1 protein). the binding properties were correlated to the conformational and chemical characteristic of peptides, as well as to the cell penetrating properties of the cpp-cargo conjugate. results show that even if certain physico-chemical properties (conformation, positive charge) govern cpp capability to interact with the model membrane, these cannot fully explain cell-permeability properties. great deals of data show that alzheimer's pathology lead to the loss of neuronal functionality and synaptic plasticity. these effects seem to be the consequence of a toxic effect of a peptides related to their ability to modify cell membrane homeostasis. in particular, a peptides, as full length or in fragments, being in oligomeric or polymeric form, could alter membrane fl uidity and compromise its functionality. we have previously demonstrated that the 25-35 fragment of a peptide a (25-35) is able to penetrate into the outer leafl ets of the membrane. furthermore, it is able to alter membrane fl uidity changing membrane response to cholesterol, and thus affecting its ability to form low fl uid membrane regions named lipid rafts. a (25-35) is considered the shortest fragment exhibiting large -sheet aggregates and retaining the toxicity of the full-length peptide. flavonoids are a group of naturally occurring, benzo--pyrone derivatives, ubiquitous in plants. they are endowed with tumor prevent activity and they act as antioxidants through a membrane mediated molecular mechanism involving the membrane ion transport. on the basis of the common ability of fl avonoids and a peptide of altering membrane fl uidity, we carried out an epr spectroscopic analysis of several fl avonoids -specifi cally quercetin, naringenin, rutin and naringin -in 1, 2-dioleoyl-sn-glycero-3-phosphocholine (dopc) vesicles. the modifi cations of the dopc physio-chemical environment were analyzed in presence of fl avonoids and beta-amyloid fragment a (25-35). our results indicate that the addition of fl avonoids induces a decrease in membrane fl uidity. this effect is retained in presence of a (25-35). thus fl avonoid compounds could be able to antagonize the effect induced by amyloid peptide on membrane fl uidity and in this respect they could be therapeutically useful as neuroprotective agents. analyse lipid peptide interactions of p59 and lipo-p59 with membrane mimicking represented by micelles and vesicles. nmr structures of p59 and lipo-p59 in mixed sds/dpc micelles are reported; the positioning of p59 and lipo-p59 peptides with respect the lipidic surface is analyzed using 5-doxyl stearic and 16-doxyl stearic acid. epr analysis is carried out in the zwitterionic dimyristoyl phosphatidylcholine and the anionic dimyristoyl phosphatidylglycerol vesicles using several different lipid spin labels. both peptides bind to lipid bilayers and trp residues and lipidic chain of lipo-p59 show a important role in the process of peptide adsorption onto the membrane, to exert its antiviral activity. studies on human cell membranes were necessary to further establish the role of membranes in these peptides mode of action. this interaction was assessed by evaluating the effects that these peptides have on the membrane dipole potential of human erythrocytes, using the fl uorescence probe di-8-anepps. in the presence of enfuvirtide or t-1249, a decrease in the di-8-anepps fl uorescence excitation ratio dependent of peptide concentration was observed. these results show that t-1249 has ten times more affi nity to the erythrocyte membrane than enfuvirtide, a factor that can be associated with the adsorption of t-1249 on cholesterol rich membranes observed on the studies with membrane model systems. moreover, as a fraction of hiv associates with erythrocytes in vivo, these cells can have a role in delivering these peptides to the viral surface. the improved clinical effi ciency of t-1249 relative to enfuvirtide may be related to its higher partition coeffi cient and ability to adsorb to rigid lipid areas on the cell surface, where most receptors are located upon membrane fusion. moreover, adsorption to the sterol-rich viral membrane helps to increase the local concentration of the inhibitor peptide at the fusion site. the platelet receptor iib 3 plays a critical role in the process of platelet aggregation and thrombus formation. upon platelet activation its conformation changes leading to an increased affi nity for fi brinogen, which forms bridges between adjacent platelets and assembles them into an aggregate. the iib 3 activation is regulated by "outside-in" and "inside-out" signaling. among the protein-protein interactions, which contribute to «inside-out» signaling, that of talin with the 3 cytoplasmic tail is the most important. it has been recently suggested that talinmediated iib 3 activation relies on the cooperative interaction of the membrane proximal (mp) and the membrane distal (md) 3 regions with talin f3 domain and that the n 744 ply 747 motif of 3 , which can be phosphorylated at y 747 , plays a critical role in this process. to evaluate the interaction of talin with the 3 tail of integrin we designed and synthesized two peptides corresponding to the md and mp parts of 3 in their carboxyfl uorescein-labeled form (md: cf-r 736 akwdtannplyke 749 and mp: cf-k 716 llitihdrke 726 ). emission and anisotropy fl uorescence spectroscopy was used to quantitatively assess the affi nities of these peptides for talin. furthermore, to challenge the role of the y 747 phosphorylation in talin-iib 3 interaction we also studied the binding of talin to the modifi ed analogue of md, cf-r 736 akwdtannpl(ptyr) ke 749 . our experiments revealed that the md and mp parts of 3 bind tightly to talin and that y 747 phosphorylation has an inhibitory effect on this binding. finally, circular dichroism studies of all peptides in aqueous solutions and mixtures with tfe were also performed in order to characterize structure-affi nity relationships. acknowledgements: gsrt and eu (epan yb/88) for fi nancial support. the infection of human cells with hiv requires two initial steps: binding of the viral glycoprotein gp120 to the receptor cd4 followed by the interaction between the v3 loop of gp120 and a seven helix transmembrane coreceptor (ccr5 on macrophages or cxcr4 on t cells). the n-type glycosylation at asn301 of the v3 loop is assumed to play a crucial role in this process. in order to understand the interaction in detail, it is important to analyze the binding of v3-glycopeptides to the membrane integrated coreceptors on an atomic scale. here, we present binding studies between multiple v3-peptides and -glycopeptides and the coreceptor ccr5 by stdd nmr and surface plasmon resonance (spr). spr experiments were carried out by immobilizing the (glyco)peptides on an spr chip by amide coupling. several concentrations of ccr5 overexpressing human osteosarcoma (hos-r5) cells were passed over the sensor surface. the role of individual amino acids in the binding process to ccr5 can be analyzed by using different v3-peptides and -glycopeptides. additionally, the observation of interactions between the glycopeptide ligands and the receptor proteins embedded in liposomes is possible by using the saturation transfer double difference (stdd) nmr technology. stdd nmr allows removal of all unwanted signals resulting from native binding processes in cells. (1) . we used ccr5 liposomes derived from hos-r5 cells. substraction of an std spectrum of ccr5 liposomes from an std spectrum of the same concentration of liposomes incubated with a ligand allows recording of clean stdd nmr spectra presenting only signals of protons of the ligand interacting with the transmembrane receptor.(2) k d values of the ligand with respect to ccr5 have been determined in series of experiments with varying ligand concentrations. we could show that the binding epitope between hiv and ccr5 is formed by the carbohydrate at asn301 as well as the peptide. this knowledge is important for the design of new inhibitors. temperature dependant methionine proximity assays highlights conformational variations occurring through the mechanism of peptidergic gpcr activation arsenault, jason; clément, martin; leduc, richard; guillemette, gaétan; lavigne, pierre; escher, emanuel university of sherbrooke, canada g protein coupled receptors are invaluable for cell signal transduction mediated by external stimulus. pharmacological efforts towards these targets are of primordial importance albeit few efforts have resulted in structural characterisation, although rational drug design necessitates such information. recent advances in crystallisation of the -adrenergic receptor have been highly insightful and such a structure corroborates our results on the human angiotensin ii type 1 receptor (hat1) using the methionin proximity assay (mpa) in identifying ligand receptor contact points. unfortunately, physical methods such as crystallography are still far from routine procedures and are limited to a static picture of a given receptor. in the present contribution we propose a more accessible method that permits analysis conformational variations of different receptor states through an energy landscape, spanning from low energy conformations to conformations at physiological temperatures. photolabelling, mpa mutants constructed on the wt receptor, the constitutively active receptor (cam) and a corresponding non-activable mutant across the temperature range reveal activation-status dependent labelling patterns. as an example, position 256 becomes signifi cantly less accessible in the cam receptor compared to the wt receptor. ligand accessibility can be classifi ed from easily accessible (low temperature photolabeling) to less accessible residues (higher temperature photolabeling). this labelling pattern can be associated to the activation status of the receptor and may allow quantifying and identifying structural changes occurring during receptor activation. such structural understanding is crucial for future endeavours in rational drug design. capillary electrophoresis for diffi cult characterization of hardly soluble polypeptides recently, we have designed and synthesized polypeptides able to stimulate the natural plant defenses. these homopeptides were obtained by ring opening polymerization of n-carboxyanhydride (nca) with several initiators. ratio nca/initiator is determinant for the length of the polymers. on the bases of elicitor activity, we identifi ed a leader, called lapp6, which results from l-ala-nca polymerization initiated by alaninol. the mixture of poly(alanine) with different lengths is diffi cult to analyze considering important solubility problems. malditof confi rmed polymerization despite limitation due to molecular discrimination during ionization. nmr in deuterated tfa was possible but only afforded the number-average degree of polymerization (dp). to obtain more detailed characterizations, we developed separation by capillary electrophoresis in new solvents mixtures based on hexafl uoroisopropanol (hfip) and water. this technique allowed us to separate the oligomers with baseline resolution. different parameters infl uencing electrophoretic separation were investigated and optimized conditions were established. this technique enabled a full characterization of the polymer distribution, with determination of number-average dp, weight-average dp and polydispersity index. the pertinence and the reliability of capillary electrophoresis for characterization of non-water soluble polypeptides have been confi rmed, with analysis of short and long peptide chains. weakly polar interactions support polypeptide structures although the sequence of a polypeptide appears to determine the secondary and tertiary structure, weak inter-residue interactions such as between aromatic side chains and other aromatic side chains, aliphatic side chains and the peptide backbone may contribute signifi cantly to the stability of the fi nal folded structure. recent advances in structural bioiformatics and computational chemistry made it possible to study precise strurctural features and energetics of these interactions in model peptides and miniproteins such as tc5b, app and c-vhp. the interaction energies of the weakly polar interactions are of the same order as of the hydrogen bonds which occur in biopolymers (15 -65 kj/ mol). furthermore, these interactions not only stabilize local secondary structures but also entire tertiary folds of miniproteins. acknowledgements: this work was supported by the nih-inbre grant (p20 rr016469) and the carpenter endowed chair in biochemistry, creighton university trusova, valeriya; gorbenko, galyna v.n. karazin kharkov national university, ukraine a number of so-called conformational diseases including neurological disorders (parkinson's, alzheimer's and huntington's diseases), type ii diabetes, spongiform encephalopathies, systemic amyloidosis, etc., are associated with the deposition in tissue of highly ordered aggregates of specifi c peptides and proteins. despite the main structural elements of amyolid fi brils (particularly, cross--structure) are well-characterized, the mechanisms of fi brillogenesis remains poorly understood. accumulating evidence substantiates the idea that formation of fi brillar structures can be initiated and modulated by peptide/protein-lipid interactions. membrane-related determinants of fi brillization are thought to involve conformational changes of the peptide/protein, increase of its local concentration at lipid-water interface, specifi c orientation of aggregating species, neutralization of the peptide/protein surface charges by anionic lipid headgroups, particular arrangement of the inserted and solvent exposed segments of the peptide/protein molecule, etc. the present study was undertaken to explore the formation of lysozyme (lz) amyloidlike fi brils in the model lipid-protein systems. lipid component of the model system was represented by liposomes prepared from zwitterionic phosphatidylcholine (pc) and anionic phosphatidylglycerol (pg) lipids in the molar ratio 4:1. fluorescence microscopy studies performed with fl uorescein-labeled and rhodamine-labeled lz showed the presence of long lz fi bers (length >80 ìm). the to elucidate the nature of the events preceding lz self-assembly into amyloid-like structures, the protein adsorption onto pc:pg vesicles was examined by monitoring fl uorescence changes of fl uorescein-labeled lysozyme. the observed sigmoidal shape of the adsorption isotherm is strongly suggestive of oligomerization of membrane-bound protein. it seems highly probable that such oligomers serve as nuclei in the membrane-assisted lysozyme fi brillogenesis structure and dynamics of photosystem ii lightharvesting complex revealed by high-resolution fticr mass spectrometric proteome analysis structure and dynamics of membrane-bound light-harvesting pigmentprotein complexes (lhcs), that collect and transmit light energy for photosynthesis and thereby play an essential role in the regulation of photosynthesis and photoprotection, were identifi ed and characterized using high-resolution fticr mass spectrometry. lhcs from photosystem ii (lhcii) were isolated from the thylakoid membrane of arabidopsis thaliana leaves after light stress treatment using sucrose density gradient centrifugation, and separated by gel fi ltration into lhcii subcomplexes. tsekova, daniela university of chemical technology and metallurgy, bulgaria formation of fi brous supramolecular complexes from l-val derivatives and their arrangement in spherulites was studied applying spectrophotometric approach and electron-microscopic observations. experiments show that boiling one and the same compound with different initial concentrations in water to completely dissolving and posterior cooling to room temperature lead to formation of stable supramolecular complexes with different sizes and properties. they behave like polymer molecules which specifi c numbers of monomers depend on the initial concentration of the boiling solution, moreover at higher supersaturations they crystallize in sperulites which is typical for crystallization of polymer molecules. metal -organic gels based on the self-assembly of peptidomimetics and cu (ii) ions peptidomimetics constructed from l-val and nicotinic/isonicotinic acid are good low molecular weight gelators and their supersaturated solutions in a number of solvents turn into gels. they make complexes with some metal ions. mixing of pure solutions of the peptidomimetic and some cu (ii) salts lead to immediate gel formation in some solvents. this kind of compounds, named metal-organic frameworks (mofs), are new class of nanoporous materials and are very promising ones for applications in catalysis, pharmaceutical industry, etc. the stability and molecular structure of these complexes in some solvents is in the process of investigation. peptide metalloconstructs often possess particular conformations and hence display increased activities and metabolic stabilities. we investigated new rgd peptide analogs cyclized through oxorhenium / oxotechnetium coordination. the rgd sequence is known to bind specifi cally to 10 of the 25 known integrins, a family of integral proteins that plays an important role in tumor neoangiogenesis, development and proliferation. several cyclic rgd pentapeptides bearing an exocyclic tc-99m oxotechnetium core have been proposed for molecular imaging, however few of them have displayed attractive selectivities and metabolic stabilities. structure, biological activity and metabolic stabilities of metallated peptides cannot be predicted. therefore, we preferred a combinatorial approach to generate a panel of tracers that may be evaluated by tumor imaging in mice. tracers were constructed from a rgd model of general formula : ns2-x1-x2-x3-rs where x1,2,3 are respectively arginine, glycine and aspartic acid analogs and r is a series of linkers that feature various lengths and geometries (ns2 is a n-bis(ethylthio) moiety). first attempts for synthesizing these peptides by the versatile ugi multicomponent reaction did not yield the peptides with suffi cient purities. a representative library of 64 peptides was obtained by standard parallel peptide synthesis and purifi cation of all members. peptides were metallated either with rhenium or technetium using standard procedures. their resistance towards mice serum, glutathione and tumor extracts was evaluated and showed that compounds containing an aminoethanethiol linker displayed higher stabilities. infl uence of the chemical environment on isomers ratios of the metallated peptides was also investigated. finally, oxorhenium peptide coordinates were assayed as specifi c ligands of integrins. their oxotechnetium equivalents were evaluated for tumor imaging in mice. degradation products of desmopressin in phosphate/ citrate buffer it shows, that introduction of unsaturated residue to the peptide chain could be useful tool to design bioactive compounds with desirable structure [8] [9] . therefore full knowledge about relation between presence of dehydroamino acid and peptide's conformation is necessary to predict biological proper and to design newdrug. for that reason we have undertook conformational investigations of numerous peptides containing two dehydroamino acid residues ( z phe, e phe, ala) in peptide chain, in different position. the investigations were based on nmr measurements (standard 2d techniques and 1d experiments, typical for detection of hydrogen bonding) and theoretical calculations. conformational preferences of investigated systems were obtained on base of roesy and noesy experiments and calculations by use of x-plor and quantum chemical calculation is concentration overload or volume overload the best strategy for synthetic peptide purifi cation? as the complexity of synthetic peptides increases so the demands on the synthesis and purifi cation increase. whilst improvements in synthesis resins and techniques enables higher purity peptides to be produced, 100% purity is not achieved. for many applications post-synthesis purifi cation is still required. methods for the purifi cation at the mg level can be relatively straightforward but where there is a need to develop methods which may be used for larger scale production there are a number of additionly considerations. the method developed must be scaleable and the economics of the process must be compatible with the fi nal product costs. when looking to develop a purifi cation method the loading will be critical to the throughput and fi nal production costs. the selection of the purifi cation media and the purifi cation conditions are two of the major infl uences on loading as these determine capacity and resolution. however, it is also important to consider the the physical properties of the pepitide -especially its solubility. as part of the purifi cation method development a loading study must be performed -increasing the amount of peptide loaded onto the column. the most common way of doing this is to increase the concentration of the sample and keep the injection volume constant, concentration overload. but this does require that the peptide is readily soluble in a solvent compatible with the hplc purifi cation conditions. alternatively volume overload can be used where the concentration of the peptide solution is kept constant and the volume purifi ed increases. the most commonly used method is concentration overload. the work presented in this poster compares the two overload strategies, concentration overload and volume overload, for the purifi cation of synthetic peptides. the suitability of the two strategies for large scale manufacture will be explored. expansion of human stem cells by passive transmembrane transfer of homeoproteins . these results clarify the effect of hoxb4 in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein to the intrinsic lympho-myeloid differentiation potential of defi ned lymphoid progenitor subsets. finally, this supports the potential use of hoxb4 for hsc and hpc expansion in a therapeutic setting, by means of direct transmembrane protein transfer. tumor selective delivery by cell-penetrating peptides systemic delivery of therapeutic agents used for cancer chemotherapy today lead to undesired side effects and toxicity. increasing the selectivity of the anti-cancer agent will not only facilitate lower dosage for equal therapeutic effect, but also decrease the frequency of drug resistance. we have studied two different targeting approaches both based on cellpenetrating peptides. the fi rst approach is a chimera between cancer homing peptides and a cpp the other is a enzyme activated prodrug design. the cyclic peptide ccpgpegagc (pega) is a homing peptide that has previously been shown to accumulate in breast tumor tissue in mice. pega peptide does not cross the plasma membrane per se; however, when attached to the cell-penetrating peptide pvec, the conjugate is taken up by different breast cancer cells in vitro. additionally, the homing capacity of the pega-pvec is conserved in vivo, where the conjugate mainly accumulates in blood vessels in breast tumor tissue and, consequently is taken up. matrix metalloproteinases (mmps) are over-expressed in a variety of tumor tissues and cell lines, and their expression is highly correlated to tumor invasion and metastasis. to exploit these characteristics, we designed a tumor cell-selective prodrug, by constructing modifi ed version of the already shown to be functional mtx-yta4 conjugate. it is an inactive pro form of a cell-penetrating peptide (nope) conjugated to the cytostatic agent metothrexate (mtx), selectively cleavable and thereby activated by mmps. characterization of hantavirus envelope structure hepojoki, jussi; strandin, tomas; vaheri, antti; lankinen, hilkka university of helsinki / haartman institute, finland hantaviruses are zoonotic viruses carried by different rodent species and if transmitted to man cause two severe diseases, the hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome to which there is no specifi c therapy. the hantavirus envelope consists of spike structures formed by surface glycoproteins gn and gc which anchorage to the viral lipid bilayer. the quaternary complexes formed by glycoproteins have not been solved. to begin with, it is important to understand the structure of virus particle in order to treat infection for example by prevention of membrane fusion. using pepspot technique the interaction site between gn and gc proteins was mapped to peptide level. to interpret interaction mapping results three dimensional (3d) models of gc protein were created. the structure of hantavirus was also investigated in this study by cryo-electronmicroscopy (cryo-em). according to our results, hantavirus spike consists of either four or eight glycoprotein units and the spike would consist of equimolar associations of both glycoproteins. overall it seems likely that the glycoproteins of hantavirus form a heterodimeric base unit analogous to semliki forest virus e1-e2 glycoprotein complex, where the interaction between e1-e2 proteins hides the fusion peptide in e1 protein and thus prevents premature fusion. isolation of a novel 24 kda protein from ginger rhizomes having anti-fungal and anti-proliferative activity gill medicinal properties of ginger have been known for long. ginger has been used in traditional indian and chinese medicine and is effective on a wide range of ailments including diarrhea, nausea, respiratory disorders, infl ammatory diseases, arthritis etc. a number of constituents and active ingredients are present in ginger. recent studies have shown the role of ginger extract in the modulation of biochemical pathways involved in chronic infl ammation and thus providing evidences for the anti-infl ammatory role of ginger. mainly the medicinal properties and anti-proliferative activity of the ginger is because of the presence of certain pungent vallinoids, viz. 6.-gingerol and 6.-paradol, as well as some other constituents like shogaols, zingerone etc. we have identifi ed and purifi ed a novel anti-fungal and anti-proliferative protein with a molecular mass of 24 kda from the crude extract of ginger rhizobium (zingiber offi cinales), belonging to the zingiberaceae family. the isolation procedure involved ion exchange chromatography using deae-cellulose and affi nity chromatography using affi -gel blue gel. crude extract was loaded on the deae-cellulose column equilibrated with 10 mm tris-hcl buffer (ph 7.0). the unadsorbed protein fraction from deae-cellulose was further loaded on affi -gel blue gel column equilibrated with 10 mm tris-hcl buffer (ph 7.0), from which the elution of adsorbed protein was done with the same buffer. the purifi ed protein of 24 kda exhibited a potent anti-fungal activity against the mycelial growth in different fungal species, for example aspergillus fumigatus. in addition, the antifungal activity is also seen against important fungi, viz, fusarium and candida species. further, the purifi ed protein also showed 60 % inhibition of cell proliferation at 10 m concentration. the anti-proliferative activity was checked on human oral cancer (kb cells). search for native interacting partners of fl uorinated amino acids using phage display the introduction of fl uorine has proven to be a successful concept for improving the biological and pharmaceutical properties of drug candidates. the unique properties of fl uorine as well as its absence from the pool of canonical amino acids make fl uorinated amino acids a promising tool in the development of peptide based drugs.(1) however, the application of fl uorinated amino acids for rational protein design requires a comprehensive knowledge of their properties within a native protein environment. our research focuses on the effects caused by single substitutions by fl uorinated amino acids within a polypeptide environment.(2) based on a parallel, heterodimeric -helical coiled coil peptide we applied phage display technology (3) to screen for preferred interaction partners of fl uorinated building blocks within the pool of the twenty canonical amino acids. three fl uorinated amino acids were introduced either at an a-or a d-position into one of the coiled coil monomers. the second coiled coil monomer was randomized at the four positions that represent the direct interaction partners of the fl uorinated amino acid within the dimerization domain. coiled coil pairing selectivity was used to determine the best binding partner out of the library. using the acylation method, fatty acyl chains are covalently linked to the free amino residues forming a stable amide bond. in this study, a novel method for acylation of proteins was developed using in situ prepared fatty acyl chloride dispersion in aqueous acetonitrile solution, which allows protein modifi cation under very mild conditions. chicken cystatin, a reversible 13 kda inhibitor of papain-like cysteine proteases, was selected as a model protein due to its high potential to inhibit intracellular cathepsins. the protein was modifi ed using fatty acyl chlorides with 6, 8, 10, 12, 14, 16, and 18 carbon atoms. based on the cell culture assays, we examined the transport properties of fatty acylated cystatin, the effectiveness of its internalization and effi ciency to inhibit intracellular enzymes, which was measured indirectly by cathepsin b inhibition. the experiments showed that acylated cystatin quickly internalized into the cells and effectively inhibited cathepsin b. in contrast, non-acylated cystatin didn't cause inhibition as it was unable to enter the cell. the permeability enhancement effect was shown to depend on the length of the attached fatty acyl chain as the strongest inhibition was caused by cystatin acylated with 18 carbon atoms long stearoyl chloride. additionally, chemical modifi cation did not infl uence the protein's immunogenicity. the results of our study provide clear evidence that fatty acylation greatly improves membrane permeabilization properties of proteins. daisogel wing takes your process separation to a higher level. custom made solution for your process peptide purifi cation problem can be easily delivered using the daisogel wing polymer grafted silica based platform. "silica or polymer?"-it used to be an evergreen debate when it came to choose the adequate stationary phase for process scale separation or purifi cation. silica based stationary phases feature much higher mechanical strength and stability, better controlled porosity and as a result higher separation effi ciency, while polymers boast wider ph range. the attempts to bring the good aspects of these opposite methods together failed so far, or failed to provide fl exible solutions. the new daisogel wing platform for silica surface polymer grafting preceding chemical modifi cation is presented. the revolutionary new technique offers high versatility: most variations of the base silica (different pore sizes or particle sizes of choice) can be combined with your choice of familiar chemical surface modifi cations to tailor make the perfect stationary phase for your given chromatography challenge. the polymer graft on the silica surface provides extreme shielding effect, the produced stationary phase displays outstanding ph stability and durability. any of your preferred and trusted regular silica surface modifi cations can be done on the top of the grafted polymer layer. you may enjoy the usual high performance separation, excellent effi ciency, high plate numbers, mechanical strength of silica based stationary phases with a dramatically extended ph range you expected so far only from polymer resins. you have the full range of choices of particle and pore sizes with the full choice of desired bonding chemistries. finally daisogel wing is here to deliver you the most versatile polymer grafted silica hybrid platform to provide the best solution for your demanding process scale peptide separations. przybylski, józef; rogala, piotr; siemaszko-przybylska, krystyna melanin, a natural compound formed in the tyrosine metabolic process. according to occurrence, melanin is divided into that present in true skin and in internal organs. the melanin synthesis and secretion process takes place in melanocytes under the control of two neurohormons: msh -melanocyte stimulating hormone and mch -melanocyte concentrating hormone. research has shown the signifi cant role of melanin in the process of breeding and storing, in regulating metabolism of fat tissue, and in carbohydrates regulation. melanin biopolymer fi nds application in it technology as neurotransmitters for non cellular intelligence. biologically active collagen constitutes the architectonics of all tissues and organs. due to its piezoelectric and dielectric properties it also provides storage and relay functions. for the needs of medical practice, transplants, pharmacy, cosmetology and information technology the key issue is to obtain a compound of melanin with biologically active collagen. this compound we named melanocollagen type i. melanocollagen type i is formed in result of protonating collagen amino acids with melanin, which yields coloured gel ranging: grey, graphite and black. /melanin (h+) + collagen melanocollagen type i/. the therapeutical capacity of biologically active collagen type i with melanin is an ideal formulation inhibiting aging and mitigating related neurovegetative and dementia symptoms, and in vivo transplantology. freeze dried melanocollagen type i retains the features of both collagen type i and melanin. it can be a formulation on its own or a supplement for in vivo and in vitro tissue engineering, biotechnology, information technology, pharmaceutical industry and cosmetology. a patent application for melanocollagen was placed with the polish patent offi ce on 05.08.2004 as p-369439, entitled: melanocollagen type i -method of obtaining. [ 1, 2 ] . 1,4-dhp lipid structure was calculated with ab initio quantum mechanics to obtain the charges for molecular dynamics with amber 8.0 force fi eld. dhp-lipid molecules were subjected to molecular dynamics from the initial structure of a periodic lipid bilayerwater box, with a small amount of excessive water on the lipid edges to ensure the mobility of lipid molecules. after 14 ns of md simulation the lipid molecules with the fatty acid tails started to squeeze from one bilayer layer to another one. after 35 ns few lipid molecules turned with their charged heads to the side of the lipid bilayer and after 100 ns a profound tubular micelle structure began to form. the tubular micellae structure becomes more perfect during the course of simulation of 300 ns. conclusion is that one of the gene transfection agent 1,4-dhp lipid structures is a tubular micellae, and we could expect that such the micellaes are capable to form lipoplex for the dna transfection or peptide delivery. introduction a newly developed hydrophilic polymer-based ion exchange chromatography column for biomolecules. ion exchange chromatography (iec) is a widely used for analysis and purifi cation of biomolecules. we have newly developed polymer-based iec column, named ymc-biopro series, specially designed for separation of proteins, peptides and nucleic acids. the completely spherical and monodispersed porous / nonporous beads (5 m), optimally packed with advanced technology, provide high theoretical plate number and symmetric peak shape. excellent resolution is achieved from the high column effi ciency coupled with the excellent selectivity of qa and sp chemistries. in this paper, we will show features and benefi ts of ymc-biopro series. james p. tam, school of biological science, nanyang technological university, 60 nanyang drive, singapore 637551. viral entry requires fusion of the viral and cellular membranes. in all class-1 envelope viruses including hiv-1, fusion is mediated by envelope glycoprotein which has a homotrimeric quaternary structure which forms a hairpin-like assembly of six-helix bundle (6hb) during the fusion event. the 6hb employs a 3-on-3 locking mechanism in which 3 hrc (heptad repeating-carboxyl region) chains crosslink 3 hrn (heptad repeating-aminal region) chains to enable membrane fusion. this locking mechanism confers avidity due to multi-chain interactions and a high genetic barrier to mutations in the viral entry strategy despite the high mutation rate of their envelope protein. this mechanism also provides inspiration to our approach in designing mutation-resistant entry inhibitors of hiv-1. t20/enfuvirtide, a synthetic hrc-peptide monomer designed to interrupt membrane fusion, is the fi rst and only fda-approved entry inhibitor against hiv-1. however, t20 becomes ineffective in 20% of aids patients due to acquired resistance to t-20 resistant during the treatment course. our inhibitor design is based on a novel quaternary protein mimetic approach. key elements include a covalent-link 3parallel-chain construct mimicking the quaternary structure of gp41 in its pre-fusion state and an inhibition mechanism mimicking the multimeric interactions found in the highly conserved 6hb formation. our hypothesis is that covalent-linked, 3-chain quaternary mimetics (called 3 mimetics) of hrc peptides can confer multi-chain binding to the hrn region in a multi-chain locking mechanism that may lead to mutation-resistant hiv fusion inhibitors. we also extend our design to 2-chain hrc mimetics (called 2 mimetics) which may bind to the hrn as a 2-on-3 locking mechanism. in contrast, t20 and other single-chain peptide inhibitors with a 1-on-3 binding mechanism lacks the advantages offered by the quaternary mimetics and is susceptible to gp41 mutations. this report will describe all three types of inhibitors as a model to further our understanding of gp41 mutations and viral fusion mechanism in developing mutation-resistant entry inhibitors. references: 1. lain et al. cancer cell costentin, beauvillain, vaudry, proc. natl. acad. sci pnas 2001, 98, 944. 1080504. references: 1. a.a.zamyatnin. fragmentimics of proteins and natural oligopeptides. biofi zika the fi rst genome sequence of an elite grapevine cultivar (pinot noir vitis vinifera l.): coping with a highly heterozygous genome the grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla the erop-moscow oligopeptide database press references: 1. alcaro galactosylated peptides, their preparation and use in autoimmune disease diagnosis new coupling reagents: development and industrial aspects assessment of new 6-cl-hobt based coupling reagents for peptide synthesis. part 1: coupling effi ciency study fast conventional synthesis of chemokine sdf-1 (1-68) on the symphony® references: 1. pardridge, w. m. drug discov.today alberto 2 ; rondina, maria 3 ; rosato maura 2 ; quintieri synthesis and biological activity of new linear and cyclic shp-1 n-sh2-ligands the sequence specifi city for most sh2 domains is dictated by the amino acids surrounding the py-residue (position 0) and the c-terminal positions relative to py (2). in contrast, the sh2 domains of shp-1 in addition to py+1 and py+3 depend on position py-2 (3). it was further demonstrated that beyond this minimal consensus sequence the positions py+4 and py+5 also signifi cantly affect the binding affi nity and specifi city of the shp-1 sh2 domains (4). for the investigation of shp-1 mediated signaling pathways, inhibitors of this phosphatase are of great interest. therefore, we focused our research on linear and cyclic peptide ligands based on the previous studies [3-5]. the new ligands were c-terminally prolongated according to the recognition determinants for py+4 and py+5. except, an additional motif designed to occupy a basic gap on the surface of the ptp-domain was introduced. the latter was predicted to impair the n-sh2-ptp dissociation process design of a directed molecular network restructuring artifi cial peptide networks by external triggering the road to non-enzymatic molecular networks boolean logic functions of a synthetic peptide network systems chemistry: logic gates, arithmetic units and network motifs in small networks peptides: the wave of the future references: 1. d'andrea l.d.; iaccarino g.; fattorusso r.; sorriento d.; carannante c.; capasso d.; trimarco b.; pedone c proc. natl. acad. sci agnieszka 1 ; gofman nir 3 ; willumeit, regine 1 1 gkss research centre, germany; 2 hasylab/desy, germany urtti novel cationic amphiphilic 1,4-dihydropyridine derivatives for dna delivery dioleoyl phosphatidylethanolamine and peg-lipid conjugates modify dna delivery mediated by 1,4-dihydropyridine amphiphiles masako 1 ; kuriyama, naohiro 2 poster abstracts lacking his-pro residues and stabilized by introduction of 2 hval resulted in an analog h-2 hval-tyr-ile-tic-oh (al-35) that was very selective for irap versus ap-n and at(1) . the targeting properties of cell penetrating peptides cell penetrating peptides (cpps) offer the ability to penetrate across the plasma membrane of mammalian cells and to carry cargoes such as proteins, oligonucleotides and liposomes. due to this, cpps have gained high attention to improve the cellular uptake of the delivery of 'biologicals'. however, most of the research that has been performed with cpps restricted to in vivo studies. preclinical investigations are rare and the clinical validation of potential of cpps is required. a series of cpps were synthesized by solid phase peptide synthesis (spps) on an abi 433a synthesizer. the stability of these peptides in human serum was determined. subsequently, the uptake was studied in the following cell lines: sw 1736, pc-3, mh wt, hno in summary, the in vitro uptake studies did not reveal great differences that might have revealed a tumor type specifi city. in vivo studies revealed neither a distinct tissue uptake or tumor specifi city. design and synthesis of a tripartate paclitaxel prodrug for melanoma therapy therapy of melanoma continues to be a challenge since, regardless of the treatment used, long-term survival is quite uncommon. in an attempt to improve the effectiveness and decrease the toxicity of anticancer chemotherapy, one useful approach might be to administer a prodrug that specifi cally releases the active cytotoxic drug at the tumor site. in this work, we describe the synthesis of a peptide conjugate of paclitaxel potentially useful in the treatment of human melanoma, and characterized by the simultaneous presence of three functional domains: a "targeting domain", an "activation sequence", and the antitumor drug paclitaxel. the "targeting domain" of the prodrug is represented by an rgd-containing cyclic peptide, able to bind selectively to alpha-v beta-3 integrin, which is known to be highly over-expressed by both metastatic human melanoma cells, and endothelial cells of tumor vessels. the "activation sequence", responsible for the selective release of the drug, is a short peptide which is cleaved specifi cally by cathepsin b, a protease highly up-regulated in malignant tumors. the results of nmr conformational studies, as well as those of biological experiments aimed at evaluating the plasma stability of the prodrug and its ability to inhibit alpha-v beta-3-mediated tumor cell adhesion to vitronectin, will be also presented. angiogenesis is a remodeling process characterized by the sprouting of new blood vessels from pre-existing ones. it occurs during embryogenesis and to a limited extent in the adult. vegf is a homodimeric protein and has been characterized as a prime regulator of angiogenesis and vasculogenesis; when cells lose the ability to control the synthesis of vegf, angiogenic disease ensues (1) . in vitro studies show that vegf is a potent and specifi c angiogenic factor involved in the development of the vascular system and in the differentiation of endothelial cells (2) . vegf biological function is mediated through binding to two receptor tyrosine kinases: the kinase domain receptor (kdr) and the fms-like tyrosine kinase (flt-1), which are localized on the cell surface of various endothelial cell types. this binding activates signal transduction and can regulate both physiological and pathological angiogenesis (3) . vegf and its receptors are overexpressed in pathological angiogenesis, making this system a potential target for therapeutic and diagnostic applications (4) . the extracellular portion of vegf receptors is comprised of 7 immunoglobulin-like domains; deletion studies have shown that the ligand binding function resides within the fi rst three domains of flt-1 and in domains 2 and 3 of kdr. actually, no structural data are known on the extracellular portion of these receptors except for the second domain of flt-15.. so, our aim is the cloning and the expression of part of extracellular domains of both vegf receptors for structural characterization and to be used in interaction studies with peptide ligands or small organic molecule. we are interested in an activation mechanism of receptor tyrosine kinases (rtks), especially, how the transmembrane (tm) and the intracellular juxtamembrane (jm) region couple ligand binding to tyrosine phopsphorylation. one of the rtks that we are working on is neu. neu (erbb2) is one of the four members of erbb receptor family. this rtk with a mutation in the transmembrane region (v664e) has been recognized as a potent transforming oncogene product. structure of the tm region dimer and its structural difference between the wild type and v664e mutant have been reported by smith et.al. (1) , providing structural constraints for modeling the tm dimer. recently, we have performed a structural study on a tm-jm region of egfr (erbb1) showing that the tm helix breaks at the membrane interface and the unfolded jm region binds electrostatically to the membrane. we also have shown that ca2+ complex with calmodulin (ca2+/cam) binds to the positively charged jm region and pulls this portion off the membrane. these results are consistent with an electrostatic engine model, postulated by mclaughlin and coworkers (2) , for the autoinhibition and activation of the egfr. in this research, we are revisiting the structure of tm-jm region of neu to see if we can apply the electrostatic engine model to neu and to see if there is a structural difference between the wild type and the mutant tm-jm sequence. here we describe structural studies on the tm-jm sequence, neu(647-693), reconstituted into bilayer vesicles. a combination of solid state nmr, infrared and fl uorescence measurements are used to draw conclusions that the unfolded jm binds electrostatically to the membrane and binding of ca2+ -calmodulin to the positively charged jm sequence can, under some conditions, reverse its charge and release it from the membrane. @the collagen triple-helix consists of three polypeptide chains, each of which takes the poly-l-proline ii form in a left-handed helix and undergoes a transition to a single coil state as the temperature increases. this characteristic structure is ascribed to the unique amino acid sequence x-y-gly, where x and y are commonly pro or 4(r)-hydroxyproline (hyp r ). @so far, various nmr studies have been done to explain the mechanism of folding and the thermal stability of the triple helical structure by using model peptides rich in imino acids. however, the complexity of nmr spectra imposed severe limitations on detailed analysis. the spectrum is complicated because of a large number of conformational isomers due to cis/trans isomerization around each imino acid causing various chemical shifts with small differences in 1h-nmr spectra. it has been demonstrated that the site directed 15n enrichment allows real time nmr monitoring of the folding of residues at specifi c locations of collagen model peptides (1) . @to approach this problem, we have applied 19f-nmr study on various collagen model peptides containing 4(r)-fl uoroproline (fpro r ). compared to 1h and 13c, 19f chemical-shifts are extremely sensitive to small environmental changes around the nuclei and show a wide dispersion of chemical shifts. as a result, we not only distinguished the signals from the triple helix and unfolded states, but also differentiated the signals at intermediates states in 1d 19f-nmr spectrum of (pro-fpro r -gly) 7 . @here, in this study, a combination of 2d nmr experiments including exsy, hoesy, and tocsy has been used to investigate detailed equilibrium properties of collagen model peptides. especially, in the 2d 19 f-exsy spectra, we successfully observed many exchange cross peaks at high temperature. the 19 f-nmr method shown here provides a clue to analyze the conformational transition, dynamics and stability of collagen triple-helix. by contrast to well-folded natural peptides, oligomers consisting of nonnatural building blocks, so-called foldamers, are highly stable towards proteolytic degradation which is a key feature in the development of peptide-based drugs. aside from the fact that foldamers consisting ofand -amino acid building blocks are able to display different secondary structures, the combination of different homologous building blocks to hybrid peptides has recently shown some promising results. [1, 2] the ab initio mo theoretical studies have shown that / -hybrid peptides composed of alternating -and amino acid building blocks might be very well suited to mimic helical conformations. (3) here we report the fi rst examples of -helical coiled coil formation with synthetic foldamers. based on the computational studies, we synthesized heterogeneous peptides by automated solid phase peptide synthesis (spps). purifi cation was carried out by hplc, and products were confi rmed by esi-tof-ms. furthermore, temperature-dependent cd spectroscopy was applied to investigate the stability of hetero-oligomers formed between, either / -or / / -hybrid peptides with a naturalhelical coiled coil peptide. in addition, the interaction potential with their natural -helical counterparts in aqueous solution were investigated by cd, and förster resonance energy transfer (fret). using reversed phase high performance liquid chromatography and two-dimensional gel electrophoresis, the lhcii proteins, lhcb 1-6 and fi brillins were effi ciently separated, and identifi ed by fticr-ms proteome analysis. some of the lhcii subcomplexes were shown to migrate from photosystem ii to photosystem i as a result of short term adaptation to changes in light intensity. in the mobile lhcii subcomplexes, decreased levels of fi brillins and a modifi ed composition of lhcii protein isoforms were identifi ed compared to the tightlybound lhcii subcomplexes. in addition, fticr-mass spectrometric analysis revealed several oxidative modifi cations of lhcii proteins (1) . a number of protein spots in 2d-gels were found to contain a mixture of proteins, illustrating the feasibility of high resolution mass spectrometry to identify proteins that remain unseparated in 2d-gels even upon extended ph gradients. phosphinic analogue of the homophenylalanyl-phenylalanine dipeptide was demonstrated to be a potent, competitive inhibitor of cytosolic leucine aminopeptidase (lap, e.c.3.4.11.1), mimicking the transition state of the reaction catalysed by the enzyme. exhibiting ki value at low nanomolar range it was ranked among the most potent inactivators of lap reported so far (1) . recently, the compound has been also successfully employed to inhibit leucyl aminopeptidase of p. falciparum, a potential target protease for the development of new antimalarials (2) . thus, its structure represented an attractive lead for the design and construction of next generations of modifi ed analogues. they were targeted towards both lap as well as a related metalloprotease -microsomal leucine aminopeptidase (apm, e.c.3.4.11.2). these achievements, including recent results of studies on synthesis and activity, will be summarized here. we recently described the synthesis of some cyclic tetrapeptides bearing imidazole side chains and we analyzed, in detail, their copper(ii) binding properties in aqueous solution. 1 the copper(ii) species obtained are of interest in relation to copper-protein active-site biomimetics. in particular, a 13-membered ring cyclic tetrapeptide c(lys-dhis-ala-his) (dk13) was synthesized by the solid-phase peptide synthesis method and its copper(ii) coordination properties were studied. 2 surprisingly, all collected data strongly support the presence, at alkaline ph, of a stable peptide/copper(iii) complex that is formed in solution by atmospheric dioxygen oxidation. in order to clarify the mechanism of the copper oxidation through the average cu-n bond distance and to get information on the local geometry around copper, depending on the peptide sequence, we have collected experimental xanes and exafs spectra. the investigated cyclopeptide/copper complex shows pre-edge peak energy position and integrated intensity consistent with those of cu 3+ model compounds. also, edge energy is consistent with the presence of trivalent copper. then, calculations performed on the exafs measures should give information on the local geometry, confi rming the square planar geometry proposed by us for this dk13/cu(iii) complex. improved expressed protein ligation method for consecutive coupling of polypeptide fragments in the post-genomic era, to support the structural and functional studies of proteins, there is a growing demand for novel methods with which a wider range of selective protein modifi cations achievable. expressed protein ligation (epl) can be the method of choice for the preparation of unique protein derivatives not available by recombinant methods when the protein fragments extend the size accessible by solid phase peptide synthesis, and when extra chemical information (i.e. a special covalent modifi cation) is introduced into a well-defi ned part of the sequence. however, this method is not generally applicable, especially in the case when the target protein derivative is assembled from three or more polypeptide fragments. we demonstrate here an improved epl method that facilitates the effective ligation of large fragments in a consecutive way. the method employs a novel protection-deprotection scheme. supported by otka f049222 and jános bolyai fellowship (cs.t.). protein ligation using protein trans-splicing , in order to facilitate protein ligation using synthetic peptides. highly effi cient fl uorescent labeling of proteins has been demonstrated in a site-specifi c manner by protein ligation using the newly designed intein. this approach could be applied for sitespecifi c modifi cation of proteins in vivo because protein trans-splicing is an autocatalytic process requiring no additional cofactor. moreover, protein trans-splicing approach could be extended to dual site-specifi c modifi cation using an additional intein. la doppel (dpl) is a glycosylphosphatidylinositol-anchored protein that exhibits 26% sequence homology with prion protein but lacks the octarepeat region. the role of prion is not yet completely known but prpc seems to be involved in cu (ii) traffi cking from synaptic clefts and in preventing neuronal oxidative damage. doppel is expressed in heart and testis and has been shown a regulator of male fertility. therefore, despite a high sequence homology and a similar three-dimensional fold, it's possible to hypothesize that the function of the two protein is not correlated. in the literature is reported that both prpc and of dpl are able to bind cu (ii) but the characterization of the complex species is well defi ned only for prpc. several binding sites of dpl are involved in the complexation with cu (ii). [1, 2] in the present work we have focused our attention to the third alpha-helix of the dpl which is the preferential binding site for the metal ion. we have synthesized two peptide fragments relative to the 122-140 sequence of the dpl. the fi rst peptide has the native sequence whereas in the second fragment the asp 124 was replaced by a asn residue. this substitution was performed to understand the role played by the carboxylate group of the asp residue in the complexation of cu (ii). cd ad nmr spectra were carried out on the two peptides in absence and presence of cu (ii) to investigate conformational features upon cu (ii) binding. finally, the complex species formed were completely characterized by using spectroscopic (nmr, cd, uv-vis, epr) and thermodynamic measurements (potentiometric titrations). three-dimensional (3d) domain swapping of proteins is a phenomenon associated with formation of oligomers and fi brils of several amyloidogenic proteins (1) . the propensity of a particular protein to undergo domain swapping depends on factors like changes in environment or presence of specifi c mutations, but also on some topological determinants. it was proposed that domain swapping-prone proteins have some common "hot spots" in their structures that facilitate the process. one of these motives are so called "hinge loops" -turns showing enhanced propensity for unfolding due to conformational constraints (2). cystatin c (hcc), main inhibitor of cysteine proteases in human body was shown to dimerize (3) and oligomerize (4) through domain swapping. hcc contains highly conserved throughout the cystatin family loop l1, connecting two beta strands which, together with the only helix in the protein fold create a structural unit that undergoes the 3d process. in order to confi rm important role of distortions in l1 loop, which are centered around valine residue in position 57 in the dimerization/ oligomerization process of hcc, we designed point mutations which should inhibit or promote aforementioned processes. as it was expected, substitution of val57 with either asparagine or aspartic acid residues abolished dimerization process almost completely. in contrast, v57p mutant shows enhanced dimerization propensity. above observations are in good agreement with theoretical studies, performed on entire hcc and its fragment encompassing the hairpin structure centered on loop l1. the expansion of the cag trinucleotide that produces polyglutamine (polyq) segments in several proteins is responsible for at least eight neurodegenerative diseases. a possible therapeutic approach would be to inhibit the polyq self-assembly process using disrupting agents. although, screening studies have identifi ed several polyq aggregation inhibitors, their mechanism of action remains unknown. this is due to the poor aqueous solubility and fast aggregation of uncharged polyq peptides, which complicate their study. the most common strategy to resolve these problems is to produce polyq stretches fl anked with charged residues. however, this strategy was found to modify the aggregation behaviour of polyqs, their aggregate structure and their affi nity for disrupting molecules. to circumvent these problems, polyq peptides containing morpholine moieties, whose charge is ph-dependent, were produced using fmoc-based chemistry on spps. following an acid treatment, the designed peptides were soluble in acidic aqueous solutions and their aggregation rate could be controlled by ph variations and followed by dynamic light scattering. furthermore, aggregation initiated at physiological ph provided uncharged fi brils allowing the evaluation of the effects of charged disrupting agents. the actions of known polyq aggregation inhibitors such as polyq-binding peptide 1 (qbp1), trehalose and congo red were studied. peptide-protein and protein-protein interactions play key roles in most biological processes, and therefore represent valuable targets for drug discovery. an approach in the search of new chemical entities able to interfere with these interactions is the use of small molecules able to mimic or to induce precise aspects of specifi c peptide secondary structures. among the secondary structure elements, reverse turns have been shown relevant in many biomolecular recognition events. thus, different efforts have been directed to the design of turn mimetics or inducers. in this sense, 2-substituted azetidine-2-carboxylates, synthesized by our group through a versatile procedure from amino acids, possess the dihedral angle restricted to approximately 70º or -70º, depending on the absolute confi guration at the asymmetric carbon. these values are similar to those reported for the central residues of main types of -and -turns, and in fact, recent studies have shown the ability of the 2,2-disubstituted azetidines to induce -turns when incorporated at the i+1 position of model peptides. to know if this conformational behavior is distinctive of these restricted amino acids, we now investigate the infl uence of the incorporation of these amino acids at the i+2 position of model peptides. with this aim, we have synthesized a series of simplifi ed tetrapeptide models, rco-l-ala-azx-nhme, in which the rco and nhme groups are simplifi cations of the i and i+3 residues of the turn, respectively. for comparative purposes, dipeptide derivatives incorporating azetidine-2-carboxylate, pro and -mepro have also been prepared. the turn inducing capacities of all these model tetrapeptides have been analyzed by molecular modelling, ¹h rmn, ft-ir and x-ray methodologies. the results have shown the importance of the , -disubstitution at the heterocyclic amino acid for stabilizing reverse turns, and the infl uence of the ring size on the induced turn type infl uence of position of dehydroamino acid in peptide chain on conformation of dehydropeptides containing two dehydroamino acid residues it is known that presence of c -c double bound in dehydroamino acid infl uences on dramatic limitation of conformational space, not only side-chain but also main-chain [1] [2] . according to the peptide's length and neighboring amino acid residues, dehydroamino acid exerts s-shaped, -turn or helical conformation in peptides [3] [4] [5] [6] [7] . angiotensin-i converting enzyme (ace) somatic form bears two catalytic domains with two zn metal ions, while the testis form bears remarkable similarity to the aminoacid sequence of ace c-catalytic domain and only one zn metal ion. however, both exhibit their hydrolytic activity to vasoactive peptides such as angiotensin i (angi) and bradykinin (bk), though with different effi cacy. in order to study experimentally the possible interaction and binding events between the ace active site(s) and known ace substrates or inhibitors, we constructed peptide-based catalytic site maquettes (csm). 46-residue peptides were synthesized through solid-phase peptide synthesis having the aminoacid sequence that correspond to the ace val380-ala425 n-domain segment and to the val978-ala1023 c-domain segment and enzyme's active sites were reconstructed through addition of zinc metal ion in solution. the resulting complexes have the metal ion bound in a native-like mode, as manifested by previously reported nmr data. the reconstituted peptides were titrated separately by captopril and angiotensin-i with a molar ratio ace:captopril/angi 1:15 and 1:5, respectively. titration was followed by 1 h nmr spectroscopy and spectral changes (chemical shifts, line broadening, etc.) were recorded and analyzed. after the end of angi titration the solution of interacting peptides was titrated with captopril and the titration was followed by 1 h nmr spectroscopy. at the end of each titration 2d homonuclear 1 h-1 h tocsy and noesy spectra were recorded. analysis of high-resolution nmr data probes the conformational variation of ace csm and peptide substrates upon interaction, and in the presence of captopril in ace-angi solutions. contemporary development in nanotechnology is fabrication of nanostructured materials using the peptide-based self-assembly. importance of peptide as monomeric components is their capacity to be engineered to mediate spontaneous supramolecular self-assembly and their inherent chemical nature that facilitates chemical and biological recognition process. tubular self-assembled peptide nanostructures are of special interest since these can serve in various applications including 'bottom-up' fabrication of molecular scaffolds, nanoelectromechanical systems and nanomachines. an important discovery by gazit group revealed that the simple dipeptide molecule of phenylalanine which is core recognition motif of the alzheimer's -amyloid polypeptide effi ciently self-assembles into well-ordered nanotubular structure (1) which could be controlled by relatively simple chemical modifi cations at its termini. based on these observations and our interest in the synthesis of softmaterials for nanotechnology, we have successfully synthesized novel monomeric units of di-peptides with ureido functional group at nterminal of various hydrophobic l-amino acids. these derivatives have been prepared by using solid phase peptide synthesis protocol with cost effective process retaining the chirality of molecule. these ureido dipeptide formed nanostructures with high yield under mild and controlled condition in aqueous media. to fully understand the molecular and supramolecular mechanism guiding the formation of nanostructures we have selected a multidisciplinary approach by combining spectroscopic studies with advanced electron microscopic techniques. the morphology of nanostructures can be controlled by using variety of l-amino acids. these novel ureido di-peptides nanotubes can be used in fabrication of biocompatible peptide-based nanostructures and they will undoubtedly have nanotechnological applications. biomolecules are excellent in hierarchically organizing a characteristic structure by self-assembly. the spontaneous assembly of biomolecules has advanced a bottom-up approach for fabrication of nanostructured materials. we have developed fabrication of controlled nanofi bers through self-assembly of simple and short de novo designedsheet peptides with unique sequences [1, 2] . the single straight nanofi ber with high regularity constructed by -sheet fi peptide (sequence: pkfkiiefep) was functionalized with proteins by biotinylated peptides (3) and using functional anchors that have binding and functional groups. conjugation of self-assembled peptide nanofi bers with biomolecules such as peptides and proteins will expand their potentialities of the nanofi bers for various applications. to identify peptides binding to the self-assembled fi peptide nanofi ber, a phage display combinatorial screening using a random peptide library was performed. after fi ve rounds of biopanning, phage pools with highly specifi c affi nities to fi nanofi bers were screened. as a result of dna sequencing after phage cloning, 12 phage clones were identifi ed. binding affi nities of these clones were quantitatively investigated by an enzyme-linked immunosorbent assay. these clones showed specifi c affi nities to fi nanofi bers, as compared with ff and vi nanofi bers having slightly differences of amino acid sequences. affi nities and specifi cities were dependent on the sequence of each peptide, indicating that different amino acid sequences contributed to peptide interactions with nanofi bers. infrared spectroscopy studies by using chemically-synthesized peptides showed that the peptide binds specifi cally to the fi nanofi ber with structural transitions. owing to their diversity in size and shape, easy access, and biocompatibility, peptides represent versatile units for the construction of h-bonded tubular assemblies and other biomimetic materials with potentially useful applications. so-called peptide nanotubes (pnts) have been obtained through multiple and complementary approaches (1). originally designed from -peptides made of d-and l-amino acids (2) , fl at macrocyclic systems forming cylindricalsheet like assemblies have diversifi ed to include oligoamides made from higher amino acid homologs as well as peptide hybrids (e.g. ,peptides). tubular sheet-like assemblies however are not restricted to oligoamides. the urea group which shares a number of features with the amide linkage, i.e. rigidity, planarity, polarity, and hydrogen bonding capacity is an interesting surrogate. we and other have demonstrated that macrocyclic biotic and abiotic n,n'-linked oligoureas have a unique propensity to self-organize into polar h-bonded nanotubes (3). partial peptide backbone n-methylation has been introduced as a general strategy to generate truncated stacks (i.e. h-bonded dimers) useful to gain access to the thermodynamics of nanotube formation (1). herein, we describe biotic macrocyclic amide/urea hybrids with partially nalkylated backbones as new candidates for the formation of h-bonded dimers. a cysteine branched pga (polyglutamic acid) hydrogel is being investigated for therapeutic peptide and protein controlled delivery. entrapment of hgh as a model protein in this hydrogel has been carried out in order to check its later release in different aqueous buffered media and stability. different physico-chemical analysis (by maldi-tof, sec-mals-ri, 1h-nmr, cd, …) of the released hgh showed no effect on destabilization of the protein. controlled release systems of proteins involve important challenges such as maintaining the protein integrity. researchers need an exhaustive understanding of protein stability issue in drug delivery formulations. common degradative reactions in proteins involve: oxidation, deamidation, stability of disulfi de bonds and aggregation, which may affect secondary and tertiary structure of proteins and thus, their biological activity. the high water content which imbibes the polymer network of hydrogels enables a good accommodation for proteins. polyamino acids have been already studied in some biotechnological applications, however, in spite of their potential, they have barely been taken into account for the development of controlled release microparticles. we show that proteins could be easily loaded into cys cross-linked pga hydrogels. the protein hgh has been loaded and released preserving its physico-chemical integrity. thus, these hydrogels are promising for their use as therapeutic protein delivery systems. totally synthetic collagen-like gels by intermolecular folding of designed peptides our goal is to develop artifi cial collagens that can use as safer and functional biomaterials. here, we report the development of collagenlike gels by means of the intermolecular folding of chemically synthesized peptides. the peptides are disulfi de-linked trimers of collagenous gly-x-y triplet repeats with self-complementary shapes. the self-complementary peptides are able to form elongated triple-helix through spontaneous intermolecular folding. upon cooling of the peptide solutions, hydrogels of peptide supramolecules formed. the gel-sol transition was appeared to be reversible, and the transition temperatures were found to be tunable by the design of the peptides. our strategy for the totally synthetic collagen will offer possibilities for the development of innovative biomaterials. recent advance in biotechnology enables us to fi nd the peptides with affi nity for nonbiological materials and with function of mineralizing inorganic materials. the use of the functional peptides is attracting a growing interest for bottom-up fabrication approaches of nanoscale devise. zinc oxide (zno), a semiconductor with a wide direct band gap, possess unique optical, acoustic, and electronic properties, so that it is one of most widely studied metal oxides for solar cells, ultra violet nanolaser, blue light-emitting diode and so on. this wide variety of applications requires various fabrications of morphologically and functionally distinct zno nanostructures. here, the peptides which are selected from the phage-displayed peptide library with a 12-mer on the surface, can bind zno particle but not other metal oxides particle, and further, the zno-binding peptides play an important role on the crystal growth of zno in its synthesis. the anti-zno peptide can assist the synthesis of zno nanoparticles from a zn(oh) 2 solution even at 4 ºc, and further, the peptide leads to the self-assembly of synthesized zno particles to fl ower-type morphologies. we describe the biomimetic zno synthesis using the artifi cial peptide with affi nity for zno. the design of nanoparticles suitably functionalized for applications in biology or medicine is an ongoing challenge for both chemists and biologists. nanoparticles such as quantum dots, gold nanoparticles or carbon based-materials offer, in addition to their remarkable physical properties, the possibility to be functionalized by biomolecules, making them suitable for sensing, detection, diagnostic, and/or therapeutic applications. recently, quantum dots have been designed for biological imaging owing to their unique size-dependent fl uorescence properties. however, their plausible toxicity still remains a major concern for in vitro and in vivo applications. nanodiamonds, (nds) are also candidates for biomedical applications considering their intrinsic or induced fl uorescence and biocompatibility. the work will describe: i) the functionalisation and the characterization of 35 nm non-fl uorescent cnds and ii) their use in toxicity and transport studies in living cells after the grafting of a fl uorescent model peptide. we chose to introduce a nonpermeant fl uorescent peptide for the tracking of the nanoparticles on or inside the cells. nanodiamonds (cnds, 35 nm) coated by silanisation or with polyelectrolyte layers have been grafted with a fl uorescent thiolated peptide via a maleimido function, leading to aqueous colloidal suspensions stable for months. for each step of the preparation, the diameter of the nds measured by dynamic light scattering (dls), the zeta potential, the amount of amino groups grafted onto the surface, as well as the stability of the different nds suspensions no cytotoxicity was observed up to 72 hours incubation with cho cells with any of the prepared (40 g/ml). their capacity to enter mammalian cells, and their localisation inside cho cells, have been ascertained by confocal microscopy, refl ected light and fl uorescence. hepatitis b virus is one of the most common problem and potential danger of all over the world and also in our country. it is known that the use of proteins and peptide antigens as vaccines has several potential advantages over whole viral or bacterial preparations. to elicit the maximum immunogenic response from synthetic peptide antigens ,it is generally necessary to bind the peptide to carrier protein. moreover, to realize the full potential of synthetic peptide antigens protein-peptide conjugate will have to be used with an adjuvant. we have developed new approches for obtaining highly immunogenic peptide conjugatessynthetic polyelectrolytes (pe) were used for the conjugation with peptide molecules in which pe carry out the carrier and adjuvant roles simultaneously. this concept drive us to create new immunogenic bioconjugates of hepatitis b surface antigenic polypeptides (hbsag) with synthetic pe. in this study we used the region 95-109 of the s gene. the synthesis of peptides was performed by explorer pls ® automated microwave synthesis workstation (cem), by using f-moc chemistry. copolymers of acrylic acid with different monomers were used as a pe for the conjugation with polypeptides. pe-peptide conjugates was synthesized by microwave assisted method. composition and structure of bioconjugates werw characterized by hplc, spectrofl uorometry and different spectrophotometric methods. protein-protein and protein-ligand interactions are among the few essential cell processes whose understanding provide us insights into fundamental events of the life cycle. aside from in silico methods, natively folded proteins are an absolute prerequisite in all current biotechnological tools used for the study of these interactions. however, the recently developed ianus peptide array has a potential to evolve in to a protein-free method for detection of protein-protein interaction sites. using this assay, protein-protein interactions could be represented by and investigated as peptide-peptide interactions using peptide pairs immobilized on a solid support.(1) . two main obstacles had to be overcome for successful implementation of this array: (i) the library size and (ii) identifi cation of interacting peptide pairs. if, for example, interactions between two small proteins of only approx. 100 amino acids each should be analyzed, a library of ca. 2500 peptide pairs would be needed to cover all possible combinations of overlapping peptides derived from these proteins. although libraries of several thousand peptides were already successfully synthesized, the library size could be reduced drastically if the binding pocket of one protein is already known or if the protein-ligand interactions are studied. up to date, two methods for the identifi cation of interacting peptides in a library have been developed in our group. both methods will be demonstrated in studies of protein-protein and protein-ligand interactions. the role of the non-helical tailpiece in myosin ii assembly the hebrew university of jerusalem, israel self-assembly of macromolecules into large complexes is often mediated by folding of disordered regions. we used peptides to investigate the role of the non-helical tailpiece from non-muscle myosin iic (nm-iic) in its self-assembly process. nm-iic is a motor protein composed of a globular motor domain in the n-terminal region and a coiled-coil rod domain in the c-terminal region, which terminates with a non-helical 47-residue tailpiece (residues 1954-2000) . nm-iic molecules undergo self-assembly into fi laments that are necessary for its contractile activity. the tailpiece has an unfolded character and participates in the assembly process of nm-iic. the n-terminal region of the tailpiece (residues 1954-1968) has a net positive charge of +3 while its c-terminal region (residues 1969-2000) has a net negative charge of -10. we designed peptides that correspond to the entire tailpiece and to its negative and positive regions. these peptides were used to study interactions with the rod, their structures in the free and bound states, and structural changes they undergo upon binding to residues 1296-1854 of nm-iic rod. fluorescence anisotropy binding studies showed that a peptide corresponding to the positive region of the tailpiece (residues 1947-1968) bound the nm-iic rod (residues 1296-1854) with an affi nity of 13 m. circular dichroism studies showed that the positively charged peptide became folded upon binding the rod, indicated by a shift of the spectral minimum from 200 nm to 220 nm upon binding. a peptide corresponding to the negative region of the tailpiece (residues 1969-2000) did not bind nm-iic (residues 1296-1854). based on our results, we suggest that the positive region of the tailpiece is required for ordering and aligning neighboring molecules by electrostatic attraction, leading to fi lament formation. our results provide molecular insight into the role of the structurally disordered tailpiece of nm-iic in the selfassembly process. stefanidakis, michael; karjalainen, katja; pasqualini, renata; arap, wadih; koivunen, erkki md anderson cancer center, united states acute myelogenous leukemias (aml) are characterized with non-random patterns of medullary and extramedullary invasion. we hypothesized that a supramolecular complex, the leukemia cell invadosome, which contains certain integrins, matrix metalloproteinases (mmps) and other as yet unidentifi ed proteins, is essential for tissue invasion and may be central to the phenotypic diversity observed in the clinic. here we show that the specifi c binding of mmp-9 to leukocyte surface ám 2 integrin is required for pericellular proteolysis and migration of amlderived cells. an effi cient antileukemia effect was obtained by peptide inhibitors that prevented prommp-9 cell surface binding, transmigration through an endothelial cell layer, and extracellular matrix degradation. notably, the functional protein anchorage between ám 2 integrin and prommp-9 described in this study does not involve the enzymatic active sites targeted by any of the existing mmp inhibitors. taken together, our results provide a biochemical working defi nition for the human leukemia invadosome. disruption of specifi c protein complexes within this supramolecular target complex may yield a new class of anti-aml drugs with anti-invasion (rather than cytotoxic) attributes. the molecular mechanisms of action of the polycationic lysine homoand heterodendrimers on functional activity of adenylyl cyclase signaling system (ac system) in the myocardium and the brain of rats were studied. the lysine homodendrimers of the third (i), the fourth (ii) and the fi fth (iii) generations, as well as the lysine heterodendrimers of the fi fth generation -[(nh 2 ) 64 (lys-glu) 32 (lys-glu) 16 (lys-glu) 8 (lys-glu) 4 (lys-glu) 2 lys-ala-ala-lys(clac)-ala-nh 2 ] (iv), [(nh 2 ) 64 (lys-ala) 32 (lys-ala) 16 (lys-ala) 8 (lys-ala) 4 (lys-ala) 2 lys-ala-lys(clac)-ala-ala-nh 2 ] (v) and [(nh 2 ) 64 (lys-gly-gly) 32 (lys-gly-gly) 16 (lys-gly-gly) 8 (lys-gly-gly) 4 (lys-gly-gly) 2 lys-gly-gly-lys(clac)-ala-ala-nh 2 ] (vi) stimulated by receptor-independent mechanism the activity of heterotrimeric g proteins, preferably of inhibitory type, and interacted with c-terminal regions of their -subunits. the homodendrimers ii and iii and heterodendrimer v were more effective g protein activators. the treatment of the membranes with pertussis toxin (inactivating g i protein), but not with cholera toxin, led to a decrease of g protein activation effect of the dendrimers. the lysine dendrimers disturbed the functional coupling of the receptors of biogenic amines and peptides hormones with g i proteins and, to a smaller extent, with g s proteins. it was illustrated by the decrease of regulatory effects of the hormones on ac activity and g protein gtp binding and by the decrease of receptor affi nity to agonists in the presence of the lysine dendrimers, as result of receptor-g protein complex dissociation. it was shown that the molecular mechanisms of the action and the g protein selectivity of the polylysine dendrimers are similar to those of mastoparan and melittin, natural toxins of insect venom, which also preferably activate g i/o proteins and inhibit g i/o -coupled signaling. acknowledgements: supported by rfbi (grant 06-04-48809) and «russian science support foundation». 4 genetics, bielefeld university, germany dna-protein interactions are a key element in the regulation of cellular processes. transcription factors are able to recognize their cognate dna sequences and regulate the expression of proteins. as a model system, the specifi c dna binding of the transcription factor phob from e. coli is investigated in single molecule experiments. structurally, phob belongs to the family of winged helix-turn-helix proteins. it is composed of a transactivation domain (amino acids 1-127) and a dna binding domain (amino acids 123-229). after phosphorylation of the transactivation domain, the protein binds to specifi c dna sequences containing a tgtca consensus sequence. different c-terminally modifi ed protein epitopes representing parts of the dna binding domain of phob were chemically synthesized using microwave assisted solid phase peptide synthesis. the binding contributions of these molecules are compared to the complete dna binding domain (127-229). this protein was purifi ed using intein mediated protein splicing, an additional cysteine was ligated to the protein by intein mediated ligation. performing single molecule force spectroscopy experiments, kinetic off-rates were obtained, and sequence specifi c dna-binding of both peptide and protein was proven in competition experiments. alanine scans of strategic residues revealed the contributions of single amino acid residues for peptides and proteins. structural investigations of the peptides, the proteins and dna/protein complexes were performed using circular dichroism measurements. this method revealed structural differences of the peptides, proteins and dna upon complex formation. furthermore, the protein/dna or peptide/dna interaction was determined by surface plasmon resonance experiments and electrophoretic mobility shift assays. acknowledgment: this project was supported by dfg (sfb 613). reversed-phase chromatography is important in the development of well-characterized peptide pharmaceuticals. several factors infl uence sensitivity, resolution, and retention for lc and lc-ms applications: bonding chemistry, pore size, particle size, column confi guration, and solvent composition. by decreasing the particle size of hplc packings, column effi ciency is increased. we demonstrate the use of short 10-mm length hplc columns packed with 1.5 m, 500 å c18 silica for ultrafast, one-minute peptide analysis with moderate backpressures (< 3000 psi) using a conventional hplc system. for more complex peptide samples, such as protein digests, on conventional hplc, a novel column confi guration packed with 1.5 m, 100 å c18 is described for 10-to-20min. separations that traditionally take 30 to 80 minutes. we also discuss fast two-minute separations on an ultra-high pressure system using a variety of 1.5 m, small pore columns with unique selectivity. "unknown-genome" proteomics-based identifi cation of a new nadp-epimerase/dehydratase from desulf. phosphitoxidans by inverted-pcr, edman-sequencing and high resolution mass spectrometry protein identifi cation in proteomics is generally based on the availability of genomic data. using amenable databases, identifi cation in "bottom-up" proteomics is often straightforward but is highly complex in the absence of genome data, which typically requires "de novo"-identifi cation approaches. we present here a new approach for identifi cation of proteins from a bacterial strain, desulfotignum phosphitoxidans, with unknown genomic background, using a combination of (i), inverted pcr of degenerate primers derived from n-terminal edman sequencing, and (ii) high resolution maldi-fticr mass spectrometric peptide mass fi ngerprint-proteomics of expressed proteins. desulfotignum phosphitoxidans is an anaerobic sulfatereducing bacterium from marine sediment in which phosphite oxidation was found crucial for energy metabolism. culturing under different growth conditions provided 4 specifi cally expressed proteins with molecular masses of ca. 40 kda in the presence of phosphate, which were subjected to 2d-gel electrophoretical separation for soluble and membrane fractions using pdquest comparative analysis. n-terminal sequences of the proteins, determined by edman analysis were used for inverted pcr of degenerate primers, and provided a series of orf candidates, one of which coded for a putative nad-dependent dehydratase. in a complementary approach, protein spots from the 2d-gels were excised, digested with lys-c protease, and digestion mixtures analysed by maldi-fticr-ms. the accurate peptide masses unequivocally matched to identify a new nad-dependant epimerase/ dehydratase. the detailed functional characterization of the new protein, and further development and application of this approach to proteome analysis with unknown genomic background, are currently in progress. based on specially treated large pore silica and enhanced with a proprietary bonding process, vydac ms reversed-phase (rp) hplc columns offer superior performance for peptides and proteins. the deamidation of human growth hormone (hgh) has been monitored for many years by rp using vydac columns. the vydac ms c4 column provides the best overall performance characteristics (recovery, resolution, and peak symmetry) for the common important assay of hgh and desamido hgh. although hydrophobic membrane proteins are particularly diffi cult to separate, the vydac ms c4 column provides better separation and recovery (up to 86% higher vs. other leading columns) for a reptilian reovirus p14 protein and myristolyated form, a component of a potentially new vaccine delivery system. separation of the trypsin digest of fetuin, a glycoprotein, exhibits improved selectivity for peptide mapping on a vydac ms c18 column compared to other c18 columns, revealing some peaks otherwise not seen. the improved selectivity for peptides on the vydac ms columns results in better primary structure defi nition and easier identifi cation of degradation products and other protein characteristics. hemoglobin peptides in mammalian tissues: facts or artifacts?yatskin, oleg; karelin, andrei; ivanov, vadim shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, russian federation comparative rp-hplc and ms analysis of peptide composition of rat brain, heart, lung and spleen acidic extracts was performed. the extracts were prepared from snap-frozen organs both in the presence and in absence of protease inhibitors (10 -6 m pepstatin a, 10 -4 m pmsf, 2 mm edta) which stabilize the peptide composition of tissue acidic homogenates. the absence of inhibitors led to appearance of novel peptides and did not affect the concentration of predominant peptide components detected in samples prepared with protease inhibitors. therefore, the latter were considered as present in the tissues prior to extraction, i.e. as endogenous. the majority of predominant peptide components were found common in the studied tissues. we believe that the presence of predominant (> 100 pmol/g) peptide components in the protease-inactivated tissue extracts can be reliably extrapolated to their in vivo presence in the tissues. the endogeneity of minor (< 10 pmol/g) components requires a separate consideration. generally, the results of peptidomic studies strongly depend on sample preparation procedures involved, making diffi cult the straightforward comparison of the results obtained by different research groups. urine is a promising source of endogenous peptide biomarkers, because the protein concentration is 1000-fold lower than in plasma but the peptide concentration is equal. urine contains small metabolites that interfere with peptide analysis and urine fl ow varies between subjects and within a day. these variations pose a challenge to urine sample preparation, which, in case of serum, has been shown to be critical for reproducibility. we aim to optimize the enrichment method for urine peptides, to characterize these and to fi nd potential biomarkers. all methods resulted in good peptide recovery if the peptide concentration was normal and metabolite content low. slightly different peptides were obtained with different methods. the df preferentially recovered hydrophilic peptides, while sec-spe recovered high molecular weight peptides better than the df-method. sec-spe provided the best peptide enrichment especially for samples with high metabolite concentration. normalization was critical especially with very dilute samples. without normalization the number of peptides detected was highly dependent on urine concentration. normalization against specifi c gravity proved the most reproducible results. we have identifi ed several peptides found in all samples and some differences between cancer and control samples. validation of these differences is currently under way. jalili, pegah 1 ; ball, haydn 2 1 sigma-aldrich, united states; 2 in order to improve the detection of phosphorylated peptides/proteins, we developed a novel protocol that involves the chemical derivatization of phosphate groups with a chemically engineered biotinylated-tag (biotintag), possessing three functional domains; a biotin group for binding to avidin, a base-labile 4-carboxy fl uorenyl methoxy-carbonyl (4-carboxy fmoc) group, and a nucleophilic sulfhydryl moiety on the side-chain of cysteine. using this approach, the derivatized, enzymatically digested peptides were selectively separated from unrelated sequences and impurities on immobilized avidin. unlike previously published phosphopeptide enrichment procedures, this approach upon treatment with mild base, liberates a covalently bound gly-cys analog of the peptide(s) of interest, exhibiting improved rp-hplc retention and ms ionization properties compared to the precursor phosphopeptide sequence. the results obtained for a model peptide akt-1 and ovalbumin protein digest, demonstrated that the method is highly specifi c and allows selective enrichment of phosphorylated peptides at low concentrations of femtomoles/ l. in search of physiological substrates of brain prolyl oligopeptidase. prolyl oligopeptidase (pop) is a serine protease, which cleaves small peptides at the carboxyl side of an internal proline residue. substance p, arginine-vasopressin, thyroliberin and gonadoliberin are proposed physiological substrates of this protease. pop has been implicated in a variety of brain processes including learning, memory, and mood regulation, as well as in pathologies such as neurodegeneration, hypertension and mood disorders. however, there is no defi nite information about the physiological substrates of pop in brain. we have determined previously that some pop inhibitors, when administrated orally or intraperitoneal, are able to cross the blood-brain barrier and inhibit pop ex vivo. in this work also inhibition duration according to the doses were determined. using this system we studied the natural occurring peptide profi le in brain tissues from inhibited animals and compare it with the profi le in control tissue. we were able to design a method in which the background peptide level was importantly decreased which allowed us to identify, with high degree of confi dence, the peptides which were changed in rat hypothalamus upon pop inhibitor treatment. conclusions about the physiological substrates of pop are discussed. [1, 2] . in the present work, we further studied the dependence of peptide production by the cells on their functional state. using rat hepatocytes as a model, we compared peptide sets generated by: (1) primary hepatocytes in a differentiated state; (2) dedifferentiated primary hepatocytes with fi broblast-like phenotype; (3) rat hepatoma cells. peptides were extracted from the cellular lysates and supernatants by solid-phase extraction and then separated by rp-hplc. peaks were analyzed with maldi-tof and/or maldi-tof/tof. acknowledgements: this work was supported by ras presidium grant "molecular and cellular biology"; russian president grant "scientifi c schools" # 5737.2008.4; rfbr grant # 08-04-00550-a. triostin a is a naturally occurring quinoxaline depsipeptide antibiotic originally isolated from streptomyces s-2-210. with its two quinoxaline moieties it bis-intercalates into dna in a gc selective manner. triostin a contains n-methylated l-valine and l-cystein. the unmethylated analog tandem binds at selectively due to a different hydrogen bonding pattern. substitution of the disulfi de bridge of tandem by an azobenzene moiety leads to photoswitchable analogs. their photoswitchability was investigated and quantifi ed using uv, cd and nmr spectroscopy as well as hplc. in order to determine the three-dimensional structure, conformational analysis by nmr spectroscopy in combination with molecular dynamics calculations was carried out. in this process, distance restraints were obtained from nmr spectra measured in dmso-d 6 and applied in distance geometry/simulated annealing followed by molecular dynamics calculations. as it is possible to distinguish between cis-and transisomer in nmr spectra due to the difference in the chemical shifts, structures of both isomers can be investigated. dna binding studies were carried out using an optical tweezers setup with -dna. furthermore, uv melting curves were recorded for the tandem derivatives in complex with dna. cd spectroscopy was applied to observe changes in dna structure upon formation of the complex. in addition to structural investigations, the antibiotic activity of the tandem derivatives was investigated in minimal inhibition concentration assays against gram-positive bacillus subtilis. acknowledgment: this work was supported by the dfg (sfb 613). k. gaus gratefully acknowledges the fi nancial support (phd grant) by the international nrw graduate school of bioinformatics and genome research. cyclic peptide nanotubes are formed by self-assembly of cyclic peptides with alternating l-and d-amino acid. however, peptide nanotube selfassembly phenomenon had not yet been made cleared. intermolecular electrostatic interaction is one of the important driving forces to assemble cyclic peptides to peptide nanotubes. here in, a series of cycilc hexapeptides, cyclo(-l-lys-gly-) 3 , cyclo(-l-glu-gly-) 3 , cyclo(-l-lys-d-ala-) 3 , cyclo(-d-glu-l-ala-) 3 , cyclo(-l-trp-d-lys-) 3 , and cyclo(-l-trp-d-glu-) 3 , were designed and synthesized by solid-phase peptide synthesis using fmoc strategy. turbidity study revealed that the abilities of self-assembly formations of the 1:1 mixtures of cyclo(-l-trp-d-lys-) 3 and cyclo(-l-trp-d-glu-) 3 in aqueous solution are extremely higher than the abilities of each individual positively or negatively charged cyclic peptides. transmission electron microscope (tem) experiments showed that mixture of cyclo(-lys-xxx-) 3 and cyclo(-glu-xxx-) 3 formed fi brous structure. tem images indicated that, the diameters of the nanotubes were approximately 2-5 nm. diameter of similar cyclic peptides was found to be 1 nm approx. these results also supported that the bundle formation of peptide nanotube. conformations of monomer cyclic hexapeptides were calculated based on nmr observation. estimated formation of peptide nanotube is consisted with the bundle structure. koèevar, nina; obermajer, nataša; kreft, samo faculty of pharmacy, slovenia therapeutic proteins offer a promising potential as effective drug compounds. however, proteins are generally poorly transported across biological membranes due to hydrophilicity. fatty acylation with reactive fatty acid derivatives represents one of the basic methods for increasing the proteins' hydrophobicity and improving membrane permeability. key: cord-004534-jqm1hxps authors: nan title: abstract date: 2009-06-09 journal: eur biophys j doi: 10.1007/s00249-009-0478-1 sha: doc_id: 4534 cord_uid: jqm1hxps nan standard proteomics techniques are unable to describe the stoichiometry, subunit interactions and organisation of assemblies since many are heterogeneous, present at low cellular abundance and frequently difficult to isolate. we have combined two existing methodologies to tackle these challenges: tandem affinity purification (tap) and nanoflow esi-ms. we use methods designed to maintain non-covalent complexes within the mass spectrometer to provide definitive evidence of interacting subunits based on the masses of complexes and subcomplexes generated by perturbation both in solution and gas phases. structural models will be presented for three oligomeric protein complexes of unknown structure: the yeast exosome and the human u1snrnp and eif3 complexes. these models will then be examined within the context of their function. recent developments in mass spectrometry have added a further dimension to our studies of protein complexes: that of their collision cross-section. using ion mobility mass spectrometry we have been able to add spatial restraints to our models validating our models with measurements of collision cross-sections. very recently we have had a considerable breakthrough which has enabled us to preserve intact membrane complexes in the gas phase . this enables us to establish lipid and nucleotide binding and to define the stoichiometry and post translational modifications within the intact transmembrane regions of a number of complexes. in vivo molecular sensing: fluorescence beyond labeling f. beltram scuola normale superiore, i-56126 pisa, italy fluorescent molecules are powerful reporter tools that have much extended the impact of optical microscopy, particularly thanks to the flexibility of genetically encoded tags. detection can now target single molecules even in the complex environment of intact live cells offering unprecedented insight on biological processes in real time in live cells and tissues. fluorescent labels, however, can do more that this. our increased ability to tailor molecule and, more in general, nanosystem properties allows us to design, produce and exploit intelligent tags that can actually analyze the cellular environment. today multifunctional nanosystems can be produced that provide a signal dependent on the value of a specific biochemical parameter. importantly these nanosystems can target specific subcellular domains and have the ability to be used also in the case of live organisms. recent results will be discussed that highlight the impact of nanobiotechnology in this context with a particular emphasis given to methods suitable for in vivo studies that can be transferred to the biomedical world. far-field optical nanoscopy s. w. hell max planck institute, göttingen, germany the resolution of a far-field optical microscope is usually limited to d = λ/ (2 n sin α) > 200 nm, with n sin α denoting the numerical aperture of the lens and λ the wavelength of light. we will discuss lens-based fluorescence microscopy concepts that feature a resolving power on the nanoscale. all these concepts share a common basis: exploiting selected (pairs of) states and transitions of the fluorescent marker to neutralize the limiting role of diffraction. specifically, the fluorophore is switched on and off, that is, between a bright and a dark state, to detect the emission of adjacent features sequentially in time. the first viable concept of this kind was stimulated emission depletion (sted) microscopy in which the fluorescence ability of the dye is switched off by stimulated emission. in the sted microscope, the extent of the region in which the molecule is able to fluoresce follows d ≈ λ/ 2 n sin α 1 + i/i s , meaning that fluorophores that are further away than d can be separated. i is the intensity that drives a fluorophore from the bright fluorescent state to the dark ground state by stimulated emission. i s depends (inversely) on the lifetime of the states. for i/i s → ∞, it follows that d → 0, meaning that the resolution can be molecular). altogether, far-field optical 'nanoscopy' is a fascinating development in optics with high relevance to the many areas of sciences, in particular the life sciences. since it has already been a key to answering important questions in biology, and owing to its simplicity and commercial availability, we expect far-field fluorescence 'nanoscopes' to enter most cell biology and many nanoscience laboratories in the near future. grabbing the cat by the tail: discrete steps by a dna packaging motor and the inter-subunit coordination in a ring-atpase c. bustamante, j. moffitt university of california, berkeley, california, u.s.a. as part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid. bacteriophage ϕ29 packages its 6.6 mm long double-stranded dna into a 42 nm dia. x 54 nm high capsid using a multimeric ring motor that belongs to the asce (additional strand, conserved e) superfamily of atpases. a number of fundamental questions remain as to the coordination of the various subunits in these multimeric rings. the portal motor in bacteriophage phi29 is ideal to investigate these questions and is a remarkable machine that must overcome entropic, electrostatic, and dna bending energies to package its genome to near-crystalline density inside the capsid. using optical tweezers, we find that this motor can work against loads of up to ∼55 piconewtons on average, making it one of the strongest molecular motors ever reported. we establish the force-velocity relationship of the motor. interestingly, the packaging rate decreases as the prohead fills, indicating that an internal pressure builds up due to dna compression attaining the value of ∼6 megapascals at the end of the packaging. we show that the chemical energy of atp is converted into mechanical work during phosphate release. using ultra-high resolution optical tweezers, we determined the step size of the motor and established the coordination of the polymerases around the ring. we propose a comprehensive model of the operation of this motor. watching proteins function in real time via timeresolved x-ray diffraction and solution scattering p. a. anfinrud laboratory of chemical physics/niddk, nih, bethesda, maryland, u.s.a. to generate a deeper understanding into the relations between protein structure, dynamics, and function, we have developed x-ray methods capable of probing changes in protein structure on time scales as short as 150 ps. this infrastructure was first developed on the id09b time-resolved x-ray beamline at the european synchrotron and radiation facility, and more recently at the id14b biocars beamline at the advanced photon source. in studies of ligand-binding heme proteins, a picosecond laser pulse first photolyzes co from the heme, then a suitably delayed picosecond x-ray pulse passes through the protein and the scattered x-rays are imaged on a 2d detector. when the sample is a protein crystal, this "pump-probe" approach recovers time-resolved diffraction "snapshots" whose corresponding electron density maps can be stitched together into movies that unveil the correlated protein motions that accompany and/or mediate ligand migration within the hydrophobic interior of the protein. when the sample is a protein solution, we recover timeresolved small-and wide-angle x-ray scattering patterns that are sensitive to changes in the size, shape, and structure of the protein. scattering studies of proteins in solution unveil structural dynamics without the constraints imposed by crystal contacts; thus, these scattering "fingerprints" complement results obtained from diffraction studies. this research was supported in part by the intramural research program of the nih, niddk dc-sign is a trans-membrane protein expressed on antigen presenting cells and recognizes pathogens like hiv-1, hepatitis c virus and ebola. by electron microscopy and near-field optical nanoscopy, we demonstrated that at the plasma membrane dc-sign is organized in well-defined nanodomains of 100-180 nm in diameter. intensity-size correlation analysis revealed remarkable heterogeneity in the nanodomains molecular packing density. we constructed and characterized several dc-sign mutated forms lacking specific molecular domains. by immunogold labeling and spatial point pattern analysis, we show that the extracellular neck domain is essential for dc-sign nanoclustering. finally, we present a model that describes the probability of a cell to have a certain number of receptors joining the contact site in the initial encounter with an external object. monte carlo simulations subsequently define the parameters that are determinant in the object-cell encounter. our results show that receptor nanoclustering is of particular importance for binding objects of sizes comparable to the nanocluster size, indicating that the nanoscale spatial organization of dc-sign is optimized for binding to virus-sized objects. imaging of mobile stable lipid rafts in the live cell plasma membrane m. brameshuber 1 , j. weghuber 1 , v. ruprecht 1 , h. stockinger 2 , g. j. schuetz 1 1 johannes kepler university linz, austria, 2 medical university of vienna, austria the organization of the cellular plasma membrane at a nanoscopic length scale is believed to affect the association of distinct sets of membrane proteins for the regulation of multiple signaling pathways. based on in vitro results, conflicting models have been proposed which postulate the existence of stable or highly dynamic platforms of membrane lipids and proteins. here we directly imaged and further characterized lipid rafts in the plasma membrane of living cho cells by single molecule tirf microscopy. using a novel recording scheme for "thinning out clusters while conserving stoichiometry of labeling" 1 , molecular homo-association of gpi-anchored mgfp was detected at 37 • c and ascribed to specific enrichment in lipid platforms. the mobile mgfp-gpi homo-associates were found to be stable on a seconds timescale and dissolved after cholesterol depletion. having confirmed the association of mgfp-gpi to stable membrane rafts, we attempted to use an externally applied marker to test this hypothesis. we used bodipy-gm1, a probe that was recently reported to be enriched in the liquid-ordered phase of plasma membrane vesicles. when applied to cho cells at different surface staining, we found that also bodipy-gm1 homo-associated in a cholesterol-dependent manner, thus providing further evidence for the existence of membrane rafts. [1] appl phys lett 87, 263903 (2005) . synapsin knock-out mice as an in vitro model of human epilepsy studied with multi-electrode arrays d. f. boido, p. farisello, p. baldelli, f. benfenati department of neuroscience and brain technologies, italian institute of technology, genova, italy mutant mice lacking synapsins (syn), a family of synaptic vesicles (sv) proteins implicated in the regulation of neurotransmitter release and synapse formation, are epileptic. the attacks appear after the third month of age and severity increases with age. several mutations of syn genes have been found in families of patients with epilepsy. we used micro-electrode arrays (meas) to study spontaneous and chemically evoked epileptiform activities in cortico-hippocampal brain slices obtained from wild-type (wt) and synko mice. 6-months old synko mice show sporadic ictal (ic) events in the entorhinal cortex. a potassium channel blocker, 4aminopyridine (4ap), elicits ic and inter-ictal (i-ic) events in both wt and synko slices. in the hippocampus of young synko (15-days old) mice, 4ap induces i-ic events at higher frequencies than in wt mice. also the frequency of ic events, mainly observed in the cortex, is higher in synko. the analysis of adult (1-year old) mice, revealed a clear age-related aggravation, which paralleled the increase in the severity of the epileptic phenotype observed in vivo. many slices from adult synko mice showed an ic event, while wt slices were refractory at this age to experience ic activity. synko mice are useful to study how neuronal network hyperexcitabilty due to mutations in sv proteins leads to the development of epileptiform activity. meas proved themselves to be useful tools to characterize the epileptic signals foci and patterns of propagation. high electron mobility transistor (hemt) structures were used to bridge the gap between the analysis of biological reactions and biophysical characterization. the combination of nanotechnological measurement approaches with biological reactions provides new possibilities for living cell examinations after exposure to ionizing radiation and basically during the irradiation experiments itself. in this transdisciplinary approach experimental data and handling of biological material enables the identification and specification of systems properties of biological responses to ionizing radiation at different hierarchical levels. gan/algan-heterostructures form a hemt with a gate very sensitive to ph-value changes and potential changes in general. to record cell membrane potentials and ion fluxes during and after irradiation experiments living cells are cultivated on the functionalised biocompatible chip surface. here, we present results of x-ray stimulated cell responses grown on gan-chip surfaces. we recorded transistor signal changes of 0.13 µa within 60 s caused by an irradiated cell monolayer. to measure cell potentials, not only after irradiation experiments but also during the irradiation itself expands the examination restrictions in an enormous way. measuring diffusion by spatial-cross-correlation e. gratton, m. a. digman laboratory for fluorescence dynamics, university of california, irvine, u.s.a. fluorescence correlation spectroscopy (fcs) has emerged as a very powerful method to study the motions of proteins both in the interior and exterior of the cell. it provides information at the single molecular level by averaging the behavior of many molecules thus achieving very good statistics. single particle tracking (spt) is also a highly sensitive technique to measure particle movement. however, the fcs method suffers in spatial resolution while the spt technique only allows for the tracking of isolated molecules. here we propose a change of paradigm in which using spatial pair cross-correlation functions we can overcome this limitation. our method measures the time a particle takes to go from one location to another by correlating the intensity fluctuations at specific points on a grid independently on how many particles are in the imaging field. therefore we can trace the average path of the particles. for example, our method could be used to detect when a protein passes the nuclear barrier and the location of the passage. this information cannot be obtained with the frap (fluorescence recovery after photobleaching) technique or the image correlation spectroscopy method. the interaction of the bax c-terminal domain with membranes j. c. gomez-fernandez, s. sanchez-bautista, a. perez-lara, s. corbalan-garcía departamento de bioquimica y biologia molecular. universidad de murcia, murcia, spain the c-terminal domain of the pro-apoptotic protein bax (bax-c) acts as a membrane anchor during the translocation to the membrane of this protein leading to programmed cell death. we have used static and mas-nmr techniques to show that the interaction of bax-c with membranes is modulated by the presence of a negatively charged phosphatidylglycerol. the width of the resonance peaks were considerably more increased by bax-c, in the presence of phosphatidylglycerol. bax-c substantially decreased the t 1 relaxation times of phosphatidylglycerol and those of phosphatidylcholine when mixtured with phosphatidylglycerol but they were not decreased when phosphatidylcholine was the only phospholipid present in the membrane. 13 c-mas-nmr showed that t 1 values were decreased when bax-c was incorporated and, when phosphatidylglycerol was also present, the decrease in t 1 affected considerably more to some carbons in the polar region. these results indicate that bax-c interacts differently with the polar part of the membrane depending on whether phosphatidylglycerol is present or not, suggesting that an electrostatic interaction of bax-c with the membrane determines the membrane disposition of this domain. fluorescence spectroscopy showed that the trp residues of bax-c were located in a microenvironment more hydrophobic when phosphatidylglycerol was present. h. g. franquelim 1 , l. m. s. loura 2 , n. santos 1 , m. castanho 1 1 instituto de medicina molecular, univ. lisboa, portugal, 2 faculdade de farmácia, univ. coimbra, portugal since the efficacy of hiv fusion inhibitors was previously reported to be related to an ability to interact with membranes, we studied the interaction of the hiv fusion inhibitor sifuvirtide, a 36 aa negatively charged peptide, with lipid vesicles. since this peptide has aromatic residues, fluorescence spectroscopy techniques were used with no need for attached probes. results showed no significant interaction with both zwitterionic fluid phase and cholesterol-enriched membranes; however extensive partition to fluid phase cationic membranes were observed. in the dppc gel phase, however, an adsorption at the surface of these membranes was detected by using a differential quenching approach with lipophilic probes, as well as by fret. moreover, the interaction with gel phase membranes seems to be specific towards pc vesicles, since no significant interaction was retrieved for membranes composed by shingomyelin and ceramide. besides fluorescence, atomic force microscopy and zeta-potential were used to further investigate this issue. our results show a selectivity and specificity of the peptide towards rigid domains, where most of the receptors are found, and help explain the importance of the interaction with membranes in the improved efficacy of sifuvirtide compared to other fusion inhibitors, by providing a local increased concentration of the peptide near the fusion site on both cellular and viral membranes. the study of molecular dynamics at the single-molecule level with fluorescence far-field optics offers new detailed insights into scientific problems, especially in living cells. unfortunately, the resolution of common far-field techniques is limited to about 200nm in the lateral direction by diffraction. in recent years, several concepts such as stimulated emission depletion microscopy (sted) have been successfully applied to overcome the diffraction barrier by exploiting the photophysical properties of fluorescent labels. we present the combination of high resolution sted microscopy with different fluorescence fluctuation techniques providing the unique ability to study molecular dynamics with high spatial (<40nm) and temporal resolution (<1ms) in living cells. using fluorescence correlation spectroscopy (fcs) and general single-molecule analysis, we were able to explore single-molecule dynamics in up to 70-fold reduced focal volumes on two-dimensional samples such as lipid membranes with excellent signal-to-noise ratios. special attention is drawn to inhomogeneous lipid diffusion on the plasma membrane of living cells 1 . by extending the available spatial scale of standard single-molecule fluorescence far-field spectroscopy techniques, our experiments outline a new way of approaching scientific problems. modulation of the properties of membrane microdomains as a control mechanism in cellular physiology p. o'shea cell biophysics group, institute of biophysics, imaging & optical science, school of biology, university of nottingham, nottingham ng7 2rd, u.k. this presentation will outline the molecular-physical rationale of how membrane microdomains may modulate the behaviour of membrane receptor systems as a controlling mechanism in cell signaling. a number of external factors that modulate these properties will be indicated that have a bearing on controlling cellular behaviour. some of key questions will be considered such as the factors that control the assembly and disassembly of microdomains, the size and numberdensity of the microdomains and the lifetime that they exist within the membrane. the technical challenges that these questions identify will also be outlined with some possible solutions. throughout this presentation, correlations will be made between theory and experiment as well as between model membrane systems and real cellular systems. structural and dynamic properties of caveolin-1 and -2 fragments at the membrane interface c. le lan 1 , j. gallay 2 , m. vincent 2 , j.-m. neumann 1 , b. de foresta 1 , n. jamin 1 1 cea, ibitecs, sb2sm, & ura cnrs 2096, gif-sur-yvette, france, 2 ibbmc, université paris-sud, umr8619-cnrs, ifr115, orsay, france caveolins are major protein components of caveolae, microdomains of the plasma membrane involved in a large number of biological functions, including signal transduction, cholesterol homeostasis and transport. the consensus topological model of caveolin-1 includes a small central intramembrane region (102-134) flanked by two cytosolic amphiphilic domains (82-101 and 135-150) which probably constitute in-plane membrane anchors. we investigated the interaction of the cav-1(94-102) juxta-membrane segment with various membrane mimics, using fluorescence, cd and nmr. this segment partitioned better in dpc and in dm/anionic lipids micelles than in dm micelles and this partitioning was coupled with the formation of an amphipathic α-helix. this amphipathic helix was located in an average shallow position, in the polar head group region of the dpc micelle, as shown by fluorescence data and intermolecular noes, with the aromatic doublet w98-f99 probably pointing towards the inside of the micelle on average. the peptide encompassing the homologous sequence of cav-2 was also localized to the dpc micelle polar head group region, in which it adopted a more stable helical conformation than cav1(94-102) . these data brings experimental support for the role of this segment as an interfacial membrane anchor. resveratrol (trans-3,4',5-trihydroxystilbene) , a phytoalexin present in grapes and its analogue piceatannol (trans-3,4,3',5'-tetrahydroxystilbene) are biologically active compounds and possess potential chemopreventive and anticancer properties. the activity of resveratrol and piceatannol can be mediated by membrane effects since structure of lipid membrane domains may play an important role in cell signalling pathways. drugs interactions with dmpc bilayers was investigated using a combination of esr spectroscopy and differential scanning calorimetry. spin probes used in epr experiment were located in different part of lipid bilayer. study was performed at temperatures below and above phase transition temperature (t m ). epr spectra were simulated and displayed with ghost condensation method. the values of ϑ and ϕ (the main and asymmetry cone angles of wobbling spin probe, respectively) were taken for free rotational space parameter (ω) calculation. the decrease of ω values was observed in the presence of both compounds and the effect was more pronounced in lipid gel phase. order parameter and correlation time were also determined and presented in form of ghost patterns. using this approach the differential influence of studied compounds on membrane heterogenity was revealed. separating hydrostatic pressure from cellular strain: development of an in vitro model system r. sulley 1 , j. whatmore 1 , c. p. winlove 2 , a. shore 1 , j. tooke 1 , r. ellis 2 , k. gooding 1 1 peninsula medical school, 2 school of physics, university of exeter, uk endothelial cells (ecs) line blood vessels & are constantly subjected to haemodynamic and mechanical stresses and strains. these stimuli are known to influence ecs, modifying their morphology, intracellular signalling & gene expression. most reported systems exposing ec to mechanical forces in vitro alter pressure & strain simultaneously, making it impossible to distinguish the two potentially independent stimuli. this distinction is particularly relevant when examining the interaction of haemodynamic forces on microvascular ecs, which are exposed to low hydrostatic pressure but significant strains. this research aims to create an in vitro system that can independently examine the effects of pressure and strain, over a range experienced by ecs in the microvasculature. human ecs are seeded (12x10ˆ4cells/cmˆ2) on to the inner surface of compliant 4mm diameter tubing. which is mounted on a perfusion rig inside a sealed, fluid-filled chamber. a continuous sinusoidal cyclical strain of 5-10% is created by a pump attached to the external chamber. lumenal pressure is generated using two hydrostatic pressure heads. validation experiments show that pressure & substrate strain can be independently varied & controlled over a physiological range. the system is now being used to investigate the effects of pathophysiological haemodynamic abnormalities on ec function. membrane potential dynamics of living cells in response to femtosecond laser irradiation n. i. smith 1 , j. ando 2 , k. fujita 2 , s. kawata 2 1 photonics advanced research center, osaka university, suita, osaka 565-0871, japan, 2 dept of applied physics, osaka university, suita, osaka 565-0871, japan the ultrashort pulsed near-infrared femtosecond laser has had a large impact in biomedical research fields and in microscopy, where it has enabled new imaging methodologies. at high intensities, the focused beam of a femtosecond laser has been used to irradiate specific locations inside a cell, often beneath the cell membrane, exploiting the inherent penetration and localized absorption that comes from the multiphoton absorption physics. this has been applied in photobleaching, photouncaging, laser surgery and other experiments where the light is used not merely for observation but is instead an integral tool to interact with the dynamics of cells, to probe and perturb the cell condition. in this talk i will discuss biological and mechanical effects that can be generated by short exposures to femtosecond laser irradiation, such as calcium waves, membrane hyperpolarization, and cell contraction. this talk will concentrate on the changes in membrane potential that can occur when the cell is subjected to focused femtosecond laser beams. both depolarization and hyperpolarization of the membrane potential could be evoked, depending on the laser parameters and on the position of the laser focus. these results have implications for the use of laser beams in microscopy, optical gene transfection, and laser nanosurgery. in a recent study we showed that the melting behavior of supported lipid bilayers (slbs) on mica can be influenced by the solution ionic strength and by the slb preparation temperature [1] . by changing these parameters we could control the coupling between the two bilayer leaflets obtaining a coupled or decoupled melting behavior. thus, we could provide evidence that the slb model system is also suited for the study of lipid/protein interactions which had been questioned in the past. then we investigated the mutual interactions between the membrane lipids (pope:popg 3:1) and the kcsa potassium ion channel by studying kcsa proteins reconstituted in slbs. in particular, we studied the melting behavior of the slb and the ion channel distribution relative to the different membrane phases by temperature controlled atomic force microscopy (afm). by decreasing the temperature we found that the proteins underwent diffusion so to be excluded from the growing solid ordered regions. further, the ion channels tended to accumulate at the domain boundaries or they aggregated in the liquid disordered phase. both effects have been suggested to affect protein function. when we started from a low temperature at which the membrane was mainly in the solid ordered phase the membrane melting processes started in the vicinity of the included ion channels. we report fluorescence recovery after photobleaching (frap) measurements performed at variable spot radius for t7-egfp-hmop receptors on sh-sy5y neuroblastoma cells in the presence of ligands. two different agonists, damgo and morphine, caused markedly different changes to receptor diffusion as compared to the basal state. like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joint semi-permeable domains, but with smaller domain size and diffusion coefficient. this effect was inhibited by pertussis toxin, suggesting that this dynamic behaviour is associated with early steps of signaling. in the presence of damgo, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. hypertonic sucrose buffer suppressed this effect which we attribute to receptor entry into clathrin-coated pits. it is likely that the observation of distinct receptor dynamics in the presence of damgo and morphine involves the agonist-selective phosphorylation of the receptor. the alteration of the physiological transcriptional program is one of the constant features of cancer cells. however, the characterization of the chromatin changes at single-gene level requires going beyond the diffraction limit affecting conventional fluorescence microscopy. the ability of molecular biology techniques to obtain a detailed view of the chromatin status at a sub-promoter resolution has to pay instead the price of averaging over a cell population. we report here the application of an approach based on high-resolution cytometry, chromatin immuno-precipitation and transcriptional profiling (dna microarray) for the characterization of the transcriptional and chromatin changes induced by the oncogenic transcription factor pml/rarα. the presentation will focus on the imaging protocol employed to observe the effects on the chromatin status and the extent of the deregulation induced on transcriptional activity in acute promyelocytic leukemia cells. this multiple-approach examination provides a further step towards the comprehension of the hierarchy of chromatin modifications leading to the establishment of a malignant transcriptional program. dna is rigid negatively charged polymer and in solution exists in extended conformation. i n vivo, volume occupied by dna must be reduced to fit to tiny space of cell nucleus. to condense dna, dna-dna electrostatic repulsion must be cut off that is achieved by interaction with cationic ligands. in binding to dna, oligocations compete with salt cations (k + , na + , mg 2+ ). description of salt dependence of oligocation-induced dna condensation is still lacking. we studied dna condensation by model oligocations, ε-oligo(l-lysines), with variation of charge from +3 to +31. combination of light scattering, uv-monitored precipitation assay and isothermal titration calorimetry allowed covering wide range of dna (c dna ) and salt (c kcl ) concentrations. salt dependence of dna condensation efficiency of the ligand, ec 50 (ligand concentration at the transition midpoint) displays two regimes: salt-independent at low c kcl and salt-dependent at higher c kcl (steep increase of ec 50 with c kcl ). simple formula describing ec 50 as function of ligand charge, c dna and dissociation constant of ligand-dna complex (k d ), was proposed. in the salt-independent regime ec 50 is defined by c dna . salt-dependence of ec 50 is rooted in the variation of k d with c kcl earlier described in ligand-dna binding studies. importance of our findings for description of chromatin is discussed. interaction between proteins from linker region of nucleosome in presence/absence of dna in solution i. b. kipenko 1 , e. v. chikhirzhina 2 , a. m. polyanichko 1 1 faculty of physics, saint-petersburg state university, russia, 2 laboratory of cell biochemistry, institute of cytology, ras, saint-petersburg, russia interactions in the linker region of the nucleosome play a key role in the structural organization of the chromatin. the most fascinating and least understood is the interplay between non-histone chromatin protein hmgb1 and a linker histone h1. it is known that both h1 and hmgb1 bind the linker region of the dna in vivo. however it is still a matter of debate as to whether these proteins assist each other or compete upon binding. the main attention in this work is paid to the investigation of the interactions between the hmgb1 and h1 proteins in physiological environment. using circular dichroism (cd) spectroscopy we have studied the interactions between hmgb1 and h1 at various hmgb1/h1 ratios (r). it has been shown that there is a cddetectable interaction between the proteins h1 and hmgb1 at r < 1. we have demonstrated that the interaction between these proteins results in changes of their secondary structure. cd indicates that the structural impact of the unordered fragments decreases while the net α-helicity of the proteins increases upon the interaction. we have also shown that large higher order structures are formed in solution. in this work we have also discussed the dna-binding properties of the hmgb1 and h1 proteins. the work was supported by a rfbr grant (09-08-01119) and the government of staint-petersburg. t. u. rodionova 1 , a. m. polyanichko 1 , v. i. vorob'ev 2 1 faculty of physics, saint-petersburg state university, russia, 2 institute of cytology of the russian academy of sciences, saint-petersburg, russia hmgb1 is a nonhistone chromosomal protein. data regarding the structure of the hmgb-proteins obtained so far are rather different. thermodynamic experiments reveal predominant α-helical structure of the proteins only at temperatures below +5 • c, i.e. under physiological conditions they are mainly disordered. despite a lot of experimental data biological role of hmgb1 still remains unclear. it is believed that the proteins perform structural functions in chromatin and participate in various regulatory processes in cell. using circular dichroism spectroscopy and dna melting analysis we have shown that hmgb1 changes its structure upon binding to dna. it was shown that at room temperature only about 25% of amino acid residues form α-helices, while in dna-hmgb1complex the degree of the α-helicity of the protein increases to approximately 50%. based on the data obtained we estimate the size of hmgb1 binding site as 80-100 b.p. we have also demonstrated that despite of strong dna-bending properties of hmgb1 its binding to dna results in increase of the double helix termostability. the authors are grateful for the financial support from the russian foundation for basic research (grants 09-08-01119, 07-04-01072) and the government of saint-petersburg. structural organization of supramolecular complexes of dna with chromosomal proteins hmgb1 and h1 a. m. polyanichko 1 , h. wieser 2 1 faculty of physics, saint-petersburg state university, russia, 2 dept. of chemistry, university of calgary, canada a combination of uv and ir absorption and circular dichroism spectroscopy together with atomic force microscopy was applied to investigate the structure and formation of large supramolecular dna-protein complexes. this combination of techniques was used to overcome limitations of uv-cd spectroscopy due to considerable light scattering in such solutions. based on the analysis of ftir and uv circular dichroism spectra and afm data the interaction of dna with highmobility group non-histone chromatin protein hmgb1 and linker histone h1 was studied. it is believed, that hmgb-domain proteins perform both structural and regulatory functions in chromatin. however, the particular mechanisms of it functioning remain unclear/ our data show that histone h1 facilitated binding of hmgb1 to dna by interacting with the sugar-phosphate backbone and binding of asp\glu amino acid residues of hmgb1. acting together, hmgb1 and h1 stimulated the assemblage of supramolecular dna-protein structures. the organization of the ternary complexes is modulated by the interactions between hmgb1 and h1 molecules. the dna-proteins interactions in the presence of metal ions were different, causing prominent dna compaction and formation of large intermolecular complexes. the work was supported by rfbr (grant 09-08-01119). biophysical properties and mechanisms of phage dna ejection t. mdzinarashvili 1 , m. khvedelidze 2 , a. ivanova 1 , t. partskhaladze 1 , n. shengelia 1 1 i. javakhishvili tbilisi state university, tbilisi, georgia, 2 institute of molecular biology and biophysics, tbilisi, georgia to determine the requirements for phage adsorption on bacterial cell and for the realizing resources of following dna ejection thermodynamic and hydrodynamic methods were employed. the temperature, bacterial membrane fragments and receptors had been chosen as such external factors. the phages with short and long tail, both contractile and noncontractile have been studied. our viscometric studies of the phage dna ejection induced by receptor by the example of t5 phage and its receptor fhua have shown that the minimum protein-to-phage ratio necessary for complete dna release is 300 to 1. the viscometric study of ddvi phage dna ejection induced by membrane fragments obtained from its host cells has shown that the environmental conditions play significant role in ejection process. both methods show that the thermally induced phage dna ejection for all investigated by us phages have shown that this process is nonenthalpic. finally from our experimental results we conclude that the start of the dna ejection process from the phage particle occurs without additional energy from either a physical or chemical source. we thank gnsf for the financial support. nitroxides induce apoptosis through caspase-3 activation and collapse of mitochondrial potential k. matczak 1 , a. koceva-chyla 1 , k. gwozdzinski 2 , z. jozwiak 1 1 department of thermobiology, 2 department of molecular biophysics, university of lodz, lodz, poland nitroxides are new class of antioxidants that have been proved to show high reactivity toward free radicals. they act as superoxide dismutase mimics dismutating superoxide anions, but can also exert pro-oxidative properties. in view of their possible dual activity nitroxides could be of great importance in medicine. we have investigated pro-apoptotic activity of pyrroline and pyrrolidine nitroxides pirolid (pd) and pirolin (pl) in human breast cancer cells. in cancer, it is the failure of malignant cells to undergo apoptosis that is crucial. using microplate fluorescence methods, we estimated kinetics of changes in mitochondrial transmembrane potential and caspase-3 activity in breast cancer cells mcf-7 treated with pirolin or pirolid. these features are connected with induction of apoptosis in some type of cancer cells. we observed steady-state increase in caspase-3 activity up to 12 h of postincubation that was followed by a decrease in the enzyme activity at 24 h. caspase-3 activation was considerably greater in cells treated with pirolid. both nitroxides also caused notable decrease in mitochondrial transmembrane potential, which suggest that they can induce apoptosis in breast cancer cells through mitochondrial pathway. o. chernyavskiy 1 , l. vannucci 2 , p. bianchini 3 , f. difato 4 , l. kubínová 1 1 dept. of biomathematics, inst. of physiology, as cr, prague, czech republic, 2 dept. of immunology, inst. of microbiology, as cr, prague, czech republic, 3 lambs, dept. of physics, university of genoa, italy, 4 dept. of neuroscience and brain technologies, the italian institute of technology, genoa, italy the second harmonic generation (shg) imaging along with confocal laser scanning microscopy in reflectance mode can be applied to imaging unstained tissues in vivo, so it can be considered as a fast and non-invasive tool for in vivo studies. murine b16f10 melanoma cells after subcutaneous inoculation in syngeneic mice were let to develop into tumor up to 15-20 mm in diameter. microscopic images were taken before and after microwave hyperthermia treatment (mwht). the microscopic images were acquired by 1-photon imaging in reflectance mode, shg imaging and 2-photon imaging of tissue autofluorescence. the evaluation of changes in the images after mwht of the tumor demonstrated changes in the architecture and organization in both the tumor capsule and tumor mass. the presented study was supported by the academy of sciences of the czech republic (grant iaa500200510, institutional research concepts no.av0z50200510, av0z50110509), and ministry of education, youth and sports of the czech republic (research program lc06063). intracellular delivery and fate of peptide-capped gold nanoparticles: towards cellular biosensors y. cesbron 1 , v. sée 1 , p. free 1 , p. nativo 1 , d. g. spiller 1 , m. r. h. white 1 , m. brust 1 , b. lounis 2 , r. lévy 1 1 liverpool institute for nanoscale science, engineering and technology, liverpool, uk, 2 université bordeaux i / cnrs, bordeaux, france gold nanoparticles (nps) have extraordinary optical properties that make them very attractive single molecule labels. although understanding their dynamic interactions with biomolecules, living cells and organisms is a prerequisite for their use as in situ sensors or actuators. while recent research has provided indications on the effect of size, shape, and surface properties of nps on their internalization by living cells, the biochemical fate of nps after internalization has been essentially unknown. here we show that peptide-capped gold nps enter mammalian cells by endocytosis. we demonstrate that the peptide layer is subsequently degraded within the endosomal compartments through peptide cleavage by the ubiquitous endosomal protease cathepsin l. preservation of the peptide layer integrity and cytosolic delivery of nps can be achieved by a combination of cathepsin inhibition and endosome disruption. this is demonstrated using a combination of distance-dependant fluorescence unquenching and photothermal heterodyne imaging. these results prove the potential of peptide-capped gold nps as cellular biosensors. current efforts focus on in-vivo labeling of nps, nanoparticle-based real-time sensing of enzyme activity in living cells, and the development of photothermal microscopy for single nanoparticle imaging in living cells. towards intravital two photon microscopy study of lymphocytes mobility in lymphonodes m. caccia 1 , l. sironi 1 , m. collini 1 , i. zanoni 2 , t. gorletta 2 , m. di gioia 2 , g. francesca 2 1 dipartimento di fisica, università di milano bicocca, italy, 2 dipartimento di biotecnologie e bioscienze, università di milano bicocca, italy during the last 30 years the edge between optical fluorescence based microscopy and the world of bio-medical research has become thinner and thinner and two photon laser scanning microscopy (tplsm) is one of the most powerful tool for immunological and medical research. one of the most limiting step in intravital microscopy is the preparation of the animal model and the number of animals to be sacrificed to get good statistics. alternative routes must be searched. we employ tplsm to explanted lymphnodes. two photon excitation and a non descanned detection mode allow to increase, respectively, the excitation and detection efficiency while the explanted organs are kept very close to the condition they experience in live animals by means of an home-made temperature controlled box surrounding the microscope and a system for the flux of physiological fluids. experiments performed on explanted lymphonodes, kept under constant flux of co 2 -o 2 saturated buffers and at 36 o c, agree with literature for what concern t-cell homing and motility. this seems to confirm that t-cells behavior in explanted organs mantained in physiological conditions is very similar to that observed in live animals. we then believe that our tplsm microscope would allow to study cell behaviors in in-vivo with high efficiency and a little technical effort. a generalized quantitative frap method with no restriction on the size of the photobleached area heterochromatin protein 1 in dna damage response -recruitment or dissociation from repair sites? j. w. dobrucki division of cell biophysics, faculty of biochem. biophys. and biotechnol., jagiellonian university, kraków, poland we studied recruitment of dna repair proteins to damage sites in live cells, by microscopy approaches, using a new method of inflicting local, sublethal damage in nuclei of live cells. oxidative damage, which was inflicted by exciting dna-intercalated ethidium with focused green light, triggered recruitment of base excision repair enzymes. surprisingly, an epigenetic regulator, heterochromatin protein 1 (hp1) (zarebski et al., 2009 ) was recruited to damage as well. hp1 is a constitutive component of heterochromatin, and plays an important role in transcriptional repression and regulation of euchromatic genes, however it was not known to be required for repair of oxidative damage. the finding of hp1 recruitment is particularly puzzling, since in another study hp1 was shown to dissociate from chromatin as a result of dna damage (ayoub et al. 2008) . technical aspects of live cell imaging that may explain these contradictory results will be discussed. in this presentation, we report a method for determining both the presence and the stoichiometry of protein complexes at pixel resolution and apply it to disassembling focal adhesions. the method is derived from fluorescence fluctuation methods that have single molecule sensitivity and is based on our previously described n&b (number and brightness) method that measures the number and brightness (aggregation state) of fluorescent molecules in every pixel of a confocal microscope image. the new method exploits the correlation of fluorescence amplitude fluctuations for two colors and detects the presence of molecular complexes and their stoichiometry. while the original n&b method was developed for one color, i.e., a single molecular species, the new method, ccn&b, extends the analysis to two colors and introduces the concept of cross-variance. this method is similar in concept to the two-color pch analysis. however, the covariancebased ccn&b method also generates pixel resolution maps of protein complexes and can be used on commercial confocal microscopes. the method is highly sensitive and has relatively high temporal resolution. we have applied this method to adhesion complexes in cells. in addition of their structural role, to link the extracellular substratum to actin filaments, they also serve as signaling centers that regulate many cellular processes including their own assembly and turnover, migration, gene expression, apoptosis, and proliferation. spatio-temporal analysis of membrane lipid remodeling during phagocytosis s. de keijzer 1 , d. kilic 1 , c. g. figdor 1 , s. grinstein 2 , a. cambi 1 1 department of tumor immunology, radboud university of nijmegen, nijmegen, the netherlands, 2 department of biochemistry, university of toronto, toronto, canada the constant threat posed by pathogens and cell debris is tackled by phagocytosis, the process through which cells engulf and destroy dangerous material. the signaling, targeting and trafficking during phagocytosis is dependent on cytoskeleton rearrangements and membrane remodeling. it is becoming increasingly evident that lipids play an important role and can affect the phagocytic response. they assemble microdomains which can act as signaling platforms and confer charge and curvature to the membrane surface promoting electrostatic attraction and retention of proteins. little is known about the mechanism(s) regulating membrane lipid remodeling during phagocytosis. here we used fluorescently labeled biosensors based on k-ras and h-ras proteins to obtain spatio-temporal information on phagosomal membrane lipid remodeling during fcreceptormediated phagocytosis. the results show that cell activation by cytokines modulates the kinetics of anionic lipids thus affecting the membrane charge and the recruitment of cytosolic proteins to the phagosomal membrane. our data emphasize the fundamental role of lipids in the generation and transduction of signals in phagocytosis, and we believe this can be extrapolated to many important processes in a cell. fluorescence correlation spectroscopy (fcs) is a useful technique for characterizing the mobility and concentration of fluorescent molecules both in vitro and in vivo. we utilize two-photon fcs to characterize the concentration and mobility of fluorescent molecules within living cells of bacillus subtilis. autocorrelation functions were measured in bacteria expressing green fluorescent protein (gfp) under the lac promoter in both nutrient rich and nutrient poor culture medium. although considerable heterogeneity was evident from cell to cell, on average, both intracellular concentration and mobility were found to be dependent upon culture medium. we also investigated bacteria expressing gfp under control of native promoters for involved in the regulation of the carbon metabolic cycle in bacillus subtilis. the gfp concentration, which should be related to promoter activity, was investigated for single cells and cell populations under different metabolic conditions. some photobleaching was observed during the course of the measurements as a decrease in the average fluorescence intensity. this is due to the small size of the bacteria (∼1 fl) and low basal expression levels of gfp (∼100 nm) in the absence of iptg. methods to take this into account during data analysis are discussed. a. fassio 1 , s. congia 2 , p. baldelli 2 , f. benfenati 2 1 dimes, university of genoa, genoa, italy, 2 dnbt , iit, genoa, italy several mutations have been discovered in the synapsin i (syn) gene in families with epilepsy, but the mechanism inducing the epileptic phenotype is unknown. syn is a protein associated with synaptic vesicles (svs) that control sv trafficking and neurotransmitter release. the syn mutations subject of this study are a non sense (ns-1) and of two missense (ms-2, ms-3) . syn knockout (ko) hippocampal neurons were transfected with the either wild type (wt) or mutated syns. all syns presented a common punctate pattern of expression at the level of axonal arborizations but, while ns-1 syn targeted to synapses as wt-syn, ms-2 and ms-3 syns reached the presynaptic terminal less efficiently. we next set up a live cell imaging experiment using synaptophysin-phluorin and evaluated the effects of ns-1, ms-2 and ms-3 syns on the size of sv pools. restoring wt-syn in syn i ko terminals led to a increase of all sv pools. restoring ms-2 and ms-3 syns resulted in a phenotype not significatively different from syn i ko background whereas restoring ns-1 syn caused a decrease of all sv pools as compared either with wt-syn or syn i ko background. these data suggest an alteration in the subcellular distribution and function of syn in patients carrying ms-2 e ms-3 mutation and a more severe effect on synaptic activity in patient carrying ns-1 mutation. imaging dynamics of dc-sign at the plasma membrane of hiv-1-stimulated dendritic cells o understanding how viruses interact with host receptors is essential for developing new antiviral strategies. dendritic cells (dcs) can efficiently capture and take up hiv-1 through multiple attachment factors, such as the c-type lectin dc-sign. however, the initial interactions between hiv-1 and this receptor on dcs are not fully understood yet. in this work, we have used single-molecule epi-tirf microscopy in combination with fluorescently labelled hiv-1 virus like particles (vlps) and dc-sign-specific antibodies to image the dynamic interaction between hiv-1 and dc-sign nanoclusters at the plasma membrane of dcs. by tracking individual trajectories of dc-sign on the cell surface of living dcs we have found heterogeneity in the modes of motion of dc-sign: some clusters are immobile whereas others move very quickly, with a few ones showing a directed motion on the cell membrane. to investigate how such motion might be correlated to dc-sign function as virus attachment factor, we have developed glass platforms functionalized with hiv-1 vlps to locally stimulate living dcs. these virus platforms have allowed us to measure dc-sign diffusion rates and motion modes over the cell surface of stimulated dcs and represent a powerful tool for studying the dynamic interactions between hiv-1 and the dc membrane. a. esposito 1 , t. tiffert 2 , j. mauritz 1 , s. schlachter 1 , j. n. skepper 2 , v. l. lew 2 , c. f. kaminski 1 1 dept. of chemical engineering and biotechnology, univ. of cambridge, u.k., 2 dept. of physiology, development and neuroscience, univ. of cambridge, u.k. plasmodium falciparum (pf ) causes the most lethal form of malaria in humans. early research exposed two paradoxes: 1) during its intraerythrocytic cycle pf permeabilizes the host cell so much that a comparably permeabilized healthy red blood cell (rbc) would lyse prematurely, and 2) pf digests far more hemoglobin than needed for its metabolism. a model of the homeostasis of a pf infected rbc suggested a common explanation of both puzzles: excess hemoglobin digestion is required to reduce the colloidosmotic pressure within the host cell thus ensuring its osmotic stability to the end of the pf asexual cycle. we investigated these predictions with direct measurements of [hb] and volumes of parasitized rbcs. reliable volume and morphological data was obtained by confocal microscopy and quantitative surface reconstruction. furthermore, we developed a new fret-based method to measure hemoglobin molecular crowding by exploiting the reduction in fluorescence lifetime of a donor fluorophore loaded in the rbc cytosol. fret imaging techniques are powerful tools for probing the biophysics of living cells. these tools provided a first validation of the colloidosmotic hypothesis and a deeper understanding of the homeostasis of the intraerythrocytic stage of pf. hiv-1 assembly and release occur at the plasma membrane of infected cells and are driven by the gag polyprotein. using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated assembly of fluorescently labeled hiv-1 at the plasma membrane of living cells with high time resolution. gag assembled into discrete clusters corresponding to single virions. after their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1-2 hours. using a photoconvertible fluorescent protein, we determined that assembly was nucleated by membrane bound gag molecules, while both membrane-bound and cytosol derived gag polyproteins contributed to the growing bud. assembly kinetics were rapid and three phases are observed. in phase i, the number of gag molecules at a budding site increases following a saturating exponential with a rate constant of ∼ 5 x 10 −3 s −1 . hence, gag assembly is complete in ∼ 200 s. in phase ii, a plateau in fluorescence intensity is observed with no exchange of gag protein. the fluorescence intensity decays in phase iii. this decay, in some cases, corresponds to the release of a virion. the time scale from the onset of assembly to release of extracellular particles was measured to be ∼1,500+/-700 s. fast 3d chromatin dynamics studied in living yeasts using a novel lab on chip technology h. hajjoul 1 , m. dilhan 1 , i. lassadi 2 , k. bystricky 2 , a. bancaud 1 1 laas-cnrs, université de toulouse, france, 2 lbme, cnrs umr 5099, toulouse, france we present a novel lab-on-chip technology for 3d particle tracking yeast abased on v-shaped mirrors, which are used to observe fluorescent specimens from multiple vantage points, providing stereo-images that can be recombined for 3d reconstruction. our technology is based on v-shaped mirrors, which are fabricated by wet etching of silicon wafers, and used as optical and fluidic components. after rigorous optical optimization of the device in vitro, the lab-on-chip is applied to study chromatin dynamics in vivo using budding yeasts as a model system. yeasts cells are visualized using gfp fused to the associated repressor protein. we confirm earlier observations that telomeric sequences, i.e. located close to chromosome ends, accumulate at the nuclear periphery, whereas genes found midway along chromosome arms are mostly present in the nuclear lumen. the dynamics of these sequences is followed in 3d with an unpreceded temporal resolution of 10 ms, showing that chromosome dynamics is non linear in the short time regime and switches to a linear regime at larger timescales. notably, this behavior is reminiscent of universal responses observed in polymer solutions, and is related to the confinement and the structure of chromosomes. this technique shows a great potential for studying dynamic processes in small living organisms. retrovirus induced remodeling of the host cell actin architecture m. gladnikoff, i. rousso department of structural biology, weizmann institute of science, rehovot, israel retrovirus budding is a key step in the virus replication cycle. yet, despite substantial progress in the structural and biochemical characterization of retroviral budding, the underlying physical mechanism remains poorly understood, primarily due to technical limitations preventing visualization of bud formation in real time. using atomic force-, fluorescence-and transmission electron microscopy we find that both hiv and moloney murine leukemia virus (mlv) remodel the actin cytoskeleton of their host cells and utilize the forces it generates to drive their assembly and budding. highly dynamic actin-filamentous structures which varied in size over the duration of budding appeared to emanate from the assembled virion. these actin structures assemble simultaneously or immediately after the beginning of budding, and disappear as soon as the nascent virus is released from the cell membrane. analysis of sections of cryo-preserved virus infected cells by tem reveals similar actin filaments structures emerging from every nascent virus. substitution of the nucleocapsid domain implicated in actin binding by a leucine-zipper domain resulted in budding of virus-like particles that was not accompanied by remodeling of the cell's cytoskeleton. notably, budding of viruses carrying the modified nucleocapsid domains was an order of magnitude slower than that of the wild type. the results of this study show that retroviruses utilize the cell cytoskeleton to expedite their assembly and budding. t. gensch institute of structural biology and biophysics (isb-1) cellular biophysics, research centre jülich, germany the determination of ion concentrations in cells -in particular in neurons -is very important for understanding cell function and life. ca 2+ is an ubiquitous messenger in almost all cell types, cl − has several roles, e.g. plays an important role in some neuronal signaling pathways including olfaction, nociception and vision. fluorescence lifetime imaging (flim) is of advantage over intensity based fluorescence microscopy, when comparisons between micro-domains of one cell or between different cells of one cell type are performed. several (organic chromophores and fluorescent protein based) ca 2+ -and cl − -sensors have been tested in culture cells with respect to their applicability in flim studies. the ca 2+ -and cl − concentration in rodent olfactory sensory neurons, dorsal root ganglion neurons and neurons of the retina is investigated by time-resolved flim with two-photon excitation. viscosity is one of the main factors which influence diffusion in condensed media. in a cell viscosity can play a role in several diffusion mediated processes, such as drug delivery and signalling. previously, alterations in viscosity in cells and organs have been linked to malfunction; however, mapping viscosity on a single-cell scale remains a challenge. we have imaged viscosity inside individual cells using novel fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent. 1, 2 this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to 100× higher than that of water. 1 we have also shown that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt). 2 we have demonstrated the effect of such viscosity increase on intracellular reactions by directly monitoring the rates of formation and decay of a short lived toxic intermediate, crucial in pdt, called singlet molecular oxygen, in light perturbed cells. characean algae, close relatives of higher plants, represent a convenient model for studying the effect of propagating electrical signals, action potentials (ap) on photosynthesis. illuminated chara corallina cells produce coordinated spatial patterns of chlorophyll fluorescence (chl fl) and extracellular ph. photosynthesis is higher in the cell regions adjacent to acid zones compared to alkaline zones. under physiological conditions, the electrically induced ap differentially and reversibly suppresses photosynthesis in the alkaline and acid regions. in this work we examined the effects of an artificial psi acceptor, methyl viologen (mv), on chl fl with imaging-pam technique. mv is a divalent cation and poorly permeates through biological membranes. the presence in the medium of mv had no effect on fl as well as on p700 + absorbance signals until the application of a single excitatory stimulus. once an ap was generated in the presence of mv, it induced irreversible inhibition of native (nadpdependent) electron flow and a strong non-photochemical fl quenching all over the cell. this indicates that ap redirects electron flow from the main pathway to the artificial acceptor. we concluded that ap generation opens access for permeation of mv from the medium to the chloroplast stroma across two membrane barriers, plasmalemma and chloroplast inner membrane. we suggested that mv might enter chara cell via plasmalemma ca 2+ -channels activated during ap. evaluation of cell damage after photodynamic and sonodynamic treatment h. kolarova, k. tomankova, s. binder, p. kolar, r. bajgar, m. strnad department of medical biophysics, faculty of medicine and dentistry, palacky university, hnevotinska 3, 775 15 olomouc, czech republic photodynamic therapy (pdt) and sonodynamic therapy (sdt) is a new, combined therapy for treating cancer. the basis of the therapy is to administer a small amount of photosensitizer and sonosensitizer, which are selectively taken up by cancer cells, and then expose the body to light and ultrasound to activate these sensitizers. when the sensitizers absorb light of an appropriate wavelength, it may cause their excitation with subsequent energy transfer to oxygen; the oxygen then becomes highly reactive in cancer cells and produces reactive oxygen species (ros). the resulting damage to organelles within malignant cells leads to tumor ablation. sdt uses an agent that is sensitive to ultrasound, allowing deeper penetration and destruction of abnormal cells. changes in human melanoma cells were evaluated using fluorescence microscope and atomic force microscopy.we focused on obtaining pictures of the topography and pictures involving elastic properties of cell surface. the production of ros was investigated with the molecular probe cm-h2dcfda, and morphological changes in cells were evaluated using fluorescence microscope. the quantitative ros production changes in relation to phthalocyanine concentration, irradiation doses and ultrasound intensity were measured by a fluororeader. activity correlation imaging: visualizing function and structure of neuronal populations s. junek 1 , t.-w. chen 1 , m. alevra 1 , d. schild 2 1 department of neurophysiology and cellular biophysics, university of göttingen, germany, 2 dfg research center for molecular physiology of the brain (cmpb), university of göttingen, germany understanding a neuronal network relies on knowledge about both the function and the structure of its neurons. while he simultaneous observation of hundreds of neurons is possible by densely staining brain tissue with functional dyes, the low contrast of these stainings does not allow the identification of neuronal processes from the raw fluorescent images. however, as neurons are known to exhibit complex temporal patterns of activity, fluctuations in signal intensity over time could be exploited to generate contrast in densely stained tissue. we demonstrate that the uniform calcium signals within individual neurons of the olfactory bulb can be exploited to visualize the morphology and projection patterns of these cells in tissue slices. as different neurons exhibit distinct time patterns, it is possible to generate a highcontrast multi-color visualization of the network's active neurons, solely based on the specificity of temporal fluctuations of a single-color calcium dye. it is thus possible to use other spectral channels for additional labelling, for example using cell-type or protein specific markers. the ability to map function and structure of neuronal populations online opens up a number of intriguing applications, such as selecting cells or cell pairs with certain functional or projection profiles for targeted recordings, ablation or stimulation. -live cell imaging investigation of the dynamics of redox elements in live cells by using fluorescence ratio microscopy g. maulucci 1 , g. pani 2 , v. labate 2 , m. mele 2 , e. panieri 2 , m. papi 1 , g. arcovito 1 , t. galeotti 2 , m. de spirito 1 1 istituto di fisica, università cattolica s. cuore, roma, italy, 2 istituto di patologia generale, università cattolica s. cuore, roma, italy oxidative stress or signaling events can affect cellular redox environment, which act simultaneously as regulator and indicator of key cellular functions in both physiological and pathological settings. by using a redox-sensitive protein (rxyfp), employed ratiometrically, it is possible to generate high resolution redox maps of cells. the spatial distributions of oxidized and reduced elements have been discriminated in human embryonic kidney cells by the deconvolution of the histograms of redox maps. by transfecting cell with glutaredoxin v (grx-v), a significant shift towards more reduced state with respect to that recovered from non-transfected cells is observed. despite such large differences, a common behaviour in the spatial distribution of reduced and oxidized couples can still be observed: oxidized population shows a pronounced localization on the cell borders, near the plasma membrane, while reduced population appears as a collection of well separated spots homogeneuosly distributed thoroughly the inner part of the cell with a mean dimension of 2 um. furthermore we observe that the role of grx-v consists in causing a shift towards reduced values of the highly reduced region, while leaving unaltered the redox-balance of the intracellular side of the plasma membrane. fluorescence resonant energy transfer (fret) is a popular approach to studying molecular interactions. fluorescence lifetime imaging (flim) provides a robust read-out of fret but has largely been restricted to fixed cells owing to relatively long acquisition times. we present a highspeed wide-field multidimensional fluorescence microscope for flim fret in live cells on second timescales for high content analysis and studying fast dynamics. the system uses a nipkow disc for optical sectioning and a time-gated image intensifier for wide-field flim. a spectrally selected supercontinuum excitation source facilitates versatile realtime flim of live cells transfected with fluorescent proteins. for cell signalling studies we have developed a multiplexed fret approach. ras activation at the plasma membrane is demonstrated using flim to read out tagrfp-raf rbd interaction with mplum-h-ras. simultaneously, a spectral ratiometric read-out is used for a second fret (cfp/yfp cameleon) probe to monitor the downstream calcium flux. to further develop multiplexed fret as a tool for imaging multiple components of cell signalling networks, we are working to include polarisation-resolved imaging for steady-state and time resolved fluorescence anisotropy measurements. monitoring gene expression via novel nucleic acid and delivery methods k. lymperopoulos 1 , c. spassova 1 , a. seefeld 1 , h. s. parekh 2 , d. p. herten 1 1 bioquant institute,heidelberg university, germany, 2 school of pharmacy, university of queensland, australia application of single-molecule and high-resolution fluorescence methods to monitor gene expression in living cells increase the demand on novel probes and delivery methods.they require fluorophores with high photostability and quantum yield and highly-efficient delivery methods that ensure the minimum interference with cell processes such as metabolism and signal transduction. we used a novel class of dendrimers with varying generations and branching factors and different number of positive charges due to different moieties and functional groups.these different properties were tested for their efficiency to transfect eukaryotic cells with fluorescent oligonucleotides (odns).different parameters (temperature,concentration of dendrimers, ratio of dendrimers and odns) were evaluated and optimised.we utilised these established optimal conditions to deliver a modified concept of smartprobes to mammalian cells targeting mrnas involved in signal pathways.we tested different mrna targets and we optimised the fluorescence signal by varying a range of parameters,namely the fluorescent label and the intrinsic properties of the smartprobe (length of the loop and stem, conformation and number of guanosines).in the near future,we plan to use these probes for monitoring gene expression levels using diffusion imaging microscopy. we present a novel near-field scanning microwave microscope (smm) capable of providing surface impedance measurements of samples with nanometric resolution. the instrument is the integration of a microwave vector network analyzer (vna) and a scanning probe microscope (afm/stm). a key point is that our software, controlling and synchronizing both the instruments, creates simultaneously images of the sample at several frequency points. this can be used to extract several features of the sample depending on the frequency. moreover, close frequencies show the same features, added to random noise. exploiting this redundancy of information, we have achieved remarkable results. we have been working on the optimization of this system for biological applications, to detect functional characteristics of cells generating a variation of their dielectric properties. this instrument offers the possibility of performing local impedance measurements on a single live cell and, if correctly calibrated, it provides also quantitative information (e.g. absolute measurements of membrane permittivity). the system was demonstrated to work on saccharomyces cerevisiae. a better model for a full test of the potentialities of the new technique is given by excitable cells, characterized by a greater variability of dielectric properties. a challenging objective could be directly imaging ion channels. hiv-1 to efficiently complete a replication cycle has to integrate its genome into the host cellular dna.after hiv-1 enters target cells,neosynthesized viral dna forms along with other proteins the pre-integration complex (pic).pics are then transported into the nucleus where integration,catalyzed by the viral integrase,takes place.hiv-1 viral particles engineered to incorporate integrase fused to egfp have proven effective to study pics within nuclei of infected cells.in this study we report the live imaging analysis of nuclear pic dynamics obtained by time-lapse microscopy.intranuclear trajectories of in-egfp-labeled pic were collected in three dimensions and examined by both mean squared displacement (msd) and cage diameter (cd) analysis.in cd the maximum distances measured between two positions occupied by a pic in a time window of 2 minutes were calculated while in our msd analysis 5-minute long trajectory segments were considered.remarkably,msd revealed the presence of an underlying active transport mechanism.to test the possible role of actin filaments,pic nuclear trafficking was analyzed in cells treated with latrunculin b (actin polymerization inhibitor).preliminary results suggest that the disruption of actin function impairs the active nuclear movement of pics. second harmonic generation microscopy reveals sarcomere contractile dynamics of cardiomyocytes n. prent 1 , c. a. greenhalgh 1 , r. o. cisek 1 , j. aus der au 2 , s. elmore 3 , j. h. van beek 3 , j. squier 2 , v. barzda 1 1 department of physics and institute for optical sciences, university of toronto, toronto, canada, 2 department of physics, colorado school of mines, golden, usa, 3 department of molecular cell physiology, vrije universiteit, amsterdam, netherlands cardiomyocytes, like other striated muscles, exhibit strong inherent second-order nonlinear optical properties that make them exemplar for live cell dynamic studies with second harmonic generation (shg) microscopy. laser scanning shg microscopy with an incident wavelength around 1µm, enables fast imaging for extended periods of time with negligible tissue damage. strong shg signal originates from the anisotropic (a-) bands which are comprised of regularly arranged myosin molecules, while actin molecules, the main constituent of the isotropic (i-) bands, produce negligible shg. consequently, the alternating bands along the myofibril are clearly visualized, therefore, enabling the determination of individual sarcomere lengths and the study of sarcomere length dynamics during macro-scale contractions. shg intensity is shown to positively correlate to sarcomere length, which leads to the development of real-time inherent force sensors for in vivo myocytes. rich sarcomere dynamics can be observed during myocyte contraction, which can be used for medical diagnostic purpose of muscular degenerative diseases. imaging cellular communication in insulin secretion ex vivo and in vivo d. w. piston vanderbilt university, nashville, tennessee, u.s.a. the islet of langerhans is the functional unit responsible for glucose-stimulated insulin secretion (gsis), and thus plays a key role in blood glucose homeostasis. the importance of the islet is demonstrated by the proven ability of islet transplants to reverse type i diabetes pathologies in human patients. we are interested in understanding the multicellular mechanisms of islet function, and their role in the regulation of blood glucose under normal and pathological conditions. in many ways, the islet appears to function as a syncytium, which exhibits synchronous behavior of membrane action potentials, ca 2+ oscillations, and pulsatile insulin secretion across all β-cells in the islet. in other ways, the islet works as individual cells, especially in the regulation of gene transcription. using our unique quantitative optical imaging methods and novel microfluidic devices, the dynamics of these molecular mechanisms can be followed quantitatively in living cells within intact islets. these investigations utilize transgenic and tissue-specific knock-out mouse models with demonstrated phenotypes, as well as traditional biochemical and molecular biological approaches. do retinal rod outer segments carry out oxydative phosphorylation? i. panfoli 1 , p. bianchini 2 , d. calzia 1 , s. ravera 1 , a. morelli 1 , a. diaspro 3 1 biology dept., university of genova, italy, 2 lambs-microscobio res.centre, university of genova, italy, 3 neuroscience and brain technology dept., i.i.t. genova, italy visual transduction in vertebrate retinal rod outer segments (os) is very energy demanding. however, atp supply in os, that are devoid of mitochondria, is still puzzling. by a proteomic analysis we identified in purified bovine os disks, proteins involved in vision, as well as the respiratory chain complexes i to iv and f 1 f o -atp synthase, whose activity was comparable to that of mitochondria and sensitive to specific inhibitors. rhodamine 123 fluorescence quenching experiments showed the presence of a proton potential difference across disks. disks consumed oxygen. confocal laser scanning and transmission electron microscopy showed that cytochrome c oxydase and atp synthase are localized on disks. mitochondrial vital dyes stained os ex vivo, and disks. rhodopsin and mitotracker fluorescence co-localized in os. data, suggestive of an aerobic metabolism in os, point to the existence of "mitochondrial inner membrane-like membranes" ectopically producing atp through oxidative phosphorylation, with respect to mitochondria. new scenarios open on the patho-physiology of many retinal diseases associated to mitochondrial dysfunction. -live cell imaging -abstracts 123 unmixing using lifetime-excitation multidimensional confocal fluorescence microscopy data s. schlachter 1 , s. schwedler 2 , a. esposito 1 , a. d. elder 1 , g. s. kaminski schierle 1 , c. f. kaminski 1 1 cambridge university, u.k., 2 universität bielefeld, germany fluorescence imaging provides a powerful tool to probe biological systems, enabling the high resolution investigation of the localization, interaction and biochemical modification of biomolecules. multidimensional imaging, in particular, is a growing application of confocal and other forms of fluorescence microscopy. it seeks to measure not only fluorescence intensity in three spatial dimensions, but also other features of fluorescence emission, such as lifetime and fluorescence spectra. although multi-dimensional fluorescence microscopy has been demonstrated before, little attention has so far been paid to the problem of data interpretation and representation when dealing with such large datasets. we present here an instrument capable of recording fluorescence lifetime and excitation data by combining tcspc for lifetime determination and a supercontinuum excitation source for extracting excitation spectra. these technologies permit the acquisition of 2d and 3d images with lifetime and excitation spectrum information at every pixel. we demonstrate a method whereby the multidimensional datasets acquired with this instrument are processed to yield biologically relevant parameters, (e.g. unmix fluorophores in a multiply labelled sample). our method relies on a global analysis approach and uses ab plots for displaying multidimensional data. we demonstrate the instrument & processing method on dye and multiply labelled biological samples. advanced neuroimaging with diffractive optical elements p. saggau baylor college of medicine, houston, texas, usa studying neural systems is complicated by the large number and small size of its cellular elements. the traditional way of exploring neuronal function by electrical monitoring with micropipettes is increasingly replaced by molecular imaging. fluorescent molecules allow optical monitoring of neuronal signaling with cellular and often subcellular resolution. in addition to monitoring activity, analyzing the functional properties of individual neurons or neuronal populations requires their controlled activation. optical approaches are well-suited, including molecular photolysis of inert precursors and light activation of engineered proteins that control ionic membrane currents. to fully utilize optical techniques for exploring neural systems requires more than adequate spatial resolution to distinguish neuronal elements and sufficient temporal resolution to induce and/or monitor neuronal signaling. because of the non-linear and non-stationary nature of the studied system it is necessary to access many neuronal sites simultaneously. in modern imaging, wide-field illumination and imageformation are often replaced by patterned excitation and nonimaging photon collection. in many instants, diffractive illumination schemes can substitute for conventional reflective or refractive designs. in particular, the availability of programmable diffractive optical elements has made this an attractive alternative, since they permit highly versatile microscope designs and support a significant increase in the overall spatio-temporal resolution. i will present an advanced imaging approach using diffractive optical elements to analyze structure and function of live neurons in brain slices and intact cortex. efficient evaluation of fret in image cytometry with acceptor photobleaching and ratiometric methods j. roszik, d. lisboa, j. szöllősi, g. vereb department of biophysics and cell biology, university of debrecen, debrecen, hungary fluorescence resonance energy transfer (fret) is a powerful technique that can be applied to study nanoscale intra-and intermolecular events and interactions of molecules in situ in biological systems. a robust, easy to use, self-controlled fret method, independent of donor and acceptor concentration and stochiometry, is acceptor photobleaching fret, which requires only simple image mathematics. another approach with more complicated calculations is the intensitybased ratiometric method, which is not based on destroying the acceptor fluorophores, making it applicable for following molecular interactions in live cells. as the need for using fret in image cytometry for evaluating molecular interactions increases, we have undertaken to develop softwares for these methods involving the calculation and usage of all correction factors needed to obtain reliable energy transfer efficiencies. correction possibilities of the acceptor photobleaching method include unwanted photobleaching of the donor, fluorescent photoproduct of the acceptor after photobleaching, cross-talk of unbleached acceptor into the donor channel and partial photobleaching of the acceptor. in the case of intensity-based ratiometric fret, we can correct for all channel cross-talks and calibrate the fret efficiency calculations. both programs provide registration and semiautomatic processing, and they are freely available. the pendrin (slc26a4) gene is responsible, when mutated, for the pendred syndrome, a recessive disorder characterized by sensorineural hearing loss often accompanied by thyroid disfunctions. pendrin is an anion exchanger. the way it works and its role in different tissues, owing to the lack of known isoforms, is matter of wide research and debate. we focused on a still unexplored pendrin function, that is most important in the inner ear: cellular volume control and cl − fluxes regulation. we used hek cells over-expressing wild type pendrin or a mutated isoform together with the eyfp. we challenged cells with hypo-osmolar solutions and followed their volume variations in time. taking advantage of the confocal optical sectioning we independently measured cell volume and fluorescence intensity. in this way, given the dependence of eyfp fluorescence from [cl − ] and ph (measured with snarf5) we could estimate at the same time cl − fluxes, volume and ph variations. the contemporary measurements of the three variables, not yet reported in living cells, allowed to assess the role of pendrin in volume regulation and evidenced its participation to cl − fluxes as compared to the mutated isoform or controls. particle tracking inside the cell largely benefits of the ability to spatially and temporally mark specific structures to follow their "signalling" over a "dark" background as made possible since the advent of the photo-activatable markers. in terms of spatial confinement of the photo-activation process, the use of multiphoton excitation provides several favourable aspects compared to single photon confocal microscopy in photomarking biological structures to be tracked: the confined excitation volumes, of the order of magnitude of subfemtoliter, due to the non-linear requirements provide a unique control of the excitation and consequently photoactivation in the 3d space. in this context photoactivation experiments can be used to assess quantitative information about the binding kinetics of a macromolecule expressed in different cellular compartments. in this work we extended to photoactivation procedures and models originally developed for the quantitative analysis of frap experiments and we evaluated, for different proteins of medical interest (rac-pagfp), the diffusive behaviour in the cytoplasm and the binding kinetics at the large endosomes. the results are compared with standard photobleaching experiments, in order to evidence the gained sensitivity obtained with photo-activatable proteins. motile plaques involved in stress fiber assembly revealed by high-speed spm for living cells k. tamura, t. mizutani, h. haga, k. kawabata division of biological sciences, graduate school of science, hokkaido university, kita-ku, sapporo, japan stress fibers, which are contractile actin cables aligned in a highly-ordered manner, are important for cell migration, mechanical support of plasma membranes and extracellular matrix organization. although stress fibers are dynamically disassembled and assembled again in cells, it is poorly understood how actin filaments are organized into cables with highly-ordered alignment for stress fiber assembly. for investigation of actin cytoskeletal dynamics during actin cable formation, we performed time-lapse observation of actin cytoskeleton in lamella of living fibroblasts by using scanning probe microscopy (spm). high-speed spm observation revealed that motile plaques defined front-side ends of new actin cables and that preexisting mesh-form actin networks were remodeled into new actin cables. directional order analysis of movement of plaques and pharmacological experiments clarified that plaques were driven by myosin-ii-based retrograde actin flow, indicating that plaques are associated with actin cytoskeleton. immunofluorescence experiments showed that plaques were localized on foci of vinculin, a component of cell-substratum adhesions, suggesting that plaques can bind to extracellular substrata via vinculin. based on these results, we propose a model for actin cable formation that motile plaques initiate remodeling of preexisting actin networks into actin cables aligned in a direction of actin flow by associating with extracellular substrata. abscisic acid (aba) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. recently, aba was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells and by human and murine pancreatic β cells. aba autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin (ptx)-sensitive receptor/g-protein complex, cyclic amp, cd38-produced cyclic adp-ribose and intracellular calcium. here, the aba receptor on human granulocytes and on rat insulinoma cells is identified as the lanthionine synthetase c-like protein lancl2. co-expression of lancl2 and cd38 in the human hela cell line reproduces the aba-signaling pathway. the ptx-sensitive g protein coupled to lancl2 is identified as g i by transfection of cd38 + /lancl2 + hela with a chimeric g protein (gα q/i ). identification of the mammalian aba receptor will enable the screening of synthetic aba antagonists as prospective new anti-inflammatory and anti-diabetic agents. investigation of post-thaw damage of s.cerevisiae yeasts using fluorescent dyes 2-dab and 3-dab t. s. dyubko 1 , i. a. buriak 2 , v. d. zinchenko 2 , i. f. kovalenko 2 1 state scientific institution "institute for single crystals", national acadey of sciences of ukraine, kharkov, ukraine, 2 institute for problems of cryobiology and cryomedicine, national academy of sciences of ukraine, kharkov, ukraine we investigate applicability of the fluorescent probes 2-dab and 3-dab available from seta biomedicals (www.setabiomedicals.com) to study the post-thaw damage of saccharomyces cerevisiae yeast cells. the leaving cells stained with these dyes have bright fluorescence in the yellow and red region, respectively. however, the freezing followed by post-thawing causes cell damage, which results in a change of fluorescence intensity. in the native living cells the dyes are preferably localized in the cell membranes and organelles which are highly fluorescent. partially damaged cells have even brighter fluorescence as compared to the living cells. however, their cell ultra-structure is not well-distinguished in the fluorescence mode. ultimately destroyed cells are almost non-fluorescent: the cell membranes are not visible in the fluorescent mode and their organelles are only weakly fluorescent. the number of intensively fluorescing cells with partially destroyed membranes, which were obtained by freezing to -20 • c, is lower compared to those frozen at -196 • c. 2-dab and 3-dab enable not only to distinguish damaged and undamaged cells, but also allow quantitative estimation of the extent of damage in membranes by cryogenic effects. the thylakoid membrane is a structured network in higher plants which is organised into stacked granal thylakoids that are interconnected by single 'stromal' lamellae. we have studied the mobility of a resident protein of the stromal lamellae, hcf106, part of the tat protein translocase. hcf106-green fluorescent protein fusion (gfp) was targeted into thylakoids and studied using photobleaching approaches. we show that small regions fail to recover significant levels of hcf106-gfp fluorescence within 3 minutes after photobleaching. autofluorescence from the photosystem ii light-harvesting complex (lhcii) in granal stacks likewise fails to recover over this time scale. although the thylakoid membrane is a single continuous entity, these data show that both hcf106-gfp and lhcii are constrained within this network. since the hcf106 homologue, tatb, is highly mobile in the escherichia coli plasma membrane, we believe that the stromal lamellae take the form of distinct domains that are effectively constrained by boundaries within the thylakoid network. cytomechanical modifications induced by drugloaded carriers uptake by breast cancer cells the novel opportunities offered by the nanotechnologies have attracted great interest in the development of novel biomaterials for targeted drug delivery in cancer research. the desired features of pharmaceutical drug delivery for intravenous administration are their small size, biodegradability, high drug content, prolonged circulation in the blood and the ability to target required areas. in this work we have compared efficacy of different type of carriers having complementary properties for pharmaceutical delivery in cancer therapy. drug nano-colloids encapsulated by combination of layer by layer (lbl) techniques and ultrasonication, phytochemical encapsulated artificial oleosomes and drug-loading clay/carbon nanotubes have been used for uptake into breast cancer cells. analysis of viscoelastic response of neoplastic cells induced by cargo-carriers uptake has been carried out by a combination of high resolution optical and scanning force microscopy techniques. furthermore, the effects of drug-reservoir carriers on the cytoskeleton (re)organisation of neoplastic cells were further investigated by confocal microscopy using different fluorescent probes. mapping diffusion by raster image correlation spectroscopy (rics) with analog detection time lapse and flow cytometry data integrated in a common cell cycle proliferation model p. ubezio, v. colombo, m. lupi, f. falcetta istituto di ricerche farmacologiche "mario negri", milano, italy we present a cell cycle simulation tool, connecting the basic proliferation process to the cell population data obtained by time lapse and flow cytometry. the computer program is a general framework within which cell cycle progression can be interactively modeled at the desired level of complexity, including g1/s/g2m cell cycle phases with variable duration, g0 phase, cell subpopulations belonging to different generations or differentiation stages, distinct block and cell death parameters for each phase. in this way, we achieved a detailed rendering of cell proliferation of normal or tumor cell populations, additionally including cytostatic and cytotoxic effects of treatments. the program gives as output simulated measures, reproducing those obtained by absolute cell counting, growth inhibition tests, flow cytometric dna histograms and cell cycle percentages, pulse continuous-labeling studies, time-lapse intermitotic times and generation-wise analyses. each technique provides a piece of information related to the underlying proliferation process, catching only some of the phenomena in play, and the measures often cannot be univocally interpreted. we used the cell cycle simulator to fit together time lapse and flow cytometry time courses measures, to achieve a detailed reconstruction of the cell cycle proliferation of an ovarian cancer cell line in vitro after x-ray exposure. -live cell imaging detection of functional modes in protein dynamics j. s. hub 1 , b. l. de groot 2 1 deptartment of cell & molecular biology, uppsala university, sweden, 2 computational biomolecular dynamics group, max-planck-institute for biophysical chemistry, göttingen, germany proteins frequently accomplish their biological function by collective atomic motions. yet the identification of a collective motions related to a specific protein function from, e.g. a molecular dynamics trajectory, is often non-trivial. here, we propose a novel technique termed`functional mode analysis' that aims to detect the collective motion that is directly related to a particular protein function. based on an ensemble of structures, together with an arbitrary`functional quantity' that quantifies the functional state of the protein, the method detects the collective motion that is maximally correlated to the functional quantity. the functional quantity could, e.g., correspond to a geometric, electrostatic, or chemical observable, or any other variable that is relevant to the function of the protein. two different correlation measures are applied. first, the pearson correlation coefficient that measures linear correlation only; and second, the mutual information that can assess any kind of interdependence. detecting the maximally correlated motion allows one to derive a model for the functional state in terms of a single collective coordinate. the new method is illustrated using various biomolecules, including a polyalanine-helix, t4 lysozyme, trp-cage, and leucine-binding protein. logic estimation of the optimum source neutron energy for bnct of brain tumors boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron-10. a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding conditions on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating 65 different source neutron energies, between 0.01 ev and 13.2 mev. mcnp4b monte carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about 30 kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. the results were discussed. v. l. cruz, j. ramos, j. martinez-salazar instituto de estructura de la materia. csic cannabinoid receptors are an important class of g protein coupled receptors. in particular cb1 and cb2 have received considerable interest because they mediate a variety of physiological responses in the central nervous and immune systems. their tertiary structure is still unknown. experimental information suggests the presence of both, an active and an inactive conformation. cb1 and cb2 share a common structural framework consisting in a seven transmembrane α-helix bundle connected by three extracellular and three intracellular loops. however, the knowledge of structural differences between both receptors may serve to design new ligands to activate/deactivate selectively only one of those receptors. in the present research, we report a multi-nanosecond molecular dynamics simulation of both receptors in solution, starting from a structure obtained by homology modelling using the x-ray determined bovine rhodopsin protein. we look for differences in the behaviour of cb1 and cb2 during the simulation process to shed light about those structural features which can be important for ligand selectivity. in this sense, we observed that cb1 tend to present a more flexible and opened helix bundle than cb2. thus, it is expected than the former receptors would present less steric hindrance for ligand binding. we expect these results will be useful to design more selective ligands. self-assembly and equilibration of bola-lipids membranes studied by molecular dynamics m. bulacu, s. j. marrink groningen university, the netherlands bola-lipids consist of two monopolar, twin-tailed lipids that are held together by chemical linkage between one or both ends of the tails from one lipid and the corresponding ends from the other one. membranes formed by these lipids or by their mixtures with monopolar lipids are known to have additional mechanical stability while retaining membrane fluidity. this is traditionally attributed to the fact that, in the membrane phase, the bola-lipids have predominantly spanning configuration (the two polar heads are positioned at opposite membrane-water interfaces) in detriment to looping configuration (both head groups are located in the same membrane-water interface). we perform molecular dynamics simulations, using the coarse grained martini forcefield. we start with self-assembly simulations of bola-lipids followed by bilayer equilibration. an artificial pore is created in the membrane that significantly increases the flip-flop mobility of the lipids and hasten equilibration. the membrane properties are characterized (area per lipid, thickness, order parameter, pressure profile) with emphasis on the spanning/looping ratio. our study can help designing new artificial membranes, with higher stability under extreme conditions. -multiscale simulation evidence for proton shuffling in a thioredoxinlike protein during catalysis d. narzi 1 , s. w. siu 1 , c. u. stirnimann 3 , j. p. grimshaw 2 , r. glockshuber 2 , g. capitani 4 , r. a. böckmann 1 1 theoretical and computational membrane biology, center for bioinformatics, saarland university, germany, 2 institute of molecular biology and biophysics, eth zürich, switzerland, 3 structural and computational unit, embl, heidelberg, germany, 4 paul scherrer institute, villigen psi, switzerland proteins of the thioredoxin (trx) superfamily catalyze disulfide-bond formation, reduction and isomerization in substrate proteins both in prokaryotic and in eukaryotic cells. all members of the trx family with thioldisulfide oxidoreductase activity contain the characteristic cys-x-x-cys motif in their active site. here, using poisson-boltzmann-based protonation-state calculations based on 100-ns molecular dynamics simulations, we investigated the catalytic mechanism of dsbl, the most oxidizing protein known to date. we observed several correlated transitions in the protonation states of the buried active-site cysteine and a neighboring lysine coupled to the exposure of the active-site thiolate. these results support the view of an internal proton shuffling mechanism during oxidation crucial for the uptake of two electrons from the substrate protein. intramolecular disulfide-bond formation is probably steered by the conformational switch facilitating interaction with the active-site thiolate. a consistent catalytic mechanism for dsbl, probably conferrable to other proteins of the same class, is presented. our results suggest a functional role of hydration entropy of active-site groups . the role of water in zn(ii)-abeta(1-16) complexes beta-amyloid (aβ) peptides are the main component of amyloid fibrils detected in the brain of alzheimer patients. fibrils display an abnormal content of cu and zn ions whose binding to aβ-peptides has been recently studied by x-ray absorption spectroscopy [1] and interpreted in terms of ab initio simulations. in order to perform such simulations it is of the utmost importance to find a compromise between the need of having a realistic description of the actual physical system and the difficulty of dealing with too many atoms and electrons. what is usually done is removing solvent (water molecules), thus studying the system in the so called "gasphase". in this work we investigate the relevance of water in the zn-aβ 1−16 coordination mode. relying on a combination of classical [2] and quantum chemistry [3] methods we find a significant difference in the zn coordination geometry depending on whether water is present or not. this information is exploited in building a full model system for subsequent car-parrinello simulations where two aβ peptides in water are in interaction in the presence of zn. [ although experiments which can directly probe the mechanical properties of proteins have only been performed recently, it is clear that the complex folds of polypeptide backbones lead to very heterogeneous mechanical behavior. this heterogeneity is likely to play an important role in protein function and interaction and it would be useful to be able to predict what mechanical (and dynamical) properties will result from a given structure. we have been using coarse-grain elastic network models to investigate this question and have found that these models are able to link specific mechanical properties to a number of functional features including enzymatic active sites, folding nuclei and changes in behavior due to point mutations. these models are also adapted to looking at complex formation between proteins and how specific recognition is achieved, while being fast enough to be applied to very large numbers of interactions. refinement of protein model structures using biasing potential replica exchange simulations s. kannan, m. zacharias school of engineering and science, jacobs university bremen, d-28759 bremen, germany comparative protein modeling of a target protein based on sequence similarity to a protein with known structure is widely used to provide structural models of proteins. frequently, the quality of the target-template sequence alignment is non-uniform along the sequence: parts can be modeled with a high confidence, whereas other parts differ strongly from the template. in principle, molecular dynamics (md) simulations can be used to refine protein model structures but it is limited by the currently accessible simulation time scales. we have used a recently developed biasing potential replica exchange (bp-rex) md method (kannan, s. zacharias, m. proteins 2007, 66, 697-70) to refine homology modeled protein structure at atomic resolution including explicit solvent. in standard rex-md simulations several replicas of a system are run in parallel at different temperatures allowing exchanges at preset time intervals. in a bp-rexmd simulation replicas are controlled by various levels of a biasing potential to reduce the energy barriers associated with peptide backbone dihedral transitions. the method requires much fewer replicas for efficient sampling compared with standard temperature rexmd. bp-rexmd simulations on several test cases starting from decoy structures deviating significantly from the native structure resulted in final structures in much closer agreement with experiment compared to conventional md simulations. m. s. p. sansom, p. j. stansfeld, c. l. wee, k. balali-mood department of biochemistry, university of oxford, u.k. coarse-grained molecular dynamics simulations may be used to probe the interactions of membrane proteins with bilayers and their component lipids on an extended (∼1 µs) timescale (1 ) . conversion to atomistic resolution allows more detailed protein/lipid interactions to be examined. this multiscale approach will be examined via three examples: (i) interactions of a small ion channel toxin (vstx1) we present a new competitive approach for the treatment of biomolecular flexibility to provide an alternative to the limitations of current methodologies such as molecular dynamics and normal mode analysis. this method, called static mode method, is based on the "induced-fit" concept and is aimed at mapping the intrinsic deformations of a biomolecule subject to any external excitations: direct mono or multi-site contact, electrical etc... the algorithm allows obtaining a set of deformations, each one corresponding to a specific interaction on a specific molecular site, in terms of force constants contained in the energy model. such a process can be used to explore the properties of single molecular intrinsic flexibility, as well as to predict molecular docking or molecule/surface interactions. from a modelling point of view, the interaction problem can be expressed in terms of reactive sites between the interacting entities, the molecular deformations being extracted from the pre-calculated static modes of each separated ones. the first applications of our method have focused on the intrinsic flexibility of biomolecules like nucleic acids and proteins. more recently, this new methodology allowed us to investigate the folding of the region 1-16 of the amyloid β-peptide, via the docking of a zinc ion on the reactive sites of the molecule. conformational study on a myelin basic protein fragment: molecular dynamics simulations in membrane e. polverini 1 , g. harauz 2 1 dipartimento di fisica, università di parma, parma, italy, 2 department of molecular and cellular biology, university of guelph, guelph, ontario, canada myelin basic protein (mbp) is a multifunctional protein of the central nervous system whose principal role is in maintaining the compactness and integrity of the myelin sheath, the multilamellar membrane wrapped around nerve axons. however, mbp also interacts with other proteins such as cytoskeletal and signalling proteins, adapting its structure to the different roles. mbp is a candidate autoantigen in the human demyelinating disease multiple sclerosis. this study investigated at atomic detail the conformation of a highly conserved central fragment of mbp, constisting of two consecutive regions with different relevant functionalities. the first one is associated with the membrane and comprises the primary immunodominant epitope in multiple sclerosis; the second one was predicted to be a ligand for sh3-domains of signalling proteins. molecular dynamics simulations were perfomed in the presence of dodecylphosphocholine micelle, starting from a structure extrapolated from experimental data (harauz and libich, curr. protein pept. sci., 2009). the results confirm the experimetal hypothesis, showing, in the micelle, a stable alpha-helix anchored to the membrane for the first region and, for the proline-rich second one, a poly-proline type ii helix pointing outwards, ready to interact with the signalling proteins. multiresolution modelling of drug and hormone permeability through a lipid bilayer traditional atomic-level (al) modelling of biomembranes is time-consuming, and hence limited in the range of systems and phenomena that can be simulated. to alleviate this problem, we designed a coarse-grain (cg) representation where each lipid molecule, in reality consisting of more than 100 atoms, is modelled with only 10 cg sites. our cg technique proves two orders of magnitude less demanding of computational resources than traditional al methodology. a unique feature of our approach is that the cg potentials are directly compatible with standard al models, thus facilitating the simulation of multiresolution systems, where the "chemically-sensitive" components (e.g., the solutes in membrane permeation studies) are modelled atomistically, while the surrounding environment is coarse-grained. in this contribution, we present a summary of our multiscale methodology, together with its application to the permeation of large molecules -drugs and steroid hormones. the calculated permeabilities are compared to the available experimental measurements and al simulation data. molecularlevel insights regarding the permeation mechanism are obtained and rationalised. -multiscale simulation the processes of life involve a variety of events that occur on different scales, ranging from a fewå/ps of the triggering steps of the biochemical reactions, up to their macroscopic effects in cells and organs. intermediate steps involve the structural rearrangement of the bio-molecules (∼nm/10-100ns), their aggregation, folding (∼10nm-µm/ µs-ms), internal cell diffusion and dynamics (µm-mm/ms-hours), evidently requiring a multi-scale modeling approach. here the multi-scale approaches are first briefly illustrated with a particular attention to the issue of matching the different resolutions, which is essential to achieve a coherent descripition. the focus is then fixed on the coarse grained (cg) models, typically spanning the nm-µm and µs-ms scale, and in particular on their minimalist -simplest and computationally cheapest -versions [1, 2] . a parameterization strategy that combines accuracy and predictive power within these models is presented here and applications are shown to relevant cases including the proteins involved in the hiv replication, the green fluorescent proteins and examples of macromolecular complexes. [1] controlled degradation of collagen is an important process in tissue remodeling and wound healing. collagenase cleaves fibrillar collagens about three quarters of the distance from the amino-terminus. even though the determination of the cleavage site and the collagenase structure took place decades ago, the mode of action of collagenase on collagen is not clear. to understand the mechanism of collagenase activity on collagen, the structure, stability and dynamics of collagen, its conformation around the cleavage site and the possibilities of conformational rearrangements between the two domains in collagenase was explored using md simulation. the results of principal component (pca) and normal mode (nm) analysis of the collagen and collagenase suggests that the c-terminal domain of collagenase recognizes the collagen, and then the n-terminal catalytic domain undergoes rearrangement on the substrate (with the help of linker regions). the sda software for carrying out brownian dynamics simulations of protein-protein diffusional association with the help of biochemical constraints is being used to predict the mode of interaction of collagen and collagenase. different conformations obtained from pca and nm analysis are being used for the docking. the predicted structures of the complex will help us to understand how collagenase recognizes and binds collagen specifically. acknowledgment: daad, germany, csir-srf india, and the klaus tschira foundation. membrane aoration by antimicrobial peptides combining atomistic and coarse-grained descriptions d. sengupta, a. j. rzepiela, n. goga, s. j. marrink university of groningen, the netherlands antimicrobial peptides (amps) comprise a large family of peptides that include small cationic peptides, such as magainins, which permeabilize lipid membranes. previous atomistic level simulations of magainin-h2 peptides show that they act by forming toroidal transmembrane pores. however, due to the atomistic level of description, these simulations were necessarily limited to small system sizes and sub-microsecond time scales. here, we study the long-time relaxation properties of these pores by evolving the systems using a coarse-grain (cg) description. the disordered nature and the topology of the atomistic pores are maintained at the cg level. the peptides sample different orientations but at any given time, only a few peptides insert into the pore. key states observed at the cg level are subsequently back-transformed to the atomistic level using a resolutionexchange protocol. the configurations sampled at the cg level are stable in the atomistic simulation. the effect of helicity on pore stability is investigated at the cg level and we find that partial helicity is required to form stable pores. we also show that the current cg scheme can be used to study spontaneous poration by magainin-h2 peptides. over-all, our simulations provide a multi-scale view of a fundamental biophysical membrane process involving a complex interplay between peptides and lipids. the varkud satellite (vs) ribozyme is the largest of the nucleolytic ribozymes, and the only one for which there is no crystal structure. we have determined the overall architecture of the complete vs ribozyme using small-angle x-ray scattering in solution. the substrate stem-loop docks into the tertiary fold of the ribozyme, allowing an intimate loop-loop interaction to occur. this brings two key nucleobases a756 and g638 into close proximity of the scissile phosphate, and we believe that these nucleobases are involved in general acid-base catalysis of the phosphoryl transfer reactions. this is supported by functional group substitution, and the ph dependence of the reaction rate for the natural and modified rna. although possessing totally different folds, the functional elements of the vs and hairpin ribozymes are topologically and mechanistically very similar if not identical. this has probably arisen by convergent evolution. other nucleolytic ribozymes have diversified to employ hydrated metal ions and even small molecules to participate in genera acid-base catalysis. by contrast, the larger ribozymes seem to have adopted a different, metalloenzyme, catalytic strategy. why these different groups have evolved different catalytic chemistries is an interesting challenge to our understanding of biocatalysis. comparison of dna and sirna binding and nuclease protection by non-viral vectors for gene delivery j. lam, k. witt, a. j. mason, l. kudsiova, m. j. lawrence pharmaceutical science division, king's college london, uk the biggest obstacle for the success of gene therapy is delivery. with the discovery of rnai, the use of sirna to regulate gene expression as potential therapy has attracted much attention recently. in contrast to dna, sirna delivery does not require nuclear entry. with one less barrier to overcome, the delivery of sirna seems to be easier. it is frequently assumed that comparable delivery strategy could be used for both dna and sirna. in fact the two types of nucleic acids are fundamentally different with distinct properties, which impact their delivery strategies. in the present study it was found that most non-viral delivery vectors including polymers, peptides and lipids were generally more efficient in binding with dna than sirna as shown in gel retardation assay. the inferior sirna binding is possibly due to the rigid structure of sirna, resulting in weaker electrostatic interaction with the cationic vectors. surprisingly, it was observed that all the vectors studied offered better nuclease protection for sirna than dna despite poorer sirna binding. either the nuclease protection for sirna is easier to achieve due to its small size, or the gel retardation assay did not truly reflect the binding efficiency as the weaker sirna complexes may dissociate during electrophoresis. not only the delivery strategy, the results between dna and sirna study must also be carefully adapted and interpreted. single molecule studies of spliceosomal rnas u2 and u6 z. guo, d. rueda department of chemistry, wayne state university, detroit, michigan, u.s.a. splicing is an essential step in the maturation reaction of mrna, in which intervening introns from exons. the spliceosome is a dynamic assembly of 5 small nuclear rnas and >150 proteins that catalyzes splicing. u2 and u6 are the only 2 snrnas strictly required for splicing. major conformational changes are expected to take place during spliceosomal assembly and catalysis. we have developed a single-molecule fluorescence assay to study the structural dynamics of a protein-free u2-u6 complex from yeast. our previous data have revealed a 2-step large amplitude conformational change of the u2-u6 complex. the 1 st step is a mg 2+ -induced conformational change where helix iii and the u6-isl are in close proximity in low mg 2+ and separated in high mg 2+ . the 2 nd step corresponds to the formation of the highly conserved helix ib. we now examine the role of the highly conserved bases in the folding dynamics of the u2-u6 complex. the data show that u80 and the acagag loop play an important role in stabilizing the interaction between helix iii and the u6-isl. we hypothesize that u80 flips out of the u6-isl and interacts with the acagag loop to bring them in close proximity. our results raise the interesting possibility that this interaction plays an important role in bringing the 5' splice site and the branched a into close proximity of u80, which may be critical for catalysis. a structure-based approach for targeting hiv-1 genomic rna dimerization initiation site s. freisz, s. bernacchi, p. dumas, e. ennifar ibmc -cnrs, strasbourg, france all retroviral genomes consist in two homologous single stranded rnas. hiv-1 dimerization initiation site (dis) is a conserved hairpin in the 5' non-coding region of the genomic rna and essential for viral infectivity. the dis initiates genome dimerization by forming a kissing-loop complex, further stabilized into an extended duplex upon interaction with the ncp7 nucleocapsid protein. x-ray structures of the dis kissing-loop and extended duplex revealed similarities with the bacterial 16s ribosomal rna a-site, which is the target of aminoglycoside antibiotics. as a result, aminoglycosides also bind the hiv-1 dis as shown by our x-ray structures of the dis kissingloop bound to aminoglycosides. using fluorescence, uvspectroscopy and microcalorimetry, we further characterized hiv-1 dis/aminoglycosides interactions. we found that the affinity of aminoglycosides for the dis was higher than for their natural target, the 16s a site. they strongly stabilize the dis kissing-loop, blocking its conversion into the duplex form. finally, we also solved x-ray structures of the dis duplex bound to aminoglycosides, revealing an important conformational change following drug binding. these structures show that it is possible to target the hiv-1 dis dimer before and after the ncp7-assisted rna maturation with the same molecule. altogether, our studies create the basis for a rationally driven design of novel potential drugs targeting the hiv-1 genome. the core protein of hepatitis c virus is a multifunctional protein of 179 aa, consisting of a hydrophilic n-terminal domain with three basic domains (bd1-bd3) responsible for the interactions with rna and a hydrophobic c-terminal domain. the n-terminal domain exhibits nucleic acid chaperone properties similar to those of the nucleocapsid protein from hiv. here, we characterized the mechanism of the chaperone properties of a peptide e corresponding to the bd2 and bd3 clusters of the n-terminal domain. to this end, we monitored the promotion by this peptide of the annealing of dtar, the dna analogue of the transactivation response element to its complementary sequence, ctar dna, taken as models. the annealing involves two second-order kinetic components that are activated by at least three orders of magnitude by peptide e. this activation was correlated with the ability of peptide e to destabilize the lower half of dtar stem. using, ctar and dtar mutants, the two kinetic components were assigned to two pathways which are connected with the fast annealing of the terminal bases of ctar to dtar and slow extended duplex formation, limited kinetically by the nucleation of central segments of ctar and dtar stems. structure and conformational dynamics of a unique dead box helicase m. rudolph 1 , m. linden 2 , r. hartmann 3 , d. klostermeier 2 1 hoffmann-la roche, basel, switzerland, 2 biozentrum, univ. of basel, switzerland, 3 univ. of marburg, germany dead box helicases couple atp hydrolysis to rna structural rearrangements. t. thermophilus hera consists of a helicase core and a c-terminal extension (cte) with a putative rnase p motif. crystal structures show that the cte is bipartite, forming a highly flexible dimerization motif with a novel fold and an additional rna-binding module. we provide a first glimpse on the orientation of an rna-binding domain outside the helicase core. in a structure-based model for the complete hera dimer, the rna-binding sites of the helicase cores face each other, allowing for inter-subunit communication. the plasticity of the dimerization motif allows for drastic changes in the juxtaposition of the helicase cores within the dimer. in single molecule fret experiments we identified fragments of the 23s rrna and rnase p rna as substrates for hera. both substrates switch the hera core to the closed conformation and stimulate the intrinsic atpase activity. rna binding is mediated by the cte, but does not require the putative rnase p motif. atp-dependent unwinding of a short helix in 23s rrna suggests a specific role for hera in ribosome assembly, in analogy to the e. coli and b. subtilis helicases dbpa and yxin. in addition, the specificity of hera for rnase p rna may be required for rnase p rna folding or rnase p assembly. simultaneous action of two hera subunits on the same rna molecule may be important for efficient rna remodeling in vivo. structural basis for the encapsidation process of turnip yellow mosaic virus m. petersen 1 , j. hansen 1 , s. s. wijmenga 2 1 nucleic acid center, department of physics and chemistry, university of southern denmark, odense, denmark, 2 physical chemistry/biophysical chemistry, radboud university, nijmegen, the netherlands formation of hairpins with internal loops with c c and c a mismatches in the 5' untranslated region is a common characteristic among the plant viruses belonging to the tymovirus genus. turnip yellow mosaic virus possesses two such hairpins, hp1 and hp2. hp1, and in particular the presence of the c c and c a mismatches in its internal loop, is important for initiation of the encapsidation of the viral genome. the encapsidation occurs under acidic conditions at the neck of invaginations of the chloroplast membrane. we have now determined the 3d structures of revertants involved in the evolutionary pathway using nmr spectroscopy. these structures reveal how the grooves in the hairpin become increasingly positively charged in successive generations of evolution. the similarity between the ccca mutant and the wild-type hairpin (hp1) is striking and explains why this mutant gives rise to a viable virus. in addition, a characteristic tilt of the backbone is observed upon occurrence of a protonated c c base pair. both the positively charged major groove and the kink in the rna backbone appear to be crucial for interactions with the viral capsid. at neutral ph, the structure of hp1 resembles the watson-crick base paired mutant which explains why encapsidation only occurs under acidic conditions. a. percot 1 , j. vergne 2 , m.-c. maurel 2 , s. lecomte 3 1 ladir, umr7075, 94320 thiais, france, 2 lbeam, upmc, 75005 paris, france, 3 cbmn, umr5248, 33607 pessac, france the existence of "rna world" as an early step in the history of life increases the interest for the characterization of these biomolecules. the studied hairpin ribozyme is a self-cleaving/ligating motif found in the minus strand of the satellite rna associated with tobacco ringspot virus. surface-enhanced raman spectroscopy (sers) was successfully used to detect sub-picomole quantities of nucleic acids. sers takes advantage of the strongly increased raman signals generated by local field enhancement near metallic (typically au and ag) nanostructures. sers spectra of dna or rna are strongly dominated by stockes modes of adenine. through an interaction of adenyl residues with silver colloid, adenyl raman signal is 10 6 times increased compared to raman scattering. in controlled conditions, sers signal is proportional to the amount of free residues adsorbed on the metal surface. upon rna cleavage, residues are unpaired and free to interact with metal. in the present study, we proposed to follow the cleavage reaction of hairpin ribozyme (hpr85) using the sers signal of the liberate adenyl residues. as the sers signal is proportional to the adenyl residues, reactivity of hr85 was monitored by measuring the raman intensity of the fragment liberated during the cleavage of hairpin. the results obtained were compared with the electrophoresis method performed on the same sample and similar results were obtained. -rna world eur biophys j (2009) the development of arrays for biomolecular recognition for a broad range of applications in biomedical diagnostics is receiving a constantly increasing attention. the design of efficient protein biochips, however, requires the optimization of protein immobilization protocols for improving the device sensitivity. moreover, innovative platforms for in-vitro detection in highly diluted, small volume samples need to be developed, for which standard micro-fabrication techniques are not suitable. therefore, the development of nano-scale platforms for protein and antibody detection is essential. we report here on a novel approach for the fabrication of multiple protein nanosensors using atomic force microscopy based nanografting and dna-directed immobilization (ddi), which takes advantage of the specific watson-crick hybridization of oligonucleotide-modified proteins to surfacebound complementary oligomers. using nanografting, single-stranded dna nano-structures with well defined local environments were fabricated on a flat surface. dna-protein conjugates were then anchored on the engineered binding sites by ddi in a single chemical operation and detected by the corresponding topographic height increase of the relevant patches. immunological assay were used to demonstrate the biochemical functionality of the immobilized proteins, proving the specificity of biomolecular recognition of our nanodevices, in the micro-molar to pico-molar concentration range. dna accessible surface area and indirect protein-dna recognition: study by bioinformatical approach o. p. boryskina 1 , m. y. tkachenko 1 , a. v. shestopalova 1 , m. y. tolstorukov 2 1 institute of radiophysics and electronics nas of ukraine, 2 harvard-partners center for genetics and genomics, usa revealing the mechanisms of protein-dna recognition is essential for understanding the regulation of many cellular processes. there is growing evidence that recognition through sequence-specific contacts (direct readout) can be enhanced by recognition via dna sequence-dependent deformability (indirect readout). the role of changes in dna accessible surface area (asa) in distorted dna configurations in complexes with proteins is not fully understood yet, even though such changes and related changes in polarity of dna surface are among key factors of indirect readout. to fill this gap we have developed a publicly-available internet database of protein-dna complexes, which integrates the data on dna conformational parameters with information on asa of dna atoms in the minor and major grooves, protna-asa. the database has been used to analyze the effect of changes in dna backbone configuration on asa of dna atoms in major and minor grooves. we observe that sugar puckering and conformation of torsion angle γ affect the accessibility of dna atoms in both grooves to a noticeable extent. we also report sequence specific preferences of the nucleotides for structural domains of γ. these results can shed new light on the mechanisms of indirect protein-dna recognition. a. arakelyan biology dpt., yerevan state university, yerevan, armenia the influence of ligand, irreversibly binding with dna, on the isotherm of adsorption of ligand binding with dna reversibly has been modeled theoretically. the isotherm of adsorption of etbr on dna in presence of cis-ddpt has been considered at low concentrations of cis-ddpt and etbr.. the isotherm of adsorption of etbr on dna has linear form in scatchard coordinates at low degrees of occupation. the comparison with experimental isotherm permits to estimate the parameters of etbr binding with dna. with taking into account the pseudo-ring structures formation with partially molten regions we consider two types of binding regions, linear and ring at low degrees of occupation. the isotherm of adsorption of etbr on dna and also variation of the number of bounded etbr molecules are calculated with taking into account these two types of binding regions. it was shown that at low concentrations cis-ddp, bounding with dna, changes the isotherm of ligands adsorption. the linear in scathcard coordinates isotherm of adsorption transforms into non-linear isotherm. the degree of transformation depends on the fraction of dna in the ring regions, on the ratio between number of etbr binding cites in the ring and linear regions, and also on the binding constants for these regions. it was shown that low concentrations of cis-ddp affect the dependence of dispersion on the concentration of ligands, changing both the magnitude of maximum and its position. tunable nanoconfinement structures for dna manipulation e. angeli, l. repetto, g. firpo, c. boragno, u. valbusa nanomed labs, advanced biotechnology center and physics department, university of genoa, italy nanostructures, such as nanochannels [1] or nanoslits [2] , have been successfully used to confine and stretch dna molecules, offering interesting opportunities of investigation on conformational changes induced by confinement, physical and biological properties, etc. the integration of these nanostructures on lab-on-chip systems has shown their great potential for applications such as dna sieving or single molecule manipulation [3] , [4] . arrays of nanochannels fabricated, using a focused ion beam (fib) on a silicon master, are replicated using elastomeric materials, such as poly(dimethylsiloxane) (pdms), and soft-lithography techniques. the cross-section of these flexible polymeric nanoconfinement structures can be reversibly and dynamically tuned, in order to vary biomolecules transport characteristics and confinement conditions of trapped dna molecules. moreover, these nanochannels, with tunable cross-section, are used to study and exploit the sieving mechanism of "entropic recoil" [5] for the separation of long dna chains. [1] mannion jt, et al., (2006 ) biophys. j., 90, 12:4538-4545. [2] jo k, et al., (2007 ) pnas, 104, 8: 2673 -2678 . [3] fu j, et al., (2007 ) nat. nanotechnol., 2: 121-128 [4] huh d, et al., (2007 nat. mater., 6: 424-428. the nucleocapsid protein ncp7 of hiv-1 is characterized by two conserved zinc fingers and plays crucial roles in the virus, through its binding to nucleic acids. ncp7 is required for efficient proviral dna synthesis, by promoting the initiation of reverse transcription and the two obligatory strand transfers. using fluorescence techniques as well as fcs and nmr, we investigated the chaperone properties of ncp7 on the primer binding sites (pbs) and transactivation response elements (tar) sequences involved in the two obligatory strand transfers. we showed that ncp7 binds mainly to the (-)pbs loop, which results in an extension of the loop and a destabilization of the upper base pair of the stem. these structural changes chaperone a kissing complex with the complementary (+)pbs loop and its further conversion into an extended duplex. in contrast, the ncp7-promoted annealing of ctar-tar results from the destabilization of the bottom of ctar stem, which favors the invasion of the tar stem. by developing new fluorescence methodologies to site-specifically characterize these interactions, we further showed that ncp7 slows down the ps to ns conformational fluctuations of its nucleic acid targets and freezes the local mobility of the bases contacted by the zinc fingers. exploring dna orientation in flow e. l. gilroy, m. r. hicks, a. rodger department of chemistry, university of warwick, uk dna is one of the most important biomolecules. in order to undertake its biological role the dna needs to fold and unfold for which its structure and flexibility are crucially important. we have studied the characteristics of dna in flow to probe its structure. flow aligned linear dichroism (ld) is a technique that uses light polarised parallel and perpendicular to an orientated sample. it can be used to measure how aligned a sample is, and the orientation of any interacting molecules. to create this alignment, the sample is placed between two concentric cylinders where one is spun to create a shear flow. the longer and more rigid the molecule is the better the orientation and the signal. as the sample is in flow there are other factors than need to be considered when using ld. these include viscosity and temperature. there are many methods to measure the viscosity of a sample of dna at varying temperatures. these include viscometer, rheometer and dynamic light scattering. the effects temperature has on viscosity and the sample itself need also to be considered. the findings of all these studies have been reported and show the significance that viscosity and temperature have on dna ld measurements. possible applications of using ld to study dna have also been discussed, showing the importance of the use of ld and in the results shown. a.-m. florescu, m. joyeux laboratoire de spectrométrie physique, université joseph fourier grenoble 1, france we present a dynamical model for simulating non-specific dna protein interactions, which is based on the "beadspring" model for dna with elastic, bending and debye-hückel electrostatic interactions, and where the protein interacts with the dna chain through electrostatic and excluded-volume forces. we study the properties of this model using a brownian dynamics algorithm that takes hydrodynamic interactions into account and obtain results that partially agree with experiments and predictions of kinetic models. for example, we show that the protein samples dna by a combination of three-dimensional diffusion in the solvent and one-dimensional sliding along the dna chain. this model evidences the presence, in a certain range of values of the effective protein charge, of facilitated diffusion, i.e. a combination of the two types of diffusion that leads to faster than 3-dimensional diffusion sampling of dna. moreover, the analysis of single sliding events shows that the number of base pairs visited during sliding is comparable to those deduced from single molecule experiments. in contrast to kinetic models, which predict an increase of the number of different base pairs visited by the protein proportional to the square root of time, our model however suggests that this number increases linearly with time until it reaches a value that is close to the total number of dna base pairs in the cell (published in j. chem. phys. 130, 015103 (2009)). use of md simulation to identify the critical radiation-induced lesions of a dna binding protein n. chalabi, s. goffinont, n. garnier, d. genest, 45071 orléans cedex 2, france a key step in the regulation of gene expression, dna structuring and dna repair is the binding of some proteins to specific dna sequences. we have shown previously that ionizing radiation destabilizes such dna-protein complexes mainly through damage to the protein. for the typical lactose operator -repressor system we have shown by fluorescence measurement and mass spectrometry that upon irradiation, all the four tyrosine residues of the dna binding domain (called headpiece) are oxidized into 3,4dihydroxyphenylalanine (dopa). a circular dichroism study revealed a global conformational change and the destabilization of the headpiece. to decide which lesion is critical for the induction of these effects, a molecular dynamics simulation study was undertaken in parallel with a site-directed mutagenesis one. each tyrosine residue of the headpiece was replaced by another amino-acid that mimics the damaged tyrosine. the most common amino-acid used in site-directed mutagenesis being alanine, we have replaced one tyrosine (7, 12, 17 or 47) in the nmr-based structure of the headpiece from pdb databank (1lqc) by an alanine. the conformational stability of each tyr→ala mutant has been studied by molecular dynamics simulation (md) and compared to that of the native sequence and of the different tyr→dopa mutants. the mammalian high mobility group protein at-hook 2 (hmga2) is a nuclear protein associated with mensenchymal cell development and differentiation. disruption of its normal expression pattern is directly linked to oncogenesis and obesity. our laboratory has utilized a variety of biochemical and biophysical methods to investigate the molecular mechanisms of hmga2 recognizing at-rich dna. using a pcr-based selex procedure, we discovered that hmga2 is a sequence-specific dna-binding protein and recognizes the following two sequences: 5'-atattcgcgawwatt-3' and 5'atattgcgcawwatt-3', where w is a or t. using a double-label emsa assay, we found that hmga2 binds to at-rich dna as a monomer. using isothermal-titrationcalorimetry, we demonstrated that hmga2 binds to at-rich dna with very high binding affinity whereby the binding of hmga2 to a-tracts is entropy-driven and to alternate at sequences is enthalpy-driven. this is a typical example of enthalpy-entropy compensation in which the hydration difference between hmga2-dna complexes is a main reason for the compensation. interestingly, the binding of hmga2 to different at-rich sequences is accompanied with a large negative heat capacity change indicating an important role of solvent displacement and charge-charge interaction in the linked folding/binding processes. partition of gibb´s free energy of drug-dna complexation we report a computation methodology, which leads to the ability to partition the gibb's free energy for the complexation reaction of aromatic drug molecules with dna. using this approach it is now possible to calculate the absolute values of the energy contributions of various physical factors to the dna binding process, whose summation gives a value that is reasonably close to the experimentallymeasured gibb's free energy of binding. application of the methodology to binding of various aromatic drugs with dna provides an answer to the question 'what forces are the main contributors to the stabilization of aromatic ligand-dna complexes' ? we report the effects of 22 khz ultrasound irradiation of double-stranded dna solutions under conditions of transient cavitation. a new method was developed for studying these effects which is based on combination of two procedures: ultrasound irradiation of the solutions of double-stranded dna fragments and subsequent analysis of the high resolution denaturing gel electrophoresis data. statistical treatment of the data on the mobility of 3'-end-labelled restriction fragments with known sequence allowed us to discover the phenomenon of sequence specific ultrasonic cleavage of dna. our analysis results in the following conclusions: 1) all steps with 5'-ward cytosine [5'-d(cpn)-3'] have significantly higher cleavage rates than others; the intensity of cleavage diminishes in the order cpg > cpa ≈ cpt > cpc; 2) the cleavage rates of all 16 steps depend on the type of flanking base pairs; 3) the cleavage rates of the complementary base pair steps are not identical. thus, subtle sequence specific conformational and physical-chemical variations modulate the reaction of sugar-phosphate single bonds on the ultrasound exposure. a theory of the mechanisms on the simultaneous binding of two aromatic drugs to dna it has long been recognized that certain combinations of dna-binding aromatic drugs, x +y, lead to synergistic biological effects. considering x as a basic ligand and y as an added ligand, the change of the integral biological response of x in the presence of y has been interpreted in terms of two mechanisms: the interceptor and protector action of y on x. this mechanisms have been characterized by two criteria, r d and a d , reflecting the removal of x from dna by y (biophys. chem., 2008, vol.132, pages 148-158 ) . in this work we develop the theory of the interceptor-protector action of a mixture of two biologically-active dna-binding aromatic drugs. the theory is based on solution of a general system of mass balance equations in the three-component system x -y -dna with respect to the two factors, r d and a d . the outcome is a set of expressions enabling estimation of the change in biological response of x on addition of y as a function of equilibrium parameters under different restrictions. the results are in good agreement with known in vitro data on caffeine+antibiotic action in leukemia cell lines. the influence of mn 2+ ions on the structure of natural calf thymus dna was studied by raman spectroscopy. measurements were done at room temperature and ph 6.2 ± 0.2, in the presence of the physiological concentration of 150 mm na + ions, and in the presence of mn 2+ concentrations that varied between 0 and 600 mm. no condensation of dna was observed. dna backbone conformational changes were not detected in the whole concentration range of mn 2+ ions as judging from the raman spectra. no evidence for dna melting was identified. binding of manganese(ii) ions to the charged phosphate groups of dna, stabilizing the double helical structure, is indicated in the spectra. as judged from the marker band of dc near 785 cm −1 , altered nucleoside conformations in dc residues are supposed to occur, in the mn 2+ concentration range of 400-600 mm. binding of divalent ions to n7 of guanine and, possibly, in a lesser extent to adenine was observed as judging from the raman marker bands at 1336, 1376, 1490 and 1578 cm −1 . toward rapid dna sequencing -ab initio study of nucleotide sandwiched between au(111) plates c. morari, d. bogdan, i. turcu national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania recently a new technique for dna sequencing based on measurement of transversal conductive properties of a single strained dna molecule has been proposed. such a method would allow single-base resolution by measuring the electrical current perpendicular to the dna backbone. until now, it is still not clear if the electrical signals obtained for the four nucleotide can be clearly distinguished by a hypothetical experimental setup. several factors -like the influence of the pentose group or the presence of water -may influence quite strongly the electrical signatures of the four bases. therefore, in order to obtain a working device, the understanding and detailed description of the conduction mechanisms through dna bases connected to a metallic electrode is essential. the goal of theoretical studies in this field is to describe the electric signatures of dna bases from a theoretical point of view. our study is focused on the detailed description of the electronic structure of dna's base pairs "squeezed" between two au plates. while such a geometrical model closely mimic the sequencing devices proposed in the literature, it allows us to compute meaningful physical properties such as density of states, charge transfer and orbital localizationby using "ab initio" methods. the results allow us to give qualitative prediction over the potential use of such a device in the dna's sequencing technology. multinuclear platinum complexes represent a new class of anticancer agents, distinct in dna binding and antitumor activity from their mononuclear counterparts. bbr3464 as a first representative of this class has undergone phase ii clinical trials for treatment of ovarian and lung cancers. the structure of this trinuclear pt drug consists of two trans-ptcl(nh 3 ) 2 units bridged by a transthe main lesions formed by multinuclear pt complexes in dna are long-range intra-and interstrand cross-links (cls) bridging two guanines separated by up to four base pairs. since interstrand cls can prevent dna strand separation interfering with critical cellular events they represent a serious obstacle in cell survival. in order to contribute to understanding the biological effects of dna interstrand cls of bbr3464, we analyzed the effect of the single, site-specific 1,4-interstrand cl formed by this metallodrug between two guanine bases on opposite strands in the 3'-3' and 5'-5' direction on the thermal stability and energetics of short dna duplexes. the results demonstrate that 1,4-interstrand cls of bbr3464 in both 3'-3' and 5'-5' directions exist as two distinct conformers that are not interconvertible and affect thermodynamic stability of the dna differently. side-by-side and end-to-end attraction of doublestranded dna c. maffeo 1 , b. luan 2 , a. aksimentiev 1 1 university of illinois at urbana-champaign, urbana, usa, 2 ibm watson research center, yorktown heights, usa genomic dna is densely packed inside cell nuclei and viral capsids. such close packing suggests that electrostatic repulsion between negatively charged dna in the condensed states is balanced by counterion-induced attraction. several theoretical models have been proposed to explain dna attraction, however, specific microscopic mechanisms are not known. here, we report all-atom molecular dynamics simulations of the effective force between double-stranded dna in side-by-side and end-to-end orientations. in the side-by-side orientation, the dna molecules were found to form a bond state in the presence of magnesium ions. in the bond state, the dna molecules contact each other via their negatively charged phosphate groups, bridged by magnesium ions. the maximum attractive force in the side-by-side orientation is about 40 pn per dna turn. in the end-to-end orientation, a strong ( > 60 pn) attractive force was observed at short (< 0.8 nm) end-to-end distances regardless of the electrolyte concentration. the presence of a phosphate group at the 5'ends of the fragments was found to direct dna end-to-end self-assembly and produce bound states resembling a continuous dna molecule. the computed potentials of the mean force suggest that the end-to-end attraction, rather than being mediated by counterions, is likely caused by hydrophobic and van der waals interactions between terminal nucleobases of the fragments. doxorubicin (dox) is an anticancer antibiotics with a four-membered ring system containing an anthraquinone chromophore, and an aminoglycoside. it has good anticancer activity against a wide spectrum of tumors and is one of the most extensively used antitumor chemotherapeutic compounds currently in clinical use. interestingly, conversion of dox to 2pyrrolinodoxorubicin analog (p-dox) exhibits 500-1000 times higher toxic effects in human breast cancer cell line (nagy, a. et al., pnas, 93 (1996 ) 2464 . molecular mechanisms responsible for this enormously enhanced cytotoxicity have not been entirely clarified. there is good evidence that a key component of the mechanism of action of dox is its intercalation into dna and the formation of dox-dna adducts. therefore, we have examined in detail, using the methods of molecular biophysics, dna interactions of p-dox in cell-free media and compared these results with the same studies focused on the parental dox. we find distinct differences between dna interactions of dox and p-dox and suggest that these differences are responsible at least in part for different biological effects of these two anticancer drugs. design of a microfluidic devices for the detection of oligonucléotides by sers designing fast and efficient analytical tools allowing the detection of free dna or rna at very low concentration within small volumes, without specific molecular labeling, remains a major issue of significance to perform diagnostic or to detect pathogen agents. our strategy is to use surface-enhanced raman scattering (sers) to probe selectively the spectral signature of each base in polynucleotides. sers takes advantage of the strongly increased raman signals of species when adsorbed on adapted silver colloids. we already demonstrated that adenyl raman signal of pa in presence of silver colloids is 10 6 times enhanced compared to bulk signal. we plan to use nanoliter droplets of uniformed size that form spontaneously in microchannels when two immiscible fluid streams merge. these tiny droplets are almost ideal reactors as they create homogeneous control condition. we will design an optimized channels network platform that result in droplets production hosting both nucleotides and silver colloids: internal fluids recirculation provide fast and efficient mixing, favoring base adsorption on silver nanoparticles. sers will be used to determine the chemical composition of the droplets. nicks represent the most common damage in dna which occurs naturally in living cells. structural properties of nicked dna fragments have been an object of numerous studies due to its special role in reparation processes. here we report experimental results covering ultrasound irradiation of nicked dna solutions. several single-stranded nicks were produced into one strand of dsdna fragments by the nicking enzyme bst9i. we have quantitatively estimated the ultrasonic cleavage rates in nicked dna fragments with known sequences using the polyacrylamide gel electrophoresis. computer analysis of the cleavage pattern in the 3'-end labeled and primarily intact strand reveal cleavage enhancement in the regions of about 10 b. p. up and down the nicks which were initially produced into complementary strand. the intensity of cleavage near the nicks is (in average) about 20 times higher than cleavage in the same sites of the intact dsdna fragments. at the same time, the cleavage rates in positions beyond the regions of the nick markedly grow weak even comparing to the sequence-specific cleavage of intact double-stranded dna fragments. thus, the presence of the nick serves as an expressive structural indignation, which exceeds modulation of the structure caused by the base-pair sequence and is capable of absorbing mechanical stresses applied to the nearby sites of the molecule. comparing the native and an irradiated lactose repressor-operateur complex by md simulation g. naudin, n. garnier, d. genest, rue charles sadron, 45071 orléans cedex 02, france the function of the e. coli lactose operon requires the binding of a protein, the tetrameric repressor, to a specific dna sequence, the operator. the formation of this complex involves the interaction of at least one protein dimer with the operator sequence. this occurs via the dna-binding domains (called headpieces) of the two constitutive monomers. we have previously shown that upon irradiation with gamma rays the complex is destabilised mainly because the repressor losses its dna binding ability. radiation-induced lesions were identified that may be responsible for this deleterious effect: all tyrosine residues of the headpieces are oxidized into 3,4-dihydroxyphenylalanine (dopa). in order to unravel the mechanisms leading to the observed destabilization of the operator-repressor complex, we compare by md simulations two complexes: 1-the native complex formed by a dimer of two headpieces and a fragment of dna with the operator sequence and 2-the damaged complex in which all tyrosines are replaced by dopa. analysis of these trajectories shows a loss of stability and binding energy as well as changes in the structure of the damaged complex with respect to the native one. by comparing precisely the evolution of the two complexes we can explain how the oxidation of the tyrosine residues of the headpieces into dopa may trigger the destabilization of the complex. local conformation of confined dna studied using emission polarization anisotropy in nanochannels with dimensions smaller than the dna radius of gyration, dna will extend along the channel. we investigate long dna confined in nanochannels with dimensions down to 50*50 nm, using fluorescence microscopy. studies of the statics and dynamics of the dna extension or position in such confinements as a function of e.g. dna contour length, degree and shape of confinement as well as ionic strength has yielded new insight into the physical properties of dna with relevance for applications in genomics and fundamental understanding of dna packaging in e.g. viruses. our work extends the field by not only studying the location of the emitting dyes along a confined dna molecule but also monitoring the polarization of the emission. we use intercalating dyes whose emission is polarized perpendicular to the dna extension axis, and by measuring the emission polarized parallel and perpendicular to the extension axis of the stretched dna, information on the local spatial distribution of the dna backbone can be obtained. we will discuss results in shallow (60 nm) and deep (180 nm) channels and describe how the technique can be used to investigate non uniform stretching of a single dna molecule. raman spectroscopy of dna modified by new antitumor nonclassical platinum complexes o. vrana, v. kohoutkova, v. brabec institute of biophysics as cr, královopolská 135, cz-61265 brno, czech republic platinum anticancer agents (cisplatin, carboplatin, oxaliplatin) are in widespread clinical use especially against testicular, ovarian and head and neck carcinomas. there is a large body of experimental evidence that dna is the critical target for the cytostatic activity of cisplatin. platinum complexes form several types of adducts, which occur in dna with a different frequency and differently distort the conformation of dna. clinically ineffective trans isomer of cisplatin (transplatin) also covalently binds to dna bases. the trans geometry in dichloridoplatinum(ii) complexes was activated by various ways. the replacement of at least one amine ligand by planar amine ligand represents example of such activation. raman spectroscopy is powerful technique for examining both structural and thermodynamic properties of nucleic acids in solution. interactions of cis-and trans-pt(ii) complexes having nonplanar heterocyclic amine ligand such as piperidine, piperazine and 4-picoline with dna have been investigated by laser raman spectroscopy. raman difference spectra reveal that the pt(ii) complexes induce great structural changes in b-dna and indicate disordering of b-dna backbone, reduction in base stacking and base pairing and specific metal interaction with acceptor sites on purine residues. acknowledgement: this work was supported by grant as cr, iqs500040581. a. v. vargiu 1 , a. magistrato 2 , p. carloni 3 , p. ruggerone 1 1 cnr-infm-slacs and physics dept., university of cagliari, cagliari, italy, 2 cnr-infm-democritos and sissa/isas, trieste, italy, 3 iit and sissa/isas, trieste, italy the minor groove of dna is the target of several anticancer agents, which interfere with replication and translocation processes, leading to cell death. the molecular recognition event is a key step to achieve detailed knowledge of the interactions behind selectivity and affinity of a ligand towards a particular nucleic acids sequence. recognition is a multiroute process which can involve many steps before the formation of the most stable adduct. in particular, many studies have pointed out the importance of events like sliding along the groove and dissociation (which is a relevant step in the translocation among different sequences) for the affinity and the specificity of minor groove binders. despite this, no studies on the dynamics of this event were reported in the literature. in this contribution i present our recent work on the subject. umbrella sampling and metadynamics were used in the framework of classical md to characterize mechanisms andfree energy profiles of molecular recognition routes by the antitumoral agents anthramycin, duocarmycin and distamycin. our results are in very good agreement with the available experimental data, and provide insights on the influence of factors like size, charge and flexibility on the molecular recognition process. amplification of oligonucleotide sequence recognition using bioresponsive hydrogels s. tierney, b. t. stokke biophysics and medical technology, dept. of physics, the norwegian university of science and technology, ntnu, no-7491 trondheim, norway we describe development and characterization of oligonucleotide functionalized hydrogels for amplifying the molecular recognition signal occurring on hybridization between dioligonucleotides. the 50 µm radius hemispherical hydrogels were integrated on a high resolution interferometric fiberoptic readout platform supporting determination changes in the optical length of the hydrogel with 2 nanometer resolution. the hydrogels were designed with hybridized dioligonucleotides grafted to the polymer network as network junctions in addition to the covalent crosslinks or oligonucleotides grafted to the network chains. the probe oligonucleotide destabilizing the junction point by displacement hybridization yielded an optical signal about five times as large as for binding within the hydrogel design with a comblike grafted oligonucleotide. the optical signal was found to be dependent on the concentration of the probe, the sequence and matching length between the probe and sensing oligonucleotide. concentration sensitivity applied as specific label-free detection of oligonucleotide is estimated to be in the nanomolar region. the current design support detection in excess of 1x10 12 sequences. amplification of the molecular recognition employing the developed oligonucleotide imprinted hydrogel for label-free sensing of probe oligonucleotide sequences or taking advantage of the oligonucleotide sequence designed as aptamers for determination of other types of molecules are discussed. tracing t-cells by paramagentic nanoparticles in the brain of the rat model of als d. bataveljic 1 , g. vanhoute 2 , g. bacic 3 , p. andjus 1 1 inst. for physiology and biochemistry, univ. of belgrade, serbia, 2 bio imaging lab, univ. of antwerpen, belgium, 3 school of physical chemistry, univ. of belgrade, serbia amyotrophic lateral sclerosis (als) is a devastating neurological disorder affecting upper and lower motoneurons. since immune disbalance is known to be an important manifestation of the disease we were particularly interested in following the labeled immune cells in the familial als rat model, hsod-1 g93a . a t2-or t1-weighted mri protocol was used with a mini surface coil placed over the skull of the anesthetized animal in a 1.5 t wide bore magnet. in order to compare this approach to standard high field small animal imaging a 7.0 t mri system was also used. there was a congruence of images of lesions in the brainstem at the two field strengths. it was confirmed with gd-dtpa contrast that the blood brain barrier (bbb) is compromised at the interbrain level. in order to study immune cell infiltration rats were i.v. injected with magnetically labeled antibodies against helper cd4+ or cytolytic cd8+ killer t cells. by combined t1, t2 and t2* weighted imaging cd4+ lymphocyte infiltration was observed in the brainstem-midbrain region while the cd8+ cells were more confined to the brainstem region. comparison of mri of labelled cd4+ vs. cd8+ lymphocytes reveals the relevant cellular inflammatory mechanism in als. the appearance of the mri signal from the latter t cell type points to the mechanism of bbb disruption as suggested from a recent study on the role of cd8+ t cells in a model of multiple sclerosis. emodin uptake study in u-87mg cells using optically trapped surface-enhanced raman scattering probes s. balint 1 , s. rao 1 , p. miskovsky 2 , d. petrov 1 1 icfo -the institute of photonic sciences, barcelona, spain, 2 department of biophysics, university of p.j.šafárik, košice, slovakia emodin (1,3,8-trihydroxy-6-methylantraquinone) is a photosensitizing pigment present in plants of herbal laxatives. emodin inhibits nuclear transcription factor-κb activation and induces free radical production in human mononuclear cells resulting in its antiviral and anti-cancer activity. the uptake and distribution of emodin inside the u-87 mg cell line is studied by combining optical tweezers and surfaceenhanced raman spectroscopy (sers). sers greatly enhances the spectrum of an otherwise weakly scattering material which is achieved by attaching nano-sized silver colloids to micron-sized dielectric beads. the distribution of emodin in the cell is studied by simultaneously trapping and exciting the sers bead and scanning it across the membrane while recording the emitted light. secondly, the beads are statically placed inside the cell and excited at certain intervals in order to track the migration of emodin through the membrane. the results give new insight in to the metabolic pathways of emodin and demonstrate a new imaging and detection technique that is fast and less invasive than current standards. acknowledgement: miin fis2008-00114 (spain), fundació cellex barcelona, nadacia spp (slovakia), apvv-0449-07 (slovakia) amphotericin b (amb) is a polyene antibiotic that has been widely used for treatment of systemic fungal infections. the main mechanism of biological mode of action of amb is considered to be associated with formation of ionic membrane pores or channels in the lipid membranes. the aim of this work was to study the influence of the k + and na + ions on the aggregation process of amb in aqueous medium. the analysis of electronic absorption and fluorescence spectra of amb shows that the increasing k + concentration have influence on the level of aggregation of the drug much more than the same amount of na + ions. this effect is especially noticed at neutral ph values. the rls technique was used to study aggregation of amb in solution, in the environment of the k + and na + ions. the application of this technique makes it possible to study the electronically coupled chromophores, especially molecular aggregates. the results of the atr-ftir and raman spectroscopic studies also support this conclusion. these results provide a better understanding of the interaction between k + and na + ions and antibiotic which has not been previously considered to be significant for biological action of amb. g-quadruplexes: combining theory with experimental spectroscopic methods guanine-rich oligonucleotides can form unique tetrameric structures with four coplanar guanine bases, known as gquadruplex motifs. the g-quadruplexes have been found in vivo in the terminal parts of telomeres and other genomic regions. ligands, specifically binding to the g-quadruplex regions inhibit telomerase activity and thus can play an important role in the cancer therapy. most recently, the potential use of these structures has been tested in biosensors and nanotechnology industry. in the present work we use the infrared spectroscopy, including the relatively novel technique of the vibrational circular dichroism (vcd), in a combination with molecular dynamics and quantum chemistry computational methods to investigate the structure and spectroscopic response of the quadruplexes formed by the d(g) 8 and selfassociated dgmp. we obtained a good agreement between the computed and experimental spectra, confirming that the proposed geometrical models are realistic. the vcd technique appears especially convenient for the studies as it can detect the liquid-crystalline phases of the g-quadruplexes by an anomalous enhancement of the signal. j. bogdanovic pristov, a. mitrovic, k. radotic, i. spasojevic institute for multidisciplinary research, belgrade, serbia fructose, due to its high antioxidative capacity, represents a significant component of non-enzymatic defense of some plants against cold-provoked oxidative stress. in the present study, we have investigated role of fructose in seasonal adaptation of picea omorika (pančić) purkinye to cold. this endemic coniferous species is exposed to subfreezing temperatures that range from -10 to -30 • c during the autumn/winter and high temperatures exceeding 30 • c during the summer. characteristic epr signal of free or weakly bound mn 2+ was used as an indicator of oxidative status of needles, since coldrelated oxidative damage leads to mn 2+ release from photosystem ii. it was observed that prooxidative conditions developed in the autumn, at the beginning of cold season, which corresponded to significant increase of fructose level. total sod, as well as mnsod activity also rose significantly higher in the autumn. observed activation of antioxidative system (non-enzymatic and enzymatic) led to adaptation of needles to cold, as oxidative status during winter was decreased and similar to the status of needles in cold-free seasons. calyx of held: sted nanoscopy of a glutamatergic terminal p. bingen 1 , t. m. staudt 2 , c. kempf 3 , h. horstmann 3 , j. engelhardt 1 , t. kuner 3 , s. w. hell 2 1 german cancer research center / bioquant, 69120 heidelberg, germany, 2 max planck institute for biophysical chemistry, 37077 göttingen, germany, 3 institute for anatomy and cell biology, university of heidelberg, 69120 heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of <50 nm inside 3 µm thick tissue sections. three-dimensional shim and 2pem to study collagen arrangement and crimping pattern p. bianchini 1 , m. franchi 3 , l. leonardi 3 , a. diaspro 2 1 lambs-ifom microscobio research centre and department of physics, university of genova, genova, italy., 2 italian institute of technology (iit), genova, italy, 3 department of human anatomical sciences and physiopathology of the locomotorapparatus, university of bologna, italy ligaments have been generally described as multifascicular structures with collagen fibre bundles cross-connecting to each other or running straight and parallel with crimps. a different collagen array and crimping pattern in different ligaments may reflect a different mechanical role. aim of this study was to relate the 3d collagen arrangement and crimping pattern by backward and forward second harmonic imaging microscopy (shim) and 2-photon excitation microscopy (2pem). shim on a laser-scanning system is a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of tissue architecture. although it is a coherent process the multiple scattering through the tissue give us the capability to acquire signal in both backward and forward direction [1] . shim and 2pem were combined in a dual-mode nonlinear microscopy to find out collagen fibre arrangement and crimping pattern. both polarization dependence and differences between forward and backward signals allowed to yield information on local structure [2] . currently used tcspc flim systems are characterised by high counting efficiency, high time resolution, and multiwavelength capability. the systems are, however, restricted to count rates on the order of a few mhz. in the majority of applications, such as fret or autofluorescence, the photostability of the samples limits the count rate to much lower values. the limitation of the count rate is therefore no problem. however, if flim is used for ion concentration measurements or imaging of chlorophyll in plants the available count rates can exceed the counting capability of a single tcspc channel. we therefore developed a flim system that uses eight fully parallel tcspc channels. by using a polychromator for spectral dispersion and a multichannel pmt for detection we obtain multi-spectral flim data at a rate of several frames per second. we will demonstrate the application of the system to dyamic changes of the fluorescence lifetime of chlorophyll in living plants. -imaging and spectroscopy scattering effects on non linear imaging of thick biological samples f. cella 1 , z. lavagnino 1 , a. diaspro 2 1 lambs-ifom, microscobio research center, university of genoa, italy, 2 iit, italian institute of technology, genoa, italy non linear optical scanning microscopy became a useful tool for living tissue imaging. biological tissues are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light travels into the sample. while performing imaging of biological scattering tissues in two photon excitation (2pe) regime, the localization of the maximum 2pe intensity was found to shift closer to the surface 1 and the imaging depth limit appears strongly limited by near surface fluorescence 2 . in this work we computed illumination and photobleaching intensity distribution 3 in order to characterize the effects induced by scattering. simulations of 2pe illumination and photobleaching intensity profiles have been performed for different scattering coefficients and at different focus depth. furthermore imaging of fluorescent immobile sample (polyelectrolyte gel) allowed to perform an experimental test on thick turbid media. results confirm that under these conditions no photobleaching effects due to scattering occur close to the surface. [1]ying et al., appl. opt. 38, (1999) . [2] theer p. and denk w, j. opt. soc. am. a. (2006) [3]mazza d. et al., appl. opt. 46 (2007) . biospectroscopic probes for real time measurement of hydrogen-deuterium exchange p. carmona 1 , m. molina 2 1 instituto de estructura de la materia (csic), madrid, spain, 2 escuela universitaria deóptica, madrid, spain isotopic exchange has long been used for the analysis of biomolecular structure and dynamics. hydrogen-deuterium exchange rates depend on ph, temperature and biomolecular environment. this is due to hydrogen bonding, low solvent accessibility, and steric blocking. time resolved measurement of hydrogen-deuterium exchange for subsequent 2d correlation spectroscopic analysis can, then, be very useful to obtain structural information from the said viewpoint. we have developed a microdialysis quartz cell for use in conjunction with raman spectroscopy to investigate hydrogen-isotope exchange reactions of biomolecules. the system requires only 40 µl volumes of the initial substrate and perturbing effluent solutions. we have obtained a d 2 o efflux rate of k d = 0.38 ± 0.008 min −1 with the greatest mwco (18 kda) used here, which involves that an exchange rate of 2.5 min −1 is the limiting rate that could be resolved with the said cell system. analogous results have been obtained using an infrared biospectroscopic microdialysis probe. the use of the method described here has the advantage of avoiding sample dilution (and subsequent signal loss) involved in the known stop flow methods. acknowledgements: the authors gratefully acknowledge financial support from the spanish ministerio de ciencia e innovación (project ctq2006-04161/bqu). polarized transient absorption to resolve electron transfer between tryptophans in dna photolyase photoactivation of dna photolyase comprises electron transfer through the chain fadh • -w382-w359-w306. photo-excited fadh • abstracts an electron from the tryptophan residue w382 in ∼30 ps (monitored by transient absorption spectroscopy). the subsequent electron transfer steps (from w359 to w382 •+ and from w306 to w359 •+ ) are difficult to resolve experimentally, because electron transfer between chemically identical species does not give rise to net absorption changes. to overcome this difficulty, we make use of the fact that polarized excitation (pulse laser) induces a preferential axis (that of the excited flavin transition) in the system (photoselection), and that w359 and w306 form different angles with that axis (known from the crystal structure). thus, polarized detection should allow distinguishing between them. using polarized "classical" transient absorption on a nanosecond time scale and the pump-probe technique on a picosecond scale, we demonstrate the feasibility of the method and provide evidence that electron transfer from w306 to w359 •+ is faster than the 30 ps time constant of the initial electron transfer from w382 to excited fadh • . synchrotron based fourier transform infrared (sr-ftir) microspectroscopy was applied to investigate apoptotic death of u-87 mg cells induced by the photosensitizer hypericin (hyp), in using different transport systems (hyp alone vs. hyp/ldl complexes) and incubation protocols. the differences between ir spectra of non-treated and hyp treated cells are mainly manifested in the positions of amide i and amide ii vibrational bands of proteins. these vibrational shifts are attributed to the protein structure changes from dominantly alfa-helix, in the non-treated cells, to beta-sheets and random coil structures, which prevail 4h and 24h after photodynamic treatment, respectively. the observed conformational changes of proteins can be explained as the consequences of the processes leading to apoptosis as was verified by flow cytometry experiments. the results confirm suggestion that ir spectroscopy can be successfully applied for the detection of early apoptotic processes. a. k. de, d. goswami indian institute of technology kanpur, india molecular fluorescence has been an indispensable tool in modern day optical imaging. one of the state-of the-art challenges in fluorescence microscopy is having better depth resolution as embodied by the confocal and multi-photon laser-scanning microscopic techniques. however, each technique bears its own limitation in having sufficient out-of-focus signal for the former while the low non-linear photon absorption cross-section for the latter. for confocal microscopy using one-photon excitation, we have shown how the clever choice of pulsed illumination instead of continuous-wave excitation leads to a gigantic enhancement in fluorescence that also has immediate applications in microscopy. moreover, single-photon illumination with ultrafast pulses leads to a novel way of achieving axial resolution along with numerous other advantageous applications e.g. reduced photo-bleaching of the chromophore. on the other hand we have thrown new insight demonstrating that the use of mode-locked laser pulses in multi-photon microscopy induces severe solvent-induced photo-thermal damage and prescribed methods to get rid of it. besides, the use of pulse pair excitation in multi-photon microscopy leads to probe and control the dynamics of fluorophores which has crucial role in selective excitation of fluorophores from quantum control perspectives. all these cutting edge research works will be presented in addition to our recent work on application of laser pulse shaping in multi-photon microscopy. interkingdom signalling in pseudomonas aeruginosa b. davis 1 , r. jenson 2 , p. williams 2 , p. o'shea 1 1 institute of biophysics, imaging and optical science, university of nottingham, u.k., 2 institute of infection, immunity and inflamation, university of nottingham, u.k. quorum sensing is the process through which some bacterial species coordinate cell-cell communication. pseudomonas aeruginosa; the pathogen responsible for over 90% of chronic lung infections and the leading cause of mortality in cystic fibrosis patients, expresses two major classes of quorum sensing molecules characterized as n-acyl homoserine lactones and 2-alkyl-4-quinolones. these compounds, in addition to their signalling roles have also been found to possess virulent properties, not only against competing species of bacteria such as staphylococcus aureus but also eukaryotic cells such as t-lymphocytes. the process of bacterial quorum signalling molecules influencing eukaryotic cell activity is termed 'interkingdom signalling'. to date it has been suggested that the quorum sensing molecules outlined above act on eukaryotic cells through interactions with an as yet unidentified plasma membrane or cytosolic receptors. this project is directed towards developing an understanding of how these compounds elicit eukaryotic response through a combination of membrane based interactions at physiologically relevant concentrations. particular emphasis is placed on studies of ligand binding with membrane microdomains in and the consequent downstream signalling are also considered. this work is significant as it will not only lead to a better understanding of pseudomonas infection, but may also lead to the discovery of new classes of agents for the treatment of infective diseases. a xas study of the sulphur environment in human neuromelanin and its synthetic analogues neuromelanin is a complex molecule accumulating in the catecholaminergic neurons that undergo a degenerative process in parkinson's disease. it was shown to play an either protective or toxic role depending on whether it is present in the intraneuronal or extraneuronal milieu. in the present study x-ray absorption spectroscopy is employed to investigate the sulphur binding mode in natural human neuromelanin, synthetic neuromelanins and in certain structurally known model compounds, namely cysteine and trichochrome c. based on comparative fits of human and synthetic neuromelanin spectra in terms of those of model compounds, the occurrence of both cysteine-and trichochrome-like sulphur coordination modes is recognized and the relative abundance of these two types of structural arrangement is determined. data on the amount of cysteine-and trichochrome-like sulphur measured in this way indicate that among the synthetic neuromelanins those produced by enzymatic oxidation are the most similar ones to natural neuromelanin. c. cremer kirchhoff-institute for physics, university of heidelberg, germany here we report on "spectral precision distance/position determination microscopy (spdm) with physically modifiable fluorochromes (spdm phymod ) to analyse the spatial distribution of single nuclear proteins and dna sequences at the macromolecular optical resolution level. like other methods of "spectrally assigned localization microscopy" (salm), spdm phymod is based on labelling 'point like' objects (single molecules) with different spectral signatures, spectrally selective registration and high precision localization monitoring by far field fluorescence microscopy. the intranuclear spatial location of single molecules was determined up to a density up to ca. 1000 molecules/µm 2 of the same type, and distances down to 15 -30 nm were nanoscopically resolved. quantum dots (qds) are semiconductor nanoparticles with increasing application as fluorescent markers in biology.we investigated structure of the cell walls of different species complexed with cdse qds using fluorescence microscopy, fluorescence spectroscopy and ftir techniques. in the experiments we used the cell walls isolated from three distinct plant species: arabidopsis thaliana, acer sp. and picea omorika. we studied both unlabeled and cdse-labeled cell walls. fluorescence spectroscopy and microscopy were used for detection of qds alone or complexed to the cell walls. emission spectra were deconvolved using the nelder-mead algorithm in matlab 6.5. we calculated approximate probability distribution (apd) for positions of spectral component maxima. there was certain difference between unlabeled cell walls and those complexed with qds. the ftir spectra also show some difference between the complexed and pure cell walls. the results show that structure was changed, but not significantly in reaction with cdse qds. these results are promising in context of use of qds as labels in cell wall studies. the characterization of the complex of cell wall structure with qds is a part of the study of nanoparticles application in investigations of plant materials. modulating the response of single neurons and neuronal networks with biophysical stimuli f. difato, a. maccione, l. berdondini, f. benfenati, a. blau italian institute of technology, department of neuroscience and brain technologies, genoa, italy during differentiation, cell processes create connections with other cells to form tissue capable of performing complex tasks. biophysical constraints provide necessary inputs for cellular organization in living organism 1 . to better understand how biophysical conditions influence tissue development, it is necessary to bridge the gap between experiments on single cells and complex tissues 2,3 . to achieve this goal we pair optical tweezers with electrophysiology measurements 4 . by adopting neuronal networks as a biological model, neuronal signal transmission can be recorded either by patchclamp electrophysiology or microelectrode arrays (meas). dissociated neurons will be cultured on meas to record neuronal network activity at different sites of the network while applying spatio-temporally defined biophysical stimuli to individual neurons. a. diaspro 1 , k. cortese 2 , p. bianchini 2 , c. gagliani 2 , c. tacchetti 2 1 iit -italian institute of technology, morego, genova, italy, 2 microscobio, university of genoa, italy correlative light/electron microscopy (clem) is becoming increasingly frequent in molecular and cellular biophysics. we successfully applied the method to analyze the 3d structure of rough and smooth russell bodies used as model systems. the major advantages of this approach are the following: (i) the ability to correlate several hundreds of events at the same time, (ii) the possibilitˆto perform 3d correlation, (iii) the potential to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the effective combination of the high data analysis capability of flm with the high precisionaccuracy of transmission electron microscopy in a clem hybrid morphometry analysis. we have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3d reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach. the relevance of the presented approach lies in two important key elements, namely: the development of optical nanoscopy methods and the potentiality for exploring different correlative frameworks like optical nanoscopy vs. optical microscopy adding scanning force microscopy techniques. multidrug resistance is a well known phenomenon which limits effectiveness in treating malignancy with chemotherapy by modifying the internalization and/or externalization flow of the drugs through the cancerous cells. combined chemotherapies, such as mvac, are therefore currently used in bladder cancer treatment. however, about 30% of patients do not respond this chemotherapy because of inherent or acquired drug resistance. we developed a non invasive predicative test on urinary cells to estimate the chemotherapy effectiveness before treatment, based on the fluorescence emission of mvac. we first studied the mvac photophysical properties in solution and using five cell lines: a drug sensitive cancer cell line mgh-u1s, its multidrug resistant subline mgh-u1r, a not tumorigenic cell line sv-huc-1, its tumorigenic counterpart mc-sv-huc t-2 and a cell line from transitional cell carcinoma t24. the results revealed a penetration and localization of the drug depending of the cell line type, allowing us to find a specific fluorescence signature for the identification of mvac resistant cells. similar data have been obtained for cytospined fixed culture cells and patients urinary cells. -imaging and spectroscopy -abstracts multilayered photoresist system as a ghost model for biological samples in confocal microscopy. l. ferrari, f. cilloco, f. r. bertani, s. selci istituto dei sistemi complessi cnr rome we have realized a bilayer photoresist system as a model to perform optical characterization in the vis-ir range of multilayered complex biological specimen. our goal is to obtain structural and spectroscopic reference parameters from the identification of reflectance pattern features within a novel project granted by miur (skintarget, ideas firb). our model is composed by a 1300 nm thick layer (shipley photoresist 1813) with a refractive index n= 1.6-1.8, wavelength dependent, and a 600 nm tick hsq (hydrogen silsesquioxane) layer with a refractive index n around 1.38, both spin-coated over a glass substrate. we present the analysis of reflectance pattern that can be obtained by a confocal laser reflective system as compared to the expected values as coming from analytical calculations, using a matrix approach, or a microscopic electromagnetic finite element analysis. interfacial roughness and consequent optical scattering are analyzed using a neural network approach. translational biophysics: multimode imaging for preclinical and clinical applications d. l. farkas cedars-sinai medical center, los angeles, usa our focus is where light and patient meet, and improvements yielding better outcomes. surgery is moving towards minimally invasive intervention, where biophotonics represents a major area of hope and growth. the translation of useful laboratory-derived knowledge into clinical practice has been hampered by the difficulty of detecting, characterizing and monitoring molecules and cells in the human body, especially dynamically. advanced bi ophotonic imaging is best suited for studying such entities, but has been lagging in clinical acceptance, in spite of major advances, and a clear need for the kind of resolution (spatial, temporal, spectral) and specificity that it alone can offer. biophysics-based new strategies are needed to address this challenge. the development of biophysical methods for translational medicine will be reviewed, with emphasis on our recent advances. our approach is a multimode one -combining methods to achieve early, quantitative detection of abnormalities. with imaging fulfilling its dual role of better describing anatomy and physiology, intrasurgical histopathologyequivalent molecular and cellular imaging is achievable in vivo, as is a closer spatio-temporal connection between imaging and intervention. some application areas to be covered: cancer (early detection by spectral reflectance/autofluorescence; progression quantitation by oct; nano-and targeted chemotherapy assessment in vivo); neurobiology (imaging fast calcium transients and alzheimer's plaques); hyperspectral mie scattering imaging for in situ displasia; design and use of an advanced multimode imaging endoscope with in vivo delineation of hirschsprung's disease for better intervention; monitoring of stem cell fate in vivo. immobilization of liposomes in a sol-gel matrix: a fluorescence confocal microscopy study r. esquembre 1 , s. n. pinto 2 , j. a. poveda 1 , m. prieto 2 , c. r. mateo 1 1 ibmc, universidad miguel hernández, elche, spain, 2 cqfm, instituto superior técnico, lisbon, portugal immobilization of liposomes shows interesting applications in protein biology, membrane biophysics and biosensor technology. previous fluorescence spectroscopy works revealed that the entrappement of zwitterionic phospholipid liposomes in a silica sol-gel matrix alters the thermodynamic properties as well as the fluidity of the lipid bilayer. interactions between the polar head of phospholipids and the porous surface of the host matrix could be responsible of such behaviour. in order to get more insight into this possibility we have immobilized, for the first time, giant unilamelar vesicles (guvs) and the shape and size of these structures as well as the possible existence of lipid domains have been visualized through fluorescence confocal microscopy. this technique allows for direct observation of the effect of encapsulation on an individual liposome, in contrast to the averaged information given by macroscopic spectroscopic techniques. liposomes composed of pure popc or dopc as well as mixtures dopc/dppc were labelled with the fluorescent probes bodipy, rhd-pe and rhd-dope. preliminary results shows that only the smallest guvs (6-10µm) survive to the encapsulation process but often with a slight loss of its sphericity, probably due to pressures suffered during the matrix gelation. however no change in the gel/fluid phase proportion has been observed for immobilized dopc/dppc guvs, regarding to solution. solvent fluctuations play a key role in controlling protein motions and functions. here, we have studied how the reaction catalyzed by the light-activated enzyme protochlorophyllide oxidoreductase (por) couple with solvent dynamics (g. durin et al, biophysical j. (2009 ) 96, 1902 . to simultaneously monitor the catalytic cycle of the enzyme and the solvent dynamics, we designed temperature-dependent uv-visible microspectrophotometry experiments, using flash-cooled nano-droplets of por. the temperature-dependant formation of the first two intermediates in the por reaction were measured, together with the solvent glass transition temperature (t g ) and the build-up of crystalline ice. we find that formation of the first intermediate occurs below t g and is not affected by solvent dynamics, whereas formation of the second intermediate occurs above t g and is influenced by solvent dynamics. these results suggest that internal protein motions drive the first step of the por reaction whereas solvent slaved motions control the second step. we propose that the concept of solvent slaving applies to complex enzymes such as por. amphotericin b (amb) is one of the main polyene antibiotics widely used to treat deep-seated fungal infections. the mechanism of biological action of amb is most probably directly related to the ability of the drug to form hydrophilic pores in the membrane core, thus affecting physiological transport of ions. the effects of amb-cu 2+ complexation are demonstrated by the electronic absorption and fluorescence spectra. the absorption spectra of amb in water (ph=7) after the injection of water solution of copper(ii)sulfate display a complex structure with hypsochromic-and bathochromic-shifted bands indicative of formation of molecular aggregates of the drug. formation of the electronically coupled chromophores of amb, especially aggregates, was analyzed at different cu 2+ concentrations by the rls (resonance light scattering) technique. intensity of the fluorescence emission spectrum (characteristic of the dimeric form of amb) decreases after the amb-cu 2+ complex formation. this effect of the formation of the amb aggregated structures by amb-cu 2+ are different from the spontaneous molecular aggregation process, as deduced from the spectroscopic analysis. morfo-functional asymmetry of the olfactory receptors of the honeybee apis mellifera l e. frasnelli 1 , g. anfora 2 , f. trona 2 , f. tessarolo 3 , r. antolini 3 , g. vallortigara 1 1 cimec, centre for mind/brain sciences, university of trento, italy, 2 iasma research and innovation center, fondazione e. mach, s. michele all´adige (tn), italy, 3 biophysics and biosignals lab., dept. of physics, university of trento, italy lateralization, i.e. the different functional specialisation of the left and right side of the brain, has been documented in many vertebrate and, recently, invertebrates species. in the honeybee apis mellifera l. olfactory memory seems to involve at first the use of the right antenna. the present study investigated physiological and anatomical differences between left and right antennae of honeybees. electroantennographic responses (eag) were recorded from the left and right antennae of 16 honeybees to linalool, a floral volatile compound, and isoamyl acetate, an alarm pheromone, at 5 doses (from 0,25 to 2500 µg). the number of sensilla on the left and right antennae was recorded by scanning electron microscopy (sem). each antenna segment, from 14 insects, was observed from 4 different viewpoints in order to image the whole antenna surface and compute the number of sensilla. the tested compounds induced higher eag responses on the right than on the left antenna at every dose. sem showed that the placoidea olfactory sensilla were slightly more abundant on the right antenna surface than on the left one. results suggest an asymmetry in the peripheral odour perception mechanism in the honeybee a. mellifera. super-resolution imaging of dna through single molecule switching of intercalating cyanine dyes c. flors, c. n. j. ravarani, d. t. f. dryden school of chemistry, university of edinburgh, u.k. a growing trend in far-field super resolution fluorescence microscopy involves the replacement of photoactivatable fluorophores by common dyes such as cy, atto or alexa [1] . it has been shown that these dyes can blink in useful timescales for single-molecule based imaging by adding suitable buffers. this strategy greatly simplifies the sample preparation and imaging scheme, enabling its application to a wider range of biological systems. we have explored if a similar approach might be useful to study dna topology using intercalating cyanine dyes such as yoyo-1. there are two main advantages of this approach: i) dna labelling with intercalating dyes is straightforward, and ii) the free dye in solution is essentially non-fluorescent, greatly reducing the fluorescence background. we show that yoyo-1 can blink in the absence of oxygen and in the presence of cysteamine, which allows its application to nanoscale imaging. we exemplify its use by imaging λ-dna and a puc19 plasmid. we also explore the compatibility of several intercalating dyes with biological systems such as enzymes or cells. our results suggest that dna intercalating dyes are a promising option for fluorescence super-resolution studies of dna topology. seeing more in total internal reflection fluorescence (tirf) microscopy r. fiolka, a. stemmer nanotechnology group, eth zürich, zürich, switzerland total internal reflection fluorescence (tirf) microscopy is an effective widefield imaging tool that selectively excites a very thin sample layer within the evanescent excitation field at the glass-water interface. lateral resolution of standard tirf microscopy is limited to approx. 240nm using green emission, which can be insufficient for a large class of biological investigations. additionally, the evanescent excitation field is prone to light scattering, creating out of focus blur in the final image. we present several techniques that address these mentioned shortcomings of standard tirf microscopy. using evanescent standing waves, the lateral resolution in tirf microscopy can be extended by a factor of 2.5, reaching 90nm. we further show techniques to reduce the blur induced by light scattering of the evanescent field. finally, we demonstrate optical sub-sectioning capabilities in tirf microscopy by acquiring several images with different penetration depth of the evanescent field and applying suitable post processing algorithms. thereby the obtainable z-resolution exceeds the classical limit of widefield microscopy, and object structures lying within the evanescent field can be reconstructed. the natural photosentizer hypericine (hyp) exhibits potent properties for tumor diagnosis and photodynamic therapy. evidences of hyp release from ldls prior to passive diffusion within cells are addressed in this study. fluorescent properties of hyp have been used for dynamic studies of its interaction with low-density lipoproteins (ldls) and u87 glioma cells. subsequent non-specific staining of intracellular membranes compartment were observed by mean of colocalization fluorescent imaging studies. it was shown, that monomers of hyp are only redistributive forms. increasing of hyp concentration leads to the formation of non-fluorescent aggregates within ldls as well as within the u87 cells, and can preclude its photosensitizing activities. in all experiments, hydrophobic character of the molecules appears as the driving force of its redistribution process. acknowledgment: this work was supported by the slovak res. and dev. agency contracts no. lpp-0337-06. we also kindly thank the synchrotron soleil for using the detection system of the disco beamline. ledgf/p75 switches from a dynamic to a tight chromatin interaction upon binding to hiv-1 integrase j. hendrix 1 , z. debyser 2 , y. engelborghs 1 1 laboratory of biomolecular dynamics, katholieke universiteit leuven, belgium, 2 laboratory of molecular virology and gene therapy, katholieke universiteit leuven, belgium human transcriptional co-activator ledgf/p75 is hijacked by hiv-1 integrase (in) during the replication of hiv. little is still known about the molecular complex of these two proteins in the living cell. in this work we first studied the cellular chromatin interaction of egfp-tagged ledgf/p75 with tunable-focus fluorescence correlation spectroscopy (tf-fcs) and show that ledgf/p75 is in equilibrium between a free brownian motion and a very slow movement on the chromatin. being dependent on the size of the laser focus, this slow movement represents a continuous associationdissociation-reassociation process that is governed by diffusion. concentration-dependent continuous photobleaching measurements (cp) furthermore revealed the existence of high-affinity chromatin binding sites. next, we co-expressed mrfp-tagged in and confirmed its intracellular interaction with ledgf/p75 by fluorescence cross-correlation spectroscopy (fccs). interestingly, cp and fluorescence recovery after photobleaching (frap) indicated that the affinity of this complex for chromatin is exceptionally high. by twophoton fluorescence lifetime imaging (2p-flim) we verified if the cellular stoichiometry was altered when the proteins were expressed together. we believe that this work is useful for the understanding and targeting of hiv-replication. biophysical identification of orf10 from clavulanic acid biosynthesis cluster as a cyp450 l. s. goto 2 , c. o. hokka 1 , j. f. lima 2 , o. r. nascimento 2 , a. p. u. araújo 2 1 grupo de engenharia bioquímica, ufscar, br, 2 grupo de biofísica molecular "sérgio mascarenhas", usp, br streptomyces clavuligerus produces the clinically important β-lactamase inhibitor clavulanic acid. biosynthesis related genes reside in three gene clusters, one of these, named clavulanic acid gene cluster, includes most of the known clavulanic acid biosynthetic enzymes. the penultimate step along clavulanic acid biosynthesis remains unclear. required transformation involves at least two events: oxidative deamination and double epimerization of (3s , 5s )-clavaminic acid into (3r, 5r)-clavaldehyde. downstream the known part of clavulanic acid cluster lays orf10 , a putative gene encoding a tentative cytochrome p450-like protein which knockout has been proven deleterious to clavulanic acid biosynthesis. should such protein exist, it would be candidate to fulfill the clavulanic acid pathway missing step. in this work, orf10 encoded protein is characterized aiming to place it as a real p450. for this task, molecular cloning and recombinant expression of orf10 were accomplished. purified protein was submitted to spectroscopic measurements such as circular dichroism and electron paramagnetic resonance which indicate p450 features, including catalytic relevant heme iron redox states and homolytic peroxide scission mechanisms. further, peroxide reaction adducts were characterized by spin trapping. this work is supported by fapesp. muscle structure and gabaergic innervations in the limbs of barnacle cyprid l. gallus 1 , s. ferrando 1 , c. gambardella 1 , a. diaspro 2 , p. bianchini 2 , p. ramoino 3 , v. piazza 4 , g. tagliafierro 1 1 libiom, dibio, università di genova, italy, 2 ifom-lambs/microscobio, difi, università di genova, italy, 3 dipteris, università di genova, italy, 4 ismar cnr, venezia, veneto, italy balanus amphitrite is a sessile crustacean that settles at the larval stage of cyprid. in this stage we studied the gabaergic innervation of limb striated muscular fibers, by immunohistochemistry. the second harmonic generation (shg) and 2-photon excitation (2pe) microscopy were used to set out the muscle structure and its relationship with nerve terminal. sections were observed at a multimodal nonlinear microscope composed by the leica clsm. the laser system used is a ti:sapphire chameleon-ultra (coherent inc, santa clara, ca, usa), tunable between 690 nm and 1040 nm and characterized by a pulse width of 140 fs delivered at a repetition rate of 90 mhz by means of a homebuilt set-up (bianchini p. and diaspro a. j biophotonics 2008 1:443-50). the z-stacks were performed in order to obtain the 3-dimensional distribution of the muscular fibers. in the posterior ganglion gad immunoreactive (ir) motor neurons were arranged in 12 clusters near the emergence of the limb nerves. gaba and gababr1 ir neuromuscular junctions (nj) were localized in the limb muscle fibers; vgat ir cells surrounded each limb muscles. these results suggest that gaba plays a key role in the regulation of limbs movement. the shg was very useful to outline the relationship between nerve terminals and limbs muscle fibers. giant unilamellar vesicles (guvs) are very useful model membrane systems to study many aspects of lipid-lipid and lipid protein interactions, particularly employing fluorescence microscopy related techniques (bagatolli 2006) . the use of this model system can be particularly useful to study aspects related with the lateral structure of bacterial membranes using the aforementioned approach (i.e. fluorescence microscopy). bacterial cells have a size close to the resolution limit of optical microscopy and details about the organization of their membranes are not easy to achieve using such technique. recently a new electroformation method to prepare guvs composed of compositionally complex mixtures under physiological conditions was developed in our laboratory (montes 2007). in the present work we further extended this electroformation method to prepare guvs composed of bacterial lipid extracts and lipopolysaccharide (lps). in our experiments we used e.coli lipid extract to prepare small vesicles containing various lps species, from smooth strains and rough mutants. suvs were used as starting point to electroform guvs using various types of buffers with high ionic strength. the successfully obtained bacterial-guvs were used to study the interaction of these membranes with known lps-binging proteins (lung surfactant protein d, sp-d) and peptides (polymyxin b). our results remark the usefulness of this particular bilayer models to perform studies mimicking bacterial membranes. mechanical properties of polymeric membranes probed by afm m. kocun 1 , i. mey 1 , w. müller 2 , m. maskos 2 , a. janshoff 1 1 georg-august universität, institute for physical chemistry, göttingen, germany, 2 johannes gutenberg universität, institute for physical chemistry, mainz, germany many biological cell functions are dependent on the mechanical properties of the membrane. polymeric membranes that mimic native cell membranes are valuable research tools which can be used to better understand the physics of biological membranes. we have investigated free standing artificial membranes prepared from polybutadiene-b-polyethylene oxide (pb-b-peo). the membranes were prepared from vesicles ruptured on porous silicon substrates. these polymeric membranes were studied by confocal laser scanning microscopy (clsm) and atomic force microscopy (afm). force indentation curves were performed on the membranes and theoretical models were used to extract elasticity constants from the results. the study of polymeric membranes can give insight to the function of biological membranes, furthermore polymeric membranes can be used to create new hybrid systems by incorporating biological (lipids, proteins), artificial (polymers, dyes) and inorganic (nanoparticles) components. site-directed spin labeling study of the lightharvesting complex cp29 the topology of the long n-terminal domain of the photosynthetic light-harvesting complex cp29 was studied using electron spin resonance (esr). cp29 is a minor antenna complex of the photosystem ii (psii), a multisubunit protein complex. wild-type cp29 protein containing a single cysteine at position 108 and nine single cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. in all cases the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. the spin label esr spectra were analyzed in terms of a multi-component spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. these results permit to trace the structural organization of the long n-terminal domain of cp29. we took the crystal structure of light-harvesting complex ii (lhcii), major antenna complex of psii available in pdb as a starting point and constructed a model for cp29 based on esr data. present opportunities and future developments in soft x-ray transmission and emission microscopy b. kaulich, a. gianoncelli, v. babin, m. kiskinova elettra -sincrotrone trieste, s.s. 14 km 163.5 in area science park, i-34012 trieste-basovizza, italy soft x-ray transmission and emission imaging and spectromicroscopy are bridging the gap to other microscopy techniques in terms of lateral resolution, penetration depth and chemical sensitivity. the novel soft x-ray spectromicroscopy approach of the twinmic instrument at the elettra synchrotron radiation facility is combining several imaging modes for morphology characterization, such as scanning, projection and full-field imaging with several contrast techniques including brightfield, darkfield, differential phase and interference contrast at sub-100 nm lateral resolution. complementary chemical information is provided by chemical imaging and micro-spectroscopy using the photon-in/photon-out xray absorption and x-ray fluorescence. unique for twinmic is the low-energy x-ray fluorescence setup operated at 280 -2200 ev photon energies, which allows simultaneous analysis of the morphology, and the distribution of light elements on cellular and sub-cellular level. in the presentation the principles of the methods used in the twinmic instruments the performance and potentials of the instrument will be demonstrated by several examples of applications in the field of human, animal and plant biology, biophysics and chemistry, physiology and genetics. the potential impact of microscopy techniques using free-electron x-ray lasers on biophysics will be illustrated by the diproi project at fermi@elettra. -imaging and spectroscopy fluorescence anisotropy and afm used as tools to characterized porin´s reconstituion in luv´s s. c. lopes, i. sousa, p. eaton, p. gameiro requimte, faculdade de ciências, universidade do porto, rua do campo alegre, 4169-007 porto, portugal a major requirement to perform structural studies with membrane proteins is to define efficient reconstitution protocols that assure, not only, a high incorporation degree in preformed liposomes, but also a protein directionality and topology that mimics its in vivo conditions. for this kind of studies, protein reconstitution in membranes systems via a detergent-mediated pathway is usually successfully adopted, since detergents are generally used in the initial isolation and purification of membrane proteins. in this study we report the reconstitution of ompf in preformed dmpc and e. coli liposomes using two different techniques for detergent removal: (1) exclusion chromatography and (2) incubation with detergent adsorbing beads. the incorporation degree was determined by bicinchoninic acid assay and fluorescence anisotropy was used to determine ompf effect on the structural order of membrane lipids. these results show that protein insertion in membranes depends both on the technique used to remove detergent and on the lipids used to prepare the liposomes. moreover more anisotropy and atomic force microscopy studies will allow a better characterization of bacterial model system membranes. the wavelength dependence of the luminescence for different sized aunp by 2-photon clsm k. li, m. schneider pharmaceutical nanotechnology, saarland university, campus a4 1, d-66123 saarbrücken, germany gold nanoparticles (aunp) with different sizes can exhibit original luminescent properties if excited with pulsed nearinfrared laser light which makes them a suitable object to be detected in biological tissues. the main obstacle is to distinguish between the object of interest (emitted light) and the autofluorescence from the sample which limits the scope of application of aunp. therefore, our aim is to characterize the luminescent properties of such nanoparticles regarding their excitation and emission. in this study, the excitation and emission spectra of aunp with different sizes below 50 nm in an excitation range of 720 nm to 900 nm were investigated. our study shows the emission spectra curves of aunp are broadband spectrum and vary with the changing of excitation wavelength from 720 nm to 900 nm. the results also suggest the minimum laser power necessary to trap the aunp depends on particle size and excitation light wavelength and a maximum power above which the particles are destroyed. in all the experiment above, to avoid the simultaneous effects from slide and cover slip is a must. rocking, tumbling, and sliding: real-time nanomotion of a membrane-bound virus p. kukura 1 , h. ewers 2 , c. mueller 1 , a. renn 1 , a. helenius 2 , v. sandoghdar 1 1 laboratory of physical chemistry, eth zurich, ch-8093 zurich, switzerland, 2 institute of biochemistry, eth zurich, ch-8093 zurich, switzerland the interaction of a virus with its receptors in the plasma membrane is decisive for its infection of cells. optical studies have revealed that after binding, virus particles move laterally on the membrane, but the complexity of the cellular environment and the drawbacks of fluorescence microscopy have prevented access to the molecular dynamics of early virus-host couplings. here, we examine a model system, in which single simian viruses (sv40) interact with their gm1 ganglioside receptors in supported lipid bilayers. we employed scattering interferometry and single molecule fluorescence localization to visualize the vectorial rotational motion of virions. at low receptor concentration, we observed sliding and tumbling of single virions during rapid lateral diffusion. in contrast, at increased receptor concentration the virions repeatedly underwent periods of standstill, reminiscent of their behavior prior to endocytosis. by an unprecedented combination of millisecond time and nanometer spatial resolutions, we revealed that during these immobile periods, the virions rock back and forth among nanoscopic spots separated by 9 nm. our insights, together with the structure of the viral capsid, suggest aggregation of receptors in nanodomains and recurrent swap of binding between receptor molecules and neighboring viral protein pentamers. herein, we have developed different membrane probes binding spontaneously to the outer plasma membrane leaflet and showing sensitivity to membrane properties such as surface charge and phase state. the first two types of probes were based on 3-hydroxyflavone fluorophore. one of them showed a high sensitivity to the surface charge and to the phase state of lipid bilayers, while the other was only sensitive to the surface charge. the third type, which was based on nile red fluorophore, was sensitive only to the phase state. surprisingly, the probes that were sensitive only to the surface charge did not respond to apoptosis, while the other two types of probes showed significant spectroscopic response to it. moreover, the latter exhibited response to cholesterol depletion, which was similar to that observed on apoptosis. thus, according to our data, the intact living cells present a remarkable fraction of the cholesterol-rich domains, while apoptotic loss of the transmembrane asymmetry decreases it dramatically. these probes represent a new tool for quantification of surface charge and cholesterol-rich domains in cell membranes. -imaging and spectroscopy due to its porosity and unique optical properties porous silicon (psi) is an attractive template to develop biomaterials and biosensors. porous silicon microcavity (psimc) structures were prepared then functionalized for covalent protein attachment of glucose oxidase (gox) or solubilized bacteriorhodopsin (br). functionalization and protein infiltration was monitored by specular reflectometry sensitive to change in refractive index, when a molecule is attached to the large internal surface of psi. protein infiltration into the porous scaffold was confirmed by edx spectroscopy and the structures were imaged by biphoton microscopy. second harmonic generation and enhanced two-photon excited fluorescence emission from porous silicon was observed when resonantly exciting the structures. in addition, when the microcavities were infiltrated with gox or br, the proteins acted as a very efficient internal two-photon-excited fluorescence emitter, hence protein infiltration enabled the in-depth visualization of the porous structure by taking advantage of the optical sectioning capacity inherent to the non linear optical microscopy technique. patterning of bio-molecules: methods for characterization of neuron-substrate interfaces we investigated how to use and improve micro printing techniques to obtain molecules patterns on cell culture substrates. with micro contact printing (µcp), we generated geometrically defined depositions of poly-d-lysine (pdl) using poly-dimethylsiloxane stamps and optimized neuronal culturing conditions. rat and mice hippocampal neurons grown on those patterns showed to be alive and functional. we then applied µcp to study axonal development by combining cell culture assays with atomic force microscopy (afm) to investigate in more detail the molecule deposition on the surface and to measure morphological changes in the growth cone (gc) during the early phases of neurite development. we found distinct shapes of the gcs depending on whether they were growing on l1 adhesion molecule patterned by indirect-µcp or on pdl coated surfaces. we also attempted to transfer such patterns on multi electrode arrays in order to constrain neuronal cell bodies on the electrode area and to improve electrophysiological recordings from neuronal networks. other patterning techniques were therefore explored using a nano-drop printing system. patterned surfaces were analyzed with afm and scanning electron microscopy to combine different approaches aimed to the improvement and characterization of the printing techniques. insights on the mechanism of electron transfer in complex i a. l. maniero 1 , c. bergamini 2 , m. bortolus 1 , r. fato 2 , s. leoni 2 , g. lenaz 2 1 università degli studi di padova, padova, italy, 2 università degli studi di bologna, bologna, italy complex i (nadh dehydrogenase) plays a central role in cellular energy production, transferring two electrons from nadh through a series of iron-sulfur clusters (fes) to ubiquinone (coenzyme q); the electron transfer is coupled to the translocation of protons across the membrane. the fes center n2 is the last acceptor in the electron-transfer chain, but the mechanism through which the enzyme couples the 1e − reduction of the fes centers to the 2e − reduction of ubiquinone (q→sq→qh 2 ) is unclear [1] . in our experiments, submitochondrial particles were treated with different inhibitors, in the absence or in the presence of different quinone analogues, and nadh addition initiated the electron transfer. we assess by epr (electron paramagnetic resonance) spectroscopy the relative abundance of the reduced n2 center and of the semiquinone radical, and coupled epr data to enzymatic activity assays and to fluorescence measurements on the effect of inhibitors on reactive oxygen species (ros) production. we identify two different classes of inhibitors showing different effects on ros production. moreover the redox state of the complex has shown to depend both on the inhibitors and on the quinone analogues. a possible mechanism of the electron transfer, that can explain the experimental findings, will be presented. conventional fluorescence microscopy suffers from a resolution limit imposed by the diffraction of light. stimulated emission depletion (sted) microscopy overcomes this resolution barrier without being limited by the wavelength by taking advantage of the photophysics of the observed sample into the image formation process, and has proven to be a powerful approach for exploring relevant biological issues. the outstanding resolution of sted microscopy is achieved by drastically minimizing the spatial extent of the focal region from which fluorescent molecules can emit signal in the sample. so far, mainly complex and relatively expensive lasers systems providing pulsed beams have been used to inhibit the fluorescence in the outer area of the focal region. here we report on the development of a new setup based on a fast beam scanning confocal microscope using compact turn-key and inexpensive continuous wave lasers. the great potential of this simple configuration is demonstrated for a selection of commonly used fluorescent markers. characterization of hepatitis b antigen particles by atomic force microscopy each year, over one million people die from hepatitis b virusrelated chronic liver disease, including cirrhosis and hepatocellular carcinoma. the major surface antigen of hepatitis b virus (hbsag) is a cysteine-rich, lipid-bound protein with 226 amino acids. recombinant hbsag produced in yeast can self-assemble into 22-nm immunogenic spherical particles that are used in licensed hepatitis b vaccines (protein/lipid ratio is 60/40 in mass). hbsag size and shape have been mainly investigated by transmission electron microscopy after negative staining of the particles. however, under these conditions, no details of the particle surface can be obtained because of the shadowing effect due to the uranium salts. here we describe new structural insights of hbsag particles using atomic force microscopy (afm) performed under physiological conditions. we applied atomic force microscopy to define structural details of the surface organization with a resolution in the nanometer range. as expected, the diameter of hbsag particles is 26,8 ± 2,5 nm in average. the surface of these particles clearly shows the presence of protuberances that most probably correspond to proteins. indeed, reduction and alkylation induces the disappearance of the protuberances. the number of the protuberances estimated from afm micrographs is about 70 per spherical particles. a biophysical study of equine herpesvirus-1 entry into cells g. mckenzie 1 , p. o'shea 2 , j. kydd 1 , c. rauch 1 1 school of veterinary medicine and science, university of nottingham, uk, 2 institute of biophysics, imaging and optical science, university of nottingham, uk equine herpesvirus-1 (ehv-1) can cause respiratory disease in young horses, varying degrees of paralysis and abortion during the later stages of pregnancy. furthermore, there has been a recent increase in the number of outbreaks involving paralysis of thoroughbred horses in training. virus disseminates rapidly after initial infection via cell associated viraemia. controlling this may prove crucial in combating the pathogenicity of the disease. we have investigated the cellular interactions of ehv-1. initially, binding of ehv-1 to fluorescein phosphatidylethanolamine (fpe)-labelled phospholipid vesicles of various compositions was examined. a variety of microscopy techniques were then employed to study the events surrounding the binding of virus to equine peripheral blood mononuclear cell (pbmc) membranes. confocal microscopy images have highlighted possible colocalisation of ehv-1 with membrane 'rafts'. total internal reflection fluorescent microscopy was then used to identify viral binding at the membrane with high contrast images, able to observe single virus particles. establishing the viral entry pathway into pbmcs would allow drugs that target this process to be employed, reducing clinical viraemia. the interpretation of in-vivo binding rate measurements allows inferring the molecular interactions that regulate cellular functions. fluorescence recovery after photobleaching (frap) is a widely used tool to quantify binding reactions in vivo. however the lack of "golden standards" for these measurements requires the measured binding rates to be validated with other techniques. we present a new fluorescence correlation spectroscopy (fcs) method to measure the in vivo bound fractions and residence times for molecules that interact with an immobile substrate. we apply this method to measure binding of mutants of the transcription factor vbp (vitellogenin binding protein) to the dna. comparison of fcs with frap results in comparable estimates of the measured diffusion constants and bound fractions. however, fcs provides an estimate of the vbp residence time at the dna, while frap does not. this limitation in the analysis of vbp is due to the larger photobleaching volume used in frap, if compared to the observation volume of an fcs experiment. in sum, we present a method to measure binding rates with fcs. substantial agreement of this method with frap is shown. however, further validation on tightly bound molecules will be necessary to assess if frap and fcs agree in the measurement of residence times. -imaging and spectroscopy we have investigated the changes in the mechanical properties of the zona pellucida (zp), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. the response of the zp to mechanical stress has been recovered according to a modified hertz model. zp of immature oocyte's shows a pure elastic behaviour. mature and fertilized oocyte's zps evidence, instead, a transition from a purely elastic behaviour, which occurs when low stress forces are applied, towards a plastic behaviour has been observed. the high critical force necessary to induce deformations, that well supports the non-covalent long interactions lifetimes of polymers, increase after the cortical reaction. afm images show that oocytes' zp surface appear to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles more wrinkled surface characterize marure oocytes with respecto to immature and fertilized oocytes from a mechanical point of view, the transition of the mature zp membrane toward fertilized zp, through the hardening process, consists in the recovery of the elasticity of the immature zp, while maintaining a plastic transition that, however, occurs with a much higher force with respect to that required in mature zp. dynamical behavior of ribose and deoxyribose supercooled water solutions s. e. pagnotta 1 , s. cerveny 1 , a. alegria 2 , j. colmenero 3 1 centro de fisica de materiales, centro mixto csic-upv/ehu, san sebastián, spain, 2 departamento de fisica de materiales, universidad del pais vasco (upv/ehu), facultad de quimica, san sebastián, spain, 3 donostia international physics center, san sebastián, spain ribose and 2'-deoxyribose are probably the most widespread monosaccharides in nature. they can be extensively found in ribonucleic acid (rna) and 2'-deoxyribonucleic acid (dna), respectively, where they form, together with a nitrogenous base and a phosphate group, a peculiar building-block structure called nucleotide. in the present work, the relaxation dynamic of ribose and deoxy-ribose water solutions at different concentrations has been studied by broadband dielectric spectroscopy and differential scanning calorimetry in the temperature range of 150-250k. two relaxation processes are observed for all the hydration levels; the slower (process i) is related to the relaxation of the whole solution whereas the faster one (process ii) is associated with the reorientation of water molecules in the mixture. as for other polymeric water solutions, dielectric data for process ii indicate the existence of a critical water concentration above which water mobility is less restricted. moreover, according with these results, atr-ftir measurements of the same sugar solutions showed an increment in the intensity of the oh stratching sub-band close to 3200 cm −1 as water content increases. the regulation of the formation of cytoskeletal protein complexes by actin-binding proteins m. nyitrai, a. vig, t. kupi, z. ujfalusi, s. barkó, g. hild university of pécs, faculty of medicine, department of biophysics, pécs, hungary in living cells various groups of proteins are associated to supramolecular actin filament structures, often in a nucleation factor dependent manner. for example, actin structures associated with formins can bind tropomyosin and profilin, while those polymerised by the nucleation of the arp2/3 complex bind cofilin and myosin i. the molecular mechanisms underlying the regulation of the formation of these protein complexes is still ambiguous. we have shown recently that formins can bind the actin filaments and change their conformational state. subsequent binding of other actin-binding proteins, such as tropomyosin and myosin, can reverse these changes. it appears that the reversal effect assumes that the actin-binding protein binds the filaments in a well-defined and specific binding site. the altered conformational state of the actin filaments observed after the binding of these proteins provides a possible explanation for the modified affinity of the filaments for other-actin binding proteins. based on the results available so far we assume that the affinities are modified differently by different nucleation factors, and the conformational changes introduced to actin by actin nucleation factors can serve as the molecular bases for the regulation of the formation of actin based intracellular protein complexes. experiments are currently in progress to test and further corroborate the existence of such regulatory mechanisms in living cells. correlation between sub-cellular distribution of photoactive drug hypericin (hyp), determined by applied delivery systems (hyp vs hyp/ldl), and mode of the cell death is addressed in this study. co-localization of hyp with mitochondria, lysosomes and golgi apparatus in u-87 mg glioma cells was determined by confocal laser scanning microscopy in using organelle specific fluorescent dyes as well as by time resolved fret experiments. flow cytometry experiments were realized to study a photodynamic effect of hyp (598 nm/4 jcm −1 ) on cells. significant differences in the proportional representation of live, apoptotic and/or necrotic cells were observed for different types of delivery systems of hyp 24 hours after hyp (5x10 −7 m) photoactivation. conclusions: i) sub-cellular distribution of hyp depends on using delivery systems, ii) the mode of cell death depends more on concentration of hyp inside cells, than on different type of delivery systems (for non selective wide-field cell illumination), iii) fluorescence lifetime is sensitive parameter to study sub-cellular distribution of hyp. novel time-resolved spectroscopic methods have been used to investigate the interactions between a fluorescently-labelled mutant of the peptide melittin and supported lipid bilayers, formed by self-assembly at a silica-water interface via vesicle deposition. time-resolved evanescent wave-induced fluorescence spectroscopy (trewifs) is a surface-selective technique in which the evanescent field from a pulsed laser source is used to photoexcite fluorescent species at an interface. the resulting fluorescence decay kinetics, measured using time-correlated single-photon counting, report on the micro-domains experienced by those fluorescent species at an interface. extending trewifs to time-resolved evanescent wave-induced fluorescence anisotropy measurements (ew-trams) provides dynamic rotational information of a fluorescent species, reporting on its mobility at an interface. presented here are trewifs and ew-trams data obtained from the fluorescence of an alexa 430-labelled melittin mutant interacting with a dipalmitoylphosphocholine bilayer at room temperature, physiological ph and ionic strength. the results provide new insights into the conformation, location and motion of cytolytic peptides interacting with cell membranes. optical tweezers are used to controllably apply forces to red blood cells and the resulting chemical and structural changes are monitored using raman spectroscopy. the forces are applied in vivo and mimic that which the cell undergoes mechanically as it passes through vessels and smaller capillaries. the first result presented will be spectroscopic evidence of a transition between the oxygenation and deoxygenation states of hemoglobin that is caused by the stretching of the red blood cell. the transition is due to mechanically induced enhancements of hemoglobin-membrane and hemoglobin neighbor-neighbor interactions. the latter, lesser known effect is further studied by modeling the electrostatic binding of two of the protein structures using molecular dynamics methods. secondly, polarized raman spectroscopy is utilized to study the packing and ordering of the hemoglobin proteins in the red blood cell as it is stretched. depolarization ratios for a number of heme group modes change, indicating that the applied force additionally packs and orders the proteins inside the cell which further demonstrates the role of cell deformation in the oxygen transport kinetics. acknowledgements: this work was supported by miin fis2008-00114 (spain) and fundació cellex barcelona fret microscopy is widely used to study protein-protein interactions in living or fixed specimens. currently, most commonly used visible fluorescent proteins for live-cell fret studies are the cerulean and venus variants of the cyan and yellow fluorescent proteins. even though this fret pair appears to be ideal for monitoring protein-protein interactions, the most commonly used fixed laser wavelengths do not excite cerulean at peak absorption. recently, we characterized an ideal donor, the monomeric teal fluorescent protein (mtfp), which is excitable using the commonly available 457 (8) opt. june/july, 2008). we used teal as a donor for various red fluorescent proteins as acceptors including tdtomato, mko2, morange2, mtagrfp, mkate. we have employed a "fret standard" genetic construct to minimize variability in separation distances and positioning of the fused donor and acceptor fps. using spectral fret imaging and fluorescence lifetime measurements in living cells expressing the fused proteins, we have characterized both sensitized acceptor emission and the change in the donor lifetime distribution as a result of quenching for each of the fused fp pairings. our results indicate that some red fps are better acceptors than others in terms of quenching the teal donor and sensitizing the emission of the acceptor indicating a fret event. s. m. perepelytsya, s. n. volkov bogolyubov institute for theoretical physics, nasu, 14-b metrologichna str., kiev, 03680, ukraine stability of the dna double helix is determined by na + counterions, neutralizing the negatively charged phosphate groups of the macromolecule backbone. but under spatial conditions they may be replaced by much heavier ions, for example cs + . to determine the influence of heavy counterions on internal dynamics of the double helix the conformational vibrations of na-and cs-dna are studied. for this purpose the model of conformational vibrations of dna with counterions is used [perepelytsya s.m., volkov s.n., eur.phys.j. e. 24, 261, 2007] . as the result the frequencies and amplitudes of vibrations for b -dna with na + and cs + counerions are calculated. the frequencies of internal modes of the double helix are about 110, 79, 58 and 15 cm −1 . the frequencies of ion-phosphate modes are about 180 and 110 cm −1 for na-and cs-dna respectively. the calculated amplitudes of vibrations show that light counterions not disturb the dna internal dynamics, but heavy counterions make move all structure elements of the double helix. using the valence-optic approach the intensities of the dna vibrations in raman spectra are calculated. the calculations show that the ion-phosphate mode in cs-dna spectra is prominent, in contrast to na-dna spectra, where it has very low intensity. obtained results describe the intensity increase of the band 100 cm −1 in cs-dna spectra that was observed in [bulavin l.a., et al., arxiv:0805.0696v1]. s. soria 1 , f. quercioli 3 , r. mercatelli 3 , f. bianco 2 , i. cacciari 2 , g. righini 2 1 centro studi e ricerche "e. fermi", p. del viminale 2, 00184 rome, italy, 2 ifac-cnr, istituto di fisica applicata "n. carrara", via madonna del piano 10, 50019 sesto fiorentino (fi), italy, 3 isc-cnr, istituto dei sistemi complessi, via madonna del piano 10, 50019 sesto fiorentino (fi), italy we report on the application of a simple white light source based on the supercontinuum generation from commercial photonic crystal fibres to confocal fluorescence microscopy and fluorescence lifetime imaging (flim) microscopy. the coherent white light can be tuned by varying the wavelength and intensity of the pump, a ti:sapphire laser. there are several advantages jn the use of sc sources: spatially coherent white radiation, tuning ranges of approximately 400 nm, high brightness, a robust compact system (potentially all-fibre) and relatively low cost. being pulsed, sc sources are suitable for flim. we have used this system for measuring foerster resonance energy transfer (fret) in order to study interactions between ions channels and proteins of membrane within lives cells resolving the quaternary structure of plague f1 capsular antigen a. soliakov 1 , j. r. harris 1 , m. r. hicks 2 , a. rodger 2 , r. woody 3 , a. watkinson 4 , j. h. lakey 1 1 cell and molecular biosciences inst., newcastle univ., uk, 2 chemistry dept., univ. of warwick, coventry, uk, 3 biochemistry and molecular biology dept., colorado state univ., usa, 4 pharmathene uk, billingham, cleveland, uk most gram-negative pathogens express multi-subunit fibres on their surfaces that mediate host cell attachment, biofilm formation, invasion of host defenses and protection against phagocytosis. here we have studied capsular antigen fraction 1 (or caf1), secreted through the conserved chaperone/usher pathway by the plague agent yersinia pestis [1] . caf1 is highly immunogenic and is used in a recombinant subunit vaccine against plague. recent immunological studies indicated vaccines containing polymeric caf1 have higher protective efficacy than vaccines containing its monomeric variant. this difference in protective efficacy was attributed to the quaternary structure. however the quaternary structure of caf1 has not been characterized [2] . in our study we have used transmission electron microscopy of negatively stained specimen and linear dichroism spectroscopy to determine the quaternary structure of recombinant caf1. whereas electron microscopy revealed morphology of caf1 fibres bound to the surface of a carbon coated grid, linear dichroism gave the orientation of subunits in flow aligned caf1 fibres in solution. our results show recombinant caf1 comprises extended linear fibres and circularized fibres, both of which have high degree of conformational freedom. despite the vast application of lambert-beer law in biology, this empirical law cannot accurately describe the light absorption process of molecules with a long-lived excited state. this family of molecules includes most fluorescent molecules, which are very important in biology. lambert-beer law is in fact only valid at very low intensity of incident light and very low concentration of chromophore. if the system doesn't meet these conditions, it falls into a nonlinear regime when it is affected by a phenomenon which we call "dynamic photobleaching": the depletion of chromophores from the first layers, due to their transition to the excited state, leads to a sub-exponential propagation of light in the medium [1] . this phenomenon leads to the necessity of a new formula for the light absorption dynamics which depends on the lifetime of the excited state of the chromophore. the predictions of the theory were successful in describing the absorption dynamics of azobenzene [2] , but now they have been tested also on a biologically relevant molecule like chlorophyll. the results indicate that the absorbance is affected by the intensity of the incident light, and it is therefore a non reliable way of determining, for example, the concentration of the molecule. annular pupil filter to improve spatial highfrequency signal to noise ratio in linear and non linear microscopy e. ronzitti 2 , v. caorsi 1 , a. diaspro 3 1 lambs, microscobio, department of physics, university of genoa, genoa, italy, 2 semm, ifom-ieo, university of milan, milan, italy, 3 neuroscience and brain technologies department, iit, genoa, italy shot-noise significantly affects and deteriorates the imaging capabilities of typical two-photon excitation and confocal laser scanning microscope, especially in biological applications where the detected signal can be remarkable slight [1] . in particular, shot-noise substantially influences the spatial high-frequency range inducing a remarkable reduction of the optical transfer bandwidth. the insertion of an annular filter on the microscope objective lens in the illumination light pathway is here proposed to retrieve the high frequencies information loss [2] .the electromagnetic interference effect induced by the filter insertion, gives a redistribution of the optical transfer function. in particular, the microscope frequency response in filter scheme exhibits an enhancement of signal to noise ratio at the high frequencies able to recover the high frequencies hampered by shot-noise [3] . the goal of biomarker studies is to develop simple noninvasive tests that identify disease states. focus is beginning to shift from identification of individual biomarkers to identification of biomarker panels comprising multiple targets of different molecular species. there are, however, no current technologies available that allow for a comprehensive and simultaneous analysis of the expression levels of multiple cellular components, i.e. proteins and rna. surface plasmon resonance (spr) polaritons are surface electromagnetic waves that propagate in a direction parallel to the interface between a metal surface and an external medium e.g., liquid. these oscillations are very sensitive to any change of this boundary, a phenomenon that has been exploited to facilitate label-free, real-time detection of biological interactions, e.g. protein binding interactions. we are utilising the power of spr to develop technologies that facilitate diagnostic procedures for complex diseases such as alzheimer's disease and chronic obstructive pulmonary disease, through identification and detection of patterns of biomolecules indicative of disease. our approach will facilitate better disease characterisation, improve early detection strategies and aid drug discovery. surface generated fluorescence detection by supercritical angle confocal microscopy d. verdes, m. rabe, s. seeger institute of physical chemistry, university of zürich, winterthurerstrasse 190, zürich, switzerland a two channel confocal microscope for surface and simultaneously in solution fluorescence detection is presented. the microscope's core element is a parabolic mirror objective that collects the fluorescence above the critical angles for total internal reflection (tirf) of the water/glass interface. an aspheric lens incorporated into the parabolic mirror is used for diffraction limited focusing and collecting the fluorescence at low angles with respect to the optical axis. this low angles excitation approach is technically straightforward and gives an advantage over high numerical objectives that require very high angles for tirf illumination. by separating the collection of the fluorescence into supercritical and subcritical angles, two detection volumes highly differing in their axial resolution are generated at the interface. the surface selectivity of the detection volume is obtained on the basis of the dipole emission profile near a dielectric interface. its angular distribution is highly anisotropic and consists of a superposition of traveling and evanescent waves, which both are detected using the parabolic mirror objective. unlike with objective tirf microscopy, the parabolic mirror objective achieves easily diffraction limited excitation/detection volume at the water/glass interface. the objective optical performance is shown by measuring the actin cytoskeleton of cultured cells, fret energy transfer within adsorbed clustered proteins and single molecules detection. differential polarization laser scanning microscopy. anisotropy in biological samples g. steinbach 1 , i. pomozi 2 , g. garab 1 1 biological research center, hungarian academy of sciences, szeged, hungary, 2 pi vision bt., budapest, hungary differential polarization (dp) spectroscopy provides unique information on the anisotropic organization of biological samples [tinoco et al. 1987 ann rev biophys biophys chem 16:319]. however, anisotropy is often a microscopic property. the dp-lsm, constructed in our laboratory enables us to image the main dp quantities: linear and circular dichroisms (ld&cd), also in fluorescence detection ( . further applications include: periodic structure of isolated amyloids, anisotropy variations in cell walls related to drought resistance, and strong anisotropy of 'artificial chlorosomes', nanorods of synthetic porphyrins. dp-lsm might thus represent a novel tool in the better understanding of highly organized molecular macroassemblies. t. m. staudt 2 , p. bingen 1 , c. kempf 3 , h. horstmann 3 , j. engelhardt 1 , t. kuner 3 , s. w. hell 2 1 german cancer research center/ bioquant, 69120 heidelberg, germany, 2 max planck institute for biophysical chemistry, 37077 göttingen, germany, 3 institute for anatomy and cell biology, university of heidelberg, 69120 heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of <50 nm inside 3 µm thick tissue sections. -imaging and spectroscopy r. worch, t. weidemann, p. schwille biotec, biophysics group, technische universität dresden, germany interleukin-4 (il-4), a small soluble protein, is a principal regulatory cytokine, playing an important role during the maturation and clonal expansion of antigen specific b-cells in mammals. at the plasma membrane, il-4 is recognized by a receptor that consists of two single spanning transmembrane proteins: a high affinity il-4r alpha chain, and a low affinity il-2r gamma chain. it is still controversial by which molecular mechanism the signaling complex is fully activated: dimerization of chains, large conformational change upon il-4 binding or a combination of both. moreover, the influence of the lipid environment in which the activation takes place is poorly characterized. to address these issues we aim to reconstitute the receptor component in artificial membrane systems to study the various mutual interactions by means of fluorescence-based techniques, mainly fluorescence correlation spectroscopy. due to reduced background in a chemically defined system this may provide details not yet accessible in the living cell. k. wicker, s. sindbert, r. heintzmann randall division of cell and molecular biophysics, king's college london, se1 1ul london, u.k. fluorescence confocal microscopy, an indispensable tool of modern biology, allows the imaging of live fluorescent specimen with high lateral as well as axial resolution. through the introduction of a sufficiently small confocal pinhole, the lateral resolution can be enhanced compared to that of a wide field microscope. however, this gain in resolution comes at the cost of a decrease in detection efficiency, as light blocked by the pinhole is lost. we present a method for improving the lateral resolution (extending work of sandeau et al. [1] ) and detection efficiency of scanning microscopes by adding an interferometer with image inversion in one of its arms to the detection pathway [2] . this surpasses the lateral resolution achievable in a conventional confocal microscope (closed pinhole) while increasing the detection efficiency substantially. point spread function measurements for a uz-interferometer (uzi) are shown. the light in this setup follows threedimensional u-and z-shaped paths and relies on reflections off planar surfaces only in order to achieve image inversion. we achieved an interference contrast of 91% for white light, and excellent agreement with theoretical predictions. g. vicidomini, a. schönle, j. keller, r. schmidt, c. von middendorff, s. w. hell max planck institute for biophysical chemistry, department of nanobiophotonics, göttingen, germany fluorescence far-field microscopy is a indispensable tool in modern life science. however, the resolution of its standard variants is limited by diffraction to ∼200nm in the focal plane and ∼600nm along the optical axis. to overcome this limit, a new class of super-resolution microscopy techniques has been designed. one example is 4pi microscopy. to obtain isotropic resolution, 4pi uses interference of the wave fronts from two opposing lenses. the point spread function (psf) of 4pi system is characterized by multiple maxima/sidelobes, which replicate the object in the image. therefore image restoration is mandatory to render unambiguous imaging. the situation is further complicated because the positions and the relative heights of the multiple maxima/sidelobes of the psf depend on the phase difference (pd) between the two wave fronts at the focus. if the refractive index of the sample varies in space, this pd becomes a function of position and 4pi image formation process looses its shift invariance. the pd function (pdf) may not even be known a-priori and must then be estimated from the image, leading to a blind image restoration problem. here, we propose a maximum a-posteriori based method to solve the problem. we either assumed a mathematical model for the pdf that depends on a small number of parameters or allowed for an arbitrary pdf but introduced a smoothing constraint. designer multicomponent lipoplexes have recently emerged as especially promising transfection candidates, since they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes [1] [2] [3] . here, we show, for the first time, that after internalization binary complexes of lower transfection potency remain in compact perinuclear endosomes, while multicomponent systems have intrinsic endosomal rupture properties that allow plasmid dna to escape from endosomes with extremely high efficiency [4] . endosomal rupture results in an extraordinarily homogeneous distribution of unbound plasmid dna throughout the cytoplasm and in the nucleus [4] . fluorescence spectroscopy and stopped-flow technique were utilized for the study of the kinetics of incorporation of hypericin (hyp), a natural photosensitizing pigment, into lowdensity lipoproteins (ldl). triphasic kinetics of hyp association with ldl was observed when solutions of hyp and ldl were mixed together. the most rapid phase of hyp incorporation is completed within tens of msec, while the slowest one lasts 10-20 min. the most of hyp molecules are incorporated into ldl in the slowest phase. the kinetics of the incorporation of hyp into ldl particles pre-loaded with hyp were also investigated. the observed decrease of the lifetime and total intensity of hyp fluorescence with the increase of the incubation time of hyp with hyp/ldl complex is a sign of the formation of aggregates and the dynamic quenching of singlet excitation state of hyp inside ldl. to study the kinetics of a transfer of hyp molecules between ldl particles, the time evolution of the stopped-flow and time-resolved fluorescence experiments were investigated after the mixing of the complex hyp/ldl=200:1 with appropriate amounts of free ldl. for each final hyp/ldl ratio the increase of the lifetime and total intensity of hyp fluorescence was observed. the half-time of this process is similar to that one of the slowest phase of hyp incorporation into free ldl. a. bosca 1 , r. magrassi 2 , g. firpo 2 , l. repetto 2 , c. boragno 2 , u. valbusa 2 1 italian institute of technology, genova, italy, 2 nanomed labs (difi-cba), genova, italy the technique of choice to measure the electrophysiological activity of neuronal cells is the so called patch-clamp method because of its precision and sensitivity; this procedure could play a major role also in the investigation of the behavior of a biological neuronal network and so represents an important tool for understanding its functionality and for screening the effects of drugs and compounds on it. the final aim of this project is the development of a planar patch clamp device suited to measure simultaneously the electrical activity of cultured neurons associated in a network. this device will be made of polymeric disposable material and will include a microfluidic perfusion system. in order to create a smooth micro sized pore in a thin polymeric membrane we exploited the prototyping capabilities of focused ion beam etching and we extended the air moulding technique by combining it with soft moulding, obtaining micro structured substrates with the requested features. we also subjected our substrates to a chemical treatment capable of rendering its surface stable and hydrophilic and we verified that it makes them suitable for neuron culturing. use of lipoamino acids for nasal delivery of therapeutic proteins c. bijani 1 , s. sice 1 , j. elezgaray 2 , c. degert 1 , o. broussaud 1 , e. j. dufourc 2 1 physica pharma, 33600 pessac, france, 2 umr 5248 cbmn, cnrs-université bordeaux1, iecb, 33607 pessac, france most of the therapeutic proteins are clinically administered through an intravenous injection several times a day / week. because the repeated injections are not convenient and cause pain in patients, an alternative route of administration is desirable such as oral or nasal. unfortunately, proteins are easily degraded by proteolytic enzymes in the gastrointestinal tract and, therefore, have a low bioavailability when administered via oral route. physica pharma has gained experience in forming sprayable solutions combining lipoamino acids (laa) and small therapeutic organic compounds with the aim to improve their intranasal absorption. in the present work we investigated the ability of laa to complex large molecule such as human erythropoietin, human growth hormone and salmon calcitonin in order to form easily sprayable colloids. these three proteins are used respectively to treat anaemia, growth problem and hypercalcemy. circular dichroism and dynamic light scattering were used to further characterize the laa-protein colloids. a specific molar ratio of laa versus protein was found for wich the proteins keep their secondary structure and have an overall isotropic size slightly increased. molecular dynamics show that proteins are indeed coated with laa. such a complex is shown to pass very easily through a culture of nasal cells growth at confluence. in this study we describe two systems based on soft matter designed for the drug delivery and for the replacement of synovial fluid in osteoarticular pathologies. (i) a new class of temperature sensitive hydrogels pva/poly(ma m nipaam n ) shaped as microspheres obtained with a water-in-water emulsion photocrosslinking reaction. microparticles of pva/poly(ma m nipaam n ) with m:n theoretical molar ratios equal to 1:0; 1:4, 1:8 have been studied in terms of average size and responsiveness to temperature characterized by confocal laser scanning microscopy (clsm), dynamic light scattering (dls) and differential scanning microcalorimetry (dsc). (ii) a physical network based on hyaluronic acid with a small extent (degree of substitution: 1%) of hydrophobic moiety grafted on the backbone, hyadd4, has been characterized in order to account for the influence of thermal treatment on the stability of the hydrogel. dynamic light scattering (dls) and small angle neutron scattering (sans) have been used for dynamic-structural characterization of hyadd4 hydrogels. diffusion of macromolecular probes has been studied by fluorescence recovery after photobleaching (frap) to study the mesoscopic texture of the hydrogel and molecular dynamic (md) simulations were used to approach the time evolution of the physical junction points and of chains clusters. multi-scale estimation of water soluble diffusivity in polysaccharide gels g. eisele 1 , s. bertini 1 , d. paganini 1 , l. piazza 2 1 ronzoni institute, milan, italy, 2 distam, university of milan, italy diffusion properties in gels play important role in food and biotechnological applications. an attractive goal is to design gels in such a way that the active molecules are delivered by the material according to specific release sequences. the transport of macromolecules within polymeric gels depends: on the obstruction effects of the surrounding gel strands, on the molecular interactions between the gel and the solute, on the interactions between the solute molecules themselves and the interactions between the solute and the solvent. physical polysaccharide gels were evaluated in this work to probe diffusion over both microscopic and macroscopic distance scales. physical gels from agar and starch were investigated by high and low resolution nmr techniques in order to characterize their structures. obstruction effects of the surrounding gel strands were considered by studying diffusion of glucose. local diffusion, due to brownian motion, was quantified by low resolution nmr spectroscopy. the fickian diffusion coefficient was measured by modelling experimental concentration-distance curves obtained by means of a two-compartment diffusion-cell. diffusion coefficients depend on the viscoelastic properties of the gel matrix and on water-polysaccharide interactions. j. l. cuellar, g. koehler, m. fischlechner, e. donath medizinische institut für physik und biophysik, leipzig, germany rather than being just pathogens, from the view point of biotechnology and materials science viruses can be regarded as nanocontainers in which nucleic acids have been enclosed in a protective self assembled protein cage. in some viruses an additional lipid-protein envelope wraps the capsid shell. the capsid is constituted of several copies of one single protein subunit or just a few, arranged in a regular fashion showing icosahedral symmetry. the mechanical properties of the cage are important for understanding the infection mechanism involving the release of the encaged genome. by means of atomic force microscopy, it becomes possible to probe such material properties by nanoindentations of single viral particles. it is very interesting to learn how strong or brittle a virus can be. here we have studied the mechanical properties of empty rubella virus particles (rlps) due response of external applied forces. we found that rlps are extremely soft comparable to that of some rubber materials. a peculiarity of the rubella virus is that the capsid is considerably smaller than the surrounding shell filling only a fraction of the lumen provided by the envelope. the envelope in rubella has a distinguishable response on the material properties of the virus. deformation and fracture of the capsid requires comparatively larger forces. our results indicate that the ph is a major factor influencing on rubella particle material properties. this can be related to the infection mechanism. pentavalent antimony (pa) (glucantime, sanofi-aventis or glaxo) are the mainstream agents of choice for leishmaniasis treatment. in therapeutic doses the pa treatment has cardiac side effects like electrocardiographic (ecg) alterations, that include qt segment prolongation, t wave flattening or inversion, inversion of st segment, p, r and t waves amplitude reductions, torsade de pointes arrhythmias and sudden death by cardiac arrest. the objective of this study was to characterize the arrhythmogenic potential of pa. we used 30 guinea-pig to assess the chronic effects of pa therapeutic dose on corrected qt interval, qt dispersion, ventricular action potential (ap) amplitude and duration at 30% and 90% of maximal repolarization and survival rate. guinea-pig received daily 30mg/kg pa or saline for 15 days. eight lead ecg were recorded before and in the last treatment day. at the end the animals were killed and the left ventricle papillary muscles excised for ap recording with the intracellular microelectrode technique. our results of chronic pa treatment showed significant increase of qrs complex duration, qt interval duration, qt dispersion and incidence of t wave flattening or inversion and arrhythmias. the ap analysis demonstrated prolongation at 90% duration. the treatment was lethal in 30% of the animals. we concluded that pa is a proarrhythmic drug that upon chronic use may causes arrhythmias and mortality by disturbances in the ventricular repolarization process. m. m. khvedelidze 1 , t. j. mdzinarashvili 2 , t. partskhaladze 1 , n. nafee 3 , m. schneider 4 1 ivane javakhishvili tbilisi state university, tbilisi, georgia, 2 institute of molecular biology and biological physics, tbilisi, georgia, 3 biopharmaceutics and pharmaceutical technology, saarbrücken, germany, 4 pharmaceutical nanotechnology, saarbrücken, germany we have studied the thermodynamical properties of chitosan-coated nanoparticles (cnp) and non-coated nanoparticles (np) and have gained some insights about plga nanoparticles' properties using supersensitive differential microcalorimetry. the experiments show that in a wide ph interval the changes in transition temperature did not take place. it was shown that such nanoparticles could be used in acidic surrounding for drug transfer. stability and their other properties are less depended on either the particles were in bidistilled or deionized water, or the suspension of particles were located in buffer. to determine the interaction of plga nanoparticles with dna. in the case of dna presence in cnp solution the calorimetric experiments show that the heat absorption peak is constricted, what biophysically means that interaction between them takes place. for more exact determination the contribution of cnp in spectrum, we have compared the spectra of pure dna with the spectrum of the same concentration dna plus cnp. the optimal ratio for dna loading onto the cnp was found to be 7:1. protein-based "epoxy-like" physical hydrogels for stem cell transplantation s. c. heilshorn, c. wong po foo, j. s. lee materials science and engineering, stanford univ., u.s.a. stem cell transplantation has emerged as a promising therapy for multiple injuries and diseases; however, cell survival after transplantation is often poor and unpredictable. we hypothesize that co-injection of stem cells encapsulated within an optimized physical hydrogel will enhance viability. whereas, current physical hydrogels require a shift in environmental conditions (e.g., ph, temperature) to initiate the sol-gel phase transition during encapsulation, our newly designed molecular-recognition gels do not require environmental triggers. instead, these "physical epoxy-like" gels consist of two components that undergo a sol-gel transition upon mixing due to specific hydrogen bonding. the gel viscoelasticity is predictably tuned through precise variation in the molecular-level design of the two components, created using recombinant protein techniques. the design of the two components is based on simple polymer physics considerations and utilizes bio-mimimcry. adult neural stem cells or mesenchymal stem cells are encapsulated within these gels with high viability at constant physiological conditions. the gels promote the growth and differentiation of neural progenitors into neuronal phenotypes, which adopt a 3d-branched morphology. the gels are further optimized for use in the central nervous system by tethering neuroprotective peptides to the gel through molecular-recognition sites. these peptides are released-on-demand by cells through the action of proteolytic enzymes. we consider the integration of the portal protein of the bacteriophage virus φ29 into lipid bilayers of giant unilamellar vesicles (guvs) membranes with the aim of constructing a functional cargo-device able to transport dna and later translocate it outwards. our nano-engineering plan consists of growing guvs from bilayer membranes built up from proteoliposomes previously prepared by extrusion. we have designed two alternative chemical routes for integrating the portal protein in the lipid bilayer, the first considering the native protein and the second a mutant modified with a hydrophobic belt made of histidine-tags. in our contribution we will present details on the different nano-engineering strategies and experimental evidence about the integration of the portal protein in the membrane with an orientation adequate to allow for functional dna translocation. p.-h. guelluy 1 , m.-p. fontaine-aupart 2 , m. hoebeke 1 1 biomedical spectroscopy, ulg, belgium, 2 laboratory of molecular photophysics, univ. of paris-sud, france ppme is a second generation photosensitizer (ps), and a promising candidate for photodynamic therapy (pdt) treatment. we have previously demonstrated that ppme can be easily and efficiently encapsulated in dmpc liposomes, used as ps-vector. we therefore compared the photophysical and photochemical properties of free and encapsulated ppme incubated with human carcinoma cells. absorption and fluorescence microspectroscopy as well as flim analysis allow evaluating the aggregation state of ppme inside the different cellular organelles and the extracellular medium. confocal microscopy established undoubtedly the colocalization of ppme, by robust probabilistic exclusion method, within mitochondrion (central siege of apoptosis). after ps activation (4h and 24h), the balance apoptosis-necrosis was double-estimated by facs device and fluorescence confocal microscopy. quantification of hydroxyl radical was purchased by spin trapping-esr spectroscopy and quenching technique. all these techniques required peculiar settings because of the fluorescence activity of ppme. these results allow to ascertain that the vectorization of ppme affords a better cellular penetration and a monomeric state of ppme. the presence of ppme inside mitochondrion orientated the cellular death in apoptosis 4h following ps-activation. but necrosis is the major actor 24h after treatment. active substrates to study mechanotransduction j. le digabel, p. hersen, b. ladoux laboratoire matière et systèmes complexes, université paris diderot -cnrs, paris, france cellular processes imply an important coordination of interactions with the extracellular medium. accumulating evidences demonstrate that cell functions can be modulated by physical factors such as the mechanical forces acting on the cells and the extracellular matrix, as well as the topography or rigidity of the matrix. these extracellular signals can be sensed by mechanosensors on the cell surface or in the cell interior to induce various cell responses. we have developed an original approach based on micro-fabricated substrates of polydimethylsiloxane (pdms) to study cell migration. we used a closely spaced array of flexible micropillars (diameter∼1?m) to map the forces exerted by cells on their substrates. in this case, the micropillars act as passive force sensors. here, we propose to analyze the cell response to an external applied stress by a well-controlled actuation of the substrate. to do so, we used magnetic pillars. such substrates allow us to modify dynamic adhesion conditions of cells and to better understand the coupling phenomena between mechanical sensing and biochemical activity of a living cell. using polyacrylamide hydrogels doped with ferromagnetic iron oxide particles or ferrofluids, we can make magnetic pillars with diameters of 5 to 10 microns while a magnetic field can be locally applied with a magnetic needle. such a technique will be helpful to study the mechanical response of cells to an external force or to local changes in their microenvironment. glucose scavenging activities of pamam dendrimers m. labieniec 1 , t. gabryelak 1 , c. watala 2 1 university of lodz, deptartment of general biophisics, lodz, poland, 2 glycation is a spontaneous, non-enzymatic modification of biomacromolecules with hexoses, mainly glucose. in terms of its pathophysiological relevance, it targets predominantly proteins, but also nucleotides and phospholipids, and is of major importance to both physiology (ageing) and pathology (diseases with a metabolic background, like diabetes). earlier we demonstrated in vivo that the administration of pamam g4 to diabetic rats resulted in a significantly reduced blood glucose, as well as the early (hba 1c ) and late (ages) protein glycation products. in this study we investigated the ability of pamam g2 (16 surface nh 2 groups) and g4 (64 nh 2 ) to inhibit glycation of proteins in plasma, and a model protein -bsa. pamam g2 and g4 competed chemically with protein nh 2 for the binding of glucose, and hampered protein glycation. in a high-glucose medium they underwent an excessive glycation themselves. this modification was more effective in pamam g2, in which surface nh 2 were more mobile and accessible. pamam modification with glucose rendered these dendrimers less polycationic in aqueous solutions. pamams neither affected bsa conformation nor formed stable complexes with a protein. we conclude that pamams are very effective glucose scavengers. thus, even less toxic pamams of lower generations, like g2, may appear useful in further medical applications as the agents attenuating the detrimental effects of sever hyperglycaemia on biomacromolecules. selective drug delivery and novel drug approaches by polyelectrolytes s. krol cbm-cluster in biomedicine, area science park, s.s. 14, 163.5 km; trieste, italy the use of polyelectrolytes in the past was mainly related to targeted drug delivery and nanoparticle preparations for medical applications. but rarely the polyelectrolytes were investigated for the own features as a drug. in our present work we use a special physical feature of cancer cell membranes as a target for a specific polycation. we found that the polycation is selectively up-taken by cancer cells (leukemia, hepatocarcinoma, cancer stem cells) while normal cells remain unaffected. another interesting application of polyelectrolytes are in form of a multilayer coating on the surface of nanogold particles. for this topic, two new approaches were developed for a drug delivery through the blood brain barrier. in the first case, high amounts of creatine were bound to the gold particles and delivered as protective agents for ischemic stroke (viota et al., j colloid interface sci. 2009, 332 (1):215-23). in the second case coated multifunctional gold particles were prepared as a drug for neurodegenerative disease on the basis of protein aggregates. the use of gold as core for coated nanoparticles offers the possibility to study our systems as "theranostics", system which are modified to recognize selectively diseased cells and carry the moieties to treat or destroy the malfunctioning cells. possible treatments can be local hyperthermal therapy by using the particles as amplifier and enhancer or photodynamic therapy with gold as a drug. m. koneracka 1 , v. zavisova 1 , m. muckova 2 , p. kopcansky 1 , a. jurikova 1 , n. tomasovicova 1 , g. lancz 1 , m. timko 1 , m. fabian 3 1 institute of experimental physics, slovak academy of sciences, košice, slovakia, 2 hameln rds a.s., modra, slovakia, 3 institute of geotechnics, slovak academy of sciences, košice, slovakia the aim of this study was to develop biodegradable and biocompatible paclitaxel loaded magnetic plga nanospheres (nps) suitable for biomedical applications. biodegradable poly(d,l-lactic-co-glycolic acid) (plga) was used as a capsulation material and the magnetic fluid was used as a magnetic carrier. incorporation of magnetic particles and drug in the plga polymer matrix was confirmed by infrared spectroscopy (ftir) and differential scanning calorimetry (dsc). the release of the drug from the prepared nps to the surroundings under the different conditions was studied also. the prepared magnetic plga nps with encapsulated paclitaxel (spherical shape, size 200 -250 nm) have good stability in the presence of high nacl concentration at 25 • c, the toxicity of prepared samples declared 3 times higher value of lethal dose ld 50 in comparison with pure paclitaxel (ld 50 = 33mg/kg) and showed the significant response to external magnetic field which is useful from the point of view to achieve pharmaceutically acceptable drug delivery systems for tumour treatment. success of human gene therapy depends upon the development of delivery vehicles or vectors, which can selectively deliver therapeutic genes to target cells with efficiency and safety. many cationic polymers have been used to condense dna by electrostatic interaction into small particles (polyplex), for protecting the dna from degradation and enhancing uptake via endocytosis. polyethylenimine (pei) appears to be one of the most advanced delivery system that can condense dna efficiently forming pei-dna polyplex complex. the physicochemical properties of different molecular weights of pei, such as condensation ability, buffer capacity, time kinetics, ftir and surface charges of the pei-dna complexes may be important factors to obtain a higher transfection efficiency of the polycation vectors. our intent in this study was to characterize pei-dna complexes to see whether these physicochemical properties have any influence on their disposition characteristics and cellular uptake process. we found that pei-dna complexes, obtained by the 25 k pei at n/p ratio > 4, were more stable in the presence of tissue culture medium & serum, and did not dissociate in nacl easily. key words: polyethylenimine, polyplex, polycations, dna, transfection, gene delivery. effect of nanopatterned substrate on neuronal growth cones activity in the last20years an increasing interest has been address to explore, at the level of the single cell, the physical interaction between neurons and micro-and nano-patterned surfaces which mimic the biological environment and can induce specific biological behavior. a consistent number of substrates have been tested (nano-grooves, pillars, gap nanowires) but the effect of nano-topographical features at subcellular level e.g. branching and pathfinding of growth cones(gc)is still unexplored. using nanoimprinting lithography we fabricated gratings on glass with grooves of variable pitch, depth and width all in the nm range. embryonic stem cell derived neurons were seeded on nanostructured and on flat control glasses. we investigate gc morphology with nm resolution by afm. a significant effect on sub-cellular architecture was observed.on nanopatterned substrates72% of gc were branched with a large number of long and thin filopodia(average length and height4.3µm and 75nm)while on control only34%of gc were branched with an higher percentage of long and thick filopodia (average length and height 6,5µm and 300nm). on the contrary we did not observe a significant influence of the nanopatterning on the alignment and elongation of neurites. in both cases the distribution of angles between axon and filopodia showed a preferential direction at30 • . in conclusion, the tested nanopatterns do not influence the neurite directions but do enhance the gc morphology and explorative activities. growth enhancement and adhesion control of pc12 on micropatterned ns-tio 2 thin film c. lenardi 1 , a. v. singh 2 , p. milani 3 1 c.i.ma.i.na., dip. di scienze molecolari applicate ai biosistemi, università di milano, italy, 2 c.i.ma.i.na., semm, european school of molecular medicine, fondazione ifom, milano, italy, 3 c.i.ma.i.na., dip. di fisica, università di milano, italy cluster assembled nanostructured titanium dioxide (ns-tio 2 ) has been explored as novel substrate for in vitro cell culture. in this work, we report micropatterned ns-tio 2 thin film as putative microdevice for neuron culture and growth. in additions, we show a simple scheme of molecular patterning of bovine serum albumin (bsa) as cell anti-adherent substrate complementary to ns-tio 2 micropattern which favors a selective spatially confined adhesion of neurons. bsa was drop coated and physisorbed over glass coverslip covered with a thin conducting layer of indium tin oxide (ito) and then pmma was spin coated over it using standard protocols. later, using combinations of e-beam lithography and pulsed microplasma cluster source (pmcs), a thin layer of ns-tio 2 was deposited over micropatterned pmma. further, lift off process enabled us to generate complementary micropatterns of hydrophobic bsa (cell repellent) and hydrophilic ns-tio 2 (cell adhesive). cell culture studies have confirmed that pc12 cells like to grow on ns-tio 2 substrate and not on bsa layer. the technique offers a novel approach for neuronal cell assay applications. gene delivery with chitosan: influence of chain length on intracellular trafficking and dissociation s. lélu 1 , c. d. l. davies 1 , n. reitan 1 , s. p. strand 2 1 department of physics, ntnu, trondheim, norway, 2 department of biotechnology, ntnu, trondheim, norway chitosan, a cationic polysaccharide presenting low cytotoxicity, is a promising nonviral gene delivery vector. chitosan complexes dna into nanoparticles, and the complexation and cell transfection efficacy are function of the chitosan/dna ratio and of the intrinsic properties of chitosan, i.e. degree of polymerization dpn, charge density, and molecular structure. nanoparticles formed by shorter chitosan (dpn31-41) mediated higher transgene expression than nanoparticles based on longer chitosans. the purpose of this work is to relate the dpn of linear chitosan with the cellular uptake, intracellular trafficking and dissociation of chitosan-pdna complexes, measured by confocal laser scanning microscopy and fluorescence correlation spectroscopy (fcs). cells were incubated with oligomers (dpn 31 and 88) either free or complexed with plasmid-dna. both chitosan oligomers seem to penetrate cell nucleus in association with or free from pdna. 24 hours after incubation, accumulation of oligomers in cell nucleus is similar for free and complexed dpn31, whereas it increases for complexed dpn88 compared to free, indicating a possible delayed dissociation of the complexes based on dpn88 in the nucleus and suggesting a dissociation of pdna-dpn31 in cytoplasm. these data are consistent with previous studies which suggested that longer chitosan chains led to tighter complexes, and hence to a delayed dissociation process and lower transfection efficiencies compared to shorter chitosans. material surface properties greatly influence dna purification and pcr yield in microsystems c. potrich 1 , l. lunelli 1 , l. marocchi 1 , l. pasquardini 1 , g. guella 2 , d. vozzi 3 , l. vanzetti 1 , p. gasparini 3 , c. pederzolli 1 1 fbk, trento, italy, 2 univ.trento, italy, 3 univ.trieste, italy modern microchip platforms integrate dna purification, target amplification by pcr and dna detection in a single device. combination of these processes minimizes sample loss and contamination problems as well as reduces analysis time and costs. different strategies are available to perform dna extraction on a chip. here we exploited amino-coated materials as a tool for specific binding of dna through the electrostatic interaction between amine groups and nucleic acids. we analyzed the ability of different treated substrates to selectively absorb/desorb the genomic dna with the aim to purify dna from unwanted components. amino-coated substrates were characterized by afm, xps, fluorescence microscopy and absorption spectroscopy to define the surface chemical and morphological properties. the distribution of the dna adsorbed on materials was homogeneous and the eluted dna was tested for pcr. same materials were analyzed for their compatibility with pcr and the use of different enzymes and reagents or proper surface treatments was employed. we established the best conditions for dna amplification in silicon/pyrex microdevices depending on the type and the fabrication method used and on the quality of reagents more than on the passivation treatment or increment in standard taq polymerase concentration. nanoporous alumina fabricated using unconventional acids for enhanced biomolecular physisorption n. patra, m. salerno, r. losso, r. cingolani italian institute of technology, genova, italy in the last decades the available porecell sizes of porous alumina have been extended, but operating conditions to obtain some pore diameters still have to be optimized, particularly below the 50 nm limit. in this range the use of biocompatible porous alumina thin films could find applications in biosensors, after functionalization by appropriate physisorbed biomolecules. while pore ordering and film growth rate are mainly influenced by the electrical parameters of voltage and current, respectively, the typical pore size primarily depends on the electrolyte. in the search for alternative anodization conditions, we have investigated several acids that have never been used before, namely gallic, lactic, propionic, and glycolic acid. the anodizations were carried out in galvanostatic conditions at fixed concentration and durations, by varying the current. atomic force microscopy was used to test the oxidized surface morphology. in particular, lactic and propionic acids demonstrated feasible. lactic acid gave best results for ∼30 ma/cm 2 current density, corresponding to roughly constant ∼15 v potential, with resulting pore diameter in the 4060 nm range, whereas propionic acid performed best for ∼5 ma/cm 2 , corresponding to ∼43 v, resulting in 7090 nm pore diameter. both kind of films looked lightgray, different from the yellowish oxalic acid porous alumina, which can be a hint of lower ion contamination. chitosan-arabic gum nanoparticles as potential vehicles for peptide delivery l. moutinho, s. rocha, m. coelho, m. c. pereira lepae, chemical engineering department, faculty of engineering, university of porto, portugal chitosan (cs) -arabic gum (ga) nanoparticles were produced by an ion-ion interaction process, using different weight ratios of polysaccharides. according to zeta potential (zp) measurements, particles are ionically stable. zp values ranged from 30 to 70 mv for cs:ga ratios of 1:3.3 and 1:0.8, respectively. cs:ga 1:5 yielded uncharged nanoparticles and aggregates, showing that, at this ratio, the negative charges of ga neutralize the positive charges of cs. the particle diameters ranged from 300 to 500 nm, as measured by dynamic light scattering (dls), and displayed a slight tendency for decreasing as the ga concentration increased. transmission electron microscopy (tem) images showed numerous spherical nanoparticles. peptides were encapsulated within the nanoparticles, by mixing them with chitosan solution prior to adding ga. this system can protect short peptides from rapid metabolization, prolonging their blood half-life. physical characterization of nanocarriers for drug delivery s. motta 1 , y. gerelli 2 , g. sandri 3 , e. ricci 3 , p. brocca 1 1 dept. of chem, biochem and biotech for medicine -university of milan, italy, 2 dept. of physics -university of parma, italy, 3 dept. of pharmaceutical chem -university of pavia, italy nanoparticles used as nanocarriers for pharmaceuticals can improve solubility and bioavailability of problematic drugs and protect labile or toxic molecules. fundamental parameters like drug encapsulation efficiency and release kinetics are tuned by the physico-chemical features of the drug/carrier complex. a challenging aspect of the pharmaceuticallyoriented issues resides in the relatively low number of molecules allowed to build up nanosystems with different properties and morphology, according to the specific drug and to the intended therapy. we have studied: 1) soybeanlecithine/chitosan nanoparticles for progesterone and tamoxifen delivery; 2) solid-lipid nanoparticles (compritol 888 ato, poloxamer 188, tween 80 and chitosan) for cyclosporine-a delivery via ophthalmic formulation. both void carriers and drug-loaded nanoparticles have been studied, to understand how the structure of the carriers can be modified by the active molecules. several non-invasive physical techniques have been used to achieve a detailed knowledge of the systems: a) dynamic light scattering for the dimensional distribution of the nanocarriers, b) zeta potential to determine surface charge, c) cryo-tem for morphological analysis d) x-ray scattering (in small angle configuration) and dsc to access information on the inner structure. . a. schollier 1 , a. halperin 3 , m. sferrazza 2 , g. fragneto 1 1 institut laue-langevin, grenoble, france, 2 universite libre de bruxelles, belgium, 3 cnrs-ujf grenoble, france protein adsorption on surfaces is responsible of several unwanted effects in technological and pharmaceutical applications: fouling of contact lenses, clotting in blood containing devices, inflammation around artificial organs for instance. those phenomena can be repressed with certain types of polymer brushes, in particular peg (polyethylene glycol) brushes, and a better understanding of the mechanism of adsorption could lead to improvements in the design of biomaterials. we have developed a theory describing this mechanism and carried out measurements to confirm the theoretical predictions [langmuir 2007, 23, 10603] . three cases are predicted: primary adsorption at the grafting surface, secondary adsorption at the outer edge of the brush, and ternary adsorption within the brush itself. we prepared samples with different grafting densities and different degrees of polymerization and used different protein size and concentrations. neutron reflectivity experiments, able to determine the structure and composition of material interfaces with a fraction of nanometer resolution, were carried out and, by using deuterated proteins (anti-freeze protein, dihydrofolate reductase and myoglobin) on different peg compositions and grafting densities, it was possible to locate the proteins in the brush and to distinguish between the different kinds of adsorption : primary adsorption is dominant for short brushes and ternary adsorption for long brushes. effect of powder air polishing on nanocomposite dental materials measured by atomic force microscopy m. salerno 1 , g. derchi 2 , a. m. genovesi 2 , l. giacomelli 2 1 italian institute of technology, genova, italy, 2 istituto stomatologico tirreno, lido di camaiore, italy in dentistry, airpolishing with glycine and bicarbonate powders is widely used to remove accumulated plaque. however, the resulting teeth and gingival surface roughness, which is a risk factor for further plaque accumulation, has to be considered. in this in vitro study the effect of the above mentioned technique on the surface of a novel nanocomposite dental material is evaluated by means of atomic force microscopy (afm). square specimens (0.5×0.5 cm 2 size) were airpolished either with glycine or bicarbonate, using different application times (2, 8, 30 sec) and distances (2, 4, 6 mm) . four specimens were evaluated for each timedistance combination, and checked versus untreated specimens (controls). the specimens were imaged with tappingmode afm at two different scan sizes (3×3 and 30×30 µm 2 ) in two different regions for each sample. the surface roughness was measured as the rms of the feature heights. for the 2 sec2 mm group airpolishing resulted in an increased surface roughness as compared to the controls; however, glycine was associated with a lower roughness than bicarbonate (glycine: 238±91 nm; bicarbonate: 436±75 nm; controls: 14±14 nm; p<0.0001 for treated specimens vs controls, p<0.001 for glycine vs bicarbonate, anova test). similar results were obtained for all the other timedistance combinations. the work comprises the design of polysaccharide based nanoparticles for drug delivery. chitosan/arabic gum and arabic gum/maltodextrin nanoparticles were prepared by polyelectrolyte complexation and spray drying [1] , respectively. dynamic light scattering characterization established that arabic gum/maltodextrin nanoparticles are more polydisperse (diameters ranging from 8 to 400 nm) than chitosan/arabic gum particles (diameters of approximately 500 nm). scanning electron microscopy measurements demonstrated that the particles are spherical and have a smooth surface. the systems are highly stable to forces up to 80 nn as observed by atomic force microscopy. due to their nature they are hydrophilic and biodegradable. the nanoparticles were used to entrap short peptide sequences and antioxidants and were proved to be efficient in maintaining the biological activity of the molecules. kyotorphin (ktp) was found in 1979 in bovine brain. despite revealing remarkable analgesic properties, analgesia was only induced after central delivery. this limited ability to cross the blood-brain barrier (bbb) combined with the unknown mechanism of action largely confines its pharmacological use. to surpass these problems, we designed new ktp derivatives: ktp-rc and ktp-rc-lipogen. biophysical studies were carried out using fluorescence methodologies to characterize the peptides' interaction with biomembrane model systems. partition coefficient quantification showed a clear preference of the derivatives towards fluid zwitterionic and anionic membranes. moreover, a relationship between anionic lipid percentage and in-depth insertion in membrane was established. additionally, studies with fluorescent probe di-8-anepps revealed different membrane-interaction profiles of morphine and ktp-derivatives, suggesting distinct actions between them. the analgesic efficacy of the compounds was studied in vivo after systemic administration in models of acute and tonic pain. unlike ktp, both ktp-rc and ktp-rc-lipogen displayed high efficacy from doses as low as 1.67 and 0.85 mg/100g, respectively, indicating bbb crossing. the observed correlation between higher partition/insertion in the membrane and enhanced analgesic action proved the biophysical rationale to be a powerful strategy for early screening in cns drug development. metabolic syndrome (ms) is now regarded as a major risk factor for cardiovascular disease. as prooxidant/antioxidant balance is disturbed in the course of this disorder, it is possible that melatonin may exhibit protective effect against oxidative damage of blood cells. ms was diagnosed according to international diabetes federation definition (2005) . the investigated group consisted of patients before and after the melatonin supplementation (5 mg per day for two months) and was compared to control group of healthy individuals on normal diet. our experiments show that erythrocytes from patient group exhibit significantly higher tbars and total cholesterol levels whereas the protein thiol concentration, na + k + atpase and glutathione peroxidase activities were decreased in comparison to those of healthy volunteers. after two month melatonin supplementation, tbars and cholesterol concentrations significantly decreased, whereas the na + k + atpase, catalase and glutathione peroxidase activities increased. the glutathione concentration was also higher. these results show that melatonin supplementation has a protective effect on erythrocytes of ms patients. action of ferritin, a nanoparticle model, on ros formation and glutamate uptake in synaptosomes t. waseem, a. alekseenko, s. fedorovich institute of biophysics and cell engineering, minsk, republic of belarus nanoparticles are currently used in medicine as agents for targeted drug delivery and imaging. however it has been demonstrated that nanoparticles induce neurodegeneration in vivo and kill neurons in vitro. the cellular and molecular bases of this phenomenon are still unclear. we have used the protein ferritin as a nanoparticle model. ferritin contains iron particles (fe 3+ ) with size 7 nm and a protein shell. we investigated how ferritin influences uptake and release of [ 14 c]glutamate and free radical formation as monitored by fluorescent dye dcfda in rat brain synaptosomes. we found that even a high concentration of ferritin (800 µg/ml) did not induce spontaneous [ 14 c]glutamate release. in contrast the same concentration of this protein inhibited [ 14 c]glutamate uptake two fold. furthermore ferritin induced intrasynaptosomal ros (reactive oxygen species) formation in a dose-dependent manner. this process was insensitive to 30 µm dpi, an inhibitor of nadph oxidase and to 10 µm cccp, a mitochondrial uncoupler. these results indicate that iron-based nanoparticles can cause ros synthesis and decrease glutamate uptake, potentially leading to neurodegeneration. functionalized carbon nanotubes 1 (cf-cnts) have a promising future as vectors for drug delivery 2 and for neuronal cell growth and communications 3 . it has been demonstrated that cnts can be employed in drug delivery systems. the toxicological properties of cnts result strictly correlated with nanotube solubility 4 . in this study we focused our attention on multi-walled cnts (mwcnts) because they are easy to manipulate and we covalently functionalize them in different ways to increase their solubility.. we grew different derivatives of a dendrimer on mwcnts surface with positive charges at the periphery, in order to study the different solubilisation properties and correlate them to the biological activity in gene therapy 5 . slowdown of 1-40 β-peptide aggregation by addition of two synthetic biocompatible polymers p. sciortino 1 , r. carrotta 1 , g. cavallaro 2 , d. bulone 1 , p. l. san biagio 1 1 ibf-cnr, palermo, italy, 2 ctf dept., university of palermo, palermo, italy fibril deposit formation of amyloid β-protein (aβ) in the brain is a hallmark of alzheimer disease (ad). fibrils formation is triggered by molecular conformational changes and protein-protein interactions involving partially unfolded regions of different aβ peptide molecules. increasing evidence suggests that toxicity is linked to diffusible aβ oligomers, which have been found in soluble brain extracts of ad patients, rather than to the insoluble fibres. new therapeutical approach, based on searching molecules capable of regulating the peptide aggregation, is currently developing. here, we study the effects on the aggregation of aβ 1-40 peptide of two different synthetic polymers with structure similar to that of a protein (α,β-polyasparthylhydrazide -pahy and α,β-polyasparthylhydrazide-polyethyleneglycol -pahy-peg). static and dynamic light scattering measurements showed that the aggregation kinetic is slowed down by the presence of both polymers. optical microscopy revealed the presence of aggregates of different dimension in all samples. transmission electron microscopy allowed to establish that all aggregates are made of fibers, as confirmed by fluorescence spectroscopy measurements on thioflavine binding. a. smorodchenko 1 , d. sittner 1 , a. rupprecht 1 , j. goyn 1 , a. seiler 2 , a. u. bräuer 1 , e. e. pohl 1 1 institute of cell biology and neurobiology, charité-universitaetsmedizin, berlin, germany, 2 german federal institute for risk assessment, zebet, berlin, germany ucp4 is a member of the mitochondrial anion transporter family and one of the three ucps (ucp2, ucp4 and ucp5) associated with the nervous system. in our previous work we have shown that ucp4 appears in brain at embryonic day 14 (e14). we hypothesized that the ucp4 expression can be related to neuronal differentiation. to prove this idea we now investigated protein and mrna levels in two different systems: (i) mouse embryonic stem cells of line d3 (mesc) and (ii) brains from pre-and postnatal mice. ucp4 was not present in undifferentiated mesc. during differentiation of mesc in neurons the expression of neuronal marker map2 and ucp4 started at the same time -as early as on day 7 in culture -and were increasing simultaneously. (ii) the comparative analysis of gene transcripts prepared from whole embryos, brains and different brain regions (neocortex, hippocampus, cerebellum) demonstrated that levels of ucp4 mrna were increasing from e8 till e19, reached an expression peak between e19 and postnatal day 0 (p0) and remained constant in adult animals. in contrast, expression of ucp5 was increasing permanently until birth, whereas ucp2 expression was invariant in time. our results suggest that ucp4 contributes to specific neuronal functions. the plant hormone abscisic acid stimulates the proliferation of human hemopoietic progenitors through the second messenger cyclic adp-ribose s. scarfì 1 , c. fresia 2 , c. ferraris 1 , s. bruzzone 2 , f. fruscione 1 , c. usai 3 , m. magnone 2 , m. podestà 4 , l. sturla 2 , l. guida 2 , g. damonte 2 , a. salis 2 , a. de flora 2 , e. zocchi 2 1 advanced biotechnology center, genova, italy, 2 dept of experimental medicine, biochemistry section, and cebr, university of genova, italy;, 3 institute of biophysics, cnr, genova, italy, 4 stem cell center, s. martino hospital, genova, italy abscisic acid (aba) is a hormone involved in pivotal physiological functions in higher plants. recently, aba was demonstrated to be produced by human granulocytes, β pancreatic cells and mesenchymal stem cells (msc) and to stimulate cell-specific functions. here we show that aba expands human hemopoietic progenitors in vitro, through a cadpr-mediated increase of the intracellular calcium concentration. incubation of cd34 + cells with micromolar aba also induces transcriptional effects, which include nf-κb nuclear translocation and transcription of cytokines encoding genes. stimulated human msc produce and release aba at concentrations sufficient to exert growth-stimulatory effects on co-cultured cd34 + cells, as demonstrated by the inhibition of colony growth in the presence of an anti-aba monoclonal antibody. these results provide a remarkable example of conservation of a stress-hormone from plants to humans and identify aba as a new hemopoietic growth factor involved in the cross-talk between hp and msc. t. golovanova, g. b. belostotskaya sechenov institute of evolutionary physiology and biochemistry, russian academy of sciences, saint petersburg, russia a subpopulation of small cells (volume 141±6 µm 3 ) have been found in the neonatal cardiac myocytes culture whereas the same parameter of the remaining cells was 819±68 µm 3 on the 1 st day. in contrast to main part of hypetrofied cardiomyocytes, the small cells were able to proliferate, form colonies and differentiate spontaneously into cardiac myocytes in the culture. on the 8 th day there were slight, slow (2-3 beats/min), arrhythmic contractions in the centre of colonies. pulsing cells were not united by common contraction and had individual beating profile. on the 11-12 th days, the colonies displayed comprehensive contractile activity with the pulsation rate 25-28 beats/min and reached 46 beats/min on the 23 d -25 th day and 58 beats/min on the 26-30 th days in the culture. it has been shown that receptors of the surface membrane and sarcoplasmic reticulum of colony cells gradually mature. by estimating the ca 2+ transition under the specific agonists action: acetylcholine, kcl and caffeine, we have detected the activity of basic structural and functional elements of excitation-contraction coupling in contractile cells inside colonies. the small cells ability to proliferate and differentiate under the influence of near mature cardiomyocytes allows us to put forward a hypothesis, that they belong to a category of resident stem cells. changes in the gating properties of na + channels were studied in es-derived neural stem (ns) cells during in vitro neuronal differentiation with the use of the whole-cell and cell-attached variants of patch-clamp technique. ns cells represent a novel stem cell population remaining stable and highly neurogenic over multiple passages.voltage-clamp recordings during neuronal differentiation of ns cells indicated significant changes in the key properties of na + currents. a voltage-gated and tetrodotoxine-sensitive na + current,absent under self-renewal conditions, was first recorded following application of differentiative agents. current density increased with time of exposure to differentiating conditions. whole-cell and single channel analysis revealed that the observed increase in current density was due at least in part to changes in steady-state activation and inactivation properties. namely, half activation potential shifted from -34 mv to -45 mv, while half-inactivation potential shifted from -94 mv to -78 mv. furthermore, a contribution to the increase in na + current density could also be given by an enhancement in channel expression, as suggested by an augmentation in the number of single channels per patch area, with increasing neuronal differentiation. interestingly, those changes in na + channel activity well correlate with the capability of ns cells to generate action potentials during in vitro neuronal differentiation. a. viale, g. bonizzi, a. cicalese, f. de franco, c. pasi, s. pece, a. orleth, p. di fiore, p. pelicci european institute of oncology, milan, italy we are characterizing the biological differences between normal and transformed scs. scs are defined by their ability to generate more scs, termed self-renewal, and to produce cells that differentiate (asymmetric cell division). scs, however, possess the ability to expand in number (i.e. during development, in adulthood after injury/disease); this increase is not accounted by asymmetric divisions, in which only one daughter cell maintains sc identity. recent findings in invertebrates indicate that scs can also generate daughter cells destined to acquire the same fate (symmetric cell division). on the other hand, sc quiescence is critical to maintain tissue homeostasis after injury. here we show increased symmetric divisions of cscs in breast tumors (due to inactivation of the p53 tumor suppressor) and dependency of leukemia development on quiescent leukemia scs (due to transcriptional up-regulation of the cell cycle inhibitor p21 by leukemia-associated fusion proteins). we suggest that sc asymmetric divisions function as a mechanism of tumor suppression, that sc quiescence is critical to the maintenance of the transformed clone and that symmetric divisions of scs permit their geometric expansion. a. uccelli department of neurosciences ophthalmology and genetics, genoa, italy stem cells are considered as a possible source of cells for tissue repair. in this scenario damaged tissues will be reconstructed by newly formed cells with the result of a recovery of lost functions. thus, the rationale for utilizing stem cells for the treatment of neurological diseases such as multiple sclerosis (ms) stemmed from the idea that they differentiate in neural cells regenerating the damaged tissues at the basis of irreversibile disability. however, while multipotential embryonic stem cells may provide in the future an optimal source of cells competent for myelin and axons repair in ms, their use is still far from being exploitable in a clinical setting. moreover, it is widely accepted that adult stem cells may in vitro transdifferentiate in neural cells but recent experimental data have challenged the biological importance of this event in vivo. nevertheless, several experimental studies have provided new evidences supporting the use of adult stem cells derived form different human tissues for the treatment of ms. in the cases of mesenchymal stem cells and neural stem cells current experimental data support an unexpected therapeutic plasticity mediated by diverse paracrine mechanisms including neuroprotection, induction of local neurogenesis and modulation of the immune response. therefore, current experimental and clinical studies support the use of stem cells for the treatment of neurological diseases such as ms. unravelling the structure of the water splitting site of photosynthesis and implications for mechanism of catalysis j. barber imperial college london, u.k. photosystem ii (psii) is a multi-subunit membrane protein complex which catalyses the oxidation of water to molecular oxygen and reducing equivalents. the reaction occurs at a catalytic centre composed of 4 mn ions and a ca ion, is thermodynamically demanding and generates highly oxidised species. unavoidable side reactions cause detrimental effects on the protein environment leading to the rapid turnover of the reaction centre d1 protein. to understand the mechanisms of water oxidation and d1 turnover structural information is required. initially the positioning of various protein subunits and their transmembrane helices were determined by electron microscopy. more recently a refined structure of the cyanobacterial psii unit has been elucidated by x-ray crystallography giving details of specific environments of the redox active cofactors. the implications of these structural studies will be discussed in relation to the unique facets of psii function, particularly the water splitting reaction. importantly this new knowledge is providing a blue print for the design of photochemical catalysts which can mimic the photosynthetic water splitting reaction and thus give hope that new technologies will emerge to provide humankind with a sustainable energy supply. photochemically controlled molecular devices and machines v. balzani department of chemistry "g. ciamician", university of bologna, italy the macroscopic concepts of a device and a machine can be extended to the molecular level. molecular-level devices and machines operate via electronic and/or nuclear rearrangements and, like macroscopic devices and machines, they need energy to operate and signals to communicate with the operator. the extension of the concepts of a device and a machine to the molecular level is of interest not only for basic research, but also for the growth of nanoscience and the development of nanotechnology. if a molecular device or machine has to work by inputs of chemical energy, it will need addition of fresh reactants ("fuel") at any step of its working cycle, with the concomitant formation of waste products that compromise the operation. currently there is an increasing interest in the use of light to power molecular devices and machines. the lecture will illustrate examples of recent achievements [1, 2, 3] , which include molecular wires, switches, plug-socket systems, extension cables, antennas, and light powered nanomotors. biophysics laboratory, institute of botany, azerbaijan national academy of sciences, baku, azerbaijan it is well known that fast component of induction curves of millisecond delayed chlorophyll fluorescence (ms-df) originates via radiative recombination of reaction center with product on the donor side of psii. in view of our previous data (j. photochemistry and photobiology b: biology 86, 2007) it was shown that partners for radiative recombination of p 680 q a − localized as a hole on the donor side of psii. depending on ph this hole might be on the camn 4 -cluster or on y z and their recombination with p 680 q a − can be monitored by the ms-df fast component. for analysis of site of damages, photoinhibition of psii particles from spinach at different ph was monitored by ms-df. during photoinhibition of psii (4000 µmol photons m −2 s −1 ) the fast component of ms-df was shown to be more stable at ph 5.5 and ratio of the fast component to the steady-state level of ms-df was approximately constant, while at ph 8.5 the fast component essentially decreased, the steady-state level increased and ratio of these components rapidly went down. it is possible concluded that strong light damaged of recombination reaction with in the presence ppbq -artificial electron acceptor -the protection of psii from photoinhibition has been observed only at acidic condition. stress factors such as heavy metals, strong light and low temperature were investigated by measurement of transient pictures of ms-df in intact plants, isolated chloroplasts and pure psii particles. the heavy metals action on psii was investigated on wheat seedlings. the targets for toxic action of al, mn and co ions were found to be a q a -q b acceptor side of psii. the damage site for cd 2+ may be partners for recombination with p680 + -depend on of medium ph -either y z or camn 4 -cluster. investigated ions (mn 2+ , al 3+ , cd 2+ and co 2+ ) have lead to reduction of chlorophyllprotein complexes pigment fund and cd 2+ and co 2+ destroyed also an apoproteins, especially of psii. the photoinhibition of psii was investigated in intact leaves of barley and maize seedlings at low and normal temperature. a very sharp reduction of intensity of ms-df second fast component, possibly y z * p680ÿq a − radiative recombination of maize seedlings was observed even after short illumination by strong light at 4 • c. ph dependence photoinhibition of chloroplasts and pure psii particles have shown that strong light damaged of camn 4 -cluster in great degree than y z . hydrogenases are considered as potential energy sources. in particular, the ability of the green alga c . reinhardtii to reduce protons to hydrogen gas upon illumination by means of a [fefe]-hydrogenase is a phenomenon of great scientific interest, as it would need only light and water to generate energy. however, the catalytic activity is strongly inhibited by the o 2 produced during photosynthesis; furthermore, the protein is expressed at very low levels, only in conditions of strict anaerobiosis. this mutually exclusive nature of o 2 and h 2 photoproduction represents a crucial problem in the development of h 2 bio-production. the study of the structurefunction relationship of [fefe] hydrogenases, which would help to clarify the molecular mechanisms underlying both h 2 production and o 2 sensitivity, requires the characterization of purified native and modified proteins, which can be obtained by site-directed mutagenesis. we expressed the algal hydrogenase in the cyanobacterium synechocystis sp. pcc 6803, which holds a bidirectional [nife]-hydrogenase with a well known maturation system (1) . we obtained two constructs to stably transform synechocystis, enabling it to express the c. reinhardtii hydrogenase in an active form. this suggests that the [nife]-hydrogenase maturation pathway is able to drive the biosynthesis of functional [fefe] enzymes. these data open new perspectives about the indispensable presence of hyde, hydf and hydg auxiliary proteins (2, 3) to obtain a correctly folded [fefe]-hydrogenase. the a2 low energy monomeric chlorophyll of cp29 to investigate the role of low energy spectral forms in energy transfer among chlorophyll protein complexes, we characterized one of the redmost chlorophylls of psii antenna complexes, the a2 of cp29. pigment stoichiometry of the wild type (cp29wt) and mutant lacking the a2 binding residue (cp29a2) reconstituted complexes confirmed the loss of a chlorophyll a. to exclude the possibility of a marked excitonic interaction of the a2 with other chlorophylls, we quantified the decrease in dipole strength after mutagenesis as a function of the frequency and performed a second derivative analysis of the difference absorption spectrum cp29wt minus cp29a2. these data, along with circular dichroism spectra of both complexes, support the idea that the a2 is a monomer in cp29, in disagreement with recent suggestion of involvement in an excitonic dimer (mozzo et al., bba 2008) . chla a2 reorganization energy and inhomogeneous broadening were then determined through a thermal broadening analysis in the 80-285 k temperature range. these parameters allowed us to calculate the förster overlap integral of the homogeneously broadened a2 bandshape, a fundamental factor for energy transfer rate estimations between chromophores. a comparison with foi of chlorophyll in solution suggests that chla a2 may connect cp29 complex to psii core favoring energy transfer from cp29 towards other inner antenna low energy chlorophylls. in oxygenic photosynthetic organisms, reaction centre complexes catalyze electron transport from water to co2 using light energy absorbed by tetrapyrrole pigments bound to so called "antenna proteins". a major challenge in performing this type of photosynthesis consists on the difficulty of operating "one electron" transport through a multi-step pathway in the chloroplast environment which is the source of oxygen of biosphere. in fact, synthesis of ros, mainly singlet oxygen and superoxide anion and consequent photoinhibition is an intrinsically unavoidable consequence of photosynthesis that must be prevented and/or controlled in order to avoid photodestruction and death. mechanisms involved include chlorophyll (chl) triplet quenching, ros scavenging and controlled heat dissipation of excess chl singlet excited states. the latter process has been studied for over 40 years with little success in elucidating its mechanism. reverse genomics and ultrafast-spectroscopy led to the proposal that the transient formation of carotenoid radical cations, followed by charge recombination might be the underlying mechanism of energy dissipation while three proteins belonging to the lhc superfamily could be the site hosting the reaction. probing the dark cycle to reveal regulatory networks controlling photosynthetic efficiency the food, fibre and fuel needs of an ever-increasing population are one of the challenges facing current society. higher plant photosynthetic efficiency is ∼1% whereas the theoretical maximum is thought to be ∼6-8% giving potential for great improvements. it is not however clear where in the photosynthetic process the additional losses are occurring. we are adopting a systems biology approach to reveal the regulatory networks that control photosynthetic efficiency, the challenge being to make quantitative measurements of the input parameters to models of the network of photosynthetic reactions, and also to identify missing physical parameters and processes. the aim being to understand how they interact with other key physiological pathways and what impact they hold over photosynthetic efficiency. we report initial results in this poster from experiments using conventional and novel proteomic methods. e.g. an optical technique based on a non-linear 2dir method is being developed for proteomic and metabolomic analysis. unlike many conventional proteomic methods, which provide information on the relative levels of various proteins and metabolites, the viability of the 2dir method as a quantitative, sensitive and high throughput proteomics platform has recently been demonstrated. in collaboration with klug this project will employ 2dir as a both a proteomic and metabolomic tool. t. morosinotto 1 , p. arnoux 2 , g. saga 1 , g. m. giacometti 1 , r. bassi 3 , d. pignol 2 1 dip. di biologia, padova, italy, 2 cea, cadarache, france, 3 dip. di biotecnologie, verona, italy violaxanthin de-epoxidase (vde) is the enzyme responsible for zeaxanthin (z) production. the synthesis of this carotenoid in plants exposed to high light conditions is an important photoprotection mechanism, enhancing excess energy dissipation and reactive oxygen species scavenging. the inactive enzyme is normally soluble but, upon activation by low ph, it binds to the thylakoids membrane, where its substrate is found. we present here the first structural data on this enzyme obtained at both acidic and neutral ph. at neutral ph, vde is monomeric with its active site occluded in the lipocalin barrel. upon acidification, the barrel opens up resulting in a functional dimerization of the enzyme. the channel linking the two active sites of the dimer can harbour the entire carotenoid substrate and thus allow the parallel de-epoxidation of the two violaxanthin β-ionone rings, making vde an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate. structural data opened the possibility to investigate deeper this enzyme and further work allowed the identification of its active site, the protein domains responsible of its membrane association as well as key residues involved in the ph dependent conformational change. hydrogen is considered the fuel of the future if produced from sun light-driven water splitting. a hydrogen economy based on genetically modified organisms, immobilized enzymes or biomimetic synthetic catalysts requires a profound knowledge of the structure and function of the respective enzymes in nature. 1 light-induced water splitting is performed by a tetranuclear manganese cluster located in photosystem (ps) ii of oxygenic photosynthesis. the protons generated by ps ii can be converted to molecular hydrogen by the enzyme hydrogenase, which is for example found in green algae and cyanobacteria. [nife]-and [fefe]-hydrogenases are the two main classes of this enzyme. they contain bridged binuclear transition metal cores, which are tuned by a special ligand environment to efficiently convert protons to hydrogen -or vice versa -via a heterolytic mechanism. 2 rhodobacter sphaeroides strain a-10 isolated from mineral springs in armenia produces h 2 with high rate in anaerobic conditions under light. in our previous work the inhibition of h 2 production by n,n' -dicyclohexylcarbodiimide (dccd), the f 0 f 1 -atpase inhibitor, was shown, and dccd-inhibited atpase activity was determined. therefore it is possible to admit a role of this atpase in h 2 production by r. sphaeroides; otherwise dccd inhibition of hydrogenase is not ruled out. in order to examine the mediatory role of proton-motive force (pmf) or proton atpase in this process transmembrane electrical potential (∆ψ) and ∆ph are determined and the atpase activity is studied in r. sphaeroides grown under light. pmf was determined under anaerobic conditions in the dark. at ph 7.5 the ∆ψ was of -94 mv and the reversed ∆ph was +30 mv, resulting in the pmf of -64 mv. but ∆ψ was not affected by dccd. moreover, adenine nucleotide phosphates (anp) content is essential for cell functioning and may result with atpase activity. the percentage of atp was calculated from the total quantity of anp in whole cells, it was 46.6 %. a relatively high concentration of adp (∼35 %) and amp (∼18 %) and accordingly low energetic charge were noted; these might be indicative for the atpase activity, a further study is required. relationship between h 2 production and atpase activity by rh. sphaeroides is suggested; possible mechanisms are discussed. identification of the sites of chlorophyll triplet quenching in relation to the structure of lhc-ii from higher plants. evidence the chl a molecules involved in the triplet-triplet energy transfer to the central luteins in trimeric lhc-ii are identified by time-resolved and pulse epr techniques. the concept of spin conservation during triplet-triplet energy transfer is exploited. the sites with the highest probability to form triplet states, which are quenched by the central luteins, are chl603 and chl612. unquenched chl triplet states are also produced by photo-excitation in the lhc-ii complex. putative sites of these triplet states are chl614, chl611, chla604 and chl613, since they do not contribute to the formation of the observed carotenoid triplet states. fluorescence lifetime spectrum of the plant photosystem ii core complex the photosystem ii kinetic model (diffusion or trap-limited) is still much debated. there is discussion about whether energy transfer from the core antenna (cp47 and cp43) to the reaction center complex (d1-d2-cyt b559) is rate-limiting (transfer to trap-limited). this study investigates this problem in isolated core particles by exploiting the different optical properties of the core antenna and the reaction center complex near 680 nm, due to p680 and an isoenergetic pheophytin. this was used as a marker feature for the reaction center complex. if the transfer to the trap-limited model were correct, assuming excited-state thermalization, the specific reaction center fluorescence decay lifetime should be shorter near 680 nm, where there is reaction center complex specificity, than at the other emission wavelengths. such a selective reaction center feature was not observed in fluorescence decay measurements. at the experimental resolution used here, we conclude that the traplimited energy transfer to the reaction center could, at the most, be 20% limiting. thus, the transfer to the trap-limited model is not supported. photosynthetic apparatus response under heat stress n. pshybytko 1 , i. divak 1 , l. kabashnikova 1 , e. lysenko 2 1 institute of biophysics and cell engineering, national academy of sciences of belarus, belarus, 2 institute of plant physiology, russian academy of sciences, russia the mechanisms of psii thermoinactivation and adaptation under high temperature impact and participation of hydrogen peroxide at these processes were studied. the twice suppression of oxygen evolving activity of thylakoids with simultaneous decrease in d1 protein content and the release of extrinsic 33 kda polypeptide from woc after 15-20 min heating at 40 o c were registered. using inhibitor analysis it was shown that thermoinduced degradation of d1 protein after 20 min heating occurred by proteases. the participation of ftsh protease in thermoinduced d1 protein degradation was observed. level of transcription of psba gene in chloroplast was raised after 20 min heating and was decreased through 1 h. the content of hydrogen peroxide was increased three times after 20 min of heating and was decreased to normal level through 1 h and was raised after 2.5 h again. it is interesting that level of peroxidation lipids products was increased after 2.5 h heating only. received data indicated that hydrogen peroxide is signal molecular at the photosynthetic apparatus under heat stress. during heating the inactivation of woc and d1 protein is occurred. as result the h 2 o 2 is generated. hydrogen peroxide as signal molecule activates transcription of psba. turnover of psii is occurred. more long heating induces degradation of proteins and lipids and h 2 o 2 represents as the destructive agent. a. nurisso 1 , m. a. morando 2 , f. j. cañada 2 , j. j. barbero 2 , a. imberty 1 1 cermav-cnrs, grenoble, france, 2 centro de investigaciones biológicas, madrid, spain the mechanism of symbiosis between legume plants and rhizobial bacteria is a relevant topic of interest since it is at the basis of the nitrogen fixation process. this mechanism is strictly related to the production of lipochitooligosaccharides, nod factors (nf), which allow the bacterial invasion into the legume host roots. in medicago truncatula, the recognition of nf from the symbiont sinorhizobium meliloti requires the nfp gene, able to encode a lysm-motif receptor-like kinase. the extracellular part of this protein is characterized by three lysm motifs: only one of them seems to be involved in the nf recognition. herein, we report an in silico investigation of different structural aspects of this process. first of all, the conformational behaviors of a new generation of nf analogues were elucidated by mds simulation. then, a homology model of the lysm2 domain from m. truncatula was proposed and compared with a model of lysm domain from pisum sativum: docking calculations of natural nf identified a common binding site in which the carbohydrate portion is the main responsible of the binding. both studies provide support for the idea that the carbohydrate part of nf plays a key role in the interaction with lysm domains, while the lipid moiety modulates ligand specificity probably interacting with a potential second receptor. interaction between frutalin with biomembrane models and its correlation with cell adhesion property frutalin is a homotetrameric lectin d-galactose (d-gal) binding that activates natural killer cells in vitro and leukocyte migration in vivo, being a potent lymphocyte stimulator. in this study we investigated the interaction of frutalin with different phospho/glyco-lipids using langmuir monolayers as biomembrane mimetic system. the results attest the specificity of the protein for the carbohydrate d-gal when attached to the biomembrane model. the adsorption kinetics for frutalin to the mixed monolayers containing glycolipids showed that the interaction depends on the presence of charged groups and on the position of the d-gal on the polar head of the glycolipid. using brewster angle microscopy (bam), we investigated the morphology of the interface for the binary mixtures containing galcer, where small domains were formed at high lipid packing, suggesting that frutalin can induce the formation of likelipid rafts domains in vitro. the results obtained with the membrane models were associated with those from fibroblast adhesion induction. the cell adhesion promoted by frutalin is in accordance with the results observed in langmuir monolayers, which probes the specificity of the interaction between the lectin and d-gal on cell-membrane surfaces. based on these results, frutalin can be considered as a promise biotechnological tool to actuate in tissue engineering regeneration. supported by fapesp, cnpq and capes aggregation and glycation processes of proteins are of peculiar interest for several scientific fields. serum albumins are widely studied proteins for their ability to self-assemble in aggregates and also to undergo to non enzymatic glycosylation in cases of diabetes. in this work we report a study on thermal aggregation of glycated bovine serum albumin (bsa) prepared with different concentrations of glucose at ph 7.4. increasing concentration of sugar modulates the effect of different glycation levels on the protein aggregation. fluorescence spectroscopy, ftir absorption, static and dynamic light scattering are used to follow the time evolution of the aggregation process and of protein conformational changes. conformational changes of secondary and tertiary structures are measured by ftir absorption; the kinetics of amide i, amide ii and amide ii' bands are monitored. the kinetics of tryptophans fluorescence give complementary information on the tertiary structure changes and on the polarity modification of the fluorophores environment. the aggregates growth is studied by dynamic light scattering measurements and rayleigh scattering peak. the results show that the partial unfolding of the protein is not affected by the glycation, while the presence of increasing amounts of glycated molecules progressively inhibits the aggregates formation. solvent occupancy analysis in ligand structure prediction: the case of galectin-1 s. di lella, m. a. martí, d. a. estrin inquimae-conicet, universidad de buenos aires, argentina formation of protein ligand complexes is a fundamental phenomenum in biochemistry. during the process, significant solvent reorganization is produced along the contact surface. using md simulations in explicit solvent combined with statistical mechanics analysis, thermodynamic properties of water molecules around proteins can be computed and analyzed in a comparative view. based on this idea, we developed a set of analysis tools to link solvation with ligand binding in a key carbohydrate binding protein, human galectin-1 (hgal-1). specifically, we defined water sites (ws) in terms of the thermodynamic properties of water molecules strongly bound to protein surfaces. we then succesfully extended the analysis of the role of the surface associated water molecules in the ligand binding and recognition process to many other carbohydrate binding proteins. our results show that the probability of finding water molecules inside the ws, p(v), with respect to the bulk density is directly correlated to the likeliness of finding an oxhydril group of the ligand in the protein-ligand complex. this information can be used to predict possible complex structures when unavailable, and suggest addition of ohcontaining functional groups to displace water from high p(v) ws to enhance drug, specially glycomimetic-drugs, protein affinity and/or specificity. glyconanoparticles: nano-biomaterials for application in biotechnology and biomedicine s. penadés laboratory of glyconanotecnology, cicbiomagune -ciber-bbn, san sebastian, spain to study and intervene in carbohydrate interactions our laboratory has developed a chemical strategy (glyconanotechnology) to produce sugar functionalized gold nanoclusters with multivalent carbohydrate display (glyconanoparticles, gnps). by combining these tools with biophysical and analytic surface techniques (itc, afm, tem, spr) we have demonstrated and evaluated ca 2+ -mediated carbohydratecarbohydrate interactions involved in cell adhesion processes. the gnp system complements other currently available multivalent systems incorporating carbohydrates and presents some advantages as: 1) exceptionally small core size; 2) high stability in water and physiological buffers; and 3) multivalency and multifunctionality with control over ligands number. specific gnps have been applied to the inhibition of melanoma metastasis in mice and as inhibitors of hiv-trans infection. magnetic gnps have also been used in cellular labelling and imaging as probes for mri. in this lecture, the glyconanotechnology strategy will be presented and some application will be highlighted. -glycobiophysics expanded ataxin-1 induces membrane mechanical instability: evidences from model systems and cells c. canale 1 , s. averaimo 2 , d. pesci 3 , d. paulis 2 , v. fortunati 3 , a. gliozzi 4 , m. mazzanti 2 , c. jodice 3 , a. relini 4 1 iit, genoa, italy, 2 university of milan, italy, 3 university of tor vergata, rome, italy, 4 university of genoa, italy spinocerebellar ataxia type 1 is a neurodegenerative disorder involving the expansion of a polyglutamine stretch beyond a threshold in the protein ataxin-1, with intracellular deposition of amyloid aggregates. ataxin-1 variants with polyglutamine stretches of pathological length are not associated to disease when the stretch is interrupted by histidines. mounting evidence suggests that ataxin-1 aggregates interact with the nuclear membrane. to get insight into the mechanisms leading to neurological disorders, we studied model lipid membranes containing an expanded pathological form of ataxin-1 or expanded normal forms interrupted by histidines. electrical measurements on planar bilayers showed the occurrence of current steps, much larger for the pathological form, indicating the formation of pore-like structures. atomic force microscopy measurements showed that the longer the polyglutamine tract, the smaller was the force required to penetrate the bilayer with the afm tip. the smallest penetration force, and then the strongest membrane destabilization, was observed for membranes containing the expanded pathological form. experiments on cos1 cells provided evidence that pathological protein aggregates damage the nuclear membrane eventually causing cell autophagy. modification by dopamine adducts links αsynuclein to oxidative stress in parkinson disease m. bisaglia 1 , e. greggio 2 , l. tosatto 1 , f. munari 1 , i. tessari 1 , p. polverino de laureto 1 , s. mammi 1 , m. r. cookson 2 , l. bubacco 1 1 university of padova, italy, 2 nih, bethesda, md, usa oxidative stress has been proposed to be involved in the pathogenesis of parkinson disease (pd). a plausible source of oxidative stress in nigral dopaminergic neurons is the redox reactions that specifically involve dopamine (da) and produce various toxic molecules, i.e., free radicals and quinone species. α-synuclein (αsyn), a small protein found in lewy bodies characteristic of pd, is also thought to be involved in the pathogenesis of pd. to investigate the possibility of a synergistic role of oxidative stress and αsyn in pd, we analyzed the modulation of da toxicity by αsyn overexpression in dopaminergic human neuroblastoma cells. our results indicate that the increased expression of αsyn enhances the cellular toxicity induced by the accumulation of intracellular da. we then correlated our results with the structural modifications induced by the oxidation products of da on αsyn by studying two potential pathways for the oxidative chemistry associated with da. the first one is the auto-oxidation reaction which leads to the concomitant formation of radical and quinone species. the second is the enzymatic oxidation, mediated by tyrosinase, which leads only to the accumulation of quinones. our data suggest a link between da and αsyn in the progression of pd and provide novel insights on both the mechanisms involved in the oxidative chemistry and the aggregation properties of αsyn. dynamic light scattering aggregation studies of aβ (1-40) peptide s. bertini, r. beretta, d. gaudesi, a. naggi, g. torri ronzoni institute, milan, italy heparan sulfate (hs) proteoglycans play a role in formation of amyloid plaques by facilitating formation of amyloid aggregates. heparins and low-molecular weight heparins which mimic hs sequences could interact with amyloid peptides putatively involved in neurodegenerative processes. the aim of this work is to study the influence of chain length and structure of heparin oligosaccharides on the aggregation of amyloid. to this purpose, the interaction with amyloid peptide aβ 1−40 with a number of heparin/heparin oligosaccharides had been investigated using dynamic light scattering (dls) which permits to determine the size and the stability of molecular aggregates in solution. as indexes of aggregation in solution, two parameters were chosen, i.e., the area under the autocorrelation function (af) and the average hydrodynamic ratio (rh). we evaluated the aggregation kinetic of different preparations of aβ and the ph of solubilization. a clear indication was obtained for the trend of aggregation of the peptide aβ 1−40 alone and in the presence of oligosaccharides. the size of the aggregate in the presence of the tetrasaccharide is definitely lower than for the peptide alone. the size of the aggregates increases with increasing size of the oligosaccharides. in fact, the octasaccharide promotes further aggregation. it is crucial for pharmaceutical protein formulations to have low aggregate content. using the monoclonal igg1 antibody rituximab as a model system, we have studied the mechanisms by which antibodies aggregate at physiological ph when incubated at 52-65 • c. light scattering showed two coupled stages: an initial fast stage followed by several hours of exponential growth of the scattered intensity. data analysis showed the fast formation of a large species, which subsequently increased in size. the aggregate number density had a maximum suggesting that aggregates increase in size by coagulation. the analysis also predicted the actual underlying increase in aggregate mass to be linear and reach saturation. this was confirmed by size-exclusion chromatography of incubated samples. in an arrhenius plot the activation energy of the first stage was similar to the unfolding energy of the ch2 domain, suggesting a pivotal role of this domain in the aggregation process. cd and fluorescence showed only minor structural changes in the temperature interval studied. we conclude that coagulation is the main mechanism driving rituximab aggregation and that aggregation is due to small structural changes. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils -abstracts 123 solid-state nmr structural studies of alzheimer's disease amyloid β(1-42) in lipid bilayers j. d. gehman 1 , a. k. mehta 2 , f. separovic 1 1 school of chemistry, bio21 institute, university of melbourne, 3010 vic, australia, 2 department of chemistry, emory university, 1515 dickey dr., 30322 atlanta, ga, usa amyloid-β peptides are believed to cause loss of nerve cell function in individuals suffering from alzheimer's disease, where evidence suggests that interaction with the cell membrane correlates strongly with cytotoxicity. previous studies report a range of different but plausible structures, which depend on the molecular environment of the peptide. this sensitivity to sample preparation suggests that the structure relevant to disease is found in lipid bilayer membranes. while model membrane vesicles are too large to be studied by conventional solution nmr, solid-state nmr is one of the few technologies available to study such systems. we present recent measurements which suggest a novel peptide structure in a lipid bilayer environment: (i) rotational-echo double-resonance (redor) distance measurements between selectively enriched 13 c-carbonyl and 15 n-amide positions constrain dihedral angles of intervening residues and suggest that at least part of the aβ(1-42) peptide folds into a β-sheet like conformation, in contrast to the helical and coiled structures in previous reports; and (ii) double quantum filtered draws measurements indicate that the extended strand does not assemble into an in-register parallel sheet as reported for amyloid fibrils. a mutation in app gene with dominant-negative effect on amyloidogenesis: a light scattering study e. del favero 1 , g. di fede 2 , f. tagliavini 2 , m. salmona 3 , l. cantù 1 1 university of milan, segrate, italy, 2 "carlo besta" national neurological institute, milan, italy, 3 istituto di ricerche farmacologiche "mario negri", milan, italy we studied the effect of a single-point mutation (a673v) on the aggregative properties of alzheimer's peptides aβ1−40. by laser light scattering it was possible to follow the early stages of aggregation of aβ1−40 wt and aβ1−40 mut (within 48 hours after sample preparation), when intermediates in the aggregation pathway, rather than the mature insoluble fibrils, are formed. results show that both the kinetics and the extent of aggregation are strongly enhanced by the mutation. on the reverse, coincubation of mutated and wild-type peptides slows down the aggregation process and results in aβ aggregates that are much more unstable against dilution. it is evident that the interaction between mutant and wild-type aβ1−40 interferes with nucleation or nucleation-dependent polymerization, hindering amyloidogenesis. these results account for the highly amyloidogenic effect of the mutation in vitro, if alone, reversely reduced by the mutated-wild type mix. also a clinical case exists that constitutes the in-vivo evidence of these two opposing effects. this finding may offer grounds for the development of therapeutic strategies, based on modified aβ peptides or peptido-mimetic compounds, for the potential treatment of alzheimer's disease. the collagen i is the major structural protein of the body and it is found organized in a hierarchical manner in the extra-cellular matrix of several tissues (bone, tendon, cornea and sclera, etc..). it is responsible for their specific architecture. it is already known that collagen i is capable of forming cholesteric liquid crystalline phases that once stabilized by a ph increase, lead to collagen matrices that mimic the organization of the organic matrix found in bone [1] . in the present work, we analyze physico-chemical ways to modulate long range organizations obtained in this liquid state. we study the effect of collagen concentration, ph (2.6 / 3.6) and acids (hydrochloride acid / acetic acid) over the liquid crystalline order. in a original approach, correlation of collagen endofluorescence and shg signals has enabled us to quantify cholesteric pitch and phase transition as a function of collagen concentration within a gradient. cholesteric pitch have proved to be very responsive to physico-chemical conditions. indeed, the results allowed us to quantify a i / cholesteric transition as a subsequent transition from cholesteric to a phase hexagonal or colummar. prion diseases are deadly neurodegenerative diseases affecting human and mammalian species. according to the 'protein-only' hypothesis, the key event in the pathogenesis is the conversion of the α-helix-rich monomer (prp c ) into a polymeric β-sheet-rich pathogenic conformer (prp sc ). we have used a combination of biophysical techniques with molecular dynamics simulations (md) to elucidate the molecular mechanisms of prp c unfolding and polymerization. under well established conditions, three β-sheet-rich soluble oligomers were generated from the partial unfolding of the monomer, which were found to form in parallel. to obtain a deeper insight into the molecular events, doublecystein mutants were designed, thus 'locking' different regions of prp. single mutations were also performed, which affected dramatically and selectively the prp oligomerization pathway. furthermore, we have now identified the minimal region that leads to the same oligomerization profile as the full-length prp, namely, h2h3. the existence of at least three distinct oligomerization pathways and the effect of single mutations reveal the conformational diversity of prp and a possible relationship with prion strain phenomena. the identification of domains involved in the conversion process may lead to a better understanding of the effect of mutations or gene polymorphism on the evolution of prion pathology. investigating toxic protein nanoclusters and their interactions with living cells s. nag, b. sahoo, a. bandyopadhyay, c. muralidharan, r. abhyankar, s. gurav, s. maiti tata institute of fundamental research, homi bhabha road, colaba, mumbai 400005, india amyloid protein aggregation is responsible for many neurodegenerative diseases, such as alzheimer's and parkinson's. it appears that quasi-stable small soluble aggregates are the key to amyloid toxicity. though amyloid aggregation appears to be a nucleation mediated process, simple nucleation theory is unable to explain the stability of these intermediate species. we investigate this issue with fluorescence correlation spectroscopy in solution and on cell membranes. our initial data, varying the ph and the ionic strength of the solution, show that a charged colloid model can explain the overall stability of these species. in addition, this understanding suggests possible ways of modulating the stability of these aggregates in solution. some of these strategies successfully decimate the stable aggregate population, and also reduce the toxicity of amyloid beta to cultured neurons. afm studies of artificial amyloid fibrils and filaments p. mesquida 1 , a. kurtossy 1 , c. macphee 2 1 department of mechanical engineering, king's college london, london, u.k., 2 school of physics, university of edinburgh, edinburgh, u.k. amyloid fibrils are beta-sheet-rich superstructures of peptides or proteins. although these aggregates have first been found in connection with protein-misfolding diseases, such as alzheimer's or parkinson's disease, there is evidence that the ability to form fibrils is a generic property of any polypeptide rather than a result of specific, disease-related amino-acid sequences. fibrils can easily be formed in vitro from nondisease-related proteins and even from synthetic peptides. these artificial fibrils can thus serve as model systems to investigate the biophysical properties of amyloid fibrils. the system presented here is built from the artificial peptide ttr 105−115 , which contains 11 amino-acids and which forms well-defined nanorods of ca 10 nm diameter. we present atomic force microscopy (afm) results of their inner morphology by chemically dissecting the rods into their constituent filaments without completely breaking up the aggregates. in contrast to the stiff rods, their constituent filaments of ca 1-2 nm diameter were much more flexible. this shows that the particular way filaments are arranged around each other can massively change the mechanical rigidity of the resulting structures in amyloid fibrils, which could be one cause of the different strains in amyloid diseases. the formation of insulin fibrils is characterized by an initial apparent lag-phase, related to the formation of oligomers, protofibrils and aggregation nuclei. afterwards, the aggregation proceeds via fibril elongation, thickening and/or flocculation, and eventual gelation. here, we focus on the formation of such a gel, made of insulin amyloid fibrils, upon incubation at high temperature and low ph. by light scattering and rheological techniques, we monitor the development of the structural, dynamical and mechanical properties of fibrillar aggregates, up to the dynamic arrest of the sample and to the appearance of a non-ergodic behaviour, which marks the onset of gelation. our experiments were able to reveal the structural details hidden in the apparent lag-phase, displaying the slow fibril nucleation and elongation. this initial stage is followed by the known exponential growth of structures of different sizes. these two kinetic stages of structural growth are mirrored by the kinetics of the viscoelastic properties and, in particular, by the growth of the elastic modulus. our results show that the appearance of a noteworthy elastic network, is associated with the initial fibril nucleation and elongation more than with the formation of large structures, which causes the eventual gelation. capturing the initial events of in-cubo crystallization of membrane proteins c. v. kulkarni 3 , o. ces 1 , s. iwata 2 , r. h. templer 1 1 chemical biology centre and department of chemistry, imperial college london, united kingdom, 2 division of molecular biosciences, imperial college london, united kingdom, 3 institute für chemie, heinrichstrasse-28, university of graz, austria lipid based methods for the crystallization of membrane proteins including in-cubo crystallization are becoming popular in recent times. however, a complete understanding of the basic principles behind these methodologies is still elusive. the crystallization of membrane proteins in the lipid cubic phases involves following major steps: removal of membrane proteins from the native membrane using detergentlike molecules, followed by their insertion and equilibration into a lipid bilayer. the crystallization itself commences with precipitant induced osmotic dehydration which in turn stimulates the nucleation that subsequently leads to a crystal growth. using time-resolved x-ray scattering (saxs) and ultraviolet spectroscopy we have been able to gain new insights into the mechanism behind protein insertion into these complex three dimensional lipid structures including information on the timescales of protein folding relative to crystal growth and the effect of protein insertion on the morphology of the surrounding lipid matrix. several proteins can form amyloid fibrils under given environmental or thermodynamic conditions that affect their native conformation. lysozyme forms amyloid fibrils upon incubation of solutions at acid ph and at about 65 • c for a few days. differential scanning calorimetry experiments confirm that lysozyme is mainly unfolded above 60 • c at ph 2. we monitored the growth of aggregate size by dynamic light scattering for some days at different temperatures. our results show that the fibrillogenesis is characterized by an initial apparent lag phase and a subsequent growth with quadratic dependence upon time of the scattering intensity. this behaviour recalls a simple kinetic model of nucleation and elongation, with nuclei in equilibrium with monomers. at the end of the incubation at high temperature, we collected atomic force microscopy images which show fibrils with a diameter of a few tens of nanometers and a length of a few microns, characterized by a periodicity along the elongation axis. interestingly, the fibrils morphology exhibits no branching or thickening. this is consistent with the non-exponential growth observed in light scattering experiments. in order to elicit the role of repulsive electrostatic interaction in protein unfolding and self-assembly we extended our study at different ph. in this work we used afm to follow the amyloidogenesis pathway of transthyretin (ttr) to form protofilaments. single-molecule force spectroscopy (smfs) of native ttr and protofilaments were also compared in order to evaluate dynamic and structural differences. we observed that this pathway proceeds through the formation of transient amorphous aggregates, followed by the occurrence of annular oligomers. although implicated in cytoxicity, the role of such oligomers within the amyloidogenesis pathway is poorly understood. we show that the annular species display a tendency to stack, forming tubular-like structures that precede the formation of protofilaments. the protofilament height and pitch resemble those of ttr amyloid reported in previous structural studies. upon solvent exchange, we also observed protofilament disassembly that revealed structures reminiscent of the initial ttr annular oligomers. smfs of protofilaments showed a time-dependent increase in the length of the manipulated structures, suggesting that associations between monomers stabilize with time. force spectra of native ttr and protofilaments contained transitions spaced by ∼4 nm, indicative of sequential unfolding of individual β-strands. based on these results a model of ttr protofilament assembly is proposed. ability to undergo amyloid aggregation is affected by protein size c. parrini 1 , h. ramshini 2 , f. chiti 2 , a. relini 1 1 physics department, university of genoa, italy, 2 biochemical sciences department, university of florence, italy short polypeptide chains, typically between 28 and 253 residues, are generally involved with the conversion from the native state into insoluble fibrillar aggregates. the low prevalence of large proteins is disproportionate with their high occurrence in the human proteome. in order to explore the propensity of large proteins to form amyloid-like fibrils, the 486-residue hexokinase-b from saccharomyces cerevisiae (yhkb) has been induced to aggregate under two separate conditions, at low ph in the presence of salts and at ph 5.5 in the presence of trifluoroethanol. such conditions are among the most promising to form amyloid-like fibrils by normally globular proteins. under both conditions yhkb aggregates very rapidly into species with significant β-sheet structure, as detected by circular dichroism, and a weak thioflavin t and congo red binding. atomic force microscopy revealed globular aggregates eventually clustering into large amorphous aggregates at low ph, while in the presence of trifluoroethanol ribbon-like structures with distinct morphology from typical amyloid fibrils were observed. they had irregular width, no twist, and were connected by thinner fibril segments perpendicular to them. in general, yhkb aggregates displayed an unusual softness, as they were very easily perturbed by the afm tip. these results suggest that inability to form amyloid fibrils may prevent large proteins from being associated with protein deposition diseases. the initial stage of proteins aggregation leading to amyloid fibrils: a saxs study m. g. ortore 1 , f. spinozzi 1 , f. carsughi 1 , t. narayanan 2 , g. irace 2 , s. vilasi 3 , p. mariani 3 1 dip. saifet, univ. pol., ancona, italy, 2 esrf, grenoble, france, 3 dip. biochim. biofis., ii univ., napoli, italy under some conditions, a protein converts from its soluble form into highly ordered aggregates called amyloid fibrils, which are associated with many human diseases. in order to tackle the prevention and treatment of these diseases, we need to understand the mechanism of the pathological aggregation of proteins. in vitro amyloid fibril formation is preceded by the formation of metastable non fibrillar forms, which are responsible for cytotoxicity underlying neurodegeneration. the molecular mechanisms leading proteins into prefibrillar aggregates are still unclear. we present a saxs study performed at id02 beamline of esrf on the apomyoglobin mutant w7fw14f, which at physiological ph, firstly aggregates in prefibrillar forms that are cytotoxic and then forms amyloid fibrils. the first stages of w7fw14f oligomerization are induced by a ph jump. data show that big changes in w7fw14f in solution happen in less than 100 ms. singular value decomposition (svd) of the data yields a set of functions, from which all the scattering curves can be reproduced. the major result of our study is the determination of the presence of different oligomers in each step of the process. hence, time-resolved saxs experiments together with the estimation of different oligomers via svd method, can be a new and useful approach to investigate the first stages of amyloidogenesis. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils study of structure/toxicity relationship of amyloids by infrared spectroscopy h. p. ta amyloid diseases (alzheimer, parkinson, type ii diabetes, prion) correlate with protein aggregation. all the proteins associated with these pathologies aggregate into amyloid fibrils with a common ß-cross structure. according to many in vitro studies, the toxicity of various amyloids seems to be linked to some intermediates formed during the aggregation pathway and to their interaction with membranes. our aim is to understand the link between structure and toxicity of amyloids by studying their interaction with model membranes. for this, we use as an amyloid model, the prion-forming domain pfd218-289 (wild type wt) of het-s, a prion protein of the fungal p. anserina. when expressed in yeast, wt is not toxic whereas one of these mutants, m8 is toxic. in vitro, m8 forms very unusual short amyloid fibers contrary to wt which polymerizes as long fibers. furthermore, atr spectra have shown that m8 is essentially assembled into mixed parallel and anti-parallel ß-sheets whereas wt displays a predominant parallel organization. we are currently studying the interaction between wt or m8 with lipid langmuir film by polarized modulation-infrared reflexion absorption spectroscopy (pm-irras). this technique allows determining the secondary structure of proteins and their orientation in the membrane. the study of interactions with different charged phospholipids is following. a. stirpe, m. pantusa, r. bartucci, l. sportelli, r. guzzi dipartimento di fisica, laboratorio di biofisica molecolare & udr cnism, università della calabria, rende (cs), italy an increasing number of experimental studies is demonstrating that the propensity of forming amyloid fibrils is not limited to proteins related to neurodegenerative diseases but it is a more general phenomenon. we present data on human serum albumin (hsa) aggregation induced by a combination of thermal and metal ions effects investigated by optical density, fluorescence and electron paramagnetic resonance (epr). the turbidity experiments as a function of temperature show that the hsa aggregation starts at about 70 • c which is higher than the denaturation temperature (∼ 60 • c). in the presence of copper and zinc metal ions the onset temperature for protein aggregation is markedly reduced as the metal:protein molar ratio is increased from 1:1 to 10:1. moreover, the copper is more effective in inducing protein aggregation compared to zinc ion. the hsa aggregation analyzed by tht fluorescence at different incubation temperatures lacks a lag phase and the kinetic traces can be fitted by double exponential functions. the tht fluorescence increase evidences the formation of protein aggregates with fibrillar features. epr experiments for the cu(ii)-hsa complex show the binding of cu(ii) in the protein native state in a square planar coordination with 4 equatorial n atoms, and is not influenced by the heat treatment of the protein. the overall results suggest that hsa aggregation is compatible with a downhill process that does not require the formation of an aggregation nucleus. protein condensation diseases -a colloid physicists viewpoint p. schurtenberger adolphe merkle institute, university of fribourg, ch-1723 marly 1, switzerland a broad class of diseases, such as cataract, alzheimer's disease and sickle-cell disease, involve protein association phenomena as an essential aspect. the basic element common to all members of this class of molecular condensation diseases is the subtle interplay between protein interactions that produces condensation into dense, frequently insoluble mesoscopic phases. among this class of diseases, cataract is particularly important as the world's leading cause of blindness. this disease is most often the consequence of an uncontrolled aggregation (or phase separation) of the proteins in the eye lens that results in a loss of its transparency. the high concentration protein mixtures present in the eye lens are normally stable and produce a high refractive index that aids the eye in adaptive focusing of light. moreover, the proteins themselves exhibit a rich variety of repulsive interactions, attractive interactions, sizes, phase transitions and self-association. these mixtures also exhibit the pathological aggregation and opacification of the cataract disease that has inspired their study. in my presentation i will illustrate how we can use a combination of small-angle neutron and xray scattering experiments combined with molecular dynamics computer simulations to identify, measure and model the molecular interactions and emergent optical and viscoelastic properties and the phase behavior of the relevant, complex cytoplasmic mixtures. fluorescence microscopy studies of iapp fibrillation at model and cellular membranes d. c. radovan 1 , n. opitz 2 , r. winter 1 1 department of physical chemistry i -biophysical chemistry, tu dortmund university, dortmund, germany, 2 max-planck institute for molecular physiology, dortmund, germany type 2 diabetes mellitus (t2dm) is characterized by islet amyloid deposition and beta cell death, the main culprit being a small 37 a.a. peptide hormone, islet amyloid polypeptide (iapp), which forms fibrils under pathological conditions. we studied the interaction of iapp with giant (guvs) and large (luvs) unilamellar vesicles as well as with ins-1e cells. by using confocal / two-photon excitation fluorescence microscopy and guvs, we tested the influence of charge (dopc:dopg 7:3) and model raft systems, displaying liquid-ordered (l o ) / liquid-disordered (l d ) phase coexistence (such as dopc:dppc:cholesterol 1:2:1), respectively, on the kinetics of iapp fibril formation. a preferential partitioning into the l d phase was observed and fibrils grew along with lipid uptake. fluorescence spectroscopy leakage experiments with carboxyfluorescein-filled luvs and the corresponding tht kinetics of iapp fibril formation were carried out as well. moreover, using the wst-1 reduction assay and fluorescence microscopy, we could show that the red wine compound resveratrol is a potent inhibitor of iapp fibrillation and its cellular toxicity on ins-1e cells, these findings highlighting the potential role of resveratrol in future clinical applications, i.e., in the treatment of t2dm. a remarkably high viscosity has been induced in aqueous solutions of lysozyme by the addition of certain structurally related organic solvents, such as tetramethylurea (tmu), dimethylsulfoxide (dmso), dimethylformamide (dmf), and hexamethylphosphortriamide. dmso-induced gelation is observed in samples fulfilling the two following requirements: (1.) lysozyme concentration in excess of 5 mm, and (2.) volume fractions of dmso exceeding 0.7. based on spectroscopic data, the whole process was characterized as consisting of two mutually independent stages. the first involves an extensive transition of the polypeptide backbone, from a predominantly helical to increased random coiled and beta-sheet structures, with the occurrence of non-orthodox protein secondary structures at regions above the solvent critical point. the second stage consists of short-lived interchain contacts leading to an entanglement of the macromolecular system as a whole. here we present a set of scattering and microrheology experiments that investigate both the structural and dynamic properties of the gels under various experimental conditions. studying alpha-synuclein aggregation by fluorescence polarization based kinetics l. tosatto, f. munari, i. tessari, g. de franceschi, m. pivato, p. polverino de laureto, m. bisaglia, l. bubacco università degli studi di padova, padova, italy alpha-synuclein (syn) is linked to parkinson's disease (pd) by two evidences: the accumulation of amyloid fibrils of the protein and the autosomal dominant forms of the disease (a53t, a30p and e46k mutants). protein oligomers seem to be the most toxic species, causing dopaminergic neuronal death, probably disrupting cell membrane (volles & lansbury, 2002) . the aggregation of syn follows a nucleation dependent mechanism, i. e., aggregation is favoured only after the formation of an oligomer composed of a critical number of monomers (wood et al., 1999) . as the constitution of the nucleus is a rare event, early aggregation stages are difficult to study. to explore the early stages of the aggregation process a fluorescence polarization (fp) based method (luk et al., 2007) was applied to study syn oligomerization. fp depends on the size of the fluorescent molecule, therefore it is suitable for the detection of oligomers formation. we measured aggregation kinetic properties under several different conditions. a 100 to 1 mixture of syn and oregon green labelled syn was used to analyze the aggregation behaviour of wild type (wt) and pd mutants. the wt protein shows the fastest aggregation rate. this aggregation process is slowed down by the presence of the chaperone 14-3-3eta in wt syn samples. finally, dimers formed by a disulfide bond at the n-terminus or c-terminus of syn, when tested for their aggregation behaviour, show a different propensity to aggregate. a. pardini 1 , r. bizzarri 2 , a. diaspro 3 , p. bianchini 3 , c. usai 1 , p. ramoino 3 , g. checcucci 1 , g. colombetti 1 1 istituto di biofisica, cnr, italy, 2 a) nest, sns, iit udr, pisa, italy; b) nest, cnr-infm, pisa, italy, 3 univiversity of genova, genova, italy the colored ciliates blepharisma japonicum and fabrea salina show photomotile responses triggered by endogenous pigments of the hypericin family. b. japonicum exists in two forms: the wild one contains red blepharismin; the other one, generated by irradiating the cells with dim visible light, blue blepharismin. b. japonicum shows step-up photophobic response, whereas f. salina, that contains fabrein, shows step-down photophobic responses and positive phototaxis. we showed by confocal microscopy that the pigments are localized not only in pigment granules, but also in the cilia. this fact implies that the structure of the pigment in the cilia differs from that in the cell body, (pigment granules are too big to fit in the axoneme) and suggests that ciliary pigments might play a decisive role in photoreception. fluorescence lifetime imaging microscopy (flim) shows that there is a spatial distribution of lifetimes, which are shorter for the pigment in the cilia. this might indicate a functional role for these pigments. furthermore, lifetimes for f. salina are always longer than those of blepharisma. in order to further characterize the structure of ciliary pigments by means of spectroscopic methods, we have also performed spatially resolved static and time-resolved fluorescence anisotropy measurements. biosensing applications of micro/nano structured silicon several topics related to porous silicon (ps) biosensing properties were carefully considered in this study. ps allows the increasing of the immobilised biomolecule number on its surface and the creation of stable covalent bonds due to its controllable chemistry. beside this, ps is suitable for electrical (conductance, impedance), electrochemical and optical amplification of the detected signal. the experimental results on the fabrication of the ps microstructures such as: (i) protein immobilization and detection using microarray technique; (ii) dna biomolecule detection by impedance or by fluorescence spectroscopy; (iii) very sensitive sers biosensors (raman signal of 11-mercaptoundecanoic acid); (iv) sensitive element for neurons in nutmix culture are in detail presented. various characterisation techniques have been used, optical and scanning electron microscopy (sem), x-ray diffraction, raman, laser fluorescence and impedance spectroscopy for investigation molecule attachment on the au/ps structures. we have demonstrated that different morphologies of ps as-prepared or coated with gold nanoparticles have an important role in biomolecule/cell detection, due to its large internal surface combined with specific optical properties, being in the same time sensing element/support for immobilization of sensing biomolecules as well as transducer for biochemical interactions. heterotrimeric g-proteins interact with their g-protein coupled receptors (gpcrs) via key binding elements comprising the c-terminal segment of the α-subunit and the two lipid anchors at the α− and γ−subunit. direct information about diffusion and interaction of gpcrs and their g-proteins is mandatory, as these properties will affect the timing of events in the complex signal transduction cascade. in the case of the photoreceptor rhodopsin, receptor packing in the membrane and the related diffusion coefficients are discussed controversially (1, 2). by using single particle tracking we show that the encounters of rhodopsin with the fluorescently labeled cterminus of the α-subunit as well as with the holo-g-protein transducin change upon rhodopsin light-activation. our results indicate confined areas of interaction for the c-terminal segment of the α-subunit with inactive rhodopsin disk membranes and less restricted diffusion of the receptor-bound cterminal segment after light-activation. this suggests dynamic short-range order in rhodopsin packing and specific structures for efficient interaction (3) in vivo, skeletal muscle actively shortens and also resists lengthening, for example when landing after a jump. we study the energy during shortening and during lengthening by measuring the rate of pi release which results from atp hydrolysis. we show here the rate of pi releases in an in vitro protocol that involves muscle stretched followed by muscle shortening. it is hypothesised that the magnitude of deceleration of pi release in the stretch phase is less than the acceleration during the release due the energy input of the motor that is used to apply the length changes. therefore the end point of pi release is similar to isometric conditions. pi release responses to a ramped stretch (5%) followed 100 ms later by a symmetrical release at low velocities (0.5 l 0 /s −1 ) were measured in permeabilised fibre bundles of rabbit psoas at 20 • c. laser diffraction and high speed video were also recorded to confirm length change. we show that the rate of pi release drops during the stretch phase, returns to isometric levels during the length hold phase, and finally accelerates during the ramped release. tracking of sarcomere length change using video analysis demonstrated that laser diffraction is unreliable at times during stretches due to lack of uniformity of the sarcomere spacing. microbial rhodopsins: receptors, channels, and pumps from a single design j. l. spudich, e. negri-spudich center for membrane biology, university of texas, houston, texas, u.s.a. the microbial rhodopsin family is comprised of ∼5000 homologous proteins containing 7 transmembrane helices forming a pocket for the chromophore retinal. most are lightdriven ion pumps ("transport rhodopsins") and others are photosensory receptors ("sensory rhodopsins"). phylogenetic analysis indicates frequent lateral gene transfer of proton pumps among prokaryotic and unicellular eukaryotic species, followed by coupling of the pump's mechanism to the cell's existing signal transduction machinery to create photosensors. this evolutionary path is strongly supported by our studies of sensory rhodopsins in various organisms which demonstrate remarkably diverse signaling mechanisms with diverse transducer partners. the best studied are the phototaxis receptors in haloarchaeal prokaryotes (sri and srii) and in eukaryotic algae (channelrhodopsins). sri and srii transmit signals by protein-protein interaction to control a phosphorylation cascade that modulates motility. channelrhodopsins are light-gated cation channels that depolarize the membrane mediating calcium ion influx into the flagellar axoneme. crystallography and molecular biophysics have begun to clarify how modifications of the same architecture enable the rhodopsins to carry out their distinctly different molecular functions. interconversions of their functions by mutation reveal the elegant simplicity by which evolution uses existing genes to create proteins with novel functions. gold colloids-fluorophore complexes for protein detection assay l. sironi 1 , s. freddi 1 , l. d'alfonso 1 , m. collini 1 , m. caccia 1 , g. tallarida 2 , s. caprioli 2 , g. chirico 1 1 dipartimento di fisica, università di milano-bicocca, italy, 2 laboratorio nazionale mdm, agrate brianza (mi), italy noble metal nanoparticles (np) are endowed with peculiar optical properties related to the surface plasmon resonances (spr). the interaction of surface plasmons of gold nanoparticles with fluorophores a few nanometers away from the surface modifies their brightness and excited-state lifetime, and this effect can be exploited to obtain nanodevices for proteinprotein recognition. we studied different types of constructs based on gold nps on which derivatives of fluorescein were bound. the interaction of this fluorophore with the gold surface plasmon resonances, mainly occurring through quenching, affects its excited-state lifetime, that is measured by fluorescence burst analysis in standard solutions. the binding of proteins to the gold nps through antigen-antibody recognition further modifies the dye excited-state lifetime. this change can therefore be used to measure the protein concentration. streptavidin-functionalized gold nps of size 5-10 nm are used to bind biotin-fluorescein and biotin-antibodies for specific proteins. we have first tested the constructs for bovine serum albumine (bsa) detection. the data reported here indicate that one can measure the concentration of bsa in solution with an apparent limit of detection of 5±2 pm. we have then extended the study to the antitumor protein p53 -p53 antibody interaction in standard solution and directly in cellular extracts. calcium transport and phototransduction in isolated rod outer segments g. rispoli dip. biologia ed evoluzione, università di ferrara, italy ca 2+ concentration in photoreceptor rod outer segment (os) strongly affects the generator potential kinetics and light adaptation. light stimuli may produce voltage changes exceeding 40 mv: since the os ca 2+ extrusion is entirely controlled by the na + :ca 2+ ,k + exchanger (nckx), it is important to assess how the nckx ion transport is affected by voltage and intracellular factors. the nkcx regulation was investigated in whole-cell recorded os, using ionic conditions that activated maximally forward and reverse exchange. in all species examined of amphibia and reptilia, the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. since hyperpolarization increases ca 2+ extrusion rate, the recovery of the dark level of ca 2+ (and of the generator potential) after light stimuli results accelerated. mg-atp doubled the size of forward and reverse exchange current without modifying their voltage dependence, indicating that mg-atp regulates the number of active exchanger sites and/or the nckx turnover number. ca 2+ jumps achieved via photolysis of caged-ca 2+ produced current transients, possibly originating from electrogenic partial reactions. no monovalent cation substituted for na + at the nckx binding sites, but rb + substituted for k + , while sr 2+ , ba 2+ , mg 2+ substituted for ca 2+ with an apparent permeability ratio of 0.78, 0.20, <0.05:1, respectively. a. pfeifer 1 , k. zikihara 2 , s. tokutomi 2 , j. heberle 1 , t. kottke 1 1 bielefeld university, bielefeld, germany, 2 osaka prefecture university, sakai, japan the blue light receptor phototropin regulates the growth of plants towards the light. it contains two light-, oxygen-, or voltage-sensitive (lov) domains and a kinase domain. lov domains bind noncovalently flavin mononucleotide (fmn) as chromophore. upon illumination, the triplet excited state of flavin reacts within few microseconds with a nearby cysteine under formation of a photoadduct, which represents the signaling state. in response to adduct formation, a jα helix adjacent to the lov2 domain dissociates and allows for autophosphorylation by the kinase domain. the mechanism of the photoreaction and the signaling pathway from fmn to the jα helix are still unclear. we have investigated the lov2 domain of arabidopsis phototropin 2 by microsecond ft-infrared spectroscopy. the difference spectrum recorded at 2 µs provides evidence that the flavin is unprotonated in the triplet excited state. therefore, a previously proposed ionic mechanism of bond formation is disfavored. changes in secondary structure were detected concomitant with adduct formation that relax with a time constant of 120 µs. this early adduct intermediate has not been previously characterized. the final adduct state is formed in milliseconds by further alterations in secondary structure. these findings raise the question of whether the early or late adduct intermediate propagate the signal to the jα helix. -photosensory biophysics the psychostimulant amphetamine increased no generation measured by epr as well as amino acid release in the rat brain nitric oxide (no) is a novel messenger that modulates many functions of the nervous system. the involvement of no in brain damage was shown mainly by indirect evidence and the data are controversial. the short half-life of no makes its direct detection difficult. we measured no generation using epr spectroscopy based on determination of the amount of paramagnetic mononitrosyl-iron complexes. the aim was to elucidate whether psychostimulant drug amphetamine (amph) modulates formation of no and lipid peroxidation (lpo) products as well as the neurotransmitter release in rat brain. the output of glutamate, aspartate, gaba and acetylcholine (ach) was monitored in striatum by microdialysis with hplc detection. amph produced 2fold elevation of no generation and lpo formation in brain areas. while amph increased the aspartate, gaba and ach release, the glutamate output was not affected. pretreatment with the neuronal nos inhibitor was highly effective in abating the rise of no and neurotransmitter levels but failed to influence the lpo intensity elicited by amph. the findings suggest that activation of no synthesis is a potent factor in the amph-induced neurotransmitter release. light scattering study of dna over a wide chain length range: comparison with wormlike model p. baeri, m. zimbone dipartimento di fisica e astronomia, università di catania, italy this work reports light scattering measurements on dna in aqueous solutions (100 mm nacl ,1mm edta and 10 mm tris-hcl buffer, ph 7.8) over a wide range of molecular weights (10 2 -10 5 base pairs) and shows that, in the above standard solvent, shorter chains ( < 10 4 base pairs) behave as a "wormlike chain" and their diffusion coefficient as obtained by dynamic light scattering measurements, confirm the prediction of standard wormlike model, whilst longer chains ( >10 4 base pairs) behave in a different manner. dynamic and static light scattering and sem analysis indicate that dna molecules 10 5 base pairs long, condense into compact structures in our solvent condition. calculations done using a wormlike model are also presented and discussed in comparison both to our experimental data and to other data reported in the literature. a. asandei, t. luchian 'alexandru i. cuza'university, faculty of physics, laboratory of biophysics and medical physics, blvd. carol i no. 11, iasi, romania amphotericin b (amb) is an antifungal antibiotic which, despite the severe side effects, is still used for the treatment of systemic fungal infections. in this study we investigated the influence of ph upon the selectivity and the transport properties of amb channels inserted in reconstituted, ergosterolcontaining zwitterionic lipid membranes. our electrophysiology experiments carried out on single and multiple amb channels prove that at ph=2.8 these channels are anion selective, whereas at neutral and alkaline ph's (ph=7 and ph=11) they become cation selective. we attribute this to the ph-dependent ionization state of the carboxyl and amino groups present at the mouth of amb molecules. surprisingly, our data reveal that the single-molecule ionic conductance of amb channels varies in a non-monotonic fashion with ph changes, which we attribute to the ph-dependent variation of the surface and dipole membrane potential. we demonstrate that when added only from one side of the membrane, in symmetrical salt solutions across the membrane and low ph values, amb channels display a strong rectifying behavior, and their insertion is strongly favored when positive potentials are present on the side of their addition. we report a detection method for the redox state of proteins which combines fret-based fluorescence/confocal microscopy on dye-labeled protein with cyclic voltammetry. by using this combined method, electron transfer properties can be revealed from protein to electrode or from redox enzyme to substrate. we applied the fluorescent detection to azurin, a blue copper protein from the bacterium ps. aeruginosa, fluorescently labeled on the n-terminus for monitoring the redox state of the protein. the dye fluorescence is quenched by energy transfer to the copper in oxidized, but not in reduced azurin. fluorescence results demonstrated that cy5-labeled wtazurin switched in fluorescence intensity by up to 85% by varying the applied potential. labeled zinc-azurin was used as a control sample and did not show any fluorescence switching. for single molecule studies wt-azurin was labeled with atto 655 dye and a mixed sam was used, with 8-hydroxy-1octanethiol as a blocking agent to prevent non-specific binding of protein on the surface, and 1,10 decanedithiol for the specific covalent binding to the protein. preliminary data show that single-molecule fluorescence switching with the potential is indeed possible. -single molecule biophysics is there a specific erythrocyte membrane receptor for fibrinogen? an atomic force microscopy approach f. a. carvalho 1 , s. connell 2 , r. a. ariëns 2 , n. c. santos 1 1 instituto de medicina molecular, univ. lisbon, portugal, 2 university of leeds, u.k. fibrinogen (fg) contributes to erythrocyte (rbc) hyperaggregation by an increase in. rbc-fb protein binding considered to be non-specific. glycoprotein α iib β 3 is a specific integrin receptor for fibrinogen on platelets. we showed that there is a single molecule interaction between fg and an unknown receptor on rbc membrane, with a lower affinity when compared with platelet binding. we evaluated if rbc-fg binding is through an integrin-like receptor or not. interactions between fg and platelet/rbc receptors were studied by force spectroscopy. force curves were performed between fg-functionalized atomic force microscope tips and rbc or platelets. to evaluate if the fg-rbc binding is calciumdependent, similar studies were performed in the presence of ca 2+ or edta. we also carried out studies in the presence of a α iib β 3 inhibitor and of methyl-ß-cyclodextrin (to disrupt lipid rafts by cholesterol depletion). rbc-fg single forces were of 300-380 pn and of 400-480 pn for platelet-fg binding in presence of calcium. a significant decrease of the platelets-fg force-rupture was obtained in the presence of edta or α iib β 3 inhibitor. significant lower fg-rbc force value was obtained in the presence of mβcd, but not in the presence of edta. conclusion: fg-rbc binding seems not to be calcium-dependent but the existence of cholesterol on rbc membrane is important. nanoscopic and spectroscopic investigation of p53-based complexes at single molecule level a. r. bizzarri biophysics and nanoscience centre, cnism, facoltà di scienze, università della tuscia, largo dell'università -01100 viterbo, italy p53 is a transcription factor that plays a widely recognized role in preventing cancer development in response to dna damage. the tumor suppressor activity of p53 involves the formation of several complexes whose detection and study, at single molecule level, could be extremely relevant to understand, in detail, the mechanisms governing the cancer defense processes, as well as to develop ultrasensitive biosensors. p53-based complexes, are investigated at molecular level, by combining atomic force microscopy (afm), atomic force spectroscopy (afs) and surface enhanced raman spectroscopy (sers) with the support of computational docking. our attention is mainly devoted to study complexes between p53 and the electron transfer azurin which has been demonstrated to interact with p53, by promoting its stabilization. a possible competition between azurin and the cellular oncogene mdm2 is also investigated. unwinding the dna helix in force clamp condition p. bianco 1 , l. bongini 2 , m. dolfi 1 , l. vincenzo 1 1 physiolab, dbe, university of florence, italy, 2 university of florence and centro interdipartimentale studio dinamiche complesse, firenze, italy we use a dual-laser optical-tweezers (dlot, smith et al., science, 1996) to define the highly cooperative conformational transition in the molecule of dna, where the natural b-dna has converted into a new overstretched conformation called s-dna (bensimon et al., phys. rev. lett., 1995; cluzel et al., science, 1996) . single molecules of double stranded λ-phage dna (in a solution with 150 mm naci, 10 mm tris-hcl, 1 mm edta, ph 8.0. and 27 • c) are stretched either in length clamp or in force clamp mode. when the dna molecule is stretched in length clamp mode with a ramp lengthening, it shows the previously described highly cooperative overstretching transition at ∼60 pn, attributed to unwinding from the b-form to the 1.7 times longer s-form. stretching the molecule in force clamp mode with a staircase of force steps at 5 s intervals (step size 1-2 pn, rise time 1-2 ms) shows, for any given clamped force f in the region of the overstretching transition, different amounts of dna elongation (∆l) with exponential time courses. the analysis of the elongation rates allows to recover all the necessary parameters for an effective two-state model able to reproduce the out-of-equilibrium properties of the system. the results imply an unwinding cooperativity of 27 bps. this value is significantly lower than that obtained assuming force independent rate constants. supported by miur, ente cassa di risparmio di firenze and itb-cnr (milano). c. m. becker 1 , a. benedix 1 , b. l. de groot 2 , a. cafisch 3 , r. a. böckmann 1 1 saarland university, saarbrücken, germany, 2 mpi for biophysical chemistry, göttingen, germany, 3 university of zürich, zürich, switzerland modifying the stability or the binding behavior of the involved proteins by mutation can influence the activity of cellular processes. for an efficient identification of possible mutation-sites a fast calculation of the free energy of proteins is crucial. here we developed a fast and reliable method (cc/pbsa) [1] for the prediction of the change in stability of proteins and binding affinity of protein-protein complexes upon mutation. the energy function of cc/pbsa is based on gas phase energies, solvation free energies and entropic contributions. the protein flexibility is taken into account by generating random conformations based on geometrical constraints only applying the concoord [2] program. we applied cc/pbsa on the tem1-blip complex, which is important in bacterial antibiotic resistance. the results of single-and double-point alanine scanning are used to detect hot spots, cooperative effects, and the corresponding energy distribution. cc/pbsa is freely accessible on our web-server: http://ccpbsa.bioinformatik.uni-saarland.de the human recombinase hrad51 is a key protein for the maintenance of genome integrity and for cancer development. this protein plays a central role is the dna strand exchange occurring during homologous recombination. here we report the polymerization and depolymerization of hrad51 on duplex dna observed with a new generation of magnetic tweezers, allowing the measurement of dna twist with a resolution of 5 • in real time. at odds with earlier claims, we show that, after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition of single proteins, involving dna twisting steps of 65±5 • . simple numerical simulations support that this mechanism is an efficient way to minimize nucleoprotein filament defects. this behavior, consisting of different stoichiometry for nucleation and growth phases, may be instrumental in vivo. fast growth would permit efficient continuation of strand exchange by rad51 alone while the limited nucleation would require additional proteins such as rad52, thus keeping this initiation step under the strict control of regulatory pathways. besides, our results combined with earlier structural information, suggest that dna is somewhat less extended (4.5 versus 5.1å per bp) and more untwisted (18.2 versus 15 • per bp) by hrad51 than by reca, and confirm a stoichiometry of 3-4 bp per protein in the hrad51-dsdna nucleoprotein filament. biofunctional micropatterned surfaces to study the spatio-temporal organisation of lfa-1 r. diez-ahedo 1 , d. normanno 1 , c. g. figdor 2 , a. cambi 2 , m. f. garcia-parajo 1 1 bionanophotonics, ciber-bbn and ibec, barcelona, spain, 2 tumor immunology, nijmegen center for molecular life sciences, the netherlands lymphocyte function associated antigen-1 (lfa-1) adhesion depends on receptor occupancy and lateral organization on the cell membrane. however, the signals and mechanisms which dynamically reorganize lfa-1 into high avidity clusters are still a subject of many studies. to obtain deeper insight on the mechanisms that control and regulate lfa-1 clustering, patterned surfaces of immobilized lfa-1 ligand areas were fabricated using microcontact printing. the diffusion of lfa-1 expressed by monocytes stretched over patterned surfaces was followed in time using single molecule tirf microscopy. single lfa-1 nanocluster trajectories on individual cells showed an increase of immobile lfa-1 fraction and a slow-down of diffusing lfa-1 on the ligand areas compared to the non-ligand areas. moreover, single-cluster intensity analysis indicated a reorganization of lfa-1 nanoclusters in microclusters upon ligand binding. finally, single particle motion analysis of lfa-1 trajectories in close neighborhood to the ligand areas showed no assisted diffusion of lfa-1 towards the adhesive regions, consistent with random ligand-encountering and binding. we are currently investigating the effect of cell membrane organizers to regulate the spatio-temporal organization of lfa-1. r. diez-ahedo et al, small, in press. optical and electrophysiological detection of single phages across a lipid membrane n. chiaruttini 1 , p. boulanger 2 , m. de frutos 3 , l. letellier 2 , u. bockelmann 1 , v. viasnoff 1 1 nanobiophysique, espci paristech, cnrs, paris, france, 2 ibbmc, université paris xi, cnrs, orsay, france, 3 lps, université paris xi, cnrs, orsay, france we present an investigation study of the ejection of single t5 bacteriophages. in vivo studies of dna ejections from the bacteriophage capsid show that the t5 genome is introduced in the bacterial host in two steps. first 8% of the genome is ejected then after a pause of a few minutes the rest is internalized. bulk in vitro studies showed that various mutants of t5 eject their genome in solution following a single or a multistep process. by immobilizing single bacteriophages on a surface and following their ejection by fluorescence microscopy we showed that in all cases the ejection occurs in one step, but some mutants seem to have a subpopulation for which the triggering signal of the ejection is transmitted more slowly to the capsid entrance. we then reconstituted the phage receptor fhua into giant liposomes and followed the ejection of the dna into the liposome by fluorescence. finally we incorporated fhua in a suspended bilayer and followed the infection of the phages through the bilayer both by fluorescence labeling and electrophysiological measurements. we will discuss the influence of the cross membrane potential on the ejection speed of the dna. helixlike pili is a prerequisite of uropathogenic e. coli to adhere to host and withstand urine flow the gram-negative uropathogenic escherichia coli (upec) bacteria, invades the urinary tract region and cause in some cases severe infections, pyelonephritis, if they can withstand the rinsing action of urine and ascend to the kidney, via the bladder and ureters. to mediate adhesion, upec express quaternary surface organelles that are assembled from ∼10 3 identical subunits into a helix-like coil, with a single adhesin located at the tip. it is believed that the single adhesin mediate attachment to host cells while the helix-like structures act as shock absorbers to dampen the irregularly shear forces induced by urine flow. to unravel the biomechanical properties of such quaternary structures, in particular in terms of their force-elongation and kinetic behavior, force-measuring optical tweezers (fmot) have been used. a plethora of different types of pili have been identified in the literature and we show, using fmot, that those dissimilarities might reflect the host environment. for example, we have found differences among pili expressed at diverse environment inside the urinary tract, which imply that pili presumably have evolved to resist specific forces under in vivo conditions. it is thus worth striving for understanding bacterial adhesion in order to figure out alternative to the over-abundance of antibiotics worldwide. single molecules studies have probed protein conformational fluctuations and monitored the oscillating activity of enzymes that remained masked in ensemble measurements. various statistical models have attempted to connect the conformational motions to the fluctuating activity of enzymes and suggested that they adopt inter-converting conformations each one of them exhibiting different catalytic activity. the main drawback of these studies is the non specific interactions that may bias the observed catalytic behavior. we have developed new innovating tools to study the behavior of single enzymes. as a first step we utilized liposomes as scaffold to confine enzymes while keeping them under physiological conditions. keeping them in their native conformation allowed us to monitor the inherent properties of their behavior. as a second step we developed a new model that accurately connects enzyme activity behavior to conformational motions. using this approach we accurately predicted the behavior of single lipases in a highly controlled environment. we modulated the enzyme's conformational mobility and activity by systematically varying its accessibility to liposomes. as we anticipated, that resulted in altering the time the enzyme spends on active conformations.our results provide new insights on interpreting the behavior of enzymes. simulations of single-molecule fret experiments on ribozymes m. hajdziona, a. molski adam mickiewicz university laboratory for dynamics of physicochemical processes, poznan, poland rnazymes are important biological molecules and that can catalyze the cleavage of their own nucleotide chains. this precess is called self splicing [1] . there are several methods to to study kinetic properties of ribozymes at the singlemolecule level. in this work we focus on fret (förster resonance energy transfer) of single, immobilized molecules. single-molecule fluorescence spectroscopy gives an insight into the behavior of individual molecules rather than the average behavior of an ensemble of molecules, which makes it possible to observe the kinetic heterogeneity. we simulated fret trajectories for single immobilized ribozymes. in our computer simulations we consider two-and three-state kinetic models motivated by actual experiments, published in [2] and [3] . we fitted the histograms of on and off times to recover the kinetic parameters of the models. we investigated the bias and standard deviation of the recovered parameters when one or two thresholds are applied to fret trajectories between adjacent states. we found that two thresholds give better parameter estimates than one-threshold analysis. references frap probes the average dynamical properties of a population of fluorescently labeled molecules whereas spt obtains these properties from observations of the trajectories of individual molecules tagged with a colloidal particle (the "bead"). when results of frap are compared with those of spt, frap yields significantly higher diffusion constants than spt. to understand the origin of this difference, we have developed and tested a model system to evaluate the influence of the bead on the dynamics of a diffusing molecule. we use a dsdna tether to attach a bead to a supported lipid bilayer (slb). the dna tether is modified at one end by cholesterol for anchoring to and diffusion in the membrane, and at the other end by biotin for tagging with a bead. with this system, we can vary several parameters: distance between slb and bead, size and nature of the bead, lipid composition of the slb, external force. we can also model the brownian motion of the bead and the hydrodynamic flow around it. we will present results using φ = 200 nm neutravidin-coated latex particles attached to an eggpc slb via a 15 bp to 90 bp dna tether. elucidation of the mechanism of the lambda bacteriophage epigenetic switch l. finzi physics department, emory university, 400 dowman dr, atlanta, ga 30322, usa the lambda bacteriophage epigenetic switch determines the growth lifestyle of the virus after infection of its host (e. coli ). it is now clear that the switch consists of a ∼2.3 kbplong dna loop mediated by the lambda repressor protein. using tethered particle microscopy (tpm), magnetic tweezers and afm, our laboratory has novel, direct evidence of loop formation and breakdown by the repressor, the first characterization of the thermodynamics and kinetics of the looping reaction and its dependence on repressor non-specific binding and dna supercoiling. these in vitro data provide insight into the different possible nucleoprotein complexes and into the lambda repressor-mediated looping mechanism which leads to predictions for that in vivo. the significance of this work consists not only of the new insight into a paradigmatic epigenetic switch that governs lysogeny vs. lysis, but also the detailed mechanics of regulatory dna loops mediated by proteins bound to multipartite operators and capable of different levels of oligomerization. mechanical force at the molecular level is involved in the action of many enzymes. for example, the phi29 dna polymerase mechanically unwinds the dna helix as it moves processively along the dna replicating one strand of the dna molecule. using optical tweezers we have developed a single molecule mechanical assay to elucidate the physical mechanism of dna unwinding by the phi29 dna polymerase as the protein replicates the dna. a single dna hairpin is hold between an optical trap and a mobile surface. as a single polymerase works on the dna hairpin, its replication and unwinding reactions can be measured in real time by measuring the change in extension in the dna polymer, revealing the fluctuations of its rate in response to the dna sequence. moreover, by gradually pulling on the opposite strands of the dna hairpin we can promote the controlled mechanical unwinding of the dna helix and determine the effect of increasing unwinding forces on the polymerization and unwinding rates. the effect of force and dna sequence on the activities of the wild type and an unwinding-deficient polymerase mutant will allow us to determine the inner workings of this molecular motor. single centrosome manipulation reveals its electric charge and associated dynamic structure we demonstrate laser manipulation of individual early drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively-charged organelle with a very low isoelectric region (3.1 ± 0.1). from this single-organelle electrophoresis, we infer an effective charge smaller or of the order of 10 3 electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. by investigating the centrosome hydrodynamic behavior, we show that its charge has a remarkable influence over its own structure. specifically, we find that the electric field which drains to the centrosome expands its structure to a physiological size a 60% larger than previous electron microscopy determinations, a self-effect which modulates its structural behavior via environmental ph. this methodology further proves useful to study the action of different environmental conditions, such as the presence of ca 2+ , over the thermally-induced dynamic structure of the centrosome. magnetic contrast neutron reflectivity delivers significant improvements in resolution for membrane structure analysis neutron reflection (nr), with its high resolution and use of contrast variation, is a unique tool to gain information on the orientation and structure of lipid bilayers in the z-axis perpendicular to the surface. to improve the use of nr we have used a highly oriented and stable layer of membrane proteins and lipids made possible by self-assembly upon a gold surface. this also provides a model of the bacterial outer membrane. as the dimensions of the layer can be predicted with accuracy, the system provides a molecular ruler for improvements in methodology and the complexity of the layer structure adds significantly to the modelling challenge. early improvements in resolution were obtained through sample preparation and gold smoothness. further improvement however required clearer discrimination between similar models. this was achieved using the new approach of magnetic contrast variation which uses a magnetic layer to provide two different scattering length densities for oppositely polarised neutrons. during this data collection the sample is unchanged. we show that this approach delivers significant improvements in data analysis and resolution of the protein, lipid and solvent structures. the method will provide a new level of understanding of membrane structure and dynamics. single molecule force spectroscopy and recognition imaging p. hinterdorfer institute for biophysics, johannes kepler university of linz, altenbergerstr. 69, a-4040 linz, austria in single molecule force spectroscopy, interaction forces of ligand-containing tips with receptors on probe surfaces are quantified. here he attachment of human rhino virus 2 (hrv2) to the cell surface, the first step in infection, was characterized. sequential binding of multiple receptors was evident from recordings of characteristic quantized force spectra. this suggests that multiple receptors bound to the virus in a timely manner. unbinding forces required to detach the virus from the cell membrane increased within a time frame of several 100 ms. the number of receptors involved in virus binding was determined and estimates for on-rate, off-rate, and equilibrium binding constant were obtained. furthermore, we show that accurate free energy values of membrane protein unfolding can be obtained from single molecule force measurements. by applying a statistical theorem developed by jarzynski, we derived equilibrium unfolding free energies of from unfolding force data acquired at different force loading-rates and temperatures. finally, we present a method for the localization of specific binding sites and epitopes with nm positional accuracy. a magnetically driven afm tip containing a ligand covalently bound via a tether molecule is oscillated at a few nm amplitude, during scanning along the surface. in this way, topography and recognition images on membranes and cell surfaces were obtained simultaneously. mapping of the forces acting on biomolecules in cell membranes has spurred the development of effective labels, e.g. organic fluorophores and nanoparticles, to track trajectories of single biomolecules [1] . standard methods use particular statistical observables, namely the mean square displacement (msd), to extract cues on the underlying dynamics. yet, msd is not an appropriate tool to access force fields and becomes easily a biased estimator in the presence of positioning noise. here, we introduce general inference methods [2] to fully exploit information hidden in the experimental trajectories, providing sharp estimates for the forces and the diffusion coefficients within membrane microdomains. rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. the inference method is then applied to infer forces and potentials acting on the receptor of the ε-toxin labelled by lanthanide-ion nanoparticles. results show a constant diffusivity inside a complex force field confining the receptor inside a specific domain. our scheme is applicable to any labelled biomolecule, and results presented here show its general relevance to the issue of membrane compartmentation and protein motion. large scale domain motions are structural rearrangements often adopted by enzymes to achieve their full functionality. understanding the mechanism responsible for such movements by means of computational approaches is of great interest especially in rational drug design, since induced fit or population shift effects are closely related aspects of the problem. unfortunately, the simulation time required to sample these events by conventional techniques such as plain molecular dynamics, is unfeasible. here, the domain motion required by adenosine kinase (ak) to achieve its (pre-)catalytic conformation was studied using well-tempered metadynamics [barducci, a. et al. phys rev lett. (2008) , 100: 020603] with path collective variables (pcvs) [branduardi, d. et al. j chem phys. (2007) , 126: 054103]. first, a low energy path for the apo form of the enzyme was obtained, and the potential of mean force along the pcvs was reconstructed. then, the large scale movement was simulated in the presence of two inhibitors known to bind to the enzyme in different conformational states. the adopted approach was proven to be successful both to understand the mechanistic features of the ak domain motion and to provide a picture of the population shift effects upon ligand binding. force measurement study of the interaction between 23s rrna and the ribosomal protein l20 single molecule force measurements enable to probe the complex structure and folding dynamics of rna molecules and furthermore to investigate the numerous interactions with proteins that affect rna folding in vivo. we use a double optical tweezers setup to study a region of the ribosomal rna 23s from e.coli and its interaction with the essential ribosomal protein l20. the apparatus permits us to probe the dynamics of the rna structure with milisecond time-resolution. first, we pull the rna and show that it mechanically unfolds in several reproducible steps. we use these results in combination with structure prediction tools to evaluate the possible structures of the rna fragment in vitro. the most probable structure exhibits a unique binding site for l20. then, force measurements are done on the rna in presence of l20. we show that the protein specifically binds the rna and stabilizes it. the binding site is recognized with a resolution of a few bases. finally, two bases are mutated on the rna fragment. in presence of these mutations, in vivo and in vitro binding of l20 to 23s rna is abolished. the single molecule approach gives an explanation of this result: the force measurements show that the mutated rna folds differently from the natural one and that it does not bind l20. control of the translocation of single dna molecules through alpha-hemolysin nanopores g. maglia, h. bayley department of chemistry, university of oxford, ox1 3ta, oxford, uk the analysis of single nucleic acid molecules by electrophoretic threading through nanopores is under intense investigation as a rapid, low cost platform for dna sequencing. biological nanopores such as staphylococcal alpha-hemolysin (hl) have an added advantage over solid-state nanopores because they can be modified by genetic engineering with atomistic precision. although we showed that all four dna bases can be identified in an immobilized ssdna molecule (1), the translocation of free dna is too quick to observe single bases. here we show that by a small increase of the net internal charge of the hl nanopore (e.g. by introducing a ring of 7 arginine residues), we have augmented the frequency of dna translocation events through the pore and dramatically lowered the voltage threshold required for dna translocation (2) . by further increasing the net positive charge of the transmembrane barrel region of the pore (e.g. by introducing 49 extra positive charges) we have also reduced the speed at which dna translocate the pore by more than two orders of magnitude. these experiments provide a means of controlling dna translocation with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification. the atomic force microscope (afm) is a tool for imaging, measuring and manipulating matter at the nanoscale. single-molecule pulling experiments give information on the thermodynamics and kinetics of biomolecules. the purpose if this work was to develop software to simulate single-molecule pulling experiments and to analyze singlemolecule pulling data. the long term objective is to asses the accuracy and precision of the parameters recovered from single-molecule pulling data.we carried out simulations for two different models [1, 2, 3] , using a wide range of loading rates. from the simulated force-extension curves we extracted the kinetic parameters and compared them with the values used for simulations. the kinetic parameters were the intrinsic rate coefficient (k 0 kramers rate), the location of transition state (x ) and the free energy of activation (∆g). we have found that the loading rates have a small effect on the recovery of the free energy of activation, but have a significant effect on the recovery of the kramers rate. we report the tracking of single myosin v molecules in their natural environment, the cell. myosin v molecules, labeled with quantum dots, are introduced into the cytoplasm of living hela cells and their motion is recorded at the single molecule level with high spatial and temporal resolution. we perform intracellular measurements of key parameters of this molecular transporter: velocity, processivity, step size and dwell time. our experiments bridge the gap between in vitro single molecule assays and the indirect measurements of the motor features deduced from the tracking of organelles in live cells. a mathematical model of neurotransmission at the input stage of the cerebellum t. nieus 1 , s. solinas 3 , l. mapelli 2 , e. d'angelo 3 1 dept. neuroscience and brain technologies, iit, italian institute of technology, genova, italy, 2 dept. of biomolecular sciences and biotechnology, milan, italy, 3 dept. physiological and pharmacological sciences and cnism, university of pavia, italy the granule cell (gc) of the cerebellum has some peculiar properties compared to other cells of the vertebrate brain. the gc has a small soma (diam=6 microm) and just a few (2 to 6) excitatory and inhibitory inputs (e&i). the e&i gc synaptic inputs are formed inside the cerebellar glomerulus, which favors neurotransmitter diffusion between neighboring sites (1) protracting postsynaptic receptor activation and (2) causing cross-tall between e&i synapses through presynaptic receptors. neurotransmitter release probability (p) can be regulated by long-term plasticity (ltp and ltd at e synapses) and by gaba b and mglu presynaptic receptors (both at e&i synapses). the p change in turn modifies shortterm plasticity, affecting the first response in a train much more than the followings. to gain insight into the role that p changes might have in computations performed at the cerebellum input stage, we have built detailed biophysical models of the e&i synapses. the model has allowed to investigate glomerular processing of high frequency inputs reaching the cerebellum. optimal synaptic transmission through the mfs inputs resulted when gos inhibited synchronously the gcs. the role of feed-forward inhibition onto synaptic transmission is under investigation. unzipping dna with a nanopore j. muzard, n. chiaruttini, u. bockelmann, v. viasnoff cnrs/espci nanobiophysique, 10 rue vauquelin, 75005 paris, france the use of proteinaceous or artificial nanometer size pores has become a promising approach for sensing biomolecules at the single molecule level. it was shown that alphahemolysin, a toxin from staphylococcus aureus, can be employed to sense the translocation of dna strands through a lipid bilayer. the pore dimension allows the electrophoretically driven passage of single stranded dna whereas double stranded structures need to open prior their translocation. we study the unzipping process of the double stranded part of dna both experimentally and theoretically. we show that in a first approximation the duplex opens progressively in a sequence dependent manner. the experimental results can be accounted for by modeling the unzipping process as a free diffusion of the unzipping fork in the energy landscape defined by the sequence of the basepaires. we then discuss the effect of the pore/dna interaction on the translocation process. we further characterize the effects of the pore geometry, showing that the pre-confinement of the dna in the pore vestibule is essential to the unzipping process. we eventually discuss the possibility to use this nanopore approach to sequentially probe rna secondary structures. we report on the interaction forces in the range 100 --400 pn determined between pairs of the lectin soybean agglutinin (sba) and a modified porcine submaxillary mucin (tn-psm) using a single-molecule approach. lectins are carbohydrate-binding proteins with biological activities related to i.e. cellular recognition, adhesion, growth and metastasis. here, dynamic force spectroscopy is used to investigate pairs of sba and tn-psm with the aim to understand the mechanisms of binding and cross-linking of multivalent lectins. the unbinding force increased from 103 pn to 402 pn with increasing force loading rate and the lifetime of the complex in the absence of applied force was 1.3 -1.9 s. published kinetic parameters describing the rate of dissociation of other sugar lectin interactions are in the range 3.3 x 10 −3 -2.5 x 10 −3 s. the long lifetime of the sba -tnpsm complex is compatible with a previously proposed "bind and jump" mechanism. this mechanism has also been suggested for lectins binding to multivalent carbohydrates and globular glycoprotein. the bind and jump mechanism is also similar to that observed for binding of proteins to dna, and suggest a common conserved binding mechanism of ligands to the two biopolymers and possibly between ligands and all biopolymers, as recently suggested. the rate of topoisomerase ii activity correlates with persistence length of dna q. shao 1 , l. finzi 1 , d. dunlap 2 1 dept. of physics, emory university, atlanta, georgia, u.s.a., 2 dept. of cell biology, emory univ. med. school, atlanta, georgia, u.s.a. type ii topoisomerases catalyze the transection of one double helical segment by another to modify the topological state of dna. reversible 5' linkages to the phosphate-sugar backbones are established on each strand of the "gate" segment to create an opening through which the enzyme drives the "transfer" segment. a recent crystal structure shows that the "gate" segment bends approximately 150 • upon binding to the enzyme. bending a stiff polymer like dna requires considerable energy and could represent the rate limiting step in the catalytic (topological) cycle. using modified deoxyribonucleotides in pcr reactions, more rigid dna fragments have been produced and used as substrates for topoisomerase ii-mediated relaxation of plectonemes introduced in single molecules using magnetic tweezers. before relaxation the persistence lengths were measured by fitting force extension data with the worm-like chain model. topoisomerase ii was found to release mechanically introduced supercoils more slowly in stiffer dna suggesting that dna bending might be a rate limiting step in topoisomerase ii activity. h. seidel 1 , t. klose 2 , h. lilie 2 , c. g. hübner 1 1 institute of physics, ratzeburger allee 160, 23538 lübeck, germany, 2 institute of biochemistry and biotechnology, kurt-mothes-str. 3, 06120 halle, germany one essential element of a virus is its protein shell, the viral capsid, which encloses the viral genome. the murine polyomavirus is a non-enveloped dna tumor virus with an icosahedral t=7d structure. besides the knowledge of the structure, it is of utter importance to understand the process of viral assembly [1] . the assembly reaction of polyoma vp1 does not show the typical sigmoidal kinetics in light scattering experiments. the apparent kinetics is of fourth order, which appears rather unrealistic. in order to gain knowledge on capsid composition during assembly beyond ensemble average, we apply methods of single molecule fluorescence, namely fluorescence correlation spectroscopy (fcs), fluorescence-intensity-distribution-analysis (fida), and single-particle-imaging (spi). the molecular brightness and the diffusion time reported by fcs are in agreement with the results from light scattering. fida as well as spi, moreover, point to a polymerization of subunits to complete capsids along the assembly pathway without pronounced intermediates. mixing experiments show that already early in the course of the assembly reaction no exchange of pentamers between capsids occurs, and that the effect of breathing [2] can be excluded for polyoma vp1. genetic information coded in dna provides complete instructions to cells regarding metabolism and proper functioning. a number of endogenous and exogenous sources can damage the genomic dna. unrepaired dna damage can give rise to mutations and may cause cell death. cells have evolved different mechanisms to repair damaged dna and to protect genetic integrity. uracil in dna occurs as a result of incorporation of dump in the place of dtmp during replication and deamination of cytocine, resulting in u:a and u:g base pairs, respectively. uracil-dna glycosylase (ung) is a dna repair protein that searches and removes uracil from dna. ung removes mismatched base by flipping it out from the base stack into the active site. the exact mechanism by which ung searches dna for uracil is unknown. therefore, in our study we use atomic force microscopy (afm) to investigate the interaction between single ung molecules with dna carrying the u:a or u:g mismatches. furthermore, we study the complexation of dna and ung protein. afm images of ung bound to dna reveal the structural changes at the level of single complex, e.g. dna kinking that may occur upon binding of ung to dna. imaging of dnaprotein complexes can provide a a new level of understanding of ung-dna interaction. atomic force microscopy (afm) has been a useful device to visualize cellular and molecular structures at a single-molecule resolution. especially, afm imaging under the trec t m (topography and recognition) mode (recognition imaging) can map a specific protein of interest within an afm image. in this study, we employed an antibody-coupled afm cantilever in recognition imaging, and dissected the structural/functional domains of α actinin-4, an actin binding protein that cross-bridges actin filaments and anchors it to integrin via tailin-vinculin-α actinin adaptor-interaction. a use of monoclonal anti-α actinin-4 antibody enabled us to map its epitope in the amino-terminal actin-binding domain within a single molecule afm image of α actinin-4. counting fluorescent molecules by photonantibunching h. ta, m. schwering, d. p. herten bioquant, heidelberg university, germany information on the stoichiometry of labelled biomolecules is highly desirable. a method called photo-antibunching has successfully been used to determine the number of fluorescence emitters in multichromophoric systems. a statistical model to estimate the number of fluorescent molecules in a confocal observation volume is established based on photonantibunching in time-correlated single-photon counting (tcspc) scheme with 4 avalanche photon diodes (apds) for detection of individual photons. numerical simulations based on realistic experimental conditions show that the model is able to estimate the number of molecules with moderate errors. experiments on immobilized double-strand dna oligonucleotides with photon stabilizing agents show promising results even when the number of molecules is ∼ 20. the proposed method allows us to monitor labelled single molecules and in the near future we plan to implement it in complex biological systems (cell extracts/live cells). single molecule afm force spectroscopy and steered molecular dynamics of contactin-4 protein j. w. strzelecki, k. mikulska, a. balter, w. nowak institute of physics, nicolaus copernicus university, grudziadzka 5, 87-100 torun, poland contactin-4 (cntn4) is an axonal cell adhesion protein that contains six igc2 and four fniii domains and is responsible for creating neural outgrowths. recent research shows that mutation of cntn4 gene may be responsible for autism, while its absence in transgenic mice results in lack of smell sense. we use a homemade afm single molecule puller and steered molecular dynamics simulation to test its elastic properties through force spectroscopy. stretching experiments show unfolding of fniii domains while igc2 domains stay coiled as they are stabilized by disulfide bonds. unfolding of contactin-4 molecule appears to play role of buffer that helps to protect neural contacts from damage when neural cells are subjected to shock or tumor. single molecule studies of the thermophilic bacillus ps3 f 1 -atpase have revealed kinetic and structural information that cannot be discerned using other methods, including the presence of 40 and 80 degree physical substeps (yasuda et al. 2001 ) and the order and kinetics of chemical substeps. we are interested in using single molecule techniques to observe the effects of mgi mutations on enzyme kinetics and torque production in f 1 from the yeast saccharomyces cerevisiae. using a high speed imaging camera, we have captured the rotation of wild-type and mutant forms of yeast f 1 -atpase. rotation data for the wild-type and preliminary data for some mgi strains will be presented. we show for the first time that at saturating atp, wild-type yeast f 1 rotates approximately four times faster than the thermophilic f 1 . kinetic and substepping behaviour in yeast appears to be similar to that observed in bacterial f 1 . -single molecule biophysics biophysical characterization of a dna gquadruplex formed in the promoter of human mef2d gene w. zhou 1 , l. ying 2 1 molecular medicine, national heart and lung institute, imperial college london, london, sw7 2az, uk, 2 chemical biology center, imperial college london, sw7 2az, uk g-quadruplex is believed to be involved in many crucial biological processes, such as the gene regulation and the maintenance of human telomere. mef2d, a member of mef2 (myocyte enhancer factor-2) family of transcription factors, regulates the response of heart to cardiac stress signals. we found that a g-rich sequence starting at -145bp of mef2d promoter can form a very stable intramolecular g-quadruplex. here we present a detailed biophysical characterization of this quadruplex. we also found that this quadruplex is more stable than its duplex form under physiological conditions by cd melting and single molecular fret. we observed subpopulations in smfret measurements possibly due to different conformations of the quadruplex. we characterized its unfolding process by monitoring the change in fret when it hybridizes to its c-rich complimentary strand. finally, we proposed several possible structures of this quadruplex based on the smfret and fluorescence dms footprinting results. this research is supported by national heart and lung institute foundation. by stretching a polymer in solution using single molecule techniques it is possible to infer about its physical properties. afm stretching experiments allow for a full characterization of the elasto-mechanical properties of the sample under study. in the presented work, single molecule afm force spectroscopy experiments were used to determine mechanical properties of a peptide obtained from exon 28 (ex28) of the human elastin gene. elastin is a protein with important mechanical properties and, in particular, it shows quasi ideal elastic behavior associated to the presence of many hydrophobic unstructured domains (such as ex28) into the protein structure. ex28 coded polymer was used as a starting point to obtain bio-materials with specialized elastomechanical functions. in particular, a mutated polypeptide based on the ex28 sequence was synthesisized with the aim of obtaining a new polymer with the same mechanical and physical properties of the native molecule but with increased aggregation properties, induced by a cross-linking reaction. afm stretching experiments were used to verify the mechanical properties of the engineered proteins at a single molecule level. the obtained results allowed not only to address this question, but also to give some insight into the first aggregation steps of the polymer towards the formation of reticulated structures. luminescent lanthanide-ion doped nanoparticles (nps) are attractive single-biomolecule labels. they are synthesized directly in water, are highly photostable, and display narrow emission without intermittency. we coupled y 0.6 eu 0.4 vo 4 nps to ε-toxins produced by clostridium perfringens, which bind to a specific receptor on mdck cells. single-molecule tracking experiments using these labels produce long (5 min) uninterrupted trajectories with temporal resolution down to 20 ms or localization precision down to 15 nm. we found that the toxin receptor exhibits confined motion in cell membrane microdomains. to analyze the receptor trajectories, we used a novel approach based on an inference method [1] . this method fully exploits the information of the ensemble of the trajectory, in contrast to the usual mean square displacement analysis. applying both techniques to recorded trajectories, we highlight the difference in extracted parameters. from the shape of the confining potential, obtained by mapping the forces inside the domain, we can deduce information about the mechanism of confinement. in combination with experiments on cholesterol depletion and cytoskeleton destruction, this technique will shed light into the nature of the membrane micropatterning. background: implantable cardioverter defibrillators (icds) are save-life device for patients at risk of sudden cardiac death, and help cardiac patients to avoid slow beat-rate by means of anti-bradichardia pacing (abp) feature. however, recent literature evidenced the presence of ventricular tachyarrhythmias (vts) immediately following abp, leading to the hypothesis that icd devices might be pro-arrhythmic. aim: study differences between pacing-associated tachyarrhythmias (pat) and other spontaneous vts. signals and methods: 165 spontaneous vt episodes are retrieved from 37 patients with icd. vts are examined and pat episodes, classified. characterization of vts is based on the analysis of 20 seconds immediately preceding vt-onset quantified by: heart cycle (hc-prevt), prevalence of premature ventricular contraction (pvcprev) and prevalence of paced beats (pprev). significant differences are evaluated by student-t or chi-square tests with p<0.05. results: 20 vt episodes from 8 patients are pat episodes (12%). cardiac activity preceding pats vs. non-pats differs: pat episodes have higher hc-prevt (p<0.01), greater pprev (p<0.001), and greater pvcprev (p<0.001). analysis also shows that pat episode often occur when the paced beat immediately follows a premature contraction. conclusion: the study indicates that new icd generations should avoid abp after premature ventricular contraction. towards mechanistical understanding of adsorption: combining technologies in situ and in real time p. h. bjöörn q-sense ab, västra frolunda, sweden a growing number of researchers in different surface related fields present evidence from more than one analytical technique when detailing their findings. thus a logic and useful development is to combine technologies for simultaneous measurements on a single sensor surface. to develop a biosensor platform such as an assay fast and accurately, mechanistical understanding of why the platform works is essential. quartz crystal microbalance-with dissipation (qcm-d) technology and instrumentation provides an open platform and enable easy and precise quantification of mass, thickness and viscoelastic properties of surface bound molecules. these parameters provide both a reference tool in assay development, but also detecting and identifying your target molecules as the combination of mass and material property info in many cases provide a unique response for a specific molecule. by combining qcm-d with other technologies, a range of useful info is put within easy reach of the researcher. recent advances will be presented where simultaneous real time and in situ measurements using qcm-d together with electrochemistry, ellipsometry and fluorescence microscopy enables manipulation of films as well as quantification of the variation of water content as a result of conformational changes in immobilized molecules. examples will include new data from the formation of protein films, polyelectrolyte multilayers and polymer brushes. intrinsic gravity versus metabolically inert infrastructure and basal metabolic rate in living mass i. r. bhattacharjee 1 , r. kashyap 2 , b. shaptadvipa 2 1 assam agricultural university, jorhat-785013, india, 2 international institute of intrinsic gravitation biology (i3gb), jorhat-785013, india 'self gravitation bio' is an emerging concept in biophysics. intrinsic or 'self' gravitational force might exert when mass increases beyond critical level in biological particle pyramid. (http://en.wikipedia.org/wiki/biomechanics of intrinsic gravity) 'metabolically inert infrastructure' (mii) consists of total body mass (body water, dissolved substances, mineral and organic deposits) and serves as storage of nutrients, transport and distribution of these materials. to act independently as living body, mii is suggested also to provide structural support to the organism with density-gradient buoyant force against intrinsic and extrinsic gravitational attraction for the biological mass. it is shown that 'amniotic fluid', 'isotonic saline to ailing patient', 'growth factors', 'cultural medium' and other 'medium matrices' act as counter-gravity mechanism for micro to macro living organisms under center-of-biomass reference frame. controversy over relationship between bmr (basal metabolic rate), rmr (resting metabolic rate), pal (physical activity level), on one hand, and mass of the living organism, on the other, (as described in rubner's 2/3 law, kleiber's 3/4 law that continued to be contested by many) can be favorably resolved substituting the concept of self gravitation bio, considering 'mass' as synonym of gravitational force under center-of-biomass reference frame. bioenergetics would be an unequal but opposite entity of self-gravity reinforced by extrinsic gravity. platelet arrest on von willebrand factor (vwf) occurs transiently via the platelet receptor gpib. depending on the combination of shear stress and surface density of vwf binding sites the platelets either only adhere, pull tethers or generate microparticles. these processes are influenced by the properties of the platelet membrane and the cytoskeleton. under shear flow conditions these platelet characteristics were examined with reflection interference contrast microscopy and a viscosimeter. in addition we quantified platelet adhesion energy and tether pulling forces. disrupting platelet f-actin had several effects. the tethers were shorter, the membrane contact area was larger, the receptor αiibβ3 density of the microparticles was depleted and no platelet spreading occurred. breaking up the microtubular system had different implications. the number of severed tethers tripled, the contact area was smaller, microparticle formation was increased and the area of spread platelets was reduced. however changes in the membrane elasticity had no effect on the platelets. our results suggest that platelet attachment and adhesion is not only determined by the platelet adhesion receptors and their cytoplasmic linker proteins, but that cytoskeletal structures have also a crucial influence on how platelets interact with thrombogenic surfaces. gene expression is orchestrated by a host of regulatory proteins that coordinate the transcription of dna to rna. regulatory proteins function by locating specific binding sequences of dna and binding to these sequences to form the transcription initiation complex. in many instances, these regulatory proteins only have several hundred copies that must efficiently locate target sequences on the genome-length dna strand. the non-specific binding of regulatory proteins to random sequences of dna is believed to permit the protein to slide along the dna in a stochastic manner. periodically, a thermal kick or an interaction with another bound protein will disengage the regulatory protein from the dna surface, leading to three-dimensional diffusion. eventually, the protein will reattach to the dna at some new location that is dictated by both the diffusivity of the protein and the dna configuration. cycling through these random events constitutes a search strategy for the target site. we build a reaction-diffusion theory of this search process in order to predict the optimal strategy for target site localization. the statistical behavior of the dna strand acts as a necessary input into the theory, and we consider several governing behaviors for the dna strand. we explore the impact of dna configuration and protein occlusion on target site localization in order to predict how protein expression will vary under different experimental conditions. a. di garbo 1 , s. alloisio 2 , s. chillemi 1 , m. nobile 2 1 istituto di biofisica cnr, via g. moruzzi 1, 56124 pisa, italy, 2 istituto di biofisica cnr, via de marini 6, 16149 genova, italy in the nervous system of vertebrates there are more glial cells than neurons: from 10 to 50 times, depending on the animal type. glial cells are not able to generate action potentials but, nevertheless they play an important role for the functioning of the different brain's area. the astrocytes are the more abundant cells of the macroglia and through their processes they modulate synaptic activity. in this contribution a biophysical neural network model consisting of a pyramidal neuron, an interneuron and the astrocyte is studied. the corresponding dynamical properties are mainly investigated by using numerical simulations. the results show that the presence of the atp and of the interneuron strongly impacts the neural activity. moreover, it is shown that the fluxes of calcium through the cellular membrane strongly affect the modulation of the neural activity arising from the astrocyte. microscopic origin of the very-low energy vibrational dynamics in proteins g. d'angelo 1 , v. conti nibali 1 , c. crupi 1 , a. paciaroni 2 , u. wanderlingh 1 1 department of physics, faculty of science, messina university, italy, 2 department of physics, faculty of science, perugia university, italy important functions of biological processes require large atomic rearrangements and conformational fluctuations. for proteins, atoms are mostly displaced along the soft directions identified by the delocalized, low frequency vibrations. the study of very low energy vibrational spectrum of proteins is therefore of particular interest. as a step toward understanding their functional role, we have investigated the low frequency vibrational motions (0.03÷10 mev) in different proteins by performing inelastic neutron scattering and low temperature (1.5÷30 k) specific heat measurements. for the first time for biological systems, the well-known boson peak found in neutron scattering spectra at ∼ 3 mev is put in relationship with a clear anomaly of the measured specific heat located at around 7 k. this departure from the debye-like behavior further expands the analogy of proteins with glassy systems. quite interestingly, in the very low sub-mev energy range and below ∼3k, we observe an additional anomalous behaviour, which could be ascribed to the existence of twolevel-systems states. increasing the hydration degree, the low energy vibrational region is drastically altered, revealing that the addition or removal of the hydrogen bond network around the protein deeply modifies the interatomic forces, affecting the character of the vibrational modes. a. cupane 1 , m. levantino 1 , m. cammarata 2 , g. schirò 1 1 department of physical and astronomical sciences, university of palermo, via archirafi 36, i 90123 palermo, italy, 2 european synchrotron radiation facility, grenoble, france our efforts in recent years have been to study in great detail the way hemoglobin works in confined geometries [1] [2] [3] . to this aim we have contributed to the development of a new experimental technique, time-resolved wide-angle x-ray scattering (tr-waxs), that enables one to watch proteins at work in solution [4, 5] . a very recent and challenging application of this technique is the study of hemoglobin functioning inside intact red blood cells (rbc). our preliminary results show that, by using laser pulses of about 150 ns, it is possible to photolyse hemoglobin inside rbcs obtaining about 40% -60% photolysis. good structural signals from hemoglobin are obtained, with limited radiation damage to the cells: this opens the possibility of studying the conformational transitions of hemoglobin in its "natural" environment. preliminary results concerning the timing of the r->t quaternary transition inside the rbc and comparison with the behaviour in dilute solution will be discussed. physical review letters and physical review e invite submission of your best work in biological physics. submissions must present significant new results in physics; topics may range from the microscopic to the macroscopic. we will provide information on the different types of articles published in the journals, on authors and referees, and on the review process. for publication in physical review letters, the work should be important and of broad interest. for rapid communications in physical review e, the work should be important for the field. biological physics papers published in physical review are indexed in medline. in an effort to bring important research to the attention of a wider community, the physical review journals have recently begun highlighting important work with viewpoints in the online publication physics. all physical review journals may be accessed through the website http://publish.aps.org/. modeling of fibrin gels using confocal microscopy, light scattering and turbidimetry f. ferri 1 , d. magatti 1 , b. cardinali 2 , a. profumo 2 , m. rocco 2 1 dipartimento di fisica e matematica, università dell'insubria, como, italy, 2 istituto nazionale per la ricerca sul cancro (ist), genova, italy fibrin gels are biological networks playing a fundamental role in blood coagulation. confocal microscopy of fibrin gels shows a collection of straight fibers, not uniformly distributed in space, connected together at low-order (3) (4) branching points. although each fiber is quite straight (mass fractal dimension d m =1), for the overall system d m >1. based on the confocal images, we generated threedimensional (3d) synthetic gels made of cylindrical sticks of diameter d, joined together at randomly distributed nodal points. the resulting 3d network is no more random but ordered on length scales of the order the average fiber length, and exhibits a fractal morphology with d m ∼1. 2-1.7 . the gel structure is analyzed by means of its 3d correlation function g 3d (r)=<n(x) n(x+r)> x , where n(x) is the gel local density. since the gel is isotropic, g 3d depends on the modulus r=|r| and can be obtained by averaging 2d correlation functions evaluated at different heights of the gel volume. from this analysis we recover the fiber diameter d (fwhm of g 3d ), the fractal dimension d m (power-law decay of g 3d ) and the gel mesh-size ξ (minimum in g 3d ). furthermore, the 3d-fourier transform of g 3d (r) gives the gel power spectrum i(q), which compares quite well with elastic light and multi-wavelength turbidimetry data taken on real gels. a model coupling vibrational and rotational motion for the dna molecule e. drigo filho, j. r. ruggiero, r. a. d. s. silva unesp -sao paulo state university, brazil a largely used mechanical model for vibrational motion of dna has been proposed by peyrard and bishop (pb). in this model, dna is represented by a pair of harmonic chains coupled by a nonlinear potential. the most frequently used nonlinear potential for this purpose is the morse potential. some extensions of the original model are proposed considering, for example, the helicoidal structure of dna. an important objective for this kind of model is to understand the phenomenon of thermal denaturation and, through this, get some knowledge about other processes such as the genetic transcription and drug intercalation. it is possible to obtain from this type of model interesting properties, such as the average stretching between base pairs as a function of the temperature using the transfer integral operator. dynamical properties of this model were also explored and several phenomenological quantities are studied, such as energy localization. in this work, we extend the original pb model by introducing rotational motions for the nucleotides. in this way, both the vibrational and rotational motion for each nucleotide are considered. the stretch of the base pairs is given by the morse potential in the same way as in the original pb model. however, the coupling between the two kinds of motion, rotation and vibration, is obtained through a nonlinear combination of them in the morse potential. a. dobovišek 1 , m. brumen 2 , p.županović 3 , d. juretić 3 1 university of maribor, faculty of natural sciences and mathematics, maribor, slovenia, 2 jožef stefan institute, ljubljana, slovenia, 3 faculty of science, university of split, split, croatia mepp is a physical principle, widely used for quantitative explanation of non equilibrium phenomena in physics, chemistry and biology [1] [2] [3] . here, we applied mepp to study two and three state reversible michaelis-menten kinetic schemes of enzymatic reactions. by applying constraints as a diffusional limit for kinetic constants, constant free energy differences between enzymatic states and constant thermodynamic force, we calculated shannon information entropy and entropy production of the entire reaction system as a function of forward rate constants. we found maxima in the net steady-state metabolic flux, total entropy production and shannon entropy for equal values of forward rate constants. moreover, for these values an analytical expression derived gives a relation between substrate and product concentrations at which enzymes operate in the optimal way. in conclusion, we demonstrated that mepp is an appropriate selection principle for evolutionary optimization of enzymes. attractive interaction between like-charged lipid surfaces mediated by spherical nanoparticles with spatially distributed charge is theoretically described by using functional density theory and mc simulations. the spatial distribution of charge within a single nanoparticle is considered by two effective charges at a finite distance. the minimization of the free energy is performed to obtain the equilibrium configuration of the system. both, the rigorous solution of the variational problem and the mc simulations show that orientational ordering of nanoparticles subject to the gradient of the electric field gives rise to an attractive interaction between charged lipid surfaces for high enough charge densities of the interacting surfaces and large enough separations between charges within the nanoparticle. the attraction is explained by orientational ordering of dimeric charges in the electric field which lowers free energy. viral genomes encode for a series of membrane proteins which are embedded or attached to the lipid membrane of the host, or penetrate them during the very first step of viral invasion. we are focussing especially to those of the former which are known to assemble and from channels or pores for small ions or substrates. with these channel forming proteins the virus manipulates the host cell interior for the benefit of its replication. strategies are developed to answer the question about the assembly process of these proteins based on the 'two stage model' and the substrate flux. only few experimental data are available to answer these questions, consequently we stress computational methods to derive the adequate answers. the methods applied are ab inito molecular dynamics (md) simulations based on density functional theory (dft) and conventional md simulations. potential routes for assembly of the three transmembrane domains of 3a protein from sars-cov will be outlined and compared with computational data from other viral membrane proteins. with a novel pore lining motif suggested for 3a, the dynamics of ions at selected positions within the putative pore will be assessed. probing the molecular mechanism of antibiotics diffusion through the ompf channel e. hajjar 1 , a. kumar 1 , m. winterhalter 2 , p. ruggerone 1 , m. ceccarelli 1 1 department of physics, university of cagliari, italy, 2 jacobs university, bremen, germany in gram-negative bacteria, the outer membrane porin-f (ompf) is the preferred entry point of antibiotics. bacteria can resist to antibiotics by altering the expression and the structures of ompf. a key feature in the structure of ompf is the presence of a constriction zone, characterized by both spatial and electrostatics restrictions. to study the process of antibiotics translocation at a molecular scale, we performed molecular dynamic simulations accelerated with the metadynamics algorithm. we studied the diffusion of antibiotics with different structural and chemical properties through ompf wild-type and variants. the free energy surface suggests faster translocation for the cephalosporins compared to the penicillins antibiotics, and also for ompf mutants compared to the wild type. further, the conservation of favored orientation and affinity sites of antibiotics inside the ompf channel reveal which specific interactions govern translocation. the calculated energy barriers and rate determining interactions for translocations compared well with the electrophysiology measurements and liposome swelling assays from our collaborators. this study demonstrates how theory and experiments can be combined to reveal the structural determinants and mechanism of ompf permeation. this will benefit to the design of antibiotics with improved transport properties. m. g. gauthier, j. bechhoefer dept. of physics, simon fraser univ., burnaby, bc, canada dna replication requires two distinct processes: the initiation of pre-licensed replication origins and the propagation of replication forks away from the fired origins. experiments indicate that these origins are triggered over the whole genome at a rate i(t) (the number of initiations per unreplicated length per time) that increases throughout most of the synthesis (s) phase, before rapidly decreasing to zero at the end of the replication process. we propose a simple model for the control of dna replication in which the rate of initiation of replication origins is controlled by the interaction with a population of ratelimiting proteins. we find the time set by reaction-diffusion kinetics for such proteins to find, bind to, and trigger a potential origin. the replication itself is modeled using a formalism resembling that used to study the kinetics of first-order phase transitions. analyzing data from xenopus frog embryos, we find that the initiation rate is reaction limited until nearly the end of replication, when it becomes diffusion limited. initiation of origins and hence i(t) is suppressed when the diffusionlimited search time dominates. we find that, in order to fit the experimental data, the interaction between dna and the rate-limiting protein must be subdiffusive. we also find that using a constant nuclear import of the limiting proteins leads to a more accurate description of the experimental data. the microsoft research -university of trento centre for computational and sisytems biology, trento, italy we propose a new method for inferring the structure of a biochemical network from the time-series of the reactant species. it consists of two parts: the first is the quantification of the correlation between the time-series profiles. correlation in time series data can be used to reveal dependencies between variables and to infer the graph of connectivity among species. the second part consists in the elimination from the connectivity graph of the relationships that have a non-null correlation coefficient, but that are not biochemically plausible. the cutting of false correlations from the graph is performed through the estimation of the parameters ("calibration") of the network. to calibrate the network for detecting null dynamics correlations, we developed the software tool kinfer (knowledge inference). based on a new probabilistic model of the variations in reaction concentrations, kinfer infers the values of the kinetic rate constants, their confidence regions and the level of noise in the input data by maximizing the likelihood to obtain the observed variations given the network model. the a priori knowledge required as input is minimal, as it consists only in the time-series of reactant concentrations. the minimal a priori knowledge and the probabilistic formulation of the calibration method make the accuracy of the predictions strong against experimental, biological and stochastic noise, and allows to use it to cut the null-dynamics edges of the connectivity graph. a. kuzmanic, b. zagrovic mediterranean institute for life sciences, split, croatia root-mean-square deviation (rmsd) is a measure used to give information on the global structure of macromolecules. for example, pairwise rmsd (prmsd) is used to assess similarity of the lowest energy nmr structures or for clustering large ensembles of structures. on the other hand, to obtain information on the local structure of a macromolecule and its dynamics, root-mean-square fluctuation (rmsf) is often used. rmsf can be calculated from md simulations, but also from experimental x-ray b-factors. since prmsd and rmsf report on different features, it is interesting to ask what the relationship between them is. first, we provide a mathematical derivation showing that, given a set of conservative assumptions, the <prmsd> rms is directly related to the <rmsf> rms and, consequently, experimental b-factors. second, we demonstrate this on structures taken from distributed-computing md simulations of the native and unfolded state of villin headpiece. both our analytical and computational results suggest a strong correlation: <rmsf> rms = [(s-1)/2s] 1/2 <prmsd> rms , where s is the number of compared structures. furthermore, if <prmsd> rms is defined as a generalized radius of gyration in the space of 3d structures and using rmsd as a measure of distance, the following identity holds: <rmsf> rms = <prmsd> rms. our results provide a basis for determining the level of structural diversity of molecular ensembles, as captured by <prmsd> rms , directly from experiment. charge migration along dna helices may be biologically important because extended electronic states could play a role in the processes of sensing of dna damage and/or dna repair via long-range charge transfer. we measured conductivity and other physical characteristics of several models of natural and diversely damaged molecules of dna. dna polymers were mimicked by various sequences of 32nucleotide-long double helices with fully watson-crick (wc) paired bases, with several central bases mismatched, and also with chemical modifications that included removal of bases from a few central nucleotides (abasic dna), and neutralization of phosphate charges by their derivatization. the model dnas were investigated by scanning tunneling microscopy, time-resolved thz spectroscopy, raman spectroscopy, circular dichroism, and modeled by molecular dynamics simulations. dna has the highest conductivity in its biologically most relevant double helical form with wc base pairs and negatively charged phosphates equilibrated with counterions. mismatches and all chemical modifications always lower the conductivity. the mechanism of charge transfer is consistent with electron or hole hopping between parallel stacked bases. these observations and data showing that the natural dna has also the most regular double helical form suggest that the continuous base stacking is critical for charge transfer. to control the passage across the bacterial cell wall nature created a large number of "nano"-channels which may act as selective gates for water soluble molecules. here we focus on porins from e. coli which control permeation through interactions with the channel surface. comparing single channel temperature dependent conductance measurements with all atom modeling allow conclusions on the mode of permeation. for example, surprisingly modeling ompf-conductance revealed not only a good agreement with the experiment over a broad range of temperature but also the selectivity for ions. the primary task of porins is to provide facilitated diffusion. we investigated facilitated diffusion of maltooligosaccharide or antibiotics. time resolved conductance measurements allows conclusion on the flux and molecular modeling identifies the limiting interactions with the surface, reveal potential barriers and pathways. exploiting the selectivity of natural or bioengineered channels has promising applications for detecting molecules, characterizing molecular interaction, sequencing dna, protein folding etc. traditionally, studies of diffusion-controlled reaction of biological macromolecules have been made in diluted solutions. however, the high concentration of macromolecules in intracellular environments results into non-specific interactions (macromolecular crowding), which have a great importance on the kinetics and thermodynamics of possible reactions that occur in these systems. in the literature there are studies concerning monte carlo (mc) simulations, giving results that are satisfactory agreement with experimental data, showing, for example, that the protein diffusion in cell cytoplasm is reduced considerably. in addition, there are mc studies about enzymatic reaction, which predict a temporal dependency of the velocity constant in macromolecular crowding. in this work, we try to compare the predicted behavior by mc simulation with the results obtained from the study of the diffusion and reaction of a model protein (alpha-chymotrypsin) using spectroscopic techniques in highly confined media in order to study experimentally the temporal dependence of its diffusion and reaction coefficients. a. paciaroni 1 , a. orecchini 1 , c. petrillo 1 , a. de francesco 2 , f. sacchetti 1 1 university of perugia, italy, 2 cnr-infm, genova, italy the single-particle and collective dynamical properties of protein hydration water have been studied by neutron scattering experiments in a wide temperature and hydration range. an unprecedented accuracy has been achieved thanks to the availability of a large amount of fully deuterated protein powder and the use of the high-flux spectrometers in5 and brisp. the protein under investigation was the maltose binding protein (mbp), which is a well-known and widely studied model of biosensor systems. we found that the low-temperature single particle dynamics of mbp hydration water shows clear features that can be traced back to amorphous systems. more in detail, its vibrational density of states is simply described as the superposition of the contributions of low-density and high-density amorphous ice. the quasielastic signal, which appears at the higher temperatures, can be excellently described with a fractional power law which may put in relationship with the peculiarities of fractal systems. quite strikingly, there is a strong similarity, on both the qualitative and quantitative point of view, with the behaviour of hydrated proteins. the collective dynamics of protein hydration water is characterised by the presence of two modes, whose dispersion curves are reminiscent of those of bulk water. however, the relevant damping factors suggest a strong similarity with glassy systems. m. malferrari 1 , f. francia 1 , s. sacquin-mora 2 , g. venturoli 1 1 università di bologna, bologna, italy, 2 cnrs upr 9080, paris, france the coupling between electron transfer and protein dynamics has been compared in reaction centers (rc) from the wild type (wt) and the carotenoid-less mutant r26, by combining brownian dynamics simulations and the kinetic analysis of charge recombination. upon incorporation of the rc into a progressively dehydrated trehalose matrix the electron transfer between the primary photoreduced quinone and the photoxidized donor accelerates progressively and becomes broadly distributed. this behaviour reflects the hindrance of protein relaxation following charge separation and the inhibition of interconversion between conformational substates. in extensively dehydrated matrices the recombination kinetics is two-times faster and three-times more distributed in the wt rc, indicating a larger inhibition of the internal protein dynamics. in line with this findings brownian dynamics simulations reveal a larger rigidity of the carotenoid-containing structure, in which a cluster of residues close to the quinone acceptors is stiffened as compared to the r26 rc. the in silico and experimental results indicate that the introduction of an internal void in the rc structure has long-range effects on the protein dynamics and that the coupling between the glassy matrix and the rc interior depends markedly on the local mechanical properties of the protein. n. maghelli 1 , v. krstic 2 , n. pavin 2 , f. julicher 2 , i. tolic-norrelykke 1 1 mpi-cbg, dresden, germany, 2 mpi-pks, dresden, germany in the fission yeast schizosaccharomyces pombe, the nucleus is positioned at the cell center. since the nucleus determines the cell division site, keeping the nucleus at the center is crucial for ensuring symmetrical cell division (1 ) . microtubules push against the cell ends and exert force on the nucleus (2 ), but how the cell regulates these forces in order to center the nucleus remains unknown. here we tackle this problem by using a combination of live cell imaging, cell manipulations by optical tweezers, and a theoretical model. we show that microtubule pushing forces can center the nucleus because of a larger number of contacts between the microtubules and the proximal cell end than the distal one. moreover, kinesin-8 motors (klp5/6) increase the rate of microtubule catastrophe (transition from growth to shrinkage) in a microtubule length-and contact-dependent manner. thus, the motor behavior results in a longer contact between a microtubule and the proximal than the distal cell end. taken together, our experimental and theoretical results provide a novel centering mechanism, where kinesin-8 motors increase the efficiency of nuclear centering. electropermeabilization is a commonly used physical method which can induce a transient permeabilization of the cell membrane allowing the entry of therapeutic molecules into the cell and is thus of great interest in the fields of cancer treatment and gene therapy [1] . however, very little is known about the mechanisms occurring at molecular level. there is clearly some microscopic reorganization of the membrane which is responsible for this change in its transverse transport properties. rather than studying the change of these transport properties, we adopt a simple strategy based on the use of giant unilamellar vesicles and videomicroscopy, as described below. we apply a series of permeabilizing electric pulses to the liposomes, and we observe a size decrease down to a critical radius beyond which their size no longer changes. this decrease in size points to the fact that during the physical processes leading to electropermeabilization, lipids are lost from the vesicles. our results published in [2] suggest different possible modes for lipid loss, which can be small vesicles, pores, or tubules formation. imaging of brain activation using core techniques as fmri, pet and synchrotron radiation in parallel c. poitry-yamate, g. margaritondo, r. gruetter ecole polytechnique fédérale de lausanne, switzerland functional magnetic resonance imaging (fmri), magnetic resonance spectroscopy (mrs), positron emission tomography (pet) and synchrotron x-ray emission imaging form a highly complementary set of imaging core technologies for studying brain function and energy metabolism. owing to differences in the information each conveys and the temporal and spatial scales on which they measure labeled substances, a single working hypothesis can be tested from different angles to provide a cross-validated, consistent and coherent explanation. the cibm is a unique research facility in europe for advancing our understanding of biomedical processes in health and disease. the housing of a 7-tesla human magnet, 9.4 and 14.1-tesla animal magnets, an animal pet imaging facility and fully-equipped neurochemistry and rf laboratories has enabled us to develop and perform well-targeted experiments around one research theme. in parallel, a longterm collaborative project using synchrotron x-ray transmission and emission imaging at elettra enables us to combine these core technologies towards understanding brain function in vitro and in vivo, from tissue to cells. a brief presentation of these methods will be followed by their application in studying the visual system from man to mouse. p. picone 1 , r. carrotta 2 , d. giacomazza 2 , m. di carlo 3 1 dip. chimica e tecnologie farmaceutiche, università di palermo, italia, 2 ibf -cnr, palermo, italia, 3 ibim -cnr, palermo, italia diabetes and alzheimer's disease are connected in a way that still is not completely known. diabetes has been implicated as a risk factor for developing alzheimer's disease. some diabetes drugs appear to decrease the cognitive decline associated with alzheimer's disease. it has been recently demonstrated that extracellular injection of insulin is able to protect neurons against a-beta amyloid cell death. one of the proposed theories to explain such an effect is that the hematic glucose levels affect the metabolism of the hippocampus, a part of the brain (associated with memory, emotion and motor skills), which is strongly damaged in alzheimer's patients. the aim of this study is to investigate the effect of insulin on the a-beta induced degeneration and oxidative stress on the neuroblastoma lan5 cell line. in particular, the present study looks into the role of insulin in the inhibition of abeta specific degenerative apoptotic pathways. preliminary results indicate that insulin dissolved in culture medium in its hexameric form (as tested by absolute scale light scattering) is able to reduce neurodegeneration induced by a-beta amyloid in a dose dependent manner. the link between diabetes and alzheimer's disease may provide new targets for future alzheimer's treatments. moreover, due to the increased incidence of diabetes in western countries, a deeper understanding of such a link is relevant in order to control the escalation in the number of people dealing with dementia. a. pelizzola dipartimento di fisica, politecnico di torino, torino, italy many features of protein folding have been shown to be described by an ising-like model (one-dimensional, with longrange, multispin interactions) whose equilibrium thermodynamics is exactly solvable [1] [2] [3] . we have generalized such a model to the problem of mechanical unfolding. the equilibrium thermodynamics is still exactly solvable, and the characteristic kinetic responses found in force ramp and force clamp experiments are well reproduced [4, 5] . unfolding and refolding pathways and intermediates can also be studied, again with good agreement with experiments [6]. applications to various proteins and rna fragments will be discussed. [ the key role of water in living systems has been widely studied in literature, along with its anomalies, consequence of the extensive three-dimensional hydrogen bonding of water molecules. moreover protein-water interactions take place at protein surface where cell water has been recognized to behave differently from bulky water. the two-states theory of water assumes that water is a mixture of microdomains of different structure and density, the low-density water (ldw) and the high-density water (hdw) domains, and ions partition selectively into ldw or hdw domains. the idea developed in this work was to explore the ordered water structure by measuring delayed luminescence (dl) from salt aqueous solutions in which water structuring is anticipated. it appeared that dl signal from salt solutions is significantly relevant when prevalence of ldw domains is foreseen, with a decay time probability distribution function characterized by a broad maximum in the microsecond range. the obtained results support the ability of dl to reveal the different properties of ldw and hdw domains induced by salt molecules. moreover, the results reveal the existence of clusters, whose characteristics strongly depend on the specific ion effects, of surprisingly long lifetimes not observed till now. this could give new insight into biological water properties. self assembly of patchy particles and dnafunctionalized dendrimers f. sciortino dipartimento di fisica e infm-cnr-soft università 'la sapienza', roma, italy i will report numerical results on the phase behavior of very simple models of patchy particles with the aim of understanding the interplay between phase separation and selfassembly and how the fraction of surface allowing for attractive interactions controls the collective behavior of the system. the case of janus particles, particles characterized by a surface divided evenly into two areas of different chemical composition, will be discussed. i will also discuss the self-assembly of a simple model for four single strands of dna tethered to a central core, and show that the model exhibits a rich phase diagram that includes at least four thermodynamically distinct amorphous phases (polyamorphism) in a one-component system. the dense phases consist of a hierarchy of interpenetrating networks, reminiscent of a woven cloth. peptide dimer motifs in the phospholipid environment -structure, interaction and molecular design p. e. schneggenburger 1 , a. beerlink 2 , t. salditt 2 , u. diederichsen 1 1 iobc, universität göttingen, germany., 2 irp, universität göttingen, germany. based on recently reported homodimeric peptide pores with a d,l-alternating configuration a novel double helical hairpin-motif of a membrane active gramicidin a analog was designed. [1, 2] the cd spectroscopic analyses of the peptide-lipid complexes revealed the structural preservation and elucidated the importance of a zwitterionic interaction of the peptide termini. [3] the peptide design was enhanced regarding the versatile functionalization with analytical probes as well as molecular recognition moieties like peptide nucleic acids (pnas) to observe the effects of aggregation and specific organization within model lipid membranes even at high peptide-to-lipid ratios. [3] x-ray reflectivity on lipid bilayer stacks in combination with heavy atom labeling and spectroscopic studies of vesicle systems provides information about the peptide structure and interaction in the native fluid state of the membrane system. [2, 3] for this, the fmoc-diiodo-allylglycine building block was created to serve as a novel iodine label pinpointing at a certain position with respect to the membrane normal. we study thermal undulations of giant unilamellar vesicles (guvs) of lipids by flickering spectroscopy. getting values for the mechanical parameters of lipid bilayers requires the experimental fluctuation spectra to be scrutinized in view of the classical helfrich's theory. pure bending modes are revealed unable in predicting the large fluctuations systematically found at high wavevectors. hybrid curvature-dilational modes have been invoked as a more efficient mode of motion in producing high curvatures. a bimodal spectrum of the thermal undulations has been theoretically developed for the shell-like topology. from this new description, two important consequences emerge a priori, the dependence of the fluctuation dynamics on either vesicle size and on bending/compression parameters. for popc and dopc vesicles containing cholesterol the experimental fluctuation spectra are well described by the new spectrum. reconciliation between experiments and theory is achieved when this bimodal spectrum is considered. the new theory opens enormous possibilities for better exploring membrane mechanics in guv models. under normal conditions, platelets circulate in the vascular system having very low interaction with each other and with other cells. the platelets become activated when the biological system is disturbed, for instance by vascular damage in which blood gets in contact with collagen. upon activation, different types of receptors/molecules are exposed on the cell membrane to support adhesion, spreading and aggregation of the platelets onto the damaged vessel. the measurement of altered platelet function is particularly important in cardiovascular diseases such as thrombosis. we are investigating biosensor technologies for the detection of functional properties of platelets. an important study is the specific and non-specific stimulation of platelets in a biosensor cartridge. we will present experimental results on biosensor platelet activation using the platelet-specific membrane markers p-selectin and gp1b. multi-joint analysis of locomotion in the first neonatal rats flown in space d. sulica, j. vinersan "carol davila" university of medicine and pharmacy, bucharest, romania the first mammalian neonatal animals in space were the rats flown on the space shuttle endeavor during a 9-day mission, sts-72. the development of locomotion in weightlessness was evaluated using two litters of neonatal rats, launched at postnatal days 7 and 15. age-and cage-matched animals were used as ground controls. free walking was videotaped from the landing day. although preliminary analysis of walking showed differences in both hindlimb and forelimb joint angles and a hyperextension of the hindlimbs was apparent, the numerical values reached the significance level only for the ankle angle measured at specific moments of the step cycle: foot contact, maximum loading with weight, foot lift and maximum flexion during swing. we report here on the behavior of all the joints during the whole step cycle, by computing the integral of these angles over the step cycle. the results were affected by the differences in the walking speed (the young animals walked faster than the controls), so we scaled the integrals by the step cycle duration. we found that, besides the ankle, the knee was also more extended throughout the whole step cycle in both groups of animals. moreover, all the joints (including the toe and the hip) were affected in the same way (hyperextended), since the differences were still significant when we added together these angles. the animals recovered slowly, with significant differences remaining after 14 days of readaptation. the effect of dextran 70 concentration on red blood cell deposit formation j. strzelecka, b. grzegorzewski department of biophysics, collegium medicum in bydgoszcz, nicolaus copernicus university 85-067 bydgoszcz, poland red blood cell (rbc) deposit formation was examined by means of an optical method. blood was obtained from healthy donors and measurements were performed at initial hematocrit 40%. the intensity of scattered light was measured during sedimentation of rbcs suspended in saline -dextran 70 solutions at different polymer concentrations (2 -6 g/dl). the changes in the intensity of the scattered light manifest rbc aggregate formation, their sedimentation and the process of deposit formation. the deposit formation curve was determined. it is shown that the concentration of dextran affects the deposit formation. an empirical model has been used to describe the experimental data. the parameters of the deposit formation curve as a function of dextran concentration are analyzed. water is essential to life and a major scientific interest lies in a detailed understanding of how it interacts with biological macromolecules in cells. we studied water dynamics in whole cells with neutron scattering [1, 2, 3] . the cellular environment is extremely crowded with macromolecules and water molecules are permanently in close contact to biological interfaces. we measured water dynamics in e. coli and human red blood cells with neutron scattering [2, 3] . the data revealed two populations of water in the cells: a major fraction which has dynamical properties similar to those of bulk water (relaxation times ∼ps) and a minor fraction in the order of ∼10% which is interpreted as bound hydration water with significantly slower dynamics (relaxation times >49 ps). in this contribution we report on investigation of model membrane dynamics by means of quasi elastic and inelastic incoherent neutron scattering and on the effect of membrane inserted pore forming peptide gramicidin. model membrane are realized by highly oriented, hydrated phospholipid bilayer stacks of dmpc (1,2-dimyristoyl-snglycero-3-phoshatidylcholine) hydrated with d2o in excess of solvent condition. the bilayer were supported on mica substrates and prepared at different concentrations of gramicidin, a 15-residue oligopeptide showing antimicrobial activity by forming pores on the membrane surfaces which allow water and small ions to permeate across the membrane. incoherent qens and ins spectra, measured on in5 and in13 spectrometer at ill, allows to obtaining information on the mean dynamics of the hydrogen atoms in the system. moreover, by proper orientations of the membrane plane respect to the scattering wave vector q, we were able to derive information on in plane and out of plane motions of the phospholipids. the using of media products for the creating attracted bioenergetic brain rhythmus advertisement v. i. vlastopulo, v. g. nikolajev str. gen. petrova 92 , app. 44, 65065 odessa, ukraine the technical efficiency of bioenergetical influence of advertisement is present with assistance of consciousness of memory at revision and hearing of advertisement on tv, radio stations, mobile phones, at supermarkets and other places. in a basis of useful model it is put a task to improve the method of creating the attracted advertisement, in which the creation of bioenergetical influence by the oscillation of not less 2 electromagnetic fields or video-images is introduced with their creating as the base on the spatial or flat structure formative macro matrix or matrixes with repeatable structure with the brain α-rhythm frequency of extreme attention and δ-rhythm frequency of meditation. the point of the patent on the device is in the bioenergetical influence increases in addition to the information influence by advertisement of pictures and audio oscillations the bioenergetical influence increases at the consciousness contribution of human memory at the moment of watching or hearing of television, radio stations, working in the internet, music in the supermarkets, banks, clubs, metro and other places. cell adhesion and motility are processes involved in fundamental biological phenomena. they imply multimolecular scaffolds as anchorage points and actin cytoskeleton filaments to build internal stress and eventually crawl onto the substrate. these processes, very dynamic by nature are out of equilibrium. we study cell adhesion on micro-patterned substrates where the introduction of a finite distance between the possible anchorage points of the cell modifies drastically the organization of the cytoskeleton and the anchorage point's distribution. because statistical quantification shows that some shapes are more likely than other, we believe they represent particular organizations of the system which should minimize the energy dissipation. we checked this hypothesis by using the cellular potts model. shapes obtained by simulation are in excellent qualitative agreement with experimental shapes. they depend on phenomenological parameters such as interaction between cells and the extra cellular matrix, a line tension and an elastic modulus. the aim of this work is to link model parameters to physico-chemical properties of cells and to establish phenomenological relations between relevant biochemical regulators controlling the cytoskeleton organization. c. canale, d. ferrera, f. benfenati, l. gasparini the italian institute of technology, 16163 genova, italy alzheimer disease (ad) is characterized by cerebral extracellular deposits of β-amyloid (aβ) fibrils. aβ aggregation is a multi-step process involving the formation of various conformational species including soluble intermediate species (i.e. aβ oligomers), protofibrils and fibrils. such aggregates may have various effects on neuronal and glial function and differentially contribute to ad neurodegeneration. aim of this study was to investigate the structural properties of distinct aβ aggregated species and dissect out their effects on neuronal viability. recombinant aβ42 and aβ40 peptides were aggregated in vitro in conditions differing by ionic strength, temperature and ph and were analyzed by gel electrophoresis, thioflavin t binding assay and atomic force microscopy (afm). afm analysis was performed using both hydrophilic and hydrophobic substrates, to analyze the full spectrum of structural species. stable low molecular weight oligomers were obtained when aβ42 was incubated at 37 • c for 5 days in low salt concentration buffer. doughnut-shaped conformational species were detected by afm alongside globular aggregates (1.6-5.2 nm height range). acidic ph promoted aggregation of aβ42 into thioflavin-positive fibrils and protofibrils. protofibrils appeared as beaded chains having a mean height of 3.65±1.49 nm. effects of aβ on viability of mouse hypppocampal neurons were assessed and correlated with their conformational features. a. bellova 1 , e. bystrenova 2 , m. koneracka 1 , p. kopcansky 1 , f. valle 2 , j. bagelova 1 , f. biscarini 2 , z. gazova 1 1 1 institute of experimental physics, slovak academy of sciences, kosice, slovakia, 2 2 ismn cnr, bologna, italy peptide amyloid aggregation is a hallmark of amyloid diseases including azheimer's disease or type ii diabetes. recent works have addressed the potential of nanoparticles to affect amyloid aggregation. the experimental data are very controversial suggesting that particle characteristics markedly influence the final effect of nanoparticles on the amyloid aggregation (initiation, acceleration or inhibition of amyloid aggregation). we investigate the ability of electrostatically stabilized magnetic nanoparticles of fe 3 o 4 to affect the amyloid aggregation of lysozyme, as a prototype amyloidogenic protein. we have used a combination of spectroscopic (tht fluorescence) and local microscopic techniques (afm). we found, that the ability of magnetic nanoparticles to inhibit formation of amyloid aggregates or destroy pre-formed amyloids exhibit concentrationdependence. the values of inhibition ic50 and depolymerization dc50 were determined suggesting that nanoparticles interfere with lysozyme aggregation at stoichiometric concentrations. the observed features make magnetic nanoparticles of potential interest as a therapeutical agent against amyloid diseases. (this work was supported by project of esf 26220120021 and by slovak academy of sciences in frame of cex nanofluid, vega grants 7055, 0056 and 0038 and eu-strp 0032652 biodot.). a. bellova 1 , l. balogova 2 , b. chelli 3 , e. bystrenova 3 , f. valle 3 , j. imrich 2 , p. kristian 2 , l. drajna 2 , j. bagelova 1 , f. biscarini 3 , z. gazova 1 1 institute of experimental physics, sas, kosice, slovakia, 2 faculty of sciences, p. j. safarik university, kosice, slovakia, 3 ismn cnr, bologna, italy numerous diseases have been linked to a common pathogenic process called amyloidosis, whereby proteins or peptide clump together to form amyloid aggregates in the body. an attractive strategy to develop therapies for these diseases seems to be reduction of polypeptide aggregation. we have tested several acridine derivatives characterized by various glycosyl groups for their potential to affect the lysozyme amyloid aggregation in vitro. the ability of glycosyl acridines to interfere with lysozyme aggregation was investigated by tht assay. we found that structure of acridine side chain is factor affecting their anti-aggregation activity significantly. for the most effective compounds the values of ic50 and dc50 were obtained. the reduction of protein aggregation was confirmed by afm. to investigate influence of the glycosyl acridines on the cell processes we examined effect of compounds on cell viability. we performed glycosyl acridines characterization by high anti-aggregation activity and low toxicity suggesting their possible application for therapeutical purpose. (this work was supported by project of esf 26220120021 and by slovak academy of sciences in frame of cex nanofluid and vega grants 7055, 0056 and 0038 and eu-strp 0032652 biodot.). a. j. beevers, a. m. dixon department of chemistry, university of warwick, uk due to the immense medical importance of proteins which span the membrane of cells, detailed molecular structural information of these systems is essential. practical difficulties in employing high-resolution structural elucidation techniques have resulted in a relative paucity of fully resolved membrane protein structures. therefore a variety of lower-resolution techniques are used to determine structural information of the transmembrane (tm) domains of proteins. one example of such a membrane protein is erbb-2, a receptor tyrosine kinase responsible for triggering cell division and which is prone to a mutation in its transmembrane domain resulting in permanent activation and oncogenic effects. we have predicted an interface for the mutated tm domain dimer using site-specific infrared spectroscopy containing a repeating sequence of ile, val and leu 1 . applying the in vivo toxcat assay to the tm domain sequence and to specific mutants of it, confirms this proposed interface whilst another proposed interface is discounted. current studies are focussing on the effect of the tm mutation to the activation of the erbb-2 receptor and to any possible change in this interface. bacteriophages are complex molecular assemblies which multiplication relies on bacteria infection. the process starts with the binding of the phage on its specific host receptor and the injection of its genome into the host cytoplasm. our work aims to determine the physical mechanisms and forces driving the dna transfer from the phage capsid. the in vitro dna ejection has been analyzed by using light scattering and gel electrophoresis measurements for three phages (t5, spp1 and lambda) belonging to the same family (syphoviridae). our results reveal two forces contributing to drive the dna transfer: the first one is originated from the pressurization due to the strong confinement of dna into the capsid; the second one comes from a pulling mechanism originated by the presence of condensed dna outside the capsid. these two contributions were characterized in in vitro conditions but they likely play a role in the in vivo transfer. the ejection kinetics was also analysed and the characteristic time of the mechanism was studied as a function of the temperature. it appears to follow an arrhenius law, allowing the determination of the activation energy that governs the transfer. the energy values are close for the different phages, suggesting that the mechanism regulating the ejection is common for a given phage family. below these general features, our studies also reveal differences between the three phages. the effect of &beta-amyloid peptide on polymer cushioned membranes s. dante 1 , r. steitz 2 , t. hauss 2 , c. canale 1 , n. a. dencher 3 1 istituto italiano di tecnologia, genova, italy, 2 bensc, helmholtz center berlin, germany, 3 tu-darmstadt germany beta-amyloid (aβ) is a peptide implicated in the neurodegenerative process characteristic of the alzheimer's disease (ad). to clarify its mechanism of action it is crucial to elucidate the interaction of aβ with the neural membrane. in previous work we demonstrated the capability of aβ to penetrate and perturb stacked lipid bilayers. in this study we considered polymer cushioned lipid bilayers as a model for neural membranes. the polymer cushion is aimed to preserve the membrane natural fluidity; it is obtained depositing charged polyelectrolites layer-by-layer; the lipid membrane is built on the top of the polymer film by fusion of unilamellar vesicles. the floating membranes were kept always in contact to the subphase. the kinetics of adsorption of the lipid double layer at the polymer/water interface was monitored by neutron reflectivity; different experimental conditions to obtain the best surface coverage were exploited. after administration of aβ to the subphase the lipid membrane still adhered to the polymer cushion, but its structure was modified by the interaction with aβ. neutron reflectivity showed a change of the scattering density profile in the direction perpendicular to the membrane plane, suggesting a penetration of aβ inside the double layer. a change in the surface morphology was detected by afm imaging; afm film-rupture experiments showed that aβ weakens the lipid packing. x. cheng 1 , r. pacheco-gomez 2 , a. rodger 1 , h. matthew 1 , d. i. roper 1 1 university of warwick, u.k., 2 university of birmingham, u.k. ftsz, the ancestral homolog of eukaryotic tubulins, is a gt-pase that assembles into a cytokinetic ring structure (z ring) essential for cell division in prokaryotic cells. the z ring also recruits other proteins (e.g. zapa, ygfe, zipa) to the division site, where they participate in formation of the septum that separates the two daughter cells. we have studied ftsz polymerization and its dynamic behaviour in real time by right angle light scattering. similar to tubulin, ftsz polymerizes into dynamic protofilaments in the presence of gtp; polymer assembly is accompanied by gtp hydrolysis. the kinetics of inorganic phosphate (p i ) released from the gtp hydrolysis have been studied as well, employing a fast and sensitive colourimetric assay. at ph 6.5, approximate 90% of the p i was released into the media within 20 minutes of gtp addition. the effects of gtp, ph, k + , and mg 2+ were studied in both cases, and the results were used to build up a model for the mechanism of fibre assembly and disassembly. ygfe, a ftsz accessory protein, is identified as a functional zapa orthologue. finally, we have studied the ygfe bundling to ftsz polymers. it strongly promotes ftsz bundling and is an inhibitor of the gtpase activity. many genomes of viruses encode small membrane spanning proteins which are proposed to modify membrane permeability for ions and small molecules. these channel or pore forming proteins are getting into the focus for antiviral therapy since they are essential for some of the viruses. one of the general themes of the mechanism of function of the proteins is to self-assemble to form the functional form. we present a study on the open reading frame (orf) 8a membrane protein encoded in structural region of human severe acute respiratory syndrome coronavirus (sars-covs). the full length orf8a protein is 39 residues long and contains a single transmembrane (tm) domain. full length protein is synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers forms cation-selective ion channels. the bilayer recordings show cation selection channel activity with a major conductance level of around 8.5 ps also at elevated temperatures (38.5 • c). in silico studies with a 22 amino acid tm domain are done to assess conformational space of the monomeric orf8a helix. with this monomeric helix homooligomeric helical bundle models are built and embedded in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycerol-3phosphatidylcholine (popc) bilayer. results of both experimental channel recordings and computational modeling show sars orf8a to act as a channel forming protein. -biomolecular self-assembly microtubules are involved in many vital processes. their rigid structure can resist high forces while their intrinsic ability to switch stochastically between growth and shrinkage phases allows them dynamically to reorganise. in cells, a sizeable network of microtubule binding proteins control and regulate microtubule dynamics. alp14 and dis1 are members of the dis1/ xmap215 family that are major players in s.pombe. the deletion phenotypes of alp14 and dis1 are similar, but nonetheless distinct. both are involved in the formation of spindles but alp14 is also involved in the maintenance of cytosolic microtubules in interphase. the restrictive temperatures of alp14-deletion and dis1-deletion mutants are different. alp14 interacts with alp7, a potential member of the tacc protein family. i am working to reconstitute alp14/dis1-dependent microtubule dynamics in vitro, using purified s. pombe tubulin. both alp14 and dis1 express well in insect cells and can be readily purified. preliminary data show that both proteins bind tubulin at low salt concentrations and that both influence the dynamics of pig brain microtubules. my goal is a complete functional analysis of alp14 and dis1, individually and in combination, to test candidate molecular mechanisms for alp14/dis1-catalysis of s. pombe microtubule dynamics. the syphoviridae coliphage t5 is a well-suited model to study the assembly of large viral capsids. biochemical and biophysical approaches were used to reconstitute in vitro the assembly pathway of its capsid. the t5 structure was recently solved from cryo-em and image reconstruction. its icosahedral capsid (t = 13) is built from the major head protein (pb8, 775 copies) forming both the pentons and hexons and from the portal protein (pb7, 12 copies) located at one vertex. its assembly proceeds by steps. pb8 and pb7 first assemble into a precursor structure called prohead i, which is converted to prohead ii by proteolysis of pb8 and pb7 by a head maturation protease. packaging of the 121 kbp dsdna is then driven through the portal pore by a molecular motor, the terminase. this promotes expansion of prohead ii leading to the mature capsid. the different assembly steps and the conformational changes accompanying capsid maturation were characterized using proheads i either self-assembled from the overproduced and purified capsid proteins or isolated from a phage mutant. these precursor capsid structures were analysed by small angle x-ray scattering. the 3d structure of prohead ii and of the expanded capsid were solved from cryo-em. our data show that the assembly process of a large icosahedral capsid can be efficiently reconstituted in vitro. amyloid beta peptide fibril formation modulated by phospholipid membranes e. hellstrand 1 , e. sparr 2 , s. linse 1 1 lund univ., department of biophysical chemistry, sweden, 2 lund univ., department of physical chemistry, sweden disease-causing amyloid fibril formation can be modulated by many factors including interactions with biological lipid membranes. an increasing amount of evidence suggests that the process of fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. alzheimer's is probably the most well-known amyloid disease and the associated amyloid beta peptide originates from the membrane incorporated amyloid precursor protein (app). we use recombinant abeta m1-40 and abeta m1-42 produced in escherichia coli, which allows us to perform large scale kinetics assays with good statistics where the amyloid formation process is followed in means of thioflavin t fluorescence. the lipid membranes are introduced in the system as large unilamellar vesicles composed of dopc, dppc and sphingomyelin, with and without incorporation of cholesterol. we find that the phase behanviour of the membrane in the vesicles has a large effect on the lag time of the amyloid formation process for both abeta m1-40 and m1-42. all membranes increase the lagtime to some degree but dppc has the largest effect. by comparing different phases we can conclude that the translational diffusion in the membrane seems to be more important than the acyl chain ordering. furthermore, electrostatics, concentration dependence and membrane addition at different time points in the amyloid formation process have been investigated. equilibrium/non-equilibrium transitions in macromolecule interactions p. dumas 1 , g. gibrat 2 , s. bernacchi 1 , e. ennifar 1 1 ibmc-cnrs, strasbourg, france, 2 llb (cea/cnrs), saclay, france usually, so called 'relaxation phenomena' occur on a fast time-scale and 'p-jump' or 't-jump' techniques are required to follow such events lasting (much) less than 100 ms. we report that, during stability studies of proteins or nucleic acids, such relaxation events can be observed on astonishing long time-scales. we first performed 'melting studies' with nucleic-acid duplexes by using linear variations of temperature (t) with time (t). we observed that even very low rates dt/dt could lead to a frozen state for temperature values below a sharp temperature range, and relaxation to equilibrium beyond that range. this allows defining a 'relaxation temperature' t r separating the two regimes. numerical simulations very accurately described the related hysteresis phenomenon observed upon a heating-cooling cycle, which is the hallmark of departure from equilibrium. analogous observations were made with protein oligomers submitted to either a variable pressure, or variable concentration in denaturant. importantly, a single theoretical frame predicts that the critical relaxation value x r (x standing indifferently for temperature, pressure or denaturant concentration) depends on ln(dx/dt). one may ask whether some thermosensor rnas known for switching on or off genetic expression by 'feeling' a temperature variation, might also 'feel' dt/dt. if true, the exact switching temperature would depend on dt/dt and faster temperature changes would increase t r . -biomolecular self-assembly the major component of amyloid plaques in the gerstmann-sträussler-scheinker disease is a prion peptide fragment from 81-82 to 144-153 residues. here, we present a structural study of prp82-146 in form of oligomers and fibrils by fourier transform infrared spectroscopy (ftir) and atomic force microscopy (afm). after incubation at 37 • c, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands and by a wide distribution of oligomer volumes. after a lag phase, a conformational rearrangement of oligomers into fibrils, with a parallel orientation of the cross β-sheet structures, was observed. by afm, different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly. in addition, we also studied thermal and random aggregation. the prp82-146 peptide was found to undergo several aggregation pathways, whose end products display different structural properties and intermolecular interactions. these findings underline the high plasticity of the prion peptide, a peculiar feature of prion proteins to overcome species barriers (natalello et al. j.mol.biol. 2008;381:1349-1361). the role of proline isomerisation in the aggregation process and fibril formation of alpha-synuclein j. meuvis 1 , m. gerard 2 , v. baekelandt 3 , y. engelborghs 1 1 lab.of biomolecular dynamics, ku leuven, belgium, 2 lab.of biochemistry, campus kortrijk, belgium, 3 lab.for neurobiology & gene therapy, ku leuven, belgium alpha-synuclein (α-syn) plays a central role in parkinson's disease. the aggregation of this protein, which contains five proline residues (p108,p117,p120,p128,p138), is accelerated in vitro by fk506 binding proteins (fkbps), a family of enzymes with a peptidyl-prolyl cis-trans isomerase activity (ppiase). fkbps catalyze the cis-trans conformational change of proline, often a rate limiting step in protein folding. to elucidate the role of the proline residues in aggregation, we constructed a mutant p(108,117,120,128,138)a α-syn . the kinetics of the aggregation of the mutant were studied with turbidity and thioflavin t fluorescence (tht). turbidity measurements show the formation of early, tht negative aggregates which is as fast for both wt and mutant. fibril formation however is faster for the proline-deficient mutant. we also studied the effect of fkbp12 on the aggregation of the mutant. although wt α-syn early aggregate formation is accelerated by the addition of 1 µm fkbp12, this effect disappears in the mutant. addition of (1pm-1µm) fkbp12 accelerates the fiber formation of wt α-syn, which is abolished in the mutant. we can conclude that α-syn fiber formation is accelerated for the proline-deficient mutant, which suggests a role for the proline residues in fiber formation. furthermore all accelerating effects of fkbp12 are abolished in the mutant which suggests that the ppiase activity of fkbp12 is responsible for the accelerating effect on the aggregation of wt α-syn. materials used as gene delivery vehicles must be able to condense dna into small sizes to facilitate transport and crossing various barriers. one of the polycations investigated for dna compaction is chitosan, which has the advantage of being safe and biodegradable. as a step towards reducing the aggregation behaviour of dna-chitosan complexes, chitosans were modified by grafting peg-chains on the backbone. it is known that the transfection efficacy depends on the chitosan chain length. additionally, the degree of pegylation might influence the condensation process. here a systematic biophysical study of pegylated chitosans and how the interplay between chitosan chain length and degree of pegylation affect the compaction of dna in terms of particle size and structure, stability in pbs and when exposed to serum, and transfection efficacy is presented. three different chain lengths of chitosans are employed, and for each sample three pegylation degrees are investigated and the properties of the dna-pegchitosan complexes compared to complexes formed when employing the original, chitosan for dna compaction. it is found pegylation of chitosans can be used to increase both the stability of the dna-chitosan complexes when exposed to serum as well as increase their transfection efficacy in hek293 cells. max-planck institute for polymer research, mainz, germany model membranes mimic the essential function of a natural membrane. however, the complexity is reduced in order to allow the study of fundamental processes. tethered membranes consist in principle of a lipid bilayer that is covalently linked to a solid support through a spacer group. this architecture allows the characterization of the membrane itself as well as of incorporated membrane proteins using surface analytical techniques. we have established a versatile system of various anchor lipids, which allow membrane formation on different surfaces. the architectures have been characterized by surface plasmon techniques, neutron reflectivity and electrochemical methods. the membranes are electrically insulating and allow for the functional incorporation of ion channel proteins. polymerizable lipids allow to pattern the membrane and to study lateral diffusion processes. furthermore, the membranes can be used as a sensing platform, where embedded membrane proteins act as actual sensing units. a. perico, s. pietronave, l. arcesi, c. d'arrigo consiglio nazionale delle ricerche (cnr), institute for macromolecular studies (ismac) the electrostatic free energy (fe) of two parallel rigid likecharged polyelectrolytes (pes) is given as a function of the separation distance. 1 for high linear charge density, z , the fe shows a minimum due to the increasing of the counterion condensation and condensation volume as the two pes approach. the interaction fe is governed by a critical linear charge density, z c , inversely proportional to the counterion valence. for highly charged pes (z > z c , like dna), the pes attract the stronger the smaller is the counterion valence, because the fe is dominated by the entropic term due to condensation of counterions in a volume displaying a maximum at short distances. for weakly charged pes (z < z c /2 ) the pes remain undercritical in the whole separation range and therefore repel. for moderately charged pes (z c /2 < z < z c ), the infinitely separated pes are undercritical but become supercritical as they approach a critical distance and charge condensation and condensation volume expansion start: in these circumstances the pes may attract if the counterion valence is sufficiently large. in the case of many dna rods, hexagonal clusters may be formed. upon interaction with hydrophobic surfaces, proteins show a tendency to expose regions that are normally buried in the hydrophobic core. unfolding is generally perceived as an undesired process in studies aimed to anchor functional proteins at surfaces. upon an upset of perspective the fine control of the unfolding/re-assembly process could be regarded as a strategy to build up molecular nanostructures for the development of organic-inorganic assemblies. we show that molecular layers patterned at the nanoscale, with longrange order properties extending over the microscopic scale, can be obtained upon adsorption of proteins onto the hydrophobic and ordered surface of pyrolytic graphite. upon adsorption, proteins lose their native folding and polypeptide chains re-assemble on the surface in a layered fashion, forming a molecular bilayer. the first layer, in contact with the substrate, and the second molecular layer show corduroy-like nanopatterns of different periodicity, with a relative orientation between the first and second layer patterns of 30 • . surface-induced protein unfolding and polypeptide chain reassembly according to a layered ordered structure is a rather general phenomenon since it is observed for different proteins irrespectively of their specific structural properties. the possibility of using these ordered molecular structures as templates for the subsequent patterned deposition of supramolecular aggregates will be discussed. understanding protein-protein interactions and assemblies to control the hierarchical building of well-ordered supramolecular structures is highly relevant to new tailormade biomaterials. we previously evidenced that contrary to native calcium-loaded α-lactalbumin (holo α-la), calcium-depleted form (apo α-la) has the ability to selfassemble with lysozyme (lys) to form different supramolecular structures in temperature-dependent manner. in the present work, the events occurring at molecular scale were explored using fluorescence techniques. fluorescence anisotropy and fluorescence lifetime measurements provide a powerful and sensitive mean to measure intermolecular interactions. we showed that lys interacts with both apo α-la and holo α-la to form oligomers, assumed to be heterodimers, at 10 • c and 45 • c. the dissociation constants for dimerization, found to be in the µm range, were sensitive to the ionic strength. correlation time calculations suggest that formed heterodimers holo α-la/lys and apo α−la/lys differed in their shape and/or conformation. such conformation differences could explain why holo α-la/lys complexes are trapped as heterodimers while the apo α-la/lys complexes have the ability to further self-assemble into previously reported various supramolecular structures. polyglutamine aggregation and neurodegeneration g. nicastro, l. masino, a. pastore national institute for medical research, the ridgeway, nw71aa london, u.k. polyglutamine (polyq) diseases are rare but dominant misfolding diseases linked to neurodegeneration. they are caused by the expansion of cag codon repeats, which encode polyq tracts in the corresponding gene products. aggregation of polyq proteins is thought to be triggered by polyq expansion but be strongly modulated by the protein context. in the attempt of understanding the molecular bases of polyq diseases, we are studying the structures, interactomes and aggregation properties of selected polyq proteins. here, we present recent work on ataxin-3, taken as a representative example of the whole family. ataxin-3 is a ubiquitin specific cysteine protease, involved in the ubiquitinproteasome pathway and known to bind poly-ubiquitin chains of four or more subunits. the enzymatic site resides in the n-terminal josephin domain of ataxin-3. we have characterized, using different biophysical techniques, the structure in solution and the aggregation properties of josephin both in isolation and in a ubiquitin complex. we demonstrate that interaction with ubiquitin strongly modulates the aggregation properties of ataxin-3 and suggest the importance of protein-protein interactions in preventing aggregation. our study also provides new insights into the molecular mechanisms which determine ataxin-3 specificity for poly-ubiquitin chains of the correct length and cross-linking. förster resonant energy transfer (fret) from an optically excited to a non-excited molecule has been widely used to probe molecular interactions in living cells. changes in the molecular makeup of a cellular region occurring during the acquisition of fluorescence images place tight constraints on the fret technology and data analysis, which could not be addressed satisfactorily until recently. we will describe a method for imaging protein complex distributions in living cells with sub-cellular spatial resolution, which relies on a spectrally resolved two-photon microscope (raicu et al, 2009, nature photon. 3: 107-113) and a simple theory of fret in oligomeric complexes (v. raicu, 2007, j. biol. phys. 33:109-127) . then, we will overview recent results on the determination of the supra-molecular structure and distributions in living cells of oligomeric complexes of some g protein-coupled receptors. observing protein aggregates on surfaces m. rabe, d. verdes, s. seeger institute of physical chemistry, university of zurich, switzerland protein aggregation is an important topic of current protein research as it is associated with several human diseases including alzheimer's disease, parkinson's disease, and type ii diabetes. although protein aggregation mechanisms and conditions have been comprehensively investigated, studies on the formation and the fate of protein aggregates in contact with solid interfaces are scarce. we have comprehensively investigated the structure of protein assemblies that form spontaneously upon protein adsorption on solid interfaces using a surface sensitive fluorescence imaging technique based on super critical angle fluorescence (saf) detection. combining this technique with fret we not only succeeded to detect protein aggregates deposited on surfaces but also to characterize their behavior in real time, i.e., their emergence, growth, or spreading. the model protein bsa, for instance, was found to exhibit a certain tendency for aggregation in the buffer solution. these protein clusters can deposit onto solid surfaces and spread resulting in a large, flat structure after some time. 1 a different possibility how protein aggregates emerge on surfaces consists of a direct deposition of protein monomers to pre existing aggregates. such a growth of protein aggregates on the surface has been observed in a model system using the protein α-synuclein, which is tightly associated with the parkinson's syndrome. we present the results of a spectroscopic ellipsometry (se) study of the adsorption process of yeast cytochrome c (ycc) on gold and graphite substrates, according to methods already applied to study the growth dynamics of organosulphur sams on gold [1] . se investigation was carried out both in situ, at room temperature during protein deposition, and ex situ. on gold, se data demonstrate the formation of an about 3-4 nm thick layer, consistent with the formation of a dense monolayer of ycc molecules, confirmed by afm inspection. both in situ and ex situ measurements were characterized by well defined spectral features related to the soret band. analysis of the fine position of this feature allowed to obtain information on the oxidation state of the iron ion of the heme group. se data suggest that proteins have preserved their native structure. a completely different adsorption mechanism was observed on highly oriented pyrolytic graphite (hopg) [2] . ex-situ se data on ycc/hopg, supported by afm observations, indicate the formation of an ultrathin molecular layer (∼0.7nm) related to complete protein unfolding at the hydrophobic surface. the role of phospholipid anisotropy in the stability of inverted hexagonal phase was considered. the equilibrium configuration of the system was determined by the minimum of the free energy involving the contribution of the isotropic and deviatoric bending and the interstitial energy of phospholipid monolayers. the shape and local interactions of a single lipid molecule were taken into account. the minimization with respect to the configuration of the lipid layers was performed by the monte carlo simulated annealing method. at high enough temperature the lipid molecules attain a shape exhibiting higher intrinsic mean and deviatoric curvatures which fits better into the inverted hexagonal phase than into the lamellar phase. for the mathematical model the advanced geometry with non-spherical cross-section of inverted hexagonal phase was calculated, resulting in lower energy in non-spherical cross-section. theoretical results are in a good agreement with the small angle x-ray scattering experimental data. for a long while the conventional view has been that alzheimer disease is brought about by the beta-amyloid fibrils found in the senile plaques, but more recently it has been suggested that the main neurotoxic species would be the soluble oligomeric species, apparently prone to interact with cell structures and macromolecules potentially inducing neuronal dysfunction. peptide-peptide interactions resulting in self-assembly phenomena of beta-amyloid yielding fibrils can be modulated and influenced by small organic molecules that might also be effective therapeutic tools to ideally target both oligomeric and fibrillar species. in this perspective, polycyclic aromatic molecules are of special interest because they might disrupt the molecular architectures precursors of beta-amyloid fibrils by means of weak, non-covalent aromatic interactions, like stacking interactions. we have performed an in vitro spectroscopic study (light scattering, circular dichroism, ftir and fluorescence) of the effects on beta-amyloid fibrillogenesis of the natural pigment hypericin extracted from hypericum perforatum. our results show that, thanks to its structural characteristics and peculiar spectroscopic features, hypericin can be easily used to in vitro monitor the appearance of initial aggregation states of beta-amyloid peptides and, more importantly, that hypericin can interfere with the early stages of polymerization process, playing the role of an aggregation inhibitor. peptaibols are peculiar peptides produced by fungi associated to plants. they are composed by 4 to 20 amminoacidic residues and exhibit antibiotic and antifungal properties. due to their amphypatic nature, they can form ion channels in biological membranes. by making use of experimental models of biological membranes (biomimetic membranes) currently employed in the laboratory of bioelectrochemistry, and models of plant membranes (corn seed root), that are used in the international laboratory of plant neurobiology (linv), we characterized synthetic peptides such as trichogin gaiv and its shorter homologues (4 and 8 residues). we studied these peptaibols in a dioleoylphosphatidylcholine monolayer supported by hg using different electrochemical techniques (ac,vc,eis). the experimental technique employed in the linv (clark microelectrode coupled to mife system) allows to measure oxygen flux in the solution contacting plant cell membranes, after treatment with different peptide concentrations. preliminary results might indicate that short peptides can influence the whole metabolism of the plant and can therefore be used as "elicitors" in order to induce an acquired systemic resistance. supported biomimetic membranes (sbm) were developed for protein-membrane interactions studies. phospholipid vesicles were chemically linked onto amine grafted gold or glass surfaces; after an osmotic choc and liposomes fusion a continuous membrane bilayer was formed. the anchoring phospholipid molecule (dspe-peg-nhs) incorporated into the vesicles allowed the formation of a water-filled compartment between the surface and the bilayer. this first sbm model was used to monitor the membranes binding properties (dependent of calcium) of the adenylate cyclase toxin (cyaa) from bordetella pertussis. the sbm model was improved in order to study the translocation of the catalytic domain of cyaa across the bilayer. naturally, the cyaa catalytic domain, when it reaches the target cell cytosol, associates with intracellular calmodulin (cam) an activator of the adenylate cyclase activity of cyaa. to mimic this biological phenomenon, cam was first immobilized on the surface (gold or glass) and in a second step membrane construction was performed over the cam layer. the formation of the biomimetic membrane onto the cam layer was monitored by spr while membrane fluidity and continuity were analysed by fluorescence. our results demonstrated the potentialities of sbm for the study of protein insertion into and translocation across biological membranes. a multi-resolution approach to the structure and function of integrin αiibβ3 m. rocco 1 , c. rosano 1 , j. w. weisel 2 , d. horita 3 , r. r. hantgan 3 1 istituto nazionale per la ricerca sul cancro (ist), genova, italy, 2 university of pennsylvania, philadelphia, pa, usa, 3 wake forest university, winston-salem, nc, usa. integrins are heterodimeric transmembrane receptors involved in mechanical anchoring and two-way signaling. each α and β subunit has a modular structure with a large extracellular portion, a single transmembrane region, and a cytoplasmic domain. integrins activation mechanism is regulated by controversial conformational changes: while crystallography revealed similar bent shapes for resting and primed extracellular region constructs, ligand binding-induced large structural rearrangements in smaller constructs suggested extension, "opening" and tails separation. in a multiresolution approach, we used experimental and computed hydrodynamics to discriminate among αiibβ3 integrin models built on x-ray, nmr, and em data. in contrast with xray data and 3d em maps, an extension is needed to match the hydrodynamics of full-length, solubilized αiibβ3; an electron tomography-based model fares better. consistent with that, and with our averaged 2d em images, a conformational change in the head region (β3 hybrid domain swingout) coupled to a simple transmembrane helices shift matches priming agents-induced frictional changes in full-length αiibβ3. our multi-resolution study thus suggests that in integrins extension and immediate tail separation are uncoupled from head domain rearrangements following activation. -biomolecular self-assembly stabilizing effects of α s1 -casein, a natively unfolded protein, on the aggregation of biomolecules a. trapani, r. carrotta, p. l. san biagio, d. bulone ibf-cnr palermo, italy α s1 -casein is one of the four types of caseins, a group of calcium phosphate-binding proteins that, in the form of micellar aggregates, makes up the largest protein component of bovine milk. the structure of α s1 -casein is that of a triblock polymer with a hydrophilic tract interposed between two hydrophobic blocks. due to the lack of a compact folded conformation, this protein can be classified as one of the intrinsically disordered (or natively unfolded) proteins. this class of proteins is known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces that partially unfolded molecules may expose to the solvent. here we present results on α s1 -casein effects on the thermally induced aggregation of gluthathione stransferase, a ligand-binding anzyme, and 1-40 β-amyloid peptide involved in alzheimer's disease. by means of light scattering and circular dichroism experiments, we attempt to reveal the molecular details of α s1 -biomolecules interaction. bacterial protein self-assembly on surfaces of well-defined chemistry s-layers are one of the most common cell envelope components of prokaryotic organisms and represent the simplest biological membrane developed during evolution. these (glyco)proteins, which can self-assemble into 2-d crystalline nanostructures on lipid films, liposomes, and polymers, play already an important role in nanobiotechnology. in this work, we present new findings concerning the recrystallization of bacterial proteins on substrates with defined chemistry. manipulation of the protein-sample interaction was carried out by changing the relative height of oh and ch 3 terminated moieties in self-assembled monolayers (sams). we have found that differences in chain length lead to: i) protein bilayer-protein monolayer transition, ii) preferential protein side adsorption, and iii) increase of the crystal lattice parameters. further manipulation of the protein-sample interaction was achieved by using silane chemistry. we will show that sample hidrophobicity speeds up recrystallization kinetics and reduces the crystalline domain size (and layer compliance). the protein-adsorbed mass per unit area on these substrates is reported for the first time. self-assembly of phenylalanine oligopeptides: insights from experimental and computational studies p. tamamis 1 , l. adler-abramovich 2 , m. reches 2 , k. marshall 3 , p. sikorski 3 , l. serpell 3 , e. gazit 2 , g. archontis 1 1 dpt. of physics, univ. of cyprus, cyprus, 2 dpt. of molecular microbiology and biotechnology, tel aviv univ., israel, 3 dpt. of biochemistry, univ. of sussex, u.k. the diphenylalanine peptide (ff) forms well ordered nanotubes and its derivatives form nano-assemblies of various morphologies with promising material applications. we demonstrate for the first time by electron and laser microscopy and ftir spectroscopy that the related, triphenylalanine peptide (fff) assembles into rather planar nanostructures, rich in β-sheet. in addition, we conduct 0.4-µs replica exchange m.d. simulations of aqueous ff and fff solutions in implicit solvent. the peptides coalesce into aggregates and participate frequently in open or ring-like linear networks, as well as elementary and network-containing structures with β-sheet characteristics. polar and nonpolar interactions, as well as the surrounding aggregate medium contribute to the network stabilities. within a network, consecutive peptides are linked by head-to-tail interactions; the aromatic sidechains of neighbor peptides assume approximately "t-shaped" orientations. these features are observed in ff crystals and could characterize early formations, or stabilize the mature nanostructures. the fff aggregates acquire higher stability and peptide-network propensity compared to the ff aggregates due to energetic contributions 1,2 . chlorophyll biosynthesis is light-dependent in angiosperms because the reduction of protochlorophyllide (pchlide) into chlorophyllide is driven by a photoenzyme, nadph:pchlide oxidoreductase (por). the unique properties of por are due to its ability to assemble into dimers and oligomers within the prolamellar body (plb) membranes of etioplasts studied mainly in leaves of dark-grown seedlings under laboratory conditions. we extended these studies to plant organs developed in the nature: cabbage heads, leaf primordia inside buds, pericarp-covered regions of sunflower cotyledons, potato tubers and seedlings germinating under the soil. in electron microscopic and fluorescence spectroscopic studies we found in many of these organs poorly developed plbs in which por was mainly in monomer state. as a consequence, the chlorophyll accumulation was slow and photo-oxidation processes occurred at illumination. in vitro we artificially induced the aggregation of por monomers into oligomers in glycerol and sucrose containing buffers. this resulted in the increase of the photoreduction rate at the expense of photo-oxidation. these results underline the importance of the self-assembly of por and the plbs in chloroplast development and chlorophyll synthesis in nature. v. vetri 1 , g. ossato 2 , v. militello 1 , m. a. digman 2 , m. leone 1 , e. gratton 2 1 dsfa, university of palermo, palermo, italy, 2 lfd, university of california, irvine, ca, usa we report an experimental study on concanavalina (cona) aggregation in live cells. in vitro, close to physiological temperature, cona readily forms fibrils involving secondary structure changes leading to β-aggregate structures. the effect of cona on cell cultures and formation of protein aggregates were measured by confocal fluorescence microscopy. in particular, we monitored protein aggregation in live cells by means n&b analysis, cross-n&b and rics. n&b showed the aggregation kinetic and the progressive formation of cona oligomers at cell surface. this suggests that, at cell membrane where local concentration is higher, nucleation sites for aggregation are provided. in parallel, the morphology of the cells changes indicating the progressive cell compaction and death. aggregation and binding of small aggregates to the cell surface were assessed by rics: it is possible to distinguish regions where small aggregates are diffusing and regions where they are bound to the cell. oligomers formation may stimulate non-specific cellular responses due to the exposure of reactive regions of protein structure and of progressive formation of cross−β structures. moreover, aggregates stoichiometry was measured during the kinetic by cross-variance n&b. the two conductive pathways of p2x7 purinergic receptor: different modulation and selectivity r. barbieri 1 , s. alloisio 1 , a. di garbo 2 , m. nobile 1 1 institute of biophysics, cnr, via de marini 6, 16149 genoa, italy, 2 institute of biophysics, cnr, via g. moruzzi 1, 56124 pisa, italy the p2x 7 purinoceptor (p2x 7 r) is an atp-gated cation channel that is able to activate a cell permeabilizing pore. p2x 7 r cytosolic c-terminal tail is thought to modulate this function. this study was aimed to characterise the biophysical properties of p2x 7 r compared to those of the variant lacking the entire c-terminus tail (trp2x 7 r) by measuring whole-cell currents and intracellular ca 2+ variations. a mathematical model is used to describe the experimental results. in p2x 7 r expressing hek-293 cells, the potent agonist 3'-o-(4-benzoyl)benzoyl adenosine 5'-triphosphate (bzatp) -evoked ionic currents depending on concentration and frequency of agonist applications. the currents were strongly inhibited by extracellular mg 2+ in a noncompetitive way. by contrast, in trp2x 7 r cells, only high bzatp concentrations elicited small currents not affected by mg 2+ . interestingly, bzatp-induced ca 2+ influx was present both in p2x 7 r and in trp2x 7 r cells, albeit in the latter the intracellular ca 2+ elevation was smaller. importantly, in trp2x 7 r the intracellular ca 2+ rise maintained a competitive mechanism of mg 2+ inhibition similar to that observed in p2x 7 r. the experimental data and the modelling findings support the tenet of a functional structure of p2x 7 r possessing two distinct conductive pathways. the review of our data on the effects of physical and chemical weak signals on physicochemical properties of water, cell volume, activity and the number of membrane proteins (receptors, ionic channels and enzymes, na + /k + pump and na + /ca 2+ exchanger), intracellular signal systems in norm and pathology (cancer and nerve disorders) would be presented. light microscopic, cell voltage-and patch-clamp, isotope, standard biochemical and genetic engineering methods were used. weak signal-induced effects on cell functional activity (intracellular enzymes activity, the number of functionally active membrane proteins) are realized by changing the physicochemical properties of extra-and intracellular aqua medium. the latter induces the modulation of na + /k + pump-induced cell hydration, which serves as a primary mechanism through which the autoregulation of pump and regulation of membrane excitability and chemosensitivity are realized. by genetic engineering method in oocytes it was shown that the correlation between na + /k + pump and na + /ca 2+ exchanger, which is realized through intracellular messenger systems, plays a crucial role in weak signals transduction in cells and determination of aging-induced increase of cell pathology. listeriolysin pore forming ability in planar lipid membranes at different ph listeriolysin o (llo) is a cholesterol-dependent cytolysin secreted by the intracellular pathogen listeria monocytogenes. its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. llo exhibits optimal cytolytic activity at low ph but it is still able to bind membranes at physiological or even slightly basic ph values in a cholesterol-dependent fashion. high cholesterol concentrations in the membrane restore the low activity of llo at high ph values. based on this broad ph activity we investigated the electrophysiological properties of pores formed by llo at room temperature and at different phs using planar lipid bilayer technique. llo is able to form pores both at ph 5.5 and 7.5 with a similar permeabilizing ability and similar heterogeneous conductances in the range of picosiemes to nanosiemens. cholesterol content directly correlates with llo activity but it does not change the pore characteristics. collectively, our results demonstrate that llo activity at physiological ph cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for listeria pathogenesis. s. aimon, g. toombes, p. bassereau institut curie, paris, france the physics of membrane/channel and channel/channel interactions is difficult to investigate in cells where it is nearly impossible to modify relevant parameters to deduce physics laws. to overcome these difficulties we built a model system in which voltage gated ion channels were reconstituted in giant unilamellar vesicles (guvs) for the first time. as a first step, we successfully expressed kvap (a voltage gated potassium channel) in e-coli. the channel was purified, fluorescently labelled and reconstituted in small liposomes. its functionality was checked with electrophysiology via fusion of these liposomes into black lipid membranes (blm). as a second step, guvs were formed from these small proteoliposomes using electroformation in a buffer containing 100 mm kcl salt. the proper incorporation of proteins into guvs was controlled using confocal microscopy. functional proteins were detected using the patch clamp technique. with our protocol, we are thus able to prepare guvs containing functional voltage-gated ion channels. one goal is now to study the effect of channel activity on its spatial distribution in these guvs. ryr activation in cultured shr cardiomyocytes at the end of the prehypertensive period the rate of [ca 2+ ] i elevation after the ryanodine receptor (ryr) activation by 4-chloro-m-cresol (4-cmc) and l-type ca 2+ channels (dhpr) activation by bay k8644 was studied in cultured (5 days) cardiomyocytes of spontaneously hypertensive (shr) and normotensive rats (wky, wistar) during 6 weeks of postnatal development. the differences in dh-prs and ryrs activities began to be evident after 3 weeks age when cicr formation has finished and became more expressed at the end of prehypertensive period (6 weeks). in response to 4-cmc (2 mm), a drastic increase in the rate of [ca 2+ ] i accumulation (2.85±0.8 times) in shr myocytes was registered after 20 days age versus a decrease in the rates of ca 2+ efflux from the sarcoplasmic reticulum of wistar and wky rat cardiomyocytes. bayk (80 µm) also induced more sharp [ca 2+ ] i elevation in shr myocytes (3.35±0.25 times) as compared with wistar (2.32±0.16 times) and wky (1.22±0.09 times) ones of the same age. our results argue that in shr and wky cardiomyocytes, as opposed to normotensive wistar rats, gradual growth of dhpr activity is observed, which follows in parallel with cicr formation in the excitation-contraction coupling during early postnatal ontogenesis, and drastic activation of ryr2 in shr myocytes after the process termination. cytochrome ba 3 from thermus thermophilus belongs to the large family of structurally related heme-copper oxidases. it accepts electrons from cytochrome c 552 at the p-side of the membrane and uses them to reduce oxygen to water. the energy released in this reaction is used for proton pumping across the membrane to form of an electrochemical proton gradient, used by the cell for formation of atp. in this work we followed the kinetics of single-electron injection into the oxidized nonrelaxed state (o h →e h ) of cytochrome ba 3 by time-resolved optical spectroscopy. two main phases of electron transfer were resolved. the first (τ∼17 µs) includes oxidation of cu a and simultaneous reduction of both low and high spin hemes. the second (τ∼420 µs) reflects reoxidation of both hemes by cu b . this is in significant contrast to the o h →e h transition of aa 3 -type oxidases, where the fastest phase is due to transient reduction of the low-spin heme a only. on the other hand, the single-electron reduction of the relaxed o state in ba 3 oxidase consisted of only rapid electron transfer from cu a to heme b, which is similar to that in aa 3 oxidase. this indicates a functional difference between the relaxed o and the pulsed o h states of cytochrome ba 3 . as opposed to the phospholamban pentamer, sarcolipin forms anion-selective channels in biomembranes l. becucci 1 , c. b. karim 3 , d. d. thomas 3 , g. veglia 2 , r. guidelli 1 1 chemistry department, florence university, 50019 florence,italy, 2 chemistry department, university of minnesota, minneapolis, mn 55455, 3 3department of biochemistry, molecular biology, and biophysics, university of minnesota, minneapolis, mn 55455 sarcolipin (sln) and the phospholamban pentamer (pln) are two membrane proteins that inhibit ca-atpase of the sarcoplasmic reticulum at low concentrations. in contrast to pln, sln stimulates maximal ca 2+ uptake rates. sln and pln were incorporated in a bilayer lipid membrane (tblm) tethered to a mercury electrode through a hydrophilic spacer. electrochemical impedance spectroscopy measurements show that sln forms channels permeable to chloride ion, weakly permeable to phosphate ion and impermeable to inorganic cations such as na + and k + . a relationship between this property of sln and its regulatory function on ca-atpase of sarcoplasmic reticulum is proposed. atp increases the permeability of a tblm incorporating sln to phosphate ion by associating to sln with an association constant of 0.1 µm. an explanation for this behavior is provided. sln can be identified with the " p i transporter" described by a.g. lee et al. conversely, both electrochemical impedance spectroscopy measurements and molecular dynamics simulations provide strong evidence that the pore of the pentameric form of pln does not act as a chloride channel. "social" domain organization and dynamics of nicotinic acetylcholine receptor at the cell membrane f. j. barrantes unesco chair biophys. & mol. neurobiology. univ. nac. sur, 8000 b. blanca, argentina a combination of experimental techniques (patch-clamp, confocal frap and fcs, single-particle tracking, highresolution fluorescence microscopy) has been used to analyze the supramolecular organization of the acetylcholine receptor (achr), the dynamics of the receptor at the cell surface, and the kinetics of receptor internalization. changes in cholesterol (chol) content affected muscle and neuronal-type achr organization and dynamics at the cell surface. chol depletion produced gain-of-function of single-channel dwell time. submicron-sized (∼250 nm) domains, stable over a period of hours at the cell membrane, could be resolved into achr "nano-clusters" with a peak size distribution of ∼55 nm by sted microscopy. chol depletion reduced the number of nanoclusters, increasing their size, and changed their supramolecular "social" organization on larger scales (0.5-3.5 microns). frap, fcs and spt experiments provided information on the dynamics of achr nanoclusters, disclosing the dependence of their mobility on chol content and cortical cytoskeleton. chol content at the plasmalemma may thus modulate cell-surface organization and dynamics of receptor domains, and fine-tune receptor channel function to temporarily compensate for acute achr losses from the cell surface. m. czaplinska, k. gwozdzinski, a. koceva-chyla division of research of structure of biopolymers university of lodz, lodz, poland doxorubicin (dox) and paclitaxel (ptx) are anticancer drugs commonly used in chemotherapy of breast cancer therapy, however, the use of these drugs is limited by the risk of developing heart failure. generation of reactive oxygen species contributes to the cardiotoxicity of doxorubicin. nitroxides are low molecular weight, stable free radicals reacting with ros and they present antioxidant properties. the aim of this study was to analyze the effects of pirolid (pd) on the oxidative stress induced by dox and taxane in mcf-7 breast cancer cell line. results from mtt test revealed that ptx is more cytotoxic towards mcf-7 cells than dox. the ic 50 was 0.3µm and 3µm, respectively. pd alone does not influence cell viability. pd in combination with both drugs did not change viability of cells. both drugs increased the level of carbonyl groups in cells. the highest level of peroxide was observed in cells incubated with dox (approx. 3-fold). nitroxide alone did not influence the level of peroxide in the whole range of concentrations. combination of pd with dox and ptx reduced the level of carbonyls depending on its concentration. pd did not affected on the level of peroxide in cells suspension. dox and ptx increased (2-fold) the level of carbonyls. pd decreased the level of peroxides in cells treated with dox and ptx. the lowest concentration of peroxide was observed at 50µm of pd. these results show that pd protect mcf-7 cells against oxidative stress induced by drugs. open channel structure of mscl from fret microscopy and simulation mechanosensitive channels open in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing and osmoregulation. here, we have determined the likely structure of the open state of the mechanosensitive channel of large conductance from e. coli (mscl) in a natural environment using a combination of patch-clamp studies, fret spectroscopy, epr data, molecular and brownian dynamics simulation. structural rearrangements of the protein are measured while controlling the state of the pore by modifying lipid bilayer morphology. fret efficiency changes can be related to distance changes using a monte carlo analysis program in conjunction with detailed orientational analysis. these measurements are used as restraints in all atom molecular simulations in order to determine the likely structure of the open state, whose probable conductance is derived from brownian dynamics simulations. transition to the open state occurs via large rearrangements throughout the protein that create a wide pore nearly 30 a in diameter. both transmembrane helices are found to line part of the pore. the n terminal helix is found to lie along the face of the membrane where it can act to sense membrane tension and directly transfer this to the pore lining helices. the method of coupling spectroscopic data with simulations is likely to be of great value for studying conformational changes in a range of membrane proteins. putative potassium channels in synechocystis sp. pcc 6803 v. checchetto, m. zanetti, g. m. giacometti, i. szabò, e. bergantino department of biology, university of padova, italy we are interested in the identification and characterization of potassium channels in the cyanobacterium synechocystis sp. pcc 6803, an organism which is considered the ancestor of plant chloroplasts. a bioinformatic screening of synechocystis proteome identified, among others, two proteins on which we focused our attention. the first one (syncak) displays sequence homology to mthk, a ca 2+ -dependent potassium channel from m . thermoautotrophicum. the second one (synk) is predicted to contain six transmembrane regions and the typical selectivity filter of all potassium channels. our goal is to understand their roles in the physiology of cyanobacteria. we cloned their coding sequences in fusion with gfp and the hybrid proteins were expressed in chinese hamster ovary cells. we evaluated the presence of both proteins in plasma membrane by fluorescence microscopy and then we proceeded to their functional characterization using patch clamp technique.this analysis will allow us to gain information about channel activity, regulation and pharmacology. we also plan to evaluate the importance of syncak and synk channels in photosynthesis. to test the hypothesis that they could be involved in regulating this process, we will produce deletion and site-specific mutants in synechocystis. finally we would also identify the homologous of these channels in the higher plant a. thaliana and obtain some information about their localization and function. j. braunagel max-planck institute for polymer research, ackermannweg 10, 55128 mainz, germany the cyclododecadepsipeptide valinomycin is composed of two amino acids (l-valine and dvaline) and two hydroxyl acids (d-α-hydroxy-isovaleric acid and l-lactic acid). they form a 36membered ring of alternating amino and hydroxyl acids. in the cyclic structure, the polar groups are oriented towards the central cavity, whereas the rest of the molecule is relatively nonpolar. this enables the complexation of ions and valinomycin acts as a selective ion transporter (k + ) in lipid membranes. when one of the amino acids is exchanged, e.g one l-valine by an l-lysine, selected functionality can be engineered into the depsipeptide while maintaining its ion conducting properties. we induced several modifications into valinomycin, i.e. a biotin binding unit or a ferrocene group to induce an electrochemical active center. the ion conducting properties of the modified ion carriers have been probed in planar lipid bilayers as well as in solid supported membranes. the role of the membrane dipole potential (ϕ d ) is of a particular interest due to a powerful impact of this potential on the membrane permeability and lipid-protein interactions. channel forming activity of gramicidin a, alamethicin, syringomycin e, hpa3 peptide, and ompf porin are influenced by ϕ d . we have studied the effect of the membrane dipole modifier, phloretin, on the properties of single channels formed by a wild-type alpha-hemolysin in planar lipid bilayers. the single channel of a ∼1000 ps conductance exhibits transitions into a number of low-conductance states as the transmembrane voltage exceeds ∼130 mv (regardless of the voltage polarity). the phloretin addition to the bathing solutions (20 µm) (after the hemolysin channel was formed in the membrane) shifts dramatically the channel voltagedependence. transitions to the low-conductance states are observed at ∼30 mv. the effect of the phloretin addition was not observed in the case when it was introduced into the bathing solution before alpha-toxin. since phloretin reduces ϕ d , the data may report on the influence of the electric potential profile on the energy of the low-conductance state of the alpha-hemolysin channel. the alternative explanation of this effect consists in a specific interaction between the phloretin and toxin channel. the work is supported in part by rfbr (# 09-04-00883), ss (# 1135.2008.4) , and the program of the ras «molecular and cell biology». atypical mechanism of conduction in potassium channels c. domene 1 , s. furini 1 1 physical and theoretical chemistry laboratory, department of chemistry, university of oxford, oxford, u.k., 2 department of electronics, computer science and systems, university of bologna, bologna, italy potassium channels can conduct k + ions with rates of up to ∼ 10 8 ions per second at physiological conditions, and they are selective to these species by a factor of 10 4 over na + ions. ion conduction has been proposed to involve transitions between two main states, with two or three k + ions occupying the selectivity filter separated by an intervening water molecule. the largest free energy barrier of such a process was reported to be of the order of 2-3kcal mol −1 . here, we present an alternative mechanism for conduction of k + in k + channels where site vacancies are involved, and we propose that coexistence of several ion permeation mechanisms is energetically possible. conduction can be described as a more anarchic phenomenon than previously characterized by the concerted translocations of k + -water-k + . we exploited the au-deposited self-assembled monolayers of the type: [−s−(ch 2 ) n −ch 3 ] (where n = 4, 9 and 15) with hydrophobically adsorbed redox protein -azurin to verify intrinsic electron transfer mechanisms according to the charge-transfer theory. the enthalpies and volumes of activation were determined through the variation of temperature (2-50 o c) and pressure (5-150 mpa) and experimental values were compared with those expected on the theoretical grounds. for the case of n = 4 the activation enthalpy definitely contains a large contribution originated from frictional-like dynamics of protein, and the activation volume has a small positive value. for n = 15 the value of activation enthalpy directly matches 1/4 of that for the reorganization energy, and the activation volume attains substantially negative value. for n = 9 we observed the intermediary performance. the whole kinetic pattern is consistent with the smooth changeover between adiabatic and nonadiabatic mechanisms of electron transfer. two gating modalities in the pore of the miniature k + channel kcv kcv is a viral protein that forms functional k + channel in heterologous systems. because of its miniature size (94 amino acids) we use kcv as a model system to study and manipulate basic properties of the k + channel pore. by analysing single-channel recordings we highlighted two voltage-dependent modalities of gating in kcv: a slow and a fast gating. the presence of a slow gating is revealed by the very low (in the order of 1-3%) mean open probability. slow gating is not related to the presence of a bundle crossing, as shown by accessibility of the cavity to mts reagents. channel opening might involve the transient formation of salt bridges between residues at the n and c termini of the channel, as suggested by mutational experiments inspired by molecular dynamics simulations of kcv. fast gating, analyzed by beta distributions, is responsible for the negative slope conductance in the single-channel i/v curve at extreme potentials and can be explained by depletion-aggravated instability of the filter region. aca8 is a type 2b caatpase with a regulatory n-terminus whose autoinhibitory action can be suppressed by binding of calmodulin (cam). aca8 n-terminus is able to bind a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. to define the role of this interaction in autoinhibition we have analysed a number of single point mutants produced by mutagenesis of aca8 e252-n345 sequence. mutation to ala of any of 6 acidic residues (e252, d273, d291, d303, e302, d332) originates an enzyme with normal activity in the presence of cam, but less camstimulated. these results highlight the relevance of a negative charge of the surface area of the small cytoplasmic loop in aca8 autoinhibition. the most deregulated mutant is d291a aca8, which is less activated also by controlled proteolysis or by acidic phospholipids; moreover, the phenotype of the d291a mutant is stronger than that of d291n aca8 suggesting a more direct involvement of this residue in autoinhibition. of the other mutants (i284a, n286a, p289a, p322a, v344a, n345a), only p322a aca8 has a basal activity higher than that of the wt. these results provide the first evidence that the small cytoplasmic loop of a type 2b caatpase plays a role in the attainment of the autoinhibited state. complex i, the first member of the respiratory chain, serves as a proton pump catalyzing transfer of two electrons from nadh to ubiquinone coupled with the translocation of four protons across the membrane. so far the mechanism of energy transduction by complex i is unknown. the nadh-binding cavity of complex i has a very prominent feature -the presence of two invariant amino acid residues, glutamate and tyrosine, that are exposed to the solvent and located in the vicinity of the fmn, the primary electron acceptor in the enzyme. it was suggested that they might be involved in the binding of nadh through interaction with its nicotinamide moiety. in this work we assessed the function of corresponding glu95 from the nuof subunit of e. coli complex i by mutation for glutamine. we showed that the negative charge of glutamate in the catalytic site is needed for the electrostatic repulsion of negatively charged phosphates of nucleotides. this process facilitates release of the product nad + and, as a result, accelerates turnover of complex i. we also found that glutamate, as one of the four negatively charged amino acid residues surrounding the isoalloxazine ring of the fmn at a distance of 4-6å, has a share of 40 mv of the overall 130 mv depression of the midpoint potential of this redox cofactor. l. erokhova 1 , p. kügler 2 , p. pohl 1 1 institute of biophysics, johannes kepler university, linz, austria, 2 ricam, austrian academy of sciences, linz, austria the mechanism of water transport through epithelia is still under debate. in the present work we tested the hypothesis of isosmolal water transport using mdck cells stably expressing the human sodium-glucose cotransporter (hsglt1). tagging hsglt1 with egfp enabled determination of its abundance in the plasma membrane by fluorescence correlation spectroscopy (fcs). by monitoring tiny shifts in the concentration of water-soluble dyes in the vicinity of epithelia, fcs also allowed assessment of the water fluxes through confluent cell monolayers grown on permeable supports. fitting the set of differential equations for the osmotic drift and for the back diffusion to the experimentally determined dye distribution permitted calculation of water flow both in the presence and in the absence of an osmotic gradient. from the calorimetric measurements of glucose transported across the cell monolayer, the water:glucose stoichiometry was derived. dividing the increment in osmotic water flux due to hsglt1 expression by the number of hsglt1 copies in the plasma membrane resulted in a single transporter water permeability p f of 4.6 x 10 −14 cm 3 /sec. thus, p f is close to the single channel water permeability of aquaporin-1. consequently, even small osmolyte concentration differences between the cytoplasm and the basolateral buffer solution are sufficient to drive a substantial water flux. m. emre, s. kavak cukurova university school of medicine, department of biophysics, balcali-adana, turkey experimental studies have shown that the at1-receptor antagonists telmisartan (tel) has a ppar-activating property, but there does not appear to be a class effect. to test telmisartan's importance, we investigated its effect on electrical activities (ea) in diabetic (d) rats. the purpose of this study was to investigate the effects of the tel on ea of diabetic papillary muscle (dpm) with stz-induced. in this study, we used four groups: (1) nondiabetic control (ndc) group (c), (2) tel-treated ndc group (c+tel), (3) diabetic group (d), and (4) tel-treated d group (d+tel). diabetes was induced by a single i.v injection of stz. in the study, membrane potential (mp) and action potential (ap) recorded after the establishment of diabetes. 1) mp was decreased significantly in both tel-treated c and d rats (from -71,4±0,7 to -64,7±0,8 mv and from -69,9±0,6 to -63,8±0,7 mv). 2) ap unchanged in d group, whereas c+tel and d+tel groups showed increase in ap compared with c and d groups. 3) repolarization time was prolonged in diabetic rats. 4) in c+tel and d+tel groups depolarization rate values increased significantly. on the other hand, in d group repolarization rate values descreased increased significantly compared to baseline values in tel solution. as a result, our data suggest that the beneficial effects of tel-treatment on the ea of the dpm appear to be due to the diminished k + currents. cytochrome c oxidase is a terminal complex (cco, complex iv) of a respiratory chain that is located in an internal membrane of mitochondria or plasma membrane of bacteria. cco is an electron transfer enzyme that reduces o 2 and uses the redox energy of the o 2 reduction for the proton translocation across the membrane. the electron-and proton-transfer generate a transmembrane electrochemical gradient (∆µh + ) that is used for atp synthesis and all other kinds of work for the cell needs. the proton translocation mechanism of cco requires 'channels' for the h + uptake and expulsion within the enzyme. the proton transfer occurs on a time-scale of micro-to-milliseconds. in order to study the proton transfer in cco, a flow-flash approach based on a time-resolved ftir spectroscopy was developed and applied. our ftir flow-flash approach (the measurement of the reaction of cco with o 2 ) allows to reach a time resolution up to tens milliseconds. with this approach and site-specific mutants of cco where the catalysis is slowed down, separate steps of the proton transfer were studied. the results showed that a unique cross-linked tyr-280 (in a combination with a time-resolved visible spectroscopy and electrometry) serves as a proton donor for the dioxygen bond cleavage during the o 2 reduction by cco. furthermore, the protolytic transitions of glu-278 -a key amino acid in the proton transfer mechanism in cco -were shown for the first time. role of calcium ions in nickel potentiation of nmda currents p. gavazzo, i. zanardi, p. guida, c. marchetti biophysics institute ,cnr, genova, italy nmda receptors are glutamate-gated channels distributed throughout the brain in the excitatory synapses and are critical for the nervous system function. they are assembled from two types of subunits, the essential nr1 and at least one nr2(a,b,c,d). nickel (ni) modulates the current flowing through nmda receptors in a different way depending on the nr2 subunit present. we have recently identified several domains of the channel involved in ni interaction, but many aspects of this modulation remain elusive. in this work we intended to determine the role of calcium (ca) ions in the potentiation induced by ni on the current through nr1/nr2b recombinant nmda receptors. when ni was applied in the presence of the physiological concentration of ca (1.8 mm) , a voltage-independent potentiation of the current was observed with a kp of 2.5 µm. this effect was progressively reduced by decreasing ca concentrations and it was no more detectable with 0.18 mm ca or in the presence of barium (ba, 0.3 mm). in this last case the effect of ni on nr1/nr2b receptors was mainly inhibitory (ki(-60mv)=156µm). therefore a physiological concentration of ca is necessary to induce ni amplification of the current. many data in the literature indicate a correlation between ca ion entrance through the channel, nmda current facilitation and cytoskeleton; however, in our experiments, the application of the actin perturbing agent cytochalasin-d did not produce major modifications in ni effect. heteromerization properties of voltage dependent potassium channels f. gambale 1 , l. pedemonte 1 , a. naso 1 , i. testa 2 , c. usai 1 , a. diaspro 2 , c. picco 1 1 istituto di biofisica, cnr, genova, italy, 2 lambs-microscobio, department of physics, genova, italy voltage-gated potassium channels are either homomeric or heteromeric tetramers composed of four α-subunits. in order to bring a contribution to the comprehension of channel heteromerization we have been investigating the properties of two plant voltage-gated k + -channels by using electrophysiological and fluorescence techniques. experiments were focussed on kdc1 and kdc2, coexpressed in xenopus laevis oocytes. kdc1, the first potassium channel cloned from daucus carota, belongs to the subfamily of α-modulatory silent channels as it doesn't form functional homomeric channels by itself. on the contrary kdc1 forms functional heteromeric channels when coexpressed with homologous subunits. kdc2, the last k + channel cloned from d. carota, belongs to the kat1 family and shares an overall identity of 63% with kat1. to correlate kdc1 functional properties with its localization in oocytes, kdc1 and/or kdc2 subunits were labelled with gfp and their properties investigated by confocal microscopy and voltage-clamp. we found that the kdc1-egfp fusion protein is not targeted to the plasma membrane unless it is coexpressed with kdc2. moreover electrophysiological experiments demonstrated that the heteromeric kdc1-kdc2 channel has altered selectivity and activation properties with respect to homomeric kdc2 channel. circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome (ards) [1] . we present a microfluidic investigation of the roles of actin organization and myosin ii activity during the different stages of leukocyte trafficking through narrow capillaries using specific drugs. the deformation rate during entry reveals that cell stiffness depends strongly on f-actin organization and hardly on myosin ii activity, supporting microfilament role in leukocyte sequestration. in the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. conversely, membrane unfolding was independent of leukocyte stiffness. the surface area of sequestered leukocytes increased by up to 160% in absence of myosin ii activity, showing the major role of molecular motors on microvilli wrinkling and zipping. finally, cell shape relaxation was largely independent of both actin organization and myosin ii activity, whereas a deformed state was required for normal trafficking through capillary segments [2] . [1] g.s. worthen et al., science, 245, 183-186 (1989) excitation-contraction coupling in skeletal and cardiac muscle is tightly regulated by the calcium release channel of the sarcoplasmic reticulum, the ryanodine receptor (ryr). we could previously show that suramin is a potent activator of the ryr via the calmodulin binding site. calmodulin shows dualistic action, i.e. activation or inhibition of the ryanodine receptor, depending on the absence or presence of ca 2+ . screening of suramin analogues identified nf676 as a use-dependent inhibitor of the skeletal muscle ryr (ryr1). here we show that nf676 inhibits high affinity [ 3 h]ryanodine binding and single channel recordings of the purified ryr1. nf676 induced a reduction of open probabilities in a concentration dependent manner, with no effect on current amplitude and unitary conductance. importantly, nf676 triggers flickering episodes of channel openings and closings before the ryr1 is frozen in a complete non-conducting state, which is fully reactivated by the ryr agonist atp. moreover, zwitterionic behaviour of nf676 facilitates plasma membrane permeation, which prevented caffeine induced ca 2+ transients in skeletal muscle cells and cardiomyocytes. conversely, ip 3 mediated ca 2+ signals were not altered by nf676. this work was supported by herzfelder'sche familienstiftung and fwf . beyond steady-state protein dynamics t. hauß 1 , j. pieper 3 , a. buchsteiner 1 , r. e. lechner 2 , n. a. dencher 2 1 helmholtz-zentrum berlin für materialien und energie, berlin, germany, 2 technische universität darmstadt, germany, 3 technische universität berlin, germany to study protein dynamics beyond steady-state experiments we have developed a novel laser-pump:neutron-probe experiment which allows us to monitor temporal changes in protein dynamics during a working cycle of a protein. protein dynamics has been extensively studied, but so far, the correlation of internal protein dynamics with the function of proteins was investigated only indirectly in steady-state experiments by variation of external parameters by variation of external parameters like temperature or hydration. the method comprises of an in-situ optical activation of a protein and a time-dependent sampling of the dymamic response using quasi-elastic neutron scattering. with the membrane protein bacteriorhodopsin, a light driven proton pump, we can demonstrate for the first time temporary alterations in the protein dynamics after triggering the working cycle. this observation is a direct proof for the functional significance of protein structural flexibility, in connection with the largescale conformational changes in the protein structure occurring during the operation of a "molecular machine". the slow vacuolar (sv) channels are ubiquitous in all tissues of higher plants. the sv channel is a non-selective cation channel permeable to both monovalent and divalent cations. sv currents recorded in a typical patch-clamp experiment require unphysiologically high cytosolic and low vacuolar calcium concentrations for full activation. we aim at looking for endogenous plant substances which might be able to modify or shift the voltage activation threshold of this channel towards more physiological conditions. flavonoid naringenin [nar] is present in all plant species where it plays a central role in the flavonoid biosynthetic pathway. nar is stored in the vacuoles in glycosylated form called naringin. when nar was added to cytosolic bath solution, we recorded a dose-dependent reversible decrease in sv channel activity. when we investigated the effect of nar on the voltage dependence of the channel, we observed that the activation threshold of the sv channel is shifted towards more positive voltages. our group has evidences that approximately 10% of the total sv current at high (e.g.> 50 mv) positive voltages is mediated by calcium. therefore, in order to verify whether nar affects both potassium and calcium conductance, we performed experiments by combining the patch clamp technique with fluorescence measurements using the fluorophore fura-2: both sv currents and calcium signals were abolished by 1 mm [nar]. determination of calcium currents in cation channels using a novel fluorescence/patch-clamp approach p. v. k. gutla, a. gradogna, a. carpaneto istituto di biofisica, consiglio nazionale delle ricerche, via de marini 6, 16149 genova, italy the patch-clamp technique combined with fura-2 fluorescence detection is suitable to investigate calcium fluxes. we used the excised patch configuration and focused the photomultiplier to the tip of the recording pipette where the fluorescent dye was present (fluoresence combined with excised patch = flep). this configuration has several advantages, i.e. absence of delay in loading the fluorophore, of interference by endogenous calcium buffers and of photobleaching. here we present an application for the determination of fractional calcium currents (pf) in a plant non-selective cation channel, showing that pf can be modulated by cytosolic calcium and potassium. flep is very efficient for measuring small calcium currents (<1 pa) of sufficiently long duration; fluorescence signals are amplified by integration in time, as the calcium/fura-2 complex accumulates at the tip of the recording pipette and diffuses slowly. we propose this technique not only for the study of calcium transport pathways, but also for other transporters of divalent cations as nickel and manganese known to quench fluorescence thus reducing both 340 and 380 nm components. moreover, using the appropriate fluorophore the technique may be extended to further ion species, e.g. bcecf for investigating proton transport pathways. ref: we introduced an original method for the monitoring of the changes in the electrostatic surface potential, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. this approach opens new way to understanding the dynamic processes within the proteins. our experiments revealed that the conformation of the na + /k + -atpase large cytoplasmic loop (c45) in the presence of the atp (without magnesium) substantially differed from the conformation in the presence of mg 2+ or mgatp or in the absence of any ligand not only in the sense of geometry but also in the sense of the electrostatic surface potential. moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. this property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain. influx of antibiotics into the periplasm of gram-negative bacteria is facilitated by porins that form channels in the outer membrane. we propose that certain natural antibiotics have been optimized by co-evolution to take advantage of the charge distribution in non-specific porins to achieve binding and thereby facilitating their uptake in bacteria. we investigate the permeation pathways of antibiotics into bacteria by reconstitution of a single porin into an artificial lipid bilayer and measuring the binding of antibiotic molecules through the time-resolved modulation of a small ion current. we have been able to characterize facilitated translocation of several antibiotics through escherichia coli and enterobacter aerogenes porins. noise analysis of ion currents through a porin in the presence of effective antibiotics revealed binding kinetics at a single molecule level. we report for the first time temperature dependent antibiotic translocation that revealed complete energy profile. combining these results with microbiological assays and molecular dynamics simulations, we conclude the molecular mechanism of antibiotic permeation. our approach may contribute to the rational design of new antibiotics against clinical bacterial strains for the most efficient delivery to target sites. mitochondria regulate ca 2+ influx and determine patterns of er ca 2+ refilling in acinar cel o. kopach, i. kruglikov, t. pivneva, n. voitenko, n. fedirko bogomoletz institute of physiology, kiev, ukraine store-operated ca 2+ entry (soce) is mediated by activation of soc-channels of plasma membrane following the emptying of endoplasmic reticulum (er) ca 2+ stores. the soce is required for calcium signaling, secretion of neurotransmitters and proteins, but the mechanisms of natural soce regulation are not well understood. we utilized several imaging methods to measure ca 2+ signals in cytoplasm ( ] mit transients, observed both in egta-and bapta-buffering inside solutions. these data suggest that ca 2+ sequestration by mit is associated with the formation of microdomains and prevents ca 2+ -dependent socc inactivation. we also found that inhibition of mit under prolonged cell stimulation resulted in complete inhibition of soce as well as decrease and deceleration of er refilling. thus, mit regulate calcium recycling and maintain the soce controlling dynamic interplay between soce and sustained er refilling under prolonged stimulation. bioelectrochemical devices composed of au electrodes coated by self-assembled monolayers (sams) of different composition and thickness are ideal systems to probe et patterns and mechanisms for redox proteins. representative proteins, cytochrome c and azurin were studied by using the combinations of four different strategies including the variation of sam thickness ([−s−(ch 2 ) n −ω], with n running over the range 3 to 20, throughout), solution viscosity (varied by adding of the viscose additive -glucose), temperature (0 to 50 o c) and hydrostatic pressure (up to 150 mpa), aiming the identification of different intrinsic et patterns and interplay between them in the framework of generalized charge-transfer theory. we demonstrated the full adiabatic (frictional) control for the case of thinner sams, the intermediate (mixed) regime, and the complete changeover to the nonadiabatic mechanism (long-range tunneling) for the case of thick sams owing to the variation of electronic coupling, in a nice agreement with theoretical predictions. h. nury 1 , f. manon 1 , b. arnou 2 , m. le maire 2 , e. pebay-peyroula 1 , c. ebel 1 1 institut de biologie structurale (ibs) cea cnrs ujf grenoble france, 2 cea ibitec cnrs ura2096 univ. paris-sud gif-sur-yvette france adp/atp carriers (aacs) are major and essential constituents of the inner mitochondrial membrane. they drive the import of adp and the export of newly synthesized atp. they were described as functional dimers from the 1980s until the structures of the aac shed doubt on this consensus. we aimed to ascertain the published biophysical data claiming that aacs are dimers and to characterize the oligomeric state of the protein before crystallization. analytical ultracentrifugation sedimentation velocity experiments clearly show that the bovine aac is a monomer in 3-laurylamido-n,n 2 dimethylpropylaminoxide (lapao), whereas in triton x-100 and reduced triton x-100, higher molecular mass species can also be identified. neutron scattering data for monomeric bovine aac in lapao does not give definite conclusions on the association state, because the large amount of detergent and lipids is imperfectly matched by contrast methods. we discuss a possible way to integrate previously published biochemical evidence in favor of assemblies, the lack of well-defined multimers that we observe, and the information from the high-resolution structures, considering supramolecular organizations of aacs within the mitochondrial membrane. local anaesthetic binding to shaker channels: role of aromatic residues j. nilsson, h. ullman, k. sahlholm, p. arhem the nobel institute for neurophysiology, department of neuroscience, karolinska institutet, se-171 77 stockholm, sweden local anaesthetic, antiepileptic and antiarrhythmic drugs acting on nav and herg channels have been assumed to bind to aromatic residues in the internal vestibule; to f1764 and y1771 in nav (ragsdale et al., 1996 and to y652 and f656 in herg (mitcheson et al., 2000) . despite a lack of such residues in kv channels, local anaesthetics, antiepileptic and antiarrhythmics bind to kv channels with a considerable affinity. to explore the role of aromatic residues for the binding we investigated the effect of bupivacaine, benzocaine, phenytoin and quinidine on shaker channels mutated to residues corresponding to the most c-terminal of the two aromatic residues in the s6 segment of the nav and the herg channels, (v473y and p474f respectively). the channels were expressed in xenopus oocytes and the currents measured with the two-electrode voltage-clamp technique. the results suggest that aromatic residues do not increase the binding affinity of the studied compounds to kv channels. rather, the affinity decreases (as reflected in typical k d values for bupivacaine on v473f, p474f and wildtype channels, being 550, 740 and 300 µm, respectively). thus, aromatic residues seem not to be necessary for high-affinity binding of the studied compounds to kv channels. how this relates to their suggested roles in the nav and herg channels, remains to be evaluated. (molina et al, 2006) . the occurrence of a similar behavior in other channels points out to clustering and coupled gating as a potentially important drug target to modulate channel activity. we have identified molecular determinants involved in single and coupled channel gating in kcsa. first, we detected that clustering and coupled gating of kcsa is modulated by anionic lipid. also, a model for the interaction between two kcsa open channels was built. the docking predicts intermolecular sites which includes the non-annular lipid binding site. this explains how an excess of anionic lipid disrupts interactions between channels, destabilizing clustering and coupled gating in kcsa. in addition, the docking model reveals molecular determinants involved in single and coupled channel gating. this interaction involves w87, which affects the neighbouring channel through specific interactions in the extracellular mouth stabilizing the selectivity filter in an open conformation. the coupled gating is also explained since this mechanism affects the opposite channel in a mutual manner. finally, mutants kcsa e71a and kcsa w87a disrupt the coupled gating of kcsa, thus, supporting the model. we think that this coupled gating phenomenon could correspond to the second gate previously detected by fluorescence methods (blunck, et al, 2006) . supported by grants from the spanish bfu2008-00602/bmc and csd2008-00005. a. kumar, e. hajjar, p. ruggerone, m. ceccarelli university of cagliari, monserrato, italy the striking presence of outer membrane (om) in gramnegative bacteria of e.coli represents a strong barrier for any molecule to penetrate inside bacteria. in particular for β-lactam antibiotics, which have their target located inside the bacteria; the first step towards reaching inner part of bacterium is the cellular uptake. ubiquitous presence of porins (such as for instance ompf, ompc) in the om, function as a channel facilitating the transport of molecules (such as, for instance antibiotics) across the om. bacteria can exhibit resistant towards antibiotics by: (i) decreasing their uptake by under-expressing the porins or/and (ii) production of inactivating enzymes such as ß-lactamases. to combat the latter mechanism ß-lactamase inhibitors (such as, sulbactam for instance) are prescribed together with the antibiotics. like the antibiotics, the inhibitors must penetrate the om, the main path being through porins. it is thus evident the biological relevance of investigating the mechanisms by which porins can regulate entry/exit across the om. to achieve this goal, molecular dynamics simulations were performed to explore the structure and dynamics of pores formed by ompf and ompc porin. from the analysis of data obtained from our simulations, we identified the key residues buried behind the l3 loop, which may play be crucial for porins to exert their biological role. as a case study, we report results about the diffusion of sulbactam through the two porins. the pentadecapeptide gramicidin forms a cation-specific ion channel in membrane environment. the two main conformations are the head-to-head helical dimer (hd) known as the channel conformation and the intertwined double helical form (dh) often referred to as non-channel conformation. in this comparative study [1] , the energetics of single potassium ion permeation by means of the potential of mean force (pmf) for both gramicidin conformations embedded in a dmpc bilayer has been addressed by molecular dynamics simulations. a significantly decreased free energy barrier by ∼25 kj/mol for potassium ion passage through dh as compared to hd is reported. favorable electrostatic side chain-cation interactions in hd are overcompensated by phospholipid-cation interactions in dh. the latter are coupled to an increased accessibility of the channel entrance in dh due to distributed tryptophans along the channel axis. this result underscores the importance of the lipid environment of this channel not only for the equilibrium between the different conformations but also for their function as cation channels. y. sawada 1 , m. murase 2 , m. sokabe 1 1 dept. physiol. nagoya univ. grad. sch. med., nagoya, japan, 2 icorp/sorst cell mechanosensing, jst, nagoya, japan the bacterial mechanosensitive channel of large conductance mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer α-helices, and its 3d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. although several models for the opening process have been proposed with molecular dynamics (md) simulations, as they do not include mscllipid interactions, it remains unclear which amino acids sense membrane tension and how the sensed force induces channel opening. we performed md simulations for the mechano-gating of mscl embedded in the lipid bilayer. upon tension in the bilayer, phe78 in the outer helix was dragged by lipids, leading to a tilting of the helices. among amino acids in the outer helix facing the bilayer, phe78 at the water-lipid interface showed the strongest interaction with lipids, thus may work as a major tension sensor. neighboring inner helices cross each other in the inner leaflet, forming the most constricted part of the pore. as tension increases, the crossings move toward the cytoplasm associated with an expansion of the constricted part. during the movement, a hydrophobic water block environment around the constricted part was broken followed by water penetration and permeation. the k + channel kcsa is an integral membrane protein from s.lividans, used as a model system for studies on ion channels and oligomeric membrane proteins. its atomic structure has been solved by x-ray diffraction, which shows an assembly of four identical subunits around a central aqueous pore, including the so-called selectivity filter. this channel is able to permeate k + at high flux rate and it is blocked by na + (physiological blocker). fluorescence, circular dichroism and fourier transform infrared experiments carried out in our laboratory demonstrated that k + and na + are able to bind to kcsa in a competitive manner. this binding is indeed associated with channel conformational changes, which seem to be related to the permeation and blockade processes. to further investigate this phenomenon we carried out a detailed study of chemical and thermal denaturation of wild-type and mutant kcsa channels. these two types of experiments were in agreement and indicate that both cations are able to stabilize the channel through conformational changes, being k + the more efficient one, even more when lipids are present. particularly, mutant channels with a structurally altered selectivity filter show that ion and lipid-induced global conformational changes are intimately associated to the conformation of this selectivity filter (conductive and non-conductive forms). modulation of the voltage-gated sodium channel nav 1.5 by rcssii, a toxin from the scorpion centruroides suffusus c. picco 1 , g. corzo 2 , l. d. possani 2 , g. prestipino 1 1 institute of biophysics, cnr, genova, italy, 2 instituto de biotecnologia, unam, cuernavaca, mexico the main cardiac voltage-gating sodium channel, na v 1.5, generates the fast depolarization of the cardiac action potential and plays a key role in cardiac conduction. its importance for normal cardiac function has been exemplified by the description of numerous naturally occurring genetic variants of the gene scn5a, which encodes na v 1.5, that are linked to various cardiac deseases. subsequently, studies of this channel localization have led to its identification in immature and denervated skeletal muscle and in the brain neurons. in our effort to identify high affinity ligands for this channel, we have investigated the effects of the recombinant cssii (rc-ssii), a four disulfide-bridged scorpion toxin isolated from the venom of the scorpion centruroides suffusus. human cardiac sodium channel α subunit scn5a was expressed in cho cells and macroscopic na + currents were recorded with patchclamp technique in whole cell configuration. the electrophysiological experiments have highlighted a strong affinity for the channel at low nanomolar concentration. compared with control conditions, rcssii toxin affects in reversible way the kinetics of activation and inactivation and marked decrease the peak na + influx. the extrusion mechanism of substrates in rnd family efflux pumps: a molecular dynamics study a. v the rnd transporters of the acrab-tolc (e.coli) and mexab-oprm (p.aeruginosa) systems are able to export structurally and chemically different substrates outside bacteria through the membrane, being responsible of multidrug resistance. on the basis of crystallographic information, an extrusion process conceived as a three-cyclic peristaltic pumping has been proposed, but further microscopically well-funded investigations are needed to understand the mechanism. using different computational methods like adaptive bias force (abf) and targeted molecular dynamics (tmd), we have investigated the mechanism of substrate uptake and pumping at a molecular level. with the first method we have investigated the passage of antibiotics from the periplasm into the internal pore of the pump, while tmd has been used to assess the effect of conformational changes on the extrusion of drugs (which have been located into one of the proposed binding pockets). comparison between the active pumps acrb and mexb (which show different resistance patterns despite their homology) provide insights into the microscopic details of their functioning. in arabidopsis thaliana there are twenty genes, grouped into three subfamilies, encoding for homologues of animal ionotropic glutamate receptors (iglrs). each protein displays a pore-forming loop, flanked by two conserved helices (plus a third c-terminal helix), a glutamate-binding domain and an n-terminal region. through pharmacological and/or genetic approaches, many physiological functions have been attributed to plant glurs, such as the regulation of cytosolic calcium, photomorphogenesis, water balance and carbon/nitrogen sensing and assimilation. according to the endosymbiotic theory, the cyanobacteria are considered to represent the precursors of the present chloroplasts. the first prokaryotic glutamate receptor (glur0) was identified in the cyanobacterium synechocystis. the putative products of the atglr3.4 and atglr3.5 genes display a possible targeting sequence for chloroplast location and show a high degree of homology with glur0. using specific antibodies and confocal microscopy, we have localized the two members of the atglr subgroup 3, glr3. 4 and glr3.5 (splicing variant) , to the chloroplast in arabidopsis and to the inner envelope membrane in spinach. electrophysiological experiments indicate the presence of an activity which is compatible with that of glutamate receptors. furthermore, oxygen evolution measurements suggest that chloroplast-located glutamate receptors may play a role in the regulation of photosynthesis. under extreme conditions many cells control their volume responding to osmotic challenges by unloading or loading solutes to recover their original volume. a faster volume regulatory role triggered by membrane tension has been disclosed for aquaporins in kidney proximal tubule cells, where aqp1 is the main water channel, using isolated brush border membranes. in conventional osmotic studies in animal cells it is common to disregard internal hydrostatic pressures because they are insignificant compared to osmotic forces. however, by using low osmolarity buffers in small radii vesicles, we detected a rise on the internal pressure that creates surface tension and causes membrane stress, with a negative outcome on aquaporin water permeability. these findings suggested a mechanism for volume regulation in kidney proximal tubule epithelia where massive solute and fluid transport occurs. to further explore aquaporin regulation by membrane tension, yeast cells were used as a model that could bare surface tension some orders of magnitude higher than animal cells due to the existence of a cell wall. the effect of increasing levels of membrane tension on yeast water channel activity was evaluated. an impairment of aquaporin activity correlated with the increase of membrane tension corroborates the volume regulatory role of aquaporin in different cells. deuterium isotope effects on fast gating of the chloride channel clc-0 g. zifarelli, a. r. murgia, p. soliani, p. michael istituto di biofisica, cnr, via de marini, 6, i-16149 genova, italy gating of the torpedo cl − channel clc-0 is modulated by intracellular and extracellular ph, but the mechanism responsible for this regulation has remained so far elusive. using inside-out patch clamp measurements we studied the dependence of the fast gate on ph int and [cl − ] int . only the closing rate, but not the opening rate showed a strong dependence on these intracellular factors. using mutagenesis we excluded several candidate residues as mediators of the ph int dependence. we propose a model in which a proton generated by the dissociation of an intrapore water molecule protonates e166 leading to channel opening. deuterium isotope effects confirm that proton transfer is rate limiting for gate opening and that channel closure depends mostly on [oh − ]. the model is in natural agreement with the finding that only the closing rate constant, but not the opening rate constant, depends on ph int and [cl − ] int . deletion of the c-terminus destabilizes phosphorylated na/k pump state containing 3na ions n. vedovato, d. c. gadsby the rockefeller university, new york, u.s.a. the na/k pump's extended c-terminus (compared to the serca ca pump's) links 3 transmembrane helices, and its truncation lowers cytoplasmic na affinity for forming the occluded e1p(na 3 ) state. here we test the effects of c-terminal truncations on interactions with external na. we deleted the last 2 (yy) or 5 (kesyy) residues in xenopus α1 β3 pumps made ouabain resistant by mutations q120r-n131d (rd) or c113y (c-y), and then used two-microelectrode voltageclamp recording in xenopus oocytes to measure pump currents as 10 mm ouabain-sensitive currents while endogenous na/k pumps were silenced with 1 µm ouabain. inhibition by external na of steady outward pump current ([k] o =15 mm) at large negative voltages was somewhat weaker in both rd and c-y pumps than in wt pumps, but was severely impaired in all c-terminal truncated pumps. consistent with this, the voltage dependence of transient charge movements under na/na exchange conditions ([k] o =0 mm) was strongly shifted to more negative potentials in the truncated pumps relative to the parent rd or c-y pumps, shifts comparable to those seen in wt pumps on decreasing [na] o several-fold. together, the results suggest that these c-terminal deletions lower the apparent affinity for external na ions to bind and become occluded in the na/k pump. the c-terminus therefore provides contacts important for stabilizing the occluded e1p(na 3 ) conformation, regardless of the route of na ion entry into the binding pocket. muscle contraction is driven by molecular motors that adapt their energy utilization according to the demands made on them. we test the hypothesis that rate constants controlling the biochemical steps involved in atp hydrolysis by myosin atpase are affected by the force of the muscle. here we use fluorescence lifetime imaging microscopy (flim) of a fluorescently labelled atp analogue to investigate changes in the environment of the myosin atpase, caused by different loads applied to skeletal muscle. single muscle fibres were subjected to cycles of stretches and releases in the presence of rigor solution and 10 µm of coumarin-labelled atp. flim acquisition was synchronised with stretch/release cycles and force measurements, which allow us to investigate the effect of strain on the lifetime of the labelled atp bound to the actomyosin complex. characterization of the fluorescence decay by a bi-exponential function resolved the time constant of two populations, namely, free fluorophore (τ 1 = 0.47 ± 0.03 ns; mean ± s.d.) and fluorescent nucleotide bound to the actomyosin complex (τ 2 = 2.21 ± 0.06 ns at low strain). these experiments showed that while the time constant of the free fluorophore did not change with force, the time constant of the fluorescent nucleotide bound to actomyosin showed a linear dependence with the force applied to the muscle of 0.43 ± 0.05 ps/kpa. neck linker docking coordinates the kinetics of kinesin´s heads i. derenyi, a. czovek, g. j. szollosi department of biological physics, eotvos university, pazmany p. stny. 1a, h-1117 budapest, hungary conventional kinesin is a two-headed motor protein, which is able to walk along microtubules processively by hydrolyzing atp. its neck linkers, which connect the two motor domains and can undergo a docking/undocking transition, are widely believed to play the key role in the coordination of the chemical cycles of the two motor domains and, consequently, in force production and directional stepping. although many experiments, often complemented with partial kinetic modeling of specific pathways, support this idea, the ultimate test of the viability of this hypothesis requires the construction of a complete kinetic model. considering the two neck linkers as entropic springs that are allowed to dock to their head domains and incorporating only the few most relevant kinetic and structural properties of the individual heads, we have developed the first detailed, thermodynamically consistent model of kinesin that can (i) explain the cooperation of the heads during walking and (ii) reproduce much of the available experimental data (speed, dwell time distribution, randomness, processivity, hydrolysis rate, etc.) under a wide range of conditions (nucleotide concentrations, loading force, neck linker length and composition, etc.). besides revealing the mechanism by which kinesin operates, our model also makes it possible to look into the experimentally inaccessible details of the mechanochemical cycle and predict how certain changes in the protein affect its motion. the positive role of noise on the transport efficiency of na, k atpase c.-h. chang 1 , t. y. tsong 2 1 institute of physics, national chiao tung university & physics division, national center for theoretical sciences, hsinchu, taiwan, 2 institute of physics, academy of sciences, taipei 115, taiwan na, k atpase is a molecular motor which is able to transport ions through cell membranes, even against the transmembrane ion concentration gradient. while in vivo this nanoscale soft machine consumes atp, it may be driven by external fluctuating electric fields, no matter they are periodic or random. theoretically, the motor conformations can be described by a conformation vector v(t) governed by a multi-dimensional kinetic equation. given an oscillating electric field with a slight fluctuation, the boltzmann distributions of these conformations will change with time. the instantaneous transported ion flux is a functional of the quasi-cyclic trajectory v(t) of this non-autonomous dynamical system. various interesting dynamical properties of this ion pump, including stochastic resonance, can be studied theoretically, some of which have good agreement with recent experimental findings. in situ measurements of the molecular motor of muscle with nanometer-microsecond resolution in a contracting muscle, arrays of the dimeric motor protein myosin ii pull the actin filament towards the centre of the sarcomere during cyclical atp driven interactions. when the external load is smaller than the array force, the sarcomere works as a motor, converting metabolic energy into mechanical work; when the external load is larger than the array force, the sarcomere acts as a brake resisting the load with reduced metabolic cost. to investigate the molecular basis of the work production and the braking action of muscle, we use sarcomere-level mechanics and x-ray interferometry in intact single cells from frog skeletal muscle. during isometric contraction, each motor bears a force of about 6 pn. during shortening against high and moderate loads, the number of myosin motors attached to actin reduces in proportion to the external load while the force per attached motor is maintained similar to the isometric value (piazzesi et al., cell 131, 784-95, 2007) . rapid stretches of 1-5 nm between each overlapping set of myosin and actin filaments in a muscle sarcomere cause the stiffness of the array of myosin motors to increase up to twice the isometric value within 2 ms (brunello et al., pnas usa 104, 20114-19, 2007) , indicating that the high resistance of active muscle to stretch is due to recruitment of the second motor domain of the myosin molecules with the first domain already attached to actin. supported by miur and ente crf (italy), nih (usa), mrc (uk), embl, esrf. kinesin-1 is a molecular motor that moves cellular cargo along microtubules. its functional mechanism is well understood for individual motors. however, the way that many kinesin-1 motor proteins bound to the same cargo move together is not. we addressed the structural basis for this phenomenon using video microscopy of single microtubulebound full-length motors and various spectroscopy methods were employed to study synthetic peptides derived from hinge-1 region. these peptides show an unexpected profile of secondary structure forming propensities. video microscopy of single microtubule-bound full-length motors reveal the sporadic occurrence of high compliance states alternating with longer-lived, low compliance states. the deletion of hinge-1abolishes transitions to the high compliance state. from the results we hypothesize that strain accumulated during multiple kinesin motility populates the high compliance state by unfolding helical secondary structure in the central hinge 1 domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiple motor situations. titin is a giant protein of vertebrate skeletal and cardiac muscles. cardiac titin is expressed in two main isoforms: short n2b (∼3000 kda) and long n2ba (∼3400 kda). we have studied changes of titin isoform composition in myocardium of hibernating ground squirrels and spontaneously hypertensive rats (shr). using electrophoresis we have revealed considerable decrease (by 2-3 times) in the content of titin relative to myosin heavy chains in shr heart as compared with that for normotensive rats. surprisingly that the data of qrt-pcr showed the increase in mrna content of n2ba and n2b-isoforms in hypertrophic heart more than 2 times in comparison with norm. we suppose that such a result is an effect of depressed translation of mrnatitin in pathology. we have observed the decrease (by 1,5 times) of total titin amount in heart of hibernating animals in comparison with that for summer active animals. however n2ba/n2b ratio in the heart upon hibernation was increased by 2 times. similar trend was not revealed for the mrna level of corresponding isoforms, although we have showed the decrease of mrna of both titin isoforms in heart of hibernating ground squirrels as compared to their content of summer animals. the decrease in total mrna level may be explained by repressed transcription or mrna degradation in the cell during hibernation. these discrepancies in protein and mrna levels may be considered as the posttranscriptional regulation of titin isoforms expression. actomyosin cross-bridges formed when the globular heads of myosins bind to actin filaments are the molecular engines that drive muscle contraction, fuelled by atp hydrolysis. critical to this process is the change in shape of the cross-bridge and the change in the interactions with actin, in response to force applied to the muscle, and to the status of the nucleotide in the binding pocket. although molecular detail is known from x-ray crystallography and biochemistry, understanding of the interplay between cross-bridge shape and chemical state requires studies in muscle fibres generating force. we use fluorescence life time imaging microscopy (flim) as a probe of the cross-bridge environment.with a fluorescent analogue of atp 1 , fluorescence life-time (flt) changes when the crossbridge binds to actin. now, we show preliminary experiments on the effect of force on flt. the essential light chain of myosin (elc) is a ∼20 kda peptide that wraps around a 9nmlong α-helix of the myosin cross-bridge known as the lever arm which tilts during force generation. using a recombinant elc, labelled with a fluorophore at a strategic cys, we replace the native elc and introduce the fluorescent elc in muscle fibres. preliminary experiments demonstrate that the elc fluorophore also is sensitive to force applied to the muscle fibre. in addition, förster resonance energy transfer occurs between the nucleotide and elc fluorophores, opening the way for studying structural changes in cross-bridges during force generation by fret. muscle contraction: pitfalls in the determination of the contractile response e. grazi dipartimento di biochimica e biologia molecolare, università di ferrara, ferrara, italy the contractile response of an active muscle depends on the load. the load is a force /cross-section. there are three fundamental dimensions: the mass, m; the space, l; and the time, t. from these three dimensions are built up all the physical dimensions. as an example the acceleration, a, is given by, a=l.t −2 . once the direction and versus are settled the modulus fully defines the physical effect of the acceleration. what about the force? the force, f, is given by, f=m.a. at variance with the acceleration, once the direction and versus are settled, the modulus does not define the physical and the biological effects of the force: the same force is generated by an infinite number of mass-acceleration couples that display different physical and biological effects. the same occurs with the load. thus defining the load that opposes the contractile force does not define the contractile system. in the studies on muscle contraction the acceleration of the load is not considered nor it is provided a way to extract this information. thus these systems are poorly defined from the physical as well as from the biological point of view. models of muscle contraction that consider explicitly both the mass and the acceleration of the load show that, at the same load, the decrease of the acceleration of the load significantly delays the pre-steady state of the contraction and decreases the stiffness of the active fibre. r. shahapure 1 , f. difato 1 , a. laio 1 , d. cojoc 3 , e. ferrari 3 , j. laishram 1 , g. bisson 1 , v. torre 2 1 int. school for advanced studies, trieste, italy, 2 iit-sissa unit, trieste, italy, 3 lab. nazionale tasc, trieste, italy polymerization of actin filaments is the main source of motility in lamellipodia and is controlled by many regulatory proteins. the underlying molecular mechanisms are only partially understood and now a determination of the dynamical properties of force generation is needed. using optical tweezers we measured with millisecond temporal resolution and pn sensitivity the force-velocity (fv) relationship and the power dissipated by lamellipodia of dorsal root ganglia neurons. when force and velocity are averaged over 3-5 s, fv relationships can be flat. on a finer time scale, random occurrence of fast growths and sub-second retractions become predominant. maximal power dissipated by lamellipodia over a silica bead with a diameter of 1 µm is 10 −16 w. due to the presence of adhesion forces, beads in close contact with a lamellipodium can seal on its membrane reducing the amplitude of brownian fluctuations often by more than 10 times. under these conditions, when lamellipodia grow and push the beads, discrete jumps varying from about 5 to 50 nm are detected. when lamellipodia retract, pulling the beads, no discrete events are observed. our results on the dynamical properties of force generation are: a) force generation is a probabilistic process; b) underlying biological events have a bandwidth up to at least 10 hz; c) fast growths of lamellipodia leading edge alternate with local retractions; d) force generation is produced in discrete steps with varying amplitude up to 0.2 pn. pushing on microtubules: dominant spindle centering mechanism in c. elegans embryo? j. pecreaux, s. redemann, a. a. hyman, j. howard mpi-cbg, pfotenhauerstr 108, 01307 dresden, germany asymmetric cell division, where the content of the two daughter cells -as well as their sizes -differ, is found in many organisms. strikingly, the spindle, first centered, starts to be displaced out of the center only in late metaphase. in c elegans embryo, the spindle rocks and is posteriorly displaced during anaphase by force generators asymmetrically localized on cell cortex. prior to anaphase onset, the spindle is usually assumed to be centered by the same pulling force. it thus requires the force generators to be carefully repressed to distribute forces symmetrically. on live embryos, we measured positional fluctuations of centrosomes during metaphase with 20 nm accuracy. fourier analysis shows an extremely accurate centering respect to the number of force generators and microtubules. furthermore, spectrum is close to a lorentzian, modeled by a spring and a dashpot, suggesting a spindle centering more likely by pushing on microtubules than pulling. deviation at high frequencies indicates a subdominant pulling force. rnai of gpr-1/2, known to control force generation, increases slightly centering accuracy; this result supports the hypothesis of an independent centering mechanism. conversely, zyg-9(rnai), a microtubule growing factor, decreases centering accuracy, modeled spring stiffness and damping modulus. conclusion: first, the spindle centering mechanism is independent of cortical pulling force generator. second, microtubules pushing is likely to center the spindle. mechanical forces are important in the regulation of cellular adhesion and migration. the focal adhesion kinase (fak) has been suggested to transduce cellular forces and govern cell migration. to obtain more insight in the functioning of fak, a fret-based optical biosensor for fak was designed to relate integrin-mediated conformational changes in its ferm domain to focal adhesion behavior during cell spreading and migration in living cells. imaging of the kinetics of ferm-based fak conformational changes in spreading cells revealed two consecutive stages of focal adhesion activation. heterogeneous ferm conformational responses were observed in individual focal adhesions of adherent motile cells, with the active ferm conformation being enriched in growing and sliding fas, but not in stable and shrinking focal adhesions. inhibition of the cellular actomyosin system revealed the involvement of rho-rock rather than mlckinduced tension signaling in the modulation of the ferm response. our results place the ferm conformational change of fak at the interface between integrin and force sensing. the time course of inorganic phosphate release in permeabilized cardiac trabeculae of the rat c. mansfield, t. west, m. a. ferenczi imperial college london, u.k. the rate of p i release was determined in permeabilized rat trabeculae. contraction was elicited at 20 • c by laser-flash photolysis of npe-caged atp, and time-resolved p i release was monitored using mdcc-pbp, a coumarin-labelled phosphate binding protein, which increases its fluorescence intensity five-fold upon p i binding. the atpase rate during the first turnover of the total crossbridges (assuming 100 µm myosin heads) was 23s −1 . the rate decreased to a steady state of 4s −1 after the eighth turnover (0.5-0.6s after activation). this steady state rate is comparable to published values of 3 -10s −1 , made ∼15s after activation using an nadh-linked enzyme assay of adp release. the advantage of using mdcc-pbp is that the control of mechanochemical coupling can be examined from the onset of force production and as it progresses toward the steady state. force production and p i release were simulated using a seven step scheme. force was attributed to the states in the sequence a.m.adp.p i ↔ a.m.adp ↔ a.m.adp, with strain sensitivity incorporated into the isomerisation of a.m.adp. the a.m.adp.p i and a.m.adp states populated rapidly as force was increasing. in contrast, the a.m.adp state accumulated slowly after the force plateau was reached and became the dominant force bearing state at the time of the eighth crossbridge turnover. experiments are on-going to examine how the distribution of a.m states changes in response to rapid length-changes. pulling as a factor in forming the heterophasic structure of immunoglobulin proteins structure of proteins of immunoglobulin superfamily: human igg3 kuc and muscle protein titin, has been investigated by methods of electron microscopy and diffraction with the use synchrotron radiation. super elasticity of titin, the protein of immunoglobulin superfamily, is a key parameter that determines the mechanical properties of muscle. however, the structural-physical mechanism of titin elasticity under tension remains poorly understood. here both tension transduction and high elasticity of titin are explained in terms of crystalline polymer physics. x-ray data suggest a model of titin as a nanoscale, morphological, aperiodical array of rigid ig-and fn3-type domains covalently-connected by conformationally variable short loops. the line group symmetry of the model can be defined as s m with axial translation τ ∞ . homologous domains would have similar stability, but the structure of different domains on stretching is subject to different forces because they have different orientations relative to the axis of the molecule. under the force influence the structure of any domain can become either rigid or flexible depending on its orientation in the titin strand. pulling geometry forms an active axial structure from latent isotropic random coil structure of titin strand. we are suddenly faced with nanophase-separated morphology of igg3 kuc. study was supported by rfbr grant 07-02-01281. strain response of myosin essential light chain in permeabilized skeletal muscle fibres d. s. ushakov, d. ibanez-garcia, t. g. west, p. m. w. french, m. a. ferenczi imperial college london, uk we applied fluorescence lifetime imaging microscopy (flim) to investigate the relation between conformation of myosin head and mechanical force in skeletal muscle fibres. recombinant myosin essential light chain (elc) was expressed in e.coli and labelled at cys-178 with coumarin. the labelled elc was exchanged with native elc in single permeabilized rabbit m.psoas fibres. fluorescence lifetime was measured using leica sp5 upright confocal microscope equipped with becker & hickl time-correlated single photon counting module and 63x 1.2 na leica planapo dipping objective, with the two-photon fluorescence excitation at 856 nm by sapphire pulsed laser. after acquiring flim images of muscle fibres in relaxed state, the solution was changed to ca-free rigor. further images were acquired in rigor with or without 0.5-1% stretch applied by a motor. both single and double exponential fluorescence decay analysis showed that the lifetime in rigor was lower compared to relaxed (about 80 ps difference for single exponential fit) and to rigor fibres under strain (about 40 ps). these data suggest a change in the microenvironment of coumarin induced by nucleotide binding and strain. this change is likely to be due to interaction between c-terminal domain of elc and the n-terminal domain of myosin heavy chain related to the lever arm re-orientation process. supported by bbsrc. alpha-synuclein and its a30p mutant affects the actin cytoskeleton structure and dynamics v. sousa 1 , s. bellani 1 , g. ronzitti 2 , f. valtorta 1 , j. meldolesi 1 , e. chieregatti 2 1 department of neuroscience, hsr, milano, italy, 2 department of neuroscience, iit, genova, italy alpha-synuclein (syn) is a soluble protein abundant in the brain, primarily enriched at pre-synapses. syn overexpression and the expression of its a30p mutant participate in the pathogenesis of parkinson's disease. many roles have been proposed for syn, including the regulation of synaptic vesicle pools and of neurotransmitter release. the actin cytoskeleton regulates many aspects of synaptic function and its dysregulation may be a cause of neurodegeneration. working both in cell-free and in vivo conditions we demonstrate that syn and the a30p mutant have different effects on the actin cytoskeleton dynamics. our results show that syn binds actin, and decreases actin polymerization rate probably by monomer sequestration. on the contrary, a30p accelerates actin polymerization in vitro and disrupts the cytoskeleton of intact cells. in particular, during dynamic cytoskeleton remodeling, a30p induces the assembly of discrete actin-rich foci. actin trapping and the impairment of filaments reassembly lead to inhibition of cell movement and of the re-establishment of cell-cell contacts. in a30p expressing cells cytoskeleton-based processes, such as cell migration and the exo/endocytic traffic are inhibited. elucidating the dynamics of syn interaction with actin may contribute to the understanding of its role in neuronal physiology as well as in neurodegeneration. on the physics of muscle contraction m. l. shur ural state university, yekaterinburg, russian federation whichever energy source is chosen as an engine, its force will decrease with increasing velocity. this is connected with a limited power of any engine. thus, we state that hill's formula is a mere sequence of the law of energy conservation. to derive a mathematical dependence "force-velocity", all the means of consumption of fuel energy should be determined -in our case, the energy of the atp hydrolyze. moreover, the conformation energy of the crossbridges attached serves as the force source as well. we state that part of the energy release transforms into the energy of oscillations of myosin proteins; the other part goes into thermal energy of the sarcoplasmic solution. interaction of the oscillating myosin system with the sarcoplasmic solution controls the process of force generation by a muscle. it is just this interaction that leads to the temperature dependence of force. the presentation is devoted to constructing a theory based on these simple considerations. a discussion and constructive critic is especially wanted. -biological motility and molecular motors meiotic nuclear oscillations in the fission yeast schizosaccharomyces pombe are crucial for proper chromosome pairing and recombination. we report a mechanism of these oscillations based on collective behavior of dynein motors linking the cell cortex and dynamic microtubules that extend from the spindle pole body in opposite directions. by combining quantitative live cell imaging and laser ablation with a theoretical description, we show that dynein dynamically redistributes in the cell in response to load forces, resulting in more dynein attached to the leading than to the trailing microtubules. the redistribution of motors introduces an asymmetry of motor forces pulling in opposite directions, leading to the generation of oscillations. our work provides the first direct in vivo observation of self-organized dynamic dynein distributions, which, due to the intrinsic motor properties, generate regular large-scale movements in the cell (1 ) m. versaevel, s. gabriele, p. damman university mons-hainaut, 7000 mons, belgium the remodeling of blood vessels in response to changes in blood flow is mainly realized by endothelial cells (ecs) that convert mechanical stimuli from flowing blood into changes in cell signaling through a process called mechanotransduction. many of the biological responses to external forces originate at two types of microscale structures: focal adhesions linking cells to their extracellular matrix and adherens junctions that link adjacent cells. this study aims to elucidate the role of the cytoskeleton, cell-matrix and cell-cell junctions in transducing fluid shear stress into intracellular signals in ecs. by using microcontact printing of proteins, we design substrates with defined adhesive islands in order to control shapes of living cells. this confinement of ecs allows to study the organization and the contractile activity of the cytoskeleton in order to redistribute their intracellular forces in response to externally applied forces. we design microfluidic channels with sizes and geometries close to small blood vessels to apply a physiological range of shear stresses on ecs. our results indicate that cells deposited on a precisely defined adhesive area inside microchannels and subjected to shear stress reorganize their cytoskeleton, their focal adhesions and adherens junctions in response to blood flow. drugs interfering with the cytoskeleton are used to underline the role of its different components in the cellular adaptation to the mechanical environment. s. mahdavi 1 , b. ranjbar 1 , s. gharibzadeh 2 , m. toosi 2 , m. javan 1 1 tarbiat modares university, tehran, iran, 2 amirkabir university of technology, tehran, iran multiple sclerosis (ms) is the main known pathology of myelinating cells. an autoimmune reaction occurs against myelin sheets of neurons, so action potential (ap) propagation along the affected nerve fibers has been destroyed and it causes various disorders. here, we propose a novel strategy for ms symptoms treatment. we modeled neuron by orcad software and simulated the action potential propagation along the axon in normal condition. our model simulated normal neuronal behavior. then we destroyed the myelin sheet as it occurs in ms and observed destroyed ap propagation as it was reported in ms disease. we investigated the effect of changes in the voltage-gated sodium channel (vgsc) threshold on the efficiency of ap propagation. the results demonstrated that reduction of vgsc threshold improves the propagation of ap by increasing the amount of sodium flux during ap propagation. although, some researches have proposed vgsc blocker as ms symptom treatment, our result suggests that the increase of sodium current produced by reduction of vgsc threshold, improves ap propagation and probably cure some ms symptoms. so, we suggest that vgsc gating modifiers can be considered as novel strategy for ms treatment. surely, this results needs to be confirmed by experimental studies. a. gradogna, e. babini, a. picollo, m. pusch istituto di biofisica, consiglio nazionale delle ricerche, via de marini 6, 16149 genova, italy clc-ka and clc-kb are highly homologous cl − channels expressed in the kidney and the inner ear where they mediate transepithelial chloride transport. both channels heteromerize with the beta subunit barttin. mutations in clc-kb and barttin genes lead to bartter's syndrome. we analyzed the modulatory effect of extracellular ca 2+ and h + on clc-k channels using the xenopus oocyte expression system. clc-ka currents increased with increasing [ca 2+ ] ext without full saturation for [ca 2+ ] ext up to 50 mm. however, in the virtual absence of ca 2+ , clc-ka currents are about 18 % of currents measured in 10 mm [ca 2+ ] ext , demonstrating that ca 2+ is not strictly essential for opening. vice versa, clc-ka was blocked by increasing the [h] + ext with an almost complete block at ph 6. among various reaction models tested, the model that best fitted all state-steady data predicts an allosteric regulation of channel opening by separate binding sites for ca 2+ and h + . moreover, the best fit suggests that one ca 2+ and two h + bind to the channel. kinetic analysis of current responses upon [ca 2+ ] ext and ph jumps confirmed the allosteric character of modulation. in support of the presence of two separate binding sites we identified several mutations that selectively altered ca 2+ or h + sensitivity. our data represent a first step towards a molecular picture of ca 2+ and proton regulation of clc-k channels and suggest that it is of physiological relevance. the extracellular matrix molecule hyaluronic acid modulates l-type voltage-dependent ca2+ channels e. dvoretskova 1 , g. kochlamazashvili 2 , o. bukalo 2 , c. henneberger 3 , d. rusakov 3 , m. schachner 2 , a. dityatev 1 1 italian institute of technology, genova, italy, 2 centre for molecular neurobiology, hamburg, germany, 3 institute of neurology, university college london, london, uk we studied the effects of hyaluronic acid (ha), a major extracellular matrix molecule, on activity of l-vdccs in a heterologous expression system and in hippocampal slices. we recorded currents mediated by a major neuronal subtype of l-vdccs (ca v 1.2c, β 1 b, and α 2 δ 1 ) expressed in cho cells. a five-minute application of 0.1 mg/ml ha potentiated l-vdcc currents at -20, -10, 0 and +10 mv by approximately 50%. analysis of boltzmann curves showed that ha increased maximal conductance rather than other parameters (v 0.5 or k ). treatment with hyaluronidase removed endogenous ha in murine hippocampal slices and specifically impaired long-term potentiation (ltp) induced at ca3-ca1 synapses by repetitive theta-burst stimulation. blockade of l-vdccs reduced ltp in control slices to the levels seen after hyaluronidase treatment. a potentiation of l-vdccs with bay k 8644 fully restored ltp after hyaluronidase treatment. removal of ha reduced ca 2+ transients elicited by backpropagating action potentials in individual dendritic shafts and spines of ca1 pyramidal cells, whereas pretreatment with nifedipine fully occluded this effect. thus, ha potentiates postsynaptic l-vdccs and by this way influences use-dependent synaptic plasticity. whole-cell patch clamp recordings from a variety of human cancer cells showed that functional voltage-gated sodium channel (vgsc) expression occurred specifically with strongly metastatic cells. in addition, where studied, this was accompanied by down-regulation of outward (mainly potassium) currents. this has led to the celex ("cellular excitability") hypothesis of cancer according to which metastatic cell membranes are excitable and this promotes their hyperactivity. importantly, the vgsc genes expressed are embryonic splice variants, which are normally developmentally regulated, hence the phenomenon is 'oncofetal'. in breast cancer, where the predominant vgsc is nav1.5 the neonatal and adult forms are significantly different and this is reflected in channel activity whereby the neonatal vgsc has much slower inactivation kinetics. the double-charge change at position 211 is critical for this difference. the slow kinetics results in much greater influx of na + into cells and one consequence of this is activation of protein kinase a. this is a tonic effect and, under steady-state resting conditions, it results in a positive feedback effect promoting post-translational trafficking of vgsc protein to plasma membrane. the unique amino acid sequence of the spliced region has enabled the production of a polyclonal blocking antibody specific to neonatal nav1.5. it is concluded that vgscs represent novel biophysical targets for clinical management of metastatic disease. m. pusch cnr, istituto di biofisica, genoa, italy clc proteins form an evolutionary conserved gene-family that comprises 9 members in mammals. four of the human clcs are passive plasma membrane cl − ion channels. the other 5 clcs are expressed in intracellular organelles. clc-4 and clc-5, mutations of which lead to dent's disease, are secondary active cl − /h + antiporters, similar to the bacterial clc-ec1, and with identical 2 cl − : 1 h + stoichiometry. cl − and h + transport activity of the exchanger clcs depends on two glut residues. mutating a 'gating glutamate' (e211 in clc-5) converts the exchanger into anion conductances. neutralizing the 'proton glutamate' (e268), but not its replacement by some other titratable groups, abolishes cl − and h + transport. noise analysis indicated that clc-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 ps, indicating a very large transport turnover. no 3 − uncouples h + transport but mutating the highly conserved s168 to p, as found in the plant no 3 − / h + antiporter atclca, led to coupled no 3 − : h + exchange. clc proteins are a fascinating example of how a very similar protein architecture can be used to provide either a passive electrodiffusive permeation pathway or a strictly coupled secondary active ion transporter. (supported by telethon italy -grant ggp08064). the role of ion dynamics in zebrafish fin regeneration the specific and directional ion transport across cell membranes or tissue layers results in differential accumulation of ions and endogenous electric currents. these phenomena have been shown to be important for vertebrate organs regeneration. however, the specific ion nature of such electric currents remains unknown, as well as the role of cellular ion dynamics during regeneration and the molecular signalling pathway that transduces electric cues into cellular responses. we use zebrafish caudal fin as an adult regeneration model to unveil the specific ion composition of the currents associated with wound healing and regeneration, using a non-invasive ion-specific scanning microprobe setup. our data suggests a role for potassium (k + ), calcium (ca 2+ ) and protons (h + ) at different stages of the regeneration process. k + and ca 2+ extracellular effluxes have both been detected during the wound healing stage. h + efflux is triggered during wound healing and is maintained throughout regeneration. we are validating these data with genetic and pharmacological approaches, as well as advanced ion imaging. overall, our results suggest ion-driven mechanisms underlie adult tissue regeneration and its comprehension may open way for new therapeutic strategies, both in regenerative and developmental medicine and in cancer therapy. cardiac effects of anabolic steroids: an electrophysiological approach anabolic androgenic steroids (aas) have been used by athletes and non athletes for almost five decades in order to improve performance. however, the illicit abuse of high-doses of aas has been attributed as a main cause of several cardiovascular disorders such as arterial hypertension, lipid profile abnormalities, heart failure, hypertrophic cardiomyopathy, arrhythmia and sudden death. the aim of this study was to investigate qt interval and transient outward potassium current (i to ) changes in rats treated with nandrolone decanoate (deca). male wistar rats received weekly 10 mg/kg of deca (n=10) or vehicle (control, n=10). electrocardiogram was recorded weekly, and qt interval was measured. after 8 weeks hearts were excised and single myocytes were isolated from the ventricles of animals. i to was recorded by means of the whole cell patch clamp technique. qt interval was larger in deca group from 4 th to 8 th week (p <0.01). analysis of i to showed a decreased current density (p <0.01) in ventricular cardiomyocytes of deca group, compared to control group. in conclusion, our results show that alterations on ventricular repolarizaton may constitute an early consequence of the chronic administration of high doses of anabolic steroids in rats, and demonstrated qt prolongation and i to density reduction, which may constitute an important marker of arrhythmia vulnerability and sudden death. -ion channels in channelopathies and cancer denitrifying bacteria control no and no 2 cytosolic levels by regulating the expression of denitrification gene clusters via redox signalling of specific transcriptional factors that may act as no sensors in vivo. a protein belonging to the subclass dnr (dissimilative nitrate respiration regulator) from pseudomonas aeruginosa has been recently suggested to be a heme containing protein. very recently the three dimensional structure of the apo-form of dnr (in the absence of heme) has been determined by x-ray crystallography, whereas the holo-form (in the presence of heme) has not yet been crystallized. we have investigated the heme local structure in solution of ferric, ferrous, co bound and no bound holo-dnr by x-ray aborption spectroscopy (xas) and we added a kinetic study of the co bound form by means of a flash photolysis setup using uv-visible absorption as a spectroscopic probe. the combination of fe k-edge xanes fingerprints and kinetic study reveal a heme pocket able to bind exogenous ligands like no and co with increased plasticity, thus supporting its role as the cofactor involved in no sensing activity. molecular examination of motifs that lead to the formation of s-nitrosylated proteins i. alicea, e. r. schreiter university of puerto rico rio piedras, san juan, puerto rico physiologically, a wide range of proteins experience structural and functional modifications after the addition of a nitric oxide (no) moiety to cysteine thiol. this posttranslational modification, known as s-nitrosylation, regulates a large number of cellular processes like vasodilatation, cell signaling and others, and the products of s-nitrosylation can be involved during the development of different human diseases. however, little is known about the mechanism by which different proteins specifically bind the no moiety to their cysteine. here we show a bio-statistical analysis of some properties of cysteine that are s-nitrosylated in different proteins, including the pk, electrostatic environment, solvent accessibility of the target cysteine and identity of surrounding amino acids. we also chose model proteins (human thioredoxin and s100 protein) to make specific targeted amino acid substitutions around selected cysteines to alter the properties described above. the reactivity and stability of these mutant proteins towards s-nitrosylation will be examined. sampling the flexibility of ppar-γ s. aci-sèche 1 , n. garnier 1 , d. genest 1 , s. bourg 2 , c. marot 3 , l. morin-allory 3 , m. genest 1 1 upr cnrs 4301, orléans, france, 2 fr pcv cnrs 2708, orléans, france, 3 umr cnrs 6005, université d´orléans, france a promising approach to consider the flexibility of proteins in docking studies consists in performing multiple rigid docking on a representative set of the receptor conformations. molecular dynamic (md) simulation is one of the best adapted methods for structural sampling, but exploring the conformational diversity of a protein is computationally expensive. we present a protocol for generating a wide range of conformational states of a receptor using restrained md and a partitioning protocol to select a few representative conformations of the binding site from this md. a way to speed up efficiently md calculation is using an implicit model to represent the solute-solvent interactions. we explore a protocol using a distance-dependant permittivity function to represent solvent effect and an ensemble of controlled restraints applied on a subset of specific atoms in order to prevent artefactual structural distortions, but preserving receptor's flexibility. ten 100ns simulations have been performed using different sets of parameters and compared to a reference 30ns simulation with explicit solvent. to select a representative set of conformations, partitioning (k -means algorithm) was applied on the ensemble of simulated conformations. this methodology was applied to the ligand binding domain of peroxysome proliferator-activated receptor-γ. 4 department of biomedical sciences, university of antwerp, antwerp, belgium the complex system of cavities identified in human neuroglobin (ngb) has been postulated to be of functional significance to the putative no dioxygenase activity of the protein. the interconnected hydrophobic cavities may support this catalytic activity by acting as reservoir for reactants and providing preferential pathways assisting product removal from the active site. we thus decided to investigate co rebinding kinetics to ngb embedded in silica gels to expose ligand migration processes in the geminate phase. encapsulation of the co complexes of reduced neuroglobin, leads to a slight increase in geminate recombination after nanosecond laser photolysis. increasing the viscosity of the medium, by soaking the gels in glycerol, completely inhibits escape of the photodissociated ligand to the solvent, and highlights a complex, multiphasic kinetic pattern. this finding can be rationalized by assuming the existence of a discrete set of temporary docking sites, capable of trapping the photodissociated ligand for very long times, up to a few ms after photolysis. g. bartolommei, f. tadini-buoninsegni, m. r. moncelli bioelectrolab -department of chemistry, university of florence, italy ion pumps are integral membrane proteins devoted to ion transport through a lipid membrane phase. the ion pumps ca-atpase and na,k-atpase are prominent members of the p-type atpases family. due to fundamental physiological roles of these proteins, they are very promising drug targets. bioelectrolab has a wide expertise in the study of ion transport by these proteins [1] . our attention has been recently focused on the interaction of these enzymes with molecules of potential pharmacological interest. frequently, drugs exert an inhibitory action on the transport activity of an ion pump, usually confining it in an inactive conformation. molecules like thapsigargin and cyclopiazonic acid belong to high (nanom) affinity inhibitors of the ca-atpase, whereas clotrimazole and curcumin are medium (microm) affinity inhibitors of both ca-atpase and na,k-atpase. for each of these compounds a mechanism of action is proposed. moreover, recent results concerning ca-atpase inhibition by clotrimazole analogues will be shown: the relevance of this type of molecules is due to their potential employment as an alternative to traditional drugs against malaria parasite. financial support of ente cassa di risparmio di firenze and of miur (prin project) is gratefully acknowledged. [1] tadini-buoninsegni f., bartolommei g., moncelli m.r., fendler k. 2008. arch. biochem. biophys. 476:75-86 (review). s. asthana 1 , s. shukla 1 , g. giliberti 1 , f. luliano 1 , m. ceccarelli 2 , r. loddu 1 , p. ruggerone 2 , p. la colla 1 1 department of biomedical science and technology, università di cagliari, cagliari, italy, 2 department of physics, università di cagliari, cagliari, italy studies of protein-inhibitors interactions are helpful to elucidate the mode of action of ligands and thereby providing clues for rational drug design. bvdv, is an important target of drug discovery activities largely because it is essential for viral replication. in bvdv rdrp no specific nni binding site has been reported till now. experimental results have shown that different class of inhibitors (benzimidazole, imidazoquinolines and pyridoxyquinolines), have resistant mutations located in the finger domain of the rdrp. all the reported mutations are spatially very close to each other. thereby, indicating that binding sites of nni's may lie in the finger domain for these different class of inhibitors.herein, we have utilized docking procedure to investigate binding sites, binding modes as well as binding affinity of different class of inhibitors.we then used all atom molecular dynamics (md) simulations to investigate the stabilizing interaction between inhibitor-receptor pairs. our md results are in good agreement with experimental data and provide deep insights into the dynamical features of the high affinity inhibitorreceptor binding. thus identifying the binding modes of our inhibitors and mechanism leading to inactivity of the enzyme can help us to build a microscopically well-funded picture of the functioning of these enzymes. we present an investigation of the molecular basis of ligand binding and reactivity of heme proteins using computer simulation. a combination of classical molecular dynamics and hybrid quantum-classical (qm-mm) calculations are applied to explore distal and proximal effects on diatomic ligand binding to the heme. trends in binding energies and in the kinetic constants are illustrated through a number of selected examples. an investigation of the interplay between ligand migration and protein dynamics obtained through classical molecular dynamics techniques in combination with advanced sampling tools is also presented to yield information about free energy profiles and possible secondary docking sites. results for truncated n hemoglobin of mycobacterium tuberculosis, presented as an illustrative example, suggest that the truncated hemoglobin n has evolved a dual-path mechanism for selective/distinct migration of o 2 and no to the heme, to achieve efficient no detoxification. finally, we present also an analysis of the molecular basis of hexacoordination in human neuroglobin, which suggest that the flexibility of the cd plays a key role in determining the ligand binding properties. thermodynamic bases of nucleoplasmin-histone complexes recognition by the nuclear transport machinery i. arregi, j. falces, s. bañuelos, m. a. urbaneja, s. g. taneva unidad de biofísica (csic/upv-ehu), departamento de bioquímica y biología molecular, universidad del país vasco, spain the nuclear transport of the chromatin remodeling (nucleoplasmin) and chromatin building (histones) proteins is mediated by importins. nucleoplasmin (np) contains a classical bipartite nuclear localization signal (nls) that is recognized by importin α, while histones present multiple sequence elements (nls-like motifs) for nuclear targeting. besides, ternary importin/np/histone complexes might represent a putative coimport pathway for nuclear import of linker (h5), nucleosomal core (h2ah2b) histones and their chaperone protein np, enhancing the histone import efficiency. to better understand np and histone recognition by the transport machinery we studied the thermodynamics of complex formation of importin α (a truncated form) and importin β with histones and np, and with np/histone binary complexes by means of isothermal titration calorimetry. data show that importins interact with the two histone types and np, and that importin and histones can simultaneously bind to np. analysis of the binding energetics reveals an enthalpy driven formation of high affinity binary and ternary complexes. we demonstrate that different amount of importin molecules can be loaded on np/binary complexes dependent on the histone type, linker or core, and the amount of the bound histones. g. breuzard, i. di maïo, p. barbier, d. allegro, c. brault, v. peyrot cro2 inserm u911, ufr de pharmacie, marseille, france since a decade, our laboratory has already studied the interaction of tau variant with tubulin (tub) or microtubules (mts) and phosphorylation process on this interaction. indeed, fret assay was a powerful tool to achieve binding parameters between tau and tub in vitro: 1/ with mts stabilized by fluorescein-coupled taxol (flutax-2) as donor and rhodamine-labelled tau (rho-tau) as acceptor, and 2/ in living cells by confocal laser scanning microscopy (clsm) with tau/α-tub fused to egfp/mcherry, respectively. results revealed 47 ± 3 % energy transfer efficiency from flutax-2 to rho-tau and a donor-to-acceptor distance of 54 ± 1å. by titration, the dissociation constant of tau was determined to 1.0 ± 0.5 µm. a cleavage procedure of αβ-tub was performed to determine the influence of the c-term tails of αβ-tub on the tau-mt interaction. no difference in distances and binding parameters was observed. clsm images displayed a heightened concentration of fluorescent tau in patches along mts. fret experiments revealed in particular higher efficiencies between gfp-tau to mcherry-α tub proteins in these locations. overall, our results suggested no involvement of the hypervariable and highly acidic c-term tails of tub in mt/tau binding. a molecular model is proposed in which flutax-2 is directly accessible to tau molecules. besides, the modified distribution of fluorescent tau could be implicated in local change of mechanical properties of mts. a. boreham 1 , k. winkler 1 , c. gebhard 2 , k. rueck-braun 2 , p. henklein 3 , e. michalsky 3 , r. preissner 3 , r. misselwitz 3 , a. ziegler 3 , u. alexiev 1 1 freie universität berlin, berlin, germany, 2 technische universität berlin, berlin, germany, 3 charité-universitätsmedizin berlin, berlin, germany peptide presentation by major histocompatibility complex (mhc) molecules is crucial for immune responses. photocontrol of peptide dynamics by means of photo-switchable peptide analogs will provide insights in peptide dynamics and its dependence on mhc polymorphism (1) (2) (3) . we have designed a hemithioindigo (mhti) photo-switch bearing peptide using the viral epitope rrrwrrltv (plmp2) as a template. incorporation of mhti in the peptide backbone should result only in a minor change of the overall peptide structure given the relaxed conformation of the zisomer. indeed, the human mhc molecule hla-b*2705 can tolerate nonpeptidic elements in plmp2, as evidenced by normal peptide binding. using the autofluorescent properties of mhti we determined the stability of the hla-b*2705/mhti-plmp2 complex. photoswitching from z →e results in a decrease of hla-complex stability. based on computer modeling this decrease is due to a reduced interaction of the peptide c-terminus with hla-b*2705. truncated hemoglobins (trhbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. three phylogenetic groups (n, o, and p) have been identified in trhbs. the crystal structure of truncated hemoglobin o of b.subtilis, does not show an evident tunnel/cavity system connecting the protein active site with the solvent, a fact that cannot be easily rationalized considering the very high oxygen association rate. moreover, resonant raman results of the co bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. for these reasons we performed classical molecular dynamics simulations of the oxy, carboxy and deoxy protein, and computed the free energy profiles associated with ligand migration to the active site. our results suggest that there is a key residue, glne11, that may present an alternate conformation in which a wide ligand migration tunnel is formed, consistently with the kinetic data. the results for the co and o 2 bound protein show also that glne11 is directly involved in the stabilization of the coordinated ligand, playing a similar role as tyrb10, and trpg8 in other trhbs. our results not only reconcile the structural data with the kinetic information, but also provide additional insight about the general behaviour of trhbs. patterned functionalization of surfaces for guided transport on molecular motors tracks m. bhagawati 1 , s. ghosh 2 , t. surrey 1 , j. piehler 2 1 department of biophysics, university of osnabrueck, osnabrueck, germany, 2 european molecular biology laboratory, cell biology and biophysics unit, heidelberg, germany chelator head groups with multiple nitrilotriacetic acid (nta) moieties have been very well characterized and successfully utilized as high affinity adapters for functional immobilization of oligohistidine tagged proteins to surfaces. we have recently established a generic method for patterning nta functionalized surfaces by selective photodestruction via a light induced fenton reaction. efficiency of different transition metal ions for catalyzing this reaction was tested. functionality of the patterned protein was confirmed using the interaction between interferonα2 and its receptor. implementation of this technique in a confocal laser scanning microscope allowed us to control surface density of binding sites, providing the possibility to vary the surface concentration of immobilized proteins in a spatially resolved manner. we also applied this approach for exploring guided transport of microtubules by kinesin selectively immobilized onto trisnta patterns. rheumatoid arthritis (ra) is an autoimmune disorder, leading to pathological damage at the level of joints, associated with the hla class ii allele hla-dr4. although etiology of ra is unknown, type ii collagen (cii) is a potential antigen candidate and it is believed that t cell responses in collagendependent ra are directed towards the immunodominant pathogenic epitope cii(261-273). despite recent advances in characterization of class ii major histocompatibility complex (mhc) and t-cell receptor (tcr) contacts in this epitope, the atomic details of tcr-cii(261-273)-mhc complex are not known. here, homology modeling and molecular docking studies have been used to derive a three-dimensional model of tcr β chains, obtained from a dr4+ subject, in complex with cii(261-273)/hla-dr4. the best complex from docking was further refined using molecular dynamics simulations for 20 ns. the proposed model represents a reasonable structural basis for understanding cii(261-273)-mhcii complex recognition by tcr and for rational design of inhibitors targeting tcr-pmhc interface. probing bio-molecular bonds with magnetic force for biosensor applications a. m. de jong 1 , a. jacob 1 , x. j. janssen 1 , j. m. van noorloos 1 , l. j. van ijzendoorn 1 , m. w. prins 2 1 eindhoven university of technology, eindhoven, the netherlands, 2 philips research laboratories, eindhoven, the netherlands we investigate new technologies to be applied in next generation biosensors, which not only measure biomarker concentrations but also probe bio-molecular interactions. the concept is based on the response of ligand-receptor pairs to an applied force or torque [1, 2] . we use functionalized magnetic beads and magnetic fields to apply translational and rotational forces on the molecular bonds. in a model experiment, polystyrene surfaces were coated with anti-biotin and beads were coated with biotin. after incubation, a constant magnetic force was applied to the beads and the number of bound beads was measured as a function of time. this was repeated for a range of forces. the dissociation rate (k o f f ) is determined for each force and k o f f at zero force is extracted from these data. rotational forces were exerted on protein-g coated beads bound to igg antibodies immobilized on a surface. rotating fields revealed an oscillating behavior, which can be understood from the balance between the applied magnetic torque and the torque due to the deformation of the biological bond. studying interactions between cop1 regulatory protein and hy5 transcription factor d. s. dalafave the college of new jersey, ewing, nj 18940, usa this work addresses important questions of protein-ligand interactions and selective protein recognition. proteinligand bindings are crucial for many cellular processes. dependable methods for predicting binding sites would lead to a better understanding of proteins' selective recognition and would, in turn, help research on fighting diseases. presented here is a study of interactions between the wd domain found in a regulatory protein cop1 and the motif v-p-e/d-φ-g (φ=hydrophobic residue) found in a transcription factor hy5. cop1 and hy5 proteins have opposing roles in developmental regulation. cop1 can repress hy5 by directly binding with it. the repression involves specific interactions between the wd domain and the motif v-p-e/d-φ-g. previous experimental research showed that mutations in the motif's v-p pair resulted in a large decrease in hy5 repression. to study effects of similar mutations, residues in the motif v-p-e/d-φ-g were systematically substituted with other residues. interactions between the mutated motif and the wd domain were studied. distributions of binding sites, bond lengths, local shape complementarity, and interaction potentials were modeled for each residue substitution. the study identified binding sites critical for the cop1-hy5 binding. some hy5 residues in the vicinity of the motif were also found to be important in the binding. the significance of the results for understanding selective protein recognition is discussed. determination of protein-ligand binding thermodynamics by thermal shift assay p. cimmperman, a. zubriene, l. baranauskiene, e. kazlauskas, j. matuliene, d. matulis laboratory of biothermodynamics and drug design, institute of biotechnology, vilnius, lithuania thermal shift assay determines the effect of ligand binding on the protein thermal denaturation equilibrium. several models have been derived to describe the general cases of protein-ligand binding. first, the model describing protein stabilization and destabilization by ligand binding to the native and unfolded states. second, the split of protein melting transitions by tightly binding ligands is presented when the transition of free protein and ligand-bound protein occur separately. mathematical equations describing the protein unfolding and ligand binding thermodynamic parameters at various temperatures are presented. the protein melting temperature shift can be determined by various techniques such as differential scanning calorimetry, circular dichroism, and intrinsic or extrinsic fluorescence. the advantages of fluorescent techniques are presented. the melting temperature shift caused by ligand binding is dependent on the thermodynamic parameters of protein unfolding and ligand binding, including enthalpy, entropy, and heat capacity, thus allowing determination of binding thermodynamics. application of the models in the design of hsp90 chaperone and carbonic anhydrase inhibitors is discussed. comparison with isothermal titration calorimetry data is presented. recent reports have shown that the bacterial redox protein azurin can enter into cancer cells and induce apoptosis by stabilizing p53. the formation of a complex between the two proteins has been demonstrated, but little is known about binding features. for the first time, we show here that azurin binds to the n-terminal region of p53 with a dissociation constant in the 5-10 µm range. trp phosphorescence lifetime measurements revealed conformational changes of azurin induced by the interaction with p53(1-63). acrylamide quenching of trp phosphorescence also indicated a significant increase of the overall flexibility of azurin upon binding to p53 . no change of the fluorescence emission of p53(1-63) was detected in the presence of azurin. the latter finding suggests that w23 of p53 is not directly involved in domain binding to azurin, indicating that the binding site is distinct from that of mdm2. the present results may assist the design of novel cancer treatments based on p53 stabilization by azurin. a. eleta 1 , r. georgieva 2 , h. bäumler 2 , j. l. toca-herrera 1 1 cic biomagune, 20009 donostia-san sebastián, spain, 2 charité-universitätsmedizin berlin, berlin, germany human serum albumin (hsa) is the most abundant nonglycosilated plasma protein in the human body. this multifunctional protein has ligand-binding and transport properties, antioxidant functions and enzymatic activity [1] . bilirubin interacts with albumin in order to be transported from the blood to the liver where it is secreted. the interaction occurs specifically in hsa i-domain of its three domains [2] . in our work, we investigate the interaction between hsa and bilirubin by quartz crystal microbalance with dissipation (qcm-d) and atomic force microscopy (afm) [1, 3] . albumin was adsorbed on negatively charged silicon oxide. however, hsa was removed after rinsing with pbs. hsa adsorbed on positively charged polyelectrolyte multilayers leads to a stable layer of surface mass density of 272 ng/cm 2 . cross linked albumin with glutaraldehyde after its adsorption on nh 2 -terminated thiols is also stable reaching surface mass density of 296 ng/cm 2 . a preliminary bilirubin adsorption results on cross linked hsa substrate show that 200 ng/cm 2 is immobilized. taking into account that hsa-bilirubin stoichiometry is 1:1, the outcome demonstrates that the 70% of hsa i-domains remain active. antimicrobial peptides (amps) are short positively charged polypeptides. they are important due to their potential to provide an alternative to conventional therapy against bacterial infections. rbpi 21 is a 21 kda peptide based on the nterminal region of the neutrophil bactericidal/permeabilityincreasing protein (bpi). it was shown that this amp possesses bactericidal effects on gram-negative bacteria and higher affinity for lipopolysaccharide (lps), neutralizing its effect. the peptide use against meningitis, is in phase iii clinical trials. here, we demonstrate that rbpi 21 promotes aggregation of negatively charged large unilamellar vesicles (luv) and lps aggregates, by dynamic light scattering, while for zwitterionic phosphatidylcholine (popc) luv the size remains unchanged. the aggregation increases with peptide concentration until peptide promotes massive aggregation followed by sample flocculation/precipitation. with the rbpi 21 -lipid interaction there is a progressive change in the zeta-potential of the luv systems and lps aggregates. luv systems composed of phosphatidylglycerol (popg) and popc:popg mixtures have higher zeta-potential variations than popc luv. for lps aggregates, rbpi 21 neutralizes the surface charge and at higher peptide concentrations overcompensates it. results demonstrate that the interaction of the peptide rbpi 21 with lps aggregates and luv systems has electrostatic and hydrophobic contributions. serratia marcescens heme acquisition system: heme transport and protein:protein interactions m. delepierre unité de rmn des biomolécules cnrs ura 2185, institut pasteur, paris, france heme transport systems in bacteria are required and might be potential target for antibacterial drugs. the heme acquisition system, has, exists in pathogenic as well as in opportunistic bacteria but only for the latter one extensive studies have been conducted, constituting as such a model system. the outer membrane receptor hasr, the central component of this system, functions in synergy with a secreted high affinity heme binding protein, the hemophore hasa. hasa extracts heme from host hemoproteins and returns it to hasr. then, the energy given by a protein complex of the inner membrane is used to allow heme entrance across the bacterial membrane and to eject the empty hemophore from the receptor. reconstitution of this heme acquisition system in e coli, overexpression and purification of its various components have allowed us to obtain sufficient amount of protein to perform nmr and biophysical studies to analyse at the molecular level the different steps of heme acquisition by hasr. protein-protein interactions (ppi) are the central pillar supporting most of biological functional activity on the molecular level. a binding event between two proteins typically consists of two stages: 1) diffusional search of the binding partners for each other, and 2) specific recognition of the compatible binding surfaces followed by the formation of the complex. we focus here on the non-specific component of ppi, which refers to all physico-chemical properties of the binding partners (such as size, charge, isoelectric point, hydrophilicity etc.) that are independent of the exact details of their binding sites, but which could in turn affect their localization or diffusional search for one another. it is known that proteins co-localize due to segregation into different cellular compartments, sequestration via anchor and scaffold proteins or even chemical modifications. we suggest that the non-specific component of ppi determines in part the co-localization and clustering of the binding partners, which then directly in a non-specific fashion influences their interactions. we examine the possibility that such signature might be encoded within the experimental 3d structures of a large set of known mutually interacting proteins. we provide preliminary evidence that this indeed may be the case, and corroborate our findings by using different statistical tests to compare those features of the known interacting partners, and ascertain correlations and commonalities between them. a link between hinge-bending domain motions and the temperature dependence of catalysis in ipmdh i. hajdú, a. szilágyi, j. kardos, p. závodszky institute of enzymology, hung acad sci, budapest, hungary enzyme function depends on specific conformational motions. since conformational flexibility strongly depends on temperature, temperature dependent enzyme kinetic studies with measurements related to dynamics can give us some insight at atomic level into these functionally relevant motions. the catalytic efficiency (k cat /k m ) of 3-isopropylmalate dehydrogenase for its substrate (ipm) has unusual temperature dependence, showing a local minimum at 35 • c. in search of an explanation, we measured the individual constants k cat and k m,ipm as a function of temperature, and found that the van't hoff plot of k m,ipm shows a sigmoid-like transition in the 20-40 • c temperature range. by means of various measurements including h-d exchange and fret, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperature above 30 • c. the thermodynamic parameters of ligand binding determined by itc as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. because the binding of ipm is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with ipm binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency. a simulation approach to multiple sclerosis: study of a peptide with a pharmaceutical potential c. guardiani 1 , s. marsili 2 , p. procacci 2 , r. livi 3 1 centro dinamiche complesse, università di firenze, italy, 2 dipartimento di chimica, università di firenze, italy, 3 dipartimento di fisica, università di firenze, italy multiple sclerosis (ms) is an autoimmune disease of the central nervous system, leading to premature death. one of the potential targets of the autoimmune reaction is the myelin protein mog that has been crystallized in complex with the 8-18c5 ms-autoantibody. the analysis of contacts and buried surface area combined with an alanine scanning computation reveals the key role of mog fragment 101-108 for the interaction with 8-18c5. a docking simulation shows that the 101-108 fragment, excised from mog, and kept in crystal-like conformation, is still capable of fitting into the binding pocket of the antibody. we then studied, through replica exchange molecular dynamics simulations, the structural equilibrium distribution of the free peptide and of a number of analogs stabilized by a disulfide bond. we found that the free peptide yields a significant fraction of crystal-like conformations and the proportion of native-like structures is further increased by the disulfide bridge. when we tried to dock the centroids of the most populated clusters to 8-18c5, we discovered the existence of a docking funnel whose bottom is populated by stable complexes where the peptide occupies the same spatial region as in the crystal. we therefore conclude that the mog 101-108 fragment may be used to develop a diagnostic assay or a drug for ms. the escherichia coli membrane insertase yidc reversibly binds its substrate pf3 coat protein u. gerken, s. winterfeld, a. kuhn institute of microbiology and molecular biology, university of hohenheim, germany the membrane insertase yidc of e. coli belongs to the oxa1 family of mitochondria and plays an essential role in facilitating the insertion and assembly of membrane proteins. we have previously shown with detergent-solubilized (c 12 pc) yidc, labelled with ans, and pf3 coat that the initial step of the membrane insertion process, the binding of the substrate pf3 coat to yidc, is reversible [biochemistry 47, 6052-6058 (2008) ]. the dissociation constant k d for that particular system is about 1 µm. in order to obtain data for the native system we used in this study membrane-reconstituted (dopc and dope/dopg) yidc. the effect of the initial binding was examined in vitro by fluorescence quenching of the 11 tryptophan (trp) residues of yidc which are highly sensitive fluorescent probes for changes of the tertiary structure. quenching of the trp fluorescence after titration with a trp-free pf3 mutant indicates a change in the yidcs tertiary structure upon binding to its substrate. the binding data show a k d value in the range of 0.5-1.8 µm. the influence of different environments (lipid membranes, ddm micelles) on the secondary structure of yidc as well as on the yidc large periplasmic domain p1 was investigated by circular dichroism (cd). the cd data show that the secondary structure of yidc changes upon reconstitution into a membranes when compared to the detergent solubilized state. particularly, the p1 domain is considerably affected by the detergent c 12 pc. b. karasulu, b. erman, o. keskin koc university, istanbul, turkey histone proteins are fundamental to the cells since they are involved in cell regulatory processes, such as chromatin regulation, gene silencing and transcription, cell cycle control, and epigenetics, which are controlled via post-transcriptional modifications of the histone protein tails. these modifications are categorized under four main groups: methylation, acetylation, ubiquitination, and phosphorylation. among them methylation has been very recently proven to be reversible with the discovery of histone demethylase proteins and this modification type has been extensively studied, because the abnormal methylation rates cause the excess proliferation of the cell, which, in turn, triggers the cancer. therefore, understanding of the details of reaction mechanisms of histone methylation/demethylation dynamics provide the required knowledge basis for preventing the abnormal methylation rates by designing proper inhibitor (drug) molecules. in this study, we display possible reaction mechanisms (such as amine oxidation via lsd1) for the demethylation of specific histone tail proteins. we try to explain the role/importance of residues that take place in or near to the reaction pocket for the demethylation reaction. we also carry out md simulations and reaction path (free energy profile) analysis using free energy perturbation (fep) method for qm/mm hybrid systems in order to compare different possible reaction pathways. the sonic hedgehog (shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors patched (ptc) and smoothened (smo) , which lead to birth defects, cancer or neurodegenerative diseases. however, little is known about ptc, the receptor of the shh protein, and the way ptc regulates smo, the receptor responsible for the transduction of the signal. to develop structure-function studies of these receptors, we expressed human ptc (hptc) in the yeast saccharomyces cerevisiae. we demonstrated that hptc expressed in a yeast membrane fraction is able to interact with its purified ligand shh, indicating that hptc is produced in yeast in its native conformational state. using surface plasmon resonance technology, we showed that fluorinated surfactants preserve the ability of hptc to interact with its ligand after purification. this is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor ptc. this work will allow the scale-up of hptc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by shh signalling dysfunction. dissecting the colicin translocon c. l. johnson 1 , a. solovyova 1 , p. callow 2 , s. a. holt 3 , l. a. clifton 3 , k. weiss 4 , j. h. lakey 1 1 inst. for cell and molecular biosciences, newcastle univ., uk, 2 inst. laue langevin, grenoble, france, 3 isis, rutherford appleton lab., didcot, uk, 4 oak ridge national lab., centre for structural molecular biology, oak ridge, usa pore-forming colicin n hijacks e. coli outer membrane protein ompf and exploits it as both a receptor and translocator to cross the outer membrane [1] . it is currently a matter of debate if the translocation route is through the ompf lumen or the interface between ompf and the lipid bilayer. recent electron microscopy data from our laboratory suggests the latter route for translocation [2] . the colicin n/ompf complex in detergent has been studied by sans to examine the translocation pathway undertaken by colicin n. by using a combination of deuterated ompf and hydrogenated colicin we have been able to derive a low resolution structure of individual proteins in the binary complex. low resolution structural studies supplemented by targeted mutagenesis and screening techniques including itc, potassium efflux assays and auc have allowed us further our understanding of colicin n translocation. dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly49a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined. here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. effect of osmolytes on the dhfr activity, structure and dynamics b. legrand 1 , s. renaud 2 , m. collen 1 , c. tascon 3 , s. bonnassie 2 , e. gautier 1 , j. mellet 1 , c. blanco 2 , e. le rumeur 1 , j.-f. hubert 1 , a. bondon 1 1 rmn-ilp, 2 duals, 3 cbp, umr cnrs 6026, univ. de rennes1, france osmolytes are small molecules accumulated by a wide variety of organisms in response to hyperosmotic stress. they contribute to save the cellular integrity and to stabilize the macromolecules from environmental stress. dihydrofolate reductase activity is inhibited by several osmolytes. we studied the impact of osmolytes on the dhfr structure and dynamics by various techniques. we observe that substrate (dhf) and cofactor (nadph) diffusions are quite different in glycerol and betaine despite similar viscosities. we demonstrate that the overall structure is maintained at high osmolyte concentrations while no direct interactions can be detected with the enzyme. the k o f f of substrate analogues decreases with increasing the osmolyte concentrations. the enzyme dynamics, in various media, has been compared with the dhfr behaviour in water described in the literature. the osmolyte impact appears only partly conditioned by its viscogenic properties which reduce the molecules diffusion and the k o f f of the product controlled by the m20 loop of the dhfr. we suggest that the osmolytes decrease m20 loop mobility. comparing the results obtained with different osmolytes, we offer a better understanding of the osmolyte nature dependence of the dhfr inhibition. estrogen receptor (er) is a well characterized member of the nuclear receptor superfamily that modulates the expression of estrogen-responsive target genes in response to estradiol and other natural and synthetic chemicals mimicking the estradiol structure. in human, two ers, erα and erβ, lied on two distinct chromosomes, are known. er exhibits several functional domains: two conserved domains, a short domain c involved in dna-binding and a large domain e/f responsible for ligand-binding and hormone-dependent transcription activation, are linked by a hinge domain d; a poorly conserved a/b domain, at the n terminus, mediating interactions with the general transcription machinery, is involved in hormone-independent transcription activation. upon estrogen binding, ers can specifically bind to a dna fragment, called estrogen response element ere, and activate the transcription. the optimal ere sequence consists of two six base-pair half-sites, aggtca, organized as inverted repeats with a three base-pair spacing. in this study, we have investigated, by fluorescence methods, the effect of kcl concentrations, on the protein conformational flexibility and the thermodynamic stability of hers -eres complexes. we show, here, that electrostatic interactions, inside hers, contribute to its conformational flexibility and its thermal stability. moreover, the specific interaction between hers and eres is poorly sensitive to changes in ionic strength, in opposite to unspecific complexes. kinetic and structural explanation for the low enantioselectivity of human 3-phosphoglycerate kinase p. lallemand 1 , j. rouhana 1 , l. chaloin 1 , b. roy 2 , s. arold 3 , t. barman 1 , c. lionne 1 1 cpbs umr 5236, 4 bd henri iv cs69033, 34965 montpellier cedex 2, france, 2 ibmm umr 5247, 3 cbs umr 5048 l-nucleosides comprise a new class of antiviral and anticancer agents that are converted to pharmacologically active nucleoside triphosphates in vivo. the last step of the cascade may be catalyzed by 3-phosphoglycerate kinase (pgk), an enzyme that has low specificity for nucleoside diphosphate: ndp + 1,3-bisphosphoglycerate ↔ ntp + 3-phosphoglycerate. here we compare the kinetics of formation of the complexes of human pgk with different d-and their mirror images, l-nucleoside diphosphates, and the effect of 3-phosphoglycerate thereon. two types of experiment were carried out: equilibrium experiments allow the estimation of dissociation constants, and stopped-flow experiments the transient kinetics of the interactions. in addition, by the rapid-quench-flow technique, we compare the kinetics of the phospho-transfer and product release steps with each d-or l-nucleotide. crystallographic and molecular modelling studies allow defining the structural reasons for the low enantioselectivity of pgk. the aim of this basic work on the mechanism of action of human pgk with non-natural nucleotides is to obtain information for the optimization of therapeutic nucleoside analogues that are poorly phosphorylated by pgk. anrs is gratefully acknowledged for financial support. a new itc assay for measuring high-and lowaffinity protein-ligand interactions g. krainer 1 , j. fanghänel 2 , s. keller 1 1 leibniz institute of molecular pharmacology (fmp), berlin, germany, 2 bayer schering pharma ag, berlin, germany isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions [1] . however, reliable determination of the dissociation constant (kd) is typically limited to the range 100 µm > kd >1 nm. nevertheless, interactions characterised by a higher or lower kd can be assessed indirectly, provided that a suitable competitive ligand is available whose kd falls within the directly accessible window [2] . unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of high-and low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-affinity (kd <1 nm) and low-affinity (kd >100 µm) protein-ligand interactions. a molecular dynamics study of the cftr nucleotide binding domains interaction v. martorana 1 , r. noto 1 , o. moran 2 1 cnr-istituto di biofisica, palermo, italy, 2 cnr-istituto di biofisica, genova, italy the cystic fibrosis transmembrane conductance regulator (cftr), the dysfunctional protein in cystic fibrosis, contains two transmembrane domains, two nucleotide-binding domains (nbds), and a regulatory domain. opening of the pore have been linked to the atp-driven tight dimerisation of nbd. we have studied the nbd1-nbd2 interactions on wild type and cystic fibrosis-related mutations by steered molecular dynamics simulations (smd). a fully solvated dimer, including the two bound atps, was separated by pulling one monomer with an external, increasing force. interestingly, the force needed to break the mutated (g511d) dimer is significantly smaller than in the wild type case. the effect of a cftr potentiator, the genistein, has also been tested by repeating the smd simulations with the small molecule docked at the interface between the two nbd domains. to test the validity of our results we have repeated the separation process for different simulation lengths and force strengths. the amount of distortion on the pulled nbd domain has also been studied. this work is partially supported by the italian cystic fibrosis foundation (prog ffc #2/2008), with the contribution of "mille bambini a via margutta"onlus"blunotte", "lega italiana fc toscana" stability and aggregation studies of human septin 3 (sept3) j. n. a. macêdo, r. c. garratt, a. p. u. araújo instituto de física de são carlos, usp, são carlos, brazil the septins are a conserved family of nucleotide binding proteins firstly identified in saccharomyces cerevisae as proteins required for the completion of the cell cycle. septins are implicated in several cellular functions such as cytokinesis, vesicle trafficking, exocytosis, cytoeskeletal dynamics, cell polarity and sperm motility. furthermore, they are associated with alzheimer's and parkinson's disease. sept3 was identified in rat brain being highly enriched in presynaptic nerve terminals. it colocalizes with synaptophysin and dynamin i and is associated with synaptic vesicle. in this work, human sept3 without its n-terminal domain (sept3gc) was expressed in e. coli and purified by affinity and size exclusion cromatographies. structural stability studies were performed with recombinant sept3gc using circular dichroism spectroscopy (cd), right-angle light scattering, and fluorescence spectroscopy. the sept3gc cd spectrum showed a conformational transition from a predominantly α-helical starting structure to one dominated by β-sheet, just above physiological temperatures. the formation of irreversible aggregates, detected by light scattering, and their ability to bind thioflavin-t suggested that sept3 forms amyloidlike structures, as has been previously observed for human septins 2 and 4. our data suggest that amyloid formation by isolated septins in vitro may be a general phenomenon whose physiological relevance needs to be further investigated. pln149 is an antimicrobial peptide produced from lactobacillus plantarum nric149. analog pln149 (pln149a) and a ser-derived (pln149s, tyr to ser replacement) were synthesized on solid phase and their interactions with biomembrane model systems and inhibitory property on s.aureus and p.aeruginosa were investigated by cd, leakage assays, fluorescence spectroscopy, and differential scanning calorimetry. both peptides share the same unordered structure-like cd spectrum in aqueous solution, but a helicoidal induction in the presence of negative vesicles were observed, however pln149s showed lower helical content than pln149a. the ser-derived peptide presented 30% decrease of its leakage activity in different liposome compositions, a threefold increase in the dissociation constant from the liposomes than pln149a, and close to 40% reduction for the inhibitory activity against p. aeruginosa growth. we can conclude that besides the electrostatic contacts between the amphipathic &alpha-helix, formed by the cationic residues from pln149 and negatively charged phospholipids, the pln149-membrane binding is a process driven for the hydrophobic interactions from non-polar residues. in this case, there was a significant contribution of the tyr residue, which must be allocated in a lipid interface, described as the preferential position to this residue in membrane proteins. supported: fapesp label free detection using deep-uv laser-based fluorescence lifetime imaging microscopy q. li, s. seeger physikalisch-chemisches institut, universität zürich, winterthurerstrasse 190, ch-8057 zürich, switzerland label free detection based on native fluorescence excited at uv region shows great potential for the life sciences. it offers simple, low-cost and fast method for sensitive detection of important biological analytes without modification. in this contribution we present a deep uv fluorescence lifetime imaging microscopy system (duv-flim) based on a picosecond deep uv laser using time-correlated single-photon counting method. the described setup is well-suited for biological applications for ultrasensitive detection. we have showed single uv dye (bmq) and single protein (ecβ gal ) molecules detection using duv-flim. further, the label free detection of protein interaction between ecβ gal and monoclonal anti-ecβ gal has been demonstrated by means of steady-state and time-resolved fluorescence spectroscopy. we achieved detection sensitivity for the ecβ gal/ anti-ecβ gal pair down to the nanomolar concentration range. we also extended this method to study the interaction of therapeutic drug porphyrin with bsa protein. fluorescence resonance energy transfer between protein and alexa fluor 350 has been investigated using duv-flim. the intrinsic fluorescence and fluorescence lifetime changes of donor biotin β-galactosidase have been measured. energy transfer efficiency and donor acceptor distance have been obtained. fluorescence images of acceptor af 350 due to fret have been observed when excited at 266 nm. the monomeric heme-containing indoleamine 2,3dioxygenase (ido) catalyses the oxidative cleavage of the indole moiety of l-tryptophan (l-trp). enhanced l-trp degradation contributes to various physiological disorders including depression or failure of the immuno-regulating system. the regulation of ido by inhibitors is extensively studied. however, the catalytic mechanism of ido on the molecular level is still unknown. in addition to l-trp, a wide range of substrates are degraded including d-tryptophan, melatonine or tryptamine. in contrast, indole or histidine do not function as substrates for ido. to understand the determinants for substrate specificity, we investigate the interaction of the heme iron, the heme-bound ligand and the substrate. we use (time-resolved) uv/visible and fourier transform infrared (ftir) spectroscopy over a wide temperature range (4 -300 k) to monitor the binding of diatomic ligands to the heme iron and the binding of different substrates to coligated ido. changes in the spectra upon addition of l-trp are analyzed and compared to those induced by other substrates or inhibitors. archaeal protoglobin 3d-structure: novel ligand diffusion paths and heme-reactivity modulation protoglobin (pgb) from methanosarcina acetivorans c2a, a strictly anaerobic methanogenic archaea, is the latest entry in the hemoglobin superfamily. our previous crystallographic studies on pgb have shown that protoglobinspecific loops and a n-terminal extension completely bury the heme within the protein matrix (1) . access of o 2 , co, and no to the heme is granted by protoglobin-specific apolar tunnels reaching the heme distal site from locations at the b/g and b/e helix interfaces. here we report structural and kinetic data on pgb mutants engineered to probe the protein structural and kinetic properties. six crystal structures (pgb mutants: ∆20 (missing 20 nter residues), y(b10)61→a, y(b10)61→w, f(b12)63→w, f(g7)145→w, i(g11)149→f) show that the mutations engineered essentially restrict access to ligand tunnel 1. an accurate molecular level description of the signaling mechanism in ca 2+ transducers necessitates the knowledge of the kinetics and energetics of conformational changes associated with ca 2+ binding to various calcium binding proteins. with this in mind, we have developed an approach that combines the laser-induced photolysis of photolabile "caged" ca 2+ compound, dm-nitrophen, with the photothermal beam deflection (pbd) technique to determine thermodynamic profiles associated with the ligand binding to calcium chelator, edta, and ca 2+ sensor, calmodulin. this approach allows us to monitor time profiles of volume and enthalpy changes on the microsecond to millisecond timescale. the initial pbd study of ca 2+ photo-relase from ca 2+ loaded dm-nitrophen reveals the presence of two phases. the first step takes place within first 20 µs upon and is associated with a volume decrease of -7 ml mol −1 and enthalpy change of 66 kcal mol −1 . on the longer timescale (τ = 200 µs), the second event with a positive volume change of 7 ml mol −1 and enthalpy change of 8 kcal mol −1 was detected. on the other hand, ca 2+ photorelease in the presence of calmodulin is accompanied with an additional phase with a distinct lifetime and volume and enthalpy changes that reflects the metal binding to calmodulin and concomitant structural changes. antimicrobial peptides: linking partition, activity and high membrane-bound concentrations antimicrobial peptides (amps) have been intensively studied at micro and macroscopic levels for over twenty years. knowledge from these two domains has, however, contributed little to a comprehensive understanding of amp action; rather, in vivo amp performance has been only remotely correlated to biophysical properties. we focus on the assessment of peptide accumulation on bacterial membranes as an example of this separation: amp-bilayer interactions have been subject to extensive biophysical characterization, but conversion of that information into educated estimates of in vivo membrane-bound amp concentrations is lacking. this has led to the overlooking of important factors for activity. using simple partition models we were able to analyze available information on amp activity and interaction with membranes to show that unexpectedly high membranebound peptide concentrations are likely in vivo and may, in some cases, be required for triggering bacterial death. e. e. schäfer 1 , s. schuy 2 , a. janshoff 1 1 georg august-university göttingen, germany, 2 johannes-gutenberg-university mainz, germany retrovirus entry into cells occurs through fusion of the lipid bilayers that surround the virus and the lipid bilayer of the host cell. fusion proteins, present on the surface of the virus membrane play an essential role in the early stage of virus entry. understanding of the molecular mechanism is important for the design and function of modern fusion inhibitors. in this project we analyze the fusion of active conformation of the envelope gp41 from the human and simian immunodeficiency viruses (hiv and siv). during the infection process gp41 undergoes a sequence of conformational changes where the n -terminal nhr develop a trimer pre-hairpin intermediate. afterwards the three chr fold back towards the central nhr and a six helix bundle is formed. this rearrangement forces the viral and the host membrane into close contact and fusion pores may induce membrane fusion. this decisive molecular step in retroviral fusion has been modeled by rational design of lipopeptide assemblies that mimic a coiled-coil structure serving as a receptor for potential antagonists. for this purpose, sslbs were functionalized in an in situ coupling reaction with peptides originating from the nhr (n-peptides) of siv and hiv to monitor the interactions with the specific chr peptides (c34 and t20). binding of antagonists to surface confined coiled-coil structures has been quantified by ellipsometry, quartz crystal microbalance and was visualized by atomic force microscopy. a. rupprecht 1 , e. sokolenko 2 , e. e. pohl 1 1 institute of cell biology and neurobiology, charité -universitätsmedizin, berlin, germany, 2 frumkin institute of physical chemistry and electrochemistry russian academy of sciences, moscow, russia the production of reactive oxygen species (ros) in mitochondria is very sensitive to the proton motive force and can be decreased by mild uncoupling, mediated e.g. by uncoupling proteins (ucp) 1 . in contrast, the activation of uncoupling proteins by ros as a negative feedback loop is a highly controversial hypothesis. ucp activation in mitochondria by 4-hydroxy-2-nonenal (hne, aldehydic product of lipid peroxidation) was first demonstrated by echtay et al. 2 . here we investigate the ability of hne to activate and/or to regulate the expression of ucp in two different systems: (i) in lipid membranes reconstituted with recombinant ucp and (ii) in primary neuronal cells. total bilayer conductance was enhanced in the presence of hne, but this effect was independent on ucp1 and ucp2. the results concerning the hne-mediated ucp expression after induction of ros-production and/or after exogenous addition of hne are discussed for three brain-associated proteins (ucp2, ucp4, ucp5) in view of their possible functions. 1. beck, v et al. 2007 . faseb j. 21:1137 -1144 . 2. echtay, k. s. et al. 2003 . t. rudack, j. schlitter, k. gerwert ruhr university, department of biophysics, nd 04 north, 44780 bochum, germany the gtpase ras p21 which is linked to the membrane via a lipid anchor, is a crucial switch in the cellular signal transduction processes that control cell growth and proliferation. docking simulations can be seriously hampered by the great difficulty to accurately estimate the ligand-protein binding affinity constant k, which is usually derived via the computation of the binding free energy ∆g. unfortunately, due to the involved logarithmic relationship, errors of less than 1.5 kcal/mol in the computation of ∆g result in about one order of magnitude inaccuracy on k. this can hinder computational methods from discriminating micromolar from nanomolar compounds. to improve the reliability of docking prediction, we make use of enhanced sampling methods, ranging from steered molecular dynamics 1 to metadynamics 2 . we also test several descriptors, such as the recently developed path collective variables 3 , to identify the most suitable reaction coordinates accounting for binding and unbinding processes. using these approaches we investigate ligand docking and undocking and we attempt to describe at an atomistic level the kinetics of binding, which we intend to exploit for drug design purposes. here, an example of application in drug design is reported. 1. isralewitz, b. et al.; j. mol. graph. model. 2001, 19 , 13-25. 2. laio, a. and parrinello, m.; pnas 2002, 99 , 12562-6. 3. branduardi, d.et al.; j. chem. phys. 2007, 126 , 054103. prediction of protein-protein complex structures using wang-landau simulations a. solernou, barcelona supercomputing center, jordi girona 29, barcelona, spain protein-protein interactions are essential in the majority of life processes, so they have keen interest in several knowledge areas. however, experimental data on protein complexes is being produced at a quite low pace in comparison to that of the individual components. thus, in recent years attention has focused into computational approaches to the proteinprotein docking problem. a variety of docking protocols have been recently reported, sharing usually the following strategy: fast rigid-body search of the interacting subunits, followed by scoring and refinement of the interfaces. although this kind of strategy has proven some good results in the capri blind test it has two main limitations. on one hand one should include full flexibility on the protein structures. on the other, the evaluation should be made with free energy calculations instead of using a scoring function. in this work we propose to search the native complex structure in the minimum of the free energy landscape. we use the coarse grained potential unres to get the potential energy of the possible conformations. they are generated changing the dihedral angles, the side chain rotamers, and lastly the mutual orientation using a new sampling protocol we have developed (rotation-based uniform sampling; rotbus). finally, the free energy calculations are performed using an omp parallelization of the wang-landau algorithm. s. shukla 1 , s. asthana 1 , g. giliberti 1 , f. luliano 1 , m. ceccarelli 2 , r. loddu 1 , p. ruggerone 2 , p. la colla 1 1 department of biomedical science and technology, università di cagliari, cagliari, italy, 2 department of physics, università di cagliari, cagliari, italy the virus encoded rdrp has emerged as a prime target in the search for specific hcv and other flaviviridae antivirals. recently, the determination of the hcv rdrp structure in complex with certain benzimidazoles has been reported, these nni's bind to the surface of the thumb domain, thereby disrupting its interaction with the finger domain, which is necessary for catalytic activity. on the other hand, we have found that, in bvdv, the mutations conferring resistance to same class of inhibitors lie in the finger domain of rdrp, indicating that the mode of inhibition of benzimidazole class of compounds is different in both hcv and bvdv rdrp. herein, we have applied docking approaches (binding orientation), molecular dynamics (md) simulations combined with metadynamics to elucidate the microscopic mechanism of the interactions between the ligand and the receptor in order to identify features barely seen in experiment. the recently designed algorithm overcomes the time scale problem by accelerating properly defined reaction coordinates. it mimics the real dynamics of a ligand staying or leaving the receptor and in doing so reconstructs the free energy surface, which in turn gives an idea of the residence time of the inhibitor in the cavity.finally we identified the binding modes and different mechanism of inhibition of benzimidazole class of compounds in rdrp of two closely related rna viruses. . c. seifert 1 , w. stacklies 2 , f. graeter 2 1 protein mechanics and evolution, bioquant, inf 267 bq0031, 69120 heidelberg, germany, 2 ag graeter, picb, 320 yueyang lu, shanghai 200031, china we use a new method that detects force distribution in proteins. based on molecular dynamic simulations, changes in inter-atomic forces are calculated, here caused by different ligands. these changes will then be analyzed to detect a signal transfer through a protein initiated by the binding of a ligand to the protein. chaperones are ubiquitous proteins, which help other proteins to fold into a native conformation. they are able to refold misfolded proteins with a great variety of mechanisms. in this project, our group focuses on molecular dynamic simulations of htpg, an e. coli homolog of the human hsp90 (heat shock protein 90kda). the full structure of htpg was published by shiau et al. in 2006, but the mechanism of how htpg performs its function is still not understood. the folding mechanism is an atp driven reaction cycle, in which the functional entity is a homodimer of htpg. we separate the cycle in three main states: apo, adp-and atpbound state. conventional molecular dynamics simulations are used to build a stable simulation system and provide structure trajectories and primary information about the behavior of htpg in its different states of the folding process. experiments indicate that the molecular movement is atp driven. we use force distribution analysis to elucidate how atp effects htpg conformation and dynamics. the small size of myoglobin makes it the preferred candidate to investigate the structure-function paradigm. in its interior five docking sites have been identified and for long time these xenon cavities have been classified as packing defects. recently, it was shown that they might be involved in ligands migration path. however, some questions regarding its role as oxygen carrier no scavenger remain yet open as well as the microscopic mechanism regulating these biological functions. in this work we made use of standard md simulations of solvated myoglobin to characterize internal cavities. our principal results is that we have found several secondary cavities showing volume and occurrence at least comparable to that of xenon cavities. in order to analyze and rationalize internal cavities we applied special cluster-analysis: we classified all cavities with respect to the position, size and occurrence ascribing them to different clusters. this analysis highlights possible intrinsic migration paths for small ligands within the protein matrix controlled by spontaneous fluctuations of the protein itself. moreover, we identified some key residues playing a fundamental role in controlling internal pathways. our suggestion that the secondary cavities constitute the preferred path for ligand escape is also supported by explicit metadynamics simulations of ligand escape. a. varga 1 , p. lallemand 2 , j. szabó 1 , p. závodszky 1 , m. vas 1 , t. barman 2 , l. chaloin 2 , c. lionne 2 1 institute of enzymology, brc, hungarian academy of sciences, budapest, hungary, 2 cnrs-université montpellier 1 -université montpellier 2, institut de biologie, umr5236, montpellier, france 3-phosphoglycerate kinase (pgk) is a promising candidate for the activation of nucleotide analogues used in antiviral and anticancer therapies. pgk is a key enzyme in glycolysis; it catalyses the reversible reaction 1,3-bisphosphoglycerate + adp ↔ 3-phosphoglycerate + atp. here we explored the catalytic role in human pgk of the highly conserved lys 215 that has been proposed to be essential for pgk function, by a transient and equilibrium kinetic study with the active site mutant k215a. by the stopped-flow method we show that the kinetics of substrate binding and the associated protein isomerization steps are fast and identical for the wild-type pgk and mutant k215a. by the use of a chemical sampling method (rapid-quench-flow) under multi and single turnover conditions and in both directions of the reaction, we show that the rate-limiting step with wild type pgk follows product formation, whereas with the mutant it is the phospho-transfer step itself that is rate limiting. these data are supported by saxs measurements which showed no direct role of lys 215 in the domain closure of the enzyme i.e. the isomerisation step of the reaction. the results are explained by the structural data of the enzyme. characterization of different recombinant nrp1 proteins and interactions with heparin k. a. uniewicz, y. ahmed, d. g. fernig school of biological sciences and centre for glycobiology, biosciences building university of liverpool, l69 7zb liverpool, u.k. neuropilin-1 (nrp1) is a mammalian membrane glycoprotein involved in tip sprouting processes like angiogenesis and neurogenesis. it has been shown that the interaction of nrp1with heparin/heparan sulfate is implicated in its enhancement of growth factor signalling, however the mechanism is not yet known. commercially available extracellular domain of nrp1 is available either as a truncated his-tagged human protein (hnrp1) or as the rat protein fused to histagged human fc and expressed as a dimer (fcrnrp1). biochemical properties such as kinetics of heparin binding and structural requirements for sugar binding together with biochemical tests of protein properties were performed in order to characterise these commercial proteins. fc rnrp1 shown high affinity to heparin (kd=2.5 nm) and required a minimum of dp10 to effectively compete with fc rnrp1 binding to immobilised heparin. competition experiments with various modified sugars show that interaction of fc rnrp1 with heparin is highly ionic and dependent on the position of sulfate groups along the heparin chain. the hnrp1 did not bind to heparin immobilised via nhs-biotin, though it did bind to some heparin affinity chromatography matrices. organic compounds of tin are among wide spread environmental pollutants. due to physical and/or chemical actions, poly-substituted alkyltins speciate into less substituted, more toxic species. tetra-or tri-alkyltins show a marked delay in their toxic action with respect to the corresponding di-and mono-alkylated analogs. it has been hypothesized that the delayed toxicity may results from the progressive dealkylation of alkyltins in more toxic di-and mono-derivatives, which bind and inhibit essential enzymes. it has been proposed that alkyltins preferentially target enzyme sulphydryl groups. previously, we showed that a nine amino acid linear peptide (i 1 lgcwcylr 9 ) containing a cxc motif is able to bind and dealkylate tri-substituted alkyltin compounds into the corresponding dialkyl derivatives. here, we investigated the time dependence of the degradation of the most common alkyltin derivatives by this peptide. we monitored the reaction kinetics using the intrinsic fluorescence of the tryptophan residue in position 5 of the peptide. we found that all of the alkyltins analyzed are progressively degraded to dialkyl derivatives, following a pseudo-enzymatic reaction mechanism. the end-point of the reactions was the formation of a covalent complex between the disubstituted alkyltin and the peptide. these data agree with the speciation profiles proposed for polysubstituted alkyltins in the environment and reveal a possible biotic degradation pathway for these toxic compounds. parkinson's disease (pd) is a multifactorial neurodegenerative condition characterized by the progressive loss of dopaminergic neurons in the substantia nigra and by the presence of intracellular inclusions, composed predominantly of fibrillar alpha-synuclein (as). post-mortem studies indicate the presence of oxidative damage in the nigral neurons. dopamine oxidation, which leads to the formation of highly reactive quinones (daq), may account for the specificity neurodegeneration observed in pd. daq have many potential protein targets for chemical modifications. among them, we focused our attention on dj-1, of which mutated forms have been found in familial cases of pd. a possible function of dj-1 is its redox-dependent chaperone activity that could prevent as aggregation and fibril formation. in the present work, we analyzed the structural and functional modification induced on dj-1 by daq. 15 n-hsqc spectra of dj-1 were recorded in the presence of different amounts of daq and chemical shift perturbations were used to identify the dj-1 residues target of daq and their relative reactivity toward daq. aggregation assays were also performed to evaluate the functional effects of the daq modifications on the chaperone activity of dj-1. rabies virus (rabv) infects neurons exclusively and causes lethal encephalitis. pathogenic rabv strains favor neuronal survival, whereas non-pathogen strains lead to neuronal apoptosis. the use of recombinant rabv showed: 1/ the g protein determined the induction of the survival or death phenotypes; 2/ the last 4 cooh amino acids of the g protein cytoplasmic domain (cytog) are critical. these residues form a binding site for pdz domain (pdz-bs). one of the 6 amino acid differences between survival and death gproteins are located in this pdz-bs. results of two-hybrid experiments showed that cytog survival interacted pdz domain of ser/thr kinases (mast), while cytog death interacted also with 3 additional pdz containing host proteins. to understand the fine structural basis for the specificity of the pdz-cytog complexes, we determined the 3d structure and the dynamics of the mast-cytog complexes by nmr. the structures, as well as the affinities and constant kinetics, of the mast2 pdz complexes with both cytog are similar. we conclude that difference by one aa in the pdz-bs of the two strains cannot drastically modify the interaction with mast2-pdz, in agreement with the two-hybrid data. preliminary results suggest that the interaction of cytog death with one additional cellular partner blurs the pro-survival signals engaging the infected cells through apoptotic trails. functional protein immobilization on glass-type surfaces s. waichman, m. bhagawati, y. podoplelova, j. piehler institute of biology, department of biophysics,university of osnabrück , barbara st.11 osnabrück, germany the immobilization of membrane proteins onto solid supports enables protein interactions and conformational changes to be probed by spectroscopic techniques under highly defined conditions. here, we present a novel method for covalent protein immobilization on glass-type surfaces using a bottom-up approach. in this approach the 4'phosphopantetheinyl group of coenzyme a (coa) was transferred to the acyl carrier protein-derived ybbr tag of the target protein by means of the phosphopantetheinyl transferase sfp. the glass-support was rendered biocompatible by coating it with an ultrathin layer of peg (polyethylene glycol), followed by functionalization with coa through maleimide chemistry. immobilization of ybbr-tagged proteins in presence of sfp was followed in real time by label-free detection using reflectance interference spectroscopy. the immobilization procedure was thus systematically optimized by means of binding specificity, enzyme activity and functionality of the immobilized protein. this approach was employed for immobilizing the type i interferon ifnα2 in order to probe ligand recognition by ifnar1 and ifnar2 and ligand-induced ternary complex formation. the versatility of this technique was further enhanced by its combination with photopatterning methods. this immobilization technique can provide a beneficial tool for bioanalytical and biophysical applications at the single molecule level. r. vijayan, p. c. biggin department of biochemistry, university of oxford, south parks road, oxford, ox1 3qu, uk ionotropic glutamate receptors mediate excitatory synaptic transmission in the brain and are heavily implicated in memory and learning as well as in numerous neuropathological conditions. one family of iglurs, the kainate receptors, show unusual sensitivity to changes in external ion species resulting in an apparent requirement for both sodium and chloride ions for activation. our recent work revealed the location and selectivity of the cation binding sites. despite this progress, it is still unclear how the cation binding sites confer sodium selectivity and how apparent affinity for chloride is influenced by the presence of cations. we have attempted to address these questions by performing extensive free energy calculations using all-atom molecular dynamics simulations. the rank order of cation binding obtained from relative binding free energy calculations is in agreement with experimental measurements of apparent affinity. these calculations also reveal that the pair of cation binding sites in the dimer interface act independently. binding free energy calculations performed using a reduced model of the binding site show that cation selectivity can been attributed to both the rigidity and high charge density of the binding sites. finally, a potential of mean force derived from umbrella sampling simulations indicate that the presence of cations stabilize the anion binding site considerably. the effect of toxins on the inorganic phosphate release during the actin filament formation a. vig, t. kupi, g. hild, m. nyitrai university of pécs, faculty of medicine, department of biophysics, szigeti str. 12, 7624 pécs, hungary actin can be found in monomeric (g-actin) and filamentous (f-actin) form in eukaryote cells under physiological circumstances. the first step of actin polymerisation is the formation of actin nuclei by atp-binding actin monomers. the next step in is the elongation when monomers are associated to the previously formed nuclei or to the ends of the growing filaments. after the association of monomers the bound atp is hydrolysed to adp.p i , and with first order kinetics the release of inorganic phosphate occures. the rate constant of the release is 0,006 s −1 , which is a slower process than the hydrolysis itself (0,02 s −1 ).phalloidin, a cyclic peptide from amanita phalloides can bind to the filaments and stabilizes their structure. jasplakinolide is another cyclic peptide from marine sponge (jaspis johnstoni) which binds actin filaments. the aim of this study was to investigate whether the binding of toxins to the newly created filaments has an effect on the kinetics of the inorganic phosphate release or not. we used absorption photometry measurements to measure the rate of phosphate release. phalloidin decreased the rate of the release substantially. although the effect of jasplakinolide was weaker, the results showed that the binding of these toxins to the actin can modify the rate of the release of inorganic phosphate from the filaments. these observations are in agreement with the molecular mechanisms by which these toxins stabilise the actin filamens. fluorescent proteins (fps) are invaluable fluorescent markers in cell biology. however, their use is often limited by photobleaching of the chromophore, notably in single-molecule, time-resolved or super-resolution imaging studies. we will present the crystallographic studies at near atomic resolution of a photo-activatable fluorescent protein irisfp that has been observed in a transient radical state en route to photobleaching. we took advantage of x-rays to populate the radical, which, under illumination with visible light, presumably forms with low probability from the triplet state. the combined x-ray diffraction and in crystallo spectroscopic data (from uv-vis and raman spectroscopies) reveal that radical formation in irisfp involves strong but reversible distortion of the chromophore, suggesting a transient loss of pi-conjugation. these results will help unravel the mechanisms of blinking and photobleaching in fps, which is of importance to rationally design variants of higher photostability. optimizing photoactivatable fluorescent proteins for live-cell imaging recently, novel fluorescent proteins (fps) have been reported which perform spectral changes in response to irradiation with light of a particular wavelength. reversibly photoactivatable proteins switch between a bright and a dim fluorescent state. this process is accompanied by photoisomerization of the protein chromophore. irreversible photoactivation results from photochemical processes within the protein, e.g., photolysis of an amino acid side chain, or an extension of the alternating π-electron system of the chromophore by a β-elimination reaction. fps have became valuable tools in live-cell imaging because they allow intracellular protein labeling by using them as fusion tag, and photoactivatable fps are powerful tools for application in novel subdiffraction imaging techniques. however, the available fps still offer potential for improvement in various ways. they frequently show a tendency to aggregate or oligomerize, incomplete chromophore maturation, fluctuating emission and low photostability. here, we will present our recent progress towards engineering the 'perfect' fp. simultaneous intracellular chloride and ph measurements using gfp-based sensor in ldcv d. arosio 3 , f. ricci 1 , l. marchetti 2 , l. albertazzi 1 , f. beltram 2 1 italian institute of technology, udr pisa, italy, 2 nest, scuola normale superiore, pisa, italy, 3 nest, infm cnr, pisa, italy chloride ion participates in many physiological functions including control of neuronal resting potential, charge balance during endosome acidification, and regulation of cell volume. as a consequence dysfunctions in regulating membrane chloride permeability lead to severe diseases including motor disorders, cystic fibrosis and epilepsy. at present processes regulating intracellular chloride ion concentrations are still widely unexplored mainly as a consequence of limiting methods to quantify chloride fluxes in living cells. in the present work a highly specific, genetically encoded sensor is developed for detecting simultaneously intracellular ph and chloride concentration. the sensor is obtained by fusion of a red fluorescent protein (dsred-monomer), insensitive to chloride and ph, to a gfp variant containing a specific chloride-binding site (gfp-chl). dsred-gfp-chl binds the chloride ion following a fluorescence static quenching mechanism, which allows measurements of intracellular ph in a chlorideindependent manner. the sensor has been successfully tested in different living cells, in a ph range 5-8 and chloride concentration up to 200 mm. for the first time, to the best of our knowledge, it allowed to measure the chloride concentration of dense core vesicles in the secretory pathway. applicability to high-throughput screening, range of validity and accuracy of time-lapse maps will be discussed. we aim to understand the basis of the photophysical changes in fluorescent proteins (fp) induced by reactive oxygen species (ros). indeed, ros might be involved in photochemistry of fp, leading to their photobleaching or photoconversion. in addition, fp may be used to investigate cellular events like phagocytosis or mitochondrial activity, where ros are naturally produced. in the latter cases, an accurate analysis of fp's fluorescence signals requires the full knowledge of reactions between ros and fp. in the future, this work may help in developing either photoresistant or photoswitchable fp and improving their use for imaging under oxidative stress conditions. using γ-radiolysis as a quantitative source of ros, we investigated the reactions of oh and o 2 − radicals on the cyan fluorescent protein (cfp) and the modifications of the cfp's photophysical properties by oh radicals were explored in detail (submitted). in order to address the corresponding chemical changes in the protein, we devised a mild proteolysis protocol that for the first time offers a peptide mass fingerprint almost covering the cfp sequence (alvarez et al. biochemistry 2009). then, we achieved the meticulous characterization of the cfp oxidation products by mass spectrometry and proposed a mechanism to account for their formation by pulse radiolysis. since the cloning of the green fluorescent protein from aequoria victoria, numerous screens have been performed to improve the brightness of this protein, its spectral variants and fluorescent proteins from other species. the improvement is evaluated by comparing fluorescence intensity of individual bacteria or colonies. in this way also expression level, folding and maturation efficiency, and thickness of the bacterial colony contribute. here we report a screening method that, in addition to fluorescence intensity, quantifies the excited state lifetime of a fluorescent protein, which is independent of intensity or expression level, and provides a direct measure for the quantum efficiency of the fluorescent protein. the novel approach was used to screen a library of cyan fluorescent protein (cfp) variants randomly mutated at position 65, 148 and 224, yielding an improved bright cyan fluorescent protein named, mposeidon, with a markedly increased fluorescence lifetime and quantum yield, increased photostability and improved fluorescence intensity in vivo. it is shown that mposeidon is the brightest cyan fluorescent protein in mammalian cells. in addition, several lifetime variants were identified that can be used for lifetime unmixing. it is demonstrated that three cfp variants can be separated and their distribution quantified in a single detection channel. a. r. faro 1 , p. carpentier 2 , d. bourgeois 3 , e. rosny 4 1 irtsv, cea, ufj, grenoble, france, 2 ibs, cea, cnrs, ufj, grenoble, france, 3 ibs, cea, cnrs, ufj, grenoble, france, 4 ibs, cnrs, ufj, grenoble, france enhanced yellow fluorescent protein (eyfp) is extensively used as a fluorescent marker. like other photo activatable fluorescence proteins (pafp), it exhibits photo-switching properties. however, the mechanism by which fluorescence can be swiched on or off upon light irradiation is not fully understood at the molecular level. the bright to dark conversion involves a protonation step and structural rearrangements of the chromophore, but it is not clear which of these two steps is the triggering event. to answer this question, we carried out photo-switching experiments at cryotemperatures. our data suggest that a photo-induced protonation step (probably in the triplet state) is the primary event in the bright to dark conversion. our results may bear relevance to other pafps, such as iris, eos, dronpa, padron or kaede. how misfolding and aggregation of proteins constitutes a toxic insult to neurons remains largely unknown. in order to obtain insight into the molecular biology of neurodegenerative disease, we have developed a number of gfp-based biosensors for the detection and quantification of cellular clearing mechanisms for aggregated proteins. the high load on these protein quality control mechanisms, and their failure to meet normal physiological demand ultimately results in a "de-compensation" condition from which the nerve cell cannot recover. our fret/flim-based bioassays visualize protein ubiquitination and degradation; proteasomal activity; foldase activity using a folding-impaired gfp mutant which gains fluorescence conditional on the upregulation of chaperone activity; chaperone binding to unfolded proteins; and autophagosome formation/lysosomal integrity via the targeted and sensitive fret-based measurement of ph changes. these sensors are employed in cellular model systems for parkinson's and alzheimer's disease, and amyotrophic lateral sclerosis (als) to delineate the molecular pathway of cellular demise, and to gain a mechanistic understanding of the toxicity of protein aggregates and the basis for the vulnerability of neurons. millisecond photo-switching dynamics of e222q gfp mutants for sensor and imaging applications m. collini 1 , v. quercioli 1 , l. d'alfonso 1 , g. baldini 1 , b. campanini 2 , s. bettati 2 , g. chirico 1 1 dipartimento di fisica, università di milano bicocca, italy, 2 dipartimento di biochimica e biologia molecolare, università di parma, parma, italy e222q mutants of the green fluorescent protein are known to possess photo-chromic properties: the anionic emission, primed by a pump 488 nm laser beam, can be switched between two levels of different brightness by irradiation with a blue, ∼ = 400 nm, probe laser light. we have studied here the amplitude and the dynamics of the brightness enhancement of the e222q mutant of gfpmut2. the fluorescence emission increases almost threefold, under saturating probe laser excitation, for pump excitation intensity in the linear regime. two characteristic activation times, estimated by means of modulated two colour fluorescence correlation spectroscopy, are detected in the 1-30 ms range, independent of solution temperature and viscosity. the brightness enhancement factor and the characteristic activation times depend markedly on the solution ph. these results indicate that this mutant can be used as a high sensitive intracellular marker for local proton concentration and for modulated excitation imaging. the advent of fluorescent proteins (fp) gave researchers the opportunity to study proteins in situ. fluorescence resonance energy transfer (fret) benefited from this. cell fixation is a commonly used approach when working with microscopy. however, we have found that fret efficiency (e) in cells transfected with cerulean and venus chimeras could not be reproducibly measured after fixation. to evaluate this problem in detail, we measured e of 3 cerulean-venus standard constructs by acceptor photobleaching fret, intensity-based ratiometric fret and flim-fret. the constructs were produced as standards (biophys j, 2006, 91, 99) with 43, 38 and 31% e values, comprising donor-acceptor separations of 5, 17 and 32 amino acids, respectively. transient transfection of the fusion plasmids was performed into hela cells and e was measured in live and pfa or methanol fixed cells. literature e values were reproduced when measuring live cells. conversely, cell fixation caused a deviation of e values. methanol fixed cells showed e between 1-5% for all the constructs. the effect of pfa fixation on both fluorescence intensity and fret varied vastly among independent experiments regardless of the measurement modality. thus, fixation should be avoided due to the effects it has on fp's fluorescence and consequently on fret efficiency. to explain the improvement in the fluorescence properties of cerulean when compared to ecfp, we have determined the x-ray crystallographic structures of these two proteins at physiological ph, and performed molecular dynamics simulations. both proteins exhibit a structural heterogeneity in the nterminal half of their seventh strand, which forms a specific set of van der waals interactions with the chromophore. the critical h148d mutation present in cerulean induces a modification of these interactions, and allows the chromophore to be more planar and better packed, albeit only intermittently. as a consequence, the probability of non-radiative decay is significantly decreased. our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins, and provide insights which should allow the design of mutants with enhanced optical properties. the fluorescence lifetime of green fluorescent protein g. jung biophysical chemistry, saarland university, 66123 saarbruecken, germany biotechnological design of the green fluorescent protein (gfp) and the discovery of other proteins boosted the development of the life sciences in the past decade. tracking protein movements and high resolution microscopy are only a few recent applications which were realized by fluorescent protein technology. among these examples, the switching between two chromophore state is exploited. our aim is to establish fluorescent proteins for bioanalytical fluorescence lifetime measurements. despite the progress in other fields, quantification with gfp still imposes practical problems [1] . in the past, we observed that the uv-light driven decarboxylation of the nearby aminoacids glu222 distorts the fluorescence lifetime of gfp [2] . we found out that this chemical reaction also occurs under excitation of the anionic chromophore state with a high quantum yield [3] . recently, we could show by time-resolved spectroscopy that, indeed, the more susceptible state for this kind of photoconversion is the anionic chromophore state [4] . suppression of this reaction therefore enables the design of autofluorescent proteins which can be used e.g. for the quantification of ions and which are beneficial as donors in fret applications. the fluorescent properties of tryptophan residues (w) in proteins are highly dependent on their immediate protein environment. however, the multi exponential decay of single w proteins is not completely understood. the most cited hypothesis contributes a multi exponential decay to the existence of several micro conformations (rotamers) of the w residue within the protein matrix. to determine rotamers we apply a method based on dead-end elimination (dee) and molecular dynamics simulations (md). low energy rotamers are calculated by dee while dynamics and further refinement is accomplished using md. the method was applied on several test cases including the protein mutant bc-csp l66e from bacillus caldolyticus, which contains a solvent exposed w residue. as resolved by x-ray crystallography, this w residue occupies two conformations. using dee and md we were able to retrieve the w conformations found in the x-ray structure. the results demonstrate the ability of the method to obtain valuable w rotamers, both for solvent shielded as exposed residues. the determined conformations were compatible with the findings based on a method using replica exchange simulations. k. nienhaus 1 , h. nar 2 , r. heilker 2 , j. wiedenmann 3 , g. u. nienhaus 4 1 inst. of biophysics, universität ulm, ulm, germany, 2 dept. of lead discovery, boehringer ingelheim, biberach/riss, germany, 3 national oceanography center, university of southampton, southampton, uk, 4 inst. of applied physics, universität karlsruhe (th), karlsruhe, germany eqfp611 is a red fluorescent protein (rfp) with the chromophore in a co-planar trans orientation, whereas the cis isomer is preferred by most other rfps. by using x-ray crystallography, we determined the structures of the dimeric variants d1eqfp611 and d2eqfp611 at high resolution (up to 1.1å). for d1eqfp611, we had previously seen a redshifted species upon irradiation with 532-nm light. concomitant changes in the raman spectrum were interpreted as evidence of a trans-cis isomerization of the chromophore. here we have combined x-ray crystallography and site-directed mutagenesis to assess whether we can create a stable fluorescent, red-shifted eqfp611 variant with a cis chromophore. in a first step, we introduced the n143s substitution. this variant, d2rfp630, is highly fluorescent, with the absorption (emission) maximum red-shifted by 24 (19) nm. with an additional s158c mutation, the chromophore is found completely in the cis form. the variant, rfp639, is highly fluorescent, with excitation and emission maxima at 588 and 639 nm. still further red shifts appear to be in reach. k. nienhaus 1 , v. adam 2 , d. bourgeois 2 , g. u. nienhaus 3 1 of biophysics, universität ulm, ulm, germany, 2 esrf, grenoble, france, 3 institute of applied physics, universität karlsruhe (th), karlsruhe, germany dendra2 is an engineered, monomeric gfp-like protein that belongs to a sub-class of fluorescent proteins undergoing irreversible photoconversion from a green-to a red-emitting state upon exposure to purple-blue light. this process occurs in the neutral state of the chromophore and is known to result from backbone cleavage accompanied by an extension of the delocalized π-electron system. we have measured the x-ray structure of green dendra2 and performed a comprehensive characterization of the optical absorption and fluorescence properties of the protein in both its green and red forms. the structure, which is very similar to those reported for the closely related proteins eosfp and kaede, revealed a local structural change next to the chromophore, involving mainly arg66 and a water molecule. we propose that this structural change explains the blue shift of the absorption and emission bands, as well as the markedly higher pks of the hydroxyphenyl moiety of the chromophore. the 20-fold enhancement of the neutral species in dendra2 at physiological ph accounts for the observed higher photoconversion yield of this protein in comparison to eosfp. photochromic green fluorescent protein mutants: chromophore states unveiled by raman spectroscopy s. luin, v. voliani, g. lanza, r. bizzarri, r. nifosì, p. amat, v. tozzini, m. serresi, f. beltram nest, scuola normale superiore, cnr-infm and italian institute of technology, pisa, italy the most widespread genetically-encodable fluorescent markers used for studies in living cells and tissues belong to the green fluorescent protein (gfp) family. reversibly switchable fluorescent proteins (rsfps) were developed that can undergo repeated transitions between different states, e.g. bright and dark forms. this property makes rsfps particularly attractive as active labels in biological systems for selective photolabeling applications or subdiffraction imaging. we shall present pre-resonant raman results unveiling the photophysical mechanism underlying the observed photochromic behavior. the variation of spectral properties before and after photoconversion of chemically-synthesized isolated chromophores under different protonation and/or isomerization have been analyzed, and compared to results obtained for the case of complete folded proteins comprising the same chromophores. experimental results have been analyzed within a time-dependent density functional theory, allowing us to assign all relevant vibrational modes. these results make it possible to discriminate between the effect of cis-trans isomerization and of diverse protonation states of the chromophore in the photoproducts of these proteins. s. luin et al, j. am. chem. soc. 131, 96-103 (2009 ). r. bizzarri et al., anal. bioanal. chem. 393, 1107 -1122 . a combined study of the interaction of outer membrane proteins with cephalosporin antibiotics m. lovelle, i. barroso, m. j. feio, p. gameiro requimte , fac. ciências, universidade do porto, portugal gram-negative bacteria characteristically are protected by an outer membrane that serves as a selective permeation barrier. most of the β-lactam antibiotics appear to penetrate the outer membrane through these non-specific channels, and it becomes important to understand the possible interactions between β-lactams and the porin. fluorescence techniques have been largely used to characterize both the conformation and the dynamic behavior of large biological structures such as membranes and proteins. the fluorescence of the tryptophan residues is quenched in the presence of the different cephalosporin antibiotics. this reaction between the excited state of the fluorophores and the drug can be described as a formation of a non-fluorescent complex. the dependence of the fluorescence intensity upon quencher concentration for static quenching is proportional to the binding constant for complex formation. since β-lactam susceptibility is closely related to the presence of these non-specific porins, minimum inhibitory concentration (mic) by micro-broth dilution in microplate were used to assess the bactericidal activity of cephalosporin antibiotics upon on escherichia coli strain bl21(de3 ) and a series of bl21(de3) mutated in different outer membrane proteins. this combined study of the interactions at single molecular level and at in vivo level provides new insights for a better understanding of the antibiotic translocation. when used in combination with e.g. a cyan fluorescent protein, keima offers the unique opportunity to perform dual color fluorescence cross-correlation spectroscopy using a single laser line to excite both fluorophores. the molecular determinants of the large stokes shift of mkeima have been characterized structurally by combining x-ray crystallography with in crystallo uv-visible absorption, fluorescence and raman spectroscopy. our results reveal a ph-dependant "reverse chromophore protonation" of mkeima, driven by the key residue asp157. moreover, the chromophore protonation state is shown to be coupled with different chromophore conformations, cis conformation at ph 3.8, and mostly trans conformation at ph 8.0. these results will help unravel the mechanisms of fluorescence in fps, which is of importance to rationally design and develop new fluorescent markers. recently reversibly switchable fluorescent proteins (rsfps) have become a new branch of the green fluorescent protein (gfp) like family. the rsfps may be reversibly switched between a fluorescent and a non-fluorescent state by irradiation with light of distinct wavelengths. the key process of this switching behaviour is a light induced change of the chromophore between a cis-and a trans-isomeric state. the proteins are becoming increasingly important for diverse applications like protein tracking, sub-diffraction resolution microscopy and data storage. based on the switching mechanism, we created novel rsfps with unique and improved characteristics. padron and bs-dronpa are two of these new switchable proteins. padron features a reversed switching mechanism as compared to all other green rsfps known to date while bsdronpa exhibits a very broad absorption spectrum extending into the uv. utilizing the characteristics of both proteins, we performed multi label single detection colour microscopy as well as dual colour sub-diffraction microscopy, the latter with a resolution of 20 nm. further, we recently introduced the first monomeric rsfp exhibiting red fluorescence: using a semi rational mutagenesis approach on the non-switchable mcherry, we generated the switchable monomeric protein rscherryrev. the use of this protein in single molecule switching microscopy allowed us to record time-lapse live-cell images of the endoplasmic reticulum with sub-diffraction resolution. a spectroscopic approach to the study of chromophoric dissolved organic matter (cdom) in the sea c. santinelli, r. lavezza, l. nannicini, a. seritti cnr-ibf, via moruzzi 1, 56124 pisa, italy dissolved organic matter (dom) in the ocean represents the largest reservoir of reactive carbon on the earth. it is produce at each level of the marine food web and it represents the food for heterotrophic bacteria, which can use the different pools of dom (labile, semi-labile, refractory) with a large range of turn-over times (from minutes to millennia). dom plays a central role in the global carbon cycle and it has a high ecological significance. chromophoric dom (cdom) is the fraction of dom capable of adsorbing light at uv and visible spectral regions. it determines the underwater light availability in open and coastal regions with important implication on primary production and biological activity. it is photodegraded to co 2 , co, with a significant impact on the role of the ocean as source and/or sink of atmospheric co 2 and to highly reactive compounds, dangerous for marine organisms. in its pool "humic-like" and "protein-like" fluorophores have been individuated. cdom optical properties (absorption and fluorescence) together with doc data collected in some key regions of the mediterranean sea will be presented and discussed, in order to (i) investigate the powerful of cdom optical properties to get information on cdom "quality" (i) asses the role of dom in carbon export at depth, (iii) underline the main unresolved question on dom and cdom dynamics in the ocean, with particular emphasis to their biological lability. mechanism and applications of photo-and redox-switchable fluorescent proteins s. j. remington, j. n. henderson, x. shu, j. lohman institute of molecular biology, university of oregon, u.s.a. photoswitchable fluorescent proteins have significant advantages over conventional fluorescent labels, and in a revolutionary application, now allow cell biologists to exceed the diffraction limit in light microscopy by a factor of ten. atomic resolution crystal structures and time resolved spectroscopy of both reversible (mtfp0.7) and irreversible (pa-gfp) photoswitchable fluorescent proteins in the light and dark states explain the long term stability of either state, as well as how illumination at the appropriate wavelength causes the molecules to switch between states. the photoswitching mechanisms will be discussed in terms of photochemistry, light induced chromophore isomerization and excited state proton transfer (espt). mutagenesis of espt pathways provide insight into the nature of the ratedetermining steps in proton transfer and lead to practical applications, such as redox-sensitive gfp biosensors. k. i. willig, b. hein, s. w. hell max-planck-institute for biophysical chemistry, goettingen, germany stimulated emission depletion (sted) nanoscopy is a light microscopic technique offering a resolution far beyond the diffraction limit: the excitation beam is overlapped with a doughnut-shaped, red-shifted sted beam, which switches off the ability of the molecules to fluoresce in the outer region of the excitation spot. scanning the nanosized focal spot through the sample renders sub-diffraction images with a sub-second frame rate. we used the yellow fluorescent protein citrine to image individual structural elements of living mammalian cells: vimentin and the tubular network, structures of the cytoskeleton, were recorded with a lateral (x,y) resolution < 50 nm inside the living cell, corresponding to a 4-fold improvement over that of a confocal microscope. also, time lapse sted imaging of dendritic spines of yfppositive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. as an alternative to fluorescent proteins we used a genetically encoded protein tag which can be labelled with modified organic dyes within living samples. thus nanoscale imaging of structures in the interior of living cells greatly expands the scope of light microscopy in cell biology. pulling membrane tubes from solid-supported lipid bilayers with atomic force microscopy j. w. armond 1 , j. v. macpherson 2 , m. s. turner 1 1 department of physics, university of warwick, u.k., 2 department of chemistry, university of warwick, u.k. information on the elastic and dynamic properties of membranes is essential for a thorough understanding of biological processes such as exocytosis, endocytosis, cell division, and pore formation. we use an atomic force microscope to pull on solid-supported lipid bilayers and observe that an energy barrier must be overcome prior to the formation of membrane tubes. we have modified elastic models of lipid bilayer vesicles to describe the free energy of a planar lipid bilayer in adhesive contact with a surface. from this model we are able to extract force-distance curves for the formation of a membrane tether, including the associated energy barrier. the experimental data can thus be understood in a quantitative fashion. this work enables the atomic force microscope as a quantitative instrument for measuring membrane pulling mechanics, and in future work will allow two-dimensional mapping of elastic properties under tension. from pores to micelles -a peptide-membrane interaction study t. l. andresen, j. r. henriksen technical university of denmark, dtu nanotech, frederiksborgvej 399, b124, 4000 roskilde, denmark. email: thomas.andresen@nanotech.dtu.dk. research in the partitioning of peptides into lipid membranes has been intense for several years. along with scattering techniques and nmr, isothermal titration calorimetry (itc) has proven to be a powerful tool for thermodynamic characterization of peptide-membrane interactions. we have studied two antimicrobial peptides, mastoparan-x and melittin, and found that these peptides induce a range of structural transitions of popc and popc/popg membrane systems at different peptide-lipid ratios. we have found that itc can be used to elucidate the threshold where transitions occur, including the threshold for pore formation and micellation. this has been achieved using a thermodynamic model based on gouy-chapman theory, which provides the partitioning constant of the peptide-membrane interaction and thereby the concentration of peptide on the membrane surface. the structural changes have furthermore been visualized by cryo-tem. we have further investigated the pore formation in detail and found that the thermodynamic parameters of pore formation can be fully characterized using a system specific temperature where the enthalpy of peptide partitioning becomes zero. this allows for an exclusive study of the pore formation process. lactoferrin is a glycoprotein with two globular lobes, with of two domains each. since its discovery, the research on antimicrobial properties has been extended to peptides derived from this protein. the largest family studied so far is known as lactoferricin b, obtained from the protein by digestion with pepsin. more recently, a new family of antimicrobial peptides derived from lactoferrin was discovered by bolcher et al and named lactoferrampin (lfampin). the original sequence of lfampin contained residues 268-284 from the n1 domain of lactoferrin. we studied 3 peptides derived from lfampin obtained by extension and/or truncation at the c-or n-terminal sides, keeping the essential characteristics, in order to unravel the main structural features responsible for antimicrobial action. the peptides were tested against model membranes. the ability to adopt helical conformation was followed by cd, the perturbation of the membrane phase transition by dsc, the energetics of interaction by isothermal titration calorimetry (itc), the partition of the peptide to the membrane by trfs and the importance of charge effects assessed by zeta potential measurements. the results are discussed and compared to the antimicrobial and hemolytic activities obtained by microbiology techniques. defensins are small cysteine-rich cationic proteins or peptides found in both vertebrates and invertebrates. they can be active against bacteria, fungi and many enveloped and non-enveloped viruses; thus, being also classified as antimicrobial peptides (amps). in the present work a comparative study between psd1 (a plant defensin with antifungic properties obtained from the garden pea pisum sativum) and hnp1 (a human neutrophils defensin) was conducted, in order to shed some light on the mechanism of action at the molecular level of these defensins. large unillamelar vesicles with different lipid compositions were used for this purpose as biomembranes model systems; namely, popc/cholesterol (characteristic of the outer leaflet of vertebrates cell membranes) and popc/ergosterol (fungal) mixtures. changes on the intrinsic fluorescence of the tryptophan residues present in these peptides upon membrane binding/insertion were followed by fluorescence spectroscopy. experimental results show the affinity of both defensins for mammalian and fungal sterols. the partitioning behavior of psd1 showed a high selectivity for ergosterol rich membranes, while hnp1 has preference for cholesterol rich membranes. preliminary characterization of atomistic computer models of galactolipid bilayers k. baczynski, m. pasenkiewicz-gierula faculty of biochemistry, biophysics and biotechnology, jagiellonian university, krakow, poland the main lipid component of thylakoid membranes are galactolipids, which constitute more than 75% of its lipid composition. the most common types of galactolipids found in thylakoid are monogalactosyldiacylglycerol(mgdg), whose headgroup consists of a single molecule of beta-d-galactose and digalactosyldiacylglycerol(dgdg), whose headgroup consists of two galactose molecules: alpha-d-galactose and beta-d-galactose linked by o-glycosidic bond majorty of thylakoid galactolipid have gamma-linolenic acid both in the sn-1 and sn-2 position. atomistic computer models of mgdg and dgdg molecules have been constructed and parametrized in the opls-aa force field. the molecules were used to construct three bilayer systems for molecular dynamics (md) simulations:(1) composed entirely of dgdg molecules,(2) composed entirely of mgdg molecules,(3) composed of a mixture of dgdg and mgdg molecules the ratio 1:2. the systems have been md simulated using the gromacs 3.3.1 package. the generated trajectories were analysed to determine a number of structural parameters among them: membrane thickness, average area per lipid, electron density profile accross the bilayer, order parameters, organisation of the bilayer/water interface as well as several dynamic parametrers like diffusion coefficients, lifetimes of conformational states, lifetimes od lipid lipid interactions. single mixed-lipid guv method reveals interaction of viper venom with lipid membranes n. m. ayvazyan, n. a. zaqaryan, n. a. ghazaryan dpt. biophysics, yerevan state university, armenia studies on the interaction of snake venom and organized lipid interfaces have been conducted using a variety of systems, including bilayer lipid membranes (blms), small and large unilamellar vesicles. giant unilamellar vesicles (guvs) with a mean diameter of 30 µm have a minimum curvature and mimic cell membranes in this respect. guvs are ideal for studying lipid/lipid and lipid/protein interactions using microscopy techniques with membrane fluorescence probes. guvs were formed from the total lipid fraction from bovine brain by the electroformation method, developed by angelova and dimitrov (1987) . vipera lebetina obtusa venom was added to the sample chamber before the vesicles were formed. the membrane fluorescence probes, ans and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. ans and pyrene allows us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. the presence of viper venom in guvs media reveals a noticable decreasing of membrane fluidity compare the control, while the binding of fluorophores with guvs modified by venom lead to appearance of channel activity. it was recognized early that the vipers' venom components preferred an organized lipid substrate near the lipid's phase transition and were particularly active against micellar lipids. these studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom. the attenuated total reflection fourier transform infrared (atr-ftir) spectroscopy is ideal for highly absorbing samples such as water suspensions or even bulk water due to the small light penetration depth. it is also suited for experiments on lipid membranes in excess water. for example, we have studied the hydrogen bonding (h-bonding) between cholesterol (ch) and phosphatidylcholine (pc) or sphingomyelin (sm), which could be important for the stabilization of the cholesterol-rich lipid domains. the atr-ftir method enabled comparison of the carbonyl band shape for pc/ch samples in excess h 2 o or d 2 o, and has confirmed similar behavior for both [1] . secondly, we were able to analyze the amide ii band for sm/ch samples in excess h 2 o. the observations confirm the presence of h-bonds between ch and n-h group in sm [2] . another example is the study of the interlamellar water structure, which could influence the water-mediated phenomena in membranes. h-bonds in interlamellar water in partially hydrated lipid multibilayers are stronger with respect to bulk water. in contrast, we show by atr-ftir spectroscopy that the h-bonds are weaker in multibilayers in excess water [3] . the bactericidal activity of antimicrobial peptides is linked to their ability to perturb the permeability of bacterial cells. they often show α-helical conformation, and many peptides have a kink in the middle of this structure, caused by pro or gly. in order to understand the role of the kink we designed various analogues of p5, in which the central pro residue was moved from its central position, or removed altogether (in analogue p5f). displacement of the pro residue caused a decrease of the antimicrobial activity, and an increase in the toxicity against erythrocytes, with the less active and more toxic peptide being p5f. fluorescence studies suggest that both p5 and p5f bind on the membrane surface. fluorescence experiments show that the activities of the two analogues correlate with their affinity for different kinds of lipid bilayers: the kinked p5 peptide has a dramatically higher affinity for negatively charged vesicles (mimicking the composition of bacterial membranes) than for neutral liposomes (similar to mammalian cells), while analogue p5f exhibits comparable affinities for both membranes. therefore, our data suggest that the central pro-induced kink is involved in selectivity, inhibiting peptide binding to the membranes of eukaryotic cells. d. behn 1 , h. hoffmeister 2 , r. witzgall 2 , c. steinem 1 1 institute for organic and biomolecular chemistry, university of göttingen, germany, 2 institute for molecular and cellular anatomy, university of regensburg, germany polycystin-2, encoded by pkd2, is an integral membrane protein with a size of 110 kda and 968 amino acids. the protein, which is known to be a ca 2+ permeable, non selective cation channel, interacts with several proteins such as polycystin-1, pigea14 or pacs-1/-2 etc. the interaction takes place through a proposed coiled-coil domain of polycystin-2 located at the c-terminus of the protein.if mutation of either pkd2 or pkd1 (gene product of polycystin-1) occurs, the interaction between the proteins is disturbed leading to increased formation of renal cysts. this autosomal polycystic kidney disease (adpkd) is one of the most common genetic diseases causing renal failure due to the enormous cyst formation. in this study, the interaction of the c-terminus of polycystin-2 with its postulated specific interaction partners has been investigated in terms of its biological relevance. binding affinities as well as kinetic parameters of the interaction were determined. in particular, the interaction of c-polycystin-1 and pigea14 immobilized on solid supported membranes with c-polycystin-2 has been quantified by means of the quartz crystal microbalance (qcm) method. a dissociation constant of about 100 nm was obtained. the results are compared with those obtained by surface plasmon resonance (spr) technique using a different immobilization strategy. correlation of the lateral structure of lipid bilayers and monolayers using two photon excitation fluorescence microscopy l. a. bagatolli membrane biophysics and biophotonics group/memphys -center for biomembrane physics, bmb, university of southern denmark most of the reported fluorescence microscopy applications on langmuir lipid films are focused in obtaining fluorescence intensity images using particular fluorescence probes. in this type of experiments the probes are generally utilized to obtain "contrast" between membrane regions (lipid domains) displaying dissimilar physical properties. the obtained information largely depends on the partition of the fluorescent probes for the lipid domains and the obtained fluorescence intensity pictures are not providing any information about the local physical properties of the lipid film. local physical properties of these distinct regions can be determined by exploring fluorescent probe related parameters such lifetime, emission shift, polarization. however these fluorescent probe's properties are almost unexplored in this type of experiments. this presentation will focus in describing a new experimental setup that includes a specially designed film balance on top of a custom built multiphoton excitation fluorescence microscope. this setup allows obtaining laur-dan gp images (1) many studies on phase separations of lipids in bilayers and their leaflet asymmetry make use of fluorescence techniques. we used a dithionite assay, steady-state fluorescence anisotropy and fluorescence resonance energy transfer (fret) for characterization. dithionite assay was performed to measure the fluorophore distribution in the inner and outer leaflets of symmetric membranes. for low concentrations, the fluorophore is distributed almost homogeneously, whereas for concentrations > 0.5 mol % it accumulates in the outer leaflet. we discuss these effects taking into account the presence of multilamellar liposomes and dynamic effects produced by higher local bending elasticities. the results point out possible artifacts in the use of fluorophores to characterize bilayers under the assumption of their homogeneous distribution. dithionite relative bilayer permeability is discussed. the results won by fret and steady-state fluorescence anisotropy regarding lipid phase transitions are in good coincidence to each other and to results reported in the literature. the photosensitizing properties of three chlorins are compared in solution and when incorporated in dioleoylphosphatidylcholine vesicules. in solution, they possess a similar efficacy to generate singlet oxygen and a similar ability to induce the peroxidation. however, the role of the photosensitizer localization within the lipidic bilayer is strongly highlighted, when chlorins are incorporated in liposomes, both by the changes in order of peroxidation efficacy but by the measurements of the photoinduced permeation of the liposomes. the results are discussed in relation with the technology of photochemical internalization, pci. then, using giant unilamellar vesicles, we asymmetrically oxidize the membranes. we observed different shape transitions, such as budding, typical of membrane curvature modifications. the asymmetry of the shape transitions are in accordance to a lowered effective spontaneous curvature of the leaflet targeted. this effect is interpreted as a decreased preferred area of the targeted leaflet compared to the other, due to the secondary products of oxidation. permeabilization of guv by photooxidation is interpret as the opening of a pore above a critical membrane tension due to the budding of vesicles. the effective spontaneous curvature of photosensitized vesicle at lysis was estimated. additionally photooxidation was shown to be fusogenic. influence of polyphenol extracts from fruits on biological and model membranes d. bonarska-kujawa, h. pruchnik, j. sarapuk, j. oszmiański, h. kleszczyńska univ. of environmental and life sciences, wroc law, poland biological activity of polyphenol extracts from apple, strawberry and chokeberry was studied. polyphenols were shown to be good antioxidants and to act as antyhemolytic agents. both the activities are the result of polyphenols incorporation into biological membranes. to check potential changes they induce in membranes some experiments were performed with the use of erythrocytes, and lipid membranes. it was found that the extracts studied induced shape transitions of erythrocytes. they were classified according to the bessis-brecher scale. strawberry extract induced mainly discocytes and discoechinocytes. populations of discocytes, echinocytes and some discoechinocytes were found on applying apple extract, while chokeberry induced mainly the formation of echinocytes and spheroechinocytes. the results evidence that the polyphenols incorporate into the external monolayer of lipid bilayer of the erythrocyte membrane. the results of the fluorimetric experiments showed that all the extracts changed fluidity in the hydrophilic part of rbc membrane. the changes observed were biggest for chokeberry extract and smallest for strawberry one. incorporation was also followed by changes in electrical resistance of black lipid membranes and in shifting the temperatures of main transition (t m ) and pretransition (t p ) in liposomes. this work was sponsored by ministry of science and education, scientific project no. n n305 337034. a. boll 1 , n. czudnochowski 2 , m. geyer 2 , c. steinem 1 1 institue of organic and biomolecular chemistry, university of göttingen, germany, 2 mpi for molecular physiology, dortmund, germany hiv-1 tat belongs to the accessory proteins of hiv and has regulatory functions. tat is concentrated in the nucleus and nucleolus of infected cells. the protein is composed of 86 amino acids with a molecular weight of 11 kda. tat is a transcriptional activator protein, which stimulates rna polymerase ii-mediated transcription elongation. therefore, tat interacts with cyclin t1 and binds to the tar rna element. tat has different domains. with respect to the interaction with lipid membranes, the most important structural motif is its basic region, including 6 arginine and 2 lysine residues. the peptide derived from this basic region belongs to the cell-penetrating peptides and is able to translocate through lipid membranes. the major aim of this study is to investigate the influence of full length hiv-1 tat on artificial lipid membranes. as a starting point solid-supported membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (popc) immobilized on silicon dioxide were used. the influence of the lipid head groups on the interaction with tat was analysed by using membranes composed of the negatively charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (pops) in a mixture with popc. fluorescently labelled tat was used to localise the protein in the membrane. the impact of tat on lipid membranes was investigated by fluorescence and atomic force microscopy, showing that it is capable of perturbing lipid membranes. g. bocchinfuso 1 , a. palleschi 1 , b. orioni 1 , g. grande 1 , f. formaggio 2 , c. toniolo 2 , y. park 3 , k. s. hahm 3 , l. stella 1 1 univ. of rome tor vergata, dept. of chemistry, italy, 2 dept. of chemistry, univ. of padova and cnr, italy, 3 chosun univ., rcpm, south korea. most antimicrobial peptides exert their activity by perturbing the permeability of bacterial membranes, but the molecular details of this process are still debated. here, we compare fluorescence experiments and molecular dynamics simulations regarding the interaction of two antimicrobial peptides (pmap-23 and trichogin ga iv) with lipid membranes, showing that their mechanisms of bilayer perturbation are significantly different. pmap-23, a cationic peptide member of the cathelicidin family, associates to the membrane close to its surface and parallel to it, and in this arrangement it causes a severe perturbation to the bilayer, both regarding its surface tension and lipid order. on the other hand, trichogin ga iv, a neutral peptide member of the peptaibol family, undergoes a transition from a surface-bound state to an inserted orientation. in the first arrangement it does not cause any strong membrane perturbation, while in the second orientation it may span the bilayer from one side to the other, despite its relatively short length, by causing a significant thinning of the membrane. lipopolysaccharide (lps) is the main component of the outer membrane of gram negative bacteria. lps is also known as endotoxin because of its potency to induce sepsis, a serious source of mortality in many clinical cases. among lps-neutralizing agents, polymyxin b (pxb) is considered as the "gold standard" due to its high efficiency of binding and detoxification of endotoxin. in this work, we have further explored the interaction of pxb to lps from the rough mutant of salmonella minnesota (re-lps) both in the gel and in the liquid crystalline phase, using isothermal titration calorimetry (itc) and fluorescence based techniques. the effect of pxb binding on lps-membrane integrity was determined with a fluorescence quenching assay treating vesicles of lps labeled with nbd-pe with dithionite. thermodynamic parameters associated with the binding process as well as binding stoichiometry were obtained from itc experiments. finally, itc was conducted with enterococcus faecalis and salmonella typhimurium, as representative examples of a gram negative and a gram positive bacterium respectively. pressure jumps -an accessible trigger for biomolecular transformations high pressure can be used to induce many structural changes in biological systems: from triggering phase changes in model membranes to causing protein unfolding, in fact any change involving a volume reduction is promoted by pressure. as well as being broadly applicable, pressure changes can be applied very quickly both up and down in contrast to other structure change triggers such as temperature jumps. fast pressure jumps allow the trigger to be decoupled from structural changes, so with fast structure probe techniques such as time resolved x-ray diffraction, the out-of-equilibrium evolution of these systems can be monitored. despite great advantages, high pressure remains under-utilised primarily due to its technical difficulty. in response to this technology vacuum a high pressure user facility based around a pressure jump cell for small and wide angle x-ray diffraction has been commissioned at beamline i22, diamond light source, uk and will be freely available to the user community. the cell is highly robust requiring virtually no user intervention during an experiment and the pressure system is computer controlled with a graphical user interface and is integrated with the beamline. pressures between 0 and 0.5 gpa are accessible and jumps can be carried out in approximately 5 ms at temperatures from -10 to 120 • c. sample changing has been made simple and fast with a dedicated sample loading port and modular sample holders allow optimisation for a broad range of samples. the transformation of vesicle and lateral distribution of mobile membrane inclusions b. bozic institute of biophysics, faculty of medicine, university of ljubljana, lipičeva 2, si-1000 ljubljana, slovenia membrane inclusions such as membrane embedded peptides or proteins exhibit curvature dependent interaction with the surrounding lipid matrix due to the mismatch between their intrinsic curvature and the local membrane curvature. this interaction causes an inhomogeneous lateral distribution of the inclusions and a corresponding adjustment of the vesicle shape. by taking into consideration that the membrane free energy includes elastic energy of the lipid bilayer and a contribution due to an inclusion-membrane interaction, the configurations of lipid vesicles with mobile inclusions have been studied theoretically. the variational problem to calculate equilibrium vesicle shapes is solved by applying a ritz method based on an expansion in spherical harmonics. in general, vesicle shapes adjust to the presence of inclusions by increasing regions with favorable curvature and decreasing regions of unfavorable curvature in such a way that the lateral distribution of inclusions becomes inhomogeneous. if the number of inclusions or the inclusion-membrane interaction exceeds a certain value, the prolate shapes become globally stable. investigating the structure of pores formed by antimicrobial peptides using epr spectroscopy m. bortolus 1 , k.-s. hahm 2 , a. l. maniero 1 1 universita' di padova, padova, italy, 2 chosun university, kwangju, south korea spin label electron paramagnetic resonance (epr) is a spectroscopical technique effective to study the molecular mobility of membrane components and the membranepeptide interactions, as the timescale of epr is optimally matched to the rotational motions of lipids in membranes. we applied epr to study the pore-forming mechanism of two antimicrobial peptides (amp) that create pores of different dimensions when interacting with liposomes. hp (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) is derived from the n-terminus of helicobacter pylori ribosomal protein l1, and hpa3 is an hp (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) analogue where gln and asp at positions 17 and 19 were substituted by trp. we studied the interaction of the two amp with zwitterionic and negatively charged vesicles, doped with phospholipids spin labelled in the lipid head or at the c5 or c10 positions; the different phospholipids allow us to study the peptide-membrane interaction at different depths relative to the membrane surface. we studied the interaction of the amp with vesicles following the influence of amp on label mobility as a function of temperature and membrane depth. we also prepared spin-labelled dmpc/dhpc bicelles, doped with lanthanide ions (dy 3+ /tm 3+ ) that allow us to macroscopically orient the system using the magnetic field of the epr spectrometer. we studied the interaction of amp with the oriented bicellar system monitoring the effect of amp on the order parameter of the phase. a. chattopadhyay centre for cellular and molecular biology, hyderabad, india we addressed the organization and dynamics of the human serotonin 1a receptor fused to enhanced yellow fluorescent protein (serotonin 1a r-eyfp) expressed in cho cells. serotonin 1a receptors are prototypical members of the gprotein coupled receptor superfamily and represent a prime target for therapeutic actions of several anxiolytic and antidepressant drugs. interestingly, we observed significant retention in fluorescence of serotonin 1a receptors upon triton x-100 treatment of intact cells at low temperature demonstrating their detergent insolubility. we analyzed the role of cholesterol in the plasma membrane organization of the serotonin 1a receptor by fluorescence recovery after photobleaching (frap) measurements with varying bleach spot sizes. our results show that lateral diffusion parameters of serotonin 1a receptors are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin 1a receptors in the plasma membrane. interestingly, results from frap measurements performed under conditions of mild cytoskeletal destabilization suggest that receptor signaling is correlated with receptor mobility, in agreement with the 'mobile receptor hypothesis'. our current work is focused on exploring the oligomerization of the receptor using photobleaching anisotropy measurements and indicates the presence of constitutive oligomers of the serotonin 1a receptor in live cells. expression and reconstitution of connexin43 in pore-suspending membranes c. carnarius 1 , s. kaufmann 2 , m. tanaka 2 , c. steinem 1 1 institute of organic and biomolecular chemistry, university of göttingen, tammannstrasse 2, 37077 göttingen, germany, 2 institute of physical chemistry, university of heidelberg, im neuenheimer feld 253, heidelberg, germany the intercellular communication and electronic coupling between adjacent cells of vertebrates are mediated by gap junctions. these proteins are composed of two connexon hemichannels, whereas each connexon consists of six connexin subunits. each subunit is characterized by two conserved motives: two extracellular loops and four transmembrane α-helices. in this study, we focused on cx43. to obtain large amounts of this protein, we expressed cx43 and cx43+gfp in a rather new expression system: dictyostelium discoideum. in contrast to human tissue cultures, the system allows for high cell densities up to 30 million cells per ml and the cells can be cultivated by fermentation. cx43+gfp was successfully visualized in d. discoideum by confocal laser scanning microscopy, where it was preferentially found in the plasma membrane.after the cells were harvested, plasma membranes were prepared and both proteins (cx43 and cx43+gfp) were verified by western blot analysis. the proteins were solubilized by addition of 5% n-octyl-β-d-glucopyranoside and purified by ion metal chelate affinity chromatography. the activities of both proteins were confirmed by a cytochrome c assay. after the purification of both proteins, they were reconstituted in µm-sized pore-suspending membranes. in the near future, we plan to determine the mobility of cx43 and cx43+gfp in these membranes by fluorescence recovery after photobleaching. o. cañadas, c. casals dpt. biochemistry and molecular biology i, and ciber enfermedades respiratorias, complutense university of madrid, madrid, spain inhaled bacterial lipopolysaccharide (lps) may incorporate into the lung surfactant monolayer. in this study, the effect of smooth lps (s-lps) on the surface activity of lung surfactant was evaluated. to that end we investigated the behavior of dppc films containing s-lps, with and without surfactant protein a (sp-a) in the subphase, using epifluorescence microscopy combined with a surface balance. our data show that s-lps injected into the subphase incorporated into dppc films forming mixed dppc/s-lps monolayers. cospread s-lps fluidized the dppc monolayer as demonstrated by epifluorescence images and changes in the compressibility modulus of the monolayer as a function of s-lps molar fraction (x s−lps ). the presence of low amounts of s-lps in the monolayer promoted early collapse, preventing high surface pressures to be reached. moreover, s-lps hampered the re-spreading of dppc molecules during dynamic compression at s-lps concentrations as small as x s−lps = 0.02. such inhibitory effects could not be relieved by repeated compression-expansion cycles or by adding surfactant protein a. however, sp-a facilitated the squeeze-out of s-lps from dppc/s-lps mixed monolayers, suggesting that sp-a is an s-lps scavenger. cholesterol displaces ceramide from its tight packing with sphingomyelin in the absence of ld phase j. v. busto, j. sot, j. requejo-isidro, f. m. goñi, a. alonso unidad de biofísica (csic-upv/ehu) and departamento de bioquímica, u. país vasco (upv/ehu), spain a set of biophysical approaches have been applied to study the phase behaviour of palmitoylsphingomyelin (psm)/cholesterol (chol) model membranes upon palmitoylceramide (pcer) addition. fluorescence spectroscopy of di-4-aneppdhq-stained psm/chol vesicles reveals no segregation of large liquid-ordered (l o ) microdomains. in contrast, the formation of disperse, compositionally homogeneous l o psm/chol (3:1) nanodomains over a psm gel (l β ) phase is proposed. dsc measurements show that pcer addition to vesicles with coexisting l o and l β phases (low [chol] ) induces the formation of large gel-like psm/pcer microdomains, coexisting with a l o psm/chol phase. the ∆h for the psm/pcer phase at high psm/(chol+pcer) ratio is close to that of the binary mixture in the absence of chol, supporting immiscibility, but no displacement, between chol and pcer-rich phases. on the contrary, both confocal microscopy of guvs and the dsc data coincide in showing that a rise in pcer increases the gel-like phase to a lower extent than in the pure psm/pcer mixture, revealing some cholinduced restriction. in the presence of a pure l o psm/chol phase (high [chol] ), pcer addition is unable to induce the formation of large psm/pcer microdomains. the present data support the role of chol as the key determinant in controlling its own displacement from l o domains by ceramide. hydroquinones modifying lipid membrane morphology c. di vitta 1 , l. marzorati 1 , v. rebbin 2 , s. s. funari 2 1 iqusp, university of são paulo, são paulo, brasil, 2 hasylab, hamburg, germany quinones are important molecules in cells. flavina, ubiquinone and coenzyme q, coq, are associated with electron transfer. coq, having a hydrophobic tail, is soluble in the internal lipid membrane of mitochondria. their reduction leads to the equivalent hydroquinones. we synthesized novel alkylthioquinones aths, to investigate their interaction with phospholipids. one or more long hydrophobic chains attached to the quinone ring alter their hydrophobicity and electron availability. we aimed at their influence on the structure of membranes. different phs highlights the charge/polarity effect on the interaction and on the morphology of the bilayer. for that, pope and popc, lipids with different charges, but identical chains were selected. x-rays diffraction on pope/2,6bath, 100/1 at different temperatures and ph, showed cubic phases coexisting with the usual phases formed by simply hydrated pope, at different phs. for investigation of the charge/polarity, we turned to the popc/2,6bath system (smaller charge/polarity). despite decrease in temperature of phase transition and dimensions of the lattice, the structures were the same as in hydrated lipid, illustrating a less significant effect than on the similar lipid pope. another additive, 2,5ath, mixed with pope showed the effect of multiple thioalky chains. no morphology change was seen, compared to pure lipid. interestingly, despite both additives differ by one thioalkyl chain only, their influence on the pope matrix is so different. cross-linking of phospholipid membranes by calcium-sensitive synaptotagmins synaptotagmins are vesicular proteins implicated in many membrane trafficking events. they are highly conserved in evolution and the mammalian family contains 16 isoforms. we now show that the tandem c2 domains of several calcium-sensitive synaptotagmin isoforms tested, including drosophila synaptotagmin, rapidly cross-link phospholipid membranes. in contrast to the tandem structure, individual c2 domains failed to trigger membrane cross-linking in several novel assays. large-scale liposomal aggregation driven by tandem c2 domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic lightscattering and both confocal and negative stain electron microscopy. high-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem c2 domains results in a constant distance of approximately 9 nm between the apposed membranes. our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem c2 domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion. membrane proteins can be challenging samples to work with and as such often require the use of multiple techniques. here we present an insight into the structure of the antimicrobial peptide melittin in lipid membranes using various techniques. we explore the conformational changes of membrane-bound melittin and its interaction with model membrane lipid systems with a series of spectroscopic methods that can be used in parallel. the techniques used include linear dichroism and ft-ir for orientation information and circular dichroism for conformation information. we use dynamic light scattering for molecular sizing, fluorescence emission for information on peptide environment, thin-layer chromatography for lipid identification, analytical ultracentrifugation to identify oligomerisation state and calorimetry to investigate the thermodynamics. we observe how the physical properties of both the peptide and the membranes affect the insertion kinetics of the peptide in the membrane. phospholipid membranes dynamics: molecular dynamics vs neutron scattering v. conti nibali 1 , m. tarek 2 , u. wanderlingh 1 , g. d'angelo 1 1 department of physics, faculty of science, university of messina, italy, 2 unité mixte de recherche cnrs/uhp 7565, université henri poincaré, nancy, france collective dynamics and single-particle dynamics of hydrated multilamellar phospholipid bilayers (1,2-dimyristoylsn-glycero-3-phosphatidylcholine, dmpc) have been studied by means of all-atom molecular dynamics simulations. here we report results of a gel phase bilayer at 283 k and of a liquid crystal phase bilayer at 303 k. coherent and incoherent dynamic structure factors and meansquare displacements have been calculated from the trajectories for both the inplane and out-of-plane lipid dynamics. moreover, the results have been compared to recent quasi-elastic and inelastic neutron scattering data. optical tweezers allow trapping of particles of different types in a wide range of sizes [1] . among these, unilamellar vesicles are of interest as they are known to be effective vectors in drug delivery and they are also studied as models for cell membranes. this work focuses on the interactions between optically confined unilamellar vesicles and their cross-linking by proteins [2] . synaptotagmins are vesicular proteins implicated in many membrane trafficking events having an accelerating effect on the membrane fusion. calcium-sensitive synaptotagmins are thought to confer calcium sensitivity to the fusion of secretory vesicles with target membranes [3] . the novel optical assay reported here allowed us to visualize the cross-linking of 600 nm liposomes mediated by synaptotagmin and calcium. the use of the optical tweezers approach to investigate the function of other fusogenic proteins related to exocytosis (i.e., snare proteins) is discussed as well. antimicrobial peptides (amps) have received increasing interest as the search for new potential antibiotics has become imperative due to increasing bacterial multiresistance. acylating the natural occurring polymyxin b (pmb) significantly enhances antibacterial activity towards two representative gram-negative bacteria e. coli and k. pneumonia compared to the nonacylated pmb. the aim of the presented work was to study various biophysical parameters, such as partitioning coefficients, zeta potentials and effective charges of a selected amp, mastoparan-x (mpx) and a propanoylated (pampx) and octanoylated (oampx) analogue, respectively. fluorescence spectroscopy, isothermal titration calorimetry and ζ-potential measurements were the main techniques used for the measurements. we employed 2 different luv model systems; a partially charged (popg/popc 1:3) and a neutral system (popc). for the neutral luvs there was an increase in partitioning with increasing length of the acyl chain, whereas partioning into the partially charged luvs was governed by a balance of hydrophobic and electrostatic contributions. the selectivity for the partially charged luvs over the neutral luvs was in the order pampx, mpx and oampx. the modeled effective charge for the peptide followed the same trend as the partitioning coefficient for the three peptides for the respective luv systems. does the lysosomal membrane need triglycerides? a spectroscopic study of a simple model membrane l. duelund 1 , k. pakkanen 2 , m. vuento 2 , j. h. ipsen 1 1 memphys -center for biomembrane physics, university of southern denmark, odense, denmark, 2 nanoscience center, university of jyväskylä, jyväskylä, finland lysosomes are intracellular organelles in which proteins and other macromolecules are degraded. morphological and functional changes in different compartments of the endocytic pathway are connected to several diseases. a crucial step in understanding biogenesis of lysosomes and their role in disease conditions, is to characterise the properties of the lysosomal membrane. by the use of tlc we have found that lysosomes contain non-neglible amounts of triglycerides (tg). to investigate how the presence of tgs could influence the lysosomal membrane, we have investigated the properties of a mixture of popc and triolein, as a simple model for the lysosomal membrane. we found the system to form two types of popc-rich membranes. these were determined as co-existing phases based on their spontaneous and stable separation and named heavy and light phase according to their sedimentation behaviour. by using epr and fluorescence spectroscopy the physical properties, including order, fluidity and water penetration, of these phases were found to differ markedly despite of their almost identical composition. the results suggest that presence of tgs on lysosomal membranes could have a crucial effect in the barrier functions and thus, the integrity of the organelle. model membranes with the capacity to align in magnetic fields such as bicelles and magnetically sensitive vesicles are of high interest, since their macroscopically alignment allows for the determination of structure, dynamics and topology of molecules within the membrane [1] . we performed 31 p solid state nmr on a magnetically sensitive lipid possessing a large positive magnetic anisotropy introduced in form of a biphenyl unit during lipid synthesis in one of its acyl chains (1-tetradecanoyl-2-(4-(4biphenyl) butanoyl)-snglycero-3-phosphocholine (tbbpc)). the phosphorus lineshapes of tbbpc mlvs studied at various magnetic fields (7.1t, 9.4t, 11.7t, 16.4t), revealed a drastic change in shape upon exposure to fields > 9.4 tesla: resonances resulting from phospholipid molecules oriented perpendicular to the magnetic field decrease whereas resonances resulting from parallel oriented molecules increase. this is a sign for magnetically induced vesicle deformation from vesicle to oblate ellipsoid shape. factors influencing this drastic deformation, such as magnetic anisotropy, membrane elasticity, lipid chain length and field dependency are discussed based on existing theories [2] . hepatitis g virus (gbv-c/hgv) is an enveloped viruses belonging to the flaviviridae family. clinical findings have suggested that in people co-infected with gbv-c/hgv and human immunodeficiency virus (hiv), delayed progression of aids has been observed (1). the mechanism by which this virus may inhibit the progression of aids remain to be elucidated. enveloped viruses acquire their lipid membranes by budding through host cellular membranes (2) , in this process; fusion peptides play an important role. study of the interaction of hgv-c peptides with lipid membranes could lead us useful information about the mechanism that takes place. in this work we present a study on the effects of e1 peptides on the fluidity of and polarizability of model membranes (dmpc and dppc liposomes) by dsc and fluorescence polarization. both techniques showed that the presence of the studied sequences in phospholipid mixtures already affected the thermotropic properties of the gel to liquid-crystalline phase transition. systems were thermodynamically characsmall angle neutron scattering (sans) studies have been performed to study the structural changes induced in membranes of vesicles prepared from phospholipid and mixed phospholipid-sterol mixtures, in the presence of different concentrations of the anti-fungal drug, amphotericin b (amb).the vesicles, sonicated to a mean size∼100nm, were prepared using dimyristoylphosphatidylcholine(dmpc) or dmpc-cholesterol or dmpc-ergosterol mixtures -with both of the mixed systems involving 30mol% sterol. analysis of the sans data show that when the concentration of amb added is just above the drug's cmc (∼1µm) there is an increase in the membrane thickness of both the dmpc-chol and dmpc-erg vesicles (both cases + 4å), but the thickness of the pure dmpc vesicle membranes remains the same as in the absence of amb. when amb is added at a concentration in excess of its cmc (∼10µm), the mixed-sterol vesicles show the same changes in membrane thickness as observed with the lower amb concentration, and the pure dmpc vesicles again remain unaffected. on the basis of these studies, therefore, there appears to be no difference in the structural changes induced by the insertion of amb into the model fungal cell membranes (mimicked by dmpc-erg vesicles and those resulting from its insertion into the model mammalian cell membranes (mimicked by dmpc-chol vesicles). the third transmembrane helix of bacteriorhodopsin, also known as phlip, is a unique model system for studying the interactions of a natural transmembrane domain with lipid membranes: depending on ph, the water-soluble peptide either adsorbs superficially or inserts as a transmembrane helix on addition of lipid vesicles [1] . published values for the free energies of these processes were based on a stoichiometric model invoking two distinct sets of binding sites [2] . however, discrepancies between data obtained from different experimental techniques and inconsistencies between experimental and expected temperature dependencies suggest that these values should be taken with caution. we therefore reassessed membrane interactions of phlip using titration calorimetry and fluorescence spectroscopy. if electrostatic effects at the membrane surface are taken into account, the data can be described quantitatively by a partition equilibrium, but not by a stoichiometric binding model. the thermodynamics of membrane partitioning differ substantially from those determined previously [2] and draw a different picture of peptide-lipid interactions. beyond deepening our insights into the first step of the two-stage model of membrane protein folding, this also sheds light on the ability of phlip to drag cargo molecules across lipid membranes. adsorption of monolysine and polylysines at the surface of lipid membranes of varied composition was studied by two methods. both methods -the electrokinetic one, measured the surface potential of liposomes, and the intramembranouse field compensation sensitive to boundary potential of planar bilayer lipid membranes -detected the positive changes of the potentials for membranes with negatively charged component (cardiolipin, cl). neither monolysine, nor polylysines adsorbed at neutral membranes (phosphatidylcholine, pc). pentalysine show the difference between these potentials for the membranes composed from cl/pc mixtures. this difference is attributed to dipole part of boundary potential and indicates the changes in lipid packing. polylysines show high affinity to the membrane and saturation with plateau. these saturation levels correspond to surface charge densities 0.005 and 0.016 c/m 2 for oligomers with 5 and 12 units, and 0.032 c/m 2 for polymers with 130 and 1435 units. these values do not depend on the ionic strength of background electrolyte but proportional to the content of negatively charged components in the lipid bilayer. the polylysine layer at the mica surface was studied by atomic-force microscope (afm) technique. it was shown that pentalysine molecules cover the surface by the layer of 0.8 nm thickness and polylysines of high molecular weight by the layer up to 4 nm. effects of lysolipids on the mechanical stability of lipid membranes j. r. henriksen 1 , l. feldborg 1 , j. h. ipsen 2 , t. l. andresen 1 1 technical university of denmark, dtu nanotech, frederiksborgvej 399, b124, 4000 roskilde, denmark., 2 university of southern denmark, department of physics and chemistry, memphys center, campusvej 55, 5230 odense m, denmark lysolipids (lpcs) and fatty acids (fas) are the natural products formed by the hydrolysis of phospholipids. lpcs and fas form micelles in solution and thus act as detergents in the presence of lipid membranes. in the present study we investigate the detergent strength of a homologous series of lysolipids (lpcs) on popc lipid membranes by use of isothermal titration calorimetry (itc) and vesicle fluctuation analysis (vfa). the membrane partition coefficient (k) and critical micelle concentration (cmc) are determined and found to obey an inverse proportionality relation (cmc x k ∼ constant). the partition coefficient and critical micelle concentration are used for the analysis of lpc's effect on the membrane bending rigidity. the dependence of the bending rigidity on the lpc membrane coverage has been analyzed in terms a phenomenological model based on continuum elastic theory which yield information about the curvature inducing properties of the lpc molecule. the results reveal: (i) an increase in the partition coefficient with lpc acyl-chain length and (ii) the degree in acyl chain mismatch between lpc and popc determines the magnitude of the membrane mechanical perturbation per lpc molecule on the membrane. patch clamp electrophysiology remains the gold-standard for ion channel research because of the information richness of the data produced. automated planar patch clamp devices, with their higher throughput and high data information content, have made the technique accessible to a wider audience. nanion technologies offers two planar patch clamp workstations combining higher throughput with high data quality. the port-a-patch records from a single cell at a time and the patchliner from up to 8 cells simultaneously with high success rates (typically 60-80%). both, the port-a-patch and the patchliner are bench top patch clamp rigs which uses a planar borosilicate glass chip for obtaining a giga-seal for electrophysiological recordings. suction applied from the underside of the chip is used to attract a single cell to the recording site without the need for optical visualization. both workstations have been successfully used for whole cell, perforated patch, and cell attached recordings as well as for guv-bilayer recordings. due to the versatility of nanion`s products it is possible to study a wide variety of ion channels including nav, kv, cav, ligandgated, herg or reconstituted proteins such as ksca, cx26, ompc. special features unique to nanion products, including but not limited to internal solution exchange and temperature control, expand the experimental possibilities. waves on lipid monolayers j. griesbauer, s. boessinger, a. wixforth, s. f. matthias univiversity of augsburg, experimental physics i, biological physics group, augsburg, germany " for the sake of illustration we shall try to provide a physical basis for the equations, but must emphasize that the interpretation given is unlikely to provide a correct picture of the membrane." [ hodgkin&huxley, 1952 ] to explain the occurrence of reversible heat production during nerve pulse propagation it has been suggested by multiple authors [wilke,kaufmann,heimburg] that travelling sound waves and not ion channels may offer a better explanation than the hodgkin&huxley theory. therefore, if sound waves are an essential feature of the nerve membrane they should also appear on lipid monolayers where ion conductivity is evidently absent. here we demonstrate that sound waves can be excited on a lipid monolayer by using a set of planar electrodes incorporated into the monolayer and driven by an alternating voltage. not only do our results indicate propagating waves on lipid monolayers in accordance with their thermodynamic predictions, but importantly, no significant attenuation is detected proposing an adiabatic phenomenon. in order to provide evidence that our physical explanation provides a correct picture of the membrane, direct detection of the waves was done, whereas a clear transduction of signals was shown. finally, the impact of toxins, neuropharmaca and anaesthetics needs to be integrated in our physical picture of the nerve membranes, what can easily be done now and could deliver a new approach for understanding their physical mechanism. our earlier studies showed that organometallic compounds (otc) in the presence of uvc radiation enhanced the degree of phosphatidylcholine (pc) liposome oxidation, whereas quercetin effectively protected the membrane. the present investigation is concerned with the effect of uvb and otc (chlorides of diphenyl-and dibutyltin, triphenyl-and tributyltin), both in combined action and separately, on oxidation of erythrocyte proteins, pc liposome and albumin. the degree of oxidation of proteins and liposomes induced by otc and radiation, also in the presence of selected antioxidants (trolox, quercetin) was determined on the basis of changes in the number of sulfhydryl groups, carbonyl groups and malone dialdehyde, respectively. the studies indicate that uvb induces pc liposome and erythrocyte oxidation, whereas in the case of albumin it causes both an increase in the number of c=o groups and free sh groups (most probably, braking sulphonic bridges). otc compounds interact with membrane biomolecules both as weak pro-and antioxidants, also in combined action (uvb plus otc). the prooxidative effects are markedly diminished by application of antioxidants. the action of quercetin results from its ability to incorporate into membranes and formation of complexes with otc (liposome>erythrocyte>albumine). this work was supported by grant n n3364 34. a phospholipase c/ sphingomyelinase from pseudomonas aeruginosa has been assayed on giant unilamellar vesicles (guv) consisting of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol, at equimolar ratios. the enzyme activity modifies the chemical composition, thus the physical properties of the bilayer, and conversely the latter influence the enzyme activity. biochemical assays of enzyme activity, together with confocal fluorescence microscopy examination of guv provide novel information about the system. the original lipid composition in the absence of enzyme gives rise to lateral phase separation of liquid-ordered and liquid-disordered domains in the guv. the two enzyme end-products, diacylglycerol and ceramide, have opposite effects on the bilayer physical properties, the former abolishes lateral phase separation, while the latter generates a new gel phase. morphological examination of individual guv shows that the enzyme binds preferentially the more fluid (or more disordered) domains, and that, in most cases, it causes the fluidification (disordering) of the other domains.. cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. to this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures (33-75 mol% cholesterol) have been measured and compared with the original mixture before lipid hydration. there is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the bilayers is much lower than expected. the maximum solubility of chol in bilayers containing saturated pc or pc with less than four unsaturations is 50-60mol%, while in polyunsaturated pc, e. g. dic18:4, and in sphingomyelin the maximum chol contents is 23mol%. a quantitative analysis of the vesicles is thus required before any experimental study is undertaken. membrane fusion assay based on poresuspending lipid bilayers i. höfer, c. steinem institute of organic and biomolecular chemistry, university of göttingen, germany membrane fusion has attracted significant interest due to its biological relevance. thus in recent years a great variety of fusion assays has been developed. since the process of membrane fusion is not yet fully understood, our aim is to develop a new fusion assay based on pore-spanning membranes, which have been proven mechanically stable, highly insulating and rather tension free. the application of these so-called micro-blms should allow simultaneous monitoring of lipid mixing, content release as well as electrical readouts. furthermore both membrane sides can be addressed individually to apply transmembrane potential or fusion modulating compounds. first results show that the micro-blms provide the opportunity to investigate lipid bilayer fusion by means of fluorescence microscopy. the formation of porespanning membranes is achieved by the painting technique of dphpc doped with oregon green dhpe dissolved in ndecane. the addition of large unilamellar vesicles doped with texas red dhpe allows direct observation of single fusion events. lipid mixing during fusion leads to a decrease of the donor fluorescence (oregon green) and an increase in acceptor fluorescence intensity (texas red) in the plane of the planar bilayer due to fret. in future work simultaneous monitoring of lipid-mixing and content release combined with electrical measurements are planned to gain further insight into different intermediate steps of membrane fusion. the kinetics of membrane-peptide folding and orientation m. r. hicks department of chemistry, university of warwick, uk. understanding interactions of peptides with lipid membranes is essential if we are to be able to design new and better antibiotic peptides. there are different steps in these interactions and following the kinetics of these processes requires that we combine the information from different biophysical techniques. here we present data on the kinetics of peptidemembrane interactions using circular dichroism to report on conformation and linear dichroism to report on orientation of the peptides in/on membranes. these are combined with other techniques such as dynamic light scattering and fluorescence spectroscopy to elucidate the mechanisms of action of the peptides. one of the most important aspects of membrane-active peptide design is that of specificity. we investigate specificity for different types of membranes by using libraries of lipids with different properties of charge, chain saturation and curvature stress. in this way one can test for effects of e.g. negatively charged head groups found in bacterial membranes or cholesterol found in animal and human membranes. using these approaches it is proposed that we will be able to modify peptide sequences, test what part of the kinetic processes are affected and subsequently use this information to design new and better antibiotics. k. kubica, h. misiak wroclaw university of technology, wroclaw, poland reversible membrane electroporation, that can be observed as a effect of electric field influence on biological membranes, finds application in cell delivery of biologically active compounds. the membrane susceptibility for creation of stable pores, depends on electric field parameters applied to the membrane as well as on the membrane environment (ionic strength, ph, temperature) and membrane molecular composition. we have already shown that the changes of van der waals lipid chain interaction effect the process of pore formation. there are known factors which increase or decrease the membrane respond to electric field. because pores appear in lipid membrane matrix it make sense to study this phenomenon using model planar lipid membranes. as lipids are very receptive on oxidizing factors we decided to study the relation between lipid oxidation and membrane reaction on electric field. with the use of four electrode galvanostat we are examining the influence of factors initiating lipid oxidation (uv, fe 2+ ) and the efficiency of some antioxidant compounds of lipid protection on electric properties of lipid membrane. the product of lipid oxidation is determined by characteristic reaction with tiobarbituric acid. we want to determine the role of polyunsaturated fatty acids on membrane properties also. results of our work will be useful in optimization of medicine distribution, aided by electric field, into cells. budding of giant phospholipid vesicles induced by β2-glycoprotein i j. kovacic, b. bozic, s. svetina institute of biophysics, faculty of medicine, university of ljubljana, 1000 ljubljana, slovenia β 2 -glycoprotein i (β 2 gpi) is classified among amphitropic proteins: in its inactive form it is dissolved in plasma and in its active form it is bound to membrane. in comparison to other amphitropic proteins it exhibits a distinct membrane interaction behavior. a patch of positively charged amino acid residues contacts anionic phospholipids via electrostatic interactions, and a hydrophobic loop anchores the protein into the outer leaflet of the membrane via hydrophobic interactions. the binding constant depends on physical properties of the membrane such as membrane lipid composition, surface potential, lipid packing density and curvature. the binding of an amphitropic protein alters the membrane's spontaneous curvature as well as the difference between the equilibrium areas of membrane leaflets. theoretical model has been developed to predict vesicles shape transformations realized by the formation of buds. it was shown that the effects are stronger for flaccid vesicles which were therefore chosen for our experimental investigations. vesicles were composed of 20 % pops and 80 % popc, and flaccidity was achieved by an osmotic adjustment. the solution containing β 2 gpi was subsequently injected to a chamber with flaccid vesicles by a syringe pump. observation took place under a phase-contrast microscope. experiments showed a concentration dependent occurrence of buds. their number and size were related to the degree of vesicle flaccidness. the interplay between detergent cohesion and peptide adsorption on the structure and dynamics of a glycophorin a tm-detergent complex j. khao, j.-p. duneau, j. n. sturgis lism, cnrs -aix marseille university, marseille, france in spite of the major interest of membrane proteins at functional, genomic and therapeutic scales, their biochemical and structural study remain challenging as they require, solubilization in detergent micelles. the complexity of this task arises from the structural dependence of membrane proteins on their anisotropic environment and in particular by a delicate balance between different physico-chemical properties. in order to study these properties in a small protein detergent complex, we have used molecular dynamics simulations on the transmembrane part of glycophorin a (gpatm) solubilized in micelles of the detergent di-hexanoyl-phosphatidylcholine(d6pc). we show that the molecular aggregates organizes to give distinct populations of detergent molecules. those molecules which loosely interact with the peptide are preferentially involved in highly cohesive inter-detergent interactions that impose a global bilayer structure to the micelle. interaction profiles of other detergents with gpatm depend upon the nature of residues along the surface of the peptide. this topology dependence leads to different modes and strength of interactions that ultimately constrain the orientation of the micelles around the peptide. this simple model illustrates how differential detergent selectivity for faces and strong constraints coming from purely environmental features could influence transmembrane helix packing, membrane protein structure and assembly. in this paper we present a study of a new family of bolaamphiphiles. these amphiphiles are unsymmetrical bolalipids having one sugar polar head and the second glycine betaine polar head. they are potentially useful for pharmaceutical or cosmetics applications as vectors for drugs. therefore it is important to investigate their self-assembled properties. the chemical variations that we introduced in this new family concern the length of the main chain that connects two polar heads as well as the length of the side chain placed on the position 1 of the sugar moiety. another variation concerns the introduction of a diacetylenic unit into the main chain in order to rigidify it. we have performed the saxs (small angle x-ray scattering) measurements on the dehydrated compounds as a function of temperature and observed the lamellar liquid crystalline structures. we also measured the saxs spectra of aqueous solutions of these compounds that have shown lamellar l α structure in all cases. these measurements are compared with polarised optical microscopy measurements that confirmed our interpretation. formation of membrane tubular structures induced by phase separation in giant vesicles y. li, r. lipowsky, r. dimova max planck institute of colloids and interfaces, science park golm, 14424 potsdam, germany tubular membrane structures (also known as tethers) exist widely in eukaryotes. abundant work has been done on tube extrusion from cells and model membranes under the application of external forces. we present a novel system allowing tube formation in the absence of external forces. aqueous solutions of two chemically dissimilar polymers, polyethylene glycol (peg) and dextran are encapsulated in giant vesicles, a cell-size model system. the exposure of vesicles to hypertonic solutions induces phase separation of the internal aqueous polymer solution. the excess membrane area created by this vesicle deflation, engages in the formation of tubular structures. membrane tube formation and phase separation are coupled processes. hydrodynamic flows and changes in the membrane spontaneous curvature during phase separation might be the driving forces for tube formation. the tubes are rather stable: without external perturbation, they can exist for several days. they prefer to be located in the peg-rich phase at low polymer concentration. at high concentration, they are absorbed at the interface of the liquid phases to lower the surface energy of the system by decreasing the contact area between the liquid phases. the membrane tubes can be retracted back to the vesicle surface by increasing the membrane tension via vesicle aspiration. membrane tubes, which can form and be retracted easily, might be relevant to lipid storage in cells. insight into the antimicrobial mechanism of a de novo auto-assembling peptide b. legrand 1 , m. laurencin 2 , e. duval 3 , c. zatylny 3 , j. henry 3 , m. baudy-floc´h 2 , a. bondon 1 1 rmn-ilp, umr cnrs 6026 -univ. de rennes1, france, 2 icmv, umr cnrs 6226 -univ. de rennes1, france, 3 pemm, umr ifremer 100 -univ. de caen, france a short (14 residues) de novo antimicrobial peptide (k4) composed of a cationic polar head and a hydrophobic tail was studied. it exhibits a broad spectrum of antimicrobial activity on bacteria. no haemolytic activity or cytotoxicity on eukaryotic cells are observed at mic. when bacteria are lysed by the k4, spherical objects are observed on the sem micrograph. k4 was structurally studied using various membrane mimetic media such as different micelles and small unilamellar vesicles (suv) of different composition. cd revealed that k4 adopt various structures (random, β-turn, α-helix) in the presence or the absence of detergents or phospholipids. nmr structures confirmed the α-helical structure of k4 hydrophobic tail in presence of sds. k4 self-assembles at high concentration as observed by sem and dls. we suggest that k4 may act as a surfactant building mixed microsomes composed of peptide and lipids. this destabilization mechanism of the bacterial membrane support the "detergent like model" previously described in the literature. oxidative stress and the membrane dipole potential; modulation with tocopherol s. le-nen-davey 1 , b. m. davis 2 , j. l. richens 2 , k. a. vere 2 , m. w. tilley 2 , p. g. petrov 1 , c. p. winlove 1 , p. o'shea 2 1 biomedical physics group, university of exeter, uk, 2 cell biophysics group, university of nottingham, uk tocopherol, a component of vitamin e well know for its antioxidant properties, has recently been thought also to influence the structure of cellular membranes. tocopherol treatment reduces hyperglycaemia induced oxidative stress and the associated endothelial dysfunction which is a precursor to the vascular complications of diabetes. tocopherol and insulin interactions are also modified by hyperglycaemia. it is unclear whether these clinically important effects arise from the anti-oxidant and/or structural properties of tocopherol. we have therefore measured the dipole and surface potentials of phosphatidylcholine vesicles containing different amounts of cholesterol, ketocholesterol and tocopherol. we have also investigated the effects of hyperglycaemia, ketocholesterol and tocopherol, alone and in combination, on microdomain formation and interactions of insulin within the membranes of cultured endothelial cells. both sets of experiments indicate that tocopherol causes significant modifications of the membrane dipole and surface potentials. the physiological significance of these changes will be discussed. t. d. lazzara 1 , a. janshoff 2 , c. steinem 1 1 georg-august university göttingen, institute for organic and biomolecular chemistry, tammannstr. 2, 37077, germany, 2 georg-august university göttingen, institute for physical chemistry, tammannstr. 6, 37077, germany cell penetrating peptides (cpp) have been shown to penetrate cellular membranes. they have been of interest for their ability to translocate not only themselves through cellular membranes, but also carry along with them, cargo as large as iron nanoparticles. the exact entry mechanism remains unclear, but has been shown to vary with peptide sequence. their translocation properties have been demonstrated through different experiments involving vesicles, cells and living animals. we plan on using nano-black lipid membranes (nano-blm), which span the pores of nanoporous materials as a model system to study the interaction between cpp and lipid membranes. the nanopores can be used as cellular containers whose interior surface can be functionalized with receptors for biotin-streptavidin recognition. we hope that this system will provide greater control over experimental variables, such as the type of cpp and lipid used, as well as provide kinetic data that can be used to evaluate cpp activity and the kinetics of cargo transport. ethanol induces phospholipid acyl chain interdigitation. while much is known, important issues remain unclear, such as the role of lipid domains. the main purpose of this study was to follow, in real time, the changes induced by ethanol on supported lipid bilayers with nano to microdomains by in situ atomic force microscopy. to this goal, a pure lipid in the fluid phase (dopc), a pure lipid in the gel phase (dppc), binary phospholipid mixtures with gel/fluid phase coexistence, and cholesterol-containing, raft -forming mixtures (dopc/dppc/cholesterol and dopc/sphingomyelin/cholesterol) were investigated. from the height differences observed upon ethanol addition to pure lipids (dppc and dopc), and to dopc/dppc mixtures, it is shown that in the binary system the interdigitation of the fluid phase occurs prior to the gel phase. however, for the lipid rafts mixtures the simultaneous interdigitation of both raft and non raft portions of the membrane is observed both in mica and silicon substrates. for all compositions studied, domain formation or rearrangement accompanied by lipid bilayer expansion occurs as a consequence of interdigitation. these results show the ability of ethanol to influence the bilayer properties in different ways according to membrane composition. ethanol may exert its biological effects by reducing bilayer thickness, and also by changing membrane proteins conformation and lateral distribution, as a consequence of the altered properties of the lipid bilayer. interactions between non-steroidal anti-inflammatory drugs and a pc/cholesterol bilayer m. markiewicz 1 , t. librowski 2 , p. serda 3 , m. pasenkiewicz-gierula 1 1 faculty of biochemistry, biophysics and biotechnology, jagiellonian university " 2 department of pharmacodynamics, medical college, jagiellonian university " 3 regional laboratory, jagiellonian university, krakow, poland. the non-steroidal anti-inflammatory drugs (nsaids) are among the most frequently prescribed and used drugs [1] . there are several side effects connected with frequent use of nsaid, mainly gastrointestinal ulcers and bleedings. a plausible molecular mechanism of these effects are direct interactions of nsaids with gastric phospholipids [2, 3] . the influence of three well-known non-steroidal antiinflammatory drugs with a diverse gastric toxicity (aspirin, ketoprofen and piroxicam) and three newly-synthesized xanthone derivatives (belonging to the nsaids) on the structure and dynamics of lipid bilayers was studied using small angle x-ray scattering and molecular dynamics simulations. the results showed some correlation between nsaid toxicity and its binding to the lipid polar groups. this binding increases membrane fluidity by reducing its density due to an increased membrane surface area. a reduced lipid packing in the membrane most likely increases gastric mucosa permeability, which can result in a decreased resistance of the gastric mucosa to luminal acid. we studied the partition of the anionic amphiphile 1pyrenesulfonate (psa) into mlv and luv (produced by extrusion), composed by zwitterionic popc and zwitterionic/anionic mixtures with pops (until 10 mol %). psa is an anionic amphiphile that mimics several xenobiotics (e.g. pharmaceuticals, pesticides) and endogenous substrates that interact with biological membranes. we found increasing b. lorenz, s. schuy, a. janshoff georg august university, göttingen, germany cell-cell and cell-virus interactions are ubiquitous in living organisms. the analysis of the forces acting between two cells or a cell and a virus gives insight into details of these interaction processes such as their stochastics, cooperativity, reversibility, and energy landscape. using colloidal probe microscopy in conjunction with solid-supported lipid bilayer techniques, we mimic the contact between two membranous compounds displaying sponge glycolipids or viral peptides on their surfaces. after spreading functionalized lipid bilayers on both -a colloidal probe and a silicon wafer surface -the molecular interactions are quantified by means of force-distance curves. by probing the dynamic interaction strength between the viral peptides n36 and c34 we aim for a deeper understanding of the role of these peptides in the complex process of the formation of the prehairpin intermediate as the key step in retroviral fusion. as far as the cellular interaction of marine sponge cells is concerned, we intend to investigate the strength, specificity, and the ca 2+ dependency of the self-recognition between sponge glycans. the systems advantages are the flexibility of the membrane composition and the control over the distribution of receptor molecules in the membranes. since adhesion in biological systems relies heavily on cluster formation within the biomembrane, we plan to mimic the clustering by "printing" interaction domains and comparing the results to homogeneous samples. development of video-rate imaging microscope using laurdan and its applications to lipid raft t. ohba, k.-i. muto, t. kiuchi, k. ohki department of physics, graduate school of science, aobaku, sendai, japan 980-8578, ohki@bio.phys.tohoku.ac.jp various biological functions are located on cell membranes, and the biomembranes maintain heterogeneity as microdomain even in its dynamic structure. existence of such domain was reported as phase-separated 'rafts' in a cell. and the bio-functions of membrane protein are affected by physical property of its surrounding lipid bilayers. laurdan is a useful fluorescence dye to monitor membrane fluidity, and we have developed an instrument to image spatial and temporal change in membrane fluidity by use of laurdan. in order to examine role of 'micro-domain' in biomembrane, we applied this imaging instrument to a giant liposome, cho cells and pc12d cells. fluorescence microscope was equipped with a home-build dual-view optical unit. microscopic image of membranes stained with laurdan is separated into an image at 440 nm and an image at 490 nm by the combination of monochromatic filters and dichroic mirrors. the each image is focused on an image plane of a ccd camera side by side. and generalized polarization (g.p.) image is calculated from those images by personal computer according to the definition of g.p. the g.p. imaging at video rate was applied to a giant liposome of composed of dmpc and dmpe in order to observe phase separation. it was also examined that specific interaction of sphingomyelin and cholesterol in living cho cells and neuritis protrusion from raft region of pc12d cells stimulated by neuron growth factor. current fluctuations in biological lipid membranes from human cell lines s. nuschele, a. wixforth, m. f. schneider university of augsburg, experimental physics 1, univer-sitätsstrasse 1, d -86159 augsburg, germany lipid membranes can undergo phase transitions at physiological temperatures. not only do single lipid component membranes show melting behaviour but also biological lipid membranes from eukaryotes as well as from prokaryotes. performing calorimetric and monolayer studies we are able to detect phase transitions in extracted membranes from different human cell lines (e.g. keratinocytes and melanoma cells). close to and in the melting transition regime we measured distinct channel like current fluctuations. the opening times of these current fluctuations can be predicted based on the lateral compressibility measured from the monolayer isotherm and agree well with the experimental data [*]. the applied method of extraction excludes the presence of functional proteins in the membrane rendering the lipid bilayer as the source of the observed current fluctuations. the molecules are composed of single hydrophobic tail and two hydrophilic aldonamide-type groupings (gluconyl c12-dga or lactobionyl c12-dla) linked by the propylene chain at the nitrogen atom. the micellization processes of c12-dga and c12-dla were studied by means of itc. the critical micelle concentrations, the enthalpies (∆h m ) and the entropies (∆s m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (∆g m 0 (hy)) were calculated. qspr analysis was also used to predict cmc of studied compounds. the interactions of c12-dga and c12-dla with model membranes (dppc and dppc/chol. bilayers) were studied by means of dsc. using quantum computations some basic molecular properties were calculated. the conformational space was explored using molecular mechanics. obtained results were compared with those for analogical compounds with single head groups, e.g. with also synthesised by us n-alkanoyl-nmethyllactitolamines (c n mela) and common sugar-based surfactants c 12 gluc and mega-10. enfuvirtide (t-20) was the first hiv-1 fusion inhibitor peptide approved for clinical use. t-1249 is an inhibitor still under evaluation. previous studies, based on tryptophan intrinsic fluorescence, showed that the peptides interact differentially with membrane model systems (luv) with different lipid compositions. studies with human blood cells were necessary to further establish the role of membranes on these peptides mode of action. an experimental strategy was applied based on the membrane dipole potential, as measured by the fluorescent probe di-8-anepps. human erythrocytes and peripheral blood mononuclear cells (pbmc) were successfully labeled. for both systems, a fusion inhibitor concentration-dependent decrease on di-8-anepps fluorescence excitation ratio (a measure of the spectral shift and dipole potential) was observed. the quantitative analysis of these variations indicated that t-1249 has an approximately ten-fold higher affinity towards erythrocyte and pbmc, when compared with enfuvirtide. this is in agreement with the previously known adsorption of t-1249 on cholesterol-rich membrane domains and with its higher partition constants. hiv associates with erythrocytes in vivo, which can constitute a route to deliver peptide to the viral membranes (also rich in cholesterol). lymphocyte membranes can concentrate and accelerate the drug interactions with its molecular target, gp41 in its exposed conformation. oxidation of low density lipoprotein (ldl) is known to be a key step in atherogenesis, leading to inflammation, proliferation and apoptosis of cells of the arterial wall. these effects are largely exerted by oxidatively fragmented phospholipids, which are highly exchangeable between cells, tissues and lipoproteins. in particular, pgpc has been identified in minimally modified ldl and has been reported to elicit a wide range of pathophysiological responses in vascular cells, e.g. the activation of apoptotic signaling pathways. we investigated here the behavior of the fluorescent oxidized pgpe-alexa647 compared to dhpe-bodipy in artificial supported lipid bilayers with different cholesterol contents. the two labeled lipids differ in the type of membrane insertion: while dhpe-bodipy is anchored to the membrane via two fatty acids, pgpe is incorporated with only one fatty acid. the second chain is an acyl fragment in sn-2 position, which represents the oxidation product of an unsaturated acyl chain. with increasing cholesterol content we observed a decrease in the diffusion coefficient for both lipids. interestingly, the diffusion of the oxidized lipid was reduced in a higher degree compared to that of the non-oxidized lipid. the calculated ratios of the diffusion constants of pgpe-alexa647 and dhpe-bodipy suggest a different type of interaction with cholesterol. membrane proteins account for a third of all proteins encoded for by the human genome, and play a vital role in a number of cellular processes. few membrane protein structures have been determined to date in comparison to soluble proteins. this discrepancy is due to experimental difficulties in preparing membrane protein samples for structural analysis. traditional techniques for studying membrane proteins by x-ray crystallography or solution nmr use detergent solubilised proteins which can differ from their native confirmations. solid-state nmr allows the study of transmembrane proteins in lipid bilayers, representing a more native like environment in which to obtain biologically relevant structural information. we have been working on the development of reliable methods for reconstitution of transmembrane peptide into liposomes using glycophorin a as a model tm protein. using reconstitution methods based on the removal of detergent by bio-beads, we have used electron microscopy to screen the ideal conditions for insertion of gpa into liposomes in preparation for mas nmr experiments. em has allowed us to identify conditions favourable for insertion of peptide into lipid vesicles and those that result in aggregation. in order to confirm the secondary structure and insertion of gpa into lipid vesicles, techniques such as cd, ocd, ftir and dls have been used to provide quantitative information in addition to the visual results from em. nonesterified fatty acids regulate a broad spectrum of metabolic activities and are involved in many physiological, pathological and/or pharmacological processes in living cells. they spontaneously transfer between donors and acceptors such as fatty acid binding proteins and lipid membranes. we focus on protein-lipid dispersions of human serum albumin (hsa) and sterically stabilized liposomes (ssl) composed of dppc and appropriate amount of peg:2000-dppe in which stearic acids (sa) are inserted either in the protein or in the ssl. exploiting the fact that hsa has a single tryptophan residue and that the intrinsic trp-fluorescence emission signal is quenced by the presence of sa, the kinetics of sa transport between hsa and peg:2000-grafted dppc membranes is studied by means of fluorescence. it is found that the transfer of sa between hsa and ssl is a first-order process and the kinetics of transfer depends on the type of donor and acceptor matrix, on the temperature (i.e., on the physical state of the lipid bilayers), and on the grafting density of the peg-lipids at the protein/lipid interface. indeed, in the absence of polymer-lipids, the rate of transfer increases with temperature in both directions of transfer and it is faster for the passage from dppc bilayers to hsa. the presence of polymer-lipids reduces the rate of transfer both in the mushroom and in the brush regime of the polymer-chains, especially for lipid membranes in the fluid phase. a. orth 1 , w. römer 2 , l. johannes 2 , c. steinem 1 1 institute for organic and biomolecular chemistry, university of goettingen, germany, 2 laboratoire trafic et signalisation, institut curie, paris, france shiga toxin (stx) from shigella dysenteriae is an ab 5 -class bacterial toxin. infections with stx lead to the haemolyticuraemic syndrome, which is known to be a major cause for renal failure at an early age. the interaction of the homopentameric b-subunit (stxb), which is responsible for binding and intracellular transport of the holotoxin, with its cellular receptor, the glycosphingolipid gb 3 , is the first step for endocytosis of the toxin. one b-subunit can bind up to 15 gb 3 -molecules to form stxb-gb 3 -clusters causing negative curvature of a membrane. the binding of stxb to giant unilamellar vesicles, composed of dopc, cholesterol and gb 3 induces tubular membrane invaginations, which were also found in experiments with energy-depleted hela-cells. in recent studies, protein and lipid reorganization processes after attaching stxb to solid supported membranes, composed of dopc/sphingomyelin/cholesterol/gb 3 have been investigated. the compaction of gb 3 -molecules led to an additional stxb-gb 3 -enriched phase, which was also observed in lipid monolayers at the air/water interface. in this study, the influence of stxb on model membranes will be investigated combining the advantages of free-standing lipid membranes with those of ssms. the impact of stxb on pore-suspending membranes will be followed by confocal laser scanning mi croscopy and atomic force microscopy. a. robaszkiewicz 1 , c. spickett 2 , p. sicinska 1 , g. bartosz 1 , m. soszynski 1 1 department of molecular biophysics, university of lodz, lodz, poland, 2 strathclyde institute of pharmacy and biomedical sciences, glasgow, u.k. hypochlorite generated in vivo under pathological conditions is a known oxidant, able to initiate lipid peroxidation process, which affects the stability of biological membranes. the aim of this study was the analysis of the products formed during the reaction of hypochlorite with phosphatidylcholines containing unsuturated fatty acid residues (1-stearoyl-2-oleoyl-, 1-stearoyl-2-linoleoyl-, 1stearoyl-2-arachidonylphosphatidylcholine) and their effects on the human erythrocytes. using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in the decrease of the rotational correlation time and rotational motion freedom of liposomes estimated by epr using spin probes (16-and 5doxylstearic acid). unilamellar chlorohydrin liposomes had lower diffusion coefficient for calcein than liposomes made of parent lipids. flow cytometry demonstrated fast incorporation of uni-and multilamellar chlorohydrin liposomes labeled with nbd-pe into erythrocytes. this effect was accompanied by the formation of the erythrocyte subpopulations of higher volume, decrease of the rate of fluorescein diacetate hydrolysis, estimated by flow cytometry, and increase of affinity and maximal velocity of the membrane enzyme acetylcholinesterase. extensive bilayer perforation coupled with the phase transition region of an anionic phospholipid k. a. riske 1 , l. q. amaral 2 , m. t. lamy 2 1 depto. biofisica, universidade federal de sao paulo, sao paulo, brazil, 2 instituto de fisica, universidade de sao paulo, sao paulo, brazil at low ionic strength dimyristoylphosphatidylglycerol (dmpg) exhibits a broad phase transition region characterized by several superimposed calorimetric peaks. peculiar properties, such as sample transparency, are observed only in the transition region. we use differential scanning calorimetry, turbidity and optical microscopy to study the narrowing of the transition region with the increase of ionic strength. upon addition of salt, the temperature extension of the transition region is reduced and the number of calorimetric peaks decreases until a single cooperative event is observed in the presence of 500 mm nacl. the transition region is always coupled with a decrease in turbidity, but a transparent region is detected within the melting process only in the presence of up to 20 mm nacl. optical microscopy of giant vesicles shows that bilayers first rupture when the transition region is reached and subsequently lose optical contrast. fluorescence microscopy reveals a blurry image in the transparent region, suggesting a different lipid self-assembly. overall sample turbidity can be related to the bilayer optical contrast. our observations are discussed in terms of the bilayer being perforated along the transition region. in the transparent region the perforation is extensive and the bilayer completely loses the optical contrast. financial support: fapesp. label-free imaging of biological membranes using surface imaging techniques j. l. richens, k.-a. vere, p. o'shea cell biophysics group, university of nottingham, nottingham, uk surface plasmon resonance (spr) is a detection technique traditionally used for specific protein detection, which is now exploited routinely as a generic label-free sensor. spr responds to changes on a metal surface conditioned to sense the binding of analytes. thus, the composition of the external medium and metal surface will be fundamental to the signal output. here we use a model phospholipid membrane system to investigate the effect of altered buffer and surface compositions on spr signals. comparisons are undertaken between continuous gold surfaces and gold nanoparticles. we demonstrate that surface electrostatics and the salt composition and molarity of a buffer all have significant impacts on spr output. the association of respiratory syncytial virus matrix protein with membrane microdomains the association of the matrix protein from respiratory syncytical virus with membranes has been characterised by tensiometry, brewster angle microscopy, and atomic force microscopy following deposition of langmuir monolayers onto modified silica substrates. association of the protein with monolayers containing phosphocholines and cholesterol leads to the formation of materials with new properties that differ from those of either of the pure components. the behaviour of the protein in monolayers rich in cholesterol and sphingomyelin exhibits a significant concentration dependence. at low concentrations, the protein exhibits a simple partioning behaviour. at higher concentrations, lipids are extruded from the monolayer below a critical surface area. these findings are discussed in relation to the recently published structure of the protein (pnas, 2009, 106, 4441-4446) , the documented formation of viral filaments during key stages of the infection cycle and the isolation of the protein from detergent-resistant membrane fractions. structure and dynamics of closed melting membranes v. m. rondelli 1 , s. santorsola 1 , p. brocca 1 , e. del favero 1 , l. cantù 1 , m. zimbone 2 1 dep. of chemistry, biochemistry and biotechnologies for medicine, university of milan, segrate, italy., 2 dep. of physics, university of catania, catania, italy. we studied the dynamical and structural properties of large unilamellar vesicles (≈ 120 nm luvs) of phospholipids (dmpc, dc 15 pc and dmpc : dc 15 pc = 1 : 1 molar mixture) in the temperature range around the chain-melting transition, ≈ 3 • c wide, with 0.1 • c resolution and 0.01 • c accuracy. small-angle (saxs) and wide-angle x-ray scattering (waxs) measurements show that across the transition the vesicle behaves as an 'evolving membrane', passing through several different states, each of them being characterized by different proportions of coexisting fluid-and gelchains molecules. noteworthy, no kinetics has been detected. on the same samples, a unique and very sensitive laser light scattering technique allows to determine the characteristic times of thermally induced shape fluctuations, connected to the elastic properties of the membranes. results indicate a clear softening of the membranes in correspondence to the chain-melting transition, as indicated by a manifold increase of the corresponding fluctuation characteristic time. meanwhile the overall size of the vesicle is not sensibly changed. this softening is likely to be due to the presence of structural defects, eventually driving to local morphological modifications. thermodynamics characterization of isolated lung surfactant assembled as lamellar bodies e. x. rodriguez 1 , r. alvarez 1 , r. barrio 2 , c. irles 3 , a. ortega 1 1 biochemistry depto., school of medicine, 2 inst. of physics unam, 3 nat. inst. of perinatology, mexico city, mexico lungs are a large extension of ∼80m 2 of one layer cells, which is structured as compacted sacs called alveoli, where lung function takes place: the gas exchange. alveolar endotelium is formed by two kinds of cells: neumocyte type i (nti), which covers ∼97% of the alveolar surface where gas exchange occurs and, ntii where lung surfactant (ls) is produced. ls are a mixture that lay over a water film in the luminal surface of the alveoli and it is thought to decrease the surface tension to avoid alveolar collapse during expiration. ls are made of phospholipids and proteins, which are determinant for the structure of ls under particular conditions of pressure and temperature. ls inside the cell is packed in organelles called lamellar bodies (lb), and is released to the water/air interphase in other conformation. in the present study we isolate lb from pig's lung and study lb thermodynamic characteristics by microcalorimetry in the presence and in the absence of structural proteins. phase transition profile of lb with and without proteins is basically the same; while ls in the alveolar lumen have a higher transition temperature (tm). mayor changes in tm are observed between lb and ls from alveolar lumen. although ls lipid composition in and outside the cell is assumed to be the same, the ground for the differences in tm under these two conditions is unknown. m. rodrigues 1 , g. rádis-baptista 3 , b. g. de la torre 2 , m. castanho 1 , d. andreu 2 , n. c. santos 1 1 instituto de medicina molecular, portugal, 2 pompeu fabra university, spain, 3 federal university of cearà, brasil nucleolar-targeting peptides (nrtps) have been recently designed by structural dissection of crotamine, a toxin from the venom of a south america rattlesnake (radis-baptista et al. 2008. j med chem 51:7041) . at µm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work is to decipher the molecular mechanism for the translocation of nrtps into cells. quantification of partition into membranes was carried out, based on intrinsic tyrosine fluorescence. the role of the bilayer phase, anionic lipids, reducing agents and peptide concentration on the extent and kinetics of partition were studied. both nrtp1 and nrtp2 exhibited high partition to popc (neutral) lipid vesicles (k p ≈3×10 3 ), which was enhanced by the anionic lipid popg for nrtp1, but not nrtp2. the peptides showed a decrease in partition for popc:cholesterol (liquid ordered state) or dppc (gel) membranes. depending on the lipid composition, the peptides either increased or decreased their quantum yield upon membrane insertion. quenching experiments with acrylamide showed no peptide aggregation in solution. once the translocation mechanism is fully understood we will test nrtps as carriers of relevant cellular cargos, evaluating their potential clinical application in drug delivery or gene therapy among other applications. the ingestion of trans fatty acids (tfa) formed during the partial hydrogenation of vegetable oils has been linked to a detrimental impact on health by an, as yet, unknown mechanism. we synthesized deuterated analogs of 1-elaidoyl-2-stearoylphosphatidylcholine (t18:1-18:0pc) that contains a single "unnatural" trans double bond and 1-oleoyl-2-stearoylphosphatidylcholine (c18:1-18:0pc) that contains a single "natural" cis double bond. solid state 2 h nmr, complemented by molecular dynamics (md) simulations, was then employed to compare molecular organization in model membranes prepared from these isomeric molecules. analysis of spectra recorded as a function of temperature reveals a higher chain melting temperature for the trans isomer, indicating tighter molecular packing in the gel state. in the liquid crystalline, however, the difference between the trans and cis isomers is subtle. order as probed by the perdeuterated [ 2 h 31 ]18:0 sn-2 chain, and corroborated by computer simulation, coincides within <5%. only in the conformation of the double bond is an appreciable difference implied. thus, our results contradict the conventional view that tfa resemble saturated fatty acids, which is >20% more ordered. (supported by acs, prf 43281-ac7.) photooxidation and lateral membrane diffusion of dipole molecules v. s. sokolov 1 , e. a. sokolenko 1 , a. a. lents 2 , p. pohl 2 1 a.n frumkin institute of physical chemistry and electrochemistry, russian academy of science, moscow, russia, 2 institut fuer biophysik, johannes kepler universitaet linz, austria the photodynamic oxidation of phloridzin, of the styryl dye di-8-anepps and of unsaturated lipids was monitored "online" by measuring the collapse of the dipole potential which has been introduced to the membrane by these molecules. their photodamage occurred at different rates when the target molecules and the singlet oxygen generating photosensitizer (phthalocyanin) were adsorbed to the same or to opposite sides of the planar lipid bilayer. the difference in the oxidation rates were attribute to singlet oxygen transport through lipid bilayer and therefore we were able to estimate the permeability of lipid bilayers to singlet oxygen. however, the apparent permeabilities derived from experiments with different targets were differ from each other. therefore, we tested the hypothesis that the lateral membrane diffusion of target molecules and oxidation product may have biased our analysis. in line with this anticipation we found that the apparent permeability is dependent on the size of the planar bilayer. the development of a new mathematical model, which takes the mobility of all reacting species in the aqueous and lipid environments into account, allowed estimation of the real membrane permeability to singlet oxygen. it appeared to be very close to that of oxygen in the ground state. a number of complex three-dimensional lyotropic liquid crystal phases are already known, such as the bicontinuous cubic phases, but so far only a single example has been found -a cubic phase of spacegroup fd3m -of a structure based upon a complex close packing of inverse micelles (1) . we now report the discovery (2) of a novel lyotropic liquid crystal phase, of space group, p6 3 /mmc, whose structure is based upon a hexagonal close packing of identical quasi-spherical inverse micelles. the model membrane system consists of a hydrated mixture of dioleoylphosphatidylcholine, dioleoylglycerol, and cholesterol. this novel phase has a number of unique features which may render it useful for a range of applications. firstly, it is the only known self-assembled lyotropic phase whose structure consists of a periodic close packing of identical inverse micelles. secondly, it is stable in excess aqueous solution, which is very important for potential biological or biomedical applications. references (1) v. luzzati, v., r. vargas, a. gulik, p. mariani, j.m. seddon, and e. rivas, biochemistry 31, 279-285 (1992) . dystrophin is a rod-shaped muscular subsarcolemal protein. its deficiency is one of the root causes of duchenne muscular dystrophy. dystrophin rod domain contains 24 homologous repeats, where the sub-domain constituted by the repeats 11 to 15 (r11-15) was reported to bind actin and membrane lipids. we analyzed the interaction of r11-15 with lipid monolayers. to better understand the assembly mechanism of this protein with lipids, we studied its adsorption behavior at the air-liquid and lipid/liquid interface using ellipsometry, surface pressure, and atomic force microscopy (afm). using two different mixtures of phospholipids, ellipsometry and pressure surface data show that r11-15 interacts with the lipid monolayers, but is inserted into the monolayer formed by dopc-dops while it lies below the monolayer of dopc-dope. this indicates that r11-15 interacts more strongly with dopc-dops than with dopc-dope. afm images show that the pressure of lipid monolayer influences the organization of r11-15. when the lipid surface pressure is 30mn/m, r11-15 forms a striking network, indicating a protein-protein interaction in addition to the protein-lipid interaction. this unique behavior of one part of the central domain of dystrophin may explain its key role in muscle cell. studies on polyphenol extracts from helichrysum l., fagopyrum mill., crataegus l. and hypericum l. were performed in order to find if they may be applied as a natural free radical scavengers protecting biological membranes against peroxidation. ghosts erythrocytes were used in the experiments. they were suspended in tris-edta solution, ph 7,4, then.irradiated with a bactericidal lamp without (control) or with a proper amount of the extracts studied. the product of lipid peroxidation was malonic dialdehyde (ma). the colour reaction of ma with thiobarbituric acid (tba) enables to determine the concentration of ma spectrophotometrically. it was found that peroxidation increased with the irradiation time. however, it significantly decreased when concentrations of poliphenols increased. the best antioxidtive property was found for hypericum l. the antioxidative efficiency sequence of the plant extracts studied was the following: hypericum l. > crataegus l. > fagopyrum mill. > helichrysum l. the results obtained indicate that polyphenol extracts exhibit excelent antioxidative properties that make them good free radical scavengers for efficient protection of biological membranes. this work was sponsored by ministry of science and education, scientific project no. n n305 337034. interaction of cationic porphyrins with neutral and negatively charged liposomes i. voszka 1 , g. corradi 2 , p. maillard 3 , h.-j. steinhoff 4 , g. csík 1 1 institute of biophysics and radiation biology, semmelweis university budapest, hungary, 2 research institute for solid state physics and optics, hungarian academy of sciences, budapest, hungary, 3 institute curie, section de biologie, orsay, france, 4 fachbereich physik, universitat osnabrück, germany porphyrin derivatives are used in photodynamic therapy of tumors. the knowledge of the photosensitizer location within the cell is important. the effect of porphyrin derivatives containing cationic side groups was examined on neutral and negatively charged liposomes by esr. the esr signal was first examined as a function of temperature and porphyrin concentration in the dark. a significant change related to the appearance of quasi free spin labels was obtained for spin probes at the 12 th carbon atom and was more expressed for the asymmetrical derivative. illuminating the samples the esr amplitude decreased for all positions of the spin probe but to different extent. the effect was more expressed in case of the symmetrical derivative, especially for label positions 5 and 12. for the asymmetrical derivative the effect changed from moderate to weak from the 5 th to the 16 th position. this indicates that the asymmetrical derivative is incorporated nearer to the lipid head groups while the symmetrical one may be located deeper in the membrane. under oxygen-free conditions both derivatives showed weaker but still pronounced effects.. single channel recording of α-hemolysin in nanopore-spanning tethered bilayer lipid membranes n. t. thet 1 , i. pfeiffer 2 , w. knoll 1 , i. köper 1 1 max planck institute for polymer research (mpi-p), ackermannweg 10, 55128 mainz, 2 austrian research centers gmbh -arc, tech gate vienna, 1220 vienna, austria artificial lipid bilayer membranes mimic biological cell membranes in many aspects and can be used to study functional processes such as ion channeling, signal transducing, transport of nutrients etc. as most of the functions of a cell are accomplished by membrane proteins, research has been ongoing in studying and characterization of membrane proteins embedded in model lipid bilayer membranes. black lipid membranes (blms) were studied for decades but their potential for practical applications is hindered, mainly due to their lack of stability and limited lifespan. recently, tethered bilayer lipid membranes (tblms) proved to have long life span of up to several months without significant changes in membrane resistance and capacitance. in order to combine advantages of blms and tblms, we have designed a membrane system which is freely spanned across a single nanopore in a silicon nitride membrane. this system not only mimics cell membranes, but also it allows control over the chemical composition of buffer with unlimited ionic reservoirs on both sides of the membrane. this freestanding tblm maintains structural stability and lifetime of up to 120 hours without significant decrease in its structural integrity and electrical sealing. for the validation of the tblm formation, we have inserted well known α-hl pores. we are able to control the amount of α-hl pores insertion and measure single channel ion transport across the tblm by α-hl pores using a capacitor feedback amplifier. the language of shape: biological reactions are dramatically affected by the shape of lipid membrane d. stamou university of copenhagen, copenhagen, denmark a plethora of biological process are taking place on the surface of lipid membranes. as a rule membranes in vivo are curved in a variety of complex geometries. here i will present a quantitave study on the influence of membrane curvature on protein-membrane and membrane-membrane interactions. to gain systematic access to a continuum of membrane curvatures we immobilized liposomes on a surface at dilute densities. using confocal fluorescence microscopy we imaged single liposomes of different size, and therefore different curvature, and monitored their interaction with a binding partner (proteins or other liposomes). i will discuss unpublished data on two important classes of biomolecular interactions that exhibited dramatic curvature dependence: a) snare-mediated docking and fusion b) anchoring of peripheral proteins. the following references provide partial information on the single-liposome assay: a. zettergren, c. gudmundsson, t. nylander, e. sparr physical chemistry1, lund university, 22100 lund, sweden there is accumulating evidence of substantial amounts of phospholipids in the cell nuclei 1 , although the function of these lipids is still not fully understood. it has been shown that the chromatin complex composed of dna, rna and proteins also includes phospholipids, and that rna colocalize with these 2 . although the rna-phospholipid interactions may have important implications to biological function, in gene therapy and in medicine, very little work has been dedicated to the characterization of rna interaction with phospholipids. the objective of this work is to investigate the adsorption behavior of short single stranded 10 bases long rna (ssrna 10 ) molecules (similar to mirna) to lipid monolayers at the air-water interface as well as to study how the presence of rna affect the domain formation in the monolayers using fluorescence microscopy. monolayer studies have shown adsorption of ssrna 10 to monolayers consisting of zwitterionic dppc as well as to monolayers consisting of cationic dodab. the adsorption behavior of these very short nucleic acids differ significantly from the adsorption process for longer nucleic acids as for example a 2000 base pairs long ds dna (dsdna 2000 ) which has been used as a reference system 3 . the presence of ssrna 10 significantly changes the compression isotherm of both dppc and dodab monolayers. plant defensins are cysteine-rich antimicrobial peptides found in various plant species. they share a common threedimensional structure, stabilized by eight disulphide-linked cysteines consisting of three antiparallel β-strands and one α-helix. most plant defensins show antifungal activity with no effect on mammalian and plant cells. expression of these peptides in plant tissue is induced by pathogen infection. the mechanism of defensin action is based on membrane permeabilization. this occurs through an interaction with high affinity binding sites on fungal membranes, resulting in alteration of membrane potential. the genes encoding for a peach ppdfn1 and a grape vvamp defensins were expressed in e. coli and purified to homogeneity. they were tested for antimicrobial activity against some fungi and showed to have an inhibitory effect on the spore germination. biophysical analysis showed that defensins were able to interact with artificial membranes. binding of defensins to membranes was dependent on lipid composition, increasing with the sphingolipids content. interaction between peptides and sphingolipids could lead to insertion of the defensins into the membrane resulting in its destabilization. extracts of the plant perilla frutescens have many uses in the asian kitchen, e.g. as a popular garnish used in sushi. the plant is also employed in eastern traditional medicine to treat a variety of ailments including colds, food allergy and depression. two of the main constituents of the plant are limonene and perilla aldehyde. by bio-oxidation, these molecules can be converted to perillic alcohol and perillic acid. these cyclic terpenes possess antibacterial and anticarcinogenic properties. the modes of action for these compounds are at present not understood, but their remarkably diverse pharmacological properties suggest that they might target the phospholipid matrix of the cellular lipid membrane. indeed, the cyclic terpenes can bind to, and alter the physiochemical properties of the lipid bilayer of the membrane, the effects of which can cascade down to several essential cellular processes. here, we use molecular dynamics (md) simulations to investigate the effect of limonene and its derivatives on the properties of lipid bilayers including the changes in acyl chain order parameters, bilayer thickness and the area per lipid. md can afford molecular-scale dynamic information, often not easily accessible from experimental measurements. this information can be used to interpret existing experimental data obtained by e.g. isothermal titration calorimetry, electron paramagnetic spectroscopy and differential scanning calorimetry. catalysis in the membrane: interfacial mechanism of phospholipase a2 h. p. wacklin 1 , r. k. thomas 2 1 institut laue langevin, grenoble, france, 2 physical and theoretical chemistry laboratory, oxford university, u.k. phospholipase a2 cleaves the sn-2 acyl chains of membrane phospholipids and performs a range of physiological functions. one of the least well understood aspects of the its mechanism is how its activity is regulated by the interaction with the substrate membrane. we have used neutron reflection to monitor changes in membrane structure during lipid hydrolysis [1, 2] . the penetration depth of pla2 depends on lipid packing and increases during the lag phase of porcine pancreatic pla2. by using a selectively deuterated lipid substrate, d31-popc, we determined the relative membrane-water partitioning of the lipid products in-situ. the lyso-lipid product partitions into the solution phase, while fatty acid accumulates in the membrane and increases the affinity of pla2 [2] . pla2 is inhibited at ph 5, which is consistent with protonation of the catalytic histidine. however, irrespective of ph, pla2 is fully activated by me-betacyclodextrin, which facilitates the release of the lyso-lipid from the enzyme-substrate complex. me-beta-cyclodextrin does not interact directly with the membrane surface or the substrate lipids, indicating that product release occurs outside the immediate membrane-water interface. diffraction limits resolution in optical microscopy, but many interesting biological problems occur on shorter (molecular) length scales. recently, methods to circumvent the diffraction limit have been presented. fluorescence photoactivation localization microscopy (fpalm) uses activation of many small subsets of photoactivatable or photoswitchable fluorescent probes (pafps) to generate images with effective resolution in the tens of nanometers. pafp molecules are photoactivated, imaged, localized, and photobleached in small numbers. the process is repeated for many subsets to build up data on thousands to many hundreds of thousands of molecules. the positions of all localized molecules are used to construct an image of the sample with resolution limited not by diffraction, but by the localization precision and molecular density. results will be shown from a variety of biological systems, including live and fixed cells expressing a variety of pafp-tagged proteins. bi-plane fpalm can image in three dimensions with demonstrated resolution of 30 nm x 30 nm x 70 nm. polarization fpalm can image both molecular positions and anisotropies simultaneously with lateral resolution of ∼20-30 nm. using these powerful capabilities, many potentially interesting biological problems can be addressed. binding of the hiv-1 ncp7 on oligonucleotides at the single-molecule level j. godet, p. didier, a. jouonang, y. arntz, y. mély laboratory of biophotonics and pharmacology, umr cnrs 7213, university of strasbourg, france the nucleocapsid protein (ncp7) of the hiv-1 is a small basic protein which plays key functions in the viral life cycle. the activity of ncp7 mainly rely on its potent rna-and dna-chaperone activities that direct the rearrangement of numerous nucleic acid molecules into their most stable conformation. two main features of nc's chaperone activity are its abilities to aggregate and destabilize nucleic acids. in addition, the rapid kinetics of ncp7 interaction with nucleic acids was recently proposed as another major component of nc's chaperone function based on the apparent correlation between an indirect measurement of the nucleic acid dissociation kinetics of ncp7 and its overall chaperone activity. but so far, no direct measurement of the on/off rates of ncp7 binding to oligonucleotides was performed. in the present work, we realized single molecule fluorescence resonance energy transfer (smfret) measurements to probe the transient interactions of a tmr-labelled ncp7 with a short cy5-labelled dna oligonucleotide confined into nanovesicles. after confirming the efficiency of the ncp7/odn complex encapsulation into the nanovesicles by fcs, the vesicles were tethered to the surface for immobilization. integrity of the entrapping vesicles attached on the surface was confirmed by afm. finally, the association and dissociation constants retrieved from these smfret measurements were discussed in the context of the dna-chaperoning activity of the protein. high-resolution spatiotemporal organization of the integrin lfa-1 m. f. garcia-parajo 1 , t. s. van zanten 1 , g.-j. bakker 1 , r. diez-ahedo 1 , a. cambi 2 , c. g. figdor 2 1 bionanophotonics group; ibec, barcelona, spain, 2 tumour immunology dept; ncmls, nijmegen, the netherlands lfa-1 is a leukocyte-specific integrin involved in different steps of the immune response. on monocytes, lfa-1 plays a key role in the regulation of monocyte-endothelial interaction during rolling, arrest and extravasation into the underlying tissue. i n-vivo experiments showed that blood borne lymphocytes can 'switch' within seconds from rolling to arrest. furthermore, tem observations of pro-active, ligandindependent nanoclusters confirmed that affinity and clustering are complementary processes required in adhesion. yet, the mechanisms leading to fast-switching remain obscure. in our group, we used a combination of single molecule fluorescence techniques to study the spatiotemporal organization of lfa-1 on monocytes. we performed optical nanoimaging of lfa-1 nanoclusters in relation to membrane rafts with a resolution of 70nm and accuracy of ∼3nm. in quiescent cells, lfa-1 nanoclusters do not associate with membrane rafts and diffuse freely on the membrane. binding of the integrin to its ligand icam-1 induces the formation of microclusters that further associate with rafts and exhibit reduced mobility, consistent with cytoskeleton interactions. our work highlights the markedly different spatiotemporal organization of lfa-1 that might explain its concerted action to form larger and stable platforms on the cell surface required for rapid and effective cell adhesion. c. ciobanasu, u. kubitscheck rheinische friedrich-wilhelms-universität bonn, germany cell penetrating peptides like the hiv1 tat peptide have the property to rapidly translocate the cell membranes and the capability to deliver a wide range of cargoes. the mechanism of the membrane translocation is still under investigation and object of considerable controversy. we applied and single-molecule and confocal laser scanning microscopy (lsm) to study peptide-membrane interactions. electron-multiplying ccd cameras yield images of single fluorescent molecules with a time resolution in the range of a few milliseconds only, which allows the tracking of fluorescently labelled peptides and lipids at bio-interfaces in realtime with a localization precision of a few nanometers. we formed giant unilamellar vesicles (guvs) from different lipid mixtures and examined their interaction with fluorescently labeled tat peptides. we found that the passive peptide internalization process depends on lipid composition, charge of the lipid bilayer, and the ionic properties of the medium. a translocation of cationic tat peptides was observed in membranes containing at least 20 mol% of lipids with a phosphatidyl ethanolamine or a high mol fraction of the phosphatidyl serine head group. in salt-free solution tat efficiently bound to guvs, however, in a physiological nacl solution tat binding was completely abrogated, but the peptides efficiently equilibrated across the guv membrane. new approaches to measure interactions in the live cell plasma membrane g. j. schütz biophysics institute, johannes kepler university linz, austria in my lecture, i will show examples how to obtain insights into the organization of the cellular nanocosm by single molecule experiments. our primary goal is an understanding of the role of such structures for immune recognition. brightness and single molecule colocalization analysis allows us to study stable or transient molecular associations in vivo. in particular, i will present results on the interaction between antigen-loaded mhc and the t cell receptor directly in the interface region of a t cell with a surrogate antigenpresenting cell. in addition, we developed a method for in vivo micropatterning of plasma membrane proteins to measure molecular interactions. the method allows identifying and characterizing interactions between an arbitrary fluorescence labeled protein ("prey") and a membrane protein ("bait") directly in living cells. cells transfected with a fluorescent fusion protein of the prey are plated on micropatterned surfaces functionalized with specific antibodies to the extracellular domain of the bait; the fluorescence copatterning is used as readout for the interaction. we applied this tool for the study of the interaction between cd4 -the major coreceptor for t cell activation -and lck, an important tyrosine kinase in early t cell signaling. in addition to the well-known zinc-clasp structure, we found strong contributions of lck membrane anchorage for the binding of the two proteins. developing a fluorescent redox sensor for monitoring metal-ion mediated catalysis in biosystems a. rybina 1 , a. kiel 1 , b. thaler 2 , a. sprödefeld 2 , r. krämer 2 , d. p. herten 1 1 bioquant and, 2 department of inorganic chemistry, heidelberg university, germany a fluorescent redox sensor is an electron photo-switching device that can be used for the characterization of the redox state of a given environment. it combines a fluorescent fragment with a redox-active unit that senses the media by a redox reaction and controls the light emitting properties of the fluorophore. such reversible sensor can help to examine the electrochemical state and changes in biological systems during biochemical processes in real time. recently a new fluorescent molecular sensor with a redox-active hydroquinoneunit covalently linked to fluorophore rhodamine b was developed. (kierat r.m. et al., bioorg. med. chem. lett., 2009 -accepted) . the reduced hydroquinone-form of the sensor is fluorescent while its oxidation to benzoquinone-derivative leads to a significant decrease of the fluorescence. although the above method shows great promise for applications in biological systems, the exact mechanism of this process is not fully understood yet. we use fluorescence spectroscopy to investigate oxidation reactions on the ensemble and single-molecule level and study kinetic rates. the proposed strategy is to use cu (ii) complex as oxidation mediator immobilized on surface via dna linker to examine the oxidation mechanism. fluorescently labeled atp as a probe of the outer mitochondrial membrane barrier: role of vdac fluorescence correlation spectroscopy (fcs) was applied for studying the distribution of fluorescently labeled atp (bodipy-atp) in isolated mitochondria. the setup and peak intensity analysis (pia) was described in our recent paper (perevoshchikova et al. 2008 biochim.biophys.acta 1778 :2182 . the binding of bodipy-atp to mitochondria was maximal in the non-energized state, whereas the addition of succinate (respiratoty substrate) or atractyloside (adenine nucleotide translocase inhibitor) led to a decrease in the binding. nadh reduced the fcs signal from bodipy-atp added to non-energized mitochondria more than nad+ did under the same conditions suggesting the control of nucleotide transport through voltage-dependent anion channel (vdac) residing in the outer membrane. konig's polyanion also decreased the bodipy-atp binding to mitochondria with the effect being reduced by alamethicin or digitonin. control experiments showed that bodipy-atp did not bind to liposomes showing minor role of unspecific binding. it was suggested that bodipy-atp in combination with fcs can be used to monitor the functional state of mitochondrial vdac which is considered to be a principal regulator of mitochondrial function. fluorescence correlation spectroscopy studies of lysozyme partition to phospholipid vesicles a. m. melo 2 , a. coutinho 2 , m. prieto 1 1 cqfm and in, ist, 1049-001 lisboa, portugal, 2 dqb, fcul, 1749-016, lisboa, portugal binding to membrane lipids has been increasingly recognized as an important step in the aggregation and cytotoxicity of several amyloidogenic proteins [1] . in addition, it has been recently reported that membranes containing negatively-charged phospholipids can also trigger rapid amyloid-like fiber formation by a variety of several nonamyloidogenic proteins, such as cytochrome c and lysozyme [2] . our study aims to elucidate the factors that govern the formation of these lipid-protein complexes. given the importance of electrostatic interactions between the proteins and the acidic phospholipids in the putative membrane-induced protein misfolding step, it is essential to first characterize quantitatively the protein partition behavior towards liposomes prepared with variable anionic lipid content. in this study, lysozyme was chosen as a model protein and fluorescence correlation spectroscopy (fcs) was used to monitor its binding to liposomes after its conjugation to alexa fluor 488. most organic dyes labelling techniques produce a mixture of populations of molecules labelled with a different number of fluorophores. the influence of this poli-dispersity of labelled molecules on the protein partition behaviour will be explored, namely the ability of the fcs technique to detect the production of non-competent membrane-binding species. t. wohland 1 , p. liu 1 , x. shi 1 , y. h. foo 1 , t. sudhaharan 2 , s.-w. chong 3 , v. korzh 3 , s. ahmed 2 1 chemistry dept., singapore nat. univ., 2 medical biology inst., singapore, 3 molecular & cell biology inst., singapore biomolecular interactions have been measured mostly under in vitro conditions because of higher accuracy and ease of measurement. however, it has become clear that the cellular environment has an important influence on these interactions. it was therefore necessary to develop new tools to allow the measurement of interactions in cells and organisms. recently, we have developed a modality of fluorescence cross-correlation spectroscopy (fccs) called single wavelength fccs (sw-fccs), which uses one-photon excitation to excite two fluorophores with overlapping absorption but separable emission spectra. sw-fccs has been used to determine e.g. dimer fractions of proteins in live cells. here we aim to extend the use of sw-fccs to cells and organisms. in the first part we determine the dissociation constants of a small rho-gtpase (cdc42) with an effector domain (crib) and two effectors (n-wasp or irsp53). in the second part we measure the binding between cdc42 and a scaffolding protein (iqgap1) in dependence of their expression levels in cho cells and in live zebrafish embryos. by using gfp/mrfp fusion proteins we can excite both fluorophores at 514 nm and detect them separately in two different wavelength channels. we quantitatively determine the dissociation constants and compare their differences in vitro, in cells, and in embryos. these experiments demonstrate that sw-fccs is a powerful biophysical tool for the quantitation of biomolecular interactions in cells and organisms. addressing plasma membrane structure at the nanoscopic length-scale s. wieser, m. axmann, g. j. schütz biophysics institute, johannes kepler university linz, altenbergerstr.69, a-4040 linz, austria there is increasing interest in a detailed understanding of the structure and dynamics of the cellular plasma membrane, primarily based on recognizing its essential role for controlling cellular signaling processes. we employed single molecule fluorescence microscopy to study diffusion of cd59, a gpi-anchored protein, in the plasma membrane of living t24 cells at sub-wavelength resolution, both on the cell body and on tunneling nanotubules connecting cells. the lateral motion of this single fluorescence labeled molecule was imaged on a millisecond time scale. within the experimental errors, no indications for confined diffusion for cd59 on the cell body in t24 cells have been found. furthermore by separating longitudinal and transversal mobility, we found isotropic diffusion behavior on the surface of tunneling nanotubules. in both studies we analyzed the mean square displacement as a function of the time-lag and the distribution of displacement steps. however, a closed analytical theory for these analysis is only available for the simplest models. to address a suspected diffusion process we reasoned that a full analytical description may not be required; it may well be sufficient to compare the experimental data with monte carlo simulations of the process. we demonstrated the working principle for this simulation based analysis for free diffusion, hop diffusion and transient binding of the tracer molecule to slowly moving receptors. n. chakroun 1 , f. fraternali 1 , m. malfois 2 , h. rezaei 3 , c. a. dreiss 1 1 king's college london, u.k., 2 diamond light source, didcot, oxfordshire, u.k., 3 inra, jouy-en-josas, france prion(prp) diseases are fatal neurodegenerative diseases affecting mammals including human and sheep.they are characterized by the accumulation of extracellular βrich fibrillar deposits of a structurally modified form (prp sc ) of the cellular prp c .despite the increasing interest for prp diseases,the mechanism of prp c /prp sc conversion is still unknown.studies on prp diseases suggest that neurotoxicity arises from small pre-fibrillar oligomers.we have used a range of biophysical techniques combined with molecular dynamics simulations (md) to resolve the oligomerization pathways of sheep prp (sprp).we have shown that under well established conditions, sprp oligomerizes into three oligomers, which form in parallel.in addition, we have now identified the minimal region of sprp leading to the same oligomerization profile of the entire sprp,namely h2h3.low resolution shapes of sprp, h2h3 and resulting oligomers have been determined by small-angle x-ray scattering.time-resolved studies have been used to follow the oligomerization of sprp and h2h3 monomers into the oligomers.the conversion of sprp sc at the molecular scale was studied by md.simulations of the h2h3 region recreating experimental conditions revealed a complete unfolding of h2 helix followed by h3 helix.these crucial steps are followed by the formation of β-sheet structures leading to a stable βrich double hairpin structure. single-molecule force spectroscopy investigation of the conformational equilibria of alphasynuclein m. brucale 1 , a. rampioni 1 , m. sandal 1 , i. tessari 2 , l. tosatto 2 , l. bubacco 2 , b. samorì 1 1 istituto di biochimica g.moruzzi, università di bologna (italy), 2 dipartimento di biologia, università di padova (italy) alpha-synuclein (asyn) is an abundant intrinsically disordered protein (idp) primarily located at presynaptic terminals. mutations in the gene encoding asyn have been linked to early-onset parkinson's disease (pd). by means of single molecule force spectroscopy (smfs) experiment, we show how the conformational equilibrium of monomeric wild type (wt) asyn shifts toward more compact structures in several unrelated conditions linked to pd pathogenicity [1] . the conformational heterogeneity of pathological alpha-syn mutants a30p, a53t and e46k has also been characterized, revealing marked differences in the conformational behaviors of the mutants with respect to wt asyn [2] . all the mutants show a distinctively higher propensity, with respect to wt, to acquire a monomeric compact conformation that is compatible with the acquiring of beta structure. the same smfs experimental methodology is then used to characterize the conformational behavior of wt and mutant asyn in a variety of conditions, in an attempt to gain insight about the multiple and contrasting parameters controlling the equilibrium. in vitro protein folding studies using chemical denaturants have contributed tremendously to our understanding of the folding thermodynamics and kinetics of water-soluble proteins. this is not the case for integral membrane proteins, which constitute about one third of all eukaryotic proteins and more than half of all validated and potential drug targets. fully reversible denaturant-induced unfolding remains limited to a few β-barrel porins, whereas the much larger and more relevant class of α-helical membrane proteins has thus far evaded this approach. we report here the first example of an α-helical membrane protein that can be unfolded completely and reversibly by a chemical denaturant: mistic, a 110-residue protein from bacillus subtilis [1] , dissociates from detergent micelles or lipid vesicles and assumes an unfolded monomeric state on titration with urea. using spectroscopic and microcalorimetric techniques, we exploited this unique property to provide (i) a quantitative comparison of membrane protein stability in different membrane-mimetic systems; (ii) an experimental test of controversial predictions [2] regarding the folding core of mistic; and (iii) a convenient setup to study the spontaneous, translocon-independent membrane insertion of this unusual membrane protein. the mechanical functioning of biological tissues is important from many viewpoints such as diseases, clothing and even food. the protein collagen makes up the greater part of these tissues, and is remarkable for its many uses in the body, however there are at least two other major components. one of the most interesting properties of these tissues is their non-linear behavior under stress. this behavior is essential to prevent a catastrophic failure such as an aneurysm. at least three major theories have been proposed within the past few years to explain this behavior, but have been impossible to verify. in order to determine a correct description of the mechanical structure of the tissue we have been using cutting edge technological solutions to address the single molecules within the extra cellular matrix. this technique combines optical methods with single molecule force spectroscopy, allowing stiffness measurements over the nanoscale as well as determining the individual protein tensions within the extra cellular matrix. the results show that this method can be used to determine the network properties even in the complicated aortic wall enabling better understanding of disease states, which in this case include marfan's syndrome and ascending aortic aneurysms. beta amyloid peptide abetapy3-42 shows a faster aggregation kinetics than abeta1-42 c. d'arrigo 1 , m. tabaton 2 , a. perico 1 1 institute for macromolecular studies, national research council, 16149 genova, italy, 2 department of neurosciences, university of genova, 16132 genova, italy we test directly the differences in the aggregation kinetics of three important beta amyloid peptides, the full-length abeta1-42 and the two n-terminal truncated and pyroglutamil modified abetapy3-42 and abetapy11-42, found in different relative concentrations in the brains in normal aging and in alzheimer disease. 1 we find by following the cd signal and the tht fluorescence of the solution in phosphate buffer, a substantial faster aggregation kinetics for abetapy3-42. this behavior is due to the particular sequence of this peptide which is also responsible of the specific oligomeric aggregation states, found by tem, during the fibrillization process which are very different from those of abeta1-42, more prone to fibril formation. in addition abetapy3-42 is found here to have an inhibitory effect on abeta1-42 fibrillogenesis, coherently with its known greater infective power. this is an indication of the important role of this peptide in the aggregation process of beta-peptides in alzheimer disease. the puzzle of the anomalously long denaturation kinetics of green fluorescent protein (gfp) mutants still is largely unveiled. in this study we have followed the effect of mutation h148g on the stability of gfpmut2 (mut2g) in the presence of guanidinium hydrochloride (gdnhcl). different techniques of fluorescence spectroscopy have been employed in order to obtain information concerning the unfolding event: time resolved fluorescence, fluorescence correlation spectroscopy (fcs), and fluorescence anisotropy. the substitution of the histidine with glycine affects protein stability versus ph: in particular mut2g kinetics is not ph dependent and at basic ph values the protein is less stable. the fluorescence properties (quantum yield, lifetime) and the rotational correlation time are unchanged during the unfolding dynamics, while the number of fluorescent proteins decreases exponentially. an extrinsic probe, bound to cysteine 48, has been employed in order to gain more insights on the unfolding process, monitoring the stability of a different region of the protein. in particular, it has been found that a softer region is present around cysteine 48 in both gfp variants, showing that the unfolding process does not follow a simple two step mechanism. recently, negatively-charged membranes were reported to catalyze amyloid fiber formation by amyloidogenic peptides/proteins and also to induce formation of "amyloidlike" fibrils by nonamyloidogenic proteins. here, we used an approach which combines steady-state and time-resolved fluorescence measurements to obtain structural information about these supramolecular assemblies and to gain insights about the factors that control their formation. by exploring a wide range of lipid concentrations, the interaction of alexa488-lysozyme with phosphatidylserine-containing membranes was found to be a complex multi-step process, critically dependent upon the protein-to-lipid molar ratio (p/l) used. upon increasing the total lipid concentration in solution, there was a balance between an increased overall protein binding to the lipid vesicles and a progressive protein dilution on the membrane surface. as the surface potential of the vesicles decreased upon increasing the protein interfacial coverage of the liposomes, the protein binding mode was found to switch from a peripheral binding of lysozyme to the anionic headgroups (at low to intermediate p/l) to a partial insertion of the basic protein into the hydrophobic core of the membrane (at a high p/l). it is hypothesized that disruption of the protein tertiary structure might be a stepwise process beginning with loosening of the structure caused by its deeper insertion in the membrane bilayer. unexpected scaling laws in the mechanical unfolding of single protein molecules m. clusel, e. i. corwin, h. lannon, j. brujic center for soft matter research, physics department, new york university, new york, ny, usa it is a question of fundamental importance to understand the response of proteins to a stretching force, particularly in the case of mechanically active proteins, such as those in muscle fibers. we aim to understand how the structure and topology of a protein affect its resilience to external forces and presumably its function. owing to recent advances in single molecule force-clamp spectroscopy using the atomic force microscope (afm), we are now able to probe the structure and dynamics of single proteins under a constant stretching force by measuring their end-to-end length over time. the probability distribution of unfolding times at a given force allows us to estimate the strength of the protein in terms of a characteristic energy barrier, while the shape of the distribution provides a window into the microscopic mechanism by which the protein breaks apart. here we show a novel scaling of the unfolding kinetics with the stretching force, which deviates significantly from the currently accepted bell model. instead, we propose a physical picture for forced unfolding that is analogous to the mechanics of fracture. v. garcía-gonzález, j. mas-oliva instituto de fisiología celular. universidad nacional autónoma de méxico. 04510 méxico, d.f. méxico. studies focused on the thermodynamic and kinetic analysis have demonstrated that transfer of neutral lipids such as cholesterol esters through an aqueous phase is a highly costly biophysical event. therefore, nature has developed a series of lipid transfer proteins such as the cholesterylester transfer protein (cetp) designed to efficiently lower the energy barrier for transfer of cholesterol-esters between lipoproteins through an aqueous environment. employing circular dichroism we evaluated the secondary structure stability of a small peptide derived from the carboxy-end of cetp (helix y ) in a wide range of ph's. the percentage of α-helix is diminished only at extreme temperatures and acidic ph's in a reversible way. we report that while a mixture of phosphatidylcholine/cholesteryl-ester forms large aggregated particles independently of ph, inclusion of helix y to the mixtures close to neutral ph's allows the formation of small micellar-like structures confirmed by dynamic light scattering and electron microscopy. these results suggest that helix y when close to physiological ph values presents the ability to organize a micellar structure around itself. this type of organization allows the process to dramatically lower the energetic barrier for lipid transfer through aqueous media, phenomenon directly related with the facilitation of lipid transfer between lipoproteins. mimicking metastasis by a novel microfluidic approach there is increasing evidence that cancer metastasis shares commonalities with thrombosis. the von-willebrand-factor (vwf), a mechanical active blood clotting protein appears to be a particular potent candidate to bridge the gap between clotting and cancer extravasation. modeling the crucial physiological conditions of the blood circulatory system, for an in situ study of blood clotting-metastasis connections is not only, absolutely necessary, but also a challenging task. here, we present acoustically driven flow as a novel microfluidics method for mimicking the blood flow. this method enjoys very beneficial advantages of possibility of handling very little volumes of fluids, together with freedom to model most of the geometries present in our microcirculatory system. one technologically challenging, yet physiologically important factor, is the hydrodynamic condition in a bifurcated vessel, where complex shear profiles arise. we present a model to mimic these conditions and discuss the impact of hydrodynamics on vwf mediated cancer cell adhesion in bifurcated vessels of our microcirculatory system. protein structural changes occurring in flows stresses inherent to viscous fluid flow have previously been associated with protein unfolding, although structural changes have not been well documented as a function of relevant hydrodynamic parameters. we have used raman spectroscopy to monitor the structure of various protein solutions in situ for multiple flow scenarios within a concentric cylinder fluidic device (1) . the flows, which ranged from circular couette to wavy taylor-couette flow, were characterised experimentally using particle image velocimetry. several proteins were observed to change conformation when exposed to these flows, although the nature of these changes was protein specific. shearing hen egg white lysozyme in water altered the protein backbone structure, while similar shear rates in a 95% glycerol, 5% water solution affected the solvent exposure of the side chain residues near the exterior of the α-domain. the solventdependent response may be due to the flow topology, viscous stress, or the surface hydration properties. comparison of spectra acquired at different time points, including before and after flow, confirmed that the observed changes are reversible and independent of fluid stress exposure time. the scripps research institute, la jolla, ca, usa. intrinsically disordered proteins are increasingly found to play major roles in cell biology and disease. we are utilizing single-molecule fluorescence methods to probe these complex and highly dynamic molecules, allowing more direct measurements of structural distributions and dynamics, while avoiding loss of information due to ensemble averaging. in one example, we investigated the structural dynamics of sup35-nm, whose regulatable amyloid formation is believed to have functional significance in yeast. using a combination of single-molecule fret as a molecular ruler, coincidence to interrogate intermolecular interactions, and correlation analysis to probe conformational dynamics, we showed that the monomeric protein populates an ensemble of compact and rapidly interconverting conformations. a particularly interesting feature of intrinsically disordered proteins is that they are relatively unstructured in isolation, but can gain stable structure by interaction with binding partners. in this context, we used single-molecule fluorescence to characterize the complex folding pathway for the parkinson's-related idp alpha-synuclein induced by binding to a lipid-mimic. this combined single-molecule fluorescence methodology provides a powerful approach for detailed studies of the coupling of folding and dynamics with interactions and biology of this important class of proteins. m. ito 1 , j. johansson 2 , r. stromberg 1 , l. nilsson 1 1 department of biosciences and nutrirtion, karolinska institutet, huddinge, sweden, 2 department of anatomy, physiology and biochemistry, swedish university of agricultural sciences, the biomedical centre, uppsala, sweden amyloid β-peptide (aβ) is a 39-42 amino acid polypeptide and known to aggregate and form insoluble amyloid fibril, which is regarded as a primary cause of alzheimer's disease (ad). the discovery of practical and effective treatments and drugs for ad has been waited eagerly. in a recent experimental study, it was suggested that stabilization of the helical conformation of the aβ middle region, which strongly favors collapsed coil formation in the extracellular environment, would reduce the aβ fibril formation. based on the experimental evidence, inhibition of the unfolding of the aβ α-helix can be a forceful strategy to repress the aβ fibril formation resulting in prevention of ad. however, the detailed mechanism of the unfolding of the aβ α-helix has remained unclear, because the x-ray or the nmr structure of the aβ α-helix in aqueous solution has not been reported due to its instability. the aim of this study is to find effective ways to inhibit the unfolding of the aβ middle region, which is a prerequisite for the aβ fibril formation. in this study, we attempted to elucidate the molecular mechanism of the aβ unfolding by molecular modeling and molecular dynamics (md) simulations. the md simulations were performed for α-helical structures of a wild-type aβ(13-26) model and a mutant aβ(13-26) model. linker average hydrophilicity as a tool to discriminate between extended and non-extended calcium binding proteins a. isvoran 1 , e. quiniou 2 , c. craescu 2 , l. mouawad 2 1 west university of timisoara, department of chemistry, pestalozzi 16, 300115 timisoara, romania, 2 inserm u759, centre universitaire paris-sud, bâtiment 112, 91405 orsay, france the ef-hand calcium binding proteins (cabps) may exist either in an extended or a compact conformation, sometimes correlated with their functions. for the cabps with know structure and function, calcium sensors are usually extended and calcium buffers compact, hence the interest to predict the form of the protein starting from its sequence. in this study we used two different procedures, the sosuidumbbell algorithm and a novel procedure that is based on the linker average hydrophilicity (lah). the two procedures were tested on 17 known-structure cabps and then applied to 59 unknown-structure centrins. the so-suidumbbell algorithm yielded the right conformations for 15 of the 17 known-structure proteins and predicted that all centrins should are compact. the lah procedure discriminated well between the extended and non-extended forms of all the known-structure cabps and it reflected well the phylogenetic classification of centrins being a simple and powerful means to discriminate between extended and nonextended forms of cabps. only few residues that constitute the linker are responsible for the form of the cabp, showing that this form is mainly governed by short-range interactions. (http://u759.curie.u-psud.fr/modelisation/lah) lipid and protein organization of hepatitis b antigen characterized by fluorescence spectroscopy v. greiner 1 , c. egelé 1 , s. oncul 1 , f. ronzon 2 , c. manin 2 , a. klymchenko 1 , y. mély 1 1 laboratoire de biophotonique et pharmacologie, umr 7213 cnrs, faculté de pharmacie, université de strasbourg, france, 2 sanofi pasteur, 1541 av. marcel mérieux, 69290 marcy l'étoile, france. hepatitis b surface antigen (hbsag) particles are 20 nm lipoprotein particles, mainly composed of the major s surface viral protein containing 13 trp residues and yeast-derived lipids. since the structure of these particles is still missing, we further characterized them by fluorescence techniques. fcs indicated that the particles diffuse mainly as monomers and contain about 80 proteins per particle. fluorescence quenching and time-resolved fluorescence experiments showed that the fluorescence signal is largely dominated by the trp residues at the protein surface. moreover, time-resolved anisotropy measurements indicate that the protein motion is restricted and that the surface trp residues exhibit both local and segmental motions. the lipid part of the particles has been studied by environment sensitive 3-hydroxyflavone probes and viscosity-sensitive dphbased probes, and compared to lipid bilayers and low density lipoproteins (ldls), taken as models. the results suggest that the lipid part of hbsag is closer to ldls than to model lipid bilayers. we present an extensive calorimetric study of bovine alphalactalbumin for various ca++ content. equilibrium dsc raw data are analyzed and the melting temperature tm, the specific heat jump deltacp, the heat of unfolding deltahm are directly extracted. the binding of calcium on the native (n) state greatly stabilizes the protein, essentially by the enthalpic difference between the unfolded (u) and n states. we show that subsequent addition of calcium in the mm range stabilizes further the n state. the equilibrium calorimetric measurements are completed with out of equilibrium stopped flow refolding kinetics by cd spectroscopy performed at different temperature and ca++ concentrations. we discuss the possible stabilization mechanisms compatible with our measurements. protein unfolding/refolding in cellular compartments: application of luciferase constructs our studies show that a reporter enzyme, firefly luciferase, can be used for evaluation of the stress-induced proteotoxicity within different cellular compartments such as the nucleus, cytoplasm or mitochondria. in transfected mammalian cells, firefly luciferase is localized in microsomes. we engineered plasmid constructs encoding luciferase with inserted specific sequences that ensure its cytoplasmic or intranuclear, or intramitochondrial localization. in addition, we fused luciferase to the green fluorescent protein (gfp) that enables to visualize patterns of the compartment-targeted product. using such gfp-labeled constructs we had a possibility to monitor protein unfolding, aggregation and refolding in the cytoplasm, nucleus and mitochondria of transfectants exposed to either stressful conditions. gfp-luciferase expressed in mammalian cells behaves as a relatively labile protein which can undergo reversible unfolding and aggregation in response to heat shock, atp depletion or action of toxic agents. in the case of cell recovery, refolding of denatured luciferase is carried out at the chaperone machine. we explored unfolding/refolding of gfp-luciferase in the cytoplasm, nucleus and mitochondria of ischemia-stressed rat cardiac cells and in several cancer cell lines treated with hyperthermia or some chemotherapeutic drugs. the results obtained have revealed intriguing correlations between the proteotoxic impact within either compartment and the viability of treated cells. amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid bilayers s. jha 1 , d. sellin 1 , r. seidel 2 , r. winter 1 1 biophysical chemistry, department of chemistry, tu dortmund university, otto-hahn str. 6, d-44227, dortmund, 2 max-planck-institute for molecular physiology, otto-hahn str. 11, d-44227, dortmund, germany the islet amyloid polypeptide (iapp), which is considered as the primary culprit for β-cell loss in type 2 diabetes mellitus patients, is synthesized in the β-cells of the pancreas from its precursor, the pro-islet amyloid polypeptide (proiapp). proiapp is co-processed in the secretory granules and co-secreted to the extracellular matrix together with insulin as iapp. here, we compare the amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. the two peptides show an enhanced amyloidogenic propensity in the presence of negatively charged membranes and similar secondary structural properties. however, proiapp shows a much less amyloidogenic propensity, probably due to the increased net charge on proiapp, compared to iapp. unlike iapp, proiapp forms small oligomeric structures at the lipid interface, having heights of ∼3.5 nm. this morphological distinction can be attributed to the presence of the pro-region, flanking the amyloidogenic iapp. the addition of proiapp to iapp marginally delays iapp fibrillation, probably by interfering with the interaction between amyloidogenic iapp cores of distinct iapp molecules. thus, it appears reasonable to speculate that the pro-region of the proiapp could serve to delay the fibrillogenesis of iapp at negatively charged lipid bilayers. the role of transmembrane domain interactions in the kinetics and folding of cpt 1 z. a. jenei 1 , k. borthwick 2 , v. a. zammit 2 , a. m. dixon 1 1 chemistry dept., univ. of warwick, coventry, uk, 2 clinicalȃsciencesȃres.ȃinst., warwick univ., coventry, uk carnitine palmitoyltransferase i (cpt 1) enzymes are polytopic integral membrane proteins in the outer membrane of mitochondria (omm). cpt 1 controls the rate of entry of long-chain fatty acids into the mitochondrial matrix for βoxidation. the two catalytically active isoforms, cpt 1a and cpt 1b, are different in their inhibitor binding kinetics and structure (interaction between n-and c-segments, interactions of transmembrane domains (tmd)). it has been suggested that inter-and intramolecular tmds interactions are important for cpt 1a, but not for cpt 1b function. cpt 1a has also been implicated in formation of oligomeric complexes through its tm segments. the study of tm helix-helix interactions in cpt 1 isoforms could lead to a better understanding of their function and inhibitor binding kinetics, and will contribute towards the design of pharmacological strategies aimed at modulating the activities of cpt 1 enzymes in conditions such as diabetes. to investigate the ability of the tmd in cpt 1 to self-associate and the order of any oligomers formed, several biochemical and biophysical techniques have been used. we found the self-association of rcpt 1a tmds (tm1, tm2) to be different as measured using the in vivo tox-cat assay. chemical cross-linking and analytical ultracentrifugation studies demonstrated formation of both trimers and hexamers by the rcpt 1a tm2 peptide. these results provide further evidence that tm2 plays role in formation of a channel in the omm. self-assembly of transmembrane domains in cpt1: role of gxxxg(a) motif in possible channel formation z. a. jenei 1 , k. borthwick 2 , v. a. zammit 2 , a. m. dixon 1 1 department of chemistry and, 2 warwick medical school, university of warwick, coventry, uk carnitine palmitoyltransferase 1a (cpt1a), a membrane protein that controls the rate of oxidation of long-chain fatty acids, is of key importance in diabetes and has recently been reported to exist as an oligomer in vivo. we have investigated full-length cpt1a and find that the protein exists as a hexamer in liver mitochondria. using mutants of cpt1a expressed in yeast mitochondria, we have localised key protein interactions in the hexamer to the transmembrane (tm) domains of the protein. detailed study of the tm domains in isolation, in both e.coli membranes and detergent micelles, demonstrated that while tm1 shows little self-assembly, tm2 displayed significant self-association. biophysical analyses of a tm2-derived synthetic peptide revealed oligomerization behaviour identical to native cpt1a in mitochondria, providing a strong link between tm2 helixhelix interactions and cpt1a hexamer formation. this is significant in light of a recent suggestion that cpt1a oligomerization may lead to formation of a channel in the mitochondrial outer membrane through which acylcarnitine gains access to the inter-membrane space. our data supports this new theory, and we go on to demonstrate experimentally the structural determinants of hexamer (channel) formation, specifically gxxxg(a) motifs in the tm domain which pack favourably in the hexamer and stabilize the oligomer. investigation of flexible loop role in structure and thermodynamic stability of firefly luciferase p. maghami, b. ranjbar, s. hosseinkhani department of biochemistry and biophysics, faculty of basic sciences, tarbiat modares university, tehran, iran protein folding, like any chemical process, consists of two fundamental components, thermodynamics and kinetics, which determine the stability of the folded state and the pathway of folding, respectively. these processes are currently too difficult to be solved de novo by purely computational methods. experimental evidence is required to simplify the problem via protein engineering .in this research, the wild type firefly luciferase (photinus pyralis) and some of its mutants were over expressed and purified. then their unfolding thermodynamics were examined, using circular dichroism and conventional fluorescence measurements. the unfolding equilibrium constant were measured over a complete rang of denaturant conditions. the measurements were shown structural and physico-chemical changes between wild-type and mutant proteins. exploring intrinsic disorder of unstructured membrane proteins by surface polymer physics intrinsically unstructured proteins (iups) are considered as a separate class within the protein world because they lack a well-defined folded structure. because iup's function is indeed directly linked to structural disorder, they are assumed to be natively unfolded. several physicochemical techniques are available to discriminate the degree of disorder. however a clear structural classification is still lacking. in this context, polymer physics emerges as a powerful tool for getting inside on the conformational abilities directly related to structural disordered of iups. in the present contribution, surface pressure and ellipsometry experiments in conjunction with polymer physics have been used to infer structural data in terms of molecular conformation and flexibility of a membrane protein essential for bacterial division, zipa. this protein has been pointed to posses a high molecular flexibility and to adopt a random coil conformation. folding dynamics of peptides studied by timeresolved infrared spectroscopy c. krejtschi 1 , o. ridderbusch 1 , r. huang 2 , l. wu 2 , t. a. keiderling 2 , k. hauser 1 1 institute of biophysics, university of frankfurt, germany, 2 department of chemistry, university of illinois at chicago, usa peptides are ideal model systems to study protein folding mechanisms. ir techniques provide both the necessary time resolution as well as the structural sensitivity. we initiate rapid heating by laser-excited ns temperature jumps (∼10 • c) and study fast ns-to-µs relaxation dynamics [1] . the dynamics of the α-helix to random coil transition of polyglutamic acid was analyzed under reversible folding/refolding ph-conditions. the observed relaxation kinetics allowed separation of the folding and unfolding process with additional use of ftir measurements in thermal equilibrium. sitespecific dynamics were monitored by use of isotopic labeling for a β-hairpin peptide whose conformation is stabilized by a hydrophobic core. various single and cross-strand isotopically labeled variants were analyzed. the isotope-edited kinetics show variations in local structural stability of the hairpin backbone. our data support a multistate dynamic behavior, and the site-specific kinetics are consistent with a hydrophobic collapse hypothesis for hairpin folding [2] . small heat shock protein hsp22 was predicted to belong to the family of intrinsically disordered proteins. one of the features of these proteins is that they do not demonstrate cooperative thermal transitions on heating. we applied different methods (dsc, ftir and intrinsic tryptophan fluorescence) to investigate the thermal unfolding of hsp22. dsc results have shown that thermal denaturation of hsp22 begins from 25 o c and occurs, with very low cooperativity, within a broad temperature region (up to 80 o c and above). the thermal unfolding of hsp22 is fully reversible. the ftir data show that heating of hsp22 from 20 to 70 o c results in complete disappearance of α-helices (from 8-12% to 0) and the decrease in β-sheets content from 42 to 30%. studies on the temperature dependences of tryptophan fluorescence have shown a significant red shift of the spectrum. these changes occurred within temperature region from 30 to 80 o c with midpoint at ∼56 o c. probably, this transition can be explained by destruction of β-sheets around trp96, the only trp residue of the α-crystallin domain of hsp22 (other 3 trp residues of hsp22 are localized in the n-terminal domain). the data obtained confirm the suggestion that hsp22 is a protein, whose significant part is intrinsically disordered. we propose that, on heating, the α-crystallin domain containing β-sheets melts at higher temperature than the n-terminal domain containing the most of α-helices. t. rosenkranz 1 , a. katranidis 1 , d. atta 1 , j. enderlein 2 , i. gregor 2 , m. grzelakowski 3 , p. rigler 3 , w. meier 3 , j. fitter 1 1 isb-2: molecular biophysics, research centre jülich, germany, 2 institute of physics, biophysics/complex systems, georg august university, göttingen, germany, 3 institut für physikalische chemie; universität basel, basel, swizerland the protein folding mechanism is the missing link in the biological flow of information from the dna to its specific function. since most of proteins within a cell consist of more than one domain studies on this protein class are of major importance. it is a common feature of multidomain proteins to aggregate under refolding conditions, which hinders a refolding. molecular encapsulation of single molecules prevents aggregation. by immobilizing the nanocapsule the observation period in a wide field microscope will be extended, so that slow or rare folding events can be detected. a major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein folding studies [1] . polymer vesicles maintain their structural integrity even under harsh unfolding conditions. furthermore the nanocontainer proved to be permeable to guanidinium hydrochloride. using encapsulated phoshoglycerate kinase, labeled with atto-655, a dye which experiences fluorescence quenching by photo-induced electron transfer (pet) with tryptophans, we demonstrate the remarkable properties of polymeric nanocontainers. applying pet as a folding probe we detected multiple unfolding/refolding transitions of single proteins. proteins frequently become irreversibly modified by carbonylation, a process of introducing the carbonyl group (carbon monoxide) in a reaction with reactive oxygen species (ros) such as superoxide, peroxide or ozone. the main targets for carbonylation in proteins are amino-acid side chains of lysine, arginine and proline. products of carbonylation are aminoadipic semialdehyde from lysine (asa) and glutamic semialdehyde (gsa) from arginine and proline. importantly, carbonylated proteins are marked for proteolysis by the proteasome, but can escape degradation and form aggregates that can be cytotoxic. carbonylation increases with the age of cells and it is associated with ageing and age related disorders such as alzheimer's disease, parkinson's disease and cancer. we have used the molecular dynamics method to study the stability of carbonylated proteins villin headpiece and ubiquitin. simulations were run after mutations of arginine, proline and lysine into gsa and asa had been performed. in addition, we have used thermodynamic integration on lysine, arginine, proline, asa and gsa residues in order to estimate their solvation free energy (related to relative hydrophobicity and hydrophilicity). our results suggest that carbonylation markedly decreases the overall stability of proteins, and that one potential reason for that may be a disruption of the balance between hydrophilic and hydrophobic regions in the protein. a. martino, d. crane, i. m. feavers, b. bolgiano division of bacteriology, national institute for biological standards and control, potters bar, uk the sensitivity to protein's secondary structure and progress in computational calculations have made circular dichroism (cd) an attractive technique to explore the optical properties of three promising vaccine candidates to neisseria meningitidis. clinical batches of a c-term deleted form of nada (genome-derived neisseria antigen -gna1994) and the fusion proteins gna2132-1030 (fp-1) and gna2091-1870 (fp-2) were therefore studied. increases in temperature and denaturant concentration on secondary structures and folding/unfolding profile were monitored by cd in the far and near uv regions and complemented by trp fluorescence spectroscopy data. furthermore, epitope conformational changes on binding activities to immune-sera were investigated. the calculated secondary structure content was in broad agreement with the available predicted or solved protein structures. upon temperature incubation, a structural transition from a highly α-helical nada to a more unordered conformation, with a mid point at ∼40 • c, was observed. fp-1 and fp-2 maintained their conformation up to 50 • c or 6m guhcl in the case of fp-1. unfolding was not always reversible. reductions in binding to monoclonal ab titrated along with increasing unfolding. folding/unfolding studies have proven useful in better understanding the solution behaviour and extent of folding of proteins. cold denaturation of yfh1 offers the clue to understand the effect of alcohols on protein stability s. r. martin 1 , v. esposito 1 , p. de los rios 2 , a. pastore 1 , p. a. temussi 3 1 national institute for medical research, the ridgeway, nw7 1aa london, u.k., 2 laboratoire de biophysique statistique, sb/itp, ecole polytechnique fédérale de lausanne (epfl), ch-1015, lausanne, switzerland, 3 dipartimento di chimica, università di napoli federico ii, via cinthia, i-80126 napoli, italy although alcohols are well known to be protein denaturants when present at high concentrations, their effect on proteins at low concentrations is much less well characterized. here we present a study of the effects of alcohols on protein stability using yfh1. exploiting the unusual property of this protein of undergoing cold denaturation around 0 • c without any ad hoc destabilization, we determined the stability curve on the basis of both high and low temperature unfolding in the presence of three commonly used alcohols: trifluoroethanol,ethanol methanol. in all cases, we observed an extended temperature range of protein stability as determined by a modest increase of the high temperature of unfolding but an appreciable decrease in the low temperature of unfolding. we suggest that alcohols, at low concentration and physiological ph, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. our results also clarify the molecular mechanism of the interaction and validate the current theoretical interpretation of the mechanism of cold denaturation. biomolecular sciences and biotechnology tor vergata moro 5, 00185 rome, italy cholesterol plays an important role in regulating the structural properties of phospholipid and non-phospholipid membranes. in this study we have applied in situ energy dispersive x-ray diffraction (edxd) to investigate the effect of cholesterol on the structure of different phospholipid and non-phospholipid oriented membranes. in detail, phosphatidylcholine (pc) bilayers and niosomal membranes, made of a non-ionic surfactant centre for bioactive chemistry, department of chemistry department of chemistry this process is initiated at nuclear envelope remnants (ners) in the presence of atp and gtp. the mvs can be divided in two main populations: mv1 and mv2. mv2 has a classical lipid composition while mv1 is enriched in phosphoinositides (pips: pi, pip, pip 2 and pip 3 ). ners have an unusual lipid composition, enriched both in cholesterol and pips. physicochemical properties of the pips were investigated as a function of ph and temperature (t) using nmr, saxs and dls to map out their phase state. pips-water dispersions are observed in lamellar, hexagonal or isotropic phases depending on t and ph. in parallel, model membranes mimicking mv1 and ners lipid composition were studied by 2 h and 31 p nmr. mv1 modelling shows a complex behaviour of pips on pc membranes: they order or disorder membranes, whereas the order of pc/pi/pip/pip 2 membrane is lower than that of pc or pc/pi membranes c. manzo, t. s. van zanten, m. f. garcia-parajo bionanophotonics group, ibec-institut de bioenginyeria de catalunya, barcelona, spain membrane proteins play a fundamental role in intra-and inter-cellular functions. in particular, the proteins lateral mobility in the fluid membrane environment is crucial for the regulation of several mechanisms, as receptor-mediated signal transduction and establishment of immunological synapses. these mechanisms are controlled through protein crowding and reduced lateral diffusion, which induce macromolecular associations and limit the application of conventional single molecule fluorescence techniques. to measure proteins mobility on living cells membrane, we developed a fluorescent correlation spectroscopy (fcs) setup in which the sample illumination is obtained through near-field scanning optical microscopy (nsom) probes. the use of nsom probes is particularly suited for the observation of dynamics on the cell membrane and overcomes the drawbacks of other techniques. in fact, through a shear-force-based position control, the probe is kept at a fixed distance from the membrane and its sub-wavelength aperture (∼100 nm) reduces the illumination area, allowing the observation of highly crowded regions of the membrane. on the basis of preliminary results, the nsom-fcs is expected to provide an additional insight on the proteins trafficking at the membrane level. the technique also presents several potential developments, as the further reduction of the illumination area and two-colors correlation. single molecule fluorescence microscopy of the store-operated calcium channel subunit orai1 j. madl, j. weghuber, d. bergmair, m. fahrner, m. muik, c. romanin, g. j. schütz johannes kepler university, institute for biophysics, linz, austria store-operated calcium entry (soce) is essential for many cellular signalling processes. the essential pore forming subunit of soce channels in the plasma membrane is orai1. here we present single molecule fluorescence microscopy of orai1 which was performed in order to directly visualize the stoichiometry of mobile orai1 pores. the protein was fluorescently labeled with monomeric gfp. a novel single molecule fluorescence approach, toccsl (thinning out clusters while conserving the stoichiometry of labeling), was used for the determination of the stoichiometry. this technique allows reducing the density of fluorescently labeled molecules without affecting the stoichiometry of labeling. density reduction is achieved by completely photobleaching a defined area within the plasma membrane; nonbleached gfp-orai1 aggregates enter the bleached region subsequently by diffusion. our data indicate that there are different populations of orai1 present in the cell: most of orai1 is located in the plasma membrane. a second population of orai1-mgfp was found to be localized in intracellular vesicles. a significant fraction of the plasma membrane orai1 exhibits a diffusive movement. we found by analyzing the bleaching characteristics of single orai1-mgfp aggregates that in resting cells mobile orai1 is predominantly dimeric. a. kobitski 1 , a. nierth 2 , m. helm 2 , a. jäschke 2 , g. u. nienhaus 3 1 university of ulm, germany, 2 university of heidelberg, germany, 3 university of karlsruhe, germany rna molecules have attracted enormous attention in recent years, and various novel roles were revealed for rna in biological processes. ribozymes are a class of rna molecules capable of catalyzing chemical reactions. we have studied a diels-alderase (dase) ribozyme, a small artificial 49-mer ribozyme, which is capable of catalyzing carbon-carbon bond formation between an anthracene diene and a maleimide dienophile in multiple turnovers. single-molecule fluorescence resonance energy transfer was employed to investigate the intramolecular dynamics of this rna molecule as a function of mg 2+ ion concentration. folding into a functional state occurs via an intermediate state, and continuous fluctuations between these two states were observed on the 100ms time-scale at the midpoint concentration of mg 2+ ions. an effect of substrates binding on the folding and catalytic reaction of the dase ribozyme is in the focus of our recent research with the ultimate goal to obtain a detailed structural view of the single-molecule conformational changes that accompany the catalytic reaction. a. katranidis 1 , r. schlesinger 1 , k. nierhaus 2 , i. gregor 3 , m. gerrits 4 , g. bueldt 1 , j. numerous studies showed that protein folding and maturation can differ substantially between de novo synthesized proteins and in vitro refolded proteins. here we present an approach employing a two color single molecule sensitive fluorescence wide-field microscope in order to visualize surface tethered fluorescently labeled ribosomes and de novo synthesized gfp molecules in real time [1] . fluorescence of co-translational folded proteins was observed from mature fluorescent gfp molecules which carry 31 additional amino acids at the c terminus remaining linked to the ribosome. thus it was possible to co-localize fluorescence from labeled ribosomes and from gfp molecules. we demonstrate that the green fluorescence protein mutant gfp emerald is produced with a characteristic time of five minutes. the fastest gfp molecules appeared already within one minute. processes precedent to chromophore formation, such as polypeptide synthesis and protein folding, are fast and last not longer than one minute. in fluorescence spectroscopy, photobleaching is a process which leads to irreversible loss of fluorescent properties of a dye molecule, usually due to photochemical reactions. it is especially important for fcs experiments on slow-diffusion systems since for high excitation intensities it can have a strong impact on fluorescence intensity correlation function. usually it is observed as apparent shortening of the mean diffusion time of the dye molecules. the behavior of tmr-labeled fd-virus rods in water solution under various excitation conditions was investigated. the experiments were conducted for low (1:1) and high (625:1) tmr:virus ratios and for increasing laser intensities. the correlation function was measured in the experiments. the results were fitted using origin software to estimate the influence of photobleaching, and compared with computer simulations. a strong effect of photobleaching was visible for rods labeled with a single dye molecule, while rods labeled with 625 tmr molecules showed little to no bleaching. a prolongation of characteristic diffusion times for highly labeled virus rods in comparison to low-labeled ones was also observed. k. toth 1 , a. gansen 1 , a. valeri 2 , v. böhm 1 , c. a. seidel 2 , j. langowski 1 1 abt. biophysik der makromoleküle, deutsches krebsforschungszentrum, heidelberg, germany, 2 lehrstuhl für molekulare physikalische chemie, heinrich heine universität, düsseldorf, germanythe nucleosome has a central role in the compaction of genomic dna and the control of dna accessibility for transcription and replication. we studied the effect of dna sequence and selective histone acetylation on the structure, stability and disassembly of the mononucleosomes. quantitative single molecule fret measurements between dyes attached to different parts of the nucleosome permitted us to detect the equilibrium between several subpopulations of reconstituted nucleosomes in solution. we obtained that the heterogeneity and stability of the samples are correlated with each other and influenced both by the dna sequence and the histone acetylation. the path of the linker dna is more sensitive to all studied effects than the dna on the core. intermediates of the disassembly pathway were identified and characterized. j. strömqvist 1 , s. johansson 1 , y. ohsugi 3 , k. andersson 2 , l. xu 1 , m. kinjo 3 , p. höglund 2 , j. widengren 1 1 experimental biomolecular physics, kth, stockholm, sweden, 2 department of microbiology and cell biology, karolinska institutet, stockholm, sweden, 3 laboratory of molecular cell dynamics, hokkaido university, sapporo, japan dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly49a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined.here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. dynamic multiple-target tracing probes spatiotemporal cartography of cell membranes in order to decipher the non random and non homogeneity of the plasma membrane organization, we had performed fluorescence correlation spectroscopy measurements on live cells. this allowed us to establish the presence of nanoscale confining structures 1 and to demonstrate their implication in signaling process 2 . complementing these studies, we present here a new analytical method, namely multiple-target tracing (mtt) 3 which takes advantage of the high resolution provided by singlemolecule sensitivity to generate dynamic maps at high densities of tracked particles. introducing deflation by subtracting detected peaks allows detecting peaks of lower intensity. we achieved an exhaustive detection of particles with performances reaching theoretical limits, and a reconnection of trajectories integrating the statistical information from past trajectories. we demonstrate the potential of this new method of analysis by applying it to the epidermal growth factor receptor labeled with quantum dots, in the plasma membrane of live cells. this has allowed us to build up a global representation of molecular dynamics in cell membranes. dual polarisation interferometry (dpi) is a surface analytical technique capable of dynamically measuring biophysical parameters of conformational change in biomolecular interactions. the technique measures three key parameters, namely layer thickness, layer density (ri) and mass, thereby enabling the resolution of conformational changes involved during binding. a number of different applications are presented. protein-protein interactions: understanding the biophysical nature of protein interactions can deliver insights into the mechanisms by which proteins interact, thereby elucidating protein function. dpi enables correlation between binding affinity and conformational change, greatly enhancing the study of structure-function relationships. lipid layers: the birefringence mode of dpi can be used to study the formation of lipid bilayers and biomolecular self-assembly. it is possible to use a combination of bilayer refringence and mass to study phase transitions associated with protein or peptide binding to the lipid bilayer. the individual stages of adsorption, absorption and micellisation can be distinguished. carbohydrate interactions: dpi uses a planar glass surface and a wide range of coupling chemistries. a carbohydratespecific surface can be used to study a wide range of biomolecular interactions, such as lectins, acidic proteins, extracellular matrix signaling and interactions with complex membranes. the experimental characterization of the elementary conformational steps constituting the protein folding pathway remains a big challenge. quenching of the triplet state of tryptophan by close contact with cysteine has been shown to provide a new tool for measuring the rate of intramolecular contact formation -one of the most elementary events in the folding process -in peptides and proteins using only natural probes. here we show an extensive study on a stabilized mutant of the second beta-hairpin of gb1 domain. steady state fluorescence and kinetics of contact formation between a natural tryptophan and a cysteine added to the c-terminal are measured for different temperatures and solvent conditions. we separately address the contributions of different structural elements to the overall stability of the hairpin. we extract kinetics parameter for contact formations in the unfolded state, formation of the hydrophobic core and tails pairing in the folded state. by means of a fragment peptide terminated with a tryptophan and a cysteine, we also estimate the structural propensity of the turn region. the data coherently combine with a simple model previously developed to describe the dynamics of unstructured chain [biophys. j. 96, 1515 (2009)], here modified with the addition of attractive interactions between specific residues. catalytic power of partially denatured enzymes: implementation of molten-globule-like states m. shushanyan 2 , d. e. khoshtariya 1 , m. makharadze 1 , t. tretyakova 2 , r. van eldik 3 1 institute of molecular biology and biophysics, gotua 12, 0160 tbilisi, georgia, 2 department of physics, i. javakhishvili tbilisi state university, 0128 tbilisi, georgia, 3 department of chemistry and pharmacy, university of erlangen-nürnberg, germany impact of nonspecific moderate denaturants, urea and dmso, on the kinetic (functional) and thermodynamic (stability) patterns of a hydrolytic enzyme, α-chymotrypsin (α-ct) has been investigated. furthermore, an impact of urea and guhcl in combination with of temperature on the kinetic pattern of carboxypeptidase a has been examined. for the case of α-ct, in particular, we have observed about tenfold increase of the apparent mickaelis constant when going from 0 to 6 m urea (25 o c), whereas the value of catalytic constant remained almost unchanged, indicating that the protein is not unfolded even under those severe conditions. the matching microcalorimetric experiments revealed that both the temperature-induced melting temperature and transition enthalpy decrease gradually with the increase of the additive concentration. with 6 m urea the peak-shaped calorimetric feature disappears totally. however, catalytic power of α-ct was preserved owing to its catalytic constant. for other studied enzyme/substrate/denaturant arrays diverse kinetic peculiarities due to mgls have been observed. rubredoxins are a class of iron-containing proteins whose biological role on electron transfer processes and metal binding is still unclear. the unfolding dynamics of the rubredoxin mutant rda51c from the mesophile desulfovibrio vulgaris (dv) was studied on the temperature range from 25 • c to 87 • c and along time at 87 • c. resonance energy transfer (ret) was used to determine the donor (d; trp37) to acceptor (a; 1,5-iaedans) distance. from 25 • c to 87 • c the d-a distance increased 4å. however the random coil expected d-a distance was only achieved after heating the protein solution during 2.5 hours at 87 • c. from uv-vis absorption data it's clear that this protein is capable of maintaining its iron-sulfur center at 90 • c. by melting the protein at the same temperature all iron-centers disintegrate and the protein unfold after 2.5 hour. the trp37 fluorescence also shifts 13 nm to the red reflecting the partial exposition of the indole ring to the solvent. from fluorescence and anisotropy decay curves a breathing type movement of the protein structure was observed between 30 • c to 60 • c without lost of significant secondary structure. this structure flexibility should play an important role on the thermal stability of the dvrd the antimicrobial peptide novicidin (nc) -modified from the sheep self defense peptide smap-29, for reduced mammalian cytotoxicity -is a cationic peptide (net charge +∼7.5) that adopts random coil structure in solution, but an α-helix in the presence of lipid vesicles. the conformation of nc in presence of the lipids dlpc, dlpg, dmpc, dmpg, dopc, dopg, dope, and dops in different combinations reveal the lipid selectivity, affinity, and phase dependent changes with varying l/p ratios and temperatures, observed from cd spectroscopy. it is understood that the conformational change is dependent on chain length and head group of the lipid. apart from the in vivo results on the nc activity, studies using qcm-d, dual polarisation interferometry, and calcein release assay reveal the kinetics and concentration dependent activity of nc in lipid bilayers and vesicles. preliminary studies on orientation of nc in various lipid environments using ssnmr, lsnmr, and molecular dynamics simulations apparently suggest that nc may form toroidal pores/detergent effects depending on the chain length of the lipids. further experiments on nc in presence of lipids using dsc, itc, ssnmr, oriented cd, ld, and confocal microscopy to determine the structure, thermal stability, orientation in lipid bilayers, and thereof the action of nc will help in proposing a comprehensive model for its mechanism of action in model membranes. dielectric method for measuring glass transition and denaturation temperatures of hydrated proteins g. e. thomas 1 , s. bone 1 , g. drago 2 1 institute for bioelectronic and molecular microsystems, bangor university, gwynedd, uk., 2 applied enzyme technology, pontypool, uk.the flexibility of protein structures is important in allowing the variety of motions, covering a wide range of magnitudes and frequencies, essential to biological activity. high frequency dielectric measurements can be used to study the flexibility of proteins by probing the relaxation of dipolar constituents of their structures. hydrated proteins exhibit a broad dielectric loss extending over the frequency range from 1mhz to 10ghz which can be decomposed into a number of constituent dispersions. one of these dispersions, with a relaxation time of ∼18ns, has been attributed to the relaxation of protein backbone peptide groups in the protein interior. in the work reported here, this dielectric dispersion was investigated as a function of temperature for the enzyme glucose oxidase. two critical temperatures were identified as the glass transition and denaturation temperatures, both of which were found to decrease with increasing protein water content. the results were consistent with a scheme in which the hydrated glassy protein undergoes a change in structural mobility at the glass transition temperature and experiences an irreversible change in conformation at a higher denaturation temperature. both glass transition and denaturation temperatures are key indicators of protein stability and are important in the production and storage of protein based pharmaceuticals. a. szymańska, k. kacprzyk, g.ślósarek department of molecular biophysics, a. mickiewicz university, umultowska 85, 61-614 poznań, poland aggregation dynamics of proteins plays an important role in molecular biology and medicine as it permits explanation of several disease states. an interesting problem is to find out in which conditions the interactions of the protein molecules lead to formation of ordered structures and in which to disordered ones. in this study, dynamic light scattering, circular dichroism and also congo red dye experiments were performed to analyze various structural states of lysozyme induced under different concentration of ethanol solvent. at low ethanol concentration the attractive interaction between the protein macromolecules dominate. after addition of more ethanol solvent, the translational diffusion coefficient was much smaller than that for lysozyme solution at zero ethanol concentration. it can be explained by the structural transformation of the polypeptide chain leading to a partially folded conformation needed for oligomerization and fibrillation process. on the basis of the cd experiments we concluded that the ethanol solvent induces changes in secondary structure of lysozyme solution. on addition of 75% v/v ethanol solution the intramolecular hydrogen bonds were destabilized. above this ethanol concentration, β -sheet were the dominant secondary structure of lysozyme in solution. the phase diagram illustrating the formation of: monomers, oligomers at various structural states, protofilament formation state, protofilament and amyloid fibrils was constructed. key: cord-018493-q24f86e9 authors: ranjan, prabhat; dey, asmita; sharma, vinod praveen; tiwari, neeraj kumar title: importance of natural proteins in infectious diseases date: 2015-08-08 journal: biomedical applications of natural proteins doi: 10.1007/978-81-322-2491-4_8 sha: doc_id: 18493 cord_uid: q24f86e9 proteins are important biomolecules, extensively involved in almost all biological processes. a number of proteins are also implicated in infectious diseases. bacterial proteins used in adhesion to host epithelium, bacterial toxins, and viral membrane glycoproteins are some of the proteins involved in infectious diseases. even components of the host innate immune system like toll-like receptors and nod-like receptors and adaptive immune components like immunoglobulins aiding in defense against pathogens are important biological proteins. chaperones like acid and heat shock proteins provide protection from high temperatures, metabolic poisons, and other stressful conditions. several natural and artificial proteins are components of vaccines, a key strategy to control fatal diseases, lacking empirical treatment. it is necessary to investigate these proteins, to develop new biomedical tools and technologies, aiding in eradication of various diseases. thus, further research should be carried out in this field, for saving and improving quality of human lives. an infectious disease is a disease that is caused by the invasion of a host by agents whose activities harm the host's tissues (that is, they cause disease) and can be transmitted to other individuals (that is, they are infectious). infectious diseases, also known as transmissible diseases or communicable diseases, comprise clinically evident illness (i.e., characteristic medical signs and/or symptoms of disease) resulting from the infection, presence, and growth of pathogenic biological agents in an individual host organism. infections are caused by infectious agents such as viruses, and prions, microorganisms such as bacteria. proteins might be involved in causing a disease as well as protecting the host from it. various membrane proteins of microorganisms (virus, bacteria, parasites, etc.), prions are responsible for causing different diseases. proteins like heat shock proteins/chaperons aid in causing as well as protecting from diseases. proteins might also be involved in playing solely protective roles against infections like various cell surface receptor proteins (toll-like receptors, nod-like receptors, etc.), which trigger protective immune responses. nowadays, various antimicrobial peptides or antibiotics are known to kill microorganisms or inhibit their growth, thus aiding in treatment of diseases. thus, proteins, one of the most incredible biomolecules, are extensively involved in almost all biological mechanisms, especially those implicated in pathophysiology of infectious diseases. the understanding of this topic is essential for developing different biomedical tools, for combating various diseases, thus, improving the quality of human life. a number of bacterial proteins are involved in its pathogenesis and virulence. bacterial infections are usually initiated by adherence of the microbe to a specific epithelial surface of the host, otherwise the organism will get removed: (1) fimbrial adhesions involved in mediating attachment of some bacteria to mammalian cell surfaces, e.g., neisseria gonorrhoeae use pilli to adhere to the mucous membrane of urethra and can resist the flushing action of urine [1, 2] . (2) nonfimbrial adhesion including the filamentous hemagglutinin of bordetella pertussia helping in attachment to respiratory epithelium, a mannose-resistant hemagglutinin produced by salmonella typhimurium and a fibrillar hemagglutinin from helecobacter pylori [3, 4] . other adhesions like protein f produced by streptococcus pyogenes, aiding in attachment to pharyngeal epithelium [5] . some extracellular bacterial proteins considered invasins like hyaluronidase, produced by streptococci, staphylococci, and clostridia, which degrades hyaluronic acid of connective tissue [6, 7] , collagenase produced by clostridium sp, which dissolves collagen framework of muscles [8] , neuraminidase produced by vibrio cholerae and shigella dysenteriae, which degrades neuraminic acid of intestinal mucosa [9] . other extracellular proteins like invasive enzymes, e.g., coagulase, contributes to the formation of fibrin walls around staphylococcal lesions [10] ; exotoxins (proteins released extracellularly), like neurotoxin (tetanus toxin, by clostridium tetani, botulinum toxin by clostridium botulinum) [11] and cytotoxins (diphtheria toxin produced by corynebacterium dipthereae) [12, 13] , also known as a-b toxins (consisting of 2 subunits: one binds to cell surface receptor and the other is transferred into the cell to damage the cell) [14] , cytolytic toxins (attacking cell constituents causing lysis) like hemolysins produced by bordetella pertussis, inducing apoptosis of host cells, super antigen toxins (e.g., superantigen, sized 22kda produced by 5-25 % of staphylococcus aureus isolates, causing toxic shock syndrome (tss) by stimulating the release of large amounts of interleukin-1, interleukin-2 and tumor necrosis factor, etc.) [15] . enterotoxins (exotoxins that act on the small intestine, generally causing massive secretion of fluid into the intestinal lumen, leading to vomiting and diarrhea) produced by vibrio cholerae, e. coli o157:h7. endotoxins (generally cell-bound toxins released only when cells are lysed) produced by most gram-negative bacteria are generally nonproteinaceous, lipopolysaccharide in nature [16] (fig. 8.1 ). viral glycoproteins, made up of carbohydrates and proteins mainly help in adherence of viruses to host cell surfaces and internalization of viral components. the addition of sugar chains or glycosylation of the protein surface-components, can happen either at asparagine, and is termed n-glycosylation, or at hydroxylysine, hydroxyproline, serine, or threonine, and is termed o-glycosylation. glycosylation is often present in proteins that are at least in part located in extracellular space. the sugar group can assist in protein folding or improve its stability. glycoproteins also aid in immune cell recognition. viral glycoproteins are composed of three parts: external/ecto-domain, which interacts with host; transmembrane segment, which spans the viral envelope membrane, typically α-helix; and endo-domain, the internal part. ecto-domain can be of two classes: class i, found in orthomyxoviruses, paramyxoviruses, retroviruses, filoviruses and coronaviruses; class ii, found in flaviviruses and alphaviruses. other types of ecto-domains that do not fit in class i and ii are glycoprotein b (gb) of herpes simplex virus (hsv) and glycoprotein g of vesicular stomatitis virus (vsv) [17] [18] [19] [20] . glycoproteins on the surface of viruses are anchored in the lipid bilayer of the envelope by means of hydrophobic bonds, and only about 30 amino acids penetrate into the virus. these transmembrane proteins have large external domains and small cytoplasmic domains. viral glycoproteins are oligomers that are associated with each other to form tetramers, etc. there are often the spikes seen on the virus surface. some glycoproteins such as the influenza hemagglutinin are anchored at both ends, thus forming a loop. in such cases a signal sequence at the amino end has been removed. there are two types of glycoproteins: external glycoprotein anchored in the envelope by a single transmembrane domain, and a short internal tail. these proteins are usually the major antigens of the virus and involved in functions such as hemagglutination, receptor binding, and membrane fusion. the other classes are channel proteins, which are mostly hydrophobic proteins that form a protein lined channel through the envelope. this protein alters permeability of the membrane (e.g., ion channel). such proteins are important in modifying the internal environment of the virus [21] [22] [23] [24] (fig. 8.2 ). the first line of defense against pathogenic microorganisms is innate immunity, and its activation is initiated by the recognition of microbial structures by pattern recognition receptors (prrs). the first and most studied class of prrs is that of a proteinaceous receptor named toll-like receptors (tlrs), named after the toll receptor of drosophila melanogaster. ten human tlrs have been identified, from tlr 1 through 10, with important roles in host defense against bacteria, viruses, and fungi [25, 26] . they recognize a large array of pathogen-associated molecular patterns (pamps), including peptidoglycan, lipoproteins, lipopeptides, phenol-soluble modulin, lipoteichoic acid, lipoarabinomannan, atypical lipopolysaccharides (lpss), porins, flagellin, heat shock proteins (hsp), lycoinositol phospholipids, glycolipids, zymosan, also nucleic acids like double-stranded rna of viruses, etc. [27, 28] . tlr family members are characterized structurally by the presence of a leucine-rich repeat (lrr) domain in their extracellular domain and a tir (toll/interleukin-1 receptor homology) domain in their intracellular region [29] . expression of tlrs is modulated by a variety of factors such as microbial invasion, microbial components, and cytokines. there are two distinct signaling pathways of tlrs: myd88-dependent and myd88-independent pathways. the myd88-dependent pathway signals via myd88, irak, and traf6 and leads to nf-κb activation. the activity of nf-κb (nuclear factor kappa-light-chain-enhancer of activated b cells) is regulated by association with i-κb, which sequesters nf-κb in the cytoplasm until phosphorylated on serine residues by the i-κb kinase (ikk) complex. this phosphorylation leads to the dissociation and nuclear translocation of nf-κb. nf-κb is a transcription factor involved in upregulation of genes responsible for both the innate and adaptive immune responses, e.g., genes involved in t cell development, maturation, and proliferation, cell apoptosis, etc. similarly, in the myd88independent pathway an adaptor molecule named tir domain-containing adaptor protein (tirap)/myd88-adaptor-like (mal) is involved (fig. 8.3 ). nucleotide-binding and oligomerization domain (nod)-like receptors (nlrs) are a group of evolutionarily conserved intracellular proteinaceous prrs that play a vital role in innate immunity and host physiology, in both plants and animals [30, 31] . in humans there are 22 known nlrs, and the association of mutations and single nucleotide polymorphisms (snps) in their genes with human diseases reflect their vital role in host defense. nlrs also play important roles in reproduction and embryonic development. the characteristic feature of nlrs is a central nod (or nacht) domain, required for oligomerization, an n-terminal homotypic protein-protein interaction domain and a c-terminal series of leucine-rich repeats (lrrs) involved in agonist sensing or ligand binding [32] . mammalian nlrs are subdivided into four subfamilies based on the variation in their n-terminal domain: nlra or class ii transactivator (ciita) contains an acid transactivation domain, nlrbs or neuronal apoptosis inhibitor proteins (naips) possess a baculovirus inhibitor of apoptosis protein repeat (bir), nlrcs have a caspase-recruitment domain (card), and nlrps a pyrin domain (pyd). nlrx1 contains a card-related x effector domain. among the nlrs, nlrp1, nlrp3, nlrp6, nlrp7, nlrp12, nlrc4, and naip have been reported to operate via inflammasomes. other nlrs such as nod1, nod2, nlrp10, nlrx1, nlrc5, and ciita do not directly engage the inflammatory caspases, but instead activate nuclear factor-kb (nf-kb), mitogen-activated protein kinases (mapks), and interferon (ifn) regulatory factors (irfs) to stimulate innate immunity [32] . nod-like receptors have been described as master regulators of innate immunity. nlrs are essential in recognition of microbial-and pathogen-associated molecular patterns (mamps and pamps), and have the ability to initiate and support robust immune responses through the formation of inflammasomes and the activation of nf-kb, irf, and mapk pathways. functions such as the enhancement of mhc transcription and presentation implicate nlrs in adaptive immunity, and their roles in reproduction indicate a broader responsibility of this gene family than previously suspected. the potency of nlrs in inducing immune defenses is vital for the host, but can also provide serious problems when dysregulation or malfunction occurs (fig. 8.4 ). the concept of "protein interaction" is generally used to describe the physical contact between proteins and their interacting partners. however, protein interactions do not always have to be physical [33] . protein interaction networks are useful resources in the abstraction of basic science knowledge and in the development of biomedical applications. therefore, protein interaction networks can elucidate the molecular basis of disease, which in turn can inform methods for prevention, diagnosis, and treatment [34] . microorganisms frequently change the ph of their own habitat by producing acidic or basic metabolic waste products. if the external ph decreases to 4.5 or lower, chaperones such as acid shock proteins and heat shock proteins are synthesized. chaperones were first discovered because they dramatically increased in concentration when cells were exposed to high temperatures, metabolic poisons, and other stressful conditions. thus many chaperones are often called heat shock proteins or stress proteins. heat shock proteins influence infectious disease processes in a number of diverse ways: they are involved in the propagation of prions, replication and morphogenesis of viruses, and resistance of parasites to chemotherapy. several heat shock proteins function as intracellular chaperones for other proteins. they play an important role in protein-protein interactions such as folding and assisting in establishment of proper protein conformation (shape) and prevention of unwanted protein aggregation. by helping to stabilize partially unfolded proteins, hsps aid in transporting proteins across membranes within the cell [35] . these proteins also appear to be important mediators of bacteria-host interactions and inflammation, the latter via interactions with cell surface molecules and structures such as toll-like receptors and lipid rafts. heat shock proteins can be expressed on the surface of infected cells, and this is likely to provide a target for the innate immune response. elevated levels of circulating hsp are present in infectious diseases and these proteins might therefore regulate inflammatory responses to pathogenic challenge on a systemic basis [36] . for many years it was believed that polypeptides would spontaneously fold into their final native shape, either as they were synthesized by ribosomes or shortly after completion of protein synthesis. although the amino acid sequence of a polypeptide does determine its final conformation, it is now clear that special helper proteins aid the newly formed or nascent polypeptide in folding to its proper shape [37] . these proteins, called molecular chaperones, recognize only unfolded polypeptides or partly denatured proteins and do not bind to normal, functional proteins. their role is essential because the cytoplasmic matrix is filled with nascent polypeptide chains and proteins. under such conditions it is quite likely that new polypeptide chains often will fold improperly and aggregate to form nonfunctional complexes. molecular chaperones are key players of the homeostasis of the proteome network and suppress incorrect folding and may reverse any incorrect folding that has already taken place [38] . therefore, their expression and the activity are tightly regulated at both the transcriptional and posttranslational level at various age-related diseases (e.g., degenerative diseases and cancer) [39] . chaperones and atp-dependent proteases collectively engaged in the first line of defense of mitochondrial protein quality control (mtpqc) system, and this way it monitors the removal of oxidatively damaged polypeptides. for example, 'lon protease' governs the removal of oxidized proteins in yeast and mammalian mitochondria [40] . many transmembrane proteins are also involved in the protection from infectious diseases. for example, interferon-induced transmembrane protein-3 (ifitm3) is a virus restriction factor mediating cellular resistance to influenza viruses and other viruses that enter cells via the acidic endosome [41] . transmembrane mucins and their o-glycans on the glycocalyx provide the transcellular barrier, a second layer of protection. cell surface glycans bind carbohydrate-binding proteins that respond to extrinsic signals and modulates pathogenic responses. apart from maintaining the homeostasis, it also restricts drug targeting of epithelial cells [42] . some of the members of gap junction (gj) family proteins like intercellular channels, which connect the cytoplasm of neighboring cells and hemichannels that connect the intra-and extracellular milieu, are also known to participate in physiologic and pathologic processes including electrical conduction, inflammation, immune system activation, tissue repair/remodeling, and response to bacterial and viral infections. however, little is known about the role of gj channels in parasite infection. a number of proteins take part in protection of human body from external harmful agents. keratin protein is a component of skin which is considered the first line of defense. this innate immunity component serves to prevent microorganisms from entering the body. lysozyme, an enzyme present in tears and saliva, and thus, another protein also confer protection to the body. likewise, almost all the elements of our immune system, e.g., immunoglobulins (antibodies), various cell surface proteins (t and b cell receptors, major histocompatibility complex, cd4, cd8, cd3, etc.), the several adaptor proteins and enzymes, involved in signal transduction pathways, are all of protein origin and crucial for combating against external infections. since peptides play a crucial role in the fundamental physiological and biochemical functions of life, they have for decades now attracted much attention for their potential therapeutic use. compared with small chemical entity drugs, peptide-based drugs possess certain favorable characteristics, including: • higher potency: peptide-based drugs generally are very active on their target receptor, which translates into a high effect at a low dose; • higher selectivity: peptides can very tightly fit to their receptors, which make them much more selective than smaller molecules. this means that peptides tend to bind only to their target receptor and therefore are less likely to be associated with serious adverse side effects; • naturally occurring biologics-better safety: peptides are naturally degraded in the blood stream by circulating enzymes to their component amino acids. as these are natural biological products, peptide drugs are also associated with less accumulation in body tissue and fewer toxicity findings also owing to their low doses. there are a few challenges associated with the use of peptides like they are generally short-lived, cannot be administered orally and have low product stability. various peptide/glycopeptides drugs are administered: vancomycin, vasopressin, insulin (recombinant), nesiritide, ceruletide, bentiromide, exenatide, etc. vaccination, composed of several natural and artificial proteins, is a key strategy for the control of various infectious diseases. many pathogens, such as streptococcus pneumoniae, haemophilus influenzae type b (hib), and neisseria meningitidis produce on their surfaces dense and complex glycan structures, which represent an optimal target for eliciting carbohydrate specific antibodies able to confer protection against those bacteria. the traditional mechanism of action of glycoconjugates has considered peptides generated from the carrier protein to be responsible for t cell help recruitment. progress of synthetic and biosynthetic methods for the preparation of glycoconjugates gives new insights for the design of improved carbohydrate-peptide conjugate vaccines [43] . similarly, staphylococcus aureus is a prominent cause of human infections worldwide and is notorious for its ability to acquire resistance to antibiotics. methicillin-resistant s. aureus (mrsa), in particular, is endemic in hospitals and is the most frequent cause of community-associated bacterial infections in the united states. s. aureus produces numerous molecules that can potentially promote immune evasion, including protein a (spa), an immunoglobulin (ig)-binding protein present on the bacterial surface and freely secreted into the extracellular environment. this finding provides the foundation to develop a vaccine that prevents severe s. aureus infections [44] . stanley b. prusiner coined the term prion in 1982. it is basically derived from the words protein and infectious [45] . the protein that prions are made of (prp) is found throughout the body, even in healthy people and animals. however, prp found in infectious material has a different structure and is resistant to proteases, the enzymes in the body that can normally break down proteins. the normal form of the protein is called prp c , while the infectious form is called prp sc -the c refers to 'cellular' or 'common' prp, while the sc refers to 'scrapie', the prototypic prion disease, occurring in sheep [46] . a prion in the scrapie form (prp sc ) is an infectious agent composed of protein in a misfolded form [47] . so, they are not considered living organisms but may propagate by transmitting a misfolded protein state. if a prion enters a healthy organism, it induces existing, properly folded proteins to convert into the disease-associated, prion form; the prion acts as a template to guide the misfolding of more proteins into prion form. these newly formed prions can then go on to convert more proteins themselves; this triggers a chain reaction that produces large amounts of the prion form [48] . this altered structure is extremely stable and accumulates in infected tissue, causing tissue damage and cell death [49] . all long-term hematopoietic stem cells express prp on their cell membrane and those hematopoietic tissues with prp-null stem cells exhibit increased sensitivity to cell depletion [50] . a naturally occurring disinfectant exists within common lichens and might actually be able to stop prions in the wild. christopher johnson and his team describe experiments with lichens, symbiotic collections of algae, fungus, and bacteria that casual observers might mistake for moss. three common species of lichens, the team has found, exude an enzyme that breaks down the prion. they showed that these lichen extracts efficiently degrade disease-associated prion protein (prptse), the probable etiological agent of the transmissible spongiform encephalopathies (tses), and suggest that some lichens could have potential to inactivate tse infectivity on the landscape or be a source for agents to degrade prions [51] . proteins are one of the most important biomolecules, involved in any biological process of all living beings, playing useful as well as harmful roles in their survival. it is necessary to investigate these proteins, and to develop new biomedical tools and technologies, which will play a key role in evasion of various diseases, thus, saving and improving quality of lives. further research is ongoing and should be carried out to explore this area of utmost concern. role of adhesin release for mucosal colonization by a bacterial pathogen bacterial adhesins: function and structure molecular aspects of bordetella pertussis pathogenesis the role of the bacterial flagellum in adhesion and virulence molecular basis of group a streptococcal virulence role of hyaluronidase in subcutaneous spread and growth of group a streptococcus acceleration of hyaluronidase production in the course of batch cultivation of clostridium perfringens can be achieved with bacteriolytic enzymes bacterial collagenases: a review neuraminidase production by vibrio cholerae o1 and other diarrheagenic bacteria mcgraw hill, new york 11. brook i (2008) current concepts in the management of clostridium tetani infection university of texas medical branch at galveston, galveston microbial toxins: current research and future trends bacterial toxins toxic shock syndrome: role of the environment, the host and the microorganism bacterial endotoxin and current concepts in the diagnosis and treatment of endotoxaemia glycoproteins encoded by varicella-zoster virus: biosynthesis, phosphorylation, and intracellular trafficking human cytomegalovirus phosphoproteins and glycoproteins and their coding regions the glycoproteins of the human herpesviruses viral glycoprotein heterogeneity-enhancement of functional diversity the role of human immunodeficiency virus type 1 envelope glycoproteins in virus infection virus receptors: implications for pathogenesis and the design of antiviral agents the influenza virus-infected host cell, a target for the immune response influenza: pathogenesis and host defense pathogen recognition and innate immunity toll-like receptors and innate immunity the repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors discrimination of bacterial lipoproteins by toll like receptor 6 toll-like receptors genomic insights into the immunesys-temoftheseaurchin conservation of nlr-triggered immunity across plant lineages functions of nod-like receptors in human diseases interactome data and databases: different types of protein interaction protein interactions and disease: computational approaches to uncover the etiology of diseases molecular chaperones-cellular machines for protein folding prokaryotic and eukaryotic heat shock proteins in infectious disease molecular chaperones in cellular protein folding heat shock proteins: molecular chaperones of protein biosynthesis molecular chaperones and proteostasis regulation during redox imbalance mitochondrial atp-dependent proteases in protection against accumulation of carbonylated proteins. mitochondrion a diverse range of gene products are effectors of the type i interferon antiviral response the ocular surface epithelial barrier and other mechanisms of mucosal protection: from allergy to infectious diseases recent mechanistic insights on glycoconjugate vaccines and future perspectives staphylococcus aureus protein a promotes immune suppression prusiner: autobiography. nobelprize.org. accessed biomedicine. a view from the top prion diseases from 10,000 feet sherris medical microbiology unraveling prion strains with cell biology and organic chemistry the structural basis of protein folding and its links with human disease prion protein is expressed on longterm repopulating hematopoietic stem cells and is important for their self-renewal degradation of the disease-associated prion protein by a serine protease from lichens key: cord-031907-ilhr3iu5 authors: nan title: isev2020 abstract book date: 2020-07-15 journal: nan doi: 10.1080/20013078.2020.1784511 sha: doc_id: 31907 cord_uid: ilhr3iu5 nan introduction: primary tumours secrete large amounts of extracellular vesicles (evs), which play critical roles in preparing distant sites for a pre-metastatic niche formation, thereby promoting metastasis and even determining metastatic organotropism. whether biogenesis, secretion rates and organotropism of evs are linked remains unknown. we have recently shown that ral gtpases control evs secretion in nematodes as well as in mouse mammary tumour cells (hyenne et al. jcb 2015) . since both rala and ralb are overexpressed or over-activated in various human cancers, we aimed to investigate the mechanisms by which these two gtpases control evs secretion and to determine how this affects metastatic progression, with a focus on breast cancer. methods: we used 4t1 mouse mammary carcinoma cells knocked down for either rala or ralb and determined their ability to induce orthotopic tumours and metastasis in a syngeneic mouse model. in vitro, we investigated ev secretion mechanisms using confocal and electron microscopy (em). evs were isolated either by uc or sec and characterized by nta, em, rna sequencing and mass spectrometry. the function of evs was assessed using a transwell assay. finally, we tracked the organotropism of fluorescently labelled evs and their capacity to induce pre-metastatic niches in mice. results: we show that rala and ralb promote lung metastasis of breast cancer cells in mice without affecting their invasive behaviours. we found that rala and ralb control the biogenesis of exosomes, by acting on the formation of multi-vesicular bodies though the phospholipase pld1. as a consequence, knock down of rala or ralb reduces the levels of secreted evs and modifies their rna and protein contents. these differences alter the pro-tumoural function of evs, as demonstrated with an in vitro permeability test. importantly, we show in vivo that evs from rala or ralb depleted cells have a decreased lung organotropism and, as a consequence, are less efficient in priming lung metastasis. finally, we show that high expression of rala or ralb is associated with a bad prognosis in human breast cancer patients. summary/conclusion: altogether, our study identifies ral gtpases as central molecules linking the mechanisms of evs secretion, their dissemination and their capacity to promote metastasis. nuclear proteins are recruited into tumour-derived extracellular vesicles upon expression of tetraspanin tspan8 introduction: tetraspanin tspan8 is a transmembrane protein that exhibits a unique expression pattern, being overexpressed in many cancer types, but undetectable in most healthy tissues. although there is increasing evidence of an effect of tspan8 in invasion, metastasis, and regulation of extracellular vesicle cargo, the molecular mechanisms of tspan8 are yet not fully understood. methods: to study the function of tspan8, we have established a fibrosarcoma model consisting of the parental cell line (ht1080) and its derivatives expressing tspan8 (ht1080-tspan8) either fused with different fluorescent tags or tag-free. life imaging, sted and storm microscopy were used to determine the intracellular localization of tspan8. co-immunoprecipitation from nuclei lysates was performed to detect direct and indirect interacting partners of tspan8. small evs were purified from cell-conditioned media using sec and subjected to mass spectroscopy and ngs for a comprehensive comparative analysis of the proteome and transcriptome of the evs. results: the results of the proteome analysis showed a strong effect in the protein cargo of evs upon tspan8 expression. remarkably, among 20 of the most regulated targets, several histones and ribosomal proteins were enriched in the evs derived from ht1080-tspan8 cells. in line with this finding, life imaging and super-resolution microscopy revealed that, while a majority of the intracellular tspan8 is located on the cell membrane or intracellular membranes, -as it is known for other tetraspanins-, a portion of tspan8 is located on the nuclear envelope. in fact, several histones co-immunoprecipitated with tspan8, indicating their interaction. summary/conclusion: our data show that the expression of tspan8 in the tumour cells greatly impacts ev cargo. moreover, localization of tspan8 on the nuclear envelope, together with the enhanced recruitment of nuclear and ribosomal proteins to the evs, suggests a new mechanism of action of tspan8. introduction: extracellular vesicles (evs) modulate tissue development, regeneration and disease through the transfer of proteins, nucleic acids and lipids between cells. currently, the mechanism of cytosolic delivery of ev cargo is largely unknown. here, we unravel how evs release their cargo in recipient cells. methods: evs were isolated from gfp-cd63 and cd63-rfp expressing hek293 t cells by ultracentrifugation. gfp-cd63 and cd63-rfp evs were added to hek293 t cells stably expressing anti-gfp fluobody and fluorescently tagged galectin-3, respectively. clem and fluorescence microscopy were employed to visualize fluorescent markers in recipient cells. bafilomycin a1 and u18666a were used to inhibit endosomal acidification and cholesterol export from lysosomes, respectively. results: fluorescent galectin-3 which binds to betagalactosides present at the luminal side of endosomes was used to detect endosomal permeabilization. the absence of galectin-3 recruitment to endosomes in presence of cd63-rfp evs showed that endosome permeabilization is not the mechanism behind ev cargo release. gfp-cd63 ev addition to cells expressing anti-gfp fluobodies resulted in the formation of fluobody punctae, reflecting cytosolic exposure of ev cargo. subsequent clem of the fluobody punctae revealed endosomes as the underlying cellular compartments from where cargo release takes place. neutralization of endosomal ph and accumulation of endosomal cholesterol blocked cargo release, showing that ev cargo release is dependent on endosomal ph and cholesterol level. summary/conclusion: we show that genetically encoded cytosolic probes and clem offer an excellent approach to study both the mechanism and efficiency of ev cargo release in cells. we provide experimental evidence that ev cargo release occurs from endosomes. funding: the research was supported by dutch technology foundation ttw and netherlands organization for scientific research nwo, de cock-hadders stichting, and erasmus mundus namaste scholarship. healthy humans. to determine cut-off values for diagnoses, reference intervals of evs in plasma are needed. to establish such reference intervals, (1) a significant number of healthy donors should be included, (2) the presence of non-ev particles, residual platelets, lipoproteins, and haemolysis should be quantified, (3) flow cytometry signals should be in si units. the long-term aim of this study is to determine reference intervals of ev concentrations in human plasma within known dynamic ranges of the detectors. methods: (1) to establish a clinical reference, we collected blood from 224 healthy volunteers and prepared platelet-free plasma. (2) we performed quality control measurements including residual platelet count, serum index, and lipid spectrum. (3) we measured all samples by flow cytometry (apogee a60-micro) and used custom software (matlab r2018b) to automate calibration of all signals and data processing. scatter signals were calibrated in comparable units of scattering crosssection (nm2) and diameter (nm). fluorescence signals were calibrated in units of molecules of equivalent soluble fluorophores (mesf). results: the quality controls showed that most residual platelet concentrations ranged from 10^5 to 10^6 per ml except for one outlier, while the serum index and lipid spectrum were normally distributed. preliminary results of the first 21 donors analysed, show that within the ev size range of 162-1,000 nm, the median concentration of cd61+ evs is 3.9•10^8 per ml (apc>150 mesf), cd62p+ evs is 1.1•10^7 per ml (pe>83 mesf), cd235a+ evs is 5.2•10^7 per ml (pe>123 mesf), and cd45+ evs is 1.8•10^7 per ml (apc>91 mesf). summary/conclusion: we have developed reliable procedures for establishing reference intervals of ev concentrations, within a well-defined size and fluorescence intensity range, in human plasma by flow cytometry. we are currently applying these procedures to 224 samples to obtain, for the first time, ev reference intervals for human plasma. funding: pol, e. van der is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. introduction: the use of extracellular vesicles for diagnostic and therapeutic applications has seen a major interest increase in recent years because of their capacity to exchange components such as nucleic acids, lipids and proteins between cells. isolation of a pure population of evs is the first step in studying their physiological functions since contamination of ev preparations with non-ev proteins can lead to incorrect conclusions about their biological activities. we have developed a new method termed tangential flow for analyte capture (tfac) using ultrathin nanomembranes to purify extracellular vesicles from pure, highly complex biological fluids such as blood plasma, resulting in a new method for extracellular vesicle purification. methods: the tff microfluidic devices are assembled through a layer stack process using patterned polydimethylsiloxane (pdms) sheets with the membranes sandwiched between top and bottom channels. undiluted plasma was tested in both normal flow filtration (nff) and tangential flow filtration (tff) modes on ultrathin nanomembranes. we have utilized a pore patterning technique called nanosphere lithography (nsl) that uses close-packing of nanoscale beads to pattern pores in an ultrathin membrane. results: nff of undiluted plasma resulted in a protein cake of~8 μm on the membrane, which prevented further transport across the membrane and evs were buried in the formed cake that were impossible to identify. however, tfac as a modified version of tff, led to capturing cd63 positive evs on the pores of the membrane with little evidence of protein fouling. nsl allows us to fabricate nanopockets (bowls with a single pore at the base) with various diameter, depth and pore diameter. using nsl, we further utilize nanopocket membranes to purify ev samples in tfac devices. this nanomanufacturing technology will allow us to pattern nanopockets with various diameter, depth and pore diameter which increases the efficiency of capturing of evs. furthermore, nanopockets can be modified and coated by specific ev markers to capture different subpopulation of evs based on size and affinity and further allows identifying the phenotypic subsets of evs by combining both size and affinity-based techniques. summary/conclusion: we have developed a method for the capture and release of nanoparticles such as evs called tfac using ultrathin nanomembranes. nsl technology can be applied to fabricate nanopockets with different physical and biochemical properties. utilizing nanopocket membranes in tfac system will allow us to separate different subpopulations of evs based on size and affinity. funding: this project was supported in part by the national science foundation (iip 1660177) to j.l.m and t.r.g., department of defence (ca170373) to j. l.m., and the national institutes of health (r35gm119623) to t.r.g. the addition of a size exclusion chromatography step to various urinary extracellular vesicle concentrating methods reveals differences in the small rna profile introduction: urinary extracellular vesicles (evs) and their rna cargo are a novel source of biomarkers for various diseases, however non-vesicular rna (e.g. associated with proteins) is also present within urine. this study aimed to identify the optimal method for isolating and enriching evs from human urine prior to small rna analysis. methods: three ev concentration methods, ultracentrifugation (uc); a precipitation-based kit (pk); and ultrafiltration (uf), were compared using 50 ml aliquots of pooled healthy volunteer urine. evs were then separated from protein contaminants by size-exclusion chromatography (sec). presence of evs was confirmed by transmission electron microscopy and western blotting, and evs were quantified using nanoparticle tracking analysis (nta). small rna content of concentrated urine and fractions obtained by sec (evs and proteins) were evaluated with the agilent bioanalyzer small rna chip. results: ev recovery following sec of concentrated samples was 35-78%, however particle: protein ratio (indicating ev purity) was approximately 10x greater after sec, regardless of the concentrating method used. uf+sec yielded the highest number of evs (per ml of urine) compared with pk+sec and uc+sec. small rna analysis from uf-concentrated urine (prior to sec treatment) identified peaks at 20 nucleotides (nt) and 60 nt. following sec, rna analysis indicated that ev fractions contained mostly small rna of~60 nt, whereas the protein factions contained small rna of 20 nt in size (consistent with mirnas). summary/conclusion: uf+sec provided the best balance between ev recovery (per ml urine) and particle: protein ratio. these data indicate that most of the 20 nt sized rnas, presumably mirnas, are not within evs in urine. ev preparations obtained after uc, pk and sec (regardless of concentrating method) contain pre-dominantly~60 nt sized small rna. these data outline the importance of removing non-vesicular proteins and rna from urine ev preparations prior to small rna analysis. funding: this research has been funded by petplan charitable trust. the use of rev for the optimization of ev separation and characterization by af4 introduction: the reproducibility of extracellular vesicle (ev) research has been hampered by the infinite number of separation and measurement techniques and the lack of appropriate reference materials (van deun et al., nat methods, 2017) . recombinant extracellular vesicles (rev) were developed as a biological reference material to overcome these limitations (geeurickx et al. nat comm 2019) . since rev have ev-like physical an biochemical characteristics and as they are trackable and distinguishable from sample ev they can be used as a spike-in material for data normalization and method development, and as a quality control. we used rev to optimize ev separation by asymmetrical flow field-flow fractionation (af4). methods: an af4 long channel column with a frit inlet driven by the eclipse system (wyatt) was coupled to a uv detector (shimadzu), mals dawn helios-ii (wyatt) and fluorescent detector (agilent). a spacer of 350 µm and a regenerated cellulose membrane of 10 kda were used. pbs supplemented with 0.02% nan3 was used as a running buffer. light scatter profiles and uv profiles were analysed as well as the fluorescent emission spectrum as the rev are gfp positive. fractions were collected and analysed by nanoparticle tracking analysis (nta) and western blot. we also estimated the repeatability and reproducibility of the af4 technique by light scatter and fluorescence profiles as well as the recovery efficiency by nta. results: in a first step 4*10^10 rev isolated from conditioned medium by a velocity gradient were injected in the af4 system to optimize the ev characterization protocol. later concentrated conditioned medium was spiked with 1*10^11 rev and injected in the af4 column to optimize ev separation from non-ev contaminants. the most optimal separation protocol was obtained by varying detector and cross-flow settings. this protocol shows elution of monodisperse particles at each time point and size distribution estimations by af4 correspond to size determination by nta and electron microscopy. summary/conclusion: we were able to optimize the af4 protocol for characterization of ev by af4 as well as for separation of ev from crude conditioned medium samples by using rev. we demonstrate that rev are suitable for method development and that af4 has high potential as an ev separation technique. comparative evaluation of ev isolation methods for ev subpopulation analysis in human urine, plasma and cell culture media liang dong, richard zieren, kengo horie, sarah amend and kenneth pienta the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: extracellular vesicles (evs) are membrane-enclosed particles of variable sizes that are released by any cell types to the extracellular space and are identified in all body fluids. a shortcoming in ev research is the lack of standardized isolation protocol for various sample types, resulting in heterogeneous outcomes in downstream analyses. in this study, we compared the ev isolation purity and efficiency among ultracentrifugation (uc), precipitation, sizeexclusion chromatography (sec) and a microfluidic tangential flow filtration device (exodisc) in human plasma, urine and cell culture media (ccm). methods: all evs were isolated by different isolation methods and characterized per misev2018 guidelines. single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence and nanoflow (nfcm) were used for single particle analysis. results: in ccm, total particle yield of exodisc was about 5 times higher than those of the rest three methods. size distribution differed per sample, but the ranges were comparable between the different isolation methods. the total protein amount of sec, precipitation and exodisc were similar which were 6-10 times higher than that of uc. uc had the highest particle-toprotein ratio followed by exodisc. precipitation and sec had low ratios. when loading 9ug of total protein for western blot, cd9, cd81, cd63 and flot1 could only be detected in uc and exodisc samples, but not precipitation or sec. sp-iris and nfcm demonstrated consistent purity findings. in urine, total particle yields of exodisc and sec were about 4 times higher than those of the rest two methods. the total protein amount of precipitation was 3 times higher than exodisc and sec, 10 times higher than uc. sec had the highest particle-to-protein ratio followed by uc and exodisc. precipitation had low ratios. in plasma, total particle yields of exodisc and precipitation were 100 times higher than those of the rest two methods. and so were the total protein amount. sec had the highest particle-to-protein ratio followed by uc. exodisc and precipitation had low ratios. western blot, sp-iris and nfcm demonstrated consistent purity findings in urine and plasma. to evaluate particle capture efficiency, we spiked a known number of density-gradient uc purified evs to each method and the recovery rate of uc, precipitation, exodisc and sec was 8.6%, 31%, 50.1% and 42%, respectively. summary/conclusion: the order of ev isolation purity in ccm is uc, exodisc, sec and precipitation. in urine it's sec, exodisc, uc and precipitation. and in plasma, this order is sec, uc, exodisc and precipitation. exodisc and sec have similar high isolation efficiency followed by precipitation. uc has low efficiency for ev capture. a capillary-channelled polymer (c-cp) fibre spin-down tip approach for the isolation and biomarker characterization of extracellular vesicles of ovarian cancer origin kaylan d. kelsey, rhonda r. powell, terri f. bruce and r. kenneth marcus clemson university, clemson, usa introduction: extracellular vesicle (evs) profiling has shown promise for disease detection through less invasive sampling (liquid biopsies). current diagnostic tools for ovarian cancer are invasive or only semiinformative. thus, use of evs could prove useful in early disease detection. demonstrated is a hydrophobic interaction chromatography (hic)-based capillarychannelled polymer (c-cp) fibre tip spin-down process for the isolation of ovarian cancer evs for use in diagnostics. methods: polyester c-cp fibre micropipette tips are employed in the isolation of evs from biological matrices including cell culture media, urine, and blood plasma in a spin-down solid-phase extraction (spe) approach. evs were isolated from standards of healthy urine origin and from skov3 cells (human ovary adenocarcinoma). the c-cp fibre isolation method (taking less than 5 mins and 10 μl sample volumes) preserves the morphology and functionality of evs as confirmed by sem, tem, and confocal fluorescence microscopy. results: the dynamic binding capacity of ev standards on a 1 cm pet c-cp fibre tip was found to be~7e11 particles (50%). the release of evs was confirmed using dot blot analysis for cd9, cd81, and cd63 tetraspanin proteins. immobilized evs were subjected to immunolabeling to allow the positive identification of a profile of ovarian cancer biomarker proteins (her2, cd24, egfr, epcam, ca125). summary/conclusion: this new ev isolation method introduces a simple capture mode, allowing for direct immuno-characterization and imaging on the fibre surface. this offers a unique and cost-effective opportunity for clinical analyses related to early detection and diagnosis of ovarian cancers (and others). the longterm goal is the creation of a rapid ev isolation and biomarker detection platform. funding: support from the national science foundation, eppley foundation for scientific research, gibson foundation, prisma health system and itor biorepository are gratefully acknowledged. development and optimization of purification method of exosomes by tangential flow filtration and ion-exchange chromatography approach tek lamichhane, ali navaei, sandeep choudhary, yonatan levinson and senthil ramaswamy cell & gene therapy r&d, lonza inc, rockville, usa introduction: extracellular vesicles (evs) such as exosomes have significant therapeutic potential, however, translation of ev-based therapies has been slowed down because of the biomanufacturing challenges. the isolation of evs, especially exosomes, is inherently challenging due to their small size, and heterogeneity in the mixture. the current isolation methods either have low recovery rate, aggregation, damaging the structure, time consuming or co-precipitation of contaminants. specially, it is difficult to process larger sized samples by centrifugation-based or immunoaffinity based methods because of the time and cost associated with these methods. methods: to overcome these roadblocks, we developed and optimized alternative purification techniques to isolate evs with higher purity and yield by using tangential flow filtration (tff) coupled with ion-exchange chromatography. we used bioreactor platform to produce evs from serum-free medium using bm-msc and hek293 s cells. bm-mscs were cultured on stirred tank bioreactors using microcarriers which provide a high surface area to volume ratio for the optimal cell growth and evs production. impellers were used to enhance mixing and maintain homogeneous culture conditions that can be easily monitored and controlled. results: depth filtration was applied for clarification of conditioned medium. we screened different types of filters during depth filtration for the best recovery of evs. tff membranes with different pore sizes were used to optimize the purity and yield of evs. because of the negatively charged nature of evs, anion exchange chromatography was chosen to capture and separate tff purified vesicles by their surface charge characteristics. we compared monolith based and membrane-based anion exchange columns to remove contaminants and purify exosomal fractions. the purity, size and presence of exosomal markers in isolated evs at each step of purification was evaluated by f-nta, nano-fcm and tetraspanins based elisa kits. summary/conclusion: in summary, our optimized methods improved the speed of isolation and purity of evs to the clinical grade. the production and isolation methods of exosomes that we developed here will be easily expandable to support large-scale and cgmp compatible bio-manufacturing in the future. use of an alternating current electrokinetic microelectrode chip to positively identify oncology, neurology, and infectious disease samples through plasma extracellular vesicle analysis juan pablo hinestrosa, jean lewis, david searson, orlando perrera, alfred kinana, heath balcer and rajaram krishnan biological dynamics, inc., san diego, usa introduction: cancer, neurological, and infectious diseases are leading causes of death, with early detection needed to improve outcomes. extracellular vesicles (evs) in the blood contain disease biomarkers, but current methods do not allow rapid analysis, and are often limited to one biomarker type. methods: we developed methods using alternating current electrokinetics (ace) to isolate evs from bloodbased samples and analyse the evs in situ with downstream assays for protein and nucleic acid biomarkers. we investigated if we could identify tuberculosis (tb) donor samples, protein and nucleic acid biomarkers in evs derived from cancer cell lines, and alzheimer's disease (ad) protein biomarker levels. results: ev isolation was confirmed by positive identification of the proteins cd9, cd63, and cd81 and measurement of ev mrnas using a direct rt-ddpcr assay. different disease models were analysed following method development. tb was used as a model for infectious disease, with 20 tb positive and 20 tb negative samples isolated on ace chips and analysed for levels of lipoarabinomannan and ag85. using a cut-off above the negatives, the auc of roc curves were 0.9975 and 1.0, respectively. for oncology, cancer cell lines were cultured and evs isolated from supernatants were spiked into human plasma for analysis. levels of pd-l1 or glypican-1 on evs were able to be measured following ace capture. additionally, dna and rna mutations known to be present in the cell lines were able to be detected using ngs and qrt-pcr, respectively. using ad samples as a neurological disease model, tau and phospho-tau t181 (p-tau t181) in human donor plasma were detected. in 8 ad and 5 healthy donor samples, p-tau t181 signal increased 134% in diseased versus healthy donors. summary/conclusion: ace chips are an innovative ev isolation and analysis platform that allow rapid disease sample detection in a wide range of studies with high sensitivity and specificity. introduction: colorectal cancer (crc) is one of the most frequent causes of cancer-related death. in the majority of crc patients, mutation in the apc gene is among the first genetic events. it leads to uncontrolled activation of the wnt pathway, and thus, to adenoma formation. some of these adenomas may then further progress to crc with the accumulation of other mutations. the 3d organoids maintain the cellular and genetic heterogeneity of in vivo tissues and haves proved to be so far the best ex vivo model of human cancers. here we analysed the ev-based communication between cancer cells and fibroblasts by i) identifying factors that substantially increase ev release from intestinal cancer cells and ii) by determining cargo components of evs that enhance tumour cell proliferation. methods: we used commercially available and patientderived fibroblasts and crc organoids. the medical research council of hungary approved all experiments with human samples and informed consent was obtained from patients. evs were studied by using antibody-coated beads, trps, nta, tem and western-blotting. we introduced apc mutation into wild type murine small intestinal organoids by crispr-cas9. results: we found that in crc patient-derived organoid cultures, small evs were preferentially secreted. we observed that apc mutation and the accumulation of the extracellular matrix component collagen critically enhanced ev secretion in intestinal organoids. furthermore, we showed that amphiregulin, present on fibroblast-derived ev, contributed to the maintenance of the intestinal stem cell pool and to cell proliferation in epidermal growth factor-dependent crc organoids. summary/conclusion: by proving the key role of mutations, collagen deposition and ev-bound amphiregulin in the release intensity and functions of the evs, we identified novel mechanisms in the progression if crc. funding: this work was funded by otka-nn 118018, by the national competitiveness and excellence program nvkp_16-0007 (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). prostate cancer-derived evs induce a pro-inflammatory phenotype in the stroma blandine f. victor a , dolores di vizio b , andrew chin c , tatyana vagner b , javier mariscal b , mandana zandian a , catherine grasso a , roberta gottlieb a and helen goodridge a a cedars-sinai medical center, los angeles, usa; b cedars-sinai medical center, west hollywood, usa; c cedars-sinai, los angeles, usa introduction: since 90% of patients with metastatic prostate cancer (pc) develop bone metastasis, identifying the mechanism that drives this process is essential. most ev research has been focused on the role of exosomes in mediating the pre-metastatic niche formation. however, most of these studies do not separate exosomes from large evs. our preliminary studies have demonstrated that a subclass of evs known as large oncosomes (lo) can reprogram prostate fibroblasts, at the primary tumour site, promoting angiogenesis and enhancing the migration and invasion of pc cells in vitro and tumour growth in vivo. the bone marrow is the initial site of entry into the bone microenvironment for disseminating tumour cells (dtcs) and is a rich source of nutrients that houses various cells types including bone marrow derived mesenchymal stem cells (bm-msc) and immune cells such as neutrophils, which have been implicated in metastasis. here we investigate the role of lo in reprogramming bm-mscs and driving bone metastasis in pc. methods: differential centrifugation, density gradient centrifugation, trps, rna sequencing, qpcr, migration assay, invasion assay, chemotaxis assay. results: we report that pc-derived evs induce distinct gene expressions changes in bm-mscs. rna-seq analysis identified inflammatory and immune regulating cytokines as top differentially expressed genes (deg) in bm-msc. moreover, lo induced a more potent response in bm-msc in comparison to exo and to non-treated controls. the genes enriched in lo treated bm-msc were associated with tumour cell motility. in agreement with the gene expression data, lo-treated bm-msc attracted migration and invasion of significantly more pc cells than exo -treated bm-mscs. in addition, the top deg expressed in ev treated bm-msc were identified as potent neutrophil chemoattractant proteins. in line with the rnaseq findings, the lotreated bm-msc demonstrated enhanced chemotaxis of neutrophils towards them in comparison with exo or vehicle-treated bm-msc. finally, we show that the observed differences in bm-msc's response to lo and exo may be mediated by distinct molecular pathways. summary/conclusion: the results from this study provide novel insight into how tumour derived evs alter the bone marrow microenvironment and how they may drive bone metastasis in prostate cancer. the αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis introduction: prostate cancer (prca) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sevs). sevs isolated from prca cell media, express the epithelial-specific αvβ6 integrin, a surface receptor for fibronectin and vitronectin. the αvβ6 integrin is not detectable in healthy prostate tissues but is highly expressed in prca. in this study, we hypothesized that αvβ6 in cancer sevs plays a crucial role in angiogenesis. methods: the sevs isolated from prca cell media were characterized by nanoparticle tracking analysis, iodixanol density gradients and expression of sev markers. the αvβ6-negative endothelial cells (hmec1) were incubated with αvβ6-positive sevs from prca cells to evaluate the transfer of αvβ6 by immunoblotting (ib) and facs. the effect of αvβ6-positive sevs on motility, tube formation and angiogenic signalling were assessed by boyden chamber, angiogenesis assays and ib in hmec1. results: we demonstrate for the first time that the αvβ6 is de novo expressed on endothelial cell surface by sevmediated protein transfer. prca cell-derived αvβ6-positive sevs, significantly promote the motility and the formation of nodes, junctions and tubules by hmec1. mechanistically, we demonstrate that hmec1 treatment with sevs from pc3 cells that endogenously express αvβ6, decreases pstat1(y701), a negative regulator of angiogenesis, while upregulating survivin, an inducer of angiogenesis. hmec1 treatment with sevs isolated from pc3 cells harbouring crispr/cas9-mediated downregulation of β6, or shrna-mediated downregulation of β6, results in increased levels of pstat1(y701). this sev treatment also results in a decrease of survivin in sevs and hmec1. summary/conclusion: overall, our findings show that αvβ6 in prostate cancer sevs regulates a novel proangiogenic signalling pathway. funding: this study was supported by nci r01-224769 (lrl); p01-140043 (lrl and dca). introduction: advanced prostate cancer (pca) is asso-introduction: extracellular vesicles (evs) are secreted from cells, and carry bioactive proteins and rna cargoes. increasing numbers of studies have identified key roles for exosomes in driving aggressive tumour behaviours, including metastasis. however, the detailed mechanisms and responsible factors in the ev cargo are still unclear. recently, immune system has been considered as an important factor in establishing and maintaining metastasis. our goal is to identify the role of head and neck squamous cell carcinoma (hnscc) derived small evs (sevs) in tumour metastasis from the study analysing the effects of sevs on metastasis and tumour immunity. methods: sevs were collected from the conditioned media of hnsccs and purified through cushioned density gradient ultracentrifugation. an orthotopic mouse model was used for the assessment of tumour angiogenesis and metastasis. moc1 (inflammationinducing rarely metastasizing murine hnscc line) and moc2 (highly metastasizing murine hnscc line) were used for this study. moc1 and moc2 cells were transplanted into mice tongues orthotopically, and moc1/moc2 derived sevs or pbs were injected into the tumour twice in a week. two weeks after tumour transplantation, mice were sacrificed and tumours were sectioned for pathological analysis and facs analysis. in facs analysis, the number and species of tumour-infiltrated immune cells were measured. results: injection of sevs from moc1 into moc2 tumours suppressed frequency of lymph node metastasis. on the other hand, injection of sevs from moc2 into moc1 tumours didn't promote metastasis. cd4 positive t-cell distribution in moc2 tumour was significantly changed by moc1 sev injection. t-cell deprivation treatment using anti-cd3 antibody increased the frequency of metastasis in moc1-sev treated moc2 tumours. from the result of proteomics analysis on moc1 and moc2 sevs, immune-regulated proteins and metastasis-suppressing proteins were observed in moc1 sevs. summary/conclusion: we find that low aggressive hnscc sevs affect metastasis of highly metastasized hnscc, and also find that changing immune cell distribution may be related to the result. this mechanism and finding contributes to understanding the possible role of hnscc sevs on metastasis as well as on the tumour immune microenvironment. funding: this work was supported by the nih under award numbers r01ca163592 and r01ca206458 to aw. desmoglein 2 enhances squamous cell carcinoma tumour development through extracellular vesicles in an il-8/mir-146a-dependent mechanism introduction: the cadherin dsg2 is a stem cell marker that is upregulated in many different cancers, including sccs, and its expression correlates with poor prognosis. dsg2 activates mitogenic signalling and plays a key role in cell proliferation, migration, and survival. we recently showed that dsg2 enhances ev release, but the mechanism by which these evs modulate tumorigenesis is not fully understood. methods: we established scc cell lines stably expressing wildtype dsg2 or a palmitoylation deficient mutant, dsg2cacs. evs were isolated by sequential ultracentrifugation, iodixanol gradient separation, or qev izon column, and analysed by nta and bca. tumour xenografts were established by subcutaneous injection of 106 cells in scid mice and monitored up to 4 weeks. cytokine profiling was determined by antibody array. mirna expression was analysed by rnaseq and confirmed by qpcr. results: dsg2 enhanced ev release by 40% and promoted a~fivefold increase in tumour size in xenograft models. tumour growth was increased when control cells were treated with a single 20 µg dose of evs. loss of palmitoylation, which altered membrane trafficking of dsg2, reduced ev release (~55%) as well as tumour development. plasma evs from xenograft mice reflected in vitro particle counts from scc cell lines. a cytokine array analysis was performed revealing that dsg2-evs were enriched with pro-inflammatory cytokines including il-8, a potent chemotactic and angiogenic factor. most importantly, il-8 was surface-bound on evs. furthermore, rnaseq revealed mir-146a, a negative regulator of il-8, to be significantly downregulated in response to dsg2. treatment with mir-146a mimic or mir-146a inhibitor decreased or increased, il-8 expression in scc cells, respectively. summary/conclusion: in summary, dsg2 plays a key role in scc tumour development by increasing ev biogenesis and downregulating mir-146a, which in turn upregulates il-8 synthesis and release which can promote invasion, angiogenesis and metastasis. funding: nih r01 introduction: stem-and progenitor cell transplantation therapy holds great promise for regenerating damaged heart tissue. several lines of evidence suggest that its efficacy is mainly caused by secreted extracellular vesicles (evs). indeed, cardiac progenitor cell (cpc)-derived evs have been shown to protect the myocardium against ischaemia/reperfusion injury in several preclinical models. however, the underlying mechanisms for cpc-ev-mediated cardioprotection remain elusive. here, we utilized the proteomic composition of cpc-evs released during different culture conditions, to unravel protein-mediated effects of cpc-evs on the endothelium. methods: cpcs were stimulated with calcium ionophore (ca ion-evs) or vehicle (control-evs) for 24 hours and evs were isolated from serum-free conditioned medium using size exclusion chromatography. ev concentration and size was assessed using nta. evs were functionally characterized based on endothelial cell activation by western blotting and an endothelial cell scratch assay. the proteomic composition of both ev conditions was profiled using mass spectrometry. cpc-ev knockouts for specific proteins were generated using crispr/cas9 technology. results: we found enhanced phosphorylation of erk1/2 and akt in endothelial cells and increased wound closure after stimulation with control-evs, but not after stimulation with ca ion-evs. proteomic analysis identified a total of 1411 ev-associated proteins, with 79 proteins uniquely expressed in control-evs. another 68 proteins were revealed as candidate proteins, based on their relative enrichment in control-evs compared with ca ion-evs. go analysis demonstrated that differentially expressed proteins were involved in vascular endothelial growth factor signalling, extracellular matrix organization and angiogenesis. to investigate the involvement of the individual candidate proteins on endothelial cell activation, knockout evs of multiple proteins were generated and functionally characterized. summary/conclusion: a specific set of ev proteins is identified that may be functionally responsible for the activation of endothelial cells upon exposure to cpc-evs. generating knockout evs for each of these proteins will help to investigate their individual roles. this may lead to a better mechanistic understanding of the use of cpc-evs as therapeutics for cardiac repair. funding: erc-2016-cog-725229 evicare grant. hypoxia enhances the therapeutic potential of human cd34+ stem cell exosomes in ischaemic hindlimb repair ischaemic cardiovascular disease. we have previously shown that human cd34+ cell-derived exosomes (cd34exo) improve perfusion and function of the ischaemic tissues. hypoxia is shown to modulate the secretion and content of exosomes in both cardiovascular and cancer research. therefore, we hypothesized that hypoxia can modulate the content and regenerative efficacy of human cd34exo. methods: cd34exo were isolated from primary human cd34+ stem cells cultured under hypoxia (1.5% o2, or normoxia (20% o2, n-cd34exo) using density gradient ultracentrifugation. cd34exo size was measured using trps, nta, and dls and surface protein expression was determined using imaging flow cytometry. function of cd34exo was assessed using cell viability, migration and matrigel tube formation assays in vitro and a mouse hind limb ischaemia model (hli) in vivo. protein content of hypoxic or normoxic cd34exo was evaluated via lc-ms/ms and 2-d -dige followed by lc-ms/ms. results: we did not observe any significant differences in size or in quantity of exosomes secreted from h-or n-cd34 cells. both h-and n-cd34exo expressed cd9, cd81 and cd63 surface markers. interestingly, h-cd34exo significantly improved cell viability, migration and tube formation of huvecs in vitro compared to n-cd34exo. in the same line, h-cd34exo also significantly improved perfusion (ratio: 0.93 ± 0.05 v 0.77 ± 0.02) and prevented ischaemic limb amputation (0% v 37.5%) as compared to n-exo (p < 0.05; n = 7-8) in a murine (balbc nude) model of hind limb ischaemia. flow cytometry and confocal microscopy indicated that h-exo was uptaken by endothelial cells in the ischaemic limb. remarkably, we detected several proteins (including a fragment of hemopexin) and mirnas (mir-210) that could be responsible for the proangiogenic and beneficial function of h-cd34exo. we have also demonstrated that removal of surface proteins diminished the pro-angiogenic function of cd34exo. summary/conclusion: hypoxia enhanced the proangiogenic and regenerative potential of cd34exo, and thus, may represent a more efficient clinical strategy for cd34exo therapy. our research is clinically important to improve therapeutic angiogenesis in diabetic and cardiovascular patients with compromised stem cell populations. hyun-ji park a , jessica r. hoffman b and michael davis b a emory university, decatur, usa; b emory university, atlanta, usa introduction: exosomes, a subset of membrane nanovesicles, transfer cellular information by passing proteins and nucleic acids between cells. exosomes have been implicated as the mechanistic unit in stem cell therapy, as inhibition of exosome synthesis abrogates the effects of cell therapy following cardiac injury. more importantly, increasing evidence indicates that mirnas (mirs) within exosomes serve as important signalling molecules to regulate inflammation, recruit stem cells, and repair diseased tissue. among exosomal mirs, mir-192 and −432 are known to decrease angiogenesis, cell migration, and increase inflammation in various types of cells. here, we investigated the inhibition of these negative mirs as a means to improve the reparative capacity of c-kit+ progenitor cell (cpcs) exosomes. methods: cpcs were isolated from three paediatric patients using magnetic-bead sorting. 2ʹ-o-methylated rna duplexes inhibited mir-192 and −432 expressions in cpcs. exosomes (inhexos) were isolated from mirinhibited cpc conditioned medium. mir expression in exosomes and cpc was quantified by qrt-pcr. migration and proliferation of mesenchymal stem cells (mscs) were assessed two days post-exosome treatment. for inflammation analysis, thp1 cells with/without tnfα exposure were treated with exosomes and the expression of il-6, −8, and −10 was quantified by qrt-pcr. finally, the angiogenic potential of inhexos was tested by tube formation of cardiac endothelial cells. results: inhibitor treatment of cpcs decreased exosomal mir-192 and −432 expression. treatment with inhexos enhanced msc migration and proliferation compared with normal cpc exosome (norexo). moreover, inhexos showed promising results for immune regulation, as tnfα-induced inflammation was decreased in thp1 exposed to inhexos for 4 h. however, tube formation capacity is slightly decreased (~20%) by inhexo compared to norexo. summary/conclusion: exosomes from mir-192 and −432-depleted cpcs may be a promising strategy for the treatment of various cardiac diseases, as they enhanced stem cell recruitment and proliferation, and regulated inflammation and angiogenesis. while other studies focus on boosting the reparative potential of exosomes by increasing positive mir and mrna cargo, the inhibition of negative mir in exosomes could be an overlooked strategy for the treatment of cardiac disease. endo-lysosomes as an alternative intracellular location for ev cargo delivery with disease relevance introduction: extracellular vesicles (ev) are lipidbilayer nanovesicles that carry macromolecules and act as paracrine vectors for cell-to-cell communication. the processes regulating ev biogenesis are largely known, whereas how ev cargo is delivered to recipient cells remains poorly understood. a simple mechanism proposed is direct ev fusion with the cell membrane that liberate cargo into the cytosol. in this study, we observed that cargo release occurs also at an alternative intracellular location and that this acquires a disease relevance. methods: ev were isolated by serial centrifugation and characterized. for uptake studies, ev were traced by labelling donor cells with a lipophilic dye or by overexpressing gfp-cd63. uptake was assessed by cytofluorimetry or by live confocal imaging. co-localization studies were performed with ectopic marker expression or by immune staining. protein-protein interaction was analysed by bi-molecular fluorescence complementation (bifc). prion-like transmission was studied using a pro-fibrillogenic tau fragment in donor cells and full-length tau in recipient cells. for quantification of subcellular localization, an automated algorithm based on machine learning was developed. lysosomal stress was monitored by nuclear translocation of tfe3 and lysotracker staining. antibodies directed against pathogenic epitopes of tau were employed to assess prionlike transmission. results: ev were taken up by recipient cells through an endocytic process and accumulated in endo-lysosomes (el). when cells were exposed to ev carrying a profibrillogenic tau, recipient cells accumulated tau within el by an autophagic process. direct interaction of ev-tau and cellular tau in el favoured the appearance of pathological epitopes. cells displaying this condition showed an increased el stress and cytotoxicity. summary/conclusion: in this study, for the first time we report that el represent a critical subcellular location where transcellular prion-like transmission mediated by ev of a neurodegeneration-associated protein occurs. thus, the degradative pathway most likely involved in the recycle of ev and endogenous proteins is highjacked in disease. these findings represent a novel mechanism for ev acting as vector for transcellular propagation of tau, which opens up new therapeutic interventions trying to halt the disease. funding: supported by gelu foundation. anti-human fab fragment of cd9 antibody prevents the endocytosis of melanoma and colon cancer-derived extracellular membrane vesicles and nuclear transfer of their cargos introduction: interfering with the mechanisms regulating intercellular communication mediated by extracellular membrane vesicles (evs) may find relevance especially in oncology where cancer cell-derived evs have an implication in the malignant transformation of tumour microenvironment. our laboratories recently demonstrated a novel intracellular pathway in which a fraction of endocytosed ev-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of rab7+ late endosomes entering into the nucleoplasmic reticulum. here, we have investigated the effect of a monovalent fab antibody against the tetraspanin cd9 (referred hereafter as cd9 fab), on the internalization of evs and nuclear transfer of their cargo proteins. chair: david r f. carter -oxford brookes university chair: neta regev-rudzi -weizmann institute of science methods: to monitor the intracellular transport of ev-associated proteins, we used bioengineered fluorescent evs containing cd9-gfp fusion protein from femx-i melanoma, sw480 colorectal cancer and bone marrow-derived mesenchymal stromal cells (msc) as donors and the same cell types as recipients. evs were enriched by differential centrifugation from 72 h serum-free conditioned media and characterized by zetaview nanoparticle tracking analysis, zetapotential and immunoblotting. cd9 fab was prepared from 5h9 hybridoma cells using the pierce fab purification kit. results: we previously demonstrated that silencing cd9 both in evs and recipient cells strongly decreased the endocytosis of evs and abolished the nuclear transfer of their cargos. here we show that cd9 fab significantly reduced the cellular uptake of cd9-gfp+ evs and the nuclear transfer of their proteins in melanoma, colorectal cancer and msc used as receptor cells in a dose-dependent manner. the effect on the nuclear transfer is probably a direct consequence of the endocytosis inhibition of evs. in contrast, the divalent, intact cd9 antibody stimulated both events. summary/conclusion: the effect of cd9 fab appears independent of the used ev-donor cell types or receptor cells, probably due to the widespread expression of cd9 both at plasma membrane and ev surface. in conclusion, by impeding intercellular communication in the tumour microenvironment, cd9 fab-mediated inhibition of ev uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-cancer therapeutic strategies. a bright, versatile reporter for multivesicular body trafficking and exosome secretion and uptake bong hwan sung, ariana von lersner, jorje guerrero, evan krystofiak, david inmann, roxanne pelletier, andries zijlstra, suzanne ponik and alissa weaver vanderbilt university, nashville, usa introduction: live imaging of exosomes is one of the required tools to understand the function of exosomes. our previous live-cell reporter, phluorin-cd63 allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. however, there were some caveats to its use, including dim fluorescence and the inability to make cell lines that stably express the protein. methods: a stabilizing mutation, m153 r is incorporated in the phluorin moiety and now exhibits stable expression in cells and superior monitoring of exosome secretion. a dual-tag reporter was created by incorporating a further ph-insensitive red fluorescent protein, mscarlet to the c-terminus of phluo_m153 r-cd63. cancer cells stably expressing the constructs were imaged using a variety of microscopy techniques in vitro as well as in vivo. purified small evs labelled with phuo_m153 r-cd63 were imaged using immunogold transmission electron microscopy (tem) and quantitated for the half-life in the blood circulation using flow cytometry. results: phluo_m153 r-cd63 and phluo_m153 r-cd63-mscarlet are exclusively detected in exosomeenrich small ev preparations. immunogold tem visualizes the phluo_m153 r tag is located on the surface of small evs. live cell imaging reveals phluo_m153 r-cd63-positive puncta left behind migrating cells suggesting the deposition consists of exosomes. those puncta and trails are not only positive for exosome markers such as cd63, alix, and tsg101 but also correspond to small evs observed by a scanning electron microscopy. the dual-tag reporter allows visualization of the exosome lifecycle, including multivesicular body (mvb) trafficking, mvb fusion, exosome uptake and endosome acidification. summary/conclusion: using phluo_m153 r-cd63 construct, we demonstrate superior visualization of exosome secretion in multiple contexts and a role of exosomes in promoting leader-follower behaviour in collective migration by observing that exosomes are secreted at the front of migrating cells and left behind in exosome trails. the dual-tag reporter allows visualization of the entire exosome lifecycle. we anticipate that these reporters will be broadly useful to investigate regulation and functions of exosome secretion and uptake in diverse physiological conditions. funding: r01gm117916, r01ca206458, 3u19ca1795 14-05s1, r01ca216248. uncovering novel genes regulating ev-mediated functional rna transfer using a crispr/cas9-based reporter system introduction: extracellular vesicles (evs) play a pivotal role in intercellular communication through functional transfer of bioactive cargo, including rna molecules. despite increasing interest in ev-mediated rna transfer, our understanding of the pathways and mechanisms regulating ev-mediated rna delivery and processing is limited due to a lack of suitable readout systems. we recently developed a novel crispr/cas9-based reporter system that allows study of ev-mediated rna transfer at single-cell resolution. here, we further validate this system by studying the role of known targets involved in ev uptake and intracellular membrane trafficking, and subsequently employ this system to uncover various novel genes that play a regulatory role in functional rna transfer. methods: we employed a novel crispr/cas9-based stoplight reporter system, in which egfp expression is activated upon functional delivery of targeting single guide rnas (sgrnas) stably expressed by donor cells. intercellular functional rna transfer was assessed by measuring egfp expression in acceptor cells using fluorescence microscopy and flow cytometry after direct co-culture, transwell co-culture, and upon addition of isolated evs. potential roles of various genes in intercellular rna transfer were assessed by rnaimediated target knockdown in acceptor cells, prior to co-culture experiments. rnai knockdown was confirmed by qpcr analysis. results: a significant activation of egfp expression was observed in acceptor cells after direct co-culture and transwell co-culture with donor cells expressing sgrnas, as well as after addition of evs from cells expressing sgrnas. reporter activation was substantially decreased after knockdown of multiple targets involved in ev uptake through endocytosis and/or intracellular membrane trafficking. based on these results, a potential role of various novel genes in intercellular rna transfer was studied in acceptor cells. these experiments uncovered various novel targets involved in ecm binding, endocytosis, intracellular membrane trafficking, as well as various rho gtpase interactors. summary/conclusion: we previously demonstrated a crispr/cas9-based reporter system that allows the study of functional delivery of small non-coding rnas with single-cell resolution. here, we show that this novel approach allows the study of specific genetic targets and pathways in ev-mediated functional rna delivery, and unravel the regulatory pathways that dictate the underlying processes. quantitative characterization of extracellular vesicle uptake and content delivery within mammalians cells gregory lavieu a , emeline bonsergent b , eleonora grisard c and clotilde théry d introduction: extracellular vesicles (evs), including exosomes, are thought to mediate intercellular communication through the transfer of biomolecules from donor to acceptor cells. occurrence of ev-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. methods: we developed a cell-based assay in which evs containing luciferase-tagged cytosolic cargo are loaded on unlabelled acceptor cells. measurement of luciferase activity associated with acceptor cells revealed ev uptake efficacy. additional cell fractionation procedure that separates membranes from cytosol revealed the occurrence of ev-content release within the cytosol of acceptor cells. results: results from dose-responses, kinetics, and temperature-block experiments suggest that ev-uptake is limited (1% spontaneous rate at 1h), does not depend on bona-fide ev-receptor, at least for the tested acceptor hela cells. yet, further characterization of this limited ev-uptake, through cell fractionation that separates membranes from cytosol, revealed the occurrence of ev-content release within the cytosol of acceptor cells. cytosolic release is inhibited by bafilomycin-a and overexpression of ifitm proteins, which prevent virus content delivery. summary/conclusion: our results show that ev-content release requires endosomal acidification and suggest the involvement of membrane fusion. funding: anr18-ce15-0008-01 and arc pja 20171206453 and pga1 rf20180206962. introduction: glioblastoma is a highly malignant brain tumour with a poor prognosis. its ability to develop therapeutic resistance result in devastating clinical outcomes. to solve the intractable problem, we need highly sensitive diagnostics that can detect the molecular changes during treatments. extracellular vesicles (evs) can be a potential biomarker to monitor treatments and the host cell ev mapping can better reflect molecular changes in the tumour immune microenvironment. we have developed a droplet-based single ev protein sequencing platform that overcomes limitations of current bulk measurement technologies, which make it difficult to discover a rare ev population in the presence of high background. methods: we multiplex protein measurements to profile hundreds of proteins at a time by using an antibody-dna conjugate and sequencing. we barcode each ev in droplets and make amplicons that are comprised of both ev barcodes and antibody barcodes for sequencing. barcoded antibodies are made using tco-tetrazine click reaction and evs are labelled with these barcoded antibodies. the labelled evs are encapsulated into droplets with barcoded beads that serve as a template for ev barcodes. we then perform extension to make amplicons that contain both ev barcodes and antibody barcodes for sequencing. results: we successfully fabricated barcoded beads using a split-pool approach and validated by observing a fluorescence decrease of the sybr green after dna strand denaturation. we used a 3-channel droplet maker to encapsulate barcoded beads, single ev, and master mix into droplets. close packing of barcoded beads allowed >95% encapsulation into droplets. both droplet and tube-based methods achieved a similar high amplification efficiency (ct < 30 for 300 evs). we confirmed the amplicon size by running a gel, which showed the right amplicon size (~150 bp) from the droplet and tube prepared samples and no signal from the negative control. summary/conclusion: the droplet-based single ev profiling platform has the ability to identify rare immune ev subtypes in the peripheral blood, which would otherwise be impossible to detect due to the copresence of abundant normal evs. this cutting-edge technique has the potential to revolutionize treatment monitoring of high-cost immunotherapies, avoid unnecessary toxicities, and enhance personalized medicine capabilities. funding: schmidt science fellows, in partnership with the rhodes trust po1 ca069246, ro1 ca204019, r21 ca236561 quantbio graduate student award at harvard university. introduction: in this study, we compared four orthogonal technologies for sizing, counting, and phenotyping of evs. the platforms were: single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence, nanofcm nanoflow (nf), nanoparticle tracking analysis (nta) with fluorescence, and microfluidic resistive pulse sensing (mrps) . results from these platforms were compared with results from standard ev characterization techniques such as transmission electron microscopy (tem) and western blot (wb). methods: human t lymphocyte h9 (high cd81, low cd63) and promonocytic u937 (low cd81, high cd63) cells were chosen for their distinct tetraspanin profiles without abnormalities that might result from genetic manipulation. evs were isolated from culture conditioned medium (ccm) by differential ultracentrifugation (duc) and size exclusion chromatography (sec) and characterized per misev2018 guidelines. synthetic particles (silica and polystyrene spheres) with known concentrations and mixed size distributions were also tested. results: particle counts from nf and mrps were consistent, while nta detected approximately one order of magnitude lower for ccm derived evs, but not for synthetic particles. sp-iris events could not be used to estimate particle concentrations. for sizing, nf, mrps, and sp-iris returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. nta detected a population of particles with a mode diameter above 100 nm. additionally, sp-iris, nf, and mrps were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. finally, for tetraspanin phenotyping, the sp-iris platform in fluorescence mode and nf were able to detect at least two markers on the same particle. summary/conclusion: based on the results of the study, we can draw conclusions about existing singleparticle analysis capabilities that may be useful for ev biomarker development and mechanistic studies. funding: this project is funded by mh118164 and ug3ca241694. importance to ev organotropism. yet, most techniques rely on bulk characterization, or are severely restricted by the diffraction limit. the exoview r100 (nanoview biosciences) combines interferometry, immunocapture, and immunofluorescence, introduced as an alternative technique to multiplex protein detection on single evs below the limit of diffraction. here, we use this technique to characterize tetraspanin multiplexing on evs and to identify spatial patterning of tetraspanins using steric hindrance of antibodies (abs). methods: evs were isolated from conditioned media from skov-3 cell culture or human serum. evs were incubated overnight on chips to allow immunocapture by anti-cd9, anti-cd63, or anti-cd81. chips were then incubated with three fluorescent abs against the same epitopes and imaged on the exoview r100. following concentration optimization, evs were tested after preincubating with carboxy-fluorescein diacetate succinimidyl ester (cfse) or fluorescent abs against tetraspanins. results: using different concentrations of evs, binding curves could be fit to characterize binding kinetics of abs. maximum concentration of evs could be identified that minimized fluorescent overlap. bright-field interferometry (detection limit~50 nm) distinguished 10x fewer bound evs than fluorescent detection, while pre-labelling evs with cfse produced 10x more detectable evs than immunofluorescence. interestingly, evs captured by one tetraspanin did not necessarily show high fluorescent detection of the same tetraspanin. upon pre-incubating evs with a single ab, vastly different expression profiles were identified, indicating significant steric hindrance between abs. furthermore, pre-incubating evs with anti-cd9 ab significantly decreased detection of cd81 with less impact on cd63. this discrepancy indicated possible spatial patterning of tetraspanins with cd9 and cd81 closely colocalizing on the ev surface. summary/conclusion: this combination of interferometry, immunocapture, and immunofluorescence produces unique information about size distribution of evs and single ev protein profile. this data corroborates that evs have distinct subpopulations of tetraspanins and indicates that tetraspanins may be spatially patterned. regulation of liver homoeostasis, regeneration and diseases by mesenchymal stem cell-derived apoptotic extracellular vesicles university of pennsylvania, philadelphia, usa introduction: billions of cells undergo apoptosis and produce apoptotic extracellular vesicles (apopevs) each day, whereas the roles of apopevs in regulating the organismal health and disease remain poorly understood. mesenchymal stem cells (mscs) emerge as critical contributors to tissue homoeostasis, while mscs suffer from apoptosis in regenerative transplantation. in this study, we investigated the function and mechanisms of msc-derived apopevs in regulating the organismal homoeostasis. methods: fas mutant (fasmut) and caspase 3 knockout (casp3-/-) mice were applied for apoptotic and apopev deficiency. mouse bone marrow mscs were cultured and apoptosis was induced by staurosporine (sts). msc-derived apopevs were collected by serial centrifuges and were infused into mouse circulation via caudal vein. tracing of apopevs were performed by radioisotope or fluorescent labelling. liver homoeostasis was evaluated at the histological and functional aspects. liver regeneration was induced by partial hepatectomy (phx). acetaminophen (apap) was used to establish acute liver drug injury. high-fat diet (hfd) was used to establish type 2 diabetes (t2d) and non-alcoholic fatty liver disease (nafld). results: after systemic injection, msc-derived apopevs migrate to liver and can be uptaken by liver macrophages and hepatocytes. fasmut and casp3-/mice develop hepatomegaly with structural disorders, which particularly reveals hepatocyte polyploidization. furthermore, fasmut and casp3-/-mice demonstrate liver glucose and lipid metabolic disorders. importantly, msc-derived apopev infusion significantly rescues structural and metabolic dysfunction in fasmut and casp3-/-mice. mechanistically, apopevs use the soluble n-ethylmaleimide-sensitive fusion protein attachment protein receptor (snare) protein for interactions with recipient organelles thus transferring signalling molecules. moreover, msc-derived apopev infusion promotes liver regeneration after phx, prevents apap-induced liver injury, and ameliorates nafld in t2d. summary/conclusion: msc-derived apopevs serve as crucial regulators of liver homoeostasis, regeneration and diseases. these findings indicate potential significant roles of apopevs in maintaining the organismal health and in developing therapeutics for diseases. (msc-sevs) mediate osteochondral regeneration in rats. however, the therapeutic effects of these msc-sevs/exosomes in restoring the mechanical competence of the repaired cartilage for joint function in a clinically relevant animal model remain to be addressed. to investigate this, we compared the structural and mechanical properties of the repaired cartilage in a rabbit model after intraarticular administration of msc-sevs and hyaluronic acid (ha) with that of ha alone, which is widely used as visco-supplementation. methods: bilateral osteochondral defects were surgically created on 17 rabbits. immediately after surgery and at days 7 and 14 post-surgery, 9 rabbits received 1ml injections of 200 µg msc-sevs and ha in both knees, and 8 rabbits received 1-ml injections of ha in both knees. at 6 and 12 weeks, macroscopic evaluation, histological scoring and compressive testing at different points on the repaired cartilage were performed. results: defects treated with msc-sev/ha showed improvements with time in macroscopic and histological scores and mechanical properties than defects treated with ha alone. in contrast, ha treated defects showed some repair at 6 weeks, but this was not sustained, as evidenced by significant deterioration of histological scores and a plateau in mechanical properties from 6 to 12 weeks. by 12 weeks, the msc-sev/ha repaired tissues demonstrated significantly better macroscopic score (10.33 vs 7.5; p < 0.001) and histological score (21.87 vs 11.11; p < 0.001). mechanical strength as measured by the young's modulus was significantly higher in the msc-sev/ha repaired cartilage than that in ha repaired tissues [defect centre (13.41 vs 3.95mpa; p = 0.001) and overall periphery (12.22 vs 3.37mpa; p = 0.012], and approximated that of the adjacent native cartilage. summary/conclusion: our findings demonstrated that msc-sevs and ha not only improved tissue morphology of the repaired cartilage but also promoted functional mechanical competence. this study establishes a clinically translatable protocol for use of msc-sevs for cartilage repair. introduction: mesenchymal stromal/stem cell (msc)exosome (mex) treatment has shown considerable promise in experimental models of bronchopulmonary dysplasia (bpd) and pulmonary hypertension (ph). mechanisms by which mex afford their beneficial effects remain incompletely understood and here, we embark into investigating them through assessment of mex biodistribution and impact on immune cell heterogeneity. methods: newborn fvb mice were exposed to hyperoxia (hyrx, 75% o2) at birth and returned to room air at postnatal day (pn) 14. mice received a bolus mex dose at pn4. adoptive transfer studies were used to determine the role of mex-educated myeloid cells in vivo. mice were harvested at pn4, 7, 14, or 28 to characterize mex biodistribution and for assessment of pulmonary parameters. results: mex therapy effectively ameliorated core features of hyrx-induced neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular remodelling and blood vessel loss. exercise capacity testing and assessment of ph showed functional improvements following mex therapy. biodistribution studies demonstrated that mex localize in the lung, where they interact with lung monocytes/macrophages. whole lung mass cytometry (cytof) revealed that mex treatment promotes a pro-homoeostatic shift in lung immune cell apportion, replenishing the early hyrx-induced depletion in pulmonary cd45+ immune cells, restoring alveolar monocyte and macrophage populations and suppressing cellular inflammation. ex vivo and in vivo analysis showed that mex promotes a "pro-resolving" ccr2-monocyte phenotype. notably, adoptive transfer of mex-educated bone marrow-derived myeloid cells (bmdmy), but not naïve bmdmy, restored alveolar architecture, blunted fibrosis, improved vascular remodelling and pulmonary blood vessel loss. summary/conclusion: mex treatment ameliorates core features of experimental bpd, restoring lung architecture, decreasing pulmonary fibrosis and vascular muscularization, ameliorating ph and improving exercise capacity. the beneficial actions of mex are associated with modulation of immune cell phenotypes, arising from mex-monocyte interaction. furthermore, adoptive transfer of mex-educated bmdmy rescued, at least in part, alveolar architecture, reduce fibrosis, improve vascular remodelling and pulmonary blood vessel loss. funding: this work was supported in part by an american thoracic society foundation grant (grw); the little giraffe foundation (grw); charles h. hood foundation major grants initiative (sk), nih r01hl146128 (sk) and united therapeutics research grant (sk and sam). immunomodulatory small extracellular vesicles derived from mesenchymal stem cells: a potential cell-free therapy for acute and chronic pulmonary vascular diseases introduction: vascular inflammation plays a critical role in acute respiratory distress syndrome (ards) and pulmonary arterial hypertension (pah). despite decades of research, there is no curative therapy for either condition. mesenchymal stem cells (mscs) have shown preclinical efficacy, mediated by release of extracellular vesicles. hence, msc-derived small extracellular vesicles (sevs) can harness the benefits of mscs with advantages in cost and safety. this study aims to evaluate the immunomodulatory effects of sevs in preclinical ards and pah. methods: msc-sevs were characterized by nanoparticle tracking analysis, electron microscopy and western blot. live fluorescence imaging measured in vitro and in vivo distribution of sevs. using a lipopolysaccharide (lps)-induced mouse model of acute lung injury (ali), a time course study of inflammatory response guided endpoint analyses. cell count and cytokines were measured in bronchoalveolar lavage fluid (balf) and histological lung injury was assessed. in ali mice, saline, mscs, msc conditioned media or sevs were administered 0.5 h post-lps. using a monocrotaline (mct)-induced rat model of pah, animals received saline or sevs at day 3. haemodynamic changes and right ventricular hypertrophy were evaluated at 3 weeks. results: msc-sevs were 72 nm in size with cd63/81 expression. pkh26-labelled sevs were taken up by endothelial cells. in the ali time course study, cell count and il1b in balf peaked at 24h post-lps, whereas il6 peaked at 10h. histology showed significant intra-alveolar cell infiltrate at 72h. msc conditioned media attenuated il1b in balf, whereas a trend towards reductions in il1b and cell count were seen from delivery of mscs and sevs. using fluorescence imaging, lung accumulation of dir-labelled sevs was highest when administered 1 h post-lps as compared to 5 h, 10 h or 24h. for pah rats, sevs reduced right ventricular systolic pressure (51.4 ± 8.3 mmhg) as compared to control (68.1 ± 0.4 mmhg; p = 0.012), whereas no changes were observed for right ventricular remodelling. summary/conclusion: these findings demonstrate the potential of msc-sevs to be used as a cell-free immunomodulatory therapy for acute and chronic lung vascular diseases. additional live and ex-vivo biodistribution studies will determine optimal timing of sev administration, tissue distribution and clearance in both ali and pah. changes in extracellular vesicle protein cargo after pro-inflammatory priming of umbilical cord mesenchymal stem cells (ucmscs) have been shown to suppress inflammatory responses in studies of autoimmune diseases. these therapeutic effects can be attributed to paracrine signalling, by which extracellular vesicles (evs) are one of the essential components. this study looks at how the culture conditions of ucmscs affects the type of evs they secrete. it also aims to identify an ev population with an anti-inflammatory potential for the treatment of autoimmune diseases. methods: ucmscs were isolated and culture expanded in a quantum® cell expansion system, then grown at 21%o2, 5%o2 and primed with a pro-inflammatory cocktail. evs were isolated from ucmsc conditioned media by differential ultracentrifugation using a sucrose cushion and characterised by transmission electron microscopy and nanoparticle tracking analysis. ev markers were analysed using a europium-based immunoassay, macsplex exosome detection kit and immunoblotting. a proximity-based extension assay was used to identify 92 inflammatory proteins in the evs. results: there was no difference in evs cultured at 21%o2, 5%o2 or with pro-inflammatory conditions when analysed for size and morphology. all evs displayed the tetraspanin markers (cd9/63/81) and internal proteins (alix, hsp70). evs from primed cells showed a > twofold increase of 5 cc chemokines and a > sixfold increase in cxcl5 and csf-1. protein cargo did not differ in evs from 21%o2 and 5%o2. summary/conclusion: this study showed that proinflammatory culture conditions alter ev protein cargo, evidenced by the increased production of chemotactic and angiogenesis associated proteins. upcoming rnaseq analysis will show if ucmsc culture conditions also affect mirna expression in evs. ongoing functional studies will determine how changes in ev cargo correlates with changes in t-cell proliferation and polarisation. funding: this work is fund by the orthopaedic institute ltd, keele university and the rjah orthopaedic hospital charity. alzheimer's disease biomarkers in plasma extracellular vesicles of neuronal origin correlate with brain pathology in mice introduction: multiple studies have shown that neuronal-derived extracellular vesicles (ndes) in blood contain alzheimer's disease (ad) biomarkers, especially tau. however, the convergent validity of tau in blood ndes in relation to brain pathology is yet to be determined. to address this, we measured total and phosphorylated tau levels in matched nde and brain tissue samples from ad mouse models. methods: we collected the cortex, hippocampus and plasma of 4 2xtg-ad, 15 3xtg-ad, and 9 wild type (total of 28 mice; 14 female, 14 male; age: mean = 8.17, sd = 1.61, 5-11 months) . plasma samples were collected retro-orbitally for 5 weeks and at euthanasia via heart puncture. ndes from the pooled serial blood collections (nde1) and the single endpoint (nde2) were immunocaptured by targeting the neuronal marker l1cam. we measured human total tau and pthr181-tau (p-tau) in ndes and cortex and hippocampus homogenates using a luminex multiarray. results: overall, there were strong positive correlations for both total tau and p-tau between ndes and brain tissues across mice types. total tau in ndes showed positive correlations with levels in the cortex and hippocampus (r = 0.6885 and 0.7026, p < 0.0001, cortex vs nde1 and nde2; r = 0.4958, p = 0.0073, hippocampus vs nde1; r = 0.6058, p = 0.0006, hippocampus vs nde2). levels of p-tau in nde1 showed positive correlations with levels in the cortex (r = 0.4640, p = 0.0155) and hippocampus (r = 0.6086, p = 0.0008); however correlations were not observed for nde2 (r = 0.1152, p = 0.6273 vs cortex; r = 0.0531, p = 0.7926 vs hippocampus). summary/conclusion: tau levels in circulating ndes reflect levels in cortex and hippocampus across ad model mice, supporting their convergent validity as "liquid biopsy" biomarkers for ad. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. exosomal ceramide mediates neurotoxicity of amyloid beta (aβ) in alzheimer's disease. ahmed elsherbini a , simone crivelli b , alexander kirov c , michael dinkins d , zhihui zhu a , haiyan qin a , sanjib karki a , priyanka tripathi a and erhard bieberich a a university of kentucky, lexington, usa; b university of kentucky, lexington, usa; c augusta university, augusta, usa; d augusta university, augusta, usa introduction: amyloid beta is a pathologic hallmark of alzheimer's disease (ad), however, the mechanism of aβ neurotoxicity is not fully understood. it has been reported that exosomes associate with aβ, but it is not clear how this association affects aβ neurotoxicity. methods: here we utilized several techniques to isolate exosomes from the sera of wild type (wt) and ad transgenic mouse model. (5xfad) as well as alzheimer's patients and healthy controls. we used exoquick, exoeasy, sequential ultracentrifugation, and size exclusion chromatography. particles' size and number were characterized by nanoparticle tracking analysis (zetaview). results: we report that the sphingolipid ceramide mediates neurotoxicity of aβ. we show that sera from ad transgenic mouse model (5xfad) and ad patients, but not the wt or healthy controls, contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide and are prone to aggregation (termed astrosomes) as confirmed by nanoparticle tracking and cluster analyses. when taken up by introduction: multiple sclerosis is the most common chronic inflammatory demyelinating disease of the central nervous system, affecting more than 2 million people worldwide. ms is a multifactorial, immunemediated disease caused by complex genetic and environmental interactions. in recent years, extracellular vesicles (evs) have been described as powerful mediators of the modulation of biological processes (e.g. inflammatory and immune response) following environmental exposures such as particulate matter (pm), and have been described altered in ms. we characterized evs in 48 patients with ms and 20 healthy subjects matched for age and gender and evaluated the effects of pm exposure on ev release patients with ms compared with controls. methods: evs isolated from blood samples were characterized by nanotracking analysis and by flow cytometry after labelling with the following markers: cd14 + (monocyte), cd61+ (platelet), cd66+ (neutrophil), cd25+ (t-reg), and cd105+ (endothelium). pm10 and pm2.5 concentrations at the residency of each subject were obtained from the regional air quality monitoring network. results: we observed decreased concentrations of cd14+ (p < 0.05), cd61+ (p < 0.003), cd66+ (p < 0.009), cd25+ (p < 0.003), and cd105+ (p < 0.003) in patients compared with controls. in cases, pm was inversely associated with cd14+ evs (pm2.5, β = −0.03; p < 0.05), cd66+ evs (pm2.5 β = −0.03; p < 0.03), and cd25+ evs (pm10 β = −0.02; p < 0.04; pm2.5, β = −0.03; p < 0.02). on the contrary, in controls pm was positively associated with cd14+ evs (pm10 β = 0.02; p < 0.03; pm2.5, β = 0.03; p < 0.02). summary/conclusion: our findings showed a different composition of blood-derived ev subpopulations in patients compared with controls. moreover, we observed that patients and controls react differently to pm exposure in terms of blood-derived ev release, suggesting the involvement of this mechanism in the modulation of both inflammatory and immune responses, and thus in ms pathogenesis. plasma neuronal and astrocyte-derived exosomes serve as biomarkers of neurodegeneration and systemic bioenergetic effects in male cynomolgus monkeys self-administrating oxycodone ashish kumar a , yixin su a , david soto-pantoja a , jingyun lee a , ravi singh a , cristina furdui a , michael nader b and gagan deep a a wake forest baptist medical center, winston-salem, usa; b wake forest baptist medical center, winston-salem, usa introduction: opioid use disorder (oud) is currently a health emergency in the usa affecting millions of people. oud is a complex issue requiring a multipronged strategy. at the biological level, there is an urgent need to understand the dynamic molecular changes and adverse effects associated with opioid addiction. here, we aimed at identifying the biosignature of brain cells-derived exosomes associated with opioid addiction in a non-human primate (nhp) model of oud in which cynomolgus monkeys perform cognitive tasks and self-administer (sa) intravenous oxycodone daily. we also characterized the systemic adverse effects of the brain cells-derived exosomes from drug-naïve and oxycodone sa monkeys. methods: we isolated total exosomes (te) by ultracentrifugation and exoquick methods from the plasma of male monkeys self-administrating oxycodone for 3 years and naive monkeys. subsequently, from the te population, we isolated neuron-derived exosomes (nde) and astrocytes-derived exosomes (ade) using surface biomarkers l1cam (l1 cell adhesion molecule) and glast (glutamate aspartate transporter), respectively. this novel method involved streptavidin coated magnetic beads and photo-cleavable (pc) biotin, providing us biologically intact exosomes useful for co-culture studies. we characterized the exosomes by nanoparticle tracking analyses (nta), western blotting, flow cytometry, immunogold labelling, transmission electron microscopy (tem), elisas and mass spectrometry. respirometric profiling in cardiac myoblasts and monocytes following exosomes treatment was performed by seahorse xf. results: the quality of isolated exosomes (te, nde, and ade) was confirmed by nta (size distribution and concentration), western blotting (e.g. cd9) and tem (size and shape). nta did not show any significant difference in exosomes size and concentration (number per ml) between control and oxycodone sa groups. flow cytometry (e.g. l1cam and glast) and immunogold labelling (cd9, cd63 and l1cam) confirmed the purity of nde and ade isolated from te. proteomics analyses of te, nde and ade identified several unique proteins present in exosomes from the oxycodone sa group. interestingly, we observed significantly higher expression of neurodegeneration markers neurofilament light protein (nfl) and alpha-synuclein in nde and ade of oxycodone sa group compared to controls. furthermore, te treatment of h9c2 cardiac myoblasts and raw264.7 monocytes significantly compromised their mitochondrial metabolism (basal and maximum respiratory capacity). summary/conclusion: these results suggest the utility of plasma exosomes as biomarkers for better understanding of the neurodegenerative and systemic effects of oxycodone addiction. funding: da049267, da017763. vesicles released during mycobacterium tuberculosis infection: immunomodulatory (glyco)lipids and role in host-pathogen interactions emilie layre, pierre boyer and jerome nigou cnrs-université paul sabatier, toulouse, france introduction: the tuberculosis disease remains one of the top 10 causes of death worldwide. mycobacterium tuberculosis (mtb) has evolved strategies to evade immune responses and to persist within the hostile intracellular environment of alveolar macrophages. the current lack of efficient anti-tuberculosis strategies is largely due to our incomplete understanding of the host-pathogen interactions of mtb infection. vesicles released by the bacillus itself (bacterial membrane vesicles, bmv) and by infected cells (host extracellular vesicles, hev) have immunomodulatory properties in vitro and when administered to animals. if vesicles likely play key role in host-pathogen interactions of the tuberculosis infection, their content in bacterial factors, their uptake, trafficking and interaction with host cells receptors remain incompletely deciphered. methods: bmv and hev have been purified by combining differential centrifugation, density gradient and exclusion chromatography. after characterization by microscopy, nanosight and western blot, their content in bacterial (glyco)lipids has been characterized by the use of high sensitivity mass spectrometry-based lipidomic approach. bmv have been tested for their capacity to activate reporter cell lines of pattern recognition receptors. in addition, fluorescent-labelled bmv have been used to study their uptake by host cells thank to super-resolution microscopy. results: we have undertaken to characterize the content, the trafficking and interaction with pattern recognition receptors of bmv and hev released during infection by mycobacteria of variable virulence. we have importantly optimized the purification of bmv showing that lipoproteins aggregates are co-purified with vesicles on density gradient. sfc-ms lipidomic analyses allowed the characterization of the repertoire of immunomodulatory bacterial lipids released by bmv and hev, which excluded a continuum between these two release pathways. preliminary, assays have shown that these vesicles are capable to interact with different pattern recognition receptors including tlr and lectins. finally, we have been able to visualize fluorescent-labelled vesicles uptake by macrophages using superresolution microscopy. summary/conclusion: during m. tuberculosis infection, the bacillus as well as infected cells release vesicles that harbour different content in immunomodulatory bacterial (glyco)lipids, including strain-specific lipids. these vesicles likely play important role in host-pathogen interactions by modulating immune response beyond the infected cells, in part through their interaction with different pattern recognition receptors. funding: fondation pour la recherche medicale, fondation fonroga. introduction: conventional diagnoses of mycobacterium tuberculosis (mtb) rely on quantifying bacteria in sputum samples, which make it incapable of measuring the body's total bacterial load and diagnosing patients that have difficulty producing sputumsuch as children and those that are hiv-positive. nanoscale (30-200 nm) outer membrane vesicles (omvs), which are shed from their bacterial cells of origin and circulate in the bloodstream, have been found to contain rich molecular information from their mother cells. despite the diagnostic potential, their nanoscale size in the presence of high background has complicated the use of these promising biomarkers for clinical diagnosis of tuberculosis. chair: amy buck -the university of edinburgh chair: cherie blenkiron -the university of auckland methods: here we report two complementary approaches to systematically discover and clinically detect mtb-derived omvs using protein and rna biomarkers. first, we employ a digital droplet elisa on whole, unprocessed samples to detect and quantify the presence of these omvs using surface protein markers. second, we have developed a platform to specifically enrich for mtb-derived omvs using our previously developed magnetic nanopore platform, wherein millions of nanofluidic devices are operated in parallel, increasing throughput relative to a single nanofluidic device by a million-fold. using this approach, we identify rnas that are specifically enriched in mtb-derived omvs and can be used to identify tb strain, infectious activity, and total body burden. results: using these platforms, we enriched for mtbderived omvs from plasma and profiled their cargo, both proteins and rna. we first determined a panel of protein biomarkers for multiplexed detection of omvs through a digital droplet sandwich elisa. we then tested our protein markers on spiked plasma samples as models for clinical tb samples. simultaneously, we performed rna sequencing and discovered a panel of rna biomarkers that are preferentially enriched in omvs. we picked ten of the most highly-expressed rna biomarkers and also tested for them on spiked plasma samples using our magnetic nanopore platform. summary/conclusion: these results demonstrate the capability of omv biomarkers in the development of novel liquid biopsy based mtb diagnostics. building on this work, we are working with clinical collaborators to test our assays on clinical samples from philadelphia and west africa. funding: nih ot08.3 introduction: a dearth of knowledge exists regarding the molecular mechanisms by which host exosomes regulate immune response to infections caused by gram-negative pathogens. to address this gap in knowledge, our laboratory has been using two wellestablished model organisms; yersinia pestis (yp), and burkholderia pseudomallei (bp). yp and bp cause the emerging human diseases plague and melioidosis respectively. currently, no licenced vaccines or highly effective therapeutics are available for either disease. methods: ex were purified from naïve u937 monocytes (exu) and infected u937 (exi) by serial centrifugation followed by sucrose density gradient purification, and characterized by tem, zetaview nanoparticle tracking, and exosome markers (cd63, tsg101, . immune responses of naïve u937 cells and response mechanisms were analysed following treatment with equivalent amounts of exi or exu (as control). these included macrophage differentiation assays, multiplex measurements of inflammatory cytokines, bacterial clearance assays, quantitative protein microarray analysis of 173 host signalling proteins, sirna knockdown of exi-induced cytokines in recipient cells, and mass spectrometry (ms) analysis of exi contents. for all assays, at least four biological replicates were performed. results: exi induce monocyte differentiation to macrophages and dramatic release of il-6, il-8 and il-10 cytokines, effects that are also seen when monocytes are infected with the bacteria. the exi also induce a substantial increase in the capacity of the recipient monocytes to clear bacteria in an il-6-dependent manner. specific host signalling molecules are strongly modulated by the exi, including p38, jak2 and alk for exi-yp, influencing the observed phenotypes. ms analysis showed lack of lps in exi-yp and demonstrated the presence of specific bacterial proteins that have antigenic properties. summary/conclusion: we have identified some of the molecular mechanisms by which exi assist the host in clearing infection. exi prime distant naïve monocytes through modulation of distinct pathways such as p38 to mount immune responses similar to when they become infected. these include differentiation to macrophages and migration to infection site for increased il-6-dependent bacterial clearance. introduction: recruitment of monocytes to sites of infection is important in restricting growth and invasion of various microorganisms such as pathogenic fungi c. albicans. beside complement supported phagocytosis and extracellular trap formation, human monocytes secrete extracellular vesicles which are crucial in cellular communication in physiology and pathophysiology as they transfer proteins, lipid, and nucleic acids. the current study attempts to shed light on immune evasion mechanisms by c. albicans via extracellular vesicles. methods: human monocytes were isolated by magnetic beads technique and extracellular vesicles were isolated using polymer precipitation or ultracentrifugation or size exclusion chromatography. vesicles were characterized by elisa, lc/ms-based proteomics, confocal laser scanning microscopy (clsm) as well as electron-and dynamic light scattering microscopy, etc. crispr-cas9 based genome editing was performed to knockout cd11b in human monocytic thp-1 cells. effect of isolated vesicles were determined by using proximity ligation assay (pla), elisa, western blot, next generation rna sequencing, qpcr, immunohistochemistry, etc. results: here we show for the first time that human blood derived monocytes alone and in a whole blood model strongly induced and released extracellular vesicles in response to the pathogenic fungus c. albicans. one induced population carried the anti-inflammatory cytokine tgfβ-1. release of these vesicles is triggered by binding of soluble β-glucan from c. albicans to the cr3 receptor on monocytes as demonstrated by crispr-cas9 based cr3 genome editing in thp-1 cells, and by using cr3 knock out mice. isolated tgf-β1-transporting vesicles reduced the inflammatory response in human m1 macrophages and in a whole blood model. the anti-inflammatory effect by tgf-β1transporting vesicles is investigated in detail and results in inhibition of il-1β gene transcription. summary/conclusion: showing that human apoptotic bodies similarly induced tgf-β1-transporting vesicles from human monocytes we hypothesize that c. albicans hijacks this new cr3-dependent anti-inflammatory vesicle pathway for immune escape. funding: this work was supported by the "deutsche forschungsgemeinschaft" transregio funginet 124 projects c4, c5, c6 and z2. introduction: to date, most research involving extracellular rnas has focused in rnas encapsulated inside extracellular vesicles (evs) or in total unfractionated biofluids. it is known that exrnas also exist outside vesicles or in lipoprotein particles. however, nonvesicular exrnas remain widely uncharacterized despite being a feasible source of contaminants in ev preparations. our interest in nonvesicular exrnas arises from the observation that some small rnas, such as specific trna-derived fragments, have much higher relative representation in this extracellular fraction. at least in part, this enrichment seems to be a consequence of their differential extracellular stability. methods: to get a representative picture of the whole set of rnas released to the extracellular nonvesicular space by cultured human cells, we inhibited extracellular degradation by adding recombinant ribonuclease inhibitor to the cell-conditioned media and studied the kinetics of rna release and degradation. high-resolution iodixanol gradients were used to separate evs from extracellular rnps or vesicle-free rna. the conversion rate between parental ncrnas and their fragments was studied by high-throughput sequencing and northern blot. results: the inhibition of extracellular rnase activity revealed the presence of full-length trnas and ribosomes in the extracellular space of a variety of malignant and non-malignant cell lines. extracellular ribosomes co-isolate with evs purified by ultracentrifugation or size-exclusion chromatography, but not with evs purified by density gradients.these ncrnas are substrates of extracellular rnases, demonstrating an extracellular biogenesis route for the formation of ncrna-derived fragments, some of which achieve remarkable stability and can be detected in biofluids. we also highlight the immunoregulatory potential of purified rna-containing extracellular complexes. summary/conclusion: in conclusion, ribonuclease inhibition dramatically shapes extracellular rna profiles and uncovers a population of extracellular ribosomes, trnas and other coding and noncoding rnas which exists outside evs. although these rnas are prone to degradation, some of their fragments can accumulate in cell culture media and in biofluids. this dynamic view of exrnas impacts our understanding of rna secretion mechanisms and may offer a window to new molecules with biomarker potential. in contrast, evs confer an rnase-protected environment and contain more full-length ncrnas (trnas, yrnas, 7sl rnas, rrnas depending on vesicle size) than their fragments. introduction: cd47 is a ubiquitously expressed membrane protein that functions as a receptor for thrombospondin-1 and the counter receptor for signal regulatory protein-α in phagocytes. high expression of cd47 is associated with a poor prognosis for some cancers. conversely, cd47 blocking agents are in clinical trials for enhancing innate and adaptive antitumor immunity in cancer patients. these studies suggest utility of cd47 as a diagnostic and prognostic biomarker and as a therapeutic target. cd47 is also expressed on extracellular vesicles (evs), and we reported that cd47 expression identifies a distinct population of evs from those that express the traditional ev markers cd63 or mhc1. cd47-, cd63-and mhc1-enriched vesicles contain distinct small rna populations (pmid: 29416092), and these differ in rna content from evs that lack any of these markers. the mechanisms by which cd47 directly or indirectly regulates which rnas are packaged into ev remain unknown. methods: to elucidate the mechanism by which cd47 regulates ev rna composition and function, we performed global mirna microarray analysis between evs produced by wild type and cd47-deficient t cells. results were further validated using real-time pcr and rna-immunoprecipitation. interactions between cd47 and exportin-1/ran complex was identified by mass spectrometry and confirmed by using co-immunoprecipitation, subcellular localization, flow cytometry, and confocal and electron microscopy. results: ev released from cd47-deficient human t cells and in cd47-/-mouse plasma were enriched in 5ʹ-7-methylguanosine-capped micrornas and mrnas that depend on the exportin-1/rangtp pathway. knockdown of cd47 in wild type cells or thrombospondin-1 treatment correspondingly enhanced levels of capped-rnas released in ev and re-expressing cd47 in null cells decreased their levels. mass spectrometry and co-immunoprecipitation identified specific interactions of cd47 with components of the exportin-1/ ran nuclear export complex and its known cargos and between the cd47 cytoplasmic adapter ubiquilin-1 and the exportin-1/ran complex. interaction of cd47 with exportin-1 was inhibited by leptomycin b, which inactivates exportin-1 and increased levels of cap-dependent rnas in ev released from wild type but not cd47-deficient cells. summary/conclusion: these findings indicate that cd47-dependent thrombospondin-1 signalling regulates cytoplasmic levels of cap-dependent rnas in t cells at least in part through ubiquilin-1-and gtpdependent physical interactions with the exportin-1/ ran transport complex, which regulate levels of specific pre-mirnas and mrnas available for sorting into evs. funding: this work was supported by the intramural research program of the nih/national cancer institute (zia sc 009172). role of membrane protein palmitoylation in extracellular vesicle biogenesis in squamous cell carcinoma introduction: desmoglein 2 (dsg2), is a palmitoylated cadherin that is involved in cell-cell adhesion. interestingly, dsg2 promotes mitogenic cell signalling and is upregulated in many cancers, including scc, contributing to poor prognosis and survivability. we recently demonstrated that dsg2 promotes ev release, but the mechanism by which dsg2 enhances ev biogenesis and role of palmitoylation is poorly understood. methods: pharmacological drug inhibitors 2-bromopalmitate, gw4869, and bafilomycin a1 were used. stable scc cell lines were established by retrovirus infection expressing gfp, wild type dsg2/gfp, or palmitoylation deficient dsg2cacs/gfp. evs were isolated by sequential ultracentrifugation and iodixanol gradient separation and analysed by nta. proteins associated with the endocytic pathway were analysed by immunofluorescence and imaged by confocal microscopy or immunoblotting and signals were quantitated using imagestudio. results: here we demonstrate that the effect of dsg2 on ev release was reduced by the palmitoylation inhibitor 2-bromopalmitate. furthermore, mutations that prevented palmitoylation (dsg2cacs) dramatically abrogated ev release by targeting of un-palmitoylated dsg2 to the lysosomes for degradation. dsg2 increased expression and subcellular localization of flot1, a membrane lipid raft protein critical for membrane invagination. dsg2 also altered membrane localization of several early (eps15 and eea1), but not late (rab7, rab11, and hrs), endocytic pathway proteins. loss of palmitoylation in the dsg2cacs mutants abrogated these effects. finally, dsg2-induced ev release was abrogated by the sphingomyelinase inhibitor gw4869 or augmented by the v-atpase inhibitor bafilomycin a1. summary/conclusion: the combined results of the drug treatments and functional mutations of dsg2 suggest that dsg2 plays a critical role in ev biogenesis by modulating proteins involved in early endosome sorting and is dependent on post-translational palmitoylation. introduction: the translation initiation factor eif4e (4e) is an oncogenic protein that is upregulated in 30% of cancers including a subgroup of acute myeloid leukaemia (aml) patients. eif4e regulates post-transcriptional rna processing including the nuclear export and/or translation of mrna transcripts. in particular, it selectively increases the expression of genes that have a prominent role in cancer progression such as myc, cyclin d1, and mcl1. furthermore, our lab pioneered studies demonstrating that a subset of 4ehigh aml patients is clinically responsive to treatment with a 4e inhibitor (ribavirin) indicating the importance of 4e in aml progression and its relevance as a therapeutic target. we investigated an as yet unexplored perspective of 4e-whether the oncogenic role of 4e is in part mediated by its function as a master regulator of vesiculation. methods: to assess mrna export and identify 4ebound mrna targets that correspond to vesiculationrelated genes and associated cargo, we used cellular fractionation and rna immunoprecipitation techniques. to determine whether 4e regulates the number of extracellular vesicles (evs) released as well as their protein and rna cargo we used nanoparticle tracking analysis (nta) as well as mass spectrometry, antibody microarrays, and rna sequencing technologies. results: eif4e upregulates cellular protein levels of the vesiculation marker cd63 by increasing its nuclear export. in addition to increased cellular expression, cd63, cd81, cd9, and flotillin-1 proteins are elevated in evs released from 4e-high cells. this is also associated with an increased release of vesicles that are 50-200 nm in size. currently, we have validated the upregulation of several receptors and cytosolic proteins in evs isolated from 4e-overexpressing cells that function in cell growth, migration, invasion, and stemness. the most abundant rnas in our ev preparations are micrornas (mirs) and we have confirmed downregulation of several of these. summary/conclusion: our work shows that 4e reprograms the vesiculation of cancer cells changing the release and cargo of evs. this may impact cellular communication and tumour biology, which we are currently addressing in functional studies. we hope that these studies will highlight novel therapeutic strategies for aml patients. intranasal administration of neural stem cells-derived extracellular vesicles promotes neurogenesis and reduces neuroinflammation and amyloid plaques in a mouse model of alzheimer's disease introduction: cognitive and memory impairments worsen with time in alzheimer's disease (ad), likely due to a progressive loss of hippocampal neurogenesis, and escalation of neuroinflammation. these changes are also accompanied by increased deposition of amyloid plaques in the brain. methods: in this study, using the 5xfad mouse model, we examined the efficacy of extracellular vesicles (evs) shed from the rat subventricular zone neural stem cells (svz-nscs) for disease modification. we first purified evs from the rat svz-nsc cultures through ion-exchange chromatography and then administered intranasally to 3-months old 5xfad mice (~75 billion/week for two weeks). two months later, the functional effects of ev treatment were quantified through a series of behavioural tests, and animals were euthanized for quantification of hippocampal neurogenesis, oxidative stress, neuroinflammation, and amyloid plaque deposition. results: in comparison to ad mice receiving vehicle, ad mice receiving nsc-evs displayed improved cognitive function to discern minor changes in the environment in an object location test, better spatial recognition memory in an object-in-place test, and improved pattern separation ability in a pattern separation test. besides, ev-treated ad mice displayed no anhedonia in a sucrose preference test. analyses of neurogenesis using the birth-dating marker 5ʹ-bromodeoxyuridine and the newly born neuron marker doublecortin revealed maintenance of a higher level of hippocampal neurogenesis in ad mice receiving evs, in comparison to vehicle-treated ad mice. moreover, analyses of brain tissues from ev-treated ad mice revealed decreased concentrations of oxidative stress markers malondialdehyde and protein carbonyls and elevated levels of antioxidants catalase and superoxide dismutase. also, the concentration of proinflammatory cytokines tumour necrosis factor-alpha and interleukin-1 beta and the extent of amyloid plaques were significantly reduced in ev treated ad mice. immunohistochemical analysis showed reduced hypertrophy of astrocytes. summary/conclusion: intranasal administration of nsc-derived evs restrains the deterioration of cognitive and mood dysfunction of ad by maintaining higher levels of neurogenesis and curtailing the progression of neuroinflammation. funding: supported by a grant from the national institute of neurological disorders and stroke (1r01ns106907-01 to a.k.s.) ot10.2 the case of mesenchymal stromal cells, opening new perspectives in the use of ipsc in regenerative medicine. the aim of this study is to evaluate the potential of ipsc-ev in the treatment of kidney disease. methods: the ipsc were generated from skin fibroblast after informed consent of healthy donors (cytotune®-ips 2.0 sendai reprogramming kit -protocol: clementino fraga filho uh 38583914.7.0000.5257/933.018). the ev were isolated from ipsc supernatants (cultured 24 h in mtesr-1 medium) by ultracentrifugation (100,000 g for 2 h at 4°c). characterization of ipsc-ev was performed using zetaview, tem, exoview™ tetraspanin kit and macsplex exosome kit. for in vitro injury, renal epithelial cells were cultured under hypoxia (1% o2). for in vivo injury, male wistar rats were submitted to bilateral renal arterial clamping (45 min) followed by reperfusion without or with injection of ipsc-ev (protocol approval: federal university of rio de janeiro -043/19). kidney damage was assessed by histological and immunohistochemistry analyses (pcna, tunel and ed-1). modulation of rna levels was assessed by rt2 profiler pcr array. results: the results show that ipsc-ev reduce renal cell death, tissue damage, macrophage infiltration, promote mitochondria protection and ameliorate renal function. the ipsc-ev mechanism of action is related to the regulation of key genes known to prevent damage caused by oxidative stress like gstk1, sod1, sod3, txn1 and txnrd2. characterization of ipsc-ev showed that ipsc-ev can carry important molecules that can support renal recovery as epcam and prominin-1. summary/conclusion: ipsc-ev presents renoprotective properties, acting on different aspects of aki. this presents a new relevant application of ipsc as a source of ev for therapeutic purpose in kidney diseases. the hospital for sick children, toronto, canada introduction: incomplete lung development, also known as pulmonary hypoplasia (ph), is a recognized cause of neonatal death. we have previously shown that experimental ph can be rescued by the administration of extracellular vesicles derived from amniotic fluid stem cells (afsc-evs) through an rna-mediated mechanism. this effect was not observed with evs derived from mesenchymal stromal cells (msc-evs) . the aim of this study was to 1) evaluate which rna species were responsible for ph rescue, and 2) to define the mechanism behind this effect. methods: evs were isolated and characterized from conditioned medium of rat afscs and rat mscs (control group) using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd63, hsp70, flo-1, and tsg101 (western). to identify the mediators of afsc-evs, we used deseq (fdr<0.01) to differentially analyse rna from: a) asfc-ev and msc-ev cargo, isolated with seramir and sequenced with nextseq. b) lung epithelial cells from rat ph lungs treated with vehicle or afsc-evs. epithelial cell rna was isolated with mirvana and sequenced with nextseq. we correlated afsc-ev cargo mirna with validated mrna targets that were downregulated after ev conditioning in lung epithelial cells. results: of the rna species contained in asfc-ev and msc-ev cargo, mirnas were the most proportionally different between the two ev populations. afsc-evs were enriched for mirnas that are critical for lung development, such as mir17~92 and their paralogues that control lung branching morphogenesis. afsc-ev administration to ph lung cells significantly downregulated 35 genes, which formed 415 mirna-mrna reported interactions. summary/conclusion: afsc-evs contain many rna species in their cargo, but mirnas are the main effectors of their ability to rescue underdeveloped foetal lungs. we have identified for the first time that afsc-ev biological effect on underdeveloped foetal lungs is in part due to the release of mir17~92 cluster. funding: cihr-sickkids foundation grant. bottom-up assembly of fully-synthetic extracellular vesicles oskar staufer a , franziska dietrich a , jochen hernandez a , martin schröter a , sebastian fabritz b , heike böhm a , ilia platzman a and joachim spatz a a max planck institute for medical research, department for cellular biophysics, jahnstraße 29, 69120 heidelberg, germany, heidelberg, germany; b department for chemical biology, max planck institute for medical research, jahnstraße 29, 69120 heidelberg, germany, germany, germany introduction: extracellular vesicles (evs) are considered as key elements for future therapeutic and diagnostic procedures. however, despite enormous research efforts to understand their physiological relevance and several greatly successful clinical trials, evs are currently not authorized for clinical routines by american or european regulation and approval agencies. this is especially because therapeutic evs are produced or isolated from cell cultures or biofluids, both of which are subjected to batch-to-batch variations and ill-defined contaminations. therefore, complementary technologies that produce evs as reproducible and defined as state of the art nanotherapeutics, would revolutionize the application of evs in clinical settings and provide the scientific community with a holistic understanding of ev-mediated signalling processes. in our study, we achieve de novo bottom-up assembly of fully synthetic evs (fsevs) that comprise identical physiological and therapeutic functionalities to natural evs. methods: we applied droplet-based microfluidic synthesis to sequentially amalgamate synthetic lipids, proteins and nucleic acids into defined vesicles that display analogous therapeutic capabilities to natural evs. fsevs were characterized by electron and confocal microscopy, dynamic light scattering and mass spectrometry and tested on organotypic models or in vivo. results: using previously described evs as "naturegiven" blueprints, we assembled several fsevs in their exact molecular composition. in particular, we produced wound-healing promoting evs composed of several exosomal proteins, lipids and micrornas and showed that their therapeutic performance on human skin wounds is equivalent to that of natural evs. besides their high molecular complexity, being composed of dozens of different molecular building-blocks, the presented fsevs are completely defined on a quantitative level. based on this, we achieved a stoichiometric understanding of cell-vesicle interactions. summary/conclusion: by applying bottom-up synthesis of fsevs for quantitative studies on ev signalling, we not only provide innovative and safe compounds for ev-therapeutics but also a vastly new perspective on the application spectrum of extracellular vesicles in fundamental research. introduction: small extracellular vesicles (sevs) contain functional molecules from their cell of origin and can enter recipient cells for intercellular communication. ifnβ has been shown to induce some lncrnas to regulate host immune response and play a major role in the positive regulation of the activity of natural killer (nk) cells. here, we aim to clarify whether ifnβ induced sevs can regulate the cytotoxicity of nk cells by transferring specific lncrnas into nk cells. methods: evs were purified from a549 with/without ifnβ treatment by serial centrifugation followed by sucrose density gradient purification. elisa assay were performed to demonstrate the cytotoxicity of nk cells. qpcr and western blot were used to verify the expression of nkp46. results: surprisingly, ifnβ induced sevs can strengthen the cytotoxicity of nk cells. through human transcriptome array (hta) we found the expression levels of 69 lncrnas were significantly changed within sevs isolated from a549 cells following ifnβ treatment. additionally we found a specific sev cargo, linc-epha6-1, acted as a competing endogenous rna (cerna) for hsa-mir-4485 which subsequently up-regulate the natural cytotoxicity receptor (nkp46) expression. furthermore, we verified over-expression of linc-epha6-1 significantly enhance the cytotoxicity of nk cells against zika virus-infected a549 cells. summary/conclusion: our results demonstrated that ifnβ-induced linc-epha6-1 wrapped in sevs can regulate the cytotoxicity of nk cells. our study provides a novel link between type i ifn and nk cells, which are two major players for the host innate immunity against pathogen infections. introduction: hiv-infected t cells release simultaneously viral particles and small extracellular vesicles (sevs) including mvb-derived exosomes and plasma membrane-derived evs. sevs and hiv share many physical and chemical characteristics, which makes their separation difficult. although several approaches have been used to obtain sevs free of virus they leave a majority of sevs within hiv preparations. for this reason, the function of sevs during hiv infection remains unclear. methods: we have developed a novel un-biased proteomic profiling approach to identify specific markers of the virus or sev subtypes released by a human t lymphoma cell line. our approach was to combine differential centrifugation of medium/small evs contained in the ccm with quantitative mass spectrometry to generate protein abundance profiles across the different sub-fractions. we generated an interactive database to define groups of proteins with similar profiles, suggesting their release in the same evs. results: we thus identified different categories of evs, which bear different surface proteins, e.g. different combinations of t cell surface markers, integrins or tetraspanins. in evs released by infected cells, we identified cellular proteins behaving like hiv proteins, and several that changed behaviour after infection, either moving towards or away from the hiv cluster. we identified two cell-derived proteins that are included in the viral particles and one that is specific of non-viral sevs that are modified by infection, and analysed their respective roles in controlling ev composition or virus infectivity. summary/conclusion: our approach presents a powerful tool for identification of common cargoes of given ev subtypes, and could be now used to identify modifications of ev composition in any given physiological or pathological situation. the encephalomyocarditis virus leader modulates autophagic pathways to promote the release of virions inside extracellular vesicles introduction: recent data indicate that naked viruses belonging to the picornaviridae family can be released from host cells via enclosure in extracellular vesicles (ev). ev cloak virus particles in a host-derived "envelope" and can thereby affect antiviral immune responses and disease severity. a better understanding of the formation and function of ev-enclosed viruses is therefore required. previously, we showed the presence of the autophagosome marker lc3 in ev isolates from encephalomyocarditis virus (emcv) infected cells, suggesting the involvement of a secretory autophagy pathway in ev-mediated virus release. however, little is known about the viral and host factors that regulate this process. here, we have assessed the role of the emcv leader, a viral protein that is dispensable for replication but is required for symptomatic disease. methods: cells were infected with wildtype virus or a mutant carrying an inactive leader. ev produced during the infection were isolated using differential ultracentrifugation and density gradient purification. ev were characterized by high resolution flow cytometry and their infectivity determined using end-point dilution assay. in addition, the fate of autophagosomes in infected cells was monitored using a reporter assay for autophagosome-lysosome fusion and analysis of the secretion of autophagosomal proteins. results: inactivation of the emcv leader strongly reduced the release of ev-enclosed virus. whereas autophagosomes are typically degraded, we show this is blocked by the leader. instead, autophagosomes fuse with the plasma membrane, as indicated by the secretion of autophagy marker lc3 during infection with wildtype but not the mutant virus. pharmacological reactivation of degradative autophagy in infected cells resulted in a strong reduction in the release of ev and ev-enclosed virus. similarly, the reduction in evenclosed virus release in the absence of the leader could be partially reversed by drugs that promote the secretion of autophagosomes. summary/conclusion: our data supports a role for secretory autophagy in the release of viruses in ev, a pathway that is regulated by the emcv leader. these findings highlight an unconventional route for ev formation that intersects with autophagosomal compartments and contributes to viral pathogenesis. introduction: zika virus (zikv) causes a public health emergency of international concern because of its correlation with microcephaly. during viral infection, the innate immune response quickly to produce some endogenous functional molecules which can prevent viral invasion or replication. extracellular vesicles (evs) contain molecules from their cell of origin under virus infection and can enter recipient cells for intercellular communication. here, we aim to clarify whether zikv induced evs can regulate viral pathogenicity by transferring specific rna. methods: evs were purified from a549 with/without zikv infection by serial centrifugation followed by sucrose density gradient purification. human transcriptome array (hta) was used to found rna expression within evs. flow cytometry was used to determine cell cycles. zikv replication was assayed by qpcr and western blot. flow cytometry was used to determine cell cycles. results: through hta we found the defensin alpha 1b (defa1b) expression was significantly increased within evs isolated from zikv infected a549 cells. additionally, we found that the extracellular defa1b but not the intracellular defa1b exerts anti-zikv effect mainly before entry step. surprisingly, up-regulate defa1b can retard cell cycles of host cell. we verified defa1b could bind with the origin recognition complex 1 (orc1) which is required to start dna replication during the cell cycle. furthermore, up-regulate defa1b decreased the orc1 level in nuclear. interestingly, evs with defa1b can internalize into recipient cells and inhibit their cell cycles. summary/conclusion: together, our results demonstrated that zikv infection can induce defa1b wrapped in evs, and defa1b not only exerts anti-zikv effect but also regulate cell cycles which may affect neurodevelopment. our study provides a novel viewpoint that defa1b act as first-line anti-viral molecules during zikv infection also correlate with neurodevelopment by retarding cell cycles. extracellular vesicles mediate bacterial-immune cell interactions during respiratory viral-bacterial co-infections sidney w. lane a , matthew hendricks b and jennifer bomberger a a university of pittsburgh, pittsburgh, usa; b university of washington, seattle, usa introduction: respiratory infections are a major cause of morbidity and mortality worldwide and host-derived extracellular vesicles (evs) play important roles in mediating these infections. during respiratory infection, evs are shown to have a modulatory effect: promoting or suppressing infection dependent on the pathogen and cell type. in the age of next-generation sequencing, we now appreciate that many respiratory infections are polymicrobial in nature, with viral-bacterial co-infections correlating with worse disease outcomes. epidemiological studies correlate acute viral infections with the increased likelihood and severity of both acute and chronic secondary bacterial infections; however, the exact mechanisms of these interactions remain poorly understood. evs have been understudied in the context of respiratory viral-bacterial coinfections; thus, their role in mediating these infections is relatively unknown. unpublished data from the lab shows that in airway epithelial cells (aecs), viral infection induces the release of evs that associate with pseudomonas aeruginosa (pa) and promote biofilm growth. here, we aim to expand upon these findings and determine how aec evs mediate pa-immune cell interactions during respiratory viral-bacterial co-infection. methods: to determine how exposure to evs impacts pa-immune cell interactions, evs were isolated from the apical secretions of aecs and co-cultured with pa. ev-treated pa was then co-cultured with macrophages to evaluate ev impact on pa uptake and survival. results: in preliminary experiments using control evs, we observed that evs associate with pa. interestingly, during co-culture with macrophages, ev-treated pa are more susceptible to phagocytosis in comparison to non-treated pa. however, after 4 hours of co-culture with macrophages, ev-treated pa are able to survive and replicate, while nontreated pa are effectively controlled by the macrophages. summary/conclusion: these findings suggest that while pa-ev association promotes pa uptake, it may ultimately enhance pa immune evasion and survival. ongoing experiments in the lab are evaluating the mechanism of pa-ev association and how evs from virus-infected aecs affect the phenotypes observed with control evs. notably, this is one of few reports of a mammalian ev influencing the pathogenesis of a bacterium; thus, results from these experiments will define the function of aec evs in regulating bacterial-immune cell interactions during respiratory co-infections. using machine learning with neuronal ev target proteins and clinical data to predict cognitive impairment in hiv infection lynn pulliam a , michael liston b , bing sun c and jared narvid d a university of california, san francisco, san francisco, usa; b veteran affairs, san francisco, usa; c ncire, san francisco, usa; d ucsf, san francisco, usa introduction: objective biomarkers are needed to assess and predict neuronal function and cognitive impairment. in people ageing with chronic infections, such as hiv, determining the mechanism of impairment will be important when therapies are available. methods: sixty plasma samples from hiv-infected people were obtained from nih-sponsored aids banks. clinical and epidemiological data were collected. all underwent neuropsychological testing and 40 were considered impaired. neuronal extracellular vesicles (nevs) were isolated from plasma and assayed for high-mobility group box 1 (hmgb1), neurofilament (nf-l) and phosphorylated tau-181 (p-tau) proteins. results: using 3 different algorithms, support vector machines (svm) performed the best with an area under the curve (auc) value of 0.82 ± 0.16. using 4 different combinations of clinical data and the 3 nev protein targets, selected clinical data and hmgb1 best predicted cognitive impairment (auc = 0.82). the most important features included cd4 count, hmgb1, nf-l and education. summary/conclusion: specific clinical features plus nev hmgb1, an inflammatory marker, were the best predictors of cognitive impairment. previous published data showed nev p-tau-181 elevated in alzheimer's disease and in this study p-tau had no importance in assessing hiv-associated cognitive impairment. nev target discovery can be improved to better identify neuronal damage, possibly to differentiate other neurodegenerative diseases and hopefully recovery after therapies are identified. in recent years, we have been able to separate and characterize extracellular vesicles (evs) from several different viruses including hiv-1, htlv-1, rift valley fever virus and ebolavirus. however, to date it is not clear whether there is a timing difference between ev and virus release from infected cells. methods: ev isolation by nanoparticle capture and differential centrifugation, ev quantification by nanoparticle tracking analysis, western blot, rt-qpcr, virus rescue assay. results: we have attempted to address the kinetics of ev and virus release from multiple-infected cells using serum starvation experiments from infected (100%) cells. these infected cells were initially put in g0 quiescent stage using serum starvation. both supernatants and cell pellets were collected postinduction release (20% fbs + pma/pha) at 0, 3, 6, 12, and 24 hours and examined for the presence of ev, autophagy and viral proteins as well as viral rna expression. results from supernatants of uninfected cells showed a peak of tetraspanin proteins (cd63, cd81, and cd9) at 6 hours and a gradual decrease of all ev associated proteins by 24 hours. however, the ev from hiv-1 infected cells showed all three tetraspanins present at 3 hours and expression gradually increased up to 24 hours. when compared to htlv-1 infected cells, the three tetraspanin proteins peaked at 6 hours and expression continued to decrease up to 24 hours. htlv-1 infected cells also showed a unique pattern of cd81 expression. autophagy associated proteins (lc3a, lc3b and p62) from uninfected cells and htlv-1 infected cells plateaued at 6 hours, whereas in hiv-1 infected cells their expression continued to increase and peaked at 24 hours. hiv-1 viral proteins (p24, gp120, nef) expression was present at 6 hours and continued to increase and peaked at 24 hours. htlv-1 proteins (p19 and gp46/61) peaked at 6 hours and gradually decreased overtime. hiv-1 and htlv-1 rna gene expression analysis was performed, and data correlated with viral protein expression. additionally, evs release was quantified and showed significant increase of ev concentration overtime in both uninfected and infected samples. finally, experiments of infectivity from 6-and 24hour supernatants were performed on three naive cells. hiv-1 supernatant 6-hour sample was found not to be infectious. however, hiv-1 was successfully rescued from 24-hour sample. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = 31). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and 10 ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine 37 distinct uev surface markers of cd9+/cd63+/cd81+ vesicles in a multiplexed bead-based manner including respective controls. the accuri c6 (bd) was utilized for data acquisition. for further misev2018-compliant characterization, cd9+/cd63+/cd81+ uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd209, all other 36 surface markers could be identified. the most abundant markers were cd9 and cd63, which were detected in 93% of samples, followed by cd133/1 (84%), cd326 (81%), cd81 and cd24 (77%, each). cd3 (42%), cd9, cd45 (39%), cd49e (32%) and cd81 showed similar relative median fluorescent intensities (rmfi), while cd63 yielded significantly higher (p = 0.009) and all other markers significantly lower rmfi (p < 0.011). tem and f-nta revealed cup-shaped vesicles (137 ± 22 nm) with 8.8 ± 7.0 e + 10 particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg101, cd9, cd63 and cd81 without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a ,esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = 40) aged 40-70 yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a 3-colour panel, which included annexin v (for the majority of circulating evs), cd41 (for platelet-derived evs) and cd105 (for endothelial-derived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and 10-yr cvd risk detected by qrisk2. results: ev numbers, as determined by nta, were strongly associated with bmi (r = 0.602, p < 0.001), blood pressure (systolic bp: r = 0.359, p = 0.023; diastolic bp: r = 0.550, p < 0.001) and plasma triacylglycerol levels (r = 0.703, p < 0.001). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = 0.330, p = 0.038). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining 49.4% of the variance for total ev numbers. an additional 9.3% of the variance in total ev numbers was predicted by diastolic bp. ancova of the 10-yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with 10-yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd4 + t cells were isolated from secondary lymphoid organs of foxp3-rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th1, th2, and th17), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, 15-day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for 5 days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th1, th2, th17) showed either no change in proliferation (th1) or decrease in proliferation (th2, th17), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < 0.0001). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th1), il4 and il13 (th2), or il17a and il17 f (th17). in contrast, exposure of tregs to cdc-evs resulted in~1000-fold increase in expression of il10, a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il-10 in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il-10. this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t32hl116273. prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from 42 patients treated with prostanoids (study group; n = 42, 50 ± 16 years, 70% female) and 38 patients not treated with prostanoids (control group; n = 38, 55 ± 19 years, 65% female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; 0.5 mm), adenosine diphosphate (adp; 6.5 µm) and thrombin receptor-activating peptide (trap; 32 µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd61+ and cd62p+; pevs), leukocytes (cd45+, levs) and endothelial cells (cd146+, eevs) were measured in plateletdepleted plasma by flow cytometry (a60-micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = 0.01) and lower concentrations of pevs and levs (p ≤ 0.05). furthermore, thrombus formation was delayed (p ≤ 0.003) and thrombus size was decreased (p = 0.008) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd61+ pevs (p = 0.04). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ 0.05) and the concentrations of pevs (p ≤ 0.08). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ 0.04). introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from 17 cf children (<6 years of age). for nanoflow cytometry, evs were stained with di-8-anepps, and with epcam, cd66b and cd115 (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. results: the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = 0.003, spearman's rho = 0.689). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = 0.001, rho = 0.857). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv15a0), emory pediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: 142 individuals were enrolled into our study, subdivided into a training (volunteer n = 27, pneumonia n = 12, sepsis n = 28) and testing cohort (volunteer n = 20, pneumonia n = 18, sepsis n = 37). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq2) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative realtime pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed 29 significantly regulated mirnas in pneumonia compared to volunteers, and 25 mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by 12 mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: 0.982, pneumonia: 0.965, sepsis: 0.992). mir-193a-5p (log2fc = 1.86, padj = 1.49e-6) and mir-542-3p (log2fc = 1.67, padj = 3.29e-5) differentiated between pneumonia and volunteers and mir-1246 (log2fc = −2.41, padj = 1.78e-04) between pneumonia and sepsis. expression levels of mir-193a-5p and mir-1246 were related to disease severity. mir-542-3p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was 73.33%. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study,3healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd63 and cd81 were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and 9 mirnas (mir-103a-p, mir-191-5p, mir-320a, mir-200b-3p, mir-185-5p, mir-223-3p, mir-21-5p, mir-155-5p, let-7 g-5p) , were evaluated by rt-qpcr. after the optimization of the methodology, 10 healthy adults subjects and 10 patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd63 and cd81. however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna-320a showed a significant increased (p = 0.02) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir-320a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf2-active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds joseph kuhn a , absara hassan b , sonali sharma b , jennifer kwong b , montaha rahman b , salma adam b , jasmine lee b , alvaro villarreal ponce b and piul rabbani b a nyu langone health, new york, usa; b nyu langone health, new york, usa introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid 2-related factor 2 (nrf2) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf2-active exosomes, mscs were incubated with potent nrf2 activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express >95% positive msc markers (cd44, cd73, cd90, and cd105) and <5% express negative markers (cd45, cd31, cd14, cd19, or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd81, cd9 and tsg101. nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of 168.0 ± 6.5nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf2-active human mscs increase exosome secretion by 54%, compared to nrf2-baseline mscs (p < 0.05). both nrf2-baseline and nrf2-active exosome treatment significantly reduced closure time to 15.5 and 14 days respectively, compared to 29.8 days for vehicle-treated wounds (p < 0.05). this reduction eliminated the delay in closure time compared to wounds of c57/b6 mice. nrf2-active exosome treatment of db/db wounds reduced closure time by a further 2.6 days compared to untreated c57/b6 wounds. at day 10, exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd31+ vessels compared to vehicle-treated wounds. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist1 is a key mediator of tumour cell metastasis.. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist1. methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc3-ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc3-ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc3-ml lines stably overexpressing or deficient in twist1. results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc3-ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc3-ml overexpressing twist1 showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist1 expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. funding: american heart association of12 symposium introduction: as researchers continue to explore the therapeutic potentials of extracellular vesicles (evs) for the treatment of many diseases, there is a growing unmet need for real-time in vivo monitoring of these therapeutic evs after they are injected into a subject to understand their safety, targeting, and effectiveness. while current optical imaging solutions like bioluminescence and fluorescence are useful for ev tracking studies in animal models, there is limited utility in clinical applications. here we present a novel ev tracking solution utilizing clinically applicable mri technology. methods: to generate trackable evs, cells were labelled with a clinically applicable novel magnetic agent. evs secreted by the labelled neural stem cells and amniotic fluid stem cells (afscs) were isolated by differential ultracentrifugation. the viability and morphology of labelled-cells were evaluated, and the in vitro mr properties of their derived evs were analysed by magnetometer. a proof of concept in vivo biodistribution study was conducted by injecting labelled evs into wt and alport mice (a model of chronic kidney disease) via retro-orbital and intra-cardiac routes and tracking them via mri at 10 min and 3 hr postinjection. results: the magnetic label did not affect the physiological characteristics of the cells. the mr detectability of labelled-evs was confirmed by in vitro/ex vivo mri phantoms. mri studies showed that homing of afsc evs to the kidney injected intra-cardiacally into alport mice were more efficient versus the retro-orbital route, and prussian blue staining of kidney sections confirmed the mr findings. introduction: a central question in ev biology is the fate of circulating ev. this can be evaluated by developing non-invasive ev bioimaging techniques in mice in order to benefit from transgenic and knock-out models. recent reports described ev biodistribution in vivo using optical (fluorescence) and nuclear imaging. but the physicochemical properties of the probes impact ev integrity, labelling efficiency, background signals and observation timecourse. methods: we developed the radiolabeling of red blood cells (rbc) and ev with [18 f]fluorodeoxyglucose (18 f-fdg). we used rbc-derived ev in their native, intact form, without pre-experimental processing (no centrifugation or filtration). we tracked 18 f-fdg in vivo by pet-scan, within seconds of ev, rbc or free 18 f-fdg injection, and during their dissemination in blood and recruitment by organs over one hour. ev and rbc biodistribution were confronted to the kinetics of free 18 f-fdg. results: we collected images of the biodistribution of rbc, and rbc-derived ev. nuclear imaging was well suited for accurate studies of ev organotropism, with high sensitivity, excellent signal-to-noise ratio, very low signal absorption by tissues and an inherent quantitative tomographic nature. ev-specific signals were mostly accumulated within minutes of injection (tail vein), in the spleen and liver, with a small part in the bone marrow (femurs). signals in other compartments were largely transient and linked to tissue perfusion and blood volume. we selected the most drastic control conditions to secure a correct interpretation of the data. this made kidneys, hearts and brains unavailable for analysis. hence the new approach came with limitations, but we describe how "free" 18 f-fdg signals can be used to draw sound conclusions for ev. summary/conclusion: we propose that three types of compartments coexist in control mice at rest: active ev-capturing organs with high capacity and specificity including the spleen, and to a lesser degree the bone marrow; passive ev-retaining organs with high capacity, including the liver; and ev-neutral organs where transient signals only mirror tissue perfusion. we also report how ev biodistribution patterns are altered in ageing animals, as an example. we hope that this novel, non-invasive, quantitative, dynamic wholebody imaging approach will help characterize native cell-derived ev and help set standards for the reproducibility of ev bioimaging in mice. funding: frm grant "biface", inserm copoc, cnrs. introduction: extracellular vesicles (ev) are important mediators of intercellular communication; however, basic principles of ev biogenesis and loading remain largely unknown. a limited repertoire of tools has thus far made these processes challenging to research. the development of an ev-transfer reporter in a genetically tractable organism such as drosophila has allowed us to study mechanisms of cargo loading in vivo and has provided us with a platform to explore fundamental aspects of ev biology. methods: we have developed a bioinformatic pipeline to analyse the properties embedded in the 3ʹutr of mrnas enriched in evs released by drosophila cells. in parallel, we have adapted a cre-loxp system for use in fruit flies that appears to be proficient to reveal the exchange of bioactive molecules between secretory and recipient cells. results: taking advantage of computational methods, we uncovered sequence motifs that preferentially appear in combinations along the 3ʹutr. these sequence motifs occur within characteristic secondary structures, in a way that is more variable and motif dependent than previously reported. identified motifs also show similarities to known binding sites for rna binding proteins; a feature potentially important for ev-loading. in parallel, we developed a drosophila in vivo system to detect cell communication in complex tissues and between different cell types. using this system, we studied the biological significance of specific sequence motifs and identified their ability to modulate mrna ev-transfer in a context dependent and evolutionarily conserved manner. summary/conclusion: in summary, we have developed a novel tool to study cell communication in complex tissues, and shown its effectiveness to study principles of ev biogenesis and loading. beyond improving our understanding of ev biology and providing a novel tool to the scientific community, we hope this knowledge will pave the way to harnessing evs as a means of remotely manipulating cell communication in many biological contexts. introduction: the idea of cross-kingdom, species and inter-individual transfer of bioactive compounds via extracellular vesicles (evs) is a recent avenue. however, the bioactivity and bioavailability of these dietary compounds upon consumption is highly debated. it has been proposed that evs from diet can absorbed by consuming organisms, be bioavailable in various organs and exert phenotypic changes. milk is the most vastly consumed beverage and is an abundant source of evs that may act as signalosomes. whether these milk-derived evs can serve as cross-species messengers and have a biological effect on host organism has been poorly understood. methods: bovine milk-derived evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. the evs were characterised by tem, nta, quantitative proteomics and rna-seq. evs were orally administered to various mice models of colorectal, breast and pancreatic cancer. primary tumour burden was monitored, and the rate of metastases was measured by imaging and qpcr. immune cells were analysed by facs. mechanistic insights were obtained using quantitative proteomics, confocal microscopy and biochemical experiments. results: we demonstrated that upon oral administration, bovine milk-derived evs were able to survive the harsh degrading conditions of the gut and be bioavailable in peripheral tissues. interestingly, oral administration of milk-derived evs reduced the primary tumour burden in various cancer models and attenuated cancer cachexia. intriguingly, despite the reduction in primary tumour growth, milk-derived evs accelerated metastasis in breast and pancreatic cancer mice models. timing of ev administration was critical as oral administration after resection of the primary tumour reversed the pro-metastatic effects of milkderived evs in breast cancer. biochemical and quantitative proteomics analysis highlighted the induction of epithelial-to-mesenchymal transition and senescence upon treatment with milk-derived evs. summary/conclusion: taken together, we were able to demonstrate the capacity of bovine milk-derived evs in mediating cross-species communication and regulating cancer progression in a context-dependent manner. bacterial membrane vesicles (mvs)a bacterial innate defence system against viral infection xiaomei yan, qian niu and ye tian xiamen university, xiamen, china (people's republic) introduction: in order to survive the constant onslaught of phage, bacteria have evolved diverse defence mechanisms that act at every stage of the phage life cycle. it has been suggested that bacterial membrane vesicles (mvs) may play a key role in innate bacterial defence against phage infection by acting as a decoy to prevent phage adsorption. nearly a decade has passed since mvs were first proposed as a decoy, but details of how bacteria utilize mvs to defend against phages remain poorly understood. here we use the laboratory-built nano-flow cytometer (nfcm) to reveal details of the interaction between mvs and phages at the single-particle level, and to provide new insights into innate defence mechanisms of mvs. methods: s. typhimurium was used as the model system. differential ultracentrifugation and density gradient centrifugation were used to isolate and purify mvs and bacteriophage p22. cryo-tem was used to determine the morphologies of mvs and phage p22. the purity of mv isolates was validated by measuring the particle concentration before and after triton x-100 treatment. monodisperse silica nanoparticles were used as the size reference standards to measure the size distribution of mvs via single-particle light scattering detection. the purity of phage p22 was verified by concurrent detection of side scatter and fluorescence signals of single phages upon nucleic acid staining by syto 82. results: by incubating mvs and af532-labelled p22, the number of phages adsorbed on single mvs were accurately quantified. we found that s. typhimurium and mvs it secretes express different affinity for phage p22 attachment. the binding ability of p22 to mvs is greater than that of bacteria. we confirmed that p22 can inject their nucleic acids into mvs, and these nucleic acids can be degraded by non-specific nucleases inside mvs for the first time. besides, by labelling the nucleic acids of mvs with syto 82, we were able to distinguish three different subpopulations of mvs. summary/conclusion: taking advantage of the superior sensitivity of nfcm in single-particle analysis, we developed a novel approach to the characterization of the interaction between mvs and phages. our study revealed that bacteria produce mvs as bait to attract viral adsorption and nucleic acids injection. funding: this research was supported by the national natural science foundation of china (grants 21934004, 21627811 and 21475112). introduction: the development of evs for therapeutic applications requires an in-depth understanding of their in vivo biodistribution and pharmacokinetic profile. in this study, we have made a comprehensive comparison of nuclear, fluorescent, and bioluminescent imaging technologies to identify the most suitable in vivo ev tracking method. methods: evs were purified from expi293 f cell supernatant by differential centrifugation followed by iodixanol density gradient separation and further characterized following misev guidelines. engineered expi293 f cells were used to generate evs carrying mcherry or nanoluc (nluc) proteins. the membrane of naïve ev was labelled with indium111(in111)-dtpa or xenolight dir post-ev isolation. ct26 tumour-bearing balb/c mice were intravenously dosed with 1 × 1011 evs followed by imaging at 1 h, 4h and 24h using spec/ct and ivis systems. tissue distribution and blood circulation profile of evs were analysed from ex vivo samples up to 24h post-injection. results: xenolight dir and (in111)-dtpa were the most suitable ev labels for live whole-body animal imaging, ex vivo organ imaging, and tissue lysate quantification. nluc was appropriate for ex vivo imaging and tissue lysates quantification, but suboptimal for live imaging with limited sensitivity. mcherry evs were found not suitable for in vivo tracking studies due to high background signal fluorescence. ex vivo organ quantification of in111-dtpa and dir showed that naïve evs mainly accumulate in liver, followed by spleen, kidneys, and lungs at 24h post-dose, with less than 5% ev exposure to the tumours. interestingly, nluc-evs accumulated mainly in the lungs, regardless of the small size of the particles injected and the absence of aggregation. blood circulation profile of in111-dtpa and nluc evs showed rapid clearance of vesicles from circulation, with 15% of injected dose detected in blood after 30 min and less than 5% after 16 h. summary/conclusion: radionuclide imaging is an excellent technology to detect evs in vivo and ex vivo with high resolution and sensitivity but requires advanced infrastructure for radiolabeling. the optical methods have limited tissue penetration and sensitivity but can be improved with the right selection of the dye. these results contribute to the understanding of the biodistribution and pharmacokinetics of evs and are highly relevant to exploiting their potential for targeted delivery to diseased tissues in vivo. symposium introduction: new methods for quantifying extracellular vesicles (evs) in complex biofluids are critically needed. we report the development of a new technology combining size exclusion chromatography (sec), a commonly used ev purification technique, with fluorescence detection of specifically labelled evs (flu-sec). methods: flu-sec was validated using red blood cell derived evs (revs). size and concentration measurements were performed by microfluidic resistive pulse sensing (mrps) using the ncs1 instrument (spectradyne llc, usa). pe-cd235a (anti-glycophorin a) and alexa647-wga (wheat germ agglutinin) were used to label revs. flu-sec experiments were performed on a liquid chromatography system using a tricorn 5/200 glass column filled with sepharose cl-2b gel (ge healthcare). results: a log-normal size distribution was obtained for revs with a mean diameter of 163.5 ± 0.7 nm and standard deviation of 30.6 ± 0.6 nm. the concentration of revs measured by mrps was 3.14*10e11 particles/ ml. the fluorescence chromatograms of the rev samples labelled with pe-cd235a and with alexa647-wga show the typical features of the separation of evs from soluble proteins with sec and enables the determination of the labelling efficiency of the markers. the linear range for quantification of evs in our experiments spans over two orders of magnitude ranging from 10e9 particles/ml to 10e11 particles/ml. the lod depends on the type of the label. in our experiments the lowest lod was 10e9 particles/ml for alexa647-wga. summary/conclusion: the results indicate that flu-sec is a quantitative technique with very good linearity over a wide range of concentrations, though the limit of detection depends largely on the employed label (sci. rep. 9, 19868, 2019) . moreover, the ratio of ev-bound and free-antibody molecules can be also determined by flu-sec, which can be used to calculate the labelling efficiency of the used marker. funding: this work was supported by the national research, development and innovation office (hungary) under grant numbers pd 121326 and nvkp_16-1-2016-0007. zv was supported by the janos bolyai research fellowship. the conan assay: purity grade and concentration of ev microlitre formulations by colloidal nanoplasmonics. (evs). control over such properties is constantly experienced by researchers to be critical for ev proper manipulation, engineering and translation. however, the need for characterization methods that strike the balance between robustness, working volume, cost and accessibility remains unmet. methods: the colorimetric nanoplasmonic (conan) assay we developed consists of a solution of gold nanoparticles (aunps) into which 1-2 μl of the ev formulation is added. the solution turns blue if the formulation is pure, while stays red if soluble exogenous single and aggregated proteins (saps) are present. the colour shift is visible by the naked eye and can be quantified by conventional uv-vis spectroscopy, providing a quantitative index of purity and an estimation the ev molar concentration (particle number). results: the assay specifically targets saps, and not the ev-related proteins, with a detection limit < 50 ng/μl (an order of magnitude higher resolution than the bradford protein assay). for pure solutions, the assay also allows for determining the ev number, as the colour shift is linearly dependent to the aunp/ev molar ratio. instead, it automatically reports if the solution bears sap contaminants, thus avoiding counting artefacts. experiments, conducted on ev separated from milk and ascaris suum culture medium, are repeatable, with an error below 20%. summary/conclusion: conan proves to be robust and reliable, while displaying appealing performances in terms of cost (inexpensive reagents, run by standard microplate reader), working volumes (1-2 μl) and time (the procedure takes less than one hour). the ability to assign a quantitative purity grade is, up to date, a unique peculiarity of this assay. finally, the assay is potentially extendable to all classes of natural and artificial lipid micro-and nanoparticles. funding: evfoundry project, horizon 2020 -future and emerging technologies (h2020-fetopen), id: 801367. marina cretich a , roberto frigerio b , alessandro strada b , greta bergamaschi b , marcella chiari c and alessandro gori c a consiglio nazionale delle ricerche (cnr), istituto di scienze e tecnologie chimiche (scitec), milano, italy; b consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy; c consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy introduction: small extracellular vesicles (sev) present fairly distinctive lipid membrane features in the extracellular environment. these include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sev membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. here we introduce the use of membrane sensing peptides as new, highly efficient ligands to directly integrate sev capturing and analysis on a microarray platform. methods: we designed and synthesized membranesensing peptide ligands as molecular baits for small ev and we demonstrate their use in a microarray platform as valuable alternative/complement to antibodies. evs from blood serum and plasma were isolated by ultracentrifugation, characterized by tem, nta, wb. samples were analysed by label-free, single particle counting and sizing on peptide microarrays coupled to fluorescence co-localization immune staining with fluorescent anti-cd9/anti-cd63/anti-cd81antibodies. results: peptide microarrays were realized using a click-chemistry strategy for optimal peptide surface orientation and used to analyse evs from human blood. membrane sensing peptides showed a capturing capacity higher than anti-tetraspanin antibodies. in addition to purified vesicles, peptide ligands were tested with pure serum showing capacity to capture evs even from complex samples. in order to get insights into the ev-peptide binding mechanism and verify whether it is directly mediated by the lipid membrane, trypsin-treated evs were captured on peptide microarrays demonstrating that binding is not directly mediated by surface associated proteins. summary/conclusion: we introduced the use of membrane sensing peptides as a novel class of molecular ligands for integrated sev isolation and analysis, reporting for the first time on peptide microarrays for extracellular vesicles. given their affinity to the membrane of small ev, these molecules can serve as general baits, enabling vesicles capturing unbiased by differential surface protein expression. these new class of molecular probes may be integrated with the use of protein markers towards improved small ev isolation and characterization. compared to proteins and antibodies, peptides are characterized by low cost of preparation, remarkable stability and ease of chemical manipulation, offering virtually unlimited possibilities for experimental design. we anticipate that this new class of ligands, may greatly enrich the molecular toolbox for ev analysis. funding: hydrogex (regione lombardia&fondazione cariplo, grant n. 2018-1720) and index (european union's horizon 2020 research and innovation programme under grant agreement n°766466) projects are acknowledged for partial financial support. high-resolution size-based profiling and morphological analysis of extracellular vesicles by scanning electron microscopy sara cavallaro a , federico pevere b , petra hååg c , kristina viktorsson c , rolf lewensohn c , jan linnros a and apurba dev b a kth royal institute of technology, stockholm, sweden; b uppsala university, uppsala, sweden; c karolinska institute, stockholm, sweden introduction: extracellular vesicles (evs) have been found to mediate intercellular communication in physiological and pathological processes. nevertheless, the understanding of evs bio-functionality remains elusive, mainly because of their high heterogeneity in molecular content, but also in size (30-2000 nm) . therefore, accurate size measurements of evs are highly desired, particularly for exploiting their full diagnostic/therapeutic potential. currently available techniques, such as nanoparticle tracking analysis (nta), cannot accurately measure evs smaller than 70 nm and are not capable to distinguish them from protein aggregates. on the contrary, electron microscopy (em) techniques allow high-resolution size-profiling and morphological analysis of evs over their whole size range. however, their low throughput combined with several long preparatory steps have prevented em from being routinely used for ev size profiling. methods: we shall present a method improvement in throughput and reproducibility of ev size-analysis by scanning em (sem). the technique is based on covalent ev capture onto a silicon wafer, using the protocol reported by cavallaro et al. up to the glutaraldehyde step. after immobilization, critical point drying (cpd) is performed to dehydrate evs before sem, while preserving their shapes. results: sem images, showing the comparison in densities of evs prepared by covalent and non-covalent coupling to substrate, indicated a good capture efficiency of our covalent protocol. the size distribution analysis showed good agreement between nta and sem for evs >80 nm. for smaller evs, sem is more sensitive than nta, thus more suitable to check the purity of ev-isolation techniques. last, atomic-force microscopy (afm) measurements was also used to validate our measurements. introduction: extracellular vesicles (evs) are membrane vesicles secreted into extracellular space, by almost all cellular populations, playing a major role in cell-to-cell communication. it has been already demonstrated that changes in luminal or surface protein cargos of these vesicles, may reflect the status of producing cells. for this reason, evs are considered as potential biomarkers in several types of diseases ranging from cancer diagnosis to heart rejection. periostin (postn) is a matricellular protein associated with evs, and its level is considered a possible biomarker, which indicate malignancy and poor clinical outcome in different types of cancer. here we extensively characterize the presence of postn associated on evs, showing how different isolation methods can drastically affect the amount of postn content in extracellular vesicles fraction. methods: serial ultracentrifugation steps or size exclusion chromatography were used to isolate evs from primary culture of cardiac progenitor cells. evs were characterize, according to misev guidelines, by western blot, nta, facs and cryotem analysis methods. postn amount, associated with evs, was analyses by western blot and elisa. furthermore, functional tests were performed on h9c2 cardiomyoblast cell line, treated with the same amount of evs from different isolation methods; cells response were analysed by western blot. results: evs, from both the isolation methods, showed tsg101, syntenin1, cd63 positivity while grp94 was absent. nta showed no differences, in terms of amount and size of particles. by facs analysis evs resulted enriched with cd63, cd9 and cd81. cryotem showed a similar morphology in the two preparations with presence of protein contaminant in the ultracentrifuge pellet. in vitro, h9c2 treated with evs showed activation of pfak after 10ʹ of treatment, this induction was 1.5 times higher in cells treated with evs isolated with ultracentrifuge compared to evs isolated with sec, confirming a drastic effect of postn protein contamination. furthermore, by phospholipase-c treatment, we found that postn is bound to evs surface through a gpi anchor. summary/conclusion: these results suggest that selection of a proper isolation method is critically relevant in evs studies, in particular when protein analysis is considered. different isolation methods dramatically influence protein amount in extracellular vesicles and consequentially their function. furthermore, in this study we show for the first time, that postn is actually bound to evs surface and not carried in their lumen as previously believed. members of the y-rna family have been detected in ev from various cell types and are among the most abundant non-coding rna types in plasma. we previously showed that shuttling of full-length y-rna into ev is modulated by tlr-activation of ev-producing immune cells. this suggested that y-rnas may have potential as biomarker for immune-related diseases. methods: we separated rna-containing structures in plasma based on differences in size, density, and resistance to protease/rnase treatment. using rt-qpcr, we quantified full-length y-rna subtypes (y1, y3, y4) in ev from various blood-related cell types cultured with or without lps-stimulation. inflammationinduced changes in y-rna were assessed in plasma samples from a human endotoxemia study. results: full-length y-rna in plasma was mainly found in ev (early sec-fractions, density 1.11-1.18 g/ml). in contrast, specific mirnas were either enriched in lpp (e.g. mir-122), in both ev and lpp (e.g. mir-16 and mir-21), or in ev (e.g. mir-150). evenclosed full-length y-rna was resistant to enzymatic degradation, while lpp-bound mirnas were degradation sensitive. we discovered that ev released by different blood cell types varied in y-rna subtype ratios. these ratios remained stable upon lps-stimulation of the ev-producing cells. in endotoxemia plasma samples, the neutrophil-specific y4/y3 ratios and pbmcspecific y3/y1 ratios changed significantly during systemic inflammation. importantly, the plasma y-rna ratios strongly correlated with the number and type of circulating immune cells during the inflammation process. summary/conclusion: cell type specific "y-rna signatures" in plasma ev can be determined without prior ev-enrichment, and may be further explored as biomarkers to diagnose inflammatory responses or other immune-related diseases. mining public ev small rna-seq data with mirev -insights into potential reference transcripts and abundant mirnas recently, extracellular membrane vesicle (mv) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. s. aureus produce membrane-bound, spherical, nano-sized, mvs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. the present study sought to examine the nature of the association between nucleic acids and mvs produced by s. aureus. we also sought to analyse the immunostimulatory potential of mvassociated rna and dna, and to evaluate receptormediated recognition of mv-associated rna and dna molecules by innate immune cells. methods: by following a stringent purification protocol, we characterized the rna and dna content of mvs produced by actively growing s. aureus. nuclease protection assays were performed to determine whether mv-associated nucleic acids are protected from degradation. we assessed the immunomodulatory potential of mv-associated rna and dna by treating cultured mouse macrophages with mvs and measuring the induction of interferon-β mrna using qpcr. introduction: urinary extracellular vesicle (uev) transcriptome could potentially reflect the kidney gene expression profile and serve as virtual/liquid biopsy. in order to explore this possibility, we performed mrna sequencing of uevs from individuals with type 1 diabetes to assess whether it can capture a "kidney enriched genes" expression signature that could lead to novel biomarker discovery for diabetic kidney disease. methods: the study included 75 type 1 diabetic individuals (41 normoalbuminuric, 15 microalbuminuric and 19 macroalbuminuric). urine samples were collected either overnight (n = 38) or during 24-hours (n = 37) and uevs were isolated from 20-30 ml of urine by differential centrifugation. the evs quality was ensured by electron microscopy (em), western blotting and ev-rnasprofiling with the bioanalyzer. isolated rnas were subjected to rna sequencing after cdna library preparation (ultra-low amount protocols) using hiseq 2000 (illumina) pair-end protocol. the association between kidney specific gene expression levels (>4 fold higher compared to other tissues, n = 53) and degree of albuminuria or glomerular filtration rate was explored. results: isolated ev quality appeared good by em and western blotting. rna quantity and quality were sufficient for sequencing of all samples with >5 million pair end reads. we detected on average expression of 12,642 genes. principal component analysis (pc) of the expression of all genes did not reveal any systematic batch differences between the overnight and 24-hour urine collections. comprehensive look-up of 53 kidney-enriched genes revealed expression of >73% (total 39) of these genes in urine evs with high expression of five kidney-specific genes (slc12a3, slc12a1, nphs2, aqp2 and slc22a12). pc analysis combining the impact of 39 kidney-enriched genes revealed that most macroalbuminuric patients clustered together along the pc1 axis, and the axis also correlated with the albumin-to-creatinine ratio (p = 0.0004) explaining 11% of the variance (p = 0.005) in the whole data set. the pc1 axis also showed correlation with hba1c (p = 0.003), but not with diabetes duration, bmi, age and egfr. introduction: due to their safety profile, tissue tropism and long-term transgene expression, adeno-associated viruses (aavs) have become the vector of choice for human gene therapy. however, pre-existing neutralizing antibodies (nabs) to many aav serotypes pose a critical challenge for the translation of gene therapies to clinic. here, we describe the use of exosomal aavs (eaav) as a robust cardiac gene delivery system that enhance transduction efficiency while shielding from pre-existing humoral immunity to the viral capsid. methods: we developed an ultracentrifugation-based purification strategy to obtain eaav specimens from aav-producing hek-293 t cells, and used electron microscopy-based visualization, confocal microscopybased colocalization studies, qpcr, immunoblotting, dynamic lights scattering, exoview technology and protease assays to characterize eaav morphology, contents and mechanism of action. we then evaluated efficiency of heart targeting for eaav9 or eaav6 and standard aav9 or aav6 encoding for egfp, mcherry or firefly luciferase in different human cell lines in vitro, in black mouse and in passive immunity nude mouse model in vivo using flow cytometry, confocal microscopy, langendorff perfusion system and methods: hlhs patients (n = 3) after glenn procedure and swine (n = 3) after pab were given rv injections of allogeneic/xenogeneic mscs. donor-specific, hla-i+, exosomes were isolated from plasma. in swine, exosomes were collected and rv fractional area change (fac) was measured post-msc-injection. in the elpis patients, exosomes were collected and outcome measurements (fac, stroke volume (sv), rv mass) were recorded 6 and 12-months post-injection. exosomal mrna, microrna (mirna), and proteins were quantified and partial least squares regression (plsr) reduced the dimensionality of the datasets to build a swine model, upon which elpis outcome predictions were made. results: multiomics analysis of swine exosome cargo revealed mirna to be the largest contributor to overall variance. in swine and elpis patients, mirnas were similarly expressed (98%, fold-change<2). plsr reduced the dimensionality of the swine mirna dataset to 50 mirnas with the highest weighted coefficients for changes in fac. pathway analysis of mirna targets revealed links to smooth muscle cell proliferation and cardiac chamber development. importantly, the swine mirna plsr model predicted elpis patient improvements in fac, sv, and rv mass with strong correlation (r > 0.9). summary/conclusion: these findings support the use of: (1) swine pab model for rv failure in hlhs, (2) circulating donor-specific msc-exosomal mirna as a novel, non-invasive biomarker of patient outcomes, and (3) introduction: evs have been shown promising potential as a drug delivery vehicle, especially nucleic acid therapeutics. however, the overall short of specificity to target cancer cells has led to low therapeutic efficacy and potential toxicity. rna nanotechnology is the bottom-up self-assembly of nanometre-scale rna architectures. we previously discovered a stable phi29 prna three-way junction (3wj) motif and used it to construct multivalent rna nanoparticles with high chemical and thermodynamic stability. the resulting arrow-shape rna nanoparticles are homogenous, uniform in size and shape, and can harbour different functionalities while retaining their tertiary folding and independent functionalities both in vitro and in vivo. this flexible platform using rna nanotechnology to achieve tumour-specific targeting has been demonstrated over the last decade. here we introduce a strategy to take advantage of both evs and rna nanotechnology to develop a versatile platform for efficient target-specific delivery of sirnas for cancer treatment. methods: we design membrane-anchoring arrowtail 3wj rna nanoparticles to display tumour targeting ligand (psma rna aptamer or egfr rna aptamer or folate) on birc5 sirnas loaded evs (fig.1 ). nanoparticles were characterized by nanoparticles tracking analysis (nta), transmission electron microscopy (tem), dynamic light scattering (dls) and atomic force microscopy (afm). evs were produced by hollowfiber bioreactor and purify by tangential flow filtration (tff) follow by ultracentrifugation. cell binding were evaluated by flowcytometry and confocal microscopy and gene knockdown effect were assay by quantity reverse transcription-pcr (qrt-pcr). formulated evs were introduced to tumour (prostate, triple negative breast cancer, colon pdx) xenograft mice by tail-vein injection and evaluate in vivo tumour inhibition. results: 1) we found the orientation of arrow-shaped rna can be used to control ligand display on evs membranes for specific cell targeting. 2) by placing membrane-anchoring cholesterol at the tail of the arrow results in display of rna aptamer or folate on the outer surface of the evs and enhance cancer cell binding and uptake. 3) taking advantage of the rna ligand for specific targeting and evs for efficient cytosolic delivery, the resulting ligand-displaying evs or plant derived evs-like nanovesicles were capable of specific delivery of sirna to cells, and efficiently blocked tumour growth in three cancer models. summary/conclusion: we developed an rna-evs based nanoparticles platform and shown the flexibility for different cancer type treatment. related publications: pi f et.al. nature nanotechnology. 2018 , 13:82. li z et.al. sci rep. 2018 introduction: extracellular vesicles (evs) contain plasma membrane surface markers that provide insights into their cell source. until now, our understanding of the circulating ev-biome has been limited by the lack of celland size-specific ev quantitation methods. we have developed and validated a multiplex nanoscale flow cytometry approach to image cell-and size-specific ev populations using a novel human "ev-lyoplate" with 3 differently coloured monoclonal antibodies per well in a 96 well plate format (n = 242 separate antibodies with isotype, stained pbs, unstained plasma, and quant-beads controls per plate). we hypothesized that platelet poor plasma samples from patients diagnosed with pancreatic cancer would have significantly different ev-biome profiles than screen negative study subjects. methods: study subjects were enrolled and sampled before clinically scheduled endoscopic ultrasoundguided biopsy (eus-fna) procedures to screen patients with symptoms of pancreatic duct obstruction who later had at least two years of clinical follow up, including surgical resection in cases of pancreatic neoplasia (n = 36) or at least one follow up clinic visit to confirm resolution of symptoms. blood samples were uniformly collected, processed, and banked per isev recommended guidelines. uniform machine (facsymphony) settings to standardize light scatter and fluorescence detection were based on commercially available beads (eg. megamix). samples were coded and randomized for testing and results were reported as the mean cell-and size-specific ev events/ul of plasma. results: clinical outcomes confirmed 10 cases of cancer and 26 screen negative controls. principle component analysis suggested that a number of different celland size-specific evs were significantly more common in the cancer cases (adjusted p-value < 0.05, with aucs >0.80), including epcam+/cd63+ events likely from cancer cells and cd41+/cd62p+/cd9+ microvesicles from platelets, among others. summary/conclusion: in this proof of principle study employing an ev-lyoplate design and nanoscale flow cytometry, we could reliably discriminate the ev-biomes in patients with cancer from negative controls. ongoing studies will determine whether these discriminators will be validated in larger cohorts and provide at least noninferior predictive value compared with the current gold standard clinical testing assay (eus-fna introduction: small cell lung cancer (sclc) is an aggressive tumour type, usually metastatic at diagnostic leading to poor overall survival. interestingly, sclc tumours are composed by distinct subpopulations of cells that cooperate as an ecosystem to drive tumour survival. since the subtype of sclc may have prognostic significance, the aim of this study was to identify surface marker proteins as biomarkers of sclc. methods: a linear discriminant analysis (lda) model, implemented in python via sci-kit learn, was used to choose the best 4 markers for distinguishing subtypes. this analysis was based on rna-seq data from a previous study. in order to identify ev-based biomarkers that would identify sclc evs and not normal evs, we excluded from this analysis proteins without a verified transmembrane domain and proteins associated with evs expected to be present in white and red blood cells, and endothelial cells (according to exocarta and vesiclepedia databases). we also prioritized proteins that could be pan markers for sclc and that might have prognostic significance. to validate our findings, we performed western blotting and flow cytometry in sclc cell lines from different subtypes. results: our rna analysis indicated that the best 4 surface markers to distinguish sclc subtypes were ceacam5, fam174a, lrfn1, epha2. immunoblot analysis validated ceacam5 and epha2 but not fam174a or lrfn1. we also found that ncam1, a commonly used sclc marker, only marks some of the subtypes. for further analysis, we chose proteins with antibodies validated for flow cytometry as our chosen biomarker platform. flow cytometry analysis of cd24 is suitable as a pan-sclc marker. however, the expression of non-ne cell lines was decreased compared to rna-seq data. summary/conclusion: protein analysis of ceacam5 and epha2 corresponded to rna-seq data. ncam was not detected as a pan marker for all sclc subtypes. however, we could see cd24 expression in all sclc subtypes, indicating it may be a useful pan marker for sclc. future studies will be performed to validate the expression of other surface markers in cells, purified evs, and plasma of sclc patients. funding: nih u01 ca224276 and nih u54 ca217450. leukobiopsyexploiting extracellular vesicle-mediated leukocyte sequestration of cancer-specific signatures introduction: in cancer, extracellular vesicles (evs) act as a unique exit mechanism for mutant and oncogenic macromolecules (proteins, rna and dna) en route from malignant cells to blood1. while this process has inspired major liquid biopsy efforts, the biology of circulating evs that carry oncogenic mutations (oncosomes)2 is still poorly characterized. it is also unclear what part (if any) of the tumour-related cell free dna (ctdna)3,4, a major liquid biopsy analyte, is linked to circulating evs and what is their fate, receptacles and biological activity. methods: we employed as series of cancer cell lines carrying mutations in major oncogenes (hras, her2, egfrviii). ev-dna was analysed by digital droplet pcr (ddpcr), along with nuclear anomalies in donor cells (dapi, electron microscopy) and transfer of dna to recipient cells of endothelial (huvec, mmbec), astrocytic (nha) or myeloid (hl60) origin. blood underwent fractionation into red blood cells (rbc), white blood cells (wbc), platelets (plt), evs (100,000g ultracentrifugation) and soluble plasma (sup)5. results: hras-mediated cellular transformation (in ras-3 cells) triggers profound changes in the structure of nuclear chromatin, which is driven into the cytoplasm and released as cargo of evs. oncogenic dna is detectable in blood fractions of tumour bearing mice. while evs, ctdna and plts contain intermediate levels of mutant dna, rbcs contain only traces of this material. the highest hras copy number per ml of blood is found in wbcs (monocytes and neutrophils), which contain more cancer dna/cell than liver, spleen and bone marrow. depletion of neutrophils using anti-ly6g antibody results in an increase in ev-and ctdna-associated mutant dna in blood, suggesting the role of these cells in regulating the circulating levels of cancer cell-derived particles. uptake of dna-containing evs impacts the phenotype of myeloid cells, which adopt thrombo-inflammatory properties. these cells also retain cancer-specific transcripts and other cargo. finally, normal astrocytes treated with oncogenic evs also exhibit phenotypic changes and signs of genomic instability including formation of micronuclei. summary/conclusion: we propose that the process of leukocyte sequestration of circulating particles containing tumour-related nucleic acids renders these cells potentially usable as a novel liquid biopsy platform (leukobiopsy) in cancer. introduction: early diagnosis of colorectal cancer (crc) and precancerous adenoma patients is of vital importance. previously we profiled small extracellular vesicles (sevs) derived mirnas isolated from plasma, proposed a new promising biomarker category of crc patients. here we further gave a full landscape of circulating sevs derived rnas to explore and evaluate sevs based rna biomarkers for early detection of both crc and adenoma patients. methods: plasma sevs were isolated from 60 participants, including 31 early-stage crc patients, 19 adenoma patients, and 10 normal controls (nc), and characterized according to misv2018 guideline. the total sevs derived rna expression profile of all participants was investigated by next-generation sequencing (ngs). weighted gene coexpression network analysis (wgcna) was performed to categorize differentially expressed rnas, and t-distributed stochastic neighbour embedding (tsne) was adopted to distinguish crc, adenoma, from nc samples with the top-ranked genes in wgcna modules. rt-qpcr validation was performed in a cohort of 120 additional participants. results: a total of 1615 rna species (including mirnas, mrnas, and lncrnas) were found differentially expressed between plasma sevs in crc and nc participants. additionally, 888 rna species were differentially expressed between plasma sevs in adenoma and nc participants. 1160 rna species were differentially expressed between plasma sevs in crc and adenoma participants. wgcna categorized all rnas into 6 modules, which exhibited different expression trends during the carcinogenesis of crc. a 60-gene combined tsne model consists of the top 10 genes in each module could perfectly classify crc, adenoma, and nc samples. a 6-gene combined tsne model consists of the top 1 gene in each module could roughly distinguish crc and adenoma from nc, with only 1 sample misclassified. rt-qpcr assays also confirmed the potential classification ability of those genes in another validation cohort of 120 participants. introduction: although the concept of systematic "liquid biopsy" using bodily fluids is simple and elegant, the path of clinical reality has been challenging. recently, numerous tissue-specific biomarkers have been discovered in evs derived from blood, urine, cerebrospinal fluid, cell culture media, and a variety of other fluids. however, tracing the lineage of evs to their tissue of origin remains challenging due to their minute amount of cargo and unavailability of matching biopsied tissue and bodily fluids from the same patient. we recently demonstrated in three separate publications (dogra et. al; smith et. al; murillo et. al) , a new device (nanodld) for ev isolations, it's comparison with current technologies, bioengineered vesicles, and a detailed study of rna types present in small/ large vesicles, lipoproteins, and ago2 protein in different biofluids. in the present study, we aim to investigate the lineage of prostate derived evs in biofluids. methods: using our chip technology, we have isolated exosomes from prostate cancer cell lines and patient tissue, blood and urine samples. after exosome isolation, small rna libraries were prepared, and sequencing is carried out at icahn school of medicine and new york genome center using illumine sequencer hiseq2500. our nanofluidic pillar array is manufactured in an sio2 mask using optical contact lithography and deep ultraviolet lithography. results: our study revealed i) rna markers, which are exclusive to their prostate tissue of origin and are secreted in evs; ii) approximately 1-3% of prostate tissue-specific rna were discovered in evs; iii) over 77% (17 of 22 rna) of literature curated prostatespecific rna signatures were detectable in serum and urine evs from pca patients; iv) evs contained over 50-70% of noncoding rna (40-60% was mirna), while tissue predominantly yielded rrna (>60%); v) finally, gene set analyses generated that over 90% of evs rna were enriched for signalling pathways, yielding mirna-associated, non-canonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively monitor prostate tissue-specific biomarkers, identify tumour-specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. summary/conclusion: in summary, we have investigated patient matched tissue, serum, and urine derived evs in prostate cancer. we present a set of 68 prostatic rna in evs, which are enriched in noncanonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively track prostatic biomarkers, identify tumour specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. a multi-model, liquid biopsy approach for diagnosing and staging pancreatic adenocarcinoma introduction: pancreatic ductal adenocarcinoma (pdac) is the third largest contributor to cancerrelated death in the usa. since there is not yet a feasible technology to diagnose pdac early in the disease, 80% of patients are diagnosed at an advanced stage. moreover, for patients with confirmed pdac, standard imaging method has low sensitivity to detect early metastatic disease, which complicates the selection of therapy. to address these challenges, there has been great interest in developing minimally-invasive, extracellular vesicle (ev) based blood tests for pdac. to this end, we have integrated measurements of tumour derivedev rna cargo with circulating proteins and cell free dna (cfdna), and use machine learning algorithms to distill this multiplexed diagnostic to 1. diagnose pdac patients from healthy and disease controls and 2. distinguish pdac patients with distance sites of metastasis to guide their treatment. we make use of our lab's magnetic nanopore isolation technique to specifically enrich for tumour derived evs directly from patient plasma. methods: we have developed a high throughput nanofluidic sorting platform, which immunomagnetically isolates individual evs from plasma using magnetic nanostructures. however, our architectures is uniquely designed for massive parallelization allowing high throughput, robust processing of ml of plasma in minutes. we performed sequencing on a discovery set of patients and controls (n = 29). subsequently, we trained our panel of biomarkers using a training set of n = 47. finally, we validated the performance of our platform using an independent blinded test set of n = 145. results: the results of a blinded test set achieved an accuracy = 92% and an auc = 0.95 on binary classification of pdac patients versus those that were healthy or disease controls. in addition, we achieved an auc = 0.85 and accuracy = 89% with sensitivity of 90% and specificity of 88% on detecting occult metastasist. summary/conclusion: we developed a highly sensitive pancreatic cancer diagnostics by combining our nanomagnetic isolation platform for tumourderived ev isolation, rna sequencing, and machine learning. we isolated tumour-derived evs and profiled their rna cargo, combined with cfdna and ca19-9 for pancreatic cancer diagnosis. the predictive panels successfully distinguished non-cancer patients from pdac patients, and nodistant metastasis patients(m0) from distant metastasis patients(m1) for appropriate treatment. the resulting auc and accuracy from the independent blinded test set outperformed any individual biomarker, showing both the benefits and the robustness of combining multiple orthogonal biomarkers for pdac diagnosis. introduction: both hypertension and diabetes exhibit significant molecular changes to the vasculature that are associated with increased cardiovascular risk. here we examined the protein composition of large evs (l-evs) isolated from the plasma of hypertensive, diabetic and healthy mice to identify common and diseasespecific molecular changes. methods: we examined circulating l-evs isolated from transgenic mice expressing active human renin in the liver (ttrhren, a model of hypertension), ove26 type 1 diabetic mice, and their wild-type (wt) littermates. at 20 weeks of age mice were sacrificed and blood samples were obtained by cardiac puncture. l-evs were isolated from platelet-free plasma via differential centrifugation and protein content was assessed via mass spectrometry (ms). results: ttrhren mice exhibited increased blood pressure compared with ove26 mice or their wt littermates. (144.2 ± 7.6 vs 123.5 ± 4.9 [ove26] vs. 114.6 ± 5.7 mmhg [wt], p < 0.05). ms identified 297 independent proteins with at least 2 peptides per protein. of these, 163 proteins were found in all groups studied, 48 were exclusive to wt mice, 23 were exclusive to ove26 mice and 4 were exclusive to ttrhren mice. in addition, 22 proteins were observed with >1.5 fold change (fc) compared to wild-type mice, and 68 proteins were reduced by >50%. amongst the top ten differentially expressed proteins, fibrinogen was upregulated in both ove26 and ttrhren mice compared with wild-type controls. similarly trem-like transcript 1, sarcoplasmic/endoplasmic reticulum calcium atpase 3 and junction plakoglobin were all downregulated in both ove26 mice and ttrhren mice suggesting molecular changes common to both conditions. conversely, arginase was up-regulated in diabetic, but not hypertensive mice while carboxypeptidase was upregulated in hypertensive but not diabetic mice. summary/conclusion: taken together, these results show that the protein composition of circulating l-evs is altered in diabetes and hypertension and that both common and disease-specific changes may be detected. further analysis of these changes may lead to the identification of novel pathways associated with the pathogenesis of vascular injury in hypertension and diabetes. funding: this study was supported by grants (to db) from the canadian institutes of health research, an ontario early researcher award, and the canada foundation for innovation. understanding the role of endothelial cell-derived apoptotic bodies in inflammatory signalling and cell clearance in an atherosclerosis model of inflammation. introduction: apoptotic bodies (apobds) are a class of large (~1-5um) evs formed during apoptotic cell disassembly, that are becoming increasingly recognized as potential mediators of intercellular communication, e.g. via the transfer of proteins and other cargoes to target cells. during the inflammatory vascular disease atherosclerosis, endothelial cell (ec) apoptosis contributes to loss of barrier function and promotes the formation of plaques in regions of ec damage. although, experimentally, ecs generate an abundance of apobds, a specific role for ec-derived apobds (ec-apobds) in the progression of atherosclerosis remains poorly defined. methods: in the present study, a detailed in vitro characterization of ec disassembly was performed via flow cytometry, confocal live cell imaging and cytokine profiling, followed by function analyses of ec-apobds using a murine in vivo model of dead cell clearance. results: characterization of ec disassembly revealed that apobd formation in ecs is regulated by rho-associated, coiled-coil-containing protein kinase 1 (rock1), a process that can be pharmacologically inhibited using a rock-1 inhibitor, thereby providing tools for functional in vivo studies. the specific cargo and role in clearance of ec-apobds were then investigated. profiling of ec-apobds was performed via cytokine antibody array to reveal that ec-apobds generated under inflammatory conditions contain high levels of pro-inflammatory cytokines including mcp-1 and il-8, suggesting a potential role for ec-apobds in the propagation of inflammation during vascular disease. furthermore, the ability of ec-apobds to be cleared from the vasculature via phagocytosis was investigated, revealing that ec-apobds can travel to distal organs to undergo clearance. summary/conclusion: these findings provide important insights into the potential functions of ec-apobds generated under both non-inflammatory and inflammatory conditions and may contribute to future studies involving the therapeutic targeting of ec disassembly for the treatment of atherosclerosis. funding: this work was supported by grants from the national health & medical research council of australia (gnt1141732, gnt1140187) adipose mesenchymal stromal cell derived evs foster cardio-renal protection in the doca-salt hypertensive rat model introduction: cardio-renal syndromes (crs) are disorders of the heart and kidneys whereby "acute or chronic dysfunction in one organ may induce dysfunction of the other". stem cell-derived extracellular vesicles (evs) mediates the protection of the kidney from development of chronic kidney disease (ckd). we here investigated the potential of adipose-mesenchymal stromal cells derived evs (asc-evs) as therapeutic tools for the treatment of crs. methods: adult wistar rats were uninephrectomized and treated with a high-na+ diet and deoxycorticosterone-acetate (doca-salt) for 8-weeks (038/15; a02/16-61-15). evs were isolated by ultracentrifugation method. ev dimension, concentration and surface markers were characterized by nta, cytofluorimetric analysis and transmission electron microscopy. to characterize the role of evs in crs, doca-salt rats were injected weekly with asc-evs. systolic blood pressure was measured by the tail-cuff method. plasma creatinine and urinary protein excretion were determined by colorimetric assays and microalbuminuria by immune turbidimetric assay. qrt-pcr and western blot were conducted to evaluate fibrosis and inflammatory-related genes and proteins in the kidney and heart of doca-salt rats. immunohistochemistry was used to confirm matrix accumulation (a-sma) and immune infiltrate (cd68+ cells). results: multiple administration of asc-evs in doca-salt rats induced a protective effect on the kidney, by reducing tubular and vascular damage. kidney function was also conserved by ev treatment as detected by the normal glomerular filtration rate and the absence of proteinuria with respect to doca-salt untreated rats. ev administration significantly decreases the pro-inflammatory molecules mcp-1 and pai1 and reduce the recruitment of macrophages in the kidney. the mitigation of the inflammatory response by asc-ev infusion consequentially affected the development of fibrosis, as detected by the decrease in collagens (col1a1, col4a1) and fibronectin (fn) expression in respect to doca-salt animals. asc-evs were able to act in multiple organs, preventing fibrosis and inflammation also in the heart, therefore alleviating blood pressure rise during the 8-weeks of treatment in doca-salt rats. summary/conclusion: our results indicate that asc-ev administrations in hypertensive-induced ckd rats promote protection from renal damage, reduction of the inflammatory response and prevention of interstitial fibrosis in the kidney. asc-evs are also able to protect the cardiac tissue and to control blood pressure increase, displaying complex and multiorgan beneficial effects. introduction: alveolar macrophages (ams) tonically secrete extracellular vesicles (evs) containing suppressor of cytokine signalling 3 (socs3) protein. uptake of socs3-containing evs by alveolar epithelial cells is critical for restraint of cytokine-induced janus kinasesignal transducer and activator of transcription 3 (jak-stat3) signalling to promote homoeostasis in the distal lung. at steady state, ams exhibit suppressed glycolytic activity, a metabolic phenotype that promotes homoeostatic function. whether this glycolytic restraint is critical for am secretion of socs3 is unknown. in fact, to our knowledge, metabolic control over release of any ev cargo has never been explored in any cellular context. methods: immortalized mouse ams (mh-s) were treated with various doses of 2-deoxy-d-glucose (2-dg) and oligomycin, inhibitors of glycolysis and oxidative phosphorylation, respectively. primary rat ams collected by lung lavage were treated with an aqueous extract of cigarette smoke (cse) with or without 2-dg. metabolic activity was measured by seahorse assay, evs were quantified by nanoparticle tracking analysis, and vesicular (>100-kda) socs3 secretion was determined by western blot of conditioned medium. additionally, ams collected from wild-type (wt) and lsl-krasg12d mice bearing lung tumours 16 weeks after intrapulmonary ad-cre were cultured ex vivo in the presence or absence of 2-dg. vesicular (>100-kda) socs3 secretion was measured by elisa. results: in a dose-dependent manner, oligomycin inhibited, whereas 2-dg enhanced, socs3 and ev release by mh-s cells. treatment of rat ams with cse (1%) attenuated secretion of socs3, an effect that coincided with increases in glycolytic activity, and co-treatment of ams with 2-dg abrogated the inhibitory effect of cse on socs3 release. finally, ams collected from lsl-krasg12d mice exhibited a deficiency in socs3 secretion relative to wt ams, an effect that was reversible by overnight culture in the presence of 2-dg. summary/conclusion: in tandem, our data generated using in vitro and in vivo approaches demonstrate that am secretion of vesicular socs3 is down-regulated by glycolysis. we speculate that metabolic control over release of ev cargoes is a phenomenon of broad biologic relevance within and outside of the lung. introduction: bacterial extracellular vesicles (ev) are described to play roles in defence and resistance, pathogenesis and stress responses. cyanobacteria pioneered oxygenic photosynthesis, and are the ancestors of modern chloroplasts. we previously described that by deleting the gene encoding tolc (δtolc) in the model cyanobacterium synechocystis sp pcc6803 (s6803), a key player in protein-mediated secretion systems, a hyper-vesiculating phenotype could be obtained. the goal of this work was to understand why δtolc hyper-vesiculates. methods: isobaric tag for relative and absolute quantitation (itraq) was used for quantitative proteomic analyses of total cell extracts. ev were isolated as follows: cells were separated from the extracellular medium (em) by centrifugation (4400 g, 10 min) and filtration (0.2 µm pore-size filters). cell-free em was concentrated using centrifugal filters (mwco of 100 kda), and later ultracentrifuged for 3 h at 100000 g. the final ev fraction was suspended in growth medium. ev characterization was performed using tem, dls, nanosight, and by the detection and quantification of lps (lipopolysaccharides). detection of specific proteins in ev was carried out by western blot. copper (cu) levels were quantified by atomic absorption spectrometry (aas). results: a large-scale quantitative proteomic analysis was performed, resulting in the identification of several metal-related proteins with differential regulation in s6803 δtolc. both wild-type (wt) and δtolc cells were then challenged with different metals. compared to the wt, δtolc showed impaired growth only when exposed to cu, a co-factor for several proteins with roles in primary metabolism. the intracellular cu levels were quantified and δtolc accumulates threefold more cu than wt cells. we then asked whether the hyper-vesiculating phenotype observed could be linked to the stress induced by cu accumulation. in ev isolated from δtolc we detected the metallochaperone copm, a periplasmic cu-binding protein involved in cu-resistance mechanisms in s6803. in addition, cu could also be detected in isolated δtolc-ev. in addition, more ev were detected when s6803 wt cells were challenged with cu, in a cu-concentration dependent manner. summary/conclusion: these results support the idea that bacterial ev represent an alternative cu-secretion mechanism to deal with cu-induced stress. funding: fct phd grant sfrh/bd/130478/2017; feder-compete2020-poci-fct project: poci-01-0145-feder-029540. juan wang and maureen barr rutgers university, human genetics institute of nj, piscataway, usa introduction: extracellular vesicles (evs) function in intercellular communication. despite their physiological importance and biomedical relevance, knowledge of ev fundamental biology is not well understood, in part due to a lack of tractable animal systems. our analysis of environmentally-released c. elegans ciliary evs provides strong evidence that nematodes package cargo in evs that mediate inter-organismal communication, in analogy to intercellular signalling in mammals. we predict that conserved mechanisms underlie ev cargo sorting, biogenesis and signalling. cilia act as cell towers to both receive extracellular signals and to send information via ciliary evs. ciliary defects result in human ciliopathies including autosomal dominant polycystic kidney disease (adpkd). adpkd is a life-threatening disease that affects 1/800 and is caused by mutations in pkd1 and pkd2, which encode polycystin-1 and −2. in c. elegans and humans, the polycystins are architecturally similar, act in the same genetic pathway, function in a sensory capacity, localize to cilia, and are shed in evs, suggesting ancient conservation. moreover, ciliary ev biogenesis and shedding is an evolutionary conserved process from algae to worms to humans. by studying how cilia make and receive evs, we aim to uncover fundamental principles of how cells communicate using evs. methods: to study ciliary ev cargo sorting and biogenesis, we use genetically-encoded fluorescent-tagged ev cargo and superresolution zeiss airyscan confocal microscopy in living animals. results: we find that cargoes are sorted into distinct populations. in cilia, kinesin-2 motors and kinesin-3 klp-6/kif28 transport different ev cargoes to the ciliary tip and generate an ev cargo enrichment zone. from here, evs are shed and released into environment in a spatially and temporally regulated manner. ciliary ev biogenesis and release is regulated by mechanical pressure and ph. our work revealsat the single cell levelthat different evs are made in response to environmental stimuli, which may be important for ev signalling properties. summary/conclusion: cells exploit the spatiallyrestricted cilium and its sophisticated transport system to generate distinct populations of ciliary evs. how these ciliary ev communicate cellular messages awaits decoding. introduction: we recently demonstrated that recycling endosomes marked by rab11a generate exosome subtypes distinct in cargos and functions from late endosomes, which we collectively term rab11-exosomes. these exosomes are preferentially released from cancer cells in response to metabolic stress and promote adaptive changes in a xenograft model. here we use comparative ev proteomics in hct116 colorectal and hela cervical cancer cell lines to identify rab11-exosome signature proteins and screen for functional effects. methods: we analysed ev preparations by mass spectrometry using tandem mass tag® labelling to identify changes in ev protein cargo in response to glutamine depletion. candidate genes were subsequently knocked down in drosophila secondary cells, which permit visualisation of rab11-exosome biogenesis using fluorescence microscopy, and in human cancer cell lines. results: we show that accessory escrt-iii proteins, chmp5, chmp1 and ist1, are enriched on glutamine-depletion-induced evs and play a selective and conserved role in generating rab11-exosomes. they are, however, not required to traffic ubiquitinated cargos into late endosomes and lysosomes. escrt-0 components, thought to regulate trafficking of ubiquitinated cargos into intraluminal vesicles, are also required to make rab11-exosomes. in flies the escrt-0, hrs, localises to the limiting membrane of rab11-endosomes. comparative proteomics reveals other proteins enriched in rab11-exosomes, which also appear to be needed to mediate this novel exosome formation mechanism. summary/conclusion: we conclude that rab11-exosome subtypes are formed via a distinct mechanism requiring accessory escrt-iii components, suggesting a route to selectively target these exosomes. introduction: the tumour microenvironment consists of a complex network of host cells embedded within extracellular matrix. communication between these cellular compartments is critical for tumour progression and exosomes have emerged as important regulators of intercellular communication. while a number of studies have implicated exosomes in cancer progression, mechanisms controlling exosome transfer are not well understood. we developed three-dimensional (3d) culture models to evaluate the role of cues provided by the extracellular matrix in exosome release and uptake. methods: exosomes were isolated from cells in two-and three-dimensional culture via ultracentrifugation and characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. exosomes were labelled with fluorescent lipophilic dyes and uptake in recipient cells quantified by flow cytometry. results: cells cultured in 2d display decreased exosome release and increased uptake compared to 3d cultured cells. exosome release in 3d culture was inhibited with the exosome release inhibitors brefeldin a and gw4869, but was not significantly altered by knockout of rab27b. in addition, disruption of polarity signals provided by 3d culture did not impact exosome release or uptake in 3d, but induction of oncogenic hras increased both secretion and uptake of exosomes through activation of pi3 k signalling. summary/conclusion: release and uptake of exosomes is altered in 3d environments. these studies help provide insight into exosome production and uptake in vivo and have potential implications for therapeutically targeting exosome release and the development of exosome based therapeutic delivery vehicles. introduction: previous studies in our lab found that expression of r345 w-fibulin-3 induces rpe to undergo emt. the purpose of current study was to characterize the extracellular vesicles (evs) in rpe cells expressing wt-fibulin-3 versus rpe cells expressing r345 w-fibulin-3 and investigate the effects of these evs on rpe cell differentiation. methods: arpe-19 cells were infected with lentivirus with luciferase-tagged wild-type (wt)-fibulin-3 or luciferase-tagged r345 w-fibulin-3. evs were isolated from the media of arpe-19 cells by conventional ultracentrifugation or density gradient ultracentrifugation. transmission electron microscopy (tem) and cryogenic electron microscopy (cryo-em) were performed to study the morphology of the evs. the amount and size distribution of evs were analysed by nanosight tracking analysis (nta). ev protein concentrations were quantified using the dctm protein assay (bio-rad). ev cargo were analysed by unbiased proteomics using lc-ms/ms with subsequent pathway analysis (advaita). migration ability was evaluated in arpe-19 cells with or without the exposure of evs by conducting scratch assays. results: morphologically, tem imaging showed concave-appearing vesicles and cryo-em imaging showed spherical vesicles with two subpopulations of evs: a small group with diameters around 30 nm and a large group with diameters around 100 nm. moreover, tem and cryo-em showed an increased amount of small evs (~30 nm) in the mutant group compared to the wt group. this result was further confirmed by nta showing that, in the mutant group, the particle size distributions were smaller than the wt evs. no significant differences were shown in ev protein concentrations per particle between wt and mutant groups. our previous data suggest that the expression of r345 w-fibulin-3 causes rpe cells to undergo emt as evidenced by upregulated emt drivers and an increased migration ability. proteomic studies showed that evs derived from arpe-19 cells overexpressing wt-fibulin-3 contain critical members of sonic hedgehog signalling (shh) and ciliary tip components, whereas evs derived from rpe cells overexpressing r345 w-fibulin-3 contain emt mediators, indicating that ev cargo reflects the phenotypic status of their parental cells. ev transplant studies showed that exposing native rpe cells to mutant rpe cell-derived evs containing emt drivers, including tgf-β-induced protein (tgfbi), vim, and smad4, leads to an enhanced migration ability of rpe cells in a dosedependent manner. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms (2d static tissue culture flask vs 3d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in 2d t-flasks and 3d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both 2d and 3d culture systems. introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies methods: pca patients and age-matched healthy controls (hc) plasma (n = 6 in each group) were used to isolate sevs with 3 different isolation methods including commercial exoquick ultra kit, qev 35 and qev 70 size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd9 and cd81 commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev35 demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev70. furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods summary/conclusion: qev 70 method demonstrated better performance in isolating relatively pure sevs from human plasma; qev35 has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev35 but with the highest non-sev protein contaminations. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd9, cd63 and cd81, it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: 1) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and 2) the assessment of subpopulation target enrichment relative to the population average by leveraging tim4 as an unbiased, lipidbased ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasisassociated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr3 (her2+), t47d and mcf-7 (er+pr+), bt549 and mda-mb-231 (triple negative) breast cancer cell lines, as well as five mda-mb-231-derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her2 was broadly detected in her2+ skbr3 evs. interestingly, her2-t47d and mcf-7 evs also expressed her2 where it was highly enriched in its epcam+ subpopulations. itg α6, β3 and β4 were only found in triple negative and organotropic evs with itg β3 and β4 differentially enriched based on the organotropism. the population average of mda-mb-231 and lung-tropic evs had high expression of itg β4, where subpopulations of cd44+ evs showed positive enrichment while cd9+ and cd63+ evs showed negative enrichment. itg α5, β3 and β4 were absent in the bone-tropic cd81+ ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb-231 evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. op2.06 = pf17.06 heparan sulphate proteoglycans are required for ev-mediated delivery of multiple growth factors sara veiga, alex shephard, alex cocks, aled clayton and jason webber cardiff university, cardiff, uk introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du145 prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified 49 targets that bind to hs-gag chains, and also 108 different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of evintroduction: methamphetamine (ma) and related amphetamine compounds, which are potent psychostimulants, are among the most commonly used illicit drugs. neuroimaging studies have revealed that chronic ma abuse can indeed cause neurodegenerative changes in the brains of human ma abusers including prominent microglial activation throughout the brain. it is still unclear how chronic inflammation caused by ma abuse leads to long-term damage to the brain. with this in mind, we are particularly interested in studying the role of extracellular vesicles (evs) in eliciting chronic inflammation in ma exposed brains. in the present study, we focus on the role of a mirna, mir-29a-3p (mir-29a) in chronic ma exposure. here, we present novel data that shows for the first time how chronic ma impacts not only the biogenesis but also the ev associated mirna cargo thereby affecting the overall health of the neurons and glial cells in the brain. methods: -density gradient centrifugation for isolation of brain-derived vesicles -characterization of bdes by western blotting, nanoparticle tracking analysis and transmission electron microscopy -quantitative rt-pcr -digital droplet pcr -confocal imaging of dendritic spines and synapses results: in the present study, we show from both in vivo and in vitro studies that chronic methamphetamine (ma) treatment alters ev biogenesis and microrna (mirna) cargo. brain-derived evs (bde) isolated from frontal grey tissue of rhesus macaques that were administered ma in a chronic regimen revealed a significant increase in both number and size. further analysis revealed increase in biogenesis genes and increased levels of mirna, mir-29a-3p (mir-29a). in situ hybridization of the frontal brain area revealed that mir-29a was exclusively expressed in microglia and neurons. further, in vitro studies revealed that ev associated mir-29a elicited not only neuronal damage but also was able to activate microglia to release pro-inflammatory cytokines thereby inducing a chronic inflammatory cycle. finally, we show that an anti-inflammatory drug was able to rescue inflammation, mir-29a levels and synaptodendritic injury. summary/conclusion: in summary, our results present for the first time show that chronic ma exposure in the brain affects ev biogenesis and mirna expression. we further confirm that mir-29a can serve as potential marker to diagnose synaptic deficits for chronic ma addiction in humans. finally, we reveal that anti-inflammatory drug could rescue the ev biogenesis and reduces the secretion of mir-29a, thereby rescues synaptodendritic injury. our data further supports the use of the anti-inflammatory drugs as therapeutic interventions for ma addiction. funding: nida funding # r01da042379 blood-borne and brain-derived ectosomes/microparticles in morphineinduced anti-nociceptive tolerance deepa ruhela, veena bhopale, ming yang, kevin yu, eric weintraub, aaron greenblatt and stephen r. thom university of maryland school of medicine, baltimore, usa introduction: opioid pain treatment is impeded because chronic administration decreases analgesia, a condition called tolerance that prompts dose escalation contributing to morbidity and mortality. inflammatory interleukin (il)-1β is required for tolerance development, so we hypothesized that pro-inflammatory extracellular vesicles (evs) play a role. methods: evs with opioid administration were assayed in mice and humans. annexin v-positive, 0.1-1 µm diameter microparticles (mps) were assessed by flow cytometry in murine and human blood and in murine deep cervical lymph nodes that drain brain glymphatics. blood-borne exosomes (<100 nm) were assayed by tunable-resistance pulse sensing (trps). anti-nociceptive tolerance following morphine administration to mice was assessed by speed of tail removal from warm water. results: repetitive morphine dosing of mice to induce anti-nociceptive tolerance increased blood-borne mps by eightfold, and by tenfold in cervical lymph nodes. mps expressed proteins specific to neutrophils, microglia, astrocytes, neurons and oligodendrocytes. il-1β content of mps increased 68-fold. administration of an il-1β antagonist to mice diminished blood and glymphatic mps elevations and abrogated tolerance induction. intravenous polyethylene glycol telomer b that lyses mps and intraperitoneal methylnaltrexone that binds peripheral opioid-mu receptors and myeloid differentiation factor-2 to inhibit toll-like receptors, inhibited mps elevations and tolerance. neutropenic mice did not develop anti-nociceptive tolerance, elevations of blood-borne mps or cervical node mps expressing microglial proteins. elevations of blood-borne exosomes were not identified based on trps analysis. patients entering treatment for opioid use disorder exhibited similar mps elevations as do tolerant mice. summary/conclusion: neutrophil-derived mps containing il-1β are required for morphine-induced antinociceptive tolerance. funding: this project was supported by grant n00014-16-1-2868 from the office of naval research and an unrestricted grant from the national foundation of emergency medicine. evs are a conveyor of toxic dipeptide repeat proteins in c9orf72 als/ ftd models thomas jefferson university, philadelphia, usa introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterized by loss of motor neurons. in als, motor symptoms initiate focally and then progress gradually, distal from the initial focus. abnormal forms of als-associated proteins are physically exchanged between neuronal cells. pathogenic als proteins like sod1, fus and tdp43 are transmitted between cells by assisted mechanisms, mainly extracellular vesicles (evs), spreading toxicity and misfolding of native proteins within the recipient cells. an intronic g4c2 aberrant nucleotide repeat expansion in c9orf72 gene is the most common genetic cause of als. translation of this expanded region occurs by a process called repeat associated non-aug (ran) translation that produces five dipeptide repeats proteins (dprs), polyga, polygp, polygr, polypa and polyga. polyga, polygr and polypr are associated with toxicity in neurons. in this work we study the recruitment of these aberrant proteins into extracellular vesicles (evs) and the potential role of these evs in spreading toxicity between cells of the central nervous system. methods: to isolate the evs from cell culture media we isolated by ultracentrifugation the larger vesicles at 21,000xg and the smaller evs at 100,000xg. number, size and fluorescence of the vesicles were analysed by fluorescent nanotrack analysis (f-nta) and by cytoflex. the protein content of the vesicles was analysed by western blot (wb). to evaluate the potential toxicity of the evs, a transwell system (tw) was employed. neuron viability was assessed using live imaging techniques. results: nsc34 were transfected with reporter constructs expressing dprs tagged with gfp protein. by f-nta, cytoflex and wb analysis we assessed that all the five dprs were loaded in both the large and the small vesicles isolated from cell culture medium. by tw, nsc34 transfected with the dprs were put in contact with primary cortical neurons (cns) transfected with synapsin driven td-tomato for live imaging purposes. we observed that polygr+ nsc34 were able to cause a significant decrease in cns viability. we also observed that polygr+ evs associated toxicity was directly dependent on polygr length. this effect was reverted reducing the number of polygr+ evs treating nsc34 with gw4869. to understand the downstream effect of polygr+ evs in recipient cells we studied tdp43 mislocalization, ran-translation and activation of the integrated stress response, finding a dysregulation of all these potentially toxic pathways in neurons treated with polygr+ vesicles. summary/conclusion: concluding, dprs are actively secreted in evs and polygr+ vesicles cause the activation of toxic mechanisms in the recipient cells, possibly contributing to the spreading of als introduction: pregnancy is the a condition that profoundly mitigates symptoms of multiple sclerosis (ms) a complex disease characterized by immune dysfunction and neurodegeneration affecting 2.3 million people worldwide. serum exosomes, released by specific cells during pregnancy, modulate the immune and central nervous system function and contribute to pregnancy-associated suppression of experimental autoimmune encephalomyelitis (eae), an induced preclinical model of ms. extracellular vesicles (evs) are the new means for communication among cells. the aim of our study was to characterize the ability of human amniotic fluid stem cells-derived evs (hasc-evs) to antigen presenting cell function thus correcting immune dysfunction in eae. methods: amniotic fluids were obtained from human 16-17-week pregnant women. hasc-evs were collected by ultra-centrifugation. evs were characterized for their specific proteins, lipids and nucleic acids expression. the ability of evs to modulate immune responses was performed in vitro, testing the ability of evs to induce a tolerogenic phenotype in mouse bone marrow derived dendritic cells, and in vivo for their potential to suppress eae, induced by immunization c57/b6 female mice with mog35-55 peptide. results: we found that hasc-evs expressed high levels of galectin-1 and promoted a significant increase of the immunoregulatory enzyme indoleamine 2,3dioxygenase-1 enzyme in dcs. moreover in in vivo experiments administration of hasc-evs significantly reduced disease severity in eae. such effect was associated with reduced neurological deficits and suppression of pathogenic t helper 17 (th17) cells, and increased percentage of regulatory t cells (treg-foxp3+) cells. summary/conclusion: our findings unravel immunoregulatory effects of evs secreted by hascs. evs may represent a novel cell-free immune regulatory and regenerative therapeutic approach that can potentially mitigate immune dysfunction and promote remyelination. association of neuronal-derived extracellular vesicles cargo with cognitive decline in late middle life introduction: alzheimer's disease (ad) is characterized by a long preclinical stage during which phosphorylated tau pathology spreads in the brain leading to clinical symptoms. pathogenic tau spreads, in part, via extracellular vesicles (evs). we and others have demonstrated that tau cargoes of neuronal-derived evs (nevs) from blood can serve as biomarkers for ad. we aimed to examine whether nev tau cargo can predict cognitive decline in late middle age by leveraging samples from participants in the wisconsin registry for alzheimer's prevention (wrap) study. methods: we blindly immunoprecipitated nevs using antibody against neuronal l1 cell adhesion molecule (l1cam) from serum samples of 146 wrap participants who were cognitively unimpaired at baseline (mean age 62.4 ± 6.3 years old; 71.2% females; 42.5% apoe4 carriers), of whom half subsequently developed cognitive decline. we measured phosphorylated (p181 and p231) and total tau in nevs using electrochemiluminescence assays. we used linear regression models to identify differences between cognitive status groups including age, sex apoe status and the cognitive status*age interaction in the model. results: at baseline, we found trends for higher p181-(p = 0.07) and p231-tau (p = 0.05) levels in future decliners compared to stable participants. further, there were significant cognitive status*age interactions for ptau231 (p < 0.01), total tau (p < 0.001) and ptau181 (p < 0.05) with higher levels with increasing age in future decliners summary/conclusion: nev tau cargo differs between late middle-aged individuals at risk for ad with and without future cognitively decline even before decline occurs, presumably due to subclinical spread of tau pathology. further nev biomarker development may allow preclinical ad diagnosis. introduction: in the brain, circulating extracellular vesicles (evs) in the cerebrospinal fluid (csf) contain a variety of signalling factors, including proteins, enzymes, and rna transcripts. while evs have been implicated in many cell-to-cell signalling contexts, the vast majority of these studies are based on findings derived from cell culture conditions. thus, the ability to identify cell typespecific ev release from cellular subpopulations within the brain represents a critical barrier in the field. methods: to address this knowledge gap, we utilized a novel transgenic mouse model to determine the release of cell-type specific evs. here we report the exomap-2 mouse, which is designed to express an exosomal green fluorescent protein in response to expression of cre recombinase. specifically, the exomap-2 transgene was inserted at the mouse h11 locus and consists of (i) a broadly expressed cag promoter/enhancer, (ii) a floxed orf encoding mts-tdtomato, (iii) an orf encoding the exosomal protein acyltya fused to mneongreen (mng), and (iv) a 3ʹ utr containing the wpre element and polyadenylation signal from the bovine growth hormone gene. results: intracranial ventricular injections of the viral vector aav-ttr-cre, which drives cre recombinase expression from the choroid plexus-specific promotor of the transthyretin gene, leads to acyltya-mng expression in the choroid plexus. moreover, we observed that these mice released mneongreen-positive evs into the cerebrospinal fluid and also visualized the vesicles in the blood. furthermore, these mice displayed an accumulation of acyltya-mng fluorescence in the medial habenula. summary/conclusion: the results indicate that choroid plexus-derived evs are trafficked to the csf and the medial habenula, and more generally, that the exomap-2 mouse can be used to follow the trafficking of tissuespecific evs into biofluids and between tissues in vivo. introduction: large-scale colorectal cancer (crc) sequencing studies have shown that 93% of all tumours had at least one mutation in proteins implicated in the wnt signalling pathway. mutations in β-catenin have often been associated with the constitutive activation of wnt signalling pathway and has been established as a major driver of crc. one of the proposed mechanisms of activating wnt signalling involves extracellular vesicles (evs) as cellular couriers to transfer wnt ligands from one cell to another. however, the association of oncogenic mutant β-catenin with evs has not been studied. subpopulations of cancer cells with different mutational loads and behavioural variations lead to intra-tumour heterogeneity methods: integrative proteogenomic analysis showed the secretion of mutant β-catenin via evs. evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. silac-based quantitative proteomics analysis, immunofluorescence, biochemical analysis, qpcr and xenograft models were employed to unveiling the role of evs carrying mutant βcatenin. results: an integrative proteogenomic analysis identified the presence of mutated β-catenin in evs secreted by colorectal cancer (crc) cells. follow up experiments established that evs released from lim1215 crc cells stimulated wnt signalling pathway in the recipient cells with wild type β-catenin. silac-based quantitative proteomics analysis confirmed the transfer of mutant β-catenin to the nucleus of the recipient (rko crc) cells. in vivo tracking of dir labelled evs in mouse implanted with rko crc cells revealed its bio distribution, confirmed the activation of wnt signalling pathway in tumour cells and increased the tumour burden. introduction: there has been a significant increase in incidence of human papillomavirus 16 (hpv16) driven oropharyngeal cancer (opc) in developed countries. there is evidence that hpv alters the molecular cargo of exosomes released by opc. emerging evidence suggests that hpv integration within the human genome is associated with both genomic and transcriptomic alterations. consistent with previous studies, the genomic viral-cellular junctions were identified using dips-pcr method in 15 (88%) saliva samples collected from hpv16-driven opc. methods: morphology and molecular features of exosomes derived from three different saliva sampling methods: unstimulated saliva; acid-stimulated saliva; and salivary oral rinses were examined using transmission electron microscopy (tem), nanoparticle tracking (nta) and western blot analysis. hpv-16 dna detection in salivary exosome was determined by using qpcr method. proteome profile of salivary exosomes derived from both cancer-free controls and hpv16-driven opc patients was characterized using liquid chromatography-electrospray ionization-tandem mass spectrometry (lc-ms/ms). results: we demonstrate that unstimulated saliva had greater abundance of exosomes when compared to the other sampling methods. three common exosome markers (cd9, cd63 and cd81) were higher in unstimulated saliva. only salivary exosomes derived from hpv-driven opc patients had a detectable level of hpv-16 dna. the proteomic signature of salivary exosome was significantly (p < 0.01) different between cancer-free controls and hpv-driven opc. we found elevated protein abundance of five main glycolytic enzymes (i.e. phosphoglycerate kinase 1 (pgk1), glyceraldehye-3-phosphate dehydrogenase (gapdh), aldolase (aldoa) and lactate dehydrogenase a (ldha) in salivary exosomes derived from opc patients, suggesting a functional role of salivary exosome in the reciprocal interplay between hpv-driven opc and glucose metabolism. summary/conclusion: our data suggest that the development of a low-cost non-invasive saliva-based test using both salivary exosomal dna and protein may offer an opportunity to detect hpv-driven opc, that may be clinically useful in managing these patients. continuous in vivo release of mast cell derived extracellular vesicles from an implanted device spreads pro-inflammatory response in mice introduction: mast cells are important players of the immune system and they secrete a wide range of mediators during bacterial infections. mast cells are also able to release extracellular vesicles (evs). here, we report that mast cells communicate with each other in vivo by evs. methods: we isolated bone marrow-derived and peritoneal mast cells from gfp-transgenic and wild type mice. evs were separated from the conditioned media of these cells cultured in the presence or absence of lipopolysaccharide (lps). evs were characterised according to the misev2018 guidelines by flow cytometry, electron and fluorescent microscopy, trps, the spv lipid and the bca protein assays. separated ev-s were cultured with naïve mast cells, and tumour necrosis factor (tnf)-α production was tested by elisa and intracellular flow cytometry. gfp+ mast cells were seeded in diffusion chambers which were implanted into the peritoneal cavities of mice enabling us to investigate the continuous in vivo release of evs. uptake of gfp+ evs and tnf-α expression of peritoneal mast cells were tested by flow cytometry and fluorescent microscopy. results: here, we showed that bacterial lps-sensing mast cells release evs that in turn, induce tnf-α expression in resting mcs in vitro. moreover, we confirmed that evs are transmitted to other peritoneal mast cells in vivo spreading the pro-inflammatory response by inducing tnf-α secretion in peritoneal mast cells. summary/conclusion: ev communication between members of the mast cell network, play an important role in spreading and escalating pro-inflammatory responses to immune stimuli. our data may provide an explanation how the relatively rare tissue resident mast cells can play key roles in diseases such as autoimmune arthritis. the ability of small extracellular vesicles (sevs) to reprogramme cancer cells is known. integrins, receptors for extracellular matrix proteins, are major players in mediating sev functions. previously, we have reported that the αvβ3 integrin is detected in sevs of prostate adenocarcinoma (prca) cells and transferred into recipient cells in a paracrine fashion; however, its role and expression have never been explored in the most aggressive forms of prca, such as neuroendocrine prca (neprca). neprca does not express androgen receptor (ar) but does express neuron-specific proteins, such as aurora kinase a, synaptophysin and neuron specific enolase, that activate pro-tumorigenic pathways independently from the ar. methods: we isolated sevs from prca c4-2b cells using iodixanol density gradients and characterized them by immunoblotting and exoview. the experiments were performed in vivo by injecting subcutaneously, in nude mice, du145 cells treated with sevs expressing or lacking the αvβ3 integrin, and in vitro, by testing anchorage-independent growth of different cell lines treated with the same sevs. discarded human tissues from prca metastasis were analysed by immunohistochemistry (ihc). results: we demonstrate that a single treatment of prca cells with sevs significantly stimulates tumour growth and anchorage-independent growth. moreover, we show that one treatment with sevs, shed from c4-2b cells that express αvβ3, but not from the control cells that lack αvβ3, induces differentiation of prca cells towards a neuroendocrine phenotype and downregulates ar. finally, our ihc analysis shows coexpression of αvβ3 integrin and synaptophysin in neprca metastatic lesions. summary/conclusion: in conclusion, our current study shows, for the first time, that αvβ3 integrin expression in donor cells generates sevs that reprogramme recipient cells towards an aggressive tumour phenotype. funding: this study was supported by nci-p01-140043, r01-224769 to lrl. introduction: exosomes are small extracellular vesicles (sevs) that carry a variety of cargoes and have been shown to promote tumour cell motility and metastasis. cell motility is influenced by dynamic formation and stability of filopodia: actin-rich protrusions that extend from the leading edge and perform directional sensing. filopodia regulators such as fascin are upregulated in multiple epithelial cancers and can promote invasive phenotypes. however, how filopodia are induced and controlled by extracellular factors is poorly understood. here, we describe a role for sevs in regulating filopodia formation and tumour cell motility. we utilized b16f1 melanoma cells and ht1080 fibrosarcoma cells for fixed-and live-cell imaging to quantify filopodia numbers and dynamics in control and exosome-deplete conditions. itraq proteomics was used to identify sev protein cargoes that contribute to filopodia formation. in vivo experiments were performed using a chick embryo model for metastasis. results: inhibition of exosome secretion in cancer cell lines, via rab27a or hrs knockdown, led to decreased filopodia numbers. specificity to sevs was demonstrated by rescue experiments in which purified sevs but not large evs rescued the filopodia phenotypes of exosome-inhibited cells. live imaging of hrs-kd cells revealed that exosome secretion regulates formation and stability of filopodia. proteomics data and molecular validation experiments identified the tgf-beta coreceptor endoglin as a key sev cargo regulating filopodia formation, cancer cell motility, and metastasis. summary/conclusion: in this study, we identified exosomal endoglin as a regulator of filopodia formation and in vivo metastasis. these data are relevant to cancer as endoglin expression is altered in many cancers. in addition, endoglin is the disease gene for hereditary haemorrhagic telangiectasia, and may influence angiogenesis. overall, our data implicate sev-carried endoglin as a key cargo regulating filopodia. astrocyte-derived ev-mediated blood-brain barrier disruption shilpa buch, ke liao, susmita sil, fang niu and guoku hu university of nebraska medical center, omaha, usa introduction: the breach of the blood-brain barrier (bbb), resulting in ensuing neuroinflammation, is a key feature of hiv-associated neurological disorders (hands). while combination antiretroviral therapy (cart) has successfully suppressed peripheral viraemia, cytotoxicity associated with the presence of viral tat protein in tissues such as the brain, remains a significant concern. our previous study has demonstrated that hiv-1 tat can induce disruption of bbb by downregulation of tight junction (tj) proteins in human brain microvascular endothelial cells (hbmecs) and that this is regulated by the autophagic pathways. methods: evs were isolated from hiv tat-stimulated mouse/human primary astrocytes using the standard differential ultracentrifugation method and characterized by transmission electron microscopy, nanosight & western blot analyses. among the various mirs dysregulated in hiv tat -stimulated astrocyte ev cargo, mir-7 was found to be upregulated by realtime pcr. confocal microscopy identified uptake of astrocytic evs by hbmecs. functional assessment of astrocytic ev uptake by hbmecs involved cell permeability using transepithelial electrical resistance as well as trans-well endothelial cell monolayer permeability assays. results: hiv-1 protein tat-mediated induction of micrornas (mirs) in astrocyte-derived extracellular vesicles (adevs) regulated the permeability of bbb by targeting the expression of tj proteins in the hbmecs. exposure of hbmecs to tat-adevs resulted in down-regulation of the tight junction protein claudin 5, resulting in increased endothelial cell monolayer paracellular permeability. microarray data of tat-adevs demonstrated upregulation of several mirs compared to that of controls, among which upregulated mir-7 was identified to target the tj proteins using ingenuity pathways analysis. increased expression of mir-7 was validated in tat exposed astrocytes and tat-adevs. adevs loaded with mir-7 oligos showed similar effects as that observed with tat-adevs in inducing permeability in hbmecs. increased expression of mir-7 with downregulation of claudin-5 was also recapitulated in microvessels isolated from the brains of doxycycline-inducible hiv-1 tat transgenic mice (itat) mice and in lysates isolated from the frontal cortices of siv+ macaques/hiv+ autopsied brains. summary/conclusion: our findings demonstrated that tat-adevs containing mir-7 as an important mediator underlying tat-mediated disruption of the bbb. introduction: endogenous exosomes and related extracellular vesicles (evs) are potent nanoparticles released by all cells tested to date. the exploitation of their unique scaffolding for engineering next-generation drug delivery systems represents a major area of academic and commercial interest. the lag in exploiting this potential is in part due to our inability to measured extent and efficiency of modification, e.g., composition and drug loading. here we report a robust pipeline of optical tweezing combined with raman spectroscopy to molecularly characterize engineered evs and quantitatively assess extent of drug loading at single particle resolution. methods: evs derived from cell culture and isolated by ultracentrifugation were fused with synthetic liposomes to create engineered evs (eevs). these eevs were formed via well-established vesicle fusion techniques, namely (1) mechanical extrusion, (2) freeze-thawing, or (3) probe-tip sonication. prior to formation, calcein was encapsulated in the liposomes and used as a surrogate for soluble drug loading. laser trapping raman spectroscopy (ltrs) was used to optically trap single evs, before and after synthetic manipulation. raman spectral analysis was used to assess trapped eevs compared to pure standards to quantify ratiometric variation in chemical composition. results: raman laser trapping experiments confirmed that each formation method results in largely varying (1) extent of fusion between evs and synthetic calceinloaded liposomes, (2) efficiency of calcein loading, and (3) particle size. we could also quantify the molar amounts of liposome vs. ev molecules for single particles, revealing a great amount of variation from particle to particle. functional membrane proteins we left intact to varying degree across fusion methods. summary/conclusion: given the rising importance of analytical tools able to characterize extent of molecular loading for engineered evs, we believe this technology will be very useful, thus warrants further investigation for eev characterization across a variety of clinical applications. funding: randy carney, phd was supported by a research scholar grant, rsg-19-116-01-cdd, from the american cancer society. extracellular vesicles containing host restrictive factor ifitm3 inhibited zika virus infection of foetuses in pregnant mice through trans-placenta delivery allen z. wu nanjing university, nanjing, china (people's republic) introduction: zika virus (zikv) infection can lead to neurological complications and foetal defects, and has attracted global public health concerns. effective treatment for zikv infection remains elusive and a preventative vaccine is not available yet. therapeutics for foetus need to overcome blood brain barriers to reach placenta and require higher safety standard. methods: in the present study, we engineered mammalian extracellular vesicles (evs) to deliver a host restrictive factor, interferon-induced transmembrane protein 3 (ifitm3), for the treatment of zikv infection. results: our results demonstrated that the engineered ifitm3-containing evs (ifitm3-exos) were overall safe to the animals and suppressed zikv viraemia by 2 log10 s in the pregnant mice. moreover, the engineered evs effectively delivered ifitm3 protein across placental barrier and suppressed overall zikv viraemia in the foetuses to the basal level with significant reduction of viraemia in key foetal organs as measured by q-pcr. mechanistic study showed that ifitm3 was delivered to the endosomes/lysosomes where it inhibits viral entry to the host cells. summary/conclusion: our study demonstrates that exosomes can act as a cross placenta drug delivery vehicle to foetus and ifitm3, an endogenous restriction factor that is highly expressed in placenta, is a potential treatment for zikv infection during pregnancy. introduction: extracellular vesicles (ev) are natural and abundant nanoparticles capable of transferring complex molecules between neighbouring and distant cell types. translational research efforts have focused on co-opting this communication mechanism to deliver exogenous payloads to treat a variety of diseases. important strategies to maximize the therapeutic potential of evs include payload loading, functionalization of the ev surface with pharmacologically active proteins, and delivery to target cells of interest. methods: through comparative proteomic analysis (lc/ms) of purified evs, we identified several highly enriched and ev-specific proteins, including a transmembrane glycoprotein (ptgfrn) belonging to the immunoglobulin superfamily. leveraging ptgfrn as a scaffold for surface display, we generated evs with functional targeting ligands, including single domain antibodies (sdabs), single chain variable fragments (scfvs), single chain fabs (scfabs), and receptor ligands, on the surface to direct ev uptake to cell types of interest. biological activity of these engineered evs was assessed in an array of in vitro and in vivo assays and compared to untargeted controls. results: we engineered evs displaying anti-clec9a scfabs to target conventional type 1 dendritic cells (cdc1s), anti-cd3 scfabs to target t cells, and cd40 ligand to target b cells. in mice, systemic administration of anti-clec9a evs resulted in a 75% increase in the percentage of cdc1 cells that take up evs over controls. anti-cd3 evs resulted in both an increase in the percentage of ev positive t cells (3.75 and 3-fold for cd4+ and cd8+) and the number of evs per cell (15 and 7-fold for cd4+ and cd8+) in the blood. furthermore, in primary mouse dendritic cells, anti-clec9a evs loaded with sting agonist achieved a 15fold greater pathway induction compared to untargeted controls. preliminary in vivo data suggest that anti-clec9a evs reduce the required sting agonist dose 10-fold to achieve efficacy and induce anti-tumour responses, compared to control evs. summary/conclusion: these results demonstrate the potential of our ev engineering platform to generate novel ev therapeutics targeted to cell types of interest for pharmacologic payload delivery. a novel method for the delivery of cell-free therapy to foetuses with congenital anomalies: a proof of principle study lina antounians, louise montalva, gabriele raffler, maria sole gaffi and augusto zani the hospital for sick children, toronto, canada introduction: antenatal cell-based therapies are currently considered invasive for the foetus. a promising cell-free strategy that holds great regenerative potential for several organs is the administration of stem cell derived evs, whose cargo contains bioactive molecules that epigenetically regulate target cells. herein, we aimed to 1) assess the ability of evs to reach foetal organs when administered to the mother intravenously or intra-amniotically; 2) compare these administration routes on normal foetuses and foetuses with a congenital anomaly. methods: evs were isolated from rat amniotic fluid stem cell conditioned medium using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd63, hsp70, flo-1, and tsg101 (western). we injected rat dams with evs stained by exoglow™-vivo or saline (control) via maternal tail vein (iv) or intra-amniotically (ia) at e20.5. ia and iv injections were performed on dams carrying normal foetuses or foetuses exposed to nitrofen to induce congenital diaphragmatic hernia. after 24h, dams and pups were sacrificed. 3d high-sensitivity optical reconstructions of whole foetuses or micro-dissected foetal organs were imaged using the ivis® spectrum imaging system. ev fluorescence signal was compared between normal (n = 27) and nitrofen-exposed (n = 45) foetuses. results: both iv and ia injection routes were successful in delivering evs to foetal organs. no fluorescent signal was detected in saline only control. ia injections yielded higher signal than iv, and evs reached more organs with ia than iv injections. ia injected evs were detected in the lungs, gastrointestinal, and urinary tract of normal and nitrofen-exposed foetuses. nitrofen exposed foetuses had higher signal than normal foetuses. summary/conclusion: this proof of concept study shows that antenatal administration of stem cell evs is feasible with different routes. although maternally administered evs cross the placenta, ia injection is more effective at reaching foetal organs. further studies are underway to reproduce these findings in experimental models of various congenital anomalies. funding: cihr-sickkids foundation grant os22.5 introduction: safe, efficient and specific nano-delivery systems are essential to the current cosmetic, nutraceutical and therapeutic medicine sectors. the ability to optimise the bioavailability, stability, and targeted cellular uptake of bioactive molecules while mitigating toxicity, immunogenicity and off-target/side effects is of the utmost priority. ves4us is a european project, which aims to develop an innovative platform for the efficient production of extracellular vesicles (evs) from microalgae, which constitute a promising renewable bioresource (www.ves4us.eu). here we present characteristics of evs from several microalgal lineages, which offer the opportunity for a potentially developing a new and scalable tailor-made biogenic nanotechnology. methods: we cultivated a number of ev-producing microalgal species and developed protocols for ev isolation both at laboratory (differential ultracentrifugation) and pilot scales (tangential flow filtration). the physico-chemical characterization of microalgal evs was carried out according to the minimal information for studies of extracellular vesicles 2018 (misev-2018 guidelines): biochemical methods to verify the presence of specific ev-biomarkers, tuned for microalgal evs; dynamic light scattering (dls) and nanoparticle tracking analysis (nta) to assess the particles number and size distribution; electronic scanning microscopy (sem), atomic force microscopy (afm), and cryo transmission electron microscopy (cryo-tem) for imaging analyses; bilayer-specific fluorescence staining (f-nta) to test the purity of ev preparation. results: we identified microalgae as a novel natural source of evs that could constitute a cost-effective and sustainable way of mass-producing them. we screened 20 strains of microalgae and generated an "ev identity card" for each, which contained a variety of ev features relating to their biophysical, biochemical and biological characteristics in line with the misev-2018. our approach will next focus on the scalable production, surface functionalization and bio-engineering of selected microalgal evs. at the same time, their bioactivity will be explored using both in vitro and in vivo biological models. summary/conclusion: the ves4us consortium is investigating the potential of microalgae as novel ev bioresources. this research will attempt to bioengineer novel naturally-derived nanocarriers, microalgal evs, suitable for the development of future cosmetics, nutraceutical or therapeutic formulations. funding: this project has received funding from the european union's horizon 2020 research and innovation programme under grant agreement no 801338. sequence-specific rna trafficking to extracellular vesicles is conserved across cell types several sequences have been identified that act as a zipcode for preferential rna targeting into ev (evtropic) or for retention in parental cells (cell-tropic) . in this work, we aimed to compare the ev-tropic capacity of specific rna sequence motifs in promoting loading into ev, across different cell models representing the main cell types found in the body. methods: immune, epithelial and mesenchymal cell lines were transiently transfected with xenogeneic c. elegans micrornas (mirnas) containing ev-tropic or cell-tropic sequences and grown in culture. ev were isolated from the supernatant by differential (ultra)centrifugation. rna was extracted from both cell pellets and isolated ev fraction, and target mirnas were quantified by digital droplet pcr. distribution of cargo mirna across cells and ev was also analysed for chimeras of ev-and cell-tropic sequences. results: the mirnas containing an ev-tropic sequence were highly enriched on the ev fraction, with 1000-10,000 higher levels than in parental cells. contrarily, cell-tropic mirnas were only 10-100 times higher in ev. no significant differences were observed in the ev loading efficiency for the various ev-tropic motifs tested. mutations in the ev-sorting motif resulted in reduced ev loading. ev-tropic sequences consistently promoted mirna loading into ev across all the cell models evaluated, suggesting conserved biological mechanisms. summary/conclusion: we showed that rna loading into ev is dependent on the presence of defined evtropic rna motifs, and that sorting mechanisms are conserved across the major cell types tested. the highest loading efficiencies resulted in 0.001 mirna copies per particle on average, suggesting a limited scope for ev-tropic motifs for therapeutic rna loading into ev. funding: as, os and eli are fellows of the astrazeneca postdoc programme. introduction: coordinated activity between pancreatic islet cells is critical for the regulation of glucose homoeostasis. chronic exposure to diabetogenic factors such as pro-inflammatory cytokines, perturb islet cell crosstalk and β-cell function in diabetes. extracellular vesicles (evs) derived from cytokine-exposed β-cells modulate physiological and pathological responses to β-cell stress. however, the mechanisms governing this process remain largely unknown. we set out to test the hypothesis that β-cell failure in diabetes is mediated in part through β-cell autocrine release of pro-inflammatory evs which promote inflammation and inhibit βcell function. methods: pro-inflammatory cytokine-exposed evs (cytoevs) were generated using conditioned media from mouse min6 β-cell line treated with diabetogenic cytokines (tnfα, il-1β, ifnγ, 48h). evs were also isolated from human type 2 diabetic (t2dm) and lean non-diabetics (lnd) plasma. gw4869 (n-smase inhibitor) was used in the presence of cytokines to determine the effect of reduced ev concentrations on the restoration of β-cell function. proteomic and rna-seq analysis was conducted on min6 β-cell cytoev (vs. control ev) and cytoev treated mouse islets, respectively. results: assessment of ev concentrations from cytoev and human t2dm plasma revealed a~twofold increase (p < .05, vs. control (ctl) and lnd ev). immunofluorescence staining of cd9 and cd63 expression was significantly elevated in human t2dm pancreas (p < .05, vs. lnd). while acute inhibition of ev formation with gw4869 (5 µm) showed significant restoration in β-cell function (glucose stimulated insulin secretion assay, gsis) in cytokine-exposed mouse and human islets (~7 and 2 fold vs. cytokines alone, p < .05). moreover, functional assessment of mouse islets exposed to cytoev (48h) resulted in suppression of gsis (~55%, vs. untreated, p < .05). identification of cytoev content through proteomic analysis revealed a significant upregulation of the chemokine, cxcl10 (~40 fold vs. ctlev) and rna-seq analysis of cytoev treated mouse islets depicted a marked upregulation of transcripts associated with cxcl10-cxcr3 signalling (p < .001) and downstream pathways (e.g. nfκb; p = .016 and jak/stat; p = .021). furthermore, inhibition of cytoev (gw4869) with cytokines markedly decreased cxcl10 (~30%) and cxcr3 receptor (~65%) expression in min6 β-cells. summary/conclusion: these data suggests that cytokines elevate cxcl10 expression in β-cell ev to enhance inflammation-induced diabetes. this is mediated through ev-autocrine release of cxcl10 consequently activating cxcr3 signalling and downstream pathways to impair β-cell function in diabetes. synergy between 15-lipoxygenase and secreted pla2 promotes inflammation by formation of tlr4 agonists from extracellular vesicles introduction: damage associated molecular patterns (damps) are endogenous ligands that induce innate immune response, thus promoting sterile inflammation. during oxidative stress, stress-derived evs (stressevs) were found to activate toll-like receptor 4 (tlr4), but the activating ligands were not fully determined. additionally, several enzymes, among them 15-lipoxygenase (15-lo) and secreted phospholipase a2 (spla2) are induced during inflammation and were suggested to promote damp formation. methods: stressevs were produced from hek293 cells exposed to 10um a23187 and isolated with ultracentrifugation. 20:4 lysopi was oxidized for 10 min with 15-lo. additionally, synevs were prepared from phospholipids (pls), oxidized with 15-lo and hydrolysed with spla2. activity was measured by qpcr and elisa on wt and tlr4-ko macrophages. 15-lo oxidized 20:4 lysopi was analysed by mass spectrometry. spla2 activity was measured in synovial fluid from rheumatoid and gout patients using fluorometric assay. k/bxn serum transfer induced arthritis model on wt and tlr4 ko mice (c57bl/6 mice) with spla2-iia injection was used (approval no. u34401-14/2019/8 by mkgp of slovenia). results: stressevs released after oxidative stress were found to activate tlr4 with a gene profile different from bacterial lipopolysaccharide (lps). stressevs, 15-lo oxidized synevs, but only 15-lo oxidized lysopls activated cytokine expression through tlr4/md-2. hydroxy, hydroperoxy and keto products of 20:4 lysopi oxidation were determined by ms and they activated the same gene pattern as stressevs. furthermore, spla2 activity, which we detected in the synovial fluid from patients, promoted formation of tlr4 agonists after 15-lo oxidation. injection of spla2-iia into mice promoted k/bxn serum induced arthritis in tlr4-dependent manner. summary/conclusion: both 15-lo and spla2 are induced during inflammation, therefore these results imply the role of oxidized lysopls in stressevs in promoting sterile inflammation through tlr4 signalling. the formation of tlr4 agonists is enzyme driven so it provides an opportunity for therapy without compromising innate immunity against pathogens. funding: h2020-msca-itn project tollerant (grant no. 642157), slovenian research agency (project no. j3-9257 to mmk, research core no. p4-0176 to rj). monocytes traffic extracellular vesicles to damaged muscle and adopt a novel immunophenotype to support muscle regeneration russell g. rogers, akbarshakh akhmerov, weixin liu, lizbeth sanchez and eduardo marbán smidt heart institute, cedars-sinai medical center, los angeles, usa introduction: extracellular vesicles (evs) are secreted membrane vesicles that carry bioactive molecules such as mirnas, mrnas, proteins, and lipids to modify recipient cell behaviour. we recently demonstrated evs secreted by cardiosphere-derived cells (cdc-evs) augment endogenous muscle regeneration in mdx mice, a model of duchenne muscular dystrophy, when delivered intravenously. in parallel, macrophages preferentially accumulate surrounding small regenerating myofibers in cdc-ev treated mdx muscle. however, it is currently unclear how intravenous cdc-evs home to dystrophic muscle and exert their therapeutic bioactivity. methods: fluorescently-labelled and unlabelled cdc-evs were infused into the contralateral femoral vein of wild-type mice with unilateral muscle injury induced by bacl2. injured and uninjured muscles were dissected 24h following infusion and subjected to optical imaging, immunohistochemistry, and confocal microscopy. this experiment was repeated using clodronate liposomes to deplete endogenous monocytes/macrophages. next, rna-seq was preformed on bone marrow-derived m1, m2, and cdc-ev (mcdc-ev) polarized macrophages from mdx mice. conditioned media (cm) from these macrophages were tested in an in vitro model of myogenesis. lastly, small rna-seq was performed on evs secreted by m1, m2, and mcdc-ev macrophages. results: when delivered intravenously, cdc-evs naturally home to injured, but not uninjured, skeletal muscle. cdc-evs were detected in the interstitium adjacent to non-muscle cells, macrophages, and within surviving myofibers. after depletion of monocytes/ macrophages by clodronate liposomes, the presence of cdc-evs in the injured muscle was attenuated. bioinformatic analyses indicate cdc-evs confer a novel immunophenotype to mdx macrophages with features of both m1 and m2. indeed, mcdc-ev cm promotes myoblast proliferation and supports myogenic differentiation. interestingly, mcdc-ev evs have a unique mirna signature and contain several mirnas with known roles in myogenesis. summary/conclusion: these data indicate circulating monocytes traffic cdc-evs to damaged muscle where they adopt a novel immunophenotype to support muscle regeneration. we propose mcdc-ev macrophages mediate their pleiotropic effects via paracrine factors, possibly including evs. introduction: microglia, the immunocompetent cells of the cns, play an important role in maintaining cellular homoeostasis in the cns. these cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. the contribution of microglial-derived extracellular vesicles (m-evs) to the maintenance of cns homoeostasis is unclear. in addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to m-evs is scarce. methods: here, we analysed the effects of m-evs produced in vitro by either tnfα-activated or non-stimulated microglia bv2 cells. we showed that m-evs are internalized by both mouse bv2 and human c20 microglia and that the uptake of m-evs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. consistently, exposure of microglia to m-evs increased the protein expression of the autophagy marker, lc3b-ii, and promoted autophagic flux in live cells. to elucidate the biological activities occurring at the transcriptional level in c20 microglia exposed to m-evs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted rna sequencing. results: inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia exposed to m-evs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. summary/conclusion: we demonstrate that in vitro produced microglial evs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homoeostasis. funding: this work was financed by hasselt university and by efro through the interreg v grensregio vlaanderen nederland project trans tech diagnostics. evaluation of plasma extracellular vesicles as biomarkers for longevity xin zhang a and virginia kraus b a laboratory medicine center, nanfang hospital, southern medical university, guangzhou, guangdong, 510515, p. r. china, guangzhou, china (people's republic); b division of rheumatology, duke molecular physiology institute, duke university school of medicine, durham, usa introduction: extracellular vesicles (evs) have emerged as key indicators and effectors of ageing. although plasma concentrations of evs decline with age, the ev biomarkers associated with ageing and longevity are not fully understood. recently, our group found an age-related decline of plasma evs associated with immune cells during normal human ageing. our study aims to evaluate the association of plasma evs with longevity. methods: plasma samples were selected from the established populations for epidemiologic studies of the elderly study subjects (n = 48): half dying within 2 years (short-lived group) and half surviving ≥10 years (long-lived group) after the blood draw; all matched for age (median age 77.3 ± 1.7 years, range 72-80), gender (50% female), and race (50% white/50% black). the samples were acquired under donor consent and irb approval of duke university. evs were separated from the plasma samples, and profiled based on the surface markers of haematopoietic stem cells (hscs), mesenchymal stem cells, immune cells, skeletal muscles, cardiac muscles and adipocytes (cd81, cd9, cd29, cd63, cd8, cd4, cd68, cd14, cd56, cd15, cd19, cd235a, cd41a, cd34, cd31, hla-abc, hla-g, hla-drdpdq, cd90, cd73, cd105, m cadherin, ryr1, ryr2, fabp4, dlk1). the percentages of evs expressing each tested molecule were determined using a high-resolution multicolour bd lsr fortessa x-20 flow cytometer as we recently reported. graphpad prism 8.0 software was used for statistical analysis. results: we found significantly increased percentages of cd9+, hla-abc+, cd31+ and cd41a+ large evs (1000-6000 nm) in the long-lived compared to the short-lived group. none of the tested surface marker expressing medium (100-1000 nm) or small (<100 nm) evs showed differential percentages between the shortand long-lived groups. summary/conclusion: evs carry surface markers from their parent cells. cd9 is expressed by hscs and immune cells. cd9 regulates homing of human cord blood cd34+ hscs, and delivers a potent cd28independent costimulatory signal to activate t cells. hla-abc, the key human immunogen, is expressed by nucleated cells and platelets. cd31 is expressed by hscs, immune cells and epithelial cells, and cd31 + plasma evs declined with age in healthy people. cd41a is expressed by hscs, megakaryocytes and platelets, and is functionally relevant for hsc maintenance and haematopoietic homoeostasis. our preliminary data suggest that hscs and immune cell associated plasma evs (cd9+, hla-abc+, cd31+, cd41a+ large evs) inform on health status related to longevity. introduction: it is anticipated that stem/progenitor cells-derived extracellular vesicles (spc-evs) will rapidly progress towards clinical studies, and the development of reproducible, efficient, scalable and costeffective process for their production is expected to boost the therapeutic applications of evs-based products. in addition, the use of defined serum-/xenogeneic(xeno)-free culture medium formulations could result in substantial improvements for spc-evs production in terms of reproducibility, stability and quality, while ensuring the approval of regulatory agencies. the main goal of this work is to develop a full-controlled manufacturing platform for the spc-evs production. methods: human mesenchymal stromal cells (msc) were expanded in a xeno-free microcarrier-based bioreactor culture system operating in fed-batch feeding mode and after 10 days the conditioned medium was collected. different methods for spc-ev isolation/purification from the msc-derived conditioned medium, including chromatography were compared and the the quality of the final product obtained was characterized by different methods according to misev, including nanoparticle tracking analysis, lipidomics and western blot. moreover fourier-transform infrared (ftir) spectroscopy was evaluated in terms of its implementation as a standard technique for the identification and characterization of evs. results: after 10 days of msc expansion under dynamic conditions, we collected 1.3 l of conditioned medium with approximately 0.5 million evs/msc. a combination of a pretreatment with a nuclease for the digestion of dna/chromatin with a purification using strong anion exchange chromatography led to the best results so far in terms of evs isolation. of notice, by ftir spectroscopy, it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture conditions tested. summary/conclusion: the platform established herein could be applied to the production of wellcharacterized spc-evs targeting their biomedical use in different settings (e.g. as drug delivery systems), as well as evs from other parental cells lines (i.e. dendritic cells) in therapeutic settings as cancer. ultrasensitive protein detection for quantification of extracellular vesicles in human biofluids enables comparison of isolation techniques dmitry ter-ovanesyan, maia norman, wendy trieu, roey lazarovits, george church and david walt wyss institute, boston, usa introduction: extracellular vesicles (evs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. one of the main challenges in studying evs and using them in diagnostics is a lack of suitable methods to quantify evs that are sensitive enough and can differentiate evs from similarly sized lipoproteins and protein aggregates. we propose using ultrasensitive single molecule array (simoa) assays to quantify evs by immunoisolating and detecting ev transmembrane proteins in microwell arrays. we developed single molecule array (simoa) assays using the quanterix hd-x analyser for the quantification of evs using the tetraspanins cd9, cd63, and cd81. simoa allows for the detection of single proteins using arrays of femtoliter wells, turning elisa into a digital immunoassay. we then used these assays, together with an additional assay for albumin, to compare commonly used ev isolation methods from plasma and cerebrospinal fluid (csf): ultracentrifugation, precipitation (exoquick), and size exclusion chromatography (sec) using the izon qev columns. we further used these assays to rapidly optimize and improve sec by comparing different sec resins and column dimensions in both plasma and csf. results: in comparing our simoa assays to traditional elisa with the same antibodies, we found that the simoa assays were more than 100 times more sensitive, detecting the tetraspanins in samples where the proteins were undetectable by elisa. given the high dynamic range and high-throughput capabilities of simoa, we were able to comprehensively compare relative ev yields and ev purity for different isolation methods of evs from plasma and csf. we provide average tetraspanin and albumin levels to directly compare the methods. we also tested different sec resins and provide data for custom sec columns that outperform izon qev and allow for fine tuning of different ratios of evs to albumin. summary/conclusion: our results highlight the utility of quantifying evs using ultrasensitive simoa assays for tetraspanins. we were able to rapidly simoa to rapidly evaluate different ev isolation methods in csf and plasma. in general, the experimental framework we present could be easily applied to evaluate new ev isolation methods, or applied to any other biological fluid. thus, we think simoa is a powerful new tool for relative ev quantitation. introduction: the protein profile of extracellular vesicle (ev) subpopulations has been shown to contain valuable disease information, notably in cancer. currently, techniques aiming to find ev proteins that associate together mainly focus on transmembrane proteins, while methods that also probe cytosolic proteins generally resort to a combination of affinity capture, elution, and lysis, which limits throughput. to allow the high-throughput analysis of both membrane and cytosolic ev proteins, we optimized a total extracellular vesicle antibody microarray (tevam) incorporating fixation and heat-induced epitope retrieval (hier), then leveraged it to perform combinatorial protein profiling of evs from colorectal cancer (crc) cell lines ht29 and sw403. methods: arrays of iggs targeting surface protein markers were incubated overnight with evs purified from cancer cell line supernatants. hier optimization was carried out through variation of buffer contents, presence or absence of prior permeabilization, as well as incubation time and temperature, for a total of 38 conditions. a431 evs, previously profiled with other methods, were used as a model during the optimization. cytosolic protein hsp90 and membrane marker egfr, both with high expression in a431 evs, were probed and the results used to compare hier conditions. following hier treatment, protein targets were detected through incubation with primary antibodies and fluorescent secondary antibodies or streptavidin. the resulting optimized tevam workflow was used to phenotype ht29 and sw403 evs through probing of trios of surface (2) and internal (1) protein targets. results: the selected tevam protocol successfully maximized hsp90 signal while minimally affecting egfr detection, enabling simultaneous analysis of surface and internal proteins. profiles of more than 450 combinations, featuring integrins, claudins, cytokines, and other key actors of cancer-relevant pathways, were obtained for ht29 and sw403 evs, revealing coexpression patterns that highlight the biomolecular heterogeneity both within and between crc cell line evs. summary/conclusion: using tevam, intra-and extravesicular proteins can be detected simultaneously in evs immobilized based on surface protein content, yielding extensive combinatorial protein profiles with significance for health and biomarker research. characterization of evs using orthogonal techniques identifies discrete ev populations from a mouse dendritic cell line bryce killingsworth a , timothy traynor b , joshua a. welsh c , aleksandra dakic a , jason savage a , kevin camphausen d , kenneth aldape a and jennifer jones a a laboratory of pathology, national cancer institute, national institutes of health, bethesda, usa; b laboratory of pathology, national cancer institute, national institutes of health, gaithersburg, usa; c laboratory of pathology, national cancer institute, national institute of health, bethesda, usa; d radiation oncology branch, national cancer institute, national institutes of health, bethesda, usa introduction: extracellular vesicles (evs) have the potential to serve as valuable biomarkers for patient response to cancer therapy. however, development of robust ev-based clinical assays relies on knowledge of ev concentration and diameter distribution. many different methods exist to measure the size and concentration of evs, and each method exhibits strengths and limitations. it is important to use orthogonal methods for determination of these important properties of ev preparations. here, we use dendritic cellderived evs to demonstrate that some ev analysis methods can give a biased interpretation of both diameter and concentration. through comparison, we highlight why orthogonal assays are essential in providing measurement reliability. methods: dc2.4 mouse dendritic cells were cultured in flasks containing a total of 1.2 l of ev-depleted media (10% fbs, centrifuged 18 hr. x 100,000 g.) when cells reached 80% confluency, conditioned media was collected, depleted of debris with two 10 min. x 2,500 g spins, and concentrated down to~5 ml using a pall jumbosep 100 kda mwco filter. the ev concentrate was purified from protein using an izon qev-10 column, with 5 ml fractions collected. the protein content of the ev-containing fractions was analysed by a280, pierce bca, and bioanalyzer. the diameter distribution of the evs was determined by nanoparticle tracking analysis (nta), resistive pulse sensing (rps), flow cytometry (fcm), and electron microscopy (em.) concentration was compared using nta, rps, and fcm. evs were further analysed by protein mass spectrometry and rna sequencing. results: we have identified two distinct populations of evs with our dc2.4 preparation, one highly abundant population with a power-law distribution, whose peak diameter is below 60 nm, and a second, less abundant population with a peak diameter at approximately 140 nm. these two distinct populations and their relative concentration were not detectable with all analysis techniques. based on cross-platform measurements, these populations appear to have distinct compositions that warrant further investigation. summary/conclusion: the use of orthogonal methods allowed the detection of two discrete populations of evs which was not possible on some platforms and would have resulted in a biased perspective of the sample composition. this work has highlighted the need for orthogonal measurements to be conducted by pairing techniques that do not have the same biases. introduction: extracellular vesicles (evs) are nanosized vesicles shed by all cells that serve vital roles in cell-to-cell communication. tumour-associated ev subpopulations vary in molecular content (lipids, proteins, nucleic acids, small molecules), enabling minimally invasive spectroscopic analysis for a wide variety of cancers. here, we use surface-enhanced raman spectroscopy (sers) in combination with a novel plasmonic substrate for global chemical composition analysis of cancerous and non-cancerous populations of evs to determine distinguishing surface characteristics. methods: evs were isolated from ovarian cancer (ovca) patient serum samples by differential ultracentrifugation. a new hybrid nanoplasmonic scaffold comprised of a microscale biosilicate diatoms embedded with silver nanoparticles (agnps) was used for sers measurements. the substrate was incubated with cysteamine to positively-charge the agnps (responsible for the sers enhancement) so that evs could attach (evs are naturally anionic). in a typical experiment, 40 μl of~108 particles/ml evs per sample were incubated with the porous substrate surface, which was inverted on a glass cover slip for raman interrogation. principle component analysis (pca) was used to compare the spectra and determine distinguishing characteristics between populations from tumour and non-tumour sources. we also trypsinized evs before sers analysis to see the extent of influence the surface molecules play in localizing the evs to the agnp "hot spots." results: a total of 8 clinical samples (7 ovca and 1 non-malignant control) were tested in combination with ovca skov-3 cell line evs. simple pca was able to separate clinical samples according to disease subtype and major peaks were identified to provide chemical content analysis. each sample exhibited inherent heterogeneity but clustered together in a distinguishable way from the others. summary/conclusion: despite innate heterogeneity within single samples (i.e., evs isolated from a single patient sample), evs isolated from clinical samples could be easily distinguished from each other using our hybrid sers substrate, with minimal sample processing, a label-free approach, and only a few microlitres of sample. our study using this novel plasmonic material demonstrates its potential for use as a component in next-generation diagnostic platforms. introduction: single-particle analysis is critical for understanding extracellular vesicle (ev) heterogeneity. yet such techniques remain technically challenging due to low detection sensitivity and presence of variable amounts of "contaminants," including lipoproteins. the high degree of structural similarity between evs and lipoproteins in size, density, and chemical composition, results in their co-isolation using any of the standard ev isolation techniques. here we introduce laser trapping raman spectroscopy (ltrs) as a wellsuited, label-free, and non-destructive tool to distinguish evs from various lipoprotein species at single particle resolution. methods: ev samples were isolated from skov-3 cell culture supernatant by differential ultracentrifugation and their raman spectra measured. as the most abundant lipoproteins in ev isolations from human biofluids are sub-micron low density lipoprotein (ldl), very low density lipoprotein (vldl), and high density lipoprotein (hdl) particles, these were purchased as pure components and also measured by ltrs. ldl and vldl were then spiked-in to isolated evs to mimic "contaminated" post-isolation ev samples. raman spectra were analysed by principal component analysis (pca) using a custom matlab script. results: ldl and vldl have been observed to adhere to ev surfaces in vitro after standard isolation techniques. we could readily distinguish pure vldl, ldl, and hdl standards according to their raman spectra. pca revealed distinction of skov-3 evs from both ldl and vldl. pca also differentiated skov-3 evs incubated with ldl from skov-3 evs incubated with vldl. extent of ldl and vldl adherence to evs could be observed and quantified. summary/conclusion: through raman and pca, classes of lipoprotein and evs can be identified and quantified when co-incubated. ltrs is a quantitative single-ev analysis technique that can be used to differentiate between lipoprotein classes and evs when incubated together. this technique allows for analysis of evs where standard isolation methods fall short. introduction: extracellular vesicles (evs) are endogenous membrane-derived vesicles that shuttle lipids, proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the cns and contributing to neurodegenerative conditions. although evs hold great potential as cns theranostic nanocarriers, the specific molecular factors that regulate neuronal ev uptake and release are currently unknown. methods: we used a combination of patch-clamp electrophysiology and ph-sensitive dye imaging to examine stimulus-evoked ev release in individual neurons in real time. results: whereas spontaneous electrical activity and the application of a high-frequency stimulus (hfs) induced a slow and prolonged fusion of multivesicular bodies (mvbs) with the plasma membrane (pm) in a subset of cells, the neurotrophic factor bfgf (basic fibroblast growth factor) greatly increased the rate of stimulusevoked mvb-pm fusion events and, consequently, the abundance of evs in the culture medium. proteomic analysis of neuronal evs demonstrated bfgf to increase the abundance of the v-snare vesicle-associated membrane protein 3 (vamp3, cellubrevin) on evs. conversely, knocking-down vamp3 in cultured neurons attenuated the effect of bfgf on ev release. introduction: heat shock proteins (hsps) function as chaperones under both normal and pathologic conditions. as chaperones they assist in protein folding, in holding protein complexes for current or future activation, and in degradation of senescent proteins for recycling of components and display for immune surveillance. during stressful situations, hsp quantities and/or activities increase as cells and tissues seek protection from insults. these insults can result in the cell surface display of hsps, which can then lead to the surface display of hsps on extracellular vesicles (evs). hsps present on the cell surface or in the extracellular space are regarded as "danger signals" in an ancient biologic paradigm. hsp-accessorized evs may act as "danger boli", carrying not only the hsps, but hundreds of components of the stressed parental cell, capable of prompting differential responses depending on the status of the recipient cell. methods: clarified/filtered plasma from patients suffering from neurologic maladies (cancer, brain injury, multiple sclerosis) was incubated with peptides designed to bind hsps. the evs congeal under these conditions and are pelleted (microfuge) and washed with increasing-stringency buffers. we lysed the evs and subjected them to metabolomic analyses (focused on lipids) or assayed them on phosphokinase arrays. results: we show that evs from the blood of patients suffering from brain tumours, or from tbi, or from ms, possess distinct metabolomes compared to blood evs from healthy donors. we found hundreds of differentially-expressed lipids amongst the patients vs the healthy donors. the levels of annotation and identification for these compounds ranges from level 4 (low, no matches in databases) to level 2 (high, annotation matches to known database components). in addition, we found differences in phosphorylated kinases as cargo in these evs between patients with matched primary vs recurrent gliomas, and among tbi/stroke patients compared to healthy donors. summary/conclusion: hsp-accessorized evs present different metabolomic and phosphokinase content which may serve as biomarkers in a "liquid biopsy" setting, but may also play roles in the pathobiology of neurologic diseases. introduction: methamphetamine (ma) has deleterious effects to both peripheral organs and the central nervous system. the rewarding properties and addictive potential of ma are correlated with increased synaptic dopamine availability following alterations in dopamine and vesicular monoamine transporter function. in rodents, ma alters brain mirna expression and the mirna content of serum extracellular vesicles (ev). here we examined plasma evs isolated from human subjects actively using ma (ma-act) for size, concentration, protein markers, and mirna content. methods: plasma samples from 10 ma-act, and 10 controls (ctl) were obtained from the methamphetamine abuse research center. plasma evs were evaluated by vesicle flow cytometry (vfc) for size, concentration, and surface protein markers. vfc antibodies included markers for a pool of tetraspanins (cd9, cd63, and cd81), platelet evs (cd41), pro-coagulant evs (annv), and red blood cell evs (cd235). next plasma ev isolated by size exclusion chromatography were analysed by qpcr on taqman® array human microrna a + b card set v3.0. fold change was calculated by δδcq between ma-act and ctl for mirna expressed in ≥ 60% of samples in at least 1 group. we identified the top 20% of ranked mirna by fstatistic; of these, the mirna of interest for ma-act were identified by at least a (i) 1.2 fold change in expression, (ii) area under the receiver operating characteristic curve of 0.75, and (iii) glass's δ of 1. for mirna of interest correlations to additional ma variables were conducted, along with ingenuity pathway analysis of predicted gene targets. tobacco use was controlled for. results: vfc data show that the size (~110 nm) and concentration (~7.5 x 1010 particles/ml) of all plasma evs is comparable between ma-act and ctl groups. in addition, the plasma evs primarily consist of tetraspa-nin+, annv+, or cd41+ evs, and to a much lesser extent cd235+ evs. of the 207 mirna expressed in ma-act and/or ctl plasma evs, there were 110 mirna that have at least a 1.2 fold increase or decrease in ma-act. 10 mirna were identified to be of interest in ma-act based on fold change, effect size and diagnostic potential, compared to ctl. further, 5 of the 10 mirna correlate with a ma associated variable, including frequency of use and age of first use. together the 10 mirna best cluster subjects based on ma-act status, not tobacco use. finally, the predicted gene targets of the 10 mirna are associated with canonical pathways linked to ma. summary/conclusion: ev mirna expression in ma-act subjects was unique to ctl participants, suggesting that ma may affect ev communication among cells. the differential mirna expression also implicates a role for evs in behavioural and physiological effects specific to ma, and suggests that there may be changes in expression of mirna that are relevant to specific drugs of addiction, as well as to a spectrum of drug-mediated addiction disorders. bone marrow-derived extracellular vesicles may alter the ageing phenotype of murine haematopoietic stem cells. sicheng wen a , jill kreiling b , mark dooner c , elaine papa c , michael del tatto c , yan cheng c , mandy pereira c , peter quesenberry c and laura r. goldberg d in natural ageing of haematopoietic stem cells (hscs) is unclear. we tested the hypothesis that bone marrowderived evs (bm-evs) can modulate the ageing hsc phenotype. methods: we flushed bone marrow from old (24-26month old) and young (6-8-week old) c57/bl6 mice and collected bm-evs by differential centrifugation (2000 × g for 30 min, supernatant collected and centrifuged 100,000 × g for 1 hour, bm-ev pellet collected and quantified by nanoparticle tracking analysis). we injected old mice with 2 x 10^9 young bm-evs via tail vein, daily x 3 days. control mice were injected with age-matched bm-evs or vehicle alone. we euthanized the mice one month post-injection, harvested whole bone marrow (wbm) and highly purified hscs (lineage negative/c-kit+/sca-1+/cd150+; lsk-slam) and tested stem cell function in competitive bone marrow transplants (4-5 recipients/group). results: at 6 months post-transplant, wbm from old mice exposed to young bm-evs exhibited a statistically significant decrease in engraftment when compared to wbm exposed to age-matched bm-evs (percent average donor chimerism ± sem: 15% ± 5% (young evs) vs. 61% ± 14% (old evs)). for lsk-slam from old mice, we observed a trend towards decreased engraftment when exposed to young bm-evs and a trend towards increased engraftment potential when exposed to old bm-evs (percent average donor chimerism ± sem: 7% ± 4% (young evs), 27% ± 10% (old evs), 15% ± 2% (vehicle)). these findings are consistent with our previous data showing that, in contrast to highly purified hscs which develop impaired stem cell function with ageing, total un-separated old wbm actually has increased engraftment capacity when compared to young wbm. of note, we found that the classic myeloid skewing by old lsk-slam was partially reversed by exposure to both young and old bm-evs. finally, consistent with the known increase in highly purified hscs with age, our preliminary data showed that old mice exposed to young bm-evs had an approximately 7-fold decrease in the number of lsk-slam cells in marrow, indicating that bm-evs may influence agerelated changes in hsc number. summary/conclusion: these preliminary data suggest bm-evs may play a role in modulating hsc ageing phenotypes, potentially altering engraftment capacity, lineage fate, and lsk-slam population size. future studies delineating the molecular mechanisms underlying these ev-mediated effects could provide key insights into normal haematopoietic ageing. funding: this work was supported by the nih grants 5p20gm119943, dk112808-01a1 and by the savit foundation. oral with poster introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr. results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom20. mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the biogenesis of evs is neutral sphingomyelinase 2 (nsmase2), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase2 is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase2 inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles. methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo [1,2-b] pyridazin-8-yl) pyrrolidin-3-yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated 5xfad mice with 10 mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay. results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, 5xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting 20% of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human post-mortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of 20 mdd subjects and 20 hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed. results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd9. tem images showed the typical cup-shaped morphology with sizes mostly between 30 and 200 nm. preliminary sequencing results revealed that mir-33a-5p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir-33a-5p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. op3.04 = ps15.04 combining nanomagnetic isolation and artificial intelligence to guide the treatment of traumatic brain injury zijian yang a , kryshawna beard b , david meaney b , danielle sandsmark b , ramon diaz-arrastia a and david issadore c a university of pennsylvania, philadelphia, usa; b university of pennsylvania, philadelphia, usa; c department of bioengineering, university of pennsylvania, philadelphia, usa introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of 7 protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores (600 nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the 7 protein biomarkers (tau, uchl-1, nfl, gfap, il6, il10, and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl1 were elevated in mtbi patients compared to controls (p < 0.05), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il6 and tnf-with tau from glur2+ evs showed 88% accuracy with 80% sensitivity and 100% specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. l1cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l1cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l1cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l1cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l1cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd9, cd63 and cd81) as well as l1cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l1cam appears. we also immunocapture l1cam from csf and plasma and perform western blots for the internal and external domains of l1cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l1cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l1cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l1cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l1cam in plasma is likely not coming from the brain. this data call into question the utility of l1cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant 3d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed 3d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in 6-month-old wt mice, (n = 6/groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for 15% of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy5y cells with n-myc amplified sk-n-be2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. op3.08 = ps15.08 introduction: quantification and characterization of single extracellular vesicles (sevs) based on surface markers can aid in dissecting the heterogeneous landscape of ev subpopulations. we and others have demonstrated the potential of imaging flow cytometry (ifc) to perform sev characterization. we recently showed release of protoporphyrin (ppix) positive sevs by 5-aminolevulinic acid (5-ala) dosed glioma cells, in vitro and in vivo. rickfels et al. also used ifc to demonstrate the enrichment of cd63+/cd81+ evs in the plasma of glioma patients. herein, we performed in vitro studies to characterize ev subfractions using 5-ala as well as ev and cns specific surface markers. methods: we use ifc to characterize evs released by glioma using 5-ala, fluorescently labelled ev (cfda-se, cd81) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd81) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that 58% are tenascin c positive, 2.7% are egfrviii positive and 1.6% are 5-ala positive. there was only a minor overlap (<16%) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs (2.5-fold), tumour specific evs (2.8-fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: f. graminearum (fgr) and f. oxysporum f. sp. vasinfectum (fov) are severe fungal pathogens of cereals and cotton, respectively. fgr and fov cause economic losses and threaten food and fibre supplies worldwide. understanding host-pathogen interactions is crucial for developing new strategies for disease control. we are determining whether extracellular vesicles (evs) have a role in the interaction between fungal pathogens and their host plant. methods: we isolated evs from fgr and fov by sizeexclusion chromatography and characterized them by nta and tem. evs from fgr and fov are between 100-300 nm and have morphology similar to evs reported for other fungi. we performed label-free quantitative proteomics to describe the protein cargo of evs from fgr and fov, including a comparative study of evs from fov grown on different media: czapek dox (cd) and saboraud's dextrose broth (sdb). results: a total of 658 proteins were detected in fgr evs and, according to prediction software effectorp, 12.5% of these were potential effectors. similarly, 70% of ev proteins do not contain signal peptide indicating that packaging into evs is a novel mechanism of secretion for these proteins. notable fgr ev proteins include lipases, proteases and synthases for toxins and chitin. fov produced evs in similar quantities in both growth media tested, but ev protein cargo differed between them. there was a 39% overlap in proteins identified in the 465 cd and the 658 sbd ev proteins. in general, ev proteins were involved in metabolism, cell wall architecture and oxidoreduction, with 15.4% and 12.9% of potential effectors, respectively. polyketide and toxin synthases, proteases and effectors were present in both types of fov evs. summary/conclusion: this new fungal ev isolation method was rapid, yielded high-quality evs, and did not submit particles to high centrifugal forces. our data revealed that both fgr and fov produce evs enriched with proteins that could alter host immune responses or facilitate fungal infection. furthermore, the protein composition of fov evs was dependant on culture conditions. this supports a potential role for fungal evs in disease progression in plants and provides the foundations to pursue the role of evs in plant-fungal interactions with the potential to identify new targets for disease control. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll 2 receptor (tlr2) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il-12 and il-6, which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells. objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow fieldflow fractionation (af4) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp-1) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk-2 epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with 50 to 50 nm (ev1) and another with 100 to 120 nm (ev2). ev1 induced more tnf-alpha, il-6, ip-10 and ccl20 than ev2. it was also more effective in promoting t. cruzi infection in epithelial cells. summary/conclusion: t. cruzi released two ev populations that affects differently host cells. identification of these evs composition might help to better understand the role of evs in the modulation of t. cruzi infection funding: fapesp, cnpq and capes op3.11 = ps15.11 commensal bacterial extracellular vesicles act as carriers for norovirus sutonuka bhar, melissa jones, annalise galbraith and mariola edelmann university of florida, gainesville, usa introduction: human norovirus (hunov) are one of the most common causes of gastroenteritis and, along with inducing morbidity and mortality by diarrhoea, have a massive economic impact resulting in approximately 60 usd billion each year in healthcare costs and missed worker productivity. development of anti-viral therapies for hunov has been hampered by the lack of robust in vitro cultivation systems. several cell types support viral replication but only produce modest amounts of virus due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays. noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr. isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and 0.2um filtration. co-purification of norovirus with the evs was checked. ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid50 assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems. detection of bacterial extracellular vesicles in blood from healthy volunteers kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of 11.5 µg/ml of plasma, as geometric means ≥11.5 µg/ml were nearly equal. hevs were detected at 48 µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f200x electron microscope and measured between 40-90 nm, and hevs were between 60-160 nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: new zealand (nz) has a population of just 4.8 million people with a remote geographical location in the pacific ocean. its unique culture, food-based industries and ethnic population make nz an invaluable place for extracellular vesicle research into all areas. however, as for many places in the world, standardization of methodologies, training and access to appropriate equipment is challenging. methods: the hub for extracellular vesicle investigations (hevi) is a virtual research centre established in 2017 with three-year seed funding from a university of auckland strategic research initiatives fund. two staff members are employed to support training, education and optimization of methods. the hevi is guided by a governance group providing valuable input from australasian experts in evs. results: since 2017 the hevi has organized 2 research symposia, 2 hands-on training days, hosted 2 international students as well as providing one-on-one training for 41 individuals from universities and institutes across nz. training is provided on multiple isolation and characterization methods and tailored to individuals access to essential equipment without bias towards individual manufacturers or techniques. travel funding has supported 38 people to attend conferences and workshops for the purposes of education, networking and research dissemination. the hevi also provides support for project design with 10 grants awarded to hevi members and a number of manuscripts in submission for publication. the embo practical course "extracellular vesicles: from biology to biomedical applications" is organized each year by a group of 4 researchers active in the ev field in collaboration with the embl advanced training center in heidelberg. the course focuses on training phd students and postdoctoral researchers who enter or are already active in the field of ev research. given the large number of methods and protocols that is being used by researchers in the ev field, the organizers aim to provide practical guidance to new researchers and teach them appropriate skills. methods: participants obtain theoretical knowledge and hands-on experience on different ev purification and characterization techniques, such as fluorescent labelling, density gradient centrifugation, size exclusion chromatography, electron microscopy, flow cytometry and nanoparticle tracking analysis and on databases like ev-track and funrich. in addition, the organizers and invited lecturers from several different research areas explain which strategies are used to understand the role of ev in biomedical applications and give an overview of the current state of the field of ev research. results: the course therefore covers a broad range of topics important in the ev field, including heterogeneity in ev subpopulations, mechanisms of ev cargo selection, ev biogenesis, pre-analytical variables, therapeutic and diagnostic use of ev, and in vivo functions of ev. group discussions are facilitated and stimulated via assignments to analyse data obtained during the practicals and to critically evaluate literature. participants also have the opportunity to present their own research during poster presentations and ask for feedback from organizers and invited lecturers. summary/conclusion: among the participants selected for the course, a large geographical distribution is reached, including researchers from the newer eu member states and from outside of europe, to ensure a broad geographical distribution of the knowledge gained during this course. introduction: on 25 october 2019, we organized the 1st lugano exoday, first initiative in the southern switzerland to bring together resident researchers and european experts in the field of extracellular vesicles (evs). the workshop, centred on "technical challenges of extracellular vesicle research" aimed to highlight technical requirements and advances in the evs area, focusing on isolation, characterization and tracking. methods: the workshop started with a lecture by dr. cecilia lässer, from the university of gothenburg. the rest of the workshop was divided in two working groups (wg), each introduced by a keynote lecture followed by presentations by young researchers and a round-table discussion. wg1, introduced by dr. mercedes tkach, from the institute curie in paris, focused on recent advances on evs characterization and isolation. wg2 was centred on evs tracking and introduced by dr. frédérik verweij, from the institute of psychiatry and neuroscience of paris. results: dr. lässer opened the workshop with a comprehensive review and introduced recent developments in the evs field. the first wg discussed different isolation methods, focusing on ultracentrifugation, size exclusion chromatography and immunoprecipitation-based techniques. supported by the keynote speakers, the participants agreed that the best approach to optimize the isolation process consists in the combination of different techniques. wg2 shared insights about new strategies to visualize and tracking evs, focusing on how to improve the routinely approaches used, defining optimal criteria for evs labelling and imaging. all the participants had an in-depth overview on the requirements and the state-of-the-art techniques currently in use for the isolation, characterization and tracking of evs. summary/conclusion: the transferable knowledge acquired during the workshop ensures participants to remain up-to-date with the advances in the field of evs. as our ultimate goal is to create a competence centre in southern switzerland around the field of evs, the workshop was an invaluable opportunity to intensify collaborations between resident laboratories and broaden the scientific exchange with laboratories of the experts hosted during the event. given the success of this first workshop we are already working to prepare the second edition and make the event a recurring appointment. funding: supported by the swiss national science foundation the role of core facilities and emerging technologies in maximizing rigour and reproducibility of ev quantification and characterization and following misev guidelines rachel derita a and andrew hoffman b a thomas jefferson university, philadelphia, usa; b university of pennsylvania school of veterinary medicine, philadelphia, usa introduction: it remains very clear in the field of extracellular vesicle (ev) research that the rapid rate of increase in publications and expansion of interdisciplinary clinical ev interest has created the need for increased standardization and access to the appropriate technologies to uphold these standards. as the first core facility in the usa with the sole intention of creating a space where users can both isolate and characterize evs, we provide a central location for the facilitation of ev research via access to multiple technologies (both established and emerging) such as resistive pulse sensing, nanoparticle tracking analysis, ultracentrifugation, high-performance liquid chromatography, flow cytometric analysis of evs and additional immune or fluorescence-based ev characteri zation techniques. methods: we surveyed a group of leading scientific investigators and researchers in varying stages of their scientific careers in the mid-atlantic region of the us. the survey data demonstrate applications of greatest current and future interest to be employed in a shared lab resource. results: the current demand is highest for isolation services, ultracentrifugation and nta, with a gradually increasing demand for immunophenotying analyses such as the exoview chip array, fluorescent nta and flow cytometry. we additionally present strategies and data-based examples of how shared resource facilities can facilitate multifactorial and rigorous ev characterization in accordance with misev guidelines, and encourage collaboration among ev researchers. summary/conclusion: in order to answer the larger remaining questions in the ev field such as the isolation of specific ev subsets, ev tracking between cells and the use of evs for biomarker discovery and drug delivery, it is essential that shared resource facilities interact not only with investigators, but with each other to integrate the necessary resources to progress. programme to assess the rigour and reproducibility of extracellular vesicle-derived analytes for cancer detection national cancer institute, rockville, usa introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par20-053, is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par16-267/ 277, successfully funded 7 r01 and 4 r21 grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, 2018); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, 2019). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, 2019). summary/conclusion: drs. sudhir srivastava and matthew young are the program directors for the par which began accepting applications on 5 january 2020. this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute introduction: early detection of cancer as well as monitoring cancer treatment are important to improve cancer care. diagnostics for cancer are mainly based on tissue biopsies and re-biopsy during treatment is challenging. moreover, current diagnostics are expensive, time-consuming and have low-throughput. therefore, liquid biopsies are expected to bring the next breakthrough in cancer diagnostics. in liquid biopsies tumour-secreted material is isolated from body fluids and subsequent analyses thereof allow for non-invasive diagnostics. one type of tumour-secreted materials are extracellular vesicles (evs), which are schedded from tumour cells. evs are surrounded by a lipid bilayer, whichs composition resembles the plasma membrane of their parental cell. as many tumours are driven by over-expression or upregulation of transmembrane proteins e.g. growth factor receptors, detection of the later on evs holds promise for early tumour detection and treatment monitoring. methods: for the immuno-pcr evs were first affinitycaptured on magnetic beads, allowing immobilization of purified evs as well as evs secreted into cell culture medium or spiked into plasma. afterwards each sample was divided and affibody-dna-conjugates directed against different targets were added. affibodies are small affinity proteins, which often are developed as high affinity binders for tumour imaging, making them suitable probes in the presented assay. after washing, the bead-ev-affibody-dna-complexes were analysed for the immobilized dna-amount via qpcr. results: via the presented immuno-pcr evs secreted from the non-small cell lung cancer cell line h1975 as well as the ovarian cancer cell line skov3 were analysed. the immuno-pcr method allowed the detection of the tumour-associated membrane receptors epidermal growth factor receptor (egfr), receptor tyrosineprotein kinase erbb2/her2 and insulin-like growth factor 1 receptor (igf1r). different levels of membrane receptors depending on the ev source and concentration were detected. summary/conclusion: the presented immuno-pcr showed to be a comparably fast and robust method for detection of tumour-associated membrane receptors on evs derived from cancer cell lines with medium through-put and is currently further developed into a method for liquid biopsy for non-small cell lung cancer patients. introduction: introduction. evs produced by cells can originate from different cellular compartments and evs in complex biofluids may originate from many different cell types. traditional biochemical analysis, which reports on the total composition of all evs in a sample can't adequately resolve this heterogeneity. single vesicle analysis methods can, if they have the necessary specificity, sensitivity and speed. flow cytometry (fc) is capable of rapid and quantitative analysis of individual particles, but conventional fc-based assays lack the specificity and sensitivity to measure individual evs. assays that combine sensitive instruments with ev-selective sample staining can measure individual evs with accuracy and precision. to better understand the nature and origins of ev diversity, we used single vesicle fc (vfc) to quantitatively measure vesicle number, size, and surface cargo expression on individual evs. methods: methods. evs in culture supernatants (293 t, a431, u87, thp-1, sh-sy5y) were used neat or enriched by standard methods including differential centrifugation or ultrafiltration. evs from platelets (plt) and red blood cells (rbc) were induced by ionophore treatment of washed cells, and measured in diluted supernatant. evs were stained with a membrane-selective dye and fluorescence-labelled antibodies using a commercial vfc assay kit (cellarcus biosciences), measured using a commercial flow cytometer (cytoflexs, beckman coulter), and data analysed using fcs express v6 (de novo software). vesicle size, fluorescence intensity, and antibody binding were calibrated using appropriate vesicle and beadbased standards and essential controls performed as recommended by the miflowcyt-ev reporting guidelines. results: results. to assess the compositional heterogeneity of evs, we first characterized the expression of tetraspanins (tss; cd9, cd63, cd81, cd82, cd151, cd53, cd231) on evs released from cultured cell line and primary cell cultures. we find quantitative differences in the expression of ts on evs from different cell types that generally reflected the expression on the cell of origin, with most ev types expressing detectable amounts at least one of the common ts molecules (cd9, cd63 or cd81) but generally not all three. in evs from some cell types, ts expression was uniform across the ev population (cd9 on evs), but evs from other cell types differentially expressed tss, with some evs expressing no detectable ts (rbc evs). intracellular cargo labelled genetically using fluorescent proteins (egfp or mneongreen) or fluorogenic enzyme substrates (cfse) were measured in individual evs and revealed distinctive associations between ev surface and internal cargo. summary/conclusion: conclusions. high resolution measurement of cargo on/in individual evs can help interpret ev heterogeneity in terms of cell of origin, signals carried, and effects on target cells. integrated omics reveal conserved and divergent modulation of cardiovascular disease by tissue-entrapped extracellular vesicles introduction: fewer than 50% of patients develop both vascular and valvular calcification, implying differential pathogenesis. while circulating extracellular vesicles (evs) act as biomarkers of cardiovascular diseases, tissue-entrapped evs are implicated in early mineralization but their contents and function are unstudied. we developed an innovative method to isolate and study evs from fibrocalcific tissue and investigated entrapped ev cargoes in human cardiovascular diseases. methods: human carotid artery endarterectomies and stenotic aortic valves were obtained from 27 donors under irb-approved informed consent. tissues underwent enzymatic digestion, ultracentrifugation, and a 15-fraction optiprep density gradient. global proteomics was performed on intact tissue, each optiprep fraction, and ev-enriched pooled fractions; the latter also underwent mirna-seq. fractionated samples were also studied by cd63 immunogold electron microscopy (tem) and nanoparticle tracking analysis (nta). high confidence mir targets were predicted by targetscan, pathway analyses utilized the biocarta/kegg/reactome databases, and protein-protein interaction networks were built in string. results: vesicle-associated pathways were increased 5.1x (p < 0.01; 15/30 vesicle-related top go terms) in proteins common to intact arteries and valves (n = 1,411). proteomics found 24 ev markers to be highly enriched in the four least-dense optiprep fractions of arteries and valves, while extracellular matrix and mitochondria were predominant in denser fractions, as confirmed by tem/nta. proteomics and mirna-seq of tissue evs quantified 1,104 proteins and 123 mir cargoes linked to 5,182 target genes. pathway networks of proteins and mir targets common to artery and valve tissue evs revealed a shared regulation of rho gtpase and mapk intracellular signalling cascades. 179 proteins and 5 mirs were significantly altered between artery and valve evs (q < 0.05); multi-omics integration found that evs differentially modulated cellular contraction and p53mediated transcriptional regulation in vascular and valvular tissue. summary/conclusion: our findings delineate a novel strategy for studying tissue-entrapped ev protein and mir cargoes and identify critical roles that tissue-resident evs play in mediating cardiovascular disease. funding: this study was supported by a research grant from kowa company (ma) and nih grants r01 hl136431, r01 hl141917 and r01 hl147095 (ea). mir-20a regulates exogenous cd82 expression on proliferation, invasion, migration and angiogenesis of gastric cancer zhengzhou university, zhengzhou, china (people's republic) introduction: to investigate the possible mechanism of mir-20a regulating the expression of exosome cd82 on proliferation, invasion, migration and angiogenesis of gastric cancer, and to study the application value of cd82 in the early diagnosis and prognosis of gastric cancer. methods: the gastric cancer cell line mgc-803 was used as the research object. the exosomes were extracted from the culture supernatant of mgc-803 by exosome extraction kit. the extracted exosomes were identified by transmission electron microscopy and western blotting. the expression of cd82 in exosomes was detected by elisa. the expression of cd82 in exosomes and cd82 in whole blood and serum were detected by western blot. they were randomly divided into blank group (mock) and mir-20a lentivirus experimental group (mir-20a group). the lentivirus control group (mir-20a/con) was transfected into cells. qrt-pcr was used to verify the status of mir-20a after transfection; western-blot was used to detect the expression of cd82 and downstream erk1/2, akt and m tor proteins; mtt assay, cell colony formation assay, transwell migration assay for cell proliferation, invasion, and migration. a nude mouse xenograft model was constructed to observe the growth of transplanted tumours,microvessel density (mvd) was detected by immunofluorescence, and distant metastasis was recorded. results: the expression of cd82 in exosomes was detected by elisa and western blot. the expressions of cd82, akt, erk1/2 and m tor in mir-20a group were significantly lower than those in mir-20a/con and mock groups. cd82 protein is positively correlated with downstream protein levels.the growth rate and cell invasion ability of mir-20a group were significantly lower than those of mir-20a/con group and mock group. the weight of the nude mice in the mock group and the mir-20a/con group decreased, while the weight loss in the mir-20a group was not significant. the tumours in the mir-20a/con group and the mock group showed invasive growth accompanied by abundant microvessels, while the mir-20a group had smaller tumour volume and uniform cell distribution. only a small amount of microangiogenesis was observed, and no obvious necrotic area was observed. summary/conclusion: mir-20a affects the proliferation, invasion, migration and angiogenesis of gastric cancer mediated by akt/erk/m tor signalling pathway by regulating the expression of exosome cd82. streamlined detection and quantification of plasma extracellular vesicles and their protein cargo by high-performance nanoscale flow cytometry and label-free mass spectrometry introduction: nanoscale flow cytometry (fc) and mass spectrometry (ms) are useful for profiling ev surface proteins and performing bulk ev proteomics, respectively. this study sought to develop pre-analytical and analytical pipelines for ev protein profiling that are applicable to clinical studies. methods: to optimize plasma ev detection and quantification by fc, modifications of instrument settings and serial dilutions of platelet-free plasma (pfp) and antibodies were tested for improved separation of signal from noise and reduction of event coincidence and swarming. the high-performance flow cytometry (hpfc) platform was used to assess the effect of time (1, 2, 3, 5, 9, 24, 48 or 120hrs) between blood draw (into acd, nacit, edta or heparin) and blood processing, on ex-vivo release of evs from blood cells. label-free ms was used to examine the intensity and breadth of identified proteins in plasma evs purified using several density and size separation methods, either manually or automated, along with various buffer conditions. results: ev event aborts were minimized at a pfp dilution, prior to staining, of 1:10 and by using a narrow cytometer window extension. target ev signals were distinct from noise and were triton x-100 labile. the most significant changes in plasma evs were associated with platelet-derived fractions, use of heparin and >5-hour delay before blood processing. yet, platelet ev numbers did not significantly change for up to 24 hrs in citrated and edta plasma. higher overall coverage of known ev proteins and a fivefold increase in number of uniquely identified proteins were observed in ms profiling of evs prepared by a combination of ultracentrifugation (uc) and manual size-exclusion chromatography (sec) compared to preparation by fplc on capto core 700/superose 6 resins. uc/sec was better than direct sec at reducing contamination by excipient plasma proteins. column buffers with trehalose increased ev protein recovery while adding protease inhibitors had minimal effect. summary/conclusion: with our optimized hpfc protocol, we established that blood ev numbers remain stable for up to 24 hrs in acd or edta and that uc+sec with trehalose-containing buffer result in high canonical ev protein recovery. we are applying these workflows to investigate cancer-associated changes in plasma ev protein cargo. the value of exosomes as a potential biomarker for devil facial tumour disease. university of tasmania, hobart, australia introduction: the tasmanian devil (sarcophilus harrisii), the largest living carnivorous marsupial is endangered because of two transmissible cancers: devil facial tumour disease (dftd) one and two. current efforts to manage dftd are hindered by the lack of a preclinical diagnostic test for dftd. detecting dftd infection is only possible once tumours are noticed, too late to stop dftd progression. a preclinal test could tell us about unknown components of dftd pathogenesis, such as latent period and host-tumour dynamics. exosomes are extracellular vesicles released by most types of cells under both physiological and pathological conditions. exosomes have utility as diagnosis and prognosis biomarkers in a range of diseases, including cancers. the aim of this study is to investigate exosomes-based approaches towards a preclinical and progression biomarker for dftd 1 and 2 in tasmanian devils. methods: exosomes were isolated from three different dftd-1, dftd-2 and devil fibroblast cell lines by sizeexclusion chromatography. likewise, exosomes were isolated from plasma of healthy and diseased devils. to determine the size and morphology of exosomes, samples were imaged with transmission electron microscopy. exosomes isolated from cell lines and devil plasma were analysed with mass spectrometry to characterise proteins and determine their differential expression between the cell origins, and healthy and diseased animals. results: this study identified the presence of myelin proteins in exosomes from dftd cells relative to fibroblasts, which are diagnostic of dftd. additionally, we found that exosomes derived from dftd-2 abundantly express the inhibitory checkpoint molecule cd200 relative to exosomes from dftd-1 cells and devil fibroblasts, indicating a potential candidate for a differential diagnosis between tumours. moreover, exosomes from dftd cells present a greater amount of proteins related with metastasis in comparison with fibroblast exosomes, such as integrins. finally, we report the protein expression profile of exosomes from healthy and diseased devils, showing clear differences between them and the presence of immunosuppressive and metastasis proteins in animals in late stages of the disease. summary/conclusion: dftd-exosomes may provide a non-invasive diagnosis tool to detect early stages of dftd in tasmanian devils to facilitate the prevention of the disease. furthermore, dftd-exosomes may have utility as a prognosis biomarker, determining late stages of the disease using a simple a blood test, which would facilitate monitoring of wild populations. this project will provide long-term benefits for the future of the devils and encourage exosome-based solutions for other future wildlife disease outbreaks. introduction: despite the increased understanding of evs, from involvement in disease pathophysiology to therapeutic delivery, improved molecular tools to track biodistribution are largely lacking. current approaches used for ev labelling lacks sensitivity and specificity. here, we have explored bioluminescent labelling of evs to achieve a highly sensitive system for absolute in vivo quantification and tracking of exogenous evs at low cost and in a high throughput manner. methods: ev-producing cells were genetically engineered to express various tetraspanin-luciferase fusion proteins. evs purified by uf-sec from these cells were characterized by nta, multiplex bead-based array, tem and wb, followed by luciferase assay to determine the labelling efficiency. for in vitro applications cell lysate from treated cells or the conditioned medium were subjected to luciferase assay. for in vivo applications two different methodologies were applied to determine biodistribution; either by non-invasive real time in vivo imaging using ivis or by luciferase assay on harvested tissues for absolute quantification of injected evs. results: we initially performed a systematic comparison of five different luciferases for endogenous labelling of evs and identified nanoluc and thermoluc as lead candidates. we applied this technology to monitor in vitro cellular uptake and observed cell type differences in cellular uptake of engineered evs. in addition, we also observed an effect of different culturing conditions on exocytosis kinetics. for in vivo application, we applied the nanoluc labelling strategy to determine the pharmacokinetics and effect of different routes of injection on ev distribution. our results indicated a rapid uptake profile of administered evs in different tissues with liver, spleen, and lungs being the primary recipients. we also observed similar results upon tracking in vivo biodistribution in real time immediately after administration. finally, we show how different subpopulations of evs differ in their in vivo biodistribution. summary/conclusion: overall, nanoluc and thermoluc labelling of evs holds great potential for various in vivo and in vitro applications. in addition, it can enable the simultaneous detection of different subpopulations of evs in vivo, which may aid in our understanding of different sub-populations and their behaviour in vivo. apart from monitoring therapeutic evs, with one simple modification this platform offers great potential for tracking tumour derived evs both in vivo and in vitro and thus could aid in the development of anti-tumour therapies. biofunctional peptide-modified extracellular vesicles for cell targeting, macropinocytosis induction, and effective intracellular delivery ikuhiko nakase department of biological science, graduate school of science, osaka prefecture university, sakai-shi, japan introduction: [introduction] in our research group, developing therapeutic techniques based on extracellular vesicles (exosomes, evs) by effective usage of peptide chemistry to deliver therapeutic/diagnostic molecules into targeted cells has been focused. in this presentation, modification techniques using biofunctional peptides such as arginine-rich cell-penetrating peptides [1] , artificial coiled-coil peptides with receptor targeting [2] , and cell-penetrating sc18 peptides [3] derived from cationic antimicrobial protein, cap18 for cancer targeting with macropinocytosis induction, on the ev membranes will be introduced. i will also show effects of lyophilization of the peptidemodified evs on their biological activity [4] . methods: [methods] cd63 (ev marker)-gfpfusion protein expressed evs were used for cellular evs uptake assessments. all biofunctional peptides were synthesized by fmoc solid-phase method. results: [results] macropinocytosis with actin reorganization has been shown to be crucial for cellular ev uptake [5] . we developed the methods for modification of arginine-rich cpps or sc18 peptides on ev membranes using chemical linker techniques, and for example, arginine-rich cpps modification can induce proteoglycan-clustering (e.g. syndecan-4) and macropinocytosis signal transduction [1] . the artificial leucine zipper peptide-modified evs recognize the peptide-tagged epidermal growth factor receptor (egfr) on targeted cells, leading to macropinocytotic cellular ev uptake [2] . in addition, lyophilization is a useful technique for long term storage, however, we found that lyophilization negatively affected biological functions of encapsulated proteins in the evs after their cellular uptake [4] . summary/conclusion: [conclusion] these techniques and findings will contribute to development for the ev-based intracellular delivery systems. reference: [1] sci. rep. 6, 34937 (2016), [2] chem. commun. 53, 317 (2017), [3] chrmmedchem. 12, 42 (2017) [4] anticancer res. 39, 6701 (2019), [5] sci. rep. 5, 10300 (2015) os28.3 multi-compartmented microvesicles: novel extracellular secretory organelles that release exosomes and extracellular vesicles introduction: extracellular vesicles (ev) bud from the plasma membrane (pm) as microvesicles (mv) or arise from the fusion of multivesicular bodies (mvb) with the pm to release intralumenal vesicles (ilv) as exosomes. the variety of bioactive molecules carried by ev imparts diverse functionality to ev in intercellular signalling. the biogenesis and extracellular release of these specialized messenger organelles is not well understood. to investigate, we studied endothelial cells that line the inside of blood vessels, known to release ev that support angiogenesis. methods: cultured human umbilical vein endothelial cells (huvec) were examined by thin-section electron microscopy (em), serial sectioning and immunogold labelling to study the structure and composition ev release sites. to obtain optimal views of cellular ultrastructure, cells were preserved by fast-freezing and a freeze-substitution. results: a potential release site was identified in em thin sections as a discrete domain, up to several microns long, on the otherwise smooth huvec pm, where numerous bulbous membrane protrusions with thin necks were clustered. the cytoplasm in these protrusions was enriched with mvb and other vesicles and appeared to be on the verge of pinching off to release multi-compartmented mv (mcmv). consistent with this notion, in the neighbouring extracellular space, a plethora of mcmv of 300-1200 nm with ultrastructural features matching the bulbous protrusions were observed, supporting the concept that mcmv bud from the release site. serial sections confirmed that these extracellular mcmv were independent of cells and not linked by nanotubes or other processes. remarkably, fusion of mvb with the mcmv membrane was directly observed, presumably caught in the act of releasing ilv (exosomes) from the mcmv. immunogold labelling for ev markers is being used to identify proteins enriched at release sites and on released mcmv. summary/conclusion: in summary, 1) mcmv bud from localized sites on the endothelial pm, 2) mcmv contain mvb, and 3) fusion of mvb to mcmv to release exosomes occurs extracellularly. mcmv can now be evaluated as a potential source of exosome and ev release that occurs after budding from the cell of origin, adding new layers of regulation to when, where and how ev are assembled and released. funding: this work was supported by the division of intramural research of the nih. one size does not fit all: overcoming barriers to successful discovery and scaled manufacturing of therapeutic extracellular vesicles jieun lee a , wei guo b , hal sternberg c , mike west d and dana larocca d a stem cell team, seoul, republic of korea; b university of pennsylvania, philadelphia, usa; c agex therapeutics inc, alameda, usa; d agex therapeutics inc., alameda, usa introduction: extracellular vesicles have tremendous intrinsic therapeutic potential. however, the limited availability of production cell lines presents a barrier to scaled ev production and novel ev discovery. indeed, ev sources have been largely confined to a handful of cell types with the vast majority consisting of msc evs. to overcome this limitation, we developed a diverse library of hundreds of clonally pure and scalable progenitor cell lines that provides an alternative resource for ev drug discovery and production. methods: we harnessed the capacity of human pluripotent stem cells (hpsc) to differentiate into virtually any cell type by subjecting hpsc to a wide variety of media and culture conditions to maximize the diversity of partially differentiated cells. the resulting heterogeneous "candidate cultures" were plated at clonal density and further selected for self renewing and scalable clones. transcriptomic analysis indicated >200 distinct progenitor lines. cell fate potentials were mapped by screening for cell type specific marker expression in various differentiation conditions. evs were produced using cgmp methods (tff and sec) and characterized by nta, trps, surface marker analysis, rna and protein content. bioactivity assays included proliferation, migration, vascular tube network formation, senolysis, and oxidative stress. results: the progenitor library contained >200 distinct lines with diverse lineage fates including various types of bone, cartilage, muscle,and fat cells, as well as all blood vessel cell types. the lines displayed much longer replicative lifespans (70-100pd) than primary cell lines like msc. clonal purity minimized phenotypic drift resulting in maintenance of cell identity, genome integrity, differentiation capacity and bioactive ev production over extended culture. evs were highly diverse in their rna and protein cargo and bioactivity displaying various degrees of migratory, proliferative, angiogenic and senolytic activity. library screening identified evs with higher angiogenic potency than primary adult stem cell evs. summary/conclusion: we demonstrated scalable and stable production of bioactive evs from a large progenitor cell library. library screening resulted in discovery of novel angiogenic and senolytic evs having diverse rna and protein cargo. we are currently creating a corresponding library of progenitor cell evs to accelerate discovery of novel evs and their production cell lines. funding: the initial establishment of the cell library was funded in part by grants from the california institute of regenerative medicine and national institutes of health. introduction: besides extreme potential in biomedical applications, extracellular vesicles (evs) are also promising candidates to expand biophysical understanding of membrane active biomolecules. their complex bilayer composition allows the better understanding of adsorbed proteins and protein coronas as well, which sets of macromolecules will likely be key for advanced ev targeted delivery. considering cargo, membrane active peptides are interesting as these can be both drugs to be delivered, but can also facilitate cargo insertion through lipid bilayers. however, at present very little is understood regarding interactions between the peptides and the ev lipid bilayer, and between peptides and membrane associated proteins on evs. methods: we have recently demonstrated, that ev membrane adsorbed proteins and their interactions can be studied by techniques such as polarized light spectroscopy, microfluidic resistive pulse sensing measurements and freeze-fraction transmission electron microscopy [1] . furthermore, initially we studied several peptides with known antimicrobial properties and found that these strongly interact with the ev surface proteins, resulting in efficient removal of some from the lipid bilayer [2] . results: here we present investigation of further evpeptide interactions also focusing on anticancer peptides, which may be promising drug candidates for targeted delivery. these studies allowed to gain insight to novel functions of several peptides, such as melittin, magainin, buforin, lasioglossin, temporin, but also provide a more detailed understanding on how ev protein coronas, or ev bilayers are affected, to such extent that they cannot exert their potential function as delivery systems. summary/conclusion: the above interactions are expected to be interesting both for applicability, i.e. for selecting suitable compounds for ev processing, and also for curiosity-driven understanding of peptide functions, and ev-biomolecule interactions. based on these we promote that peptide -ev interactions will receive increased focus in ev-engineering. introduction: our late-breaking finding is the identification of a non-coding rna (ncrna) in extracellular vesicles (evs) from neuronal cells that is a natural antisense transcript for the dbh gene and associated with epigenetic changes and gene silencing. dna methylation in neurons is involved in memory and neurological disorders (science 2018 361 (6409)). earlier work found that during chronic brain infection with toxoplasma gondii induced a decrease in norepinephrine levels and expression of the host dbh gene; and the decrease is correlated with behaviours linked to noradrenergic signalling (infect immun. 2019 87 (2); infect immun. 2016 84 (2861)). dbh catalyzes the production of norepinephrine from dopamine in noradrenergic neurons. we found that evs from infected cultures suppress transcription of the dbh gene and hypermethylation of the gene in noradrenergic cells in vitro. in this study, we identify a ncrna in the evs from infected neuronal cells. methods: neuronal cells were induced by infection with toxoplasma gondii and evs purified on sucrose gradients. evs were characterised by electron microscopy and used to treat rat and human neuronal cells and expression levels of dbh mrna and nascent dbh gene transcription were measured. induced evs were injected into the locus coeruleus of rats and dbh gene expression was monitored. rna purified from evs was screened for natural antisense transcripts (nats) by strand-specific rt-pcr. results: we found that evs purified from infected neuronal cultures induced transcriptional gene silencing (tgs) and dna methylation of dbh in recipient neuronal cells. the induced evs down-regulated dbh gene expression >200-fold and induced dna hypermethylation of the dbh gene. this could be induced in the brains of recipient rats by intracerebral injection of evs. using a panel of strand-specific primers, antisense transcripts for the dbh gene were identified in infected cells. this permitted us to examine the rna in purified evs and identify a lncrna in evs selective for evs from infected cultures. summary/conclusion: this is the first study to find a specific neurotransmitter antisense lncrna in evs associated with transcriptional gene silencing and epigenetic changes in the gene. this represents a different type of neuron-to-neuron signalling than the classic chemical and electrical neurotransmission. the findings will enhance our understanding of neurological disorders (ie. schizophrenia, epilepsy, drug addiction) and how memory works. human cd4 + t regulatory-derived extracellular vesicles and associated micrornas: role in cell-to-cell communication and involvement in the loss of immune tolerance during multiple sclerosis introduction: an impairment of immune tolerance is a determining factor in multiple sclerosis (ms) and dysregulation of cd4 + t regulatory (treg) cell function is believed to be a major pathogenic factor. micrornas (mirnas) released by treg cells in association with extracellular vesicles (evs) have been shown to participate in the block of pathological immune responses by inhibiting the growth and cytokine production of cd4 + t conventional (tconv) cells, but the molecular mechanism is still poorly characterized. aim of the present work was to evaluate whether treg cell-derived ev-associated mirna signature is dysregulated in ms and whether this defect may play a role in the development of autoimmunity. methods: human treg cells isolated from blood of naïve to treatment relapsing-remitting ms patients and healthy controls were in vitro stimulated and released evs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, electron microscopy and flow cytometry. evassociated mirnas were quantified by traditional rt-qpcr and droplet digital pcr for absolute quantification. the actual ev-mediated passage of rna molecules from cell to cell was followed through rnaspecific fluorescent staining and treg-derived ev effect on tconv cell transcriptome was evaluated by rnaseq. results: in healthy conditions, the treatment of tconv cells with treg-derived evs was shown to cause the specific repression of genes involved in the proteasome-dependent proteolytic process, known to be crucial for t cell activation. in ms, treg-derived evs may have lost this capability as a direct consequence of a significantly decreased expression of mir-142-3p, able to target key factors of the proteasome system. summary/conclusion: our results unveil a novel molecular mechanism for treg-mediated maintenance of self-tolerance based on ev-associated mir-142-3p and its potential alteration in human autoimmunity. funding: fondazione italiana sclerosi multipla, fism, # 2016/r/10 and # 2018/r/4 revealing the proteome of brain derived extracellular vesicles isolated from human amyotrophic lateral sclerosis post-mortem tissues. introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterised by the deposition of misfolded proteins in the motor cortex and motor neurons. although a multitude of als-associated proteins have been identified, few have been associated with extracellular vesicle (ev) trafficking, a form of inter-cellular communication. additionally, the role of evs in als is undetermined, specifically in relation to pathogenic stress granule formation, a response to cellular stress involving aggregation of non-coding rnas and their rna binding proteins. therefore, this study aimed to determine the proteome of brain derived small extracellular vesicles (bdevs) isolated from als subjects and identify novel alsassociated deregulated proteins and their potential contributions to pathogenic pathways in als. methods: bdevs were isolated from human post-mortem als (n = 10) and control (n = 5) motor cortex brain tissues through an ultracentrifugation protocol (vella et al., 2017) . following thorough characterisation, bdevs that successfully met the minimum criteria required by the international society for extracellular vesicles were classified as evs. the bdevs subsequently underwent mass spectrometry analysis on the thermo scientific q-exactive hf with ultimate 3000 rslcnano. proteins identified to be statistically significant differentially expressed then underwent validation by western blotting. results: a panel of 16 statistically significant differentially packaged proteins were identified in the als bdevs. this included several up-regulated rna binding proteins and a down-regulated cell adhesion molecule; dhx30, stau1 and vcam1, respectively. pathway analysis revealed that the bdevs were enriched in proteins associated with stress granule dynamics, exosomal and lysosomal pathways. summary/conclusion: the identification of the rna binding proteins in the als bdevs suggests there may be a relationship between als-associated stress granules and als bdev packaging. the packaging of stress granule associated rna binding proteins into als bdevs may be an attempt by the cells to compensate for lysosomal dysfunction caused by stress granule accumulation, a feature of als. thus, these results highlight a potentially novel role for evs in the pathogenesis of als for long-term cultivation . the whole cultivation process of tissue preparation, cultivation, and cryopreservation has been established using strict serum-free conditions under a good manufacturing practice. long-term-cultivated hmnpcs retained stemness and hmnpcs have excellent differentiation efficiency into dopaminergic neurons. hmnpcs reversed impaired motor function in a rodent model of parkinson's disease (pd). based on the promising results in animal experiments, the clinical trial is under way (nct01860794). multiple-system atrophy (msa) is one of fatal neurodegenerative diseases with a combination of progressive autonomic nervous system disorders, parkinson's syndrome, and cerebellar pyramid syndrome. there are three types of msa such as msa-a, msa-c, and msa-p. in case of a msa-p type, it is difficult to diagnose due to the similarity of symptoms with parkinson's disease (pd). methods: in vitro and in vivo animal msa model were established and rotational behavioural was performed. npc cells were isolated and cultured based on moon et al. mirna sequencing (bgi) was performed and several bioinformatics analyses were done. results: based on the finding that hmnpcs exhibited therapeutic effects on pd, we hypothesize that hmnpcs will have a therapeutic effect on msa-p, where sympotoms are largely common with pd. as expected, transplanted hmnpcs survived, integrated, and differentiated in to dopamine neurons in the host brain, consequently leading to the functional recovery in the msa-p model. to further investigate the therapeutic key factors of hmnpcs in msa-p, mirna sequencing of the extracellular vesicles (evs) secreted from hmnpcs was performed. we found that mir-451a highly expressed in the npc-derived evs is one of key regulators of inflammatory response via nfkb pathway. we further experimentally demonstrated that mir-451a had anti-inflammatory effect on cells of msa-p condition such that the level of cx3cl1 expression and its receptor, cx3cr1 were both decreased in the msa-p modelled cells and in severe inflammatory environment in msa brain. summary/conclusion: our study first showed that mir-451a in hmnpcs-evs is one of key therapeutic factors for the recovery of brain damage through immuno-modulation in msa-p. introduction: oxidative insults are known to be involved in the pathophysiology of alzheimer's disease (ad). we have previously demonstrated that some blood-based redox-signature were associated to the cognitive scores in mild cognitive impairment patients and in ad (perrotte et al., 2019) . the aim of this study was (1) to evidence the presence of some oxidative markers in circulating extracellular vesicles (evs), and (2) to compare to their plasma levels. methods: plasma samples from healthy, mci and ad patients were from the memory clinic of sherbrooke (québec, canada). ad patients were stratified in three groups (moderate, mild and severe) according to the mmse and moca scores. total plasma extracellular vesicles (pevs) were isolated from plasma with the total exosome isolation reagent (invitrogen™ by life technologies inc.). pevs were then characterized by electronic microscopy, nta, dls and western blot. antioxidants apolipoprotein j, d (apo j, apod), the glyoxalase-1 and protein carbonyls were determined by western blot. results: in pevs, we found that apo d levels were higher in mci patients but not in ad patients. protein carbonyls levels were higher later, in pevs from moderate and severe ad while apo j levels were not different in pevs from the five groups of patients. in plasma, the pattern of apo j and apo d was different. the levels of apo d was not different in the five groups of patients while apo j levels were elevated in mci and in all ad groups. protein carbonyls were higher earlier from mild ad group, earlier than in pevs. the levels of the detoxifying enzyme glyoxalase-1 were higher in pevs than in plasma and were significantly decreased in early ad as compared to control subjects and mci summary/conclusion: these results demonstrate a differential regulation of redox homoeostasis in plasma and in pevs from ad patients. funding: acknowledgements: this work was supported by the chaire louise & andré on alzheimer's disease, foundation armand-frappier (cr) and cihr grant (tf). carlos j. nogueras-ortiz a , pavan bhargava b , sol kim b , francheska delgado-peraza a , peter calabresi b and dimitrios kapogiannis a a laboratory of clinical investigation, national institutes of ageing, baltimore, usa; b department of neurology, johns hopkins university school of medicine, baltimore, usa introduction: multiple sclerosis (ms) is a neurological disorder characterized by white matter demyelination and extensive synaptic pathology. recent studies have shown synaptic loss in the grey matter of ms brains in the absence of demyelinating lesions which could account for disease progression independent of demyelinating episodes. opsonization of synapses with complement components is a mechanism by which phagocytic cells normally prune synapses, but, when occurring in excess, it may underlie pathologic synapse loss. we sought to identify blood-borne biomarkers of hypothesized complement-mediated synaptic loss in ms using circulating neuronal-enriched and astrocytic-enriched extracellular vesicles (nevs and aevs). methods: nevs and aevs were immunocaptured in parallel from the plasma of 60 ms patients (45 with relapsing remitting, 15 with progressive ms) and 31 healthy controls, targeting the neuronal-specific marker l1cam and the astrocyte-specific marker glast, respectively. we measured the protein levels of preand post-synaptic proteins synaptopodin and synaptophysin in nevs using elisas and multiple complement cascade components (c1q, c3, c3b/ic3b, c4, c5, c5a, c9, factor b, factor h) in aevs using a luminex array. results: synaptopodin and synaptophysin protein levels in nevs of ms patients compared to controls were markedly reduced (2.5-fold; p < 0.0001 for both), whereas multiple complement components in ms aevs were markedly increased (c1q: 2.5-fold change; c3: 1.25-fold change; c3b/ic3b: twofold change; c5: 1.4-fold change; c5a: 1.4-fold change; factor: 1.5-fold change; p < 0.0001); differences were not observed in total circulating evs or neat plasma. strikingly, we found the nev-associated synaptopodin/synaptophysin and the aev-associated complement levels to be negatively correlated in people with ms (synaptopodin vs: c1q, r = −0.7 and p < 0.0001; c5, r = −0.6 and p < 0.0001; factor h, r = −0.46 and p < 0.0002/synaptophysin vs: c1q, r = −0.75 and p < 0.0001; c5, r = −0.65 and p < 0.0001; factor h, r = −0.52 and p < 0.0002), but not in controls. summary/conclusion: circulating evs provide markers of synaptic loss and complement activation in ms and suggest a link between astrocytic complement production and synaptic decline. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. methylglyoxal and glyoxal affect the protein cargoes in neuronal-derived extracellular vesicles introduction: advanced glycation end-products (ages) and their receptor rages are known to be involved in the pathogenesis of alzheimer's disease (ad). methylglyoxal (mg) or glyoxal (go) are the precursors of ages and particularly n-(1-carboxymethyl)-l-lysine (cml), the most abundant ages. mg induced tau hyperphosphorylation and causes hippocampal damage and memory impairment in mice. the aim of our study was to analyse the effects of mg and go on the neuroprotective, neurotrophic factors, inflammatory and neurodegenerative markers in the human cell line sk-n-sh and their release into the neuronal derived-evs. methods: briefly, sk-n-sh cells were incubated in fbs free media with mg and go (0.5 mm) for 24 hours. neuronal derived-evs (nevs) from culture media were isolated as previously described (haddad et al. 2019 ). nevs were characterized by electronic microscopy, nta and by western blot. cellular and nevs concentrations of bdnf, prgn, nse, app, mmp9, angptl-4, lcn2, ptx2, s100b, rage, dj-1 and alpha synuclein were determined by a luminex assay from r&d systems, inc. aβ1-40, aβ1-42, ptau t181 and total tau levels were measured also with luminex assay from emd millipore corp. results: we found that both ages precursors, at non toxic concentration, reduced the neuronal levels of nse with no effect on bdnf, ptrx-2, lcn-2, dj-1, on neurodegenerative markers and on cml. go decreased the levels of prgn, app, angpl-4 while the expressions of mmp-9 and angpl-4 were, respectively lower and higher in the presence of mg. mg and go greatly reduced the release of lcn-2 by neuronal cells in nevs. bdnf and prgn in nevs were reduced in the presence of go. both mg and go did not modify the release of nse, app, mmp9, agntl-4, ptx-2, dj-1, aβ, ptau and cml in nevs. summary/conclusion: our data demonstrated that mg and go differently affect the content of some protein cargoes in nevs and suggest that targeting mg and go may be a promising therapeutic strategy to prevent neurodegeneration. introduction: peripherally circulating brain-derived extracellular vesicles (evs) and their encapsulated rnas may serve as biomarkers for hiv-associated neurocognitive disorders (hand). however, rates of cigarette smoking are significantly higher in hiv+ individuals than the general population, and smoking can modulate the expression of these markers. to better understand how cigarette smoke might modulate rna expression and ev release, we examined several cnsderived cell lines, representing astrocytes (u87 mg), microglia (sv40), and oligodendrocytes (hog). methods: cigarette smoke extract (cse) was prepared by bubbling through culture medium using a standardized and published method. all cell types were exposed to either 0% or 50% cse for 24 hours. cell viability was assessed by musetm cell analyser, and evs were isolated from culture conditioned media (ccm) by size exclusion chromatography. the void (fractions 1-6), ev (7-10), and protein (11-14) enriched fractions were pooled and concentrated. evs were characterized by transmission electron microscopy (tem), microfluidic resistive pulse sensing, and western blotting. total rna was isolated from cells and circular rna (circrna) expression was assessed with a circrna microarray. results: in response to cse exposure, cell viability was only slightly reduced for all cell types. tem images validate the presence of vesicles in the ev fractions, and their absence in the void and protein fractions. spectradyne particle counts indicated cse exposure substantially increased the ccm particle count in the ev fraction when compared with control. the presence of expected ev markers (cd63, cd81, and tsg101) in the ev fractions, and their absence in the void and protein fractions was observed via western blot. intracellular circrna expression was significantly altered in all three cell lines. summary/conclusion: cns cells display physiologic responses to cse that include vesiculation pathways and significant alterations in circrna expression. we are now studying the effects of cse exposure on circrna expression in released evs. funding: this work is supported by da040385, da047807, and ai144997. a method for exosomal rna extraction from paired human brain and blood specimens emily n. moya a , lillian wilkins a , esther cheng a , lisa linares b , brian kopell b , navneet dogra c , bojan losic a and alexander charney a a icahn school of medicine at mount sinai, new york, usa; b mount sinai hospital, new york, usa; c department of genetics and genomic sciences, department of pathology, icahn school of medicine, mount sinai, new york, usa introduction: diagnosis and treatment of neuropsychiatric disorders has made little progress in the last half-century likely in large part due to the absence of a scalable technique to profile the complex biological activity of the brain in a living person. exosomes are nanovesicles 30-150 nm in size that mediate intercellular communication and contain proteins, lipids, and nucleic acids. it has been shown that brain derived exosomes can be found in peripheral blood, but determining whether peripheral exosomes truly reflect ongoing brain processes has to date not been possible due to the absence of paired living brain and blood specimens. here, we present a novel method for paired sampling of the dorsolateral prefrontal cortex (dlpfc) and peripheral blood from living human subjects for exosomal rna profiling. methods: informed consent, approved by the irb at the icahn school of medicine at mount sinai, was obtained for patients undergoing deep brain stimulation (dbs). paired brain and blood specimens were collected from 8 patients at two deep brain stimulation (dbs) electrode implantation procedures: left hemisphere followed by right hemisphere (total of 30 samples). we developed protocols to profile rna from exosomes of brain tissue extracellular matrix (ecm) and peripheral blood. exosomes were isolated via our in-house protocol using ultracentrifugation. rna was then extracted from the exosomes using the qiagen mirneasy mini kit protocol. quality control (qc) was performed to determine whether rna obtained was sufficient for next-generation sequencing. results: we demonstrate the safety of a novel procedure to sample the brain in living human subjects. bioanalyzer traces and qc data show a mean total rna of 14.83 ng (range 1.72-137.17 ng) and no samples fell below the threshold required for library preparation and sequencing (10 pg) determined by inhouse optimization on the smart-seq v4 ultra low input kit. summary/conclusion: to our knowledge, we have performed the first study to sample pairs of dlpfc and blood from living human subjects for exosomal rna for subsequent next-generation sequencing. ongoing analyses by our group promise to establish peripheral exosomal rna transcripts reflective of brain activity. this non-invasive approach to probing neurobiology in the living human brain may facilitate the development of exosome-based diagnostics for neuropsychiatric disorders. introduction: the relationship between obesity and dementia is complex. while obesity in middle age triples the risk of dementia 30 years later, many patients with alzheimer's disease (ad) are cachectic, and a decline in adiposity portends progression of dementia. this suggests adipose-derived factors are important to nervous system homoeostasis. we previously showed that adipocyte-derived small extracellular vesicles (ad-sevs) induce pathologies critical to developing obesity-related diseases and may provide a mechanistic link between adiposity and dementia. we hypothesized that altered expression of ad-sev micrornas involved in neurodegenerative pathways is associated with more severe cognitive impairment methods: we studied serum and cerebrospinal fluid (csf) from 19 participants with ad and 14 non-ad controls. ad-sevs were isolated from samples by precipitation and immunoselection. ad-sev microrna expression was profiled in both biofluids and compared. results: serum and csf microrna expression correlated strongly (r2 = 0.98). in serum, 189 micrornas were differentially expressed by a fold change ≥|1.1| in the ad and control groups (p ≤ 0.1) and 251 micrornas were differentially expressed in csf. using ingenuity pathways analysis, we identified mrnas expressed in nervous system tissue that are targeted by the differentially expressed micrornas. the mrnas map to 145 diseases and functions; neuronal cell death, neurodegeneration, and neuronal growth and developmental pathways are highly represented. of the 189 differentially expressed micrornas in serum, 6 were moderately correlated with participants' score on the mini-mental state exam, a test of cognitive function (rs = |0.488-0.609|). as validation, rencell cx cortical derived neuronal stem cells had decreased doubling time when exposed to ad-sevs from obese adipose tissue in vitro. summary/conclusion: these findings support our hypothesis that altered expression of circulating ad-sev micrornas are involved in neurodegenerative pathways associated with cognitive impairment. these findings support using serum ad-sevs as a surrogate for csf ad-sevs. functional validation is underway to define the connection between ad-sevs and ad. understanding the link between obesity and ad is crucial as the population ages and the global obesity epidemic grows. funding: supported by uw adrc (nih:p50ag005136) expression of extracellular vesicles after acute traumatic brain injury: an exploratory flow-cytometry study introduction: coagulation derangements related to disseminated intravascular coagulation (dic) are common after tbi and contribute to secondary neural injury. extracellular vesicles (evs) are released from all cell types, including platelets, endothelium, and lymphocytes, which are responsible for dic. we hypothesized that specialized flow cytometry techniques could identify a unique ev signature of dic in acute tbi. methods: using a modified flow cytometry instrument for detection of small particles, fluorescence panels were created to assess for evs from endothelial cells (cd144, cd105), platelets (cd31, cd62p, cd41a, cd42b), and erythrocytes (cd235) as well as brain biomarkers (s100b, uchl-1, gfap, tau and nse) and t-lymphocytes (cd3, cd4, cd8, cd31). samples were prepared in trucount tubes to determine volume and treated with triton to confirm presence of evs. results: 13/17 study patients and 16/20 controls were male. 76% of study patients presented with a glasgow coma scale of 15. in the hypercoagulability panel, of the 10 subsets with statistically significant differential expression, 4 involved s100b+ and were elevated in patients. platelet-derived cd41a evs and uch-l1 evs were significantly elevated in controls in 7 ev subsets identified in the brain-specific panel. finally, cd3+/31+ evs, derived from t-cells and identified in the endothelial/t cell panel, are significantly lower in patients suggesting cns recruitment. summary/conclusion: endothelial and platelet/erythrocyte evs may be elevated early after tbi. s100bcarrying evs are significantly elevated in circulation of tbi patients; if reproducible, this signature profile may be informative for diagnosis and risk stratification. further study is warranted to evaluate whether this expression correlates with secondary microvascular brain injury. funding: intramural award from the university of pennsylvania enrichment of mir-451a in cns extracellular vesicles following impairment of the blood brain barrier nasser nassiri koopaei a , ekram-ahmed chowdhury b , lais da silva a , jinmai jiang a , behnam noorani b , ulrich bickel b and thomas d. schmittgen a a university of florida, gainesville, usa; b texas tech university, amarilo, usa introduction: extracellular rnas (exrnas) are present in essentially all biofluids and include all types of rna including mirna. to enhance their stability outside of the cell, exrnas are bound within ribonucleoprotein complexes or packaged into extracellular vesicles (evs). the blood brain barrier (bbb) is a dynamic interface between the systemic circulation and the cns and is responsible for maintaining a stable extracellular environment for cns cells. the intent of this study was to determine if evs and their contents are transferred from the peripheral circulation to the cns under conditions of an impaired bbb. methods: the bbb of mice was disrupted by hyperosmolar mannitol injections. to validate that the bbb has been disrupted with mannitol, intravenously-dosed [13 c]-sucrose was increased in the forebrain by 14fold with mannitol compared to sham treated mice. evs were isolated from the forebrain, hindbrain and spinal cord following gentle tissue lysis and differential ultracentrifugation. evs were validated by nta, tem and western blotting. mir-451a, a mirna that is highly abundant in erythrocytes, was measured in the evs by qpcr. results: qpcr showed that mir-451a in cns tissue evs increased with mannitol treatment in the forebrain, hindbrain and spinal cord by 15-, 1.6-and twofold respectively. qpcr analysis of mrna from reported mir-451a target genes showed reduced target gene expression with mannitol. summary/conclusion: we demonstrate that evs containing mir-451a, a highly abundant mirna present within erythrocytes and erythrocyte evs, is enhanced in the cns upon bbb disruption. astrocyte-derived extracellular vesicles in morphine tolerance guoku hu, rong ma, naseer kutchy, yuetong zhao, susmita sil and shilpa buch university of nebraska medical center, omaha, usa introduction: opiates, such as morphine are used extensively in the clinical setting owing to their beneficial effects. paradoxically, however, the prolonged use of morphine often results in the development of tolerance, drug addiction, and ultimately leading to various comorbidities associated with drug abuse. although great efforts have been made, at present there is no treatment. the sonic hedgehog (shh) plays a key role in brain development, and brain cells fine-tuning processes such as their proliferation, patterning, and fate specification recent findings have demonstrated that inhibition of the shh signalling prevents morphine tolerance in rodent models. we thus hypothesize that extracellular vesicles (evs) derived from morphine exposed astrocytes and their cargo such as shh are critical for the development of morphine tolerance. methods: mice were received either saline or chronic morphine injection with escalating doses of morphine for 5 days (subcutaneously; 10 mg/kg, day 1, 20 mg/kg days 2-3, and 40 mg/kg days [4] [5] . the development of tolerance was assessed by measuring the tail-flick latency using tail flick analgesia metre (le7106, harvard apparatus). evs were isolated using either differential ultracentrifugation from astrocyte conditioned media or gradient ultracentrifugation from brain tissues. western blotting and qpcr were performed to determine the expression/activation of shh signalling pathway components. results: our data showed that the levels of shh protein were upregulated in morphine exposed astrocytederived extracellular vesicles (morphine-adevs). furthermore, shh containing morphine-adevs activated shh signalling in astrocytes. our in vivo study further demonstrated the upregulation of shh, as well as the activation of shh signalling, in astrocytes of morphine-administered mice. summary/conclusion: these findings thus demonstrated an autocrine mechanism for shh pathway activation in astrocytes associated with morphine tolerance. these findings could pave the way for the development of shh signalling pathway targeted strategies in the prevention and treatment for substance use disorders. biophotonics-based platforms for the evaluation of circulating extracellular vesicles as biomarkers of neurodegeneration in alzheimer's disease silvia picciolini a , cristiano carlomagno a , alice gualerzi a , monia cabinio a , francesca baglio a and marzi bedoni b a irccs fondazione don carlo gnocchi, milan, italy; b irccs fondazione don carlo gnocchi, milano, italy introduction: in the search for novel and non-invasive biomarkers of alzheimer's disease (ad), both circulating brain-derived extracellular vesicles (evs) and whole serum represent a valuable integration of the currently used classification system. to face the technological challenge of evs and serum analysis, we propose the use of biophotonics techniques as reliable, sensitive, fast and label free methods, potentially useful in tailoring pharmacological and rehabilitation treatments. methods: circulating evs, isolated by sec, and serum samples were collected from 10 healthy subjects (hc) and 10 ad patients. all subjects were asked to complete montreal cognitive assessment scale and mri examination. surface plasmon resonance (spr) was performed in order to detect evs coming from neurons, astrocytes, oligodendrocytes and microglia and to characterize each of them for the amount of ganglioside m1 (gm1), aβ and tspo expressed on their surface. serum analysis was performed using a raman microscope through the surface enhanced raman spectroscopy (sers) effect by mixing serum with ag nanoparticles. the pearson's correlation index was used to assess the linear correlation between spri data and clinical, mri data and data obtained from multivariate analysis (mva) of sers spectra. results: the spr analysis of evs showed that the selected bioactive molecules are differently loaded on neural ev populations and that their amount is increased on total evs in ad patients compared to hc. we observed a significant correlation between mva data from sers and the presence of aβ on neuronal and microglial evs and of tspo on neural evs, measured with the spr array. summary/conclusion: thanks to our methodological innovation we have verified the potentiality of evs as ad biomarkers, correlating biophotonics blood-based analysis with clinical data. this platform could provide a powerful tool for the evaluation of ad neurodegeneration. funding: the study was supported by the italian ministry of health (ricerca corrente 2017-2018 to irccs fondazione don carlo gnocchi). raman profiling of extracellular vesicles as new blood-based biomarker for brain disorders: focus on parkinson's disease introduction: extracellular vesicles (evs) play a pivotal role in brain homoeostasis and intercellular communication in both physiological and pathological conditions. in parkinson's disease (pd), evs are key players in the transfer of α-synuclein, with blood evs reported to undergo proteomic modifications. nonetheless, the detection and characterization of the ev cargo is technologically challenging, limiting the use of evs as biomarkers so far. herein, we propose raman spectroscopy for the label-free, bulk characterization of blood evs in pd patients. methods: evs were isolated by sec and ultracentrifugation from the serum of 18 healthy subjects (hc) and 22 pd patients. in all patients, the severity of pd was evaluated with the unified parkinson's disease rating scale (updrs) part iii and with hoehn and yahr scores (hy). after proper ev characterization following misev2018 guidelines, raman analysis was performed. the raman microspectroscope was used with a 532 nm laser in the spectral ranges 600-1800 cm-1 and 2600-3200 cm-1. data from hc and pd patients were compared by multivariate statistical analysis (pca-lda). results: the raman analysis of evs highlighted differences in the biochemical profile of the two groups, with the main variations in the spectral regions related to proteins, lipids and saccharides. a preliminary estimate of the accuracy of raman profiling of blood evs for pd diagnosis was obtained, demonstrating an accuracy of 71%. even more interestingly, we demonstrated the correlation between the raman spectra and the clinical scales (updrs and hy) used to stratify pd patients. summary/conclusion: in conclusion, the biochemical signature of blood evs can be detected by raman spectroscopy in pd patients and the ev spectral modifications can be related to their clinical status. these data suggest the possibility to use the raman profile of circulating evs as a biomarker for brain disorders, complementary to other specific molecular markers. funding: the study was supported by the italian ministry of health (ricerca corrente 2017 to irccs fondazione don carlo gnocchi) impact of circulating extracellular vesicles on brain functions and behaviours introduction: peripheral immune alterations have been described in psychiatric disorders such as schizophrenia, depression, and autistic spectrum disorders. in addition, behavioural changes have been observed in various immunodeficient animal models. however, the mechanisms by which peripheral immune system influences brain development and function are not well understood. in this study, we explored the mechanisms by which circulating extracellular vesicles (evs) mediate immune-brain communication and influence mouse behaviours. methods: mice deficient for rag1 or rag2 gene (rag ko mice) were used as a model to study the effects of loss of adaptive immune cells (t and b cells) on brain cellular phenotypes and behaviours. circulating evs were collected from their sera and analysed by using electron microscopy, nanoparticle tracking assay, and western blotting. brain cellular phenotypes were assessed by immunofluorescent staining and gene expression analysis. behavioural phenotypes of rag ko and wt mice were examined in social interaction test. in vivo transfer of evs was performed to see its effects on behavioural alterations of rag ko mice. results: rag ko mice displayed social behavioural deficits, accompanying by enhance c-fos immunoreactivity and altered microglia morphology in the medial prefrontal cortex (mpfc). circulating evs were also affected in these mice and lacked the expression of markers for t cells. a set of micrornas (mirnas) in circulating evs were diminished in rag ko mice. in vivo transfer of circulating evs rescues the social behavioural deficits of rag ko mice and ameliorate the c-fos immunoreactivities in mpfc of rag ko mice. summary/conclusion: our data showed that circulating ev profiles were altered in mice lacking adaptive immune cells and, accordingly, showing social behavioural deficits. notably, our in vivo experiments suggest that circulating evs may contribute to social behaviours. further study will provide a novel biological insight into the mechanisms underlying peripheral-to-brain immune communication via evs. introduction: the involvement of neuroinflammation on ageing process is widely recognized. extracellular vesicles (evs), such as exosomes, are able to cross the blood-brain barrier and were related to neuroinflammation. in this context, evs have been considered a potential mechanism of spreading molecules, including micrornas (mirnas) that can promote mrna degradation or inhibit translation of their targets. our aim was to investigate the mirna profile of circulating total evs during ageing process and their impact on canonical pathways. methods: the local ethics committee (comissão de ética no uso de animais -ufrgs; n 29818) approved all animal procedures and experimental conditions. plasma was obtained from wistar rats (3 and 21 months-old) and total evs were isolated. ev microrna isolation and microarray expression analysis was performed to determine the predicted regulation of targeted mrnas. results: the analysis of global microrna expression revealed 48 differentially expressed micrornas (p < 0.05; fold change of ≥ |1.1|); 18 mirnas were up-regulated and 30 were down-regulated in circulating total evs from aged animals compared to youngadult ones. a conservative filter was applied on ingenuity pathway analysis (ipa) and only experimentally validated and highly conserved predicted mrna targets were used. ipa showed that neuroinflammation signalling is ranked among the top canonical pathway impacted by differentially expressed micrornas and is upregulated in aged animals (p < 0.0001; z-score: 3.413). the differentially expressed mirnas impacted 32 molecules in the neuroinflammation pathway. interestingly, the ion channel grin2b is predicted to be up regulated and is a target of many evs mirnas; in accordance with our results grin2b was previously related to neurodegenerative diseases. moreover, let-7a-5p is predicted to be downregulated and target all the 32 molecules of the neuroinflammation signalling pathway. previous studies have correlated let-7a-5p and neurodegenerative diseases. summary/conclusion: our data suggest that circulating total evs cargo, specifically mirnas, are altered by ageing and impact neuroinflammation pathway, suggesting the involvement evs mirna on ageinginduced susceptibility of neurodegenerative diseases. introduction: bidirectional cell-cell communication via paracrine mechanisms is critical for wound healing. a new paradigm involving exosome-borne distinctive repertoire of cargo such as mirnas has emerged as a predominant mechanism of cellular communication at the site of injury. unlike other shedding vesicles of similar size, exosomes selectively package mirna by sumoylation of heterogeneous nuclear ribonucleoprotein (hnrnp). methods: keratinocyte-derived exosomes (exoκ) were genetically labelled with fluorescent reporter (gfp) using tissue nanotransfection. purified, gfp-labelled exoκ were isolated from dorsal murine skin and wound-edge tissue by differential ultracentrifugation followed by affinity selection using magnetic beads. distributions of intact exosome were analysed using a prototype jarrold-geometry charge-detection mass spectrometer to directly measure differences in particle mass and charge distributions. complementary ms and ion mobility spectrometry (ims)-ms experiments have been used to characterize surface glycans and glycopeptides. to selectively inhibit mirna packaging within the exoκ in vivo, ph-responsive targeted sirna functionalized lipid nanocarriers (tlnκ) were designed using materials that have prior history of fda approval for human use. results: an increase in mass/charge ratio with glycan binding sites on the surface of wound-edge exoκ were observed compared to dorsal skin exoκ. wound-edge exoκ were selectively taken up by the macrophages in the granulation tissue (n = 6). keratinocyte targeting sirnahnrnp functionalized lipid nanocarriers (tlnκ) were designed with encapsulation efficiency of 94.3%. application of tlnκ encapsulating sirna of hnrnp (tlnκ/si-hnrnp) to murine dorsal woundedge significantly inhibited the expression of hnrnp by 80% in epidermis compared to control (tlnκ/sicontrol)(n = 10). moreover, mice treated with tlnκ/si-hnrnp showed impaired barrier function, with significant presence of macrophage in granulation tissue at day 10, suggesting impaired conversion of macrophage in the granulation tissue. summary/conclusion: this work provides a novel insight wherein exosomes of keratinocyte lineage are recognized as a major contributor that directs macrophage conversion in granulation tissue for wound healing. multifaced effects of milk-exosome (mi-exo) as modulator of scar-free wound healing gna ahn, hyo-won yoon, yang-hoon kim and ji-young ahn chungbuk national university, cheong-ju, republic of korea introduction: recently, milk exosome (mi-exo) has been focused particularly on the possibility of oral distribution for therapeutic agents. however, studies related to the cosmeceutical effects associated with mi-exo are fairly limited. the purpose of this study is to suggest the anti-oxidant and antiinflammatory effect of mi-exo and possibility that can be induced by scar free healing by micro rna in mi-exo. methods: the characteristics of the extracted mi-exo were verified by size measurement, morphological characteristics through cryo-em and western blot. for antioxidant experiments, an abts assay was performed. next, mrna expression through four major cytokines (tnfα, il-6, cox-2, inos) was used to evaluate anti-inflammatory effects. finally, cell migration assay was performed to confirm the effect of scar-free healing and the detection of mir-30b in mi-exo and vegf mrna expression confirmed. results: mi-exo using 1% acetic acid extraction showed the highest yield. the average size of the exosomes is approximately 110 nm, confirmed the typical double membrane vesicle. as a result of antioxidant experiments, it was confirmed that the treatment of exosomes of 10^10 particles showed about 65% antioxidant activity. when 10^10 particles were treated, rna expression of cytokines showed about 2 times more inhibitory effect than control. elisa test results also confirmed that the concentration-dependent decrease. the activation of the raw cell less proceeded as the treated mi-exo increased. the cell scratch assay cells did not close the cells as the number of milk exosomes increased (wound closing % of 10^10 particle = 4.7%). and mir-30b in milk exosomes was detected at ct value = 22. summary/conclusion: the antioxidant and antiinflammatory effects of mi-exo showed the greatest efficacy when 10^10 particles were treated. in addition, it induced to scar free healing rather than wound healing. mi-exo has great potential as a superior natural material in the future cosmeceutical field. extracellular vesicles in human milk expose tissue factor and promote coagulation introduction: tissue factor (tf), a transmembrane protein, initiates coagulation by binding and activating coagulation factor vii (fvii). tf is associated with extracellular vesicles (evs) in saliva and urine, but it is unknown whether also human milk (hm) contains evs exposing coagulant tf. methods: hm was collected from six healthy nursing women with informed consent. evs were isolated by ultracentrifugation and size exclusion chromatography (sec). the presence of tf antigen exposing evs was studied by western blot, flow cytometry, cryo-electron microscopy (cryo-em), and surface plasmon resonance imaging (spri). the ability of tf exposing evs to trigger coagulation was investigated with a plasma fibrin generation test (fgt), performed in the absence or presence of antibodies against tf or fvii(a). results: addition of hm to plasma shortened the plasma clotting time, even when hm was highly diluted. after ultracentrifugation of hm, both tf antigen and tf activity were detected in the ev-containing pellet. after sec, tf antigen and tf activity were present in the ev-containing fractions 8 and 9. the presence of tf-exposing evs in these sec fractions was confirmed by western blot (cd9, cd63 and tf), flow cytometry, spri, and fgt. in addition, the presence of evs in hm was confirmed by cryo-em. scalable isolation of evs from different probiotic strains with potential as cosmetic ingredients laura soriano-romaní, joaquin espí and begoña ruiz ainia, paterna, spain introduction: extracellular vesicles (evs) are increasing their application in a number of fields. recently, it has been shown that skin health may be affected not only by commensal skin bacteria, but also by the evs that they secrete. however, because most of the efforts have been directed to the characterization and evaluation of evs, the scaling up of the production process remains a bottleneck at the industrial level. in this work, the goal was to evaluate the potential applications of evs produced by different probiotic strains commonly used in the cosmetic field, considering the economic and technical viability of the process. methods: to meet our goal, a standardized workflow was defined to isolate evs from probiotic strains such as lactobacillus and bifidobacterium species, that have demonstrated cutaneous immuno-regulatory effects. the different bacterial strains were produced under standard culture conditions. to isolate the secreted bacterial evs, different chromatographic techniques were performed starting from clarified growth medium. then, evs were evaluated in vitro for a number of biological effects related with skin health. results: the ev yields obtained after downstream processing were calculated for each strain and isolation technique by means of nanoparticle tracking analysis (nta) and total protein content. moreover, evs were visualized by electron microscopy. the in vitro evaluation of isolated evs was based on changes in the expression of five biomarkers related with anti-ageing, anti-inflammatory and whitening effects using distinct skin cell types to identify possible cosmetic claims that could be associated to each probiotic source. summary/conclusion: the potential of evs obtained from probiotic strains as cosmetic active cell-free ingredients was preliminarily assessed with this work, where the process yield and cosmetic function were evaluated. however, additional experiments will be needed in order to increase and optimize the productivity of each step of the ev manufacturing process. acerola derived exosome-like nanovesicles enhances the repair of ultraviolet b-induced dna damage in cultured skin fibroblasts tomohiro umezu, masakatsu takanashi, yoshiki murakami and masahiko kuroda tokyo medical university, shinjyuku, japan introduction: acerola (melpighia emarginata dc.) is a fruit is known to contain not only high amounts of ascorbic acid but also various nutritional components such as carotenoids and polyphenols. previous reports showed the acerola juices are able to confer protection against ultraviolet radiation b (uvb), to improve barrier function of skin. uvb is the main cause of dna damage in epidermal cells, generating several types of pro-mutagenic lesions, like cyclobutene prymidine dimers (cpds) and prymidine (6-4) prymidinone photoproducts (6-4pps): if not repaired, this dna damage leads to skin cancer. in this study, we investigated the biological property of the acerola derived exosome-like nanovesicles (adens), aiming to clarify the involvement of adens in repair of uv-induced dna damage. methods: normal human dermal fibroblasts (nhdfs) were purchased from lonza inc. the exosome-like nanovesicles were isolated from acerola juices using exoeasy maxi kit (qiagen). the morphology and size distribution of adens were checked by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta, nanosight lm10, malvern). nhdfs were exposed to uvb (1 mj/cm2) with pre-or post-adens. effect of uvb was assessed by examining cell viability, cell morphology, and dna damage levels through biochemical assays, microscopy and protein expression studies. results: purified adens were compatible with nta or tem for assessing the nanovesicle size range and concentration (200-400 nm). when nhdfs were added with adens and incubated at 37°c for 48 h, there was no effect of adens on cell proliferation of nhdfs. we found that adens treatment to uvb exposed nhdfs significantly reduced cpds and 6-4pps dna adduct formation. present results showed that aden treatment prevented uvb induced dna damage in nhdfs. summary/conclusion: we confirm that adens have the effect of repairing dna damage caused by uvb. these results provide that adens can be a new source to protect human skin from uv-induced skin cancer. introduction: introduction: despite the development of a variety of therapies, complex wounds resulting from disease, surgical intervention, or trauma remain a major source of morbidity. extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) have been shown to improve wound healing, especially via enhanced wound angiogenesis. however, despite their clearly established potential, evs have limitations that may limit clinical relevancy, such as low potency. hypothesis: increased expression of pro-angiogenic lncrna hotair within msc evs enhances their proangiogenic effects and thus their wound healing properties. methods: methods: hotair was overexpressed in human dermal microvascular endothelial cells (hdmecs) to determine any molecular or functional pro-angiogenic effects. anti-angiogenic mirnas and angiogenic mrna levels were quantified by rt-qpcr. effects of hotair on proliferation of hdmecs was also determined. hotair was then loaded into msc evs by delivering a cmv-based hotair plasmid to mscs for endogenous loading via a concentration gradient. evs were collected by differential centrifugation. hotair content within evs was confirmed by gel electrophoresis and rt-qpcr. effects on migration of hdmecs by hotair-loaded msc evs were determined using a scratch assay. results: results: overexpression of hotair decreased mir-29 c and mir-107, while increasing vegf and hif-1a. hdmec proliferation was also increased in hdmecs overexpressing hotair (p < 0.01). hotair was visually confirmed in hotair-loaded msc evs by gel electrophoresis, but was undetectable in unmodified msc evs. rt-qpcr confirmed a 900-fold increase of hotair compared to control msc evs. hdmecs showed a more statistically significant rate of gap closure when treated with hotair-loaded evs (p < 0.01) than compared to control msc evs (p < 0.05). summary/conclusion: summary: loading lncrna hotair into msc evs is achievable by a concentration gradient-dependent method and offers potential to enhance the angiogenic properties of msc evs. nanomaterial labelling of exosomes for cell biology introduction: exosomes are vesicles secreted by many, if not all, cell types and have been known about for decades. among larger micro vesicles that are produced directly from the cell membrane, the small (30-150 nm), exosomes are similar in size to a virus surrounded by a lipid bilayer. we and others have demonstrated that exosomes contain proteins, lipids, rna, and dna, making them promising materials for diagnosing and treating diseases, including many cancers such as brain cancer. in addition, exosomes from neurons and glial cells represent a novel type of intercellular communication. however, their size makes them hard to track with traditional fluorescence microscopy. to address this, we developed photothermal microscopy (ptm), which uses gold nanomaterial labelling to track exosomes' interaction with and effect on cells/tissue. methods: exosomes secreted by tumour cells and general exosomes found in the blood were isolated using differential ultracentrifugation or a commercially available kit (invitrogen). next, the exosomes were characterized by (tem), (nta), and western blotting to determine shape, size, morphology and the protein profile in the exosomal membrane. after characterization, the exosomes were labelled with gold nanoparticles via sonication. next, the samples were washed, and the exosomes were labelled with fluorescence dye to stain the membrane. after staining and labelling, the exosomes were added to u87 cells in culture and incubated for 3 h. they were then fixed by 4% paraformldehyde and imaged by ptm. results: ptm found that exosome-cell interactions are exosome-type dependent, as u87 cells took up exosomes from other u87 cells but not human serum exosomes. this suggests that exosome uptake is a selective process and depends on the source of the donor cells. summary/conclusion: exosomes can be labelled with gold nanoparticles via sonication then successfully tracked by ptm to study the effect of exosome source on exosome-cell interactions and communication. cells incubated with u87 exosomes took the vesicles up rapidly, while cells incubated with serum exosomes had little uptake. ptm will help us design selective exosome-based strategies to treat different conditions, including brain cancer and cns damage. funding: nsf epscor riii award 1457888. loading of goat´s whey extracellular vesicles with spiked microrna and curcumin as an strategy for developing new nanocarriers for acellular therapies introduction: extracellular vesicles (ev) are involved in cell signalling and are present in a variety of cell secretions such as milk, from which enormous amount of ev can be purified, thus milk is an attractive raw material for scaling up ev production for therapeutic, cosmetic or other uses. here we isolated evs from the whey fraction of goat´s milk and demonstrated that such evs can be loaded with molecules like polyphenols and mirna. methods: to achieve this, milk was collected from lactating goats and fractionated by acidification and centrifugation into whey and caseins. evs were purified from the former fraction by serial centrifugation and precipitation with commercial kit (total isolation/ thermo fisher) and characterized by electron transmission microscopy (tem), western blot to identify surface markers and measurement of size through nanotracking analysis. once isolated, evs were loaded with different concentration of a spiked synthetic mir39 or with the polyphenol curcumin. mirna or curcumin were co-incubated over night with evs at 4oc, precipitated and purified as described above, with an additional washing and precipitation for curcumin. concentration of mirna uploaded by evs was quantified using mir39 specific qpcr. curcumin was measured using a spectrophotometer at 420 nm. results: evs isolated from whey had an average size of 120 nm, were positive for hsp70, cd9 and alix. in tem, evs were identified with their natural conformation and corresponding size to exosomes. qpcr showed a significant difference of expression of mir39 in relation to control (loaded with shame) and the negative control (p < 0.05). curcumin presence was also confirmed after washing and precipitacion. summary/conclusion: in conclusion, milk evs and exosomes can be loaded with mirna and a polyphenol and can be used as alternative nanocarrier for acellular therapies. introduction: extracellular vesicles (evs) are cellderived lipid membrane nanoparticles that serve as messengers of intercellular communication, transferring bioactive molecules to recipient cells. evs have a natural therapeutic potential with high flexibility and biosafety for employing natural and synthetic biomolecules as therapeutic delivery vehicles. considering the importance of evs, their isolation methods are still a bottleneck. to get insights into the tissue-specific cargo in vivo for complete exploitation of evs as therapeutic, biomarker and diagnostic tools, ev purification methods are critical. the aim of the study was brought about to develop an efficient ev purification method both in vitro and in vivo and to further investigate function of evs in cellular senescence. methods: to isolate tissue-specific evs in vivo we developed recombinant evs by genetically fusing snorkel-tag to the cd81. the snorkel-tag enables on-column protease treatment for purifying evs which does not rely on traditional immunoaffinity purification protocols using low ph or high salts solutions. results: we systematically evaluated the purification of evs harbouring snorkel-tag by employing different methodologies. our findings suggest that evs harbouring snorkel-tag indeed can be purified at high purity without altering ev biophysical properties. furthermore, we expressed cd81-snorkel-tag under p16ink4a promoter and were able to purify evs derived from senescent cells. summary/conclusion: finally, we are developing an in vivo model with cd81-snorkel-tag under p16ink4a promoter. this will provide us detail insights into the ev cargo secreted from senescent derived cells, by purifying evs harbouring snorkel-tag under pathophysiological conditions, allowing us to develop biomarkers and therapeutic tools. summarized, we have here developed novel tool for studying content and function of evs in the context of ageing and disease. this tool will now pave the way for studying the molecular mechanisms underlying these ev functions in vivo. funding: this work was funded by the austrian science fund phd program biotopebiomolecular technolgy of proteins (w1224). engineering exosomes with gata-4 jie xu, christian paul, yi-gang wang and meifeng xu university of cincinnati, cincinnati, usa introduction: exosomes, are small vesicles (30-150 nm) secreted from cells that can transport and deliver of their components such as lipids, proteins, dna, mrna, and mirna to target cells. gata-4, a cardiac transcription factor, has been shown to regulate differentiation, proliferation, and survival of a wide range of cell types. delivering gata-4 protein into ischaemic tissues may be one of the most straightforward approaches to improve cardiac function following myocardial infarction. here, exosomes were engineered with gata-4 by infusing gata-4 with exosome targeting peptide. methods: the open reading frame of mouse gata-4 cdna was ligated to xpack lentivirus vector (xpack-gata-4) and plvx-ef1-ires-pouro lentivirus vector (plvx-gata-4), respectively. hek 293 cells were transduced by lentivirus, then exosomes were isolated from conditioned medium of hek293 cells using ultracentrifugation. exosomes were identified using transmission electronic microscope (tem), and the expression of gata-4 was semi-quantified using western blot. the internalization of exosomes was tracked via treating bend3 cells with exosomes pre-labelled with pkh26. introduction: chinese hamster ovary (cho) cells have dominated as the mammalian cell host for the manufacture of humanized biologics, in part owing to their genomic plasticity and robust growth in suspension culture. there is great interest surrounding the use of extracellular vesicles (evs) as novel therapeutics owing to their capacity to deliver bioactive molecules. however, much remains unknown about the mechanisms involved in ev cargo loading, limiting their development as novel biologics. to this end, we have engineered cho cells to stably express constructs enabling loading of gfp into evs. methods: tetraspanins are established markers of ev identity. accordingly, cd81 was selected as a tethering point to generate evs with gfp cargo and constructs were generated via golden gate assembly. cho cells were stably transfected by electroporation and expression was verified with fluorescence microscopy and western blotting. growth in batch culture was monitored to establish maximum viable cell densities for ev harvest and recovered evs were characterized by nanoparticle tracking analysis (nta). finally, uptake of gfp-evs was studied using time-lapse fluorescence imaging in co-culture experiments. results: strong localization of cd81-gfp was observed at the cell membrane and blotting confirmed intact tetraspanin fusion present at the expected molecular weight. additionally, cells were confirmed to retain high gfp expression post-cryopreservation. stable cell pools were able to reach viable densities greater than 7 million cells/ml in batch culture and nta allowed for detection of gfp cargo even prior to ev isolation. evmediated transfer of functional gfp to recipient cells was found to occur over a period of hours. introduction: extracellular vesicles (evs) are considered promising for therapeutic applications. evs resemble the cell membrane, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. despite the promising potential of evs for therapeutic applications, robust manufacturing processes that would increase the scalability and consistency of ev production are still lacking. methods: in this work, evs were produced by mesenchymal stromal cells (msc), isolated from different human tissue sources (bone marrow, umbilical cord matrix and adipose tissue). msc were selected as these cells allow for a scalable production of evs, while displaying low immunogenicity. a vertical-wheel™ bioreactor system was implemented for the production of msc-derived evs and compared with traditional static systems. the obtained ev products were characterized by nanoparticle tracking analysis, atomic force microscopy, zeta potential and western blot. results: the bioreactor system allowed to obtain evs at higher concentration and productivity, as well as more homogeneous size distribution profiles, when compared to traditional static culture systems. functional studies were performed using breast cancer and lung cancer cell lines. proliferation assays allowed to determine the dose-response profiles of these cell lines when exposed to msc-derived evs. a bell-shaped profile was observed for most cases, since raising the ev concentration lead to increased cell proliferation until a certain point (25-50 µg/ml), after which cell proliferation was attenuated with increasing ev concentrations. summary/conclusion: the bioreactor culture system allowed a substantial improvement in the production of msc-derived evs, while the obtained dose-response profiles will be valuable to determine the most appropriate ev concentrations for anticancer drug delivery. overall, we demonstrate that this culture system is able to robustly manufacture human msc-derived evs in a scalable manner towards the development of novel therapeutic products such as anticancer drug delivery systems. biodistribution and cellular location of inhaled exosomes and liposomes in the lung introduction: increasing evidence reveals the potential role of extracellular vesicles, such as exosomes and liposomes, in lung regenerative medicine for the treatment of lung diseases. encapsulation and delivery of potential rna and microrna targets into liposomes and exosomes are attractive drug delivery methods, but remain difficult to deliver to the pulmonary parenchyma to reach target lung cell types. here, we demonstrate effective delivery and cellular uptake of exosomes and liposomes to the pulmonary parenchyma via inhalation treatment in a murine model of idiopathic pulmonary fibrosis. methods: human lung stem cells (lscs) were generated and expanded from healthy whole lung donors. lsc-exosomes were purified via ultrafiltration and diilabelled using vybrant☐ labelling solution according to the manufacturer's instructions. dsred-labelled liposomes were generated using lipofectamine™ rnaimax transfection reagent and block-it™ alexa fluor™ red fluorescent control according to the manufacturer's instructions. lsc-exosomes and liposomes were delivered via nebulization to cd1 mice with bleomycin-induced pulmonary fibrosis. exosome and liposome delivery and biodistribution were visualized 4-and 24-hours post-treatment through histological analysis. the study was approved by the institutional animal care and use committee of north carolina state university and complied with all national and state ethical standards. results: exosome and liposome delivery to the pulmonary parenchyma was confirmed by the presence of dii and dsred fluorescence in lung histological sections that penetrated the mucus-lined respiratory epithelium. more exosomes and liposomes surpass mucus-lined surfaces 24-hours post-treatment compared to 4-hours post-treatment. fluorescent colocalization of exosomes and liposomes with alveolar type i cells, alveolar type ii cells, basal lung cells, and cd68 + macrophages was observed through immunohistochemistry analysis. more exosomes and liposomes colocalize with these cell types 24-hours post-treatment compared to 4-hours post-treatment. summary/conclusion: lsc-exosomes and liposomes penetrate the mucus-lined respiratory epithelium and reach the pulmonary parenchyma through inhalation treatment. lsc-exosomes and liposomes are uptaken by alveolar epithelial cells, basal cells, and interstitial macrophages with improved biodistribution 24-hours post-treatment. funding: this study was supported by the nc state chancellor's innovation fund. transfection reagent artefact accounts for some reports of extracellular vesicle function codiak biosciences, cambridge, usa introduction: extracellular vesicle (ev) functions are frequently investigated by transiently transfecting cells with plasmid dna to produce evs modified with protein(s) or nucleic acid(s) of interest. however, evs and the dna-complexes used to transduce cells are physically similar, raising the possibility that they may co-purify during differential ultracentrifugation, the most common ev isolation procedure. activities attributed to evs may therefore be due to contaminating dna -transfection reagent complex. methods: ev producing cells were transiently transfected with plasmid dna encoding gene-editing or split enzymes fused to ev-targeting protein sequences. differential and density gradient ultracentrifugation were used to purify evs from cell culture supernatant or dna lipoplexes from cell-free culture media. protein expression and localization to evs was confirmed by western blot. cell lines stably expressing fluorescent or luminescent reporters were used to assess functional enzyme delivery in recipient reporter cells. results: reporter cells treated with ultracentrifuge pellet material (ucp) from media of transiently transfected cells showed robust and reproducible signal, however fractionating the ucp with an iodixanol density gradient revealed that reporter activity was associated with high-density fractions that were depleted in evs. ucp isolated from identical transfection conditions, but lacking cells (and exosomes), showed identical biological activity levels and distribution in iodixanol gradients, suggesting that the activity was due to contaminating transfection reagent complexes and not evs. serial media changes on ev producing cells post-transfection did not significantly reduce ucp activity on reporter cells. treatment with nucleases did not digest complexed dna, did not significantly reduce dna levels in the ucp as measured by qpcr, and did not decrease activity in reporter cells treated with ucp from either transfected cells or no-cell controls. summary/conclusion: we find that dna-transfection reagent complexes are not separated from evs using differential ultracentrifugation and that common approaches to remove such complexes, including media exchanges and nuclease treatment, are ineffective. due to the pernicious nature of the dna-complex in these cellular assays, it is likely that some reports of ev function are likely artefacts produced by contaminating dna-complexes. we find that density gradient centrifugation can effectively separate evs and dnacomplexes, highlighting the importance of validating elimination of contaminating transfection reagent complexes when using transient transfection to interrogate ev function. chair: suresh mathivanan -la trobe university cancer stem cell-derived exosomes: potential biomarkers for early diagnosis and prognosis in pancreatic cancer introduction: pancreatic cancer (paca) is the most deadly manlignancy, due to late daignosis and early metastatic spread, which prohibits surgery. it is urgently for relaible, early detection. research shows that tumour-derived exosomes, which had been present in the blood in the early stage of tumour formation and before metastasis, is the vanguard forces of tumour formation and metastasis; cancer stem cell-derived exosomes (csc-exos) has stronger migration ability, so the detection of blood csc-exos for early diagnosis and monitoring of progress for paca has great research potential and the value of application. methods: protein markers were selected according to expression in exosomes of paca cell line culture supernatants, but not healthy donors' serum-exosomes. according to these preselections, serum-exosomes were tested by flow cytometry for the pancreatic cancer stem cell marker cd44v6 and tspan8. results: the majority (95%) of patients with paca and patients with nonpa-malignancies reacted with anti-cd44v6 and anti-tspan8. serum-exosomes of healthy donors' and patients with non-malignant diseases were not reactive. recovery was tumour grading and staging independent including early stages. introduction: chronic traumatic encephalopathy (cte) is a tauopathy that affects individuals with a history of mild repetitive brain injury frequently seen in contact sports. initial neuropathologic change of cte include perivascular deposition of phosphorylated tau (p-tau) in cortical neurons and, in later stages, the formation of neurofibrillary tangles in neurons throughout the brain. extracellular vesicles (ev) are known to carry neuropathogenic molecules in neurodegenerative disease and able to cross the blood brain barrier. we therefore examined the protein composition of ev separated from cerebrospinal fluid (csf) and plasma in former national football league (nfl) players with cognitive dysfunction, and an agematched control group with no history of contact sports. methods: evs were separated from csf and plasma from former nfl players (n = 4, 14) and controls (n = 5, 12) by affinity separation method or size exclusion chromatography, respectively. the ev protein profiling was characterized by simoa for tau and ptau and mass spectrometry. the protein data was analysed for ev enrichment, differentially expressed proteins, pathway analysis and correlation with cognitive function, head impact and tau/p-tau levels by biostatistics and bioinformatics. results: the level of total tau and p-tau in csf evs was not significantly changed, but significantly elevated in plasma evs from former nfl players. the 95 proteins were commonly identified between the paired plasma-csf from the same patients, but there was no significant correlation with disease status. collagen alpha-3(vi) chain (col6a3), −1(vi) chain (col6a1) and reelin (reln) were differentially expressed in former nfl players' plasma evs. a combination of these 3 proteins in plasma ev can distinguish former nfl players from controls with 85% accuracy by machine learning. summary/conclusion: the interacting plasma-csf ev proteomes provide an original resource to ev biomarker development for neurodegenerative disease, and col6a3, reln and col6a1 in plasma evs can be potential biomarker for monitoring the cte development. density-based fractionation of urine to unravel the proteome landscape of extracellular vesicles in prostate cancer introduction: current diagnostic tests are unable to discriminate indolent from aggressive prostate cancer (pca), leading to overdiagnosis and overtreatment, and an intense interest in biomarkers to improve clinical decision making. urine is considered an ideal proximal fluid for biomarker identification in pca due to its direct contact with the urogenital system. the discovery and translation of extracellular vesicle (ev) content into pca biomarkers remains challenging due to the difficulty of obtaining urinary ev (uev) with high specificity. methods: we developed a step-by-step protocol to separate uev by orthogonal implementation of ultrafiltration and bottom-up density gradient centrifugation (bu-odg). we implemented complementary particle and protein measurements to identify uev (lower density) and protein rich fractions (higher density) and assess the performance of bu-odg (specificity, efficiency and reproducibility). using mass spectrometry-based proteomics we interrogated uev and protein rich fractions from matched urine and radical prostatectomy tissue samples from pca patients (n = 4), and urine from men with pca prior to (n = 12) and after local treatment (n = 10), benign prostatic hyperplasia (n = 12) and other urological cancers (n = 11). results: bu-odg separated uev from soluble proteins and tamm-horsfall protein (thp) complexes with high specificity and reproducibility, outperforming differential ultracentrifugation, exoquick and size-exclusion chromatography. comparison of the uev proteome from men with benign or malignant prostate disease, allowed us to expand the known human uev proteome and identify a pca specific uev proteome not uncovered by the analysis of the protein rich fraction. proteomic analysis of ev separated from prostate tumour interstitial fluid and matched uev confirmed pca specificity of the uev proteome. analysis of the uev proteome from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uev reflecting their respective cancer tissues of origin. summary/conclusion: we identified hundreds of previously undetected proteins in uev of pca patients and developed a powerful toolbox to map uev and protein rich fractions, ultimately supporting biomarker discovery for urological cancers. immunoglobulin a coating of faeces-derived bacterial vesicles as a marker of inflammatory bowel disease in humans nader kameli a , frank stassen b , heike becker c , john penders c , daisy jonkers d and paul savelkoul b introduction: iga is the most abundant antibody in mucosal secretions and plays a crucial role in maintaining the balance between the host and the gastrointestinal microbiome. recent studies suggested that pronounced iga coating is especially prominent among inflammatory commensals which drive intestinal disease. membrane vesicles (mvs, nano-sized particles released by bacteria) have also been found to interact with the host and modulate development and function of the immune system. however, their interaction with iga has not been studied yet. here we developed a method to isolate and characterize the mvs from faecal samples and checked for possible differences in iga coating patterns of mvs in health and disease. methods: mvs were isolated by using a combination of ultrafiltration and size exclusion chromatography from faecal samples of 6 healthy controls (hc), 6 patients with active crohn disease (cd) and 6 cd patients in a remissive state. quantification and verification have been done with tunable resistive pulse sensing (trpsbased analysis) bead-based flow-cytometer (bbfc) and transmission electron microscope (tem). mvs were selected with specific antibodies for capturing (gram +: lta, gram-: ompa) followed by pe-conjugated anti-human iga antibodies as detection. results: we could successfully isolate 1*109-5*109 particles/ml from 500 mg of faeces. bbfc in combination with trps provide a valuable method for (semi-)quantitative measurements of mixed populations. intriguingly, remarkable differences were found between iga coating mvs derived from healthy controls and active and remissive cd patients as mvs derived from healthy controls were significantly more coated compare to both cd patient groups. in details, for selected g-ve derived mvs: 60% of the total population of mvs derived from hc were coated, 20% from remissive cd patients, and <5% of active cd patients; and for selected g+ ve derived mvs: 55% of the total population of mvs derived from hc were coated, 34% from remissive cd patients, and 25% of active cd patients. (data are represented as the mean). summary/conclusion: here we demonstrate for the first time that mv isolated from the faecal samples are also coated with iga, and surprisingly mvs from healthy volunteers were more densely coated than mvs from diseased patients. the possible consequence of this difference remains to be determined in future studies. monitoring altered tetraspanin and psma expression in prostate cancer derived extracellular vesicles via advanced image flow cytometry (isx) lukas w. prause a , christopher millan b , natalie hensky c , tullio sulser c and daniel eberli c a universityhospital zurich, zurich, switzerland; b university of zurich hospital, schlieren, switzerland; c university of zurich hospital, zurich, switzerland introduction: new diagnostic and therapeutic options for patients with prostate cancer are urgently needed. prostate-specific membrane antigen (psma)-based imaging and therapy are increasingly used for prostate cancer management. unfortunately, as a membrane protein, psma is not found as a soluble protein in the blood and therefore has limited utility as a diagnostic biomarker. however, psma has reportedly been observed as a cargo protein of prostate cancer-derived extracellular vesicles (evs). we demonstrate altered psma expression on evs derived from prostate cancer cell cultures (c4-2, lncap) in response to novel next-generation androgen receptor inhibitor (enzalutamide), a standard chemotherapy agent (docetaxel), a novel experimental nonsteroidal antiandrogen (epi-001) that binds covalently to the n-terminal domain of the androgen receptor and dihydrotestosterone (dht). additionally, evs were isolated from the plasma of prostate cancer patients who participated in the prococ biobank campaign at the usz. plasma was taken and stored from patients both pre-and post-prostatectomy. results: transmission electron microscopy, nanoparticle tracking analysis and simple western (wes) analysis show stable size distribution and amount of evs produced by treated and non-treated cells. using advanced image-based flow cytometry, altered tatraspanin and psma expression could be detected in evs isolated from cell culture supernatants of lncap and c4-2 prostate cancer cells following their treatment. summary/conclusion: measuring psma expression on extracellular vesicles might pave the way to use image flow cytometry of evs to develop a blood based diagnostic test for prostate cancer patients with a wide range of possible applications including: 1) monitoring response to therapy and, 2) early indications of potential relapse. funding: vontobel fondation. proteomic profiling of human neural cells derived extracellular vesicles to identify human brain cell-type specific markers introduction: alzheimer's disease (ad) is a common neurodegenerative brain disease which affects appropriately 30 million patients worldwide. one of the major challenges in ad is to develop reliable biomarkers for early diagnosis and disease-modifying therapies, especially before the clinical symptoms. extracellular vesicles (evs) carry cargos of proteins, lipids and nucleic acids. there was no comprehensive characterization of evs isolated from specific brain cell types, which may be useful for cell type-specific biomarkers. the purpose of this study is to isolate evs from human induced pluripotent stem cell (ipsc)derived brain cells for proteomic profiling and characterization of cell type-specific molecules. methods: human ipscs-derived neurons, microglia and primary cultured astrocytes were differentiated in ev-depleted media. the evs were isolated by differential centrifugation combined with size exclusion chromatography, followed by characterization using nanoparticle tracking analysis and mass spectrometry. the proteomic data were subjected to bioinformatics analysis results: we identified 153 proteins from neuronderived ev (nde), 215 proteins from microgliaderived ev (mde) and 380 proteins from astrocytederived ev (ade) by proteomics. gene ontology analysis indicated that most of these proteins are associated with evs. furthermore, 15, 48 and 251 proteins are present individually in ndes, mdes and ades. among them, high levels of atp1a3 and syt1 in ndes, itgam and cd300a in mdes, and eaat1 and gfap in ades were found, all of which are typically and highly expressed in the original cells. summary/conclusion: our results provide us the potential candidates for cell-type specific ev markers, which will be helpful to develop non-invasive tools to enrich ev originating from specific brain cells and may lead to the development of new biomarkers for neurodegenerative disorders. ) are a tremendous resource for extracellular vesicle (ev) research, but they are heavily focussed on mammalian evs, i.e. evs from humans and laboratory animals, where protein cargoes are well characterised, and a wide selection of antibodies are commercially available. protein markers can be used to identify and define the types of mammalian ev and to determine the presence of any contaminants that might confound functional studies. similar resources are not as readily available for bacterial evs as these are not as well characterised, commercially available antibodies are much less abundant and immunological variation between different bacterial species (and there are 1 trillion bacterial species on planet earth!) means that each species, strain, or group of related species may require different antibodies. methods: to identify quality markers for bacterial evs, we have characterised the proteome of cells, crude evs (ultracentrifuge pellet from cell free culture supernatant) and size exclusion chromatography or density gradient centrifugation purified evs from two different (pathogenic vs probiotic) strains of escherichia coli grown under two different environmental conditions, and one strain of mycobacterium marinum grown in one medium. results: our results identify a selection of proteins enriched in purified ev preparations, and proteins that are depleted after purification steps. summary/conclusion: our results allow the identification of potential markers for ev purity and non-ev contaminants, but also highlight the variability in bacterial ev preparations and suggest potential targets that can be used to investigate the heterogeneity of bacterial ev populations. introduction: recent findings indicate an increase in mid-life mortality rates in the usa and persistent, significant race-related health disparities exemplified by differential mortality rates. this suggests that exploring new molecular markers that may be linked to mortality could provide novel insights into factors that are driving mortality rates. accumulating data suggests that extracellular vesicles (evs) circulating in blood may be potential biomarkers of age-related disease. evs are nano-sized membranous vesicles that bear molecular cargo and mediate intercellular communication between different cells and tissues. little is known about whether ev characteristics differ by race or whether evs are associated with clinically relevant mortality risk factors. methods: in this cross-sectional study, plasma evs were isolated from middle-aged african american (aa) and white males and females. results: we report no significant differences in ev size or concentration with race or sex. there were significantly higher ev levels of phospho-p53, total p53, cleaved caspase 3, erk1/2 and phospho-akt in whites compared to aas. higher ev levels of phospho-igf-1r were found in females compared to males. we examined ev characteristics and protein cargo in the context of well-established clinical mortality risk factors. ev concentration was significantly, and positively, associated with several mortality markers including, high-sensitivity c-reactive protein (hscrp), homoeostatic model assessment of insulin resistance (homa-ir), alkaline phosphatase, pulse pressure, body mass index, and waist circumference. the relationship of ev concentration and cargo with mortality markers differs by race. summary/conclusion: our data show that ev-associated proteins can differ by race and sex and are associated with mortality risk factors. this study provides insight into the characterization of evs in middle-aged aas and whites, which may aid in the development of ev-based diagnostics. funding: this study was supported by the national institute on ageing intramural research program of the national institutes of health. repurposing specialised cell-free dna blood collection tubes for extracellular vesicle isolation introduction: liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. however, many plasma processing protocols have been designed with a single biomarker in mind. here we investigate whether specialised dna blood stabiliser tubes could be repurposed for the analysis of extracellular vesicles (evs). methods: peripheral blood (n = 3) was collected into k3-edta, roche or streck cell-free dna (cfdna) blood collection tubes and processed using sequential centrifugation immediately or after storage for 3 days. microev were collected from platelet poor plasma by 10,000 g centrifugation and nanoevs isolated using size exclusion chromatography. particle size and counts were assessed by nanoparticle tracking analysis, protein by bca assay and dot blotting for blood cell surface proteins. results: major variations in micro and nanoevs were seen with delayed time to processing. nanoev counts did not change with processing delay or tube collection type but the associated protein amount increased, indicative of cell lysis or activation. the protein was predominantly derived from from platelets (cd61) and red blood cells (cd235a). the increase in associated protein was seen more in the k3-edta and streck tubes indicating that the roche tubes may offer improved cell stability. conversely, microevs increased in both quantity and protein content with delay to processing indicative of both lysis and cell activation, irrespective of tube type. epithelial cell surface marker epcam abundance remained the same across conditions in both micro and nanoevs demonstrating that epcam+ evs were stable. summary/conclusion: specialised cfdna collection tubes can be repurposed for micro and nanoev analysis, however simple counting or using protein quantity as a surrogate of ev number may be confounded by pre-analytical processing. the evs would be suitable for disease selective ev subtype analysis if the molecular target of interest is not present in blood cells. introduction: nutrigenomics and nutrigenetics have been defined as the effect of nutrients on gene expression and genetic variation on dietary response, respectively. here, we propose the isolation and characterization of exosomes from donors carrying different alleles of hla-dqa1 and hla-dqb1, to investigate their involvement in coeliac disease (cd) management. methods: a chilean population (n = 30) was investigated for snps mutations in hla class ii alleles associated to cd predisposition (as well as other mutations related to other food intolerances), using the genochip food technology. exosomes have been isolated from donors' serum by ultracentrifugation and characterized by sds-page, western blotting (cd63 and cd9), and transmission electron microscopy. exosomes were also studied for their interleukins (il-6 and il-1ra) content. results: among the studied population, 86% present at least one of the alleles leading to cd development and 60% carry alleles encoding for αand β-chains heterodimers associated with very high risk to develop cd. in parallel, isolated exosomes from donors with low to extremely high risk for cd showed high il-1ra content (108.8 ± 15.91 to 148.8 ± 12.37), as the persons were not following any treatment. however, values of il-1ra decrease in exosomes isolated form persons receiving treatment for cd. a relationship between exosomes' content and genetic susceptibility for cd has been observed, which may suggest their possible use as biomarkers for cd as the diagnostic of this disease is still a big issue. summary/conclusion: until this point of this underway project, we demonstrate the existence of a relationship between the exosomes' content in il-1ra and genetic susceptibility for cd. furthermore, the genetic predisposition to cd could also modulate the gut colonization process, another important player in intestinal homoeostasis. in the next step, extracellular vesicles from gut microbiota will be isolated and analysed to determine their role in cd management. nasibeh karimi a , razieh dalir fardouei a , jan lötvall a and cecilia lässer b a krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, göteborg, sweden; b krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, gothenburg, sweden introduction: the ability to isolate extracellular vesicles (evs) from blood is vital in the development of evs as disease biomarkers. both serum and plasma can be used but few studies have compared them in terms of amount and type of evs. we have previously developed a method to isolate evs from plasma with minimal contamination of lipoprotein particles (karimi et al 2018) . the aim of this study was to compare the presence of different subpopulations of evs in plasma and serum. methods: blood was collected from healthy subjects, from which plasma and serum were isolated. evs were isolated using a combination of density cushion and size exclusion chromatography (sec) (protocol 1) or a combination of density cushion and density gradient (protocol 2) or immune-capturing (anti-cd63, anti-cd9 and anti-cd81 beads) (protocol 3). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, electron microscopy (em), exoview, flow cytometry and mass spectrometry (lc-ms/ms). results: as determined by nta and protein measurement more evs could be isolated from plasma with protocol 1 and the majority of the vesicles were cd9/ cd41a positive as determined with exoview and western blot. additionally, flow cytometry and western blot showed that more cd9/cd41a positive evs where also identified with protocol 3. furthermore, western blot showed increased amount of cd41a in plasma samples in protocol 2. when labelled evs were spiked in freshly collected blood, no difference in recovery was seen for plasma and serum. summary/conclusion: this study shows that a larger amount of evs could be isolated from plasma compared to serum when three different isolation methods were used. firstly, this suggests that more evs are present in plasma. secondly, it suggests that these vesicles are probably released by platelets and that evs are not trapped in the clot during serum formation. future studies are needed to answer how this affects the use of blood-derived evs as biomarkers from serum and plasma. tumour-derived extracellular vesicles contain distinct integrin proteins stephanie n. hurwitz a and david g. meckes b a university of pennsylvania, philadelphia, usa; b florida state university, tallahassee, usa introduction: cargo profiling, including proteomic analyses, of tumour cell-derived extracellular vesicles (evs) may provide ripe opportunities for further understanding cancer growth, drug resistance, and metastatic behaviour. accumulating data suggest that cancer-derived evs contain membrane-bound integrin proteins which may aid in cell detachment, migration, and homing to future metastatic niches. we have previously published an extensive proteomic profile of secreted vesicles from the nci-60 panel of human cancer cells. methods: here, we further examine the distinct integrin components in these cancer-derived evs, and additionally profile evs released from benign epithelial cells by liquid chromatography and tandem mass spectrometry for comparison. results: we demonstrate the enrichment of integrin receptors in cancer evs compared to vesicles secreted from benign epithelial cells. total ev integrin levels, including the quantity of integrins α6, αv, and β1 correlate with tumour stage across a variety of epithelial cancer cells. in particular, integrin α6 also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumours. other integrins including α4, αl, and β2 are enriched in vesicles derived from leukaemia cells, and may provide a means to distinguish haematopoietic cell-derived evs. summary/conclusion: this study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumour stage. differential expression and selective packaging of integrins into evs may contribute to further understanding the development and progression of tumour growth and metastasis across a variety of cancer types. effect of nicotine and menthol on cytochrome p450 and antioxidant enzymes in rat plasma-derived extracellular vesicles introduction: tobacco products such as e-cigarettes pose potential adverse health effects caused by direct exposure to aerosolized nicotine, flavorants such as menthol, and other particulates. here, we aimed to study the hypothesis that whether nicotine and menthol modulate nicotine-metabolizing cytochrome p450 2a6 (cyp2a6), antioxidant enzymes (aoes), sod1 and catalase in plasma extracellular vesicles (evs). modulation of these enzymes would eventually lead to nicotine-induced toxicity and hiv-1 pathogenesis via evs-based cell-cell interactions. methods: we isolated and characterized evs from rat plasma before and after nicotine self-administration (nic) with audiovisual cue (av) and menthol and characterized using ev markers according to the isev guidelines. protein associated with cyp2a6, sod1, and catalase were quantified by western blot. results: we measured size, total protein, and acetylcholine esterase activity of evs and found no significance difference in these characteristics before and after nic. to investigate the effect av, menthol alone or in combination in the absence and presence of nic, first we evaluated the expression of ev markers cd9 and cd63. the results showed menthol and av together increased the levels of cd9 (p ≤ 0.05), the marker of small vesicles, in the presence of nic. the nic with menthol and av showed a pattern of increased levels of small vesicle but could not reach to significance. next, we demonstrated that the nic with av increased the level of sod1 (p ≤ 0.05), which showed a pattern of increased levels of catalase and cypa6, though statistically non-significant. the expression of nicotine receptor did not change under any conditions used. the results showed an increased level of cyp2a6 (p ≤ 0.01), sod1 (p ≤ 0.05), and catalase (p ≤ 0.05) in plasma evs in the menthol-nic group compared to menthol group only. nic group with a combined av and menthol, showed further increase in the levels of cyp2a6 (p ≤ 0.01), and catalase (p ≤ 0.05). further analysis of plasma evs on inflammatory cytokines/chemokines in these groups, and the effect of plasma evs on nicotine-induced toxicity and hiv pathogenesis are underway. summary/conclusion: nicotine administration increased, though not statistically significant, the levels of circulatory evs. moreover, the study provided evidence that nicotine in the presence of menthol, av, and/or menthol+av increased nicotine-metabolizing cyp2a6 in all the groups and aoes in specific groups. funding: we thank the national institute on drug abuse (grant #da047178, da-047638) for supporting our work. introduction: biomarker discovery in breast cancer (bc) is a clinical need for therapeutics and non-invasive diagnostics. tumour exosomes are involved in premetastatic niche formation and drug resistance and represent a source of non-invasive biomarkers. the identification of tumour exosomal biomarkers provides, not only, the possibility to discriminate patient groups also potential targets to control cancer progression that could be exploited to develop innovate bc therapeutic strategies. methods: we have performed a comparative differenti. al proteomic profile of four bc cell lines and their derived-exosomes, representative of the most relevant bc subtypes in clinic to search non-invasive biomarker candidates. then, we have carried on two bioinformatics approaches: 1) protein association network analysis interaction (string) and 2) pathway inference analysis (hipathia), to characterize the functional profiling for each bc subtype. results: we have found differentially-expressed proteins, in both cells and exosomes, that include indicators of invasion, metastasis, angiogenesis and drug resistance. exosome proteome profile reflects their different bc cell origin suggesting potential indicators of bc subtype. further, bioinformatics analysis reveals a differential role of exosomes in bc signalling pathways in recipient cells, according to their protein cargo and cell origin. summary/conclusion: our results show a set of cells and exosome proteins that highly discriminate bc subtypes and may significantly contribute to further studies for the design of bc biomarker predictor to stratify bc patients and the development of novel therapeutic strategies. funding: a set of potential biomarkers to discriminate breast cancer subtypes. circulatory evs as potential biomarkers of hiv-drug abuse interactions and neurological dysfunction in hiv-infected subjects and alcohol/ tobacco users sunitha kodidela a , kelli gerth a , namita sinha a , asit kumar b , prashant kumar a and santosh kumar a a uthsc, memphis, usa; b university of tennessee health science center, memphis, usa introduction: abuse of alcohol and tobacco can exacerbate hiv pathogenesis and its associated complications. further, the diagnosis of neurocognitive disorders associated with hiv infection and drug abuse using csf or neuroimaging are invasive or expensive methods, respectively. therefore, extracellular vesicles (evs) can serve as reliable non-invasive markers due to their bidirectional transport of cargo from the brain to the systemic circulation. hence, we aimed to study the specific evs proteins, which are altered in both hiv and drug abusers to identify a physiological marker to indicate the immune status and neuronal dysfunction of hiv-positive drug abusers. methods: evs were isolated from plasma of the following subjects: a) healthy b) hiv c) alcohol drinkers d) cigarette smokers e) hiv+alcohol drinkers f) hiv +cigarette smokers. quantitative proteomic profiling of evs was performed by mass spectrometry and potential ev proteins associated with neuronal dysfunction were quantified by westernblot. results: the evs were characterized according to the isev guidelines. a total of 343 proteins were detected in evs of all the study groups. comparison of proteins among all the study groups revealed that hemopexin was significantly altered in hiv+drinkers compared to drinkers and hiv subjects. further, our study is the first to show properdin expression in plasma evs, which was decreased in hiv+smokers and hiv+drinkers compared to hiv patients. though we couldn't identify the few other cns-specific proteins, g-fap and l1-cam, associated with neuronal dysfunction in plasma evs by mass spectrometry, we could detect those by westernblot. the protein expression of gfap (p < 0.01) was significantly enhanced in plasma evs obtained from hiv-positive subjects and drinkers compared to healthy subjects, suggesting enhanced activation of astrocytes in those subjects. the l1cam expression was found to be significantly elevated in smokers (p < 0.05). both gfap and l1cam levels were not further elevated in hiv+smokers compared to hiv+nonsubstance users. summary/conclusion: the present findings suggest that hemopexin, and properdin show potential as markers for hiv-drug abuse interactions. further, astrocytic and neuronal-specific markers (gfap and l1cam) can be packaged in evs and circulate in plasma, which is further elevated in the presence of hiv infection, alcohol, and/or tobacco and thus may represent as potential biomarkers for neurological dysfunction in those subjects. funding: we thank the national institute on drug abuse (da047178) for supporting our work. . electrochemical detection of mirna-21-5p introduction: micrornas (mirnas) are small, single-stranded, non-coding rna species that regulate gene expression post-transcriptionally, and are transported by extracellular vesicles (evs). they play an essential role in biological processes, such as development, cell proliferation, apoptosis, stress response and tumorigenesis. thus, mirnas are considered relevant biomarkers in health. more particularly, mirna-21-5p is expressed in neurons after traumatic brain injury, being expectably transported to peripheral fluids by brain evs that cross the blood-brain barrier. the main goal of this work is to develop an electrochemical biosensor for the detection of mirna-21-5p in serum. methods: overall, the experimental assembly of the biosensor was made in three stages. the first one consisted in the electrodeposition of aunps, the second one in the incubation of anti-mirna21-5p on the carbon screen-s printed electrodes and the final stage in the incubation of mercaptosuccinic acid for blocking unspecific bindings. the probe was hybridized with the target mirna21-5p by a consecutive incubation of several standard solutions. each modification was evaluated with cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis) and square wave voltammetry (swv). the electrochemical behaviour of the biosensor was followed in all steps by monitoring the electron transfer features of a standard redox system. the redox probe selected for this purpose was [fe (cn)6]4-/[fe(cn)6]3-. results: the results indicated that the electrodeposition of gold was more effective for −1.5 v for 600 s and could lead to better signals upon anti-mirna-21-5p hybridization. summary/conclusion: in general, the experiments showed increasing charged transfer resistance upon the incubation of higher concentrations of mirna-21-5p. in these experiments, ev concentration is a critical variable that must be carefully controlled to ensure scientific rigour and reproducibility: without controlling for concentration (dose), experimental outcomes will exhibit excess variability that could mask important biological discoveries. in this study, three orthogonal methods are compared for accuracy in ev quantification: microfluidic resistive pulse sensing (mrps) and nanoparticle tracking analysis (nta) were compared to each other and relative to the gold standard method, transmission electron microscopy (tem). the ability of nta to accurately measure particle concentration is shown to depend on the polydispersity of the sample itself. results validate the accuracy of mrps and emphasize the importance of using orthogonal techniques to quantify evs. methods: reference urinary vesicles were prepared and analysed with the three methods and the relative concentration accuracy of nta and mrps were compared as a function of particle size. the hypothesis that nta concentration accuracy was impeded by sample polydispersity was tested using polystyrene bead mixtures having a range of polydispersity. a theoretical argument based on fundamental physics explains the experimental observations. results: tem and mrps measurements of the evs were in excellent agreement and showed a broad, polydisperse particle size distribution with no peak on the measured size range (50 nm -400 nm diameter). nta differed significantly from tem and mrps by reporting a steep decrease in measured concentration below about 150 nm that resulted in a peak in the reported particle size distribution. bead measurements confirmed the hypothesis to be tested: sample polydispersity significantly affects the ability of the nta method to accurately measure concentration, even for particles as large as 150 nm diameter. summary/conclusion: these experiments validate mrps as an accurate method for quantifying evs and highlight the importance of using orthogonal measurement methods in accordance with misev2018 guidelines. clinically relevant synthetic reference materials to standardize concentration measurements of extracellular vesicles: state-of-the-art and future prospects introduction: there is an unmet need to standardize concentration measurements of extracellular vesicles (evs). flow cytometry is the clinically most applicable method, but the currently available reference materials for calibration are insufficient. for example, the refractive index (ri) between standard particles and evs substantially differs, whereas concentration and fluorescence calibration particles are too bright. the goal of this study is to ascertain the most desired properties of reference materials to standardise ev measurements. methods: an online survey was prepared within the meves ii project to measure the desired size, concentration range, optical properties, choice of fluorochromes, and stability of synthetic ev reference materials for flow cytometry (fcm) measurements. besides the desired properties of ev reference particles, also the available instrumentation was assessed in the survey, which was sent to the members of the stakeholder committee of metves ii project and members of the ev flow cytometry working group. results: the most desired size, concentration, and ri range for ev reference particles is 50 nm to 500 nm, 10e7 to 10e10 1/ml, and 1.35-1.45, respectively. based on mie-theory evaluation of the sensitivity of the available instruments, none of the respondents would be able to detect 50 nm particles with ri = 1.35 with their current instruments. regarding fluorescence intensity, the most desired range according to the responses is from 10 molecules of equivalent soluble fluorochromes (mesf) to 10 000 mesf. considering the sizes of evs and fluorescent labels, the maximal mesf that can be obtained for ev reference particles with 50 nm diameter and high molecular mass fluorescent dyes is in the range of several hundreds. typical antigen densities on evs fall below 100 copies per ev with 50 nm diameter, i.e. mesf values above 100 are probably not physiologically relevant in this size range. summary/conclusion: a part of the desired properties of ev reference materials precludes either their physical feasibility of production or their detection at most currently available fcms, meaning that the intended reference materials will be future-proofed. funding: this work was supported under 18hlt01 metves ii project by the european metrology programme for innovation and research (empir). the empir initiative is co-funded by the european union's horizon 2020 research and innovation programme and the empir participating states. comparison of production and activity of amniotic fluid stem cell extracellular vesicles from 3d hollow fibre bioreactor and 2d culture. culture conditions may affect ev composition and potency. here we compare production, potency, identity and therapeutic potential of evs collected from cells grown in culture dish (2d) versus hfbr (3d). methods: human clonal afsc were derived from patient-consented amniotic fluids. 1x10e6 hafsc were seeded in 2d (145cm2), and 1.6x10e7 hafsc on a small 20kd mwco hfbr (fibercell-c2025d, 450cm2) with fibronectin coating; both cultured in chang medium with 20% of es-fbs, starved for 24 hr and then evs collected. the effect of harvest frequency was tested (8hrs, 24 hr, 72hrs,1 wk). 2d-evs and 3d-evs were compared by nanosight, potency assay (by wb), identity (by exoview analysis) and therapeutic effect (in vivo in an animal model of kidney disease, alport syndrome). results: 2d production was~5.5x10e9 ev/ml/24 hrs while 3d was~2.8x10e10 ev/ml (first four 24 hrs) and 4.4x10e10 ev/ml (two days of hourly harvests). very little difference in ev concentration and very similar size distribution (~130 nm) were observed during harvest intervals; possibly indicating either significant ev re-uptake or inhibition of ev secretion dependent upon free ev in the supernatant. 3d-evs trapped vegf (an in vitro established potency assay) as efficiently as 2d-evs, and expressed cd9, cd81, cd63, cd80, cd86 and vegfr1 as 2d-evs. summary/conclusion: 3d-evs had comparable properties and bio-activity to 2d-evs, but the hfbr produced 10x more evs. hfbr cell culture conditions for hafsc still need optimization, however an available 1.2 m2 cartridge provides a 50x scale up potential. the hfbr, a cgmp closed system, can produce sufficient numbers of ev to support pre-clinical and clinical applications with at least similar properties to evs produced by conventional 2d methods. funding: -intramural funding -intramural ev core pilot funding demonstration of high gain mode in combination with imaging flow cytometry for improved ev analysis luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. in recent years, the importance of evs has become apparent, as they are key mediators of intercellular communication. however, quantifying and characterizing evs in a reproducible and reliable manner is challenging due to their small sizeexosomes range from 30 to 100 nm in diameter. it is well-known that flow cytometers were originally designed to measure and detect cells, and due to the quantitative power flow cytometry offers, there has been a push to quantify and characterize evs using flow cytometric methods. however, these systems have not been designed to measure objects smaller than a cell. methods: here, we describe the use of high gain mode on the amnis® imagestream® imaging flow cytometer to address the challenges of measuring small particles. in this new high gain mode, the charge-coupled device (ccd)-camera is manually adjusted to higher gain settings, increasing the signal obtained from the ev. object thresholds and masking have also been adjusted to better identify and detect small particles. results: preliminary results using murine leukaemia virus-sfgfp reference particles have shown up to a fivefold increase in the number of gfp-positive objects collected in high gain mode, when compared to standard gain on the imagestream system. summary/conclusion: in this study, we demonstrate improved small particle detection, including evs, using this new high gain mode on the imagestream imaging flow cytometer. distance-controlled accelerated catalysed hairpin dna circuit for multiple and sensitive detection of exosomes-associated mirnas introduction: sensitive and simultaneous monitoring of multiplexed exosome-associated rnas is of great value for early cancer diagnosis remains a challenge. methods: here, we report a simple, multiple and sensitive exosomes-associated multiplex mirnas detection method that uses distance-controlled accelerated catalysed hairpin dna circuit (chdc) system without any complex operation or enzymatic amplification. the distance-controlled accelerated chdc can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting mirnas without any transfection means. results: we show that distance-controlled accelerated chdc strategy with signal amplification capability could selectively and sensitively identify low level rnas in serum evs, distinguishing patients with early-and late-stage breast cancer from healthy donors and patients with benign breast disease. summary/conclusion: this simple, accurate, sensitive, and cost-effective liquid biopsy by the distance-controlled accelerated chdc method is potent to be developed as a non-invasive breast cancer diagnostic assay for clinical applications. impact of isolation methods on biophysical heterogeneity of single extracellular vesicles university of california los angeles, ca, los angeles, usa introduction: current biophysical analysis of extracellular vesicles (evs) typically encompasses particle density and size distribution determinations using various techniques. however, variabilities in ev isolation methods and the structural complexity of these biological-nanoparticles (sub-100 nm) necessitate more rigorous nanoscale biophysical characterization of single evs to facilitate more reliable and comparable evbased assays. methods: combining atomic force microscopy (afm), super-resolution optical and conventional particle sizing light scatter and microfluidic techniques, we compared the unique sub-nanometre scale biophysical properties of breast cancer cell-derived ev isolates obtained using different isolation methods. results: afm and dstorm particle size distributions showed coherent unimodal and bimodal particle size populations in centrifugation and immune-affinity isolates respectively. more importantly, afm imaging revealed striking differences in nanoscale morphology, surface undulations, and vesicle-to-non-vesicle ratios among ev isolates from different isolation methods. our findings demonstrate the effectiveness of orthogonal high-resolution biophysical characteristics of single evs, not discernable via particle size distributions and counts alone. summary/conclusion: the identified nanoscale biophysical characteristics of ev isolates represent a strategic and complementary framework to resolve differences in the heterogeneity and purity of evs from introduction: extracellular vesicle (ev) concentrations measured by flow cytometry are incomparable. to improve comparability, the metves ii consortium is developing traceable reference materials and procedures, which require validation by test samples. in previous interlaboratory comparison studies, however, a main source of variation was introduced by pre-analytical variables and measurement artefacts introduced by test samples. to minimize variation introduced by test samples, our aim is to develop off-the-shelf biological test samples containing pre-labelled evs. methods: human urine and plasma were collected from healthy donors. evs were labelled with lactadherin-fitc, isolated by size-exclusion chromatography to remove free dye and minimize swarm detection, and mixed with dimethyl sulphoxide (dmso), exocap or trehalose, frozen in liquid nitrogen and stored at −80°c . after thawing, ev concentrations were measured by a calibrated flow cytometer (apogee a60-micro). results: compared to the ev concentrations measured in fresh plasma and urine, the concentrations decreased 27% in plasma (p = 0.04; mean of the 3 cryopreservation agents) and 35% in urine (p = 0.05) after one day of storage. after 5 months of cryopreservation, the concentration of plasma evs decreased 2% (dmso and exocap) and 8.5% (trehalose) compared to one day of storage, whereas the concentration of urine evs decreased 6% (exocap) and 18% (dmso and trehalose). summary/conclusion: we have developed ready-touse, pre-labelled human evs that are stable up to 5 months and dedicated for use in interlaboratory comparison studies. to further increase stability, other cryopreservation agents will be tested. our biological test samples will be key to validate the new reference materials and procedures developed by metves ii in 2021. funding: this project has received funding from the empir program co-financed by the participating states and from the european union's horizon 2020 research and innovation program. understanding intracellular fate of ev-delivered content introduction: despite much work performed on evaluating the potential effects of extracellular vesicles (evs), the functional uptake of their cargo is still controversial. this project aimed to demonstrate that ev content (protein and mrna) is protected and can be subsequently transferred with functional activity into recipient cells, while also developing a tool to assess and quantify functional ev uptake. methods: fusion proteins used were mitochondrial localized coxviii-cfp-nanoluc(cox) and nuclear localized h2b-rfp-nanoluc(h2b). results: hek293 t cell-derived evs protected cox proteins from proteinase k digestion while demonstrating significantly improved efficiency of uptake when compared to free protein, as measured by bioluminescence that was still detectable in recipient cells 96 hrs post-ev-exposure. to confirm functional uptake, recipient cells exposed to evs containing h2b for 72 hrs were imaged and some recipient cells manifested fluorescent red nuclei. to demonstrate the presence of functional mrna within evs, producer cells were transfected for such a duration as not to have detectable levels of protein in the evs while still containing detectable levels of mrna (qpcr) even after rnasea treatment. transfer of these evs to hela cells showed an increase in expression of h2b which was blocked by cyclohexamide, confirming translation of the mrna (2.2kb). to determine if recycling of ev delivered proteins occurs, recipient hela cells were exposed to evs containing cox for 72 hrs. all extracellular evs were removed and cells were trypsinized (0.25% for 30 min) to remove any non-internalized cox protein. 48 hrs later, evs (cd63+ and cd9+) released from cells contained cox suggesting recycling of protein or possibly recycling of entire evs. lastly, an assay was developed to measure functional ev uptake. nanoluc protein was split in two and fused to mturquoise2 (n65) or mscarlet-i(66 c). expression of each fragment alone exhibited non-detectable levels of luminescence while expressing both together had a significantly increased signal. delivery of either fragment within an ev to a cell expressing the corresponding fragment worked as confirmation and quantification of ev uptake (hek293, u87, hela cells). summary/conclusion: this study robustly demonstrates ev delivery of functional mrna and protein to cells, while also establishing a simple assay to quantify and validate functional ev uptake. theoretical model of ev losses due to adsorption on the tube walls. application for immunomagnetic detection of the vesicles introduction: short-term storage of unfrozen samples of vesicles, mainly at 4°c, overnight or during a couple of days is rather common laboratory practice. however, it was found to lead to significant losses of vesicle concentration supposedly due to adsorption on the walls of the tube. the present work develops a theoretical model intended to describe the vesicle adsorption process. the experimental validation of the model was made using method of immunomagnetic precipitation. methods: the theoretical model considers the "diffusion-limited" case of vesicles storage. the maximal adsorption capacity of the surface of contact between the tube and the solution is given as the number of vesicles in hexagonally packed monolayer. for experiment, the vesicles were purified from ht29 cell culture supernatant by differential centrifugation, aliquoted and kept at −80 c. further the aliquots were consequently unfrozen, and placed into the tubes with different surface treatment and kept at +4 c. the kinetics of vesicles loss was measured by anti cd9 immunomagnetic capturing followed by cd81, epcam and cd166 staining and flow cytometry. results: the model allows the estimation of the adsorption-associated losses as dependent on initial vesicles concentration, volume of the solution, tube geometry, the storage temperature and duration case of quiet vesicles storage (without mixing) and also accounts an expected effect of active agitation of the solution (ev-beads complexes formation). theoretical calculations were illustrated by analysis of ev at different storage conditions and during reaction of immunomagnetic precipitation of the vesicles. summary/conclusion: it was demonstrated that application of tubes surface treatment allows increasing sensitivity of immunomagnetic precipitation method to 2x10^5 for cd81+, 5x10^5 for epcam+ and 2x10^8 for cd166+ vesicles. introduction: it is now largely accepted that the intestinal microbiota plays a key role in intestinal bowel diseases (ibd). an imbalance in the composition and diversity of the intestinal microbiota (i.e. dysbiosis) of patients has been repeatedly pointed out by several teams. there are also indications that extracellular vesicles produced by bacteria and exosomes produced by epithelial cells might be increased in this family of diseases. methods: in order to differentiate healthy and ibd faecal samples on the basis of their vesicle profiles, we want to develop a means to enumerate rapidly particles in faecal samples, based on interferometric microscopy. the videodrop technology, developed by myriade, relies on the creation of single beam interferences between two signals from the same light path by nanoparticles such as small vesicles. it will permit to compare on large scales the viral load of healthy subjects and ibd patients. results: this fast and easy-to-use device was compared to the nta on several types of eukaryotic and prokaryotic vesicles and our preliminary results are encouraging. introduction: small extracellular vesicles (sevs) produced by mesenchymal stromal cells (msc-sevs) may be useful in cell-free therapies for immunomodulation and tissue regeneration. methods: to characterize msc-sevs produced ex vivo, human bone marrow mscs were cultured in mesencult-acf plus (macfp), an ev-free and animal component-free culture medium for 3 days and spent medium collected to isolate sevs by ultracentrifugation (uc). analyses of sevs were performed by nanoparticle-tracking analysis (nta), western blot (wb), and human umbilical vein endothelial cell (huvec) tube formation assay. results: analysis of fresh uncultured macfp by uc, nta and wb for cd63, cd81, and cd9 confirmed the absence of sevs. msc-sevs isolated from spent macfp by uc ranged from 80-150 nm in size and were positive for cd63, cd9, and cd81 proteins. these sevs could be stored at −80°c for >4 months in solution or lyophilized with minimal loss based on nta and wb analysis. the msc-sevs contained the msc-associated micrornas let7a, mir21, and mir26a as per qpcr analysis. the biological function of ex vivo isolated msc-sevs was assessed using a human umbilical vein endothelial cell (huvec) tube formation assay. huvecs treated with msc-sevs generated tubes as early as 6 h after seeding, which were not observed in control huvec cultures until 15 h. moreover, the number of branch points present in such tube structures was >fourfold higher in huvec cultures (n = 5) supplemented with msc-sevs versus control, with the former lasting >60 h and the latter lasting <50 h in culture. direct comparison of the performance of macfp medium to media containing non-depleted or ev-depleted foetal bovine serum demonstrated that only mscs cultured in macfp (n = 3) were able to expand robustly with a doubling time of 1.1, 2.1 and 8.9 days in these media, respectively. lastly, methods for isolating sevs using newly developed easysep-ev™ magnetic separation kits and size exclusion columns will be presented. summary/conclusion: taken together, these data demonstrate that msc-sevs can be produced in high yield in macfp medium and that these possess similar physical, phenotypic and functional characteristics as sevs in vivo. funding: this work was privately funded by stemcell technologies inc. introduction: extracellular vesicles (evs) are heterogeneous group of small vesicular structures released by different types of cells, including stem cells (scs). as recent studies demonstrate that they may enclose bioactive content and transfer it into the target cells, growing interest is placed on the utilization of evs in the field of biomedical research. however, there is still lack of standardized methods of evs characterization. as an example, typical flow cytometry-based protocols, commonly used for cells phenotyping, may be inadequate for the characterization of evs as particles with size close to the detection limit of conventional cytometers. thus, the aim of this study was to optimize and compare the use of different flow cytometry platforms for the multiparameter analysis of evs isolated from different types of scs populations. methods: ev samples were obtained by ultracentrifugation of conditioned media collected from selected scs types, including human induced pluripotent scs (ips) and mesenchymal scs (mscs). next, several high resolution flow cytometry systems: cytoflex, apogee (a50 and a60 micro-plus) and image stream mk ii were employed to compare their sensitivity and resolution, as well as influence of "swarm" effect. furthermore, we examined evs phenotype, including expression of tetraspanins and other surface markers. results: our results have revealed that tested flow cytometry systems may be utilized for the phenotypic characterization of evs secreted by scs populations. however, the conventional staining and gating strategy protocols have to be thoroughly optimized. additionally, depending on a type of tested cytometer, we have demonstrated the difference in a "swarm" effect and its influence on obtained results regarding evs phenotype. finally, imaging flow cytometry platform was also employed to visualize evs on the single particle level. summary/conclusion: in conclusion, we have demonstrated that tested high-resolution flow cytometry platforms are convenient methods for the multiparameter characterization of evs produced by different types of scs populations. however, careful selection of particular measurement parameters should be performed depending on a type of employed system. funding: this study was funded by ncbr grant strategmed iii (strategmed3/303570/7/ ncbr/2017) to ezs. evaluation of atcc's exosomes from cell culture supernatant as reference standards in research and development. introduction: exosomes are subcellular particles 30-150 nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. exosomes are stable carriers of cell-free cargo in the form of dna, rna, and proteins, thereby making them an attractive candidate for diagnostic and therapeutic applications. however, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards in extracellular vesicle research (ev). methods: exosomes were isolated from cell culture supernatants of different atcc cell lines including stem cells and cancer cell lines representing the most prevalent cancer types -prostate, colorectal, breast, lung, cervical and glioblastoma, using tangential flow filtration (tff). these exosomes underwent sterility and mycoplasma tests as a part of their quality control. the morphology and size distribution of these exosomes were evaluated through multiple strategies including nanoparticle tracking analysis (nta), asymmetrical flow field-flow fractionation (af4), cryo-electron microscopy (cryo-em) and spectra dynetm particle analyser. exosome surface markers were also analysed through multiple strategies such as electro chemiluminescent elisa, flow cytometry and western blotting. also, stem cell exosomes and cancer exosomes were further evaluated for functionality through in vitro functional assays including migration assay, angiogenesis and anchorage independent growth assay. results: our optimized tff method resulted in high yields of > 1 × 1010 exosomes/ml and average protein equivalent of more than 1 mg/ml. more than 90% of the exosomes population had an average size distribution of 50-200 nm and median size of 110 nm confirmed through a number of different size distribution instruments. although cell line dependent, we were able to obtain similar expression levels of different cell surface markers including tetraspanins (cd63, cd81, cd9) when evaluated through different methods. our functional data demonstrated stem cell exosomes were functionally active in promoting cell migration and tubule formation. additionally, cancer cell exosomes were found to promote a malignant phenotype in an anchorage independent growth assay. summary/conclusion: collectively, we demonstrated our ability to reproducibly manufacture production-scale batches of exosomes from multiple different cell types. our purified exosomes are of high yield, meet well-established quality control specifications, and are robust in maintaining size distribution, surface marker expression, and functionality in vitro. therefore, they can serve as ideal reference materials that can support different ev-based research applications. exo-cise: extracellular vesicles enriched from plasma post-exercise promotes myogenesis and neurogenesis bianca paris a , yaomeng liu a , vicente pagalday-vergara a , julie davies b , priya samuel a , ayman abu seer a , johnny collett a , laura gathercole a , ken howells a , karl j. morten b , zhidao xia a , daniel anthony b , david r f. carter a , helen dawes a and ryan c. pink a a oxford brookes university, oxford, uk; b university of oxford, oxford, uk introduction: physical activity brings about a widespread physiological response and elicits the beneficial adaptation of several tissues and organs. furthermore, regular participation in physical activity reduces the risk of developing major non-communicable diseases such as cardiovascular disease, diabetes, cancer, osteoporosis, and dementia. two important processes known to occur following physical activity are myogenesis and neurogenesis; both of which involve the activation and proliferation of specialised tissue-resident stem cells. the molecular mechanisms regulating these processes following exercise are poorly understood to date. here, we investigated the contribution of extracellular vesicles, which are released into the circulation after exercise, to benefit adult myogenesis and neurogenesis. methods: small extracellular vesicles were enriched from the blood of healthy participants before and following maximum and moderate intensity exercise. differentiation and proliferation using a range of methods was measured following vesicle treatment onto primary myoblasts and neuronal primary exvivo stem cells. activation of key cellular pathways were measured. results: we show significant proliferation and differentiation changes of both stem cell types. this is independent of extraction method, extracellular vesicle depleted fractions and is interestingly conserved across mammalian species. remarkably, we see an age-related effect. summary/conclusion: this advocates that short single bouts of exercise may promote myogenesis and neurogenesis via systemic signalling of extracellular vesicles which opens an interesting field in endogenous ev therapies. show promise as a cell-based therapy for retinal degeneration. while clinical trials are ongoing, the potential of extracellular vesicles (evs) as biomarkers for monitoring eye health and disease is not well studied. this study characterized the ev surface profile and cargo of hipsc-rpe to offer a baseline assessment in normal and disease conditions. moreover, we evaluated the importance of pnpla6, a gene involved in membrane integrity and when mutated causes retinal degeneration, in ev biogenesis and secretion. methods: evs were isolated from serum-free culture medium of hips-rpe and identified with nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis of exosomal markers, including alix, tsg101, and cd63. surface marker detection and proteomic profiling were completed using an ev surface marker kit and mass spectrometry, respectively. small interfering rna targeting pnpla6 was used to knockdown the expression in hipsc-rpe and evs were characterized. results: nanoparticle tracking analysis confirmed the presence of both microvesicles (>150 nm) and exosomes (<150 nm) by size distribution and the concentration of evs (1x108 particles/ml) from rpe. tem displayed typical morphological characteristics of evs. the presence of known ev markers, alix, tsg101, and cd63 was confirmed via immunoblot and flow cytometry. surveillance of ev surface markers revealed enrichment of epithelial markers (cd326) and stem cell markers (cd133/1) that depict donor cell origin and functional proteins including integrin-binding (cd29) and tgf-beta receptors (cd105). in addition, proteomic analysis revealed regulators of inflammation and rpe function, including hemopexin, clusterin, complement factor i, and pigment epithelium-derived factor. furthermore, reduction in pnpla6 expression reduced vesicle secretion and vesicle size compared to non-targeting controls. introduction: vascular endothelial growth factor (vegf) is a potent angiogenic factor and was first described as an essential growth factor for vascular endothelial cells. vegf plays a role in normal physiological functions such as bone formation, haematopoiesis, wound healing, and development. mesenchymal stem cell (msc) was found to secretes potential growth factors such as vegf when cultured in vitro. however there are some beliefs that foetal bovine serum (fbs) which usually used as serum in cell culture content vegf. methods: msc seeded in in 96-well plate in with concentration of 5,000 cell/well. cells were incubated for 24 hours and fasted for another 16 hours using only dmem. cells were treated with complete medium consist of dmem and 10% fbs. culture medium were collected after 5, 12, and 24 hours after treatment. cell were culture in 37ºc dan 5% co2. vegf concentration was detected using elisa technique. results: vegf concentration was not found in fbs which do not contact with msc. an increasing of vegf concentration in time-dependent manner was shown when culture medium was used in msc cell culture in normoxic condition. the result of vegf concentration when culture 5, 12, and 24 hours were 74.022 pg/ml, 76.67 pg/ml, and 93.58 pg/ml, respectively. the mechanism of msc release growth factor is still under investigated. however, the classic growth factors and cytokines serves paracrine control molecules which were important in regenerative medicine. vegf was found to be an important molecules in angiogenesis process and determine the fate of cells. summary/conclusion: msc secreted vegf and concentration increased in time-dependent manner. isolation and characterization of exosomes from canine stem cells introduction: unlike induced disease models using laboratory animals, naturally occurring disease models display pathophysiologic attributes that are more similar to human diseases. unfortunately these models are underutilized in translational regenerative medicine research. this is partly due to the slow development of species-specific experimental therapeutics to investigate comparative efficacy. thus, we set out to isolate and characterize exosomes from canine adipose-derived mesenchymal stem cells (cad-msc) to use as a comparative therapeutic in dogs. to accomplish this, we optimized an isolation and purification strategy and characterized their molecular properties. methods: exosomes were isolated by sequential centrifugation and subsequent ultrafiltration. the proteome was characterized by tandem mass tag (tmt) mass spectrometry and the mirna cargo was identified using a canine specific pcr array with subsequent target and enrichment analysis using targetscan and the panther platform, respectively. also, nanoparticle tracking analysis and transmission electron microscopy were used to determine exosome size and structure. to investigate bioactivity, we measured the ability of exosomes to inhibit collagen production in an in vitro model of fibrosis. results: exosomes were purified by ultrafiltration using a 100 kda cut-off. proteomic analysis by tmt mass spectrometry identified 1262 unique proteins. 90% of the exocarta top 100 were identified from this list. additionally, we identified the mirna cargo within exosomes and found 27 highly expressed mirnas. enrichment analysis identified multiple pathways of probable regulation including angiogenesis (fold enrichment = 3.1; p < 0.0001) and transforming growth factor-beta (tgfb) signalling (fold enrichment = 4.1; p < 0.0001). exosome size was quantified to be 119.7 ± 3.4 nm with a modal average of 98 nm. lastly, in the presence of exosomes, tgfb stimulated fibroblasts deposited 32.2% less collagen than vehicle controls (p = 0.036). summary/conclusion: in summary, cad-mscs exosomes display structural and functional features comparable to stem cell derived exosomes from other species. use of these exosomes in naturally occurring disease canine models may provide superior predictive value for human clinical trials. funding: support provided by the ccah, school of veterinary medicine, uc davis. mesenchymal stem cells-derived exosomes promote in vitro the progression of triple negative breast cancer cells introduction: mesenchymal stem cells (mscs) are multipotent stromal cells and have been described as key regulators of different aspects of tumour physiology. in tumour pathogenesis, mscs can integrate the tumour microenvironment after recruitment and are able to interact with cancer cells to promote tumour modifications by affecting epithelial-tomesenchymal transition (emt). it was revealed that exosomes derived from mscs are critical players in the tumour niche. exosomes are a novel way of cellto-cell communication and play crucial roles in the majority of pathways that contribute and affect response to therapy, cell-adhesion molecules and the progression of tumour cells. because of the known importance of this communication we decided to investigate the implication of mscs with triple negative breast cancer (tnbc) cell lines as well as exosomal profiles between the experimental conditions. methods: the interactions of mscs with triple negative breast cancer cell lines (mda-mb-231 and hs578t) was performed by coculturing mscs (or tnbc cell lines) with exosomes derived from tnbc cell lines (or mscs). physical characterization of isolated exosomes was performed followed by their molecular investigations. cell proliferation was detected by mtt assay and migration was analysed by wound healing assay using 2d cultures. moreover, we also used 3d culture to assess the exosomes uptake and to observe their capability of internalization into a 3d structure. the alterations in expression level of some transcripts (mrnas and mirnas) and protein profile were investigated by qrt-pcr, western blot and immunofluorescence staining. results: we found that mscs-derived exosomes are actively incorporated by triple negative breast cancer cell lines (2d culture). in coculture, in tnbc cells the expression level of mesenchymal markers and emt markers (e-cadherin, vimentin) at mrna and at protein levels, as well as mirna-derived exosomes targeting mesenchymal genes were significantly affected. using bioinformatics tools, we highlighted the important biological processes which were activated by promoting tumour modifications. in addition, using 3d culture we provided a comprehensive understanding regarding exosomes internalization in 3d structures, which closely mimics in vivo conditions, compared to 2d culture. summary/conclusion: in this work, we focus on the investigation of mscs-derived exosomes in order to highlight their implication in several biological processes, including tumour proliferation and progression of triple negative breast cancer cells. all these alterations affect the response to therapy and should be considered for developing efficient therapeutic strategies. natural killer cell-derived extracellular vesicles have a potent anti-leukaemic effect and selectively target the cancer stem cell subpopulation introduction: natural killer (nk) cells of the immune system recognize and kill tumour cells. extracellular vesicles (evs) secreted from nk cells are capable of killing tumour cells independent of the cell to cell contact required for nk cell activation. cancer is a leading cause of death, primarily due to metastasis and recurrence. cancer stem cells (csc) within tumours are resistant to chemotherapy and immune attack, and cause metastasis and relapse. identification of the cancer types killed by nk evs is limited, and the effect of nk evs on cscs has not been described. here we determine whether nk-derived evs kill a myeloid leukaemia cell line and its csc subpopulation. methods: nk evs were isolated from our nk cell line, nk3.3, derived from normal human lymphocytes. nk3.3 evs were characterized by immunoblotting, proteomics, and next generation rna sequencing. human k562 leukaemia cells were treated with nk3.3 evs in vitro and analysed for proliferation and markers of cell death. results: nk3.3 evs contain ev-associated proteins alix, cd63, hsp70, and tsg101, nk effector molecules perforin, granzymes a and b, granulysin and nklam/ rnf19b, an e3 ubiquitin ligase required for maximal nk cytotoxicity, and tumour suppressor mir-186. nk3.3 ev treatment of k562 significantly decreased its expression of proliferation markers cd71 and ki67, and increased the frequency of apoptotic and necrotic cells, paralleled by elevated levels of active caspases −3 and −7. non-tumorigenic cells were unaffected by nk ev treatment. most notably, nk3.3 ev treatment significantly reduced the frequency of k562 cells highly expressing aldh, a csc marker. summary/conclusion: nk3.3-derived evs have a robust anti-tumour effect on k562 myeloid leukaemia cells and selectively target the csc population, suggesting they may circumvent the evasion and resistance mechanisms used by cscs. nk3.3 evs therefore have introduction: due to their potential as a key bioactive agent in regenerative medicine applications, mscderived extracellular vesicles (msc-evs) are increasingly being investigated as a clinical therapy. manufacturing that generates enough evs for product development and clinical doses is currently a limitation in the field and clearly a scalable manufacturing solution will be necessary for successful translation. moreover, a complementary approach that increases the ev productivity, i.e. the number of evs produced per cell, could further help to accelerate the development of msc-evs as a therapy. methods: we developed a process that leverages a series of new cell culture reagents to couple to our established cell-media system for scalable manufacturing of msc-evs. briefly, human bone marrow-or umbilical cord-derived mscs were rapidly expanded under xeno-free conditions (i.e. >150x expansion within 10 days). cultures were then switched to our proprietary ev collection medium and evs were harvested for up to three additional days. at the end of culture, the evs in the conditioned media were concentrated using a tangential flow filtration (tff) system. to increase the productivity of mscs, two medium supplements were developed that increased ev yield by either increasing the number of evs generated per cell in a shortened culture process or increasing the number of collected evs by lengthening the ev collection culture period. results: this scalable msc-ev manufacturing method was implemented in both 2d flask and 3d bioreactor culture and generated over 2,000 particles per cell in 2d and over 4,000 particles per cell in 3d. with the addition of a medium supplement to increase evs produced per cell, the ev productivity was increased >2x after 24 hrs. alternatively, ev productivity was also increased >2x by addition of the medium supplement that extended ev collection culture period. summary/conclusion: msc-ev success in clinical translation will be reliant on a manufacturing method that can scalably and reliably generate large amounts of evs. these results present one such solution. furthermore, increasing ev productivity, for instance by medium supplements that increase evs per cell or lengthen culture times could further address the limitation of generating the evs required for development and translation of clinical therapies. simplifying scalable msc ev production in a microcarrier-based bioreactor system divya patel, josephine lembong, katrina adlerz, jon rowley and taby ahsan roosterbio inc, frederick, usa introduction: the growpt0ing numbers of msc-ev clinical applications drives the need for a scalable msc-ev production platform. while most msc-evs are generated while cells are attached to tissue culture plastic, such 2d cultures cannot be scaled up to meet the yields necessary for commercialization of ev-based therapeutics. we have shown that 3d bioreactors can be used to generate msc-evs and that paradigm can be scaled directly in terms of yield from the 3 to 15 l scales. the technical expertise of seeding cells onto microcarriers for expansion in bioreactors, however, requires technical expertise not available to all those in the ev field. therefore, our goal here is to simplify and expedite the ev collection process in bioreactors by cryopreserving cells on microcarriers, such that end users can merely thaw and then collect msc-evs. methods: mscs were expanded in 2d and then seeded on three different microcarriers and cultured in a bioreactor for 5 days. when confluent, cells on microcarriers were cryopreserved. to evaluate the microcarriers and the cryopreservation protocol, the cells-microcarriers were thawed, cultured in a bioreactor in growth media for 24 hours, then in ev collection media for 3 additional days. cell recovery and ev production upon thaw was evaluated and compared to ev collection from fresh, non-cryopreserved cells. results: total cell counts 24 hrs post thaw were comparable to those before cryopreservation and to fresh samples prior to ev collection. following 3-day ev collection, concentration of particles collected from cryopreserved cells on microcarriers were similar to those collected from the fresh cells (2e9 particles/ml). this process was validated for two different microcarriers using two separate cryopreservation solutions. summary/conclusion: our results show that cryopreserved hmscs on microcarriers can support ev collection in a 3d bioreactor process with a particle yield that is comparable to those collected from fresh cells. this cryopreserved product can simplify ev production, reducing cost and time by removing process steps associated with the hmsc expansion, with in a paradigm suitable for scale-up. the whitening, anti-wrinkle, and wound-healing effects of extracellular vesicles from orbicularis oculi muscle-derived stem cells. introduction: skeletal muscle-derived stem cells possess potent therapeutic activities in the treatment of muscle-related disorders. in our study, we tried to isolate and characterize orbicularis oculi muscle (orm)-derived stem cells (orm-scs) from the discarded human tissues which were obtained from the ocular surgery-subjected patients. we also prepared the natural extracellular vesicles (evs) from the cultured orm-scs and assessed the their therapeutic actitities including the skin whitening, anti-wrinkle, and wound healing effects. methods: we isolated the orm-scs from the patients subjected to ocular surgery and characterized the orm-scs by analysing cell morphology, proliferation, expression levels of the cell surface and stemness-associated markers, and tri-lineage differentiation and colony-forming capacities, confirming the stemness properties of the orm-scs. then, we prepared the natural evs from the orm-scs via the centrifugation and filtration of the media supernatants and their therapeutic activity was investigated. results: the isolated orm-scs showed spindle-like morphology and positive expression of cd105, cd 90, and cd73, but they were negative in expression of cd45 and cd34. the orm-scs showed the capacity of osteogenic, adipogenic, and chondrogenic differentiations. the evs from orm-scs (orm-sc-evs) possessed the apparent inhibitory effect on the melanin synthesis in b16f10 cells by blocking the tyrosinase activity, although orm-sc-evs treatment did not dramatically change the expression level of melanogenesisrelated genes, such as microphthalmia-associated transcription factors (mitf), tyrosinase (tyr), tyrosinaserelated protein1(tyrp-1), and tyrp-2. in addition, we confirmed that orm-sc-evs could stimulate skin cell migration and increase the expression level of antiwrinkle related genes and wound-healing properties. summary/conclusion: this study revealed the stem cell property of orm-scs and the whitening, antiwrinkle, and wound healing effects of orm-sc-evs, suggesting that orm-scs and orm-sc-evs can be successfully used for stem cell-based ev therapy and cosmetics, by regulation the melanogenesis, wrinkle, and wound. funding: this work was supported by grants from the national research foundation (nrf) funded by the korean government (2019m3a9h1030682). use of stem cell extracellular vesicles as a holistic approach towards cns repair introduction: neurological diseases and disorders are leading causes of death and disability worldwide. many of these pathologies are associated with high levels of neuroinflammation and irreparable tissue damage. we have previously shown that extracellular vesicles (evs) from infected cells contain viral by products (noncoding rnas and proteins) and that these evs can exert deleterious effects on recipient cells1-3. therefore, in the context of neurotrophic viruses evs may contribute to or perpetuate processes relating to neuroinflammation and neurodegeneration. due to their multipotent properties, stem cells have broad applications for tissue repair; additionally, stem cells have been shown to possess both immunomodulatory and neuroprotective properties. in recent years it has been well-established that stem cell evs play a critical role in the functionality associated with stem cells. the diverse biological cargo contained within these vesicles are proposed to mediate their effects and, to date, the reparative and regenerative effects of stem cell evs have been demonstrated in a wide range of cell types. while a high potential for their therapeutic use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how they may regulate cells of the central nervous system (cns). methods: we have isolated and recovered high yields of evs from large scale cultures of both induced pluripotent stem cells (ipscs) and mesenchymal stem cells (mscs) using tangential flow filtration. our ev characterization includes both phenotypic (size, tetraspanin expression) and biochemical assays. ev functionality has also been assessed in vitro utilizing several cellbased assays related to cellular viability, migration, angiogenesis, and immunomodulation in both healthy and damaged recipient cells with relevance to the cns. results: our data suggests that evs from different sources of stem cells display unique phenotypes, exhibit differential association with various cytokines, proteins, and long non-coding rnas, and have the ability to significantly enhance processes that are critical for cellular repair4. lastly, utilizing an ipsc-derived neurosphere model, we have observed a robust uptake of stem cell evs and have found that these evs are able to effectively penetrate these 3d structures. summary/conclusion: collectively, these results highlight the "holistic" properties of stem cell evs by demonstrating their ability to partially reverse or reduce damage in various cell types. funding: this work was supported by national institutes of health (nih) grants ai078859, ai074410, ai127351-01, ai043894, and ns099029 to fk and r33 ca206937 and r01ar068436 to lal. the effect of cell culture media on extracellular vesicle secretion from mesenchymal stem cells and human pluripotent stem cell-derived neurons introduction: cell culture media and its supplements are known to affect the secretion and isolation of extracellular vesicles (evs) from cell cultures. identification of these effects is crucial especially when planning to use evs as therapeutic agents. here, we investigated the effect of cell culture media on ev yield from human mesenchymal stem cells (mscs) and human pluripotent stem cell (hpsc)derived neurons. methods: evs were collected from cell-conditioned media (ccm) and no cell control (ncc) media using size-exclusion chromatography (sec). mscs were cultured in dmem/f12:neurobasal medium or in opti-mem reduced serum medium, both supplemented with exosome-depleted foetal bovine serum (fbs). the ev yield from hpsc-derived neurons was compared at two maturation time points (day 46 and 60), in dmem/f12:neurobasal or in opti-mem, with and without 3-hour kcl stimulation. sec fractions were analysed by nanoparticle tracking analysis (nta), protein concentration assay and blinded transmission electron microscopy (tem). results: ccm samples had a clear peak of evs in sec fractions 7-10, which was not detected with ncc. interestingly, a second population of evs eluted in sec fractions 13-17 in both ccm and ncc, indicating presence of evs in exosome-depleted fbs. moreover, this second population differed largely between used media batches. culture medium had no significant effect on msc ev yield (dmem: 8.30e+08 particles/ ml, opti-mem: 9.61e+08 particles/ml). with neuronal cultures, no significant differences in ev yield were found between culture media or cell maturation time points. in contrast to earlier findings, 3-hour stimulation of neurons by kcl resulted in significantly smaller ev yield compared to non-stimulated controls (stimulated: 5.28e+10 particles/ml, non-stimulated: 1.43e+11 particles/ml, p < 0.02). summary/conclusion: our results indicate that exosome depleted-media are not entirely devoid of vesicles, which can cause bias in downstream analyses. however, sec is a good method to separate cellsecreted evs from the contaminating medium-derived evs. culture medium did not affect the number of evs secreted by mscs or neurons; instead, we observed larger differences between media batches. this data emphasizes the importance of analysing the ncc as negative control in all cell culture experiments. mouse mesoangioblast stem cell extracellular vesicles are able to influence macrophage cell activity maria magdalena barreca a and fabiana geraci b a dept stebicef university of palermo, palermo, italy, palermo, italy; b dept stebicef, university of palermo, italy, palermo, italy introduction: it is largely demonstrated that stem cells release extracellular vesicles (evs) that are able to modify target cell behaviour. interestingly, there is a bidirectional signalling exchange between stem cell evs and damaged cells. moreover, it is well known that macrophages, could also play a role in wound repair and tissue regeneration. it was also demonstrated that stem cell evs are involved in immune cell regulation. for this reason, today takes hold the idea that evs could replace stem cells in regenerative medicine. the aim of our work was to evaluate if evs released by mouse mesoangioblast stem cells (a6) could have a role in immune cell regulation. specifically, we have investigated the possible a6 ev effect on murine macrophages (raw264.7) in terms of cell proliferation, migration and phagocytic ability, and cytokines/chemokine release. methods: a6 evs were collected from conditioned milieu by ultracentrifugation. raw 264.7 cell proliferation with or without a6 evs was evaluated via cfse assay. scratch test was performed to assay their migration ability. to study raw264.7 cell phagocytosis they were treated with 2 μm beads. finally, cytokine array was used to monitor their secretion after ev treatment. results: we have found that a6 evs inhibited macrophage proliferation as proved by a proliferation index significantly reduced after ev treatment. simultaneously, we have noticed that evs increases raw264.7 migration ability. furthermore, a6 evs are able to increase macrophage phagocytic activity. as it is known that hsp70 is involved in for macrophagic activity increase and a6 evs express hsp70 on their surface, we performed phagocytosis assays assay by blocking the protein or its receptor tlr2, tlr4 and cd91. our data demonstrated that a6 evs increase phagocytosis through hsp70 and its receptors. we have also proved that a6 evs modify the expression pattern of cytokines/chemokines released in the extracellular milieu by raw264.7 cells. in particular, we observed an increase in anti inflammatory cytokines, and a decrease in some inflammatory ones, suggesting that evs could polarize macrophages towards an anti inflammatory m2 phenotype. summary/conclusion: in conclusions, our data show that a6 evs influence macrophage activity and additional studies could provide a new insight into understanding the underlying potential of evs in tissue regeneration. 1 and 2) . in a healthy kidney, the polycystins localize to renal cilia. mutations that abrogate ciliary localization of pkd2 (yet preserve its channel function) also cause cysts. besides cilia, pkd2 is also found in other subcellular locations including extracellular vesicles (evs) of human urine. how dysfunction of pkd2 trafficking and localization leads to the kidney pathology remains unknown. pkd2 is evolutionarily conserved across all members of eumetazoa. in c. elegans, pkd-2 is exclusively expressed in ciliated male-specific neurons, where it is trafficked to cilia and evs. gfp-tagged pkd-2-containing evs play a signalling role in inter-organismal communication between animals. conservation of polycystin-2 cellular localization between worm and human suggests that their network of molecular interactions may also be conserved. we propose that pkd-2 plays distinct roles in cilia versus ciliary evs. methods: to understand the role of evs in c. elegans inter-organismal signalling, we aim to identify the pkd-2-associated ev proteome, transcriptome, and metabolome. we established a pipeline for fluorescent labelling and tracking specific ev cargoes in a living animal using super-resolution microscopy. we used fluorescence of the pkd-2 carrying evs to optimize biochemical procedures for their enrichment. results: our initial analysis revealed two populations of pkd-2-carrying evs that differ in their densities: 1.11-1.12 versus 1.14 g/ml. we are currently characterizing these two distinct populations using transmission electron microscopy and refining our enrichment procedure for protein identification by mass spectrometry, sequencing of their rna cargoes and metabolome analysis. summary/conclusion: what function human pkd2 plays within the cilia and within the urinary evs is not well understood. identification of molecular mediators of c. elegans pkd-2 ev signalling will inform on the interactome of human pkd2 and its function in cilia versus evs. introduction: ectosomes play roles in many physiological and pathophysiological processes, and their precise is dependent on molecular cargo and parent cell type. a single cell can release distinct subpopulations of evs enriched with different molecular cargo, which adds complexity to elucidating cargo sorting and biogenesis mechanisms. in the nematode c. elegans, ectosomes bud from sensory neuron cilia and are released into the environment to modulate animal behaviour. methods: c. elegans is genetically tractable and optically transparent, allowing for live imaging of fluorescently tagged ev cargo. we express all tagged cargo at endogenous levels, adding physiological relevancy. results: we discovered that the calcium homoeostasis modulator ion channel clhm-1 localizes to cilia of ev-releasing neurons and observed gfp-tagged clhm-1 in ciliary evs. using super resolution microscopy, we imaged evs released from animals coexpressing tdtomato-tagged clhm-1 and gfp-tagged pkd-2 (another vesicle cargo) in the same neurons. while the two proteins colocalize in the cilia, clhm-1::tdtomato and pkd-2::gfp rarely colocalize in evs. this indicates that separate subpopulations of evs are being released from the same neurons. to determine how the clhm-1 subpopulation is formed, we are investigating candidate genes. anoh-1, a homolog of the ca2+ scramblase tmem16f, localizes to neuron cilia and induces phosphatidylserine exposure on the outer membrane leaflet. in anoh-1 mutants, the number of clhm-1::gfp evs released is significantly decreased but the number of pkd-2::gfp evs does not significantly change. in addition, i am using facs to isolate clhm-1 and pkd-2 containing evs and analysing the respective proteomes with lc-ms/ms. summary/conclusion: we are elucidating mechanisms that give rise to distinct subpopulations of ciliary evs in c. elegans and defining cargoes being enriched in these ev subpopulations to gain insight into ev cargo sorting and biogenesis mechanisms in ciliated neurons. ceramide accumulation induces exosome secretion through lysosomal protein laptm4b kohei yuyama, hui sun and yasuyuki igarashi hokkaido university, sapporo, japan introduction: exosomes, a type of extracellular vesicles originated from multivesicular bodies (mvb), are important carriers of cellular molecules and have critical roles in intracellular communication in both health and disease. ceramides (cer) are implicated in biogenesis of exosome, however the molecular machinery that mediates exosome secretion remains obscure. lysosome-associated protein transmembrane-4b (laptm4b) is a lysosome/late endosome-resident transmembrane protein, which has been reported to bind cer. we demonstrate here that laptm4b is involved in the exosome secretion, which are induced by exogenous cer treatment or lysosomal ceramidase inhibition in cultured neuronal cells. methods: neuroblastoma sh-sy5y cells were treated with cer (porcine brain-derived cer or synthetic d18:1/c2:0~c24:0 cer) for 24 h. exosomes were isolated from the culture supernatants by sequential centrifugation and their amounts were measured using ps capture exosome elisa kit. to analyse mvb transport, mvb and recycling endosomes are visualized with gfp-cd63 and rab11 immunostaining, respectively. results: we found that exogenous treatment of cer, especially those with c16 and c18 fatty acids, resulted in a marked increase in exosome secretion. in addition, lysosomal cer accumulation induced by acid ceramidase inhibition also accelerated exosome production. knockdown of laptm4b significantly prevented the ceramide-dependent exosome release. in addition, we showed that these cer loading promoted colocalization of cd63-positive mvb with rab11-positive recycling endosomes, further demonstrated that laptm4b knockdown cancelled the cer-dependent increase of the colocalization. summary/conclusion: these data suggest that lysosomal cer binds to laptm4b and promote the transport of mvb to plasma membrane, resulting in an increase of exosome secretion in neuronal cells. chloroquine-mediated lysosomal inhibition alters composition and function of cancer-derived extracellular vesicles jing xu a , kevin yang a , shane colborne a , elham hosseini-beheshti b , gregg morin a , emma guns b and sharon m. gorski a a bc cancer, vancouver, canada; b the vancouver prostate centre, vancouver, canada introduction: small extracellular vesicles (sev) are signalling entities released by many types of eukaryotic cells. sev are of special interest in cancer due to their reported roles in modulating the cancer microenvironment and facilitating cancer cell invasion. macroautophagy (hereafter autophagy) is a catabolic process well-known for the recycling of cytosolic cargos through lysosome-mediated degradation. in this study, we profiled the changes in sev content and function under lysosome inhibition and investigated the involvement of autophagy machinery in sev content. methods: chloroquine (cq) was used to inhibit lysosomal degradation and autophagy turnover in triplenegative breast cancer (tnbc) cell lines. sev were collected via precipitation after pre-clearing and concentration of conditioned media. western blotting, nanosight and transmission electron microscopy were used to profile sev. quantitative mass spectrometry was used to characterize cq-induced changes in the sev proteome. antibody-conjugated magnetic beads were used in immunoprecipitation of sev. results: cq treatment did not substantially alter the physical properties of tnbc-derived sev. however, cq treatment altered the sev proteome and growth effects of sev on normal and endothelial recipient cells. cq treatment induced co-localization of mammalian atg8 proteins with endolysosomal markers in the cytoplasm, which coincided with an enrichment of atg8 s and their adaptor proteins in sev. cq-induced enrichment of atg8 s in sev required lipidation, and occured preferentially in one subset of sev. summary/conclusion: our study reveals changes in the content and function of cancer cell-derived sev in response to perturbation of intracellular trafficking pathways, demonstrates the flexibility and heterogeneity of sev composition, and has implications for cq efficacy in therapeutic settings. introduction: introduction: argonaute 2 (ago2) is the essential component of the rna-induced silencing complex (risc) that binds mirnas and promotes mrna degradation. extracellular vesicle (ev)-carried mirnas have been shown to influence gene expression and functional phenotypes in recipient cells. many investigators have found ago2 in evs and it is postulated that ago2 is a major transporter of mirnas into small evs (sevs), such as exosomes. others have reported extracellular ago2 that is non-vesicular. we set out to evaluate the effect of growth factor signalling and serum contamination on the detection of ago2 in sevs. methods: methods: wildtype kras colorectal cancer cells, dks8, were conditioned with 3 different culture media (serum-free dmem, dmem supplemented with ev-depleted fbs, and opti-mem). evs were purified from conditioned media by cushion-density gradient ultracentrifugation. western blot analysis of dks8 total cell lysates, large evs and density gradient fractions was performed, probing for ago2 and ev marker proteins. the size and concentration of the evs were determined by particle metrix analysis. results: results: in all conditions, we found the highest abundance of sevs in fractions 6 and 7, as assessed by western blot analysis. ago2 was detected in the same fractions as sevs in both the serum-free dmem and opti-mem conditions, although the levels of ago2 was higher in the serum-free dmem fractions compared to that of opti-mem. in contrast, ago2 was present in both vesicular and non-vesicular fractions in the dmem supplemented with ev-depleted fbs condition. no significant differences were observed in the size and number of evs collected in the three conditioning methods. summary/conclusion: summary/conclusion: the presence or absence of ago2 in evs has been controversial. multiple factors may affect the ability to detect vesicular ago2, including serum and growth factors in the conditioned media that may provide sources of extravesicular ago2 and also regulate the trafficking of ago2 into vesicles. introduction: cancer-associated glycosphingolipids have been utilized as tumour markers and targets of cancer therapy. we have investigated roles of gangliosides in cancers, and clarified that cancerassociated gangliosides enhance malignant properties of cells by forming complexes with membrane molecules in lipid rafts. in this study, we analysed contents of gangliosides and membrane molecules on extracellular vesicles (ecvs) secreted from melanoma cell lines. methods: melanoma cell lines with various ganglioside patterns were used for isolation of ecvs. gangliosidemodified melanomas with genetic engineering were also used. genetic modification was done by cdnas of ganglioside synthase genes. ecvs were collected by ultra-centrifugation, or by tim4-beads. contents in ecvs were analysed by immunoblotting or flow cytometry. roles of lipid rafts in the generation and secretion of ecvs were analysed by treating cells with 1 mm methyl β-cyclodextrin. results: using 4 melanoma cell lines, ecvs were isolated by ultra-centrifugation, and their sizes were analysed by nanosight. all samples showed uniform sizes between 100 and 200 nm. protein amounts in ecvs were measured, showing heterogeneous levels at 10~80 μg/100 ml. then, gangliosides expressed on ecvs from these cell lines were analysed using tim4beads and flow cytometry. gd3 and gd2 were detected on ecvs almost proportionally with expression levels of those gangliosides on the cell surface. then, immunoblotting was performed to analyse integrin levels in ecvs from transfectant cells expressing high levels of gd3, showing increased levels of integrins in ecvs from gd3+ cells compared with those from gd3-cell lines. integrin levels in cell lysates from these cells (gd3+ and gd3cells) were almost equivalent. treatment of a gd3-expressing melanoma cell line by 1 mm methyl β-cyclodextrin resulted in marked reduction of secreted ecvs and amounts of tsg101 in them. summary/conclusion: ganglioside expression patterns on melanoma cells were well reflected in the expression of gangliosides on ecvs. these results as well as increased levels of integrins in ecvs from gd3 + cells suggest that gangliosides and lipid rafts are involved in the generation and secretion of ecvs. introduction: hypoxia, or low oxygen tension, is a common feature associated with tumour growth and is known to regulate tumour cell function, especially through rewiring of cell metabolism. however, how hypoxia influences tumour cell interactions with surrounding cells is not fully elucidated. we sought to evaluate how hypoxia alters metabolite and metabolism-associated mirna packaging in exosomes. methods: exosomes were isolated from 4t1 breast cancer cells cultured in normoxia (21% o2) and hypoxia (5% o2) via ultracentrifugation, optiprep gradients, and size exclusion chromatography. exosomes were further characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. metabolite and mirna profiling was performed on exosomes and exosome-producing cells in normoxia and hypoxia. results: secretion of exosomes was increased under hypoxic conditions. metabolite profiling revealed alterations in metabolites specific to exosomes derived from hypoxic cells. profiling of exosomal mirna showed packaging of metabolism-related mirna into exosomes derived from hypoxic cells. summary/conclusion: hypoxia alters the metabolite and mirna profiles of cancer cells, with selective packaging of these molecules into exosomes. we identified metabolites and mirna that are depleted and enriched in exosomes compared to cells. these studies identify hypoxia-associated shifts in exosome cargo, providing insight into exosome cargo packaging with potential implications for understanding how cancer cell-derived exosomes regulate recipient cell function. lysosomotropic agents prompts the release of extracellular vesicles carrying autophagy-associated markers: evidence of a general mechanism of secretion driven by lysosomal impairment introduction: drug-induced lysosomal storage disorders (lsds) are due to the transient intracellular accumulation, mostly of phospholipids, into multilamellar inclusion bodies within late endosomal/lysosomal compartment. they represent a major side-effect for many drugs of several pharmacological categories. most lsds inducers are cationic amphiphilic drug (cad), but the molecular mechanisms leading to accumulation of undigested substrates are unknown. extracellular vesicles (evs) have been implicated in cell waste disposal, but it is unclear whether they might be involved in extracellular release of undigested substrates. methods: to investigate this aspect, we developed hek cells stably expressing the fluorescent fusion proteins egfp-cd63 and mcherry-cd63, separated evs by differential ultracentrifugation and quantified by evassociated fluorescence and nta particle count. results: evs released by these models upon treatment with drugs inducing the accumulation of phospholipids (amiodarone) or glycosaminoglycans (tilorone), showed the release of fluorescent medium/large evs (10k fraction) and small evs (100k fraction), whose size and distribution were similar to the same vesicles released by control cells, but enhanced the recovery of medium/large evs and to a lower extent of small evs, analysis of evs associated markers revealed a dosedependent increase of autophagy-associated markers in medium/large and small evs. similar results were obtained when autophagic flux was impaired by drugs raising lysosomal ph by different mechanisms, such as chloroquine and bafilomycin, but not when autophagic flux was stimulated by drugs such as curcumin or overexpression of the endosomal/lysosomal regulator tfeb. summary/conclusion: overall results show that impairment of autophagic flux, either by indigested substrates or higher lysosomal ph, is associated with an increased release evs enriched in autophagy markers, compatible with autophagomes and/or amphisomes, unravelling a connection with secretory autophagy. tomofumi yamamoto a , yusuke yamawaki b , yutaka hattori c and takahiro ochiya a a tokyo medical university, shinjuku-ku, japan; b national cancer center research institute, chuo-ku, japan; c keio university faculty of pharmacy, minato-ku, japan introduction: multiple myeloma (mm) is a haematological tumour. last decade, the prognosis of mm has improved by the development of therapeutic drugs; however, mm cells acquire drug resistance by longterm exposure of these therapeutic drugs. one of the possible explanations of drug resistance is that cells with drug resistance transmit information to other mm cells and their microenvironmental cells. although the elucidation of the mechanism of drug resistance in mm have been desired, it remains poorly understood. methods: in order to understand the mechanism of drug resistance in mm, lenalidomide resistant cell lines were established by long-term exposure of low concentration of lenalidomide. drug resistance was assessed by mts assay and caspase assay. the amount of ev was measured by exoscreen, which is ultra-sensitive detection method of evs by measuring surface protein of evs, such as, cd9 and cd63 (yoshioka et al., nat commun., 2015) . to identify the genes which involved in drug resistance, rna sequence among the drug-resistant cell lines and their parental cell lines was performed. results: firstly, characterization of these cells was confirmed. we found that all of the lenalidomide resistant cell lines secreted more evs than their parental cell lines. in addition to this, the size of ev derived from resistant cells are smaller than those of parental cells. next, we collected evs from resistant cells and parental cells by using ultracentrifugation, and added them to parental cells in the presence of lethal dose of lenalidomide. compared with ev derived from parental cell lines, the evs derived from lenalidomide resistant cell lines increased a number of living parental cells. these results suggested that the evs derived from lenalidomide resistant cells can affect the lenalidomide sensitive cells. as a result of rna sequence, several genes highly expressed in resistant cell line we found, which associated with lysosome pathway. among them, attenuating the sort1 and lamp2 genes could significantly reduce the ev secretion in mm cells, leading to enhance the lenalidomide sensitivity. summary/conclusion: our results showed that ev secretion via sort1 or lamp2 could induce the drug resistance in mm. study on biological stimulate mechanism of stem cell-derived exosome generation by nanoparticles introduction: mesenchymal stem cells (mscs) are pluripotent stromal cells known to release extracellular vesicles (evs) containing various growth factors and antioxidants that can positively affect surrounding cells. nanoscale msc-derived evs, such as exosomes, have been developed as bio-stable nano-type materials, but had low yield and were difficult to quantify. we hypothesized that the mechanism of nanoparticleenhanced exosome production would stimulate intracellular molecules. the aim of this study was to elucidate the molecular mechanisms of exosome generation by comparing the internalization of surface-modified positively charged nanoparticles and exosome generation from mscs. methods: mesenchymal stem cells (mscs) were cultured in mem-alpha with 10% fbs and 1× antibiotics. the positively charged nanoparticles were synthesized by poly-lactide-co-glicolide (plga) and polyethylenimine (pei) with cy5.5 for tracking nanoparticles. all of the exosome image were identified using an electron microscope. additionally, it was confirmed the internalization of the nanoparticles by if. the primary antibodies used were anti-eea1, anti-rab7 and anti-gm130. in order to prove the development of exosomes, rt-pcr using autophagy-related mrna was performed. real-time rt-pcr was performed using the applied biosystems sequence detection system 7900. lastly, mirna from msc-derived exosome analysed automatically in the affymetrix data extraction protocol using the provided affymetrix genechip® command console® software (agcc). all statistical testing and visualization of differentially expressed genes was conducted using r statistical language 3.3.3 results: we determined that rab7, located in the mvb and autolysosomal membrane, was increased upon exosome expression and was associated with autophagosome formation. these results suggested that nanoparticles migrated to lysosomes during treatment; however, intracellular exosome-forming factors were stimulated during endosomal maturation simultaneously. summary/conclusion: therefore, msc-derived exosome research using nanoparticles is useful for increasing exosome yield and the discovery of nanoparticleinduced genetic factors. theoretical description of formation of extracellular vesicles by budding of membrane introduction: understanding mechanisms of extracellular vesicles (evs) formation is of utmost importance for their effective use in science, medicine and technology. in particular, the discovery of universal mechanisms explaining the phenomena taking place in vesiculation appears to be crucial and highly warranted. mammalian erythrocytes and giant phospholipid vesicles have been largely used as model systems to study principles of membrane budding and vesiculation. the mechanisms conveniently studied in these simple systems are then generalized to other types of biological membranes. we present a theoretical description of membrane budding and compare the theoretically obtained shapes with the observed ones. methods: in accordance with the fluid crystal mosaic model, membrane is considered as composed of constituents (inclusions) subjected to the local curvature field created by surrounding constituents. constituents can attain different in-plane orientations in the membrane which correspond to different energies. the thermal motion oposes the complete orientational ordering. the single-constituent energy expresses a mismatch of the curvature of the membrane at the position of the constituent and the intrinsic principal curvatures of the constituent and inplane orientation of their principal axes. the free energy of the whole membrane is obtained by summing up (integration) the contributions of the constituents and using methods of statistical physics, and minimized by using numerical methods. results: to outline the principle of (outward and inward) budding, respective sequences of shapes corresponding to a formation of one (outward and inward) spherical bud were calculated by minimization of the free energy. also the corresponding shapes observed in evs (imaged by electron microscopy) and in erythrocytes and giant phospholipid vesicles (imaged by optical microscopy) are shown. it can be seen that theoretically calculated shapes and experimentally observed ones agree well over up to 3 orders of magnitude (the order of the size of giant phospholipid vesicles is between 1 and 100 micro metres, in erythrocytes it is about 5 micro metres and in evs it is about 100 nanometres). summary/conclusion: budding of the membrane is an universal mechanism in formation of external and internal vesicles. introduction: the release of extracellular vesicles (evs) from cells is important for many cellular mechanisms both in normal physiology and in disease. arrdc4 (arrestin domain containing protein 4) is an adaptor protein known to facilitate the ubiquitination of target substrates by nedd4 family ubiquitin ligases. it also traffics cargo to extracellular vesicles. previous studies show the involvement of arrdc4 in the trafficking of the divalent metal ion transporter dmt1 to evs in a ubiquitin-dependent manner, and we aimed to further understand this mechanism. methods: we performed mass spectrometry to identify ubiquitinated lysine residues in arrdc4. we then generated arrdc4 wt and lysine mutant clones and expressed these in cells to determine the effect on ev biogenesis and protein trafficking. results: mass spectrometry data identified 5 potential ubiquitinated lysine residues. out of these, lysine 270 appeared to be the most important for arrdc4 function. arrdc4k270 r mutation caused a decrease in the number of ev released by the cell compared to arrdc4 wt, and a reduction in trafficking of dmt1 to evs. furthermore, we also observed a decrease in dmt1 activity and an increase in its intracellular degradation in the presence of arrdc4k270 r. k270 also appeared to be ubiquitinated with k29 polyubiquitin chains by the ubiquitin ligase smurf1. summary/conclusion: our data suggests that k29 polyubiquitin chains are the signal for arrdc4mediated ev biogenesis and protein trafficking, and loss of this signal causes cargo to be rerouted to intracellular degradation mechanisms. chair: tanina arab -department of molecular and comparative pathobiology, johns hopkins university school of medicine a 3d-printed model to represent the structure and nature of extracellular vesicles, for public engagement and education events. christian burton a , sara veiga a , jason webber a , kate milward a , muireann ni bhaoighill a , lauren evans a , andreia de almeida b , rachel j. errington a and aled clayton a a cardiff university, cardiff, uk; b cardiff university, research associate, uk introduction: explaining the field of extracellular vesicles to the lay public and young audiences can often be challenging. whilst diagrams and images of evs may be helpful, conveying clearly the shape and composition of an ev by these means is not always a success. whilst many members of the audience may be familiar with concepts of cells and related structures, others will find such discussions very abstract and challenging. in order to aid interactions with lay audiences we embarked on the design of a physical hand-held plastic model, representing a typical ev. incorporating flexibility in the design allowing the community to adapt it to showcase their own research. the second goal was to ensure manufacturability using widely available 3dprinting technologies. methods: the basic model design was conceived by dr c. burton, and iteratively developed using solidworks, 2019, then exported for use in any cad environment (stl format). a model showing a halved ev hemisphere, with a visible lipid-bilayer was developed. attachable rings allow trans-membrane-molecules to be represented, current designs include mhc class-i, hspgs, integrins, tetraspanins and supported by handouts accompanying the models. intraluminal cargo is included via removeable "pegs", and examples representing rna or simple globular proteins, and a template has been created. results: the design is free and open source, and available to the community at: https://www.thingiverse. com/thing:3986565. instructions for 3d printing are available from the uk extracellular vesicle society website; https://www.ukev.org.uk/public-engagementmaterials/. models have been produced using entrylevel 3d printers and trialled at engagement events with good early responses. summary/conclusion: the authors hope the community will use and develop this 3d-model design and that the approach provides an additional and helpful tool for educating audiences about the complexities and roles of evs in biology and disease. centrifugal filtration-sec is promising for extracellular vesicle isolation from d492 and d492her2+ breast epithelial cell lines introduction: despite recent developments in breast cancer therapy, there is still need for a more targeted approach. extracellular vesicles (evs), endogenous nanovesicles released from human cells, are an attractive choice as nanodrug carriers due to their size, stability and their unique targeting specificity. the aim of this study was to determine if centrifugal filtration (cf) combined with size exclusion chromatography (cf-sec) would be useful for ev isolation from two epithelial breast cell lines d492 and d492her2+, representing the tissue of interest, and the amount of cell culture needed to get measurable ev concentrations. methods: cell culture media (without serum) from the immortalized breast epithelial cell lines d492 and d492her2+ was concentrated with centrifugal filtration (cf) followed by isolation with size-exclusion chromatography (sec) using hiprep 16/60 sephacryl s-400 column run with äkta start (280 nm), 240 min runs. each fraction (4-5 ml) was collected with fraction collector. dulbecco's particle free pbs was used as mobile phase. the resulting particles were analysed with nanoparticle tracking analysis (nta, nanosight ns300, camera gain 10, static mode, capture time 90 sec), western blotting (wb), microbca and transmission electron microscopy (tem, samples fixed with 2% formaldehyse and stained with 2% uranyl acetate, run at 80kv). results: although sec did not show any prevalent peaks from early eluting regions previously shown to contain extracellular vesicles, these fractions (f1-f3, 40-130 min) were collected from d492her2+ cell culture medium. interestingly, both nta and tem suggest that f2 and f3 contained evs as the isolated particles measured 65 and 58 nm, respectively and tem revealed spherical particles 20-50 nm in diameter. wb was unable to detect the ev associated protein alix (but was present in the whole cell lysate). soluble proteins and protein aggregates eluted late in the sec chromatogram (180 min), with protein analysis (microbca), tem and wb confirming their presence. summary/conclusion: cf-sec is a promising method for ev isolation for pharmaceutical applications, but further work is needed to optimize the isolation process using äkta start for these cell lines. customer stories from the ev core of university of helsinki introduction: the ev core, world's first ev-dedicated technology platform established in 2016, is a joint venture of two extracellular vesicle (ev) research laboratories at university of helsinki. as an academic research/service facility, the ev core provides infrastructure, state-of-the-art and emerging ev-technologies for research groups, hospitals, companies and authorities in the ev-field. the ev core provides ev isolation, purification and characterization services and offers contacts to downstream analyses in other core facilities based on optimized ev protocols. here, we present and discuss the customer experiences and prospects with the aim to further develop ev core services. methods: our most wanted services are nanoparticle tracking analysis, electron microscopy, ev isolation and rna isolation and consultation. currently, the key down-stream analysis methods are (mi)rna sequencing, metabolomics, flow cytometry and functional assays. results: we present the stories from our customers starting with their research questions and need for the ev expertise/consultation and equipment. next, we show how the projects advanced and what types of ev core -derived or other downstream services helped them to achieve their aims. in the end, we will acknowledge the customers experience and current status of their research. summary/conclusion: narratives of customer stories are an effective starting point for fruitful discussions about the current status and next developments in the young ev service field. recent isev workshops: open, reproducible and standardized ev research (ghent, 2019) and evs in immunology (buenos aires, 2020) introduction: since its founding in 2012, isev has sought to further extracellular vesicle research in various ways including scientific meetings. these events encompass annual meetings as well as smaller, topically focused workshops, with the first isev workshop (on rna and evs) organized in new york city in october, 2012. in december, 2019, the workshop "open, reproducible, and standardized ev research" was held in ghent, belgium. in march, 2020, the workshop, "evs in immunology" was held in buenos aires, argentina, with a preceding education day. methods: the international organizing committees of the 2019 and 2020 isev workshops prepared scientific programs around key themes of ev rigour and standardization (ghent, belgium, 2019 workshop) and evs in immunology (buenos aires, argentina, 2020 workshop). abstract and application submissions were invited. applications were reviewed and ranked by panels of ev experts for each event, and participants were invited. results: the 2019 and 2020 workshops assembled a total of more than 100 individuals for talks and discussions around the themes of rigour and standardization and evs in immunology. the buenos aires workshop was preceded by an education day, coordinated by the isev executive committees for education and science and meetings. during these two workshops, poster presentations were permitted for the first time, affording additional presentation and interaction opportunities. the rigour and standardization workshop also featured real-time discussant polling to facilitate discussion. summary/conclusion: isev workshops such as those addressing rigour and standardization (ghent, 2019) and evs in immunology (buenos aires, 2020) continue to provide opportunities for focused discussion of small groups of experts on key topics in the field. often followed by published products, isev workshops help to lead and coordinate progress in ev science. for future isev workshops, educational activities may again expand the reach of each event, while poster sessions and app-driven real-time responses should be considered for enhanced interactions and participant canvassing. ev journal club: exchanging pizza for a worldwide audience during covid-19 kenneth w. witwer johns hopkins university school of medicine, baltimore, usa introduction: a monthly journal club focused on extracellular vesicle science was established at johns hopkins university in 2016, featuring lunch and presentations by academic and industry participants. when covid-19 prevented in-person meetings beginning in march, 2020, the journal club was converted to a virtual, weekly format on the popular online meeting app zoom. the journal club has persisted despite initial problems with online vandalism. most sessions are also made public on a youtube channel, https:// www.youtube.com/c/extracellularvesicleclub. methods: weekly ev club sessions are arranged by the host. most focus on a specific manuscript related to evs, but some weeks feature presentations of published or soon-to-be-published research by the presenting authors. sessions are advertised one week to several days in advance on social media platforms such as linkedin, twitter, and facebook, asking interested parties to sign up to join a mailing list via surveymonkey. the log-in information is then sent to the mailing list. upon clicking the link, participants are placed in a virtual waiting room for vetting by the host and volunteers. after admission, all parties but the host and presenter are muted to avoid distractions. questions and comments may be placed in a chat box. contributions are monitored and compiled by the host and volunteers to build a question-and-answer session at the end of the presentation. recorded sessions-with or without editing as needed-are placed on the youtube channel for additional access. results: despite initial problems with online vandalism known as "zoombombing," the journal club has continued weekly during the covid-19 shutdown in the host country (us). an audience of between 80 and 150 individuals is typical. participants typically ask more questions than can be answered in a one-hour time frame. the online format also allows for debate-style events and polling of the audience. summary/conclusion: this ev journal club is an example of how online tools can be used to facilitate international scientific interactions. further development of such formats could provide alternative approaches for isev activities in the science, education, and communication areas. the study aim is to assess whether the exposure to pm10 and pm2,5, chosen as paradigmatic environmental stressors, could modify the composition of nasal microbiota (nm) and extracellular vesicle (ev9 signalling network, showing a role in allergic ar exacerbation). methods: nm analysis were performed on v3-v4 16 s rrna gene regions amplified from upper-airway tracts of 25 ar cases and 25 healthy individual controls to perform nm analyses. ev size, concentration and cellular origin for each subject were assessed by nanoparticle tracking analysis (nta) and flow-cytometry (fc). information on daily pm10 and pm2,5 concentrations at the municipality of residence in the 7 days preceding nasal sampling (i.e. day −1 to day −7) was assigned to each subject by arcgis software. multivariable and logistic analyses were applied on nm, nta and fc outcomes. results: when taxonomy composition was considered, in controls actinobacteria (50.8%) was the most represented, followed by firmicutes (34.7%) and proteobacteria (12.8%) while in cases proteobacteria were 38.8%, actinobacteria were 37.1% and firmicutes were 23.4%. cases showed a higher concentration of all the investigated ev types, derived from platelets (cd61 +), activated endothelium (cd62e+), monocytes (cd14+), eosinophils (cd294+), neutrophils (cd177+), mastocytes (cd203 c+), epithelial cells (epcam+), gram+ bacteria (lipoteichoic acid+), gram-bacteria (lps+). the effect was greatest in the case of mastocytes evs which were increased 2.5fold in cases versus controls (p < 0.001). evs were modified by pm exposure at several time lags. in particular, a negative association between pm10 and eosinophil evs was observed (beta = −0,016; pvalue = 0,017). as we clustered subjects according to their nm, we observed this variable was a strong effect modifier of the association between pm exposure and ev release. summary/conclusion: our findings start to provide an insight on the effect of air pollution on evs, taking into account the effect of nm, in patients with ar. further research is necessary to disentangle the mechanism exerted by inhaled pollutants in modulating evs and nm, and therefore ar exacerbation. funding: gsk investigator sponsered study aryl hydrocarbon receptor activation induces the expression of specific microrrnas in th17 cells that are release into extracellular vesicules and associated with arthritis introduction: in rheumatoid arthritis (ra), an autoimmune disorder characterized by a chronic sinovial inflammation, smoking is a major risk factor contributing to disease progression, and poor response to therapy. th17 cell is actively involved in worsening smooking-associates inflammation mediated by aryl hydrocarbon receptor (ahr), a cytoplasmic transcription factor involved in xenobiotic metabolism. both, ahr and th17 cells, has important implications during ra development. considering that cigarette smoke is a potent epigenetic modifier, we hypothesized that ahr activation, by cigarette components, would transcribe specific micrornas in th17 cells as a molecular mechanism to exacerbate inflammation in arthritis. methods: microrna expression was evaluated by largescale approach or real-time pcr. c57/bl6 and ahr null mice were submitted to arthritis experimental models and exposed or not to cigarrete smoke (ethical committee approved 048/2012). extracellular vesicles (evs) were isolated by ultracentrifugation, and characterized by western blot and nanosight. rankl-induced osteoclasts (ocs) differentiation in vitro was stained for trap. inhibition of mirnas were performed using anti-mirs transfection. results: we identified a specific group of mirnas induced in th17 cells after ahr activation. during arthritis progression, the micrornas are expressed and increases after exposure to cigarette smoke. in the absence of ahr their levels were drastically reduced. interestingly, we found that these micrornas are released by th17 cells into evs, and are able to promote osteoclastogenesis. ocs differentiation in vitro increases in the presence of th17-derived evs, and this process is reduced in the absence of micrornas. summary/conclusion: microrna-mediated gene regulation plays crucial roles in the immune system functions, and their abnormal expression is highly correlated with the pathogenesis of ra. evs are known to function in cell-to-cell communication and are able to transmit their contents and cause changes in the target cell. our findings demonstrate a new molecular mechanism by which cigarette smoke could aggravate inflammation in arthritis; through the activation of ahr receptor in th17 cells, inducing the transcription of specific micrornas that are released into evs, and act as pro-inflammatory mediators. introduction: chagas disease (cd) is caused by the flagellated protozoan t. cruzi. trypomastigote forms are capable of releasing extracellular vesicles (evs) that contain the major surface molecules of the parasite. the parasite has a complex life cycle that leads to it a rapid adaptation in the environmental changes in the hosts. however, the effects of stress on on evs release are not completely understood. objetive: we evaluated the release of evs by trypomastigotes incubated under different stress conditions and the immunomodulatory role of these evs in pre-activated bone marrow-derived macrophages (bmdm). methods: nanoparticle tracking analysis (nta) and scanning electron microscopy (sem) showed an increase in evs releasing by trypomastigotes at 4°c under acidic conditions, evs released was affected and triggered amastigogenesis process. results: treatment with sodium azide (nan3) also caused changes in the release of evs regarding size and concentration. nitrosative stress caused by sodium nitrite (in culture medium mildly acidic, ph 5.5; in this condition nano2 releases nitric oxide) stimulated an increase in production of evs by t. cruzi. when the parasites were treated with 100 nm s-nitrosoglutathione (snog), we observed a reduction in size and concentration of vesiculate material by trypomastigotes. at a higher snog concentration (100 µm), the concentration of the vesiculate material increased. t. cruzi-derived evs exposed to stress conditions increased the expression of inos, arg 1, il-12 and il-23 genes in ifn-γ and lps pre-activated bmms. summary/conclusion: results suggest that the viability and/or integrity of the parasite are necessary for the evs releasing. in those in vitro conditions they triggered a proinflammatory response in host cells. this may be a strategy developed by the parasite to favour its establishment in the host. funding: fapesp, cnpq, capes and fapemig ppm-x 00102/6. immuno-toxicological evaluation of human mesenchymal stem cellintroduction: mesenchymal stem cells (mscs) have been widely used to the field of autoimmune diseases or tissue regeneration therapy. recently, many research groups have reported that mscs showed their ability via secreted paracrine mediators including extracellular vesicles (evs) rather than cell-to-cell contact. mscs mainly exist on bone marrow, peripheral blood, umbilical cord and adipose and can mostly secrete evs. it has emerged that evs alone are responsible for the therapeutic effect of mscs in plenty of animal diseases models. hence, msc-derived evs may be used as an alternative msc-based therapy in regenerative medicine. methods: as part of safety programme for human therapeutics, we performed immunotoxicological assessment of evs obtained from human mscs (hevs) in mice and human peripheral blood mononuclear cells (hpbmcs). firstly, mice were treated intravenously with a negative control, a positive control (lps; 0.4 mg/kg), or low-dose (1x10e8 paticles/head) and high-dose (1x10e9 paticles/ head) of hevs every other day for 10 days and then analysed lymphocyte subsets from collected spleen by facs. next, we treated the evs on hpbmcs for 3 days with low conc. (1x10e8 particles/ml), high conc. (1x10e9 particles/ml), pma/ionomycin as a cell activator or cpt (10 μm) as an apoptotic inducer. annexin v/pi and csfe were analysed by facs. results: as a result, splenic nk cells and b cells were slightly increased about 2~7% in hevs-treated mice, without biological significance, compared with a positive control (lps) as an immunogenicity inducer. and there were no effects on serum levels of inflammatory cytokines in mice. in addition, hevs had no cytotoxic effect on hpbmcs at both low and high conc. under the culture medium with evs-depleted fbs, the hevs appeared minimal anti-apoptotic effect on hpbmcs. for the cfse assay, the hevs showed slight proliferation on hpbmcs and pbmc activation induced by pma/ionomycin. summary/conclusion: in conclusion, the hevs have little immuno-toxicological effects in mice and hpbmcs. further detailed studies to elucidate immunological response of hevs for development of human therapeutics are needed. funding: this research was supported by a grant (18172mfds173) from ministry of food and drug safety. investigation of immune response to mesenchymal stem cell-derived extracellular vesicles in the cancer setting introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) are thought to be a fingerprint of the secreting cell and therefore may retain the cancer targeting and immune privilege of mscs. thus msc-evs hold immense potential as tumour-targeted therapeutics for breast cancer. the aim of this study was to determine whether msc-ev administration in tumour bearing immunocompetent animals would initiate an immune response. methods: evs were isolated from conditioned media of both human and murine bone marrow-derived mscs through sequential differential centrifugation, microfiltration and ultracentrifugation. evs were characterized by nanoparticle tracking analysis (nta), western blot and transmission electron microscopy (tem). 1 x 10(8) human or murine msc-evs were administered intravenously into 4t1 breast tumour bearing balb/c mice (n = 6) and healthy controls (n = 6). tumour tissue, draining lymph node and spleen were then harvested, dissociated and flow cytometry performed targeting markers associated with a range of immune cells including t-cells, macrophages and natural killer (nk) cells. results: evs were successfully isolated from murine and human mscs with the appropriate size of small evs (sevs: 30-150 nm) and morphology including a lipid bilayer observed by tem. evs expressed tetraspanins cd63, cd81, cd82; cytosolic protein tsg101 and were negative for calnexin. ev concentrations ranged from 4.08 x 10(9) -6.6 x 10(9)/ml. in order to study a range of immune cell populations two antibody panels were created using complimentary fluorescent dyes. the proportion of t-cells (cd4+, cd8+, cd25+), neutrophils (gr-1+, ly-6 c+), dendritic cells (cd11 c+), macrophages (cd11b+, mhci+, mhcii+), nk cells (cd27+) and b cells (cd19+) remained stable in the tumour, draining lymph node and spleen of all tumour-bearing animals that received either human or murine msc-evs, with no significant change observed in any category. summary/conclusion: the data presented supports the hypothesis that msc-evs retain the immune privilege of the secretory cell, with human cell-derived evs illiciting no immune response in mice. this is encouraging and reinforces the potential for use of msc-evs in the therapeutic setting. introduction: mycobacterium avium (m. avium) is a slow growth rate non-tuberculous mycobacterium (ntm). m. avium infection is a severe global health problem. but the mechanisms of pathogenicity of m. avium are poorly understood. outer membrane vesicles (omvs) that traverse the cell wall and contain a varied bioactive components inculding dna, rna, protein and toxins. previous studies have suggested that these omvs are produced in vitro and during animal infection, but the role of omvs secretion during the interaction of m. avium with host cells remains unknown. methods: in this study, m. avium were grown in middlebrook 7h9 medium (m7h9) supplemented with 10% (v/v) oadc enrichment and 0.5% (v/v) glycero. m. avium omvs were isolated by ultracentrifugation method. characterization of omvs by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). the raw 264.7 murine macrophages were incubated with the m. avium omvs to analyse inflammatory response and production of nitric oxide (no) and reactive oxygen species (ros) of macrophage. results: in this study, we demonstrate by fluorescence microscopy that murine macrophages can phagocytosis omvs produced by m. avium. incubation of m. avium omvs with murine macrophages resulted in increased levels of extracellular tumour necrosis factor alpha (tnf-α), interleukin-1β (il-1β), terleukin-6 (il-6) and interleukin-12 (il-12). meanwhile omvs stimulated macrophages produce no and ros. introduction: hospital associated venous thromboembolism (ha-vte) in paediatric patients is the second most common contributor to harm in hospitalized children. platelet-endothelial interactions are integral to the formation of vte, especially in inflammatory conditions such as sepsis. small extracellular vesicles (sevs) have the ability to reprogramme target cell phenotypes via their microrna contents and are known to contribute to vte formation. we hypothesize that sepsis alters platelet-derived sev micrornas capable of net upregulation of vascular endothelial procoagulant and downregulation of anticoagulant pathways. methods: using a precipitation solution and size exclusion chromatography, we isolated sevs from platelet poor plasma of children admitted to the paediatric intensive care unit for sepsis and from healthy controls. we positively selected platelet-derived sevs using immunomagnetic isolation for cd42b platelet antigen and confirmed selection using flow cytometry. microrna was profiled using affymetrix genechip mirna 4.0 array. results: microrna from 9 sepsis patients (median age 7.0 years; iqr:1.2-13 and 77% female) with a median psofa score of 6 (iqr:2.5-10) and from 5 healthy controls (median age 9 years; iqr:6.5-13.5 and 20% female) was isolated and compared. in septic vs. healthy patients 30 mirnas were differentially expressed (false discovery rate (fdr)<0.05; fold change ≥|1.5|) affecting 921 mrna pathways. in septic children, pathways affecting chemotaxis and cell movement of leukocytes were predicted to be activated with z-scores ≥2. summary/conclusion: we developed a method to successfully isolate platelet-derived sevs. sepsis alters the platelet-derived sev microrna profile in paediatric patients with sepsis. these micrornas are predicted to target chemotaxis and cell movement pathways, important contributors in the formation of ha-vte. further analysis into specifically targeted pathways should be conducted as a potential target for the prevention of ha-vte in sepsis. introduction: sjögren´s syndrome (ss) is a systemic autoimmune disease that mainly affects salivary and lacrimal glands. mechanisms of ss pathogenesis are poorly understood. it is thought that inflammation leads to destruction of exocrine glands, however the triggers of autoimmunity and the mechanisms by which inflammation drives immunopathology are not characterized. our work identifies t cell-exosomederived mir-142-3p as a pathogenic driver of immunopathology in ss. micrornas (mirnas) are endogenous small noncoding rna molecules that regulate the expression of target genes through translational repression of mrnas. through transcriptomic profiling studies our group had previously documented a significant upregulation of mir-142-3p in patient ss tissues and in serum exosomes. methods: structured search for target genes of mir-142-3p involved in salivary gland (sg) physiology was performed with mirdip 4. serca2b, ryr2 and ac9 were selected for further validation and functional analysis. binding of the mirna was confirmed by luciferase reporter assays in hsg cell lines and human-derived primary epithelial cells. the mrna and protein levels of serca2b, ryr2 and ac9 were determined by qpcr and western blot, respectively. to investigate the cell-specific distribution of mir-142-3p in relation to the expression levels of serca2b, ryr2, and ac9, a double fluorescent in situ hybridization was performed. ca2+ signalling and camp levels were measured using fluorescent sensor. to isolate exosomes, the t cell medium and serum of ss-patients and healthy volunteers (hv) were collected. results: we show that mir-142-3p is over-expression in the sgs of ss-patients. next, we demonstrated that mir-142-3p is contained in exosomes in serum of sspatients significantly more than serum of hv. we also show that activated t cells secrete exosomes containing mir-142-3p which transfer into glandular cells and affecting intracellular ca2+ signalling, camp production and protein production by mir-142-3p targets (serca2b, ryr2 and ac9). summary/conclusion: this study provides evidence for a functional role of the mir-142-3p in ss pathogenesis and promotes the concept that t cell-activation directly may impair epithelial cell function through secretion of mi-rna containing exosomes. treg-derived il35-coated extracellular vesicles promote infectious tolerance (p35) subunits, yet the forms that il35 assumes and its role in peripheral tolerance, remain elusive. methods: we induce cba-specific, il35-producing t regulatory (treg) cells in tregebi3 wt c57bl/6 reporter mice, and identify il35 producers by expression of ebi3tdtom gene reporter, plus ebi3 and p35 proteins. results: curiously, both subunits of il35 were displayed on the surface of tolerogen-specific foxp3 + and foxp3neg (itr35) t cells. furthermore, il35 producers, although rare, secrete ebi3 and p35 on extracellular vesicles (ev) targeting a 25-to 100-fold higher number of t and b lymphocytes, causing them to acquire surface il35. this surface il35 is absent when ev/exosome production was inhibited, or if ebi3 is genetically deleted in treg cells. summary/conclusion: the unique ability of ev to coat bystander lymphocytes with il35, promoting exhaustion in, and secondary suppression by, non-treg cells, identifies a novel mechanism of infectious tolerance. funding: nih grants r01-ai119140-03 (to w.j.b.), r01 ca203689 and p30 ca047904 (to d.a.a.v.) and the university of wisconsin carbone cancer center support grant p30 ca014520. unique formulated dual targeting antigen specific and delivered mirna-150 gene regulating exosomes acting at the immune synapse to induce apc-derived secondary suppressive exosomes introduction: an exosome-apc circuit we uncovered may be applicable beyond skin immunity we study in mice. methods: high antigen dose tolerized cd8 + t cells make suppressive antigen-specific exosomes due to chosen surface antibody light chains that enable targeting antigen presenting cells (apc) antigen-specifically for delivery of also chosen inhibitory mirna-150 to mediate specific functional gene alterations. results: both antigen and gene specificity aspects are lent to naïve but activated exosomes by simple in vitro incubations alone. for mechanism, these primary exosomes bind antigen peptides in mhc on apc that in turn make secondary suppressive exosomes that act peptide/mhc-specifically on the effector t cells at the immune synapse. they transfer another mirna for strong prolonged inhibition of active delayed-type hypersensitivity (dth) for days even, when the primary mirna-150-pos exosomes are administered orally at the height of the in vivo response, in a physiological dose. summary/conclusion: it is shown possible to induce therapeutic exosomes with ag targeting of choice due to placed ab on the surface and that also target specific gene functions of acceptor cells due to carriage of a selected mirna. this dual ag and gene-specific therapy has applications in treatment of cancer, autoimmunity and allergies. introduction: previously, our group characterized distinct populations of extracellular vesicle (ev) released from neutrophilic granulocytes: ev formed spontaneously (sev) and upon activation with opsonized particles (aev). the aev differs in protein cargo and its ability to inhibit bacterial growth. we described that mac-1 integrin (cr3 receptor) plays key role in the aev production and extracellular calcium supply is crucial in this signalization. in the present work, our aim was to investigate whether mac-1 activation or casignalling on their own are sufficient for the initiation of the aev biogeneis. methods: we isolated neutrophil derived evs from peripheral human blood and murine bone marrow by two-step centrifugation and filtration. we tested the effect of ca-ionophore and examined the ev production on c3bi coated surface and in soluble form. we quantified the vesicles by flow cytometry and determined their protein content by bradford assay. we examined their antibacterial effect in parallel with optical density-based measurement and our flow cytometry based method. results: on c3bi coated surface, we observed an increased ev production, and these evs possessed antibacterial capacity. however, in soluble condition, c3bi did not induce further ev production, and these evs did not show any antibacterial property. we found that ca-ionophore initiated ev formation, but these ev did not show antibacterial effect. we observed ev production increase after ca-ionofore treatment both in the presence and in the absence of extracellular ca. the ca-ionophore slightly increased the opsonized particle induced ev production, but did not potentiate their antibacterial capacity. summary/conclusion: mac-1 activation is not just crucial, but sufficient in initiation of the aev biogenesis. clustering of this receptor is required. while the ca-signal is crucial, it is not sufficient in the generation of aevs. extracellular vesicles and their microrna cargo in retinal health and degeneration: mediators of homoeostasis, and immune modulation yvette s. m. wooff, adrian cioanca, riemke aggio-bruce, joshua chu-tan, ulrike schumann and riccardo natoli the australian national university, canberra, australia introduction: photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (amd). however, the molecular mechanisms regulating these biological processes are largely unknown. extracellular vesicles (ev) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. evs, including exosomes, encapsulate and transfer microrna (mirna) to recipient cells and in this way can modulate the environment of recipient cells. dysregulation of evs however is correlated to a loss of cellular homoeostasis and increased inflammation. in this work we investigated the role of isolated retinal small-medium sized ev (s-mev) in the regulation of homoeostasis and immune modulation in both the healthy and degenerating retina. methods: isolated s-mev from healthy and degenerative (photo-oxidative damaged) mouse retinas were characterized using dynamic light scattering, transmission electron microscopy and western blot, and quantified using nanotracking analysis. small rna-seq was used to characterize the mirna cargo of retinal s-mev isolated from healthy and degenerating retinas. finally, the effect of exosome inhibition on s-mev-mediated immune modulation was investigated using systemic daily administration of exosome inhibitor gw4869 and analysed by in situ hybridization of s-mev-abundant mirna. electroretinography and immunohistochemistry were performed to assess functional and morphological changes to the retina as a result of exosome depletion. results: our results demonstrated an inverse correlation between s-mev concentration and photoreceptor survival, with decreased s-mev numbers following retinal degeneration. small rna-seq revealed that s-mevs contained uniquely enriched mirnas, however no differential composition in s-mev mirna cargo following photo-oxidative damage was observed. exosome inhibition using gw4869 exacerbated photoreceptor degeneration, with reduced retinal function and increased levels of inflammation and cell death seen following photo-oxidative damage. further, reduced translocation of the photoreceptor-derived s-mev was demonstrated following exosome-inhibition in photo-oxidative damaged mice. summary/conclusion: taken together, we propose that retinal s-mev and their mirna cargo play an essential role in maintaining retinal homoeostasis through immune-modulation, and have the potential to be targeted using gene therapy for retinal degenerative diseases. impacts of agricultural dust exposure on human lung-resident mesenchymal stromal/stem cells and their extracellular vesicles introduction: agricultural dust is considered a high-risk occupational hazard by the cdc, with impacts reaching throughout the communities surrounding these industries, leading to increased incidence of respiratory illness and disease among individuals within this occupation and these communities. lung-resident mesenchymal stromal/stem cells (lr-msc) have an important role in maintaining homoeostasis in the lung, and mediating pro-and anti-inflammatory effects, particularly during exposure to inhaled irritants, like agricultural dust. one way in which these lr-msc promote lung homoeostasis is through the release of extracellular vesicles (ev), with a variety of cargo that elicit changes among target cells. we hypothesize that exposure to agricultural dust modifies the quantity and cargo of ev released by lr-msc to promote lung tissue homoeostasis. methods: primary human lung-resident mesenchymal stromal cells were exposed to extracts of dusts collected from swine confinement facilities (de) for 8 or 48hrs and the media from these exposures were collected and enriched for ev by opti-prep density gradient ultracentrifugation. the quantity of these ev were assessed by nanoparticle tracking analysis. additionally, cytokine and chemokine release by lr-msc were analysed by enzyme-linked immuno assays. results: as assessed at 48 hr following treatment, deexposed lr-msc released pro-inflammatory cytokines, il-6 and il-8, with il-8 release reaching statistical significance at 0.1%, 0.5%, and 1% de concentrations (p = 0.0367, <0.0001, and <0.0001 respectively) and il-6 trending a similar dose response but only statistically significant at 1% de (p = <0.0001). de exposure of lr-msc also induced changes in the lr-msc-derived ev populations when compared to vehicle control, where lr-msc released significantly more ev in the 5 and 10% iodixanol fractions (p = <0.0001 and 0.0219, respectively) at 8 hr following de treatment. alternatively, there were significantly less ev in the 20 and 40% density fractions in the media of deexposed lr-msc versus vehicle control. summary/conclusion: following exposure to agricultural dusts, lr-msc-derived ev populations more likely consist of exosomes and ectosomes, which play an important role in promoting lung tissue homoeostasis during exposure-related pulmonary inflammation. introduction: during analyses of single extracellular vesicles (evs) by flow cytometry (fcm), particles below the detection limit may exceed the trigger threshold, which is called swarm detection and generates false-positive counts. serial dilutions are recommended to find the minimal dilution for which swarm detection is absent. however, because particle concentrations in plasma vary, the optimal dilution differs >100-fold between donors, but it is unfeasible to do serial dilutions for each clinical sample. therefore, our aims are to (1) develop a faster method to avoid swarm detection, and (2) increase the number of detected evs per second. methods: we measured serial dilutions of cd61stained evs in platelet free plasma (pfp), with and without spiking of fitc beads, by fcm (apogee a60-micro). we triggered either on side scatter or fluorescence. results: for scatter triggering with our fcm, swarm detection consistently occurred for plasma samples exceeding a (total particle) count rate of 4,100-4,200 events/s. the cd61+ evs concentration scaled linearly over 1.5 orders of magnitude of the dilution and most donors required >1000-fold dilution to avoid swarm detection, thereby reducing cd61+ ev counts. for fluorescence triggering, the cd61+ evs concentration scaled linearly over >3 orders of magnitude of the dilution. for all donors, swarm detection was absent after 32-fold dilution (relative to pure plasma). the count rates of cd61+ evs were 30-100-fold higher compared to scatter triggering. the spiked fitc beads confirmed that the median signals remained constant. summary/conclusion: we have developed two clinically applicable ways to avoid swarm detection. for scatter triggering, the count rate provides direct feedback on the presence of swarm detection in plasma samples. for fluorescence triggering, swarm detection was absent for all plasma samples diluted ≥32-fold and compared to scatter triggering, count rates of cd61 + evs were 30-100 fold higher, thereby improving statistical significance. funding: edwin van der pol is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. benchmarking flow cytometric analysis of nanoparticles: a cross-platform study for single extracellular vesicle detection introduction: despite flow cytometry being widely used to analyse cells in suspension, most commercial instruments lack sensitivity when measuring nanoparticles (nps) and extracellular vesicles (evs). furthermore, the use of appropriate reference materials (rms) for calibration and quality control are essential to compare results acquired with different instruments. to work towards successful clinical applications for ev biomarker profiling, benchmarking studies including state-of-the-art flow cytometers are required. we here investigated the ability of three different flow cytometers to detect nps and evs. methods: the instrument sensitivity of light scattering detection was evaluated by using synthetic nps of different sizes and refractive indices. fluorescent calibration was investigated by using molecules of equivalent soluble fluorophores (mesf) beads. biological recombinant evs (revs) were used to validate the detection and quantification of fluorescent evs in a side-by-side cross-platform study using an n30 nanoflow analyser (nanofcm), an optimized bd influx and a cytoflex lx. results: we found that when light scatter based detection was used, the nanofcm detected the smallest non-fluorescent nps, the bd influx was able to provide reliable fsc information from the smallest detected nps and the cytoflex performance was greatly improved by the use of violet-ssc. biological revs showed that the nanofcm could clearly resolve fluorescent evs while the bd influx and cytoflex were unable to fully resolve revs from background, although fluorescence threshold improved detection. in addition, our findings revealed that different concentrations are required to ensure single ev detection in these platforms. summary/conclusion: we identified several strengths and limitations for each platform with respect to single ev analysis. furthermore, our results showed that proper calibration and rms are of utmost importance to ensure reliable interpretation of ev flow cytometric data. caution when using membrane dyes for sequential extracellular vesicle analysis diana pham, michael wong, desmond pink and john lewis nanostics, edmonton, canada introduction: confirmation that particles detected by microflow cytometry are actually extracellular vesicles (evs), or at least membranous in composition, can be achieved through a variety of methods. positively staining particles with a membrane dye strongly suggest that the particle contains a membrane; loss of stain (or detection) after detergent solubilization of the membrane-dyed particles provides even stronger evidence that the particles were evs. it is important to recognize that the labelling protocol provided by the membrane dye manufacturer may not be ideal for all types of evcontaining biological samples, such as blood, urine, semen etc.). removal of excess dye from stained evs is very difficult and can be impractical depending on the nature of the experiment. however, this means that the potential for excess dye to contaminate subsequent sampling is high. therefore, it is important to determine optimal working concentrations and labelling conditions when using membrane dyes for ev detection to understand properties that may impact your analyses. methods: to assess the utility of membrane dyes, titration curves were generated to determine the optimal working concentrations of membrane dyes for ev detection in conditioned media and human serum samples. once the optimal concentration was determined the potential of dye carry-over from sample to sample during microflow cytometry detection was evaluated by tracking dye positive (dye+) particles in phosphate buffered saline (pbs) blanks and matched, unlabelled, sample replicates. results: we found that optimal concentration of any membrane dye is dependent on sample type. even with the inclusion of system washes to prevent sample carryover, there was carryover of low amounts of dye+ particles into sequentially analysed pbs blanks. if unstained samples were analysed following a stained sample, excess dye (or at least dye+ events) appeared in the data. a sample concentration effect was also seen; samples of lower concentrations were more susceptible to dye carryover. summary/conclusion: when using membrane dyes to stain evs in biological samples, especially if an autosampler is employed to run a series of tests, it is critical to determine the optimal concentration of dye for each type of sample, as excess dye can carry over to the next sample in the queue. in addition, determining the necessary steps to clean any excess dye following each sample run will improve the accuracy of ev detection and analyses. funding: nanostics alberta innovates alberta cancer foundation correlation between size and protein expression of single exosomes by combined atomic force and fluorescence microscopy introduction: there are no universal markers of extracellular vesicles, but often they are identified by the presence of tetraspanins in their membrane. based on this, products have been developed to precipitate or quantify evs by acting upon cd9, cd81, and cd63. however, evs also carry proteins from their parent cells, and capturing evs based their presence allows for a more complete understanding of vesicle heterogeneity from a single cell type, and for evs derived from specific tissues to be enriched from other biofluids in support of biomarker assessment. for example, evs derived from the brain could be captured from the general population of serum evs for better assessment of cargo associated with proteinopathy. the goal of this study was to identify specific antibodies to capture and label evs bearing the neural markers cd171, snap25, α-synuclein, tau, and ncam. methods: the targets were overexpressed in hek293 t cells through transient transfection of plasmids (origene). media was conditioned for 24-48 hours, and then centrifuged to remove cell debris. cell lysates and concentrated conditioned media (cm) were analysed by western blot. unpurified cm, or cm after performing size exclusion chromatography (sec, izon), were analysed in the exoview r100 system. diluted cm was incubated on custom antibody microarray chips overnight. then the chips were labelled with a cocktail of 3 labelled antibodies, washed and imaged. vesicles were counted, sized, and phenotyped. next, commercially available pooled human csf was analysed in a similar fashion to determine their abundance in a relevant biofluid. results: multiple antibody clones were tested in different combinations for capture and labelling for the five different neuronal enriched proteins of interest, and optimal combinations were identified. some markers were identified on particles > 50 nm in size that were negative for tetraspanins, while others colocalized with tetraspanins. through comparing permeabilized and intact evs with and without sec to remove non-vesicular proteins, we found that tau could be on the vesicle surface, within the vesicle, and free in solution. summary/conclusion: the exoview platform can be customized to enable the detection of proteins of interest and to determine whether they are on the ev surface, intravesicular, or non-ev associated. methods: forty non-smoking male and female subjects (40-70y) at moderate risk for cvd were recruited for the study. evs from platelet-free plasma (pfp) were isolated using size exclusion chromatography (sec). the concentration and size distribution of evs were measured by nanoparticle tracking analysis (nta) and flow cytometry (fcm). three ev markers, including annexin v for the circulating phosphatidylserinepositive (ps+) evs, cd41 for platelet-derived evs and cd105 for endothelial-derived evs were used for phenotyping. in addition, coagulation and fibrinolysis were assessed using a thrombodynamics analyser (hemacore). platelet aggregation to determine platelet function was assessed by a high-throughput platelet function assay with a wide range of concentrations of agonists, including adenine diphosphate (adp), collagen-related peptides (crp-xl), epinephrine, thrombin receptor activating peptide (trap-6) and u46619. the association between thrombogenic risk markers for cvd and ev numbers was tested by pearson's correlation coefficient and linear regression model using the statistical program, spss. results: circulating ev concentration with threshold of 71 nm, measured by nta, were positively associated with coagulation-related risk markers, including rate of clot growth (r = 0.568; p = 0.01) and clot size at 30 min (r = 0.480; p = 0.002). ps+ evs derived from endothelial cells, determined by fcm, were negatively associated with lysis onset time (r = −0.410; p = 0.009), whereas they were found positively correlated with lysis progression (r = 0.417; p = 0.007). both mean and mode size of cevs, detected by nta, were significantly correlated with u46619-induced platelet aggregation (r = −0.338; p = 0.033, r = −0.382; p = 0.015, respectively). summary/conclusion: in subjects at moderate risk for cvd, cev numbers were positively related to rate of clot growth and clot size and size of cevs was negatively related to platelet activity. higher numbers of endothelial cell-derived ps+ cevs were associated with lower rates of fibrinolysis. this suggests that cevs promote clot growth and reduce fibrinolysis, and may therefore be an indicator for greater risk of cvd. beyond stem cells: extracellular vesicles from human induced pluripotent stem cells (hipsc) and hipsc-cardiomyocytes as therapeutic approaches for heart failure introduction: heart failure is caused by a variety of underlying diseases, the most common being myocardial infarction. initially regarded as an alternative to pharmacological approaches, stem cell transplantation has failed to demonstrate clinically meaningful results. instead, it has become increasingly apparent that the therapeutic effects of transplanted cells are largely mediated by their secretome, while mounting evidence suggests extracellular vesicles (evs) play a major role in cardiac repair. within this framework, evs from human induced pluripotent stem cells (hipsc) and hipsc-derived cardiomyocytes (hipsc-cm), hold a tremendous potential to treat cardiovascular disease. we isolated evs from conditioned culture media at key stages of the hipsc-cm differentiation and maturation processes, i.e. from hipsc (hipsc-ev), cardiac progenitors (cpc-ev), immature (cmi-ev) and mature (cmm-ev) cardiomyocytes, with the aim of studying their potential role as therapeutics, and whether their effectiveness was influenced by the state of their parent cell. methods: hipsc were differentiated into cardiomyocytes in a 3d culture approach, using the protocols developed by our group. ev isolation was performed on an iodixanol density gradient, and the evs were characterized in terms of particle size and particle size distribution, presence of ev-specific markers, and imaging through transmission electron microscopy. functional studies were performed using human umbilical vein endothelial cells (huvecs) to evaluate evuptake, cell migration and angiogenesis. results: evs from all hipsc and cardiac derivatives presented a typical cup-shaped morphology and expressed cd63 and cd81. ev yield varied along differentiation, with a minimum for cpc and a maximum for cmi. pkh26-labelled evs were uptake by huvecs, and colocalized with calnexin, a protein from the endoplasmic reticulum. wound healing assays showed an increased cell migration in huvecs treated with cardiomyocyte-derived evs, in comparison with control evs isolated from foetal bovine serum. summary/conclusion: our findings suggest a different ev secretion profile along cm differentiation and maturation, with preliminary assays showing ev functionality. ongoing work aims at elucidating the possible differences in function and cargo amongst these types of evs. endothelial cells differentially load and secrete extracellular vesiclederived micrornas into apical and basolateral compartments this may play a role in microcalcification in calcific aortic valve disease (cavd), but this is poorly understood. annexin a1 is thought to be a marker of membrane-derived evs, but because it can be found on the cytoplasmic or extracellular side of the plasma membrane, its localization within or on the surface of evs is unclear. the goal of this study was to determine whether annexin a1 is found on the surface of evs in two cell lines relevant to cavd, and develop an assay that can be used to determine whether this changes under pathogenic conditions. methods: evs were isolated by differential ultracentrifugation from the conditioned medium (cm) of smooth muscle cells (smc) and valvular interstitial cells (vic). total protein in the cell lysates and ev pellets was analysed by western blot. evs from cells treated with control sirna or anxa1-sirna were enumerated and phenotyped using the exoview r100 platform. evs with surface expression of cd9, cd81, cd63, and annexin a1 were captured using a customized antibody microarray chip. then evs were labelled with fluorescent antibodies to assess ev number, size, and colocalization of ev proteins. the knockdown of annexin a1 allowed us to assess the specificity of the selected annexin a1 antibody. results: the ev fraction was positive for cd9, and lacked markers of other vesicle types. western blot on the ev pellet and supernatant in ± edta indicated that there is annexin a1 both on the surface of and within the evs. using the antibody microarray chips, numerous annexin a1+ evs were captured on the annexin a1 spots from the control cm, and there was a marked decrease in capture and labelling from anxa1-sirna treated cells. under both conditions, vesicles were also captured on tetraspanin probes, with the greatest number captured on cd9, then cd63 and cd81. there was a significant population of annexin a1+ evs that was negative for tetraspanins. summary/conclusion: annexin a1 is found on the surface of evs. the assay developed in collaboration with nanoview biosciences is well suited for assessing the number and phenotype of annexin a1+ evs derived from smc and vic cell lines, which could provide a useful method for understanding ev populations in cavd patient cell lines. funding: this work was supported by hl136432 and hl147095. possibility of exosomal micrornas associated with chronic limb-threatening ischaemia, the end stage of atherosclerosis, as a promising biomarker introduction: chronic limb-threatening ischaemia (clti), the end stage of peripheral artery disease (pad), has poor prognosis and is attributed to lifestyle disease. with increasing of atherosclerotic disease all over the world, establishment of biomarker for should play a pivotal role for early detection and preventing aggravation of the disease. the aim of this study is to explore the possibility of liquid biopsy for atherosclerotic disease by analysis of clti-associated exosomal micrornas. methods: clti due to pad was diagnosed by anklebrachial blood pressure index, skin perfusion pressure (<40 mmhg) and angiography. ten preoperative clti patients and 10 control patients without pad were analysed (all patients with diabetes and 50% of patients had end-stage renal failure [esrd] ). to identify biomarkers associated with clti, exosomes were extracted from patient's serum after ultracentrifugation and total rna including small rna was isolated from the exosomes. the expression profile of exosomal micrornas associated with clti were evaluated using a next generation sequencing. results: forty-three exosomal mirnas associated with clti were identified. intriguingly, these mirnas were clearly categorized with esrd, which was well known as end-stage of life-style disease: these were stratified into 20 micrornas for esrd patients and 23 micrornas for non-esrd patients. since esrd is the most important factor significantly related to patient's prognosis in clti, exosomal micrornas reflected patient's comorbidity onto the expression profile. summary/conclusion: a portion of the expression profile of exosomal micrornas associated with clti was identified. exosomal microrna could be a biomarker to stratify patient's condition along with their comorbidities and is very promising for individualized diagnosis in atherosclerotic diseases with risk diversity. postoperative plasma exosomal mir-21 and mir-133a signature in patients with left ventricular reverse remodelling after surgical mitral valve repair underwent implantation of a prosthetic mitral ring. lv remodelling was assessed by cardiac magnetic resonance imaging and pexos were isolated by optimized ultracentrifugation before surgery (t0) and six months after surgery (t1). isolated pexos were quantified by nanoparticle tracking analysis and mir-1, mir-21, mir-133a, and mir-208a were measured by rt-qpcr. the same analysis was performed on healthy subjects with normal cardiac function (n = 8). local ethical committee approved the study (emigrate study, approval n°1529) and informed consent was obtained from all patients. results: pexos levels at t0 were lower (−32%, p = 0.02) in patients with worst postoperative lv function, while they were higher at t1 (+31%, p = 0.03) in patients with reversed lv remodelling after surgery. at t1, the increase in pexos levels was associated to decreased heart mass index (−13%, p = 0.02) and higher levels of exosomal mir-21 (+78%, p = 0.02) and mir-133a (+69%, p = 0.05) were detected in patients with improved lv function. summary/conclusion: higher postoperative levels of pexos delivering mir-21 and 133a depict lv reverse remodelling after surgical mitral valve repair. monitoring of exosomal micrornas cargo might predict postoperative outcome in patients with mr. expression of lipocalin-2 (lcn2) in circulating extracellular vesicles (evs) and femoral plaque-derived evs of peripheral arterial disease patients. introduction: clinically, the drug resistance situation of acinetobacter baumannii is becoming increasingly serious, and its drug resistance has become a difficult problem for nosocomial infection and clinical treatment. in view of the relatively slow development of antibacterial drugs, exploring the resistance mechanism of acinetobacter baumannii is of great significance to improve bacterial resistance and help clinical treatment. studies have shown that outer membrane vesicles (omvs) can transmit resistance genes to mediate the spread of drug resistance, and recent studies have confirmed that high expression of efflux pumps play an important role in the multidrug resistance of a. baumannii. in this study, we want to explore whether the outer membrane vesicles of acinetobacter baumannii can transfer the efflux pump related substances. methods: first, ultracentrifugation and density gradient centrifugation were used to extract the omvs of acinetobacter baumannii antimicrobial-sensitive strains (atcc19606) and antimicrobial-resistant strains. then, nanoparticle tracking analysis (nta) technology was used to analyse the particle size and distribution range of omvs. transmission electron microscopy (tem) was used to identify their morphology and structure. bradford method was used to determine the protein concentration of omvs. next, the omvs of antimicrobial-resistant strains were incubated with the antimicrobial-sensitive strains and then the drug susceptibility test was done to determine whether omvs of antimicrobial-resistant strains could transmit antimicrobial-resistance information to the antimicrobial-sensitive strains. finally, pcr, qpcr and mass spectrometry were used to determine whether the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. results: nanoparticle tracking analysis (nta) detected the concentration and size distribution of omvs of acinetobacter baumannii strains. it showed that the extracted omvs have a relatively uniform particle size and a size between 100-250 nm. tem showed that omvs had a typical vesicle structure. omvs coculture experiments showed that omvs of the antimicrobial-resistant strains can indeed pass resistance to the antimicrobial-sensitive strains. and the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. summary/conclusion: omvs of the antimicrobialresistant strains can indeed pass resistance to the antimicrobial-sensitive strains. the cause of acquiring antimicrobial resistance in sensitive strains may be caused by resistant strains passing efflux pump-related genes or proteins to sensitive strains. characterization of melanocytic extracellular vesicles during ageing of the choroid kelly coutant a , léo piquet a , nathan schoonjans b , philippe gros-louis a , julie bérubé c , stéphanie proulx a , alain r. brisson d and solange landreville a a université laval, quebec city, canada; b université de lille, lille, france; c centre de recherche du chu de québec-université laval, quebec city, canada; d université de bordeaux, bordeaux, france introduction: the choroid is located at the backside of the light-sensitive retina and is highly vascularized. it contains pigmented melanocytes, and their melanin protects them against oxidative stress. since ageing reduces the number of melanosomes in melanocytes and generates a stiffer extracellular environment, our hypothesis is that surrounding choroidal cells and the retinal pigment epithelium (rpe) are subject to more oxidative stress-related damages. this study aimed to characterize evs released by human choroidal melanocytes in the context of intercellular cooperation during ocular ageing. methods: melanocytic evs were recovered from the conditioned culture medium of young/old melanocytes grown on hydrogels of varying stiffness (0.5-20 kpa) by differential centrifugation. the concentration and size distribution of melanocytic evs were determined by high-sensitivity flow cytometry. cryo-transmission electron microscopy combined with receptor-specific gold labelling were used to reveal their morphology, size and phenotype. the relative abundance of 8 surface markers was evaluated with the exo-check exosome antibody array. the uptake of fluorescent melanocytic evs by the rpe and choroidal endothelial cells was assessed by confocal microscopy. results: choroidal melanocytes released evs positive for annexin-5 and the tetraspanin cd63. young melanocytes produced more annexin-5 positive evs and evs larger than 500 nm compared to older donors. the stromal stiffness impacted the concentration and size of melanocytic evs. we confirmed the uptake of melanocytic evs by endothelial and rpe cells. summary/conclusion: evs from choroidal melanocytes are internalized by surrounding endothelial cells and rpe. age-related stressors modify the phenotype of melanocytic evs. the identification of melanocytic factors that can protect retina/choroid cells from oxidative stress-induced cell death could lead to more efficient therapy for patients suffering from dry agerelated macular degeneration. introduction: owing to their proposed biocompatibility and ability to cross biological barriers, evs represent an attractive therapeutic delivery platform. however, evs are eminently heterogeneous. a better understanding of ev heterogeneity and its origins will allow for improved design of ev-based therapeutics. ev heterogeneity is mainly studied by focusing on distinct ev subpopulations. other sources of heterogeneity, such as heterogeneity within ev secreting cells themselves, have been investigated in lesser detail. in this study, we assessed the phenotypic drift of cell derived evs to explore the origins of ev heterogeneity and its potential impact. methods: three independent samples of two mda-mb-231 breast cancer cell sub-clones were cultured for six weeks. evs were harvested weekly and analysed using the macsplex exosome flow cytometry kit. at two time points the proteome of evs was analysed by lc-ms/ms mass spectrometry with subsequent gene ontology and reactome pathway analysis. results: the expression of over 600 proteins was deregulated in evs derived from the two different cell clones. many de-regulated proteins were associated with biological processes predicted to affect potential ev toxicity (platelet activation, neutrophil degranulation, blood coagulation) and ev biological activity (antigen presentation, inflammation, tgf-beta/ mtor/wnt signalling). more surprisingly, within only two weeks, over 400 ev proteins, many associated with immune modulation, apoptosis, interleukins, cytokines and cell signalling pathways (including those affecting t-cell/b-cell receptors) were de-regulated between the two ev isolation time points. summary/conclusion: results suggest that temporal changes can be observed in the ev proteome (potentially by clonal drift, epigenetic changes or cellular genomic instability) over short time periods. these changes could cause significant differences in biological effects and delivery capabilities between evs harvested from the same cells at different time points and conditions. in vivo tracking and biodistribution analysis of mesenchymal stem cellderived extracellular vesicles in a radiation injury murine model introduction: recent studies indicated that extracellular vesicles (evs) play key roles in intercellular communication and have great potential for clinical application. understanding the biodistribution of evs is therefore essential. our previous works have shown the ability of mesenchymal stem cell (msc)derived evs to protect haematopoietic cells from radiation damage. in this study, we evaluated the biodistribution of msc-evs in a radiated mouse model. methods: human msc-evs were harvested by ultracentrifugation and labelled with did lipid dye. the reliability of the labelling evs was confirmed by sucrose gradient fractionation analysis. the distribution of evs in radiation-exposed mice after ev intravenous administration were evaluated by fluorescence molecular tomography and further confirmed by flow cytometry and confocal microscopy analysis. results: we observed that did labelled msc-evs appeared highest in liver and spleen, lower in bone marrow in tibias, femurs, and spine, and were undetectable in heart, kidney and lung. we found the significantly increased msc-ev accumulation in spleen and bone marrow post-radiation appeared with an increase of uptake of msc-ev by cd11b+ and f4/80 + cells, but not b220+ cells, compared to those organs from non-irradiated mice. however, there was a predominant ev accumulation in lung and less accumulation in spleen and liver; in mice infused with human lung fibroblast cell derived evs (lfc-evs) and there was no significant lfc-evs accumulation change in the spleen or liver after radiation. we further found that increasing levels of irradiation caused a selective increase in vesicle homing to marrow and spleen. this accumulation of msc-evs at the site of injured bone marrow could be detected as early as 1 hour after msc-ev injection and was not significantly different between 2 and 24 hrs. post-msc-ev injection. summary/conclusion: this study indicated the specific accumulation of ms-evs at the site of injury of haematopoietic tissue in radiation injury mice. funding: this work was supported by the nih grants 5uh2tr000880, 3uh3tr000880-03 s1, 5p20gm11 9943, and 5t32hl116249. linking fat to colorectal cancer: extracellular vesicle crosstalk sheffield hallam university, sheffield, uk introduction: colorectal cancer is the third most common cancer worldwide, and fourth leading cause of malignancy related mortality. understanding the mechanisms of its growth and metastasis is key to elucidating new therapeutic targets and developing treatments in the clinical setting. epidemiological evidence indicates an increased risk of cancer in obese patients, pointing to bidirectional communication between colon and adipose cells. extracellular vesicles (evs) are small membrane enclosed packages released by cells, capable of transporting bioactive cargo from donor to recipient cells and inducing phenotypic changes. adipocytes are a key component of the tumour microenvironment and interactions between adipose tissue and tumour cells may be important in the growth and metastasis of cancer. in this study, we investigate the effects of colorectal cancer evs on adipocytes in vitro, and potential induction of dedifferentiation to a more fibroblastic, pro-inflammatory phenotype. methods: evs were isolated from sw480 and ht29 human colorectal cancer cell lines by differential ultracentrifugation and mature adipocytes generated by differentiation of the sgbs human pre-adipocyte cell line. adipocytes were treated with evs and their lipid content measured by oil red o to determine loss of lipids. inflammatory cytokine profile was measured by elisa to assess any increase in pro-inflammatory behaviour, and expression of late adipogenesis markers were determined by western blot. results: ev treatment was shown to reduce lipid accumulation in adipocytes, with up to 80% reduction in lipids observed at the 50 µg/ml dose. treatment was also shown to reduce the expression of late adipogenesis markers, and increase secreted levels of proinflammatory cytokines il-6 and il-8 by over 3 fold and 10 fold respectively. these results provide evidence for colorectal cancer derived ev involvement in the dedifferentiation observed in cancer associated adipocytes in vivo, displaying an altered phenotype, releasing lipid energy stores to fuel tumour growth and increasing pro-inflammatory signalling. summary/conclusion: studies have shown colorectal cancer evs may be involved in signalling which induces functional changes in cells within the tumour microenvironment. our work indicates that ev mediated dedifferentiation of resident adipocytes may potentially contribute to a microenvironment favouring cancer cell growth and metastasis. further work aims to elucidate the specific ev cargo which mediates these effects. introduction: ageing is a major risk factor for many human diseases. it is a complex process that progressively compromises most of the biological functions of the organisms, resulting in an increased susceptibility to disease and death. senescence is a cellular phenotype characterized by a stable cell cycle arrest. senescent cells are accumulated in the body during ageing. it contributes to develop age-related diseases and cancer. the alteration in intercellular communication with age has been demonstrated to be due to senescent cells developing a phenomenon denominated senescenceassociated secretory phenotype (sasp). exosomes are small extracellular vesicles (sev) (30-120 nm) of endocytic origin whereas microvesicles are formed by shedding of the plasma membrane. they contain nucleic acids, proteins and lipid that generally reflect the status of the parental cell and can influence the behaviour of neighbouring cells. methods: in this study, we demonstrated that the small extracellular vesicles (sev) contribute for transmitting paracrine senescence to proliferative cells firstly, we evaluated the presence of exosome-like particles in the sev from senescent cells by detection of exosome markers (alix, tsg101 and cd63), transmission electronic microscopy (tem) and nanoparticle tracking analysis (nta). to determine that sev from senescent cells are mediators of the paracrine senescence, we performed functional assays using cre-loxp reporter system and high-throughput results: besides, we confirmed at a single-cell level that the proliferative cells internalizing sev from senescent cells activate senescence process using the cre-reporter system. sev protein analysis from senescent cells by mass spectrometry (ms) and validation of top candidates using a functional sirna screen identify interferon induced transmembrane protein 3 (ifitm3), a component of non-canonical interferon (ifn) pathway, as partially responsible for transmitting senescence to proliferative cells. summary/conclusion: in conclusion, we found that sev are regulators of paracrine senescence and ifitm3 contained in senescent sev has an important role in the intercellular communication mediated through sev during cellular senescence1. bin wu a , lei guan a , ye xu a , likang chin a , ting li a , youhai chen a , gordon mills b , jinqi ren a , ravi radhakrishnan a , rebecca wells a and wei guo a a university of pennsylvania, philadelphia, usa; b oregon health & science university, portland, usa introduction: extracellular matrix (ecm) remodelling and stiffening are associated with solid tumour progression. stiff ecm promotes cell proliferation, epithelial-to-mesenchymal transition (emt), metastasis and chemoresistance. hepatocellular carcinoma (hcc) appears frequently in patients with liver cirrhosis or fibrosis while the mechanism remains unclear. exosomes have been determined to serve as messengers to mediate intercellular communication and influence the extracellular. tumour-derived exosomes have been shown to influence tumour progression, metastasis, drug resistance, angiogenesis and immune regulation. thus, determining whether exosomes provide a mechanism by which stiff matrix modulates tumour microenvironment for tumour progression opens a new way to understand cirrhosis and oncogenesis. here we identified the molecular mechanism of matrix stiffening induced exosome secretion and showed the different effect of exosomes induced by soft or stiff matrix on tumorigenesis. methods: huh7 cells were cultured on acrylamide gels with the stiffness was modulated to 500 pa (soft) or 10 k pa (stiff). the exosomes in conditioned media were collected and analysed by nanoparticle trafficking analysis (nta) and immunoblotting. protein expression level in cells was screened by reverse phase protein array (rppa). inhibitor or shrna were used to inhibit target proteins function. in vitro phosphorylation and gef assay were used to verify rabin8 phosphorylation and activation. exosomes from cells on soft or stiff matrix were injected into mice to study their effect on tumour growth. results: (1) stiff matrix promoted exosomes secretion. (2) akt was activated by stiff matrix and was required for exosome secretion. summary/conclusion: matrix stiffening promotes exosome secretion via akt-rabin8-rab8 pathway, contributing to tumorigenesis. tridimensional fibroblast culture revealed a novel exososome-dependent extracellular matrix secretion mechanism vincent clément a , bastien paré b , cassandra goulet a , thiéry de serres-bérard a , stéphane bolduc a , françois berthod a and françois gros-louis a a université laval, québec, canada; b norgen biotek corp., thorold, canada introduction: the extracellular matrix (ecm) is constituted of a variety of proteins and polysaccharides that are secreted locally and assembled into a thick 3d meshwork to provide biophysical and biochemical support to the surrounding cells, and regulate numerous cellular functions such as adhesion, migration and proliferation. dysregulation of ecm components or aberrant ecm remodelling can lead to various pathologies, as well as to play important roles in wound healing. although ecm secretion pathways are still largely unknown, the current paradigm is that ecmassociated proteins are synthesized in the endoplasmic reticulum and transported via the endosomes to the golgi apparatus en route to the cell surface and released by exocytosis. methods: to study ecm secretion pathway, we used 3dimensional (3d) cultured fibroblasts. this culture method technique has been used widely to generate tissue-engineered self-assembled stromal tissues, free of exogenous materials, and rely on long-term supplementation of sodium ascorbate into the culture medium. non-cancerous fibroblasts, grown in conventional two-dimensional (2d) cellular cultures, are known to be a poor source of secreted exosomes when compared to cancerous fibroblasts. results: here, we provide evidence that non-cancerous dermal fibroblasts can secrete high amounts of exosomes, containing different ecm proteins, when cultivated in a 3d fashion. we also demonstrated that dermal fibroblast-derived exosomes had the capacity to travel from one cell to another, induce cellular migration and promote wound healing. summary/conclusion: altogether, these findings reveal a novel exosome-dependent ecm deposition mechanism and suggest that the use of 3d-fibroblast cellular culture may emerge as an innovative approach in precision medicine to better study the role of patient-derived exosomes and ecm proteins in the establishment of cellular microenvironment in health and disease. anthony yan-tang. wu a , charles lai, yun-chieh sung b , steven t. chou c , vanessa guo c , jasper c. chien c , john j. ko c , alan l. yang c , ju-chen chuang c , hsi-chien huang b , syuan wu c , meng-ru ho d , maria ericsson e , wan-wan lin f , koji ueda g , yunching chen h , chantal hoi yin cheung i and hsueh-fen juan j introduction: bionanoparticles including extracellular vesicles and exomeres (collectively termed evs), have been shown to play significant roles in diseases and therapeutic applications. however, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a limited suitable method. methods: we developed a bioluminescence resonance energy transfer (bret)-based reporter, palmgret, to enable pan-bionanoparticle labelling ranging from exomeres (< 50 nm) to small (< 200 nm) and medium and large (> 200 nm) evs and larger evs (> 50 nm). results: palmgret emits robust, sustained signals and allows the visualization, tracking and quantification of bionanoparticles from whole-animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, bret, and fluorescence. using palmgret, we show that evs released by lung metastatic hepatocellular carcinoma (hcc) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. ev proteomics identified hcc-ev lung tropic protein candidates associated with cancer progression, in which slco2a1 and clic1 expression on non-tropic evs conferred lung-tropism, while cd13 gave spleen tropism. our results further demonstrate that redirected lung tropism decreases ev distribution to the liver, whereas the spleen tropism significantly reduces over time delivery to most major organs distribution including the liver and kidney. summary/conclusion: we established a multimodal and multi-resolution palmbret method to enable pan-bionanoparticle labelling and imaging and therefore quantification in live cells, whole animals, and preserved tissues. the method can resolve the intricate spatiotemporal dynamics of evs. palmgret revealed that evs derived from lung metastatic hcc are lung tropic, and the tropism can be conferred to non-lungtropic ev-293 t by decorating evs with identified hcc-ev membrane proteins. importantly, the enhanced ev delivery to tropic organs also significantly alters its distribution to other major organs. our findings suggest that the dynamics of ev biodistribution and targeted design should be investigated at the organ systems level in ev biology and therapeutic developments, respectively. tracking mesenchymal stem cell-derived extracellular vesicles (evs) in a in vivo cancer model introduction: small extracellular vesicles (sevs) are nanoparticles (30-120mn) encircled by a phospholipid bilayer, derived from the endocytic pathway and released by all cells. sevs have an inherent role in cell communication and deliver cargo to target cells. mesenchymal stem cells (mscs) and have a natural ability to home to tumours and metastases while avoiding the host immune response. it is hypothesised that msc derived sevs (msc-sevs) also possess tumourhoming and immune-evading capacities therefore could provide a novel targeted delivery vehicle for treatment of cancer. it is imperative to elucidate msc-sevs migratory itinerary in vivo to support translation to the clinical setting. methods: this study aimed to image the interaction of labelled msc-sevs with cancer cells in real time in vivo. sevs were isolated from wildtype mscs and mscs with stably expressing red fluorescent protein (rfp) (via lentivirus) by the combined techniques of differential centrifugation, microfiltration and ultracentrifugation. isolated sevs were extensively characterised by transmission electron microscopy (tem), nanoparticle tracking analysis and western blot. nod scid gamma (nsg) mice with dorsal skinfold window chamber (dsfwc) were injected with either mda-mb-231 luciferase (luc) expressing cells or ht-29-luc cells. bioluminescence imaging was performed to confirm tumour formation. a dose of 1x10^7 msc-rfp-sevs was directly added to the window chamber and rfp expression detected using a microscope with rfp filter attachments. 8x10^7 evs were incubated with the radionuclide, technetium-99m tagged duramycin (99 mtc-dur) for 30 minutes at room temperature. excess radiolabel was removed using exosome spin column (invitrogen™). the 99 mtc-dur-sevs were then added directly to the window chamber and charged particle imaging carried out. results: 18 hours post-administration; the rfp signal was localised at the tumour site. radiolabelled sev signal could be detected 10 minutes and 4 hours after administration. msc-sevs were successfully detected at the tumour site following direct administration using two different tagging and imaging approaches. summary/conclusion: this promising preliminary data supports the potential of this approach for tracking msc-sev migration in vivo. future studies will investigate systemic tracking of msc-sev migration. vaughn garcia 1 ; aejez sayeed 2 ; rachel derita 1 ; shiv ram krishn 3 ; peter a. introduction: tumor-derived small extracellular vesicles (sevs) have emerged recently as mediators of tumorigenesis. however, the role of sevs in response to irradiation, a widely used therapy in prostate cancer, is not fully understood. methods: our study involved the tramp mouse model of prostate cancer. we used plasma sevs isolated using differential ultra-centrifugation and further isolated using iodixanol gradient fractionation. we also used nanoparticle tracking analysis (nta) to analyze sevs. mouse pelvises were irradiated using 10 gy, for 5 consecutive days. results: we first observed that upon pelvic irradiation of tramp mice, the levels of the signaling oncogene c-src are reduced in plasma-derived sevs, while the average size of sevs is increased from 50-100nms to 70-250nms. furthermore, we show that the sevs from irradiated cells lose the ability to stimulate anchorage independent growth and migration of recipient cancer cells. additionally, sevs from irradiated mice increase the amount of dna damage in recipient cancer cells. summary/conclusion: overall, our data show that irradiation of tramp mice (and prostate cancer cells) significantly reduces the pro-metastatic and pro-anchorage-independent growth potential of sevs when tested on human cells. changes to the composition and behavior of a cancer cell sev population via radiation therapy offers promise for future therapeutic approaches for prostate cancer. introduction: there are emerging physiological and pathological functions of extracellular vesicles (evs) in neurodegenerative diseases including alzheimer's disease (ad). brain derived-evs contain pathogenic proteins, such as tau, amyloid beta (aβ), which have been reported to contribute to cell-to-cell propagation in those diseases. investigation of the brain-derived ev cargo, therefore, is important to further understand the mechanisms of progression in neurodegenerative diseases. we developed the ev separation method from unfixed frozen mouse and human brain tissues and assessed the protein composition. methods: to establish the ev separation method, we separated evs from frozen mouse brain tissue using sucrose density gradient ultracentrifugation (sg-uc) or size exclusion chromatography to compare the results from the particle number, morphology and protein profiling by nta, tem and mass spectrometry. evs were then separated from cortical grey matter of ad (n = 20) and control (n = 18) by sg-uc. tau and aβ in the evs were measured by immunoassay. differentially expressed ev proteins were observed by quantitative proteomics employing machine learning. results: the separated evs were enriched in ev molecules and devoid of contaminant proteins by sg-uc, showing our method was successful. the levels of ps396 tau and aβ1-42 were significantly increased in ad evs. annexin a5 (anxa5), neurosecretory protein vgf, neuronal membrane glycoprotein m6-a (gpm6a), and alpha-centractin (actz) were differentially expressed in ad evs. a combination of these 4 proteins were confirmed to predict ad with the 88% accuracy by machine learning. summary/conclusion: these data suggest our method were suitable for the separation of brain-derived evs and ev anxa5, vgf, gpm6a and actz can be potential biomarkers for monitoring the progression of ad. edta stabilizes the concentrations of extracellular vesicles during blood collection introduction: to establish reliable biorepositories for research on extracellular vesicles (evs) as disease biomarkers, the release of evs during blood collection and handling must be avoided. currently, citrate is recommended as the anticoagulant for blood ev research, but citrate does not inhibit the release of evs from activated platelets. the release of platelet-derived evs excludes pneumatic tube transport and makes assays time dependent, thereby limiting clinical compatibility. therefore, we aim to stabilize the release of platelet ev concentrations. methods: blood samples were collected from healthy individuals and subjected to common circumstances known to induce platelet activation. blood was (i) incubated with or without thrombin receptor-activating peptide 6 (trap; n = 7), a potent platelet activator, (ii) send to the lab by a routine blood transport (pneumatic tube system; n = 3), and (iii) stored at room temperature or at 4°c for 6 hours (n = 3). the concentrations of evs from platelets (cd61+), activated platelets (p-selectin+), erythrocytes (cd235a+), and leukocytes (cd45+) were determined by flow cytometry (apogee a60-micro). results: following activation by trap, concentrations of platelet-derived and activated platelet-derived evs increased 5.8-fold and 10.4-fold in citrate-anticoagulated blood, compared to 1.4-fold and 2.0-fold in edta-anticoagulated blood (edta vs citrate: p = 0.018 and p = 0.043, respectively). preliminary data show that during pneumatic tube transport and routine sample handling, both platelet-and activated platelet-derived evs were more stable in edta compared to citrate. the concentrations of evs from erythrocytes and leukocytes were unaffected under all studied conditions. summary/conclusion: to conclude, edta stabilizes platelet ev concentrations during and after blood collection, which would facilitate pneumatic tube transport, enhance reliability and thereby improves the establishment of reliable biorepositories for ev research. introduction: cancer-cell secreted extracellular vesicles, called exosomes, are an emerging biomarker for cancer liquid biopsy. profiling of cancer-associated exosomes usually required lengthy, and multi-step procedures; therefore simple and easy-setup sensing methods are urgently needed for diagnosing cancer in a timely manner. chirality, the foundational property of all biomolecules, including exosomal proteins, can be utilized for exosome detection and differentiation using recent advances in chiral nanostructures. we found that microfluidic sensors can be successfully implemented for successful detection of cancer-associated exosomes taking advantage of unusually high circular dichroism (cd) of chiral gold nanoparticles (aunps). circular dichroism-based exosome (cdexo) detection utilizes chiroplasmonic enhancement of cd signatures of cancer-associated exosomes. we first synthesized donut-shaped aunps conjugated with l-cysteine and immobilized the aunps on a glass slide using a layer-by-layer assembly. the aunps on slide glass were surface functionalized by the standard biotin-avidin reaction after mua treatment. biotinylated annexin v marker, targeting phosphatidylserine (ps) expression on cancer-associated exosomes, was conjugated to the aunp surface. 5 μl of exosome samples from cancer cells (a549 and h3255) or normal cells (mrc5) were injected into the pdms microfluidic device and incubated for 30 minutes. the cd signal before and after exosome exposure was monitored, compared, and systematically analysed as a rapid technique for the detection of exosomes with high sensitivity. results: we showed that the cdexo signals from cancer exosomes showed 26.6 folds absolute cd peak value change and 3.41 folds shift, respectively, compared to that of healthy exosomes. importantly, the cdexo sensing method takes less than 10 mins in terms of total scanning time and requires minimal sample volumes. from the preclinical studies using 7 blood samples from cancer patients and healthy donors, we found that cancer patients show stronger band shift and signal change comparing to that of healthy donors, implying our platform could be used for cancer diagnosis. summary/conclusion: this new versatile and sensitive method based on chiroplasmonic exosome detection paves the way to profiling disease-associated exosomes in a timely manner for minimal volumes of liquid biopsies. ev classification and fractionation strategy using surface charge labelling takanori ichiki a , hiroaki takehara a , hirofumi shiono b and hiromi kuramochi a a the university of tokyo, bunkyo, japan; b innovation center of nanomedicine, bunkyo, japan introduction: the development of new classification technology is required based on the evaluation of physicochemical properties of exosome surfaces and the diversity of constituent molecules. in this presentation, we present the electric charge activated exosome sorting platform comprising microfluidic device technology and electric charge labelling technique. methods: the single nanoparticle analysis platform, which has been developed by our research group, images rayleigh scattered light (elastically scattered light) obtained by irradiating nanoparticles with convergent laser light and provides information of individual particles by image processing. the method that utilizes electrokinetic phenomena, unlike the method using fluorescent labels, measures the properties of the particle surface without serious difficulty in principle even if the particle size is on the order of tens nanometres, and further enables to perform fractionation. since the number of particles usually handled in exosome research or its envisioned application is enormous, it is not realistic to take an approach such as a cell sorter in which particles are sequentially manipulated one by one following the measurement results of individual particles. results: particles receive attraction or repulsion by an external field according to the charge density on the surface, so there is no need to control the external force, and it is possible to design a device that can autonomously fractionate particles according to the difference in zeta potential. summary/conclusion: in conclusion, we have proposed and demonstrated the new concept of electric charge activated ev sorter. funding: this research was partially supported by the center of innovation program (coi stream) from the japan science and technology agency. high throughput exosome analysis by using reversible microfluidic electrochemical sensor system introduction: exosome is one of the important extracellular vesicles (evs) released from parental cells and it contains various types of molecular cargos from its original cell including proteins, messenger rna (mrna), and micro rna (mirnas) [1] . the exosomes have recently emerged as biomarkers for early stage cancer detection because the number of exosomes originated from cancerous cells are significantly higher than those from normal cells [2] . since many different types of exosomes exist in the whole blood, it is necessary to isolate and detect disease-specific exosomes. for this reason, the isolation and the detection of exosomes is an important research issue and has been studied by many groups. however, limitations such as low throughput and low recovery still make it difficult to use exosomes in diagnostics and therapeutics. methods: in this study, we developed an integrated microfluidic electrochemical biosensor to extract plasma from whole blood and subsequently detect cancer related exosomes in a continuous manner. this consists of two parts. the first part is a channel for extracting plasma containing exosomes from whole blood, and the second part is a channel combined with an electrochemical sensor for multiple detection of various exosomes in the extracted plasma. previously, a multi-orifice flow fractionation (moff) channel that consists of a series of expansion and contraction structures has been developed in our group. in this channel, the blood cells are moved to sides of channels by hydrodynamic forces and then are eliminated to outlets. at this time, the plasma is moved to the electrochemical sensor part, the exosomes in the plasma are captured to the electrodes immobilized with the specific antibodies and are quantified the amount of cancer-related exosomes. results: using this chip, blood cells were eliminated from the whole blood with over 90% of separation efficiency at 225 µl/min flow rate and exosomes were collected continuously with high recovery (~70%). in order to quantify various types of exosomes, a labelfree electrochemical biosensor with electrochemical impedance spectroscopy (eis) was used for the continuous detection of exosomes. the limit of detection was 1x10^6 exosomes/ml. summary/conclusion: the developed device is an integrated device capable of separating exosomes from whole blood with high purity and quantitating exosomes through the electrochemical sensor in a continuous manner. , 1800528, 2019) . the development of highthroughput techniques capable of simultaneously monitoring physical and biochemical properties of evs would significantly simplify and accelerate the characterization process. in this context, microfluidic technology is emerging as an attractive platform. here, we present a microfluidic device based on the combination of diffusion sizing and multi-wavelength fluorescence detection to simultaneously provide information on ev size, concentration and composition. methods: the diffusion of evs in the microfluidic channel provides information on their size distribution, and four different staining protocols with high signalto-noise ratios track different ev native molecules. evs are separated from unbound fluorophores directly during the microfluidic analysis, therefore avoiding the need for sample pretreatments and allowing to operate the device as a single-step immunoassay. results: the microfluidic device coupled with complementary staining techniques allows to individually detect and size particle populations with different ev components such as lipids, primary amines and the ev marker cd63. we demonstrate that this approach can probe the abundance of ev-specific markers and impurities such as lipoproteins with high throughput and low sample consumption. summary/conclusion: we present a microfluidic technique capable of characterizing and quantifying evs at low costs, in a time-scale of minutes and requiring only up to 2 µl of non-pretreated sample. this method is an important complementary tool to the current array of biophysical methods for ev characterization, in particular for high-throughput screening applications. funding: h2020-eu.1.2.1-fet open programme via the grant agreement 801338. immunomagnetic isolation of specific subpopulations of exosomes for liquid biopsy via nano-architected porous materials introduction: exosomes offer the potential to reveal significant biological information in many areas of clinical importance by virtue of their rna contents and protein surface markers. this abstract reports the fabrication of a device for high throughput targeted immunomagnetic capture of exosomes via the use of highly-ordered nano-architected porous metal lattice materials. methods: we have invented a fabrication technique to precisely make millions of nanoscale exosome sorting devices that can operate on unprocessed plasma. each nanoscale device can precisely sort targeted exosomes from background vesicles but is too slow for practical use individually. however, the operation of millions of these devices in parallel preserves the precision of nanoscale sorting while also enabling high throughput and robust use on raw plasma samples. the metal lattice within which these devices are contained is assembled via metal electroplating onto a selfassembled polystyrene bead lattice with face-centred cubic (fcc) symmetry with 600 nanometre pores. the devices feature a conformally-coated layer of nickel-iron with gold passivation atop a base layer of nickel, resulting in a lattice of millions of nanoscale pores capable of magnetic sorting of exosomes tagged via surface-marker-based immunomagnetic labelling with magnetic nanoparticles. results: compared to our previous work on immunomagnetic exosome capture via commercial track-etched membranes (tempo), this device offers superior capture due to increased surface pore density (>25x) and three-dimensional pore density (>1000x) alongside lower required sample volume due to decreased noncapturing volume in the device. finite-element analysis simulations show that strong magnetophoretic traps emerge at the pore boundaries in this structure between higher-permeability metals such as nickeliron permalloy and the lower-permeability sample fluid in the device. preliminary experimental data shows that this device can isolate iron nanoparticles in solution with >100x enrichment from input and 49x capture efficacy versus tempo. summary/conclusion: current methods of exosome isolation such as ultracentrifugation and column chromatography all suffer from low throughput and limited yield. the application of inverse opal materials towards exosome capture offers the potential for isolation of specific exosome populations from very low clinical sample volumes or sparse biological signals. micropatterned growth surface topography affects extracellular vesicle production colin l. hisey a , james hearn b , yohanes nursalim a , vanessa chang a , cherie blenkiron a and lawrence w. chamley a a the university of auckland, auckland, new zealand; b university of auckland, grafton, new zealand introduction: extracellular vesicles are micro and nanoscale packages released by all cells and play an important role in cell-to-cell communication by shuttling biomolecules to nearby and distant cells. however, producing enough evs for many in vitro studies using conventional tissue culture techniques can be challenging, and despite the success of some bioreactors in increasing ev-production, it is still unknown how many independent culture conditions like growth surface topography can alter the production and content of evs. methods: standard 150 mm petri dishes were patterned with 2 µm tall polystyrene microtracks spaced by 5, 10 and 20 µm across a 100 mm area using standard microfabrication techniques including photolithography, soft lithography and microtransfer printing. the micropatterns were characterized with sem and profilometry, then activated with oxygen plasma and uv sterilized. mdamb231 cells were seeded onto patterned and smooth (control) dishes and grown in serum-free media for the final 48 hours of culture. evs were isolated using sequential ultracentrifugation of conditioned media and characterized using nta, tem and western blot. cell morphology was imaged using immunocytochemistry and single cell migration was characterized using time-lapse microscopy and manual single cell tracking in fiji. results: we demonstrate the simple and repeatable fabrication of microtracks across a large surface area in order to culture cells on topographically patterned growth surfaces. furthermore, we show that the 5 µm spacing produced significantly more evs than other patterns as well as the highest cell aspect ratio and average single cell migration speed (p < .05). summary/conclusion: these findings have implications in both biomanufacturing of evs and potentially in enhancing the biomimicry of evs produced in vitro. however, further experimentation to assess the differences in cargo on patterned growth surface topographies compared to conventional methods is still required. funding: this project was funded by the maurice and phyllis paykel trust. using miscroscale thermophoresis and surface plasmon resonance to measure the interactions of extracellular vesicles mst is a quick method, easy to handle, has a low sample consumption, has no limitation on molecule size, and enables measurements in solution, either in various buffers or complex biological liquids. these properties make mst an interesting tool for research of extracellular vesicles (evs); therefore, our aim is to apply this method to evs. methods: evs were isolated from jurkat cell line by differential centrifugation. microscale thermophoresis (mst) and surface plasmon resonance (spr) were used to analyse the interaction between antibody and evs. results: we have demonstrated that interactions of evs with antibodies could be analysed by mst. however, the tiny glass capillaries for sample mounting represent a challenge due to adhesion of evs to their surface. we have tested commercial capillaries as well as prepared capillaries in house coated by liposomes or bovine serum albumin. the interactions between evs and antibodies were confirmed by surface plasmon resonance (spr), which is an established method for studying the interactions of evs. introduction: the isolation of extracellular vesicles (evs) from cell culture supernatants and complex body fluids, such as blood and urine, is of high importance for ev research as well as for future medical applications in diagnostics and therapy. nevertheless, it is still challenging to reach the desired recovery, purity and specificity due to many manual and time intensive sample preparation steps. conventional centrifugation for ev isolation or sample preparation prior to affinity-based separation methods can damage evs and cells, leading to misinterpretation of results or inactive evs. alternative field flow fractionation methods employing acoustic fields are highly promising, but so far limited to laboratory usage, based on a complex (moulding) fabrication and/or hardly reproducible. here, we present an innovative surface acoustic wave (saw)-based acoustofluidic device for gentle sorting of cells and particles. methods: our device consists of interdigital transducers patterned on a piezoelectric substrate generating saw propagating on the substrate surface. upon interaction of saw with our on-chip structured, fluid-loaden microchannels, an acoustic pressure field is developed across the fluid wherein particles are suspended. this pressure field can be employed to simply manipulate cells and particles based on their intrinsic properties, such as size, density and compressibility in continuous flow. the device is manufactured using precise and low-cost microtechnological methods and is suitable for reproducible mass fabrication. results: we demonstrated the separation of blood components, i.e. the sorting of erythrocytes and thrombocytes. furthermore, we could also show results on thrombocyte activation indicating a gentle separation without damaging these shear-sensitive cells, as well as first results on plasma separation from whole blood samples and nanoparticle sorting. summary/conclusion: our unique acoustofluidic sorting technology for complex suspensions has the potential to overcome the need for time-effective, cheap and gentle separation of evs. funding: this work was supported by efre infrapro project "champ: chip-based acoustofluidic medtech platform". nanophotonic platform for cancer-associated exosomal microrna detection introduction: exosomes have an important role in intercellular communication at physiological and pathological processes. their cargo includes micrornas (mirs), single-stranded non-coding rnas, involved in alterations on recipient cells, such as development of tumourous phenotype and metastasis. more particularly, mir-21 excels due to its association with several cancers. determining exosomal mirs as cancer indicators demands selective and accurate methods, which are not currently available or entail high costs. colorimetric photonic-based assays are a promising label-free alternative, which dismisses complex apparatus for signal reading since biorecognition is detected by colour change. moreover, the clinical and economic systems have also been demanding a decrease on the green footprint of biosensors, requirement fulfilled with naturally derived biomaterials. methods: herein, the biosensor is constructed on a biopolymer matrix to meet the requirements of an eco-friendly disposable device, and it is based on a photonic structure obtained by imprinting a nanopattern on the polymer surface. then, the surface is functionalized with the complementary oligonucleotide sequence of mir-21 as sensing probe. a labelfree detection is thus envisioned and the sensor performance is evaluated by changes in the optical properties when the target is present. results: the combination of biological materials conducted to a biosensor support with great flexibility and low water permeability, allowing easy surface functionalization. the self-reporting ability of the photonicbased sensor enables high intensity colours detected by naked eye. summary/conclusion: the alliance with the high selectivity of oligonucleotide hybridization is expected to offer great exosomal mir-21 recognition ability and an optimistic perspective for utilization in clinical setups. funding: the authors acknowledge the financial support from the european commission/h2020, through mindgap/fet-open/ga829040 project. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = 31). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and 10 ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine 37 distinct uev surface markers of cd9+/cd63+/cd81+ vesicles in a multiplexed bead-based manner including respective controls. the accuri c6 (bd) was utilized for data acquisition. for further misev2018-compliant characterization, cd9+/cd63+/cd81+ uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd209, all other 36 surface markers could be identified. the most abundant markers were cd9 and cd63, which were detected in 93% of samples, followed by cd133/1 (84%), cd326 (81%), cd81 and cd24 (77%, each). cd3 (42%), cd9, cd45 (39%), cd49e (32%) and cd81 showed similar relative median fluorescent intensities (rmfi), while cd63 yielded significantly higher (p = 0.009) and all other markers significantly lower rmfi (p < 0.011). tem and f-nta revealed cup-shaped vesicles (137 ± 22 nm) with 8.8 ± 7.0 e + 10 particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg101, cd9, cd63 and cd81 without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a , esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = 40) aged 40-70 yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a 3colour panel, which included annexin v (for the majority of circulating evs), cd41 (for plateletderived evs) and cd105 (for endothelialderived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and 10-yr cvd risk detected by qrisk2. results: ev numbers, as determined by nta, were strongly associated with bmi (r = 0.602, p < 0.001), blood pressure (systolic bp: r = 0.359, p = 0.023; diastolic bp: r = 0.550, p < 0.001) and plasma triacylglycerol levels (r = 0.703, p < 0.001). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = 0.330, p = 0.038). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining 49.4% of the variance for total ev numbers. an additional 9.3% of the variance in total ev numbers was predicted by diastolic bp. ancova of the 10-yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with 10-yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd4 + t cells were isolated from secondary lymphoid organs of foxp3-rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th1, th2, and th17), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, 15-day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for 5 days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th1, th2, th17) showed either no change in proliferation (th1) or decrease in proliferation (th2, th17), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < 0.0001). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th1), il4 and il13 (th2), or il17a and il17 f (th17). in contrast, exposure of tregs to cdc-evs resulted in~1000-fold increase in expression of il10, a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il-10 in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il-10. this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t32hl116273 prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from 42 patients treated with prostanoids (study group; n = 42, 50 ± 16 years, 70% female) and 38 patients not treated with prostanoids (control group; n = 38, 55 ± 19 years, 65% female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; 0.5 mm), adenosine diphosphate (adp; 6.5 µm) and thrombin receptor-activating peptide (trap; 32 µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd61+ and cd62p+; pevs), leukocytes (cd45+, levs) and endothelial cells (cd146+, eevs) were measured in plateletdepleted plasma by flow cytometry (a60-micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = 0.01) and lower concentrations of pevs and levs (p ≤ 0.05). furthermore, thrombus formation was delayed (p ≤ 0.003) and thrombus size was decreased (p = 0.008) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd61+ pevs (p = 0.04). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ 0.05) and the concentrations of pevs (p ≤ 0.08). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ 0.04). summary/conclusion: patients with pah treated with prostanoids have increased risk of bleeding both due to impaired platelet aggregation, ev release and thrombus formation, compared to patients not treated with prostanoids. antiplatelet effect of prostanoids varies: whereas epoprostenol decreases the release of pevs, treprostinil and iloprost impair platelet aggregation. funding: ag is supported by the national science centre, research project preludium 2018/31/n/ nz7/02260. evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. nanoflow cytometry identifies an imbalance of epithelium-and neutrophil-derived extracellular vesicles in the airway environment of paediatric cystic fibrosis patients brian dobosh, vincent giacalone, milton brown, lucas silva, lokesh guglani and rabindra tirouvanziam emory university, atlanta, usa introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from 17 cf children (<6 years of age). for nanoflow cytometry, evs were stained with di-8-anepps, and with epcam, cd66b and cd115 (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = 0.003, spearman's rho = 0.689). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = 0.001, rho = 0.857). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv15a0), emory paediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: 142 individuals were enrolled into our study, subdivided into a training (volunteer n = 27, pneumonia n = 12, sepsis n = 28) and testing cohort (volunteer n = 20, pneumonia n = 18, sepsis n = 37). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq2) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative real-time pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed 29 significantly regulated mirnas in pneumonia compared to volunteers, and 25 mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by 12 mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: 0.982, pneumonia: 0.965, sepsis: 0.992). mir-193a-5p (log2fc = 1.86, padj = 1.49e-6) and mir-542-3p (log2fc = 1.67, padj = 3.29e-5) differentiated between pneumonia and volunteers and mir-1246 (log2fc = −2.41, padj = 1.78e-04) between pneumonia and sepsis. expression levels of mir-193a-5p and mir-1246 were related to disease severity. mir-542-3p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was 73.33%. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study,3healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd63 and cd81 were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and 9 mirnas (mir-103a-p, mir-191-5p, mir-320a, mir-200b-3p, mir-185-5p, mir-223-3p, mir-21-5p, mir-155-5p, let-7 g-5p), were evaluated by rt-qpcr. after the optimization of the methodology, 10 healthy adults subjects and 10 patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd63 and cd81. however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna-320a showed a significant increased (p = 0.02) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir-320a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf2-active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid 2-related factor 2 (nrf2) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf2-active exosomes, mscs were incubated with potent nrf2 activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express >95% positive msc markers (cd44, cd73, cd90, and cd105) and <5% express negative markers (cd45, cd31, cd14, cd19, or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd81, cd9 and tsg101. nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of 168.0 ± 6.5 nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf2-active human mscs increase exosome secretion by 54%, compared to nrf2-baseline mscs (p < 0.05). both nrf2-baseline and nrf2-active exosome treatment significantly reduced closure time to 15.5 and 14 days respectively, compared to 29.8 days for vehicle-treated wounds (p < 0.05). this reduction eliminated the delay in closure time compared to wounds of c57/b6 mice. nrf2-active exosome treatment of db/db wounds reduced closure time by a further 2.6 days compared to untreated c57/b6 wounds. at day 10, exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd31+ vessels compared to vehicle-treated wounds. summary/conclusion: enhancing nrf2 function in mscs multiplies exosome yield. our results demonstrate exosome-based therapies hold tremendous promise and warrant further investigation for rapid translation. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist1 is a key mediator of tumour cell metastasis. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist1. methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc3-ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc3-ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc3-ml lines stably overexpressing or deficient in twist1. results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc3-ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc3-ml overexpressing twist1 showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist1 expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. introduction: exercise is associated with various health benefits, including the prevention and management of obesity and cardiometabolic risk factors. however, a strong heterogeneity in the adaptive response to exercise training exists. differential response to exercise training might be mediated by myokines (proteins, nucleic acids, metabolites) that can be released directly into the systemic circulation, or packaged within extracellular vesicles (evs). the objective of this study was to evaluate if changes in evs after acute aerobic exercise (ae) were associated with the responders phenotype following 6-week resistance exercise (re) training. methods: this is a secondary analysis of plasma samples from the exit trial (clinical trial #02204670). eleven sedentary obese youth (15.7 ± 0.5 years, bmi ≥ 95th percentile underwent an acute bout of ae (60% heart rate reserve, 45 min). blood was collected before [time (at) −15, 0 min], during [at15, 30, 45 min], and after [15 min (at60), 75 min (at120)] exercise. afterwards, youth participated in 6-week re programme, and were categorized into responders or non-responders (nr) based on changes in insulin sensitivity (above or below 50 percentile). primary outcome: evs were isolated using size exclusion chromatography (izon®) at baseline (at0), immediately after ae (at45) and after recovery (at120). ev protein concentration, size, and zeta potential were analysed in a single-blind fashion. results: responders had larger evs (~141.1 nm) as opposed to nrs (~97.3 nm) at at0 (p < 0.05) and this pattern was maintained at at45 and at120, though not significant (p = 0.1). nrs displayed differential ev size distribution (peaks at 100 nm or 300 nm), while ev distribution was highest at 250 nm in responders. no difference in average zeta potential or total ev protein yield was observed between groups. an increase in ev yield with exercise time and recovery was observed in both groups. summary/conclusion: our preliminary data suggest that ev size is significantly increased after an acute bout of ae in obese youth responders. further research to delineate the role of evs as predictors of exercise adaptation is warranted. funding: funded by dream and research manitoba. using dual-fluorescent reporter mice to track tissue-specific extracellular vesicles andrea estrada, gabriella hehn, zackary valenti, christopher allen, nicole kruh-garcia and dan s. lark colorado state university, fort collins, usa introduction: extracellular vesicles (evs) from tissues like skeletal muscle (skm) and adipose tissue (at) have been implicated in human disease but are understudied. skm is likely a major player in ev biology as it accounts for~35% of total body mass. tools to define cellular ev origin are needed because tissues like skm are comprised of a variety of cell types. here, we describe our ongoing efforts using the dual fluorescent mg/mt mouse as a tool to analyse skm-myocyte derived evs. methods: wild-type (wt) and mg/mt mice were used for these studies. mg/mt mouse cells express membrane-tagged red (mt) or green (mg) fluorescent protein in the absence or presence of cre, respectively. we made skm myocyte mg expressing mice using a mouse expressing cre on the human skeletal actin promoter. blood was collected via cardiac puncture and platelet-free plasma was obtained via centrifugation. plasma evs were isolated using exoquick, exoquick-tc or size exclusion chromatography. skm and at were dissected into~5mm chunks, placed in serum-free dmem and incubated for 24 hours. tissuederived evs were isolated using exoquick-tc. ev abundance was determined with a horiba viewsizer. individual evs were analysed with a cytek aurora spectral flow cytometer. settings were optimized using polystyrene beads and spectral unmixing was performed to allow detection of mg and mt. results: in wt mice, skm releases >100 times more evs than adipose tissue per unit of mass (p < 0.01 using paired student's t-test). since skm is also a major component of total body mass, these data further emphasize the importance of skm-derived evs. skmderived evs from wt mice were not fluorescent (<0.1% of events). evs from mg/mt mouse skm overwhelmingly expressed mg (>95% of events) with negligible (< 1%) expression of mt. at-derived evs robustly expressed mt but lacked mg. summary/conclusion: these data provide "proof-ofprinciple" that mg and mt are readily incorporated into evs secreted ex vivo. surprisingly however, plasma evs from mg/mt mice expressed very little mg (~3%) or mt (~4%). this observation was confirmed with three separate isolation techniques. we are currently exploring possibilities to explain this finding, including: 1) modification of evs post-secretion, 2) clearance of fluorescent evs by the liver or 3) that evs secreted from tissues remain predominantly in the interstitial space. funding: this work was supported by an innovative project award from the american heart association (18ipa34110052) to dsl. endothelial cd36 delivery of fa loaded extracellular vesicles is critical for thermogenesis. introduction: membrane cd36 facilitates tissue fatty acid (fa) uptake. we recently found that endothelial cell (ec) cd36 controls muscle and adipose tissue fa uptake, and influences the tissue's metabolic phenotype. the mechanism for cd36-facilitated fa uptake is unknown. here we examined the role of ec cd36 in thermogenesis and in fa delivery to brown fat tissue. methods: adult male mice were housed individually, restricted from food during acute (4 hr) cold exposure (4°c) with core temperature monitored every 30 minutes. after 4 hours, animals were sacrificed and samples collected for analysis. for cellular studies, human microvascular (lonza) or primary murine microvascular ec were used. for primary cells, crude cell pellets from lung homogenates were purified using mouse-cd31 magnetic beads (miltenyi). for microscopy studies, alkyne fa (cayman) was added to cells and to enable visualization of internalized fas, click chemistry (invitrogen) used to label alkyne-fa with alexa 568. for radioactive studies, primary lung ec were serum starved for 5 hrs and incubated overnight with 3h-oleic acid bound to fa-free bsa (2:1 ratio). media was collected, clarified by centrifugation to remove microvesicles and debris. small extracellular vesicles (sevs) were isolated from clarified media using total exosome isolation reagent (invitrogen) and counted for radioactivity. results: basal core body temperatures are similar in mice lacking ec cd36 (eccd36-/-) compared to controls (cd36fl/fl). however, during cold exposure at 4°c , eccd36-/-are unable to maintain body temperature (p < 0.001). plasma free fa are higher in cold exposed eccd36-/-indicating fa clearance by brown fat is impaired. mitochondrial function and expression of thermogenic and mitochondrial genes in brown fat from eccd36-/-and cd36fl/fl mice were similar. these data suggested that endothelial delivery of fas is necessary for thermogenic maintenance of body temperature. to examine fa handling by ecs we used alkyne fas to visualize the process. we found that fas are transferred by microvascular ec through caveolae-mediated transcytosis involving src signalling and cav-1 phosphorylation. the internalized cav-1 and cd36 positive vesicles containing fas are released as sevs. to determine the dependence of cd36 on this process, we treated primary microvascular ec with radiolabeled fa and found that sevs secreted by cd36-/-cells contain less labelled-fa (p = 0.05). summary/conclusion: endothelial delivery of fa is critical for thermogenesis. our working model for the mechanism of fa uptake by brown adipose tissue is the following: endothelial cells transfer the fa through caveolae-mediated transcytosis and secrete small extracellular vesicles (sevs) that help deliver fas to brown adipocytes. funding: this work is supported by nih grants dk111175 and dk056341. introduction: diet-induced obesity modifies intestinal permeability leading to bacteria infiltration and to a decrease in the number of immune cells protecting mucosa. as orange consumption is beneficial for human health and used in preventive medicine, we determined whether orange juice-derived nanovesicles (onv) might be recommended as nutritional strategies for the treatment of intestinal complications associated with obesity. methods: onv isolated from fresh orange juices were characterized by lipidomic, metabolomic, microscopy, nta and for their stability during digestion. intestinal barrier (ib = caco-2 cells+ht-29 cells differentiated with oleic acid) were treated with onv and co-cultured with adipocytes to monitor ib fat absorption and release. obesity was induced in mice fed for 12 weeks with a high-fat high-sucrose diet (hfhs mice vs standart chow diet mice). then half of the hfhs mice were gavaged with 150 micrograms/day for 4 weeks. results: onv did not modify high-fat high-sucrose diet-induced obesity and insulin resistance but reversed diet-induced gut modifications. six hours post-gavage, onv accumulated preferentially in jejunum involved in lipid absorption. in jejunum, and no other intestinal region, onv increased villi size, restored immune response and decreased barrier permeability in hfhsd mice. in addition, onv-treated mice had increased expressions of acat2, angptl4 and dgat1, but a decreased expression of fabp2, fatp4, mtp vs hfhsd animals, which indicated that fat absorption, tg synthesis and chylomicron release were strongly reduced. similarly to other plant-derived nanovesicles, these results were likely associated with onv lipid and metabolite compositions (strong enrichment in bioactive phospholipids: pe, pa, pc, pi and leucine) as onv did not resist to harsh digestive conditions in vitro and were poorly incorporated in enterocytes. as the effects of onv on the decrease in tg content and epithelial cell growth were also observed in vitro, gut microbiota unlikely participate to these effects. summary/conclusion: onv are important bioactive compounds of orange juice and for the first time we demonstrated that they can modulate lipid metabolism in the intestinal barrier associated with morphological changes. interestingly onv treatment targets mtp and angptl4 mrnas, 2 therapeutic intestinal targets to reduce plasma lipids and for attenuating inflammation in gastrointestinal diseases. therefore, onv might be used to reduce the development of dyslipidemia-associated diseases and to restore intestinal functions in obese patients. funding: olga triballat institut; benjamin delessert institut, inrae institut. association, structure, and function of fibronectin in extracellular vesicles from hepatocytes xinlei li, ruju chen, sherri kemper and david brigstock nationwide children's hospital, columbus, usa introduction: we have shown that extracellular vesicles from normal hepatocytes have anti-fibrogenic activity and that they preferentially bind to hepatic stellate cells (hscs, the principal fibrosis-causing cell in the liver) and hepatocytes. in this study, our goal was to determine the molecular nature of the ev components involved in cell binding. fibronectin (fn1) is a key component of extracellular matrix, functioning in processes including cell adhesion, differentiation, and wound healing. two types of fn1 are present in vertebrates, of which the soluble plasma fn1 is derived principally from hepatocytes, while cell-associated fn1 is produced by numerous cell types. here we describe a novel function of plasma fn1 in facilitating binding of hepatocyte evs to target cells. methods: differential ultracentrifugation was used to collect evs released by parental mouse aml12 hepatocytes, fn1 ko aml12 cells in which fn1 was ablated using crispr-cas9, primary human or mouse hepatocytes, or human hepg2 cells, or from human or mouse serum. evs were characterized by nanosight tracking analysis (nta), western blot, iodixanol gradient ultracentrifugation, and mass spectrometry. the binding efficiency of pkh26-labelled evs from parental (ev-hep) or fn1 ko (ev-hepfn1 ko) aml12 cells was analysed in hepatocytes or hscs. swiss webster mice were injected with ccl4 for five weeks to induce liver fibrosis, with some mice also receiving i. p. administration of ev-hep or ev-hepfn1 ko over the last two weeks, followed by determination of hepatic fibrogenic genes by qrt-pcr. results: ev-hep or ev-hepfn1 ko were 50-200 nm in diameter and positive for common ev markers (cd63, cd9, flotillin-1). mass spectrometry showed that fn1 was the most abundant protein in ev-hep and comprised principally the plasma form. the abundant presence of ev fn1 was verified by western blot and co-immunoprecipitation with anti-cd9 or antiflotillin-1. western blot showed that fn1 was also abundant in evs from primary human or mouse hepatocytes, hepg2 cells, and human or mouse serum. fn1 and ev-hep co-sedimented at a density of~1.15 g/ml. ev-hepfn1 ko yield and size-range were similar to those of ev-hep, suggesting that ev biogenesis is fn1-independent. as compared to ev-hep, the binding of ev-hepfn1 ko to target cells was highly reduced whereas ev binding was independent of fn1 expression by the target cells themselves. both ev-hepfn1 ko and ev-hep were anti-fibrogenic in vivo but only ev-hep attenuated collagen 1⍺1 expression in mouse hscs in vitro. summary/conclusion: fn1 is abundantly associated with hepatocyte evs and facilitates ev binding to target hepatocytes or hscs. additional studies are needed to clarify the functional role of fn1 in mediating ev-hep anti-fibrogenic actions in vitro or in vivo. elevated glucose increases soluble and aggregated forms of human islet amyloid polypeptide in islet-derived extracellular vesicles -implications in type 2 diabetes and islet transplantation introduction: type 2 diabetes (t2d) is characterized by reduced beta cell mass and function. islet amyloid, formed by aggregation of human islet amyloid polypeptide (hiapp), contributes to progressive beta cell loss in t2d. amyloid also forms in human islets during pre-transplant culture and following transplantation in patients with type 1 diabetes (t1d) which is associated with graft failure. the cellular mechanisms underlying islet amyloid formation are still unclear. in this study, we examined the potential role of islet-derived extracellular vesicles (ev) in the clearance of soluble and aggregated (pro)iapp species from beta cells and amyloid formation. methods: human islets isolated from cadaveric pancreatic donors (n = 4 donors) and wild-type or hiappexpressing (hiapp+) transgenic mouse islets (n = 4/ group) were cultured in normal (5.5 mm) or elevated (11.1 mm) glucose to form amyloid. ev (exosomes) were isolated from culture medium using classical centrifugation and ultracentrifugation. purified ev were analysed by nanoparticle tracking analysis. western blot analysis and double immunogold transmission electron microscopy were performed to verify the presence of ev markers as well as (pro)hiapp species and oligomers (aggregates). results: human islets formed amyloid during culture with elevated glucose which was associated with progressive beta cell apoptosis. (pro)iapp species were detectable in ev released from human islets cultured in normal and elevated glucose. the latter markedly increased (pro)iapp content in islet-derived ev. interestingly, hiapp aggregates (oligomers) were present in the majority of ev released from human islets cultured in elevated glucose but were not detectable in islets cultured with normal glucose. similarly, ev released from hiapp+ mouse islets which formed amyloid during culture had higher (pro)iapp content compared to wild-type islet-derived ev. moreover, hiapp oligomers were present in ev derived from hiapp+ islets but not wt islets. summary/conclusion: in summary, our data show that (pro)iapp species are present in islet-derived ev and that elevated glucose increases (pro)hiapp and its aggregates in ev released from islets. islet-derived ev may play a key role in the process of amyloid formation in t2d and human islet grafts. funding: university of manitoba research grants program (urgp). on. contraction, but not glycolysis, regulates the size of skeletal muscle evs secreted ex vivo. colorado state university, fort collins, usa introduction: skeletal muscle (skm) is a metabolically active tissue and accounts for~35% of total human body mass. acute exercise increases secretion of extracellular vesicles (evs), but the mechanisms responsible are unknown. muscle contraction increases the demand for atp which requires intercellular communication in order to adapt. we hypothesized that this "metabolic stress" during contraction increases skm ev secretion. methods: we tested our hypothesis using an ex vivo ev secretion assay. all studies were approved by the colorado state university institutional animal care and use committee. vastus medialis muscle (skm) from male c57bl/6 j mice (n = 6) or female mt/mg mice (n = 6) was cut into~5 mg pieces and added to 12 well plates (~50 mg/well) filled with 1 ml of serum-free dmem and placed in a cell culture incubator at 37 c for 24 hours. skm from male mice was treated with 2-deoxyglucose (2-dg) (0.1 nm -100 mm) to induce metabolic stress via inhibition of glycolysis. skm from female mice was treated with 10um of blebbistatin (bleb), a contraction inhibitor. after incubation, skm mass was measured and conditioned media was centrifuged (3,000 x g for 15 min) to remove cell debris. evs were isolated using exoquick-tc. nta was performed on isolated evs using a horiba viewsizer 3000. ev secretion was normalized to tissue mass and culture media volume then reported as ([particle]/ml/mg tissue). statistical comparisons for 2-dg experiments were made using a repeated measures 1-way anova. bleb experiments were analysed using a paired student's t-test. results: there was a trend towards greater ev abundance (p = 0.07) as a function of 2-dg treatment, but no effect on ev diameter (p = 0.37). bleb treatment did not alter ev abundance (p = 0.69), but significantly reduced ev mean diameter (p = 0.007; 11% decrease; dmso: 114.9 ± 5.1 vs. bleb: 103.9 ± 4.2). summary/conclusion: contrary to our hypothesis, inhibition of glycolysis with 2-dg did not stimulate skm ev secretion. however, bleb did appear to promote the release of small evs and/or inhibit secretion of larger evs. ongoing efforts are focused on testing other metabolic stressors and defining how blebbistatin promotes small ev secretion. funding: american heart association grant to dsl (ipa34110052). introduction: extracellular vesicles (evs, exosomes) are nanovesicles (30-200 nm) secreted from various types of cells. because of vesicular encapsulation of mirnas and enzymes, the evs play crucial roles in cell-to-cell communication by delivering these functional molecules to other cells [1] . on the other hand, the evs are highly expected as next generation therapeutic tools due to pharmaceutical advantages such as controlled immunogenicity, effective usage of cell-tocell communication routes, artificial modification and encapsulation of functional molecules. however, cellular targeting and uptake efficacy of the evs are insufficient to be utilized as therapeutic tools [2, 3] . in this study, we newly developed evs decorated with cellpenetrating sc18 or (sc18)2 peptides, which are derived from the c-terminal domain of the cationic antimicrobial protein, cap18, because the peptides can be efficiently internalized by breast cancer cells. [4, 5] . methods: all peptides were prepared by fmoc-solid phase synthesis. secreted evs from cd63-gfp stably expressing hela cells were isolated by ultracentrifugation. cellular uptake of evs was analysed using a flow cytometer and a confocal laser microscope. encapsulation of saponin in the ev was conducted by electroporation. results: sc18 peptide is known as one of cell-penetrating peptides, and branched structure of sc18 peptides, (sc18)2, further enhances the cellular uptake [5] . in this research, we examined the effects of the peptide modification on cellular ev uptake, and modification of the sc18 or (sc18)2 peptides on ev membranes was conducted via stearyl moiety. as our results, increased macropinocytotic cellular uptake by modification of the peptides was successfully attained. especially, the modification of (sc18)2 peptides showed higher cellular uptake and macropinocytosis induction efficacy than that of sc18 peptides. in addition, anticancer protein, saporin toxin-encapsulated evs modified with the (sc18)2 peptides significantly enhanced their biological activity with dependency of glycosaminoglycan expression on targeted cells. summary/conclusion: the cell-penetrating (sc18)2 peptide-modified evs shows high abilities to be effectively internalized by cells and are applicable for intracellular delivery of therapeutic molecules. this study is expected to contribute to development of intracellular delivery techniques based on evs. [ introduction: rna therapeutics possess high potential which is yet to be realised, largely due to difficulties involved in delivery to the cytoplasm of target cells. extracellular vesicles (evs) possess numerous features that may help overcome this hurdle and have emerged as a promising rna delivery vehicle candidate. despite extensive research into the engineering of evs for rna delivery, little is known about how their intrinsic rna delivery efficiency compares to current synthetic rna delivery systems. using a novel crispr/cas9 based rna transfer fluorescent stoplight reporter system, we here compared the delivery efficiency of evs to state-of-the-art dlin-mc3-dma lipid nanoparticles (lnps). methods: evs were isolated from mda-mb-231 cells expressing either a targeting or non-targeting control sgrna and applied to hek293 t stoplight+ reporter cells. lnps containing targeting sgrna were titrated onto hek293 t stoplight+ reporter cells to determine the minimum effective dose. lnp and ev particles were characterized using nanoparticle tracking analysis, dynamic light scattering and zeta potential analysis. sgrna copy number was determined using rt-qpcr. results: evs were 140 ± 7 nm in diameter as measured by dls and possessed a negative surface charge of −25.3 ± 3.3 mv. rt-qpcr and nta analysis indicated that sgrna ev loading was low, with only 1 in 3.8e05 ± 3.5e05 evs containing a single sgrna copy. nevertheless, evs containing targeting sgrna induced significant reporter activation while evs containing non-targeting sgrna did not. lnps were 51 ± 0.6 nm in diameter and possessed a neutral charge. these particles also induced significant reporter activation when loaded with targeting sgrna. when delivered via evs, only between 4 to 54 sgrna copies per cell were required to induce statistically significant reporter activation. in contrast, the minimal effective sgrna dose when delivered by lnps was considerably higher at approximately 9e03 copies per cell. summary/conclusion: mda-mb-231 evs deliver rna in a highly efficient manner and are functional at sgrna concentrations several orders of magnitude lower than those required for lnp mediated delivery. this underlines the potential of evs as rna delivery vehicles and highlights the need to study the mechanisms by which evs achieve their efficiency in order for improved development of rna therapeutics. the role of circulating extracellular vesicles in patients with chronic chagas disease introduction: chagas disease is a neglected tropical disease (ntd) caused by the flagellated protozoan trypanosoma cruzi. it is a major public health problem in latin america, and it is now expanding over the globe through immigration of infected individuals. eukaryotic cells release extracellular vesicles (evs) that circulate in body fluids and have an important roles in intercellular communication, both in physiological and pathological conditions. objectives. our study proposes to characterize and to compare the circulating evs isolated from plasma of the chronic chagas disease (ccd) patients with healthy individuals (controls). methods: peripheral blood was collected from patients and controls in the presence of edta and evs enriched from plasma by differential ultracentrifugation. the obtained evs were characterized and quantified by nanoparticle tracking analysis (nta) and added to human thp-1 cells. after 48 h, the cell supernatants were analysed by elisa for the presence of cytokines. results: lower amounts of evs were obtained from ccd patients in comparison with control individuals. however, the same amount of evs of ccd were more capable of inducing cytokines such as ifn-gamma and il-17 in relation to controls. summary/conclusion: although less evs are present in the blood of ccd, these evs induce high inflammatory reactions on macrophages suggesting a possible role of these evs in the establishment of chronic disease. funding: supported by fapesp, cnpq and capes. extracellular vesiclesa trojan horse for therapeutic agent delivery introduction: extracellular vesicles (evs) may prove to be one of the optimal payload carriers for therapeutic agents. while they travel through the extracellular space, the ev's lipid membrane layer shields their luminal cargo from deleterious external factors. when autologous evs are used to protect this therapeutic cargo, little immunogenic effects are expected compared to viral vectors and artificial structures, such as liposomes. their usage is potentially manifold, and they are ubiquitously present in all body tissues and fluids. the key is to develop a manageable ev loading agent for adoptive transfer therapies. methods: to exploit the unique properties of evs, highly positively charged proteins were used to load them with multiple biomolecules, such as a cas9 protein or dicer substrate dsrna as a functional payload and to improve their apparently inadequate natural ability to deposit cargo into the cytoplasm of recipient cells. results: highly positively charged proteins can associate with and/or diffuse through a phospholipid bilayer (thompson et al. 2012) . when these kinds of charged proteins are mixed with isolated evs in vitro, they are loaded into the evs. the positive charge of the protein has the advantage that it can associate with negatively charged agents, such as rna species, and aids the associated molecule to also incorporate into the ev. moreover, the positive charge of the protein helps with cargo delivery, and thus overcoming the bottleneck of the ev's cargo to escape the endosome post-uptake in a recipient cell. self-quenching fluorescent lipid dyes demonstrated that discharge of the highly positive ev cargo into the cytoplasm is concomitant with lipid mixing between the membrane of evs and the membrane of the recipient cell. when egfp-expressing microglia were exposed to evs loaded with a dicer substrate dsrna able to silence egfp via the positively charged protein, the uptake of dicer substrate dsrna was concomitant with a decrease in egfp expression in the microglia. a similar result was achieved when evs were loaded with cas9 protein conjugated to the highly positively charged protein. post-uptake of these cas9-loaded evs, microglia expressing anti-egfp sgrna (single guide rna) lead to decreased egfp expression. summary/conclusion: our ev delivery technology has the capability of delivering multiple biomolecules, such as protein and rna cargo and demonstrates postuptake of the ev functionality of the ev delivered cargo in the recipient cell. hybrid extracellular vesiclesbiomimetic tool for drug delivery to repair endothelial cell dysfunction introduction: traditional drug delivery systems (dds) are usually based on liposomes, micelles or dendrimers. unfortunately, many dds cause side effects including organ toxicity and/or unexpected immune response. in living organisms, extracellular vesicles (evs) are responsible for delivering biologically active molecules to distant cells. in vitro loading of therapeutic compounds into evs is still not effective and needs developing new strategies. for these reasons we aimed to design hybrid extracellular vesicles (hevs) with high loading capacity for dds. methods: for hev synthesis, we used human endothelial derived evs. using freeze/thawing method we fused them with liposomes composed of cholesterol and one of the three lipids: dopc, sphingomyelin or phosphatidylserine. to confirm membrane fusion, we applied a spectroscopy ruler -fret (förster resonance energy transfer) and cryotem imaging technique. we characterized hevs using nta (for size distribution evaluation), dls (zeta potential) and western blot (for detection of evs markers). we evaluated loading efficiency using calcein as a model drug. additionally, we performed cytotoxicity tests. results: in the cryotem imaging, pure and homogenous hev population with a diameter of 65 ± 15 nm was detected. additionally, we observed changes in zeta potential and in size distribution after fusion. fret measurements showed increased fusion efficiency with the increasing number of freeze/thawing cycles and dependence on a lipid-to-protein ratio in evs. additionally, hev had higher loading efficiency than liposomes and sole evs and that their internalization by endothelial cells did not cause a cytotoxic effect. summary/conclusion: based on cryo-tem and fret, we confirmed that our fusion method of hybrid evs is effective and can be applied as a delivery platform for dds to endothelial cells. response to a range of stressors. the functional activity of these evs in recipient cells may, in part, be driven by changes to their biological cargoes. however, the molecular details of the underlying ev biogenesis and loading processes, and how this may vary in different conditions, is poorly understood. methods: we first studied the effect of oxidative stress on the functional activity of evs in recipient cells using cell viability and mitochondrial membrane potential assays in drosophila s2r+ cells. we then carried out total rna sequencing of ev and cellular rna under three stress conditions and compared results to existing data in mouse cells. further to this we have used a bioinformatic pipeline to identify sequence motifs enriched in evs under stress. results: functional assays indicated changes to cell viability and mitochondrial membrane potential in recipient cells, which were donor cell-stress dependent. subsequent characterisation of rna showed an enrichment of ribosomal rna in evs relative to cells, but no significant changes to other biotypes. comparative analysis has also uncovered a set of genes enriched in evs under oxidative stress, and a further subset whose enrichment may be evolutionarily conserved in mouse. we also identified potential ev-loading motifs which may assist in rna loading specifically under stress. summary/conclusion: we have shown that evs derived from oxidatively stressed cells show dose-dependent differences in rna cargo and identified potential sequence motifs that may have a role in its loading. we are now validating the biological significance of these findings by combining different in vivo approaches in drosophila. this will enable us to gain insights into the basic mechanisms which govern ev loading in different contexts, and ultimately the molecular mechanisms underlying ev-mediated intercellular communication. ishai luz a , bibek bhatta a , kanaga sabapathy b and tomer cooks c a ben-gurion university of the negev, beer-sheva, israel; b national cancer centre, singapore, singapore, singapore; c ben-gurion university, beer sheva, israel introduction: mutations in tp53 are considered one of the most frequent genetic alterations in human cancer. besides the abrogation of the wild-type (wt) p53-mediated tumour suppression, a distinct set of missense mutations was reported to endow mutant p53 proteins with novel activities termed gain-of-function (gof). even though mutations in tp53 are typically thought to arise in the tumour cells rather than in the stroma, the non-cell-autonomous effects of these mutants over the tumour microenvironment are poorly understood. in the presented studies, focusing on colon cancer as well as on lung cancer microbiome, we investigated intercellular interactions mediated by exosomes and outer membrane vesicles (omvs) in the context of cancers harbouring mutant p53. methods: p results: in the colon, tumour cells harbouring mutp53 were found to exert a non-cell-autonomous effect over macrophages. when exposed to tumour cells harbouring mutp53, monocytes became polarized towards a distinguished subset of macrophages characterized by tams-related markers. the mutant p53 affected tam were characterized as tnf-αlow/il-10 high, over expressing cd-206 and cd163, with decreased phagocytic ability and increased invasion and matrix degradation potency. investigating the exosomal transfer from mutp53 tumour cells to macrophages, revealed a mutp53-specific mirs signature led by mir-1246 promoting the tam phenotype and creating an invasive front together with tumour cells. mir-1246 was also found to be the top mutp53-associated mir in a cohort of 57 human colorectal resected tumours. separately, in two lung cancer cohorts, we identified a signature of microbiome members associated with p53 mutations. acidovorax temperans, a gram negative bacterium, was found to be abundant in tumours of patients with mutant p53. we found a significant increase in tumour volume in animals inoculated with acidovorax temperans as compared to sham treated animals, and increased lung weight as a percent of total body weight. these preliminary data indicate that acidovorax temperans contributes to lung tumorigenesis in the presence of activated k-ras and mutant p53. omvs shed by acidovorax temperans promoted inflammatory signalling in lung carcinoma cells and elevated cd47 expression on tumour cells and sirpα levels on macrophages. summary/conclusion: altogether, these findings are consistent with a microenvironmental role for specific "hot-spot" gof p53 mutants tightening the interaction between the tumour cell and the immune compartment in colon cancer. in both colon and lung cancer, mutant p53 facilitates cellular interactions within the tumour microenvironmets mediated by vesicles. funding: intramural funding from the national cancer institute, national institutes of health. lori zacharoff and mohamed el-naggar university of southern california, los angeles, usa introduction: the metal respiring bacterium s. oneidensis creates outer membrane extensions and outer membrane vesicles that are sculpted by the novel bar domain protein bdpa. these vesicles and extensions incorporate mutliheme cytochromes involved in extracellular electron transfer to metals and electrodes. however, the physiological relevance of incorporating these cytochromes into the higher order 3d architecture of a vesicle or extension is unknown. given that bar domains serve as a protein sorting mechanism in eukaryotes, we investigated the pathway crosstalk between bdpa and outer membrane multiheme cytochromes as means to understand the physiological significance of membrane architecture. methods: o this end, vesicle morphology and content was measured using dry weights, dynamic light scattering, fluorescence microscopy and comparative proteomics from wild type s. oneidensis and deletion strains. results: cells lacking bdpa make large amorphous vesicles that are dense with protein. in contrast, a strain lacking outer membrane cytochromes recruits less total protein into smaller vesicles. proteomics to show that both bdpa and multiheme cytochromes are involved in recruiting other proteins to outer membrane vesicles and have a reciprocal relationship. summary/conclusion: in the absence of bdpa, protein crowding has to become the main driving force of vesiculation and bdpa is essential for efficient incorporation of cytochromes. however, multiheme cytochromes are not only vesicular cargo, but are also important for shaping and loading vesicles. both of these situations make it clear that vesicles play a role in increasing the respiratory surface area of s. oneidensis cells. moving forward, we hope to be able to control bdpa and cytochrome levels for selective recruitment of technologically relevant payloads. introduction: fascioliasis caused by fasciola hepatica represents a major economic loss and clinical burden in cattle farming worldwide. extracellular vesicles (evs) contain pathogen-derived molecules that represent novel biomarkers of disease. in the present study, we have identified potential new biomarkers of f. hepatica infection in evs present in sera of infected cattle. methods: parasites and sera were obtained from local abattoirs (valencia, spain, and medellin, colombia, respectively). 38 sera from infected and 32 from healthy animals. parasites were cultured, and evs obtained by sizeexclusion chromatography (sec) and characterized by nta, tem and proteomic profiling. recombinant proteins from f. hepatica evs (enolase and fh16.5 tegumentary protein) were produced, and coupled to magnetic beads. measurement of bovine igg antibodies was performed using luminex bead array technology. results: a total of 239 proteins were identified associated with evs as shown by the presence of typical ev-markers (tsg101, alix, cd63). two parasite proteins, enolase and the fh16.5 tegumentary protein were produced as recombinant proteins and used for detection of cattle igg employing luminex bead array technology. interestingly, significant differences were found in the fluorescence values of both recombinant proteins allowing discrimination between sera from infected and non-infected cattle. the use of the fh16.5 protein generated a highly significant difference between the two groups (p value = 0.003614); as did enolase (p value was 0.02294). summary/conclusion: this study demonstrates the usefulness of ev proteins as new biomarkers for early diagnosis of helminth infections using multiplex assays, a technology that may also be applied to other parasite ev molecules. life stage-specific glycosylation of schistosome-derived extracellular vesicles introduction: glycans play an essential role in pathogen-host interactions. larvae and adult worms from schistosoma mansoni release distinct subsets of glycoconjugates as excretory/secretory (es) products. extracellular vesicles (evs) are also among the es products. we recently found that schistosomuladerived evs are glycosylated and bind human dendritic cells via c-type lectin receptor (clr) dc-sign, leading to increased il-10 and il-12 release. here we investigated the glycosylation profile of evs released by s. mansoni adult worms, compared this to schistosomula evs, and addressed how this may affect parasite-host interactions via clrs. methods: evs from cultured s. mansoni parasites were obtained by ultracentrifugation and purified with iodixanol density gradients. isolated evs were analysed by nta and cryo em. n-glycan and lipid glycan content was determined by mass spectrometry. density gradient fractions with evs were loaded onto sds-page gels followed by western blot (wb) analysis using anti-glycan monoclonal antibodies (mabs). results: cryo em showed that adult worm evs lacked the long thin filaments that are characteristic for schistosomula evs. additionally, in contrast to schistosomula evs, glycolipids could not be detected in the adult worm evs. mass spectrometry analysis showed that the most abundant n-glycans in the adult worm evs contained galnacβ1-4glcnac (lacdinac, ldn) motifs, which correspond to previously published overall glycan profiles of this specific life stage. other differences in ev glycosylation between the two life stages were observed by wb using anti-glycan mabs: adult worm evs showed a paucimannosidic glycan motif whereas in the schistosomula evs galβ1-4(fucα1-3) glcnac (lewis x) was detected in line with previous ms analysis. introduction: phloem plays a central role in plant function, as it is the responsible for the translocation of photoassimilates from source-to sink-organs, and a long-distance route for signals distribution. due to the sap high nutrient content, sieve elements are primary target for plant pathogens and pests. in this work we aimed to isolate and characterize extracellular vesicles (evs) from cucumis melo phloem sap, derived from plant either exposed or not to the melon aphid, aphis gossypii (hemiptera: aphididae). methods: phloem exudates from 5-week-old melon plants, either uninfested or infested with adults of a. gossypii (n = 15, 4 replicates each), were collected by cutting the stem with a sterile razor blade between first and second expanded leaf from the top. evs were isolated by size exclusion chromatography, and analysed by nanoparticle tracking analysis (nta) and transmission electron microscopy. evs proteome was determined by quantitative mass spectrometry. results: evs from phloem sap were successfully isolated in every condition. no significant differences were detected among distinct samples, neither in particle concentration and size by nta, nor in protein concentration. most importantly, a total of 381 different proteins were identified in phloem sap evs, including 152 present in exosome databases (exocarta). on top of that, 44 differentially expressed proteins were identified in evs derived from aphid infested or uninfested plants (p value < 0.05). summary/conclusion: understanding how plants trigger their defences against pests and pathogens is important to develop new control measures. the characterization of several proteins in evs from the phloem sap provide valuable information on long distance signalling in plants. moreover, as plants lack an immune system comparable to animals, the different protein content in phloem sap evs after exposure to aphids could indicate their important role in delivering inducible defences against invading pests and pathogens. extracellular vesicles from nematode species heligmosomoides bakeri and trichuris muris contain distinct small rnas that could enable niche specificity in the host introduction: gastrointestinal nematodes are extremely prevalent parasites that infect most animals and 24% of human population. their success as parasites is attributed to their ability to secrete diverse molecules that modulate the host immune system. extracellular vesicles (evs) are one of the immune modulatory compounds they release that directly modulate host cells. our goal is to understand how the small rna (srna) cargo underpins ev function, using a comparative analysis of ev cargo from diverse nematode species. methods: we first compared how different ev isolation methodologies (ultracentrifuge (uc), size fractionation, sucrose gradient floatation) effect the small rnas detected in h. bakeri evs using different library preparation kits (cleantag, truseq), with or without polyphosphatase treatment. we then compared this to small rna libraries from t. muris evs using comparable methods, uc ev purification, with or without polyphosphatase treatment and using the cleantag library preparation kit. results: evs from both species contained mirnas, however the mirna gene familes in h. bakeri and t. muris evs are distinct. the mirna content detected in ev samples collected by different purification protocols is robust. the largest difference in detected mirnas was found when comparing different library preparation kits. although both h. bakeri evs and t. muris evs were dominated by srnas derived from intergenic or repetitive elements in the parasite genomes, only in h. bakeri evs were these secondary sirnas. summary/conclusion: h. bakeri and t. muris evs contain distinct small rna cargos, which may underpin their ability to colonise different host niches, and/or modulate the host immune system differently. t. muris evs do not contain secondary sirnas, in contrast to h. bakeri, however they are dominated by srnas derived from intergenic or repetitive regions. comparative analysis of helminth evs could help pinpoint the srnas involved in cross-species communication. please provide any keywords if applicable.: nematode, cross-species communication, small rna introduction: extracellular vesicles (evs) are secreted from various cells including cancer cells and known to contain protein and small rnas including mirna isoforms (isomirs). therefore we also focused on isomirs including other small non-coding rnas for biomarker discovery. although liquid biopsies using small rnas are promising biomarkers for early detection of cancer, current approaches to detecting and analysing mirnas in the blood are still inadequate. artificial intelligence (ai) data analysis may provide better algorisms for diagnosing cancer. methods: small rnas were isolated from serum or purified evs using a mirneasy mini kit (qiagen) and quantified by using the ion s5™ next-generation sequencing system. (thermo fisher scientific). evs were purified using total exosome isolation reagent (invitrogen™). ai data analysis was performed using jmp® genomics and datarobot enterprise ai platform. results: three small rnas, isomir of mir-21-5p, mir-23a-3p, and trf-lys (ttt) were significantly upregulated in breast cancer patients compared with the healthy cancer-free individual. the combination algorithm using these three small rnas allows for a more accurate diagnosis of the area under the curve (auc) 0.92. to test the possibilities that these small rnas are derived from cancer cells, we isolated evs from the serum and performed ngs analysis to profiled serum small rnas in evs. interestingly we found that two small rnas, mir-21-5p and mir-23a-3p, also high in breast cancer evs, indicating that these small rnas were expected to be derived from cancer cells. in oesophagus cancer, we also performed ngs analysis and identified twenty-four mir/isomirs candidates for diagnostic biomarkers. a multiple regression model selected mir-30a-5p and two isomirs (mir-574-3p and mir-205-5p) . the auc of the panel index was 0.95. we also performed ai data analysis and discovered the novel algorisms that can diagnose breast and oesophagus cancer more accurately. summary/conclusion: we demonstrated combinations of circulating non-coding rnas containing evs potentially useful for the detection of early-stage breast and oesophagus cancers. in addition, the datarobot enterprise ai platform enables us to the more accurate diagnosis of cancers at the early stage. identification of novel ev-associated mirnas as toxic biomarkers in mouse introduction: recent findings reveal that extracellular vesicles (evs), secreted from cells, are circulating in the blood. evs are classified into exosomes (40-120 nm), microvesicles (50-1,000 nm) and apoptotic bodies (500-2,000 nm). evs contain mrnas, micrornas, and dnas and have the ability to transfer them from cell to cell. recently, especially in humans, the diagnostic accuracy of tumour cell type-specific evs as biomarkers is more than 90%. in addition, micrornas contained in the evs are being identified as specific biomarkers in blood for chemical-induced inflammation and organ damage. therefore, micrornas contained in the evs released into the blood from tissues and organs in response to adverse events such as chemical substances and medicine are expected to be useful as novel biomarkers for toxicity assessment. in this study, we aimed to identify target organs by comprehensive analysis of ev rnas in the blood of mice after chemical exposure to establish a highly sensitive "next generation type" toxicity test for chemical substances and medicine using ev rna in blood as a biomarker. methods: all animal studies were conducted in accordance with the helsinki declaration and the guidelines approved by the animal care committee of the national institute of health sciences. c57bl/6 j male mice (12 weeks) were orally dosed with ccl4 (vehicle, 7, 70 mg/kg). serum were separated from blood after 2, 4, 8 and 24 hours after ccl4 administration. the serum was centrifuged at 10,000 x g to remove cellular debris and subsequently ultracentrifuged 100,000 x g. the pellet is resuspended in pbs and ultracentrifuged 100,000 x g again. the comprehensive small rna-seq of collected evs were performed according to the manufacture's protocols. results: we succeeded in isolating more than 10 novel small rnas, which could be used as novel highly sensitive biomarkers for hepatotoxicity due to carbon tetrachloride (ccl4: 7 mg/kg & 70 mg/ kg). well known hepatotoxicity biomarkers, mirna-122 and mirna-192 were upregulated more than 1000-fold in the administration of 70 mg/kg ccl4, but not responded in the administration of 7 mg/kg ccl4. summary/conclusion: these results suggest that mir-122 and mir-192 are mainly released from liver to blood directly only in the administration of 70 mg/kg ccl4, while novel more sensitive hepatotoxicity biomarkers which responded in the administration of both 7 mg/kg and 70 mg/kg ccl4 should be included in the ev. our novel biomarkers will accelerate a rapid evaluation of chemical substances and medicine in nonclinical safety evaluation. introduction: advancements in sequencing technologies have allowed analysis of the genomic landscape of cancer using circulating cell-free(cf) dna. however, cfdna does not originate only from tumour cells. we recently demonstrated that most of the dna circulating in plasma of cancer patients is associated with large evs (l-evs), and that l-ev-associated dna reflects genomic aberrations of the cells from which l-evs arise. since l-evs are specifically released by tumour cells, we explore their potential to report cancer-specific genomic alterations in patient plasma and compare it to cfdna. methods: differential ultracentrifugation, tunable resistive pulse sensing, qubit dsdna high sensitivity assay, capillary electrophoresis, whole exome sequencing (1500-2000x), targeted sequencing (qiaseqtm), flow cytometry. results: we show here that l-evs in the size range of >1 micrometre are present exclusively in plasma obtained from cancer patients and absent in plasma from healthy donors. in agreement with this finding, double-stranded(ds) dna is detected only in l-ev fractions of patient plasma and not in those obtained from healthy donor plasma using the same protocol. we also demonstrate that the fragments of dsdna associated with circulating l-ev are larger in comparison with cfdna (>10,000 bp versus~170 bp). a large-scale analysis of l-ev dna obtained from plasma of patients with metastatic castration-resistant prostate cancer (mcrpc) as well as with non-small cell lung cancer (nsclc) demonstrates that dna associated with circulating l-evs reports cancer-specific genomic alterations in both types of cancer. we further investigate if l-evassociated dna is intra-or extravesicular and demonstrate that it is present in both forms. we finally compare the purity of the tumour signal in intravesicular l-ev dna, total l-ev dna, and cfdna obtained from patient plasma. summary/conclusion: our results demonstrate that circulating l-evs contain high quality, large molecular weight dna that contains cancer-specific genomic alterations, supporting the use of l-evs as a source of tumour-derived dna in plasma. introduction: epidermal growth factor receptor (egfr) mutation driven lung adenocarcinoma (ac) represents a unique subgroup that lends itself to treatment with oral egfr tyrosine kinase inhibitors. current methods that are used to detect these mutations (e.g. l858 r or the resistance mutation t790 m) involve invasive tumour biopsies or blood circulating tumour dna (ctdna) and cell free dna (cfdna). the sensitivity of blood ctdna and cfdna is limited by the frequency of genomic alterations in the egfr gene; additionally, ctdna does not reflect changes in the egfr protein, against which novel therapies are in development. there remains a need to develop bloodbased biomarkers that can circumvent these disadvantages and replace the more standard, invasive tumour biopsies. we propose the study of exosomes for treatment monitoring as well as to identify egfr resistance related genomic and proteomic changes. methods: we enrolled 10 patients with metastatic lung ac: 8 with egfr mutations and 2 without (control). from the patients with egfr mutant lung ac, we processed 25 blood samples through the patients' treatment course, using ultracentrifugation to isolate exosomes. we then used both droplet digital pcr (ddpcr) to test exosomal rna (exorna) for the mutation of interest and western blots to test protein resulting from exon 19 deletion or l858 r mutations. results: from 6 patients with egfr exon 19 deletion mutations, we detected identical mutations in exorna from 15/19 samples. exorna based mutational load increased and mirrored clinical progression in 2 patients. three patients whose cancer remained stable demonstrated a decrease in their exorna. one patient had blood drawn only at 2 points and was therefore not plotted. exorna from 2 patients with l858 r and t790 m mutations demonstrated the corresponding mutations; however, exorna did not mirror their disease course. we also demonstrated mutant egfr protein presence in exosomes from 3 patients. finally, we tested cfdna for egfr mutations from four matched samples using ddpcr. we detected matched mutations in exosomes in all four, while cfdna mutations were only detected in 2/4 patients. summary/conclusion: in summary, we detected egfr mutations in 19/23 exosome samples isolated from metastatic lung ac. our results set the stage for optimization of exorna methods and inform future experiments relating to exosomal cargo in patients with egfr mutant lung ac. identification of plasma-derived, ev-based biomarkers for glioblastoma introduction: glioblastoma multiforme (gbm) is the most malignant and aggressive primary brain cancer in adults, with an incidence of 3.2 per 100,000 people. currently, diagnosis is only performed via histopathological investigation of a tissue sample from a gbm lesion, complemented with molecular diagnostics for identification of select biomarkers. mri is the standard of care for follow-up and monitoring of treatment response. therefore, development of a "liquid biopsy" to obtain disease-relevant information from patient's body fluids is highly desirable. methods: we present the results from a clinical study in which extracellular vesicle (ev)-derived mrnas and long non-coding rnas were profiled from the plasma of gbm patients and control individuals. we obtained plasma from 8 patients at the time of initial diagnosis, and 8 matched controls by sex and age. ev-associated rna was isolated from 1-2 ml plasma and rna-seq was performed using our proprietary pipeline. sequencing data was analysed for differential gene expression. results: we observed 95 mrnas as differentially abundant between gbm and control samples, with 82 mrnas enriched in gbm samples and 13 mrnas enriched in control samples (p < 0.05). correlation based on differentially abundant mrnas separated gbm and control samples into two unique populations. eight differentially expressed mrnas were previously identified as part of the mesenchymal gbm subtype. these data, while preliminary, provide a potential basis for the further development of a noninvasive gbm gene panel test. summary/conclusion: we have identified a novel rna signature for gbm from plasma derived evs, which differs from previously identified biomarkers isolated from tissue. further work will refine this signature to enable detection, characterization, and patient monitoring for gbm with minimally invasive techniques. introduction: sjogren's syndrome (ss) is a systemic autoimmune disease in which inflammation progressively damages the moisture producing glands of the afflicted. 4 million americans are estimated to be suffering from the disease, 90% of which are women with an average age of 40. overlapping symptoms with other health conditions and co-morbidities make ss particularly difficult to diagnose, with average time to diagnosis of 3 years. saliva exosomal rna profiling has been primarily focused on small rnas and has been limited thus far due to the large contribution of sequencing reads from the oral microbiome. a noninvasive saliva exosomal rna (exorna) based test capable of diagnosis would be highly desirable. methods: we began by first developing a novel long rna-seq workflow to selectively enrich and profile human exosomal mrnas and long non-coding rnas (lncrnas) from saliva. we then profiled salivary exorna obtained from 7 ss patients and 7 healthy matched controls. finally, we performed differential gene expression analysis to obtain an exorna signature for ss. results: rna-seq data analysis demonstrated highly efficient enrichment of human transcriptome, with over 80% of reads mapping to the transcriptome. further rna biotype analysis showed over 75% of transcriptome reads mapped to protein coding genes and lncrnas. we detected over 10,000 mrnas and approx.1000 lncrnas. differential expression analysis (dex) of ss vs. healthy control exorna identified 455 upregulated genes, including 415 mrnas and 37 lncrnas (p < 0.05). 120 genes were found to be downregulated in ss, including 114 mrnas and 6 lncrnas. gene ontology analysis of dex genes revealed enrichment of genes involved in various immune system related pathways. most importantly, principal component analysis (pca) resulted in clear separation of ss patients from healthy controls. summary/conclusion: our optimized rna-seq workflow enables saliva-based liquid biopsy for biomarker discovery. the gene signature identified in this ongoing study could potentially provide a non-invasive molecular means of diagnosing sjogren's syndrome. introduction: increasing embryo implantation rates has become one of the greatest challenges in assisted reproduction techniques. usually an endometrial biopsy is done to identify a receptive endometrium, which prevents embryo transfer in the same cycle, as it is detrimental for the implantation. the implantation is a complex process, which requires a synchrony between the development of the embryo and the endometrium, but also, an adequate embryo-endometrial cross talk. the presence of extracellular vesicles (evs) as mediators of this communication has been describe in the endometrial fluid. therefore, we hypothesize that the molecular analysis of the content of the evs and companion molecules from endometrial fluid could be a non-invasive method to recognize an implantative endometrium and consequently improve the implantation rates. methods: the objective is to define a simple, sensitive and reproducible non-invasive ev-based method that allow the quick identification of an implantative endometrium by means of mirna analysis. for the establishment of a robust methodology for analysing evs from endometrial fluid in clinical settings, where the sample is limited and no sophisticated equipment is available, five different methodologies were compared in triplicate. two of them consisted in the direct extraction of rna while in the other three, before the rna extraction an enrichment of evs was done. smallrnaseq was performed to determine the most efficient method. once the best method was selected, it was applied in a set of real samples with different implantation outcome. the content of mirnas (mainly associated with evs) of endometrial fluid samples from women in whom the implantation was successful (n = 15) and unsuccessful (n = 15) were analysed. results: our results show that the protocols with a previous enrichment step of evs obtained a higher mirna expression. the results obtained from the differential analysis of the set of samples with different implantation outcome are being analysed and it is expected that the results will be available by the time this communication is presented. summary/conclusion: this work demonstrates that it is possible to obtain and analyse evs and evs-associated mirnas from a small volume of endometrial fluid samples, which allows the use of ev-mirnas as a low-invasive biomarkers for the detection of an implantative endometrium. funding: jip is supported by a predoctoral grant from the basque government. small rna cargo of evs is affected by hormone treatment in prostate cancer introduction: small rnas are recently reported as a regulator for prostate cancer progression to castration-resistant disease. our previous work has shown that evs protein cargo is affected by male steroid hormone, dihydrotestosterone (dht). in this study, we assess the small rna cargo of evs in response to androgen manipulations. methods: androgen receptor-positive lncaps are grown in css medium to deplete the androgens. media were then replaced with vesicle-depleted css medium ± 10 nm dihydrotestosterone (dht) ± 10 µm enzalutamide (enz) for 48 h. evs were isolated using sequential ultracentrifugation (2000 g for 20 min, 10,000 g for 30 min, 100,000 g for 2 h), washed once in pbs. protein and rna were collected from both parent cells and conditioned medium to allow direct comparison between s-evs cargo and cells. small rna ngs libraries were prepared using the illumina's truseq small rna library prep kit and single-end sequenced at a read length of 50 nucleotides (nt). fastq library files were processed using a custom-designed pipeline. adapters were removed using the cutadapt tool, trimmed reads were mapped with high stringency against ribosomal sequences using bowtie2. snorna and trna fragments were identified using the flaimapper software. remaining reads were mapped against the human genome hg38 using bowtie2. results: we found that the presence or absence of androgens does not significantly change the amount of total rna in small evs (s-evs). however, hormone stimulation altered the small rna content of s-evs, in parallel with our previous published data on ev protein cargo. dht increased the abundance of snorna in cells, while a reduction of snornas was observed in the s-evs fraction. interestingly, dht induced the formation of cell filopodia that are not inhibited by androgen inhibitor enzalutamide. pathway analysis indicates the p53 mediated regulation driven by mirnas found in s-evs upon exposure to dht. the expression profile of snorna and trna fragments in dht treated cells resembles results from clinical prostate cancer specimens. summary/conclusion: our findings show that androgen manipulation alters both s-ev derived protein and rna cargo. changes in the s-ev rna profile due to treatment with androgens are not identical to small rna profiles in parental cells, indicating a specific sorting mechanism of s-ev small rna upon androgen manipulation. further, dht induces the formation of cell filopodia irrespective of enzalutamide, suggesting cargo selection of s-evs. we conclude that small rna ev cargo can be utilised to as prostate cancer biomarkers in androgen targeted treatments. introduction: cancer immunotherapy, such as pd-l1 blockade, is a method to eliminate cancer cells. ectopic expression of pd-l1, on the surface of tumour cells, has been associated with tumour persistence and as an important predictor of therapy response. a test that, specifically and accurately, detects pd-l1 is critically important in order to identify patients that would benefit from these treatments. emerging evidence has shown that extracellular vesicles (ev) can carry immune checkpoint molecules, such as pd-l1, and whose expression have been correlated with tumour immunity response. with a multitude of commercially available antibodies identifying appropriate clones and associated assay is important in order to standardize the diagnostic modality used. methods: pd-l1 expressing cancer cell lines were used to generate evs. pd-l1-myc vector was transfected to generate an overexpression system. exoview® sensors containing different anti-pd-l1 clones were generated. samples (cell derived and plasma) were incubated on chips to allow the antibody to bind the antigen on the ev. after incubation, chips were immunolabeled with fluorescently labelled antibodies against pdl-1 or ev associated markers. exoview r100 reader was used to enumerate the evs captured on the sensor surface and analyse the expression of pdl-1 on single vesicle through fluorescence imaging. immunoprecipitation and mass spectrometry (ip/ms) were employed as an orthogonal method to verify the specificity of the assay. results: to study the detection efficiency of the antibodies, engineered pd-l1-myc evs were used. under these circumstances, all the tested antibodies were able to capture evs. when testing endogenous pd-l1 positive evs from different cancer cell lines, only 28.8 and 73 10 clones consistently bound to evs. in addition, evs derived from plasma demonstrated to be positive for pd-l1, however, only clone 28.8 was able to immobilize these evs. the results suggested that clone 28.8 could be a potential pd-l1 antibody to detect pd-l1 positive evs originating from various sources. to confirm these results, and assure the specificity of the antibody targeted ip/ms was employed. summary/conclusion: in combination with the exoview platform, anti-pd-l1 antibodies can be screened and potentially used to generate a non-invasive ev-specific assay that could detect this protein in patients. differences in extracellular vesicle protein cargo is dependent on head and neck squamous cell carcinoma cell of origin university of michigan, ann arbour, usa introduction: head and neck squamous cell carcinoma (hnscc) is the sixth most common, eighth most fatal cancer worldwide and includes cancers of the oropharynx, larynx, hypopharynx, and oral cavity. in 2017, there were over 49,000 new cases and 9,700 deaths estimated in the usa alone. despite recent advances in treatment, including radiation, chemotherapy, surgery, concurrent chemoradiation, and immunotherapy, many tumours develop resistance and progress. patients develop metastases or tumours recur locally or regionally; the 5-year overall survival rate for hnscc is only 40-50%. factors that contribute to poor survival for patients with hnscc include late stage diagnosis, lack of reliable markers for early stage detection, high level of biologic heterogeneity, and local recurrence and distant metastases after treatment. methods: this study used 8 representative hpv-positive and hpv-negative hnscc cell lines, one hpvtransformed cell line. and two non-cancer oral keratinocyte cell lines. evs were isolated using differential ultracentrifugation and peg precipitation/ultracentrifugation. evs were characterized by tem, nta, and wes protein analysis for reported ev markers. ev and whole cell lysates were assessed by lc-ms/ms analysis using the tandem mass tag-10plex kit. cluster analysis was performed on the fold-change peptide spectrum matches (psm) for the evs from the hnscc lines compared to the evs from the normal keratinocyte line (noksi). protein was measured using a capillarybased electrophoresis instrument. results: cd9 and annexinv were detected in all of the ev lysates tested, while calnexin was detected in all of the whole cell lysates and none of the ev samples tested. selected proteins stat3, hla-a, tenascin, e-cadherin, β catenin, cytokeratin 19, epha2, and cd59, and hpv-related markers p16, p53, rb, cyclin d1, and egfr were tested using the wes platform. evs from hpv-positive cell lines showed higher protein levels compared to evs from hpv-negative cell lines in stat3, hla-a, and tenascin. only kert19 demonstrated lower protein levels in evs from hpvnegative cell lines. of the common hpv-associated hnscc markers: egfr, p53, rb, cyclin d1 and p16, only egfr was positive in any the evs tested. the remaining proteins queried, e-cadherin, β catenin, epha2 and cd59 showed varying protein levels in evs from both hpv positive and hpv-negative cell lines. summary/conclusion: our findings suggest that these proteins may be potential hnscc ev markers that may be 1) selectively included in ev cargo for export from the cell as a strategy for metastasis, tumour cell survival, or modification of tumour microenvironment, or 2) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. validation of antibodies on western blot for extracellular vesicles from biological human samples and cancer cell conditioned media the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: one of the major challenges in extracellular vesicles (evs) research is to prove the particles that are isolated are true evs, rather than other co-isolated contaminants, like lipoproteins. isev recommends using multiple assays to characterize evs. this study aims to validate the positive and negative protein markers for extracellular vesicles from plasma, urine and prostate cancer cell conditioned media (ccm). methods: membrane and cytosolic fractions of mcf7 cells served as positive and negative controls for all antibodies validated. evs were isolated from plasma of healthy volunteers, urine of healthy volunteers and ccm of pc-3 cells using differential ultracentrifugation. eight protein markers were assessed: positive markers cd63, cd81, cd9, flotillin1 (flot1), alix and tumour susceptibility gene 101 (tsg101), negative marker calnexin (canx), and contaminant markers apo-a1 for plasma and thp for urine. tetraspanins are small transmembrane proteins expressed in evs. flot1 is membrane protein that forms microdomains in the plasma. alix and tsg101, an accessory protein of the endosomal sorting complex required for transport, are involved in the biogenesis of evs. they are positive markers for evs. canx is in the membrane of the endoplasmic reticulum. apolipoprotein-a1 (apo-a1) is the protein components of lipoproteins, therefore it is marker of contamination for plasma ev. tamm-horsfall protein (thp) is contamination marker for urine ev, because it is most abundant protein in human urine. results: all antibodies were validated in the correct positive and negative control, thus confirmed as usable and reliable antibodies for western blot. in plasma ev, cd63, cd9, cd81 and flot1 were positive and canx and apo-a1 were negative. in urine evs, cd9, cd63, flot-1, alix and tsg101 were positive and canx and thp were negative. in ccm evs, cd9, cd63, flot1, alix and tsg101 were positive and canx was negative. summary/conclusion: we confirmed a high degree of ev purity from 3 sample types: urine, plasma, and ccm. of particular importance, we confirmed that evs isolated from biologic patient samples, plasma and urine, had low contamination. future work will use these methods to confirm purity of ev samples prior to addition analysis, such as examining ev cargo and biologic significance. proteomic study of mesenchymal stem cells derived exosomes modified using mir. introduction: the project we are working on is to modify the immunogenic profile of human cmms from the umbilical cord stroma through its stable transfection with anti-mir-21-5p, and therefore of the exosomes that these cells generate, for use in free-cell therapy to treat inflammatory process. methods: evs released from a primary culture of human umbilical cord mesenchymal stem cells and from primary culture of human umbilical cord mesenchymal stem cells mir21-/-modified through stable lentiviral transfection were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (tem) and measured by nanoparticles tracking analysis (nta). protein extraction from evs was made using ripa buffer and after checking protein integrity the total ev proteins. we performed a shotgun proteomic study using a tmt (10-plex) label of the total mir21-/-exosomes protein comparing it with normal exosomes. after labelling the ltq-orbitrap platform of proteored was needed for fraction injections and data acquisition. proteome discoverer 2.2 (thermo) was used for protein processing and quantification. results: a total of 1.861 proteins were identified at least with a unique peptide and we have able to establish the proteomic profile of mir21-/-exosomes against normal exosomes. we found out several protein modulated by mir21 and related to inflammation. summary/conclusion: we have able to establish the proteomic profile of mir21-/-exosomes against normal exosomes focusing on proteins involving inflammation process. all those results seem indicate that exosomes could be modified, which could be used as an anti-inflammatory free-cell therapy. funding: proteored concept test project grant. a novel extracellular vesicle isolation method used to discover urine liver disease biomarkers introduction: hepatocellular carcinoma (hcc) is the 6th most common cancer worldwide and the 3rd most common cause of cancer death; additionally, its incidence is increasing. while outcomes for early hcc are superior to those for late stage disease, early detection of hcc remains a challenge. current guidelines have suboptimal sensitivity and specificity. in this pilot study, we hypothesize that urine extracellular vesicles (evs) may identify candidate biomarkers towards the development of an inexpensive, widely accessible screening assay for the early detection of hcc. methods: urine samples from 20 healthy subjects, 19 subjects with cirrhosis, and 19 subjects with cirrhosis plus hcc were collected and processed using ymatrix columns to isolate ev-associated protein and mirna. protein was analysed using a tandem mass tag method on a thermo scientific orbitrap fusion mass spectrometer with comet/paws and edger processing. mirna was analysed using a targeted firefly microarray from abcam. differential expression and predictive modelling for the presence of hcc and cirrhosis was performed to identify candidate mirna and protein biomarkers. results: for mirna, 39 samples were eligible for analysis after low expression filtering. we used pair-wise ratios of 34 cancer-associated mirnas by gradient boosting of decision trees to develop a predictive model for hcc. our best model had a sensitivity and specificity of 0.93 and 0.92 respectively using 6 mirnas to distinguish hcc from cirrhosis. all samples were eligible for protein analysis. based on differential expression and biologic relevance, we identified 10 protein candidate biomarkers. interestingly, we found liver-selective proteins and known hcc/cirrhosis plasma/tissue markers, demonstrating proof-of-concept for the method. summary/conclusion: urine extracellular vesicles contain liver-selective proteins and known liver disease serum biomarkers as well as novel mirna and protein biomarkers that are significantly up-regulated in disease samples. the described candidate biomolecules may be easily accessible biomarkers with which to develop a sensitive and specific universal screening diagnostic for the early detection of cirrhosis and hcc. introduction: the peptidergic g-protein coupled receptors (gpcrs) are cell-signalling transmembrane proteins, which in their native form comprise of seven segments embedded in the cell membrane. this structural advancement is believed to be maintained in extracellular vesicles (evs). in autoimmune diseases, the presence of autoantibodies towards gpcrs is not uncommon, and to detect plasma autoantibodies, evs carrying gpcr will be used as template in a novel microarray screening tool. methods: purified evs from hek293 cells were printed on different types of surfaces; polymer coated glass slides and hydrophilic and hydrophobic plastic 96 well plates. five different print buffers were tested in a multiplex assays. spots containing evs were stained with biotinylated antibodies (cd9, cd63, cd81, adrβ2, hsp70, epcam and flotilin-1) followed by binding of cy5-labelled streptavidin and visualized microarray scanner. results: the outcome of these experiments was promising, as some of the chosen printing buffers showed increased tendencies to bind evs. the ev presence was verified with a panel of markers known to be present on small evs. in addition, the ev content of the adrenergic beta-2 receptor (adrβ2-receptor), which is a gpcr of interest in autoimmune diseases, was verified in some of the experimental setups. summary/conclusion: the approach of using evs as template in a screening tool possesses the potential to easily screen for autoimmune illness markers in diagnostic purposes. using the microarray technology allows the screening to be multivariate, specific and highly sensitive. circadian variation of extracellular vesicles secreted in urine: analysis of time point collection and normalization strategy. introduction: urinary extracellular vesicles (uevs) are an ideal source of biomarkers for kidney and urogenital diseases. despite the great deal of interest generated by uevs, little is known about its collection time and normalization approach. the majority of the studies on uevs focus on spot urine collection based on the assumption that it accurately reflects the renal function, although time point of collection is not standardized. therefore the practice to collect spot urine does not allow for calculating and standardizing accurately the uev excretion rate which may vary during the day. in addition, no research has been carried out yet to show the quantitative and qualitative difference of uevs between spot urine and 24 h collections.the aim of this study is to compare uevs excreted in all single voids during a 24 hour collection period and compare it with 24 hour collection performed. methods: uevs were enriched by differential centrifugation and electron microscopy, western blot, nanoparticle tracking analysis, tuneable resistive pulse sensing and imaging flow cytometry were used to quantify uevs and associated markers variation during the 24 hour. creatinine, urine osmolality and particle concentration were used to normalize the assessed analytes. results: electron microscopy showed a heterogeneous population of evs and western blot confirmed the presence of ev markers (tsg101, alix and cd9). rna was extracted by a column-based method (mirna extraction kit qiagen) and cel-39 mirna was spiked in each sample. a multiparametric detection of nephron markers podocalyxin, aquaporin-2 and uevs pan tetraspanins (cd9 + dc63 + cd81) was performed utilizing imaging flow cytometry. whereas the uev composition did not change across the 24 hours analysis, the quantity of uevs and related markers fluctuated during the day depending on the hydration and excretion rate.the results of a 24 hour urine collection reflected the average results of all single voids over a 24 hr period. creatinine and particle count normalization failed to normalize "outliers". summary/conclusion: this study represents the very first report which compares single void urine versus 24 hour uev analysis. we concluded that the 24 hour collection is the preferred choice for a robust and rigorous assessment of uevs and its associated markers. porcine body fluids differ in small extracellular vesicle counts: comparison of blood plasma, seminal plasma and cerebrospinal fluid as vesicle sources for proteomic analyses helena kupcova skalnikova a , jakub cervenka b , jaromir novak a , karolina turnovcova c , bozena levinska a , jana juhasova a , stefan juhas a and petr vodicka a a institute of animal physiology and genetics, czech academy of sciences, libechov, czech republic; b institute of animal physiology and genetics cas, v. v. i. libechov, libechov, czech republic; c analysis tools were used to identify in silico biological pathways and functions governed by detected mirnas. expression of putative targets of selected mirnas was tested using qpcr after in vitro delivery of uterine evs to ptr cells. results: careful characterisation confirmed that uterine lumen is enriched with a diverse population of evs caring mirnas. interestingly, 36 out of 79 detected mirnas showed difference in abundance between tested days of pregnancy and half of them was exclusively detected on d16. identified mirnas were characterized as potent regulators of cellular development, growth, proliferation, and movement, in addition to their involvement in organismal and embryonic development. the expression of 20 genes identified as a possible mirna targets was tested after evs delivery to ptr cells in vitro. both down-(e.g., ptger4) and up-regulated (e.g., lifr) genes were found (p < 0.05); involved in the same molecular and cellular functions enriched by detected mirnas. methods: evs were harvested from wild type and arrdc4-/-epididymal cells using differential ultracentrifugation, then characterised using nanoparticle tracking analysis and transmission electron microscopy. sperm motility was measured using computer assisted sperm analysis and imagej. fertilisation capacity was measured using the following assays: capacitation-associated tyrosine phosphorylation, calcium ionophore induced acrosome reaction, zona pellucida binding assay and in vitro fertilization with time-lapse imaging of embryo development. immunohistochemistry was also used to visualise two pronuclei formation and blastocyst morphology. arrdc4-/-sperm was supplemented with wild type evs in the above assays to assess whether they could restore function. results: sperm from arrdc4-/-mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as evidenced by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and production of two-cell embryos. we observed a significant reduction in ev production by arrdc4-/-epididymal epithelial cells, and addition of wild type evs to arrdc4-/-sperm dampens the acrosome reaction and restores zona pellucida binding. introduction: gestational diabetes (gdm) is among the most common pregnancy complications. despite treatment, up to 25% of pregnancies complicated by gdm result in infants being born large-for-gestational-age (lga). this not only causes problems at birth but predisposes offspring to developing cardio-metabolic disease in adulthood. there are no treatments for lga as the cause is unclear, although it is associated with altered placental vascular development. micrornas (mirnas) regulate placental development; they are produced within cells but can be released into the circulation inside evs, which in turn can be transported into target cells and tissues to influence cellular processes. we aimed to characterise circulating evs in pregnancies complicated by gdm-lga and determine if ev-derived mirnas have the potential to influence placental development. methods: maternal serum and plasma samples were collected from women with pregnancies complicated by gdm at 24-32 weeks gestation; placental tissue was collected at delivery and birth outcomes recorded. serum and plasma evs were isolated and characterised by electron microscopy (shape), nanoparticle tracking analysis (nta; size/concentration), and western blotting (ev-enriched proteins). mirna qpcr arrays were performed on evs. mirnas were quantified in placental tissue via qpcr. results: em and western blotting confirmed isolation of evs and nta revealed no significant difference in size/ concentration in gdm-lga pregnancies (n = 7) compared to gdm-aga (n = 13; p > 0.05). several ev mirnas were altered in maternal circulation in gdm-lga compared to gdm-aga (n = 7/group; >twofoldchange; p < 0.05), including four skeletal muscle-specific "myomirs": mir-1-3p, mir-133a-3p, mir-133b, and mir-499a-3p (all increased). all four myomirs were present in placenta but only mir-1-3p was significantly altered in gdm-lga compared to gdm-aga (n = 12-14/group; p < 0.05). summary/conclusion: ev-bound myomirs could have predictive value for aberrant foetal growth in cases of gdm. mir-1-3p regulates vascular development in other systems, so we propose that mir-1-3p contributes to lga by influencing placental vascular development, however further work is required to establish this. introduction: seminal plasma is particularly rich in extra cellular vesicles. myelinosomes are membranous organelles described throughout the seminiferous epithelium of the testis but never reported in semen. the aim of this study was to look for the presence of myelinosome vesicles in human seminal plasma. methods: because of the viscosity of seminal gel and its water-holding capacity, classical transmission electron microscopy does not seem to be an optimal technique to reveal the presence of myelinosomes in this fluid. cryo-electron microscopy is a technique that allows visualization of nanosized structures without prior fixation or addition of heavy metals for contrast. the sample is therefore visualized as close to its native state as possible. using standard myelinosome preparation from tm4 sertoli cells, we first analysed the appearance of "standard" native myelinosomes by cryo em and then compared it with the vesicles from human seminal plasma samples. results: we have specified by cry-em the morphological aspect of "standard" myelinosomes isolated from the culture media of tm4 sertoli cells. the vesicles with the same morphological appearance were revealed in human seminal plasma specimens. summary/conclusion: myelinosomes are membranous organelles found in the seminiferous epithelium of the testis and secreted by the somatic sertoli cells in the lumen of the seminiferous tubules.the preparations from human seminal plasma contains a population of large ev (average diameter 200 nm) whose morphological appearance resemble those of myelinosomes. defining the specific biomarkers and functionalities of myelinosomes in human seminal plasma are the concerns to be addressed in our further research. introduction: more than one million patients worldwide suffer from tuberous sclerosis complex (tsc) and have mutations in either tsc1 or tsc2 genes. together, the tsc proteins regulate mtorc1 activity. all tsc patient post-mortem samples exhibit renal disease and 40% of patients with tsc experience a premature loss of renal function. mouse and human studies are incongruity with the second somatic hit mechanism of disease, because of the low percentage of cystic cells exhibiting loss of tsc expression. we posited that the loss of a tsc protein expression may alter extracellular vesicle (ev) biology and contribute to disease. methods: we used crispr/cas9 to disrupt the tsc2 gene in mouse inner medullary collecting duct (mimcd) cells, and isolated evs using gel filtration from the isogenic cell lines. we characterized the evs using tunable resistive pulse sensing (trps), dynamic light scattering (dls), transition electron microscopy (tem), and wester blot analysis. we further performed mass spectroscopy on the ev proteins. results: loss of the tsc2 gene in mimcd cells induced a greater than three-fold increase in ev production compared to the same cells having an intact tsc axis. electron microscopy confirmed the purity and spherical shape of evs. both trps and dls demonstrated that the isolated evs possessed a heterogenous size distribution. approximately 90% of the evs were in the 100-250 nm size range. western blot analysis using proteins isolated from the evs revealed the cellular proteins alix and tsg101, the transmembrane proteins cd63, cd81 and cd9, and the primary cilia-related hedgehog signalling-related proteins arl13b. proteomic analysis of evs identified a significant difference between the tsc2-intact and tsc2-deleted cells that correlated well with the increased production. summary/conclusion: evs may be involved in tissue homoeostasis and cause disease by overproduction and altered protein content. the evs released by renal cyst epithelia in tsc complex may serve as a tool to discover the mechanism of tsc cystogenesis and in developing potential therapeutic strategies. introduction: we have shown that evs derived from amniotic fluid stem cells (afsc) of mouse origin present therapeutic effect in an animal model of chronic kidney disease, alport syndrome (as). in light of clinical translation, we isolated afsc-evs of human origin, characterized their cargo and evaluated thier therapeutic effect in vivo. methods: human clonal afsc were derived from amniotic fluid collected after volunteer donors provided consent. evs were obtained from afsc and identity and purity were assessed by rna-seq and proteomics. potency of hafsc-evs was evaluated by performing in vivo studies. ev biodistribution was evaluated by mri and therapeutic effect by measuring renal function and mice life-span. bulk rna-seq was performed on glomeruli obtained from injected and non-injected mice to identify potential ev regulating targets. results: proteomic profiling identified 675 intact proteins and rna-seq data identified 2,535 mirs in hafsc-evs. hafsc-ev "fingerprint" was assessed by performing go analysis on the 100 most highly expressed proteins and mirs. the results identified pathways involved in tissue homoeostasis such as mtor pathway, tgfβ and vegf pathways. when injected in vivo into as mice, biodistribution studies showed that hafsc-evs localized in the kidney, corrected proteinuria. no side effects (including teratoma) were noted in the treated mice. rna-seq of glomeruli obtained from treated as mice showed similar gene expression patterns to wilt type mice, by cluster analysis. our data indicated that hevs highly modulated pathways involved in collagen and matrix deposition remodelling, in addition to downstream targets of vegf, fgf, tnf, angiotensin and preserved glomerular cells structure and function. summary/conclusion: our protocol for hevs derivation is reproducible and allows derivation of ev lots with the same identity (specific cargo of proteins and mirs) and potency (present therapeutic effect in as). hafsc-evs modulated signalling pathways that are central to maintaining glomerular homoeostasis and preserved glomeruli structure with improved kidney function. this suggests the possibility of using hafsc-evs as a new therapeutic option for treating renal failure in humans. introduction: recent studies have shown that stem cell-derived extracellular vesicles (msc-ev) therapy improves renal outcomes in models of acute ad chronic renal disease. however, to better investigate the molecular mechanisms of ev-induced regeneration, and to define new ev sources, devices that mimic 3d organ architecture and flow conditions are needed. the aim of our work is to evaluate the regenerative potential of naïve and engineered ev in a millifluidic in vitro 3d model of glomerular damage in continuous perfusion. methods: methods: we set a millifluidic in vitro 3d model of glomerular filtration, a three-layers structure composed by human podocytes and glomerular endothelial cells, and, in between, of a basement membrane of collagen type iv. the barrier thus formed is set up inside a bioreactor, in a closed milli-fluidic circuit in which fluid flows continuously at a certain flow rate. we reproduced different pathological conditions and tested the localization and effect of evs in a dynamic system. : results: we obtained a standardized protocol and an adequate configuration of the milli-fluidic circuit subject to continuous reperfusion. renal damage was induced by doxorubicin or by hypoxia-reperfusion injury. we evaluated uptake, cargo transfer and effect of naïve and mirna engineered msc-evs or of klotho engineered ineffective evs administered into the dynamic co-culture system. evs were able to pass through the system and to deliver to podocytes proregenerative factors, promoting survival and limiting permeability. introduction: worldwide, renal cell carcinoma (rcc) is 8th most common cancer in men and 10th most common in women. new biomarkers are needed to aid rcc-diagnosis, provide prognostic information, and to predict response to modern targeted therapies. extracellular vesicles (evs) are an emerging source of cancer biomarkers because all cells, including cancer cells, secrete evs into biofluids as blood and urine. however, benign cells contribute to ev populations isolated from blood and urine reducing the diseasespecificity. we have developed a protocol for ev isolation directly from human rcc tissue that can increase tumour-specificity of biomarkers. methods: we obtained technical and biological replicates from normal kidney tissue and clear cell rcc tissue. serum-free media was incubated with the specimens. a combination of differential centrifugation, filtration, and ultracentrifugation was used for ev isolation. evs were quantitated using two methods, allowing for comparison between nanosight ns300 and nanofcm. tem was used to determine presence of intact vesicles in the ev samples. presence of ev introduction: urothelial carcinoma (uc) is a malignant cancer that affects the urothelial cells, representing 90% of all bladder tumours. at diagnosis 75% of bladder cancers are non-muscle invasive tumours. importantly, upon transurethral resection of the bladder tumour, nearly 35-80% of these patients will experience disease relapse and 10-20% will progress to muscle invasive tumour, requiring thereby, a rigorous and expensive follow-up. currently, this is performed through the frequent use of highly invasive cystoscopy and the low sensitivity urine cytology. thus, innovative liquid biopsy-based biomarkers that circumvent these drawbacks are highly desirable for improved uc clinical management. here, we aim to implement a protocol for the isolation and characterization of extracellular vesicles (evs) from uc patients' urine samples. methods: a two-step protocol involving ultracentrifugation (uct) and by size-exclusion chromatography (sec) was optimized for urine samples. the isolated urine-derived evs from 9 uc patients were then characterized according to their size, concentration (nta), morphology (tem), protein amount (lowry method), presence of ev-associated and disease-associated protein markers (western blot). results: isolated urinary evs from uc patients had a size ranging from 50nm to 400 nm with characteristic ev morphology, express ev-associated markers as cd63 and hsp70 and were negative for cell debris markers. the recovery yield and purity of isolated evs following each isolation technique was characterized. upon uct, sec was required to deplete most of the ev-associated thp and albumin protein contaminants. some disease-associated protein markers were highly enriched in isolated urinary evs compared to crude urine. summary/conclusion: taken together, these results indicate that a two-step ev isolation protocol was properly implemented and validated in uc patients' urine samples. notably, several ev-associated disease biomarkers were detected in the urine of uc patients. this ev-based liquid biopsy might provide the means for real-time monitoring of residual disease and relapse in uc patients. introduction: glioblastoma multiforme (gbm) is a very aggressive type of brain tumour. different gbm molecular subtypes (proneural, mesenchymal and classical) often co-coexist within the same tumour, with the mesenchymal subtype driving the tumour progression. recently, our lab demonstrated that the cargo of extracellular vesicles (evs) could mirror the molecular background of the gbm cells from which they were derived. altogether, we believe that gbm cell-derived evs can be directly involved in the expansion of the mesenchymal signature in tumours, thus supporting gbm aggressiveness. methods: non-mesenchymal (t98 & u138) gbm cells were "primed" using evs derived from mesenchymallike (u87 & ln18) gbm cells. ev-primed gbm cells were then co-cultured with their non-primed counterparts to determine whether the mesenchymal signature can "spread" from cell to cell via evs. effect on cell proliferation, migration and invasion (in hyaluronic acid hydrogels) was assessed following ev treatment and co-culture. the expression of mesenchymal gbm markers was measured by western blotting. further mass spectrometry analysis of cell and ev content was undertaken to describe potential underlying mechanisms. results: co-culture with ev-primed gbm cells significantly increased proliferation and hydrogel invasiveness of non-mesenchymal cells. interestingly, the stimulating effect of co-culture was even stronger on the proliferation of ev-primed gbm cells. moreover, further proteomic analysis revealed that expression of mesenchymal gbm markers such as cd44 was increased in non-mesenchymal cells following coculture. summary/conclusion: our data suggest that evs from mesenchymal gbm cells can be uptaken by gbm cells from different subtypes, thus stimulating tumour progression. overall, we think the present study provides with new insights for the understanding of gbm recurrence and the development of potential therapeutic strategies. introduction: triple-negative breast cancer (tnbc) is the most aggressive form of breast cancer. previously we reported that the heterogenous population of evs released from tnbc cells promotes the growth and aggression of recipient cells. here we investigated if, by using compounds proposed to inhibit ev release i.e. calpeptin and y27632 (to block those budding at cell membrane) and gw4869 and manumycin a (to block evs from mvbs), we could reduce the associated transmission of aggressive phenotype. methods: evs were separated from medium conditioned by tnbc cell line hs578ts(i)8, using a discontinuous optiprep density gradient, after the cells were treatment for 48 hrs with the compounds listed above. evs (pooled fractions 3-9 with a density range of 1.03-1.16 g/ml) were characterised by nta, bca, lipid assay, immunoblot, tem and flow cytometry. to investigate the functional effects of the evs released, proliferation and migration assays were performed on hs578t and mda-mb-468 cells using the ev to cell ratios of 1 × 105 evs/3x103 cells, 1 × 106 evs/3x103 cells, 1 × 107 evs/3x103 cells to evaluate doseresponse. ev-track id ev190109 (score of 88%). results: gw4869 significantly (p = 0.035) decreased ev release from hs578 ts(i)8 cells. manumycin a and a combination of calpeptin and y27632 (combo) decreased ev release, but significance was not reached. conversely, calpeptin and y27632 actually increased ev release; but not significantly. of the reduced numbers of evs released following gw4869 treatment, hla-dr+ evs were significantly (p = 0.032) enriched. none of the evs analysed significantly changed hs578 t or mda-mb-468 growth rates. however, evs from cells treated with calpeptin (p = 0.007), gw4869 (p = 0.025), manumycin a (p = 0.01) and combo (p = 0.001) caused significant reduction in mda-mb-468 migration compared to the effects of evs from untreated cells. similarly, ev from cells treated with gw4869 (p = 0.011), and combo (p = 0.018) caused significant reduction in hs578 t migration. summary/conclusion: while gw4869 was the only compound that caused a significant decrease in quantities of ev released, the evs that continued to be released following treatment with gw4869 or calpeptin and y27632 significantly reduced migration of both recipient cell lines. funding: phd funding: 1252 tcd scholarship and carrick therapeutics ltd extracellular vesicles from highly metastatic lung cancer cells induce barrier impairment, permeability, and epithelial-to-mesenchymal plasticity in a 16-day mature bronchial epithelium purdue university, west lafayette, usa introduction: epithelial-to-mesenchymal (emt) transition plays an integral role in cancer metastasis, which is responsible for as much as 90% of cancer mortality. cancer exosomes induce emt in bronchial epithelial cells, however, the epithelial cells inhibit emt when allowed to form a mature epithelial barrier with apicalbasal polarity. it is not known if cancer-derived extracellular vesicles (evs) can induce emt and more importantly, barrier disruption in a mature epithelium. here, we show that evs from a highly metastatic lung cancer cell line (calu6) are) are not only sufficient to induce emt in non-tumorigenic bronchail epithelial cells (beas-2b), but are also capable of disrupting a 16-day mature bronchial epithelial barrier by significantly reducing teer, inducing sixfold increase in permeability and complete loss of e-cadherin at cellcell tight junctions. methods: beas-2b and calu6 evs were characterized using electron microscopy, nanosight and western blotting for exosome-specific features. for permeability studies, beas-2b cells were cultured in transwell for 16 days to establish an intact epitheliumconfirmed by measuring teer (trans-epithelial electrical resistance). intact beas-2b monolayers were treated with calu6 evs at 1, 10 and 20 μg/ml for 24 hrs, and barrier intactness and permeability were evaluated by measuring teer, apical-basolateral translocation of dextran beads and confocal imaging of tight junctions (e-cadherin). for emt experiments, beas-2b cells treated with calu6 evs at 1 and 10 μg/ml were evaluated for ecadherin and vimentin levels by qrt-pcr and western blot after 48 hrs. results: beas-2b and calu6 evs were enriched in 50-200 nm size range, and cd9 and cd81 were enriched in the ev fraction in contrast to the cell lysate and vice versa for gp96. calu6 evs significantly impaired 16day mature beas-2b monolayer's barrier properties, which at the highest dose caused 33% reduction in teer from 27.0 ± 2.5 to 18.2 ± 3.2 ω.cm2 (n = 4). this was further confirmed by~sixfold increase in dextran beads' apical-basolateral translocation in 30 min (14.8 ± 7 ng/ml in control vs 87.3 ± 51 ng/ml in treated) (n = 3) and complete loss of e-cadherin expression at cell-cell tight junctions (n = 3). at the transcript level, calu6 evs induced significant downregulation of e-cadherin by 36% and upregulation of vimentin (mesenchymal marker) twofold (n = 3) in beas-2b cells, indicating transition into mesenchymal phenotype. summary/conclusion: we demonstrated the involvement of evs derived from highly metastatic lung cancer cells in inducing emt in bronchial epithelial cells and epithelial barrier disruptionthe initial stage of the intravasation process. grp78 plays a crucial role in the extracellular vesicle-promoted radioresistance of irradiated head and neck cancer cells introduction: small evs released from irradiated head and neck squamous cell carcinoma (hnscc) cells increase resistance of recipient hnscc cells to radiation in vitro. we have identified the glucose-regulated protein 78 (grp78), a chaperone protein of the hsp70 family which is involved in cellular stress responses and associated with worse survival in head and neck cancer patients, as an essential component of the ev-mediated radioresistance. methods: small evs were isolated from conditioned medium from irradiated and non-irradiated bhy hnscc cells by combined microfiltration (0.22 µm) and differential ultracentrifugation. grp78 surface expression was measured by proteomic analysis, immunoblotting and bead-facs. radiation resistance of bhy cells was determined by a clonogenic survival assay. results: increased grp78 was identified on the surface of evs from irradiated cells. the increase in ev grp78 correlated with increased grp78 expression at the donor cell surface. the grp78 content of recipient cells also increased upon transfer of evs from irradiated, but not non-irradiated cells, ultimately leading to enhanced cell survival. to check a potential role of elevated grp78 in radiation resistance we overexpressed grp78. here the modest (3x) overexpression of grp78 was sufficient to confer an enhanced radioresistant phenotype to the bhy cells. a correlation between grp78-dependent increase of radioresistance and activation of the akt pathway is yet to be determined. summary/conclusion: our results suggest a pivotal role for ev-transferred grp78 in modulating the radiation response of recipient hnscc cells. radiation directly increases the cellular and vesicular grp78 levels, and subsequent ev-mediated transfer leads to enhanced grp78 levels and radioresistance in recipient cells. this study provides new mechanistic insights into the effects of evs in radiation response and elucidates an interesting target protein and novel strategies for the improvement of radiotherapy. 3d modelling of ev release in progressing prostate cancer introduction: the modelling of cancer progression should be capable to translate acquired knowledge of cell behaviour to the real human body conditions. however, the extracellular vesicles (evs) isolated from 2d cell models are commonly exploited in research. taking into account the specificity of the prostate cancer (pc) environment, and a strong need of early diagnosis of castrate-resistance by prostate cancer (crpc) patients, we suggest in-depth profiling of different ev subtypes isolated from 3d culture as a new tool to model the progressing pc. methods: cells from hormone-resistant prostate carcinoma 22-rv1 line were cultured in 2d and 3d conditions, using 3d coseedistm. acd plasma controlled for haemolysis and remaining platelets was taken from patients with pc and crpc. the 40 fractions of 4 ev subtypes from cell culture and plasma were obtained by differential centrifugation (dc) followed by iodixanol density gradient purification. each of the fractions was measured by nanoparticle tracking analysis (nta), tunable resistive pulse sensing (trps) followed by elisa. for that, cd63 and cd9 were used as ev markers, apob and apoa1 for lipoprotein contaminants control, and cd3, cd41 and psma as tissue-specific biomarkers for determination of fractions containing evs of different origin. ev-contained fractions were subjected to next generation sequencing (ngs). results: in 3d conditions, the 22-rv1 cells produce up to 1000-times higher ev number than in 2d. size and density distribution of evs derived from 3d cultures but not of 2d resembled plasma evs. size distribution and biomarker expression among different ev subtypes allowed distinguishing between pc and cprcderived samples, indicating a potential to translate these results into clinics for early cprc detection. summary/conclusion: this work demonstrates a new approach to study the secretome of a progressing pc under 3d conditions. the profiles of ev subtypes produced by cancer cells growing in a 3d spatial architecture resemble the profiles of plasma evs and can serve a useful tool for the establishment of new biomarkers. introduction: renal cell carcinoma (rcc) is the most common primary renal neoplasm, with over 80,000 cases in the us alone each year. early detection of rcc leads to consistently better patient outcomes, and extracellular vesicles (evs) isolated from patient samples may prove to be a valuable clinical tool in the future. evs are abundant in blood and urine and show a large amount of heterogeneity but are difficult to analyse due to their small size and difficulty in isolation. here, we employ a multiparametric analysis of ev surface markers to identify a set of markers that may prove clinically relevant in future studies. methods: rcc cell lines vok111, vok130, and vok151 were cultured in flasks containing 500 ml of ev-depleted media (10% fbs, centrifuged 18 hr x 100,000 g). when cells reached~75% confluency, the conditioned media was collected and spun at 2,500 g for 10 mins two times to deplete any remaining debris, leaving~450 ml of media. this media was concentrated to a final volume of~7 ml using a pall jumbosep 100 kda mwco filter. this concentrate was purified from protein by using an izon qev-10 column, collecting 5 ml fractions. protein content of each fraction was analysed using a280 absorbance while concentration and diameter distribution were determined through nanoparticle tracking analysis (nta). pooled samples made of the three most concentrated fractions were concentrated to a final volume of~190 µl using the pall microsep 100 kda filter and then used for analysis in the miltenyi macsplex exosome kit. flow cytometric data were generated by the cytoflex s and analysed using flowjo and mpapass software. these positive signals were verified through bead-only controls and titrations. results: the mpapass software allowed for heatmap generation, data reduction, clustering and visualization of expression patterns. of the 11 detection antibodies used across 39 capture beads, cd276, cd26, cd82, beta-2 microglobulin, and cd151 were found to be prevalent in these rcc evs. these markers were found to be co-expressed particularly with cd63, cd81, and cd29. summary/conclusion: the use of multiplex analysis allowed for detection of five distinctive surface markers found to be prevalent in evs collected from rcc cell lines. these results demonstrate the utility of multiplex analysis and mpapass software for identifying potential markers of interest and provide proteins that are worth exploring further. the next steps to this work will be developing custom multiplex arrays that tailor capture and detection of evs specifically for rcc pathology. low molecular weight protein tyrosine phosphatase (lmwptp) carried by colorectal cancer cells-derived extracellular vesicles as a player in tumour-educated human fibroblast university of campinas -unicamp, campinas, brazil introduction: extracellular vesicles (evs) are doublemembrane-bound nanovesicles released by cells playing a key role as mediators of intercellular communication. low molecular weight protein tyrosine phosphatase (lmwptp) is upregulated in several cancers type, including colorectal cancer (crc), and it has been correlated with aggressiveness, chemoresistance and poor prognostic. methods: the aim of this study was to determine whether crc cells release lmwptp-enriched-evs and influence tumour microenvironment-associated cells as a representative tumour education. crc cells, hct116 and ht29, were cultured in serum-free medium for 24 hours. conditioned medium was concentrated by ultrafiltration (mwco 10 kda) and evs were isolated by total exosome isolation reagent (invitrogen). evs were characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and western blotting (wb). lmwptp levels were analysed by wb and sandwich-elisa. to evaluate tumour education, hff-1 fibroblasts were used as recipient cells. the uptake of evs (pkh26 fluorescently labelled evs), proliferation (viability) and migration (wound healing assay) were analysed in a co-culture model of crc-derived evs and hff-1. results: nta showed a higher concentration of evs released by ht29. hct116 and ht29 evs displayed a mean diameter around 140 nm and a cup-shaped morphology. isolated evs were positive for evs-markers cd81 and tsg101 and negative for gm130 a non-evs marker. ht29 lineage as well as derived-evs are lmwptp-enriched in comparison to hct116 cells and evs. upon incubation, fluorescently hct116 and ht29 derived evs were internalized into hff-1 cells in a perinuclear region. evs derived from both cells increased the viability and proliferation of hff-1 cells. intriguingly, evs derived from ht29 promoted cell migration. summary/conclusion: in conclusion, for the first time, we showed that lmwptp can be carried by evs derived from crc cells and lmwptp-enriched-evs can modulate biological aspects of hff-1 fibroblast. overall, our findings point lmwptp out as important player in tumour-educated fibroblast. exosomal mir-181a inhibition by vincristine and prednisone in paediatric acute lymphoblastic leukaemia. introduction: vincristine and prednisone are standard agents in treatment of paediatric acute lymphocytic leukaemia (p-all). mechanistically, vincristine induces apoptosis by blocking microtubules formation, while prednisone binds to cytoplasmic receptors and inhibits dna synthesis, both of which lead to apoptosis. the effect of these agents on exosomal micro-rna expression and its functional regulation is not yet investigated. elevated levels of mir-181a in circulating exosomes (nanoparticles) has been shown to lead to progression in several cancers, including all. we have previously shown that leukaemia-derived exosomes induce leukaemia cell proliferation via up-regulating of mir-181a expression and silencing of exosomal mir-181a reverses this exosomeinduced cell proliferating effect. the objective is to investigate the effect of vincristine and prednisone on exosomal mi-r181a expression in all. methods: jm1, sup-b15, and nalm-6 leukaemic cell lines were treated in vitro with vincristine (0.1 to 4.0 µm) and prednisone (0.1 to 12.0 µm) in exo-free medium and apoptosis was measured by mts assay. total rna of exposed cell lines was isolated and cdna was prepared for mir-181a analysis. expression of mir-181a was analysed by q-pcr. exosomes from conditioned medium of exposed cell lines were isolated by ultracentrifugation method. purity and particle size of exosomes were confirmed by western blot and nanoparticle tracking analysis (nta) assay respectively. total exosomal rna was isolated from exosomes (exo-rna) by trizol method. synthesis of cdna was carried out with the miscript ii rt kit (qiagen). results: vincristine and prednisone promote apoptosis in leukaemia cell lines (jm1 and sup-b15) in a dosedependent manner. both cellular and exosomal mir-181a expression was down-regulated by vincristine and prednisone exposure in all three leukaemia cell lines (jm1, sup-b15, and nalm-6). these observations demonstrate that cellular mir-181a down regulation in the parental cells is stable and can be transferred to exosomes, confirming the concept that exosomes are the fingerprint of parent cells. summary/conclusion: our data suggest that the vincristine and prednisone anti-proliferative effect in p-all maybe induced by another yet unexplored pathway, that suppresses mir-181a at a cellular and exosomal level in p-all, resulting in apoptosis. funding: this project is supported by the dimartino family foundation. secreted extracellular vesicles from renal cell carcinoma cells anatoliy samoylenko, artem zhyvolozhnyi, eslam abdelrady, naveed ahmad, genevieve bart and seppo vainio oulu university, oulu, finland introduction: clear cell renal cell carcinoma (ccrcc) represents the most common form of kidney cancer and is among the most lethal of all genitourinary cancers. despite surgery and medication therapy, most patients with metastatic ccrcc have a poor prognosis. intratumoural hypoxia is a key factor involved in renal cancer progression and it is known to promote secretion of evs by many types of tumour cells. methods: rcc-derived renca cells, embryonic kidney derived ub cells, and primary mouse hepatocytes were used in the study. evs were purified from cell culture media by gradient ultracentrifugation, sequential ultracentrifugation and exo-spin™ columns. before ev isolation cells were kept for 24 h either under normoxia or hypoxia (1% oxygen). evs were analysed by transmission electron microscopy with negative staining and immunolabeling, by nanoparticle tracking analysis (nta) and western blotting. cells proliferation and viability were assayed by live cell imaging using incucyte zoom (essen bioscience), cell metabolic activity by seahorse xf analyser (agilent), rna expression by qpcr and ddpcr. proteins were identified by ultra-performance liquid chromatography-mass spectrometry (uplc-ms). rna libraries were made using nebnext small rna library prep kit, and sequenced on nextseq550 (illumina). results: we showed that hypoxia induced production of evs by rcc cells, and characterized differences in protein and rna content of evs generated by renca cells cultured under normoxic and hypoxic conditions. we also showed that rcc-produced vesicles modify key features of tumorigenesis (gene expression, metabolic activity, motility, and growth) of target cells. these data were obtained by using two target cell types: model mouse kidney cells and primary mouse hepatocytes, which represent typical site of rcc metastasis with an exceptionally poor prognosis. we proposed that a possible mechanism of ev action in rcc is related to changes in caveolin-1 function. we also tracked renca-derived evs in a chick embryo model and in a novel kidney organoid co-culture assay developed by our group (xu et al., 2017) . summary/conclusion: hypoxia may influence tumorigenic properties of rcc by changing rates of production and composition of evs. funding: the study was supported by finnish cancer foundation grants. exosomes synthesizing her2 mirna and engineered to adhere to her2 on tumour cells surface exhibit enhanced anti-tumour activity introduction: exosomes are small extracellular vesicles averaging 100-150 nm in diameter. they serve as a means of intercellular communication. typically they consist of structural proteins as well as selected proteins, mirnas, mrnas, and long noncoding rnas. thus in an earlier report this laboratory designed a mirna targeting a major herpes simplex virus regulatory protein. as predicted by the nucleotide packaging signal the mirnas were packed in exosomes and on exposure to infected cells significantly reduced virus yields. her2 (human epidermal growth factor receptor 2) plays an important role in the neoplasia of some breast cancers. the protein is exhibited on the cell surface and is the target of therapeutic antibodies. methods: firstly, we report on the construction of a mirna targeting the synthesis of her2 both in cells constitutively expressing her2 and in cells transfected with a plasmid encoding her2. secondly, we report that the mirna targeting the synthesis of her2 reduced the viability of her2 positive cancer cells both in cell culture and in implanted tumours. lastly, we enhanced the anti-tumour activity of the exosomes by binding to the exosome surface a ligand with affinity for the her2 on the surface of tumour cells. the 293-mir-her2 exosomes package with mirna designed to block her2 synthesis and deliver to cells. these exosomes kill cancer cells dependent on her2 for survival but have no effect on cells lacking her2 or which were engineered to have her2 but do not depend on it for survival. the 293-mir-xs-her2 exosomes carry in addition a peptide which enables the exosome to adhere her2 on the surface of the cancer cells. in consequence, these exosomes preferentially enter and kill cells exhibiting her2 on their surface. the exosomes with 293-mir-xs-her2 are significantly more effective in shrinking the size of her2-positive tumours implanted in mice than the 293-mir-her2 exosomes. summary/conclusion: our studies indicate that exosomes carrying mirna against her2 have no effect on her2 negative cells it was nevertheless desirable to increase the uptake of exosomes carrying the her2 mirnas by her2-positive tumour cells. to this end we modified the exosomes to exhibit on their surface a peptide that bound the exosomes to the her2 on the surface of cancer cells. in consequence, we significantly enhanced the uptake of exosomes carrying the mirnas directed against her2 by her2 positive cells. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd2015071414385495, shenzhen science and innovation commission project grants jcyj20170411 094933148, jcyj20180306173333907 to shenzhen international institute for biomedical research. systematic characterization of ovarian cancer-derived exosomes unveil mirnas interfering with cd8 + t cell activation introduction: cd8+ tumour-infiltrating lymphocytes (til) have been widely reported to correlate with cancer patient survival, including ovarian cancer. even with the presence of tils, immunotherapy has limited success in ovarian cancer. understanding the interaction between cd8+ til and tumour cells is thus important. our hypothesis is that tumour-derived exosomes are released and taken up by cd8+ til such that specific mirnas contained within modulate physiological processes that inhibit cd8 + t cell activation. we aim to identify mirnas carried in tumour-derived exosomes that inhibit cd8 + t cell activation in ovarian cancer. methods: we purified exosomes from nine ovarian cancer cell lines and stocked in high concentration. interferon-gamma (ifn-gamma expression screening was performed after 3 days of co-incubation of tumour derived exosomes, cd8 + t cells, and activators in conditioned medium. cell counts and viability were tested by trypan blue staining at day 0 and day 3. rna-seq for exosomes were generated to identify mirnas critical in differentiation effects on cd8 + t cell activations. microrna target matching uncovered target mrnas while enriched pathway analysis predicted potential signalling pathways involved. results: our ifn-gamma screening results indicated the exosomes exhibit different behaviours in interfering cd8 + t cell activation owing to different donors. exosomes derived from peo.1 and ovca432 cells have consistent polarized results in ifn-gamma expression. exosomes derived from peo.1 remained a low ifn-gamma expression and from ovca432 stayed at relatively high level. small rnas profiling analysis between the two cell lines identified 56 mirnas (p < 0.05), and 13 mirnas have been reported with validated targeting information, and 10 out of 13 have targets involved in immune signalling. 210 mrna targets were uncovered by target matching. cmap search identified complex connections among mrnas with the top 20 enriched pathways actively involved in cell cycle and immune related behaviours. summary/conclusion: our ifn-gamma screening identified crucial mirnas in ovarian cancer exosomes interfering cd8 + t cell activation. computational modelling on both experimental and public multiomics datasets predicted promising signalling pathways of tumour-immune crosstalk for functional validation. irradiation of breast cancer cells alters the quality of dna cargo in the exosomes that they produce sheila spada, paul zumbo, doron betel, tuo zhang, nils-petter rudqvist and sandra demaria weill cornell medicine, new york, usa introduction: irradiation of breast cancer cells with an immunogenic dose (8gyx3) leads to accumulation of cytosolic dna that is sensed by cgas leading to interferon type i (ifn-i) signalling via cgas/sting pathway [1] [2] [3] . we previously showed that tumour-derived exosomes (tex) secreted by irradiated (8gyx3) (rt-tex) but not untreated (ut-tex) tsa carcinoma cells carry dna that stimulates the production of ifn-i in recipient dendritic cells (dc) via the cgas/ sting pathway [4] . moreover, mice vaccination using rt-tex, but not ut-tex, elicited anti-tumour immune response inhibiting tumour growth [4] . here, we hypothesized that the differential ability of rt-tex and ut-tex to activate ifn-i in recipient dcs is due to qualitative differences in dna cargo of rt-tex compare to ut-tex. methods: the length of dna purified from tex and from the cytosolic fraction of tsa cells was measured by agilent bioanalyzer. the dna cargo of tex was analysed by whole-genome sequencing (wgs) and whole-genome bisulphite sequencing. the percentage of methylation of total dna in tsa cells was quantified by 5-methyl cytosine dna elisa kit. results: dna fragments with size between 60 and 250 bp were enriched in rt-tex compared to ut-tex, as well as in the cytosolic fraction of irradiated compared to mock-treated tsa cells. wgs revealed that the entire genome was represented in tex dna cargo, regardless of rt. more than 99% of tex dna was of nuclear origin, but mitochondrial dna was increased in rt-tex. interestingly, we found that rt decreases the level of methylation in both exosomal and total dna in tsa cells compared to the controls. summary/conclusion: these data support the hypothesis that immunogenic rt alters some characteristics of the exosomal dna cargo, mirroring molecular changes occurring in parent irradiated breast cancer cells. the enrichment in dna fragments of 60-250 bp in rt-tex is intriguing considering that cgas is optimally activated by dna in this length range [5] . we are currently investigating which features of the cargo dna that differ between ut-tex and rt-tex may explain the differential ability to induce ifn-i pathway activation in recipient dcs. the identification of a dna signature associated with the ability of tex to activate the cgas/sting pathway could provide a circulating biomarker of the rt-driven immunogenic tumour response. introduction: triple negative breast cancer (tnbc) is among the most difficult cancer subtypes to treat and continues to cause a high number of cancer-related deaths annually. extracellular vesicles (evs) transfer cell type-specific cargo and have important implications in disease initiation, therapy and outcome. upon treatment of cancer cells with low-dose chemotherapy, released evs are able to transfer phenotypic traits to other cancer cells. new treatment strategies for tnbc, like inhibitors of the er stress pathway (ire1) might impact on ev biogenesis, cargo delivery and response of cells in the cancer microenvironment. our aim is to identify immune modulatory alterations in breast cancer cells and cancer derived evs upon treatment with inhibitors of the er stress pathway. methods: human tnbc cell lines were treated with ire1 inhibitor mkc8866 and cells were analysed for immune modulatory surface markers, like hla-i, b7-h molecules and different integrins. mitochondrial and lysosomal activities were investigated by the use of a mito-and lysotracker and analysed by imagestream (isx) technology. extracellular vesicles were isolated from cell culture supernatants by sequential centrifugation, quantified by nanoparticle tracking (nta) and characterized by exosome bead array. single ev analysis of total cell free supernatants and of isolated evs was performed by isx and marker positive evs were quantified for absolute fluorescence signals and total amount by objectives/ml. ev uptake into t cells was investigated by the use of different ev labelling strategies. results: several immune relevant surface markers (hla-i and cd54) are downmodulated by ire1 inhibition across different cell lines. cell surface expressed cd63 and b7-h3 show cell line specific downmodulation profiles upon ire1 inhibitor treatment. other immunomodulatory marker such as b7-h1 and b7-h4, integrin cd29, cell adhesion-promoting cd146 and stemness/metastasis marker (cd44 and ssea) are unaltered on ire1 treated breast cancer cells. cancer cell derived evs were tetraspanin positive (cd9, cd63, cd81), similar in number and showed differential expression of immune markers upon ire1 treatment. mitochondrial and lysosomal activities were unaltered under ire1 inhibition, whereas cell proliferation was diminished. no breast cancer-derived ev uptake of externally labelled evs into healthy t cells could be detected. summary/conclusion: ongoing analyses focus on the multicolour analysis of multiple markers on single evs by imaging flow cytometry and on the functional impact of cancer derived evs on t cells delivered by ev receptor binding. funding: dagmar quandt is supported by the sfi (cúram research centre, 13/rc/2073), the european regional development fund and the dr. werner jackstädt-stiftung. chair: uta erdbrügger -university of virginia chair: larry harshyne -thomas jefferson university comparison of three isolation protocols to search extracellular vesicles signature in sickle cell disease patients introduction: sickle cell disease (scd) is an inherited disorder characterized by chronic haemolysis and continuous activation of different cell types. extracellular vesicles (evs) were described to be at increased levels in scd patient's plasma compared to healthy subjects and were associated with several clinical manifestations such as leg ulcers and stroke. scd patient's plasma has increased concentrations of haem, free-hb and other proteins and lipoproteins as chronic haemolysis consequence. here, we report the comparison of three mostly used isolation protocols to search ev signature in scd patient's plasma by flow cytometry. methods: blood samples were obtained from scd patients (n = 3) following wisgrill et al., (2016) protocol. three different ev isolation protocols were used: differential centrifugation (dc), ultracentrifugation (uc) and size-exclusion chromatography (sec). lactadherin and calcein-am were used to detect phosphatidylserine (ps)+ vesicles and membrane integrity, respectively. platelet-derived evs (pevs), endothelialderived evs (eevs), leucocyte-derived evs (levs) and monocyte-derived evs (mevs) were quantified. silica beads were used to define evs gate and samples were acquired in the cytoflex cytometer platform. results: the quantification of pevs in uc, dc and sec samples was, respectively, 31x106, 8,5x106 and 9,7x106 events/ml mean, eevs was 6,4x106, 1 × 106 and 4,3x106 events/ml mean, levs was 2x106, 6 × 105 and 1,3x106events/ml mean and mevs 5,7x105, 6,5x105 and 3,7x105 events/ml mean. uc samples demonstrated a higher concentration of evs, which could be more useful to functional studies than dc and sec, however, it took more time to separate than dc. dc was the fastest method to separate evs from plasma, being useful to study large patients cohorts, but showed the smallest overall number of evs. sec also demonstrated high capability to detect evs in plasma and the possibility of obtaining a purer sample, although it is the most expensive and time-consuming method among all tested. all evs populations were detected in the three protocols tested. summary/conclusion: in summary, all protocols tested were efficiently to detect evs in scd patient's plasma and the definition of the best protocol may vary based on the research aim and time and budget available. funding: fapesp 2014/00984-3. gabrielle lapping-carr, joanna gemel, yifan mao and eric beyer university of chicago, chicago, usa introduction: aberrant cell-cell interactions involving the endothelium are central to the pathophysiology of sickle cell disease (scd), including acute chest syndrome (acs), a deadly and unpredictable complication. we previously demonstrated that the plasma of scd patients contains increased circulating small extracellular vesicles (evs) compared to controls and that those vesicles can disrupt endothelial integrity in vitro by affecting adherens junctions and ve-cadherin. the current study was designed to examine the effects of those evs on other cellular junctions including tight (zonula occludens 1, zo-1) and gap junctions (con-nexin43, cx43) and to test the hypothesis that the junctions would be more severely affected by evs isolated from patients during an episode of acs than by ones isolated from the same patient at baseline. methods: we identified subjects with scd in our biobank who had plasma isolated at baseline and at the beginning of an admission for acs. evs were isolated from platelet free plasma using established methodologies. to determine the effects on endothelium, cultures of human microvascular endothelial cells were treated with evs for 48 h and studied by immunofluorescence, immunoblotting and rt-qpcr. gap junction-mediated intercellular communication was assessed following microinjection of lucifer yellow and neurobiotin. results: the distribution and abundance of zo-1 at the plasma membrane were minimally affected by scd evs. while baseline evs did not affect the distribution of cx43, evs isolated during an episode of acs caused loss of cx43 from the plasma membrane. the integrated intensity of cx43 membrane staining was decreased bỹ 20% following treatment with acs evs. cx43 protein decreased on average by 32%, cx43 mrna levels by 21% and neurobiotin transfer by 67-94% in cells treated with acs evs, compared to baseline evs. summary/conclusion: circulating evs in scd affect multiple components of endothelial junctions. gap junctions composed of cx43 are the most sensitive of the cellcell junctions, since their abundance and function are reduced by acs evs even when the endothelial monolayer appears intact. cx43-mediated intercellular communication may be an early and sensitive event in the endothelial disturbance caused by evs in scd patients. funding: nih ul1 tr000430, comer hospital rbc race funds, ted mullin fund. the effects of platelet concentrate storage time on extracellular vesicle interactions associated with fibrin clot formation in-vitro jamie nash a , christine saunders b , amanda davies a and philip james a a cardiff metropolitan university, cardiff, uk; b welsh blood service, velindre university nhs trust, cardiff, uk introduction: platelet concentrates (pcs) have been utilised for decades to prevent bleeding in thrombocytopenic patients and to stop active bleeding. the storage of pcs however is a logistical challenge due to the limited 7 day shelf life under standard conditions. during storage, platelets undergo a number of mechanical and biochemical changes contributing to the short shelf life of a pc. these changes are collectively known as the platelet storage lesion. platelet extracellular vesicles (pevs) are known to increase throughout pc storage, due to an increase in platelet activation. as pevs have previously been shown to be pro-coagulant and increase in annexin v binding over pc storage. the aim was to investigate the effect of pc storage time on extracellular vesicle interactions on fibrin clot formation. methods: pcs were sampled on alternate days up to 10 days of storage and centrifuged to achieve acellular plasma. the plasma was subjected to ultracentrifugation (100,000xg) to pellet evs. the size and concentration of evs was assessed using nanoparticle tracking analysis software, followed by a western blot to confirm evs were of platelet origin. the pevs were added at a fixed number to a control pooled plasma sample with added thrombin and tissue plasminogen activator. the time to clot and 50% lysis time were recorded by using the turbidometry of the plasma over time. results: evs isolated from the pc were confirmed to be of platelet origin by western blot using cd41 as a marker of platelet origin and cd9 as an ev marker. pevs caused a significant increase effect on the fibrin clot formation (p < 0.001) when compared to the control plasma. pevs also had a significant effect (p < 0.01) on the fibrinolysis time, extending the time taken to lyse the clot. characterization of mirna from serum derived exosomes in a mouse tibia fracture model of introduction: complex regional pain syndrome (crps) is a debilitating chronic disease that occurs after trauma to the periphery and is intimately associated with nerve injury. its presentation is often described as an injury that is disproportional to the inciting event and manifests neuropathic pain, systemic inflammation, and immune dysregulation. owing in part to our poor understanding of disease aetiology, current treatments for crps are insufficient and as a disease of exclusion there is a lack of quantitative diagnostic markers. exosomes are small extracellular vesicles (sevs) 30-100 nm in size which provide a means of cellular communication through their cargo molecules (protein, mirna, mrna, lipids) , and have demonstrated promise in uncovering mechanisms of disease manifestation and identifying potential diagnostic markers. we have shown previously that crps patients have differential expression of several mirnas in serum derived sevs as compared to healthy controls, but little is known on how this compares to the established mouse tibia fracture model of crps. methods: mice undergoing fracture were anesthetized and subjected to a unilateral tibia fracture followed by casting of the injured limb. after confirming the establishment of pain hypersensitivity, serum samples were collected from fracture model and control mice three weeks post-injury. sevs were isolated by differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna-seq analysis is being performed to identify differentially expressed mirnas. results: nanoparticle tracking analysis showed no significant difference in the number or size of sevs present in the serum from the fracture model and control mice. rna-seq is ongoing and differential mirna expression in sevs from fracture model will be compared to control samples. comparative studies identifying mirnas that are common between crps patients and the rodent model will facilitate the development of correlational outcomes between preclinical and human studies. summary/conclusion: identification of similarities and differences between crps patients and animal models will aid in directing future studies at clinically relevant aspects of crps aetiology and identifying potential diagnostic markers for crps patients. extracellular vesicle-based liquid biopsy in acute myeloid leukaemia: a reliable source of residual disease biomarkers? introduction: acute myeloid leukaemia (aml) is an haematopoietic stem cell disorder with a poor 5-year survival rate. monitoring of measurable residual disease (mrd) in aml patients receiving chemotherapeutic treatment is useful to assess therapy response and predict relapse. indeed, many different leukaemia associated immunophenotypic protein markers (laips) are presently useful to detect mrd. nevertheless, their analysis currently requires invasive bone marrow aspirates, thus severely hindering real-time monitoring of the disease. therefore, alternative peripheral blood-based methods are highly desirable for an easy, real-time and costeffective monitoring of aml progression. this work aims was to assess the feasibility of a peripheral blood ev-based liquid biopsy method for aml disease monitoring, based on the detection of laips with a known negative impact on the prognosis of aml. methods: the profile of evs isolated from 12 paired samples from aml patients' blood plasma collected at diagnosis, complete remission (and some at relapse) was compared and correlated with clinical data. for that, a size-exclusion chromatography (sec) method was optimized to isolate the circulating evs from the blood plasma. the evs of the 12 paired aml patients' blood samples were then characterized according to their size (dls/nta), morphology (tem), proteinto-lipid ratio (lowry/sulpho phosphovanillin assay), surface charge (zeta-sizer) and protein cargo (western blot). results: sec allowed the isolation of size-resolved plasmaderived evs from the peripheral blood of aml patients. isolated evs had a size ranging from 30 nm to 300 nm with an intact morphology, expressing ev-associated markers such as hsp70, cd63, cd81 and cd9. size-resolved evs also had a differential expression of mitofilin, actinin-4, syntenin-1 and annexin-xi proteins. several laips were detected in the isolated evs and their relative abundance changed throughout the stage of the disease. summary/conclusion: our preliminary data shows that aml patients' circulating evs carry relevant immunophenotypic protein markers, which might predict aml clinical outcome. introduction: cell plasticity regulated by the balance between the epithelial-to-mesenchymal transition (emt) and met is critical in the metastatic cascade. extracellular vesicles (evs) may play an important role in this balance by shuttling molecular cargos into recipient cells. this study aims to evaluate the feasibility of profiling mrnas of parental prostate cancer (pca) cells with different phenotypes and their daughter evs using the nanostring low rna input ncounter assay. methods: pc3-epi and pc3-emt cell lines representing epithelial and mesenchymal phenotype, respectively, were generated from original pc3 cell line. the cell culture supernatant was first pre-cleared for any dead cells and debris by centrifugation at 1000 × g for 20 min. without disturbing the pellet, the supernatant was then transferred to a fresh ultracentrifuge tube and centrifuged at 10,000 × g for 20 min at 4°c. the remaining supernatant was then centrifuged to isolate the evs at 100,000 × g for 120 min at 4°c. the evs pellet was further washed in 1× pbs followed by a second centrifugation at 100,000 × g for 120 min at 4°c. the final evs pellet was resuspended in 1× pbs for subsequent characterization (transmission electron microscopy, nanoparticle tracking analysis and western blot) and ncounter assays. the total rna of cells and their daughter evs were assayed by the ncounter pancancer progression panel to determine expression of 770 selected mrnas. the nanostring ncounter low rna input kit with the multiplex 770gene primer pool was used for the pre-amplification of mrna and overnight hybridization with the pancancer progression panel. each sample type was submitted to the assay in biological triplicate. results: when comparing all 12 samples, eisen cluster analysis separated all the cells and all evs into two groups, regardless of their phenotypes. in subgroup analysis, the expression patterns between pc3-epi and pc3-emt cells were significantly different. clec2b, kdr, crip2, il13ra2, cc2d1b were significantly upregulated in pc3-emt cells, while cxcl8, epcam, esrp1, tgfb2, cdh1, s100a14, ovol2 were significantly downregulated in pc3-emt cells. the expression patterns between pc3-epi and pc3-emt evs were also significantly different. tbx1, cav1, col4a1, slc35a3, myc, itgb2, timp4, camk2b, ptgds, p3h2, itgb6, vim, stat3 were all significantly downregulated in pc3-emt cell derived evs. summary/conclusion: the nanostring low rna input ncounter assay can provide reliable mrna expression profiling of evs. the mrna expression patterns are very different between cells and their daughter evs. both cells and evs with different phenotypes have different gene expressions. cancer cell-derived evs containing alphav beta6 integrin regulate cd163, il-6 and il-10 levels in peripheral blood mononuclear cells introduction: extracellular vesicles (evs) mediate communication in the tumour microenvironment and play an important role in cancer progression. previously, we have shown the enrichment of alphav beta6 integrin in small extracellular vesicles (sevs) isolated by differential ultracentrifugation and iodixanol density gradient from pc3 prostate cancer cells. we have also shown in the past that alphav beta6-positive sevs induce peripheral blood mononuclear cell (pbmc) polarization by increasing the expression of pro-tumorigenic m2 markers, such as cd163 and cd204. finally, we have demonstrated that down-regulation of alphav beta6 integrin up-regulates the stat1-interferon stimulated genes (isgs) pathway in cancer cells and in sevs released by them. methods: in order to investigate whether prostate cancer cell-derived vesicular stat1 has a causal effect in pbmc polarization, we down-regulated alphav beta6 and stat1 in prostate cancer cells derived sevs using sirna as well as crispr-cas9 strategies. the sevs isolated from these cells were used to analyse m2 polarization by measuring the levels of cd163 in pbmc. the results show that sevs lacking alphav beta6 inhibit cd163 levels in pbmc in a stat1-independent manner. analysis of cytokines released by pbmc upon incubation with sevs lacking alphav beta6, show that pbmc selectively up-regulate the levels of il-10 and il-6, which are predominantly anti-tumorigenic cytokines. in contrast, sevs lacking alphav beta6 do not upregulate pro-angiogenic cytokines, such as vegf. summary/conclusion: these findings suggest that cancer cell-derived sevs containing alphav beta6 integrin promote a pro-tumorigenic pbmc phenotype in the tumour microenvironment by regulating cd163, il-6 and il-10 levels. introduction: the recognition of donor-mhc molecules by recipient t cells triggers the immune response leading to rejection of allografts. our recent studies have documented the presence of high numbers of recipient apcs displaying donor-mhc molecules (cross-dressed) on their surface in the lymphoid organs of mice after skin, heart or pancreatic islet transplantation. in addition, we have reported that acquisition of allogeneic mhc molecules by host apcs (mhc crossdressing) is mediated by donor-derived extracellular vesicles (evs) trafficking through blood and lymphatic vessels (marino et al. science immunology, 2016) . in the present study, we investigated the ability of allogeneic evs and allo-mhc-cross-dressed cells to initiate a t cell alloresponse in vitro and in vivo. methods: evs were isolated (using differential centrifugation) from balb/c bone marrow derived dendritic cells (bmdcs). these evs were used to cross-dress b6 splenocytes in vitro. the transfer of donor mhc class i and ii on b6 cells was analysed by imaging flow cytometry. next, t cells from b6 mice were cultured in vitro with either allogeneic bmdc-derived balb/c evs or b6 spleen cells crossdressed with allogeneic balb/c mhc. alternatively, 2 × 109 balb/c or b6 bm derived evs or 20 × 106 balb/c bm cells were injected iv to b6 mice. in both cases, the t cell response was assessed by activation markers detection, infg production and cell proliferation. results: apcs cross-dressed with allogeneic mhc molecules can trigger a pro-inflammatory direct alloresponse by t cells in vitro and in vivo. on the other hand, allogeneic evs alone were only able to induce early t cell activation but not proliferation in vitro. furthermore, injection of mice with allogeneic evs alone could induce some but suboptimal alloresponse in vivo and only when administered with complete freund's adjuvant. summary/conclusion: blocking donor evs release and subsequent recipient apc cross-dressing may represent a promising target to selectively inhibit anti-donor t cell inflammatory responses thus achieving long-term allograft survival. funding: r01dk115618. antifungal antibiotic activity of outer membrane vesicles from adherent lysobacter enzymogenes c3 against therapeutic and biocontrol targets. rutgers university, new brunswick, usa introduction: lysobacter enzymogenes is a predatory gram negative bacterial species being studied for biocontrol activity against fungi. planktonic l. enzymogenes c3 produces outer membrane vesicles (omv) harbouring small molecule antifungal antibiotics (meers et al. 2018) . we show here that the more biologically relevant surface-associated c3 exerts remote antifungal activity via omv as well. the results have important consequences regarding the natural mechanism of biocontrol of fungal pathogens by c3 as well as isolation and delivery of therapeutically relevant antifungal compounds. methods: omv were isolated from scraped adherent c3 culture on agar by similar methods to meers et al 2018. omv were stained in some cases with fluorogenic syto 9 dna stain for microscopic observation. fungal growth was monitored via turbidity readings in liquid culture or photomicrographs on agar. c3 was also grown on polycarbonate filter membranes with defined pore sizes to monitor growth of fungal cells on the opposite side. vesicles were also labelled with an amine-reactive probe alexa-555 and washed 4x by sedimentation. binding of labelled omv to fungal cells was observed by epifluorescence microscopy. results: syto 9-stained vesicles from surface-adherent c3 were similar to previously observed~130 nm vesicles (meers et al., 2018) . the isolated vesicles inhibited growth of saccharomyces cerevisiae or candida albicans in liquid cultures at similar potency and were active against the filamentous species fusarium subglutinans grown on agar or maize leaves. c3 cultures grown on filters with 400 nm pore size but not 30 nm were able to inhibit the hyphal growth of f. subglutinans on the opposite side. similarly c3 on filters with a 200 nm pore size were able to inhibit growth of c. albicans. observation of fluorescently-labelled c3 omv after interaction with c. albicans showed binding specifically to hyphae or pseudohyphae and for f. subglutinans to the growing hyphal tips. summary/conclusion: the omv of c3 specifically bind and inhibit the growth of fungal hyphae of various species without direct c3 cell contact. these data elucidate mechanisms of biocontrol and suggest strategies for production of therapeutic antifungal antibiotics. meers et al. elucidating the cellular uptake and tissue distribution mechanism of cell derived vesicles, a novel therapeutic carrier hui-chong lau a , jae young kim a , jin-hee park a , jun-sik yoon a , min jung kang a and seung wook oh b a mdimune inc, seoul, republic of korea; b mdimune inc, seattle, usa introduction: cell derived vesicles (cdvs) are emerging as a novel therapeutic carrier. one of the crucial factors in the development and therapeutic applications of cdvs is to understand the precise mechanism by which vesicles find and enter the target cells. in this study, we aim to investigate the uptake mechanism of cdvs produced from natural killer (nk) cells using a manufacturing process established at mdimune inc. both in vitro uptake assay and in vivo distribution analysis were performed to provide precise insights into how cdv exert its effect at the cellular level. methods: nk cells were mainly used to produce cdvs. breast cancer cells, bt549, and human and rodent endothelial cells, with a varying degree of icam-1 expression, were used to determine the effect of lfa-1 expressed on the surface of nk-cdvs in cellular uptake using facs and confocal imaging analysis. next, various inhibitors for uptake pathways, such as phagocytosis, dynamin dependent endocytosis, and receptor mediated endocytosis, were used to understand the underlying mechanism of cellular uptake of cdvs. biodistribution profile of cdvs were characterized using both normal and tumour xenograft models by ivis imaging. results: using a recently established manufacturing process, we demonstrate that nk-cdvs can efficiently enter the target cells. this study also shows that the cellular uptake depends on the molecular interaction between icam-1 and lfa-1. in vivo distribution profile of nk-cdvs are also assessed using various tumour models. furthermore, we present a cellular uptake mechanism involved in the entrance of cdvs into the target cells. summary/conclusion: this study demonstrates that the cdvs produced at the manufacturing scale can be easily taken up by cells via specific cellular pathways. this finding will facilitate the development of more efficient therapeutics for cancer and other debilitating diseases. myofibroblasts-derived microvesicles increase dermal fibroblasts collagen production through plgf-1 syrine arif, sebastien larochelle and véronique j. moulin chu de québec -université laval, loex, québec, canada introduction: a proper wound healing of the skin involves angiogenesis, extracellular matrix (ecm) remodelling and re-epithelialization. these three mechanisms require well-organized interactions between different cell populations. a key role in this context is played by myofibroblasts (wmyo), a cell population mainly differentiated from dermal fibroblasts. these cells contract wound edges and synthesize new ecm. we previously showed that myofibroblasts predominantly produces microvesicles (mvs) and can favour angiogenesis. however, proteomic analysis of mvs from our previous studies indicated some molecules that can potentially be implicated in ecm remodelling. in this study, we evaluated whether myofibroblasts-derived mvs could affect dermal fibroblasts who are highly responsible for ecm regulation. methods: mvs were isolated by differential centrifugation of medium collected from wmyo cells. number and size of mvs were characterized by transmission electron microscopy and nanosizer. multiplex assays of 45 cytokines were evaluated in mvs samples, wmyo and mvs-depleted medium. to examine the interaction of mvs with fibroblasts, we evaluated the uptake of mvs isolated from wmyo transduced with a fluorescent protein. we then treated fibroblasts cultures with mvs or a selected cytokine for 5 days and evaluated collagen production. lastly, we neutralized the selected cytokine in mvs samples before evaluating collagen production. results: plgf-1 was the cytokine detected in mvs samples in large amount (0.88 ± 0.63 pg/µg proteins in mvs). fibroblasts treated with mvs or plgf-1 significantly stimulated pro-collagen i level production with a fold change of 1.80 ± 0.18 and 2.07 ± 0.18. moreover, the neutralization of plgf-1 present in mvs significantly inhibited the production of pro-collagen i by dermal fibroblasts. summary/conclusion: our results indicated that mvs influence fibroblasts pro-collagen production through plgf-1 signalling. funding: this work was supported by natural sciences and engineering research council of canada (nserc) (rgpin2014-04404); les fonds de recherche du québec-santé (frqs) via the research centre funding grant; the quebec cell and tissue and gene therapy network-thécell (a thematic network supported by frqs). structural insights on fusion mechanisms of extracellular vesicles with model plasma membranes introduction: extracellular vesicles (evs) represent a potent intercellular communication system. while their functional biological properties are more and more investigated, the biophysical aspects of their interaction with recipient cells are often overlooked. small size (30 to a few hundred nanometres in diameter) of evs and their heterogeneous origin still pose a great challenge for their isolation, quantification and biophysical/biochemical characterization. in particular the complex network of interactions between differently classified evs and recipient cells remains to be further revealed. here we deeply investigate the fusion mechanism between evs and a model plasma membrane system by an interplay of different structural/morphological techniques to get a molecular description of the interaction helping to clarify the role of different membrane compartments on the evs uptake mechanism. standardized protocols and good manufacturing practice conditions were employed to derive highly stable vesicles of defined size and reproducible molecular profiles from umbilical cord multipotent mesenchymal stem (stromal) cells. after a thorough biophysical and biochemical characterization of evs non-contact liquid imaging atomic force microscopy (afm) and, in parallel, neutron reflectometry (nr), as well as small angle neutron scattering (sans) experiments were performed on evs to determine their interaction with model plasma membranes in the form both of supported lipid bilayers and suspended unilamellar vesicles of variably complex composition. results: we observed that evs tend to fuse with the model membranes with a preferential interaction with the external layer of the fluid membrane. moreover we revealed a stronger interaction with the liquid ordered domains, strengthening the hypothesis of a critical role of lipid rafts in fusion mechanisms. summary/conclusion: our results on the analysis of the interaction of evs with artificial lipid membranes could provide insights on the internalization mechanisms of evs. the approach shown here can be further extended to convey incremental complexity, adding glycolipid and membrane proteins to the model lipid bilayers. this approach combined with data on the specific biological function of each ev subpopulation as retrieved by standard functional assays, will turn useful to select the crucial molecular aspects of evs internalization by cells. introduction: platelet-derived extracellular vesicles (pev) are the most abundant circulating extracellular vesicle (ev) and exhibit platelet-like properties, hence the original term "platelet dust". direct phenotyping of ev surface markers within biofluids is challenging often requiring time-intensive purification steps that can significantly alter resultant ev population characteristics. the exoview™ (nanoview biosciences) specifically captures ev sub-populations and was used to characterise the ev content of platelet free plasma (pfp) and a potential novel haemostatic agent designed for the treatment of severe trauma and haemorrhage, platelet enhanced plasma (pep). methods: freeze-thaw cycling of platelet rich plasma/ expired platelet concentrates was followed by centrifugation to remove platelet remnants and yeilded pep. pfp controls were prepared by double centrifugation (2000 g for 20 minuntes followed by 13,000 g for 2 minutes). rotational thromboelastometry (rotem) and calibrated automated thrombography (cat) were used to assess ev driven haemostasis and thrombin generation. a dilutional and hypothermic model of coagulopathy was designed to assess pep. ev capture arrays comprised of anti-cd41, anti-cd63, anti-cd81 and anti-cd9 were used (exoview™, nanoview biosciences). captured vesicles underewent interferometric imaging and were quantified, sized and further probed with fluorescent tetraspanin markers, annexin-v and intravesicular markers. results: pep is highly procoagulant, exhibits enhanced thrombin generation and can restore haemostasis in a dilutional model of coagulopatic whole blood. pep can be generated from expired platelet concentrates, potentially allowing for upscalable production. the predominant vesicle population were pev with a large cd41/cd9 population that contained a smaller subpopulation of phosphatidyserine positive procoagulant vesicles. pfp as expected has a much lower number of pev and a cd81 positive ev population. summary/conclusion: pep is a unique resuscitation fluid containing high pev levels for the potential treatment of severe trauma and haemorrhage. exoview measurements can be performed in unpurified plasma and may be useful for measuring circulating ev in health and disease. funding: defence and security accelerator, dstl therapeutic effect of exosomes in mice model of autism daniel offen a , reut horev a , nisim perets a , ehud marom b , uri danon b and yona gefen b a tel aviv university, tel aviv, israel; b stem cell medicine ltd., jerusalem, israel introduction: during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. we have previously shown that intranasal administration of msc-exo, cross the bbb and significantly ameliorate autistic-like behavioural phenotype in btbr and shank3 animal models of autism, representing a potential therapeutic strategy to reduce symptoms of autism spectrum disorder (asd). our objective is to study the mechanism of action and the cellular pathways in which the msc-exo activate their target, we performed rna sequencing analysis of primary neurons isolated from shank3 mice treated with msc-exo. methods: primary neuronal cell cultures were prepared from newborn shank3 homozygotes mice model of autism. cultures were treated with msc-exo (10^7 particles/ul), isolated from human adipocytes, followed by rna sequencing. the alterations in gene expression between the treated and intact neurons were analysed for gene ontology and pathways and were also compared to proteomics analysis of the msc-exo in order to find regulatory proteins that may lead to these differences. results: bioinformatic analysis revealed several up-regulators proteins that might be responsible for the increase in anti-inflammatory and protective factors seen in the mice neurons treated with msc-exo. one of them is bdnf which is known as an essential growth factor responsible for neuroprotection and neurogenesis. importantly, no difference in the genetic expression of cancer-related genes was identified following msc-exo treatment indicating for their safety. summary/conclusion: our data suggest that adipocytederived msc-exo carry therapeutic potential in asd via alternation in gene-expression related mainly to immuno-modulation, reduce neuroinflammation and increase neuroprotection and neurogenesis. the beneficial effects of the exosomes treatment in mice models is being translated into a novel, easy to administer, a therapeutic strategy to reduce the symptoms of asd. introduction: autologous blood-derived products gain increasing focus in regenerative medicine, especially in orthopaedics and osteoarthritis therapy. this disease is characterised by cartilage degradation and inflammation among other symptoms, which are targeted by conventional therapies, but genuine cartilage regeneration is rarely achieved. citrate-anticoagulated platelet rich plasma (cprp) is often clinically applied to stimulate soft and hard tissue healing. recently, cell-free alternatives to cprp including hyperacute serum (hypact™ serum) have been developed. cprp and hypact™ serum contain specific profiles of growth factors, however, they also contain extracellular vesicles (evs) that harbour signal molecules including mirna. methods: evs were enriched by ultracentrifugation (uc) followed by size exclusion chromatography (sec) to obtain purified evs. particle size and concentration of each fraction was measured by nanoparticle tracking analysis (nta). fractions with the highest amount of particles were pooled and concentrated via uc, before mirna expression was assessed via screening with a panel of 372 mirna-specific primer pairs by rt-qpcr. presence of evs was confirmed by cryoelectron microscopy. results: the ev concentration tended to be lower in hypact™ serum than in cprp as determined via nta. similarly, lower diversity of mirna species was found in hypact™ serum than cprp evs. around 90% of detected mirnas were found in both blood products, whereas only 30% of mirnas were shared between evs from cprp and hypact™ serum. while mirnas such as mir-101 were consistently depleted in evs compared to the corresponding blood product, others like mir-27a were in enriched in hypact™ evs, but not cprp evs, indicating release of specific mirnas via evs in response to clotting. summary/conclusion: although the purification resulted in high loss of evs, we identified specific mirnas enriched in evs from cprp and hypact™ serum. their functional spectrum with respect to osteoarthritis therapy focuses on inhibition of inflammation, inhibition of tissue remodelling via matrix degrading enzymes as well as preventing senescence. this renders blood product derived evs as interesting candidates for in vitro and in vivo testing with respect to cartilage regeneration. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst3-f-5030664/003-2017. protective role of shiitake mushroom-derived exosome-like nanoparticles in d-galactosamine and lipopolysaccharide-induced acute liver injury in mice baolong liu, xingyi chen and jiujiu yu university of nebraska lincoln, lincoln, usa introduction: fulminant hepatic failure (fhf) is a rare, life-threatening liver disease with poor prognosis. new therapeutic interventions are urgently needed to treat this disease. administration of d-galactosamine (galn) and a low dose of lipopolysaccharide (lps) triggers acute liver damage in mice, which simulates many clinical features of fhf in humans and therefore is widely used to investigate the molecular mechanisms and potential therapeutic interventions of fhf. recently, suppression of the nucleotide binding domain and leucine rich repeat related (nlr) family, pyrin domain containing 3 (nlrp3) inflammasome was shown to alleviate the severity of lps/galn-induced liver injury in animal models. therefore, the goal of this study was to identify food-derived exosome-like nanoparticles (elns) with anti-nlrp3 inflammasome function to potentially control fhf. methods: seven commonly consumed mushrooms were used to extract elns, which were examined for anti-nlrp3 inflammasome activities in primary macrophages. results: it was found that these mushrooms contained elns composed of biomolecules including rnas, proteins, and lipids. among these mushroom-derived elns, only shiitake mushroom-derived elns (s-elns) strongly inhibited nlrp3 inflammasome activation by blocking the inflammasome assembly. this inhibitory effect was specific for the nlrp3 inflammasome because s-elns had no impact on activation of the absent in melanoma 2 (aim2) inflammasome. s-elns also inhibited the secretion of interleukin (il)-6 and both protein and mrna levels of the il1b gene in macrophages. remarkably, pre-treatment of s-elns protected mice from lps/galn-induced acute liver injury. summary/conclusion: therefore, s-elns, identified as potent inhibitors of the nlrp3 inflammasome, represent a new class of agents with the potential to combat fhf. approaches to assess clinically available exosomes' quality and safety introduction: recent adverse events resultant from an exosome product use in a nebraska clinic, highlight the importance of assuring product quality and safety standards. an often-overlooked safety risk is ancillary reagents remaining within a finished product. when processes to obtain exosomes utilize cow proteins such as fbs or bovine sera albumin, failure to adequately remove these can result in significant adverse allergic reactions. we evaluated 3 different exosome products to test the hypothesis that purity of some products may not be consistent with actual product quality and safety profiles claimed. methods: three different exosome products (manufacturer a, b, and c) were prepared per their instructions for use. sample source identity was blinded from assaying scientists. an independent cro service was used to conduct the experiments to ensure unbiased assay execution and data collection. exosome suspensions were sampled undiluted for bovine protein content using commercially available bovine secretome protein arrays from ray biotech. a total of 30 different proteins found in bovine serum were quantified. results: six of 30 proteins were not detected in any sample. 8 of 24 array antibodies were found to cross react with human antigens. of the 16 bovine proteins that were acceptable for analysis, manufacturers a, b, and c exosomes contained 15 of 16 proteins,13 of 16 proteins, and 0 of 16 proteins, respectively. concentrations of individual bovine proteins ranged from 0.2 to 1,220.1 ng/ml. summary/conclusion: these results indicate manufactures a and b are selling potentially dangerous products. the successful implementation of exosome products into the clinic requires equivalent demonstrations of safety and quality. this requires adopting strict quality standards and safety testing during their production. physicians must require safety data prior to clinical use. engineering pro-healing ev cargo using a closed-system bioreactor. introduction: chronic wounds, including diabetic ulcers and pressure ulcers, are difficult and expensive to treat. while tissue engineering approaches have largely failed as a viable treatment for chronic wounds, we hypothesize that stem cell-derived extracellular vesicles (evs) may provide several unique advantages. zenbio, inc has developed a methodology to generate commercial-scale stem cell-derived exosomes using a closedsystem hollow fibre bioreactor capable of continuous ev production. additionally, we have shown that by manipulating the cellular environment, we can improve the pro-healing capacity of the evs.this technology leverages the complex healing capabilities of stem cells without the obstacles of replicating cells. methods: we have demonstrated that a mild heat shock resulted in evs enriched for stress-response proteins and increased pro-healing activities in vitro. we extended this innovative approach to include stimulating adipose stem cells with combinations of heat shock and growth factors to generate differential extracellular vesicle packaging that enhances pro-healing activity. to monitor reproducibility across lots and batches, we rigorously characterized tuned evs for particle size and number as well as surface marker and cargo composition. results: our results using tuned evs showed efficacy using cellular models of inflammation, motility, vascularization, collagen production and metalloprotease activity. we utilized an established murine model of pressure ulcers to assess the in vivo efficacy of the tuned evs. these studies showed a single injection into the wound site activated a more rapid wound closure, increased collagen deposition and reduced dermal thickness compared to saline control. summary/conclusion: these data strongly support our hypothesis that evs may be selectively modified to improve their wound healing activity by modulating the culture or tissue microenvironment. future studies will use chronic wound models to determine optimal dosing and routes of administration. introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) can reduce inflammation, promote healing and improve organ function thereby providing a potential "cell-free" therapy. prior to clinical translation, there is a critical need to synthesize existing preclinical evidence supporting their efficacy. this systematic review provides the most comprehensive evidence map of methods, safety and efficacy for msc-ev research to date. methods: medline and embase were systematically searched for in vivo interventional studies using msc-evs. two reviewers extracted data for: 1) methodology, 2) study design, 3) intervention details and 4) efficacy/ adverse events. results: after screening 754 articles, 206 studies met our eligibility criteria. msc-evs were used to treat a variety of diseases including renal (16%), neurological (12%) and cardiac (10%) conditions. benefits were described in 95% of studies across all organ systems and adverse effects were seen in only three studies; two showing tumour growth. however, several key methodological concerns were evident. based on size criteria for ev subtypes (exosomes/small evs~30-150 nm, microvesicles~150-1000 nm) only 60% of studies used appropriate nomenclature. ultracentrifugation (70%) and isolation kits (23%) were the most common isolation methods despite marked differences in yield and purity. evs were inconsistently dosed by protein (68%), particle number (16%) or cell count (4%), hindering inter-study comparisons. two-thirds of studies used xenogeneic evs suggesting immunocompatibility. techniques to determine size, protein markers and morphology was highly heterogeneous, and only 12 and 4 studies met the characterization standards recommended in the misev 2014 and 2018 guidelines, respectively. finally, 50% of studies did not incorporate randomization which represents a high risk for bias and only a quarter performed biodistribution studies. summary/conclusion: this systematic review reveals extensive heterogeneity in methods and intervention details for animal studies of msc-evs. nonetheless, nearly all studies showed significant benefits in a wide range of distinct conditions. the knowledge gaps we identified highlight important opportunities for improving preclinical design and the need for more standardized approaches in this growing field of ev therapeutics. msc-exosomes as next generation therapeutics for atopic dermatitis exocobio inc, seoul, republic of korea introduction: atopic dermatitis (ad) is a systemic inflammatory disease with unknown cause. recent approval of a targeted therapy, dupilumab, opens new era of ad management. however, current therapeutic options for ad are only targeting inflammation, a component of ad vicious cycle including itching and barrier disruption. human mesenchymal stem cells (mscs) have been highlighted as a novel therapy for suppressing allergic progress of ad in clinical studies. unfortunately, phase iii clinical study of human umbilical cord blood mscs for ad was failed with unknown reason. previously, our group reported that exosomes derived from human adipose tissue-derived mscs (asc-exosomes) alleviated the pathological symptoms in a murine ad model with concomitant reduction of inflammation. methods: our group has further investigated the therapeutic effects of human asc-exosomes in an alternative murine ad model with skin barrier defects. large scale isolation of asc-exosomes was performed by tangential flow filtration and isolated asc-exosomes were characterized according to the recommendation by the isev. the protein and lipid cargo were also analysed. results: we found that asc-exosomes induced restoration of skin barrier by inducing de novo lipid synthesis and reduced the levels of multiple inflammatory cytokines. in addition, asc-exosomes suppressed the expression of itching-causing cytokines. transcriptomic analysis of ad skin lesions revealed that asc-exosomes reversed the abnormal expression of genes functioning in skin barrier function, lipid metabolism, and cell cycle. summary/conclusion: taken together, asc-exosomes could be a promising cell-free therapeutic option for the treatment of ad, which affecting inflammation, skin barrier function, and itching. cell derived vesicles: unravelling the science of novel vesicles with therapeutic promises introduction: cell derived vesicles (cdvs) are nanosized vesicles produced by serially extruding cells through small pores. a growing number of studies have implicated their therapeutic potentials, with superior yield compared to other extracellular vesicles (evs). however, two key objectives remain to be accomplished to demonstrate the utility of cdvs in clinical applications. first, a manufacturing process has to be developed to allow a large-scale production of cdvs. next, these novel vesicles need to be thoroughly characterized at multiple levels. methods: manufacturing-scale extruders were developed to allow extrusion of large volume of cell suspension in a single process. cdvs with approximately 150-200 nm in diameter were obtained by a serial extrusion. crude samples were then purified using the tangential flow filtration method to further remove cellular impurities. finally, physical and biochemical characteristics of purified cdvs were analysed using dls, nta, cryo-em, and facs analysis. additionally, cdvs were subject to multi-omics profiling to comprehend our understanding in molecular contents of cdvs. both mesenchymal stem cells (mscs) and natural killer (nk) cells were used for this study. results: in this study, we first demonstrate that the large-scale extruder efficiently produce cdvs with consistent quality at the scale that are compatible for clinical applications. surface marker and membrane composition analyses show that the cdvs are primarily formed using plasma membrane of source cells, with characteristic cellular markers enriched on the surface. comprehensive profiling of molecular components reveals the unique properties of cdvs as well as the underlying mechanism of formation of cdvs. summary/conclusion: recently, we have established a manufacturing process to enable clinical applications of cdvs. this study also highlights key molecular features of cdvs that can be harnessed to offer a powerful tool for regenerative and anticancer medicine. antifibrotic properties of extracellular vesicles derived from human induced pluripotent stem cells introduction: fibrosis is a pathological condition resulting from abnormal healing of various tissues. it is triggered by activation of fibroblasts and their subsequent transition to myofibroblast. in consequence, excessive deposition of extracellular matrix proteins leads to impaired organ function. to revert this process, we employed extracellular vesicles (evs) derived from human induced pluripotent stem cells (hipscs). as a model system, we used human cardiac fibroblasts (hcfs), since heart fibrosis constitutes a serious socioeconomic problem worldwide. methods: we isolated evs from conditioned media from three hipsc lines using ultrafiltration combined with size exclusion chromatography methods. next, we analysed the evs by nanosight, transmission electron microscopy, mass spectrometry and western blot methods. finally, we treated tgf-b-stimulated hcfs with hipsc-evs and evaluated expression of fibrosisrelated genes using real-time qpcr, western blot and fluorescence microscopy. results: we detected anti-fibrotic properties of hipsc-evs exerted on hcfs pre-stimulated with tgf-b. the evs significantly decreased the expression levels of acta2, fn, tnc, snai2, col1a1 and reduced the number of myofibroblasts. the canonical profibrotic tgf-b-dependent smad2/3 pathway was significantly attenuated in response to ev-treatment. summary/conclusion: in this study we demonstrated strong anti-fibrotic function of hipsc-evs. our findings can further be exploited for future medical applications to treat fibrotic diseases, such as heart fibrosis. funding: this work was supported by the project sonata12: umo-2016/23/d/nz3/01310 from the national science centre of poland to sbw. induced pluripotent stem cells-derived extracellular vesicles ameliorates d-galactosamine and lipopolysaccharide induced acute liver failure tianjin third central hospital affiliated to nankai university, tianjin, china (people's republic) introduction: liver failure is among the most causes of death in patients with liver disease. promoting liver regeneration will help patients with liver failure recover on their own. extracellular vesicles (evs) can released by induced pluripotent stem cells (ipscs) through paracrine effects and play a pivotal role in inter-cellular communication in the treatment of disease. in this study, we investigated whether the ipscs-evs have therapeutic effects on acute liver failure. methods: the ipscs-evs were isolated by ultracentrifugation and identified using nanoparticle tracking analysis, transmission electron microscopy and western blotting. the isolated ipscs-evs were administrated d-galactosamine-injured heprg cells in vitro and tail intravenously injected into d-galactosamine and lipopolysaccharide induced acute liver failure model mice in vivo, respectively. the anti-apoptosis role and potential mechanism were evaluated using flow cytometry and immunofluorescence staining. and alanine transaminase (alt) and aspartate transaminase (ast) in serum, h&e staining and tunel staining were explored the effect of ipscs-evs on liver injured and liver function. finally, high throughput sequencing of small rnas was performed to investigate mirna expression profiles in ipscs-evs and ipscs. results: the ipscs-evs that were all 50-200 nm, doublelayered and oval or round cellular vesicles and expressed the marker proteins cd63, tsg101 and hsp70. in vitro, the ipscs-evs treatment inhibited heprg apoptosis induced with d-galactosamine in a time-and dosedependent manner and promote the proliferation of hepatic stem cells. in vivo results showed that ipscs-evs significantly alleviated liver failure, improved liver function and prolonged the survival period. tunel assay showed that ipscs-evs suppress apoptosis of hepatocytes. moreover, mirna expression profiles analysis found that mir17-92a cluster and mir302-367 cluster were enriched in ipscs-evs and ipscs. summary/conclusion: these findings indicated that ipscs-evs could ameliorate d-galactosamine and lipopolysaccharide induced acute liver failure to attenuate hepatocyte apoptosis, which will be benefit for therapy of liver disease in the future. msc-derived extracellular vesicles promote human cartilage regeneration by control of autophagy introduction: osteoarthritis (oa) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. recently, allogeneic human mesenchymal stromal/stem cells (msc) entered clinical trials as a novel therapy for oa. increasing evidence suggests that therapeutic efficacy of msc depends on paracrine signalling. here we investigated the role of bone marrow msc-derived extracellular vesicles (bmmsc-evs), an important component of msc secretome, in cartilage repair. methods: to test the effect of bmmsc-evs on oa cartilage inflammation the tnf-alpha-stimulated human oa chondrocytes were treated with bmmsc-evs and inflammatory gene expression was measured by qrt-pcr after 48 h. to access the impact of bmmsc-evs on cartilage regeneration the bmmsc-evs were added to the regeneration cultures of oa chondrocytes, which were analysed after 4 weeks for glycosaminoglycan content by dmmb and qrt-pcr. paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-o) and type ii collagen (immunostaining). results: we show that bmmsc-evs promote cartilage regeneration in vitro. treatment of oa chondrocytes with bmmsc-evs induces production of proteoglycans and type ii collagen and promotes proliferation of these cells. msc-evs also inhibit the adverse effects of inflammatory mediators on cartilage homoeostasis. our data show that bmmsc-evs downregulate tnfalpha induced expression of pro-inflammatory cox-2, pro-inflammatory interleukins and collagenase activity in oa chondrocytes. the anti-inflammatory effect of bmmsc-evs involves the inhibition of nfκb signalling, activation of which is an important component of oa pathology. autophagy, a cellular homoeostatic mechanism for the removal of dysfunctional cellular organelles and macromolecules, is essential to maintaining chondrocytes survival and differentiation. the expression of autophagy regulators is reduced in osteoarthritic joints, which is also accompanied by increased chondrocyte apoptosis. our preliminary data indicate that bmmsc-evs carry mrna of natural autophagy inducers and promote autophagy in oa chondrocytes. therefore, we hypothesize that msc-evs exert their beneficial effects on cartilage regeneration by restoring the expression of autophagy regulators. summary/conclusion: in summary, our findings indicate that bmmsc-evs have ability to promote oa cartilage repair by reducing the inflammatory response and stimulation of oa chondrocytes to produce extracellular matrix, the essential processes for restoring and maintaining cartilage homoeostasis. thus, msc-evs hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis. large-scale preparations of small extracellular vesicles from conditioned media of mesenchymal stromal cells modulate therapeutic impacts on a newly established graft-versus-host-disease model in batch dependent manners introduction: extracellular vesicles (evs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (mscs) can suppress acute inflammatory cues in a variety of different diseases, including graft-versus-host disease (gvhd) and ischaemic stroke. furthermore, they can promote regeneration of affected tissues. following a successful clinical treatment attempt of a steroid refractory gvhd patient, we intend to optimize msc-ev production strategies for further clinical applications. as we observed functional differences of independent msc-ev preparations in vitro, we aimed to adopt an in vivo gvhd model for the more advanced functional testing of different msc-ev preparations. methods: to this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (iir). gvhd was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine t cells. if not treated otherwise, myeloablated mice developed severe gvhd symptoms. results: the gvhd symptoms were effectively suppressed, when msc-ev preparations were applied at 3 consecutive days, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay. msc-ev preparations lacking in vitro immune modulating activities, however, hardly improved the symptoms of the gvhd mice. thus, our results demonstrate that not all msc-ev preparations harvested from adult bone marrow-derived mscs contain the same therapeutic potential. summary/conclusion: thus, successful transplantation of msc-evs into the clinics requires a platform allowing identification of msc-ev preparations with sufficient therapeutic, most probably immune modulating activities. funding: this research was funded by sevrit leitmarkt lifescience.nrw ls-1-1-051 g. introduction: malnutrition impacts approximately 50 million children worldwide and is linked to 45% of global mortality in children below the age of five. severe acute malnutrition (sam) is associated with intestinal barrier breakdown and epithelial atrophy. extracellular vesicles including exosomes (evs; 30-150 nm) can travel to distant target cells through biofluids including milk. since milk-derived evs are known to induce intestinal stem cell proliferation, this study aimed to examine their potential efficacy in improving malnutrition-induced atrophy of intestinal mucosa and barrier dysfunction. methods: mice were fed either a control (18%) or a low protein (1%) diet for 14 days to induce malnutrition. from day 10 to 14, they received either bovine milk evs enriched using differential ultracentrifugation and sucrose gradient purification or control gavage and were sacrificed on day 15, 4 hours after a fluorescein isothiocyanate (fitc) dose. tissue and blood were collected for histological and epithelial barrier function analyses. results: mice fed low protein diet developed intestinal villus atrophy and barrier dysfunction. despite continued low protein diet feeding, milk ev administration improved intestinal permeability, intestinal architecture and cellular proliferation. summary/conclusion: our results suggest that evs enriched from milk should be further explored as a valuable adjuvant therapy to standard clinical management of malnourished children with high risk of morbidity and mortality. funding: cb was generously awarded a catalyst grant from the centre for global child health at the hospital for sick children to support this work. the impact of spheroids culture on mesenchymal stem cells and ev production introduction: mesenchymal stem/stromal cells (mscs) are now widely believed as bio-factories releasing bioactive products responsible for their therapeutic effect, i.e. cytokines, chemokines, and extracellular vesicles (evs). mscs are highly sensitive to physical stimuli from their surrounding microenvironment and can change their characteristics in response to their environment. the application of 3d spheroids cell culture allows mscs to adapt to their cellular niche environment which, in turn, influences their paracrine signalling activity. we aim to determine how 2d and 3d culture microenvironments can modulate the ev production and investigate their anti-fibrotic activity. methods: for 2d culture, bone marrow-derived mscs were cultured on standard tissue culture plastic. for 3d culture, mscs were aggregated into spheroids using non-adherent 96-well plates and cultured with addition of 0.25% methylcellulose. to collect conditioned media, both 2d and 3d mscs were cultured using serum free medium for 4 days. evs were isolated by serial ultracentrifugation and were characterised on exoview platform which allows simultaneous detection of particle size and expression of cd81/cd63/cd9. cell lysates were collected for mirna isolation and qrt-pcr was performed to analyse expression of candidate mirnas. to model the progress of lung fibrosis, human lung fibroblasts (hlfs) were cultured with tgf-β1 to induce fibroblast activation, subsequently exposed to 2d and 3d evs, and collagen production was measured. further, 2d and 3d msc-evs were added into human lung mscs isolated from healthy and ipf patients and cell proliferation was assessed using mts assay. results: 2d and 3d msc-evs have similar ev characteristics in terms of particle size and ev tetraspanin markers expression. exoview analysis showed expressions of cd81/cd63/cd9 and average particle diameters of <100 nm. on a cellular level, we identified a panel of anti/pro-fibrotic mirnas which are differentially expressed in 2d and 3d mscs. 2d and 3d msc-evs have similar anti-fibrotic activity shown by their ability to reduce collagen deposition in hlf cultures. both 2d and 3d msc-evs could promote cell proliferation on ipf lung mscs but no overall effect on healthy lung mscs. summary/conclusion: this concept of engineering the cellular microenvironment to promote ev production is as yet untouched and we foresee that in 3d cultures, we can culture mscs for longer timeframe and therefore maximising the overall ev production process. the outcome presents future potential for 3d culture of msc to increase the efficiency and feasibility of scalable ev production. outer membrane vesicles from photobacterium damselae subsp. piscicida: characterization and antigenic potential introduction: photobacterium damselae piscicida (phdp) is a gram-negative bacterium that causes a septicaemia in > 20 fish species worldwide. it represents a major drawback for aquaculture, whose importance has been sharply growing as a food supplier. given the phdp massive mortality and widespread antibiotic resistance, an effective vaccine is highly needed. extracellular products (ecps) have an essential role in phdp virulence, containing important antigens. however, the ecps' identity remain undisclosed. in our efforts to dissect their composition, we found that they contain high amounts of outer membrane vesicles (omvs). these particles are potent weapons for bacteria and are being explored in the field of vaccinology, since omvs present antigens in native conformations and are strongly immunogenic, without requiring adjuvants. this potential associated to the urgent need for an anti-phdp vaccine prompted us to isolate and characterize the omvs shed by phdp. methods: in order to harvest high amounts of pure phdp omvs, a reproducible optimized protocol was developed: the bacteria-free supernatant from a phdp overnight culture is concentrated, dialysed and ultracentrifuged to collect the omvs. results: analysis of the obtained omvs preparations by transmission electronic microscopy and dynamic light scattering indicate that the main population of vesicles has sizes around 30-50 nm. proteomic analysis of the vesicles revealed the presence of the apoptogenic ab toxin aip56 that is known to play a major role in phdp virulence, a putative pore-forming toxin, a putative adhesin/invasin and several outer membrane proteins (omps), including a 64kda omp, predicted to be involved in iron acquisition, and 5 other omps (18-34kda), with an ompa-like structure that may act as adhesins. moreover, preliminary in vivo studies suggest that some of those proteins may have important roles for virulence, since injection of knock-out strains in sea bass induced a decreased mortality comparing to the wt strains. summary/conclusion: our findings suggest that omvs are a promising vaccine candidate and we are currently studying their biological activities and determining the antigenic potential of the identified proteins. introduction: whole body exposure to high doses of ionizing radiation (ir) can potentially be lethal if radiation injury is not diagnosed and treated expeditiously. when considering a non-invasive approach for the identification of biomarkers of ir exposure, we and others have studied molecules in plasma, serum, saliva, and urine. however, these matrices can potentially have significant background noise, obscuring potential biomarkers of biological importance. extracellular vesicles (evs) are fast becoming a platform for biomarker discovery in radiation research as well as in other pathologies. however, no groups have investigated the use of metabolomics to analyse evs derived from urine in the context of ir exposure. furthermore, the dominant protocols for ev isolation from urine require a large (up to 30 ml) amount of starting volume, which may not be available for many studies. the aim of this study was to optimize ev isolation from rat urine and assess radiation-induced alterations in urine ev number and metabolic content. methods: as a proof of concept, we compared and optimized several ev isolation methods on small volumes of urine from male wag/rijcmcr rats exposed to 0 gy or 13 gy x-rays to the whole body except the hind leg. starting with either 500 µl or 1000 µl of urine, we isolated evs using ultracentrifugation (uc) with filtration, size exclusion chromatography (sec), and a proprietary bead-based isolation method developed by a 3rd party provider. ev samples were characterized using nanoparticle tracking analysis. metabolomics profiles were measured using lc-qtof-ms. results: we found that sec resulted in the highest yield of evs from as little as 500 µl of urine, while uc was the poorest performing. lc-qtof-ms analysis revealed that sec and uc had the most consistent identification of features, whereas the bead-based method contained artefacts likely as a result of the extraction method. we next used sec to isolate evs from a larger cohort of rats exposed to ir and analysed with ms. ev metabolic content will eb related to differences in survival and organ function between sham and irradiated groups. summary/conclusion: we conclude that sec is the preferred method for isolating evs from small volumes of urine for broad-based mass spectrometric analysis, and that the ev metabolome may be a sensitive and specific early indicator of radiation injury. introduction: there is growing evidence that contents (including rna and proteins) of exosomes may serve as biomarkers for early diagnosis and prognostic prediction of cancers. here we aim to identify potential protein markers for oesophageal cancer. methods: using our newly developed label-free exosome automated preparation system (leaps), exosomes were isolated from 20 ml culture medium of various oesophageal cancer cells with different differentiation profiles and different sources of metastasis. exosomes from 20 µl plasma of cancer patients at different clinical stages or with/without relapse and healthy controls were also prepared by leaps. matrix-assisted laser desorption ionization time-of-flight mass spectrometry (maldi-tof ms) was employed to directly analyse exosomes. protein identities of exosomal fingerprint peaks were tentatively assigned by correlation with top-down and bottom-up proteomics. results: start from 20 ml culture medium or 20 µl plasma, high-quality exosomes rapidly isolated by leaps are sufficient for maldi-tof mass spectrometry. it seemed that poorly differentiated cells showed more exosome release. maldi-tof ms fingerprints of exosomes in cells is cell line specific. ms profiles from poorly differentiated cells showed more peaks than that from highly differentiated cells. fingerprints also allowed classification of cancer cell lines through software mathematical analysis. we identified different numbers of significantly differentially expressed peaks in exosomes of various cancer cells. fingerprints of exosomes derived from the poorly differentiated cells showed more elevated peaks. top four peaks (2,427 m/z, 3,596 m/z, 4,458 m/z, 4 ,537 m/z) were commonly down-regulated in exosomes of most cancer cells. top four protein peaks (2,268 m/z, 5,713 m/z, 6,255 m/z, 6,337 m/z) that might be correlated to the differentiation profile of cancer cells were also identified. maldi-tof ms detection of exosomes in the plasma and clarifying identities of potential biomarker peaks will be done in the future. summary/conclusion: the combination of leaps and maldi-tof mass spectrometry provides a fast and high-throughput tool for exosomal marker discovery. potential biomarker identified in exosomes derived from oesophageal cancer cells or from plasma of cancer patients by this tool might be useful in cancer diagnosis and prognosis. fraction-based proteomic profiling of serum extracellular vesicles derived from cervical cancer patients introduction: current evidence indicates that extracellular vesicles (evs) can release from most of cell types and affect adjacent or distant cells by circulating in all bodily fluids. proteomic analysis of evs from clinical samples is complicated by the low abundance of ev proteins relative to highly abundant circulating proteins. size exclusion chromatography (sec) has been overcome as a method to deplete protein contaminants and enrich evs. methods: we collected serum of healthy women and cervical cancer patients with stage i-iii and then counted concentration and size distribution of the evs using nanoparticle tracking analysis (nta). differential ultracentrifugation combined with sec was used to isolate and purify evs from contaminant proteins. isolated evs were investigated their characteristic based on morphology using transmission electron microscope (tem) and on expression of cd63, cd81, cd9 protein markers using western blot analysis. fraction no.8-10 of isolated evs in among sample groups were profiled by nano-liquid chromatography tandem mass spectrometry (nanolc-ms/ms) analysis. results: nta shows that the concentration of evs is increased in patients compared with healthy women. proteome profiles of evs isolated by sec were compared in each fraction. moreover, we detected molecular evidence for fraction-specific molecular pathways in connection with cancer progression and complied a set of protein signatures that closely reflect the associated clinical pathophysiology. summary/conclusion: these unique features in each fraction among sample groups would be the informative considering in order to select for further analysis as in vitro. introduction: recently, diagnostic biomarkers from exosomes by proteomic analysis have been reported, but it is required to optimize the isolation protocol to screen out more effective biomarkers. for serum-originated exosomes, it has been also reported to isolate them selectively, however, it is observed that a different method resulted in different protein profiles in 2-d gel electrophoresis. methods: we isolated exosomes by two discrete methods, using ultracentrifugation and magnetic separation. before ultracentrifugation and magnetic separation, precipitation using polymer materials was perforemd. the isolation of exosomes by these two methods followed by comparison of their size, total vesicle number, morphology, and protein markers. to identify protein biomarkers, proteomic analyis using 2-d gel electrophoresis was performed. results: both methods induced enrichment of exosome-specific proteins, but protein profiles in each exosome fraction was totally different. the protein profiles showd that the magnetic seperation following a polymer-based precipitation step was more efficient to screen out candidate biomarkers, which showed nearly 200 protein profiles originated from exosomes. summary/conclusion: in our study, magnetic separation of exosomes from serum fraction was optimized for 2-d gel electrophoresis to observe identifiable biomarkers. an extracellular small rna-seq data processing pipeline optimized for high-performance computing chenghao zhu and angela zivkovic department of nutrition, uc davis, davis, usa introduction: a variety of rna species is found in extracellular biofluids such as blood, bile, and urine, carried by extracellular particles including extracellular vesicles (evs) and lipoproteins (e.g high density lipoproteins (hdls)). the extracellular rna (exrna) carried by evs and hdls is of great interest for two reasons: 1) the exrna within different carriers could be diagnostic of the state of the tissues from which the particles originate, and 2) exrna has been shown to affect gene expression in target cells. although the origin and functions of exrnas remain largely unknown, there is growing interest in exrna research for the development of diagnostics and new therapeutic targets. small rna sequencing is widely used to estimate the abundance of exrnas in biofluid samples. here we present a data processing pipeline for extracellular small rna sequencing. sequencing data are pre-processed through quality control, and then aligned to the endogenous genome to obtain the gene counts for various rna biotypes, including microrna, trna, rrna, piwi-interacting rna, long non-coding rna (lncrna) and protein coding rna. it also aligns sequencing reads to exogenous databases, including the ribosomal rna sequence database silva, and all sequenced bacteria genomes available on ensembl, to estimate the abundance of exogenous genes. results: we analysed a publicly available small rna-seq dataset of hdl from three systemic lupus erythematosus (sle) patients and three healthy controls using this pipeline. the mirna hsd-mir-93, lncrna al137186.2 and ac108050.1 were elevated in sle patients compared to controls. exogenous rna reads mapped to bacteroidetes were also elevated in sle patients. summary/conclusion: our pipeline is able to process exrna sequencing data and estimate the abundance of major exrna species, as well as exogenous rna taxonomy. the pipeline is optimized for the job scheduler slurm, and can therefore utilize the full computational power of high-performance computers. the pipeline is publicly available on github (www.github. com/zhuchcn/excernapipeline). introduction: ibd is a chronic hyperinflammatory disorder that severely compromises the intestines. the aetiology of ibd is poorly understood. however, it has been associated with a dysregulation of the immune system and gut microbiota and with genetic and environmental factors. cumulative evidence indicates that evs play an essential role in modulating immune responses. recent research suggests that evs derived from dendritic cells, saliva and intestinal epithelial cells may be involved in the progression of ibd inflammation. however, little is known about the contribution of immune cells-derived evs with this pathology. the goal of this study is to shed light on the contribution of pbmc-derived evs on ibd pathogenesis. here we characterized and compared the composition of evs derived from pbmcs of 3 ibd patients and 1 healthy control. evs were isolated by differential centrifugations from the supernatant of pbmc activated with cd3-cd28 beads for 6 days in serum-free media. size and concentration were analysed using a nano sight 300 instrument, while the presence of known evs markers (cd63, cd81, hsp70) was analysed by immunoblotting. whole evs proteome was performed by ms/ms and functional-enrichment analysis was done using funrich with uniprot database. results: proteomics analyses identified a total of 1299 proteins in the four groups. of those, 673 (51.8%) were present in both the ibd patients and control. this group of protein was composed of several ras-related proteins, eukaryotic initiation factors, granzyme, cd9, tubulin, and serpins among others. patients' evs shared 46 proteins in common such as proteasome subunit beta type-10, t cell receptor beta, and the amine oxidase containing copper 3. interestingly, each patient sample had a unique group of proteins. among these are myeloperoxidase, neutrophil elastase, proteasome subunit alpha type-3, and signalling lymphocytic activation molecule (slamf1). summary/conclusion: these preliminary studies show that the ev composition from pbmcs of ibd patients is specific and differs from a healthy control. this exclusive composition has the potential to be used as a biomarker for diagnostics and progression of the disease, and it could also provide new insights into our understanding of the cellular pathways involved in the pathogenesis of ibd. the studies were performed with corresponding irb approvals. proteomic analysis of exosomes isolated using precipitation and columnbased approaches introduction: exosomes are a subtype of small extracellular vesicles (evs) involved in various physiological and pathological processes with huge potential as biomarker resources or as therapeutic tools. although several exosome isolation approaches are available, complementary studies focusing on optimizing the methods for human bloodderived exosomes isolation and method-specific comparative exosomal proteomic profiles will be of clinical value. methods: blood-derived evs were isolated through precipitation-and column-based methods and characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. serumderived exosomal proteomes were analysed by mass spectrometry (ms). the resulting proteomes were then overlapped with the proteomes obtained from exosome-related databases, to determine the % of similar content. in addition, bioinformatic analysis, including gene ontology (go) was carried out. results: both methodologies tested isolated particles with the expected morphology and size range, although the column-based method isolated a higher number of particles. about 75% of the exosomal proteins identified through ms overlapped with the proteomes extracted from the databases. go terms were similar for the proteomes isolated from the column-and precipitationbased methodologies. the top 3 go terms identified for molecular function were ion binding, peptidase activity and enzyme regulator activity and for biological process were immune system process, transport and response to stress. further, partial least square analysis revealed a clear segregation of proteomes obtained by the distint methodologies and complementary statistical analysis revealed the proteins differently expressed. summary/conclusion: no major differences were found in the top 3 biological processes and molecular function based on go analysis. nonetheless, the two approaches result in different evs yields and significant proteome differences were identified. characterization of distinct methods for blood-derived exosomes isolation can be useful in the context of evs potential in disease diagnostics/therapeutics. introduction: we and others are developing biomarkers for neurodegenerative diseases using neuronalenriched evs immunocaptured from a suspension of total plasma evs. here we assess how the isolation method for total evs affects the yield, purity and enrichment of neuronal evs. methods: for n = 5 subjects, total evs were isolated by ev precipitation solution (exq), ev precipitation solution plus bipartite resin columns (exu) and size exclusion chromatography (qev) from 0.5, 0.5 and 2.7 ml plasma, respectively. then, neuronal-enriched evs were immunoprecipitated using anti-l1cam antibody. in total and l1cam evs, we measured particle concentration by nanoparticle tracking analysis, protein concentration, and novel multiplex electrochemiluminescence immunoassays for tetraspanins cd81, cd63 and cd9 on intact evs. results: for total evs, yield followed the order of exq > qev > exu, assessed by particle (p < 0.0001) and protein concentrations (p < 0.001). l1cam evs immunocaptured after exq showed 16-fold higher particle (p < 0.001) and fivefold higher protein (p < 0.01) concentrations compared to l1cam evs after exu, and 41-fold higher particle (p < 0.001) and 11-fold higher protein (p < 0.001) concentration compared to l1cam evs after qev. l1cam evs after ev precipitation (exq) showed 48, 35 and 30-fold higher cd81, cd63, and cd9 concentrations (p < 0.001) compared to l1cam evs after exu, and 59, 43 and 36-fold higher cd81, cd63, and cd9 concentrations (p < 0.001) compared to l1cam evs after qev. l1cam evs following 3 different methods had equal purity assessed by ratios of particle/protein concentrations (p = ns), and tetraspanin/particle concentrations (p = ns). summary/conclusion: l1cam ev immunocapture preceded by exq exceeded the yield of immunocapture preceded by exu or qev. recovered l1cam evs showed equal purity by particle/protein and tetraspanin/particle metrics. neuronal enrichment results will be available by the time of isev. immunoprecipitation following exq, often considered impure, purifies final isolates as effectively as more onerous methods typically considered purer. balancing sensitivity, purity and scalability is essential for implementation of blood biomarkers in the clinical setting and may be achieved by combining techniques. funding: this research was supported in part by the intramural research program of the nih, national institute on aging. characterisation of breath exosomes: towards non-invasive diagnosis deanna ayupova a , renee goreham b and paul teesdale-spittle c a school of chemical and physcial sciences, victoria univeristy of wellington, wellington, new zealand; b university of newcastle, newcastle, australia; c school of biological sciences, victoria univeristy of wellington, wellington, new zealand introduction: breath-derived exosomes present new potential for non-invasive diagnosis of lung cancer. however, breath-derived exosomes have not been well characterized and methodology for their purification has not been optimised. in order to exploit their potential for diagnosis, it is first necessary to develop methods that reproducibly provide high quality pure exosomes from breath. in this study, we optimise methods for their isolation and characterise them in comparison to exosomes derived from cell culture models. methods: in order to characterize exosomes from exhaled breath condensate (ebc) it was first necessary to optimize methods for isolation of pure, intact, and high quality exosomes. to this end, isolation methods were optimised on cell-derived exosomes and then applied to ebc, yielding high quality exosomes from size exclusion chromatography (sec). ebc exosomes were compared with those from a549 and wi-cells using dls, tem, and cryo-sem. an immunoblotting-grid technique was used to validate the presence of exosome-specific markers cd63 and cd81. protein content of exosomes were quantified and compared. results: sec-based isolation was more effective at isolation of pure and intact exosomes than ultracentrifugation, with the highest purity exosomes obtained in the middle fractions of the exosome-containing eluate. exosomes from ebc had a size range (45-100 nm), protein content (20-40 ug/ml) and molecular markers typical of cell-derived exosomes. summary/conclusion: breath-derived exosomes isolated through size exclusion chromatography are sufficiently pure for diagnostic purposes and are phenotypically similar to exosomes derived from other sources. we foresee their use in non-invasive diagnostics for lung cancer as an important future application. ligand-based exosome affinity purification (leap) is a rapid and reproducible method for the enrichment of functional evs introduction: platelet-derived extracellular vesicles (pevs) represent the next generation of therapeutic biologics as they enable a more refined and targeted approach when compared to crude blood derivatives currently used for treating diseases such as cancer, thrombocytopenia and chronic wounds. however, development of an ev-based therapeutic is hindered by the lack of a scalable, validated and reproducible purification process. in this study, pevs were isolated from activated platelet concentrates and purified using exopharm's ligand-based exosome affinity purification (leap) technology to produce a functionally active ev therapeutic. methods: platelet concentrates (n = 20) were obtained from the australian red cross blood service and were activated by exopharm's proprietary process. activation was verified by measuring cd62p using flow cytometry. the resulting platelet releasate (500 ml) was subjected to leap purification to isolate pevs. for characterization, protein concentration was determined by a bicinchoninic acid assay, microfluidic resistive pulse sensing (mrps) was used to perform a particle count and transmission electron microscopy (tem) enabled visualization of ev morphology. key ev markers were detected using mass spectrometry (ms) and western blots. to confirm biological activity, human dermal fibroblasts were subjected to serum starvation for 16 hours before treatment with pevs (15 µg/ml). cell growth was recorded by the real-time xcelligence system and differences in proliferation were statistically analysed using a one-way anova. results: mrps and tem both revealed isolated pevs to be 100-300 nm in size. the final product was positive for platelet markers (cd41, cd62p) and key ev markers (tsg101, alix, cd63). treatment with purified pevs significantly increased proliferation in serumstarved fibroblasts over 48 hours. summary/conclusion: exopharm's leap technology is a rapid and reproducible purification process which produces pevs that adhere to misev 2018 guidelines and are functionally active. funding: all funding was through exopharm ltd (asx:ex1) a novel but simple method to obtain purified exosomes by one-step ultracentrifugation introduction: exosomes are extracellular vesicles (evs) that are derived from endosome membrane. they are usually 50-150 nm in diameter, actively secreted in most living cells. originally, exosomes were thought to act as cellular garbage disposals. recent studies showed that exosomes not only can serve as biomarkers for diagnosis, but also can be used as an ideal delivery vehicle for drugs in therapeutics. exosomes are natural carrier for mrna, mirna, sirna, protein, dna and peptide for long distance intercellular communication. isolation of exosomes is challenging due to their small size and heterogeneity. traditional differential ultracentrifugation method is still the gold standard for exosome purification. to further explore the potentials of exosomes being as the therapeutic delivery vehicle or diagnostic reagent, it is an essential step to purify them in high quality at high yield. methods: here, we report a novel method to obtain intact shape, high-quality and high purity exosomes with one-step ultracentrifugation by using "exojuice". results: data of nanoparticle tracking analysis (nta) and western blotting showed "exojuice" can yield exosomes with a simpler method to obtain higher purity exosomes in comparison to previous method of cushion ultracentrifugation using optiprep. summary/conclusion: our method can be used to purify exosomes from cell culture medium, serum, urine, saliva, and other biofluids. a straightforward device to extract apoplastic fluid from succulent fruits for higher purity of extracellular vesicles introduction: edible plants are emerging as a sustainable source for extracellular vesicle (ev)-based drug delivery vehicles. however, current isolation methods (e.g. grinding or squeezing) may cause destruction of plants' biostructures, and in turn leads to unwanted effects in downstream applications and complicates the study of nanovesiclescell. therefore, we designed a simple device that allows the extraction of apoplastic fluid (af) from succulent fruits, facilitating ev isolation as well as effective downstream applications. methods: an inner filter tube was designed to extract af with a determined membrane pore size. af was collected by low-speed centrifugation method and then filtered to eliminate the impurities from the cytoplasm and damaged cells. minced juice (mj) was homogenized by a blender and then centrifuged to remove large fragments. subsequently, the differential centrifugation method was employed to extract evs from af and mj. fourier-transform infrared spectroscopy (ftir), nanoparticle tracking analysis (nta), and transmission electron microscopy (tem) were performed to discriminate af, mj and their evs. results: the "spectroscopic" protein-to-lipid (p/l) ratio of af (1.23 ± 0.03) is significantly lower than that in mj (1.27 ± 0.01), showing the higher lipid contents in af, which may result from the loss of lipids in mj obtained from grinding or juicing methods. similarly, ftir showed the difference in p/l ratio between af and its evs (1.23 ± 0.03 and 1.29 ± 0.02, respectively). nta showed the sharper peak and smaller vesicle size in the following order: mj (173.0 ± 21.3 nm), af (154 ± 11.0 nm), af-derived evs collected at 40,000 × g and 10,000 × g (108.5 ± 2.4 nm and 95.7 ± 3.1 nm, respectively). furthermore, tem study indicated that the collected evs exhibited a typical lipid bilayer of extracellular nanovesicles. summary/conclusion: by using a reusable filter device, we successfully isolated af from succulent fruits, paving the way to collect plant evs without an interference of significant biodestruction or damaged cells, hence improving the purity of evs and facilitating downstream applications. moreover, this method is straightforward, reproductive, and can be potentially used in a large-scale production. method to simultaneously capture multiple classes of intact extracellular rna carriers including extracellular vesicles and lipoprotein particles introduction: extracellular particles including extracellular vesicles (evs), lipoproteins, and free proteins are carriers of extracellular rna (exrna), which has been shown to regulate cellular function. because these particles have different physiological origins, they have different rna signatures, so the first step to understanding the biology of exrna is to isolate individual particle fractions with high purity and efficiency. current methods for isolating evs are optimized for increased yields and purity of ev fractions but typically require multiple millilitres of starting plasma and do not capture the other exrna carrier particle types. methods that can capture evs from low starting plasma volumes and can also capture other exrna carriers simultaneously are needed for analysing samples from previously conducted large cohort studies, biorepositories, and in populations where sample volume is limiting. methods: we have developed a method adapted from lipoprotein isolation that requires only 500 µl of starting plasma, and uses brief ultracentrifugation (uc) followed by fast protein liquid chromatography (fplc) to capture 6 classes of purified exrna carriers including evs, ldl, hdl, lipidated albumin, proteins, and vldl/chylomicrons. we have validated successful capture of evs by microfluidic resistive pulse sensing (mrps, spectradyne), transmission electron microscopy (tem), and single particle interferometric reflectance imaging system (sp-iris; exoview) with optional fluorescence. results: we have observed 1.02 × 1010 particles per ml from a 1 ml fplc fraction of evs measured from 25,000 events by mrps, confirming that evs are being captured by this method. there were also 1.26 × 1010 particles/ml and 2.70 × 1010 particles/ml in the two subsequent 1 ml fractions that are known to contain lipoprotein particles, though these were measured from 1,500 events each. by tem we confirmed these observations that evs are eluting before lipoprotein particles with some evs eluting later in fractions containing lipoproteins. summary/conclusion: these results confirm the efficacy of the method in isolating multiple exrna carrier fractions simultaneously from a single 500ul plasma sample, making it amenable for the analysis of exrna in samples from large cohort studies, biorepositories, and vulnerable populations such as the elderly and young children. funding: nih/nia r01ag062240; nih ug3 ca241694-01 optimizing the isolation of placental mesenchymal stromal cell-derived extracellular vesicles in a 3d bioreactor system leora goldbloom-helzner a and aijun wang baaaaa a uc davis, davis, usa; b uc davis medical center, sacramento, usa introduction: extracellular vesicles (evs) derived from placental mesenchymal stromal cells (pmscs) have the potential to provide neuroprotection at sites of injury. however, a rate limiting step in ev research is the low yield, high technical time, and high cost of current isolation procedures. to address this inefficiency, we cultured pmscs in a unique bioreactor system to increase the absolute yield of evs per ml of media and per cell. future studies will determine if this system can improve pmsc ev yield without altering the demonstrated neuroprotective properties of pmsc-evs. methods: pmscs were cultured in the bioreactor for ten weeks. ev-conditioned media was collected weekly and evs were isolated through differential centrifugation. nanoparticle tracking analysis (nta) measured ev size and concentration. western blots tested for normal ev markers (cd9, cd63, and cd81, calnexin(-)) and enzyme-linked immunosorbent assays (elisa) measured levels of characteristic growth factors in conditioned media including vascular endothelial growth factor (vegf), brain-derived neurotrophic factor (bdnf), and hepatocyte growth factor (hgf). results: evs remained consistent until week eight, after which a decrease in both ev size and concentration was seen. western blots revealed normal positive expressions of cd9, cd63, and cd81 and negative expressions of calnexin. levels of vegf, bndf, and hgf were comparable after 10 weeks. cost analysis revealed an overall increase in ev yield for shorter labour time and lower material cost. summary/conclusion: this initial study uses a bioreactor system for a unique source of cells and has brought us closer to optimizing pmsc ev isolation protocols for increased yield, lower cost and time commitment, and maintained sample purity. preliminary data suggests the ev phenotype and cell secretome are consistent with those present in current culture settings. future experiments will assess the preserved neuroprotective properties of the pmsc evs. a novel method for isolating extracellular vesicles from cell culture media and plasma using polyethylenimine introduction: due to their ability to transport dna, rna, and protein cargoes between cells, extracellular vesicles (evs) are becoming popular for biomarker discovery as well as for therapeutic delivery. here we describe the development of a novel precipitation method for the isolation of evs from cell culture media and plasma that is based on polyethylenimine (pei), an inexpensive, water-soluble, and biocompatible cationic polymer. pei is a group of hydrophilic cationic polymers that are synthesized as either linear or branched forms of varying molecular masses (1,000 to 750,000 da) and are widely used in the biomedical field as a coating and transfection agent. methods: linear and branched pei of varying molecular weights (mw) were tested for their ability to precipitate evs from either conditioned culture media (ccm) or human plasma. isolated evs were characterized by western blotting and nanoparticle tracking analysis (nta). the small rna profile of evs isolated using pei from human plasma was analysed by ngs and ev-specific mirnas were confirmed by digital droplet pcr (ddpcr). mass spectrometry (ms) was used to analyse the proteome of pei-captured evs from plasma. hek293 cells producing gfp+ evs were used to optimize conditions for release of evs from both linear and branched pei by fluorescent spectrophotometry and flow cytometry measure-ments. results: linear and branched pei were both able to precipitate evs as determined by western blotting for ev protein markers; however, branched pei with mw > 10,000 da and linear pei with mw > 25,000 da were more efficient for ev precipitation than lower mw forms. despite its known ability to bind nucleic acids pei was unable to capture cell-free dna from plasma, although rna and in particular ev-associated mirnas such as mir-142-3p were recovered. ms revealed that pei enriches extracellular exosome proteins from plasma. evs captured from ccm by pei could be released from the complex using heparin or high salt conditions. summary/conclusion: pei has an unexpected preference for associating with evs compared to nucleic acids in complex biological samples and has a hitherto unrecognized application for ev precipitation. introduction: there is ongoing debate about which is the most appropriate method for isolation of evs, with most labs using some combination of differential ultracentrifugation (uc), size-exclusion chromatography (sec), and/or density gradient ultracentrifugation (dg). here we applied a surface-enhanced raman spectroscopy (sers) analysis platform to compare chemical composition of the isolate from each method against lipoprotein standards to assess the relative purity of the ev preps. methods: 2-3 ml of plasma was separated from whole blood collected from head and neck cancer patients. each sample was split into 3 batches and evs were isolated by either uc, sec, or dg. following isolation, samples were incubated on commercial sers substrates and raman spectra were collected. lipoprotein standards were purchased and also measured for comparison. using principle component analysis (pca), spectra were analysed for chemical variability. results: sers analysis of sec, uc, and dg isolated evs were chemically distinguishable using simple pca. the chemical changes could in large part be attributed to fitting the differences in spectra to lipoprotein standards. we found that uc isolated populations clustered with the high-density lipoproteins (hdl), sec populations with the low-and very low-density lipoproteins (ldl, vldl), and dg populations were more variable, but mainly clustered together with the highdensity-lipoproteins (hdl). summary/conclusion: this set of experiments matches our expectation that various lipoprotein would contaminate ev preps according to their relative size and density distributions. no single isolation method could separate pure ev samples. this study also illustrates the utility of label-free sers analysis for rapid chemical characterization of evs. bioreactors: lessons to develop an extracellular vesicle factory vanessa chang, priscila dauros-singorenko, lawrence w. chamley, colin l. the university of auckland, auckland, new zealand introduction: high density mammalian cell culture systems (bioreactors) provide valuable advantage for large scale production of secreted products such as extracellular vesicles (ev). however, optimisation of design selection, handling and operational costs can be quite challenging. here we provide our experience with a celline bioreactor system. methods: cultures of 6 adherent cell lines were established in celline ad 1000 bioreactors and propagated for up to 19 weeks. media was changed twice weekly and cells shed into serum-free conditioned medium were counted and assessed for viability. nanoevs were isolated by sequential centrifugation (2000 g -10,000 g -100,000 g) and size exclusion chromatography (sec). nanoevs were characterised in their protein (bca) and particle (nanoparticle tracking analysis) amount, ev markers (western blotting) and morphology (transmission electron microscopy, tem). results: the viability of shed cells varied between cell lines and through time, suggesting a changing dynamic during reactor establishment and continuous growth phases, that was specific to each cell line. hdfa, bt20 and bt474 consistently shed mainly dead cells (60-100%), as opposed to mcf7 and mda-mb-231 which predominantly shed live cells. sec fractionation of nanoevs identified a dominate ev-rich peak and significant quantities of smaller proteins, highlighting the need for further purification. nanoev yields from each 3-4 day culture averaged 2-7 × 1010 particles, representative of yields obtained from cells grown in 8 to 28 conventional t175 tissue culture flasks. ev markers and tem confirmed the protein profiles and morphology of evs obtained from bioreactors. summary/conclusion: high density bioreactor cultures offer a physiologically relevant, cost and space efficient approach to produce significant amounts of evs, providing sufficient material for numerous experimental uses. in our hands, with careful twice weekly management, they can be propagated for up to 19 weeks without significant changes to the evs. introduction: extracellular vesicles (evs) have potential applications for clinical theranostics. ultracentri-fugation is most commonly adopted to the evs isolation, which is recommended as a gold standard method. however, ultracentrifugation is time-consuming and expensive equipment requirement, resulting in the coisolation of contaminants such as protein aggregates. therefore, our aim is to develop a rapid and efficient platform to isolate heterogonous evs based on the insertion of lipid molecules into the evs membrane to avoid co-isolation of non-membranous protein particles. methods: herein, a defected nanoscale functional metal organic framework (mof) was constructed as an efficient platform for evs isolation. typically, one single-stranded dna was designed and modified with a phosphate group at the 5ʹ-end and cholesterol at the 3ʹ-end to form a capture dna named phosphate−dna−cholesterol (pdc). the phosphate group forms a strong covalent bond with the designed defeated site of zr (iv) in mof uio-66-nh2 and the cholesterol inserts into the phospholipid bilayer to capture evs without non-membranous particles contamination. the formed mof−phosphate−dna −cholesterol−evs (mof@pdc@evs) system was further treated with dnase i for dna hydrolysis to give high pure evs. results: a rapid and efficient isolation platform of evs based on a defected mof functionalized with phosphate-dna-cholesterol (mof@pdc) has been constructed successfully. compared with ultracentrifugation, mof@pdc platform promises to isolate size heterogeneous evs i) without non-membranous particles contamination, maintaining evs intact membrane structure, protein components, and biological functions; ii) with the ability to capture evs with 78% isolation efficiency; iii) makes evs isolation process simple and fast, which could be finished in 40 minutes without requirement of the expensive equipment. summary/conclusion: in conclusion, this rapid and efficient platform is suitable for isolation evs from biological fluid for downstream protein analysis. this work opens a new perspective in mof-based separation researches and may shed light on further studies towards evs isolation. introduction: incorporation of pharmacologically active molecules on the surface or the lumen of extracellular vesicles (evs) is an important strategy for maximizing the therapeutic potential of evs. genetic engineering of producer cells by introducing dna through random or site-specific integration are promising strategies for creating engineered evs. longterm stability with consistent transgene expression in the ev producer cells and therapeutic potency of resulting engineered evs are crucial for biomanufacturing. we present a comprehensive study to investigate stability of transgene expression and potency of two potential therapeutic engineered evs derived from stably selected pools transfected by either random integration (ri) or site-specific integration (ssi). methods: producer cells were engineered to make evs displaying interleukin 12 (il12) or interferon gamma (ifng) by ri or nuclease-mediated ssi into aavs1 locus. following puromycin (puro) selection, longterm cellular stability and transgene expression without selective pressure was investigated. evs were generated from stable cell pools at 0, 1, and 2 months post-thaw and purified by density gradient ultracentrifugation. purified evs were biochemically characterized by nta, bca, western blot, and cholesterol quantitation. transgene expression and biological activity of evs displaying il12 and ifng were assessed by alphalisa and in vitro reporter assays. results: transfection by ssi resulted in faster recovery in puro selection compared to ri. all stable cell pools, regardless of integration method, resulted in comparable cell culture performance, ev yield, and lipid and protein content at all time points tested. the engineered evs also demonstrated long-term stability of il12 and ifng transgene expression and in vitro activity from both integration strategies. summary/conclusion: both methods for generating stable cell lines were comparable in terms of cell stability, transgene expression, ev titre and potency, with ssi having the advantage of speed, allowing for more rapid iteration cycle times. thus, both methods are suitable for the precision engineering of therapeutic evs. this work demonstrates feasibility to manufacture therapeutic engineered evs from stable cells from either integration strategy for clinical development. transport of outer membrane vesicles as a model therapeutic delivery system in pathogenic and commensal bacteria introduction: outer membrane vesicles (omvs) in gram-negative bacteria have been shown to be important carriers of biomolecules, including toxins and other virulence factors, peptidoglycan, and nucleic acids. it has been shown that omvs play an important role in the delivery of these biomolecules to host cells and bacterial cells. while many thorough studies have explored omv delivery to host cells, few studies have explored the mechanisms of delivery of omvs to bacterial cells. our goal was to study the delivery of omvs to other bacterial cells. specifically, we were studying the oral pathogen aggregatibacter actinomycetemcomitans (a.a.), a gram-negative organism associated with localized aggressive periodontitis, to study the process by which vesicles from this organism communicate with other bacterial cells. overall, we want to understand the roles specific surface components of omvs play in the transport of these omvs to other bacterial cells. methods: we studied omvs from two strains of a.a.: jp2, a highly pathogenic strain, and 33384, a natural commensal strain. af488-labelled omvs were incubated with fresh bacterial cultures. association of the omvs with the bacterial cells was quantified using flow cytometry. to examine the role of surface-associated dna in this process, dna was digested with dnase, and the amount of surface-bound dna was quantified with the membrane impermeable dna stain, toto-1. results: using flow cytometry, we observed jp2 omvs were delivered to 33,384 cells, and at a lesser amount to jp2 cells. alternatively, 33,384 omvs associated readily with jp2 cells, more than to 33,384 cells. this suggests that the delivery of omvs to bacterial cells may be a targeted delivery mechanism. furthermore, we hypothesized surface-associated dna may play a role in this interaction. we next digested the surface-associated dna on the omvs with dnase, and observed a decrease in association between the omvs and bacterial cells. this supports our hypothesis that dna on the surface of the omvs plays a role in association. current experiments are investigating this interaction in more detail. summary/conclusion: we have demonstrated that omvs are selectively delivered to bacterial cells, and surface-associated dna plays a role in this process. we propose to investigate this process to further understand omvs delivery to bacterial cells. funding: r03de025275 & r21de027769. utilizing a gaucher's disease cell line for the evaluation of a novel exosome-based replacement therapy annie k. brown a , jiayi zhang b , brendan lawler b and biao lu b a santa clara university, san jose, usa; b santa clara university, santa clara, usa introduction: engineered nano-scale exosomes have great potential as new and targeted delivery vehicles for the treatment of gaucher's disease, the most common lysosomal storage disease. recently, we have reported the design, production, and isolation of exosomes loaded with lysosomal β-glucocerebrosidase (gba). people suffering from gaucher's disease do not have functional gba, which results in toxic build-up of undegraded substrates within the cell. methods: to evaluate the efficacy of this exosomebased therapy, a human gaucher's disease model is required. here, we have utilized near-haploid human cells (hap1) modified via crispr-cas9 to model gaucher's disease in vitro. these cells contain a 479bp insertion in the 6th exon of the gba gene, resulting in non-functional gba. pcr, enzyme activity assays, and flow cytometry have been employed to confirm the diseased genotype and phenotype. results: characterization of gba-knock out cells shows a total loss of gba enzyme activity. further characterization demonstrates a normal growth rate but an increased number of lysosomes, indicating a diseased phenotype. summary/conclusion: the utilization of a human gba-knock out cell line will enable the evaluation of the efficacy of our engineered exosomes. disease models will be an important resource for the evaluation of new biologic therapeutics, including exosomes. funding: we would like to acknowledge the santa clara university school of engineering for their support. thrxosomes: a novel exosomes based theranostic for lung cancer introduction: chemotherapy is the first-line of treatment for lung cancer. however, inefficient bio-distribution and reduced accumulation of drugs in the tumour results in treatment failure. therefore, improved drug delivery and diagnostic systems are warranted. herein, we propose a novel theranostic system "thrxosomes" where exosomes are loaded with super paramagnetic iron nanoparticles (spions) conjugated to an anticancer drug via a phresponsive linker for controlled release. we hypothesize that thrxosomes will exert profound anticancer tumour activity that can be concurrently be monitored by magnetic resonance imaging (mri). methods: thrxosomes were produced by combining normal human lung fibroblast (mrc9) cell-derived exosomes with spions conjugated to and anti-cancer drug (chemodrug or mirna) via a ph cleavable linker. the physical and biological properties of thrxosomes were determined using transmission electronic microscopy (tem), nanotracker-analysis (nta), inductively coupled plasma mass spectrometry (icpms), western blotting, cell viability, and mri. results: exosomes used in preparing thrxosomes were 130 nm in size with a typical lipid bilayer structure, and were positive for cd63, cd81, flotillin and negative for annexin a1 confirming presence and purity of exosomes. charge analysis, tem, and icmps data showed successful loading of spion-drug conjugate. biological studies showed selective and enhanced drug release under acidic condition (ph 5.5) compared to drug release at ph 7.2. cell uptake and viability studies demonstrated increased uptake and killing of thrxosome-treated human a549 lung cancer cells compared to mrc-9 cells. in vivo studies demonstrated accumulation and detection of spions by mri in in-situ tumours of a549 tumour-bearing mice. summary/conclusion: our study demonstrates thrxosomes will produce profound anticancer activity in lung cancer that is measurable by mri. exosome-modified nanoparticles as an alternative delivery system for small rnas in cancer therapy petro zhupanyn a , alexander ewe b , thomas büch c and achim aigner a a independent division for clinical pharmacology at rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany; b dr., leipzig, germany; c rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany introduction: gene knockdown by rna interference (rnai) is an alternative, non-invasive method for inhibiting proliferation or promoting apoptosis in tumour cells. this technique allows the specific targeting of key signalling proteins or mutated genes. most of the available transfection compounds suffer from rather profound cytotoxicity in vitro. the aim of our study was to establish a novel targeted small nucleic acid delivery system to the cells, with good cellular biocompatibility and applicability for in vivo studies. for this aim, we used native, cell own vesicles-exosomes. since exosomes are known to transport peptides and different rnas between cells and tissues, these unique, small extracellular vesicles (ev) may also be useful as transport vehicles for therapeutic sirna. methods: as detected by multiple cell surface protein expression analysis, exosomes carry specific surface expression markers, allowing the cellular uptake by the most of tissues. we established an ev purification protocol from tumour cell culture supernatants and a strategy for the efficient ev loading with our test sirnas or antimirs. here we used the combination of polyethylenimine (pei)-complexation of the rnas with ultrasound treatment for their loading into the evs. our ev-modified, ultrasound-treated nanoparticles were tested in vitro by measuring knockdown efficacies in luciferase reporter cell lines or by rt-qpcr gene expression analysis. results: more efficient cellular sirna uptake was observed upon ev-modification of our pei/rna nanoparticles, accompanied by efficient inhibition of gene expression. biological efficacies were retained also after storage for several days at room temperature. the monitoring of the ev-based particles by facs revealed a different time resolution of cellular uptake and nucleic acid release compared to the classically formulated peinanoparticles. in an in vivo therapy study in tumour xenograft-bearing mice, high biocompatibility, significant biological knock-down and tumour inhibition were observed after injection of anti-survivin sirnas formulated in our ecv-modified pei nanoparticles. summary/conclusion: our data demonstrate the usability of ecv-modified nanoparticles as efficient delivery system for small rnas in cancer therapy. microglial extracellular vesicles as therapeutic vector for neuroinflammation giulia marostica a , annamaria finardi b and roberto furlan a a san raffaele scientific institute, milan, italy; b san raffaele scientific institute, milan, italy introduction: microglia is considered an eligible target against the progressive multiple sclerosis (ms), but currently available therapies do not allow its efficient targeting. as many cell types, microglia communicate with the neighbouring cells through a complex system of extracellular vesicles (evs) exchange. recently my group described that microglia derived-evs, engineered to encapsulate il4, are taken up by microglia itself, mediating a phenotype switch to a protective phenotype. in vivo studies suggest that these evs can ameliorate established neuroinflammation, thus making them a promising drug-delivery tool to target cns in ms. my project focuses on understanding the mechanism of action and the signalling pathway of evs delivery and to exploit this knowledge to specifically deliver different potential therapeutic molecules. for this purpose, we decided to characterize the evs through trps technology. methods: a murine microglia cell line (bv2) was engineered to stably overproduce endogenous il4. this cell line was cultured in exosome-depleted rpmi and stimulated with pma(20 mg/ml) for 30 min. evs isolation was carried out by collecting supernatant and subjecting it to consequential centrifugation of 300 g,10 min, rt and 2000 g,20 min, 4°c. the resulting supernatant was filtered (5 µm) and ultracentrifuged at 100,000 g for 2h at 4°c. the evs pellet was re-suspended in ice-cold pbs. the evs analysis with trps shows two populations of evs, one with a mean diameter of 60-80 nm and a broad zeta potential ranging from −10 mv to −60 mv, while the second population has a mean diameter of 120-140 nm and a zeta potential of −10/-20 mv. this difference can be consistent with the different pathway formation of exosomes and microvesicles. we demonstrated in vivo the strong phenotypic change induced by our evs to resting microglia in a dose-and time-dependent effect. then, impairing the physiological procedure of the endosome acidification, the effect of our evs on recipient cells is higher. thus, suggesting an endocytic pathway for the internalization of the vesicles. we further demonstrate with gradient ultracentrifugation the capability of our formulation to vehicle endogenous il4 inside the vesicles. even if some protein is co-purified in the procedure, we know that the half-life of this cytokine is too short to elicit a strong in vivo response. consequently, we assume that the anti-inflammatory effect of our evs in vivo is a result of the il4 internalized in our formulation. summary/conclusion: these data help us understand more in detail the process of internalization and phenotype change mediated by these evs. our next goals are to discriminate between different internalization pathways and further validate the efficacy of our therapy on the eae mouse model. targeting il-3rα on tumour-derived endothelial cells blunts metastatic spread of triple negative breast cancer via extracellular vesicle reprogramming introduction: the lack of an approved targeted therapy and the early onset of metastasis highlight the need for new treatments for triple-negative breast cancer (tnbc) patients. interleukin-3 acts as an autocrine factor for tumour-endothelial-cells (tec), and exerts pro-angiogenic paracrine action via extracellular vesicles (nevs). il-3rα blockade on tec changes tec-ev (anti-il-3r-evs) microrna cargo and promotes the regression of established tumour vessels. as tec are the doorway for "drug" entry into tumours, we have aimed to assess whether il-3r blockade on tec impacts tumour progression via their unique ev cargo. methods: 27 human tnbc samples, mda-mb-231, mda-mb-453 and mcf10 cell lines were evaluated for the expression of il-3rα. nevs and anti-il-3r-evs were characterized by electron-microscopy, macsplex-exosome-kit and western blot. proliferation, migration, apoptosis and sphere formation were evaluated. scid mice were used for in vivo experiments. results: we noticed that, besides tec and inflammatory cells, tumour cells from 55.5% of the human tnbc samples expressed il-3rα. mda-mb-231 and mda-mb-453, but not mda10 cells, expressed il-3rα. in vitro, nevs provide survival and migratory signals, while anti-il-3 r-evs promoted apoptosis as well as reduced cell viability and migration of human tnbc cell lines. in vivo anti-il-3 r-ev treatment induced vessel regression in established tumours formed of mda-mb-231 cells and almost abolished the spread of liver and lung metastasis. moreover, decreased β-catenin and twist1 were found in tumours from animals treated with anti-il-3 r-evs. in addition, anti-il-3 r-evs reduced lung metastasis that was generated via the intravenous injection of mda-mb-231 cells. nevs that were depleted of mir-24-3p (antago-mir-24-3p-evs) were effective as anti-il-3 r-evs in down-regulating twist1 and reducing lung-vessel density and metastatic lesions in vivo. summary/conclusion: overall, these data provide the first evidence that il-3 rα is highly expressed in tnbc cells, tec and inflammatory cells, and that il-3 rα blockade on tec impacts tumour progression. introduction: high-grade serous ovarian cancer (hgsoc) is the deadliest gynaecologic cancer. its lethality is explained for late diagnosis at advanced stages and frequent recurrences despite achieving complete response with standard therapy. most of recurrences occurs at abdominal cavity with multiple metastasis. therefore, identifying key determinants of metastatic process remains as priority to find better therapies. current evidence assigns a central role of the exosomes in conditioning the metastatic niche in epithelial cancers. recently, we demonstrated that statins reduce metastasis in hgsoc in preclinical models. here, we decided to study the effects of statins on hgsoc-derived exosomes and its capability to condition the metastatic niche. methods: exosomes were isolated from heya8 ovarian cancer cell line and primary tissue cultures established from advanced-stage hgsocs (with signed informed consent and irb approval) by differential ultracentrifugation and quantified by nanoparticle tracking analysis (nta). enriched-cancer initiating cells (cic) spheroids were established from heya8 cells by using stem-selecting conditions. the paracrine effect of exosomes on cic migration/invasion was studied using either 3d migration or boyden chamber invasion assays. previous to exosome isolation, heya8 cells were treated with simvastatin (5um, 24 h) or solvent and proteins involved in exosome biogenesis/uptake (alix, tsg101), its trafficking (rab7a, rab27a) and in conditioning the metastatic niche (emmprin) were measured by immunoblotting. results: exosomes isolated from heya8 cells or hgsocs enhance the metastatic potential of heya8established spheroids in 3d migration or boyden chamber invasion assays. upon simvastatin treatment, we observed a significant reduction in migration/invasion induced by equivalent number of exosomes in heya8-derived cics. under same treatment, we observed a significant decrease in protein levels of alix and tsg101 and an increase in the inactive forms of rab7a and rab27a in heya8 cells. we also observed a decrease in emmprin levels in heya8-derived exosomes. summary/conclusion: here, we demonstrated a paracrine effect of hgsoc-derived exosomes that favour the metastasis process. in addition, we demonstrated that simvastatin reduces metastasis induced by cancerderived exosomes. such an effect is partially explained by changes in the expression of proteins involved in exosome biogenesis/uptake, its endocytic trafficking and in the content of proteins conditioning the metastatic niche. thus, simvastatin arises as potential therapeutic target to improve outcomes in this disease. funding: this research was supported by fondecyt granted to mauricio a. cuello label-free optical imaging and characterization of cancer-associated extracellular vesicles in tissues introduction: cancer-associated extracellular vesicles (evs) visualized in the tumour microenvironment have been identified as a potential biomarker for cancer-related tissue changes. analyses of evs have traditionally been performed in cells or isolated evs, with no temporal or spatial information that could be critically important for elucidating their roles in carcinogenesis. since the unperturbed distribution and organization of evs in the tumour microenvironment is associated with their cellular function and can potentially serve as a diagnostic and prognostic biomarker, there is a strong need for visualizing evs in freshly isolated tissue specimens. currently, only fluorescent labelling methods enable visualization and tracking of evs. we used a custom label-free multimodal multiphoton optical imaging system to detect and characterize evs and classify them using their optical signatures both in isolated tissues and in situ tumours. methods: label-free multimodal multiphoton imaging was used to provide simultaneous, co-registered structural and functional images of evs in untreated samples. heterogeneous populations of evs could be identified from their unique optical signatures. results: the intrinsic metabolic and structural properties of evs enabled reliable visualization and optical characterization of evs from cell cultures and in situ imaging of tumour-bearing rats. unique optical signatures were then used for identification of cancer-related evs in tissues from human breast cancer patients, and their density was found to be highly correlated with clinical diagnosis. in the current study, evs were isolated from urine of tumour-bearing dogs, and urine and serum from breast cancer patients. analysis of ev content showed higher concentration of nad(p)h in evs isolated from cancer subjects, than from healthy subjects, which reflects the reprogramming of cellular metabolism in carcinogenesis. summary/conclusion: these results suggest a potential label-free optical methodology to detect and characterize evs by their optical signatures, which can be utilized as possible diagnostic and prognostic biomarkers for cancer. funding: this research was conducted under protocols approved by the iacuc and irb at the university of illinois and carle foundation hospital, and supported by funding from nih. novel potential anticancer therapies based on interference with nuclear entry of cancer cell-derived extracellular vesicle components in recipient cells introduction: the intercellular communication mediated by extracellular vesicles (evs) in the tumour microenvironment plays an important role in tumour progression. experimental evidence indicates that evs derived from highly metastatic cells influence the behaviour of less aggressive cancer cells. we have previously described a novel intracellular pathway where a fraction of endocytosed ev-associated proteins and nucleic acids is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into nucleoplasmic reticulum (nr). here, we better characterize this pathway and report that it is required for the induction of an aggressive behaviour induced by evs released from highly metastatic sw620 colon cancer cells in isogenic primary cancer cells. methods: super resolution-structured illumination microscopy and magnetic-based co-immunoisolation studies were employed to identify the protein components of the nuclear pathway and to monitor the entry of ev-containing late endosomes into the nucleoplasmic reticulum. human sw480 carcinoma cells expressing er-gfp and rab7-rfp were exposed to evs from sw620 cells and then live imaged. results: we have previously reported that the tripartite protein complex, containing vap-a, orp3 and rab7 orchestrates the localization of ev-carrying late endosomes into nr. we now report that silencing of orp3 or vap-a, but not its homologue vap-b, reverses the pro-metastatic changes induced by evs isolated from metastatic cells on their non-metastatic counterpart, including transition to an ameboid phenotype, cell rounding and blebbing. moreover, we found that certain nuclear pore complex proteins and importin-beta1 are co-immunoisolated with orp3, vap-a and rab7 suggesting the formation of a large protein complex at the entry of nuclear pores. summary/conclusion: interfering with the mechanisms regulating this novel intracellular pathway may find therapeutic applications particularly in ev field and oncology. educated osteoblasts regulate breast cancer proliferation via small extracellular vesicles thomas jefferson university, philadelphia, usa introduction: breast cancer commonly traffics to bone, where breast cancer cells (bccs) can survive undetected for years before metastatic outgrowth. in bone, bccs interact with surrounding stromal cells, including osteoblasts (obs), to shape the metastatic niche. our lab discovered there are at least two subpopulations of obs in the bone-tumour niche, based on protein marker expression. one group, "educated osteoblasts" (eos) have engaged in crosstalk with bccs whereas another group, naïve obs, have not. we have novel evidence that eos regulate bcc proliferation. the purpose of this study was to determine if extracellular vesicles (evs) produced by eos play a role in regulating bcc proliferation. we hypothesized evs produced by eos would decrease bcc proliferation. methods: eo-derived small evs from culture media were isolated via ultracentrifugation and characterized evs for size, protein marker expression, and density floatation to validate the purity of ev samples. the functionality of eo-derived evs on bcc proliferation was examined using edu and checkpoint proteins p21 and p27. bcc protection from chemotherapy induced cell death was also examined. results: we found that evs produced by eos, but not naïve obs, decreased both triple negative and erpositive bcc proliferation in a concentration dependent manner. furthermore, using an edu assay, we found that exposure to eo-derived evs induces bccs to undergo cell cycle arrest. interestingly, the cell cycle arrest was reversible and bcc proliferation was restored upon removal of eo-derived evs. in addition, exposure to eo-derived evs leads to increases in bcc expression of the g1 checkpoint proteins, p21 and p27. we next wanted to investigate proliferative signalling pathways that may be deregulated in bccs following exposure to eo-derived evs. we found that eoderived evs reduce bcc levels of erk1/2. because our data indicate eo-derived evs induce sustained cell cycle arrest in bccs, we desired to know if eo-derived evs protected bccs from chemotherapy-induced cell death. we found that bccs exposed to eo-derived evs and the chemotherapy drug, doxorubicin, have decreased cell death compared to bccs exposed only to doxorubicin. summary/conclusion: altogether, our data suggest eos play a crucial role in bone-tumour microenvironment by regulating bcc proliferation. funding: supported by nih r00 ca178177 and commonwealth of pennsylvania -department of health sap 4100072566 for kmb. phosphorylation of tyrosine 23 in annexin a2 is essential for its association with exosomes and for imparting invasive and proliferative capacity to other cells priyanka prakash desai a , pankaj chaudhary b , xiangle sun b and jamboor vishwanatha a a unt health science center at fort worth, fort worth, usa; b unt health science center, fort worth, usa introduction: triple negative breast cancer (tnbc) accounts for 15%-20% of all breast cancer cases. the lack of targeted-based therapies highlights the importance of studying tnbc. elevated levels of annexin a2 (anxa2), a ca+2-dependent phospholipid binding protein, has been correlated with worse overall survival in tnbc patients. our previous data implicate that exosomal anxa2 is involved in creating a pre-metastatic niche and facilitating metastasis in tnbc. moreover, n-terminal phosphorylation of tyrosine (tyr) 23 in anxa2 has been implicated in regulating several anxa2 activities in cancer progression. here, we demonstrated that n-terminal phosphorylation of anxa2 at tyr23 is important for its association with exosomes which imparts invasive and proliferative phenotype to other cells. hence, dissecting the regulatory pathway will be critical for verifying the value of anxa2 as a therapeutic, diagnostic or prognostic marker in tnbc. methods: pn1-egfp plasmids expressing the constitutive phosphomimetic (anxa2-y23e) and non-phosphomimetic mutant (anxa2-y23 f) gene expressing mutation at tyr23 site were overexpressed in mda-mb-231 tnbc cells. mutant cells were experimentally validated for anxa2 specific functions like migration, invasion and proliferation. exosomes were isolated from the mutant phosphomimetic (exo-anxa2-y23e-gfp) and non-phosphomimetic (exo-anxa2-y23 f-gfp) cells and were analysed for exosomal surface expression of anxa2 by immunoprecipitation and flowcytometry. cal-148 breast adenocarcinoma epithelial cells were treated with exo-anxa2-y23e-gfp and exo-anxa2-y23 f-gfp to analyse the rate of invasion and proliferation by transwell invasion and proliferation assay, respectively. transfer of exosomal anxa2 in cal-148 was studied using immunofluorescence and its implications on signalling pathways were studied by western blot. results: mda-mb-231 phosphomimetic tnbc mutant cells showed increased migratory, invasive and proliferative capacity compared to non-phosphomimetic tnbc mutant cells. exo-anxa2-y23e-gfp had elevated surface anxa2 expression compared to exo-anxa2-y23 f-gfp. cal-148 cells treated with exo-anxa2-y23e-gfp showed high migratory, invasive and proliferative characteristics, with a higher expression of p-anxa2(tyr23), p-src(tyr416) and p-paxillin(tyr31) compared to exo-anxa2-y23 f-gfp treated cells. summary/conclusion: n-terminal phosphorylation of tyr23 in anxa2 in mda-mb-231 tnbc cells (phosphomimetic mutant cells) is essential for its association with exosomes and for conferring increased invasive and proliferative capacity to other breast cancer cells. funding: the above study is funded by national institute of health ro1ca220273 and nimhd's u54md006882 to dr.j.k.vishwanatha. a novel method for epithelial-derived extracellular vesicle isolation and enrichment in patients with advanced prostate cancer arpit rao, helene barcelo and bharat thyagarajan university of minnesota, minneapolis, usa introduction: evaluation of changes in prostate cancer biology is difficult due to presence of lymph nodal or bony metastatic disease in a majority of patients. a number of liquid biopsy assays have shown clinical utility in prostate cancer, but are limited by low sensitivity (e.g. circulating tumour cells-based assays) or inability to perform transcriptome sequencing (cellfree dna-based assays). epithelial-derived extracellular vesicles (epi-ev)-based assays are uniquely positioned overcome both these limitations as evs are abundantly secreted into the blood and have rnacargo that mirrors the cell of origin. however, a reliable method to enrich for epi-evs is currently lacking. methods: plasma was isolated from the peripheral blood collected from 15 patients with metastatic prostate cancer enrolled in an institutional biobanking study before initiation of systemic antineoplastic therapy. evs were isolated from 500 µl of plasma using a commercially available qev 35 size exclusion column (izon inc.). without subjecting the evs to any physical stressors such as centrifugation, cd61 magnetic beads were used to fractionate the evs into cd61+ (platelet derived) and cd61-(non-platelet derived) fractions. multiparameter flow cytometry was used to evaluate evs that expressed cd9 and epcam and were negative for calnexin. nanotracking analysis (nta) was used to quantify both total ev and cd61+ and cd61fractions in all patient samples. the average ± standard deviation of total evs obtained from the 15 patients was 6.39x10^11 ± 4.60x10^11 evs/ml of plasma (coefficient of variation [cv] : 72%) while the average and standard deviation of cd61-evs was 1.24x10^10 ± 3.37x10^10 (cv: 271%). the cd61-ev fraction represented a variable amount of the total evs in prostate cancer patients ranging from 0.0001% to 32.21%. multiparameter flow cytometry showed that over 80% of total evs were cd9+ and calnexin-, suggesting an endosomal origin for a vast majority of the evs in these plasma samples. however, the proportion of evs expressing epcam (marker of epi-evs) was higher among the cd61-fraction (5% -30%) as compared to the cd61+ fraction (0.04% -1%). summary/conclusion: our novel method was able to isolate and enrich the epi-ev from the plasma of advanced prostate cancer patients. correlation between clinical characteristics and ev quantity is being evaluated to identify the reason(s) for large variations in cd61-ev fraction. future studies are planned to use our method in improving the sensitivity of ev-based assays and increase the rna yield to facilitate transcriptome sequencing. funding: this work was funded by grants from randy shaver community and research fund, minnetonka, mn. exosomes drive medulloblastoma metastasis in a mmp2 and emmprin dependent manner introduction: recurrent/metastatic medulloblastoma (mb) is a devastating disease with an abysmal prognosis of less than 10% 5-year survival. the secretion of extracellular vesicles (evs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. matrix metalloproteinases (mmps) are enzymes secreted by tumour cells that can potentiate their dissemination by modification of the extracellular matrix. we hypothesise that exosomal mmp2 and its inducer emmprin could enhance metastasis of mb. methods: proliferation, invasion and migration assays were used to evaluate the phenotypic behaviour of primary cell lines pre-treated with metastatic tumour cell-derived exosomes. gelatin zymography and western blotting were performed to confirm mmp2 functional activity in cell lines and exosomes. nanoscale flow cytometry was used to measure surface exosomal emmprin levels. exosomal mmp2 and emmprin were modulated at the rna level. results: number of exosomes is directly related to the migratory behaviour of parental mb cell lines (p < 0.01). notably, functional exosomal mmp2 and emmprin levels also correlate with this. furthermore, exosomes from metastatic cell lines conferred enhanced migration and invasion on their matched isogenic primary (non-metastatic) cell line pair bỹ 3.8-fold (p < 0.01). exosomes from metastatic cell lines also conferred increased migration on poorly migratory foetal neuronal stem cells. summary/conclusion: together this data suggests that exosomal mmp2 and emmprin may promote medulloblastoma metastasis and supports analysis of exosomal mmp2 and emmprin levels in patient cerebral spinal fluid samples. introduction: exosomes secreted from cancer cells harbour the potential to regulate intracellular signalling and promote metastasis. wherein, metastasis suppressor genes (msgs) play a pivotal role in regulating such signalling cascades. however, the regulation gets hampered due to low expression of msgs under metastatic conditions. nm23-h1, product of first identified metastasis suppressor gene nme1, is significantly downregulated under metastatic conditions. nm23-h1 serves as a regulator of small gtpases. several evidences have highlighted an involvement of small gtpases (such as rab5, rab7 and rab27) in the biogenesis of exosomes. in addition, bacterial homolog of nm23 has been shown to interact with rab5 and rab7. however, experimental evidence supporting a relationship between exosomes and nm23-h1 is lacking. our current focus is to deduce the relationship between exosomes and msgs. methods: breast cancer cell lines were used to assess the effect of exosomes isolated from highly metastatic cells (mda-mb-231 cells) on lower/non metastatic cells (mcf-7 cells). nme1 was overexpressed in mda-mb-231 cells and subsequently used to isolate exosome fractions. equivalent amount of isolated exosome fractions from mda-mb-231 cells and mda-mb-231/nme1 cells were utilized to access their effect on migration and difference in exosome markers. results: we observed an enrichment of nm23-h1 in the exosomes isolated from mda-mb-231 cells upon overexpression of nme1. proteinase k protection assay confirmed the packaging of nm23-h1 inside the exosomes isolated from mda-mb-231/nme1 cells and excluded the possibility of membrane association of nm23-h1. additionally, overexpression of nm23-h1 led to a significant reduction in the ability of mda-mb-231 exosomes to stimulate movement of mcf-7 cells as confirmed by wound healing assays. our data also highlights a clear reduction in the protein levels of exosome markers such as cd63, cd9 and alix in the exosome fraction isolated from mda-mb-231/nme1 cells as compared to mda-mb-231 cells. interestingly, rab7a, a protein involved in the endosome-lysosome fusion was also present in lower amount in the exosomes isolated from nm23-h1 overexpressing cells. summary/conclusion: our data highlights an antimigratory effect of nm23-h1 via exosomes. these findings support a regulatory role of nm23-h1 in the packaging or release of exosomes in highly metastatic breast cancer cells, and further suggest that metastasis suppressor proteins may be involved in the regulation or packaging of exosomes. additional studies will be required to decipher the downstream signalling of nm23-h1 which affects the biogenesis of exosomes as well as to assess the effect of nm23-h1 overexpression on the content of exosomes. these insights could help us delineate the complex exosome biogenesis pathway and provide new potential drug targets for exosome regulation. introduction: exosomes (exs) are emerging as novel players in the beneficial effects induced by exercise on vascular diseases. our recent study has revealed that moderate exercise enhances the function of circulating endothelial progenitor cell-derived exosomes (cepc-exs) on protecting endothelial cells against hypoxia injury. in this study, we aimed to investigate whether exercise-regulated cepc-exs contribute to the beneficial effects of exercise on ischaemic stroke (is). methods: c57bl/6 mice performed moderate treadmill exercise (10 m/min, 4-wks) before is induced by middle cerebral artery occlusion surgery. acute injury was evaluated at day 2 by determining neurologic deficit, infarct volume, cell apoptosis in the penumbra and neurologic recovery was assessed by determining angiogenesis/neurogenesis, sensorimotor functions at day 28. the correlations of cepc-exs and their carried mir-126 with neurological parameters were analysed. the underlying mechanism of the effects of cepc-exs isolated from exercised mice was explored in a hypoxia neuron model. cellular mir-126 level, apoptosis, axon growth ability and gene expressions (cas-3, bdnf and akt) were measured. results: 1) exercised mice had a smaller infarct volume on day 2, which was associated with decreased cell apoptosis and cleaved cas-3 level, and a higher microvessel density than those in control; 2) the elevated cepc-ex level positively correlated with tepc-exs in ischaemic brain of exercised mice on day 2. the upregulated mir-126 level positively correlated with the numbers of tepc-exs in ischaemic brain; 3) the numbers of cepc-exs and their carried mir-126 level negatively correlated with the infarct volume, cell apoptosis and positively correlated with the microvessel density in the peri-infarct area on day 2; 4) exercised mice had decreased infarct volume, increased microvessel density, promoted angiogenesis/neurogenesis and improved sensorimotor functions on day 28, accompanying with upregulated levels of bdnf, p-trkb/trkb and p-akt/akt; 5) cepc-exs of exercised mice protected neurons against hypoxia-induced apoptosis and compromised axon growth ability which were blocked by mir-126 and pi3 k inhibitors. summary/conclusion: our data suggest that the protective effects of moderate exercise intervention on the brain against mcao-induced ischaemic injury are ascribed to cepc-exs and their carried mir-126. funding: this work was supported by american heart association (18post33990433) and nih (1r01ns 102720). syndecan-1 regulates alveolar type 2 epithelial cell senescence mediating through extracellular vesicles during lung fibroproliferation tanyalak parimon a , changfu yao a , adam aziz a , stephanie bora a , marilia zuttion1 zuttion a , dianhu jiang a , melanie koenigshoff b , cory hogaboam a , paul nobel a , barry stripp a and peter chen a a cedars-sinai medical center, los angeles, usa; b university of colorado, denver, usa introduction: alveolar type 2 epithelial cell (at2) senescence is implicated in the pathogenesis of lung fibrosis, a progressive fatal condition. syndecan-1, a heparan sulphate proteoglycan, is overexpressed by at2 cells of human idiopathic pulmonary fibrosis (ipf) and bleomycin-injured wt mice and the overexpression of syndecan-1 is profibrotic. moreover, syndecan-1 deficient (sdc1-/-) mice had less lung fibrosis after bleomycin injury. we reported that extracellular vesicles (evs) in bronchoalveolar lavage (bal) of bleomycin-injured wt mice augmented lung fibrosis whereas the sdc1-/--bal-evs attenuated the process. moreover, wt-bal-evs expressed lower level of anti-fibrotic mirnas (mir-34b-5p, −142-3p, −144-3p, and −503-5p) compared to the sdc1-/-bal-evs. these mirnas targeted genes in the cellular senescence pathway indicating that syndecan-1 altered microrna profiles in the bal-evs to promote cellular senescence during lung fibrogenesis. we investigate how syndecan-1 regulates at2 senescence through evs. methods: bleomycin was intratracheally given into wt and sdc1-/-mice. at day 21, lungs were processed for single-cell rna sequencing (scrnaseq) and western blot (wb). evs were isolated using ultrafiltration centrifugation method. human (a549) and mouse (mle-15) lung epithelial cell lines were used for in vitro experiments. results: scrnaseq analysis indicated while bleomycin stimulated an overexpression of cellular senescencespecific genes on at2 cells of wt mice, these genes were significantly downregulated on sdc1-/-at2 cells. senescence proteins, p16 and p21, were also less expressed in the lungs of sdc1-/-than of the wt mice by wb. to determine the functional effects of evs in bal, a549 cells were treated with human ipf or control lung wash-evs and evaluated for beta-galactosidase activity. we found that ipf-evs markedly increased beta-galactosidase enzymatic activity. corroborating with these data, bleomycin-injured bal-wt-evs also significantly upregulated senescence marker, p21, by wb on mle15 cells whereas sdc1-/--bal-evs inhibited p21 expression. summary/conclusion: our data indicate that syndecan-1 regulates lung fibrosis through the senescence signalling pathway on at2 cells. furthermore, syndecan-1 controls at2 senescence mediating through extracellular vesicles in the bal. lastly, the most likely cargo molecules mediating this process are micrornas. immortalized cardiosphere-derived cell ev-associated pirna, imev-pi, protects against ischaemic injury in the heart alessandra ciullo, ahmed ibrahim, liang li, chang li, weixin liu and eduardo marbán smidt heart institute, cedars sinai medical center, los angeles, usa introduction: cardiosphere-derived cells (cdcs) are a population of heart-derived progenitors with demonstrated therapeutic efficacy in preclinical and clinical settings. cdcs function by secreting extracellular vesicles (evs), lipid-bilayer nanoparticles laden with bioactive molecules. recently our group developed a strategy for immortalizing cdcs (imcdc) that retains their therapeutic potential and enhances cdc function indirectly through their secreted evs. imcdc show a different rna content(mirna, mrna, rrna, trna and pirna) compared to primary cdc. in particular, we focus on piwi rnas (pirnas), small rnas bound by piwi proteins, important regulators of both the epigenome and transcriptome. we seek to explore the role of a pirna highly enriched in imcdc-evs (imev-pi). methods: evs are prepared by conditioning cells for 24 hrs in serum-free basal media, in hypoxic culture. after 24 hrs conditioned medium is cleared of cellular debris and evs isolated using ultrafiltration by centrifugation (ufc). fractions were analysed in terms of particle size, number, and concentration and pirna content. in vitro, bone marrow derived-macrophages (bmdm) were exposed to imcdc-ev, imev-pi and control and transcriptomic profile and potentially activated pathways were assessed. in vivo, 8-10 week-old wistar-kyoto female rats received 10^10 imcdc-ev, imev-pi, scramble or vehicle intracoronary 20 minutes after ischaemiareperfusion(i/r). cardiac troponin i levels, scar size and monocytes were assessed at 24 and 48 hrs. results: by small-rna sequencing analysis we found that pirnas are enriched in both cdc-ev and imcdc-ev. imcdc show a different pirna composition compared to primary cdc. imev-pi was identified as one of the most highly-expressed non-coding rnas (the number of reads were 35x higher in imcdc-ev compared to cdc-ev). in vitro, imexo-pi-conditioned bmdm exhibit a different transcriptomic profile compared with control, with upregulation of pathways involved in the inflammatory response, cell death, and cell-to cell signalling. in vivo, imev-pi is cardioprotective, as shown by reduced scar size and lower cardiac troponin levels compared to vehicleand scramble-injected animals at 48 hrs post i/r. imev-pi only minimally alters neutrophil counts profile in blood but it alters monocytes profile with a decreased number at 24 hrs and an increase at 48 hrs. summary/conclusion: we posit that imev-pi is a key determinant of imcdc-ev therapeutic efficacy. our results indicate that target cells may be macrophages/ monocytes, given that imev-pi exposure modifies their composition and mrna profile both in vitro and in vivo. introduction: extracellular vesicles (exosomes, evs) are cell membrane particles (30-200 nm) secreted by virtually cells. during intercellular communication in the body, secreted evs play crucial roles by carrying functional biomolecules (e.g., micrornas and enzymes) into other cells to affect cellular function, including disease progression and tissue regenerations. literature previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of evs. the activation of growth factor receptors, such as epidermal growth factor receptor (egfr), induces macropinocytosis. in this study, we demonstrated the effects of evs on demal papilla and hair follicle regeneration. methods: identification of distinct nanoparticles and subsets of extracellular vesicles from umbilical cord blood stem cell by asymmetric flow field-flow fractionation. results: the effects of evs from umbilical cord blood stem cell on the propagation of demal papilla and hair follicle regeneration were observed. summary/conclusion: the enhancement of extracellular vesicles from umbilicalcord blood stem cell the propagation of demal papilla and hair follicle regeneration were observed and confirmed. mechanisms of host resistance to plasma membrane damage induced by pneumolysin attack introduction: bacterial pore-forming toxins (pfts) are major virulence factors produced by pathogens. pfts target host plasma membrane (pm) and create transmembrane pores, which allow uncontrolled flux of ions and small molecules across the pm disrupting cellular homoeostasis. to survive, cells display poorly understood repair mechanisms to recover the cell homoeostasis. several mechanisms were proposed to participate in cell recovery: exocytosis of cortical lysosomes; endocytosis of pfts pores; pm blebbing and shedding. methods: we used increasing concentrations of purified ply to intoxicate cells. pm permeability was assessed by flow cytometry using propidium iodide dye. cytoskeleton rearrangements were investigated by confocal immunofluorescence microscopy. extracellular vesicles released during pm repair were isolated by high-speed centrifugation and characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and mass spectrometry/ liquid chromatography analysis. results: ply triggers a complete reorganization of the actomyosin cytoskeleton inducing the formation of cortical actomyosin bundles at sites of pm remodelling. these structures assemble upon loss of pm integrity and disassemble as pm recovers. we detected the release of microvesicles during the recovery of pm integrity. vesicle population is heterogeneous with sizes ranging from 100 to 500 nm, with the majority of them measuring 100-200 nm. vesicle proteomic analysis revealed that they contain ply, suggesting they participate in pore removal, proteins involved in vesicle trafficking, pm repair and exosome biogenesis. summary/conclusion: our data demonstrate that cells are able to recover from the damage induced by sublytic concentrations of ply. actomyosin cytoskeleton undergo massive changes with the assembly of cortical bundles possibly at sites of pm damage. we showed that cells produced extracellular vesicles during the process of repair. we are now focusing on understanding the biogenesis of those vesicles and its importance during the process of repair. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms (2d static tissue culture flask vs 3d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in 2d t-flasks and 3d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both 2d and 3d culture systems. msc-ev were characterized according to misev 2018 guidelines, using techniques as nta, protein and lipid quantification, western blot, imaging and fourier-transform infrared spectroscopy (ftir). the results indicate that msc-ev derived from different sources/donors have similar size distribution, however, ev yields tend to be higher for the 3d culture system. of notice, several spectral regions were identified by ftir, enabling the detection of differences in the biomolecules present in msc-ev, msc-conditioned media and cells produced under different conditions. summary/conclusion: in summary, this study contributes to the establishment of a scalable process for msc-ev production. evaluation of three different isolation methods for small extracellular vesicles from human plasma in prostate cancer diagnosis introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies. methods: pca patients and age-matched healthy controls (hc) plasma (n = 6 in each group) were used to isolate sevs with 3 different isolation methods including commercial exoquick ultra kit, qev 35 and qev 70 size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd9 and cd81 commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination. results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev35 demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev70. furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods. summary/conclusion: qev 70 method demonstrated better performance in isolating relatively pure sevs from human plasma; qev35 has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev35 but with the highest non-sev protein contaminations. people can choose higher sev content or higher sev purity according to the downstream analysis. moreover, sevs may also be used for treatment monitoring, as recent studies suggested that the expression levels of certain markers may change during therapy, reflecting tumour response. for cancer diagnostics and therapeutic purposes in clinical settings, it is important to have a device which allows multiplexed measurements, in order to scan a large number of markers simultaneously and compare the expression levels of different patients, or same patients at different treatment stages, in a time efficient manner. methods: herein, we propose a multiplexed platform for label-free detection and surface protein profiling of sevs. the technique is based on the electrokinetic phenomena of streaming current and zeta potential (\zeta*) and measures the\zeta* change upon sev binding on functionalized microcapillary surfaces. for the purpose, we used sevs derived from lung cancer cells. in its current form, the platform can measure up to 5 channels simultaneously, however, it can be further expanded. results: having demonstrated that our electrokinetic sensor successfully detects sevs in a specific way, we tested its ability to measure the expression level of membrane proteins. the analysis showed that it could detect differences in the expressions of egfr on sevs, with a sensitivity of 10%. we then extended the platform for multiplexed analysis, by connecting and measuring four capillaries, functionalized with different capture probes, simultaneously. for the purpose, we targeted specific tumour markers, i.e. egfr, and exosomal tetraspanin family proteins, such as cd9 and cd63. the results showed successful multiplexed ev detection. summary/conclusion: being the sensor suitable for multiplexed sev detection, we shall present our investigation on a set of pleural effusion samples collected from a cohort of lung-cancer patients with different genetic makeup. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd9, cd63 and cd81, it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: 1) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and 2) the assessment of subpopulation target enrichment relative to the population average by leveraging tim4 as an unbiased, lipid-based ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasis-associated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr3 (her2+), t47d and mcf-7 (er+pr+), bt549 and mda-mb-231 (triple negative) breast cancer cell lines, as well as five mda-mb-231-derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her2 was broadly detected in her2+ skbr3 evs. interestingly, her2-t47d and mcf-7 evs also expressed her2 where it was highly enriched in its epcam+ subpopulations. itg α6, β3 and β4 were only found in triple negative and organotropic evs with itg β3 and β4 differentially enriched based on the organotropism. the population average of mda-mb-231 and lung-tropic evs had high expression of itg β4, where subpopulations of cd44+ evs showed positive enrichment while cd9+ and cd63 + evs showed negative enrichment. itg α5, β3 and β4 were absent in the bone-tropic cd81+ ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb-231 evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du145 prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified 49 targets that bind to hs-gag chains, and also 108 different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev-associated hspgs will result in attenuation of ev induction of a tumour-supporting fibroblast phenotype. introduction: ovarian cancer (oc) is the fifth leading cause of cancer-related death in women, partly due to difficulty in early diagnosis. extracellular vesicles (evs) show promise for use in early diagnostics of oc. here, evs from cervical mucus (cm) of ovarian cancer patients were used for discovery of oc biomarkers for diagnostics. machine learning was used to mine ev mirna data to develop an oc biomarker panel (validation via the cancer genome atlas). examination of the mirna targets reveal that the panel is a sufficiently accurate predictor of oc. methods: evs from the cm of 48 patients (15 highgrade serous, 24 low-grade, 7 benign) were isolated for small rna-sequencing. the top differentially expressed mirnas were used in a random forest and "voom" (variance modelling at the observational level) model. unsupervised approaches were used and then vetted against patient symptomology data. a tcga ovarian cancer dataset (n = 100) was used for validation. results: an oc biomarker panel of 10 micrornas (voom: 96.55% accuracy; random forest: 88% accuracy) was generated. the panel consists of members from the mir-200 family and the mir-16 family, among others. the mirna targets are associated with molecular functions and pathways specific in oc progression. summary/conclusion: our method has identified ev mirna biomarkers that may be crucial for early, noninvasive detection of oc. data science has been used to develop a feedback system integrating biochemical experiments, smaller datasets, and previously available data to identify and verify a biomarker panel for oc diagnostics. introduction: liver disease has become a significant cause of morbidity and mortality among hiv patients. alcohol exposure can further exacerbate liver damage by activating hepatic stellate cells (hscs), leading to hepatic fibrosis or cirrhosis, often seen at all levels of alcohol exposure among people with hiv. due to the potentiating effects of alcohol on hiv-induced hepatocytes (hep) damage, as well as the effect of ethanol in hsc-mediated extracellular remodelling, it is imperative to understand the interplay of hep and hscs. here, we focus on the exosomes released by hiv-and ethanol exposed hep and how these exosomes modulate the functional behaviour of hscs. methods: human hepatocyte huh7.5cyp2e1 [hepatoma cells stably transfected with cyp2e1 designated as rlw cells] were infected with hiv in the presence or absence of alcohol metabolite, acetaldehyde using the acetaldehyde-generating system (ags). the conditioned medium was collected from 4 groups of cells: untreated, hiv-, ags-and hiv+ags. quantification of exosomes number and size were evaluated with zetaview or nanosight and further characterized for exosome markers following the guideline from minimal information for studies of evs 2018 (misev2018). the human hepatic stellate lx-2 cell line was exposed to hepatocyte-derived exosomes and assessed for the activation using pro-inflammatory markers il-1β, il-6, tnfα, and fibrotic markers acta2, and timp1 using quantitative pcr. we also analysed exosome mirna content in primary human hepatocytes (phh), which potentially regulates the function of recipient cells by "programming" their inflammation/fibrosis status. the network analysis for mrna and mirna were carried out using gene ontology consortium, and mirror 2.0 and david bioinformatics resources 6.8. results: ags treatment further enhanced the release of hiv-induced exosome from hepatocytes. size distribution assessed by zeta view or nanosight revealed that approximately 85-90% of particles distributed in the range of 50 to 200 nm, with a peak at~90 nm. enriched expression of hiv protein p24 was observed in fractions f9-f12. western blotting of hepatocytederived exosome demonstrated positivity for exosome-enriched proteins alix, tsg 101 and cd9 specifically in f2-f8 fractions and negative for endoplasmic reticulum protein calnexin. the uptake of hepatocytederived exosomes by hscs was apparent as demonstrated by immunofluorescence. the internalization of hepatocyte-exosome induced activation of hscs as evidenced by increased expression of pro-inflammatory il-1β, il-6, tnfα markers in the latter cells. summary/conclusion: we conclude that ags treatment in hiv-infected hepatocytes potentiates the release of exosomes, which, following uptake by the hscs, leads to their activation. funding: this work is supported by nih-1 r01 aa027189-01a1. antimicrobial peptide ll-37 induces neutrophil-derived extracellular vesicles with antibacterial potential and protects murine sepsis yumi kumagai, taisuke murakami, kyoko kuwahara and isao nagaoka juntendo university, bunkyo-ku, japan introduction: extracellular vesicles (evs) released from immune cells or other host cells upon microbial infection modulate the immune response and thereby regulate the infection. sepsis is a life-threatening multiple organ dysfunction caused by systemic dysregulated inflammatory response to infection. nevertheless, numerous therapeutic trails concerning immune dysfunction have still been disappointing outcomes. we have previously shown that ll-37, a human cathelicidin antimicrobial peptide, improves the survival of caecal ligation and puncture (clp) septic mice. here, we investigated the induction of ev release by ll-37 and functions of ll-37-induced evs in murine sepsis. methods: evs were isolated from peritoneal exudates of clp mice and the supernatant of ll-37-stimulated mouse bone marrow neutrophils by differential centrifugation or size exclusion chromatography. isolated evs were analysed by flow cytometry, western blotting, and nano particle analysis. neutrophil-derived evs were injected into clp mice to assess the protective function of evs against septic mice. the antibacterial activity of evs was evaluated by incubating with escherichia coli. results: in clp mice, ll-37 augmented the level of evs. evs from ll-37-injected clp mice contained higher amounts of neutrophil-derived antibacterial proteins (lactoferrin and cramp, cathelicidin-related antimicrobial peptide) and exhibited higher antibacterial activity compared to evs from pbs-injected clp mice. furthermore, ll-37 stimulated mouse bone marrow neutrophils to release evs with antibacterial potential, and administration of the ll-37-induced evs reduced the bacterial load and improved the survival of clp mice. summary/conclusion: ll-37 induces the release of antimicrobial evs from neutrophils in clp mice, thereby reducing the bacterial load and protecting mice from lethal septic condition. identification of mirna profiles of serum exosomes in active tuberculosis introduction: tuberculosis (tb) has exceeded hiv as the most lethal infectious disease globally for two consecutive years, mainly due to difficulties in achieving early and definitive diagnosis, and timely treatment. exosomes carrying rna, particularly mirna, have demonstrated their functional and diagnostic potential in diseases including tb. however, few published studies have explored whether exosomal mirnas could be used for diagnosis of tb. thus, more systematic and comprehensive study of exosomal mirnas with regard to their potential as non-invasive tb biomarkers is still urgently needed. methods: we searched the gene expression omnibus database for datasets published before december 2019, and performed meta-analysis on available exosomal mirna profile data for healthy control (hc) and active tb clinical specimens . reprocessing next generation sequencing data under uniform parameters and utilizing state-of-the-art bioinformatics analysis. results: we identified many distinct up-regulated and down-regulated differentially expressed exosomal mirna across multiple studies, and further screened the top 10, which might provide a potential panel for differentiation of hc and tb. we classified all differentially expressed mirnas into six expression patterns and identified two persistently up-regulated mirna (hsa-mir-140-3p, and hsa-mir-423-3p) as potential markers during tb progression. moreover, the differential expressed exosomal genes that we screened from the datasets were consistent with the genes overlapped with predicted mrna targets of differentially expressed mirna. pathway and function analysis further demonstrated down-regulated signalling pathways/immune response and up-regulated metabolism and apoptosis/necrosis. introduction: trypanosoma cruzi is a protozoan parasite that causes chagas disease, a relevant source of morbidity in latin america, which has spread to many countries as result of immigration of the people from endemic areas. many studies have been showed that trypomastigote forms of t. cruzi release extracellular vesicles (ev) that increase parasite infection. objectives. here, we aim to test if previous immunization with evs in adjuvant can generate a protective immune response by decreasing the effects of evs in experimental chagas disease. methods: female balb/c mice were immunized by intra peritoneal (ip) administration with 5 × 105 or 107 evs isolated from trypomastigotes forms, with aluminium hydroxide adjuvant (aloh). injections were administered intravenous in 3 doses during 45 days (15 days interval). after immunization, mice were infected intra-peritoneally with 500 trypomastigotes forms. parasitaemia was quantified by counting motile parasites in fresh blood sample drawn from lateral tail veins. mortality and weight were analysed during the infection. in control group, the mice were immunized with aioh. results: the immunization with evs with aloh decreased the blood parasitaemia and the animals survived, while all animals died in the group aloh alone. the animals immunized with evs had an increase of f4/80+ cd11b+ and cd80/cd86 expression in cells isolated from the peritoneum. summary/conclusion: these results indicate that t. cruzi ev antigens can induce an immune response that controls the development and establishment of the experimental chagas disease. introduction: acinetobacter baumannii (ab) is a nosocomial pathogen, of major concern due to its multidrug resistance (mdr) and the recent appearance of hyper-virulent strains in the clinical setting. the world health organization included ab as a critical priority pathogen for the development of novel antibiotics. ab pathogenesis is associated with a multitude of potential virulence factors (vf) that remain poorly characterized. there is growing evidence that outer membrane vesicles (omv) are used as vehicles to transport bacterial proteins that contribute to set up the conditions for the infections. in the present work we studied the physiopathology of mdr ab. we focused on the contribution of non-characterized outer membrane proteins (omps) associated to omvs, with special focus on lipoproteins (lp). methods: we conducted a bioinformatic prediction using available datasets to construct a list of omv-associated omps putatively acting as vf in ab5075. seven genes were selected and the corresponding mutants were obtained from manoil lab collection. physiological analyses of the mutants were performed, and the involvement of the selected proteins in ab pathogenesis was evaluated by adherence, invasion, and cytotoxicity assays on human lung cells a549. results: biochemical analysis indicated similar growth rates in rich media, as well as similar levels of omv production for all the mutants as compared to wt. also, no differences in susceptibility to chaotropic agents were observed, indicating no alteration of the om function as a general permeability barrier. all mutants similarly reduced a549 cell viability, but to a lesser extent than the wt. moreover, three of them exhibited less adhesion and invasion compared to the wt, and omv isolated from these mutants displayed variable levels of cytotoxicity. summary/conclusion: these results suggest roles for the mutant gene products in ab pathogenesis and contribute to the better understanding of ab virulence mechanisms, revealing novel possible targets for therapeutic development. funding: agencia nacional de promoción científica y tecnológica (anpcyt, pict 2017-3536) medicine, nanfang hospital, southern medical university, guangzhou, 510515, china, guangzhou, china (people's republic); d zhujiang hospital, southern medical university, guangzhou, china, guangzhou, china (people's republic) introduction: talaromyces marneffei (t. marneffei) grows as a mycelial form in the environment but multiplies rapidly as a yeast form in the host and within macrophages. the yeast can cause disseminated and progressive infections or lethal talaromycosis. but the mechanisms of pathogenicity of t. marneffei are poorly understood. fungal extracellular vesicles (evs) have previously been shown to transmit a proinflammatory message to macrophages. however, the characteristics and effects of t. marneffei evs on the progress of infection have not yet been investigated. methods: in this study, evs of t. marneffei yeasts were isolated by ultracentrifugation method. evs were detected and confirmed by electron microscopy and nanoparticle tracking analysis (nta). the raw 264.7 murine macrophages were incubated with the t. marneffei vesicles to observe the changes of macrophage morphology and function, especially in inflammatory response. the proteins, dnas, rnas of t. marneffei vesicles were respectively removed with protease, dnase and rnase. all treated evs were used to incubate with murine macrophages observe the effect on macrophages in inflammatory response. results: we observed that evs secreted by t. marneffei have a typical spherical shape with a diameter of 30 to 200 nm. t. marneffei evs were internalized by raw 264.7 murine macrophages and promoted the production of no and proinflammatory cytokine by macrophages in a dose-dependent manner. t. marneffei evs stimulate macrophages to generate reactive oxygen species (ros). addition of t. marneffei evs to macrophages also promoted transcription of the m1-polarization marker cd86 and diminish that of the m2 markers cd206. incubation of t. marneffei vesicles with murine macrophages resulted in increased levels of extracellular interleukin-1β(il-1β), interleukin-6 (il-6) and interleukin-12 (il-12). the proinflammatory effect of vesicles was weakened when the proteins of the vesicles were destroyed. in contrast, no similar changes were observed in degraded dna and rna. summary/conclusion: our results indicate that the extracellular vesicles of t. marneffei can stimulate macrophage towards to m1 polarization phenotype and promote proinflammatory function. plasma-derived extracellular vesicles as potential biomarkers in chronic chagas disease patients introduction: chagas disease (cd), caused by the parasite trypanosoma cruzi (t. cruzi), is a neglected tropical disease affecting about 8 million people worldwide. currently, one of the main clinical problems is the lack of effective biomarkers for therapeutic response and disease prognosis during chronic infections. in that context, extracellular vesicles (evs) are raising attention as novel, minimally invasive, and inexpensive method for diagnostic and screening of diseases, as well as a new source to identify new biomarkers. the main objective of this study is to use evs derived from biological fluids of chronic cd patients for identifying novel biomarkers, specifically in the context of therapeutic response and disease prognosis. methods: plasma, saliva and urine from a cohort of chronic cd patients are being collected before and at the end of benznidazole treatment. as negative controls, healthy donors have been also included. the purification and characterization of the evs was performed by size exclusion chromatography, followed by nanoparticle tracking analysis, bead-based flow cytometry assay and transmission electron microscopy. a proteomic analysis of the evs was also performed. results: proteins associated with evs secreted by infective t. cruzi have been previously identified in cell culture, but never in human samples. our results, based on the analysis of a single heart-transplanted patient with chronic cd, showed the presence of t. cruzi and human proteins specifically associated with plasma-derived evs. noticeably, several human and parasite proteins identified in evs obtained from plasma samples, were present or upregulated before chemotherapy and were absent or downregulated following treatment. currently, proteomics analyses are being performed with higher numbers of cd plasma samples. summary/conclusion: to the best of our knowledge, this is the first proteomic profiling of plasma-derived evs from a heart-transplanted patient with chronic cd. these results thus open the possibility of using evs from biological fluids as a tool for the identification of new biomarker candidates in chronic cd. these biomarkers are essential for assessing disease introduction: eukaryotic cells communicate with one another through multiple pathways. an established route of communication between eukaryotic cells is via the production of a range of different membrane bound signalling "packages", called extracellular vesicles (evs). evs are produced by all domains of life and carry proteins, nucleic acid (rna and dna), and other biological material, travelling between cells and around the body to deliver a range of chemical messages. bacteria can also produce evs that communicate with each other to coordinate population behaviour, as well as with eukaryotic cells to stimulate host defence or induce tolerance. here i investigate the poorly explored axis where evs are the vehicle for communication between eukaryotic cells and bacteria. methods: as a first step, i have isolated evs from tissue cultured eukaryotic cells grown in advanced rpmi media with minimal ev-depleted fbs. nanoevs were isolated from spent culture media using sequential centrifugation (2,000 × g, 10,000 × g) and concentration (100 kda filter) before purifying using size exclusion chromatography columns. nanoev-rich fractions were pooled based on particle (nanoparticle tracking analysis) and protein quantity data. nanoevs were characterised by electron microscopy and expression of exosomal markers. eukaryotic nanoevs were then characterised in their effect upon the growth of escherichia coli as a model bacterium, also grown in tissue culture media to mimic relevant in vivo conditions. results: further experiments with increased dosages are required to determine the effect of human evs on bacteria. summary/conclusion: our work will investigate whether human evs communicate with the resident and pathogenic microbiota, while examining the mechanisms behind this communication. escherichia coli pathogenic bacteria commensal bacteria hydrogen sulphide (h2 s) derived extracellular vesicles: a potential protective role in response to respiratory syncytial virus (rsv) infection methods: evs were isolated from untreated (control evs) and gyy4137 treated (gyy-evs) a549 cells, a human alveolar type ii-like epithelial cell line. evs were purified using a two-step enrichment procedure. evs were characterized using particle sizing (size and concentration) and western blot for the ev markers. electron microscopy and immunofluorescence staining were used to investigate presence of multivesicular bodies (mvbs), evs precursors, in both groups. recipient a549 cells were cultured for 24 hours in the presence or absence of control-or gyy-evs, then infected with rsv for 24 hours. viral titres by plaque assay were measured in recipient infected a549 cells. results: we confirmed the presence and purity of our evs. we found that gyy4137 reduced the particles number of evs, but did not change ev size. a549 cells treated with gyy4137 showed an accumulation of mvbs/lysosomes-like structures, as well as an increase in cd63 expression, a mvbs marker, compared to untreated cells. recipient a549 cells treated with gyy-evs showed lower viral replication than control ev-treated cells in response to rsv infection. we are currently investigating the potential mechanism for this observation and characterizing the rna cargo composition of gyy-evs. summary/conclusion: no vaccine or effective treatment is currently available for rsv. cellular pretreatment with gyy-evs reduced the rsv replication in airway epithelial recipient cells, suggesting that h2 s could exert its antiviral activity in the context of rsv infection potentially through modulation of ev composition. therefore, gyy-evs could represent a future novel pharmacological approach for ameliorating virus-induced lung disease. effects of extracellular vesicle-mediated transmission on reoviridae infection results: taken together, these data suggest that multiple particles of reovirus and rotavirus egress in large, virus-modulated evs, and that transmission in evs increases segment complementation compared to transmission as free particles. summary/conclusion: these discoveries may be broadly applicable to viruses that travel in evs and will contribute to general principles of virus transmission and diversification. continued studies will illuminate the specific cellular pathways reovirus and rotavirus utilize for successful egress. these pathways may prove to be critical targets for the improvement of vaccines and oncolytic therapy. multiparameter flow cytometry analysis of the human spleen and its interaction with plasma-derived evs from plasmodium vivax patients introduction: the spleen is a secondary lymph organ that filters blood and elicits immune responses against blood-borne pathogens, such as malaria parasites. extracellular vesicles (evs) are membrane-bound particles involved in intercellular communication. evs play several roles in malaria ranging from modulation of immune responses to induction of vascular alterations. here, we report the first integrated characterization of human spleen cells using multiparameter flow cytometry (mfc) describing subpopulations of splenic leukocytes and red blood cells (rbcs), and studied their interaction with plasma-derived evs from p. vivax patients (pvevs). methods: human spleens were obtained from organ transplantation donors. myeloid, lymphoid, erythroid and haematopoietic stem cells (hscs) were immunophenotyped by mfc. t cells, dendritic cells (dcs) and rbcs were enriched by density centrifugation and immunomagnetic isolation. pvevs and healthy donors evs (hevs) were purified by size-exclusion chromatography (sec) and characterized by bead-based flow cytometry. enriched evs were labelled with fluorescent lipophilic dyes and incubated with total splenocytes or enriched populations. evs-cells interaction was assessed by flow cytometry. results: human spleen immunophenotyping showed that cd45+ cells included b (30%), cd4 + t (16%), cd8 + t (10%), nk (6%) and nkt (2%) lymphocytes. myeloid cells comprised neutrophils (16%), monocytes (2%) and dcs (0.3%). erythrocytes represented 70% whereas, unexpectedly, reticulocytes were 0.93% of total cells. in addition, we also detected hscs, which accounted for 0.02%. sec separated evs from the bulk of soluble plasma proteins as shown by the enrichment of cd63, cd5 l and cd71 markers. interaction studies showed an increased proportion of t cells (cd4 + 3-fold and cd8 + 4-fold), monocytes (1.5-fold) , b cells (2.3-fold) and erythrocytes (threefold) interacting with pvevs as compared to hevs. summary/conclusion: the integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and evs. a larger proportion of monocytes, t and b lymphocytes as well as erythrocytes was found to interact with pvevs compared to hevs. future functional studies of these interactions can unveil pathophysiological processes involving the spleen in vivax malaria. neuroblastoma-secreted exosomes carrying mir-375 promote osteogenic differentiation of bone marrow mesenchymal stromal cells introduction: bone marrow (bm) is the major target organ for neuroblastoma (nb) metastasis and its involvement is associated with poor outcome. yet, the mechanism by which nb cells invade bm is largely unknown. tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (mscs) have been recognized as a fundamental part of the associated tumour stroma. here, we explore the potential role of nb-derived exosomes in induction of a pro-osteogenic phenotype on bm-mscs. introduction: extracellular vesicles (evs) are nanosized particles delimited by a lipid bilayer which transfer functional molecular cargos from the cells of origin to target cells. this intercellular crosstalk controls both physiological and pathological conditions. given their presence in body fluids and their characteristics, these nanocarriers might be potentially used in diagnostics and/or therapy. breast cancer is the most frequently diagnosed malignancy and ranks as the leading cause of cancer mortality in women worldwide; the triple negative breast cancer, in particular, is the most aggressive subtype with a poor prognosis. since it is recognized that cell stiffness of cancer cells play a crucial role during the metastatic spreading, we set ourselves the goal of clarify the effects and the activity of small-evs (i.e. with a diameter below 200 nm) in metastatic breast cancer, with a special attention on their correlation with the biomechanical properties of cells. methods: functional assays were performed on the non-invasive mcf7 breast cancer cell line, before and after the cellular uptake of small-evs originating from the invasive mda-mb-231 triple negative breast cancer cell line. the mechanical properties (cell stiffness, cytoskeleton organization and focal adhesions) of mcf7 cells were investigated before and after the vesicle uptake. results: the uptake of small-evs derived from mda-mb-231 significantly reduces the young's modulus values of mcf7 cell line making them more invasive. moreover actin and focal adhesion variations were observed in mcf7 cells before and after small-ev's uptake, suggesting a molecular rearrangement inside mcf7 cells upon uptake. summary/conclusion: our results evidence that small-evs play a key role in altering biomechanical properties of target cells and underline their relevance in cell-cell crosstalk. our approach is very promising to identify new molecular mechanisms through which evs perform their oncogenic function. stratification of angiogenic or non-angiogenic lesions in colorectal cancer liver metastases patients using extracellular vesicle mirna introduction: colorectal carcinoma (crc) is the second leading cause of cancer death in the western world. over 50% of the crc patients develop liver metastasis (lm) and 90% will die from metastatic disease. in the current clinical setting, liver resection provides the only possible cure, but only 20% of crclm patients are resectable. the combination of angiogenic inhibitors with chemotherapy is used to downsize crclm with the goal of converting unresectable patients to resectable ones. however, only 10-15% of these patients can be successfully converted to a resectable state. we have no way of identifying those crclm patients that would respond/benefit to the addition of anti-angiogenic therapies (e.g. bevacizumab: bev)). proper stratification of patients into angiogenic inhibitor responders and non-responders will permit a proper assessment of the efficacy of angiogenic inhibitors. crclm forms 2 distinct histopathological growth patterns (hgp): angiogenic (desmoplastic) and nonangiogenic (replacement) hgp. we demonstrated that crclm patients with predominant angiogenic lesions receiving bev plus chemotherapy have a more than double 5-year overall survival compared to patients with non-angiogenic lesions. therefore, nonangiogenic lesions do not respond to angiogenic inhibitors. our study focuses on stratifying angiogenic vs non-angiogenic lesions of crclm through extracellular vesicle mirnas. we are using two approaches in the selection of mirnas to target: 1. text mining of published ev mirna from crclm patients; and 2. differentially expressed mirnas present in tumour tissue from both lesion types, we have obtained by sequencing 50-60 patients. these two strategies will generate a list of mirnas that we will target using qpcr on plasma-derived ev mirna from the patients used in approach 2, where we have classified the lesions in the patients. preliminary data on 10 patients will be presented. methods: ev isolation was performed using the gold standard centrifugation method. rnaseq and qpcr are used to generate the expression profile for angiogenic vs non-angiogenic type of crclm. results: the research is under progress. summary/conclusion: the research is under progress. the introduction: it is known that bone metastasis causes a reduction in the quality of life of cancer patients due to fractures and nerve compression. therefore, it is important to elucidate the mechanism of bone metastasis and develop new treatments. metastatic bone tumours occur at particularly high rates in cancers of the prostate, breast, and lung. in this study, we focused on extracellular vesicles (evs) in bone metastasis, and investigated that the role of evs derived from cancer cells in osteolysis. methods: the prostate, breast, and lung cancer cellderived evs were added to osteoclast precursors with rankls. the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase (trap) stain and by measuring the expression level of osteoclast markers using by qrt-pcr. a proteome analysis (lc-ms/ms) and sirna approaches were used to identify molecules which are responsible for promotion of osteoclast differentiation in the prostate cancer cellderived evs. to investigate whether the molecules are suitable for the detection of bone metastasis in serum evs, we isolated evs from serum of prostate cancer patients, and analysed the protein level of the molecules by western blot analysis. results: we found that the prostate cancer and lung cancer-derived evs significantly promoted the rankl-stimulated osteoclast differentiation. our analysis revealed that cub domain-containing protein 1 (cdcp1), which is a membrane protein on the prostate cancer cell-derived evs, was responsible for promotion of osteoclast differentiation. moreover, cdcp1 was markedly detected in the evs-derived from serum of prostate cancer patients who had bone metastasis than that of normal subjects. we also found that cdcp1 exits on the breast and lung cancer cell-derived evs. summary/conclusion: we showed that the evsderived from bone metastatic tumours have a role in activation of osteoclastogenesis. moreover, we revealed that cdcp1 in the evs is responsible for promoting of osteoclast differentiation. these evs could be the novel diagnostic and therapeutic target for bone metastasis. increased expression of chemokine receptor cxcr4 in non-invasive colorectal cancer cells after incorporation of platelet-derived extracellular vesicles. introduction: blood platelets and platelet-derived extracellular vesicles (p-evs) play a crucial role in tumour growth and metastasis. p-evs, also referred to as platelet microparticles, are recognized as a carrier for proteins and nucleic acids that control cell-to-cell communication, mediate the formation of metastatic niches and affect tumour invasion and metastasis. among the other factors, p-evs contain the chemokine receptor cxcr4, known as a co-receptor for hiv entry but also regarded as important in cancer development due to the importance of cxcr4/cxcl12 signalling. overexpression of cxcr4 was reported in various, especially in invasive cancers, including colorectal cancer (crc). crc, the third most commonly diagnosed cancer, is usually diagnosed at the late stage and patient's death is mainly related to metastasis. increased levels of cxcr4 has been reported as a poor prognostic factor for survival of crc patients and its blocking has been suggested as therapeutic approach. the aim of this study was to analyse the effect of p-evs on the levels of cxcr4 in crc cells on various epithelial-to-mesenchymal transition stage. methods: we used crc cell lines ht29 and sw620, which represent distant invasive potential and different phenotypes, epithelial and strongly mesenchymal, respectively. p-evs were isolated from outdated concentrates of human blood platelets after activation by thrombin in the presence of calcium ions, by subsequent centrifugation and ultracentrifugation. the p-evs were labelled using pkh67 fluorescent dye to visualize their uptake into cell lines by confocal microscopy. we also quantified the levels of cxcr4 in ht29 and sw620 by western blot analysis. the effect of p-evs uptake on the migration of crc cells was studied by "wound healing" method. results: we found that the levels of cxcr4 in crc lines used in the study were correlated with their emt stage. we show here that p-evs released by activated platelets were incorporated into both ht29 and sw620 cell lines. the expression of cxcr4 in ht29 was increased after the uptake of p-evs. additionally we observed that migration rate of ht29 cells with incorporated p-evs was elevated as compared to control cells. summary/conclusion: we posit that circulating p-evs can be incorporated into yet not invasive crc cells to significantly increase the level of cxcr4 receptors and that may lead to the their more invasive characteristics. introduction: for cancer therapy it is important to identify markers and key processes induced during cancer progression. one of them is epithelialmesenchymal transition (emt) which is associated with cell acquisition of invasiveness, stem cell characteristics and resistance to apoptosis and therapy. also the extracellular vesicles (evs) released from tumour cells, which can be taken up by cells constituting pre-metastatic niches, can alter cancer progression by promoting cells' reprogramming. our group has recently reported that snail transcription factor, a key factor of emt, when overexpressed in crc ht29 cells, drives their early emt and alters the expression of microrna (mirs). in the present study we analysed the mirs profile of evs released from those cells. methods: evs from three ht29 clones stably overexpressing snail and from control ht29-pcdna were isolated by differential centrifugation and ultracentrifugation of conditioned media after 24 h of culturing in serum-free medium. total rna was isolated and nextgeneration sequencing (ngs) analysis of the mirnas was performed followed by gene ontology ( introduction: prostate cancer (pca) is the most common malignant tumour in male urinary system and osteoblastic bone metastasis is the most observed metastasis in prostate cancer patients. it has been demonstrated that circulating micrornas contained in extracellular vesicles are potential early biomarkers and therapy targets for many diseases. however, the potential role of micrornas in prostate cancer bone metastasis, is not yet to be fully explored. methods: after isolation and purification evs using ultracentrifugation from conditioned media of bone metastatic co-opting prostate cancer cells and normal cells, total rna was extracted. subsequent to library preparation and small rna-seq, differential gene expression analysis was performed. data were filtered by mean mirna expression of ≥ 50 reads, two fold up or down regulation between 2.5 − 7.5 and adjusted pvalue ≤ 0.05. the uptake of pca-sevs was performed. three candidate mirnas (has-mir-200 c-3p; has-mir-1275; has-mir-383-5p) were internalized and osteoblast differentiation were detected by qpcr, histochemical staining and protein activity detection. results: total reads of mirnas in bone metastatic co-opting pca-evs exceeded significantly than that in normal evs (p < 0.001), indicating that mirnas delivered by pca cells play critical role in pca bone metastasis. pca-cm enhanced osteoblast differentiation and can be reversed by gw4869. the uptake of pca-evs by mc3t3-e1 was efficient. the high expression of the three candidate mirnas in pca-evs was verified by qpcr. all the three candidate mirnas promoted osteogenesis, verified by mrna expression of osteoblastic markers (alp, ocn, runx2, osx), alp activity, alp staining and aliza red s staining. summary/conclusion: these findings suggest that mirna cargos in pca-evs play a pivotal role in the development of osteoblastic bone metastasis of pca, which can be potential early biomarkers and therapy targets for prostate cancer bone metastasis. funding: this work was supported by grants from the national natural science foundation of china (81872347); xijing hospital science and technology foundation project (xjzt19ptk14). introduction: retinoblastoma (rb) is the most common intraocular cancer of childhood. despite recent advances in conservative treatment have greatly improved the visual outcome, local tumour control remain difficult in presence of massive vitreous seeding. thus, the identification of new biomarkers is crucial to design more effective therapeutic approaches. traditional biopsy has long been considered unsafe in rb, due to the risk of extraocular spread. exosomes, nano-sized vesicles containing nucleic acids and proteins, represent an interesting alternative to detect tumour-associated biomarkers. the aim of this study was to determine the protein signature of exosomes derived from rb tumours (rbt) and vitreous seeding (rbvs) primary cell lines. methods: exosomes from 4 rbt (hsjd-rbt1, hsjd-rbt2, hsjd-rbt5, hsjd-rbt14) and 3 rbvs (hsjd-rbvs1, hsjd-rbvs3, hsjd-rbvs10) cell lines were isolated by high speed ultracentrifugation. vesicles number and size were confirmed by nanosight and scanning electron microscopy. protein content was analysed by bicinchonic-acid assay and high resolution mass spectrometry. results: a total of 5666 proteins were identified. among these, 5223 and 3637 were expressed in exosomes rbt and one rbvs group respectively. gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified identified in rbvs exosomes upregulated proteins specifically related to invasion and metastasis such as proteins involved in extracellular matrix (ecm) remodelling and interaction, resistance to anoikis and metabolism/catabolism of glucose and aminoacids. summary/conclusion: in conclusion, in this study, we isolated exosomes from rb primary tumour and vitreous seeding cell lines and characterized their content with a proteomic approach. this is the first evidence describing a proteomic exosome signature specifically associated with vitreous seeding in rb. this characterization may represent a starting point for future analyses that allow defining exosomal markers as promising diagnostic and potential prognostic markers in rb as well as therapeutic targets. activation of hepatic stellate cells by extracellular vesicles released by uveal melanoma cells introduction: uveal melanoma (um) is the main intraocular tumour in adults, and is particularly resistant to treatments when disseminated to the liver. our hypothesis is that extracellular vesicles (evs) released by the primary tumour are priming the liver stroma for metastatic cell colonization by activating hepatic stellate cells (hstecs). this study aimed to characterize evs from um cells, and to determine their interactions with liver cells. methods: evs were isolated from cell lines derived from ocular tumours and liver metastases by differential centrifugation. their concentration/diameter range were determined by high-sensitivity flow cytometry. cryo-tem combined with receptor-specific gold labelling was used to reveal the morphology/size of melanomic evs. the presence of melanoma and ev markers was assessed by western blotting. the internalization of fluorescent melanomic evs in hstecs and their subsequent activation were assessed by confocal imaging using alpha-smooth muscle actin (alpha-sma) and phalloidin stainings. ev impact on invasion was measured with a tumour spheroid model embedded in extracellular matrix. melanomic evs were inoculated into the retro-orbital sinus of immunodeficient mice to study their selective organ distribution. results: melanomic evs were positive for annexin-5, tetraspanins, as well as some melanoma markers. stellate cells with internalized melanomic evs expressed more alpha-sma, reflecting their activation. adding evs on tumour spheroids increased the invasion process. melanomic evs were localized into different murine organs, but mainly into the liver, as observed by in vivo fluorescent imaging. introduction: exosomes are being tested for their use as therapeutic agents in degenerative and chronic diseases. however, the optimal source of exosomes is currently under investigation. amniotic fluid (af) is a naturally-rich source of exosomes that is easily obtained for use in regenerative medicine. organicell flow™ is a minimally-manipulated, acellular product derived from human af and consist of over 300 cytokines/chemokines as well as exosomes derived from the amniotic membrane and surrounding tissues. we characterized the exosome fraction of our product to elucidate the protein cargo of af exosomes and demonstrate the therapeutic potential as a novel regenerative therapy. methods: the exosome fraction of our product was analysed using nanosight nanoparticle imaging and macsplex exosome surface marker array analysis. exosomes were precipitated using size-exclusion filtration followed by ultracentrifugation from 3 independent products (in triplicate) and subjected to protein lysis and preparation for mass spectrometry analysis using the easy nlc 1000 and q exactive instruments. tune (version 2.9) and xcalibur (version 4.1) was used to collect data while proteome discoverer (version 2.2) was used to analyse data. protein expression lists were created by merging the 3 sample replicates together and commonly expressed proteins were determined using vinny 2.0 vin diagram analysis. webgestalt tool kit classification system was used to identify top protein function and pathway hits. results: organicell flow™ contain a mean concentration of 5.24x10^11 particles/ml (n = 12) with a mean mode size of 125.2nm (n = 12). surface marker analysis confirms the presence of exosome associated proteins cd63, cd81, and cd9 in addition to a high expression of cd133 (n = 3). the completed analysis revealed 1225 commonly detected proteins across 3 products. the top molecular functions of identified proteins included protein-binding, ion-binding, and nucleic acid-binding with enzymes, transcription regulators, and transporter proteins representing the most abundant protein groups. pathway enrichment analysis revealed top hits for integrin, pdgf, and p53 pathways. a deeper dive into the enzyme category of the protein cargo further demonstrates the presence of proteins that promote dna repair such as dna polymerase (beta and lambda), telomerase reverse transcriptase, and brca1. summary/conclusion: organicell flow™ characterization demonstrates the therapeutic potential of afderived exosomes. proteomic analysis revealed protein cargo that may regulate various growth factor and cellcycle associated pathways. furthermore, the presence of dna damage response proteins suggests a possible mechanism for induction of cellular repair. generation of car-t and γδt cell-derived exosomes for future cell free immunotherapies γδt cells are a subset of t cells with dual innate and adaptive qualities. this duality provides various advantages over their more studied and used counterpart, αβt cells. in the present study, we sought to compare the immunotherapeutic potential of car-t cell and γδt cell-derived exosomes as novel cell-free based alternatives. methods: cd19-targeting car-t cells were obtained following the isolation, expansion and transduction of αβt cells using a lentiviral vector bearing the car construct. γδt cells were isolated and expanded from peripheral blood mononuclear cells (pbmcs) following innate or adaptive stimulation. exosomes from both cell sources were isolated after a 3-day culture in serum-free media using ultracentrifugation-based methods. exosomes were characterized by nanoparticle tracking analysis (determination of size) and western blot assays (detection of the appropriate surface markers). nalm-6 (b cell precursor leukaemia) cells were used as target cells for assessment of exosome cytotoxic/ killing function. car-t cell and γδt cell-derived exosomes were incubated at 1000 particles/target cell for 24-hours. total viable cell counts were assessed via imaging-based cytometry (nc-3000) utilizing acridine orange and dapi staining. results: exosomes derived from γδt cells activated via innate mechanisms showed significant killing of nalm-6 as compared to exosomes from non-activated or adaptively activated γδt cells. in comparison, car-t cell-derived exosomes showed minor killing capabilities of the target cells. summary/conclusion: here, we report for the first time that exosomes derived from cd19 car-t cells and innately activated-γδt cells show/exert inhibitory action on nalm-6 cells. further studies are currently underway to identify the underlying mechanism(s) responsible. introduction: age-related cognitive dysfunction is associated with increased oxidative stress, low-level chronic neuroinflammation, and waned hippocampal neurogenesis in the brain. from this perspective, biologics capable of modulating oxidative stress and neuroinflammation, and stimulating neural stem cell activity in the brain might be useful as anti-ageing interventions. methods: we investigated the efficacy of intranasal administration of extracellular vesicles (evs) generated from cultures of rat subventricular zone neural stem cells (svz-nscs) in the middle-aged mice to alleviate cognitive and mood dysfunction, increased oxidative stress, neuroinflammation, and neurogenesis decline in old age. mice were treated intranasally with nsc-evs once weekly for three weeks (50 billion per administration) starting from 18.5 months of age. a month later, the animals were examined for cognitive, memory, and mood function using multiple behavioural tests, and brain tissues were examined for oxidative stress, neuroinflammation, and neurogenesis. results: object-based tests revealed that aged animals receiving vehicle displayed cognitive impairments for discerning minor changes in the environment as well as for distinguishing similar but not identical experiences. these animals also exhibited spatial memory dysfunction and anhedonia. in contrast, aged animals receiving nsc-evs showed improved cognitive and mood function. biochemical analyses of brain tissues revealed that nsc-ev treatment normalized elevated concentrations of oxidative stress markers malondialdehyde and protein carbonyls and the proinflammatory cytokine interleukin-1 beta. moreover, nsc-ev treatment stimulated increased production of antiinflammatory protein interleukin-10 and the antioxidant superoxide dismutase. immunohistochemical analysis revealed modulation of neuroinflammation typified by reduced activity of reactive astrocytes and activated microglia and improved hippocampal neurogenesis. summary/conclusion: the results suggest that the intranasal administration of nsc-evs is a promising approach for maintaining better cognitive and mood function in ageing through modulation of oxidative stress, neuroinflammation, and neurogenesis. funding: supported by a grant from the national institute of neurological disorders and stroke (1r01ns106907-01 to a.k.s.) chemically modified myocytes-derived evs for the treatment of cardiac fibrosis. marta prieto-vila a , asao muranaka a and takahiro ochiya b a tokyo medical university, tokyo, japan; b tokyo medical university, shinjuku-ku, japan introduction: myocardial fibrosis is a disorder that may occur after cardiac injure due to a malfunction of the cardiac remodelling. fibroblasts resident in myocardium are erroneously activated causing an excessive accumulation of extracellular matrix, which decreases cardiac function and eventually, leads to death. it is known that cardiomyocytes communicate with the surrounding cells such as fibroblast and endothelial cells by extracellular vesicles (evs). the loss of this communication is thought to play a central role in cardiac fibrosis. therefore, cardiomyocytes-derived evs may be a promising a cell-free system for the treatment of fibrosis inhibition. methods: a novel culture medium was stablished to improve the expansion of primary cardiac myocytes. this was tested using two commercially available primary myocytes cell lines. evs were collected by serial ultracentrifuges, and their effect on fibrosis was tested. for that, prior to any treatment, and to mimic fibrosis, primary cardiac fibroblast were activated overnight with tgfβ. results: by the use of a defined conjunct of chemicals, mature cardiomyocytes culture was highly improved to ensure a high collection of evs. terminal differentiation markers, as well as senesce apparition was delayed in comparison to predetermined culture medium. interestingly, those primary cells secreted a rather large amount of evs, which expressed common evs membrane marker. tgfβ-treated cardiac fibroblasts were co-cultured with myocytes showing a decrease of fibroblast activation markers both at mrna and protein levels. similar results were found when activated fibroblast were treated with evs. summary/conclusion: our findings indicate that the use of evs derived from chemically modified myocytes is a promising treatment for ischaemic myocardial fibrosis. however, further molecular experiments have to be done to identify the molecules within evs responsible for the inactivation of fibroblast. evaluation of osteoinductive and anti-inflammatory properties of spinederived exosomes renaud sicard a , tania del rivero b , jonathan messer c , shabnam namin c and timothy ganey c a vivex, biologics, inc., miami, usa; b vivex, biologics, inc., miami, usa; c vivex, biologics, inc., miami, usa introduction: over the last 2 decades, mesenchymal stem cell-derived exosomes have been shown to play a crucial role in a myriad of cell function such as extracellular matrix synthesis, proliferation, differentiation or cell migration. biological sources of exosome (heterogeneous or homogeneous cell population, serum, urine etc.) have a direct influence on the content of their cargo and their therapeutic application and potential. in this study, we evaluated exosomes excreted from cadaveric spine-derived cells. we hypothesized that exosomes derived from a bone source such as the spine, will drive the osteogenic differentiation of progenitor cells. we also investigated their effects on inflammation in nucleus pulposus cells using an in-vitro assay. methods: after their isolation and characterization, exosomes derived from cadaveric human spines were assayed for osteoinductive properties. a c2c12 myoblast cell line was treated with different concentrations of exosomes and expression of alkaline phosphatase was measured after 5 days incubation. treatment with bmp-4 was used as positive control. anti-inflammatory properties were assessed by incubating tnf-treated nucleus pulposus cells with exosomes for 3 days. qpcr analysis of mrna expression of inflammatory cytokines (il-6, il1-beta, il-8) metalloproteinases (mmp3 and adamts4), and apoptotic genes (bax, bcl2) was used to determine the effects of exosomes on inflammation. results: spine-derived exosomes positively expressed the exosome flow cytometry markers tested (cd81, cd63 and cd9). the mean number of exosomes per microgram of protein was 3.31 ± 2.33 x 108 indicating a relatively high purity. osteoinductive (oi) testing was performed using different concentrations of exosomes. the oi index of treatment of c2c12 cells with bmp-4, 2 x 108, 1 x 109, 2 x 109, 5 × 109 or 1 × 1010 exosomes alone was 28. 5, 1.0, 3.7, 7.4, 11.8 and 27.6 respectively. anti-inflammatory properties of exosome are currently being assessed and will be presented at the time of the poster presentation. summary/conclusion: administering exosomes alone or in combination with an exogenous scaffold has the potential to repair injured tissue and to restore bone function. the clinical significance of this application is aimed to promote the patients' bone healing process and provide a cell-free therapeutic platform that is safe and effective. administration of human mesenchymal stem cell derived extracellular vesicles modulates the abnormal plasticity of newly born neurons and neuroinflammation in a rat model of status epilepticus maheedhar kodali a , daniel gitai b , dong ki kim a , mariam atobiloye a , bing shuai c , sahithi attaluri c , raghavendra upadhya c , leelavathi n madhu a , olagide w. castro a , darwin j. prockop a and ashok k. shetty c decline in the percentage of newly born neurons displaying basal dendrites. besides, ev treated animals displayed higher percentages of resting microglia (ramified microglia), reduced percentages of activated microglia (microglia expressing iba-1 and cd68), in comparison to animals receiving vehicle after se. interestingly, diminished abnormal plasticity of newly born neurons was accompanied by the preservation of interneurons positive for reelin; a protein believed to guide newly born neurons to their correct locations. summary/conclusion: the results suggest that even a low dose in administration of msc-derived evs after se can limit neurons loss, dampen the abnormal plasticity of newly born neurons, and modulate the activation of microglia. introduction: autism spectrum disorders (asd) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. they have been steadily increasing in children over the past several years, with no effective treatment. two percent of all asd patients are suffering from a disorder caused by a mutation in the shank3 gene. shank3 is an important synaptic protein, disruption of this gene directly leads to cognitive and motor impairments. during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. here we test three different autistic mice models. btbr as a multifactorial mice model of autism and two different shank3 mutated mice. the first is a complete deletion of exon 22 (22q13.3) and the second is a specific insertion mutation of guanine to 3680 position in the gene (insg3680) that leads to stop codon. methods: exosomes were isolated using differential centrifugation protocol and characterized using the 2018misev guideline recommendations. each animal received an intranasal administration of 20ul containing 107 exosomes/µl. for intravenous administration, the same number of exosomes, were used, injected in 100µl. results: all three animal models showed significant improvement in their autistic behavioural phenotypes following intranasal administration. the improvement seems to be dose-dependent and was better achieved via intranasal vs intravenous administration. biodistribution of msc-exo showed accumulation in the brain within 72 hours, yet the reduction of the signal was observed in the kidneys, heart and lungs. summary/conclusion: our data suggest that exosomes derived from adipose msc, carry a therapeutic potential in asd, via non-invasive intranasal administration in three different mice models. these data further emphasize our potential therapeutic strategy to reduce symptoms of autism in clinical trials. funding: stem cell medicine ltd. israel. equine tendon injury treatment by evs: an in vitro study introduction: current treatment options for tendinopathies (chronic, painful tendon disorders), are not able to restore the functional properties of native tendons. hence, new treatment options are sought. the efficacy of mesenchymal stem cells (mscs) therapies, which combined with a rehabilitation programme including controlled exercise is the current gold standard in equine tendon treatment, has been shown to be largely due to the cells´paracrine activity. the aim of this study was therefore to evaluate the effect of bone marrow msc derived autologous and allogeneic conditioned medium (cm, full secretome) and their extracellular vesicles (evs) on "tendon healing" in vitro. methods: to compare the "therapeutic" effect of msc derived evs and cm, a standardized scratch assay (wound healing assay) was performed. cm from equine tenocytes, ev depleted medium and medium with or without fcs served as controls. tendons and bone marrow aspirates were obtained from three horses (6, 19 and 23 years) which were euthanized for reasons unrelated to this study. mscs were isolated by ficoll density gradient centrifugation and tenocytes were obtained by migration from tendon explants. for cm and ev production, cells were cultured in ev depleted medium. evs were harvested by a stepwise ultracentrifugation approach and characterized by nanoparticle tracking analysis (nta), western blot (cd9, cd63) and transmission-electron microscopy (tem). results: western blot, nta and tem confirmed successful isolation of evs from equine mscs. the strongest positive effect on wound healing (fastest gap closure) was achieved by msc-cm (p < 0.0071). the gap closure achieved with msc-evs was slower than with msc-cm (p < 0.7985) but faster than with cm of tenocytes (p < 0.8289). donor specific differences in wound healing capability were shown for both autologous and allogeneic application. summary/conclusion: treatment with msc-cm resulted in significantly faster wound healing of adult tenocytes in vitro than msc-evs or tenocyte-cm. mscs donor age shows a significant effect on gap closure following autologous but not allogeneic administration. ev-enriched secretome fraction from gmp-compatible, scalable, human ipsc-derived cardiac progenitors improve heart function in chronic heart failure mice introduction: we have shown that research-use-only grade (res) human ipsc-derived cardiac progenitors (cpcres) can produce a secretome whose small-evenriched fraction (svf) can treat chronic heart failure (chf) in mice. gmp-compatible, scalable processes for a cpc-derived svf suitable for human therapeutic use is needed. methods: ipsc-derived cpc were produced and cultured using gmp-compatible, scalable processes (cpctx). media without cells were "cultured" in parallel for "virgin media" controls (mv). cpcres were cultured as previously described. as a proof of concept, svfs were isolated from conditioned media by ultracentrifugation: cpctx-ev, cpcres-ev and mv. particle size distributions/concentrations (nanoparticle tracking analysis), protein levels (bsa), and the presence of cd-63 (elisa) were determined. in vitro activity was assessed by huvec scratch wound healing assay, and by rat and human cardiomyocyte (cm) survival assays. c57bl/6 mice in chf received echoguided myocardial injection of pbs vehicle control (60ul, n = 11), cpctx-ev (60ul, n = 11), or cpcres-ev (45ul, n = 11). change in cardiac function was assessed by echocardiography. results: cpctx-ev particle sizes were polydisperse (mode~72 nm) at a concentration of~1.6e11 particles/ml (~2,600 particles/cell) and~0.975mu cd63/ug protein. cpctx-ev increased wound healing, human cm survival, and rat cm survival in vitro by 4.75x, 1.4x, and 2x, respectively over mv controls. in chf mice, significantly less cpctx-ev mice, and less cpcres-ev mice had severely progressive heart failure (left ventricular end systolic volume, lvesv, increased >14%) than pbs control mice (pbs vs cpctx-ev, p < 0.05; pbs vs cpcres-ev, p < 0.056), and the average ejection fraction of the pbs group deteriorated 2.5x more than the cpctx-ev group (−4% vs −1.6%, respectively; ns). summary/conclusion: we have a process for cpc differentiation and production of conditioned media suitable for use in human clinical trials from which can be made an svf with the potential to treat chf, possibly through re-vascularization or preservation of cm viability. introduction: exosomes are nanoscale vesicles that mediate cell-to-cell communication via exchanging molecular cargo. mesenchymal stem cell (mscs) modification towards an osteogenic path can occur by uptake of exosomes from other cells. it is less clear whether vesicle placement in the absence of cells will facilitate site-specific delivery through acellular transfer of osteogenic activity. an electrospun fleece was combined with bone marrow-derived exosomes in the absence of cells to evaluate osteoinductive potential that might be thermo-stable and be used in a biologically neutral collagen carrier. comparisons were made of standard laboratory assay of osteoinductivity (oi), and in vivo expression in a mouse calvarial defect model. methods: electrospun type-i collagen was prepared with and without hydroxyapatite (ha) (spinplant gmbh, leipzig) as a foundation base for application of the bone marrow-derived exosomes. individual discs of the collagen enhanced scaffolds (3-mm) were prepared and placed in a mouse calvarial skull defect. animals were followed for 4 and 8 weeks. exosomes were isolated from qualified cadaveric human spines by differential ultracentrifugation. microscopic observation, quantitative assessment of oi with an alkaline phosphatase assay, and flow cytometry were used to evaluate the composition, the hybrid nature of the addition to the nano-collagen fibres. a fluorescent protein reporter transgenic mouse model expressing osteocalcin, type-i collagen, phex, and sp7 (osterix) was evaluated at 4 and 8 weeks to determine bone formation across the defect. results: alp activity on the scaffold with ha demonstrated an approximate tenfold increase to that of the collagen scaffold alone. while a dose-dependent effect, with higher doses of exosomes resulting in a greater amount of alkaline phosphatase expression, expression that exceeded that of the 50ng bmp-4 control. dose escalation from 1.5, 2, and 4e9 resulted in similar increases in expression that was statistically greater with the combination of the fleece with the exosome component. bone formation in the mouse calvaium did not demonstrate gap closure at 4 or at 8 weeks, but did demonstrate enhanced osteoclastivity and robust bone remodelling at the margins of the defect. summary/conclusion: bone marrow-derived exosomes dried into an electrospun fibrillar collagen demonstrated in vitro osteoinductive potential that might provide site-specific placement that could enhance biologic potential. with the capacity for ambient temperature storage, the provision of site-specific placement becomes a technical consideration. placement of the human tissue derived exosomes in a transgenic mouse calvarial defect model did not demonstrate bridging bone across the defect. exosomes loaded with pten-interfering rna enables functional recovery in rats after complete spinal cord transection daniel offen a , nisim perets a , shaowei guo b , oshra betzer c , rachela popovtzer c and shulamit levenberg b a tel aviv university, tel aviv, israel; b technion, haifa, israel, haifa, israel; c bar ilan university, israel, ramt gan, israel introduction: complete spinal cord transection is a debilitating disease that usually leads to permanent functional impairments, with various complications and limited spontaneous recovery. the current investigation of molecular mechanisms controlling axon regeneration, (e.g., signalling networks and environmental cues), led to new strategies to enhance axonal regeneration. we have previously shown that intranasal administration of mesenchymal stem cells derived exosomes (msc-exo), cross the blood-brain barrier and significantly ameliorate motor and behavioural phenotype in several animal models of neurotrauma and neuropsychiatric disorders. methods: msc-exo were isolated from human bone marrow and were loaded with phosphatase and tensin homolog small interfering rna (pten-sirna). the exosomes were given intranasally to rats two hours after complete spinal cord transaction. eight weeks later we followed the motor function and histology and electrophysiology study was performed in order to reveal the connectivity and the biochemical changes in the treated rats. results: we demonstrate that intranasal (in) administrations of msc-derived exosomes could penetrate the blood-brain barrier, home selectively to spinal cord lesion via chemotaxis, and integrated in neurons within the lesion. furthermore, in rats with complete spinal cord transection, msc-exo loaded with pten-sirna silenced pten protein expression in the lesion and promoted robust axonal regeneration and angiogenesis, companied with decreased astrogliosis and microgliosis. moreover, the intranasal treatment partially restored electrophysiological and structural integrity, and most importantly, enabled the remarkable functional recovery and significant improvement in their movements. summary/conclusion: this rapid, non-invasive, approach, using cell-free nano-swimmers carrying molecules to target pathophysiological mechanisms suggest novel strategy for clinical translation to spinal cord injury and beyond. a novel umbilical cord derived wharton's jelly formulation for regenerative medicine applications introduction: musculoskeletal injuries have traditionally been treated with activity-modification, physical therapy, pharmacological agents and surgical procedures. these modalities have limitations, as well as potential side-effects. over the last decade, there has been an increased interest in the use of biologics for regenerative medicine applications (rma), including umbilical cord (uc) derived wharton's jelly (wj). despite this increase, there is insufficient literature assessing the amount of growth factors, cytokines, hyaluronic acid (ha) and extracellular vesicles (ev) including exosomes in these products. the purpose of this study was to develop a novel wj formulation and evaluate the presence of growth factors, cytokines, ha and ev including exosomes. methods: wj was isolated from human-uc obtained from consenting c-section donors and formulated into an injectable form. randomly selected samples from different batches were analysed for sterility testing and quantified for presence of growth factors, cytokines, ha and particles in ev size range. the results showed all samples passed the sterility test. growth factors including igfbp 1, 2, 3, 4 and 6, tgfα, pdgf-aa were detected. expression of several immunomodulatory cytokines, rantes, il-6 r, il-16, were also detected. expression of pro-inflammatory cytokines mcsfr, mip-1a; anti-inflammatory cytokines tnf-ri, tnf-rii, il-1 ra; and homoeostatic cytokines timp-1 and timp-2 were observed. cytokines associated with wound-healing, icam-1, g-csf, gdf-15, and regenerative properties, gh were also expressed. high concentrations of ha were observed. particles in the ev size range (30-150 nm) were detected and were enclosed by the membrane, indicative of true ev. summary/conclusion: our results confirmed the presence of numerous growth factors, cytokines, ha and ev in the wj formulation. more studies are underway to confirm the presence of exosomes in detected ev using exosome-specific markers. we believe the presence of multiple factors within one wj formulation may play a role in reducing inflammation, pain and augment healing of musculoskeletal injuries. this offers a potential expanded use for rma. funding: this study was funded by biointegrate llc, new york, ny, usa. collagen sponge loaded with mesenchymal stem cell-derived small extracellular vesicles promote robust bone regeneration shang jiunn chuah a , chee weng yong a , jacob ren jie chew a , ruenn chai lai b , yi ann cheow a , raymond chung wen wong a , asher ah tong lim a , sai kiang lim c and wei seong toh d introduction: mesenchymal stem cell (msc) therapy has demonstrated effective bone regeneration in clinical studies. however, the therapeutic efficacy of mscs have been attributed to the secretion of extracellular vesicles (evs), particularly 50-200 nm small evs (sevs). here, we investigate the efficacy of msc-sevs loaded in collagen sponge in the regeneration of critical-sized calvarial defects in immunocompetent rats. methods: sevs were isolated from conditioned medium of human mscs and stored at −20 c. calvarial defects of 8-mm diameter were surgically created on thirty-two 10-week-old male sprague-dawley rats. these rats were then randomly assigned to 2 groups (n = 16 rats/group): defects treated with collagen sponge containing 100 μg of sevs in 100 μl saline (cs/sevs) and defects treated with control collagen sponge containing an equivalent volume of saline (cs/control). at 1 and 8-week post-surgery, the calvarial bone samples was harvested for analyses by micro-computed tomography (micro-ct), histology, immunohistochemistry and histomorphometry. results: at 1-week post-surgery, micro-ct analysis showed little bone formation at the defect site in both cs/sevs and cs/control groups. no statistical differences were observed in micro-ct and histology scores in both groups. interestingly, cs/sevs group showed significantly higher osteocalcin (ocn)+ area of 5.7 ± 2.4% than that of cs/control group (2.1 ± 1.3%; p = 0.003). cd31+ microvessels at sizes ≤50 µm and >50 µm in cs/sevs group (34.4 ± 2.9 and 8.6 ± 2.1 microvessels/hpf) were also significantly higher than that of cs/control (18.8 ± 1.1 and 5.2 ± 1.4 microvessels/hpf; p = 0.002 and p = 0.038 respectively). by 8 weeks, cs/sevs group displayed enhanced new bone formation that completely bridged the calvaria defect. in contrast, rats in cs/control showed limited bone formation. consequently, cs/ sevs group displayed a micro-ct score of 3.9 ± 0.2 which was significantly better than that of cs/control group (2.5 ± 0.8; p = 0.001). cs/sevs group also exhibited >twofold increase in bone volume, and improved bone quality with higher trabecular thickness and number, and smaller separation (p < 0.01), compared to cs/control group. consistently, cs/sevs group displayed a significantly better histology score of 5.4 ± 1.0 than that of cs/control (2.6 ± 1.8; p = 0.001). moreover, cs/sevs group showed significantly higher ocn+ area of 48.9 ± 7.9% than that of cs/control group (20.4 ± 4.4%; p = 0.004). summary/conclusion: this study demonstrates that single-stage implantation of collagen sponge loaded with ready-to-use msc sevs can promote robust bone regeneration in a rat calvarial defect model. funding: national university of singapore, r221000114114, national medical research council singapore, r221000123213. immunomodulatory potential of extracellular vesicles derived from mesenchymal stromal cells introduction: extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) are promising new agents in regenerative medicine and immunotherapy. considering that independent msc-ev preparations might differ in their therapeutic function, we have set up a functional assay allowing testing for the potential immunomodulatory properties of independent msc-ev preparations. methods: human peripheral blood-derived mononuclear cells (pbmcs) were pooled from up to 12 different healthy donors warranting high allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. after thawing, mixed pbmcs were cultured for 5 days in the absence or presence of msc-evs. thereafter, cell morphologies were documented, supernatants were harvested for cytokines quantification and cells were phenotypically characterized by flow cytometry. by analysing the expression of a collection of different lineage and activation markers, we selected a panel of antigens apparently being regulated by msc-ev preparations considered to be therapeutically active. results: we observed that in the presence of active msc-ev preparations more cd14+ (monocytes) are recovered from the mlr assay than in corresponding control samples. focusing on t cells, we learned that active msc-ev preparations reduced the content of cd4 and cd8 t cells expressing activation markers like cd54 and cd25. summary/conclusion: the mlr assay allows elaborated functional testing of immunomodulatory activities of given msc-ev preparations. currently, we are comparing the immune modulatory capabilities of evs derived from distinct sources and optimize the marker panel to distinguish discrete immune cell subtypes such as different cd4 cell types, i.e. th1, th2, th17 and tregs. extracellular vesicles in platelet-rich plasma: dependency on sample processing zala jan a , saba battelino b , darja božič c , matej hočevar d , ales iglič e , marko jeran c , manca pajnič a , ljubiša pađen a , domen vozel f and veronika kralj-iglič a introduction: platelet-rich plasma (prp) proved effective in regenerative medicine. numerous protocols for its preparation and application are available in the published literature. prp possesses important immune, haemostasis and regenerative factors, however, the mechanisms of their action are yet poorly understood. extracellular vesicles (evs) could be one of the important factors that would contribute to the beneficial effects of preparations. this study was performed as a part of a registered randomised controlled clinical trial (nr: nct04281901). prp was used to treat chronic middle ear inflammations. here we present the results of prp analyses from 29 blood samples of volunteers with no record of disease. methods: plasma obtained from 20 ml of blood was depleted of erythrocytes and enriched with other particles by repetitive centrifugation of samples. flow cytometry (fcm) was employed to monitor particle contents (cells and smaller particles) throughout the sample processing. the platelet gate was divided into two parts: intact platelets and smaller particles. identity and morphology of particles in the preparations were examined by scanning electron microscopy (sem). standard laboratory tests of blood were performed. results: sem images revealed the presence of heterogeneous population of particles in the preparation of prp, most of which were activated and partially fragmented platelets. the population of smaller particles measured with fcm, was identified as evs. the erythrocyte sedimentation rate was statistically significantly correlated to the volume of plasma obtained in the initial centrifugation step (r = 0,70, p < 0,001) and to the concentration of evs (r = 0,82; p < 0,0001). time from sample collection to the preparation of prp was negatively correlated with the concentration of platelets in prp and positively with the concentration of evs (r = 0,75, p < 0,001). platelet concentration in preparation samples was found to depend on the concentration of platelets in the blood and parameters of sample processing connected with larger centrifugal and shear forces on the samples during centrifugation. these include: sample volume, the size and shape of the centrifuge tube and the distance of the sample from the rotor axis. summary/conclusion: evs are gradually forming upon activation and degradation of cells in the sample throughout the sample processing. optimal processing may importantly contribute to the healing properties of preparation. funding: authors acknowledge support from the european union's horizon 2020 research and innovation program under grant agreement no. 801338 (ves4us project) and slovenian research agency (arrs, grants p2-0232, p3-0388, j1-9162). satellite cell-derived extracellular vesicles as a therapeutic for mitochondrial dysfunction in duchenne muscular dystrophy duchenne muscular dystrophy (dmd). sc-derived extracellular vesicles (sc-evs) may unlock the therapeutic potential of scs by overcoming these limitations. to investigate their therapeutic potential, we assessed the ability of sc-evs to reverse mitochondrial dysfunction, a key pathological feature of dmd, in oxidatively-damaged c2c12 and primary dmd myotubes. methods: scs from c57 mice were isolated and cultured. evs were isolated from the supernatant of scs via polyethylene glycol precipitation and characterized using nanoparticle tracking analysis. the ability of sc-evs to deliver protein cargo to c2c12 myotubes, and the localization of the cargo once delivered, were analysed using fluorescence microscopy. to examine sc-ev potential to restore the function of damaged mitochondria, c2c12 myotubes were treated with 50 µm h2o2 for 24 h followed by treatment with 3.12x108 sc-evs for 24 h. separately, cultured dystrophic myotubes were treated with 3.12 × 108 evs every 24 h for 72 h. in both sets of experiments, maximal oxygen consumption rate (max ocr) was measured via seahorse xf cell mito stress test. where appropriate, a t-test was performed to test for statistical significance (p < 0.05). results: based on estimated cell number and ev quantification, each sc released approximately 2.35 × 105 ± 3.10x104 evs/day. evs delivered protein cargo into myotubes within 2 h. fluorescent labelling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. incubation of myotubes with h2o2 resulted in a 42% decline in max ocr relative to untreated myotubes. subsequent treatment with sc-evs resulted in a 76% increase in max ocr. treatment of undamaged myotubes with sc-evs had no effect on max ocr. primary dmd myotubes treated with sc-evs showed a 78% increase in max ocr relative to untreated dmd myotubes. summary/conclusion: sc-evs rapidly deliver proteins into myotubes, much of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged and dystrophic myotubes. introduction: flow cytometry has been used extensively for analysis of ev particles stained with fluorescent antibodies directed to the known cell surface markers. quantitation of the surface markers in terms of the number of molecules or the number of antibodies bound per specific marker has remained one of the largest challenges in the ev research field. changes in instrument setup as well as changes in fluorescent antibodies from different vendors, all impact the relative mfi values for the same ev sample. in this work we report a standardization method of quantitating extra-cellular vesicle surface markers with mesf liposomes. methods: liposomes labelled with fitc fluorescent dye were prepared with a bd proprietary technology. dynamic light scattering analysis was used for size determination of the liposomes. bd facsaria™ fusion system, modified with a small particle side scatter module (sp ssc), was used for analysis of the labelled liposomes by flow cytometry. results: we created a set of 180 nm fitc-modified liposomes of various fluorescent intensities with a known number of fitc molecules incorporated in each liposome intensity. the mfi values of each liposome population (intensity) had a linear relationship to the amount of fitc used for labelling the liposome nanoparticles, suggesting that no self-quenching of fitc fluorescence had occurred. the number for the fitc fluorophores for each liposome intensity was expressed in the units of molecules of equivalents soluble fluorochrome (mesf). a plot of mesf vs. the fluorescent intensity of the liposomes (mfi values) obtained from flow cytometry analysis provided a calibration curve, from which the fluorescent intensity (mfi value) of a stained ev sample can be converted to the number of fluorophores bound (mesf value) to the surface of the ev particles. summary/conclusion: by this approach, the mfi values of stained ev particles are converted to standardized mesf values that are independent of instrument variation, resulting in further improvement of inter-laboratory standardization. furthermore, utilization of liposomes with similar size and refractive index to ev particles simplifies the data evaluation and improves the accuracy of ev surface marker quantitation by flow cytometry. currently, other fluorescent dyes are being explored to expand the utility of mesf liposomes with other fluorescent colours. measuring cholesterol as a high-throughput method for quantifying extracellular vesicles introduction: the extracellular vesicle (ev) field currently lacks a high-throughput method for accurately quantifying evs in solution. ev quantification has traditionally relied on nanoparticle tracking analysis (nta), which is time intensive and indiscriminately counts non-ev particles, such as membrane fragments and protein aggregates. we have rigorously assessed two commercially available methods for measuring cholesterol, a major lipid component of the ev lipid bilayer, and evaluated the utility of these assays to quantify evs in minimally processed samples. methods: the amplex® red cholesterol assay and cedex bio ht were used to quantify cholesterol in ev samples via enzymatic oxidation, with dynamic ranges of 80-8,000 ng/ml and 4-800 µl/ml, respectively. samples throughout various stages of purification were analysed, from clarified cell culture medium to highly purified evs separated on an iodixanol gradient. we evaluated several pre-processing methods, to remove non-ev cholesterol content prior to analysis. results: the amplex® and cedex bio ht assays were found to perform comparably for quantifying cholesterol in purified evs (r2 = 0.92). importantly, cholesterol quantification on purified ev samples, ranging from 1e11 to 4e13 particles/ml, correlated well with nta measurements (r2 = 0.96). both 45 µm filtration or an additional 16,000 rcf centrifugation step following clarification removed cholesterol associated with cellular debris or other non-ev sources, allowing for accurate quantification of conditioned medium samples or ultracentrifugation pellets (ucp) instead of needing to rigorously purify samples with an iodixanol density gradient. summary/conclusion: cholesterol quantitation can be used to accurately estimate ev concentration, allowing for rapid characterization of samples from clarified cell culture supernatant to highly purified evs. this highthroughput analytical capability may enable more comprehensive assessment of methods to boost ev yield through mass screening of cell culture conditions. optimization of nanoparticle tracking analysis of extracellular vesicles isolated from plasma and bronchopulmonary lavage fluid of patients with non-small cell lung cancer introduction: recent studies show that tumourderived extracellular vesicles (evs) greatly influence the tumour microenvironment and impact the therapy. in non-small cell lung cancer (nsclc), bronchopulmonary lavage fluid (balf) appears to be a good source of tumour-derived evs, providing more accurate information about the tumour microenvironment than evs from plasma. so far there is a lack of accurate and standardized methods for ev quantification. fluorescence nanoparticle tracking analysis (fl-nta) is an emerging method of ev-analysis, allowing discrimination of evs and exosomes from impurities. here we perform an optimization of the fl-nta method to compare evs from plasma and balf of nsclc patients and healthy controls (nc). methods: evs were isolated using homemade sizeexclusion chromatography (sec) columns (plasma) and ultrafiltration or differential ultracentrifugation (balf). nta was performed using zetaview pmx220 (particle metrix) after ev-staining with membrane dyes or fluorescence-labelled antibodies against typical ev-marker (cd9, cd81, cd63). results: nta scatter measurements showed a higher total particle concentration in plasma than in balf. however, membrane-specific staining showed a much greater purity of ev-preparations from balf, where nearly 100% of the particles detected in scatter mode showed positive membrane-staining. in contrast, only around 40-50% of particles in the plasma ev-preparations were positive for the membrane dyes. fluorescence-staining for ev surface marker requires further optimization to obtain reproducible results. summary/conclusion: classical nta using only the scatter mode fails to discriminate between evs, lipoproteins and protein aggregates. for ev-analysis from complex biofluids like plasma, fla-nta and staining for specific ev marker is necessary to receive reliable data. balf seems to be a better source of tumourderived evs than plasma, since the obtained ev-preparations show a higher purity. improving conditions for fluorescence-staining and nta measurement of evs from plasma and balf of nsclc patients will provide an additional method for quantifying and phenotyping of evs. introduction: the exoviewer platform currently enables the user to capture extracellular vesicles (ev) by means of surface antigen-specific antibodies (e.g. targeting tetraspanins), making possible the enumeration of individual particles using single-particle interferometric reflectance imaging sensor (sp-iris, interferometric) imaging as well as fluorescence. currently, through interferometric imaging particles smaller than 50 nm cannot be detected, while fluorescently stained ev smaller than 50 nm can be well resolved. further, it is conceivable that small ev contain antigen numbers in the single digits, making antigen-specific immunostaining a challenge. to further characterize ev populations of different sizes and surface marker composition, it would be highly advantageous to target the vesicular nature of the detected particles linked to a fluorescence readout. methods: the goal of this project is to detect ev with a probe that is ubiquitously distributed across the surface (or lumen) of the vesicle. small (30-150 nm) ev present fairly distinctive lipid membrane features in the extracellular environment, turning the ev membrane into a "universal" marker, and as such may serve as an alternative marker that is complementary to canonical ev surface markers. results: here we present data on successfully staining ev with the membrane dye di-8-anepps (di-8) and the luminal dye calcein-am. we demonstrate that ev from different sources can be efficiently stained with either dye, allowing the quantitative characterization of ev in an unbiased manner using exoviewer's fluorescence mode. while both dyes certainly have their own unique strengths, they exhibit the wanted linear correlation of ev staining versus concentration. further, both dyes are compatible with subsequent immunostaining applications, allowing the user to target specific surface or luminal markers (di-8). summary/conclusion: while a large-panel screening featuring other powerful dyes is continuously ongoing, the current data support the notion of providing the experimenter with a reference for total particle count and at the same time fully exploring the larger dynamic range of the fluorescence mode. moreover, the universal probe will enable the user to correlate intensity and particle size measurements, thereby significantly improving the exoviewer platform and its applications. membrane labelling is essential for the identification and quantification of extracellular vesicles via facs introduction: extracellular vesicle (ev) research is challenged by the lack of standard protocols to identify and distinguish between exosomes and ectosomes being released via exocytosis or plasma membrane shedding, respectively. analysis of small ev populations requires high-resolution technology and can be further improved using fluorescent labels such as carboxyfluorescein diacetate succinimidyl ester (cfse). at the inner leaflet of the plasma membrane, cfse is cleaved enzymatically resulting in covalent binding of the dye. in this study we optimized the conditions for membrane labelling of evs and their subsequent detection by flow cytometry to obtain a maximum yield of intact evs. methods: using sequential centrifugation, we separated ev subpopulations from supernatants of colo 357 pancreas carcinoma cells based on size and mass. after 10,000x g centrifugation, we reconstituted evs from the pellet. we used cfse for ev detection and analysed the expression of tetraspanins by facs to confirm the lipid bilayer structure. furthermore, we determined size distribution of evs by nanoparticle tracking analysis (nta) and electron microscopy. detecting evs as cfse+ events, we quantified our samples and investigated the impact of threshold adjustment on ev quantification. results: after high speed centrifugation of cell free supernatants, we identified cfse+ events as evs, which appeared as round structures under the microscope, and ranged from 80 to 40 nm in size. interestingly, tetraspanin markers cd9 and cd81 were detectable only on a subpopulation of purified evs, suggesting heterogeneity of our preparations. for sufficient labelling of evs, minimal temperature variations and short incubation times correlated with ev stability. of note, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of labelled evs and hence, is central for data comparability. summary/conclusion: protocol standardization is of major importance for the use of evs as diagnostic markers in liquid biopsies. funding: this project has been supported in part by annelise-asmussen foundation, luebeck (grant 180802), leo pharma germany (grant 180208). surface plasmon field-enhanced fluorescence spectroscopy (spfs) system for quantitative and qualitative extracellular vesicles total evaluation without any sample pretreatment introduction: the function of extracellular vesicle (ev) is interested in the immunology and oncology fields as a key transmitter for cellular communication. however, the conventional ev evaluation methods are required complicated evs preconcentration from the sample, its leads ev analysis uncertainty. in this study, we applied the spfs highly sensitive automated system for quantitative and qualitative ev evaluation without any sample pre-concentration and preparation step. methods: spfs automated system and plastic disposable sensor had been developed by konica minolta corporation in house. anti-membrane protein (cd9, cd63, cd83) antibody was chemically bonded on hydrophilic polymer which was immobilized through the gold thin film on the spfs sensor. the concentration of standard ev materials was evaluated by the qnano system before using. ev detection without preconcentrating was achieved by sandwich immunoassay step in microchannel round-trip flow reaction (tat 120 min) with the spfs system, and elisa was adapted as a conventional standard method. after spfs highly sensitive fluorescent measurements step, extracted and detected ev were effectively recovered by using the recovery buffer reaction. results: the ev sensitivity performance between spfs and elisa clearly showed a significant difference, and the lod of spfs (8.3 particles/μl) method was estimated 3000 times superior to the lod of conventional elisa (26,000 particles/μl). the spfs calibration curve showed a wide dynamic range at least over 5 logs as an additional specificity. spfs method also showed fine results in the dilution linearity test with high reproducibility under the serum/plasma sample condition. the data for recovery test of ev expected us that highly accurate measurement can be guaranteed under the condition of dilution about 10 times or less even in the whole blood sample. after the spfs measurement, extracted ev on the spfs sensor chip could be effectively recovered and could be analysed nucleic acid which contains micro rna. summary/conclusion: spfs system might have great potential for quantitative and qualitative ev evaluation. our strategy with spfs system for ev proteomic and genomic profiling will be possible for applying to ev quality control as well as a novel biomarker development. identification of a novel compound that inhibits small ev secretion and tumour progression by a sensitive elisa screening. yunfei ma a , takeshi yoshida a , duc tuan nguyen a , kazutaka matoba b , katsuhiko kida b , taito nishino b and rikinari hanayama c a kanazawa university, kanazawa, japan; b nissan chemical corporation, tokyo, japan; c wpi nano life science institute, kanazawa university, kanazawa, japan introduction: small evs from tumour cells are known to promote tumour progression, therefore, it is expected to develop drugs that regulate small ev secretion, which can be used in clinical applications. methods: to identify such regulators, we first developed a sensitive elisa system for the quantification of small ev secretion using a high-affinity ev binding protein tim4. by using this elisa system, we screened for small compounds that promote or inhibit small ev secretion using a drug-repositioning compound library (about 1,600 compounds). results: as a result, we identified eight promoters and two inhibitors, including compound a, which significantly reduced small ev secretion from various cell types without affecting cell growth. we further investigated the effects of compound a on a mouse model of osteosarcoma and found that compound a suppressed tumour progression efficiently. summary/conclusion: these data suggest that compound a would be useful not only for the characterization of small ev function but also for the clinical therapy against tumour progression, by inhibiting small ev secretion. introduction: for many years it was believed that several proteins such as cd63, cd9 and flotillin-1 were unique for exosomes, however recent studies have shown that several of these markers also can be present in other subpopulations of evs (kowal et al pnas 2016) . furthermore, few markers have been identified as uniquely present in microvesicles. the aim of this study was to in depth compare the proteome of microvesicles and exosomes. methods: mda-mb-231-luc-d3h1, -d3 h2ln and -bmd2a were cultured in ev-depleted media. microvesicles (16,500 x g, 20 min) and exosomes (118,000 x g 2.5 h) were isolated using a combination of differential ultracentrifugation and a density cushion (~1.1 g/ml). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, and electron microscopy (em). quantitative mass spectrometry (tmt-lc-ms/ms) was used to identify differently enriched proteins in microvesicles and exosomes (n = 3 x 3 cell lines). results: in total 6493 proteins were quantified, with 4851 being quantified in all samples. in total 818 and 1567 proteins were significantly upregulated in exosomes and microvesicles, respectively. go terms associated with the proteins significantly upregulated in exosomes were "extracellular exosome" and "plasma membrane", while the microvesicle proteome was associated with "membrane" and mitochondrion". in exosomes tetraspanins, annexins, escrt and rab proteins were significantly upregulated. in contrast, proteins that were upregulated in microvesicles were involved in protein translocation into the mitochondrial membrane (timm and tomm proteins), in cytokinesis, and in micos complex. however, flotillin-1 was not differently expressed in the ev subtypes. summary/conclusion: this study identifies several proteins to be differently enriched in exosomes and microvesicles. several of the proteins suggest recently by kowal and colleagues, such as adam10 and mitofilin could be validated. additionally several novel proteins could be identified. identifying markers separating microvesicles and exosomes is of high importance for the ev field and future studies will have to validate them also in other cells to determine if they are generic. introduction: the cellular elements composing the lining of brain ventricles have drawn much attention from neuroscientists, especially the role of subependymal cells in neurogenesis, but the role of ependymal cells in brain function and disease is still neglected. our objective is to study the morphological aspects of rat brain ventricles and the ependymal cells as analysed by transmission and field emission scanning microscopy in normal or ischaemic rats. methods: for this purpose, male wistar rats were submitted to 10 minutes of global brain ischaemia and divided into two groups: a) sham-operated animals and b) saline-treated ischaemic animals. all animals were allowed to survive for seven days. all procedures were approved by the ethics committee of the federal university of são paulo (2018/7633081117). transmission and scanning electron microscopic analysis of lateral brain ventricles were done in buffered 2,5% glutaraldehyde/2%formaldehyde perfused brains. cerebrospinal fluid was collected for nta analysis. results: the morphological characterization of brain ventricle revealed a slight rarefaction of ciliary tufts of animals submitted to ischaemia when compared to normal animals. field emission electron microscopy revealed the secretion of vesicles by the ependymal cilia in the lateral ventricle. size and concentration of particles in the cerebrospinal fluid was confirmed by nta and transmission electron microscopy. summary/conclusion: our results are unprecedented and bring innovative potential regarding the role of extracellular vesicles in both the physiology and pathogenesis of the nervous system. these data may also contribute to the development of new technologies for diagnosis and therapy of chronic degenerative diseases. introduction: the function of mitochondria relies on precise and effective quality controls. neurons have high metabolic demands and employ multiple mechanisms to ensure functional mitochondria. we investigated mitochondrial vesiclesa less understood quality control mechanism for mitochondriaand assessed the effect of cellular stress. methods: we surveyed mitochondrial vesicles in rat and planaria brains with electron microscopy. we quantified these vesicles with serial-section electron microscopy (fib-sem). we also conducted confocal microscopy with airyscan analysis of cultured neurons expressing fluorescently tagged mitochondrial markers. results: electron microscopy showed the ultrastructure of various types of mitochondrial vesicles. serial-section electron microscopy revealed the 3d ultrastructure of mitochondrial vesicles and their prevalence in neurons. confocal microscopic analysis showed increased numbers of mitochondrial vesicles in neurons under mild stress. summary/conclusion: our findings provide direct structural evidence for mitochondrial vesicles in neurons and their abundance in response to neuronal stress. their detection in the extracellular compartment (evidence for which is expected to be presented by the time of isev) may allow for development of biomarkers for mitochondrial health, with relevance to numerous pathologic conditions. from endosomes, might be involved in the impairment of rna, specific feature of als disease. combining high-resolution flow cytometry and surface marker analysis using an automated platform to study extracellular vesicle in cerebrospinal fluid unity health toronto, toronto, canada introduction: there is growing enthusiasm that extracellular vesicles (evs) carry the potential for a variety of applications in medicine. as biomarkers, evs may aid clinicians in the evaluation of diagnoses, disease progression, or even response to therapy. however, proper characterization of the amount, size, and phenotype of evs in a given sample remains challenging due to their sub-micrometre size and heterogeneity. over the last years, technologies, including high-sensitivity flow cytometry and automated platforms that simultaneously assess ev amount, size, and phenotype, have matured, providing new opportunities to study evs for future clinical applications. using such technologies to analyse cerebrospinal fluid (csf), which is in direct contact with the brain and spinal cord, may yield valuable insights into neurological disease processes. while there is often uncertainty about the exact source of evs in a biological sample, cd171 has emerged as a surface marker that suggests a neuronal origin. methods: csf samples that had been stored at -80 degrees celsius for advanced biomarker studies were analysed using two distinct approaches. a becton, dickinson and company (bd) aria iii flow cytometer was converted into using violet side scatter (ssc) for improved detection of evs with 405 instead of 488 nm ssc. for the combined analysis of amount, size, and phenotype, samples were analysed with the nanoview bio r100 platform. phenotype analysis included probing for the classic tetraspanins associated with exosomes (cd9, cd63, cd81) and the neural cell adhesion molecule l1 (cd171). results: flow of csf samples showed similar vesicle counts in control vs. disease and an increase of counts in later disease stages when neurodegeneration is thought to be more prominent. all csf samples showed some binding to classic exosomal markers (cd9, cd63, cd81). the sample taken at the latest time point showed relatively high vesicle counts, overall larger vesicle size, and abundant cd171 binding. interestingly, the cd171 positive evs were not positive for any of the classic exosomal markers (cd9, cd63, and cd81). summary/conclusion: this data supports the notion that analysing the amount, size, and surface markers of evs in csf can reveal intriguing dynamics in such basic ev characteristics over time and suggests important differences between ev populations in different disease stages. while previous studies indicated that cd171 could identify an ev to be of neuronal origin, it remains to be determined whether such specific surface markers will emerge as clinically relevant tools to support the evaluation of people affected by neurological diseases. a distinct microrna signature in plasma derived small extracellular vesicles of different neurodegenerative diseases introduction: exploring identifying robust biomarkers is essential for early diagnosis of neurodegenerative diseases. blood stream transports large (levs) and small extracellular vesicles (sevs), which are extracellular vesicles of different sizes and biological functions that are transported in blood. aim of our study was to investigate mrna/mirna signatures in plasma derived levs and sevs of amyotrophic lateral sclerosis (als), alzheimer's disease (ad), parkinson's disease (pdpd), fronto-temporal dementia (ftd) and alzheimer's disease (ad) patients. methods: levs and sevs were isolated from plasma of patients and healthy volunteers (ctr) by ultracentrifugation and rna was extracted. whole transcriptome and mirna libraries were prepared with truseq stranded total rna kit and truseq small rna library kit (illumina). results: our data suggested that the rna cargo in levs and sevs varies among different diseases. mirna analysis in sevs provided the most informative disease specific signatures, while whole transcriptome analysis did not show any specific signature. als was characterized by a small but specific group of circulating mirnas. mirnas profiling revealed that pd and ftd can be subgrouped in two classes while ad appears to be a homogeneous disease population. furthermore, mirnas profiling show the presence of overlaps in the signatures between the analysed diseases. mirna profiling in levs is similar to that observed in sevs, although in levs the overall differences between diseases are less marked. summary/conclusion: in this study we have demonstrated that mirnas are the most interesting subpopulation of transcripts transported by plasma derived sevs since they discriminate a disease from the other and they can provide a signature for each neurodegenerative diseases. may be linked with apoe genotype, we investigated the possible effect of apoe genotype on brain-derived evs (bdevs) and their protein and rna molecular cargo. methods: cortical brain tissues of ad patients with different apoe genotypes [ε2/ε3 (n = 5), ε3/ε3 (5), ε3/ε4 (6), ε4/ε4 (6)] and non-ad controls (n = 7) were obtained. bdevs were separated by size exclusion chromatography plus ultracentrifugation (uc) and characterized per misev2018. proteins were analysed by mass spectrometry. after protein identification, data were normalized using the cyclicloess method and analysed by principal component analysis (pca). nested factorial design highlighted differentially expressed proteins. rna from bdevs was extracted by mirneasy mini kit. small rna libraries were constructed using the ion total rna-seq kit and sequenced on the ion torrent s5™ using ion™ 540 chips. reads were aligned to human reference transcriptomes using bowtie. differential gene expression was quantified by edger and limma. results: among 28 proteins dysregulated in ad bd-sevs, several have reported roles in ad, e.g., microtubule-associated protein tau and peroxiredoxin-6. regarding apoe genotypes, 16 proteins were differentially expressed between ε4 carriers (ε3/ε4 and ε4/ε4) with non ε4 carriers (ε2/ε3 and ε3/ε3). however, ev markers did not differ by apoe genotype. in contrast to protein cargo of bdevs, the overall small rna expression pattern was similar among ad patients with different apoe alleles and non-ad patients. only a few mirnas showed different abundance level between ε2/ε3 and ε4/ε4 groups, or between ad and non-ad groups. summary/conclusion: bdevs carry proteins and mirnas related to ad development and apoe genotypes. further verification of protein and rna expression in brain and plasma derived evs may reveal mechanisms of ev function in neuroinflammation and develop biomarkers for ad disease. funding: this project was funded by mh118164. efficient pathology spread by extracellular vesicles from human brain tissues in mouse brain and tissue cultured neurons: transmission and propagation to gabaergic neurons however, whether human brain-derived evs induce tau pathology has not yet been characterized in the mouse brain. here, we assess the mechanisms of disease spread after intrahippocampal injection of human brainderived evs into the aged mouse model. methods: ev-enriched fractions were isolated from unfixed frozen human brain samples from ad, prodromal ad (pad), control (ctrl) cases, and tau knockout (tko) mouse brains. isolated evs containing 300 pg of human total tau were sterotaxically injected into the right outer molecular layer of the dentate gyrus of 18 months-old c57bl/6 female mice. 4.5 months after the injection, hippocampal slices were prepared for whole-cell patch clamp recordings of ca1 pyramidal neurons were undertakent. hippocampi were analysed with immunohistochemistry using phosphorylated-tau (p-tau) epitopes including at8. evs were examined for protein composition by protein mass-spectroscopy, the neuronal uptake in vitro, and structural analysis by the atomic force microscopy (afm). results: semiquantitative brain-wide immunohistochemistry of p-tau revealed that inoculation of ad or pad-evs induced tau propagation throughout the hippocampus, including the dentate gyrus, ca3 and ca1 subregions. at8 was localized primarily in gad67 + gabaergic neurons in pad and ad evs groups, accompanied with reduced amplitude of inhibitory postsynaptic currents and excitatory-inhibitory ratio in amplitube of postsynaptic currents in ca1 pyramidal neurons in pad evs. afm analysis showed higher density of tau oligomers in both ad and pad evs while only ad evs showed significantly higher neuronal uptake compared to ctrl evs. finally, proteomic analysis showed that ad evs are enriched in disease and glia-related molecules compared to ctrl evs, which may contribute to their enhanced neuronal uptake. summary/conclusion: intracranial injection of ad or pad evs induced p-tau accumulation primarily in gabaergic neurons throughout the hippocampus, resulted in higher uptake by neurons, and tau oligomer conformation, indicating of their pathogenic potency as seeding factors. gabaergic neuronal dysfunction in the hippocampal neuronal circuitry reported in early ad brains could be attributed to specific ev mediated tau propagation in this cell type, a phenomenon meriting further investigation and validation. funding: nih rf1ag054199, nih r01ag054672, nih r56ag057469, cure alzheimer's fund, brightfocus foundation, curepsp, coins for alzheimer's research trust introduction: extracellular vesicles (evs) are released by cells of the central nervous system as a result of injury, including mild traumatic brain injury (mtbi). since mtbi may alter circulating levels of evs, this study aimed to investigate differences in circulating ev numbers between contact sport athletes with and without acute mtbi. methods: circulating evs containing cd63 (cd63 + ev), cd81 (cd81+ ev), and neural cell adhesion molecule (l1cam+ev) were analysed in young, male athletes with or without mtbi (18-29 yo, n = 6 per group). sodium citrate-treated blood samples were obtained from athletes with mtbi within 48-hours of injury and from control athletes free of mtbi for one year. athletes were best matched for age and history of prior mtbi. samples were double-centrifuged to obtain platelet-poor plasma and stored at −80°c until analysed. quantification of evs was performed using a spectral flow cytometer. the study was approved by temple university's irb, and all athletes provided written informed consent. results: mann-whitney u tests showed that population percentages of small size (179-304 nm) cd63 + ev, cd81+ ev and l1cam+evs were significantly higher in mtbi athletes (mean rank: 8.0, 9.5, 9. 3) than controls (mean rank: 3.6, 3.5, 3.7) (u = 3.0, p = 0.03; u = 0.0, p > 0.01; u = 1.0, p > 0.01, respectively). population percentages of large size (500-800 nm) cd63+ ev, cd81+ ev and l1cam+evs were also significantly higher in mtbi athletes (mean rank: 8. 2, 9.2, 9.5) than controls (mean rank: 3.4, 3.8, 3.5) (u = 2.0, p = 0.02; u = 2.0, p > 0.01; u = 0.0, p > 0.01, respectively). there were no significant differences between percentages of evs associated with blood brain barrier function (cd144+ ev) or platelets (cd42a+ev) among mtbi athletes or controls. introduction: parkinson's disease (pd) is characterized by clinical heterogeneity, different rates of progression and absence of definitive biomarkers. extracellular vesicles (evs) are easily isolated from plasma and play a central role in intercellular communication which is highly relevant for inflammatory processes implicated in protein misfolding-related neurodegenerative disorders. thus, we characterized distinctive plasmatic ev subpopulations of pd and atypical parkinsonisms (ap) patients, with the aim to identify candidate biomarkers among evs surface membraneproteins. methods: plasmatic evs were collected from 27 pd, 19 matched healthy controls (hc), 9 ap with multiple system atrophy (msa) and 9 ap with tauopathies (ap-tau). evs were quantified by nanoparticle tracking analysis. the expression of 37 ev-surface markers, related to inflammatory and immune cells, were measured by macsplex and correlated to clinical scales. a diagnostic model based on ev markers expression was built via supervised machine learning algorithms and validated in an external cohort (10 pd, 20 hc, 5 msa, 5 ap-tau). the cantonal ethics committee approved the study protocol. all enrolled subjects gave written informed consent. results: pd showed the highest ev concentration compared to others groups. pd and msa displayed a greater pool of overexpressed immune markers compared to ap-tau. ev antigens correlate to cognitive impairment and disease gravity in pd and msa. the roc curve analysis of a compound ev marker showed optimal diagnostic performance for pd (auc 0.908; sensitivity 96.3%, specificity 78.9%) and msa (auc 0.974; sensi-tivity100%,specificity94.7%)andgoodaccuracyforap-tau (auc 0.718; sensitivity 77.8%, specificity 89.5%). a diagnostic model based on ev markers expression, cor-rectlyclassified88.9%ofpatientswithreliablediagnostic performance after validation in an external cohort (77% of accuracy). summary/conclusion: this analysis of multiple immune surface markers of circulating evs in pd and ap well captured the clinical heterogeneity of pd and showed optimal diagnostic performance. furtherly it suggests a different immune dysregulation in pd and msa vs. ap-tau, to be confirmed by functional analysis in experimental models of disease. funding: supported by abreoc. separation and characterization of extracellular vesicles from human cerebrospinal fluid introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off 3,000), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions (3 + 4) after the sec void volume as evaluated by detection of cd63 and cd9 markers (immunoblotting) and annexin a2 (peptide mapping by nanolc-ms/ms). there, nanoparticles around 150 nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between 55 and 165 nm, with average diameter 94 ± 31 nm. cd63 was visualized by immunocytochemistry at the surface of ev around 80 nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin-3 binding protein (lgals3bp or 90 k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between 15 and 60 nm; some of those nanoparticles had ring-like appearance. occasionally 90 k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of 90 k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from 90 k nanoparticles to investigate their individual physiological relevance and biomarker potential. introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off 3,000), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions (3 + 4) after the sec void volume as evaluated by detection of cd63 and cd9 markers (immunoblotting) and annexin a2 (peptide mapping by nanolc-ms/ms). there, nanoparticles around 150 nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between 55 and 165 nm, with average diameter 94 ± 31 nm. cd63 was visualized by immunocytochemistry at the surface of ev around 80 nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin-3 binding protein (lgals3bp or 90 k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between 15 and 60 nm; some of those nanoparticles had ring-like appearance. occasionally 90 k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of 90 k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from 90 k nanoparticles to investigate their individual physiological relevance and biomarker potential. release of extracellular vesicles from platelets requires platelet-platelet interaction aleksandra gąsecka a , naomi c. buntsma b , sytske talsma c , krzysztof j. filipiak d , rienk nieuwland e and edwin van der pol f introduction: arterial thrombosis is a major and global cause of human death and disability, but a biomarker for early-diagnosis of thrombosis is absent. platelet activation and aggregation are the first steps of plateletrich thrombus formation, but their relative contribution to platelet extracellular vesicles (pevs) release is unknown. methods: to study the relation between pev release and platelet interaction (aggregation), citrate-anticoagulated whole blood (wb) from healthy donors was diluted 2, 4, 8, 16 and 32-fold and activated by 30 μm thrombin-receptor activating peptide (trap). in addition, undiluted wb and 10-fold diluted wb, which totally blocked pev release, were activated with various trap concentrations. concentrations of pevs (cd61 + and cd61+, cd62p + >1000 nm) and activated platelets (cd61+, cd62p+ >1000 nm) were measured by flow cytometry (apogee a60-micro). platelet aggregation was assessed using impedance aggregometry. results: a 10-fold dilution of wb blocked both aggregation and the release of pevs. compared to baseline, activation of undiluted wb with trap increased the concentrations of cd61 + 2.2-fold and cd61+-cd62p + pevs 7.2-fold. the concentration of cd61+ (r2 = 0.71) and cd61+-cd62p+ (r2 = 0.78) pevs as well as platelet aggregation (r2 = 0.8) scaled inversely (reciprocal) with the dilution of wb. further, we found a linear correlation between the % of activated platelets and the concentration of cd61+ (r2 = 0.80) and cd61 +, cd62p+ (r2 = 0.64) pevs in undiluted wb, which was absent in 10-fold diluted blood (r2 < 0.05). summary/conclusion: the absence of aggregation and pev release upon platelet activation in 10-fold diluted blood shows that aggregation directly depends on the distance between platelets, which is confirmed by the reciprocal relationship between pev release and blood dilution. because pevs are only released when platelet activation is followed by aggregation, pevs are a potential early biomarker of thrombosis. funding: ag is supported by the national science centre, research programme preludium 2018/31/ n/nz7/02260. evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. age-dependent alteration in concentration and size distribution of extracellular vesicles in plasma of normotensive and hypertensive rats kosuke otani, muneyoshi okada and hideyuki yamawaki laboratory of veterinary pharmacology, school of veterinary medicine, kitasato university, towada, japan introduction: spontaneously hypertensive rats (shr) are the most widely used animal model of human essential hypertension. we previously reported that plasma small extracellular vesicles (sevs) in shr regulate systolic blood pressure, however, the mechanism has not been clarified. in the present study, we compared the concentration and size distribution of plasma evs (sevs and large evs) from young and aged normotensive wistar kyoto rats (wky) and shr. methods: heparin-anticoagulated plasma was collected from male wky and shr at 5~7-(young) and 15-(aged) week-old. large evs were isolated from the plasma by centrifugation (10000 x g). sevs were isolated by ultracentrifugation (164,071 x g) following precipitation with polyethylene-glycol. the concentration and size distribution of sevs and large evs were measured by a tunable resistive pulse sensing analysis. results: there was no significant difference in the total concentration of plasma sevs between wky and shr or between young and aged rats. the mean diameter of plasma sevs from aged rats was larger than that from young rats in both wky and shr. also, the number of particles with a diameter of smaller than 150 nm in plasma sevs from aged rats was lower than that from young rats. the concentration of plasma large evs from aged rats was higher than that from young rats in both wky and shr. there was no significant difference in the size distribution of plasma large evs between wky and shr or between young and aged rats. summary/conclusion: the present results for the first time demonstrate that the concentration of plasma large-sized evs is increased by ageing, while there is no difference in the concertation and size distribution of evs between wky and shr. further research is required to clarify the cause of age-dependent alternation in plasma ev size distribution and its physiological meaning. microrna profiling of circulating extracellular vesicles is involved with susceptibility to age-related diseases: relevance to cardiovascular signalling in ageing process ionara rodrigues siqueira a , laura cechinel b , rachael batabyal c and robert freishtat c a universidade federal do rio grande do sul (ufrgs), porto alegre, brazil; b universidade federal do rio grande do sul, porto alegre, brazil; c children's national hospital, washington, usa introduction: ageing represents a central risk factor for several diseases, such as cardiovascular diseases. our hypothesis is that extracellular vesicles (evs) can be potential mechanism of spreading molecules, such as micrornas, involved with susceptibility to chronic age-related diseases and geriatric syndromes. in this context, the role of micrornas in age-induced detrimental changes in the cardiovascular system has been suggested. although evs can protect micrornas from endogenous rnases and internalization of these vesicles into cells is involved with cell communication, delivering micrornas even to distant tissues, the relationships between evs micrornas profile and chronic age-related diseases has not been evaluated. our aim was to investigate the microrna profile of circulating evs during ageing process and their downstream signalling pathways. methods: the ethics committee (ceua -comissão de ética no uso de animais -ufrgs; nr. 29,818) approved all animal procedures and experimental conditions. male wistar rats of 3-and 21-month-old were used, and plasma was obtained from the trunk blood. evs were isolated with exoquick following the manufacturer's instructions. microrna was isolated from evs and then amplified. microrna was labelled using the flashtag biotin hsr rna labelling kit and profiled on affymetrix genechip microrna 4.0 arrays. ingenuity pathway analysis (ipa) was used to identify pathways regulated by significantly altered micrornas. results: microarray analysis revealed 728 micrornas. of these micrornas, 48 were differentially expressed between aged and young-adult animals, 18 micrornas were significantly upregulated and 30 were downregulated in aged animals compared to young adult (p < 0.05; fold change of |1.1|). a conservative filter was applied on ipa and only experimentally validated and highly conserved predicted mrna targets for each microrna was used. ipa analysis showed that cardiac hypertrophic signalling is ranked as highly predicted targets for these differentially expressed micrornas (p < 0.0001). moreover, ipa demonstrated that this canonical pathway is upregulated in aged animals when compared to young adult. in addition to cardiac hypertrophic signalling, other relevant cardiovascular canonical pathways, such as endothelin-1 signalling and intrinsic prothrombin activation pathway have predicted targets. summary/conclusion: our results showed for the first time that micrornas profile in circulating evs has a potential role to drive heart senescence and consequent cardiac diseases which represents the leading cause of death. introduction: introduction: the vascular endothelium and smooth muscle form adjacent cellular layers that comprise part of the vascular wall. here, we examined the extent to which extracellular vesicles (evs) vesicles participate in endothelial-vascular smooth muscle cell communication. methods: methods: evs were collected from rat aortic endothelial and smooth muscle cell serumfree media by ultracentrifugation. vesicle morphology, size and concentration were evaluated by transmission electron microscopy and nanoparticle tracking analysis. endothelial cell and vascular smooth muscle cell cultures were subjected to various concentrations of evs for various times. functional assays were performed. results: results: western blot as well as shot gun proteomic analyses revealed sets of proteins common to both endothelial-and smooth muscle-derived ev as well as proteins unique to each vascular cell type. functionally, endothelial-derived evs stimulated vascular cell adhesion molecule-1 (vcam-1) expression and enhanced leukocyte adhesion in vascular smooth muscle cells while smooth muscle evs did not elicit similar effects in endothelial cells. evs from endothelial cells also induced protein synthesis and senescenceassociated β galactosidase activity in vascular smooth muscle cells. proteomic analysis of vascular smooth muscle cells following exposure to endothelial cellderived evs revealed upregulation of several proteins including pro-inflammatory molecules, high-mobility group box (hmgb) 1 and hmgb2. pharmacological blockade of hmgb1 and hmgb2 and sirna depletion of hmgb1 in smooth muscle cells attenuated nfkb (p65) phosphorylation and nuclear translocation, vcam-1 expression and leukocyte adhesion induced by endothelial cell evs. summary/conclusion: conclusions: these data suggest that endothelial cell-derived evs can enhance signalling pathways that induce a pro inflammatory in vascular smooth muscle cells. introduction: graft patency is one of the major determinants of long-term outcome following coronary artery bypass graft surgery (cabg). biomarkers, if indicative of the underlying pathophysiological mechanisms, would suggest strategies to limit graft failure. many studies have generated compelling data on the sensitivity of mvs as biomarkers of cardiovascular disease progression and events. the mv usefulness in cabg has been tested only in a study that highlighted their importance in surgical haemostasis. no information is so far available on the association between the amount or pattern of circulating mvs and cabg outcome. we aimed to evaluate whether mv pre-operative signature could predict mid-term graft failure. methods: this was a nested case-control substudy of the coronary bypass grafting: factors related to late events and graft patency (cage) study that enrolled 330 patients undergoing elective cabg. of these, 179 underwent coronary computed tomography angiography 18 months post-surgery showing 24% graft occlusion. flow cytometry mv analysis was performed in 60 patients (30/group with occluded [cases] and patent [controls] grafts) on plasma samples collected the day before surgery and at follow-up. results: before surgery, cases had two-fold (p = 0.020) and four-fold (p = 0.042) more activated platelet-derived and tf+ mvs, respectively than controls. the mv thrombin generation capacity was also significantly greater (p < 0.05). this mv signature predicted graft occlusion (auc of 0.897 [95%ci: 0,77-0,96], p = 0.02). by using a mv-score (0-6), the or for re-occlusion for a score above 3 was 16.3 (95% ci 4.1-65.3, p < 0.001). summary/conclusion: the pre-operative signature of mvs is an independent predictor of mid-term graft occlusion in cabg patients and a cumulative mvscore stratifies patient's risk. since the mv signature mirrors platelet activation, patients with a high mvscore would benefit from a personalized antiplatelet therapy. exosomes from engineered immortalized human heart cells improve ventricular function and attenuate fibrosis in mice with arrhythmogenic cardiomyopathy yen-nien lin, lizbeth sanchez, rui zhang, thassio ricardo ribeiro mesquita, chang li, ahmed ibrahim, eduardo marbán and eugenio cingolani heart institude, cedars sinai medical center, los angeles, usa introduction: arrhythmogenic cardiomyopathy (ac) is characterized by progressive loss of cardiomyocytes and fibrofatty tissue replacement. currently, there is no effective treatment for this disease. exosomes (imexos) secreted by heart stromal cells, engineered to be immortal and overexpressing β-catenin, exert anti-inflammatory and anti-fibrotic effects and improve ventricular function in models of ischaemic injury (ibrahim et al., nature bme 2019). methods: to investigate the effectiveness of imexos in a murine model of ac, four-week old homozygous dsg2 knockout (dsgko) mice and wild type (wt, age-and strain-matched) mice were compared. dsgko mice were randomized to receive weekly imexos or vehicle via intravenous injection for 4 weeks. neonatal rat ventricular myocyte (nrvm) proliferation and apoptotic assays were performed to explore potential effects of exosomes. results: biodistribution studies of dir-labelled imexos revealed some cardiac uptake, along with strong signals in spleen. at 4 weeks, dsgko mice which had received intravenous imexos showed improved cardiac function (echocardiographic ejection fraction 73 ± 2 vs 59 ± 5% in vehicle mice, p = 0.03), with an underlying attenuation in myocardial fibrosis by histology. electrophysiology test showed shorter qrs duration (4.0 ± 2.0 ms imexo vs 6.5 ± 2.9 ms vehicle, p = 0.02) and effective refractory period. programmed ventricular stimulation showed dsgko mice which had received imexos were remarkably less prone to ventricular tachycardia induction (10.0 ± 32% vs 55.0 ± 52% in vehicle, p = 0.031). in vitro study showed nrvm exposed to imexos for 2 days exhibited higher brdu expression relative to vehicle group, and less annexin-v expression after oxidative stress induced by 10-minute illumination with 254 nm uv. summary/conclusion: intravenous administration of imexos improved cardiac function, reduced cardiac fibrosis, and suppressed arrhythmogenesis in ac. our findings motivate clinical testing of imexos in ac, an orphan disease with great unmet medical need. funding: nih r01 hl124074 (to em) cardiac-derived extracellular vesicles contribute to communication between heart and brain in chronic heart failure (chf) and target nrf2/ are signalling changhai tian a , lie gao b and irving zucker b a department of cellular and integrative physiology, university of nebraska medical center, omaha, usa; b department of cellular and integrative physiology, university of nebraska medical center, omaha, usa introduction: mirnas regulate the translation of proteins that are involved in redox homoeostasis in the heart and brain. intra-and/or inter-organ communication takes place by multiple mechanisms including extracellular vesicular (ev) transport. our previous studies suggested that cardiac derived mirna-enriched evs contribute to the dysregulation of nrf2/antioxidant enzyme (are) signalling in the myocardium via intercellular cross-talk, and result in the decreased nrf2/are signalling in the sympatho-regulatory areas of the brain in chf. however, it is unclear if cardiac derived evs circulate to the central nervous system evoking sympatho-excitation by disrupting central redox homoeostasis. methods: cardiac-specific membrane gfp+ mice were generated to track the brain distribution of cardiac evs in rats with chf (coronary ligation). the isolation and characterization of evs were carried out by differential ultracentrifugation, tem, nanosight, western blotting, and qrt-pcr. transfection, labelling, and microinjection of evs into the rostral ventrolateral medulla (rvlm) were performed. results: nrf2 protein was reduced in the rvlm of chf rats consistent with an upregulation of nrf2-targeting mirnas. nrf2-targeting mirnas were enriched in cardiac and circulating evs of chf rats. nrf2-targeting and cardiac-specific mirnas were abundant in brain-derived evs. circulating evs were taken up by neurons in sympatho-regulatory areas of the brain. mirna-enriched evs from chf animals increased sympathetic tone which was prevented by a cocktail of nrf2-targeting mirna inhibitors. summary/conclusion: myocardial infarction-induced mirna-enriched evs mediate the inter-organ crosstalk between heart and brain in the oxidative regulation of sympathetic outflow through targeting the nrf2/ are signalling pathway. these findings suggest that cardiac-derived ev mirnas targeting nrf2/are signalling may act as an endocrine signalling mediator of chf that has potential as a novel therapeutic target. introduction: a fine-tuned communication between cardiac cells is vital to maintain myocardial integrity and contractility. not only an impairment of gap junction (gj)-mediated intercellular communication, but also defects in ev-mediated communication have been associated with ischaemic heart disease, a major causative factor of heart failure. we have previously shown that cx43, the main ventricular gj protein, assembles into channels at the evs surface, mediating the release of vesicle content into target cells.the main objective of this work was to characterize the signals underlying protein sorting into extracellular vesicles (evs) in a human pathophysiological context, using connexin43 (cx43) as a model substrate. methods: animal models of ischaemia/reperfusion (i/ r) injury by ligation of the left anterior descending coronary artery, ex vivo and in vitro ischaemia models and human patients were used to investigate the secretion of ev-cx43. results: release of cx43 was downregulated in circulating vesicles from i/r-injured mice and patients with st-segment elevation myocardial infarction, as well as in intracardiac and cardiomyocyte-derived evs. additionally, we show that ubiquitin signalled the release of cx43 in basal conditions but appeared to be dispensable during ischaemia. depletion of the autophagy adaptor p62 partially restored the secretion of cx43, suggesting an interplay between ischaemiainduced cx43 degradation and secretion. summary/conclusion: overall, we demonstrated that ischaemia impairs the sorting of cx43 into evs, which may ultimately affect long-distance communication. through the identification of the underlying molecular mechanisms and players, these results pave the way towards the development of innovative diagnostic and therapeutic strategies for cardiovascular disorders. introduction: remote ischaemic conditioning is a cardioprotective intervention which protects the heart against ischaemia/reperfusion injury. transient activation of toll-like receptor 4 (tlr4) and its downstream regulators (tnfα and il-6) have been implicated in cardioprotective interventions. extracellular vesicles (evs) play a role in cardioprotection through the activation of the tlrs. however, since isolation of evs in high amounts with suitable purity from blood is a challenge, our aim was to develop a cellular model system from which tlr-inducing, cardioprotective evs can be isolated in a reproducible manner. methods: ev release from hek293 cells was induced by calcium-ionophore a23187. evs were characterized, cytoprotection by evs against simulated ischaemia/ reperfusion injury and its mechanism were investigated in h9c2 and ac16 cell lines. results: a23187 induction of hek293 cell induced ev release and the isolates contained mostly large evs. evs decreased cytotoxicity and apoptosis due to 16 h ischaemia followed by 2 h reperfusion in h9c2 and ac16 cells in a dose-dependent manner. evs activated tlr4 and its downstream signalling pathway in h9c2 and ac16 cells as well as the expression of cytoprotective haem oxigenase 1 (ho-1) in h9c2 cells. summary/conclusion: a23187-induced evs exert cytoprotection in h9c2 and ac16 cells by inducing tlr4 signalling and ho1 expression. therefore, evs released via calcium-ionophore treatment may serve as a basis of an efficient carpdioprotective therapy. introduction: biliary strictures may be benign or malignant. the major malignant causes of biliary stricture are a primary cholangiocarcinoma (cca) or pancreatic ductal adenocarcinoma (pdac). there is ongoing debate about adequate diagnostics in biliary strictures of unknown aetiology. micrornas (mirnas) are small non-coding rnas important in tumourigenesis. mirna have been found to be enriched in exosomes, small membrane-bound extracellular vesicles (ev) of endocytic origin, which is a novel pathway for intercellular signalling within the tumour microenvironment and have been implicated in loco-regional pre-metastatic niche formation. this project aims to investigate circulating-free and ev mirnas as biomarkers that can aid diagnosis in patients with a biliary stricture. we will (1) isolate and characterise evs in plasma and bile from patients with benign and malignant biliary strictures (i.e. pancreaticobiliary cancers); and (2) identify differentially expressed circulating-free and ev mirnas in plasma and bile suitable for detecting malignancy. methods: sample size (n = 126) was calculated for a study power of 90% and α error of 5% for the ability of extracellular mirnas to discriminate benign from malignant biliary strictures. prospective matched plasma and bile samples will be collected from patients with benign (n = 63) and malignant (n = 63) biliary strictures undergoing endoscopic retrograde cholangiopancreatography (ercp). evs will be isolated from the biofluids by ultracentrifugation and/or size exclusion chromatography and then characterised (tem, nta and immunoblotting). circulating-free and ev-associated mirnas will be profiled using small rna sequencing. extracellular mirna "signatures" will then be validated by rt-qpcr, and diagnostic accuracy confirmed (sensitivity, specificity, auc). results: evs derived from patient samples have been characterised using nta, western blotting and tem. sec derived evs appear to be more well-defined than uc evs with marker positivity for cd63, cd81 and cd9. ongoing work will be focused on rna profiles of evs from both malignant and benign cohorts. summary/conclusion: there is currently no effective method to differentiate benign from malignant biliary strictures. novel plasma and bile circulating-free and ev-associated mirna biomarkers may improve the speed and accuracy of diagnosis, resulting in considerable patient benefits. furthermore, as little is known about the ev-associated function of these tumours, candidate ev-mirnas could be taken from "bedside to bench" and their function further investigated using in vivo, vitro and silico models. introduction: urine is a source of extracellular rna (exrna) biomarkers that can be obtained non-invasively throughout pregnancy. several studies have profiled extracellular mirnas in biofluids during pregnancy, but few have profiled extracellular mrnas (ex-mrnas) in urine. objective: to optimize methods for ex-mrna isolation and rna-seq library preparation from urine of healthy pregnant and non-pregnant females. methods: rna was isolated from pooled non-pregnant urine using kits based on ev precipitation (mircury exosome kit for csf/urine, seramir), ev affinity purification (exorneasy), and protein precipitation (mirneasy serum/plasma advanced). next, long (>200nt) and short rnas were isolated from ev enriched urine of pregnant (n = 5) and non-pregnant (n = 5) individuals using the mircury kit followed by the mirneasy micro kit. rna-seq libraries were prepared using the smart-seq v4 ultra low input rna (oligo(dt) priming) and the smarter stranded total rna-seq kit v2 -pico input (random priming) methods (takara). preliminary data were obtained using the illumina miseq, and aligned using star v.2.7.3.a. results: overall, rna isolation using mircury followed by the smart-seq v4 library preparation kit yielded the highest % of mapped reads: 42% in pooled non-pregnant, 27% in individual non-pregnant, and 31% in individual pregnant urine. for rna extracted using the mircury kit, the smart-seq v4 libraries had higher % of mapped mrna reads compared to pico libraries (p < 0.05, t-test). in contrast for mirneasy advanced it was reversed (38% vs 21%). summary/conclusion: early results from low-depth sequencing show the highest mrna mapping rates for mircury followed by the smart-seq v4 kit. high-depth sequencing data are now being generated, which will enable us to perform detailed comparisons of different rna species from the rna profiles obtained using different library preparations and rna isolation methods from urine of pregnant and non-pregnant subjects. funding: this study was funded by nih 7 k99 hd096125-02, nih u01 hl126494, and a ucsd igm-illumina mini-grant. il-2 mutein-induced changes of exosomal mirna cargo in a humanized mouse model emily lurier, erik sampson, patrick halvey, mike cianci and katalin kis-toth pandion therapeutics, cambridge, usa introduction: regulatory t cells (tregs) are key contributors to immune homoeostasis. decreased number and/or function of these cells are frequent features of many autoimmune diseases linked to the development of tissue inflammation. while interleukin-2 (il-2) is essential for pan t cell proliferation and performance, low dose il-2 treatment has been shown to preferentially affect tregs and is being evaluated as an intervention in autoimmune diseases. pt101 is a novel il-2 mutein fc fusion molecule (il-2 m) designed to selectively engage with tregs. using a humanized nod-scid il2rn-null (nsg) mouse model we have shown that pt101 expanded tregs without significant effects on other immune cells. we have also shown that tregs from pt101-dosed humanized mice exhibit increased expression of foxp3 and cd25, and demethylation of foxp3 and ctla-4 genes, suggesting enhanced function and stability. in the current study we investigated the mirna content of plasma exosomes isolated from pt101-or vehicle-treated mice in order to identify treg specific mirnas from the il-2 m treated animals. methods: cd34+ haematopoietic stem cell humanized nsg mice were dosed once subcutaneously with pt101 or vehicle. plasma samples from 8 mice were collected at day 7 and exosome isolation was conducted using the exoquick method. small rna was extracted and quantified using the bioanalyzer small rna assay. an illumina nextseq instrument was used for library preparation and sequencing with 75bp single end reads at an approximate depth of 10-15 million reads per sample. raw sequences were mapped to human genome grch37 and analysed via a pipeline provided by the university of california santa cruz. results: rna within the exosomes from vehicle and il-2 m-treated groups was mostly comprised of mirna and trna. plasma was pooled from 8 animals per treatment group and differential expression was determined using a twofold change cut-off. we found that pt101 treatment actively altered the mirna content of plasma exosomes, compared to exosomes from vehicle-treated mice. many of the differentially expressed mirnas are involved in immunoregulation. summary/conclusion: plasma exosomes from pt101treated humanized mice encapsulated treatment-specific mirnas which can potentially be used as systemic biomarkers of treg expansion and function. identification of potential biomarkers in microglial specific exosomes isolated from prion-infected serum introduction: transmissible spongiform encephalopathies (tse) are neurodegenerative disorders caused by the misfolding of the cellular prion protein (prpc) to the beta-sheet rich abnormal prion protein (prpsc). prpsc aggregates in the brain and causes amyloid plaques, neuronal loss, spongiform degeneration and microglial activation. currently, definitive diagnosis of tse diseases is only confirmed post-mortem thus a diagnostic test in accessible body fluid is of interest. exosomes are a good resource for biomarker discovery since they cross the blood-brain barrier easily and contain protein, lipids and nucleic acids from the cells of origin. the goal of this study was to look at biomarkers from brain-originating exosomes (specifically microglia) isolated in the serum of prion-infected animals. methods: westerns and nanoparticle tracking analysis (nta) were used to look at the composition of microglial-specific exosomes. as proof of principle, exosomes were isolated from a microglial cell line (bv2 cells). a cd63 antibody was labelled with a fluorophore and binding to exosomes was visualized via nta. exosomes were isolated from serum of both prioninfected and mock-infected mice throughout disease course. a macrophage specific antibody (f4/80) was bound to beads which were used to isolate exosomes which includes those of microglial origin. microrna was extracted from these exosomes and next-generation sequencing (ngs) was performed using the illumina platform. clc genomics workbench was used for bioinformatics analysis. results: microglial and macrophage proteins (tmem119 and iba1) were identified in exosomes isolated from bv2 cells and prion-infected mouse serum. macrophage exosomes were isolated via a novel antibody-bead based system. results of the ngs analysis of the microrna isolated from these exosomes indicated a series of mirna that could differentiate between control and infected samples as well as age-specific markers. summary/conclusion: to our knowledge, this is the first time microglial-specific exosomes have been isolated from prion-infected serum from early and end stage disease. the results of this analysis could facilitate the diagnosis of prion disease in easily-accessible biofluids pre-mortem. comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease introduction: urinary extracellular vesicles (uevs) are emerging as a source for early biomarkers of kidney damage, holding the potential to replace the conventional invasive techniques including kidney biopsy. several methods are available for uev isolation. our aim was to compare different workflows and isolation by hydrostatic filtration dialysis (hfd), ultracentrifugation (uc) and a kit based isolation method for their subsequent use in mirna-seq and rna-seq for biomarker discovery in diabetic kidney disease. methods: type 1 diabetic patients (t1d) with macroalbuminuria and normoalbuminuric healthy controls were included in the study. sample collection and all experiments were performed in accordance with the declaration of helsinki. evs were isolated from 10-50 ml of 24 h urine collections by uc, hfd, or a commercially available kit (purification based on spin column chromatography, urine exosome purification and rna isolation midi kit, norgen biotech, canada) each with different established urine clarification steps. quality control of the evs was performed with negative staining em, nta and western blotting. isolated rnas were profiled with bioanalyzer pico kit and subjected to mirna and mrna sequencing. for rna-seq, cdna library was prepared using smart-seq v4 ultra low input rna kit for sequencing (takara bio, japan). rna-seq was performed using hiseq 2000 (illumina). mirna-seq library was prepared using qiaseq mirna library kit (qiagen, germany). mirna-seq was performed on the illumina hiseq 4000 platform (illumina). results: our data showed that uev yield, morphology and size distribution were closely similar in hfd and uc preparations, while lower yields were obtained using the kit. by western blot, ev markers were detectable in samples isolated by hfd and uc but not readily in samples isolated with the kit. tamm-horsfall protein was detected in all the samples and albumin levels appeared higher in hfd and kit isolated samples relative to uc samples. the number of paired-end reads for rna-seq in hfd and uc samples (in both > 5 m) were closely similar. instead, rna reads were lower than 2 m for the kit samples. for mirna-seq, the number of reads as well as the molecular biotype distribution were similar for the three methods. by principal component analysis of the rna-seq data, we observed that hfd and uc grouped together showing similarities. however, for mirna-seq data such similarities were not obvious. this suggests that the three different workflows and isolation principles may enrich different mirna-rich uev preparation components. summary/conclusion: our transcriptomics data shows that hfd and uc are suitable methods to isolate uevs for mirna-seq and rna-seq. the kit based method appears better suited for mirna-seq. introduction: exosomes contain a variety of biomolecules including dna. knowledge of cfdna distribution and localization in bioliquid is important for understanding both biological function of cfdna and exosomes. some publications state that a large proportion of plasma cfdna is localized in exosomes. to quantify cfdna content in free vs. exosomal form in human plasma, urine, and saliva, we employed subx technology, which allows affinity capture dna via phosphates groups of the polynucleotide chain and exosomes via membrane surface phosphate moiety clusters. subx is a proprietary compound that can simultaneously bind to both cfdna and exosomes in bioliquids, thus allowing precipitation of the [subx-dna/ subx-exosomes] complexes without ultracentrifugation. methods: detection of subx-dna and exosomes binding was done by measurement of particle sizes using zetasizer nano zs and nanosight ns300. the samples were processed with the subx exo-dna isolation kit following the standard protocols. dna, protein and lipid concentrations were measured by fluorescent assays using qubit fluorometer. results: subx efficiently and selectively captures and co-precipitates cfdna and exosomes directly from bioliquids. exosomes are easily extracted from the pellet in exosome reconstitution buffer (erb), followed by subsequent isolation of tightly bound cfdna from the subx pellet. erb does not extract dna form the [subx -dna] pellet and thus does not contaminate reconstituted exosomes with cfdna. thus, we separate two distinct types of extracellular materialintact exosomes and purified cfdna in a single protocol from the same sample. over 90% of dna in plasma and urine exist as a free circulating pool, while in saliva up to 30% is associated with exosomes. thus, cfdna distribution is probably bioliquid-specific and must be evaluated by methods that eliminate cfdna-outer exosomal membrane aggregation. summary/conclusion: subx technology is suitable for simultaneous isolation of both cfdna and exosomes from the same bioliquid sample. subx separates cfdna fragments non-specifically attached to the outer lipid layers of the exosome membrane from the true intra-exosomal cfdna. in contrast, salting-out peg technique is associated with aggregation of macromolecules and vesicles and thus leads to overestimation of exosome-associated polymers content, including cfdna. tracing extrachromosomal dna inheritance patterns in glioblastoma using crispr eunhee yi, amit gujar, hoon kim, albert cheng and roel verhaak jackson laboratory for genomic medicine, farmington, usa introduction: glioblastoma multiforme (gbm) is the most lethal brain tumour; it is characterized by poor response to standard post-resection radiation and cytotoxic therapy, resulting in a dismal prognosis with a five-year survival rate of 10%. recurrence after therapy for gbm is unavoidable. there are substantial differences among the cells of gbm tumours in the abundance and types of genetic material. this heterogeneity likely is the major cause of therapy failure, the development of treatment resistance, and ultimately recurrence. a recent study has suggested that the amount of a particular type of dnaextrachromosomal dna (ecdna)differs substantially among different gbm tumours, and differs within a given gbm tumour over time. despite the speculation that ecdna is a key factor of tumour heterogeneity, how ecdna is propagated and distributed amongand how it behaves withincancer cells is completely unknown. methods: to address this gap in knowledge, this study focused on developing a novel cytogenetic crisprbased tool that enables visualization and tracking ecdna behaviour in live gbm cells. results: we found breakpoint sequences resulting from genome rearrangements during ecdna formation by performing computational analysis from whole genome sequencing data. and each breakpoint was regarded as a unique target sequence for ecdnaspecific labelling. the uniqueness of each breakpoint was validated by breakpoint-pcr (bp-pcr). furthermore, the location and the amount of each breakpoint were observed by breakpoint-fish (bp-fish) analysis in gbm cells. summary/conclusion: this results will be strong evidence to make ecdna-specific crispr system in further research. tracing ecdna dynamics will provide new insight into the impact of ecdna on cancer evolution. introduction: small extracellular vesicles (sevs) are 30-150 nm vesicles that mediate intercellular communication by transferring rna and proteins to the recipient cells. these cargo molecules are selectively sorted into sevs and mirror the physiological state of the donor cells. given that sevs can cross the bloodbrain barrier and their composition can change in neurological disorders, there is an increasing interest in elucidating the molecular signatures of sevs in circulation as disease biomarkers. however, circulating sevs are derived from multiple cellular sources and determining their source is challenging. information on sev composition can be beneficial in predicting whether these sevs are released predominantly from central nervous system cells. we hypothesized that differentially expressed mirnas between neuronal sevs and astrocytic sevs could be used as cell-typespecific signatures. methods: small extracellular vesicles were isolated from cell culture media of postnatal mouse primary neurons and astrocytes using differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna from neurons, astrocytes, and their respective sevs were used for transcriptome and small rna sequencing. results: we observed that only a subset of cellular mirnas was packaged into sevs; differential expression of specific mirnas between sevs and their corresponding cells suggest that cells employ special mechanisms to sort mirnas into sevs. these mechanisms could be celltype specific since neuronal sevs showed a different mirna profile compared to astrocytic sevs. exomotifs, the short sequence motifs that control the loading of rna into sevs, were present in differentially expressed mirnas. we also observed that five rnabinding proteins, which are associated with passive or active rna sorting into sevs, were differentially expressed between neuronal and astrocytic cells. summary/conclusion: mirna signatures of sevs from neurons and astrocytes could be beneficial in determining if these cell types contribute to the alterations of sev composition in circulation in neurological disorders. cell-type-specific selectivity in rna loading might be attributed to the differential expression of rna-binding proteins. introduction: analytes present in the extracellular fraction of bodily fluids (ex. blood, urine) have utility as a tool for uncovering the molecular landscape of tumours and hold great potential for discovery of individualized cancer medicine. urine, being noninvasive as a sample type, has an obvious advantage over blood when used for liquid biopsy purposes. however, potential for microbial proliferation and the labile nature of host cells and extracellular vesicles (evs) at the point of sample collection/transport to the lab drives the need for stabilization of urine samples. development of such sample stabilization opens up capability for the detection of various biomarkers present in the extracellular fraction to be used in liquid biopsy. this is of particular concern as studies around urinary analytes for cancer diagnosis, progression and therapeutic effect are rapidly expanding in cohort sizes. multi-site collections and at-clinic collections are increasingly prohibitive for large scale recruitment and also lead to variability in the time between collection and processing. methods: in this study, we have analysed two commercially available ev extraction kits and compared them with ultracentrifugation technique for size, concentration and specificity of the isolated evs from human urine samples with and without our proprietary preservation solution using nanoparticle tracking analysis and western blot analysis for exosomal membrane markers. ev rna contents in various urine fractions (first morning first void, random first void and midstream) were compared using rt-qpcr assay to provide better understanding of the collection techniques and fractionations that are ideal for ev research work. results: in our current work, we have bench-marked human urine collection and ev extraction in order to provide recommendations in standardization of sample acquisition and processing for urinary ev studies. we have utilized these standardization in order to develop a novel and efficient sample stabilization principle for preservation of evs and ev rna in urine samples during an ambient temperature hold. summary/conclusion: taken together, we have established a framework for evaluating technologies and techniques in the ev sample processing space, which can be utilized by other research groups. vn96-isolated plasma extracellular vesicles improve tumour mutation detection by next-generation sequencing compared to cell-free dna and correlate with tissue biopsy of nsclc patients introduction: liquid biopsy is a minimally-invasive diagnostic method that detects circulating biomarkers and has the potential to improve access to molecular profiling for nsclc patients when tissue biopsy material is unavailable or insufficient. although isolation of cell-free dna (cfdna) from plasma is the standard liquid biopsy method for detecting dna mutations in cancer patients, the sensitivity can be highly variable. vn96 is a 27 amino acid peptide with an affinity for heat shock proteins that are exposed on the surface of extracellular vesicles (evs); peptide-ev aggregates readily sediment using a benchtop centrifuge and therefore the vn96 peptide provides a rapid, clinically-amenable procedure for ev isolation. in this study, we determine whether isolation of evs from nsclc patient plasma improves the sensitivity of single nucleotide variants (snvs) detection compared to cfdna and correlate genetic changes observed by liquid biopsy with tumour ffpe tissue biopsy. methods: blood was collected from stage iii/iv nsclc patients with informed consent in either edta or cell-free dna bct® collection tubes and plasma was harvested within 30 minutes. total nucleic acid (tna) was extracted from either vn96-isolated evs from edta plasma or directly from plasma collected in edta or cell-free dna bct® tubes (cfdna). snvs were detected by next-generation sequencing (ngs) results: vn96 isolation of evs from plasma resulted in higher recovery of dna than cfdna isolation. the snvs detected in both ev-dna and cfdna correlated well with those reported in matched ffpe tumour tissue using ngs, including 100% specificity for egfr mutations. no improvement in snv detection was observed using cell-free dna bct® collection tubes compared to edta tubes. isolation of evs with the vn96 peptide prior to sequencing improved a number of ngs parameters including library yield, total reads, median read coverage and molecular coverage, resulting in improved sensitivity of snv detection. summary/conclusion: in summary, our research demonstrates that vn96-based ev isolation is useful for molecular profiling of nsclc patients for whom tissue biopsy is not an option, thereby improving access to molecular profiling and targeted therapies. funding: atlantic canada opportunities agency novel markers for neuroendocrine prostate cancer divya bhagirath a , michael liston b , theresa akoto a and sharanjot saini a a augusta university, augusta, usa; b veteran affairs, san francisco, usa introduction: prostate cancer (pca) is fuelled by androgens and androgen receptor (ar) signalling. therefore, ablation of ar signalling by androgen deprivation therapy (adt) is the goal of first-line therapy that results in cancer regression initially. however, two to three years post-adt, the disease develops into castration-resistant prostate cancer (crpc). as a second-line of therapy, next generation of ar pathway inhibitors (api) such as enzalutamide (enz) are used that are effective initially followed by emergence of drug resistance. a subset of api-resistant tumours emerges to an ar independent state via undergoing a trans-differentiation to neuroendocrine lineage, a process referred to as neuroendocrine differentiation (ned). due to lack of ar signalling, these pca variants, referred to as neuroendocrine prostate cancer (nepc), are impervious to anti-androgen therapy and constitute an aggressive variant of advanced crpc with poor prognosis. currently, there is a lack of effective molecular biomarkers for predicting api therapy resistance and emergence of therapy-induced ned. methods: exosomes/evs were isolated from sera of a patient cohort with/without ned. the study was conducted in accordance with ethical guidelines of us common rule and was approved by the institutional committee on human research. written informed consent was obtained from all patients. following extensive characterization of evs by electron microscopy, nanosight tracking analyses and western blotting of exosomal markers, small rna sequencing was carried out on illumina hiseq platform to identify differentially expressed transcripts. machine learning algorithms were applied to clinical sequencing data to train a "mirna classifier". further, we probed the proteomic profile of exosomes isolated from nepc cellular model nci-h660 and enzalutamide resistant crpc cell lines by mass spectrometry. results: we identified that transition from crpc-adenocarcinomas to neuroendocrine states is associated with significant ev-mirna dysregulation, with a specific dysregulation in certain mirna families. with the application of machine learning algorithm, we identified an ev-based "molecular classifier" that can robustly stratify crpc-ne tumours from crpc-adenocarcinomas. proteomic analyses identified novel nepc-specific, glycosylated proteins that can be exploited for nepc diagnosis. summary/conclusion: our data suggest that ev mirna and protein profile can predict neuroendocrine differentiation in advanced castration-resistant prostate cancer patients. exosomal mrna in diagnosis strategy for hepatocellular carcinoma aleksandr abramov, alisa petkevich, vadim pospelov and pavel ogurtsov peoples' friendship university of russia (rudn university), moscow, russia introduction: exosomal cargo is informative source illustrating the genetic events happening in cells, what can be especially advantageous in case of cancer development for disease progression or treatment effectiveness monitoring. methods: 10 plasma samples of hepatocellular carcinoma (hcc) patients, 10 plasma samples of patients with liver cirrhosis 3-4 on the hepatitis c virus (hcv) background, 5 healthy donors' plasma samples. exosomes were isolated with ultracentrifugation, western blot (cd63, cd9) was performed. total mrna was isolated with exosomal rna isolation kit, norgen biotec corp. sequencing was carried out on a minion sequencer. housekeeping genes (gapdh, b2 m, actb, tuba1a). detected mutations were confirmed by real-time pcr with specific highly sensitive lna probes. results: significant changes in expression levels were identified for 50 genes in hcc and liver cirrhosis groups (increasing up to x200 compared to control samples and decreasing up to no detected expression). in 6 out of 10 patients with hcc mutant burden was significant increased compared to mutant burden in groups with cirrhotic samples. in 8 out of 10 patients with hcc increased expression for mrna line-1 was identified compared to cirrhotic patients. summary/conclusion: exosomal mrna expression levels may serve as a prognostic and diagnostic marker for patients with liver cirrhosis caused by hcv for hcc risk development. funding: research is supported with federal funds "5-100" circulating extracellular vesicle signatures in small cell lung cancer michela saviana a , giulia romano a , giovanni nigita b , robin toft a , patricia le a , kai wang c , mario acunzo a and patrick nana-sinkam a a virginia commonwealth university, richmond, usa; b the ohio state university, columbus, usa; c institute for systems biology, seattle, usa introduction: lung cancer is the leading cause of cancer deaths worldwide and classified primarily as either non-small cell lung cancer (nsclc) or small cell lung cancer (sclc). compared to nsclc, sclc has a faster growth rate, earlier widespread metastasis, and shorter overall survival. the early diagnosis of sclc and the development of novel therapeutics have proven challenging. thus, progression and recurrence rates remain high. non-invasive methods for cancer detection are increasingly being used to inform clinical decision making. extracellular vesicles (evs) have recently emerged as potential carriers of genetic contents such as micrornas (mirs) to induce reprogramming of components of the microenvironment in cancer initiation and progression. moreover, extracellular mirs expression profiles have been shown to have signatures related to tumour classification, diagnosis, and progression. methods: we selected a cohort of patients divided into 4 groups: high-risk smokers, adenocarcinomas, squamous carcinomas, and sclc. we extracted total circulating ev and plasma rna from plasma (38 patients in total) and rna from plasma in a separate group (24 patients in total). utilizing both next-generation sequencing (ngs) and nanostring platforms, we analysed for global microrna (mirs) expression patterns. candidate mirs were then validated by qrt-pcr. results: we identified several deregulated mirs in both evs and plasma of sclc patients compared to the other groups. for evs, we validated mir-1285-5p as a significant biomarker for the late stage of sclc compared to controls. in the case of plasma, we validated the upregulation of mir-375 in sclc compared to controls. summary/conclusion: our results indicate that a potential combination of plasma (mir-375) and ev-based (mir-1285-5p) mirs be valuable biomarkers for sclc detection and serve as a basis for a non-invasive sclc classifier. funding: virginia commonwealth university, doim -nih/nci introduction: the isolation of evs from milk is technically challenging due to the complexity of milk. currently used separation procedures allow for the removal of milk fat globules and cells (by low speed centrifugation of fresh milk), removal (by acidification), or disruption (by addition of edta) of casein micelles. using these protocols the integrity, composition and targeting of bovine milk evs has been evaluated and has led to believe that milk evs might withstand these conditions. however, the effects on functionality of milk evs (i.e. immunomodulatory properties) after processing and isolation have not been studied. therefore, we have set up an in vitro culture system using a human t cell line that allows for the rapid screening of milk ev functionality. methods: fresh bovine milk was defatted and cells were removed after 2x3,000 g centrifugation, followed by differential centrifugation at 5,000 g and 10,000 g. this milk was either subjected to acidification with hcl, or edta was added, or the milk supernatant remained untouched. top down optiprep density gradient separation followed by sec was used to further purify evs. these highly purified milk evs were added to human jurkat t cells, which were simultaneously stimulated using anti-cd3 and anti-cd28 antibodies. after 24 h t cell activation was measured by il-2 cytokine production. results: precipitation or disruption of casein micelles allowed for the substantial removal of proteins during isolation compared to directly isolated evs, which aids in the purification of milk evs. in vitro analysis revealed that in the presence of directly isolated, or edta isolated milk evs, jurkat cells were suppressed in their activation as measured by il-2 production. remarkably, evs isolated from hcl-acidified milk were impaired in their suppressive capacity to inhibit il-2 production. summary/conclusion: although casein removal from bovine milk greatly improves purity of isolated milk evs, the detrimental effects on ev functionality should be considered. interestingly, evs exposed to acidic conditions lost their ability to modulate t cell activation, which is in contrast with the general believe that milk evs could withstand the gastro-intestinal tract. funding: this work is funded by the european union's horizon 2020 framework programme under the grant fetopen-801367 evfoundry. optimising methods for separation and characterisation of extracellular vesicles from skim milk and infant milk formula introduction: infant milk formula (imf) is intended to impart nutrition to infants, similar to breast milk. however, although industrial imf production involves harsh treatment, potential consequences on extracellular vesicles (ev) in imf are not yet established. this study aimed to optimise methods for separating evs from imf and skim milk (sm) and to characterise the evs in accordance with misev2018. methods: sm and imf were either not treated (nt) or treated with acetic (aa) or hcl acid (isoelectric precipitation, ip), to remove caseins. samples were then subjected to differential ultracentrifugation (duc) or gradient ultracentrifugation using iodixanol solution (guc). for duc, 38 ml samples were centrifuged at 12 k g, 35 k g, 75 k g, 100 k g and 200k g sequentially for 75 min each and pellets re-suspended in 1 ml pbs. preparation of agarose microspheres for high-efficient separation of extracellular vesicles cheng-tai chen, chien-an chen, carolyn yen and nien-tzu chou industrial technology research institute, chutung, taiwan (republic of china) introduction: size exclusion chromatography (sec) is becoming a widely used technique for separating of extracellular vesicles. various commercially available products were launched on the market, however, their separation efficiencies were not fully disclosed. herein, novel porous agarose microspheres with the tunable diameter and pore size were synthesized by emulsion reaction. the performance was evaluated and compared with commercial products. the modified sec column packing materials were shown to exhibit advantages for rapid, high-recovery and high-purity separation of extracellular vesicles from cell culture-conditioned medium and human plasma. methods: the homemade sec column was packed by gravity flow. 100 μl of the sample was loaded and the pbs buffer was used as eluent. 24 factions were collected and analysed by cd9/cd9 sandwich elisa assay and by micro bca assay for determining respectively extracellular vesicles and total protein content. results: agarose microspheres were prepared by emulsification. the particle size can be controlled by the types and concentrations of surfactants. the product was collected by desired screen meshes and used as packing materials of the sec column. our results showed that the extracellular vesicles were clearly separated from proteins. more than 99.8% of proteins were removed while the recovery of extracellular vesicles was close to 70%, which is much higher than 32% of the commercial product. the total separation time was less than 15 min. summary/conclusion: we have established an approach for generating spherical agarose microspheres as packing materials of homemade sec columns, which are capable of separating extracellular vesicles from complex samples with high efficiency. further validations with additional samples are currently ongoing. immunomagnetic sequential ultrafiltration (isuf) platform for enrichment and purification of extracellular vesicles from large and small volumes of biofluid eduardo reategui, jingjing zhang, luong t. h. nguyen, richard hickey, nicole walters and andre f. palmer the ohio state university, columbus, usa introduction: evs derived from tumour cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. however, compromises have to be made when using a particular technology/methodology for the isolation of evs. currently, there is a trade-off between sample volume and specificity in ev isolation technologies that limits quantitative molecular analysis of ev contents, ultimately impacting the utility of evs in cancer diagnostics. here, we present an approach called immunomagnetic sequential ultrafiltration (isuf). our platform combines ultrafiltration and immunoaffinity separation. using isuf, we demonstrate that small or large volumes of biofluid can be processed (~100 µl or > 100 ml) while concomitantly removing 99.9 % contaminating proteins. we also processed serum from breast cancer patients enabling the characterization of different tumour and immune biomarkers on the isolated evs. methods: human samples were collected under an approved irb. size distribution and concentration of evs were measured using a tunable resistive pulse sensing (trps) method. ev proteins and rnas were extracted and quantified using a bca protein assay and uv spectroscopy. isuf and other ev isolation methods were compared for ev concentration, protein, and rna quantity. results: 50 ml of cell culture media (ccm), 0.5 ml serum, and 100 ml urine samples were processed with the isuf platform and recovered in 100 µl. for all cases, evs were enriched with recovery efficiency greater than 95%. the processing time for a 100 ml sample was 120 min with over 99% of purity. we compared ev concentration and purity isolate from 0.5 ml serum using isuf and other commercially available methods, isuf demonstrated superior performance on isolating evs at high concentrations and purities. analysis of total rna amounts in the isolated evs using different methods was corresponding to higher ev recovery efficiency of isuf. we also compared protein and rna levels of evs enriched with isuf present in urine and serum samples from the same donors (n = 10), and we found that for the same number of evs, the ev rna concentration from both biofluids showed no significant difference. finally, we have processed serum samples from 10 metastatic breast cancer patients and demonstrated that their isolated evs have expression levels of her2, cd24 and mir21 biomarkers at significantly higher levels than healthy controls. summary/conclusion: the isuf platform can be scale-down or -up to work with small or large volumes of biofluids for the isolation of evs. using the isuf platform with clinical samples shows the potential of our platform to be used for cancer diagnosis or monitoring treatment response. funding: national institutes of health (nih) grants ug3tr002884 (e.r.); r01hl 126945, r01hl138116, and r01eb021926 (afp). challenges in exosomes isolation from primary biological samples derived from multiple myeloma patients introduction: multiple myeloma (mm) remains incurable despite advances in its treatment and research progress on the crosstalk between mm and surrounding host cells. exosomes are important regulators of the cellular niche. their importance for diagnostic and therapeutic applications has been proven in many cancers. in this context we hypothesized that a better understanding of the molecular role and features of mm-derived exosomes would provide a basis for their use for both risk stratification and as predictive biomarkers of response to anti-mm drugs already in use in clinical settings, given the optimization and validity of their isolation/purification method. methods: exosomes were isolated from human mm cell lines (hmcls) supernatants and peripheral blood plasma (pbpl) isolated from healthy donors, mm and mgus (monoclonal gammopathy of undetermined significance) patients. both fresh and frozen samples were tested. we evaluated 3 commercially-available kits, density-based separation and ultracentrifugation. results: higher purity and recovery, evaluated by western blotting, nanoparticle tracking analysis and electron microscopy, were observed for supernatant density-based purification and for pbpl resin-based isolation. exploring the function of mm-derived exosomes, we observed an increase in proliferation of the immortalized stromal cell (sc) line hs5 treated with exosomes when compared to untreated cells, and a higher increase in proliferation of scs treated with mm-exosomes when compared to exosomes derived from normal and mgus pbpl samples. summary/conclusion: the method of isolation represents a critical step in the study of exosomes as many factors can affect the purity, yield and downstream application. our data demonstrated that density and resin-based isolation methods provided functional mm-derived exosomes with proliferative effects on scs. altogether our findings may serve as a guide to choose exosome isolation methods for mm studies. further optimization steps, including albumin-depletion from plasma samples and use/type of serum in cell cultures, should be taken into consideration when planning proteomics and genomics as downstream applications. funding: australian government rtp and monash departmental scholarship. a rigorous method for exosome isolation from post-mortem eyes introduction: in order to determine and validate the tissue-specific content of extracellular vesicles (evs) in biofluids, robust ev isolation methods from tissues must be developed. however, to date very few rigorous methods to isolate or enrich for intact evs from tissues have been reported. we present a comprehensive exosome isolation method with a sufficient level of characterization to unequivocally demonstrate true ev identity from ex vivo eyes. methods: iodixanol (optiprep) buoyant density gradient ultracentrifugation (dguc), cushioned dguc (c-dguc), and our newly developed c-dguc immunocapture (c-dguc-ip) method were used to compare yield and enrichment of exosomes isolated from porcine eyes between 3 to 5 hours post-mortem. yield was assessed by nanoparticle tracking analysis (nta) and immunoblotting for exosomal markers along with total protein quantitation. enrichment was assessed by comparison of exosomal markers, ocular-specific markers and known contaminant markers, plus in-depth proteomic mass spectrometry analyses. results: high enrichment of posterior eyecup small evs (sev) were achieved by dguc and c-dguc, with c-dguc resulting in an eightfold increase in yield by nta and two to fivefold increases of exosomal protein markers such as syntenin-1 and cd81 by immuno-blotting compared to dguc. interestingly, in-depth proteomic analyses revealed that a majority of these sevs with densities of 1.07-1.11 g/ml isolated by dguc and c-dguc were likely of endoplasmic reticulum (er) and golgi origin, suggesting er-to-golgi transport vesicles resulting from post-mortem tissue cell rupture. in order to enrich further for sevs (including exosomes) we subjected sevs isolated by c-dguc to anti-cd81 immunocapture. the resulting sev proteome was enriched 1.5-to 4-fold for bona fide sev and exosome markers compared to c-dguc. summary/conclusion: the c-dguc method provides an enhanced yield and purity of sevs and exosomes from ex vivo eye tissue. however, to avoid significant contamination with er and golgi-derived vesicles from postmortem eyes, a final ev-specific immunocapture step is required to achieve sufficient purity for subsequent analyses. our highly rigorous method paves the way for identification and validation of ocular-derived exosomes in blood and their potential use as eye disease biomarkers. characterization of the extraction of extracellular vesicles using a lab-ona-disc filtration system introduction: personalized treatment for cancer is a promising way to face the multiplicity of the disease, to increase the efficacy of drugs and to decrease their toxicity. as part of this strategy, liquid biopsy explores a new non-invasive approach to diagnose cancer, guide treatment and monitor its efficacy. extracellular vesicles (evs) are nanometric lipid bilayers micelles with high potential as biomarkers. they are involved in the transfer of information (proteins, rna and dna) between cells. evs include a broad spectrum of particle sizes, from the tens to thousands of nanometres. the isolation of evs from complex matrices is the first step of any protocol and is particularly important for the reproducibility and fidelity of the results presented, as it could bring bias in further analysis. in order to explore the heterogeneity of evs, a full characterization (physical and biological) of the extracted evs is needed. we evaluate and compare 3 evs purification methods, including ultracentrifugation, sizeexclusion chromatography (sec) column and an emerging microfluidic technology: labspinner filtration labon-a-disc device isolating evs between two filters of 600 and 20nm. methods: a431 cell supernatant was used as a model matrix. we compared three methods of extraction of evs: ultracentrifugation with two cycles of 1 h10 at 110,000 g at 4 degrees celsius (rotor type 70ti, beckman floor ultracentrifuge optima l90 k), qev size exclusion chromatography columns from izon (qevoriginal/70 nm) and lab-on-a-disc filtration system (labspinner, exodisc c). evs characterization was conducted with nta (nanosightns500), trps (izon), nanodrop (spectrometernd1000), tem (fei tecnai 12 120 kv) and custom micro-immuno-assay. results: in this study, we characterize a filtration system made of two serial filters of 600 nm and 20 nm pores for isolation of evs. compared to ultracentrifugation and chromatography columns, yield of extraction is up to 10 times higher and the size of the extracted particles is smaller. tem imaging was used for assessment of the quality of the extracted evs. however, albumin concentration measurement tends to show that the purity of the solution is decreased. the immuno-labelling analysis shows that the proteomic signature of the extracted evs differs according to the extraction methods. the new filtration technology seems to give us access to a broader range of evs compared to standard methods. summary/conclusion: in this study, we characterized 3 purification methods including lab-on-a-disc filtration, and were able to demonstrate an increase of the concentration of evs by a factor of 10, a decrease of the size of the accessible extracted particles and access to new proteomic signatures. funding: we acknowledge the support of génome québec and action marie skłodowska-curie. effects of sample processing on isolation of extracellular vesicles from blood plasma by centrifugation darja božič a , matej hočevar b , veno kononenko c , marko jeran a , urška štibler d , immacolata fiume e , manca pajnič f , ljubiša pađen f , ksenija kogej g , damjana drobne c , ales iglič h , gabriella pocsfalvi i , veronika kralj-iglič f and darja bozic j introduction: the isolation of extracellular vesicles (ev) from body fluids is still controversial and the poor understanding of vesicle stability and effects of sample processing is probably one of the core issues preventing the breakthrough of this field into applicative practices. methods: we performed an in-depth study of sample changes in blood, blood plasma and samples throughout the increasing speed of centrifugation, considering the number, size, contents and shape of particles in the isolates. flow cytometry, light scattering, mass spectrometry and scanning electron microscopy were employed to reveal the properties of material in the samples. results: the particles of size about 100-500 nm with characteristic topology of membrane vesicles without internal structure were observed by the scanning electron microscope only in ev isolates prepared from fresh blood sample. inspection of the tube surface in which the isolation took place suggests that those particles are likely formed from activated platelets tearing at the tube wall due to the centrifugal pull. the isolates prepared from frozen blood plasma prepared by centrifugation with different forces contained different amounts of particles with similar protein contents, predominated by highly abundant human plasma proteins, including albumins and immunoglobulins. some lipoprotein clearance and fibronectin precipitation were however observed through increased speed and time of centrifugation. summary/conclusion: the results of this study [1] contribute to the understanding of stability and dynamics of membrane particles. the reported evidence provides the support for viewing ev isolates as a product, shaped by uniqueness of the starting samples and the thermal and mechanical stress applied upon processing. we believe this kind of insights strengthen our ability of reading the story of evs. introduction: apoptosis is a form of programmed cell death with diverse roles in the tumour microenvironment and emerging data show that, besides its role in tumour suppression, it can also promote oncogenic proliferation. highly aggressive tumours such as burkitt lymphoma (bl) show high levels of apoptosis, which has a diagnostic and prognostic value for classifying and staging the disease. we hypothesize that amongst other elements, extracellular vesicles (ev) are key mediators of apoptotic cell-derived tumour microenvironment signals. here, we report on ev released in vitro by apoptotic bl cells (apo-ev) in relation to their potential use as cancer biomarkers. methods: basic physical properties of apo-ev such as structure, size distribution, surface charge and membrane fluidity are discussed using cryo electron microscopy (em) and tomography, nanoparticle tracking analysis, dynamic light scattering and fluorescence anisotropy respectively. for phenotypic analysis we apply immunocapture and flow cytometry, immunogold labelling on transmission em, fluorescence microscopy and quantitative pcr. in addition, we study the interaction of apo-ev with blood components such as platelets, leucocytes and red cells, in order to understand their effects in the circulation and therefore their potential for analysis in blood samples. results: looking at the differences between apo-and non-apo-ev, apo-ev have larger diameter, while structurally are not different. however, we have identified distinct apo-ev markers such as active caspase 3 and histones, or dna and small non-coding rna-y. there is also strong interaction of ev with platelets and leucocytes but not with red cells, indicating potential routes of transfer of ev cargo in the circulation. summary/conclusion: it is concluded that for the characterization of the heterogenous ev populations, combination of multiple techniques is often required, and also, understanding the strengths and limitations of each method is essential for choosing the appropriate set of analytical tools. finally, we consider that monitoring free circulating apo-ev or blood cells with which they have interacted is a promising approach to improve cancer diagnosis, prognosis and evaluation of therapeutic response. casting a small netrin: functional roles of a novel surface factor on stroma-derived extracellular vesicles in pancreatic cancer kristopher s. raghavan a , ralph francescone b , janusz franco-barraza b and edna cuckierman b a drexel university; fox chase cancer center, philadelphia, usa; b fox chase cancer center, philadelphia, usa introduction: pancreatic ductal adenocarcinoma (pdac) is a devastating disease driven and supported by changes in its microenvironment, or stroma. here we dissect the intercellular communication that exists between the primary stromal component, cancer-associated fibroblasts (cafs) and pdac. pdac communicates with its microenvironment, in part, through the exchange of specific types of extracellular vesicles (evs). specifically, we focus on the mechanism by which caf-secreted evs support pdac survival, with an additional goal to identify biomarkers suitable to generate a future "liquid biopsy" test for early pdac detection and prognosis. methods: evs are isolated from patient-derived pdac-associated fibroblasts via differential ultracentrifugation and validated by isev standards. human pdac cell lines used as recipient cells are treated with caf-evs to assess their role in supporting pdac survival. recombinant proteins, neutralizing peptides, and non-functional mutant proteins are used to block ev interaction with target cells. results: we observe sub-types of caf-evs containing unique surface receptors. one ev sub-population of interest contains a novel surface protein (nsp) expressed on the plasma membrane of pancreatic cafs, but not their healthy counterparts. further, pdac cells up-regulate nsp's lone binding partner, suggesting a role for these factors in pdac-selective ev uptake. functional assays designed to test pdac viability suggest these nsp(+)-evs protect pdac cells from programmed cell death as a result of physiological stress. this ev-mediated survival benefit can also be inhibited by blocking the interaction of nsp and its binding partner, suggesting the engagement of these two factors is necessary for cafs to support pdac via evs. pursuing our biomarker goal we confirm stromal nsp expression increases during early panin stages prior to tumour development, and we are currently seeking to validate nsp(+)-evs in blood of pdac patients. summary/conclusion: this research shines light on a novel mechanism of tumour-stroma communication that may be crucial for cancer progression during early disease stages and a potential target for disrupting the supportive role of the tumour microenvironment. additionally, we describe a sub-population of nsp (+)-evs that have the potential to serve as biomarkers for identifying pdac development. exosomes carry distinct mirnas that drive medulloblastoma progression introduction: extracellular vesicles (evs) represent an ideal source of functional biomarkers due to their role in intercellular communication and their ability to protect cargo, including rna, from degradation. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the bloodbrain-barrier. here we characterised the rna of exosomes isolated from medulloblastoma cell lines, with the aim of investigating exosomal rna cargo as potential functional biomarkers for medulloblastoma. methods: exosomes derived from a panel of matched (original tumour and metastasis) medulloblastoma cell lines were isolated and characterised by nanosight, electron microscopy, western blotting and nanoscale flow cytometry. exosomal mirna and mrna from our matched cell lines and foetal neuronal stem cells, which were used as a normal control, were analysed by rna-sequencing technology. results: based on hierarchical clustering, malignant derived exosomes were distinctly separated from normal control exosomes. mirna profiling revealed several established oncomirs identified in our malignant derived exosomes compared to control samples. using interaction pathway analysis, we identified that our malignant exosomes carry numerous mirnas implicated in migration, proliferation, cellular adhesion and tumour growth. several previously identified oncomirs were also identified to be present at higher levels in metastatic exosomes compared to primary and normal, including hsa-mir-455-3p and hsa-mir-92a-3p. summary/conclusion: this study shows that exosomes from mb cells carry a distinct mirna cargo which could enhance medulloblastoma progression. the use of circulating exosomes as markers of metastatic disease could be an innovative and powerful noninvasive tool. introduction: inflammatory changes in the bone marrow (bm) and suppression of haematopoietic stem and progenitor cell (hspc) function during acute myeloid leukaemia (aml) significantly contribute to patient morbidity and mortality. our laboratory has previously shown that aml-derived extracellular vesicle (ev-aml) trafficking confers a state of enforced quiescence and leads to lasting dna damage in hspcs. here we explore the underlying cause. specifically, we hypothesize that ev-aml incite inflammatory regulators as potential mediators of dna damage. methods: as a validated model of aml, we utilized the murine tib49 cell line as a source of ev-aml. ev-previous work has indicated that mirnas, notably mir-146a and mir-155, play a critical role in scc tumour development. evs are membrane-bound vesicles involved in cell-cell communication carrying actively sorted cargo, protected from degradation. the potential pathways these vesicular mirnas modulate and the implication they have on cancer biology is under active investigation. we have previously shown that the cadherin dsg2, a stem cell marker, modulates ev release. dsg2 is upregulated in a number of cancers, including scc, and correlates with poor prognosis. here we aim to elucidate the impact of ev-associated mirnas in sccs by bioinformatic analysis. methods: scc cells stably expressing dsg2 were generated and evs isolated by sequential ultracentrifugation. total cellular and ev rna was isolated by mirneasy, analysed using rnaseq and identified by grch37 alignment. results were confirmed by qpcr. altered pathways based on targets were identified using mirnet and kegg pathway analysis. potential cancerassociated cytokine targets were confirmed by antibody array. results: rnaseq revealed 87 cellular and 15 ev mirnas that were differentially expressed in response to dsg2 with 7 overlapping. the highest altered mirnas were validated by qpcr. kegg pathway analysis determined that these mirnas have the highest number of shared targets in cancer, cell cycle, and p53 signalling pathways. interestingly, mir-155 was upregulated while mir-146a was dramatically downregulated in evs. targets of mir-146a, icam-1, il-6, and il-8, cytokines critical for cancer progression were upregulated. summary/conclusion: these results suggest that the mirna content of evs is tightly regulated. by altering the mirna profile, dsg2 contributes to the pathogenicity of these evs by increasing levels of cytokines important for cancer stem cell renewal and metastasis. in addition, these mirnas may serve as non-invasive diagnostic markers for sccs. funding: nih r01 cancer cells grown in 3d release distinct extracellular vesicles during tumour growth and invasion jens c. luoto, sara bengs, leila coelho rato, lea sistonen and eva henriksson åbo akademi university, turku, finland introduction: cancer cells secrete extracellular vesicles (evs) that affect tumour progression. the characteristics of evs produced during tumour growth and invasion are however poorly understood. in this study, we identify the composition and characteristics of evs produced by noninvasive and invasive tumours and correlate these characteristics with the invasive status of the tumour. for that purpose, we established a protocol for isolating evs from extracellular matrix (ecm)-based three-dimensional (3d) cancer cell cultures. methods: human prostate cancer pc3 cells were grown in 3d cultures using ecm-based hydrogel, in standard 2d culture conditions and in bioreactor. evs were isolated from these cultures with differential and density gradient centrifugation. the isolated evs were characterized with nanoparticle tracking analyses, electron microscopy, immunoblotting and mass spectrometry (ms). results: our results demonstrate that 3d ecm-based hydrogel cell cultures secrete evs that can be isolated from both the conditioned media and the hydrogel. the invasive 3d cultivated pc3 organoids were found to secrete large amounts of evs compared to the non-invasive organoids. interestingly, our ms results revealed that non-invasive and invasive organoids secrete evs with partially distinct protein cargo. summary/conclusion: we have established a novel protocol for ev production in a 3d cell culture system utilizing ecm-based hydrogel, in which invasive tumour growth can be mimicked. our method allows the specific isolation and characterization of evs derived from different stages of 3d culture, such as non-invasive and invasive organoids. importantly, we found that tumour-derived evs change in composition during the tumour progression. taken together, our method can be used to define the distinct ev characteristics involved in cancer invasion. we previously showed extracellular vesicles (evs) to be causally involved in transmitting drug resistance. this study aimed to evaluate compounds proposed to reduce/block ev release. specifically, we selected calpeptin and y2763 (proposed to inhibit evs budding from the cell membrane) and manumycin a and gw4869 (proposed to inhibit evs deriving from mvbs). associated effects on -and consequences of-ev release were then investigated. methods: suitable compounds concentrations that were non-toxic to cells were first selected by performing cytotoxicity assay and flow cytometry (fc). conditioned medium (cm) was collected from docetaxel-resistant pc3 (pc3 rd) cells after 48 h incubation in dfbs-medium with or without the 4 compounds. evs were separated from tangential flow filtration concentrated cm using optiprep density gradient. 1.03-1.16 g/ml fractions were then pooled and washed. evs were characterised using nta, immunoblot, tem and lipid assay and fc. influences on growth and migration, of evs continuing to be released (at 1x105evs/3x103cells, 1x107evs/3x103cells), were evaluated on recipient du145 and 22rv1 cells. evtrack id ev190113, score 88% results: calpeptin and y27632, alone and in combination, did not significantly affect quantities of evs released. however, gw4869 significantly (p < 0.004) increased quantities of released evs, of a larger size; very high protein to lipid ratio; and carrying grp94 compared to control evs (p < 0.006). this effect was reverted when gw4869 was combined with manumycin a (p < 0.004). following all compounds treatments, 1x105evs/ 3x103cells inhibited 22 rv1 proliferation (p < 0.0014), while at 1x107evs/3x103cells only evs from manumycin a (p < 0.05) and y27632 (p < 0.00036) treated cells reduce 22 rv1 proliferation. evs following gw4869 treatment significantly (p < 0.001) inhibited du145 migration compared to bulk non-treated control and compared to the effect obtained using the entire pool of evs (p < 0.001). summary/conclusion: while none of the 4 proposed inhibitors significantly reduced ev release, the resulting evs were less potent in transmitting aggressive behaviour, such as proliferation and migration, to receiving cell lines. patient-derived organoids represent a novel tool to study the effect of intra-tumoral heterogeneity on ev release in non-small cell lung cancer introduction: lung adenocarcinoma (luad) is the leading cause of cancer-related death with a low 5-year survival. although the importance of intra-tumoral cellular heterogeneity of solid tumors in the clinical outcome and treatment is emerging, proper models to study its effects on ev release and cargo in human tissues still lack. the 3d organoid technology maintains the cellular and genetic heterogeneity of in vivo tissues and has proved to be so far the best ex vivo model of human cancers. by using patient-derived and mouse organoids we set out i) to compare the ev release from normal and tumor tissues and ii) to follow changes in ev secretion when the relative ratio of tumor cell subpopulations is shifted. methods: we used mouse and luad patient-derived normal and tumor organoids. the medical research council of hungary approved our experiments with human samples and informed consent was obtained from patients. evs were detected by antibody-coated beads, nta and tem. intra-organoid heterogeneity was proved by immunostaining and rt-qpcr. results: we provide evidence that both mouse and human normal organoids contain all the bronchiolar cell types. interestingly, luad organoids selected for tp53 mutation contained not only ki67+ proliferating cells, but differentiated cell types as well. furthermore, all the lung organoid cultures produced evs and this was shifted to the smaller size range. interestingly however, when modifying the proportion of organoid cell types, we observed an increased ev release when more ki67+ proliferating cells were present both in normal and in luad samples. summary/conclusion: our data show that patientderived lung organoids represent a novel model to study the role of intra-tissue heterogeneity in ev functions in the humans, leading to improved diagnosis. funding: this work was funded by the national competitiveness and excellence program nvkp_16-0007 (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). exosome mediate heart-adipocyte communication after myocardial ischaemia/reperfusion and impairs adipocyte endocrine function yajing wang, lu gan, dina xie, wayne lau, theodore christopher, bernard lopez and xinliang ma thomas jefferson university, philadelphia, usa introduction: by incompletely understood mechanisms, mi patients sustain systemic metabolic disorder. adipocytes are an important cellular type regulating energy homoeostasis. the impact of mi upon adipocyte function remains unknown. exosomes (exo) are critical vehicles mediating organ-organ communication. however, whether and how exo may mediate post-mi cardiomyocyte/ adipocyte communication have not been previously investigated. methods: adult male mice were subjected to mi/r. serum exo were isolated 3 hours after r and incubated with 3t3 l cells for 24 hours. the effects of exo upon adipocyte function were determined. results: compared to control, mi/r exo significantly altered the expression of 17 genes known to be important in adipocyte function. go analysis revealed that genes associated with endoplasmic reticulum (er) function and adipocyte endocrine function are the primary two pathways altered by mi/r exo. venn analysis identified 11 mi-rnas as cardiac-enriched, adipocyte-poor, and er function-related mirnas. rt-qpcr confirmed the mir-23a/27a/24-2 family members are the most markedly increased mi-rnas in mi/r exo. incubation of 3t3 l cells with mi-r27a mimic significantly downregulated edem3, dsba-l, and pparn, and upregulated perk and chop. conversely, mi-r27a inhibitor significantly decreased the impact of mi/r exo upon er function genes. additional studies demonstrated edem3 and pparγ (two critical molecules maintaining er function and adipocyte endocrine function) to be direct targets of mi-r27a. one of the most significant endocrine molecules of adipocyte origin, adiponectin is regulated by pparn at the transcriptional level and by dsba-l at the post-translational level. we next determined whether mi/r exo may affect adiponectin expression/ assembly. incubation of 3t3 l cells with mi/r exo significantly inhibited total and high molecular weight adiponectin expression, an effect blocked by mir27a mimic. finally, in vivo administration of gw4869 (exo biogenesis inhibitor) or mir27a inhibitor attenuated adipocyte er dysfunction and restored plasma adiponectin level in mi/r animals. summary/conclusion: we demonstrate for the first time that mi/r causes significant adipocyte er and endocrine dysfunction by exo mediated cardiomyocyte/adipocyte communication via mir-23a/27a/ 24-2. funding: nih and american diabetes association pancreatic cancer cell extracellular vesicles drastically alter the behaviour of recipient normal pancreas cells charles p. hinzman a , yaoxiang li a , meth jayatilake a , jose trevino b , partha banerjee a and amrita cheema a a georgetown university medical center, washington, usa; b university of florida health science center, gainesville, usa introduction: pancreatic cancer (paca) is predicted to become the 3rd leading cause of cancer-related deaths by 2030. patients diagnosed with pancreatic ductal adenocarcinoma (pdac) have a 5-year survival ratẽ 9%. detection of pre-neoplastic lesions can potentially improve survival. however, there is currently no screening test for early stage detection. importantly, paca tumours are 90% non-tumorigenic cells. a better understanding of early paca oncogenesis is needed. cancer cells shed extracellular vesicles (evs) that are internalized by neighbouring and distant cells to induce a myriad of cancer progression events. we hypothesize that in early paca oncogenesis, evs mediate a behavioural change in surrounding normal cells, leading to the formation of this unique stroma. the purpose of this study was to develop a model to examine the phenotypic changes undergone by normal human pancreas cells when they are exposed to paca cell evs. methods: evs were isolated using differential ultracentrifugation with filtration from established (panc-1, sw-1990, capan-1 and miapaca-2) and patientderived xenograft (ppcl-46 and ppcl-68) paca cell lines. cells were grown using ev-depleted fbs. ev isolations were validated and quantified using transmission electron microscopy, quantitative elisa, immunoblot and nanoparticle tracking analysis. normal pancreas cells (htert-hpne and hpde-h6c7) were co-cultured with cancer cell evs for 24-48 hours. metabolic activity was measured using a mito stress test on a seahorse xfe96 extracellular flux analyser. results: we discovered that normal cells undergo vast behavioural transformations, including significant morphological changes, increased proliferation and an uncharacteristic invasive capability, when co-cultured with paca cell evs. these responses were ev dose dependent. further, paca cell evs metabolically reprogrammed normal cells, causing a bioenergetic switch, from a quiescent, aerobic profile to a highly energetic and glycolytic profile. summary/conclusion: our results indicate that paca cell evs confer enormous transformational properties to normal human pancreas cells in vitro. we hypothesize that evs impart distinct transformational properties to normal cells in vivo and this influence could unveil novel mechanisms regulating cancer onset and progression. these signals may be detectable before progression of early-stage paca to pdac, leading to the development of assays for earlier diagnosis in patients. further studies are underway to identify the biochemical mediators of these changes. plasma extracellular vesicles-mirnas released by hypoxic cells are associated to pro-tumorigenic and immunosuppressive microenvironment in lung cancer introduction: extracellular vesicles (ev) containing specific subset of functional biomolecules, such as micrornas (mirnas) are released by all cell types. it has been widely demonstrated that ev-mirnas from cancer cells can manipulate the tumour microenvironment modulating the gene expression of recipient cells. we previously identified a three levels risk classifier (msc) based on 24 plasma-mirnas associated with lung cancer development and prognosis. the aim of this study was to investigate the potential role of ev-24 mirnas as mediators of pro-tumorigenic features. methods: evs were isolated from plasma of heavysmoker individuals with high (mscpos) or low (mscneg) risk of lung cancer by differential centrifugation method. purity of evs isolated was confirmed by sizing using nta and tem analysis and expression of ev-enriched proteins. mirna levels were analysed by dpcr. in vitro and in vivo analyses were used to assess the biological effect of plasma evs on different recipient cells. results: levels of mirnas in evs correlated with determination of whole plasma (80% of correlation with msc classifier). mscpos-evs stimulated 2d and 3d proliferation of non-tumorigenic epithelial cells through c-myc transfer into recipient cells. furthermore, mscpos-evs increased the ability of huvec to form tubular structures compared to mscneg-evs. in vivo co-injection of mscpos-evstreated huvec with a549 lung cancer cells resulted in an increase of tumour growth compared to mscneg-evs-treated huvec. mir-126 modulation in evs with mirna mimics or in recipient cells using mirna inhibitors demonstrated that this mirna is implicated in the autocrine proangiogenic modulation of huvec phenotype. mscpos-evs induced m2 polarization of macrophages both in vitro and in vivo. we demonstrated using synthetic oligonucleotides that mir-320 is responsible for the immunosuppressive modulation of these cells. regarding the potential origin of evs in mscpos individuals, we observed that hypoxia stimulated the secretion of evs containing c-myc from fibroblasts, mir-126-evs from endothelial cells and mir-320-evs from granulocytes. summary/conclusion: these data show that plasma evs of high-risk individuals display pro-tumorigenic features, as documented by their ability to induce a pro-angiogenic and immunosuppressive microenvironment suggesting an involvement of evs in lung cancer development. exploration of ev-associated metastatic targets on melanoma cells introduction: cancer cells secrete evs that may harbour metastatic proteins. previous studies have demonstrated the decrease of cd63 tetraspanin expression in melanoma cells are correlated with enhancing its metastatic potential. however, other proteins, such as cd44, cd47, met and nrp2 which are overexpressed in melanoma cells, are also associated with the spread of cancer. in this study, we sought to investigate the expression of metastatic proteins in evs derived from murine melanoma b16f10 lineage. methods: b16f10 cells were cultured in dmem supplemented with 10% fbs and antibiotics. the cells supernatant were harvested each 24 hours, filtered through 0.22 µm and ultracentrifuged at 110000 x g for 90 min at 4ºc to pellet evs. next, size and concentration was determined using nanoparticle tracking analyses technique, and the morphology of evs were analysed by negative-staining transmission electron microscopy (tem). the ev's surface protein were characterized by flow cytometry and protein content was profiled by mass spectrometry. results: our flow cytometry results have shown the presence of tetraspanins markers cd63, cd9 and cd81 on vesicle´s surface. in addition, our assay demonstrated a diminished expression of cd63 molecule in comparison to cd9 and cd81. we have profiled melanoma-evs by mass spectrometry, identifying the presence of proteins that may be associated to metastasis, such as cd44, cd47, met and nrp2. summary/conclusion: these preliminary results are consistent with the literature and suggest that melanoma-derived evs harbour proteins, which may contribute to promote tumour metastasis. in our next step, we plan to generate b16f10 lineages harbouring shrna vectors, in order to knockdown gene expression of cd63, cd44, cd47, met and nrp2 to investigate the metastatic potential in vitro, in comparison to parental cells. we also may use syngeneic mice models to explore metastatic potential of genetically modified b16 f10-derived cells. introduction: overexpression of her2 occurs in2 0% of breast cancers and confers aggressive behaviour and poorer prognosis. thankfully, a number of drugs such as neratinib have been developed to target her2, potentially providing substantial benefit for many patients. nevertheless, it is estimated that up to 70% of patients with her2-overexpressing tumours do not gain benefit, as a result of innate or acquired drug-resistance. this study aimed to investigate if nano-sized membrane-surrounded extracellular vesicles (evs) released from drug-sensitive and drug-resistant cells reflect the her2 status of their cells of origin and thus have potential as minimallyinvasive biomarkers. methods: evs were isolated from conditioned media (cm) of her2-positive cell lines (hcc1954 and skbr3) and their neratinib-resistant counterparts (hcc1954-nr and skbr3-nr) that we developed in our laboratory. evs from cm of a triple-negative breast cancer (tnbc) cell line variant, hs578 ts(i)8, were evaluated as negative control for her2. in brief, cm was centrifuged at 300 g, for 10 min x3 to get rid of any cells. the supernatant was then centrifuged at 200,000 g for 6 h at 4ºc to collect evs. the resulting evs were washed in pbs, centrifuged as before, and resuspended in 100 μl pbs. ev quantities were estimated by nanoparticle tracking analysis (nts). ev lysates were characterised by immunoblots, for established positive and negative ev markers. particle concentration as well as size distribution of evs were measured using the zetaview (particle metrix, germany). surface proteins on single evs were analysed by highresolution flow cytometry (fc), using an amnis imagestreamx mark ii. all data was submitted to ev-track (ev-track id: ev190112). results: neratinib-resistant cell line variants were found to have reduced her2 protein expression compared to their respective drug-sensitive counterparts. neratinib-resistant cell line variants released fewer evs, when normalised per number of secreting cells, compared to their-drug sensitive counterparts. furthermore, evs from drug-sensitive cells carried her2, while those from drug-resistant cells lacked her2 (similar to the evs from the tnbc cells); reflecting the her2 status of their cells of origin. summary/conclusion: this study indicates that a reduction in her2 protein expression is a mechanism by which cancer cells manifest resistance to her2-targeted drugs (i.e. by making fewer her2 receptors available on the cells surface to accommodate the drugs activity). furthermore, ev-carried her2 seems to reflect the her2 status of their cells of origin. this suggest that analysis of her2 on evs in the peripheral circulation may help predict response to her2-targeted drugs. thus, analysis of evs in appropriate cohorts of patients' specimens is warranted. introduction: rab27a, a small gtpase involved in exosome biogenesis by regulating mve docking at the plasma membrane, and its expression level highly correlated with ohsv replication ability in vitro. oncolytic viruses is a newly promising therapeutic agent for cancer treatment. however, more than 50% of tumours naturally showed highly resistant to oncolytic viruses for unknown reasons. uncovering the underlying mechanisms of resistance to ohsv can offer potential therapeutic targets to enhance ohsv activity to kill tumour cells. in addition, it will give new insights into the identification of therapeutic biomarkers, which can be used to predict patient response to ohsv in clinical. methods: deep-sequencing data showed that lower expression level of rab27a is present in ohsv resistant tumour cells compared to that in sensitive tumour cells. then an ohsv resistant mc38 tumour cell line was established by repeated injections with ohsv in mc38 tumour-bear mouse model. lastly, it was verified that ohsv resistance is associated with a downregulation of rab27a and overexpression of rab27a can rescue ohsv replication. results: 1) the lower expression level of rab27a is shown in ohsv resistant tumour cells compared to that in sensitive tumour cells shows in deep-sequencing data. 2) furthermore, we established an ohsv resistant mc38 tumour cell line by repeated injections with ohsv in mc38 tumour-bear mouse model. similarly, in ohsv resistant mc38 cell line, rab27a expression was lower than parental mc38 cells. and the release of exosomes and virus was decreased in ohsv resistant mc38 cell line. these results were confirmed by rab27a sirna knockdown. 3) we verified that in ohsv naturally resistant human cancer cell lines, ohsv resistance is associated with a downregulation of rab27a and overexpression of rab27a can rescue ohsv replication. summary/conclusion: downregulation of rab27a expression restricts the efficiency of ohsv replication and the spreading ability of the released progeny virus which also provide rab27a as a potential target and biomarker for ohsv cancer therapy. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd2015071414385495, shenzhen science and innovation commission project grants jcyj201 70411094933148, jcyj20180306173333907 to shenzhen international institute for biomedical research. inactivation of emilin-1 by proteolysis and secretion in extracellular vesicles favours melanoma progression and metastasis introduction: studies have demonstrated that melanoma-derived extracellular vesicles (evs) home in distal organs and sentinel lymph nodes favouring metastasis. although lymph node metastases are themselves rarely life threatening, they could be considered as one of the first step of metastasis in many cancer types. therefore, defining the mechanisms involved in lymph node metastasis and pre-metastatic niche formation in lymph nodes could bring the clue to block the metastatic process from the beginning. methods: we have characterized secreted exosomes from a panel of mouse melanoma models representative of low metastatic potential (b16-f1), high metastatic potential (b16-f10) and lymph node metastasis (b16-f1 r2). we analysed the rna expression in cells and protein content of exosomes derived from mouse melanoma lymph node metastatic models by rna sequencing and mass spectrometry respectively. we validated expression by western-blot, qpcr and immunofluorescence. to define the mechanism of emilin-1 secretion cells were treated with the ev secretion inhibitor (non-competitive inhibitor of sphingomyelinase), gw4869. cell viability and cell cycle assays with an overexpression model (b16-f1-emilin-1) were also performed. in vivo experiments based on subcutaneous and intrafootpad injection for studying the role of this protein during melanoma progression were performed to define the relevance of our findings in vivo. human paraffin samples were analysed for emilin-1 expression by immunohistochemistry. results: we found a signature of over-expressed genes and hyper-secreted proteins in exosomes related to lymph node metastasis in the b16 mouse melanoma model. among them, we found that emilin-1, a protein with an important function in lymph node physiology, was hyper-secreted in exosomes. interestingly, we found that emilin-1 is degraded and secreted in exosomes as a mechanism favouring metastasis. further, we found that emilin-1 has a tumour suppressive-like role regulating negatively cell migration. importantly, our in vivo studies demonstrate that emilin-1 overexpression reduced primary tumour growth and metastasis in mouse melanoma models. analysis in human melanoma samples showed that cells expressing high levels of emilin-1 are reduced in metastatic lesions. summary/conclusion: overall, our analysis suggests that the inactivation of emilin-1 by proteolysis and secretion in exosomes reduce its intrinsic tumour suppressive activities in melanoma favouring tumour progression and metastasis. funding: this work was supported by grants from mineco (saf2014 54541 r), asociación española contra el cáncer, fundación de investigación oncológica fero and mineco-severo ochoa predoctoral program. introduction: the amplification of erbb2 gene and the consequent overexpression of the encoded protein her2 have an important role in breast cancer classification at diagnosis and subsequent treatment decision with the anti-her2 monoclonal antibody trastuzumab. fish and ihc have been used so far to detect erbb2 gene amplification and her2 protein overexpression respectively in tissue biopsies. in this context, a major goal for liquid biopsies is to take advantage of the information carried by circulating tumourderived materials (such as extracellular vesicles (evs) and cell free dna (cfdna)) to noninvasively detect erbb2 gene status in the blood. however, the isolation of diverse tumour-derived materials from a single aliquot of patients' plasma and the accurate detection of cancer biomarkers is still challenging. methods: by adopting a recently published nickelbased evs isolation (nbi) protocol1 that allows for recovery of cfdna after evs isolation, we generated a high-sensitivity molecular assay to accurately detect erbb2 amplification and consequent her2 overexpression on a limited volume of plasma (1.5 ml) collected from breast cancer patients (stage i, ii and iii) at diagnosis. results: 1) we detected erbb2 amplifications by droplet digital pcr (ddpcr) on the cfdna isolated from the plasma of erbb2 positive patients; 2) we confirmed her2 overexpression on a subset of patients by setting up an antibody-based affinity reaction designed to detect her2 protein on the surface of the isolated evs; 3) we succeeded in the quantification of her2 transcripts enclosed within evs by performing ddpcr in samples of patients showing a range of circulating tumourderived material. the specificity and sensitivity of these novel methodological assays were tested on a cohort of healthy individuals (n = 20) and on a cohort of her2 positive (n = 20) or her2 negative breast cancer patients (tnbc; n = 20). summary/conclusion: here we report a pilot study on a novel multimodal method for erbb2 detection from a minimal amount of plasma. this approach integrates information from cfdna with evs-derived rna and proteins analysis. this proof of concept may ultimately translate into relevant clinical applications for disease diagnosis as well as for therapy selection and monitoring of disease progression. introduction: the biological and medical importance of exosomes recognized over the last decade has given rise to a crucial need for the discrimination between true evs and co-purified nanoparticles, such as lipoproteins, protein aggregates or debris. additionally, it is imperative to develop methods to identify and characterize ev sub-populations. considering ev biology and the reliability of labelled biomolecules, we developed both exogenous and endogenous labelling protocols, taking advantage of different dye properties which can target a multitude of compartments. this approach reveals key aspects of ev structure and integrity. methods: nanosized evs/exosomes were purified by size exclusion chromatography (sec) from model cell lines and human plasma. diverse dyes were orthogonally evaluated through different single particle and bulk analysis technologies, such as high-resolution cytometry, nanoparticle tracking analysis and plate fluorimeter. concomitant profiling of specific ev subpopulations was optimized using antibodies (abs) against tetraspanins and cell type specific targets and assessed by single particle analysis and elisa. to assess specificity of labelling protocols we used specific controls such as recombinant rfp expressing vesicles and purified lipoproteins. results: our ev staining protocols allowed for high labelling efficiency and unprecedent ev discrimination, quantification and characterization by combining single particle analysis and bulk measurements in simple matrices (saline buffers) and in complex biofluids (i.e. plasma). different approaches have diverse and complementary advantages (costs, capacity, sensitivity, informative readout) for implementation in research and diagnostic development flows, directly feeding in-house r&d and qc pipeline. summary/conclusion: overall, fluorescent evs are versatile and valuable tracers that can be applied in the optimization of pre-analytical and purification protocols, selection of target biomarkers and diagnostic assay calibration and validation. funding: endevor (por-fse 2014-2020) region tuscany and exosomics r&d program. subpopulations of tissue-derived extracellular vesiclesmethodological evaluation for vesicle size measurement introduction: introduction: subpopulations of extracellular vesicle (evs) are commonly classified by their different size, however, the ev size cut off is still under discussion. the aim of the study was to evaluate size range of six ev subpopulations using three methods: electron microscopy (em), nanoparticle tracking analysis (nta) and exoview™. methods: methods: ev subpopulations were isolated from melanoma tissues by a centrifugation based protocol recently established in our lab. large and small evs (levs and sevs) were isolated with differential ultracentrifugation and these were further separated into low and high-density fractions (ld and hd). size of ev subpopulations was then evaluated by: em introduction: small-extracellular vesicles have an important role in cell metabolism and cell-to-cell communication. moreover, sevs when secreted from cells are capable to act as mediators of various neurological diseases. however, sevs show heterogeneity and this may impact their functions. therefore, to characterize sevs at a single-particle level is important to better detail the associated biological activity. in this scenario, we innovatively propose the structured illumination microscopy (sim) as a technique able to complement the non-optical methods (transmission electron microscopy, tem) to analyse single sevs and their markers. methods: human plasma sevs were separated from healthy cognitive control (ctrl), mild cognitive impairment (mci) and demented subjects. the sevscontaining pellet was resuspended in 4% paraformaldehyde or 2% glutaraldehyde solutions. for sim, sevsenriched preparations were washed with the blocking solution and incubated with the primary antibody (l1cam). the secondary fluolabelled antibody was then added. between the steps with the blocking solution, the primary and secondary antibodies, two ultracentrifuge steps were performed. the image acquisition was done on a nikon sim system with a 100x oil immersion objective. sevs were imaged with a 3d-sim acquisition protocol. tem was performed on 300 mesh formvar copper-coated grids. sevs-enriched preparations were incubated first with the blocking solution and then, immunogold-labelled for cd63. samples were counterstained with uranyl acetate and observed under a jeol 1010 ex electron microscope. data were recorded with a morada digital camera system. participation to the present study was approved by relevant local ethics committee of mendrisio and lugano hospital and written informed consents were obtained from subjects. results: for sim methodology, only vesicles in the range from 180 to 190 nm were detected, as the final resolution achieved was 160 nm. instead, for tem, sevs under 100 nm were identified. none of these methods provided relevant information about possible difference in morphology of the ctrl-, mci or demented subjects-derived sevs. summary/conclusion: even if both methods identified cd63 or l1cam-positive vesicles, sim resolution and the complexity of the protocol represent some disadvantages respect to tem, that may be the first choice screening technique for evs analysis to be then completed by sim for particular tasks. fabrication of nanopore structures via conformal metal-film deposition for ev sensing kwanjung kim a , seung-min han b , soo-hyun kim c and sung-wook nam d luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. these evsreleased under normal physiological conditions as well as in the pathogenesis of neurological, vascular, haematological, and autoimmune diseaseshave been shown to transfer biological molecules such as protein and rna between cells, potentially transmitting signals. to understand more about these signalling mechanisms, there is a need for detecting and quantifying evs with cargo protein and rna in a reproducible and reliable manner. however, this has been challenging due to the small size of evs (ranging from 30 to 100 nm in diameter), and the lack of specific staining reagents. methods: here, we utilize the amnis® cellstream® flow cytometer, which enables high-throughput flow cytometry with increased sensitivity for detecting small particles. we demonstrate that a charge-coupled device (ccd)-based, time-delay-integration image capturing system can be used to detect and quantify evs and their cargo labelled with exoglow™-protein or exoglow™-rna. results: in this study, we show flow cytometry data quantifying ev samples that have been labelled with cargo markers for proteins and rna. the ev cargo contents along with the appropriate control samples will be shown. summary/conclusion: the ccd based detection of the cellstream flow cytometer has the sensitivity to quantify evs and their cargo. single ev imaging reveals novel ev biomarkers and dna cargo siobhan m. king a , ricardo bastos a and andras miklosi b a oni, oxford, uk; b oni (oxford nanoimaging ltd), oxford, uk introduction: extracellular vesicles (evs) are cellderived membrane-bound particles that range in size from 30-1000 nm and carry active molecules such as dna, rna and proteins. upon secretion, evs can execute many biological functions such as initiating intracellular communication or regulating immune responses. depending on their origin evs have different characteristics and cargo, making them attractive candidates for early diagnostic and therapeutic applications. however due to their small size and heterogeneity, direct visualization and characterization of the surface markers expressed remains a challenge since these vesicles are below the resolution limit of standard light microscopy. methods: here, we describe a method that provides size analysis of single-evs, which falls below the diffraction limit of light. this was done with purified evs, immunostained using fluorescently labelled primary antibodies raised against ev surface markers (cd63, cd81, cd9), specific cargo such as dna and probed for tissue specific cargo. characterization of the molecular content and structural properties of surfaceimmobilized evs was performed using single-molecule localisation microscopy (smlm) on the nanoimager platform. results: multicolour smlm was used to detect up to three ev biomarkers showing successful characterization of the molecular signature for different ev subpopulations. the distribution of novel components on urinary evs were visualized for the first time using this approach. in addition, smlm revealed the presence of dna on both the surface and also as a cargo inside evs isolated from tumour cell culture media, which was validated using complementary biochemical characterization. summary/conclusion: smlm is a powerful technique for single-ev analysis and characterization. visualization of single-urinary evs enabled accurate sizing and further insights into novel components expressed on the subpopulation's membrane surface. together, the data demonstrates that the quantitative abilities of smlm can significantly enhance our understanding of evs, as structure, phenotypes, and cargoes can now be successfully resolved. introduction: working skeletal muscle is a common site for injury due to unaccustomed exercise with or without underlying pathology. direct analysis of skm injury requires invasive tissue biopsies. circulating extracellular vesicles (evs) are abundant in blood and have been shown to be enriched in microrna; profiles of which may reflect the state of tissues. evs may therefore serve as a non-invasive indicator of muscle injury and regenerative processes in vivo. methods: two consecutive bouts of muscle-damaging exercise (plyometric jumping and downhill running) were performed by 9 healthy male volunteers. serum creatine kinase (ck) and plasma evs were analysed at baseline, 2 and 24 hr post-exercise. perceived muscle pain (pmp) was assessed at 2 and 24 hr post-exercise. large evs were isolated using a 10 000 g centrifugation step, and small evs were isolated using qev columns. ev-enriched isolates were visualized using tem, and size and numbers were quantified using nta. based on nta results the highest particle fractions (7-10) were pooled for rna analysis. qpcr was done on plasma, large evs and small evs. a group of muscle and immune cell-important mirs were analysed by means of normalization to an exogenous control. results: ck and pmp increased post-exercise, providing evidence for muscle damage. tem revealed an abundant and heterogeneous pool of evs. a concomitant abundance of evs was seen with nta (mean = 1.17 x 1010 particles/ml plasma). mean ev diameters were 194 ± 22 nm across all timepoints. no change in ev size nor number was seen over time, however, mir-31 decreased at 24 hr when compared to 2 hr in the small ev isolate only. plasma displayed an immediate increase in myomirs-1 and −206 at 2 hr, which returned to baseline at 24 hr. in contrast, myomirs-208b and 486 remained elevated over the 24 hr period. myomir-133b and 486, as well as immune-mirs, did not change in evs or plasma as a result of the intervention. summary/conclusion: the decrease in mir-31 in small evs at 24 hr is consistent with previous data. no decrease in mir-31 in large evs suggests specific packaging and hence a specific response to the muscle damage in small evs. more changes occurred in plasma myomirs suggesting less specific passive leakage into circulation from damaged cell membranes. funding: south african national research foundation pulsed electromagnetic fields potentiate the paracrine function of mesenchymal stem cells for cartilage regeneration yingnan wu a , dinesh parate a , eng hin lee a , zheng yang a and alfredo franco-obregón b a national university of singpaore tissue engineering program, yll school of medicine., national university of singapore, singapore, singapore; b biolonic currents electromagnetic pulsing systems laboratory, biceps, national 11 university of singapore, singapore, singapore introduction: the mesenchymal stem cell (msc) secretome, via the combined actions of its plethora of biologically active factors, is capable of orchestrating the regenerative responses of numerous tissues by both eliciting and amplifying biological responses within recipient cells. mscs are "environmentally-responsive" to local microenvironmental cues and biophysical perturbations, influencing their differentiation as well as secretion of bioactive factors. we have previously shown that exposures of mscs to pulsed electromagnetic fields (pemfs) enhanced msc chondrogenesis. here, we investigate the influence of pemf exposure over the paracrine activity of mscs and its significance to cartilage regeneration. also, the subsequent extracellular vesicles analysis and isolation are processed for the understanding of how the pemfs affect stem cell evs and consequent differentiation induction. methods: conditioned medium (cm) was generated from mscs subjected to either 3d or 2d culturing platforms, with or without pemf exposure. the paracrine effects of cm over chondrocytes and msc chondrogenesis, migration and proliferation, as well as the inflammatory status and induced apoptosis in chondrocytes and mscs was assessed. the cms which have significant effects during chondrogenesis will be analysed by protein and mirna studies. results: we show that the benefits of magnetic field stimulation over msc-derived chondrogenesis can be partly ascribed to its ability to modulate the msc secretome. mscs cultured on either 2d or 3d platforms displayed distinct magnetic sensitivities, whereby mscs grown in 2d or 3d platforms responded most favourably to pemf exposure at 2 mt and 3 mt amplitudes, respectively. ten minutes of pemf exposure was sufficient to substantially augment the chondrogenic potential of msc-derived cm generated from either platform. furthermore, pemf-induced cm was capable of enhancing the migration of chondrocytes and mscs as well as mitigating cellular inflammation and apoptosis. the cms protein results in the significant promotion chondrogenesis condition showed an increase in proliferation and anti-inflammatory cytokines. summary/conclusion: the findings reported here demonstrate that pemf-stimulation is capable of modulating the paracrine function of mscs for the enhancement and re-establishment of cartilage regeneration in states of cellular stress. the pemf-induced modulation of the msc-derived paracrine function for directed biological responses 1 in recipient cells or tissues has broad clinical and practical ramifications with high translational value across numerous clinical application. effects of extracellular vesicles from blood derivatives on osteoarthritic chondrocytes within an inflammation model introduction: the degenerative disease osteoarthritis (oa) is one of the leading causes of disability especially of elderly people. besides various treatment options depending on the severity of the cartilage degradation, the application of blood derived products such as platelet rich plasma (prp) are getting more and more popular in clinical practice due to its high concentration of platelets and the perceived high growth factor levels. drawbacks of using prp include high donor variability, discrepancies among preparation protocols and the presence of cells (platelets, leukocytes) which can evoke cellular processes, especially inflammation, when injected into the diseased tissue. one possibility is to isolate only extracellular vesicles (evs) from blood derivatives to overcome these problems. in the current study the effects of evs isolated from blood derivatives on oa chondrocytes within an inflammation model was investigated. methods: cd14 positive primary monocytes were isolated from citrate anticoagulated whole blood by magnetic bead sorting. monocytes were differentiated into resting m0 macrophages and activated into m1 macrophages according to published protocols. elisa measurements verified successful differentiation and activation as il1β and tnfα levels increased. as control, thp1 monocytes were used. patient-derived oa chondrocytes were grown in 6 well plates and co-cultivated with activated m1 macrophages which were seeded into thincerts and added to the 6 wells representing the inflammation model. furthermore, cells were treated for 48 hours with media containing fcs, ev depleted fcs or evs isolated from prp or hypact serum. results: successful differentiation and activation of monocytes (thp1 and primary monocytes) into m1 macrophages was demonstrated by elevated levels of the inflammatory cytokines il1β and tnfα. within the inflammation model (co-culture of oa chondrocytes with m1 macrophages), addition of evs isolated from prp or hypact serum resulted in decreased secretion levels of il1β and tnfα compared to media supplemented with either fcs or ev depleted fcs. summary/conclusion: taken together, evs from blood derived products might be chondroprotective and anti-inflammatory mediators which protect cartilage from being degraded during oa. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst3-f-5030664/003-2017. 1α,25(oh)2d3 regulates growth cartilage matrix vesicle micrornas niels asmussen, michael mcclure, zhao lin, zvi schwartz and barbara boyan virginia commonwealth university, richmond, usa introduction: matrix vesicles (mvs) are small (50-150 nm in diameter) lipid bound extracellular organelles isolated from calcifying tissues including the growth zone (gc) of growth plate cartilage. 1α,25 (oh)2 vitamin d3 (1α,25) is a regulator of gc chondrocytes and the mvs they produce. these mvs are key players in the mineralization process and are selectively enriched with enzymes and growth factors. we found that mvs are also selectively enriched with micrornas (mir), including mir-22, mir-122 and mir-451. the aim of this study was to determine the regulatory role of 1α,25 in the packaging of mirna in mvs by gc cells. methods: gc cells were isolated by enzymatic digestion from costochondral gc cartilage harvested from 5 wk-old male sprague dawley rats (iacuc approved). confluent fourth passage gc cell cultures were treated with 10-8 m 1α,25 or vehicle for 24 h. media were removed, cell monolayers digested with trypsin and cells and mvs isolated by differential ultracentrifugation. rna was precipitated from cells and mvs. small rnaseq data were trimmed, aligned and counted before undergoing differential expression analysis. experimental groups had an n = 3 per variable. significant differences (p < 0.05) were determined using r v 3.6.2. results: 1α,25 treatment altered expression of 30 mv mirs compared to control mvs, whereas 5 cell mirnas were differentially expressed. 62.1% of significantly up or down regulated mir found in mvs overlapped between 1α,25 and vehicle groups with the remaining being uniquely differentially expressed. 1α,25 increased mv mir-150 and decreased mir-384-3p two mirs known to regulate osteoblast proliferation (150 increases, 384 decreases). summary/conclusion: 1α,25 regulates gc chondrocyte and mv behaviour and this study demonstrates that it also impacts the mir packaging within mvs. mir discovered in mvs have been demonstrated to impact chondrocyte behaviour and the present study indicates that 1α,25 regulates the growth plate through mir delivered by mvs. introduction: increasing evidence has proposed extracellular vesicles (evs) as mediators of many of the therapeutic features of mesenchymal stromal cells (msc) that have been widely studied in clinical trials over the last years. these evs have been recognized as nanocarriers of important biological information, which play a central role in cell-to-cell communication. in this context, evs can be used as an alternative to a cell-based therapy, with reduced risks. the present work aimed to evaluate the impact of different culture conditions on the msc-derived evs molecular composition through fourier-transform infrared (ftir) spectroscopy. methods: evs derived from msc from different sources, expanded in two different culture media ((xenogeneic -free (xf) vs serum-containing medium (fbs)) were characterized by ftir spectroscopy, a highly sensitive, fast and high throughput technique. moreover, principal component analysis (pca) of preprocessed ftir spectra of purified evs was conducted, enabling the evaluation of the replica variance of the evs chemical fingerprint in a reduced dimensionality space. for that, different pre-processing methods were studied as baseline correction, standard normal variation and first and second derivative. results: evs secreted by mscs cultured with serumcontaining medium presented a more homogenous chemical fingerprint than evs obtained with xf medium. the regression vector of the pca enabled to identified relevant spectral bands that enabled the separation of samples in the score-plot of the previous analysis. ratios between these spectral bands were determined, since these attenuate artefacts due to cell quantity and baseline distortions underneath each band. statistically inference analysis of the ratios of spectral bands were conducted, by comparing the equality of the means of the populations using appropriate hypothesis tests and considering the significance level of 5%. it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture medium used for msc expansion and the msc donor. summary/conclusion: this work is a step forward into understanding how different culture conditions affect msc-derived evs characteristics. funding: fundação para a ciência e tecnologia (ptdc/equ-equ/31651/2017, uidb/04565/2020). performance qualification for microflow cytometers: understanding technical limitations to improve your research desmond pink a , michael wong a , diana pham a , renjith pillai a , leanne stifanyk b , sylvia koch b , rebecca hiebert a , oliver kenyon c and john lewis a a nanostics, edmonton, canada; b dynalife, edmonton, canada; c apogeeflow systems, hemel hempstead, uk introduction: as microflow cytometry and other techniques mature as validated modalities for analysing extracellular vesicles (ev), there has been a concerted effort to improve reproducibility . in order for this reproducibility to occur there has to be a critical understanding of advantages and limitations for each technology. for microflow cytometry, several instruments are available to analyse evs. each platform has different limitations as well as advantages over other platforms. to provide the optimal data for your specific research, it is critical to understand the limitations of your platform. to accurately define these limitations, a performance qualification (pq) of your instrument should be undertaken. methods: an apogee a60 platform was used in these experiments. experiments were designed with expected ranges and cut-offs for acceptance criteria.initial tests included autosampling of a 96 well plate with either single or double aspiration, single sample reproducibility and linearity proportional to flow rate. other experiments designed to show machine performance included minimal time to achieve valid data, sample volume required for double aspiration, determination of coincidence; detection sensitivity using a spiked sample; flow rate stability for extended periods (5-30 minutes) . tests should also be performed to determine carryover at a range of sample concentrations. if present, the means to remove contaminating samples should be determined. any performance tests should be applicable to any instrument in the field. results: auto-sampling helped demonstrate consistent data; reproducibility of total events and biomarkers was 2-8% c.v. detected bead concentrations were linear with flow rates between 0.75 and 10.5 ul/min. double well aspirations provided similar data with aspirations between 60-80ul. valid data was achieved for a low abundant target (~200-400 events/ul) after only 30 s, <10%c.v. detection sensitivity was determined to be~1/400,000. carryover ranges were determined in the presence of nominal unstained serum. an optimal number of machine washes was determined. some membrane stains, such as cell mask and cfse require much more rigorous cleaning to remove stain carryover. summary/conclusion: to improve data reproducibility, performance qualification of any instrument is key. operational limitations help define optimal performance parameters of any technology. understanding the types of experiments to perform for your particular type of characterization technology depends on the requirements you set for your research. a good performance test should be applicable to any related instrument in the field. funding: funding provided by nanostics, the alberta cancer foundation, and alberta innovates. introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par20-053, is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par16-267/ 277, successfully funded 7 r01 and 4 r21 grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, 2018); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, 2019). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, 2019). summary/conclusion: drs. sudhir srivastava and matthew young are the programme directors for the par which began accepting applications on 5 january 2020. this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute. introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of 7 protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores (600 nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the 7 protein biomarkers (tau, uchl-1, nfl, gfap, il6, il10, and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl1 were elevated in mtbi patients compared to controls (p < 0.05), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il6 and tnf-with tau from glur2+ evs showed 88% accuracy with 80% sensitivity and 100% specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. ps15.05 = op3.05 l1cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma maia norman, dmitry ter-ovanesyan, wendy trieu and david walt wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l1cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l1cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l1cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l1cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd9, cd63 and cd81) as well as l1cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l1cam appears. we also immunocapture l1cam from csf and plasma and perform western blots for the internal and external domains of l1cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l1cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l1cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l1cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l1cam in plasma is likely not coming from the brain. this data call into question the utility of l1cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant 3d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed 3d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in 6-month-old wt mice, (n = 6/groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for 15% of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and nonamplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy5y cells with n-myc amplified sk-n-be2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. ps15.08 = op3.08 results: murine ctl evs were broadly divided into two populations that were eluted at low salt (l-s: 0.15 m-0.3 m nacl) and high salt (h-s: 0.3 m-0.5 m nacl) concentrations. l-s ctl evs were abundant in late endosome-related proteins, integrins, rabs, and effective mirnas, indicating exosome characteristics, and had biological activity for preventing tumour metastasis after depletion of tumoural mesenchymal cell populations by intratumoral administration (see seo et al., nat. commun. 9: 435, 2018) . contrary, h-s ctl evs were rich in dna, core histones, ribosomal proteins, cytoskeleton proteins, and housekeeping proteins, considering microvesicles and apoptotic bodies, and easily phagocytosed by a kupffer cell line (kup5: kitani et al., results immunol. 4: 68-74. 2014 ). in addition, there were noticeable differences between ls and h-s ctl evs in the negative zeta potential width and membrane glycan structure. summary/conclusion: thus, ion exchange can be an optimal mass fractionation method for discriminating bioactive exosomes from cargos for nucleic acids in evs. funding: cryotem was conducted in nara institute of science and technology (naist), supported by nanotechnology platform program (synthesis of molecules and materials: 2019 #04) of the ministry of education, culture, sports, science and technology (mext). this work was supported by grants from the japan agency for medical research and development (translational research network program (nagoya univ. seeds a64)) and the japan science and technology agency (crest [jpmjcr17h2]). clic4 is essential for breast cancer metastatic competence and predicts disease outcome introduction: metastatic breast cancer is a consequence of complex interactions between cancer cells and the host. clic4, a member of a conserved gene family in the glutathione-s-transferase superfamily, mediates crosstalk between tumour and host in breast cancer. tcga and metabric data indicated that elevated clic4 expression was associated with breast cancers from young women, those with poor prognosis, and those with early stage metastatic disease. methods: since bulk tumour analysis does not distinguish between cancer and host stromal cells, we used genetic modifications of established syngeneic breast cancer mouse models to evaluate the contributions of clic4 in the host or tumour cells to develop metastases. results: experimentally, the essential clic4 host contributions for metastatic competence were related to circulating levels of pro-metastatic soluble factors, neoangiogenesis, tumour cell attachment to lung tissue, myofibroblast differentiation, and leukocyte migration. clic4 was detected as cargo in circulating extracellular vesicles (evs) from breast cancer patients. similarly, circulating evs from tumour-bearing mice have abundant clic4 in comparison to those from mice bearing tumours that lack clic4. tumour cells released evs that induced myofibroblast conversion of wildtype but not clic4 ablated lung fibroblasts. summary/conclusion: these results illuminate clic4 expression as a prognostic marker for breast cancer patients, and experimentally, clic4 is a critical host factor for metastatic competence and potential target within host tissues for anti-metastatic therapy. funding: this work was supported by the intramural program of the national cancer institute under project zia bc 005445. the application of flow cytometry in an ev-based liquid biopsy for the detection of cancer multidrug resistance in myeloma gabriele de rubis a , krishna sunkara a , sabna rajeev krishnan and mary bebawy b a laboratory of cancer cell biology and therapeutics, discipline of pharmacy, graduate school of health, the university of technology sydney, australia, sydney, australia; b the university of technology sydney, sydney, australia introduction: multiple myeloma (mm) is an incurable cancer of bone-marrow plasma cells. it is characterized by unpredictable and highly variable therapeutic response and poor survival, attributed to the development of multidrug resistance (mdr) to chemotherapy. presently, no clinical procedures allow for a continuous, minimally invasive monitoring of mdr. we identified unique extracellular vesicle (ev) populations in the blood of myeloma patients, which serve as biomarkers of disease evolution and mdr to combination chemotherapy. we describe approaches used to optimise the use of flow cytometry (fcm) for ev summary/conclusion: although further investigation is required, our results potentially promise an effective and inexpensive priming agent (i.e., ethanol) for the production of anti-inflammatory msc-evs. this, combined with the significant increase in yield via 3d dynamic culture, presents practical solutions to both ev manufacturing scalability and potency issues. donor source affects potency of mesenchymal stem cell-derived extracellular vesicles introduction: mesenchymal stem cell (msc) therapies have been heavily investigated for their utility in applications such as wound healing and regenerative medicine due to their angiogenic, immunomodulatory and anti-apoptotic effects. recently, msc-derived extracellular vesicles (evs) have been implicated as primary effectors in msc-based therapies via protein and nucleic acid cargo transfer to patient cells. msc evs represent a superior alternative to msc-based therapies, as they lack the ability to replicate and are much smaller in size, circumventing related safety concerns such as immunogenicity, teratoma formation and blood vessel occlusion. however, a key drawback with msc therapies in general is their variable therapeutic potency, which is dependent on donor source. as a cell derived therapeutic, this crucial limitation is hypothesized to exist in msc evs as well. here, we demonstrate the varying bioactivities of isolated msc evs from differing donors and tissue sources. methods: six separate msc lines were obtained from different donors, with three msc lines derived from donor adipose tissue, and the other three from the bone marrow of separate individuals. evs were isolated from each msc line at passage 3 via differential centrifugation and ultrafiltration. these isolated msc evs were then characterized for size/concentration via nanoparticle tracking analysis, and ev markers (tsg101, alix, cd63) via western blot. pro-vascularization capacities of msc evs were determined by a gap closure assay using human umbilical cord vein endothelial cells (huvecs). results: characterization of msc evs revealed similar sizes and ev marker expression across donor groups, frontiers in chemistry, submitted funding: this work was funded by the momentum programme (lp2016-2), by the national competitiveness and excellence program catalan institution for research and advanced studies (icrea) proteomic profiling of retinoblastoma-derived exosomes reveals potential biomarkers of vitreous seeding angel montero carcaboso g , andrea petretto b , franco locatelli a and angela di giannatale a a department of paediatric haematology/oncology and cell and gene therapy, irccs, ospedale pediatrico bambino gesù ps08: separation and concentration a laboratory of clinical biophysics, faculty of health sciences ps10: diverse ev biomarkers chair: pia siljander -faculty of biological and environmental sciences urinary evs were isolated using low vacuum filtration method followed by ultracentrifugation. raman spectra of urinary evs were recorded using a renishaw invia raman spectrometer. data analysis was performed using principal component analysis (pca) and hierarchical cluster analysis (hca). the size distribution and morphology of evs were analysed by transmission electron microscopy and nanoparticle tracking analysis methods. results: average raman spectra obtained for urinary evs from studied groups showed differences in intensities of specific bands in the region of 400-1800 cm-1. we found significant correlations between mean area under curve (auc) calculated for raman bands (phenylalanine, dna, proteins, lipids and amide i) and selected clinical parameters such as: egfr, serum creatinine, glucose, urine creatinine. chemometric methods showed spectral pattern responsible for separation between studied groups. nta measurements visualized evs with size of 204.7 ± 96.5 nm. summary/conclusion: our results showed that characteristic raman spectra of urinary evs are promising candidates for new, non-invasive biomarkers for dkd isolation of circulating extracellular vesicles and cfdna allows for erbb2 detection in a single aliquot of breast cancer patients plasma michela notarangelo a , mattia barbareschi b , antonella ferro c , orazio caffo c , vito d'agostino a and francesca demichelis d a department of cellular results: results: tissue-derived large and small evs showed difference in size (mean 142 nm vs 75 nm) when examined by em, whereas nta and exoview™ were unable to show a clear difference between the populations (nta: mean 125.7 nm vs 122 nm nta can only detected vesicles above 70 nm and exoview™ only measures vesicles between 50-200 nm. of the three different methods, em analysis of single vesicles visualized in a significant number of micrographs was the only one able to distinguish ev subpopulations by size. funding: funding: swedish research council knut och alice wallenberg foundation imaging of human plasma-derived small-extracellular vesicles using transmission electron microscopy and structured illumination microscopy mitovesicles: a new extracellular vesicle of mitochondrial origin altered in ageing and neurodegeneration alldred b , chris goulbourne b , hediye erdjument-bromage d , monika pawlik b , mitsuo saito e , mariko saito f , stephen d. ginsberg b an in vitro and in vivo perspective on the role of erythrocyte-derived extracellular vesicles in parkinson's disease pathology frédéric calon c , éric boilard f and francesca cicchetti b a centre de recherche du chu de québec and faculté de médecine, département de psychiatrie & neurosciences département de microbiologie-infectiologie et d'immunologie evidences on microalgal extracellular vesicles: a morphological assessment antonella bongiovanni i , ales iglič j and veronika kralj-iglič j a laboratory of clinical biophysics, faculty of health sciences a faculty of dentistry, national university of singapore, singapore, singapore, singapore; b institute of medical biology, agency for science, technology and research, singapore, singapore, singapore; c exosome of cancer-associated fibroblast induce anti-cancer drug-resistance of nsclc so-young kim a and yeon-ju lee b a chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea; b chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea introduction: the understanding of interaction mechanisms between cancer cells and the tumour microenvironment (tme) is crucial for developing therapies that can arrest tumour progression and metastasis. cafs are the major constituent of the tme in many cancers. recent studies indicate that exosomes harbour the potential to regulate proliferation, survival and immune status in recipient cells. most of the current studies are focused on cancer cell secreted exosomes; and little is known about cafderived exosomes and their influence on cancer cells. methods: nsclc cell lines (pc9gr) and mrc5 (normal fibroblast cells) were grown in culture with exosome-free fbs. cutured media was filtrated by tangential flow filtration systems. exosomes in supernatant were isolated with the exoquick-tc™ system. considering the important role of cell extrinsic factors on cell growth and survival, we assessed whether factors contained in the mpa exosome could affect proliferation and survival of recipient cancer cells. cells were then treated with 1 μm osimertinib or pbs for 3 days prior to cell quantification of live cells.to investigate mechanisms of resistance to osimertinib mediated by ma or mpa-exosome in nsclc cell lines, we test cell viability by crystal violet assay in trametinib or osimertinib treated after pretreated ma or mpa-exosome, pc9gr during 6 days. we will investigate how mpa-exsomes activate erk signalling pathway in pc9gr cells to induce antitumor effects by western blot. results: mpa exosome increased proliferation of pc9gr cells by more than 50% compared to control pbs. pc9gr cells grown in mpa-exosome and subsequently treated with osimertinib showed a significant increase in cell survival compared to pc9gr cells grown in ma-exosome. osimertinib is used to treat egfr-mutant non-small cell lung cancer (nsclc) with tyrosine kinase inhibitor resistance mediated by the egfr t790 m mutation.these data show that "mrc5-pc9gr-crosstalk factors" affect proliferation and adaptive drug resistance of cancer cells. mpa-exosome mediates erk signalling activation and attenuated after treatment of 1um osimertinib. summary/conclusion: cafs support cancer growth and invasion. co-cultured nsclc with mrc5 lung fibroblast increased cell viability and exosomal mir-21 through the tgf-ß pathway in treatment osimertinib. exosomal mir-21 up-regulation in cocultured nsclc with mrc-5 induced drug resistance to drug-induced apoptosis. thus, exosomal mir-21 expression in co-cultured nsclc with mrc5 may support drug tolerance persister cells. introduction: neural stem cell (nsc) therapy has shown promise for brain repair after injury or disease mostly through bystander effects. nevertheless, the translation of nscs derived from human induced pluripotent stem cells (hipscs) to the clinic remain constrained due to safety issues, which include immunogenic risks, tumorigenesis potential, and incomplete differentiation. a way to avoid these issues is by using extracellular vesicles (evs) generated from nscs, as nsc-evs likely have similar neuroprotective properties as nscs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. however, this would require reliable purification and characterization processes, and testing of evs for composition and biological properties. methods: we generated evs from hipsc-derived nscs using a combination of ion-exchange chromatography (iex) and size-exclusion chromatography (sec) and investigated their composition through small rna sequencing and proteomics. we also performed in vitro and in vivo experiments to determine their biological and functional properties. results: iex and sec facilitated purification of hipsc-nsc evs nearly to homogeneity, which expressed ev markers such as cd9, cd63, cd81, and alix with a mean size of 145 nm. small rna sequencing revealed enrichment of mirnas related to different neuroprotective signalling pathways and diverse metabolic functions consistent with their role in cell-cell communication. the proteomic analysis identified >1,000 proteins, including ev markers and many other proteins involved in central nervous system function and cellular processes. the evs also displayed antiinflammatory activity in an in vitro mouse macrophage assay. intranasal (in) administration of nsc-evs resulted in their rapid incorporation by neurons, microglia, and astrocytes in virtually all regions of the brain. functionally, in administration of nsc-evs reduced inflammatory activity in the brain in a model of status epilepticus, and increased hippocampal neurogenesis in the adult brain. summary/conclusion: biologically active evs with antiinflammatory and neurogenic properties could be purified and harvested from hipsc-nscs. such evs also contain many mirnas and proteins that are of interest for brain repair after injury or disease. funding: supported by a grant from the national institute of neurological disorders and stroke (1r01ns106907-01 to a.k.s.) introduction: extracellular vesicles (evs) generated from human bone marrow-derived mesenchymal stem cells (hmscs) display anti-inflammatory and neuroprotective properties. our recent study has shown that intranasally (in) administered hmsc-evs incorporate into significant percentages of neurons and microglia in virtually all regions of the intact as well as the injured forebrain within 6 hours (kodali et al., int j mol sci, 2019) . in this study, using a rat model, we investigated the efficacy of a low dose of hmsc-evs administered intranasally for alleviating the abnormal plasticity of newly born neurons and the activation of microglia after se. methods: approximately 10 billion evs were dispensed bilaterally into both nostrils of young f344 rats that experienced two hours of kainate-induced se. animals were euthanized seven days after se, and brain tissue sections were processed for immunohistochemical staining of neun (a neuronal marker), dcx (a marker of newly born neurons), iba-1 (a microglial marker), and parvalbumin (pv) and reelin (markers of subclasses of interneurons). in addition, activated microglia were quantified using iba-1 and cd68 dual immunofluorescence. results: in administration of evs reduced the seinduced loss of pyramidal neurons in the hippocampal ca1 subfield. also, ev administration after se maintained higher levels of pv+ interneurons in the dentate gyrus. furthermore, ev treatment after se modulated abnormal neurogenesis, which was evidenced by a the role of small extracellular vesicles in chronic neuropathic pain zhucheng lin a , renee jean-toussaint b , yuzhen tian b , ahmet sacan a and seena ajit b a drexel university, philadelphia, usa; b drexel university college of medicine, philadelphia, usa introduction: chronic pain is the most prevalent, disabling, and expensive public health condition in the usa. exosomes are 30-150 nm extracellular vesicles that can transport rnas, proteins, and lipid mediators to recipient cells via circulation. exosomes can be beneficial or harmful depending on their source and contents. we hypothesized that the composition of small extracellular vesicles (sevs) can be altered following nerve injury and these alterations can provide insight into how the body responds to neuropathic pain. methods: to characterize changes following nerve injury, small extracellular vesicles (sevs) were purified by ultracentrifugation from mouse serum four weeks after spared nerve injury (sni) or sham surgery. mirna profiling and proteomics analysis using tandem mass spectrometry were performed to determine differential expression of mirnas and protein cargo respectively. for in vivo studies, sevs were administrated intrathecally into the mouse lumbar region. animals were evaluated for mechanical and thermal hypersensitivity over 21 days after injection. results: our mirna profiling showed a distinct mirna signature in sni model compared to sham control. proteomics analysis detected 274 gene products. of these, 24 were unique to sni model. neuropathic pain can induce the activation of the complement cascade and we observed significant upregulation of complement component 5a (c5a) in sevs from sni model. intercellular adhesion molecule 1 (icam-1), required for the leukocyte recruitment, adhesion and homing of exosomes was also upregulated in sevs from sni model compared to sham control. administration of sevs from sni model increased paw withdraw threshold in naïve recipient mice and inflammatory pain model, indicating a protective role for sevs in attenuating chronic pain. summary/conclusion: our preliminary studies suggest a critical role for sevs cargo in regulating pain. additional studies are ongoing to determine the functional significance of alterations in sevs composition using mouse models of pain. introduction: amyotrophic lateral sclerosis (als) is a progressive adult-onset neurodegenerative disease caused by selective motor neurons (mns) death. the rapid disease progression strongly suggests that cell-tocell spreading of noxious factors could take place in als pathogenesis. extracellular vesicles could potentially spread the disease. in this study, we characterized large (levs) and small extracellular vesicles (sevs) isolated from plasma of sporadic als patients and healthy controls and determined their different composition in order to understand their neuroprotective or neurotoxic role in als pathogenesis. methods: levs and sevs were isolated from plasma of 40 als patients and 30 healthy volunteers by differential centrifugation and characterized by nanosight ns300. cd45, cd31, cd61, cd235a and annexin v were used for flow cytometry. sod1, tdp43, fus protein level was investigated by western blot. for raman spectroscopy, evs were dried on top of a caf2 slide and raman spectra were acquired using a 633 nm laser line. mirna libraries were prepared by truseq small rna library kit (illumina). results: the mean size both for levs and for sevs resulted increased in als patients compared to controls. levs derived from als patients were enriched in sod-1, tdp-43 and fus proteins compared to ctrls. sevs showed a distinct spectral pattern from levs. in addition, levs of als patients were richer in lipids and had less intense bands relative to aromatic aminoacids compared to healthy controls. we also found a great presence of leukocyte derived levs (lmvs) in als patients compared to ad patients and healthy donors and significant correlation with the progression rate of the disease. on the other hand, mirna and rna whole transcriptome sequencing identified a specific signature of mirnas in plasma derived sevs of als patients compared to a group of healthy controls and three neurological groups of control. summary/conclusion: these data may suggest that levs derived from als patients, enriched in lipids and toxic proteins, might play a role in prion-like propagation and immunity of als disease, while sevs, deriving ps05.07 introduction: dendritic spines are actin-rich structures at the postsynaptic sites of most excitatory synapses in the central nervous system. they are highly important structures for higher brain functions such as learning and memory. several live imaging studies have shown that long, thin, actinrich protrusions called dendritic filopodia are precursors of dendritic spines in hippocampal and cortical neurons. so far, many intracellular factors that regulate filopodia formation have been identified. however, extracellular mechanisms of filopodia formation are largely unknown. also, detailed molecular mechanisms by which astrocyte secreted factors regulate synaptogenesis are not well understood. small extracellular vesicles (sevs)/exosomes have potential to regulate filopodia, spine and synapse formation in autocrine or paracrine manner due to their unique cargo composition. here, we examine role of exosomes in filopodia, spine and synapse formation. methods: primary rat hippocampal and cortical neurons were transiently transfected with the multivesicular body (mvb) docking regulator gfp-rab27b or with shrnas against the exosome secretion and biogenesis regulators rab27b and hrs. transfected neurons were immunostained for synaptic proteins and analysed for filopodia at day in vitro (div) 6 or spines at div12. for rescue experiments, exosomes were isolated using differential ultracentrifugation method from conditioned media of div9 cortical neurons or primary astrocytes and characterized for their size, common protein markers and morphology. results: here, we find that mvb docking factor gfp-rab27b localizes to both the tips and bases of actin-rich filopodia and spines in primary neurons. furthermore, genetic regulation of exosome secretion by overexpression or knockdown of rab27b or hrs leads to respective increases or decreases in the number of filopodia, spines and synapses. the defects of exosome-inhibited neurons in filopodia density are rescued by add-back of neuronal exosomes. additionally, treatment of primary neurons with exosomes isolated from primary astrocyte cultures leads to enhanced spine and synapse formation. summary/conclusion: these results indicate that autocrine and paracrine communication via exosomes are a key part of the process of neuronal filopodia, spine and synapse formation. effects of apolipoprotein e genotype on protein and small rna profiles of brain tissue-derived extracellular vesicles of alzheimer's disease patients introduction: multiple sclerosis (ms) is the most frequent chronic inflammatory disease of the young adult central nervous system. nevertheless, the pathogenesis remains largely unknown. it is therefore relevant to better characterise in cerebrospinal fluid (csf), which irrigates the brain, novel bioactive compounds whose dysregulation could be involved in ms pathology. the concentration of extracellular vesicles (evs) has been already found affected in ms patient fluids but the content in bioactive molecules, particularly the micrornas (mirnas), remains barely investigated. the mirna are short oligonucleotides that are major posttranscriptional regulators and we previously showed the dysregulation of specific mirnas in csf of ms patients. evs can potentiate mirna effects by allowing remote action through the shuttling within biological fluids such as csf while providing a protection from circulating rnase. nevertheless, csf remains a challenging fluid to analyse due to limited access, low volume and presence of lipoproteins (other putative mirna carrier) that can be co-isolated with evs. methods: we performed a comparative analysis of ev isolation from csf by size-exclusion chromatography (sec), density-gradient ultracentrifugation, ultrafiltration or chemical precipitation (chemp) to determine the optimal technique(s) to enrich ev. results: sec applied on csf of control patients showed optimal ev purification with sufficient evs from 0.5 ml of csf for downstream ev characterization. furthermore, we were able to isolate mirnas from csf and determined their enrichment in evs by rnase-sensitivity treatments. finally, we have combined chemp and sec to enable a fast and largescale isolation of evs from > 5 ml of csf, which successfully provided an increase in particles detected by nanoparticle tracking analysis. we are currently characterising the particles to confirm that they are purified evs, cleared from contaminants. summary/conclusion: this work opens perspective to analyse evs from ms patients and to determine whether mirnas participates in ms pathogenesis through their transit in evs. funding: fondation louvain, charcot foundation. differences in circulating number of extracellular vesicles between contact sport athletes with and without acute mtbi: a pilot study meghan rath a , jacqueline sayoc a , soo-young choi a , karlee burns b , aja corchado c , jane mcdevitt b , jingwie wu d , ryan tierney b , michael selzer e , xiaoxuan fan f and joon-young park a for bottom-up guc, increasing iodixanol gradients with 2.3 ml of samples were centrifuged at 186 k g for 18 h. fractions were then pooled based on densities (1.1-1.2 g/ml). bca and sds-page were used to analyse total protein; nanoparticle tracking (nta) and transmission electron microscopy (tem) for ev presence; and immunoblotting and imaging flow cytometry (ifcm) to evaluate ev specific markers. (ev-track id: ev190096). results: immunoblotting showed absence of actinin4 from all samples, while cd63 and tsg101 were detected for all samples; apart from imf_ip. nt_samples were not analysed reliably by nta and ifcm, due to the high concentration of casein micelles present (~10^14/ml in milk) that otherwise would be co-counted with evs. as expected, following ip, which most efficiently removed casein micelles, bca showed that samples had lowest total protein. this was confirmed by sds-page. thus, most effects were then focused on the ip casein-depleted samples. ifcm indicated that, post-guc, sm_ip evs had significantly (p < 0.05) more cd81-positive particles/ml of milk vs all other guc and 200 kduc samples. while there were no significant differences in sizes of ev separated from sm or imf, directly comparing the ip pre-treated samples, sm had significantly (p < 0.01) higher quantities of evs when compared to imf. additionally, tem indicated that evs separated from sm by guc were intact with limited background debris, whereas those separated from sm by duc and all imf evs were not. summary/conclusion: in conclusion, regardless of the method used, imf has fewer intact evs compared to sm. also, to obtain purest sm evs, ip followed by guc separation is optimal. introduction: extracellular vesicles (evs) exist as subpopulations with heterogeneous content. the surface heterogeneity of evs may reflect differences in functionality between ev subpopulations, as interactions with recipient cells may differ between ev subpopulations with different surface profiles. however, it is currently challenging to study functional differences between ev subpopulations due to the lack of suitable techniques to purify intact evs based on their surface signature. here, we showcase a novel capture-and-release platform to enrich intact ev subpopulations by their surface profile and compare their characteristics. methods: mda-mb-231 and skov-3 cell-derived evs were isolated using size exclusion chromatography. ev subpopulations were enriched based on surface markers cd9, cd63, cd81 or phosphatidylserine (ps) using a novel magnetic bead-based capture-and-release platform. obtained evs were characterized by transmission electron microscopy (tem), nanoparticle tracking analysis (nta) and western blotting. evs were fluorescently labelled using pkh67 and celltracker deep red (ctdr) and their uptake by recipient cells was examined using flow cytometry. results: western blot analysis showed that ev subpopulations enriched for the selected tetraspanins and ps were successfully isolated using a novel capture-andrelease platform. interestingly, evs isolated based on ps exposure (ps+) lacked most canonical ev markers. all ev subpopulations showed intact, cup-shaped morphology when analysed by tem, but contained less protein contaminants compared to the initial ev isolate. ps+ evs were slightly larger than other ev subpopulations when analysed by tem and nta. to test the capacity of ev subpopulations to interact with recipient cells, evs were labelled with pkh67 and ctdr prior to subpopulation fractionation. after fractionation, ps+ evs showed a significantly higher ctdr/pkh67 ratio than other ev subpopulations as determined by fluorescence spectroscopy, suggesting higher esterase activity of ps+ evs compared to other tested subpopulations. furthermore, mda-mb-231derived evs isolated based on cd9 and cd81 expression were taken up more efficiently by hmec-1 and mda-mb-231 cells than evs isolated based on presence of cd63 or ps. summary/conclusion: using a novel technology to isolate ev subpopulations based on their surface profile, we here show that composition and cellular uptake efficiency differs between ev subpopulations. theoretically, this technology is applicable to any surface marker of interest, allowing its use to further establish ev surface-functionality relationships and enrich evs with desirable characteristics for therapeutic purposes. funding: this work was supported by a veni grant (no. 17296) of the dutch research council (nwo). aml were harvested from tib49 cells cultured in evfree medium using serial ultracentrifugation. hspc (ksl; lin-sca1+ ckit+) clonogenicity and inflammatory responses were assessed using colony-forming unit (cfu) assay and real-time polymerase-chain reaction, respectively. ifn-alpha receptor 1 (ifnar1) expression and intracellular reactive oxygen species (ros) levels were assessed by flow cytometry. dna damage were assessed by quantifying nuclear γ-h2ax using immunofluorescent microscopy. results: similar to evs derived from aml patients, tib49 ev-aml elicited double-stranded breaks in hspcs, and actively suppressed hspc clonogenicity. transcriptional profiling revealed that exposure to ev-aml induced the upregulation of several inflammatory mediators in hspcs, including isg15, il-6, ifnα, ch25 h. inflammatory signalling triggered by ev-aml did not depend on ifnα signalling as evident from suppression of clonogenicity in ifnar1-null hspcs as well as the lack of evs-induced stat1 phosphorylation or ifnar1 downregulation. instead, we found increased levels of ros following ev-aml exposure. summary/conclusion: our findings support a model whereby ev-aml inflammatory signalling and oxidative stress lead to dna damage in hspcs. introduction: basic leucine zipper atf-like transcription factor 2 (batf2) is implicated in inflammatory response and anti-tumour effects. although the tumour suppressive function of batf2 has been reported, its extracellular role in maintaining a non-supportive cancer microenvironment has not been explored. methods: in this study, we established gbm orthotopic and subcutaneous tumour models in nude and balb/c mice and flow cytometry analysis determined the batf2 inhibitory effects of mdscs recruiting. we used transwell assay to determine batf2-positive evs (evs-batf2) inhibitory of the chemotaxis of myeloid-derived suppressor cells (mdscs) in vitro. in addition, exo-counter detection during the development of the gbm-batf2 model to demonstrate evs-batf2 crosstalk with distant tissues. amd3100 blocking in tumour model confirms that evs-batf2 dominated by the sdf-1a/cxcr4 signalling pathway. in addition, exo-counter detection of evs in 32 pairs of gliomas in different stages proposes plasma-evsbatf2 (plevs-batf2) as a prognostic marker. results: we found that tumour-derived evs-batf2 regulate crosstalk between glioma cells and tumour microenvironment by inhibiting mdscs recruitment. evs-batf2 can be detected in plasma and bone marrow of glioma-bearing mice, this provides direct evidence that glioma-derived evs can communicate with distant site by crossing blood-brain barrier. besides, evs-batf2 injection significantly reduced sdf-1α expression in the tumour tissues. after blocking sdf-1α signalling by amd3100, the inhibitory effects of batf2 overexpression on mdscs recruitment were rescued. evs-batf2 inhibit mdscs recruiting and secreting mmp2, mmp9, and vegfa which promote gbm progression. strikingly, exo-counter detection of evs in 32 pairs of gliomas in different stages reveals that the number of plevs-batf2 can distinguish stage iii-iv glioma from stage i-ii glioma and healthy donors. summary/conclusion: our results suggest that evs-batf2 may be an effective circulating biomarker associated with glioma progression. of note, we are the first to determine the regulatory role of evs-batf2 in regulating tumour microenvironment and propose plevs-batf2 as a prognostic marker predicting glioma progression and candidate target for gbm therapy. introduction: electrofluidics is an emerging technology of combining electronics and nanofluidics. one important device in electrofluidics is an ion transistor in which the ionic current through a nanopore is regulated by gate voltage bias. here, we suggest a fabrication method of nanopore by introducing focused ion beam (fib) and atomic layer deposition (ald) to sense extracellular vesicle (ev) via metal electrode structures. methods: we deposited 100 nm-thick silicon-nitrite layers on both sides of silicon wafer by low-pressure chemical vapour deposition (lpcvd). we fabricated rectangular patterns by photolithography followed by reactive ion etching (rie) on the backside of the wafer. anisotropic silicon etching by koh was performed. the front side of the chip was patterned by photolithography followed by ti/au deposition for the fabrication of electrode structures. we drilled 50~100 nm pores in the si3n4 membrane by fib. by the ald process, we deposited highly-conformal metal film, either platinum (pt) or ruthenium (ru) to shrink nanopores by a self limiting process. results: we expect that the ion current through the nanopore is efficiently controlled by the gate-surrounding structures. the nanopore ion transistor can be used to count the number of evs. summary/conclusion: we suggest a fabrication method of nanopore ion transistors by introducing focused ion beam (fib) and atomic layer deposition (ald). this device will be applicable for single ev sensing. introduction: extracellular vesicles (evs) are key players in cell-cell communication and increasing evidence has shown that evs function in cancer by promoting cancer cell motility and metastasis. analysing tumour-derived evs in biofluids is attractive because it would be a novel approach to a non-invasive liquid biopsy. unfortunately, evs are highly heterogeneous. they vary greatly in size, lipid composition, and cargo and are difficult to distinguish from other small particles in complex biofluids. we have developed a novel flow cytometry method to generate a distinct ev fingerprint to profile biological specimens. methods: evs from cell culture media (purified and unpurified) and biological fluids (plasma and urine) were detected by flow cytometry using features on individual evs produced by intrinsic (cd63-phluorin) and extrinsic (lipophilic dye, di-8-anepps, and antibodies) fluorescent labels. ev subpopulations were visualized with dimensional reduction (t-sne and umap) of 20-150 features that defined the vesicle size, shape, and fluorescent emission spectra associated with the fluorescent marker. unsupervised density based clustering (hdbscan) in conjunction with supervised machine learning (xgboost) was subsequently used to define subpopulations. we refer to this method as "ev fingerprinting". results: ev fingerprinting was successfully used to detect evs in complex biological specimens and trace their differential enrichment through conventional purification methods. evs were readily distinguished from protein complexes, lipoproteins and non-lipid particles. calibration with externally validated purified ev, as well as size, lipid, and fluorescence standards enabled ev fingerprinting as a rigorous and reproducible method for resolving heterogenous ev samples. ev fingerprinting applied to conditioned medium from tumour cells and biological fluids from cancer patients reveals unique ev profiles generated by cancer, further supporting the potential of ev fingerprinting as a liquid biopsy. summary/conclusion: our single-ev analysis approach characterizes whole ev populations in complex biological fluids without the need for purification, reducing time intensive purification protocols and subsequent sample loss, permitting efficient analysis of liquid biopsy samples. detection and quantification of extracellular vesicles with cargo protein and rna using the amnis® cellstream® flow cytometer introduction: the particle size distribution (psd) of extracellular vesicles (evs) is commonly measured by tunable resistive pulse sensing (trps) and nanoparticle tracking analysis (nta). both trps and nta have limitations that hamper the accurate measurement of the psd of evs, specifically in the size range from 50 to 100 nm. an alternative technique for measuring the psd of evs is micro-fluidic resistive pulse sensing (mrps). because a standard operating procedure (sop) for characterizing evs by mrps is absent, we aim to establish a reliable sop to ensure reproducible psd measurements of evs by mrps. methods: measurements (n = 3) of red-blood cell, prostate cancer cell line supernatant, and human urine and plasma evs were acquired in 10 × 10 s acquisitions. two microfluidic cartridges were used to study a dynamic range of 50-450 nm. samples were diluted into phosphate buffered saline with different concentrations of tween 20 or bsa. because the excess of particles affects the detection limit, serial dilutions were performed to find the optimal dilution for each sample. data were evaluated using data viewer software. results: the optimal dilution was determined for each sample by maximizing the particle rate and minimizing the measurement time while preserving a robust detection limit of 50 or 65 nm. moreover, we developed a procedure to optimize the peak filter settings of data viewer by fitting data to normal distributions and identifying threshold values for signal-to-noise ratio, symmetry, and transit time within 99% confidence. summary/conclusion: we recommend to use 0.1% w/ v bsa in dpbs as sample diluent, because tween 20 affects evs as confirmed by flow cytometry. by using orthogonal techniques and well-characterized biological test samples, we developed and validated a sop for ev detection by mrps, thereby making mrps a valuable tool for ev researchers. real-time measurements of extracellular vesicles binding kinetics achieved through interferometric imaging in a multiplexed microarray modality introduction: extracellular vesicles are very promising diagnostic biomarkers. as a matter of fact, the properties of these biological nanoparticles depend on the health conditions of each individual. however, experiments that involve evs phenotyping are time consuming, due to 12 h-or overnight incubations. in order to get accurate results, maximizing binding efficiency is a necessity; that normally involves ensuring the saturation of the capture reaction, which can result in an unnecessarily long incubation time. with the ability of labelfree kinetic binding measurements using interferometric reflectance sensing in a microfluidic chamber, we perform an optimization of the incubation time in different flow conditions, while demonstrating a new way of multiplexing for real-time evs specific capture and detection.methods: all the real-time binding measurements were performed with the interferometric reflectance imaging sensor (iris). iris chips were first coated with an organic polymer (mcp-2), which provides an active surface for probe immobilization. then, antibodies against cd9, cd81, cd63 markers were spotted at different densities in a microarray modality. the chips were then encapsulated with a glass window to form a microfluidic chamber that allows for imaging the sensor surface. samples of hek-derived extracellular vesicles were flowed across the sensor surface in the iris system and real-time images were acquired. incubation was performed at different flow rates, and in static and stopflow modalities. results: in this work, we focus on the specific capture of evs under different flow conditions to achieve an optimization of the incubation time. indeed, through the acquisition of real time binding data, we are able to precisely monitor the equilibrium point of the capture reaction. in this configuration of iris, low magnification optics allow for simultaneous detection of binding on hundreds of capture ligand spots. therefore, surface probes (surface density and specificity) as well as assay conditions can be optimized. we report on the optimization of antibodies against cd9, cd81, and cd63 markers. since the sensor chips are identical to the single-particle detection assays developed by nanoview biosciences, the optimization of binding assays will directly impact the phenotyping of individual exosomes. summary/conclusion: our method proved to be very efficient in optimizing the most crucial aspects concerning evs captureflow conditions, incubation time, surface density and sample concentration. introduction: diabetes is a life treating diseases extending its impairing influence on more than 500 billion of people around the world within upcoming 20 years. the most harmful complication generating high treatment and social costs is diabetic nephropathy, which develops in about 10% of patients suffering diabetes. still we do not have an effective and direct prognostic biomarker to diagnose renal complications in the primary stage of renal disease. methods: extracellular vesicles were concentrated from diabetic patients' urine and washed to perform spectral analysis: fourier transform infrared spectroscopy (ftir), based on the molecular absorption of electromagnetic radiation in the infrared region of the spectrum in a range from 400 cm-1 to 4000 cm-1 and raman spectroscopy (rs) as a technique based on inelastic scattering of monochromatic light. both techniques provide information on the chemical structure of compounds by identifying functional groups with high molecular specificity. results: average spectral signature obtained for evs from urine samples of patients in the different stage of kidney damage allowed distinguishing specific bands, representative for amide (i/ii), lipids, cholesterol and nucleic acids. spectral parameters correlated with a clinical stage and a commonly used indicator of renal function (creatinine) in diabetic patients. summary/conclusion: infrared and raman spectroscopy are promising tools to diagnose and monitor renal function in diabetes. introduction: several existing bioanalytical strategies for purifying and characterizing exosomes have allowed for fundamental progress to be made. mixtures of evs can be enriched for exosomes by techniques such as ultracentrifugation and size-exclusion chromatography. but, these processes require large amounts of material that are often difficult to obtain and many different types of particles have similar sizes and densities. it is likely that unique subfractions within enriched samples exist, particularly in complex biological matrices such as blood, urine or milk which remain difficult to characterize and isolate with existing analytical technologies. methods: bovine milk exosomes were isolated via differential ultracentrifugation and resolubilized in 100 mm ammonium acetate. these data were recorded using charge detection mass spectrometry (cdms). in cdms, individual particles are reflected back and forth through an electrostatic ion trap where they pass through a sensitive charge detector. each time a trapped particle enters and exits the detector, its charge (z) and mass-to-charge (m/z) ratio is measured. mass distributions are generated by multiplying the m/z values by the charge measured for each ion and binning the resulting masses.results: the masses of particles in a bovine milk extracellular vesicle (ev) preparation enriched for exosomes were directly determined for the first time by cdms. particle masses and charges span a wide range from m~2 to~90 mda and z~50 to~1300 e and are highly dependent upon the conditions used to extract and isolate the evs. in total, 57,350 particles were detected from eight cdms measurements. a simple two-dimensional gaussian model suggests that eight unique subpopulations of particles may be resolvable based on charge and mass. complementary em and proteomics analyses confirm that samples are enriched for exosomes. particles associated with the s1, s2, and s3 families that are centred at~3.5,~5.9, and~8.3 mda, respectively, appear too small to be ascribed to exosomes. the remaining 45,229 (79%) particles detected by cdms are within the mass range expected for exosomes. while cdms measurements are at an early stage of development, this approach appears to provide a new physical basis for separating and characterizing ev particles. summary/conclusion: this work describes a novel biophysical approach for measuring and characterizing the masses and charges of the extremely heterogenous population of exosomes and other extracellular particles enriched in bovine milk. as new sample preparation methods, aimed at purifying specific types of exosomes from different cell lines, tissues, and other body fluids continue to evolve, rapid and sensitive cdms measurements of the physical properties of mass and charge may become an important means of assessing the efficacy of different protocols. funding: nih (r01 gm131100-01). bab is supported by indiana university quantitative chemical biology fellowship (t32gm131994). in situ detection of exosomal microrna-10b by fusion with liposomeencapsulated nanomotor introduction: breast cancer is the most common cause of cancer-associated death in women and has raised global health concerns. early diagnosis and treatment are crucial to improve the prognosis and survival rate of breast cancer patients. liquid biopsy is expected to provide a strategy for early diagnosis of breast cancer. exosomes have been regarded as novel liquid biopsy biomarkers due to their stable cargo of rnas, lipids, and proteins from their origin cells. exosomal micro (mi)rnas have recently been recognized as promising indicators of cancer occurrence and progression. however, most of the reported exosomal mirna detection methods require the lysis or extraction process, which increases the possibility of sample loss. in situ detection strategies avoid interference from body fluid. in this study, we developed a gold nanomotor fluorescence platform based on liposome fusion for breast cancer exosomal mirna in situ detection. the exosomal mirna detection platform was constructed using a gold nanomotor (detector) and liposomes (carrier). the dnazyme amplification sequences which could be especially triggered by mirna-10b were identified by sds-page before modified on gold nano-motor and the capacity of the nanomotor was assessed using synthetic target sequence, breast cancer cell mda-mb-231, mirna-10b-encapsulated anionic liposomes, and mirna-10b-expressing exosomes. three kinds of liposomes were synthesized, characterized, and assessed for loading ability. membrane fusion effect was evaluated by confocal laser scanning microscopy (clsm) and nanoflow cytometry. the performance of this method to discriminate between breast cancer patients and healthy individuals was investigated. results: the chosen dnazyme amplification sequences transformed "locked" status to "cleavable" status on target addition, releasing a fluorescence signal. the modified gold nanomotor showed a ten times higher fluorescence signal in the presence of mirna-10b than the background and no noticeable fluorescence changes from a single-base-mismatch sequence. moreover, among the three different liposomes, cationic liposomes exhibited great stability, high loading efficiency, and excellent membrane fusion effect. furthermore, the fluorescent experiments confirmed that cationic liposomes could load and transfer the nanomotors into exosomes for mirna-10b detection. finally, we were able to distinguish breast cancer patients and healthy individuals by sensing exosomal mirna-10b directly from plasma samples without exosome isolation. summary/conclusion: a separation-free and sensitive assay based on dnazyme amplification technique and membrane fusion effect was established for breast cancer-derived exosomal mirna-10b detection, which could be a promising tool for the liquid biopsy of breast cancer. isolation of exosomes by membrane affinity column increases non-exosomal rna recovery in comparison to differential ultracentrifugation introduction: exosome-based liquid biopsy is a potential aid in the diagnosis and prognosis of cancer patients. however, in order to incorporate exosomes into clinical routine, there is a need to compare different isolation methods. here we analysed the impact, in exosomal rna yield, of two intermediate recovery/ intermediate specificity methods: differential ultracentrifugation (ucd) and a membrane-affinity column (mac) kit. although mac has a faster performance which is more suitable to the clinic, we found that ucd results in a higher recovery of exosomes and less contaminating non-exosomal rna.methods: exosomes were enriched by mac and ucd from identical volumes of human plasma (12,000xg, 34 min/0.22 ) m filtration/110,000xg, 5 h)(n = 7) and lymphoma conditioned medium(300xg, 10 min/200xg, 20 min/1000xg, 30 min/120,000xg 1.5 h/120,000xg, 1 h) (n = 3). all exosomes were characterized by nanoparticle tracking analysis (nta), immunoblotting of cd63/cd9/flotilin/alix and electron microscopy (tem). exosome pellets were pre-treated with proteinase k (1 mg/ml/56°c/10 min) and rnase a (2 mg/ ml/37°c/20 min) before phenol-chloroform/glycogen rna extraction. rna yield was measured by both fluorometer and bioanalyzer.results: isolation of exosomes by ucd, in both plasma and medium, resulted in a higher yield in comparison to mac. this was shown by an augmented intensity of marker bands in the ucd samples (p = 0.005, n = 4) as well as by an increased number of exosomes in tem.in contrast, mac final exosomal fraction (from both plasma and medium), resulted in a 13-fold and 200fold increase in rna, respectively, in comparison to ucd when measured by fluorometer. this was confirmed by bioanalyzer. introduction: there is a need for better techniques for characterizing ev populations. we developed a sensitive multiplexed electrochemiluminescence (ecl)based assay format to characterize evs in cell-conditioned medium (ccm) and human biofluids. here we use the format to analyse ev samples for the presence of 66 ev surface proteins, and to identify changes in ev phenotype associated with different cell lines, purification methods and growth conditions. methods: multiplex plates were prepared on msd's u-plex® platform with antibodies for 66 putative evsurface proteins. each well displayed an array of nine specific capture antibodies and a negative control antibody. evs from samples were captured on the arrays and then detected with a cocktail of anti-tetraspanin antibodies (cd9, cd63 and cd81) conjugated to an ecl label. three distinct cell types were grown at two sites, msd and atcc. resulting ccm were each purified by four common methods: tangential flow filtration, peg-based precipitation, size-exclusion chromatography and centrifugal ultrafiltration. all samples were also assayed without purification.results: fifty-five of the surface markers were detected on intact evs from at least one evaluated cell type. datasets were analysed using correlation matrices, hierarchical clustering, and machine learning. for each cell type, when comparing unpurified ccm grown at different sites or evs prepared by different purification methods, we typically observed correlations above 0.9, indicating that the purification methods did not introduce bias to ev phenotypes, and that the assay format can provide robust phenotypic information without any purification of evs. two unsupervised clustering analyseshierarchical clustering and t-distributed stochastic neighbour embeddingboth generated wellseparated clusters for each of the cell types, regardless of purification method or source. summary/conclusion: we developed multiplex ev surface marker assays and demonstrated their use for multimarker ev phenotyping. this flexible format enables rapid assay development for new ev subpopulations with or without sample purification. these results also demonstrate ev surface marker phenotyping via multiplex ecl assays may be used to distinguish ev populations from various cell types, and characterize bias introduced by purification. detection of misev recommended ev protein-markers using automated western blotting method for isolation of evs and a simple western blotting platform for automated protein separation and immunodetection of misev-recommended proteins.methods: total evs were isolated by affinity-membrane spin columns from pre-filtered 0.5-4 ml plasma or 2-20 ml urine, respectively. intact vesicles were eluted and the ev-depleted biofluid fraction was collected from the flow-through. a small fraction (4 μl) was analysed by a simple western blot workflow providing automated capillary electrophoresis-based protein separation and immunodetection, characterizing each fraction for presence or absence of misevrecommended proteins.results: a range of specific antibodies were identified and the ev fractions were shown to be enriched in evproteins, whereas contaminating non-ev proteins were significantly reduced. isolation of evs was necessary to allow detection of the low abundant ev protein markers, whereas non-ev proteins were readily detectable both in the neat biofluids and in the ev-depleted flowthrough. we characterized the effect of washing on the purity of ev isolates and defined the dynamic range of the workflow using titrations of input volume of both plasma and urine ev isolations. summary/conclusion: simple western blotting protocols were established for quality control of isolated evs in accordance with misev-guidelines. evs isolated using affinity-membrane spin columns were shown to be enriched in ev markers and depleted for non-ev proteins. al-pha beads: a library of extracellular vesicle-associated metalloproteinase biosensors (adams) and a disintegrin and metalloproteinase with thrombospondin motifs (adamtss) are highly promising cancer biomarker candidates that have complex roles in cancer pathogenesis and metastasis. importantly, within the context of lung cancer, the detection of adam10 proteolytic activity might be more informative than the level of adam10 protein.therefore, the development of low-cost metalloproteinase biosensors could serve as useful biomarker research tools. methods: to this end, we developed advanced proteolytic detector polyhydroxyalkanoates (al-pha) beadsa library of biodegradable, biopolymer-based protease biosensors. broadly, these biosensors utilise phac-reporter fusion proteins that are bound to microbially manufactured bioplastic beads. these phac-fusions also incorporate specific protease cleavage sites. in the presence of a specific protease, reporter proteins are cleaved off of the al-pha beadsresulting in a loss of bead fluorescence that can be measured using flow cytometry. these biosensors were assayed using either metalloproteinases, conditioned media or evs from in vitro cancer models.results: human metalloproteinase recognition motifs were identified in the literature and a total of 70 different al-pha bead biosensors were designed. a control, tev-specific biosensor detected 0. introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr.results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom20. mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the 374 isev2020 abstract book biogenesis of evs is neutral sphingomyelinase 2 (nsmase2), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase2 is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase2 inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles.methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo [1,2-b] pyridazin-8-yl) pyrrolidin-3-yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated 5xfad mice with 10 mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay.results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, 5xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting 20% of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human postmortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of 20 mdd subjects and 20 hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed.results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd9. tem images showed the typical cup-shaped morphology with sizes mostly between 30 and 200 nm. preliminary sequencing results revealed that mir-33a-5p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir-33a-5p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. methods: we use ifc to characterize evs released by glioma using 5-ala, fluorescently labelled ev (cfda-se, cd81) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd81) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that 58% are tenascin c positive, 2.7% are egfrviii positive and 1.6% are 5-ala positive. there was only a minor overlap (<16%) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs (2.5-fold), tumour specific evs (2.8-fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll 2 receptor (tlr2) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il-12 and il-6, which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells.objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow field-flow fractionation (af4) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp-1) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk-2 epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with 50 to 50 nm (ev1) and another with 100 to 120 nm (ev2). ev1 induced more tnf-alpha, il-6, ip-10 and ccl20 than ev2. it was also more effective in promoting t. cruzi infection in epithelial cells. due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays.noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr.isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and 0.2um filtration. co-purification of norovirus with the evs was checked.ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid50 assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems.ps15.12 = op3.12 kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of 11.5 µg/ml of plasma, as geometric means ≥11.5 µg/ml were nearly equal. hevs were detected at 48 µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f200x electron microscope and measured between 40-90 nm, and hevs were between 60-160 nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: nasopharyngeal carcinoma (npc) is characterized by a large presence of regulatory t cells (tregs) and the production of tumour-derived exosomes with immunosuppressive properties. our team showed that npc-derived exosomes favour the suppressive activity and recruitment of human tregs via ccl20 chemokine, thus contributing to npc immune escape (mrizak et al., jnci, 2015) . more recently, our team has shown that npc-exosomes could induce tregs by altering the maturation of dendritic cells (dcs) and promoting tolerogenic dendritic cells (tdcs) (renaud et al., herpas congress 2017). our main objectives in this study are (i) to define and compare the metabolic status of mature dendritic cells (mdcs), control tdcs and tdcs generated in the presence of npc-exosomes (exocnptdc) and (ii) to evaluate the chemoattractive potential of npc-exosomes on exocnptdcs, and notably to investigate the involvement of ccl20 in this recruitment. methods: dcs are generated from human monocytes in the presence or absence of npc-exosomes. the maturation status of dcs was evaluated at a phenotypic level by studying the expression of maturation markers using flow cytometry and at a functional level by analysing cytokines secretion using elisa. this cytokine analyse has been performed in both conditions, on treated dcs and during co-culture assays of autologous cd3 t lymphocytes with treated dcs. in a second step, a mitochondrial metabolic and glycolytic study was performed using the seahorse technology (ocr and ecar measurement). finally, the chemoattractive potential of npc-derived exosomes on the different induced dcs was analysed (i) using boyden chamber chemoattraction assays or real-time videomicroscopy (chemotaxis µslide ibidi) and (ii) using rt-qpcr analysis of the receptor expression of ccl20 (ccr6).results: npc-exosomes alter dc maturation, which gives rise to tolerogenic dcs that favour the induction of tregs. in addition, the metabolic analysis of dcs seems to put foward a specific metabolic signature of the tdcs induced by npc-exosomes. and finally, chemoattraction assay suggests that npc-exosomes preferentially attract tdcs and exocnptdcs in a ccl20dependant manner. summary/conclusion: taken together our results should allow us to characterize the major role of npc tumour exosomes on the maturation and the recruitment of dc and so identify them as anti-tumoural therapeutic targets. cytotoxic t lymphocyte ev that prevents tumour metastasis by collapse of tumoural mesenchymal stroma is classified into exosome, but not microvesicle or apoptotic body.naohiro seo a , junko nakamura a , tsuguhiro kaneda a , takanori ichiki b , asako shimoda c , kazunari akiyoshi c and hiroshi shiku a a mie university graduate school of medicine, mie, japan; b the university of tokyo, bunkyo, japan; c kyoto university, kyoto, japanintroduction: recently, instead of ultracentrifugation, development of new preparation protocol is demanded for research of reliable bioactivity and drug discovery of extracellular vesicles (evs). in this study, we propose a novel method for large scale preparation of highperformance extracellular vesicles focusing on membrane negative charge. methods: murine cytotoxic t lymphocyte (ctl) evs in supernatant were concentrated more than 20 times at over 97% purity without leaking by 750 kda mwco ultrafiltration, and subjected to ion exchange deae column chromatography after replacing with pbs. after ion exchange, evs were characterized by bca assay, nta assay, cryotem observation, proteome analysis, dna content measurement, mirna microarray analysis, zeta potential measurement, lectin array analysis, and target cell analysis.biomarker detection and analysis and detail strategies for cross-platform analytical validation. methods: we conducted a cross-platform analysis using two commercially available flow cytometers designed for ev detection. scatter resolution, enumeration accuracy and precision were determined across both platforms by analysing submicron silica beads (apogeemix, 180-1300 nm) of known concentration.we detected large evs, as established by reference size beads, electron microscopy, expression of phosphatidylserine and the presence of integral membrane proteins of cell of origin. we analysed evs isolated from plasma by high-speed centrifugation (18,900 g) as well performing analysis by direct plasma labelling followed by validation by detergent lysis of vesicular constituents. a clinical operating range was defined which ensures linearity and avoids swarm detection. we observed comparable scatter resolution, enumeration accuracy (error ≤15%) and precision (cv ≤5%) across both platforms used. we defined two ev size gates: a "latex" gate (300 to 1100 nm polystyrene latex beads), and a "silica" gate (180 to 1300 nm silica beads) for evs at the lower end of our size range of interest. to improve detection sensitivity, we identified common contributors to signal noise and applied workflow strategies to minimize these. finally, we identified linear ranges which avoid swarm detection, and which ensures reproducible ev counts (cv <20%) across both instruments. summary/conclusion: we present an optimised, standardised and cross-platform reproducible working protocol which supports the use of fcm in an ev-based liquid biopsy application. funding: the project is funded by spark oceania and uts innovation commercialisation seed fund scheme to mb. metabolomic profiling of serum and exosomes isolated from head and neck cancer patients after radiotherapy introduction: cancer radiotherapy (rt) induces the response of the whole body that could be detected at the blood level. searching for new molecular signatures which could correlate with treatment response in cancer patients is of particular importance. radiation-induced changes in proteome and transcriptome of serum have been widely described. however, metabolomic changes in serum, exosomes and other classes of small extracellular vesicles (ev) of cancer patients after rt have not been given as much attention. metabolomics of serum and ev of cancer patients could provide a valuable insight into the response of both tumour and whole organism to the treatment. the aim of the study was to compare serum and ev metabolomic profiles in head and neck cancer (hnc) patients before and after rt. methods: serum samples from 10 hnc patients were taken before (a) and after (b) rt. 10 healthy volunteers were used as a control group (c). ev were isolated from 1 ml of serum using size-exclusion chromatography (sec). selected sec fractions were subjected to extraction of metabolites. a mixture of meoh/h2o was used for extraction of metabolites from serum and ev samples. samples were analysed by gas chromatography-mass spectrometry (gc-ms).the study protocol adhered to the tenets of the declaration of helsinki and was approved by the bioethical committee of the maria skłodowska-curie national research institute of oncology, branch gliwice, poland (permit nr. do/dgp/493/4/06/1/2016/g). results: an untargeted gc-ms-based approach allowed the detection of 189 metabolites in serum samples and 50 exosomal small molecules, of which 32 joint. the identified compounds included amino acids, fatty acids, carboxylic acids, sugars, and others. there were 49 metabolites which levels discriminated compared groups (a,b,c) of serum samples and 12 compounds that discriminate the ev isolated from hnc serum before and after rt from hc. summary/conclusion: rt caused significant changes in levels of serum and ev metabolites witch are involved in amino acid metabolism, lipids metabolism, energy metabolism and oxidative stress response. capable of contributing to intercellular communication and metastasis. numerous studies have focused on elucidating their role in cancer progression. we recently showed that sevs isolated from pancreatic cancer cells can function as an initiator in malignant cell transformation. here, using a mass spectrometry (ms)-based proteomics approach, we analysed the differences in the protein cargo of sevs secreted from normal pancreatic and cancer cells to better understand their biological characteristics. methods: sevs were isolated from 3 human pancreatic cancer cell lines (capan-2, mia paca-2, and panc-1) and normal pancreatic epithelial cells (hpde) using a combined ultrafiltration-ultracentrifugation method coupled with a sucrose density gradient purification. proteomic profiling of sevs was carried out using an lc-ms/ms method. protein identification from resulting ms/ms spectra was conducted using proteome database search software followed by gene ontology (go) enrichment and reactome pathway analysis.results: a total of 4,907 unique proteins were identified confidently across the combined samples. the proteins present in all four sev types (1,135 proteins) consist of general housekeeping proteins. 348 proteins were uniquely found in all cancer sevs but not in the normal hpde sevs. this group contains an enrichment of proteins that function in the endosomal compartment of cells responsible for vesicle formation and secretion and suggest their important role in driving the increased production of sevs from cancer cells relative to normal cells. moreover, this group includes a set of proteins that have been implicated in malignant cell transformation, consistent with our previous work showing that each of the cancer sevs analysed here could initiate malignant transformation of nih/3 t3 cells. conversely, there were 313 proteins uniquely found in normal hpde sevs. this group includes a number of immune response proteins that are not found in any of the pancreatic cancer cell sevs. summary/conclusion: the differences in the proteomes of cancer and normal sevs may be indicative of their varying roles in cell transformation and helpful in delineating the types of evs that are being produced. in addition, these differences point towards their potential value as cancer biomarkers. proteomic profile of tumour-derived exosomes in plasma of melanoma patients introduction: in the past years, extracellular vesicles (evs) have attracted considerable interest due to their ability to provide valuable diagnostic information from liquid biopsies. the high abundance in all bodily fluids and their cargo stability confers evs the potential as a powerful tool to not only obtain novel biomarkers from inaccessible tissues, therapy response and monitoring, but also to reduce infection risks of conventional highly invasive biopsies. virtually all cells continuously release vesicles into the extracellular environment, diverse in size, content and features depending on the biogenesis, origin and function. this heterogeneity adds a layer of complexity when attempting to isolate and characterize tissue-specific vesicles. methods: hence, we aimed to use a immunomagnetic capture approach for prostate-derived evs from cell culture supernatants, with further investigation into human plasma and urine samples. analysis was performed by nanoparticle tracking analysis, western blotting and electron microscopy. additionally, an in-house spotted antibody microarray is in development. here, we intend to detect different ev sub-populations based on their surface markers. results: isolated immunocaptured ev populations based on the classical ev marker cd9 show an increased signal for the luminal protein tsg101. ev populations targeting the tissue-specific marker prostate specific membrane antigen (psma), were found positive for tsg101 in a lower extent indicating a subpopulation of evs. the microarray uses less than 100 µl of sample (concentrated cell culture supernatant, human plasma, urine) and leads to a faster characterization within 3 h for ev surface marker as compared to western blot. summary/conclusion: immunomagnetic isolation might be a promising approach for liquid biopsy and thereby the microarray could be valuable to identify potential capture targets. the current design for 6 different surface marker from 16 samples simultaneously could be easily extended for sample size and surface profiling allowing for a more economical way to multiplex samples. paving the way for implementing a feasible and reliable technique for assessing urinary extracellular vesicles as biomarkers for bladder cancer in clinical practice introduction: extracellular vesicles (ev) in urine have been proposed as biomarkers for bladder cancer (bc). however, at present there are no standardized methods for ev isolation or urine sampling. our goal was to evaluate the ev isolation performance between different methods, the effect of the sampling time and the importance of urinary creatinine (ucr) normalization. methods: two urine samples of 120 ml were collected from 5 patients with non muscle-invasive bc: one from the first micturition and another from any time of the day. twenty ml were used for ucr measurement and 100 ml were used for ev isolation by either precipitation with polyethylene glycol (peg), concentration by filtration (uf, centricon plus-70, 10 k, millipore), sepharose size exclusion column (sec), or combinations of these methods. additionally, the effect of protease inhibitors (pi) and dtt treatment after collection or during processing was analysed. size and number of particles were evaluated by nanosight and the presence of exosomal markers was evaluated by western blot. results: among the methods evaluated, uf + sec showed the best performance retrieving the highest number of particles in the range of 50-200 nm, and the highest protein expression of exosomal proteins. uf alone showed the highest concentration of ev, but with a tendency to isolate larger particles. particle concentration was positively correlated with ucr, reflecting the importance of ucr normalization before journal of extracellular vesicles 385 comparing between patients. finally, no differences in the performance according to the time of collection, nor in the use of pi or dtt were observed. summary/conclusion: uf + sec gave the highest ev yield and was not affected by the time of urine collection. the use of pi and dtt can be avoided, and normalization to ucr should be considered when implementing this technique for assessing evs as biomarkers for bc in clinical practice. funding: pida 2019. the introduction: human tumours, including pancreatic ductal adenocarcinoma (pdac), often harbour a subpopulation of cancer cells with extra centrosomes. we found, that these cells secrete an increased number of small extracellular vesicles (sevs), within the 20-120 nm size range. sevs play a role in cancer signalling and progression and are widely studied for their diagnostic potential. we aim to understand the role of sevs secreted by cells with extra centrosomes in shaping pdac-associated stroma, particularly fibrosis. methods: to study the sev mediated changes in the pdac microenvironment, we purified sevs through serial ultracentrifugation and size exclusion chromatography, characterised the content through silac-based proteomics, and assessed phenotypic changes in pancreatic stellate cells (pscs) and extracellular matrix (ecm) production through immunofluorescence staining. results: our data indicates, that the sevs secreted by cells with extra centrosomes are exosomes due to their endocytic origin, and we found, that they can activate pscs, key mediators of fibrosis in pdac. indeed, we observed an increased level of collagen i produced by pscs activated by sevs from cells with extra centrosomes as compared to cells without extra centrosomes. interestingly, we found, that psc activation through sevs is not mediated by tgf-β, assessed by the level of nuclear smad2 accumulation downstream of tgfβ activation, suggesting a novel mechanism of pscs activation. summary/conclusion: pdac cells with extra centrosomes contribute to a novel type of psc reprogramming, which could alter their ecm deposition and contribute to the extensive fibrosis observed in pdac. we are currently characterising the signalling pathways associated with sev mediated psc activation and how it impacts padc progression to better understand the role of centrosome amplification in the cancer-stromal crosstalk. exosomal carboxypeptidase e confers and cpe-shrna loaded exosomes inhibit growth and invasion of hepatocellular carcinoma cells. methods: exosomes were isolated from the culture media of high metastic hcc97 h cells and incubated with low metastatic hcc97 l cells. in other experiments, cpe-shna loaded exosomes from hek293cells were incubated with hcc97 h cells. the recipient cells were analysed for proliferation using mtt assay, colony formation, and matrigel invasion. results: analysis of exosomes derived from hcc97 h cells revealed cpe-wt mrna and protein. exosomes released from hcc97 h cells were able to enhance proliferation and invasion of hcc97 l cells. when cpe expression was suppressed in the hcc97 h cells before exosome isolation, the exosomes had no effect on proliferation and invasion. these data demonstrate the ability of exosomes to confer growth and invasion in hcc cells and the role of exosomal cpe in driving the process.previously it was shown that down-regulation of cpe expression by shrna can reverse tumour growth and metastasis in an hcc mouse model. we therefore loaded cpe-shrna into exosomes by infecting hek293 (human embryonic kidney) cells with adenovirus carrying cpe-shrna-gfp. these modified 386 isev2020 abstract book exosomes were used to transfer cpe-shrna to hcc97 h cells, resulting in significant reduction in proliferation and colony-forming ability of these cells. cpe-shrna loaded exosomes were found to down-regulate the expression of cyclin d1 and c-myc, two genes with high relavance to tumour growth and metastasis. summary/conclusion: our results demonstrate the ability of exosomal cpe to enhance proliferation and invasion in low metastatic hcc cells and the potential to use shrna loaded exosomes to target cpe as a therapeutic strategy to treat liver cancer.funding: intramural program of the eunice kennedy shriver national institute of child health and human development, and national cancer institute, national institutes of health, bethesda, md. 20892. stress hormones promote prostate cancer aggressiveness through modulation of mir-628-5p expression and exosome release north carolina central university, durham, usa introduction: despite proactive screening and steady declines in mortality, prostate cancer (pca) remains one of the most prevalent cancers among men. evidence suggests that chronic activation of stress signalling pathways can result in an altered mirnas transcriptome and affect exosomal content and release. here, we study the interaction between leptin and mir-628-5p expression, previously shown to be downregulated in pca patients. in addition, explored the effect of stress hormones cortisol and leptin on exosomal release and content from pca cells.methods: we utilized normal prostate cell line rwpe-1, and pca cells pc3, lncap and mda-pca-2b. proliferation of cells treated with leptin in the presence or absence of mir-628-5p mimic or negative control was assessed by mtt, colony formation, wound healing, and expression of targets affected by mir-628-5p was assessed by western blotting. moreover, exosomes were isolated via differential centrifugation from pca cells treated with leptin or cortisol and exosome number was determined by nanotracking analysis. exosome content was determined by western blotting and proteomic analysis by mass spectrometry.results: we observed that leptin significantly decreased expression of mir-628-5p in rwpe-1 cells.co-treatment with mir-628-5p mimic and leptin abrogated these effects in a cell dependent manner. we also observed that co-treatment with leptin affected mir-628-5p target jag1 and other molecules involved in epithelial to mesenchymal transition. in parallel, we demonstrated that cortisol increases exosome secretion particularly in pc3 cell exosomes with a 2.6-fold increase at 5 nm cortisol compared to untreated. western blotting revealed the presence of gr in exosomes particularly at 5 nm cortisol. summary/conclusion: understanding epigenetic regulation through mirnas and exosomes may be the key to understand stress hormone influence in pca progression. these findings suggest that stress hormones effectively affect mir-628-5p expression and exosomal release and signalling.introduction: extracellular vesicles (evs) are promising drug delivery vehicles for therapeutic microrna (mirna). for the loading of exogenous cargo, researchers broadly seek to either manipulate the evs directly or the cell that produce them. electroporation, sonication, and direct ev transfection are common methods that work by physical disruption or irreversible chemical addition, which may irreparably damage the molecules intended for therapy. on the other hand, transfection into the producer cells is a simple option that does not imperil ev integrity.methods: there are multiple factors that contribute to ev loading efficiency, including transfection reagent used, timing, and dosage. thus, we sought to establish a basic protocol and improve understanding of the underlying dynamics involved in a basic system consisting of hek293 t cells and mir-146a-5p mimic.results: in this work, we examined how different reagents lead to variable ev loading. then we looked at variable dosages, specifically the relationship between rna amount added to reagent, amount present in cell, and amount exported to evs. summary/conclusion: these results will help future studies produce evs with exogenously loaded small rna, and suggest future optimizations. funding: national institutes of health. r01 and t32 (host pathogen interactions at university of maryland). we report a single ev trapping method via aptamermediated assembly between au nanoparticle (aunp) and au superlattice template. we propose a chip-based ev trapping technique based on semiconductor processes. methods: we introduce aptamer coated au nanoparticle (aunp) and au superlattices as a template to capture evs. first, we fabricated poly(methyl methacrylate) (pmma) hole pattern on au-coated si substrates by using electron beam lithography (ebl). we designed 200 nm-diameter hole patterns to capture one ev in each hole. to connect the aunp and the au superlattice template, we used an aptamer molecule as a linker strand. also, to capture individual evs, the aptamer molecule is designed to have a hairpin structure to specifically bind to cd63, a protein marker of ev. we modified 5ʹ-terminal and 3ʹ-terminal of the cd63 aptamer with thiol group for the formation of self-assembly monolayer (sam) on both aunp and au superlattice surface. results: first, we coat the cd63 aptamer on the surface of aunp. afterwards, we load the aptamer-coated aunp into au superlattice template. ev solution is specifically bound to cd63 aptamer. after washing step, each ev is expected to locate within a single hole due to the size confinement of the hole. to separate the evs from the aptamer, we use restriction enzyme, bamhi, to recognize specific dna sequence and cleave them. summary/conclusion: in this report, we propose a aunp -linked au superlattice chip by aptamer molecules for trapping evs. we selected cd63 aptamer for specifically binding with cd63 in evs. in addition, we designed cd63 aptamer as a linker strand to connect introduction: a hallmark of platelet activation is the release of internal granules as extracellular vesicles/ microparticles. thrombolux is a dynamic-light-scattering-based (dls) instrument that was developed for use in clinical setting to check for platelet activation before transfusion. compared to traditional dls, the thrombolux requires no cleaning (single-use capillary) and requires very little sample (70 µl). hence the thrombolux may be a useful instrument beyond platelet pack test in blood transfusion laboratory. we have evaluated its use as an in-process monitoring tool for industrial ev manufacturing, for both quantifying cells (input) and evs (output). methods: the thrombolux was used to test the activation status of expired platelet packs (donated by arcbs for research purpose). the readout was compared with platelet swirling test and flow cytometry data (surface marker). furthermore, the thrombolux was also tested for process development and ev manufacturing monitoring purposes at different stages of the process for its ability to rapidly obtain particle presence and size information on evs. time to result was also compared between different particle analysis methods. results: the thrombolux was a better predictor of platelet packs variability compared to the traditional platelet swirling method. however, we did not observe a strong correlation between the activation status and the flow cytometry-based activation marker data. the thrombolux was able to provide a useful estimation of particle presence and sizing of evs in-process.results are obtained rapidly, within minutes, with minimal sample prep. summary/conclusion: although we did not observe a significant direct correlation between flow cytometry activation data and the % microparticles (within a small sample size), the thrombolux has shown potential to become a useful tool for in-process monitoring for ev manufacturing and other ev research, in particular through its speed and ease of use. funding: all funding was through exopharm ltd (asx:ex1). secreted introduction: a major manufacturing challenge related to exosome bioprocessing is that of robust and scalable purification. as efforts to translate exosomes into clinics grows, the more important the design of quality systems which can reproducibly purify the product becomes. the current gold-standard, ultracentrifugation, was adopted from the viral vaccine industry, but remains imperfect in terms of scale up and manufacturing due to labour and time intensive process requirements. in order to follow the preferential adoption of more standard bioprocesses, as previously achieved by the viral vaccine industry, we show the development of two monolith chromatography steps which can be used to purify exosomes from a clinically relevant, allogeneic stem cell product (ctx0e03). methods: t-flask expansion of ctx0e03 cells was performed to yield batches of 5-15 l of conditioned medium. the medium was subsequently clarified by benchtop centrifugation, and concentrated into a crude concentrate by tangential flow filtration [tff], using a combination of 0.22 µm dead-end filtration prior to concentration in a 300kda hollow-fibre tff system. tff retentate was loaded onto 1 ml hic or aex monoliths, for further purification. potency was assessed by a fibroblast wound healing assay in vitro. results: exosome presence was verified in the tff material by detection of cd 81 and cd 63. exosomes recovered in this manner could achieve full wound closure in vitro over 72 hours, when dosed at 20 µg. further purification by monolith chromatography showed high levels of reduction of albumin, detected by western blot, as well as heightened ratios of particles to both total protein, and total dna. the results indicate that neither aex nor hic steps cause detrimental loss to product function, either alone or in combination with one another. introduction: custom-made platelet pellet lysate (ppl) and heat-treated ppl (hppl) exert strong neuroprotective effects of neurotoxin-exposed dopaminergic luhmes neuronal cell culture. this effect is significantly enhanced using hppl, which was also highly protective of th-expressing neurons in mice parkinson's disease (pd) model. introduction: there is a critical unmet medical need for new therapies to treat age-related diseases including cardiovascular diseases such as stroke. exosome derived from stem cells have shown intrinsic therapeutic potential in a variety of animal models of ischaemic diseases. we have identified scalable exosome production cell lines (purestem) as a source of angiogenic exosomes and are aiming to generate good manufacturing practice (gmp) grade therapeutic exosomes that can effectively mediate angiogenesis and tissue regeneration. we are developing exosome production and purification protocols that combine methods of tangential filtration flow (tff) and size exclusion chromatography (sec). the particle number and size were measured by both tunable resistive pulse sensing (trps) as well as nanoparticle tracking analysis (nta) for comparison. exosomes were characterized by detection of exosome surface markers and absence of cellular markers. purity was assessed by measuring particles per ug of total protein content. the angiogenic activity of purestem-exosomes was assessed using live-cell imaging to measure endothelial wound-healing and tube formation assays. we further investigated the molecular cargo of purestem-exosomes by screening mirnas targets, rna-seq analysis, and mass spectrometry analysis.results: the isolated purestem-exosomes using our developed protocols were highly purified, resulting purity in the range of 1e10-5e10 particles/ug. we selected angiogenic exosome-producing cell lines from our purestem library by screening for functional activity and characterizing their molecular cargo. we found that purestem progenitor-derived exosomes showed higher angiogenic potency than primary mesenchymal stem cell (msc)-derived exosomes. furthermore, angiogenic micrornas such as mir-126 were enriched in purestem-exosomes from certain producer cell lines. summary/conclusion: these data demonstrate the potential for using purestem lines as a highly scalable source of therapeutic exosomes. we were able to obtain highly pure exosomes that retain their angiogenic activity. we anticipate that purestem-exosomes will be a valuable resource for developing ev therapies for stroke and other ischaemic diseases. we have developed purification methodologies aimed at achieving a robust and scalable exosome production compatible with gmp for clinical grade purestem-exosomes. these developments have great potential as therapeutic agents for future preclinical in animal model of stroke and clinical trials. neuronal introduction: the hallmark of parkinson's disease (pd) is a-synuclein accumulation, predominantly in dopaminergic neurons, causing neurodegeneration. pd is also associated with insulin resistance, a condition characterized by phosphorylated insulin receptor substrate-1 (irs-1). besides motor symptoms, some pd patients develop mild cognitive impairment (pd-mci) or dementia (pd-d). given the importance for prognosis, there is an urgent need to develop biomarkers for distinguishing pd with normal cognition (pd-n) from pd-mci/d. neuronal-origin extracellular vesicles (nevs) contain cell signalling and pathogenic proteins (including a-synuclein), which may serve as biomarkers for alzheimer's disease, pd and other dementias.methods: from 0.5 ml of plasma from 104 pd-n, 83 pd-mci, and 39 pd-d patients, we immunocaptured nevs using anti-l1cam antibody. then, irs-1pser312 and irs-1ptyr20 and a-synuclein were measured in nevs using electrochemiluminescence immunoassays.results: a-synuclein was lower in pd-mci and pd-d compared to pd-n (p < 0.005) and significantly decreased with increasing motor symptom severity measured by mds-updrs iii score (p = 0.005). irs-1pser312 was lower in pd-d than in pd-n. irs-1ptyr20 significantly decreased with increasing mds-updrs iii score (p < 0.005). no biomarker was associated with disease duration. summary/conclusion: pd patients with cognitive impairment exhibited lower nev levels of a-synuclein than cognitively intact pd patients, whereas a-synuclein and irs-1ptyr20 were inversely associated with pd motor symptom severity. additional biomarkers and measurements will be available by the time of isev. plasma nevs is a valuable tool for discovering biomarkers in pd and investigating aspects of disease progression. introduction: despite decades-long advancement in transplant medicine, there is a necessity for personalized approach regarding early kidney allograft injury recognition and immunosuppression therapy towards improved transplant outcomes. biopsy, a gold standard for assessment of kidney allograft injury, cannot be serially used for the diagnosis of subclinical injury due to it's invasiveness and possible sampling errors. instead, urine is easily obtainable and bearing extracellular vesicles (evs), potential carriers of pathological signals related to kidney injury. our aim was to set up a urinary ev (uev) isolation protocol that would allow consistent and reliable identification of their characteristics and cargo. methods: second morning urine sample (25 ml) was collected from 7 patients and processed within 4 hours. oxalate precipitation, ph and dilution variability, uromodulin polymerization and high protein content were taken into account. isolated evs were defined by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). uev specific proteins and mirnas were analysed by western blot and qpcr, respectively. results: the optimal protocol relied on low speed urine centrifugation (2.000 x g, rt) for cell removal and storage at −80°c prior to further analyses. after urine thawing at rt, added edta averted cryoprecipitate and uromodulin polymer formation, while concentrated pbs neutralized the ph. filtration through 0.22 µm pores was used for large particle removal, while centrifugal 100 kda membrane units (amicon®, milipore) served for sample concentration followed by particle separation on sizeexclusion chromatography (sec; qevoriginal, izon q). protein vacant sec fractions (as rated at a280) were pooled and concentrated to a volume of 70 µl. tem micrographs revealed high sample purity and cup-shaped morphology of uevs. as per nta results, the average mean size of evs was 129,9 nm with concentration range of 1 × 109 particles/ml of starting urine. uevs were positive for the tested marker proteins hsc70, flotillin, tubulin, gadph and cd63. qpcr verified mirna presence in uevs, with ct for mir let-7i at 20. summary/conclusion: we successfully isolated pure uevs. the set up protocol will be used to assess uevs as non-invasive biomarkers of allograft injury in kidney transplant recipients. astrocyte-derived extracellular vesicles regulate dendritic spine formation and neuronal network connectivity introduction: recent advancements in the biology of extracellular vesicles have begun to implicate glial released microvesicles as mediators of glia to neuron communication, suggesting that alterations in the release and/or composition of astrocyte microvesicles could impact neuronal function. methods: astrocytes were allowed to constitutively release extracellular vesicles (adev-cr), or stimulated with atp (adev-atp). adevs were isolated by ultracentrifugation followed by proteomic analysis. we developed a normative whole transcriptome database using primary neurons exposed to adev-cr, and identified changes in neuronal gene expression produced by exposure of neurons to adev-atp. we identified a number of pathways associated with the biological response of synapse, spine and neurite outgrowth that were regulated by adev-atp. the molecular cargo of adev-atp responsible for regulating synaptic functions in neurons were characterized by biochemical, molecular, and functional assays. results: adev-atp enhanced the maturation of dendritic spines and produced functional enhancements in neuronal activity and network connectivity. the mechanism for this effect involved the delivery of integrin-4 1 and epha2 that were enriched in adev-atp. integrin-4 1 facilitated binding of adevs to the neuronal surface, and epha2-receptor signalled through ephrin to the tyrosine kinase erbb2/ 4 that regulated the phosphorylation and activation of trkb without increasing expression of the natural ligands bdnf or ntf3. this direct activation of trkb increased the expression of the synaptic scaffolding proteins disc1, arc, and cplx3 to promote the maturation of dendritic spines. this increase in mature dendritic spines was associated with increased neuronal activity and network connectivity demonstrating a functional strengthening of synapses. summary/conclusion: these data identify a molecular mechanism whereby modifications in adev protein cargo produced by the stimulation of astrocytes with atp regulates synaptic maturation through activation of trkb in a manner independent of growth factors. stephanie kronstadt and steven m. jay university of maryland, college park, college park, usa introduction: mesenchymal stem cell extracellular vesicles (msc-evs) have been shown to have an immunosuppressive effect in both autoimmune and inflammatory disorders. despite this, clinical translation of ev therapies is hindered by potentially low potency in vivo and the lack of a scalable biomanufacturing process. cell culture parameters are critical in modulating both yield and bioactivity of evs. thus, we hypothesized that the combination of chemical priming and 3d dynamic culture would enhance the yield and potency of immunosuppressive msc-evs. methods: bone marrow-derived mscs cultured in flasks were chemically primed using ethanol or curcumin. mscs were also cultured using a 3d-printed scaffold-perfusion bioreactor using a flow rate of 5 ml/min. anti-inflammatory effects were assessed following application of msc-evs to lipopolysaccharide (lps)-stimulated murine macrophages. subsequent inhibition of the production of the pro-inflammatory cytokine il-6, quantified using an elisa, was used to characterize evs as anti-inflammatory. in addition, both chemical priming and the bioreactor will be simultaneously utilized to potentially uncover any synergistic effects on ev immunomodulation abilities. nanoparticle tracking analysis (nta) was used to assess ev size and concentration while protein mass was measured via a bca assay. results: preliminary data suggests that priming mscs with 100 µm ethanol for 24 hours prior to ev collection results in a strong inhibition of il-6 production in stimulated murine macrophages. nta revealed that msc-ev yield increased by about two orders of magnitude in the bioreactor (1.40e12 ± 7.92e10) when compared with flasks (2.28e10 ± 2.81e9). protein measurements also indicated that ev production in the bioreactor (~7600 µg) was much greater compared with production in the flasks (~2400 µg). additionally, average protein content per ev was reduced in the bioreactor when compared with flask evs. regardless of tissue source. furthermore, comparison of adipose tissue-derived (ad) msc evs from three donors indicates varying pro-vascularization bioactivity between those donors evaluated in vitro via gap closure assay. similar results were observed for the bone marrow-derived (bm) msc ev donor groups. summary/conclusion: this work highlights the need for screening of donor derived-mscs before use for therapeutic ev production. additionally, standardized criteria for msc donor selection are needed before isolated msc evs can be used as a large-scale, repeatable therapeutic treatment. analysis of extracellular vesicle populations from malaria-infected erythrocytes by field-flow fractionation reveal distinct sub-sets alicia rojas a , paula abou-karam a , anna rivkin a , yael fridmann-sirkis b , yifat ofir-birin c and neta regev-rudzi c a department of biochemical sciences, weizmann institute of sciences, rehovot, israel, rehovot, israel; b wis, rehovot, israel; c weizmann institute of science, rehovot, israel introduction: malaria is one the most devastating infectious disease in the world and plasmodium falciparum (pf) represents the deadliest species. this parasite invades human red blood cells (rbcs) and releases extracellular vesicles (evs) carrying dna, rna and protein cargo components which are involved in the pathogenesis of the disease. recently, it has been shown in mammalian systems that evs are subdivided into different subpopulations, each with a distinct biological function. however, it is still unknown whether pfinfected rbcs (pf-evs) release different ev subpopulations with distinct cargo. methods: we isolated evs from pf-infected and uninfected rbcs, pf-evs or ui-evs, respectively, using differential centrifugation. the ev pellet was subjected to field flow fractionation (fff). the different subpopulations were collected, concentrated with size-exclusion filters and evaluated by nanoparticle tracking analysis. additionally, the presence of ev markers (sr1 and hsp90) were examined by western blot analysis. results: the fff analysis showed four particle subpopulations derived from the pf-evs and five in the ui-evs. the first three subpopulations were similar in their detection signals in both samples, but the fourth subpopulation was consistently higher in ui-evs than in pf-evs. moreover, hsp90 was detected in subpopulations 3 and 4 of both pf-evs and ui-evs, whereas sr1 only in subpopulation 3. isev2020 abstract book summary/conclusion: pf-ev and ui-ev have similar separation profiles and proteins markers in their subpopulations, consistent with the fact that both samples are derived from host rbcs. additional data regarding the dna and rna cargo, as well as microscopic observations of the pf-ev and ui-ev subpopulations is necessary. this will clarify how malaria parasites sort their components into evs and which fractions are associated to immune evasion and pathogenesis. we have established a small size laboratory production of the microalgae culture in order to harvest the extracellular vesicles (evs) for pharmaceutical and medical uses. in this work we report on globular particles in the isolates from media of microalgae of two types, that we recognize as evs. we observed changes in their production at different temperatures and conditions. methods: samples were fixed by various combinations of aldehyde fixatives and/or osmium tetroxide. they were dehydrated in a graded series of ethanol, hexamethyldisilazane, and air dried. they were au/pd coated for inspection with scanning electron microscopes (sem) crossbeam 550 fib-sem gemini ii (zeiss, germany) and jsm-6500 f field emission scanning electron microscope (jeol ltd., tokyo, japan). results: microalgae were incubated overnight at 22°c and 37°c in growth medium and in growth medium supplemented with detergent. the samples obtained from the microalgae culture contained particles that we recognized as extracellular vesicles, however, these particles do not correspond to characteristic shapes of membrane enclosed entities without internal structure. increased temperature and/or presence of surfactant (triton x-100 and sodium dodecyl sulphate) stimulated formation of evs of different shapes and sizes. the isolates of these samples were rich with evs. in the presence of surfactant, the cell-walls detached from the cell and collapsed upon dehydration. this was documented by sem. summary/conclusion: focused ion beam technique revealed complex internal structure of the algae. it seems from the shapes of the observed structures that the particles deposited on the surface of the microalgae do not derive from budding of the membrane surface, but are instead shed by the cells from the cell interior upon the rupture of the cell wall. key: cord-308043-h0knm8y4 authors: hussey, séamus; travassos, leonardo h.; jones, nicola l. title: autophagy as an emerging dimension to adaptive and innate immunity date: 2009-08-31 journal: seminars in immunology doi: 10.1016/j.smim.2009.05.004 sha: doc_id: 308043 cord_uid: h0knm8y4 abstract autophagy is an evolutionary conserved cellular process during which cytoplasmic material is engulfed in double membrane vacuoles that then fuse with lysosomes, ultimately degrading their cargo. emerging evidence, however, now suggests that autophagy can form part of our innate and adaptive immune defense programs. recent studies have identified pattern recognition molecules as mediators of this process and shown that intracellular pathogens can interact with and even manipulate autophagy. recent translational evidence has also implicated autophagy in the pathogenesis of several immune-mediated diseases, including crohn disease. in this review, we present autophagy in the context of its role as an immune system component and effector and speculate on imminent and future research directions in this field. throughout evolution, facets of our cellular biology have been conserved and adapted. one process at the forefront of homeostasis and environmental interaction is autophagy. this complex process in eukaryotic cells involves the trafficking of cellular elements from the cytosol to the lysosome wherein they are degraded and processed. ongoing developments in this field point to an inextricable link between autophagy and the innate and adaptive immune system. in this review, we summarize the pertinent research findings to date and suggest future research directions in this dynamic field, especially with respect to pattern recognition receptor interaction. three sub-types of autophagy have been described-chaperonemediated autophagy, microautophagy and macroautophagy (hereafter called autophagy). the term 'autophagy' was first suggested by de duve over 45 years ago [1] . lamellar vesicles that encapsulated portions of the cytosol and organelle remnants had been described in early electron microscopy studies as vacuoles and lysosomes, and were speculated to arise from focal cytoplasmic degradation [2] [3] [4] . such vesicles bore the hallmarks of what are now termed 'autophagosomes', the characteristic vacuoles synonymous with autophagy. metabolic manipulation was shown to affect autophagy induction, demonstrating that autophagy was a malleable rather than a static process. the catabolic hormone glucagon and deprivation of amino acids and nutrients were shown to induce autophagy while insulin and certain exogenous amino acids impaired autophagy and proteolysis, defining a role for autophagy in adaptation to cellular stresses [2, [5] [6] [7] [8] [9] [10] [11] . the complexity of signaling molecules that influence autophagy is an ongoing focus of research and will be discussed later. the stepwise process of autophagosome biogenesis is a cornerstone of autophagy. over thirty governing autophagy genes (atg) and their proteins (atg) have been identified in elegant studies in yeast species [12] [13] [14] . while not all mammalian orthologs have been identified, some have numerous mammalian paralogs with striking similarities in structure and/or function to their yeast counterparts [15, 16] . ultra-structural studies of autophagosome membranes have shown that they harbor relatively few transmembrane proteins and are thinner than other cellular membranes e.g. the plasma membrane [17] . the earliest identifiable structure in the sequence of autophagosome formation is the diskshaped, isolation membrane or phagophore (fig. 1) . once formed, this membrane progressively elongates, encircling its cytosolic target, e.g. bacterium, within a portion of the cytosol, eventually sealing to complete the autophagosome. speculation continues whether the foundation template for the isolation membrane originates from the endoplasmic reticulum, golgi, mitochondria, a pre-formed organelle membrane or even de novo [18] [19] [20] [21] [22] [23] [24] [25] . the molecular mechanisms that lead to isolation membrane appearance continue to be elucidated in both yeast and mammalian cell systems. two ubiquitin-like conjugation systems are pivotal to autophagosome formation and completion. the first system modifies a core autophagy protein-microtubule-associated protein 1 light chain 3 (lc3). multiple paralogs of atg8 exist in mammals -lc3a, lc3b, gate16, gabarap -hereafter referred to collectively as lc3 [26, 27] . lc3 has a diffuse cytosolic distribution. it is cleaved at its c-terminus by the cysteine protease atg4 and in turn undergoes sequential ubiquitin-like modifications by the e1-like enzyme, atg7, and the e2-like enzyme, atg3, to form lc3-1. the c-terminal carboxyl group of lc3-i is ultimately conjugated to the amine of phosphatidylethanolamine, forming lc3-ii. this lipidation of lc3-i to form lc3-ii is notable in that lc3-ii is exclusively found on autophagosome membranes. the conjugated yeast ortholog of lc3, atg8, is known to have membrane tethering properties, which may explain one of its roles in autophagosome formation [28] . atg4 also deconjugates lc3-ii on the autophagosome membrane, releasing lc3, highlighting the plasticity of this process. the multifunctional protein p62 interacts with both ubiquitinated proteins and lc3, whereby it is incorporated into autophagosomes. p62 accumulates during autophagy inhibition and has been implicated in targeting proteins to autophagosomes, although it is not itself essential for autophagosome formation [29] . in the second conjugation system, the ubiquitin-like autophagy protein atg12 is covalently conjugated to atg5 via its c-terminal glycine, forming the dimeric atg12-atg5 complex, following ubiquitin-like reactions involving atg7 and atg10. the autophagy scaffold protein atg16l1 is then conjugated to atg5 via its nterminus, forming the atg12-atg5-atg16l1 complex. the atg16l1 complex self-multimerizes, forming large 800 kda complexes. these are found in the cytosol and on the evolving isolation membrane, and are likely necessary for the ultimate conjugation of lc3-i. fujita et al. showed that the atg16l1 complex behaves as an e3-like enzyme and targets lc3-i to its membrane site of lipid conjugation [30] . the atg16l1 complexes dissociate from autophagosome membranes as they near completion. other essential groups of autophagy proteins participate in isolation membrane formation. the mammalian autophagy proteins ulk1 (unc-51-like kinase), fip200 (focal adhesion kinase family interacting protein) and atg13 were recently identified in a complex which subsequently co-localized at the nascent isolation membrane on autophagy induction, similar to the atg1-atg13-atg17 complex in yeast [31] . the c-terminus of ulk-1 binds to fip200 and atg13, and ulk-1 also interacts with lc3 [32] . mammalian studies of the trans-membrane protein atg9 have similarly underscored its essential role early in autophagosome formation. it associates with the trans-golgi network, late endosomes, lc3, the rab-gtpase proteins (rab7 and rab9) and re-distributes following autophagy induction, localizing to the nascent autophagosome [33] . yeast atg9 was recently shown to self-multimerise via its c-terminus, facilitating its intra-cellular trafficking, independent of other autophagy proteins. this novel finding suggests a potential further role for such atg9 complexes in contributing to early isolation membrane formation [25] . the discovery of the target of rapamycin in yeast (tor) and mammalian cells (mtor) led to significant advances in understanding autophagy regulation, through the family of phosphatidylinositol kinase-related kinases [34] [35] [36] . these signaling networks are involved in broad cellular functions from metabolic responses to growth and proliferation. the key serine/threonine kinase, akt, links the mtor and phosphatidylinositol-3 kinase (pi3k) pathways which are activated by a diverse array of stimuli, including cytokine receptors and toll-like receptors (tlr) [37] . following receptor activation, class-i pi3ks are recruited by receptor adaptor molecules to phosphorylate phosphatidylinositol-4,5-bisphosphate, which in turn phosphorylates and activates akt [38, 39] . the mtor complexes, down-stream positive effectors of akt, integrate multiple cellular signals, including those from growth factors, amino acids and intracellular atp. mtor activation increases cellular anabolic activity and protein translation [40] [41] [42] . autophagy is under negative regulation by activated akt and mtor [43] . recently, mtor was shown to phosphorylate and therefore inhibit the ulk kinase-complex activity, disrupting autophagosome formation [44, 45] . rapamycin inhibition of mtor and amino acid deprivation reversed these effects. mtor may further affect autophagy through its control of autophagy gene transcription [40, 46] . the class iii pi3k enzyme, vps34 (vacuolar protein sorting 34), solely phosphorylates phosphatidylinositol and is involved in regulating vesicular trafficking, nutrient sensing and autophagy [47, 48] . the pharmacological agent 3-methyadenine (3-ma) inhibits its function in vitro. together with vps15 (another kinase), beclin-1, uvrag (ultraviolet radiation resistance associated gene) and ambra-1, vps34 forms a multiprotein complex that is necessary for early stages of autophagosome biogenesis and can up-regulate autophagy overall [49] [50] [51] . however, its seemingly paradoxical role in signal transduction to the mtor complex following amino acid sensing suggests that its signaling function may depend on the nature of its interacting protein complexes [52, 53] . beclin-1, a tumor suppressor protein, is itself also involved in modulating autophagy through its interaction with bcl-2, an anti-apoptotic protein that inhibits both autophagy and apoptosis. the beclin-1/bcl-2 interaction is an evolutionary conserved phenomenon, the balance of which determines either up-or downregulation of autophagy. silencing or over-expression of bcl-2 was shown to enhance or suppress starvation-induced autophagy respectively [54] . these effects were specifically dependent on beclin-1/bcl-2 interaction, suggesting that nutrient sensing affects the equilibrium of the beclin-1/bcl-2 interaction. bcl-2 dominant interactions with beclin-1 likely disrupt beclin-1/vps34 complex formation, leading to autophagy suppression, although the mechanism has not been fully elucidated. recently, the toll-like receptor (tlr) signaling molecules myd88 and trif were shown to modulate the beclin-1/bcl-2 interaction, enhancing their interaction with beclin-1 to induce autophagy [55] . a myriad of other signal transduction and effector molecules influence autophagy regulation, directly or indirectly. the akt and jnk pathways have been shown to enhance or reduce expression of lc3 and beclin-1 in response to tumor necrosis factor-␣ (tnf-␣) and insulin-like growth factor-1 respectively [56] . the mammalian transcription factor, nfb, is a key regulator of gene expression, modulating physiological processes including inflammation, apoptosis and also autophagy. tnf␣-induced nfb activation suppresses autophagy, while nfb suppression enhances starvation-induced autophagy [57, 58] . nfb may signal through mtor activation or by affecting enhanced bcl-2 expression to modulate autophagy. autophagy itself may in turn influence nfb activity since it is involved in degradation of ib kinase, the upstream activator of nfb, through association with the heatshock protein, hsp90 [59, 60] . reactive oxygen species (ros) are highly reactive molecules generated from mitochondrial respiratory activity and the products of oxidase enzymes, including nadph oxidase, and are capable of modulating autophagy [61, 62] . atg4 is redox-regulated via a conserved cysteine residue and, furthermore, starvation-induced autophagy depends on h 2 o 2 signaling [62] . starvation lead to local h 2 o 2 formation, partly dependent on class iii pi3k activity, and anti-oxidant treatment in vitro attenuated autophagy induction. recently, a transgenic mouse model harboring a mutant form of super-oxide dismutase, a key anti-oxidant enzyme, also showed increased autophagic activity due to ros accumulation [63] . evidence also suggests that autophagic (type ii) cell death may stem from ros accumulation, as seen following in vitro treatments with tnf␣ and lps [57, 64] . microbial invasion of the cytosol presents a serious challenge to our innate defenses, including autophagy. while several agents succumb to autophagic destruction (xenophagy), others have evolved mechanisms of autophagy evasion and manipulation. various gram+ and gram− bacteria, viruses and protazoa are known autophagy targets ( table 1 ). the mechanisms by which microbes are selectively sequestered in autophagosomes remain elusive and a combination of host and microbial factors are likely to be necessary. microbial molecular motifs themselves may solicit autophagosome formation. alternatively, the up-regulation of autophagy through activating multiple pattern recognition receptors could culminate in xenophagy or perhaps organelle or compartmental damage may lead to targeting by autophagic machinery. microbial factors may be of equal importance for autophagy activation. for example, group a streptococcus (gas) is sequestered in autophagosomes following escape from its early endosomal compartment into the cytosol [65] . lysosomal degradation of bacteria-containing autophagosomes ensues, effects not observed in autophagy deficient cells. strains of gas lacking the streptolysin o toxin remain within endosomes and avoid autophagic destruction indicating a role for streptolysin o in induction of autophagy. the gram− human diarrheal agent shigella flexneri is a highly adapted pathogen harboring a type iii secretion system (ttss) for delivery of its effector proteins to host cells. in epithelial cells, wild-type (wt) strains secreting the effector icsb are capable of evading entrapment in autophagosomes, in comparison to mutant strains lacking icsb [66] . interestingly, icsb did not appear to confer autophagy protection in a subsequent study in murine marrow derived macrophages, suggesting a cell-type specific phenomenon [67] . the intracellular bacterium burkholderia pseudomallei, also avoids autophagic destruction in murine macrophages through secretion of its ttss-delivered effector protein bopa, which shares some homology with icsb [68] . salmonella enterica serovar typhimurium resides within salmonella-containing vacuoles following intracellular invasion. salmonella employs its ttss to disrupt these vacuoles, facilitating cytoplasmic entry. autophagy promptly contributes to subsequent restriction of intracellular proliferation by targeting bacteria from damaged vacuoles-effects that were dependent on a functioning ttss and reversed in autophagy deficient cells [69, 70] . listeria monocytogenes, a gram+ bacillus, replicates within the host cytoplasm following phagosome escape, evading autophagic destruction [71, 72] . the virulence factors listeriolysin o, acta and phospholipase c were recently shown to be of importance in modulating listeria-containing phagosomal compartments, blocking lysosomal degradation and facilitating replication and survival [73, 74] . mycobacterium tuberculosis has adapted to survive within host macrophages by interfering with and blocking phagosome fusion with lysosomes [75] . autophagy up-regulation with rapamycin or ifn-␥ overcame this evasion, and lead to phagosome degradation [76, 77] . secreted bacterial toxins are themselves capable of interacting with the autophagy pathway. the non-invasive pathogen, vibrio cholerae, causes a potentially fatal secretory diarrhea. its secreted exotoxin, vcc, induces vacuole formation consistent with autophagy induction [78] . furthermore, cell viability was adversely affected following autophagy inhibition, suggesting that in this situation, autophagy may defend against cell toxicity. our group has recently reported autophagy induction following infection with helicobacter pylori, which was dependent on the vacuolating cytotoxin, vaca. autophagy limited the stability of intracellular vaca, again suggesting a cytoprotective function of autophagy in response to secreted toxins [79] . viruses also interact with autophagy. the herpes virus hsv-1 evades autophagy in part through beclin-1 inhibition by its neurovirulence protein icp34.5 [80] . poliovirus manipulates autophagic machinery following cellular infection, as evidenced by a marked reduction in viral release following pharmacological inhibition of autophagy and sirna silencing of key autophagy proteins [81] . rotavirus has been suggested to harness the autophagic apparatus to facilitate replication. its enterotoxin, nsp4, was found to co-localize with lc3+ structures on immunofluorescence microscopy, and the study authors speculate that nsp4 may interfere with autophagosome-lysosome fusion, enabling viral recruitment of autophagosomes as replication niches [82] . the antiviral protein kinase, pkr, participates in viral induced autophagy, functioning upstream of beclin-1. it is possible that other viruses which inhibit pkr function, including influenza and ebstein-barr virus, may in turn inhibit autophagy to enhance their own survival [83, 84] . the possibility of a cell-type dependent autophagy response to viral infection was suggested by coronavirus studies, wherein mouse hepatitis virus replication was impaired in atg5 −/− stem cells, but not in atg5 −/− embryonic fibroblasts or marrow derived macrophages [85] [86] [87] . these diverse examples of autophagy-microbial interactions underpin the conserved primary innate role of autophagy as an anti-microbial, protective mechanism and how certain pathogenic organisms have evolved to recognize and commandeer this process for their own advantage. the innate immune system is responsible for the early detection and destruction of pathogens. this first line of defense relies mostly on a set of receptors called pattern recognition molecules (prm) that sense molecular motifs that are common to a wide range of pathogens, triggering different signaling cascades that culminate with the elimination of pathogens and the initiation of an adaptive response [87, 88] . the findings that autophagy can specifically target cytosolic pathogens immediately prompted the investigation of the role of prm in the autophagic detection and elimination of intracellular microbes. the tlrs are transmembrane proteins, mostly located at the cell surface, with a toll-il-1 receptor (tir) domain facing the cytosol. this domain is able to recruit four different adapter molecules: the myeloid differentiation primary response protein 88 (myd88), the tir domain-containing adaptor protein (tirap, also called myd88 adaptor-like-mal), the tir domain-containing adaptor-inducing ifn-␤-trif, also called tir-domain-containing adaptor molecule 1-ticam-1) and the trif-related adaptor molecule (tram or ticam2) [88, 89] . as we will see in this section, recent data suggest that induction of autophagy after tlr engagement requires the recruitment of specific adaptors (fig. 2) . eissa and colleagues provided the first evidence that tlrs are able to trigger an autophagic response by showing the formation of numerous autophagosomes in response to lps stimulation in the murine macrophage raw264.7 cell line [90] . furthermore, silencing tlr4 using rna interference resulted in significant reduction in autophagosomes. the tlr4-induced autophagic response was dependent on p38, rip1 and trif-, but not myd88. as tlr4 can use fig. 1 . autophagosome biogenesis. the earliest identifiable structure in the initiation (nucleation) sequence of autophagosome formation is the crescent-shaped isolation membrane or phagophore. key elements include atg9, the ulk1-fip200-atg13 complex, lc3-ii, the atg12-atg5-atg16l complex. once formed, this membrane progressively elongates (elongation), encircling its cytosolic target, e.g. bacterium, within a portion of the cytosol. the membrane tips fuse and eventually seal, forming the autophagosome (completion). the autophagosome may fuse with the endosomal compartment, forming an amphisome, prior to its ultimate maturation step, whereby its outer membrane fuses with the lysosome to form an autolysosome (also termed autophagolysosome). this facilitates degradation, processing and recycling of the contents of the autophagosome. both myd88 and trif adapter molecules for downstream signaling, the authors proposed that by recruiting both signaling cascades, tlr4 could promote both a fast phagocytic response (through myd88) and a slower autophagic response (via trif). other tlr family members have also been implicated in the control of autophagy (fig. 2) . deretic and colleagues have recently demonstrated that when raw264.7 macrophages were stimulated with a panel of tlr ligands such as pam 3 csk 4 (tlr2), flagellin (tlr5), cpg dna (tlr9), poly (i:c) (tlr3), lps (tlr4) and ssrna (tlr7), the latter three were able to up regulate autophagy [91] . in contrast to tlr3 (that recruits only trif) and tlr4 (that recruits both myd88 and trif), tlr7 recruits only myd88, suggesting that myd88 may trigger autophagy after tlr7 activation. however, tlr9 activation by cpg dna also activates myd88 but did not induce autophagy. therefore, a simple analysis of which downstream adaptor protein is recruited by tlrs does not fully explain the induction of autophagy by some pathogen associated molecular patterns (pamps) and not others. the mechanistic explanation is still elusive and seemingly conflicting evidence remains difficult to reconcile. for example, tlr7 recruitment of myd88 also leads to the activation nfb, which is thought to inhibit autophagy [57] . trif-dependent signaling leads to the induction of type i interferon, which was previously shown not to affect autophagy [76, 88] . two recent studies have proposed a mechanism by which tlrs might regulate autophagy. kehrl and shi demonstrated that not only trif, but also myd88 targets beclin1 and reduces its binding to bcl-2, upon stimulation with an array of tlr ligands [54, 55] . alternatively, wagner and colleagues observed that tlr activation leads to the activation of mtor, which in turn interacts with the adaptor proteins myd88 and interferon-regulatory factors (irfs) 5 and 7, thus controlling the transcription of cytokines such as tnf-␣, il-10, il12, type i interferons but, surprisingly, not il-1␤ [92] . these lines of evidence suggest a more elaborate tlr control of autophagy whereby tlr-adapter molecules interact with proteins from the autophagic pathway rather than by simply activating the classic hierarchical signaling cascades described heretofore. in contrast with the general notion that tlr ligands up regulate autophagy, green and colleagues suggested a model in which some tlrs, when engaged by their cognate ligands, usurp the autophagic pathway, recruiting lc3 to the phagosome membrane instead of forming classic autophagosomes [93] . however, as pointed out by the authors, it is not possible to exclude the possibility that the lc3 recruited to phagosomes has its origin in rapidly forming autophagosomes. if confirmed, these data would have a deep impact on the understanding on the role of autophagy in the enhancement of antigen presentation for example. pamp recognition as an autophagy trigger seems to be an evolutionary conserved feature. in drosophila, peptidoglycan-recognition protein (pgrp) family members sense peptidoglycan (pg) from gram-negative bacteria [94] . one of the pgrp family members, pgrp-le was recently implicated in pg sensing and induction of autophagy upon infection with l. monocytogenes thereby leading to clearance of bacteria [95] . cytosolic prms have also been implicated in regulation of autophagy. suzuki and colleagues demonstrated that ipaf, a nod-like protein previously shown to sense flagellin, down regulates autophagy during infection with the non-flagellated bacterium s. flexneri [67] . the down regulation of autophagy did not involve the asc adapter protein, normally required for the induction of il-1␤ after ipaf activation. one can speculate that nod proteins, which sense pg in mammalian cells, may play a similar role in the regulation of autophagy. up-coming studies addressing this question are eagerly awaited. in summary, the data above suggest a dynamic interaction between receptors from the innate immune system and regulation of autophagy. additional studies with knockout mice are now needed in order to demonstrate a definitive role for tlr-or nlr in autophagy during infection with pathogens known to activate specific prms. fig. 2 . tlr activation triggers autophagy. lps triggers autophagy after recruitment of trif (also rip1 and p38, not shown) and myd88. the latter seems to interact with beclin-1, reducing its binding to the anti-autophagic molecule bcl-2. tlr2 engagement induces the incorporation of lc3 to phagosomes (unkown mechanism). viruses are able to induce autophagy through tlr3, rig-i (dsrna) or tlr7/8-myd88 (ssrna). conventional dcs sense viral ligads through the rig-i/mavs axis to secrete type i interferon, while the conjugate atg5/12 seems to be a down regulator of such response. plasmocytoid dcs deliver tlr7 ligands from the cytosol to the compartments containing tlr7 using basal autophagy. ipaf inhibits autophagy through an unclear mechanism. in the last few years, autophagy induction has been frequently reported as a consequence of innate immune system activation. however, there is compelling evidence that the relationship between autophagy and the immune system is reciprocal. cytokines from the innate and adaptive systems regulate autophagy by different mechanisms. two of the prototypical th1 cytokines, ifn-␥ and tnf-␣, were shown to up-regulate autophagy. gutierrez and colleagues first demonstrated that mouse macrophages harboring mycobacterium within phagosomes were able to clear bacteria after stimulation with ifn-␥ in an autophagy-dependent manner [76] . follow up studies implicated gtpases in this process. the mouse genome contains 23 different immunity-related gtpases, most of which respond to ifn-␥ stimulation and play a role in the defense against intracellular pathogens via a mechanism which is as yet unclear [96] . the studies from deretic's group showed that both mouse immunity-related gtpase (irgm1) and its human ortholog irgm are the key molecules driving the induction of autophagy upon ifn-␥ stimulation, leading to the clearance of mycobacterium from infected macrophages [76, 97] . the other th1 cytokine shown to stimulate autophagy is tnf-␣. codogno and colleagues observed that cells stimulated with tnf-␣ are committed to die when nfb is blocked [57, 59, 60] . these findings are of great interest as the activation of autophagy may represent a way to overcome the resistance of cancer cells to anticancer drugs targeting nfb. in contrast to the autophagy enhancing effect of some th1 cytokines, th2 cytokines such as il-4 and il-13, seem to counteract starvation and ifn-␥-induced autophagy by different pathways [97] . while il-4 and il-13 block starvation-induced autophagy by activating the akt-mtor axis, these cytokines inhibit ifn-␥induced autophagy in an akt-independent but stat6-dependent manner. the regulation of cytokine secretion by autophagy, has also been reported. jounai and colleagues demonstrated that in response to infection with rna viruses or immunostimulatory rna, ifn-␤ levels were increased in atg5 knockout embryonic fibroblasts [98] . the authors demonstrated that the atg5-atg12 conjugate negatively regulates the antiviral immune response by interacting with the rig-i-like receptor (s protein retinoic acid-inducible gene i (rig-i) and ifn-␤ promoter stimulator 1 (ips-1) thus, implying autophagy contributes to viral replication. iwasaki and colleagues showed that in autophagy-impaired cells the increased cytokine secretion in response to immune-stimulatory rna is due to the accumulation of defective mitochondria and consequent ips-1 and ros accumulation, further strengthening the importance of autophagy in the maintenance of cellular homeostasis [99] . autophagy has also been proposed to regulate cytokine secretion in crohn disease. crohn disease (cd) is a chronic inflammatory intestinal disease with a complex and multifactorial etiology. several recent independent genome wide association studies have implicated a number of heretofore unappreciated biological pathways in cd pathogenesis, including autophagy [100] [101] [102] [103] . since the identification of a non-synonymous single nucleotide polymorphism in the atg16l1 gene as a causal risk variant for cd, several groups have sought to elucidate its functional impact on development of cd. akira and colleagues generated mice lacking the coiled-coil domain of atg16l1 and observed aberrant il-1␤ secretion upon lps stimulation of fetal derived liver macrophages. in contrast to previous studies, lps did not induce autophagy in control macrophages indicating the enhanced il-1␤ was not due to disruption of lps-mediated autophagy [104] . chimeric mice with atg16l1-deficient hematopoietic cells had an unremarkable baseline intestinal phenotype, but displayed increased susceptibility to dss-induced colitis compared with controls. even though this study used mice expressing a truncated form of atg16l1, rather than the atg16l1 risk allele, the results point to the importance of functional autophagy machinery for normal intestinal function. using an alternative mouse model hypomorphic for atg16l1 protein expression, cadwell and colleagues noted paneth cell-specific abnormalities including degenerating mitochondria, loss of lysozyme granule integrity and absence of apical microvilli [105] . parallel findings were observed when intestinal atg5 expression was suppressed. transcriptional profiling analysis revealed that, among other differences, transcripts for the adipocytokines leptin and adiponectin were highly enriched. similar increased expression profiles were observed previously in patients with cd [106, 107] . the above findings, while not specific to atg16l1 suppression, underscore the importance of autophagy pathway integrity to normal paneth cell function. interestingly, atg7 knockout of pancreatic islet cells resulted in abnormal cellular morphology on em, including mitochondrial swelling, distension of the endoplasmic reticulum and a paucity of insulin granules when compared with controls [108] . it remains unclear why paneth cells, above others, are susceptible to autophagy interference and how autophagy is involved in maintaining integrity of its lysozyme exocytosis pathway. however, the interaction between autophagy and multivesicular body biogenesis may provide a potential explanation for abnormal granule formation and exocytosis. once again, the paneth cell is placed at the convergence of several innate immune pathway aberrations and cd pathogenesis. translational clinical data are keenly awaited. the products of the two main cellular degradation systems -the proteasome and the lysosome -are not merely unwanted material but are, instead, key molecules utilized to instruct the immune system. this instruction step is achieved by the presentation of these products to cells from both innate and adaptive immune systems. cd8 + t cells monitor mainly cytosolic and nuclear antigens degraded by the proteasome (a large cytosolic enzyme complex) and loaded into mhc class i. in contrast, cd4 + t cells respond to extracellular or membrane peptides generated by lysosomal degradation and presented in the context of mhc class ii at the cell surface [109, 110] . however, this paradigm has been challenged by the demonstration that dendritic cells (dcs) are also capable of presenting extracellular antigens on mhc class i, and not just on mhc class ii as initially thought, through a mechanism called crosspresentation. cross-presentation allows dcs to instruct also cd8 + t cells, generating a more efficient t-cell response [111] . functional evidence for the presentation of endogenous antigens on mhc class ii was first provided by long and colleagues, who demonstrated that measles and influenza antigens could be presented in the context of mhc class ii [112, 113] . indeed, the affinity purification of mhc class ii from epstein-barr virus (ebv)transformed b lymphoblastoid cells, murine b cell lymphoma and myeloid cells showed that more than 20% of natural mhc class ii ligands had their origin in intracellular proteins [109] . together these studies suggested that an alternative and unknown route could deliver antigens from the cytosolic compartment for presentation on mhc class ii. knecht and colleagues were the first to suggest a role for autophagy in this process by showing that glyceraldehyde-3-phosphate dehydrogenase, an important source of human mhc class ii ligands, is degraded via chaperone-mediated autophagy [114] . additionally, peptides from two atg8 homologues, lc3 and gabarap, have been isolated from human and mouse mhc class ii molecules, respectively, providing further support to the notion of autophagy as an alternative route for delivery of cytosolic antigens for mhc class ii. more direct evidence came from studies using pharmacological inhibition of macroautophagy with pi3k inhibitors (such as 3-ma and wortmannin), which are thought to block the sequestration step of autophagy. stockinger and colleagues demonstrated that over-expressed c5 was processed and loaded onto mhc class ii in an autophagy-dependent manner, as the loading was decreased in the presence of 3-ma [114] . a similar approach was used to show that an endogenously expressed bacterial peptide, neor (neomycinphosphotransferase ii), was sequestered in autophagosomes and processed in endosomal/lysosomal compartments for loading onto mhc class ii. brossart and colleagues also used pharmacological inhibition to demonstrate that dcs electroporated with rna coding for the tumor-associated antigen muc-1 requires not only lysosomal antigen degradation and processing, but also autophagy in order to prime cd4 + t cells [115] . further evidence that autophagy contributes to mhc class ii presentation came from munz and colleagues, in which they analyzed the endogenous mhc class ii processing of the nuclear antigen 1 (ebna 1) from ebv, the dominant ebv-latent antigen for cd4 + t cell. inhibition of autophagy by atg12sirna in ebv-transformed b cells reduced recognition by ebna1-specific cd4 + t cells [116] . in another study, the same group demonstrated that the fusion of influenza matrix protein 1 (mp1) with atg8/lc3 drives this molecule to autophagosomes in different cell types and enhances recognition by antigen specific cd4+ t cells [117] . knockdown of atg12 confirmed that the localization of the fusion proteins with mhc class ii molecules was dependent of autophagy. importantly, these results represent great potential for vaccine design, since targeting antigens to autophagosomes induces a more robust t cell response. in support of this contention, jagannath et al. demonstrated that induction of autophagy enhances bcg vaccine efficacy in a murine model [118] . in contrast to model or viral antigens, very little is known about bacterial antigens requiring autophagy for proper presentation on mhc class ii. so far, only the 85b antigen from m. tuberculosis was shown to be presented more efficiently on mhc class ii upon induction of autophagy [119] . accordingly, atg6 silencing dampened this process, while rapamycin treatment enhanced priming of 85b-specific cd4 + t cells, strongly suggesting a role for autophagy in mhc class ii presentation of antigens of bacterial origin. even though studies with other bacterial models are lacking, it is possible to speculate a role for autophagy in mhc class ii presentation during infections with bacteria that escape from phagosomes, such as l. monocytogenes autophagy is steadily emerging 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enhanced by autophagy protective role of autophagy against vibrio cholerae cytolysin, a pore-forming toxin from v. cholerae effect of helicobacter pylori's vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells hsv-1 icp34, 5 confers neurovirulence by targeting the beclin 1 autophagy protein subversion of cellular autophagosomal machinery by rna viruses rotavirus nsp4 induces a novel vesicular compartment regulated by calcium and associated with viroplasms identification of a lytic-cycle epstein-barr virus gene product that can regulate pkr activation binding of the influenza virus ns1 protein to double-stranded rna inhibits the activation of the protein kinase that phosphorylates the elf-2 translation initiation factor coronavirus replication complex formation utilizes components of cellular autophagy coronavirus replication does not require the autophagy gene atg5 nod-like proteins in inflammation and disease pathogen recognition and innate immunity intracellular pattern recognition receptors in the host response signaling pathway of autophagy associated with innate immunity toll-like receptors control autophagy mammalian target of rapamycin (mtor) orchestrates the defense program of innate immune cells toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis pgrp-lc and pgrp-le have essential yet distinct functions in the drosophila immune response to monomeric dap-type peptidoglycan autophagic control of listeria through intracellular innate immune recognition in drosophila autophagy and pattern recognition receptors in innate immunity human irgm induces autophagy to eliminate intracellular mycobacteria the atg5 atg12 conjugate associates with innate antiviral immune responses absence of autophagy results in reactive oxygen species-dependent amplification of rlr signalling genomewide association study identifies new susceptibility loci for crohn disease and implicates autophagy in disease pathogenesis a genome-wide association scan of nonsynonymous snps identifies a susceptibility variant for crohn disease in atg16l1 sequence variants in the autophagy gene irgm and multiple other replicating loci contribute to crohn's disease susceptibility genomewide association defines more than 30 distinct susceptibility loci for crohn's disease loss of the autophagy protein atg16l1 enhances endotoxin-induced il-1beta production a key role for autophagy and the autophagy gene atg16l1 in mouse and human intestinal paneth cells overexpression of leptin mrna in mesenteric adipose tissue in inflammatory bowel diseases production of adiponectin, an anti-inflammatory protein, in mesenteric adipose tissue in crohn's disease loss of autophagy diminishes pancreatic beta cell mass and function with resultant hyperglycemia syf-peithi: database for mhc ligands and peptide motifs rapid degradation of a large fraction of newly synthesized proteins by proteasomes autophagy in cd4+ t-cell immunity and tolerance hla class iirestricted presentation of cytoplasmic measles virus antigens to cytotoxic t cells an endogenous processing pathway in vaccinia virus-infected cells for presentation of cytoplasmic antigens to class ii-restricted t cells excessive degradation of intracellular protein in macrophages prevents presentation in the context of major histocompatibility complex class ii molecules processing and presentation of hla class i and ii epitopes by dendritic cells after transfection with in vitro-transcribed muc1 rna endogenous mhc class ii processing of a viral nuclear antigen after autophagy antigen-loading compartments for major histocompatibility complex class ii molecules continuously receive input from autophagosomes autophagy enhances the efficacy of bcg vaccine by increasing peptide presentation in mouse dendritic cells enhancing immunity through autophagy funding sources: s.h. is supported by a research fellowship award from cihr, canadian association of gastroenterology and crohn's and colitis foundation of canada. l.h.t is supported by a research fellowship award from cihr. n.l.j. is supported by operating grants from cihr and ccfc. key: cord-022940-atbjwpo5 authors: nan title: poster sessions date: 2016-09-07 journal: febs j doi: 10.1111/febs.13808 sha: doc_id: 22940 cord_uid: atbjwpo5 nan ** each poster has been given a unique number beginning with the letter p; the next part relates to the session in which the poster will be presented. moreover, klf5 is also acting on cellular processes such as cell migration, apoptosis, inflammation, angiogenesis and differentiation. previous studies showed a novel role for klf5 as a regulator of proliferation and differentiation in skeletal muscle stem cells. detecting klf5 at the protein level harbored technical obstacles. commercially available antibodies exhibited low affinity, low specificity and failed to recognize post-translationally modified forms that are directly relevant to the function. thus, these obstacles prevent further functional protein studies such as western blots, protein co-immunoprecipitation and chromatin immunoprecipitation (chip) assays. therefore, we used crispr/cas system to establish a stable cell line which carry v5 epitope tag into the n-terminal of klf5 gene. insertion into the target side of klf5 gene via crispr-cas9 system provided an opportunity to overwhelm the above mentioned obstacles. v5 epitope tag would not interfere with the function of the klf5 and also enable us to recognize endogenous klf5 via anti-v5 antibody in the mouse myoblast cell lines (c2c12). we confirmed the targeted insertion into the exon 1 of the klf5 gene both at the dna and protein levels. the conformational dynamics of structural domains plays an important role in functioning of many proteins. the reca proteins from e. coli are known to be the central catalyst of homologous recombination and repair in bacteria. it forms a helical filament on ssdna capable to bind homologous dsdna and catalysis of the exchange of the complementary strand. significant mobility if its c-terminal domain has been observed experimentally by cryo-electron microscopy. however its potential significance for reca protein activities still remains unclear. in this work we investigated this question by construction of a mutant reca protein with artificial disulfide bridge between central and c-terminal domains. the wild type protein has no disulfide bonds, and therefore its native mobility can be restored in vitro, by addition of b-mercaptoethanol. our data suggest that the s-s bridge decreases both the rate of atp hydrolysis in vitro and the e. coli resistance to uv in vivo. thus, our experimental results indicate that the flexibility of the c-terminal domain significantly affects recombination activity of reca protein in vivo and in vitro. hydroxiurea (hu) is an inhibitor of ribonucleotide reductasethe enzyme that catalyzes the process of free dntps synthesis in living cells. treating cells with hu causes diminishment of the nucleotide pool. as a result, single-stranded dna regions are generated, which leads to s-phase checkpoint activation. the progression of replication forks is blocked and the completion of dna replication is prevented. this results in s-phase cell arrest. nevertheless, our results demonstrate that after prolonged hu treatment, the saccharomyces cerevisiae cells seam to escape the arrest and continue the progression of their cell cycle. we show that when cells re-enter the cell cycle, mrc1, but not ctf4 is detached from chromatin. our data also shows that meanwhile, rad53 checkpoint activity is diminished in order to allow s-phase checkpoint escape and completion of the cell cycle. moreover, cells not only continue the cell cycle, but steadily surmount in the presence of hu. all this data indicates that cells have made the decision to compromise s-phase checkpoint and to adapt to the novel environmental conditions in order to survive. as both mrc1 and ctf4 are known to be responsible for polymerase and helicase harmonization during replicative arrest, our data indicates that mrc1 has a more specific role in the process of adaptation. our data demonstrates that mrc1 is a leading protein to regulate the stability of s-phase checkpoint arrested replication forks. zinc finger domain is the most common dna binding domain in metazoa. almost 100 drosophila proteins with c2h2 zinc fingers also have zinc finger associated domain (zad). several proteins with zad (zw5, pita and zipic) were found to interact with cp190 and act as insulator proteins. for some of the zad-containing proteins (for example, weekle and grauzone) it was shown that their zad domains can form dimers with each other. the ability of these proteins to dimerize appears to be especially important in the light of the model suggesting that dna-binding insulator proteins can support genome looping and organization of chromatin structure via interaction with each other. in this work we aimed to understand the role that zadmediated protein-protein interactions play in maintenance of dna loops, focusing on proteins: zw5, pita, zipic and cg6808. first, we performed co-precipitation and yeast two hybrid assays to confirm dimerization of isolated zads in vitro. we observed that only zads from the same protein can specifically interact with each other (homodimerization) and they are unable to interact with zads from different proteins (heterodimerization). we confirmed homo-but not heterodimerization of zads in vivo with coimmunoprecipitation experiments in s2 cells. furthermore, we found that zad proteins can support longdistance interactions in transgenic constructs in flies. using model system with cg6808 protein, we demonstrated that zad is essential for these interactions. proteins without zad cannot maintain loop formation. finally, analysis of chip-seq experiments for zw5, pita, zipic and cg6808 revealed that binding sites of zad proteins often overlap with regions of inter-chromosomal contacts known from hi-c experiments. we conclude that zad-containing proteins can support longdistance genomic interactions and dimerization of zads is necessary for these interactions. this study was supported by the russian science foundation (project №14-24-00166). over the years a large body of clinical knowledge has accumulated on pharmacological effects of drugs on thyroid function. antipsychotics are administered over long periods in humans; therefore their possible adverse side effects should be taken into consideration. the aim of this study is to evaluate the effects of haloperidol and clozapine on plasma t3 and t4 concentrations in adult male wistar rats. fifty rats aged between 14 and 15 weeks (270 ae 30 g) were divided into five groups (n = 10 in each group), and drugs were administered each day intraperitoneally (ip) for 28 days. the first group was a sham group. the other four groups were considered as low and high treatment doses of the drugs. after a one-week habituation period, animals was administered haloperidol (0.05 mg/kg, n = 10 and 2 mg/kg, n = 10) and clozapine (0.5 mg/kg, n = 10 and 20 mg/kg, n = 10). the rats were anesthetized with ether, and bloods were collected by direct cardiac puncture 24 hours after the last injection. the t3 and t4 plasma concentration levels were analyzed with chemiluminescent immunoassay. statistical analysis was performed with ibm spss v20.0. kruskal-wallis and bonferroni tests were used. t4 plasma concentration levels significantly differ between sham (median=8.25 mg/kg) and haloperidol (2 mg/kg) (median=7. 00 mg/kg), haloperidol (0.05 mg/kg) (me-dian=7.70 mg/kg) and clozapine (20 mg/kg) (median=8.32 mg/ kg), haloperidol (2 mg/kg) (median= 7.00 mg/kg) and clozapine (20 mg/kg) (median= 8.32 mg/kg) groups (p < 0.05). however, no significant differences between the groups regarding to t3 plasma levels were observed. in conclusion, haloperidol and clozapine increased the t4 plasma concentrations, but didn't have any significant effect on t3 plasma concentrations. p-02.09.1-003 isolation of lipase producing strains of bacillus obtained from olive wastewater and screening for substrate specificity in this work, wastewater samples of an olive factory from yusufeli (artvin, turkey) were collected carefully. after a centrifugation period of samples, supernatants were applied to a 0.45 lm filter and incubated on lb agar medium for 24 hours. based on differencies of colony morphologies, 13 isolates were selected and purified for identification. 16s lipase activity assay was carried out by rhodamine b. all of the 13 strains exhibited lipase activity. for determining the substrat specificity of isolates, 5 different substrates were used; 4nitrophenyl-butyrate, 4-nitrophenyl-caprylate, 4-nitrophenyl-laurate, 4-nitrophenyl-myristate, 4-nitrophenyl-palmitate. results were measured spectrophotometrically at 405 nm. all of the strains hydrolyzed 4-nitrophenyl-butirat, while there was no activity with 4-nitrophenyl-palmitate. bacillus sp. l3 was the most efficient strain that hydrolyzed all of the substrates. the gene encoding for lipase of bacillus sp. l3 will be cloned and expressed for more analyses and industrial applications. p-02.09.1-004 some quantitative aspects of hair follicle layers differentiation e. vsevolodov 1,2 , v. golichenkov 3 , a. mussayeva 1,2 , i. latypov 2 1 llc "kazcytogen", almaty, kazakhstan, 2 "institute of general genetics and cytology" sc mes, almaty, kazakhstan, 3 lomonosov's moscow state university, moscow, russia in the course of stable hair growth the differentiation of hair bulb cambium cells to several layers with dissimilar cytochemistry and morphology takes place. this means the activation of different genes in the cells of different layers. depending upon the hair diameter some layers may be absent (medulla in the thin hairs). the hair diameters of the carpet sheep breeds vary widely even within the same square mm of the skin. we compared the different layers thicknesses proportions for the follicles with varying hair diameters. the follicle layers were measured on 155 microphotos of transverse histological sections of the follicles made under the standard magnification. all follicles belonged to the same skin biopsy. the measurements were made at the levels just below the fissure separating the hair and inner root sheath appeared. the empirical regressions of the layers thicknesses and of ratios of different layers against hair diameters were counted. the computer model was made on the basis of these regressions which allowed to obtain the absolute parameters of the layers as well as ratios of these parameters for every chosen hair diameter. using this model we found an essential trend in changing the proportions in relative layers dimensions as we choose the follicles with more and more thick hair. when we change the follicles with 30 mcm hair diameter for those with the hair diameter 100 mcm the ratio of hair medulla diameter to hair diameter increases from 0.07 to 0.76. the ratio of hair diameter to the diameter of inner root sheath increased from 0.70 to 0.84. it means that the thicker is the hair the higher proportion of cells produced by cambium are spent to build innermost layers (medulla layer within the hair or hair within the complexinner root sheath + hair). these data may throw some light on positional information mechanism of layers differentiation. lignin is a heterogeneous polymer that constitutes 30% of woody plant cell walls. microorganisms that degrade lignin are fungi, actinomycetes and to a lesser extent, bacteria. in case of industrial applications, the use of fungi is not feasible due to the structural hindrance caused by fungal filaments, requirement of particular culture conditions such as humidity, aeration which are not compatible with industrial processing environments. bacteria are worthy of being studied for their ligninolytic potential due to their immense environmental adaptability. environmental concerns and increasingly stringent emissions standards have led the pulp and paper industry to devise ways to decrease the level of chlorinated lignin residues in its effluents through both production process changes and improved treatment technologies. bleaching with the enzymes is the most promising because the enzymes may be very efficient, and can be used under industrial conditions. the main objective of this study was to investigate the adequency of klebsiella pnuemonia gst (glutathione-s-transferase) pretreatment for bleaching of calabrian pine kraft pulp. for this purpose the following conditions were investigated: enzyme loadings from 3 to 10 u/g pulp basis and the consistecy of the pulp was between 3 and 10%. enzyme at the desired concentration was added to the pulp and the mixture was incubated at 40°c for 2 hours. after the enzymatic pretreatment to determine the optimum conditions the kappa number of all reactions were analyzed according to tappi standarts. as a result of this study we determine the optimum conditons as 5% pulp consistecy, 8u/g enzyme for pulp treatment. after the enzymatic treatment carried out under optimum conditions we are planning to submit a short bleaching sequence and analyze for physical properties such as viscosity and brightness. owing to this bleacing sequence we are going to able to compare the enzymatic and chemical treatments of pulp in bleaching prosess. p-02.09.1-006 biochemical characterization of lipase from bacillus subtilis strain a10 from olive waste water f. ay sal, m. kac ßagan, s. c ß anakc ßi, a. o. beld€ uz karadeniz technical university, trabzon, turkey lipases (triacylglycerol acyl hydrolases, ec 3.1.1.3) are regarded as mild and environment-friendly biocatalysts for triacylglycerols hydrolysis. in addition to this hydrolytic reaction, they also catalyze reverse reactions of esterification, transesterification, and interesterification in non-aqueous environments. substrate, stereo-, regio-and enantio-specificities, and chiral selectivity are certain unique attributes of lipases that make them industrially attractive. these properties are often exploited in the manufacturing of detergent formulations, synthesis of fine chemicals, useful esters and peptides, food processing, paper manufacturing, degreasing of leather as well as in bioremediation. in this study, lipase from bacillus subtilis strain a10 is partially purified and characterized. bacillus subtilis strain a10 is isolated from olive factory from soke (aydin, turkey) and identified with 16s rrna analysis. lipase activity is screened on petri supplemented with rhodamine b. bacteria was grown in lb medium supplemented with 1% olive oil (vol/vol) for 48 hour at 37°c. after incubation, cells were harvested by centrifugation at 11,000 rpm for 5 minutes, resuspended in 50 mm tris-hcl (ph 8.0) buffer, followed by sonication with sartorius labsonic m to release intracellular proteins. q-sepharose is used as ionexchange column chromatography for lipase purification. effects of temperature on activity and stability were determined spectrophotometrically using p-nitrophenyl laurate as the substrate. effects of ph on activity and stability were also determined. the effects of various metal ions and other reagents on the hydrolytic activity were assayed at 37°c. the enzyme was active and stable in the broad ph range of 5.0-10.0 and temperature range of 24-50°c. bacillus subtilis strain a10 have high lipolytic activity. after cloning this enzyme to an expression vector and detailed characterization, this may suggests its usefulness in industrial applications. p-02.09.1-007 investigation of pin1 as a nuclear factor one binding partner s. saritas, a. e. yilmaz, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey the nuclear factor one (nfi) proteins are important regulators of gene expression in the developing embryo and in adult stem cell niches. this transcription factor family has four members: nfia, nfib, nfic, and nfix. nfi proteins bind a consensus sequence on gene regulatory regions as homo or heterodimers. each member of nfi family has a highly conserved n-terminal dna binding and dimerization domain and a diverse proline rich c-terminal transcriptional activation/repression domain. as knockouts of nfi genes display distinct developmental phenotypes, we hypothesized that specificity of nfi protein function may arise from their interactions with binding partners. a yeast-two hybrid screen identified protein interacting with never in mitosis a1 (pin1) as a potential nfib interactor. pin1 is a ubiquitously expressed protein that specifically recognizes and binds to a phospho-serine or a phospho-threonine followed by a proline (ps/pt-p motif), and catalyzes isomerization of peptidyl-prolyl bonds. interestingly, both n-terminal and c-terminal domains of four nfi isoforms contain several conserved putative ps/pt-p motifs and some of these are reportedly phosphorylated. we looked for nfi pin1 interactions in vitro by gst-pulldown and co-immunoprecipitation assays. while gst-pin1 fusion protein interacts with all of four nfi isoforms, it binds nfib most strongly, nfia and nfic moderately, nfix most weakly. moreover, deletion of the cterminal domain leads to loss of nfi affinity for pin1 implicating this domain in nfi-pin1 interactions. co-immunoprecipitation assays where we co-expressed various epitope tagged nfi and pin1 proteins in hek 293t cells showed that pin1 precipitates nfib, as well as other nfi isoforms and nfib can, in turn, precipitate pin1. we are currently carrying out site-directed mutagenesis on nfib to identify the specific residues that pin1 recognizes. we will further explore if this interaction regulates nfi function during embryonic development. that pre-adapt migrating fish to the life in seawater. among others, smoltification induces intense growth of fish that enter the ocean at a size where risk of predation is significantly reduced. skeletal muscle growth depends on a tightly controlled balance between protein synthesis and degradation. protein synthesis driven by hormone regulation is well studied in smoltified atlantic salmon; while less is known on protein degradation occurring via a number of pathways including cytosolic ubiquitin-proteasome system and calcium dependent calpains. the aim of this study was to compare calpain and proteasome enzymatic activities in the skeletal muscles of s. salar parr, pre-smolts and smolts. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from indera river (kola peninsula, russia). our results demonstrated the significant differences in studied protease activity levels between parr and smolts. calpain and proteasome activities in s. salar smolt muscles showed a significant drop compared with that of parr. the negative correlation between proteases activity levels in the muscle tissue and overall fish growth rate was shown. so, our data indicated life stage specificity in skeletal muscle protein degradation capacity in migrating fish. we suppose that intense muscle growth in s. salar pre-smolts is supported by various mechanisms including accelerated muscle protein accretion through the reduction of protease activities. obtained results enhance our knowledge in the mechanisms of atlantic salmon smoltification. the work was supported by the russian scientific foundation, project no. 14-24-00102. p-02.09. [1] [2] [3] [4] [5] [6] [7] [8] [9] the sociodemographic characteristics of the pregnant women who double and triple prenatal screening test h. d€ ulger, s. yabanci€ un meram medical faculty, n.e.university, konya, turkey double and triple prenatal screening tests which are applicable during first and second trimesters of pregnancy predict existent abnormalities at early stage. the aim of this study is to investigate the relationship between positive results of double and triple tests, further confirmatory tests during prenatal phase, postnatal status of babies and maternal age. in this study, double and triple test results of pregnant women who were admitted to meram faculty of medicine during 2009-2013 period were scanned from archive and test results indicating risk were detected. from these results, those which were above cut-off values for down syndrome, trisomy 18, open spina bifida were determined. a questionnaire was carried out with voluntary participants by reaching to these individuals. positive-negative result ratio of all double and triple test results and sociodemographic features such as age, occupation, presence of consanguineous marriage were investigated. all data from archive and answers from survey questions were assessed statistically. participants of the study were 18 to 46 years old and their average age was 29.89 ae 6.56. 219 ofthem (69.30%) were under 35 years of age whereas 97 of them (30.70%) were above 35 years of age. number of pregnancies were scaling between 1 to 13 with an average of 2.56 ae 1.56. 207 of 316 mothers (65.50%) were not undergone amniocentesis, whereas 6 babies with chromosomal abnormalities were detected among 109 mothers who were undergone amniocentesis. in conclusion, there may be regional, sociological and such that reasons for those who were not undergone amniocentesis despite positive double and triple test results. 6 (5.50%) chromosomal abnormalities were detected among pregnancies with increased risk assessment with positive double and triple results. p-02.09. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] the effects of oil on the growth and development of amphibians l. sutuyeva, t. shalakhmetova, a. ondassynova al-farabi kazakh national university, almaty, kazakhstan currently, the pollution of ecosystems by oil and oil products is increasing everywhere. the oil gets into water and ground during oil production, transportation and accidents. as a result, terrestrial animals and hydrobionts are exposed to oil contamination. thus, populations of animals decline. it can be assumed that the most sensitive to the effects of pollutants are animals in early stages of development. amphibians have established themselves as the most convenient bioindicator species. since lake frog (rana ridibunda) and green toad (bufo viridis) are the bioindicator species in kazakhstan, the study of the effects of oil on their larvae was carried out. we used water-soluble fraction of the oil from zhanazhol field (aktobe region) in our test. the larvae of control group were kept in pure water, and larvae of test groupsin aquariums with 0.05, 0.5 and 1% concentrations of the oil fractions. the concentrations were chosen in accordance with the level of pollution of kazakhstan's water bodies with oil. mortality of larvae, morphometric parameters and morphogenesis were studied. it was found that high mortality of larvae is the most visible reaction when exposed to oil. this indicator rose noticeably depending on the doses (0.05, 0.5% and 1%) in both species with percentages 16%, 51% and 78% in r. ridibunda and 22%, 61% and 83% in b. viridis, respectively, while in the control group it was about 10%. furthermore, delayed larval development was detected. thus, the larvae from the control and 0.05% oil group reached gosner stage (gs) 36, tadpoles from 0.5% and 1% groups were at gs-32 and gs-29, respectively, by the 30th day of life. moreover, behavioral abnormalities (sluggish movements) and decreased sensitivity to mechanical stress (touch) were observed under the influence of high concentrations of oil fractions. thus, oil in low concentrations alters the growth and development of tadpoles of anurans, and causes their increased mortality in high concentrations. p-02.09.1-011 effect of catechin loaded plga nanoparticles on glioma cell line histone h1t is a linker histone which binds to dna and contribute in chromatin condensation as well as regulation of specific genes through spermatogenesis. replacement of this histone h1 subtype and hyperacetylation of histone h4 tail, facilitate the replacement of histones with sperm chromatin condensing proteins of tnps and prms. ethical approval and informed patient consent was gained from 12 infertile men referred to royan institute. testicular biopsies were collected from patients through assisted reproductive techniques (art) procedure. based on pathological results samples were classified into the following three subgroups: obstructive azoospermia (as positive control), complete maturation arrest and sertoli cell only syndrome (negative control). chromatin of tissues evaluated for presence/absence of histone h1t protein in regulatory regions of tnps and prms genes using chip-real time pcr. results showed lower incorporation of h1t protein on regulatory regions of tnps and prms genes in two spermatogenic failure group versus positive control. in this study, it can be concluded that the decreased levels of h1t histone variant in testis tissues and failure in chromatin condensation have significant association with male infertility. p-02.09.1-013 serum dickkopf-1 levels in obese children and adolescents that obesity is detrimental to bone health despite potential positive effects of mechanical loading conferred by increased body mass on bones. the wnt/b-catenin pathway is essential for normal osteogenesis. serum dickkopf-1 (dkk-1) is one of the most important inhibitors of the wnt//b-catenin pathway. the aim of this study was to investigate the serum dkk-1 levels in obese and non-obese children and adolescents. materials and methods: the study included 30 obese children and adolescents (14 males and 16 females) aged from 7 to 17 years and 30 healthy normal-weight controls (13 males and 17 females) aged from 6 to 17 years. serum dkk-1 levels were measured by elisa method using commercially available kit. results: body mass index of the obese children was significantly higher than that of non-obese children (p = 0.000). however, there was no significant difference between dkk-1 levels of the groups. (these results are preliminary and the study is continuing). discussion and conclusion: our result showed that serum dkk-1 levels were not changed in obese children and adolescents. p-02.09.1-014 transcriptional regulation of cdo by nuclear factor one proteins b. kutay, c. lektemur, v. g€ uler, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey nuclear factor one (nfi) transcription factors play important roles in regulation of central nervous system development. three of the four members of nfi family, nfia, nfib, and nfix are expressed in neural progenitors, as well as neurons and glia in the embryo. inactivation of these genes in mice show that they function in development of neocortex and hippocampus in the forebrain, cerebellum, spinal cord and precerebellar nuclei of the hindbrain, regulating neurogenesis, gliogenesis, as well as neuronal migration, axonal outgrowth and guidance. all three neural specific nfis are expressed in precerebellar neuroprogenitors, however, only deletion of nfib leads to a delay in development of precerebellar neurons. investigation of misregulated genes in nfib à/à precerebellar neuroprogenitors identified cell adhesion associated, oncogene regulated (cdo) as a potential downstream target of nfib. interestingly, this gene has been reported to be upregulated in nfia à/à hippocampus as well. cdo, a cell surface glycoprotein of the ig superfamily, has been found to regulate neurogenesis in vivo, is highly expressed in the developing brain and can induce neural differentiation by promoting heterodimerization of basic helix loop helix transcription factors with e proteins. bioinformatic analysis of the 5 kb human cdo promoter region identified five nfi binding sites: one cluster in the first 1 kb region, another in the 3.5 kb upstream region. electrophoretic mobility shift and supershift assays showed that nfib binds to all five sites. furthermore, nfib, along with the other neural nfis, inhibits the proximal cdo promoter driven luciferase activity by up to 85% in hek293t cells. preliminary data indicate that nfis bind to sites in both clusters in human neural stem cells (hnscs) suggesting that these sites are functional in vivo. we are currently investigating this possibility through nfi overexpression and silencing experiments that will examine regulation of cdo in hnscs. differentiation. the aim of this study is to investigate bdnf and drd2/ankk1 gene variants in eos development. in this study, 111 eos patients and 138 healthy controls were used. genomic dna extraction was performed from peripheral blood leukocytes. drd2/ankk1 taq1a (rs1800497) and bdnf val66met (rs6265) polymorphisms were determined by real-time polymerase chain reaction (rt-pcr). positive and negative syndrome scale (panss) was used to determine eos severity. for drd2/ankk1 rs1800497 polymorphism, there was a significant difference in the genotype frequencies between patients and controls for the co-dominant model (p = 0.05, or = 1.723; 95% ci: 0.996-2.98). however, no significant relationship was observed in the genotype frequencies of bdnf val66met polymorphism between eos patients and controls (p = 0.489). these results indicate that, drd2/ankk1 rs1800497 genotypes may affect eos development. however, bdnf val66met polymorphism may not be associated with eos. lack of association of bdnf val66met polymorphism may be due to limited number of patients. our findings need to be confirmed by further studies. various dyes used in the textile industry are discharged in large quantities to the receiving environment in the manufacturing process. this is the beginning of a process that is difficult to compensate for environmental and human health. therefore, contaminated areas should be cleaned. in addition, technologies with high polluting potential should be integrated with biological approach and thereby the impurities consisting of dyes should be reduced. in this experiment; burdirect black meta konz (c.i. direct black 38) was intended to decolorization using laccase. firstly, enzymatic decolorization of the dye was determined using spectrophotometry. the wavelengths of maximum absorption of burdirect black meta konz (c.i. direct black 38) was determined between 200 and 800 nm. then, optimization studies have been done. for optimization studies; dye concentration, laccase activity, ph, buffer concentration, temperature, mediator effect, mediator concentration and time parameters were determined. lastly, in optimal conditions, atr-ftir and gc-ms analyzes of ensuring decolorization of dye were analyzed. decolorization of burdirect black meta konz (c.i. direct black 38) was performed successfully and the absence of any metobolite in the decolorization medium has been provided by atr-ftir and gc-ms analyzes. assessing in terms of application, it can be easily applied by provided the reaction conditions in textile factories. laccase is a tool of decolorization of dyes in environmental friendly process. thus for the development of spermatids into mature sperm able to fertilize the oocyte.one of the causes of male infertility is in fact impaired sperm fertilization capacity due to sperm chromatin abnormalities and aberrant protamine replacement.recent research has focused on protamine biology,including protamine gene and protein structure,mechanisms of protamine expression regulation and involvement of the protamines in male fertility.various studies reported abnormal expressions of protamine (prm) genes in sperm of infertile men.the aim of the study is to investigate the gene expression of prm1, prm2 and their relationship with defective spermatogenesis. materials and methods: this study has been performed on 50 infertile and 3 fertile turkish men.total rna was extracted from the sperm pellet using trizol reagent.after rna extraction and cdna synthesis,real-time quantitative polymerase chain reaction (rt-qpcr) was used to determine the expression of prm1 and prm2. results: distinct levels of spermatozoal prm1 and prm2 mrna were found in infertile patients compared to fertile control groups.we found that the mrna levels of prm1 was reduced in 23 (%47), and the mrna levels of prm2 was reduced in 33 (%64) out of 50 infertile patients.in the current study,we found statistical significant association between the prm1 expression and infertility (p < 0.05).although prm2 gene expression was decreased in most of infertile patients compared to fertile control groups,the differences between the groups were statistically insignificant (p > 0.05). discussion: the results of the study suggested that, the protamine expressions which were associated with spermatogenesis may be important in infertility treatment. further studies are required in a large series of different populations to clarify the role both prm1 and prm2 themselves and their mrna expression on male fertility. the study was conducted to characterize the processes of muscle growth in atlantic salmon (salmo salar l.) of different ages inhabited rivers indera and varzuga (kola peninsula, russia) in summer and autumn. the expression levels of genes myosin heavy chain myhc, myostatin (mstn), and myogenic regulatory factors myf5, myogenin) in white muscle were studied in salmon parr of age groups 0+, 1+, 2+ in june and october. the changes in expression levels of mrfs, myhc and mstn indicating the extent of hyperplasia, hypertrophy, and restriction of muscle growth at different ages of parr were revealed. the pattern of age-related changes differed between seasons. especially, the expression of genes myod, myogenin and myhc peaked in yearling parr (1+) in summer, that indicated the high rate of hyperplastic and hypertrophic muscle growth in yearlings (1+). at the same time, the mstn expression level, the negative regulator of muscle growth, was highest in parr at age 1+. possibly, it is the necessary regulation mechanism to attenuate hyperplasia and hypertrophy and control muscle growth. in autumn, the expression level of myhc and myogenin were higher in salmon of age 0+ and 1+ then in 2+, indicating the higher intensity of hypertrophy in parr at both first ages in comparison to 2+. there was no differences in expression level of myod, myf5 and mstn between age groups in autumn. moreover, the expression levels of genes studied were lower in autumn than in summer. thus, it indicated the decrease of muscle protein synthesis and muscle growth rate in autumn. these findings expand knowledge on age-and season-related features of muscle development in young atlantic salmon in their natural habitat. the study was supported by the grant of the russian science foundation no. 14-24-00102. p-02.09.1-020 lmp2 and lmp7 gene polymorphisms in the southeastern anatolia population of turkey d. mihc ßioglu 1 , f. ozbas gerceker 2 1 sanko university, gaziantep, turkey, 2 gaziantep € universitesi, gaziantep, turkey introduction: the low molecular weight polypeptide 2(lmp2) and low molecular weight polypeptide 7(lmp7) genes are located in the class ii region of the major histocompatability complex (mhc) locus on chromosome 6. these genes encode peptides forming the large components of the proteosome complex which degrades short-lived cytoplasmic proteins. due to the significant role of lmp products antigen presentation, these genes can be accepted as strong candidates of susceptibility factors for different diseases. population genetic studies can also contribute to understanding of the possible role of lmp gene polymorphisms. the aim of this study was to determine the allele and genotype frequencies of the lmp2 and lmp7 gene polymorphisms in southeastern anatolia population and to compare these with the frequencies in other populations previously reported. material and methods: a total of 110 healthy and unrelated individuals participated in this study. polymorphism analyses were done by polymerase chain reaction (pcr)-restriction fragment length polymorphism (rflp) method and allele/ genotype frequencies of lmp2 and lmp7 genes were determined. results: a deviation from the hardy-weinberg equilibrium (v2 = 17.97,p < 0.05) was found for the genotype distribution of lmp2 gene polymorphism, while the lmp7 genotypes found to be distributed (v2 = 0.43,p > 0.05). discussion: available allele frequency data for different populations were used to calculate genetic distances and to construct a neighbor-joining tree. among the included populations, nahuas (mexico) population was found to have the lowest genetic distance from the southeastern anatolia-turkey population. conclusion: it can be concluded that, more studies using different types of genetic markers are needed to clarify the filogenetic relationships of southeastern anatolia population with other populations and also the number of population studies on lmp2 and lmp7 genes should be increased to understand their effects as a genetic marker. p-02.09.1-021 investigation of in vitro antioxidant activity of quercetin loaded plga nanoparticles pharmacological effects. but its usage is restricted because of low aqueous solubility, poor bioavailability, poor permeability and instability in physiological medium. these problems can be overcome with encapsulation of quercetin into nanocarriers such as biodegradable plga based nanoparticles. polymeric nanoparticles which have 1-1000 nm particle size and providing controlled released of biological active agent are prepared by using biodegradable and biocompatible polymers. in this study, encapsulation of quercetin molecules into plga nanoparticles was carried with using the single emulsion (w/o) solvent evaporation method. size measurements of the obtained nanoparticles were performed by zetasizer and their size were found 882. 1; 189.25; 436 .9 nm respectively. the morphological features were examined by sem images. antioxidant activities of q5, q7 ve q10 nanoformulations have been investigated by dpph and no (nitric oxide) methods. it is thought that the nanoparticular formulations that is developed in this study can be useful model for the other antioxidant molecules and will provide a significant contribution to the food and pharmaceutical industry. "this research has been supported by yıldız technical university scientific research projects coordination department. project number: 2014-07-04-gep03". p-02.09. the effect of environmental enrichment on spatial memory and certain nmdars, and 5ht2a expressions in rat pups introduction: the aim of the study was to investigate the effect of environmental enrichment exposed during whole childhood on spatial learning and memory and certain nmdars, and 5ht2a in the hippocampi of pups. materials and methods: four-weeks old, male, weaning rats were randomised into 2 groups as enviromental enrichment (ee, n = 12) and standard cage control (scc,n = 12) groups. eeg housed in an enriched environment and sccg were kept in standard cages for 8 weeks. following the experiment the rats were trained and tested in the morris water maze (mwm) , open field test (oft) and forced swim test (fst) in order to assess the neurobehavioural effects of ee. nr2a, nr2b, 5ht2a protein levels were analyzed by western blotting from hippocampi of rats. results: the positive effect of ee was seen at the learning phase in the mwm as 'latency to locate the hidden platform' between groups thoughout the 4 training days showed that eeg located the hidden platform significantly earlier than sccg on days 2, 3 (p = 0.006, p < 0.0001). also eeg significantly spent lower time in the outer zone of the maze on days 2, 3 which was the sign of low anxiety level (p = 0.011, p = 0.049). the parameters of oft which indicated increased locomotion, exploration and low anxiety were significantly higher in eeg (p < 0.05), in fst comparison of groups showed no difference (p > 0.05). the levels of nr2b and 5 ht2a were significantly increased as compared to sccg as well (p < 0.0001, p = 0.003). discussion & conclusion: these findings showed that exposure to ee throughout the whole childhood causes several neurobehavioural effects like increased exploration and low anxiety. these effects may lead to improvement in speed of learning. increase in the nr2b and 5ht2a concentrations which are the receptors that are related to learning and memory in the hippocampi accompanied these changes which may be basis of the neurobehavioural improvements or may provide contribution to positive neurobehavioural effects. p-02.09.1-023 effects of monosodium glutamate exposure during prepubertal term on several biochemical parameters in rats h. i. b€ uy€ ukbayram, d. kumbul doguc ß, i. ilhan, a. y. ismail s€ uleyman demirel university, isparta, turkey monosodium glutamate, which is commonly used in processed foods as flavor enhancer, is considered 'generally recognised as safe' by fda; however many studies have revealed the negative effects of msg.we aimed to evaluate the effects of msg in childhood on several serum parameters. sixty-six rats, (4 weeks old) were divided into 3 groups as control (cg, n = 22; 11 + 11, male+female) , experiment 1 (msg-low dose, e1g, n = 22; 11 + 11, male+female) and experiment 2 (msg-high dose, e2g, n = 22; 11 + 11) groups. msg was administered at 25 mg/kg/d to e1g, 2.5 g/kg/d to e2g for 6 weeks by oral gavage. the rats were sacrified and blood samples were collected from aorta. the blood samples were centrifuged, the serum samples were separated and glucose, alt, total protein, albumin, creatinine, cholesterole and triglyceride levels were analysed by beckmann au 5800 autoanalyser. level of total protein was significantly increased in e1g and e2g groups when compared to cg (p < 0.05). level of alb€ umine was also increased in both egs but significant difference was seen in e2g as compared to cg. creatinine levels were significantly increased in egs when compared to cg (p < 0.05). although the glucose levels in both egs were increased, the increase in e2g was statistically significant (p < 0.05). the alt levels of in egs were also increased but the significant increase was seen in e2g (p < 0.05). the effect of msg seem to be dose dependent and especially effect on carbonhydrate metabolism. increasing doses caused increase in glucose level, and tendency to glucose intolerance. increasing doses of msg also caused increase in creatinine and urea. another apparent effect of msg was detected on alt activity. in conclusion the negative effect of msg on glucose level, liver and kidney functions depends on daily dose intake. consumption of msg seem to be inevitable it has to be restrained in children otherwise early metabolic problems may be future problems for these children. (700 mg/kg) + tartrazine (750 mg/kg) + brilliant blue fcf (600 mg/kg) + ponceau 4r (70 mg/kg) + azorubine (400 mg/kg) + indigotine (500 mg/kg) + erythrosine (10 mg/kg). artificial food color mixture were administered to g 2 and g 3 and drinking water was applied simultaneously to g 1 by oral gavage per day for 3 weeks. after application all rats were sacrificed, the total oxidant (tos)/antioxidant (tas) capacity were analyzed in rats' brain, liver, kidney homogenate and serum with rel tos-tas diagnostics assay kit.the statistical analysis was carried out by using kruskal wallis test. tas and tos levels in liver homogenate were not found significantly different between all groups (p > 0.05). in serum and kidney and brain homogenate, tas levels were not significantly different between all groups. tos levels in g 3 were higher than g 1 and g 2 in serum and kidney and brain homogenate (p < 0.05). exposure to synthetic food colors may increase oxidative stres in vitale organs such as brain, kidney in female rats. these alterations differ according to organ and dose. parallel with increasing trends on healthy eating habits, consumption of prebiotics and probiotic microorganisms have been popular due to their benefits on human health. functional dairy foods such as probiotic yoghurt and cheese are the most common foods including probiotic microorganisms. due to some considerations such as standardization and quality in bulk production, starter cultures are used in industrialised fermentative food production to start fermentation. starter culture basically refers the microorganisms which induce and maintain fermentation of the fermentative foods and starter cultures including probiotic microorganisms are called as probiotic starter cultures. in this study, probiotic cheese starter cultures as a microbial community were investigated using computational systems biology tools. a metabolic network model of probiotic cheese starter culture was reconstructed using microbial community network modeling approach. literature-based genome-scale metabolic models of commonly used lactic acid bacteria were used for the microbial community metabolic model. the microbial community metabolic model simulated metabolic interactions of the microorganisms in the probiotic starter culture. metabolic flux values computed by the metabolic network model also predicted the metabolic pattern of the glycolysis (conversion of lactose), lipolysis (conversion of fat) and especially amino acid catabolism which are associated cheese flavor metabolism. simulations obtained by metabolic network-based analysis of cheese starter cultures can also be used for other fields like genetic engineering, upstream processing of the functional cheese production. p-03.01.1-002 er quality control protein network in cf to modulate f508del-cftr rescued phase ii xenobiotic metabolizing enzymes convert parent compounds into more hydrophilic metabolite by catalyzing conjugation reactions including glutathione and amino acid conjugation, glucuronidation, sulfation and acetylation. this study was aimed to describe the best cell line model for studying phase ii xenobiotic metabolizing nqo1 and gst-pi enzymes. for this purpose, mrna and protein expression of nqo1 and gst-pi enzymes were analyzed in ht29 and sw620 (colon); hepg2 and huh7 (liver); pnt1a and pc3 (prostate) cell lines by qrt-pcr and western blotting techniques, respectively. protein expression analysis revealed that nqo1 protein was expressed in all cell lines and relative protein expression is highest in the hepg2 (100%) and pnt1a (97%) while huh7 (31.9%) showed relatively low expression of nqo1. in addition, nqo1 mrna expression was relatively high in ht29 (1.68 fold) and pnt1a (1.92 fold) when compared with liver cell line hepg2 (1.00 fold). gst-pi protein expression was found very high in huh7 (100%) while there was no expression in hepg2. gst-pi mrna expression was relatively higher in pnt1a (3.56 fold) and ht29 (2.47 fold) when compared with huh7 (1.00 fold). according to these results, choosing the best cell line as model depends on the purpose of the research. for studying metabolism of a chemical by nqo1 and gst-pi or effect of a chemical on translational regulation of these enzymes, it is better to consider protein expression of the cell lines for choosing best model. however, if the aim is to study effect of a chemical on transcriptional regulation of these enzymes, it is better to choose a cell line that expressing highest mrna of gene of interest. in conclusion, considering both mrna and protein expression levels together, the best model cell lines for studying phase ii xenobiotic metabolizing nqo1 and gst-pi are ht29 and huh7, respectively. p-03.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] the studies on the pancreatic cells' surface glycoconjugates profiles in rats fed with high fat with lectin labelling methods by flouresans microscopy y. mater, s. beyhan ozdas gebze technical university, kocaeli, turkey in this study, the backbone of the cellular adhesion-recognition mechanism, located in the cell membrane. the study material selected pancreas tissue, has a privileged structures. the pancreas is one of the main organs to aid in digestion. the pancreas functions as an exocrine gland and role in digestion. in addition, the pancreas also functions as an endocrine gland, secreting several hormones into the blood that control the blood levels of glucose and other nutrients. due to the pancreas have been selected for this unique feature. thus, different types of cells in the same sample will be able to study the structure of the surface glycoconjugates. generally researches about determination of carbohydrates in the cell, glycoproteins or/and glycolipids are cut with enzymes. next step, the oligosaccharide mixture obtained, than establishing the complete structure of oligosaccharides and polysaccharides requires determination of branching positions, the sequence in each branch, the configuration of each monosaccharide unit, and the positions of the glycosidic links. this is a more complex problem than protein and nucleic acid analysis. these processes are indispensable for the understanding of the chemical structure of the sugar. whereas in cells using labeled lectins specific sugars, it is possible to accurately determine. in this study, was used triticum vulgaris (wga) labeled with fluorescein (fitc). thus, the cells located on the cell surface and neu5ac (sialic acid) for wga sugar residues were investigated. according to preliminary results of this study, wga labeled with fitc is specifically binding of these sugars. when this study is completed, the differences of sugar on the surface of different type of cells in the pancreas can be distinguished in micrographs. thus, in the cells of the pancreas, the sugar units involved in adhesion-recognition can be possible to determine specifically. large scale gene networks could be topologically analyzed in order to obtain possible global system-level structure cancer gene co-expression networks can have lower connectivity as compared to normal samples. using colorectal tissue gene expression datasets, we observed that tumor specific networks are less connected than normal networks. functional enrichment analysis suggested that cell cycle genes and methylation-associated cell adhesion genes can specifically play a role in the connectivity loss of carcinoma samples. literature confirmation provided a gene network including significant genes playing roles in the intersections between cell cycle, cell adhesion, and cell skeleton dynamics. this network can provide novel insight to our understanding of the molecular mechanisms of colorectal cancer. p-03.01.1-012 tf-mirna circuits specific to epithelial cancers y. oztemur, a. aydos, b. gur dedeoglu ankara university biotechnology institute, ankara, turkey cancer is the most common cause of death in the world but there are still a lot of uncertainties about the exact mechanism taking roles in regulation of it. cancers can be classified according to cell type; in which they start. carcinomas are the most prevalent types of cancer and start in epithelial tissues. they are also named as epithelial cancers (ecs) and make up about 85 out of every 100 cancers. over the past few years, many studies are concentrated on mirnas, which have emerged as important regulators of gene expression like transcription factors (tfs). tfs are regulators at transcriptional level while mirnas are post-transcriptional regulatory key-elements. otherwise the transcription of mrnas and mirnas are known to be regulated by tfs and tfs are the targets of mirnas. therefore, it is crucial to characterize the relation of tfs, mirnas and their targets by building circuits in diseases such as in ecs. for this study, mirna and mrna expression studies including epithelial tumors and normal samples searched in geo and array express microarray databases. 4 mrna studies and 4 mirna study, which were designed for 4 different ecs (breast, lung, ovary and colorectal) were selected to be analyzed. differentially expressed (de) mrnas and mirnas between epithelial tumors and normal samples were extracted (p ≤ 0.05, 2 fold change). among de genes, transcription factors and mirnas were identified and listed for epithelial tumor vs. normal comparison. circuit analysis resulted with remarkable circuit, which was common for all the types of ecs that includes klf4 transcription factor and hsa-mir-145. in the literature hsa-mir-145 and klf4 are known as important regulators in different types of cancer, which indicated that the motifs involving tfs and mirnas might be useful for understanding the regulation of ecs. as a conclusion finding out new and common circuits may aid us in predicting new or alternative diagnostic and/or prognostic biomarkers for ecs. mesenchymal stem cells (mscs) are multipotent stromal cells that can differentiate into a variety of cell types which are used in cell therapy. although they are the most attractive cell type for cell therapy studies, primary mscs lose their differentiation potential with increasing time in culture and passage so they are of limited use. due to this disadvantage, msc lines are more suitable for in vitro researches owing to their immortality. in this study we compared primary bone marrow-derived msc (bm-msc) with bone marrow derived msc line (rcb2153) in terms of cell characteristics and gene expression profiles to determine the functional differences among mscs types. firstly, mscs were identified by using cd29, cd70, cd90 and cd105 as positive markers and cd34 as a negative marker. gene expression profilling was investigated using affymetrix hg-u133-plus2 arrays. the significant go biological process terms and kegg pathway enrichment analyses of the identified degs were performed using david (p < 0.001, fold change≥2). the analysis showed similar pathway clustering in both cell types. the resulting quantitave transcriptome of 754 genes were identified that differentially expressed in msc line versus primer mscs (538 upregulated and 216 down-regulated). functional classification of changed genes was mainly clustered in cell cycle, cell death and mismatch repair. kegg pathway analysis revealed that the genes were significantly enriched in pathways including "cell cycle, dna replication and focal adhesion" pathways. in conclusion, our results indicate that msc lines can be used instead of primary mscs. these quatitative results provide an important basis to adapt cell lines to more closely resemble physiological conditions as oppossed to animal experimentation. this could help to minimize the use of animals in research. p-03.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] association between loss of 18q21, gain of 20q13.33 and progression in sporadic colorectal cancer n. belder 1 , b. savas 2 , m. a. kuzu 2 , i. pak 3 , h. s€ umer c ß elebi 1 , a. ensari 2 , h. € ozdag 1 1 biotechnology institute, ankara university, ankara, turkey, 2 school of medicine, ankara university, ankara, turkey, 3 ankara oncology training and research hospital, ankara, turkey colorectal cancer (crc) is one of the most diagnosed cancer and the third leading cause of cancer deaths throughout the world. identifying of copy number variation (cnv) profiles between early and late stage cancers can be useful to understand the progression and aggressiveness of cancer. the main goal of this study was to construct a comprehensive insight of association between cnv and sporadic crc stages in order to identify novel candidate targets which may contribute to tumor progression. affymetrix 6.0 genechip snp arrays were used for characterization of cnvs in tumor and matched normal formalin-fixed, paraffin-embedded (ffpe) tissues from 5 stage i, 17 stage ii and 25 stage iii samples. paired cnv analyses were performed using partek genomic suite 6.6 and genomic segmentation algorithm was performed using a minimum of 10 markers per segment, a signal-to-noise ratio of 0.3 and the cut-off value for the gain and loss was set of 2 ae 0.3. the adjusted p-value ≤0.05 were considered to be significant. whole genome cnv analysis revealed that amplification of 20q13.33 with 4 genes was found the most frequent (76.5%) in stage ii tumors. the most frequent (36%) amplifications were 13q12.2 and 7p22.2 in stage iii tumors. while deletion of chromosome 18q21.2 in stage iii with a frequency of 36% was found the most frequent loss, deletion of 18q21.1 was seen the most frequent (64.7%) in stage ii tumors. two tumor suppressor genes smad2 and smad4 which are found in these deletion regions were common genes between stage ii and stage iii. our results showed that gain of 20q13.33 might have a significant role in the progression of cancer. loss of 18q21 comprising two tumor suppressor genes is also another important finding. 18q21 loss can be a significant prognostic value in colorectal cancer even though validation of target genes requires additional study and larger sample size. this work was supported by tubi-tak project no:109s477. p-03.01.1-016 meta-analysis based mirna signature discriminates cervical cancer from normal samples a. yucel polat, y. oztemur, a. aydos, b . gur dedeoglu biotechnology institute, ankara university, ankara, turkey gynaecological cancers are common problems in female health. squamous cell carcinoma (scc) is a type of these malignancies. this tumor type is derived from pre-cancerous lesions, which is called cervical intraepithelial neoplasia (cin). cin is classified as cin1, cin2 and cin3 according to their dysplasia grade in the cervical tissues. mirnas are small non-coding rnas that were shown to have important roles in the development and progression of various cancers. the aim of this study is identifying mir-nas, which are playing a part in progression of cervical lesions by a ranking based meta-analysis approach. two mrna and three mirna expression studies, which include normal, cin1, cin3 and scc samples were selected from arrayexpress and gene expression omnibus (geo) databases. three mirna studies were combined with anova dependent ranking based meta-analysis program which was developed in our laboratory to find out a mirna signature that can discriminate cin1, cin3 and scc samples from normal samples. the top five mirnas with the highest ranks in meta-list were selected for further analysis. predicted targets of these mirnas were identified by mirdb target prediction tool. additionally two mrna datasets were selected for mirna-target validation studies. common genes, which were obtained from meta-mirna targets and differentially expressed genes between normal and cin1, cin3 and scc groups from two independent studies, were identified and they were subjected to pathway enrichment analysis. pathway enrichment analysis that was performed with 338 common genes showed that these targets were significantly enriched (p < 0.05) in especially cell proliferation, cell survival and cell cycle pathways, which are the key players of cancer development and progression. the meta-analysis results together with validation analysis of their targets may point out the potential roles of mirnas as biomarkers for the diagnosis and the treatment of cervical cancer. p-03.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] the hypoglycaemic and regenerative activity of thymbra spicata in alloxanized-diabetic rats thymbra spicata (labiatae), a carvacrol and thymol containing plant, is one of the medicinal herbs used by diabetic individuals to reduce blood glucose in turkey. we investigated the hypoglycaemic and anti-lipemic effects of the aqueous extract prepared from dried leaves and flowers of this plant in alloxanized-diabetic rat model. rats were divided as: diabetic control (group 1), dia-betic+glibenclamide (group 2), diabetic+plant extract (group 3), untreated control (group 4) and control+plant extract (group 5) groups (n = 6 for each group). serum glucose, lipid levels and body weight changes were mesasured and pancreas and liver histology of the rats were examined. each rat in all groups were administered the plant extract (30 mg), and the reference drug glibenclamide (2 mg/kg) by gastric gavage every day for 8 weeks. in group 1, blood glucose, serum alt, ast, triglyceride, cholesterol and ldl cholesterol levels increased while body weights decreased. in group 2, serum glucose, alt, ast, triglyseride and hba1c levels decreased compared to group 1 while cholesterol and ldl levels were high. in group 3, serum alt, ast, trigliserit, cholesterol, ldl levels decreased significantly but serum glucose and hba1c were higher compared to group 4. body weights increased except group 1 and hdl levels were not altered. histologically degenerative changes observed on pancreas of group 1 were decreased in groups 2 and 3. there was no difference on liver histology of the groups. in conclusion, thymbra spicata showed a protective and regenerative effect on diabetic pancreas. the hypo-lipidemic effect of the plant extract was also more effective than glibenclamide possibly due to the flavonoids, saponins and triterpenoids contents in the extract. its hypoglycaemic and protective activity should be tested for different doses and extract preparations and for longer periods. our study suggests that thymbra spicata is an excellent candidate for future studies on diabetes mellitus. with three different transcriptome data sets from the public gene expression omnibus database: time dependent data of dphop mutant, dargr mutant and wild type strain. the dynamic data spanned both primary and secondary phases of the metabolism. statistical results of transcriptome data were used for reporter metabolite analysis and reporter pathway analysis, which identify the metabolites (or pathways) with a significant coordinated transcriptional change in response to gene deletion perturbation in phosphate and nitrogen metabolisms. further, the production of actinorhodin, a pharmaceutically important compound, was modeled in the two deletion strains by calculating the metabolic fluxes subject to transcriptional level constraints on enzyme-coding genes. the metabolic switch from primary to secondary metabolism was highlighted in terms of the activity of pathways and fluxes as a result of the computational analyses in this work, leading to a better understanding of the role of phosphate and nitrogen metabolisms in increasing production levels. introduction: as a member of legume family licorice (glycyrrhiza glabra l.) has been widely used by human kind for many years as food constituent. especially by folks in rural sites licorice consumed widely. beside food constituent licorice has been used for medical purposes as well. licorice found effective with scientific datas on peptic skin infections, ulcers, inflammation, eczema, alzheimer disease, liver disease, and cancers. it also has been used as natural sweetener and food additive for preparing candies, chewing gum and beverage since ancient times. like all other medicine it has not been free of adverse event or toxicological effects. material and methods: alcoholic extracts of plant obtained by maceration process. for in vitro examination of anti-oxidant profile of licorice dpph free radical scavenging, abts cation radical scavenging and cupric ion reducing antioxidant capacity assay applied. application of extract made by oral route to rats for a week. anti-oxidant profile has been evaluated by myeloperoxidase (mpo), arylesterase (ares), total oxidative stress (tos) and total antioxidant status (tas) of serum levels. determination of toxicological effects alt, ast, ldh and alp values studied. histological investigation applied on liver and kidney tissues. results and discussion: results compared with control and standarts. antioxidant potential of licorice has been observed by in-vitro assays. serum mpo and ares values also compared with in-vitro results and correlation between them has evaluated. toxicological investigations made after evaluation of ast, alt, ldh and alp values. conclusion: in vitro assays has showed that licorice has potential anti-oxidant effect. investigation revealed that a mild toxic effect of licorice by biochemical tests. toxicological profile compared with control group and alt, ast values found slightly decreased and a mild elevation has been seen in ldh and alp values. for further and detailed investigation is needed. p-03.01.1-020 on the applications of a metabolic network model of mesenchymal stem cells h. fouladiha, s. a. marashi, m. a. shokrgozar, m. farokhi mesenchymal stem cells (mscs) have several applications in tissue engineering and regenerative medicine. mscs can be very useful in stem cell therapy, because they can be isolated bone marrow or adipose of an adult. these cells have also been used as gene or protein carriers. therefore, maintaining them in a desire metabolic state has been the subject of several studies. here, we have used a genome scale metabolic network model of bone marrow derived mscs for exploring the metabolism of these cells. then, we try to validate the computational results by experimenal tests. we analyzed metabolic fluxes in order to increase stem cell proliferation using the metabolic model. consequently, the experimental results were in consistency with computational results. in the present work, the applicability of the metabolic model was successfully approved. therefore, this metabolic model can be useful in biomedical researches of stem cells. p-03.01.1-021 qtl analysis for body weight and fatness in bxd recombinant inbred mouse strains a. dogan 1 , c. neuschl 2 , r. alberts 3 , g. a. brockmann 2 1 school of medicine, istanbul kemerburgaz university, istanbul, turkey, 2 department for crop and animal sciences, humboldt-universit€ at zu berlin, berlin, germany, 3 helmholtz-zentrum f€ ur infektionsforschung, braunschweig, germany genetic variation in body weight and composition is under the influence of many genes and have different genetic architectures. in the present study, the genetic factors contributing to body weight and fatness were examined under energy rich feeding conditions. growth traits, lean and fat weight, fat mass gain were analyzed to map qtls in a set of bxd ri strains. genome-wide analyses were revealed several genomic loci that control body weight and associated bodily changes in a sex and age-specific manner. the genetic data provided evidence for significant qtls on chromosome (chr) 4, 5, 14, and 16. most likely candidate genes within or near the regions with the highest significance levels were identified. the genes 1700048f04rik, gbe1, a830060n17, and four genes cenpc1, stap1, uba6, gnrhr for example, are suggested as most likely positional candidates accounting for the qtl effects on chr 5 for fat mass, on chr 16 for fat mass gain and on chr 16 for lean weight, and chr 16 for body weight, respectively. our results showed that body composition and fatness are highly complex that many genetic factors regulating and suggested candidate genes, which may help for studies of human fatness. related to serotonergic and gaba systems in response to hormonal changes. the nutrients involved in neurotransmitter synthesis may be the cause of relationship between diet and premenstrual syndrome. therefore, the aim of this study was to investigate the effect of various nutrients and premenstrual syndrome. this study was conducted to 29 healthy women aged 20-37 years. participants were asked to fill in premenstrual assessment form. dietary intakes (three days in each phases) were recorded during premenstrual, menstrual and postmenstrual phases. energy, protein, amino acids, iron, calcium, and magnesium intakes were estimated. statistical analyses were performed using the spss software. friedman tests were conducted and differences were considered to be statistically significant for p-values lower than 0.05. 60.9% of the participants reported premenstrual symptoms and premenstrual symptoms related nutrient intake were increased in these women. it was determined that energy (p = 0.03) and protein (p = 0.001) intakes were higher in the premenstrual phase. during premenstrual phase; tyrosine (p = 0.002), isoleucine (p = 0.001), leucine (p = 0.001), lysine (p = 0.008), methionine (p = 0.008), cysteine (p = 0.008), tryptophan (p = 0.000), and glutamic acid (p = 0.005) intakes were higher than other phases. likewise, iron intake was higher on premenstrual phase (p = 0.054). on the other hand, intake of other potential premenstrual syndrome related nutrients like fat, cholesterol, calcium, magnesium, and vitamin b6 were not significantly different within the menstrual phases. amino acids including tyrosine, tryptophan, glutamine, and vitamin b6 are involved in neurotransmitter synthesis and might be related to premenstrual symptoms. consequently, elevated intakes of dietary protein and some amino acids during premenstrual phase may be related to premenstrual syndrome symptoms. until now far uv cd spectra of only two potexviruses were published. the papaya mosaic virus (papmv) spectrum, measured by leclerc and co-authors contained no obvious anomalies and was similar to the spectrum of isolated papmv coat protein (cp). but measured 30 years earlier by homer and goodmanfar uv cd spectrum of potato virus x (pvx) itself had anomalous character and differed strongly from the spectrum of isolated pvx cp. in the present work we measured far uv cd spectra for two more members of potexvirusgenus: alternanthera mosaic virus (altmv) and potato aucuba mosaic virus (pamv) and their free cps. the altmv virion and altmv cp spectra were similar to each other and to the spectra of papmv and its cp. the pamv spectrum resembled the pvx spectrum in anomalously low ellipticity of the negative band at 208 nm, but in contrast to pvx, did not have additional peak at 228 nm. homologous modeling showed that cp of the three viruses is very similar in the core structure, and the observed difference may be explained by differences in disordered parts of proteins. possible reasons of potexvirus structural variability are discussed and it is suggested that the intravirus potexvirus cps may assume different conformations in different virions of the same preparation or even along the length of one virus particle. this work was supported by the russian science foundation (grant 14-24-00007). the antimicrobial potential of different phenolics was tested on pectobacterium in search of possible mode of action. in this respect, biofilm formation, exoenzyme activity, gene expression and virulence on its natural host (potato, cabbage, calla lily) were performed. also computational approach to show interaction between phenolic compounds and target protein was carried out using docking tools. the virulence determinants of pectobacterium were significantly impaired, at compound concentrations that did not affect bacterial cell growth. these observations suggested a mechanism which specifically interferes with bacterial virulence. since, these virulence determinants in pectobacterium are controlled by quorum sensing (qs), we focused on the effect of phenolics on the qs system in pectobacteria. the study revealed an inhibiting effect of the tested compounds on the expression level of central qs system and controlled genes, using qrt-pcr. also, there was a prominent reduction in the level of qs signal molecules n-acyl-homoserine lactone (ahl) accumulation. in addition infection capability was also practically blocked, which was completely recovered by application of exogenous-ahl. these results were supported by a potential interaction of plant phenolics with qs targets, as shown by molecular docking tool. collectively, results suggest the potential interference of phenolic compounds with qs central components (expi/expr proteins). moreover, it holds potential for future development of control measures against pectobacterium, and possibly other pathogens with similar mode of virulence. saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including double-stranded rna (dsrna) viruses. the l-a dsrna virus family of s. cerevisiae is widely distributed in nature. several versions of l-a virus are described and new ones continue to be discovered. some s. cerevisiae strains along with l-a dsrna possess smaller dsrnas, called m satellites. these dsrnas encode a sole secretable protein, known as k1, k2, k28 and k-lus toxin. l-a genome encodes the gag major structural protein and gag-pol fusion protein, formed by ribosomal frameshifting. gag-pol has transcriptase and replicase activities are necessary for maintenance of both l-a and m satellite dsrnas. so far, it's not known whether certain l-a virus has evolved to maintain a distinct type of satellite dsrna or this phenomenon lacks inherent specificity. we developed universal strategy to obtain full length l-a and m dsrna genomes from s. cerevisiae. complete viral dsrna genomes can now be cloned, as evidenced by l-a-28 dsrna, analyzed and sequenced directly from any yeast strain by means of enzymatic manipulations on total or fractioned rna content. we have identified previously undescribed l-a variant from different yeast strains specifically associated with certain type of m satellites. moreover, we identified for the first time full 5 0 -utr and 3 0 -utr sequences of m2 satellite. highly conserved sequence regions along with variable fragments were discovered at protein level, revealing clear trend to form clusters among different l-a gag-pol proteins. the obtained data suggest that each l-a virus variant can specifically maintain a distinct type of satellite dsrna. p-04.01.1-004 physic-chemical characterization of plga adjuvants for immunization per os t. chudina, d. kolybo palladin institute of biochemisry of the national academy of sciences of ukraine (nasu), kyiv, ukraine antibodies against diphtheria toxin play the most important role in the immunity against corynebacterium diphtheriae. all current diphtheria vaccines have parenteral route of administration. undoubtedly, oral administration of antigens would be the most patient-friendly way of immunization. however, the efficacy of free antigens oral administration is limited by their degradation in the gastrointestinal tract and poor absorption by m-cells. biodegradable and biocompatible polymers, like poly (d,l lactide-co-glycolide) (plga), are widely used for the design of mucosal immunizing agents. importantly, that the way of particle preparation plays an important role in plga biodegradation and antigen release. the aim of this work was to characterize the main physic-chemical properties of two types of plga particles: with immobilized antigen (plga 1) and with encapsulated antigen (plga 2) . we have prepared two types of plga particles containing egfp-sbb proteins (non-toxic recombinant fragment b of dt fused with egfp). the antigen loading efficiency of particles was determined based on the ratio of protein concentration in solution before and after loading and shown better results for plga 2 particles (plga 1 -72.05%, plga 2 -90.02%). the flow cytometry results demonstrated that 99% of plga1 particles conjugated with egfp-sbb, and only 92.2% of plga 2 particles conjugated with protein.the particle sizes had the slight difference by the results of two different techniques (ntanumber based, the software tracks individual particles; dls -scattering intensity weighted), however demonstrate similar patterns. dls data showed that the mean plga 1 particles size was 203.3 nm and plga 2 -211.6 nm. nta data also showed that mean plga 1 particles size a little smaller than plga 2 (183.8 nm and 192.8 nm respectively) . demonstrated differences in the properties of synthesized particles may have an influence on the immunogenicity of the used for oral immunization antigen. p-04.01.1-005 a suitable system for studying the functionality of a plasmodial protein in mammalian cell lines cho-mt58, a mutant cell line was proved to be an appropriate tool for investigating intracellular function of cct. in this cell line, the endogenous cct activity decreases dramatically at 40°c, blocking membrane synthesis and ultimately leading to apoptosis. we have studied the rescuing potential of pfcct in cho-mt58 cells with the isogenic cho-k1 cells as a control. cells after transient transfection were incubated at 40°c and then analysed by facs using the fluorescence of egfp fused to pfcct. the proportion of cells undergoing apoptosis was determined by propidium-iodide staining. we have demonstrated for the first time that heterologously expressed pfcct is able to complement endogenous cct activity in mammalian cells. thus, a suitable system has been established for functional investigation of structural elements of pfcct. in order to reveal the role of different protein sequences in enzymatic function, we redesigned the structural gene of pfcct obtaining a modular system where different domains are easy to be removed or exchanged. here we designed a series of different truncation and deletion constructs to reveal the role of plasmodium specific sequences. in parallel, heterologous expression experiments of different constructs in the mutant cho-mt58 and the wild type control cell lines are performed to validate the reported model system. p-04.01.1-006 host-pathogen interactions: is there a relationship between tlr 4 polymorphisms and tuberculosis in a group of turkish patients? introduction: tuberculosis (tb) is a global health problem and according to world health organization (who) each year more than 2 million individuals die from tb and each year20,000 cases of tb are notified in turkey. malatya is the third largest city in east anatolian region of turkey and tb incidence rate is higher (28.5/100,000) comparing to the general population of the country. for this reason it is important to determine the factors that lead to tb in this population. disease agent can stay in the latent phase for long periods of time after infecting the individuals. while some infected individuals show the symptoms some others never do and even 90% of these never develop clinical disease. various mechanisms take place during the host response to infectious agents. toll-like receptor (tlr) genes are shown to be candidate genes in these responses. materials and methods: in this study 49 tb patients and 50 healthy controls were included. tlr 4 genotyping for rs4986790, rs4986791 was performed by using a commercial taqman snp genotyping assay kit. data were summarized by count and percent. hardy-weinberg equilibrium was tested by chi-square distribution with 1 df. differences between groups due to allelic and genotypic distributions were analyzed by pearson's exact or fisher's exact tests. in all comparisons significance level was considered to be 0.05. results: the single nucleotide polymorphisms (snps) which were subject of this study haven't been screened in turkish population earlier. no significant association was found between tb and the snps we screened in our group of patients. discussion and conclusion: unlike other populations results we couldn't find a significant association between the disease and the genotypes of our patients. the study should be performed in bigger populations in order to confirm the results. p-04.01.1-007 lytic action of bacteriophages as a tool for the obtaining of images p. boltovets 1 , r. radutny 2 , t. shevchenko 3 1 institute of semiconductor physics nas of ukraine, kyiv, ukraine, 2 scientific and technical center of advanced technologies nas of ukraine, kyiv, ukraine, 3 taras shevchenko national univercity of kyiv, kyiv, ukraine obtaining of images by different types of bacteria now became a very special branch of skill at the interface between science and art. however the authors did not found any scientific article, where bacterial lawn was used as the background and the image was formed by the lytic action of the virus (bacteriophage). whereas the mentioned approach could be used not only with artistic aims but for the practical use. the aim of this work was to demonstrate a possibility to obtain the image on the bacterial lawn by the lytic action of the bacteriophage. the bacterial lawn was obtained by the standard metod using the 1.5% agar with the nutrient medium and the 0.7% agar containing escherichia coli culture. stencils with the preparation of the bacteriophage t4 were applied. samples were incubated during the twenty-four hours at +37°c. after that stencils were removed and the samples were stained by coomassie blue r-250 or fuchsine (with further fixation by the 7% acetic acid). several approaches to obtain the image by the lytic action of the virus were applied. first of all stencils made from printing paper and filter paper were compared. it was demonstrated, that the use of filter paper stensil allows to obtain more accurate and controllable images, than the use of the printing paper stensil. in the next series of the experiment the possibility of the reversed stencil use (where the image is formed not by the lytic zone but by the zone of bacterial growth) was demonstrated. also the possibility of the partial staining of the obtained image was explored. it gives an opportunity to obtain polychrome images using available colorants. summarizing the above it should be noted, that it was the first time when the graphical image was obtained by the lytic action of the virus on bacteria. this approach could be used not only for the artistic aims but as well for the practical use, for example, for the restriction of the action of microorganisms in out-of-theway places. burgdorferi the identification and characterization of possible antigens is essential for the improvement of current laboratory diagnostics for lyme disease and vaccine development. in this study, several recombinant b. burgdorferi outer surface proteins have been obtained and their antigenic properties have been evaluated in an effort to characterize novel immunodominant antigens. because b. afzelii and b. garinii are the most prevalent species in latvian ticks, proteins with conserved domains were included in this study. a panel of serum samples of lyme disease patients with early and disseminate disease stage was used. the controls were matched by age and sex to the patients and represented the same geographic area. the results show that proteins of several b. burgdorferi gene families have properties with respect to their candidacy as a subunit assay for a novel lyme disease immunodiagnostic. especially, the difference in their size in a range on the western blot assay may provide good discrimination between protein bands. however, they have potential for diagnosis if used in combination with other antigens but not as a "stand alone" test. in conclusions, this study showed the existing challenges in serological testing of early lyme disease. the conservation of the sequence of antigen between species of b. burgdorferi complex is essential for the most successful serodiagnostic marker candidate. the presence of homologous proteins in treponema species could lead to the cross reactivity in syphilis patients, and should be carefully evaluated. antimicrobial resistance is one of the greatest challenges in modern medicine. there is a pressing need for better understanding of the specific mechanisms that contribute to resistance to optimize existing therapies. in 2013 in georgia extended-spectrum beta-lactamase (esbl)-producing e. coli strain was isolated from the post-surgical sample obtained from gallbladder of the patients with chronic calculous cholecystitis which belongs to the sequence type 23 (st23) complexes with ctx-m 55 gene. is this strain characterized by other differences on a proteome level? are antibiotics against which the strain is resistant inducing the changes in bacterial proteome? the present work was aimed (i) to study the differences on a proteome level (i) between e. coli 92-1917/13-g and attc e. coli-reference strain and (ii) to compare the proteomes of 1917 strain at two conditions: with and without antibiotics. 1917 strain was grown in the presence of three antibiotics: rocephin (ceftriaxone), fortum (ceftazydym) and claforan (cefotaxime sodium) together. proteomic expression was analyzed using two-dimensional gel electrophoresis and mass spectrometry. significant differences were found for several proteins, including putative abc trnsporter arginine protein 2, cystine-binding periplasmic protein, fkbp-type peptidyl-prolyl cis-trans isomerase, outer membrane protein a, d-galactose binding periplasmic protein and some others. the importance of these differences for anti-microbial resistance will be discussed. p-04.01.1-010 molecular characterization of resistance and virulence features in staphylococcus aureus clinical strains isolated from cutanaeus lesions in patients with drug adverse reactions i. lupu 1 , i. gheorghe 2, 3 , m. popa 2, 3 , a. ion 3 , m. mihai 1 , v. lazar 2, 3 , m. c. chifiriuc 2, 3 1 carol davila" university of medicine and pharmacy, bucharest, romania, 2 research institute of the university of bucharest-icub, bucharest, romania, 3 faculty of biology, university of bucharest, bucharest, romania patients treated with epidermal growth factor inhibitors often experience cutaneous adverse reactions. however, the infectious complications of these toxic effects and the contribution of specific pathogens, such as the community emergent methicillin resistant staphylococcus aureus strains. the present study was aimed to identify the types of sccmec and virulence genes profile in clinical s. aureus isolated from cutaneous lesions of different severity degrees in patients with dermatologic toxic effects. this study was conducted on a total of 42 s. aureus clinical strains isolated in 2016 from acneiform reactions pustulae and periungual lesions in patients with drug cutaneous adverse reactions. multiplex pcr was performed on genomic dna from isolates in order to identify the sccmeccentral elements and the virulence genes: bbp (bone bound sialoprotein), ebps (elastinbinding protein), fnbb, fnba (fibronectin-binding proteins), fib, clfa, clfb (clumping factors a and b), cna (collagen-binding protein), luk-pv (panton-valentine leucocidin), hlg (haemolysin), tst (toxic shock toxin). the mrsa phenotype was genetically confirmed by the presence of meci gene in case of 19.04%, meca in 14.28%, sscmec type ivd element in 11.90%, ccrb2 in 7.17% and sccmec types i, iii, iv in 4.76% of the studied s. aureus strains. regarding the virulence genes encountered in s. aureus strains, the most frequent was clfa (90.47% of the isolates), followed by clfb (88.09%), fib (35.71%), hlg (16.66%) and bbp (14.28%). these results confirm the high prevalence of mec i and sscmec type iv elements, usually encountered in communityacquired mrsa strains, in cutaneous isolates from patients with dermatologic toxic effects. more data on the virulence and genetic background of these local strains are needed to appropriately assess the risk of such infections and avoid the inappropriate administration of beta-lactams. p-04.01.1-011 analysis of toxicogenic properties of staphylococcus aureus strains isolated from cows with subclinical form of mastitis in the central area of russian federation. pore-forming toxins and enterotoxins), which are present in s. aureus strains isolated from clinically healthy cows. staphylococcus strains were isolated from cow's milk. disk diffusion method was used to determine the sensitivity to antibiotics. pcr analysis was used for detection of meca, mecc genes and genes of toxins. investigated strains were resistant to oxacillin (14%), vancomycin (8%) . it was found that all strains, which contain meca and mecc genes, showed resistant to more than 5 antibiotics. it was determined that among the investigated strains 13% contained meca, 13% -mecc, 9% contained both meca and mecc. some strains contained genes of panton-valentine leukocidin (pvl) or alpha-hemolysin and several strains contained both types of genes. enterotoxin a (sea) gene was detected in 26.7% of cases, sed -5%, seg -10%, sei -35%. genes of staphylococcal toxins b, c, e, h were not found. the presence of phenol soluble modulin biosynthesis genes was determined: genes of alpha peptide synthesis were found in 93% of strains, beta peptide toxin genes in 73%, delta toxin gene in 100%. it was determined, that clinically healthy animals are carriers of s. aureus strains that cause mastitis. high level of antibiotic resistance was found in strains containing meca and mecc genes. the major part of the strains carried genes of phenol soluble modulin biosynthesis. the role of phenol soluble modulins as well as of pvl and alpha-hemolyzin in the development of mastitis is not completely clear. we conclude that pore-forming toxins have dominant role in the latent form of mastitis. p-04.01.1-012 impact of lactoferrin on the hydrophobicity and adherence to the inert substratum of staphylococcus aureus strains isolated from patients with cutaneous drug reaction skin healing is a complex biological process that requires the involvement of different cell types and humoral effectors. one of the main factors are aggravating and delaying the healing process is represented by the supra-infection with pathogenic or opportunistic microorganisms that grow in specialized consortia embedded in a self-produced extracellular polymeric matrix, called biofilms, which are extremely resistant to any antimicrobials and host immune response. lactoferrin (lf) is an ironbinding glycoprotein which promotes skin healing by enhancing the initial inflammatory phase, but also by inducing an overabundant immune response. the aim of this study was to investigate the influence of lf, one of the main components of innate, humoral anti-infectious immunity on some microbial features, involved in the first steps of the infectious process, such as hydrophobicity and adherence of staphylococcus aureus strains isolated from maculo-pustular lesions in patients with adverse reactions to epidermal growth factor inhibitors. for hydrophobicity measurement the bacterial suspensions were grown in the presence or absence of lf, and then, the "microbial adherence to hydrocarbons test" (math) was performed. the capacity to develop biofilms on inert substrata and the influence of lf on this feature was spectrophotometrically quantified using an adapted microtiter method, after crystal violet staining. our results showed that lf decreased the hydrophobicity and limited the biofilm development of all 42 s. aureus tested strains, in a dose and time dependent manner. the decreasing effect on the microbial hydrophobicity was accompanied by a lowering effect on the adhesion of microbial strains to the inert substratum. in conclusion these observations indicate that lf exhibits a wound pro-healing effect, by limiting the microbial colonization and biofilm formation and thus, the occurrence of infectious complications of skin lesion. p-04.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] host-specificity determinants of bacteriophage vb_ecom_fv3 considered vehicles of s.aureus intoxication in humans throughout the world. the objective of the present study was to assess the presence of enterotoxigenic and methicillin-resistant s. aureus in water buffalo milk and dairy products. a total of 100 water buffalo milk and 100 dairy products (50 water buffalo cream and 50 water buffalo cheese) were collected from different dairy farms, smallholders and local bazaars in samsun, turkey. all samples were analyzed using the standard procedure en iso 6888-1 and isolates were confirmed for the presence of the 16s rrna and nuc gene by polymerase chain reaction. s. aureus was identified in 30 of 100 water buffalo milk (30%), 9 of 50 water buffalo cream (18%), and 17 of 50 water buffalo cheese (34%). a total of 99 isolates were confirmed as s. aureus by pcr. genotypic methicillin resistance was evaluated using pcr for the meca gene. out of 99 isolates, 9 (9%) were found to be methicillin resistant (meca gene positive) by pcr. the enterotoxigenic s. aureus was identified in 12 out of 99 (12%) isolates by the mpcr technique. five isolates produced staphylococcal enterotoxins sea (5/12; 41.6%), two isolates produced sec (2/12; 16 .6%), one isolate produced (1/12; 8 .3%) sed, one isolate produced (1/12; 8.3%) see and three isolates produced sec+sed (3/12; 25%) . none of samples were positive for seb. in conclusion, the presence of enterotoxigenic and methicillin-resistant s. aureus in milk and dairy products is of significant for public health concern and also these enterotoxin genes sea and sed are predominant toxins that can cause staphylococcus intoxication in humans. this study was funded by ondokuz mayıs university, samsun, turkey, scientific research project programs (project no: pyo. vet -1904.12 .004) and this article was part of a phd thesis. p-04.01.1-015 identification and biochemical characterization of an immune modulating protein from helicobacter pylori b. kaplan t€ urk€ oz faculty of engineering, department of food engineering, ege university, izmir, turkey helicobacter pylori is able to achieve persistent infection with minimal immune response. the first line of defence during h.pylori infection is through gastric epithelial cells which present toll like receptors (tlr). a family of bacterial proteins which share homology with the toll/il-1 receptor (tir) domain were identified. the structure of btpa from brucella showed that bacterial tir proteins (btp) mimick human tir domain proteins and act on myd88 signaling pathways to suppress tlr signaling. h.pylori might also produce a similar protein. a putative h. pylori tir protein was found based on sequence homology and the corresponding gene; hp1437; was cloned in fusion with an n terminal cleavable 6his-tag. the recombinant protein, 6his-1437 was purified using nickel affinity chromatography. 1437 was subjected to limited proteolysis and the bands were analyzed by peptide mass fingerprinting (pmf). oligomerization of 1437 was investigated by in vitro pull-down and size-exclusion chromatography. 1437, a 239 amino acid protein, has a predicted c terminal tir domain similar to other btps and sequence alignments verified the presence of tir domain signature regions. recombinant 6his-1437 was produced with a yield of 10 mg/l culture. a structurally stable 25 kda fragment was obtained from limited proteolysis which contained the tir domain as verified by pmf. in vitro pull down assays showed 1437 interacts with itself forming dimers as shown by size-exclusion chromatography. tir domain proteins function by interacting with themselves and other tir domains. our results showed that 1437 also form dimers, supporting that it is a btp. current research is focused on solving the structure of 1437 and investigating its interaction with myd88. 1437 might play a direct role in reduced immune response against h.pylori by binding to myd88 analogous to other btps. further characterization of 1437 will provide the first solid evidence of presence of a tir domain protein in h.pylori. p-04.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] lipopolysaccharides with different lipid a acylation status from vibrio cholerae and campylobacter jejuni contribute differently to il6 production by bone marrow-derived macrophages k. korneev 1,2 , e. sviriaeva 1, 2 lipid a is a biologically active part of lipopolysaccharide (lps) from gram-negative bacteria that is responsible for the activation of the innate immunity through interaction with toll-like receptor 4 (tlr4) and subsequent production of proinflammatory cytokines. bacteria frequently transform their lipid a so that its recognition by tlr4 is not sufficient for induction of effective antibacterial immune response. we compared biological activity of various lps from pathogenic bacteria vibrio cholerae and campylobacter jejuni. we purified r-form lps for each strain by hydrophobic chromatography. the biological activity of lps preparations was evaluated by their ability to activate production of proinflammatory cytokine il6 by bone marrow-derived macrophages from c57bl/6 mice, using tlr4-deficient macrophages to control for specificity of tlr4 signaling. lps from e. coli and inactive lps from f. tularensis were used as positive and negative controls. lps from v. cholerae demonstrated biological activity similar to that of lps from e. coli, consistent with the presence of highly acylated lipid a in both strains. however, the former was a slightly weaker activator than the latter, because lipid a from v. cholerae had on average shorter acyl chains. lipid a from c. jejuni had on average longer acyl groups than in e. coli, while degree of acylation was lower, and as a result its lipid a displayed significantly lower biological activity. our study demonstrates importance of functional groups of lipid a in the ability of lps to activate production of il6 by macrophages. in line with our previous reports, we confirmed a direct correlation between biological activity of various lps species with their lipid a acylation status: the biological activity increases with increase in the length and in the number of the acyl chains. excess proinflammatory cytokine production through tlr4 activation can cause sepsis, while inefficient activation may result in the failure to clear bacteria. clostridium perfringens phospholipase c (cpplc) is the most toxic extracellular enzyme produced by this bacterium and it is an essential virulence factor in the pathogenesis of gas gangrene. cpplc may lead to cell lysis at concentrations that causes extensive degradation of plasma membrane phospholipids. however, at sublytic concentrations it induces cytotoxicity without causing evident membrane damage. the results of this work demonstrated that the cytotoxic effect of cpplc requires its internalization and the activation of the mek-erk pathway. cpplc internalizartion occurs through a dynamin-dependent mechanism and in a time progressive process: first, cpplc colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. the results also showed that cpplc requires endocytosis in order to activate mek-erk, because treatment with the dynamin inhibitor, dynasore, prevents cpplc endocytosis, erk 1/2 activation and cytotoxcity. cholesterol sequestration as well as inhibition of actin polymerization also prevents cpplc internalization and cytotoxocity, involving endocytosis in the signaling events required for cpplc cytotoxic effect. once internalized, cpplc induces reactive oxygen species production through the activation of pkc, mek/erk and nfjb dependent pathways. inhibition of either of these signaling pathways prevents cpplc's cytotoxic effect. in addition, it was demonstrated that nfjb inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of cpplc in mice. these data provide new insights about the mode of action of this bacterial phospholipase c, previously considered to act only locally on cell membrane. understanding the role of these signaling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridial myonecrosis. p-04.01.1-020 apoptosis induced by clostridium perfringens phospholipase c is mediated by reactive oxygen species m. flores-d ıaz 1 , l. monturiol-gross 1 , m. j. pineda padilla 1 , c. araya-castillo 1 , a. alape-gir on 1 bacterial phospholipases are lipolytic esterases surface associated or secreted by a wide variety of bacterial pathogens. clostridium perfringens, the most broadly distributed pathogen in nature, secretes a prototype phospholipase c (plc), also called a-toxin, which plays a key role in the pathogenesis of gas gangrene. this toxin causes death to cultured cells and extensive myonecrosis when injected intramuscularly in experimental animals. the results of the present study showed that c. perfringens plc (3-5 ng/ml) induces morphological and biochemical changes characteristic of apoptosis in cultured cells, as determined by scanning electron microscopy. nuclei condensation and fragmentation were observed by fluorescence microscopy and a typical ladder fragmentation pattern of genomic dna was detected by dna in agarose gels. cell death was prevented by the caspases inhibitors z-devd-fmk and z-vad-fmk. c. perfringens plc induces oxidative stress in cultured cells as determined by fluorescence microscopy and flow cytometry using the membrane permeable probe dcfda. different antioxidants including the gluthation precursor nac, several iron chelators and the free radical scavengers tiron and edaravone prevent cell death induced by c. perfringens plc in cultured cells or in mice challenged intramuscularly with 1.2 lg of that toxin. thus, this work provides compelling evidence that superoxide, hydrogen peroxide, and the hydroxyl radical are involved in the cytotoxic and myotoxic effects of c. perfringens plc. furthermore, the data demonstrated that edaravone, a clinically used hydroxyl radical trap, reduced the myonecrosis and the mortality caused by c. perfringens in a murine model of gas gangrene, induced by intramuscular bacterial injection of 10 8 bacteria. this knowledge provides new insights for the development of novel therapies to reduce tissue damage during clostridial myonecrosis. lectins are ubiquitous proteins able to recognize mono-and oligosaccharides with high specificity and low affinity. lectins do not have any catalytic activity, unlike enzymes, and they are not products of the immune system in contrast to antibodies. lectins play a crucial role in cell interactions on molecular level showing their importance in various physiological and pathophysiological processes as well as both mutualistic and parasitic interactions between microorganism and hosts. photorhabdus luminescens is a gram-negative bacterium from the family enterobacteriaceae. the bacteria have a complex life cycle that involves mutualistic and pathogenic interaction with two different invertebrate hosts. it is highly pathogenic towards insect larvae. in addition, p. luminescens lives in the intestine of infective juveniles of nematode heterorhabditis bacteriophora, together forming an effective entomopathogenic complex. we have identified several soluble lectins produced by p.luminescens. in this study, we focus on 4 proteins from p. luminescens, which show a high sequence homology with each other. a wide range of methods was used for structural and functional studies of photorhabdus lectins, e.g. surface plasmon resonance, isothermal titration calorimetry, analytical ultracentrifugation and x-ray crystallography. all lectins from p.luminescens recognize l-fucose and d-mannose. despite being closely related, they differ in fine binding specificities. to determine their biological function, knock-out mutants of p. luminescens are being prepared to study its interaction with axenic nematodes and insect larvae. breast cancer is the major disease of women in developed countries occuring predominantly after the age of 65. triple negative breast cancer (tnbc) is a typical subtype of epithelial breast cancer which lacks estrogen receptor (er), progesterone receptor (pr) and human epidermal growth factor receptor 2 (her2) all together. although various researches have been focused on characterizing tnbc and enlightening different molecular markers with the aim of improving the overall outcome, currently the sole affective therapy action for tnbc is chemotherapy. thus chemoresistance is the main clinical challange and accounts for 90% of failures in terms of treating the disease. multidrug resistance (mdr) is defined as simultaneous resistance towards the drugs which do or do not demonstrate structural resemblance and have different effects on their molecular targets. p-glycoprotein (p-gp) is a membrane protein coded by abcb1 (mdr-1) gene. p-gp is an atp-dependent pump which pumps a wide range of drugs out of the cells including chemotherapeutic agents such as doxorubicin (dox) and pactilaxel. in the present study, tnbc cell line mda-mb-231 was treated with increasing doses of dox, cell viability was examined with srb assay and development of mdr was investigated through mdr assay and rt-pcr. results demonstrated that cell viabiliy decreased significantly with the treatment of higher doses. mdr was shown to be increased when cells were treated with 50, 200 and 800 nm of the drug respectively along with 15 lm of p-gp inhibitor verapamil. rt-pcr results were obtained to be consistent with mdr assay results and indicated increased mdr-1 gene expression with the treatment of dox. especially after 400 nm of dox treatment, mdr-1 was overexpressed to be 59 fold when compared to control. in conclusion, it was demonstrated that mda-mb-231 cells have shown to display elevated resistance to higher doses of dox. p-05.01.1-005 targeting dna damage response pathway in cancer cells under heat stress and the mechanical effect of ultrasound y. furusawa 1 , t. kondo 2 1 toyama prefectural university, imizu-shi, japan, 2 university of toyama, toyama, japan ultrasound (us) has been widely utilized for diagnosis and therapy in many medical fields. the biophysical modes of us are divided into three classes, thermal, cavitation and non-thermal non-cavitation effects. in clinical use for cancer therapy, the thermal effect was utilized for hyperthermia therapy with focusing us on cancer to rise the temperature from 41°c to 44°c, or further which could induce thermal ablation of cancers. cavitation leads to a variety of mechanical stress such as shear stress, shock wave, high pressure, and chemical stress such as free radical formation, both of which have been inferred to act simultaneously on all biological materials. it has been indicated that us induces cell killing, cell lysis, loss of viability, and loss of clonogenicity. recently, we found that heat stress as well as us without thermal effect induce not only dna single-strand breaks but also dna double-strand breaks, a most cytotoxic region of dna, in chromatin dna detected by both gammah2ax staining and neutral comet assay. in response to the stresses which induce dna damage, the dna damage sensor protein kinase, ataxia telangiectasia mutated (atm), atm and rad3 related (atr), and dna-dependent protein kinase (dna-pk) become activated form to initiate signal transduction pathways activating cell-cycle checkpoints, dna repair, and apoptosis. the molecules consisting of dna damage response pathway were expected as therapeutic targets because defects in the response to dna damage agents can be lethal. this work was designed to explore the possible therapeutic targets of the molecules in dna damage response pathways for future us-aided therapy. finally, several kinases (e.g., checkpoint kinase) on dna damage response pathway seems to be the targets for hyperthermia and us therapy. (ural branch) , ekaterinburg, russia, 2 shemyakin and ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia based on the recently synthesized (s)-(2-aminopurin-6-yl) amino acids (gly, ala, val, phe, pro), we obtained a series of novel modified nucleosides using the transglycosylation reaction. for the first time, it has been demonstrated that the corresponding nucleobases are good substrates for the genetically engineered recombinant e. coli purine nucleoside phosphorylase (conversion to nucleosides reached 90-98%). nucleosides, such as ribosides, 2-deoxyribosides, and arabinosides were obtained in high yields (70-88%). it has been found that yield in the transglycosylation reaction does not depend on the structure of the amino acid fragment. the nucleosides synthesized are considered as potential inhibitors of intracellular adenosine deaminase (ad), the increasing activity of which is observed in hepatitis, cirrhosis, hemochromatosis, obstructive jaundice, prostate and bladder cancer, hemolytic anemia, rheumatic and typhoid fever, gout, and cooley's anemia. cytotoxicity of the synthesized nucleosides was tested in the jurkat (model of human t-lymphoblastic leukemia) and el-4 (model of mice t-lymphoblastic leukemia) cell lines. the compounds studied did not exhibit cytotoxic activity compared to the activity of the known antitumor agent nelarabin. the work was financially supported by the russian science foundation (grant 14-13-01077). p-05.01.1-007 dna binding, dna cleavage, antimicrobial activities, antimutagenic and anticancer studies of a schiff base and its complexes n. yildirim 1 , n. demir 2 , m. yildiz 3 1 health services vocational school, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 2 department of biology, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 3 department of chemistry, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. the rational design and synthesis of new schiff bases and their metal complexes have been drawing great interest because of their diverse biological and pharmaceutical activities. so, exploring and designing novel molecules that have biological activities and capable of interacting with nucleic acids has a great significance for disease defence and to discover new dna-targeted anticancer drugs for chemotherapy. in this study, we report the synthesis and characterization of a novel schiff base and its ni(ii) and cu(ii) complexes. the minimal inhibitory concentration (mic) of the compounds was screened in vitro against bacteria and yeast cultures using broth micro dilution test. dna binding and dna cleavage activity of the compounds were investigated by uv-vis spectroscopy and agarose gel electrophoresis. antimutagenic activity of compounds were tested in the absence of microsomal enzymes (s9-). also, cytotoxicity of the compounds against hepg2 cell lines was assayed by the mtt (3-(4,5-dimethylthyazolyl-2)-2,5-diphenyltetrazolium bromide) method. consequently, uv-vis spectroscopy studies indicated that the compounds interact with calf thymus dna (ct-dna) via intercalative binding mode. dna cleavage activity studies showed that the cu(ii) complex can effectively cleave pbr322 plasmid dna. compounds inhibited the base pair mutation with high inhibition rate in the absence of s9. also, schiff base complex had cytotoxic activity towards hepg2 cell line, that it was found to be more potent than the control cisplatin. p-05.01.1-008 single particle electron tomography of rnap elongation complex, stalled at position +24 genome in vivo is constantly exposed to the damaging effects of the environment. single-strand breaks (ssbs) are the most frequently occurring dna lesions. accumulation of unrepaired ssbs can interfere with the cells metabolism and increase genomic instability. in vivo, ssbs are repaired in specific pathway, but, in eukaryotic nuclei, dna is organized in chromatin that could affect the accessibility of lesions to sensor proteins. breaks in a template strand induce arrest of rna polymerase ii (polii) in vitro and in vivo and can be revealed in a transcription-dependent manner. our recent biochemical studies identified two key intermediates formed during transcription through a nucleosome by rnap that are nearly homogeneous, active and stable by biochemical criteria (complexes stalled after entering 24 or 42 bp into the nucleosome; ec+24 or ec+41, respectively). hear we produced two complexes, both stalled in the +24 position, one without break in the dna, and the other with introduced ssb at position +12 of a non-template dna strand. complexes were purified using affinity chromatography and applied to a carbon-coated, glow-discharged em grid. tomographic studies were performed at ae70°in a jeol microscope at 200 kv accelerated voltage. images were recorded using a gatan ccd camera. image analysis was performed using the imod software. the resulting structure of the ec+24 complex with no break in dna consist of two domains, connected by a single dna string. the complex with a break introduced into the dna has a more compact appearance and its two domains were connected by two dna strings, thus forming an intranucleosomal dna loop. our data suggest that ssbs in a non-template strand can induce the formation of stable non-productive transcription intermediate. the inhibitory effect of ssbs onto transcription may suggest a possible mechanism for their recognition in vivo with a transcription-dependent pathway. this work has been supported by the rsf grant #14-24-00031. colorectal cancer (crc) is one of the leading causes of cancerrelated deaths in the developed countries. according to 2014 who report new incidence rate of crc in turkey is 6.5% among other cancer types. owing to difficulty of the low allele frequency variations detection, genetic association profiles of crc have not been entirely identified. low allele frequency variations mlh1 à93g>a (rs1800734) promotor substitution, mlh1 415g>c (rs28930073) exonic substitution, mthfr c677t (rs1801133) and apc 1307 t>a (rs1801155) were investigated in this study. these 4 snps "rs1800734, rs28930073, rs1801133, rs180115" are located on 1p36.3, 5q21, 3p22 respectively. colonoscopic investigations were performed on both cancer and control group. the 4 snps were genotyped using kompetitive allele specific pcr technology in 1014 cases and 805 healthy controls. statistical analysis was carried out with cochran-armitage chi-square test. in this study these 3 of the 4 snps in mlh1, mthfr genes were examined for the first time in turkish sporadic crc cases. statistical analysis showed no significant association within our turkish sporadic crc population. percentage of mlh1 à93aa genotype in group aged ≥ 65 was found to be 5.7% in cancer versus 2% in control group. moreover apc 1307a, mlh1 415c alleles were detected only 1 and 3 allele respectively. previously, apc 1307a allele was determined in 53.3% of a turkish cohort. however in the present study apc 1307a allele was detected on 1 allele only. studies showed mlh1 à93 promoter variation as a risk factor for microsatellite instabile crc but for the current study this data is not available. in spite of literature mthfr c677t and mlh1 415g>c snps were not found to be associated with sporadic crc in turkish population. this research demonstrates that importance of population based studies in multifactorial disease. p-05.01.1-010 excision of damaged bases from transcription intermediates by fpg/nei superfamily dna glycosylases k. makasheva, d. zharkov sb ras institute of chemical biology and fundamental medicine, novosibirsk, russia oxidative lesions are abundant due to constant presence of reactive oxygen species in living cells. repair of oxidative base lesions is initiated by dna glycosylases. for example, bacterial fpg and nei dna glycosylases excise oxidized purines and pyrimidines, respectively, from dna. their human homologs, neil1 and neil2, have been reported to show preference towards oxidized lesions in dna bubbles. from these observations, it had been hypothesized that neil proteins may be involved in the repair of lesions in dna bubbles generated during transcription. however, it is not presently clear how neils would behave on bubbles more closely resembling transcription intermediates (e. g., containing the rna strand), and bacterial homologs fpg and nei had never been investigated with bubble substrates. we have studied excision of either 8-oxoguanine (8-oxog) or 5,6-dihydrouracil (dhu) by e. coli fpg and nei and human neil1 and neil2 from single-strand oligonucleotides, perfect duplexes, bubbles with different number of unpaired bases (6 to 30), d-loops with dna or rna and from complexes with rna polymerase. fpg, neil1 and neil2 efficiently excised dhu located inside a bubble. fpg and neil1 was generally more active than neil2 in excision of 8-oxog from ssdna and bubbles. nei, on the other hand, was active only on dhu located in dsdna (either perfect duplex or dna/dna d-loop). fpg and neil1 also have shown activity in d-loops with rna. the presence of an additional unpaired 5 0 -tail of the third strand of d-loops didn't affect the glycosylases activity. the activity of fpg was observed in pre-assembled transcriptional complexes with e. coli rna polymerase and depended on the position of the lesion in the transcription bubble, possibly reflecting local accessibility of the lesion within the elongation complex. this work was supported by rsf (14-24-00093). nucleotide excision repair (ner) is a multistep process that eliminates a wide range of lesions in dna, including uv photoproducts and base modifications by many carcinogenic and chemotherapeutic agents. one of the advanced approaches to ner process investigation is based on reproducing the repair reaction by mixing protein extracts from mammalian cells with model linear dnas, bearing lesions. long linear dnas (137 bp) containing efficiently recognized and processed by ner system lesions (fluoro-azidobenzoyl photoactive lesion fab-dc, nonnucleoside lesions nflu and nant) in both strands have been synthesized. we have demonstrated that dnas containing closely positioned lesions in the both strands represent difficult-to-repair (fab-dc/nflu(+4), fab-dc/nflu(à3)) or unrepairable (nflu/nflu (+4), nflu/nflu(à3), nant/nflu(+4), nant/nflu(à3)) structures. besides, it has been shown that model dnas bearing 2 bulky lesions in opposite positions (fab-dc/nflu(0), nflu/nflu(0)) represent unrepairable structure as well. the model substrates with increasing distance between lesions in the duplex demonstrated the full recovery of substrate properties in ner process (fab-dc/nflu(+8), fab-dc/nflu(à10), fab-dc/nflu(à21), nflu/nflu (+8), and nant/nflu(+8)), whereas the level of specific excision from nflu/nflu(à10), nflu/nflu(à21) and nant/nflu(à10), nant/nflu(à21) was approximately 50% of the nflu/dg or nant/dg dna respectively. it has been shown that modified dna-duplex (54 bp) with fab-dc has decreased structurally dependent affinity for xpc-hr23b compared to duplexes containing lesions in both strands being analyzed (fab-dc/dg, fab-dc/nflu(+4), fab-dc/nflu (à3), fab-dc/nflu(+8), fab-dc/nflu(à10), fab-dc/nflu(-21)) and increased compared to umdna. the data provide an argument that the ner system of higher eukaryotes recognizes and eliminates injured dna fragments on a multi-criteria basis. it is well known that dna plays crucial role in the biological system because of including all the genetic information for cellular function. therefore, the interaction of molecules with dna has gained interest in the medicinal chemistry to explore new anticancer agent. photodynamic therapy which is alternative cancer treatment method depends on free radicals and singlet oxygen to destroy tumor tissue via necrosis and apoptosis. phthalocyanines (pcs) are used for photodynamic therapy because of their absorption of high wavelength light ability and they have high triplet quantum state yields and long lifetimes in triplet states. also they do not have any toxic effect without light. in this study the novel synthesized 4[2-(2morpholin-4-ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanines were used. the potential properties of phthalocyanine compounds for photodynamic therapy were purposed to reveal by the preliminary work. for this aim, the mode of dna binding, photocleavage and topoisomerase i inhibition of these compounds were investigated. 4[2-(2-morpholin-4-ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanine compounds have been synthesized. the interaction of novel pcs compounds with calf thymus (ct) dna was investigated by using uv-vis spectroscopy, thermal denaturation studies and viscosity measurements. additionally, dna photocleavage and topoisomerase i inhibition studies were performed to pbr322 dna by using agarose gel electrophoresis. the interaction studies indicated that pcs compounds powerfully bound via an intercalation mechanism with ct-dna. these compounds showed efficiently dna photocleavage under irradiation at 650 nm. the all of pcs inhibited topoisomerase i in a dose-dependent manner. all the experimental studies showed that pc compounds might be used agents for photodynamic therapy. p-05.01.1-014 target search by base excision repair dna glycosylases e. dyatlova, g. mechetin, d. zharkov institute of chemical biology and fundamental medicine, novosibirsk, russia the problem of rapid target search in dna is faced by transcription factors, restriction endonucleases, dna repair enzymes and other sequence-or structure-specific dna-binding proteins. theoretically, the fastest target search in dna can be achieved by combining one-dimensional diffusion along the dna contour (processive search) and three-dimensional diffusion (distributive search). the balance between these search modes depends on many factors affecting dna-protein interactions, such as the presence of mono-and divalent cations, competing proteins, crowding effect, etc. presently, the mechanisms of target search are understood only for a handful of enzymes. we have recently developed an assay to study target search by dna repair enzymes, based on cleavage of oligonucleotide substrate containing two targets. thus, the distance between the targets can be precisely controlled, and any modification can be introduced into dna. subsequently, the probability of correlated cleavage (p cc ) is estimated, reflecting the efficiency of enzyme transfer between the specific sites. in this work, we have investigated five repair enzymes: e. coli endonuclease viii (nei), its human homologs neil1 and neil2, and uracil-dna-glycosylases (ung) from e. coli and vaccinia virus. as expected, p cc of all enzymes depended on the ionic strength of the solution and the presence of mg 2+ . ung from vaccinia virus was the most sensitive to these factors, raising questions about its proficiency as a suggested processivity factor of viral dna polymerase. nei, neil1 and neil2 showed a peak of p cc at low but non-zero ionic strength indicating that nonpolar interactions contribute to binding of these proteins to nonspecific dna. this conclusion was also supported by analyzing amino acid conservation in the catalytic core of nei. introduction of bulky fluorescent group between two specific sites greatly reduced the ability of glycosylases to slide along dna. this work was supported by rsf (14-24-00093). p-05.01.1-015 does causes 1800 mhz magnetic field application kras and p53 mutations in colon?: occurences histopatologically and microbiologically changes in colon determination of kirsten rat sarcoma (kras) and p53 gene mutations in colon. materials and methods: in this study, three groups were prepared as control,sham and electromagnetic field (emf) group.1800 mhz radiofrequency (rf) radiation was produced by using an electromagnetic energy generator.the emf group rats were exposed to electromagnetic field for 12 weeks as 45 minutes per day.at the end of experiments, rats were sacrificed under ethyl ether anesthesia and the rat colons were dissected.fecal speciments were collected.fecal dna (for detection of fusobacterium and bacteroides) and colonic dna (for detection of kras and p53 mutations) were isolated.rt-pcr tchnique was used for detection of bacterias and mutations. results: no any differences was observed histopathologically between control and sham groups.erosions and partial losses were observed at mucosal epitelium in the emf group.the corrupted gland structure, the mucosal edema and the inflammatory cell infiltration were observed.the amout of collagen was increased and fibrosis was detected in emf group.goblet cell number decreased statistically significant when compared to control and sham groups (p < 0.05).the amount of fusobacterium increased significantly in emf group compared to controls.the difference was not detected between groups in the amount of bacteroides.all the samples analysed for kras and tp53 mutations in the colon tissue were found to be wild type.no significant difference was observed between the control group and the emf applied group. discussion and conclusion: in conclusions,for 12 weeks 45 minute/day exposure to 1800 mhz emf caused histopatological damage in rat colon.the amount of fusobacterium is increased.emf exposure did not caused to kras and p53 mutations in colon tissue. p-05.01.1-016 synthesis, antimicrobial activity, genotoxicity, dna binding and dna cleavage studies of new glycine methyl ester derivative schiff base there has been an increasing focus on the binding study of small molecules to dna during the last decades, since dna is an important genetic substance in organisms. therefore, the current growing interest in small molecules that are capable of binding and cleaving dna is related to their utility in the design and development of synthetic restriction enzymes, new drugs, dna agents, and also to their ability to probe the structure of dna itself. in recent years, schiff bases have found increased application in pharmaceutical research, organic synthesis, and bio-processes. schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. in this study, we report the synthesis and characterization of a novel glycine methyl ester derivative schiff base. the minimal inhibitory concentration (mic) of the compound was screened in vitro against bacteria and yeast cultures using broth micro dilution tests. antimutagenic activity of compound was tested in the absence of metabolic activation. also, dna binding and dna cleavage were investigated of compound by uv-vis spectroscopy and agarose gel electrophoresis respectively. consequently, this compound differs significantly in its activity against tested microorganisms. this difference may be attributed to the fact that the cell wall in gram-positive bacteria is a single layer, whereas the gram-negative bacteria cell wall is a multilayered structure, and the yeast cell wall is quite complex. the compound inhibited the base pair mutation in the absence of s9 with high inhibition rate. uv-vis spectroscopy studies of the interactions between the compound and calf thymus dna (ct-dna) showed that the compound interacts with dna via intercalative binding. to date a large number of the sequences in the human genome (g4 motifs) with the potential to form a spatial structure, gquadruplexes is known. g4 motifs were found in the promoter regions of most of the known oncogenes. recent experimental studies have shown that genome instability directly related to the non-canonical dna structures, including g-quadruplexes. in this work we study the distribution of somatic snvs within the g4 motifs in tumor samples with the aim to identify involvement of the motifs in the process of mutagenesis in pancreatic cancer. using the access kindly provided by the international icgc consortium to the database, we analyzed 68 samples of pancreatic ductal adenocarcinoma and 27 samples of pancreatic endocrine neoplasms. we considered only the promoter regions as the richest with g-quadruplex motifs. we found that quadruplex sequences have the ability to focus somatic snvs. this could be explained by the errors of polymerase during replication through secondary dna structures. furthermore, the snvs occur much more often in loops of g4 motifs than in g blocks, without changing the motive. in addition, t>g(a>c) and t>c(a>g) substitutions occur significantly more likely in loops which in turn stabilize the g-quadruplex structure. the cancer-related mutations tend to increasing the length of g blocks. the conservation of g4 motifs may indicate an important functional significance of g-quadruplex structures in human genome. supported by project no. 16-14-10396 of the russian science foundation. background: multiple myeloma (mm) is a rare, leading to bone destruction and marrow failure, largely incurable malignant disease of plasma cells. anemia (mostly normocytic normochromic) is seen in most patients. mean platelet volume (mpv) is a laboratory marker of platelet function and activity, the most accurate measure of platelet size. the aim of this study was to investigate the mean platelet volume (mpv) values in this disease. materials and methods: whole blood samples were collected from 60 healthy controls and 105 patients with mm. the mean age for controls and patients were 54.5 ae 8.5 and 57.4 ae 8.1 years, respectively. mpv levels were calculated with cancer is a chronic disease in the world which is the second leading cause of death, after cardiovascular diseases. benzimidazoles have been known to act as antiproliferative or anticancer agents in chemotherapeutic drug research area. in this regard we aimed to investigate the cytotoxic and apoptotic properties of novel benzimidazole derivatives bearing pyridyl/pyrimidinyl piperazine moiety against a549 lung adenocarcinoma cells. a549 lung adenocarcinoma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by mtt assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic 50 values of the compounds were determined for a549 cell line. compounds 4, 7 and 12 which were including 4-chlorophenyl, 4-nitrophenyl on pyridine ring; 4-fluorophenyl on pyrimidine moiety, had significant cytotoxic activity with ic 50 values lower than 55.33 ae 23.40 lg/ml. compound 12 showed the highest cytotoxic activity with a ic 50 value of 16.67 ae 2.24 lg/ml, whereas cisplatin ic 50 values were 14.0 ae 2.0 lg/ml lg/ml against a549 cells. cytotoxic activity of compound 4 and 7 with a ic 50 value were 55.3 ae 23.4 and 21.7 ae 8.4 lg/ml, respectively. also, compound 12 showed the highest population of early apoptotic cells (39.4%) of the tested compounds which was 5.88-fold higher than for cisplatin. compound 7 produced a comparable population of apoptotic cells with a percentage of 9.5%, respectively according to cisplatin's percentage of 6.2%. it was determined that synthesized compounds 4, 7 and 12 had considerable anticancer activity against a549 cell lines compared to cisplatin. compound 12 including 4-florophenyl on pyrimidine ring was the most cytotoxic compound against the a549 cell line. our study results demonstrated that compound 7, 12 also induced apopototic pathway on a549 cells. p-05.01. in vitro/in vivo antimitotic activity and structure-activity relationships of new glaziovianin a isoflavone series glaziovianin a (gva), isolated from the leaves of the astelia glazioviana, demonstrated cytotoxicity, disrupting microtubule structure and dynamics of hl-60 cells. the aim of the present work was to devise a concise synthetic route toward gva and its derivatives in order to expand structure-activity relationship studies and to investigate their anti-mitotic effect. a concise six-step protocol for the synthesis of gva and its alkoxyphenyl derivatives 9 starting with readily available plant metabolites from dill and parsley seeds was developed. the sea urchin embryo tests confirmed that gva directly affects tubulin/ microtubule dynamics and structure. the b-ring substitution pattern of gva derivatives exhibited strong effects on activity. according to the assay results, the anti-mitotic activity decreased in the following order: gva > myristicin ≥ 3,4,5-trimethoxyphenyl = 4-methoxyphenyl > dillapiol > 3-methoxyphenyl> 3,4dimethoxyphenyl > 2,3,4,5-tetramethoxyphenyl derivatives. a methylenedioxy moiety was essential for the activity of compounds substituted with four b-ring alkoxy groups. the mts assay of the limited panel of cancer cell lines shows that gva displayed the highest inhibitory activity, with ic50 values ranging from 0.27 (a375 cells) to 2.2 lm (mda-mb-231 cells). compounds, containing 3,4,5-trimethoxy and apiol-derived b-rings, respectively, were less active. other isoflavones did not affect cancer cell growth up to 10 lm. anti-proliferative effects of isoflavones 9 observed in both the sea urchin embryo model and human cancer cell lines correlated well. importantly, none of the synthesized isoflavones 9 demonstrated cytotoxicity in human pbmcs, up to 10 lm. in summary, gva and its analogues were synthesized via a scalable six-step reaction sequence. the gva and its analogues containing 3,4,5-trimethoxy and apiol-derived b-rings were found to be promising anti-mitotic microtubule destabilizing agents with low toxicity against human pbmcs. bag-1 is a multifunctional protein which has interactions with a number of cellular proteins; nuclear hormone receptors, bcl-2, hsp70/hsc70 family, growth hormone receptors, raf-1, ubiquitin machinery and dna to regulate cell survival. for this reason, bag-1 is a critical molecular player in the regulation of cell survival signaling and apoptosis mechanism. elevated expression levels of bag-1 are associated with progression of cancer. in the treatment of breast cancer, silencing tools as a promising combined therapy strategies in the presence of classical chemotherapeutics gain importance to investigate interaction networks of cell death and survival signaling pathways. therefore, we aim to understand potential role of bag-1 silencing in the treatment of breast cancer cells with apoptotic agents; cisplatin or paclitaxel. our results showed that, silencing of bag-1 enhanced cisplatin or paclitaxel-induced apoptosis in mcf-7 cells by down-regulating antiapoptotic and upregulating proapoptotic bcl-2 family proteins, changes on cell cycle, upregulation on subg1 phase, activating caspases and cleavage of parp. in addition, knockdown of antiapoptotic bag-1 has a suppressive role in pi3k and akt signaling pathway in mcf-7 breast cancer cells through inhibition of akt phosphorylation and downregulation on pi3k. investigation targets of akt pathway showed that mtor cell survival pathway also affected through bag-1 silencing. bag-1 silencing inhibited mtor signaling via downregulating both rictor and raptor proteins which are the members of rapamycininsensitive mtorc2 and rapamycin-sensitive mtorc1 complexes, respectively. knockdown strategies of bag-1 is important to enlighten the network interactions of bag-1 and clarify its interaction partners in the cells. therefore utilization of bag-1 targeted strategies might further increase therapeutic efficiency of drugs through inhibiting cell survival machinery in the treatment of metastatic breast cancer. p-05.01.1-024 biological activity evaluation of new 3,5,6trisubstituted triazine derivatives bearing different heterocyclic rings against lung cancer cell lines l. yurttas, g. akalin c ß iftc ßi, h. e. temel, b. demir anadolu university, eskisehir, turkey cancer is one of the major death causing disease worldwide. among the various cell types occurs on different organs, lung cancer is one leading cause of cancer death accounting for approximately 26% of all female and 28% of all male cancer deaths in 2013. the resistance development, cytotoxicity and inadequacy are the main encountered problems by the treatment with existing chemotherapeutic agents. therefore, there is continuous need to discover new active and non-toxic molecules. 1-[4-(5,6-bis(4-substituted phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl]-2-[benzimidazole/benzoxazole/benzothiazole-2-yl)thio]ethanone (1-9) derivatives were synthesized with a four-step synthetic procedure using toluil, anisil and 4-chlorobenzil as starting materials. the anticancer activity of the compounds was evaluated using the methods mtt (3-(4,5-dimethylthiazol-2-yl )-2,5-diphenyltetrazolium bromide), brdu (bromodeoxyuridine) assays and flow cytometric analysis against lung cancer cell lines. the lipoxygenase enzyme inhibition activity of the compounds were also investigated using the method described by baylac and racine. compounds was found to have (inhibition concentration) ic 50 values between 135-500 lg/ml. the early and late apoptotic cell percentage was determined as 6.6 for compound 8 by flow cytometric analysis. the lox inhibition activity was found 48.35 ae 3.08 for compound 1. compound 8 bearing 8-chlorobenzil and benzoxazole moieties was found as the most active compound when we evaluate anticancer potential of all compounds. the lox enzyme inhibition was indicated for the compound 1 including methyl substituent on phenyl rings. the dna synthesis inhibition of the compounds has been still studied at the concentrations ic 50 /2, ic 50 and ic 50 x2. p-05.01.1-025 single amino acid substitutions and deletions modulate the drp-lyase activity of human dna polymerase iota n. miropolskaya, i. petushkov, a. kulbachinskiy, a. makarova institute of molecular genetics, moscow, russia dna polymerase iota (pol ι) is a y-family dna polymerase that possesses an unusual combination of properties. due to the special organization of the active site pol ι has a very low accuracy of dna synthesis but possesses an ability to bypass a variety of dna lesions. in addition to the dna polymerization activity, human pol ι also possesses an intrinsic 5 0 -deoxyribose phosphate (drp)-lyase activity. removal of the drp group is a pivotal step in base excision repair (ber) in vivo. although pol b plays a key role in the drp group cleavage and dna synthesis during ber, pol ι was shown to complement the in vitro single-nucleotide ber deficiency of pol b null cell extracts and was suggested to be involved in ber under oxidative stress. the drp-lyase active site in pol ι is still not known. to address the mechanism of the drp-lyase activity of pol ι we obtained a series of pol ι mutant variants including point mutations of conserved lysine residues and deletions in different locations. we purified human pol ι variants from yeast saccharomyces cerevisiae and tested the effect of mutations on the cleavage of an internal 5 0 -drp group in oligonucleotide dna substrates in the presence or absence for me 2+ ions. the experiments revealed several point amino acids substitutions that significantly affected the drp-lyase activity of pol ι, thus suggesting a possible location of the drp-lyase active site. furthermore, we showed that deletions in the n-terminus of pol ι and metal ions modulate its drp-lyase activity, which may play an important role in the regulation of pol ι activities in vivo. this work was supported by russian foundation for basic research grants 15-04-08398-a and 15-34-70002-mol-a-mos and by the russian academy of sciences presidium program in molecular and cellular biology. rosmarinus officinalis, commonly known as rosemary, is an aromatic plant belongs to lamiaceae family. from past to now, rosemary have been used as a traditional medicine to cure for various illnesses such as diabetes, rheumatism and cancer. recent studies have shown that rosemary is effective for various cancer types. in this study we aimed to investigate the effect of rosemary in glioblastoma cells (gbm) by comparison with etoposide and the effect of rosemary by concurrent application with the etoposide. gbm cells (u87 mg) were seeded into the 24 well plates and cultured with dmem supplemented with 10% fetal bovine serum. rosmarinus officinalis tea was prepared just as traditional usage and filter sterilized. at the second day of the culture rosemary in 1/75 (v/v) dilution ratio was given to first group, 40 lm etoposide was given to second group, 1/75 (v/v) diluted rosemary and 40 lm etoposide together were given to third group. after one day incubation cell viability was measured by neutral red assay. it was observed that rosemary reduced the viability of gbm cells by nearly %38, etoposide reduced the viability by nearly % 49 and rosemary with the etoposide reduced the viability by nearly %50. the results showed that rosemary was able to reduce the viability of gbm cells but hadn't got an increasing or inhibiting potential over the etoposide's cytotoxic effect. from our previous studies we know that rosemary increases the proliferation of mouse embryonic fibroblasts. it is considered that rosemary might have a protection potential from dna damages and when rosemary is used with etoposide during the cancer treatment, it might reduce the side effects on healthy cells. in conclusion rosemary promises hope for developing new cancer treatment strategies and reducing the side effects of chemotherapeutics. for further studies it is aimed to examine the effects of rosemary with other chemotherapeutics and if rosemary has got a protection potential from the genotoxic stress. morpholine moiety has been found to be an excellent pharmacophore in medicinal chemistry and a number of molecules possessing morpholine skeleton are the clinically approved drugs. in this present study, we aimed to investigate the possible underlying apoptotic mechanism for the cytotoxicity of new morpholine dithiocarbamate derivatives bearing 1-(2-aryl-2-oxoethyl)-2-substituted benzimidazole moiety on c6 glioma. c6 glioma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by cell proliferation analysis using standard (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (mtt) assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic50 values of the compounds were determined for c6 cell line. compounds 1, 2, 8, 9, and 10 , which were including hydrogen, 4-methyl, 3-methoxy, 3-chloro and 3-floro substituents on phenyl acetyl moiety, had significant cytotoxic activity with ic 50 values lower than 55 lg/ml. compound 10 showed the highest cytotoxic activity with a ic 50 value of 28 lg/ml, whereas cisplatin ic 50 values were 15 lg/ml against c6 cells. cytotoxic activity of compound 1, 2, 8, 9 and 10 with a ic 50 value were 42, 55, 50 and 30 lg/ml, respectively. compound 2, 9 and 10 showed the highest population of early apoptotic cells as 11.5, 10.1, and 10.3% respectively compared to cisplatin (6.2%). also, compounds caused dna synthesis inhibition depend on their ic50 values by brdu assay. conclusions: it was concluded that synthesized compounds had considerable anticancer activity against c6 cell lines. however, compound 2, 9 and 10 including 4-methyl, 3-chloro and 3-floro substituents were the most active compounds against the c6 cell line. also our study results showed that compound 2, 9, 10 induced apoptosis in c6 glioma cells. rutin is a glycosided flavonoid and known to have antioxidant and anti-inflammatory properties.trail induces the apoptosis of tumor cells and has no significant toxic effect on normal cells. although trail is a promising anticancer agent, trail resistance is a major barrier to effective cancer therapy. this study was conducted to examine the utility of the combined use of rutin and trail in prostate cancer cells. pc-3 and du145 prostate cancer cells were treated with rutin (1-1000um) and/or trail (20 ng/ml), cell viability and migration were examined. cell viability was determined by trypan blue exclusion and mtt assay. cell migration was determined by wound healing assay. furthermore, lactate dehydrogenases (ldh) levels of medium were determined as biochemical markers of cell viability. pc-3 and du-145 prostate cancer cells were treated with rutin for 24 and 48 hours incubation and ic 50 doses for 48 hours incubation were determined 250um and 1000um respectively. treatment with rutin, pc-3 cells is more sensitive than du145 cells. rutin and rutin plus trail inhibit prostate cancer cell growth in a dose-dependent manner. treatment with trail has no effect at inhibiting growth of pc-3 and du145 prostate cancer cells. the combination of rutin and trail elicit a synergistic antitumor effect on pc-3 and du145 prostate cancer cells. there is a significant increased in rutin and rutin+trail treatments group of ldh activities with respect to control and trail group. conclusion: present data show that rutin efficiently enhanced trail effects in prostate cancer cells. combined treatment with rutin and trail is more effective than the individual treatments of trail at inhibiting growth of prostate cancer cells. p-05.01.1-030 determination of antigenotoxic, proliferative and cytotoxic properties of ellagic acid since ancient time, people use plant for traditional treatment. plants or fruits are produced different type of secondary metabolites. particularly phenolic phytochemicals from plants play an important role in the prevention and treatment of radical damage by inactivating the reactive oxygen compounds due to their antioxidant properties. however, the structure and the activities of many herbal products are not fully elucidated yet and there are several studies about the toxicity of herbal antioxidants and their possible risks to human health. ellagic acid, phenolic compounds, is an important substance. ellagic acid is a naturally occurring plant phenol found in numerous fruits, including blackberries, raspberries, strawberries, cranberries, walnuts, pecans, pomegranates and wolfberries. different researchers give some information about the biological activities of ellagic acid. in this study, we aimed to determine the cytotoxic, proliferative and antigenotoxic effects of ellagic acid, which is phenolic compounds found in natural products. cytotoxic effects of ellagic acid on huvec is investigated by lactate dehydrogenase (ldh) and cell proliferation (wst-1) methods; and antigenotoxic effects against ccl 4 on human lymphocytes is investigated by single cell gel electrophoresis (comet) methods. the rusults showed that high concentration (100 and 200 lm) of ellagic acid has cytotoxic and mutagenic effects, but showed antiproliferative effects. on the contrary, low concentrations (4, 8, 12.5 lm) of ellagic acid has anticytotoxic and antimutagenic effects. as a conclusion, low concentrations of ellagic acid might be use treatment of some disease. but high concentrations of ellagic acid constitute a risk factors for people. keywords: cytotoxicity, antiproliferation, wst-1, ldh, rtca-sp the constitutive nuclear factor kappa b (nf-kb) activation is widely found in diverse types of hematologic malignancies such as acute myeloid leukemia (aml) and chronic myeloid leukemia (cml) as well as solid tumors. inhibition of nf-kb signaling via proteasome inhibitors such as bortezomib can induce apoptosis in myeloid leukemia cell lines. however it is not clear whether the cytotoxic effects of bortezomib on myeloid leukemia cell lines is due to direct inhibition of nf-kb or another pathway, such as dna damage. in this study, cml cell line k562 and aml cell line hl-60 were treated with bortezomib (bor) , etoposide (eto) and camptothecin (cpt) alone or in dual combination with these drugs, following by measuring the effects on cell viability, apoptosis and signal pathways. the effect on cell viability was determined using the mtt assay. the data were used in combination index and isobologram analysis. the expression levels of apoptototic genes (bcl2, bax and caspase 3), the related dna damage genes (atm and atr) and the involved genes in nf-kb signaling (rela and p50) were determined by real time rt-pcr. we showed that combinations of bor with topoisomerase inhibitors (cpt and eto) exhibited synergistic cytotoxic effect in k562 cell line but not in hl-60 cell line. the combination treatment increased apoptosis and dna damage response. dnadamage-sensing kinases were detected in k562 and hl-60 cells following treatment with bor as similar as topoisomerase inhibitors. bor increased the mrna levels of atm and atr dramatically, which indicated active dna damage in the myeloid cell lines. furthermore, bor induced apoptotic cell death by decreasing bcl2 and increasing bax and caspase 3 levels. these effects of bor were observed to correlated with increasing the p65 expression levels. this study on the mechanism of action of bor indicates that this compound affects several pathways involved in the control of cell cycle progression, apoptosis and dna damage. p-05.01.1-032 analysis of molecular cytogenetic alterations in gastric and colon carcinoma by array-based comparative genomic hybridization (array cgh) introduction: genomic dna regions are frequently lost or gained during tumor progression. we aimed to evaluate tumor samples of patients with gastric cancer and colorectal carcinoma to show these genetic alterations by array-based comparative genomic hybridization (array cgh) method. materials and methods: dna isolation was performed from the tumor samples obtained from sixteen patients with primary gastric adenocarcinoma and twelve patients with colon adenocarcinoma. then, agarose gel electrophoresis was performed in those dna samples. following electrophoresis of dna, array cgh procedure was performed to four patients with gastric adenocarcinoma and three patients with colon adenocarcinoma who had dna breaks with 100-200 kb. results: after array-cgh study, many common genetic changes in gastric and colon cancer genome were determined. in gastric cancer dna samples, common losses were detected in chromosome 1p34. 1, 1p13.3, 1q22, 3q29, 5p13.2, 6q24.1, 7q11.23, 7q22.1, 8q 12.1, 9p12, 11q13.1, 12 q24.31, 14q32.31, 16q22.1, 17q21.2, 19q13.3, and 20q11 .23, and also common gains were detected in chromosome 3p26.1, 6q27, 8q12.1, 15q11.2 and xq25. in colon cancer dna samples, common losses were detected in chromosome 1p34. 1, 3q29, 7p22.2, 7p11.21, 9q34.11, 12q24.31, 17q21.2, 17q25.1, 19p13.3, 19p13.33, 20q11.21, and 20q11.23 , and also common gains were detected.in chromosome 14q32. 33, xp22.33, xp22.11, xp13.3, xp11.1 and xq25 . both in gastric and colon cancer dna samples, common losses were detected in chromosome 12q24. 31, 17q21.2, and 19p13.3 , and common gains were detected in xq25. discussion and conclusion: we think that these common changes, generally in dna loss areas harboring tumor suppressor genes and dna gain areas harboring oncogenes, may important in gastrointestinal tumorigenesis. the dna of every cell is under a constant attack by various mutagenic factors which damage the dna and can cause cell cycle arrest and even cell death. accumulation of dna damage is the basis for cancer development and one of the reasons for aging of the organisms. in order to preserve the integrity of its dna cells have evolved an impressive array of dna repair pathways, which are precisely coordinated with the progression of the cell cycle. one of the first events at the site of dna damage is poly(adp-ribose) polymerase 1 (parp1) recruitment which is a sensor for single strand breaks in dna. parp1 catalyzes the synthesis of poly(adp-ribose) or par which is needed for the recruitment of many other dna repair proteins by means of par-binding domains. we used high speed confocal spinning-disk microscopy of living cells to obtain precise kinetics of recruitment of par-dependent proteins to the sites of laser induced dna damage. our results show that the investigated par-dependent proteins are recruited to dna damage sites in the matter of seconds, they reach peak intensities for 20 to 30 seconds after damage infliction and start dissociating. the recruitment of the proteins is entirely dependent on par because addition of parp inhibitor abbrogated their recruitment. the use of spinning-disk microscopy of living cells allowed us to obtain the kinetics of recruitment of the studied proteins to the sites of dna damage. the results are consistent with the fact that parp1 and par-dependent proteins are quickly recruited to damage sites and generation of par is essential for other dna repair protein recruitment. the precise kinetic curves may serve as a basis for investigating how they will change or if they will change at all when cells are put in different conditions or treated with various chemical substances affecting dna metabolism and repair. introduction: chronic myeloid leukemia (cml) is a myeloproliferative disease associated with reciprocal translocation between chromosomes 9 and 22. bcr-abl fusion gene which exhibits constitutively active tyrosine kinase activity has a main role in cml. the tyrosine kinase inhibitor imatinib is used as a first line treatment in cml patients, but imatinib resistance leads to failure in therapy. the application of imatinib in combination with other anticancer agents may be a strategy to increase the antileukemic effect of imatinib. in this study, we have investigated the antiproliferative effect two novel agents: a benzamide derivative xt5 and a benzoxazole derivative xt2b in combination with imatinib. these molecules were investigated in imatinib-sensitive (k562s) and imatinib-resistant (k562r) cml cell lines. materials and methods: antiproliferative and apoptotic effects were assessed by mtt assays and flow-cytometry, respectively. we also evaluated the effects of these compounds on the expression of apoptosis-related genes bax, bcl-2, bad, bim, bcl-xl and mcl1 by real-time quantitative pcr. results: treatment of k562 cells with xt5 increased the expression levels of the pro-apoptotic genes bax, bad and bim in both sensitive and resistant cells. however, xt2b was not found to have similar effects on k562r and k562s cells. combined application of xt5 increased cell death in the mtt assay. mtt assay demonstrated that ic50 for xt5 treated cells in k562r with imatinib (ic50 = 3.5) is lower than k562r without imatinib (ic50 = 8.5). discussion and conclusion: our results showed that combining xt5 with imatinib has more antiproliferative and apoptotic effect on a cml cell line. as a result combination of xt5 with imatinib can be an alternative approach to overcome imatinib resistance. introduction: the mmr(mismatche repair) system recognizes base-base mismatches and insertion or deletion loops in doublestranded dna, and it degrades the error-containing region of the newly synthesized strand, allowing the polymerase to correctly resynthesize the second strand according to the template sequence. the human mmr system includes the mlh1 and msh2. alteration in expression or a defect in mlh1 or msh2 can cause resistance to anti-cancer drugs used in chemotherapy. the attempt of the mmr system to detect drug induced dna damage, triggers the activation of apoptosis, a mechanism which may enhance the cytotoxicity of chemotherapy. loss of the mmr system would make the neoplastic cell less able to initiate apoptosis. inability to initiate apoptosis could be a mechanism of resistance to drugs. chronic myeloid leukemia (cml) is a clonal disease originating from aberrations in hematopoietic stem cell. imatinib, a tyrosine kinase inhibitor has significantly improved clinical outcome for cml patients. however, patients develop resistance when the disease progresses to the blast phase (bp) and there are several mechanisms involved in imatinib resistance. in this study we investigated the role of mmr system in imatinib resistance. materials and methods: k562s (sensitive) and k562r (resistance) were grown in rpmi-1640. k562r cells were maintained in rpmi-1640 medium supplemented with 5 lm imatinib rna isolation, cdna synthesis, rt-pcr was performed respectively. results: the results demonstrated that expression of mlh1 in k562r cells is dramatically lower than equal amount of imatinib treated k562s cells, whereas msh2 expression level did not change in both cell lines. conclusion: it can be suggested that alteration and down-regulation of mlh1 genes leads to imatinib resistance. p-05.01.1-037 characterization of interaction between rad51 inhibitor dids and human serum albumin d. velic, s. henry, c. charlier, m. popova, p. weigel, j. masson, i. nabiev, f. fleury cnrs/university of nantes, nantes, france 4 0 -diisothiocyanostilbene-2,2 0 -disulfonic acid (dids) has been largely used during the last 30 years for its inhibitory effect on anion transporters and channels. more recently, ishida and colleagues have described a possible mechanism by which dids inhibits rad51-mediated homologous pairing and strand exchange, key processes in dna repair by homologous recombination. thus, dids could act as a potential revertant of radioand chemo-resistance in cancer cells, which is the major cause of failure during therapeutic protocols. new drugs targeting rad51 protein have since been developed with potential use for medical applications. in this context, we attempted to determine the behaviour of dids towards blood and plasma proteins such as serum albumins. firstly, we analysed the effects of several environmental factors such as solvent polarity, which may affect the stability of the molecule. secondly, we analysed the spectroscopic properties of dids in the presence of human or bovine serum albumin proteins. uv-visible absorption, circular dichroism, fluorescence spectroscopy and isothermal calorimetry were used. here we show for the first time that dids can interact with both serum albumins. we have also determined the characteristics of these interactions. the comparison of several dids derivatives led us to identify the essential chemical moiety of this compound involved in the interaction. moreover, by using site competition approaches we show that the main binding site for this molecule is in subdomain ib of the protein. these findings show that the binding of dids to serum albumin proteins may change the equilibrium between the free and bound dids forms, thereby affecting its bioavailability and efficiency against the rad51 recombinase protein. p-05.01.1-038 mechanism of tap73 beta-mdm2 autoregulation p73 is a transcription factor which is the member of a p53 family. it regulates many cellular processes, such as apoptosis, cell cycle, and senescence. in contrast to p53, p73 is rarely mutated in tumors and elevated p73 expression is observed in many types of cancers including hepatocellular carcinoma, neuroblastoma, and lung. defining regulatory mechanisms which control p73 protein abundance and activity will be crucial for the development of new therapeutic strategies for cancers. mdm2 is known as the key player in regulation stability and activity of p53. in addition, p53 induces mdm2 transcriptional activity, and caspase-2, 3 activations which cleave mdm2 n-terminal at asp 367. cleaved form of mdm2 binds p53 and promotes its stabilization. mdm2 suggested as a candidate to modulate p73 activity and stability too. however, an interaction between p73 and mdm2 has not defined well. in this study, we aimed to analyze the role of mdm2 in p73 stability. to define this relationship, firstly, we overexpressed the tap73beta isoform using trex system in hep3b. tap73 beta and mdm2 protein levels were determined by western blot. to examine whether mdm2 mediate tap73 beta protein degradation by the proteasomes, cells were treated with proteasome inhibitor, mg132 for 4 hours prior to analysis. previous studies showed that p53-induced caspase-2 and caspase-3 activation cleaves mdm2. considering this, we firstly examined caspase-3 activation by western blot in hep3b tap73 beta cells. then we analyzed expression of cleaved mdm2 and tap73 beta levels following caspase inhibitor, z-vad-fmk treatment. as a conclusion, tap73 beta-induced full-length mdm-2 expression. furthermore, tap73 beta enhanced cleavage of mdm2 via increased caspase-3 activation. in addition, inhibition of caspase-3 activation caused a decrease in cleaved-mdm2 levels in parallel with tap73 beta expression repression. our results suggested positive regulation between mdm2-tap73 beta. hepatocellular carcinoma (hcc) is one of the most common type of liver cancer and third leading cause of cancer related deaths in worldwide. discovery of new targets is important in survival of hcc patients. p73 is a transcription factor which is the member of p53 family. it has two promoters; while p1 promoter expresses apoptotic ta isoforms, p2 promoter expresses anti-apoptotic dn isoforms. in addition, alternative splicing in c terminal creates many isoforms of ta and dn p73. it has been shown that both tap73 and dnp73 isoforms are expressed in hcc patient tissue and cell lines. the ratio between tap73 and dnp73 affects the apoptotic response, drug response and prognosis. accordingly, identification of the role of p73 and its targets are important in discovery of new treatment strategies in hcc. to understand the role of p73 isoforms in hcc, firstly we performed mtt assays following dna-damaging drugs and multikinase inhibitor, sorafenib treatment to categorize hcc cell lines as resistant or sensitive. after that, we analyzed the expression levels of tap73 isoforms via western blot in all hcc cell lines. then we overexpressed the tap73beta isoform using trex system in hep3b and snu449 cells. these two clones were analyzed for dna damaging drug response by mtt, cell cycle and apoptosis by flow cytometry, and tumor formation by in vitro and in vivo experiments. in scope of our study; 1. only tap73 alpha isoform is expressed in a few hcc cell lines. 2. there is no correlation between basal expression of p73 isoforms and drug responses in hcc cell lines. 3. there is no change in expression of p73 isoforms after treatment of drugs. 4. we showed that the ectopic expression of tap73beta in hep3b arrested the cell cycle in g1/ s and decreased the colony formation. therefore, the capacity of tumor formation of the cells dramatically decreased in scid mice. as a result, we revealed that tap73 beta play role in tumor formation, cell cycle arrest, dna damage responses in hcc. p-05.01.1-040 biochemical characterization of exonuclease iii-family ap endonuclease point mutants reveals role of conserved amino acid residues in the nir-specific enzymes a. mursalimov, z. koshenov, t. yeleussizov, m. redrejo-rodriguez, a. ishchenko, b. t. matkarimov, m. saparbaev national laboratory astana, astana, kazakhstan oxidative dna damage caused by reactive oxygen species is believed to be a major type of endogenous cellular damage. oxidatively damaged dna bases are substrates for two overlapping repair pathways: dna glycosylase-initiated base excision (ber) and apurinic/apyrimidinic (ap) endonuclease-initiated nucleotide incision repair (nir). in the ber pathway, an ap endonuclease cleaves dna at ap sites and 3 0 -blocking moieties generated by dna glycosylases, whereas in the nir pathway, the same ap endonuclease incises dna 5 0 to a number of oxidized bases. majority of characterized ap endonucleases possess classic ber activities and about half of them are able to catalyze nir activity. at present, the molecular basis of dna substrate specificities of various ap endonucleases remains unclear. here, we examined amino-acid sequence requirement of the nir activity of human major ap endonuclease 1 (ape1). amino acid sequence alignment of various ap endonucleases including e coli exonuclease iii (xth), human ape1 and archaeal mth212 revealed conserved amino acid residues in the nir-specific ap endonucleases ape1, mth212 and exoa that are absent in xth. based on these data, we constructed four ape1 point mutants y128h, n174q, g231s and t268d and examined their dna substrate specificities. results obtained from biochemical characterization of ape1 mutants are discussed in the light of the evolutionary conserved dna repair functions of ap endonucleases and whether these functions can be mutationally separated from. since its discovery some 40 years ago, cisplatin has evolved for its efficacy in one of the most used drugs in treatment of various cancer types. huge effort was invested in understanding the action of cisplatin and development of more potent drugs. they target mainly neighboring purine bases of nuclear dna forming covalent intra-or inter-strand cross-links that affect inhibition of replication and transcription, cell cycle arrest, and attempted repair of the damaged nucleotides. if such damage cannot be removed the cell dies. we have studied the details of the binding site of the short oligonucleotide modified by a platinum compound using complementary solution techniques used in modern structural biology, including raman spectroscopy with dft calculations aided interpretation of the obtained vibrational spectra. moreover, the calculated structure of the dna duplex was verified using saxs (small angle x-ray scattering) curve. in our contribution, we will present an nmr structure of a dna cross-linked with a cisplatin derivative containing a cyclohexane ring. at this atomic level resolution, structural features probably influencing cytostatic effects are described and compared with previously published structures. common structural features of previously determined structures are: a significant roll (25-60°) of the guanine bases involved in the cross-link, bending and unwinding of the double helix at the site of cross-link and orientation towards the major groove. also, the platinum-guanine plane angle varies between 19 and 54°. although the experimental structures were often used as the starting models for molecular dynamics (md) simulations, results of these md still leave many questions unresolved. the results of this research have been acquired within ceitec 2020 (lq1601) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. p-05.01.1-043 ercc2/xpd polymorphisms and colorectal cancer risk: a case control study in a north eastern iranian population j. mehrzad islamic azad university, neyshabur, iran excision repair cross-complimentary group 2 (ercc2) is one of the important dna repair genes.ercc2 codon 751 and 312 polymorphisms has been shown to modulate cancer risk. we therefore assessed the relationship between the ercc2 polymorphisms and the susceptibility to colorectal cancer in a case-control study. there were 105 lung cancer cases and matched healthy controls in this study. information concerning demographic and risk factors was obtained, each person donated 2 ml blood for biomarker testing. ercc2 genotypes were determined by t-arms-pcr method. all of the statistical analyses were performed with spss (v 20.0). there was significant difference between the frequencies of ercc2 polymorphism in cancer cases and controls (p < 0.05). the frequencies of ercc2 751 gln allele were 6.2% in controls and 13.8% in cancer cases. the individuals with lys/gln+gln/gln combined genotype were at an increased risk for lung cancer as compared with those carrying the lys/lys genotype (adjusted or=2.80, 95%=ci 1.21à6.48). the above findings indicate that the genetic polymorphism in the ercc2 codon 751 is associated with the risk of colorectal cancer in an iranian population (neyshabur citizenship). peptide pore blockers are potent tools to study structure and function of potassium voltage-gated channels (kv). kcsa-kv1.x chimeras, in which a ligand-binding site of eukaryotic kv-channel is inserted into bacterial kcsa channel, mimic properly the pore domain of kv-channels. a fluorescence-based approach to study the binding of peptide blockers with kcsa-kv1.1-kcsa-kv1.3 chimeras was developed by us. this approach rested on high-level expression of kcsa-kv1.x chimeras in e.coli inner membrane, binding of fluorescently-labeled toxin at the surface of the spheroplast and analysis of competitive binding of studied ligands by laser scanning confocal microscopy (lscm). here we report on a new analytical system for search and study of kv1.6-channel blockers that combines bl21 (de3) cells expressing kcsa-kv1.6 and rhodamine-labelled agitoxin 2 (rh-agtx2) as a fluorescent probe. by tuning cultivation conditions, the high-level of membrane expression of kcsa-kv1.6 was achieved. it was found that lowering both the growth temperature and the concentration of inducer resulted in significant increase in membrane-embedded kcsa-kv1.6. for system validation, wellknown kv1 channel blockers were studied by the method of competitive binding, and equilibrium dissociation constants were estimated for agtx2, osk1, and kaliotoxin. a new system was applied to study molecular determinants of peptide-kv1.6 channel binding using a number of agtx2 mutants constructed by us, whose affinities to kcsakv1.6 were measured. a new bioengineering fluorescent system is a robust and sensitive assay for assessing the binding activity of kv1.6 channel blockers. it can be used to study interaction interfaces of toxinchannel complexes, to search for novel peptide blockers and to develop new potent and selective kv1.6-blockers for scientific and medical purposes. the work was supported by the grant 14-14-00239 from russian science foundation. asparagus racemosus root extracts (ar) have been exhibited to show a wide range of pharmacological benefits. in this study, liposomes of ar were developed and assessed their physicochemical properties and anti-inflammatory activity in monocytic leukemia cell line (thp-1). liposomes containing ratios of ar to lipid and phosphatidylcholine to cholesterol ratio were synthesized by thin-film hydration (tf), reverse-phase evaporation (rev), and polyol dilution (pd). the in vitro anti-inflammatory activity was assessed in terms of inhibition of tumor necrosis factor alpha (tnf-a) in lipopolysaccharide activated thp-1 by elisa. the size of ar liposomes prepared by tf were larger, whereas those prepared by rev and pd were smaller. ar to lipid ratio was shown to have no influence on particle size, whereas zeta potential enhanced with increasing ar to lipid ratio. ar liposomes with lipid ratio of 1:5 achieved the highest value of entrapment efficiency and were at the highest with polyol dilution method. ar was found to have no toxic effects on thp-1 cells. the anti-inflammatory activities of ar and ar liposomes in terms of tnf-a in thp-1 cells were was exhibited to possess the highest values of around 52% at ar concentration of 1 lg/ml and % tnf-a inhibition tended to decline with the increasing amount of ar. this result may be attributed to the increased amount of liposomal particles being uptaken into the cells as a result of the increasing ar concentrations. it can be suggested that ar liposomes could be an alternative choice of topical/transdermal drug delivery for anti-inflammatory activity. p-mis-002 inhibition of ire1 signaling enzyme increases the expression of tumor suppressor genes and modifies their hypoxic regulation in u87 glioma cells d. tsymbal, o. minchenko palladin institute of biochemistry of the national academy of sciences of ukraine (nasu), kyiv, ukraine gliomas constitute one of the most aggressive groups of malignant neoplasms with poor survival prognosis and scarce therapeutic options. plentiful studies have proven the connection between endoplasmic reticulum stress and malignant growth. we have studied the effect of inhibition of ire1 (inositol requiring enzyme 1), which is a central mediator of endoplasmic reticulum stress and controls cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in u87 glioma cells. it was shown that inhibition of ire1 leads to up-regulation of the expression of krt18, cd24, mest, cenpu, myl9, ing1, ing2, mybl1, and mybl2 genes at the mrna level in u87 glioma cells, with more profound changes for mest, mybl1, and cd24 genes. hypoxia leads to up-regulation of the expression of cd24, ing1, and ing2 genes and to down-regulationof krt18 gene in glioma cells. at the same time, inhibition of ire1 modifies the effect of hypoxia on the expression of all studied genes: suppresses effect of hypoxia on ing1 gene, eliminates hypoxic regulation of krt18, cd24, and ing2 genes in glioma cells. the present study demonstrates that inhibition of ire1 enhances the expression of all studied genes and modifies the hypoxic regulation of these gene expressions in gene specific manner and thus possibly contributes to slower glioma cell proliferation, but several aspects of this regulation remain to be further clarified. amplification and clonig of dna polymerase 1 (pol1) of thermus scotoductus k1 isolated from an armenian goethermal spring a. saghatelyan, h. panosyan, a. trchounian, n. birkeland yerevan state university, yerevan, armenia the most important enzyme ''mined'' from thermophilic microorganisms is dna polymerase, which widely used in molecular biological studies. although dna polymerase produced by thermus aquaticus (taq polymerase) was launched into the market long back, isolation of more processive, reliable and stable dna polymerases from other species is a demand. the purpose of this work was to amplify and clone the pol1 gene of t. scotoductus strain k1 recently isolated from an armenian geothermal spring. the draft genome sequence of strain k1 was deposited under accession number ljjr00000000.1. genomic dna was isolated using genelute bacterial genomic dna kit. primers for the pol1 gene were designed manually. the gene was amplified using pfu polymerase, and amplicons (~2.5 kb) were ligated into the pet-21b(+) vector (novagen) and transformed into chemically competent top10 escherichia coli. inserts were sequenced with t7prom and t7term primers, which showed that the gene sequence was correct and in the right reading frame and could be expressed in mesophilic e.coli. dna polymerases patented form different species of thermus are mostly comparable, suggesting that only limited natural variations in taq-like dna polymerase may be discovered. the pol1 gene from k1 shares 99% and 83% similarity with pol1 of t. scotoductus sa-01 (2.0 kb) and t. aquaticus, respectively. although the difference is not huge at sequence level, possible functional differences (e.g. stability, proofreading activity, resistance to different pcr inhibitors etc.) may occur. therefore, it is important to express and purify dna polymerase from strain k1 for further investigations. peptide ligands of the immunoglobulin g fc region identified by screening phage libraries and site-directed mutagenesis n. kruljec, p. molek, t. bratkovic young researcher, ljubljana, slovenia affinity chromatography based on immunoglobulin (ig)-binding proteins, such as staphylococcal protein a and streptococcal protein g, typically represents the initial step in therapeutic antibody purification process. however, this approach suffers from high cost, poor ligand stability and the requirement for relatively harsh elution conditions that can negatively impact activity and immunogenicity of antibodies. compared to protein ligands, peptides represent an interesting alternative due to higher stability and less expensive production. furthermore, the expected lower affinity for immunoglobulins should allow for elution under milder conditions. the aim of our research was to identify short peptide ligands for the fc region of human iggs. we have screened three commercially available phage display libraries of random cyclic and linear peptides for binding to human fc region in solution using an optimized biopanning approach. five non-homologous linear peptides were shown to specifically interact with the fc portion of immunoglobulins as verified by a set of phage elisa assays. individual phage-displayed peptides were able to recognize specific subclasses of igg. the highest-affinity peptide (12l-19fc), which competed for fc binding with protein a, was subjected to mutagenesis studies. we displayed on phage several variants of 12l-19fc with individual amino acid residues exchanged for alanine as well fragments of the parent peptide of different lengths and evaluated binding to fc with phage elisa to identify the minimal binding motif. binding characteristics of the minimized peptide were further analyzed using spr biosensor. the details will be disclosed at the meeting. diverse effects of ganoderma lucidum in combination with tamoxifen citrate and doxorubicin in mcf-7 breast cancer cells ganoderma lucidum, an edible medicinal fungus, has been known with its anti-metastatic, anti-carcinogenic bioactivities and widely used in asian countries in complementary and alternative medicine. however, there is no information regarding its combined usage with tamoxifen and doxorubicin in breast cancer treatment. we investigated the interactions between ganoderma lucidum and tamoxifen or doxorubicin in mcf-7 human estrogen receptor positive breast cancer cell line. anti-proliferative properties of six extracts were assessed by wst-8 method. the most effective extract in inhibition of mcf-7 cell viability was then evaluated in terms of its anti-metastatic activity by boyden chamber assay. apoptosis and cell cycle assays were performed by flow cytometry. ganoderma lucidum ether extract (g.ether) was the most effective extract on inhibition of cell viability among others with ic (50) values of 100 lg/ml and 12.82 lg/ml at 48 h. and 72 h. respectively. we found that g.ether is capable of inducing apoptosis and changing cell cycle dynamics. however, incubation with g.ether did not affect mcf-7 cell motility significantly. we then assessed the interactions between g.ether and tamoxifen or doxorubicin in mcf-7 cells. the interactions between g.ether and cancer therapeutics were examined by combination index analysis and macsynergy ii software. interestingly, g.ether increased the anti-proliferative effect of tamoxifen although exhibited strong antagonism with doxorubicin in mcf-7 cell line. testing the best matrix/analyte combination for maldi tof mass spectrometric detection of steroid hormones, amino acids, vitamins and carbohydrates in spite of numerous advantages, there are serious drawbacks of the application of matrix assisted laser desorption/ionization time-of-flight mass spectrometry (maldi tof ms) for smallmolecule analyses (below 500 da) and quantification. the main problem is the background interference from commonly used maldi matrix materials. the aim of this work is to evaluate maldi tof mass spectra of physiologically relevant small molecules: steroid hormones, vitamins, amino acids and carbohydrates, acquired with several organic, traditional matrices. small volume, 0.5 ll, of each sample solution (testosterone, progesterone, estradiol, l-cysteine, l-alanine, dl-methionine, glutathione, d-(+)-glucose, d-(+)-maltose, vitamin a, vitamin e) was mixed on the sample plate with the same volume of organic matrix solutions (dhb, thap, chca, 9-aa). for each molecule/matrix pair, we determined quantitative and qualitative parameters of ms analysis. to calculate within day and day-today variation we used excel tools (anova tests). in addition, homogeneity of the sample/matrix distribution on the target was also calculated and expressed as the coefficient of variation of a series of measurements. our results show selectivity of the detection of individual molecules related with the matrix applied. the statistical analysis of certain molecule/matrix pairs gave within and day-to-day variations less than 15%. additionally, homogeneity of the sample/ matrix mixture distribution on the target plate was with some matrices, also less than 15%. some of the used matrices have a great potential for the analysis of small molecules with good analytical parameters, with low variations and high homogeneity of samples on the maldi target plate. these results hold potential for quantification of metabolically-significant small molecules and are very promising for future applications of maldi tof ms analyses. stress causes different expression of mitochondrial biogenesis markers in rat steroid-producing cells of adrenal gland and testes i. starovlah, s. radovic, t. kostic, s. andric faculty of science univeristy of novi sad, novi sad, serbia functional mitochondria of steroid producing cells of adrenal cortex and leydig cells of testes are essential for steroid hormones biosynthesis and regulation. the aim of this study was to determine transcriptional profile of mitochondrial biogenesis markers in adrenal cortex and leydig cells by applying in vivo and in vitro studies. immobilization stress (imo), was performed for 2 hours daily for one (1ximo), two (2ximo) or ten (10ximo) consecutive days. in in vitro studies, primary cultures of purified leydig cells from undisturbed rats were stimulated with stress hormone adrenaline, propranolol (nonselective b-adrs-blocker) and prazosin (the selective a1-adrs antagonist). rq-pcr results showed that the transcription of the main regulator of mitochondrial biogenesis, ppargc1a and ppargc1b, significantly decreased in adrenal cortex of 10ximo rats. oppositely, the significant increase of the same transcript was registered in leydig cells from the same rats. in parallel, transcription of ucp1, the mediator of regulated proton leak, decreased in adrenal cortex, but increased in leydig cells of the same group of rats. incubation of leydig cells with adrenaline, increased transcription of the main markers of mitochondrial biogenesis (ppargc1a, ppargc1b, nrf1 and nrf2a). nonselective b-adrsblocker attenuated this effect. the selective a1-adrs antagonist did not change adrenaline-induced stimulation of ppargc1a, ppargc1b, nrf1 and nrf2a transcription in leydig cells, indicating that the most of the effects are probably mediated by b-adrenergic receptors, not by a1-adrs of leydig cells. in summary, the results suggest that reduction of transcription of mitochondrial biogenesis markers could be a possible mechanism that protects body from excessive glucocorticoid production from adrenal glands in stress conditions, while at the same time stimulation of mitochondrial biogenesis markers transcription in leydig cells could serve as mechanism to preserve testosterone production. p-mis-008 generation of new mitochondria is possible protection mechanism of basal steroidogenesis in leydig cells s. radovic, i. gak, t. kostic, s. andric faculty of science, university of novi sad, novi sad, serbia mitochondria are the most important component of stress response in all cells and for steroid-hormones-producing cells they are the starting point for steroid biosynthesis. here we investigated the parameters of mitochondrial biogenesis in these cells from rats exposed to the psychophysical stress by immobilization (imo). imo stress was applied for 2 hours daily for one (1ximo), two (2ximo) or ten (10ximo) days.hormone levels were measured employing eia, elisa kit or ria. mitochondrial membrane potential (δwm) was measured by tmre fluorescence, mitochondrial mass was detected by quantitative analysis of mitotracker-green fluorescence as well as relative intensity of fluorescence, since number of mitochondria and mitochondrial architecture were defined using transmission electron microscopy. relative gene expression and proteins analyses were performed by rq-pcr and western blot. there was positive correlation between δw m of leydig cells and androgens production of leydig cells. both of them were reduced in all stressed rats but partially recovered in 10ximo group. the mitochondrial mass in leydig cells from 10ximo group was increased. transmission electron microscopy analyses showed that acute and two times repeated stress altered architecture of mitochondrial cristae, while 10ximo increased number of mitochondria and recovered mitochondrial architecture. there was significant increase in the expression of the all markers of mitochondrial biogenesis in leydig cells from 10ximo rats compared with other groups. accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. supporting the evidence that stress, a constant factor in life of humans, induces mitochondrial biogenesis in leydig cells, our results indicate this mechanism probably protects the basal steroid production in stress conditions. targeting survival pathways in leukemic cells through synergism of metformin and thymoquinone u. glamoclija, m. suljagic international university of sarajevo, sarajevo, bosnia and herzegovina generation of resistance to current treatment options is common problem in the therapy of many hematological malignancies. combined therapies utilizing compounds with low toxicity that act synergistically, are proposed to overcome this problem. metformin and thymoquinone (tq) are two molecules which have proven safety profile and represent potential candidates for treatment of hematological malignancies. there are more than 150 clinical trials, at different stages, exploring metformin anticancer activity. metformin activates amp activated protein kinase (ampk) leading to inhibition of the mammalian target of rapamycin (mtor) and induction of apoptosis in different cancers. however, human leukemic cells with increased basal protein kinase b (akt) phosphorylation were shown to be resistant to metformin-induced apoptosis. it was found that activity of metformin can be enhanced by combination with akt and/or nuclear factor 'kappa-lightchain-enhancer' of activated b-cells (nf-jb) inhibitors. tq is phytochemical compound that has shown inhibitory capacity on both of these targets. wst-1 assay was used to evaluate the effects of metformin and tq in dhl4 (b cell lymphoma) and k562 (chronic myelogenous leukemia) cell lines. compusyn software was used in order to calculate the combination index (ci). the ci value indicates whether two drugs have synergistic (ci<1), additive (ci=1) or antagonistic effects (ci>1). we have shown that separately, metformin and tq, exhibit dose dependent inhibition of dhl4 and k562 cells. in combinatorial study with fixed constant ratio and simultaneous drug exposure, in dhl4 and k562 cell lines, ci values were 0.68 and 0.66, respectively. to our knowledge, this is the first report showing synergistic effects of metformin and tq in lymphoma and chronic myelogenous leukemia derived cell lines. these promising data are currently being investigated in order to obtain the insight into their molecular mechanisms. for the last decade many methods of calculating and analysing the physical characteristics of dna has been developed. these methods allow to estimate distributions of free energy, propensity to bend, stress-induced duplex destabilization (sidd), electrostatic potential (ep) etc. and most of them have been used for prediction of genomic regulatory site positions. the main idea of such approach is that proteins recognize genome regulatory sites by these physical and chemical properties, so the physical characteristics are used to predict the location of regulatory sites. most of the characteristics mentioned above describe properties of dna at equilibrium or steady state, but we propose to use characteristics of internal dna dynamics. in this work we used the coarse-grained model of dna, developed recently, to simulate dynamics of the dna open states. with this model we were able to calculate trajectories of the open states moving along the molecule and their dynamical characteristics, such as: open state activation energy, size, half decay time and sound velocity in dna. we use distribution of four dynamical characteristics around transcription start site of experimentally found e.coli promoters taken from regulon db to organise them in stable clusters. clusterization was made with ward method and consensus clustering technique was applied to clusterization results for analysis of its consistency. the same procedure was applied to equilibrium dna characteristics for comparison. distribution of go functions among clusters was also analysed. stable promoter clusters obtained with different physical properties share some similarity. it was not surprise that clusters obtained with dynamical characteristics of dna more similar to sidd clusters then to ep clusters. the data highlights the possible role of dna dynamical properties in transcription initiation and its applicability to promoter identification together with other physical and textual properties of dna. chromium complex with 5-hydroxyflavone acts on metabolic pathways the development of novel therapeutic strategies for obesity treatment are urgently required as obesity is currently the main leading cause in type ii diabetes and insulin resistance. among natural compounds, flavonoids have recently gained interest due to their positive role in maintaining blood glucose levels and insulin secretion. their association with trace elements, wellknown for their capacity in increasing the efficiency of insulin, might potentiate flavonoids biological effects. in this context, the aim of our study was to investigate the in vitro changes in energetic metabolism related genes expression profile in the presence of a chromium complex with 5-hydroxyflavone. dna microarray technology was used for a large scale screening of differentially expressed genes in human adipose stem cells (hascs) after 3 weeks of adipogenic induction in the presence of the chromium complex with 5-hydroxyflavone. moreover, perilipin expression was assessed by flowcytometry. the chromium complex with primuletin negatively regulates the expression of key genes involved in adipogenesis and also modulates the expression of the genes associated with triglyceride synthesis and subsequent fat storage in mature adipocytes. consequently, the chromium complex with 5-hydroxyflavone can be further employed in studies on animal models to investigate the possible improvement of metabolic disorders. deinococcus radiodurans is a highly radioresistant and stress-resistant bacterium. despite extensive studies, the mechanisms of transcription regulation that contribute to the stress-resistance are still poorly understood. d. radiodurans encodes multipe stress-related proteins including three members of the gre-family of transcription factors: grea, gfh1 and gfh2. while grea is a universal bacterial factor that stimulates rna cleavage by rna polymerase (rnap), the functions of lineage-specific gfh proteins remain unknown. we cloned, expressed and purified d. radiodurans rnap and gfh factors and their mutant variants and analyzed their properties using various in vitro transcription approaches. we tested gfh effects on rnap activity in promoter, elongation and termination complexes assembled on natural and synthetic dna templates under different conditions. we found that the gfh factors strongly enhance site-specific pausing and intrinsic transcription termination by d. radiodurans rnap but do not act on active transcription complexes and do not compete with the grea factor. uniquely, the pause-stimulatory activity of gfh is greatly enhanced by manganese ions, which are accumulated in d. radiodurans cells under stress conditions, and is modulated by the secondary rna structure. we revealed functionally important regions in the gfh factors and the rnap active site involved in transcriptional pausing. we propose that gfh factors inhibit rna extension in paused complexes through binding within the secondary rnap channel, coordinating metal ions in the rnap active site and stabilizing an inactive enzyme conformation. this may serve as a sensitive mechanism to regulate transcription under stress conditions and coordinate it with dna repair and replication. our data suggest that gre and gfh proteins target different structural states of the transcription elongation complex and reveal functional diversity of the factors that bind within the secondary channel of rnap. from planktonic to biofilm state of growth, flagella formation is turned off, and the production of fimbriae and extracellular polysaccharides is activated. bola protein is widespread in nature and has been associated with several cellular processes. using high-troughput techniques we showed that bola protein is a new bacterial transcription factor, which regulates the switch between motile and sessile lifestyle. it negatively modulates flagellar biosynthesis and swimming capacity in escherichia coli. moreover, bola overexpression favors biofilm development, involving fimbriae-like adhesins and curli production. our recent results show that bola action in these pathways is related with cdi-gmp a relevant intracellular signaling molecule involved in biofilm formation. we demonstrate that bola contributes to a fine-tuned expression of different diguanylate cyclases and phosphodiesterases and c-di-gmp has a negative influence in the bola mrna transcription. herein we propose that bola is a key player in motile/adhesive transcriptional switch, contributing to a fine-tuned regulation of these important pathways. background: deep venous thrombosis (dvt) is an important health problem worldwide. its pathophysiology is multicausal and involves environmental, genetic and acquired factors. factor v leiden (fvl), prothrombin g20210a (pt g20210a), and methylenetetrahydrofolate reductase (mthfr) gene mutations are to predispose to venous thrombosis. the aim of this study was to compare the frequency of fvl, pt g20210a and mthfr polymorphisms between patients with dvt and healthy controls. methods: this study was conducted at the bozok university hospital. total 220 participants were included in this study, 126 patients with dvt and 94 healthy blood donors. in order to identify fvl, pt g20210a, mthfr c677t and mthfr a1298c, the polymerase chain reaction (pcr) method was utilized combined with the amplification refractory mutation system. results: in 126 patients fvl was present in 54 (42.8%) patients while in controls fvl was present in only 18 (19.1%). frequency of fvl was significantly higher in cases as compared to controls (p < 0.05). pt g20210a mutation was present in 13 patients (10.3%) and in 5 healthy participants (5.3%). mthfr c677t and mthfr a1298c polymorphisms were almost equally distributed among patients and healthy participants. however, the concomitant presence of fvl and double heterozygous polymorphisms of mthfr c677t/a1298c was found in 11 patients (8.7%) and in 2 healthy controls (2.1%), showing significant association with deep venous thrombosis. conclusion: in this study, the frequencies of fvl and pt g20210a polymorphisms were found significantly higher in patients with dvt than those in healthy participants. thus, fvl and pt g20210a polymorphisms have a contributory role on the development of dvt in contrast, mthfr c677t and mthfr a1298c genotypes were not associated with a predisposition to development of dvt. but, a combination of double heterozygous polymorphisms of mthfr c677t/a1298c with fvl may be associated with increased risk of dvt. p-mis-016 self-assembling micellar clusters comprising drugs, nanoparticles and fluorescent compounds for bilogical applications when designing drug carriers, the drug-carrier ratio is an important consideration, because the use of wrong drug-carriers relation can result in toxicity as a consequence of poor metabolism and elimination of the carriers. solubility problem of various substances also plays an important role in many aspects of fundamental science and practical field. specifically, it is an important parameter as well as bioavailability, which determines the required concentration of drug in the body needed to achieve a pharmacological response. among the variety of solubilization methods micellar solubilization is widely used as an alternative to the dissolution of poorly soluble drugs. here, we show a specific approach based on sequential selfassembly of nonionoc detergent micelles (t 9 100, tx114) followed by enacpsulation of various nanoparticles (noble metals, magnet etc.), drugs, fluorescent compounds leads to the formation of stable micellar nano-amd microcomplexes. we propose 3 ways of micellar clusterisation. in the first one micelles are modified by semi-hydrophobic chelator followed by addition of metal ion to make cross-linking. the second way is similar to the first one and suggests application of the metal complex with incresed denticity instead of naked metal ion, and the third one involves micelles clusterisation by semi-hydrophobyc metal complex directly. therefore, one can stabilize micellar network by means of 'interactions on interface': semi-hydrophobyc metal complexes are embedded inside micelle due to hydrophobyc interactions. hydrophobic fluorescent compounds-loaded micellar complexes demonstrates better optical response in aqueous media without crystallization. such obtaining clusters are also very flexible and can be modified by nanoparticles to obtain various nanocomposites, such as fluoromagnetic clusters. this work was supported by russian foundation of basic research grants no. 15-43-03214 r_center_a (2015 no. 15-43-03214 r_center_a ( -2016 no. 15-43-03214 r_center_a ( ) and 15-33-20002 mol_a_ved (2015 no. 15-43-03214 r_center_a ( -2016 . lamellipodia and membrane blebs utilize different signalling pathways to induce directional movement of walker carcinosarcoma wc 256 cells in a physiological electric field clear if those reactions are mediated by similar mechanisms. to establish that, we performed proteomic analysis and subsequent investigation of the role of differential signalling pathways in electrotaxis of cells representing various strategies of movement. cells were exposed to ef in galvanotaxis apparatus and their reaction was recorded. in some experiments cells were pre-incubated with erk1/2 or btk-1 inhibitors. the phosphorylation of erk1/2 and btk-1 was determined by western blot analysis. proteomic analysis was performed by ultimate 3000rs lc nanosystem coupled with a q-exactive mass spectrometer. both blebbing (bc) and lamellipodial (lc) cells show cathodal migration in a physiological ef (1 v/cm). comparative analysis of bc and lc cells proteomes revealed about 150 differential proteins. functional analysis in ingenuity analysis pathway allowed to determine the statistically significant signalling pathways in which these proteins are engaged. among the most distinctively regulated pathways are tec kinase and erk/ mapk signalling activated in lc but not bc. it was found that btk-1 is required for directional movement of lc but not for bc cells. moreover, ef induced stronger and faster btk-1 phosphorylation in lc than bc cells. in contrast erk 1/2 activity was not necessary for electrotaxis of lc cells and ef did not induce erk 1/2 phosphorylation. our results reveal that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of wc256 cells but it is mediated by different signalling pathways. this work was supported by a grant from the national science centre 2012/07/b/nz3/02909, poland. newborn screening for congenital hypothyroidism in turkey: a regional evaluation € o. demirelce 1 , n. y. saral 1 , f. b. aksungar 1,2 , a. coskun 1,2 , m. serteser 1,2 , i. unsal 1,2 1 acibadem labmed, istanbul, turkey, 2 acibadem university, istanbul, turkey congenital hypothroidism (ch) is the most common congenital endocrine disorder and the most important cause of preventable mental retardation. it is important to begin the treatment within 2 weeks before the development of brain damage. tsh based newborn screening programs are shown to be useful for implementing early treatment of ch. in this study, regional results of ch screening program in turkey between 2006 and 2015 were assessed retrospectively. we have evaluated the results of marmara, central anatolia, aegean and mediterranean regions in which our laboratories are located. screening was based on tsh determination in dried blood spot specimens. tsh limits determined to be 10 lu/ml for cut off point and 25lu/ml for clinical decision point. tsh was measured using enzyme immune assay (eia). blood spot tsh data for 65857 newborns during this time period were evaluated. permanent or transient ch was determined according to the results of thyroid function tests. confirmed ch cases were based on local endocrinologists' report and initiation of thyroxine treatment. the frequency of neonatal tsh levels were found to be under the cut off level of 10lu/ml in 63513 (96.44%), between 10 and 25lu/ml in 2287 (3.47%) and above the level of 25lu/ml in 57 (0.09%) babies, respectively. recall rate was 3.5%. ch cases of neonatal tsh levels greater than 25lu/ml were 26. the incidence of ch of this group was 1:2532. there were no significant differences in the number of congenital hypothyroidism between males and females (p > 0.05). the preliminary results of our study indicate that the incidence of ch in our region is higher than the worldwide reports as has been proved by preceding studies. iodine deficiency, dyshormonogenesis, highly consanguineous population, may contribute to the high incidence of ch in turkey. newborn screening of ch must be developed for detecting true cases and tsh cut off point must be reviewed for decreasing redundant recall rate. in silico analysis of the first complete genome sequence of lactobacillus acidipiscis species k. papadimitriou 1 , m. kazou 1 , v. alexandraki 1 , b. pot 2 , e. tsakalidou 1 1 agricultural university of athens, athens, greece, 2 institut pasteur de lille, lille, france introduction: lactic acid bacteria (lab) constitute a significant group of microorganisms for the food industry, as they play a key role in food fermentation and consequently in human health. lactobacillus acidipiscis aca-dc 1533 is a gram-positive, motile, rod-shaped lab isolated from traditional greek kopanisti cheese. here we present the in silico analysis of the first complete genome sequence of l. acidipiscis in order to explore the biology of the species. materials and methods: sequencing of l. acidipiscis genome was performed using the hiseq 2000 and pacbio rsii sequencing platform technologies and the genome assembly was validated against an nhei optical map of the l. acidipiscis genome. protein-coding sequences were predicted by glimmer, rrna genes by rnammer and trna genes by the trnascan-se server. potential genomic islands were detected using the island-viewer software tool, prophage regions by phast and the subsystem-based annotation by rast server. finally, the circular representation of l. acidipiscis genome keyed to the cog groups was constructed by cgview server. results: the sequencing analysis resulted in one continuous genomic scaffold of 2,678,726 bp with a g+c content of 39.75%. the genome contains 2,525 protein-coding genes on the chromosome covering up to 82.09% of the genome sequence, 63 trna and 18 rrna. according to the subsystem-based annotation, 1,562 protein-coding genes were assigned to 310 metabolic subsystems. the most abundant of the subsystems are related to carbohydrates (n = 287, 18.37% of total protein-coding genes) and protein metabolism (n = 173, 11.08% of total protein-coding genes). furthermore, three prophage regions were detected; one intact (43.5kb), one incomplete (14.7 kb) and one questionable (29.3 kb). discussion and conclusion: the whole genome analysis of l. acidipiscis aca-dc 1533 provided interesting information about a not well-studied species. investigation of serum irisin levels of patients with metformin taking new onset type 2 diabetes mellitus increases glucose tolerance and energy expenditure and improves carbohydrate homeostasis. metformin is a biguanidine class antidiabetic drug which inhibits liver gluconeogenesis and decreases insulin resistance and is frequently recommended in treatment of new onset type 2 diabetes mellitus (t2dm). irisin has a role in the regulation of energy metabolism pathways and its level in blood of persons with t2dm has been reported to decrease. regarding this relationship, it was aimed to reveal the effect of metformin on serum irisin levels. 20 patients with impaired oral glucose tolerance test were included to this investigation. they were recommended to take metformin and to change their life style, such as exercise and diet. their blood were taken at the beginning and after 1 month. also, a healthy control group (n = 20) was formed from persons with similar age and sexual distribution as the patient group. irisin levels of their sera were measured by enzyme-linked immunosorbent assay (elisa) method. statistical evaluation of the measurements showed no significant difference (p = 0.780) between the irisin levels of the patients at the beginning and after 1 month treatment. a similar result was found between the control and the treated groups (p = 0.170), while a significant difference (p = 0.002) was observed between the control and untreated patients groups. the results obtained from this study do not show a clear and significant change in the blood irisin levels of the patients with new onset t2dm taking metformin together with life style change. a longer period of treatment and a higher number of patients may be needed for more reliable results. thermodynamics of dna ligands binding at specific sites of telomeric g-quadruplex dna g-quadruplexes are a perspective target for anticancer therapy. for example stabilization of the telomeric g-quadruplex dna formed by single-stranded ends of the chromosomes leads to inhibition of telomerase, which is active in 90% of cancer cells. similarly, small molecules targeted to a specific g-quadruplex would inhibit various cellular processes. stoichiometry and affinity of interaction of these compounds to dna is determined by specific structural motifs within a g-quadruplex. rational design of novel chemical compounds requires an in depth knowledge of interactions between known ligands and g-quadruplex structures. experimental methods that are used for determination of thermodynamic binding parameters, such as isothermal titration calorimetry, differential scanning calorimetry, ultraviolet absorption and circular dichroism spectroscopy provide a collective characteristic for all of the ligand molecules bound to dna, while the information on ligand affinity to individual dna binding sites is lost. we propose a complimentary method for detailed analysis of thermodynamic parameters of ligand binding based on the introduction of fluorescent probes in the structure of g-quadruplex. monitoring fluorescence quenching of the fluorescent labels allows to derive binding constants of the dna-ligand interaction at a specific binding site. temperature dependence of the fluorescence quenching determines the thermodynamics of the dnaligand complex formation. since only a proximal ligand is able to quench the fluorescence, this method allows characterization of the ligand binding to a particular site the g-quadruplex structure. the study was supported by project no. 16-14-10396 of the russian science foundation. the correlation between biochemical and dynamic surface tension parameters of calves blood serum during the animal ontogenesis, as well as by various pathologies or poor diet, the imbalance of protein, mineral, lipid components is observed (the changes in all parameters of biological liquids are accompanied of these metabolism peculiarities). the dynamic surface tension (dst) of serum essentially depends on these factors and (in combination with the biochemical parameters) can provide the valuable information for evaluation of the physiological and biochemical status of the organism (can be used as an express test for animal diagnostics in future). the aim of the work was to study dst and biochemical parameters of calve serum, as well as their correlations, as the main indicators of the animals. ) of calve serum were in the range of the normal values for healthy animals and can be considered as reference data for animal science and practice. the obtained results enable us to establish correlations between the dst and biochemical parameters of calves serum. this work was supported by the russian scientific foundation (grant 14-16-00046). the middle strong correlations of dst values of calves serum with the level of total protein, albumin, billirubin, some enzymes and cholesterol, whereas only weak correlations with the other biochemical parameters (urea, calcium, magnesium, phosphorus, etc.) were found. in the veterinary science and practice such correlations are important for the estimation of the organism physiologicaland biochemical status, for general inspections of cattle before vaccination (immunization) or slaughter, for "quick separation" of healthy and ill animals in the case of infection, etc. role of protein kinase c in the regulation of astrocytic glutamine transporter sn1 in ammonia-exposed mouse cortical astrocytes (bisi; 1 lm). total pkc activity was analyzed by a direct pkc assay and phosphoserine detection by western blot (wb) analysis. protein level of sn1 and sn2, second astrocytic gln transporter belonging to system n, in a membrane fraction was also analyzed. the total uptake and system n-mediated (l-ala and l-leu-inhibitable) gln uptake was tested. treatment of astrocytes with ammonia resulted in a decrease of pkc activity, whereas pma treatment increased pkc activity in ammonia-independent way. bisi treatment reversed fully, and ammonia partially, the pma-induced pkc activity. pma treatment resulted in only a slight decrease in sn1 protein level in both control and ammonia-treated astrocytes, while a decrease of total and system n-mediated gln uptake were noted in control astrocytes, an effect not exacerbated by ammonia. in turn, cotreatment with pma and bisi reversed the decrease of total gln uptake and showed tendency towards increase in system nmediated gln transport. the results suggest that: a) ammonia changes the dominating direction of system n transport from release to uptake, which may be related to decreased phosphorylation or to alterations in relative phosphorylation by different pkc isoforms. this inference remains to be verified in further studies; b) changes in system n transporter function induced by ammonia appear to involve mechanisms other than changes in transporter expression. evidence for human ghrelin ghs-r1a and orexin ox1 heteroreceptor complex formation in a heterologous system ghrelin and orexin are two peptides implicated in the regulation of energy balance and modulation of food-related motivation at the level of the midbrain dopamine reward system. their function in the hypothalamic arcuate nucleus and the ventral tegmental area (vta) has already been described, but the modulation at the level of receptors remains unclear. the action of these peptides is mediated by g-protein-coupled receptors (gpcrs): ghrelin 1a and 1b (ghs-r1 a , ghs-r1 b ) for ghrelin, and orexin 1 and 2 (ox 1 , ox 2 ) for orexin. traditional approaches to know the mechanism of neurotransmission of dopaminergic neurons in the mesolimbic system have focused on targeting neuronal receptors as single entities. from the discovery that gpcrs for neuromodulators may form heteroreceptor complexes, our hypothesis is that ghrelin and orexin receptors may interact and form novel functional units that may specifically participate in the central regulation of food intake and energy balance. as a proof of concept we have investigated the potential of human ghs-r1 a and ox 1 receptors to form heterocomplexes. formation of ghs-r1 a -ox 1 receptor heteromers in transfected hek293t cells was detected by bioluminescence resonance energy transfer (bret) and proximity ligation (pla) assays. furthermore, a negative crosstalk was identified in cells co-expressing both receptors by assessing mitogen-activated protein kinase (mapk) and adenylyl cyclase (camp) pathways, and by a label-free dynamic mass redistribution assay. experiments in sources endogenously expressing ghs-r1 a and ox 1 receptors are needed to know the functional relevance of the heteromer. from the negative crosstalk here identified, it is tempting to speculate that ghs-r1 a -ox 1 receptor heteromers are important players in mediating the response to the combination of different orexigenic signals. lysosomal storage diseases which are related to deficiency of specific lysosomal hydrolases resulted to clinical aspects due to accumulation of substrates in different tissues. since dried blood spot (dbs) is non-invasive, low-cost, easy transportable, acceptable enzyme stability compared to leucocyte and/or fibroblast culture, it's recommended as a first screening test. however the false positive rate with dbs sample is higher compared to other samples. we aimed to investigate any possible effect of leucocyte number on enzyme activity in dried blood samples in a retrospective study. we re-evaluated the lysosomal enzyme activity results in regard to leucocyte number among data within last 1 year. enzyme activities had measured by using fluorometric and lc msms method. we determined the correlations between the lysosomal enzyme activities of alpha glycosidase, glycocerebrosidase, alpha galactosidase, sphingomyelinase, galactocerebrosidase and alpha-l-iduronidase in healthy population (n = 220). while glycocerebrosidase and galactocerebrosidase positively correlated with the number of neutrophils, alpha galactosidase, sphingomyelinase and alpha-l-iduronidase positively correlated with the number of lymphocytes. alpha glycosidase activity showed a correlation both lymphocytes and neutrophils. the patients having the glycocerebrosidase enzyme activity which was lower than 0.6 nmol/ml/hour (which is accepted as the cut off value to recall the patients) existed significantly lower number of leukocyte, lymphocyte and neutrophil compared to those of patients having higher enzyme activity than 0.6. our data indicated that the enzyme activity in dried blood samples including low leucocyte number might be found lower than reference intervals resulting in false positive diagnosis. therefore we suggest that the laboratory scientists should evaluate the number of leucocyte levels while they were interpreting data. using dna-markers for estimation of genetical variability of two kazakh sheep breeds a. mussayeva 1,2 , b. bekmanov 1,2 , a. amirgalieva 2 , k. dosybaev 1,2 , z. orazymbetova 1,2 , r. zhapbasov 2 , a. zhomartov 2 , n. zumadillaev 3 , n. zumadillaev 3 1 llp "kazcytogen", almaty, kazakhstan, 2 "institute of general genetics and cytology" sc mes, almaty, kazakhstan, 3 branch "scientific research institute of sheep" llp "kazakh research institute of animal husbandry and feed", almaty, kazakhstan to compare the frequencies of different microsatellite loci in sheep breeds subpopulations genomic structure of edilbay and kazakh archaromerinos was investigated. different methods for homogeneity testing of two breeds were elaborated. inter simple sequence repeats (issr) pcr analysis of the breeds studied displayed species and breed specific fragments with different frequencies (population frequency more than 0.4) there were found rarely met fragments (frequency lower than 0.4). the combinations of these fragments present the specific issr-spectra which arrange genofond profiles of breeds. using panels of microsatellits (recommended by isag) 2 breeds (5 populations) were characterized. informative value and resolving capacity of the sum of 32 str-loci were estimated. wide polymorphism of alleles length was demonstrated both when the breeds were compared and within the breeds. 10 informative markers were chosen for both two breeds, 7 markers being used for both breeds, while other 3 markers were informative for one of the breed only. when the animals of one breed were compared unique alleles which were met only within one of populations were of much interest. for example the allele 174 of bm1824 was met in birlik population of edilbay breed as often as in 59% of animals while in two other populations there were no this allele. in kumtekey population one can meet 70% animals having particular locus (dyms1), while in the other population (cf ablay) this locus was not met at all. basing on genetical distances obtained using fragment analysis phylogenetic relationships between populations were estimated. so for example edilbay population of cf ajar has the larger distance from two other populations (birlik and bayserke-agro) than each of them from one another. two subpopulations of kazakh arkharomerinos breed (cf kumtekei and cf ablay) also have the genetical difference. how preeclampsia affects oxidant status and antiinflammatory potential of breast milk? preeclampsia is a pregnancy syndrome associated with hypertension, proteinuria and edema, leading to maternal morbidity/mortality and preterm delivery. in this study we aimed to investigate if the breast milk of preeclamptic mothers is effected in oxidative status and anti-inflammatory activity in comparison to the breast milk of mothers with healthy pregnancies. for the aim of the study, hyaluronidase and myeloperoxidase activities (mpo), total oxidant status (tos), total antioxidant status (tas), oxidative stress index (osi) and tbars levels were measured in breast milks of 20 preeclamptic mothers and 20 mothers with healthy pregnancies as control group. when the control group and preeclamptic group were compared, hyaluronidase activity, tas, tos and osi levels showed statistically significant differences in the preeclamptic group. hyaluronidase activity was significantly higher in the preeclamptic mothers' breast milk (734 vs 594 u/ml, p = 0.046). while tos levels were significantly higher in the preeclampsia group (7.48 vs 4.51 lmol/l, p = 0.001), the tas levels were significantly higher in the control breast milks (0.557 vs 0.813 mmol/l, p = 0.011). as expected osi levels (tos/tas ratio) were significantly higher in the preeclampsia group. even though the mean levels were higher in preeclamptic group, the difference in mpo activities and tbars levels did not show statistic significance. oxidant status parameters also suggest that preeclampsia effects in both ways by increasing oxidant status and also decreasing antioxidant capacity shifting the balance to the increased oxidant stress side. as the results showed that the preeclampsia group had higher hyaluronidase activity, this can be interpreted as preeclamptic mothers' milk have higher inflammatory potential as this enzyme enhances inflammation by catalyzing the depolymerization of certain acidic glycosaminoglcans. p-mis-030 investigation of relationship between postprandial lipemia and erythrocyte membrane cholesterol level postprandial lipemia is a metabolic condition related to an increase in plasma triglycerides. remnant-like lipoprotein particles are predominant in postprandial phase and they play an important role in development of atherosclerosis. cholesterol is a prominent component of erythrocyte membranes and regulates the membrane functions such as viscosity and permeability. free cholesterol derived from erythrocytes is thought to participate in the atherosclerotic plaque formation. in the current study, it was aimed to investigate the relationship between postprandial lipemia and erythrocyte membrane cholesterol level in healthy subjects. study group included 87 subjects (39 female and 48 male with age range of 18-55 years). then these individuals were divided into three groups according to the values of area under curve (auc) calculated by using triglyceride levels at the fasting state and at 2nd, 4th and 6th hours after the high fat diet (ottt). lipid and erythrocyte membrane cholesterol (emc) values were compared between groups with low and high ottt response. while tc, tg, ldl-c and emc were significantly higher, hdl-c was significantly lower in high ottt response group than low ottt response group. it was not observed any statistically significant difference when compared emc values between women and men study groups. on the other part, it was seen positive correlation between emc and auc (r = 0.33, p = 0.002), tg (r = 0.26, p = 0.016), tc (r = 0.30, p = 0.005), ldl-c (r = 0.29, p = 0.007) in the total study group. it was concluded that, postprandial lipemia may show atherosclerotic tendency not only with atherogenic lipid profile but also with increasing emc. p-mis-031 eu-openscreen: the european infrastructure for chemical biology b. stechmann, p. gribbon eu-openscreen, fmp leibniz institute for molecular pharmacology, berlin, germany small molecules that can be applied as chemical 'tool' compounds (or 'probes') have become indispensable in basic research for the elucidation of fundamental biological mechanisms. they act directly with the protein-of-interest and often allow for the interrogation of biological processes that cannot be properly studied with traditional genetic or rna interference approaches. eu-openscreen (www.eu-openscreen.eu) is the largest emerging academic chemical biology research infrastructure initiative in europe and will provide access for molecular and cell biologists to screening infrastructure, well-characterized highquality chemical libraries, and facilities for medicinal chemistry services for compound optimization. molecular biologists who have a robust and suitable biological assay and are interested in collaboratively developing chemical tool compounds to validate their targets-of-interest are welcome to work with eu-openscreen. selected assays are screened against a collection of more than 100,000 compounds, incl. confirmatory and counter screening, ic/ec50 determination, sar (structure-activity relationships) and qc of confirmed hit compounds. eu-openscreen will start operations in 2017, but it can already look back on a growing number of transnational activities: joint screening projects, exchange of local compound libraries, development of new design principles for its compound collection; exchange of experimental data through its pilot database etc. steps towards an arthrobacter nicotinovorans based biotechnology for production of 6hidroxy-nicotine as the archetypal agonist of nachr, nicotine stands up as a powerful scaffold for developing new alzheimer disease therapeutic agents in form of nicotine derivatives. in this context, arthrobacter nicotinovorans pao1 and its wide range of nicotinederivatives produced when grown on nicotine have a huge biotechnological potential. indeed, the metabolic intermediate 6-hydroxy-nicotine (6hnic) produced by a. nicotinovorans pao1 was shown to bind to the nachrs, and by modulating their function, to sustain spatial memory formation in a rat model of ad. the current work presents the first attempts to produce and isolate 6hnic by using a genetically engineered a. nicotinovorans strain. the growth and the 6hnic accumulation were compared for two strains: 1. a. nicotinovorans pao1 wild type strain and 2. a genetically engineered a. nicotinovorans pao1 strain (part2ndh) containing the nicotine-dehydrogenase (ndh) genes cloned in the nicotine inducible part2 vector. the growth curves were followed spectrophotometrically. the consumption of nicotine and accumulation of 6hnic were monitored by hplc using a mn nucleodur 100-3 c18ec column and 0.1 m sulfuric acid at a flow rate of 1 ml/minute. the growth curve of the part2ndh strain shows that the bacteria grow slower when compared with the wt. as a result, in the wt strain, the nicotine is quickly depleted from the medium and only low amounts of 6hnic are observed. although the sds-page analysis of the total protein extracts from the part2ndh strain did not show clear signs of ndh overexpression, the enzyme is produced and is active, allowing a 5 fold accumulation of 6hnic in the growth medium. the first attempts to purify ndh from the part2ndh strain using imac were nevertheless unsuccessful. in conclusion, using the part2ndh strain for 6hnic production is feasible. further improvements of the growth condition and strain are envisioned (i.e. knocking the ndh downstream genes; adding inhibitors for the downstream enzymes). studies on the impact of butyrylcholinesterase (bche) on the symptoms and progression of cognitive impairments like alzheimer's disease (ad) or other neurodegenerative disruptions speak in favour of selective bche inhibitors as a new approach in future ad pharmacotherapy. some derivatives of quinine and quinidine, present in the cinchona species bark, have already been identified as selective bche inhibitors with respect to acetylcholinesterase (ache); therefore, further investigation of these compounds might result in promising leads for enhanced anti-ad drugs. we synthesised ten quaternary derivatives of cinchonines and their corresponding pseudo-enantiomeric cinchonidines. quaternization of quinuclidine moiety was carried out with groups diverse in size: methyl and differently meta and para substituted benzyl groups. all of the compounds were prepared in good yields, characterized by standard analytical spectroscopy methods, and were tested for their bche and ache inhibition potency. the inhibition potency of the compounds was defined by the dissociation constants of the enzymeàinhibitor complex (ki). all of the tested compounds reversibly inhibited both human bche and ache. the compounds inhibited bche with ki constants in the range of 0.04-30 lm, and ache in the range 2.5-70 lm. five cinchonidines displayed a 95-510 times higher inhibition selectivity to bche over ache, and four of them were potent bche inhibitors with ki constants up to 100 nm. bche affinity toward the studied compounds depended on the size of the substituent on the nitrogen of the quinuclidinium part of the molecule and on the resonance stabilization of the substituent at the quaternized nitrogen. based on the presented results, cinchonidine cd-(pbr) can be pointed out as a potent and selective bche inhibitor that could be considered for further research in alzheimer disease pharmacotherapy. exposure to nmda (100 lm) for 72 h increased the expression of kir4.1 mrna and decreased that aqp4-and gs mrna. the expression of kir4.1 was decreased by 72 h exposure to glu (2 mm) and tnfa (50 ng/ml). at 8 h incubation, nmda induced a decrease of kir4.1 expression in the presence but not in the absence of calcium in the medium. nmda did not alter the expression of nmda receptor subunits. tnfa increased the expression of the nr1 subunit, and decreased that of nr2b mrna. glu decreased the expression of 3 out of 7 subunits. the study demonstrates, to our knowledge for the first time, that prolonged exposure of astrocytes to nmda alters the expression of mrna coding for critical astrocytic proteins. the dependence of the decrease of kir4.1 mrna expression on extracellular calcium suggests the ionotropic nature of nmda receptor stimulation. the effects of nmda receptor stimulation occurred by a mechanism bypassing changes in subunit composition of the nmda receptor. experiments are under way to establish whether the tnfa-induced changes in the expression of nmda receptor subunits contribute to modulation of nmda receptor stimulation by inflammation. the importance of education in reducing preanalytical errors the preanalytical phase includes the request of test, the preparation of patient, the obtaining of sample from the patient, the transport of the sample to the laboratory, and the pretreatment of sample. the preparation of patient and the obtaining of sample are considered as the most common error sources. in order to reduce preanalytical errors, we aimed to provide training for phlebotomists and to also determine their knowledge level about the preanalytical phase before and after these training. it was given the training related with preanalytic phases to 130 pediatric nurses and 350 adult nurses, other phlebotomists in march. the surveys which are made before and after the training were consisted of 20 questions that are related with demographyic features and preanalytic phases. in order to determine the effects of training to the preanalytic phase, the preanalytic error rates before (in february) and after (in april) traning was calculated with the formula of: (the number of rejected samples/the number of total samples)x100. the average age of participants was 35 ae 9 years. it was not found significant difference between their correct answers rate before the training and the education degree of the participants. the correct answer rate before the training was 59% and after the training it was 92%, which showed an increase of 55%. the preanalytic error rates which were 0.61% in february were decreased to 0.40% in april. in our study, the positive results were obtained through the training aimed to reduce the preanalytical errors. by providing regular training to the phlebotomists and also providing pretraining to the beginners, the updating of their information about preanalytic phase can be achieved. in this way, the loss of labor and economic related to preanalytical errors can be avoided and the accurate results can be obtained in short time. curcumin, the active compound of turmeric (curcuma longa) has antiinflammatory, antioxidative and antitumour effects. unfortunately, curcumin has a poor absorption and low stability. both can be solved by encapsulation of curcumin using a proper technique like electrospray. it was reported that piperin, the active compound of black pepper, enhances the intestinal absorption of curcumin and thus its bioavailability. due to these facts it was aimed in this study to nanoencapsulate turmeric extract in order to enhance its absorption and stability. for that purpose, it was encapsulated with the maize protein zein, chitosane and black pepper extract by varying the voltage and flow rate of electrospray and the concentration of the compounds. the nanocapsules were characterised by measuring their particle size and with help of sem photographs. the particle size of the final nanocapsule formulation was 550 nm and had a sufficient stability over a period of 6 months, visually determined. by encapsulating turmeric extract into double layer nanocapsules with help of black pepper extract, zein and chitosane, the turmeric extract could be protected from degradation, which was observed for the pure turmeric extract in form of clearing its yellow colour. analysis of the human genome reveals that potential g-quadruplex sequences are enriched in promoters of the oncogenes. growing body of evidence suggests that g-quadruplexes (g4) may play putative roles in various biological processes, such as the regulation of gene expression. consequently, targeting the oncogenic g-quadruplexes using small molecules is an alternative strategy for the potential treatment of cancers. porphyrin derivatives are promising class of drug in this respect, being nucleic acids binders and generators of reactive oxygen species under visual light irradiation. interaction between porphyrin derivatives and g4 dna from oncogene promoter region has been studied in vitro. we applied chemical probing, circular dichroism spectroscopy and uv melting techniques in order to map the oxidized bases, monitor structural rearrangements and evaluate stability of the resulting dna structures. specifically, we observed that g4 dna is considerably more susceptible to lightinduced modification than duplex dna; 5 0 -terminal tetrads of the g4 dna are preferably oxidized; structural changes induced by oxidation result in decrease of the thermodynamic stability of the g4 dna. irreversibility of these effects on dna make porphyrin derivatives perspective lead compounds for rational design of ligands targeting human oncogenes. the study was financially supported by project no. 16-14-10396 from the russian science foundation. resistin levels in denervated obese rats n. saglam 1 , t. ahmedi rendi 1 , c. kahraman 2 , a. alver 1 1 department of medical biochemistry, faculty of medicine, karadeniz technical university, trabzon, turkey, 2 school of health, d€ uzce university, d€ uzce, turkey the sympathetic nervous system is an important factor affecting the metabolic and secretory function of the white adipose tissue. resistin is mainly expressed by mononuclear cells, also it is expressed by adipocytes, pancreatic cells, and muscle. resistin induces insulin resistance and glucose intolerance in mice. resistin plasma levels depend on fat depots size and sex. resistin levels decrease in short-term fasting in mice, then it increase refeeding. also, it increase as a response to fed with the high fat diet. in our study we aimed to determination of the effect of high-fat diet and denervation on serum resistin levels in rats. in this study 4 experimental groups were formed each consisted of 8 rats. during 10 weeks, first two groups are fed with high-fat diet and other two groups are fed with standart diet which they purchased from research diets company. at the beginning of the feeding periods, retroperitoneal fat tissiues of animals assigned to the first and the third groups were denervated. second and fourth groups were not denervated. at the end of the 10 week feeding periods, blood collected from rats and blood resistin concentration was determinated by elisa. in denervated and fed with high fat diet groups serum resistin levels higher than control groups (p < 0.05). according to our literature research, there are no studies demonstrating the relationship between resistin and the sympathetic nervous system. also, denervation may lead to increase in serum resistin levels. the amount of resistin is possibly reduced by b -adrenergic activation. in conclusion, it was concluded that there is differences on serum resistin levels depending on diet in bilateral denervation of retroperitoneal fat tissues of rats. stress activated protein kinases regulates the ribosomal frameshift rate in est3 gene, encoding subunit of telomerase s. t€ urkel, s. sarica uludag university, bursa, turkey est3 gene (ever shorter telomere) of s. cerevisiae encodes one of the essential subunits of telomerase enzyme. expression of est3 gene is regulated at the translation level by +1 programmed ribosomal frameshift (prf). it is known that the physiological stresses affect telomere length. in this study, we have investigated the effects of stress activated protein kinases snf1p (ampk) and gcn2p (eif2 kinase) on the prf rate in est3 gene. prf rate of est3 gene was quantified in plasmid based expression system. expression vectors were transformed in to the wild type and mutant yeast strains that deleted for snf1, or gcn2 genes. yeast cells were grown in normal conditions or subjected to acid stress, osmotic stress, or glucose limitations to activate protein kinases gcn2p, and snf1p, respectively. prf rate of est3 gene was measured as 13% in the normal growth conditions in the wild type cells. but, the prf rate of the wild type strain grown in glucose limited conditions decreased more than 10-fold, giving less than 1% prf rate. contrary to glucose limitation, osmotic or acid stress activated frameshift rate by 2-fold in the wild type cells and prf rate increased to 25%. when the prf rate was analyzed in gcn2 and snf1 mutants, frame shift rate of est3 was 4-5% in normal growth conditions. when these mutants were subjected to acidic or osmotic stress, prf rate activated slightly. we have also shown that gcn1p and gcn20p, positive regulator of gcn2p, is also essential for the regulation of prf in est3 in response to stress conditions. it is clear that the basal level expression of est3 is highly dependent on the gcn2p kinase complex. gcn2p is also associates with ribosomes, indicating that gcn2p might have a significant function in connecting the stress signals to biosynthesis of the full length est3 peptide. this regulation might also link the biosynthesis of functional telomerase and telomere replications to cell physiology through protein kinases such as snf1p and gcn2p. inflammation might have a role in erosive esophagitis but not in non-erosive reflux disease the relationship between inflammatory activation mechanisms and acid-peptic injured esophageal tissue is not clear. we evaluated whether there are differences between inflammation and tight junctional proteins such as e-cadherine among subtypes of gastroesophageal reflux disease. the aim of this study was to investigate any possible role of inflammation in pathologic mechanism of reflux disease by determining the inflammatory markers in injured esophageal tissue as well as serum of patients. three groups (erosive-ee, n = 18; nonerosive-nerd, n = 12; healthy controls-hc, n = 13) were evaluated with upper gastrointestinal endoscopy. the esophageal biopsies and blood samples were collected. serum e-cadherine levels, nfkb, chitotirosidase (chit), myeloperoxidase (mpo) activities in serum and homogenized tissues were determined. nkfb levels in tissue was significantly higher in subjects with ee (4.9 ae 2.53 ng/mg.prt) versus hc (2.95 ae 1.51 ng/mg.prt, p = 0.018). mpo tissue activities in ee group were significantly lower (0.07 ae 0.06 u/mg.prt) than hc (0.23 ae 0.22 u/mg.prt, p = 0.025) while mpo serum levels were higher in ee (1.15 ae 1.63 ul) versus hc (0.56 ae 0.69 ul, p = 0.045). tissue chit levels were three fold increased in ee versus hc (p = 0.071). none of these measurements showed any differences in nerd group. nfkb and mpo levels had a negative correlation (r=à0.408, p = 0.005) in tissue. nfkb and ecad levels had a positive correlation in serum (r = 0.642, p < 0.0001). inflammatory process might play a pivotal role in injured mechanism only in erosive esophagitis but not in nerd. noninflammatory mechanisms might be responsible such as hypersensitivity in patients with non-erosive reflux disease. d-dimer (a fibrin degradation product) test is used to aid in the diagnosis of intravascular coagulation. the aim of this study is to investigate the correlation between d-dimer levels and other inflammatory markers including procalcitonin. anonymized data on d-dimer, fibrinogen, hscrp, wbc, neutrophil% (neut%) and procalcitonin levels from 50,107 patients (mean age ae sd, 53.37 ae 20.6) were used for the correlation (excel analyze-it v4.60.4) and linear regression (pasw statistics 18 v18.0) analysis between the measured parameters. there was a significant (p < 0.05) age-dependent increase in d-dimer levels between different age groups. patients with the highest d-dimer levels were also found to have an increased frequency of hscrp levels. d-dimer levels showed a significant correlation with hscrp, wbc and neut%. a model describing the positive association between these parameters were built. the resulting equation is as follows: d-dimer = (hscrp*0.054) + (0.011*age) + (0.006*wbc) + (0.04*neut%)à0.929. correlation analysis between procalcitonin and d-dimer levels gave pearson's correlation coefficient of 0.159. our results suggest that the age-dependent variations should be taken into account while interpreting d-dimer test results. in addition, neut% ratio was found to be the most important parameter for estimating d-dimer levels. our equation can be used when the d-dimer test is not available or for control purposes only. in the field of cancer research great hope lies in finding more powerful and selective way for the direct elimination of cancer cells. this task can be solved by means of nanobiotechnology. recent progress in this field has arisen interest in a carbon nanostructurefullerene c 60 . fullerene exhibits not only unique physico-chemical properties and biological activity but also a significant potential to serve as a nanocarrier for selective drug delivery into cancer cells. the aim of this study is to analyze a unique tool for cancer therapy. the main idea is realized by the non-covalent conjugation of c 60 with the well-known anticancer drug -doxorubicin (dox). two types of conjugate with different c 60 -dox ratio (1:1 and 2:1) were studied. conjugates absorbance and fluorescence, size distribution as well as a mass data were recorded utilizing optical and analytical equipment (microplate reader, zetasizer, lc-ms/ms and maldi-tof). in vitro studies were performed including evaluation of c 60 -dox conjugate effects on human leukemic cells (jurkat, ccrf-cem, thp1 ad molt-16) viability. conjugates accumulation and distribution within cancer cells was monitored using fluorescent microscopy accompanied with fluorescence-activated cell sorting. it was evidently proven that both c 60 -dox conjugates were stable and could be used as reliable candidates for biological application. cellular accumulation and distribution studies showed that conjugation of dox with fullerene promoted its entry into leukemic cells. accumulation of dox in the form of conjugates within cancer cells was intensified compared to the free drug. the results show that conjugated dox is more cytotoxic and the value of its ic50 are lower compared with the free dox. obtained results confirm nanocarrier function of fullerene c 60 and the perspective of its application for optimization of doxorubicin efficiency against leukemic cells. comperative investigation of protective effects of tea and tea-related wastes on reducing potaential of h2o2-induced erythrocytes tea processing waste (tpw) formed during the tea production process in tea factories is up to 50,000 tones/year in turkey. tpw is one of the abundant available phenolic biomass among plantal wastes. in this study, black and green teas and their wastes were used. the aim of the study is to determinate the phenolic content and the radical scavenging activities of the samples, and to measure their effects on hydrogen peroxide-induced erythrocyte damage due to analyzing the reducing potential of erythrocyte involving glutathione reductase (gr), glutathione peroxidase (gpx) activities and reduced glutathione (gsh) content. total polyphenol content of samples was determined as mg catechine per dry mass by using folin-ciocalteau reactive and dpph radical scavenging activity was estimated by cuendet method as equivalent catechine standard. in erythrocyte, gsh level was measured by method of sedlak and lindsay while gr and gpx activities were assayed by the methods of bergmeyer and beutler, respectively. the highest phenolic content was observed in green tea and its wastes (p < 0.01) whereas black fiber waste had the lowest phenolic content. therefore, the highest radical scavenging activity and gsh level were detected in green tea and its wastes (p < 0.01). erythrocyte with the extracts of the teas and their wastes had the similar enzyme activities for both gpx and gr. in sum, the teas and wastes have antioxidant activity but, green tea and its leaf waste hade higher antioxidant activity than other samples. the tea wastes might be evaluated as many of protective health products, particularly in cosmetic fields thus, these by-products no application for any area is expected to become an economical value. fluorouracil (5-fu) is a chemotherapeutic drug classified as an "antimetabolite". it works through irreversible inhibition of thymidylate synthase. chemical derivatization of 5-fu with carbohydrtates is being investigated widely in order to enhance its bioavailability, therapeutic efficiency and to reduce its toxicity. however, water solubility of the newly derived compounds is usually very low. so, in order to obtain a pharmaceutically relevant formulation they need to be formulated appropriately. in this study, we prepared micellar delivery system for the new tetra-o-acetylglycose derivative of 5-fu synthesized via "click reaction", namely f1-[{1 0 -(2″,3″,4″,6″-tetra-o-acetyl-b-dglycopyronosyl)-1 0 h-1 0 ,2 0 ,3 0 -triazole-4 0 -yl}methyl]5-fluorouracil. since the water solubility of this compound is very limited, we tested its solubility in several pharmaceutically relevant solvents by visual estimation after stiring increasing amount of the compound in 1 ml of solvent for 48 h. to estimate the carcinogenic potential of this compound, salmonella/microsome mutagenicity assay (ames test) was performed in four histidine-requiring strains of s. typhimurium, tester strains ta98, ta 1537 (for the detection of frameshift mutations) ta100 and ta 1535 (for detection of base pair substitutions) according to the oecd guideline 471. the drug was solubilized (500 lg/ml) with no precipitation in lutrol ò -f68/ethanol/water (2.25:2.25:5.5 , wt/wt) micelles (7.3 ae 1.0 nm). the results of ames test were negative so the compound neither produced frame shift mutations nor base pair mutations in s. typhimurium strains. the results imply that the new compound can be dissolved in aqueous micellar delivery system in order to be used for further studies, and that it was not mutagenic in the tested s. typhimurium strains. in conclusion, the formulation of the newly synthesized compound is not carcinogenic, and can be evaluated for anticancer activity in vitro and in vivo. integral metabolism parameters of dairy goats during reproductive cycle periods d. solovyeva, e. zarudnaya, s. zaitsev moscow savmb, moscow, russia study of the goat metabolism at different periods of the reproductive cycle allows to correct feeding ration, to increase the age of the productive use of animals and to receive high-quality products. the aim of the work was to determine the metabolic parameters of blood serum of goats, expressed in terms of biochemical parameters and interfacial tensiometry and study their relationship to metabolic processes in the body goats depending on the age and the period of the reproductive cycle. the 90 healthy goats were divided into 5 groups. the dynamic surface tension (dst) parameters were obtained from dependences of a surface tension (r) vs. time (t): at t?0 (r 0 ), at t = 0.02 s (r 1 ), t = 1 s (r 2 ) and t?∞ (r 3 ). this work was supported by the russian scientific foundation (14-16-00046). all animals had 4-5% fat content. the contents of total protein (5.4%), albumin (8.4%) and urea (8.8%) are higher for the lactating animals as compared to the normal goat values. the levels of total cholesterol (18.0%) and creatinine (6.2%) are higher for the lactating animals. in lactating animals have the highest level of, which along with high phosphorus level talks about the intensification of energy processes during lactation. the correlations were found between the biochemical and dst parameters of the goat blood: lipids or cholesterol levels with r 0 (r = à0.40), r 1 (r = à0.72), r 2 (r = à0.89); total protein or albumin levels with r 1 (r = à0.42), r 2 (r = à0.56), r 3 (r = à0.90); aminotransferase activity with r 2 (r = à0.47), r 3 (r = à0.63). the correlations were found between the total protein and albumin levels with k 0 (r = 0.54), k 1 (r = 0.44); glucose levels and r 1 (r = 0.47), r 2 (r = 0.43). thus, the dst and biochemical parameters of goats have strong correlation relationships that are important for biomedical and veterinary applications. the relation of the severity of atherosclerotic disease with oxidative stress in patients with stable coronary artery disease h. sezen harran university, sanliurfa, turkey introduction: because, to the best of our knowledge, the relationship of total oxidant status (tos) and total antioxidant status (tas) with the severity of stable coronary artery disease (cad) has not been investigated in the literature so far, the present study was conducted to address this issue. materials and methods: this study consisted of 349 consecutive patients and controls who underwent coronary angiography. for each patient, the total gensiniscore (gs) was calculated andthose with a gs of >20 were classified as the high gs group (hgg), and those with a gs less than 20 were defined as the low gs group (lgg). the total oxidant status (tos) and total antioxidant status (tas) levels were measured using the erelmethod. the osi, which is an indicator of the oxidative balance, was calculated as the percentage ratio of tos to tas. results: the tas was lower in the hgg than lgg. the tos and osi were higher in the hgg than lgg. the correlation analysis showed that gs was negatively associated with the tas and positively with the tos and the osi. the multivariate analysis showed that age, tos, and hdl-c were independent variables for a high gs. the cut-off level of 10.9 lmol h 2 o 2 equiv./ l for serum tos levels predicted high gs with a sensitivity of 70% and a specificity of 56%. discussion: information on the severity of atherosclerosis is requiredtopredicttheprognosis of an individualpatientandtodetermine the proper treatment modality. the gs system has beenproventodemonstratethe severity of atherosclerotic disease. inthepresentstudy, thepatientswith a high gs had increasedlevels of oxidants. inaddition, tos was an independentindicator of theseverity of atherosclerosis. the optimal cut-offvaluefor tos topredict high-gens score was 10.9 (sensitivity 70% and specificity 56%). conclusions: the results suggest that the severity of atherosclerosis in stable cad is associated with increased oxidative status. evaluation of roemerine as a multidrug resistance pump inhibitor f. g. avci 1 , c. velioglu 1 , e. recber 1 , c. unsal 2 , g. gulsoy 2 , b. sariyar akbulut 1 1 marmara university, istanbul, turkey, 2 istanbul university, istanbul, turkey efflux by multidrug resistance (mdr) pumps is a common defense mechanism used against antimicrobials. by pumping the drugs out, these pumps significantly reduce the efficacy of drugs. one approach to overcome this limitation is offered by the combinatorial therapies where drugs are co-administered with together with pump inhibitors. by simply preventing the efflux of the drug, the presence of inhibitors enhance drug efficacy. (-)-roemerine is an aporphine type alkaloid with significant antibacterial (against bacillus cereus, escherichia coli) and antifungal (against candida strains) activities. interestingly, (-)-roemerine was also found to enhance the cytotoxic response mediated by vinblastine in multidrug-resistant kb-v1 cells. in the same study, this finding was linked to its possible interaction with p-glycoprotein, a eukaryotic mdr pump. taking this finding as the starting point, the current study investigates the potential of roemerine as an inhibitor of the p-glycoprotein homologue pump, bmra, in bacillus subtilis 168. the antimicrobial agent berberine was used as the model agent since its efficacy is reduced by efflux through mdr pumps. to this end, bacillus subtilis 168 cells were subjected to 100 lg/ml berberine, a value well below the mic. this concentration only slightly retarded growth for 2 hours but then cells resumed their regular growth. upon addition of 25 lg/ml (-)-roemerine to the bacillus subtilis 168 cells treated with berberine, growth pattern changed, indicating possible interaction with bmra. further investigation for the change in the expression of bmra was achieved with real time pcr analysis. glucose oxidase is an enzyme that catalyzes the oxidation of glucose to d-glucono-1,5-lactone and hydrogen peroxide. we hypothesized that enzyme would cause a double negative effect on cancer cells, by reducing the presence of glucose in cancer microenvironment and producing reactive oxygen species. to increase enzyme stability and enhance cellular uptake we encapsulated the enzyme with a thin acrylamide layer. the purpose of this work was to optimize the synthesis of these glucose oxidase nanoparticles and investigate their effect on cancer cells. nanoparticles containing single glucose oxidase were synthesized in two steps; first by introducing the vinyl groups onto the surface of enzyme by acyloylation followed by polymerization step with acrylamide monomers. encapsulated enzymes are approximately 150 nm in size and retain most of their activity. after the optimization of nanoparticles, the anticancer potency of these nanoparticles was in vitro tested in mcf-7 breast cancer cell line. according to results, both nanoparticles and free enzyme are capable of inhibiting viability of cancer cells in a similar manner at very low concentrations. currently we are investigating mechanisms involved in this viability inhibition. initial results demonstrated that glucose supplement does not rescue cells from death induced by the activity of glucose oxidase, suggesting an oxidative stress related cause of inhibition. further studies are required to elucidate the exact mechanism. until now there is no determined advantage of glucose oxidase encapsulation against proteolysis. however, encapsulation may induce the accumulation of enzyme in cancer microenvironment. furthermore results suggest that glucose oxidase has a high effect on the viability of mcf-7 breast cancer cells indicating that this enzyme may have a potential use in cancer treatment. studies on the interaction of human phospholipid scramblase 1 with c-terminal domain of topoisomerase iia u. sivagnanam, s. n. gummadi applied and industrial microbiology lab, bhupat and jyoti mehta school of biosciences, indian institute of technology madras, chennai, india human phospholipid scramblase 1 (hplscr1) is a multifunctional protein that plays key roles in several cellular processes including apoptosis, tumorigenesis, anti-viral defense, cell signalling and several protein-protein interactions. it has been shown that hplscr1 interacts with the c-terminal domain of topoisomerase iia (topo iia) and enhances its decatenation activity in vitro. the interacting region in topo iia was identified but till date, no reports exist on the binding region in hplscr1. this study aims to identify the region of hplscr1 that interacts with topo iia. to identify the topo iia interacting sites in hplscr1, nterminal deletion constructs of hplscr1 viz δ25-hplscr1, δ50-hplscr1, δ75-hplscr1, δ100-hplscr1 and δ160-hplscr1 were generated by pcr, cloned, overexpressed and purified to homogeneity using ni 2+ -nta purification. the cterminal domain (ctd) of topo iia was cloned in pgex6p-1 and was expressed as a gst fusion protein. gst pull down assays will be performed with the deletion constructs of hplscr1 and the gst-ctd-topo iia. the binding region in hplscr1 will be confirmed by peptide competition assays. our initial results show that the decatenation activity of topo iia was enhanced when the topo ii was pretreated with hplscr1. δ100-hplscr1 did not show any enhancement of the decatenation activity compared to full length hplscr1. hence, the binding region could be in the 1-100 region of hplscr1. further deletions were done in the 1-100 region of hplscr1 as described earlier. gst-pull down assays and decatenation assays will be performed for the deletion constructs to narrow down the region of hplscr1 that binds to topo iia. we conclude that hplscr1 interacts with and enhances the activity of topo iia and the 1-100 region of hplscr1 is critical for enhancement of decatenation activity. further work is under progress to identify the exact topo iia binding region of hplscr1 and the physiological relevance of this interaction in the cell. a. ugur kurtoglu 1 , v. aslan 2 , e. kurtoglu 2 1 department of biochemistry, antalya education and research hospital, antalya, turkey, 2 department of hematology, antalya education and research hospital, antalya, turkey beta-thalassemia is a common autosomal recessive disorder resulting from over 200 different mutations of the beta-globin genes. our aim was to creat a mutation map of beta thalassemia in province of antalya, turkey. in this study, mutation analysis of a total 150 of beta-thalassemia patients followed up at the thalassemia center of the antalya education and research hospital, antalya, turkey, were included. according to our results, the ivs 1.110 is the most frequent mutation type in our province same as other geographical regions of turkey. the most frequent mutations in heterozygous or homozygous patients are ivs 1.110, ivs 1.6, ivs 2.1 and ivs 1.1. our results indicate the importance of micromaping and epidemiology studies of thalassemia, which will assist in establishing the national prevention and control program in turkey. keywords: beta-thalassemia, beta-globin gene, mutation p-mis-052 investigation of the in vitro effects of some antibiotics on the purified beta-glucosidases from the rat liver n. t€ urkmen 1 , h. kara 2 1 karadeniz technical university medical biochemistry department, trabzon, turkey, 2 balikesir university veterinary faculty biochemistry department, balikesir, turkey beta-glucosidases catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in b-d-glucosides and oligosaccharides.b-glucosidases are widely distributed in the living world.b-glucosidases which in mammals, primarily found in the liver and kidneys;lysosomal b-glucosidase (gba1),non-lysosomal b-glucosidase (gba2), cytosolic b-glucosidase (gba3),intestinal lactase-phloriz the hydrolase (lph). liver tissues of wistar-albino rats were homogenized with homogenizer in the extraction buffer and crude extract was obtained after centrifugation.ammoniumsulfate precipitation range designated crude extract was purified by sepharose 4b-ltyrosine-1-naphthylamine hydrophobic gel.commercially availabled antibiotics were prepared with substrate buffer.it was investigated inhbition effects of cefuroximesodium, ampicillin-sulbactam, amoxicillin trihydrate/potassium clavulanate, cefazolin sodium, gentamicin sulfate and ceftriaxone disodium antibiotics onto gba2.inhibition types and k i values of related antibiotics were determined with p-npg substrate.lineweaver-burk plot was used for that purpose. rat liver gba2 was purified at 30.2-fold with 43.4% yield.gba2 was illustrated 58 and 110 kda at sds-page.ic 50 value of ampicillin/sulbactam antibiotic for gba2 was found 62.97 mg/ml with competitive type inhibition and other antibiotics didn't inhibit. purfication methods are being used in the literature for the purified b-glucosidase from different sources.purified gba2 was illustrated 58 and 110 kda at sds-page.about molecular weight of bglucosidases is presented different information in the literat€ ure. this has been reported because of acid beta glucosidases are abnormal migration at the acrylamide or agarose gels.it was investigated inhbition effects of various antibiotics onto purified gba2.ampicillin/sulbactam antibiotic inhibited to purfied gba2 at the competitive type.similiar antibiotics studies have been made in the literature for different enzymes. effect of glutamine on insulin resistance and endoplasmic reticulum stress g. aydogdu 1 , p. b. sermikli 1 , a. abbasi taghidizaj 2 , e. yilmaz 2 1 ankara university, institute of science, ankara, turkey, 2 ankara university, biotechnology institute, ankara, turkey obesity and diseases are one of the most important public health problems of the world.excess fat storage in adipocytes leads to the release of increased amounts of non-esterified fatty acids, glycerol, hormones, cytokines, which are factors involved in the development of insulin resistance that cause type 2 diabetes. one of the major differences between obese and lean individuals is the amino acid concentration in the circulation. although there are many studies about the amino acid metabolism associated with insulin resistance in obese individuals, the effect of glutamine metabolism in insulin resistance mechanisms are not well understood yet. glutamine can be used as fuel and its levels in tissues and circulation can regulate cell responsiveness to insulin and cellular metabolism. therefore, glutamine is a potentially important factor that might help us better understand insulin resistance and type 2 diabetes. to determine whether glutamine effect on insulin resistance and endoplasmic reticulum stress, 3t3-l1 cell is treated with different concentration of glutamine and analyzed by western blot for er stress markers. our results indicated that glutamine reduced endoplasmic reticulum stress and related with that attenuated insulin resistance. in case of transport of amino acids, insulin resistance, how it is affected when we have the information about the important tips on energy requirements and metabolism reach insulin resistance and type-2 diabetes treatment is likely to reveal a possible new targets. how does different lead levels affect tsh, ft3, ft4, vitamin b12 and folate? e. ozkan ankara occupational disease hospital, ankara, turkey exposure to heavy metals is increasing with the industrialization of society. one of the most intense exposure to heavy metals is pb on this issue. this study was aimed to determine the relationship between different blood pb levels and serum thyroid hormones (th), vit b12, folate. the cases were 20-65 years old, male individuals who admitted to our hospital between april 2012-march 2016 for periodic inspections because of occupational exposure to pb. the parameters of the cases were retrospectively retrieved. according to their pb levels, exposed workers (n: 9400) were divided into four subgroups; group (g) 1: 0-9.99 lg/dl, g 2: 10-19.99, g 3: 20-39.99, g 4: ≥40 . from these, the number of cases whose th levels were measured (n:3894) given respectively; g 1:3062, g 2:178, g 3:334, g 4:275 cases. also the number of cases whose vit b12 and folate levels were measured (n:2807) given respectively; g 1:2139, g 2:143, g 3:276,g 4:249 cases. levels of pb were determined by icp-ms. th, vit b12, folate were determined by cmia. between the groups formed according to pb levels, there was no significant difference in terms of average t3, tsh and vitamin b12 (p > 0.05). on the other hand there was statistically significant difference between t4 and group 1,3,4 (p < 0.05) but there was no difference between group 2 (p > 0.05). the average folate belongs to the first group was about 10% higher than the other 3 groups, and found that the difference was statistically significant (p < 0.05). there are many publications which have various results between pb levels and t3,t4, tsh. but this study is important to compare the effect of different levels of pb. up to day there was no publication about the relation between different pb levels and vit b12, folate. it was seen that there was no significant clinical relation between different pb levels and thyroid parameters, vit b12. but the low levels of folate in the high pb levels groups shows us that we need further studies about this relationship. fluorescent study of in meso crystallization of membrane proteins with the introduction of membrane protein in meso crystallization 30 years ago by landau and rosenbusch, a new era of membrane protein structural research has emerged (1). since that time this method became associated with a number of major breakthroughs in the field (2) including exceptional successes in structural studies of microbial rhodopsins and g-protein coupled receptors (3) . here we used fluorescence microscopy to study in meso crystallization process of bacteriorhodopsin. several observations provide new insights into the in meso crystallization process. the crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. these observations demonstrate an ostwald ripening mechanism of the in meso crystal growth. the depletion zone formed around the growing crystal is consistent with the previously proposed analogy relating in meso crystallization with the crystallization in a microgravity convection-free environment. this work is supported by rsf 14-14-00995. (1) landau, e. m.; rosenbusch, j. p. proc. natl. acad. sci. u. s. a. 1996, 93, 14532à14535. (2) caffrey, m. acta crystallogr., sect. f: struct. biol. commun. 2015, 71, 3-18. (3) katritch v., cherezov v., stevens r.c. (2013) . annu rev pharmacol toxicol 53, 531-556. p-mis-058 stamp1 is critical for both ar and mtor signaling in prostate cancer cells x. sheng, y. jin, f. saatcioglu university of oslo, oslo, norway androgen receptor (ar) signaling plays a central role in the initiation and progression of prostate cancer (pca), including when the disease progresses to castration-resistant pca (crpc). the second central signaling pathway in pca, similar to various other cancers, is the pi3k-akt-mtor signaling. importantly, these two oncogenic pathways cross-regulate each other in pca cells by reciprocal feedback, thereby maintaining tumor cell survival even when one is suppressed. we have previously identified that the six transmembrane protein of prostate 1 (stamp1) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk mapk signaling. human pca cell lines lncap and vcap were used in the study. colony formation, soft-agar growth, prostatosphere formation assays were performed. for in vivo xenograft experiment, the cells were implanted subcutaneously into the flanks of nude mice. here, we show that stamp1 knockdown caused defects in colony formation, anchorage-independent growth and prostatosphere formation in lncap and vcap cells both in vitro, as well as tumor formation and growth in vivo. this may be due to the impaired ar and mtor signaling in these cells upon stamp1 knockdown. interestingly, in the crpc cell line 22rv1, where-stamp1 knockdown did not affect mtor signaling, there was a remarkable repression of tumor take rate and growth. these results clearly indicate that stamp1 is essential for both ar and mtor signaling, and is crucial for pca growth in vitro and in vivo. however, the detailed molecular mechanism requires further investigation. taken together, these data unveil a critical role for stamp1 in coordinating the ar and mtor signaling pathways in pca cells, solidifying the basis for its pro-survival effects in pca, including in advanced disease. quantification of 2d thin layer chromatograms using 2d gel analysis software and gel documentation system o. kaynar, m. ileriturk, d. kaynar, s. kurt ataturk university, erzurum, turkey introduction: thin layer chromatography (tlc) is an important chromatographic technique that is widely used as a cost-effective method for rapid-sensitive analysis of compounds in plants, animals, and humans. however, one dimentional (1d) tlc is not sufficient for the separation of complex compounds. therefore, two-dimentional (2d) tlc was developed. the quantitative evaluation of plates are performed with tlc scanners or documentation systems. however, these systems specific for 1d plates, and cannot be adapted to quantitative evaluations of 2d plates. in this study, the applicability of the gel documentation systems and 2d analysis software for the analysis of 2d tlc plates were examined. material and method: 2d tlc of lipids: 1st dimension: methyl acetate/n-propanol/chloroform/methanol/0.25% kcl (25/ 25/28/10/7 v/v); 2nd dimension: chloroform/methanol/acetic acid/ water (90/40/12/2 v/v); detection: charring. 2d tlc of aminoacids: 1st dimension: 1.5% (v/v) formic acid; 2nd dimension: toluene/glacial acetic acid (10:1 v/v); detection: uv. phospholipid and aminoacid standards, each include 6 different classes were developed by 2d tlc. plates visualized with biorad geldoc xr, and band volumes on plates were calculated with biorad pdquest 2d gel analysis software. for the method validationa) 5 plates containing same 6 lipid classes were developed in the same day, and results were used for the calculation of intra-assay cv;cv% = average of each sample standard deviation/mean of sample 9 100 b) 6 plates containing same 6 lipid classes were developed in 6 different days, and results were used for the calculation of interassay cv; cv% = standard deviation of each sample average/mean of the plates 9 100 results: volume of each phospholipid and aminoacid had less than 10% intra and inter-assay cv. conclusion: gel documentation system with 2d gel analysis software can be used for the quantitative analysis of the 2d tl plates both at uv and visible light. the role of na + k + atpase activity in the vasodilatory effect of n-acetylcysteine introduction: spasm occurred at the stage of and after the preparation of arterial grafts used in coronary artery bypass surgery (cabg) is effective on morbidity and mortality in the first 24 hours of postoperative patients. n-acetyl cysteine (nac) that vasodilatory effect is known,may be considered as a suppressor agent for vasospasm developing during cabg. however, for the prevention of complications that may arise during or after cabg mechanism of these vasodilatory effects should be described. this study was aimed to investigate the role of atpase enzyme on the vasodilatory effect of nac. materials and methods: in this study, 28 adult male wistar albino rats were used. rats were separated into four groups as control rats (g1), 2 mm nac (g2), 5 mm nac (g3) and 10 mm nac (g4). a portion of the thoracic aorta isolated from rats was used for the relaxation response recording, and the other portion was used for measurement of nakatpase activity. isolated smooth muscle rings are suspended in the 20 ml organ bath containing krebs solution for relaxation responses. in all groups, level of smooth muscle contraction were allowed to reach a plateau by adding 60 mm kcl to the organ bath. then, in the first 10 minutes of application relaxation responses which created by adding nac to the medium were recorded and the maximum relaxation responses were measured. nak atpase activity was determined using the mazzanti method. groups means were compared by one-way analysis of variance (anova). the threshold for statistical significance was set at .05. results: the contraction force decreased in all nac dose groups compared to control group and this reduction was statistically significant (p < 0.05). similarly, nak atpase activity is also decreased in a dose dependent manner (p < 0.05). the findings obtained in this study suggest that vasodilator effect of nac formed in thoracic aortic smooth muscle was associated with the activity of the enzyme na k atpase. in the presented study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic activity. a bacillus uk69, which was isolated from the soil samples taken from farmland on kahramanmaras sutcu imam university campus, showed high keratinolytic activity when cultured on feather meal medium. the enzyme activity was studied in the ph range of 5.0-12.0. the optimum temperature for keratinase activity was investigated by varying the incubation temperature between 20°c and 80°c. optimum keratinolytic activity was observed at 60°c and ph 10.5. the enzyme was stable at 60°c. the activity was investigated in the presence of some chemicals, including sds, tween 80, dmso, triton x-100, edta, nacl, zncl2, cacl2, glucose. the keratinolytic activity was inhibited by all chemicals tested to some degree. the molecular weight of keratinase was determined by polyacrylamide gel (10%) using standard molecular weight marker and estimated about 37 kda by sds-page. the keratinase isolated from bacillus uk69 could be used in biotechnological processes i.e. feather degradation, wastewater treatment and in industrial processes, such as detergent, food and leather industries. introduction: hemoglobinopathies, including thalassemia, abnormal hemoglobins, constitutes a major group of inherited disorders of hemoglobin synthesis. the reduced or absent of the beta (b) or alpha (a) globin chains of the adult human hemoglobin molecule results beta or alpha thalassemias, leading to imbalanced a-globin/non a-globin chains. the aim of this study was to give a quik desicion with a/b-globin mrna ratio for sequencing of a or b gene, when the anemia is not detectable. materials and methods: mrna and cdna extraction of 25 bthalassemia and 15 a-(including two of 3.7 kb del./hbs) thalassemia subjects and normal controls were accomplished using the high pure rna isolation kit and transcriptor first strand cdna synthesis kit, respectively, following the manufacturer's instructions. we used cdna as a template in the real-time pcr amplification using primers specific for a, b globin genes. amplification was performed in a lightcycler ò 480 instrument. the a/b-globin mrna ratio of each sample was calculated based on the 2 àddct method. results: a/b-globin mrna ratios calculated in a-thalassemia subjects relative to normal control as a result of numbers of defective a-globin genes. the a/b-globin mrna ratio was found higher in b-thalassemia subjects. coinheritance of a-thalassemia in hb s subjects concluded a stabile a/b-globin mrna ratio as per a-thalassemia or b-thalassemia subjects. discussion and conclusion: instability in a/b-globin chains is a significant factor of thalassemia disease severity and can be used before deciding type of gene sequencing when the anemia is not detectable. this study indicates that imbalance in globin gene expression could be demonstrated by measuring a/b-globin mrna ratio, which was conveniently and accurately determined by qrt-pcr and give an information about globin gene function which gene should be correct to investigate an individual for globin gene mutation. p-mis-067 self-assembling peptides mimic supramolecular biochirality r. garifullin 1,2 , m. o. guler 1 1 bilkent university unam, institute of materials science and nanotechnology, ankara, turkey, 2 kazan federal university, institute of fundamental medicine and biology, kazan, russia supramolecular chirality is rooted in asymmetric spatial arrangement of structural elements (e.g. molecules or units with higher hierarchy). self-assembled systems giving rise to this kind of chirality are of great importance because they closely resemble natural biological systems and potentially can lead to new advanced functional materials. in the process of self-assembly, both molecularly chiral and achiral structural units can organize into chiral nanostructures. chiral arrangement of chromophore molecules in space is known to result in emergence of chiroptical properties of a chromophore. organization of pigment-protein complexes into macrodomains in green plants gives rise to biochirality emanating from long-range chiral order of complexes. owing to this order, macrodomains start to absorb circularly polarized light intensively and thus exhibit huge circular dichroism (cd) signal. in our study, a simple approach which was aimed at mimicking the biochirality phenomenon makes use of self-assembling peptide amphiphiles and their interactions with pyrene chromophore. designed peptide amphiphiles are capable of self-assembly into nanofibers with chiral interior, which in principle gives an opportunity to achieve long-range chiral order. two modes of interactioncovalent and noncovalentwere utilized in order to induce supramolecular chirality. covalent interaction mode included direct covalent attachment of pyrene to peptide sequence. upon self-assembly of peptide amphiphile into nanofibers intense circular dichroism phenomenon was observed. noncovalent interaction mode envisioned encapsulation of pyrene molecules in the hydrophobic core of nanofibers of another peptide amphiphile. co-assembly of peptide amphiphile and pyrene molecules led to chiral order and intense cd signal. in addition, it was possible to control the sign of cd signals by using either of peptide isomers, l or d. p-mis-068 pon1 activity in hdl subgroups of obese, overweight and normal weight subjects objective: the aims of this study were isolation of hdl-c subgroups by using precipitation method, determination of pon-1 activity in both total and hdl3 subgroups, and evaluation of performance characteristics of pon-1 activity measurement method in newly diagnosed obese, overweight and normal subjects. material and methods: the study population consists of newly diagnosed 71 obese, 40 overweight and 30 normal subjects. fasting morning blood samples were taken from all study groups. hdl3 subgroup was obtained by heparin-mn-dextran sulphate precipitation method and cholesterol was measured with direct (homegenous) hdl-c method. hdl2-c concentrations were calculated with the subtraction of hdl3-c from total hdl-c. hdl3-c and total pon-1 activity were determined by using eckerson method. non-hdl3 pon-1 activitiy was calculated with subtraction of hdl3 pon-1 activity from total pon-1 activity. results: total hdl-c, hdl2-c and hdl3-c concentrations and the activity of total pon-1 and hdl3 pon-1 were found lower in obesity according to overweight and normal subjects (p < 0.001). negative correlations were found between body mass index and hdl3-c, total pon-1 and hdl3 pon-1 (r = à0.244, p < 0.005; r = à0.247, p < 0.005; r = à0.199, p < 0.05, respectively). conclusion: our findings indicated that hdl-c metabolism and lipoprotein associated antioxidant defense mechanisms were adversely affected with obesity. in conclusion we think that precipitation method using for separating hdl3 subgroup, is simple and cost effective for routine applications in clinical laboratories. besides hdl3-c measurements, pon1 activity, measurement of total and hdl3-c subgroup might be helpful to evaluate the atherosclerotic process in obese subjects. keywords: obesity, body mass index, paraoxonase, hdl subgroup, cholesterol p-mis-069 hepatitis e virus antibody prevelance among persons who work with animals in north cyprus introductions: hepatitis e infection is a major cause of viral hepatitis in many developing countries. the objectives of the present study was to determine the seroprevelance of hev infection in peoples who work with animals in northern cyprus. materials and methods: prevelance of hev infection were determined in study 4 group population: persons without occupational exposure to animals; persons who work with animals; veterinarian and butcher. a total of 400 blood samples were collected. all serum samples were tested elisa using a commercially available kit according to the manufacturer's instructions. ti-test were used for istatistical analyses. p > 0.05 was accepted as significant value. results: in a study of 400 blood donors (334 male, 66 female), the overall prevelance of anti-hev igg antibodies were 3.0%. the blood samples were collected 5 different areas. the prevelance of anti-hev igm antibodies was 0.25% and he was 44 years and acting a butcher during 20 years. the prevelance of anti-hev igg of women were approximately two fold higher than men. no significant difference in anti-hev prevelance was observed between the age of the blood donors. according to the anti-hev igg prevelance, the without occupation expose to animal animal were 1%, the animal husbandry were 7% and the veterians and the butcher were 2% were found. discussion: the prevelance of anti-hev in the north cyprus (3%) was found low such as the prevelance of the turkey (5%). the prevelance of anti-hev igg in animal husbandry were higher that the other groups because of they may be more spend of time and contact with animals. the prevelance of igm results suggested that the possibility of outbreaks may be low in north cyprus. conclusion: this study was the first seroprevelance analysis of north cyprus according to the population number.the further studies could be included the seroprevelance of anti-hev from the animals. most errors in the clinical laboratory occur in the preanalytical phase the aim of this study was to investigate the causes and rates of rejected samples, regarding to certain test groups in our laboratory. this study was designed on the rejected samples between january 2015 and january 2016. clinical chemistry, coagulation, hormone, cardiac markers, total urine evaluation and other (ethanol level, hba1c, hb electrophoresis, neonatal bilirubin, drug level, blood gas, fecal occult blood) test groups were included. the total number of specimen and rejected samples was obtained from the hospital information system retrospectively. types of inappropriateness were evaluated as follows: erroneous coding, clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen. it was determined that 873343 blood samples were sent to our laboratory in one-year period. 0.37% of them were rejected because of preanalytical errors. erroneous coding was found as the most common rejection cause (33%). rejection rates of clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen were found to be 11%, 15%, 19%, 2%, 4% and 16% respectively. in our study, erroneous coding was the most common cause of preanalytical errors. education of medical secretaries is relevant and important as can be seen in the decrease of sample errors and the resulting quality improvement. glycosylated hemoglobin test (hba1c) is important for screening, diagnosing, and monitoring diabetes and prediabetes. however, hba1c levels may dependent on patient ethnicity suggesting that the diagnostic cut-offs should be evaluated for specific populations. therefore, our aim in this study was to evaluate the efficiency of hba1c for predicting diabetes in comparison to oral glucose tolerance test (ogtt) results for turkish population. the study included anonymous lab results (acibadem labmed laboratories in turkey) of 6270 patients (3913 female, 2351 male) aging 40.4 ae 11.6 years (15-86) who had an initial diagnosis of diabetes. glucose and insulin levels during ogtt were measured after the initial administration of 75 g sugar (0hour), 1-hour and 2-hour. these parameters were statistically analyzed in comparison to simultaneous hba1c results. glucose measurements at 1 hour had better distinction power (p < 0.05) between these individual groups than initial and 2-hour glucose measurements. the average hba1c (%) levels for healthy, pre-diabetic and diabetic individuals were 5.4 ae 0.4, 5.7 ae 0.4 and 6.2 ae 0.7, respectively. roc curve analysis showed 23.4% sensitivity and 98.2% specificity for the clinically accepted hba1c cut-off value of 6.5%. hba1c cut-off value of 5.9% had a higher sensitivity of 67.8% and comparable specificity of 85.7%. the highest discrimination power between healthy, pre-diabetic and diabetic individuals was observed at glucose concentration at 1-hour after sugar administration in ogtt test as opposed 2-hours generally used for diagnosis. low sensitivity was observed for the clinically adapted 6.5% cut-off value of hba1c. the cut-off value of 5.9% for hba1c was found to be more sensitive with comparable specificity than the 6.5% cut-off values for diabetes screening in our population. our results suggest that 5.9% for hba1c should be considered for diabetes cutoff value for turkish population. induction of the glutathione-dependent detoxification capacity is involved in the hepatoprotective effect of silymarin against acetaminophen-induced hepatotoxicity y. kim, d. kwon, c. ahn seoul national university, seoul, south korea recent findings in this laboratory showed that silymarin was capable of promoting hepatic glutathione (gsh) synthesis via a modification of the transsulfuration reactions in the liver. to investigate its pharmacological significance, we examined the hepatoprotective effect of silymarin against liver injury induced by acetaminophen (apap). adult male mice were treated with silymarin (200 mg/kg, po) every 12 hours for a total of 3 doses prior to an apap challenge (500 mg/kg, ip). the apap-induced liver injury was assessed by histopathological examination and measurement of changes in plasma enzyme activities, lipid peroxidation and formation of nitrotyrosine protein adducts in the liver. plasma levels of apap and its major metabolites were monitored for 24 hours to estimate the metabolic transformation of apap. also protein and activity of the major cyp subtypes involved in the metabolic activation of apap into a toxic metabolite were determined in liver of the mice treated with silymarin only. silymarin pretreatment attenuated the apap-induced liver injury significantly when determined 24 hours later. plasma concentrations of apap, apap-glucuronide or apap-sulfate in plasma were not changed, but thiol conjugates of apap, such as apap-glutathione, apap-cysteine and apap-n-acetylcysteine, were elevated significantly in the mice pretreated with silymarin. however, silymarin treatment did not affect protein expression of cyp2e1, cyp1a2, or cyp3a11 in the liver. also hepatic microsomal enzyme activities measured using p-nitrophenol, ethoxyresorufin and erythromycin as substrates, were not increased by silymarin, indicating that the elevation of apap-thiol conjugates should be attributed to an augmentation of the gsh conjugation capacity. it is suggested that silymarin may protect the liver against an electrophilic substance-induced toxicity by increasing gsh availability which would enhance the detoxifying capacity of liver cells. prostate cancer (pca) is the second leading cause of death among men in western countries. we have previously found that the six transmembrane protein of prostate 1 (stamp1) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk/mapk signaling. we also found that stamp1 is highly mobile in pca cells and shuttles between the plasma membrane and the golgi, often found in vesiculotubular structures in the cytosol. using advanced imaging techniques, we have now characterized the trafficking of stamp1 from the plasma membrane to early endosomes in lncap cells, by analysing its dynamic targeting to the three main endocytosis pathways: clathrin-mediated endocytosis, caveolae/lipid rafts, and the arf6-dependent pathway. we found that stamp1 fused to cyan fluorescent protein (cfp-stamp1) is present at the plasma membrane where it accumulates in punctate structures. live cell confocal imaging showed that these puncta were dynamic over time indicating that stamp1 may be constitutively delivered to the plasma membrane and removed from it by endocytosis. co-expression of cfp-stamp1 with various fluorescent protein markers revealed that cfp-stamp1 puncta corresponded to lipid rafts that were labelled with caveolin-1-rfp or antibodies against flotillin. live cell imaging showed that cfp-stamp1 and caveolin-1-rfp disappeared at the same time from the same region of the plasma membrane suggesting that lipid rafts are likely to be responsible for stamp1 internalization. notably, stamp1 was absent from other endocytosis structures such as clathrin-coated pits/vesicles. further work is needed to determine whether stamp1 internalization is required for its function, such as its link to erk signaling, and whether interference with lipid rafts influences stamp1 effects on pca cell proliferation and survival. antithrombin-iii, mpv and plasma total homocysteine levels in behcet's disease introduction: behcet's disease is a multi-systemic and chronic inflammatory vasculitis of unknown etiology characterized by recurrent oral and genital ulcers, uveitis, arthritis, arterial aneurysms, venous thrombosis and skin lesions. platelet indices such as mean platelet volume (mpv) is a standart indicator of platelet function in disease pathophysiology. antithrombin, a glycoprotein synthesized in the liver, is the major plasma inhibitor of thrombin thus modulating blood coagulation. antithrombi-iii (at-iii) is a enzyme even moderate deficiency significantly increases the risk of thrombosis. homocysteine (hcy), that is formed during the metabolism of methionine. several clinical studies have clarified that elevated blood hcy levels are related to atherosclerotic disease. in our study, we investigated ovocystatin is one of the best characterized members of cystatin superfamily of protease inhibitors, and it has been frequently used for pathophysiological studies as the model protein, representative for this superfamily. its application has been supported by high structural similarity to human cystatin c as well as several common biological activities. as regard to biological activity, cystatins, including ovocystatin, are best characterized as inhibitors of cysteine proteases of papain family (c1), such as cathepsins b, h, l and s. these inhibitors participate in intra-and extracellular control of proteolytic events, both in physiological and pathological states. in the recent decade also new activities of cystatins, not assigned to inhibition of papain-like cysteine cathepsins, were found. these activities are associated with an alternative active center for legumain-type proteases in the molecule. here we report a chemical modification of ovocystatin that disables the anti-papain activity of the inhibitor but does not affect its anti-legumain activity. the chemical knockout has been obtained by reaction with 2-hydroxy-5-nitrobenzyl bromide (hnbb) that covalently modifies the trp104 residue in the molecule. the reaction has been monitored by uv-vis and fluorescence spectroscopy. the anti-papain activity of the inhibitor has been measured colorimetrically against bana as a substrate. the anti-legumain activity was assessed fluorometrically using z-ala-ala-asn-amc. the reacted inhibitor exhibited an additional, characteristic for hnbb, band at 410 nm in uv-vis scan. accordingly, an ablation of trp fluorescence was also observed. the molecule fully retained the anti-legumain activity, while only residual antipapain activity (10%) was observed. the modified ovocystatin can be a useful molecular tool for studying the physiological and pathological processes specifically associated with legumain activity. 3 departments of medicine (hematology/oncology) and biochemistry and molecular biology, university of louisville, james graham brown cancer center, louisville, ky, united states 6-phosphofructo-2-kinase/fructose-2,6-bisphospatase (pfkfb) family of enzymes are responsible for the conversion of fructose-6-phosphate (f6p) to fructose-2,6-bisphosphate (f2,6bp) and vice versa, and f2,6bp is an allosteric activator of phosphofructokinase-1 (pfk1), a rate-limiting enzyme of glycolysis. among the four identified pfkfb isozymes (pfkfb1-4), pfkfb2 is the least studied isozyme in human cancers. there exists two different splice variants of pfkfb2, variant-1 and variant-2, coding two different isoforms, isoform a and b, respectively. in this study, we first analyzed the effect of k-ras(g12d)induced oncogenic transformation on pfkfb2 expression in pancreatic duct cells. we found that oncogenic k-ras induction in immortalized pancreatic duct cells (ipde) was associated with decreases in total pfkfb2 mrna and protein expressions (mrna; ipde: 1 ae 0.15; ipde+kras: 0.78 ae 0.24 and protein; ipde: 1 ipde+kras: 0.55). we then, checked individual expressions of splice variants and observed that while pfkfb2 splice variant-1 (p2-v1) expression was reduced by k-ras induction (ipde:100; ipde+kras:81.50), pfkfb2 splice variant-2 (p2-v2) expression was increased (ipde:100; ipde+kras:125.70). then, we checked effects of p2-v1 and p2-v2 on glycolytic phenotype of ipde and ipde+kras cells. over-expression of pfkfb2 variants increased f2,6bp concentration (p2-v1: 1.96; p2-v2: 1.72 fold; compared to empty vec), glucose uptake (p2-v1: 16%; p2-v2: 30%) and glycolysis (p2-v1: 20%; p2-v2: 30%) in ipde+k-ras cells. we next analyzed the subcellular localizations of pfkfb2 isoforms and observed that both pfkfb2 isozymes localize to the nucleus, with more prominent nuclear localization of p2-v1 compared to p2-v2. also, nuclear localization ratio of p2-v2 increases after oncogenic transformation with mutant k-ras. taken together, these results suggest that pfkfb2 may have a role in the glycolytic phenotype of pancreatic cancers characterized with hyperactive k-ras signaling. effects of p38 map kinase inhibitors on mda-mb-231 cell line introduction: p38 mapk phosphorylates serine and/or threonine residues of the target proteins. the activation of p38 mapk leads to cell growth, differentiation, survival or apoptosis. in this study, we tested the effect p38 mapk sb203580 and sb202190 on mda-mb-231 cells to further elucidate the controversial role of p38 mapk on cell proliferation or cell migration. materials and methods: mda-mb-231 cancer line was cultured in rpmi-1640 supplemented with 10% fbs. the cytotoxic and cell migration effects of sb203580 and sb202190 inhibitors were tested by mtt assay and wound assay, respectively. the effects of both inhibitors on proliferation and adhesion of md-mb-231 cells were determined by icelligence system. results: it was found that sb202190 p38 map kinase inhibitor was more effective than sb203580. however, no significant effects of low doses of 1 lm and 5 lm of both inhibitors were seen on cell proliferation as compared to the dmso-treated control cells for up to 96 hours as determined by icelligence system. on the other hand, both sb203580 and sb202190 significantly prevented cell proliferation at a concentration of 50 lm. both sb203580 and sb202190 significantly reduced cell migration in a time-dependent manner at a concentration of 50 lm. then, we tested whether each p38 mapk inhibitors have any effect on cell adhesion during a treatment period of 3 hours using icelligence system. only 50 lm concentration of sb202190 reduced cell adhesion for about 1.5 hour (p < 0.001). conclusion: p38 mapk inhibitors sb203580 and sb202190 differentially affect cell proliferation, survival and migration. acknowledgements: this study is financially supported by dumlupınar university, scientific research project no 2015-85. mutagenicity of a series efficacious benzoxazine derivativesa new approach to evaluate ames test data e. foto 1 , f. zilifdar 1 , s. yilmaz 2 , t. sarac ßbasi 1 , i. yalc ßin 2 , n. diril 1 1 hacettepe university, ankara, turkey, 2 ankara university, ankara, turkey testing safety of drug candidates is as crucial as evaluating their efficacy in early drug development. we previously synthesized a series of 1,4-benzoxazine-3-one derivatives showing significant antimicrobial, in vitro anticancer, topoisomerase i inhibitory activities and studied their several mechanisms of action. in this present study, we have evaluated mutagenic activities of these compounds and their potential metabolites. moreover, we aimed to develop a new statistical algorithm available for structureactivity relationship analysis to identify the regions responsible for the activity. to evaluate mutagenicity of the compounds, ames salmonella/microsome test was used. salmonella typhimurium ta98 and ta100 strains were used to detect for frameshift and basepair substitution mutagens, respectively. additionally, mutagenicity of potential metabolites of them were evaluated by adding metabolic activation system (s9) which was prepared from a pool of male sprague dawley rats. results were evaluated with student's-t test. following regression model estimation analysis, we detected minimum mutagenic doses of all tested compounds for generating a 3d-common features pharmacophore model with hiphop method. according to the results, only bs12, bs13, bs16 and bs17 exhibited strong mutagenic effects on both strains in the presence and absence of s9. additionally bs10, bs7, bs1 and bs15 (in the absence of the s9), bs18, bs4 and bs7 (in the presence of the s9) showed weak mutagenic effects on ta98. hiphop analysis results revealed that mutagenicity was increased in the presence of aromatic desactivating groups which might form hydrogen bonds at the position of r3 and hydrophobic groups at the position of r2 of the benzene ring in the structure of benzoxazine. the new statistical approach developed in this study can be useful for assessing the ames test data available for structure activity relationship analyses. background: recently more than thirty different diseases can screen simultaneously with expanded newborn screening (nbs) programs by tandem ms.expanded nbs with tandem ms is performed routinely at akdeniz university hospital central laboratory since 2008.the aim of this study was to evaluate our nbs results with some second-tier and confirmatory tests. materials and methods: nbs results (n = 1863) were evaluated in dried blood samples which sent to our laboratory for the study between august 2014 and august 2015. electrospray ionisation (esi)triple quadrupole mass spectrometer (shimadzu lc-ms/ms 8030,japan) was used for nbs analysis,acylcarnitine and amino acid profile were screened with mrm (multiple reaction monitoring) spectrum within 2 minutes.second-tier tests were performed as urine organic acid analysis by gas chromatography-mass spectrometry (gc-ms),plasma and urine quantitative amino acid analysis by high pressure liquid chromatography (hplc).pathological nbs results were assessed in three separate groups as amino acid metabolism disorders, fatty acid oxidation defects and organic acidemias. results: metabolic diseases were found in 69 (3.70%) patients by the second-tier tests performed.there were detected amino acid metabolism disorders in 13,organic acidemia in 42,fatty acid oxidation defects in 14 patients. conclusions: the reason of high positive results in our laboratory could explain that our study includes both screening and monitoring of previously diagnosed metabolic patients.nbs is performed in only a few centers in turkey although there were the national screening programs included nbs in many foreign countries.more expanded nbs programmes in our country is required to start treatment of patients before irreversible damage is not occured. although many reports indicate the involvement of calpain in several human pathologies, it is not yet clarified how the protease can recognize the substrates to digest and how can escape to its natural inhibitor calpastatin. answers to these questions have been obtained by identifying specific intracellular localizations of calpain and its substrates and analyzing the interactions of the protease with calpastatin. these studies were carried out using human sknbe neuroblastoma cells. protein-protein interactions and intracellular localization of calpain and the related proteins were determined by immunoprecipitation and isolation of membrane microdomains. we have observed that small amounts of calpain-1 are localized in lipid rafts microdomains together with n-methyl-d-aspartate receptor (nmdar) containing nr1/nr2b subunits. immunoprecipitation experiments have demonstrated that nmdar containing nr1/nr2b subunits, calpain-1, hsp90 and neuronal nitric oxide synthase (nnos) but not calpastatin and calpain-2 are present in specific protein complexes. thus, in this localization calpain activity is regulated by hsp90 that reduces the affinity for ca 2+ of the protease. cell stimulation with nmdar agonists induces calpain activation that specifically cleaves the subunits nr2b of the receptor promoting changes in lipid rafts organization and internalization of nmdar without affecting cell viability. moreover, in these conditions, also nnos is digested and converted in the active form by calpain-1. our data suggest a physiological role of calpain-1 at specific cell sites. the protease inserted in lipid rafts microdomains is in strict contact with its targets and escapes to calpastatin which is not inserted in these structures. following an increase in ca 2+ influx, the activated protease regulated by hsp90, promotes the removal of nmdar from the plasma membranes, decreasing ca 2+ entrance through this receptor-channel and protecting cells from ca 2+ overloading. tissue transglutaminase 2 (tg2) is a multifunctional protein complex that can act as a crosslinking enzyme, gtpase/atpase, protein kinase and protein disulfide isomerase. at the cell surface, tg2 was shown to be involved in adhesion, migration, invasion, growth, epithelial mesenchymal transition and hence implied in the metastatic development of many different tumor types. renal cell cancer (rcc) is one of the most common type of cancer in adult males that generally grows as a single tumor within a kidney. our previous findings indicate that the increased expression of tg2 in rcc results in tumor metastasis with a significant decrease in disease-and cancer-specific survival outcome. herewith, the role of tg2 in cell migration of rcc was investigated in this study by transducing the model rcc mouse cell line renca with a series of tg2 mutant constructs. renca cells were transduced by lentiviral particles encoding wttg2, transaminase-defective tg2-c277s form with low gtpbinding affinity, gtp-binding deficient form tg2-r580a, and transaminase-inactive tg2-w241a. in order to investigate the role of tg2 transamidating and gtpase activity in cell migration, scattering assay was used where 7 colonies for each mutant clone was followed for a time interval of 24 hours. our results showed that non-transduced control and tg2-c277s mutant renca cells demonstrated a similar migration pattern with a 20% of scatter activity. on the other hand, 52% colonies formed by renca cells overexpressing wttg2 and tg2-w241a mutant scattered away from each other. a small insignificant increase in scattering was seen in 28% of the total number of 10 colonies for renca cells overexpressing tg-r580a construct. data from this study supports that gtp-binding activity of tg2 is the drive force in migration driven scattering of renca cells, suggesting that inhibitors targeting the gtp-binding activity of tg2 may serve as a new therapeutic approach in the treatment of rcc. background: in this study, we aimed to investigate the relationship between level of vitamin d with subclinical hypothyroidism and subclinical hyperthyroidism. material and metod: study groups planned as three groups such as euthyroid (n = 11966), subclinical hypothyroid (n = 1040), subclinical hyperthyroid (n = 795). serum tsh, free t4 (ft4) and free t3 (ft3) levels were determined by chemiluminescence immunoassay and serum 25-hydroxy (oh) vitamin d 25 (oh) d level were determined by liquid chromatography-tandem mass spectrometry. euthyroidism was defined as a normal level of tsh (range, 0.4 to 4.2 miu/l), ft4 (range, 0.8 to 2.3 ng/dl) and ft3 (range, 2.3 to 4.2 ng/dl). subclinical hypothyroidism is defined as an elevated serum tsh level associated with normal total or free t4 and t3 levels. subclinical hyperthyroidism is defined as low serum tsh levels associated with normal free t4 and free t3 levels. results: subclinical hyperthyroid group had significantly higher 25 (oh) vitamin d levels compared to the euthyroid and subclinical hypothyroid groups (p < 0.05). 25 (oh) vitamin d levels in subclinical hypothyroid group was not statistically significant when compared with the euthyroid group. food processing wastes provide carbon sources in high amounts for fermentative microorganisms to produce energy. converting carbon-rich biomass into bioethanol through fermentation by microorganisms both provides energy requirement for humankind and also decrease pollutant gases like co 2 , no x and so x (ghorbani et al., 2011) . fermentation processes for bio-ethanol production could be achieved by saccharomyces cerevisiae, zymomonas mobilis, and escherichia coli. bacterial hemoglobin (vitreoscilla hemoglobin, vhb) is the first and best characterized prokaryotic hemoglobin molecule. the function of vhb is supporting the cellular respiration through binding to oxygen at microaerobic environment, transferring it to the terminal respiration oxidases (geckil et al., 2004) and thus improving growth and productivity of the microorganisms. in this study, e.coli strains fbr5, ts3 and ts4 were used as ethanologenic microorganisms. expression of vhb in ts3 is lower than in ts4 strain. for the efficient ethanol production effect of different inoculum sizes, sugar species and sugar concentrations in the growth medium were investigated. vhb expression increased effectively the viability of ts3 strain by up to 1.8x10 9 cfu per ml of fructose (3%, w/v) supplemented lb medium starting with small inoculum for fermentation. this indicates that vgb expression should be at the certain level to maintain sufficient the cell growth for ethanol production. geckil h, gencer s (2004) . production of l-asparaginase in enterobacter aerogenes expressing vitreoscilla hemoglobin for efficient oxygen uptake. applied microbiology and biotechnology 63: 691-697. ghorbani, f., younesi, h., sari, a. e., najafpour, g. (2011) . cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by saccharomyces cerevisiae. ethanol production from dairy industry by product using bacterial hemoglobin t. sar, g. seker, a. g. erman, m. yesilcimen akbas gebze technical university, depertment of molecular biology and genetics, kocaeli, turkey bioethanol production from biomass has a great potential to reduce greenhouse gases emissions. ethanol has several applications in industries (chemical, medical, pharmaceutical, food etc.) in the form of raw material, solvent and fuel. one of the most abundant liquid wastes is cheese whey generated from dairy industries. whey powder is concentrated form of whey and contains lactose and also protein, lipid, minerals and vitamins. vitreoscilla hemoglobin (vhb) is the first bacterial hemoglobin. the main function of this molecule is to improve oxygen transfer to cellular oxidases and thus supporting cellular growth and productivity at low oxygen levels (kallio et al. 1994) . in this work, e. coli strains fbr5, ts3 (low level vhb expressing) and ts4 (high level vhb expressing) were used as ethanol producing microorganisms. fermentation medium containing whey powder supplemented with lb material was inoculated with these strains and incubated for 48 hours at 37°c and 180 rpm in a 1000 ml erlenmayer flask. the ethanol production was improved over 300% by using lower vhb expressing strain. the ethanol levels (v/v, %) were determined as 1.08, 4.99 and 1.51 for fbr5, ts3 and ts4 strains respectively. it is shown that the certain levels of vhb could be useful tool to increase the growth and productivity of ethanol from dairy industry wastes. kallio p.t., kim d.j., tsai p.s. and bailey j.e. (1994) . bioethanol is usually produced from cellulose, hemicellulose and lignin. the lignocellulosic wastes should be hydrolysed into fermentable sugars by using enzymes or dilute acids before microbial fermentation. acidic hydrolysis methodology is cheaper than enzymatic hydrolysis but it can cause production of some inhibitors like aliphatic acids, which affect the growth of microorganisms. vitreoscilla hemoglobin (vhb) is the first described prokaryotic hemoglobin. the recombinant strains carrying vgb gene (e. coli, p. aureginosa) which encodes vhb showed increased bacterial growth, productivity of metabolites compared to untransformant counterparts under low oxygen concentrations [nasr et al., 2001; geckil et al., 2004] . in this study, ethanologic e. coli strains fbr5, its derivative strains ts3 (vgb+) and ts4 (vgb+) were used. ts4 was constructed in such that it could express more vhb than ts3. bioethanol production by these strains in presence of lignocellulosic hydrolysates derived inhibitors was investigated. different acetic acid concentrations (2.5-10 mm) were used as inhibitors from lignocellulose hydrolysate. 10.0 mm acetic acid was used as an inhibitor. the growth of vhb expressing ts3 and ts4 strains was inhibited about 31% after 48 hours fermentation time. strain fbr5 was inhibited as high as 76% by using the same inhibitor including growth medium. it was shown that the expression of vhb could improve growth and productivity in presence of lignocellulosic inhibitors. differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. however, excessive adipogenesis is closely linked to the development of obesity. thus, any drug or chemical that can inhibit adipogenesis may have preventive and/or therapeutic potential against obesity and related diseases. azd1208, an inhibitor of the family of pim kinases, is known for anti-cancer activity. here we investigated the effect of azd1208 on adipogenesis in 3t3-l1 preadipocytes. notably, azd1208 treatment led to a concentration-dependent inhibition of both lipid accumulation and triglyceride (tg) synthesis during the differentiation of 3t3-l1 preadipocytes into adipocytes with no cytotoxicity. on mechanistic levels, azd1208 strongly reduced not only the expression levels of ccaat/enhancer-binding protein-a (c/ebp-a), peroxisome proliferator-activated receptor-c (ppar-c), fatty acid synthase (fas), and perilipin a but also the phosphorylation levels of signal transducer and activator of transcription-3 (stat-3) during adipocyte differentiation. furthermore, azd1208 largely decreased leptin, but not adiponectin, mrna expressions during adipocyte differentiation. collectively, these results demonstrate that azd1208 inhibits adipogenesis in 3t3-l1 preadipocytes and the inhibition is largely attributable to the reduced expression and/or phosphorylation levels of c/ebp-a, ppar-c, fas, perilipin a, and stat-3. effect of intrauterin exposure to artificial food colourings on dna damage in rats in many research genotoxic potential of food additives has been investigated. however there are few findings about the effect of artificial food colourings (afc) on dna. in this experimental study, we aimed to analyze whether in utero exposed artificial food colourings would have effect on dna and cause damage.thirteen female rats were included to the study which were equally divided into two groups as control (cg, n = 15) and food colouring (fcg, n = 15) groups. a mixture of nine food colours were given daily to fcg by oral gavage from preconception to birth. no adverse effect level (noael) of artificial food colourings for each colouring was administered to fcg. three months after the birth, 6 offspring from each group were selected randomly as control (cg) and experiment (eg) groups. then they were sacrified under anesthesia. for performing the alkaline comet comet assay leukocytes were seperated from whole blood samples. the alkaline comet assay was performed. the extent of dna damage was assessed from the length of dna migration derived by subtracting the diameter of the nucleus from the total length of the image and graded into 5 categories and these grades were converted into arbitrary unit (au). differences between the means of data were compared by independent samples t test. the results were given as the meanaesd, p values of less than 0.05 were considered as statistically significant. although the extent of dna damage was higher in eg, the comparison of experiment (13.50 ae 1.76) and control (11.66 ae 1.36) groups showed no statistical difference (p = 0.072). relationship between glucocorticoid receptor gene polymorphisms and recurrent depression l. aydogan, i. benli, z. c. ozmen, i. butun gaziosmanpasa university medical faculty, department of biochemistry, tokat, turkey objective: sensitivity to glucocorticoids varies between individuals and these differences have been implicated in the etiology of psychiatric diseases such as depression. recent studies have found relationship between common glucocorticoid receptor (gr) gene (nr3c1) polymorphisms and unipolar or bipolar depression. the nr3c1 gene is a candidate gene affecting depressive disorder risk and management. the aim of the present study was to evaluate the relative distribution of specific polymorphisms of nr3c1 (bcl1 and rs33388) in recurrent depressive disorder (rdd) patients. methods: our study included 100 volunteers with recurrent depressive disorder and 100 healthy individuals without any mental illness. depression was assessed by hamilton and madrs depression scale. nr3c1 gene polymorphisms were detected by real-time pcr, with hybridization probe method. allele and genotype frequencies at two loci (bcli and rs33388) were investigated in rdd patients and controls. results: genotype distribution among rdd patients and the control group for bcl-1 (g/c) were as follows: cc 3% and 5%, gc 27% and 34%, gg 70% and 61%, respectively. there was not a significant difference when the frequency of the allele (p = 0.204) and genotype frequency (p = 0.382) were compared between the patients and the control. genotype distribution in the rs33388region (a/t) of the patients and controls were tt 29% and 25%, ta 49% and 54%, aa 22% and 21%, respectively. allele frequency (p = 0.841) and the genotype frequencies (p = 0.754) were not significantly different among the groups. conclusion: numerous nr3c1 gene polymorphisms were previously reported in association with modification of depressive disorders. the results of our study showed no association between gr genotype and recurrent depressive disorder. nr3c1 polymorphism does not play a role in the development of recurrent depressive disorder. thymoquinone (tq) has been shown to supress the proliferation of various tumor cells, while it is minimally toxic to normal cells. the aim of this study is to investigate the potential therapeutic effects of tq on cell proliferation, apoptosis, invasion, migration, colony formation and wound-healing in sh-sy5y human neuroblastoma cell line. sh-sy5y cell line treated with 5-400 lm tq by solving medium for 24, 48 and 72 h considering a time-and dose-dependent manner. the cytotoxic effect of tq was determined by mtt method. total rna was isolated by trizol reagent. cdna synthesis was performed by using commercial kit. mdm2, p53, p21, akt, pten, cdk4, cyclin d1, caspase-3, -8, -9, -10, bcl-2, bax, parp, bcl-xl, bid, dr4, dr5, puma, noxa, mmp-2, -9, timp-1, -2 and gapdh gene expression profiles were analysed by real-time pcr method. effects of tq in sh-sy5y cells on invasion, colony formation and cell migration were detected by matrigel-chamber, colony formation assay and woundhealing assay, respectively. statistical analysis were performed with rt 2 profiles array data analysis by using student's t test. ic 50 value of tq in sh-sy5y cells was detected as 15 lm at 48 th hours. by rt-pcr results, it was determined that tq caused a decrease in the expression of mdm2, akt, cdk4, cyclin d1, bcl-2 and mmp-2. it is also observed that tq caused a significant increase in the expression of p53, pten, caspase-3, -10, bid, dr4, puma, noxa and timp-1. it was also found that tq in sh-sy5y cells suppressed invasion, migration and colony formation by using matrigel invasion chamber, wound healing and colony formation assay, respectively. in conclusion, we demonstrate that tq significantly effect cell cycle, apoptosis, invasion, migration and colony formation of sh-sy5y cells. tq may be a potential candidate as chemotherapeutic agent for the treatment of neuroblastoma. more studies have to be performed to profile the mechanisms and genome wide effects of tq to prove its therapeutic potential. dna aptamers can achieve a very high affinity to the target due to the potential of developing broad target-binding interface. however, classic strategy selection of aptamer binders is a challenging task requiring multiple rounds of panning and post-selection optimization. we have developed fast and convenient technique for the selection of dna aptamers based on the offrate selection and tandem affinity purification (tap). we constructed and produced in e.coli recombinant chimeric protein, comprising two affinity tags (his6 and gst) separated from each other and from the target protein (anthrax protective antigen domain iv, padiv) by sumo protease recognition polypeptide and synthetic cleavage site for the anthrax lethal factor (lf). the protein bound to aptamer library is first captured by imac resin, cleaved by sumo protease, captured by gst resin and eluted by lf following the lines of the tap method. the gst-captured aptamer-target complexes were subjected to the off-rate selection using soluble padiv as the competitor. multiple selection rounds are cumbersome and can result in carryover. high abundance of moderate affinity aptamers in the resulting pools obtained by classic selection approaches suggests that the procedure to counter-select them at the beginning of panning is needed. reduction of the contact duration between the aptamer library and the target was crucial for efficient selection of high-affinity binders. on the other hand, tap prevents contamination, and bundled with the off-rate selection, allows for clean isolation of high-affinity binders with affinity in the low nanomolar range. the developed technique is applicable for efficient selection of high affinity dna binders to soluble recombinant proteins and their fragments. dna aptamers obtained will be further used for the development of diagnostic and therapeutic tools for the detection and treatment of anthrax. the work was supported by russian science foundation research grant no. 14-15-00630. the role of macab efflux pump in protection of serratia marcescens against antibiotics and oxidative stress the emergence of bacterial multi-drug resistance is a growing problem of public health worldwide. bacterial drug efflux pumps are membrane protein complexes that function to expulse drugs from the cell. they play a crucial role in the rising rates of antibiotic therapy failures. the homolog of macrolide-specific pump macab was identified in opportunistic pathogen serratia marcescens and was used in this study to characterize its role in protection against antimicrobials and other processes beyond the active efflux of antibiotics. here we used method of serial dilutions to determine minimum inhibitory concentration (mic) for s. marcescens sm6 wild type (wt) and its isogenic δmacab mutant strains. we also used h 2 o 2 survival assay to evaluate the ability of wt and the mutant strain to withstand an oxidative stress. finally, we used b-galactosidase assay to evaluate macab promotor activation in the reporter strain and followed macab expression by western blotting analysis using macab-6xhis strain. we show that in contrary to its e. coli homolog, macab pump in s. marcescens is not involved in the protection against macrolides but instead it is required for protection against aminoglycosides. we further show that similar to its salmonella typhimurium homolog, s. marcescens macab is essential for protection of bacteria against h 2 o 2 . transcriptional analyses demonstrate that while low level of macab promotor activity can be detected after 2 hours of growth in lb-broth there is at least 5-fold increase in expression in response to the presence of h 2 o 2 . on the protein level macab can be detected starting from 3 hours of growth in lb-broth and it reaches maximum expression on 16 hour of growth. our data suggest that macab pump in s. marcescens is involved in protection of bacteria against aminoglycoside antibiotics and is crucial for protection against reactive oxygen species. we are currently working on identification of macab substrate with anti-h 2 o 2 properties. antiproliferative and apoptotic effects of noscapine on mcf-7 and mda-mb-231 human breast cancer cell lines approximately 10-17% of breast cancers are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. these are most aggressive tumor and a clinical problem because of lack of targeted therapies. noscapine is an alkaloid from opium. noscapine is a microtubule-interfering agent. it causes mitotic arrest, induces apoptosis. in this study, we aimed to investigate the effects of noscapine in mcf-7 and mda-mb-231 human breast cancer cell lines. the cytotoxic effects of docetaxel, tamoxifen, and noscapine on the mcf-7 and mda-mb-231 cell lines were analyzed by roche xcelligence system. the cells were cultured in 10% fetal bovine serum containing dulbecco's modified eagle medium at 37°c in a humidified atmosphere containing 5% co 2 . 24 h after seeding, the cells were treated with 4 different doses of docetaxel (12.5 to 100 nm), tamoxifen (12.5 to 100 lm), and noscapine (12.5 to 100 lm). cultured cells were harvested, fixed with 10% formalin, and centrifuged. pellet was blocked, fixed, and embedded in paraffin. paraffin-embedded cells blocks were sectioned at 4 lm thickness and stained with h&e, ki-67, bcl-2, cyclin-d1, and bax. sides were assessed under a light microscope. quantification of the analyzed proteins were evaluated by the percentage of positive cells. all drugs showed cytotoxic effects on both cell lines. all drugs inhibited the proliferation of breast cancer cells, but effects were dependent on time and dose. all drugs were especially more effective on mcf-7 cells. immunohistochemical examinations revealed that tamoxifen was more effective on mcf-7 cells, hovewer docetaxel and noscapine were more effective on mda-mb-231 cells. tamoxifen has more apoptotic and antiproliferative effects on mcf-7 cells. docetaxel and noscapine showed more apoptotic and antiproliferative effects on mda-mb-231 cells. noscapine may be an effective anticancer agent due to antiproliferative and apoptotic effects on breast cancer cells. negative selection of dna aptamers to reduce non-specific binding in solid-phase-based selection procedures carryover by binders specific to the components of the selection system can be a serious issue in hampering the aptamer selection campaign. solution-or "mass"-based techniques still cannot substitute classic phase-separation strategies. one approach to prevent selection of "passenger" phase-specific (plastic, beads) or blocking agent specific aptamer species is their depletion from the initial library pool. our aim was to develop the universal technique for removal of such aptamers exemplified by bsa-and casein-specific binders, while preserving the initial library complexity. the dna aptamer library was subjected to three rounds of depletion using magnetic beads with covalently attached casein and bsa. to ensure high depletion efficiency, beads were pelleted in a 15-ml centrifuge tube by a neodymium magnet through a 10-cm cushion of 20% sucrose, thus preventing weakly bound aptamers from re-populating the library. high complexity of the input library helped to avoid pcr amplification after depletion rounds preventing the library bias introduced by dna amplification. the depletion effciency was confirmed by real-time pcr. resulting oligonucleotide sub-library was analyzed for binding to the targets using solid-phase real-time pcr assay. we have shown that three rounds of panning under the conditions employed provided full depletion of the initial dna pool from nucleic acid structures capable of binding to protein competitors and hampering the process of aptamer selection. we compared selection efficiency of aptamers specific to type a botulinum neurotoxin light chain in depleted vs undepleted library. the yield of the target-specific aptamers was 10-fold higher in the library subjected to the depletion procedure. removal of undesired binders from aptamer libraries appears an important step of solid-phase selex procedure. it can become a useful approach in optimizing solid-phase selex. the work was supported by russian science foundation research grant no. 14-15-00630. epithelial mesenchymal transition (emt) is a critical trans-differentiation program driving cancer metastasis. patients showing signs of emt or presence of distant metastasis have poor prognosis. another well-known feature of decreased cancer-associated survival is the lack of anti-cancer immune responses. thus we hypothesized that the emt and anti-tumor response should be linked via altered secretion of soluble factors by metastatic cells. all cell lines were grown in dmem. emt status of crc cell lines were assessed by investigating canonical markers of emt. cytokine/chemokine expression of crc cells was performed using r&d systems antibody arrays and validated using ccl5 sandwich elisa and rt-pcr. the mechanism of action of zeb1/2 on ccl5 promoter has been studied by luciferace assay and chip. ccl5 coding region was cloned into pcdna3.1 and stably transfected into dld-1 cells. ccl5 deficient ct26 cells were generated using lentivirual shrna transduction. cells overexpressing or knock/down ccl5 were injected orthotopically into mice. t lymphocyte (til) infiltration in respect to ccl5 and sip1 expression was studied using ihc or flow cytometry. emt status catagorised 13 crc cell lines into epithelial, intermediate epithelial, intermediate mesechymal and mesenchymal. cytokine/chemokine antibody arrays showed a significant increase in ccl5 in induced dld-sip1 cells. elisa, multiplex assays and rt-pcr confirmed a significant increase of secreted ccl5 in the induced dld-sip1 cells as well as mesenchymal crc cells as compared to epithelial ones (p = 0.027). promoter studies showed that zeb1/2 bind to ccl5 promoter and and activate ccl5 gene expression. no metastasis was observed for dld-1 cells overexpressing ccl5 but significant alterations of tumour associated lymphocytes were identified in syngeneic orthotopic crc models. our data shows that ccl5 is up-regulated by emt inducing transcription factor sip1, and mesenchymal (metastatic) crc cells secrete significantly more ccl5 compared to epithelial (non-metastatic) ones. ccl5 did not induce emt per se but abundant secretion of ccl5 by metastatic crc cells was a crucial regulator of immune infiltrate in crc. inhibiting ccl5 in metastatic crc may have a therapeutic potential. barley (hordeum vulgare l.) belongs to the grass family, poaceae (gramineae). it is the fourth most important cereal crop after wheat, maize and rice and is among the top ten crop plants in the world. talbina was used to be recommended for the sick and for one who is grieving over a dead person. talbina is made by adding one or two tablespoon of barley flour (must be 100 percent wholegrain barley flour) to one-and-a-half cups of water and placed on low heat for 10-15 minutes (optional: add milk or yoghurt and sweeten with honey). the main objectives of this investigation were determine the a-tocopherol contents and antimutagenicity activity of talbina (hordeum vulgare l.). our results showed that the total tocopherol content was in the range of 0.25 to 1.03 lmol/g fw. talbina extract was shown to have greater antimutagenic activity observed in the 2500 lg/plate concentration s. typhimurium ta98. at all the doses antimutagenic response was significant at (p < 0.01) against both the strains with a percent mutagenicity decrease from 40 to 25 for ta98 followed by ta100 with percent antimutagenicity from 30 to 11. the results of the study concluded that talbina is a better antimutagenic agent than vitamin e and combination of vitamins did not produce any synergistic activity. the compounds containing thiadiazoles have diverse applications as antifungals, anticancer agents, antibacterial, antiinflammatory drugs, antidepressants and carbonic anhydrase inhibitors according to literature. in this study some novel thiadiazole compounds [(1,4,10,13)-tetrathia[4.4] (2,5)-1,3,4-thiadiazolophane; (4,16)dioxo-1,7,13,19)-tetrathia[7.7](2,5)-1,3,4-thiadiazolophane; (4,7,19,22)-tetraoxo(1, 10, 16, 25 )-tetrathia[10.10](2,5)-1,3,4-thiadiazolophane and (4,7,10,22,25,28)-hexaoxo(1, 13, 19 ,31)-tetrathia [13.13] (2,5)-1,3,4-thiadiazolophane] were used to evaluate the cytotoxicity on healthy human lymphocytes and the antibacterial activities. cytotoxicity tests were perfomed using mts assay and the trypan blue test. cells were incubated with the compounds for 72 hours. at the end of the each 24 hour, cell vitality was assessed by measuring the absorbance (490 nm) of each well using a microplate reader for mts assay. in addition, viability percents of the cells were determined after trypan blue test. as a result, the compounds showed cytotoxicity in a dose dependent manner. for the concentrations of 1:1000 of 0.5 mg/ml, the cytotoxic effect was eliminated. also, antioxidant capacity was determined using 2,2-diphenyl-1-picrylhydrazyl (dpph) reagent. moreover, the antibacterial activities of the compounds were analyzed using a microdilution test against e. coli and s.aureus. compounds having various concentrations showed different antibacterial effects against these two bacteria. arabidopsis thaliana ecotypes vary in their ability to utilize organic p substrates insufficient quantity of inorganic phosphorus in soil is an evergrowing problem that affects many fields of agriculture. unlike inorganic phosphates, organic phosphorus compounds are very common in many soil types, but plants are often unable to efficiently utilize them. to better characterize the extent of natural variation in the ability of the model plant arabidopsis thaliana to grow on organic phosphorus compounds, we grew 19 arabidopsis ecotypes on several organic and inorganic sources of phosphorus. plants were grown in liquid or solid media containing naphosphate, phytate and atp as the sole supply of phosphorus or in absence of any phosphorus source. after several weeks of growth, plants were assayed for changes in their morphological and physiological characteristics. phytate was shown to be the least preferred source of phosphorus compared to inorganic phosphate and atp. the rate of biomass accumulation in all ecotypes decreased in the following order from inorganic phosphate to atp to phytate. lateral root formation was markedly reduced in the absence of any phosphorus source or in the presence of phytate. we also showed that phosphomonoesterase activity in intact roots increased when plants were grown on atp and phytate. overall phosphorus content in leaves and roots was similar when plants were grown on atp or inorganic phosphate, but it was markedly reduced on phytate. substantial differences between ecotypes were also observed in root length, p content in ash and phosphomonoesterase activity in intact roots. our analysis of the ability of arabidopsis ecotypes to grow on several different phosphorus sources provides a unique opportunity to investigate the degree of natural variation in this plant's ability to adapt to different nutritional environments. analysis of many important morphological and physiological changes observed in these plants can further extend our understanding of the full range of plant responses to phosphorus availability. laboratory tests are important in terms of confirmation of diagnosis given by clinics and implementation of appropriate treatment protocols for patients. laboratory tests used by the clinics have been increased in parallel with time.there are many reasons for increased use of the test such as increase of elderly population, increase in standard of care, lack of information and shortening of turn around time. unnecessary laboratory testing also constitute one of the reasons for increased use of laboratory tests. in our study we aimed to investigate the unnecessary laboratory testing for fpsa test. fpsa tests which are ordered with total psa tests that values of less than 4 ng/ml or greater than 10 ng/ml were accepted as inappropriate initial testing. 9759 fpsa tests were evaluated as unnecessary laboratory testing. the clinic which ordered the maximum unnecessary laboratory testing with 3315 was urology within all the clinics. although to the restrictions about the ordering of total psa and fpsa tests there were no decrease in the number of unnecessary laboratory testing. unnecessary usage of laboratory testing may cause increase of false positive results, increase in the use of invasive testing, unnecessary drug consumption and increase of healtcare costs. some precautions may be effective in reducing unnecessary tests such as to inform clinicians about the cost of laboratory tests, to increase the clinician education programs and to develop usage of disease specific diagnostic algorithms about test ordering. local clinical validation of blood collection tubes although the tubes with gel and clot activator are widely used due to the advantages, there are ongoing discussions about the effects of the blood collection tube on clinical outcomes in the analysis of biochemical parameters. therefore, we aimed to prove the local clinical validation of the new produced blood collection tubes with low-volume. the blood samples of 40 patients who referred to the hospital phlebotomy unit were collected using holder into the 3 different tubes. first tube was 5 ml glass tube and with no additive, second was 5 ml tube with gel separator, third was 1 ml tube with gel separator. serum was separated and immediadiately analysed for 25 biochemical parameters. the difference between the analyte amounts in the different tubes was evaluated using paired t-test. the clinical significance was evaluated using significant change limit. bias (%) between the other tubes with the reference tube was also evaluated according to the ''allowable total error". when we compared the other test tubes to a glass tube which was assumed reference tube, total protein, albumin, amylase, calcium, triglyceride, cholesterol, hdl-cholesterol, total and direct bilirubin, iron, gamma glutamyl transferase, magnesium, phosphorus results were statistically significant. but the results of all the analytes were within the significant changes limit and the allowable total error was not significant. while a biochemical parameters have analysed, it may be absorbed into the gel and this may caused from factors such as the chemical structure of the gel, analyte itself, the residence time in the gel, storage temperature and volume of the sample e.g. as well as the leaking of gel material to the sample was reported to be another factor for affecting the analysis. despite these factors, we observed that neither gel-clot activator tube with low nor high volume affect the clinical results. the research of the frequency of interference in thyroid function tests interference is defined as the effect of substance in the sample which changes the correct value of laboratory results. the frequency of interference in immune techniques is varied. the frequency of interference depends on population of the study, technique for detecting the reaction and researcher's method. unexpected or inconsistent results with clinical findings should suggest the possibility of interference. in this study it is aimed to investigate the frequency of interference in thyroid function tests (tsh, ft3, ft4) which are the most common requested laboratory tests. thyroid function tests of 47915 patients are analyzed in ankara numune education and research hospital in october 2014-may 2015. five samples which had the incompatible results with clinical findings are re-evaluated just because of the suspicion of interference. the detection of interference included; repetition of test via different immune techniques, serial dilution, polyethylene glycol (peg) precipitation and incubation with heterophilic blocking tubes (hbt). the results of two different immune techniques and before/ after incubation with hbt showed no significant difference. linear curves had observed in serial dilution. after peg precipitation; below 40% of recovery had obtained in one sample, therefore it is interpreted as macro-tsh. the frequency of interference in thyroid function tests for 8-month study period was 0.01%. no information is found about the best test for defining the cross reaction. it is also aforethought that interference should not be excluded by using any single procedure. p-mis-105 development of polyclonal and monoclonal antibodies against fatty acid binding protein 4 (fabp4/ap2) a. abbasi taghidizaj, g. aydogdu, b. p. sermikli, e. yilmaz ankara university, ankara, turkey recombinant proteins and antibodies can be use for therapeutic or diagnostic purposes which produced in many different host organisms. the technique for the production of immortal cell making single antibody, fusing target antibody-forming b lymphocyte precursor with a suitable myeloma cells. the fused hybrid cells (called hybridomas), as a cancer cell will reproduce rapidly and will produce large amounts of the desired antibodies. fatty acid binding protein (fabp4) is a well characterized intracellular lipid transport protein and plays a key role in the intracellular fatty acid transport and adipose tissue metabolism. fabp4 as a adipokine that regulates glucose homeostasis and has various features for metabolic syndrome associated with obesity. in this study, production of monoclonal antibodies against immunogenic fabp4 protein made by recombinant dna technology. recombinant his-fabp4 was expressed in e.coli and purified. balb/c mice used for immunization and serum anti-fabp4 antibodies determined by enzyme-linked immunosorbent assay (elisa). hybridoma cells created by fusion of splenocytes and myeloma partner cells. after selection of antibody producing cell clones, injecting hybridomas into the peritoneal cavity in balb/c mice ascites fluids was obtained. we have selected fifteen hybridoma clones that produced antibodies specific for fabp4, as shown by western blotting and immunocytochemistry. as a result we produced mabs that will be useful for the scientific community working on fatty acid binding proteins and lipid metabolism. in near future, therapeutic approach for this antibody maybe a possibility in metabolic syndrome. thioridazine, an anti-psychotic drug, inhibits migration, invasion and epithelial mesenchymal transition in breast cancer cell lines thioridazine (thz), an antipsychotic drug, exhibits anti-angiogenic effects on breast cancer cell lines. however the mechanistic insight in exerting antiangiogenic effect is not clearly understood. the objective was to investigate the role of thz in epithelialmesenchymal transition (emt) by using cell migration assay, scratch assay, western blot (wb) and immunocytochemistry. thz treatment reduced cell viability on mda-mb-231, mcf-7 and cd44 + /cd24-cells and ic50 values of thz were found to be 9 lm, 16.4 lm and 18 lm respectively, at 24 hours. invasion potency of mcf-7, cd44 + /cd24-and mda-mb-231 cells were determined as 29%, 24%, 16.5% when compared to relevant treatment controls. migration potency of mcf-7, cd44 + /cd24-and mda-mb-231 cells was determined as 28.5%, 63.2%, 61% respectively. among the three cell lines mda-mb-231 cells display enhanced invasive and migration ability when compared to other cell lines. western blotting results demonstrate that thz significantly increases e-cadherin, cytokeratin-18, b-catenin, while inhibiting n-cadherin, vimentin, fibronectin. immunocytochemistry studies revealed decrease in e cadherin and a concomitant increase in vimentin level for all three cell lines upon treatment with thz. moreover thz significantly inhibited the cell migration, invasion and emt in mda-mb-231, mcf-7 and cd44 + /cd24 cell lines by suppressing mesenchymal markers. in conclusion, these data suggest that thz might be a novel anti-proliferative and anti-metastatic agent for treatment of breast cancer. effect of seasonal temperature and humidity on urine density in children environmental heat and humidity are important factors affecting hydration status in childhood. hereby, we aimed to investigate the effects of seasonal climate changes on urine density of children living in mediterranean climate, cyprus. 1700 healthy 0-18 year children's (850 girls, 850 boys) age, sex and urine density results were collected retrospectively for three consecutive years. the correlation of urine density with each seasonal and 12 months' average temperature and humidity has been analysed. the urine density results had a positive correlation with temperature (r = 0.083, p = 0.001) and a negative correlation with humidity (r= à0.072, p = 0.003). mean urine density in spring was higher than that of autumn (p = 0.02) and winter (p = 0.00). mean value of summer was higher than autumn (p = 0.03) and winter (p = 0.00). 0-24 months age group had lower urine density. evaluation of urine density based on gender and puberty revealed no statistically significant difference. seasonal mediterranean climate changes have an impact on urine density in children which may affect hydration status especially in infants < 2 yrs of age. during high temperature seasons ensuring adequate water intake is essential in this age group in mediterranean climate. p-mis-108 implementation related to the use of antibiotics and data sources by community pharmacists in north cyprus as the resistance to antibiotics is gaining importance in today's world;the solution to this problem is possible through a common consciousness of the doctor who prescribes antibiotics,the pharmacist who sells and the patient who consumes antibiotics. irrational use of drugs is an economic and medical problem in many developed and developing countries around the world.the aim of this study is to determine the sales ratio of non-prescription antibiotics in pharmacies which is the biggest category of the antibiotic group sold as well as the indications that lead to its' prescription. eighty-four pharmacies out of 168 pharmacies located in north cyprus were involved in the study with 50%stratified systematic sampling, questionnaires were filled and a consent form was signed by the participating pharmacists. the pharmacists involved in the study stated that non-prescribed antibiotics were demanded from the pharmacists and all except two (97.6%),responded positively to this demand. it has also been identified in the study that 41.5% of the daily sale of antibiotics in the first half of the year 2014 was non-prescribed. the most purchased antibiotics either with or without prescription was found to be the penicillin and its derivatives with 76.2% and upper respiratory tract with 86.9%. when the level of selfawareness of the pharmacists was examined, the rate is found in north cyprus to be (41.5%),compared with the studies conducted in greece,italy,malta and spain 47% and egypt 50.4%that designated the non prescribed antibiotics purchased from the public pharmacies. the rate of sale of non-prescribed antibiotics in north cyprus has been found to be at a higher level compared to the rates in many developed and developing countries. furthermore, the upper respiratory tract infections are amongst the most common viral causes which lead to a high consumption of both prescribed and non-prescribed antibiotics. this study was supported by turkish viral hepatitis prevention society. acrylamide has cytotoxic, antiproliferative and apoptotic effects on human lung adeno carcinoma cell line a549 acrylamide (aa), a widespread substance in many fields, forms in foods during high temperature processing such as baking, roasting, frying. aa is a potent neurotoxic, genotoxic and clastogenic agent being a strong electrophile and forming adduct with biological molecules or potent nucleophiles. up to now, several studies confirmed the toxicity of acrylamide to several organs. on the other hand, aa is reported to have inhibition effects both on proliferation and differentiation of different cancer cells in a time and dose-dependent manner. in addition, natural and synthetic acrylamide derivatives are also used as potent anti-cancer agents. moreover, inhibition concentration (ic50) values of aa against these cancer cells have not been investigated in detail yet. thus, the goal of this study is to investigate the cytotoxicity of aa on a549 cells including with ultrastructural and morphological effects. ic50 value of aa on a549 cells for 24 h was detected with mtt (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide) colorimetric assay. we evaluated morphological changes under confocal microscopy and ultrastructural changes under transmission electron microscopy (tem). our results demonstrate that aa inhibits the proliferation of a549 cells in dose-dependent manner and ic50 on a549 cells was found to be 4.6 mm for 24 hours. confocal microscopy evaluations showed that aa caused nuclear condensations, fragmentations, cytoskeleton lacerations and membrane blebbing. tem results revealed membrane blebbing, chromatin condensations and cell shrinkage. although aa is a probable carcinogen substance, it drastically inhibited cell viability in dose-dependent manner. from microscopic assessments, aa is suggested to induce apoptosis in a549 cells. in conclusion, the present study confirms the high potential of aa for cytotoxic, antiproliferative and apoptotic activity on a549 cells. however, appropriate aa dose is critical to prevent its possible adverse effects. effect of hemolysis and lipemia on some immunochemical tests in beckman coulter unicell dxi 800 immunoassay analyzer c. yilmaz, s. yildiz, m. senes, v. fidanci, d. y€ ucel ankara training and research hospital, ankara, turkey the aim of the study was to investigate the effects of in vitro hemolysis and lipemia on 25 immunoassays studied by the beckman coulter unicell dxi 800 immunoassay analyzer. we prepared a serum pool without hemolysis, lipemia and icterus. baseline serum pool concentrations of 25 tests were measured by the beckman coulter unicell dxi 800. d _ ifferent serum pools, six for hemolysis and five for lipemia, were spiked with increasing concentrations of hemoglobin (0.6, 1.2, 2.4, 4.5, 6.6 and 8.6 g/l hemoglobin) and intralipid (0.625, 1.25, 2.5, 5 and 10 g/l intralipid). the hemolysate was prepared by osmotic shock method. intralipid (20%, baxter, deerfield, il) was used to mimic the effect of lipemia. the hemolysis (h), lipemia (l) and icterus (i) indices were measured on beckman coulter au 5800. after spiking the pools, the 25 tests were measured again in duplicate on beckman-coulter dxi 800 analyzer. a change of 10% from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. we observed a positive interference due to hemolysis for folat, vitamin b12, testosterone and by lipemia for cortisol. there was a negative interference of hemolysis for ca 19.9, ca 125, ca 15.3, insulin, pth and e2, and of lipemia for progesterone, ca 19.9, vitamin b12 and pth. we found clinically significant effect (>total analytical error) of hemolysis on folate and insulin, and lipemia on cortisol. investigation of the effect of two different p38mapk inhibitors in rats subjected to isoproterenol-induced acute myocardial injury: an experimental study objective: acute myocardial infarction is a serious acute condition. in the current study, we aimed to investigate the possible effect of two different mitogen-activated protein kinase (p38mapk) inhibitors in rats subjected to isoproterenol (iso)induced myocardial injury. materials and methods: a total of 32 male wistar-albino rats were equally and randomly seperated into four groups as follows: control, iso, iso plus sb203580 andiso plus tak-715. treatment agents were orally administered and myocardial injury was induced by subcutaneous injection of iso. serum cardiac troponin-i (ctni), ischemia modified albumin (ima), heart fatty acid binding protein (hfabp) levels and paraoxonase-1 (pon-1) activity, tissue tos (total oxidant status), tas (total antioxidant status), tt (total thiol), tumor necrosis factor-a (tnf-a) levels, superoxide dismutase (sod) and glutathione peroxidase (gsh-px) activity levels were measured. tissue mrna levels of nf-jb, p38 mapk and nuclear factor erythroid 2-related factor 2 (nrf2) were analyzed. heart tissues were also immunohistochemically and histopathologically evaluated. results: both compounds have led to a decrement in myocardial damage, apoptosis, ctni, ima, hfabp, tos, and tnf-a levels, nf-jb, p38 mapk, phosphorylated c-jun n-terminal protein kinase (pjnk 1/2) expressions. on the other hand, the applied treatment increased sod, gsh-px, tas and tt levels, as well as phosphorylated extracellular signal-regulated kinase (perk 1/2) and nrf2 expressions. conclusion: data established from the current study suggest that administered agents have protective effect against cardiac injury induced by iso, which was more prominent in rats received sb203580 treatment. p38mapk inhibitors may constitute a useful choice as cardioprotective agents due to their antiinflammatory, antioxidant and anti-apoptotic effects. keywords: _ isoproterenol, myocardial infarction, myocardial ischemia, p38 mitogen-activated protein kinases, sb203580, tak-715. silicosis composes the vast majority of occupational lung diseases. silicosis, caused by inhalation of crystalline silica, is a chronic lung disease characterized by parenchymal nodules and pulmonary fibrosis. the susceptibility of patients with silicosis to infection is thought to be due to toxic effects of silica on pulmonary macrophages. ada activity is considered as a nonspecific marker of t cell activation and cellular immunity. this study aimed to compare the serum ada activity in silicosis patients with spirometric values. in this study there were 35 males in each groups which contained patients with silicosis (group 1), individuals having similar symptoms with silicosis from same occupational area (group 2) and healthy subjects (group 3). routine hematological and biochemical parameters were also measured. the serum ada activity and spirometric values (fev1, fev1%, fev1/fvc, fev1/ fvc%, fef25-75 and fef25-75%) were compared. the average age of group1, 2 and 3 are 38.5 ae 10.6, 39.5 ae 2 and 51 ae 10.5 years, respectively. there was a significant difference between group 1 and 3 in terms of the ada level (p < 0.05). there was a negative correlation between ada activity and fev1, fev1%, fev1/fvc, fev1/fvc%, fef25-75 values. elevated serum ada activity has been shown in many diseases with induced cellular immunity. despite initially toxic effects were lead to a little immunological reaction in patients with silicosis, continuation of this immunological response is important in some chronic manifestations of silicosis. the release of chemotactic factors and inflammatory mediators cause the migration of polymorphonuclear leukocytes, t lymphocytes and macrophages. in this study, the ada activity was significantly higher in patients with silicosis than others. increased immunity in patients with silicosis is being considered, increasing ada activity might be help of earlier recognition of these patients and to take better quality of life. atlantic salmon (salmo salar l.) is an important model system in evolutionary and conservation biology that provides fundamental knowledge into population persistence, adaptive response and the effects of anthropogenic change. the role of behavioral and body size variation in environmental adaptation of atlantic salmon is well known, by contrast, the underlying biochemical mechanisms are largely unknown. intracellular proteases, such as cathepsins b and d in lysosomes and calpains and proteasome in cytosol, due to their metabolic and regulatory role may contribute to phenotyping speciation of salmon young. we examined the activity of intracellular proteolytic enzymes in skeletal muscles of atlantic salmon parr from two local habitats of the varzuga river (the main channel and small tributaries) differing in hydrological and feeding parameters. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from varzuga river (kola peninsula, russia). it is known that salmon parr originated from a common hatch became phenotypically divergent during the settle in the biotopes. reliable difference in studied enzyme activities in the salmon parr from two local habitats was found; furthermore, calpain and cathepsin b proteolytic activities were found to negatively correlate with parr body size. muscle proteolytic activity data support an idea on protease contribution to environmentally-driven adaptation and speciation process in fish. the work was supported by the russian scientific foundation, project no. 14-24-00102. the phylogenetic analyses of anthriscus (apiacea) species from turkey based on non-coding "trn" regions of chloroplast genome p. yilmaz sancar 1 , m. tekin 2 , s. civelek 1 1 firat university, elazig, turkey, 2 cumhuriyet university, sivas, turkey anthriscus pers. (apiaceae) species belongs to apiaceae family and is represented by 16 genus on the world and by 8 genus in turkey. anhriscus species are used extensively for treatment various disease such as asthma, alzheimer and show anti-tumoral, anti-microbial, antioxidant features. for determining exact species which treat disease it is necessary sorting species correctly with molecular markers to support morphological features. anthriscus species were defined by examining insufficient quantity of samples in turkey flora. besides, no detailed study was found in our country after flora study. for this reason a revision study was made with the aim of solving some systematical problems in 2013 by tekin. the result of the study provided important contribution to the systematic of the species in turkey. however a molecular study was also required for building the obtained results on a more solid ground. in this study, the aim to reveal systematic and phylogenetic relationship among species of anthriscus in turkey, by using trnl-f region in chloroplast genome. dna was isolated by ctab method and amplified in pcr by using e-f primaries. the obtained data was evaluated by mega 7.0 program and phylogenetic tree was prepared by using maximum likelihood method. according to the phylogenetic tree that we prepared by using the sequence line up of trnl-f section, it was observed that a. cerefolium, a. caucalis and a. tenerrima species completed their speciation and an isolation with other species in terms of speciation was provided. it was also observed that a. kotchi, a. sylvestris subs. sylvestris, a. sylvestris subs. nemarosa and a. lamprocarpa'nın provided hybridization among themselves but they did not complete their speciation. it was determined that a.lamprocarpa var. chelikhii which is one of the two different varieties of a. lamprocarpa is actually a new sub-species. this fact was supported by molecular data obtained from the study we made after morphologic data. introduction: excessive production of androstenedione can becaused by defects of adrenal steroid biosynthesis, tumors of ovarian and adrenal origin, polycystic ovarian syndrome, increased peripheral sensitivity to androgens, and increased peripheral production of androgens. most epidemiologic studies use enzyme-linked immunosorbentassay (elisa) to measure sex steroid hormones because they have acceptable turnaround times and arerelatively inexpensive. mass spectrometry-based methods are currently the most specific quantitative analytical methods for steroid determination. mass spectrometry methods are independent of matrix effects or cross-reactivity. in this study, a new liquid chromatography-tandem mass spectrometry (lc-ms/ms) method was developed. materials and methods: for serum androstenedione measurement, 50 ll of internal standard (d5-11 deoxycortizol) in methanol was added to 250 ll standart or serum and centrifuged at 4.500 rpm for 10 minutes to remove the precipitated proteins. supernatant was transferred to clean tubes and this procedure was performed twice. the supernatant was collected and dried under a nitrogen gas flow at 60 • c and dissolved in mobile phase. 60 ll was injected in to the ultra performance liquid chromatography analytical column for chromatography. elisa study was conducted with drg (lot. no. 50k074) brand kit. results: method comporison between lc-ms/ms and elisa was found slope value 18,412, intercept value à22.87 and r² value 0.1033. the regressione quation was elisa= à2.861782 + 4.905103 lc-ms/ms. discussion and conclusion: method comparison study presented higher results in elisa compared to lc-ms/ms. in our opinion, this might due to the interference in elisa systems. our lc-ms/ms method allows rapid, sensitive and specific determination of androgens in plasma and serum.the specificity of liquid chromatography-tandem mass spectrometry (lc-ms/ ms) offers advantages over immunoassays. heparins play an important role in cell growth, differentiation, migration and invasion. however, the molecular mechanisms of heparin mediated cellular behaviors are not well defined. to determine the effect of heparin on gene expression, we performed a cdna microarray in a hepatocellular carcinoma cell line and found that heparin regulates transcription of genes involved in glucose metabolism. in this study, we showed a new role of heparin in the regulation of thioredoxin interacting protein, which is a major regulator of glucose metabolism, in hepatocellular carcinoma cell lines. we determined the importance of a unique carbohydrate response element located on its promoter for the heparin-induced activation of thioredoxin-interacting protein and the modulatory role of heparin on nuclear accumulation of carbohydrate response element associated proteins. we showed the importance of heparin mediated histone modifications and downregulation of enhancer of zeste 2 polycomb repressive complex 2 expression for heparin mediated overexpression of thioredoxininteracting protein. when we tested biological significance of these data; we observed that cells overexpressing thioredoxininteracting protein are less adhesive and proliferative, however they have a higher migration and invasion ability. interestingly, heparin treatment increased thioredoxin-interacting protein expression in liver of diabetic rats. in conclusion, our results show that heparin activates thioredoxin-interacting protein expression in liver and hepatocellular carcinoma cells and provide the first evidences of regulatory roles of heparin on carbohydrate response element associated factors. this study will contribute future understanding of the effect of heparin on glucose metabolism and glucose independent overexpression of thioredoxin-interacting protein during hepatocarcinogenesis. prolidase activity in chronic obstructive pulmonary disease and asthma t. g€ uc ßl€ u 1 , h. s€ urer 1 , g. bilgin 2 , d. y€ ucel 1 1 ankara training and research hospital, medical biochemistry department, ankara, turkey, 2 ankara training and research hospital, chest diseases department, ankara, turkey chronic obstructive pulmonary disease (copd) is a consequence of an underlying chronic inflammatory disorder of the airways that is usually progressive and causes dysregulation in the metabolism of collagen. and asthma is a disease where there is an accumulation of collagen in the reticular basal membrane of the airway leading to chronic inflammation. prolidase has an important role in the recycling of proline for collagen synthesis and cell growth. we measured and compared prolidase activity in healthy individuals with copd and asthma patients to find out that whether its activity might reflect disturbances of collagen metabolism in the patients. 60 patients with copd, 60 patients with asthma and 20 healthy control subjects with similar age range and sex were included in our study. the patient and control groups do not have any other chronic disease. serum prolidase activity was measured in the patient and control groups. ferritin and alpha-1 antitrypsin concentrations were also compared. there was no significant difference between serum prolidaz activities of asthma and copd patients. serum prolidase activities of both copd and asthma patients were significantly lower than those of the control subjects (p < 0.05). there was no significant difference for ferritine and alpha-1 antityripsin levels between the groups. the prolidase activity is significantly lower in asthma and copd patients comparing with control subjects. the collagen metabolism may be undergone to a change in these patients. hence, there may be an effect on the accumulation of collagen in the reticular basal membrane. the results suggest that collagen turnover are altered by the development of copd and asthma in human lungs, and prolidase activity may reflect disturbances of collagen metabolism in these pulmonary diseases. monitoring serum prolidase activity may be useful in evaluating fibrotic processes and in the chronic inflammatory lung diseases in human. acyclovir molecule in the active site of e. coli purine nucleoside phosphorylase (on the basis of x-ray study) i. kuranova 1,2 , v. timofeev 1,2 , n. zhukhlistova 1 , y. abramchik 3 , t. muravieva 3 , r. esipov 3 1 shubnikov institute of crystallography of fsrc "crystallography and photonics" ras, moscow, russia, 2 national research centre "kurchatov institute", moscow, russia, 3 shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia e. coli purine nucleoside phosphorylase (pnp), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family i of hexameric pnps. due to key role in the purine sulvage pathway pnps are attractive targets for drug design against some pathogens. they also used widely in biotechnology for the synthesis of nucleoside analogues as well as for the activation of the prodrugs in anti-cancer gene therapies. the acyclovir (acv), acyclic derivative of guanosine, is antiviral drug for the treatment of some human viral infections. the crystalline complex of e. coli pnp with acyclovir was prepared by co-crystallization using counter diffusion in capillary through the gel layer. the set of x-ray data at 100 k from single crystal grown in space (sp. group p6 1 22) was collected on the spring-8 synchrotron-radiation facility (japan) and the structure was solved at 2.32 a resolution, using the molecular replacement method (pdb id 5i3c). acv molecule was located in the nucleoside binding pocket of the enzyme in two conformations. the phosphate binding site was occupied by so4 ion. the hydrogen bonds network and hydrophobic interactions stabilising acv molecule in the active site as well as the conformational changes upon ligand binding were described. the comparison of e. coli pnp/acyclovir complex and the similar complexes of bacillus subtilis pnp (pdb id 4da7) and human pnp (pdb id 1pwy) allowed to establish the peculiarities of acv binding of in the e. coli enzyme. gonadotropins are glycoprotein hormones that regulate normal growth, sexual development, and reproductive function. these are large, up to 40 kda proteins, which are synthesized and secreted by the gonadotropic cells of the anterior pituitary gland. these hormones may vary in the level of glycosylation depending on the tissue and the metabolism cycles. follicle-stimulating hormone (fsh) and upon binding to fsh receptor, a g-protein coupled receptor (gpcr), regulates the development, growth, pubertal maturation, and reproductive processes of the body. human chorionic gonadotropin (hcg) and luteinizing hormone (lh) act via a shared gpcr (lh receptor) and regulate mechanisms essential for ovulation, early pregnancy and placental function in females as well as spermatogenesis and testosterone production in males. activation of gpcrs by these hormones can be measured by monitoring formation of cellular cyclic adenosine monophosphate (camp). the level on camp was measured using a f€ orster resonance energy transfer (fret)-based biosensor tepacvv (h74) kindly provided by dr, kees jalink. the biosensor was expressed using the developed bacmam gene delivery system (recombinant baculoviruses carrying the transgene under a strong mammalian promoter). kgn cells expressing the fsh receptor and cos7 cells expressing the lh receptor served as study objects. monitoring of specific gpcr activation in living cells, allows detection of only the biologically active agonists, which has real impact in quantification of large hormones. differences in levels of hormone glycosylation may affect their biological function. investigation of this phenomena is planned for near future. detection of biological activity of gonadotropins is of importance for pharmaceutical industry, where today the concentration of recombinant proteins is mostly estimated using immunological assays only. development of a colorimetric aptasensor for the detection of peanut allergen protein ara h 1 in food samples b. bora ege university, izmir, turkey food allergy, especially peanut allergy is a life-threatening problem, and severe reactions against these foods can be observed. since unintnded consumption of non-labeled foods is the most dangerous risk, any residual allergen protein should be tested and labeled by the manufacturers. an aptamer based colorimetric test is a powerful alternative to commercially available rt-pcr and elisa test kits. the main objective of this study is to develop an aptamer based colorimetric test fort he detection of major peanut allergen protein ara h 1. ara h 1 aptamer was used to recognize any residual peanut major allergen protein ara h 1 in food samples. recombinant ara h 1 protein was produced and puirifed to be used as a target. ara h 1 aptamer was used in combination with a blocking sequence, to prevent non-specific binding event, a biotinylated complementary strand to the blocking sequence, and finally strp-hrp interaction in order to facilitate colorimetric reaction. optimal blocking sequence length was optimized and introduced to the 3 0 site of aptamer sequence to construct an aptamer-hairpin structure. liberation of the blocking sequence allows biotinylated complementary strand to bind to the blocking sequence and consequently str-hrp conjugate to achieve color development that is proportional to the target concentration. since, the aptasensor will be used for the detection of ara h 1 in food samples, total protein extraction from chocolate samples was also optimized. in order to lower the detection limit of aptasensor, aptamer coupled magnetic bead based pre-enrichment assay was aslo optimized for the total protein extraction. as a result, a sensitive, fast and reliable aptamer based colorimetric assay was developed for the detection of peanut allergen protein from food samples. moreover, the assay has the advantages like ease of application and low cost which makes the assay a promising and a powerful alternative to commercially available rt-pcr and elisa tests. the association between lipid parameters and waist circumference in female university students in turkey s. ozen, a. cort sanko university, department of nutrition and dietetics, gaziantep, turkey a high waist circumference is associated with an increased risk for type 2 diabetes, dyslipidemia, hypertension, and cvd in patients with a bmi in a range between 25 and 34.9 kg/m 2 . monitoring changes in waist circumference may be helpful, in addition to measuring bmi, since it can provide an estimate of increased abdominal fat even in the absence of a change in bmi. objective of the study was to find an association between plasma lipid profile and anthropometric parameters (waist circumference percentage of body fat and body mass index (bmi)) in abdominal obesity in turkish university students. lipid profile and anthropometric parameters of obesity were studied in a sample of 30 women. students with high bmi (>24) had higher values of low-density lipoprotein (ldl), triglycerides (tg) and cholesterol (c) than students with low bmi (<24) but these differences were not significant. high-density lipoprotein (hdl) levels were non-significantly higher in low bmi (<24) student group. waist circumference, percentage of body fat was higher in high bmi (>24) group than low bmi (<24) group. waist circumference, percentage of body fat was positively correlated with bmi in both samples (bmi (>24) and bmi (<24)). students were grouped depend on their waist circumference. healty individuals who had lower than 80 cm waist circumference had decreased tg levels compared to cardiovascular risk group who had higher waist circumference than 80 cm. this study shows an association between waist circumference, percentage of body fat, body mass index and lipid parameters in young female university students. with regard to the relationship, the screening females for central obesity to prevention of cardiovascular disease are recommended. a new biotechnological product from propolis with low allergen: anti-inflammatory effect propolis is extensively used in food industry due to its special medical properies (antioxidant, antimicrobial, antiseptic, antibacterial, anti-inflammatory and antimutagenic effects). even these positive properties it may cause some allergic reactions in consumers with allergic predispositons. previously, we demonstrated that biotransformation of propolis by some special strains of lactobacillus plantarum (10, 8014, aatc strains) might decrease the allergenic molecules in propolis. in this study, we aimed to investigate the effect of biotransformation of popolis on it's antiinflammatory activities. before biotransformation, propolis samples were treated with different solutions (10% ethanol and polyethylene glycol -peg 40%) and different method (ultrasonic treatment 300 w/25 o c/ 30 minutes) in order to facilitate solvation of solid samples which are very dense and not suitable for fermentation. fermantations were performed at 30 o c/48 hours under constant agitation conditions. the anti-inflammatory activity was determined in-vitro conditions using hyaluronidase's analysis and the xanthine oxidase activity. the highest inhibition (%) of radicals produced by xanthine oxidase was determined in solid samples treated by peg prior to biotransformation and using of l.plantarum 8014 strain during fermentation (88.43%), followed by liquid samples treated by ultrasonic method prior to transformation (86.21%). concernig the results of hyaluronidase activity (%) inhibitions, the best value were determined in the solid sample treated by peg prior to biotransformation and using of l.plantarum 8014 strain during fermentation (93.93%). results indicated that the anti-inflammatory activities of analysed samples are quite high and depending of used extraction methods prior the biotransformation and used specific strain of l.plantarum could be optimized in terms of other required parameters. faceanti-mullerian hormone is not predictive for poor neonatal outcome aim: anti-mullerian hormone (amh) is a growth factor specific to ovaries. it is commonly used to predict ovarian reserve and outcomes of fertility treatments. recently, low levels of amh have been shown to be related to hypertensive diseases of the pregnancy and the risk of preterm labor. the aim of this study was to investigate the diagnostic performance of amh levels of mothers to predict poor neonatal outcome in term pregnancies and the relationship between amh and birthweights of the newborns. materials and methods: 187 patients, having delivery beyond 37 weeks, and who did not have any other medical problems were included in the study. the patients had normal 50 g. oral glucose tolerance test results. they were divided as 3 groups, based on their newborns' birthweight as "2500 g. and 4000 g.". level of amh was determined by elisa method. results: there was not any relation with the amh of the mothers and the poor neonatal outcome of the newborns, in all 3 groups. also no siginificant difference was observed in amh levels of the patients having delivery in early term and late term periods. when the patients of the same group were evaluated; amh levels were irrelevant to age, gravidy, delivery week, body mass index, the weight gain during pregnancy, and poor neonatal outcome. conclusion: amh is not a predictive factor for poor neonatal outcome and it is not a determinant of the weight of the newborn. objectives: the aim of the study was to investigate the effects of differing amounts of hemolysis on serum high sensitvity troponin i (hs-tni), ck-mb mass and myoglobin measurements. materials and methods: we prepared serum pools having troponin i, ck-mb and myoglobulin concentrations at low (5.33 ng/l,1.5 ng/ml and 34.3 ng/ml respectively), normal (39.25 ng/l, 3 ng/ml, 197.9 ng/l respectively) and high (7345 ng/l, 22 ng/ml, 574 g/ml respectively) values. the osmotic shock method was utilized to prepare a hemolysate. hemolysate was added into serum pools increasing concentrations of hemoglobin (0.65, 1.3, 2.5, 5, 7.26 and 9 .42 g/l hemoglobin). troponin i, ck-mb (mass) and myoglobin concentrations were measured in duplicate by beckman coulter access 2 analyzer. the hemolysis indices were measured on beckman coulter au 5800. a change of 10% from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. results: we found a positive interference due to hemolysis for ck-mb (mass) at low concentrations (1.5 ng/ml), and a negative interference for myoglobin at low concentrations (34.3 ng/ml) and high concentrations (574 ng/ml). conclusions: ck-mb increase and myoglobin decrease in hemolyzed samples with hemoglobin ≥9.42 g/l, but the bias might not be clinically significant (< total analytical error) in samples. a retrospective study to determine a reliable marker for selective screening of pompe disease lysosomal storage diseases (lsd) are rare inherited metabolic disorders caused as consequence of a deficiency in a specific enzyme required for lysosomal function. pompe disease is one of these disorders with deficiency of a-1,4 glycosidase enzyme with an incidence of 1:4,500-1:33,000. as enzyme replacement therapies are available nowadays, early diagnosis is crucial and selective screening is a rational method to reach pompe patients among people who administer to healthcare with lsd suspected symptoms. this study aims to examine the relationship between basic biochemistry parameters and a-1,4-glycosidase activities retrospectively, in order to find a key parameter for selective screening of pompe disease. for this reason a-1,4glycosidase, creatine kinase (ck), creatine kinase-mb (ck-mb) activities calcium, phosphate levels of those who had been suspected to be lsd patients and administered to our laboratory for analysis are examined retrospectively. out of 134 patients's examined,14 of them were diagnosed with pompe disease depending on clinical findings & low a-1,4glycosidase activity. enzyme activities of pompe patients were 0.337 nmol/ml/hour as lsd suspected patients'activities had a mean of 2.78 nmol/ml/hour (p = 0.00).comparison of ck activity was compared results showed significant difference between pompe patients and lsd suspected patients. even though ck activity levels of the lsd suspected patients were much higher (400vs41-171u/l) than reference interval, the levels of the pompe disease patients' were still more than twice of the lsd suspected group (956vs400u/l, p = 0.02). ck-mb, ca, p levels didn't show a significant difference. a strong (-) correlation (p = 0.005 r=à0.240) was observed between a-1,4-glycosidase and ck activities (n:134). selective screening is a rational way to diagnose rare diseases. this study's results show that ck activity can be used as a key parameter to determine patients for selective screening of pompe disease within lsd suspected population. the functional effect of stem cells on the reproductive organs infertitility is considered as a major health problem of recent century. importance of stem cell is increasing so it is searched new features and supposed to be involved in the infertitility treatment where oxidative stress and apoptosis play importany role. we aimed to investigate the beneficial effect of the stem cells related to free radicals and cell death on testis and ovary. biopcy model of wound healing was created in the rat testis and ovary with ppd syringe where stem cells were delivered by injection. rats were divided into four groups including controls, sham, wound healing and wound healing with stem cell. after the creation of the wound, bone marrow-derived mesenchymal stem cells from the tibia of the mature rats and medium were administered to ovaries and testes. following the applications, ovary and testis samples were investigated for oxidative stress and apoptosis by immunohistochemistry. in comparison with the medium and stem cell applications without a medium support, it was meaningfully determined that healing effect in testicles and ovaries were spotted specifically on the seven day. tissues were analysed for these staining by h-score and h-score results were determined using one-way anova test statistically. our results show the positive effects which clinic applications can bring by displaying the great contribution of the stem cell application in the treatment of testicle and ovary damage. these findings suggest that transplantation of the mesenchymal stem cells may help to promote better enviroment for the reproductive organs by the effect on oxidative stress and apoptosis. the further studies of these results in the molecular level can lead the way to solve the problem of infertility, to increase the percentage of success in the ivf and icsi techniques and more importantly to perform a differentiation from a somatic cell to a germ cell. the antimicrobial activity of 2 (5h)-furanone derivative on staphylococcus aureus nosocomial infections caused by methicillin-resistant staphylococcus aureus strains are known to be a reason of many infectious diseases like osteomyelitis, endocarditis, sepsis etc. being organized in biofilms these bacteria become extremely resistant to antimicrobials and host immune system leading to difficulties in treatments. here we report the effect of 2 (5h)-furanone derivative possessing sulfonyl group and l-menthol moiety (f105) on biofilms formed by s. aureus atcc29213 and mrsa cells. while exhibiting relatively high minimal inhibiting concentration -mic (16 mg/l), clear synergy with a number of antibiotics was found in the checkerboard assay. thus, in the presence of 2 mg/l of f105 the mic of kanamycin was decreased 4-fold, and the mics of both erythromycin and ampicillin were lowered 2-fold. at the concentration of 80 mg/l f105 also completely inhibited the biofilm formation by s. aureus; the cell growth was suppressed by two orders of magnitude as judged by differential fluorescent staining with syto9 and propidium iodide. the addition of f105 to preformed 24 h-old biofilms increased the fraction of red-stained (dead) cells of both s. aureus atcc29213 and mrsa strains uniformly throughout the whole profile of the biofilm. the quantitative analysis of clsm microphotographs revealed that f105 at concentration of 80 mg/l led to death of up to 98% of biofilm-embedded cells. this fact suggests that f105 efficiently penetrates into the biofilm matrix and kills the cells without visible damage of biofilm structure. in summary, furanone f105 seems to be a promising compound for drugs design to treat biofilm-embedded s. aureus. this work is supported by the russian science foundation, project №15-14-00046 and the german academic exchange service (№91531398). pneumonia is an inflammatory lung disease which can be associated with inadequacy of host defense system and the proliferation of various pathogenic microorganisms into the lower respiratory tract. community acquired pneumonia (cap) is one of the leading causes of death in elderly. the incidence of pneumonia in people aged 65 and over is 3-5 times more than young adults. creactive protein (crp) is an acute-phase protein of hepatic origin that increases following interleukin-6 secretion by t cells and macrophages. procalcitonin (pct) is a peptide precursor of the hormone calcitonin, the latter being involved with calcium homeostasis. it is composed of 116 amino acids and is produced by parafollicular cells (c cells) of the thyroid and by the neuroendocrine cells of the lung and the intestine. the level of pct rises in a response to a proinflammatory stimulus, especially of bacterial origin. the aim of this study was to compare crp and pct levels in young and elderly patients with pneumonia. recently diagnosed 43 young and 46 elderly patients with pneumonia and their respective aged matched controls (n = 41, n = 42) were enrolled this study. crp and pct levels were by immunoturbidometric and by elisa methods respectively. crp and pct levels for young control and patients and elderly control and patients respectively are 2.32 ae 2.25 mg/l, 0.26 ae 0.13 ng/ml, 20.71 ae 34.61 mg/l, 0.64 ae 1.09 ng/ml, 2.32 ae 2.14 mg/l, 0.27 ae 0.07 ng/ml and 31.41 ae 31.0 mg/l, 0.49 ae 0.83 ng/ml. young patients with pneumonia have significantly higher crp and pct levels than their controls (p < 0.001 and p < 0.028). elderly patients with pneumonia have significantly higher crp levels than their controls (p < 0.001). crp and pct are important markers in the diagnosis of pneumonia. effect of serum albumin concentration on total and ionized calcium z. adiyaman, c. yilmaz, s. a. peker, d. y€ ucel ankara training and research hospital, ankara, turkey objective: the aim of the study is to investigate in vitro effect of albumin concentration on total and ionized calcium concentrations. materials and methods: a serum pool with low albumin (3.10 g/dl) and normal calcium (9.79 mg/dl) concentrations was prepared from leftover sera. from this serum pool, two parts, each of 10 ml were aliquoted. purified albumin, 0.3 g, was added to one of these pools and albumin concentration was determined as 6.1 g/dl. the low and high albumin pools were mixed at different ratios and pools with 3.89, 4.78, and 5.37 g/dl albumin concentrations. total calcium and albumin concentrations of these 5 pools were measured at a beckman-coulter au5800 analyzer and ionized calcium was measured at a radiometer abl 800 blood gas analyzer in triplicate. total and ionized calcium concentrations were evaluated as compared to those of the original pool with an albumin concentration of 3.10 g/dl. results: total calcium concentrations are increased with the increasing albumin concentrations: 1.2%, 1.58%, 2.85%, and 3.59%, respectively. whereas, ionized calcium concentrations were decreased with increasing albumin: 3.0%, 6.1%, 7.4%, and 8.6%, respectively. conclusions: when total allowable error limits based on biological variation were considered, total calcium concentrations are significantly increased at >5 g/dl albumin concentrations. ionized calcium is significantly affected by 0.8 g/dl and over albumin concentrations. a regression equation based on albumin concentration may be useful for corrected ionized calcium concentrations. relationship between lipoprotein (a) and hba1c in patients with type ii diabetes , is a complex lipoprotein consisting of ldl and apolipoprotein(a). lp(a) is a risk factor for coronary artery disease and stroke. the relationship between lp(a) and diabetes mellitus is not clear. in this study, the relationship between lp(a) and glycemic parameters such as hba1c and fasting glucose concentration was investigated. lp(a), hba1c, fasting glucose, triglyceride, total cholesterol, ldl-and hdl-cholesterol concentrations were screened retrospectively from july 2013 to july 2016. there were 1831 patients with these test results at the same time. the patients were grouped according to hba1c values: group i < 5.7% (n = 468), group ii 5.7-6.4% (n = 739), and group iii >6.5% (n = 624). the relationship between these parameters were statistically within each group and all groups. there was not a statistically significant difference between the lp(a) concentrations of group i and group ii. lp(a) concentrations of group i and ii were significantly higher than those of group iii.. _ in total, lp (a) was negatively correlated with hba1c (r = 0.09; p < 0.01), but there was not a significant correlation with fasting blood glucose. _ in groups, there was a significant and negative correlation between lp(a) and fasting glucose in only group i. the negative correlation between lp(a) and glycemic parameters is interesting in patients with diabetes. despite lp(a) is an independent risk factor for cardiovascular diseases, on the contrary to expectations, lp(a) concentrations are decreased in diabetes. effect of blood collection through intravenous lines on hemolysis erroneous results are one of the most important causes of medical errors and may lead to unnecessary investigations or inappropriate interventions. total testing process consists of preanalytical, analytical and postanalytical phases. hemolyzed specimens that one of the most common source of preanalytical errors are frequently observed in laboratory practice and associated with incorrect laboratory results. blood collection through intravenous lines frequently results in hemolysis especially at eds and icus. in this study, we aimed to compare the effect of blood drawing by using bd luer-lock adapters and injector on the hemolysis rates at the ed. 60 patients who has been admitted to the ed were included in this study. all samples were drawn from newly inserted iv lines. the first blood sample was drawn with injector and the second one was drawn with luer-lock adapters to vacuum tubes. after the centrifugation routine chemistry tests and hemolysis indices were analysed on a beckman coulter au680 analyzer for each serum tube. the statistical significance of differences between two tubes was calculated with paired samples t test and statistical significance was accepted as p < 0.05. there were statistically significant differences between the two groups of tubes for the following parameters: ldh, ck, ast, k + , total bilirubin, protein, albumin, alp, calcium and hemolysis index (p < 0.05). the use of luer-lock adapters instead of injector could reduce the hemolysis rate. because of it reduces false results and unnecessary investigations, this approach will be more appropriate and cost-effective in ed. hemolysis and test rejection: are we following a reliable process? introduction: in laboratories, some blood samples are rejected due to hemolysis. we usually cancel only some of the tests that are affected by hemolysis. however, the frequency of the test cancellation may be relative. each test is affected in different degrees of hemolysis; some of them are not even affected at all. in this study, we aim to investigate unnecessary cancellations and explain the relationship between hemolysis and test results according to their kit inserts. materials and methods: we measured hemoglobin levels of 50 hemolyzed serum using drabkin method (abbott). interference studies are conducted using clsi protocol nccls ep7-p is written in kit inserts. target values (100%) and their change due to different degree of hemolysis have been defined. results: hb concentration ranges of hemolyzed sera were found from 44 to 849 mg/dl. according to kit inserts, aspartate aminotransferase (ast) test results deviate 5.7% from the target when the degrees of hb are 62 mg/dl. when the degree of hemoglobin is 125 mg/dl, the test strays about 11.6%. potassium levels increase (108%) at 125 mg/dl hb while this increase reaches to 17.9% at 250 mg/dl hb. sodium, calcium, ck, crea, total bil, lipase are not significantly affected even at 2000 mg/dl. in lactate dehydrogenase (ldh) tests, test reporting is not allowed at any hemolysis level. alt increases 11%, at the 1000 mg/dl hb. ast and potassium results were excluded from patients' reports even though those samples had low hb. some of them were reported despite of excess hemolysis. some tests are even blocked without ever being studied. discussion: prior to the approval of the lab specialist, technicians decide whether to cancel the tests affected by the hemolysis according to the visible hemolysis based on their personal knowledge. conclusion: we should use the hemolysis index, in which standards would be defined via guidelines. this way, all technicians and specialists could know which results are false. the dna-binding hu-proteins are present in all bacteria and belong to the family of nucleoid-associated proteins. these proteins can be considered precursors to eukaryotic histones. gene knockout of hu-proteins partially inhibits the growth of bacteria, their ability to resist various stressing factors and in some cases leads to their death. since the spatial structure of hu-proteins is highly conserved it is possible to create inhibitors that will affect them in a broad spectrum of pathogenic bacteria. in the present work the preparation of the recombinant hu protein from mycoplasma gallisepticum, crystallization of this protein, and x-ray diffraction study of this protein has been reported. the crystallization conditions for studying protein were found by the hanging-drop vapor-diffusion method. found conditions have been adapted to the counther-diffusion method in the capillary. the x-ray diffaction dataset from grown crystals have been collected using synchrotron radiation. 3d-structure of the hu protein from mycoplasma gallisepticum have been determined with 3a resolution. structural features of the investigated protein are described. this work is supported by russian scientific fund (15-14-00063). a novel sensitive disposable indium tin oxide (ito)-based electrochemical immunosensor was developed for simple, rapid and sensitive biomonitoring of sox2. sox2 is a cancer biomarker and used for detecting of small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, skin cancer, prostate cancer, and breast cancer. in this study, indium thin oxide (ito) thin film was used as working electrode. carboxyethylsilanetriol was also used for electrode modifying so as to obtain self-assembled monolayers. the formed self-assembled monolayers were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (edc)/n-hydroxysuccinimide (nhs) chemistry. edc was used as a heterobifunctional crosslinker. nhs was used in conjunction with the crosslinker edc. anti-sox2 antibody was used as a biorecognition element and it was covalently immobilized onto the ito electrode modified with carboxyethylsilanetriol. immobiliztion steps were characterized by cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis), and scanning electron microscopy (sem). the optimal immobilization conditions for the best sensitivity of the new immunosensor were investigated. under optimal conditions, this immunosensor demonstrated a wide linear range (0.0025-2 pg/ml) with a detection limit as low as 0.02 ng/ml sox2. furthermore, the developed sox2 immunosensor had good storage stability, repeatability and reproducibility. in this work, we successfully fabricated disposable ito thin film based electrodes for sensing the interaction between sox2 antigen and anti-sox2 antibody by electrochemical impedance spectroscopy and cyclic voltammetry. and our developed immunosensor has an acceptable performances for the detection of sox2 antigen, exhibits low detection limit, has selective and reproducible results in immunoreaction analysis. we are thankful for the support from t € ub _ itak (the scientific and technological research council of turkey, project number: 113 z 678). applying multiple linear regression model to determine the relationship between anti mullerian hormone with age, luteinizing hormone, follicle stimulating hormone and estradiol: a data mining study introduction: anti mullerian hormone (amh) has a widely used in our life because it is a good indicator of reproductive age to estimate the time of menopause. the purpose of this retrospective data mining study is the estimate of ovarian reserve by using amh and determines relationship between other indicators which are luteinizing hormone (lh), follicle stimulating hormone (fsh), estradiol and age. materials and methods: 25.294 women members were included this retrospective data mining study who were applying to acıbadem labmed laboratory. multiple regression analysis of age related changes of amh (18-45) and lh, fsh and estradiol were investigated. beckman gen ii elisa kit was used for amh and the technique of electrochemiluminescence and roche elecsys cobas analyzer were used for the measure of other hormones. results: amh shows meaningful correlation between lh, fsh, estradiol and age but also seen there is no correlation between progesterone. after the multiple linear regression analysiz amh= 11.018-(0.220 9 age)à(0.066 9 fsh) + (0.044 9 lh)à(0.004 9 estradiol) is detected and the model's r 2 = 0.627 is also detected. conclusion: nowadays there are lots of methodology were developed the estimate the function of ovary and biological age of ovarian. age, fsh, lh and estradiol show ovarian reserve by indirectly. this study shows the mathematical relationship between amh and the other indicators and results are thought to lead to future developments. antioxidant and anticancer effect of artemisia absinthium extract on colon and endometrium adenocarcinoma cells plants have always been among the common sources of medicines that have many phytochemicals with various bioactivities, including antioxidant and anticancer activities artemisia absinthium (ar) has been used as an antipyretic, antiseptic, anthelmintic, tonic, diuretic, and for the treatment of stomachaches in turkish folk medicine. this study aimed to investigate antioxidant, cytotoxic, genotoxic and apoptotic effect of methanol extracts of ar activities on the human colon (dld-1) and endometrium (ecc-1) adenocarcinoma cell line. total phenolic, flavonoid content, and antioxidant activities were determined using suitable methods (abts, cuprac i.e). cytotoxic effects of ar on cells were determined by mtt and neutral red uptake assays. genotoxicity was evaluated by comet assay and, apoptosis induction were detected by apoptosis elisa and acridine orange staining methods at the half maximal inhibitory concentrations (ic50) levels. it was determined that extract have shown antioxidant activity in all tests and that they could be considered as a source of natural antioxidants. cytotoxic effects were concentration-time dependent. specifically, apoptotic and genotoxic effect increased at 100 and 200 lg/ml concentrations by 48 hours. we found that ar extract had antiproliferative, genotoxic and apoptotic effects on the human cancer cell lines dld-1 and ecc-1. however, further studies at molecular level are required to support our findings and to elucidate chemopreventive and chemotherapeutic effects of ar on colon and endometrium cancers. keywords: artemisia absinthium, antioxidant, anticancer, apoptosis, genotoxicity introduction: colorectal cancer is considered as a major gastrointestinal. this cancer is the second cancer related cause of death after lung cancer in worldwide. we designed a vaccine chimeric including cea and ca19-9 against colorectal cancer (ce-ca). materials and methods: the construct were analyzed by bioinformatics softwares. in this study, the ce-ca gene was optimized using the codon bias of e.coli and synthesized by biomatik company. then construct (ce-ca) was cloned into an expression vector and recombinant constructs transferred to e.coli bl21de bacterium and desired recombinant protein was expressed. recombinant protein was purified using ni-nta affinity chromatography. the content of secondary structures was obtained by circular dichroism (cd) spectrum. then recombinant protein was confirmed using western blot analysis and indirect elisa method. results: sds-page analysis showed that the recombinant protein was highly expressed and purified. western blot analysis confirmed recombinant protein. also cd spectrum confirmed predicted structures by bioinformatics tools. the elisa results showed significantly high affinity toward recombinant ce-ca protein. discussion: based on many studies, cea as potential immunogenic candidate could be considered in vaccine studies. also ca19-9 is a cell-surface antigen that has significant increase of expression in colorectal cancer, thus as marker of colorectal cancer. based in available data, these two antigens, in combination can provide specificity for production of colorectal cancer vaccine. conclusion: these findings suggest that ce-ca as potential immunogenic candidate which could be considered in future vaccine studies and detection of colorectal cancer. flow cytometric cell cycle and apoptosis analyses of some wild animal species a. tas, e. koban bostanlar tubitak, marmara research center (mrc), genetic engineering and biotechnology institute (gebi), animal genetic and reproductive biology laboratory, kocaeli, turkey cell biobanking; more specifically cryopreservation of biological diversity, is promising as a tool to preserve wild animals as well as domestic ones via nuclear transfer. in this study, we investigated the viability and cell cycle characteristics of 5 wild animal species (fallow deer, red deer, wild sheep, wolf, wild goat). auricular tissue samples were maintained in pbs+2%psa. tissues were seeded on 35 mm petri dishes containing dmem/high glucose supplemented with 20% (v/v) fcs and incubated 5%co2 in air at 95% relative humidity and at 37°c. after seeding, the medium was unchanged for 7 days and then it was changed in every 2 days for 25 days at maximum. once the cells were obtained; flow cytometric cell cycle and apoptosis analyses were done. in terms of apoptosis, all the groups showed high viability rates (over 95%) in culture when compared with the negative control (38%). the cell cycle comparisons were made between serum-starved cells and roscovitine treated cells, both for which untreated cells were used as control, which revealed different results for different species. there was no difference found between serum-starved cells and roscovitine treated cells for red deer and wolf. the serum-starved cells resulted in higher g0/g1 phase for fallow deer and wild goat. on the contrary, roscovitine treated cells resulted in higher g0/g1 phase for wild sheep. as a result; the cells obtained from wild animals had high viability and g0/g1 phase rates. therefore, they may serve as a donor cell source for nuclear transfer studies.(grant: tubitak kamag, turkey, 109g016). the interaction of different types of antibiotics with endothelial cells in the presence of nanoparticles the interaction of nanomaterials with cells and lipid bilayers is critical in many applications such as phototherapy, imaging, and drug/gene delivery. the aim of this study was to investigate the interaction of nanoparticles (fe 3 o 4 ) or nanoparticles fused with different antibiotics with cell membranes in order to reveal changes in the membrane organization. endothelial cells were used to determine the effect of different antibiotics (gentamicin, kanamycin, amikacin, penicillin, polymyxin, neomycin, cefotaxime, bacitracin, moxicillin, erythromycin, streptomycin and vancomycin) on the membrane organization. for recording the anisotropy of cell suspensions treated with antibiotics or nanoparticles fused with antibiotics we used 1-4-trimethyl-6-phenyl 1, 3, 5 hexatrien p-toluenesulfonate (tma-dph). we decided to use nanoparticles fused with antibiotics because they contain small amounts of antibiotics which makes them less toxic than simple antibiotics,which is very important in patients with genetic diseases such as cystic fibrosis, that should be treated with antibiotics for a long time. our results showed that at temperatures between 31 and 35°c simple nanoparticles decreased the membrane fluidity. at physiological temperatures (37-39°c) nanoparticles fused with antibiotics (gentamicin, vancomicin, cefotaxim, bacitracin, amoxicillin) increase more the membrane rigidity compare with simple antibiotics or nanoparticles.erythromycin, polymyxin and penicillin increase the membrane rigidity at 37°c, and at 39°c the same effect was obtained in the presence of nanoparticles fused with these antibiotics,suggesting that the nanoparticles are dependent to temperature for penetrating the membrane. in conclusion the membrane fluidity does not depend on antibiotics types, the modification are present in many antibiotics irrespective of class type.the presence of nanoparticles fused with antibiotics is very important for long term treatment. objectives: hypertension is an important cardiovascular risk factor for the development of atrial fibrillation (af). increased atrial electromechanical coupling time interval measured by tissue doppler is accepted as an important factor for prediction of af development in hypertensive patients. monoamine oxidases (maos), are enzymes which catalyze the oxidation of monoamines. 8-isoprostane is considered as an indicator of oxidative stress. mao activity and 8-isoprostane levels were measured in some diseases. however, there are no information on 8-isoprostane levels and mao activity in newly diagnosed patients with stage 1 hypertension has not been observed in a study of literature. aim: this is the first study, we aimed to evaluate the levels of mao and 8-isoprostane in newly diagnosed patients with stage 1 hypertension. the study included 60 newly diagnosed stage 1 hypertensive patients with no other systemic disease. 30 patients were selected as randomized (17 women, 13 men; range of age 46-74 years) and 30 healthy individuals as control (15 women, 15 men; range of age 44-72 years). all the underwent tissue doppler echocardiographic examination. blood samples were taken from patients and controls and, the levels of mao and 8-isoprostane in serum samples were measured by elisa. results: baseline blood pressures, electrocardiographic and echocardiographic findings, and atrial electromechanical coupling were similar in both groups (p > 0.05). compared to the control group, the activity of mao and 8-isoprostane levels were found significantly higher in patients (p < 0.05). conclusion: increased 8-isoprostane level indicate that there is oxidative stress in newly diagnosed patients with stage 1 hypertension. also, increased mao activity may be biochemical biomarkers for the diagnosis of hypertension. keywords: hypertension, monoamine oxidase, 8-isoprostane p-mis-147 determining the indirect reference intervals for complete blood count parameters in bursa, turkey reference intervals (ris) for laboratory test results are defined as the most commonly used diagnostic tool in medicine. therefore, careful determination of ris by the laboratory for use is a very important task. although c28-a3 guideline recommends the direct ris (dris) calculated from healthy subjects, ris can be calculated from laboratory data which are called as indirect ris (iris). the study was carried out at the central laboratory for clinical chemistry, teaching and research (uludag university, bursa, turkey) . the results of the laboratory analyses from 142,591 males, 215,658 females, stored for approximately one year, were used for statistical analysis. data for hospitalized patients and for ambulatory patients from the intensive care unit were eliminated. furthermore, we used evidence based criteria to enrich the health-related values. a modified bhattacharya procedure was used to estimate the iris from hospital patient data. the nested anova was used to evaluate variations among genders and ages. cell dyn analyzer (abbott diagnostics, il, us) was used for the measurements of complete blood count. the obtained iris were also compared the dris determined in our previous ri study and the ris suggested by the manufacturer. we found that the ris of rbc, hb and hct required strong gender partition and calculated the ris of rbc, hb and hct separately. the observed iris for wbc, sub-fractions of wbc and plt in both genders are in good accordance with the dris reported in previous study. age-related changes were noted for rbc, hb, and hct. the calculated iris for rbc, mcv and rdw are different from the ris suggested by the manufacturer. we believe that, using this relatively easy technique, every laboratory can produce its own iris, divided, where possible, according to sex and age and according to local conditions. these ranges can be complementary to dris obtained for reference individuals according to the ifcc recommendations. the principal sigma 70 subunit, involved in transcription of most house-keeping genes in escherichia coli, was also shown to induce rnap pausing during transcription elongation, by interacting with promoter-like motifs in the transcribed dna. such pauses were proposed to play important roles in the regulation of phage and cellular genes. e. coli contains six alternative s subunits but little is known about their ability to induce transcriptional pausing. we expressed and purified alternative s subunits of the sigma 70 family and tested their effects on transcription elongation in vitro on natural and synthetic dna templates containing consensus promoter motifs. the structure of the paused complexes was analyzed by dna footprinting methods. in vivo analysis of transcription was performed using reporter genes placed under the control of corresponding promoters. we demonstrated that the stationary phase sigma 38 subunit induced efficient rnap pausing on both synthetic and natural dna templates containing promoter-like motifs in initially transcribed regions. in contrast, the sigma 32 and sigma 28 subunits did not affect rna elongation. we showed that the sigma 38 -induced pausing depends on sigma contacts with both nontemplate dna strand and rnap core. the pausing results in formation of backtracked transcription elongation complexes which can be reactivated by gre factors that stimulate rna cleavage by rnap. our results for the first time reveal transcriptional pausing induced by an alternative s subunit. analysis of sigma 38 -dependent promoters shows that a substantial fraction of them contains potential pause-inducing motifs suggesting that such pausing may be a widespread phenomenon. we propose that sigma 38 -dependent pauses may play important roles in genetic regulation and modulate the binding of transcription repressors or activators to promoter regions. the crosstalk between streptococcus pneumoniae rnase r, ribosomes and translation c. b arria, s. domingues, c. arraiano instituto de tecnologia qu ımica e biol ogica, lisbon, portugal ribonucleases (rnases) are enzymes that ensure maturation, degradation and quality control of rna thus, contributing to the maintenance of the optimal amount of each transcript in the cells. escherichia coli rnb family of enzymes is present in all domains of life and includes rnase r, rnase ii and the eukaryotic rrp44/dis3, dis3l1 and dis3l2 proteins. in streptococcus pneumoniae only rnase r was identified. rnase r, encoded by the rnr gene, hydrolyzes rnas starting from the 3 0 end. rnase r level is increased in several stress conditions such as heat shock, stationary phase or cold shock, conditions in which most of the proteins translation is blocked. moreover, rnase r is the only exoribonuclease able to degrade highly structured rnas without the help of a helicase which is critical at low temperatures. here, we investigated the role of this enzyme by comparing the wild type strain with an rnr mutant strain. for that purpose we performed northern blots analysis of transcripts involved in translation. also, we investigated rnase r connection to the ribosome and polysome fractions using sucrose gradient polysome separation and western blots. in this study, we highlight the importance of s. pneumoniae rnase r in translation. we show that this enzyme interacts with ribosomes mostly with the 30s subunit at 37°c. moreover, in the absence of this enzyme we have observed a decrease in the amount of the 70s ribosomal subunit, concomitantly with the increase of 30s and 50s subunits. rnase r seems also to modulate the amount of the elongation factors ef-tu and ef-g transcripts. nevertheless, preliminary results further suggest other roles of rnase r in translation. modified nucleotides are present in many rna species in all domains of life. the biosynthetic pathways of such nucleotides are well studied. however, much less is known about the degradation of rnas and the salvage of modified nucleotides, their respective nucleosides or heterocyclic bases. using an e. coli uracil auxotrophic strain, we screened the metagenomic libraries for genes, which would allow the conversion of 2-thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. we show that a novel gene encoding previously uncharacterized domain of unknown function (duf) is responsible for such phenotype. we have purified this recombinant protein and demonstrated that it contains a fe-s cluster. the substitution of cysteines, which have been predicted to bind such clusters, with alanines abolished the growth phenotype. we conclude that this domain is required for conversion of 2-thiouracil into uracil in vivo. this work is supported by the research council of lithuania (lmt, mip-103/2015 modified nucleotides are present in almost all classes of rna. they have great chemical diversity and are critical for rna folding, stability, interaction with cellular proteins and thereby for various cellular processes such as translation, stress response, and signaling pathways. biosynthesis of pyrimidine nucleotides and their modified derivatives in rna is well studied. nonetheless, not much is known about the cellular degradation of these compounds and the enzymes catalyzing such processes. using an e. coli uracil auxotrophic strain, we screened metagenomic libraries for genes encoding isocytosine deaminases. three novel genes were obtained, one of which encodes a protein similar to 8oxoguanine deaminases. the other two encode proteins resembling hydroxydechloroatrazine ethylaminohydrolases. we confirmed that these proteins are functional in vivo, allowing growth of e.coli on minimal medium with isocytosine. we also demonstrated that such purified recombinant enzymes catalyze the conversion of isocytosine, but not cytosine, into uracil in vitro. natural products display special attributes in the treatment and prevention of various human diseases, including cancer. a significant number of organic compounds from plants exhibit anticancer properties as attested by in vitro and in vivo studies. emerging evidence supporting the antineoplastic activity of natural compounds has rendered them promising agents in the fight against cancer. in this study, skin from limnio grape, a red greek grape variety that is indigenous to the greek island of lemnos, was extracted using mixtures of methanol, water and acetone; the apoptosis-inducing properties of these extracts were studied in the human ovarian malignant adenocarcinoma cell lines tov-21g and tov-112d. for this purpose, tov-21g and tov-112d cells were treated with limnio grape skin extracts at a range of concentrations, at 37°c, for 24, 48 and 72 hours. untreated cells incubated for the same time intervals served as controls. cell viability was determined by measuring metabolic activity (colorimetric mtt assay) and observing cell membrane integrity (cell staining with trypan blue). after the determination of the optimal concentration of the extract, total rna was extracted from treated and untreated (control) tov-21g and tov-112d cells. after determination of rna concentration and subsequent first-strand cdna synthesis, mrna expression analysis of apoptosis-related genes was performed with rt-pcr using gene-specific primers. an increasing percentage of non-viable cells was observed by increasing cell exposure time and extract concentration. distinct modulations of the expression of apoptosis-related genes at the mrna level were also observed, mainly concerning bcl2, bclx, bax, bak1 and bcl2l12, along apoptosis induction. in conclusion, the cytotoxic properties of limnio grape skin extracts against ovarian malignant adenocarcinoma cells merit further investigation. the intrinsic apoptotic pathway seems to be the major mechanism of action induced by these plant extracts. almost all eukaryotic mrnas are polyadenylated by a complex machinery that recognizes the poly (a) signal, cleaves the mrna and adds the poly (a) tail. 70% of human genes harbor multiple poly (a) signals. alternative polyadenylation (apa) generates transcript isoforms with different 3 0 utr (untranslated region) lengths due to the use of proximal or distal poly (a) signals. hence, tightly regulated apa has been observed in normal physiological settings as well as in diseases. considering that 3 0 utr shortening cases have been linked to increased protein levels, we hypothesized deregulated apa to be one of the potential cancer related mechanisms. we investigated the 3 0 utr alterations in er(+) breast cancer patients and cell models compared to normal breast tissue, using gene expression data and a probe-based quantification tool, apadetect. based on means of proximal to distal probe sets, slr (short-long ratio) were calculated as an indication apa. significance analysis of microarrays (sam) determined significant genes. the gse numbers of the datasets are gse2034, gse7390 and gse9761. we analyzed two datasets of er(+) breast cancer patient samples (n = 209, n = 135) compared to normal breast tissue (n = 82) using apadetect and sam. a total of 184 3 0 utr shortening and 253 3 0 utr lengthening events were detected in breast cancer samples compared to normal breast tissue. ontology analysis suggested almost all the 3 0 utr shortening genes were proliferation related and were indeed reported to be upregulated in breast cancer. to further investigate the connection between apa and era status, we used data from a cell line model; wild type or era transfected mda-mb-231 cells that are otherwise of triple negative nature. our results suggested that most of the genes are 3 0 utr shortened or lengthened via direct binding of era to dna. our results suggest involvement of apa mechanisms in era action mechanisms. possible link between era regulated transcription and apa remains to be elucidated. contamination of nucleic acids (na) as a result of na extraction protocols may result an inaccurate measurement of dna copy number. agarose gel electrophoresis and spectrophotometric methods are commonly used to check dna purity. however, the resolution of these methods may not be good enough for special applications such as determination of dna copy number and separation of base pairs (bp) that are close in their bp number. in this study, we have developed a new method for separating na's ranging between 75-20000 bp also detecting the impurities in dna solution in 1%, 5% and 10% ratios to the dna of interest. the developed method was validated using the in-house dna fragments of 100, 150 and 200 bp. the dna mixture analyzed using analytical hitachi elite lachrom hplc using the guard and analytical columns tskgel dna-npr, 2.5 lm, 4.6 mm id 9 0.5 cm and tskgel dna-npr, 2.5 lm, 4.6 mm id 9 7.5 cm, respectively. the validation of the analysis was performed by running each sample five times on three different days. the linearity of the detector response was established by plotting a graph to quantity versus area of 200 bp dna. the lod and loq were then measured by calculating the minimum level at which analyte can be readily detected and quantified. the ratios calculated with hplc were compared to the ratios calculated by quant-it kit. recovery values were calculated for each measurement and the uncertainty were calculated for each ratio. the method was found linear for 200 bp in the range of 0.4 ng to 800 ng dna with the regression coefficient of r 2 =0. 9992. lod and loq for the 200 bp dna was found to be 0.39 ng and 1.56 ng, respectively. the recovery values for the 1%, 5% and 10% impurity ratios were found 101.74. 97.41 and 99.47, respectively. the purity of the synthetic dna was determined by hplc and related uncertainty was calculated. the developed method is a simple alternative to electrophoresis and spectrophotometric methods with higher resolution and separation range. physical and chemical factors can disturb the conformation of proteins maturing within the cellular secretory pathway. in response to unfolded proteins the cell activates several stress signaling and adaptive response mechanisms. the aim of our study was to investigate small non-coding rnas as the potential regulators of cellular response to unfolded proteins (upr). for this, we conduct the next generation sequencing of small rna and transcriptome analysis of mrna from jurkat cells exposed to dithiothreitol (dtt), which reduces protein disulfide bounds. analysis of mirnas reveals the differential expression of 104 mirnas. we observe a decrease in the normalized amount of reads aligned to mirna loci in stressed cells. affymetrix analysis with subsequent gsea reveals downregulation of reactome mirna biogenesis pathway (fdr = 0.096). the length distribution of small rnas revealed 32 nt-peak corresponding to trna-derived fragments, amount of which was increased by 2.6-fold under dtt treatment. the trna isotypes that gave rise to almost 76% and 86% of all 32nt rna fragments in stressed and control cells, respectively, include glycine, glutamic acid, aspartic acid and valine. the vast majority of 32nt fragments produced from these trnas are precisely phased 5 0 halves with the characteristic cleavage patterns generated by rnase a angiogenin (ang). observed upregulation of tirna in stressed cells is accompanied with upregulation of ang mrna and down-regulation of angiogenin inhibitor 1 (rnh1). we speculate that translational repression, associated with observed tirna, is an additional mechanism of reducing global protein synthesis in response to dtt-induced stress. collectively, our findings reveal the increase in tirna, the differential regulation of mirna expression together with the global mirna downregulation as the most prominent small rnome reprogramming events and possible fine-tuned levels of post-transcriptional regulation upon dtt-induced cellular stress response. global gene expression changes after spinal cord injury j. k. hyun 1,2,3 , j. kim 1,2 , j. y. hong 1 1 dankook university, cheonan, south korea, 2 institute of tissue regeneration engineering (itren), cheonan, south korea, 3 the neuronal regeneration is hardly achieved spontaneously after spinal cord injury (sci), and the restoration of somatic and autonomic functions after sci is also challenging in the clinical field. the pathophysiology of sci is extremely complex and many in vitro and in vivo studies continued to report opposite results each other in spite of the same treatments, therefore a fundamental analysis such as an extensive assay of global gene expression is required to find a way for spinal cord regeneration. in this study, we aimed to detect the changes of global gene expression after spinal cord contusion in rats according to the time sequence. the spinal cord tissues at contusion site were sequenced after spinal cord contusion in rats using rna-sequencing technology. for time sequence analysis, five time points was determined; 1 hour, 1 day, 1 week, 1 month and 3 months after spinal cord contusion, and sham operated rats at each time point were used as controls. quantitative rt-pcr analysis was also performed to validate expression changes of candidate genes in each category. we found that the pattern of changes in gene expression at acute and subacute stages was quite different from that at chronic stage, especially genes associated with with neurotrophin signaling and apoptosis pathways. most of gene expression levels of inflammatory cell markers were increased and peak during acute stage (1 hour to 1 week) and maintained until chronic stage. some of regeneration-associated genes (rags) including brain derived neurotrophic factor, glial cell derived neurotrophic factor and ciliary neurotrophic factor were increased at 1 hour or 1 day after sci. we concluded that the information of gene expression level according to the time sequence after sci might be useful to determine treatment strategies for spinal cord regeneration especially in chronic stage. p-01.02.2-011 3 0 utr length isoform generation profile in a differentiation model alternative polyadenylation (apa) is the regulated selection of a specific poly(a) signal among other proximal and/or distal signals on the 3 0 utrs (untranslated region) for the endolytic cleavage and addition of a poly(a) tail to form the mature mrna. consequently, position of the poly(a) site determines the length of the 3 0 utr which is known to harbor microrna and rna binding protein sites. such apa isoforms have already been linked to altered protein levels and even functions. therefore we hypothesized apa to be one of the mechanisms to generate isoform diversity in proliferating and differentiated cells to better understand the molecular basis of cancer. we used a combinatorial in silico and in vitro approach to analyze a well known enterocyte differentiation model; caco-2 cells. initially we analyzed gene expression datasets for the proliferative and differentiated caco-2 cells using a probe based apa detection tool. to better understand the significance and to validate these results, we used proliferating and differentiated (day10) caco-2 cells and tested sample apa events by rt-qpcr. 3 0 utr isoforms were identified by using 3 0 race pcr. we identified 43 genes (32% of all apa events) to undergo 3 0 utr shortening in differentiated cells compared to proliferating cells. on the contrary 91 genes (68% of all apa events) went through 3 0 utr lengthening events. several genes have been validated to follow the pattern that was seen in apa detection tool so far. to begin understanding the mechanism behind these observations, we are investigating potential inducers of apa during the complex events of differentiation. our next aim will be to further validate and investigate the consequence of such isoform generation events both in the context of differentiation in colon cancer cells. recognition of phosphorylated threonine-4 of rna polymerase ii c-terminal domain by 3 0end processing apparatus o. jasnovidova, m. krejcikova, k. kubicek, r. stefl central european institute of technology, masaryk university, brno, czech republic rna polymerase ii has evolved an array of heptad repeats with the consensus sequence y1-s2-p3-t4-s5-p6-s7 at the c-terminal domain (ctd) of its largest subunit, rpb1. phosphorylation of serines (s2, s5, and s7) and tyrosine-1 orchestrate the binding of rna processing and transcription factors in the site of transcription. several recent studies showed that also threonine-4 site can be phosphorylated which has a number of functional consequences. to reveal the structural basis for the recognition of threonine-4 phosphorylated ctd, we set out to investigate several proteins factors that were implicated with a high levels of threonine-4 ctd phosphomarks using integrative structural biology. one of them, a factor involved in the 3 0 -end processing and transcription termination, showed a high affinity to the phosphothreonine ctd. using nuclear magnetic resonance spectroscopy (nmr), we determined its structure bound to the ctd phosphorylated at threonine-4 that reveals a direct read-out of the phosphothreonine. altogether, our data provides the first insights into the recognition of this poorly understood ctd mark that plays important role in the ctd code of rna polymerase ii. the results of this research have been acquired within ceitec 2020 (lq1601) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. introduction: the treatment of brain tumor glioblastoma (gbm) is still one of the greatest challenge. anti-inflammatory drug indomethacin (ind) mainly acting through the inhibition of cyclooxygenase (cox) has also anti-cancer activity including brain tumors. the aim was to investigate how ind effects an immortality enzyme telomerases' activity. materials and methods: monolayer and spheroid cultures of t98g human gbm cell line were used to evaluate the effects of ind (100 lm) on cell proliferation, viability, apoptosis, cell cycle, camp levels, the levels of apoptotic and anti-apoptotic proteins, morphology (sem) and ultrastructure (tem) for 72 hours. results were analyzed using the student's t-test. results: ind decreased cell proliferation (p72 < 0.01), cell viability (p72 < 0.000001), cell rate at s phase (p72 < 0.000001) and g2 + m phase (p72 < 0.001), camp levels (p72 < 0.01), the levels of pdgfr-a (p72 < 0.001), mrp-1 (p72 < 0.05), nf-jb (p72 < 0.01) and cox-2 (p72 < 0.01) in comparison to control group. ind mildly increased apoptosis (p72 < 0.001) and caspase-3 levels (p72 < 0.01). interestingly, ind increased htert levels (142%, p24 < 0.001; 154%, p72 < 0.0001). sem evaluation showed that ind led to decreased and shortened microvilli, the lost of cell interactions and the conversion of many cell shapes from spindle to oval. many cell remnants in the intercellular area, intact cell membranes, many dense lipid droplets and few autophagic vacuols in the cytoplasm were observed under tem. discussion and conclusions: the effect of ind on telomerase activity can only be found in 8 publications at pubmed research that they only showed its' inhibitory effect in colon, gastric, head and neck cancers. in contrast to previous studies, it was shown for the first time that ind increased telomerase activity in gbm cells and this increase was independent from cox-2 and other tested factors. p-02.02.2-002 interaction between fibrinogen and insulin-like growth factor binding protein-1 under physiologic conditions and influence of diabetes mellitus type 2 on this interaction n. gligorijevic, o. nedic institute for the application of nuclear energy, university of belgrade, belgrade, serbia fibrinogen is plasma glycoprotein and principle participant in blood coagulation. it interacts with many proteins, including insulin-like growth factor binding proteins (igfbps). one of them, igfbp-1, is controlled by insulin. metabolic changes due to diabetes mellitus (dm) affect igfbp-1. besides glucose regulation, igfbp-1 stimulates wound healing. we have investigated complexes formed between fibrinogen and igfbp-1, their change in dm type 2 (dm2) patients and involvement in fibrin clot. samples from adult healthy persons and dm2 patients were studied: plasma, isolated fibrinogen and fibrin. the amount of igfbp-1/fibrinogen complexes was determined using immunoblotting. immunoprecipitation and lectin affinity chromatography were used to confirm interaction between fibrinogen and igfbp-1. in vitro incubation of fibrinogen with excess glucose or methylgyoxal (mgo) was employed to demonstrate influence of glyco-oxidation on complexes. results have shown that igfbp-1/fibrinogen complexes can be differentiated from igfbp-1 oligomers and igfbp-1/alpha-2macroglobulin complexes. the amount of igfbp-1/fibrinogen complexes was lower in patients with dm2. complexes participated in fibrin clot formation, the amount being significantly lower in patients' samples. the quantity of igfbp-1 monomer in fibrin clot was greater in patients' samples. in vitro experiments revealed that complexes undergo glyco-oxidative modifications leading to their reduced formation, cross-linking and increased acidity (faster electrophoretic movement). isolated fibrinogen from patients with dm2 was additionally able to bind exogenous igfbp-1. since igfbp-1 stimulates wound healing, directly and by delivering igfs, igfbp-1/fibrinogen complexes may be seen as igfbp-1 storage instrument, ready to participate in fibrin formation and to assist in damage repair. reduction of complexes due to glyco-oxidative stress in patients with dm may be part of the mechanism responsible for impaired coagulation process. human interferon gamma (hifnc) is a proinflammatory cytokine involved in the regulation of nearly all phases of immune and inflammatory responses. its abnormal expression is associated with the aetiology of many inflammatory and autoimmune diseases. recently we have been exploring the idea to counteract the over-expression of the endogenous hifnc by competitive inhibition with inactive hifnc mutants. they are designed to have preserved affinity to the hifnc receptor, but to be deprived in their capability to trigger the intracellular signal transduction. to this end a library of mutants was created and two potential hifnc antagonists were selected for further investigations: a single point mutant k88q (q substitution for k in position 88) and a double mutant with additional substitution in the n-terminus. both mutants and the wild type hifnc were expressed in e. coli employing the established by us methodology for large scale production of aggregation-prone proteins in soluble native form. the purified mutants were screened for interferon activity (antiproliferative assay), binding affinity (isothermal titration calorimetry) and ability to compete with the wild type for the hifnc receptor (competition assay on wish cells). the selected mutants demonstrated 100 (single mutant) and 1000 (double mutant) times lower antiproliferative activity than the wild type. measuring the binding thermodynamic parameters, we proved that the receptor binding affinity of both mutants was preserved, which is an indication for their potential to compete with the wild type hifnc for its receptor. finally, the biological assay performed on wish cells showed a distinct dose-dependent competition between the wild type hifnc and the mutants. based on the results presented in this study we conclude that the two hifnc mutants are potential candidates for autoimmune therapy based on selective suppression of the endogenous hifnc activity. mesencephalic astrocyte-derived neurotrophic factor (manf) is an er (endoplasmic reticulum) stress-inducible protein and widely expressed in mammalian tissues. it has been identified as a secretory protein that protects cells against er stress-induced damage. er-stress is one of the main mechanisms that play a role in ischemia/reperfusion (i/r)-induced renal injury. recent studies demonstrated that manf can protect cardiac myocytes and cortical neurons against i/r-induced injury. moreover, it has been suggested that it has a restorative effect in ischemic injury. nevertheless, the function of manf in i/r-induced renal injury is still not known. in the present study, we investigated the function of manf by manipulation its expression level in ischemic acute renal failure model established in proximal tubular kidney cells (hk-2 cells). for this purpose, the cells were transfected with either manf sirna or manf encoding plasmids for silencing or over-expression of manf, respectively. then, the cells were exposed to hypoxia-reperfusion (h/r) induction for indicated times. evaluations of cell viability were determined with wst-1 reagent. the changes in protein levels of h/r-induced stress markers were analyzed byimmunoblotting. the results showed that the overexpression of manf has provided a significant resistance to h/r-induced cell death, whereas silencing of manf has rendered the cells more susceptible to death. it was also determined that the pretreatment of cells with manf conditioned medium caused a decrease in cell death. additionally, oxidative/nitrosative stress (os/ns) and er stress levels were decreased with over-expression of manf and increased by silencing of manf in hk-2 cells. taken together, our study suggests that manf may have a protective role against h/r-induced renal cell injury, possibly through the reducing effects on os/ns and er stress. p-02.02.2-005 his-flag tag as a fusion partner in insect expression systemgain or loss? e. krachmarova 1 , m. tileva 1 , k. maskos 2 , i. ivanov 1 , g. nacheva 1 1 institute of molecular biology "roumen tsanev", sofia, bulgaria, 2 proteros biostructures, martinsried, germany human interferon gamma (hifnc) is a glycoprotein playing major role in the regulation of innate and adaptive immunity. glycosylation is not essential for hifnc activity but is important for its stability, half-life and protease resistance in blood. the commonly used hifnc in therapy and research is produced in e. coli and therefore is not glycosylated. bearing in mind the above mentioned shortcomings of the non-glycosylated hifnc we expressed it in mammalian cells and transgenic mice, however very low yields were achieved. to obtain glycosylated hifnc, here we employed a secretory expression of n-terminal his-flag fusion protein in baculovirus-infected insect high five ò cells. this small hydrophilic tag is designed to not affect the proper folding of the target protein and to facilitate the detection and purification procedures. in parallel the same fusion was expressed in e. coli cells. the fusion proteins were purified to high degree of purity by affinity and size-exclusion chromatography. bioassay carried out on wish cells showed that the antiproliferative activity of both fusion proteins was 500 times lower than that of the native hifnc. this result shows that, in contrast to the generally hold view, the n-terminal his-flag tag interferes with the biological activity of hifnc despite of the protein glycosylation. in order to restore the biological activity we attempted to remove the his-flag tag enzymatically. surprisingly, we found that the fusion protein obtained from insect cells was resistant to enterokinase, independently of the enzyme source and experimental conditions, whereas the protein isolated from e. coli was susceptible and the tag-free protein showed fully restored biological activity. we are prone to explain the enterokinase resistance of the fusion protein from insect cells with either the specific conformation of the glycosylated protein or with the interaction of the carbohydrate residues with the enzymatic activity of the enterokinase. p-02.02.2-006 development of fluorescence assay for highthroughput screening system based on flow cytometry for directed evolution of cellobiose dehydrogenase cellobiose dehydrogenase (cdh) is an enzyme produced by phanerochaete chrysosporium and it has been already successfully cloned in other organisms. one of the most important roles of cdh is removing products of cellulose degradation. cdh is very important for biofuel and biosensor industry. for improvements of enzyme properties we have used directed evolution. the most important step is to develop screening system that reflects properties of interest. screening in microtiter plates (mtp) is expensive, time-consuming and has low throughput with a small number of variants detected (10 3 -10 4 in months). the aim of this work was the development of screening system for mutant libraries of cdh expressed on surface of yeast cells based on fluorescent enzymatic assay and flow cytometry. the screening method should be capable of screening cellobiose dehydrogenase variants mutated for higher activity and higher thermostability by error prone pcr. the fluorescent assay was beta-galactosidase (ec 3.2.1.23) also known as lactase is the enzyme that typically catalyzes hydrolysis of beta-1,4-d-galactosidic linkages in beta-d-galactosides, including disaccharide lactose, with glucose and galactose as end reaction products. this enzyme is able to catalyze synthesis of oligosaccharides, in particular galactooligosaccharides via galactosyl transfer reaction. arthrobacter sulfonivorans beta-galactosidase of unique for prokaryotes extracellular localization may find application in food industry for manufacturing lactose-free dairy products and in pharmacology as bioactive principle of medicines prescribed for patients suffering from lactase deficiency. the study was aimed at cloning of the gene encoding a. sulfonivorans beta-galactosidase, purification and characterization of the enzyme. a novel extracellular beta-galactosidase from a. sulfonivorans was recovered with an overall 207-fold purification, a 7.7% yield and specific activity 16 300 uámg à1 protein. the subunit molecular mass of the enzyme determined by sds-page analysis equalled 125 kda. it was found that the enzyme displays pi 5.35, prefers ortho-nitrophenyl-beta-galactoside as substrate (km 27 mm) and shows maximum activity at 40°c and at ph 7.5-9.5. the beta-galactosidase gene was isolated from the genomic dna library of a. sulfonivorans, sequenced, cloned and deposited in the genbank database under accession number km277894.1. it was established that the gene carries an open reading frame consisting of 3132 bp (1043 amino acids) and encodes beta-galactosidase referred to glycosyl hydrolase family 2 (cazy database). p-02.02.2-009 different splice-forms of tdrd7 protein mutated in cataract's and glaucoma's interacts with s6k1/2 o. skorokhod, v. filonenko department of cellular signalling, institute of molecular biology and genetics nas of ukraine, kyiv, ukraine ribosomal s6 kinases (s6k) are important players in cellular pi3k/mtor signalling network, deregulation of which has been associated with methabolic disorders, inflammation and cancer. previously we had identified a novel binding partner of s6k1 -tdrd7 (trap). tdrd7 is a scaffold protein detected in complexes involved in the regulation of cytoskeleton dynamics, mrna transport, protein translation, non-coding pirnas processing, transposons silensing. it was reported recently that mutations in human tdrd7 result in cataract and glaucoma formation, defined by elevated intraocular pressure (iop) and optic nerve damage. the aim of our study was to confirm s6k-tdrd7 interaplay and study its role in cells. bioinformatical analysis of tdrd7 sequence revealed the presence of potential phosphorylation sites of s6k2. using in vitro kinase assay, we have demonstrated that recombinant s6k2 phosphorylate 3 from 5 fragments of tdrd7. formation of s6k2-tdrd7 complexes in vivo was further confirmed by coimmunoprecipitation using anti-s6k2 and anti-tdrd7 antibodies generated previously in rat brain lysates. this interaction was further confirmed by confocal microscopy, oleksandr had shown that tdrd7 co-localize with s6k2 in hepg2 cells, predominantly in perinuclear region, enreached for one of the tdrd7 isoforms identified previously. moreover, we have detected that c-terminal synthetic peptides of s6k2 with methylated arg interfere with tdrd7 from hepg2 lysates. the physiological characteristics of s6k2-tdrd7 interaction and the role of this complex formation in neuropathology's development need further investigation. many biological function of placenta are performed not just a set of individual proteins, but also different oligomeric structures and complexes. herewith, activities of complexes may considerably differ from activities of individual proteins. therefore, identification and characterization of placental multi-protein complexes is an important step to understanding the placenta function. the aim of the present work was to investigate a composition and biological functions of the very stable high molecular mass multi-protein complexes (spc) from placenta of healthy mother. we isolated spcs (~1000 kda) from the soluble fraction of three human placentas. light scattering measurements and gel filtration showed that the spc is stable in presence salts, acetonitrile and triton x-100 in high concentrations, but efficiently dissociates in the presence of 8 m urea and 50 mm edta. such a stable complex is unlikely to be a random associate of different proteins. it was shown the spc includes a number of proteins with molecular weights of 2 to 180 kda. several protein components of the spc were identified, including serum albumin, transferrin, iggs, annexin a5 and other proteins. serum albumin, transferrin and protein with molecular weight 14,1 kda are the main proteins of the complex. it was shown high the spcs from three placentas possesses dnsase and catalase activities. an addition, investigation of cytotoxic effect on human cancerous cell lines has shown that the spcs reveal high cytotoxicity. antibody-cytochrome b5 fusion protein, characterization and applications for antibody development process antibodies have recently become an essential tool being a part of immunodiagnostics, therapeutics and as a valuable instrument in life science research. an enormous number of options utilizing a various tags were used to create a universal antigen-binding domain, which can be easily detectable, highly soluble and might be produced in high yields with low costs, but no multipurpose solution exists yet. we addressed the question whether a single tag could be found for enhancing solubility of recombinant fab antibody fragment and providing its detection and accurate quantification by rather simple method. a new application for hemeproteincytochrome b 5 as the antibodies fusion partner were proposed. we have constructed of recombinant fab antibody fragment cytochrome b 5 fusion protein. we have shown that cytochrome b 5 enhance expression of fab antibodies fragments in bacterial system, and could be a versatile tool for recombinant proteins folding, redox (oxidation) state studies and for their precise concentration determination in the turbid solutions. fusion fab-b5 protein has a stable red color and characteristic absorbance spectrum with the maximum absorbance at 413 nm in oxidized environment. cytochrome b 5 change its spectrum maximum depending on environmental redox potential and its folded state, so one can track these events in real time spectrophotometrically. binding activities of fab-b 5 fusion protein and hybridoma secreted immunoglobulin were measured by biolayer interferometry and elisa. no significant difference between them was revealed. due to this feature we can distinguish the chimeric protein of interest in complex mixtures and control the process of recombinant proteins expression and purification in real-time. besides, cytochrome b 5 fusion tags multiples recombinant antibody yield (from 2 to 3 times) and doesn't affect antigen-binding properties. the bb125-135 site of fibrin molecule is the site of fibrin protofibrils lateral association l. urvant palladin institute of biochemistry nas of ukraine, kyiv, ukraine previously we showed that fibrin-specific monoclonal antibody i-3c (monab i-3c) inhibited the fibrin protofibrils lateral association. we suggested that the epitope of monab i-3c in bb118-134 of coiled-coil region of fibrin molecule coincides with the site involved in fibrin protofibrils lateral association. the aim of this study was to localize the site of protofibrils lateral association in fibrin molecule using the synthetic peptides bb121-138, bb125-135 and both their scrambled version, and bb109-126 peptide. monab i-3c was isolated from hybridoma culture medium by affinity chromatography on fibrin-sepharose. turbidity analysis was used to study the effect of synthetic peptides on fibrin polymerization. the interaction between peptides and monab i-3c was investigated by spr method using plasmon-6 device. we investigated the effect of synthetic peptides which corresponded to amino acide sequences of fibrin molecule bb109-126, bb121-138, bb125-135, and the scrambled versions of bb121-138 and bb125-135 peptides on a binding to monab i-3c and on the fibrin polymerization process. in spr analysis was showed that bb121-138 and bb125-135 peptides, but not their scrambled version, binds to monab i-3c, immobilized to a chip. turbidity data showed that only bb121-138 and bb125-135 peptides caused the 2-fold decrease of the rate of the lateral association of protofibryls at the concentration 2.2 9 10 à4 m and 2.7 9 10 à4 m, respectively. both of them decreased the final clot turbidity. our data let us to suggest that the bb125-135 site is the site that involved in protofibryls lateral association. it has been recently shown that irisin immunoreactivity was altered in gastrointestinal cancers. as known hematological malignancies was one of the most common malignancies through world, but no study was present how irisin was changed in this type of cancers. therefore, purpose of this was to investigate how immunoreactivity to irisin was altered in hematological malignancies (blood cancers). we used an antibody from phoenix to demonstrate how a 12 kda band after deglycosylation of irisin altered in hematological malignancies. here we first time showed that irisin tissue immunoreactivity from acute lymphoblastic leukemia (all) and acute myelogenous leukemia (aml) patients was increased when compared with unaffected biological tissue parts. from the immune-histochemical (ihc) investigations it is concluded that hematological tissue and blood cells may be another source of irisin and increased with cancer, thus this finding might help to enlighten pathophysiology of hematological malignancies. the value of urine neutrophil gelatinaseassociated lipocalin (ngal) in acute heart failure n. serdarevic clinical centre, sarajevo, bosnia and herzegovina introduction: renal dysfunction is very common in heart failure (hf) and neutrophil gelatinase-associated lipocalin (ngal) is used as an early marker of acute renal tubular injury. recent studies have been reporting that ngal is inhibitor of inactivation of matrix metalloproteinases (mmp-9) which results in enhanced proteolytic activity with prolonged effects on collagen degradation. due to its relation to extracellular matrix degradation in myocardium and infammation, we hypothesized possible increased ngal expression in hf besides it renal dysfunction etiology. patients and methods: in study were included 30 patients hospitalised with signs and symtoms of ahf. urine samples for ngal analysis were collected at admission and analysed by the chemiluminescent microparticle immunoassay (cmia) for the quantitative determination of neutrophil gelatinase-associated lipocalin in human urine (abbott, architect analyzer). refferent range for urine ngal is 0-135 ng/ml. on admission blood samples for bnp (brain natriuretic peptide) analysis were drawn and tested by architect bnp chemiluminescent microparticle immunoassay (cmia), abbott laboratory. results: the mean age of the patients (male= 30, female= 30) was 68.86 years (sd 10.92 years). among them 18 (30%) patients was diagnosed as a hf-pef (hf with preserved ejection fraction) while 42 (70%) as a hf-ref (hf with reduced ejection fraction). mean bnp values was 1616.71 pg/ml (sd 1511.15 pg/ml) and mean lvef was 33.66% (sd 14.19%). mean urine ngal was 60.91 ng/ml (sd 78.72 ng/ml). we found significantly positive, but weak correlation among ngal and bnp only by pearson correlation test (r = 0.139, p = 0.464, wilcoxon signed rank test z = à4.782 p < 0.5). conclusion: bnp levels are elevated in hf with reduced and preserved ejection fraction. urine ngal is not elevated in acute heart failure, but it is slightly positively correlated with serum bnp values. converging evidence implicates the intermediate and medial mesopallium (imm) of the domestic chick forebrain in memory for a visual imprinting stimulus. a number of learning-related changes have been found in plasma membrane and mitochondrial proteins of imm. for broader analysis of these changes we employed two-dimensional gel electrophoresis/mass spectrometry approach and identified differentially expressed proteins in membrane-mitochondrial fraction of the imm across chicks with different estimated levels of imprinting 24 h after training. we further inquired whether the amounts of those proteins in the imm and a control region (posterior pole of the nidopallium, ppn) are correlated with memory for the imprinting stimulus. learning-related increase in the amounts of the following proteins was demonstrated in the left imm, but not in the right imm or left and right ppn: (i) membrane cognin;(ii) a protein resembling the p32 subunit of splicing factor sf2;(iii) voltage dependent anionic channel-1;(iv) dynamin-1; (v) heterogeneous nuclear ribonucleoprotein a2/b1. obtained results indicate that the molecular processes involved in learning and memory of imprinting cover a wide range of cellular activities, including stabilization of protein structures, increased mrna trafficking, synaptic vesicle recycling and specific changes in the mitochondrial proteome. the aim of this work is to study the substrate and inhibitory properties of uridine derivatives in the reactions catalyzed by e.coli up in order to shed some light on the substrate's conformation in the productive complex with the enzyme. we studied the e.coli up-catalyzed phosphorolysis of uridine and its derivatives modified in the heterocyclic base and the sugar moiety. the kinetic constants (km, ki, kcat ) of the phosphorolysis reaction of near 30 uridine derivatives were determined. the combined kinetic (nnna, 35, 2016) and structural data (acta crystallogr., d70, 3310, 2014) provide clear evidence that up binds uridine in the most energetically unfavorable conformation, which, to the best of our knowledge, has no precedents in the enzymes of nucleic acid metabolism. this is possible due to multiple interactions between the substrate and the protein environment (active site residues) mainly through hydrogen bonds. these results are important for understanding the mechanism of action of this class of enzymes. an analysis of the conformations of nucleosides in solution and rotational barriers suggests that the energy difference between the ground state of uridine and uridine complexed with up may be high as 63-69 kj/mol. the binding in a high-energy conformation results in the weakening of the glycosidic bond. the observed conformation of uridine complexed with sulfate (mimetic of phosphate) may be very similar to its conformation in the transient state. until now, foxp1 (forkhead box p1) has been identified as a tumor suppressor in several correlation studies in breast cancer. although, foxp1 is defined as a transcriptional repressor that interacts with other transcription factors in various mechanistic studies, there is no study that explains its repressor functions in breast cancer biology. here we demonstrate the repressor function of foxp1 on nfat (nuclear factor of activated t cells) and the migratory effect of this repression in mda mb 231 breast cancer cells. we performed co-immunoprecipitation experiments for the investigation of protein-protein interaction between two transcription factors. protein-protein interaction on dna was investigated with emsa and transcriptional effects of foxp1 on nfat, lusiferase reporter assay was performed. wound healing assay was used to analyse the effects of overexpression of foxp1 on tumor cell migration. our results showed that foxp1 has protein-protein interaction with nfat on dna and enhances breast cancer cell migration by repressing nfat transcriptional activity and foxp1 shows oncogenic function by regulating breast cancer cell motility. introduction: phosphodiesterase 5 (pde5) is one of phosphodiesterase lead to hydrolyzing cgmp.the cgmp signaling pathway has an important role in proliferation of cells. previous studies showed pde5 was increased in cell lines cancers thus pde5 inhibitors can used as efficacious therapeutic option for treatment of cancers. the current study was to investigation the effect of hydroalcoholic achillea.wilhelmsii extract (hawe) on the pde5 gene expression and cgmp signaling in the mcf-7 er + and mda-mb-468 er à . methods and materials: the ed50 of the hawe on both cell lines were examined by using mtt viability test then the expression of pde5 and cgmp concentration were measured in timedependent manner (in the ed50) by real-time rt-pcr and colorimetric assay respectively. results: treatment with the hawe showed, 25 lg/ml is ed50 for both cell lines and the hawe lead to reduction in pde5 mrna expression and evaluation of intracellular cgmp showed an increase pattern in the time-dependent manner. conclusion: our results showed that the hawe has anti-proliferative property in the mcf-7 and mda-mb-468, cell lines of breast cancer through the cgmp pathway, these data suggested that the hawe can be potential source for the isolation of effective anti-proliferative molecules. keywords: achillea.wilhelmsii, breast cancer, anti-proliferative, phosphodiesterase, cgmp signaling pathway. outer membrane protein g (ompg) is a stable monomeric porin having 14-stranded beta barrel form from e.coli. its exact function is not fully understood; however, it allows the passage of molecules up to 900 da in neutral ph but the pore is closed by going through a conformational change under ph 5.5. as being monomeric and having ph-dependent gating characters, it is suitable for biosensor and targeted drug delivery applications. an attempt on ompg is to create a larger pore while its stability is undisturbed. ompg-16s is obtained by adding 38 amino acids to the primary chain in order to have a 16-stranded beta barrel porin. ompg-16sl is formed by further adding 6 amino acids to loop l6 and by replacing 7 lysines with arginines. ompg-16s and ompg-16sl mutants are investigated by fourier transform infrared spectroscopy (ftir) and compared with ompg-wild type (wt) in terms of ph-dependent conformational changes and thermal stability. each mutant is prepared in na-phosphate buffer pd 5.5/7.5 and infrared spectra are recorded. further, temperature profiling are recorded for the range between 22 to 106°c. results show that both mutants are responsive to ph changes. while turning the ph from acidic to neutral, beta sheet signals shift to lower wavenumbers showing difference in secondary structure, implying the existence of closed and open states. on the other hand, mutant proteins show structural differences compared with the wt protein. porins are known for their remarkable thermal stability. the mutans retain this character by having transition temperature of $ 75°c, although this is less than the wt transition at $ 85°c. in conclusion, two mutants show signs of open and closed states as ompg-wt and even if the mutants are less stable than ompg-wt. this study shows that the attempted alterations in ompg structure are successful in terms of ph-response but it needs improvement in terms of stability when necessary. nad is a key factor in the regulation of mitochondrial metabolism. besides its vital role as redox carrier, nad serves as substrate for protein adp-ribosylation and deacetylation, modifications which modulate enzyme activities in mitochondria. these functions depend on how nad levels are maintained in this organelle. in human cells, mitochondrial nad is segregated from the cytosolic pool and can be synthesized from nmn, which is probably imported into the matrix. here, we tested whether the nudix pyrophosphatase nudt13 participates in the regulation of the mitochondrial nad pool. this enzyme has a predicted nadh pyrophosphatase zinc ribbon domain and a mitochondrial targeting sequence at its n-terminus. however, it has not yet been functionally characterized. we overexpressed nudt13 endowed with a c-terminal flag-epitope in human cells. to evaluate changes in the mitochondrial nad concentration, we used a reporter system which includes the overexpression of the catalytic domain of poly(adpribose) polymerase 1 (parp1) within the organelles (mitoparp). thereby mitochondrial nad is converted into protein-bound poly(adp-ribose) (par). the extent of par formation correlates with the mitochondrial nad availability and is detected by western blotting. our results established that nudt13 is indeed a mitochondrial protein, as it was localized exclusively to these organelles. moreover, when nudt13 was overexpressed along with the mitoparp detector system, a dramatic decrease of par was observed. the obtained results indicate that nudt13 is enzymatically active upon overexpression in the mitochondrial matrix and that it might cleave nad, thereby modulating its organellar level. however, at this point we cannot exclude the possibility of direct par cleavage by nudt13. further characterization of nudt13 will define its substrate specificity and clarify its role in mitochondrial metabolism. the incidence of increase in colorectal cancer (crc) worldwide has become a major health problem. early diagnosis and treatment of crcs are of importance for improving survival. in the present study, it was aimed to investigate chemopreventive effect of rosmarinic acid and evaluate the angiogenesis process in azoxymethane (aom)-induced crc model. male sprague-dawley rats were randomly divided into a control group, aom-induced rat colorectal cancer group (15 mg/kg body weight aom; ip, weekly for four weeks), and rosmarinic acid (5 mg/kg body weight; oral, daily for four weeks)-treated group. in addition to the standart diet of the all groups 15.8% peanut oil was added throughout the experiment. the all rats were sacrificed at the end of 30 weeks. biochemical examinations were performed in rat plasma. histopathological adenocarcinoma rates were observed in 87.5% of aom group. the incidence of adenocarcinoma was showed a reduction in the treatment group. significant increases in plasma tos and mcp-1 levels were found in the aom group compared to controls. these increases were reduced in the treatment groups but no significant. a significant increase was detected in tas levels in the treatment group when compared to the aom group. significant decreases in plasma adiponectin levels were found in the aom and the treatment groups compared to controls. in conclusion, treatment with rosmarinic acid reduced the occurrence of inflammation and was helped to maintain the oxidant-antioxidant balance in the model of aom-induced rat colon cancer. mitochondrial genome, while being strongly reduced in course of evolution, still codes for several proteins. the vast majority of them are components of the respiratory chain complexes. to produce these proteins, the system of mitochondrial translation is presented in the organelles, which is in common close to that in bacteria. translation initiation in bacterial cells is orchestrated by three protein factors called if1, if2 and if3. the orthologs of the two latter proteins are commonly found in mitochondria. however, mitochondrial if3 could not been identified in several groups of organisms, including s.cerevisiae, for a long time. recently we have shown that baker's yeast protein aim23p possesses a function of mtif3. however, the mitochondrial translation has not been stopped in the yeast strain without aim23p which is surprising taking into account the fact that if3 is obligatory for the translation in bacterial systems. instead of blocking of mitochondrial protein synthesis in absence of aim23p, we observed the translational imbalance: the synthesis rate of the complex v subunits was increased while the synthesis rate of the complex iv subunits was repressed. thus, in addition to its general role in translation initiation, aim23p might specifically affect the biosynthesis of individual mitochondrial-encoded protein species. our genetic experiments have revealed that, indeed, aim23p is almost indispensable for cox1p synthesis, and that it affects the translation of cox1 mrna through its 5 0 -utr, like classical mitochondrial translational activators. this is in accordance with our measurements of complex iv activity which is several times less in yeast lacking aim23 gene than in the wild-type. taken together, our results point on the multiple role of aim23p in mitochondrial translation: in addition to its function as mitochondrial if3, it specifically regulates the amount of complex iv subunits and its activity. p-02.02.2-024 the circulating betatrophin and irisin levels in polycystic ovary syndrome patients with and without insulin resistance introduction: polycystic ovary syndrome (pcos) is the most common endocrine/metabolic disease in women around the world, characterized by oligo-or anovulation, polycystic ovary, and/or hyper-androgenism. insulin resistance (ir) and obesity are common findings in patients with pcos. irisin is a recently identified myokine secreted from skeletal muscle in response to physical activity. irisin has been postulated to induce the differentiation of white fat tissue into brown fat tissue. betatrophin is a currently discovered new hormone proposed to stimulate b-cell proliferation. in this study we investigated the levels of irisin and betatrophin in pcos patients. materials and methods: our study group was consisted of 40 patients with pcos and 20 healthy volunteers. patients group was divided into two subgroups according to presence of ir. (pcos+ir and pcos-ir). the oral glucose tolerance test (ogtt) and the homeostatic model assessment (homa-ir) were performed to assess glucose tolerance and insulin sensitivity. irisin and betatrophin levels were measured by elisa method. results: circulating irisin was significantly higher in the pco-s+ir subgroup than the control group (p < 0.022). circulating betatrophin was significantly lower in both patients subgroups than the control group (p < 0.008). there was no negative or positive correlation between irisin and betatrophin levels. discussion: these data suggest that irisin and betatrophin may act a role together in the ir mechanism in pcos patients. butyrylcholinesterase (bche) synthesized in liver has long been associated with hyperlipidemia, type 2 diabetes and obesity. there are also reports on bche knockout mice becoming obese. the exact involvement of how bche interacts with lipids is still not clear. previously we displayed a correlation between leptin, waist circumference, fat mass and bche levels. recently, we have also shown that bche overexpression in hepg2 cells is regulated by alpha linoleic acid. as the next approach on the analysis of lipid metabolism and bche interaction, we considered the capability of bche to hydrolyze lipids. human serum bche was purified by subsequent deae-tris-acryl m and procainamide chromatography. the purified bche was utilized in a modified acid lipase assay with the acid lipase substrate 4-methylumbelliferyl palmitate (4-mu-palmitate). as the second alternative substrate trioleic acid was utilized. the triolein hydrolysis was measured by the nefa kit. verification that bche hydrolysis of these lipid substrates was not due to another esterase was done by iso-ompa inhibition studies. also, lectin binding studies with bche and rca were carried out to rule out non-specific esterase activity. using purified human serum bche and hepatic lipase as control enzyme we found that bche is able to hydrolyze the acid lipase substrate 4-methylumbelliferyl palmitate (4-mu-palmitate). we found that bche hydrolyses this molecule at ph 8 rather better than at ph 7.4. at ph values, purified human bche has a km value that was 10 times bigger than that of human pancreatic lipase. with the bigger molecule the triolein, the difference between the km values of bche and pancreatic lipase was smaller. bche seems to hydrolyze triolein with an efficacy comparable to approximately 25% that of human pancreatic lipase. our results display that another function of bche may be its lipid hydrolyzing activity. p-02.02.2-026 determination of regional reference ranges for erythropoietin with laboratory data mining serum erythropoietin (epo) levels are the main regulator factor of erythrocyte production and increase in response to hypoxia. our region is a location dominated by hypoxic conditions due to the high attitude. in this study we aimed to investigate the mean serum epo levels in the living conditions of our region. two hundred and eighty epo results from our laboratory data whose hemoglobin levels were normal were evaluated in the study. mean serum epo levels were analyzed via chemiluminescence method in beckman coulter dxi 800 auto analyzer. the epo levels of samples was 12.26 ae 9.32 mul/ml (ranged between 3.00 and 48.57) mul/ml. when we performed ae 1 sd for the studies population we determined normal serum epo levels were as 2.94-21.58 mul/ml. the upper limit determined by our results was 17% higher than that of determined by the manufacturer as 18.5 mul/ml and the lower limit determined by our results was 17% higher than that of determined by the manufacturer as 2.59 mul/ml. normal serum epo levels were considerable for our region and the upper and lower limits were higher than those of determined by the manufacturer. more detailed studies considering the physical properties of participants including a higher number participants are necessary. subclinical hypothyroidism is the precursor to hypothyroidism because it has a tendency to transform into hypothyroidism. subclinical hypothyroidism is considered one of the risk factors causing metabolic syndrome. metabolic syndrome can be characterized by plasma level of apelin released from adipocytes. in the present study, we aimed to measure serum apelin level of patients with subclinical hypothyroidism and compare them with serum apelin level from healthy individuals. our study group included 31 patients diagnosed with subclinical hypothyroidism and 23 healthy volunteers. serum samples were obtained from each participant for the measurement of apelin. these were then stored at à20•c until the time of analysis. serum apelin concentrations were determined using an enzymelinked immunosorbent assay. the mean serum apelin levels of subclinical hypothyroidism and control groups were 79 ng/l, control group 60 ng/l respectively. there was no statistically significant difference in terms of the mean apelin levels between the groups (p > 0.05). apelin levels didn't show significant correlation with bmi (p > 0.05). in the present study, no significant difference of serum apelin level was observed between patients with subclinical hypothyroidism and healthy control subjects. however, the apelin levels were higher in the patients with subclinical hypothyroidism than in the control group. the possible relationship between thyroid hormones and apelins is critical to understanding the etiopathogenesis of metabolic disorders. the mitochondrial erv1/mia40 import system does not impact cytosolic fe-s cluster protein maturation and iron regulation erv1 is a sulfhydryl oxidase that partners with the import receptor mia40 to import small cysteine-rich proteins into the mitochondrial intermembrane space. it has also been suggested that erv1 has an additional role in maturation of cytosolic fe-s cluster proteins and regulation of iron homeostasis in s. cerevisiae. however, these studies were performed on one particular erv1 mutant strain (erv1-1) that we discovered has additional defects in glutathione (gsh) metabolism. since gsh is required for iron regulation and cytosolic fe-s cluster assembly, this complicates our understanding of erv1 0 s role in these processes. we discovered that the erv1-1 strain originally tested for fe-s cluster defects was the only strain to exhibit defects in the cytosolic fe-s enzymes. mitochondrial and cytosolic fe-s protein activities in the other erv1 and mia40 mutants tested were similar to the wt control. in addition, while all the erv1 and mia40 mutants tested exhibit temperature-dependent defects in mia40 oxidation, only the erv1-1 strain has significantly reduced gsh levels and more oxidized gsh: gssg redox state. we determined that the cause of gsh depletion in the erv1-1 strain is an additional mutation in the gene encoding the glutathione biosynthesis enzyme (gsh1) that compromises gsh1 protein folding and/or stability. to address whether gsh deficiency in the erv1-1 mutant is the underlying cause for the cytosolic fe-s cluster defects and iron misregulation for this strain, we measured fe-s protein activity, iron-regulated gene expression, and iron accumulation in erv1 and mia40 mutant strains. only the erv1-1 strain exhibited iron misregulation and accumulation of mitochondrial iron, while exogenous gsh rescued these defects. these results demonstrate that the defects in cytosolic fe-s enzymes and iron homeostasis in erv1-1 are due to gsh depletion and neither erv1 nor mia40 play significant roles in cytosolic fe-s cluster assembly and iron homeostasis. human c-peptide is a 31 amino acid polypeptide, which is secreted into blood from b-cells in the pancreas where pro-insulin undergoes a post translational modification and cleaved into insulin and c-peptide. human c-peptide concentrations in blood plasma and urine reflect the level of insulin resistance associated b-cell function and can point out insulin secretory failure. the reference intervals in blood plasma and urine are 0.5-10.0 ng/ml and 40-150 ng/ml respectively. c-peptide measurement in urine and plasma provides a guide for therapy in diabetes. this study describes a method for the development and validation of picaa (peptide impurity corrected amino acid analysis) method for the determination of the purity of the human c-peptide which could be used as a reference material to measure cpeptide concentrations in plasma. two different methods were performed for the picaa; aaa-id-ms/ms for quantification of constituent amino acids following hydrolysis of the material and rp-hplc-esi-tof ms for determination of the peptide related impurities. the result of the aaa id ms/ms method was corrected for the amino acids originating from the impurities. id ms/ms-aaa was performed with zivak ò hplc and zivak ò tandem gold triple quadrupole ms equipped with a phenomenex ez:faast 4l aaa column (250 9 2 mm i.d). the mobile phase was composed of, a: 20 mm ammonium formate (af) in water, b: 10 mm af in acetonitrile (acn). the intact peptide analysis was performed by a hitachi lachrome elite hplc and bruker microtof-q mass spectrometer equipped with a capcell pak mg-ii c18 column (150 9 2 mm i.d., 5 mm particle size). the purity of the synthetic c-peptide was determined by picaa analysis and related uncertainty was calculated. traceability to si was established using the amino acid standards of which the purity was determined by tub _ itak ume using qnmr analysis. picaa is a simpler alternative to the full mass balance approach which requires large quantities of the peptide material. p-02.02.2-034 heat shock proteins: complementary therapies in brain tumors with viscum album e. onay-ucar, s. n. biltekin 1 istanbul university, faculty of science, department of molecular biology and genetics, vezneciler, istanbul, turkey cancer is one of the lethal diseases in the world. different cancer types possess overexpressed hsps levels. viscum album extracts with their anticancer and antioxidant properties are being used in cancer therapies. biochemical composition of this plant is known to vary its features depending on the host trees and time of harvest. in our previous study, it has been found that v.album inhibited hsp27 expression and induced caspase-dependent apoptosis in c6 rat gliomas. the aim of the current study is to find out whether different v.album extracts have different effects on hsps expression level and apoptosis in c6 glioma cell line or not. in this study, three different extracts of v.album were compared for their potential inhibition effects on hsps. the cytotoxic effects of extracts have been determined via mtt test. different experiment groups were set up subjected to heat shock and/or incubated without any heat shock application. overexpression of hsps was induced by heat shock at 42°c for 1 h in c6 cells. expression levels of hsps were determined by western blot analysis. the apoptosis inducing effect was also evaluated via caspase-3 activation in c6 glioma cells. pretreatment of the cells with non-toxic dose (10 lg/ml) of v.album extracts prior to heat shock, reduced significantly the expression levels of hsps. similarly, pretreatment with the extracts prior to heat shock increased apoptosis via caspase-3 activation in c6 glioma cells. these results will be utilized in the determination of the relation between extract composition and stress protein expressions. these results suggest that different extracts of v.album are able to down regulate expression of hsps, and induce apoptosis. this warrants further exploration as a potential resource of bioactive compounds that can be used in cancer therapy. future studies targeting hsps for the development of chemosensitizers may help improve the treatment of cancer in combinational therapy. biological drugs (biologics) are the fastest-growing category of therapeutics among those approved by the agencies for drugs regulation. most biologics are proteins designed for parenteral use. however, proteins are characterized by poor pharmacokinetic and safety profiles. peg-coating (poly-ethylene glycol coating) of biologics provides several benefits, including an increased half-life related to reduced renal clearance, an increased stability to degradation, and a reduced immunogenic/antigenic response. preservation of the three-dimensional structure and activity of the pegylated form is a strict requirement for human use. the recombinant proteins used for this studies (as-sod, superoxide dismutase; mmp12, matrix metalloproteases 12; ansii, l-asparaginase ii) were cloned and then over-expressed in escherichia coli. pegylation reactions were performed using commercial reagents. all the protein samples were purified and analyzed by solution and solid-state nmr (fields from 700 mhz to 950 mhz). we developed new protocols to prepare samples of pegylated proteins, demonstrating that solid-state nmr spectra of exceptionally good quality can be obtained for pegylated proteins in the sedimented state (obtained by either ultracentrifugation or rehydration of freeze-dried samples); surprisingly, sedimentation of pegylated proteins to this end has never been attempted. the spectral quality is comparable toor better thanthat of the corresponding crystalline samples. the excellent quality of the solid-state nmr spectra would make it possible to perform extensive resonance assignment and even a conventional full structure determination of biologics. the proposed method is based on the comparison of a standard twodimensional solid-state nmr spectrum of the sedimented pegylated protein with that of the crystalline state of the native proteinfor which the x-ray structure is available. all eukaryotic creatures hereditarily have natural defense mechanisms and are protected from the infections with this defense mechanism. antimicrobial peptides (amp) contain 10-50 amino acid content, are positively charged with amphipathic feature. the antimicrobial activities of amps are thought to be depended on the microbiocidal effects by binding to the surface of microorganisms and creating pores in their membranes. defensins are both effector and mediator small antimicrobial peptides of the immune system. these peptides in cationic and amphipathic structure have broad spectrum antibacterial, antifungal and antiviral features. defensins regulate the innate and acquired immune systems by suppressing proinflammatory responses during infection. mammals have three structural subfamilies of defensins. these show differences according to the trisulfide arrays in their structure and are classified as a,b, h defensins. human beings have tissue-specific six functional a defensins. human hnp-1 and hnp-4 encoded by defa1, defa3 and defa4 genes are firstly expressed in neutrophils. human hdp5 and hdp6 encoded by defa5 and defa6 are firstly expressed in paneth cells in the intestines and play important role in the defense and homeostasis. human beings have many pseudogenes such as defap and deftp in addition to these functional genes. according to literature data, defensins play an important role in defense against microbial placements on mucosal surfaces. in addition, the antimicrobial spectra of defensins include gram negative and gram positive bacteria, fungi and viruses. in addition to their antimicrobial efficiency, they can accelerate the wound healing due to their mitogenic effects on epithelium cells and fibroblasts. bile salt hydrolase (bsh) enzyme catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts into amino acid residues and the free bile acids that reduce cholesterol. however, some intestinal bacteria have an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid having potential harmful side effects to the host. the catalytic mechanism and substrate preference of such bsh enzyme is not clear. in this study, bsh gene from lactobacillus plantarum gd2 strain was cloned, expressed, characterized in escherichia coli blr(de3) strain, and then val-58 and phe-129 amino acids, supposed to be responsible for substrate preference, were substituted for met-58 and ile-129 amino acids respectively by site directed mutagenesis. the hydrolysis activities and stability of the mutant recombinant bsh (mrbsh) enzymes were examined along with six different bile acids by ninhydrin assay and sds-page respectively. ninhydrin test results indicated that wild-type recombinant bsh (wrbsh) hydrolyzed six major human bile salts with an apparent preference towards glycine-conjugated to tauro-conjugated bile salts. however, the activities of mrbsh/phe129ile enzyme are 29%, 23%, 13%, 14%, 0% and 0% of the activity of wrbsh against to glycocholic acid (gca), glycodeoxycholic acid (gdca), glycochenodeoxycholic acid (gcdca), taurocholic acid (tca), taurodeoxycholic acid (tdca) and taurochenodexycholic acid (tcdca) respectively. the activities of val58met mrbsh enzyme are 79%, 93%, 74%, 43%, 44% and 28% of wrbsh against to gca, gdca, gcdca, tca, tdca and tcdca respectively. our findings support the suggestion that bsh enzymes recognize their substrates predominantly at the amino acid moieties and not at the cholate moieties. however, further pcr-based site-directed mutagenesis and structure-driven computational and theoretical approaches are required for the precise determination of their substrate specificities and the selection of probiotic bacteria. we deposited bacteriorhodopsin in purple membranes under applied electrical field onto ito (indium tin oxide) support. purple membranes film, highly oriented in one direction, was placed between two ito electrodes. we studied dependence of electrical properties of these films on light illumination. we argue that this setup can be used for functional studies of microbial rhodopsins. in opposite to already published results where this system was used as a photocondensor for studying functional properties of bacteriorhodopsin, we studied electric properties of such systems and we found strong light dependence of resistivity of bacteriorhodopsin in purple membranes films. optogenetics is already used in study of neuronal cells cooperation in vitro and in vivo by means of microbial rhodopsinsion pumps and channels incorporated in membranes of neurons changing their electrical potential while receiving a light quantum by laser or led source. best perspectives optogenetics will give after successful transfer to medical applications, such as the treatment of blindness, treatment of disorders like parkinson's disease etc. but to achieve these we need a broad set of tools, optogenetics tools, highly specialized to solve specific problems of neurophysiology. to the creation of such tools our work is dedicated. new optogenetic tools can be made by mutations in existing ones altering their properties (mainly spectral characteristics, selectivity and conductivity) or some promising mutations in conserved residues can be found in existing organisms. a halophilic archaeon halosimplex carlsbadens is a host of protein of our interest. according to the theoretical data based on the alignment with br and the 3d structure model of this novel protein, we suppose this protein functions like the light-driven h+ pump: all the key residues are the same or at worst have the similar properties, except one in the position 96leucine instead of the aspartic acid. a gram-positive bacteria deinococcus-thermus phylum syntheses rhodopsin with substitution of this aspartic acid to alanine. sphingomonas paucimobilis has rhodopsin where aspardic acid in position 96 is changed to serine residue. and one yet uncharacterised guillardia theta rhodopsin even has the same as br motif (d85, t89, and d96) but according to alignment is closer to chr2 even the last one motif is e85, t89, n96. it is expected that all of them will show us new properties. though the further experimental data are essential. the work is supported by rsf 16-15-00242. evaluation of some thymus proteins in patients with crimean congo hemorrhagic fever i. b€ ut€ un 1 , s. sahin 1 , f. duygu 2 1 university of gaziosmanpasa, department of biochemistry, tokat, turkey, 2 oncology education and reasearch center, ankara, turkey crimean congo hemorrhagic fever (cchf) is a tick-borne viral zoonotic disease. it has a high fatal rate (%5-30). tokat is one of the cities having the most reported cchf cases, in turkey. clinical presentation of the disease varies widely among patients. thymic peptides are small molecules synthesized by thymic epithelial cells. they play role in the immune response, as well as anti-inflammatory process. fourty patients referring to the hospital with tick-contact history and/or presenting clinical manifestations consistent withcchf and with positive pcr results for cchf virus in blood samples were included to the study. the wbc and platelet values at application and before the patients were discharged were recorded. the healthy control group consisted of age and gender matched healthy volunteer adults free of any chronic disease. thymosin alpha1 (ta1), thymuline and thymosin beta4 (tb4) were studied by the elisa method in this study. biochemical parameters were also analysed. ast and alt values were significantly higher (p < 0.01) and plt and wbc levels were significantly lower in the cchf group (p < 0.05). levels of tf, ta1 and tb4 were found to be significantly higher in cchf (p < 0.01). there was no mortality during the study period. duration of hospitalization was 6.73 ae 2.74 days. levels of tb4 were significantly correlated with duration of hospitalization (r= à0.351, p = 0.036). alt levels were significantly correlated with tf levels (r = 0.413, p = 0.010). 17 patients received ffp and apheresis for the supportive treatment, while 5 patients received only ffp and 2 patients got only apheresis. 16 patients did not get any of these blood products. there was not a statistically significant differences in thymus peptides among these treatment groups (p > 0.05). we report 40 survived cchf patients with elevated thymic peptides. pathogenesis of cchf has many points to be highlighted. thymic peptides may play role in the clinical situation of the patients with the disease. the effect of methocarbamol on the peroxiadse activity of human erythrocyte hemoglobin hemoglobin is released to blood circulation, after red blood cells lysis. it is carried in circulation by binding to haptoglobin. in normal persons, no free hemoglobin is observed in the blood, because most of hemoglobin is in the form of haptoglobin complex. in some diseases that are accompanied by hemolysis, the amount of released hemoglobin is higher than its complementary haptoglobin. as a result, free hemoglobin appears in the blood, which is a toxic compound for these patients. free hemoglobin has been showed to have peroxidase activity and considered a pseudoenzyme. in this research, the effect of methocarbamol on the peroxidase activity of human hemoglobin was studied. our results showed that the drug inhibited the pseudoenzyme by un-competitive inhibition. both k m and v max decreased by increasing the drug concentration. k i and ic 50 values were determined as 6 and 10 mm, respectively. molecular docking results showed that methocarbamol did not attach to heme group directly. a hydrogen bond connected nh 2 of carbamate group of methocarbamol to the carboxyl group of asp126 side chain. two other hydrogen bonds could be also observed between hydroxyl group of the drug and ser102 and ser133 residues of the pseudoenzyme. p-02.02.2-042 dca reduces viability and down regulates mapk protein activations in human malignant mesothelioma cells and pericardium. microarray analyze results performed in mm patients revealed that one of the most prominent changes is upregulation of many genes involved in glycolysis and the krebs cycle. dichloroacetate (dca) is an inhibitor of pyruvate dehydrogenase kinase (pdk) that enhances the oxidative activity of cells by activating pyruvate dehydrogenase (pdh) in mitochondria. dca has shown as a promising anti-neoplastic agent that re-sensitizes cancer cells to apoptosis. the aim of this study is to elucidate the coupling between pdk inhibition and mm cell proliferation and cell cycle. human malignant mesothelioma (spc212) cell line was used as a model for dca treatments. cell viability was measured by mts assay; mapk protein activations and expressions were assessed by western blotting; cell cycle profile was analyzed by flow cytometry. statistical analysis was performed by utilizing one-way anova test. results showed that dca reduced viability of spc212 cells in a concentration and time dependent manner. protein analysis indicated that mapk pathway was down regulated at concentrations greater than 50 mm. moreover, primary cell cycle analysis has indicated arrestment at g2/m phase in 24 hours. our findings corroborate with recent reports where dca treatments resulted in reduction of viability and g0/g1 and g2/ m arrest in other cell lines. abnormalities in mapk signalling play a critical role in the progression of cancer. here, we showed for the first time that dca decreased mapk activation in 24 h. our results suggest that dca is an anti-prolifertive agents for mm cells in vitro. however, it requres extra analysis with other mesothelial cells. future study will focus on investigating relation between mapk and mitochondrial apoptosis. adrenomedullin (adm) is a vasodilator peptide consisting of 52 amino acids. adm is synthesized in many tissues. and is a biologically active peptide that has various effects including vasodilatation, the regulation of vascular endothelial function and adjusting adipogenesis. hypoxia inducible factor 1 alpha (hif 1a) is a subunit of a heterodimeric transcription factor hypoxia inducible factor 1. it is the master transcriptional regulator of cellular and developmental response to hypoxia. the dysregulation and overexpression of hif1a by either hypoxia or genetic alternations have been heavily implicated in cancer biology as well as a number of other pathophysiologies. in our research, the adm and hif 1-a levels in heart, kidney and lung tissues of rats were investigated in control, hypoxia, control+adm and hypoxia+adm groups. rats in hypoxia groups were provided hypoxic environment containing of 10-12% oxygen and 88-90% nitrogen for 1 week. rats in adm groups were injected intraperitoneally in a dose of 1.25 nmol/kg for four days before the collection of the tissues. the control group was oxygenated with normal air. the control and treatment groups were formed from 7-8 animals and adm, hif 1-a levels were measured in taken tissues with immunoassay method. the aim of this study was to investigate the reaction of the organism when exposed to hypoxic conditions and the effect of adm over hif 1-a level. adm levels and hif 1-a in heart tissue were found decreased in hypoxia group, and adm levels increased in hipoxia+adm group. hif 1-a levels decreased in hypoxi+adm group. adm levels in liver tissue were found decreased in hipoxia and control+adm groups than control group. hif 1-a levels were higher in control+adm group. adm has a role in angiogenic process, and our experiment showed that adm reacts earlier than hif 1-a, and affects its synthesis. organism increases its vascularization as a reaction to hypoxic condition, and adm treatment may provide a rapid adjustment. p-02.02.2-044 covalent conjugation and characterization of immunogenic protein of toxoplasma gondii and polyacryclic acid as vaccine candidate r. c ß akir koc ß yildiz technical university, department of bioengineering, istanbul, turkey toxoplasmosis is a major medical and veterinary disease caused by toxoplasma gondii which infect approximately half of the world's population. this infectious disease especially gains importance in pregnant women and immunodeficient individuals. also t. gondii infection has economic importance. however, there are only one attenuated-live t. gondii vaccine for veterinary uses and no vaccine against t. gondii is available for humans. therefore development of an effective vaccine would be extremely valuable for preventing disease in human and veterinary medicine. subunit vaccines are very attractive vaccine candidates but there is low antigenicity problem when they are used alone. polymers themselves don't stimulate immune response while they used with antigenic structure of various infective agents enhance immune response because when proteins are covalently conjugated with hydrophilic polymers, (1) their circulatory-lives and stability (in different ph and temperature values) enhance (2) binding to proteases and clearance by the reticuloendothelial-system decreases. in this study, immunogenic protein of t.gondii and polyacrylic acid with immune stimulant properties was covalently conjugated and conjugation was demonstrated by size-exclusion chromatography (sec) and fluorescence spectroscopy. it is significant to detect time of death in case of a sudden death for medical and legal concerns. there is no known method that can be used for post mortem time detection. based on this deficiency pmi detection in narrow time frame is a big problem. in this study, we aimed to investigate and determine timedependent expressional changes of apoptotic markers by western blot technique. 14 postmortem skeletal muscle were analyzed 12hour periods in first 36-hour after death. 2nd and 3rd 12-hour periods were statistically significant (p < 0.005). keywords: post mortem interval, time of death, apoptosis. hyaluronidases are excessively found in nature and involved in numerous biological functions. hyaluronidases primarily degrade hyaluronic acid (ha) and have significant role in fertilization during acrosomal reactions. therefore, the measurement of hyaluronidase enzyme activity may provide valuable information about acrosomal function and the fertilizing ability of the sperm. the aim of this study was to investigate the semen hyaluronidase enzyme activity changes among four different sheep breeds (akkaraman, suffolk, merino, and kıvırcık). in this research, ten ram testis tissues from each sheep breed, a total of 40, were cut and collected on ice. ovine testicular hyaluronidase of four different sheep breeds was purified from a crude ammonium sulfate-precipitated fraction of an extract of ram testis. the semen hyaluronidase enzyme activity differences between the sheep breeds were examined by spectrophotometrically monitoring the appearance of ha at 232 nm. analysis of variance test was used to examine the possible mean differences among the four different sheep breeds. the observed mean differences in enzyme units for kıvırcık, suffolk, akkaraman, and merino were as follows 891.60, 817.60, 729.40, and 681.60, respectively. the observed mean differences in absorbance values for kıvırcık, suffolk, akkaraman, and merino were as follows 0.4455, 0.4088, 0.3648, and 0.3408, respectively. the results showed that the observed mean differences in enzyme units and absorbance values among the four different sheep breeds were not statistically significant. despite that, in average kıvırcık had higher values for the activity of each sample and yet it had the smallest values for standard deviation. therefore, in order to achieve higher enzyme activity and more homogenous samples kıvırcık breed should be preferred. what is extra to learn from protein drying measurements? hydrations of soluble proteins are crucial for their functionality. therefore elucidating the details of protein hydration is still of interest in the proteins' action mechanisms. this is the motivation of the present study. in order to study protein hydration, changing concentrations of the well-studied serum albumin protein was measured with the spectroscopic techniques like uv-vis and ft-ir spectroscopy. spectral data is analysed and calculations were performed on the data to extract the relevant changes in the protein. experimental parameters' variation in association with the spectral changes implies the involvement of protein structure and hydrogen bonding in the drying process. the protein's reactions may not be merely a feature of the protein structure in the common sense but it could be related directly to the protein hydration states as well. this is understandable since it is already known that enzymatic proteins lose their functionality when they are dried while this drying may or may not involve dramatic structural changes. on the other hand, here it is claimed that the role of water in gaining the functionality that was lost in the dried state is not just about enhanced diffusion processes and the dynamicity but could be related to the functionality of water in the energy transfer processes as well. investigating the cellular effects of the aldoketo reductases akr1b1 and akr1b10 in hct-116 colon cancer cells b. taskoparan, e. g. seza, m. s. ceyhan, s. banerjee middle east technical university, ankara, turkey aldo-keto reductases (akr) are nad(p)h dependent oxidoreductases are best characterized as glucose reducing agents, and have been implicated in diabetic pathophysiology. increased expression of akr has been associated with tumors of lung, breast, prostate, cervix, ovarian and colon. two members of the akr superfamily that have been associated with different cancer types are akr1b1; aldo-keto reductase family 1, member b1, and akr1b10; aldo-keto reductase family 1, member b10. both are 36-kda cytosolic reductases that are similar in both amino acid sequence identity (71%) and tertiary structure with the (a/b) 8 barrel topology. while hct-116, a colorectal cancer cell line, cells expresses akr1b1 robustly, there is no expression of akr1b10. in this study, we have stably knocked down akr1b1 through shrna technology and overexpressed akr1b10 in hct-116 cells. comparisons were made with a known akr inhibitor sorbinil. with the knock down of akr1b1, we have observed reduced cellular proliferation, enhanced apoptosis, delay in cell cycle progression, reduced expression of mitogenic proteins and a decrease in activation of the inflammatory transcription factor nuclear factor kappa b (nf-kappab). interestingly, although akr1b10 overexpression did not affect cell proliferation, apoptosis or cell cycle, some effect was observed with nf-kb signaling. our data indicate that, although closely related, akr1b1 and akr1b10 have very different contributions towards signaling pathways in colorectal cancer. comparison of different nisin quantification methods and optimization of nisin production by lactococcus lactis z. girgin ersoy, g. demir, m. f. cesur, s. tunca gedik gebze technical university, kocaeli, turkey nisin, which is produced by certain strains of lactococcus lactis, is the only bacteriocin approved by world health organization (who) as a food additive. it prevents the growth of foodborne bacteria which cause food spoilage. nisin research and applications necessitates developing an accurate and reproducible method for its quantification. the agar diffusion bioassay is the most widely used method for quantifying nisin, although it has limitations especially diffusion-related difficulties of the active substance. in the present study, "agar diffusion bioassay", "enumeration of colony forming units", "colorimetric assay" and "flow cytometry" methods were compared with each other to determine antibacterial activity of nisin on micrococcus luteus. moreover, this study also covers the results about the effect of different cultural conditions to optimize nisin production by l. lactis. galactose, lactose and their combination in m17 medium (ph 7) boosted nisin production at 25°c, as the addition of 1.5 lg/ml hemin into the fermentation broth. to our knowledge, this is the first study showing the usage of "flow cytometry" method to determine nisin activity of fermentation broth filtrates. p-02.02.2-051 coronaviral nucleocapsid protein is an antiviral target for drug development institute of genomics and bioinformatics, national chung hsing university, taichung, taiwan between 2003 and 2004, the severe acute respiratory syndrome (sars)-cov caused a worldwide epidemic and had a significant economic impact in the countries affected by the outbreak. recently, the middle east respiratory syndrome human coronavirus (mers-cov) was found in patients with severe acute respiratory tract infections in the middle east and south korea. as is true for all coronavirus infections, there are no efficacious therapies currently available against coronaviral diseases, making the development of anti-coronavirus compounds a priority. the cov genome consists of positive-sense, single-stranded rna approximately 30 kb, and it contains several genes encoding several structural and non-structural proteins that are required for progeny virion production with a conserved order. the n proteins exist in the center of the viral particle and represent a helical structure complex. nucleocapsid protein is most abundant structural protein of covs, binds the viral rna genome to form the virion core, leading to the formation of a ribonucleoprotein (rnp) complex or to a long helical nucleocapsid structure, that is important for maintaining the rna in an ordered conformation for replication and transcription. the cov n protein is also involved in the regulation of cellular processes, such as gene transcription, interferon inhibition, actin reorganization, host cell cycle progression, and apoptosis. two strategies to inhibit oligomeric n protein function have been reported. the first strategy is to discover antiviral agents that target the rna-binding site. the second one is to impair normal n protein function by interfering with monomer-oligomer equilibrium. our recent studies suggest that n proteins in infections caused by coronaviruses will be useful antiviral drug targets because they serve many critical functions during the viral life cycle. post-translational modification of vascular endothelial growth factor (vegf) in colon cancer cells s. tunc ßer, e. solel, s. banerjee middle east technical university, ankara, turkey vascular endothelial growth factor a (vegf-a), commonly referred as vegf, is a potent secreted mitogen crucial for tumor initiation and progression. the gene for vegf is translated into a number of splice isoforms that lead to 121, 165, 189 and 206 amino acid proteins, with different receptor-binding and matrixbinding properties. in the present study, we discuss the functional significance of post-translational modification/processing of vegf 165 isoform in hct-116 colon cancer cells. we also focus on the role of calcium in the post-translational modification of vegf 165 . we show that vegf 165 undergoes n-linked glycosylation in hct-116 cells. perturbation of cellular calcium may affect vegf driven malignant phenotypes. p-02.02.2-053 novel methods for modulating the activity of bcl2 family proteins in apoptosis p. rowell, j. miles, a. wilson, t. edwards apoptosis, also known as programmed cell death, is an essential cellular process, but is implicated in several human diseases, including diabetes and cancer, when it is up-or down-regulated respectively. bcl-2 family proteins are major players in the control of intrinsic, or mitochondrial apoptosis; they respond to intracellular stress signals, function through protein-protein interactions and converge on the mitochondrial outer membrane to cause membrane permeabilisation, release of cytotoxic molecules, and initiation of an apoptotic cascade that leads to cellular demise. our work aims to identify molecules able to bind and modulate the activity of several key players in the bcl-2 family, including the pro-survival members bcl-2, bcl xl and mcl1, and the death promoting family member bax. adhirons, novel non-antibody peptide display scaffolds developed at the university of leeds, have been used to construct a phage display library containing over 10 10 clones, and form a key part of the strategy to identify such molecular modulators. adhirons able to selectively bind individual bcl-2 family members have been identified, in vitro assays carried out to test for modulatory activity, and xray crystallography used to elucidate details of how they interact with their target proteins. more recently, studies have been carried out to identify adhirons able to target multiple bcl-2 family members, with the aim of selectively inhibiting defined groups of proteins in cells. this work provides opportunities to differentiate the activities carried out by different bcl-2 family proteins in apoptosis, enabling us to better understand how their dysregulation contributes to human disease. biophysical and evolutionary study of the structural flexibility of adp-dependent sugar kinases from mesophilic and psychrophilic archaea r. zamora 1 , v. castro-fern andez 1 , c. a. ramirez-sarmiento 1 , e. a. komives 2 , v. guixe 1 1 facultad de ciencias, universidad de chile, santiago, chile, 2 department of chemistry and biochemistry, university of california, san diego, united states of america the capability of extremophiles microorganisms to live at low temperatures is mainly attributed to the high structural flexibility of its enzymes. several sequence and structure features have been associated to a high structural flexibility that enables metabolic processes to occur at low temperatures. during evolution, the general mechanism adopted by these enzymes has been to reduce the free energy of the transition state rather than the michaelis constant, k m . increased structural flexibility and decreased affinity for its substrates in psychrophilic enzymes is compensated by an increase in the catalytic rate, k cat . few psychrophilic enzymes have been reported to performance the optimization of their catalytic efficiency (k cat /k m ) by decreasing k m values. we use the adp-dependent kinase sugar family of archaea as a model, to identify particular structure and sequence features of a psychrophilic enzyme that would make this enzyme more flexible than their thermostable homologues. we characterize the bifunctional psychrophilic enzyme phosphofructokinase/glucokinase from methanococcoides burtonii (mbpfk-gk) and the bifunctional mesophilic enzyme phosphofructokinase/glucokinase from methanococcus maripaludis (mmpfk-gk) by spectroscopic, biophysical and computational techniques. the comparison showed that the absence of two ion pairs is primarily responsible for the increased structural flexibility accounted in the psychrophilic model. this increase in structural flexibility is reflected in the exponential increase in the k m values with temperature. additionally, we reconstruct the sequences of all ancestral enzymes between the current enzymes and their last common ancestor, which was used to trace the occurrence of these electrostatic interactions during evolution in the adp-dependent sugar kinase family. our results suggest that electrostatic interactions are a dominant feature in the transition from psychrophilic to thermophilic environments. fondecyt 1150460. elucidating the domain swapping mechanism of the forkhead domain of human foxp1 fox transcription factors control gene transcription during key processes, such as embryogenesis and immunity, and feature a conserved dna-binding domain known as forkhead. while most forkhead domains are monomeric, solved structures of members from the p subfamily (foxp) show that they can oligomerize by domain swapping (ds), a mechanism where protein segments are exchanged between subunits leading to an intertwined dimer. the biological relevance of ds in foxp has been stressed by disease-causing mutations that impair this process. however, for many proteins ds takes days to occur and requires global protein unfolding. thus, the current mechanism impedes a conciliation of the occurrence of ds of foxp1 in a biological context. here, we elucidate the biological feasibility of this process by biophysically characterizing the ds mechanism of the forkhead domain of foxp1 using size exclusion chromatography (sec), circular dichroism, and hydrogen-deuterium exchange mass spectrometry (hdxms). our results show that ds of foxp1 occurs at micromolar protein concentrations, within hours and is energetically favored. remarkably, dimeric foxp1 follows a threestate n 2 « 2i « 2u folding mechanism, where dimer dissociation into a monomeric intermediate (i) precedes protein unfolding, in contrast to other ds proteins. using sec and hdxms, we show that the i state of foxp1 largely resembles the native state, but has a larger hydrodynamic radius and a higher deuterium uptake in regions that maintain the compact monomer, suggesting that the i state is an 'open' conformation en route of ds. finally, we compared the local flexibility of the dimer and monomer of foxp1, showing that only the hinge region connecting ds segments exhibits different deuteration rates. our results show that ds in foxp1 follows an unusual threestate folding mechanism that proceeds through transient structural changes rather than needing protein unfolding as in most ds proteins. (fondecyt 1130510, 11140601). p-02.02.2-057 the sustained release of growth factor proteins following their implantation in tissue engineering j. jang bone tissue engineering has become a promising approach for bone regeneration. however, insufficient vascularization during bone regeneration, particularly with large bone defects, results in poor and unsustainable bone formation due to central necrosis. therefore, vascularization following implantation in vivo is essential to the successful formation of new bone tissue. we evaluated the release profile of vegf from bgs using a novel fluorescence-based retention assay, which revealed that vegf loaded on bgs can be released in a sustained manner without an initial burst (near zero-order cumulative release) with a controlled release rate of 13.6% per week for up to 7 weeks. in contrast, an elisa-based release assay showed vegf to have an early burst-release profile for the first week. however, the biological activity of vegf released from the bgs was preserved over the 7-week release period, which is consistent with the sustainedrelease profile observed in the fluorescence-based retention assay. we developed a novel fluorescence-based retention assay to evaluate the release of vegf from bgs. this fluorescence-based retention assay, which detects the vegf that remains on bgs, reveals that vegf loaded on bgs can be released in a sustained manner, with a minimal initial burst, for up to 7 weeks. these results indicate that the sustained biological activity of the vegf released from bgs over the full 7-week period promotes bone regeneration, and suggest its potential use for bone tissue engineering. irisin is a recently discovered myokine which regulates energy metabolism and is associated overweight. we aimed to evaluate serum irisin levels in the patients with morbid obesity. a total of sixty patients with morbidly obese and thirty healthy control subjects were included in this study. all participants were measured body weight and height, the lipid profile, and plasma glucose, hba1c, insulin and irisin levels. irisin levels were measured by elisa method. serum irisin levels were significantly lower in morbidly obese patients than healthy controls (p < 0.05). there was no statistically significant difference in terms of age or gender. serum irisin was negatively correlated with bmi, insulin levels, and homa-ir (p = 0.006, p = 0.046, p = 0.048, respectively). our study revealed lower irisin levels in morbidly obese patients with respect to control subjects. the lower irisin levels observed in morbidly obese patients might suggest a loss of browning of subcutaneous adipose tissues. p-02.02.2-059 pka inhibition restores adenosine uptake in renal tubular epithelial cells under high d-glucose conditions w. garrido, j. catal an, s. alarc on, r. san mart ın universidad austral de chile, valdivia, chile introduction: diabetic nephropathy (dn) is the leading a cause of end-stage renal failure whose pathogenesis must to be elucidated. the progression of dn has been associated with elevated levels of adenosine. extracellular adenosine availability is regulated by the activity of the equilibrative nucleoside transporters (ents). due to the ents have putative sites of phosphorylation our objective was to evaluate the role of pka and ck2 kinases on ents activity. methods: adenosine uptake (10 lm adenosine, 60s, 22°c) was assayed in hk2 cells preincubated with 5 mm or 25 mm d-glucose for 24 h and exposed to 10 lm 8-br camp, 10 lmh89 or 100lm tbb for 1 h. plasma membrane and intracellular proteins were fractionated by the biotinylation method and ent1 and ent2 contents were quantified by western blot. results: high d-glucose in hk2 cells inhibited the uptake of adenosine. this effect was reversed using a pka inhibitor (h89) through an increased ent2 uptake activity. we noticed this pka inhibitor did not regulate the plasma membrane localization of ent1 or ent2 under normal d-glucose (5 mm) or high d-glucose conditions (25 mm). also, we saw that tbb (ck2 inhibitor) decreased the activity of ent1 and ent2 under normal glucose conditions, decreasing the localization of ent1 at cell surface, while the membrane localization of ent2 decreases under the effect of tbb and high d-glucose conditions. conclusions: pka inhibition reversed the effect of high d-glucose, increasing the uptake of adenosine mediated by ent2. this could be a new target for the restoration of adenosine levels in dn. relation between serum lipo (a), plasma fibrinogen, red cell distribution width and mean platelet volume in healthy adult men the aim of this study was to investigate the relationship between the serum lipo (a) and plasma fibrinogen, red cell distribution width (rdw) and mean platelet volume (mpv) in healthy adult men. for this purpose, 37 healthy adult men have normal physical examination and laboratory findings and not use any drug were included to the study. serum lipo (a) levels and hematologic parameters (fibrinogen, rdw-sd and mpv) were measured by autoanalyzer and commercial kits. the mean of the age of the persons was 27.2; body mass index was 24.2; serum lipo (a) level was 0.21 and plasma fibrinogen level was 1.61. there was significant positive correlation between the serum lipo (a) and plasma fibrinogen levels (r = 0.246; p = 0.025), significant positive correlation between the serum lipo (a) and rdw-sd (r = 0.267; p = 0.004) and significant negative correlation between lipo (a) and mpv (r= à0.205; p = 0.027). the plasma fibrinogen and the serum lipo (a) levels have been known as the risk factors for cad (coronary artery disease) increase together in healthy adult men. similar findings have been reported in cad patients. it has reported that elevated rdw is associated with intracoronary thrombotic burden and may be associated with the severity and instability of acute myocardial infarction. in addition, mpv is predictor of severe atherosclerosis and may be used for the prediction and identification of cardiac risks in cad patients. our findings show that elevated rdw and decreased mpv may predict to increased risk of cad in the future, in healthy adult men. follicular fluid is rich in peptides, which significantly influence the growing oocyte. due to existence of a link between kisspeptin (metastin) cells and gonadal steroids kisspeptin might manipulate the gonadotropin axis and folliculogenesis. in this context, the study was planned to investigate for the first time that the follicular fluid (ff) and serum concentration of kisspeptin in high and poor responders undergoing in vitro fertilization (ivf)/intracytoplasmic sperm injection (icsi). biological samples were collected from twenty infertile women with polycystic ovary syndrome (pcos) and 20 poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone (gnrh) antagonist protocol for ivf/icsi treatment. kisspeptin concentrations were measured in serum and follicular fluid by using elisa, whereas fsh and lh levels were detected by routine laboratory methods. it was found that kisspeptin levels were significantly lower in serum and follicular fluid of infertile women with pcos. kisspeptin levels were correlated with fsh and lh level in infertile women with pcos. it can be concluded that low level of kisspeptin might inhibit gnrh release that might cause to the inhibition of fsh and lh release and might disrupt folliculogenesis. decline in serum and ff levels of kisspeptin might be possible cause of anovulation and subfertility in pcos subjects. cryptococcus albidus d24 is a newly identified yeast isolates from petroleum area in _ izmir as a lipase producer. the molecular weight of the enzyme is 36.31 kda as found. optimum temperature was 40°c and half-life times were 180, 27, 9 and 0.59 min at 30, 40, 50 and 60°c, respectively. optimum ph value was 8.0. however, it shows significant ph stability at ph values 4.0 and lower. the existence of acetone in the solution as a solvent enhanced lipase activity. cryptococcus albidus d24 lipase was able to catalyze the esterification reaction between fructose and palmitic acid to produce fructose palmitate using acetone as the solvent. due to its stability in organic solvents, we propose that in order to increase the yield of fructose palmitate, we could immobilize d24 lipase. therefore, the effect of immobilization on kinetic parameters of d24 lipase was investigated. different immobilization materials and methods were used to find efficient support materials for d24 lipase immobilization. additionally, fructose palmitate production processes will be optimized with immobilized lipase. introduction: the diagnosis of osteoarthritis (oa) is based on clinical symptoms and radiographic findings. it is known that the pathologic changes at the molecular level in the joint cartilage tissue start before symptoms appear in oa. c1q tumor necrosis factor-related protein 1 (ctrp-1), c1q tumor necrosis factor-related protein 3 (ctrp-3) and kallistatin are related to many different cellular processes including bone and cartilage tissue metabolism. the aim of this study was to investigate any probable association between the serum ctrp-1, ctrp-3 and kallistatin levels and diagnosis and radiologic staging of knee oa patients. materials and methods: this study included 60 patients with knee oa and 30 healthy individuals for control purposes. the patient group was divided into four stages radiologically. ctrp-1, ctrp-3 and kallistatin levels were measured in serum samples of patient and control groups with elisa method, and the differences between the groups were analyzed with statistical methods. results: the levels of serum ctrp-1 in the patient group were significantly higher than in the control group (p = 0.001), serum ctrp-3 and kallistatin levels were not statistically different (in order of p = 0.251, p = 0.160). in the patient group, there was not a significant difference between serum ctrp-1, ctrp-3 and kallistatin levels and radiologic stages (respectively p = 0.811, p = 0.715, p = 0.202). there was a significant positive correlation between the radiologic stage and patient's age, body mass index, western ontario and mcmaster universities arthritis index and visual analogue scale values (respectively p = 0.001, r = 0.510; p = 0.010, r = 0.331; p = 0.001, r = 0.683; p = 0.001, r = 775). discussion and conclusion: serum ctrp-1 levels were detected significantly increased in patients with knee oa, but there was no significant difference in ctrp-3 and kallistatin levels. there was not a significant association between the radiologic stage and levels of ctrp-1, ctrp-3 and kallistatin. enzymes in microorganisms, especially termophilic bacteria are more attractive in biotechnology and molecular biology due to the higher catalytic activity. turkey is rich in geothermal resources and it is important to determine unknown microbial content of geothermal sources. in this study, water and sludge samples were taken from ayder, kizilcahamam and gonen hotsprings. firstly, ph, temperature, salt concentration, gram reaction, mobility, endospore formation, oxidase and catalase tests were carried out as conventional characterization. molecular characterizations of isolates were achieved by fames, rep-pcr and 16s rrna sequencing. finally, test isolates were evaluated according to enzyme production capability by petri dish. as result of conventional tests, isolates were determined as gram positive, mobile-rod shaped, aerobic, oxidase, catalase and endospore positive. the growth range for ph and temperature of the isolates were determined as 5-9 and 50-65°c. in consequence of the salt test, the test isolates were grown at 2-10% nacl. 19 of thermophilic isolates were selected by rep-pcr and according to 16s rrna sequencing analysis test isolates were belonging to bacillus, geobacillus, anoxybacillus and brevibacillus genus at a range of 98-99%. enzyme tests showed that, some of the isolates were able to produce protease (f17, f31, f76, f6, f99 and f19), amylase (f41, f76, f42, f98 and f10), cellulase (f17, f41, f31, f6, f99 and f19), xylanase (f17, f31, f76, f99 and f19) and lipase (f17, f31, f6, f99 and f19). it can be concluded that, geothermal regions are rich in bacillus and related genera. fame analysis was particularly insufficient for diagnosis of thermophilic microorganisms, but rep-pcr was successful in separation of organisms at species and even subspecies levels. most of our bacterial isolates have industrially important enzyme production capacities. it is a pioneer result to use bacteria for industrial applications which need higher temperatures. p-02.02.2-065 warburg effect was investigated by studying various metabolic molecules and assays in mammalian cell lines z. i. kanbagli, b. kiratli, h. cimen yeditepe university, istanbul, turkey majority of the different cancer cells switch their metabolism from oxidative phosphorylation pathway to glycolytic pathway; in order to meet excessive energy requirement, which is also called warburg effect. acetylation is one of the most crucial post-translational modifications playing key roles in metabolic reprograming. in this study, the relationship between acetylation dependent changes in energy metabolism and apoptotic pathways were investigated in pnt1a, du145, hela, hep3b, hek293t, shsy5y. immunoblotting experiments were applied by using antibodies against acetylated-lysine to examine the changes in overall acetylome. candidate proteins displaying elevated acetylation was identified with mass spectrometry based-proteomic analysis. glucose transporter 4 (glut4) was used to detect insulin-stimulated glucose transport, total oxphos rodent antibody cocktail to identify variations in complexes which are responsible for most of the atp production. caspases (casp-3, -9) to unreveal different activation levels of apoptotic pathway among the cell lines. mitochondrial membrane potential was measured by using rho-damine123 by employing confocal microscopy. the expression level of respiratory chain complex iv subunit mtco1 and casp-3 was higher in hek293t compared to other cell lines. casp-9 was upregulated in cancer cell lines, mostly in hep3b. glut-4 levels were downregulated in cancer cell lines in contrast to healthy cell lines. findings imply that these proteins might have significant roles leading to variable metabolic and apoptotic activity of each cell line during energy production. due to the results, mtco1 might be important in adaptation of different cell lines to regulate the overall respiratory chain complex activity. reduction in glut4 level demonstrates insulin desensitization in cancer cell lines, which might lead to metabolic defects in these cells. besides, since p53 has a repressive effect on glut4, it also can lead us to study about p53 levels. the effect of inhibition of pi3k/akt/mtor signaling pathway on receptor tyrosine kinase expression in breast cancer cells g. tunali, a. l. dogan department of basic oncology, hacettepe university cancer institute, ankara, turkey the increased expression and activation of receptor tyrosine kinases (rtk) (egfr, her2, her3) play important roles in breast cancer pathogenesis. her2/her3 interaction is the most potent heterodimer and it causes oncogenic pi3k/akt activation. inhibitors of pi3k/akt pathway (akt inhibitor and pi3k/mtor dual inhibitors) lead to increase in rtk levels and activities while blocking signaling pathway. in this study, the time dependent effect of dual pi3k/akt inhibitor pi-103 on receptor tyrosine kinase expressions' in breast cancer cells was investigated. two breast cancer cell lines, mda-mb-468 cells (which has egfr overexpression and pten deficiency) and skbr-3 cells (which has her2 overexpression) were evaluated for the effect of dual inhibitor. these cells were treated with dual pi3k/akt inhibitor pi-103 for different time periods (1-24 h) . egfr, her2 her3 total rtks expression and pi3k/akt pathway inhibition (p-akt and p-p70s6k expression) were evaluated by western blot. in mda-mb-468 cells, there were significant decrease in p-akt and p-p70s6k proteins' expression during the first 3 h. this inhibition was followed by reactivation of the signaling pathway after 6 h. in skbr-3 cells, p-akt and p-p70s6k proteins' expression were significantly decreased during the first 6 h. the pi3k/ akt signaling pathway in these cells were reactivated after 12 h. basal expression of egfr and her3 in mda-mb-468 cells and basal expression of her2 and her3 in skbr-3 cells were found to be very high. transient inhibition of akt and mtor protein kinase activation in tumor cells followed by reactivation of signaling pathway did not result in a time-dependent difference on egfr, her2 and her3 expression levels. these results suggest that dual pi3k/mtor inhibiton by pi-103 may trigger receptor tyrosine kinase reactivation due to the signal distruption without affecting total protein expression level. site-specific bioorthogonal reactions are one of the significant tools for discovering different aspects of biological systems in live cells. the reactions should be highly stable and rapid in physiological conditions. various chemical tools can be used in bioorthogonal reactions to monitor biological systems, therapeutics, microscopy and diagnostic applications in live cells. synthetic covalent chemistry in the study of biological systems has been used to label biomolecules selectively in their native environment. for example, small synthetic fluorophores can be added to biomolecules without disturbing other molecular biological pathways. aldehydes or ketone-based functional handles can be attached onto protein at specific sites via chemoenzymatic reactions. labeling of carboxy terminus of a-tubulin has been successfully studied in our previous studies by replacement of wild type tyrosine with unnatural amino acid 3-formyltyrosine in the presence of tubulin tyrosine ligase enzyme (ttl)-as its role can suggest whether certain cancer cells might grow more aggressively than others. in this work, we highlight the synthesis and spectroscopic properties of azacoumarin chemical probes to study tubulin-tubulin tyrosine ligase (ttl) system in live cells. significant increase in fluorescence quantum yield or a red shift on absorption and emission maxima is observed when the conjugated product is formed. bioorthogonal fluorescent labeling is such a favorable reaction to perform rapid kinetics, localization and high site-specificity in cell environment. newly synthesized azacoumarin fluorophores should therefore not only be useful for studying ttlbased biological systems, but also would enable broad range of high-yielding and fast diagnostics for future biolabeling applications in biochemistry, cell biology and beyond. binase is an extracellular ribonuclease from bacillus pumilus which shows antiviral and antitumor activities in cell cultures. however, the action of binase on intracellular functions and processes has not yet been identified. here, for the first time we report the whole transcriptome analysis of binase-treated human lung adenocarcinoma epithelial a549 cells. a plasmid-based reverse genetics system and colorimetric cell viability assay were used to identify the binase internalization and binase cytotoxicity towards a549 cell line, respectively. for the whole cell transcriptome analysis a549 cells were treated with 100 lg/ml of binase for 24 h followed by mrna extraction and library preparation. sequencing was performed on solid 5500xl wildfire next-generation sequencer. we found that binase internalized into a549 cells after 2 h of incubation. the binase at 100 lg/ml was absolutely non-cytotoxic towards a549 cell line and was active in the cell culture medium during 48 h incubation. the analysis of rna-seq data showed that among 13 thousands of protein coding transcripts 791 transcripts were up and down regulated by binase, among them 79 transcripts were induced and repressed. binase repressed the production of s100a16 and tnxb which act cancer biomarkers, scn8a and drd4 which play a crucial role in cancer metastasis and responsible for pediatric tumors, respectively. the induction of transmembrane protein transcript abcb11 by binase can help binase to internalize into the cell as abc transporters are often account for transporting drugs across the cellular membrane. binase induced the production of nlrp3, rasgrp1and alpk2 transcripts which can activate apoptosis, cytokine or t cell activation in cancer cells. thus, binase exerts different effects in cancer cells. the rnaseq data obtained will help to understand the mechanism of binase anticancer action. .72) is a specific group of phosphatases capable of hydrolyzing myo-inositol 1,2,3,4,5,6-hexakisphosphate (phytate) with the formation of less phosphorylated inositol derivatives (from mono-to pentaphosphate). three major types of phytases are recognized on the basis of the first phosphate group hydrolyzed by the enzymes: 3-phytase, 4/6-phytase, and 5-phytase. due to the stereospecific way of phosphate release from the phytate molecule by the action of phytases, these enzymes by themselves and their composition may serve as a potential alternative for production of myo-inositol phosphate isomers with therapeutic properties. chemical synthesis of these compounds is inefficient and costly. pantoea sp. strain 3.5.1 showing high phytase activity was isolated from the forest soil sample of the republic of tatarstan, russia. in this study the main objective was the cloning and expression of pantoea sp. 3.5.1 phytase gene in e. coli. first, we amplified the phytase gene (agpp) from the genomic dna of the bacteria using specific primers phmh_dir and phmh_rev. size of phytase gene corresponded to 1729 base pairs. during the optimization of amplification conditions it was found that the optimum temperature for primer annealing was 66°c. this temperature increases the specificity and efficiency of annealing. then the pcr-product of agpp gene was cloned into the pbad myc/ his vector first. on the next step we carried out subcloning of the agpp into a pet28a expression vector. multicopy plasmid pet28a/agpp contained the sequence of the phytase gene of pantoea sp. 3.5.1 under t7 promoter. the corresponding recombinant protein was expressed in e. coli as a fusion with a 6 his-tag and was detected by western blotting. recombinant phytase was purified via affine chromotography on the ni-nta column and displayed high phosphomonoesterase and phytase activities. bag-1 (bcl-2 associated athanogene) is a multifunctional protein that interacts with diverse array of cellular targets and modulates a wide range of cellular processes, including proliferation, cell survival, transcription, apoptosis, metastasis and motility. in human cells bag-1 exists as three major isoforms (bag-1s, bag-1m and bag-1l) derived by alternative translation initiation from a single mrna, which allows interactions with various molecular targets such as hsp70/hsc70 molecular chaperones, components of the ubiquitylation/proteasome machinery, bcl-2, raf-1 kinase, nuclear hormone receptors and dna. our work aims to investigate how altered bag-1 expression levels affect cell survival in mda-mb-231 (er, pr and her2/neu negative) breast cancer cell lines. we first cloned bag-1l gene to a cloning vector, later we transfected mda-mb-231 cells for overexpression of bag-1. we also used bag-1 sirna to silence bag-1 gene. western blot analysis was applied to demonstrate relative expression levels of bag-1, its interacting partners and certain proteins which are important for apoptosis pathway. we performed xtt cell viability assay for bag-1 overexpressed cells to checkbag-1's impact on cell survival, and observed enhanced survival rates on cells compared to that of the untreated cells with bag-1 overexpression. in addition, our study revealed that once bag-1 forms a complex with c-raf/b-raf/hsp70/akt/bcl-2, modulation of cell survival was observed. we believe that once the exact localization and involved molecular mechanisms of bag-1 and its isoforms are found, the role of each bag-1 isoform in cell survival can be understood better. this can further provide routes to study tumor development. the aim of this study is testing the recombinant glp1 encapsulation into a biocompatible material. we also tested if it can be a therapeutic candidate drug for type 2 diabetes. the incretin hormones, which are also named as endogenic peptide hormones have become a more attractive therapy for type 2 diabetes because of different physiological effects. in circulation, glp1 is cleaved by ddp4 in a very short time. if glp1 can be protected from cleaving, the effective time of glp1 would be increased and by this way it can replace the therapy of insulin. chemical synthesis methods of peptides are limited because of low efficiency and high cost. the production of peptides by recombinant e. coli is an alternative way because of effective production, simplicity and low cost. however, the major disadvantage derived from the recombinant e. coli is the frequent formation of inclusion body. for that reason, extra methods are needed for obtaining soluble recombinant peptides. glutathione s-transferase (gst) tag is commonly used as affinity and solubility tag to improve the solubility of recombinant peptides. in this study, we cloned and heterologously produced glp1 using the gst fusion system in e. coli. affinity purification of recombinant protein was achieved by using glutathione immobilized columns. characterization of the gst-tagged glp1 was performed by sds-page. the purity of fusion protein was found to be 65%, as confirmed by glp1 elisa kit. then, the fusion protein was encapsulated in a chitosan coated polygalacturonic acid. the different ph stability and in vitro release tests also in different phs was studied. morganella morganii is an opportunistic pathogen capable of causing a wide range of clinical infections. it is known that microbial metalloproteases play an important role in the development of bacterial infections. thus, investigation of m. morganii metalloproteases has a particular interest. bacteria were grown in lb medium at 37°c. as a bioinformatics tool blast was used. for molecular biological experiments, thermo scientific kits and sibenzyme enzymes were used. pbad/myc-his plasmid was used as an expression vector. bacterial transformation was carried out by heat shock method. bacterial cells were disrupted by sonication. gene expression products were analyzed by western blotting. to analyze the actinolytic activity of bacterial extracts sds-page electrophoresis was used. the putative metalloprotease gene (an cp004345.1) has been found in the genome of annotated strain of m. morganii kt. its amino acid sequence has partial homology (37%) with actin specific metalloproteases grimelysin from s. grimesii and protealysin from s. proteamaculans. using specific primers the gene with 99% homology was identified in the genome of clinical isolate of m. morganii 4. rt-pcr analysis showed that this gene had the maximum expression at 48 h of growth. in addition, the cellular extract of m. morganii 4 had small actinolytic activity. cloning of the gene into e. coli dh5a cells led to the synthesis of the 35 kda protein. it is known that the highest expression of serratia proteases is observed at 48 h of growth, and the molecular weight of the mature proteins is 32 kda. it was shown that metalloprotease gene of m. morganii 4 expressed at the same time of growth. moreover, the recombinant e. coli cells synthesized protein with the similar weight (35 kda) which perhaps is a mature form of the metalloprotease from m. morganii. as a result, in the genome of m. morganii 4 the metalloprotease with similar properties to grimelysin and protealysin proteases was identified. the preliminary characterization of p-ii like protein glnk from lactobacillus brevis z. iskhakova 1 , d. zhuravleva 1 , a. laykov 1 , k. forchhammer 2 , a. kayumov 1 1 kazan federal university, kazan, russia, 2 eberhard-karls university tuebingen, tuebingen, germany the p-ii proteins in bacteria, archaea and plants regulate the activity of a variety of proteins in response to specific metabolic signals which affect their structural state and interaction ability. among various bacteria belonging to lactobacillus only some species have genes encoding pii protein in the genome. here we report the preliminary characterization of pii-like protein lbrglnk from lactobacillus brevis. while the amino acid sequence alignment revealed only 50-70% homology of lbrglnk with other well studied pii proteins, lbrglnk also has the atp binding motive gdgk. trimeric structure of lbrglnk was confirmed in vitro by size exclusion chromatography, suggesting possible similarities of lbrglnk properties with pii proteins. the isothermal titration calorimetry revealed a preferential binding of adp (kd = 50 lm) over atp (kd = 357 lm) suggesting that they compete for binding to lbrglnk. neither 2-oxoglutaric acid nor other nucleotides were interacting with lbrglnk in itc measurements. the mutation gly91ala in the atp binding motive completely abolished the interaction with both adp and atp. the pull down of lbrglnk with l. brevis cell extract allowed identification of chaperonin grol, transketolase and glnr-like transcriptional regulator from merr family as most probable partner proteins for interactions with lbrglnk. this work was supported by the russian foundation for basic research (project no. 15-04-02583a background: hemophilia is a bleeding disorder due to the deficiency in coagulation factors viii (hemophilia a) or ix (hemophilia b). hemophilia patients are essentially treated with intravenous replacement of the missing or dysfunctional factors fviii or fix by recombinant proteins. these therapies often induce the generation of acquired antibodies, and thus, novel approaches are needed. most recent hemophilia strategies target the tissue factor pathway inhibitor (tfpi), which is the major inhibitor of the coagulation cascade, particularly of the extrinsic tenase complex. anti-tfpi agents have been empirically developed such as aptamers, peptides, monoclonal antibodies. we have followed a structure-based strategy, to design a mutated fxa that would show more affinity for tfpi, and thus trap this inhibitor. tfpi exists as two isoforms are folded as multi-kunitz domains related by linkers. the second kunitz type domain of tfpi (tfpi-k2) is known to bind the catalytic site of fxa. methods: the molecular complex of tfpi-k2-fxa was modeled and submitted to molecular dynamics (md), allowing the identification of low-spots interaction. modified fxa with theoretically stronger interaction with tfpi-k2 were predicted using md. the mutants and wild type proteins were expressed in hek cells, and their processing status was checked. they were tested by western blotting, by chromogenic activity using a specific substrate of fxa, by thrombin generation assay in fviii depleted plasma. finally, their binding to a tfpi-k2 peptide array was compared. results: the mutants showed better efficiency to restore thrombin generation in plasmas from hemophiliacs and displayed stronger binding to tfpi-k2 than the wild type fxa. conclusions: the proof of concept of the synergistic approaches of md and mutagenesis was obtained and an efficient tfpi trap was designed. the mutated fxa is a candidate for a new hemophilia therapy. organophosphorus acid anhydride (op) nerve agents are potent inhibitors which disrupt the mechanisms of neural transmission. organophosphorus acid anhydrolase (opaa; e.c.3.1.8.2) is a class of enzyme that potentially acts on phosphorus anhydride bonds, reported intracellularly in diverse organisms, albeit notably the enzyme belongs to alteromonas species are more extensively studied. whereas mass-transfer problem is a major issue in native whole cell biocatalysis, new anchor system derived from the n-terminal domain of ice-nucleation protein from pseudomonas syringe inav (inav-n) was used for the first time to display opaa onto the cell surface. tracing of the recombinant protein and its activity assay showed a successful presentation of opaa and its significant ability for biodegradation of organophosphorus compounds. further studies on bacterial fractions confirmed that opaa is remarkably located on the outer membrane. the specific activity of recombinant bacteria to degrade diisopropylfluorophosphate (dfp) was measured at 260 u/mg of cell wet weight, which almost all was observed in the outer membrane fraction. recombinant cells could also degrade chlorpyrifos (cp) compound in 195.6 u/mg activity. it can be concluded that inav-n anchor is efficient for targeting opaa on the cell surface and can effectively eliminate the masstransfer problem in native whole cell bioconversion system. proper spatial and temporal organization of proteins involved in cell signal transduction is crucial for the specific and efficient information transfer. scaffold proteins coordinate the action of signaling molecules by their physical binding and organization in multiprotein complex assemblies. multiple protein binding is often mediated through intrinsically disordered regions of the scaffold, where the interaction epitopes are defined by linear peptide motifs. using a hub scaffolding protein axin as a paradigm, we have employed peptide microarray technique to identify the binding epitopes for axin interaction partners at high resolution. this enabled us to design axin-derived peptides corresponding to the respective binding epitopes that compete for the interaction in vitro. by transfection of chemically stabilized competitive peptides directly into the cells, we have shown the effect of specific interaction blocking on axin-mediated signaling in vivo. our data demonstrate a proof of concept for a rational design of inhibitors of protein-protein interactions that allow specific intervention with single function of the targeted protein (i.e. recruitment to the axin complex). contrary to the inhibitors that completely disrupt the protein function (e.g. inhibition of a kinase catalytic site), this approach provides a tool for investigating specific action within the axin complex, while the other cellular functions of the targeted protein remain preserved. the results of this research have been acquired within cei-tec 2020 (lq1601) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. de novo design of an artificial biocatalyst using immunoglobulin template became rather routine procedure due to the achievements of molecular biology and crystallography. recently the 'reactibody' approach was developed based on the chemical selection of catalytic repertoires from immunoglobulin library followed by expression of these biocatalysts in eukaryotic system. in this study we structurally characterize the a5 antibody, its kappa and lambda variants, in order to understand the difference on the active site between a5 and a17 which although there are two antibodies sharing very high homology and sequence identity their active residues are located in a different region of the antibody. the structures of the a17 antibody kappa and lambda variants have been already determined, there was no structural information though about the a5 antibody. the structural analysis revealed dramatically different angle in position of nucleophilic residue tyr33 and area of solvent accessible surface. the structural difference of active center reflects on kinetics of the a5 organophosphate modification. both variants of antibody bind with organophosphate throw induce-fit mechanism, but rate of the step of induce fitting is different (k obs are 35 s à1 and 16 s à1 for a5kappa and a5lambda respectively). this observation may hint at novel means of regulation of velocity and specificity of artificial biocatalysts. this study was supported by grant #rfmefi61614x0009. translation elongation factor 1ba (eef1ba) is a component of a heavy form of translation elongation factor 1 (eef1h). it functions as a catalyst of gdp/gtp exchange in translation elongation factor 1a (eef1a) restoring its active conformation appropriate for aminoacylated trna binding. eef1ba forms a tight complex with translation elongation factor 1bc (eef1bc) via the n-terminal domain, while its c-terminal domain executes the catalytic activity. eef1bc has been shown to enhance the attributed to the c-terminal domain catalytic activity of eef1ba. this suggests that the eef1ba n-terminal domain may influence the guanine nucleotide exchange process. to test this hypothesis we prepared a set of n-terminal truncated forms of human eef1ba and checked their activity in the guanine nucleotide exchange assay on both isoforms of eef1a, eef1a1 and eef1a2. we showed that recombinant eef1ba is a non-globular monomeric protein in solution with an elongated shape by analytical ultracentrifugation approach. the truncation of the dispensable for the catalytic activity n-terminal domain of eef1ba resulted in significant acceleration of the rate of guanine nucleotide exchange in eef1a2 comparing to full-length eef1ba. similar effect on the catalytic activity of eef1ba was observed after its interaction with eef1bc. in contrast, the effect of full-length eef1ba and its truncated forms on the rate of guanine nucleotide exchange in eef1a1 was similar but relatively modest compared to eef1a2. this can be explained by higher rate of spontaneous gdp dissociation from eef1a1 comparing to eef1a2 and lower affinity of eef1a1 to eef1ba. thus, we propose that the n-terminal domain of eef1ba via flexible linker region may interfere with eef1a binding to the cterminal catalytic domain that results in a decrease of the overall rate of guanine nucleotide exchange reaction. the formation of a tight complex between eef1bc and eef1ba n-terminal domains abolishes this inhibitory effect. p-02.02.2-079 assessment of quantitative proteomics results in large-scale data-independent with fragmentation spectra reproducibility measure reduces variation and allows to use lowintensity signals organisms with reduced genomes that lack the vast majority of transcriptional or translational regulation systems tend to adapt to changing environment with a variety of subtle changes in protein abundances. as soon as relative changes for most proteins fall below 50%, the power of traditional label-free proteomic analysis rapidly becomes insufficient for robust profiling of hundreds of samples. intoduction of frament-by-fragment and sample-by-sample signal quality assesment in mrm and dia experiments helps to increase accuracy of methods and at the same time reintroduce cases which could have been excluded during bulk quality assessment due to lower signal-to-noise ratio for several fragments. 240 samples of mycoplasma gallisepticum were acquired in data-independent manner on sciex tripletof 5600+ mass-spectrometer (swath acquisition) during the year. the samples were produced from mycoplasma gallisepticum culture cultivated at different temperatures. the signals for each fragment were extracted with vendor-supplied software with the theoretical fragment ions for each peptides instead of spectral library. the results were used for relative protein quantitation in two manners the first conventional method included direct use of peptides with top 3 most intense signals. the second included selection of peptides and ions for quantification for each pair of samples based on the reproducibility of fragmentation patterns after computing the areas of chromatographic peaks for each ion. spectral angle was used as a distance measure for fragmentation patterns for clustering. further, a base set of detected ions was selected for each peptide and a subset for comparison of each pair of runs. the first method resulted in quantitation of 354 proteins across all samples with variation across lc-ms replicates was 21% on average, and the second approach led to quantitation of 515 proteins in total, 390 of them across 75% of samples, all with the variation about 11% on average. p-02.02.2-080 interaction of plasminogen fragments k 1-3 and k 5 with fibrin fragment dd t. yatsenko, v. rybachuk, l. kapustianenko, s. kharchenko, o. yusova, t. grinenko palladin institute of biochemistry of nas of ukraine, kyiv, ukraine introduction: plasminogen interaction with specific binding sites in c-terminal d-domains of fibrin molecule initiates the activation process of proenzyme and subsequent fibrin clot lysis. the sites are exposed under fibrin polymerization. plasminogen kringle domains ensure the proenzyme interactions with fibrin clot. in this study, we investigated the binding of human plasminogen kringle fragments k 1-3 and k 5 with human fibrin fragment dd and their effect on glu-plasminogen interaction with dd. results: kringle-containing fragments k 1-3 and k 5 reduce plasminogen activation by tissue-type activator on fibrin fragment dd. the level of glu-plasminogen binding to dd is decreased by 50-60% in the presence of k 1-3 and k 5. fragments k 1-3 and k 5 have high affinity to fibrin fragment dd (dissociation constant is 0.02 lm for k 1-3 and 0.054 lm for k 5). analysis of k 1-3 and k 5 binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with c-c-chains of fragment dd. k 1-3 interacts with complex of fragment dd-immobilized k 5 as well as k 5 with complex of fragment dd-immobilized k 1-3. the plasminogen fragments do not displace each other from binding sites located in fibrin fragment dd, but can compete for the interaction. analysis of k 1-3 and k 5 binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with aand c-chains of fragment dd. conclusions: widely known specific plasminogen-binding site located in aa148-160 region of fibrin molecule is not a single binding sequence of fibrin peripheral domains or plasminogenbinding site is not linear and contains amino acid residues from other polypeptide chains of fibrin d-domains. fibrin fragment dd contains different binding sites for plasminogen kringle fragments k 1-3 and k 5, which can be located close to each other. possible plasminogen kringle-binding sites are located in aand c-chains. p-02.02.2-081 implementation of budded baculovirus particles for characterization of ligand binding to g protein-coupled receptors a. allikalt, a. rinken university of tartu, tartu, estonia g protein-coupled receptors (gpcrs) constitute the largest class of membrane receptors involved in regulation of signal transduction into the cell in response to various extracellular stimuli. for that reason, gpcrs have become important targets for variety of drugs. as these receptors are present in native tissues at very low concentrations, efficient recombinant expression systems are needed to produce sufficient amounts of protein. we have shown that budded baculovirus particles, which display gpcrs on their surfaces can be used as a source of receptors for the investigation of ligand-receptor interactions. this expression system can be used for radioligand binding assay as well as for fluorescence anisotropy-based assay (fa). we have validated the system with budded baculovirus particles produced in spodoptera frugiperda (sf9) cells expressing human dopamine d 1 receptors using [ 3 h]sch-23390 and bodipy-fl-skf-83566 as reporter ligands for corresponding assays. this system has many advantages, for example good signal to noise ratio, homogeneity of the receptor, high expression levels and long-term stability of the receptor preparation. fa method allowed real-time monitoring of reporter ligand binding in the absence and presence of different dopaminergic ligands, giving information about their kinetic properties. association, as well as dissociation of the bodipy-fl-skf-83566 itself were rapid with an apparent half-life of t 1/2 = 38.5 ae 0.3 s for association (2 nm) and t 1/2 = 73.4 ae 3.8 s for dissociation. we determined the pharmacological profiles of different dopaminergic ligands in displacement binding assays with membranes of sf9 cells or budded baculovirus particles. the data were in good agreement for both membrane preparations tested in radioligand binding as well as in fa assay. obtained results indicate that budded baculovirus particles can be proposed as a source of gpcrs for performing fluorescence anisotropy as well as radioligand binding assays. gastrointestinal (gi) cancer includes a variety of cancer types affecting the structures and functions of the gi system, encompassing the gi tract and the accessory organs of digestion, from the esophagus, stomach, biliary system and pancreas to the intestine, rectum and anus. despite the significant advances however, much remains to be learned in the spectrum of gi cancer. several investigators have shown that both gas6 and its receptors, axl, sky, and mer are expressed in various types of cancers. however, the expression level of gas6 in gi cancer remains unclear. the aim of the study was to determine and compare plasma gas6 levels in gi cancer patients. 15 female and 27 male patients were included in the study (n = 42): 21 colorectal, 8 gastric, 4 pancreatic, 3 liver, 2 ampullary, 2 gall bladder and 2 esophageal. from all gi cancer patients, 2 ml venous blood was collected in citrate tubes before surgery. blood samples were centrifuged at 3000 g for 10 min, and plasma samples were carefully removed and stored in à80°c prior to use. the level of plasma gas6 was measured using a commercial developmental elisa kit (r&d systems, minneapolis, mn) which is validated by our laboratory. plasma gas6 levels in cancer patients were determined as follows: 1.84 ae 1.1 ng/ml in colorectal; 1.16 ae 1.1 ng/ml in gastric; 1.63 ae 0.61 ng/ml in pancreatic; 3.91 ae 0.89 ng/ml in liver; 2.15 ae 0.26 ng/ml in ampullary; 1.34 ae 0.46 ng/ml in gall bladder and 2.32 ae 1.7 ng/ml in esophageal cancer. preliminary findings indicate that there is a relation between gi cancers and plasma gas6 levels. taken together, these results suggest that gas6 may be a candidate biomarker for diagnostic use in gi cancer. inhibition of gas6 would be an attractive therapeutic target for slow down the progression of gi cancer. monday 5 september 12:30-14:30 computational biology p-03.03.2-001 computational approaches as an assay for blactam hydrolysis in class a b-lactamases c. tooke university of bristol, bristol, united kingdom b-lactam hydrolysing enzymes, in particular carbapenem-hydrolysing enzymes, are an increasing clinical threat. herein we show that molecular dynamics (md) and combined quantum mechanics/molecular mechanics (qm/mm) approaches are a predictive tool of carbepenemase activity in class a b-lactamases. b-lactam drugs are the most prescribed class of antibiotics worldwide, especially in the treatment of gram-negative pathogens such as klebsiella pneumoniae and escherichia coli. these organisms produce b-lactamases, enzymes which hydrolyse the b-lactam ring, a key resistance mechanism. class a b-lactamases have the ability to hydrolyse carbapenems, termed 'last resort' antibiotics. in particular, the kpc (klebsiella pneumoniae carbapenemase) family are the most clinically important, and recently identified natural kpc variants show increased hydrolytic activity against ceftazidime, a third generation cephalosporin. here we use computational simulations of b-lactam hydrolysis by b-lactamases. in particular, molecular dynamics (md) combined with qm/mm approaches have been used to model the deacylation of the carbapenem meropenem across 8 class a b-lactamases. this method has been extended to model cephalosporin hydrolysis across class a b-lactamases, including kpc variants. these approaches calculated the free energy barriers and correctly distinguished carbapenemases from carbapenem-inhibited enzymes. preliminary results suggest this protocol is also a predictive tool for ceftazidime hydrolysis. further, md simulations of 5 kpc variants (single and double amino acid changes) were analysed to identify structural changes in the active site, highlighting that variants differ in the size of the active site opening, corresponding with experimentally derived kcat values. these computational assays provide a predictive tool of b-lactam hydrolysis and has potential to provide insights into important mechanistic differences both across class a b-lactamases and within the same families. p-03.03.2-002 computational design of a novel polyglutamic dendrimer-based platform as an anticancer therapeutic approach poly (glutamic acid) (pg)-dendrimer as potential nanocarriers for cancer therapies, to specifically deliver tumor associated antigens (taa)mannosamine and melanato target cells and to modulate cancer antigen intracellular trafficking within the cytoplasm to promote an efficient and selective antitumor immunotherapeutic effect. the theoretical structures were obtained using x-plor software. the molecular dynamics simulation of pg-g4-dendrimer and taas was performed using desmond. the electronic properties of the structures were determined by semi-empirical methods using mopac. docking studies of taa to pg-g4-dendrimer to mannose receptor (mr1) were performed using hex 8.0.0 software. taa lumo atoms were conjugated to homo atoms of pg-g4 dendrimer using maestro software. results showed that pg-g4-dendrimer displays 64 carboxylic end groups available for covalent interaction with taas. the homo molecular orbitals of the dendrimer was located on the a-carbon of the carboxylic acid groups from backbone chain and it preferentially interacts with lumo molecular orbitals of amine group from taas. no differences in the gap energy of homo/lumo of all pg-g4-conjugates. taas bind preferentially to a-carbon of cooh of backbone chain instead of cooh from side chain. docking results showed that majority of taa conjugated pg-g4-dendrimer binds to the core of the mr1 receptor. increasing of the number of mannosamine conjugated to pg-g4-dendrimer more close and stable is the conjugated to the receptor. this system shows promising results as a novel functionalized pg-dendrimers for cancer therapy. theileria parva is one of the the economically important protozoan of the theileria genus belong to apicomplexa phylum which include plasmodium spp. and toxoplasma gondii, causative agents of malaria and toxoplasmosis respectively. this parasite is the disease agent of tick-borne east coast fever (ecf) ranks first among the tick-borne diseases of cattle in sub-saharan africa. the disease caused by the parasite affects a large proportion of domestic and wild animals and leads serious economic losses in the world. major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. thus, it is important to develop an efficient and affordable antitheilerial agent. for this aim, 1-deoxy-d-xylulose-5 phosphate reductoisomerase (dxr) which subjected to identify novel drug aganist malaria and toxoplasmosis, of theileria parva was selected as potential target for improving novel inhibitors aganist ecf. a computational molecular modeling approach was conducted to determine the 3d structure of tpdxr by phyre2. energy minimisation and root mean square deviation (rmsd) was performed by 3drefine and superpose servers. to ensure the quality of modelling, stereochemistry, energy profile and residue environment of modelled structure were checked by different servers and possible ligand binding pockets were identified by metapocket 2.0 server a reliable 3d model for dxr from t. parva was modeled by using 3au9 as a template. the ca rmsd and the backbone rmsd deviations for the model and the template crystal structure were found to be 0.85 and 0.86 a, respectively. the ramachandran plot for the predicted model by rampage reveals that model shows an acceptable stereochemistry. top three considered possible binding pockets have been identified. these results have important implications for future screens aimed at finding new and safe molecular entities active against tpdxr through docking studies. p-03.03.2-004 molecular binding profile of protoberberine alkoloids on amyloid precursor proteincleaving enzyme 1 (bace1) as a drug candidate for alzheimer's diseases g. yalcin 1 , i. yildiz 2 1 biotechnology institute, ankara university, ankara, turkey, 2 department of pharmaceutical chemistry, faculty of pharmacy, ankara university, ankara, turkey alzheimer's disease (ad) is the most prevalent neurodegenerative disorder that leads to dementia and nowadays over 46 million people live with dementia worldwide. because of the prevalence and economic burden of the disease, drug development studies have picked up speed and scientists especially focused on natural products. ad is basically characterized with tau hyperphosphorylation and accumulation of amyloid b (ab) proteins. ab proteins are generated from sequential cleavages of amyloid precursor protein (app) by b and c secretases, and b-site app cleaving enzyme 1 (bace1) is a b secretase essential for ab production. the alkaloids represent a very extensive group of secondary metabolites, with diverse structures, distribution in nature and important pharmacological activities. protoberberine alkaloids, which belongs to isoquinoline alkaloid class, are widely arranged in many species of the berberidaceae, annonaceae, fumariaceae, papaveraceae, and other plant families. recent searches showed that some of the protoberberine alkaloids such as berberine, palmatine, jatrorrhizine, columbamine, magnoflorine prevents the progress of neurodegenerative disorder. however, the mechanisms of them are not absolutely clear. therefore, we have aimed to elucidate the binding and affect mechanism of these alkaloids on the bace1 open and closed forms in here. for this purpose, molecular docking studies were applied for these natural products to the both forms of bace1 by using autodock vina and it was subjected to explicit solvent simulations by amber molecular dynamic package. our preliminary studies indicate that gly34, thr72, gln73, phe108, tyr198, lys224, thr232, arg235, thr329 residues of binding pocket have affiliations with all of the mentioned alkaloids and the binding of them generates alterations on closed form of bace1. the complexity of animal milk needs to apply numerous approaches and methods for its investigations. an understanding of the processes occurring in the milk can be used, for example, for quality control of the products. fat and protein are main components of milk which have a significant influence at its colloid properties, such as dynamic surface tension (dst). the application of regression-correlation analysis to milk data enables to develop a reliable quantitative model. the aim of our investigation was to perform the regression analysis to establish the relationship between above-mentioned parameters. for this purpose, we used a statistical software packages r version 3.1.2. dst was determined by bpa -1p tensiometer. milk fat (f) and protein (p) contents were measured by analyzer bentley 150. this work was supported by the russian scientific foundation (grant 14-16-00046). obtained formulas characterized the degree of influence of fat and protein contents of a milk sample for each of the dst parameters (r 0 , r 1 , r 2 , r 3 , k 0 , k 1 ): r 0 = 61.1538 + 0.8908 * p à 1.2953 * f r 1 = 63.3297 + 0.5658 * p à 1.4132 * f r 2 = 55.4274 + 0.4585 * p à 1.0143 * f r 3 = 45.6265 + 0.34634 * p + 0.05197 * f k 0 = 6.0986 + 0.1543 * p + 0.1351 * f k 1 = 10.7832 à 0.1470 * p à 1.0302 * f these formulas show that the maximum total effect of fat and protein contents influences at r 0 and r 1 . a significant coefficient (> 1) before the fat is observed in the formula, which describes the value of the tilt of final part of the tensiogram (k 1 ). the resulting regression equations have fundamental importance. with their help it is possible to calculate the dst parameters without their experimental determination, positioning fat and protein contents data. obtained dst parameters promote more complete characterization of the properties of the milk that may be used for dairy products. p-03.03.2-006 molecular studies of scorpion toxin and its mutants interactions with voltage-gated potassium channels the voltage-gated potassium kv1.3 channel is mostly expressed in neurons and immune cells. its blockage has a high therapeutic potential, for example, specific inhibitor shk toxin is undergoing clinical trials on psoriasis. goal of the current study was an interface analysis in complexes of hybrid channel kcsa-kv1.3 with peptide blockers agitoxin and its mutant forms. 3d structure was generated by homology modeling method using complex of mutated kcsa channel with charybdotoxin (pdb-code 2a9h) as a template and equilibrated by molecular dynamic simulation in gromacs software. analysis of hydrophobic and stacking interactions, hydrogen and ionic bonds of the toxin and potassium channels was performed for representative frames with optimal toxin orientations using program platinum and apbs software package. we performed contacts energy characteristics estimation to predict key toxin residues for binding process and possible mutation sites for changing selectivity against kv1.x channels. the results of investigation are in good agreement with the experimental values of binding constants, obtained by competitive binding assays. results of the conducted investigation may find an application in fundamental science and drug design. the research was supported by the russian science foundation grant no. 14-14-00239. simulations were performed using the supercomputing center of lomonosov moscow state university. p-03.03.2-007 homology modeling and molecular docking study of the paraoxonase-1 and its polymorphic variants q/r 192 and m/l 55 for non-statin lipid lowering drugs paraxonase-1 (pon1) enzyme is an hdl associated ester hydrolase exhibiting paraoxonase, arylesterase and lactonase activity, and reduces the formation of atherosclerosis blocking the ldl oxidation and reducing levels of oxidized lipids. in this study, molecular docking approach and molecular dynamics simulation were applied for finding the affinity of non-statin lipid-lowering drugs to pon1 and its polymorphic structures pon1 q/r 192 and m/l 55. fibrates (bezafibrate, ciprofibrate, clofibrate, fenofibrate, gemfibrozil), phytosterols (beta-sitosterol, brassicasterol, campesterol, stigmasterol) and other lipid lowering drugs (ezetimibe, niacin, orlistat, probucol, and sibutramine) was obtained from pubchem database. x-ray crystallographic structure of human pon1 and its polymorphic variants pon1 q/r 192 and m/l 55 was generated via 'modeller', homology modelling software, from human-rabbit hybrid x-ray crystal structure of pon1 (pdb code: 3sre). 10 ns molecular dynamic simulation analysis was performed using gromacs 4.5.5. affinity of lipid lowering drugs to pon1 and its polymorphic variants was predicted by molecular docking approach using autodock 4.2 suite. unlike other lipid lowering drugs that they have negative δg values for affinity, probucol, orlistat and betasterol was calculated by positive δg values (18.7, 12.3 and 1.4 kcal/mol). these values suggest that they may have no affinity to pon1 q/r 192 polymorphic structure. in all drug groups, brassicasterol and stigmasterol to pon1-m/l 55 and sibutramine to pon1 q/r 192 were calculated as the highest affinity. in generally, phytosterols predicted by high affinity to pon1 and m/l 55 polymorphic structures in comparison to other lipid lowering drugs. our study demonstrated that phytosterols predicted as high affinity compounds on pon1 structures may reduce the activity of antioxidant pon1 enzyme. this study need to be supported by in vitro and in vivo detailed studies. prolactin and its cognate receptor, prolactin receptor (prlr), are involved in over 300 distinct functions in mammalians. the mammalian prlr gene consists of 10-13 exons and several 5 0 and 3 0 regulatory sequences. in this study, gaps and annotation errors in the rat prlr gene were corrected by comparing the genomes of mammals and rodents and new putative exons were identified. the rat prlr gene sequences from two different sources (rnor_6.0, nc_005101.4 and rn_celera, ac_000070.1) were used and primary analysis showed that both sequences contain several gaps (varying from 0.35 to 4 kbp), corresponding to about 5.6% (10-11 kbp) of the gene. using the rat known prlr mrna exon sequences, it was found that the rnor_6.0 prlr gene has two exon-10 (one is about 2 kbp long and the other immediately after this). comparisons of mammalian and rodent prlr gene structures showed that the 2 kbp stretch is an assembly artifact. by comparing both gene sequences (and also other available rodent prlr genes), the gaps in the rat prlr gene were reduced from 5.6% to 3.8% (from 11 kbp to 7 kbp). functional annotation of the gene revealed that r. norvegicus prlr gene could have two more additional exons, exon-12 and -13, similar to mus musculus prlr gene. in mammals, prlr mrnas contain non-protein coding exons in the 5 0 utr (exon-1 and -2). in rats, there are 5 exon-1 variants, resulting from alternative promotor usages. studies on the rat and mouse prlr genes revealed that both rodents share 4 common non-protein coding exon-1 variants. in conclusion, it is found that the rnor_6.0 version of the prlr gene has the highest number of unidentified base pairs (corresponding to 5.6% of the gene) and the second exon-10 is the assembly artifact. the rat prlr genes in both databases have several gaps and our corrected version is the best available and characterized form of the rat prlr gene. in silico affinities of some statins to paraoxonase-1 enzyme the structure of the statins (atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin) was obtained from pub chem database, and x-ray crystal structure of pon1 (pdb id:3sre) from protein data bank. modeller software was used for homology modeling of pon1 and its polymorphic variants that's called as pon1 q/ r 192 and m/l 55. amino acid sequence of human serum pon1 (uniprot: p27169) was used as the modeller template. all molecular dynamics simulations were carried out with gro-macs 4.5.5 software. molecular docking calculations on each of the polymorphic structure of the pon1 was performed with auto dock4.2. suite. for each substrate, y71 residue showed open conformation in pon1 and m/l 55 polymorphic structures while q/r 192 polymorphic structure showed closed conformation. in comparison between structures of pon1 variants, in most cases statins had lower affinity to q/r 192 polymorphic structure than to the other variant. in this study, among statins, atorvastatin showed lowest but simvastatin highest affinities to pon1. by considering that the high affinity drugs can have reducing effect of pon1 activities, it may be more appropriate to use the low affinity statins in hyperlipidemia treatment. however, these findings need to be supported with in vivo and in vitro studies. p-03.03.2-010 self-assembly of lipidoids for sirna uptake and release mechanisms studied by molecular dynamics simulations o. acar 1 , d. alpay 2 , a. r. atilgan 1 , c. atilgan 1 1 sabanci university, istanbul, turkey, 2 northwestern university, evanston, united states small interference rna (sirna) has the ability to bind a specific mrna which provides silencing of selected genes. nanocarriers made out of self-assembled lipidoids encapsulate sirna and deliver them into target cells effectively. in this study, a library of lipidoid structures is constructed and studied by molecular dynamics (md) simulations in different solvents, including sodium acetate, to ferret out their self-assembling mechanisms. the effect of the protonation state of the head group of lipidoids on the final shape of the self-assembled carrier is also studied. we further examine the role of the size of hydrocarbon tails in the packing. we study the final topology and the geometry of the self-assembled lipidoids both in the presence and in the absence of sirna. we find that stable clusters form with as few as 20 chains. for lipidoids having neutral head groups, clusters are in the form of dense bundles, while those with charged head groups form spherical capsids which are depleted of the salt on the inside and having a salt rich phase on the outside. in the self-assembled structure, lipidoids are arranged so as to expose the nitrogen and oxygen atoms to the solvent. while partial capsids with these properties also form at lower lipidoid numbers, 200 chains are necessary to form a fully closed capsid. in the presence of the sirna, the capsid assembles around the nucleotide. the free energy to remove the sirna from the assembly is calculated via repeated steered md calculations utilizing jarzynski's equality relating it to the irreversible work along and ensemble of trajectories. we therefore determine an optimal tail length for the most stable nanostructure, paving the way for designing nanocarriers with high efficacy. milk is one of the most valuable products for humans and attracts a lot of interest of researchers in various fields such as biochemistry, biology, food science and technology. the methods of milk study are quite varied. we chose the combination of the ultrasonic and dynamic surface tension (dst) measurements with the possible correlations among the obtained parameters. the aim of this work is to study the correlation between the parameters of milk, such as a content of fat, protein, lactose, minerals, dry milk solids and dst parameters. for this purpose we used milk analyzer 'klever-1m' and tensiometer 'bpa-1p'. three groups of animals were formed from clinically healthy holstein cows at the age of 4-5 years according to the fat content in the milk sample. group i -5 cows (milk fat content 4.01 ae 0.41%), group ii -6 cows (milk fat content 3.32 ae 0.14%), group iii -11 cows (milk fat content 2.73 ae 0.20%). this work was supported by the russian scientific foundation (grant 14-16-00046). the biochemical parameters of the milk samples of all three groups are in the range of the 'normal' values for healthy holstein cows: protein content varies from 3.0% to 4.2%, lactose and mineral content varies from 4.6% to 0.7%, respectively. the dst parameters (r1, r2 and k0) for the group i have strong positive correlations with the fat content in the studied milk samples. at the same time for the groups ii and iii the fat content in the milk indicates only medium positive and weak positive correlations with the r1, r2 and k0. obtained absolute values of the dst parameters of the milk samples showed some differences between all three groups. thus, the dst parameters are changing in direct proportion to fat content in the milk sample that can be explain by the primarily role of the milk lipids in the formation of the water/fat surfaces (such as fat globules, lipid-protein particles, etc.). p-03.03.2-012 exploration of allosteric paths in caspase molecules using energy dissipation e. n. bingol, o. sercinoglu, p. ozbek sarica marmara university, istanbul, turkey caspases are highly regulated aspartate-specific cysteine proteases that have major roles in programmed cell death; apoptosis. effector caspases are at the terminal step of the pathway, hence they are considered as death switches. with the discovery of the presence of allosteric sites, these molecules attracted the attention of the pharmaceutical studies and became drug targets. as a result of the binding of small molecules to the dimeric interface, active site loops are shifted to an unfavorable position. this is associated with a network between distal allosteric sites and the active site loop. an energy dissipation model was applied in order to analyze this matter in further detail. perturbation of specific residues enable us to determine a possible signaling network in proteins using external energy as an input, while focusing on the dispersion of this energy between residues throughout the structure. molecular dynamics simulations were performed with and without energy perturbation using namd software with charmm27 force field. energy perturbation was applied by increasing the velocity of a chosen residue at the desired time step of the initial md simulation. energy change of each residue was calculated upon the application of perturbation. as a result, residue response times, corresponding to the time of the response of a residue after the perturbation of another chosen residue, are obtained. combining reponse time data with a residue interaction network, it is possible to construct a final network that shows the communication started by perturbation within the molecule. it is shown that perturbation of allosteric sites result in the disruption of the catalytic sites given in literature. our findings support this and also gives a little detail of the possible communication between distal allosteric site and the active site loops. this finding enables the usage of this methodology for similar structures where the exact allosteric mechanism is yet not known. p-03.03.2-013 effect of complex mammalian membrane models with multiple membrane components on ras protein nanoclustering a. farcas 1,2 , l. buimaga-iarinca 2 , c. floare 2 , l. janosi 2 1 faculty of physics, babes-bolyai university, cluj-napoca, romania, 2 national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania ras proteins are essential for the cellular signal transduction that regulates cell proliferation and differentiation and act as binary switches between gdp and gtp forms. a wide range of human tumors are associated with defective ras protein signaling. the production of permanently activated ras proteins is correlated with mutations in ras genes. experiments and computer simulations have shown that membrane-bound ras proteins form nonoverlapping dynamic nanosized subdomains (nanoclusters) in activation state-/isoform-dependent manner. we performed coarse-grained molecular dynamics simulations to investigate the effect of complex mammalian membrane models on formation and evolution of ras nanoclusters. a fundamental part of the plasma membrane is the phospholipids bilayer, which contains phosphatidyl-choline (pc), phosphatidylethanolamine (pe), phosphatidyl-serine (ps), sphingomyelin (sm) and cholesterol (chol). the nature of lipid-lipid and protein-lipid interactions was studied in binary (pc:chol) and quinary mixtures (pc:pe:ps:sm:chol). because the polar lipids are not uniformly distributed between the two leaflets of the membrane, the construction of the plasma membrane with five-component lipid mixtures took into account the asymmetry between the outer and inner mono-layers. the phospholipids chain saturation (combined with the presence of cholesterol) constitute the dominant factor in phase separation and was, therefore, modeled in different lipid tail combinations for various headgroups. using microsecond timescale simulations of membraneembedded ras proteins, we have shown that the nanoclusters are spontaneously forming dynamic structures whose behavioral characteristics is modulated not only by the ras isoform, but also by the complexity of the membrane model. furthermore, we showed that variations in the plasma membrane lipid composition have important implications in the localization of ras protein nanoclusters. optogenetics comprises biological methods to achieve gain or loss of function of well-defined events in specific cells of living tissue by means of targetable control tools that respond to light and deliver the effector function. microbial rhodopsins (mrs) have been established as powerful light-sensitive tools for optogenetics. acting as ion pumps or channels, mrs are used to induce cell (de)polarization to control neuronal activity in a wide range of living organisms. mrs are membrane proteins found in a large clade of organisms, including eukaryotes, bacteria, and archaea. they share a common architecture of 7 transmembrane a-helices and a covalently linked retinal, which is employed to absorb photons for energy conversion or the initiation of cellular signaling. major efforts are put into screening of natural and generating of synthetic mrs with desirable properties for optogenetics, e.g. ion selectivity. however, experimental study of mrs is difficult and resource consuming owing to, among other factors, low expression levels and protein stability. thus, there is a need in developing of computational tools for identification of mrs with desirable properties. we used non-redundant atomic structures of mrs taken from protein data bank to develop a set of numerical descriptors that reflects functional properties of mrs. then, we calculated the descriptors for non-redundant sequences of mrs with known function taken from the uniprot database, resulting in the feature matrix. we applied the support vector machine and the 5fold cross-validation procedure, using the feature matrix as the training set. as a result, we obtained the classifier that discriminates mrs in terms of the ion selectivity, e.g. na + , h + , or cl à pumps, with high precision. finally, we used the derived classifier on a test set of proteins and identified mrs for the further experiment in vivo. rational design of peptides with required stability and functional activity properties becomes a real instrument for the new generation drug development. the reca bacterial protein (and human rad51 homolog) is considered to be the central catalyst of homologous recombination, a mechanism essential for the accurate repair of double-strand dna breaks. dna repair via homologous recombination requires reca nucleoprotein filaments assembly. using seqopt (http://mml.spbstu.ru/seqopt/), a novel method for a-helix sequence optimization we present the successful design of peptide sequences capable to maintain a very stable a-helix structure and to inhibit reca activity. novel a-helical 18 amino acids peptide is constructed based on reca-dna complex structure. we observed in vitro inhibition of reca atp hydrolysis, dna strand exchange reaction and reca filament formation. also, we observed lower e. coli resistance to uv and sos-response suppression in vivo. computational identification of promiscous enzyme activity for the morita-baylis-hillman reaction k. ozturk, s. sayin, n. celebi olcum yeditepe university, istanbul, turkey enzyme promiscuity attracts considerable attention in terms of enzyme evolution, protein engineering and biocatalysis. especially, development of highly efficient novel biocatalysts starting from promiscuous enzymes that have the catalytic machinery to perform desired chemistry is an intense area of research in recent years. in this work, we computationally explored the catalytic promiscuity of natural enzymes for the synthesis of morita-baylis-hillman (mbh) adducts, which display antitumoral activity against human cervical cancer cells, by mining structural protein databases using quantum mechanically optimized theoretical active site models (theozymes). catalytic interactions in the active sites of selected hit proteins with potential mbh activity were evaluated in solvated dynamic environment using molecular dynamics simulations. computational screening of the protein data bank for the quantum mechanically determined optimal arrangement of catalytic functional groups for the target mbh reaction successfully identified an enzyme with experimentally determined promiscuous mbh activity. ras proteins mediate a wide variety of signal transduction pathways that regulate cell growth, proliferation and differentiation. these proteins are small gtpases that act as binary switches between gdp-bound 'off' and gtp-bound 'on' states. oncogenic point mutations of ras are associated with~15% of all cancers and up to 90% in specific tumors and many developmental disorders. both experimental and in silico results showed that the membrane-bound ras proteins form non-overlapping dynamic nanosized subdomains called nanoclusters in an activation state-/ isoform-dependent manner. we performed coarse-grained molecular dynamics simulations in order to investigate the formation and evolution of ras nanoclusters in mammalian model membranes. ras proteins were inserted into the cytoplasmic side of the plasma membrane model (di-c16:0-phosphatidyl-choline: di-18:2-phosphatidyl-choline: cholesterol 5:3:2) where they formed highly dynamic nanoclusters, both in size and in composition. furhermore, we found that the presence of ras protein nanoclusters has a significant impact on the model membrane behavior. properties such as phase behavior, diffusion coefficient, surface tension and lipid tails order parameter are also influenced by the temperature variation of the model membrane. we have investigated dynamics in three different crystal forms of ubiquitin, as well as ubiquitin in solution, with particular emphasis on (i) conformational exchange between b turn type i and ii in the region 51-54 and (ii) rocking dynamics where protein molecules as a whole undergo subtle reorientational motion within the confines of the crystal lattice. experimentally, both motional processes have been probed using relaxation dispersion techniques, including recently developed near-rotary-resonance dipolar relaxation dispersion experiments. thereby it has been determined that rocking motion in one of the crystal forms (pdb id 3n30) occurs on the time scale of tens of microseconds, whereas the conformational exchange has characteristic time constant of ca. 100 ls. using molecular dynamics simulations, we have shown that the similarity of motional time scales is not accidental: bi↔bii exchange and rocking motion appear to be coupled. we have investigated the mechanisms of this coupling and predicted a number of point mutations that are expected to abrogate (or enhance) rocking. the crystals of ubiquitin containing these mutations have been modeled in silico. we have also investigated the interactions (in particular, crystal contacts) that control the balance between bi and bii conformations in different crystal forms. finally, we have used md simulations as a basis for chemical shift calculations and illustrated how relaxation dispersion effects can emerge as a function of bi↔bii exchange in conjunction with the rocking motion. the md simulation study was supported by rsf grant 15-14-20038. serine/threonine kinases are attractive targets in targeted cancer therapy due to their overexpression in several forms of cancer. flavonoids are highly bioactive plant secondary metabolites that are important in human health due to their antioxidant property. quercetin, a natural flavonoid derivative, has been shown to regulate several signal transduction pathways and is in phase i clinical trial as an anticancer drug. this study explored the inhibitory potential of quercetin and its derivatives using in silico methods like molecular docking and molecular dynamics simulations. quercetin and its derivatives were observed to bind to several serine/threonine kinase family proteins with binding energy significantly better than other known inhibitors and commercially available drugs. this study thus sheds light on the atomic level interactions that define the polypharmacological nature of quercetin and its ability to interfere with a number of cancer pathways. introduction: noninvasive prenatal diagnosis (nipd) of the fetal rhd status by rhd genotyping of the maternal plasma was initially applied in alloimmunized pregnant women. fetal rhesus d status detection for management of rhd incompatibility using circulating cell-free fetal dna from maternal plasma or serum is now accepted by many obstetricians in europe as reliable and useful. the aim of the study was to detect fetal rhd specific antibodies in maternal plasma using a nanopolimer based electrochemical biosensor. materials and methods: a three-electrode system, consisting of a gold electrode, an ag/agcl reference electrode and a pt counter electrode, was accommodated in a 10-ml electrochemical cell. anilin and jelatin were used for immobilization of rhd antibody. the polimerization was occured at 319 nm uv light. antibodies of rhd antigen were detected using differential pulse method at between 0.4 and 0.6 v potentials by observing the differentiations in the current values. results: the rhd status of the fetus was predicted in 20 rhdnegative pregnant women (8-36th week of pregnancy). rhd antibody were detected in maternal bood using biosensor in 15 of the fetuses. the results were confirmed with real-time pcr. the fetuses found rhd (+) for exon 5 and 7 of rhd gene by multiplex real-time pcr. discussion and conclusion: biosensors based studies might be useful, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses. we found that more advantages in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, specific, economic, practical and less time-consuming. fetal rhd detection at low concentrations and in the early week of pregnancy is possible with this method. p-03.03.2-022 investigation of phylogeography of cricotopus sylvestris (diptera: chironomidae) using mitochondrial and nuclear molecular markers the family chironomidae is one of the most widely distributed insect families of diptera, and this family is distributed in all continents and all habitats from the tropics to the arctic in lakes, streams and puddles. in this study, we aimed to determine the dispersal of c. sylvestris using molecular phylogenetic markers not only in turkey but in the world and to reveal from where this species may have entered to turkey in the past. c. sylvestris larvae were collected from 8 lakes across turkey. after total genomic dna extraction from body of larvae, fragments of two mitochondrial genes, cytochrome c oxidase subunit i (coi) and cytochrome b (cytb), and one nuclear gene, carbomyl phosphate synthase domain (cps) of cad, were amplified and sequenced. in addition, several sequences of these three genes of c. sylvestris from different countries of different continents such as south corea, japan, canada, denmark, and sweden were obtained from genbank. all sequences were aligned using mega 6 and bioedit version 7.0.9.0 and were used for phylogenetic analyses. neighbour-joining (nj) tree was created in mega 6 and paup 4.0b10 with 1000 bootstrap replicates. maximum likelihood (ml) analysis was performed in raxmlgui 1.0 using gtrgamma model with 1000 bootstrap replicates. beast v1.8.0 was used for bayesian analysis. our phylogenetic analyses indicated that the japanese, south corean and american c. sylvestris were different from european and turkish members. turkish members of c. sylvestris were closely related to european ones according to our bayesian, nj and ml analyses. in turkish members, c. sylvestris collected from hazar and c ß ıldır lake was more ancient than those from marmara, sapanca, c ß ıldır, aygır, beys ßehir, e girdir and sıhke lakes. in conclusion, our results clearly suggest that several transoceanic dispersal events among the continents may have occurred and that the entrance of turkish c. sylvestris to turkey may have been from southeast and northeast of the country. metagenomics is providing great help to explore world of unculturable microorganisms in the natural samples to enhance our knowledge about microbial diversity. here, we have performed metagenomic analysis of fresh water lake bacterial community using 454 pyrosequencing techniques. we have observed a wide array of bacteria from phylum proteobacteria and family enterobacteriaceae as well as very few viruses from podoviridae, siphoviridae and unclassified phages. we have conducted a metagenomics analysis with the primary focus on the examination of the community of bacteria in a fresh water lake ecosystem. roche gs flx software gave us total 156 253 reads (with an average read length of 795.274 bp). there were 15 226 contigs having > 100 bp sequence length whereas 10 481 contigs with > 500 bp sequence length. for further analysis we have taken contigs with > 500 bp only. further, we have analyzed the microbial community composition using blastn/blastx against nt/nr databases with e-value cutoff of 10 à5 . ≥70% of total contigs were mapped to the reference with ≥60% contig match coverage. the community analysis revealed that domain bacteria is predominantly present (99.8%) in the water sample, followed by eukaryota (0.02%), viruses (0.08%) and other sequences (0.02%). most abundant phyla was proteobacteria (99.8%) and the most dominant family was enterobacteriaceae (89%) followed by xanthomonadaceae (10%), vibrionaceae (0.4%), pasteurellaceae (0.1%), shewanellaceae (0.07%). we performed functional analysis of all 5974 contigs using rapid annotation using subsystems technology (rast) 4 which detected 15 319 coding sequences and 197 rnas in 619 subsystems. among the classified cds from rast showed major cds hits for enzymes involved in the subsystems amino acids and derivatives and the carbohydrate metabolism. the great diversity of microorganisms present in the lake may reflect the human activity in the area. maldi-tof mass spectrometry is a ubiquitous and widespread tool for protein identification. once the protein sequence is unavailable, unambiguous identification cannot be performed, and predictability is limited by the presence of sequenced homologous proteins. we present a statistical approach to predict a number of structural, localization and functional properties of unknown proteins by direct analysis of mass distribution shapes of their post-cleavage fragments obtained from maldi-tof mass spectrometry data. secondary structure of proteins is best predicted by their specific cleavage at the inertial hydropathy group amino acid residues (filmv), with thermolysin (afilmv) being the closest commercially available reagent, leading to distinguishing between proteins with presumably ahelixes or b-sheets with 90% accuracy. cellular localization of proteins is best predicted by their specific cleavage at the external hydropathy group amino acid residues (dehknqr), exemplified by gluc(phosphate)+lysc(dek) cleavage. protein location in the cell membrane and its localization character (monotopic/ transmembrane, single-pass/multi-pass transmembrane) are predictable with~75% accuracy by this single cleavage, with optimal combination of 3-4 cleavages improving the accuracy tõ 80%. functional prediction of proteins is the best among membrane-associated proteins with characteristic structural conformations. we attribute the differences in the mass distribution shapes to the characteristic clustering of amino acids residues with respective hydropathy properties that are involved in the formation of 3d structural conformations of proteins. the suggested approach allows for a non-parametric statistical prediction of uncharacterized proteins from their maldi-tof mass spectrometry data without knowledge or reconstruction of their primary sequence. potential applications include proteomic studies of organisms with unavailable genomic sequences and highly variable proteins analysis. the cancer genome atlas (tcga) represents a comprehensive database of genomic, transcriptomic and epigenetic alterations across more than 20 tumor types. earlier we developed cross-hub tool aimed at multi-way analysis of tcga data in the context of gene expression regulation. in the present work, the software was updated with new features that are described below. crosshub is a python-based application. one of the features of crosshub is the combining tcga rna-seq co-expression analysis to encode chip-seq data in order to reveal most possible transcription factor (tf) targets and coupling mirna-mrna co-expression to several algorithms of mirna target prediction in order to enhance its efficacy. the key feature of the updated crosshub version is the analysis of the associations between expression ratio of tf to its targets and tf mutation status. this allows identification of tfs that are functionally (in)activated with driver mutations in a particular cancer type. the second novel feature of crosshub is the analysis of associations between 'tf-to-targets' expression ratio and tumor characteristics (tnm classification, pathological stage), patient follow-up, etc. in turn, this analysis may result in the identification of 'tf-targets' functional relations that are important for disease progression, tumor invasion, response to chemotherapy. thus, crosshub was supplemented with new features that can be useful for comprehensive tcga data analysis. the updated version of crosshub is freely available at http://sourceforge.net/ projects/crosshub/. this work was financially supported by the russian foundation for basic research (grants 15-04-08731, 16-16-00114 and 15-34-70055) and ras presidium program 'molecular and cellular biology'. p-03.03.2-026 mutations leading to increased rnase production and streptomycin resistance in bacillus pumilus bacillus pumilus strain 3-19 which was derived from soil-isolated b. pumilus 7p using chemical mutagenesis is characterized by resistance to streptomycin (str, up to 500 lg/ml) and ability to produce extracellular enzymes in quantities almost 10-fold higher than the parent strain. these features make the 3-19 strain suitable for industrial production of rnase (binase) which is known for its antitumour and antiviral properties and can be used as an rna-degrading tool in molecular biology. the whole genomes of both mutant and wild-type b. pumilus strains were sequenced recently. to reveal the exact genetic features responsible for rnase overproduction and str resistance we have fulfilled comparative genome analysis of b. pumilus 7p and 3-19 strains. facilities of rast server, edgar platform and additional bioinformatics tools (plasmid finder, prophinder, bl2seq) were used. it is found that both b. pumilus genomes under study contain an intact prophage, while only wild-type strain bears a 6 kb cryptic plasmid. none of the systems is inactivated in mutant strain according to the results of metabolic reconstruction. 3.4% of total cdss differ in 3-19 strain in comparison to 7p one, 36% of them are hypothetical. the altered genes are involved in membrane transport, cell wall composition, chemotaxis, spore formation, carbohydrate metabolism, dna metabolism, translation and transcription regulation. mutation (k56n) leading to str resistance is identified in 30s ribosomal protein s12p. regulatory and coding regions of binase gene have no modifications. candidate genes which can account for binase overproduction are selected. mutation k56n is classical in str resistance and leads to enhancement of decoding accuracy while decreasing elongation speed. rnase overproduction is brought about by non-specialized mechanism since other hydrolases are also overproduced in mutant strain. genes encoding extracellular serine protease, sporulation initiation phosphotransferase f, gnat-family acetyltransferase and cell wall modifying enzymes are reported previously to increase production of degradative enzymes. the action of encoded by them proteins lead to increase of stability and release of secreted proteins to environment and to derepression of their transcription from negative regulators bacillus pumilus strain 7p has been identified on its ability to produce ribonuclease and different extracellular proteases. in order to increase inherent biosynthesis of proteases the 7p strain was screened on culture medium supplemented by streptomycin. derivative b. pumilus strain 3-19 gains the resistance to streptomycin and also shows the increased ribonuclease activity. we used genomes of both these strains to explore streptomycin susceptibility and increased activity of hydrolytic enzymes. whole-genome shotgun sequencing was performed using a combination of pyrosequencing and ion semiconductor sequencing, which provided 23x (7p) and 30x (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) overall genome coverage. assembled genome sequences of 7p and 3-19 strains included 9 and 8 scaffolds > 500 bp with a calculated genome size of 3 577 758 bp and 3 572 739 bp, respectively. the gc content was 42%. both draft genomes have been deposited at genbank (jojx00000000.2 for 7p and jhud00000000.2 for [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . detailed comparative genomic analyses of strains have been performed. we calculated average nucleotide identity (ani) values between the genomes of our strains and 9 completed b. pumilus genomes deposited in ncbi database. two b. pumilus strains (sh-b9 and safr-032) revealed the max. ani value (95.17% and 94.65%, respectively). b. pumilus sh-b9 strain has been used as a reference for snp calling in strains 7p and 3-19. 3828 snps for the 7p strain and 862 for the 3-19 strain were classified as nonsynonymous variants. 20 radical nucleotide substitutions from the 3-19 genome were not found in 7p genome. among them, the mutation in the 56 codon of rpsl gene (coding 30s ribosomal protein s12) is probably associated with resistance to streptomycin. also, two mutations in rpob and nusa genes (coding rna polymerase and transcription termination factor rho, respectively) may be related to increased enzymes activity. both our strains contain 143 protease-coding genes. twelve of them are encoding extracellular proteases. here we propose an algorithm that can predict an antibodies mutant forms with desired specificity. this algorithm allows to determine the position and type of amino acid residue for mutagenesis. approach is based on a hybrid method of quantum and molecular mechanics (qm/mm) that allows to understand the reaction mechanism and the role of active center amino acids. catalytic antibody a17, that is able to hydrolyze pesticide paraoxon, was selected as a model. however, the hydrolysis efficiency of paraoxon by a17 antibody is only 9 m à1 min à1 , that is insufficient for using this antibody as antidote. the main fundamental goal of our study is to determine the necessary conditions for improving the binding reaction of paraoxon by catalytic antibody a17. the hybrid qm/mm method allows to study the reaction mechanism of interaction of a17 with paraoxon. it was shown that the reaction proceeds via the classical s n 2 mechanism. the key step of the reaction is the proton transfer from the catalytic residue tyr-37 to paraoxon. qm/mm approach determines position for mutagenesis -leu-47 in light chain. for one of the mutant in this position -leu47argwere predicted (i) increased probability of formation of a hydrogen bond between the catalytic moiety and paraoxon compared to the wild type antibody and (ii) smaller value of the diffusion coefficient, which reflects the best positioning of paraoxon in the active center. steady-state kinetic analysis shows that leu47arg exhibits a 70-fold increase in k 2 /k d compared to a17 (90 m à1 min à1 vs. 1.35 m à1 min à1 ). double mutant leu47arg/ser35ala also has improved constants of interaction with paraoxon in comparison with the wild type antibody, however, a single mutant leu47arg still binds paraoxon three times better, that may be due to the fact that the serine in 35 position increase the nucleophilicity of tyr37. thus, our results are in line with our computed predictions. this work was supported by rfmefi60414x0069. due to high prices of meat and meat products, low quality raw materials like offal tissues are commonly used in turkey. in the retrospect of the studies for evaluating and detection of unwanted tissues in the sample is basic histological examination. the light microscopy techniques are very strong method if a researcher qualification is enough. a new researcher-independent method must be developed. therefore, different tissues and organs constitute of unique mrna and protein. our method is based on this event, so the antigenic sites of the tissues can be detected by selected antibodies. the first set of the antibodies are for detecting muscle and adipose, consist on muscle actin and adipose triglyceride lipase. this set is used for calibration on standard meat sample. the second set of the antibodies are detecting of offal tissues, consist on trrap and casein. anti-casein antibody is selected because the mammary gland usage in grinded-meat is very common. immuno-staining started with hier (heat mediated epitope retrieval), then classical ihc method applied to slides with dab-chromogen. after all process completed the slides were photographed by las (leica application suite) on microscope. the capture settings were remained same on both sets. image capture size is 1392x1040 pixels and field of view (fov) is 449 9 335 lm. all the image files were converted to binary for threshold operation. the threshold values of first set and second set were calculated and their ratios were compared. the formula is based on the distribution (dst) of pixel intensity (int) over threshold (thrs) values on all fov (axis: the results are good enough to detect the unwanted micro-structures on 80% raw meat and 20% offal tissue. calculations proofed with imagejò. future application of this method and opencv-based software algorithm is to port the source code to a single board computer (sbc) with a digital microscope connected. monday 5 september 12:30-14:30 mechanisms of pro-inflammatory diseases p-04.02.2-001 the effects of raas inhibition in rate limiting step by aliskiren on testicular torsion injury in rats testis torsion is a urological emergency condition that results in necrosis of the testis if the condition is not treated. unfortunately treatment of testis torsion is not fully understood, therefore clinical and experimental studies are performed continuously. reninangiotensin-aldosterone system (raas) contributed to pathophysiology of several diseases. aliskiren (als) inhibits the renin on the first step of this system. our aim is to investigate the protective effect of aliskiren on unilateral testis damage caused by experimental testis torsion and detorsion. the forty-eight rats were separated into eight groups of six animals: sham, sham+als 200 mg/kg (oral) group, torsion group (tor), torsion/detorsion group (tor/det), tor+als 100 mg/kg (oral) group, tor+als 200 mg/kg (oral) group, tor/det+als 100 mg/kg (oral) group, tor/det+als 200 mg/kg (oral) group. in the tor and tor/det groups, the left testes were rotated 720°clockwise together with the spermatic cord and tunica vaginalis in the scrotal space. the left testes of the rats were subjected to torsion and detorsion during 2 h. after experimental procedures, testicular tissues were examined by histopathologic and molecular analyses. the il-1b and inos mrna expressions were increased in tor and tor/det groups when compared with sham group. both doses of aliskiren administration decreased these expressions in tor/det groups. the stereological results revealed that aliskiren administration promote the numerical density of mature spermatids in tor and tor/det groups. the numerical densities of tor/det+als100 and tor/det+als200 groups were similar and these two groups have significant difference when compared to the tor and tor/det groups. the administration of als may be useful for preventing ischemic damage on unilateral testes injury in rats. this study supported by a tubitak 3001 project, coded 114s048. 3 ) has recently been recognized as a potent immunomodulator which acts through regulation of gene expression involved in immunity response thus affecting various inflammatory and autoimmune diseases. the study was aimed at investigating hepatoprotective role of d 3 in vdr-mediated regulation of pro-inflammatory factors in diabetic liver. materials and methods: type 1 diabetes was induced in male c57bl/j6 mice by i.p. injection of high-dose stz (150 mg/kg b.w.). after 2 weeks of stz-induced diabetes animals were treated with/without d 3 (15 iu/mouse per os) for 8 weeks. blood serum 25ohd 3 was assessed by elisa. rel-a, vpf, inos and vdr expression was measured by qrt-pcr and/or western-blot. results and discussion: diabetes caused two-fold reduction of serum 25ohd 3 level, indicative of d 3 deficiency. significant alterations in d 3 -endocrine system were found as is evident from reduced expression of cyp27a1, cyp2r1, vdbp and vdr at transcriptional and translational levels. these changes were accompanied by a marked increase in mrna and protein levels of inflammation markers rel-a, vpf and inos in hepatic tissue of diabetic mice. diabetes also led to structural lesions in liver tissue. complete restoration of 25ohd 3 content and partial normalization of liver tissue structure were achieved after d 3 treatment. d 3 administration partially normalized expression of cytochromes involved in d 3 metabolism and hepatic pro-inflammatory factors. d 3 treatment prevented overexpression of rel-a and phosphorylated p65/rel-a translocation to hepatocellular nuclei that is most likely mediated through 1,25(oh) 2 d 3 and vdr. conclusion: study confirmed that diabetes-induced liver abnormalities are associated with chronic inflammation that can be linked to impaired d 3 metabolism and deficiency. our findings demonstrate protective vdr-mediated effect of vitamin d 3 against diabetes-induced liver injury. p-04.02.2-003 lavandula stoechas extract increased glucose uptake and protein levels of key signaling molecules in insulin resistant c2c12 muscle cells s. savranoglu 1 , h. ipek 2 , s. arslan 3 , h. delig€ oz 4 , a. r. t€ ufekc ßi 5 , i. demirtas 5 , t. boyunegmez t€ umer 6 1 graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 2 graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 3 deparment of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, 4 department of chemical engineering, faculty of engineering, pamukkale university, denizli, turkey, 5 department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, turkey, 6 department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: the aim of this is to identify remedial effects of lavandula stoechas, anatolian traditional medicine, against metabolic disorders developed on the ground of insulin resistance. ethyl acetate extract (eae) of l. stoechas was investigated in c2c12 myotubes which were made insulin resistant by free fatty acid (ffa) treatment, for its effects on glucose uptake and as well as on the activation of akt-1 (by pakt/ akt ratio) molecule which plays a central role in insulin signaling through serine (473) phosphorylation. in addition, the protein level of lipoprotein lipase (lpl) enzyme was also evaluated. material and methods: c2c12 cells were made insulin resistant by palmitic acid (ffa) and effects of eae on p-akt (ser473)/akt ratio and lpl level were determined by sds-page/western blot. the effect of eae on glucose uptake in insulin resistant cells were determined by the 2-deoxyglucose uptake assay. results: eae at 25 and 50 lg/ml significantly increased the glucose uptake 120 and 182% compared to insulin resistant control cells. metformin at 2 mm increased this parameter up to 132%. eae increased pakt ser473/akt level 43-37% and lpl expression 50-92% for 25 and 50 lg/ml, in insulin resistant myotubes, respectively (p < 0.05). discussion: eae of l. stoechas improved impaired insulin sensitivity through both enhancing glucose uptake and activation of akt1 molecule through ser473 phosphorylation. in addition, it also considerably increased lpl level which has very important function in lipid metabolism. conclusion: overall, results demonstrated that l. stoechas contain phytochemicals which can be effective for the prevention and also treatment of insulin resistance and associated conditions. our research group is on the way for the identification of these 'bioactive' molecules with bioassay guided fractionation studies. tubitak (projectid: 112t442) supports this study. achievement of complete pain control is very difficult task, which requires a search for new molecular targets during the analgesic substances development. considering the importance of glial cells and their signaling molecules, development of new gliotropic therapeutic methods is a promising direction in pain treatment. polyunsaturated fatty acids, including docosahexaenoic acid demonstrating anti-inflammatory and antioxidant activity are of considerable interest. docosahexaenoic acid (dha, 22:6 n à 3) analgesic activity was studied using a chronic constriction injury (cci) rat model. animals were subcutaneously injected with dha emulsion at a dose of 4.5 mg/kg (125 mm/kg) daily during 2 weeks after surgery. collection of material for subsequent immunohistochemistry investigation was performed on day 28. we clearly demonstrated that the activation of neurokinin neurotransmission and nnos synthesis are coincided with the astroglial activation in the spinal cord dorsal horn (scdh) superficial lamina during neuropathic pain development. however, the detailed mechanisms of interaction between substance p (sp)-, no-ergic systems and astrocytes in the spinal cord remain to be elucidated. systemic administration of dha to cci animals reduced neurogenic pain intensity and duration, leading to an earlier stabilization of paw weight distribution and preventing the development of degenerative changes in denervated limb. this drug treatment reduced the level of the sp-and no-ergic neurotransmission and decreased astrocytosis in the scdh superficial lamina. thus, the ability of dha to affect nociception is a promising and safe alternative to current pharmaceutical therapeutics. immunohistochemistry studies carried out with the russian science foundation financial support (agreement no. 14-50-00034), obtaining dha and all manipulations with animals of the material was funded by rfbr according to the research project no. 16-34-00023 mol_a. p-04.02.2-005 circulating endothelial-derived apoptotic microparticles and aopps are related to highsensitive troponin t in patients with chronic hepatitis c infection the aim of this study was to evaluate non-standard risk factors for cardiovascular events, such as endothelial dysfunction assessed by endothelial-derived microparticles (emps) (cd144 +/ cd31 + ), advanced oxidation protein products (aopps), and low-grade inflammation, that are potentially associated with elevated levels of high-sensitivity troponin t (hs-tnt) and n-terminal pro-brain natriuretic peptide (nt-probnp) in patients with chronic hepatitis c (chc). methods and results: eighty-six chc patients and 60 healthy control subjects were enrolled in the study. circulating levels of hs-tnt, nt-probnp, aopps-albumin (the ratio of aopps to albumin content), emps (cd144 +/ cd31 + ), hs-crp, and tnf-a were assessed. compared with chc patients, the chc patients with diabetes mellitus (dm) had higher levels of emps (cd144 +/ cd31 + ) and aopps-alb, which were associated with elevated hs-tnt levels (≥13.3 pg/ml). nt-probnp positively correlated with tnf-a level in all chc patients and this correlation was stronger in diabetic patients. in multivariate logistic regression analysis, the independent factors associated with the presence of elevated hs-tnt levels were the presence of dm (p < 0.001) as well as high levels of aopps-alb, apoptotic emps (cd144 + /cd31 + /an-v + ), and nt-probnp (p = 0.04, p = 0.03, p = 0.04 respectively). conclusion: the prevalence of elevated hs-tnt were increased significantly in the diabetic patients with chronic hepatitis c. hs-tnt was related to non-standard risk factors for cardiovascular events, and circulating endothelial-derived apoptotic microparticles (cd144 + /cd31 + /an-v + ) level was an independent predictor for elevated hs-tnt levels, potentially indicating some abnormalities in the myocardium. apnea; and healthy individuals; and assessing the connection between the pain and the dimension of the sleep disorder. material and methods: 60 patients who were diagnosed with obstructive sleep apnea and 40 healthy individuals who were similar in terms of age and gender were included in this study. the patients, who were diagnosed with obstructive sleep apnea with the examination and sleep tests, were assessed according to the 1990 american college of rheumatology (acr) criteria in terms of fms. serum d vitamin level was measured by using the ultra performance liquid chromatography method. findings: when the fibromyalgia syndrome and obstructive sleep apnea and pure obstructive sleep apnea patient groups are compared with the control group, the vitamin d level was found to be low at a significant level (p = 0.038, p = 0.001, respectively). no significant difference was found between the vitamin d levels in fibromyalgia syndrome, obstructive sleep apnea and pure obstructive sleep apnea patient groups. a negative correlation was found between the number of the sensitive points and vitamin d levels in fibromyalgia syndrome patients (p = 0.013). results: it has been concluded that the obstructive sleep apnea and fibromyalgia syndrome patients have low vitamin d levels, and this situation must be considered in treatment modalities. on the other hand, the results obtained in the study make us consider that vitamin d metabolism is not influential in the pathogenesis of the fibromyalgia syndrome and obstructive sleep apnea togetherness. p-04.02.2-007 decreased chitotriosidase activity and levels in familial mediterranean fever discussion: familial mediterranean fever is an inflammatory disease. several cytokines and inflammatory mediators are playing role on pathogenesis of the disease. although ıt has been demonstrated that the increased concentrations of cht in patients with fmf. we found lower cht activity and concentrations in patients with fmf. conclusion: serum cht enzyme activity and concentrations may not be considered as a biomarker in fmf patients taking colchicine. new studies are needed to evaluate the changes of the enzyme activity, concentration and the role of cht in patients with colchicines negative patients. chronic hyperglycemic state leads to an increase in subclinical systemic inflammatory response in diabetes mellitus type 2 (dmt2) patients. inflammation-based scores, neutrophil to lymphocyte ratio (nlr), platelet to lymphocyte ratio (plr) and red blood cell distribution width to platelet ratio (rpr) are biomarkers able to quantify systemic inflammation. the aim of the study was to investigate association of the inflammation-based scores with short-and long-term glycemic control markers, and whether they could be used as indicators of glucoregulation in dmt2 patients. the cross-sectional study included 92 dmt2 patients, treated at the primary health care centre zenica from december 2015 to april 2016, distributed into groups according to glycated hemoglobin (hba1c) values: a (n = 59, hba1c ≤7.0%) and b (n = 33, hba1c > 7.0%). complete blood cell count, fasting blood glucose (fbg) and hba1c measurements were determined at the primary health care centre zenica and at the department of laboratory diagnostics, cantonal hospital zenica by standard laboratory methods. all statistical tests were performed using spss 19.0. p values fasting blood glucose and hba1c were significantly higher in the group b compared to the group a (p < 0.0005). there was no significant difference of nlr, plr and rpr between the groups (p = 0.50; p = 0.220; p = 0.525, respectively). significant correlation of inflammation-based scores with fbg and hba1c was found only between plr and hba1c in the group a of dmt2 patients (r = 0.328, p = 0.011). inflammation-based scores could gather meaningful clinical information, either diagnostic or prognostic, on a variety of hyperglycemic, inflammatory, cardiovascular and thrombotic disorders. since there was no statistically significant difference of nlr, plr and rpr between dmt2 patients with good and poor glycemic control, we conclude that these scores could not be used as indicators of glucoregulation in dmt2 patients. chronic inflamation plays a central role in the development and progression of diabetes and in the pathogenesis of its comlications. the neutrophil-lymphocyte ratio (nlr) and platelet-lymphocyte ratio (plr) are indicators of subclinical inflamation. mean platelet volume (mpv) is one of the platelet function indices that reflects the platelet production rate and stimulation. we investigated the association of nlr, plr and mpv with prediabetes and type 2 diabetes mellitus (t2dm) and determine whether or not these are reliable markers for diagnosis. we evalueted 76 people's results who were carried out oral glucose tolerance test (ogtt). acording to 2-h values of plasma glucose in the ogtt; 1. group (normal glucose tolerance: ngt): under 140 mg/dl (n = 42), 2. group (prediabetic: impared glucose tolerance (igt)): ranging from 140 mg/dl to 199 mg/dl (n = 25), 3. group (firstly diagnosed diabetic by ogtt): above 200 mg/dl (n = 9). 4. group is clear diabetic without complication (taking treatment) group (n = 34). we compered nlr, plr, mpv and some biochemical markers between four groups. there are significantly differences between all groups in nlr (p = 0.004) and plr (p = 0.021) values. nlr values are significantly higher in prediabetic (1. it is recognized that a chronic low-grade inflammation and an activation of the immune system are involved in the pathogenesis of insulin resistance and type 2 diabetes mellitus (t2d). this study aimed to analyze the long-term impact of altered metabolism in female t2d patients at the level of mediators of inflammatory response. this study included 65 femalet2d patients and 107 control subjects, which were recruited at the clinical center university of sarajevo and the general hospital tesanj. in this study the effects of glycemic control on markers of the inflammatory response crp, fibrinogen, leukocytes, sedimentation, and cytokine il-6, were analyzed. all subjects included in this study were free of evidence infections, surgery, thyroid disease, polycystic ovarian syndrome, active liver and kidney damage. all biochemical analyses were performed by employing standard ifcc protocols. results from this study demonstrated significant increase of fibrinogen (p = 0.0001), crp (p = 0.001), il-6 (p = 0.013), leukocytes (p = 0.0001) and sedimentation rate (p = 0.008) in female t2d population compared to control subjects. interestingly, a significant correlation was shown between crp and hba1c (p = 0.035), il-6 and glucose (0.032), il-6 and bmi (0.007). in our study, female t2d compared to the healthy population had significantly higher levels of fibrinogen, leukocytes, il6, crp and sedimentation. other studies conducted in female population associated elevated levels of il-6 and crp with t2d independent of other risk factors for diabetes. crp being most robust predictor of diabetes. studies have shown that crp is an important predictor of t2d for the female but not the male population. thus, our data suggest that inflammation play an important role in the pathogenesis in female diabetic population. a more detailed study on a far larger number of subjects should point out fact if they can effectively be used as biomarkers in the primary prevention of t2d in this population. objectives: bone and mineral metabolism disorders hold an important place among the complications after renal transplantation. the purpose of this study was to demonstrate the relationship between vitamin d, calcium, phosphorus metabolism with graft function and to measure 1,25(oh) 2 d 3 levels with lc-ms/ ms in renal transplant recipients. design and methods: this study included 30 renal transplant recipients (10 female, 20 male; mean age: 40.30 ae 12.86) from living related donors were transplanted. blood samples were collected immediately before and after transplantation at month 6. serum creatinine, bun, calcium, phosphorus, alkaline phosphatase, glucose, albumin, pth, 25(oh)d and 1,25(oh) 2 d 3 levels were measured. gfr values were estimated by ckd-epi. plasma 1,25(oh) 2 d 3 levels were determined in a lcms-8040 triple quadrupole tandem mass spectrometer (shimadzu corporation, japan) by mrm. spss 20.0 software was used for statistical analysis. results: although plasma 1,25(oh) 2 d 3 levels significantly increased (p = 0.0001), we did not find any significant differences for serum 25(oh)d levels after transplantation. when posttransplant levels of serum phosphorus, pth, creatinin, bun and alp levels were found to be significantly decreased (p = 0.0001, p = 0.011 for alp), we observed significantly higher calcium and gfr values (p = 0.0001). vitamin d insufficiency was present 13.3%, deficiency 36.7%, severe deficiency 50% before transplantation, insufficiency was also seen 26.7%, deficiency 50%, severe deficiency 23.3% after transplantation at month 6. conclusions: in our study, all patients were found to vitamin d deficiency and insufficiency. determination of vitamin d deficiency and consequently treatment with vitamin d supplements could lead to better graft surveys. free fatty acids (ffa) represent important link between obesity, insulin resistance, type 2 diabetes (t2d), and dyslipidemia. increased adiposity, as approximated by body mass index (bmi), correlates well with increased serum levels of leptin-adipocyte derived hormone implicated in the regulation of adipose mass and alterations in insulin action and secretion. the main objective of the present study was to investigate the potential association of serum ffas with leptin levels in healthy and newly diagnosed type 2 diabetic subjects. this study involved 13 newly diagnosed type 2 diabetics and 13 healthy subjects. all participants in the study were free of evidence of hepatitis, viral infection or active liver and kidney injury. for biochemical analyses of glucose, glycosylated hemoglobin (hba1c), and lipid profile, standard ifcc protocols were used. analysis of free fatty acids (ffas) was done by gas chromatography, while serum leptin levels were determined by the elisa kit. in addition to the expected differences in glucose, hba1c, and bmi, our results also showed significant differences in leptin, myristoleic, palmitic, linolenic, arachidic, and arachidonic acids between t2d and control subjects. in healthy subjects, a significant correlation was demonstrated between glucose and lauric, arachidic, arachidonic acid levels, body weight, and bmi. newly diagnosed diabetics showed significant association between glucose and lauric, myristoleic and linolenic acid levels; with leptin being associated with myristic and palmitoleic acid levels. interestingly, in all participants, significant association was found between glucose and hba1c, glucose and leptin, myristoleic, arachidic, and bmi as well as between leptin, arachidic acid, and bmi. thus, our data point out association of different types of ffas with leptin levels in newly diagnosed type 2 diabetics. however, further studies should be done in larger number of patients to confirm our results. rheumatoid arthritis (ra) and ankylosing spondylitis (as) are chronic inflammatory diseases with distinct clinical manifestations in many ways. the aim of this study is to evaluate the serum levels of molecules which may be used as markers for angiogenesis and vascular leakage in the processes of two clinically different pictures, ra and as. 30 ra patients, 30 as patients and 30 healthy volunteers with mean age of 30-50 were included in the study. serum levels of vegf, angiopoetin-2 and tie-2 were measured by enzymelinked immuno-sorbentassay (elisa) using a commercially available kit. serum nitricoxide levels were evaluated by the griessreaction. serum vegf, ang-2 and no levels were significantly higher in the as group ml, p < 0.001; p < 0.001; p < 0.05). no differences were found between as and ra for tie-2 (p > 0.05). vegf, ang-2, tie-2 and no levels were positively correlated in both as and ra patients (p < 0.001), but no correlation was detected between clinically activation index das-28, basda _ i scores and laboratory measurements such as sedimentation, crp and anti-ccp (p > 0.05). when the diagnostic performance of the parameters were evaluated with the roc analysisonly the performance of the ang-2 in as patients was sufficient (auc (95% cl): 0.718, p < 0.05). elevation of angiogenic factors in the serums of as and ra patients supports the role of angiogenesis in the etiopathogenesis of these diseases. however, lack of relationship between disease activity leads to not to use these factors as a marker for clinical follow-up. only ang-2 measurements may be useful for the differantial diagnosis. the evaluation of ischemia modified albumin as an early biomarker of acute myocardial infarction introduction: acute myocardial infarction (ami), remains a leading cause of morbidity and mortality worldwide. early diagnosis of ami is very important because early treatment may reduce the extent of injury to the myocardium. currently, biomarkers of myocardial necrosis such as myoglobin, ck-mb and troponins are highly sensitive and exhibit good specificity. however, these biomarkers increase after tissue injury, approximately 4-6 h after the cardiac event and detect only the consequences of prolonged ischemia. recently, ischemia modified albumin (ima) has been assessed and found to be very useful for the diagnosis of myocardial ischemia and it is considered as a serum biomarker. the aim of the present study was to evaluate the serum level of ima and determine the relation between patients with ami and control group, in order to verify its potential as a novel marker for early detection of mi. materials and methods: the study was performed with 25 patients and 37 healthy controls. blood samples from all subjects were collected by venipuncture in plain tubes, and immediately centrifuged at 4000 g for 10 min at 4°c. the serum samples were stored at à20°c until analysis. the serum levels of ima were determined using the cusabio biotech human ischemia modified albumin, elisa kit according to manufacturer's instructions. the results are given as international units/milliliter (iu/ml). results: our findings revealed that ima showed no significant difference between the groups. conclusion: our results suggest that ima assay is not a sensitive marker for early detection of ischemic hearth disease and cannot be used alone for the diagnosis of ami. prospective studies are needed to identify ima's potential as a biomarker for ami. p-04.02.2-015 neutrophil-to-lymphocyte ratio and platelet-tolymphocyte ratio in polycystic ovary syndrome polycystic ovary syndrome is a complex and multifactorial disease with metabolic dysfunction and the etiopathogenesis is not well established. emerging data suggest that adiposity and chronic low-grade inflammation are involved in the development of the metabolic dysfunction. neutrophil-to-lymphocyte ratio (nlr) and platelet-to-lymphocyte ratio (plr) have recently been investigated as two new inflammatory markers used in the assessment of systemic inflammation in many diseases. the purpose of the study was to investigate their relation with pcos patients. the study population consisted of 44 patients with polycystic ovary syndrome and 23 healthy women controls. nlr and plr obtained by dividing absolute neutrophil to absolute lymphocyte count and absolute platelet count to absolute lymphocyte count, respectively. the neutrophil count (4.41 ae 1.6 vs. 3.77 ae 1.23, p < 0.05) and platelet count (320.34 ae 55.11 vs. 283.96 ae 52.17, p < 0.05) were higher in patients with pcos compared to the control group. lymphocyte count was 2.08 ae 0.43 in pcos patient and 2.30 ae 0.57 in control group. the nlr and plr of pcos patients were significantly higher compared to those of the controls (2.18 ae 0.92, 1.69 ae 0.57 p < 0.05, 158.31 ae 32.85, 128.59 ae 31.94 p < 0.05, respectively). in this study we found nlr and plr were significantly increased in patients with pcos compared to healty control. nlr and plr were two useful inflammatory markers for assessment of patients with pcos. imbalance in neurotransmission in conjunction with neuroinflammation contribute to neurological dysfunction observed during acute liver failure (alf). own observations indicate that alf in a mouse model is associated with altered expression and/or intracellular distribution of synaptic proteins. since neutralization of tgf-b1 appears to improve the neurological score in alf mice, we examined the possibility that increased levels of tgf-b1, caused by liver damage, may affect the expression of selected synaptic proteins. expression and/or cytoplasmic vs. membrane distribution of a number of functionally critical synaptic proteins in cerebral cortex and blood tgf-b1 were measured in c57bl6 mice with alf induced by single i.p. injection of aom (100 mg/kg of b.w.) and after neutralization of tgf-b1 induced by single i.p. injection of ab-tgf-b1 (1 mg/kg) 2 h before aom injection. in alf mice, blood tgf-b1 was increased, and the expression of presynaptic proteins: synaptophysin and synaptotagmin was increased in the cytosolic (s2) fraction by~45% and 30%, respectively, but was slightly depressed in the membrane (p2) fraction by~20% and~15%. aom induced an increase of postsynaptic proteins: psd-95 and nnos by~40% in p2 fraction. tgf-b1 neutralization resulted in a reduction in the expression of presynaptic proteins by~30% in s2 fraction and~20% in p2 fraction, in control animals and normalized their amount in the cytosolic fraction after aom injection, but was ineffective with regard to psd-95 and nnos. the results indicate that in alf mouse, neutralization of cytokine tgf-b1 normalizes synaptophysin and synaptotagmin expression in the synaptoplasm, without affecting their synaptic membrane content. effect of tgf-b1neutralization appear to be confined to the presynapse. 25% of acute pancreatitis (ap) patients develop severe acute pancreatitis (sap), which is is resulted in multiple organ dysfunction syndrome. an extensive inflammatory response occurs due to inflammatory mediators synthesized and secreted during sap. since preventing the inflammation in sap is important in the prognosis of the disease, new drug candidates having strong antiinflammatory effects will provide a new concept for therapeutic strategies against acute pancreatitis. non-steroidal anti-inflammatory drugs (nsaids) show their effects by inhibiting cyclooxygenases (cox-1 and cox-2) and they play an important role in the pathogenesis of acute pancreatitis. since conventional nsaids inhibit both cox-1 and cox-2, they have serious side effects on gastrointestinal system. therefore, new highly selective cox-2 inhibitors having fewer side effects are needed. in the present study, selective cox-2 inhibitory activities and cytotoxic effects of new series of 2-benzoxazolinone and thiazolo [3,2-b ]-1,2,4-triazole derivatives previously synthesized as specific cox-2 inhibitors with no side effects on gastrointestinal system were investigated. permeability of the compounds was tested by pampa using caco-2 cells. compounds were found highly selective, non-toxic and permeable. ap was induced in rats via retrograde injection of stc into the pancreatic duct system. rats were pre-treated with saline or celecoxib or the new compounds before stc injection and sacrificed 24 h later. the severity of ap was evaluated using biochemical and histopathological analyses. edema, inflammation, hemorrhage and acinar cell necrosis were detected in the pancreatic tissue of sap group. sap was remarkably increased serum lactate dehydrogenase, ast, alt, lipase and amylase activities and serum tnf-a, il-1b, il-2, il-6 and il-8 levels. tissue myeloperoxidse activity was also increased. pretreatment with the novel compounds reserved all these biochemical and histopathological parameters. alopecia areata (aa) is an inflammatory disease which is affects hair follicles, and sometimes nails. it is suggested that cytokinemediated immunity plays an important role in etiopathogenesis of aa. this study was planned to evaluate the serum ykl-40 and tgf-b1 levels of patients with aa. 40 patients with aa and 40 healthy volunteers were recruited into the study. fasting venous blood samples were collected from the participants and serum was obtained by centrifugation. serum ykl-40 and tgf-b1 levels were measured by enzyme linked immunosorbent assay (elisa). serum tgf-b1 levels in the patient group were significantly lower compared to the control group whereas serum ykl-40 levels were significantly higher in patient group. tgf-b1 levels of men and women with aa were found to be significantly lower than that of controls. while serum ykl-40 level of male control group is higher than the male patients, there were no significant differences between women groups. the increased serum ykl-40 levels in aa patients suggests that ykl-40 plays a crucial role in the pathogenesis of aa. arterial immune mediated inflammation participates centrally in all stages of the development of atherosclerosis, from the initial lesion to the end-stage thrombotic complications. although emerging evidence supports augmented cardiovascular morbidity and mortality in cutaneous psoriasis (psc) and psoriatic arthritis (psa) as compared to the general population its underlying mechanism is poorly understood. here we analyzed the inflammatory burden in recent onset of psa patients without traditional cardiovascular risk factors (cvrf) in a transversal study measuring carotid intima media thickness (cimt) (measured with ecodoppler), and proatherogenic inflammatory molecular markers like c-reactive protein (crp), interleukin 6 (il-6), and soluble intercellular adhesion molecule-1 (sicam-1) in comparison with control patients. cimt values are similar in psa (0, 59 ae 0.045*) and psc (0, 62 ae 0.10) patients. however, both of them were significant increase compared with control (0.436 ae 0, 05). regarding inflammatory markers il-6 serum levels in patients with aps was higher than pcs (18 ae 2.9) and healthy controls (16, 3 ae 2.5) but the difference did not achieve statistical significance (*p > 0.05). on other hand mean of sicam-1, value from patients with recent onset of psa is significant higher than controls. psc remain without significant changes compared to control (*p > 0.05). in addition mean value from patients with recent onset of psa is significantly higher than in controls (*p < 0.05) and psc group. overall, preliminary findings suggest for the first time that patients with early psa, without evident traditional cvrf have significant increased values of cimt, sicam-1 crp against the general population control group. this data strongly supports that early cv molecular markers are increased after the first symptoms and signs of this disease even in the absence of traditional cardiovascular risk factors. furthermore, this give new windows for a proper treatment. p-04.02.2-020 protective effect of trail against proinflammatory cytokines on pancreatic beta cells correlated with decrease in dr5 and increase in dcr1 expressions universitesi, antalya, turkey introduction: proinflammatory cytokines are known to have destructive effects on beta cells, which contribute to type 1 diabetes (t1d) development. the combinatory effects of three of these cytokines in particular, namely tnf-alpha (tnf-a), ifngamma (ifn-c), and il-1beta (il-1b), are claimed to render beta cells prone to t cell-mediated destruction. the recently identified anti-inflammatory feature of tnf-related apoptosis-inducing ligand (trail), its possible protective role in this process. in this study, the effects of applications of trail with tnf-a, ifn-ᵞ, and il-1b on beta cell viability and correlation of these effects with trail receptor expression patterns were investigated. methods: glucose-responsive insulin-secreting nit-1 mouse beta cell lines were treated with tnf-a, ifn-ᵞ, il-1b, and soluble trail (strail) individually and in various combinations. cell viabilities were determined at 24 and 48 h by mtt assay. trail ligand and receptor expression profiles on nit-1 cells, and alterations in receptor expression levels following cytokine applications were determined by western blotting analysis. results: trail treatment did not have any cytotoxic effects on nit-1 beta cells at 48 h, while increasing cell viability following il-1/ifn/trail and il-1/tnf/trail combined applications. substantial levels of death receptor 5 (dr5) expression were detected on nit-1 cells before applications, yet it displayed decreased levels at 48 h of trail treatment. lower levels of decoy receptor 1 (dcr1) expression detected prior to treatments increased significantly in contrast. discussion: the fact that trail co-treatment with tnf-a, ifn-ᵞ and il-1b increased cell viability in nit-1 beta cell lines along with reduction in dr5 death receptor expression and an increase in the decoy receptor dcr1 expression, points out to a possible protective effect of trail in insulitis, and strengthens its potential as a putative therapeutic molecule in prevention of beta cell loss. behc ßet's syndrome (bs) is a multisystemic inflammatory disorder with a strong and complex genetic background. being a prevalent disorder both in turkey and also in the ancient trade road 'silk road' countries, bs is an important cause of impairment and disability owing to its chronic and relapsing nature. besides, bs is reported to be an important cause of mortality among the young male patients. while the epidemiology of bs is substantially well documented, currently, the etiology, the molecular mechanisms underlying its pathogenesis, and the classification of the disorder remain to be elucidated. our aim was to disclose the disease mechanisms at molecular level in turkish bs patients by obtaining, comparing, and analysing the transcriptome data of bs patients with age and gender matched healthy controls. for this purpose, by using the affymetrix hg u133 plus 2.0 microarrays, peripheral blood cell mrna profiles of 30 bs patients (b) and 15 matched healthy controls (c) were obtained. following bioinformatics, gene ontology, and pathway analysis, validation experiments of the identified prominent mrnas were performed by qrt-pcr methodology. the comparison of b vs. c yielded differentially expressed gene numbers of 30 and 625 for the chosen fold changes of 2.0 and 1.5 respectively (p ≤ 0.05 for both). during gene ontology and pathway analysis, immune system process, immune system diseases, systemic lupus erythematosus, arthritis, and intestinal immune network for iga production categories/pathways were significantly enriched. clustering analysis revealed a molecular signature which accurately distinguished b and c samples, while the qrt-pcr analysis successfully validated the chosen mrna transcripts. this study documented differential expression of a large number of immune system and immune disease related genes in bs patients. the uncovering of the molecular disease mechanisms of bs will point to novel candidate molecules to be targeted for the treatment of the disorder. obesity is a public health problem in developed countries and worldwide with increasing prevalence through a relationship primarily with atherosclerotic cardiovascular diseases as well as several metabolic disturbances such as increased insulin resistance and diabetes. although several studies identified obesity as an independent risk factor for atherosclerotic cardiovascular diseases, the mechanism underlying the increased cardiovascular risk in obese patients has not been clearly delineated. adma, no, endothelin-1 and homocysteine are an indicator of endothel disfunction that plays an important role in the pathophysiology of atherosclerosis. in our study, obese children and the control group were compared in terms of adma, no, endothelin-1 and homocysteine, we also investigated whether there is a correlation between these parameters. 58 obese and 30 healthy children, participated in the study. when the obese group was compared to the healthy controls, the adma level of the obese group were significantly higher than those of the control group but there was no statistically significant difference in no, endothelin-1 and homocysteine. increased adma level might trigger the pathogenesis of atherosclerosis starting from the childhood years onward. that is why controlling obesity in this age group with diet and other treatment modalities will prevent the mortality and morbidities that will be seen in adult years. inh deficiency leads to the formation of bradykinin causing to dilation of blood vessels. furthermore, the study conducted by shagdarsuren, on the damage done by c1-esterase, demonsrates that the complement system and triglyceride levels are affected. we investigated lipid oxidation and fetuin a levels in patients with c1 _ inh deficiency. materials and methods: 44 people with c1 _ inh and 44 people without any illnesses were taken into the study. fetuin a was studied using an el _ isa kit from raybio (usa). ferrous ion oxidation-xylenol orange test was used to find looh serum levels. sh (free thiol groups) test was studied with regards to ellmans method modified by hu. _ ibm spss 20.0 was used for statistical results. results: in assessments made between the healthy and the illness groups, there was significant differences in the levels of fetuin (p = 0.035), looh (p = 0.000) and sh (p = 0.047). when pearson correlation analysis was performed, we detected a significant positive correlation between fetuin a and looh levels (r: +0.326) discussion and conclusion: in these patients, lipids is secreted from the adipose tissue. in response, anti-atherosclerotic fetuin a levels were risen. patients also possessed increased lipid peroxidation, this increase shows positive correlation with fetuin a levels. in conclusion, we identified that sh with antioxidant properties have increased levels. aim: high fructose corn syrups are found in soft drinks, juice beverages, breakfast cereals, most of the processed foods. it has been shown that high dose of fructose intake may lead to a reduction in the number of hepatocytes, deterioration of liver function, increasing reactive oxygen species and liver steatosis. the aim of this study was to explore whether caffeic acid phenethyl ester (cape) has any potential protective effect on high fructose diet-induced fatty liver model. materials and method: totally fifty rats were divided into five groups. control group, %10 fructose administered group, cape group, %10 fructose + cape administered group and ethanol group. after 6 weeks, liver oxidant and antioxidant status, and blood tnf alpha, il-6, and il-8, tissue nfkb levels were quantified. protein levels were investigated against, nfkb and p-nfkb antibodies and normalized and analyzed against b-actin antibody by western blotting. results: serum tnf-alpha, il-6, il-8 levels were found to be increased in fructose group compared with the control group (p < 0.05). in liver tissue of 10% fructose administered group, mda, protein carbonyls and no levels were higher than control group. however sod activity did not show any difference among the groups. in the fructose administered group, caspase 3 showed liver apoptosis and was considered as positive. acquired data revealed that nfkb protein level was decreased in the presence of cape while increment in nfkb protein level was observed in the fructose administered group compared with control group. in case of pnfkb antibody, increment observed in fructose only and both cape and fructose administered groups, respectively. in cape only administered group, there was a decrement in the level of pnfkb protein. conclusion: depending on further analysis, experimental findings are expected to implicate the role of cape as a protective agent on high fructose diet-induced fatty liver model in relation of inos, nfkb and p-nfkb pathways. the investigating association of hepcidin levels with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction a. g. agg€ ul 1 , n. uzun 1 , e. c ß inar tanriverdi 2 , h. € un 1 1 agri ibrahim cecen university, agri, turkey, 2 nenehatun maternity hospital, erzurum, turkey this study was designed to investigate hepcidin levels and their associations with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction (iugr). a total of 88 pregnant women were included in this study. pregnant volunteers were divided into two groups (30 healthy pregnant women and 58 pregnant women with iugr). serum hepcidin, total free iron, ferritin, transferrin, transferrin receptor and interleukin-6 (il-6) levels were measured by elisa. also, hemoglobin (hb) and c-reactive protein (crp) levels were determined in serum samples from the healthy pregnant women and the pregnant women with iugr. there were significant differences in hepcidin, ferritin, transferrin receptor, crp and il-6 levels between healthy pregnant women and pregnant women with iugr. hepcidin, ferritin, crp and il-6 levels in pregnant women with iugr were significantly higher than healthy pregnant women (p). the mediators of systemic inflammation in lipopolysaccharide-induced neonatal sepsis rat model sepsis is an excessive inflammatory response that causes shock, multi-organ failure and high mortality. foreign bacterias and lipopolysaccharides lead to stimulation of endothelial cells to produce biologically active mediators such as proinflammatory cytokines and chemokines, cell adhesion molecules, and growth factors. then these mediators could be act on targets, which were involved in the initiation of systemic inflammation in neonatal sepsis. our aim was to indicate a protective role of thalidomide and etanercept, which have anti-tnf-a activity on systemic inflammatory response in lipopolysaccharide (lps)-induced neonatal sepsis rat samples. thirty 7-day-old wistar rats were randomly divided into five groups: a control group that received normal saline, a sepsis group that received lps, thalidomide, etanercept and both thalidomide and etanercept treatment group that were administered with therapeutic agents 6 hrs after lps injection. the rats were sacrificed at 24 hrs after lps or normal saline injection (n = 6). hepatic tissue tnf-a, il-6, icam-1 and pdgf levels were determined by enzyme-linked immuno sorbent assay (elisa) method in all groups. in sepsis group, tissue tnf-a, il-6, icam-1 and pdgf levels were statistically significantly higher than in controls (p < 0.001). at same time, pretreatment with both thalidomide and etanercept were found statistically dramatically decreases the levels of tnf-a, il-6, and pdgf when compared to sepsis group (p < 0.001). there were no significant differences in the icam-1 levels between the all treatment groups and the sepsis group. higher liver tissue tnf-a, il-6, icam-1 and pdgf levels are associated with severe bacterial infection. these proinflammatory cytokines and angiogenic factors may be important in the endothelial dysregulation seen in sepsis. therapeutic agents used in the present study can be help to avoid devastating effects of neonatal sepsis. n-stearoylethanolamine (nse)is saturated minor compound of natural origin that represents the large family of signaling lipids n-acylethanolamines, which belong to endocannabinoid system. considering the crosstalk between obesity-induced inflammatory response and its key role in synaptic dysfunction and neurodegeneration, our current study aimed to investigate the biological effect of nse on brain tissue under high fat diet-induced insulin resistance. previously we found that nse administration to insulin resistant rats caused normalization of liver and pancreas lipid composition followed by the improvement of glucose tolerance and insulin sensitivity (decline in serum insulin level and homa-ir value). moreover, this effect of nse correlated with inhibition of nf-kb translocation into the nucleus of peritoneal macrophages and decreased pool of serum tnfa level in obesity-induced insulin resistant rats. further experiments showed that fat overload triggered significant reduction in the level of main phospholipids (phosphatidylethanolamine, phosphatidylcholine and sphingomyelin), while there were no changes in cholesterol content. nse at a dose of 50 mg/kg during 2 weeks of administration to insulin resistant rats showed a tendency to restore the phospholipid level that was accompanied by increased neural cell survival (91%) compared to rats without treatment (84%). neuroinflammation accompanied by intensive reactive oxygen species (ros) production impairs neurotransmission in a wide range of neurodegenerative pathologies. flow cytometry is used for quantitative analysis of global dna methylation, but fluorescence microscopy is mostly preferred to qualitatively reveal intranuclear localisation of dna methylation and its copattern with other markers. both methods use a similar immunostaining protocol. in this study, we aimed to compare these methods concerning the detection of the global amount of dna methylation. for this, mouse embryonic fibroblasts were cultured either with or without phenol red and then stained for dna methylation or positive controls (histone, betaactin, phosphoakt) by specific antibodies, or nonspecific control antibodies. some cells were incubated with trypan blue before or after the addition of antibodies. fluorescence intensities were measured by the green fluorescence channel (530/30 nm). autofluorescence spectrum of cells was analysed, and fluorescence channel used for dna methylation detection was changed to red (650 nm lp). a poor discrimination between signal and noise was detected due to cellular autofluorescence interfering with specific detection of dna methylation by flow cytometry but not by microscopy. it was also the case for the other markers examined. conventional advances to reduce autofluorescence such using phenol red free culture media or trypan blue quenching were not effective, but using the red channel regarding autofluorescence spectra allows detecting specific staining of dna methylation by flow cytometry. but, green channel did work well for microscopy analysis. findings show that flow cytometry detection of dna methylation requires much attention to quench cellular autofluorescence compared to detection by fluorescence microscopy. one reason could be that flow cytometry detects all cellular content, but manual image-based analysis can exclude cytosolic components. these results suggest the usability of flow cytometry and microscopy as complimentary methods for dna methylation detection, but optimisation to reduce autofluorescence is crucial for flow cytometry. objectives: lung cancers are divided in two main groups as small cell lung cancer (sclc) and non-small cell lung cancer (nsclc) . docetaxel (dtx) and cisplatine are chemotherapeutic that has an anti-tumor activity against various solid tumors. the growing resistance against dtx and cisplatine (cis) still continues to be the biggest obstacle for the treatment success of nsclc patients. deguelin (deg.) is a natural plant derivative and has an encouraging activity against a lot of human cancers. the comparison of the treatment activity of the separate and combined usage of deg., which is a potential chemotherapeutic agent, and dtx, cis which are used in standard treatment, is aimed in this study. material-method: the ic50 doses of dtx, cis and deg. on the a549 and h1299 nsclccell lines were determined via the cell vitality tests in our study. the active concentrations determined were applied to nsclc cell lines as deg., dtx, cis and their combinations. the impacts of the medicine are studied by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, colony formation, migration and angiogenesis analyzes on the treated cells and measuring the oxidative stress index (osi). statistical analyse program, rstudio (v.0.98.501) and the r-script language were used to examine the differences between the agents. the states in which the pvalue was lower than 0.05 were accepted as statistically meaningful. results: we found that deg. has pro-apoptotic, anti-migratory and cytotoxic potential on lung cancer cells. deg. amplified cis and dtx-related anti-cancer efficacy (increased apoptotic cell content and cytotoxicity, reduced migration). also, deguelin pretreatment sensitized the cells dtx-treatment (reduced ic50 values). these effects were remarkable in p53-mutant cells. conclusion: deguelin, solely, has anti-cancer potential on nsclccells. both deguelin pre-treatment and combinantion with standart chemotherapeutics result in enhanced anticancer efficacy. the 83% of the lung cancers are non-small cell lung cancers (nsclc). despite docetaxel (dtx) and cisplatine (cddp) are agents used in the standard treatments of these patients and the recent improvements in the treatments, the response and remission rates observed on the patients are relatively nominal. selenium (se) is an essential diet component and is introduced to have a preventive impact on different levels of cancer. the aim of our study is to investigate the impacts of selenium addition on anticancer feature and tumor prevention before or/and during nsclc standard treatment. the ic50 doses of dtx, cddp and selenium on the a549 and h1299 (p53 mutant) nsclc cell lines were determined via the cell vitality tests in our study. the active concentrations determined and the stipulated available concentrations were applied to cell lines as dtx, cddp, se combinations. the impacts were compared by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, western blot analyzes on the treated cells and measuring the oxidative stress index (osi) and thioredoksin reductase activity. selenium pre-treatments reduced dtx-related ic50 concentrations at lower doses in both nsclc cells. however, cddprelated ic50 concentrations reduced dose-dependent manner. selenium supplementation also altered cell-cycle charactheristics at several concentrations and combination regimens. the remarkably higher osi values were observed after dtx treatment and osi levels were found to be lower in selenium pre-treated nsclc cells. selenium sensitizes nsclc cells to dtx treatment at lower concentrations. however, this effect is obtained dose-dependent fashion for cddp regimen. breast cancer is the most common female malignancy worldwide. human epidermal growth factor receptor 2 (her2) is overexpressed in 30% of breast cancers in association with aggressive phenotypes. the prognosis of metastatic breast cancer remains poor in spite of advances in therapy. as such, her2 has long been studied as a potential target for anticancer drugs. the modulation of intracellular signaling pathways leads to altered cell metabolism that triggers tumorigenesis and adapts cells to cancer cell metabolism. this characteristic hallmark of cancer metabolism is known as warburg effect meaning energy production via enhanced glycolysis. despite of several studies in breast cancer metabolism, little detail exists on the link between her2 overexpression and warburg effect. we have committed examining the nature of aerobic glycolysis in her2 overexpression. in breast cancer cell line mcf7, her2 overexpression (mcf-her2) results in mitochondrial dysfunction with low mitochondrial membrane potential (δᴪm) and ros accumulation. intracellular iron levels are also higher in mcf7-her2 cells than vector control (mcf7-vec). additionally, mcf7-her2 cells show enhanced levels of atp and lactate in association with increase in glucose levels. we have found that complex i activity increases in mcf7-her2 and decreases in knockdown of her2 in hcc1954 cells that is her2 positive breast tumor cell line. based on these results, we conclude that there is a link between her2 overexpression and metabolic indicators of warburg effect. expression and methylation analysis revealed 11 microrna genes deregulated by methylation and new potential target genes of mir-375 and mir-127-5p in breast cancer micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to assess the contribution of methylation to expression level alterations of 20 mirna genes and to search for novel potential targets of these mirnas. to analyze alterations in expression we used qpcr technique with 2 references (rnu6, rnu48) and 30 paired (tumor/normal) breast cancer (bc) samples. for methylation analysis a methylation specific pcr and the same set of bc samples were used. significant downregulation was shown for mir-125b-5p, -129-5p, -132-3p, -193a-5p, -34b-3p, -212-3p, -127-5p, and -17-5p (p ≤ 0.05, fisher's exact test) in bc. we observed 10 mirna genes to be hypermethylated and mir-191hypomethylated. hypermethylation for 3 of these mirna genes was shown for the first time: mir-132, -137, and -1258 (41-34% of bc cases). a significant correlation between methylation and expression alterations was revealed for 5 mirnas with downregulation: mir-125b-5p, -129-5p, -132-3p, -193a-5p, and -34b-3p (spearman's correlation coefficient (rs) was in the range à0.71 to à0.81, p ≤ 0.01), and for 3 mirnas with both scene (down-and upregulation) as well: mir-148a-3p, -203a, and -375 (rs = à0.72 to à0.93, p ≤ 0.01). comparative analysis of the data on expression alterations of 20 mirna genes and 6 protein-coding genes, which were predicted as targets by mirwalk 2.0, revealed the negative correlation between expression levels for some potential mirna-mrna interaction pairs. for example, for pairs mir-375/rhoa, mir-375/rassf1(a), mir-127-5p/dapk1 (rs = à038 to à0.46, p ≤ 0.05). thus, both mirnas and methylation affect regulatory networks in bc. novel potential mirna-mrna interaction pairs could be useful in the development of bc therapy approach. this work was financially supported by grant 14-15-00654 from the russian science foundation. the authors thank the n.n. blokhin cancer research center for tissue samples. clear cell renal cell cancer (ccrcc) with metastases has pour prognosis: 5-year survival is about 9%. micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to find out mirnas which methylation contributed to ccrcc progression, including metastasis, and to reveal potential target genes of these mirnas. to analyze methylation status, we used a methylation specific pcr as a method and a representative set of 70 paired (tumor/ normal) ccrcc samples. we also used 19 post-mortal renal tissues from individuals without cancer history as additional control. for expression analysis we used qpcr method and 45 paired ccrcc samples. we observed 9 mirna genes (mir-124a-1/-2/-3, -9-1, -9-3, -34b/c, -129-2, -193a, -107) to be hypermethylated, (p ≤ 0.05, fisher's exact test), 3 mirna genes to be hypomethylated and mir-203a with both scene (hyper-and hypomethylation was detected). methylation of 7 mirna genes (mir-124a-2/-3, -34b/c, -129-2, -107, -148a, -203a) correlated with advanced stage and/or tumor size and/or dedifferentiation. hypermethylation of mir-129-2, mir-203a, and mir-107 significantly correlated with metastasis presence (p < 0.05, fisher's exact test). besides, preliminary data revealed the positive correlation between hypermethylation of mir-129-2 and up-regulation of 3p protein-coding genes: rarb(2), rhoa, nkiras1, and chl1, which were predicted as targets by mirwalk 2.0 (spearman's correlation coefficients (r s ) was in the range 0.35-0.53, p ≤ 0.05). in conclusion, novel supposed interactions of mir-129-2 with target genes could be useful as missing chains in signaling pathways. tests for hypermethylation of mir-129-2, mir-203a, and mir-107 could be suggested as markers of metastasis and pour prognosis of ccrcc. because of difficulty in diagnosis and treatment hc is a clinical problem: early symptoms of hc are often non-specific and surgical resection is the only curative treatment for hc. it is well known that epigenetic alterations are linked to cancer development. the purpose of this study was to determine potential mechanisms of epigenetic regulation of genes related to energy metabolism in hc. we have performed bioinformatics analysis of the cancer genome atlas (tcga) project rna-seq data with crosshub software and found a number of genes involved in glycolysis and differentially expressed in cholangiocarcinoma. qpcr analysis revealed significantly decreased expression of pgm1 and eno3 genes in a majority of hc samples which were known as up-regulated in other human cancers according to the literature date. on the basis of tcga methylation dataset (450k illumina microarrays) we supposed that cpg methylation of pgm1 and eno3 promoters may play a role in their inactivation. using bisulfite sequencing study we identified several regions within the gene promoters (pgm1:~750 bp and 330 bp upstream tss; eno3:~750 bp downstream tss) that are frequently methylated in hc samples (up to 60%, 12/20) with down-regulated pgm1 and eno3 expression. thus, we demonstrated frequent and significant pgm1 and eno3 down-regulation associated with hypermethylation of the specific regions within the gene promoters in hc. the pattern of pgm1 and eno3 gene promoter methylation suggests a possibility of ones to be used for the hc diagnosis and development new strategies for therapy. this work was financially supported by grant mк-8047.2016 .4 from the president of the russian federation. the work was performed using the equipment of eimb ras 'genome' center. introduction: the development of stomach cancer is a multifactorial and complex process and includes multiple epigenetic, genetic alterations and dietary/non-dietary factors. iodine as an antioxidant may play a protective role against gastric cancer. the aim of this study was to investigate the changes in iodine level in rat with stomach cancer induced by n-methyl-n 0 -nitro-n-nitrosoguanidine (mnng). materials and methods: a total of 62 sprague dawley rats were randomly divided into six groups. 55 rats were administered with mnng (200 lg/ml) by oral gavage on days 0, 14 and 21 to initiate stomach cancer. during the experiment, 28 rats died and those surviving were sacrificed in the 3rd, 5th, 7th, 10th and 12th months of the experimental period (group i, ii, iii, iv, v, respectively). the control group (group vi) contains 7 rat which are given only food and water for 12 months. the stomach tissue was examined histopathologically. and also, iodine levels in stomach tissue was determined using the foss method. results: a decrease in iodine level was determined in stomach cancer tissue of rats in group i-v compared with normal healthy stomach tissue in group vi. when the control (group vi) iodine level was taken as % baseline, the % iodine levels of all groups were determined as follows 71.78, 52.79, 40.76, 25.33 and 11.96 for groups i-v, respectively. the pathological diagnosis of gastric cancer was adenocarcinoma. discussion and conclusion: the iodine levels of group i were higher than those of group ii (p < 0.01) and of groups iii, iv and v (p < 0.001) and also were lower than in the control group (p < 0.001). iodine deficiency as one of the risk factors of stomach cancer strongly supports the necessity for the application of effective iodine prophylaxis in the areas with iodine deficiency. iodine supplementation might be useful in stomach cancer therapy and therefore, further research is warranted. this study was supported by ataturk university (project number: 2005/147). effect of water extract of turkish propolis on mitochondrial membrane potential in human laryngeal epidermoid carcinoma cell lines propolis is the generic name for the resinous substance collected by honeybees from the buds of various plant sources and it is used by bees to seal holes in their honeycombs, smooth out the internal walls, and protect the entrance of bee hive against intruders. the aim of this study is to investigate what kind of changes the turkish propolis cause on mitochondrial membrane potential (mmp) of human laryngeal epidermoid cell lines (hep-2), by considering its anticancer features. water extract of turkish propolis (wep, 1-3 mg/ml) and ethanolic extract of turkish propolis (eep, 0.075-3 mg/ml) were prepared and incubated with hep-2 cell lines (24, 48, and 72 h). mmp was investigated with a flourometric method by using dioc 6 (3,3 0 -dihexyloxacarbocyanine iodide). the most significant mmp decrease was seen on 72rd hour. both wep and eep extracts at all concentrations decrease mmp according to that of control. the recent studies have shown that propolis extracts have induced apoptotic cell death by decreasing mitochondrial membrane potential in various cancer cells. it was concluded that both wep and eep decreased mitochondrial membrane potentials on hep-2 cell series according to control (0 concentration) depending concentration and time. there are numerous transcription factors involved in the regulation of the inducible gene expression. thus, transcription of proinflammatory genes, steroid hormone receptors, etc. is controlled by the group of factors triggering gene expression which includes nf-kb. another group of factors is involved in the formation of the structure of the chromatin of the inducible genes regulatory regions, providing competence for gene expression. it is expected that this group of factors includes the proteins of nf1 (nuclear factor 1) family. there are few data suggesting that the nf1 factors maintain potentially active state of the chromatin of the hormone-dependent gene promoter regions. these findings initiated studies of the correlation between presence of the nf1 transcription factors on the chromatin of a gene regulatory region and the functional state of the gene in vivo. as a model we chose the rat tryptophan dioxygenase (tdo) gene which is expressed tissue-specifically in the liver under control of glucocorticoid hormones. three constitutive dnase i-hypersensitive regions are identified in the regulatory region of this gene. to conduct the study we used rat liver and kidney. the basic methods were electrophoretic mobility shift assay (emsa), immunoblotting assay and chromatin immunoprecipitation combined with real-time pcr (chip-qpcr). using emsa we found that the proteins of nf1 family interact with the constitutive dnase i-hypersensitive regions in vitro. immunoblotting assay of the protein fraction from rat liver used in emsa experiments showed the presence of the nf1-b1 isoform. chip-qpcr revealed statistically significant differences in the level of the factor nf1 enrichment of the tdo gene regulatory region between the rat liver and kidneys at p < 0.05. these data suggest the involvement of the nf1 proteins in the formation of the chromatin structure of the rat tdo gene promoter region. reciprocal (9;22) translocation and bcr-abl fusion protein that is responsible for developing leukemia are observed in more than 95% of chronic myeloid leukemia (cml) cases. epigallocathecin-3-gallate (egcg) is a green-tea flavonoid and egcg is proposed as a natural anti-cancer agent. histone modifications which contain histone deacetylases (hdac) and histone acetyltransferases (hat) are parts of epigenetic regulations. hdacs play important roles in different human malignancies including leukemia via activation of abnormal signaling pathways. hdac inhibitors have become remarkable therapeutic molecules for malignancies. the aim of this study is to determine the expression changes of leukemia-related hdacs with the treatment of egcg in k-562 cells. the cytotoxic effect of egcg on k-562 cells was determined in time and dose dependent manner by wst-1 analysis. total rna was isolated from k-562 cells. reverse transcription procedure was performed for cdna synthesis and gene expressions were detected by rt-qpcr. the expression level of hdac4, hdac6, hdac11 gene that supports cell proliferation was down-regulated 2.29, 2.56, 3.81 folds in k-562 cells treated with ic50 dose of egcg, according to control, respectively. our current findings suggest that is a polyphenol egcg may be a hopeful agent in treatment of cml by hdac inhibitory effect. chronic lymphocytic leukemia (cll) is a disorder of morphologically mature but immunologically less mature lymphocytes and is manifested by progressive accumulation of these cells in the blood, bone marrow, and lymphatic tissues. carbonic anhydrase (ca) is a metalloenzyme which is widely distributed in the living world, and it is essential for the regulation of acid-base balance. anti-ca antibodies have been reported in many disorders, such as systemic lupus erythematosus, sj€ ogren's syndrome, rheumatoid arthritis, endometriosis, idiopathic chronic pancreatitis, type 1 diabetes and graves' disease. the goal of this study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in cll. 38 patients with cll and 39 healthy controls were included in the study and ca i and ii autoantibody levels were investigated by elisa. the ca i autoantibody levels of cll group were significantly higher than the healthy group (p = 0.006) while there was no statistical difference between serum ca ii autoantibody levels of the groups (p = 0.301). we found a significant positive correlation between hemoglobin and hemotocrit levels in patients with cll (r = 0.931, p = 0.0001). cut-off value of 0.072 absu for anti-ca i was associated with 92% sensitivity and 41% specificity and a cut-off value of 0.047 absu for anti-ca ii was associated with 48% sensitivity and 85% specificity for predicting cll. the ca i autoantibody levels in patients with cll were found higher compared to control group and the results suggest that ca i autoantibody may be involved in the pathogenesis of cll. genetic and epigenetic aberrations can lead to the activation of oncogenes and inactivation of tumor-suppressor genes (tsgs) followed by the development of malignant tumors. in the present work we evaluated the frequency of alterations of cpg island methylation and dna copy number in 47 paired (tumor/normal) breast cancer (bc) samples using comparative dna hybridization on noti-microarrays and original niman software. the microarrays contained 180 noti-clones associated with 188 chromosome 3 genes. expression alterations were assessed with the use of qpcr technique, ddct method and original atg software. in total, 35 noti-sites with high (15-38% of cases) hypermethylation/deletion (hm/d) frequency were revealed in bc. among genes associated with these sites, there are both known tsgs and tsg-candidates (aldh1l1, vhl, ctdspl, etc.) as well as genes, which involvement in breast oncogenesis was shown for the first time (lrrn1, foxp1, prickle2, etc.). noti-microarray data were verified selectively using bisulfite sequencing for vhl, nkiras1, itga9, lrrc3b, and ctdspl genes. several genes with high hm/d frequency (aldh1l1, ephb1, itga9, and ropn1) were tested for expression alterations using qpcr. frequent (57-90% of cases) and significant (> 2-fold) down-regulation was shown for all of them in bc. the most significant expression loss was observed for aldh1l1 geneon the average 50-fold mrna level decrease in 90% of samples. the involvement of the majority of genes with high hm/d frequency in breast oncogenesis was shown for the first time. these genes are novel tsg-candidates in bc. functional hypermethylation associated with expression loss was shown for aldh1l1, ephb1, itga9, and ropn1 genes thereby strengthening the speculation on tumor suppressor abilities of these genes. methylation and expression analyses of genes, that were revealed by noti-microarrays, were financially supported by grant 14-15-00654 from the russian science foundation. functional hypermethylation of a number of chromosome 3 genes was revealed in colon cancer using noti-microarrays cancer is a disease of genome caused by genetic and epigenetic aberrations. noti-microarrays, that were developed by prof. e.r. zabarovsky, is a unique tool that allows us to simultaneously detect hypermethylation of cpg islands and dna deletionstwo major reasons of inactivation of tumor suppressor genes (tsgs). in the present work, the frequency of chromosome 3 genetic and epigenetic alterations in colon cancer (cc) was evaluated. noti-microarrays, that contained 180 noti-clones associated with 188 chromosome 3 genes, were used for comparative (tumor/normal) hybridization of dna from 24 paired cc samples. data analysis was performed using original niman software. expression alterations were evaluated using qpcr technique and original atg software. in total, 24 noti-sites with 20% and above hypermethylation/ deletion (hm/d) frequency were revealed in cc. among genes associated with these sites, there are several known tsgs and tsg-candidates (for example, vhl, ctdspl, and itga9), but for the majority of genes, involvement in colon oncogenesis was shown for the first time (for example, lrrn1, nbeal2, and ube2e2). the highest hm/d frequency was observed for ankrd28, nkiras1/rpl15, itga9, cmtm6, and gor-asp1/ttc21a genes -38-42%. expression alterations were evaluated for 3 genes with high hm/d frequency (plcl2, prickle2, and ppp2r3a) and significant mrna level decrease (> 2-fold) associated with hypermethylation was shown for all of them in the majority of samples. a number of novel potential tsg-candidates was revealed in cc. functional hypermethylation associated with expression decrease was shown for plcl2, prickle2, and ppp2r3a genes thereby enhancing the suggestion on tumor suppressor function of these genes. this work was financially supported by in many countries, radon is the second leading cause of lung cancer, which accounts from 3% to 14% of cases. it is obvious that the population of all the developed and industrial countries in the world spend most f their time, almost 80%. therefore it is necessary to explore the obtained radiation dose, because of the presence of radon in a room due to the radon emanation from the soil and exhalation from a variety of building materials. the developed countries solve this problem of radon pollution as well as create a special monitoring services. the paper presents some data of 8 genes molecular-genetic analysis from patients with lung cancer who live in almaty located in a foothill area of tectonic faults. the object of research were blood samples obtained from patients diagnosed with lung cancer who are receiving a treatment at the almaty oncology center and living in the city of almaty, where the level of radon activity exceeds the norm approved by the international commission on radiation safety. as a control group relatively people living in the plains, characterized by a lower radon emanation have been considered. to determine mutations in the genes polymerase chain reaction with a subsequent analysis of restriction fragment length polymorphism has been conducted. the pcr products were subjected to hydrolysis by bstni restriction endonucleases haeiii, ras i. disturbances in the genes under consideration to variour types of cancer development. the analysis showed that examinees do not have mutations in the kras gene codons 12-13, which corresponds to a control group consisting of 90 people living in the city of balkhash. on the whole, molecular genetic studies have shown that 44 examined patients do not have mutations in the kras gene. one mutation was been found in the egfr gene. aim: polyps are abnormal growths of tissue that can be found in gastro intestinal system. they are most often found in the colon and rectum. most polyps are noncancerous (benign) however, because of abnormal cell growth, they can eventually become cancerous. the aim of this study is to determine the concentrations of trace element contents in colon and rectum polyp tissues and whether there is any relationship between polyp tissue element levels and the disease. material and method: the present study was conducted on total of 82 individuals including 57 patients and 25 healthy subjects. while receiving normal intestinal tissue from healthy control group; from the patient group both normal tissue and polyp samples were taken during colonoscopy procedure. the concentrations of the elements (al, cr, mn, fe, co, ni, cu, zn, as, se, ag, cd, hg and pb) were determined with induced coupled plasma-mass spectrometer. results: the mean concentrations of cr, mn, ni, se and ag in colorectal polyp tissues of patients were significantly higher than in colorectal tissues of control subjects (p is less than 0.05). on the other hand the mean concentration of cd and pb in colorectal polyp tissues of patients were significantly lower than in control colorectal tissues of control subjects (p is less than 0.05). there was no any significant difference between the groups in terms of concentrations of al, fe, co, zn, as and hg (p is more than 0.05). conclusion: the differences found in some elements between polyps and a control tissues may provide an indication about the role of trace elements in the early stage (polyps) in the colon carcinogenic process and encourages further studies to confirm the involvement of such elements in neoplastic processes. the use of herbal medicines is steadily growing, with approximately 40% of the population use herbs to treat various illnesses in the western world. vitex agnus-castus has been used since ancient times as a remedy. the aim of this study was to investigate the in vitro anticancer activities of vitexagnus-castus oil. for this purpose, the cytotoxicity of vitexagnus-castus oil in sh-sy5y cells was investigated by crystal violet staining. ec50 was found to be 0.5%(w/w) vitexagnus-castus oil for this cell line. this dose was applied to the cell for 24 h, and the cells were harvested for further studies. vitex agnus-castus oil treatment increased bax and p38 mrna levels. on the other hand, bcl-2, bcl2 l1, erk-2, jnk, caspase 3 and 8mrna expression levels were reduced significantly withvitexagnus-castus oil treatment while p53 and pten remained unchanged. these results indicate that another effector caspase such as caspase 6 or 7 may be involved apoptosis process which remains to be elucidated. moreover, mapk pathways, p38 and erk, may be involved in vitexagnus-castus oil induced apoptosis in sh-sy5y cells. these initial observations suggest that this agent might not be useful in treating cancers. further detailed studies should be carried out to elucidate the exact mechanism of vitexagnus-castus oil in neuroblastoma cell lines. melanoma is a skin cancer with a melanocyte origin that can occur in any part of the body that contain melanocytes. while melanoma is less common than other skin cancers, it causes the majority of deaths related to skin cancer. several gene expression databases have shown that interferon regulatory factor 4 (irf4) is upregulated in melanomas, and genome wide association studies linked variation at irf4 locus with skin cancers. irf4 was first identified to have roles in lymphocyte development and function. studies have identified a 'non-oncogene addiction' of malignant cells to irf4 in various hematopoietic cancer types. the aim of this study is to investigate the role of irf4 in melanoma cell lines. lentiviral vectors were used to reduce irf4 levels in melanoma cell lines. a gfp competition assay was performed to study the competitive fitness of melanoma cells with irf4 knockdown (gfp positive cells) over melanoma cells with normal irf4 levels (gfp negative cells). cell cycle profiles were investigated in melanoma cells with irf4 knockdown by propidium iodide staining. migration potential was assessed as well by wound healing assay. our preliminary data showed a decreased competitive fitness for cells with decreased irf4 levels. cell cycle profiling showed increased g0/g1 and decreased g2/m levels in irf4 knockdown cells compared to controls. wound healing assay results showed no difference between controls for cells with reduced irf4 levels. taken together, these results indicate that irf4 knockdown affects the melanoma cell lines' survival and cell cycle profile, suggesting a non-oncogene addiction of melanomas to irf4. these observations are largely similar to previous observations in hematopoietic cancers. unravelling the role of irf4 in melanoma will increase our knowledge about melanoma development and progression and thereby may lead to targeted therapy in melanoma treatment. humans are exposed to various chemicals having beneficial or toxic effects at a time in their daily lives. 7,12-dimethylbenz[a] anthracene (dmba) is a carcinogenic compound produced during the incomplete combustion of carbon-containing compounds. endosulfan is an organochlorine pesticide used against insects on food. morin is an antioxidant, antiinflammatory and chemoprotective flavonoid. this study is aimed to determine the effect of morin in the presence of dmba and endosulfan. for this purpose, 56 adult wistar male rats weighing 170-255 g were randomly selected and divided into eight groups. 25 mg/kg body weight (b.wt.) morin and 5.0 mg/kg b.wt. endosulfan were given to morin and endosulfan treated groups three times in a week. the rats in dmba treated groups were gavaged with 30.0 mg/kg b.wt. dmba three times during the administration period (54 days). cytochrome p4501a (cyp1a) associated 7-ethoxyresorufin o-deethylase (erod) and glutathione s-transferase (gst) activities were measured in rat liver cytosols and microsomes. in addition, liver tissues were evaluated by histopathological analysis. erod activities of control, morin, endosulfan, dmba, morin+endosulfan, morin+dmba, dmba+endosulfan and morin+dmba+endosulfan groups were 71 ae 7, 112 ae 6, 114 ae 8, 126 ae 6, 121 ae 9, 156 ae 12, 151 ae 15 and 185 ae 13 pmol/min/mg protein, respectively. all treatments increased erod activities. gst activities of these groups were 365 ae 14, 430 ae 27, 365 ae 24, 651 ae 57, 460 ae 10, 577 ae 25, 585 ae 23 and 649 ae 33 nmol/min/mg protein, respectively. histopathological studies showed that endosulfan and dmba induced inflammation in the liver tissues and morin reduced their effects. in conclusion, morin treatment increased the metabolism of dmba and endosulfan by inducing cyp1a activity. gst activities of morin+dmba+endosulfan group were not significantly different from those of dmba group. histopathological studies indicated that morin administration reduced the toxic effect of endosulfan and dmba in the liver cells. hepatocellular carcinoma (hcc) is the sixth most common cancer and third most frequent cause of cancer-related death worldwide. molecular mechanisms of hepatocarcinogenesis is still unclear. the impairment of epigenetic mechanisms is implicated in the development of multiple cancers, including hcc. transforming growth factor-beta has been shown to play both tumorsuppressive and tumor promoting roles. transforming growth factor-beta signaling pathway involves activation of smad2 and smad3 by the type i receptor and formation of smad2/3/4 heteromeric complexes that enter the nucleus to regulate transcription. 3-deazaneplanocin a is an inhibitor of the histone methyltransferase ezh2. we aimed to reveal the effect of 3-deazaneplanocin a on transforming growth factor-beta /smad pathway in hepg2 cell line. hepg2, a human liver cancer cell line cultured in dulbecco's minimal essential medium supplemented with 10% fbs. the cells were seeded the day before 3-deazaneplanocin a administration and then the cells were treated with 5 lm 3-deazaneplanocin a for 3 days. expression levels of genes were analyzed by roche lightcyclerò 480. gapdh was used as housekeeping gene. apoptosis assay was performed by the muse annexin v and dead cell assay kit. the unpaired t-test was used to compare variables and p < 0.05 was accepted as statistically significant. 3-deazaneplanocin treatment was significantly reduced transforming growth factor-beta, smads 2-7 in hepg2 cells (p < 0.05). we also found that 3-deazaneplanocin induces apoptosis in treated cell line (p < 0.05). as a result, 3-deazaneplanocin a may take place in treatment of hepatocellular cancer by its inhibitory effect on transforming growth factor-beta /smad pathway and inducing apoptosis in liver cancer cells. brefeldin a (bfa) is a lactone antibiotic first isolated from the fungus eupenicillium brefeldianium. bfa inhibits the transport of secreted proteins from endoplasmic reticulum (er) to golgi apparatus, leading to disruption of golgi function, accumulation of unfolded and not fully incompletely processed proteins in er. bfa also inhibits cell proliferation, phosphorylation and migration of cancer cells. therefore in this study, we investigated the effects of bfa on breast cancer cell proliferation of various phenotypes. in we observed that bfa inhibited the proliferation of all three phenotypes of breast cancer cells, but the effects of bfa were seen at different times and doses. according to time and dose, bfa was observed more effective to mcf-7 compared to other cell lines. physiological, pathological and physical factors. moreover, nlr may represent the two opposing inflammatory and immune pathways that exist together in cancer patients. we aimed to investigate nlr in breast cancer in our population. methods: using data retrieved from the medical records, 66 women diagnosed primary breast cancer met our study inclusion criteria as they had a complete blood count with leukocyte differential performed before any anti-cancer therapy. and 44 women with benign mammary neoplasm/disease, followed up in the outpatient clinics of mammary disease and confirmed with sonographical/histopathological examination, made up our controls. exclusion criteria included laboratory evidence of white blood cells count (wbc) > 10.5 9 10 9 /l. differential leukocyte counts were obtained by bc 6800 (mindray medical international ltd., china), we examined wbc, neutrophil, lymphocyte, platelet counts, and hematocrite, nlr, mean platelet volume values. results: although there is lack of evaluation of tumor-associated neutrophils and lymphocytes, higher nlr median values and lower lymphocyte mean counts (lymphopenia) were shown in women with breast cancer (p < 0.0001). there was a weak negative correlation in breast cancer between nlr values and platelet counts (r s = à0.274; p = 0.026). holmboe] is distributed throughout southern mediterranean europe from spain to the eastern mediterranean on anatolian peninsula of turkey. present study was designed to investigate the in vitro anti-cancer activities of turkish black pine essential oil. the essential oil was extracted by steam-hydrodistillation and its chemical composition analyzed by gc-ms. the major components of the essential oil were a-pinene, b-pinene and trans-b-caryophyllene, respectively. the crystal violet staining method was used to investigate the cytotoxicity of essential oil in sh-sy5y cells. ec50 was found to be 0.75% (w/w) essential oil for sh-sy5y cells. neuroblastoma cells were incubated at 37°c for 24 h. after 24 h, cells were harvested for further studies. bax and p38 mrna levels were significantly elevated in essential oiltreated cells. on the other hand, bcl-2, bcl2 l1, casp-3, casp-8, erk-2 and jnk expression were significantly downregulated. unlike these proteins, p53 and pten mrnas were not changed. in this study, apoptosis was enhanced by turkish black pine essential oil treatment which was activated by the involvement of another effector caspase subfamily, like casp-6 and casp-7. additionally, erk and p38 mapks may be associated with upregulation of the level of bax. based on these results, we suggest that p. nigra subsp. pallasiana essential oil might not be well-suited in cancer treatment. however, further detailed research is necessary to establish the exact role of p. nigra subsp. pallasiana essential oil in sh-sy5y cells. p-05.02.2-026 the protective effect of newly derivatized compound naringenin-oxime and relative to naringenin against cisplatin-induced nephrotoxicity and genotoxicity in rat background: the aim of this study was to evaluate the possible protective effect potentials of newly derivatized compound naringenin-oxime (ng-ox) relative to efficacy of free naringenin (ng) on cisplatin (cis) induced nephrotoxicity and genotoxiticity in rat. methods: totally, fifty six male wistar albino rats were equally divided into eight groups as follows: control; cis treatment (7 mg/kg b.w., i.p.), ng and ng-ox (20 mg/kg b.w., i.p daily for 10 days) alone treatment; cis + ng (20 or 40 mg/kg b.w., i.p daily for 10 days) and cis+ng-ox (20 or 40 mg/kg b.w., i.p daily for 10 days) combination treatment. at the end of the study total antioxidant capacity (tac) levels, total oxidant status (tos), lipid peroxidation (lpo), total thiol, catalase (cat) were studied in homogenate kidney. peripheral lymphocyte cell dna damage was investigated with comet assay results: the results suggest that cis induces oxidative stress resulting in increased tos and lpo reduction thiol, tac and cat in kidney and increased peripheral lymphocyte cell dna damage. the treatment with naringenin and naringenin oxime alone or with cis treatment showed a protective effect against the toxic influence of cp on peroxidation of the membrane lipids and an altering of the total thiol status in the kidney of rats. from our results we conclude that naringenin and naringenin oxime functions as a potent antioxidant and suggest that it can control cp-induced nephrotoxicity and genototoxicity and ng-ox was found more protective than that of ng on cisplatin induced toxicity in rats. keywords: naringenin, naringenin-oxime, antinephrotoxic, antigenotoxic, comet assay. introduction: oxidative damage is considered to play a pivotal role in ageing, several degenerative diseases, and carcinogenesis. lung cancer is the most common type of cancer, resulting in over 1.3 million deaths each year worldwide. accurate and reliable determination of superoxide radicals has been widely investigated using spectrophotometric, electrochemical, amperometric, polarimetric, piezoelectric technologies. among these methods, electrochemical detection is a most promising approach to achieve accurate, separate and rapid superoxide radicals monitoring with using biosensor system. materials and methods: we used a new technic for detecting superoxide radicals in samples. superoxide dismutase (sod) enzyme immobilized on the surface of gold electrode with the help of gelatin, bovin serum albumin (bsa) and glutaraldehyde (ga) crosslinker. for the biosensor preparing benzoquinone selected as a mediator in working buffer and measurements were carried out at à0.7 v. result: for the optimization studies, effect of the bsa, gelatin, glutaraldehyde, ph, buffer concentration on biosensor response. characterization of the biosensor commitment to the work process and answer reproducibility were evaluated. the analytical characteristic of the biosensor were evaluated by measuring the steady state current response to superoxide radical concentrations. the electrochemical response of the enzyme electrode was linearity gradually leveled of at higher concentration. we found that crosslinking of the sod (e.c.1.15.1.1) with glutaraldehyde could be achieved over a wide range of relative mole ratios in 50 mm phosphate buffer at ph 7.5, glutaraldehyde concentration of %2.5. discuss and conclusion: in this study, a new technique for developed sod biosensors has been developed, which features effective combination of sod/gelatin/bsa/ga modified electrode, trapping of sod and glutaraldehyde cross-linking. this technique is reliable and cost effective. the effect of astaxanthin on apoptosis and cell arrest in u87 brain cancer cell line f. s€ og€ utl€ u, b. € ozmen yelken, c ß . kayabasi, a. asik, s. gonca, r. gasimli, s. yilmaz s€ usl€ uer, c ß . biray avci, c. g€ und€ uz department of medical biology, izmir, turkey a brain tumor is a collection, or mass, of abnormal cells in your brain. brain tumors can be cancerous (malignant) or non-cancerous (benign). the brain is one of the least accessible organ that active pharmacological compounds cannot be delivered. the two physiological barriers control and block the entry and exit of endogenous, exogenous compounds. one of these is the bloodbrain barrier and the other is the blood-cerebrospinal fluid barrier. this structures maintain protection of the brain. when there is a cancer case, it can lead to problem. astaxanthin with potent antioxidant properties can cross blood-brain barrier. in our study, we aimed to evaluate the effects of astaxanthin on apoptosis, cell cycle and also migration in brain cancer cell line. in present study, xcelligence real-time cell analyzer was used so as to determine cytotoxic effect of astaxanthin in u87 cell line. changes of apoptosis and cell cycle in u87 cell line exposured to ic 50 dose of astaxanthin (19.5 nm-80 lm) are detected with annexin v-egfp apoptosis detection kit and cycle test plus dna reagent kit with facs, respectively. the result of apoptosis and cell cycle test was analysed in flow cytometry. the group to which active substance was not treated was used as controlled. the wound healing assay performed in order to measure migration ability of u87 cell line to which astaxanthin was treated or not. ic 50 dose of astaxanthin was calculated as 9.20 lm at 48 h by xcelligence rtca sp based on time and dose. astaxanthin decreased the migration ability at rate of 25% in u87 cells treated by ic 50 dose of astaxanthin. astaxanthin had no apoptotic effect on viability in u87 cell line and astaxanthin caused an increase of g2/m phase arrest (1.15 fold) and s phase arrest (1.41 fold). astaxanthin has cytotoxic effects in brain cancer. it determined that astaxantin decreases cell cycle potential at g2/m even a little. the effect of anticancer of astaxanthin should be researched further. interferon regulatory factor 4 (irf4) is a critical transcription factor in development and survival of different cell types including immune cells and melanocytes. furthermore, it has been demonstrated that irf4 expression levels are elevated in several lymphoid cancers, and irf4 is one of the key transcription factors for the survival of these cancers. several genome-wide association studies identified irf4-linked genetic variants to increased melanoma incidence. in addition results from our lab and elsewhere have shown high levels of irf4 expression in melanoma cell lines. furthermore our preliminary results suggest melanoma cells are sensitive to irf4 expression levels. however, there are no published studies about irf4 target genes in melanoma cells. in this study, we are investigating the genome-wide target genes of irf4 in melanoma cell lines via high-throughput sequencing of immunoprecipitated chromatin (chip-seq). we have identified possible irf4 binding regions in loci with known key roles in development of melanocytes from neural-crest cells. one such key factor is mitf, which is the master regulator in melanocyte development and also plays critical roles in melanoma. integrating chip-seq and rna-seq data suggests irf4 as a transcriptional regulator of genes related to progression of melanoma. objectives: aim of this study was to evaluate prognostic importance of selected laboratory parameters (c-reactive protein (crp), gama glutamiltransferaz, ferritin (fer), potassium, chloride, calcium, phosphorus, magnesium, total protein, aspartat aminotransferaz, alanin aminotransferaz (alt), ifn-c, il-6, tnf-a) in non-small cell lung cancer (nsclc). material and methods: 20 patients with nsclc who were treated with chemoradiotherapy (crt) prospectively evaluated. all patients were newly diagnosed tumour. heparinized blood samples were taken from the patients before and after the completion of crt. fer analyzed by chemiluminescence method on beckman coulter dxi 800; ifn-c, il-6, tnf-a were analyzed with elisa kits (boster biological technology) and other biochemical parameters analyzed on abbott architect c16000. post-crt and pre-crt levels compared with survival. results: the lr cox regression analysis revealed that pre-crt ferritin was significantly associated with survival of patients with nsclc (hazard ratio (hr) = 1.007, p = 0.023, 95%ci; 1.001-1.013). it was also demonstrated by lr cox regression analysis, high levels of pre-crt crp was associated with worse outcome of patients (hr = 1.017, 95%ci;1.001-1.032, p = 0.035). after crt, mean alt level was determined as 16.71. there was survival difference in nsclc patients with high post-crt alt (hr = 0.912, 95%ci; 0.841-0.990, p = 0.027). conclusions: there exists a clinically relevant relationship between pre-crt fer concentration and the prognosis of survival in patients with nsclc. elevated fer is the result of inflammation rather than body iron overload. ferritin showed negative correlation with survival so it could be a useful biomarker to indicate bad prognosis of the patients with nsclc. additionally, crp which is easy to detect and feasible for the use in the routine clinical practice should be considered in the prognosis of nsclc patients. keywords: ferritin, nonsmall cell lung cancer, survival, c-reactive protein. epigenetic therapy tries to reverse the aberrations followed to the disruption of the balance of the epigenetic signaling ways through the use of natural and synthetic compounds, active on specific targets, such as dna methyltransferases (dnmts). we previously synthesized some benzoxazole and benzamide derivatives which might have anticancer activities on account of their heterocyclic structure. our studies showed that not only these compounds caused selective cytotoxicity towards cancer cells (hela) with little or no toxicity on normal cells (l929) but also were not genotoxic. in this study, we aimed to test whether these compounds changed global demethylation profile of normal and cancer cells. we used methylation specific comet assay (msc assay) to determine global methylation levels of cells. cells were treated with the tested compounds at ic 50 concentrations for 48 h. slides were prepared as did in alkaline comet assay, then they were incubated with methylation specific restriction enzymes (mspi, hpaii) before electrophoresis. differences in global methylation levels between nontreated control cells and cells treated with compounds were compared by using tail moment data. 5-aza-c, a demethylating agent, was used as reference drug. msc assay results revealed that none of the tested 9 compounds caused hypermethylation on both cell lines. however, global methylation levels decreased statistically (p < 0.05) through both cells treated with c-2 and c-8. only c-3 decreased methylation level on l929 but not on hela. consequently, c-2 and c-8 caused demethylation on hela cells similarly with 5-aza-c at low concentrations. for the reason that dna methylation is regulated mainly dnmt enzymes in the cell, c-2 and c-8 might cause global demethylation in the cell by inhibiting dnmt activity. further studies will be done to support this prediction. overall, macrophages and some subtypes of lymphoid cells are found in tumour stroma. these cells secrete a variety of growth factors, proinflammatory cytokines and chemokines, esp. tnf-a, il-1b and il-6, causing the formation of inflammatory microenvironment around tumour cells. tnf-a and il-1b signaling increases activity of nf-kb pathway. at the same time, il-6, triggers jak-stat signaling pathway, which effector is stat3. nf-kb and stat3 activity facilitates hyperexpression of mir-nas mir-155, mir-181 and mir-21 as well as down-regulates expression of mirnas mir-15/16, mir-199 and let-7. this investigation aims to identify in what way these shifts in mirnaome can lead to epigenome reorganization supporting the cell transformation. mirna targets within gene transcripts were predicted in silico using targetscan software. transcripts of hdac2/4/8/9 and sirt1/5 genes encoding histone deacetylases carry targets for at least one of up-regulated mirnas mir-155, mir-181 or mir-21. also, these mirnas can silence ezh1, mll, mll3, nsd1, setd6/7/8, smyd1, suv39h2 genes encoding histone methyltransferases. mirna mir-21 suppresses gene encoding de novo dna methyltransferase dnmt3b. at the same time, down-regulation of mirna mir-15/16 can allow hyperexpression of gene encoding acetyltransferase elp3. these shifts impair dna and histone methylation, cause the increase of overall level of chromatin acetylation and expression and, therefore, create epigenetic background for reactivation of silent transposons, oncogenes as well as other genes important for cell transformation. immune system can paradoxically facilitate the tumour growth instead of healing. cancer-related inflammation leads to the mir-naome and epigenome shifts contributing to the tumour promotion and progression. lysine acetylation is one of the key mechanisms to regulate chromatin structure and transcriptional activation. acetyl-lysine modifications are recognized by bromodomains, which are small interaction modules found on diverse proteins including histones. among these acetyl-lysine reader proteins is the family of the bet (bromodomain and extra-terminal) proteins which contain tandem bromodomains (bd1 and bd2). the recent discovery of potent and specific inhibitors for the bet family proteins has stimulated intensive research activity in diverse therapeutic areas, especially in oncology, where bet proteins regulate the expression of key oncogenes and anti-apoptootic proteins. several bet inhibitors are currently in clinical trials and reported to exhibit promising clinical activities. however, pleiotropic nature of bet proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. here, we report the recent progress in the development of bet inhibitors in korea research institute of chemical technology (krict). we have identified the bet inhibitors with a novel scaffold different from the previously reported diazepine and azepine scaffolds and specific for first bromodomains (bd1s). a medicinal chemistry effort is currently made to optimize the pharmacokinetic properties of these lead compounds for further drug development. the experimental data from the biochemical and cell-based assays for these bd1-selective bet inhibitors will be presented. family of small c-terminal domain serine phosphatases (scp), which includes ctdspl, ctdsp1, and ctdsp2, plays a regulatory role in a number of vital processes. in particular, it is shown that ctdspl is capable to activate the retinoblastoma protein (rb) which is well-known tumor suppressor and one of the key cell cycle regulators. although the question on whether ctdsp1 and ctdsp2 dephosphorylate rb is open, high similarity of sequences and three-dimensional structures of phosphatases may indicate the similar function of these enzymes. in the current study expression of scp genes was evaluated by quantitative pcr in 28 non-small cell lung cancer (nsclc) samples. using original crosshub software, that combines an analysis of high-throughput sequencing data of the cancer genome atlas project (tcga) and databases of mrna-mirna interactions (targetscan, mirtarbase, etc), the involvement of mir-183-96-182 microrna cluster in co-regulation of ctdspl/1/2 genes in nsclc was predicted. the significant (5-fold on the average) and simultaneous decrease of mrna levels of ctdspl/1/2 genes was revealed in the majority of nsclc samples (82%, 23/28). such unidirectional expression change and strong positive correlation between phosphatase expression levels (r s = 0.53-0.62, p ≤ 0.01) allowed us to suggest a common mechanism of their inactivation. we evaluated the expression of predicted co-regulators of scp gene expression, mir-183-96-182 family, in examined nsclc samples. as a result, the simultaneously increased levels of all three mir-nas in most nsclc samples (82%, 23/28) and negative correlation with phosphatase gene expression was shown. the results suggest the ability of investigated phosphatases to exhibit tumor-suppressive activity and the involvement of mir-183-96-182 micrornas in the regulation of rb protein activity via inactivation of ctdspl/1/2 in nsclc. cancer is one of the leading causes of death in all around the world. cancer is defined as a disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. tumor markers are substances that are produced either directly by the tumor or as an effect of the tumor on healthy tissue. tumor markers can be used for screening, determining prognosis and monitoring effectiveness of therapy and disease recurrence. the aim of this study is to investigate the frequency of tumor markers orders and the appropriateness of these requests. laboratory information systems data for 2015 were reviewed. for 2015, a total of 35874 patients and 55539 tumor marker requests were included. carbohydrate antigen 19-9, cancer antigen 125, cancer antigen 15-3, prostate specific antigen, alphafetoprotein and carcinoembryonic antigen were measured by chemiluminescence method. according to the data from the year of 2015, both positive tumor markertest resultsratio and the positive patient ratio were 14%. in the patients group with increased marker levels, 48% of the patients had no history of cancer. in the patients group with tumor marker levels in referenceranges, 40% patients with diagnosed cancer history in remission. the ratios of positive tumor markers were 13% forca 19-9, 19% for ca 125, 7.9% for psa, 25%for ca 15-3, 8.4%for afp, and 9.56% for cea. in conclusion; unnecessary test requests increase laboratory work load and health expenses. laboratory and clinical staff collaboration is crucial to increase the appropriate use of tumor markers. dna methylation is an epigenetic modification that is involved in both normal biological and disease states. hypermethylation of promoter regions of tumor suppressor genes have a role in tumor development. therefore, the measurement of promoter methylation of genes can be used for diagnosis and prognosis purposes of cancer. to detect dna methylation alterations in a sample (biopsy, blood, saliva, etc.), sensitive detection systems and optimization of the methods are needed. as a part of a collaboration project between national metrology institute of korea (kriss) and national metrology institute of turkiye (tubitak ume). dna methylation status of apc and gstp1 genes were studied. dna methylation measurements were performed using stepone real-time pcr system and results were analyzed using hrm (high resolution melting) software. the parameters effecting the quantification of dna methylation were found as primers, annealing temperature, pcr cycle number, fluorescence dye and the commercial dna methylation standards used for quantification of dna methylation. since, the accurate measurement of dna methylation is very critical in early diagnosis of cancer and choosing the right therapy, optimization of the method is required. cancer is a disease that includes heterogenic and complex molecular changes. anti-carcinogenic effects of resveratrol, a natural polyphenol, have been proved in a variety of cancer cells. considering the effects of resveratrol, the influence of the signal transduction pathways in the presence or absence of p53 of colon cancer cells is gaining importance. our aim was to investigate the effects of resveratrol in the presence or absence of p53 on cell viability, apoptotic cell death ratio and fold changes of proliferative or anti-proliferative gene expressions, which may have important effects on colon cancer, in hct116 colon carcinoma cells. ic50 doses of resveratrol were determined by wst-1 assay. the apoptotic cell death ratios in treatments of resveratrol were determined by annexin-v-fitc/pi assay for flow cytomety . the changes of ccnd1, fra1, ppard, egfr, birc5, pcna, mcl1, stat3, fos, jun, p27, atf4, trail, puma, gadd45a, rb1, faslg, tnf, socs3 , stat1 gene expressions were evaluated by real time pcr. all data were statistically analyzed by student's t test. our research has revealed that resveratrol (60 lm) causes decrease in cell viability and increase in apoptotic cell death in hct116 p53(+/+) and hct116 p53(à/à) cells significantly (p < 0.05). the fold changes of the gene expressions have shown that resveratrol has significant (p < 0.05) and different effects on the expressions of the genes related with the existence of p53 in hct116 cell lines. therefore we proposed that resveratrol might show proliferative or apoptotic effects related with p53 mutation of colon cancer cells and we predicted that unconscious consumption of resveratrol in colon cancer patient might cause adverse effects. introduction: colorectal cancer (crc) is the third most common cancer worldwide. alterations in methylation profiles of tumor suppressor genes (tsgs) have been recognized as a key mechanism in colorectal cancers. in the current study, we investigated the hypermethylation status of 25 tsgs in colorectal cancer tissues. materials and methods: formalin-fixed paraffin-embedded (ffpe) tissue samples obtained from patients with crc. methylation specific-multiplex ligation dependent probe amplification (ms-mlpa) technique was used to assess the methylation status of 25 tsgs. the findings were evaluated in terms of age, mortality, survival, positive lymph node status, lymphovascular invasion, and perineural invasion. results: hypermethylation-detected patients and hypermethylation-undetected patients were called as group 1 and group 2, respectively. hypermethylation was detected in atm, cdkn2a, and gata5 genes. mortality rate was (12.5%) in group 1 and group 2 (p > 0.05). mean 5-years survival rate in group 1 was 45 ae 3 months and mean 5-years survival in group 2 was 44 ae 3 months (p > 0.05). positive median lymph node count was 10 ae 2 for group 1 and 5 ae 1 for group 2 and the difference was statistically significant (p < 0.05). frequencies of perineural invasion and lymphovascular invasion rate in two groups were 25% (p > 0.05). discussion and conclusion: our findings suggest that tsg hypermethylation found in crc patients may increase the lymph node metastasis. further investigations with larger sample size are required to support our results. boron (b) is known to be important for cell replication and development, but the underlying mechanism remains obscure. recently b has also become important in some specific anticancer processes. some recent reports advise using of some boron compounds for the treatment of specific forms of cancer. for instance, boron-based drugs (bortezomib) are now being developed for use as therapeutic agents with anticancer activities and several other boron-based compounds are in various phases of clinical trials. it has been shown that bortezomib disrupts the regulation of cell cycle and induces apoptosis in both hematologic and solid tumor malignancies except for colon carcinoma. colorectal cancer (crc) is the third most common cancer in men and the second in women, accounting for 10% of all tumour types worldwide. cytotoxic effects of boron compounds on crc cells and changing of its effects related with p53 mutation, which is mutated 50% of cancer cases, have not take part in literature yet. for this purpose; the aim of the study was designed to investigate the effects of borax pentahydrate and disodium pentaborate decahydrate compounds on cell viability, apoptotic cell death ratio and parp protein expressions in p53 (+/+) and p53 (à/à) hct116 colon carcinoma cells lines. the effects of the boron compounds on cell viability were assessed by xtt assay and apoptotic effects and parp protein expression of the compounds were evaluated by flow cytometry and western blot analysis respectively. our results showed that borax pentahydrate (6 mm) and disodium pentaborate decahydrate (12 mm) significantly causes nearly 50% reduction of cell viability at 48 h (p < 0.05). apoptotic cell death ratios and parp expressions revealed that both of the compounds might have a potential for a candidate of anticancer agent. epithelial-mesenchymal transition (emt) is a significant event for metastasis, and could be mediated by several pathways such as pi3k/akt, map kinases and many epigenetic regulators. satb2 is an epigenetic regulator involved in emt and osteoblastic differentiation. since preliminary results indicate that there is a crosstalk between p38 and akt pathways in nsclc cells, we aimed to determine whether this crosstalk has a regulatory effects on emt and satb2 expression in nsclc cells. we used a549 and h1650 cells as a model to evaluate the effects of the crosstalk between p38 and akt on emt of nsclc cells. therefore, cell culture, inhibition of p38 activation via sb203580, transient expression assay for (ca-akt), western blot analysis, sirna transfection for satb2, wound healing and invasion assay were performed in this study. firstly, the expression statues of e-cadherin, satb2, p-p38, p38, p-akt and akt was examined in a549 and h1650 cells by western blot analysis. we observed that e-cadherin and satb2 are downregulated in a549 cells (highly active p38, lowly active akt) compared to h1650 cells (lowly active p38, highly active akt), suggesting that e-cadherin and satb2 are associated with the crosstalk between p38 and akt pathways. our results demonstrated that p38 inhibition in a549 cells leads to decreased pten expression and subsequently increased akt activation. then, we found that p38 inhibition upregulated satb2 expression, and reversed emt in a549 cells. furthermore, alone satb2 knockdown is sufficient to induce emt, and prevented the effects of p38 inhibition on emt. all these results strongly indicate that the crosstalk between p38 and akt pathways might determine satb2 expression and epithelial characters of nsclc cells, and satb2 is a critical epigenetic regulator for emt in nsclc cells. therefore, it is also need to explore how p38 and akt signalling pathways could regulate satb2 expression. this work was supported by tubitak (114s007, 215z283). introduction: lung cancer is a disease characterized by uncontrolled cell growth in the lung tissues. the most common causes of lung cancer are tobacco smoke, radon gas, asbestos, air pollution, and genetic factors. nitric oxide (no) has potential mutagenic and carcinogenic activity and may play important roles in lung cancer. endothelial no, synthesized from l-arginine by endothelial no synthase (enos), inhibits apoptosis and promotes angiogenesis and tumor cell proliferation. the aim of the present study was to examine the possible relationship between enos gene intron 4 vntr and exon 7-g894t (glu298asp) the stressful ecosystems exert strong adaptive pressure and proteins that facilitate these adaptation processes are candidate drug targets. nucleotides are the core of biochemical pathway required for cancer cell growth and replication and genetic changes will lead in oscillation in their pools. although it is questionable whether the warburg effect actually causes cancer, impairing dglucose uptake and metabolism induces oxidative metabolism. lproline (lproline) homeostasis is critical in a constellation of human diseases, in parametabolic linkage between cancer, epigenetics (phang et al. 2013 ) and bioenergetics (pallotta 2013) where degradation and biosynthesis are robustly affected by oncogenes or suppressor genes that can modulate intermediates involved in epigenetic regulation. lproline-fueled mitochondrial metabolism involves the oxidative conversion to l-glutamate by a flavin dependent lproline dehydrogenase/oxidase and a nad +dependent l-d 1 -pyrroline-5-carboxylate dehydrogenase. in saccharomyces cerevisiae an important test tube, put1p and put2p respectively help cells to respond to changes in the nutritional microenvironment by initiating lproline breakdown after mitochondrial uptake (pallotta 2014) . in this preclinical study, low molecular weight compounds were tested for inhibiting lproline mitochondrial transport and put1p/put2p catalytic activities. thus, in seeking for natural bioactive compounds targeting lproline pathway and its substrate channeling (becker's group 2016), we report data using in silico screening and in vitro researches in saccharomyces cerevisiae with genetic background atcc18790 but different phenotypic landscape induced by nutritional stress/ ph changes. cells vitality, dψ measurements, nad(p) + /nad(p) h pool and flavine turnover were determined in spectrofluorimeter microplater reader and via hplc (pallotta et al. 1998 (pallotta et al. , 1999 (pallotta et al. , 2004 pallotta 2011; di martino pallotta 2011) thus in supporting of future cancer therapies with decreasing side effects. evaluation of lymphocyte to monocyte ratio (lmr) in patients with colorectal cancer introduction: inflammation may play an important role in cancer progression and a high neutrophil to lymphocyte ratio (nlr) has been reported to be a poor prognostic indicator in several malignancies. the aim of this retrospective study was to evaluate the prognostic value of nlr, lymphocyte to monocyte ratio (lmr) and platelet to lymphocyte ratio (plr) in patients with colorectal cancer (crc). : 107 patients who were diagnosed with colorectal cancer between january 2010 and january 2016; were evaluated retrospectively. the cutoff value was determined using receiver operating characteristics curve analysis. survival analysis was performed using the kaplan-meier method and log-rank test. the cox proportional hazard model was used to identify the influence of factors related to survival. (tnm stage, tumor differentiation, age, tumour size and lmr) results: receiver operating characteristic curves showed that lmr was superior to plr and nlr as a predictive factor in patients with colorectal cancer. the cutoff value for lmr was 1.82. cancer-specific survival was not significantly different between the high-and low-lmr groups (p = 0.786). age was identified as independent prognostic factor in colorectal cancer (hazard ratio: 3.50; 95% confidence interval: 3.14-351.82; p = 0.004). discussion and conclusion: our preliminary study showed that the lmr was not an independent prognostic factor in crc patients, but additional large sample sized prospective studies will be needed to confirm these findings. the aim of this study is to investigate the effects of luteolin treatment on enzymatic activity of arginase, and ornithine and polyamine levels (putrescine, spermidine spermine) in serum and cancer tissues of ehrlich ascites breast cancer model. balb/c female mice were divided randomly into following groups: healthy control, healthy treatment, cancer control, treatment 1 and treatment 2. 0.2 ml ehrlich ascites tumor cells was inoculated subcutaneously to medial part of left hind leg. healthy treatment and treatment 1 groups, and the treatment group 2 were given 5 mg/kg and 10 mg/kg dose of luteolin, intraperitoneally, for a 12 days period, respectively. luteolin has a hydroxylated flavonoid structure and shows potent antioxidant, anti-inflammatory, and anticarcinogenic properties. luteolin not only leads to cell death in various tumors by suppressing cell survival pathways and stimulating apoptotic pathways, but also sensitize them to cytotoxic therapy. supporting various previous studies, tumor implantation to healthy mice resulted in statistically significant elevation of serum arginase and polyamine levels (p < 0.05) indicating the tumor cells as the main source of this production. furthermore, luteolin treatment abolished this increase in serum arginase and polyamine levels (p < 0.05). tissue measurements of arginase and polyamine levels indicated that luteolin treatment resulted with an increase in these parameters of tumor tissue while the serum levels of them showed a significant decrease. our results revealed that increased tissue arginase and polyamine levels might be related with estrogenic agonistic effect of luteolin on utilized tumor model in this experiment; and decreased serum levels of these parameters while there is a significant increase of them in tissue levels might be a result of a suppression of polyamine efflux from the tumor tissue by inhibitory effect of luteolin on plasma membrane polyamine transporters. hepatocellular carcinoma (hcc) is the third most common cause of cancer-related deaths. around 30-40% of hcc patients are diagnosed at an early stage of the disease. hepatic resection, liver transplantation are common strategies in hcc treatment. even if, most of the patients present advanced-stage tumors and have a restricted survival rate. for the reason, resistance against existing tumor stress conditions have been demonstrated in hcc. hypoxia, hyperglycemia are general stress sources in hcc and result in aggressive cell phenotype, resistance to apoptosis and therapeutic drugs. thioredoxin interacting protein (txnip) regulates cellular responses under stress conditions. over-expression of txnip results activation of oxidative stress and apoptosis. in cancer models txnip is considered as a tumor suppressor gene. however, its role in the development, progression of hcc and mechanisms behind it warrant further investigation. in this study expression levels of txnip were examined in 12 hcc cell lines by rt-pcr and western blotting. txnip expression was significantly high in poorly-differentiated snu-182, snu-387 and snu-423 than the well-differentiated hcc cell lines such as huh-7, hepg2 and plc/prf/5. besides, expression of txnip was examined in 16 non-hcc and 79 hcc tissue samples by immunohistochemical staining. txnip positivity was observed in 40% of well and 80% of poor differantiated hcc tissues. however, no txnip positivity was observed in non-hcc tissues. to investigate whether txnip might be involved in biological responses such as cell proliferation, motility and invasion, we used overexpression and silencing strategies. overexpression of txnip minimally inhibited adhesion and proliferation, whereas boyden-chamber motility and invasion assay showed that invasiveness of cells were increased. our findings suggest that txnip expression is increased in hcc and txnip over-expression is important for invasive phenotype during hepatocarcinogenesis. cardiovascular diseases are the leading cause of morbidity and mortality in the western world. it was shown that ischemic tolerance of the heart can be enhanced not only by ischemic or pharmacological conditioning (pre-and postconditioning), but also by adaptation to chronic hypoxia. different studies have indicated that these cardioprotective phenomena may at least partly share the same signaling pathways. the jak/stat signaling pathway has been demonstrated to participate in the development of cardioprotection by conditiong apparently through the inhibition of gsk-3b. the aim of our present study was to determine whether this pathway also takes part in cardioprotection induced by adaptation to chronic hypoxia. we investigated the effect of inhibitor of jak2 kinase (ag-490) on myocardial infarct size and the jak2/stat3 signalling pathway and other effector molecules that may participate in cardioprotection conferred by adaptation to hypoxia. adult male rats were adapted to intermittent normobaric hypoxia (10% o 2 , 3 weeks, 8 h/day) and part of them recieved ag-490 (3 mg/kg) 15 min before ischemia. control rats were kept under normoxia. infarct size was assessed in isolated perfused hearts. relative expression of the key components of the jak2/stat3 signalling system and other proteins was detected using western blotting. preliminary data indicate that administration of the jak2 inhibitor ag-490 caused a significant increase in infarct size in hypoxic rats. western blot analysis revealed changes in phosphorylation of jak2, stat3 and some other proteins involved in cardioprotection (akt, erk1/2, gsk3b). these results suggest that the jak/stat signaling pathway could participate in the development of a cardioprotective phenotype in rats exposed to chronic hypoxia. however, further research will be needed to clarify in more detail the role of this signalling pathway in the cardioprotective mechanism. p-06.01.2-002 detrimental effect of hypertension on myocardium was reversed by liver x receptor agonist gw3965 hypertension is a cardiovascular disease that causes functional and structural changes in the heart. nuclear liver x receptors (lxrs) are involved in the control of cholesterol and lipid metabolism. however, effect of lxr activation on the hypertensive heart is not well characterized. in this study, the effects of lxr agonist gw3965 on hypertension-induced damage of myocardium were investigated. hypertension was induced by deoxycorticosterone acetate (doca) injection (20 mg/kg, twice a week) following the unilateral nephrectomy in male 8-week-old wistar albino rats for 6 weeks. blood pressure was measured by using tail-cuff method. gw3965 (10 mg/kg/day, i.p.) was administered last 1 week. expression of various markers (grp78, perk, p-perk, ikb-a, nf-kb p65, tnf-a, bax, bcl-2, mmp-2) in the ventricular tissue were examined by western blotting. inflammation and fibrosis were evaluated in histopathological examination. gw3965 treatment reduced systolic blood pressure of hypertensive animals. expressions of endoplasmic reticulum stress markers grp78 and p-perk were increased by hypertension and gw3965 treatment reversed them. hypertension-induced increase in nuclear nf-kb p65 expression and decrease in ikb-a expression were reversed by gw3965 treatment. while bcl-2 expression was lower, bax level was higher than control in the hypertensive animals. in hypertensive group, fibrosis marker mmp-2 expression was augmented and gw3965 treatment reversed this elevation. hypertension-induced increase in interstitial and perivascular collagen deposition and inflammatory cell infiltration in left ventricle were prevented by gw3965 treatment. these results suggest that lxr activation by gw3965 restored the hypertension-induced structural changes of heart in the doca-salt hypertension. methylphenidate (mph) is a psychostimulant prescribed for the treatment of attention deficit hyperactivity disorder (adhd), one of the most common neurobehavioral disorders of childhood and adolescence. in fact, despite the widespread use of mph the full comprehension of its cellular/molecular mechanisms is still elusive, including its effect on blood-brain barrier (bbb). this barrier is a key structure in the central nervous system since it protects the brain and its dysfunction has been described as a critical event in several brain diseases. thus, the aim of the present study was to clarify the effects of mph on the bbb function in both physiological and adhd conditions. for that, we used a rat model of adhd, the spontaneously hypertensive (shr) rats, and wistar kyoto (wky) animals as inbred comparator strain. also, to mimic a clinical dosing schedule for adhd treatment, rats were administered for monday-friday with vehicle or mph (1.5 mg/kg/day or 5 mg/ kg/day, per os) from p28-p55. chronic mph treatment (5 mg/kg/day) promoted cortical bbb permeability in both wky and shr animals; however, more prominent in wky rats. this effect can be explained by the downregulation of claudin-5 and collagen-iv, tight junction and basal lamina protein, respectively. noteworthy, wky animals also showed an increase in the expression of caveolin-1 and in both vascular cell and intercelular adhesion molecules. these bbb alterations led to subsequent infiltration of peripheral immune cells, including cd169 + macrophages. furthermore, hippocampal bbb disruption was only observed in wky rats with 5 mg/kg of mph. here, mph decreased collagen iv expression and upregulated caveolin-1, with no alterations in claudin-5. overall, our results show that chronic exposure to mph can led to brain vascular alterations particularly under physiological conditions. this highlights the importance of an appropriate mph dose regimen for adhd, and also that mph misuse can have a negative effect. regulators of g proteins signaling (rgs) serve several cellular functions varying from tolerance, dependence, neuroprotection, transcription and tumorgenesis. despite their initial role as gtpase activating proteins, evidence suggests that rgs proteins are localized in the nucleus, interact with transcription factors thus regulating transcriptional responses. it was shown that rgs4 directly interacts with and interferes in opioid receptor (or) signaling. rgs4 is mostly expressed in brain and is implicated with brain structural alterations; however, the molecular mechanisms of how rgs4 could be involved in cellular differential functions remains unclear. based on these observations we examined whether rgs4 can regulate transcriptional responses mediated by the stat5b transcription factor. isolated neural stem cells from rgs4 à/à mice were immunostained for the mitosis marker ph3 and the mrna levels of antiapoptotic genes were determined. proliferation assays were performed with brdu staining in neuro2a cells stably expressing rgs4. the functional assays of stat5b transcriptional activation were performed in hek293 expressing either the erythropoietin receptor (epo-r) or the delta opioid receptor (d-or). the present data demonstrate that rgs4 blocks stat5b phosphorylation and transcriptional activation by interfering in stat5b heterodimerization upon epo-r or d-or activation triggered by cytokines or opioids administration. lack of functional rgs4 results in increased mrna levels of stat5b target genes such as the members of the bcl anti-apoptotic family bcl-2, bcl-6 and bcl-xl. this upregulation of stat5b inducible gene transcription results in an increased proliferation rate of neural stem cells. this study demonstrates for the first time a non-canonical function of rgs4 in stat5b mediated transcriptional responses and a novel selective role of rgs4 in transcription. role of the pre-molten globule structure in amyloid fibril formation a. eshaghi department of biology, faculty of science, islamic azad university, mashhad branch, mashhad, iran the major factor that caused extensive research on the protein fibrillation is their crucial roles in important diseases known as the amyloidosis diseases. neurodegenerative diseases, including alzheimer's, parkinson's, diabetes and huntington are the most important types of this disease. understanding the mechanisms of fibril formation and ways of treatment can be useful in reducing this type of disorder. in this project, the fibrillation of carbonic anhydrase protein was investigated as a model. carbonic anhydrase creates two stable intermediated known as pre-molten and molten globule, in different ph solution. this protein at ph between ph 3-4 molten globule structures was formed while the pre-molten form took place under ph 3. in our tests at ph 3.5 when the protein in molten globule structures only the amorphous aggregates were formed. instead, at ph 2.4 in pre-molten globule structure amyloid fibrils formed in the protein. there some reports, indicated the protein from pre-molten globule structure go toward amyloid assembly. even intrinsically unstructured proteins such as alpha-synuclein first took a structure similar to pre-molten globule and then made amyloid fibrils. it seems pre-molten globule structure have the major role in promoting to amyloid fibrils. perhaps drugs that prevent the formation of premolten globule structure have an important role in inhibiting amyloid fibrils. identification of compounds preventing the biochemical changes that underlie the epileptogenesis process and understanding the mechanism of their action is of great importance. we have previously shown that myo-inositol (mi) daily treatment for 28 days prevents certain biochemical changes that are triggered by kainic acid (ka)-induced status epilepticus (se), [1, 2] . however in these studies we have not detected any effects of mi on the first day after se. in the presented study we broadened our research and focused on ka induced other early molecular and morphological changes and influence of mi treatment on these changes. the increase in the amount of voltage-dependent anionic channel-1 (vdac-1), mitochondrial-plasma membrane cofilin and caspase-3 activity was observed in the hippocampus of ka treated rats. administration of mi 4 h later after ka treatment abolishes these changes, whereas under the same time schedule diazepam treatment has no significant influence. the number of neuronal cells in ca1 and ca3 subfields of hippocampus is decreased after ka induced se and mi post-treatment significantly lessens this reduction. no significant changes are observed in the neocortex. obtained results indicate that mi post-treatment after ka induced se could successfully target the biochemical processes involved in apoptosis, reduces cell loss and can be successfully used in the future for translational research. references 1. r. 2010. neuroscience letters, vol. 468, no. 3, pp. 277-281. 2. r. solomonia, et al; 2013. cell. mol. neurobiol. vol. 33, no extracellular deposits of amyloid-b peptide (ab) in brain parenchyma via proteolytic processing of amyloid precursor protein (app) are one of the typical characteristics of alzheimer's disease (ad). these aggregates mainly occur as a result of an increase in ab production or a decrease in its degradation. it was found that the neurotoxicity of ab aggregates is accelerated by acetylcholinesterase (ache). besides, ab-ache complex has a prominent neurodegenerative effect in brain. thus, cholinesterase inhibition and preventing ab production are current treatment strategies for ad. recent studies have shown that methylene blue (metb), a cholinesterase inhibitor with phenothiazine structure, inhibits the formation of amyloid plaques and neurofibrillary tangles. azure b, the major metabolite of metb, has been shown to inhibit ache and bche with ic50 values of 0.486 lm and 1.99 lm, respectively. in the present study, we tested whether azure b, may effectively lower the levels of ab 40/42 . we treated chinese hamster ovary cells, which stably express human wild type app and presenilin-1 (ps70) with 0-15 mm azure b or vehicle for 24 h. to determine the effect of azure b treatment on ab 40/42 levels, we used separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. azure b treated ps70 cells were also assessed by propidium iodide in flow cytometry for cellular toxicity. we observed a significant decrease in both extracellular ab 40 and ab 42 levels with a dose range treatment of azure b in ps70 cells. ab 40 levels were reduced by 89.2% in 10 lm and 94.1% in 15 lm azure b-treated cells when compared to control. additionally, ab 42 levels were decreased by 83% in 10 lm and 93.5% in 15 lm azure b-treated cells when compared to control. overall, these preliminary results suggest that azure b may have beneficial effects for the treatment of ad. the effect of green silver nanoparticles (agnps) on the amyloid formation in alphalactalbumin and chaperone action of alphacasein a. ghahghaei, m. dehvari, j. valizadeh formation and deposition of protein fibrillar aggregates in the tissues is associated with several neurodegenerative diseases such as alzheimer's and parkinson's disorders. molecular chaperones are a family of proteins that are believed to have ability for inhibiting protein aggregation. in the present study the effect of different concentrations of green synthesis silver nanoparticles (agnps) from pulicaria undulate l. on the amyloid formation of a-lactalbumin (a-la) and chaperone action of a-casein have been investigated. the effects of the agnps were determined using light scattering absorption, tht binding assay, intrinsic fluorescence assay, ans binding assay, cd spectroscopy and sds-page. light scattering and tht assay results showed that agnps have the ability in preventing aggregation of a-la in a concentration-dependent manner. consistent with these results, sds-page results represented that by increasing the concentration of agnps the adsorption and interaction between agnps and protein have increased. light scattering and tht assay results, also, revealed that the amyloid fibrilation decreased in the presence of both agnps and a s -casein compared to presence of a s -casein alone. fluorescence results, however, show that agnps have no effect on the chaperone ability of a-casein and in fact they perform their protection of protein aggregation action independently. consistent with the above experiments, cd spectroscopy also revealed that agnps have decreased structural changes in reduced a-la in absence and presence of a-casein, both the tertiary and the secondary structure of the proteins. our finding represented that agnps have preventing effects on protein aggregation and have no effect on the chaperone ability of a s -casein. in the main, results of this study show that biosynthesized agnps mediated by >pulicaria undulate l. maybe could be affective as a therapeutic agent for inhibiting aggregation in treatment of amyloidosis disorders. pink1 is a mitochondrial kinase with multiple cellular functions. while loss-of-function mutations of pink1 gene lead to early onset parkinson's disease, its over-expresion is associated with cancer development. parkinson is a multifactorial neurogenerative disease, with a complex aethiology including various cellular stressors. it is now known that genotoxic stress also triggers the release of soluble factors able to induce changes in neighboring cells enhancing the initial lesions, process known as bystander phenomena. althrough the mechanisms are still unclear, recent studies point towards a role for mitochondria in this process. our work investigates pink1 role in intracellular and intercellular stress response, comparatively in various models: fibroblasts (mefs) and neuroblastoma (sh-sy5y) used as a tumoral model or differentiated to a neuronal phenotype. pink1 role in this process was analyzed using genetically engineered pink1 deficient cells exposed to a genotoxic agent, bleomycin. the modified cell lines showed a reduced level of basal atp production. pink1 proved to be involved in cellular vulnerability to stress. despite differences in cellular sensibility between our models, genotoxic treatment of pink1 deficient cells induced consistently higher lesions compared to corresponding wild type variant. pink1 deficient cells showed altered intercellular signaling of stress, impairing bystander phenomena induction, by suppression of signal formation in treated cells, but also by altering the capacity to respond to the signals in neighboring cells. our hypothesis is that pink1 contributes to the management of cellular stress being involved in bystander transmission of detrimental effects through intercellular communication. this is determined mainly by its role in maintaining mitochondrial homeostasy and atp levels, pink1 deficient cells lacking the amount of energy required for rapid dna repair and stress signaling transmission. p-09.02.2-008 intranasal administration of synthetic fragments from receptor for advanced glycation end products prevents memory loss in olfactory bulbectomized mice the receptor for advanced glycation end products (rage) is a member of the immunoglobulin protein superfamily. activation of rage causes brain inflammation, oxidative stress and secretion of beta-amyloid that has been recognized as an essential phase in the development of alzheimer's disease. it is known that the receptor soluble isoform (srage) which lacks the transmembrane and cytosolic domains binds to ligands and prevents negative effects of the receptor activation in in vivo and in vitro experiments. we proposed that potential ligand-binding peptide fragments from srage would demonstrate the same biological activity. we have selected and synthesized 10 peptide fragments from unstructured surface-exposed regions of rage. synthetic peptides were intranasally administrated into olfactory bulbectomized (obe) mice with neurodegeneration of alzheimer's type. we have found that only administration of rage fragment (60-76) effectively prevents the obe murine memory from impairment, leads to decrease of beta-amyloid level and blocks the development of neuronal pathology in the brain of experimental mice. six overlapping fragments of rage (60-76) peptide were synthesized in order to find a site, responding for the therapeutic effect. tests in obe mice carried out with these fragments showed that only the n-terminal part of the molecule is responsible for preventing obe mice memory from impairment. all fragments which do not include n-terminal 60-61 dipeptide have been fully inactive in these experiments. we have proposed that active peptides can interact with beta-amyloid or s100b protein preventing these ligands from binding with rage. this interaction can inhibit the development of neurodegeneration. the aim of this study was to examine effects of social isolation, enriched environment and exercise on learning in rats. the study included 36 female 25 day old wistar rats. the rats were randomly divided into four different groups; control, exercise, social isolation and the enriched environment groups. the social isolation group and the enriched environment group were housed under their specific conditions and the exercise group and the control group were housed in standard conditions during 6 weeks. the rats in the exercise group swam for 6 weeks. after 6 weeks, the rats were evaluated in the morris water maze. brain and blood samples were taken and the hippocampus tissue was dissected. bdnf and ngf levels were measured in these samples. in conlusion, while enriched environment was a positive effect on spatial learning, social isolation was a negative effect on spatial learning and increase thigmotactic behaviors. according to the analysis results ngf and bdnf levels in the hippocampus and plasma did not change with environmental conditions and exercise. time of exposure to social isolation, procedures of the enriched environment, time of exposure to the environment, type and duration of exercise and gender may affect the results. alzheimer's disease (ad) was characterized by dementia that typically begins with subtle recognition failure and poor memory. it slowly becomes more severe and, eventually, incapacitating. the cholinergic system seemed particularly susceptible to synapse loss, especially in cortical regions associated with memory and executive function (1) . recent studies showed that the main cause of the loss of cognitive functions in ad patients was a continuous decline of the cholinergic neurotransmission in cortical and other regions of the human brain (2) . acetylcholinesterase (ache) and butyrylcholinesterase (bche) are hydrolytic enzymes that act on acetylcholine (ach) to terminate its actions in the synaptic cleft by cleaving the neurotransmitter to choline and acetate. both enzymes are present in the brain and detected in neurofibrillary tangles and neuritic plaques. it was suggested that ache predominates in the healthy brain, with bche considered to play aminor role in regulating brain ach levels. however, bche activity progressively increases in patients with ad, while ache activity remains unchanged or declines. both enzymes therefore represent legitimate therapeutic targets for ameliorating the cholinergic deficit considered to be responsible for the declines in cognitive, behavioral, and global functioning characteristics of ad (3). we initiated a study to screen their acetylcholinesterase (ache, ec 3.1.1.7) inhibitory activities, which are the key enzymes taking place in pathogenesis of ad. newly synthesized chiral benzimidazole derivatives with thioure structure showed ic 50 values in the range of 11.55-36.00 nm for ache. this study was financed by turkish research council-tubi-tak (kbag 115z837). p-09.02.2-012 f3-a13 h, novel fingolimod derivative, activates camp-dependent signalling pathway in sk-n-sh cell line g. celik turgut 1 , a. sen 1 , d. doyduk 2 , y. yildirir 2 1 department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, 2 department of chemistry, faculty of sciences, gazi university, ankara, turkey fty720, a sphingosine 1-phosphate (s1p) receptor modulator, is the first oral disease-modifying therapy to be approved for the treatment of relapsing-remitting multiple sclerosis. in this study, we have synthesized and characterized novel derivative of fty720, namely f3-a13 h, and have determined its underlying camp regulation in sk-n-sh cell lines. for this purpose, we first determined the regulation of the camp response element (cre) activity and camp concentration by f3-a13 h along with fty720 using pgl4.29 luciferase reporter assay and camp immunoassay, respectively. then, we have determined their effect on camp/pka-related gene expression profiles using custom arrays along with fty720 treatment at non-toxic doses. it was found that f3-a13 h significantly activate cre and increase camp concentration in the sk-n-sh cell line, indicating that it activates camp pathway through cell surface receptors as fty720 does. furthermore, f3-a13 h modulates the expression of the pathway related genes that are important in camp signaling pathway. in summary, our data demonstrate that the novel fty720 derivative act as a modulator of camp ultimately by influencing the gene expression via the camp and downstream transcription factor cre pathway. in conclusion, f3-a13 h might contribute future therapies for multiple sclerosis. alzheimer disease (ad) results in memory impairment and accompanied by neuroinflammation, cholinergic deficit and amyloid-beta (ab 1-42 ) accumulation in brain. we found that bacterial lipopolysaccharide (lps) injections or mice immunizations with extracellular a7 nicotinic acetylcholine receptor (a7 1-208 nachr) domain resulted in astrogliosis, decrease of a7 nachr density, accumulation of ab 1-42 in brain and episodic memory impairment. the aim was to reveal main event triggering ad-like symptoms development. c57bl/6 mice were injected with lps, immunized with recombinant a7 1-208 or a7 1-208 endoglycosidase treated to remove carbohydrates. two immunizations with 3 week interval were performed. control mice obtained complete freund's adjuvant injections. mice were tested for memory performance, blood sera were examined for presence and fine specificity of a7 1-208 -specific antibodies and brain preparations were studied for a7 nachr, ab 1-42 and il-6 levels. the original a7 1-208 ('glyc') was more immunogenic than 'deglyc', and their epitopes were recognized with different efficiency. in contrast to lps and 'glyc' a7 1-208 immunization with 'deglyc' a7 1-208 did not stimulate il-6 elevation in brain and had no proinflammatory effect. immunizations with 'glyc' or 'deglyc' a7 resulted in similar a7 nachrs decrease and ab 1-42 accumulation in brain and significant episodic memory decline comparable to those after lps injections. a7 nachr interacts directly with amyloid-beta precursor protein and facilitates its proper processing and metabolism. our data indicate that decrease of a7 nachr density caused by a7 1-208 -specific antibody is critical for ab 1-42 accumulation and episodic memory impairment while pro-inflammatory capacity of a7 1-208 -specific antibody plays secondary role in ad-like symptoms development. in vitro antioxidant and antiacetylcholinesterase activity of achillea millefolium alzheimer diseases (ad) is a neurodegenerative condition without a current effective treatment. increase in reactive oxygen species and lipid peroxidation or decrease in total antioxidant capacity causes oxidative stress-induced tissue damage. it has been suggested that decrease in oxidative stress and inhibition of acetylcholinesterase (ache) activity play a major role in the prevention and slowing of cognitive symptoms of ad. recently, studies have been directed for the discovery of medicinal plants and natural substances that are known to have natural antioxidants. achillea millefolium (a. millefolium) is a traditional herbal medicine that contains natural compounds with antioxidant activity and has been used as a carminative, diuretic, menstrual regulator and wound healer, however the mechanism of its actions are unclear. the aim of our study was to investigate the effects of a. millefolium extracts on free radical production, acetylcholinesterase (ache) activity and lipid peroxidation in vitro. methanol (me) and ethanol (ee) extracts of a. millefolium were prepared to determine (a) in vitro antioxidant capacity (by using 2,2-diphenyl-1-picrylhydrazyl assay, radical scavenging activity, phosphomolibdenum-reducing antioxidant power, ferricreducing antioxidant power, and total phenolic-total flavonoid contents), (b) effects on ache kinetics (by using a colorimetric assay) and (c) effects on sodium nitroprusside-induced lipid peroxidation in mice brain homogenates. me showed a higher antioxidant activity compared with ee in the biochemical assays tested. similarly, me demonstrated significant inhibition of ache activity that was potent than ee. both extracts dose-dependently decreased malondialdehyde content in mice brain homogenates suggesting a strong inhibition of lipid peroxidation. these results showed that a. millefolium has a high antioxidant capacity and antiache activity, indicating a potential use as an adjuvant therapy in ad. b-cells are known to play a key role in multiple sclerosis (ms) progression and autoimmune response. cxcr5 is the main b-cell chemokine receptor that under normal conditions directs their migration to specific areas of secondary lymphoid organs. in ms, areas of demyelinating lesions have been reported to attract bcells due to overexpression of cxcl13, the cxcr5 ligand. we aimed to determine whether snp rs630923 located in the promoter of cxcr5 gene and associated with high risk of multiple sclerosis could have a direct effect on of cxcr5 promoter activity. mef2c binding to dna was assessed using pull-down assay. b-cell stimulation was performed using lps, pma and ionomycin. activities of variants of cxcr5 promoters containing different rs630923 alleles were estimated using luciferase reporter assay. we determined that minor rs630923 allele creates functional mef2c-binding site within one of the regions required for the basal activity of the cxcr5 promoter. cxcr5 promoter containing minor rs630923 variant that is statistically associated with low risk of ms showed significantly decreased activity in stimulated human b-lymphoblastoid cell lines. mef2c has been reported to play an essential role in b-cell survival and b-cell responses. we determined mef2c as the main regulator of rs630923-dependent modulation of cxcr5 promoter activity in b-lymphoblastoid cell lines. this link may be directly related to pathogenic b-cell activities in multiple sclerosis. introduction: parkinson's disease (pd) is the second most common neurodegenerative progressive brain disease with increasing prevalence in aging population. the etiopathogenesis involves many cellular procesess, but is not fully elucidated yet. treatment of pd is based on levodopa and dopamine agonists, but mao-b inhibitors, comt inhibitors, amantadine or anticholinergics may be used as initial monotherapy or as adjuvant therapy. treatment related adverse drug reactions (adrs) are frequent, but cannot be predicted and/or prevented. non-motor adrs, such as nausea, somnolence, hallucinations and hypotension are frequent in dopamine agonist therapy, while dyskinesias along with motor fluctuations are the most common late adrs with levodopa. the aim of our study is to combine clinical data with genetic and epigenetic biomarkers in the algorithm for personalized approach to pd management. materials and methods: we are planning a clinical study to assess the combined impact of selected clinical, genetic and epigenetic factors on the progression of pd, adrs and treatment response. our study will have a retrospective and prospective arm. we will collect peripheral blood samples of pd patients and clinical data. single nucleotide polymorphisms (snps) in the genes involved in dopamine, neurotransmitter and drug metabolism and transport, receptors and signalling pathways will be genotyped. snps within inflammatory, neurodevelopmental, antioxidative defense, synaptic transmission and immune response pathways will also be analysed. in the prospective arm we will isolate the exosomes and check their mirna content at the time of diagnosis and after the treatment initiation. the combined effects of clinical, genetic and epigenetic factors will be analyzed using lasso penalized regression analysis. conclusions: we hope to identify genetic and/or epigenetic biomarkers that may predict progression of pd, adrs and treatment response and may support personalized tratment of pd. most evidence indicates that g protein-coupled receptors form heteromers between them and with other receptors. by allosteric mechanisms, them acquire a multiplicity of unique pharmacological and functional properties. recently, we discovered that dopamine d1 receptors (d1r) and histamine h3 receptors (h3r) form heteromers through which h3r ligands can inhibit d1r function. d1rs also physically interact and modulate ionotropic glutamate nmda receptors (nmdar). in the present work, we investigated if nmdar, d1r and h3r form a heterotrimeric complex in brain. the heteromer expression was studied in slices from both rat and mouse brain cortex by co-immunoprecipitation (co-ip) and proximity ligation assays (pla). the ability of d1r and h3r to interact with nmdar in transfected hek cells was analyzed by bioluminescence resonance energy transfer (bret) with bimolecular fluorescence complementation (bifc) experiments. heteromer properties were studied by analyzing erk1/2 phosphorylation and cell death in cortical slices. endogenous d1r-h3r heteromers were detected in rat and mouse cortical slices, where h3r ligands decreased d1r signaling (erk1/2 pathway) and were also able to block the cell death induced by overstimulation of either d1r or nmdar. by bret experiments in transfected hek cells, we demonstrated that both d1r and h3r form heteromers with nmdar subunit 1a in the presence of subunit 2b. d1r-h3r-nmdar heteromers were detected by bret with bifc. endogenous d1r-h3r-nmdar heteromers were observed in rat and mouse cortex by pla. many systems, including the glutamatergic and dopaminergic, are involved in neurodegeneration. our innovative finding is that d1r, h3r and nmdar form heteromers that may be a point of intervention for cognitive disorders in neurodegeneration. d1r-h3r-nmdar heteromers are expressed in brain cortex and a complex interaction exists between protomers in the heteromer, where h3r ligands act as a 'molecular brake' for d1r and nmdar signaling. studies conducted on obesity and hfd (high fat diet) revealed hypothalamus have crucial roles on development of metabolic diseases. after chronic over nutrition or high fat diet, as a neurodegenerative condition, premise inflammation, neural stress and development of functional impairments are observed. these studies generally focused on changes in neurons, but it's effects on brain vessels are still unknown. in this study, as a neuronal damage infrastructure, changes in hypothalamic vascularity investigated. experiment initiated with 5 weeks old total 40 male wistar rats. in order to acquire obese phenotype, the rats were fed either cafeteria diet as hfd, or normal/chow (standard diet, sd) for 9 months. intravenous glucose tolerance tests performed before sacrification. animals were exposed for 10 s to co2 and then decapitation was performed with guillotine. isolated brains were directly immersed into liquid nitrogen and stored at à80°c. the hypothalamic sections were acquired with the cryostat instrument at different. immunofluorescence was performed on serial sections through the hypothalamus using the antibody smi-71 and cd31. changes in tight junction (tj) proteins (occluding and zone occludin-1) are evaluated via western blot (wb) analysis. the hfd-treated consumed significantly more food than did control animals, when examining average food consumption per day and rats that received the hfd diet weighted significantly more at the end of 9 month diet treatment. there were no differences acquired for glucose tolerance tests. however, after hfd treatment, wb analysis have shown that tj proteins decreased even if hypothalamic micro vessel number increased and smi-71 staining have shown that increased. our primary results have shown that hfd diet can affect hypothalamic vascularity and such changes might initiate neurodegenerations and functional impairments as observed in neuroretinal degeneration in relation to vasculopathy in diabetic patients. defects of mitochondrial trafficking are common problem in many neurodegenerative diseases. its dysregulation can contribute to changes in bioenergetics profile of the cell and can lead to cell death. in our study we investigate distribution of mitochondria and their transport in primary fibroblasts derived from patients with sporadic form of alzheimer's (ad) and parkinson's (pd) diseases. our data revealed that in the most cases the velocity of mitochondrial movement is lower in ad and pd cells in comparison to the control. the most intense differences between ad, pd patients and control group are observed in the case of movement of large mitochondria. owing to the fact that mitochondrial trafficking depends on mitochondrial state, we investigated the 'age' of mitochondria. we observed a diminished mitochondrial turnover in ad and pd fibroblast. evaluation of the mitochondrial distribution within the cell in all 3 groups (ad, pd and control) showed that in the perinuclear area are accumulated 'old' and 'worn out' mitochondria, probably dedicated to remove from the cell. because mitochondrial biogenesis, shape and size depends on fusion/fission proteins we assessed their level within the cell. to summarise, our results revealed alterations in mitochondrial trafficking in fibroblasts derived from patients with alzheimer's and parkinson's diseases in comparison to the healthy control cells. carbonic anhydrases (cas, ec 4.2.1.1) is a zinc metalloenzyme that catalyzes the reversible reactions of co 2 and water. carbonic anhydrases (cas, ec 4.2.1.1) form a family of metalloenzymes that play an important function in various physiological and pathological processes. therefore, many researchers work in this field in order to design and synthesize new drugs. carbonic anhydrase activators are important as much as inhibitors. caas have polar groups to make hydrogen bond in the main body and the activation property of enzyme increaase in this way. caas are have polar groups to make hydrogen bond in the main body and the activation property increaase in this way. furthermore, recent studies suggest that ca activation may provide a novel therapy for alzheimer's disease. in this study ca activators are determined. human carbonic anhydrase isozymes ca i and ca ii are isolated from human blood erythrocyte. hca-i and hca-ii isoenzymes were purified using sepharose-4b-l tyrosine-sulfanilamide affinity colum. finally, hca-i and hca-ii isoenzymes were eluted with appropriate elution buffers. enzyme purity was checked by sds-page. the enzyme activity system contained 0.05 m tris-so 4 ph 7.4, r-nitro phenol in 1 ml total volume. effects of some macrocyclic thiacrown ethers derivates were investigated. enzyme activities were measured at constant substrate and different activator concentrations to find ac 50 value. these compounds are thought to be useful for treating alzheimer's disease. introduction: gender differences in stress models are not studied in detail. we compared different stress conditions on brain bdnf levels, in social isolation (sit) and predator scent tests (pst) in rats. bdnf levels in cortex, hippocampus and amygdala were compared, effects of chronic fluoxetine (flu) treatment were evaluated. methods: 128 rats were used. for sit, animals were kept individually for 1 month and for pst, rats were exposed to dirty cat litter for 10 min at the first day of 1 month stress. flu was given (5 mg/kg, ip) through stress. controls, stress and treated groups were evaluated in elevated plus maze (epm), anxiety scores were calculated. brain bdnf levels were determined in cortex, hippocampus and amygdala by western blot. p < 0.05 were considered significant. results: sit and pst induced anxiety in both male and female rats, females having greater anxiety scores than males (p < 0.05). flu restored anxiety scores in both sexes (p < 0.01) in two settings. male and female rats exhibited reduced cortical bdnf levels in sit (p < 0.001). pst reduced cortical bdnf in females, but increased in males. hippocampal bdnf expression was lowered in sit (p < 0.01) and pst (p < 0.001) in both sexes. female rats had 40% lower bdnf expression than males in amygdala in sit. flu did not restore cortical bdnf in females in both tests, but reduced incresed bdnf levels in males (p < 0.001). flu did not restore reduced brain bdnf in males in hippocampus and amygdala, but restored in hippocampus, in females. discussion: our findings indicate that sex differences must be considered in studies related to mood disorders of animal models, and suggest that bdnf expression in different brain regions are altered differentially in a gender-dependent manner in rats. antianxiety effect of flu is not mediated through increasing bdnf activity in cortex in both genders. increased bdnf in hippocampus and amygdala may reflect its antidepressant effect in female rats, but not in males. perineuronal nets (pnns) are special forms of neural extracellular matrix found around neuron bodies and neurites. hyaluronan and proteoglycan link protein1 (hapln1) is one of the major elements of pnns. hapln1 interacts with tenascins and aggrecan which are other essential pnns components. in most of neurodegenerative disorders caused by neuritogenesis defects, disrupted pnns structure and decreased expression of hapln1 were observed. however, the role of hapln1 in neural differentiation is unknown. the aim of this study is to determine mrna and protein levels of hapln1 during differentiation using pc12 cell line as a neural differentation model, derived from rat pheochromocytoma. after pc12 cells were stimulated to differentiate into neurons by nerve growth factor on days 3, 5 and 7; cells were collected, qrt-pcr and western blot were performed. also, in order to find out whether there is a physical interaction between hapln1 and proteins related to neuritogenesis defects, spinal muscular atrophy (sma) was used as a neurodegenerative disease model. therefore, a detailed hapln1 and survival motor protein 1 (smn1) network analysis were performed in-silico. as a result, we analyzed 3 fold increase in hapln1 mrna level compared to undifferentiated state. on the other hand, a decrease in protein level was detected. this decrease in cellular hapln1 level suggests that, hapln1 is required for formation of pnns structure, thus secreted to extracellular environment at early stage of differentiation. in addition, according to in-silico analysis, an indirect path between hapln1 and smn1 through fibulin2 (fbln2) was detected. fbln2 was also found to be an interaction partner between different matrix molecules such as aggrecan and hapln1 which form a macromolecular meshwork. the results of this study will pave the way for investigating the role of hapln1 and fbln2 in neurodegenerative disease models. also it will help us to understand the mechanism of neuritogenesis defects. determination of properties of bone marrow and tissue-derived mesenchymal stromal/stem cell population in neurofibromatosis type 1 patients neurofibromas, complex tumors deriving from schwann cells and containing fibroblasts, vascular structures and mast cells, are part of the clinical picture in nf1. the risk of malignancy is increased in nf1, wound healing is delayed and keloid formation is frequent. because multiple tissues are involved in malignant and non-malignant manifestations of nf1, we considered the mesenchymal stem/stromal cells (msc) carrying the nf1 mutation might play a role in the microenvironment. mscs affect the biological behaviour of other cells: they alter their proliferation, apoptosis and migration through various secreted growth factors, cytokines, chemokines, or by direct contact. we examined the msc of nf1 patients. methods: the adipogenic and osteogenic differentiation potential of mscs from nf1 and healthy subjects was examined in vitro and by rt-pcr. msc's migration potential was measured in the scracth assay. mscs' interaction with schwann cells and their effect on tumorigenesis was examined in co-culture by apoptosis markers on flow cytometry. results: nf1-mscs' adipogenic and osteogenic differentiation potential was lower than healthy controls as assessed by staining aizerin red s and oil red o and rt-pcr for osteopontin and collagen1. mscs cultured from dermal neurofibroma showed faster closing of the scratch compared to the same patient's normal and caf e au lait skin. on the other hand, mscs obtained from plexiform neurofibroma healed late, while mscs derived from the same patient's caf e au lait skin showed the fastest healing. hepatic encephalopathy with ammonium ions accumulation is accompanied by some disorder in the brain due to toxic material concentration being usually detoxified in the liver. one of the reasons for hyperammoniemia could be some imbalance in brain glutamine metabolisation induced by the key enzymes glutamine transferases (gts), which catalyze the reaction of glutamine transamination resulting in neurotoxic product of a-ketoglutaramate (akgm). akgm is hydrolyzed to a-ketoglutarate and ammonia by x-amidases. in the study, the dynamics of the enzymes activity in the tissues and biological liquids of experimental animals with hepatic dysfunction induced by thioacetamide (taa) was under investigation. white laboratory rats of wistar line (female, weight of 140 g) chronically intoxicated with hepatotrophic toxine of taa. every 2 weeks, some biological samples were collected to assess gt-k and x-amidase activities. x-amidase activity was the highest in the kidney tissue in the control and decreased by 70% in the experimental group. in the experimental hepatic x-amidase activity decreased by 240% compared to those in the control. the average x-amidase activity in the blood serum (0.015 nmols/ mg/min) and in the brain (0.005 nmols/mg/min) differed faintly. maximal gt-k activities were revealed in the kidneys; in the controls, it was about 250% higher than those in the experimental animals. the difference between average enzyme activities in the liver of the control and experimental animals reached 350%. the average gt-k activities in the blood serum and brain of the control and experimental animals were rather similar. the decrease in x-amidase and gt-k activities obtained in the study during hepatic pathology development could testify to imbalance of glutamine metabolism, possibly aimed at declining the level of akgm neurotoxicant under the hepatic dysfunction. acknowledgments: supported by the russian federation ministry of science and education (grant no rfme-fi60414x0116). wilson disease is an autosomal recessive disorder of copper metabolism characterized as neurodegeneration and liver abnormalities. it is caused by defects in the atp7b gene. atp7b is responsible for the sequestration of cu into secretory vesicles, and this function is exhibited by the orthologous ccc2p in the yeast. we aimed to characterize clinically-relevant novel mutations of p.t788i, p.v1036i and p.r1038g-fsx8 in yeast lacking the ccc2 gene. the patients with these mutations have copper storage abnormalities in different parts of their bodies; p.t788i mutation mainly affects the liver and the nervous system, p.v1036i mutation affects the nervous system, and p.r1038g-fsx83 mutation causes damages to the liver. to better understand the effects of these mutations on normal functions of atp7b, we cloned human atp7b gene onto a yeast expression vector and created the same mutations by site directed mutagenesis. then, wild type and mutated forms of atp7b genes were transformed into yeast cells lacking the homologous ccc2 gene for functional comparison. first, we analyzed the expression of atp7b and its variants in yeast cells by a real time pcr approach and western blot to make sure that transformed cells express the plasmids. expression of human wild type atp7b gene in ccc2d mutant yeast restored the growth deficiency and copper transport activity, however, expression of the mutant forms did not restore the copper transport functions and only partially supported the cell growth. our data support that p.t788i, p.v1036i and p.r1038g-fsx83 mutations cause functional deficiency in atp7b functions and suggest that these residues are important for normal atp7b function. in recent years, attempts were made to develop miniaturized potentiometric biosensors which is particularly important to reduce the amount of enzyme and reagents needed. the miniaturization of a biosensor is possible by using an all solid-state polymer membrane ion selective electrode which is cheap, easy to prepare and allow micro-sized construction. the use of all solidstate polymer membrane ion selective electrode as the basic sensing element also has the advantage of providing biosensors that are easy to fabricate, exhibit rapid response and have long life-times. they are also mechanically stable and allow flow-through configuration. genetical and chemical modifications for the alteration of enzyme molecule characteristics are gained considerable importance. enzymes can be modified chemically by using water-soluble polymers or some chemicals. conjugates of natural and synthetic macromolecules with enzymes provides wide usage in medicine and in many fields of biotechnology. in this study, enzyme-polymer conjugates with different molar ratios were synthesized using urease enzyme. in this study micro sized potentiometric urea sensitive biosensor has been developed in which urease-polymer conjugates were immobilized on polymeric membrane ammonium ion selective electrodes whether pvc or derivatized-pvc via glutaraldehyde cross linking reaction. biosensor is not include inner reference electrode and inner reference solution. potentiometric performance of biosensor will be examined with a computer-controlled measurement system designated. the most important features of the obtained micro sized urea biosensor by using enzyme-polymer conjugations were being highly sensitive, having long life-time, easily built at a low cost, and having short response time when compared with conventional potentiometric urea biosensor. also, these biosensors were easily built at micro-construction. this study was supported by grant from the tub _ itak research fund (project number: 114z138). creative drama technique as a new tool to increase enthusiasm and to achieve learning objective for medical students e. y. sozmen 1,2 , e. erem 3 1 faculty of medicine, ege university, izmir, turkey, 2 center for continuing education, ege university, izmir, turkey, 3 recently drama in education techniques have been implemented successfully in education program of primary and secondary high school and positive effects of these techniques on learning ability and attitude of students have been shown. the aim of this study was to organize an education program based on drama in education techniques in a special module of ege university medical faculty and to test any effect of this technique on achievement of learning objectives and student's perspectives on drama. the special module program was on the oxidative stress and antioxidants. the program covered the drama in education sessions (improvisation, role play, game) linked with learning objectives (understanding of free radical generation and free radical reactions in body, evaluation of the effect of free radical reactions in diseases as well as increase the ability usage of scientific information), laboratory work (antioxidant activity determination) and searching a special scientific topic on literature. 12 students (in 3rd year of faculty) who had taken theoretical lecture on this subject a year ago, participated in this special module. the opinions of the students on the program were obtained through a questionnaire form and the increase in knowledge was evaluated via pre/posttest. the mean of pretest point was 2.7/10, that increased to 7.4/10 in the posttest evaluation. 50% of students pointed that they enjoyed participating in drama activities in the pre-questionnaire, this rate was 100% in the final questionnaire. they all remarked that implementation of drama in education was beneficial for their communication skill, helping them to learn more about science and increased their enthusiasm to learn and discuss the scientific information. although the preparation process might take more time and need to hard work for teachers, we concluded that the drama methods as a new tool to increase of participant's interest might be proposed for students in higher education. laboratory-based performance assessment in medical education: an opportunity for connection between scientists and medical students h. tuncel cerrahpasa medical faculty, istanbul university, istanbul, turkey number of medical students who interested in basic medical sciences is declining and medical sciences literacy is falling, it is crucial to develop ways for students and scientists to connect. students need to know that science is an intensely human endeavor, and scientists need mechanisms to bring that truth to the community at large. based on continued interest and experience on the part of faculty, and on student feedback, the development of a more effective and stimulating interactive learning tool was undertaken. an in-depth knowledge of laboratory medicine principles is vital to all practicing physicians. great variation exists in the ways that medical students learn the principles of laboratory medicine. there are a number of programs for electronic media that emphasize laboratory-related skills. some of these are appropriate for medical students in the clinical years. programs that teach skills in common laboratory procedures, such as interpretation of peripheral blood smears and microscopic examination of urine sediment, have been shown to improve student performance. to ensure that important principles are addressed, medical schools should establish goals and objectives specifically related to laboratory medicine and experiment with optimal teaching and assessment methods. we also hope that this study will inspire dialogue among primary care and specialist physicians as to the proper degree of education in this area. ideally, it will encourage scientific studies that address evidence-based possibilities for improving critical laboratory medicine educational outcomes, that is, the training of physicians who optimally use laboratory diagnostics and therapeutics. engaging medical students in scholarly scientific activities and producing clinically competent and research-oriented medical workforces are essential demands, particularly in developing countries. an experimental special study module for medical undergraduate students: learning western blot analysis and detection of b-actin protein expression in tissue and cell culture samples learning, introduce basic principles of laboratory research and to present the results.b-actin is one of six actin isoforms which is mainly expressed in all eukaryotic cells. western blotting is a widely used laboratory technique to determine specific proteins and to evaluate protein expression in tissues and cells. in our study, different concentrations of rat spleen homogenates (25, 50, 75 lg/well) and 50 lg protein/well of human lung and ovary cancer cell lysates were used. the proteins were seperated by 10% sds-page, transferred to pvdf membrane, incubated with specific b-actin antibody and then with hrp-conjugated antibody. protein bands were detected with ecl and densitometric analysis of proteins was quantified by imagej software. differences in protein band intensities were compared using one way anova.a value of p < 0.05 was considered statistically significant. we detected b-actin expression in rat spleen homogenates, human lung and ovary cancer cell lysates, as a 43 kd protein. the protein band intensities were in correlation with protein concentrations. the highest concentration resulted in the highest signal intensity in rat spleen homogenates.b-actin level was higher in ovary cancer cells than in lung cancer cells, although both proteins were loaded equally. the feedback showed that students were very satisfied with this laboratory ssm. they developed their independent study skills, planned a research, worked in a laboratory, learned and performed a technique, western blotting. the hands-on experience is very important for medical undergraduate students for their future scientific career. three-dimensional structure of truncated human kv10. voltage-gated potassium channel kv10.2 belongs to the ether-ago-go family. it has been proved that its mutants are involved in development of neurological diseases and some types of tumor. directed drug design needs knowledge of details of the threedimensional channel structure. the members of the kv10-12 subfamilies are characterized by extremely long n-and c-terminal intracellular tails, which possess a number of structural domains. the n-terminal pas domain in kv10 plays an important role in activation, and is thought to alter the rate of deactivation, possibly by binding at or near the s4-s5 linker at the inner mouth of the pore. here we present an improved 3d structure of the truncated human kv10.2 channel, obtained by single particle em. this channel lacks its cytoplasmic pas domain but it still forms tetrameric particles. earlier we showed that the full length kv10.2 channel is build according to the 'hanging gondola' type, and its cytoplasmic and transmembrane parts are connected by linkers. the cytoplasmic part includes the interconnecting pas and cnbd domains. deletion of the pas domain leads to the conformational change in the cytoplasmic part of the channel. for interpretation of the 3d structures we used homology modeling and molecular dynamics simulation. there are several templates available to the moment including eag domain-cnbhd complex of the mouse eag1 channel, full-length shaker potassium channel kv1.2, c-linker/cnbhd of zelk channels and others. but there are still no templates for many fragments that led to necessity of partial de novo modeling. analysis of molecular trajectory allowed estimating dynamical characteristics of channel, supposing interdomain interactions. results of the conducted investigation have a great interest at both the academic and the industrial levels. the objective of this task program is to enable students to gain scientific attitude and skills for them to be able to deal with the problems they'll confront in future research careers. it's been emphasized in various studies that medical students are keen on conducting scientific research, and it's been stated that they need to be supported in this respect, as they lack the adequate fund of knowledge. this study aims to share information throughout a 5-year performance of the research training program (rtp) conducted at ege university, faculty of medicine. rtp is an educational program applied in addition/parallel to the bachelor's degrees for establishing scientific thought, attitude, proceeding and knowledge of the willing medical students, and enabling them to adopt study skills. the dynamic program produced by the rtp committee in 2011 has been developing each and every year via feedback received from the students. an operating principles, a manual for advisor and an introductory booklet have been laid out. applications are being accepted at the end of first academic year, making announcement to the freshmen in advance. students are being admitted to the program, taking the assessment criterias into consideration. second and third year medical students attend the didactic part of the program during the terms devoted to special study modules. thereafter, they go through the project management phase, and accomplish their projects under supervision of a faculty member until their graduation. 12 first graduates of the program, accomplishing their projects, received their certificates at the graduation ceremony of 2015 graduation. currently, there are 61 students in total from all classes who perpetuate their studies within the program. an inventive training pattern of ege university faculty of medicine, rtp experience is being maintained as a dynamic process and successfully keeps the students advised of conducting scientific researches, cultivating scientific awareness. objectives: objectives selection and verification of blood collection tubes is an important preanalytical issue in clinical laboratories. in this study comparison with the reference glass tube of ayset plastic tubes containing separator gel and assessment for routine clinical chemistry laboratory testing in samples were aimed. materials and methods: thirty-four volunteers were included in the study. samples were taken into two different tubes by two experienced technologists according to the clsi protocol [tube1: z (becton dickinson and company, franklin lakes, nj, usa); tube2: ayset (lot10069, turkey)]. glucose, urea, creatinine, ast, alt, total-cholesterol, triglycerides and high density lipoprotein-cholesterol were analyzed subsequently (olympus au2700) and randomised samples stored at 2-8°c for 24 and 48 h. 0th hour sample was analyzed immediately after collection and accepted as the reference for the comparison of the other samples. a paired t-test and wilcoxon signed rank sum test were used to test the significance of differences between the reference tube and test tubes. results: the difference between the results of reference tube and test tubes for glukose, urea, creatinine, ast, alt, total cholesterol, tg and hdl-cholesterol at 0, 24 and 48 h were statistically no significant (p = 0.09, p = 0.07, p = 0.20, p = 0.16, p = 0.13, p = 0.09, p = 0.05, p = 0.54, p = 0.58, p = 0.46, p = 0.19, p = 0.13, p = 0.66, p = 0.72, p = 0.34, p = 0.11, p = 0.16, p = 0.06, p = 0.08, p = 0.39, p = 0.23, p = 0.63, p = 0.58, p = 0.54, respectively). conclusions: no significant difference was observed between ayset tubes' results and the reference tube's results. e. akin c ß akir d€ uzen laboratuvarlar grubu, istanbul, turkey insulin resistance underlies the development of obesity which is a global health problem. obesity is a main concern of scientists because it's associated with type 2 diabetes, hypertension and some cancers. recently, inflammation centered mechanisms are deeply investigated as well as the effects of anti-inflammatory diets which are highly rich in vitamins, minerals, fibers and healthy oils. these diets are proposed to inhibit or supress the secretion of the inflammatory mediators and also improve the intestinal microflora. the aim in this study is to highlight the increasing trend of publications in regard to insulin resistance and inflammation based obesity along with the effects of antiinflammatory diets used for its treatment in the last decade. we performed a pubmed search with key words of 'obesity and insulin resistance and inflammation' (01/01/2005-15/05/ 2016) (search #1). besides, we performed another search with key words of '(anti-inflammatory diet or dietary supplement) and (obesity or insulin resistance)' (search #2) to highlight the value of anti-inflammatory diets and dietary supplements in combating inflammation based obesity and insulin resistance. search #1 revealed 6763 articles; of these 341 were clinical trials, 18 were observational studies. human studies were 4 552 while animal studies were 2 580. overall, there were 2 229 review articles and 5 meta-analysis in the field. search #2 revealed 4 255 articles of which 796 were clinical trials, 958 were review articles, 46 were meta-analysis. human studies were 2 610 and other animal studies were 1 931. the relationship of metabolic diseases with a low grade inflammatory state has opened a new area of research to understand the consequent causes of inflammation in the human body. the increasing scientific data in the field indicates that antiinflammatory diets may serve as powerful tools to solve the inflammation and the consequent insulin resistance and obesity. medical and biological illustrations for life sciences education: is 'a picture worth a thousand words' in visualizing medicine and science? medical and biological illustrations (mbi) convey complex ideas with just an image and they are powerful intersections of science and art. the clarification of complex pathways via illustrations can be effective means in education as they help the student to visualize the biomolecular world and understand the mechanisms. our aim is to illustrate how a mbi is developed over the example of mechanism of insulin action, via the phosphoinositide (pi) 3 kinase-protein kinase b (akt) pathway. organising one's thoughts and clarifying relationships and then using the optimal complexity level to illustrate the pathway most clearly are the basics of mbi. thus, we made a thorough investigation of insulin mechanism on glucose uptake in skeletal muscle and adipose tissue; a biochemical process that includes insulin receptor (ir), ir substrate, pi3 kinase, pi-dependent kinase 1 and akt. then, we found the 3d structures of molecules via protein databanks and accordingly created drawings and diagrams of each component in both molecular and macrolevels by adobe photoshopò software. graphics tablets and a compatible pc were also used in the production phase. the use of computer hardware/software enables unlimited detail in images and provides the flexibility that classical drawing techniques can not. thus, the final diagram clarifies the underlying molecular mechanisms of a biochemical pathway along with the physiologic actions. recent improvements of computer technology have resulted in the creation, and reproduction of high-quality lower cost medical art. mbi's can be used in education to explain concepts/pathways to students to enhance learning. similarly, mbi's are great tools to show mechanism/procedures to enhance patients' understanding of their medical condition. considering their unquestionable contribution to education, research and patient care, the creation of mbi's should be promoted as a graduate level course and the discipline should be represented at academic level. biochemistry is a compelling field with broad applications in many disciplines like medicine, dentistry, pharmacy and bioengineering. biochemical research increasingly combines ideas from genetics, molecular biology, etc. and collaborates with many disciplines. our objective is to conduct a scientometric analysis of the last decade's postgraduate theses in the field of biochemistry (ptb) in regard to number, collaborations, subject area distribution, etc. to discover the characteristics and trends in turkey. we searched the turkish higher education council's theses database (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) which includes master of science (msc.), doctorate (ph.d.) and specialization (s) theses in all disciplines. an electronic search with the keyword of 'biochemistry' (in the thesis subject area) was conducted, thus it brought all theses either in the biochemistry discipline or theses in other disciplines but have a biochemistry component. we performed data cleaning and further quantitative analyses in excel. we also executed word count analysis on the titles of theses to derive the main subject areas in ptb. of the total of 6374 ptb (2215 s, 2781 msc, 1339 phd theses) 37.6% was in natural sciences while 62.4% was in health sciences. the theses output-growth measured by the compund annual growth rate was 82% over the 10-years. the top 3 clinical disciplines in collaboration with biochemistry were pediatrics, surgery and cardiology, and the top 3 science disciplines were biotechnology, bioengineering and biology. oxidants-stress and antioxidants (1008), endocrine-metabolism (655) and enzymology (608) were the top research areas in ptb, followed by genetics (305) and cancer (252). scientometrics is a powerful tool to understand the direction of science and research. our ptb analysis indicated that prominent areas like stem cell, biosensors, geriatrics are somewhat lagging in turkish biochemistry research while postgraduate education and research in total is growing fast with sound collaborations. the 1st turkish in vitro diagnostic symposium evaluation objective: in vitro diagnostic (ivd) medical laboratory tests and the equipment are closely related the public health, patient safety and the safety of all who utilize these tests. it depends on auditing of the process from the production to the consumption of these materials, that they do not pose a risk to individuals and society. upon these basic requirements; 'turkish in vitro diagnostic symposium: medical laboratory tests' was held in february 2016, organized by the cooperation of turkish biochemical society branch of izmir, and dokuz eylul university health sciences institute. it was intended to shed light on some questions such as, what is the place and importance of ivd in turkey? what are the responsibilities of educational institutions?, what is the role of ministry of health? to put across the conditions of preparing a substructure that may provide achieving success in producing ivd medical devices and in this sector, in our country. material-method: 39 invited speakers attended the symposium, along with the participation of both as lecturers and attendees; ministry of health, turkish standards institution; representatives of manufacturer enterprises; representatives of enterprises manufacturing in turkey; scientists conducting considerable researches on health technology; students and representatives from some of the non-governmental organizations. in addition to the presentations, gathering up the written opinions of the participants, a report was prepared. results: the symposium that lasted for 3 days was realized with 120 participants in total,55 of which from universities;38 representatives of their companies;17 from chamber-institute-public establishments and 10 of which from public hospitals. 94 of the participants were from izmir,26 of them were coming from out of izmir. conclusion: at the end of the symposium,40% of the participants gave feedback. among the feedback selected; 86.2% of the participants evaluated the symposium overall, as successful. 75.6% of them found the symposium successful with regard to its scientific content. their feedback were 55.2% positive in terms of the symposium's contribution to the situation assessment on ivd in turkey, and 44.8% of them stated they would consider participating in the second of the ivd symposium if it is to be organized. perceptions of molecular life science master's students on their scientific and academic competencies and prospective plans for professional development the master's education (me) in molecular life sciences (mls) is aimed at strengthening the knowledge and skills base of the young scientist, preparing him for the competitive academia/industry positions. the rapidly developing pace of science and research forces the master's student (ms) to play a central role in monitoring and guiding his scientific education and professional development (pd). thus, the aim of our study was to examine the perceptions of ms of mls, regarding their scientific and academic competencies. with this data, we planned to analyse if this awareness channels ms to take action and/or matches with their prospective plans. we developed a 15 item online survey with 3 sections (demographic data, current data-contributions of me, competencies and prospective data-action for pd, future plans) and distributed it via e-mail to various postgraduate institutes. at the end of the 10-day period, 138 ms students (in the thesis phase) answered the questionnaire (female: 66.7%, male: 33.3%). the most highly rated activities that contributed to their scientific knowledge and skills gained in the me were laboratory work (73%), visits with their mentors (70%) and theoretical lessons (65%). ms expressed low levels of sufficiency both in theoretical scientific knowledge and laboratory skills (only 43% and 33% sufficiency, respectively). communication skills (80%) and team work (78%) were rated as the highest professional competencies followed by literature search and research planning (both 73%). it was striking that ms perceived themselves as quite insufficient in scientific writing (50%), data analysis (60%) and project writing (61%) while proficiency in english (55%) was the first area they wanted to take action. despite their perceptions of insufficiencies in many areas, a majority (69%) wanted to continue to phd education. these and similar surveys may lead to an increase in selfawareness in ms and the data may contribute to the revision of me. the report of the 1st turkey in vitro diagnostic symposium results the following aspects and suggestions took place in the results report prepared after the symposium: about the national ivd-tc r&d, production, forming quality assurance and innovation strategy and policy the cooperation of the university and the industry is not sufficient most of the industrialists cannot take enough advantage of the support provided by the institutions like tubitak, the ministry of science, technology and industry and the ministry of development the statistics on the ivd-tc in turkey should be carried out as soon as possible national standards should be determined in parallel with the international standards the vat rate of the exported raw materials that would be used in ivd production should be decreased. about the education and training, the job titles and positions the related graduate programs, which would focus on all steps of the whole life cycle related to the ivd-tcs one by one, are not widespread there is no 'postdoc' application in turkey. 'postdoc' staff is needed for insufficient component human resources the lecturers should not be restricted to one discipline only graduate programs on laboratory medicine are needed to be established and spread, in order to train component labor specified on the ivd-tc about research and development there are almost no researches related to product development. this should be associated with the education and training institutions about the research centers currently, there is a real infrastructure on health technology in turkey, but there are difficulties in its instituonalism the insufficient cooperation of the university and the industry does not allow the inventions to turn into products the cooperation supports of r&d, being restricted to tech-nopark and r&d centers, are open fields for improvement p-edu-014 phd training in medical education: career profiles and satisfaction levels of graduates from dokuz eylul university graduate school of health sciences this survey was carried out within the scope of special study modules that is entitled 'phd training in medical sciences' by a group of medical students in deu. the purpose of the survey to investigate the members of health sciences that have successfully completed their phd training in terms of the levels of satisfaction and the status of their career. from this scope we generated two hypothesis: we expected that graduate phd graduates are mostly involved in academy and find their satisfaction levels at moderate level as to phd education. the study was designed as cross-sectional. we reached 166 phd graduates who had graduated from deu graduate school of health sciences between 1991 and 2002 from 12 different departments via e-mail. the survey was included 27 questions, which were prepared in the light of the existing literature. among the 166 phd graduates, 55 (30%) participated in the study. through this survey, perception of phd students on supervisors' scientific and educational abilities, opinions on phd training, productivity of phd training, number of articles published, their position and related satisfaction levels after graduation were investigated. according to the results, more than half of the graduates (%52.7) are well satisficed from the education they had taken. beside this, interestingly we found that %94.5 of graduates prefers staying in the academic positions and %64.8 of them sustains their communication with their supervisors. in conclusion, most of phd graduates were contented with phd training and their career profiles. as a result of this survey, we produced a novel and precise contribution to the literature. in a further study, this survey may extend to other parts of turkey and compile the results in order to get more accurate and inclusive data. phd is an international degree that is reached by conducting an original research after finishing bachelor or master's degree. doctoral degree (phd) can open the door of academic career; on the other hand, a person with a doctoral degree is equipped to carry out important work in research, industry, or public sector. today, gradually increasing number of phd students have brought some problems in phd training. the purpose of this study is to investigate and review activities that have been done by the following international organizations: orpheus: (organization for phd education in biomedicine and health sciences) eua-cde: european university association-council on doctoral education. febs education committee: federation of european biochemical societies these three organizations have done workshops on phd training to pay attention to the following points: *a phd student must take some courses and trainings outside her/his institute, should not be limited to the institute. *the phd training programme should include transferable skills courses. *clinicians, if involved in phd training, should be given free time from their clinic duties. *with regards to potential problems with the supervisor, the institute should provide the student an advisory system. *students should be encouraged to participate in the management of doctoral programmes in the institute. *the students should be given be opportunity to select their own supervisor (thus their thesis area). the phd training has gained quality thanks to these organizations. it is advised that graduate school of health sciences should follow the recommendations and report from these organizations. itsn1 and itsn2 are genes encoded adaptor proteins with multiple isoforms participating in clathrin-mediated endocytosis (cme), mapk signaling and reorganization of actin cytoskeleton. changes in itsns expression can lead to different neurodegenerative disorders and cancers. to date little is known about regulation of itsn genes on posttranscriptional level. the aim of our work was to predict and experimentally confirm target sites for micrornas that could potentially regulate itsns expression. using 8 web servers we analyzed 3 0 utrs of short and long isoforms of human itsn mrnas and found conservative target sites for mir-34, mir-19, mir-129, mir-103/107, mir-194, mir-181 and mir-30 in 3 0 utr of itsn1-s, predicted by 5 servers, mir-203 predicted by 5 servers for itsn1-l, and mir-153, mir-148/152, mir-27, mir-144 and mir-128 predicted by 5-6 servers for itsn2-l. to elucidate potential impact on cme, mapk signaling and actin cytoskeleton regulation by these mirnas we performed enrichment analysis by diana-mirpath server and found that mir-34, mir-19, mir-103/107, mir-181, mir-30 and mir-148 were highly enriched for all analyzed pathways. using regrna 2.0 and scan for motifs services we predicted 12 types of different regulatory elements in 3 0 utr of itsn1 and itsn2: k-box and brd-box, musashi binding element for rbps musashi1 and musashi2, gu-rich element (gre) and au-rich elements (are) that regulate mrna decay. to confirm itsn1-s regulation by micrornas we cloned 3 0 utr of itsn1-s into luciferase reporter vector and transfect hek293 cells by this construction and mir-181a, mir-30a and mir-19a. for mir-181 transfected cells, we found 25-40% decrease of expression of itsn1-s 3 0 utr-bearing construction. for other mirs we did not obtain strong reproducible effect in luciferase assay. these data may confirm mir-181 target site in 3 0 utr of itsn1-s mrna but needed additional research. objective: stroke is an acute neurological disorder that is mostly caused by ischemia in central neural system. 30% of stroked patients lose their life in 1 year, and remaining 1/3 of the living patients continue to their lives as dependent to others. nihss and mrs are two scales which are used in prognosis studies because they can show stroke intensity and after stroke functional recovery. microrna's which have effects on transcription and posttranscription gene regulations are small rna molecules. their roles have been investigated on pathophysiology and treatment of diseases. in this study, it was aimed to detect changes in blood serum levels of mir-146a, -155, -210, 181b, -31, 126, -92a and let-7f of ischemic stroke patients and to investigate role in predicting prognosis methods: 21 patients diagnosed by acute ischemic stroke admitted to neurology service of g€ oztepe hospital and 16 healthy individual were included in the study. after stroke patients' blood samples were taken periodically in 1st day, 1st week, and 3rd month, and at the same time nihss and mrs scores were determined. set 8 mirna blood serum levels were measured by rt pcr results: when compared to the control group, we found that after stroke 1st day peripheric blood levels of mir-146a,-31,-92a and let-7f were significantly low; when 1st week and 1st day serum records were compared there was a significant increase in mir-126 level; and when 1st week and 3rd month records were compared we noted that there was a significant increase in mir-146a,-126 and let-7f levels. from prognosis point of view; after ischemic stroke measurements showed that mir-181b in the 1st day, mir-146a and mir-210 in the 1st week showed positive significant correlation with 3rd month mrs scores (p = 0.002, p = 0.05, p = 0.002, respectively) conclusion: according to the outcomes of this study, after stroke 1st day mir-181b, 1st week mir-146a and 1st week mir-210 levels can be stipulated to use in predicting patients' 3rd month prognosis p-01.03.3-004 inhibitory rna aptamer against lambda ci repressor showed the transcriptional activator activity s. ohuchi, b. suess tu darmstadt, darmstadt, germany because of the variety of functionalities on gene regulation and the easiness of molecular engineering, functional rnas are promising parts for the construction of genetic circuits. artificial affinity rna, or rna aptamer, is one of such genetic parts. in the previous study, an inhibitory rna aptamer against a repressor protein, tetr, was developed as a transcriptional activator [1] . the expression of this aptamer abolishes the repressor activity of tetr, resulting in the elevated gene expression under the control of tetr. because of simplicity of the mechanism, similar transcriptional activators can be generated by using rna aptamers against other repressor proteins. here, we examined the generation of an activator based on an rna aptamer against one of the most frequently applied repressor proteins, lambda phage ci. in vitro selection (selex) was performed targeting a recombinant ci protein employing an rna pool containing 40-nucleotides of a random region. after 6 rounds of selex, the pool rna showed the affinity, as well as the inhibitory activity, against ci in vitro. then, rna aptamers with the transcriptional activator activity were screened from the enriched pool in vivo employing a reporter plasmid on which the expression of a reporter gene, lacz, is repressed by ci. when the variants of the rna pool were transformed to e. coli cells harboring the reporter plasmid, about half of the transformants showed the elevated reporter expression. interestingly, all of these desired rna clones shared the same sequence. quantitative analysis indicated that 35-fold induction of the reporter expression was achieved upon the aptamer expression. our results suggested that diversity of artificial transcriptional activators can be extended by employing rna aptamers against repressor proteins to broaden parts for the construction of genetic circuits. [1] hunsicker, et al. (2009) an rna aptamer that induces transcription. chem. biol., 16: 173-180. p-01.03.3-006 microrna expression signatures between non-atherosclerotic plaque and atherosclerotic plaque in cad with humans, and parallels whole blood rnas and represent a new important class of gene regulators. the present study was designed (i) to investigate the mirna expression profile in human atherosclerotic plaques from peripheral arteries aorta as compared to non-atherosclerotic left internal mamary artery (lima); (ii) to examine the expression levels of mirna in whole with correlation mirnas of aorta tissue. material and methods: thirty-one patients with cad were enrolled in study. lima and aorta tissue samples were obtained during coronary artery bypass surgery. whole blood samples were collected before cabg surgery. each patient with cad was obtained from whole blood, aorta and limas tissues. these tissue samples were immediately soaked in rnalater solution and homogenized using a magnalyser. the rna was extracted using the trizol reagent and the mirneasyò mini-kit. the expression profiles of 738 mirnas were evaluated using highthroughput qrt-pcr. results: we found that mir-497-3p was expressed only in aorta. mir-431-5p and 433 were expressed both aorta and whole blood. 6 mirnas were significantly up-regulated in aorta when compared to lima tissue (fc > 2). 59 mirnas were significantly down-regulated in aorta compared to lima. conclusion: in conclusion, our study suggests that mir-497-3p, mir-431-5p and 433 might be a potential for cardiovascular disease development. also mir-431-5p and 433 might serve as novel non-invasive biomarkers for cad p-01.03.3-007 mir193b regulates cell proliferation and colony formation in pancreatic ductal adenocarcinoma n. gurbuz, e. isildar due to the strong metastatic potential, pancreatic cancer has the highest mortality rate of all major cancers. therefore, the investigators are in urgent need of developing the new alternative therapeutic approaches for the prevention of pancreatic cancer. mirnas, small noncoding rnas, regulates as an inducer or inhibitor in expression of key mediators related molecular mechanisms in cancer promotion. to investigate the effect of mir193b on pancreatic ductal adenocarcinoma cells, we performed the cell viability and clonogenic assays by mts and crystal violet dye, respectively, in panc-1 and miapaca-2 cells transfected with mir193b mimic. our data revealed that mir193b led to decrease the cell viability depending on enhanced mir193b doses, which are 25, 50, 100 and 150 nm, as the ratio of % 23, 60, 82 and 87, respectively, in miapaca-2 cells and as the ratio of % 40, 58, 77 and 89, respectively, in panc-1 cells compared with control condition of each cell. this inhibition mediated by mir193b was also obtained in colony formation both of pancreatic cancer cells. when the induced effect of mir193b on the death of pancreatic cancer cells was compared with gemcitabine, which is currently used as a clinical drug for pancreatic cancer patients, we determined that mir193b was more effective than gemcitabine. based on our findings, it is clearly shown that mir193b serves as a tumor suppressor in pancreatic ductal adenocarcinoma cells. we strongly believed that mir193b gene therapy might be more effective and targeted approach than classical gemcitabine therapy for pancreatic cancer patients. *this work was supported by tubitak (the scientific and technological research council of turkey) grant 114s501. breast cancer is the most common cause of cancer death in women. trastuzumab is a therapeutic agent frequently used against her2+ breast cancers, which has role in approximately 20% of invasive breast cancers. with the discovery of their activity in cancerogenesis, micrornas (mirnas) have become potential candidates to mediate therapeutic actions by targeting genes that are effective in drug response. recent studies have showed that mirnas are induced by targeted therapies. in this study, we aim to find out mirna-mediated mechanism, which is driven by common trastuzumab responsive micro-rnas in her2+ breast cancer. for this purpose, the common trastuzumab responsive mirnas were determined in treated bt474 and skbr3 cells by microarray profiling. two datasets were intersected to find out common mirnas for both cell lines. the overlapping predicted targets of common mirnas were provided by two different mirna-target prediction databases and then a mirna-gene network was built in cytoscape by using networkanalyzer plugin. the most shared target genes were chosen to be analyzed in the ebi-embl gene expression atlas for their expression patterns in breast cancer. 25 common mirnas were found to have overlapped targets in two target prediction algorithms that were used to build the mirna-gene regulatory network. 14 overlapped targets were determined as the most shared genes in the mirna-gene network. expression pattern of each shared gene in the gene expression atlas showed that 12 out of 14 the most shared target genes were strongly dysregulated in several breast cancer types. our results suggest that mirnas might show a common response to the targeted therapies and network analysis can be profitable to have a better explanation of the regulatory mechanisms between drug responsive mirnas and their target genes. revealing the mirna-potential target interactions might provide novel key players that mediate the mechanisms of action in drug treatment. chronic myeloid leukemia (cml) is a clonal disease of primitive pluripotent stem cells that identified with a specific t(9;22) reciprocal translocation that encoding bcr-abl oncoprotein. resveratrol (res) is a natural phytoalexin found in grapes and induces apoptosis, erythroid differentiation and autophagy in leukemic cells. micrornas are small, single strand, non-coding rna molecules that regulate post-transcriptional gene expression via disrupting the stabilization of target transcripts or inhibiting protein translation. in our study we aimed to determine cytotoxic effect of res in k562 human cml cell line and to evaluate the expressions of mirnas that are associated with genetics of leukemia after treatment with res; to investigate target genes of mirnas which show significant expression alterations and molecular mechanisms of res treatment. k562 cells were treated with 100 lm (ic50 dose) res during 72 h and cytotoxicity was evaluated by using wst-1 assay. the rt-qpcr is used for mirna and gene expression analysis. results showed that; res up-regulated tumor suppressor mir-214-3p level 2.87 fold and significantly downregulated hdac1 gene expression (p = 0.003) and upregulated p62/ sqsmt1 gene expression (p = 0.001), according to the control cells.p62/sqstm1 interacts with lc3 and plays role as a critical player in the autophagic degradation of the bcr-abl fusion protein. our findings showed that resveratrol acts as a hdac inhibitor targeting hdac1 and p62/sqsmt1 gene expression level. treatment with hdac inhibitors results apoptosis, cellcycle arrest, cell differentiation, anti-angiogenesis and autophagy. downregulation of hdac1 provides post-translational modification for expression of tumor supressor genes and leads to cell cycle arrest and increases apoptosis. these results could be linked to hdac1 dependent induction of gene associated with autophagy like p62/sqsmt1 and resveratrol could be a therapeutic candidate as a hdac inhibitor for cml treatment. the mirna used in this study are hsa-mir-145, hsa-let-7a, hsa-mir-29b and hsa-mir-155. thereafter, we bought pre-mirnas and their mirnas commercially. we apply them to the a549 cell line in different combination and different concentrations. these mirnas applied solely onto cells or in combination as; four of them, let7 + 145, let7 + 29b, let7 + 155, 145 + 29b, 145 + 155, 145 + let7 + 29b, 155 + let7 + 29b. the cell viability was detected by wst-1 kit in a 96 well plate elisa reader. cells were seeded as 10 000 per well, mirnas incubated with cells for 24 h in an appropriate atmosphere. according to our results some combinations and mirnas didn't alter viability, however 145 + 29b and 145 + 155 combination increased the cell viability dramatically. on the other hand let7 + 29b and 155 only applications decreased the cell viability. the other applications' viability results are not different from the control cells significantly. in this study, we used a549 cell line also called non-small lung cancer (nsclc) cell line and possibly effective mirnas on lung cancer. it is important to exhibit the mirna combinations should be effective on cancer cells' viability. the prospect combinations were determined which is crucial to develop new strategies for cancer treatment. competing endogenous rnas (cernas) act as molecular sponges for the same pool of micrornas through their mirna response elements (mre), titrate mirna levels and thereby regulate gene expression post-transcriptionally. smad interacting protein 1 (sip1), a member of the zeb family is a regulator of epithelial-to-mesenchymal transition (emt) program, which is active during embryogenesis and tumor invasion and metastasis. hence, we investigated the regulation of sip1 by cernas in hepatocellular carcinoma (hcc) cells. among hundreds of sip1 cernas listed at competive endogenous mrna database (cerdb), 14 transcripts (pten, zeb1, ptch1, creb5, acvr2b, enah, robo2, erbb4, e2f3, foxo1, rictor, klf3, ets1, cdk6) sharing at least 9 common mre sites with sip1 were selected and their expression in 9 hcc cell lines were determined by qrt-pcr. ets1 was found to be highly correlated with sip1 in hcc. furthermore, repressing sip1 expression by shrna in hcc cells resulted in decreased expression of ets1, pten and zeb1. our results suggest a possible bidirectional and post-transcriptional regulation of sip1 and its cer-nas in hcc. a meta-analysis for the identification of common microrna signatures in colorectal cancer n. sahar 1,2 , n. belder, h. ozdag 1 biotechnology institute, ankara university, ankara, turkey, 2 comsats institute of information technology, islamabad, pakistan colorectal carcinogenesis (crc) has quite frequent incidence and mortality rates worldwide, despite being studied for decades now. new biomarkers are needed to be identified in addition to the existing ones, due to heterogeneous nature of this disease. the regulatory molecular machinery of a cell, including micrornas (mirnas), contributes to this heterogeneity upon aberrant expression. herein, for a mechanistic understanding of differential gene expression in crc tissue we analyzed mirna expression profiles of 78 crc tumors against 62 normal colorectal mucosa samples, using raw data from e-mtab-752 and e-geod-35834 (affymetrix microarrays), and gse35982 and e-mtab-813 (agilent microarrays) datasets obtained from gene expression omnibus and arrayexpress. raw samples were normalized (different platforms separately) using quartile normalization in brb-array-tools. differential expression of mirnas was identified using cut-off values of p ≤ 0.05, fold change ≥ 1.5 and stringent false discovery rates. mirtarbase and mirwalk2.0 databases were explored to identify validated targets. we found thirty (including mir-21 and mir-183) and thirteen (mir-1, mir-139, mir-375, etc.) mirnas commonly upregulated and downregulated respectively, in both affymetrix and agilent microarray results. predicted targets of these mirnas frequently belong to pathways related to cancer like b-catenin, phosphoinositol-3kinase, and transforming growth factor-b, to name few. moreover, the target genes were significantly enriched in clusters related to cell cycle, cell differentiation and regulation of apoptosis. these promising results will further be compared with differentially expressed gene profiles from a cohort of turkish crc patients. hence the integrated study will refine the panel of diagnostic and prognostic crc markers. hsa-mir-x modulates motility and invasion in triple breast cancer cell line s. noyan 1 , h. g€ urdal 2 , b. g€ ur dedeoglu 1 1 biotechnology institute, ankara university, ankara, turkey, 2 department of pharmacology, ankara university, ankara, turkey breast cancer is a heterogeneous disease and expression levels of certain receptors have demonstrated subtypes which characterize clinically distinct breast tumors. a triple-negative phenotype lacks expression of er, her2 and pr and is known as basallike carcinoma. micrornas are a class of small non-coding rnas that participate in the gene expression in many biological processes. e-cadherin is an important mediator of adhesion in epithelial tissues, and loss of e-cadherin can play a critical role in tumor invasive behavior. another key player of cell integrity pip (3, 4, 5) triphosphate is generated at the leading edge of the cell and leads to cell polarization. pip3 is generated by hydrolysis of pip (4,5) bisphosphate, which is synthesized by pip5k1. any dysregulation in these molecules may support the invasive behavior of the cells. the aim of this study is to find out the role of mirna precursor (hsa-mir-x) in invasion and motility in triple negative breast cancer cells. in this study a triple-negative breast cancer cell line bt-20 was transfected with hsa-mir-x or scrambled control sirna. to check its role in motility and invasion, wound healing and invasion assays were performed respectively. cell invasion was monitored over a period of 24 h by xcelligence real-time cell analyzer using a double-plate and measuring impedance-based signals. additionally emt markers were analyzed by qrt-pcr to explain the molecular mechanisms beneath motility and invasion. we observed that cell motility and cell invasion diminished after transfection of bt-20 cells with mimic for hsa-mir-x. furthermore, qrt-pcr experiments indicated that transfection of hsa-mir-x decreased the expression level of pip5k1c while increasing the e-cadherin expression level. wound healing and invasion assays together with qrt-pcr results support the role of hsa-mir-x in cell motility and invasion. this process might be explained via e-cadherin mediated met or gsk-3-beta related inhibition of invasion. expression level of five micrornas as diagnostic markers in glioblastoma situated in the main brain lobes, but can also be found in other brain regions. while microrna (mirna) as non-coding rnas, play a crucial function in the post-transcriptional regulation of gene expression by mrna degradation or translational repression. in the present study, we aimed to investigate the contribution of gene expression of the five mirnas and to unravel their role in brain tumor cell lines, the mirnas to the risk of gbm tumor. the five gbm cell lines (crl-2365, crl-2366, crl-2948, crl-1690 and htb-15) were evaluated with non-malignant (normal) brain cell line (hcn-2) . determinations of expression level of five mirnas (mir-21, mir-101, mir-138, mir-196, and mir-222) were evaluated by monitoring quantitative rt-pcr (qrt-pcr) technique. the expression levels of four mirnas (mir-101, mir-138, mir-196, and mir-222) were significantly decreased while the expression level of mir-21 was increased in gbm cell lines according to hcn-2 cell line. consequently, these five mirnas can potentially be used as biomarkers for gbm tumor; further studies are mandatory to a better understand and confirm our preliminary findings. background: noncoding rna are known to be crucial molecules with diverse regulatory roles in neural oncology and neurodegenerative disease. the recent study suggested that lncrna anril play role in the development of neuroblastoma and alzheimer disease via binding disease-specific micrornas. material and methods: we used lncrnadisease, hmdd v2.0, mir2disease to predict lncrna-and mir-associated disease in our study. in addition, we utilize tardetscan to search lncrna-mirna interaction. results: disruptions of lncrna anril expression (also named as cdkn2b-as, locus cdkn2a/b (ink4/arf), chromosome 9p21) have been associated with the development of neuroblastoma and alzheimer disease. here, we predicted interactions between noncoding transcripts encoded by locus cdkn2a/b and their molecular partners -microrna. anril can act as decoy while containing sequences that mimic mirna target sites to titer these mirs away from their primary targets thereby act as molecular sponge. using targetscan 7.0, we predicted target sites for hsa-mir-15-p/16-p/195-p/424-p/497-p/6838-p and hsa-mir-125-5p/4319 in anril 3 0 utr. then, we used hmdd v2.0 and mir2disease databases to define if any of these mirs participate in alzheimer disease and neuroblastoma. according to both databases, mir-125 is implicated to alzheimer disease and mir-15 to neuroblastoma. as soon as anril participate in the development of both abovementioned disorders and can have microrna sponge activity, it could potentially positively regulate mir-125 and mir-15 targets by competing with them for micro-rna binding sites thus restoring the expression of target genes. in our further research we plan to experimentally validate predicted microrna sites in anril 3 0 utr. conclusions: we predicted sites for mir-125 and mir-15 in 3 0 utr of anril lncrna that could uncover its possible sponge activity in the development of neuroblastoma and alzheimer disease. aim: matrine excracted from saphora flavescens root and demonsrated that indicates pro-apoptotic and anti-proliferative effect in many types of cancer. acute lymphoblastic leukemia (all) is an acute form of leukemia, or cancer of the white blood cells which characterized by the overproduction and accumulation of lymphoblasts. mirnas play important roles in deregulated cell death mechanisms. we aimed to investigate the effects of critical mirnas during the development of matrine resistance on all cell line ccrf-cem. material-method: ccrf-cem cells were treated with different (0.5-3 mg/ml) concentrations for 72 h and cell viability measurements were carried out with xcelligence device to determine the cytotoxic effects of matrine. mirnas were extracted from treated and untreated ccrf-cem cells using the mirna isolation kit. cdna was synthesised using allin-one first strand cdna synthesis kit. expressions of 44 selected mirnas were analysed with miprofiletm custom mirna qpcr array using the applied biosystem 7500 fast real-time pcr system. results: according to the cytotoxicity assay, it was determined that 'treatment with increasing concentrations of matrine, decreased the viability of the ccrf-cem cell line. expression levels of 44 different mirnas were studied for indicated passages in two replicates. our results showed that hsa-mir-376b-3p (-37,099 fold, p = 0.008), hsamir-106-3p (-16,6795 fold, p = 0.028), hsa-mir-20a-3p (-15,926 fold p = 0.0148), hsa-mir-519a-3p (-11,7398 fold, p = 0.00534), hsa-mir-204-5p (-10,9536 fold, p = 0.0012), hsamir-30b-5p (-9,0631 fold, p = 0.0221), hsa-mir-15b-5p (-8,8971 fold, p = 0.0339), hsamir-106b-5p (-8,8561 fold, p = 0.021), hsa-mir-885-3p (-8,6139 fold, p = 0.00006), hsamir-30a-5p (-8,594 fold, p = 0.009) expression were decreased during the development of matrine resistance. conclusion: these data suggested that especially hsa-mir-376b-3p plays a critical role in the matrine response. ericd (e2f1-regulated inhibitor of cell death) is a newly found lncrna. it is located at 8q24.3. it has two exons and its transcript size is 1745 bp. ericd is regulated by e2f (transcription factor 2) and modulates the cellular response to dna damage. arid3a is a family member of the at rich interaction domain (arid) dna-binding proteins that participate in diverse biological processes like development, cell cycle control, chromatin remodeling and epigenetic regulation. both, ericd and arid3a have just opposite roles in apoptosis in case of dna damage indicating a probability of reciprocal interaction between each other. till now, there is no work related to the interaction between lncrna and arid3a in cancers. herein we try to find a probable interactive role between these in cancers. in this study, 12 different cancer cell lines, 1 osteoblast cell line and 19 different types of normal human tissues rnas were selected for expression analysis of ericd and arid3a. after rna isolation, cdna was converted from their rnas. expression profile analysis of ericd and arid3a in different cancer cell lines and normal tissues was done using imagej program for semiquantitative and 2 (-ddct) method for quantitative rt-pcr. among used cancer cell lines, ericd was highly expressed and arid3a had lower expression in u-2os (osteosarcoma), a-172 (glioblastoma) and a549 (lung cancer). at the same time, ericd expression was lower and arid3a had high expression in hfob 1.19 (osteoblast cell line) and normal tissues like brain and lung . both ericd and arid3a are cell cycle regulated and are commonly regulated by e2f. they have just opposite roles in apoptosis during dna damage. these two genes have a probability of reciprocal interaction between each other in cancer. our results indicate that both ericd and arid3a might have opposite roles in lung cancer, glioblastoma and osteosarcoma. ericd and arid3a might act as cancer promoting and tumor suppressor genes respectively in these cancers. the importance of mirna expressions in infertilty implantation process is controlled with endometrium, factors secreted by the embryos and in accordance with these factors embryo and/or endometrium via receptors on. more than 700 human microrna (mirnas) that are small noncoding rnas were shown to play an important role in intracelluler cycle regulation in both normal and pathological conditions. in this study we aim to identify mirnas and controlling molecules expressions in different time period of endometrium in fertile and infertile cases. the endometrial samples were taken from fertile and infertile patients in proliferation and early secretion periods. the samples are fixed and stained either with hematoxylen-eosin for morphological analysis or with immunohistochemistry for distributions of anti-dicer, anti-drosha, anti-eif2a and anti-eif2c. mir-17-5p, mir-23a, mir-23b, mir-542-3p, mir-21, mir-199a*, mir-705, mir-20a, mir-26a, mir-125b, mir-200a/b/c were analyzed with qrt-pcr. while dicer immunoreactivity was detected weakly only proliferation phase of fertile group, this immunoreactivity were detected strongly in both proliferation and early secretory phases of infertile group. drosha immunoreactivitiy was also weakly detected in the proliferation phase of fertile group, it was moderately detected in both proliferation and early secretory phases of infertile group. eif2a immunoreactivitiy was similar in each groups but there were a few differences between fertile and infertile group. eif2c immunoreactivitiy was negative in all groups. mir-21, mir-199a* and mir-23a were highly expressed in proliferation phase of fertile group, mir-23a and mir-125b were highly expressed in early secretion phase of infertile group. in conclusion, dicer and drosha immunoreactivities and different expression of mirna's were detected in all groups. implantation problems may be reason for different mirna expression which controlling with dicer and drosha in the infertile endometrium in both proliferation and early secretory phases. wheat is an important agricultural crop with an over 615.8 million metric tons harvesting capacity annually. drought and salinity are environmental stress factors that affect yield and quality of wheat, dramatically. there are different defense mechanisms against these stress conditions in plants. altering gene expression profiles by micrornas at post-transcriptional level is one of the most conserved mechanisms among plants. micrornas are an extensive class of noncoding rnas, approximately 22 nucleotide length which regulates the expression of genes by binding to the 3 0 -untranslated regions of specific mrnas. micrornas implicated under salt and drought stress have widely been reported in numerous plant species and wheat genomes in the last years; however, studies on einkorn wheat (triticum monococcum spp. monococcum) are not yet available. the goal of this study is identification of conserved micrornas from einkorn wheat using next generation sequencing technology and bioinformatic analysis. in this study, small rna molecules were extracted from pooled plant samples grown under normal, drought and salinity conditions. sequencing analysis revealed 75 164 unique small rna sequences obtained from 15 139 448 raw reads. after bioinformatic analysis based on comparative genomics approaches, we identified 168 putative mature microrna sequences belonging to 142 distinct microrna families. since chromosomal sequence data is not available for triticum monococcum spp. monococcum, we used available sequences from triticum urartu, a close relative, as template to extract precursor microrna sequences. 111 of precursor sequences showing 100% homology to triticum urartu genome were analyzed for secondary structure prediction using mfold software. data provided in this study is critical to investigate transcriptional regulation of genes involved in stress metabolism in einkorn wheat. the role of vim-as1, a natural antisense transcript, in cancer coding or non-coding transcript. in this regard, vim-as1 is nats located antisense to vimentin gene. in the present study, we aimed to determine tissue and cell line distribution of vim-as1. materials and method: for the tissue expressions analysis, human total rna master panel ii (containing 20 different human tissue samples) was used. total number 14 cancer cell lines and 5 normal cell lines included in the study. for the expression analysis rt-pcr and qpcr methods were used. results: as a result, expression levels of vim-as1 were found to be tissue specific. particularly, while vim-as1 was highly expressed in lung and thyroid gland tissues, its expression was not observed brain, stomach and adrenal gland tissues. also, vim-as1 was also found to be differentially expressed in cancer cell lines. vim-as1 expression was found to be lost in cal29, pc3, and hct116 and highly diminished a549 cancer cells. also, it is found to be highly expressed in bcpap, panc1 and beas2b cells. discussion: results of the current study suggest that vim-as1 may have significant role in the regulation of vim gene in thyroid gland tissues, as it is highly expressed in both thyroid gland tissues and bcpap thyroid cancer cells. moreover, vim-as1 and vim axis can be involved in the formation of lung tumors because vim-as1 was highly expressed in normal lung tissues and beas2b cells and expressed very low levels in a549 lung cancer cells. lung cancer is the leading cause of cancer related deaths in the world and approximately 90% patients with lung cancer ultimately die from metastatic disease. metastasis is the most dangerous step of cancer. in our recently published work showed that akt/nf-kb pathway is continuously active and induces cellular invasion and pten suppresses cellular invasion via inhibition of akt/nf-kb pathway. in this study we aimed to show nf-kb mediated induction of mirna expression can responsible for inducing nsclc invasion. we used chromatin immunoprecipitation (chip) assay kit for detection of tnf-a induced nf-kb mediated mirnas. therefore, h1299 and pc14 cells treated by tnf-a (30 ng/ml) for chip assay. chromatin regions, reading with chip-seq, were analyzed using bioinformatics tools. we also performed additional bioinformatics search to find nf-kb related mirnas which potentially take a role in nsclc invasion. we investigated the effects of mirna which determined at the bioinformatics analysis results on invasion using invasion chamber method. we found 16 mirnas which potentially induced by nf-kb and related with nsclc invasion. our invasion results indicate that mir-548a-3p, mir-548as-3p, mir-8078, mir-1915, mir-6814-3p, mir-548q mimics can induce cellular invasion on h1299, mir-548v, mir-548 h-5p, mir-138-5p, mir-548a-3p, mir-548as-3p, mir-8078 mimics can induce cellular invasion on pc14. we also verified our results by qrt-pcr, because we want to sure that mirnas which can induce invasion, can also transcriptionally regulated by nf-kb or not. we found that mir-548q, mir-548a-3p, mir-584as-3p, mir-1915, mir-8078 in h1299, mir-138-5p, mir-548a-3p, mir-548as-3p, mir-8078 in pc14 can induce cellular invasion by nf-kb. as a conclusion, our investigation indicate that nf-kb can induce nsclc invasion via mir-548a-3p, mir-548as-3p and mir-8078. (this study is supported by tub _ itak grand number 112s636). to understand of the molecular mechanisms of cellular invasion is very important for fight against cancer mechanisms and first step of invasion is emt. cells change phenotypical and genotypical in emt progress. in this study, we focussed on the explore molecular mechanism of tnf-alpha induced cellular invasion of nsclc. we use western blot, qrt pcr and mirna transfect methods for this purpose. we find that tnf-alfa treatment reduce the expression of pten and induce e cadherin expression in a549 cells. when we inhibit nf-kb activity by p65 targeted sirna tnf-alpha can not reduce pten expression means that tnf-alpha inhibits pten expression through on nf-kb. because tnf/nf-kb pathway change the transcriptional level of mir-548as and pten 3 0 untranslated region has recognition site for mir-548as. therefore; we transfected a549 cells by mir-548as. mir-548as transfection leads to inhibition of pten expression. our results indicate that tnf-alpha mediated activation of nf-kb can inhibit pten expression on induction of emt through on mir-548as. (this study is supported by tubitak grand nummber 112s636). introduction: the corpus luteum (cl) is an ephemeral tissue whose regulated secretion of progesterone is essential for maintenance and/or timely termination of pregnancy in rodents. our previous finding that cl of pregnant rats does not possess fas/ fasl system suggests that this tissue may undergo autophagic, but not apoptotic, cell death during regression. we here investigated the presence of autophagic system in cl and its any implications in rodent pregnancy and parturition. materials and methods: lc3 (-i and -ii) expression in cl was estimated by western blot analysis in comparison with progesterone secretion and luteal mass throughout pregnancy. lc3 was also tested by immunocytochemistry. functional implication of autophagy in this tissue was examined by local treatment with bafilomycin a1 (autophagy and v-atpase inhibitor, 6.23 pg/ 0.1 ml/ovary) on day 19 of pregnancy. reproductive, biochemical, and morphological outcomes were evaluated. results: while the expression of cytosol-associated lc3-i was not significantly altered throughout pregnancy, that of autophagosome-associated lc3-ii increased significantly from day 15, showed a peak on day 21, and decreased on day 23 (day of normal parturition). lc3-ii / i ratio had positive correlations with steroidogenic activity and cell size. immunoreactive lc3 was found to be present in the cytosol of steroidogenic cells and showed marked aggregation in cells on day 21. in vivo treatment with bafilomycin a1 resulted in unaltered delivery, but in significant reductions in steroidogenic cell size and neutrophil infiltration compared to vehicle-treated control groups. discussion and conclusion: the ratio of lc3-ii / i in cl was markedly up-regulated in the late phase of pregnancy. manifestation of this autophagy parameter was temporally matched with further structural and functional activation of cl. autophagy may contribute to activation, but not regression, of rodent cl and thus their female reproduction. apoptotic and necrotic effects of low dose bisphenol a in shsy5y neuroblastoma cells b. ayazg€ ok, t. t€ uyl€ u k€ uc ߀ ukkilinc ß faculty of pharmacy, hacettepe university, ankara, turkey bisphenol a (bpa) is a commonly used chemical in industry to make plastics. 'low-dose' term has been expressed for the first time in studies with bpa in 2001. the value of low dose bpa was received as <1lm. in this study, matrix metalloproteinases (mmps) together with their tissue inhibitors (timps), involved in tissue remodeling after i-131 therapy, have been examined in 51 patients (8m/46f) with ptc and 38 (3m/38f) with ptc+ht. peripheral blood samples were collected just before and, subsequently, at 4 days after i-131 administration (3.7 gbq). ptc+ht patients had positive titers of anti-thyroglobulin autoantibodies (tgab). the serum levels of tgab, mmp-2, mmp-9, timp-1 and timp-2 were measured by elisa. there were no significant changes in serum concentrations of mmp-2, timp-2 and mmp-2/timp-2 ratio after i-131 in the two groups. in ptc patients, i-131 administration resulted in an increase with 26% in timp-1 level (p = 0.005) and a reduction with 44% in mmp-9/timp-1 ratio (p = 0.003). in ptc+ht patients it has been observed an increase with 18% in tgab level (p = 0.001), 5% in mmp-9/timp-1 ratio (p = 0.003) and unchanged timp-1 serum concentration. tgab titers were positively correlated with mmp-9/timp-1 ratio (r = 0.51, p < 0.001). our data suggest that radioiodine therapy for ptc patients, but not for ptc+ht, modulates the balance of mmp-9/timp-1 for anti-invasion and anti-migration by augmenting timp-1. in criminal cases, the determination of the time of death of the bodies step in the analysis of events is making a big contribution. today, forensic and molecular methods utilized time of death rather than provide clear information offers potential death time interval. principally, 'time of death' is a term that should be avoided. in forensic science, the postmortem 'interval' is the term to be considered. nowadays, there is no precise molecular method that can be used alone in the determination of pmi. aim of this study is to analyze the usability of ecm components, adamts1,5 and 9 and mmp1,2 and 9 to determine pmi. for this purpose, with iliopsoas muscle tissues provided by ethical board of the ankara institute of forensic medicine, 21 cases have been included in this study, divided into 3 groups according to their pmis: '0-12 h', '12-18 h' and '18-27 h'. from these tissues, western blotting technique is used to analyse the expression of adamts1,5 and 9 and mmp1,2 and 9. it is determined that when pmi increases; adamts-1 and 9 amounts decrease. on the other hand adamts-5 amounts are examined to increased related to the interval., especially on the '12-18 h' dataset. additionally, considering metalloprotease levels, mmp-2 and 9 amounts decrease as pmi increases. unlike mmp-2 and 9; mmp-1 levels increase proportional to the interval, especially on the '18-27 h' dataset. these results are the first data on pmi determination from iliopsoas muscle tissue. in this study, it is suggested that adamts-5 and mmp-1, proteases responsible for ecm degradation, can be used to determine pmi as markers. here we present the first observations of postmortem variation of mmp and adamts activities in skeletal muscle. in recently, popular mmps and adamts s pathways of the relationship between time-dependent changes to investigate the post mortem time interval determination to shed light on the future. the role of functional polymorphisms of matrix metalloproteinases 2 and 9 in spontaneous abortion samples capillaries, which are responsible for maintenance of implantation and placental nutrition, have major effects on mechanisms underlying abortion. matrix metalloproteinases (mmps) function in various cellular pathways. they play a role in patholohical conditions, metastasis; as well as normal physiological functions like tissue and bone regeneration, physiologic function of uterus, ovulation, embryogenesis and embryo implantation. mmp2 and mmp9 (gelatinases) have key roles at organisation of extracellular matrix and affect endometrial implantation. previous studies have shown that mmp2 -1306c>t and -735c>t polymorphisms cause loss of gene function, and mmp9 -1562c>t polymorphism causes a decrease in gene expression, and also gene expression levels are lower in abortion specimens, compared to control specimens. the goal of this study was to investigate the potential effects of functional mmp2 -735c>t and -1306c>t polymorphisms, and mmp9 -1562c>t polymorphism on etiology of abortion. restriction fragment length polymorphism (rflp) method was used to analyze the polymorphisms those evaluated in the study. study group consisted of samples collected from 80 spontaneous abortion specimens, and control group consisted of peripheral blood blood samples collected from 100 healthy subjects. the risk of abortion was 2.2-fold higher in patients with heterozygous -1306c>t polymorphism (p = 0.043). combined genotype analysis showed that the risk of abortion was 3.7-fold higher for patients with normal -735c>t polymorphism, and heterozygous -1306c>t polymorphism (p = 0.021). functional polymorphisms of mmp2 and mmp9 may have a role in etiology of abortion. p-02.07.5-011 changes in the specific extracellular matrix proteins and expression of adamts proteinases and effects of hypoxia in the adriamycin-induced renal fibrosis model renal fibrosis is a common cause of renal dysfunction with chronic kidney diseases. this process is characterized by excessive production of extracellular matrix (ecm) or inhibition of ecm degradation. adamts proteinases, which are widely presented in mammals, have very critical roles in ecm remodeling. we aimed to study the role of adamts proteinases in the pathogenesis of renal fibrosis and the effets of hypoxia by studying adamts expressions in rat kidney after adriamycin (adr) administration. adr was administered intravenously in consequtive two doses (2.5 and 4 mg/kg) to the rats. in addition to control and adr groups, another rats were assigned into three groups as sham, 15 min and 45 min ischemia-reperfusion. after 2 months following the first dose, the expression of adamtss were determined in the renal tissues using western blot analysis. additionally, renal nephropathy and fibrosis were assessed pathological and immuno-histochemical staining methods. in the adr group rats developed renal dysfuntion and tissue damage in favor of renal fibrosis pathologically. it is observed that occurred remarkable changes in the expression of adamtss. it is showed that hypoxia and hypoxia time enhanced tubulointerstitial fibrosis in the rat kidney tissues. expression differences were absorved also in the hypoxia groups, and especially the expression of adamts-1 and -15 genes were showed an increase in various rates. the restricted and different expression pattern of adamts protein profiles in the adr-induced renal fibrosis suggest that adamts family members are related with development and progression of fibrosis. moreover, our findings raise the possibility that the hypoxia promotes renal fibrogenesis. the monitoring of adamts proteinases might contribute to the early diagnosis of renal fibrosis and chronic kidney disease. adams (a disintegrin and metalloproteas) are transmembrane proteins that contain a pro-domain, a metalloprotease, a disintegrin, a cysteine-rich, an epidermal-growth factor like and a transmembrane domain, as well as a c-terminal cytoplasmic tail. they are involved in cell adhesion and they have protease activities. previous studies showed that some adam proteins act in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis, embryonic tgf-a release and the inflammatory response. although there are more researches about adam proteins, still the function of all adam proteins remain unclear. we aimed to investigate the potential link of infertilty with adam9, -10, and -17. in this study twenty four patients were included. the patients were classified as normozoospermia (ns; n = 8), oligozoospermia (os; n = 8), azoospermia (as; n = 8) groups. adam9, -10 and -17 protein levels in infertility indviduals were analysed by western blot. adam9 protein level was 1.7 fold lower in the os and as groups than in the ns group. adam10 protein level was 1.36 fold higher in the as group than in the ns group. adam17 protein level was 2 fold lower in the as group accourding to ns group. we observed no change between protein level of adam10 and adam17 of os and ns groups . in conclusion, adam proteins may have a potential role in male infertility. our study is a preliminary and first study on this issue. keywords: adam, infertility. the role of tissue metalloproteinase inhibitors thymus chemokine and thrombospondin-1 on prognosis of crimean-congo hemorrhagic fever s. bakir, m. bakir, s. ersan, a. engin cumhuriyet university, sivas, turkey crimean-congo hemorrhagic fever (cchf) is a disease which is caused by an arbovirus carried by ticks and characterized by the sudden onset of high fever, severe headache, dizziness, back and abdominal pain. cchf pathogenesis is still not resolved today to fully open. therefore, in this study, to determine the level of tck-1, timp-1 and tsp serum samples obtained from cchf patients and the control group is intended to be examined for the pathogenesis and prognosis of the disease. the study sample was created 45 patients with diagnosis of cchf. 45 healthy volunteers were chosen control group matched for gender and similar to in terms of age. tsp, tpc-1 and timp-1 levels of patients and a control group were analyzed using the human elisa kits. serum timp-1 tck-1 and tsp levels in cchf patients were measured significantly higher than the in the control group. cchf pathogenesis of today still have not provided fully open. therefore, it reveals the importance of this work. in our study, presence of high timp-1, tck-1 and tsp levels indicate important direction for pathogenesis and prognosis of cchf disease. p-02.07.5-015 activation of calpain 1 and protein kinase ca promotes a constitutive expression and release of matrix metalloproteinase 9 in peripheral blood mononuclear cells from cystic fibrosis patients matrix metalloproteinase 9 (mmp9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies, including cystic fibrosis. we have studied if the alteration in intracellular ca 2+ homeostasis, observed in peripheral blood mononuclear cells (pbmcs), isolated from cystic fibrosis (cf) patients homozygous for deletion of phenylalanine 508 in gene of cystic fibrosis transmembrane conductance regulator (f508del-cftr), could be involved in the abnormal presence of mmp9 in the extracellular fluids of cf patients. pbmcs were isolated from 23 healthy donors and 26 cf patients homozygous for f508del-cftr. mmp9 levels were evaluated following 2 h of cell incubation. our investigations show that all cf pbmcs analyzed constitutively express and release high levels of mmp9; conversely, in pbmcs from healthy donors, expression and secretion of mmp9 are undetectable but both events can be evoked, after 12 h of cell culture, by a possible paracrine stimulation. we have demonstrated that in cf and 12 h-cultured control pbmcs mmp9 secretion is prevented by chelation of intracellular ca 2+ and mediated by the concomitant activation of calpain and protein kinase ca (pkca) and also that mmp9 expression is mediated by the sequential activation of pkc and extracellular signal-regulated protein kinases 1 and 2 (erk1/2). moreover, the rescue of active f508del-cftr reduces the extent of mmp9 secretion, correlating the channel defect to the [ca 2+ ] i dysregulation of cf pbmcs. our results indicate that the high level of intracellular ca 2+ concentration in cf pbmcs, promoting the aberrant activation of both calpain and pkca, induces a constitutive release of mmp9. these data characterize new alterations in mononuclear leukocytes of cf patients that may be of primary importance in the progression of the disease and indicate that pbmcs may contribute to the accumulation of mmp9 in fluids of cf patients. p-02.07.5-016 aebp1/aclp is upregulated in differentiation, injury repair and fibrotic degeneration of skeletal muscle c. € ozdemir 1,2 , u. akpulat 1 , i. onbasilar 3 , c ß . kocaefe 1 1 department of medical biology, faculty of medicine, hacettepe university, ankara, turkey, 2 department of stem cell, institute of health, hacettepe university, ankara, turkey, 3 laboratory animal breeding and research unit, faculty of medicine, hacettepe university, ankara, turkey aebp1/aclp is an ambiguous gene with several attributed functions and cellular events, adipogenic differentiation, cell adhesion, pattern development and fibrosis are the well-understood. aebp1 is the short isoform that acts as a transcriptional repressor by targeting the ap2 promoter and aclp, which is the long isoform that harbors a leader sequence that directs the peptide to the extracellular compartment. the latter is known to be associated with development of the connective tissue, injury repair and fibrosis in certain pathological conditions. aebp1/aclp displays a moderate expression in skeletal muscle where the role is not known. we have investigated the spatial and temporal expression of aebp1/aclp in defined models of skeletal muscle differentiation, injury repair and fibrosis. aebp1/aclp expression is present in steady state dividing myoblasts. this basal expression is upregulated 4 folds upon the induction of differentiation in c2c12 cells. considering that differentiation and post-natal injury repair share several common aspects, we also investigated the expression of aebp1/aclp in acute injury-repair model. in the course of cardiotoxin induced injury, aebp1/aclp expression peaks up to 5 folds in the 6th day of regeneration. this time point concomitantly corresponds to tissue remodelling. since aebp1/aclp is also known to be associated with fibrotic events in chronic pathological conditions, we also have investigated its expression in tenotomy induced skeletal muscle degeneration which mimics endomysial and perimysial fibrosis. aebp1/aclp expression is upregulated up to 10 folds in early time-point samples. these results depict a novel role for aebp1/aclp in extracellular remodeling of the skeletal muscle during injury repair as well as fibrotic degeneration. the source of this expression might come from fibroadipogenic precursers which reside in endomisial area of muscle. our current efforts are focused on presenting of this endomysial expression. the mprbp gene from b. pumilus 3-19 encoding a novel secreted metalloproteinase was identified. based on the primary structure the enzyme has been classified as metzincin metalloproteinase that combines the features of two families: astacin and adamalysin. representatives of the adamalysin family previously have not been described for bacilli. the aim of the work was to elucidate the mechanisms of the gene expression regulation of a new bacillus pumilus 3-19 extracellular metalloendopeptidase. promoter region analysis revealed the presence of potential binding sites for transcription factors spo0a (sporulation) and degu (biodegradation). study of mprbp expression in strain defective in regulatory proteins degs and degu shows that the productivity of metalloproteinase biosynthesis decline in average 60% compared with the strain with a complete degs-degu system. we also studied mprbp expression in strains with a mutation in the gene degu, leading to stabilization of degu~p protein. it is known, that this mutation leads to a multiple increase in the gene expression level, positively regulated by degs-degu system. our data shows a 10-fold increase in metalloproteinase productivity in the recombinant strain. thus, deg-system participates in control of the proteinase synthesis but not only in the regulation of mprbp gene expression. the mprbp expression in the strain deficient in regulatory protein spo0a remained at the level with expression in the strain with the complete spo0a. a similar pattern we observed in the study of mprbp gene expression in strains defective in other spore-specific regulatory proteins (spo0b, spo0f, spo0k, spo0j, sigf, sigh, sigk). these data indicate that mprbp gene expression is free of spo-regulatory proteins. on this basis, we concluded that the expression of metalloproteinase gene is not correlated with the sporulation. p-02.07.5-019 paricalcitol attenuate activity and expression of matrix metalloproteinases in a rat model of renal ischemia-reperfusion injury matrix metalloproteinases (mmps) are endopeptidases involved in the degradation of extracellular matrix. they have been postulated to have a role in the pathogenesis of ischemia-reperfusion injury (iri). in the present study, we investigated the effect of paricalcitol, a synthetic vitamin d analog, on mmps in renal iri. 21 wistar albino rats were divided into three groups: sham operated, ischemia-reperfusion, and paricalcitol-pretreated. iri model was induced by bilateral clamping of renal arteries for 45 min followed by 24 h of reperfusion. the analysis of serum creatinine levels and activities/expressions of mmp-2 and -9 were performed after 24 h of iri. the effects of paricalcitol on activities and expressions of mmp-2 and mmp-9 levels were investigated by gelatin zymography and immunohistochemistry, respectively. the pathological examinations were performed to score tubular damage by light microscopy. creatinine levels increased significantly in the iri group. rats in the paricalcitolpretreated group showed significant decrease in expressions and activities of mmp-2 and mmp-9 during iri. moreover, pathological examinations displayed significantly lower score of tubular damage in paricalcitol-pretreated group. in conclusion, paricalcitol attenuated iri by downregulating the expressions and activities of mmp-2 and -9. p-02.07.5-020 the changes of matrix metalloproteinase 2, 9 activity and hyaluronic acid level in rat's heart and serum under cadmium influence o. fomenko 1 , o. shaulska 1 , y. kot 2 , g. ushakova 3 , a. shevtsova 1 1 dnipropetrovsk medical academy, dnipropetrovsk, ukraine, 2 kharkiv national university, kharkiv, ukraine, 3 dnipropetrovsk national university, dnipropetrovsk, ukraine the changes in the molecular mechanisms of the extracellular matrix degradation under toxic factors are not well known. the main goal of work was the investigation of the mmp2 and mmp9 activity and hyaluronic acid level in the heart and blood serum under cadmium influence at different doses. the 18 wister rats divided to 3 groups were used for the experiment. cdcl 2 x2.5h 2 o in doses 0.1 lg/kg and 1 lg/kg was given to rats intragastrically in drinking water during 36 days. the rats were decapitated under isoflurane anesthesia according to ethical rules; the heart was quickly removed. the relative activity [in arbitrary units (au)] of pro-and active forms of mmp9 and mmp2, total protein (tp) and hyaluronic acid levels were calculated. it was shown that low doses of exogenous cadmium (0.1 lg/ kg) lead to reduced activity of pro-and active forms of mmp9 in myocardium (7.3 ae 0.6 au/mgtp and 7.1 ae 0.6 au/mgtp compare to the 9.67 ae 0.4 au/mgtp and 9.7 ae 0.5 au/mgtp in the control rats accordingly) and in serum (0.95 ae 0.2 au/mgtp and 0.35 ae 0.05 au/mgtp compare to the 1.54 ae 0.05 au/mgtp and 1.49 ae 0.05 au/mgtp in the control rats accordingly), but pro-mmp2 activity in heart was increased (14.5 ae 1.6 au/mgtp compare to the 9.8 ae 0.6 au/mgtp in the control rats); level of ha was decreased in both tissues (0.69 ae 0.16 lg/ml and 3.63 ae 0.3 lg/ml compare to the 1.0 ae 0.13 lg/ml and 3.91 ae 0.3 lg/ml in the control rats accordingly). high doses of cadmium (1 lg/kg) caused a reliable increase of both gelatinase activity in the myocardium: mmp2 increased from 9.65 ae 0.4 au/mgtp to 14.1 ae 0.8 au/mgtp, prommp9to 12.6 ae 1.5 au/mgtp, mmp9to 15.4 ae 1.6 au/mgtp. ha level was increased in serum (4.28 ae 0.1 lg/ml) and decreased in heart (0.49 ae 0.09 lg/ml). the results indicate the dose-dependent and tissue-specific effect of cadmium on mmp-depended protein degradation and level of hyaluronic acid. a disintegrin-like and metalloproteinase domain with thrompospondin-1 repeats (adamts) are a large family of proteoglycanase that show proteolytic activity towards proteoglycans like aggrecan, brevican, neurocan, and versican. interleukin-33 (il-33) is an il-1 cytokine family member that uniquely plays a role as a cytokine and nuclear factor. it is released by necrotic epithelial cells and activated innate immune cells as an alarming danger signal. adamts and il-33 implicated in brain cancer pathogenesis. we aimed to seek the amount of adamts15 in u118 proteolytic enzymes are able to speed up wound healing by removal of the necrotic tissues and fibrin. several investigations have reported that proteases damage also the microbial biofilms formed by opportunistic bacteria including staphylococci on surfaces of chronic and acute dermal wounds. therefore, proteases are seemingly perspective enzymes for biofilm eradication by hydrolysis of both matrix proteins and adhesins, proteins providing cells attachment onto solid surface and other bacteria, as well as by the cleavage of signalling peptides of intercellular communication of gram-positive bacteria. here we report that ficin, a plant protease, efficiently degrades the structural components of biofilm matrix formed by s. aureus and s. epidermidis at concentrations of 10 lg/ml while trypsin and chimotrypsin are used as 1-2 mg/ml solution. the spatial structure of the biofilm was analyzed by atomic force microscopy. after ficin treatment, the biofilm structure became porous, with reduced viscosity. the congo red staining of the treated biofilms confirmed the hydrolysis of the protein component of the matrix. moreover, the biofilm treatment with ficin increased the antimicrobial efficiency of ciprofloxacin against biofilm-embedded cells of s. aureus and s. epidermidis. while 24 h antibiotic treatment did not lead to the increase of dead cells of neither s. aureus nor s. epidermidis embedded into the biofilm matrix, in the presence of ficin the fraction of viable cells decreased significantly. accordingly, soluble ficin appears beneficial for outer wound treatment biofilm eradication and reduces the reinfection risk. the wound-healing activity of ficin requires further investigations. this work was supported by the russian science foundation (project no 15-14-00046). resveratrol (resv) is an antioxidant that belongs to the group of plant compounds, called polyphenols. resv is defined as an antimicrobial substance that is produced by several plants (red grape skin, peanuts and berries) to protect them from rough environments like excessive ultraviolet light, infections and climate changes. as an antioxidant, this polyphenol protects the body against cardio-vascular and cancer diseases. besides, resv has anti-inflammatory, neuro-protective, anti-diabetic and other pharmacological effects. although the positive pleiotropic effects of this polyphenol are well documented, there is a huge need to understand its influence on the biophysical properties of lipid bilayer. in the present work, the interaction of resv with membranes composed of palmitoyl-docosahexaenoyl phosphatidylcholine (pdpc) or palmitoyl-oleoyl phosphatidylcholine (popc), sphingomyelin (sm) and cholesterol (ch) was investigated by means of fluorescence spectroscopy. generalized polarization of the fluorescent probe laurdan (gp) as a function of temperature was used to probe the changes in the fluidity of lipid bilayer induced by different resv concentration. the obtained results showed decreased lipid ordering from 50 to 100 lmol resv and opposite effect from 200 to 500 lmol in pdpc/sm/ch mixtures as compared to the control without resv. the interaction of resv with popc/sm/ch mixtures caused only an increase in the lipid ordering as a function of resv amount. popc and pdpc have the same head group but different fatty acid chains at sn-2. since resv changes the physicochemical properties of lipid bilayer by different ways one might suggest that the interaction of polyphenol with the membrane depends on the level of fatty acid unsaturation. this specific effect of resv on lipid organization could be related to its unique properties to prevent the cell from oxidative stress. neurodegeneration is the umbrella term for the deseases including progressive loss of structure or function up to death of neurons. beta-amyliod peptide is proteolytic fragment of the amyloid protein. the spontaneously formation of selective, voltage-dependent, ion-permeable channels in the lipid bilayers was reported as one of amyliod peptide toxicity mechanisms. the aim of our study was the investigation of the influence of the flavonoids (phloretin, phlorizin, quercetin and genistein) on the membrane activity of amyliod peptides. virtually solvent-free bilayer lipid membranes were prepared from mixtures of phospholipids in 0.1 m kcl (ph 7.4) using monolayer-opposition technique. using spectrofluorimetry we estimated prepared from phospholipids by extrusion the liposomal membrane permeability for calcein. we found that the addition of phloretin into membrane bathing solution led to an signicant increase in the channel forming activity of fragments 25-35 of amyloid peptide, fragment 25-35 of [gly35]-amyloid peptide and 106-126 of human prion protein. addition of other flavonoids did not cause any changes in the steady-state amyloid-induced current. it was found that the effect was caused by electrostatic interaction with the peptide. we found that time course of amyloid induced leakage calcein from liposome's is characterized by two components: the fast one is related to sorption of b-amyloid peptide on the membrane and the slow one is related to the oligomerization of the peptides on the surface of the lipid bilayer. addition of the phloretin simultaneously with b-amyloid peptide to the suspension of liposomes caused significant reduction in time parameters characterizing fast and slow components. from this results we can proposed that phloretin compensates the positive charge of the b-amyloid peptides and leads to the changes in their oligomerization status. the study was supported in part by rfs (14-14-00565) and sp-69.2015.4. ferritin nanocarriers: a focus on a metal-based drug encapsulated inside the protein cavity s. ciambellotti 1 , c. bernacchioni 2 , f. scaletti 3 , p. turano 1 1 cerm (magnetic resonance centre), florence, italy, 2 department of biochemical sciences, university of florence, florence, italy, 3 chemistry department 'ugo schiff', florence, italy ferritin is one of the main player involved in the iron metabolism. the biological role of ferritin is the storage of iron atoms inside the cavity preventing the uncontrolled accumulation of toxic species inside cells. ferritins are polymers constituted by 24 subunits that self-assemble giving rise to an almost spherical nanocage. in vertebrates, ferritins are formed by two distinct subunits, the heavy chain (h), with oxidoreductase activity, and the light chain (l) without catalytic activity. ferritins are promising nanocarriers for the delivery of contrast agents for diagnosis and of drugs for therapeutic purpose. their endogenous origin and the possibility to stabilize and protect the enclosed cargo inside the cavity, make ferritin a biocompatible vehicle. moreover, there are specific receptors on cells that recognize and incorporate ferritin by endocytosis, prospecting a kind of targeted-delivery. following the increasing interest in nanotechnology, we studied the interaction between different isoforms of ferritin with an antimetastatic drug, called nami-a, which is the first ruthenium derived anti-cancer drug to have entered clinical evaluation. this molecule is a metal-based prodrug that can release the metal ion ligands. here, we describe nami-a hydrolysis in the presence of recombinant homopolymers of ferritin followed spectrophotometrically. thanks to characteristic time dependent changes of spectral profile in the uv-visible region, we could detect differences in the hydrolysis process. the formation of a ru-adduct with hferritin was established by uv-visible and circular dichroism spectroscopies, as well as by kinetics measurements that showed inhibition of the ferroxidase activity of h-ferritin. crystallization trials are in progress to identify the binding site of ru by solving the x-ray structure of the complex. finally, we planned to test the cytotoxicity of ferritins pre-incubated with nami-a, using different cancer cell lines. a. cort 1 , t. ozben 2 , a. sansone 3 , s. barata-vallejo 4 , c. chatgilialoglu 5 , c. ferreri 3 1 sanko university, gaziantep, turkey, 2 akdeniz university, antalya, turkey, 3 cnr, bologna, italy, 4 universidad de buenos aires, buenos aires, argentina, 5 national center for scientific research 'demokritos', athens, greece liposomes as biomimetic models of cell membranes were used for examining some novel aspects of drug-metal induced reactivity with unsaturated lipids under oxidative conditions. the chemical basis of cis to trans transformation was ascertained by liposome experiments, using bleomycin-iron complexes in the presence of thiol as a reducing agent that by incubation at 37°c gave rise to the thiyl radical-catalysed double bond isomerisation of membrane phospholipids. the effect of oxygen and reagent concentrations on the reaction outcome was studied. as a chemical biology model for antitumoral strategies, liposomes highlight the role of cell membranes, which are not spectators but important targets of the drug effect, with synergic roles for chemotherapeutic effects. indeed, fatty acid recruitment and membrane formation are attracting a lot of interest in cancer, and in this context the loss of the natural cis geometry and the oxidationinduced lipid remodelling are worthy of deeper studies in antitumoral strategies. furthermore, the interaction between drugs and lipids can be suggestive of novel aspects of chemical reactivity for liposome carriers when circulating in vivo. gpr17 is a g-protein coupled receptor (gpcr), expressed in cells of brain, heart and kidney. it is related to the leukotriene and purinergic subfamilies of the rhodopsin-like class a gpcrs. gpr17 plays controversial role in the brain and spinal cord cells recovery after injuries. it is assumed that gpr17 is one of the cell death regulators immediately following an injury but at later stages it also takes part in tissue regeneration. there are also data implying some role of gpr17 in glucose homeostasis. drugs targeting gpr17 may help with treatments of multiple sclerosis and ischemia. the damage of rat's brain in artificially induced ischemia disease decreased after gpr17 inhibition. in addition, gpr17 takes part in myelin sheath formation, the lack of which is known to be the reason of multiple sclerosis. to better understand functional role of gpr17 and enable design of more efficient ligands we initiated structural studies of this receptor. to improve receptor stability and facilitate crystallization we created a series of chimeric constructs using different fusion partnerssmall soluble proteins inserted into the native amino acid sequence. preliminary experiments were carried out to evaluate the influence of different ligands on the receptor stability and cell surface expression in insect sf9 cells. this work was supported by russian federal target sterols are significant for the structural and dynamical features of cell membranes. among them, cholesterol is the major sterol in mammalian cell membranes whereas stigmasterol is the predominant sterol in plant membranes. stigmasterol varies structurally from cholesterol in having both an ethyl group at carbon 24 and an additional trans double bond between carbons 22 and 23. dimyristoylphosphatidylcholine (dmpc) is a widely studied synthetic lipid, which has a neutral (zwitterionic) pc headgroup and two symmetrical 14-carbon fatty acyl chains. the studies on the interactions of cholesterol and stigmasterol with dmpc membranes at molecular level are very limited. in the present study, a calorimetric comparison of the effects of the animal sterol cholesterol and the plant sterol stigmasterol on zwitterionic dimyristoylphosphatidylcholine (dmpc) multilamellar vesicles (mlvs) was investigated for the first time by using differential scanning calorimetry (dsc). our dsc studies indicate that with the inclusion of increasing cholesterol and stigmasterol concentrations from 5 to 40 mol% into pure dmpc mlvs, the pretransition disappears, the main phase transition shifts to lower temperatures and then disappears at cholesterol and stigmasterol concentration above 25 mol%. the main phase transition enthalpy (dh) is progressively reduced whereas full width at half maximum (dt ½ ) gradually increases with increasing cholesterol and stigmasterol concentrations. moreover, the main phase transition peak consists of overlapping sharp and broad components, indicating the hydrocarbon chain melting of sterol-poor and sterol-rich dmpc domains, respectively. in conclusion, this study shows clearly that both cholesterol and stigmasterol interact effectively with dmpc membranes and cause changes in their structural and functional properties. p-03.03.3-007 trh receptor mobility in the plasma membrane is affected by its interaction with its cognate signaling molecules and by cholesterol depletion r. moravcova, b. melkes, j. novotny department of physiology, faculty of science, charles university in prague, prague, czech republic g protein-coupled receptors (gpcrs) play a fundamental role in transferring information from extracellular environment to the cell interior. some gpcrs are supposed to form signaling complexes with their cognate g proteins and possibly other accessory proteins, which may facilitate the activation of g proteins and thus accelerate signal transmission. here we investigated the role of some components of thyrotropin-releasing hormone (trh) receptor signaling in hek293 cells stably expressing yfp-tagged trh receptor using fluorescence recovery after photobleaching (frap). we observed significant changes in values of the diffusion coefficients if expression of b 2 -arrestin or gb 2 subunit were suppressed by sirna. results of our frap experiments indicated significant difference between control and trh-treated cells as the diffusion coefficient markedly decreased after agonist stimulation. on the other hand, the same decline of the diffusion coefficient value was found after silencing with sirna and subsequent treatment with trh for most of the screened proteins. treatment of cells with 10 à5 m trh led to fast internalization of trh receptor, which was revealed by real-time confocal microscopy. it is known that cholesterol is an essential component of the cell membranes and it exerts modulatory effects on the functioning of various membrane proteins. disruption of plasma membrane integrity by cholesterol depletion using b-cyclodextrin significantly reduced the apparent diffusion coefficient values. interestingly, addition of trh to cells treated with b-cyclodextrin did not further reduced trh receptor mobility. it can be concluded that stimulation with agonist and/or sirna silencing of some components of the trh receptor signaling cascade significantly affects the mobility of trh receptor in the plasma membrane. p-03.03.3-008 l-opioid receptor mobility in the plasma membrane is diversely affected by biased ligands b. melkes, l. hejnova, j. novotny opioid receptors belong to the large family of g protein-coupled receptors (gpcrs), which are currently considered among the most important targets for pharmacological manipulations. during the past few years, a great deal of attention has been devoted to biased agonism. this phenomenon reflects the ability of different ligands to selectively affect the functioning of some gpcrs so they can display marked differences in their efficacies for g protein-or b-arrestin-mediated signaling. here we decided to investigate the effect of different agonists of the l-opioid receptor (l-or) on the lateral mobility of this receptor in the plasma membrane of hek293 cells which were stably transfected with l-or tagged with yellow fluorescent protein (l-or-yfp). it has been found previously that damgo stimulates g-protein-dependent signaling, endomorphine 2 stimulates arrestin-dependent signaling and morphine does not show any significant bias towards these two signaling pathways. in our experiments, we used the fluorescence recovery after photobleaching (frap) method to estimate the diffusion coefficients of l-or-yfp in the resting state and after addition of the above mentioned specific agonists. we observed that addition of damgo increased the value of the diffusion coefficient and addition of endomorphin 2 decreased the value of diffusion coefficient of l-or-yfp. addition of morphine or the l-or antagonist naloxone did not change the value of the diffusion coefficient compared to the resting state. these results indicate that different biased agonists of l-or may differently affect the mobility of this receptor in the plasma membrane. these findings provide new insights into the dynamics of l-or in the plasma membrane and contribute to better understanding of the mechanism of biased agonism at gpcrs, which is of central importance for receptor homeostasis and fine regulation of receptor activity. color tuning and adding potassium selectivity to a light-driven sodium pump k. kovalev 1,2 , v. polovinkin 1,2,3 , v. shevchenko 1,2 , v. gordeliy 1,2,3 1 moscow institute of physics and technology, dolgoprudniy, russia, 2 research centre j€ ulich, j€ ulich, germany, 3 institut de biologie structurale, universit e grenoble alpes, cnrs, and cea, grenoble, france recently a light-driven sodium pump has been discovered, characterized and tested as an inhibitory optogenetic tool. sodium pumping rhodopsin from dokdonia eikasta kr2 has an absorption maximum at 523 nm at ph 7.5 and to create more redshifted variants we analyzed available structures of the kr2 (pdb codes: 4xtl, 4xtn) and did the rational mutagenesis of residues in the retinal proximity region (i.e. m149, g153 and s254). the mutants of kr2 under investigation were: m149a, g153v, m149a/g153v, s254a, s254f, s254g, s254l, s254m, s254n, s254t, s254v, s254y. the protein mutants were expressed in escherichia coli c41 strain, expression was induced by the addition of 1 mm isopropyl b-d-1-thiogalactopyranoside. the cells were then washed trice with unbuffered 100 mm nacl or kcl solution. finally, the ph changes in cell suspensions (od600 = 8.0) were monitored. ph changes upon the addition of 30 lm of protonophore carbonylcyanide m-chlorophenylhydrazone were also measured. the following mutants completely abolished the protein function and were not used for further characterization: s254f, s254l, s254m, s254n, s254v. the remaining mutants have shown sodium pumping activity and s254a mutant has gained an additional potassium pumping activity. all functionally active mutants were purified using ni-affinity chromatography and the absorption spectra were collected for them at ph 7.5 (50 mm na/na-pi, 100 mm nacl). m149a mutant absorption maximum is blue-shifted to 519 nm. g153v and m149a/g153vblue-shift to 470 nm. s254a, a potassium pumping variant,red-shift to 545 nm. s254g, s254yred-shift to 545 nm. s254tno change in absorption maximum position. based on structural analysis of kr2 we discovered another potassium pumping variant and provided the variants with absorption maximum blue-shift up to 53 nm and red-shift up to 22 nm. human endothelin receptors belong to the g-protein coupled receptors (gpcrs) superfamily. this class is pharmacologically important, with more than 40% drugs targeting it. the human endothelin system, which includes endothelin receptors types a and b (etb and eta), plays a highly important role in the blood pressure regulation. endothelium cells produce peptides, known as endothelins 1-4, which activate endothelin receptors and launch cascades of reactions that lead to vasoconstriction or vasodilatation depending on the receptor subtype and the tissue. additionally, endothelin receptors take part in such processes as transmission of nerve impulses, development of neural crest, and regulation of acid-base-salt balance in kidneys. in order to stabilize etb receptor for crystallization trials we introduced a compact soluble protein, apocytochrome b564ril (bril), is the third extracellular loop of the receptor. bril is known to be an effective crystallization driver for gpcrs. the engineered protein was expressed using baculovirus system in sf9 insect cells, purified and subject to variety of pre-crystallization assays. localization of the overexpressed protein in insect cells was visualized via the confocal microscopy. thermal stability of the protein in the presence and absence of ligands was measured by the thermal shift assay. finally, the mobility of the receptor in lipidic cubic phase (lcp) at many different conditions was probed by the lcp-frap (fluorescence recovery after photobleaching) assay. these tests showed that the obtained protein is thermally stable, functionally active and diffuses well in lcp at certain conditions, making it a suitable candidate for proceeding to in meso crystallization trials. this work was supported by the russian science foundation (project no. 16-14-10273). mitochondrial oxidative phosphorylation is the key metabolic pathway for the production of atp. mitochondrial respiratory chain (rc) defects are some of the most commonly diagnosed inborn errors of metabolism with a diverse spectrum of clinical phenotypes. the aim of the study is to evaluate the rc enzyme activities and histopathological findings in muscle biopsies of patients with suspected mitochondrial disease. muscle biopsy samples were collected from 48 pediatric patients. the samples were homogenized in seth buffer using a glass/glass homogenizer. the activities of citrate synthase (cs) and rc enzymes (complex i, ii-iii, and iv) were measured in tissue homogenates by kinetic spectrophotometric assays by schimadzu uv spectrophotometer (uv-1800). non-collagen protein was determined by the modified lowry assay. activities of complex (c) i, ii-iii and iv (cox) were normalized by cs. histopathological investigations included h&e, modified gomori trichrome, periodic acid schiff, oil-red-o, nadh, sdh, cox and atpase stains. deficiency of rc complexes was detected in 39 biopsies (81%). c iv deficiency was most common (60%), followed by c i (40%) and c ii-iii (27%). multiple complex deficiency was present in 40% and isolated deficiency was present in 42% of the biopsies (20% c i, 20% c ii-iii, 60% c iv). cs activity was elevated in 44% of the biopsies. decreased c i/cs, c ii-iii/cs and c iv/cs ratio was observed in 44%, 42% and 52%, respectively. comparing with histological data, biochemical analysis revealed additional findings in 50% of biopsies. complex iv deficiency, either isolated or accompanied by other complex deficiencies, is most common in our cohort. rc analysis may reveal additional findings to histopathological results and careful clinical investigation with correlation of clinical, biochemical and histopathological data is mandatory for the challenging diagnosis of mitochondrial disorders in childhood. investigation of adipocytokines, activity of glut and na + /k + -atpase in rats fed glucose, fructose, starch-based sugars objective: all over the world, shows a significant increase in obesity and diabetes. intake of foods that contain fructose, glucose and starch-based sugar is a potential risk for metabolic syndrome. obesity and diabetes are important effects of high fructose corn syrup (hfcs). we aimed to research, activity of na + /k + -atpase in addition to glucose transporter (glut) 2, resistin, adiponectin and other biochemical markers in rats fed glucose, fructose and starch-based sugars. materials and methods: study was performed on rats and 3 groups were included in the study. rats were fed with chows that were given either normal diet for control group (70% carbohydrate, 20% protein and 10% fat), high fructose (70% carbohydrate (87% fructose and 13% starch), 20% protein and 10% fat), or high sucrose (70% carbohydrate (87% sucrose and 13% starch), 20% protein and 10% fat). rats were fed with chows for 8 weeks. in this process, the weight of the rats were followed. at the end of the experiment, blood is taken in all groups. level of hba1c, glucose, resistin and adiponectin were studied. glut2 and na + /k + -atpase activity were studied in the liver tissue. results: a significant increase in adiponectin levels were determined in rats fed both hfcs and sucrose (p < 0.001). a significant decrease in level of na + /k + -atpase activity were determined in rats fed both hfcs and sucrose (p < 0.001). there was no significant differance level of hba1c, glucose, resistin and glut2 in rats fed sucrose or hfcs (p > 0.05). conclusions: fructose-rich diet has an effect on changes in the atpase activity and is a major risk factor for obesity. keywords: adiponectin, fructose, glucose, high-fructose corn syrup, na + /k + -atpase, resistin. p-03.03.3-014 nucleic acid-biomembrane lipid selfassemblies: from primordial soup to novel genome organization model and cellular nonviral nanotherapies f. k. demirsoy 1 , n. eruygur 2 , e. s€ uleymanoglu 3 1 biotechnology central laboratory, biotechnology institute, ankara university, ankara, turkey, 2 department of pharmacognosy, faculty of pharmacy, cumhuriyet university, sivas, turkey, 3 department of pharmaceutical chemistry, faculty of pharmacy, gazi university, ankara, turkey turkey nucleic acid-cell membrane complexes has attracted research interest as models in gene regulation, cell cycle and division, as biosensors designs, as well as in molecular evolution. zwitterionic phospholipids, complexed with polyribonucleotides by divalent metal cations (mg2+) are considered as genosome vehicles. their formations are studied by spectroscopic, thermodynamic, interfacial and microscopic approaches to build thermodynamic and kinetic models of their structural transitions. dna forms a mg2+-driven ternary complexes with neutral liposomes both at the airwater interfaces and at vesicle surfaces. the described self-assemblies form relevant models for nuclear pore complexes and their further implications in gene expression and functions. such membrane contacts could be considered also in prokaryotic nucleoids important in bacterial segregation, whereas in eukaryotes these complexes can be regarded as focal points for transcription-translationtranslocation processes. the effects of ozone/oxygen mixture on citrate synthase and mitochondrial complex activities of striated muscle tissue of healthy mice we investigated the effects of ozone/oxygen mixture and oxygen on citrate synthase (cs) and succinate dehydrogenase (sdh) activities of striated muscle tissue of healthy mice. thirty-six mice were included the study. firstly muscle samples were taken from all mice's left thigh muscle in under anesthesia (group 0). secondly mice were randomly divided in four groups as: group 1: oxygen was given once at 3 days for 7 days, group 2: ozone/oxygen was given once at 3 days for 7 days, group 3: oxygen was given once at 3 days for 30 days, group 4: ozone/oxygen was given once at 3 days for 30 days. ozone/oxygen mixture and oxygen were given at a dose of 1 mg/ kg groups (1) (2) (3) (4) . after treatment animals were sacrificed, and muscle samples were taken and stored in à80°c for until the analyses. muscle tissues were homogenized in 0.05 m tris-hci and 0.15 m kci. cs and sdh activities were measured with spectrophotometer. cs and sdh activities were expressed as lmol/min/g tissue. cs and sdh activity results were given as mean ae sd. cs activity has been found in group 0 (37.8 ae 11.7), group 1 (46.9 ae 10.8), group 2 (34.7 ae 6.8), group 3 (32.9 ae 7.5) and group 4 (33.9 ae 8.5). sdh activity has been found in group 0 (2.50 ae 1.03), group 1 (2.37 ae 0.77), group 2 (2.52 ae 1.45), group 3 (2.24 ae 1.06) and group 4 (2.59 ae 1.25). there was no statistically significant difference among all groups in terms of cs (p > 0.130) and sdh activities (p > 0.997). there was no difference between all groups in terms of inflammation, muscle fiber size, regeneration or necrosis. vascular structures, connective tissues, lipid and glycogen content of fibers were normal. cytochrome oxidase activity was also normal. ratio of ragged blue fibers of all groups were less than 0.3%, so they were scored as 1. there was no difference among groups for ragged red fiber content. we have not found a significant effect of ozone/oxygen mixture and oxygen on cs and sdh activities of striated muscle tissue of healthy mice. lipidic cubic phases (lcps) consist of bicontinuous lipid bilayers and water channels. lpcs are widely used for membrane proteins crystallization and further determination of their spatial structures by means of x-ray crystallography. this approach was successfully used for studying g-protein coupled receptors structures. usually crystallization initiates by adding the precipitants (buffers with high salt concentrations). here we propose to use photo-switchable analogs of 1-monoolein to change lattice parameter of lpc. we synthetized a number of novel diazo-analogs of 1-monoolein. their structures were confirmed by nmr-spectroscopy and mass-spectrometry. being incorporated in phospholipid vesicles or detergent micelles they subjected to trans-to cis-isomerization under the light exposure at 365 nm. also we characterized the lcp's structures and properties by small-angle x-ray scattering on the rigaku instrument. one of the synthetized compounds, 3-(4-{-[4-(octyloxy) phenyl] diazenyl} phenoxy) propane-1,2-diol (1% mol), was incorporated into the 1-monoolein cubic phase. according to small-angle x-ray scattering data the structure of the monoolein cubic-pn3 m phase with lattice parameter 106.3 angstrem was not affected by insertion of this photo-switchable monoolein's analog. after the light exposure at 365 nm we observed trans-cis-isomerization. in the same time the cubic-pn3 m phase was not changed but the lattice parameter reduced to 98.8 angstrem. this effect on monoolein lpc is similar to the addition of a precipitant to initiate protein crystallization process. the spontaneous return to the initial lattice parameter was completed after 3 days in dark. thus, we demonstrated the possible controlling of the monoolein cubic phase lattice parameters by adding the photo-switchable diazo-derivatives of monoglyceride analogs, which can be used for crystallization of membrane proteins. evaluation of certain protein and phosphoprotein expression levels by using western blot technique in head and neck squamous cell carcinoma a. kalayci yigin 1 , t. cora 2 1 cerrahpasa faculty of medicine, istanbul university, istanbul, turkey, 2 faculty of medicine, selcuk university, konya, turkey introduction: head and neck squamous cell carcinoma (hnscc) is a primer tumor type in head and neck cancers, characterized by aggressiveness, early recurrence and metastasis. while alcohol and smoking play an important role at pathogenesis of disease, deregulation of some signaling pathways, genes and protein levels related to these pathways are considered as important at contribution of development of hnscc. materials and methods: in this study, protein and phosphoprotein expression levels of the frequently phosphorylated sites (egfr, pegfr, igf-ir, pigf-ir, pdgfrb, ppgdfrb, pten, ppten, akt and pakt) were investigated by using a western blot to confirm the expression of mrna in the context of protein levels at normal-tumor tissues of hnscc and sccl-mt1 that is a hnscc tumor cell line and hek-293 that is a normal cell line. results: as a result of western blot analysis egfr, pdgfrb and igf-1r were detected as highly overexpressed cell surface receptors in tumor tissues of hnscc. discussion and conclusion: the findings of this study revealed the overexpression of other cell surface receptors as well as egfr in hnscc. in conclution, potential pathways were identified by determining the cell surface receptors overexpressed in hnscc, these data support each other and may be important in pathogenesis of hnscc. introduction: the investigation of final products of lipid peroxidation is considered as the main mechanism involved in development of pathogenesis, diagnostics and prognosis of various parasitic illnesses. materials and methods: the concentration of lp-ap in the blood was determined in the study group considered of 129 women (65%), and 69 men (35%). results: before antiparasitic treatment, women infected with g. intestinalis showed a statistically significant 1.5 times increase of gpx activity levels; and 1.7 times ada level increase compared to the control group. after the treatment, the cat activity showed a sharp increase, whereas the ada activity decreased by 1.4 times, compared to the average level before the treatment. the results of the blood samples of the infected men with giardiasis, show the statistically significant increase in the level of all the studied parameters of lipid peroxidation, except the total primary production (tpp). the exception was the mda level, remaining significantly increased, in contrast to the control group and to the condition after antiparasitic treatment. in infected men, the level of cat activity was more than 5.8 times higher than that noted in control group. after treatment the levels of ada activity and gpx returned to the values of the control group, while the level of cat activity remained elevated. conclusion: an accumulation of primary and secondary metabolites in the course of giardiasis seems to confirm its involvement in the induction of oxidative-antioxidative homeostasis. antiparasitic treatment in giardiasis leads to normalization of the ap parameters studied in women and men, except the mda content in the blood of men. therefore, additional antioxidant treatment is advised for the antiparasitic therapy of men. in vitro effects of ethanol on rat brain synaptosome and dose-dependent antioxidative role of boric acid ethanol is a psychoactive drug that is very large and used frequently today. it has suppressive effects brain's comminication pathway. depending on its acute or chronic use and the dose, ethanol increase membrane fluidity and it causes oxidative stress. this study deals with the in vitro effects of ethanol toxicity (200 mm) and potential protective effect of different doses of boric acid (ba) (5, 10 and 25 mm) on rat brain synaptosomes. with this aim, five male spraque dawley rats are killed by decapitation under anesthesia. after the frontal cortexes of the rats are taken out, each of them is divided into four pieces. these pieces were used as a sample in five groups (control, ethanol, ethanol+5 mm ba, ethanol+10 mm ba, ethanol+25 mm ba) which include six samples. the synaptosomal fractions are prepared by the homogenization of the frontal cortex pieces and centrifugation for each samples. as markers of ethanol-induced oxidative stress in the synaptosome of the rats, malondialdehyde (mda), nitric oxide (no) and catalase (cat) levels were measured. mda levels in the ethahol group were a quantity increased compared with those in the control group but it unchanged significantly as statistically (p < 0.05). however the mda level in the ethanol+ boric acid (25 mm) group was shown to be significantly decreased compared with that in the ethanol group (p < 0.05). the cat activity of the ethanol group was significantly higher than that in the control group and cat activity of the ba (5 mm,25 mm) groups were close compared with control groups (p < 0.01). no levels in ethanol groups were decreased but unchanged compared with control groups as statistically. neverthless, no levels in ethanol+ boric acid (25 mm) groups were increased (p < 0.05). these results demonstrate that ethanol (200 mm) is capable of triggering damage to rat brain synaptosome and ba could be influential in antioxidant mechanisms against oxidative stress resulting from ethanol exposure. acute myeloid leukemia (aml) is the most common form of acute leukemia in adults and its incidence increases with age. carbonic anhydrases (cas) are zinc-containing enzymes. these enzymes catalyze a very simple physiological reaction, the inter conversion between carbon dioxide and the bicarbonate ion, and are thus involved in crucial physiological processes connected with respiration and transport of co 2 /bicarbonate between metabolizing tissues and lungs, ph and co 2 homeostasis, electrolyte secretion in a variety of tissues/organs, and biosynthetic reactions and many other physiologic or pathologic processes including reproductive tract. investigation of autoantibodies in aml patients have been popular research area in recent years. the aim of the current study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in the serum of subjects with aml based on the information and considerations of autoimmune relation of acute myeloid leukemia. anti-ca i and ii antibody levels were investigated by enzyme-linked immunosorbent assay method (elisa) in serum samples of thirty patients with aml and thirty healthy peers. anti-ca i and ii antibody titers of aml group were significantly higher compared with the control group (p = 0.000), (p = 0.034), respectively. we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in patients with aml (r = 0.613, p = 0.000). we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in women and the men (r = 0.851, p = 0.000), (r = 0.503, p = 0.080), respectively. at an anti-ca i cut-off point of 0.123 absu, sensitivity was 80% and specificity 100%. at an anti-ca ii cut-off point of 0.097 absu, sensitivity was 60% and specificity 77%. the ca i and ca ii autoantibody levels in patients with aml were found higher compared to control group and the results suggest that ca i and ca ii autoantibodies may be involved in the pathogenesis of aml. aim: behc ßet's disease (bd) is a systemic autoimmune disease. recurrent oral and genital mucosal ulcers, uveitis, and skin lesions are characteristic findings for bd. platelet-lymphocyte ratio reflects a novel marker for romatological diseases. the aim of this study was to investigate the platelet/lymphocyte ratio in behc ßet's disease. methods: whole blood samples were collected from 50 healthy control and 21 patients with behc ßet's disease. the mean age for controls and patients were 40 ae 1.0 and 37 ae 13, respectively (p = 0.639). patients with chronic disease and inflammatory disorders were excluded. thrombocyte and lymphocyte counts were analyzed with abbott cell dyne heamotolgy analyzer. statistical analysis was performed with ibm spss v20. results: platelet counts were higher but not statistically significant in patients with behc ßet's disease compared to control group (289 ae 100 vs. 257 ae 57) (p = 0.088). lymphocyte counts were lower in patients with behc ßet's disease compared to control group (2.56 ae 0.58 vs. 2.69 ae 0.85) (p = 0.513). platelet/lymphocyte ratio was higher but not statistically elevated in patients with behc ßet's disease compared to control group (118 ae 41 vs. 106 ae 49) (p = 0.344) conclusions: platelet/lymphocyte ratio (nlr) and mean platelet volume (mpv) as inflammatory markers recently became popular because of their simplicity, cost effectivity and their advantages to predict clinical prognosis of specific diseases. according to this study's results, platelet/lymphocyte ratio must be analyzed in vast scale patient populations to identify the disease. objectives: systemic lupus erythematosus (sle) is a chronic relapsing autoimmune disease characterized by production of autoantibodies against a series of nuclear antigens and by chronic inflammation. in recent years, neutrophil-lymphocyte ratio (nlr) was determined to be a good indicator of inflammatory status. nlr can be easily calculated from a whole blood count. introduction: neuroblastoma, an embryonal malignancy, is the most common extracranial solid tumor of childhood. untreated neuroblastoma tumors and cell lines are reported to have reduced hla class i expression, rendering them potentially susceptible to natural killer cell killing due to lack of engagement of hla class i-specific inhibitory killer cell immunoglobulin-like receptors (kirs). the aim of this study was to investigate whether kir genes could influence the risk of neuroblastoma and prognosis of the patients. materials and methods: study group consisted of 50 neuroblastoma patients (15 male, 21 female, median age: 36 months) followed at the pediatric oncology clinic of c ß ukurova university medical faculty. control group consisted of 100 healthy children. 14 patients had stage 1, 2, 3 or 4s disease, 36 patients had stage 4 disease. 16 different kir genes were analysed by sequence specific oligonucleotide probe (ssop) analyses. statistical analysis were done using fisher's exact test. results: compared to the control group, neuroblastoma patients had lower expression of activating kir gene, kir2ds3 (p = 0.005), and higher expression of inhibitory kir gene 2dl3 (p = 0.038). additionally kir2ds3 genes were more common in patients with early stages (stage 1, 2, 3 or 4s) (p = 0.023) and kir2dl3 genes were more common in patients with stage 4 (p = 0.044). furthermore, there were no statistically significant differences between the rate of aa and bx genotypes and their centromeric/ telomeric regions of patients and controls. discussion: kir2dl3 gene can have a triggering effect in neuroblastoma pathogenesis; whereas kir2ds3 can have a protective role. investigating nk cell infiltration and kir receptors in neuroblastoma tissue samples will give more insight to the pathogenesis p-04.03.3-008 neuroprotective and immunomodulatory effects of urtica urens s. arslan 1 , g. terzioglu 1 , b. kabalay 1 , a. r. tufekci 2 , a. sen 1 , i. demirtas 2 1 department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, 2 deparment of chemistry, faculty of arts and sciences, c ß ankiri karatekin university, c ß ankiri, turkey urtica urens (small stinging nettle) has been used for medical purposes in turkey as an alternative therapy. it has been used in the treatment of inflammation that is early, non-specific immune reaction to tissue damage or pathogen invasion, plays an important role in the initiation of neurodegenerative disorders such as multiple sclerosis. however, there are limited studies that investigate anti-inflammatory activity of urtica. therefore, aim of this study is to find out theanti-inflammatory effect of chloroform extract in caco-2 cell line. for this purpose, firstly, chloroform extract of urtica leaves was prepared. chemical composition of extract was determined by lc-ms. the effect of chloroform extract on selected pro-inflammatory and inflammatory proteins such as tumor necrosis factor-a (tnfa), nuclear factor kappa b (nf-jb), c-x-c motif chemokine 10 (cxcl10), and 11 (cxcl11) were determined. whole genome transcriptome analysis was performed by using human ht-12 v4 beadchip. extract treatment caused 35% and 40% increases in protein and mrna levels of nf-jb, respectively. on the other hand, tnf-a protein and mrna levels decreased significantly (24% and 12%, respectively). similarly, cxcl10 and cxcl11 mrna levels decreased 45% and 38%. transcriptome analysis showed that 233 probes were significantly changed (p < 0.05). pathway analysis revealed that the extract altered a group of genes involved in immune response, calcium ion homeostasis and transport, potassium channel complex, g-protein coupled receptor protein signalling pathway, etc. it is well established that calcium is very critical for brain cell death and formation of many brain disease including multiple sclerosis. these observations suggests that urtica maybe used in neurodegenerative diseases. in order to further test this hypothesis experimental autoimmune encephalomyelitis experimentsand activity guided fractionations have been still continuing. this work is supported by tubitak 111t515. p-04.03.3-009 linear low molecular weight a-1,6-glucan from bifidobacterium bifidum bim b-733d is implicated in pathogenesis of celiac disease the bifidobacteria are recognized as human commensals and widely used as probiotics. earlier, we have found (kiseleva et al., benef. microbes, 2013, 4(4) : 375-391) that bifidobacterium bifidum bim b-733d contains low molecular mass (5-7 kda) a-1,6glucans (g anti-tpo and g anti-tg ) that interact selectively with human autoantibodies to thyroid peroxidase (anti-tpo) and thyroglobulin (anti-tg), recognized markers of autoimmune thyroid disease (atd). the aim of the study was isolation and identification of b. bifidum bim b-733d biopolymers (bps) interacting selectively with autoantibodies to tissue transglutaminase (anti-ttg) and antibodies to gliadins (anti-gl), recognized markers of celiac disease (cd). we used affinity chromatography with anti-gl, size exclusion chromatography, 1 h and 13 c nmr spectroscopy, elisa with immobilized bps, tissue transglutaminase (ttg) and gliadins (gl) as positive controls. the bp isolated by affinity chromatography with anti-gl (as more available marker of cd) and size exclusion chromatography was identified by two-dimensional nmr spectroscopy as 5-7 kda linear a-1,6-glucan identical to g anti-tpo and g anti-tg . the functional activity of the bp named g anti-gl , viz., ability to interact selectively with anti-ttg and/or anti-gl was proven by elisa with (i) serum samples of cd patients containing either both anti-ttg and anti-gl without anti-tpo and anti-tg or anti-gl without anti-ttg, anti-tpo and anti-tg vs. serum samples of healthy donors without four types of antibodies and (ii) pure anti-gl vs. pure total igg (without anti-ttg, anti-gl, anti-tpo, anti-tg). since (i) serum samples of cd patients do not contain anti-ttg without anti-gl and (ii) pure anti-gl isolated by affinity chromatography with gliadins (gl) cross reacts with tissue transglutaminase (ttg), additional studies with pure anti-ttg are necessary to find out which of the two antibodies, anti-ttg and anti-gl, bind g anti-gl . in conclusion, we proved that b. bifidum bim b-733d cells contain linear low molecular mass a-1,6-glucan, g anti-gl , that interacts selectively with anti-ttg and/or anti-gl. since g anti-gl is identical to earlier found g anti-tpo and g anti-tg , we hypothesize that the a-1,6-glucan is implicated in pathogenesis of both autoimmune diseases, cd and atd. influences of elevated serum ferritin levels on insulin resistance and non-insulin-dependent diabetes mellitus (niddm) have predicted either because of increased body iron stores or influenced by several inflammatory diseases. low serum 25 hydroxyvitamin d is known to perturb cellular function in many tissues, including the endocrine pancreas, which are involved in obesity and niddm. we planned to investigate the association between 25hydroxyvitamin d with hematologic parameteres and iron status in obesity vs. diabetic patients. study groups consist of control, non-diabetic obese, obese-diabetic and lean-diabetic groups. serum triglycerides, total cholesterol, ldl-c, hdl-c, fasting glucose, hba1c, uric acid, creatinine, ggt,25-hydroxyvitamin d, insulin, crp, esr, total blood count and iron status. apart from the three parameters, there were no significant difference (p > 0.05) between groups. serum ferritin and mchc levels were significantly higher in lean-diabetic patients (p < 0.05). on the other hand, rdw are determined to be significantly lower (p < 0.001) in the non-diabetic obese group. no difference was detected in 25-hydroxyvitamin d levels between the control and the study groups (p > 0.05). non-diabetic obese patients had significantly (p < 0.05) higher levels of tg and lower levels of hdl compared to obese-diabetics. insulin levels were higher in nondiabetic obese and obese-diabetics than lean-diabetics (p < 0.05). this study provides evidence that lean diabetic patients show higher ferritin and mchc levels than obese patients. the increase in serum ferritin and mchc levels is related with altered iron metabolism at cellular level. at late mitosis, the mother cell divides, leaving two daughter cells connected by a thin intercellular bridge (icb). during abscission of the icb, the ingression of the cleavage furrow is formed, and the central spindle microtubules are compacted into the structure known as midbody (mb). the mb is situated within the icb, with the abscission usually occurring at one side of the mb. as a result, only one daughter cell inherits the post-mitotic mb. these mbs can then either accumulate in the cytoplasm or be degraded. recent studies have identified mbs as novel signaling platforms regulating stem cell fate and proliferation. indeed, stem cells as well as cancer cells were shown to accumulate post-mitotic mbs, resulting in reprogramming of the cell fate and conversion to highly-proliferative, stem cell-like phenotypes. it has been proposed that regulated macroautophagy may be playing a key role in mediating pots-mitotic mb degradation. therefore, the experimental approach involved studying the dynamics and function of a protein known as fyco1, which associates with postmitotic mbs and may regulate their degradation. in this study we identified fyco1 as a protein, which associates with post-mitotic mbs and may regulate their degradation. interestingly, fyco1 is also known to be present on autophagosomes, and overexpression of fyco1 can induce the formation of enlarged lc3-containing autophagocytic structures. here we demonstrate that fyco1 knock-down leads to defects in autophagic mb degradation, and that fyco1 functions by targeting endocytic membranes to the autophagic phagophore during early stages of mb degradation. additionally, we showed that fyco1 depletion leads to increased proliferation and cell growth in soft agar. based on all these data, we hypothesize that fyco1 mediates selective mbs degradation via endosome-dependent extension of the phagophore around the post-mitotic mbs, and that mbs may be the regulators of cancer proliferation and progression. p-05.03.3-002 proliferative effect of hypericine on human skin fibroblast cells and identification of the mechanism of action in molecular level to drawbacks associated with efficiency and viral genome integration. in order to improve reprogramming efficiency and compensate for viral transduction, new chemicals have been explored through ipsc research. the aim of this study was to investigate the proliferative effect of hypericine on human skin fibroblast cells (sf) in-vitro, and to identify the mechanism of action in molecular level. the proliferation was measured using the clonogenic and dimethylthiazol diphenyltetrazolium bromide (mtt) assays. real-time quantitative polymerase chain reaction (qrt-pcr) was performed to detect the mrna levels of cyclins (d1 and b1) and cell cycle controller genes (p53 and p21). sf cells were treated with different doses (1 nm-100 lm) of hypericine for 24 h and 48 h. a significant cell proliferation was observed in moderate concentrations (0.1-15 lm; %110-%134), but at high concentrations (25-50 lm) cytotoxic effects emerged in sf cells (ic50 = 23.62 m, r 2 = 0.915). qrt-pcr results revealed that the most proliferative dose of hypericine (15 lm) stimulates cyclin d1. the anti-proliferative activity of hypericine was accompanied by inhibition of cyclin b1 mrna, whereas it induced expression of p53 and p21 genes, and thus apoptosis was observed by dna laddering at the same dose (50 lm). overall results suggested that hypericine can compensate for viral transduction and improve reprogramming efficiency of ipscs by enforcing them in g1 phase. hence we report that hypericine can be a good candidate component for cocktails produced to trigger ipsc proliferation. glioblastoma (gbm) is the deadliest brain tumor. the mean survival time of gbm patients is approximately 12 months, increasing to 14 months after treatment with temozolomide, which is the gold standard chemotherapy. the resistance of gbm to chemotherapy seems to be associated with the blood-brain barrier (bbb) that limits the delivery of chemotherapeutics, and the presence of a population of cells that expresses stem cell-like properties, which are known to be chemo-and radioresistant,5 the glioblastoma stem cells (gscs). the difficulties imposed by these two factors could be reduced by the use of a targeted drugdelivery liposome-based strategy that allows bbb passage and reduces the side effects of chemotherapeutics. the present study evaluated the ability of the f3 peptide-targeted ph-sensitive lipid-based nanoparticle containing doxorubicin (dxr) to target gscs and non-gscs. we evaluated the expression of cell-surface nucleolin by flow cytometry, as well as of stem cell-like markers, in two gbm cell lines. we also determined the ability of gbm cell lines to specifically uptake the f3 peptide-targeted ph-sensitive lipid-based nanoparticles, by flow cytometry, and correlated it with the expression of stem cell-like markers. moreover, to ascertain the impact of intracellular delivery of chemotherapeutic drugs into gbm cell lines, cytotoxicity was further assessed by the mtt assay. our results showed that the f3 peptide-targeted ph-sensitive lipid-based nanoparticles successfully targeted glioblastoma cells and particularly gscs. in addition, the results also provided evidence of the nucleolin overexpression-dependency of this strategy, emphasizing the need to adapt the therapeutic strategy to the individual patient. this study showed that f3-targeted ph-sensitive liposomes may constitute an appropriate strategy to overcome the chemoresistance associated with glioblastoma cells. p-05.03.3-004 leukemic cell plasticiy as a resistance mechanism towards tyrosine kinase inhibitors chronic myelogenous leukemia (cml) is a hematopoietic stem cell disease characterized by the t(9;22)(q34;q11) translocation, which encodes the chimeric tyrosine kinase onco-protein, bcr-abl. the tyrosine kinase inhibitor (tki) imatinib is the first-line treatment for patients with cml. unfortunately drug resistance is one of the main problems observed. while secondary resistance is associated with bcr-abl kinase domain mutations, oncogene amplification and mechanisms interfering with intra-cellular drug concentrations; primary resistance mechanisms haven't been elucidated. we generated high dose imatinib-resistant k562 subclones (k562-ir) by clonal selection to study primary resistance mechanisms in vitro. drug resistance was shown by caspase 3 and annexin v/pi assays. we also showed cellular uptake and function of imatinib with western blot technics. k562-ir cells are not only resistant to imatinib but also to 2nd, 3rd generation tyrosine kinase inhibitors. we demonstrated that k562-ir cells have a highly adherent character, proliferate slowly and are resistant to drug-induced senescence. microarray analysis revealed that k562-ir cells differentially express tissue/organ developement and differentiation genes at high levels. we showed that k562-ir cells forms intact tumor spheroids in 3d cell culture conditions which is a marker of tumor initiating potential. cell surface maker analyses and protein analyses of k562-ir cell population, points towards an epithelial-mesenchymal plastic cell capable of adopting different morphologies. we hypotizied that imatinib and other tyrosine kinase inhibitors may cause the gain of phenotypic plasticity potential in leukemic cells, by interfering with signalling pathways; which in itself may lead to therapy resistance. hypoxia has multiple effects on cancer cells, which are critically involved in tumor progression. hypoxia leads to changes in tumor cell metabolism and can promote cancer cell survival, invasion and metastasis by its critically important role on maintenance of cancer stem cell (csc) phenotype. in this research, human cd133 + cscs isolated from human osteosarcoma cell line saos-2 using macs magnetic separation technique were characterized, and their stemness properties under hypoxic (1% o 2 ) and normoxic (21% o 2 ) conditions were compared in two and three dimensional culture conditions. two different 3d culture techniques (nanofibrous bacterial cellulose scaffolds and scaffold free microtissues) were used to evaluate effects of hypoxia on csc behavior, and the results were compared with the cell behavior in classical 2d culture systems. the morphologies of cells were examined by scanning electron microscopy (sem); rt-pcr and immunocytochemistry staining were used to examine the cancer stem cell phenotype maintenance under hypoxic and normoxic conditions. it is shown that hypoxia supports the expression of stemness markers such as oct3/4, nanog and sox2 compared to normoxic conditions in 3d cultures. although similar effects of hypoxia were observed in 2d cultured cscs, the expression levels of stem cell phenotypeindicative markers were significantly lower on 2d compared to 3d culture systems. this study is seen as an introduction to develop a more relevant 3d hypoxic cancer stem cell based tumor model to study csc behavior and tumor genesis in vitro for testing of novel cancer stem cell therapeutics and to understand signal transduction in cancer stem cells. prostate cancer (pca) is the second most frequent cause of cancer-specific mortality in the world. cancer stem cells (cscs) are a subpopulation of cells that involved in drug resistance, metastasis and recurrence of cancers. the efficacy of natural flavanone apigenin on cell survival, apoptosis and migration of cscs were evaluated. cd44 + cscs were isolated from human pca pc3 cells using a magnetic-activated cell sorting system. pc3 and cscs were treated with different concentrations of apigenin, docetaxel and combinations of the two agents for 48 h. apigenin dose dependently inhibited cscs and pc3 cell viability, and this was accompanied with a significant increase of the cell cycle inhibitors p21 and p27 (kip1). the flavonoid significantly induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mrna expressions of caspases-8, -3 and tnfa, but failed to regulate the intrinsic pathway as determined by the bax, cytochrome c and apaf-1 in cscs. in contrast to cscs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of bax, cytochrome c and caspase-3 while caspase-8, tnf-a and bcl-2 levels remained unchanged in pc3 cells. the ability of apigenin to inhibit the proliferation of cscs through apoptosis was confirmed by tali image-based cytometer. the flavanone strongly suppressed the migration rate of cscs compared to untreated cells. significant downregulation of mmp-2 and -9 exhibits the ability of apigenin treatment to suppress invasion. the expressions of pi3k/akt and nf-kb p105/ p50 were significantly decreased after 48 h apigenin treatment. taken together, these data demonstrated that flavonoid apigenin is an invaluable chemopreventive compound that inhibits proliferation, invasion and the stemness properties of cscs. this study was funded by the scientific and technological research council of turkey (tubitak, grant no. 115s356). (pi3k), are frequently found in patients with severe early-onset segmental overgrowth. whilst differences in timing and location of the founder mutation are likely to explain part of the observed disease heterogeneity, it is less clear whether and how quantitative differences in the strength and timing of pi3k activity contribute to phenotypic variability. our aim is to characterise pik3ca mutant-specific signalling as well as to explore the effects of varying the strength and/or temporal pattern of pi3k activation on downstream output specificity in the cell. we are currently employing crispr/cas9mediated gene editing in human induced pluripotent stem cells to generate isogenic disease models of three such activating pik3ca mutations. these cells will be used for signalome profiling by reverse-phase protein arrays (rppa) to compare and contrast mutant-dependent alterations to candidate signalling networks. in parallel, ongoing efforts focus on developing an endogenously expressed optogenetic p110a, allowing precise spatiotemporal control over pi3k signaling to unravel the extent to which pi3kdependent phenotypes are determined by strength of activation and/or dynamic encoding. ultimately, the outcome of this research will yield novel insight into fundamental aspects of pi3k signalling and potentially aid the development of targeted therapies for human diseases of pi3k hyperactivation. e. gov, n. kaya, k. y. arga cancer stem cells (csc) have been proposed to be the cancer initiating cells. because of their highly tumorigenic and drug resistant properties, cscs offer significant potential for developing novel anticancer drugs and therapeutic strategies. in the present study we analysed eight gene expression datasets for breast, ovarian, lung cancer and glioblastoma by comparing gene expression levels between stem cells and tumor cells and integrating them with genome scale biological networks. consequently, mutual molecular signatures (i.e: differential expressed genes, transcription factor, mirna) and biological characteristics were determined via integrative analyses, which might be feasible to uncover the mutual biological mechanism insights behind the cscs. it was identified twenty mutual differential expressed genes in four cancer types; jun and klf6 as transcription factors, egfr and cdk14 as receptors come into prominence as mutual signatures. molecules and pathways that were related to mapk, wnt, p53 signaling and pathways in cancer were the common indicators in csc types. our results provided similarities in gene expression profiles of various cscs and gave clues about the seed of tumorigenesis. this study proposed signatures and pathways that could be considered as effective therapeutic approaches in further experimental and clinical applications to eliminate subpopulation of csc. colorectal cancer (crc) is one of the leading causes of mortality worldwide. metastasis is associated with the presence of circulating tumor cells (ctcs) in the peripheral blood of cancer patients. ctc cut-off values have been shown to predict for poorer overall survival in metastatic breast (≥5), prostate (≥5), and colorectal (≥3) cancer based on assessment of 7.5 ml of blood. in our study, ctcs were detected in blood samples of colorectal cancer patients, using with our modified convenient method for the strategies of ctc enrichment and detection. 7.5 ml peripheral blood samples were firstly collected and peripheral blood mononuclear cells (pbmcs) were isolated from the fresh blood samples by ficoll gradient separation. next, the leukocytes in pbmcs were removed by magnetic microbeads conjugated with cd45 for a negative selection. finally, the retained cells were labeled with anti-epithelial cell adhesion molecule (anti-epcam), cytokeratins (ck8, ck19) and the leukocyte-specific marker as anti-cd45. all samples were analyzed by bd facs aria iii flow cytometry. in total, 10 patients and 7 healthy people were included in this study. the results showed that ctcs were not detected in the blood samples of healhty volunteers, but 3-13 ctcs were detected with ck14, 15, 16, 19-based gating strategy in the blood samples of colorectal cancer patients. it is accepted that the cut off value is 3 ctcs for colorectal cancer and ctc is negative if it is below this value or ctc is considered as a positive, if it is equal to or above this value, which might be an indication for poor prognosis. thus ctc's detection may serve a representative surrogate tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. cells were grown in culture flasks in a humidified incubator at 37°c with 5% co 2 and were used at the proliferation and confluent stages. cultured cells were exposed to the pemf and prfe. the proliferations of the cells are measured by mtt assay for the effect of emf on the cancer cells. on the other hand the wound healing was investigated by closure of the wound by the cell proliferation with cell morphology using inverted microscope images. the proliferation decreased significantly by the effect of pemf on the semi confluent mcf-7 and mda-mb-231 cells. this effect was observed more prominent on mcf-7. considering prfe therapy this effect is much more pronounced especially for mda-mb-231 comparing with pemf. the phase contrast observations of these results were consistent with mtt analyses. similarly, this effect was seen less for pemf but the proliferation was more suppressed with prfe on the wound models. it was considered that the emf applications could be effective in cancer cells, but this effect has not been studied how it occurs in invasive cancers. in our cell culture study, the appropriate emf applications were found to be effective though the inhibition of proliferation of cancer cells even in invasive cancer but with lower effect. this means that emf applications may support the existing treatment methods of cancer patients and even people who suffer from invasive cancer. metastasis is the one of the most known causes of death in patients diagnosed with cancer. circulating tumor cells (ctcs) are shed from primary tumors and circulating in the bloodstream, and thought to play a key role in metastasis. a hypothesis that ctcs may contribute to metastasis was first introduced in the mid 19th century by thomas ashworth, an austrilian pathologist. in today's research, identification and molecular characterization of ctcs are thought to be a novel target for treatment of cancer and a key factor to understand the metastatic process. existing methods of ctc capture based on the cell search system, flow cytometers, laser scanning cytometers instruments, fiber-optic array scanning technology (fast), isolation by size of epithelial tumor cells (iset), and definition fluorescence scanning microscopy. ctcs are increasingly considered as a 'liquid biopsy' and when liquid biopsy is compared to tumor tissue biopsy, liquid biopsy for ctcs detection can be carried out routinely in patients due to accessibility and ease of blood collection. also, primary tumor sampling may not reflect the actual metastatic conditions, ctcs are thought to be a novel tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. with futher works, ctcs may be used as liquid biopsies and it might provide better understanding metastatic process, new approaches in cancer diagnostics and treatment. mesenchymal stem cells (mscs) are distributed all over the organism as a source of tissue formation and regeneration. glucose is vital for the proliferation and differentiation of mscs. glucose uptake is mediated by specific glucose transporters of two families, the na-coupled glucose transporters (sglt) and glucose transporter facilitators (glut). the presence and function of glut proteins in human placental amnion derived mscs (hamscs) is unknown. we aimed to investigate the presence of glut1, glut3, glut4 proteins and genes in hamscs isolated from term placentas. mscs were isolated from human term placenta amniotic membrane, the characterization of cells were provided by flow cytometry. mscs were used to assess their chondrogenic, osteogenic and adipogenic differentiation potential. the expression of glut1, glut3 and glut4 proteins was detected in hamscs by immunofluorescence. glut1, glut3, glut4 protein and gene expression in these cells were investigated by western blot and real-time pcr, respectively. flow cytometry analysis results of isolated cells showed that they were positive for cd44, cd90, cd73, cd105 (mesenchymal stem cell markers) and hematopoietic markers cd34, cd11b, cd19, cd44 and hla-dr were negative. the presence of glut1, glut3, glut4 proteins and genes were identified in hamscs. in this study, for the first time in literature, glut1, glut3 and glut4 gene and protein presence was determined in hamscs. therefore, gluts could mediate glucose transport in human amniotic membrane mscs. proliferation and differentiation of mscs in vitro are still not optimized. further studies are required to clarify the complex mechanisms regulating the relationship between glucose and mesenchymal stem cells. disclosure of this relationship may provide a better understanding of glucose-related pathologies such as diabetes. tumors have hierarchically organized heterogeneous cell populations and a small subpopulation of cells, termed cancer stem cells (cscs), is responsible for tumor initiation, maintenance as well as drug resistance. therefore, killing the cscs along with the other cancer cells is gaining an importance. in the present study, it was aimed to evaluate the cytotoxic and apoptotic activity of a novel platin (pt) (ii) complex [pt(hepy) 2 cl 2 ] on mammospheres obtained from mcf-7 human breast cancer line. elevated expression of stemness markers were determined by western blotting. cytotoxicity was assessed using the atp viability assay. effect of the pt (ii) complex on the formation and development of mammospheres was analyzed with sphere formation (sfa) assay. apoptosis was determined via cytofluorimetric analysis (caspase 3/7 activity, annexin-v-fitc and bcl-2 activity) as well as gene expression analysis. cytotoxicity was confirmed with the atp viability assay after the treatment with zvad-fmk (an apoptosis inhibitor) and necrostatin (a necroptosis inhibitor). in addition, alterations in mitochondrial membrane potential were evaluated by jc-1 staining. mammospheres exhibited increased oct-4 and sox2 (stemness markers) expressions compared to parental mcf-7 cells. cytotoxicity by pt (ii) complex was evident in a dose-dependent fashion (1.56-100 lm) . pt (ii) complex significantly prevented mammosphere formation and disrupted mammosphere structure in a dose-dependent manner. pt (ii)-induced apoptosis was determined based on the presence of caspase 3/7 activity, annexin-v-fitc positivity and bcl-2 inactivation. apoptosis was also confirmed with increased tnfrsf10a and hrk gene expressions. in addition to apoptosis, necroptosis was also present as evidenced with increased mlkl expression. mitochondrial membrane was depolarized. in conclusion, the pt (ii) complex seems to be a powerful apoptosis-inducing compound on cancer stem cells, thereby warrants further in vivo experiments. cancer is a disease which arises from destruction of growth and proliferation mechanisms in cells and is the second leading cause of death worldwide [1] . in the development of primary cancers, the head and neck cancer is accounting for approximately 550.000 new cases annually around the world [2] . laryngeal cancer is a type of head and neck cancer in which malignant cells arise from the mucosal tissues of the larynx [3] . cancer might spread from primary tumor by getting into the lymph and blood vessel system and forms secondary tumor. greater than 90% of deaths in cancer patients are attributed to metastasis [4] . circulating tumor cells (ctc's) provide an opportunity to understand the metastatic process of cancer patients. identification and molecular characterization of ctc's in the peripheral blood of cancer patients is a promising research area in the field of biomarker development and novel treatment targeting in today's cancer research [5] . the detection of ctc methods include cell search system, flow cytometry, high-definition fluorescence scanning microscopy, fiber-optic array scanning technology, isolation by size of epithelial tumor cells, and laser scanning cytometers [6] . in our study, 7.5 ml of peripheral blood samples were collected from larynx cancer patients and healthy volunteers and the samples were analyzed by bd facs aria iii flow cytometry via biomarkers epcam, ck19, ck8 for positive selection and cd45 for negative selection [7] . according to the results of our study; ctcs were detected in larynx cancer patients by our newly modified method whereas there was no ctc's detection in the samples of controls. thus, this study may provide us monitoring of the treatment process of larynx cancer and this method might be used as diagnostic, prognostic, and predictive biomarkers in cancer therapy as a liquid biopsy. prostate cancer is the second most common cancer and the fifth leading cause of death from cancer in men 1 . circulating tumor cells (ctcs) present in the peripheral circulation of cancer patients with different solid malignancies including prostate cancer and have a potential as a liquid biopsy to monitor disease progression and response to therapies at cell and molecular level 2 . one of the general methods in ctc detection is flow cytometry 3 . radical prostatectomy is the most frequently applied procedure in the surgical management of localized prostate cancer. in this surgical operation, the surgeon removes the entire prostate gland with the seminal vesicles. a radical prostatectomy procedure can be done using the da vinci robotic system (intuitive surgical, sunnyvale, ca, usa) 4 . robotic surgery has been suggested to have fewer complications, lower risk of infections and shorter recovery period following robotic radical prostatectomy 5,6 . in this study, our aim was to detect ctcs before and after robotic radical prostatectomy in clinical localized prostate cancer patients. the ctc detection study was performed with our modified method in which 7.5 ml of peripheral blood samples were collected from each prostate cancer patient and healthy individual; the samples, using with biomarkers epcam, ck19, ck8 for positive selection and cd45 for negative selection, were analyzed by bd facs aria iii flow cytometry 7 . according to our results, we detected ctcs in the peripheral blood samples of prostate cancer patients before robotic radical prostatectomy. however, following this surgical procedure no ctc or decreased number of ctss was detected. our study might contribute to understand disease progression after robotic radical prostatectomy in clinically localized prostate cancer patients that warrants further research. keywords: circulating tumor cells, prostate cancer, flow cytometry, robotic radical prostatectomy. p-05.03.3-017 determination of effect cytotoxic, apoptotic, caspace-3 activity and mrna expression levels of apoptototic related genes of vulpinic acid on breast cancer cell lines n. kilic ß, s. aras, d. cansaran-duman ankara university, ankara, turkey breast cancer is the most common cancer types in women. several drugs used to treat breast cancer patients are developing resistance to the treatment for this reason success rate falls. therefore the discovery of alternative therapeutic agent and molecular detection of anticarcinogen effect because of treatment for cancer patients may be a source of hope for the contributions. in this study, different concentrations (1.562, 3.125, 6.25, 12.5, 25, 50 , 100 lm) vulpinic acid (va) lichen seconder metabolite was determined to cytotoxic, apoptotic effect and caspase-3 activity in breast cancer cells (mda-mb-231, mcf7, bt-474, sk-br3) and normal cell (mcf12a). in addition to the quantitative real-time pcr (qrt-pcr) using apoptose specific primers (tp-53, bcl-2, bax, birc-5, gapdh, caspase-3, caspase-7, caspase-8, caspase-9) and sybr green dye were performed to determine expression patterns of transcript level in cancer cell lines, using gapdh as a reference gene. the antiproliferative characterization of va effects identification of the gene set at molecular level and we determination role of va on apoptotic pathway. according to our study, va is demonstrated significantly (p < 0.05) effect cytotoxic, apoptotic, caspase-3 activity. beside this, dose dependent expression patterns decreased apoptose spesific genes (except of bcl-2) mrna levels from six to eleven fold change more than untreated va cell lines. va will be used as candidate molecule for effective treatment on breast cancer in the future. glycosylation largely determines the variety and functions of proteins. paucimannose, a mannosidic n-glycoepitope has long been thought to be specific for plants and invertebrates. recently, it has also been detected in mammalsin physiological conditions (stem cells) and in pathophysiological conditions (inflammation and cancer). in glioblastoma cells, paucimannose also seems to play a role in cell proliferation. glioblastoma is the most frequent brain tumor in adults with poor prognosis due to a lack of suitable treatments. we hypothesize that paucimannose could be a promising new biomarker as it is otherwise rarely found in mammals. therefore, paucimannose levels were investigated in different glioblastoma cell lines differing in their proliferation rate and tumorigenicity. the highest paucimannose levels were detected in low proliferating, nontumorigenic cells. furthermore, we found that modulation of paucimannose function by application of a specific antibody regulated cell proliferation and the capability of cells to form colonies in soft agar. these data support a functional role of paucimannosidic epitopes in tumorigenic processes. glioblastoma multiforme (gbm) is the most lethal type of malignant brain tumors. recently, gbm stem cells (gscs) have been studied in great deal and accepted that they have a legitimate role in tumor formation, development, chemo-resistance and recurrence. in this study, it is aimed to investigate new therapeutic targets within apoptosis related molecules to select and eliminate cd133+ gscs effectively. ten primary gbm cells were isolated from gbm tissue samples and they were cultured among with the 4 gbm cell lines (u87, u138, u118 and t98). cd133+ and cd133à cells were seperated by macs method via anti-cd133 (ac133) antibody from cultured cells and cell lines. rna isolation from cd133+ and (à) cells, cdna synthesis was performed. finally, by performing pcr array, mrna expression levels of 88 genes were detected. proper results were collected and analysed statistically. according to the results of pcr array; it has been found that cd133+ cells express approximately 223 fold tnfrsf21 and 20 fold tnfsf7 when they are compared with control cells. tnfsf7 binds to cd27 that is expressed on the surface of tcells. cd27 does not have a death domain, instead it has a cytoplasmic tail which binds to trafs. trafs act as adaptor molecules that are related with jnk and nf-jb signalling pathways. tnfrsf21 (dr6) is a death receptor which are known for transmitting the pro-apoptotic signals from outside to the inside of the cell. it negatively regulates t-cell activation and the release of few cytokines. as a conclusion, tnfsf7 and tnfrsf21 both are found on immune system cells, mostly on t-cells, which may mean that gbm stem cells act as a immune system cells to avoid the elimination by the immune system. to conclude, acting as an immune system cell and promoting survival via tnfsf7 and tnfrsf21, these molecules may be essential markers to target cd133+ gbm stem cells. the effect of docetaxel on p53, sin3a and mdm2 gene expression in mcf-7 breast cell line docetaxel is a cytotoxin effective in treating breast cancer. it stabilizes microtubules and causes catastrophic cell cycle arrest in g2/m. it also initiate signaling through cell death pathways that result in programmed cell death. in this study, it was aimed to investigate apoptotic and cytotoxic effects of docetaxel has on the mcf-7 breast cancer cells line. in this study, mcf-7 breast cell line was applied different doses docetaxel (10 nm, 100 nm, 1 lm, 10 lm, 100 lm) as 24 h and 48 h. mtt analysis was performed to the mcf-7 breast cancer line in control group and groups of docetaxel. afterwards, evaluation of apoptosis by tunel and levels of p53, sin3a and mdm2 gene expression by real-time pcr were determined in an order. it was observed cell variable was significant lower in docetaxel groups compared to control group (p < 0.05) in 24 h as mtt analysis. the lowest cell viabilty was determinated in group applied 100 lm docetaxel. while the lowest positive cell density was determinated in control group, it was observed apoptotic cell density gradually increased with increasing docetaxel concentration in groups treated docetaxel (p < 0.05). the highest p53, si3a and mdm2 expressions were apperared in 100 nm docetaxel group compared to control group. human alpha-fetoprotein (afp) and afp receptor binding domain (afprbd) are able to bind and internalize effectively by wide range of human tumor cells and tissues. as other vector molecules afprbd has insufficient quantity of chemical groups which can be conjugated with drugs or diagnostic agents. conjugation of vector molecules with macromolecular polymer carriers like dendrimers aims to solve this problem. our study describes influence of afprbd-dendrimer-doxorubicin conjugate surface charge on intracellular trafficking routes and toxicity. the amineterminated (g2) and acetyl-terminated (g2 12 ) 2nd generation pamam dendrimers carrying doxorubicin (dox) were used to synthesize conjugates with afprbd. unmodified by afprbd g2 and g2 12 dendrimer derivates labeled with dox were absorbed by the cells at 37°c with different efficiency. g2 12 -dox derivate characterized much slower internalization rate than nonacetylated g2-dox. only g2 12 -dox shown partial colocalization with lysosomal marker lamp2 after 4 h of incubation. internalization of afprbd-g2-dox and afprbd-g2 12 -dox did not show significant difference. at the same time, both conjugates contained afprbd wyкy almost fully associated with lamp2 already after 30 min of incubation. cytotoxicity results revealed that ic50 levels of g2 12 -dox and afprbd-g2 12 -dox coincided and demonstrated a bit higher activity against sensitive to dox skov3 and resistant skvlb cells than afprbd-g2-dox conjugate after 72 h of incubation. at the same time, after 1 h of incubation afprbd-g2-dox and afprbd-g2 12 -dox were much more than g2 12 -dox and g2-dox. we may conclude that there is significant difference in ways of dendrimers internalization by tumor cells depending on nature of surface chemical groups. on the other hand, chemical modification of dendrimer conjugated with does not afprbd influence dramatically on the protein trafficking and resulting cytotoxic effect. russian scientific foundation supported this study (no. 15-15-10013 1.40) , a key enzyme in glycolysis, catalyzes conversion of phosphoenolpyruvate (pep) into pyruvate with regeneration of adenosine triphosphate (atp). the key regulator of the metabolic alterations found in tumor cells is the glycolytic isoenzyme pyruvate kinase type m2 that is generally expressed in all proliferating cells and overexpressed in all tumor cells investigated to date. during carcinogenesis a shift in the pyruvate kinase isoenzyme equipment always takes place, such that the tissue-specific isoenzymes disappear, and m2-pk is expressed. breast carcinoma, the third most common cancer worldwide, accounts for the highest morbidity and mortality. breast cancer tissue analysis confirmed the upregulation of m2-pk in breast cancer, and high m2-pk levels were associated with poor prognosis of breast cancer patients. materials and methods: poly hema (mac) nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. pyruvate immobilization was actualysed with cross linking reagent glutaraldehyde. biosensor was developed by preparing pottasiumferrociyanide, selected as a mediator. results: cyclic voltammograms have been carried out at between~0.4 and 0.6 v potentials vs. ag/agci. m2-pk activty was detected by using differential pulse method at between 0.3 and à0.25 v potentials by observing the differentiations in the current values. in the optimization studies, some parameters such as optimum ph, temperature, concentration of glutaraldehyde and p-hema-mac, were investigated. discussion and conclusion: the method developed for the measurement of the tumor m2-pk activity by using biosensor. we found that more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. piruvat kinase tumor m2-pk activity determination at low concentrations is possible with this method. p-05.03.3-023 tie2/tek: a potential biomarker for targeting glioblastoma stem cells role in angiogenesis, endothelial cell survival, proliferation, migration and adhesion. therefore, tie2/tek could be a potential target for therapeutic strategies directed against glioblastoma stem cells and their microenvironment. in this study, we investigated the gene expression levels of tie2/tek in both cd133+ gscs and cd133à gbm cells. gbm primary cells were freshly isolated from glioblastoma tissue samples and cultured in dmem supplemented with 10% fetal calf serum and 1% penicillin-streptomycin at 37°c in 5% co 2 -humidified incubator. we isolated cd133+ and cd133à cells from 10 gbm primary cells using macs system. following rna isolation from healthy brain tissues, cd133à and cd133+ cells, cdna synthesis was performed. finally, according to microarray protocol, cell surface marker panel array was applied. expression levels were analyzed using the delta delta ct method. statistical analysis was performed using spss software for windows version 13.0. tie2/tek gene expression was determined as 50.07 fold higher in cd133+ gscs than normal brain tissue (p < 0.05). morever it was determined 7.52 fold higher compared to normal brain tissue in cd133à (p < 0.05). according to our results tie2/tek expression was higher in gscs, indicating that tie2/ tek may be a potential marker for targeting cancer stem cells in gbm. this research has been supported by the scientific and technological research council of turkey (no:114s189). adenosine inhibited the breast cancer stemlike cell population through erk1/2 pathway s. m. jafari, m. aghaie cancer stem cells (cscs) are immortal tumor-initiating cells that can self-renew and drive tumorigenesis in various cancers, including breast cancer and others solid cancers. in a study indicated that extracellular atp reduces tumor sphere growth and cancer stem cell population. but at present, there are no reports available in literature on the effect of adenosine on breast cancer stem cells. in this study we evaluated the effect of adenosine inhibition and its mechanism of action in breast cancer stem cells isolated from breast cancer cell lines. our result showed that adenosine significant reduces breast cancer stem cell population. reduction of erk1/2 protein levels was also observed after treatment cancer cells with adenosine. in conclusion, our results indicate that adenosine decreases the breast cancer stem-like cell population through erk1/2 pathway. taxanes are commonly used for the treatment of many cancers as chemotherapeutic drugs that resistance to these agents has become a major clinical obstacle. taxane based chemotherapy drugs such as paclitaxel, docetaxel and cabazitaxel bind microtubules and inhibit to microtubule polymerization appear to stimulate programmed cell death. taxane-resistance to cancer has not been clearly in progression and development of drug resistance. multiple mechanisms are involved in the drug efflux proteins as multidrug resistance protein, differences in amino acid sequences among the b-tubulin isotypes. we investigated taxane resistance with different doses of paclitaxel, docetaxel and cabazitaxel in prostate cancer stem cells. we compared the expression level of apoptotic proteins, and its functional role in resistance mechanisms in cd44 + /cd133 + prostate cancer cell lines. taxane drugs were categorized as concentration-dependent or time-dependent. cabazitaxel caused a time-dependent and dose-dependent reduction in cell viability in all tested cell lines. resistance activity was consistently higher in docetaxel in prostate cancer cells compared with the other drugs. there are many different response of clonogenic formation cd44 + /cd133 + cells with resistance to docetaxel, paclitaxel and cabazitaxel in prostate cancer stem cells. the innate of prostate cancer resistances are important characterization steps and critically limits treatment outcomes therefore novel drugs must be focus on antiresistance and molecular based combinations. mesenchymal stem cells (mscs) are self-renewing cells with ability to differentiate into organized, functional network of cells. mscs isolated from various tissues including adipose tissues, bone marrow, umbilical cord, placenta and pancreas have different differentiation and proliferation potential. good knowledge of the metabolism and proliferation mechanisms of stem cells is required for stem cell therapies. glucose is an important molecule in the culture of stem cells. glucose concentration affects the differentiation and proliferation potential of stem cells. the aim of the study was to investigate the proliferation status by identifying the proliferating cell nuclear antigen (pcna) expression under normoglycemic and hyperglycemic conditions in mscs. mscs were isolated from human term placenta amniotic membrane. characterization of the isolated cells was performed using flow cytometry. chondrogenic, osteogenic and adipogenic differentiation potential of these cells were investigated. characterized cells were cultured in normoglycemic and hyperglycemic conditions for 24 and 48 h and the expression of pcna protein expression in these cells were investigated by western blot. flow cytometry analysis showed that isolated cells were positive with mesenchymal stem cell markers cd44, cd90, cd73, cd105 and negative with hematopoietic markers cd34, cd11b, cd19, cd44 and hla-dr. western blot result of pcna protein expression statistically significantly increased in human amniotic membrane mscs under hyperglycemic conditions for 24 and 48 h culture. the glucose content of stem cell medium is important because glucose is an effective molecule of the proliferation of stem cells. proliferation of mscs in vitro are still not optimized. when the relationship between glucose and stem cells be understood, it will provide a better understanding for the glucose-related pathologies such as diabetes during pregnancy. prostate cancer (pca) is the second most common type of cancer among men in the world. it is revealed that some gene, protein and metabolite sets control the pca, however the whole metabolomics changes are not completely understood yet. pca is common among older men, and this is an important health problem in developed countries. sarcosine is the n-methyl derivative of the glycine amino acid. glycine n-methyl transferase produces sarcosine from glycine. besides, it is metabolized to glycine by sarcosine dehydrogenase. in 2009, high level of sarcosine in urine was associated with pca by sreekumar et al. they identified sarcosine as a pca biomarker that was significantly increased in urine during prostate cancer progression to metastasis. following this study, several studies have been published indicating sarcosine as a pca biomarker. in our study, a preliminary biosensor system was fabricated for determination of sarcosine in urine by using sarcosine oxidase. sarcosine oxidase was immobilized on au electrode surface using gelatin as an immobilization matrix. glutaraldehyde was used as a cross-linking agent to avoid the loss of the enzymegelatin mixture. optimization and characterization studies were carried out. sarcosine concentrations were detected carefully with the developed biosensor system. the fabricated preliminary biosensor is a promising system that can allow lower detection limits after surface modifications. activation of the epithelial-mesenchymal transition (emt) program in tumor cells is associated with invasiveness and stemness. recent studies implicate emt-inducing molecules in reprogramming energy metabolism. the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (pfkfb3) regulates glycolysis by producing fructose 2,6-bisphosphate (f2,6bp). given that pfkfb3 is induced by several established emt-inducers in tumor cells, e.g. hif-1a and ras, we hypothesized that pfkfb3 may be involved in regulation of the emt in tumor cells. silencing of pfkfb3 in pancreatic adenocarcinoma cell lines panc1 and s2vp10 was achieved using specific sirna molecules. mrna and protein expressions of the cdh1 gene (encoding e-cadherin, an established epithelial marker), as well as zeb1 and snai1 genes, by real-time quantitative (q)-pcr and western blot, respectively. immunfluorescence analysis was performed to visualize e-cadherin protein expression on plasma membrane. in order to test the effect of pfkfb3 on the invasive ability of the cells, a matrigel invasion assay was performed. ectopic expression of zeb1 was achived by transfecting cells with a plasmid carrying zeb1 cdna. cells that were depleted of pfkfb3 exhibited markedly increased cdh1 mrna and e-cadherin protein expressions and reduced snai1 and zeb1 mrna expressions. immunfluorescence analysis confirmed the upregulation of the e-cadherin protein on plasma membrane. silencing of pfkfb3 caused approximately 40% reduction in matrigel invasion, compared to non-targeting sirna. inhibition of the matrigel invasion caused by pfkfb3 depletion does not appear to be associated with reduced zeb1 expression, as ectopic expression of zeb1 did not reverse the effect of pfkfb3 silencing on invasion. taken together, these data suggest that pfkfb3 may be required for the maintenance of the mesenchymal phenotype and associated traits in pancreatic adenocarcinoma cell lines. introduction: leukemias are neoplasms that arise from hematopoietic cells initially proliferate in the bone marrow, and then disseminated in the peripheral blood, spleen, lymph nodes and eventually to other tissues. lymphomas occur primarily in the lymph nodes, but can be extended in peripheral blood and bone marrow infiltrate. aim: to determine the values of haematological parameters the control and test groups. to determine the prevalence of types of chronic leukemia in relation to the experimental group. compare haematological parameters in relation to the type of chronic leukemia. materials and methods: a prospective-retrospective study included subjects who were made laboratory hematology in oj clinical chemistry and biochemistry ukcs. blood tests conducted on the hematology analyzer siemens advia 2120 hematology system and abbott cell dyn 3700 and microscopic analysis of the peripheral blood smear. results and discussion: according to the age of respondents test group was established mild form of anemia, a red blood cell count is totaled 3.69 ae 1.13x10 12 , which is signifycantly lower compared to the control group. the average number of leukocytes was significantly higher in subjects studied groups and amounted to 136.91x10 9 , with a maximum value of 580 x10 9 . in the peripheral blood of patients with chronic leucosis has established a significantly higher number of cells compared to the control group (p = 0.001), while the number of monocytes was a significantly smaller. in the group of patients with chronic leukosis largest number had chronic lymphocytic leukemia (70%), and chronic myeloid leukemia had 30% of respondents. conclusion: subjects with cll were statistically older than patients with cml, and as regards the gender structure, men have dominated in cll and cml in women. white bloodline was found that the number of leukocytes in both forms of chronic leukemia high above the reference value. p-05.03.3-032 effect of enzymatic and non-enzymatic isolation methods of endometrial stem cells on their cell proliferative potential and mesenchymal stem cell characteristics human endometrial stem cells (hescs) are responsible for the monthly renewal of the basal layer of the human endometrium by facilitating stromal and vascular regeneration. in this study, hescs were isolated with three different isolation methods including non-enzymatic and enzymatic digestion using trypsin and collagenase type 1. the effect of these three isolation methods on the acquisition of mesenchymal stem cells (msc) and on hesc proliferative potential was evaluated through flow cytometric analysis of cd surface markers and wst-1 tetrazolium salt assay. our findings indicate that hescs isolated with these three methods have statistically similar cell proliferation rate at 24 h time point. however, at 48 h time point, hescs isolated with the non-enzymatic and collagenase type 1 method displayed a higher expansion in cell number when compared to the hescs isolated with trypsin. the late passage of hescs isolated with non-enzymatic and trypsin methods showed the highest proliferation rate in comparison to the hescs obtained via collagenase type 1 isolation method at 24 h, 48 h and 72 h. the three isolation methods for the early passages of hescs had a resemblance in their msc profile with no significant difference. on the other hand, late passage hescs isolated using trypsin non-enzymatic method showed a higher cd31and lower cd44 profile. moreover, late passage of hescs isolated with non-enzymatic method displayed a significant reduction in their cell surface cd90, cd73, and cd105 surface expression levels. only hescs isolated with collagenase type 1 did not present a significant shift in their mesenchymal cd marker profile from early to late passages, taken together results from this study suggest that the longterm maintenance of mesenchymal markers can only be achieved in cell isolation with collagenase type 1, while non-enzymatic method is more suitable to obtain higher msc cell yield for immediate use. hepatocellular carcinoma (hcc) abundantly arises on the viral and/or chemical-induced cirrhosis in liver. cirrhosis is defined as one of the premalignant stage hcc in which microenvironmental changes occurred such as uncontrolled production of collagen type i and activation of hepatocyte growth factor (hgf)/c-met signaling. it has been shown that epcam+/cd133+ subpopulation of cells isolated from hcc tissue can initiate tumor at very low concentration in xenograft model and behaves as hepatic cancer stem cells. however, the molecular mechanisms supporting hepatic stem cell activation are not well understood and knowledge about the role of hgf/c-met pathway in this process is not clear. in this study, we aimed to define effect of collagen type i and hgf induction on the cell behaviours of epcam+/ cd133. epcam+/cd133+ cells were sorted by magnetic separation from huh-7 cells. then proliferation and invasion of cells were analyzed under the hgf induction as well as branching morphogenesis in vitro. after hgf stimulation, phosphorylation level of c-met increased in epcam+/cd133+ subpopulation. moreover, presence of collagen type i enhanced significantly effect of hgf stimulation in the invasion of epcam+/cd133+ cells. we also have showed that hgf stimulation increased branching tubulogenesis capacity of epcam+/cd133+ subpopulation while it did not effect proliferation of cells. these effects of hgf reverted by c-met inhibitor, su11274, in vitro. all these findings showed that hgf and collagen type i regulates aggressive phenotype as microenvironmental changes via induction of invasiveness of epcam+/cd133+ subpopulation of huh-7. in conclusion, we showed that hgf/c-met signaling causes to get more metastatic phenotype based on invasion and tubulogenesis in epcam+/cd133+ hepatic cancer stem cells in hcc and it might be possible to use c-met inhibitors to target hepatic cancer stem cells during hepatocarcinogenesis. endometriosis is defined by the migration of endometrial mesenchymal stem cells (emscs) into the peritoneal cavity or other site of body rather than uterus in a retrograde fashion. its previously known intracellular crosslinking enzyme called tissue transglutaminase (tg2) was shown to play important roles in the extracellular matrix (ecm) modelling, fibrosis, cell adhesion and migration. we have hypothesized that tg2 might be expressed in emscs and take part in the formation of endometriosis. the difference in the proliferation capacity of emsc isolated from endometrial tissue with/without endometriosis was determined using wst-1 assay and tg2 activity and expression levels were analysed by btc assay and rt-pcr. the biosynthesis and activity for mmp-2 and -9 were investigated with zymography and rt-pcr, respectively. although tg2 activity was found to be 50% less in emscs isolated from endometriotic tissue, these cells showed 9 times higher tg2 protein expression than those isolated from the control tissue without endometriosis. emscs from endometriotic tissue have 2.3 times higher tg2 and 10.3 fold higher itgb1 mrna levels when compared to the cells of healthy group. similar results were observed in sdc-4 gene expression with a 2.5fold increase. endometriotic emscs demonstrated an average of 11.5-fold increase in the mmp-2 activity while a onefold increase was evident in mmp-9 activity when compared to the healthy emscs. emscs from patient group possessed a higher proliferative ability in comparison to that of healthy subjects within 96 h. the fact that emscs from the control tissue showed lower tg2 protein levels with a high enzyme activity suggested that tg2 might be important in the development of endometriosis not only by destabilizing ecm but also enhancing the cell migration. in this context, the upregulation of tg2 along with itgb1 and sdc4 was evident in emscs of endometriosis which was possibly associated with the increase in the activity of mmp-2 and -9. recent studies have indicated that pluripotent stem cells and some stromal stem cells such as mesenchymal stem cells (msc) are metabolically different from their differentiated counterparts. in this study, the cellular mechanisms controlling metabolic changes in stem cells was investigated using wharton jelly mesenchymal/stromal stem cells (wj-mssc). wj-msscs were isolated by the explant method and cultured in dmem-f12 with 10% fbs. endothelial differentiation was induced by the addition of vegf, egf, insulin and hydrocortisone for 6 days. neuronal differentiation was achieved by using commercial neuronal differentiation medium (millipore) for 3 days. in parallel experiments, cellular metabolic activity such as lactate production was measured. the msc characterization was performed by flow cytometry using antibodies against cd90, cd105, cd73 and cd44 (bd human msc analysis kit). the differentiation process was followed by measuring the expression of cd31, cd34 for endothelial and gfap, neu and tyrozine hydroxylase proteins for neuronal cells by immunofluorescence. for gene expression, nanog, cd34 and gapdh genes were analyzed by rt-pcr. differentiation stimuli to endothelial or neuronal cells resulted in a significant decrease in msc marker proteins. expression of stem cell markers other than cd73 were decreased to 2-20%. differentiation induced the expression of cd31, cd34 for endothelial and gfap and neu proteins for neuronal cells. in vitro lactate production was decreased following differentiation in both lineages. neuronal differentiation increased glucose consumption by~48% and the extracellular calcium concentration of these cells was significantly lower than synchronous undifferentiated cells. glycolytic activity is decreased during in vitro differentiation of wj-msscs. metabolic reprogramming and glucose uptake of cells may be an early indicator of the differentiation process in wj-msscs, supporting the view on their metabolic plasticity. store-operated ca 2+ entry (soce) activated by depletion of intracellular ca +2 stores has been shown to control intracellular ca 2+ homeostasis in many physiological and pathological events. stromal interactive protein, stim1, as endoplasmic reticulum (er) ca +2 sensor and orai protein as pore-forming subunit of soc channels play crucial roles in the activation of soce channels. stim1 and orai were reported to have pathophysiological roles especially in hepatocellular carcinoma (hcc). anticancer chemotherapy frequently falls back because of these tumor-initiating subpopulations, tentatively called 'cancer stem cells'. the purpose of this study was to investigate the roles of stim1 and orai on soce in differentiation of huh-7 hccs expressing epcam and cd133 surface adhesion molecules (epcam + cd133 + ). epcam + cd133 + subpopulations in huh-7 cells were separated via flow cytometry and transfected with stim1 and orai-1 over-expressing (oe) plasmids. expression levels were confirmed by rt-pcr. changes in intracellular ca 2+ concentration were monitored via dual wavelength spectrofluorimeter in fura 2-loaded cells. in epcam + cd133 + cells, er ca 2+ release increased without any change in soce compared to that of epcam à cd133 à cells. similar results were observed in stim1-oe epcam + cd133 + cells. on the other hand, increase in orai1 has no effect on either parameter. cancer is globally one of the most death causes. recently, huge improvements occurred in the cancer diagnosis and treatment due to advanced technology, however recurrence occurs almost 30-40% of patients and their survival times decreases. in this study, we aimed to investigate of relationship between the cancer stem cells which are strongly associated with chemotherapy and radiotherapy resistance and recurrence with the non-classical mhc i antigens which have immunosuppressive properties. for this purpose, we immunohistochemically evaluated the expression patterns of cd133, cd44, nanog, oct3/4, hla-g and hla-e in the advanced stage colorectal, gastric and breast cancer and also non malign biopsy samples. we detected that the cancer stem cell markers cd133, cd44, nanog and oct3/4 significantly increased in the advanced stage cancer tissues. however, the immunosuppressive hla-g and hla-e expressions increased only in the colorectal and gastric tumor tissue. in addition to the presence of cancer stem cell like cells in the tumor tissues, increased expressions of hla-g and hla-e may indicate an immune evasive adaptation of tumor cells. according to our findings, the hla-g and hla-e may be potential therapy targets to elimination of cancer stem cells of colorectal and gastric cancers. however, more detailed studies are needed to support our findings and also to determinate of clinical values of these markers. endocannabinoids increase sdf-1 release from human mesenchymal stem cells s. k€ ose 1 , f. aerts kaya 1 , d. uc ßkan c ß etinkaya 1 , p. korkusuz 2 1 stem cell research and application center, hacettepe university, ankara, turkey, 2 department of histology and embryology, hacettepe university, ankara, turkey lipid-structured endocannabinoids are endogenous morphine ligands and present widespread receptor-mediated effects at physiological and pathological levels on the nervous system as well as many other systems. these effects are partially realized through mechanisms affecting cell growth, differentiation, apoptosis and migration at the molecular level. the hematopoietic progenitor cells (hpcs) and mesenchymal stem cells (mscs) form a distinct niche in bone marrow where they interact with each other in harmony. the stromal cell-derived factor 1 (sdf-1/cxcl12) is a chemotactic factor in bone marrow and is released from mscs and their receptor cxcr4 is found in hpcs. with these rationale in mind, we asked if hpcs and msc interaction mediates sdf-1 release via endocannabinoidal system. bone marrow mscs obtained from healthy donors and passage 3 mscs were induced with 200 ng/ml lipopolysaccaride (lps) for 4 h. antagonists for cb1 (am281) and cb2 (am630) receptors were added to cultures for 4 days. after incubation with antagonists msc culture supernatants collected and processed with human sdf-1 beta in elisa medium. analyses demonstrated direct decreasing effect of endocannabinoid receptor antagonists on sdf-1 beta release from bone marrow mscs. in conclusion, endocannabinoidal system regulates sdf-1 release on mscs and directly act on hpcs mobilization in bone marrow microenvironment (niche). this may have a clinical implication on therapeutic mobilization strategies for hscs in hematology clinical applications. implantation is invasion of the embryo into the endometrium and occurs in three stages apposition, adhesion and invasion, via the complex cellular and molecular mechanisms. during these stages, both of maternal endometrium and embryo should be appropriate for the implantation which is the beginning of pregnancy. receptivity of uterine consists in the existence of growth factors such as tgfbeta-1, igfr1, vegf. it is indicated that damages of factors relesead from endometrium and blastocyst prevent implantation. recently, stem cells can be obtained from many sources to use for therapeutic purposes and mesenchymal stem cells derived from bone marrow are the most studied. in our study, it was aimed to investigate molecules play a role in blastocyst implantation after bone marrow derived mesenchymal stem cell application into the rat endometriyum. female rats were divided into three groups which were saline (sf, n:7), media (m, n:7), stem cell in media (m+bmsc, n:7). after vaginal smear technique, female rats in estrous cylcle were injected into the uterine and periton 200 ll saline, 200 ll culture media and 1 9 10 6 cell/200 ll culture media. the pregnant female rats on the 7 day were sacrified and uterine samples removed and were stained with heamatoxylin-eosin histochemically and anti-tgfbeta-1, anti-igfr1, anti-vegf and anti-pcna immunohistochemically and obseved under light microscope. h-score results were determined using one-way anova test statistically. it was found that intraperitoneal administration of stem cells with media, was increased tgfbeta-1, igfr1, vegf and pcna parameters when compared with the intrauterine administration of stem cells. in this study, it was revealed that distribution of molecules play role in implantation were changed due to stem cell application. it is supposed that stem cell treatments can be cured the molecules caused infertility. many unconventional biochemical factors remain to be investigated for their potential effects on stem cells. among others, endogenous gasotransmitter h 2 s, generated from l-cysteine and organosulfur-compounds (oscs) metabolisms, plays very important roles in the central nervous, respiratory and cardiovascular system. slow-releasing h 2 s donors are viewed as powerful tools for basic studies and innovative pharmaco-therapeutic agents for cardiovascular and neurodegenerative diseases. exogenous h 2 s administration is able to promptly scavenge ros, activate myocardial k atp channels and increase pro-cell survival signaling, very likely activating erk and phosphatidylinositol 3-kinase (pi3k)/akt pathways. the effects of h 2 s-releasing agents on the growth of stem cells are not yet widely investigated. therefore, stem cell therapy combined with h 2 s may have great clinical relevance in cell-based therapy for regenerative medicine. the effects of slow-releasing h 2 s agents on the in vitro cell growth and differentiation of human lin à sca1 + cardiac progenitor cells (hcpc) were here studied. in particular, the effects of h 2 s-releasing agents, such as na 2 s, gyy4137 and water-soluble gsh-garlic conjugates (gsgaws), on the cell viability and differentiation of hcpc were here investigated by colorimetric assay, immune-fluorescence microscopy and western-blotting analysis. the treatment with slow-releasing h 2 s donors increased the cell proliferation in a concentration dependent manner respect to the control. moreover, the treatment with gsgaws led to an up-regulation of the expression of proteins involved in the cell adhesion and differentiation processes. these preliminary results highlight on the effects of this gasotransmitter on the stem/progenitor cells and on the possibility to develop functional 3d-systems for cardiac tissue repair, that take into account the relevant biological role of h 2 s in the cardiovascular system. p-06.02.5-002 investigation of the protective effect of boric acid and omega-3 fatty acid in model of acute myocardial infarction changes in myocardial rats ischemic heart disease being the most common cause of the mortality and morbidity in worldwide commonly results from the occlusion or narrowing of the coronary arteries by atheromatous plaque and thus is named as coronary artery disease. male sprague dawley rats were used in the present study. rats were divided into 5 groups with 10 rats in each: control, mi, mi+boric acid, mi+omega-3 and mi+boric acid+omega-3 groups. control rats were treated with 2 ml/day saline, boric acid-treated rats received 100 mg/kg/day boric acid and omega-3-treated rats received 800 mg/kg/day for 28 days by oral gavage. for the experimental mi model, 200 mg/kg izoproterenol-hcl (iso) was administered subcutaneously two times with a 24-h interval in the last 2 days of the boric acid and/or omega-3 treatments. twelve hours after the second dose of iso, general anesthesia was induced. under general anesthesia and spontaneous respiration, ecg recordings were obtained by using a computerized data recording and analysis system (mp100, biopar) and d-ii recordings were used in the analysis. compared to the control group, serum ck-mb, bnp and tnf-a levels were higher in mi group (p < 0.001, p < 0.001 and p < 0.01 respectively). in the heart tissue homogenate, biochemically measured calpain activation and mda were increased (p < 0.01 and p < 0.001, respectively) and pon1 levels were decreased (p < 0.05). according to the ecg recordings, st wave and heart rate were found to be decreased (p < 0.001 and p < 0.001, respectively). on the other hand, all above mentioned parameters were found to be improved in rats treated with boric acid and/or omega-3 after induction of mi. moreover, histological analysis including light microscopy and tem revealed a significant histological improvement in rats treated with boric acid and/or omega-3 after induction of mi. results of the present study suggest that omega-3 and/or boric acid treatment significantly decreases the cellular damage in mi. this is study is aimed at measuring the level of serum heart-type fatty acid binding protein (h-fabp) in patients presenting with diabetic ketoacidosis (dka) and diabetic ketosis (dk) and to determine its role in identifying early period cardiac ischemia by comparing this level with the level of a control group at a comparable age this study was planned to be a prospective study and it included 35 patients diagnosed with dka, 20 patients diagnosed with dk and 20 voluntary pediatric and adolescent healthy control subjects. the h-fabp, creatine kinase-mb (ck-mb) and troponin-i levels were studied in patients with dka and dk as well as in the control group at the time of presentation. for dka patients, their h-fabp values were measured once again after acidosis correction and compared with the values they had at the time of presentation there were no differences among groups in terms of sex, age, height and weight. no statistically significant differences were found among groups with respect to troponin-i values (0.06 ae 0.08, 0.07 ae 0.04 0.04 ae 0.04; p = 0.457). no statistically significant differences were found among groups with respect to ck-mb values (1.48 ae 0.91, 1.55 ae 0.9, 2.09 ae 1.37; p = 0.229). the h-fabp values of dka patients at the time of presentation were found to be statistically significantly higher than those of dk patients and control group (1.17 ae 0.79; 0.79 ae 0.5 0.69 ae 0.36; p = 0.006). the h-fabp value of the dka group at the time of presentation was found to be statistically significantly higher than the value at hour 36 after acidosis correction (1.17 ae 0.79; 0.55 ae 0.28; p = 0.0001) the fact that h-fabp levels were found to be high in pediatric patients diagnosed with dka at the time of presentation suggested that myocardial ischemia had been triggered. in diabetic patients, every ketoacidosis attack may lead to cardiac ischemia, thereby accelerating progress to necrosis. in conclusion, we would like to propose h-fabp as a potential marker for indicating myocardial ischemia. p-06.02.5-004 genome-wide analysis of hypoxic stress response in human cardiomyocytes stress in human cardiomyocytes on a genome-wide scale remains poorly understood. this study aimed to identify the gene expression patterns of adaptive response of the human cardiomyocytes (hcm) to hypoxic stress. in vitro experimental models of hypoxia mimicking in-vivo coronary ischemia, are useful tools to identify molecular pathways involved in myocardial ischemia. in the current study, we cultured ac16-hcms in dmem/f12 with 10%fbs. to simulate hypoxia model, cardiomyocytes were plated in hypoxia chamber (1%o 2 , 5%co 2 , 94%n 2 ) for 3, 6, 12, 24 h and the control group was incubated in normal conditions (5%co 2 , 95%o 2 ). cell viability was determined using mttassay. annexin-v assay was used to monitor apoptosis. gene expression profiling was analysed with affymetrix-hg-u133-plus-2 arrays. following bioinformatic and statistical analyses differentially expressed genes (deg) were classified according to gene ontology using david and kegg pathway analysis tools. according to mtt, annexin-v and hif gene expression results, hypoxia time was determined as 3 h. we identified 649 genes (279 down-regulated and 370 up-regulated) (p < 0.001, fold change ≥1.5) were differentially expressed in hypoxic-ac16 vs. ac16. degs were mainly clustered in cell proliferation, regulation of cell death, cell adhesion and response to stress. furthermore, transcriptome analyses revealed that 'metabolic, cytokinecytokine receptor interaction, hif-1 signaling, tgf-beta, cell cycle and apoptosis' pathways were involved in the hypoxic stress response of human cardiomyocytes. this study provides molecular information regarding gene expression reprogramming of human myocardial hypoxia. the pathways identified in this study may pave the road for translational medicine. this study was supported by tub _ itak project number 111s189. autologous ips cells after reprogrammed into endothelial progenitor cells (epcs) may offer several advantages in the treatment of cardiovascular disorders because of their cardiogenic and vasculogenic differentiation potential. to reach that purpose, we differentiated and characterized mouse ips cells into flk1 + , a well-recognized epc marker. further maturation of epc was characterized by the expression of cd31 and cd133 markers. purified ips cells were differentiated into flk1 + cells with the use of differentiation medium on type iv collagen-coated dishes in the absence of lif. we then analyzed flk1 gene expression and protein levels with qrt-pcr, western blot and immunocytochemical methods on days 2.5 to 7.5. flk1 + cells isolated with macs system and then recultured these cells in differentiation medium with vegf to induce epc cells. following induction, cd31 and cd133 gene expression and protein levels were analyzed with genomic and proteomic methods. after isolating these cells and aggregate overnight, we cultured cells in three-dimensional condition in collagen type i and used differentiated medium including vegf and egf. we found that flk1 expressing cell number reached to a peak level (24%) on day 5.5 followed by a progressive decline subsequently. in the second step, cd31 and cd133 positive cells were generated and enriched during day 4 of induction. we showed optimal time for harvesting flk1 + cells is day 5.5 of initial differentiation. following isolation of flk1 + progenitor cells they were further matured into functional epcs by vegf within 4 days of induction. additionally for evaluation of angiogenic potantial differentiated cells, we monitored epcs behavior along vascular formation in 3d culture. our work demonstrates that epcs could be successfully derived from ips cells and these cells have vascular formation and angiogenic potential in 3d culture. epc drived ips cells play important role in the treatment of cardiovascular disease. p-06.02.5-006 electrophysiological, biochemical and genotoxic effects of luna experience on heart tissue in rat model pesticides are widely used for the control of agricultural, industrial and domestic pests. however, the uncontrolled use of pesticides has diverse effects on ecological system and public health. fungicides are one of the pesticide type used to kill fungi or fungal spores. in this study, the effect of different doses of luna experience, a fungicide, on the cardiac electrophysiology and genotoxicity in rats were investigated. among five groups (5 mg, 10 mg, 20 mg, control and positive control for comet assay) treatment groups received by gavage doses of luna experience for 30 days. electrical activity of heart were recorded using electrophysiological recording techniques. tissue activities of paraoxanase (pon) and arylesterase (are) and level of malondialdehyde (mda) were measured using biochemical methods. comet assay was performed on heart tissue. we calculated genetic damage index (gdi) and damaged cell percent (dcp) from comet assay. it was observed that there is a significant decrease in heart rate in all treated groups as compared with control group (p < 0.05). amplitude of p wave and qrs complex did not change (p > 0.05). in all treated groups, statistically significant differences were found for values of pon, are, mda, gdi and dcp when compared to control group (p < 0.05). according to our results, exposure to different doses luna experience have a probable hazard potential for the cardiac system. the macrocyclic cage complexes iron (ii) clathrochelates are of the interest due to their bioactivity; they are able to inhibit t-7 rna polymerase, possess toxicity to leukemia cells hl-60 and suppress amyloid fibril formation. their binding to serum albumins was reported; the extreme binding affinity to albumins is observed for the compounds bearing carboxy groups. upon this interaction, clathrochelates quench protein intrinsic fluorescence and gain optical activity inducing circular dichroism (cd) signal in 350-600 nm region. here we examine the effect of spatial arrangement (isomery of substituents) of clathrochelates on their binding to globular proteins. we study 6 bis-substituted clathrochelates bearing two same or different isomers (ortho-/meta-/para-) of carboxyphenylsulfid groups. their interaction with bovine (bsa) and human serum albumins, b-lactoglobulin and lysozyme are explored by cd and protein fluorescence quenching method. the binding of compounds to albumins evoked the cd bands of the same shape, but their intensities vary up to 45 times depending on substituents isomery. in the presence of b-lactoglobulin, the intensities, shape, and positions of the induced cdbands differ for the compounds with different isomer groups. the cd bands induced by the lysozyme in the case of di-para substituted clathrochelate are shifted relatively to the bands of other isomeric compounds. the pronounced quenching of protein fluorescence by clathrochelates was observed only in the case of bsa, its intensity depends on the geometry of substituents (9-17 times). the different spatial arrangement (isomery) of carboxyphenylsulfid substituents in clathrochelates causes the distinctions in both their cd-signal induced by interaction with proteins and their effect on the protein fluorescence. the geometry of ribbed substituents is important for their binding to biomolecules (particularly proteins) and is suggested to determine the structure of the formed guest-host complex. 3d bioprinting is a new technology that revolutionized the field of tissue engineering and regenerative medicine, allowing reconstruction of living tissue and organs preferably using the patient's own cells. using a 3d printer we can design biological structures by controlling exact deposition of cells, growth factors and extracellular molecules in a spatially-controlled manner. the aim of this study was to evaluate the differentiation of human amniotic fluid stem cells (afsc) into endothelial progenitor cells using a bioinkò hydrogel photopolymerized in a 3d network resembling vascular tissue. characterization of afsc was performed by flow cytometry, followed by sorting of the cd177 + stem cell subpopulation. cd117 + stem cells were stained with cell tracker red cmtpx and then mixed with bioinkò hydrogel. printing was done using a 150 lm diameter needle, under 1 bar pressure, and 150 mm/min speed. the network models with define distance apart were printed and analyzed by fluorescent microscopy. mtt test was used to evaluate the viability of the cd117 + stem cells. our results showed that afsc remained viable as shown by mtt assay. the fluorescent microscopy images confirm the viability biochemical test showing that the cd117 + cells viability is maintained after 7 days of cultured in bioinkò hydrogel. furthermore, histological section of hydrogel showed that cells have a relatively uniform distribution forming network interactions between cells. flow cytometry assay showed that cd117+ cells expressed endothelial markers such as cd31, cd105, cd133, cd144 and vegfr2. in conclusion 3d printers are useful tools for creating three-dimensional scaffolds that mimics the cell microenvironment where different types of cells could proliferate, differentiate and crosslink with each other forming tissue-like structures. this study aims to reveal the biocompatibility, biodistribution and immunomodulatory impact on the production of inflammatory citokines of magnetite (fe3o4) nanoparticles functionalized with natural compounds with proved antimicrobial and immunomodulatory effects. co-precipitation synthesized fe3o4 were functionalized with plant-derived compounds: eucalyptol, carvone, limonene and b-pinene. characterization was done by ir, sem and hr tem, while in vitro biocompatibility was tested using endothelial human cells (fluorescence microscopy and proliferation assay). in vivo biodistribution was tested in a balbc mouse model at 2 and 7 days post-intraperitoneal injection, followed by experimental organ removal. tissue sections obtained from vital organs were stained with hematoxylin-eosin. production of inflammatory cytokines was assessed by elisa. results demonstrated that, at concentrations of 500 lg/ml, all prepared nanosystems have a good biocompatibility in vitro and in vivo, allowing the development of cultured cells and also not affecting any visible behavior and organ morphology of the mice. microscopy evaluation of the organs sections revealed that nanoparticles are not present in vital organs such as brain, heart, kidney and liver, but aggregates were visible in the lungs and spleen. at 7 days post-injection no visible aggregated were found in the lungs, few dark-brown nanoparticles clusters being visible in the red pulp of spleen. elisa results revealed that fe3o4 functionalized with carvone and limonene significantly stimulated the production of il-2, il-10 and il-6, while reducing the production of tnfa. other nanosystems din not impact significantly on the cytokine production. functional fe3o4 nanoparticles are efficient drug delivery shuttles, able to stabilize pharmacological compounds, such as plant-derived bioactives, and their biocompatibility, specific biodistribution and limited immunomodulatory effects recommend their use in pharmacological formulations. p-07.01.3-004 new approach for cell imaging with fluorescent carbon nanoparticles m. dekaliuk, k. pyrshev, o. demchenko palladin institute of biochemistry, kiev, ukraine in the nanotechnology field, much interest was focused on the new carbon nanomaterials for cell imaging. recently discovered inorganic carbon nanoparticles ('c-dots') due to their excellent fluorescence characteristics and biocompatibility have ample opportunities for their use in imaging and functional transformations in living cells. their distinctive features, such as high brightness, small sizes, high biocompatibility, small negative charge on the surface and very easy methods of their preparation present a good alternative to other nanoscale materials. the focus of our research was to determine the possibility of using c-dots as the easily available probes for apoptotic cells detection. the carbon nanoparticles were prepared from alanine, citric acid, urea, etc by hydrothermal treatment at 180 c. the studies were performed with adherent epithelial vero and hela cell lines (atcc). with these tools we demonstrate that both native and apoptotic cells can be easily visualized. the cdots uptake occurs probably by endocytosis, which allows for much larger their number to accumulate in apoptotic cells. using the different methods of sample preparation, they show the ability for labeling various structural compartments of the cell. for living cells there are the intracellular vesicles and lysosomes. in contrast, in fixed cells the nucleus is labeled preferentially. the fact that apoptotic cells accumulate strongly increased amount of cdots can be efficiently used in flow cytometry for characterizing the cell populations regarding the relative amount of apoptotic cells in different experimental conditions. the application of such cheap and easily accessible nanoparticles provides more opportunities to simplify the popular methods of cell labeling and detection. previously, our studies showed the possibility of using these nanoscale fluorophores for super resolution method sofi. a new electrochemical microbial biosensor for the fast detecting of dopamine and epinephrine based on candida tropicalis immobilized in a carbon paste electrode (cpe) modified with single wall carbon nanotube (swcnt) was described in this paper. the immobilized cells were used as a source of polyphenol oxidase (ppo) to develop voltammetric epinephrine and dopamine biosensor. voltammetric determination of phenolic compounds like epinephrine and dopamine is a simple technique available. direct oxidation of phenols can be used, but the oxidation potentials of this compounds are similar and they can not be detected distinctively. another possibility is the use of biosensors based on the polyphenol oxidase (tyrosinase) enzyme that oxidises the phenolic compounds into their corresponding quinones. by this way phenolic compounds that epinephrine and dopamine that used in this study were detected at the different potential. the effect of varying the amounts of swcnt and microorganism on the response to epinephrine was investigated to find the optimum composition of the sensor. the effects of ph and temperature were also examined. increases in biosensor responses were linearly related to dopamine concentrations between 0.1 and 1.0 mm and epinephrine concentrations between 0.01 and 1.0 mm. limits of detection of the biosensor for dopamine and epinephrine were calculated to be 0.021 and 0.0029 mm, respectively. finally, proposed systems were applied to epinephrine and dopamine analysis in pharmaceutical drugs. objective: it has started a long time ago to search for a material that can replace blood. this material does not require special storage conditions, independently of the recipient's blood group and can be applied to all individual. milk, casein derivatives, starch, saline and ringer were used for this aim in the past. the determination of toxic effect of natural hemoglobin (hb) on human, researchers have focused on development modified blood. in this work, the development of an artificial biomaterial alternative of blood for using as preoperative and operative aims was aimed. material and methods: in our study, ultrapure hb molecules are immobilized on triethanolamine coated magnetic nanoparticles using various techniques. prepared nanoparticles were characterized by ft-ir, ctem, xrd and cyclic voltammetry (cv). the cytotoxic effects of artificial blood were tried on mtt cell proliferation. results: the characteristic peaks of hemoglobin were obtained from ft-ir spectra differently from support. particles size is concluded by using debye-scherrer equation as > 80 nm from xrd spectra. sem and ctem images supported xrd result. cv results showed that hb molecule has à0.418 v cathodic potential against ag / agcl standard electrode. significant differences were not observed in the mtt results (p < 0.01). conclusion: the nanoparticles were obtained in accordance with the intended desired method. it is determined that the hemoglobin molecules give the same potential with natural blood even after 3 weeks of immobilization and carrying oxygen as natural blood. there are statistical differences between results of mtt tests due to used concentration. but, it is considered that decantation advantage of the artificial blood minimized cytotoxic effects. proteoglycans are among the most abundant molecules of the inter-cellular structure and they are present in extracellular matrices of connective tissues. these glycosylated proteins contain one or more (gag) chains that are covalently attached to the core protein and their hydrodynamic function is mainly due to the physicochemical characteristics of this gag component which provides hydration and swelling pressure to the tissue. gag levels excreted via urine are used as a marker to monitor different diseases (chronic renal disease, renal fibrosis, glomerular filtration abnormalities, bladder stones, breast and lung cancers, hypertension and diabetes, etc.) besides the well known mucopolysaccharidoses. however, their detection by using chromatographic methods is hard, because of the high polarity of negative charges and different functional groups such as acetyl sulfates that generate microheterogenity. in this study, we developed molecularly imprinted chromatographic hplc columns for specific heparan sulfate (hsa), chondroitin sulfate (cs) and dermatan sulfate (ds) detection in urine. positively charged acrylamide monomers were first polymerized by precipitation polymerization, to produce polymers which will show specific recognition for gag's via electrostatic interactions and hydrogen bond formation. these gag selective polymers were then filled in the steel hplc columns and columns eluents were chemically degraded. degradation products of gag's were examined offline column coupled with tandem mass spectrometry. the results showed that our imprinted columns separated gag's specifically and sensitively. thus, urine gag's can be specifically determined by using a gag specific molecularly imprinted column. in this study internal standart weren't used because the matrix effect was lower than 5% for each urine samples. %cv of ds, cs and hsa was calculated as; supported lipid bilayers (slb) were started to be used for cell culture studies to focus on cell adhesion, cell signaling etc. testing the stability of slbs is essential to utilize them as cell culture platforms. in this study, the stability of phosphatidylcholine (pc) lipid bilayers on glass was investigated under milli-q water, phosphate buffer saline (pbs) and dulbecco's modified eagle medium culture (dme) medium supplemented with/without serum. the stability was also checked by enriching slb with different lipids. pc-liposomes were prepared by hydrating the dried thin lipid film with pbs and then by extruding the suspension through a polycarbonate membrane. a negatively charged phospholipid, phosphatidylserine (ps, 25%); a positively charged phospholipid, dotap (50%) and cholesterol (25%) were also used for liposome preparation. liposomes were fluorescently labelled and series of slb imaging were taken for a week. in all experiments in milli-q water and pbs, the stability was conserved for 7 days. pc bilayers in medium supplemented with serum showed hole formations on the second day and their number and size increased rapidly in time. when the bilayers were prepared in medium without serum, disruption was lowered but not completely removed as a result of other factors in medium. cholesterol providing an increased rigidity to the membrane caused higher stability. positively charged bilayer structures also showed increased stability. this can be explained by decreased mobility of bilayer as a result of electrostatic interaction between positively charged molecules and negatively charged glass surfaces. decreased mobility decreases the interactions within the medium. lastly, negatively charged bilayers did not show high stability. strong repulsive forces between the negatively charged surface and bilayer probably prevented the integrity of the bilayer and increased the deformation. in recent years the use of biopolymers has gained priority in tissue engineering and biotechnology, both as dressing material and for enhancing treatment efficiency. there is a demand for new biopolymers designed with protease inhibitors and antimicrobials. ll-37 is an important antimicrobial peptide in human skin and exhibits a broad spectrum of antimicrobial activity against bacteria, fungi, and viral pathogens. using lignin which is an abundant carbohydrate polymer in nature and a polyacrylic acid, we prepared a polymer film by plastifying caprolactone and polyacyrlic acid. films were actified to immobilize ll-37. the structure was elucidated in terms of its functional groups by fourier transform infrared spectroscopy (ftir), and the morphology of the film was characterized by scanning electron microscopy (sem) before and after the immobilization process. the amount of ll-37 immobilized was determined by elisa method. 99.9% of ll-37 peptide was successfully immobilized onto the films. antimicrobial activity was determined in the film samples by quantitative antimicrobial activity method. according to the results, ll-37 immobilized film samples were effective on test organisms; gram-positive staphylococcus aureus and gram-negative escherichia coli. in bio-compatibility assays, the ability to support tissue cell integration was detected by using 3t3 mouse fibroblasts. samples were examined under transverse microscope, non-immobilized sample showed a huge cellular death, whereas ll-37 immobilized film had identical cellular growth with the control group. this dual functional film with enhanced antibacterial properties and increased tissue cell compatibility may be used to design new materials for various types of biological applications. p-07.01. [3] [4] [5] [6] [7] [8] [9] [10] in vitro modulation of the cross-talk between macrophages and osteoblasts by titania nanotube-modified ti surfaces p. neacsu, a. mazare, p. schmuki, a. cimpean bone remodeling is a dynamic process that maintains a fine balance between bone formation and resorption, and is highly influenced by the inflammatory state of the local microenvironment. therefore, a proper modulation in the cellular interactions and cytokine expression is a promising approach to achieve enhanced bone healing. as the biomaterials surface has a major impact on cellular behavior, the goal of the current study was to investigate the influence of tio 2 nanotube-modified ti surfaces (ti/tio 2 ) on the cross-talk between raw 264.7 macrophages (mf) and mc3t3-e1 preosteoblasts (ob) in mono-and co-culture systems in comparison with flat ti (cpti). raw 264.7 and mc3t3-e1 cells were seeded on the test surfaces and grown in standard culture conditions for various periods of time. for co-culture studies, the cells were cultivated using a transwell system. inflammatory mediators released by raw 264.7 cells were measured using elisa technique, while the ob capacity to produce calcified bone matrix was evaluated by alizarin red staining. in co-cultures, lps-stimulated tnf-a, il-6 and mcp-1 release was significantly increased at 24 h, while after 7 days only il-6 exhibited higher amounts when compared with mf cultures alone. moreover, the secretion of these mediators by cells exposed to ti/tio 2 was diminished, especially in lps evoked conditions. also, alizarin red staining demonstrated the presence of calcium deposits when ob were co-cultured with mf for 24 h and 7 days, whereas the presence of the mf for 4 weeks significantly inhibited mineralization. on ti/tio 2 surface elevated calcified matrix was observed, as compared with cpti. this study reveals that the overall effect of inflammation suppression induced by ti/tio 2 may contribute to the enhanced mineralization. also, chronic inflammation may inhibit or delay the regeneration process. therefore, an adequate modulation of mf and ob interactions is vital for the biomaterials success in stimulating bone regeneration. p-07.01.3-011 synthesis and characterization of the branched magnetic polymer for drug delivery systems t. tarhan 1,2 , b. tural 2 , s. tural 2 1 mardin artuklu university, mardin, turkey, 2 dicle university, diyarbakir, turkey magnetic nanoparticles (mnp) have gained a lot of attention in biomedical and industrial applications due to their biocompatibility, easy of surface modification and magnetic properties. magnetic nanoparticles can be utilized in versatile ways, very similar to those of nanoparticles in general. however, the magnetic properties of these particles add a new dimension where they can be manipulated upon application of an external magnetic field. this property opens up new applications where drugs that are attached to a magnetic particle to be targeted in the body using a magnetic field. often, targeting is achieved by attaching a molecule that recognizes another molecule that is specific to the desired target area. in recent years, the development of the systems in which drug is delivered magnetically to the target is drawing considerable attention since it is a current issue. it is possible to eliminate the most of the problems caused by high doses of chemotherapy by using the magnetic drug delivery systems. therefore, it is important to design delivery systems with high drug loading capacity. it is necessary to increase the number of reactive groups on the surface of nanoparticles in order to increase drug loading capacity. in this study, we synthesized a novel magnetic surface for drug delivery systems. magnetic dextran-nta (md-nta) was synthesized by using magnetic o-carboxymethyl dextran (ocmd) and nana-bis (carboxymethyl) -l-lysine hydrate (nta) in order to increase the number of reactive carboxyl groups on the surface of biocompatible and biodegradable magnetic dextran. magnetic material (md-nta) which was prepared and characterized by the analysis of transmission electron microscopy (tem), scanning electron microscope (sem), vibrating sample magnetometer (vsm), fourier transform infrared spectroscopy (ftir) and x-ray photoelectron spectroscopy (xps). there are three subtypes of the tgf-b protein that has been reported to be involved in tissue repair process; scar tissue formation has been reported on tissues that has been affected by tgf-b1 and 2 due to high collagen synthesis. on the other hand the other isoform tgf-b3, suppresses the dense collagen production caused by tgf-b1 and prevents the scar formation. to be able to use these growth factors local or iv route, new drug transport systems are needed to protect the bioactivity during the treatment and controlled release. for this purpose poly(lactic-co-glycolic) acid polymer which is widely used in controlled release systems was chosen as the matrix material. aim of the project was to design, formulate, prepare and optimize tgf-b3 loaded plga nanoparticular and/or plga polymeric film drug delivery systems and to test their effect on cell proliferation. tgf-b3 loaded nanoparticles was prepared with emulsion-solvent evaporation method; whereas polymeric film systems was prepared with film castingsolvent evaporation method. following the preparation tgf-b3 loaded drug delivery systems was characterized. quantification and in vitro release of the growth factor tgf-b3 was studied with elisa. hepg2 cell line was used on mtt cell proliferation assay for both tgf-b3 loaded nanoparticles and films on a time course study. nanoparticles and films were prepared and loading efficiency of the nanoparticles were found to be 42.42%. particle size, zeta index and polydispersity index for this formulation were determined as 204.9 ae 10.3 nm, 0.0381 mw and 0.380, respectively. thickness of the prepared films were 286 ae 20.16 nm. additionaly prepared nanoparticles and films were found non-toxic. tgf-b3 nanoparticles and films which were prepared in this study are planned to be used as an effective treatment strategy for wound healing after injury. this project was supported by grand 112s541 from the scientific and technological research council of turkey (tubitak). polyvinylpyrrolidone (pvp) is a biodegradable material and natural polymeric biomaterial in such studies. ganoderma lucidum is a natural material containing triterpenes, polysaccharides, adenosine, polypeptides, and amino acids. these constituents have been shown to exhibit anti-cancer properties, enhance and regulate immunity, resist oxidation and ageing, and promote metabolism and cell proliferation. composites of polyvinylpyrrolidone (pvp) have been prepared by solution intercalation method using ganoderma lucidum at different loading amounts. the characterization of pvp/ ganoderma lucidum composites was made by x-ray diffraction (xrd) and scanning electron microscopy (sem); the interactions between ganoderma lucidum and pvp was determined by ftir-atr; the thermal stability was determined by simultaneous tg/dta. hemocompatibility of the prepared composite samples were investigated by a 96-well plate spectrophotometer. in addition, contact angles and antimicrobial activity of biomaterials were also determined. ftir-atr confirms interactions formed between ganoderma lucidum and pvp. xrd and sem results give evidence that ganoderma lucidum was well dispersed and homogenously in the pvp matrix. thermogravimetric analysis indicated that introduction of clay to the polymer network resulted in an increase in thermal stability. the results of in vitro hemocompatibility test were showed that pvp/ ganoderma lucidum composites are used as biomaterial. the development of synthetic materials, textured polymers and metals and their increasing use in medicine make research of biomaterials' hemocompatibility very relevant. composite material is a multi-phase system consisted of matrix material and reinforcing material. matrix material is a continuous phase and reinforcing material is a dispersed phase. the main two bioactive components of ganoderma lucidum can be broadly grouped into triterpenes and polysaccharides. despite triterpenes and polysaccharides being widely known as the major active ingredients at anti-cancer effect. this study describes the synthesis and characterisation of biocomposites of different molecular weight of peg (polyethylene glycol) as matrix with ganoderma lucidum as a filling material at different loading (%1, %2.5, %5 wt). the composites have been prepared by solution intercalation method using ground and sieved ganoderma lucidum at 25 micron scale. the characterization of composites was made by x-ray diffraction (xrd), scanning electron microscopy (sem) and fourier transform infrared attenuated total reflectance (ftir-atr) also in this study the hemocompatibility and antibactarial properties of composite investigated. when xrd and ftir-atr results discussed, all of the composites using the different loading amunt of ganoderma lucidum (%1, %2.5 and %5 wt) were shown a homogen distribution in the matrix (peg). and an interaction have occured between matrix and filling material. the sem photos have confirmed these results. peg and composites have been detected as hemocompatible. these results showed that they can be used as biomaterials. p-07.01.3-016 evaluation of the genotoxic potential of some nanocomposites by comet assay b. yilmaz 1 , s. dogan 1 , s. celikler kasimogullari 2 1 department of molecular biology and genetics, balikesir university, balikesir, turkey, 2 department of biology, uludag university, bursa, turkey due to its similar nature to the bone, nanohydroxyapatite is a biocompatible particle and poly(methyl methacrylate) (pmma) is a polymer that has been used in dentistry and orthopedic applications for years. in this study, genotoxic potential of pmma/nanohydroxyapatite nanocomposite films composed of polymers having different molecular weights and nanohydroxyapatite fillers in different concentrations (1, 2.5 and 5%) were investigated by comet assay which is a kind of gel electrophoresis that can be used to measure dna damage in individual cells. if the dna is damaged we expect broken ends to migrate apart from the head. at the end of the assay performed after incubation with lymphocytes of healthy humans, we measured the dna damage index (ddi) and percentage of damaged cells (pdc). in addition, to prove the morphological properties of the nanocomposites scanning electron microscope was used and an interaction between the matrix and nanoparticles with a homogeneous dispersion was observed. protein adsorption on stimuli-responsive mixed pdmaema/peo polymer brushes a. bratek-skicki 1,2 , c. dupont-gillain 1 1 universit e catholique de louvain, louvain-la-neuve, belgium, 2 j. haber institute of catalysis and surface chemistry, polish academy of sciences, krakow, poland smart polymer brushes are made of macromolecules that are sensitive to stimuli from the external environment, including ph, ionic strength, temperature, etc. when stimuli-responsive polymer brushes are introduced onto material surfaces, their properties can be adjusted by tuning the environmental stimuli. these brushes can find promising applications across many areas of research, including surface science, nanotechnology, and biotechnology. in our work, the adsorption of human serum albumin (hsa, molecular weight of 66.5 kda, isoelectric point ip at ph 4.7) and lysozyme (lys, molecular weight of 14.3 kda, ip~11) was studied on polymer brushes composed of poly(ethylene oxide) (peo) and poly (2-(dimethylamine) ethyl methacrylate) (pdmaema). peo is a protein-repellent polymer and pdmaema is a polyelectrolyte bearing a variable density of positive charges depending on ph. a gold substrate was modified by these thiolated polymers according to the 'grafting to' method. the obtained polymer brushes were characterized by xray photoelectron spectroscopy, static contact angle measurements and atomic force microscopy. polymer brush formation and protein adsorption were monitored by quartz crystal microbalance. surface characterization of the mixed brushes revealed the presence of both polymers at the surface. conformational changes of pdmaema/peo brushes were experimentally evidenced, and the results indicated that the brushes collapse at ph 9.00 (pdmaema is neutral in such conditions) and were swollen at ph 3.5 (pdmaema is positively charged). protein adsorption was performed at different ph values (3.5-9 .0) and salt concentrations (0.001-0.15m). it was shown that pdmaema has a high affinity to hsa at ph above its isoelectric point. however, the adsorption of positively charged lysozyme in a wide range of ph was not observed. these results indicate that pdmaema/peo brushes are promising candidates for selective adsorption from a mixture of proteins. clay-polymer nanocomposites (cpn) developed in recent years as a new type of inorganic-organic hybrid materials that were conceived for medical uses such as tissue engineering or drug delivery [1] , [2] . the understanding of the structure and physico-chemical properties of cpn is a first step in the investigation of biomaterials, but their potential in this respect is determined by their interaction with living tissue components. in this study, pure kaolinite was intercalated with dimethyl sulfoxide (dmso) and then intercalated kaolinite was modified pyridine, 2-amino pyridine and 2,6-diamino pyridine to expand the interlayer basal spacing. modified kaolinite samples as filler and poly(vinyl chloride) (pvc) polymer as matrix were used in the nanocomposite synthesis. nanocomposites of pvc have been prepared by solvent blending method using thf as a solvent. the material characterizations were carried out by xrd, afm, ftir-atr, dta/tg and dsc. the xrd results reveal the formation of intercalation/exfoliation of modified kaolinite in the pvc matrix. ftir and afm results confirm the presence of nanomaterial in kaolinite/pvc nanocomposites. tga data show that the modified kaolinit/pvc nanocomposites have significant enhanced thermal stability. the glass transition temperature (tg) of pvc nanocomposites is higher than that of pure pvc. in addition, the antimicrobial activity of clay-polymer composites were also determined. introduction: polyhydroxyalkanoates (phas) are biocompatible and biodegradable materials obtained from microorganisms. they are produced in the cytoplasm of several bacteria as energy reserve. the physical properties of poly(3-hydroxybutyrate) (phb), which is from the group of phas, make it a competitive source to petrochemical plastics. phb has potential in order to be used in a variety of application fields such as packaging industry, printing materials, agriculture and food industry. furthermore, phb meets expectations for tissue engineering applications, since it is biocompatible, biodegradable, non-toxic and has good mechanical properties. although its many advantages, blending approach could be needed in order to fulfill all expectations of a material. due to its flexibility, polycaprolactone (pcl) is a promising candidate to be blended with phb. the aim of this study is to construct a scaffold by using phb produced by extreme alkaliphilic b. marmarensis gmbe 72 t (dsm 21297) and commercial pcl as components and investigate its properties. materials and methods: electrospinning method was used in order to construct scaffolds from blend polymer solution containing phb from b. marmarensis and commercial pcl. results: nanofiber structures were observed on scanning electron microscope (sem) images and fourier transform infrared resonance (ftir) analyses have shown characteristic peaks for both phb and pcl. discussion and conclusion: phb could be blended with other polymers in order to enrich its properties. in addition, nanofiber structure of electrospun phb-pcl blend makes it a rewarding material as scaffold for several tissue engineering applications. q fever is a zoonotic disease that is encountered widely around the world, the most common acute form of q fever shows the following symptoms; a sudden fever, shivering, lassitude, headache, anorexia. because this disease does not show specific symptoms its diagnosis is possible with laboratory tests. current diagnostic kits lack effectiveness; this is why the main goal of our studies is to come up with a new diagnostic kit that does not have disadvantages that current diagnostic kits show. with this goal, nine mile i strain (rsa 493), s serologic virulent phase i, were obtained from slovak science academy, virology institute for rickettsia reference and research from who co-operation centre. these cells were purified and lipopolysaccharide (lps) isolation from coxiella burnetii was performed. the polymeric carrier, poly (n-vinyl-2-pyrrolidone-co-acrylic acid) [p(vp-co-aa)] was synthesized and characterized. physical complexes of obtained lps and p(vp-co-aa) with varying ratios. ternary complexes of lps-cu 2+ -p(vp-co-aa) were also synthesized with copper metal mediation. structure and interaction of lipopolysaccharide-p(vp-co-aa) complexes were investigated with zeta-sizer device using zeta potential analysis and ftir spectrophotometry according to the ratios of components, reaction environment conditions and chemical structure of the polymer. the best complex ratio according to analysis results will be used in the future studies for obtaining monoclonal antibodies which will be an important step for obtaining more effective and stable diagnostic kits that can be used for q fever. this 2513 in this study; new types of water soluble polymer-biomolecule conjugates were synthesized using covalent bonding techniques between polymers and co-polymers (varying monomers of polyacrylic acid and acrylic acid) with peptides. different compositions of polymers, varying ratios of biomolecule/polymer and different molecular weight of polymer has yielded new types of bioconjugates. conjugation mechanism, composition and structure were investigated with various physicochemical methods (uv, ftir, hplc, gpc, etc.). the peptide used in this study was the antigenic peptide epitope of sheep-goat pox disease (eakssiakhfslwksyadadiknsenk). whether this peptide series was bound to polymers or whether it was bound to polymer-protein carrier; peptide-specific immunogens that were capable of producing antibodies were synthesised. it is thought that using polymer-peptide bioconjugates that contain just peptide will yield a higher peptide-specific immunogenicity compared to traditional adjuvants. in vitro and in vivo investigations of bioconjugates effectivity is planned to be done in the future studies. p-07.01.3-026 bioinert fluorinated ethylene-propylene copolymer modified for keratinocyte adhesion surface properties are crucial when adhesion of a cell to a polymeric material is required for a biomedical application. one of the methods for polymer surface tailoring is argon plasma treatment. this simple and reproducible method alters the surface properties such as roughness and wettability without affecting the bulk properties of the material. for the modification of the bioinert fluorinated ethylene-propylene (fep), related to teflonò, we employed argon plasma treatment with the powers of 3 and 8 w for 20-240 s. the human keratinocytes of the hacat cell line served as a model cell line for biocompatibility testing. we studied adhesion, proliferation and morphology of the cells on modified fep matrices as well as controls (pristine fep and standard polystyrene tissue culture dish) by means of fluorescence microscopy. further morphological details were acquired by scanning electron microscopy. furthermore, fluorescence microscopy with immunochemical labelling was used to determine the size and distribution of focal adhesions in cells grown on the modified matrices. the overall effect of the matrices on metabolic activity of cultured hacat cells was also evaluated using the wst-1 reagent. the ar plasma treatment of fep matrices improved significantly cell adhesion and proliferation and promoted spreading of the hacat cells compared to the pristine fep, on which cells were not able to spread properly. also, increased metabolic activity rates for cells cultured on modified matrices were found in comparison to the pristine fep. altogether, we found that ar plasma treatment improved the surface properties of fep to such extent, that it allows cultivation of adherent cells on its surface. we therefore propose that ar plasma treatment is a useful method for fep surface modification. p-07.01.3-027 graphene oxide enriched biomaterials display potential for tissue engineering applications tissue engineering (te) requires more efficient systems that favor local tissue regeneration with minimum cytotoxicity. materials based on natural compounds ensure biocompatibility and have better effects for regeneration. graphene oxide (go) has been shown to enhance cell adhesion and to improve the rate of cell differentiation. in this context, the aim of this study was to evaluate if the addition of different concentrations (0.25-1 wt.%) of graphene oxide (go) improves the properties of cellulose acetate (ca) materials for biocompatibility and cell differentiation, in the prospective of using these films for te applications. go-containing ca films were tested for cytocompatibility by quantitative and fluorescence microscopy assays, and compared to the ca control. cell cytoskeleton architecture in contact with biomaterials was revealed by confocal microscopy. furthermore, bioconstructs were exposed to in vitro osteogenic and adipogenic induction and monitored for 21 days. histological stainings were performed to validate differentiation. osteogenic and adipogenic markers gene expressions were assessed via qpcr. ca/go 1 wt.% revealed best biocompatibility among all tested scaffolds. adhesion proved to be dependent on the percentage of go in material's composition. cells cultivated on ca/ go 1 wt.% expressed adipogenic and osteogenic markers earlier than cells cultivated on materials with lower go content. differentiation markers displayed an increasing profile of gene expression from 7 to 21 days post induction, with higher levels registered for materials with high go content as compared to films with low go content and to the control. go added to ca materials positively influenced cell survival, proliferation and cell differentiation. ca/go films represent potential candidates for te applications. the design of appropriate scaffolds remains one of the most important challenges for te. current idea is that the cell-scaffold interaction could drive cell differentiation and be linked to gene expression and protein organization. therefore, their quality is essential and should favour cell attachment, growth, migration, in situ vascularization and release of biochemical and physical factors able to address the cell fate. moreover, for an ideal scaffolding material an adequate and interconnected porosity is relevant for facilitating cell spreading and colonization of the inner layers. a combination of optimal mechanical and biochemical properties were here utilized to design a 3d composite hydrogel scaffold (3d-chs) in order to favour cell-scaffold interactions and promote a functional differentiation of human lin à sca1 + cardiac progenitor cells (hcpc). the biocompatible peg-diacrylate (pegda) was used to prepare hybrid protein-pegda hydrogels with embedded albumin-microspheres (ms) as protein component. ms were able to modulate the mechanical and biological behavior of the scaffold acting as air-reservoir, porogen agents and potential carriers of biomolecules. an increase of the hcpc viability in the ms-concentration dependent manner was observed. moreover, the microarchitecture of the 3d scaffold also plays a key role in the stability and functionality of cellularcomposite systems. therefore, pegda-honeycomb structures were fabricated using microstereolithography process and the hcpc viability and adhesion to the microstructures were assessed. 3d-chss were synthesized embedding honeycombstructures into ms-pegda hydrogel and the effects on cell proliferation, cell-cell interactions and cellular alignment were investigated. these results could be of relevant interest for expanding the knowledge on cell-scaffold interaction processes and to promote the development and the application of 3d-chs for tissue regeneration using the emerging bioprinting technologies p-07.01.3-029 gene expressions of mesenchymal stem cells after osteogenic induction on ceraform bone substitute a. kilic s€ uloglu, e. karacaoglu, h. akel, c ß . karaaslan hacettepe university, ankara, turkey ceraformò, is a synthetic calcium phosphate ceramic with the chemical composition of hydroxyapatite 65% and tricalcium phosphate 35%, with 60-85% pore volume and 100-400 lm pore diameter. in this study adipose tissue derived mesenchymal stem cells were differentiated into osteoblast cells and loaded on cer-aformò. in order to improve cell adherence, ceraformò was covered with fibronectin. the cells were cultivated for 28-day period by osteogenic induction medium. days 1, 7, 14, 21 and 28 were selected as specific intervals for incubations. total rna was isolated and cdna was synthesized. differences in the expression of runt-related transcription factor 2 (runx2), bone morphogenetic protein-2 (bmp-2), and osteocalcin (ocn) were determined by qpcr. the peptidylprolyl isomerase a (ppia) gene was used as an internal control. according to the qpcr results, ocn gene expression was observed on the day 14th, continued to increase in day 28. bmp-2 gene expression was increased in 14, 21, 28 day compared to 7 day. on the other hand, runx2 gene expression was increased only on days 21 and 28. these findings pointed out that the osteogenic induction was successfully activated on fn coated bone material. therefore, these results can be used in bone injury treatment and related disorders. p-07.01.3-030 on the in vitro cytotoxicity of graphene oxide nanomaterials v. grumezescu 1 , i. negut 1 , f. sima 1 , e. axente 1 , l. e. sima 2 1 national institute for lasers, plasma and radiation physics, magurele, romania, 2 institute of biochemistry, romanian academy, bucharest, romania during the last decade, graphene and its derivates have proven unique physicochemical properties, several applications being continuously developed. among them, electronic, catalytic, mechanical, optical, and magnetic properties have attracted huge interests. however, the merging of graphene and graphene oxide (go) with biotechnology is still in its infancy, many challenges remaining unexplored. potential applications are related to biosensors, drug delivery or gene therapy and cells imaging. in order to use gos as drug release matrices for cancer cells targeting, it is necessary to ensure that these molecules do not affect normal cells within tissues. it was shown that the cytotoxicity of graphene nanomaterials is highly dependent on surface functionalization. studies suggest that pristine and reduced go with fewer surface functional groups tend to be more toxic than go. in striking contrast, it has been reported that functionalized graphenes, can significantly reduce the cytotoxicity even at relatively high concentrations. in this study, we report on the comparison between go and protein functionalized go when submitted to in vitro cytotoxicity tests. bovine serum albumin (bsa) was used for the noncovalent go surface conjugation. three case-studies were investigated: aqueous nano-colloids consisting of serial dilutions of both go and go-bsa conjugates, dropcasted thin films and laser-assembled thin films on glass substrates. safe concentration windows were identified by live/dead staining and mts assays for different human melanoma cell lines, while melanocytes and human dermal fibroblasts were used as normality controls. the predominant melanoma subtype is represented by cells bearing braf (v600e) activating mutation. with a view to target this specific melanoma subpopulation, we embedded braf inhibitors into go laser-deposited scaffolds and tested their anti-tumoral effect. our results evidence the high potential of these nanomaterials for biomedical applications. osteoporosis is a skeletal disorder associated with low bone mass and increase in bone fragility due to increased osteoclastic activity. binding of rank ligand to its receptor on osteoclast precursor cells results in the osteoclast differentiation. sirna is a dsrna, used to inhibit the translation of the target gene. the aim of the study is to develop an injectable sirna-delivery system targeted to the bone for osteoporosis treatment. pei (polyethyleneimine) and rank complex was loaded into poly(lactic acid-co-glycolic acid) (plga) nanocapsules which are bound to hydroxyapatite (hap)-specific elastin-like protein (elp) for targeting to bone tissue. plga nanocapsules were prepared by w/o/w double emulsion technique. affinity of elp to hap was determined by ftir. elp was coated on the nanocapsules by using the transition temperature of elp. elp on plga nanocapsules were crosslinked by genipin and binding of elp on plga nanocapsules were studied by xps and tem. the optimum ratio of n (pei) to p (sirna) in the complexes to be loaded into plga nanocapsules were studied by etbr staining and zeta potential measurements with varying n/p ratios and finally pei-dnaoligo encapsulation efficiency of the capsules was determined by picogreen reagent. xps results of elp treated plga (elp-plga) nanoparticles indicated the presence of nitrogen atom (11.7%) in the sample which appeared as a fuzzy halo in tem micrographs. n/p ratios up to 5 show negatively charged particles. when n/p was 5, the zeta potential of complex was neutralized which also resulted in larger particles compared to the others. zeta potential moved to positive values when n/p was higher than 5. the migration of polyplexes with different n/p ratios (1-10) was analyzed by gel electrophoresis. dnaoligo complexes show the same patterns of complexation wih that of sirna and thus were used in the encapsulation efficiency studies instead of sirna. the encapsulation efficiency of pei-dnaoligo in plga nanocapsules was 29%. tuesday 6 september 12:30-14:30 aging p-09.03.3-001 novel benzenesulfonamides exhibit low toxicity on zebrafish embryonic development and selectively inhibit carbonic anhydrase ix with nanomolar affinity in xenopus oocytes introduction: the toxic effects of two recently discovered inhibitors (vd12-09 and vd11-4-2) that selectively and with extraordinary strong, picomolar affinity bind to human carbonic anhydrase (ca) ix, an anticancer target, were investigated on zebrafish embryonic development and in xenopus laevis oocytes. zebrafish has emerged as a promising animal model to evaluate the toxicity of the drug candidates. xenopus oocytes do not natively possess any ca activity and thus became a convenient in vivo model system to study the ph effects and the selectivity of synthetic ca inhibitors. materials and methods: morphological changes in zebrafish treated with the compounds were studied by light-field microscopy and histological analysis. ca activity in xenopus oocytes was monitored by measuring ph in the cytosol and at the outer membrane surface and confirmed by mass spectrometry of lysed oocytes. the toxicity studies showed lc50 values to be 13 lm for vd12-09, 120 lm for vd11-4-2 and 9 lm for ethoxzolamide (eza), a non-selective ca inhibitor commonly used in clinic. the zebrafish exposed to lc50 doses of vd12-09 and vd11-4-2 showed fewer phenotypic abnormalities and less morphological changes compared to the zebrafish treated with the corresponding dose of eza. vd11-4-2 exhibited 10-25 nm ic50 for both intracellularly and extracellularly expressed ca ix in xenopus oocytes while exhibiting strong selectivity over ca ii, ca iv and ca xii. discussion: interestingly, the compounds exhibited 10-fold lower toxicity, induced fewer side effects in zebrafish than eza and the amount of vd11-4-2 needed to cause complete inhibition of ca ix enzymatic activity in xenopus oocytes was 30-fold lower than eza. conclusions: vd compounds did not lead to deleterious effects on the zebrafish embryonic development and reached the ic50 of 10 nm for ca ix in xenopus oocytes. the compounds could be further developed as anticancer drugs. cacybp/sip is present in various cells and tissues, both normal and pathological. in normal tissues, e.g. stomach or colon, cacybp/sip is weakly or barely detected whereas in gastric or colon cancer this protein is expressed at a high level. there are also data indicating that the level of cacybp/sip expression correlates with tumor metastatic potency and multidrug resistance. taking into consideration data that suggest association of cacybp/sip with many vital cellular processes, in this work we decided to investigate the possible mechanism involved in regulation of cacybp/sip gene expression, mainly by transcription factors and, on the other hand, the influence of cacybp/sip on the expression of other genes. we have shown that nfat (nuclear factor of activated t cells) influences the cacybp/sip gene expression and that overexpression of cacybp/sip has an effect on the level of ap-1 and on the activity of nfat and ap-1 transcription factors. by analyzing the cacybp/sip gene promoter sequence we also found potential binding sites for transcription factors from the stat family, which are involved in interferon signaling. microarray data indicate that indeed overexpression of cacybp/sip affects levels of the stat proteins as well as of some interferons and interleukins. based on functional analysis we have found many genes the products of which are involved in immune response. to analyze in more detail the influence of an altered level of cacybp/sip on interferon signaling pathways as well as on factors involved in expression of interleukins, including nfkappab, we plan to apply methods such as luciferase assay, real-time pcr or immunocytochemistry. one of the pathological hallmarks of alzheimer's disease (ad) is the neuritic plaques occurred as a result of the extracellular accumulation of aß peptides formed from amyloid precursor protein (app) via the ß-amyloidogenic pathway. aß42 is more prone to aggregation to form plaques and more toxic to neurons than aß40. in addition to change in app metabolism, the decline in levels of neurotransmitter acetylcholine and cholinergic dysfunction are also observed in ad. thus, current strategies for ad treatment focus on compounds with inhibitory effect on cholinesterases as well as preventive effect on aß aggregation. in our earlier studies, toluidine blue o (tbo), a phenothiazine dye, was shown to be a highly effective inhibitor of cholinesterases with k i values in nm range. we also found that intracellular app and aß42 levels are reduced in human neuroblastoma cells after treatment with tbo. additionally, an earlier study revealed that tbo has a selective inhibitory effect on tau aggregation, the other pathological characteristic of ad. the aim of this study was to investigate whether tbo may effectively lower the level of extracellular aß40/42 in an ad-like cellular model. chinese hamster ovary cells that express human wild type app and presenilin 1, namely ps70, were treated with a dose range of tbo (0-15 lm) or vehicle control for 24 h. after treatment, aß40/42 levels in cell culture media were assayed by separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. besides, biocompatibility of tbo was evaluated in the ps70 cells using cell viability assay for flow cytometry. strikingly, all dose ranges of tbo inhibited both aß40 and aß42 secreted into the cell culture media. significant reduction for both aß species was evident at 5 lm (p < 0.05), 10 lm (p < 0.001), and 15 lm (p < 0.001) of tbo vs. vehicle control. in conclusion, these results support the idea that tbo may be used as a therapeutic in ad. monitoring the changes of key molecules participating in the osmo-regulatory response of nucleus pulposus intervertebral disc cells during stress-induced senescence e. mavrogonatou, d. kletsas ncsr 'demokritos', athens, greece introduction: intervertebral disc cells are faced with a harsh extracellular milieu characterized by hyperosmotic conditions, nutrient and oxygen deficiency because of the absence of vascularization and oxidative stress due to the accumulation of their metabolism's by-products. we have previously shown that high osmolality is anti-proliferative for disc cells through the activation of the g1 and g2 cell cycle checkpoints by p53 and p38 mapk, respectively. in addition, we have shown the participation of nine solute transporters, with the a1 subunit of na + /k + -atpase being central in this response. here we assessed the changes in the expression of these key osmo-regulatory molecules during in vitro stress-induced senescence. materials and methods: changes in cell cycle progression were assessed using flow cytometry; overall transcriptional alterations were assessed by whole-genome arrays; differences in expression at the mrna and protein level were revealed by quantitative rt-pcr and western blotting, respectively; knocking-down of selected proteins was performed by sirna. results: high osmolality led to the differential expression of > 200 genes, including nine genes encoding transporters. p38 mapk and p53 were demonstrated to differently participate in the regulation of the aforementioned transporters, while knocking-down of three selected transporters had a distinct outcome on the overall cellular response towards hyperosmotic stress. these molecules were found to show differences in their expression in senescent cells. discussion: given that the presence of senescent cells has been demonstrated in the intervertebral disc in vivo and could most probably attributed to the prevailing stressful conditions, here we showed differences in the expression profile of known key molecules for osmo-adaptation during senescence. conclusion: understanding disc cells' physiology is of outmost importance when designing cell-based therapies for disc degenerative disorders. p-09.03.3-005 smad specific e3 ubiquitin protein ligase 2 (smurf2) and its potential effects on inhibitory transmission in aging adams 1, 2, 4, 5 1 interdisciplinary program in neuroscience, bilkent university, ankara, turkey, 2 national nanotechnology research center (unam), bilkent university, ankara, turkey, 3 department of molecular biology and genetics, bilkent university, ankara, turkey, 4 molecular biology and genetics zebrafish facility, bilkent university, ankara, turkey, 5 department of psychology, bilkent university, ankara, turkey smad specific e3 ubiquitin protein ligase 2 (smurf2) is part of the tgf-b signaling pathway associated with cellular proliferation, differentiation, genomic stability and senescence. moreover, smurf2, via its downstream partners, may regulate inhibitory synaptic transmission. our research group previously found that the smurf2 transcript is significantly higher in old zebrafish brains. thus, smurf2 may alter inhibitory synaptic transmission in aged animals. the focus of this study was to examine age-related changes in smurf2 protein levels and related key inhibitory synaptic proteins; gephyrin (gep), a scaffolding protein for gaba receptors, and gaba a , an ionotropic gaba receptor subtype. additionally, the levels of those proteins were studied in a mutant zebrafish line, which lacks acetylcholinesterase (ache) and is suggested to be a delayed aging model. whole brain tissues were isolated from young, middle-aged and old male and female zebrafish brains (ab/wildtype strain), as well as from old male and female ache mutant zebrafish (ache sb55/+ ). animals were maintained and raised in standard conditions. the extracted brain tissue was homogenized in ripa buffer and subjected to western blot analysis to determine differences in the relative protein expression levels. our preliminary data indicated that smurf2 and gep levels remain stable in the aging brain (p = 0.301, p = 0.335), and in the ache mutants gep levels are increased compared to the wildtype controls (p = 0.001). further analysis of the relationships between smurf2 and gaba a levels and brain aging is ongoing. we predicted that alterations in smurf2 levels would parallel changes in key synaptic inhibitory proteins during the aging process, which was the case for the gep levels. while smurf2 may regulate inhibitory synaptic transmission, the exact roles of those synaptic proteins in the context of normal and delayed brain aging are not known well-understood and the subject of continuing study. in recent years, express the hypothesis that aged individuals are vulnerable to infectious and other inflammatory agents and they become more prone to develop majority of severe age pathologies, including cardiovascular and oncology diseases, neurodegenerative diseases, type 2 diabetes mellitus and inflammatory diseases, etc. one of the central components of immune response is the family of toll like receptors (tlr). there are several opinions that single nucleotide polymorphisms (snp) leading to a loss of function of the respective tlrs can be associated with age and increase the risk of age related diseases, especially cardiovascular diseases (cvd). however, many available studies focusing on tlr snps and cvd are with conflicting results. the aim of this study was to assess the potential interaction between genetic variants of tlr2 and tlr4 and ischemic heart disease (ihd) in kazakhstan population over 45 years old. we evaluated 148 patients with ihd and 144 healthy subjects aged 45 years and over (ethnical kazakhs and russians living in republic of kazakhstan). polymorphic loci of the genes tlr2 rs5743708 and tlr4 rs4986790 were genotyped by pcr with subsequent restriction analysis. our results indicated that the genotype and allele frequencies of tlr2 (arg753gln) and tlr4 (asp299gly) were not significantly different between the 2 groups (p ≥ 0.05). statistical analysis didn't elicit any association between studied gene polymorphisms and predisposition to ihd in individuals over 45 years old (p ≥ 0.05). for these polymorphisms, age, fasting blood sugar and serum lipid levels were not also significantly different among different genotypes in the ihd and control groups. in conclusion, the data shows that there is no interaction between tlr2 and tlr4 and ischemic heart disease (ihd) in kazakhstan population over 45 years old. we plan to include other types of polymorphisms in tlr 2 and tlr 4 genes and increase the volume of patient cohort in our future studies. p-09.03.3-007 evaluation of prognosis with total oxidant/ antioxidant status and some oxidative stress parameters in patients with acute ischemic stroke stroke is the third most common cause of death after coronary heart disease and cancer. strokes are classified into two groups according to their pathology: ischemic stroke and hemorrhagic stroke. ischemic strokes make up 87% and hemorrhagic strokes 13% of all strokes. during ischemic stroke, oxidative stress has been shown to play a major role in the occurrence and progression, formed oxidants also affect cell membranes and genetic material such asdna, rna, and various enzymatic events, and they lead to cell damage. some studies have shown oxidant-antioxidant status but have not shown the relationship with prognosis. this study has investigated the relationship between prognosis and total oxidant/antioxidant status and biochemical parameters in patients with acute ischemic stroke 58 patients, with acute ischemic stroke and 37 healthy controls we reenrolled in the study. blood samples were taken within 1st and 7th days, and after 3rd months in the patient group for analysing serum total oxidant status (tos), antioxidant status (tas), catalase, arylesterase, and thiol. prognosis was evaluated with national institutes of health stroke scale (nihss) and-modifiedrankinscale (mrs) scores. there was no significantly difference between groups by means of serum tas, tos and catalase levels. but arylesterase (p: 0.07) and thiol (p: 0.031) levels were significantly higher in first 24 h blood samplingthancontrolgroup. statistically significant negative correlation was observed between the 3rd month values of tos and nihss score (r = 0.410, p = 0.037). but there was no correlation between mrs scores and serum tas, tos, catalase, thiol and arylesterase. similarly, our findings suggested some serum oxidant levels were increased in acute ischemic stroke patients and total oxidant status might be used in evaluation of prognosis but larger studies are needed. p-09.03.3-008 amylin and preptin regulate glucose homeostasis in infertile women with polycystic ovary syndrome and poor responders undergoing ivf/icsi disrupted glucose homeostasis leads not only metabolic disturbance such as polycystic ovary syndrome (pcos), but also influences oocyte growing. this study was designed to evaluate follicular fluid (ff) and serum levels of glucoregulatory hormones, amylin and preptin, in infertile women with pcos and poor responders undergoing ivf/icsi. human follicular and serum were obtained from 20 infertile women with pcos and 20 poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone antagonist protocol for ivf/icsi treatment. ff and serum amylin and preptin levels were measured by elisa. it was found that ff and serum amylin and preptin were lower in infertile women with pcos when compared with poor responder participants. ff amylin and preptin concentrations were lower than that of the serum amylin and preptin concentrations. decreased follicular fluid amylin and preptin levels suggest that amylin and preptin may have a physiological role in follicular maturation via controlling local glucose homeostasis. despite high serum levels of amylin and preptin in pcos their low concentration within the follicle may be main culprit of defective folliculogenesis seen in pcos subjects. similar to insulin resistance in pcos subjects existence of amylin and preptin resistance support the critical role of both peptides in follicular maturation in pcos. keywords: follicular fluid; amylin; preptin; polycystic ovary syndrome; infertility. the transcription initiation on p3 promoter of xp10 bacteriophage in presence of p7 protein, a modulator of rna-polymerase activity a. shadrin g.k. skryabin institute of biochemistry and physiology of microorganisms, ras pushchino, russia many bacteriophages are able to manage the transcription system of their bacterial host for their own needs. for example, bacteriophage xp10, in the early stages of infection of xanthomonas oryzae inhibits transcriptional activity of bacterial rna-polymerase on majority of promoters via p7 protein, except of bacteriophage p3 promoter responsible for expression of the bacteriophage 'middle' class genes. the focus of this work is to study the mechanism of action of p7 protein in the transcription initiation and identification of the role of the individual elements of p3 promoter of xp10 bacteriophage, enabling x. oryzae rna-polymerase escapes inhibition by p7 protein. we have designed a set of promoter probes representing the combination of sequences of p7-resistant p3 promoter and p7sensitive t5n25 promoter. using fret-based assay it was shown that the truncated probes corresponding to promoter dna downstream à26 position, relative to the transcription initiation start site, did not lead to dissociation of the sigma-factor. longer probes, containing à35 promoter element, induce dissociation sigma-factor. the in vitro transcription experiments show that the deletion of region 4, a sigma-factor domain responsible for interaction with à35 promoter element during the transcription initiation, is not critical for inhibition of rna-polymerase by p7 protein. promoter probe with up-element of p3 promoter had affinity to x. oryzae rna-polymerase a several times higher than a probe containing the consensus up-element for e. coli rna-polymerase . summing up the results, it seems like the transcription initiation on p3 promoter of bacteriophage xp10 can escape inhibition by p7 protein through a high affinity interaction between the up-element and c-terminal domains of the alpha subunit of rna-polymerase x. oryzae. p-09.03.3-010 distribution of soluble form of glial fibrillar acidic protein in the different areas of gerbils brain during development and aging y. kovalchuk, g. ushakova oles honchar dniepropetrovsk national university, dniepropetrovsk, ukraine astrocytes are the most abundant cell type within the cns and play an important role in cns homeostasis and function. glial fibrillary acidic protein (gfap) forms the main astrocytic intermediate filament (if). the overall level gfap in different parts of the brain uneven and depends on the number of astrocyte cells. gfap is very sensitive to any kind of neurodegenerative diseases and aging. during aging, a glial reaction is observed in the human brain, as well as in rat and mouse brains. the aim of our study was to investigate the quantitative astrocytes-specific protein gfap in different areas of the gerbils brain at the first stages of postnatal development and aging. for the study 30 gerbils brains were used and divided into 5 groups (n = 6): 1: newborn animals (1 day), 2-4: 30, 90 and 180 days respectively, 5: animals aged 2 years. the animals were decapitated under mild anesthesia (thiopental), with isolated brain three divisions: the cerebellum, thalamus and hippocampus, which are then used to produce cytosolic protein fractions. the level of gfap in the obtained fractions were determined according to the method of competitive elisa. newborn gerbils found no significant content of soluble form of glial fibrillar acidic protein in all investigated parts of the brain, and a sharp increase of amount within 30 days (in cerebellumamounted to 1.12 ae 0.03 lg/100 mg tissue; to 90-180 days increased to 1.67 ae 0.11 lg/100 mg tissue, and began to grow again in older individuals aged 2 years). unlike the cerebellum, the level of sgfap in hippocampus and thalamus reached the maximum at 30 days p.d. (1.5-1.7 lg/ 100 mg tissue), and unchanged for 180 days. these results revealed that the most intensive development of astrocytes in the cerebellum to 90 p.d. of gerbils, and in the thalamus and hippocampus are formed within the first month of life. the plastid-nucleus located protein whiry1 acts as an upstream regulator of leaf senescence binding to the promoter of senescence associated genes (sags) like senescence marker gene hvs40. in order to investigate the impact of whirly1 on drought stress-induced senescence, transgenic barley plants with a knockdown of whirly1 (hvwhy1kd) were grown under untreated and drought stress conditions. the leaf senescence evolution was monitored by physiological parameters and gene expression studies of senescence and drought stress related genes. to reveal the epigenetic indexing at hvs40 at onset of drought-induced senescence in wild type (wt) and hvwhy1kd lines, stress-responsive loading with histone modifications at 6 gene regions of hvs40 (2 regions in the promoter, one region around translation start site and 3 regions located in the gene body) was analysed by chip and quantified by rtq-pcr. in barley, drought treatment caused acceleration of leaf senescence in wildtype (wt) plants, whereas why1kd lines showed a staygreen phenotype. expression of senescence-associated and drought stress responsive genes expression was delayed in hvwhy1kd indicating that whirly1 protein acts as an upstream regulator of drought stress-induced senescence. the chip results showed that drought treatment is causing in wt a significant increase in the levels of h3k9ac all over the analyzed gene regions, correlating with a massive induction of hvs40 expression, while drought stress caused no substantial increase of h3k9ac in why1kd plants. the results suggest that drought induced expression of hvs40 is under epigenetic control, and furthermore that why1 is involved in this epigenetic control level. oncolytic viral therapy is based on the capabilities of selective lysis of tumor cells and is a prospective trend in cancer disease treatment. in vitro experiments showed that plant rhabdoviruses does not have any direct cytotoxic effect upon sarcoma 37 cells, causes induction of apoptosis in these cells and does not pose any threat to somatic cells of warm-blooded animals, which makes it possible to use this virus for therapy of malignant neoplasms. buckwheat burn virus (bbv), the prototypic member of the family rhabdoviridae, contains surface glycoprotein and which is lectin-active. its carbohydrate branch can aid adhesion of lymphocytes to tumor cells. the present study has addressed the effect of bbv on cancer cell viability. all studies were carried out after 1 week of inoculated with erlich cancernome (2 9 10 6 cells/animal, i. p.) in 2 months male balb/c mice treated at once with or without plant extract with bbv (15 mg/kg, i. p.). by fluorescent microscopy and using two due staining by acredine orange and propidium iodide it was found that in the 3rd day of administration of bbv lead to increasing of necrotic and apoptotic cells on 45% and 4% respectively versus to untreated group. at the same time the viability of investigated cells was impaired too and according to flow cytometry analysis using propidium iodide the amount of dead cells was elevated by fivefold (17.7% versus 3.5% in untreated group). also as was shown in previously reports bbv decreased activity of macrophages in the early stages after injection and it may have a positive effect when using this drug in tumor therapy. when using this drug appears to slow down the possibility of a sharp activation of macrophages, and as a consequence of the development of cytotoxic effect will be prolonged. key words: rhabdoviruses, buckwheat burn virus, cancer, cell viability. plants are considered as one of most promising sources for new antimicrobials, based on the evidence of their use in folk medicine to treat various infectious diseases since ancient times. despite relatively small area size, armenia has large diversity of flora with many endemic species. the main goal of this study was the screening of various parts of 28 herbs (widely being used in armenian folk medicine) for their antimicrobial activities in order to select most prospective plants for further comprehensive studies. plant crude extracts were obtained with maceration technique using five solvents: water, methanol, chloroform, acetone and hexane. agar well diffusion assay was used to evaluate antimicrobial properties of plant crude extracts at 500 lg/ml concentration against escherichia coli vkpm-m17, pseudomonas aeruginosa grp3, bacillus subtilis wt-a1, salmonella typhimurium mdc 1754 and staphylococcus aureus mdc 5233, candida albicans wt-174 and candida guilliermondii hp-17. statistical analysis was done using graphpad prism 5.03. crude extracts of all tested plant materials expressed antimicrobial activity against at least one test strain. most of the tested extracts inhibited growth of both gram-negative and gram-positive bacteria. in contrast, only some plant materials exhibited inhibitory activity against yeast strains. according to obtained data sanguisorba officinalis, rumex confertus, hypericum alpestre, lilium armenum and agrimonia eupatoria possessed the highest and broadest antimicrobial activity. moreover, the results showed that acetone was the most effective solvent for solubilizing antimicrobial compounds from plant materials followed by methanol, chloroform, hexane and water. the results demonstrated high antimicrobial activity of medicinal plants used in armenian traditional medicine. five plant species were selected for further comprehensive studies. besides, acetone was proposed as efficient solvent in antimicrobial screening protocols. p-02.08.5-004 effects of aluminum stress on photosystem-i apoprotein a2 gene (psab) transcription level in lichen xanthoria parietina (l.) th. fr. unal € ozakc ßa ege university, izmir, turkey in this study the effects of shortrerm aluminium (al) toxicity on the lichen xanthoria parietina (l.) th.fr. were investigated at physiological and transcriptional level. lichen thalli were treated with alcl 2 in different doses (0.25, 0.5, 1 and 5 mm). lipid peroxidation and chlorophyll integrity were determined by spectrophotometer. expression level of psab gene was also investigated. chlorophyll a content was significantly (p ˂ 0.05) decreased after 48 hours treatment with 1 mm and 5 mm of al, while chlorophyll b content was increased significantly due to treatment with increased concentration of aluminum. also treatment with 0.25 and 0.5 mm al for 24 hours increased the gene expression level of psab by 35.6% and 21.3% respectively. our results indicated that aluminum treatment has decreased the chlorophyll biosynthesis and increased the lipid peroxidation depending on time and concentration. this study also demonstrates that the psi can be readily photo-inhibited by aluminum stress. in conclusion, 5 mm al exposure for 48 hours could damage the electron transport in photosystem i. p-02.08.5-005 nigella sativa reduces paracetamol-induced nephrotoxicity and oxidative stress in rats: biochemical evaluation background: nigella sativa l. (ranunculaceae) (ns) is traditionally used to treat many conditions such as inflammation. this study evaluates the effects of ns seeds ethanol extract in paracetamol-induced acute nephrotoxicity in rats. material method: forty-eight female wistar albino rats were divided into eight groups: i = sham; ii = sham + 1000 mg/kg ns; iii = sham + 140 mg/kg (n-acetyl cysteine) nac; iv = 2 g/ kg paracetamol; v = 2 g/kg paracetamol + 140 mg/kg nac; vi, vii and viii = 2 g/kg paracetamol + 250, 500 and 1000 mg/kg ns, respectively. paracetamol administration (oral) was carried out 1 h after ns and nac administrations (oral), and all animals were sacrificed 24 h later. result: urea and creatinine levels were determined in serum, while glutathione, malondialdehyde levels and superoxide dismutase activity were determined in the kidney tissues. there were significant increases in the serum levels of urea and creatinine in the paracetamol-administered group. serum levels of urea and creatinine were decreased in all groups administered ns with paracetamol. ns administration dramatically restored sod, gsh, and mda levels in the kidneys. conclusion: the results suggest ns has a significant nephroprotective activity on paracetamol-induced nephrotoxicity. it may be suggested that the antiinflammatory and antioxidant effects of ns ethanolic extract originated from different compounds of its black seeds. p-02.08.5-006 the study of problems of preservation of the birches e. shadenova 1,2 , e. zhumabekov 2 , m. sembekov 2 , m. burchaeva 2 1 institute of general genetic and cytology, almaty, 2 laboratory of genetics and reproduction of forest culture, institute of general genetics and cytology, almaty, kazakhstan nature of deciduous trees have a whole range of various medicinal properties. instead of synthetic hormone substitutes, you can use medicinal infusions and decoctions of natural phytohormones are widely used in both folk and professional medicine. one of these plants is birch, its young leaves and buds. however, they also must be used with caution because overdose of these compounds is very dangerous, not only can you not get the desired effect, but also face the opposite of his action. in our research to mass replication of plants (different types of birches (betula ajanensis, yarmolenko, jacguemontii, maximowiczii, ulmifolia, middendorffii, kelleriana, tianschanica)) we use nutritional medium excluding the application of phyto promoters in order to prevent mutation. the object of research serve as the old, the sick, being on the verge of extinction, mature trees as explant meristema. since from the moment of calling experience and most cultivation occurs at nutritional medium without hormones. as a result of molecular analysis we get without virus, genetically identical plants. molecular certification of different types of birches of interest, both in terms of organizing, and in terms of selection and genetic improvement of valuable forms, identification of lines selected from natural populations and clones obtained in vitro. relationship between clones and installed parent form by comparing profiles amplific pcr products using issr-marking. according to the results of carried out works really recovered clones obtained from one source tree, indicating the potential for certification of clones studied forms of birches pcr. a study performed in the framework of the state grant project "conservation of breeding valuable species of birches". p-02.08.5-007 fractionated triterpenoid glycosides from sea cucumber inhibit invasion and metastasis in human cancer cells sea cucumbers are slow-motioned invertebrates. holothuria polii delle chiaje, 1824 is widely distributed sea cucumber in _ izmir coastline (turkey). it secretes saponins i.e. triterpenoid glycosides (ttg) as secondary metabolites. the aim of this study is to evaluate anti-invasive and anti-migrative effects of fractionated ttgs obtained from h. polii on ht-29, t84 and upci-scc-131 cancer cell lines. the semi-purified ttgs was extracted from h. polii collected from coast of _ izmir-dikili. the four different fractions (fraction a-d) were collected by using hplc (high-performance liquid chromatography) and characterizated with maldi-ms/ ms. the fractions obtained from h. polii extract include holothurin a (1243.50 m/z) and 24-dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer (1227.50 m/z). anti-invasive and anti-migrative effects of the fractions on the cancer cell lines were detected with xcelligence rtca dp system. the results showed that fraction a-d inhibited migration and invasion of human cancer cell lines at 6th and 12th hours compared to control group. this study shows that holothurin a, 24dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer could be evaluated as promising anti-cancer agents for human cancers. acknowledgement: the authors acknowledged the scientific and technological research council of turkey (t € ub _ itak) for financial support (114z211). p-02.08.5-008 alternative splicing regulation of sr proteins in response to environmental stress in chinese cabbage serine/arginine-rich protein (sr protein) family, which acts as rna-binding protein, plays a major role in post-transcriptional regulation of pre-mrna, such as alternative splicing (as). these proteins cause pleiotropic effect by regulating as of pre-mrna in a tissue and developmental stage-specific and stress-responsive manner in arabidopsis. here, we identified 31 genes encoding sr proteins in chinese cabbage (brassica rapa chiifu-401) from brassica database and analyzed their phylogenetic relationship. b. rapa has 31 types of sr protein that are classified into common (sr, rsz and sc) and plant specific (scl, rs2z, rs and sr-like) subfamily similar with arabidopsis. interestingly, the as pattern of most sr genes changed at the late stage (14 and 21 days after germination). to verify the correlation between sr genes and environmental stress, we screened the as pattern of sr genes to various abiotic stress using rt-pcr and a microarray analysis. in particular, the expression level and the as pattern of bra015576 and bra018581 were affected significantly by heat stress. these results suggest that the as regulation by sr protein correlates with adaptation mechanism to the environmental stress in chinese cabbage. p-02.08.5-010 characterization of recombinant prolyl oligopeptidase from myxococcus xanthus and potential use in gluten hydrolysis e. k. kocaazorbaz, f. zihnioglu faculty of science, biochemistry department, ege university, izmir, turkey a recombinant prolyl oligopeptidase from myxococcus xanthus was purified with a specific activity of 224 u mg(-1) by using nickel-metal-chelate affinity chromatography and gel permeation chromatography. the recombinant enzyme had a monomeric molecular weight of 70 kda. its isoelectric point, determined by two dimension polyacryl-amide gel electrophoresis, was close to 6.3. the optimum ph and temperature was estimated as 7.5 and 37°c, respectively. the purified enzyme was stable from ph 6.0-8.5 and able to thermal stability up to 37°c. the k(m) and v(max) values were 0.2 mm and 3.42 micromol/ min per mg. the enzyme exhibited hydrolytic activity for suc-gly-ala-pna, suc-gly-pro-pna, z-gly-pro-pna, igf-1, substance p, whereas no activity for h-gly-pro-pna, h-val-ala-pna, h-arg-pro-pna, h-ala-pro-pna, glu-ala-pna, pro-pna, leu-pna. its proteolytic activity was inhibited by activesite inhibitors of serine protease, z-pro-prolinal pmsf, and metal ions, cd 2+ and hg 2+. the potential use of the enzyme was tested by the hydrolysis of the wheat gluten. the resulting gluten hydrolysate were characterized by means of their antioxidant, antibacterial, trypsin inhibition and prolyl oligopeptidase inhibition activities. keywords: serine protease, prolyl oligopeptidase, bioactive peptides, 2,4,6-trinitrobenzene sulfonic acid. proteomic analysis is probably the best approach to analyze seed germination. however, it is difficult to analyze complex samples and there are many obstacles that must be faced in order to achieve a reasonable proteome coverage. for example, the barley (hordeum vulgare) genome was fully sequenced in 2012, but the uniprot database contains less than 1000 reviewed sequences, which is approximately 16-fold less than for arabidopsis thaliana. here, to improve the barley proteome coverage, we employed several fractionation methods including polyethylene glycol precipitation, strong cation exchange chromatography, off-gel separation, sds-page and acetonitrile elution gradient. proteomic analyses were performed using an lc-ms-based analyses and an uhr q-tof mass spectrometer. the candidate peptides were targeted via selected reaction monitoring (srm) and triplestage quadrupole (tsq) mass spectrometer. in total, 4092 proteins were identified, which represents a three-to four-fold increase compared with the standard shotgun analysis of the same sample. out of these, 2240 were only accessible by one of the techniques and, besides, the detection limits were not similar. we hypothesized that an srm-based targeted analysis will allow detection and quantitation of most of these proteins, even without the application of proteome fractionation. we can conclude that all peptides from the library with ms/ms spectra of the total intensity above 10,000 are easily detectable in the total protein extracts. p-02.08.5-012 transcriptome sequencing based identification of alternative oxidase genes in white waterlily, nymphaea alba alternative oxidases (aoxs) are the terminal oxidases in the respiratory electron transport chain of plants. they reduce molecular oxygen to water with low proton translocation across the inner mitochondrial membrane. in plants, aoxs increase local tissue temperature to release volatile compounds thereby attracting pollinator insects and regulation of mitochondrial retrograde signaling pathway. regulation of retrograde signaling pathway is currently under investigation to improve cultivation studies in many plants. water lilies are aquatic ornamental and economically valuable plants classified under nymphaea family. nymphaea alba, white water-lily, has a special focus since its applications in landscaping of parks and gardens, farming as vegetable and medical applications. however, cultivation of n. alba is a challenging process. we hypothesized that by controlling alternative oxidases, success rate can be increased for n. alba cultivation. to identify alternative oxidase encoding genes in n. alba, we performed transcriptome analysis. by using transcriptome analysis data, aox gene sequences, subcellular localization of aox proteins and structural modelling of aox proteins were predicted. in 272934 transcripts, database search with trinotate tool revealed 77 transcripts with aox domains characterized in known alternative oxidases. blast analysis of these 77 sequences with known aox proteins revealed three distinct aox genes (nalba-aox1, nalba-aox2 and nalba-aox4). after subcellular localization analysis of three identified aox proteins by using targetp server tool, nalba-aox1, nalba-aox2 are predicted as mitochondrial while nalba-aox4 is localized in chloroplasts. template based structural modelling results showed that all identifed proteins are statistically similar to known structure models of corresponding aoxs. most environmental contaminants have toxic and mutagenic effects on living organisms as a result of the activation of free radical formation and inhibition of reparation activity. it is becoming relevant to search for protectors of natural origin from the effects of xenobiotics. many biologically active substances (bas) of inartificial origin are found to be antioxidants and can increase the body's resistance to the toxic and mutagenic effects of a wide range of pollutants. the aim of the study was to investigate the antioxidant and antimutagenic properties of bas from medicinal plants limonium gmelinii (plumbaginaceae) and inula britannica (compositae). the antioxidant potential of plant extracts was determined by the activity of superoxide dismutase (sod), catalase, and the content of malonic dialdehyde. mutagenic and anti-mutagenic properties of the extracts were determined in the test by counting chromosomal aberrations in root meristem of barley seeds. barley seeds were treated with an aqueous solution of unsymmetrical dimethyl hydrazine (udmh), which is highly toxic i class hazardous material, well known pro-oxidant. the results showed that udmh enhanced the process of lipid peroxidation and decreased the mitotic activity. treatment of barley seeds with extracts from i. britannica and l. gmelinii and their germination in the presence of stress factors stimulated antioxidant defenses in the primary roots of barley seeds. increase of the activity of sod and catalase, and reduction of peroxidation level of lipids were observed. cytogenetic study showed no mutagenic activity in plant extracts. when effects of plant extracts and udmh were combined there was a significant reduction in the frequency of structural mutations, induced by the toxicant. conclusion about the presence of the antioxidant and antimutagenic activity in the studied plant extracts is made. the work done within the framework of the mes project (no. gr 0115rk00378). p-02.08.5-014 comparative analysis of cytokinin dehydrogenase inhibition and trans-zeatin treatment in arabidopsis seedlings j. nov ak 1 , v. koukalov a 1 , z. medvedov a 1 , c. martin 1 , j. hradilov a 1 , l. sp ıchal 2 , b. brzobohat y 1 1 mendel university in brno, brno, 2 palack y university in olomouc and centre of the region han a, olomouc, czech republic cytokinins are plant hormones regulating many processes during plant life ranging from germination to senescence. manipulation of cytokinin levels and their impact on plant vitality, production and ability to defend against stresses is in great interest of agriculture. in this work we focused on comparison of inhibitor of the cytokinin degradation incyde (2-chloro-6-(3-methoxyphenyl)aminopurine) and exogenous application of trans-zeatin on arabidopsis thaliana seedlings. transcripts of genes regulating cytokinin metabolism were analysed by rt-qpcr analysis. classical cytokinin root essay revealed that incyde effect is comparable to that of trans-zeatin in a similar concentration-dependent manner. besides a negative effect on the primary root length, both substances induce flavonoid accumulation and an increase in the root hairs formation. histochemical staining of transgenic plants expressing glucuronidase (gus) under cytokinin-responsive promoter of arr5 gene revealed increased gus activity in cotyledons following incyde treatment suggesting diverse localization of cytokinin modulation upon trans-zeatin and incyde treatment, respectively. possible molecular differences originating in different cytokinin population and distribution following trans-zeatin or incyde treatments were monitored on the level of gene expression and via an lc-ms proteome analysis in roots and shoots of 14-day-old plantlets. rt-qpcr analysis revealed an alteration in cytokinin metabolism that could explain observed differences on the proteome level between incyde and trans-zeatin treated seedlings. pharmacologically inhibited cytokinin degradation could be very efficient tool for modulation of cytokinin levels. interestingly, the application of incyde and trans-zeatin shows a contrasting spatial and temporal pattern on molecular levels. incyde represents potent growth regulator with interesting properties useful for agriculture. p-02.08.5-015 the expression yield of prokaryotic alphaamylase is significantly magnified by molecular cloning techniques randomly hydrolyzing glycosidic bond alpha-amylase has been traditionally employed in bread and similar industries. in that regard, increasing the overall expression level of the enzyme is a crucial concern in biotechnology. to reach the goal, appropriate alpha-amylase producing species and expression vector were carefully selected. therefore, genome of bacillus subtilis was extracted and amplified by polymerase chain reaction (pcr) using specifically designed primers. subsequently, the extracted gene was inserted in expression vector pht43 and transferred to e. coli as intermediate host followed by bacillus subtilis host replacement. the recombinant vector was expressed in bacillus subtilis and the expression was evaluated by agarose gel electrophoresis. relative purification of the recombinant enzyme was performed by 50 kda filtration to remove impurities. to identify the biochemical characteristics, starch was used as specific substrate to measure enzyme activity and the enzyme was exposed to various ph and temperatures. the extra-cellular expression of alpha-amylase enzyme was successfully elevated by 5 folds in comparison to the native enzyme. the optimum temperature and ph for the enzyme was carefully determined as 70°c and 6, respectively. the enzyme was stable at 50°c, but thermal stability was dramatically decreased at higher temperatures up to 70°c. kinetic parameters were also measured; vmax was 1.998 u/ml min and km was 3.998 mg/ml. it is concluded that the elevated expression extent of recombinant alpha-amylase together with appropriate qualifications could make the clone a good choice for various industrial applications. flax seedlings of cultivars tmp1919, lira and lines g-1071/ 4_o, g-1071/4_k were treated for 4 and 24 hours with 500 lm alcl 3 solution or distilled water (control). twelve small rna libraries were constructed and sequenced using illumina gaiix. to identify known mirnas, obtained sequences were aligned with mirnas from mirbase (http://www.mirbase.org/). fold change value was calculated to identify up-and down-regulated mirnas under al stress. in total, about 40 million raw reads were obtained and 109 conserved mirnas from 26 families were identified. significant expression alterations in flax plants under al treatment were shown for mir319 and mir390. expression level of mir319 was varied in similar way in resistant and susceptible to al genotypes: mir319 was up-regulated after 4 hours of alcl 3 exposure and down-regulated after 24 hours. mir390 expression was increased after 4 hours of alcl 3 exposure and decreased after 24 hours in susceptible to al flax genotypes (lira, g-1071/4_o), while in resistant genotypes (tmp1919, g-1071/4_k) mir390 level was decreased after both 4 and 24 hours of al treatment. in other plant species, mir319 and mir390 were identified as al-responsive. mir319 targets mrna of tcp (teosinte branched/cycloidea/pcf) transcription factors, which control plant growth. mir390 targets mrna of tas3 protein, which regulates lateral root growth via degradation of arfs (auxin response factors). in flax, the involvement of mir319 and mir390 in response to al stress was shown for the first time. moreover, we revealed diverse expression alterations of mir390 in susceptible and resistant to al genotypes. this work was financially supported by grant 16-16-00114 from the russian science foundation. p-02.08.5-017 association genetics of phenylalanine ammonia lyase (pal) and cinnamyl alcohol dehydrogenase (cad) enzymes involved in lignin biosynthesis of european black poplar (populus nigra) b. taskiran, z. kaya middle east technical university, ankara, turkey populus nigra l. are considered as one of the most economically significant forest trees with respect to production of wood, biomass, and other wood-based products. while wood quality and biomass are directly associated with high cellulose content, lignin emerges as an undesirable polymer for both pulp and biofuel manufacturing industries. the aim of the study is by choosing the superior and eliminating the inferior clones to make a contribution to woody feedstock development and to improve wood quality of populus nigra. to estimate association genetics of pal and cad enzymes which have important functions in lignin biosynthesis, the important germplasms of populus nigra has been sampled from 3 year old poplar trees (285 clones x 2 replicates x 2 ramets) which were grown in behic ßbey plantation clone bank in ankara. additionally, five commercially registered clones and six foreign clones were included to the study to make comparison. the average mean values of cellulose, lignin and glucose content were calculated as 21.8 ae 16.29 lg/ml, 23 ae 4.64 lg/ml, and 35 ae 9.71 lg/ml, respectively. even though for pal and cad enzymes, data gathering process have been still resuming, particular clones have been separated from all in terms of pal and cad activities as expected. key words: populus, poplar, lignin, pal, cad, genetic variation, feedstock p-02.08.5-018 proteomic analysis of the molecular mechanisms of the response of plant seeds to pre-sowing treatment by stressors seed treatment with non-ionizing low-level radiation (nr), such as cold plasma (cp) or electromagnetic field (ef), is a modern eco-agricultural technology for stimulation of plant germination and performance. the molecular determinants of seed response to these treatments are not established and no genomic studies of plant seed response to nr have been reported. we studied the effects of pre-sowing seed treatment, using vacuum (7 min), radio-frequency ef (5-15 min) and cp (2-7 min), on germination and growth of non-oilseed helianthus annuus. to gain an insight into the molecular mechanisms underlying effect of nr on sunflower seed germination and dormancy, we estimated changes induced in the balance of plant hormones and differential protein expression. the results of the germination tests and estimation of seedling morphology showed that response develops in time and is stronger when sowing is performed in 7 days in comparison to 3 days after seed treatment. the 2d dige analysis revealed 38 differentially expressed proteoforms in kernels of seeds treated with cp or ef. proteins involved in biological processes of seed maturation, response to stress, response to abscisic acid stimulus, processes of organonitrogen compound metabolism and glucose catabolism were identified. while expression patterns for majority of the proteins were highly specific to cp and ef treated seed kernels, accumulation of several proteoforms of seed storage proteins (ssp), including vincilin-like, miraculin-like protein and albumin-8 were common for both experimental groups. this suggested that response to nr treatment could be at least partially associated to function of ssps in response to oxidative stress that protects proteins required for seed germination and seedling formation. variation of abundance of distinct proteoforms of helianthinin, vicilin-like and 11s globulin-like ssps suggested that post-translational modifications are involved in regulation of the function of ssps. p-02.08.5-019 suppression of lipopolysaccharide-induced inflammatory responses in raw 264.7 macrophages by tuber extract of cyclamen l. turkey is a prominent centre of plant diversity, being the meeting point of three main floristic zones. geophytes which have underground storage organs such as, tubers, bulbs and rhizomes. cyclamen l. is a tuberous geophyte traditionally used by some people for treating whooping cough, headaches or sinusitis, and confirmed to have antioxidant, analgesic and anti-inflammatory properties by several reports. a prolonged inflammatory response is often associated with chronic diseases such as cancer, arthritis and autoimmune disorders. recently, plant based products are used as an alternative and complementary treatment of these diseases. in this respect, the present study was aimed to determine the effects of three cyclamen tuber extracts on lps-induced inflammatory responses of murine raw 264.7 macrophages. firstly, c. cilicium (endemic), c. pseudibericum (endemic) and c. graecum subsp. anatolicum were collected from different localities of turkey. the tubers of plants were air-dried and grounded to fine powder and then extracted with ethanol. cell viability assay was performed to evaluate the nontoxic concentration in cell line by mtt assay. several measurements were performed including tnf-a, no and il-8 concentration assay by elisa after treatment compared to non treated cells to determine the anti-inflammatory activity. also, tnf-a and inos mrna levels were evaluated by quantitative rt-pcr. the cytotoxic activity which is considered safe on raw.264.7 cell were found as 0.5-5 lg/ml. studied cyclamen taxa inhibited tnf-a and il-8 release on lps stimulated-raw.264.7 in a concentration-dependent manner. among the three cyclamen tuber extracts evaluated, the highest nitrite-associated no inhibitory activity was obtained from c. pseudibericum compared to other two cyclamen l. taxa. collectively, these results suggest that cyclamen tuber extracts possess anti-inflammatory properties. p-02.08.5-020 in vitro hypoglycemic activity of ziziphus jujuba recent reports have indicated that continuous treatment with nutritional jujuba (ziziphus jujuba miller) fruit extracts in diabetic rats improved glucose utilization and produced a significant decrease in the blood glucose. in the present study, hypoglycemic activity of z. jujuba was investigated using various in vitro techniques. the hypoglycemic effect of z. jujuba in phosphate buffered saline which grown in balıkesir was studied by measuring glucose adsorption, glucose diffusion and glucose uptake by yeast cells. the glucose content in the solution measured by spectrophotometrically with commercially kits. the adsorption capacity of the z. jujuba was found to be directly proportional to the molar concentration of glucose. the glucose binding capacity of extract increased in higher glucose concentratrations. there was significant differences were observed between the adsorption capacities of z. jujuba and control samples (p < 0.05). the rate of glucose diffusion was directly proportional to the time. diffusion rate was significantly lower in the solution containing z. jujuba compared to control (p < 0.05). the extract demonstrated significant inhibitory effects on movement of glucose into external solution across dialysis membrane compared to control. the rate of glucose transport across cell membrane in yeast cells was observed to be inversely proportional to the molar glucose concentration. z. jujuba inhibited glucose transport across the yeast cells. the results showed that z. jujuba reduced glucose levels at least by three mechanisms. first by increasing glucose adsorption capacity during postprandial hyperglycemia; second by retarding glucose diffusion rate and third, at the cellular level by inhibiting glucose transport across the cell membrane. all of these decreased the absorption of glucose in the intestinal cells and the concentration of postprandial serum glucose. p-02.08.5-021 cucurbitacin b increased the anticancer effect of imatinib mesylate through inhibiton of matrix metalloproteinase-2 expression in colorectal cancer cells f. bakar ankara university, ankara, turkey several natural products have been investigated for their anticancer effects. among these, cucurbitacin b (cub) has been reported as its inhibitory effects on cancer cell proliferation. matrix metalloproteinases (mmps) belong to endopeptidase family and they are received as potential biomarkers for several types of cancer. the aim of this study is to investigate the effect of cub in combination with imatinib mesylate (im) on mmp-2 mrna expression of human sw480 colorectal carcinoma cells. the cytotoxicity analysis was performed via mtt assay. muse cytofluorimetric analysis system was performed to evaluate apoptotic cell population. the mmp-2 mrna expression was determined by quantitative real-time pcr. this study was supported by scientific and technological research council of turkey grant, sbag-114s871. data obtained from the cell culture experiments were expressed as mean ae sd and one-way anova test was applied for multiple comparisons. cub alone significantly inhibited cell growth at 10 lm and higher concentrations. the most potent effect was observed in cub-im combination treatment with 3.51 lm ic 50 value. in cub-im treated group, the apoptotic effect was higher than cub and im treated groups. cub-im induced apoptosis significantly at 10 lm concentration when compared to control and 1 lm (p < 0.05). cub alone showed inhibitory effects on mmp-2 mrna expression at 1 lm and higher doses significantly (p < 0.05). the results showed that the combination treatment of cub with imatinib synergistically inhibited human sw480 cell growth and induced apoptosis by increasing the anti-histone antibodybound nucleosom levels and annexin v binding. although cub could inhibit mmp-2 expression alone at higher treatment doses, it enhanced the inhibitory effect of im on mmp-2 synergistically in a dose dependent manner. in conclusion, this study suggests that cub combined with imatinib mesylate may enhance the effects of chemotherapy in patients with colorectal cancer. plants are most important parts of natural resources that alternatively referred to synthetic drugs for reasons such as being less side effects and lower costs. ziziphus jujuba miller (z. jujuba), a plant used in traditional medicine, is one of the most important ziziphus species belonging to rhamnacea family. the fruit and seeds of this plant are used different purposes such as antiinflammatory, antioxidant, immune-stimulant and wound healer. in this study, we investigated the antibacterial effects of z. jujuba. the aims of this study were to screen the antibacterial activity of z. jujuba. the extract was obtained from z. jujuba fruits pulverized with the aid of ball mill using 50% aqueous-ethanol solution. extracts were screened for antimicrobial activity against six different standard strains of bacteria by determining minimum inhibitory concentration (mic) according to clsi criteria. serial dilutions are made between 64 mg/ml and 0.031 mg/ml concentration range. the lowest concentration of wells that no visible growth has been accepted as mic value. materials in the mic and lower concentrated wells were transferred to 5% sheep blood agar petri dishes for calculation of minimal bactericidal concentration (mbc). the lowest concentration that no colony formation has been accepted as mbc value. jujuba showed the most potent effect on strain of s. aureus atcc 29213 is gram-positive cocci (mic: 2 mg/ml). the mic values of other gram-positive bacteria s. aureus atcc 43300, e. faecalis atcc 51219, l. monocytogenes f 1483 and m. smegmatis cmm 2067 were detected as 8, 16, 8 and 8 mg/ml respectively. mic values of gram-negative bacilli were detected as >64 mg/ml. consequently, z. jujuba was found to be effective on grampositive cocci bacteria (s. aureus atcc 29213, s. aureus atcc 43300 and e. faecalis atcc 51219). the strongest effect was observed on s. aureus atcc 29213 strain. in contrast, extract showed less effect on gram-negative bacilli. p-02.08.5-023 selective cytotoxic effect of morus rubra extract in human lung cancer cells through enhancing apoptosis and cell cycle arrest cancer is a disease that develops as a result of unlimited proliferation of abnormal cells that occurs due to loss of control over the mechanisms of normal growth and differentiation of cells. morus rubra, known as "red mulberry" belongs to family of moraceae. for many years, the fruits of morus species have been used to treat many diseases in traditional medicine. biological effects of morus species is predominantly attributed to its content of polyphenolic compounds. many studies have evaluated the cytotoxic effects of different morus species, but there is no study about cytotoxic effect of m. rubra. in this study, we aimed to evaluate the cytotoxic effect of m. rubra extract in human lung cancer cells (a549) with regard to apoptosis, cell cycle and mitochondrial membrane potential. cytotoxic effect of m. rubra extract on human lung cancer cells was determined using mtt assay. then, mechanisms of cytotoxic activity of m. rubra extract on a549 cells were examined in terms of cell cycle, apoptosis and mitochondrial membrane potential using flow cytometric methods. m. rubra extract exhibited selective toxicity against a549 cells compared to normal foreskin fibroblast cells. we determined that m. rubra extract increased cell cycle arrest at g 1 phase, the level of apoptotic cells and decreased mitochondrial membrane potential in a549 cells. our results showed that m. rubra extract has pro-apoptotic and antiproliferative effect in a549 cells. further studies are also needed to fully mechanisms underlying this effect of m. rubra extract. p-02.08.5-024 dipeptidyl peptidase iv inhibitory activity of arctium tomentosum l. a. zeytunluoglu 1 , f. zihnioglu 2 1 denizli vocational school of technical sciences, pamukkale university, denizli, 2 department of biochemistry, faculty of science, aegean university, izmir, turkey type 2 diabetes mellitus (t2dm) is rapidly growing metabolic syndrome of multiple aetiologies causing hyperglycaemia with insulin resistance at cellular level. a novel approach in the treatment of t2dm is based on preventing of rapid inactivation of the incretin hormone glucagon-like peptide-1 (glp-1) and glucose-dependent insulinotropic polypeptide (gip) by dipeptidyl peptidase-iv enzyme. in this study; dipeptidyl peptidase iv (dpp-iv; ec 3.4.14.5) inhibitory activity of the aqueous and methanolic extracts of arctium tomentosum leaves were successfully tested in vitro conditions. our study revealed that both aqueous and methanolic extracts obtained from test material had a significant dppiv enzyme inhibitory activity in changing ratio. the ic 50 values were also determined by nonlinear regression curve fit using graph pad prism 5.0 with appropriately diluted of lyophilized arctium tomentosum. diprotein-a (ile-pro-ile) was used as reference inhibitor. a. tomentosum aqueous extracts showed ic 50 4.6 lg/ml while the standard (positive control) diprotin a displayed the ic 50 value of 10.1 lg/ml. this study demonstrates that a. tomentosum aqueous extracts could be a good lead for further development as a new antidiabetic agent. p-02.08.5-026 dna recognition determinants of arabidopsis thaliana b3 transcription factors g. sasnauskas, k. kauneckaite, k. lapenas, v. siksnys institute of biotechnology, vilnius university, vilnius, lithuania transcription, one of the most important cellular processes, is regulated by transcription factors (tf), proteins that often directly interact with gene promoter sequences. tf binding to dna is mediated by various dna binding domains. the b3 tfs constitute a large, plant-specific protein family (approx. 10% of all tf proteins in the flowering plants), which is characterized by the presence of one or several small (approx. 110 amino acids) b3 dna binding domains. currently the b3 tfs are divided into four groups (lec2-abi3/val, arf, rav and rem). the preferred recognition sites were identified for representatives of all groups except the rem family. currently, only a single structure of a dna-bound b3 domain (arf1, arf family) is available, thus the mechanism of site-specific dna recognition by the lec2-abi3/val and rav b3 domains remains poorly understood. based on the arf1-dna structure (pdb 4ldx) we have built homology models of dna-bound b3 domains from a. thaliana abi3 (lec2-abi3/val family) and nga1 (rav family) transcription factors, mutated putative dna-interacting amino acid residues and characterized the dna binding ability of the purified mutants using electrophoretic mobility shift assay and a set of radiolabelled dna substrates carrying various variants of the optimal recognition site. we confirm the importance of several positively charged amino acid residues, which are conserved between the abi3/ nga1 b3 domains and structurally related dna-binding domains of bacterial restriction endonucleases ecorii, bfii and ngoavii; furthermore, we identify residues in the 'n-arm' and 'c-arm' loops that may be involved in specific interactions with the dna bases. our results therefore help us refine the homology models of the dna-bound b3 domains and in the future may help us predict the dna binding properties of currently uncharacterized b3 domains. p-02.08.5-027 immunohistochemical analysis of inhibitory effects of origanum hypericifolium oil on dipeptidyl peptidase iv in streptozotocininduced diabetic rats p. ili 1 , a. zeytunluoglu 2 1 denizli health services vocational high school, pamukkale university, denizli, 2 denizli vocational school of technical sciences, pamukkale university, denizli, turkey diabetes mellitus (dm) is a serious metabolic disorder with micro-and macro-vascular complications that result in a significant morbidity and mortality. glp-1 and gip have significant role in pancreatic beta cells and prevention of inactivation of them by dipeptidyl peptidase iv (dpp iv) inhibition is a novel approach to treatment of dm. origanum hypericifolium (lamiaceae) is an endemic turkish plant and its essential oil is mainly composed of monoterpenes including carvacrol and thymol. streptozotocin (stz) is used to induce diabetes in rats. the aim of this study is to investigate the inhibitory effects of o. hypericifolium essential oil on the dpp iv in stz-induced diabetic rats. the animals (female spraque-dawley rats) were assigned to four groups (group 1: control, group 2: stzinduced diabetic, group 3: o. hypericifolium injected, group 4: stz-induced diabetic and o. hypericifolium injected). dm was experimentally induced in groups 2 and 4 by a single intraperitoneal injection of stz at a dose of 45 mg/kg body weight. in groups 3 and 4, rats were intraperitoneally injected with o. hypericifolium oil at a daily dose of 1 ml/kg body weight for 42 consecutive days. at the end of the experimental period, all animals were sacrificed by cervical dislocation under ether anesthesia and liver and kidney tissues of each animal were rapidly excised. tissues were fixed in sainte-marie fixative. after routine histological processes, samples were embedded in paraffin, immunohistochemical staining for dpp iv was performed on sections and then they were photographed. the immunohistochemical reaction intensity differences were observed between the groups. in conclusion, the immunohistochemical distribution of dpp iv in the tissues that the test oil was applied in the diabetic rats may be important for the investigation of the inhibitory effects of oil on the enzyme. moreover, our findings suggest that o. hypericifolium oil may be used for prevention of diabetic diseases. introduction: all eukaryotic cells need microtubules for purposes of nuclear and cell division, organization of intracellular structure, and intracellular transportation, as well as ciliary and flagellar motility. microtubules are made of polymerized a/btubulin subunits. mec17 is important for microtubules, because it encodes the enzyme that adds acetyl groups to lysine 40 (k40) of tubulin. k40 is largely conserved in a-tubulins of many eukaryotes, and acetylation is thought to stabilize microtubule structure. in algae, the effect of acetylation by mec17 on flagellar motility and phototaxis has not been tested previously. materials and methods: in this study, mec17 mutant chlamydomonas reinhardtii cells were compared to wild-type cells to see the effect on flagellar motility and phototaxis. we tested phototaxis, eyespot size and quantity under the microscopy. in addition to this, we fixed cells and examined them by immunofluorescence microscopy using antibodies to tubulin, acetylated tubulin, and photoreceptor. results: we observed that some mec17 mutant cells contain more than one eyespot. we detected no acetylated-tubulin (ac-tub) by immunofluorescence. the cells still phototax and have normal motility discussion and conclusion: interestingly, mec17 cells still have the ability to phototax and they have normal flagellar motility, even though they contain occasional additional eyespots and no ac-tub. chlorella vulgaris as a model system for screening of plant growth modulators p. volynchuk 1 , e. marusich 1 , r. chuprov-netochin 1 , j. neskorodov 2 , y. mishutkina 2 , s. leonov 1 1 life sciences center, moscow institute of physics and technology, dolgoprudny, 2 center "bioengineering", russian academy of sciences, moscow, russia the discovery of new plant growth modulators became extremely important task as an alternative approach to overcome plants resistance to herbicides and pesticides, which leads to harmful action on plants and land rising, environmental and ecological problems. small molecules provide agricultural biotechnology with valuable tools, which help to circumvent the need for genetic engineering and offer unique benefits to modulate plant growth and development. we developed a system to explore molecular modes of action of plant growth modulators using chlorella vulgaris model. our model allows applying high content screening approach in 96well plate format for fast and robust effect assessment of large number of tested modulators. chlorella v. was grown in climate chamber under optimized constant temperature (20°c) and light conditions (16:8 hours/light:dark). modulating effect of tested compounds was estimated by spectrophotometric measurement of microalgae density at the beginning of the experiment (start point-0.1od) and 48 hours later. to validate our system we used known cytokinins and auxins (10 mm) as positive controls of growth stimulation. we showed that in presence of each compound the density of chlorella v. was increased in 7-11 times range, compared with only 2 times increase in control group. eight new chemicals (10 lm), which demonstrated modulation effect on nicotianatabacum l. pollen and arabidopsis thaliana models, were tested on developed chlorella v. model. positive controls showed no stimulating effect at this concentration, while tested compounds were confirmed as hits and increased the density up to 400%. we demonstrated that developed model system, based on chlorella v., is an effective system for primary screening of plant growth modulators. the main advantages of this system are short time of assay, simplicity of performance, possibility of automation and low cost. selected hits can be recommended as perspective candidates for future test on crop field. sunflower is under a big threat of downy mildew which is a fungal disease caused by plasmopara halstedii. the disease can cause up to an 80% yield loss in sunflower production. downy mildew resistance genes (r) denoted as pl has been discovered to date in sunflower. in recent years, single nucleotide polymorphism (snp) markers have become widely used in plant breeding programs. in this study snp markers have been currently developing for pl 6 , pl 8 , pl 13 , pl arg genes by competitive allele specific pcr (kasp) assay which enables bi-allelic scoring of snps and insertions/deletions (indels) at specific loci. in total 66 sequence tagged site (sts) sequences from ncbi were aligned for pl 6 , pl 8 , pl 13 , pl arg genes to identify conserved regions for each gene. based on the conserved regions, specific pcr primers were designed in order to make sequencing of these genes in five crosses and their f 2 progenies. sequence data will be used to design an allele specific primer maches the target snp and amplifies the target region with the common reverse primer provided by kasp genotyping assay. snp markers linked to pl genes which are being developed in this study, have the potential to be used in marker assisted selection (mas) for sunflower breeding programs. p-02.08.5-031 investigation of the antidiabetic effects of hibiscus sabdariffa, teucrium polium and myrtus communis in hepg2 cells line s. altundag 1,2 1 pamukkale university, denizli, 2 istanbul medeniyet university, istanbul, turkey some antidiabetic plants currently are used in alternative treatment of type ii diabetes. hibiscus sabdariffa, myrtus communis and teucirum polium plants are also known for their antidiabetic properties. hibiscus sabdariffa, myrtus communis and teucrium polium; depending on the impact on hepg -2 cells to investigate the possible mechanisms of type ii diabetes with researches on glucolysis and glucogenesis pathways gene expressions (pk m , glut-2,pepck). plants were obtained in dried state from reliable herbalists in denizli and mersin. plants treated with the extractor device. plants obtained aqueous extract was subjected to lyophilization process. human cancer cells have been used throughout the study. the cytotoxicity of the cells was measured by elisa plate reader . total rna was isolated using trızol ò solution was carried out according to the instructions of the manufacturer's (thermo scientific) recommended procedures were performed, but we have to optimize our own laboratory conditions. pk m , glut-2,pepck genes were synthesized by b _ ioneer. during our study the activation of certain genes (pk m , glut-2,pepck) were examined by real time pcr. in our study hibiscus sabdariffa, mrytus communis and teucrium polium plant to extract applied hepg -2 cell line in the gluconeogenesis and glycolysis pathways in involved in some important genes (pepck, pk m , glut-2) analyzed the expression levels . teucrum polium plant extract is applied in hepg -2 cells the glycolysis pathway genes (pk m glut-2) an increased expression also genes of gluconeogenesis pathway (pepck) were not decreased. however hibiscus sabdariffa and the expression of genes involved in glycolysis and gluconeogenesis pathway mrytus communis plants were observed to have a full effect as diabetic or hypoglycemic . in this context, it is considered that the plant teucrum polium on the line hepg -2 cells showed antidiabetic effect. p-02.08.5-032 purification of b-glucosidase from malatya apricot (prunus armeniaca l.) seeds and some of its biochemical properties h. kara 1 , s. sinan 2 , z. ekmekci 2 , y. turan 2 1 university of balikesir faculty of veterinary, balikesir, 2 university of balikesir faculty of arts and sciences, balikesir, turkey introduction: b-glucosidases are one of the key enzymes in carbohydrate metabolism and located in glycosyl hydrolases (ec 3.2.1) family. plant b-glucosidases have biotechnological significance as they are effective on glycosidic bonds of flavor and aroma precursors in plants. b-glucosidases that located in fruit seeds are important because they affect the amygdalin. aim of this study is purification and partially characterization of b-glucosidase from malatya apricot seeds. materials and methods: apricot seeds were homogenized with extraction buffer to prepare of crude extract. the enzyme protein was precipitated with 60% ammonium sulfate then purified by hydrophobic interaction chromatography using sepharose 4b-ltyrosine-1-naptylamine gel. para-nitrophenyl b-d-glucopyranoside (p-npglc) was used as substrate to determine biochemical properties of the enzyme. the optimum ph was determined using buffers between ph 2-12 and thermal optima was determined using 25-85°c temperature range. inhibitory effects was determined with 1 mm substances. results: the enzyme was 11.1-fold purified with yield of 14%. purified b-glucosidase from apricot seed was visualized about 60 kda molecular weight on sds-page. the kinetic parameters were determined against p-npglc substrate as km and vmax values of 2.5 mm and 58.1 eu, respectively. the optimum ph and temperature were determined 5.0 and 55°c respectively. effects of cacl 2 , kcl, nacl, mgcl 2 , k 2 so 4 , na 2 so 4 , cuso 4 , fecl 3 , pb(ii) acetate, agno 3 , zncl 2 and glucose on purified enzyme activity were investigated. kcl, na 2 so 4, pb(ii) acetat and cuso 4 reduced the enzyme activity. discussion and conclusion: in this study, b-glucosidase was purified from malatya apricot seed and some of its biochemical properties were determined. because this enzyme has pharmaceutical importance hydrolyzing amygdalin. the results showed that immobilized almond b-glucosidase was used to break amygdalin and release -cn compound that effective to shrink cancer mass. p-02.08.5-033 a new affinity gel for purifing polyphenol oxidase enzyme a. erg€ un 1,2 , o. arslan 2 1 balikesir university, science and technology application and research center, balikesir, 2 department of medical chemistry, faculty of science, balikesir university, balikesir, turkey polyphenol oxidase (ppo) enzyme, sometimes called as phenol oxidase, catecholase, phenolase, catechol oxidase or tyrosinase, is considered to be an o-dipenol. ppo (ec 1.14.18.1), a multifunctional copper containing metalloenzyme, is widely distributed in nature. ppo exists in many kinds of plants and fungi, such as banana, mushroom, butter lettuce, napoleon grape, potato, coffee, marula fruit, artichoke heads, longan fruit, tobacco, wheat flour. in this study, a novel affinity chromatography gel was synthesized for purifing ppo enzyme. the affinity chromatography gel was synthesized by coupling aniline as a specer arm to cnbr activeted sepharose-4b. then, p-amino benzoic acid was coupled to aniline as a ligand. ppo was purified from musa sapientum var. cavendishii (banana) by using sepharose-4b-aniline p-amino benzoic acid affinity chromatography gel. % 4.49 yield and 33.4 fold purification were achieved. sds-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent mw of 35 kda. the v max and k m of the purified enzyme were determined 42,628 u/ml min and 9.27 mm, respectively. p-02.08.5-036 phenolic content and antioxidant capacity of gamma irradiated olive leaves m. e. diken 1 , b. kocat€ urk 2 , s. dogan 1 , h. tuner 1 1 balikesir university, balikesir, 2 kavram vocational school, istanbul, turkey in this study, dried in diffirent ways (such as microwave, infrared, convection heaters and under normal atmospheric conditions) olive leaves has been used as experimental material. radiation has been applied to dried olive leaves in three diffirent dosages in room temparature. the amount of radiation to be implemented to the samples have 3, 5, 10 kgy/min. in this study, how gamma rays (radiation) effects phenolic components, total phenolic content and antioxidant capacity of dried olive leaves has been determined. the phenolic components, total phenolic content and antioxidant capacity were analysed with hplc, folin ciocalteu and dpph radical scavenging method, respectivelty. gamma rays is well known as a decontamination method for many foodstuffs and plant materials, being an environment friendly and effective technology to resolve technical problems in trade and commercialization. for this reason, nowadays it is utilized as an alternative method of sterilization. gamma rays are of ionizing radiation. when ionizing radiation interacts with matter, generally it causes a break in the molecular bonds and/or breaks the bonds between the molecules. these intermediates have unpaired electron and called free radical. gamma radiation or the radiation-induced free radicals would break or cause damage to the dna molecules of living organisms. gamma irradiation is predict to change phenolic content and antioxidant capacity in living tissues. phenolic compounds are secondary plant metabolites naturally present in fruits and vegetables. in recent years there has been a growing interest in food phenolics because of their potential health benefits mainly due to their antioxidant and free radical scavenging activity. despite the controversy about potential risks posed by genetically modified plants on human health, environment and microorganisms, cultivation area of these crops increases day by day. this increment has revealed concerns especially related to hgt. hgt studies indicated that antibiotic resistance genes in gm plants have a potential to transfer to soil microorganisms. in this study, hgt of widely used genetic elements such as regulatory sequences, from transgenic plants to bacteria was investigated. three soybean feed and four seed examples from turkish feed manufacturers' association were used for genetic analysis based on foreign gene determination. gmo analysis were conducted by camv 35s promoter-specific pcr in genomic dnas. in gm positive samples, genomic dnas sheared into appropriate fragment size by ultrasonication for the purpose of bacterial transformation. presence of 35s promotor region in fragmented dnas was proved by pcr. escherichia coli dh5a strain was transformed by fragmented dna samples according to cacl 2 method. 35s promotor sequence screened by using pcr in bacterial genomic dnas. as a result of gm screening, all feed and three of the seed samples were found to be transgenic. ultrasonication conditions were optimized for shearing dna's to 450-500 bp for bacterial transformation. fragmented dnas confirmed for carrying intact 35s promotor sequence. none of bacterial genomic dnas were found to be 35s-positive. according to the transformation results, absence of 35s promotor sequence in all tested bacterial genomic dnas, proved the dna samples belonging to gm plants can not transfer into compotent e. coli dh5 under laboratory conditions. for verification of this finding, transformation studies will continue with acinetobacter baylyi bd413 strain which is naturally competent soil bacterium for natural transformation. we believe that all of our findings will contribute to constitute transformation system which can be used as model in hgt studies. p-02.08.5-038 preliminary studies on differential methylation in a and d sub-genomes of upland cotton (gossypium hirsutum l.) four species of cotton (gossypium l.) provide raw materials for the textile industry. among the four species, two have diploid genome and another two have tetraploid genome. tetraploid genome consists of a and d sub-genomes. a sub-genome belongs to asian cotton while d-sub genome belongs to american cotton. previous studies revealed that d sub-genome of gossypium species contributes to the superior yield and quality of tetraploid gossypium l. species (atdt). dna cytosine methylation of four regions of dna sequences located on a and d sub-genomes of gossypium hirsutum l. texas marker 1 (tm-1) was investigated using bisulfite sequencing technique. among the regions studied two could not be located on subgenomes due to sequence identity match between a and d subgenomes. on the other hand two dna regions could be located on a and d sub-genomes using the blast searches. some of the dna sequences located on different sub-genomes showed polymorphic nucleotides including c/t and g/a polymorphisms. in silico analysis indicated that some alleles located on different sub-genomes of cotton have c/t and g/a polymorphisms. c/t polymorphisms between the sub-genomes could be thought as unmethylated cytosine using the bisulfite sequencing technique. this indicated that an extra attention needs to be paid in dna total cytosine methylation studies in polyploid species such as cotton using bisulfite sequencing, methylation sensitive amplification polymorphism (msap), whole-genome bisulfite sequencing (wgbs). otherwise, t in c/t polymorphism between the subgenomes could be thought as unmethylated cytosine. based on the two genomic regions we could conclude that a sub-genome may be more methylated than d sub-genome. differential methylation of genes located on different sub-genomes may provide another mechanism responsible for differential gene expression of genes located on different sub-genomes. p-02.08.5-039 cleaved minisatellite locus (cml) markers for fingerprinting of cotton cultivars grown in turkey e. u. gocer, m. karaca akdeniz university, antalya, turkey after their discovery by alec jeffreys in 1984, minisatellites have been used in genetic studies of many organisms. minisatellites, also called variable number tandem repeats (vntrs), are composed of arrays of longer repeats mostly dispersed throughout heterochromatin (centromeres and telomeres). direct amplification of minisatellite regions of dna using a single core primer is a powerful method to amplify minisatellites (damd-pcr). although the damd-pcr technique has been applied to many plant species, the level of polymorphisms in cotton (gossypium l.) is very low due to very narrow turkish cotton genetic base. the objective of this study was to improve the level of polymorphisms by cleaving minisatellite loci by restriction enzyme digestion. genomic dna samples of twenty-one turkish cultivars, pima 3-79, tm-1 and ps-7 were extracted. twenty-one minisatellite primers were screened using the damd-pcr technique. monomorphic amplicons were digested using several restriction enzymes. three to five micro liters of amplified products were digested with various restriction enzymes. digested products of minisatellite loci were separated in 3% high resolution agarose gels. comparison studies of digested and undigested markers revealed that cleaved minisatellite markers showed polymorphisms in those cotton lines that could not be differentiated by microsatellite and minisatellite markers. this approach was called cleaved minisatellite locus markers (cml). the amplification reactions of minisatellites used touch-down (td) cycling conditions. the use of td offered a simple and rapid means of optimizing polymerase chain reaction (pcr), increased specificity, sensitivity, and efficiency without the need for lengthy optimizations of minisatellite primers. the cml markers were obtained at a 55°c annealing temperature, which is a temperature higher than those used in random amplified polymorphic dna (rapd) inter-simple sequence repeat (issr) markers. p-02.08.5-040 association between cytosine methylation and tissue specific expression of microsatellites a. g. ince, m. karaca akdeniz university, antalya, turkey heritable covalent modification of dna, rna or protein without altering their primary sequences is defined epigenetics. because all biological events are influenced by epigenetics, it is one of the most important fields in science. dna methylation is one of the most important epigenetic mechanisms. dna cytosine methylation is a process by which methyl groups are added to cytosine bases of dna. microsatellites, also knows simple sequence repeats are dna sequences consisting of 1-6 nucleotide repeats. there is a large body of information regarding the relationship between microsatellite instability and abnormal gene expression, and between dna methylation and altered gene expression. however, there is limited information on cytosine methylation of microsatellites. in the present study, we investigated whether there is any association between cytosine methylation and tissue specific expression of microsatellites. genomic dna samples of various tissues and developmental stages of pepper line demre sivrisi (capsicum annuum l.) and cotton line tm-1 (gossypium hirsutum l.) were extracted. cdna samples were synthesized using mrna expressed in pepper tissues. cytosine methylation levels were investigated using bisulfite sequencing methods. screening studies of microsatellites revealed that some genes containing microsatellites were differentially expressed in tissues and developmental stages of pepper. microsatellite containing genes that expressed differently among tissues had also showed different methylation levels in cg, chg and chh (where h refers to a, c or t) contexts. methylation level differences between microsatellites were also observed. as far as our knowledge, it is the first report on differential expression of genes containing methylated microsatellites. p-02.08.5-041 pcr-lg: an alternative way to assign the chromosome location of genes/markers in cotton a. aydin, m. karaca akdeniz university, antalya, turkey assignment of genes and dna markers on chromosomes is very important in life sciences, especially for plant breeding and medicine. there are several methods for the assignment of a gene or dna sequence to a specific location on a chromosome. for example, the most widely used technique is the assignment of fluorescently-labeled gene or dna sequences (markers) on chromosomes using the fluorescently-labeled gene (for instance, fish technique). another example is the construction of a genetic map (linkage map) which orders the targeted genes along the dna strand based on recombination frequency. sequencing is the most precise technique in which coding (gene-containing) and noncoding dna region of genes could be located on a chromosome molecule. aneuploid lines could also be used to locate genes in a specific chromosome, but maintenance of these lines is difficult. here we report the use of chromosome substitution lines to indirectly locate genes/markers on chromosomes. we used chromosome substitution lines (csls) that carry a chromosome pair or chromosome arms from gossypium barbadense l. while the rest of chromosomes belong to g. hirsutum l. a total of 10 chls, a homozygous cotton line tm-1 and a double haploid line pima 3-79 were used as plant materials. twenty microsatellite primer pairs were utilized in touch-down polymerase chain reactions. we developed a method, called polymerase chain reaction to locate gene (pcr-lg), to assign genes/markers on chromosome or chromosome arm. with the use of pcr-lg approach any polymorphic genes/markers between tm-1 and pima 3-79 (g. hirsutum and g. barbadense) could be assigned to a chromosome or chromosome arm. results indicated that if 26 csls were used any polymorphic markers (genes) with known primer pairs could be assigned to cotton chromosomes. although we used cotton chromosome substitution lines to validate the proposed technique, it could be applicable all the species that have chromosome substitution lines. p-02.08.5-042 pecularities of genome variability of antarctic hairgrass deschampsia antarctica desv. from the maritime antarctic deschampsia antarctica desv. (poaceae) is the only grass species native to the antarctic region, adapted to harsh environmental conditions. however, reasons for its unique success remain unexplored. stressful environmental factors can influence a plant genome and cause changes in the chromosome number and morphology and increase genetic variation. therefore, the purpose of our research was to explore alterations in the d. antarctica genome both at the chromosomal and molecular levels and to investigate species genome stability using in vitro tissue cultures. plants used for the study were grown in vitro from seeds collected in the argentine islands region during 2008-2014. chromosome number was determined in plant root apical meristems and specimens prepared from tissue cultures. rrna genes localization were determined using the fish technique. moleculargenetic analysis was performed using pcr with polymorphic issr-primers. new forms of chromosome polymorphism, associated with aneuploidy (2n = 13-27), polyploidy (2n = 13-39) and the occurrence of additional b-chromosomes (2n = 26 + 1-3b), were observed. fish analysis also confirmed that genotypes with a different chromosome numbers varied in the number of 5s rdna and 25s rdna sites. assessment of genetic variation demonstrated a low level of diversity: differences between the plants with different chromosome numbers do not exceed the level of within-population variation. cytology analysis of d. antarctica cultured tissues revealed aneuploidy (up to 60.6 %) with predominance cells with diploid and near-diploid chromosome number irrespective of plant's initial karyotype (diploid, mixoploid or polyploid). we assume that discovered intraspecies chromosomal polymorphism is a manifestation of quick genome reaction to harsh antarctic conditions. whereas the results of molecular-genetic analysis and study of cell cultures of this species suggests on the relative stability of d. antarctica genome. p-02.08.5-043 angiotensin converting enzyme inhibitory activity of morchella esculenta (l.) pers a. zeytunluoglu 1 , i. arslan 2 1 biomedical equipment technology, pamukkale university, denizli, turkey, denizli, 2 biomedical engineering, pamukkale university, denizli, denizli, turkey hypertension is a multi-aetiological, chronic pathophysiology that leads to multi-organ dysfunctions like cardiovascular diseases, strokes, and renal complications. natural extracts play an important role in traditional medicines for the treatment of hypertension and are also an essential resource for new drug discovery. mushrooms are use as therapeutics in alternative and complementary medicine as functional food because of contain a large number of biologically active components that offer health benefits and protection against many degenerative diseases. morchella esculenta is one of the most highly priced edible mushrooms worldwide. it contains a wide range of active constituents which include tocopherols, carotenoids, organic acids, polysaccarides and phenolic compounds which exhibit a wide range of medicinal and pharmacological properties including anti-microbial, anti-inflammatory, immunustimulatory, antitumor and antioxidant. in this study; the in vitro angiotensin converting enzyme-i (ace-i) inhibitory activity of m. esculenta peptides were generated by alcalase hydrolysis were studied. the 5 kda < peptides < 10 kda in the ultrafiltration fractions displayed highest ace inhibition (85.9 ae 5.09% at 140 lg/ml). the results indicate that m. esculenta derived peptides may have potential as functional food ingredients in the prevention and management of hypertension. p-02.08.5-046 modulations of antioxidant enzymes, gsts and catalase, by salvia absconditiflora in hepg2 cell line d. irtem kartal 1 , a. altay 2 1 yuzuncuyil university, van, 2 erzincan € university, erzincan, turkey oxidative stress is considered to play a important role in the pathogenesis of aging and several degenerative diseases, such as cancer. in order to cope with an excess of free radicals produced upon oxidative stress, humans have developed several mechanisms for maintain redox homeostasis. these protective mechanisms either scavenge or detoxify ros, block their production, and include enzymatic and nonenzymatic antioxidant defenses. in enzymatic defenses include glutathione s-transferases and catalase enzymes. many epidemiological studies have revealed that there is a strong correlation between consumption of polyphenol-rich foods and the prevention of certain diseases like cancer, cardiovascular diseases and aging. phenolic compounds are abundant in all plants. so, they form an integral part of the human diet. salvia species, commonly known as sage, have been used since ancient times for more than 60 different ailments ranging from aches to epilepsy. there are around 900 species of salvia, 95 of which are represented in turkey including salvia absconditiflora. in this study, s. absconditiflora collected from metu campus (ankara, turkey) is extracted with methanol and water. effects of the water and methanol extracts on the mrna expressions of antioxidant enzymes gstm1 and catalase in hepg2 cells were investigated by q-rt-pcr technique. it was also monitored the effects of the extracts on the enzyme activities of gsts and catalase by spectrophotometrically. water and methanol extracts decreased gsts mrna expression in hepg2 cells for 48 hours and 72 hours incubation and methanol extract decrease catalase mrna expression for only 72 hours incubation. on the other hand, extracts highly increased the gsts and catalase activities in both hour incubation. overall, these results indicate that s. absconditiflora and/or its components have regulatory activities on antioxidant enzymes and they may have a potential as a therapeutic agent in the treatment of cancer. p-02.08.5-047 transcriptomics and proteomics approach to drought stress mechanism in wheat b. cevher keskin tubitak marmara research center genetic engineering & biotech. institute, kocaeli, turkey birsen cevher keskin, yasemin yıldızhan, oktay kulen, bayram y€ uksel, selma onarıcı, _ ismail t€ urkan, as ßkım hediye sekmen, u gur sezerman, bu gra € ozer identification of novel stress-responsive genes and their role in drought response is an important area for the improvement of the crops. drought-related genes were investigated in leaves and roots of three wheat genotypes after different drought stress treatments by rna sequencing (rna seq) technology and de novo assembly was performed before comparative transcriptome analysis. analyzing 311 gigabases of 100 bp paired end illumina reads from a hexaploid wheat poly(a) rna library, we identified common and new differentially expressed transcripts. selected differentially expressed genes were confirmed by qrt-pcr. we also performed root proteome analysis with nano electrospray ionization source coupled to a high-performance liquid chromatography system (nanouplcàesiàqtofàms) to identify drought-related proteins. totally 191 proteins were differentially expressed in root tissues of tolerant and non-tolerant wheat genotypes. responses of antioxidative defense system to drought stress were comparatively studied in the same wheat cultivars. similarities between protein and rna levels help increase our confidence in novel biomarkers, differences may also reveal other post-transcriptional regulatory junctures. all these analyses will allow us to get a better idea about the possible role of these genes in the drought-response mechanism. the drought-related genes that are functionally characterized could be introduced into agronomically important wheat cultivars. this work offers a resource for accelerating drought-related gene discovery and improving this important crop. p-02.08.5-048 isolation and characterization of a hexose converter from olive s. altunok 1 , e. d€ undar 2 1 department of biology, university of balikesir, balikesir, 2 department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey introduction: hexose sugars are key components of glycolysis and photosynthesis. the genes regulating their conversion into one another, is therefore, of great importance for the control of carbon metabolism. in this study, we report isolation and characterization of a cdna associated with conversion of hexoses in olive. the cdna putatively named aldolase based on bioinformatic and experimental analyses. material-methods: characterization based on nucleotide and amino acids were conducted using bioinformatic tools such as nucleotide and protein blast, bioedit, primer3, finchtv, clc genomic workbench, expasy, targetp, sosui and web promoter scan. comparison of the genomic and cdna sequence of the gene and detailed bioinformatic analyses including cellular location, hydropathy analysis, amino acid-nucleotide composition and predicted 3d structure were also conducted using the bioinformatics tools mentioned above. temporal expression pattern of the putative aldolase were conducted using real-time pcr experiments. sds and western blot analyses were completed while biochemical analyses are ongoing. oleocanthal is an important secondary metabolite that has been reported to be useful against important human diseases including cancer. the aim of this study was to identify and characterize the key biochemical and genetic components of oleocanthal biosynthesis. to determine the biochemical components of the pathway, multiple olive cultivars along with their spatial and temporal points were determined. the expression levels of multiple candidate genes were also aimed via real-time pcr. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes with that of other organisms) were conducted on ncbi web page. phylogenetic tree construction, amino acid composition analysis, nucleotide composition analysis, hydropathy analysis and translations through expasy were conducted. primer3 was used to design forward and reverse primers to amplify the target genes from different olive tissues at different times. analysis of the first candiate gene with bioedit program revealed that a+t ratio was more than g+c according to the nucleotide composition analysis. according to amino acid composition analysis isoleucine, lysine and leucine were more than other amino acids while kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. abundance of hydrophobic amino acids (leucine and isolecine) along with an abundant hydrophilic amino acid (lysine) suggest the existance of hydrophobic pockets in the protein which may mean a membrane bound protein or a sitoplasmic protein with a strong hydrophobic core. the molecular weight of the protein was 56 kda with a pi of 9.21. the protein was found to have a signal peptide. according to the sosui gramn , intracellular localization was found to be in the inner membrane. analysis of other candidate genes contiunes. acknowledgements: this study was supported by tubitak with grant number 110o108. key words: olive, olea europaea l., secologanin, polymorphism, allele diversity p-02.08.5-050 antioxidant potentials of propolis and its bioactive components, and their effects on cyp2e1 gene expression in ht-29 adenocarcinoma cell line a. altay 1 , d. irtem kartal 2 1 erzincan € university, erzincan, 2 y€ uz€ unc€ u yil university, van, turkey propolis is a resinous mixture that is collected by honeybees from plants, and is combined with beeswax and secretions from the bee's salivary glands plus some pollen. it is a rich mixture of polyphenols, flavonoid aglycones, phenolic acids and their esters. it has been used in traditional medicine for thousands of years because of these ingredients. propolis, just like honey, has been the subject of many studies due to its antimicrobial, antifungal, antiviral and hepatoprotective activities. the cytochrome p450 enzymes are involved in phase i xenobiotic and drug metabolism. cyp2e1 is present in high levels in human tumors demonstrated by qrt-pcr and immunohistochemical studies. in this study, propolis collected from datc ßa (mugla, turkey) is extracted with 70% ethanol. phenolic contents of the propolis were measured by lc-ms/ms. cytotoxic effects of the propolis on ht-29 human colon adenocarcinoma cell lines were examined via xtt colorimetric cell proliferation assay. effects of propolis extract and its main bioactive component caffeic acid on the expression of phase i detoxification enzyme cyp2e1 in ht-29 cells were investigated bu using q-rt-pcr technique. ic 50 values for dpph radicals scavenging activities of the extract was calculated as 0.042 mg/ml. tpc and tfc were determined as 168.6 mg gae/g extract and 141.8 mg qe/g extract, respectively. caffeic acid content of the extract was measured as 10.6 lg/g extract. ic 50 values for xtt assay in 48 hours incubation with the extract and caffeic acid were evaluated as 1.16 mg/ ml and 0.149 mg/ml, respectively. propolis extract and its main phenolic content caffeic acid significantly dicreased cyp2e1 mrna expression with 2.4 and 5.3 fold, respectively in ht-29 human colorectal adenocarcinoma cells for 48 hours incubation. overall, these results indicate that propolis and/or its components have regulatory activities on cyp2e1 expression and they may have a potential as a therapeutic agent in the treatment of cancer. p-02.08.5-051 effects of histone h3 lysine 9 inhibition on gene expression profile in tobacco (nicotiana tabacum l.) differentiation is the characteristic of multicellular organism. cellular dedifferentiation underlies topical issues in biology such as reprogramming in stem cell research, regeneration and nuclear cloning, and has common features in plants and animals. the state of dedifferentiation is evidenced by changes in cell morphology, genome organization and the pattern of gene expression as well as by the capability of plant tissues to differentiate into multiple types of cells depending on the type of stimulus applied. chromatin reorganization appears to be a fundamental theme in cellular dedifferentiation and reentry into the cell cycle both in plants and animals. the chemical modifications of histone proteins which are structural units of the nucleosome, generate 'codes' for the recruitment of proteins or protein complexes that affect chromatin structure and gene expression according to 'histone code' hypothesis. methylation of histone h3 at lysine 9 residue by specific methyltransferases suv39h1 in humans and kyp/suvh4 in arabidopsis generates a'code' for the recruitment of hp1 proteins. in this study, to enhance the dedifferentiation efficiency, chaetocin which inhibits suv39h1 has been used at the germination stage of tobacco seeds in in vitro conditions. our results showed that chaetocin induced callus formation from leaf discs of tobacco in the early stage of the inhibitor application. chaetocin can enhance reprogramming of plant cells in seed development treatment as callus induction. it is known that the formation of callus which is the non-differentiated cell community in plants, is a consequence of the changes in the gene expression profile. it has been found that epigenetic modifications play a crucial role in some of these changes. the definition of the genes related to callus formation by the inhibitation of an epigenetic modification -h3k9 methylation-in tobacco might be used to bear on various dedifferentiation-driven cellular processes. induction of tumor cell death by the use of some phytochemicals that consumed through diet, and derived from medicinal plants opens up new horizons for cancer treatment researches. rheum ribes species, which is studied in this research, is one of the commonly used herbs in pharmacological researches. the high content of phenolic compounds in r. ribes extracts were known to be responsible for the high antioxidant and antibacterial activities. our aim in this study is to assess cytotoxic and apoptotic changes by way of implementing methanol extract of the rheum ribes (root) to the mcf-7 breast cancer cell line. cytotoxic effect of rheum ribes extract was evaluated by using the xtt (2,3-bis(2-metoksi-4-nitro-5-sulfofenil)-2h-tetrazolyum) test. in order to determine the dose of ic50, plant extracts were applied as time and dose dependent in the range of 10-500ug. in 72nd hour, the ic50 value is determined as 400ug. to examine the apoptotic effects of the extract, total rnas were isolated from dose group and the control cells firstly, then cdnas were synthesized. expression profile of the target genes(caspase-3, caspase-7, caspase-8, caspase-9, bax, bcl-2, fas) are determined by qpcr. according to the results, when the control group compared with the cells, it was determined that, while 17.33 and 3.05-fold respectively increase in the gene expressions of caspase-3 and caspase-7 of dose group cells, 15.45-fold decrease in the gene expressions of bcl-2. no significant difference was observed in the other genes examined. based on the obtained data, we believe that methanol extract of the rheum ribes induces apoptosis by activating intrinsic pathway. as a result, this plant species can be a new and effective therapeutic candidate for the medical world in search of alternative anti-cancer approaches, and could shed light on the work to be done in this area. p-02.08.5-053 the effect of ferulic acid and 5-fluorouracil combination on apoptosis in pc-3 human prostate cancer cells prostate cancer is quite often seen in industrialized countries and has the second most common death rate due to cancer after lung cancer in men. 5-fluorouracil (5-fu) is a pyrimidine analog and cell cycle-targeting drug by inhibiting dna synthesis. it has been widely used for treatment of several cancers such as gastric, colorectal, and breast cancers. phenolic compounds found in foods are potential antioxidants of harmful oxidative processes related to cancer and also important due to induction of different mechanism such as apoptosis. ferulic acid (fa; 4-hydroxy-3-methoxycinnamic acid), a phenolic compound, is abundant in fruits and vegetables. the purpose of this study was to investigate combination effect of fa and 5-fu on apoptosis in pc-3 human prostate cancer cell line. the effects of 5-fu, fa, and combination of both of them on cell viability were determined by xtt method. total rna isolation was conducted using trizol reagent. expressions of important genes in apoptosis including casp3, casp7, casp8, casp9, bcl2, bax, fas and cycs were investigated in four groups by qpcr. the ic 50 doses of fa and 5-fu were found to be 300 lm and 60 lm for 48 hours in pc-3 cells, respectively. in order to determine combination effect, pc-3 cells were treated with <ic 50 doses (200 lm fa and 40 lm 5-fu). according to qpcr results, a significant increase in the expressions of bax and cycs genes and a decrease in the expression of bcl2 were observed in the fa group, compared with the control group cells. after the treatment with 5-fu, the expression of casp8 was significantly increased compared with the control group. furthermore, combination of fa and 5-fu significantly increased expression of casp7, casp8, fas and cycs genes. in conclusion, comparing with the single treatments, it was observed that combination of fa and 5-fu affected expression of different genes in apoptosis in pc-3 cell line. p-02.08.5-054 combination of ferulic acid and gemcitabine affects expression of some apoptotic genes in pc-3 human prostate cancer cells prostate cancer, the second causing of cancer-related death in men, is an important cancer type due to the late age of onset, slow the progression, high incidence. gemcitabine is an effective agent in several cancer types including non-small cell lung cancer, pancreatic, bladder and breast cancer. it is known that gemcitabine is used single agent or as a part of combination treatment in prostate cancer therapy. nowadays, new treatment methods have been tested for cancer therapy including natural compounds with low toxicity. ferulic acid (fa) one of these agents, an abundant phenolic compound found in various fruit and vegetables. in this study, objective was to investigate combination effect of ferulic acid and gemcitabine on apoptosis in pc-3 human prostate cancer cell line. cell viability was determined by using xtt method after the treatment with gemcitabine, fa and combination of both of them. total rna was isolated with trizol reagent. expressions of casp3, casp7, casp8, casp9, bcl2, bax, fas and cycs genes are important in apoptosis, were evaluated in four groups by qpcr. the ic 50 doses of fa and gemcitabine were found to be 300 lm and 50 lm for 48 hours in pc-3 cells, respectively. for determination of combination effect, pc-3 cells were treated with <ic 50 doses (200 lm fa and 35 lm gemcitabine). when compared with the control group, qpcr results illustrated, a significant increase in the expressions of bax and cycs genes whereas a decrease in the expression of bcl2 gene in the fa treatment group. after the treatment with gemcitabine, the expression of casp3 and fas genes were significantly increased compared with the control group. furthermore, combination of fa and gemcitabine significantly increased expression of casp3, casp7, casp8, fas and cycs genes. in conclusion, it was observed that combination of fa and gemcitabine affected different genes in apoptosis compared with the single treatments in pc-3 human prostate cancer cell line. pistacia lentiscus linn. is widely distributed in mediterranean europe, morocco and turkey. the resin part of its known as mastic gum and plant called as mastic tree. it has been referred to over centuries as having medicinal properties to treat a variety of diseases. the aim of the present study are to evaluate antioxidant, cytotoxic and antimicrobial activities of heptane, chloroform and methanol extracts from p. lentiscus leaves and mastic gum. the antioxidant activities of chloroform and methanol extracts were assayed with various methods, including tpc, dpph free radical and abts radical cation scavenging activity. also, cytotoxicity of extracts was evaluated and screened against human mcf-7, hela, a549, and ht-29 cancer cell lines and nih-3t3 cells. the viability of cells in 100 lg/ml concentration of extracts was evaluated using mtt assay. antimicrobial activity of extracts were investigated against s aureus, s epidermidis, e coli, p aeruginosa, p vulgaris, k pneumoniae, c albicans, c glabrata, c guilliermondii, c tropicalis, c parapsilosis test organims by the agar well diffusion and broth microdilution methods . according to our results, the methanol extract of p. lentiscus leaves showed the highest dpph free radical scavenging activity (ic50 = 0.16 ae 0.02 mg/ml) and abts radical cation scavenging activity (1.33 ae 0.06 mg/ml). this study also exhibited that methanol extract of p. lentiscus leaves had the highest tpc (126.7 ae 5.7 mg gallic acid equivalents/g extract). the hexane extract of mastic gum showed antifungal activity against all of the tested candida species (6-16 mm / <0.25-8 mg/ml). the methanol extracts of p. lentiscus leaves showed the highest antimicrobial activity against all of the tested bacteria except k pneumoniae strain (8-14 mm/>256 mg/ml). on the other hand, p. lentiscus showed no cytotoxicity against cancer and normal cells. the results suggested that p. lentiscus may be natural source of antioxidant and antimicrobial activities. p-02.08.5-056 antibacterial activity of and chemical composition of alcoholic extract of marjoram against some human pathogens m. ahanjan, m. rahbar mazandaran university of medical sciences, sari, iran herbs enjoy a unique value and importance in sustaining healthy communities in terms of disease prevention (1) . in this regard, marjoram is a plant of the mint family which has antibacterial properties on microorganisms (2) . the current study aims to investigate the anti-microbial activity of the alcohol extracts (i.e., methanol or ethanol) of marjoram plants on the bacteria of staphylococcus (atcc: 25923), aureus e. coli (atcc: 25922), and salmonella enterica (atcc: 13076) and p. aeroginosa through utilizing disk diffusion method. also, the minimum inhibitory concentration and the minimum bactericidal concentrationwere measured through tube. the measurement of minimum inhibitory concentration and minimum bactericidal concentration of ethanol and methanol extracts on e.coli were equal with 100 and 120 milligrams per milliliter, subsequently. moreover, the measurement of the minimum inhibitory concentration and of the minimum inhibitory concentration of marjoram ethanol extraction on staphylococcus aurous was reported to be 90 milligrams and 100 milligrams per milliliter, subsequently. in addition, the amount of ethanol and methanol extracts on salmonella enteric and p. aeroginosa was equal with 80 and 90 milligrams per milliliter, subsequently. the results showed that marjoram alcoholic extract enjoy antibacterial properties. also, among the alcoholic extracts, the ethanol extract has demonstrated to be the most effective extract on salmonella enterica and e. coli and p. aeroginosa. p-02.08.5-057 molecular analysis of serine/arginine rich sc35 splicing factor from olive b. bas 1 , e. d€ undar 2 1 depertmant of biology, university of balikesir, balikesir, 2 department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey olive (olea europaea l.) is an evergreen fruit tree adapted to mediterranean climate and rich in tannins, essential oils and organic acids. serine/arginine rich splicing factors are essential in seqeunce specific splicing of pre-mrnas. in this study we report molecular characterization of an serine/arginine rich sc35 splicing factor (oesarsc35sf) that was isolated from a cdna library constructed from olive pedicels. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes from other organisms) were conducted on ncbi web page. amino acid composition analysis, nucleotide composition analysis, hydropathy analysis, open reading frame determiantion, through, molecular weight and the isoelectric points calculations were conducted using online expasy software. primer3 was used to design forward and reverse primers to amplify the target gene from different olive tissues at different times. analysis with bioedit program revealed that a+t ratio was more than that of g+c. oesarsc35sf was a protein consisting of 267 amino acids. as expected, amino acid composition analysis revealed that serines and arginines were more than other amino acids. kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. the molecular weight of the protein was 30 kda with an isoelectric point (pi) of 11.5. the protein was found to have a signal peptide. according to the predotar analysis results, intracellular localization was found to be in the mitochondrial. the combined results suggest oesarsc35sf might function as splicing factor as its homologs from the other plants. confirming this hypothesis with futher experimental characterization including biochemical function analysis continues. acknowledgements: this study was supported by tub _ itak with grant number 110o108. keywords: olea europaea l., oesarsc35sf, alternative splcing, pre-mrna splicing, bioedit, pedicel specific cdnas. p-02.08.5-058 application of three-phase partitioning for the purification of peroxidase from kiwano (cucumis metuliferus) z. denli 1 , g. arabaci 2 1 kto karatay university faculty of medicine, konya, 2 faculty of arts and sciences, sakarya university, sakarya, turkey peroxidases are enzymes able to catalyze reduction of h 2 o 2 and oxidize various substrates. the kiwano is an oval shaped fruit which has an orange skin with lots of tiny horns. in this study, peroxidase isolated from kiwano is purified with three-phase partitioning (tpp). kiwano fruit was homogenized and obtained crude enzyme extract. the extract was saturated with 50% (w/v) ammonium sulfate ((nh 4 ) 2 so 4 ) and added t-butanol with the ratio of 1:1.5 (v/v). the lower and interfacial layer was collected. the influence of percent saturations of (nh 4 ) 2 so 4 (50, 65, 75, 85, 95%) and tbutanol ratios (1:0.5, 1:1, 1:1.5, 1:2) to the partitioning behaviour of peroxidase were analyzed. after dialyzed, the interfacial and lower phases were measured for peroxidase activity and protein content. the protein pattern of the peroxidases was evaluated by using gel electrophoresis. peroxidase activity recovery and the purification fold of interfacial and lower phases were 117.4, 1.7% and 0.84, 0.03. therefore, other experiments were continued with interfacial phase. at constant t-butanol with the ratio of 1:1.5, the enzyme activity recovery and purification fold of interfacial phase for saturations of (nh 4 ) 2 so 4 (50, 65, 75, 85%) were 40.9, 43.2, 39.2, 135% and 0.37, 0.31, 0.4, 0.91 . the interfacial phase was not dissolved in 95% (nh 4 ) 2 so 4 . at constant 85% (nh 4 ) 2 so 4 , the enzyme activity recovery and purification fold of interfacial phase for t-butanol ratio (1:0.5, 1:1, 1:1.5, 1:2) were 61.6, 48. 1, 127.6, 126.1% and 1.79, 0.6, 1.84, 1.41 . finally, at optimum conditions (% 85 (nh 4 ) 2 so 4 , t-butanol 1:1.5) after dialyzed interfacial phase, the enzyme activity recovery and the purification fold were 138.8% and 4.46. the results of gel electrophoresis showed that the molecular weight of enzyme was between 30-60 kda. the applications of tpp gave the maximum recovery of 138.8% and 4.46-fold purification. as a result, for purfication of peroxidases, tpp is a rapid, simple and economical technique. accumulating the most robust genes and proteins in elite genotypes without any harmful effect on the potential plant yield is an urgent need to enhance productivity under various stressors. among the stressors, drought is a major challenge for agricultural productivity. brachypodium distachyon, with its close relationship to agriculturally and economically important crops, is an important model plant species. although ongoing transcriptomic analyses in brachypodium distachyon available, proteomic analyses are required to obtain an integrated picture of drought response. in the current study, a comprehensive proteome analysis was conducted on brachypodium leaves under increasing levels of drought stress. to screen gradual changes upon drought stress, brachypodium leaves subjected to drought treatment for 4, 8 and 12 days were collected for each treatment day. the cellular responses were investigated through a proteomic approach involving two dimensional difference gel electrophoresis and subsequent combined tandem mass spectrometry. for the validation of transcriptional expression, the genes encoding selected proteins were examined through quantitative real-time pcr. spot detection on cye-dyed gels revealed a total of 497 distinct spots in brachypodium protein repertoire. a total of 13 differentially expressed proteins (deps), with at least 2-fold changes in abundance, were identified by mass spectrometry and classified according to their functions. the biological functions of deps included roles in photosynthesis, protein folding, antioxidant mechanism and metabolic processes, highlighting the significant degree of overlapping between metabolic alterations induced by drought stress. identified proteins in this study and understanding the molecular mechanisms of drought response and defense mechanisms in plants will contribute to the researches on development of drought tolerant crop species. p-02.08.5-060 immunohistochemical and electron microscopy investigation of tmv-based chimeric virus particles carrying conserved influenza antigen in nicotiana benthamiana recently we obtained and partially characterized set of viral vectors based on tobacco mosaic virus (tmv) genome displaying conserved influenza a m2e epitope from matrix m2 protein. purified chimeric virus particles (cvp) conferred protection of mice against lethal homologous and heterologous influenza virus challenge. we revealed significant difference in symptoms of infections caused by tmv-m2e recombinant viruses containing cysteine (cys) to serine (ser) or alanine (ala) substitutions in the human consensus m2e sequence. accumulation level of m2e-ala recombinant coat protein was significantly higher than m2e-cys/ ser (ratio 5:1). tmv-m2e-ser infection, in contrast to ala mutant, suspended growth and development of nicotiana benthamiana. non-inoculated leaves (14 d.p.i.) were fixed with ethanol and histological sections were incubated with mouse serum to m2e and secondary antibodies conjugated with either hrpo or fitc. cvps of all three mutants were detected in epidermal and stomata cells as well as in sieve elements and minor veins. electron microscopy analysis of mesophyll cells revealed typical rigid helical particles. cys/ser mutants mostly accumulated within ground cytoplasm as aggregates of discrete tubules in parallel arrangement, which were not delimited by lipid membranes. we discovered huge amount of cvps in the cytoplasm and lesser amount diffused in the central vacuole. essential part of ala particles was located in the cytoplasm, but mentioned aggregates were not found and only insignificant number of virions was revealed in vacuole. unlike wild-type tmv, none of the mutants was revealed in chloroplasts. diameters of cvps were as follows: sersingle particles in cytoplasm 13 ae 1. cyanidin is a natural anthocyanidin found in a variety of fruits (grapes, blackberry, blueberry, cherry and cranberry etc.) and vegetables (red cabbage, red onion). this polyphenolic compound is a flavonoid with significant antioxidant activity. cyanidin and its glycosides have vasoprotective effects and can interfere with inflammation, carcinogenesis, obesity, and diabetes. one important role of the macrophages is the release of pro-inflammatory mediators, such as nitric oxide, various cytokines, in response to activation signals, including chemical mediators, cytokines, and bacterial lipopolysaccharide (lps). in this study, we investigated the role of cyanidin chloride in inflammation. anti-inflammatory effects of cyanidin chloride were examined in lps-stimulated murine raw 264.7 macrophages. we observed the level of various inflammation markers such as nitric oxide (no), inducible no synthase (inos), cyclooxygenase-2 (cox-2), tumor necrosis factor-a (tnf-a) and interleukin-8 (il-8) under lps treatment with or without cyanidin chloride. cyanidin chloride inhibited not only no production but also the expression of cox-2 and inos, without any cytotoxicity. cyanidin chloride also attenuated pro-inflammatory cytokines and other inflammation-related markers such as il-8 in a dosedependent manner. in conclusion, cyanidin chloride may be beneficial for the prevention and treatment of anti-inflammatory diseases. p-02.08.5-062 the investigation of centranthus longiflorus plant extacts effects on cell proliferation and apoptosis activity in the cell lines of mcf-7 breast cancer h. askin ataturk university, erzurum, turkey introduction: in the u.s., breast cancer is the second most common cancer in women after skin cancer. current treatment of cancer can be done by surgery, chemotherapy, and radiation therapy. in addition, there is widespread use of complementary and alternative medicine in developed countries. plants and plant extracts play a critical role in the research into new anticancerogenic agents. centranthus longiflorus (cl) is used in alternative medicine for sedative and antispasmodic purposes. a plant of turkish origin, centranthus longiflorus used as traditional turkish medicine have remained uninvestigated for familial hypercholesterolemia, diabetes, coronary artery disease and cancer for their in vitro biological activity despite their use for sleep disorders. in this study, growth-inhibiting and pro-apoptotic effects of hexane, ethyl acetate and ethanol extracts of cl in mcf-7 breast cancer cell line were investigated. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from cl was analyzed using a gc-ms system. dose-and time-dependent cytotoxic and apoptotic effects of cl were evaluated by mtt cell proliferation kit and cell death detection elisa kit, respectively. manufecturer's protocol was followed for analyses. then, apoptotic genes; caspase3, bax and p53 and antiapoptotic genes; bcl-2 and pi3 expression levels were determined by rt pcr. results: according to our results, cytotoxic effect on mcf-7 cell was only observed in 20 and 50 lg/ml doses of cl. however, any of the application doses showed an apoptotic effect on mcf-7 cells. they exhibited a necrotic effect rather than the apoptotic effect. although alterations in expression levels of these genes were determined, this alterations was statistically insignificant. discussion and conclusion: consequently, we can say that cl have a cytotoxic effect on mcf-7 breast cancer cell lines. p-02.08.5-063 reduction of the chloroplast genome and the loss of photosynthetic pathways in the mycoheterotrophic plant monotropa hypopitys, as revealed by genome and transcriptome sequencing e. gruzdev, a. mardanov, a. beletsky, v. kadnikov, e. kochieva, n. ravin, k. skryabin institute of bioengineering, research center of biotechnology of the russian academy of sciences, moscow, russia genomes of parasitic plants represent interesting model systems to study effects of relaxed selective pressure on photosynthetic function. previous genomic studies of nonphotosynthetic plants revealed reduction of their chloroplast genomes, but the corresponding changes in their nuclear genomes are less known. here we present the data on the transcriptome and the chloroplast genome of the non-photosynthetic mycoheterotrophic plant monotropa hypopitys. the chloroplast genomes were sequenced for two specimens of m. hypopitys, collected in different regions of russia. the cpdnas are 34,800 bp (mon-2kalr) and 35,336 bp (mon-1volr) long and rearranged with respect to each other. both genomes contains genes encoding ribosomal proteins, infa, matk, and 4 ribosomal rna genes. 17 and 19 trna genes were predicted in two cpdna. genes encoding nadh dehydrogenase, plastid rna polymerase, all genes related to photosynthetic apparatus, clpp, ycf1, ycf2, accd, and some genes for ribosomal proteins are missing or became pseudogenes. the reduction of gene content is associated with extensive gene order rearrangement and the lack of inverted repeats. overall, the size and gene content of m. hypopitys cpdna indicates that it is close to the end of plastid genome degradation process. in order to get insights into the changes in the nuclear genome associated with the transition to nonphotosynthetic lifestyle, we sequenced and assembled the transcriptome of m. hypopitys. as expected for holoparasites, we did not found transcripts for the nuclear genes encoding the components of photosynthetic machinery, including photosystem i and ii, cytochrome b6f complex, and ribulose bisphosphate carboxylase. contrary to the holoparasitic plant phelipanche aegyptiaca, almost all genes of chlorophyll biosynthesis pathway from protoporphyrin ix were not found in the m. hypopitys transcriptome. this work was supported by the rsf grant 14-24-00175 and rfbr grant 14-14-00749 (mon-1volr cpdna sequencing). introduction: beta-sitosterol is a substance found in plants. chemists call it a plant sterol ester. it is found in fruits, vegetables, nuts, and seeds. it is used to make medicine. beta-sitosterol is used for heart disease and high cholesterol. the federal food and drug administration (fda) allows manufacturers to claim that foods containing plant sterol esters such as beta-sitosterol are for reducing the risk of coronary heart disease (chd). centranthus longiflorus (cl) is used in alternative medicine for sleep disorders. a plant of turkish origin, cl used as folk medicine have remained uninvestigated for familial hypercholesterolemia, coronary artery disease and preventing colon cancer for their in vitro biological activity despite their use for sleep disorders. we investigated of the chemical constituents from dried aerial parts of centranthus longiflorus. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from the aerial parts of cl was analyzed using a perkin-elmer gc-ms system. results: ten compounds were obtained and identified as butanoic acid, hexadecanoic acid (palmitic acid), 7-methyl-z-tetradecen-1-ol acetate, octadecanoic acid (stearic acid), diisobutyl phthalate, 9-octadecenamide, octacosane, nonacosane, alfa amyrin and beta sitosterol. the latter two were obtained in all extraction (hexane, ethyl acetate and ethanol). discussion and conclusion: all of these compounds are isolated from centranthus longiflorus for the first time. these findings may shed light on the design of new drugs, the cholesterollowering effect. p-02.08.5-065 role of lutein for the high light-induced inhibition of photosystem ii related reactions in thylakoid membranes of arabidopsis thaliana, wt and lut2 k. dobrev, d. stanoeva, m. velichkova, a. v. popova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthetic reactions taking place in thylakoid membranes of higher plants are extremely sensitive towards different environmental stress conditions such as high and low temperature, high light intensity, uv radiation etc. carotenoids are intrinsic component of photosynthetic pigment-protein complexes and are involved in performing multiple important functions. their role of accessory pigments in absorbing sun light, participation in photoprotection via dissipation of excess absorbed light, deactivating of stress-induced reactive oxygen species and structural role are well documented and recognized. the role of lack of lutein in high light-induced alterations in structural organization and functional activity of the main pigment-protein complexes was evaluated using isolated thylakoid membranes of arabidopsis thaliana, wt and mutant lut2, deficient in lutein, subjected to photoinhibitory treatment for different periods of time. alterations in photochemical activity of photosystem i and photosystem ii were determined by a clark-type electrode in the presence of exogenous electron donors and acceptors. activity of oxygenevolving complex and of the grana and stroma situated photosystem ii reaction centers was evaluated by determination of flash oxygen yields and initial oxygen burst under constant light without donors and acceptors. low-temperature (77k) fluorescence was applied for unraveling of light-induced alterations in energy transfer and interaction between the main pigment-protein complexes. maximal quantum efficiency of psii was registered by pulse amplitude modulated fluorescence method. results obtained are discussed in respect to the importance of lutein for the organization and sensitivity of photosynthetic apparatus towards high light intensity treatment. modern agriculture relies heavily on phosphate rock fertilizer to improve phosphorus availability in many soils, but this approach is not sustainable long-term. phytate (myo-inositol hexakisphosphate) is an organic phosphorus compound often present in many soils. however, phytate can not be utilized by most plants, and its accumulation in soil leads to substantial ecological problems. phytases are enzymes that hydrolyze phytate and release inorganic phosphate. many microorganisms such as bacteria and fungi synthesize highly diverse phytases which are suitable for plant biotechnology. generation of transgenic plants expressing phytases of bacterial origin has been proposed as one option to improve plant phosphorus nutrition. in this study, we generated and characterized transgenic arabidopsis thaliana plants expressing a modified phytase gene paphyc from pantoea sp. under strong camv35s promoter. three individual transgenic a. thaliana lines expressing the bacterial phytase gene, as well as negative control plants harboring the camv35s promoter alone were identified. expression of phytase in plants was verified at both transcription and translation levels. phytase-expressing plants grown on media with phytate as the sole source of phosphorus demonstrated better than wild-type growth rates, shoot dry mass, shoot phosphorus content, as well as higher phytase activity in cell-wall extracts. overall, we show that plants expressing bacterial phytase are capable of better growth on phytate as the only source of phosphorus in laboratory conditions. further research investigating the applicability of using bacterial phytase expression to improve plant growth in soil is necessary to evaluate the different routes of solving the phosphorus deficiency problem in agriculture. p-02.08.5-067 the role of elevated temperature in photoinhibition and recovery of photosystem ii in thylakoid membranes from arabidopsis thaliana a. faik, m. gerganova, m. velitchkova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthesis of higher plants is the principle process to transform light energy into biochemical usable energy. in nature, plants are exposed to the environment where light, temperature, uv-b radiation varied and very often their extreme values that are unfavorable for effective performance of photosynthetic reactions. plant are developed various strategies to cope with stress including radical scavenging enzyme system, accumulation of protective compounds, etc. pigment-protein complexes of photosystem i and photosystem ii and their light harvesting antenna, situated within thylakoid membranes, are involved in the primary reactions of photosynthesis -absorption of light, charge separation and electron transport. photosynthetic process is sensitive towards higher than optimal temperatures, the photosystem ii and oxygen evolving complexes being extremely sensitive to elevation of temperature. in present work pam fluorescence was applied to evaluate the effect of long term action of elevated temperature (38/30°c) on the quantum yield of photosystem ii, non-photochemical quenching and rdf, the latter quantifying the photosynthetic process. in addition, the activity of oxygen evolving complex was determined polarographically in the presence of exogenous electron acceptor 1,4 -benzoquinone. sds-page electrophoresis and western blot were applied to determine the damage of d1 -reaction center protein of photosystem ii. alterations of mutual organization within photosystem ii complex and its antenna and of energy interaction between them were followed by analysis of 77k steady state chlorophyll fluorescence spectra. the simultaneous application of high temperature and high light intensity resulted in a well pronounced reduction of non-photochemical quenching that restore to the initial values after recovery for 5 days at optimal conditions. d1 was also restored while quantum efficiency of photosystem ii did not recuperate to initial values. p-02.08.5-068 reorganization of the main pigment-protein complexes in thylakoid membranes from tomato (solanum lycopersicum) during long term exposure to elevated temperature the changes of earth climate resulted in unfavorable environment for plants. depending on the duration of influence of stress factors, the response of plants includes short and long term acclimation. the population, structure and organization of pigmentprotein complexes within thylakoid membranes are dynamic and flexible, thus providing for the acclimation of the photosynthetic apparatus to the changed environment. the main pigment-protein complexes, involved in energy transduction, are photosystem i, photosystem ii and light harvesting complexes. they are separated in grana and stroma regions of thylakoid membranes but it is well established that they can rearrange as a result of alterations of light intensity, temperature increase and decrease in order to balance the perception and utilization of excitation energy. in present work the effect of long term action of elevated temperature on organization and stoichiometry of main pigmentprotein complexes in the thylakoid membranes from tomato plants (solanum lycopersicum cv. m82) was investigated. three weeks old tomato grown at optimal conditions (22/20°c day/ night temperature and light intensity 250 lmol/m 2 /s) plants were exposed for 2 and 6 days to elevated temperature at 38/30°c. by means of blue-native electrophoresis the effect elevated temperature on the populations of psii (dimmer and monomers) and lhcii (monomers and trimers) was estimated and compared with the same parameters for control plants. the ability of plants to recover from this treatment was checked after 5 days under optimal conditions. the changes of content of chlorophylls and carotenoids were determined at every stage of treatment. based on the results obtained it can be concluded that one of the mechanisms for regulating the energy balance and maintenance of efficient photosynthetic process involves a change in the organization and stoichiometry of the photosystem ii and oligomer state of light harvesting complex ii. aim of the study, was to evaluate the anti-tumor effects of silymarin, curcumin and propolis on leptin-induced mcf-7 cells. mcf-7 cells were incubated various concentrations leptin (physiological, obesity and pharmacologically doses; respectively 10, 100 and 1000 ng/ml) . then different doses of silymarin (10, 20, 40, 80 lm), curcumin (10, 20, 40, 60 lm) and propolis (0.063, 0.125, 0.25, 0.5 mg/ml) were added. after 24, 48, 72 and 96 hours incubation periods 3 different area images were taken digital camera. then using dye release reagent we determined the intensity of apoptosis via colorimetric determination by elisa reader. absorbance was directly proportional the number of apoptotic cells (biocolor cell-apo percentageapoptosisassay). also, we examined the effect of these natural products on proliferation rate of leptin-induced mcf-7 cells for 1, 2, 3 and 4 hours (biovision cell proliferation kit) all experiments were carried out 3 different days, at least 3 times. all of three compounds were stimulate the apoptosis at all time points and all different doses of leptin. the differences was statistically significant at the level of p < 0.05 between 24 and 48 hours. it was found that there were not seen much cells at 72 and 96 hours time points. we thought that most of the cells were gone necrosis instead of apoptosis. the best effective doses on apoptosis of propolis was 0.063 mg/ml, silymarin and curcumin were 20 lm. also, we evaluated the effects of on proliferation rate the mcf-7 cells, we found that only propolis was effective of inhibiting proliferation at all doses of leptin induced mcf-7 cells in 1 hour. we hope this study will be a guide for the further studies in anti-cancer agent development field and show that the natural origin substances cause cancer cells apoptosis and provide targeted treatment for cancer therapy. p-02.08.5-070 investigation of some lichen-derived substances' cosmetic potential for skin protection against ultraviolet b due to the depletion of the stratospheric ozone layer and chronic exposure, the occurrence of various skin diseases have been increased in recent decades. thence, people and cosmetic companies have progressively given more importance natural sunscreen products for the protection from harmful sun rays, especially ultraviolet b rays. we, therefore, isolated some lichen-derived substances; 3-hydroxyphysodic acid and protolichesterinic acid from hypogymnia tubulosa and cetraria aculeate, respectively. chemical characterization and identification of the isolated lichen substances were accomplished by using ftir, 1 h-nmr and melting point analyses. the theoretical uv-vis spectra and 3d conformations of the isolated compounds were determined by using the gaussian 03 software with hf theory at the b3lyp/3-21g level. the dark toxicities and ultraviolet b protection capacities of the substances were lighted up as previously described [1, 2] on hacat human keratinocyte cell line by using mtt cell viability and ldh cellular membrane degradation assays. the obtained results from the assays showed that protolichesterinic acid has a more dark toxic activity on keratinocyte cells than 3hydroxyphysodic acid, and the toxic activities were found sufficient as much as 80% at the highest doses of the substances; 400 lm. however, it was observed that the cytotoxicity of the substances were reduced at the rate of approximately 15% by the irradiation. consequently, we think that the substances block the ultraviolet b rays but their cytotoxic feature is an important limitation to their usage in cosmetic industry. references [1] varol, m., t€ urk, a., candan, m., tay, t., koparal, a. t. (2016) photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes, phytotheraphy research 30:9-15. [2] varol, m., tay, t., candan, m., t€ urk, a., koparal, a. t. (2015) a great effort and financial supports have been consumed to explore and design novel sun protection factors due to the unhindered increase of malignant and non-malignant skin diseases caused by the chronic exposure and depletion of the stratospheric ozone layer. the testing of naturally produced compounds seems to be the best and inexpensive way to search for potentially photoprotective substances. on the other hand, as photo-resistant species, lichens are still poorly exploited. norstictic acid was, therefore, isolated from the acetone extract of pleurosticta acetabulum. ftir, 1 h-nmr and melting point analyses were performed to identify the chemical features of norstictic acid. gaussian 03 software with hf theory at the b3lyp/3-21g level was also performed to determine the theoretical uv-vis spectrum and 3d conformation of the isolated compound. the dark cytotoxicity and ultraviolet b-protection capacity of norstictic acid were comparatively tested as previously described [1, 2] by using mtt cell viability and ldh cellular membrane degradation assays. as a result of the experiments, it is observed that norstictic acid has a dark-cytotoxicity as less as 25% at the highest dose of the substance; 400 lm. however, ultraviolet b-induced damage on human keratinocytes was blocked by the lower concentrations of norstictic acid as 25, 50 and 100-lm, and 20% of cells were protected according to the control experiments of irradiated cells. consequently, we think that norstictic acid might be employed as a sun protection factor at the low concentrations, and further studies should be performed. years, researchers have focused on the lichen acids because of their biological activities. it is also suggested that lichens can be used as anticancer agents. vulpinic acid, an important lichen seconder metabolite, has antimicrobial activity and strong antimutagenic, anticancer and antioxidant capacity. nanotechnology has the potential to offer solutions to current obstacles in cancer therapies, because of its unique size (1-100 nm) and large surfaceto-volume ratios. so, in this study we aimed to determine the cytotoxic and proliferative effects of vulpinic acid and magnetic nanoparticles loaded with vulpinic acid (fe 3 there are four species of wild rice known around the world. zizania aquatica l., zizania palustris l. and zizania texana hitche are found in north america, whereas zizania latifolia (griseb) turcz) is native to east asia. cwr mainly grows in the areas along the yangzi river and the huai river in china without any cultivation and domestication. cwr was an ancient grain that has been used in chinese herbal medicine to treat a variety of ailments associated with nutrition, including gastrointestinal disorders and diabetes. our previous studies have demonstrated that consuming chinese wild rice can significantly improve blood lipid profiles and ameliorate high-fat/cholesterol diet-induced insulin resistance. however, compared to the well studied common dietary white rice, active composition and the associated proteomic information of chinese wild rice have yet to be investigated. in this study, we compared and analysed the different proteins between chinese wild rice and white rice by proteomics method. our study provides insights and experimental evidence for further exploration of this ancient medical food in disease prevention and therapy. the homology between cwr and n22 is 64%, but significant differences also exist between the two. we gained new insight by analyzing the biological function of the high reliability (credibility score 67 or higher, p < 0.05) peptide mass fingerprint of cwr2-de electrophoresis revealed differences in protein composition between cwr and n22. information obtained from the pmf indicates that glutelin precursor, caffeoyl coenzyme a (coa) omethyltransferase and putative bithoraxoid-like protein can provide gene evidences for its biological function. p-02.08.5-075 mir396 and growth-regulating factors interaction during maize leaf growth under low-temperature stress g. aktug, f. aydinoglu gebze technical university, kocaeli, turkey microrna (mirna) genes are a class of non-coding small rnas about 21 nucleotide-long which are revealed as regulators of plant growth and stress responses. the mirna mir396 targets and regulates growth-regulating factors (grfs) which are plant specific transcription factors family and this regulation machinery is conserved among plant species. plant growth is a result of cell division and expansion which took place as spatial gradient zones throughout maize leaf which are meristem, elongation and mature zones. cells proliferate in meristem, migrate to elongation zone and finally reach to mature zone to get its final size. it has been shown that mir396 affects cell division by regulating grfs and changes leaf size which are determined by cell number and cell size of leaf. this study aims to investigate the role of mir396 and grfs interaction during maize leaf growth under low-temperature stress. maize seedlings were grown under low-night temperature for stress treatment to generate growth retardation and control conditions as well to make comparative analysis. length of the third and fourth leaves of seedlings was measured every day and leaf elongation rate was calculated to observe stress effects on the leaves. growth zones of fourth leaves were harvested during steady-state growth phase for determining expression level of mir396s and their target by q-rt-pcr. we mined 8 mir396 genes sharing sequence similarity and 8 grf targets. the expression analyses of mir396s and grf5 are proceeding for different growth zones. in conclusion, this is the first study investigating the regulation network between mir396s and grf5 in different developmental stages of maize leaf under low-temperature stress. oeigpd cdna was isolated from a cdna library we constructed from olive pedicels. homology searches for nucleotide, amino acids and alternative open reading frames were conducted utilizing blastn, blastp, and blastx, respectively. nucleotide sequences of homologous genes from other plants were aligned using bioedit and the number of snps were detected. the alignment was then used to generate a phylogenetic tree using mega7 program. another alignment with amino acid sequences of the homologues proteins was also generated to construct a phylogenetic tree displaying oeigpd's position among other plants. various aspects of oeigpd including amino acid composition, hydropathy analysis, isoelectric point (pi) and three dimentional structure of the protein were determined using online software at expasy. multiple primer pairs to amplify the full length open reading frame of the gene, to clone the gene into the expressing vector plate51, and to detect expression through real-time pcr were designed using primer3. amino acid composition analysis revealed that oeigpd contained serine, arginine and isoleucine predominantly while hydropathy analysis suggested it was an hydrophilic protein. isoelectric point (pi) of the protein was calculated as 4.97. the molecular weight of the protein was calculated as 11 kda. analyses continue to determine the polymorphism of oeigpd among olive cultivars, and biochemical function of the gene in olive. p-02.08.5-077 cytotoxic effect of fractionated triterpenoid glycosides from holothuria polii in human cancer cells sea cucumbers are the members of class holothuroidea and they have more than 1100 described living species all around the world. sea cucumbers secrete special secondary metabolites from their body walls and they are called triterpene glycosides (tggs). in this study, cytotoxic activity of fractionated ttgs from h. polii on different cancer cell lines were carried out. h. polii delle chiaje, 1824 samples were collected from dikili-_ izmir. the semipurified extracts were fractioned by using hplc. four different fractions (fraction a-d) were obtained. in order to characterize the fractions, maldi-tof/ms was used. the cytotoxic activity of the fraction a-d were tested on ht-29, t84 and upci-scc-131 cell lines by using xcelligence rtca sp system. the cells were treated with three different concentrations of the fractions for 48 hours. the cell index data were compared with the control group. ic50 values of the fractions for three cell lines were calculated. according to the results, the fractions have holothurin a (1243.50 m/z), 24-dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer (1227.50 m/z). the fraction d was the most effective on all cell lines with ic50 value of 10.46 ae 0.18 mg/l, 11.24 ae 0.57 mg/l and 11.13 ae 0.29 mg/l for ht-29, upci-scc-131 and t84, respectively. in conclusion, sea cucumber ttgs are promising agents for colon adenocarcinoma, oral squamous cell carcinoma and colorectal carcinoma (metastatic) treatment. p-02.08.5-078 effect of horse-chestnut (aesculus hippocastanum) seed extract on matrix metalloproteinases during diabetic wound healing impaired wound-healing in diabetics is a major public health problem. the expression and activation of matrix metalloproteinases (mmps) are also impaired in diabetic wounds according to previous studies. their main function is to degrade the various components of the extracellular matrix. also, they participate physiological processes such as inflammation, angiogenesis, tissue remodeling. horse-chestnut seeds (hc) are rich in saponins and flavonoids. it has been shown that hc has antiinflammatory, antioedema, vessel protective, and free radical scavenging properties. the aim of this study is to determine with molecular signs on cutaneous wound healing effects of the ethanol (50%) extract of hc (hce) seed in rats by excision wound model. this study was conducted on diabetic wistar albino rats, which were injected by a single dose (50 mg/kg i.p.) streptozotocin. diabetic treatment rats were applied topically 15% (w/w) ointment with hce and control rats were applied topically simple ointment, once a day during the experimental period. the gene expression levels of mmp-1, mmp-9 by qpcr and levels of nitric oxide (no), hydroxyproline and malondialdehyde in wound tissue investigated at the end of 3rd, 7th, and 14th days. wound closure was also measured. the hydroxyproline and no levels were significantly increased in the hce treated group versus control after the 3rd and 7th days. the malondialdehyde levels were significantly lower in the treatment group. mmp1 gene (associated with collagen processing and reepithelialization) expression levels in hce treated rats were increased in the 7th day while it was reduced in 14th day. mmp 9 gene (associated with inflammation and gelatinase) expression levels in hce treated rats were decreased in 7th, and 14th days compared to the control. these findings indicate that hce accelerated the cutaneous wound healing process in diabetic rats via mmp1 and mmp9 regulation. p-02.08.5-079 isolation and molecular molecular characterization of vps39/vam6 from olive b. celikkaya, e. dundar molecular biology and genetic at balikesir university, balikesir, turkey vps39/vam6 promotes aggregating and fusing of endosomes and lysosomes. it is a component of a protein complex that is found in vacuole membranes. this gene has been studied from various organisms including humans, drosophila, arabidopsis and rice. no studies on olive vps39/vam6, however, have been reported. the aim of this study is to report information of olive vps39/vam6 including expression pattern and biochemical characterization. vps39/vam6 was isolated from a cdna library we constructed from fruited olive leaves in july. to determine the putative name of the cdna, blast analyses were conducted for nucleotide, open reading frame and amino acid sequence comparisons. bioedit program was used to determine the nucleotide and amino acid composition along with its molecular weight and isoelectric point (pi). hydropathy analysis was conducted using kyte and doolittle program. phylogenetic analysis was done using mega7. cellular localization of the product was predicted using sosui gramn. the three dimentional structure of the protein was calculated using i-tasser and compared to previously known structures using cn3d. the blast and bioedit analyses revealed vps39/vam6 had 836 base pairs coding 120 amino acids with a molecular weight of 13.38 kda, and pi of 6.39. the at/gc ratio was very high (1.5) comparing to its homologs from other plants suggesting to expect significant differences of this gene's function from the others. amino acid composition analysis revealed high rates of serine, leusine and isoleucine indicating a hydrophobic property of the protein. the hydrophobic feature was confirmed by kyte and doolittle analysis while the cellular location was revealed to be extracellular. the hydrophobic nature despite extracellular location suggests it is a membrane associated protein which was confirmed by transmembrane domain analysis. as expected no signal peptide was detected. the 3d structure of the protein was similar to its previously reported homologs. p-02.08.5-080 isolation and characterization of the ribosomal l1 protein from olive s. cinarli, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey despite as a ribosomal protein, l1 is known as the inhibitor of the cellular aging gene and it has been reported to have roles in apoptosis. the ribosomal l1 protein is larger than the lsu of ribosome and contains 2 domains as 2-layered alpha/beta domain and 3-layered alpha/beta domain. in ribosomes, it functions in translocation and orientation of trnas. although the ribosomal l1 (rl1) gene has been studied in many plants, reports on olive rl1 (oerl1) are very rare. this study presents molecular characterization of rl1 gene from olive. oerl1 was isolated from a cdna library we constructed from unfruited olive leaves in july. homology analyses were conducted using blast programs. nucleotide and amino acid compositions, molecular weight, isoelectric point (pi) and at/gc ratio were determined using bioedit and expasy programs. cellular location of the l1 protein was determined using sosui-gramn program. signal peptide detection, transmembrane domain detection, three dimensional (3d) structure analysis, and phylogenetic analysis were conducted using signalp 4.1, thhmm, i-tasser/cn3d and mega7, respectively. oel1 was found to have an open reading frame of 909 base pairs coding 220 amino acids that constitutes a molecular weight of 24.9 kda and a high pi of 9.87. lysine, leucine and valine had higher rates. the hydrophilic nature suggested by kyte and doolittle analysis despite high rates of leucine and valine suggests an amphipathic nature of the protein that can bind to both hydrophilic and hydrophobic proteins and / or function in both media. a 1.59 at/gc rate is significant comparing to that of its homologs from other plants. sitoplasmic location predicted by sosui-grann is in agreement with the hypothesis suggesting an amphyphatic nature for oerl1. likewise, no signal peptide was detected and it was predicted to have at least one transmembrane domain. further characterization of oerl1 with respect to expression pattern and biochemical function continues. p-02.08.5-081 isolation and characterization of an olive splicing factor 3b subunit 1 m. nurcin, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey splicing factor 3b subunit 1 (sf3b1) functions in the regulation of translation and gene expression. sf3b1 forms u2 small nuclear ribonucleoprotein complex (u2 snrnp). splicing factors 3a and 3b binds pre-mrna at the 5 0 site of the intron branching point. this binding joins u2 snrnp to pre-mrna. although sf3b1 from various plants have been widely studied, no studies on olive sf3b1 (oesf3b1) have been reported. this study reports information on various aspects of oesf3b1. oesf3b1 was obtained from a cdna library we constructed from fruited olive leaves in december. it was putatively identified as a splicing factor using blastn, blastp and blastx. to determine wheter oesfb1 was a sitoplasmic protein, sosui gramn was used. tmhmm was used to detect any transmembrane domains while signal peptide analysis was conducted by signalp. i-tasser and cn3d were used to generate the calculated 3d structure and to compare it with experimentally generated models, respectively. nucleotide and amino acid compositions along with the calculated molecular weight and isoelectric point (pi) were analyzed using bioedit and online expasy software. the phylogenetic trees revealed genetic relationship of olive among other plants based on oesfb1. the orf contained 1950 nucleotides coding 254 amino acids that produce a 28.2 kda peptide with a pi of 5.94. alanine, valine and leucine were found at high ratios suggesting a hydrophobicity which was also predicted by kyte and doolittle analysis. the at rich property of oesf3b1 is not unusual comparing to most plant genes. cellular localization of the gene was suggested to be in mitochondria with no signal peptide indicating oesf3b1 could be synthesizing in mitochondria. the predicted 3d structure of oesf3b1 was similar to experimentally produced structures while some hydrophobic pockets were predicted. further characterization of the gene with respect to temporal and spatial expression pattern and biochemical function continues. p-02.08.5-082 kafirin profile of turkish originated sorghum populations r. temizg€ ul, s. yilmaz, m. kaplan, t. akar department of biology, faculty of science, erciyes university, kayseri, turkey sorghum bicolor l. is the fifth important crop in the world with its high photosynthetic activity and resistance to unfavourable conditions as high temperature, drought, salt, and ph changes. sorghum has attracted great interest due to its intensive usage both as human and animal nutrition, and contribution to resistance against many diseases. some proteins of sorghum exerts reducing effect on nutrient digestion through making connetions with other proteins and/or carbohydrates. kafirin proteins have the highest proportion in grain with a range of 77-82%. they are grouped into a (23-25 kda), b (18 kda), c (28 kda) and d (13 kda) subunits depending on molecular weight, solubility and structure. in the current study, kafirin proteins from turkey originated 282 sorghum populations were acquired through sequential extraction; first, non-prolamines were removed through application of 5% naci concentration, and second, kafirins were obtained using tertiary butanol (60%) and reducing agents. sds-page was conducted for seperating and visualising the subunits of kafirins. the a, b, c, and d subunits of populations were respectively estimated as 97, 85, 95, and 60%. of the total proteins, 74% was identified as a, 12% b, 12% c, 1% d, and %1 non-prolamines. non-prolamin group of proteins were visualised as 18 different bands ranging from 15.6 to 230 kda. c and b group of proteins were only viewed when treated with reducing agents as 2-me and dtt suggesting that they are connected with complex cross-links. however, a group of proteins visualized without using these agents due to not having intra molecular disulphide bridges and inter molecular cross-links. non prolamins, except for 47.8, 45.7, 21.7, 20.3 and 15.6 kda, were able visualised in the presence of reducing agents. transcriptomic analysis of the genes encoding analysed proteins needs to be elucidated for better understanding of the genetic diversity and biochemical characteristics of sorghum. p-02.08.5-083 untargeted metabolomic profiling of romanian and uk tomatoes varieties by high performance liquid chromatography coupled with mass spectrometry c. socaciu 1,2 1 university of agricultural sciences and veterinary medicine, cluj-napoca, 2 center for applied biotechnology ccd-biodiatech at proplanta ltd, cluj-napoca, romania tomato flesh is a rich source of many phytochemicals of high nutritional value, including a large variety of carotenoid derivatives with health promoting properties. metabolomics became the most adequate technology for an accurate chemotaxonomic classification and discriminations between different varieties, based on untargeted profiling or targeted, quantitative analysis. different varieties of tomatoes (b-carotene-rich, lycopene-rich, ketocarotenoid-rich) cultivated in romania and uk were comparatively studied using enriched fractions obtained by a preliminary fractionation of the whole pulp homogenate. two methods were applied for carotenoid extraction: a mixture of hexane/ethanol (1) and chloroform/methanol (2) . the dried extracts were dissolved in ethyl acetate and analyzed by uv-vis spectrometry and hplc-esi(+)qtof-ms (bruker gmbh). the base-peak chromatograms were processed by specific biostatistics software (data analysis and profile analysis) and the molecular identification were determined by comparison with the data base lipidomics gateway (www.lipidmaps.org). the content of carotenoids were significantly higher using extraction (2) , ranging from 4.5 to 7.6 mg/100 g. the major carotenoid derivatives, were represented by lycopene, hidroxy-lycopene, all-trans or cis-beta-carotene, echinenone, all-trans retinyl palmitate, but also sterols, phospholipids, di/tri glycerides and ceramides. the romanian varieties were more rich in polar carotenoids and lipids, in general, while the uk tomatoes proved to be enriched in non-polar derivatives, especially esterified carotenoids, keto-carotenoids and glycerides. new molecules were identified, as good discriminatory markers of each tomato variety. acknowledgements. this work was supported by a grant of the romanian national authority for scientific research and innovation, cccdi -uefiscdi, project nr. 16/2015, pncdi3. glycogen is a multi-branched polysaccharide that serves as the main form of glucose storage in the body, where the main reserves are in the liver and muscle. it has been observed that glycogen metabolism is altered in many tumor types, and that glycogen content is inversely correlated with proliferation rate. in addition, it has recently been described that when glycogen accumulation is forced in glioblastoma u87 cells in hypoxia, senescence is induced and tumor growth is inhibited in vivo. our laboratory has various animal models with different parts of the glycogen metabolism pathway affected. most notably, we have two animal models lacking glycogen: muscle glycogen synthase (gys1 ko) and liver glycogen synthase (gys2 ko) knockout animals. we isolated mouse embryonic fibroblasts (mefs) from gys1 ko to perform replicative senescence assays. in addition, we induced hepatocellular carcinomas in gys2 ko animals via n-nitrosodiethylamine (den) injections in order to track tumorigenesis in animals lacking hepatic glycogen. lastly, we performed partial hepatectomies (phx), which involves the resection of two thirds of the liver, on gys2 kos to evaluate the effect of the lack of glycogen on hepatocyte proliferation. interestingly, we have observed that glycogen levels are increased in human and mouse fibroblasts under replicative senescence, and that mefs depleted of glycogen bypass senescence and immortalize faster than wts. we have also demonstrated that senescence pathways are down regulated in mefs lacking glycogen. furthermore, gys2 kos treated with den show higher tumor burden and mortality than controls. we also evaluated the effect of glycogen on hepatocyte proliferation after phx. gys2 ko mice present faster proliferation and liver regeneration rates, when compared to wt counterparts. collectively, our preliminary data suggest that glycogen metabolism plays a crucial role in the regulation of cell cycle in both physiological and pathological states. it is established that pineal is involved in circadian regulation of testosterone secretion from leydig cells. however, the precise routes of this regulatory involvement are still unknown. as cgmp has been also regarded as modulator of steroidogenesis we sought to study the effects of pineal removal on the circadian pattern of cgmp variations and expression of the genes that encode elements of no-cgmp signaling pathway in adult rat leydig cells. the analysis was performed on testicular leydig cells obtained from pinealectomised and shame pinealectomized rats, in six time point during 24 hours. the pinealectomy was confirmed by serum melatonin eia measurement. the androgen levels were measured by ria; cgmp by eia and gene expression was quantified by rq-pcr. all results were analyzed by cosinor method. data revealed circadian transcriptional pattern of nos2, nos3 (genes encoded no producers) and pde5a (gene for cgmp remover) in leydig cells from adult rats. pinealectomy significantly increased expression of nos2 which lost rhythm and increased and delayed amplitude of nos3 expression. further, pinealectomy initiated cyclic transcription of gucy1b3 and noncyclic transcription of gucy1a3 (genes encoded cgmp producers) and increased mesor and amplitude of pde5 transcription. the transcription of prkg1, the main effector in this signaling pathway was not affected with pineal abolition. additionally, pinealectomy did not influence the circadian transcription profile of coxi2 or other investigated genes (coxi1, nrf1, nrf2a, pgc1a) related to mitochondrial function and biogenesis. finally pinealectomy reversed phase of circadian cgmp oscillation in leydig cells, increased amplitude and slightly advanced peak of serum testosterone oscillation. results suggested pineal influence on circadian rhythm of no-cgmp signaling in leydig cells. further studies based on these data are needed to better understand the relationship between pineal and circadian rhythm of testosterone production. influenza is a contagious respiratory infection caused by a variety of influenza viruses. neuraminidase inhibitors is a new class of antiviral drugs that inhibit influenza viruses. the most popular antiviral agents is oseltamivir, having a commercial name of tamiflu, within anti-influenza antivirals. as well as tamiflu is a member of neuraminidase inhibitor group drug. therefore, this study was performed to determine the effect of tamiflu on cultured human peripheral blood lymphocytes. material and methods: for examining the presence of the indirect mutagenic effect of oseltamivir in iver s9 fraction mix was used. cells were treated with 0.5, 1 and 2 lg/ml oseltamivir, the tamiflu capsule ingradient, for 24 or 48 hours in the absence or presence of an exogenous metabolic activation system. the test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. on the other hand, some concentrations of tamiflu induced sce and also decreased significantly the proliferation index (p ˂ 0.05) in the absence of s9 mix. result: tamiflu did not induce significant increases of ca or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but week sce induction was observed. on the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the s9 mix. discuss and conclusion: tamiflu weakly induced sce at the highest concentration with/without added s9 mix in cultured human peripheral lympocytes. it could be assumed to be a sce inducer. sces can be increased by several agents that attack dna. tamiflu decreased the proliferation index and nuclear division index at some concentrations thus interferring it as being weakly cytotoxic, though this effect disappeared in the presence of s9 mix applications. this finding is important for showing the inefficiency of tamiflu metabolites on the cell cycle. introduction: chronic renal failure as a result of the progression of diabetic nephropathy is the main cause of mortality in patients with type 1 diabetes. chronic hemodialysis is a life-saving therapy for patients with strong renal disorders. the main goal of hemodialysis is toxins removal from the patient. the monitoring of hemodialysis is the best way for biomedical evaluation of correctness and efficiency of this clinical treatment. according to the published data, the markers of development of diabetes complicated with renal failure are increased levels of glucose, urea and creatinine in the patient blood. today colorimetric and spectrometric methods are most commonly used for determination of the above metabolites in biological samples. however, these methods are complex in application, have low selectivity, and require pretreatment of samples. materials and methods: we propose for levels of glucose, urea and creatinine detection the potentiometric multibiosensor based on ph-sensitive field-effect transistors and immobilized enzymes developed in our laboratory. results: we developed a potentiometric multibiosensor and studied its main analytical characteristics. linear dynamic ranges of determination of substrates were following: 0.08 -1 mm of glucose, 0.1-10 mm of urea, and 0.02 -2 mm of creatinine. it was shown that the potentiometric multibiosensor had good reproducibility, and its bioselective elements were working independently from each other, because test of substrates cross-selectivity was negative. discussion and conclusion: very sensitive, fast and selective multibiosensor for simultaneous measurement of three metabolites in a single cycle based on ph-sensitive field-effect transistors and immobilized enzymes is developed. the developed potentiometric multibiosensor was verified by quantitative analysis of glucose, urea and creatinine in blood serum of patients with diabetic nephropathy. p-03.04.4-002 ph-dependent interaction of asymmetrically charged peptides with a protein nanopore over the past two decades, the ability to use natural or artificial nanopores to probe at uni-molecular level the structural and kinetic features of various bio-molecules (peptides, dna, rna) was successfully achieved. the operating principles of the nanopore-based single-molecule technique are simple: the single macromolecule capture, entry and subsequent translocations through a free-standing, voltage-biased nanopore, depend upon the physico-chemical and topological features of the analyte. the concentration, identity volume and charge of the analyte are then deduced from the analysis of the stochastic current blockade events caused by the trafficked analyte across the nanopore. herein, we used the a-hemolysin (a-hl) nanopore and set up an experimental model providing efficient control of a-hl-peptide interactions, in the presence of a ph gradient across the nanopore. for this, we engineered a 36 amino acids long peptide containing a neutral asparagines-containing sequence, flanked by oppositely charged aminoacid patches at the n-(glutamic acids) and c-termini (arginines), whose length was set as to span a single a-hl protein. when the ph of the solution in contact to the a-hl's b-barrel opening is changed from neutral to acidic values, the electrostatic interactions between the protein's mouth and either the n-or c-terminus end of the peptide occurs, and this influences strongly the dynamics of a peptide translocating the nanopore. we further proved that during the same experiment, peptide entry into the nanopore can be set to occur with either n-or c-terminus end head on, by simply changing the sign of the transmembrane potential across the nanopore. nanopores are emerging as a powerful and broadly applicable tool in biophysics, which allows one to study the features of charged macromolecules under confinement. a few noteworthy examples are: determining the electrophoretic mobility, effective charge and diffusion coefficients of charged molecules; exploring the folding and unfolding of peptides and proteins; analyzing biopolymers trafficking, protein transport, dna translocation, rna and dna sensing and sequencing. herein, we employ single molecule analysis techniques using a wild-type ahemolysin (a-hl) protein nanopore to study the capture and translocation behavior of a short cationic peptide (20 amino acids in length) at an extremely low ph value. our experiments revealed that an effective absorbing field is created by the electroosmotic flow, against the electrophoretic force, which enables the peptide capture inside the nanopore. furthermore, our findings show that the trajectory of a single peptide can be experimentally visualized and the main steps determined: the peptide capture, reversible translocation across the pore's vestibule and lumen regions, and the peptide release from the nanopore. also, the kinetic analysis of the main steps observed allowed us to describe the free energy profile of the peptide interactions with the protein nanopore. the presented work provides evidence for the ability of controlling the dynamics of a single-peptide, its capture and passage inside a a-hl nanopore, that underlie the processes naturally occurring in cells, thus proving a powerful approach for probing single molecule biophysics phenomena, in general. changes in the physical conditions of the cancer microenvironment driven by elevated tissue growth and angiogenesis, may introduce exposure of laminar fluid flow, which effect the key factors of cancer, such as progression, immune-escaping and metastasis. conventional experimental models fail to mimic the physical cues on tumor microenvironment. microfluidic culture techniques allow precise control of fluids, simultaneous manipulation and analysis of cultured cancer cells. here, we present a platform that can be used for the investigation of the role of flow mediated mechanical stimuli on cancer cells. microfluidic cell culture platform was fabricated using polymethyl methacrylate and double-sided adhesive films with 25 9 41 mm dimensions. ovary adenocarcinoma cells (efo-27 and onco-dg-1) were used for the optimization of the platform. to understand the fluid and gas distribution patterns, specific modeling was performed. dynamic microfluidic cell culture and static conventional cell culture conditions were compared for the differences of cancer cell phenotype, such as proliferation, viability, epithelial-mesenchymal transition. we confirmed that, the proliferation and viability of cancer cells are increasing under dynamic fluid flow. the proliferation rate of ovary adenocarcinoma cells was correlated with the increase of fluid flow rate. immunocytochemical analysis showed that fluid flow causes decrease in e-cadherin expression, and increase in n-cadherin and vimentin expressions, which indicate mesenchymal phenotype of cancer cells. our results showed that, cancer cells present different characteristics due to fluid flow of tumor microenvironment. to understand the role of physical dynamics by using microfluidic culture techniques, is a key to elucidate the mechanisms underlying disease progression, and may lead to new diagnostics and therapeutic approaches. (this study was funded by turkish scientific and technical research council (tubitak-214s334). high-sensitive detection of low-affinity antibodies by immuno-pcr with supramolecular olygonucleotide-streptavidin complex detection of low affinity antibodies in blood sera and cell surface outwashes is important both in the study of molecules that bind to cellular receptors (circulating tumor cell masking antibody, for example) and medicine (diagnosis of allergy). low affinity igm and ige antibodies can not sometimes be determined by conventional methods. we using supramolecular oligonucleotide-streptavidin complex formed from single-stranded synthetic oligonucleotide (60 n) contains biotin on 5'-and 3'-ends, and sterptavidin in molar ratio 1:1. this complex represents a structure with equivalent electrophoretic mobility of 600 bp dna and preferred "valency" of streptavidin is 2. this universal immuno-pcr approach make it possible to increase a signal by using several oligonucleotides per one antibody. after the method optimization we achieved 100-1000 times highter sensitivity than elisa. to reduce the matrix effect we used 100-500 fold dilutions of sera samples. this approach achieved a significant advantage, because it allows working with small-volume samples (need only 2 mkl of serum sample). antibodies to the disaccharide galb1-3glcnac (le c ) are typical of the natural antibodies. the igm anti-le c antibodies are found in almost healthy people without the epitope specificity variation. we have shown that the concentration of igm anti-le c antibodies was higher (p ≤ 0.0005) for health donor sera (n = 45; 31.3 ae 4.3 pg/ml) compared with sera from patients with breast cancer (n = 38; 17.0 ae 4.1 pg/ml). sensitivity of igm anti-le c antibodies detection was 100 pg/sample (50 mkl) ie 6.7 9 10 7 molecules. thus for the immuno-pcr detection of antibodies the 10 3 -10 4 tumor cells are sufficient. such amount of cells seems to be a realistic one for detection of antibodies masking circulating tumor cells. this study was supported by a grant from russian science foundation (#16-14-00136) and by russian federation president scholarships donated to d. yu. riazantsev (# sp4413.2015.4 bacterial pathogen detection and identification is of crucial importance for disease diagnosis, bacterial contamination surveys and water quality assessment. we propose herein a novel method for bacterial detection based on the interaction of single gram-negative bacterial cells (i.e.: escherichia coli and pseudomonas aeruginosa) with an ahemolysin (a-hl) protein nanopore embedded in a reconstituted lipid bilayer, at neutral ph. as a consequence of an applied voltage, the negatively charged bacteria suspended in saline buffer solution are electrophoretically driven towards the pore opening, inducing reversible blockages in the ionic current through a-hl. experiments were also performed in the presence of an antimicrobial peptide, cma3, as well as in acidic environment. statistical analysis of the frequency and duration of blockage events allowed us to discriminate between the two types of bacteria. the frequency of interactions was higher for escherichia coli with respect to pseudomonas aeruginosa. adsorption of cma3 peptides on the membrane of bacteria increased the frequency of interactions with the pore, contrary to the expected effect induced by lowering the net surface charge of the cells. in experiments performed at ph = 4, the frequency of blockage events was found to be two orders of magnitude higher, with longer interaction life-times. the net negative charge (7 uncompensated aspartate residues) localized at the entrance of the pore contributes an additional electrostatic repulsion interaction between negatively charged bacterial cells and a-hl. thus, adsorption of cationic peptides at the interface will reduce this repulsive interaction. the same effect was recorded at ph = 4, when the aspartate residues are partially protonated, confirming our understanding of the previously observed results. this method could be further developed and integrated with other techniques, making nanopore-based systems a fast and reliable bacterial detection and identification tool. this study was performed to analyze the effects of tunicamycin (tm) and taurohyodeoxycholic acid (tudca) on thle-3 cells. cells were treated with tm to induce endoplasmic reticulum (er) stress and tudca was administered as an er stress inhibitor. cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations of either tudca, tm or both. thle3 cells were cultured in fibronectin, bovine collagen i and bovine serum albumin coated plates. cell lines were grown in begm media supplemented with epidermal growth factor, phosphoethanolamine, fetal bovine serum, 100 u of penicillinstreptomycin and maintained in a humidified incubator at 37°c and a 5% co 2 atmosphere. cell viability was measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay kit. cells were grown to confluence in 96well plates and incubated with 1 ll/ml dmso, 1-10 lg/ml tm, 0.1-5 mm tudca, or 10 lg/ml tm + 0.1-1 mm tudca for 18-48 hours. control cells were prepared in plates containing only medium. at the end of the incubation period, mtt was added to each well and incubation was carried out for 4 hours at 37°c. formazan production was expressed as a percentage of the values obtained from control cells. at all hours of incubation neither dmso or 1 mm tudca was cytotoxic. at 24 and 48 hours incubations 5 mm tudca and 10 lg/ml tm + 1 mm tudca were significantly cytotoxic compared to control, dmso and 1 mm tudca groups. treatment of cells with 0.5 mm tudca 8 hours before administrating 10 ug/ml tm significantly decreased the cytotoxic effect of tm. we conclude that tudca may show cytotoxic effects at 1 mm concentration when treated with tm. therefore 0.5 mm of tudca, administered 8 hours before tm treatment should be applied to protect against er stress. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; 214s223). recent studies reveals that history of preeclampsia is an independent risk factor for cardiac events and stroke. lipoprotein-associated phospholipase a2 (lp-pla2) is a vascular inflammatory marker associated with cardiovascular diseases (cvd). we hypothesize that vascular inflammation (lp-pla2 mass, activity, index) related genetic variations (pla2g7) increase the risk for developing future cardiovascular disease in women with pe. a group of 150 preeclamptic patients and 50 normal pregnant women were recruited from university of istanbul, cerrahpasa medical school, department of gynecology and obstetrics included into the study. the control group was matched for maternal and gestational age at time point of sampling. preeclamptic patients were starified into two groups; early-onset and late-onset according to the 32 gestational weeks. enzyme-linked immunosorbent assay procedure was used to determine the serum lp-pla2 mass level. lp-pla2 activity were determined by kinetic method. plag7 snp genotyping performed by using the sequenom massarray iplex. the rs1805017 tt genotype had a higher lp-pla2 index (p = 0.015) for early onset preeclampsia, cc genotype had a higher lp-pla2 mass and lp-pla2 index for late onset preeclampsia. no difference were found for control. the rs1809381475 gg genotype had higher lp-pla2 mass and index for late onset preeclampsia (p = 0.008, p = 0.012 respectively). stepwise logistic regression analysis performed to identify cardiovascular disease related variables that independently and significantly contributed to the presence of alleles of rs1805017 and rs1809381475 snps in early, late onset preeclampsia and control group. only lp-pla2 mass was independently and significantly associated with both snps in early onset preeclampsia. the association between lp-pla2 mass, index and rs1809381475, rs1805017 snps might be useful genetic markers to adress future cvd risk in patients with preeclampsia. introduction: b-thalassemia is one of the most monogenic autosomal recessive disorder characterized by defective production of the b-chain of hemoglobin. definition of the b-globin genotype is necessary for genetic counselling in the carriers, and for predicting prognosis and management options in the patients with thalassemia. dna-based diagnosis of b-thalassemias routinely relies on polymerase chain reaction (pcr) and gel electrophoresis. the aim of this study is to develop a new procedure, a dna-based piezoelectric biosensor, for the detection of b-thalassemia ivsi-110 mutation, the most common b-thalassemia mutation in turkey. materials and methods: b-globin gene of genomic dna isolated from whole blood, was amplified by pcr. bioactive layer was constituted by binding 2-hidroxymetacrilate metacriloamidoscystein (hema-mac) nanoploymers on the gold electrode's surface. single oligonucleotide probes specific for ivsi-110 mutation of b-thalassemia were attached to the nanopolymer via reactive cross-linker glutaraldehyde. the measurements were executed by piezoelectric resonance frequency which is caused by binding of pcr products in media with single oligonucleotide probe on the electrode surface. the results were confirmed by the conventional molecular method as arms. results: the piezoelectric resonance frequencies obtained by hybridization of the pcr products on bioactive layer were found 216 ae 12, 273 ae 6, and 321 ae 9 hz for the samples of normal b-globin, heterozygote, and homozygote of ivsi-110 mutation, respectively. discuss and conclusion: the developed biosensor serves as a specific result to ivsi-110 mutation. it could accurately discriminate between normal and ivsi-110 mutation samples. because of low costs, fast results, specificity and high detection/information effectiveness as compared with conventional methods, we can be offered this techique as an alternative to conventional molecular methods. the increasing use of nano-sized materials in the last several years has compelled the scientific community to investigate the potential hazards of these unique and useful materials. one of the most widely used nanoparticles is titanium dioxide. the objective of the research is to investigate the alterations in molecular and cellular responses in culture of primary lymphocytes to tio2 nps. human lymphocytes isolated from heparinized blood of healthy individuals were exposed to tio2 nanoparticles. viability, ros generation, the changes in the expression of genes encoding proinflammatory mediators tnf-a, il-1b and il-8 and dna damage were assessed. human lymphocytes were incubated with nanoparticles of different concentrations and viability was determined in 24 and 48 hours after treatment, respectively. cell viability was decreased by a treatment with nanoparticles in both a time-and concentration-dependent manners. the ability of tio2 to induce ros formation in lymphocytes was evaluated using dcf fluorescence as a reporter of oxidant production. the fluorescence intensity of oxidized dcf was increased in cells treated with nps. this means that ros generation occurred in response to the treatment with tio2. to investigate the expression level of mrna related to the inflammation responses in human lymphocytes real-time pcr was performed. the expression of il-1b, il-8 and tnf-a genes were increased by the exposure to nanoparticles of 10, 20 and 40 lg/ml for 24-48 hours. tio2 nanoparticles were shown to induce the dose-dependent fragmentation of dna strands. much evidence of hazardous health effects of nps has been reported. in this study, viability was reduced under the exposure to tio2. oxidative stress was elevated by the treatment with tio2 nps. oxidative stress can also trigger inflammation signals. induced by exposure to nanoparticles they may cause the translocation to the nucleus of transcription factors, which regulate proinflammatory genes, such as tnf-a, il-1b, il-8. background: endothelial cells (ec) represent one of the primary targets of the major pro-inflammatory cytokinetumor necrosis factor (tnf). development of the new approaches for the treatment of acute and chronic inflammatory conditions, including the strategies aimed to tnf neutralization, requires the usage of the adequate cellular models closely resembling the properties of the endothelium. the endothelium-derived ea.hy926 cell line expresses several inflammation and neoangiogenesis markers in response to activation factors however their expression can differ from the patterns demonstrated by primary ec. the aim of the current study was to compare the expression of the known endothelial cellular markers including receptor of vascular endothelial growth factor-2 (vegfr2) and a v b 3 -integrin on 2d and 3d cultures (spheroids) of ea.hy926. methods: the ea.hy926 cell line was used with permission from dr. edgell. the cells were cultivated in the presence of tnf (25 ng/ml) or vegf a (25 ng/ml) for 5 hours. mrna was isolated using rneasy kit from qiagen and reverse-transcribed with revertaid kit (fermentas). rt-pcr was performed with specific primers. expression of vegfr2 and a v b 3 -integrin was visualized by confocal microscopy using specific monoclonal antibodies and previously developed fluorescent hybrid proteins. results: the expression of a v b 3 -integrin and vegfr-2 increased on the 3d culture compared to 2d according to confocal microscopy and rt-pcr. the aforecited methods revealed elevated expression of a v b 3 -integrin in the 3d culture of the ea.hy926 cell line activated with tnf. also increased expression of vegfr2 in the 3d culture activated with vegf a. then by confocal microscopy, we analyzed our fluorescent hybrid proteins that bind a v b 3 -integrin and vegfr2 on the surface of 2d and 3d cultures as well as antibodies with fluorescent label. conclusions: 3d cultures of the ea.hy926 cell line represent a promising model for the inflammation studies. tumor necrosis factor (tnf) is a trimeric cytokine associated with the inflammatory response to tissue injury and found to possess a key role in rheumatoid arthritis pathogenesis. spd 304 is a highly toxic recently discovered tnf inhibitor that promotes trimer dissociation and lead to the inactivation of the protein. according to the traditional anti-tnf treatment of ra, we aim at extracellular inhibition of this pro inflammatory cytokine as an effective therapy. the project plan comprises design, synthesis and validation of candidate inhibitors (measurement of dissociation constant and aqueous solubility). because of the elevated percentage of insoluble compounds a solubility enhancement protocol has been developed. the experimental procedure was the following:: a. drug design. identification of novel drug compounds are based on two approaches: i) structure based drug design using the 3d structure of tnf and ii) design of more potent and less toxic spd304 analogues. b. drug synthesis. a series of spd304 analogues were in house synthesized while novel candidates discovered by in silico approaches were commercially available. the purity of the majority of the compounds exceeded 90%. c. solubility measurement and enhancement. samples were incubated under specific conditions that can enhance aqueous solubility and solubility measurement with a direct uv method pursued. d. measurement of the dissociation constant. a fluorescence binding assay was used in order to evaluate the inhibitory activity of the compounds. from our results it can be concluded that dmso, peg3350 and b-cyclodextrin can be used for solubility enhancement without interfering with fluorescence assay. however peg3350 -in contrast to dmso-is not suitable for isothermic titration calorimetry measurements. dissolution procedure also plays a crucial role in the levels of solubility reached. finally, it has been shown that some of the studied spd304 derivatives have better dissociation constants than spd304. the effect of exercises on serum bmp-6 levels of knee osteoarthritis cytokines. more complicated approaches are expected to focus on molecular proteins as bone morphogenetic proteins (bmps) of the transforming growth factor (tgf)-beta superfamily. bmps associated with many cellular functions, such as proliferation, differentiation, and apoptosis. bmp-6 is significantly important for the endochondral bone formation. inflamation can induced serum bmp levels in oa patients. the aim of this study is to evaluate the clinical findings of oa patients after the isokinetic exercise together with the serum levels of bmp-6 to sustain the molecular approaches for treatments. a total of 36 patients were included in this study. the groups are formed as follows: group 1, oa patients before the exercise; group 2, oa patients after the exercise; group 3, oa patients before the isokinetic exercise; group 4, oa patients after the isokinetic exercise clinical and biochemical findings were evaluated before and after 3 weeks of the exercise programme. self reported severity of pain was measured using the 100 mm visual analog scale (vas), womac scores were calculated and isokinetic knee muscle strength testing was measured using cybex dynamometer that a standardized protocol previously described was applied in a subject-specific range of motion. serum bmp-6 levels of all patients were studied by elisa method. results represented a better vas and womac scores for all exercise groups after treatment. the serum bmp-6 levels were significantly decreased in group 2 compared to group 1 (222.94 ae 44.66; 246.49 ae 38 .15 respectively, p < 0.01) and in group 4 compared to group 3 (246.74 ae 32.26; 256.23 ae 33.11 respectively, p < 0.05). there is not any statistically differences between group 2 and group 4 (p > 0.05). as a conclusion, the decreased serum levels of bmp-6 may be suggested as a biochemical marker for oa patients during exercise programmes. p-04.04.4-006 tnf-a blokade efficiently reduced severe intestinal damage in necrotizing enterocolitis c. tayman, s. aydemir, i. yakut, u. serkant, a. c ß iftc ßi g€ olbasi devlet hospital, ankara, turkey objectives: to ascertain the beneficial effects of infliximab an inhibitor of tumor necrosis factor alpha (tnf-a) on the development of nec in an experimental nec rat model. material and methods: thirty newborn sprague-dawley rats were randomly divided into three groups as nec, nec+ infliximab, and control. nec was induced by enteral formula feeding, exposure to hypoxia-hyperoxia and cold stress. pups in the nec+ infliximab group were administered infliximab at a dose of 10 mg/kg daily by intraperitoneal route from the first day until the end of the study. all pups were sacrificed on the 5th day. proximal colon and ileum were excised for histopathologic, immunohistochemical (tunel and caspase-3), and biochemical evaluation, including, total antioxidant status (tas), total oxidant status (tos), malonaldehyde (mda), and myeloperoxdase (mpo) and tnf-a activities. results: we observed better clinical sickness scores, weight gain, and survival rate in the nec+ infliximab group compared to the nec group (p < .05). histopathological and apoptosis examination (tunel and immunohistochemical evaluation for caspase-3) revealed lower damage in the nec+ infliximab group compared to the damage in the nec group (p < .01). tissue mda, mpo, tnf-a levels, and tos were significantly decreased in the nec+infliximab group, whereas tas was significantly increased in the nec + infliximab group (p < .01). conclusion: tnf-a blockade with infliximab efficiently reduced the intestinal injury and preserve the intestinal tissues from severe intestinal damage by its complex mechanisms on nec. therefore, it may be an alternative option for the treatment of nec.keywords: tnf-a; infliximab; necrotizing enterocolitis; newborn; protection; rat; treatment p-04.04.4-007 short-term diabetes causes cardiovascular inflammation: anti-inflammatory effect of resveratrol introduction: diabetes is a metabolic dysfunction and has been associated with various disorders including inflammation, cardiomyopathy and coronary artery disease. inflammation is a protective mechanism elicited by the host in response to infection, injury, and tissue damage. the aim of this study was to investigate the effect of intraperitoneally resveratrol administration on cardiac and vascular function in diabetic rats. materials and methods: diabetes was induced in sprague-dawley rats by using injection of streptozotocin (55 mg/kg, i.p.). rats were divided into group i: control, ii: control/20 mg/kg resveratrol; iii: diabetic/vehicle; and iv: diabetic/20 mg/kg resveratrol. histopathological examinations with masson's trichrome and verhoeff-van gieson staining were carried out to reveal cardiac and vascular tissue damage and inflammation. in addition to plasma glucose and cardiac & vascular mda levels were measured by standard enzymatic kits while tnf-a, il-1b, il-18 (mbl) were analyzed by elisa kit. results: final body weight decreased in all groups compared to control. in the diabetic rats, plasma glucose and vascular mda levels were enhanced while cardiac mda was unchanged compared to control. vascular tnf-a, il-1b and mbl and cardiac mbl were increased in the diabetic groups compared to control. discussion and conclusion: it has been found that resveratrol has greatly normalized altered parameters. taken together, resveratrol partly improved cardiac and vascular inflammation induced by diabetes. this may be due to the healing activity of resveratrol on pro-inflammatory markers. p-04.04.4-008 cytokine network is critical in growth hormone-induced resistance mechanism against curcumin which modulates jak/stat/ socs pathway in mda-mb-231 and mcf-7 breast cancer cells m. c ß elik, a. c ß oker g€ urkan, e. damla arisan, p. obakan yerlikaya, n. palavan unsal t.c. istanbul k€ ult€ ur university, istanbul, turkey curcumin (diferuloylmethane), a polyphenolic compound that triggers apoptotic cell death in various cancer cells such as prostate, colon, melanoma and breast cancer. a pituitary-derived hormone, growth hormone (gh) play role in elongation and differentiation of ductal epithelia into the breast terminal and buds. in this study, our aim is to determine the role of inflammation in curcumin induced apoptotic cell death via acting on jak/stat/socs pathway in wt and gh+ mda-mb-231 and mcf-7 breast cancer cell lines. according to mtt cell viability assay curcumin triggers cell viability loss in time and dose dependent manner in mda-mb-231 wt and mda-mb-231 gh+ breast cancer cell lines, respectively. selected concentrations of curcumin as 20 lm (for mcf-7) and 25 lm (for mda-mb-231) decreased cell proliferation and induced apoptosis through causing jak2 dephoshorylation, stat1, 3, 5 dimerization and acting on socs proteins expression in each cell lines. in addition, activated jak/stat/socs pathway, via forced gh expression has been suppressed following curcumin treatment for 24 hours. 20 lm curcumin-induced apoptotic cell death via dephosphorylating jak2 at tyr1007/1008 residues and decreased phospho-stat1, 3 level in both breast cancer cell lines. although curcumin dephosphorylated stat5 in both mda-mb-231 and mcf-7 wt cells, no significant effect has been observed in mda-mb-213 gh+ and mcf-7 gh+ cell lines. in consequence, although forced gh expression induced cell proliferation in mcf-7 and mda-mb-231 breast cancer cells, curcumin overcame gh-mediated resistance mechanism via acting on jak/ stat/socs signaling, which is related to pparg-induced inflammation. acknowledgment breast cancer is one of the highest cancer type among women worldwide. various enviromental and genetic factors such as age, gender, family history, metabolic diseases and gene mutations are involved in the breast cancer pathogenesis. growth hormone (gh), a pituitary derived hormone, has essential role on postnatal growth and development. it is also established that signalling route of gh and its receptor (ghr) activity is increased in different cancer types. curcumin, a nutraceutical deriatives from rhizomes of turmeric (curcuma longa), has potential therapeutic activity against cancer cells, including breast cancer. curcumin inhibits proliferation of cancer cells such as prostate, colon, melanoma, cervical and breast cancer via induction of apoptosis and inflammation. stat5, a major downstream target of gh/ghr signalling, is related to survival, proliferation and differentiation. in this study, our aim was to investigate curcumin-induced apoptotic cell death in gh overexpressed mda-mb-453 breast cancer cells via jak-stat/socs signalling and inflammatory response profile. according to mtt cell viability assay, curcumin decreased cell viability in time and dose dependent manner in wt and gh+ mda-mb-453 breast cancer cell lines. we found that 20 lm curcumin-decreased in apoptotic cell death through inactivity at jak2 which led to dimerization of stat1, stat3, stat5. concomitantly, curcumin affected stat regulating socs proteins in mda-mb-453 breast cancer cell line. in addition, we demonstrated that 20 lm curcumin induced pparg expression and altered inflammatory cytokine signalling cascade. consequently, although gh overexpression led to agressive profile in mda-mb-453 breast cancer cells, curcumin overcame this resistance. inflammation is involved in many systemic disturbances, including osteoarthicular or skin diseases, coordinating the signaling network that contributes to tissue injuries. the aim of our study is to reveal pro-inflammatory messengers at the cutaneous barrier (keratinocytes, fibroblasts, endothelial cells), simulating the dermal impact of active principles, especially polyphenols and flavones from vegetal sources: salvia officinalis, asculum hippocastanum and calendula officinalis. we focused on il6 and il8 cytokines as main mediators of inflammation progression, correlated in keratinocytes with il1a as skin irritation indicator and vegf as pro-angiogenic factor, as well as in endothelial cells with icam-1 and vcam-1 adhesion molecules expression. in order to in vitro mimic the inflammatory conditions, we used targeted stimuli for each type of cells: for fibroblasts and endothelial cells -tnfa, a systemic stimulus, single or combined with pma that activates protein kinase c and up regulates nadph oxidase, which lead to superoxide anion production; for keratinocytescontrolled uv-a and uv-b radiation, simulating the solar damages or potential uv interactions with active principles in light exposed skin. the main analysis technique was flow cytometry: beads bases assay for soluble factors and fluorescent antibodies staining. our results prove the different involvement of polyphenols and flavones in the anti-inflammatory mechanisms, depending of the vegetal source: active principles from salvia officinalis induce a strong inhibition of il6 and il8 in tnfa stimulated keratinocytes, fibroblasts and endothelial cells, reduce the icam-1 over-expression but have no effects on irradiated keratinocytes; biocomplexes from asculum hippocastanum inhibit only il8 release in stimulated fibroblasts, but protect keratinocytes from uv-a and uv-b radiation; compounds from calendula officinalis are active on il8 signaling in fibroblasts and counteracts only uv-b inflammation. ischemia and/or reperfusion injury is one of the most common causes of acute renal failure. ischemia-reperfusion associated with thrombolytic therapy, organ transplantation, coronary angioplasty, aortic cross-clamping, or cardiopulmonary bypass results in local and systemic inflammation. within the endothelium, ischemia produces expression of proinflammatory gene products (e.g. cytokines) and bioactive agents (e.g., endothelin), while preventing other "protective" gene products (e.g., thrombomodulin) and bioactive agents (e.g. nitric oxide). therefore, ischemia induces a proinflammatory state that increases tissue vulnerability to further injury on reperfusion. this experimental study was designed to investigate the protective effect of salvia l. extracts on kidneys from i/r injury. salvia lamiaceae have been used for treatment of some illnesses in turkish folk medicine. forty spraque dawley rats were divided into 5 groups (n = 8). right nephrectomy was performed to all groups. group i: control group; group ii: i/r group; group iii: i/r + 50 mg/kg salvia l.group; group iv: i/r + 100 mg/kg salvia l. group; group v: i/r + 50 mg/kg rosmarinic acid. group. salvia l. and rosmarinic acid for 7 days was given single dose as a gavage. 60 minutes ischemia, 60 minutes reperfusion were applied to groups except control. intracardiac blood samples were taken, high sensitive crp (hscrp), tumor necrosis factor-a (tnf-a), interleukin (il)-6 and interleukin ib (il-1b) levels were detected. serum hscrp levels were also determined in our clinical laboratory using routine standard methods. serum tnf-a, il-6 and il-ib levels were evaluated using an enzyme-linked immunosorbent assay technique. mean values were evaluated by statistical analysis. serum hscrp, tnf-a, il-6, and il-1b concentrations were significantly increased after renal i/r as compared to the control group. our treatment group 50 mg/kg salvia l. and 50 mg/kg rosmarinic acid especially 100 mg/kg of salvia l. were found to show a protective effect against renal structure and function. we concluded that salvia l. extracts could be beneficial in the treatment of renal ischemic injury. but 100 mg/kg salvia l. extract were more effective than 50 mg/kg salvia l. extract and used as synthetic 50 mg/kg rosmarinic acid. acne vulgaris is a common chronic inflammatory skin disease of unknown etiology. excess levels of secretory phospholipase a2 (spla2) contributes to inflammatory diseases and studies indicate that lipoprotein lipase (lpl) has differential effects on several inflammatory pathways. the aim of the present study was to assess serum activity of spla2, lpl and evaluate changes in circulating protein levels of angiopoietin-like protein 3 (angptl3), angptl4, cyclooxygenase (cox) and prostaglandin e2 (pge2). serum from 21 control subjects and 31 acne vulgaris patients with moderate and severe disease was evaluated for levels of spla2, cox, pge2, lpl, angptl3 and angptl4. disease activity was determined according to the national health service (nhs) lambeth and southwark clinical commissioning group guidelines for the management of acne. lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods using autoanalyzers (beckman coulter au5800 clinical chemistry and unicel dxi 800 immunoassay systems). serum levels of spla2 and lpl were significantly increased in acne vulgaris patients compared to age and gender matched controls. no significant differences were found for cox, pge2, angptl3 and angptl4 levels between acne vulgaris patients and controls. the results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly increased serum spla2 activity. increased lpl activity in serum of acne vulgaris can be protective in patients through its anti-dyslipidemic actions. to our best knowledge, this is the first study investigating spla2, lpl, angptl3 and angptl4 levels in acne vulgaris. future studies are aimed to understand the regulation of spla2 and lpl expression in acne vulgaris patients. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; #115s940). p-04.04.4-013 8-ohdg and hogg1 levels are as an oxidative dna damage markers in acne vulgaris treated with isotretinoin h. ecevit, m. izmirli, b. gogebakan, e. rifaioglu, d. sonmez, b. bulbul sen, t. sen, h. m. okuyan mustafa kemal university, hatay, turkey acne vulgaris is a skin disease that characterized by comedones, papules, pustules, nodules and cysts at face, back and body skin. isotretinoin is one of the treatment agents in acne vulgaris. about 4 weeks after drug treatment, the amount of sebum which is produced by sebaceous gland reduces keratinization disorder and the number of propionibacterium acnes normalizes. however, isotretinoin is known that has a wide range of side effects. in recent studies, isotretinoin treatment has been shown to increase the oxidative stress. 8-hydroxy-2 0 -deoxyguanosine (8-ohdg), an important indicator of oxidative dna damage, hydroxyl ion is bound at the 8th carbon of guanine. this structure is repaired through a base excision repair mechanism and the human 8-oxoguanine dna glycosylase 1 (hogg1) plays a key role in this processes. in this study we aimed to evaluate the dna damage and it's repair in acne vulgaris before and after 6 months of isotretinoin treatment by measuring 8-ohdg and hogg1 levels. the current study includes 43 acne vulgaris patients who are diagnosed in mustafa kemal university, department of dermatology. 8-ohdg and hogg1 levels were measured by enzymelinked immunosorbent assay (elisa) method for before and after 6 months of isotretinoin treatment. the commercial elisa kits (cloud-clone corp; usa and cell biolabs; usa) were used for the assessment of hogg1 and 8-ohdg, respectively.both 8-ohdg (p as a conclusion, isotretinoin increases dna damage and high serum 8-ohdg and hogg1 levels as a result of isotretinoin treatment may effect on the amount of reactive oxygen species. the pineal gland is a circumventricular organ which serves as a major neuroendocrine gland in the brain. its primary function is the production of melatonin which is controlled by signals from the suprachiasmatic nucleus. melatonin codes the length of the night and it is well recognized for its anti-inflammatory effects. lipopolysaccharide (lps) is the essential component in the outer surface membrane of gram-negative bacteria and act as a strong stimulator of natural and innate immunity in all eukaryotic species. furthermore, lps reduces melatonin synthesis and induces the expression of the serine protease inhibitor 3 (spi-3) in the stat3-mediated manner in pinealocytes. however, the precise function of stat3 in the cell signaling in the pineal gland is not yet known. here we investigated the effect of inhibition of stat3 on lps-induced changes in melatonin levels, expression of arylalkylamine n-acetyltransferase (aa-nat) and spi-3 in the pineal gland. experiments were performed in vitro using organotypic and primary cultures prepared from the rat pineal glands. levels of melatonin and spi-3 were determined from tissue homogenate by enzyme-linked immunosorbent assay (elisa). the pinealocytes were used to carry out sirna stat3 transfection. the successful transfection and subsequent decline in stat3 expression levels were proved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the changes in synthesis of aa-nat and spi-3 were studied by rt-pcr. in conclusion, lipopolysaccharide can affect the immunomodulators secreted by the pineal gland. the clarification of the effect of inhibition of stat3 on those immunomodulators is important from the clinical point of view because inhibitors of stat3 are nowadays used as tumour suppressors. silica nanoparticles have a great potential for a variety of industrial, diagnostic and therapeutic applications. in this study, we have evaluated the in vitro effects of amorphous silica nanoparticles (7 nm) using human lung mrc-5 fibroblast as model. cells were exposed to 62.5 lg/ml silica nanoparticles for 24, 48 and 72 hours. the cytotoxic and inflammatory response, and matrix metalloproteinase expression were examined. the pro-inflammatory cytokine il-1b, il-6, il-8, tumor necrosis factor (tnf-a), matrix metalloproteinases (mmp-2, mmp-9, mmp-1) and tissue inhibitor of metalloproteinase-1 (timp-1) were analyzed by western blot method. cytotoxicity was evaluated by lactate dehydrogenase (ldh) released into the culture medium by damaged cells. the level of ldh activity was increased after exposure to silica nanoparticles, in a time-dependent manner compared to control. the protein expression of il-1, il-6, il-8 and tnf-a as well as of mmp-1 and timp-1, was up-regulated whereas those of mmp-2, mmp-9 was down-regulated after 48 and 72 hours respectively. in conclusion, our data indicate that amorphous silica nanoparticles generate a cytotoxic and inflammatory response, as well as an imbalance in extracellular matrix due to the differential regulation of mmps and tissue inhibitor of metalloproteinase-1 in mrc-cells after 48 and 72 hours. p-04.04.4-017 association of fto gene variant (rs8050136) with markers of t2dm and obesity in population from bosnia and herzegovina and kosovo fto (fat mass and obesity-associated gene), recently discovered in a genome-wide association study for type 2 diabetes (t2d) encodes a 2-oxoglutarate-dependent nucleic acid demethylase and is mainly expressed in the hypothalamus. this gene may play important role in the management of energy homeostasis, nucleic acid demethylation, and regulation of body fat masse by lipolysis. the aim of this study was to analyze the association of this single nucleotide polymorphisms (snps) with clinical and biochemical parameters of obesity, t2d, prediabetes and at the level of healthy population from bosnia and herzegovina (bh). the study included 638 patients with t2d and prediabetes and 360 healthy controls both sexes, aged from 40 up to 65 years. patients were recruited at the clinical centre university of sarajevo, university hospital of clinical centre in banja luka, general hospital in te sanj and health centre in prizren. genotyping of analyzed polymorphism was performed by rt-pcr method in cooperation with the department of clinical chemistry, faculty of pharmacy, university of ljubljana (ljubljana, slovenia) and university hospital of charles university (hradec kralove, czech republic). our results did not show significant differences in genotype frequencies of the analyzed polymorphisms between patients with t2d, pre-diabetes and healthy population also, results of logistic regression analyses did not show significant association of risk a allele of fto gene polymorphism -rs8050136 with increased risk of t2d (or = 1.084, 95% ci 0.758-1.551, p = 0.659). a allele was significantly associated with higher values of hba1c, insulin, homa ir index, diastolic blood pressure and higher levels of inflammatory markers (fibrinogen and leukocytes). interestingly, a tendency of association of a allele with higher values of obesity markers (bmi, waist and hip circumference) was noted. further studies are needed on a larger population in order to confirm these results. the water extract of capparis ovata (cowe) has been shown to be used as an alternative medicine for the treatment of multiple sclerosis (ms). cowe was further fractionated and studied for additional anti-neuroinflammatory effects in sh-sy5y cells. for this purpose, the dichloromethane sub-fraction of the cowe extract was tested for its anti-inflammatory effects on selected anti-inflammatory genes believed to be important in ms pathophysiology using sh-sy5y cells. cell viability was assessed using lactate dehydrogenase (ldh) activity in the media conditioned by the crystal violet cell staining. in these cells, levels of the tumor necrosis factor-a (tnfa), nuclear factor kappa-lightchain-enhancer of activated b cells (nf-jb1), glial fibrillary acidic protein (gfap), c-x-c motif chemokine 9 and 10 (cxcl9, cxcl10), matrix metalloproteinase 9 (mmp9), chemokine (c-c) motif 5 (ccl5) and tyrosine-protein phosphatase non-receptor type 11 (ptpn11) were determined by quantitative reverse transcriptase-pcr assay (qrt-pcr). we have found out that the dichloromethane sub-fraction of cowe effectively inhibited the expression of all of the genes given above in sh-sy5y cells. thus, phytochemicals present in the dichloromethane sub-fraction of the cowe extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology. studies are underway to identify the individual compound(s) in this subextract of the cowe extract contributing to these effects. this work is supported by tubitak 112s187 and pamukkale university paubap 2014fbe051. p-04.04.4-019 apigenin and luteoline were identified as active anti-inflammatory constitutents of lavandula stoeachas by bioassay guided fractionation h. ipek 1 , s. savranoglu 2 , a. r. t€ ufekc ßi 3 , f. g€ ul 3 , i. demirtas 3 , t. boyunegmez t€ umer 4 1 graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, 2 graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, 3 department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, 4 department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: lavandula stoechas, in the genus of lavender, has distinct therapeutic uses among anatolian people. rather than worldwide use of its essential oil in aromatherapy, specifically the aqeous portion as decoction has been traditionally used in anatolia against the components of metabolic syndrome, all of which share a state of chronic inflammation as an underlying cause. the anti-inflammatory constiutents of l. stoechas were isolated using a bioassay guided fractionation in lipopolysaccharide (lps) inflammed raw 264.7 macrophages. materials and methods: an aqeous extract was partitioned into ethyl acetate (eae) and n-butanol fractions. the eae, determined as bioactive extract was seperated into 12 subfractions by column chromatography. e6 was identified as active subfraction subjected to sephadex column to get pure compounds which were then applied to nmr, ir, and uv analyses for structure determination. in raw 264.7 cells, the effects of extracts/fractions/subfractions/compounds on lps induced no production was determined by using griess method. the potential inhibitory effects of each compound on lps induced inos expression were determined by qpcr and western blot. results: p-coumaric acid, apigenin and luteoline were found in the e6, and the first two compounds appeared to be primarily responsible for the anti-inflammatory activity. apigenin and luteoline at 50 lm decreased no production 66 and 80%-ic 50: 56 and 26 lm-by inhibiting inos gene expression 84 and 88% as well as protein expression 94 and 99%, respectively (p < 0.05). conclusion: this is the first time that luteoline and apigenin have been found in eae of l. stoechas, and the anti-inflammatory properties of the eae can be attributed, at least in part, to the presence of these two compounds. we are on the way to gain further insight for the action mechanism of these two active principles as anti-inflammatory agent. tubitak (project id:112t442) support this work. the role of tip60 in the inflammation process immun response generates the first line of host defense during inflammation and plays an important role inducing pro-inflammatory response by generating early response against pathogens. il-6 (interleukin 6) is one of the pro-inflamatory cytokines and its expression increases during the infection to activate the jak/ stat pathway. jak/stat pathway is regulated by hamp (hepcidin antimicrobial peptide). our previous study, we reported that hamp gene expression was decreased in liver-specific tip60 conditional knockout mice, so we thought that tip60 may have a direct or indirect role on inflammation mechanism. tip60 (tat interacting protein, 60 kda) is a member of the myst enzyme family of histone acetyltransferases (hats) and plays an important role in multiple function including cellular signaling, dna repair, cell cycle and apoptosis. in this study, the quantitative gene and protein expression of il-6 were investigated by using taqman real time pcr, western blot and immunohistochemistry analysis in control group, lpsinduced inflammation group and liver-specific tip60 conditional knockout group mouse liver. according to our preliminary results, the gene and protein expression of il-6 was increased in lps-induced inflammation group (p < 0.001, p < 0.001) and liver-specific tip60 conditional knockout group mouse liver (p < 0.05, p < 0.01). our initial data suggest that tip60 may be essential for the inflammation process. this work was funded by grants from the scientific and technological research council of turkey (tubi-tak) (grant number: 114z277). although intracellular reactive oxygen species (ros) level is necessary to maintain cellular homeostasis, elevated intracellular ros level with the impact of unfavorable environmental conditions leads to oxidative stress that may cause damage to dna, proteins and lipids. in case of inflammation, organism seeks to provide cellular homeostatis by increasing ros levels via antioxidant molecules and enzymes. therefore, it was thought that there can be a direct or indirect relation between inflammation and oxidative stress. in this study, inflammation was performed by intraperitoneal injection of lipopolysaccharide (lps). the gene expression and activity of antioxidant enzyme including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione s-transferase (gst), glutathione reductase (gr) and glucose 6-phosphate dehydrogenase (g6pd). additionally, any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h 2 o 2 ) level are accepted as an indication for the accumulation of ros, the relative levels of them were also studied. to show our inflammation model was performed in mouse kidney with lps treatment or not, the expression of interleukin (il6), which is accepted as a inflammation marker, was investigated by real time pcr. the expression of il6 was significantly increased in lps treated group. while the level of mda and h 2 o 2 was elevated in lps treated group, gssg was decreased. no changes was seen for gsh level. the correlation was observed between enzymatic and molecular levels. while the gen expression and the enzyme activity of sod, cat, gst, gr, and g6pd were decreased, gpx was increased with inflammation. in conclusion, increasing ros level was observed in the inflammation process and, the antioxidant system was affected at the molecular and protein level. this work was funded by grants from the scientific and technological research council of turkey (tubitak) (grant number: 114z277). the aim of our study is to evaluate effect of vitamin d levels on hemogram parameters including neutrophil %, lymphocyte %, neutrophil % / lymphocyte % ratio (nlr) and mean platelet volume (mpv) in behcet's patients. fifty eight patients with diagnosis of behcet that applied to selcuk university faculty of medicine department of dermatology are recruited to the study. clinical and laboratory characteristics of the patients were obtained from hospital automation. t test was used to examine the differences between the parameters. p < 0.05 was taken to be statistically significant. there was a statistically significant difference between vitamin d values and age (p = 0.036) whereas difference was not significant between vitamin d and neutrophil %, lymphocyte %, nlr, mpv values. according to the literature, there are a lot of studies that show the relationship between vitamin d and hemogram parameters. however, contrary to the previous studies, we were unable to find any significant relationship between vitamin d and these hemogram parameters. these results serve the idea that the effects of vitamin d on the hematopoietic system should be further investigated experimentally and clinically. crimean-congo hemorrhagic fever is a tick-borne disease caused by the arbovirus and characterized by a sudden onset of high fever, severe headache, dizziness, back and abdominal pains. the exact pathogenesis of cchf has not been clarified yet. the aim of this study, clinical cases of cchf in cu, se and zn is to examine the relationship between the concentration of trace elements. the study sample consisted of 30 patients which have been diagnosed with cchf. matched for gender, 30 healthy volunteers were similar to the control group according to age. the patients and control groups, serum cu, zn and se levels were analyzed using atomic absorption spectrophotometer. cchf patients in the group, cu zn and se serum levels were significantly lower compared with the control group. in our study, the cofactor of the antioxidant enzyme cu, zn and se elements were lower. this shows us in cchf disease, a decrease in antioxidant enzyme activity, and suggest that they contribute to the immune system's degradation. p-04.04.4-024 inhibitors of mdm2 ubiquitin ligase as prospective modulators of autoimmunity e. bulatov, a. valiullina, r. sayarova, a. rizvanov kazan federal university, kazan, russia ubiquitin-proteasome system is seen as a pool of promising protein targets for therapeutic impact in many human diseases. mdm2 is an e3 ubiquitin ligase widely studied due to its wellknown role in cancerit negatively regulates p53 oncosuppressor that mediates apoptosis in tumour cells. inhibitors of p53/ mdm2 interaction have long been known as potential anticancer therapeutics. however, recent advances in the field suggest that both mdm2 and p53 might be playing a substantial role in autoimmune processes. we used a small molecule p53/mdm2 inhibitor nutlin-3a to test the effect of p53 activation on peripheral blood mononuclear cells (pbmcs) from both healthy volunteers and patients diagnosed with multiple sclerosis. in our study we employed a variety of molecular biology methods, such as immunoblotting, real-time pcr, mts cell proliferation assay, fluorescence flow cytometry and confocal microscopy. we demonstrated that disruption of p53/mdm2 interaction by nutlin-3a alters the p53 levels and also affects the lymphocyte subpopulations within pbmcs. our findings suggest that p53/mdm2 interaction inhibitors can potentially be used as prospective modulators of immune response in autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus and other. the study was funded by rfbr research grant 16-34-60213 mol_a_dk. can ykl-40 be an inflammatory biomarker in vitamin d deficiency? object: the tumor necrosis factor (tnf) was found to be cytotoxic to tumor cells and to induce tumor regression in mice. except for one member, all receptors to the tnf superfamily bind tnf-related ligands and act mostly on inflammatory system. there are currently 19 tnf superfamily ligands. tnf superfamily ligands share several common features. tnf ligands are generally type ii transmembrane proteins whose extracellular domains are be divided by enzyme to create cytokines. the tnf superfamily currently consists of 29 receptors. tnf family receptors are type i or type iii transmembrane proteins that contain multiple extracellular domains. in this study, we investigated to presence, differences and effects of tnf superfamily and receptors genes in human and mice by using bioinformatics techniques. methods: the nucleotide and amino acid sequence of each protein in human and mice was determined using t-blast-n for homologous sequences. homologous sequences of human tnf family genes found an automated procedure by using psi-blast. the secondary structure of and three-dimensional of the protein were analyzed by psipred and ffas server. netcglyc 1.0 and netphos 2.0 program were used for post-translocation modifications. the web apoptosis database was also used for the lists of domains, proteins containing these domains and their associated homologs. results and conclusion: humans tnf ligands have 17 genes encoding proteins that contain a conserved carboxy-terminal domain. this family of proteins is highly conserved between humans and mice. humans contain 29 genes encoding tnffamily receptors. sequence data from the ncbi databases demonstrated the presence of 24 mouse tnf-family receptors with orthologs in humans and one additional receptor found only in mice. the differences and similarities in the tnfs genes in humans and mice will provide information for understanding the utility and limitations of the mouse models of disease and comparing of immunology outcomes. the development of left ventricular remodeling after acute myocardial infarction is a predictor of shock. the genetic influence on cardiac remodeling, and shock in the early period after acute myocardial infarction are unclear. the aim of the present study was to investigate the relationship between angiotensin converting enzyme (ace) gene polymorphism and modified shock index (msi) in the early period in patients with acute anterior myocardial infarction. overall 140 patients with a first acute ami were included in this study. dna was isolated from peripheral leukocytes. the id status was determined by pcr. based on the polymorphisms of the ace gene, they were classified into 2 groups: deletion/deletion (dd) genotype (group 1, n = 57), insertion/deletion (id), insertion/insertion (ii) genotypes (group 2, n = 83). blood pressure and pulse measurements were performed in all patients within 10 minutes admitted to coronary care unit. msi was defined as heart rate (hr) divided by mean arterial pressure (map). echocardiographic examinations were performed in accordance with the recommendations of the american echocardiography committee. one-way analysis of variance (anova) and chi-square analyses were used to compare differences among subjects with different genotypes. the study was approved by the local ethics committee, and each patient gave a written consent. there were no significant differences among clinical parameters of patients. msi was significantly higher in patients who have ace dd genotype than in patients who have ace id / ii genotypes (1.13 ae 0.52 and, 0.85 ae 0.37, p < 0.05). presentation time hypotension or developing hypotension during admission was reported to be an important predictor of intensive care unit admission besides other vital sign measurements. our results suggested that, ace gene i/d polymorphisms d allele may affect modified shock index in patients with a first acute anterior mi. glucocorticoids (gcs) are widely used in medicine, despite their side effects, e.g. osteoporosis. however, precise molecular mechanisms of gc action, especially on bone marrow (bm) cells, remain controversial. given the osteoprotective role of vitamin d 3 , the aim of our study was to examine prednisolone-induced changes in the rank (receptor activator of nuclear factor kappa-b)/rankl (rank ligand)/opg (osteoprotegerin) pathway of rat bm depending on the state of vitamin d endocrine system. female wistar rats received prednisolone (5 mg/kg b.w.) with and without 100 iu of d 3 (for 30 days). the levels of rank, rankl, opg, 1a-hydroxylase (cyp27b1) in bm were determined by western blotting. vitamin d 3 receptor (vdr) and rankl mrnas were measured by quantitative rt-pcr. 25ohd 3 content in the serum was assayed by elisa. rankand vdr-positive bm cells were quantified using flow cytometry and visualized by confocal microscopy. prednisolone induced a marked increase in rankl and rank levels, while opg level was shown to decrease. this reflects disturbances in cytokine-mediated regulation of bm progenitor cell function. data from flow cytometry indicated a significant growth in the number of rank-positive cells (hematopoietic osteoclast precursors) compared to control. these changes were accompanied by a decrease in the levels of vdr and cyp27b1, which is responsible for 1,25(oh) 2 d 3 synthesis, in bm and 25ohd 3 content in serum. co-localization of vdr and rank in mono-and multinuclear bm cells was observed, indicating a close relation between vitamin d 3 and rank/ rankl/opg pathway. vitamin d 3 co-administration prevented prednisolone-induced changes in bm cells through restoration of vitamin d 3 bioavailability and vdr signaling that resulted in a reduction of the osteoclast progenitor pool in bm. thus, prednisolone-induced imbalance in rank/rankl/ opg system components is associated with impairments of vitamin d endocrine system in bm and can be ameliorated by vitamin d 3 treatment. p-08.01.4-002 heat shock pathway in response to different stress factors the heat shock response is an emergency pathway of the cell, which mediates repair and protection from cellular stress and therefore guarantees the survival of the cell. this stress can range from heat or hypoxia to chemicals and heavy metals. it is highly conserved in all eukaryotic cells and plays an important role during atypical conditions. due to its high complexity, the pathway is not yet completely understood. most important, after activation of the pathway, is the refolding of proteins or, in case of severe misfolding, the depletion of proteins to maintain proteostasis. heat shock factor 1 encoded by the hsf1 gene is known as the main switch point in heat shock regulation. after activation it trimerizes and binds to heat shock elements in target gene promoters. one of these promoters is the hspa1a promoter (hsp72 promoter). the promoter was analyzed by dismantling it to its functional parts. especially three elements, the heat shock elements, were in the focus of this work. in first place parts of the promoter were multimerized and combined with different reporters, like luciferase, by cloning. also mutations in the natural promoter were designed by cloning. the focus now is on the heat shock elements, where hsf1 can bind as a trimer. the idea is that these different elements have various effects on different stressors like heat, chemicals (geldanamycine as hsp90 inhibitor, mg132 as proteasome inhibitor) or heavy metals (cadmium, arsenic, zinc) . this was tested on cells transiently transfected with those promoter variants. for promising variants stable cell lines were created. in these stable cell lines further experiments on mrna level can be conducted. in the last months experiments with the crispr/cas9 system were started. furthermore, experiments on transcriptional (qpcr) and translational (dual-luciferase assay) levels were done as well. in the end we hope to get a clear picture on the regulation of the hspa1a promoter by different stress factors. invasive cancer cells form membrane protrusions, invadopodia, that facilitate cell invasion and metastasis. key players invadopodia include the adaptor proteins tks4 and tks5, the actin regulators cortactin, wip and n-wasp, the kinase src and others. in spite that in the last two decades significant advances in our knowledge of the structure and development of invadopodia have been made, detailed mechanisms they are functioning is not yet available. we have identified a series of new tks4 binding partners including adaptor proteins itsn1, itsn2, crk and grb2, kinase src, amph1, bin1, plcg1 and also another member of the tks family -tks5. it may indicate the possible role of tks4 in transport and sorting of cell vesicles. current data are supported by interaction with the proteins of amph1 and bin1, as their main functions are membrane trafficking and remodeling. adaptor proteins crk, grb2 and itsns are important for the actin cytoskeleton rearrangements, endocytosis and signal transduction. moreover, we have identified and characterized new tks4 isoform -tks4-beta. we suggested that an active state of tks4 is regulated via intramolecular interactions between its proline-rich motifs and own sh3-domains. we have shown the interaction between itsns and other prominent component of invadopodia wip. data from immunofluorescent analysis revealed co-localization of itsn1 and wip at the sites of invadopodia formation and in clathrin-coated pits. we have also demonstrated that the key protein itsn1 and wip and n-wasp can form a complex in cells. together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify itsns as scaffold proteins involved in this process. we have shown the interaction between itsns and other verprolin family members cr16 and wire which play an important role in the reorganization of the actin cytoskeleton. we have demonstrated that cr16 and wire interact with sh3domains of itsns in complex with actin. p-08.01.4-004 correlation between proteomic and phenazine profile of pseudomonas sp. phenazines are widely known compounds with huge variativity of biological activities which are produced by pseudomonas sp. and some other bacteria species. the results of our work shows the correlation between the changes of proteomic profile of pseudomonas aeruginosa caused by a mutagenesis and the secondary metabolism of antibiotics (phenazine) profile. different strains of pseudomonas aeruginosa were obtained using mutagenesis, after that bacterial cells were destroyed by ultrasound. protein-containing fractions were isolated using methanol-chloroform method as well as phenazines compounds were extracted from culture media using liquid phase extraction. obtained proteome was analysed by shotgun-proteomics technique. as the result of the liquid phase extraction phenazine compounds were mainly extracted to the organic phase. this phase was evaporated and re-dissolved in 100% methanol. after sample preparation obtained solutions were analyzed by hplc-agilent 1290 with quadrupole tof mass-detector. results of the analysis were compared with the library of known phenazine compounds mass-spectras generated by cfm-id online resource. obtained phenazine profiles were compared with each other and correlation with the changes in proteome was analyzed. received results promote better understanding of mechanisms of phenazine production. this data opens possibilities for targeted changes in the methabolic pathway in order to obtain phenazine compound with required biological activity. insulators are genomic elements which block enhancer-promoter interaction and prevent spreading of heterochromatin. cp190 protein is an integral component of most known drosophila insulators, it interacts directly with ctcf and pita dna-binding insulator proteins using dimeric btb-domain, but function of cp190 within insulators still remains to be elucidated. recently we described an interaction between cp190 btb-domain and cterminal domain of ctcf insulator dna-binding protein, subsequent deletion analysis allowed us to isolate 50aa fragment within ctcf c-terminal domain sufficient for interaction with cp190 btb, but deletions of flanking regions also lead to the loss of interaction with cp190 in vivo. at the same time crosslinking experiments suggest that a dimer of btb interacts with one molecule of ctcf, presuming that it could recognize two peptide fragments within ctcf c-terminal domain. we solved crystal structure of btb-domain from cp190 insulator protein at 1. 3 a resolution. overall structure is similar to other btb-domains. cp190 btb-domain has peptide-binding groove similar to that previously found in bcl6 btb domain. inspection of btb-domain surface revealed several possible binding sites for polypeptide fragments from ctcf protein. based on these observations a set of point mutations within peptide-binding groove of btb-domain has been designed and we tested ctcf-interaction abilities of these mutants using gst pull-down assay and yeast two-hybrid assay. the most significant impact was found with alanine-substitutions of hydrophobic residues whereas substitutions of hydrophilic amino acids were less effective. therefore our results support that cp190 btb-domain recognizes ctcf protein using peptide-binding groove. this study was supported by the russian science foundation (project №14-24-00166). p-08.01.4-006 comparative study of the fatty acid composition of lipids in the raw meat samples obtained from hybrid sheep one of the most important tasks in the animal biology and husbandry is to clarify the role of animal genetic diversity in providing nutrients to the diversity of animal products. the objective of our work was to study the chemical compositions of raw meat samples obtained from domestic (group i -purebred romanov sheep) and hybrid sheep (group ii -f 3 hybrids of romanov sheep with 12.5% of argali blood). the significant changes in fatty acid composition of the lipid fraction from the fat and muscle tissue of the hybrid sheep as compared to the control were found. the content of saturated fatty acids (sfas) in the fat samples of the hybrid animals was by 12.16% lower (55.12 ae 2.30%, p < 0.01), but polyunsaturated (pufas) or monounsaturated fatty acids (mufas) contents were by 0.64% and 9.43% higher (10.10 ae 0.43 and 28.78 ae 1.77 (p < 0.01), respectively) as compared to purebred romanov sheep. the most pronounced changes were found for palmitic acid (decreased from 27.70% to 14.24%) and for oleic, linoleic, arachidonic acids (increased from 20.37%, 5.58%, 0.35% to 29.98%, 6.10%, 0.70%, respectively). the last two acids together with the linolenic acids belong to the so-called essential acids and very important for the animal metabolism. a similar trend was observed on the composition of the lipid fraction of muscle tissue. sfas, pufas and mufas content in muscle tissue of hybrid sheep was 53.38 ae 0.34, 9.39 ae 1.00 and 34.16 ae 0.39%, that was 11.17% lower (p < 0.001), and 4.13% and 4.67% higher (p < 0.01) compared to purebred romanov sheep. these results emphasized the difference of the pufas/sfas ratios in fat and muscle tissues, respectively) and characterized the biological value of the lipid fraction of fat and muscle tissue. the obtained data gave evidence of the positive changes in the fatty acid compositions of the lipid fractions for the hybrid animals as compared to the purebred sheep. supported by the russian scientific foundation, no. 14-36-00039. foodborne illnesses resulting from the consumption of agricultural commodities contaminated with enteric pathogens are an increasing problem around the world. while various possibilities of produce contamination with pathogens exist, the global warming combined with a widespread use of animal manure in agriculture will likely contribute to an increased number of such outbreaks. thus, phages isolated from different agroecosystems may prove to be useful in detection/biocontrol of enterobacteria in produce. during the investigation of the impact of global warming on the diversity and co-evolutionary dynamics between microorganisms and viruses in lithuanian agroecosystems, a novel enterobacteria phage vb_ecos_nbd2 (nbd2) was isolated from agricultural soil using e. coli novablue for phage propagation. nbd2 genomic dna was isolated from cscl-purified phage particles, and was subjected to illumina dna sequencing. nbd2 is a virulent siphovirus that has a low-temperature plating profile (fails to form plaques at a temperature >30°c). the genome of nbd2 is $ 52 kb long, and has a total of 87 probable protein-encoding genes as well as 1 gene for trna ser . the genome analysis revealed that 20 nbd2 orfs encode unique proteins that have no reliable identity to database entries. among the orfs that encode proteins with matches to those in other sequenced genomes, 64 are similar to proteins from phages that infect different members of enterobacteriaceae, while 3 nbd2 orfs are most similar to those from bacteria. based on the similarity to biologically defined proteins, 32 nbd2 orfs were given a putative functional annotation, including 15 genes coding for morphogenesis-related proteins, as well as 14 associated with dna replication, recombination, and repair. phylogenetic analysis revealed that enterobacteria phage nbd2 is distantly related to phages belonging to the subfamily tunavirinae. this research was funded by a grant (no. sit-7/ 2015) from the research council of lithuania. p-08.01.4-009 the antibiotic novobiocin affects the composition of the escherichia coli proteome n. e. arenas 1 , j. williamson 2 , v. schw€ ammle 2 , s. douthwaite 2 1 universidad de cundinamarca, cundinamarca, colombia, 2 university of southern denmark, odense, denmark novobiocin (nov) is an aminocoumarin which competitively inhibits the atp binding site in the gyrase-b subunit of prokaryotic topoisomerase ii. nov remains a therapeutic choice for treating infections with bacterial pathogens that are resistant to more commonly used drugs. the aim of this study is assess the proteomic response of e. coli strain upon nov treatment. minimum inhibitory concentrations of nov were measured by standard assays. three different e. coli strains (as19, as19-rlma::aph and b) were grown aerobically in nutrient rich lb media at 37°c during one hour. the whole cell proteome (five biological replicates in each sample,) was assessed by lc-ms by using tmt labelling protocol. raw files were imported to proteome discoverer (thermo fisher scientific) and searched together with mascot against the uniprot e. coli reference proteome. mics for nov were determined to be >1000-fold higher the wild-type b-strain of e. coli than for the hypersusceptible as19 strains (1 lg/ml). whole genome comparison of the b and as19 strains were characterized by an increase in proteasome components (6 proteins), chaperones (3), error-prone dna polymerase components (4), ribosomal hibernation factors (3), heat shock response (2), electron transport coupled proton transport (8), pentose phosphate pathway (9), flagellar assembly (4), oxidative phosphorylation (10) and tca cycle (8). whereas ribosomal proteins (45), aminoacyl-trna synthetases (7), rnases (6), abc transporters (17), mismatch repair (3) and sec secretion pathway (4) were significantly down-regulated upon nov treatment. the three e. coli strains respond similarly upon nov treatment and their proteomes showed upregulation of heat shock response with changes in the components of translation and transcription, the proteasome and atp biosynthesis. the changes observed can be used to define the processes that are required for antibiotic tolerance and survival of e. coli against aminocoumarin antibiotics. postnatal growth is under control of pituitary derived hormone, growth hormone (gh) that triggers bone, fat tissue growth and development via acting on protein, carbohydrate and fat metabolism. gh functions on postnatal development by jak2/stat5 signaling following gh:gh receptor (ghr) dimerization. isolated growth hormone deficiency (ighd) is a medical condition of insufficient production of growth hormone (gh) that is caused by mutations on gh-n gene in different ethnic origin children. various mutations within gh has been determined in different populations so far, and glutamic acid to glycine (e33g), asparagine to aspartic acid (n47d), threonine to alanin (t-24a) missense mutations, alanine to serine (a13s) substitution, tryptophan to stop codon (w-7x), gaaa insertion in intron 1 of gh-n gene and both intron 1 (+83c) and deletion of 166. amino acid of gh protein phenylalanine (f166del) mutations were detected in turkish ighd children. the potential role of these mutations on cell growth, proliferation, emt via acting on gh signaling pathway has not been observed yet. all these mutations were performed on wild type gh-n gene inserted pc3.1 vector by site-direct mutagenesis and stable cell line of each gh gene mutations were generated by neomycin selection. although w-7x, e33g, f166del, a13s and n47d mutations suppresses gh signaling via acting on either jak2 dephosphorylation or stat5 downregulation, t-24a, gaaa insertion and deletion of +83c mutations have no significant effect on gh signaling. in addition, each mutation lead different growth suppression effect and colony formation potential and intracellular polyamine levels and odc expression profiles were essential role in emt potential of hek293 cell lines. as a result, w-7x, e33g, f166del, a13s and n47d mutations prevented gh signaling and cell growth and differentiation via polyamine metabolism. pulmonary embolism (pe) is a common cardiovascular emergency and affects a large number of patients. acute pe-induced oxidative stress can lead to the accumulation of specific nitroproteins that may play a role in disease progression. the impact of nitration of a single tyrosine residue often has broad implications on the activity of biologically critical proteins, which has become increasingly related to pathological conditions. in this study, we used a proteomic approach to analyze nitrated serum proteins in patients diagnosed with acute pe and healthy controls. nitrotyrosine (no2tyr)-containing proteins were immunoprecipitated from serum with a no2tyr affinity sorbent. precipitated proteins were separated by sds-page and visualized by coomassie blue staining and western blotting with mouse monoclonal anti-no2tyr antibody. among the numerous immunoreactive bands observed in disease patients, the 138 kda protein band was in-gel digested and analyzed by maldi-tof mass spectrometry (ms). mass fingerprint data sets obtained from the peptide fragment ions matched human collagen alpha-1 (iii) chain (co3a1_hu-man) with mascot algorithm analysis giving a score of 65 (p < 0.05). collagen alpha-1(iii) chain is a fibrillar collagen that is found in extensible connective tissues such as skin, lung, and the vascular system. altered metabolism of collagen and its excessive deposition in the matrix of the connective tissue is a hallmark of chronic interstitial lung diseases. collagen can be measured in serum and bronchoalveolar lavage fluid from patients with numerous chronic interstitial lung diseases. given these considerations, future studies are aimed understand the relevance of no2tyr modifications in co3a1 relating to changes in protein structure and function. recent studies have shown that the genes involved in dislipidemia represent potential loci to be associated with diabetes as a disease. recent genome wide association (gwa) studies have associated rs1260326 in gckr gene and rs4846914 in galnt2 gene with parameters of t2d and diabetic dyslipidemia. in this study, the association of these single nucleotide polymorphisms (snps) with t2d and dyslipidemia was tested in the population from bosnia and herzegovina (bh). our study involved 352 patients with t2d and 156 healthy subjects. biochemical and anthropometric parameters were measured in all participants. after dna extraction, sequenom iplex platform was used for the analysis of galnt2 polymorphism (rs4846914), while polymorphism in gckr (rs1260326) gene was analyzed by using real time pcr. our results demonstrated significant association of gckr rs 1260326 variant with waist circumference (p = 0.003) and fasting glucose levels (p = 0.003) in the control group. no such association was demonstrated for rs4846914 galnt2 gene. in the group of diabetic patients, significant association of gckr rs1260326 variant with levels of bilirubin (p = 0.004) and rs4846914 galnt2 variant with hba1c (p = 0.013) and triglyceride levels (p = 0.043) was also demonstrated. our results suggest an association of variations of gckr and galnt2 genes with specific markers of t2d and dyslipidemia. further studies would be needed in order to confirm these genetic effects in other ethnic groups as well. osteoporosis is the most common metabolic bone disorder affecting the normal bone turnover with low bone mineral density (bmd) and risk of fragility fractures. polymorphisms at the sp1 binding site of the collagen type 1 a1 (col1a1) gene is associated with low bmd. we examined the distribution of col1a1 gene polymorphism in 50 young osteoporotic women and in control group in turkish population. patients had low bmd with t score ≤2.5 sd and controls was 25 healthy women (35-57 years). mean age (51.28 ae 5.8) and (46.56 ae 6.15) respectively. the bmd, as g/cm 2 , was measured in the hip and the lumbar spine (l2-l4) with (dexa). dna was isolated from blood. col1a1 gene was analysed with genomica clinical array system. the x 2 test was used to compare allele and genotype frequences between patients and controls. mean of t score in patients was à3.06 ae 0.42. mean bmd (as g/cm 2 ) was 0.708 ae 0.073, and (1.009 ae 0.823) genotype distribution were18(36%) ss, 31 (%62)ss, 1(%2)ss for patients, and 12(48)ss, 9(36)ss, 4(16)ss for control . patients had 33(%66)s allele, 17(34%) s allele, controls had 67(67%)s allele, 33 (33%)s allele. when genotypes and bmd were compared in patients, there was no significant correlation between osteoporosis and genotypes. the allelic distribution was not significant between patients and controls p > 0.05. genotypic distribution in patients were significantly different. patients had a higher frequency of the ss(%62) than controls (ss %36) p < 0.05. this study shows that high prevalences of the ss genotype at the col1a1 locus, in osteoporosis . _ it is possible that the presence of the s allele causes variation col1a1 and col1a2 mrna's producing abnormal collagene protein. since collagen protein is major protein of bone, it is to be expected that a defect in this protein will produce bone fragility. col1a1 gene should be detected early to initiate preventative therapy for bone health. the biological activity of nigella sativa seeds is mainly attributed to its essential oil component which is pre-dominantly (30-48%) thymoquinone (tq). therapeutic effect of tq was exhibited in many diseases including inflammation, cancer, sepsis, atherosclerosis and diabetes. tq has been reported to exhibit antiproliferative effects on cell lines derived from breast, colon, ovary, larynx, lung, myeloblastic leukemia, and osteosarcoma and inhibited hormone refractory prostate cancer. tq induces apoptosis in tumor cells by suppressing nf-jb, akt activation, and extracellular signal-regulated kinase signaling pathways and also inhibits tumor angiogenesis. the aim of this study was to evaluate the anti tumor effects of tq on hepatoma cells. these antitumor assays include cell viability assay, clonogenic assay, scratch assay and molecular expression studies of death related genes. cells were treated with different concentration of tq in hep3b for cell proliferation by mtt and clonogenic assay. in addition, the metastatic character of tq was investigated by scratch assay in hep3b at 3-6 and 24 hours. the effect of tq was also evaluated at mrna level by real-time-pcr. tq was treated on the hep3b cells in three different concentration, namely 75-50 and 37.5 lm. tq showed the cell cytotoxicity in concentration and time dependent manner. the scratch assay revealed no healing in the scratched area due to the decreased cell viability. maximum permissible dose was 50 lm. proapoptotic genes, bax and bad, and autophagy genes, beclin-1 and lc3, were upregulated in hep3b cells after 24 hours treatment in contrast, antiapoptotic gene, bcl-2, expression level was decreased for hep3b cells after 24 hours. p-08.01.4-019 association of irs1 genetic variation with type 2 diabetes and insulin resistance in patients from bosnia and herzegovina insulin receptor substrate-1 (irs1) encodes the irs1 protein, a substrate for the insulin receptor tyrosine kinase and has a critical role in insulin-stimulated signaling pathways. previous studies showed that irs1 single nucleotide polymorphisms (snps) were associated with type 2 diabetes mellitus (t2d). this is the first study performed in a population from bosnia and herzegovina (bh) in which we examined the association of rs7578326 (g>a), rs2943641 (t>c) and rs4675095 (a>t) with t2d risk and related traits. our study involved 437 t2d patients and 252 healthy subjects. biochemical parameters, including but not limited to insulin, homa-ir, hba1c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was done by spss 23. our results demonstrated a significant difference in frequency of rs4675095 (p < 0.001) and rs7578326 (p = 0.021) snps between t2d patients and control subjects. interestingly, here we showed a significant association of irs1 rs4675095 risk t allele with increased insulin levels (p < 0.001) and homa-ir (p < 0.001) in t2d patients. similarly, rs7578326 variant was also associated with the same markers of insulin resistance in diabetic patients, i.e. insulin levels (p = 0.029) and homa-ir (p = 0.025). no such association was demonstrated for rs2943641. however, this irs1 variant was associated with changes in lipoprotein levels, where risk c allele increased vldl (p = 0.006) and decreased hdl levels. our results suggest that irs1 variants are associated with t2d susceptibility in bh population, thus confirming similar findings in other population cohorts. furthermore, the associations of these variants with markers of insulin resistance and dyslipidemic metabolic changes point to their role as potential t2d biomarkers. the adra2a gene encodes alpha-2a adrenergic receptor which mediates adrenergic suppression of insulin. a genetic variant in adra2a was recently associated with defective b-cell function. the objective of this study was to analyze association of two adra2a polymorphisms (rs553668 a>g and rs10885122 g>t) with type 2 diabetes (t2d) and its related traits. in this study we have included 437 t2d patients and 252 healthy subjects from bosnia and herzegovina (bh). biochemical parameters, including but not limited to insulin, homa-ir, hba1c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was performed by ibm spss statistics 23 software. our data showed that frequencies of both, rs10885122 and rs553668, variants were not significantly different between t2d and control subjects. however, rs553668 risk a allele appear to increase insulin levels (p = 0.0002) and homa-ir index (p = 0.00001). furthermore, this variant also seems to affect vldl levels (p = 0.004) and waist circumference (p = 0.02) in diabetic patients. the genotype analysis of rs10885122 variant demonstrated that risk g allele decreased hdl (p = 0.0004) and increased ldl levels (p = 0.014), as well as affected the waist circumference (p = 0.02) in diabetic patients. interestingly, haplotype analysis demonstrated the association of rs553668a / rs10885122g with higher homa-ir index. here we demonstrated that although both, rs10885122 and rs553668, adra2a polymorphisms were not associated with t2d risk in our cohort, they were associated with markers of dyslipidemic perturbations and insulin resistance in diabetic patients. further studies in larger cohorts are needed in order to explore these possible interactions and confirm our findings. smad-interacting protein 1 (sip1), also known as zeb2 is a member of zeb family transcription factors and was shown to regulate epithelial-to-mesenchymal transition, cell cycle, cellular senescence and cancer stemness. bipartite zing finger motifs at amino and carboxyl termini of the sip1 mediate its binding to ebox sequences in the genome. however, there are only limited data about sip1 target genes. by using a home-made anti-sip1 monoclonal antibody, clone 6e5, we conducted chip-seq in three hepatocellular carcinoma cell lines, namely snu398, plc/prf/5 and sk-hep-1 and found receptor tyrosine kinase-like orphan receptor 1 (ror1) as one of the targets. sip1 dnabinding consensus motif cacctg was found at +1 kb, +30 kb and +50 kb from ror1 transcription start site. chip experiments validated sip1 binding to all consensus motifs in the ror1 gene region. interestingly, the strongest enrichment was at +50 kb suggesting that long-range interactions play an important role in the regulation of ror1 by sip1. sip1 knockdown by shrna in high-sip1 expressing snu398 cells resulted in the repression of ror1 expression. ror1 is expressed in embryogenesis and fetal life, and is absent within most of adult normal tissues. however, overexpression of ror1 was observed in many human cancers, from hematological malignancies to solid epithelial tumors. ror1-positive cancer cells have enhanced proliferation, invasion and metastasis capacities, show resistance to apoptotic stimuli and display cancer stem cell characteristics. therefore, sip1 and ror1 act in similar pathophysiological processes. our finding that ror1 is regulated by sip1 at least in hepatocellular carcinoma cells adds another level of complexity to the molecular mechanisms of proliferation, invasion and stemness of cancer cells. hepatocellular carcinoma (hcc) is the most prevalent primary liver cancer and is one of the leading causes of cancer related deaths. smad interacting protein 1 (sip1), a member of the zeb family of emt inducers, is involved in cellular proliferation, senescence, invasion and metastasis in human tumors. however, genes regulated by sip1 in hcc are yet to be identified. we conducted a chip-seq study in high-sip1 expressing hcc cell line snu398 by using a home-made anti-sip1 antibody, clone 6e5. among 509 annotated genes, we selected six1 for further studies because of its increased expression in multiple cancers and its association with poor prognosis. sip1 dna-binding motif cacctg was found at -3 kb from transcription start site of six1 gene. chip qpcr experiment validated sip1 binding to this region with 19,7 fold enrichment. compared to healthy liver, six1 transcripts were upregulated in 8 of 9 hcc cell lines included in this study. knockdown of sip1 by shrna in snu398 cells caused upregulation of six1. immunohistochemistry studies in hcc tissue arrays showed increased expression of six1 in tumors and inverse association with sip1 expression in a tumor grade dependent manner. therefore, our results strongly suggest an inverse correlation of sip1 and six1 in hcc bone mineral density (bmd) and bone turnover are under genetic control and variations in the vitamin d receptor (vdr) are related to bmd. bmd is known to be affected by 25 -hydroxy vitamin d (25 (oh)d) and intact parathyroid hormon (ipth) levels. we aimed to determine correlation blood levels of vitamin d (vitd), ipth, and vdr gene effect in healthy turkish women. the subjects were 25 healthy women in age 35-57 years. the bmd was measured as a t score in the lumbar spine (l2-l4) with dexa. all subjects had normal t score between (-1.0 to 1.4) sd. vitd was measured by lc-20-at shimadzu. ipth was measured by chemiluminescence method, dna was isolated from blood. the fok i (vdrf-foki) and bsmi (vdrb-bsmi) polymorphisms of vdr gene was analysed with genomica clinical array system. the mean vitd level was (21.06 ae 14.54) lg/l, mean plasma ipth level was (57.96 ae 30.49) pg/ml. pearson correlation test showed no relation of vit d with bmd. there was moderately negative correlation between ipth and bmd (r = à0.3062). genotype distribution and allele frequency of subjects were as follows: 21(84%) ff), 1(4%) ff, 3 (12%) ff genotype in vdrf -fok1 gene, 7 (28%) bb, 14 (56%) bb, 4 (16%) bb in vdrb-bsmi gene. allele frequencies were f: 86%, f:14%; b:56%, b:44%. when fok1 and bsmi were combined, 52%(ff-bb) and 20% (ff-bb) were found as the most frequent genotypes. bsmi frequency was in hardy weinberg equilibrium (p > 0.5). but foki was not (p = 0). it was found that vit d, ipth levels and bmd were in normal levels in all carriers of ff genotype and in combined (ff-bb) type carrying healthy women (52%). the association vdr genotype and bmd may be different in various ethnic and geographical groups. therefore it is worthwhile to assess vdr polymorphism among turkish population. these type of distribution studies of vdr in healthy and in osteoporotic women may enlighten to earlier diagnosis and treatment planning. p-08.01.4-026 determination of hb a1c values in beta thalassemia f. g€ uzelg€ ul 1 , g. s. seydel 2 , a. e. yalin 3 , e. s€ onmez 1 , k. aksoy 1 1 c ß ukurova university, adana, 2 nigde university, nigde, 3 mersin university, mersin, turkey introduction: hemoglobinopathies are most commonly seen hereditary blood diseases worldwide. our aim was to compare the hba1c values measured on cation-exchange high performance liquid chromatography (hplc) in beta thalassemia cases. materials and methods: we collected 3 ml of whole blood k 3 edta containing tubes from forty-nine cases. arms, rflp and dna sequence analysis methodologies were carried out for determination of beta thalassemia mutations. hb a1c values were measured using the agilent 1100 hplc system. results: forty-nine diabetic and non-diabetic patients were diagnosed with beta thalassemias: twenty-one ivs1-110/ivs1-110, one ivs1-1/ivs1-1, one ivs1-5/ivs1-5, two ivs1-6/ivs1-6, two ivs2-1/ivs2-1, one fsc5/fsc5, one fsc44/fsc44, two -30/-30, two cd8/cd8, one cd36-37/cd36-37, one cd8-9/cd8-9, two cd82/cd83-g/ cd82/cd83-g, two ivs1-110/ ivs1-6, one ivs1-110/ ivs2-1, one ivs1-110/fsc44, one ivs1-110/cd39, one ivs1-110/cd8, one ivs1-110/cd8-9, one ivs1-110/ivs1-5, one ivs1-6/ ivs2-1, one ivs1-6/ ivs1-25, one ivs2-745/cd8 and one fsc5/cd37. cases were classified as diabetic (6), prediabetic (11) and non-diabetic (32) introduction: members of aurora kinase family aurora a, b and c are conservative kinases of cell cycle which are encoded by genes aura, aurb and aurc respectively. overexpression of aura and aurb was found in human cancers, especially in prostate cancer. moreover, there is the evidence that aurb interacts with one of the major oncogenic kinases -braf. little is known about implication of aurc in cancer, but it was demonstrated, that it can overlap aurb function and shares its location. we studied expression of genes of these kinases in urine of prostate cancer patients aiming to evaluate their involvement in this disease and their potential as tumor markers. materials and methods: 22 urine samples from patients with prostate cancer were gathered after prostate massage before surgical invasion. we used urine samples from 6 healthy men as control. we obtained cells from each urine sample by centrifugation and isolated rna using standard approach with phenol and guanidine thiocyanate. cdna was synthesized and taken to qpcr reactions. data was statistically analysed. results: expression of all studied genes was detected in urine of patients with prostate cancer and of healthy men. expression of aurb and aurc in cancer samples each was higher than expression of aura. the cumulative expression aurb and aurc was higher than expression of aura in 13 samples from 22. we observed positive correlation between expression of aurc and braf (rs = 0.548, p = 0.01). discussion and conclusion: previous investigation showed, that for normal prostate tissue 90% of aurora family expression was presented by aura. we suppose that presence of aurb and aurc cumulative overexpression means presence of cell cycle deviations in prostate tissue of these patients and might be further studied as prognostic marker. in this study we first showed the correlation between aurc and other carcinogenic kinase braf expression, which opens the perspective for investigation of role of aurc in carcinogenesis. bacillus marmarensis sp. nov. is an extreme obligate alkaliphile isolated from mushroom compost near marmara region of turkey. it can survive at extreme ph values up to 12.5. based on its genome sequence, metabolic pathways for 7 proteases, 6 amylases, 2 cellulases, 1 lipase, n-butanol and a biodegradable plastic poly-b-hydroxybutyrate were annotated. in addition to being a potential extracellular hydrolase producer, its ability to survive in the high ph range of 8.0 to12.5 makes it an attractive microorganism for different industrial applications. in the current study, the adaptation strategy of b. marmarensis sp. nov. to alkaline conditions was investigated using proteomic tools. the organism was grown at two different ph values, 10.0 and ph 12.0. for extraction of whole cell proteins, cells were disrupted with mp bio fast prep device. protein extracts were treated with protease inhibitors and a nuclease mix. salts were removed using a cleanup kit. obtained proteins were separated based of their isoelectric points in the first dimension and then based on their molecular weights in the second dimension. proteins maps of cells grown at these two extreme ph values showed significant differences in protein expression for alkaline adaptation. p-08.01.4-029 biochemical and proteomic analyses of normal human astrocytes and glioblastoma exposed to dichloroacetate treatment f. c. atilgan, h. cimen yeditepe university, istanbul, turkey glioblastoma (gbm) is an aggressive malignant tumor composed of astrocytes in brain tissue. gbm cells utilize glycolysis rather than oxidative phosphorylation to support rapid growth rate which is called warburg effect. dichloroacetate (dca) is an antiglycolytic agent that inhibits pyruvate dehydrogenase kinase (pdk) activity and induces apoptosis via normalizing the mitochondrial activity. this study aimed to demonstrate the metabolic alterations between the normal human astrocytes (nha) and gbm cell lines which are exposed to dca, and to identify the differentially expressed proteins by ms-based proteomic analyses. nha cell line, u87mg and u373 as gbm human cell lines were examined through analyzing the alterations in the glycolysis metabolism upon dca treatment by measuring the variations in the pyruvate levels, lactate dehydrogenase a, pdk3. mts was performed to investigate the effect of dca treatment on cell viability. immunoblotting of pgc1-a, oxphos complexes, and mitotracker green staining was employed to reveal the mitochondrial differences between normal and the cancer cells, and upon dca treatment of these cells. proteomic analyses were utilized for the identification of candidate proteins depending on the acetylation status. in this study, compared to nha, the pyruvate and ldha levels were elevated and pdk3 levels in u87mg were reduced by 14%. due to mts results, ≤10 mm dca treatment showed significant decrease in gbm cells compared to nha cells. immunoblotting and mitotracker green staining results showed increase in mitochondrial mass. elevation in the pyruvate and ldha levels and reduction in pdk3 level in u87mg and u373 cells indicates glycolysis dependent metabolic switch in energy metabolism. proteomic analyses demonstrate that most of the differentially expressed proteins comprised of metabolic enzymes. this study provides novel information about metabolic alterations existing between nha and gbm, which can inspire further studies for therapeutic applications. kidney stone is a complex disease resulting from environmental as well as hereditary factors and principally composes of approximately 75% calcium oxalate (caox) crystals, which are formed through a multi-step process. vitamin d receptor (vdr) gene encodes the nuclear hormone receptor for vitamin d3; downstream targets of this gene are chiefly contributed in mineral metabolism though the receptor regulates a variety of other metabolic pathways. calcium sensing receptor casr plays an important role in sustaining mineral ion homeostasis. the aim of this study is to profile the expression level of vdr and calcium sensing receptor (casr) genes and to unravel their role in rat kidney stone induced by ethylene glycol, in order to explain the underlying molecular mechanisms. total rna were extracted from paired sample before and after ethylene glycol treated of 50 rats. the mrna expression level of vdr and casr gene were measured employing quantitative rt-pcr (qrt-pcr). the mrna expression levels of both genes were significantly down-regulated according to before treated. in conclusion, our data suggest reduced mrna expression in vdr and casr genes might be a risk factor for kidney stone formation. further studies are necessary to verify these findings in different ethnic groups. p-08.01.4-031 apj receptor a445c gene polymorphism in turkish patients with coronary artery disease against different models of expected frequencies/counts to understand the evolutionary dynamics of saars in proteins. we obtained from ensembl the assemblies of genomes/proteomes of human and nonhuman primates (chimpanzee, gorilla, and rhesus monkey), rodents (mouse and rat), and birds (chicken and zebrafinch). the expected probabilities for the occurrence of saars based on their nucleotide frequencies in coding regions and amino acid frequencies in individual protein sequences or across the whole proteome were compared with the observed repeat occurrences. we found that with all three methods and in all eight species the correlation between observed and expected repeat counts decreased above a saar length threshold. the percentage of saar proteins for each amino acid also exhibited variability among species when both the repeat length and counts were taken into account. however, clustering based on saar characteristics generally reflected the known phylogenetic relationships between species. our comprehensive bioinformatics analyses reveal that saars show amino acid-specific occurrence patterns with respect to species as well as saar length. tissue proteins play important roles in biological metabolic processes. the qualitative and quantitative analysis of tissue proteins facilitates the understanding of molecular mechanisms that differentiate between physiologic and pathologic states. health and research institutions routinely prepare formalin-fixed paraffinembedded (ffpe) tissue blocks for histopathology. proteomics on ffpe tissue still requires standardization of tissue solubilization processes to overcome variability in protein extraction results. our aim is to compare the proteomic studies of fresh frozen and ffpe rat renal tissues. fresh frozen and ffpe preparations from renal tissues were included in this study. an adult rat was sacrificed and the dissected kidneys were divided two equal section. one immediately frozen in phosphate buffer, and the other tissue specimen not thicker than 5 mm to allow rapid penetration of the fixative put in 10% buffered formalin for 48 hours. the fresh frozen tissue was dissolved and homogenised in the cold phosphate buffer solution containing protease inhibitors. paraffin blocks were performed from formalin fixed tissue specimens. we have extracted the protein from the ffpe tissues using our previously verified method. we have utilized electrophoresis three times to compare protein yield, number, intracellular and intercellular of homogenised samples obtained from ffpe and fresh frozen kidney samples. the number of proteins identified from fresh frozen kidney tissue has generally been shown to be increased compared with ffpe tissue. decrease of the qualitative results in electrophoretic bands was found similar in all replicative studies. ffpe tissues undergo extensive cross linking between protein/ dna/rna molecules during formalin fixation, which creates inter-molecular crosslinks. on the other hand, ffpe tissues represent a valuable resource to carry out retrospective studies aimed to biomarker discovery in kidney cancer as well as other kidney diseases. background: development of atrial fibrillation (af) during the course of chronic primary mitral regurgitation (mr) is common and represents complex molecular mechanisms. however, the gene expression profile of human atrial fibrillation (af) in the setting of chronic primary mr remains uncharacterized. in the current study, we aimed to compare the gene expression profiles of patients with severe degenerative mr in sinus rhythm (sr) and af. methods: left and right atrial tissue samples were obtained from patients with chronic primary severe mr in permanent af (n = 30) and sinus rhythm (n = 30). we performed a novel micro-dissection technique for thin sections of atrial tissue samples and immediately fresh froze intra-operatively. transcriptomes of left and right atrial appendages of degenerative mitral regurgitation patients with sr versus af were compared by microarray analysis on affymetrix hgu-133 plus 2 platform. bioinformatics, data mining and pathway analyses were conducted on partek gs and webgestalt. genome-wide gene expression profiles were compared between af and sr groups among 54.675 transcripts representing 38.500 well-characterized human genes. differentially regulated genes were evaluated according to fold change (fc ≥ 1.5) with a p-value ≤0.05. results: most remarkable pathways altered in af atrial tissues compared to sr group, were extracellular matrix-receptor interaction; mapk, adipocytokine, and calcium signaling; apoptosis and cardiac muscle contraction pathways. conclusions: this is the first human study of comparative transcriptomics in left and right atrial tissues of patients with af versus sr associated with severe degenerative mr. the main findings of this multidisciplinary translational research provide novel candidate targets for the treatment and prevention of af. in order to acquire iron under iron-limiting growth conditions, bacteria employ specific mechanisms such as production and secretion of siderophores. siderophores are low molecular metalchelating compounds that contribute not only to iron scavenging, but also participate in other important processes including oxidative stress response and cell signaling. serratia marcescens, gramnegative bacterium, could be found in various environments, including wastewater, plant rhizosphere and hospital setting where s. marcescens can cause serious life-threatening infections. in this study, we performed a detailed characterization of the siderophores of the clinically important pigment-free s. marcescens strain sr41-8000 and environmental pigment-producing s. marcescens strain sm6. bioinformatic analysis of these genomes by antismash software revealed the presence of several clusters involved in non-ribosomal peptides synthesis (nrps). we found four nrps clusters in genome of s. marcescens sm6. cluster 1 has a low level of identity to enterobactin gene cluster typical for bacteria producing catechol-like siderophores. second cluster has only 4% of identity to xantholipin biosynthetic gene cluster. clusters 3 and 4 of nrps genes of s. marcescens sm6 did not show any homology to known nrps clusters. in contrast, the genome of s. marcescens sr41-8000 contains only one genetic cluster of nrps genes. this cluster does not have similarity to any of the known bacterial nrps genes. thus, genetic analysis of two isolates of s. marcescens allowed us to identify nrps genetic clusters and showed that the repertoire of these genes is different between strains. we hypothesized that the strain isolated from environment has competitive advantage over clinical isolate due to genetic diversity of siderophores. on the other side, clinical isolate has specific genetic cluster of siderophores which may promote s. marcescens growth and adaptation to the extreme niches present in medical facilities. p-08.01.4-037 the first glance on the genome's structure and activity in hibernator edible dormouse hibernation is a unique adaptive way of survival in extreme environmental conditions where mammals decrease their metabolic rate and demonstrate physical inactivity for prolonged periods of time (up to 6-8 months). remarkably, some hibernating animals have a long average lifespan and the ability to avoid muscle atrophy caused by disuse or immobilization. to identify main molecular pathways behind the protective musculoskeletal adaptation and genome structure in hibernator edible dormouse (glis glis), whole-genome analysis of mrna expression in muscles (m. soleus and m. edl) and lumbar spinal cord samples was conducted. three groups of the dormice: 1) active animals 2) hibernated animals and 3) animals immobilized for 2 weeks in laboratory, were examined. rna libraries have been sequenced using hiseq 2500 illumina platform. coupled with genome dna sequencing provided x10 coverage of the estimated genome, we have assembled de novo transcriptome of the dormice. differentially expressed genes in response to immobilization and hibernation were determined. transcriptional program of these phenotypes was similar. pathways enriched by differentially expressed genes were identified. gene expression of the key muscle proteins and muscle atrophy markers was analyzed. muscle-specific e3-ubiquitin ligases murf1 and mafbx revealed no changes in mrna expression. our study represents the first attempt to elucidate changes in transcription profiles of skeletal muscles and spinal cord during hibernation and hypokinesia in edible dormice. in corroboration to the gene expression data, they demonstrated minimal morphological evidence for muscle disuse atrophy during physical inactivity. edible dormice, thus, can be considered as a novel model organisms in investigation of the genetic mechanisms of hibernation and prevention of muscle atrophy. the work is performed according to the russian government program of competitive growth of kfu and supported by rfbr jsps_a no. 14-04-92116. in response to diverse environmental cues bacteria form complex structured communities called biofilms. the metabolic pathways activated by these cues are remarkably different depending on the species studied. however, they all lead to the formation of an extracellular matrix that holds the cells together. non-pathogenic gram-positive spore-forming soil b. subtilis strain is recognized as a model system for the study of biofilms. to discover the pathways regulating biofilm formation in b. subtilis, we studied the natural isolate of b. subtilis strain 168, and constructed the recombinant strains with knocked out genes of following regulatory proteins: abrb (global transcriptional regulator), degu (two-component response regulator of signal transduction system degs-degu), ccpa (regulator of carbon catabolism) and spooa (regulator of sporulation). in the minimal medium broth b. subtilis 168 wild-type strain forms biofilm with its maximum on 48th hour of culture growth. ph-optimum for biofilms formation by the wild-type strain is in the range of 7.4-8.0. the temperature optimum is in the range from 22°c to 45°c. this corresponds to the natural conditions of the b. subtilis habitat in rhizosphere. the level of biofilm formation by regulatory mutant strains with deleted abrb, degu, ccpa, spooa genes is on average 40% lower than by the wild-type strain. this indicates that global regulatory system controlls biofilm formation process. statistically significant differences in the levels of biofilm formation between regulatory mutants haven't been identified. ph and temperature optima of mutant strains are the same as for the wildtype strain -7,4-8 and 22°c -45°c respectively. the crataegus genus which is a member of rosaceae family, has approximately 200 species worldwide and 24 species in turkey. all plant species in this genus have the common name "hawthorn". crataegus microphylla (c. microphylla) c. koch which is characterised by having erect sepals in fruit and smaller leaves in comparison with the other species, is one of the wild edible fruits in turkey. crataegus species have been used as food and also in folk medicine for the treatment of various diseases. for this purpose, the potential biological properties of crataegus microphylla were aimed to reveal by the preliminary work. in this study, prevention of oxidative dna damage using supercoiled pbr322 plasmid dna, acetylcholinesterase, tyrosinase, a-glucosidase inhibition and antioxidant effects: 2,2diphenyl-1-picrylhydrazyl radical scavenging effect, phosphomolibdenum-reducing antioxidant power, ferric-reducing antioxidant power with total phenolic and total flavonoid contents of the c. microphylla leaves, stem barks and fruits that extracted with ethanol, methanol and water were investigated. the experiments of oxidative dna damage studies and antioxidant activities of c. microphylla extracts showed that methanol and ethanol extracts possessed a strong ability to prevent dna damage and significantly antioxidant activities. methanol extracts of stem barks from c. microphylla exhibited the highest acetylcholinesterase and tyrosinase activities (48.86 ae 4.06% and 85.57 ae 2.01%, respectively), at 200 lg/ml. in addition, ethanol extract of leaves from c. microphylla inhibited the a-glucosidase activity significantly when compared to acarbose. this study explained significant antioxidant, enzyme inhibitory, hypoglycemic, and neuroprotective activities of methanolic or ethanolic extracts prepared with stem bark and leaf from c. microphylla and also strong ability to prevent dna damage that corresponded to antioxidant potential of methanol extracts of leaf and stem bark. the yarrowia lipolytica species (yl) is nonconventional yeast widely used for recombinant protein expression due to its system of post-translation protein modification, which is the most similar to that of higher eukaryotes. yl appears the promising producer of recombinant proteins with much more complicated molecules compared to those of prokaryotic producers. however, an important feature for a producer strain of recombinant proteins is the genes, the expression of which undertakes under controlled conditions, and consequently, search of new effective inducible promoters in the yl genome is of great interest. proteome analysis of the yl cells grown at different ph values (4.0, 5.5, 9.0) showed that under alkaline conditions the amount of mitochondrial porine vdac (voltage dependent anion channel), one of the most abundant protein of the mitochondrial outer membrane, increased significantly. vdac is supposed to let reactive oxygen species out of mitochondria protecting the cell against oxidative stress. therefore, the por1 gene expression, encoding vdac should increase in the stress conditions. the promoter of the por1 gene was used to construct some new expression systems based on yl w29. a new genetic construct bearing a reporter bgalactosidase gene under control of the por1 promoter. b-galactosidase activity was assayed in the cells grown in various ph conditions and exposed to exogenous oxidants such as hydrogen peroxide, menadione, and methyl viologen. it was shown, that in h 2 o 2 and methyl viologen treated cells b-galactosidase activity increased 1.5-2.0 -fold reaching its maximum in the cells, grown at ph of 9.0. thus, we demonstrated high inducibility of the por1 promoter, which is essential for effective action of the expression system based on it and potency of application for transformed lines of producers. acknowledgments: supported by the russian foundation for basic research (grant no 16-34-00634 mol_a). aspergillus nidulans is able to detoxify and catabolize the toxic proline analogue, lazetidine-2-carboxylic acid in nature, azc serves as a plant protectant against infections and consumption. we have obtained evidence that azc is not only non-toxic for the model ascomycete aspergillus nidulans, but it can be utilized as a poor nitrogen source. in order to elucidate the molecular mechanism underlying azc detoxification, we have constructed and studied a. nidulans strains deleted in the cognate genes involved in azc detoxification in pseudomonas and saccharomyces cerevisiae. these genes, found by in silico analysis, encode a putative hydrolase, acha, and an azc acetyltransferase, ngn2, respectively. gene deletion was accomplished through double crossover. a cassette containing the~1500 bp 5' and 3' flanking sequences of each gene, with the afpyrg gene as a selection marker, was contructed. crossing the achad and the ngn2d strains isolated the achad ngn2d double mutant strain. rt-pcr was used for gene expression analysis in the wild type strain, area-loss of function and crea-derepressed mutant strains. our results clearly show that azc can be used as a poor nitrogen source by a. nidulans. this utilization requires a) acha, a putative azc hydrolase, and b) a fully active gaba catabolic pathway, as lack of either amdr or gata abolishes azc utilization. most importantly, the double mutant, achad ngn2d, shows azc toxicity, suggesting that ngn2 is a true orthologue of mpr1, able to detoxify azc, a phenotype that can be observed only in the absence of acha. as a final point, ngn2 was shown to be induced by the presence of azc and and to be under nitro the spatial genome organization plays a great role in the maintenance of the nuclear architecture and regulation of all processes occurring in the nucleus. this system is controlled by a set of special proteins having an architectural function. however, the mechanisms of their action remain unknown. among these proteins are, in particular, zad-domain-containing proteins. zinc finger-associated domain (zad) is a ubiquitous motif of c2h2 zinc finger proteins of drosophila. genes that encode zad proteins are specific for and expanded in the genomes of insects. only a few zad-encoding genes have known functions, and the role of zad is being discussed. up to date there was only one known crystal structure of zad-domain from drosophila transcription factor grauzone (grauzad). here, we present for the first time the crystal structure of the zad-domain of serendipity-d transcriptional activator of the egg-polarity gene bicoid. zad-domain was cloned, overexpressed in e. coli, purified and the structure was solved at 3.5 a by mad technique. detailed analysis of the structure proved that the protein exists in dimeric form and revealed unique spatial organization of the protein, different from those for grauzad. this work is supported in part by russian ministry of education and science grant (14.616.21.0066). mycoplasma gallisepticum is a convenient model object for studying the regulation of transcription because it has a reduced genome, lack of cell wall and many metabolic pathways and also easy to culture and non-pathogenic to humans. due to the nature of the genomic organization and the loss of many of the known regulators, the effect of disrupting the function of some proteins may be a useful tool for studying the regulation of transcription. the gene expression study was performed on agilent onecolor microarray with custom design and random-t7 polymerase primer for cdna synthesis. microarray represents 3366 probes for 678 orf including genes and ncrna. in this work, we have investigated the effect of changes in the level of gene expression of m. gallisepticum for two different types of conditions: a genetic knock-out mutants and the cell response to treatment with sub-lethal concentrations of antibiotics. we characterized transcription of m. gallisepticum when the cell responses to dysfunction of proteins with metabolic potential, possible regulators of expression, in violation of permeability of membrane by cccp, inhibition of ribosomal synthesis by tetracycline, dna gyrase by novobiocin and atp synthase by oligomycin. the data obtained allow to characterize the transcriptional response under different conditions and to identify groups of genes that change expression together. major transcriptional changes were observed in the response of cells under cccp treatment due to uncoupling of the proton gradient and further reducing the membrane potential, as well as under novobiocin treatment due to changing the topology of dna. global problem of oil pollution forces scientists to search for a new safe remediation technologies constantly. careful attention is paid to bacteria, some of which possess additional biotechnologically valuable properties, such as utilization of hydrocarbons and production of biofurfactants. in this regard, we carried out proteogenomic characterization of tsukamurella tyrosinosolvens strain ps2, which was isolated from chemical sludge and capable for alkane degradation and biosurfactant production. whole genome of the strain was sequenced on the miseq (illumina) platform, assembled and annotated. proteome on mineral medium with glucose, sucrose and hexadecane as a sole carbon and energy source was studied. shotgun proteomics approach was performed on hybrid chromatography-mass spectrometry machine (maxis impact). alkane oxidation genes (alkane-1-monooxygenase, rubredoxin and rubredoxin-reductase) under genome sequence, as well as two pathways of trehalose synthesis and genes for mycolic acids production were found. emulsification activity of cell-free culture liquid was about four times higher on hexadecane in comparison with sugars. proteomic profile was different at various culture conditions. all glycolysis genes, beginning with glucose-6-phosphate isomerase to pyruvate kinase, were found on the media with sugar. the medium with hexadecane helped to reveal enzymes involved in the beta-oxidation of fatty acids, for example 2,4-dienoyl-coa reductase, 3-ketoacyl-coa thiolase and enzymes of the initial mycolic acid synthesis pathways. thus we have established that the strain t. tyrosinosolvens ps2 utilizes sugar by glycolysis. also, the bacterium is capable for alkane oxidation followed by beta-oxidation of fatty acids. based on the proteogenomic data, we assume that the bacterium is able to synthesize trehalose lipids, namely, trehalose mycolates. obtained results could be useful to create conditions for increased biosurfactants production. gestational diabetes mellitus (gdm) is a glucose intolerance firstly diagnosed during pregnancy. in this study, we aimed to investigate the association between serum adiponectin, resistin levels and insulin resistance in gestational diabetic patients. a total of 80 patients; 40 healthy pregnant women (control group) and 40 pregnant women diagnosed with gdm (gdm group) were included in this study. serum adiponectin, resistin, glucose, insulin, hba1c levels and lipid parameters were measured. insulin resistance index homa-ir values were calculated. in this study, serum glucose, insulin, hba1c levels and homa-ir were significantly higher in gdm group compared to the control group (p = 0.038, p = 0.011, p = 0.001, p = 0.008, respectively). serum adiponectin levels were significantly lower (p < 0.001); whereas serum resistin levels were significantly higher (p = 0.004) in gdm group than in the control group. it can be concluded that resistin contributes to the formation of insulin resistance, adiponectin plays an important role in the regulation of this resistance and they also have effects on gdm pathophysiology. hematological cancers including acute myeloblastic leukemia (aml) and acute lymphoblastic leukemia (all) in terms of incidence and mortality, are the second most important cancer type in turkey. numerous studies show that cancer patients respond differently to treatment thus supporting the idea of personalized therapy need for individuals. renin angiotensin system (ras) have key roles in aml and all progression and it has been shown by many studies suggests that these system's genes might be good biomarkers for aml and all personalized therapy. we aimed to identify ras gene based homogeneous subgroups of acute leukemia and determine the most effective chemotherapoetic agent for each subgroup. after validation and verification of the results, more effective drugs can be recommended for the use in clinics for chemotherapy of aml and all. results of our preliminary studies showed that we are able to identify subgroups of aml and all as well as correlating each existing subgroup with fda approved drugs. considering the long and highly cost process of developing new drugs for cancer treatment makes the present study all the more valuable. in addition, there is a serious need for change in aml and all therapy since there is no highly effective chemotherapy protocol available for their treatment. welcome trust sanger (wts) and cancer cell line encyclopedia (ccle) databases will be used to determine subgroups of aml and all based on ras genes or whole genome expression using standard deviation and hierarchical clustering analysis. the most effective drugs for each subgroup will be identified using pearson's r correlation analysis with drug sensitivity data (ic50, ic50, amax, aare, etc.) available in same databases. further validation tests will be performed by in vitro validation using aml and all cell lines: drug sensitivity profiles will be determined and gene expression will be shown by q-rt-pcr. p-08.02.5-003 functional polymorphisms of ephx2 in a turkish population h. pinarbasi, i. sari cumhuriyet university, sivas, turkey soluble epoxide hydrolase (seh; ec 3.3.3.2) is encoded by ephx2 and catalyses the degradation of endogenous fatty acid epoxides generated by cyp450 epoxygenases. these fatty acid epoxides such as epoxyeicosatrienoic acids (eets) have been shown to posses vasodilator, anti-inflammatory, anti-platelet, anti-hypertensive, anti-apoptotic, anti-thrombotic and natriuretic effects. it has been reported that eet levels are associated with hypertension, stroke and cardiovascular diseases. individual differences in the ephx2 gene that affect the seh activity may alter the circulating levels of eets. k55r and r287q polymorphisms have been known to cause increased and decreased seh activity, respectively. therefore we aimed to determine the genotype frequencies of these two polymorphisms in a turkish population. k55r and r287q polymorphisms were determined by the real time pcr using double-dye hydrolysis probes or pcr-rflp method. the observed genotype frequencies for k55r polymorphism were 80.8% wild type (aa) and 19.2% polymorphic genotype (ag+gg) and for r287q polymorphism 81.4% wild type and 18.6% polymorphic genotype (ga+aa). the genotype distributions for both polymorphisms were in hardy-weinberg equilibrium. pregnancy is one of manifestations for thrombophilia factors, which in its turn leads to various complications of its course. one of the markers of hereditary thrombophilia is mutations in the folate cycle mtr, mtrr and mthfr genes. insufficient intake of folate during pregnancy disrupts the functioning of the genome, leading to miscarriage, violation of embryogenesis and various fetal malformations. however, results of studies on the role of hereditary thrombophilia in the occurrence of complications during pregnancy are rather contradictory. aim of this study was to determine the frequency of alleles and polymorphic variants of folate cycle genes mtr a2756g, mtrr a66g and mthfr c677t in women of kazakh ethnic group with pregnancy complications. we used real-time pcr. blood samples for dna isolation were obtained from 129 pregnant women. the main group consisted of women (n = 90) which had a history of two or more pregnancy complications in the form of pre-eclampsia, eclampsia, missed abortion, miscarriage, and etc. control group consisted of women (n = 39) with two or more normal pregnancy outcomes, and had no complications during pregnancy in history. average age of women in experimental group was 32.0 ae 0.50 years compared with control of the age 33.6 ae 0.33. the analysis of the frequency distribution of alleles of genes in experimental group of women with complications of pregnancy revealed no significant differences relative to the control group. analysis of the distribution of polymorphic variants of folate cycle genes showed significant difference between the study and control groups in the occurrence frequency of heterozygotes for the mutant allele g in the gene mtrr a66g (or = 2.89, ci 95% = 1.25-6.71; v 2 = 6.376, p < 0, 05). no significant differences in alleles between homozygous wild-type and homozygous mutant alleles were observed. this work was funded by the mes kazakhstan (gr 0115rk00287 project number). p-08.02.5-005 a study on the association between rs6918698 polymorphism in connective tissue growth factor gene and pseudoexfoliation syndrome pseudoexfoliation syndrome (pes) is a disorder of the extracellular matrix characterized by the production and progressive accumulation of an abnormal fibrillary material in many ocular tissues. pes prevalence is 11.3% above the age 40 in turkey. since pes is characterized by excessive synthesis of elastic microfibrillar components throughout the body, growth factors can have important roles in the pathophysiology of pes. human connective tissue growth factor (ctgf) is a protein expressed in a variety of tissues, including the anterior chamber of the eye. ctgf coding gene has several genetic polymorphisms. rs6918698 g/c single nucleotide polymorphism (snp) is found at position à945, in promoter region. the presence of a c allele for rs6918698 is critical for transcriptional suppression of the ctgf gene which would reduce ctgf production. aim of this study was to investigate if there is any association between pes and rs6918698 polymorphism of the ctgf gene. study population consisted of 60 patients with pes and 60 controls. blood samples were collected by g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. genomic dnas were isolated from whole blood samples using manual dna isolation. the frequency of ctgf rs6918698 polymorphic allele g was 0.442 in patients, and 0.450 in controls (0.967, p = 1.000). distribution of genotypes was gg: 31.7%, gc: 48.3% and cc: 20.0% among patients, while gg: 35%, gc: 40.0% and cc: 25.0% (or = 1.162, p = 0.689) in controls. statistical analysis showed that there is no significant relationship between ctgf rs6918698 snp and pes. these are the preliminary findings of a research project which is the first study analyzing the relationship between ctgf rs6918698 snp and pes. this work did not point out a role for ctgf rs6918698 in the risk for pes. a significant relationship might be found when the study population is enlarged. p-08.02.5-006 evaluation of rs11136000 single nucleotide polymorphism of clusterin gene in pseuodoexfoliation syndrome risk pseuodoexfoliation syndrome (pes), an age-related systemic disorder, is characterized by production and accumulation of abnormal fibrillar extracellular material in anterior structures of the eye. clusterin (clu) is a multifunctional glycoprotein produced and secreted by almost all cell types and is found in all body fluids and in accumulated pes material. under cellular stress conditions, clu provides inhibition of stress-induced precipitation and aggregation of misfolded proteins. clu expression level in pes patients is unexpectedly low and this could be due to single nucleotide polymorphisms (snp) on the gene coding for clu. rs11136000 c/t polymorphism has been found to be associated with alzheimer's disease and pathophysiology of alzheimer and pes are similar. this study aimed to determine whether rs11136000 snp of clu gene have a role in the development of pes. study population consisted of 60 patients with pes and 60 controls. blood samples were obtained from g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genomic dnas were isolated from whole blood of subjects using manual dna isolation. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. t allele frequency of pes patients was 0.425 and that of controls was 0.442 (0.934, p = 0.794). the distribution of genotypes was cc: 30.0%, tc: 55.0% and tt: 15.0% among patients while cc: 28.3%, tc: 55.0% and tt: 16.7% (0.922, p = 0.841) in controls. there was no statistically significant difference between pes patients and controls in terms of tt genotype and t allele frequency. these are the preliminary findings of a research project which is the first study analyzing the relationship between clu rs11136000 snp and pes in turkish population. this work did not point out a relation for polymorphic genotype in the risk for pes. however, a relationship between clu rs11136000 polymorphism and pes can be found when we enlarge the study population. the tumor suppressor tp53 is the most frequently mutated gene in head neck squamous cell carcinoma cancer and represents a known transcription factor and tumor suppressor gene that regulates different microrna and target genes. the aim of our work is to construct the transcriptional and post-transcriptional network regulated by tp53 and to evaluate the difference at mrna and protein expression levels of the tp53 target genes in hpv negative head and neck squamous cell carcinoma (hnscc) patients with distinct tp53 mutation states and to elucidate the molecular mechanism that underlie the poor prognosis of tp53 mutation. to show the tp53 mutation landscape and its prognostic relevance for survival, we used cbioportal for cancer genomic analysis. we downloaded mutational profiles of 243 hpv negative hnscc patients. employing different databases we constructed the tp53 regulatory network. and then, to evaluate the effect on mrna, protein and microrna regulated by tp53 we used the mrna and protein expression profiles of patients from tcga. our results show that hotspot, truncating and missense mutations have statistical significance in the univariate analysis. the tp53 regulatory network show the involvement of important target involved in the progression of hnscc and the deregulation of protein expression of an important key epigenetic modifier ezh2 was significantly associated with tp53 mutational state. ezh2 is a member of the polycomb group protein enhancer zeste homolog 2 which is known to be directly repressed by tp53 and indirectly by the activation of mir-200a and mir-200b. we found a significant up-regulation of ezh2 that depend from tp53 mutation. it is important to understand the difference in mrna and protein expression of tp53 regulatory network that could depend from its mutational state. this finding suggest that ezh2 might be a potential therapeutic target for hnscc. p-08.02.5-008 next generation sequencing based approach for monitoring of minimal residual disease in acute lymphoblastic leukemia minimal residual disease (mrd) monitoring is widely used to evaluate efficiency of chemotherapy and to choose a strategy for further treatment in acute lymphoblastic leukemia (all). the most commonly used approaches for mrd detection are based on flow cytometry and qpcr. these methods have several important limitations including insufficient sensitivity, complicated experimental setup and false positivity. the newly developed next generation sequencing (ngs) approaches could overcome the existing limitations in mrd monitoring. here we describe a new mrd monitoring approach based on targeted deep sequencing of malignant rearrangements. first, we identified bcr/tcr rearrangements specific for the leukemic clones in initial bone marrow samples of 10 all patients. for this, we used sanger sequencing of the products of 19 multiplex pcr, performed with bcr/tcr specific primers combined according to the optimal frequency distribution of v/j-genes in healthy donors. second, we analyzed concentration of malignant clone rearrangements, identified at the first step, in dna samples obtained from bone marrow after 36 days of treatment. for this purpose, we performed ngs of 10 libraries for each identified leukemic rearrangement. four libraries were amplicons of bcr or tcr gene rearrangements generated using characteristic v and j segment specific primer combination. six additional libraries were amplicons of the same primer combination from the same dna sample which contained initial leukemic dna spike-in (in concentrations corresponding to 1 per 100, 1000 and 10,000 cells) for a calibration curve generation. using this approach, we analyzed 7 all clone specific rearrangements for three patients and calculated concentration of the leukemic clones by using the calibration curve. for one patient we didn't find any leukemic cells and for two patients we found 1 leukemic cell per 100,000 analyzed cells. znf365 is considered as a transcriptional target for p53 and plays an important role in the homologous recombination, mitosis, centrosome dynamics. as was shown by gwas some snps in znf365 have strong association with the risk of breast cancer (bc). however, it was unclear whether the same snps are associated with risk of bc in kazakhstan. therefore two polymorphisms (rs10995190, rs10761659) of znf365 were investigated in kazakh population in this study. the present case-control study was carried out with participation of 444 kazakh females with bc and 390 cancer-free donors. additionally, subtypes of bc, stratified by estrogen receptor (er+/à), progesterone receptor (pr+/à) and human epidermal growth factor receptor 2 (her2+/à) status were estimated. pearson p-value, odds ratio, 95% confidence interval tests were applied to data analysis. significant differences were found in allele frequency and genotype distribution at rs10995190 locus in znf365 between the patients and control groups (p = 0.013 for allele; p = 0.007 for genotype). moreover, significant association with bc was revealed for rs10995190 after dividing patients according to er+/ à, pr+/à and her2+/à status of the tumor. the g allele was associated with er+ (p = 0.01, or = 1.69, 95%ci:1.11-2.56), pr+ (p = 0.01, or = 1.78, 95%ci:1.13-2.79) and gg genotype with her2-bc carriers (p = 0.02, or = 1.66, 95%ci:1.04-2.63). also, g allele can be considered as a risk factors in er+/ pr+/her2-luminal type of tumor (p = 0.028, or = 1.79, 95% ci:1.06-3.05). our findings correlate with the data of several gwas where the association of the rs10995190 polymorphism with higher mammographic density and the risk of breast cancer have been shown. the obtained results allow us to consider g allele and gg genotype of rs10995190 as a marker of bc risk with predictive value, restricted to er, pr and her2 status of the tumor in the kazakh population. breast cancer (bc) is the most common cancer among women in most of countries. alternative variants of low-penetrance genes such as fgfr2 (rs2981582), tox3 (rs3803662), map3k1 (rs889312), lsp1 (rs381798) are shown to have high frequency in north america, south-east asia, australia, europe populations and a multiplicative effect on the development of bc. in this study was investigated assosiasion between alleles/genotypes combinations of these genes with increase/decrease of bc risk. the case-control study included 625 bc patients and 672 healthy women from kazakh and russian populations. genotyping was performed by pcr-rflp methods. combined effect of allele and genotype variations in four different genes on bc risk was assessed by apsampler algorithm. the fisher exact test, odds ratio (or) with 95% confidence intervals (95% ci) were applied to data analysis. according to obtained results combinations of allele c of tox3 rs3803662 and a of map3k1 rs889312 (p = 0.006, or = 2.2), also allele c of tox3 rs3803662 and c of lsp1 rs381798 (p = 0.02, or = 1.47) associated with increased bc risk in the russian population. consequently, combinations with c allele of tox3 rs3803662 contribute significantly to bc risk with p-value = 0.04, or = 1.86. on the contrary, tt genotype of tox3 rs3803662 with p = 0.04, or = 0.53 and its combination with allele t of lsp1 rs381798 with p = 0.03, or = 0.47 determine a bc risk reduction in russian population. in addition, a risk combination of allele c of lsp1 rs381798 and a of map3k1 rs889312 was found in kazakh population (p = 0.02, or = 1.35). studies have shown that a genetic predisposition to bc can be determined by the cumulative effect of individual alleles and genotypes and possible epistatic interactions of studied genes. obtained combinations of alleles and genotypes can be considered as complex genetic markers of bc and may be used as predictive. cancer that is caused by excessive proliferation of cells and reduced apoptosis is a pathological condition. currently, studies that are comitted with breast cancer are great important early detection and diagnosis of breast cancer. after the discovery of cisplatin as chemoterapy drug, new transition metal based complexes have been developed for treatment of cancer. in this study, anti-cancer activity of azo-azomethide ligand and its mononuclear metal complexes is studied on human cancer cell lines (mcf-7) and mouse fibroblast (l929) cell lines. cells were studied four different concentrations (12,5; 50; 75; 100 lm). xtt (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2h-tetrazolium-5-carboxanilide) protocol was applied after 24 and 48 hours. in our study 10% fetal bovine serum (fbs), 1% l-glutamine, 100 iu/ml penicillin and 10 mg/ml streptomycin in dmem (low glucose) were used. cancer cell lines in dmem medium is produced in 95% humidity and 5% co 2 incubator at 37°c. anti-cancer activity of synthesized complexes were determined on mcf-7 and l929. in the biological activity studies, synthesized compounds showed higher anticancer activity than positive control (5-fu). finally, our new synthesized complexes can be suggested that potent ajan for anti-tumuour for breast cancer drugs. large interindividual differences in response to chemotherapy present an important issue in cancer treatment. malignant mesothelioma (mm) is an aggressive tumor with poor prognosis, treated mostly with gemcitabine/cisplatin (gem/cis) or pemetrexed/cisplatin (pmx/cis) chemotherapy. as both clinical characteristics and genetic variability may affect treatment outcome, our aim was to construct and validate clinical-pharmacogenetic prediction models of treatment outcome in mm for both chemotherapy regimens and to develop an algorithm for genotype-based treatment recommendations. clinical-pharmacogenetic models were built on 71 gem/cistreated and 57 pmx/cis-treated mm patients. pharmacogenetic scores were assigned by rounding the regression coefficients. gem/cis model was validated on 66 independent mm patients. model predicting outcome of gem/cis chemotherapy included crp level, histological type, performance status, rrm1 rs1042927, ercc2 rs13181, ercc1 rs3212986, and xrcc1 rs25487. values ranged between 0 and 3.4; cutoff value of 0.75 had sensitivity of 0.62 and specificity of 0.81. patients with higher score had shorter progression-free and overall survival (p < 0.001). in the validation group, positive predictive value was 0.74 and negative predictive value was 0.56. model predicting outcome of pmx/cis chemotherapy included crp level, mthfd1 rs2236225, and abcc2 rs2273697 with scores ranging between 0 and 3.9. cutoff value of 2.7 had sensitivity of 0.75 and specificity of 0.61. patients with higher score had lower probability of good response and shorter progression-free survival (p < 0.001). clinical-pharmacogenetic models could enable stratification of mm patients based on their probability of response to gem/cis or pmx/cis and improve treatment outcome. this approach could be used for translation of pharmacogenetic testing to clinical practice as it would facilitate the selection of the best treatment option for each patient. p-08.02.5-014 evaluation of anti-diabetic potential of circiliol and circilineol using caco2 cell line diabetes mellitus is a metabolic disorder, and many people are suffering from this disease in the worldwide. oral hypoglycemic agents such as sulfonylureas and biguanides are currently used for the treatment. however, studies searching for a more effective anti-diabetic agents are being carried out continıously. based on that we aim to investigate the potential anti-diabetic effects of circilineol and circiliol isolated from teucrium alyssifolium extract, using in vitro cell culture models. for this purpose, the anti-diabetic actions were investigated by applying model caco2 (colorectal adenocarcinoma) cell line. we determined the level of ag (alpha-glucosidase), sglt1 (sodium-glucose transporter-1) and glut1-5 and glucose transport. neither ag activity nor sglt1 activity was increased with either circiliol or circilineol treatment in caco2 cells compared to positive control. similarly, neither the activity nor the expression level of glut1*-5 was increased in caco2 cell line with either circiliol or circilineol treatment relative to control. in conclusion, these results strongly suggest that circiliol and circilineol do not possess any anti-diabetic potentials.supported by tubitak 114z640 and paubap 2014fbe029 the purpose of this study was to characterize and assess the impact of a novel magnetite (fe3o4) nanosystem functionalized with the natural origin compound eugenol (e) on the pseudomonas aeruginosa virulence, biofilm formation and qs signaling in order to advance research aimed to find alternative and personalized therapeutic approaches for severe infections produced by this opportunistic pathogen. fe3o4 nanoparticles were obtained by a co-precipitation method and functionalized with analytical purity e. functionalized nanoparticles (fe3o4@e) were characterized by ir, sem, tga and hr tem. one laboratory and 9 p. aeruginosa clinical isolates were utilized in the study. growth and biofilm formation were assessed by an adapted microdilution method followed by absorbance reads and viable count analysis in dynamics (6, 12, 24 and 48 hours of treatment). soluble virulence factors production was assessed by enzyme activity evaluation of bacteria grown on specific media. the expression of qs core genes was analyzed by qrt-pcr and a luminescence assay. results demonstrated that the average size of the obtained nanosystem ranges 5-9 nm, particles are relatively homogenous and have a low tendency to form aggregates. subinhibitory concentrations of fe3o4@e limited biofilms formation in a time and strain dependent manner, and significantly inhibited the production of toxin pore forming enzymes (haemolysins and lipases) in most strains. the expression of lasi and lasr genes was three fold downregulated, while the expression of pqsr was upregulated in planktonic cultures suggesting that pqs signaling may be involved in virulence modulation after nanoparticle stimulation. the modulation of bacterial virulence and molecular signaling by functional nanoparticles utilized in subinhibitory amounts offer valuable perspectives to develop personalized antimicrobial approaches based on molecular communication control that clearly modulate pathogenicity and progression of the infectious process. p-08.02.5-016 specimen processing and handling for plasma ammonia measurement b. sarac ßligil 1,2 , s. abus ßo glu 2 , f. aky€ urek 2 , b. € ozt€ urk 2 1 selc ßuk medical school, konya, 2 biochemistry department, selcuk university faculty of medicine, konya, turkey objectives: ammonia requires special processing and handling conditions due to its' unstability. in this study, our aim to investigate a preanalytical factor (delayed analyze) affecting plasma ammonia measurement. design and methods: blood samples were obtained from 20 healthy volunteers. for determining different handling and storage conditions, the following protocols were applied: first protocol (a) transportation on ice and separation (centrifugation at 0°c) within 15 minutes of collection and analyze immediately. second protocol (b) transportation on ice and separation (centrifugation at 4°c) within 30 minutes and analyse refrigerated 2-8°c 2 hours (a1, b1) and 4 hours (a2, b2). all plasma ammonia levels was analyzed enzimatic glutamate dehiydrogenase methods by abbott architect c 8000 clinical chemistry analyzer. results: there were statistically alterations in all protocols compared to first protocol. prolonged centrifugation time for plasma ammonia lead to have higher results (38.6 versus 29.75 lg/dl, p < 0.001). in all protocols including a1, a2, b1, b2 also cause an elevation in plasma ammonia results (35.7, 37.4, 39.5 and 41.2; p = 0.032, p < 0.001, p < 0.001, p < 0.001, respectively). conclusions: ammonia concentration in the blood sample increases over time due to high concentrations in cells as erythrocyte or platelets (three fold). blood samples collected for ammonia determination should be stored on ice, and measured immediately. the wnt/b-catenin signaling pathway has been considered to be a factor in the development and progression of colorectal cancer. many studies have demonstrated that the presence of mutations or polymorphisms in ctnnb1(gene encoding b-catenin; mostly mutations in exon 3) can lead to aberrant activation of wnt/bcatenin signaling at the onset of various types of malignancies, including colorectal cancer. the aim of our study was to assess ctnnb1alteration in the patients with colorectal cancer and compared their tumor and normal tissues. a total of 30 paraffin-embedded colorectal tumor specimens were obtained from department of pathology in cerrahpasa medical faculty. also a total 30 paraffin-embedded normal tissue was used from same cases as a control group. ten-micrometer-thick tissue sections were placed on a glass slide and stained with he. then the tissue sections were dehydrated in graded ethanol solutions and dried without a cover glass. dna was extracted from the tissues with 100 ll of extraction buffer at 55°c over night. the tubes were boiled for 10 min to inactivate the proteinase k. ctnnb1 exon 3 was amplified by pcr. sscp is used to observe any difference between the 2 groups. genomic dna was isolated as described above. 10 ll of each pcr products mixed with 10 ll denaturating buffer and were denatured by heating at 95°c for 10 minutes in, and then were rapidly cooled on ice. the denatured pcr samples are run on 12% acrylamide/ bis gel in 0.59 tbe buffer for 3.5 hours at 200 v at room temperature with water cooling system. the gel was stained with silver staining method. migration and adhesion involve continuous modulation of cell motility, beta-catenin play major roles. beta-catenin gene alterations frequency range between 0 and 16% in colorectal cancer according to the different published studies. in our study no significant differences were found in the ctnnb1 exon 3 between the tumor group and normal groups. p-08.02.5-018 theranostic approach in agressive recurrent meningiomafirst experience in turkey m. o. demirkol 1 , b. uc ßar 2 1 koc ß university, istanbul, 2 american hospital, istanbul, turkey meningiomas arise from the meningothelial cells of the arachnoid membranes of the leptomeninx, which are attached to the inner layer of the dura mater. meningiomas can be classified into three grades (i-iii): grade i meningiomas which are benign, exhibit slow growth; grade ii (atypical) and grade iii (anaplastic) meningiomas, which have a much more aggressive clinical behaviour. meningiomas express non-steroid hormones, including somatostatin. in the brain, somatostatin-a cyclic tetradecapeptide neuropeptide-is believed to act as a neurotransmitter and neuromodulator. somatostatin performs its physiological functions by binding to specific receptors (sstri-sstrv). sstr2 exhibit high affinity for octreotate (tate). tate is a polar, watersoluble peptide that does not penetrate the intact bbb (brain-blood barrier). pet and scintigraphic imagings can only demonstrate somatostatin receptor positive intracranial lesions if the bbb is disrupted. in this aim, dotatate (dota-dphe1, tyr3-octreotate, tate) has been labelled with the positron emitter 68 ga and the beta and gama emitter 177 lu. in this case, we conducted a study to evaluate peptide receptor radionuclide therapy (prrt) planning based on pet/ct imaging of meningioma in the department of nuclear medicine and molecular imaging at the amerikan hospital. [ 68 ga] dota 0 -tyr 3 -oc-pet/ct has been established as the imaging modality of choice for the diagnosis and management of patient with skull-base malign meningioma (rapid progress -to 41 9 48 9 52 mm. from 31 9 37 9 35 mm. in 20 d.-after fifth operation). due to its high sstr2 selectivity, [ 177 lu]-tate showed significantly lower uptake/dose delivered to normal tissues, gatate-pet represents the imaging strategy of choice for an accurate assessment of sstr2 expression levels. although some studies have not shown a clear advantage over pet/ct, there is some evidence that it will have an advantage in selected body sites such as the head and neck, liver, and the pelvis. p-08.02.5-019 cardiovascular diseases can be treated by using 'tetr-odd-vp16' and 'hre' hypoxia inducible systems a. celik 1 , t. kaya 2 , s. cigdem 1 , m. g€ und€ uz 1 1 department of medical genetic, turgut ozal university, ankara, 2 faculty of medicine, turgut ozal university, ankara, turkey ischemia is an insufficient supply of blood to a tissue or organ, usually due to a blocked artery by a blood clot. up to now, the number one cause of death worldwide is caused by ischemia and related conditions such as heart attack or stroke. hif-1a is a transcription activator that functions as a master regulator of oxygen homeostasis. hif-1a protein levels increase under hypoxic conditions as a result of decreased o2-dependent prolyl-hydroxylation, ubiqutination and degradation. we aimed to break up clots in blood vessels and to prevent damage caused by ischemia by using hypoxia inducible systems. we added oxygen dependent degredation domain (odd) of hif1a between and in front of tetr dna binding domain and vp16 transactivation domain, so that tetr-odd-vp16 or odd-tetr-vp16 could activate transcription of tissue plasminogen activator (tpa) controlled by tetracycline response element (tre), in a hif1a independent manner. in addition, we also designed tissue plasminogen activator (tpa) under control of hypoxia response element (hre) of hif1a target genes. western blotting and immunofluorescence assay results showed the expression and nuclear localization of tetr-odd-vp16 and odd-tetr-vp16 constructs under hypoxic conditions, but not normoxic. in addition, using fluorometric reporter systems and tpa enzymatic assay we proved functionality of these constructs under hypoxic conditions. final apporoach to our project is predicting kinetic enzymatic activity of tpa during break up blood clots by using matlab. the results of the present investigation showed, the developed hypoxia responsible systems that can be engineered into endothelial cells to prevent ischemia related cardiovascular diseases. p-08.02.5-020 osteogenic potential assessment of some original scaffolds with magnetic properties new advances in bone tissue engineering demand the development of materials that can not only replace bone, but also regenerate the damaged tissue based on external or even internal stimulus. magnetic materials inside bone scaffolds are known to be a promoting factor for bone healing especially when the therapy is accompanied by application of external magnetic stimulation. based on a recent report, the presence of iron oxide in hydroxyapatite can improve the radio opacity and osteoblast proliferation. in this view, this study focuses on the development of silk fibroin-magnetite biocomposites for potential uses in bone tissue bioenegineering. such novel composites possess good mechanical properties, biocompatibility and biomineralization potential by in vitro tests and could become smart arhiectures, able to stimulate bone regeneration. a new culture model was developed by exposing a 3d cell/ scaffold bioconstruct to a continuous magnetic field during 3 weeks of osteogenic induction. in this view, mc3t3-e1 murine osteoblastes progenitor cells were seeded inside the novel silk fibroin-magnetite biocomposites and subjected to osteogenesys in a magnetic field during 21 days. osteogenic specific markers were evaluated every week in the presence and absence of the field. our results showed that the osteogenic marker's expression started earlier when mc3t3-e1 cells were exposed to the magnetic field. consequently, in our experimental model, the magnetic field had a benefic effect on the osteogenic differentiation process as mc3t3-e1 cells differentiated more efficiently in its presence. these results suggest that the bone healing process could be improved in the presence of a magnetic field. nevertheless, further in vivo studies on animal model should be employed for validation. p-08.02.5-021 impact of physical activity performed on different times of day on cardiac and skeletal muscle damage in trained and untrained male subjects s. algul, m. kara, o. ozcelik y€ uz€ unc€ u yil university, van, turkey introduction: physical activity elevates creatine kinase (ck) and creatine kinase myocardial band (ck-mb) levels which have been considered to be an indirect marker of skeletal and cardiac muscles damage. purpose: impact of physical activity performed on different times of day on serum levels of ck and ck-mb were investigated in trained and untrained male subjects. materials and methods: trained (n = 10, 18.3 ae 0.1 yr, 61.4 ae 2.3 kg) and untrained (n = 10, 18.6 ae 0.1 yr, 63.2 ae 2.4 kg) subjects performed three soccer matches (60 min) in field (30 m versus 50 m) in morning (m), afternoon (a) and at night (n) on separate days. the study protocol was approved by the local ethics committee. venous blood samples were taken at onset and at end of match. serum ck and ck-mb levels are measured using autoanalyser. data are expressed as mean ae s.e., compared by wilcoxon-signed rank and mann-whitney u-tests. p < 0.05 was accepted as statistically significant. results: ck and ck-mb levels increased in three matches in both groups (p < 0.05). importantly, there were significant increases in ck-mb levels in a and n exercises compared to m exercise (p < 0.05) in trained (10.6 ae 1.6 u/l versus 14.2 ae 1.9 u/l, 35% (m) 9.7 ae 1.3 u/l versus 18 ae 2.6 u/l, 66% (a) 9.5 ae 1.1 u/l versus 16.3 ae 2.0 u/l, 103% (n) and also untrained groups (8.6 ae 0.8 u/l versus 11.5 ae 0.8 u/l, 42% (m), 7.3 ae 0.5 u/l versus 11.9 ae 0.9 u/l, 85% (a) 7.9 ae 0.9 u/l versus 13.8 ae 0.9 u/l, 76% (n). discussion: increased metabolic stress or muscle damage during physical exercise elevate serum ck and ck-mb levels. however, higher percentage of increase in ck-mb levels in a and n exercise may reflects additional increases in cardiac muscle stress despite the similar skeletal muscle stress as indicated by ck levels. conclusion: considering the observation of higher percentage increase in ck-mb levels in untrained and also trained subjects, the caution should be taken while performing an exercise in a and n time especially in subjects who has cardiac weakness. p-08.02.5-022 regional assessment of hematological and discrimination indices of complete blood count for beta-thalassemia screening beta-thalassemia is one of the most common genetic abnormality causing health problems worldwide. blood count and film of beta thalassemia trait and iron deficiency anemia have similar features. therefore, several simple screening indices have been developed for differentiating between these diseases. it was to asaimed to assess the hematological parameters and discrimination indices in patients with betathalassemia trait who admitted to our hospital. the parameters of complete blood count (cbc) in 141 subjects (51 males and 90 females) diagnosed by mutational analysis (pcr, gene amplification, dna sequencing) between 2013 and 2015, were retrospectively screened and the thalassemia status of patients was assessed in terms of discrimination indices (eng-land&fraser (ef), green&king (gk), mentzer (m), ricerca (r), shine&lal (s-l), srivastava (s), ehsani and sirdah). the percentages of being above the cut off value were detected by ef 29.72%, gk 30.4%, m 26.35%, r 6.75%, s-l 8.78%, s 35.13%, ehsani 25.67% and sirdah 29.72%. the percentages of falsely negatives for the indices of ricerca and shine&lal were lower than others. morever, when the first three common mutations of our study were considered, 5 out of 23, 7 out of 21 and 4 out of 21 patients were up to the cut off values in terms of e&f, g&k, m, s, ehsani and sirdah indices for ivs-i-110 (g>a), ivsii-1(g>a) and heterozygous codon 8 deletion (-aa), respectively. the molecular diagnosis and prenatal detection for families at risk is important because of the difficulties of treatment in this disease. however, the use of discrimination indices may be valuable for distinguishing of thalassemia trait from iron defiency anemia when the equipment of molecular diagnosis are limited. in our study, ricerca and shine&lal had lower falsely negative results than others. nevertheless, further studies to detect diagnostic perfomance of discriminant indices should be conducted. p-08.02.5-023 novel therapeutic agents in the development of effective drug combinations to treat glioma s. avdieiev 1 , l. gera 2 , r. hodges 2 1 institute of molecular biology and genetics, kyiv, ukraine, 2 university of colorado, aurora, co, united states glial tumors are driven by multiple molecular aberrations that cannot be controlled by a single targeted agent. so, it is possible to expect that the combined multitarget anti-cancer therapy aimed simultaneously at different elements of tumor formation mechanisms will be more effective and will promote the extension of patients' life. to find out which drug combinations will enable the development of therapeutic regimens with improved effectiveness and decreased toxicity, the cytotoxic effects of several bradykinin antagonists (ba) were analyzed for different glioblastoma (gb) cell lines. among all the ba under investigation, bkm-570 appeared to be the most effective, with ic50 values of 4 lm and 3.3 lm in rat glioma c6 and human glioblastoma u251 cell lines, respectively. bkm-570 suppressed erk1/2 and akt1 phosphorylation in u251 cells. temozolomide (tmz), the firstline anti-gliomic drug used in clinics, has only a temporary positive effect and severe side effects in gb patients. we showed that the combination of bkm-570 and tmz led to significant potentiation of tmz cytotoxicity at sub-therapeutic concentrations. recombinant proteins with cytotoxic properties are promising agents for complex therapeutic applications. we revealed that the glioma-associated protein chi3l2 inhibited the viability of u251 cells more effectively than tmz. furthermore, the combination of chi3l2 and bkm-570 resulted in an additive cytotoxic effect. chi3l2-mediated decrease of cell viability was associated with a g1/s transition arrest. chi3l2 provoked the dramatic reduction of prb phosphorylation and a significant decrease of cyclin d1 expression, as well as a substantial increase in p53 level. in addition to the accumulation of p53, we observed the upregulation of cdk inhibitor p21. therefore, g1/s arrest in chi3l2-treated cells could be realized via activation of prb, downregulation of cyclin d, and activation of p53. harmful hereditary mutations in brca1 and brca2 are one of the most important risk factors of breast cancer. the aim of this study is to determine the mutations which are associated with breast cancer in people which is diagnosed breast cancer and/or have breast cancer-diagnosed family members by sanger sequencing and thus provide predictive and prognostic utility. our ongoing study is present the genetic variations in brca1 and brca2 genes in breast cancer-diagnosed 12 patients, that one of them is male, and 1 person yet healthy whose brca2 was sequenced by sanger sequencing. the data were analyzed by using seqscape software 2.6 and detected variations were compared with literature. in brca1, we determined 16 different benign genetic variations and 1 variation with unknown significance and 1 variation which has not in literature. in brca2 gene of 12 patient and 1 healthy person,14 benign variations,2 variations with unknown significance,7 variations which has not in literature and 1 mutation were determined. this mutation is c.3189-3192delgtca and is located in occr. c.9257-74a>c variation in brca2 gene, was determined in only male patient.c.32t>a variation in exon2 of brca2, was observed in only the youngest patient who has no family member with breast cancer and healthy person. while this variation takes place in literature as variation with 'uncertain clinical significance', an in silico program mutation taster speculated as 'disease causing' for this variation. also, almost all of variations with 'unknown significance' literature knowledge were determined in only one and different cases. this situation increases the possibility of being pathogenic of this variations. the our findings until now can contribute to variations with uncertain clinical significance in the literature. also the variations that have not in the literature but we suggest the possible relation with breast cancer as an estimate may be added to literature by expanding the study. p-08.02.5-025 inhibition of the recombinant human butyrylcholinesterase with paraoxon and coumarin analog of soman organophosphorus compounds (op) represent a class of extremely toxic chemicals that are used as warfare agents. uncontrolled utilization of op is highly dangerous due to their high potential to be efficient poisons in terrorist attacks. current therapy of op poisoning include intravenous administration of atropine and acetylcholine reactivators however, it does not completely eliminate brain damage effects. alternative experimental therapy against op poisoning is utilization of bioscavengers that irreversibly react with op and rapidly inactivate them. recombinant human butyrylcholinesterase (rhbche) is one of the most promising candidates as bioscavenger due to its pharmacokinetic characteristics and broad spectrum of op neutralizing activity. here we investigated in vitro inhibition of rhbche with two model oppesticide paraoxon (pox) and coumarin analog of soman (gd c ). both op lead to rapid and irreversible inactivation of rhbche that was monitored using ellman assay and fluorescence measurements. bimolecular inhibition rate constants dramatically differ between pox and gd c that could be explained by steric hindrance in soman analog. the next steps forward creation of catalytic bioscavengers based on rhbche should be done based on mechanisms of op-rhbche interactions. this work was performed in frame of grant rfme-fi60414x0069. non-hodgkin lymphomas (nhls) represent a heterogeneous group of malignancies that arise from the lymphoid system. at the present time exist a lot of drugs for the nhls therapy, but mostly all of them are unsafe and there is no consensus regarding the best treatment protocol. to increase the efficacy and safety of therapeutic b-lymphocyte depletion in lymphomas and leukemia's it would be preferable to induce the death of pathological b cells without affecting normal b cells to prevent side effects. similar to other types of cancer, nhls arise by a multistep accumulation of genetic aberrations that induce a selective growth advantage of the malignant clone. all b-cells of organism have a unique cell surface markerantigen b-cell receptor (bcr). we generate novel approach for personalized non-hodgkin lymphomas therapy based on peptide specific to malignant cells surface receptor. for this purpose we designed new lentiviral peptide library screening technique based on fluorescent reporter cells system. herein 7aa peptide library was used for screening of nhl's malignant receptor agonist. patients' lymph nodes biopsy samples mrna was used as a source of malignant bcr nucleotide sequence. variable domain of the lymphoma bcr was used for chimeric receptor generation, where bcr vh/vl part responsible for agonist recognition and bottom part of receptor was retranslate signal to the reporter gene. in this embodiment of the method, very large numbers of candidate 7aa peptides expressing lentivirus and eukaryotic reporter cells are packaged together in a format where each is capable of replication, thereby forging a direct link between genotype and phenotype. after four rounds of screening we discover peptides specifically interacted with malignant bcr's. selected peptide ligands were fused with chimeric antigen receptor for expression on t-cells. modified tcells selectively eliminate nhl's malignant cells ex vivo. this work was supported by grant rfmefi60714x0061. introduction: pesticides are used to prevent damage of unwanted insects, rodents, plant, moss and other pests. excessive use of pesticides may cause adverse effects in animals and humans. chlorpyrifosethylene (cpe) is an organophosphate pesticide, used in many agricultural products such as figs, cherries, olives worldwide; caused acute poisoning and chronically oxidative stress. rosadamascena mill (rosaceae) is a rose, used for production of rosewater (rw) and rose oil worldwide. rosaceae products are consumed in food and cosmetic industries. materials and methods: in this study, we investigated that cpe and rw effects on kidney tissues of rats. this study was included 32 adult male rats, divided into 4 groups. each group included 8 rats. i.group: control (regular feed), ii.group: cpe (0.3 mg/kg/day), iii.group: rw (100 mg/kg/day) and iv.group: cpe (0.3 mg/kg/day) + rw (100 mg/kg/day). following 15 days, kidneys were taken after sacrificed. analyzes were performed that malondialdehyde (mda), nitric oxide (no) as oxidant; superoxide dismutase (sod), glutathione reductase (gr) as antioxidant parameters. results: as compared with control, mda and no levels in cpe were a significant increase was determined (p conclusion: cpe is shown that significant increase on oxidant parameters, but significant decrease on antioxidant parameters. rw occurs opposite situation. similarly results of cpe, rw + cpe increased oxidant parameters, but decreased antioxidant parameters. these changes are lower than only cpe. these results showed that positive effects both rw and rw + cpe increasing on antioxidant parameters, also decreasing on oxidant parameters. we provide the comparative analysis of the reduced glutathione (gsh), reactive oxygen species (ros), a-tocopherol levels, an intracellular labile zn 2+ pool and esterase activity of red blood cells of patients with diagnosed components of the metabolic syndrome (ms)arterial hypertension and diabetes mellitus type ii (ah + dm + ). as the comparison groups were selected patients with one diagnosed component of msarterial hypertension (ah + dm à ) or without any diagnosed component of ms (ah -dm -). patients of all investigated groups were at the hospital treatment with a diagnosis coronary heart disease (chd) ii degree. human blood was obtained from normal donors and patients with chd ii stage. cytosolic esterase activity was assessed using calcein-am test. ros level was evaluated using cm-h 2 dcf-da. gsh level was estimated using lowry method. an intracellular labile zn 2+ pool was assessed using fluozin-3-am. investigations were performed on the specord m40, hplc system lc-20 prominence (shimadzu) and facscantoii (bd). a significant decrease of the intracellular level of labile zn 2+ in erythrocytes of patients with ah + dm + compare with ah + dm à and ah à dm à was shown. this fact confirms our assumption concerning the important role of zinc homeostasis in the etiopathogenesis of diabetes mellitus type ii. a direct relationship between the intracellular zn 2+ level modification and erythrocytes esterase activity of patients with chd ii degree was observed. moreover, in ah + dm + group of patients this relation was more marked. the unidirectional alteration in the erythrocytes redox state (gsh and a-tocopherol levels reduction, ros formation activation) was revealed at the whole of investigated chd patients groups (ah + dm + , ah + dm à , ah à dm à ). however, the pathological erythrocytes response on in vitro action of the different antioxidants (n-acetylcysteine, ascorbic acid, a-tocopherol, quercetin) had a diverse character that can be a significant test under antioxidant therapy prescription. it is known that long-term social isolation and the disorder of natural circadian rhythm is considered an important stress factors, which cause a variety of metabolic and mental disorders. it should be noted that the impact of the stresses takes up a larger area, according to, review of the action of their mechanism is one of the topical issues of modern science. it is estimated that as a result of stress the metabolic processes change in the organizm. because of this, we've studied the functionality of the antioxidant system in laboratory rat heart muscle cells and blood under psycho-emotional stress. it was found that quantitative changes of nitric oxide (no) was initiated the process of lpo, which caused oxidative stress in the cells and decreased antioxidant enzymes activity, such as catalase,sod, gpx and gr. the results suggested that psycho-emotional stress was accompanied by oxidative stress, causing a reduction in the intensity of energy metabolism in cardiac muscle cells, which was further strengthened by the fact that the activity of the enzymes involved in atp synthesis in mitochondria was reduced. also, we've studied exogenous creatine positive and negative affects on energy metabolism and blood lipid spectrum. based on this, we proposed that psychological stress is one of the factors contributing to the development of various cardiac diseases. the importance of free radical lipid transformations, which differ from the peroxidation processes, was pointed out for the first time in our laboratory studies. we have found that ros can induce free radical destruction processes of sphingolipids with c-c-bond cleavage [lipids, 2011, 46:271-276; lipid insights, 8, 1-9] . in case of acylated sphingolipids, they can undergo decomposition with c-c-bond cleavage upon direct uv irradiation [photochem. photobiol., 2012, 88:899-903] . it was of interest to establish the possibility of photosensitized decomposition reactions of not acylated sphingolipids, which do not absorb an ultraviolet. in this work we studied photosensitized reactions of sphingosines containing a free amino group, and low molecular compounds, which simulate their structure, such as aminoalcohols (serinol). as photosensitizers, the salts of transition metals, hydrogen peroxide, and acetone were used. oxygen was removed by bubbling with argon to reduce the probability of side reactions during photolysis of sphingolipids, such as oxidation processes (including oxygen reactions with alkyl radicals). we have shown that the action of uv-radiation on aminoalcohols and sphingosines in aqueous solutions in the presence of photosensitizers induces their destruction with c-c bond rupture. the main carbonyl product of sphingosines free radical destruction was an unsaturated aldehyde -2-hexadecanal. it was found, that 2-hexadecenal possesses a wide spectrum of biological activity: it promotes reorganization of the cell cytoskeleton and modifies the redox state of the cells [febs journal 282 (suppl. 1), abstracts: mem. biol. s5, lipid signaling & dynamics, p. 235.] . the results of this study can expand the frontier of research regarding free radical lipid damage, which could contribute to a better understanding of the origins of diseases associated with the activation of free radical processes in living organisms. structural basis for the 14-3-3 proteindependent inhibition of apoptosis signalregulating kinase 1 protein kinase ask1 (apoptosis signal-regulating kinase 1) is a member of the mitogen-activated protein kinase kinase kinase (map3k) family that plays a crucial role in immune and stress responses. the activity of ask1 is regulated through homo-oligomerization and interaction with other proteins including the 14-3-3 protein which binds to the phosphorylated motif located at the c-terminus of the kinase domain of ask1 and suppresses its catalytic activity through unknown mechanism. under stress conditions, ask1 is dephosphorylated at ser966 and the 14-3-3 protein dissociates. this dissociation is then one of the factors that lead to the activation of ask1. we performed low-resolution structural analysis of the kinase domain of ask1 (ask1-cd) bound to 14-3-3 using chemical cross-linking, analytical ultracentrifugation and small angle x-ray scattering. the low-resolution structural analysis shows that ask1-cd binds to the 14-3-3 protein in two to two stoichiometry through a small binding interface involving surface of 14-3-3 outside its central channel and several regions from the c-lobe of ask1-cd. the complex is dynamic and conformationally heterogeneous. phosphorus nmr and time-resolved fluorescence measurements, together with low-resolution structural analysis, indicate that binding of ask1-cd to 14-3-3 modulates conformation of ask1's activation segment. these results suggest that the 14-3-3 binding suppresses the catalytic activity of ask1 through direct structural modulation of its activation segment. our study provides new insight into the interaction between the kinase domain of ask1 and 14-3-3 and offers a plausible structural explanation for the 14-3-3 protein-dependent inhibition of ask1 kinase activity. introduction: thymoquinone (2-methyl-5-isopropyl-1,4-benzoquinone, tq) exerts a great antitumor activity against different types of cancer cells. a growing body of evidence indicates that reactive oxygen species (ros) generation followed by modulation of the akt and mapk pathways is a general mechanisms underlying the tq antitumor action. however, the data of tq effects on the mapk pathway are conflicting. to date, the activation or inhibition of the mapk protein family seems to depend on the cell type and on the tq concentration used. in order to elucidate the antitumor potential of tq against gliomas and the underlying molecular mechanism, tq influence on c6 rat glioma cells functioning was studied. results: it has been shown that the cultivation of c6 cells with tq in concentrations of 10 -100 lm during 24 hours strongly inhibits cell proliferation and induces cell death with id 50 of 60 lm. at the same time, tq induces ros generation and intracellular gsh depletion in a dose-dependent manner, that is followed by the mitochondrial potential decrease. interestingly, ros production has only cytoplasmic, but not mitochondrial origin in cells challenged with tq at the concentrations up to 100 lm. two-electron reduction of tq by dt-diaphorase attenuates tq anticancer efficiency whereby inhibition of dt-diaphorase by dicumarol increases tq-induced c6 cell death by 25 %. we analyzed mapk pathways involvement in c6 cells growth inhibition at tq treatment. it has been shown that inhibition of the erk pathway by pd98059 and jnk pathway by sp600125 does not influence on tq-induced effects. on the contrary, the specific phosphoinositide-3-kinase (pi3k) inhibitor (ly294002) abrogates tq-induced growth arrest. conclusion: antitumor effects of thymoquinone on c6 glioma cells is a result of ros generation and intracellular gsh depletion, that is followed by mitochondria disfunction, and growth arrest via pi3k pathway. assessment of oxidative stress and antioxidant defense activity parameters in patients with hiv-infection is of great importance, especially for hiv-positive women of reproductive age planning to have children. data of 53 women of reproductive age with hiv-infection analyzed: patients with hiv-monoinfection (n = 26) and patients with co-infection (hiv and hepatitis b and / or c) (n = 27). as a control we used the data of healthy women (n = 28). serum and hemolysate used as material for the study. lpo-aod products were determined by spectrophotometric and fluorometric methods. average value of initial lpo products -diene conjugates was significantly increased in the group with hiv-co-infected compared to control (1.57 times; p = 0.001) and group with hivmonoinfection (1.4-fold; p = 0.027). the level of secondary products -ketodienes and conjugated trienes increased in patients of both groups compared to control (2.16 times (p = 0.0002) and 2.43-fold (p < 0.0001), respectively). at the same time isolated double bonds and tba-active products content showed no significant changes (p > 0, 05). total antioxidant activity parameter decreased 1.28 fold (p = 0.0273) in the group with hiv-monoinfection compared to control. decrease in activity of the primary antioxidant enzyme -superoxide dismutase (p = 0.0077, compared to the control and p = 0.0035, compared with the group with hiv-monoinfection) and the level of a-tocopherol (1.22-fold to control and 1.25 fold to hiv-monoinfection) was detected in the group with hiv-coinfection. 1.29-fold higher content of retinol in hiv-coinfection group (compared to the control) revealed. in women with hiv-coinfection the oxidative stress was significantly higher than in women with hiv-monoinfection. suggested to include antioxidant supplements in the complex pathogenetic therapy in women with hiv-coinfection (hiv and hepatitis b and / or c), which will contribute to women's ability to bear children. p-09.04.4-008 acute different doses of malathion induce cholinesterase inhibition, glucogenic enzymes and histopathological change in rat liver malathion, which is an organophosphorus compound, is a widely used pesticide all over the world. despite its benefits, malathion has many toxic effects on many tissues including liver. we designed to evaluate the acute different doses of malathion on cholinesterase (che) inhibition, gluconeogenic enzymes and histopathological change of rat liver. for this purpose 4 groups were formed. rats in group 1 served as control group animals which were only given corn oil. group 2, group 3 and group 4 were administered 100, 200, 400 mg/kg of malathion, respectively, dissolved in corn oil by oral gavage. the rats were sacrificed after 24 hours following administration and the livers of rats were removed. the liver che, alanine aminotransferase (alt), aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) were studied using autoanalyzer. histopathological investigation was performed using microscope. the liver che activities of group 2, group 3 and group 4 were inhibition percentage of 53%, 43%, and 51% respectively, comparison of group 1's che activity. the liver alt, ast and ldh increased in group 3 and group 4 compare to group 1 and group 2 (p < 0.013). we also observed that there was vacuolar and hydropic degeneration in liver of group 4. according to our result, acute administrations of malathion result in hepatotoxic effects with increasing doses. background and aims: an imbalance between free radicals and antioxidants is closely linked to the onset of an acute myocardial infarction (ami). the aim of this study was to investigate the antioxidant status and the lipid peroxidation in patients admitted to hospital immediately after ami. methods: the study population comprised 19 patients with ami and 14 healthy subjects. patients that had an acute myocardial infarction (ami) less than 72 hours since onset were selected for this study. antioxidant status was assessed by lactonase activity of paraoxonase (pon1 dhc), trolox equivalent antioxidant capacity (teac) and plasma uric acid level. malondialdehyde was used as marker of lipid peroxidation. results: compare with the control group, the levels of mda and pon1 dhc were significantly higher in group with ami (p < 0.005, respectively p < 0.001). elevated levels of mda (p < 0.005) were found in patients with ami compare with the control subjects. ami patients had also statistically significant reduced levels of teac (p < 0.005) comparative with healthy subjects. no statistically significance was found for plasma uric acid level in subjects from our study. conclusion: a high lipid peroxidation correlated with a decreased teac activity suggest an exacerbated oxidative stress in subjects admitted to hospital immediately after an ami. p-09.04.4-010 dealing with copper toxicity: new insights into copper detoxification in yeast copper (cu), an essential metal, is a double-edged sword, as its essential nature is counterbalanced by the toxic effect that it can exert on cells when not properly controlled. as such, organisms have evolved defence mechanisms against cu toxicity, and in the yeast saccharomyces cerevisiae, the transcription factor ace1 orchestrates several of those mechanisms, by activating cu-detoxification genes. in s. cerevisiae iron (fe) uptake is partially dependent on cu, as the membrane multicopper-oxidase fet3, which is part of the high-affinity iron uptake system, requires cu as a cofactor. aft1, the low iron-sensing transcription factor in yeast, is known to regulate the expression of fet3 gene. however, we found that a strain lacking fet3 is more sensitive to cu surplus conditions than a strain carrying the aft1 gene disrupted. this finding suggests that under such conditions another regulator came into play and controls fet3 gene expression. we next evaluated whether ace1 could be the alternative regulator of fet3 under cu excess. to test this hypothesis we first constructed the double mutant aft1ace1 and assayed its fitness under cu surplus. as expected, the double mutant is much more sensitive to cu than the single aft1 or ace1 mutants. we next monitored the expression of fet3 gene in cells lacking ace1, using yeast-one hybrid and qrt-pcr approaches. our data clearly indicates that fet3 expression is dependent on ace1 when cu is overly abundant. altogether our data unveil a novel mechanism of cu detoxification relying on the activation of fet3 by ace1 in an aft1independent way. experiments to understand the consequences of this regulation in terms of cu detoxification are currently being undertaken. in joint degeneracy, reactive oxygen species manifest their toxicity both through intrinsic reactivity and the inflammatory process activation, leading to cartilage dysfunctions, injuries of matrix proteins and cytokines stimulation. the study is focused on the identification of oxidative balance modulation (enzymes and oxygen free radicals) by a bioactive extract obtained from small sea fish. the cellular support was represented by the chondrocyte line chon-001 derived from human long bones (atcc ò crl-2846 tm ), that assure the reproducibility of a standardised biological system. the oxidative stress was induced through stimulation with il1b, a cytokine-factor that promotes the protein catabolism and also with tnfa, the initiator of pro-inflammatory activation, combined with pma, the activator of protein-kinase c, triggering of oxygen reactive species generating cascades. the antioxidant effect was compared with known antioxidants: vitamin c, x3 fatty acid, n-acetyl -cysteine. the acellular antioxidant/antiradical screening was done using two complementary techniques for total antioxidant status evaluation and completed by measuring cellular catalase (cat) and superoxidedismutase (sod) activity, correlated with intracellular hydrogen-peroxide and superoxide anion monitoring through flow cytometry. the antioxidant properties of the bioactive extract proved in acellular systems are confirmed at cellular level by the involvement of the product in the enzymatic cascade cat -sod, potentiating the catalytic action of the enzymes, and by the decline of both intracellular reactive oxygen species (the hydrogen peroxide decrease with 50%, the superoxide anion is reduced with 31% compared with the stimulated control). the demonstrated antioxidant synergy assures a complete cellular protection induced by the small sea fish extract in human condrocytes. introduction: alzheimer's disease (ad) is a progressive neurodegenerative disorder characterized by memory loss, cognitive impairment. oxidative stress is a contributory factor in ad pathogenesis. glutathione (gsh) is the main antioxidant cellular defence. the ratio of gsh/gssg shows the redox status of gsh, and plays important role in maintaining intracellular redox homeostasis. the current study was carried out to determine oxidative dna damage and ratio of gsh/gssg which plays an important role in protection of target molecules from oxidation in the patients with ad. methods: the study subjects were consisted of patients with ad (n = 67) and age matched control group (n = 42) who were treated and followed in the cerrahpasa medical faculty hospital, department of neurology and department of internal medicine/ geriatrics. dna strand breaks and h 2 o 2 -induced dna damage were determined in lymphocyte dna with comet assay. the gsh and gssg levels in the erythrocyte lysates were measured by using a commercial spectrophotometric kit. the ratio of gsh/gssg were calculated. statistical analysis was performed with spss 22 software package. results: dna strand breaks and h 2 o 2 -induced dna damage were found to be higher (p = 0.000 for all), the ratio of gsh/gssg was found to be lower (p = 0.024) in the ad group than control group. there was no significant difference between male and female for dna strand breaks and h 2 o 2 -induced dna damage in the ad group, but ratio of gsh/gssg were higher in male when compared with female (p = 0.045). no significant difference was found between the men of ad group and men of the control group for gsh/gssg ratio whereas women of the ad have a lower gsh/gssg ration than those in the women of the control group (p = 0.009). conclusion: increased systemic oxidative dna damage and dna susceptibility to oxidation may be resulted from diminished gsh/gssg ratio in ad patients. this finding shows the importance of antioxidant support in ad management. p-09.04.4-013 validation of a liquid chromatography-tandem mass spectrometry method for the measurement of lipid peroxidation product 8iso-prostaglandin f2a in urine m. kant 1 , m. akıs ß 1 , h. _ is ßlekel 1,2 1 department of medical biochemistry, school of medicine, dokuz eylul university, izmir, 2 department of molecular medicine, school of medicine, dokuz eylul university, izmir turkey 8-iso-prostaglandin f 2a (8-iso-pgf 2a ) is a reliable indicator of lipid peroxidation resulting from oxidative lipid damage and is postulated as a gold standard biomarker for the evaluation of oxidative stress. the aim of this study was to validate non-invasive and highly accurate stable isotope dilution-multiple reaction monitoring liquid chromatography-tandem mass spectrometry (sid-mrm lc-ms/ms) method for identification and quantification of 8-iso-pgf 2a . twenty four hour urine samples were collected from healthy volunteers at medical school of dokuz eylul university for analytical performance studies. lc-ms/ms analyses were performed on conventional hplc coupled to a triple quadrupole ion trap mass spectrometer equipped with a turboionspray tm source. analyst software version 1.5 were used for data analyses. mrm transitions used were m/z? 250/164 for 8-iso-pgf 2a and m/z? 255/169 for 8-iso-pgf 2a-d 4 . analytical performance of the method was evaluated by linearity, selectivity, sensitivity, precision and accuracy studies using pure standards and samples extracted from urine of healthy volunteers. the linear calibration range for 8-iso-pgf 2a was determined as 20 ng/ml by using standards ranging from 0.25-50 ng/ml. analytical sensitivity of the method was determined by lod with s/n of 3 and loq with s/n of 10. lod and loq for 8iso-pgf 2a were 2.4 9 10 à2 and 38 9 10 à2 ng/ml, respectively. the assay stability and reproducibility were assessed by the precision and accuracy of intra-and interday measurements (n = 10). the intra-and interday cvs for 8-iso-pgf 2a were 4.5% and 1.2%, respectively. sid-mrm lc-ms/ms method for absolute quantification of 8-iso-pgf 2a was optimized and validated in our laboratory and therefore this highly accurate method can successfully be applied to clinical patient samples. p-09.04.4-015 synergistic anticancer effects of sulforaphane and cisplatin through the induction of apoptosis and autophagy following oxidative stress in malignant mesothelioma cells malignant mesothelioma is characterized by poor responsiveness to current chemotherapeutic drugs, usually owing to high resistance to apoptosis. here, we investigated chemosensitizing effects of phytochemical resveratrol, in combination with cisplatin, on malignant meothelioma cells. cell viability was evaluated using mtt assay. cell apoptosis was detected with dapi staining, caspase 3/7 activity assay and flow cytometry. cell cycle distributions, ros levels and mitochondrial membrane potential were determined using flow cytometry. the expression of cell cycle-, apoptosis-, and autophage-related proteins was measured with western blotting. the combination treatment of cisplatin and resveratrol (cis/res) synergistically induced apoptosis, as evidenced by typical cell morphological changes, the appearance of a sub-g 0 /g 1 peak, an increase in the annexin v(+) cells and the cleavage of caspase-3 and parp. cis/res increased ros production and depolarization of mitochondrial membrane potential with an increase in the bax/bcl-2 ratio. these changes were attenuated by pre-treatment with n-acetylcysteine, suggesting that cis/res induced apoptosis through oxidative mitochondrial damage. compared with msto-211h cells, cis/res was less efficient in killing h-2452 cells. h-2452 cells exhibited an enhanced autophagy to cis/res, as observed by an increase in viable cells exhibiting intense lysotracker red staining and up-regulation of beclin-1 and lc3a. inhibition of autophagy by bafilomycin a1 rendered cells more sensitive to cis/res-induced cytotoxicity and this was associated with induction of apoptosis. these data indicate that the increased resistance of h-2452 cells to cis/res is closely related to the activation of self-defensive autophagy, and provide the rationale for targeting the autophagy regulation in the treatment of malignant meothelioma. stress oxidative induced by chemotherapy with cyclophosphamide (cp) causes vulnerability in sperm and decline of fertility. this study was aimed to evaluate the role of ethyl pyruvate (ep) in the amelioration of fertility and growth of primitive embryo in animals that received cp. 24 adult male mice (6-8 weeks) were randomly divided into 3 groups: control group received normal saline (0. 2 ml/day, ip), cp group received cp (15 mg/kg/week,¬ ip), the cp+ep group received ep (40 mg/kg/day, ip) along with cp, were treated for 35 days. 8 mice from each group were arranged for evaluation of sperm quality and in vitro fertilization (ivf) too. afterward, the separated oocytes from 72 ovulation stimulated mice were conducted to evaluation of ivf and embryo development. the results revealed that cp caused notable decrease in percentage of fertilization in cp group, but administration of ep along with cp caused an increase in the percentage of fertilization in comparison to cp group. the percentage of the two cell zygotes in cp+ep group, and the percentage of blastocysts in control and cp+ep groups were higher than that in cp group (p < 0.05). results showed that the total number of arrested embryos in control and cp+ep groups was decreased in comparison to cp group (p < 0.05). the percentage of arrested embryos type i, ii, and iii in cp+ep group was decreased in comparison to cp group, but that decrease was significant only in types i and ii (p < 0.05). the average motility, viability, nucleus maturity and sperm morphology were decreased significantly in cp group in comparison with control and ep groups, whereas ep caused significant increase of these parameters (p < 0.05). also, the percentage of dna damage was increased significantly in cp group in comparison with control and ep groups (p < 0.05). the results of this study indicated that the ethyl pyruvate was able to suppress free radicals and enhance the ivf and quality of sperm in cp treated animals. malathion, which is a member of organophosphate chemical family, is used to control pests and is a widely used pesticide all over the world. however it is also known to be highly toxic on many tissues including pancreas. to test this we set 4 groups to administer a single dose of malathion dissolved in corn oil via oral gavage at the doses of 100 mg/kg (group 2), 200 mg/kg (group 3) and 400 mg/kg (group 4). only plain corn oil was given to control group (group 1). the rats were sacrificed 24 hours after administration of the chemical and the pancreases of rats were removed. in an attempt to evaluate the dose dependent response, we measured amylase and lipase activities, insulin, malondialdehyde (mda), total oxidant status (tos) levels in rat pancreases. all of the parameters were measured spectrophotometrically. we found that pancreas insulin levels significantly increased in group 4 compare to group 1, besides the insulin levels of group 3 and group 4 were significantly higher than group 2 (p < 0.013). pancreas amylase and lipase activities significantly decreased in group 3 and group 4 compare to group 1 and group 2 (p < 0.013). however, there was no significant change in pancreas mda and tos levels (p > 0.05). according to correlation analysis, when pancreas amylase levels declined, lipase levels were decreased simultaneously and there was a strong positive correlation between them (p < 0.01). in addition, when the comparison was evaluated as a binary, while pancreas amylase and lipase levels diminished, pancreas insulin levels increased and a strong negative correlation between them was found (p < 0.01). according to our result, acute administrations of malathion leads to alterations of insulin, amylase, and lipase levels with a dose dependent manner, while it does not to change oxidant status. the aim of this study is to evaluate the potential toxic effects of mancozeb on the stress biomarkers such as catalase (cat) activity, malondialdehyde (mda) level and protein levels in the brain tissue of zebrafish (danio rerio). mancozeb, is a synthetic fungicide contaminating aquatic environments as a potential toxic pollutants, was investigated in the present study for acute toxicity. zebrafish groups (m-low and m-high) were exposed to different doses of mancozeb (5 mg l-1 and 7.5 mg l-1) for 120 hours except the control group. catalase (cat) activity, malondialdehyde (mda) level and total protein levels were determined by spectrophotometer. the results showed that cat activity and mda levels were decreased in all experiment groups. protein levels were increased in experiment groups when compared to the control group. in conclusion, the changes in the cat activity and mda levels were time and as well as mancozeb dose-dependent. furthermore, the biochemical parameters of mancozeb exposure on zebrafish, showed that mancozeb has significant effect on the zebrafish and/or aquatic organisims. paracetamol (para), which is antipyretic and analgesic, is widely used around the world. paracetamol can be recommended for moderate or mild pains especially in pregnancy as an analgesic. it is known that, paracetamol can cause hepatotoxicity or nephrotoxicity. we were aimed that in this study to show potential hepatoprotective and nephroprotective effect of betaine against long term paracetamol using at therapeutic doses. it has been prepared 3 groups, control, para and para+-betain groups. paracetamol and betaine were administered by gavage to pregnant rats, from first day to the last day of pregnancy (aprox. 21 day). 2 ml physiological saline (%0.9 nacl solutions), 30 mg/kg/day paracetamol, 30 mg/kg/day paracetamol and 800 mg/kg/day betain was given by orally to control, para and para+betain groups respectively. the day of the birth, newborn rats anesthetized by ether and after decapitated. 8 newborn rat's liver and kidney tissues used for biochemical analysis [malondialdehyde (mda), reduced glutathione (gsh), nitric oxide (no) and paraoxonase-arylesterase (pon-are)] and 5 rat's liver and kidney tissues used for histological studies. we showed that, mda and no levels was significantly increased, while pon activities decreased. on the other hand gsh levels and are activities was decreased but these decline wasn't significant in the liver and kidney para group. these biochemical results showed hepatotoxicity and nephrotoxicity in neonates which can be formed in long term maternal paracetamol using at therapeutic doses. also our histological findings was support these biochemical results. we used betaine against potential hepatotoxic and nephrotoxic effect of long term maternal paracetamol using at therapeutic doses for neonates. betaine has antioxidant properties and also used as a methyl donor for transsulfuration reactions in the cell. biochemical and histological examinations showed that betaine protected the tissue injury relatively. p-09.04.4-020 lipid rafts are involved in modulation of ca 2+ responses induced by glutoxim and molixan in macrophages pharmacological analogues of oxidized glutathione (gssg) disulfide-containing drugs glutoxim ò (gssg disodium salt with dmetal nanoaddition, «pharma-vam», st. petersburg) and molixan ò (complex of glutoxim with inosine nucleoside) have found clinical application as a wide range immunomodulators and hemostimulators. however, the cellular and molecular mechanisms of these drugs action are still unclear. previously we showed for the first time that glutoxim and molixan cause biphasic intracellular ca 2+ concentration ([ca 2+ ] i ) increase due to ca 2+ mobilization from thapsigargin-sensitive ca 2+ stores and subsequent store-dependent ca 2+ entry in rat peritoneal macrophages. it is known that key proteins involved in ca 2+ signaling are localized in discrete plasma membrane lipid rafts domains. lipid rafts are cholesterol and sphingolipids enriched microdomains that function as unique signal transduction platforms. thus, the aim of the present work was to elucidate the possible involvement of lipid rafts in glutoxim and molixan effects on [ca 2+ ] i in macrophages. [ca 2+ ] i measurements were performed with fura-2am microfluorimetry. to examine the involvement of lipid rafts in the effect of gssg-based drugs on [ca 2+ ] i we used methyl-bcyclodextrin (mbcd), widely used to remove cholesterol from membranes, thus disrupting the lipid raft domains. we have shown for the first time that macrophage preincubation with 10 mm mbcd for 1 hours before molixan addition causes significant inhibition of both ca 2+ mobilization (on average, by 77.6 ae 9.2%) and subsequent ca 2+ entry (on average, by 82.3 ae 10.5%), induced by molixan. similar results were obtained in experiments with glutoxim. thus, we have demonstrated for the first time that mbcd significantly decreases both phases of glutoxim-or molixan-induced ca 2+ responses in macrophages. the results suggest that intact rafts are required to initiate complex signaling cascade activated by glutoxim or molixan and leading to [ca 2+ ] i increase in macrophages. plant-derived natural substances (phytochemicals) with potent pro-apoptotic activity towards cancer cells in vitro are considered as promising nutraceuticals in anticancer therapy. nevertheless, due to their relatively low bioavailability, administration of high doses of nutraceuticals that are not achievable in vivo seems to exert potentially negligible physiological effects in clinical trials. thus, there is a need for revealing novel cytophysiological effects of low doses of phytochemicals towards cancer cells. in the present study, we have considered thirty one nutraceuticals and selected four phytochemicals acting at low micromolar range (5 to 20 lm) against phenotypically different mcf-7, mda-mb-231 and sk-br-3 breast cancer cells, namely diosmin, sulforaphane, ursolic acid and betulinic acid. nutraceuticals inhibited cell proliferation and caused changes in the cell cycle that was accompanied by elevated levels of p53, p21, p16 and/or p27. apoptosis (annexin v staining, multicaspase and mitopotential assays) was observed exclusively when nutraceuticals were used at the concentration of 20 lm, whereas at the concentration of 5 lm, stress-induced premature senescence was noticed (sa-b-gal activity). nutraceuticals diminished the levels of glut-1 and selected glycolytic enzymes. nutraceuticals promoted oxidative and nitrosative stress as judged by increased production of total reactive oxygen species, total and mitochondrial superoxide, nitric oxide and protein carbonylation. nutraceuticals also stimulated dna single and double strand breaks that was accompanied by atm phosphorylation and to a lesser extent by histone h2ax phosphorylation and 53bp1 foci formation. taken together, several responses to nutraceutical treatment were observed in breast cancer cells that may reflect the heterogeneous nature of cancer cell populations. this study was supported by grant from the national science center, 2013/11/d/nz7/00939. nucleolus is thought to be a stress sensor and oxidative and ribotoxic stimuli may cause the inhibition of rrna synthesis by the inactivation of transcription factor tif-ia/rrn3 that is accompanied by the relocation of nucleolar proteins and p53-based cell cycle arrest and/or apoptosis. as nutraceutical-mediated modulation of nucleolar activity may be considered an attractive anticancer strategy, in the present study, we have investigated the effects of three selected nutraceuticals, namely sulforaphane, ursolic acid and betulinic acid on nucleolus state in three breast cancer cell lines (mcf-7, mda-mb-231 and sk-br-3). we found that low dose nutraceutical treatment resulted in p21-mediated inhibition of cell proliferation, a decrease in rrna synthesis, shifts in lamin a/c and b1 pools, changes in the nucleolar protein levels and their carbonylation, and changes in nucleolus size and number. breast cancer cells differed in erk activity that resulted in different patterns of erk activation/inhibition, phosphorylation status of s6 and autophagy induction upon nutraceutical stimulation. nutraceuticals also affected dna methylation parameters, namely the levels of dnmt1, dnmt3a and dnmt3b that resulted in both global dna hypo-and hypermethylation. taken together, after nutraceutical treatment, nucleolus-centered cellular response was revealed in breast cancer cells of different phenotypic characteristic that may be considered a potential target of anticancer therapy. this study was supported by grant from the national science center, 2013/11/d/nz7/00939. p-09.04.4-023 rate of apoptosis in human macrophages infected with leishmania tropica promastigotes infection of the cells with parasites or exposing cells to heat stress induces a cellular stress. in the present study human macrophages are infected with leishmania tropica promastigotes and exposed to heat stress. the measurement of cytoplasmic histone-associated dna-fragments was carried out using elisa technique. visualization of apoptotic cells was performed by the terminal deoxynucleotidyl transferase dutp nick end labeling staining method (tunel). degree of oxidative stress on cell is evaluated by measuring nitric oxide (no), malondialdehyde (mda), reducte glutathion (gsh) levels and superoxide dismutase (sod) activities. results of the elisa technique showed that infection of macrophages with promastigotes induced apoptosis rate significantly (p < 0.001), heat stress however decreased the rate of apoptosis in infected macrophages remarkably (p < 0.001). high levels of apoptosis rate in infected macrophages and drastic decdrease in apoptosis in heat subjected macrophages infected with promastigotes are confirmed by visualisation of apoptotic cells using tunel method. levels of glutathion (gsh) in infected macrophages decreased significantly (p < 0.05), while malondialdehyde (mda) levels increased notably (p < 0.05). however, no statistical significant alterations were detected in the nitric oxide (no) values and superoxide dismutase (sod) activities. results of the present study indicates that infection of human macrophages with leishmania tropica induces a cellular stress response, characterized by decreased values of gsh and increased levels of mda. increased rate of apoptosis in infected macrophages may be due to the increased cellular stress caused by leishmania tropica amastigotes. decreased rate of apoptosis measured in heat exposed macrophages infected with promastigotes indicates an extention in life span of macrophages. measurements of the parameters characterizing the redox and inflammatory processes in blood are essential for diagnosis and prognosis of type 2 diabetes mellitus (t2dm), but also for monitoring the effectiveness of medical treatments. along with other biochemical effects, hormonal imbalance leads to modified transport function of erythrocytes due to changes in enzyme systems involved in upholding of cellular homeostasis through a rapid degradation of altered proteins, being the second line of defense against the free radicals, which degrade and eliminate the damaged molecules. some of these enzymes are hemoglobin peroxidase (pa) and esterase (ea). the aim of this research study was to identify new parameters with a potential role of biochemical markers in t2dm like hemoglobin peroxidase and esterase activity from erythrocyte. our data showed that pathological processes involved in t2dm imply an increased enzymatic activity of pa (73.85%), which correlates with increased levels of ea (47.9%) and glycated hemoglobin (hba1c) (13.51%), compared with control group. the variables hba1c, pa and ea are correlated: the identified pearson correlation coefficients r = 0.637 and r = 0.573 respectively, have an associated probability of p < 0.001 and p < 0.006 a value that indicates a strong positive correlation between the dependent variables pa and ea and independent variable hba1c. based on these results we concluded that together with glycosylated hemoglobin assay, all the studied parameters can be successfully used as extra test for diabetes associated with oxidative stress and disorders in erythrocyte functions or blood rheology. the radioprotective effects of propolis and caffeic acid phenethyl ester on radiationinduced oxidative/nitrosative stress in brain tissue s. taysi 1 , e. demir 2 , k. cinar 3 1 gaziantep university, school of medicine, gaziantep, 2 haran university, sanliurfa, 3 department of neurosurgery, medical school, gaziantep, turkey head and neck cancer patients treated with radiotherapy suffer severe side effects during and following their treatment. efforts to decrease toxicity of irradiation to normal tissue, organs and cells have led to searching for cytoprotective agent. investigations for effective and non-toxic compounds with radioprotective capability led to increasing interest in antioxidant such as propolis and caffeic acid phenethyl ester (cape). the aim of this study was to evaluate the antioxidant and radioprotective effects of propolis and cape on radiation-induced oxidative/nitrosative stress in the brain tissue. fourty sprague-dawley rats were randomly divided into five groups. group 1 (irradiation (ir) + propolis) received total cranium irradiation and propolis was given orally through an orogastric tube daily. group 2 (ir+cape) received total cranium irradiation plus cape, was dissolved in dimethyl sulfoxide (dmso) just before giving to the rats, intraperitoneally (ip) every day. group 3 (ir) received 5 gy of gamma irradiation as a single dose to total cranium plus 1 ml saline daily. group 4 received daily plain dmso. group 5 received daily plain saline. at the end of the 10 day time period, xanthine oxidase (xo), nitric oxide synthase (nos) activities, nitric oxide (no•) and peroxynitrite (onoo -) levels were significantly higher in ir group compared to all other groups. in conclusion, the results suggest the radioprotective ability of propolis and cape involving prevention of radiation-induced oxidative/ nitrosative damage. p-09.04.4-027 role of leptin and adiponectin in obesityassociated oxidative stress e. becer 1 , a. c ß irakoglu 2 1 near east university, nicosia, cyprus, 2 istanbul university, istanbul, turkey objective: increased oxidative stress is one of the major characteristics of obesity and obesity-related complications. adipokines also induce the production of reactive oxygen species and generating oxidative stress in physiological and pathological conditions of obesity. the aim of this study was to determine the association of leptin and adiponectin levels with body mass index, lipid parameters and oxidative stress biomarkers in obesity. methods: the study included 150 obese and 120 non-obese subjects. plasma leptin and adiponectin levels (ng/ml) were measured using commercially available enzyme-linked immunosorbent assay kits. serum lipid, superoxide dismutase, malondialdehyde and antropometric parameters were measured. results: obese and non-obese subjects did not differ in age, while plasma glucose, total cholesterol, triglycerides, ldl cholesterol and leptin levels were significantly higher and mean hdl cholesterol and adiponectin levels were significantly lower in obese than non-obese subjects. the plasma leptin (p < 0.001) and adiponectin (p = 0.003) levels were significantly correlated with bmi in both obese and non-obese subjects. in obese subjects, leptin levels were significantly correlated with superoxide dismutase (p < 0.01) and malondialdehyde (p < 0.01). strikingly, adiponectin was significantly correlated with superoxide dismutase (p = 0.02) and malondialdehyde (p = 0.0012) levels in obese group. conclusion: our results suggest that leptin and adiponectin levels are associated with defective antioxidant status and increased lipid peroxidation which may have implications in the development of obesity related health problems. clinical trials of biologically active plant substances show a significant preventive effect in cancer, cardiovascular diseases and peptic ulcer disease in the form of both nutritional supplements and therapeutic intervention. anthocyanins contained in dark berries show great antioxidant potential, with the most important including a reduction in oxidative degradation of lipids or tyrosine oxidation by peroxynitrite. our previous studies of the antioxidant properties of extracts from vaccinium corymbosum, aronia melanocarpa and sambucus nigra, however, indicated that their activities largely depend on the method of extraction. while quantitative determination of anthocyanins pointed to a disproportionately larger content of anthocyanins in isolates from lyophylized berries, their scavenging activities against hydroxyl radicals was surprisingly the lowest. inflammatory processes, vascular damage, atherosclerosis and others are caused by oxidativenitrosative stress, so we tested their efficiency to scavenge no degradation products. we found that only purified extracts of lyophilized berries showed the most significant effects against no degradation products, with efficacy of around 50%. an extract from aronia showed greater than 60% efficiency, and a net ethanol extract from all the berries showed a 30% effect. cleaned ethanol extracts showed the lowest effects, while aronia initiated production at a concentration of 25 mg/l. conversely, all acetone extracts consistently initiated no degradation products. these findings are in complete contrast to their determined action against reactive oxygen species. in summary, it follows that a particularly adjusted lyophilized extract of the berries could be responsible for the increased biological activity of no and the observed biological and pharmacological activities of anthocyanins in circulatory disorders. the study was supported by grant vega 1/0782/15 and 1/0018/16. p-09.04.4-032 attenuation of dysfunction, oxidative stress and apoptosis by resveratrol in benzo(a)pyrene exposed ins1-e 832/13 pancreatic beta cell line s. c ß elik, b. baysal faculty of medicine, afyon kocatepe university, afyonkarahisar, turkey diabetes is one of the most important problems in the world. this disease is a very important health problem due to affects many different organs and systems. it has been well established that, environmental pollutants had deleterious effects on glucose metabolism, and caused insulin resistance and type 2 diabetes. with this investigation, it was aimed to investigate the effects of benzo(a)pyrene on pancreatic beta cells and treatment affects of resveratrol. in this study, rat ins-1e beta cell line was used. after reaching the appropriate number of cells culture operations by cell culture, benzo(a)pyrene (20 lm, 24 hours) application have been made after resveratrol (80 lm, 48 hours) application. after incubations oxidative stress, insulin secretion (in cell and in medium), cell proliferation and apoptosis were analysed in cells by biochemical and molecular techniques. b(a)p application resulted in no increase and resveratrol also increased this level of no. resveratrol increased the tas levels decrased by b(a)p, and tos levels were also increased by resveratrol interestingly. osi levels determined with tas and tos levels, has no significant change between groups. gsh levels were decreased by b(a)p while resveratrol increased its' level to control level. mrna expression levels of beta cell functions related genes ins-1, ins-2 and sirt-1 were increased by resveratrol treatment. insulin levels in cell and in medium were increased after resveratrol treatment. mrna expression level of foxo-1 gene was increased while pdx-1 was decreased by resveratrol treatmeant. b(a)p suppressed the mrna expression of p53 gene, but resveratrol increased. the effects of b(a)p on pancreatic beta cells and the protective effects of resveratrol on this cells were investigated in vitro with this research firstly. the results obtained from this research showed that oxidative changes, functional impairment and carcinogenetic effects of b(a) p in pancreatic beta cells could be blocked by resveratrol. the protective effect of vitamin e (alphatocopherol) on ischemia-reperfusion injury in rat liver ischemia-reperfusion (i/r) process is usually used during transplantation and resection of the liver but liver dysfunction and cellular death due to lack of oxygen in tissues, changes in the balance of oxidant/antioxidant in favor of oxidants, and inflammatory response are inevitable during this process. in the present study, it was aimed to investigate whether vitamin e(alpha-tocopherol), an antioxidant agent, has a protective effect against liver ischemia reperfusion injury in rats by using morphometric methods. for this purpose, 21 adult sprague-dawley male rats were divided into 3 groups as; control, ischemia / reperfusion (i/r), and vitamin e+ischemia/reperfusion (evit +i/r). in experimental groups, the total hepatic ischemia was applied for 45 minutes followed by a 24 hour of reperfusion. in the treatment group, 7 days before ischemia 40 mg / kg dose of vitamin e was administered intraperitoneally once a day. after the termination of the reperfusion, the rats were perfused by cardiac way and liver tissues were dissected. following volume and weight calculations, the livers were subjected to the standard histological preparation methods and embedded in paraffin. serial sections at 5 lm thickness were obtained from these blocks, stained with hematoxylineosin, and analyzed with morphometric methods. in light microscopic examinations of the i/r group, irregularity in lobules, dilatation in central veins and sinusoids, extensive areas of necrosis and pycnotic nuclei were seen in hepatocytes. the volume density of sinusoids to liver parenchyma was estimated as 16% in the control group, whereas it was 36% in the i/r group. this value was decreased to 32% in the evit + i/ r group. however, no significant difference was found among the groups in the lobule area calculated by the point counting method. these results show that intraperitoneal vitamin e administration for 7 days prior to ischemia partially inhibits damage caused by ischemia-reperfusion injury in the liver. the leaves, fruit and bark of annona muricata, a member of the annonaceae family, are commonly used in the traditional medicine of tropical and subtropical regions. in recent years, many studies have shown their anti-cancer, anti-convulsant, antiarthritic, anti-parasitic, anti-malarial, and anti-diabetic activities. it should be noted that these characteristics have been described using different types of extracts from different parts of the plant. our studies have focused on the systematic characterization of activities most easily accessible from an aqueous extract of leaves, with hitherto documented antihypertensive and hepatoprotective effects. we found that the extract shows almost 54% efficiency against hydroxyl radicals. with increasing concentrations, the effectiveness weakened, reaching a second peak (40%) at a concentration of 50 mg/l. the scavenging activity against no degradation products maintained a continuously increasing trend with a maximum at a concentration of 100 mg/l. surprisingly, the extract initiated peroxynitrite production in a similar trend, except at 50 mg/l, where it scavenged peroxynitrites with relatively high efficiency, up to 34%. these findings are consistent with the elevated levels of reduced glutathione detected after incubation of liver mitochondria with extract to a maximum concentration of 50 mg/l, with subsequent sharp decline. the activity of glutathione s-transferase was decreased, although not significantly. this indicates a reduction of metabolic processes of compounds, allowing their action over a longer period of time. in a live system, even antihypertensive effects can be observed. however, a significant outflow of gsh to create the gsh adducts of active substances, and particularly s-nitrosoglutathione from increased production of peroxynitrites, can cause liver toxicity. the study was supported by grant vega 1/0782/15 and 1/0018/ 16. the role of polyamine metabolism in curcumin induced apoptosis via reactive oxygene species (ros) generation in growth hormone (gh) overexpressed t47d breast cancer cells r. genc ß, a. coker gurkan, e. d. arisan, p. obakan yerlikaya, n. palavan unsal, n. palavan unsal t.c istanbul k€ ult€ ur € universitesi, istanbul, turkey autocrine growth hormone (gh) signaling triggers cell proliferation, growth, metastasis and drug resistance in cancer cells. polyamines (pas) play an essential role in cell cycle, proliferation, growth and carcinogenesis of various cancer types such as prostate, colon and breast cancer. odc (ornithine decarboxylase) is the key enzyme in the pa biosynthetic pathway that is under control of antizyme (az) and antizyme inhibitor (azi) activity. pa catabolic enzymes polyamine oxidase (pao) and spermine/spermidine acetyle transferase (ssat) by-products triggers reactive oxygene species (ros) generation and apoptotic cell death. curcumin decreased cell viability in dose and time dependent manner in t47d wt and gh+ cells. although forced gh expression induced cell proliferation, 20 lm curcumin inhibited cell invasion. curcumin (20 lm) induced apoptosis by acting on intrinsic and extrinsic pathway in both cell lines. moreover, curcumin supressed odc, azi expression in wt and gh+ t47d cancer cells. although curcumin decreased az expression in t47d wt cancer cell, increased az expression was determined in t47d gh cancer cell. pao and ssat expressions were upregulated in t47d gh+ cells. concomitantly, putrescine levels were increased in t47d gh+ cancer cell compared to wt cells and curcumin depleted spermidine and spermine levels in wt and gh + t47d cells. curcumin induced-apoptotic cell death via ros generation and co-treatment of n-acetyl cysteine (nac) overcame curcumin effect. conclusion, forced gh expression triggers cell proliferation and growth via acting on polyamine metabolism and curcumin-triggered ros generation was prevented by nac treatment in t47d wt and gh+ breast cancer cells. acknowledgment: this study was supported by tubitak 1001 research project (project no: 113z791). the radio-protective effects of propolis and nigella sativa oil on oxidative/nitrosative stress in liver tissue of rats exposed to total head irradiation s. taysi our purpose is to investigate propolis and nigella sativa oil (nso) for their antioxidant effects on the liver tissue of rats exposed to ionizing radiation. a total of 32 sprague-dawley rats were divided into five groups to test the radioprotective effectiveness of of propolis and nso administered by orogastric tube. appropriate control group was also studied. biochemical parameters in liver tissue of rats were determined by spectrophotometer. xanthine oxidase (xo), nitric oxide synthase (nos), superoxide dismutase (sod) activities, nitric oxide (no•) and malondialdehyde (mda) levels were higher in ir group while glutathione-s-transferase (gst), glutathione peroxidase (gsh-px) level in the ir group were lower in this group when compared to the other groups. gst activity in ir plus propolis group was statistically higher than in all other groups. propolis and nso clearly protect liver tissue from radiationinduced oxidative stress. the effects of royal jelly on the antioxidant parameters in the breast tissues of the rats with breast carcinoma treated with paclitaxel or not effects of royal jelly on the breast tissue antioxidant parameters in rats with breast carcinoma treated with paclitaxel or not. 8-12 weeks old female sprague-dawley rats (n = 37) included in current study were divided into 5 groups: control group (n = 8) with healthy rats; breast cancer group (n = 8); breast cancer group (n = 7) treated with 15 mg/kg paclitaxel injection (once a week for 3 weeks); breast cancer group (n = 7) treated with 100 mg/kg royal jelly (by oral gavage for 30 days); and finally breast cancer group (n = 7) treated 100 mg/kg royal jelly in addition to 15 mg/kg paclitaxel injection. rats with breast carcinoma was obtained at 150th days after a single dose injection of 50 mg/kg n-methyl-n-nitrosourea (mnu). all cancer groups were followed by 30 days with treatment of paclitaxel and/or royal jelly. the antioxidant parameters in rat breast tissues, superoxide dismutase (sod) and catalase (cat) activities were determined by spectrophotometric colorimetric methods and glutathione (gsh) by high performance liquid chromatography (hplc). all the antioxidant parameters decreased in breast cancer group without any treatment (p < 0.05). but, statistically non significant increases were observed by paclitaxel and royal jelly treatment (p > 0.05). this study indicated that royal jelly supplementation can not be sufficient to increase the antioxidant parameters in breast cancer. we are going to continue to identify the effects of royal jelly on breast cancer detail. the effects of n-acetylcysteine on microsomal and serum paraoxonase 1 activities at high fat diet induced obese rats obesity is a chronic disease that develops from the interaction between genotype and environmental factors and increase in the accumulation of body fat. it is related with glucose and lipid metabolism disorders, cardiovascular diseases and increased oxidative stress. paraoxanase 1 (pon1) is an enzyme which plays a protective role in oxidative stress, inflammation and liver diseases. it has been suggested that pon1 has protective effects against high fat diet induced obesity and obesity related disorders. n-asetylcysteine (nac) is a potent antioxidant due to its ability to stimulate glutathione synthesis. the aim of this study was to evaluate the microsomal and serum pon1 enzyme activities (paraoxonase, arylesterase and lactonase) at high fat diet induced obese rats in the presence of nac. this study consisted of control, obese and nac-supplemented obese (2 g/l nac) groups. eighteen sprague-dawley rats were randomized into three groups (n = 6). control rats were fed by standart food and obese and nac groups were fed by high fat diet. the microsomal and serum paraoxonase, arylesterase and lactonase activities were determined by colorimetric methods. serum paraoxonase, arylesterase and lactonase activities decreased in obese and nac groups when compared to control groups. on the other hand microsomal paraoxonase, arylesterase and lactonase activities increased in nac group when compared to control and obese groups. however there was no statistically significant difference between the groups. it has been concluded that the microsomal and serum paraoxonase 1 enzyme activities did not change at high fat diet induced obese rats in the presence of n-asetylcysteine. reactive oxygen species, playing an active role in the early and late course of acute pancreatitis, lead to dysfunction of cell membrane and releasing of lysosomal enzymes, and thereby to the injury in pancreatic cells. gallic acid, found in vegetables such as green tea, is an active component which has antioxidant, antiinflammatory, antiviral, anticancer activities. the aim of this study was to investigate the effects of gallic acid in experimental acute necrotizing pancreatitis (anp) model in rats. eighteen male sprague-dawley rats were divided into three groups (6 rats in each group). group 1: sham + saline; group 2: anp induced by intraductal glycodeoxycholic acid and intravenous cerulein; and group 3: anp + gallic acid (100 mg/kg/day, intraperitoneal). at the end of 18th hours, pancreas histopathology was examined. the levels of serum amylase as a diagnostic marker of pancreatitis, interleukin-6 (il-6), total antioxidant status (tas), nitrite + nitrate, total thiols as antioxidant marker and thiobarbituric acid reactive substances (tbars) to measure malondialdehyde (lipid peroxidation product) were determined by spectrophotometric methods. serum amylase, il-6, plasma tbars levels were significantly higher but total thiols levels were lower than sham group in anp group without treatment (p < 0.05). however; tas and nitrite + nitrate levels did not show any significant difference (p > 0.05). on the other hand, while serum amylase, il-6 and tbars levels were lower, total thiols levels higher in gallic acid treatment group than in the untreated anp group, but statistically insignificant (p > 0.05). in conclusion, gallic acid treatment is beneficial but not sufficient to treat the acute necrotizing pancreatitis in rats. p-09.04.4-043 evaluation of oxidant/antioxidant system parameters, il-6 and il-10 levels in amniotic fluid of pregnancies with down sydrome b. _ imge erg€ uder 1 , s. bahsi 1 , t. bahsi 2 , v. topc ßu 2 , a. bakir 2 1 ankara universty faculty of medicinel, ankara, 2 zekai tahir burak research and training, hospital genetic center, ankara, turkey introduction and aim: down sydrome (ds) can be diagnosed at high-risk of down syndrome pregnancies by invasive prenatal testing. in this study we aimed to demonstrate antioxidant/oxidant system markers, il6 and il10 levels in amniotic fluid samples of pregnancies affected by ds. materials and methods: for this purpose we collected amniotic fluid samples from 18 pregnancies affected by down sydrome and 36 normal healthy pregnancies who applied to zekai tahir burak research and training hospital genetic center and were proceeded with amniocenthesis. in the amniotic fluid samples; malondialdehyde (mda), superoxide dismutase (sod), glutathion peroksidase (gsh-px) xhantine oksidase (xo), catalase (cat), adenozine deaminase (ada), nitric oxide (no), nitric oxide senthase (nos) enzymatic activities were evaluated by spectrophotometric methods, il6 and il10 levels are evaluated by elisa. for statistical analysis student's t-test and spearman corralation analusis are used. results: it was found that sod levels are significantly elevated in study group compared to control group (p < 0.05). besides this, in study group, cat and il-6 levels are found singnificantly lower than control group (p < 0.05). we couldn't find any significant difference between two groups in terms of mda, gsh-px, xo, no, nos, ada ve il-10 levels (p > 0.05). discussion and conclusion: there is an important decrease in inflammation compared to normal pregnancie in the amniotic fluid of pregnancies having ds. based on these results, sod enzyme may be used as a marker for prenatal diagnosis of ds. for this purpose these experiments should be tried on larger sample groups. the aim of our work was to compare prooxidant and antioxidant properties of linalool, which is the oxygenated monoterpene compound reported to be a major volatile component of the essential oil of several aromatic species, in hep g2 cells. cytotoxicity of linalool was assayed by celltiter-blue ò cell viability assay. malondialdehyde levels result in membrane damage in hep g2 cells were assayed by using fluorometric method. hep g2 cells were incubated with linalool at 24, 48 and 72 hours. the viability of hep g2 cells decreased in a manner dependent upon concentration and incubation time. the ic 50 values were calculated 81.5 lg/ml (24 hours), 72.7 lg/ml (48 hours) and 64.7 lg/ml (72 hours). but, cell viability of hep g2 cells increased when the cells preincubated with linalool at lower concentrations (˂ic 50 ) against h 2 o 2 cytotoxicity. membrane-damaging effects of linalool were increased with accelerating concentrations. on the other hand, membrane damaging effect of h 2 o 2 was decreased when the cells preincubated with linalool before h 2 o 2 incubation. oxygenated monoterpene linalool had both prooxidant and antioxidant properties showing membrane damaging and protective effects on hep g2 cells depend on concentration. postprandial lipemia is primarily characterized by increasing triglyceride levels after the lipid rich meal. postprandial lipemia may cause oxidative stress by increasing free radical production and increasing oxidative stress could be responsible for the development of many diseases. plasma oxidant-antioxidant status was evaluated in healthy individuals with postprandial hypertriglyceridemia generated by performing oral triglyceride tolerence test (ottt). the study group included 86 subjects (38 female and 48 male). ferric reducing ability of plasma (frap), total thiol and thiobarbituric acid reactive substances (tbars) levels were determined by colorimetric methods at fasting and 2nd, 4th and 6th hours following ottt. the levels of frap and thiol were significantly higher in males than females (p = 0.0001 and 0.042, respectively). thiol levels decreased significantly in both gender at postprandial 2nd, 4th and 6th hours as compared with fasting condition (p = 0.0001). while tbars levels increased at postprandial 2nd hour, that was only significant for male individuals (p = 0.0001). it has been concluded that postprandial lipemia may change oxidant-antioxidant balance in favor of oxidants and gender is an important criteria while assessing the oxidant-antioxidant status in postprandial lipemia p-09.04.4-046 ischemia modified albumin and c-reactive protein levels in prediabetes prediabetes is a state of abnormal glucose homeostasis characterized by the presence of impaired fasting glucose, impaired glucose tolerance, or both. the aim of this study was assess serum ischemia modified albumin (ima) in prediabetes and determine its correlation with other risk factors for chronic complications such as inflammation and hyperglycemia. glucose, insulin, total cholesterol, hdl cholesterol, triglycerides, albumin, c-reactive protein (crp) and ima were measured in 30 patients with prediabetes and 30 controls. prediabetes patients had higher levels of ima and crp in comparison with control subjects but there was no significant difference between groups for ima. significant positive correlation was observed between crp and fasting glucose, insulin. there was no significant correlation between ima levels and the parameters tested. we have shown higher level of crp in prediabetes. these results support the hypothesis that chronic inflammation may be involved in development of hyperglycaemia. p-09.04.4-048 the effects of s _ io 2 nanoparticles of rat uterine smooth muscle specially used in textile field sio 2 nanoparticles on uterus smooth muscle was aimed to be researched. in this study 64 wistar albino female rats were used. rats were separated in 4 groups as control, dose 1 (250 lg/ml), dose 2 (500 lg/ml) and dose 3 (1000 lg/ml). nanoparticle's size was chosen as 20 nm. preparations of four groups were evaluated for biochemical and histological examinations. all isolated uterine smooth muscle stripts except the controls were treated with sio 2 for two hours. in biochemical examinations in order to evaluate oxidative stress level of malondialdehit (mda), activity of superoxide dysmutase (sod) and glutathione peroxidase (gsh-px) were measured. in histological examinations via electron microscope ultrastructure of uterus was examined as well as apoptotic cells detected with immunofluorescent labeling method. intergroups differences were defined by statistical analysis. while mda level increased depending on the dosage, sod level was decreased depending on the dosage. gsh-px rate was decreased for each dosage with respect to control. however, no significant difference is detected between groups. in electron microscopic examination no changes were observed in uterus ultrastructure with compare to control. however, in immunofluorescent labeling it was detected that apoptosis increased in dosage groups with compare to control group. as a result, it was thought that application of sio 2 nanoparticles, in 20 nm size and in 250, 500 and 1000 lg/ml dosages caused of oxidative stress and apoptosis. this results suggested that sio 2 has toxic effects on uterine smooth muscle. uterine myomas are the most common benign pelvic tumors arising from myometrium. they are rarely associated with mortality but responsible for significant morbidity and have adverse effects on quality of life especially in reproductive age women. reactive oxygen species and superoxide dismutases, as well as sex steroids play important roles in the reproductive physiology processes. in addition, oxidative stress and impaired antioxidant defense system have been linked to pathophysiology of various diseases including malignant gynecological disorders. clinical investigations indicate that women with myoma may have increased risk of developing malignant tumors particularly sarcomas. the present study aimed to investigate the possible role of oxidant and antioxidant status in myomas. blood and urine samples of 21 myoma patients were collected. activities of erythrocyte antioxidant enzymes [copper-zinc superoxide dismutase (cuzn-sod), catalase (cat), glutathione peroxidase (gpx1)] and levels of lipid peroxidation biomarkers [plasma malondialdehyde (mda) and urine 8-epi-prostaglandin f 2a (8-epi-pgf2a)] were determined. the results were compared with those of 21 controls. the groups were matched in terms of age, body mass index, smoking habit, coexisting chronic diseases, menopausal status and sex steroid hormone levels. all antioxidant enzyme activities were higher (37% for cuzn-sod, p = 0.003; 52% for cat, p0.05) and the levels of lipid peroxidation biomarkers were lower (%59 for mda, p = 0.011 and 43% for 8-epi-pgf2a, p > 0.05) in myoma patients compared to controls. correlation analyses showed a significant negative correlation between erythrocyte cuznsod activity and plasma mda levels (r = -0.431, p = 0.005). the decreased lipid peroxidation may be the consequence of elevated antioxidant enzyme activities and the data suggests a protective role of activated antioxidants especially cuznsod and cat in patients with myoma. p-09.04.4-050 investigation of ischemia-modified albumin levels in patient with acute limb ischemia introduction: acute limb ischemia commonly occurs as a result of embolus caused by cardiac origin and which may end up with limb loss or even death if left untreated. thrombosis are usually seen where the arteries give branches and tendency to atherosclerosis is more serious at these sites. involvement of several arteries in either embolus or in situ thrombosis limits the adequacy of collateral circulation. restriction of blood flow due to arterial stenosis or occlusion often leads patients to complain of muscle pain on walking. any further reduction in blood flow causes ischemic pain at rest, which affects the foot. early recognition may prevent limb loss or death. ischemia can alter the capacity of the amino terminus of the albumin to bind free metal atoms such as cobalt, copper and nickel. this new, chemically changed albumin is called ischemia modified albumin (ima). ima is a sensitive marker of myocardial ischemia, skeletal muscle ischemia, pulmonary embolism, mesenteric ischemia and stroke. therefore, in this study it was aimed to investigate the ima level in acute limb ischemia. materials and methods: in this study, 24 patients with acute limb ischemia (li group; mean age 62 years) and 34 healthy individuals (control group; mean age 42 years) were included. ima levels were detected in control and li group by elisa (organo teknika, avusturya) using ima el _ isa kit. results: ima values were compared with nonparametric methods mann whitney u test, and significantly decreased ima level was statistically significantly different between li group and control group (p < 0.001). conclusion: there is a significant increase in serum ima in limb ischemia, so that alterations also might be clinically useful in the diagnosis of limb ischemia, but should be supported with further studies. object: polycystic ovary syndrome (pcos) is a multifaceted disorder with a pathogenetic pathway that is not fully understood yet. apart from hormonal derangements, insulin signaling defects and adipose tissue dysfunction, oxidative stress, defined as an imbalance derived from excessive formation of oxidants in the presence of limited antioxidants defenses, has been actively implicated in the etiology of the syndrome. the aim of this study was to determine of serum myeloperoxidase activity (mpo), paraoxonase 1 activity (pon1) and arylesterase activity (are) in patients with pcos. material and methods: the study was carried out on 150 women consisted of 103 patients with pcos and 47 healthy ones as control. serum pon1 activities were measured spectrophotometrically using diethyl-p-nitrophenylphosphate as substrate. phenylacetate was used as substrate for are measurement, and are activity was determined by measuring absorbance of the resulting phenol at 270 nm. molar absorptivity coefficients were used in the calculation of pon and are activities as 1 nmol phenol/ml serum/min. result: the mpo and are activities were significantly lower in the patient groups when compared with the control group (72.09 ae 60.66 -119.74 ae 90.81 u/ml p < 0.001, 47.43 ae 12.21 -83.93 ae 26.38 u/ml p < 0.001, recpectively). the pon1 activities are higher in the patient group (145.55 ae 98.23 u/ml) compared to the control group (153.33 ae 101.90 u/ml) are found, but are not statistically significant. conclusion: lower serum mpo and are activities might contribute to the increased susceptibility for the development of diseases risk in women with pcos. because free oxygen radicals are thought to contribute to the complication of many chronic diseases, the pcos may be related to oxidative stress. subclinical hypothyroidism, defined as an elevated serum thyroid stimulating hormone level associated with serum thyroid hormone concentrations within the reference range. free radicalmediated oxidative stress has been implicated in the pathogenesis of thyroid disorders. the ischemia-modified albumin (ima) has been proposed as a marker of protein oxidative damage, which has been found to reflect hypoxic stress. this study aimed to investigate the influence of subclinical hypothyroidism on serum ima levels. thirty-one subclinical hypothyroidism patients and 27 control subjects were enrolled in the study. albumin, ima were measured and ima/albumin ratio was calculated. to determine the ima levels the measurement method based on albumin cobalt binding assay was used. serum ima levels of patients with subclinical hypothyroidism were 0.58 ae 0.08 absu, ima levels of control subjects were 0.49 ae 0.08 absu. ima levels were significantly higher in patients with subclinical hypothyroidism patients than in control subjects (p < 0.0001). when ima values were normalized for albumin concentrations, the ima/albumin ratio was also significantly elevated in patient group compared to control group (p < 0.01). ima levels are increased in patients with thyroid dysfunction. elevated levels of ima can be a clinically useful marker of protein oxidative damage in subclinical hypothyroidism. p-09.04.4-054 the effects on endothelial dysfunction of quercetin in streptozocin-induced diabetic rats excessively produced in pathologic conditions. ultimately, imbalance between oxidants and antioxidants results with oxidative stress (os). in this study, we investigated some os parameters in standard (20% protein, 70% (7% sucrose) carbohydrate, 10% lipid) and sucrose (20% protein, 70% (35% sucrose) carbohydrate, 10% lipid) diet fed bdnf heterozygous mice liver tissues. the male c57bl6 strain wild type (+/+) and bdnf heterozygous (+/à) mice (5 weeks) were obtained. the animals were fed ad libitum by special standard and sucrose diets. twenty four mice were divided into four groups and each group consist six mice. all mice were fed for 16 weeks. first group involved in c57bl6 wild type mice and fed by standard diet. second group contained c57bl6 bdnf heterozygous mice and fed by standard diet. third group consisted c57bl6 wild type mice and fed by sucrose diet. fourth group involved in c57bl6 heterozygous mice and fed by sucrose diet. in first group, mda levels, sod and cat activities were higher than other groups. in second group, cat activities were lower than other groups. but, we could not find any statistically significant differences between all groups about mda, sod, cat levels in bdnf heterozygous mice liver tissues. in conclusion, standard and sucrose diet feeding may not affect mda, sod and cat levels in bdnf heterozygous mice liver tissues. brain-derived neurotrophic factor (bdnf) is member of neurotrophin family which plays critical roles in the development, differention, survival, maintenance of the central and peripheral nervous systems. bdnf also contributes to food intake and body weight control. bdnf heterozygous mice display increased body weight and mild hyperphagia. expression of bdnf is not limited to the brain, it also express some peripheral tissues like adipose tissue, liver, kidney, skeletal muscle, heart. even though roles of bdnf are well known relatively in central nervous systems, effects of this protein is not clear in peripheral tissues. as mentioned before, it is expressed in organs involved in energy, lipid and glucose homeostasis, including the liver, adipose and muscle tissues, but its role there remains unclear. in this study, we aimed to investigate role of bdnf on liver oxidative stress parameters in heterozygous mice model fed fat diet induced obese mice. in this study, we used c57bl/6 mice inbred strain wild type and bdnf heterozygous (+/à) mice. animals were divided to two groups: wild type (n = 5) and bdnf heterozygote mice (n = 5). the animals were fed ad libitum by high-fat diet during 4 month. weight gain was recorded every 15th days. in liver tissues were measured malondialdehyde (mda), superoxide dismutase (sod) and catalase (cat) by spectrophotometric methods. liver mda levels decreased in obese bdnf heterozygous mice compared to obese wild type group and statistically significant difference between groups. bdnf heterozygous mice cat activities were higher than the other group and this difference was statistically significant. there was no statistically significant difference between the groups in terms of sod activities. it has been concluded that the mda levels and sod enzymes activities changed at high-fat diet induced obese bdnf heterozygous mice compared to wild type mice liver tissues. p-09.04.4-060 disturbances of microelements profile in serum of overweight/obese adult females with acute and persistent pro-inflammatory chlamydia pneumoniae infection p-09.04.4-061 determination of reactive oxygen species induced dna damage using modified cupric reducing antioxidant capacity (cuprac) colorimetric method s. uzunboy, s. demirci c ß ekic ß, r. apak department of chemistry, istanbul university, faculty of engineering, istanbul, turkey reactive oxygen species (ros) term is a common name of a group of species. hydroxyl radical and singlet oxygen can be taken into account as ros samples. ros may be formed as a result of endogenous or exogenous reasons. although ros have some beneficial functions, they should be balanced by antioxidants (aox). excessive amounts of ros can attack biological macromolecules including dna. dna damage is usually related with mutagenic and carcinogenic changes. that's why determination of dna damage is so important and there are a great many studies in literature comprising different techniques. one of the most common of them is the 'comet assay'. but application of the method and interpretation of the results is not easy. investigation of certain dna damage products is also very common. these methods usually need expensive instrumentation such as using tandem mass spectrometry. on the other hand, depicting total dna damage on a certain product may cause misinterpretations. in the presented study, dna was decomposed by hydroxyl radicals produced by fenton method. in the study while dna is not cuprac reactive the oxidation products can react with the cuprac reagent. the effect of aox was also investigated. for this purpose, selected aox compounds were added to the reaction medium. because of their radical scavenging effect, the cuprac absorbance decreased in the presence of aox. in the presence and absence of aox, absorbance differences were calculated. the calibration graphs between final concentration and absorbance differences were drawn for each aox. gallic acid was determined as the most effective one among the tested aox samples. for statistical comparison with the presented study, tbars was used as reference method. direct use of dna as a probe material to determine oxidative damage may be an advantage to understand dna hazard in biological systems. the proposed method can be applied in all laboratories having a spectrometer as a cost-effective and simple procedure. p-09.04.4-062 effects of alpha-1 antagonists on oxidative system of rat heart tissue benign prostate hyperplasia is a progressive process occurring in the stromal and epithelial components of the prostate. alpha-1 receptor blocking agents are used for relaxation of the smooth muscles in the prostatic stroma. our aim was to investigate the effects of alpha-1 antagonists on oxidative system of rat heart tissue. 33 male wistar albino rats were divided into 5 groups randomly. 1) tamsulosin (1 mg/kd/day), 2) terazosin (5 mg/kg/day), 3) doksazosin (25 mg/kg/day), 4) alfuzosin (10 mg/kg/day), 5) control. all drugs were administered every other day single doze via oral. rats were sacrificed after 30 days. heart tissue was taken for biochemical analysis. malondialdehyde (mda), nitric oxide (no), protein carbonyl (pc) levels and superoxide dismutase (sod), glutathione peroxidase (gsh-px) enzyme activities were determined in supernatant samples. there was not an significant difference between terazosin, doxazosin, alfuzosin, tamsulosin groups in means of sod, mda and gsh-px levels. no levels were significantly different between tamsulosin group and the control group (p = 0.004). in addition, tamsulosin group and terazosin group were also significantly different (p = 0.032). according to these results we can say that tamsulosin group had higher no levels than control and terazosin group. tamsulosin's enhancer effect on no levels leads to relaxation of the heart muscle and vascular relaxation, and so fewer side effects than other alpha antagonists. the effect of rat liver tissue radical metabolism and the protective role of hippophae rhamnoides l. on cold and immobilization stress model cold and immobilization stress is a widely used model for study the changes that occur on oxidant-antioxidant balance. hippophae rhamnoides l. (seabuckthorn; sbt) a unique and valuable plant has recently gained worldwide attention, mainly for its medicinal and nutritional potential. this study was aimed to investigate the protective role of sbt which is a natural herbal product with high antioxidant content on oxidative and nitrosative stress induced by cold and immobilization stress in rats. 32 wistar albino rats were divided into 4 groups randomly. control (i.p. physiological saline), sbt (i.p. 200 mg/kg/48 hours sbt), stress (i.p. physiological saline; 6 hours cold + immobilization stress) and sbt + stress (i.p. 200 mg/kg/48 hours sbt; 6 hours cold + immobilization stress) groups were formed. 3nitrotyrosine levels were determined by elisa whereas total antioxidant capacity, total thiol, total glutathione, nitrite-nitrate levels and superoxide dismutase and glutathione peroxidase activities were measured by colorimetric methods. sbt + stress group nitrite-nitrat (p = 0.0001), total glutathione (p = 0.0001) levels and glutathione peroxidase activities (p = 0.0001) were found to be significantly higher whereas superoxide dismutase activity was found to be lower (p = 0.007) when compared to stress group. there was no significant differences between stress group total thiol and total antioxidant capacity levels compared with stress + sbt group. stress + sbt group oxidative and nitrosative stress marker 3-nitrotyrosine level was found to be significantly higher when compared with control group (p = 0.002) whereas there was no significant differences between stress and stress + sbt groups. all this data show that sbt has antioxidant properties on cold and immobilization induced oxidative and nitrosative stress in rat liver tissue. obesity is a major health problem with growing incidence and accompanying complications. its relation with diminished cognitive functions was reported. this study aims to evaluate the effects of obesity induced oxidative stress and metabolic alterations on the cognitive functions of children and adults. 33 children and adolescents with obesity (age: 8-18); and 33 age and gender matched healthy subjects were enrolled. all subjects completed the battery tests of cnsvs via computer. the scores were compared by using commercial software (ibm spss statistics 19). biochemical parameters, malondialdehyde (mda) and protein carbonyl (pc) levels were estimated. mda and pc levels were significantly higher in subjects with obesity (0.78 ae 0.16 lmol/l;198.30 ae 84.45 nmol/ml) than the controls (0.5 ae 0.10 lmol/l; 125.35 ae 43.52 nmol/ml) (<0.001). there was statistically significant difference between study and control groups on all cognitive performance domains. significant correlation was detected between mda, pc levels and the cognition indexes. children with obesity should be evaluated for the cognitive functions, together with the metabolic follow-up. obesity induced oxidative stress may be the reason of the diminished cognition in children as well as the changes in the lipid profile and inflammation, but we need larger study groups to lighten these complex process. p-09.04.4-067 relative contribution of nitric oxide synthase (nos) isoforms to oxidative/nitrosative stress in the cerebral cortex of rat with acute liver failure (alf) acute liver failure (alf) is associated with deregulation of nmda/cgmp/no signaling and oxidative/nitrosative stress in the brain. however, the relative roles of the different nos isoforms and the mechanisms underlying alterations in their activities during alf are not fully clear. here we investigated gene and protein expression of nos isoforms, nos activity, enos uncoupling and total no production in cerebral cortex of rats with thioacetamide (taa)-induced alf. sprague dawley rats (250-280 g) received three i.p. injections of taa (300 mg/kg) at 24 hours intervals. the brain cortex expression nos isoforms (enos/inos/nnos) was measured by real-time pcr and western blot, nos activity was tested by monitoring the conversion of radiolabeled arginine to citrulline. reactive oxygen species (ros) were quantified in the presence of nos substrate l-arginine, using the carboxy-h 2 dcfda probe. no was measured with the griess procedure. the enos expression was decreased, whereas the enos dimmer/monomer ratio and nnos/inos expression were elevated in taa treated rats. while the total nos activity was decreased, the inos activity was elevated and no concentration tended to increase. ros production was elevated by taa. unspecific nos inhibitors l-name and l-nna attenuated ros production in both control and taa rats, but with higher efficiency in the latter case. ca 2+ chelation had almost the same effect as pharmacological nos inhibition suggesting that ca 2+ -independent inos activity is not the main source of ros. incubation with high dose of tetrahydrobiopterin (bh4) with which is critical for enos dimerization and subsequent no production also reduced ros production indicating the enos uncoupling phenomenon in taa cortex. the study points to enos downregulation due to lowered protein expression and uncoupling as a novel mechanism contributing to enhanced superoxide o 2 anion formation, and confirms the role of inos/nnos in enhancing no synthesis in alf-affected brain. introduction: diabetes mellitus (dm) is an endocrine disorder of world which is characterized by altered blood glucose levels and related complications including hepatic injury. myrtus communis l. (mc) is widely used by diabetic patients in the folk medicine of turkey as well as they are used worlwide. it is known that of leaves, oil and fruit of myrtus communis l. (mc) have therapeutic effects on diabetes mellitus (dm). this study was aimed to analyze the possible antidiabetic and hepatoprotective effects of mc berries in streptozotocin (stz) induced diabetic rats. materials and methods: a total of thirty rats composed of six groups as each included five rats were used. 40 mg/kg stz was injected once to animals to induce dm. after stz injection, rats were exposed to three different ethanol extracts of mc berries (250, 500 and 1000 mg/kg) by oral gavage during 14 days. alanine aminotransferase (alt) and aspartate aminotransferase (ast) levels were determined in serum and glutathione (gsh), malondialdehyde (mda) levels and superoxide dismutase (sod) activity were determined in liver tissue. results: mc administration provided significant reducement in the altered serum glucose, ast and alt levels in all diabetic groups. mc extract showed significant antioxidant activity by altering sod activity and gsh level and reducing mda levels in diabetic rats compared to controls (p < 0.05). serum ast and alt levels were reduced by mc administration in all diabetic groups. mc administration provided significance increment in sod activity and gsh level, and significant reduction in mda levels compared to controls (p < 0.05). the maximum hypoglycemic and antioxidant effects were observed at 1000 mg/kg dose of mc. background: human serum paraoxonase 1 (pon1) is a calcium dependent esterase that hydrolyzes organophosphates and also arylesters such as phenyl acetate. pon1 prevents ldl oxidation by hydrolyzing lipid peroxides. pon1 is inhibited by various chelating agents, heavy metal ions, and sulfhydryl reagents. in our study we investigated the effect of calcium on ldl oxidation of purified pon1 q192r isoenzymes. methods: pon1 q192r isoenzymes were partially purified from human serum. both allozymic forms were treated by preincubation with 1 mm edta for 15 minutes. ldl oxidation was induced by copper ions. formation of thiobarbituric acid-reacting substances (tbars) was used as a measure of lipid peroxidation. homocysteine thiolactonase (htlase activity) and arylesterase activities were measured spectrophotometrically by using homocysteine thiolactone and phenylacetate as the substrates. results: addition of 1 mm edta to partially purified hdl-pon1 q192r isoenzymes inhibited 100% of htlase and arylesterase activities. inactivation of pon1 for arylesterase/htlase activity by the addition of edta did not reduce the abilities of both allozymic forms in protecting ldl from oxidation. conclusion: ca +2 -dependent inhibition of pon1 q192r arylesterase/htlase by using the metal chelator edta, did not alter pon1's ability to inhibit ldl oxidation. pon1's ability to protect ldl from oxidation may not require calcium. p-09.04.4-070 evaluation of cholinesterase inhibitory effect, anti-radical and anti-lipid peroxidation activities of mentha pulegium i. hamad 1, 2 1 college of applied medical sciences, aljouf university, aljouf, saudi arabia, 2 faculty of medicine, bahri university, khartoum, sudan introduction: many studies indicated that intake of dietary and medicinal plants is effective in preventing or suppressing many diseases, therefore, there is a growing interest in plant'sbioactive compounds. mentha pulegium, is widely used in gulf countries in herbal teas or as additives in commercial spice mixtures for many foods to offer aroma and flavor. the aim of this study is to investigate the in vitro radical activity, the total phenol and flavonoid content, anti-lipid peroxidation and the cholinesterase inhibitory effects of mentha pulegium methanol extract. methods: the acetylcholinesterase and butyrylcholinesterase inhibitory potentials of extracts, were evaluated by colorimetric assay. the in vitro antioxidant activity was measured by dpph assay, the total phenols content was measured by folin-ciocalteau assay, the flavonoids content by the alcl 3 colorimetric method, and the protective effect of menthe mentha pulegium extracts against lipid peroxidation was evaluated using a liposome oxidation system. results: the methanol extract showed a scavenging activity nearly equivalent to vitamin c which is attributed to its high phenolic and flavonoid contents. the extract possessed protective effect against lipid peroxidation in a dose dependent manner. the methanol extract shows very little anticholinesterase activity as compared to the standard compound, physostigmine. conclusion: results presented here indicate that mentha pulegiumpossess strong antioxidant activity and protective effects and they can therefore be used as a natural additive in food, cosmetic and pharmaceutical industries. type 2 diabetes mellitus is a long term metabolic disorder that is characterized by hyperglycemia and insulin resistance. because of the hyperglycemia and free radicals, diabetes can cause cellular instability. micronuclei is a sensitive indicator of genetic damage and a marker of dna damage. micronuclei is also a morphological marker of chromosomal instability. in this study, we aimed to evaluate the frequencies of micronuclei in papanicolaou stained buccal cells of type 2 diabetic patients. a total of 30 type 2 diabetic patients and 30 healthy individuals were involved into our study. buccal smear samples that belong to these individuals were stained by using papanicolaou method for cytologic examination and the stained slides were evaluated by light microscopy (olympus bx-51). cells with micronuclei in each papanicolaou stained buccal smear sample were counted under light microscopy. the frequency of micronucleated epithelial cells were seen as significantly higher in type 2 diabetic patients than the control group (p < 0.05). one of the boron compounds is sodium perborate tetrahydrate (nabo 3 .4h 2 o), which the most widely used solid peroxygen compounds. it is used in safety bleach formulations, detergents and tooth powders. as known these products are commonly used in daily life. however, the actions on blood antioxidant defenses of sodium tetraborate against reactive oxygen species are not identified yet. it is reported that oxidative stress caused by ros damages. in this study, we searched enzyme activities of superoxide dismutase (sod), catalase (cat), glutathione-s-transferase (gst), glutathione reductase (gr), glutathione peroxidase (gsh-px) and glucose-6-phosphate dehydrogenase (g6pd) also the effect sodium perborate tetra hydrate on activities of these enzymes from human erythrocyte under in vitro conditions. according to our findings sodium perborate tetrahydrate caused significant (p < 0.05) increasing in the cat activity from red blood cell. the other antioxidant enzyme activities (sod, gst, gr, gsh-px and g6pd) did not show any changing by influence of sodium perborate tetrahydrate. metabolism of obese individuals could be exposed to risk of chronic low-grade pro-inflammatory effect and oxidative stress. some inflammatory and oxidative markers have been studied recently. plasma total antioxidant status (tas) and total oxidant status (tos) parameters can be non-invasive markers of diseases such as fatty liver disease, laparoscopic procedures (pneumoperitoneum), accompanying inflammatory condition like urinary tract infection, diabetic neuropathy, chronic hepatitis. the study groups have been comprised of two groups with normal to over-weight children. tas and tos levels were detected and the oxidative stress index (osi) was computed as a marker of the grade of oxidative stress. the over-weight group displayed higher levels of fasting glucose, insulin resistance, the body mass index. also, we know that insulin resistance leads to increased lipolysis and free fatty acid output. higher tos as well as crp is related to the group, also lower tas than other group is shown. crp levels in plasma were positively correlated with insulin and glucose levels. in addition, there was a significant relationship between osi and insulin resistance in the over-weight group. tas and tos are together more accurate sings of oxidative and antioxidative status of people. as well as a raise over weight-related subclinical inflammation and a fall antioxidant capacity is significant even in children. this condition may eventually develop the risk of long-term vascular damage. the effects of hydrogen peroxide pretreatment on antioxidant enzyme activities in calli tissues of two eggplant genotypes under salinity o. yasarkan 1 , e. aky€ uz 1 , g. baysal furtana 2 , s. s. ellialtioglu 3 , r. tipirdamaz 4 1 nezahat g€ okyigit botanic garden, istanbul, 2 department of biology, faculty of sciences, gazi university, ankara, 3 department of horticulture, faculty of agriculture, ankara university, ankara, 4 department of biology, faculty of sciences, hacettepe university, ankara, turkey the effects of hydrogen peroxide (h 2 o 2 ) pre-treatment on catalase (cat) and superoxide dismutase (sod) were investigated and lipid peroxidation measured as malondialdehyde (mda) content of the calli from salt-sensitive (artvin) and salt-tolerant (mardin) eggplant genotypes under salinity stress. the seeds from each genotypes were germinated on ms medium for 4 weeks and hypocotyl tissues from these plantlets were used as explants for calli induction on ms medium including 1 mg/l 2,4-d and 0.1 mg/l kinetin. as for the pre-treatment, the subcultured calli tissues were transplanted on the mediums containing 50 and 100 lm h 2 o 2 for 48 hours and then transplanted on the mediums including 150 mm nacl for 24 hours. antioxidant enzyme analysis and mda measurement was carried out for the control, nacl-only, h 2 o 2 -only and h 2 o 2 pre-treated tissues. pre-treatment with h 2 o 2 decreased the deleterious effects of salt stress on mda contents. in comparison with salt stressed groups, h 2 o 2 pre-treatment with or without nacl reduced mda content especially in artvin. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in 100 lm h 2 o 2 + nacl groups on each genotypes. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in 100 lm h 2 o 2 + nacl groups on each genotypes. the result showed pre-treatment of 100 lm h 2 o 2 induced acclimation of the plants to salinity. in addition, 100 lm h 2 o 2 pre-treatment, as a stress signal, could trigger the activation of antioxidant enzymes in calli and in this way alleviated the oxidative damage in calli growth under salinity. the investigation of effects of ghrelin and cannabinoid cb1 receptor agonist and antagonist on oxidant and antioxidant mechanisms on brain tissues of penicillininduced epileptic rats the aim of this study is to investigate the individual effects of ghrelin and cannabinoid type1 (cb1) receptor agonist acea, the antagonist am-251 and the interaction of these two different systems on oxidant and antioxidant systems in the brain, cerebellum and brain stem tissues of penicillin-induced epileptic rats. in this study 56 male wistar albino rats were used weighing 20-250 g. each group was consisted of 8 rats. study groups: 1: control, 2:penicilin(500 iu), 3:penicillin(500 iu) + ghrelin(1 lg), 4:penicillin(500 iu) + am-251(0.25 lg), 5:penicilin (500 iu) + acea(7.5 lg), 6:penicillin(500 iu) + am-251(0.25 lg) + ghrelin (1 lg), 7:penicillin(500 iu) + acea(7.5 lg) + ghrelin(1 lg). than the levels of mda, gpx and sod are measured in plasma and tissue samples of these rats. penicillin was found to be induced lipid peroxidation in the brain, cerebellum and brain stem tissues in our study. ghrelin and acea, which both have anticonvulsant effects, were shown to be effective in reversing the oxidative damage caused by penicillin and proconvulsant am251 was found to further increase the oxidative stress caused by penicillin in these tissues. ghrelin also was found to suppress the oxidative stress caused by am251 in the cerebellum tissue but it did not contribute to antioxidant effects produced by acea. since the role of oxidative stress in epilepsy has been established, it may be suggested that ghrelin and acea may have anticonvulsant effects via their antioxidant features. the discovery of inhibitors for enzymes that metabolize endogenous ghrelin and cannabinoids through new studies may contribute to the improvement of seizure resistance in epilepsy. accelerated atherosclerosis in patients with ankylosing spondylitis (as) give rise to increased cardiovascular morbidity and mortality. endothelial dysfunction could be the initial process in the development of atherosclerosis. human endothelial cell-specific molecule-1 (endocan) is a novel human endothelial cell-specific molecule. therefore, we assessed serum endocan levels and carotid intimamedia thickness (cimt) as a surrogate marker of atherosclerosis in patients with as. a total of 57 patients with a diagnosis of as according to newyork ctriteria and 50 control subjects were included in our study. serum endocan, interleukin-6 (il-6), tumor necrozis factor-a (tnf-a), c reactive protein (crp) and cimt were measured in all participants. serum endocan, il-6, tnf-a levels were measured with elisa. the other parameters were done by routine biochemical methods. as patients exhibited increased serum endocan levels and cimt compared to matched controls (p < 0.05). whereas, serum il-6, tnf-a were similar between grous. in patient with as, there were no significant differences between active and inactive patients by means of il-6, tnf-a, endocan and cimt. in as group, cimt correlated with disease duration and age (r = 0.597, p = 0.000; r = 0.721, p = 0.000). we could not find any significant correlation between serum endocan levels and parameters studied. our study shows increased cimt in as patients without traditional risk factors such as increased bmi, lipid profile compared to controls. although we found increased circulating endocan levels in patients with as, the other factors could affect increased atherosclerosis in this population because of lack of correlation between endocan and cimt. probable biomarkers could be related to increased cimt in patients with as should be investigated in larger study groups. keywords: ankylosing spondylitis, atherosclerosis, carotid intima media thickness, endocan p-09.04.4-079 investigation of pentose phosphate pathway and oxidative stress in erthrocyte infected babesia ovis a. bildik, t. karagenc ß, p. a. ulutas, n. aysul, h. aksit adnan menderes university, aydin, turkey introduction: babesia infections occur in cattle, sheep, goat, horse, dog, cat pig and rodents. in this study, the effects of babesia ovis living and present in the erythrocytes to glucose metabolism was researched. at the same time, biochemical parameters were also associated with parasitemia. materials and methods: babesia ovis (israel) cell culture was provided from dr. abel martin gonz alez oliva (portugal). culture passaged 48 or 72 hours according to parasitemia state (8-10%). biochemical analyses were performed in erythrocyte culture in which parasitemia between 1% and 16%. cell counts and hemoglobin concentration of erythrocytes culture suspension were measured at cell counter instrument and than it was washed 3 times with physiological saline, erythrocyte suspensions were stored at-80oc analysis. gssg (oxidize glutathione), gsh (reducte glutathione), nadph, glukoz 6 p dehydrogenaz, gshpx (glutathione peroxidase), gshrx (glutathione reductase) were determined by commercial kits. all experiments were done in duplicate, the results were calculated by the number of erythrocytes. results: parasitemia was positively correlated with gsh, nadph and gshrx (p < 0.05). a correlation between other biochemical parameters was not observed. discussion: the pentose phosphate pathway in erythrocytes has an important role such as to provide pentose sugar required for the synthesis of nucleic acid, to reduce glutathione, to produce nadph and to protect from methemoglobin accumulation. in studie sthat naturally infected erythrocytes with babesia parasites, it was seen to be caused to oxidative stress, however gsh results in these investigation were obtained differently . conclusion: according to the results of this study that performed in vitro, it can be suggest that their glutathione metabolism and pentose phosphate pathway of parasites may active.key words: babesiosis, gsh, gssg, nadph, g6pdh, gshpx, gshrx p-09.04.4-080 in vitro protective effect of betaine on peroxidative injury caused by ethanol and aspirin exposure on rat brain synaptosomes i. sogut 1 , g. kanbak 2 1 istanbul bilim university vocational school of health services, istanbul, 2 eskisehir osmangazi university medical school department of biochemistry, eskisehir, turkey aspirin intake of specific daily doses are advised by doctors to postmenapausal women and men above 40 years of age to prevent heart attack and even cancer in recent times. in this study, the aim is to investigate the in vitro cytototoxic effects of different doses of ethanol (50 mm, 100 mm ve 200 mm) alone and together with 100 lg/ml aspirin, and possible protective role of 1 mm betaine on rat brain synaptosomes. 15 male sprague dawley rat forebrains were divided into equal pieces and pooled to form 10 study groups. synaptosomal fractions extracted from pooled rat brains were incubated with different doses of ethanol, aspirin and betaine, and malondialdehyde (mda) levels, an important indicator of cellular damage, were measured. a significant increase (p < 0.05) was observed in mda level of 200 mm ethanol group compared to control group. different doses of ethanol (50 mm, 100 mm ve 200 mm) + aspirin exposure significantly increased (p < 0.001) mda levels compared to controls, whereas betaine administration significantly decreased (p < 0.001) lipid peroxidation caused by ethanol + aspirin treatment. we conclude that ethanol and ethanol + aspirin administration increases lipid peroxidation in rat brain synaptosomes while betaine helps prevent this peroxidative membrane injury.keywords: aspirin, betaine, ethanol, malondialdehyde (mda) p-09.04.4-081 analyses of mitochondrial biogenesis in hepatocellular carcinoma treated with berberine f. aygenli, h. c ß imen yeditepe proteomics and mass spectrometry group (yediprot), genetics and bioengineering, yeditepe university, istanbul, turkey objective: berberine (bbr) has been demonstrated to have anticancer activities against various cancer types, particularly hepatoma. in this project, we aimed to reveal the effect of bbr treatment on mitochondrial biogenesis through sirtuins and hif-1a in hepatocellular carcinoma cell line, hep3b under hypoxia. method: hep3b cells were subjected to normoxia (21% o 2 ) and hypoxia (1% o 2 ) in the presence or absence of bbr treatment. the amount of bbr was optimized via cell viability (mts) assay under normoxia. then, immunoblotting experiments were performed to identify the effect of bbr on hif-1a, pgc-1a, and sirtuins involved in mitochondrial stress. the variation in the oxphos complexes and the level of reactive oxygen species (ros) were also measured to investigate the effect of bbr on mitochondrial energy stress state. results: here, we present that cell viability was significantly decreased at 25 lm. bbr treatment has shown significant reduction in hif-1a and sirt6 which responsible for up-regulation of glycolysis. also, succinate dehydrogenase (cii) and cytochrome c oxidoreductase (ciii) of the oxphos complexes were downregulated without any change in nadh dehydrogenase (ci) or atp synthase (cv). bbr significantly abolished to oxidative stress under hypoxia, which was demonstrated as a reduction in the level of reactive oxygen species by decreasing on sirt3 expression. bbr induces the overexpression of sirt1 and its deacetylated-pgc-1a, which might be an indicator of being a potent protective agent against hypoxia by normalizing mitochondrial function and inducing mitophagy in impaired mitochondria caused by deficiency of glycolysis and oxphos. conclusion: detailed information about the communication between hif-1a and sirtuins and their relation to mitochondrial energy production was provided with the alteration of their activity by bbr treatment. it is highly expected that bbr and its derivatives might become important during the development of supplemental therapies. introduction: reactive oxygen species are involved in a variety of biological phenomena, such as carcinogenesis, inflammation and aging. among the targets of ros, dna appears most important in tumor biology since it is firmly established that cancer is a genetic disease. ros induce several kinds of dna damage, including strand breakage and dna-protein cross-linkage. fruit of zizyphus jujuba, a traditional chinese herb widely consumed in asian countries, has been reported to possess several vital biological activities. this study intends to evaluate their antioxidant activity on glioblastoma cells. materials methods: cell survival was quantified by colorimetric mtt assay. human gliblastoma cells were pretreated with 100 lm h 2 o 2 after 30 minutes 100 lm ziziphus jujuba essential oil was added to the cells for three hours. then, the cell homogenates were taken and glutathione, total oxidant and total antioxidant capacity and nitric oxide levels were estimated using spectrophotometric methods. results: ziziphus jujuba treated cells could prevent intracellular glutathione from being depleted following an exposure of h 2 o 2 . also our data suggest that ziziphus jujuba is effective in preventing h 2 o 2 induced oxidative stress and nitric oxide levels. discussion: some research showed that h 2 o 2 was over produced in the pathological process of acute and chronic neuronal toxicity, the toxicity effect of b-amyloid on the cultured neuron and neuronal cell line was mediated by h 2 o 2 . the traditional medicine recommend several medicinal plants for providing relief from various inflammatory diseases. many research has been reported that the essential oil from seeds of helping to prevent the oxidative stress and neuronal diseases in brain. introduction: toxicity by oxygen radicals has been recommended as a major cause of cancer, heart disease, and aging. oxygen radicals and other oxidants appear to be toxic in large part because they start the chain reaction of lipid peroxidation. most of the analytical techniques for peroxide determination are generally time consuming and not very suitable for routine or on line analysis. we aimed to design a new biosensor for rapid determining of oxidant agent hydrogen peroxide. materials and methods: all reagents were of analytical grade unless stated otherwise, and were purchased from sigma aldrich. firstly, the 2-hidroxymetacrilate metacriloamidoscystein nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. free nh 2 groups of catalase enzyme make schiff bases between nanopolymer's carbonyl groups, then immobilization was actualysed with cross linking reagent glutaraldehyde. we developed a biosensor system preparing ferrociyanide, selected as a mediator, in the buffer solution. results: polyhemamac nanopolymer and catalase complex were immobilized by glutaraldehite to construct a hydrogen peroxide biosensor. the responses of the biosensor are therefore proportional to the oxidation peaks of the complex at +0.019 v potential. the cyclic voltammograms obtained from the experiments showed that, pottasiumferrociyanide mediator complex positively affected the biosensor responses for hydrogen peroxide determination. discussion and conclusion: as a result of this study, the method developed by the catalase enzyme electrode was found to be more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. since biosensor technology provides economical, practical, specific and sensitive results for the determination of hydrogen peroxide, it was improved very efficiently. p-09.04.4-085 impact of amoxicillin, gentamicin and cefazolin sodium antibiotics on antioxidant gene expression and enzymatic activities in mouse liver p. g€ uller 1 , h. budak 2 , m. sisecioglu 2 , m. c ß iftci 3 1 department of chemistry, faculty of science, atat€ urk university, erzurum, 2 department of molecular biology and genetics, faculty of science, atat€ urk university, erzurum, 3 department of chemistry, faculty of arts and sciences, bing€ ol university, bing€ ol, turkey reactive oxygen species (ros) are highly reactive molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. living organisms have the antioxidant defence systems to block harmful effects of ros. the imbalance between oxidants and antioxidants is termed oxidative stress. the antioxidant defence mechanisms are divided into two groups as enzymatic and nonenzymatic defences. enzymatic defence mechanisms consist of enzymes like superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glucose-6-phosphate dehydrogenase (g6pd) and glutathione s-transferase (gst). the present study was designed to determine the effects of gentamicin, amoxicillin and cefazolin sodium antibiotics on the hepatic antioxidant system and to determine any possible correlation between enzymatic and molecular levels. for this reason, effects of these antibiotics on the transcription of the antioxidant system has been investigated by real time pcr, and then the enzyme activity of these enzymes have been measured in whole liver homogenate obtained from control group and the drug administered groups mice. our results demonstrate that administering antibiotics led to crucial inhibition of all antioxidant enzyme activity. while significant transcriptional activation for sod and cat was seen in the gentamicin treated group, the transcription of gst and g6pd was decreased. however transcriptional activation was seen for sod and cat in amoxicillin administered group, the transcription of gst was decreased as compared with the control group. in the cefazolin sodium treated group, while cat and gst transcription were elevated significantly, the expression of sod and g6pd were decreased. in conclusion, gentamicin, amoxicillin and cefazolin sodium affect the hepatic antioxidant system at the molecular and protein level. this work was supported by scientific research project of ataturk university of turkey (grant no: 2013/296). p-09.04.4-086 protective effects of curcumin supplementation on oxidant/antioxidant system changes created by organic phosphorus pesticide poisoning organic phosphorus pesticides (opp), widely used in agriculture or as insecticides in home, cause adverse health effects. chlorpyrifos is one of the most commonly used opp. we aimed to investigate the possible protective effects of curcumin (cur) supplementation, the principal curcuminoid of turmeric, on poisoning symptoms and oxidant/antioxidant system changes caused by chlorpyrifos. adult sprague-dawley rats were used. cur (30, 100 and 300 mg/kg) were administered orally for 5 days. on the sixth day, chlorpyrifos (279 mg/kg, s.c.) was administered. twenty four hours after chlorpyrifos administration, body weights, locomotor activities and body temperatures of rats were measured. following the measurements, rats were decapitated and the blood, brain and liver tissue samples were taken and prepared for the biochemical and histopathological measurements. chlorpyrifos administration increased the malondialdehyde (mda) levels but decreased catalase (cat), superoxide dismutase (sod), glutathione reductase (grx) concentrations and reduced/oxidized glutathione (gsh/gssg) ratio in the blood samples, brain and liver tissues compared with the control group (p < 0.05-0.001). the concentration of advanced oxidation protein products (aopp) were increased only in the brain tissue after chlorpyrifos administration (p < 0.001). cur administration reduced all of these changes (p < 0.05-0.001). similarly, cur at the doses of 300 mg/kg reduced the decreases in body weight, body temperature and locomotor activity with chlorpyrifos (p < 0.001). additionally, the histopathological damage scores induced by chlorpyrifos (p < 0.05-0.01) were decreased by the administration of cur (p < 0.05-0.01). our findings suggest that cur supplementation can ameliorate the poisoning effects of chlorpyrifos via supporting the antioxidant mechanisms and cur could be used for protective purposes against oxidative stress and tissue damage caused by chlorpyrifos. the effect of ogtt applied for screening in pregnancy on adenosine deaminase and xanthine oxidase activity in normal and prediabetic pregnant women z. c. ozmen, k. deveci, i. benli department of biochemistry, gaziosmanpasa university medical faculty, tokat, turkey objective: it was reported that the activities of adenosine deaminase (ada) were different in normal pregnant women and pregnant women with gestational diabetes mellitus (gdm). it was also stated that the activity of xanthine oxidase (xo) was increased in pregnant women with gdm. the objective of this study was to evaluate if glucose have effects on oxidative stress in prediabetic women by affecting ada and ox after 50 g ogtt which was applied in pregnant women for screening. methods: the serum specimens of 39 pregnant women who applied to the outpatient clinic of the obstetrics and gynecology department and had 50 g ogtt, were used in this study. ada and xo activities were analyzed in the serum specimens taken from the normal (n = 20) and prediabetic pregnant women (n = 19) in the 0th and 60th minutes of ogtt. ada and xo activities were measured with the spectrophotometric method and the u/l enzyme activity was calculated. findings: there was no significant difference between the 0th and 60th minutes regarding the ada activities in the normal and prediabetic pregnant woman groups. however, we observed a significant difference between 0th and 60th minutes regarding the xo enzyme activity in normal pregnant women (p = 0.001). in normal pregnant women, the median xo enzyme activity in the 0th minute was 0.29 (0.11-1.33) u/l and it was 0.8 (0.25-2.26) u/l in the 60th minute. nevertheless, there was no correlation between the xo activity and glucose level. as to the prediabetic pregnant women, there was no significant difference between the xo enzyme activities in 0th and 60th minutes. the results of our study showed that the xo activity increased as a response to ogtt in the normal pregnant women compared with the prediabetic pregnant women. this finding made us think that the oxidative stress caused by ogtt did not affect the xo response in prediabetic pregnant women and that there would be some adaptive mechanisms against the chronic exposure to high level glucose. rainbow trout (oncorhynchus mykiss) aquaculture continuously increases in turkey. the objective of the present study is to increase the productivity in fish farming of rainbow trout just via intervention in physical cases without the effects of any chemicals and investigate whether this conditions cause oxidative stress. in this experiment eight tanks were used and 44 rainbow trout larvae were placed in each tank and these tanks were illuminated with light in different wavelengths; natural sunlight, and incandescent long-wave (red light), medium-wave (green light) and shortwave (blue light) led lights. the experiment took 64 days. biochemical changes in rainbow trout exposed to light in different wavelengths (red, green, blue) were analysed via the variations in gr, gst, g6pd, gpx, sod and cat enzyme activities, which are significant for enzymatic antioxidant defence system and in ache activity, which plays an important role on central nervous system. maximum activity change in liver tissue was observed for gst and g6pd enzymes in fish grown under green light and for sod enzyme in fish grown under blue light. in gill tissue, sod and g6pd activities were affected the most, and in brain tissue, these were gst and sod activities. it was observed that the average weight of the fish increased 1.2 times under red and blue lights and 1.3 times under green light. the highest weight increase was observed under green light, however, antioxidant enzyme activities increased in the liver and gills and decreased in the brain tissue under this light condition. in conclusion, it was observed that productivity was 1.2 times under red light when compared with control group and it was determined that the fish grown under red light can tolerate oxidative stress more than other wavelength. p-09.04.4-089 effect of nigella sativa on biliary obstructioninduced oxidative stress and apoptosis in rats human safety concerns, since that these agents may not only cause acute toxicity via inhibition of acetylcholinesterase but they can also induce delayed toxicity in the nervous system. a key interest to the current work is the potential correlation between gene expression and cytoskeletal protein changes in differentiating neural cells exposed to sub-lethal neurite outgrowth inhibitory concentrations of specific ops, which was addressed by analysing the underlying changes in the levels of cytoskeletal gene expression and protein levels. to assess the molecular effects of op exposure, phenyl saligenin phosphate (psp), chlorpyrifos (cpf) and its metabolite chlorpyrifos oxon (cpfo) were applied at the point of induction of differentiation of rat c6 glioma and mouse n2a neuroblastoma cells and incubated for 24 hours. at sub-lethal concentrations (1, 3, 10 lm) all three ops used in this study were able to inhibit the development of neurites with no significant effect on cell viability, as determined by neurite outgrowth and mtt reduction assays. to understand the possible effects of ops on cytoskeletal gene expression, primers for genes encoding glial fibrillary acidic protein (gfap), biii tubulin, growth associated protein 43 (gap43) and neurofilament heavy chain (nefh) were optimized for qpcr analysis and the levels of the corresponding proteins were detected by western blot analysis. exposure to ops caused in most cases a reduction in the levels of cytoskeletal proteins, and the results from qrt-pcr analysis also indicated reductions in the gene expression of gfap in c6 cells, and of nefh and biii tubulin in n2a cells, in a dose dependent manner. thus, the observed changes in protein levels are at least partly due to altered gene expression. curcumin is extracted from a perennial herbaceous plant known as curcuma longa. in recent years, considerable interest has been focused on curcumin due to its use to treat a wide variety of disorders without any side effects. earlier studies have shown that curcumin has anti-apoptotic, anti-inflammatory, antiproliferative, antiangiogenic, anticancer and antiplatelet activities. the goal of the present study was to investigate the effects of curcumin on peroxy radical-induced oxidative changes in human platelets. 15 healthy volunteers were enrolled in the study. none of the study participants were on anticoagulation therapy. citrated venous blood samples were centrifuged at 200 g for 10 minute to obtain platelet-rich plasma (prp). the platelet pellet was washed and suspended with tris-nacl buffer. then, platelets were incubated with h 2 o 2 absence and presence of curcumin (50-500 lg/ ml) for 1 hours at 37°c. to determine the preventive effects of curcumin on the oxidative stress and apoptosis induced by peroxy radicals in human platelets were determined by measuring levels of of lipid peroxidation, total antioxidant capacity, caspase 3, 8 and 9 activities, and mitochondrial membrane potential. additionally, we also studied the effects curcumin on platelet aggregation induced by adp. pre-treatment of platelets with curcumin caused a marked reduction in oxidative stress, activation and apoptotic markers in a dose-dependent manner. on the other hand, pre-treatment of platelets with increasing doses of curcumin resulted in inhibition of platelet aggregation induced by adp. in the light of our findings, we suggest that curcumin may have a therapeutic potential to prevent platelet activation related disorders. people have been using mushrooms in the treatment of diseases as well as food, for centuries. most of the edible and inedible mushroom species were used in important medical studies and their effects were begun to be used in the treatment of diseases. this study focuses on the hepatoprotective effects of tricholoma anatolicum, which is endemic specie in turkey, against oxidative stress based on hydrogen peroxide (h 2 o 2 ) on hepg2 liver cancer cell line. t. anatolicum used in this study was extracted with the help of ultrasonication and fraction methods. then the cytostatic effects of extracts on hepg2 cells were explored and their hepatoprotective effects were determined. moreover, various concentrations of aqueous extract (ehta) of t. anatolicum were determined by hepg2 cells's 24-48-72 hours effect analysis on their cellular morphology, xtt and real-time cell analysis in of xcelligence device. ehta extract's cell pathway (apoptosis and necrosis) effects on hepg2 cells were determined with flow cytometry method with the help of annexin v-apc and 7aad fluorescent dye. finally, the phenolic compounds found in ehta extracts were determined with the help of hplc methods. according to xtt cytotoxicity analysis, the ehta extract values were determined as follows: 24 hours ic 50 > 2000 lg/ml, 72 hours ic 50 . furthermore, according to the real-time cell analysis made with xcelligence, ehta extracts were found to be; 24 hours ic 50 = 241.539 lg/ml, 48 hours ic 50 = 418.135 lg/ml, 72 hours ic 50 = 285.694 lg/ml. increasing concentrations of ehta extracts were determined to direct hepg2 cells to apoptosis. moreover, considering the hplc analysis -according to the reference point of 100 mg in 100 g sample-within ehta extracts, catechins and vanillic acid peaked. the final results revealed that t. anatolicum's effect on hepg2 was cytostatic at low doses, and cytotoxic at high doses. p-09.04.4-093 relationship between serum ceruloplasmin levels and coronary blood flow background: there is growing evidence that oxidative stres plays an important role in the development of the slow coronary flow (scf) phenomenon. ceruloplasmin (cp) is a copper containing metalloenyzme which has antioxidant functionthrough its ferroxidese 1 activity and is associated with cardiovascular diseases. we aimed to investigate the relationship between scf and serum cp level. methods: patients who underwent elective coronary angiography and had no significant epicardial coronary disease were included in the study. patients who had thrombolysis in myocardial infarction frame counts (tfcs) above the normal cutoffs were considered to have scf and those within normal limits were considered to have normal coronary flow (ncf). a total of 90 patients (55 subject as scf and 35 subjects as ncf) were analyzed. 5 ml blood samples were taken from the groups to study ceruloplasmin activity. serum ceruloplasmin levels were determined spectrophotometrically. results: the serum cp levels were statistically lower in scf group than in the ncf group (414 ae 79 versus 469 ae 99 ng/ml, p = 0.009). also there was a significant correlation between serum cp levels and tfcs (r = à0.378, p = 0.013). conclusion: the findings of this study suggests that patients with scf had lower serum cp levels correlated with tfcs. we concluded that reduced serum cp levels might represent a biochemical marker of scf. introduction: sleeve gastrectomy (sg) has been used for the surgical treatment of morbid obesity, as a first step or definitive treatment. alterations of thyroid hormones in gastrointestinal surgery were previously studied. the aim of the present study was to determine the effects of triiodothyronine (t3) supplementation on oxidative stress parameters in anastomotic tissue level. materials and methods: twenty-four male wistar albino rats were divided into control (n 12), and experimental (n 12) groups. rats were underwent a sg, with a hand-sewn suture. experimental group rats received a single dose of t3 (400 mg/100 g) in postoperative day. rats were sacrificed on postoperative day 7. serum thyroid stimulating hormone (tsh), free t3 (ft3), and free thyroxine (ft4) were analysed using elisa. each tissue was homogenized in ice-cold pbs (ph: 7.4) and centrifuged at 2000 rpm for 20 minutes (4°c) to avoid contamination with cellular debris. the supernatants were used to measure total oxidant status (tos), total antioxidant status (tas), nitric oxide (no) and malondialdehyde (mda) levels. all tissue parameters were analysed by spectrophotometric methods. oxidative stress index (osi) values were calculated. results: rats given t3 hormone had not decline in ft3 levels compared with the control groups. a significant decrease in ft4 levels was found in t3 given rats on postoperative day 7. whereas tissue tos levels did not alter by thyroid hormone treatment, tas levels significantly decreased. osi values were not statistically different in tissues. tissue no levels were also similar in both groups. mda levels increased in t3 given rats compared with the control group. discussion and conclusion: this study showed that anastomosis after sleeve gastrectomy is associated with decreased ft4 level. although tos levels and osi values were similar in both groups, t3 supplementation induced lipid peroxidation by increasing tissue mda levels that might deplete tissue antioxidant level. reactive oxygen species (ros) are reactive chemical molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. although intracellular ros level is essential molecules for the signal transduction pathways, elevated intracellular level of ros leads to oxidative stress that causes damage to dna, proteins and lipids. therefore, excessive ros levels have to be eliminated by antioxidant defence systems. tip60 (tat interacting protein, 60 kda) is a histone acetyltransferases (hats) that catalyses multiple functions in metabolism such as dna repair, apoptosis, etc. we thought that if tip60 has a role in signal transduction and apoptosis, it might have direct or indirect relationship with the antioxidant system. the present study was designed to determine the impact of tip60 gene on the hepatic antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), and glutathione reductase (gr) both gene and protein level. for this reason, quantitative gene expression analysis on the antioxidant system has been investigated by real time pcr, quantitative protein expression has been investigated by western blot analysis, and then the activity of these enzymes have been measured in whole liver homogenate collected from control and liver-specific tip60 conditional knockout mice. additionally, since any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h 2 o 2 ) level in the cell might be an indication for the accumulation of ros, the relative levels of them were also studied. our data showed that the absence of tip60 affects the antioxidan system both gene and protein level. in conclusion, our initial data suggest that tip60 may be essential for the (ros) homeostasis and redox regulation. curcumin is a major chemical component produced from the rhizome of the plant curcuma longa. lt has been demonstrated that curcumin has an antioxidant, anti-inflammatory, and antiproliferative effects and, protects tissues against ischemia/reperfusion (i/ r) injury. i/r has detrimental effects on transplanted organs including uteri. the major consequence of l/r injury is oxidative stress leading to the generation of ros. uterine transplantation (ut) has been gaining popularity around the worid in the past few years. the aim of our study was to examine the antiapoptotic effects of curcumin on uterine l/r injury. the rats were randomized into three groups of seven rats each, group i consisted of rats that did not receive any treatment, group ll exposed to 0.5 hour of lschemia and 1 hour of reperfusion, group iii of rats that received intraperitonealy curcumin (200 mg/kg) 0.5 hour before the induction of i/r. then, the rat uterine tissue levels of mda, tac, and activities of caspase 3, 8 and 9 were measured. furthermore, the apoptotic index was determined immunohistochemically by the tunel method using light microscopy. biochemical analysis results showed that curcumin decreased the mda and caspase-3 and 9 ieveis, and increased the uterine tissue levels of tac but, caspase 8 activity did not changed by curcumin suggesting that curcumin induces apoptosis via intrinsic apoptotic pathway. on the other hand, an high apoptotic index was observed in i/r group (27.37 ae 11.56%) and decreased after treatment with curcumin (8.69 ae 7.49%). in conclusion, we demonstrated the protective effect of curcumin on apoptosis immediately after reperfusion induction in uteri and we can say that curcumin could improve ir injury and decrease apoptotic index. we propose that curcumin may be a novel approach for improvement of uteri i/r injury. glutathione and the related enzymes belong to the defence system of the tissues against chemical and oxidative stress. these enzymes especially glutathione s-transferase are often overexpressed in tumor cells and are regarded as a contributor to their drug resistance and are thought to play an important role in cancer progression. the purpose of this study is to evaluate the protective effects of chlorophylline as an antioxidant molecule which has inhibitory effects on gst p1-1 on chemically-induced breast cancer model. in our previous work, we had observed that this molecule led to proliferation in breast cancer cells. in this study, n-methyl-n-nitrosourea (mnu) used for inducing carcinogenesis in eighteen, 21-day-old female sprague-dawley rats. chlorophylline and mnu solutions were injected intraperitoneally when the rats were 21, 28, 35 and 42 days old. their weight and tumor diameters were measured throughout the 5 months study period. at the end of the study, all animals were sacrificed and determined both glutathione levels and related enzymes activities (gluathione s transferase, glutathione reductase and glutathione peroxidase) in tumor and tissues such as liver, kidney, heart and spleen were studied and analyzed. as a result, in breast cancer model, glutathione and related enzyme activities were protected by chlorophylline treatment whereas mnu made them decreased compared to the control group. in conclusion, chlorophylline with antioxidant features decreased the toxic effect of mnu by regeneration of glutathione and enhancement of its related enzyme activities. the use of antioxidant molecules, because of proliferative effects and defence-oriented behaviours, should be discussed in cancer therapy. p-09.04.4-100 effect of overexpression of bacillus catalase on lactococcus lactis nisin production z. girgin ersoy, s. tunca gedik gebze technical university, kocaeli, turkey nisin, has been used commercially (e234) in food preservation for approximately 40 years. it's the only bacteriocin which is approved by world health organization as a food additive. the fundamental problem that limits nisin usage in food preservation is low product yield by producer strains. because of high commercial potential of nisin, studies about increasing the production efficiency of nisin is kept in the forefront in recent years. since nisin biosynthesis and bacterial growth are occuring in parallel to each other, conditions that promote growth are also expected to encourage nisin production. it is known that, when supplied with exogenous heme, lactococcus lactis cells can respire under aerobic conditions and produce higher energy which in turn cause higher biomass. however, aerobic conditions also cause oxidative stress since catalase enzyme, which detoxify hydrogen peroxide, is absent in l. lactis. in this study, to complete the missing component of the defence mechanism of l. lactis, catalase (kate) gene of aerobic bacterium bacillus subtilis was overexpressed in facultative anaerobe l. lactis cells. for this, kate gene of b. subtilis was amplified by polymerase chain reaction (pcr). plasmid constructions were established in e. coli by using an e. coli-l. lactis shuttle vector and then the recombinant plasmid was transferred to l. lactis cells by electroporation. the presence of catalase activity in the recombinant strain grown on the solid medium was first detected by dropping hydrogen peroxide directly on the cells, then with enzyme assays. fermentation studies are going on to determine nisin production of the recombinant strain. to the best of our knowledge, this study presents the first preliminary results that shows the effect of overexpression of catalase gene on nisin production. cancer is among the leading causes of morbidity and mortality worldwide. chemotherapy is one of the major cancer treatment strategies. remarkably, natural products have garnered increased attention in the chemotherapy drug discovery field because they are biologically friendly and have high therapeutic effects. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of humic acid. the present study investigated anticancer effects of ha in several human cancer cell lines. ha was purchased from sigma-aldrich. in this study, we used several human cancer cell lines: the human breast cancer cell line, mcf-7, colon cancer cell line, ht-29, lung adenocarcinoma cell line, a549, and servical cancer cell line, hela. the cells were maintained in dmem medium supplemented with 10% heatinactivated fbs and 1% penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at 37•c. five different concentrations (100 ug/ml, 50 ug/ml, 25 ug/ ml, 10 ug/ml, 5 ug/ml) were prepared using a stock solution of ha. the cell proliferation and migration was measured. on the other hand, the apoptotic mechanisms induced by ha in cancer cells were investigated using "apoptosis antibody array kit". the effects of ha on cancer cell lines were evaluated over 72 hours. according to our results, ha induced a decrease in ht-29, a549 and hela cell numbers in a dose-dependent and time-dependent manner. contrary to this, ha induced proliferation of mcf-7 cells in dose dependent manner. ha inhibited cell migration in a dose dependent manner except mcf-7 cell line. it was also determined apoptotic pathways in cancer cells induced by ha. it was concluded that ha has an inhibitory effect on certain some cancers. since the effect of ha on tumor progression is unknown, further studies are needed to clarify the rol of ha on cancer activity. p-09.04.4-102 chronic immobilization stress in rats: fluoxetine and amisulpride protects against chronic immobilization stress-induced biochemical alterations in the present study, the effects of amisulpride and fluoxetine on serum total sialic acid (tsa) and lipid bound sialic acid (lsa) levels was investigated in the rats exposed to chronic immobilization stress. the study was administered using 40 male wistar albino rats weighing 200-250 g. rats were divided into five groups (n = 8/ group). group i comprised the control group, group ii was exposed with saline + immobilization stress (30 minutes daily immobilization stress for 15 days and 0.5 ml saline was administered perorally 30 minutes before immobilization), group iii was exposed amisulpride (10 mg/kg/day) + immobilization stress, group iv was exposed fluoxetine (10 mg/kg/day) + immobilization stress and v. group was exposed amisulpride (10 mg/kg/ day) + fluoxetine (10 mg/kg/day) + immobilization stress. statistical analysis showed that the saline + stress, amisulpride + stress and amisulpride + fluoxetine + stress groups was significantly higher than the control group with regards to tsa levels (p < 0.05). whereas, the fluoxetine group was significantly lower than the group regarding tsa levels (p < 0.05). on the other hand, saline group was significantly higher than the control group with regards to lsa level (p < 0.05). whereas, no significant differences in lsa levels were observed in the amisulpride, fluoxetine and amisulpride + fluoxetine groups, as compared to the control group (p > 0.05). the present study demonstrated beneficial effect of fluoxetine and amisulpride on the concentration levels of lsa and tsa in stress. p-09.04.4-104 protective effect of borax and boric acid on total sialic acid and lipid-bound sialic acid levels against 3-methylcholanthrene and benzo(a)pyrene induced oxidative stress in rats s. ekin 1 , g. oto, f. g€ ok, y. karakus, d. yildiz 1 y€ uz€ unc€ u yil university, van, turkey the present study was performed to investigate total sialic acid (tsa) and lipid bound sialic acid (lsa) levels as possible in vivo chemoprotective effect of borax (bx) and boric acid (ba) again-st3-methylcholanthrene (3-mc) and benzo(a)pyrene (b(a)p) induced oxidative stress in rats. the rats were divided into nine groups of six rats each. group i: control, untreated animals were given % 0.9 nacl, group ii: the b(a)p were administered 25 mg/kg via ip. four times. group iii: the 3-mc-treated animals were administered 25 mg/kg via ip. four times, group iv: ba was given 300 mg/l/day with water. group v: bx was given 300 mg/l/day with water. group vi: b(a)p 25 mg/kg via ip four times + ba 300 mg/l/day dosage with water. group vii: 3-mc 25 mg/kg via ip four times + ba 300 mg/l/day with water. group viii: b(a)p 25 mg/kg via ip four times + bx 300 mg/l/ day dosage with water. group ix: 3-mc 25 mg/kg via ip four times + bx 300 mg/l/day with water. the experimental period was continued for 150 days. statistical analysis showed that the 3-mc + ba group was significantly higher than the control group with regards to tsa and lsa levels p < 0.001, p < 0.001,p p-09.04.4-105 effects of aluminum exposure on trace elements in rat tissues b. ozturk kurt, s. ozdemir department of biophysics, cerrahpasa medical faculty, istanbul university, istanbul, turkey aluminum (al) is the most abundant metal and the third most abundant element in the earth's crust. people are constantly exposed to al which is found in most rocks, soils, waters, air and foods, due to a result of an increase in industrialization and improving technology practices. the study was designed to examine the possible effects of aluminum exposure in different durations on trace elements in rat tissues. twenty-four healthy male wistar rats weighed 180-200 g were randomly divided into three groups: control group (gc) received only drinking water, short-term group (gs) and long-term group (gl). the study groups were orally exposed to 40 mg/kg body weight alcl 3 in drinking water for 8 and 16 weeks, respectively. at the end of the treatment period, rats were sacrificed and the kidney, liver, brain and cerebellum tissues were removed to analyse the levels of al, ar, b, ni, si, cr, cu, fe, mg, mn, se, cu and zn by icp-oes. the statistically significant increase were determined in cerebellum al, cu, as, b and cr levels in gl according to the gc. while as levels were statistically increased, ni levels were decreased in gl in the kidney and liver. while cu, mg and cr levels were higher, se and b levels were lower in the gs than gc in the brain. there were no significant difference in si and mn levels. as a result of our study, it may be concluded that al accumulation may lead to changes in tissue trace element levels. tacal, o., 266 tacal, ö., 321 take, g., 412 takic, m.m., 417 taldykbayev, z., 416 talim, b., 292 tamashevski, a., 382 tamer, f., 144 tamer, l., 245, 279, 395 taneva, s., 209 taniyan, g., 275, 306 tanrisev, m., 288 tanriverdi, e.c., 245 tanriverdi, k., 279 tarhan, m., 161 tarhan, t., 315 tartar, s., 387 tas, a., 204, 293 taskin, a., 406 taskin, t., 339 taskiran, e., 271 taskiran, b., 328 taskoparan, b., 221 taspinar, r., 301, 302 tastan, ö., 271 tatli, ö., 340 tauraite, d., 206 tay, t., 344 tayman, c., 354 taysi, s., 389, 392 teker, h.t., 270 tekes, s., 361 tekin neijman, s., 410 tekin, m.h., 188, 297 tekin, g., 191, 256 tekin, m., 196 tekin, n., 155 tekin, g., 256 telci, d., 187, 307 telefoncu, a., 151 temel, h.e., 158, 159, 160 temelie, m., 267 temizgül, r., 347 temlyakova, e., 168 teplova, v., 372 terashima, r., 284 tercan avci, s., 197 terekhov, s., 381 tereshenko, o., 304 terzi gulel, g., 149 terzi, e., 300, 302 terzi, e., 301 terzioglu, g., 296 terzioglu, o., 272 testoni, g., 348 tetik vardarli, a., 282 tevdoradze, t., 148 tevzadze, l., 148 tezcan, ö., 308 tezcanli kaymaz, b., 282 thielens, n., 228 thomaidou, d., 265 thomas, a., 228 thornton, j.d., 216 ticea, a.c., 168 tikhonova, a., 287 tileva, m., 209, 210 timofeev, v., 198, 202 timofeev, v., 202 timofeeva, e., 383 tipirdamaz, r., 401 tiryaki, m., 300, 302 tok, m., 140 tokay, e., 365 toker, a., 360, 402 tokgun, o., 284 toksoy öner, e., 138 tokyol, ç., 390 toman, r., 317 tomasi, a., 410 tombul, n., 333, 337 tooke, c., 231 topaloglu, h., 292 topbas, m., 200 topcu, b., 247 topcu, c., 292 topcu, g., 144, 358, 377 topçu, v., 393 topcu, c., 417 topçuoglu, c., 193, 416 topel, h., 197, 264 toprak, b., 223 toprak, m.s., 351 torac, e., 384 tosner, z., 383 tosun, m., 308, 354 toy, h., 143 toymentseva, a., 239 tozkoparan, b., 246 trabulus, d.c., 256 trantirek, l., 229 trantirkova, s., 229 trchounian, a., 165, 324 trizna, e., 200, 289 troshagina, d., 371 tro t, m., 269 truncaite, l., 149, 363 tsakalidou, e., 170 tsarkov, d., 233 tsarkova, m., 233, 235 tsigara, e.g., 206 tsigara, m.g., 206 tsverava, l., 266 tsvetkova, e., 253 tsyba, l., 362 tsyganov, d., 158 tsymbal, d., 165 tüfekçi, a.r., 241, 358 tufekci, a.r., 296 tufenkci, h., 139 tuglu, m.i., 300, 309 tuglu, i., 200 tuli, a., 142, 182, 237, 403 tuli, a., 352 tulubas, f., 247 tunali, g., 226 tunca gedik, s., 143, 408 tunca gedik, s., 221 tunçdemir, m., 396 tuncel, h., 378 tuncel, h., 273 tunçer, s., 222 tuncer, e., 161, 261 tuncer, z.s., 395 tuncer, b., 133 tuncer, e., 273 tuncez akyurek, f., 185 tunçez akyürek, f., 359 tuner, h., 333 tüney kizilkaya, i., 325 tural, b., 315 tural, s., 315 turan, i., 330 turan, m., 161, 261 turan, v., 173 turan, y., 333 turan, c., 411 turano, p., 217, 290 türel, s., 329, 341 turhan, t., 193, 242 turhan, y., 316, 317 turk, s., 373 turkan, a., 219 türkcan kayhan, c., 135 turkekul, k., 299 türkel, s., 176 turkeri, o.n., 245 turkeri, n., 141 turkkan, b., 257 turkmen, s., 401 türkmen, n., 180 turkon, h., 223 tutar, y., 161 tüten, a., 351 tutkun, e., 196, 275 tüylü küçükkilinç, t., 285 tuz, m.u., 321 twardovska, m., 335 tyapkina, o., 371 tzartos, s., 268 photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes evaluation of the sunscreen lichen substances usnic acid and atranorin pleiotropic anticancer activity of selected nutraceuticals against mcf-7 bucharest, 3 national institute for marine research and development "grigore-antipa uk in some adult and elderly populations the acute and/or persistent infection with the common intracellular respiratory pathogen chlamydia pneumoniae (chl) may be associated with increased risk of developing obesity or cardiovascular disorders. thus, 19 microelements modifying oxidative stress status were determined by icp-ms/ms in the hno 3 diluted serum samples collected from chl-positive adult females (n = 39) living in urbanized area in poland. chl infection was confirmed by igg+ antibody elisa and real-time pcr assay. all females were classified under their body-mass index values to the normal-weight (nw), over-weight (ow) and obese group (ob) although there are many drugs currently used in the treatment of peptic ulcer, such a drug providing radical treatment without side effects is not available. since oxidative stress is involved in peptic ulceration, this study was designed to investigate antiulcerogenic and antioxidant effects of hippophae rhamnoides l. ether extract on indomethacine-induced stomach ulcer in rats. materials and methods: thirty-five sprague dawley male rats (weights ranging 180-220 g) were randomly divided into 7 groups, as each composed of 5 rats. after hippophae rhamnoides l. leaf ether extracts of 100 mg/kg, 250 mg/kg and 500 mg/kg doses and 20 mg/kg doses of famotidin orally administered, 25 mg/kg doses of indomethacine were orally applied to rats in order to make ulcer. on the sixth hour of indomethacin administration all rats were sacrificed using thiopental (50 mg/kg). the stomachs were removed, and ulcer areas were evaluated macroscopically. superoxide dismutase activity (sod), glutathione (gsh) and malondialdehyde (mda) levels in stomach tissues of rats were determined by elisa method with respective kits conclusions: we can conclude that the ether extract of hippophae rhamnoides l. leaves reduces free radical formation and has antiulcerogenic effects on stomach tissue control group; 2 hours torsion/4 hours detorsion group (t/d); all other groups were saturated for four days egcg, cape and egcg+cape (10lml/kg). sections were taken from bouine's-fixed and paraffin-embedded testicular tissue blocks and stained with h&e. immunohistochemistry was applied for the detection of pi3k, akt and mtorc. intensities were evaluated as mild (1), moderate (2) or strong (3). serum 8ohdg, plasma mda levels were analyzed using elisa method. results were analyzed by anova statistical test. testis samples in control group exhibited normal histological morphology. disorganization and separation of seminiferous tubule cells and accompanying interstitial edema and vessel dilation were while mda level decreased significantly in cape+egcg group, 8ohdg level showed significant increase in cape group. in conclusion, cape and egcg exerted protective effects on tt. effects may be achieved through pi3k/ akt/mtorc pathway involved in cell proliferation, angiogenesis, apoptosis. prophylactic use of egcg prior to tt surgery improved testicular morphology, therefore could prevent destructive effects of tt lmol/l), c18 (0.58 ae 0.278 lmol/l)), respectively. conclusions: this study is important for konya region, mitochondrial fatty acid b-oxidation disorders studies subject areas. this study is the first study to assess acylcarnitine levels of patients living in our region. we believe that our results will be useful for future studies. key words: acylcarnitine, mass spectrometry, dried blood spot p-09.04.4-137 binding of fas and cu(ii) ions to hsa changes its cys34 thiol group antioxidant capacity and carbonylation pattern with methylglyoxal binding of cu(ii) ion (0.1 mol/mol hsa) led to increase of k' value if fish oil extract was present, but for other fas k' value decreased. the content of free hsacys34-sh decreased for 10% after cu(ii) ion binding, and during 24 hours incubation at 37°c, it was further decreased for 10% (stearic acid, mixfas) and 20% (myristic, fish oil extract, oleic acid). carbonylation of fa-hsa-cu(ii) complexes with mg (20 mol/ mol hsa), lead to decrease in cys34-sh content depending on fa present: 30%-35% for myristic and stearic acid, 40% for oleic acid and mixfas and 40% for fish oil extract. carbonylation of fa-hsa-cu complexes could contribute to further enhancement of oxidative and carbonyl stress in diabetes as well as other diseases 151 pirto ek p-05.03.3-007 anti-proliferative and inducing apoptosis of the hydro alcholic achelia. wilhelmsii extract on human breast adenocarcinoma cell lines mcf-7 and mda-mb-468 background: vitamin d deficiency is associated with several conditions and/or diseases like inflammation, atherosclerosis, cardiovascular disease and mortality. several studies showed that lower vitamin d levels were associated with high serum levels of inflammatory biomarkers. ykl-40 is a glycoprotein, secreted by macrophages, neutrophils and different cell types. it is also associated with inflammation and pathological tissue remodeling. in this study, we aimed to evaluate relationship between the vitamin d deficiency and ykl-40 levels. methods: our study group includes 45 subjects with vitamin d deficiency (group 1) and 40 age and sex-matched healthy subjects with normal serum levels of vitamin d (group 2). plasma 25 (oh) vitamin d levels were measured with liquid chromatography-tandem mass spectrometry (lc-ms/ms) method. plasma ykl-40 analysis was performed by elisa. serum hs-crp levels were measured with nephelometric method. results: plasma vitamin d levels below 20 ng/ml were accepted as vitamin d deficiency. although we could not find any significant differences by means of serum hs-crp levels between groups (p > 0.05), plasma ykl-40 levels were significantly higher in group 1 than group2 (p < 0.05). conclusions: in literature, vitamin d deficiency is associated with inflammation. in our study, we found similar hs-crp levels between groups and higher ykl-40 levels in group 1. vitamin d deficiency may be related to increased ykl-40 levels in terms of causing chronic inflammation.keywords: vitamin d deficiency, ykl-40, inflammation. evaluation and comparison of tnf-family ligands and receptors genes in mice and humans by bioinformatics techniques stance called plaque builds up inside the coronary arteries. apelin is a novel endogenous peptide with inotropic and vasodilatory properties and is the ligand for the angiotensin receptor-like 1 (apj) receptor. we aimed to determine genotype and allele frequencies of apj receptor a445c gene polymorphism in turkish patients with cad and healthy controls by rflp-pcr. this study was performed on 159 unrelated cad patients and 62 healthy controls. we obtained aa, ac and cc genotype frequencies in cad patients as 41.5%, 49.1% and 9.4%, respectively. in the control group, frequencies of genotypes were found as 35.5% for aa, 48.4% for ac and 16.1% for cc. we did not observe difference in apj receptor a445c polymorphism between cad patients and healthy controls (v 2 = 2.178; df = 2; p = 0.336). the a allele was encountered in 66% (210) of the cad and 59.7% (74) of the controls. the c allele was seen in 34% (108) of the cad and 40.3% (50) of the controls. allele frequencies were not significantly different between groups (v 2 = 1.57; df = 1; p = 0.225). the frequencies of apj receptor a445c genotype were not significantly different between control and patients. individuals with cc genotypes had significantly higher weight, systolic and diastolic blood pressure levels and systolic blood pressure than other genotypes, p ≤ 0.05. in addition, hdl-c level was found decreased, but this reduction was not statistically significant. contrarily, the low levels of weight, sbp, dbp and tc were statistically significant in the subjects with aa genotype in cad. in conclusion, cc genotype carriers may have more risk than other genotypes in the development of hypertension in cad. we suggest that this polymorphism may not be a marker of cad, but it may cause useful in function of the apelin/apj system and may be a genetic predisposing factor for diagnostic processes and can be helpfull in finding new treatment strategies. p-08.01.4-032 comparative genomics/proteomics analyses of single amino acid repeat containing proteins across different vertebrate taxa a. g. keskus, o. konu department of molecular biology and genetics, bilkent university, ankara, turkeyconsecutive runs of single amino acids lead to overrepresentation of certain physicochemical properties in protein sequences. researchers also demonstrated a link between single amino acid repeat (saar) containing proteins and neurodegenerative diseases as well as biological functions. moreover, saar frequencies were shown to vary across species based on selected orthologous proteomes and/or proteins. hence, analysis of whole proteomes across multiple vertebrate taxa may provide additional species-and sequence-specific trends for saars. in addition, there is a need for testing the observed saar occurrencesthe aim of this study is to evaluate the effect of quercetin (q) on liver injury secondary to cerulein induced-acute pancreatitis (ap). for this reason, rats were randomly divided into four groups (8 rats for each group) control group received physiological saline, four time and dimethyl sulfoxide, two time, at 1 hours intervals, intraperitoneally (i.p.). cerulein group received cerulein (50 lg/ kg-rat weight, in physiological saline), four times, and dimethyl sulfoxide (1%), two times, at 1 hour intervals, i.p. quercetin pretreatment (q+cer) group received quercetin (100 mg/kg-rat weight, in dimethyl sulfoxide) one time, one hour before cerulein treatment and physiological saline, one time, six hour after cerulein treatment. quercetin post-treatment (cer+q) group received dimethyl sulfoxide, one time, one hour before cerulein treatment and quercetin, one time, six hour after cerulein treatment. cerulein treatment increased significantly vascular congestion in hepatic cells. quercetin treatment also decreased significantly vascular congestion. the liver mda and carbonyl levels in cerulein group were significantly higher than the control group (p < 0.01, p < 0.001, respectively). the mda and carbonyl levels in q+cer group decreased significantly compared to the cer group (p < 0.001). the mda, carbonyl, mpo levels in cer+q group were significantly lower than the cer group (p < 0.001). the gssg/gsh ratio of q+cer and cer+q groups were significantly lower than the cer group (p < 0.05, p < 0.001, respectively). the sod activity in cer group was significantly lower than the control group, but the sod activity in q+cer and cer+q groups was significantly higher than the cer group (p < 0.05).this study shows that quercetin treatment was reduced the severity of liver injury secondary to cerulein induced-ap as reflected by changes in the parameters of hepatic oxidant and antioxidant. p-09.04.4-029 identification of water extract of propolis components by using different columns in gas chromatography-mass spectrometry propolis is a natural material obtained by honey bees from various plants. protective effect of propolis against damages of free radicals is due to different compounds within propolis. the aim of this study is to identify qualitatively and quantitatively the chemical composition of water extract of propolis (wep) provided by erzurum region using rtx-1 and rtx-5 ms column of gas chromatography-mass spectrometry (gc-ms) and to compare with two columns.in this study, wep of 25 mg/ml was prepared, cleared by membrane filter of 0.45 lm and freezed at à80°c. then, it was lyophilized up to dry form and derivatized to apply for gaseous form. 7 mg of dry extract was reacted with 350 ll pyridin + 700 ll bis-trimethylsilyl trifluoroacetamide (bstfa) mixture including 1% trimethylchlorosilane (tmcs) and incubated for 30 minutes at 100°c. all analyses were performed with shimadzu gcms-qp2010 ultra by using a flame ionisation detector (fid). rtx-1 and rtx-5 ms capillary columns and helium for carrier gas at a flow time of 1 ml/minute were used. injection was applied on split mode at 250°c. derivatized propolis sample was injected as 2 ll, initial column temperature was adjusted as 60°c, then increased to 240°c with increments of 3°c. total analysis time was determined as 62 minutes. relative percent amount of separated compounds was calculated from total ion chromatogram with computerised integrator. all components were defined by using nist and wiley libraries.peaks obtained from rtx-1 column were much more than those of rtx-5 ms. on the other hand, analyses performing with both two columns have similar carbohydrate, aromatic acid and other acid contents.consequently chemical constituents of wep were determined qualitatively and quantitatively with gc-ms. it was concluded that rtx-1 column among both columns differentiating for polarities may produce more compounds in the propolis analysis. introduction: aquatic environment can be mostly contaminated by mixtures of metals. biochemical parameters have gain importance to characterize the effects of metals on aquatic organisms. glutathione s-transferase (gst) and its substrate glutathione (gsh) are important parameters of antioxidant defense system of fish metabolism due to their vital role in xenobiotic conjugation. objective: the goal of the current study to evaluate the changes in gst and gsh levels in response to cd, zn and cd+zn effects after 14 and 28 exposure days. materials and methods: fish were obtained from cukurova university fish culturing pools (turkey). fish were exposed to 1.0 lg/ml of cd (cdcl 2 .h 2 o) and zn (zncl 2 ), and their mixture, for 0, 14 and 28 days. at the end of the exposure period, liver tissues were dissected and homogenized in a phosphate buffer (ph 7.4). homogenates were centrifuged at 10,000 g (30 min, +4°c). supernatants were stored at à80°c until the analysis. one-way anova was used to compare data (mean ae se) followed by duncan's test (p < 0.05). results: gst and gsh changes were recorded as decreases after all metal exposures at day 14. although 28 day exposure was found as less effective, combined effects caused significant decreases in gst and gsh levels. also longer exposure durations were appeared to be more effective in that situation. conclusions: significant decreases in gst and gsh levels could be occurred due to increased reactive oxygen species caused by metals particularly their combined effects. metal type, their single and combined effects and also exposure duration should be also taken into account when considering the antioxidant system response. gst and gsh might be considered as sensitive biomarker in toxicity assessment of metal mixtures.financial support: this study was supported by a grant from c ß ukurova university (turkey).background/aims: the activation of lectin-like oxidized low density lipoprotein receptor-1 (lox-1) on endothelial cells leads to intracellular oxidative stress and inflammation and a feed-forward cycle of injury in diabetes, since both oxidized low density lipoprotein (oxldls) and glucose increase lox-1 expression. quercetin (qr) is part of a subclass of flavonoids called flavonols. polyphenolic compounds affect the development of atherosclerosis not only through antioxidant properties but also by modulation of serum lipids, thereby influencing the immune and inflammatory processes associated with the development of atherogenic diseases. we investigated the effects of dietary qr on endothelial dysfunction mediated by oxidized low density lipoprotein (oxldl)/lectin-like oxidized low density lipoprotein receptor-1 (lox-1) in animal model of type 2 diabetes mellitus. methods: we compared 4 groups of male adult wistar albino rats: a control group, an untreated diabetic group, diabetic groups treated with qr, and qr group. diabetes was induced by a single injection of stz (50 mg/kg). animals were kept in standard condition. in 30 day after inducing diabetic, serum was collected for biochemical parameters. glucose, lipid profiles, microalbuminuria, oxldl and lox-1 levels were determined. results: serum triglyceride, ldl, vldl levels in diabetic control group (without treatment) was significantly higher than control group (normoglycemic untreated group). supplementation with quercetin decreased serum total cholesterol and increased hdl-cholesterol compared with the control group. serum oxldl and lox-1 levels in diabetic control group (without treatment) were significantly higher than control group (normoglycemic untreated group). conclusions: consumption of quercetin reduced oxldl and lox-1 levels. thus, quercetin could be effective in improving hyperglycemia, dyslipidemia, and endothelial damage in type 2 diabetes. p-09.04.4-055 investigation of some oxidative stress parameters in bdnf heterozygous mice liver tissue a. bodur 1 , i. ince 1 , i. abidin 2 , a. alver 1 objectives: the aim of this study was to evaluate the possible protective effects of nigella sativa (ns) against cholestatic oxidative stress and liver damage in the common bile duct ligated rats. methods: a total of 24 male wistar albino rats were divided into three groups:sham control, bile duct ligation (bdl) and bdl+received ns; each group contain 8 animals. the rats in ns treated groups were given ns (0.2 ml/kg) once a day orally for 4 weeks starting 3 days prior to bdl operation. results: the changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells, and neutrophil infiltration into the widened portal areas were observed in bdl group. treatment of bdl with ns attenuated alterations in liver histology. the alpha smooth muscle actin (a-sma), transforming growth factor beta (tgf-b1) and the activity of tunel in the bdl were observed to be reduced with the ns treatment. bdl significantly increased the tissue hydroxyproline (hp) content, malondialdehyde (mda) levels, and decreased the antioxidant enzyme (superoxide dismutase (sod) and glutathione peroxidase (gpx)) activities. ns treatment significantly decreased the elevated tissue hp content and mda levels and prevented the inhibition of sod and gpx enzymes in the tissues. conclusion: the data indicate that ns attenuates bdl-induced cholestatic liver injury, bile duct proliferation, fibrosis, apoptosis and oxidative stress. the hepatoprotective effect of ns is associated with antioxidative potential. organophosphorous compounds (ops) are used widely as pesticides for agricultural purposes, as oil additives or as flame retardants. however, due to their widespread use, there are major introduction: in this study, the antiulcerogenic effect of a water extract (cawe) obtained from a spices sample, cinnamomum aromaticum, was investigated using indomethacin-induced ulcer models in rats. materials and methods: experimental groups consisted of six rats. antiulcerogenic activities of 50, 100, 200 and 400 mg/kg body wt. doses of the cawe were determined by comparing the negative (treated only with indomethacin) and positive (famotidine) control groups. results and discussion: although all doses of the cawe showed significant antiulcerogenic activity as compared to negative control groups, the highest activity was observed with 400 mg/kg body wt. doses (45%). the cawe showed similarly antioxidant activity when compared with trolox and ascorbic acids used as positive antioxidants. in addition, the activities of catalase (cat) and myeloperoxidase (mpo) enzymes were determined in the stomach tissues of rats and compared with those of the negative and positive control groups to expose the effects of these enzymes on antiulcerogenic activity. the enzymatic activities of cat and mpo and lipid peroxidation (lpo) level in indomethacin-administrated tissues were increased significantly by indomethacin in comparison to control groups. these enzymes and lpo level were decreased, however, by the cawe. in contrast to lpo level, cat and mpo activities, glutathione (gsh) level was decreased by indomethacin and increased by all doses of cawe and famotidine. the present results indicate that the cawe has a protective effect in indomethacin-induced ulcers, which can be attributed to its antioxidant potential. introduction: thiol groups (-sh) are important anti-oxidants and essential molecules protecting organism against the harmful effects of oxidative stress. the aim of our study is to evalute thiol-disulphide homeostasis with a novel and automated method in patients with prostate cancer (pc) before and after radical prostatectomy (rp). material and methods: 18 patients with prostate cancer and 17 healty control subjects were enrolled into the study. plasma samples were collected from patients before rp and 6 months after the rp operation. thiol-disulphide homeostasis was determined with a recently developed novel method. prostate specific antigen, albumin, total thiol, native thiol, disulphide and total antioxidant status (tas) were evaulated and compared between the groups. results: native thiol levels were 419.8 ae 54.87 lmol/l in the control group, 350.7 ae 46.35 lmol/l in the patients before rp, and 364.3 ae 46.88 lmol/l in patients after rp. native thiol, total thiol and tas levels were significantly higher in the control group than the patients before rp (p values < 0.001). native thiol, total thiol and tas levels were higher 6 months after rp compared to before rp in patients, but these changes were not significant statictically (p values 0.3, 0.3 and 0.09 respectively). discussion and conclusion: our study demonstrated that antioxidant defense mechanism was weakened as indicated by the decreased thiol levels in the patients with pc. increased oxidative stress in prostate cancer patients may cause metabolic disturbance and have a role in the pathogenesis of prostate cancer. p-09.04.4-108 is there any relation between 50 g oral glucose challenge test and serum total oxidant-antioxidant status in pregnant woman? the purpose of this study was to test the hypothesis that any degree of antepartum screening for gestational diabetes mellitus with oral 50 g glucose challenge test (gct) should be associated with oxidant-antioxidant status.in this prospectif study, oral glucose challenge test was applied to 25 pregnant women aged 25-40 years and at 24-28 weeks of gestation. plasma glucose concentrations were measured initial, 1 hour and in addition to test 2 hours after ingestion of 50 g glucose. at the same time serum insulin, cortisol, total antioxidant status (tas), total oxidant status (tos) levels were measured and the oxidative stress index (osi) was calculated.ten pregnant women (forty percent) had a positive glucose challenge test (gct). a positive moderate relation with initial and 1 hour serum total antioxidant status (tas) levels (r = 0.68) and the oxidative stress index (osi) (r = 0.67) was found. there was a positive weak correlation with initial and 2 hours total oxidant status (tos) levels (r = 0.22) but statistically significance difference was not found (p > 0.05).in this study after ingestion of 50 g glucose serum total antioxidant status (tas), serum oxidant status levels (tos) and serum oxidative stress index (osi) levels were higher than the initial levels.the results of this study suggest that antepartum screening for gestational diabetes mellitus with 50 g oral glucose challenge test (gct) weakly associated with oxidant-antioxidant status and to confirm this results the longer follow-up studies with more participants are necessary.wheat (triticum ssp.), cultivated for centuries in the middle-east, central asia, europe, and north-africa, is one leading staple crops around the world, and its marginally grown ancestor einkorn (triticum monococcum ssp. monococcum), possesses rich gene resources for wheat improvement and have bioactive compounds reducing and preventing chronic diseases such as diabetes, cancer, alzheimer, and cardio vascular diseases, beside their nutritional properties. however, as more attention has been given to wheat cultivars with strong gluten, protein content, starch composition, and resistance to biotic and abiotic stresses in bread wheat and yellow-colored pasta product in durum wheat health compounds such as fibers, phytochemicals, and bioactives have been underestimated so far. the aim of this study was, then, to examine the total phenolics and flavonoids, quantify their phenolic acids, a-tocopherol by high performance liquid chromatography (hplc), and their 2,2-diphenyl-1-picrylhydrazyl (dpph) scavenging activity of bread (triticum aestivum l.), durum (triticum turgidum ssp. durum desf.) wheat cultivars and einkorn (triticum monococcum ssp. monococcum) wheat populations collected from different provinces (bolu and kastamonu) of turkey. ferulic acid (148.67-764.04-lg/g), p-coumaric (5.06-54.09-lg/g), and total phenolic content (ranged 2.06-8.11-lmol gae/g) of einkorn populations were significantly higher than bread and durum wheat cultivars. results suggested the possibility of production of einkorn wheat populations, and hopefully cultivars rich in particular health beneficial component(s) may provide benefit to the consumers. in addition, higher phenolic content of einkorn may offer novel wheat genetic resources for the improvement of new wheat cultivars and the development of wheat-based functional foods. oxidative damage due to ischemia and acute kidney injury (aki) after coronary artery bypass graft (cabg) surgery are the leading complication during this process. in the kidney, ischemia/ reperfusion injury contributes to aki that is a clinical syndrome with rapid kidney dysfunction and high mortality rates. some animal and clinical studies have demonstrated an increase in serum and urinary neutrophil gelatinase-associated lipocalin (ngal) expression after renal ischemic injury.in this study, our aim was to investigate the relationship betwwen ngal and oxidative stress parameters due to ischemia caused by total perfusion time (tpt) in patients who have undergone on-pump cabg. materials and methods: the study was conducted in 30 patients who received on-pump cabg at university of istanbul, cerrahpasa medical faculty, department of cardiovascular surgery. blood samples were collected prior to surgery and after 2 hours following the termination of cardiac pulmonary bypass (cpb) . following centrifugation, serum samples were separeted and stored at -80 c until analysis. serum ngal, ima (ischemia modified alb€ umin), pco (protein carbonyl), nt (nitrotyrosine), lpd (lipid peroxide) levels were determined by elisa procedure. results and discussion: serum ngal, pco, nt levels in after 2 hours following cpb were significantly higher than the before surgery (p < 0.001, p < 0.001, p serum ngal levels in after 2 hours following cpb was found to have positive correlation with ima, pco, nt, and lpo levels (r = 0.59, p < 0.01; r = 0.77, p < 0.001; r = 0.68, p < 0.001; r = 0.77, p < 0.001 respectively). ngal levels were positively correlated with total perfusion time after cbp (r = 0.37, p < 0.05).the results of our study show that, increased ngal levels 2 hours after cpb were positively correlated with oxidative stress parameters and total perfusion time. investigation of the effects of thymoquinone against indomethacine induced gastric damage in rats introduction: incidence and high cost of acute stomach mucosa damages make this issue a very interesting issue for study. for this reason, it is aimed to investigate the effects of different dosage of thymoquinone (tq) against indomethacine induced gastric damage in rats. material and method: in our study, six groups of 36 wistrar male rats were used. groups are named as healthy, ind control, famotidine (40 mg/kg) and three different doses of tq (0.5, 1 and 2 mg/kg). while any treatment or drug administration will done on healthy group, model was generated to other groups by giving fam or tq with tap water via oral gavage. 5 min later, 25 mg/kg ind administrated to each rat. animals were sacrified about 6 hour later and stomach samples of each groups were collected for macroscopic study and gsh levels measurements. results: lower doses of tq is more effective and all tq groups exhibit reduced ulcer region with respect to the ind control group. gsh level of ind control group is lower than healthy group. the gsh level of tq, especially in lower doses, and fam groups statistically exhibit an increase in gsh level. conclusion: it is observed that ind induced gastric damage cause ulcer and increase in free radical. it is determined that lower doses of tq (0.5, 1 and 2 mg/kg) is also exhibit a protective effect on ind induced model. it is tought that quinone in tq structure have a strong redox feature and this feature clean up the free radicals caused by ind, it reduces the oxidative stress and protect the stomach from ulcer. anzer honey is the most famous honey in turkey with many endemic species flowers. the anzer plateau is located rize province of eastern black sea region. in this study, antioxidant and anti-hyaluronidase and anti-urease activities were investigated of the plateau bee pollen. the antioxidant capacity was determined by total phenolic content (tpc), total flavonoids contents (tfc), ferric reducing antioxidant power (frap) and 2,2diphenyl-1-picrylhydrazyl (dpph) radical scavenging activity. atherosclerosis is the leading cause of mortality worldwide, and as a chronic inflammatory disease, caused by a complex interplay between inflammatory and oxidative events.quercetin, a plant derived flavonoid and a well-known antioxidant, has shown great promises with regards to its protective effects against oxidative stress.however due to its physicochemical properties, the optimum pharmacokinetic behavior is a challenging issue.herein, we aimed to fabricate quercetin loaded solid lipid nanoparticle (quer-sln) to improve the bioavailability and therapeutics efficiency. furthermore the in-vitro capacity of quer-sln for ameliorating tnf-a induced oxidative stress in human endothelial vein cell (huvec) was evaluated. quer-slns were prepared by simple hot homogenizing method and characterized by means of drug loading (dl), encapsulation efficiency (ee), cytotoxicity, size, zeta potential and morphology. antioxidant activity of plain quercetin and quer-slns were then investigated using intracellular reactive oxygen species (ros) detection method (dcfh-da assay) by facs flowcytometryin conclusion, the results here showed superior control of oxidative stress by quercetin nanosystem as compared to plain quercetin. precirol based slns as a biocompatible/biodegradable lipid, may provide a novel drug delivery system for quercetin with improved beneficiary impact in atherosclerosis. objective: zinc is known as an antioxidant essential trace element. we aimed to evaluate the dose-dependent effects of zinc on the oxidant-antioxidant system in liver, kidney and brain tissues of rats and the histological alterations in the absence of oxidative stress (os). material and methods: thirty-nine female weighing about 150 gr wistar albino rats were divided into four experimental groups as ad libitum (al) diet (control), al diet + 3 mg/kg zn sulfate (low dose; group 1), al diet + 12 mg/kg zn sulfate (middle dose; group 2) and al diet + 25 mg/kg zn sulfate (high dose; group 3). zn sulfate solutions were administered 0.2 ml/day orally for 13 days and in day 14 rats were sacrificed and tissues were excised for detecting malondialdehyde (mda), advanced oxidation protein products (aopp), superoxide dismutase (sod), glutathione peroxidase (gsh-px), glutathione reductase (gr), and glutathione-s-transferase (gst) activities. histological evaluation was also performed to confirm the effects of zinc. results: in liver tissues aopp levels decreased in all groups receiving zinc as compared to the control group. liver mda levels were increased in group 1 and 3; sod and gsh-px levels were both increased while gst levels were decreased in all groups compared to control. gr levels were increased only in group 2. in kidney; aopp level was decreased only in group 1 and sod level was only decreased in group 2 as compared to control while gr levels were increased in all doses of zinc. in brain; aopp, gsh-px and gr levels were decreased in all groups receiving zinc as compared to control group. sod activity in brain tissues was increased by the administration of middle dose of zinc (group 2). gst level was decreased in only group 1 conclusions: the biochemical and histological findings of this study suggest that zinc has various effects on liver, kidney and brain tissues in the absence of os.key words: zinc, liver, kidney, brain introduction: this study aimed to investigate the effect of diabetes mellitus (dm) on oxidative stress and antioxidant capacity in humour aqueous (ha) and venous serum using total antioxidant capacity (tac) and total oxidative stress (tos) levels in serum and ha in cataract patients. materials and methods: in this study patients were divided into two groups. group 1 was composed of patients with type 2 dm and cataract and group 2 was composed of patients with cataracts who are not accompanied by dm and cataract patients who are not accompanied by systemic diseases. each group consisted of 20 patients, totally 40 patients were included in the study. the ha which was collected from the eyes at the beginning of the cataract surgery and venous blood serum collected from the same patients were analyzed. in both groups, ha and serum tac and tos levels were measured with elisa. results: : serum tac levels in the dm group were significantly lower than in the control group (p < 0.05). tos serum levels in dm group was statistically higher than the control group (p < 0.05). differences between tac and tos levels were not statistically significant when compared the two groups' ha results (p > 0.05). group 1, divided into two subgroups according to their hba1c levels, there was no statistically significant difference between the subgroups when hba1c levels were compared with the relationship between serum and ha's tac and tos levels (p > 0.05). there was not an association between the gender, age and the levels of tac-tos in both groups (p > 0.05). discussion and conclusion: presences of dm is the only risk factor for increase of oxidative stress and decrease of anti-oxidant capacity in patients without a systemic complication of dm and diabetic retinopathy. in our study, diabetic patients without retinopathy showed similar ha tos and tac levels to healthy individuals, this finding indicates that blood-aqueous barrier is protected in these patients. the effect of ferulic acid against testicular ischemia/reperfusion injury in rats u. sac ßik 1 , g. erbil 1 , z. c ß avdar 2 , c. ural 2 1 department of histology and embriyology, school of medicine, dokuz eyl€ ul university, izmir, 2 department of molecular medicine, health science of institute, dokuz eyl€ ul university, izmir, turkeytestis torsion is one of the urologic emergencies occurring frequently in neonatal and adolescent period. testis is sensitive to ischemia/reperfusion (i/r) injury and, therefore, ischemia and consecutive reperfusion cause an enhanced formation of reactive oxygen species (ros) that result in testicular cell damage and apoptosis. ferulic acid, known as an antioxsidant, is a phenolic acid found in seeds and leaves of the plants. we aimed to investigate potential protective effect of ferulic acid against testis i/r injury.thirty five wistar rats were randomly divided into 5 groups; control, ethyl alcohol, ischemia, i/r, i/r-ferulic acid groups. animals were exposed to 2 hours of ischemia followed by 2 hours of reperfusion. ferulic acid was administered (100 mg/ kg) before reperfusion intravenously. testicular cell damage was examined by h-e staining and pas. tunnel, active caspase-3, inos and mpo were evaluated by immunostaining. malondialdehyde (mda), glutathione (gsh) levels, glutathione peroxidase (gpx) and superoxide dismutase (sod) activities were assessed by biochemical methods.histological evaluation showed that ferulic acid pretreatment reduced significantly testicular cell damage and decreased tun-nel, caspase 3 positive cells; inos and also mpo expression. in addition, ferulic acid administration decreased significantly the mda levels increased by i/r. morever, ferulic acid increased significantly the sod activity levels, which was decreased by i/r. there were no statistically significant differences in the levels of gsh and gpx activity in all groups.the present results suggest that ferulic acid is a potentially beneficial agent in protecting testicular i/r. background: in heart failure (hf), angiotensin antagonists (aa), beta-blockers (bb), spironolactone, diuretics and acetylsalicylic acid are often used. top 3 pharmaceutical groups reduce mortality. on the increased oxidative stress (os) in patients with hf, it is known to have beneficial effects of certain groups of drugs. however, the net effect of these drugs in os is unknown. the aim of this study was to investigate the effects of drugs used in hf on os. materials and methods: 133 patients were included in the study. all of the patients had systolic heart failure and all of them were under treatment. drugs used by the patients were recorded. the levels of total antioxidant status (tos), total oxidant status (tas), the enzymatic activity of ceruloplasmin, paraoxonase-1 and arylestherase were measured according to erel's method. serum total thiol levels were measured with sh modified hu method and the lipid hydroperoxide levels were measured with the ferrous ion oxidation xylenol orange assay. the percentage tos / tas was determined as osi. results: in patients treated with acetylsalicylic acid (asa), spironolactone, beta blocker and furosemide, there were increased tos, decreased tas and osi (p < 0.05). in patients treated with angiotensin blockers, increased tas and looh, and decreased sh were found (p < 0.05). in patients treated with nitrates and ccb, tos and osi were found decreased. correlation analysis showed that increased tas correlated with the use of angiotensin blockers, asa, furosemide and beta blocker positively; and with the tos and osi level correlated with the use of spironolactone, asa spironolactone and furosemide (p < 0.05). conclusion: current medical agents that are being used in hf are effective in reducing os in hf patients. one of the effective mechanisms to reduce the mortality of some of these drugs may decrease os.key words: heart failure, drug use, oxidative stress p-09.04.4-119 across adjacent ring formed titanium phthalocyanine-mediated photodynamic therapy alters and degrades filamentous actin cytoskeleton and internal membranes photodynamic therapy (pdt) is widely accepted as a promising and minimally invasive treatment strategy due to its applicability on a wide range of cancer diseases. this clinically approved treatment method relies on the dramatic production of singlet oxygen and reactive oxygen species (ros) in target tissue to evoke apoptotic cell death [1] . we, therefore, focused on the intracellular ros accumulation, internal membrane degradation, filamentous actin cytoskeleton alteration and nucleus morphology changes induced by pdt-mediated across adjacent ring formed titanium phthalocyanine which was previously synthesized bis(ethane-1,1p-phenol-2,2-p-phenoxy) phthalocyaninatotitanium (iv).characterization of the synthesized metallophthalocyanine was accomplished by using uv-vis, ir, 1 h-nmr and maldi-tofmass spectroscopies. the dark and pdt-mediated activities of bare and phosphonolipids (max. 5%) charged titanium phthalocyanine (0.625, 1.25, 2.5, 5 and 10 lm) were determined on a549 human lung carcinoma and hacat human keratinocyte cell lines by using intracellular ros assay, dioc(6), tritc-phalloidin and dapi staining protocols. waltmann pdt 1200 l was used as the non-toxic light source at 100 j/cm 2 fluence and 150 mw/ cm 2 fluence rate. the experiments showed that pdt-mediated titanium phthalocyanine leads to significant and concentration dependent reactive oxygen species accumulation. moreover, internal membrane degradation, apoptotic bodies on nucleus and filamentous actin cytoskeleton alteration were observed. consequently, the activity mechanism of pdt-mediated titanium phthalocyanine seems to be in a tight relationship with ros accumulation-mediated internal membrane degradation, filamentous actin cytoskeleton alteration and apoptotic pathways activation. introduction: retinal vein occlusion (rvo) is a common retinal vascular disorder that can affect visual acuity and cause blindness in elder population. sulphur containing aminoacids such as cysteine (cys), cysteinylglycine, glutathione, homocysteine and c-glutamylcysteine are reported to be associated with the pathogenesis of rvo. thiols are organosulfur compounds that are formed of a carbon-bonded sulfhydryl group. sulphur containing aminoacids slightly contribute the composition of plasma thiol pool. thiols can undergo oxidation reaction via oxidants and form disulphide bonds our purpose is to research the relationship between a novel oxidative stress marker serum dynamic thioldisulphide homeostasis and retinal vein occlusion. materials and methods: 22 rvo patients and 22 controls were included in the study. native thiol, total thiol, disulphide levels are measured in the serum samples of rvo and control group by using an automated method described by erel et al. also disulphide/native thiol and disulphide/total thiol ratios were calculated. results: there were no significant difference between the rvo and control group in native thiol, total thiol, disulphide disulphide/native thiol and disulphide/total thiol ratios.(p > 0.05 for all) conclusion: our study is the first report evaluating the dynamic thiol-disulphide homeostasis in rvo patients by a newly developed method by erel et al. further large sample sized studies investigating the levels of sulphur containing aminoacids may additionally be planned to verify this study. purpose: the purpose of this study was to evaluate markers of systemic oxidative stress and antioxidant capacity in subjects with severity of osas. methods: a total of 106 osa patients were included in the study (18 controls, 14 with mild, 14 with moderate, and 60 with severe osa). patients were grouped according to apnea-hypopnea index (ahi) as mild, moderate and severe osa. patients with ahi<5 served as control group. known risk factors for oxidative stress, such as age, sex, obesity, smoking, hypelipidemia, and hypertension, were investigated as possible confounding factors. plasma arylesterase, total oxidative stress (tos), total antioxidant capacity (tac), total thiol, catalase (cat) levels were measured for all patients. results: the mean age was 52.49 ae 12.9 years and 40.6% (43/ 106) of the study population was female. plasma arylesterase, tos, tac, total thiol, and cat plasma values were not different between mild, moderate, severe osa groups and controls (p > 0.05). catalase levels were significantly lower in women patients with severe osa compared to healthy women controls (p < 0.05). there was a negative correlation between ahi and serum total thiol levels (r = à0.289, p < 0.05) in severe osa groups. conclusionthe present prospective study provides evidence that osa might be associated with decreased antioxidant burden possibly via catalase way. results: the sera 8-ohdg, mda and il-6 were significantly higher in diabetic group than control group (p < 0.05). although there was a notable positive correlation between mda and 8-ohdg, there was no a relationship between 8-ohdg or mda with il-6. discussion: in agreement with previous studies our data illustrated that high levels of oxidative stress is associated with increased production of oxidized lipids and nucleobases in diabetic patients compared to control group. also enhanced proinflammatory cytokine, il-6, induced inflammmation in these patients.conclusion: oxidative stress and inflammation play pivotal roles in the development of diabetes and can cause major complications in dm. so we suggest that early detection of these measurable indicatores can help to diagnosis the severity or presence of some complication in diabet. the effect of quercetin on erythrocyte glucose-6-phosphate dehydrogenase enzyme activity in ethanol treated rats in this study, we aimed to evaluate the effects of ethanol on erythrocyte (g-6-p-d) enzyme activity and the effects of quercetin on erythrocyte g-6-p-d activity in the recovery of the effects of ethanol.rats were randomly divided into four groups. the control group (n = 6) received physiological saline. the quercetin group (n = 7) received quercetin (120 mg/kg/ day) via i.g. route the alcohol group (n = 7) received ethanol (80% v/v, 1 ml/day) via i.g. route. the alcohol + quercetin group (n = 5) received 1 ml of ethanol (80% v/v) 2 hours after quercetin treatment (120 mg/ kg/day). experimental procedures were peformed for 30 days. erythrocyte g-6-p-d activity was found to be higher in the quercetin group than those in the alcohol group (p < 0.001). in the alcohol group, the erythrocyte g-6-p-d activity was found to be significantly decreased than those in the control group (p < 0.001). statistically significant differences were observed in erythrocyte g-6-p-d activity between the alcohol group and the alcohol + quercetin group (p < 0.001).as a conclusion, our results demonstrate that ethanol decreased erythrocyte g6pd activity and quercetin was found to be beneficial in the prevention of toxic effect raised by ethanol.key words: erythrocyte, ethanol, g6pd, oxidative stress, quercetin p-09.04.4-126 effects of the sulphasalazine to the cerebral hypoxia reperfusion injury in rat background: cerebral ischemia/ reperfusion (i/r) injury is still a difficult process to treat and rehabilitate today. this study was designed to investigate beneficial effects of sulfasalazine in cerebral i/r injury in rat. methods: except control group (n = 5), 20 wistar albino rats were divided into four groups for acute and chronic stage investigation of i/r injury, and temporary aneurysm clips were attempted to both internal carotid arteries for duration of 30 minutes. four hours later, except control, sham-a, sham-c groups, 40 mg/kg once a day sulfasalazine was administered to animals, orally. animals were sacrified and then necrotic neuronal cells of hippocampal ca1, ca2, and ca3 region, and cortical necrotic neurons, perivascular edema, pyknotic neuronal cells, irregularities of intercellular organization (iio) were counted and scaled histopathologically. tissue il-1b, il-6, malonyldialdehyde (mda), myeloperoxidation (mpo), no, and tnf-a levels were measured by using elisa, too. results: sulfasalazine could reduce perivascular edema, iio, cortical and hippocampal neuronal cell death in both stages. it could decreased mda in acute stage, but not reduce il-1b, il-6, mpo, no, and tnfa levels. it could increased il-1b levels in chronic stage but not affect to il-6, mpo, mda, no, tnf-a levels.conclusion: sulfasalazine could improve histopathological architecture of hypoxic tissue in both stages of i/r injury. it could inhibit lipid peroxidation cascades in acute stage but not affect to tissue mpo, no, il-6, and tnf-a levels in any stage in rat. these results suggested that therapeutic mechanisms of sulfasalazine should be investigated by using more specific laboratory methods in future studies.key words: antiinflamatory, cerebral hypoxia reperfusion injury, sulfasalazine, stroke. camel and horse milk xanthine oxidase (xo) was found to catalyze the reduction of nitrate and nitrite to nitric oxide (no) under aerobic condition. to date, mammalian nitrate reductase (nar) and nitrite reductase (nir) have not been identified. no, a gas, is found to control a seemingly, limitless range of functions in animals.one assay was used to determine nar and nir activities of milk xo: (1) nitrite formation from nitrate by nar, and (2) nitrite utilization by nir. nitrite concentrations were determined by using sulfanylamide and n-(1-naphtyl)-ethylenediamine, which form red color measured at 485 nm.these activities of the milk xo require nadh as a physiological electron donor. high xo, nar and nir activities are detected only after heat treatment (80°c, 5 min) of the fresh milk in the presence of molybdate. in both camel and horse milk nar activity of xo was almost two times higher than its nir activity. it is well known that xo can be reversibly converted from the dehydrogenase form to the oxidase through the oxidation of sulfhydryl groups. cysteine and, to a lesser extent, glutathione increased nir activity of milk xo but not its nar activity. the mechanism of this increase of nir activity remains unclear and is currently under study. substitution of tungsten for molybdenum under above conditions gave no detectable nar and nir activity of milk xo. the molybdenum site-directed inhibitor, tungsten inhibited in a dose-dependent manner. therefore, nitrate and nitrite are clear to interact with mo center of xo.camel and horse milk are traditional drinks in central asia and kazakhstan. therefore, it is very important that xo provide a mechanism for generation of no in camel and horse milk where nitric oxide synthase, no producing enzyme, does not exist. p-09.04.4-128 the influence of phytomedicine on metabolic processes of white rats undergone to ionized radiation the study of peroxide lipids oxidation (plo) process is used as one of stability parameters of organism's changes and as a key mechanism for understanding of adaptation reactions and of pathogenesis of different diseases. it's determined by high biological activity of products which are formed in the plo reactions, in this relation lipids with high contents of fat acids play important role. to investigate the influence of phytomedicine eminium regelii on the metabolic processes (peroxide lipids oxidation) of white rats' organism in conditions of ionized radiation.the animals were exposed to ionizing radiation (gamma-radiation 60 co) on the radiotherapeutic equipment teragam in a dose of 6 gy and received phytomedicine eminium regelii in a dose of 2.5 mg/kg orally within 14 days following the ionizing radiation exposure. gamma-rays caused the increase of lipid peroxidation (lpo) primary (dc) and secondary products' (mda) concentrations in spleen, liver, thymus and adrenal glands.treatment by phytomedicine resulted in contents of dc decreased in 3 times in spleen, in 7 times in thymus, in 5 times in adrenal glands, in liver in 4 times, in lymph nodes of small intestine it in 3 times. mda decreased in liver up to 6 and 3 times, in spleen in 3.2 times, in thymus in 9 times, in liver in 6 times, in adrenal glands in 12 time, no changes in lymph nodes of small intestine.the effect of phytomedicine treatment of organisms exposed to sublethal dozes of gamma-radiation results in the lpo primary and secondary products concentrations decrease in spleen, liver, thymus and adrenal glands. p-09.04.4-131 evaluation of serum levels of ischemia modified albumin (ima) in bipolar disorder patients k. € unal 1 , c. topc ßuoglu 2 , m. cingi 3 1 clinic of biochemistry, ankara polatli duatepe public hospital, ankara, 2 clinic of biochemistry, ankara numune training and research hospital, ankara, 3 clinic of psychiatry, ankara numune training and research hospital, ankara, turkey introduction: bipolar disorder is one of the most debilitating psychiatric disorders characterized by disruptive episodes of mania/hypomania and depression. considering the complex role of biological and environmental factors in the etiology of affective disorders; recent studies have focused on oxidative stress, which may damage nerve cell components and take part in pathophysiology. aim of our study is to contribute these data about oxidative stress in bipolar disorder, by detecting ischemia modified albumin (ima) levels of bipolar disorder patients in remission and also by comparing these results with healthy controls. methods: study population consisted of 35 patients meeting the diagnostic and statistical manual of mental disorders, fifth edition (dsm-5) criteria for bipolar disorder i. 36 healthy subjects were included as control group (hc). serum ischemia modified albumin (ima) levels of all participants were determined. results: statistical analysis on serum ischemia modified albumin (ima) levels did not show any significant difference between bipolar disorder patients in remission and healthy controls.conclusion: studies on oxidative stress in bipolar disorder have reached controversial results up till now. in this study, no statistically significant difference was detected between oxidative parameters of bipolar disorder patients in remission and healthy controls. in order to evaluate oxidative stress in bipolar disorder comprehensively, further studies are needed. keywords: bipolar disorder, ischemia modified albumin (ima), oxidative stress p-09.04.4-133 xanthine oxidase and adenosine deaminase activity in patients with familial mediterranean fever (fmf) objective: fmf is an autosomal recessive dissease which is characterized by recurrent fever and inflammation of serous membranes. in this study we measured serum adenosine deaminase (ada) and xanthine oxidase (xo) levels in fmf cases. method: serum ada levels were measured with a sensitive colorimetric method described by giusti and xo levels were analysed by the method of worthington in 30 fmf patients and 30 healthy controls. results: there was a significant difference in xo and ada levels between controls and cases. ada and xo levels were higher in patents with fmf. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of ha. the present study was undertaken in order to evaluate the anticancer properties of the ha using a prostate cancer and osteosarcome cell lines pc-3, sjsa1 as an in vitro model system.ha was purchased from sigma-aldrich. the cells were maintained in dmem medium supplemented with 10% heat-inactivated fbs and 1% penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at 37•c. five different concentrations (100 ug/ml, 50 ug/ml, 25 ug/ml, 10 ug/ ml, 5 ug/ml) were prepared using a stock solution of ha. we measured cell proliferation and migration to understand of progression effects of ha in pc-3 and sjsa1 cell lines in vitro.according to our results, ha treatment caused cytotoxicity and induced cell death in vitro in pc-3 cells with an ic50 value of 67.9 lg/ml. contrary to this, ha induced proliferation of sjsa1 cells in dose dependent manner. ha demonstrated the highest proliferatif activity against sjsa1 cells with an ic50 value of 100 > lg/ml. on the other hand, cell migration was reduced in pc-3 cell line and interestingly, migration was accelerated in sjsa1 cell line.our study may provide new insights into the regulatory effect of ha in cancer, but further studies are needed to clarify the role of ha in cancer pathogenesis. the febs journal 283 (suppl. 1) (2016) key: cord-330668-7aw17jf8 authors: chen, cheng-chang; krüger, jens; sramala, issara; hsu, hao-jen; henklein, peter; chen, yi-ming arthur; fischer, wolfgang b. title: orf8a of sars-cov forms an ion channel: experiments and molecular dynamics simulations date: 2011-02-28 journal: biochimica et biophysica acta (bba) biomembranes doi: 10.1016/j.bbamem.2010.08.004 sha: doc_id: 330668 cord_uid: 7aw17jf8 abstract orf8a protein is 39 residues long and contains a single transmembrane domain. the protein is synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers that forms cation-selective ion channels with a main conductance level of 8.9±0.8ps at elevated temperature (38.5°c). computational modeling studies including multi nanosecond molecular dynamics simulations in a hydrated popc lipid bilayer are done with a 22 amino acid transmembrane helix to predict a putative homooligomeric helical bundle model. a structural model of a pentameric bundle is proposed with cysteines, serines and threonines facing the pore. coronavirus (co-v) is the causal agent responsible for the severe acute respiratory syndrome (sars) [1, 2] . the 29.7 kb genome structure of sars-cov contains a single positive stranded rna harboring 14 open reading frames (orfs) which are translated into a large polyprotein which is processed by viral encoded protease to derive the individual proteins [3, 4] . one of the structural proteins from orf8a encodes a membrane-associated protein with 39 amino acids and contains a single transmembrane domain (tmd) with a length of approximately 22 residues [5] . orf 8a has been proposed not only to enhance viral replication but also to induce apoptosis through a mitochondria-dependent pathway [5] . its topology and biological function remains yet unknown. viral channel forming proteins (vcps) participate in several viral functions. they change chemical or electrochemical gradients by altering ion permeability of the lipid membrane and modulate cellular response by interacting with membrane proteins of the host [6] [7] [8] [9] . several vcps have been detected as ion channels, especially when reconstituted into lipid bilayers. vcps are suggested to be encoded in the genomes of enveloped and naked viruses and also chlorella plant virus pbcv-1. vcps, like host channels and pore forming microbial peptides [10] [11] [12] [13] , have to assemble to form homo oligomeric bundles. they are found to consist of approximately 80 to 100 amino acids. exceptions emerge with the discovery of 3a from sars-cov with a length of 274 amino acids [14] and, based on this study, of orf8a with 39 amino acids. in the presented study, we demonstrate experimentally that orf8a forms channels when reconstituted into artificial lipid bilayers at elevated temperature (38.5°c) . it is suggested that orf8a 3-20 is part of the tmd and multi nanosecond molecular dynamic simulations of orf8a 3-20 within a popc bilayer are undertaken. a set of different oligomeric bundle models raging from 4 to 6 units is generated. the generation of computational bundle models follows the 'two stage model' [15, 16] . in short, this model suggests that the secondary structure of a protein is generated prior to its tertiary or quaternary structure. the identification and structural characterization of the new ion channel protein -orf8a from sars-covwill be helpful for the design of novel drugs against sars. orf8a, mkllivltci 10 slcscictvv 20 qrcasnkphv 30 ledpckvq 39 was synthesized using solid phase peptide synthesis on a 433a synthesizer using fmoc-chemistry and hbtu as coupling reagent. the following side-chain-protecting groups were used: t-butyl for ser and thr, trityl for cys, his, asn and gln, boc for lys and pbf for arg. after completion of the synthesis, the peptide was cleaved from the resin using 95% tfa/water and 3% triisopropylsilane for 3 h at room temperature. after cleavage the crude peptide was precipitated with ether and lyophilized. the crude peptide was purified by preparative rp-hplc on a 21 × 300 mm kromasil column with a flow rate of 70 ml/ min. peaks were detected by uv at 220 nm and the separated product was identified by mass spectrometry. channel recordings of orf8a were made with the protein reconstituted into lipid bilayer mixture of pope (1-palmitoyl-2oleoyl-sn-glycero-3-phosphoethanolamine) and dopc (1,2-dioleoylsn-glycero-3-phosphocholine) (1:4 (volume), total lipid 5 mg/ml). lipids were dissolved in chloroform, dried under n 2 gas and resuspended in pentane and decane 9:1. approximately 1 μl lipid suspension was brushed over the delrin cup aperture (diameter 150 μl) and dried. then 3 μl of peptide dissolved in trifluoroethanol (tfe) were added onto the aperture in the same way as the lipids. the lipid bilayer was formed by raising the level of the buffer (300 mm or 30 mm kcl, 5 mm k-hepes, ph = 7.05) of both trans-(ground) and cis-side (head stage). the current response was recorded using planar lipid bilayer workstation from warner instruments with a bc-353 amplifier and 1440a data acquisition system. temperature was set to 38.5°c using a bipolar temperature controller model cl-100 from warner instruments. a constant positive voltage of 70 mv (cis-side) was applied during lipid bilayer formation to achieve an asymmetric orientation of the peptides within the bilayer [17] . records were filtered with 10 hz using a bessel-8-pole low pass filter, digitized at 10 hz and stored for further analysis. experiments have been repeated in the presence of the reducing agent dithiothreitol (dtt) with the peptide dissolved in tfe containing 50 mm dtt. the application of several transmembrane (tm) prediction tools: das [18] , tmhmm [19] , tmpred [20] , split4.0 [21] and sosui [22] , which assume helical tm segments, have been used to detect the tm helical segments of the protein. an ideal helix of tmd of orf8a (llivltci 10 slcscictvv 20 , 8a 3-20 ) has been created using integrated protein builder of moe (www.chemcomp.com). the helix was embedded into a lipid bilayer (popc). overlapping lipid molecules were removed using the moe suite. the lipid-peptide system was then further processed using gromacs-3.3 (www.gromacs.org). the topology and structure of the popc bilayer was prepared as described elsewhere [23] . the lipid-peptide system was hydrated and a short minimization routine using steepest descent and conjugate gradients followed to remove unfavorable interactions. a short equilibration (500 ps) followed with the peptide restrained at the cα atoms. at this stage the lipids were expected to surround the peptide adequately. in the production run (15 ns) all components were fully unrestrained. the program g_covar from the gromacs-3.3 package was used to perform principal component analysis (pca). the covariance matrix of positional variation was computed for the full 10 ns simulation length for the main-chain, based on the square fit of the cα-atoms of tm residues 3-20. the rotational and translational motions were removed by fitting the peptide structure of each time frame to the initial structure. the derived averaged monomer structure was then used in moe to generate tetrameric (tbms), pentameric (pbms) and hexameric bundle models (hbms) [24] by creating symmetric copies of monomeric units around a central pore axis. degrees of freedom such as the rotational angle, inter-helical distance and tilt angle (further on referred to as 'tilt') were changed systematically. to sample the whole conformational space of the bundles each of the degrees of freedom is varied stepwise (inter-helical distance 0.1 å, rotational angle 2°and tilt 2°). for each position the side chains were linked with the backbone. side chain conformation is chosen to be the most likely one for the given backbone position and referenced in the moe library. a short energy minimization (15 steps of steepest decent) followed the linking. consequently the potential energy was calculated using the engh-huber force field implemented in moe. more than one hundred thousand conformations are generated and stored in a database for further analysis. before embedding low energy models into lipid bilayers two amino acids residues of the protein were added at the n and c termini of each of the helices in each bundle model to account for the consequences of their interaction with the lipid bilayer during the simulation. short ideal helices from met-1 to ile-5 and thr-18 to arg-22 were generated. they were aligned and superimposed with those residues from the bundle models. those residues of the short helices which overlapped with the residues of the bundle models have been deleted and the remaining residues of the short helices conjoined with residues of the bundle models. this finally delivered bundle models of 8a . the selected bundle models were then embedded into a pre-equilibrated (70 ns) hydrated popc bilayer [23] . gromacs-3.3 with the gromos96 (ffg45a3) force field was used for the simulations with an integration step size of 2 fs. the temperature of the peptide, lipid and the water molecules were individually coupled to a berendsen thermostat with a coupling time of 0.1 ps. isotropic pressure coupling was used with a coupling time of 1.0 ps and a compressibility of 4.5e-5 bar −1 . long range electrostatics was calculated using the particle-mesh ewald (pme) algorithm with grid dimensions of 0.12 nm and interpolation order 4. lennard-jones and short-range coulomb interactions were cut off at 1.4 and 0.8 nm, respectively. the simulation boxes contained around 26,011 atoms for a b fig. 1 . identification of the putative membrane-spanning domain of the 39 amino acid sequence of orf8a using secondary structure prediction tools (a). amino acids chosen to model a helical tmd are shown in italics. orf8a 2-20 ideal helix including the bold residues shown in its 'gaussian contact' representation (moe) (b). hydrophilic and hydrophobic residues are highlighted in black and light gray, respectively. the tetrameric models (protein 828 atoms, lipids 5824 atoms, water molecules 19,359 atoms), 26,218 atoms for the pentameric model (protein 1035 atoms) and 26,425 atoms for the hexameric models (protein 1242 atoms). calculations of the root mean square deviations (rmsd) were based on the cα atoms. the simulations were run on a dell precision 490n workstation and a 28 core opteron based compute cluster with infiniband interconnects. all pictures were generated using xmgrace, vmd-1.8.6 and moe-2007.09. application of several tm prediction tools suggests a range of amino acids within the n terminal part of the protein (fig. 1a) . the longest stretch is suggested by sosui from lys-2 to ala-28 and the shortest stretch by das (val-5 to thr-18). all other programs place the start of the helix between residues lys-2 to ile-4 and the end between thr-18 to ala-28. for the experimental and computational investigations a consensus length of 18 residues from leu-3 to val-20 is chosen. channel recordings of sars orf8a at a kcl concentration of 300 mm at 38.5°c show frequent openings ( fig. 2a) . the temperature has been chosen to simulate elevated body temperature occurring during fever. the lipid composition and their individual components have been proven successful in previous investigations [25] . dopc is also successfully used in bilayer recordings [26] and is known to reduce packing density and enhance the integration of the peptide into the bilayers [27] . the calculated conductance histograms of the recorded data enlighten a major conductance state of 8.8 ± 0.8 ps (fig. 2b ) and an ohmic behavior (fig. 2c) . experiments have also been conducted in the presence of dtt (fig. s1a) , since the sequence of the peptide contains four cysteines (fig. 1a) . the overall behavior of the collected data remains the same as for the experiments without dtt, with a mean conductance of 8.9 ± 0.1 ps (fig. s1b ) and an ohmic channel behavior (fig. s1c ). all data in common is an increased open duration of the channels at negative potential. ion selectivity of orf8a was evaluated with asymmetric buffer concentration (ten-fold in trans chamber) which shifts the potassium reversal potential independent of the presence of dtt to approximately 30-40 mv (figs. 2d and s1d) . the shift is indicative for a weak cation-selective channel. the data show a mean conductance of 8.3 ± 0.1 ps (fig. 2e ) and 8.6 ± 0.1 ps in the presence of dtt (fig. s1e ) and ohmic behavior (figs. 2f and s1f). the idealized monomeric tm helix based on the consensus sequence leu-3 to val-20 (fig. 1a) shows clustering of hydrophilic residues (thr-8, ser-11, ser-14 and thr-18) on one side suggesting that the four hydrophilic amino acids form the lumen of the pore in a homooligomeric helical bundle channel model. four cysteines are forming a diagonal arrangement excluding any possibility for interhelical cys-bridges (fig. 1b) . the generation of a series of assembled pore models follows a protocol which was reported earlier [23, 24] . a single ideal helix (8a [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] ) is embedded in a hydrated lipid bilayer and undergoes a 10 ns md simulation. an averaged helix from this simulation is generated based on a pca analysis. calculating the eigenvector quantifies the magnitude of the functional motion of the protein. averaging over all structures of the first few eigenvectors delivers a structure of the helix which is the most likely one. this structure is then used to build the bundle models. the use of the truncated part of 8a, 8a [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] , is rationalized by the understanding that the hydrophobic part of the tmd guides the assembly. in the current assembly protocol any unfavorable interactions due to hydrophilic residues at either end of the helix would be over emphasized. with 8a 3-20 residues like lys-2 and arg-22 are not included during assembly. protein-protein interactions in respect to their potential and their individual three degrees of freedom are calculated. with decreasing inter-helical distance the energy values reach a minimum around 9.5 å for the tbms and pbms (fig. 3a) . for both models some low energy models can also be observed around 8.5 å. the hbms have favorable energy values for inter-helical distances between 10 å and 11 å (fig. 3a, left column) . the lowest energy conformation in a minimum is seen as representative for similar conformations clustering around it. energy versus rotational angle correlation plots show three minima for all models which are separated by 150-160° (fig. 3a , middle column). each of the models adopts a minimum energy conformation for a left-handed tilt of around + 25°to +35° (fig. 3a , right column). for the right handed models a minimum is present at −10°and around −35°. all bundles in common are rings of cysteines (cys-13, -15, and -17), threonines (thr-8), serines (ser-11) and arginines (arg-22, for pbm2 and pbm3 only) facing the lumen of the pore (fig. 4) . all hydrophilic and polarizable residues are separated by stretches of hydrophobic residues of various lengths. hbm1 and hbm3 exhibit the longest hydrophobic stretches within the lumen. three low energy pore models for each of the oligomers have been chosen for another 15 ns md simulation to further minimize the proposed bundle models. the rmsd plots for all models rise during the first nanosecond to level off at around 1 to 3 å (fig. 3b) . in one of the tbms (tbm3) the spread of the rmsd values for the individual helices of the bundle is minimal, indicative for the helices retaining its starting structure (fig. 3b, upper trace) . for pbms a moderate spread is observed (fig. 3b, middle trace) whilst for the hbms the largest spread can be detected (fig. 3b, lower trace) . consequently, minor conformational adjustments away from the assembled structure occur. the tilt angles averaged over the last 5 ns of each simulation are in the order of +30°to + 40°and −25°to −30°( table 1) . the values are almost unchanged in respect to the values found during the assembly process (fig. 3a) . the tilts show the lowest deviations for tbms and pbms. the averaged inter-helical distance of the tbms is between 6-7 å (table 1) . for the pbms the inter-helical distance is larger with values of 8 to 11 å. in the hbms the distance is around 9 å, but shows large standard deviation. visual inspection of all bundles reveals that most of the bundles fail to retain a circular pore assembly except tbm3, pbm2 and pbm3. the averaged minimum pore radii for the models are calculated (hole) to be (4.4 ± 0.2) å for pbm2 and (3.5 ± 0.4) å for pbm3 ( table 1 ). the minimum pore radius can be located with an accuracy of ±2.3 å for pbm2 and ±2.7 å for pbm3. for tbm3 the pore radius is calculated to be below 1.5 å over the entire length of the pore which is too small to harbor any water molecules. as a result, water filled bundles of the tmd of orf8a should adopt approximately a +40°tilt with a distance between the helices in respect to their long axis of 11 å (fig. 5 for pbm2). with this large tilt hydrophilic residues thr-8 and ser-11 as well as cys-15 of one helix form the hydrophilic stretch, whilst ser-14 and thr-18 are positioned at an inter-helical site. lys-2 remains in an outward position 'snorkling' for the aqueous phase and the lipid head groups. the side chains of all the arginines (arg-22) move from an all inward facing position to such a position that they point either outside the pore or towards the helix interface interacting with gln-21 of the neighboring helix. all assemblies in common is also a line of cysteines (cys-9, -13, and -17) in each helix at the outside of the bundle (fig. 5, lower left) . the hour glass shape of the water column is shown in fig. 5 (lower right). the model underpins the necessity of a balanced pattern with alternating hydrophilic and hydrophobic residues mantling the pore to allow the existence of a continuous water column through the pore. the calculated conductance data of the ofr8a protein and its weak selectivity for cations is in accordance with findings of other viral channel forming proteins [25, [28] [29] [30] . selectivity has so far been reported for viral proteins with a single (e.g. vpu from hiv-1) or double tm topology (e.g. p7 from hcv). even though assuming that selectivity is coupled with flexibility and gating [31] it may be argued that with the helical topologies of a single and eventually double tmd no highly selective channels can be formed via self-assembly. in the protocol for the assembly of the tm helices of 8a a combination of molecular dynamics simulations with a fine grained positioning routine is used [23, 24] . the positioning routine moves the helix on the bases of the backbone atoms to a specific position and consequently generates the side chains for each particular position. in this protocol a vacuum force field (engh-huber [32] ) has been used. in another study, helix assembly undertaken in vacuum have been compared with data derived from md simulations in a hydrated lipid bilayer and reported to explore the same conformational space [33] . this makes the use of vacuum conditions in combination with a fine grained search of the conformational space, a powerful tool [34] . applying md simulations after the assembly step aims to address for the specific environment of the bundle structure such as phospholipid head group region, hydrophobic slab as well as the hydrophilic environment within the lumen of the pore. this protocol is along side of other protocols used to model tm helix assembly and bundle formation [35] [36] [37] [38] [39] . in some of these protocols a limited number of model structures is generated [35, 36] and in other methods larger numbers of models are generated through extended simulation protocols and assessed [37, 38] . all protocols so far do not account for kinetics of the assembly of larger units such as bundles or pores. the pore-lining pattern discovered in the model supports models from other viral channel forming proteins which have been derived using more biased model generation protocols in the past [40, 41] . based on experimental findings hydrophilic residues such as threonine, serine and charged residues such as aspartic and glutamic acids [42] as well as arginines and lysines, are seen as essential porelining motif for ion channels [43, 44] and viral channel forming proteins [6, 25, 45] . a cysteine residue has been suggested to be at least transiently part of the pore-lining motif of the human concentrative nucleoside transporter 3 [46] . for the transient receptor potential (trp) channels conserved cysteine residues within the pore region have been reported to be essential for channel function [47] . also the ampa receptor has a cysteine facing the lumen of the pore of the channel [48] . the cysteines at the outside of the bundles may be able to lead to covalent linkages with host cell proteins or possibly an attachment to lipid rafts or other membrane based host cell factors. also some of the threonines and serines are not solely pore lining but enable stabilize inter-helical interaction, a feature which has been suggested by modeling other bundle systems [49] . the results indicate that the helices in the pentameric bundles are strongly tilted having the largest inter-helical distance. a straightening will not allow a water filled column to exist. the lower tilt angles of the tetra and hexameric bundles match nmr spectroscopic data on equivalent single peptides in equivalent lipids [50, 51] and consecutive md simulations [51] . a series of md simulations on the tmd of vpu in various lipid bilayer systems have shown that there is no simple correlation between tilt angle and lipid thickness [23] . often the tilt shows rather large fluctuations and a mismatch is compensated by an alternating kink angle. with lys-2 and arg-22 and their flexible and extended side chains [52] at either end of the tmd a large tilt in the pentameric assembly is by all means possible for the 8a peptide table 1 interhelical distances and tilt angles calculated between the centers of mass of each helix based on the cα backbone atoms. values are averaged over the last 5 ns of the 15 ns md simulation runs. minimum pore radius (mpr) is calculated as an average over six bundle models taken at 10, 11, 12, 13, 14 and 15 ns using the program hole [44] . bundle. computational studies on a model including the extramembrane parts are necessary to further evaluate the shape of the 'proper' bundle. one the bases of the applied assembly protocol a pentameric bundle is harboring a column of water molecules during the extended equilibration of a md simulation. all other models lose their circular assembly structure by approaching each other as if the pore is 'squeezed'. a similar situation has been reported also for simulations on the tmds of vpu from hiv-1 [53] . the size of the pore in the pbms would allow the physiological relevant ions to pass. the existence of a continuous water column in a particular bundle model may of course not be the exclusive criteria. functional studies will be the next level of screening. there is no possibility for the two bundles pbm2 and pbm3 to interchange based on the energy landscape. it only needs minor changes in the tilt and distance towards the central axis, but the rotational change of almost 180°has to cross a larger barrier (fig. 3a , middle column) in order to transform pbm2 into pbm3. it needs further computational functional analysis to discriminate between the models such as simulating the passage of an ion through each of the pore and evaluating the potential of the mean force [41] . at this stage it is suggested that both models would be present if generated under the present experimental conditions. this may reflect the situation in vivo, assuming that the proteins first diffuse by themselves freely for a short while until they are finally assembled. in case of an instant assembly at the site of in vivo production in the endoplasmic reticulum, the conformational available space would be possibly limited and a single model may emerge. the protein 8a encoded by sars-cov has a single tmd being able to form weak cation-selective channels most likely in a pentameric homomeric assembly. computational models of the assembled tmd reveal that a pentameric bundle is most likely to harbor a continuous water column. in such a bundle model a polarizable cysteine residue and hydrophilic residues such as serines and threonines are facing the pore. the putative water filled pentameric bundle of the tmd of 8a should be made of helices positioned in an approximately 11 å distance in respect to their helical long axis and with a tilt of about +40°. supplementary materials related to this article can be found online at doi:10.1016/j.bbamem.2010.08.004. sars -beginning to understand a new virus a novel coronavirus associated with severe acute respiratory syndrome isolation and characterization of viruses related to the sars coronavirus from animals in southern china viral ion channels: structure and function structure-function correlates of vpu, a membrane protein of hiv-1 viral channel forming proteins membrane composition determines paradixin's mechanism of bilayer disruption structure and orientation of pardaxin determined by nmr experiments in model membranes alamethicin aggregation in lipid membranes nmr structure of pardaxin, a pore-forming antimicrobial peptide, in lipopolysaccharide micelles. mechanism of outer membrane permeabilization severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release membrane protein folding and oligomerization: the two-stage model membrane protein folding: beyond the two stage model towards a mechanism of function of the viral ion channel vpu from hiv-1 prediction of transmembrane alpha-helices in procariotic membrane proteins: the dense alignment surface method a hidden markov model for predicting transmembrane helices in protein sequences tmbase -a database of membrane spanning protein segments basic charge clusters and prediction of membrane protein topology sosui: classification and secondary structure prediction system for membrane proteins exploring the conformational space of vpu from hiv-1: a versatile and adaptable protein assembly of viral membrane proteins biophysical characterisation of vpu from hiv-1 suggests a channel-pore dualism melittin induced voltage-dependent conductance in dopc lipid bilayers the role played by lipids unsaturation upon the membrane interaction of the heliobacter pylori hp(2-20) antimicrobial peptide analogue hpa3 identification of an ion channel activity of the vpu transmembrane domain and its involvement in the regulation of virus release from hiv-1-infected cells the vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels cation-selective ion channels formed by p7 of hepatitis c virus are blocked by hexamethylene amiloride activation-dependent subconductance levels in the drk1 k channel suggest a subunit basis for ion permeation and gating accurate bond and angle parameter for x-ray proteinstructure refinement molecular dynamics simulations of the erbb-2 transmembrane domain within an explicite membrane environment: comparison with vacuum simulations mapping the energy surface of transmembrane helixhelix interactions experimentally based orientational refinement of membrane protein models: a structure for the influenza a m2 h + channel the structure of the hiv-1 vpu ion channel: modelling and simulation studies membrane assembly of simple helix homo-oligomers studied via molecular dynamics simulations side-chain contributions to membrane protein structure and stability solving the membrane protein folding problem bundles consisting of extended transmembrane segments of vpu from hiv-1: computer simulations and conductance measurements reconstructing potentials of mean force from short steered molecular dynamics simulations of vpu from hiv-1 structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel evidence that the m2 membrane-spanning region lines the ion channel pore of the nicotinic receptor bundles of amphipathic transmembrane a-helices as a structural motif for ion-conducting channel proteins: studies on sodium channels and acetylcholine receptors alphavirus 6k proteins form ion channels a proton-mediated conformational shift identifies a mobile pore-lining cysteine residue (cys-561) in human concentrative nucleoside transporter 3 conserved cysteine residues in the pore region are obligatory for human trpm2 channel function channel-lining residues of the ampa receptor m2 segment: structural environment of the q/r site and identification of the selectivity filter solid-state nmr and molecular dynamics simulations reveal the oligomeric ion-channels of tm2-gaba a stabilized by intermolecular hydrogen bonding tilt angle of a trans-membrane helix is determined by hydrophobic mismatch structure, topology, and tilt of cell-signaling peptides containing nuclear localization sequences in membrane bilayers determined by solid-state nmr and molecular dynamics simulation studies snorkeling of lysine side chains in transmembrane helices: how easy can it get? simulation of the hiv-1 vpu transmembrane domain as a pentameric bundle wbf acknowledges the national yang-ming university and the government of taiwan for financial support (aim of excellence program), as well as the national science council of taiwan (nsc). jk acknowledges a fellowship granted jointly by the alexander von humboldt-foundation and nsc.